JP2006117648A - Anticancer agent containing tamoxifen analogue as active ingredient - Google Patents
Anticancer agent containing tamoxifen analogue as active ingredient Download PDFInfo
- Publication number
- JP2006117648A JP2006117648A JP2005274471A JP2005274471A JP2006117648A JP 2006117648 A JP2006117648 A JP 2006117648A JP 2005274471 A JP2005274471 A JP 2005274471A JP 2005274471 A JP2005274471 A JP 2005274471A JP 2006117648 A JP2006117648 A JP 2006117648A
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- Prior art keywords
- formula
- compound
- tamoxifen
- compound represented
- active ingredient
- 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.)
- Granted
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- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 13
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- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
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- 125000002252 acyl group Chemical group 0.000 claims description 3
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Abstract
Description
本発明は、タモキシフェン類縁体を有効成分として含有する抗ガン剤、タモキシフェン類縁体の製造方法、及び新規タモキシフェン類縁体に関する。 The present invention relates to an anticancer agent containing a tamoxifen analog as an active ingredient, a method for producing a tamoxifen analog, and a novel tamoxifen analog.
タモキシフェンは、以下の構造を有する化合物であり、ホルモン依存性乳ガンの治療薬として用いられてきた。 Tamoxifen is a compound having the following structure and has been used as a therapeutic agent for hormone-dependent breast cancer.
タモキシフェンは、組織特異的にエストロゲン性、あるいは抗エストロゲン性作用を有し、乳房細胞においては、抗エストロゲン作用を有することが知られている。
エストロゲンは、特に乳ガン細胞のエストロゲン受容体に結合し、ガン細胞の増殖を促進するが、タモキシフェンは、エストロゲンと競合的にエストロゲン受容体と結合することにより、エストロゲンとエストロゲン受容体の結合を阻止し、ガン細胞の増殖を抑制する。
このような作用を有するタモキシフェンにおいては、現在まで様々な類縁体が合成されており、これら類縁体を例示すると、４−ヒドロキシタモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、イドキシフェン等が挙げられる。
Tamoxifen is known to have an estrogenic or anti-estrogenic action in a tissue-specific manner and to have an anti-estrogenic action in breast cells.
Estrogen specifically binds to the estrogen receptor of breast cancer cells and promotes the growth of cancer cells, while tamoxifen blocks the binding of estrogen and estrogen receptor by binding to estrogen receptor competitively with estrogen. Suppresses the growth of cancer cells.
In tamoxifen having such an action, various analogs have been synthesized so far. Examples of these analogs include 4-hydroxy tamoxifen, 4-bromo tamoxifen, 3-iodo tamoxifen, idoxifene and the like.
また、これらタモキシフェン類縁体を合成するための製法についても、様々なものが提案され、本発明者等も、オレフィン部位を有するタモキシフェン類縁体の前駆体を、異性化触媒として酸性物質あるいはアルカリ性物質を使用して転移させることにより、安価かつ効率的にタモキシフェン類縁体を合成する方法を開発している（特許文献１）。
しかし、これら従来のタモキシフェン類縁体においては、幾何異性体としてＺ体、Ｅ体が存在し、Ｚ体のみが抗ガン作用を有するので、Ｚ体を分離する等の手段が必要であり、また、これらタモキシフェン誘導体は、もっぱらホルモン依存性乳ガンの治療剤として用いられており、他のガンについても有効であるとの実証はまったくなされていない。
In addition, various methods for synthesizing these tamoxifen analogs have been proposed, and the present inventors have also used an acidic substance or an alkaline substance as an isomerization catalyst by using a precursor of a tamoxifen analog having an olefin moiety. A method for synthesizing tamoxifen analogs inexpensively and efficiently by using them and transferring them has been developed (Patent Document 1).
However, in these conventional tamoxifen analogs, Z-form and E-form exist as geometrical isomers, and only the Z-form has an anti-cancer action, and thus means such as separation of the Z-form is necessary. These tamoxifen derivatives have been used exclusively as therapeutic agents for hormone-dependent breast cancer and have not been proven to be effective for other cancers.
本発明の課題は、従来のタモキシフェン類縁体よりも、より立体構造的にエストロゲンと近似するとともに、さらに、効率的に製造し得るタモキシフェン類縁体を見いだし、その薬理作用を明らかにして、新たな抗ガン剤を提供するとともに、該類縁体の製造手段として、極めて効率的な手段を提供することにある。 The problem of the present invention is to find a tamoxifen analog that can be more efficiently produced and more efficiently approximated to estrogen than conventional tamoxifen analogs, and to clarify the pharmacological action of the analog and to provide a new anti-tumor. An object is to provide a cancer agent and to provide a very efficient means for producing the analog.
本発明者らは、鋭意研究の結果、以下の（１）に示されるタモキシフェン類縁体が、エストロゲンと立体構造的に近似することを見いだし、さらに、該タモキシフェン類縁体が、特に白血病細胞に対し優れた細胞障害性を有するという全く予想できない作用を有し、該タモキシフェン誘導体が抗ガン剤としてきわめて有用であるという知見を得るとともに、該タモキシフェン類縁体が、その構造上の特徴によって、Ｚ体の分離等の手段が不要であり、きわめて効率的に製造可能であることを確認した。また、上記タモキシフェン類縁体の作用メカニズムの解明を進めた結果、これらタモキシフェン類縁体が、血管新生を抑制するという驚くべき知見を得て、本発明を完成するに至った。
すなわち、本発明は、以下の（１）〜（９）のとおりである。
As a result of intensive studies, the present inventors have found that the tamoxifen analog shown in the following (1) is three-dimensionally similar to estrogen, and further, the tamoxifen analog is particularly excellent for leukemia cells. In addition to the fact that the tamoxifen derivative is extremely useful as an anticancer agent, the tamoxifen analog is separated from the Z-form by its structural characteristics. Thus, it was confirmed that it was possible to manufacture very efficiently. Moreover, as a result of advancing the elucidation of the mechanism of action of the above tamoxifen analogs, the inventors have obtained a surprising finding that these tamoxifen analogs suppress angiogenesis, and have completed the present invention.
That is, this invention is as the following (1)-(9).
（１）：以下の式（Ｉ）で表わされる化合物を有効成分として含有することを特徴とする、抗ガン剤。 (1): An anti-cancer agent comprising a compound represented by the following formula (I) as an active ingredient.
（但し、式中Ｒ１及びＲ２は、水素又はそれぞれ同一あるいは異なるアルキル基を表わし、Ｒ１とＲ２は一緒になって環を形成してもよい。また、ｎは０を含む整数を表わす。）
（２）：有効成分が以下の式（II）で表わされる化合物である、前記第（１）項に記載の抗ガン剤。
(However, in the formula, R 1 and R 2 each represent hydrogen or the same or different alkyl group, and R 1 and R 2 may combine to form a ring. Also, n represents an integer including 0. Represents.)
(2): The anticancer agent according to item (1), wherein the active ingredient is a compound represented by the following formula (II).
（３）：有効成分が以下の式（III）で表わされる化合物である、前記第（１）項に記載の抗ガン剤。
(3) The anticancer agent according to item (1), wherein the active ingredient is a compound represented by the following formula (III).
（４）：有効成分が以下の式（IＶ）で表される化合物である、前記第（１）項に記載の抗ガン剤。
(4) The anticancer agent according to item (1), wherein the active ingredient is a compound represented by the following formula (IV).
（５）：上記式（Ｉ）〜（IＶ）のいずれかで表される化合物を有効成分として含有することを特徴とする、血管新生抑制剤。
（６）：以下の式（III）で表わされる、化合物。
(5): An angiogenesis inhibitor comprising a compound represented by any one of the above formulas (I) to (IV) as an active ingredient.
(6): A compound represented by the following formula (III).
（７）：以下の式（IＶ）で表される、化合物。
(7): A compound represented by the following formula (IV).
（８）：式（７）で表わされる化合物と、式（８）で表わされる化合物とを塩基の存在下で反応させることを特徴とする、前記第（１）項に記載の式（Ｉ）の化合物の製造方法。
(8): A compound represented by formula (7) and a compound represented by formula (8) are reacted in the presence of a base. A method for producing the compound.
（但し、式中Ｒ１及びＲ２は、水素又はそれぞれ同一あるいは異なるアルキル基を表わし、Ｒ１とＲ２は一緒になって環を形成してもよい。また、Ｒ３はハロゲン原子、アルキル硫酸エステル残基又はトリフルオロメタン硫酸エステル残基を表わし、ｎは０を含む整数を表わす。）
（９）：式（７）の化合物が、下記の式（１）、（２）及び（３）で表わされる化合物を酸触媒の存在下で反応させて、下記の式（４）の化合物を生成させ、以下順に、加水分解反応、２重結合のマイグレーション化反応及びアルコキシ基の脱アルキル反応を行なうことにより製造されたものである、前記第（８）項に記載の製造方法。
(However, in the formula, R 1 and R 2 each represent hydrogen or the same or different alkyl group, and R 1 and R 2 may be combined to form a ring. R 3 is a halogen atom or an alkyl group. Represents a sulfate ester residue or a trifluoromethane sulfate ester residue, and n represents an integer including 0.)
(9): A compound of the formula (7) is reacted with a compound represented by the following formulas (1), (2) and (3) in the presence of an acid catalyst to give a compound of the following formula (4): The production method according to item (8), which is produced by performing hydrolysis reaction, double bond migration reaction, and alkoxy group dealkylation reaction in this order.
（但し、式中Ｒ４はアルカノイル基を表わす。）
(In the formula, R 4 represents an alkanoyl group.)
（但し、式中Ｒ５はアルキル基を表わす。）
(In the formula, R 5 represents an alkyl group.)
（但し、式中Ｒ４及びＲ５は上記と同様の置換基を表わす。）
なお、上記式（II）の化合物を、以下リダイフェンＡといい、式（III）、（IＶ）の化合物をそれぞれリダイフェンＢ、リダイフェンＣという。
(However, in the formula, R 4 and R 5 represent the same substituents as described above.)
The compound of the above formula (II) is hereinafter referred to as “redifen A”, and the compounds of the formulas (III) and (IV) are referred to as “redifen B” and “redifen C”, respectively.
本発明のタモキシフェン類縁体は、より立体構造的にエストロゲンと近似し、特に白血病細胞に対して、その増殖を極めて効果的に抑制する。このことは、ホルモン依存性乳ガン治療剤として知られていた、タモキシフェン類縁体の作用効果としては、全く予想外のものである。また、これらタモキシフェン類縁体は、血管新生抑制作用を有する。血管新生は、ガン、糖尿病性網膜症、あるいは慢性関節リュウマチ等数多くの疾患の発生あるいは悪化に必要不可欠であることが明らかにされており、特にガンによる血管新生は、ガン細胞の増殖、湿潤、転位の原因でもある。したがって、この点で、本発明のタモキシフェン類縁体は、これら疾患、特にガンの新規な治療法の開発等において大いに寄与するものである。
一方、本発明のタモキシフェン類縁体は対称型の置換アミノエチルオキシフェニル基を有し、したがって幾何異性体が存在しないため、幾何異性体の分割あるいは立体特異的な合成工程が不要で、１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（式（７）の化合物）から１工程で、極めて効率的に製造できるという利点がある。
The tamoxifen analog of the present invention more closely approximates estrogen in a three-dimensional structure, and suppresses the proliferation thereof particularly effectively against leukemia cells. This is completely unexpected as the effect of the tamoxifen analog, which has been known as a therapeutic agent for hormone-dependent breast cancer. Moreover, these tamoxifen analogs have an angiogenesis inhibitory action. Angiogenesis has been shown to be essential for the development or exacerbation of numerous diseases such as cancer, diabetic retinopathy, or rheumatoid arthritis. It is also a cause of dislocation. Therefore, in this respect, the tamoxifen analog of the present invention greatly contributes to the development of a novel therapeutic method for these diseases, particularly cancer.
On the other hand, the tamoxifen analog of the present invention has a symmetric type substituted aminoethyloxyphenyl group, and therefore there is no geometric isomer, so no geometric isomer resolution or stereospecific synthesis step is required. There is an advantage that it can be produced very efficiently from -bis (4-hydroxyphenyl) -2-phenyl-1-butene (compound of formula (7)) in one step.
本発明のタモキシフェン誘導体は、以下の式（Ｉ）で表わされ、 The tamoxifen derivative of the present invention is represented by the following formula (I):
（但し、式中Ｒ１及びＲ２は、水素又はそれぞれ同一あるいは異なるアルキル基を表わし、Ｒ１とＲ２は一緒になって環を形成してもよい。また、ｎは０を含む整数を表わす。）
その化学構造上の特徴は、ブテンの末端２重結合の炭素原子に２つの同一置換基を有するフェニル基が置換されている点にあり、これにより幾何異性体が存在しない。
これに対して、上記式（ａ）で表わされるタモキシフェンは、ブテンの末端２重結合の炭素原子に、互いに置換基の異なるフェニル基が置換しているため、幾何異性体が存在する。タモキシフェンの活性はそのうちのＺ体にあるので、このＺ体を得るための工程を必要としていた。この点は、以下に示される従来のタモキシフェン類縁体も幾何異性体が存在する点で同様である。
上記式（Ｉ）中、Ｒ１及びＲ２が形成する環としては、例えばピロリジン環、ピリミジン環、モルフォリン環等が挙げられる。また、ｎは、例えば１〜３０、好ましくは１〜１０、より好ましくは１〜５である。
(However, in the formula, R 1 and R 2 each represent hydrogen or the same or different alkyl group, and R 1 and R 2 may combine to form a ring. Also, n represents an integer including 0. Represents.)
The chemical structure is characterized in that a phenyl group having two identical substituents is substituted on the carbon atom of the terminal double bond of butene, whereby there is no geometric isomer.
On the other hand, tamoxifen represented by the above formula (a) has a geometric isomer because a phenyl group having a different substituent is substituted on the carbon atom of the terminal double bond of butene. Since the activity of tamoxifen is in the Z form, a process for obtaining this Z form was required. This is the same in that the conventional tamoxifen analog shown below also has geometric isomers.
In the above formula (I), examples of the ring formed by R 1 and R 2 include a pyrrolidine ring, a pyrimidine ring, and a morpholine ring. Moreover, n is 1-30, for example, Preferably it is 1-10, More preferably, it is 1-5.
本発明のタモキシフェン類縁体の構造は、上記製法上の利点に加えて、立体構造的に他の類縁体よりもよりエストロゲンに類似しているため、エストロゲン受容体に対して、高い親和性を有し、エストロゲンの受容体との結合を効果的に阻止するものと思われるが、特に白血病細胞に対して優れた細胞障害性を有することは全く予想外のことである。
図１は、本発明のタモキシフェン類縁体の一種である１，１−ビス（ジメチルアミノエトキシフェニル）２−フェニル−１−ブテンの立体構造を模式的に示したものである。本発明のタモキシフェン類縁体であるリダイフェンＡ（１，１−ビス（ジメチルアミノエトキシフェニル）２−フェニル−１−ブテン）と、タモキシフェン、イドキシフェンとにおいて、ブテン部分の２重結合面に対する各置換、非置換フェニル基が形成するａ，ｂ，ｃの２面角を、表１に示す。
また、エストラジオールにおける図１に示す２面角ａ、ｂ、ｃを表１に併せて示す。
The structure of the tamoxifen analog of the present invention has a high affinity for the estrogen receptor because it is more structurally similar to estrogen than other analogs, in addition to the above-mentioned manufacturing advantages. However, although it seems to effectively block the binding of estrogen to the receptor, it is quite unexpected to have excellent cytotoxicity especially against leukemia cells.
FIG. 1 schematically shows the three-dimensional structure of 1,1-bis (dimethylaminoethoxyphenyl) 2-phenyl-1-butene, which is a kind of tamoxifen analog of the present invention. In the tamoxifen analog of the present invention, reififene A (1,1-bis (dimethylaminoethoxyphenyl) 2-phenyl-1-butene), tamoxifen, and idoxifene, each substitution on the double bond surface of the butene moiety, Table 1 shows the dihedral angles of a, b, and c formed by the substituted phenyl group.
Further, dihedral angles a, b, and c shown in FIG. 1 for estradiol are also shown in Table 1.
表１に示したように、本発明のタモキシフェン類縁体は、ａの２面角がエストラジオールのそれと一致し、また、他の類縁体に比べｃの２面角が最も小さいので平面性が高く、エストラジオールの構造に近い。
As shown in Table 1, the tamoxifen analog of the present invention has high planarity because the dihedral angle of a matches that of estradiol, and the dihedral angle of c is the smallest compared to other analogs. Close to the structure of estradiol.
本発明のタモキシフェン誘導体のうち式（II）で示される化合物は公知物質であるが、該化合物の抗ガン作用は実証されていない。一方、式（III）、（IＶ）で表わされる化合物は、新規化合物である。
これら本発明のタモキシフェン化合物の製法について、図２を参照して以下に説明する。本発明の式Ｉで表わされる化合物は、直接的には、式（７）の化合物（１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテンと、一般式（８）の化合物をそれぞれＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）、ジメチルスルホキシド（ＤＭＳＯ）等の極性溶媒からなる反応溶媒中において、触媒として、例えば、水素化ナトリウム等の塩基性化合物の存在下反応せしめることにより得られる（図２中、工程５）。
この工程の触媒としては、上記水素化ナトリウムに限らず、例えば、アルカリ金属、アルカリ金属塩等の塩基性化合物、遷移金属塩等の遷移金属触媒などが挙げられる。
これらは、１種単独で使用してもよく、２種以上を併用してもよい。
反応溶媒としてはＤＭＳＯ（ジメチルスルホキシド）、ＤＭＦ（Ｎ，Ｎ−ジメチルホルムアミド）、ＤＭＥ（１，２−ジメトキシエタン）等の極性溶媒の他、さらにヘキサン、ベンゼン、石油エーテル等の非極性溶媒等も使用でき、これら溶媒は１種単独で使用してもよく、２種以上を併用しても良い。
Among the tamoxifen derivatives of the present invention, the compound represented by the formula (II) is a known substance, but the anticancer action of the compound has not been demonstrated. On the other hand, the compounds represented by the formulas (III) and (IV) are novel compounds.
The method for producing these tamoxifen compounds of the present invention will be described below with reference to FIG. The compound represented by the formula I of the present invention is directly represented by the compound of the formula (7) (1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene and the compound of the general formula (8) Obtained by reacting the compound in the presence of a basic compound such as sodium hydride as a catalyst in a reaction solvent comprising a polar solvent such as N, N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). (
The catalyst in this step is not limited to sodium hydride, and examples thereof include basic compounds such as alkali metals and alkali metal salts, and transition metal catalysts such as transition metal salts.
These may be used alone or in combination of two or more.
As a reaction solvent, in addition to polar solvents such as DMSO (dimethyl sulfoxide), DMF (N, N-dimethylformamide), DME (1,2-dimethoxyethane), nonpolar solvents such as hexane, benzene, petroleum ether, etc. These solvents may be used alone or in combination of two or more.
この製法によれば、例えば、リダイフェンＡ（式（II）の化合物）及びリダイフェンＣ（式（IＶ）の化合物）の収率は８０％以上であり、リダイフェンＢ（式（III）の化合物）の収率は１００％に近い。また、他の本発明のタモキシフェン類縁体もこの反応方式を利用することにより、高収率で得られる。
したがって、式（７）の化合物（１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテンを原料化合物として用いることにより、きわめて高収率で、効率よく本発明のタモキシフェン化合物を製造することができる。
この式（７）の化合物は、例えば、図２に示される方法（工程１〜４）で合成可能である、この図２の方法について以下に説明する。ただし、本発明においては、この図２の方法のみに限定されない。
According to this production method, for example, the yield of Ridefen A (compound of formula (II)) and Ridefen C (compound of formula (IV)) is 80% or more, and the reidifene B (compound of formula (III)) The yield is close to 100%. Also, other tamoxifen analogs of the present invention can be obtained in high yield by utilizing this reaction method.
Therefore, by using the compound of formula (7) (1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene as a raw material compound, the tamoxifen compound of the present invention can be efficiently produced in a very high yield. Can be manufactured.
The compound of formula (7) can be synthesized, for example, by the method shown in FIG. 2 (
〔工程１〕
４−ピバロイルオキシベンズアルデヒド等の４位の水酸基がアルカノイル基等で保護された式（１）の化合物、アニソール等の式（３）の化合物、及び１−フェニル−３−トリメチルシリル−１−プロペン（式（２）の化合物）を、例えば、トリメチルシリルトリフルオロメタンスルフォネート（ＴＭＳＯＴｆ）等を触媒としてＨｆＣｌ４等のルイス酸あるいはプロトン酸等の存在下、反応させ、式（４）の化合物を生成させる。この工程においては、触媒として上記ＴＭＳＯＴｆの他トリメチルシリルクロリド等も使用でき、ルイス酸としては上記の他、Ｈｆ（ＯＴｆ）４，ＴｉＣｌ４，ＴｉＣｌ２（ＯＴｆ）２等の第４属金属塩、ＡｌＣｌ３，ＢＣｌ３，Ｓｃ（ＯＴｆ）３等の第３属金属塩、ＳｎＣｌ２，Ｓｎ（ＯＴｆ）２等の第２属金属塩等が使用できる。またプロトン酸としては塩酸、硫酸、硝酸、臭化水素酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸等も使用可能である。これらは一種単独で使用してもよく、また２種以上併用してもよい。
[Step 1]
A compound of formula (1) in which the 4-position hydroxyl group such as 4-pivaloyloxybenzaldehyde is protected with an alkanoyl group, a compound of formula (3) such as anisole, and 1-phenyl-3-trimethylsilyl-1-propene (Compound of formula (2)) is reacted with, for example, trimethylsilyl trifluoromethanesulfonate (TMSOTf) or the like in the presence of a Lewis acid such as HfCl 4 or a protonic acid to produce a compound of formula (4) Let In this step, TMSOTf as well as trimethylsilyl chloride and the like can be used as a catalyst, and Lewis acid can be used in addition to the above,
〔工程２〕
ついで、式（４）の化合物を、例えばジメチルスルホキシド（ＤＭＳＯ）等の溶媒の存在下、触媒として、例えば、カリウム第３級ブトキシド等の塩基性化合物を使用して加水分解し、一般式（５）の化合物を得る。
この加水分解工程において使用する触媒としては、塩基性化合物に限らず、酸性化合物及び遷移金属触媒も使用できる。
触媒として使用する塩基性化合物としては、例えば、アルカリ金属あるいはアルカリ金属塩等が挙げられ、酸性化合物としては、例えば、塩酸、硫酸、硝酸、臭化水素酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸、あるいは上記の各種ルイス酸が挙げられる。また、遷移金属触媒としては各種遷移金属塩等が挙げられる。これらは、１種単独で使用してもよく、２種以上を併用しても良い。
また、使用する溶媒としては、上記ＤＭＳＯの他、Ｎ，Ｎ−ジメチルホルムアミド、１，２−ジメトキシエタン等の極性溶媒であってもよく、また、ヘキサン、ベンゼン、ジクロロメタン等の非極性溶媒も用いることができる。これらは一種単独で使用してもよく、また２種以上併用してもよい。
[Step 2]
Next, the compound of the formula (4) is hydrolyzed using a basic compound such as potassium tertiary butoxide as a catalyst in the presence of a solvent such as dimethyl sulfoxide (DMSO), for example. ) Is obtained.
The catalyst used in this hydrolysis step is not limited to a basic compound, and an acidic compound and a transition metal catalyst can also be used.
Examples of the basic compound used as the catalyst include alkali metals or alkali metal salts. Examples of the acidic compound include hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, Or various said Lewis acids are mentioned. Moreover, various transition metal salts etc. are mentioned as a transition metal catalyst. These may be used alone or in combination of two or more.
Moreover, as a solvent to be used, polar solvents such as N, N-dimethylformamide and 1,2-dimethoxyethane may be used in addition to the above DMSO, and nonpolar solvents such as hexane, benzene and dichloromethane are also used. be able to. These may be used alone or in combination of two or more.
〔工程３〕
式（５）の化合物に対して、溶媒として、例えば、ジメチルスルホキシド（ＤＭＳＯ）の存在下、触媒としてより多量のカリウム第３級ブトキシド等を使用して、２重結合の転移反応（マイグレーション）を行ない、一般式（６）の化合物を得る。
この工程の触媒としては、上記カリウム第３級ブトキシドの他、例えば、アルカリ金属あるいはアルカリ金属塩等の塩基性化合物、例えば、塩酸、硫酸、硝酸、臭化水素酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸、あるいは上記の各種ルイス酸等の酸性化合物、および各種遷移金属塩等の遷移金属触媒が用いられる。これらは、１種単独で使用してもよく、２種以上を併用しても良い。
また、使用する溶媒としては、上記ＤＭＳＯの他、Ｎ，Ｎ−ジメチルホルムアミド、１，２−ジメトキシエタン等の極性溶媒であってもよく、また、ヘキサン、ベンゼン、ジクロロメタン等の非極性溶媒も用いることができる。これらは一種単独で使用してもよくまた２種以上併用してもよい。
この工程３の反応系は触媒の使用量及び反応温度をのぞいて、工程２の反応系と同様であり、工程２において、あらかじめ触媒量を多くし、反応温度を高めに設定（例えば５０℃）すれば、工程２と工程３を同時に行なうことができる。
なお、式（６）の化合物においては、幾何異性体として、Ｚ体、Ｅ体が存在するが、本発明においてはこれらを分離することなく、次工程の原料化合物としてそのまま使用できる。
[Step 3]
For the compound of the formula (5), as a solvent, for example, in the presence of dimethyl sulfoxide (DMSO), a larger amount of potassium tertiary butoxide or the like is used as a catalyst to perform a double bond transfer reaction (migration). And the compound of general formula (6) is obtained.
As a catalyst in this step, in addition to the above-mentioned potassium tertiary butoxide, for example, basic compounds such as alkali metals or alkali metal salts such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, trifluoroacetic acid, trifluoromethanesulfone Acid or acidic compounds such as the above-mentioned various Lewis acids and transition metal catalysts such as various transition metal salts are used. These may be used alone or in combination of two or more.
Moreover, as a solvent to be used, polar solvents such as N, N-dimethylformamide and 1,2-dimethoxyethane may be used in addition to the above DMSO, and nonpolar solvents such as hexane, benzene and dichloromethane are also used. be able to. These may be used alone or in combination of two or more.
The reaction system of
In the compound of formula (6), there are Z-form and E-form as geometric isomers, but in the present invention, these can be used as they are as raw material compounds in the next step without separation.
〔工程４〕
図２中、一般式（６）の化合物を例えば、ジクロロメタン等からなる溶媒中で、触媒として臭素化硼素等のルイス酸の存在下、アルコキシ基のアルキル基を脱離させ、式（７）の化合物を得る。
工程４における、触媒としては、上記臭素化硼素の他、上記各種ルイス酸等が使用できるが、さらに、例えば、塩酸、硫酸、硝酸、臭化水素酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸等の酸性化合物、あるいはアルカリ金属、アルカリ金属塩等の塩基性化合物、遷移金属塩等の遷移金属触媒も用いることができる。これらは、１種単独で使用してもよく、２種以上を併用してもよい。
また、反応溶媒としては、上記ジクロロメタンの他の非極性溶媒の他、ＤＭＳＯ（ジメチルスルホキシド）、ＤＭＦ（Ｎ，Ｎ−ジメチルホルムアミド）、ＤＭＥ（１，２−ジメトキシエタン）等の極性溶媒が用いられる。また、他の非極性溶媒としては、ヘキサン、ベンゼン等が挙げられる。これら溶媒は１種単独で使用してもよく、２種以上を併用しても良い。
一方、本発明のタモキシフェン類縁体は、ガン細胞、特に白血病細胞の細胞傷害剤として有用であり、そのＩＣ５０値は、タモキシフェンあるいは従来のタモキシフェン類縁体に比較して半分以下であり、極めて有効である。さらに、本発明のタモキシフェン類縁体は低濃度で血管新生抑制作用を有する。これに対して従来のタモキシフェンあるいはその類縁体は、このような血管新生抑制作用はないかあるいはあっても極めて低い。この点で本発明のタモキシフェン類縁体は特異的である。
特に、ガンについていえば、腫瘍は血管と結ばれていない状態では、１〜２ｍｍ３以上に成長することができないが、腫瘍は自ら血管新生促進因子を産生して血管新生を誘導し、ガン細胞の増殖、湿潤に必要な、酸素、栄養素を補給するための新生血管網を構築する。そして、ガン細胞は、この新生血管網を通じて転位する。したがって、血管新生を抑制する本発明のタモキシフェンフェン類縁体は、このようなガンの増殖、湿潤、転位を抑制する新しいガン治療法の確立に大いに貢献する。
[Step 4]
In FIG. 2, the compound of the general formula (6) is eliminated in the presence of a Lewis acid such as boron bromide as a catalyst in a solvent such as dichloromethane, A compound is obtained.
As the catalyst in
As the reaction solvent, polar solvents such as DMSO (dimethyl sulfoxide), DMF (N, N-dimethylformamide), DME (1,2-dimethoxyethane), etc. are used in addition to the above non-polar solvents other than dichloromethane. . Examples of other nonpolar solvents include hexane and benzene. These solvents may be used alone or in combination of two or more.
On the other hand, the tamoxifen analog of the present invention is useful as a cytotoxic agent for cancer cells, particularly leukemia cells, and its IC 50 value is less than half that of tamoxifen or conventional tamoxifen analogs, which is extremely effective. is there. Furthermore, the tamoxifen analog of the present invention has an anti-angiogenic action at a low concentration. In contrast, conventional tamoxifen or its analogs have no or even no such angiogenesis-inhibiting action. In this respect, the tamoxifen analog of the present invention is specific.
In particular, when it comes to cancer, the tumor cannot grow to 1 to 2 mm 3 or more in a state where it is not connected to blood vessels, but the tumor itself produces angiogenesis-promoting factor to induce angiogenesis, and cancer cells A new blood vessel network is constructed to supply oxygen and nutrients necessary for the growth and wetting of the body. And cancer cells translocate through this neovascular network. Therefore, the tamoxifenfen analog of the present invention that suppresses angiogenesis greatly contributes to the establishment of a new cancer therapy that suppresses such cancer growth, wetting, and translocation.
以下に本発明の実施例を、図３を参照しつつ示すが、本発明はこれら実施例により限定されるものではない。
〔実施例１〕
＜中間体１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（図３中、式（７）の化合物）の製造＞
（１）：反応容器に、酸触媒としてＨｆＣｌ４を８０．１ｍｇ（１当量）を加えて０℃に維持しながら、アニソール０．５ｍｌ、アニソール１ｍｌにトリメチルシリルトリフルオロメタンスルフォネート（ＴＭＳＯＴｆ）２７．８ｍｇ（０．５当量）を溶解した溶液、４−ピバロイルオキシベンズアルデヒド（図３中、式（１）の化合物）５１．６ｍｇ（０．２５ｍＭ）、及びアニソール１ｍｌに１−フェニル−３−トリメチルシリル−１−プロペン（図２、中式（２）の化合物）５７．１ｍｇ（１．２当量）を溶解した溶液を順次加えた。なお、アニソールの合計使用量は２．５ｍｌである。ついで、室温で反応を行い２時間経過後、反応生成物の一部に対し、薄層クロマトグラフィー（ＴＬＣ）（展開溶媒；ｎ−ヘキサン／酢酸エチル＝１０／１）を行って原料化合物が検出されなくなったことを確認した後、反応系を０℃に冷却するとともに、ＮａＨＣＯ３水溶液を加えて反応を終了させた。
この後、反応生成物をエーテルで抽出した後、有機層を水および飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ヘキサン／酢酸エチル＝１０／１）による精製を行ない、４−（４−ピバロイルオキシフェニル）−４−（４−メトキシフェニル）−３−フェニル−１−ブテン（図３中、式（４）の化合物）を６４．７ｍｇ得た。収率は６２％であった。
Examples of the present invention will be described below with reference to FIG. 3, but the present invention is not limited to these examples.
[Example 1]
<Production of
(1): While adding 80.1 mg (1 equivalent) of HfCl4 as an acid catalyst to the reaction vessel and maintaining at 0 ° C., 0.5 ml of anisole and 17.8 ml of trimethylsilyl trifluoromethanesulfonate (TMSOTf) are added to 1 ml. (0.5 equivalent) dissolved solution, 4-pivaloyloxybenzaldehyde (a compound of the formula (1) in FIG. 3) 51.6 mg (0.25 mM), and 1 ml of anisole with 1-phenyl-3-trimethylsilyl A solution in which 57.1 mg (1.2 equivalents) of -1-propene (compound of formula (2) in FIG. 2) was dissolved was sequentially added. The total amount of anisole used is 2.5 ml. Next, the reaction was carried out at room temperature, and after 2 hours, a part of the reaction product was subjected to thin layer chromatography (TLC) (developing solvent: n-hexane / ethyl acetate = 10/1) to detect the starting compound. After confirming that the reaction was stopped, the reaction system was cooled to 0 ° C. and an aqueous NaHCO 3 solution was added to terminate the reaction.
Thereafter, the reaction product was extracted with ether, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the organic layer, purification by preparative thin layer chromatography (developing solvent; hexane / ethyl acetate = 10/1) is performed, and 4- (4-pivaloyloxyphenyl) -4- (4-methoxyphenyl) is obtained. 64.7 mg of -3-phenyl-1-butene (compound of formula (4) in FIG. 3) was obtained. The yield was 62%.
（２）：反応容器に上記工程（１）において得られた、４−（４−ピバロイルオキシフェニル）−４−（４−メトキシフェニル）−３−フェニル−１−ブテン（図３中、式（４）の化合物）５７．２ｍｇ（０．１３８ｍＭ）、カリウム第３級ブトキシド（ｔＢｕＯＫ）１５．５ｍｇ、及びジメチルスルホキシド（ＤＭＳＯ）０．６９ｍｌ（０．２Ｍ）を加えて、室温で１５分反応させ、ピバロイル基を加水分解した。次いで、反応系を０℃に冷却するとともに塩化アンモニウム水溶液を加え反応を終了させた。
反応生成物を酢酸エチルで抽出した後、有機層を水および飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ヘキサン／酢酸エチル＝３／１）による精製を行ない、４−（４−ヒドロキシフェニル）−４−（４−メトキシフェニル）−３−フェニル−１−ブテン（図３中、式（５）の化合物）を４６．７ｍｇ得た。収率は定量的であった。
(2): 4- (4-Pivaloyloxyphenyl) -4- (4-methoxyphenyl) -3-phenyl-1-butene (in FIG. 3) obtained in the step (1) in the reaction vessel. 57.2 mg (0.138 mM) of the compound of formula (4), 15.5 mg of potassium tertiary butoxide (tBuOK), and 0.69 ml (0.2 M) of dimethyl sulfoxide (DMSO) were added, and the mixture was stirred for 15 minutes at room temperature. The pivaloyl group was hydrolyzed by reacting. Next, the reaction system was cooled to 0 ° C. and an aqueous ammonium chloride solution was added to complete the reaction.
The reaction product was extracted with ethyl acetate, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After the organic layer is concentrated, purification by preparative thin layer chromatography (developing solvent; hexane / ethyl acetate = 3/1) is performed, and 4- (4-hydroxyphenyl) -4- (4-methoxyphenyl) -3- 46.7 mg of phenyl-1-butene (compound of formula (5) in FIG. 3) was obtained. The yield was quantitative.
（３）：反応容器に上記工程（２）において得られた、４−（４−ヒドロキシフェニル）−４−（４−メトキシフェニル）−３−フェニル−１−ブテン（図３中、式（５）の化合物）４５．７ｍｇ、ジメチルスルホキシド（ＤＭＳＯ）１ｍｌ（０．１３８Ｍ）を加え反応系の温度を５０℃に加温し、さらに、カリウム第３級ブトキシド２８３．９ｍｇ（１８当量）を加えて、２重結合転移反応を行った。５０℃で３０分維持した後、反応生成物の一部に対し薄層クロマトグラフィー（展開溶媒；ｎ−ヘキサン／酢酸エチル＝３／１）を行って、上記式（３）の化合物に対応するスポットが検出されなくなったことを確認した後、反応系を０℃に冷却するとともに塩化アンモニウム水溶液を加え反応を終了させた。
ついで、反応生成物を酢酸エチルで抽出した後、有機層を水および飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ヘキサン／酢酸エチル＝３／１）による精製を行ない、１−（４−ヒドロキシフェニル）−１−（４−メトキシフェニル）−２−フェニル−１−ブテン（図３中、式（６）の化合物）、を３７．２ｍｇ得た。収率は８１％であった。
(3): 4- (4-hydroxyphenyl) -4- (4-methoxyphenyl) -3-phenyl-1-butene obtained in the above step (2) in the reaction vessel (in FIG. ) 45.7 mg, dimethyl sulfoxide (DMSO) 1 ml (0.138 M) was added, and the temperature of the reaction system was increased to 50 ° C., and potassium tertiary butoxide 283.9 mg (18 equivalents) was further added. A double bond transfer reaction was performed. After maintaining at 50 ° C. for 30 minutes, a part of the reaction product is subjected to thin layer chromatography (developing solvent; n-hexane / ethyl acetate = 3/1) to correspond to the compound of the above formula (3). After confirming that no spots were detected, the reaction system was cooled to 0 ° C. and an aqueous ammonium chloride solution was added to terminate the reaction.
Subsequently, after extracting the reaction product with ethyl acetate, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the organic layer, purification by preparative thin layer chromatography (developing solvent; hexane / ethyl acetate = 3/1) is performed, and 1- (4-hydroxyphenyl) -1- (4-methoxyphenyl) -2- 37.2 mg of phenyl-1-butene (the compound of formula (6) in FIG. 3) was obtained. The yield was 81%.
（４）：反応容器に上記工程（３）で得られた、１−（４−ヒドロキシフェニル）−１−（４−メトキシフェニル）−２−フェニル−１−ブテン（図３中、式６の化合物）１４５．２ｍｇ（０．４３９ｍＭ）とジクロロメタン２．２ｍｌを加え、反応系を−７８℃に冷却した。次いで臭素化硼素（ＢＢｒ３）１０当量を含むヘプタン１Ｍ溶液４．３９ｍｌを加え、１時間維持した。次いで反応温度を０℃に上昇させた。さらに３時間経過後、反応生成物の一部に対し、薄層クロマトグラフィー（展開溶媒；ｎ−ヘキサン／酢酸エチル＝３／１）を行って、上記式（６）の化合物に対応するスポットが検出されなくなったことを確認した後、ＮａＨＣＯ３水溶液を加え、反応を終了させた。
反応生成物をジクロロメタンで抽出した後、有機層を水および飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、生じた固体を塩化メチレンに溶解し、再結晶させ、１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（図３中、式（７）の化合物）中の結晶６１．４ｍｇを得た。一方、液相を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ヘキサン／酢酸エチル＝３／１）による精製を行ない、中間体１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテンを５８．４ｍｇ得た。前者の結晶との合計は１１９．６ｍｇであり、収率は８６％であった。
(4): 1- (4-hydroxyphenyl) -1- (4-methoxyphenyl) -2-phenyl-1-butene (in FIG. 3, of formula 6) obtained in the above step (3) in a reaction vessel. Compound) 145.2 mg (0.439 mM) and dichloromethane 2.2 ml were added, and the reaction system was cooled to -78 ° C. Next, 4.39 ml of 1M heptane solution containing 10 equivalents of boron bromide (BBr 3 ) was added and maintained for 1 hour. The reaction temperature was then raised to 0 ° C. After a further 3 hours, thin layer chromatography (developing solvent; n-hexane / ethyl acetate = 3/1) was performed on a part of the reaction product, and spots corresponding to the compound of the above formula (6) were found. After confirming that it was no longer detected, an aqueous NaHCO 3 solution was added to terminate the reaction.
After the reaction product was extracted with dichloromethane, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentration of the organic layer, the resulting solid was dissolved in methylene chloride, recrystallized, and 1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene (compound of formula (7) in FIG. 3). ) 61.4 mg of crystals were obtained. On the other hand, after concentrating the liquid phase, purification by preparative thin layer chromatography (developing solvent; hexane / ethyl acetate = 3/1) was performed, and the intermediate 1,1-bis (4-hydroxyphenyl) -2-phenyl- 58.4 mg of 1-butene was obtained. The total with the former crystals was 119.6 mg, and the yield was 86%.
上記各工程で得られた化合物のＮＭＲ測定結果を以下に示す。
式（４）の化合物
1H NMR(CDCl3)σ= 7.32-6.75(m, 11H, Ph), 6.57(d, J=8.6Hz, 2H, Ph), 5.94-5.84(m, 1H, 3-H), 4.90-4.80(m, 1H, 4-H), 4.25(d, J=11.6 HZ, 1H, 1-H), 4.06(dd, J=7.1, 9.6 Hz, 1H, 2-H), 3.66(s, 3H, MeO), 1.31(s, 9H, PivO)
The NMR measurement results of the compounds obtained in the above steps are shown below.
Compound of formula (4)
1 H NMR (CDCl 3 ) σ = 7.32-6.75 (m, 11H, Ph), 6.57 (d, J = 8.6Hz, 2H, Ph), 5.94-5.84 (m, 1H, 3-H), 4.90-4.80 (m, 1H, 4-H), 4.25 (d, J = 11.6 HZ, 1H, 1-H), 4.06 (dd, J = 7.1, 9.6 Hz, 1H, 2-H), 3.66 (s, 3H, MeO), 1.31 (s, 9H, PivO)
式（６）の化合物（Ｚ体とＥ体の等量混合物）
1H NMR(CDCl3) σ=7.27-6.45(m, 13H, Ph),3.82 and 3.67(s, 3H, MeO), 2.48(q, J=7.3Hz, 2H, 3-H), 0.93 and 0.92(t, J=7.3Hz, 3H, 4-H)
Compound of formula (6) (equal mixture of Z-form and E-form)
1 H NMR (CDCl 3 ) σ = 7.27-6.45 (m, 13H, Ph), 3.82 and 3.67 (s, 3H, MeO), 2.48 (q, J = 7.3Hz, 2H, 3-H), 0.93 and 0.92 (t, J = 7.3Hz, 3H, 4-H)
式（７）の化合物（１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン）
1H NMR(CDCl3)σ=7.29-6.45(m, 13H, Ph), 2.48(q, J=7.6Hz, 2H, 3-H), 0.92(t, J=7.6 Hz, 3H, 4-H)
Compound of formula (7) (1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene)
1 H NMR (CDCl 3 ) σ = 7.29-6.45 (m, 13H, Ph), 2.48 (q, J = 7.6Hz, 2H, 3-H), 0.92 (t, J = 7.6 Hz, 3H, 4-H )
〔実施例２〕
＜リダイフェンＡの製造＞
反応容器に、６０重量％水素化ナトリウム（ＮａＨ）含有オイル６３．２ｍｇ（水素化ナトリウム２０当量）を加え、これを石油エーテルで洗浄した後乾燥させた。次に、Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）０．８ｍｌ（０．１Ｍ）を投入し、０℃に保った。次いで、実施例１で得られた、１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（図３中、式（７）の化合物）２５ｍｇ（０．０７９ｍＭ）を反応容器に加え、室温で１５分維持した後、０℃に冷却し、１−ジメチルアミノ−２−クロロエタン塩酸塩（図３中式（８）の化合物）１１３．８ｍｇ（１０当量）を加え、５０℃で６時間反応させた後、反応系を０℃に冷却するとともに、塩化アンモニウム水溶液を加え、反応を終了させた。反応生成物をジクロロメタンで抽出し、有機層を水及び飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ベンゼン／トリエチルアミン＝９／１）を行ない、リダイフェンＡ（１，１−ビス〔４−（２−ジメチルアミノエトキシ）フェニル〕−２−フェニル−１−ブテン；図３中、式（II）の化合物）３０．０ｍｇを得た。収率は８３％であった。
得られた化合物のＮＭＲ測定結果は以下のとおりである。
1H NMR(CDCl3)σ=7.19-6.45(m, 13H, PH), 4.01 and 3.85(t, J=5.9 Hz, 4H, OCH2), 2.67 and 2.57(t, J=5.9Hz, 4H, NCH2), 2.40(q, J=7.3Hz, 2H, 3-H), 2.28 and 2.21(s, 12H, NMe2), 0.85(t, J=7.3Hz, 3H, 4-H)
[Example 2]
<Manufacturing of Redefen A>
To the reaction vessel was added 63.2 mg (20 equivalents of sodium hydride) of an oil containing 60 wt% sodium hydride (NaH), which was washed with petroleum ether and dried. Next, 0.8 ml (0.1 M) of N, N-dimethylformamide (DMF) was added and kept at 0 ° C. Next, 25 mg (0.079 mM) of 1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene (compound of formula (7) in FIG. 3) obtained in Example 1 was added to the reaction vessel. After maintaining at room temperature for 15 minutes, the mixture was cooled to 0 ° C., and 113.8 mg (10 equivalents) of 1-dimethylamino-2-chloroethane hydrochloride (compound of formula (8) in FIG. 3) was added at 50 ° C. After reacting for 6 hours, the reaction system was cooled to 0 ° C. and an aqueous ammonium chloride solution was added to terminate the reaction. The reaction product was extracted with dichloromethane, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the organic layer, preparative thin layer chromatography (developing solvent; benzene / triethylamine = 9/1) was performed to obtain ridefen A (1,1-bis [4- (2-dimethylaminoethoxy) phenyl] -2- 30.0 mg of phenyl-1-butene (compound of formula (II) in FIG. 3) was obtained. The yield was 83%.
The NMR measurement results of the obtained compound are as follows.
1 H NMR (CDCl3) σ = 7.19-6.45 (m, 13H, PH), 4.01 and 3.85 (t, J = 5.9 Hz, 4H, OCH 2 ), 2.67 and 2.57 (t, J = 5.9 Hz, 4H, NCH 2), 2.40 (q, J = 7.3Hz, 2H, 3H), 2.28 and 2.21 (s, 12H, NMe 2), 0.85 (t, J = 7.3Hz, 3H, 4-H)
〔実施例３〕
＜リダイフェンＢの製造＞
反応容器に、６０重量％水素化ナトリウム（ＮａＨ）含有オイル１２６．４ｍｇ（水素化ナトリウム２０当量）を加え、これを石油エーテルで洗浄した後乾燥した。次にＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）を１．５８ｍｌ（０．１Ｍ）を投入し、０℃に保った。次いで、実施例１で得られた、１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（図３中、式（７）の化合物）５０ｍｇ（０．１５８ｍＭ）を反応容器に加え、室温で１５分維持した後、０℃に冷却し、１−（２−クロロエチル）−ピロリジン塩酸塩（図３中、式（９）の化合物）５０ｍｇ（１０当量）を加えて５０℃で１２時間反応を行った。反応生成物の一部に対し、薄層クロマトグラフィー（展開溶媒；メタノール／ジクロロエタン＝１／１９を行なって、上記式７の化合物に対応するスポットが検出されなくなったことを確認した後、反応系を０℃に冷却するとともに、塩化アンモニウム水溶液を加えて終了させた。
次いで、反応生成物をジクロロメタンで抽出した後、有機層を水及び飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、分取薄層クロマトグラフィー（展開溶媒；ベンゼン／トリエチルアミン＝１９／１）による精製を行い、リダイフェンＢ（１，１−ビス［４−（２−ピロリジン−１−イル−エトキシ）フェニル］−２−フェニル−１−ブテン；図３中式（III）の化合物）７９．７ｍｇを得た。収率は９９％であった。
得られた化合物のＮＭＲ測定結果は以下のとおりである。
1H NMR(CDCl3)σ=7.18-6.45(m, 13H, Ph), 4.09 and 3.89 (t, J= 5.9 Hz, 4H, OCH2), 2.84 and 2.73 (t, J= 5.9 Hz, 4H, NCH2), 2.58-2.47 (m, 8H, N(CH2CH2)2), 2.40 (q, J = 7.3 Hz, 2H, 3-H), 1.76-1.18 (m, 8H, N(CH2CH2)2), 0.85 (t, J= 7.3 Hz, 3H, 4-H)
Example 3
<Manufacturing of Redefen B>
To the reaction vessel was added 126.4 mg (20 equivalents of sodium hydride) of an oil containing 60% by weight sodium hydride (NaH), which was washed with petroleum ether and dried. Next, 1.58 ml (0.1 M) of N, N-dimethylformamide (DMF) was added and kept at 0 ° C. Next, 50 mg (0.158 mM) of 1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene (compound of formula (7) in FIG. 3) obtained in Example 1 was added to the reaction vessel. The mixture was maintained at room temperature for 15 minutes, cooled to 0 ° C., and 50 mg (10 equivalents) of 1- (2-chloroethyl) -pyrrolidine hydrochloride (the compound of formula (9) in FIG. 3) was added to 50 ° C. The reaction was carried out for 12 hours. A portion of the reaction product was subjected to thin layer chromatography (developing solvent; methanol / dichloroethane = 1/19), and after confirming that spots corresponding to the compound of
Subsequently, after extracting the reaction product with dichloromethane, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the organic layer, purification by preparative thin layer chromatography (developing solvent; benzene / triethylamine = 19/1) was carried out to obtain ridifien B (1,1-bis [4- (2-pyrrolidin-1-yl-ethoxy). ) Phenyl] -2-phenyl-1-butene; compound of formula (III) in FIG. The yield was 99%.
The NMR measurement results of the obtained compound are as follows.
1 H NMR (CDCl3) σ = 7.18-6.45 (m, 13H, Ph), 4.09 and 3.89 (t, J = 5.9 Hz, 4H, OCH 2 ), 2.84 and 2.73 (t, J = 5.9 Hz, 4H, NCH 2 ), 2.58-2.47 (m, 8H, N (CH 2 CH 2 ) 2 ), 2.40 (q, J = 7.3 Hz, 2H, 3-H), 1.76-1.18 (m, 8H, N (CH 2 CH 2 ) 2 ), 0.85 (t, J = 7.3 Hz, 3H, 4-H)
＜リダイフェンＣの製造＞
反応容器に、６０重量％水素化ナトリウム（ＮａＨ）含有オイル５０．４ｍｇ（水素化ナトリウム２０当量）を加え、これを石油エーテルで洗浄した後乾燥させた。次に、Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）０．６ｍｌ（０．１Ｍ）を投入し、０℃に保った。実施例１で得られた、１，１−ビス（４−ヒドロキシフェニル）−２−フェニル−１−ブテン（図３中、式（７）の化合物）２０ｍｇ（０．０６３２ｍＭ）を反応容器に加え、室温で１５分維持した後、０℃に冷却し、１−（２−クロロエチル）−ピペリジン塩酸塩１１６．４ｍｇ（１０当量）を加え、５０℃で６時間反応させた後、反応系を０℃に冷却するとともに、塩化アンモニウム水溶液を加え、反応を終了させた。反応生成物をジクロロメタンで抽出し、有機層を水および飽和食塩水で洗浄し、有機層を無水硫酸ナトリウムで乾燥した。有機層を濃縮後、薄層クロマトグラフィー（展開溶媒；クロロホルム／メタノール＝９／１）を行ないリダイフェンＣ（１，１−ビス［４−（２−ピペリジン−１−イル−エトキシ）フェニル］−２−フェニル−１−ブテン；図３中式（IＶ）の化合物）３０ｍｇを得た。収率は８８％であった。
得られた化合物のＮＭＲ測定結果は以下の通りである。
1HNMR(CDCl3)σ＝7.19-6.52(m, 13H, Ph), 4.13 and 3.97(t, J＝6.2 Hz, 4H, OCH2), 2.80 and 2.70(t, J＝6.2 Hz, 4H, NCH2), 2.57-2.43(m, 8H, N(CH2CH2)2), 2.49(q, J＝7.3Hz, 2H, 3-H), 1.67-1.54(m, 4H, N(CH2CH2)2), 1.50-1.42(m, 4H, N(CH2CH2)2CH2), 0.92(t, J＝7.3 Hz, 3H, 4-H)
<Manufacturing of Redefen C>
To the reaction vessel, 50.4 mg of 60 wt% sodium hydride (NaH) -containing oil (20 equivalents of sodium hydride) was added, washed with petroleum ether and dried. Next, 0.6 ml (0.1 M) of N, N-dimethylformamide (DMF) was added and kept at 0 ° C. 20 mg (0.0632 mM) of 1,1-bis (4-hydroxyphenyl) -2-phenyl-1-butene (compound of formula (7) in FIG. 3) obtained in Example 1 was added to the reaction vessel. The mixture was maintained at room temperature for 15 minutes, cooled to 0 ° C., 16.4 mg (10 equivalents) of 1- (2-chloroethyl) -piperidine hydrochloride was added, and the mixture was reacted at 50 ° C. for 6 hours. While cooling to 0 ° C., an aqueous ammonium chloride solution was added to terminate the reaction. The reaction product was extracted with dichloromethane, the organic layer was washed with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After concentrating the organic layer, thin layer chromatography (developing solvent; chloroform / methanol = 9/1) was performed, and Ridefen C (1,1-bis [4- (2-piperidin-1-yl-ethoxy) phenyl] -2 -Phenyl-1-butene (compound of formula (IV) in FIG. 3) 30 mg was obtained. The yield was 88%.
The NMR measurement results of the obtained compound are as follows.
1 HNMR (CDCl3) σ = 7.19-6.52 (m, 13H, Ph), 4.13 and 3.97 (t, J = 6.2 Hz, 4H, OCH 2 ), 2.80 and 2.70 (t, J = 6.2 Hz, 4H, NCH 2 ), 2.57-2.43 (m, 8H, N (CH 2 CH 2 ) 2 ), 2.49 (q, J = 7.3Hz, 2H, 3-H), 1.67-1.54 (m, 4H, N (CH 2 CH 2 ) 2 ), 1.50-1.42 (m, 4H, N (CH 2 CH 2 ) 2 CH 2 ), 0.92 (t, J = 7.3 Hz, 3H, 4-H)
〔実施例４〕
前骨髄性白血病ＨＬ−６０細胞を（２×１０５ｃ／ｍｌ）を９６穴プレートにまき、タモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、イドタモキシフェン、リダイフェンＡ及びリダイフェンＢを、各々、finalで、０，１，５，１０，２０μｇ／ｍＬになるように添加し５％ＣＯ２インキュベータ内で３７℃培養した。測定１時間前にfinal０．５ｍｇ／ｍｌＭＴＴ（３−（４，５−ジメチル−チアゾール−２−イル）−２，５−ジフェニルテトラゾリウム ブロマイド）を添加し、さらに１時間培養した。
上記前骨髄性白血病ＨＬ−６０細胞に対する上記各薬剤の細胞障害性は、該細胞のミトコンドリア中のコハク酸デヒドロゲナーゼ活性を測定することにより行ない、該酵素活性の測定は、コハク酸デヒドロゲナーゼによるＭＴＴのテトラゾリウム環の開裂に伴う不溶性の暗青色ホルマザン色素の生成量を、上記培養後、遠心して上澄みを除き、沈殿として残った上記色素をＤＭＳＯで溶解し、マイクロプレートリーダーでＯＤ５７０を測定することにより行った。なお、ＭＴＴアッセイは、多検体にわたる細胞死を判定するための分析法としてよく知られているものである。
Example 4
Promyelocytic leukemia HL-60 cells (2 × 10 5 c / ml) are seeded in a 96-well plate, and tamoxifen, 4-bromotamoxifen, 3-iodotamoxifen, idotamoxifen, ridifefen A and ridifefen B are each final. And added to 0, 1, 5, 10, 20 μg / mL, and cultured at 37 ° C. in a 5% CO 2 incubator. One hour before the measurement, final 0.5 mg / ml MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added, and further cultured for 1 hour.
The cytotoxicity of each of the above-mentioned drugs against the promyelocytic leukemia HL-60 cells is measured by measuring the succinate dehydrogenase activity in the mitochondria of the cells. The enzyme activity is measured by tetrazolium of MTT by succinate dehydrogenase. The amount of insoluble dark blue formazan dye produced by ring cleavage was determined by centrifuging the supernatant after removing the supernatant, dissolving the dye remaining as a precipitate in DMSO, and measuring OD 570 with a microplate reader. It was. The MTT assay is well known as an analysis method for determining cell death across multiple specimens.
結果を図４に示す。また、該コハク酸デヒドロゲナーゼ活性が５０％に低下する各薬剤量を図４から求め、細胞障害性のＩＣ５０値とした。使用した上記各薬剤のＩＣ５０値を以下の表２に示す。 The results are shown in FIG. In addition, the amount of each drug at which the succinate dehydrogenase activity was reduced to 50% was determined from FIG. 4 and used as the cytotoxic IC 50 value. The IC 50 values for each of the above drugs used are shown in Table 2 below.
以上の結果から明らかなようにリダイフェンＡ及びリダイフェンＢの細胞障害性は、顕著に高く、本発明のタモキシフェン類縁体誘導体は極めて効果的にガン細胞を死滅又は増殖抑制させることが明らかである。 As is clear from the above results, the cytotoxicity of Ridefen A and Ridefen B is remarkably high, and it is clear that the tamoxifen analog derivative of the present invention kills or suppresses proliferation of cancer cells very effectively.
以上の結果から明らかなようにリダイフェンＡ及びリダイフェンＢの細胞障害性は、顕著に高く、本発明のタモキシフェン類縁体誘導体は極めて効果的にガン細胞を死滅又は増殖抑制させることが明らかである。 As is clear from the above results, the cytotoxicity of Ridefen A and Ridefen B is remarkably high, and it is clear that the tamoxifen analog derivative of the present invention kills or suppresses proliferation of cancer cells very effectively.
〔実施例５〕
＜白血病Ｔ細胞であるJurkat細胞に対するリダイフェンＡ、Ｂのアポトーシス誘導＞
（１）細胞傷害性の計測
Jurkat細胞を、１０％ＦＣＳ（牛胎児血清）を含有するＲＰＭＩ１６４０培地で、３７℃、５％ＣＯ２の条件下で培養し、２×１０５cells/ml濃度になるように調製した。
この２×１０５cells/ml濃度に調製したJurkat細胞試料溶液を９６穴プレートの各ウエルに９０μｌずつ分注し、さらに、該細胞試料を含有する各ウエルに、リダイフェンＡ、リダイフェンＢ、タモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、イドキシフェンをそれぞれ１０μＭ、２０μＭ及び３０μＭ添加した。
これら全てを入れ終わった後２時間インキュベレトした、２時間後インキュベーターから取り出し、各ウエルに、Cell Counting Kit-8（和光純薬工業社製）の反応試薬（ＷＳＴ−８）を１０μｌづつ加え、再び２ｈインキュベートした。その後マイクロプレートリーダー４５０ｎｍで吸光度を測定した。結果を図５に示す。
Cell Counting Kit-8使用による解析法は、細胞試料のＮＡＤ（Ｐ）Ｈ活性を測ることにより、試料内の生細胞数を割り出すものであり、ＮＡＤ（Ｐ）Ｈ活性は試料中の生細胞数とほぼ比例関係にある。図５の結果によれば、リダイフェンＡ及びＢの極めて低い濃度で細胞傷害を引き起こしており、その細胞傷害性は、タモキシフェン及びその他のタモキシフェン類縁体に比べ遙かに高いことが分かる。また、タモキシフェンあるいはその類縁体は、乳ガン細胞膜上のエストロゲンレセプター（ER)に結合することにより、アポトーシスを誘導するとされているが、この実験で使用した白血病Ｔ細胞であるJurkat細胞は、エストロゲンレセプターを有していないものである（ER-negative)。それにもかかわらず、該細胞に対して本発明のリダイフェンＡ、リダイフェンＢが高い細胞傷害性を示したことは、特筆すべきことである。
また、上記図５の結果に基づき、リダイフェンＡ、リダイフェンＢ、タモキシフェン及びその他の類縁体のＩＣ５０を算出した。これら薬剤のＩＣ５０を表３に示す。ＴＡＭ３（３−ヨードタモキシフェン）は４０μＭまで添加しても障害性を有さなかった。
Example 5
<Induction of apoptosis of Ridefen A and B against Jurkat cells, which are leukemia T cells>
(1) Measurement of cytotoxicity
Jurkat cells were cultured in RPMI 1640 medium containing 10% FCS (fetal calf serum) under conditions of 37 ° C. and 5% CO 2 to prepare a concentration of 2 × 10 5 cells / ml.
90 μl of this Jurkat cell sample solution prepared to a concentration of 2 × 10 5 cells / ml was dispensed into each well of a 96-well plate, and each well containing the cell sample was subjected to Rifafen A, Ridaifen B, Tamoxifen, 4-Bromotamoxifen, 3-iodotamoxifen, and idoxifene were added at 10 μM, 20 μM, and 30 μM, respectively.
After all of these were added, the mixture was incubated for 2 hours and removed from the incubator after 2 hours. To each well, 10 μl of a reaction reagent (WST-8) of Cell Counting Kit-8 (manufactured by Wako Pure Chemical Industries, Ltd.) was added, Incubated again for 2 h. Thereafter, the absorbance was measured with a microplate reader at 450 nm. The results are shown in FIG.
The analysis method using Cell Counting Kit-8 is to determine the number of living cells in the sample by measuring the NAD (P) H activity of the cell sample, and the NAD (P) H activity is the number of living cells in the sample. Is almost proportional. According to the results of FIG. 5, it can be seen that cytotoxicity is caused by extremely low concentrations of rifafen A and B, and the cytotoxicity is much higher than that of tamoxifen and other tamoxifen analogs. Tamoxifen or its analogs are said to induce apoptosis by binding to the estrogen receptor (ER) on the breast cancer cell membrane, but the Jurkat cell, which is the leukemia T cell used in this experiment, has an estrogen receptor. It does not have (ER-negative). Nevertheless, it is noteworthy that the reifenfen A and redefenf B of the present invention showed high cytotoxicity against the cells.
In addition, based on the results of FIG. 5 above, IC50s of Ridefen A, Redifen B, Tamoxifen and other analogs were calculated. The IC 50 for these drugs is shown in Table 3. TAM3 (3-iodotamoxifen) was not impaired even when added up to 40 μM.
（２）アポトーシスに伴うＤＮＡ断片化の確認
上記（１）と同様に培養して得たJurkat細胞を２×１０６cellsづつ、２×１０５cells/ml濃度で９ｃｍシャーレに培地とともに分注し、リダイフェンＡ、リダイフェンＢ、タモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、及びイドキシフェンをそれぞれ以下の濃度で加え、４時間反応させた。
リダイフェンＡ；０．５μＭ、１μＭ、２μＭ
リダイフェンＢ；０．５μＭ、１μＭ、２μＭ、５μＭ
タモキシフェン；５μＭ、1０μＭ、２０μＭ、３０μＭ
４−ブロモタモキシフェン；５μＭ、1０μＭ、１５μＭ、２０μＭ
３−ヨードタモキシフェン；１０μＭ、２０μＭ、３０μＭ、４０μＭ
イドキシフェン；０．５μＭ、１μＭ、２μＭ、５μＭ
なお、これら薬剤の濃度は、上記（１）で算出された各薬剤のＩＣ５０に合わせて設定したものである（３−ヨードタモキシフェンは上限を４０μＭに設定）。
次いで、該反応液をプロテナーゼＫ（ProteinaseK）、RNaseAで処理し、ＤＮＡを抽出した。次いで、抽出されたＤＮＡについて０．９％アガロースゲル電気泳動を行ない。ＵＶトランスイルミネータで観察し、写真撮影した。
この電気泳動写真を図６に示す。なお、電気泳動写真における数字は、上記薬剤の濃度を示し、Ｃは上記薬剤無添加の場合を示す。
この電気泳動の結果によれば、リダイフェンＡ、リダイフェンＢを加えた場合においては、無添加、タモキシフェン及びその他の類縁体を加えた場合に比べ、より低濃度でJurkat細胞において、アポトーシス特有のＤＮＡ断片化が起こっていることが分かる。
(2) Confirmation of DNA fragmentation accompanying apoptosis Jurkat cells obtained by culturing in the same manner as in (1) above were dispensed with 2 × 10 6 cells at a concentration of 2 × 10 5 cells / ml into a 9 cm petri dish together with the medium. , Ridifefen A, ridifefen B, tamoxifen, 4-bromo tamoxifen, 3-iodo tamoxifen, and idoxifene were added at the following concentrations, respectively, and reacted for 4 hours.
Ridefen A; 0.5 μM, 1 μM, 2 μM
Ridefen B; 0.5 μM, 1 μM, 2 μM, 5 μM
Tamoxifen; 5 μM, 10 μM, 20 μM, 30 μM
4-bromotamoxifen; 5 μM, 10 μM, 15 μM, 20 μM
3-iodotamoxifen; 10 μM, 20 μM, 30 μM, 40 μM
Idoxifene; 0.5 μM, 1 μM, 2 μM, 5 μM
The concentration of these agents are those set in accordance with the IC 50 of each drug was calculated in (1) (3-iodo tamoxifen set an upper limit on the 40 [mu] M).
Next, the reaction solution was treated with proteinase K (Proteinase K) and RNase A to extract DNA. The extracted DNA is then subjected to 0.9% agarose gel electrophoresis. The photo was taken with a UV transilluminator.
This electrophoresis photograph is shown in FIG. The numbers in the electrophoretic photographs indicate the concentration of the drug, and C indicates the case where the drug is not added.
According to the results of this electrophoresis, DNA fragments peculiar to apoptosis were observed in Jurkat cells at a lower concentration when Rifafen A and Rifafen B were added than when they were not added, and Tamoxifen and other analogs were added. You can see that the process is happening.
（３）ギムザ染色
上記（２）と同様の操作でリダイフェンＡ、リダイフェンＢ、タモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、及びイドキシフェンをそれぞれ上記（１）で算出されたＩＣ５０の濃度（表３）で加え（３−ヨードタモキシフェンは４０μＭ）、４時間反応させた後、ギムザ染色を行い、Jurkat細胞の形態変化を顕微鏡で観察した。その結果を図７に示す。
これによれば、リダイフェンＡ、リダイフェンＢを加えた場合においては、無添加、タモキシフェン及びその他の類縁体を加えた場合に比べ、より低濃度で顕著な核の凝縮が起こっており、Jurkat細胞に対して顕著な細胞傷害性を有することが分かる。
(3) Giemsa staining The concentration of IC 50 calculated in the above (1) (Table) for each of Redifen A, Redifen B, Tamoxifen, 4-bromotamoxifen, 3-iodotamoxifen, and Idoxifen by the same operation as in (2) above. 3) (3-iodotamoxifen is 40 μM), after 4 hours of reaction, Giemsa staining was performed, and morphological changes of Jurkat cells were observed with a microscope. The result is shown in FIG.
According to this, when REDIFEN A and REDIFEN B are added, significant nuclear condensation occurs at a lower concentration than when no addition, tamoxifen and other analogs are added. It can be seen that it has significant cytotoxicity.
（４）キャスパーゼ−３（Caspase-3)の活性測定
上記（１）と同様に培養して得たJurkat細胞を２×１０６cellsづつ、２×１０５cells/ml濃度で９ｃｍシャーレに入れ、リダイフェンＡ、リダイフェンＢ、タモキシフェン、４−ブロモタモキシフェン、３−ヨードタモキシフェン、及びイドキシフェンをそれぞれ上記（１）のＩＣ５０濃度（表３）で加え、反応させた（３−ヨードタモキシフェンは４０μＭ）。この反応において、それぞれ０時間、１時間、２時間、３時間及び４時間反応させた場合の各細胞を回収し、RIPAbufferに懸濁し、該懸濁液にキャスパーゼ-３の人工基質であるAc−DEVD−pNAを加え、Ac−DEVD−pNAの切断により生じるpNAの吸光度を405nmの波長で測定した。また、その際のタンパク質量をBCA protein Assayで測定して、キャスパーゼ-３の比活性（Unit/mg）を算出し、上記反応０時間のときの比活性を１とした場合の各時間における比活性を求めた。結果を図８のグラフに示す。
キャスパーゼ−３はアポトーシスに特異的なプロテアーゼであり、本発明のリダイフェンＡ及びＢ、並びにタモキシフェン、４−ブロモタモキシフェン、及びイドキシフェンは、いずれも反応時間を長くすると、キャスパーゼ-３の比活性が増大するという同様の傾向を示しているが、本発明のリダイフェンＡ及びＢは極めて低濃度でキャスパーゼ−３の比活性を増大せしめている。
(4) Activity measurement of caspase-3 (Caspase-3) Jurkat cells obtained by culturing in the same manner as in (1) above were placed in 2 × 10 6 cells in a 9 cm petri dish at a concentration of 2 × 10 5 cells / ml, Redifen A, Redifen B, Tamoxifen, 4-bromotamoxifen, 3-iodotamoxifen, and idoxifene were added and reacted at the IC 50 concentration (Table 3) of (1) above (3-iodotamoxifen was 40 μM). In this reaction, each cell when reacted for 0 hour, 1 hour, 2 hours, 3 hours, and 4 hours, respectively, was collected and suspended in RIPA buffer, and Ac- s an artificial substrate of caspase-3 was suspended in the suspension. DEVD-pNA was added, and the absorbance of pNA generated by cleavage of Ac-DEVD-pNA was measured at a wavelength of 405 nm. In addition, the amount of protein at that time was measured by BCA protein Assay, and the specific activity (Unit / mg) of caspase-3 was calculated. Activity was sought. The results are shown in the graph of FIG.
Caspase-3 is a protease specific for apoptosis, and the specific activities of caspase-3 are increased when the reaction times are increased for all of ridififene A and B of the present invention and tamoxifen, 4-bromotamoxifen, and idoxifene. However, the ridifene A and B of the present invention increase the specific activity of caspase-3 at a very low concentration.
これらの実験結果をまとめると、タモキシフェン及び３−ヨードタモキシフェンを除くその類縁体は、ER-nagative細胞にもアポトーシスを誘導するが、特にこの中でリダイフェンＡおよびＢは、極めて顕著なアポトーシス誘導能を有し、乳ガン以外の抗ガン剤としても極めて有用であるといえる。 Summarizing the results of these experiments, tamoxifen and its analogs except 3-iodotamoxifen induce apoptosis in ER-nagative cells, among which ridefen A and B have a very significant ability to induce apoptosis. It can be said that it is extremely useful as an anticancer agent other than breast cancer.
〔実施例６〕
＜リダイフェンＡ、Ｂ、Ｃによる血管新生抑制＞
１．ＣＡＭ（鶏胚漿尿膜法）による血管新生抑制活性の検定
（１）リダイフェンＡの活性検定
ａ．サンプルの調製
５％ＥＶ(ethylene-vinyl acetate copolymer 40)にリダイフェンＡを溶かし、10μｌをガラスシャーレに滴下し、風乾させ、次に、スパーテルを用いて、風乾したフィルム状のEVを球形のペレットにして、一晩以上、冷凍庫保存した。
ｂ．実験
鶏卵を３７℃で培養し、培養４日後に、気室上部及び鶏卵側部の卵殻の２ヶ所に錐で穴を開け、側部の穴より約４ｍｌの卵白を注射器で吸引除去した。また、上部の穴より駒込ピペット用シリコンスポイトをあてて吸引し、気室上部に仮気室を作り、仮気室上の卵殻膜を除去し、窓を作り、これらの穴をオートクレーブしたアルミ箔でキャップした。
培養５日後に、上記ａで作成したＥＶペレットを漿尿膜上にのせ、再びアルミ箔でキャップした。
培養７日後に、観察しやすいように卵殻を取り除き、窓を広げ、さらに、血管網を見やすくするために漿尿膜内に脂肪乳剤を適当量注入し、漿尿膜上の血管網を実体顕微鏡下で観察した。この観察において３ｍｍ以上の無血管領域がある場合を血管新生を抑制するものと判定した。
結果を以下の表４、及び図９及び図１０に示す。なお、上記ａ．のサンプルの調製においてリダイフェンＡを使用しない他は上記と同様に実験を行なった結果もコントロールとして併せて示す。これらの結果により、リダイフェンＡは、血管新生抑制作用を示すことが明らかとなった。
Example 6
<Angiogenesis suppression by Ridefen A, B, C>
1. Assay for angiogenesis inhibitory activity by CAM (chick embryo chorioallantoic membrane method) (1) Activity assay for ridefen A a. Sample Preparation Dissolve Ridefen A in 5% EV (ethylene-vinyl acetate copolymer 40), add 10 μl to a glass petri dish, air dry, and then use a spatula to turn the air-dried EV into a spherical pellet. And kept in the freezer for more than one night.
b. Experiment Chicken eggs were cultured at 37 ° C., and after 4 days of culture, holes were drilled in two places on the upper shell of the air chamber and the eggshells on the side of the eggs, and about 4 ml of egg white was removed by suction with a syringe. In addition, a silicon dropper for Komagome pipette is applied through the upper hole to create a temporary air chamber above the air chamber, the eggshell membrane on the temporary air chamber is removed, windows are created, and these holes are autoclaved aluminum foil. Capped with.
After 5 days of culture, the EV pellet prepared in a above was placed on the chorioallantoic membrane and capped with aluminum foil again.
After 7 days of culturing, the eggshell is removed for easy observation, the window is widened, and an appropriate amount of fat emulsion is injected into the chorioallantoic membrane to make the vascular network easy to see, and the vascular network on the chorioallantoic membrane is examined with a stereomicroscope. Observed below. In this observation, a case where there was an avascular region of 3 mm or more was determined to suppress angiogenesis.
The results are shown in Table 4 below and FIGS. 9 and 10. The a. The results of experiments similar to those described above are also shown as a control, except that Ridefen A is not used in the preparation of this sample. From these results, it was revealed that Ridefen A exhibits an angiogenesis inhibitory action.
ａ．サンプルの調製
２％メチルセルロース溶液にエタノールに溶かしたリダイフェンＢ、Ｃをそれぞれを混合し（エタノールの終濃度５％）、テフロンロッドの上に２０μｌずつ滴下したあと、風乾してディスク状にする。
これをピンセットでテフロンロッドからはがし、シャーレに入れて、一晩以上冷凍庫保存した。
ｂ．実験
このようにして得られた、サンプルディスクをそれぞれ用いて、上記（１）ｂ．と同様の実験を行った。結果を表５、表６、及び図１１、図１２に示す。
This was removed from the teflon rod with tweezers, placed in a petri dish, and stored in a freezer for more than one night.
b. Experiment Using each of the sample disks thus obtained, (1) b. The same experiment was conducted. The results are shown in Tables 5 and 6, and FIGS.
〔実施例７〕
ＭＴＴアッセイによる血管内皮細胞生存率の測定
ａ．リダイフェンＡについての測定
ＢＡＥＣ（ウシ冠動脈血管内皮細胞）を１×１０５cell/mlになるように調製し、９６穴プレートに１００μｌ/wellづつ播き、３７℃、５％ＣＯ２下で２４時間培養した後、培養上清を除去し、リダイフェンＡ、タモキシフェン、４−ブロモタモキシフェン及びイドキシフェンを、それぞれ各濃度で含有するＭＥＭ培地に交換し、さらに２４時間培養した後、ＭＴＴ保存液（ＭＴＴ ５mg/mlin PBS(-) ）を１．１μｌ／ｗｅｌｌづつ添加した。１時間後、上澄みを除去しＤＭＳＯを１００μｌ／Ｗｅｌｌづつ加え、マイクロプレートリーダー（５７５ｎｍ）で吸光度を測定した。結果を図１３に示す。
ｂ．リダイフェンＢ、Ｃについての測定
ＢＡＥＣ（ウシ冠動脈血管内皮細胞）を１×１０４cell/mlになるように調製した他は、上記Ａと同様にして吸光度を測定した。結果を図１４に示す。
ＭＴＴアッセイは、実施例４に示したように細胞数の測定を、ミトコンドリアの脱水素酵素活性を測定することにより行うものであり、図１３、図１４に示されるように、タモキシフェンＡ，Ｂ、Ｃはいずれも低濃度で（５μＭ以下）でウシ冠動脈血管内皮細胞の生残数を０にしているのに対し、タモキシフェン、４−ブロモタモキシフェン、イドキシフェンは、１０μＭでもかなりのウシ冠動脈血管内皮細胞の生残していることが分かる。すなわち、本発明のタモキシフェン類縁体は、タモキシフェンあるいはその類縁体の中で特異的に血管内皮細胞の細胞死を誘導する。
血管新生においては、血管内皮細胞の増殖を伴う。したがって、この結果は本発明のタモキシフェン類縁体の血管新生抑制作用を裏付けるものである。
Example 7
Measurement of vascular endothelial cell viability by MTT assay a. Measurement for Ridefen A BAEC (Bovine Coronary Artery Endothelial Cells) was prepared at 1 × 10 5 cells / ml, seeded at 100 μl / well in a 96-well plate, and cultured at 37 ° C. under 5% CO 2 for 24 hours. After that, the culture supernatant was removed, and ridifene A, tamoxifen, 4-bromotamoxifen and idoxifene were replaced with MEM medium containing each concentration, and further cultured for 24 hours, and then MTT preservation solution (
b. Measurement of Ridefen B and C Absorbance was measured in the same manner as A described above except that BAEC (bovine coronary artery endothelial cells) was prepared to 1 × 10 4 cells / ml. The results are shown in FIG.
In the MTT assay, the number of cells is measured by measuring the mitochondrial dehydrogenase activity as shown in Example 4, and as shown in FIGS. 13 and 14, tamoxifen A, B, C is a low concentration (less than 5 μM) and the number of surviving bovine coronary vascular endothelial cells is 0, whereas tamoxifen, 4-bromotamoxifen, and idoxifene have significant bovine coronary vascular endothelial cells even at 10 μM. You can see that it survives. That is, the tamoxifen analog of the present invention induces cell death of vascular endothelial cells specifically in tamoxifen or its analogs.
Angiogenesis involves the proliferation of vascular endothelial cells. Therefore, this result supports the angiogenesis inhibitory action of the tamoxifen analog of the present invention.
Claims (9)
（但し、式中Ｒ１及びＲ２は、水素又はそれぞれ同一あるいは異なるアルキル基を表わし、Ｒ１とＲ２は一緒になって環を形成してもよい。また、ｎは０を含む整数を表わす。） An anti-cancer agent comprising a compound represented by the following formula (I) as an active ingredient.
(However, in the formula, R 1 and R 2 each represent hydrogen or the same or different alkyl group, and R 1 and R 2 may combine to form a ring. Also, n represents an integer including 0. Represents.)
（但し、式中Ｒ１及びＲ２は、水素又はそれぞれ同一あるいは異なるアルキル基を表わし、Ｒ１とＲ２は一緒になって環を形成してもよい。また、Ｒ３はハロゲン原子、アルキル硫酸エステル残基又はトリフルオロメタン硫酸エステル残基を表わし、ｎは０を含む整数を表わす。） The method for producing a compound of formula (I) according to claim 1, wherein the compound represented by formula (7) and the compound represented by formula (8) are reacted in the presence of a base.
(However, in the formula, R 1 and R 2 each represent hydrogen or the same or different alkyl group, and R 1 and R 2 may be combined to form a ring. R 3 is a halogen atom or an alkyl group. Represents a sulfate ester residue or a trifluoromethane sulfate ester residue, and n represents an integer including 0.)
（但し、式中Ｒ４はアルカノイル基を表わす。）
（但し、式中Ｒ５はアルキル基を表わす。）
（但し、式中Ｒ４及びＲ５は上記と同様の置換基を表わす。）
The compound of the formula (7) is reacted with compounds represented by the following formulas (1), (2) and (3) in the presence of an acid catalyst to produce a compound of the following formula (4), The production method according to claim 8, which is produced by sequentially performing a hydrolysis reaction, a migration reaction of a double bond, and a dealkylation reaction of an alkoxy group.
(In the formula, R 4 represents an alkanoyl group.)
(In the formula, R 5 represents an alkyl group.)
(However, in the formula, R 4 and R 5 represent the same substituents as described above.)
Priority Applications (1)
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| JP2008094836A (en) * | 2006-09-13 | 2008-04-24 | Tokyo Univ Of Science | Proteasome inhibitor which contains tomoxifen analogue as an active ingredient |
| WO2009035020A1 (en) | 2007-09-11 | 2009-03-19 | Tokyo University Of Science Educational Foundation Administrative Organization | Novel dihydronaphthalene compound and use thereof |
| WO2013165005A1 (en) * | 2012-05-01 | 2013-11-07 | 学校法人東京理科大学 | Novel compound, method for producing same, and antitumor agent |
| WO2014061821A1 (en) * | 2012-10-16 | 2014-04-24 | Tokyo University Of Science Foundation | Grb10 interacting gyf protein 2 modulator |
| JP2016222659A (en) * | 2015-05-27 | 2016-12-28 | 学校法人関西文理総合学園 | peptide |
| WO2021075147A1 (en) * | 2019-10-15 | 2021-04-22 | 学校法人東京理科大学 | Brap2 action enhancer |
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2005
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| JP2008094836A (en) * | 2006-09-13 | 2008-04-24 | Tokyo Univ Of Science | Proteasome inhibitor which contains tomoxifen analogue as an active ingredient |
| JP2014167005A (en) * | 2007-09-11 | 2014-09-11 | Tokyo Univ Of Science | Proteasome inhibitor |
| EP2192108A1 (en) * | 2007-09-11 | 2010-06-02 | Tokyo University Of Science Educational Foundation Administrative Organization | Novel dihydronaphthalene compound and use thereof |
| EP2192108A4 (en) * | 2007-09-11 | 2010-12-01 | Univ Tokyo Sci Educ Found | Novel dihydronaphthalene compound and use thereof |
| JPWO2009035020A1 (en) * | 2007-09-11 | 2010-12-24 | 学校法人東京理科大学 | Novel dihydronaphthalene compounds and uses thereof |
| US8183235B2 (en) | 2007-09-11 | 2012-05-22 | Tokyo University Of Science Educational Foundation Administrative Organization | Dihydronaphthalene compound and use thereof |
| JP5565667B2 (en) * | 2007-09-11 | 2014-08-06 | 学校法人東京理科大学 | Novel dihydronaphthalene compounds and uses thereof |
| WO2009035020A1 (en) | 2007-09-11 | 2009-03-19 | Tokyo University Of Science Educational Foundation Administrative Organization | Novel dihydronaphthalene compound and use thereof |
| WO2013165005A1 (en) * | 2012-05-01 | 2013-11-07 | 学校法人東京理科大学 | Novel compound, method for producing same, and antitumor agent |
| JPWO2013165005A1 (en) * | 2012-05-01 | 2015-12-24 | 学校法人東京理科大学 | NOVEL COMPOUND, PROCESS FOR PRODUCING THE SAME, AND ANTI-CANCER AGENT |
| WO2014061821A1 (en) * | 2012-10-16 | 2014-04-24 | Tokyo University Of Science Foundation | Grb10 interacting gyf protein 2 modulator |
| JP2016222659A (en) * | 2015-05-27 | 2016-12-28 | 学校法人関西文理総合学園 | peptide |
| WO2021075147A1 (en) * | 2019-10-15 | 2021-04-22 | 学校法人東京理科大学 | Brap2 action enhancer |
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