JPWO2004066994A1 - Anticancer drug - Google Patents

Abstract

An anticancer agent comprising a compound represented by the general formula (1): (wherein R1 to R9 are as defined in the specification) or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

  The present invention relates to an anticancer agent and a method for treating cancer.

Conventionally, it has been known that some substances extracted from plants have an anti-cancer action or a carcinogenesis-preventing action, and many studies have been conducted on components obtained from plant extracts. For example, St. A substance obtained from a plant called John's worst has been reported to show an antitumor effect (Christoph et al., Oncogene, 2002, 21, 1242-1250). In addition, among substances obtained from the genus Carophyllum, there are substances effective for cancer prevention (Ito et al., Journal of Natural Products, 2002, 65 (3), 267-272), substances having cytotoxicity (David et al., Phytochemistry, 2001, 58, 571-575), and substances that have an inhibitory effect on AIDS (Claud et al., Bioorganic & Medicinal Chemistry Letters, 1998, 8, 3475-3478). Has been reported. The structures of the substances reported in these documents vary, and their relationship with function or action is not clear.
On the other hand, in the field of chemical synthesis, various drugs useful as anticancer agents have been developed. Various anti-cancer agents and the like that exhibit high cell growth inhibitory activity have been developed so far. However, some of these are also toxic to normal cells, and many of them do not have sufficient suitability for actual administration to patients. In cancer, the relationship between the onset mechanism and p53 has been pointed out, and many anticancer agents that exert an effect by inducing p53 have been developed. Many have not shown widespread effects on different types of cancer to the extent desired. Furthermore, for the purpose of reducing synergistic effects and side effects, multi-drug therapy using a combination of a plurality of anticancer agents has also been carried out, but P-glycoprotein is induced, and the cells have multi-drug resistance (MDR). In many cases, even when an anticancer agent is administered to such a patient, the drug concentration in the cancer cells decreases, and an effective effect cannot be obtained.
From these facts, development of a new anticancer agent having a mechanism different from the conventional one, effective for multidrug resistant cancer cells, and having a large safety range has been expected.
An object of the present invention is to provide an anticancer agent having a mechanism different from the conventional one, effective for multi-drug resistant cancer cells, and having a large safety range.
Another object of the present invention is to provide a method for treating cancer.

FIG. 1 shows the structure of 15 compounds obtained from Calophyllum Brasiliensis.
FIG. 2 shows the results of examining the growth inhibitory action of BV173 cells at the concentrations of 0 μM, 1 μM, 5 μM, 10 μM and 30 μM for the 15 compounds shown in FIG.
FIG. 3 shows the results of measuring the cell growth inhibitory action of Compound 7 (GUT-70) on five types of leukemia cell lines (BV173, SEM, NALM6, HL60 and K562). In addition, (♦) indicates a measurement result at a concentration of 0 μM, (■) indicates a concentration of 0.5 μM, (□) indicates a concentration of 1.0 μM, (◇) indicates a concentration of 5.0 μM, and (×) indicates a measurement result at a concentration of 10 μM.
FIG. 4 shows the result of morphological observation of apoptosis by Compound 7. 4A shows the observation results before treatment with Compound 7, and FIG. 4B shows the observation results after treatment with Compound 7, respectively.
FIG. 5 shows the results of measuring the relationship between cell cycle and apoptosis by TUNEL assay. FIG. 5A shows the results regarding apoptosis induction when GUT-70 is not treated, and FIG. 5B shows the results regarding apoptosis induction when treated with GUT-70 5 μM for 24 hours.
FIG. 6 shows the results of examining temporal changes of p21 ^{WAF1 / CIPL} , p27 ^Kip1 , p53 and p57 in leukemia cell lines NALM6 cells and BV173 cells treated with Compound 7.
FIG. 7A shows the results of comparing the resistance of daunorubicin (DNR) to P-glycoprotein high-expressing cells K562 / D1-9 and parental K562 cells not expressing P-glycoprotein. FIG. 7B shows the results of comparing the resistance of Compound 7 to P-glycoprotein high-expressing cells K562 / D1-9 and the parent strain K562 cells in which P-glycoprotein is not highly expressed.
FIG. 8 shows the results of examining the toxicity of Compound 7 on normal cells using normal hematopoietic progenitor cells CFU-GM and BFU-E.
FIG. 9 shows the results of examining the effect of Compound 7 (GUT-70) on human small cell lung cancer.

（式中、Ｒ^１、Ｒ^２は、それぞれ独立に水素原子、アルキル基、シクロアルキル基又は置換されていてもよいアリール基を表し、或いは、ＣＲ^１Ｒ^２は、Ｃ＝Ｏを表し；
Ｒ^３、Ｒ^４は、それぞれ独立に水素原子、ハロゲン原子、シアノ基、ヒドロキシ基、アルキル基、シクロアルキル基、置換されていてもよいアリール基、アルコキシ基、アシル基、カルボキシル基、アルコキシカルボニル基、アミノ基、モノアルキルアミノ基、ジアルキルアミノ基又はアシルアミノ基を表し；
Ｒ^５、Ｒ^８、Ｒ^９は、それぞれ独立に水素原子、ハロゲン原子、シアノ基、ヒドロキシ基、アルキル基、シクロアルキル基、置換されていてもよいアリール基、アルコキシ基、アシル基、カルボキシル基、アルコキシカルボニル基、置換されていてもよいアリールオキシ基、チオール基、アルキルチオ基、置換されていてもよいアリールチオ基、アルキルスルホニル基、置換されていてもよいアリールスルホニル基、アルキルアミノカルボニル基、置換されていてもよいアリールアミノカルボニル基、アルキルアミノスルホニル基、置換されていてもよいアリールアミノスルホニル基、アルコキシカルボニル基、置換されていてもよいアリールオキシカルボニル基、アミノ基、モノアルキルアミノ基、ジアルキルアミノ基、アシルアミノ基、アルキルスルホニルアミノ基又は置換されていてもよいアリールスルホニルアミノ基を表し；
Ｒ^６は、アルキル基、シクロアルキル基、置換されていてもよいアリール基、アルコキシ基、アシル基、カルボキシル基、アルコキシカルボニル基又は下記一般式（２）

で表される構造を表し；
Ｒ^７は、Ｏ、ＮＨ又はＮＲ^１３を表し；
Ｒ^１０は、水素原子、アルキル基、シクロアルキル基又は置換されていてもよいアリール基を表し；
Ｒ^１１、Ｒ^１２は、それぞれ独立に水素原子、アルキル基、シクロアルキル基、置換されていてもよいアリール基を表し；
Ｒ^１３は、アルキル基、シクロアルキル基または置換されていてもよいアリール基を表す）
で表される化合物またはその薬学的に許容される塩を有効成分とする抗癌剤。
２．Ｒ^７が酸素原子である項１に記載の抗癌剤。
３．Ｒ^５がメトキシ基である項１に記載の抗癌剤。
４．Ｒ^９がアルキル基又は置換されていてもよいアリール基である項１に記載の抗癌剤。
５．Ｒ^９がｎ−プロピル基である項１に記載の抗癌剤。
６．Ｒ^６が、下記一般式（２）

（式中、Ｒ^１０、Ｒ^１１およびＲ^１２は、前記に定義されるとおりである）
で表される基である項１に記載の抗癌剤。
７．下記一般式（３）

で表される項１に記載の抗癌剤。
８．項１〜７のいずれかに記載の化合物の有効量を投与することを特徴とする癌の治療方法。
９．癌が、急性骨髄性白血病、急性リンパ性白血病、悪性リンパ腫、絨毛癌、多発性骨髄腫、軟部腫瘍、小細胞肺ガン、慢性骨髄性白血病、甲状腺髄様癌、骨肉腫、頭頸部癌、食道癌、非小細胞肺ガン、大腸癌、胃癌、胆道癌、脳腫瘍、悪性黒色腫、腎臓癌、膵臓癌、肝臓癌からなる群より選択される項８に記載の方法。
１０．癌が、急性骨髄性白血病、急性リンパ性白血病、悪性リンパ腫、慢性骨髄性白血病、小細胞肺ガンからなる群より選択される項９に記載の方法。
１１．癌が多剤耐性（ＭＤＲ）の癌である項８に記載の方法。
本発明は、好ましくは一般式（３）で表される化合物を有効成分として含有する抗癌剤、並びに該化合物を癌患者に投与する癌の治療方法に係るものである。
更に好ましくは、急性骨髄性白血病、急性リンパ性白血病、悪性リンパ腫、絨毛癌、多発性骨髄腫、軟部腫瘍、小細胞肺ガン、慢性骨髄性白血病、甲状腺髄様癌、骨肉腫、頭頸部癌、食道癌、非小細胞肺ガン、大腸癌、胃癌、胆道癌、脳腫瘍、悪性黒色腫、腎臓癌、膵臓癌、肝臓癌からなる群より選択される疾患を治療するための、一般式（３）で表される化合物を有効成分として含有する抗癌剤に係るものである。
また、本発明は、好ましくは、一般式（３）で表される化合物の抗癌有効量を投与することを特徴とする癌の治療方法に係るものである。
更に好ましくは、一般式（３）で表される化合物の抗癌有効量を投与することを特徴とする、急性骨髄性白血病、急性リンパ性白血病、悪性リンパ腫、絨毛癌、多発性骨髄腫、軟部腫瘍、小細胞肺ガン、慢性骨髄性白血病、甲状腺髄様癌、骨肉腫、頭頸部癌、食道癌、非小細胞肺ガン、大腸癌、胃癌、胆道癌、脳腫瘍、悪性黒色腫、腎臓癌、膵臓癌、肝臓癌からなる群より選択される疾患の治療方法に係るものである。
以下、本発明を詳細に説明する。
抗癌化合物の構造
本発明の抗癌剤の有効成分は、下記一般式（１）で表される構造を有する。
一般式（１）

（式中、Ｒ^１〜Ｒ^９は、前記に定義されるとおりである。）
Ｒ^１〜Ｒ^１３において、アルキル基は、置換又は非置換の直鎖状或いは分岐状のものを含み、炭素数は通常は１〜１８、好ましくは１〜８、更に好ましくは１〜３であり、例えば、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、ｎ−ペンチル、イソペンチル、ヘキシル、ヘプチル、オクチル、ノニル及びデシルが挙げられる。
シクロアルキル基は、置換又は非置換のものを含み、炭素数も特に限定されないが、通常は３〜１０、好ましくは３〜７である。シクロアルキル基の具体例としては、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル及びシクロヘプチルが挙げられる。
アリール基としては、「アリール基」とは、５又は６員の芳香族炭化水素環からなる単環又は多環系の基を意味し、具体例としては、フェニル、ナフチル、フルオレニル、アントリル、ビフェニリル、テトラヒドロナフチル、クロマニル、２，３−ジヒドロ−１，４−ジオキサナフタレニル、インダニル及びフェナントリルが挙げられる。
置換基を有するアリール基の置換基の数は１〜３個、好ましくは１または２個であり、該置換基としては、メチル、エチル、メトキシ、エトキシ、アミノ、メチルアミノ、ジメチルアミノ、シアノ、ニトロ、フッ素、塩素、臭素、トリフルオロメチル、ヒドロキシ、カルボキシル、メトキシカルボニル、エトキシカルボニル、プロポキシカルボニル、ブトキシカルボニル、ＣＯＮＨ２，アセチルが例示される。置換アリール基の例示としては、フェニル、２−、３−若しくは４−フルオロフェニル、２−、３−若しくは４−クロロフェニル、２−、３−若しくは４−ブロモフェニル、２，４−ジフルオロフェニル、２，３−、２，４−、３，４−、３，５−若しくは２，６−ジクロロフェニル、３−クロロ−４−フルオロフェニル、４−イソプロピルフェニル、２，６−ジメチルフェニル、２−、３−若しくは４−トリフルオロメチルフェニル、４−メトキシフェニル、４−トリフルオロメトキシフェニル、２−、３−若しくは４−ジメチルアミノフェニル、２−、３−若しくは４−ニトロフェニル、４−スルファモイルフェニルが例示される。
アルコキシ基としては、−ＯＲ’（Ｒ’は前記アルキル基である）で表される基が挙げられ、例えば、メトキシ、エトキシ、ｎ−プロポキシ、イソプロポキシ、ｎ−ブトキシ、イソブトキシ、ｓｅｃ−ブトキシ、ｔ−ブトキシが例示される。
アシル基としては、−ＣＯＲ’’（Ｒ’’は前記アルキル基または前記置換されていてもよいアリール基である）で表される基が挙げられ、例えば、アセチル、プロピオニル、ベンゾイルが例示される。
ハロゲン原子としては、フッ素、塩素、臭素、ヨウ素が挙げられる。
アシルアミノ基としては、−ＮＨＣＯＲ’’（Ｒ’’は前記アルキル基または前記置換されていてもよいアリール基である）で表される基が挙げられ、例えば、アセチルアミノ、プロピオニルアミノ、ブチリルアミノ、ベンゾイルアミノが挙げられる。
アルコキシカルボニル基としては、−ＣＯＯＲ’（Ｒ’は前記アルキル基である）で表される基が挙げられ、例えば、メトキシカルボニル、エトキシカルボニル、ｎ−プロポキシカルボニル、イソプロポキシカルボニル、ｎ−ブトキシカルボニル、イソブトキシカルボニル、ｓｅｃ−ブトキシカルボニル、ｔ−ブトキシカルボニルが例示される。
置換されていてもよいアリールオキシ基としては、−Ｏ−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルオキシ、ナフチルオキシが挙げられる。
アルキルチオ基としては、−Ｓ−（アルキル基）で表される基が挙げられ、例えばメチルチオ、エチルチオ、ｎ−プロピルチオ、イソプロピルチオ、ｎ−ブチルチオ、イソブチルチオ、ｔｅｒｔ−ブチルチオが挙げられる。
置換されていてもよいアリールチオ基としては、−Ｓ−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルチオ、ナフチルチオが挙げられる。
モノアルキルアミノ基としては、前記アルキルでモノ置換されたアミノ基、例えばメチルアミノカルボニル、エチルアミノカルボニル、ｎ−プロピルアミノカルボニル、イソプロピルアミノカルボニル、ｎ−ブチルアミノカルボニル、イソブチルアミノカルボニル、ｔｅｒｔ−ブチルアミノカルボニル、ｎ−ペンチルアミノカルボニル、イソペンチルアミノカルボニル、ヘキシルアミノカルボニルが挙げられる。
ジアルキルアミノ基としては、前記アルキルでジ置換されたアミノ基、例えばジメチルアミノカルボニル、ジエチルアミノカルボニル、ジｎ−プロピルアミノカルボニル、ジイソプロピルアミノカルボニル、ジｎ−ブチルアミノカルボニル、ジイソブチルアミノカルボニル、ジｔｅｒｔ−ブチルアミノカルボニル、ジｎ−ペンチルアミノカルボニル、ジイソペンチルアミノカルボニル、ジヘキシルアミノカルボニルが挙げられる。
アルキルスルホニル基としては、−ＳＯ_２−（アルキル基）で表される基が挙げられ、例えばメチルスルホニル、エチルスルホニル、ｎ−プロピルスルホニル、イソプロピルスルホニル、ｎ−ブチルスルホニル、イソブチルスルホニル、ｔｅｒｔ−ブチルスルホニルが挙げられる。
置換されていてもよいアリールスルホニル基としては、−ＳＯ_２−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルスルホニル、ナフチルスルホニルが挙げられる。
アルキルアミノカルボニル基としては、前記アルキルでモノ置換またはジ置換されたアミノカルボニル基が挙げられ、前記アルキルでモノ置換されたアミノ基としては、メチルアミノカルボニル、エチルアミノカルボニル、ｎ−プロピルアミノカルボニル、イソプロピルアミノカルボニル、ｎ−ブチルアミノカルボニル、イソブチルアミノカルボニル、ｔｅｒｔ−ブチルアミノカルボニル、ｎ−ペンチルアミノカルボニル、イソペンチルアミノカルボニル、ヘキシルアミノカルボニルが挙げられ、前記アルキルでジ置換されたアミノカルボニル基としては、ジメチルアミノカルボニル、ジエチルアミノカルボニル、ジｎ−プロピルアミノカルボニル、ジイソプロピルアミノカルボニル、ジｎ−ブチルアミノカルボニル、ジイソブチルアミノカルボニル、ジｔｅｒｔ−ブチルアミノカルボニル、ジｎ−ペンチルアミノカルボニル、ジイソペンチルアミノカルボニル、ジヘキシルアミノカルボニルが挙げられる。
置換されていてもよいアリールアミノカルボニル基としては、−ＣＯＮＨ−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルアミノカルボニル、ナフチルアミノカルボニルが挙げられる。
アルキルアミノスルホニル基としては、前記アルキルでモノ置換またはジ置換されたアミノスルホニル基が挙げられ、前記アルキルでモノ置換されたアミノ基としては、メチルアミノスルホニル、エチルアミノスルホニル、ｎ−プロピルアミノスルホニル、イソプロピルアミノスルホニル、ｎ−ブチルアミノスルホニル、イソブチルアミノスルホニル、ｔｅｒｔ−ブチルアミノスルホニル、ｎ−ペンチルアミノスルホニル、イソペンチルアミノスルホニル、ヘキシルアミノスルホニルが挙げられ、前記アルキルでジ置換されたアミノスルホニル基としては、ジメチルアミノスルホニル、ジエチルアミノスルホニル、ジｎ−プロピルアミノスルホニル、ジイソプロピルアミノスルホニル、ジｎ−ブチルアミノスルホニル、ジイソブチルアミノスルホニル、ジｔｅｒｔ−ブチルアミノスルホニル、ジｎ−ペンチルアミノスルホニル、ジイソペンチルアミノスルホニル、ジヘキシルアミノスルホニルが挙げられる。
置換されていてもよいアリールアミノスルホニル基としては、−ＳＯ_２ＮＨ−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルアミノスルホニル、ナフチルアミノスルホニルが挙げられる。
置換されていてもよいアリールオキシカルボニル基としては、−ＣＯＯ−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルオキシカルボニル、ナフチルオキシカルボニルが挙げられる。
アルキルスルホニルアミノ基としては、−ＮＨＳＯ_２−（アルキル基）で表される基が挙げられ、例えばメチルスルホニルアミノ、エチルスルホニルアミノ、ｎ−プロピルスルホニルアミノ、イソプロピルスルホニルアミノ、ｎ−ブチルスルホニルアミノ、イソブチルスルホニルアミノ、ｔｅｒｔ−ブチルスルホニルアミノ、ｎ−ペンチルスルホニルアミノ、イソペンチルスルホニルアミノ、ヘキシルスルホニルアミノが挙げられ、
置換されていてもよいアリールスルホニルアミノ基としては、−ＮＨＳＯ_２−（置換されていてもよいアリール基）で表される基が挙げられ、例えばフェニルスルホニルアミノ、ナフチルスルホニルアミノが挙げられる。
本発明の一般式（１）で表される化合物に関し、Ｒ^７は酸素原子（Ｏ）が好ましい。
Ｒ^５はメトキシ基（−ＯＣＨ_３）が好ましい。
Ｒ^９はアルキル基又はアリール基が好ましく、特にｎ−プロピル基が好ましい。
また、Ｒ^６は、一般式（２）

（式中、Ｒ^１０、Ｒ^１１およびＲ^１２は、前記に定義されるとおりである）
で表される基が好ましい。
本発明においては、一般式（３）

で表される化合物（５−メトキシ−２，２−ジメチル−６−（２−メチル−１−オキソ−２−ブテニル）−１０−プロピル−２Ｈ，８Ｈ−ベンゾ［１，２−ｂ；３，４−ｂ’］ジピラン−８−オン（５−ｍｅｔｈｏｘｙ−２，２−ｄｉｍｅｔｈｙｌ−６−（２−ｍｅｔｈｔｌ−１−ｏｘｏ−２−ｂｕｔｅｎｙｌ）−１０−ｐｒｏｐｙｌ−２Ｈ，８Ｈ−ｂｅｎｚｏ［１，２−ｂ；３，４−ｂ’］ｄｉｐｙｒａｎ−８−ｏｎｅ））が抗癌剤として好ましい。
この一般式（３）の化合物は、特に優れた癌細胞増殖抑制作用を有する。
本発明の一般式（１）で表される化合物の薬学的に許容される塩は、Ｒ^３〜Ｒ^６、Ｒ^８，Ｒ^９のいずれかがカルボキシル基（ＣＯＯＨ）あるいはアミノ基、モノ−またはジ−アルキルアミノ基を有する場合、薬学的に許容される塩を調製できる。薬学的に許容されるカルボキシル基の塩としては、アルカリ金属塩（ナトリウム塩、カリウム塩、リチウム塩）が例示され、薬学的に許容されるアミノ基、モノ−またはジ−アルキルアミノ基の塩としては、硫酸塩、塩酸塩、臭化水素酸塩、硝酸鉛、リン酸塩などの無機酸塩、フマル酸塩、マレイン酸塩、コハク酸塩、メタンスルホン酸塩、トルエンスルホン酸塩などの有機酸塩が挙げられる。このような薬学的に許容される塩は、本発明の化合物とアルカリ金属の炭酸塩または炭酸水素塩、アルカリ金属水酸化物、あるいは、無機酸または有機酸を作用させることにより得ることができる。
一般式（１）の化合物の製造方法
上記化合物は、種々の方法で適宜調製することができ、化学的に合成してもよく、また、適当な植物から抽出などにより分離してもよい。
（ｉ）化学的合成方法
化学的に合成する方法としては、適宜公知の方法を用いることができる。
例えば、以下のような方法が挙げられる。
下記一般式（４）

（式中、Ｒ^１〜Ｒ^５は、前記に定義されるとおりである）
で表される化合物、又は、下記一般式（５）

（式中、Ｒ^１〜Ｒ^５は、前記に定義されるとおりである。Ｚは水素原子またはＲ^１３（Ｒ^１３は前記定義されるとおりである）を示す。）
で表される化合物と、下記一般式（６）

（式中、Ｒ^８及びＲ^９は、前記に定義されるとおりである）
で表される化合物とを、濃硫酸を用いて加熱反応させて、
それぞれ、下記一般式（７）

（式中、Ｒ^１〜Ｒ^５、Ｒ^８及びＲ^９は、前記に定義されるとおりである）
で表される化合物、又は、下記一般式（８）

（式中、Ｒ^１〜Ｒ^５、Ｒ^８、Ｒ^９及びＺは、前記に定義されるとおりである）
で表される化合物を得る。
次いで、一般式（７）又は（８）で表される化合物に、例えば、下記一般式（９）

で表される化合物を、フリーデル・クラフツ（Ｆｒｉｅｄｅｌ−Ｃｒａｆｔｓ）触媒を用いて反応させることによって、下記一般式（１）

（式中、Ｒ^１〜Ｒ^９は、前記に定義されるとおりである）
で表される化合物を得る。
Ｒ^６が一般式（２）で表される構造の化合物は、一般式（９）で表される化合物として、下記一般式（１０）

（式中、Ｒ^１０〜Ｒ^１２は、前記に定義されるとおりである）
で表される化合物を、フリーデル・クラフツ（Ｆｒｉｅｄｅｌ−Ｃｒａｆｔｓ）触媒を用いてアシル化反応させて得ることができる。
フリーデル・クラフツ（Ｆｒｉｅｄｅｌ−Ｃｒａｆｔｓ）触媒としては、例えば、ＡｌＣｌ_３、ＢＦ_３、ＺｎＣｌ_２、ＳｎＣｌ_４などが挙げられる。
一般式（４）〜（１０）で表される化合物は、いずれも、汎用の方法により得ることができ、市販品がある場合には、それをそのまま用いればよい。
斯くして得られる本発明の化合物は、通常、溶解、抽出、分液、傾斜、濾過、濃縮、薄層クロマトグラフィー、カラムクロマトグラフィー、ガスクロマトグラフィー、高速液体クロマトグラフィー、蒸留、昇華、結晶化などの方法を単独又は組み合わせて、適宜精製を行う。
上記と同様の方法において、所望の置換基を有する中間物を用い、又は適当な官能基を適宜導入することによって、各種化合物を得ることができる。
例えば、一般式（４）において、Ｒ^１、Ｒ^２がメチル基、Ｒ^３、Ｒ^４が水素、Ｒ^５が−ＯＣＨ_３基である化合物を用い、一般式（６）において、Ｒ^８が水素、Ｒ^９が−ＣＨ_２ＣＨ_２ＣＨ_３基である化合物を用い、一般式（１０）において、Ｒ^１０が水素、Ｒ^１１、Ｒ^１２がメチル基である化合物を用いる場合には、
下記一般式（３）

で表される化合物を得ることができる。
（ｉｉ）植物からの抽出方法
本願発明の医薬組成物の有効成分となる化合物は、適当な植物から抽出して得ることもできる。植物としては、オトギリ草科（Ｇｕｔｔｉｆｅｒａｅ）の植物が例示できる。オトギリ草科の植物では、キャロフィラム（Ｃａｌｏｐｈｙｌｌｕｍ）属のもの、例えば、Ｃａｌｏｐｈｙｌｌｕｍ Ｂｒａｓｉｌｉｅｎｓｉｓ又はＣａｌｏｐｈｙｌｌｕｍ ｉｎｏｐｈｙｌｌｕｍなどが好適であり、特に、Ｃａｌｏｐｈｙｌｌｕｍ Ｂｒａｓｉｌｉｅｎｓｉｓが好適に用いられる。
植物から該化合物を抽出して得る方法は、適宜公知の方法を用いることができ、特に限定されないが、例えば、植物の樹皮などを粉砕し、有機溶媒で抽出する方法などを用いることができる。
具体的には、次のように行うことができる。Ｃａｌｏｐｈｙｌｌｕｍ Ｂｒａｓｉｌｉｅｎｓｉｓ又はＣａｌｏｐｈｙｌｌｕｍ ｉｎｏｐｈｙｌｌｕｍの乾燥した樹皮の粉砕物に、適当な有機溶媒、例えば、アセトン、ヘキサン、メタノール、エタノール等の低級アルコールなどを適宜組み合わせて用いて、室温で抽出した後、低圧下溶媒を蒸発する。更にクロマトグラフィーやろ過、抽出など、適宜分離精製を行って、所望の化合物を得る。
例えば、一般式（３）の化合物は、Ｃａｌｏｐｈｙｌｌｕｍ Ｂｒａｓｉｌｉｅｎｓｉｓ（Ｇｕｔｔｉｆｅｒａｅ）の植物抽出物を分離精製して得ることができる。
医薬組成物（医薬製剤）の製法
一般式（１）の化合物またはその塩は、薬学的に許容される担体と配合し、錠剤、カプセル剤、顆粒剤、散剤などの固形製剤；シロップ剤、注射剤などの液状製剤、貼付剤、軟膏剤、硬膏剤などの経皮吸収剤、吸入剤、坐剤として、適宜製剤化することができる。
本発明の医薬組成物は、経口または非経口投与され、上記化合物を１種単独で用いてもよく、又は２種以上を併用して用いてもよい。
薬学的に許容される担体としては、製剤素材として慣用されている各種有機あるいは無機担体物質を用いることができる。具体的には、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤、液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤などを配合することができる。また必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤などの製剤添加物を用いることもできる。
賦形剤の好適な例としては、例えば乳糖、白糖、Ｄ−マンニトール、デンプン、結晶セルロース、軽質無水ケイ酸などが挙げられる。
滑沢剤の好適な例としては、例えばステアリン酸マグネシウム、ステアリン酸カルシウム、タルク、コロイドシリカなどが挙げられる。
結合剤の好適な例としては、例えば結晶セルロース、白糖、Ｄ−マンニトール、デキストリン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドンなどが挙げられる。
崩壊剤の好適な例としては、例えばデンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウムなどが挙げられる。
溶剤の好適な例としては、例えば注射用水、アルコール、プロピレングリコール、マクロゴール、ゴマ油、トウモロコシ油などが挙げられる。
溶解補助剤の好適な例としては、例えばポリエチレングリコール、プロピレングリコール、Ｄ−マンニトール、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウムなどが挙げられる。
懸濁化剤の好適な例としては、例えばステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリンなどの界面活性剤；ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロースなどの親水性高分子などが挙げられる。
等張化剤の好適な例としては、例えば塩化ナトリウム、グリセリン、Ｄ−マンニトールなどが挙げられる。
緩衝剤の好適な例としては、例えばリン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などが挙げられる。
無痛化剤の好適な例としては、例えばベンジルアルコールなどが挙げられる。防腐剤の好適な例としては、例えば、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸などが挙げられる。
抗酸化剤の好適な例としては、例えば亜硫酸塩、アスコルビン酸などが挙げられる。
対象疾患
本発明の抗癌剤は、各種の癌の治療に適用することができる。
癌の種類は、特に限定されないが、例えば、急性骨髄性白血病、急性リンパ性白血病、悪性リンパ腫、絨毛癌、多発性骨髄腫、軟部腫瘍、小細胞肺ガン、慢性骨髄性白血病、甲状腺髄様癌、骨肉腫、頭頸部癌、食道癌、非小細胞肺ガン、大腸癌、胃癌、胆道癌、脳腫瘍、悪性黒色腫、腎臓癌、膵臓癌、肝臓癌が挙げられる。
本発明の抗癌剤は、特に、急性骨髄性白血病、急性リンパ性白血病、小細胞肺ガン、に有効である。中でも急性骨髄性白血病、急性リンパ性白血病、小細胞肺ガンに好適に用いられる。
本発明の抗癌剤の投与条件及び投与方法は、疾患の種類や患者の状態又は適用部位などに応じて、適宜設定することができる。
具体的には、投与量は、動物の種類や大きさ（または体重）、症状等に応じて適宜調節する。ヒト又はヒト以外の動物に投与する場合には、約０．１−１００ｍｇ／ｋｇ（体重）程度、好ましくは約１．０−１０ｍｇ／ｋｇ（体重）程度、更に好ましくは約１．０−３．０ｍｇ／ｋｇ（体重）程度の量で投与する。また投与方法も、最適の治療効果が得られるように適宜調節でき、例えば、毎日数回に分けて投与でき、治療中に危険な状態が生じた場合には、その状態に応じて投与量や投与回数を適宜減らすこともできる。
本願発明の抗癌剤は、所望の効果を得るために、単独で投与することもでき、また他の抗癌剤、化学療法剤、抗炎症剤または免疫療法剤などと適宜組み合わせて用いることもできる。
本発明の抗癌剤と組み合わせて用いる他の抗癌剤としては、例えば、代謝拮抗剤、アルキル化薬、白金系抗癌剤、トポイソメラーゼ阻害剤、抗癌性抗生物質、チロシンキナーゼ阻害剤、ヒト化抗体が挙げられる。The inventor has conducted extensive studies with the main purpose of solving the above-described problems. As a result, it has been found that a compound having a specific structure exhibits a remarkable cancer cell growth inhibitory effect, is effective against multidrug-resistant cancer cells, and has a large safety margin. Over time, the present invention has been completed.
That is, the present invention relates to the following anticancer agent and cancer treatment method.
1. The following general formula (1)

(Wherein R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group or an optionally substituted aryl group, or CR ¹ R ² represents C═O;
R ³ and R ⁴ each independently represents a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, or an alkoxycarbonyl group. Represents an amino group, a monoalkylamino group, a dialkylamino group or an acylamino group;
R ⁵ , R ⁸ and R ⁹ are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, Alkoxycarbonyl group, aryloxy group which may be substituted, thiol group, alkylthio group, arylthio group which may be substituted, alkylsulfonyl group, arylsulfonyl group which may be substituted, alkylaminocarbonyl group, substituted Optionally substituted arylaminocarbonyl group, alkylaminosulfonyl group, optionally substituted arylaminosulfonyl group, alkoxycarbonyl group, optionally substituted aryloxycarbonyl group, amino group, monoalkylamino group, dialkylamino Group, acylamino Represents an alkylsulfonylamino group or an optionally substituted arylsulfonylamino group;
R ⁶ represents an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, an alkoxycarbonyl group, or the following general formula (2)

Represents the structure represented by
R ⁷ represents O, NH or NR ¹³ ;
R ¹⁰ represents a hydrogen atom, an alkyl group, a cycloalkyl group or an aryl group which may be substituted;
R ¹¹ and R ¹² each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, or an optionally substituted aryl group;
R ¹³ represents an alkyl group, a cycloalkyl group, or an optionally substituted aryl group)
Or a pharmaceutically acceptable salt thereof as an active ingredient.
2. Item 2. The anticancer agent according to Item 1, wherein R ⁷ is an oxygen atom.
3. Item 2. The anticancer agent according to Item 1, wherein R ⁵ is a methoxy group.
4). Item 2. The anticancer agent according to Item 1, wherein R ⁹ is an alkyl group or an optionally substituted aryl group.
5). Item 2. The anticancer agent according to Item 1, wherein R ⁹ is an n-propyl group.
6). R ⁶ represents the following general formula (2)

(Wherein R ¹⁰ , R ¹¹ and R ¹² are as defined above).
Item 2. The anticancer agent according to Item 1, which is a group represented by:
7). The following general formula (3)

Item 2. The anticancer agent according to Item 1, represented by:
8). Item 8. A method for treating cancer, comprising administering an effective amount of the compound according to any one of Items 1 to 7.
9. Cancer is acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid cancer, osteosarcoma, head and neck cancer, esophagus Item 9. The method according to Item 8, selected from the group consisting of cancer, non-small cell lung cancer, colon cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, pancreatic cancer, and liver cancer.
10. Item 10. The method according to Item 9, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, chronic myelogenous leukemia, and small cell lung cancer.
11. Item 9. The method according to Item 8, wherein the cancer is multidrug resistant (MDR) cancer.
The present invention preferably relates to an anticancer agent containing a compound represented by the general formula (3) as an active ingredient, and a cancer treatment method in which the compound is administered to a cancer patient.
More preferably, acute myeloid leukemia, acute lymphoblastic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, thyroid medullary cancer, osteosarcoma, head and neck cancer, General formula (3) for treating a disease selected from the group consisting of esophageal cancer, non-small cell lung cancer, colon cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, pancreatic cancer, liver cancer It relates to an anticancer agent containing a compound represented by the above as an active ingredient.
In addition, the present invention preferably relates to a method for treating cancer, comprising administering an anticancer effective amount of a compound represented by the general formula (3).
More preferably, an anticancer effective amount of a compound represented by the general formula (3) is administered, characterized in that acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft part Tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid cancer, osteosarcoma, head and neck cancer, esophageal cancer, non-small cell lung cancer, colon cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, The present invention relates to a method for treating a disease selected from the group consisting of pancreatic cancer and liver cancer.
Hereinafter, the present invention will be described in detail.
Structure of anticancer compound The active ingredient of the anticancer agent of the present invention has a structure represented by the following general formula (1).
General formula (1)

(Wherein R ^{1 to} R ⁹ are as defined above.)
In R ^{1 to} R ¹³ , the alkyl group includes a substituted or unsubstituted linear or branched group, and the number of carbon atoms is usually 1 to 18, preferably 1 to 8, and more preferably 1 to 3. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl, nonyl and decyl.
The cycloalkyl group includes a substituted or unsubstituted group, and the number of carbon atoms is not particularly limited, but is usually 3 to 10, preferably 3 to 7. Specific examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
As the aryl group, the “aryl group” means a monocyclic or polycyclic group composed of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples include phenyl, naphthyl, fluorenyl, anthryl, biphenylyl. , Tetrahydronaphthyl, chromanyl, 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl and phenanthryl.
The number of substituents of the aryl group having a substituent is 1 to 3, preferably 1 or 2, and examples of the substituent include methyl, ethyl, methoxy, ethoxy, amino, methylamino, dimethylamino, cyano, Examples include nitro, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, carboxyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, CONH2, and acetyl. Examples of substituted aryl groups include phenyl, 2-, 3- or 4-fluorophenyl, 2-, 3- or 4-chlorophenyl, 2-, 3- or 4-bromophenyl, 2,4-difluorophenyl, 2 , 3-, 2,4-, 3,4-, 3,5- or 2,6-dichlorophenyl, 3-chloro-4-fluorophenyl, 4-isopropylphenyl, 2,6-dimethylphenyl, 2-3 -Or 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 2-, 3- or 4-dimethylaminophenyl, 2-, 3- or 4-nitrophenyl, 4-sulfamoylphenyl Is exemplified.
Examples of the alkoxy group include a group represented by —OR ′ (R ′ is the alkyl group). For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-Butoxy is exemplified.
Examples of the acyl group include a group represented by —COR ″ (R ″ is the alkyl group or the optionally substituted aryl group), and examples thereof include acetyl, propionyl, and benzoyl. .
Examples of the halogen atom include fluorine, chlorine, bromine and iodine.
Examples of the acylamino group include a group represented by —NHCOR ″ (where R ″ is the alkyl group or the optionally substituted aryl group), and examples thereof include acetylamino, propionylamino, butyrylamino, benzoyl. Amino is mentioned.
Examples of the alkoxycarbonyl group include a group represented by —COOR ′ (R ′ is the alkyl group), and examples thereof include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, Examples are isobutoxycarbonyl, sec-butoxycarbonyl and t-butoxycarbonyl.
Examples of the optionally substituted aryloxy group include a group represented by -O- (an optionally substituted aryl group), and examples thereof include phenyloxy and naphthyloxy.
Examples of the alkylthio group include a group represented by -S- (alkyl group), and examples thereof include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, and tert-butylthio.
Examples of the optionally substituted arylthio group include a group represented by -S- (an optionally substituted aryl group), and examples thereof include phenylthio and naphthylthio.
Examples of the monoalkylamino group include amino groups monosubstituted with the above alkyl, such as methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, n-butylaminocarbonyl, isobutylaminocarbonyl, tert-butylamino. Examples include carbonyl, n-pentylaminocarbonyl, isopentylaminocarbonyl, hexylaminocarbonyl.
Examples of the dialkylamino group include amino groups disubstituted with the above alkyl, such as dimethylaminocarbonyl, diethylaminocarbonyl, di-n-propylaminocarbonyl, diisopropylaminocarbonyl, di-n-butylaminocarbonyl, diisobutylaminocarbonyl, ditert-butyl. Examples include aminocarbonyl, di-n-pentylaminocarbonyl, diisopentylaminocarbonyl, and dihexylaminocarbonyl.
Examples of the alkylsulfonyl group include a group represented by —SO ₂ — (alkyl group), for example, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, isobutylsulfonyl, tert-butylsulfonyl. Is mentioned.
Examples of the optionally substituted arylsulfonyl group include a group represented by —SO ₂ — (optionally substituted aryl group), and examples thereof include phenylsulfonyl and naphthylsulfonyl.
Examples of the alkylaminocarbonyl group include aminocarbonyl groups mono-substituted or di-substituted with the alkyl. Examples of the amino group mono-substituted with alkyl include methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, Examples include isopropylaminocarbonyl, n-butylaminocarbonyl, isobutylaminocarbonyl, tert-butylaminocarbonyl, n-pentylaminocarbonyl, isopentylaminocarbonyl, and hexylaminocarbonyl. Examples of the aminocarbonyl group disubstituted with alkyl include Dimethylaminocarbonyl, diethylaminocarbonyl, di-n-propylaminocarbonyl, diisopropylaminocarbonyl, di-n-butylaminocarbonyl, diisobutylaminocarbonyl Boniru, di tert- butyl aminocarbonyl, di n- pentyl aminocarbonyl, di isopentyl aminocarbonyl include dihexyl aminocarbonyl.
Examples of the optionally substituted arylaminocarbonyl group include a group represented by -CONH- (an optionally substituted aryl group), and examples thereof include phenylaminocarbonyl and naphthylaminocarbonyl.
Examples of the alkylaminosulfonyl group include an aminosulfonyl group mono-substituted or di-substituted with the alkyl, and examples of the amino group mono-substituted with the alkyl include methylaminosulfonyl, ethylaminosulfonyl, n-propylaminosulfonyl, Examples include isopropylaminosulfonyl, n-butylaminosulfonyl, isobutylaminosulfonyl, tert-butylaminosulfonyl, n-pentylaminosulfonyl, isopentylaminosulfonyl, hexylaminosulfonyl, and examples of the aminosulfonyl group di-substituted with alkyl. , Dimethylaminosulfonyl, diethylaminosulfonyl, di-n-propylaminosulfonyl, diisopropylaminosulfonyl, di-n-butylaminosulfonyl, diisobutylaminos Honiru, di tert- butyl aminosulfonyl, di n- pentyl aminosulfonyl, di isopentyl aminosulfonyl include dihexyl aminosulfonyl.
Examples of the optionally substituted arylaminosulfonyl group include a group represented by —SO ₂ NH— (an optionally substituted aryl group), and examples thereof include phenylaminosulfonyl and naphthylaminosulfonyl.
Examples of the optionally substituted aryloxycarbonyl group include a group represented by —COO— (an optionally substituted aryl group), and examples thereof include phenyloxycarbonyl and naphthyloxycarbonyl.
Examples of the alkylsulfonylamino group include a group represented by —NHSO ₂ — (alkyl group). For example, methylsulfonylamino, ethylsulfonylamino, n-propylsulfonylamino, isopropylsulfonylamino, n-butylsulfonylamino, isobutyl Sulfonylamino, tert-butylsulfonylamino, n-pentylsulfonylamino, isopentylsulfonylamino, hexylsulfonylamino,
Examples of the optionally substituted arylsulfonylamino group include a group represented by —NHSO ₂ — (an optionally substituted aryl group), and examples thereof include phenylsulfonylamino and naphthylsulfonylamino.
Regarding the compound represented by the general formula (1) of the present invention, R ⁷ is preferably an oxygen atom (O).
R ⁵ is preferably a methoxy group (—OCH ₃ ).
R ⁹ is preferably an alkyl group or an aryl group, and particularly preferably an n-propyl group.
R ⁶ represents the general formula (2)

(Wherein R ¹⁰ , R ¹¹ and R ¹² are as defined above).
The group represented by these is preferable.
In the present invention, the general formula (3)

(5-methoxy-2,2-dimethyl-6- (2-methyl-1-oxo-2-butenyl) -10-propyl-2H, 8H-benzo [1,2-b; 4-b ′] dipyran-8-one (5-methoxy-2,2-dimethyl-6- (2-methyl-1-oxo-2-butenyl) -10-propyl-2H, 8H-benzo [1,2 -B; 3,4-b '] dipyran-8-one)) is preferred as an anticancer agent.
This compound of the general formula (3) has a particularly excellent cancer cell proliferation inhibitory action.
The pharmaceutically acceptable salt of the compound represented by the general formula (1) of the present invention is any one of R ^{3 to} R ⁶ , R ⁸ and R ⁹ is a carboxyl group (COOH) or an amino group, mono- or When it has a di-alkylamino group, a pharmaceutically acceptable salt can be prepared. Examples of the pharmaceutically acceptable salt of the carboxyl group include alkali metal salts (sodium salt, potassium salt, lithium salt), and pharmaceutically acceptable amino group, mono- or di-alkylamino group salts. Are organic salts such as sulfates, hydrochlorides, hydrobromides, lead nitrates, phosphates, and other inorganic salts, fumarate, maleate, succinate, methanesulfonate, toluenesulfonate, etc. Acid salts. Such a pharmaceutically acceptable salt can be obtained by reacting the compound of the present invention with an alkali metal carbonate or bicarbonate, an alkali metal hydroxide, or an inorganic acid or an organic acid.
Method for Producing Compound of General Formula (1) The above compound can be appropriately prepared by various methods, chemically synthesized, or separated from a suitable plant by extraction or the like.
(I) Chemical synthesis method As a method of chemically synthesizing, a known method can be appropriately used.
For example, the following methods are mentioned.
The following general formula (4)

(Wherein R ^{1 to} R ⁵ are as defined above)
Or a compound represented by the following general formula (5)

(Wherein R ^{1 to} R ⁵ are as defined above. Z represents a hydrogen atom or R ¹³ (R ¹³ is as defined above).)
And a compound represented by the following general formula (6)

Wherein R ⁸ and R ⁹ are as defined above.
The compound represented by is heated and reacted with concentrated sulfuric acid,
Each of the following general formula (7)

(Wherein R ^{1 to} R ⁵ , R ⁸ and R ⁹ are as defined above).
Or a compound represented by the following general formula (8)

(Wherein R ^{1 to} R ⁵ , R ⁸ , R ⁹ and Z are as defined above).
To obtain a compound represented by:
Next, the compound represented by the general formula (7) or (8) is converted into, for example, the following general formula (9).

Is reacted with a Friedel-Crafts catalyst to produce a compound represented by the following general formula (1):

(Wherein R ^{1 to} R ⁹ are as defined above)
To obtain a compound represented by:
A compound having a structure in which R ⁶ is represented by the general formula (2) is represented by the following general formula (10) as a compound represented by the general formula (9)

(Wherein R ^{10 to} R ¹² are as defined above)
Can be obtained by an acylation reaction using a Friedel-Crafts catalyst.
Examples of Friedel-Crafts catalysts include AlCl ₃ , BF ₃ , ZnCl ₂ , SnCl _{4 and the} like.
Any of the compounds represented by the general formulas (4) to (10) can be obtained by a general-purpose method. If there is a commercially available product, it may be used as it is.
The compound of the present invention thus obtained is usually dissolved, extracted, separated, gradient, filtered, concentrated, thin layer chromatography, column chromatography, gas chromatography, high performance liquid chromatography, distillation, sublimation, crystallization. These methods are used alone or in combination for appropriate purification.
In the same manner as described above, various compounds can be obtained by using an intermediate having a desired substituent or by appropriately introducing an appropriate functional group.
For example, in the general formula (4), a compound in which R ¹ and R ² are methyl groups, R ³ and R ⁴ are hydrogen, and R ⁵ is —OCH ₃ group is used. In the general formula (6), R ⁸ is hydrogen. , R ⁹ is a —CH ₂ CH ₂ CH ₃ group, and in the general formula (10), a compound in which R ¹⁰ is hydrogen and R ¹¹ and R ¹² are methyl groups is used,
The following general formula (3)

Can be obtained.
(Ii) Extraction method from plant The compound which becomes an active ingredient of the pharmaceutical composition of this invention can also be obtained by extracting from a suitable plant. An example of the plant is a plant of Gutiferae. Among the plants of the herbaceous family, those belonging to the genus Calophyllum, such as Calophyllum Brasiliensis or Calophyllum inophilyllum, are preferred, and Calophyllum Brasiliensis is particularly preferred.
A method of extracting the compound from the plant can be appropriately selected from known methods, and is not particularly limited. For example, a method of pulverizing a plant bark or the like and extracting it with an organic solvent can be used.
Specifically, it can be performed as follows. After extracting at room temperature using a suitable organic solvent, for example, a lower alcohol such as acetone, hexane, methanol, ethanol, etc., in combination with a pulverized product of dried bark of Calophyllum Brasiliensis or Calophyllum inophilylum, the solvent at low pressure is used. Evaporate. Further, the desired compound is obtained by performing appropriate separation and purification such as chromatography, filtration and extraction.
For example, the compound of the general formula (3) can be obtained by separating and purifying a plant extract of Calophyllum Brasiliensis (Gutiferae).
Preparation of pharmaceutical composition (pharmaceutical preparation) The compound of general formula (1) or a salt thereof is blended with a pharmaceutically acceptable carrier, solid preparation such as tablets, capsules, granules, powders; syrup, injection It can be appropriately formulated as a liquid preparation such as an agent, a transdermal absorbent such as a patch, an ointment, a plaster, an inhalant, or a suppository.
The pharmaceutical composition of the present invention is administered orally or parenterally, and the above compounds may be used alone or in combination of two or more.
As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials can be used. Specifically, excipients, lubricants, binders, disintegrants in solid preparations, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffering agents, soothing agents, etc. can do. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.
Preferable examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.
Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.
Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.
Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
Preferable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; polyvinyl alcohol, polyvinyl Examples thereof include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.
Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate, citrate and the like.
Preferable examples of the soothing agent include benzyl alcohol. Preferable examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbic acid.
Target Disease The anticancer agent of the present invention can be applied to the treatment of various cancers.
The type of cancer is not particularly limited. For example, acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid cancer , Osteosarcoma, head and neck cancer, esophageal cancer, non-small cell lung cancer, colon cancer, stomach cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, pancreatic cancer, liver cancer.
The anticancer agent of the present invention is particularly effective for acute myeloid leukemia, acute lymphocytic leukemia, and small cell lung cancer. Among them, it is preferably used for acute myeloid leukemia, acute lymphocytic leukemia, and small cell lung cancer.
The administration conditions and administration method of the anticancer agent of the present invention can be appropriately set according to the type of disease, the patient's condition or application site.
Specifically, the dose is appropriately adjusted according to the type and size (or body weight) of animals, symptoms and the like. When administered to a human or non-human animal, about 0.1-100 mg / kg (body weight), preferably about 1.0-10 mg / kg (body weight), more preferably about 1.0-3. Administer in an amount of about 0 mg / kg (body weight). Also, the administration method can be adjusted as appropriate to obtain the optimal therapeutic effect.For example, it can be administered in several divided doses every day, and if a dangerous state occurs during treatment, the dosage or The number of administrations can also be reduced as appropriate.
The anticancer agent of the present invention can be administered alone to obtain a desired effect, or can be used in appropriate combination with other anticancer agents, chemotherapeutic agents, anti-inflammatory agents, immunotherapeutic agents, and the like.
Examples of other anticancer agents used in combination with the anticancer agent of the present invention include antimetabolites, alkylating agents, platinum-based anticancer agents, topoisomerase inhibitors, anticancer antibiotics, tyrosine kinase inhibitors, and humanized antibodies.

Hereinafter, although an example, a test example, etc. are given and the present invention is explained still more concretely, the present invention is not limited to these examples.
Compound Preparation Using Calophyllum Brasiliensis collected in Brazil, several compounds were obtained in the following procedure. First, Calophyllum Brasiliensis dried bark (630 g) was pulverized, and then subjected to cold extraction three times using acetone (3 L). The obtained acetone extract was concentrated under reduced pressure, and the residue was used as an acetone extract (19.26 g). When this acetone extract was subjected to Si gel column chromatography using hexane-acetone as an eluent, Compound 7 (GUT-70) (137.8 mg) was obtained from the 85:15 eluate. As a result of carrying out the same treatment using hexane-acetone, acetone, CH ₂ Cl ₂ -MeOH, and methanol as an eluent, 15 kinds of compounds including Compound 7 were obtained. The structure of the obtained 15 compounds is shown in FIG.
Compound 7 (GUT-70) was obtained from Palmer, C.I. J. et al. Josephs, J .; L. Tetrahedron Lett. 1994, 35, 5363-5366, and references therein. The structures of other compounds were also identified based on known literature.
Cell Preparation Human leukemia cells BV173, SUPB15, NALM6, HL60 and SEM were obtained from the American Type Culture Collection. In addition, human small cell lung cancer cell lines SBC-1, SBC-3, SBC-5, N427, N69, LUT-13O, H82, NCI345, and LUT134B were used in the experiments. Human leukemia cell lines BV173, SUPB15, NALM6, HL60, and SEM, and human small cell lung cancer cell lines SBC-1, SBC-3, SBC-5, N427, N69, LUT-13O, H82, NCI345, LUT134B are inactivated. In RPMI-1640 culture medium (GibcoBRL) supplemented with 10% FCS (Hyclone), the cells were maintained in a 37 ° C., 5% CO ₂ humidified culture. SEM was cultured in Iscove broth (GibcoBRL) supplemented with inactivated 10% FCS. P-glycoprotein high expression multidrug resistance (MDR) K562 / D1-9 cells were maintained in RPMI-1640 culture medium supplemented with 10% FCS and 0.1 μM daunorubicin (DNR).
In Vitro Cell Proliferation Activity Measurement Cell proliferation was determined by counting the number of cells using trypan blue dye exclusion method and modified MTT assay using SF reagent (Nacalai Tesque). And decided. Leukemia cells were cultured as 2 × 10 ⁴ cells per well in 100 μl medium in flat bottom 96-well plates (Greiner labortechnik) and cultured with various concentrations of compounds for 72 hours. In order to examine the effect of GUT-70 (compound 7) in cells expressing high P-glycoprotein, K562 / D1-9 cells were cultured for 72 hours at various concentrations for GUT-70 or DNR under the same conditions. The average of 6 data was used for each. A linear relationship was seen between MTT assay and cell number.
Western Blot Analysis Protein samples were separated by SDS / PAGE and electroblotted on nitrocellulose membrane (Amersham Bioscience). The membrane was saturated with TBST (25 mM Tris.HCl pH 7.8, 140 mM NaCl, 0.1% (vol / vol) Tween 20) solution containing 5% non-fat dry milk, and rabbit polyclonal p21 ^{WAF1 / CIPL} , p27 ^Kip1 , And incubated overnight with p53 antibody (diluted 1/1000). It was then washed thoroughly with TBST and incubated with donkey anti-rabbit IgG complexed with horseradish peroxidase (Santa Cruz Biotech.) For 1 hour. Detection was performed with an amplification chemifluorescence kit (Amersham Biosciences).
Apoptosis induction analysis NALM6 cells treated with untreated NALM6 and GUT-70 for 12 and 24 hours were examined for apoptosis induction. May-Giemsa staining was used to study the whole morphology, and terminal deoxynucleotidyl transferase (TUNEL) assay was used to detect apoptosis. After drug treatment, 5 × 10 ⁴ cells were washed with PBS (pH 7.3) and resuspended in the same buffer. The cytospin preparation of the cell suspension was fixed and stained with May-Giemsa staining solution. Cell morphology was determined with a light microscope. The TUNEL assay was performed using an apoptosis detection kit (R & D systems, Wiesbaden, Germany) according to the instructions. Briefly, NALM6 cells were resuspended in 3.7% formaldehyde in PBS for 10 minutes on ice and rinsed with PBS. The fixed cells were then incubated in cytonin for 30 minutes at room temperature and washed with labeling buffer. The cells were resuspended with 37 μl of labeling mix (dNTPPMix, Mn ²⁺ , TdT, TdT labeling buffer) at 37 ° C. for 1 hour and the reaction was stopped with stop buffer. It was then resuspended for 10 minutes at room temperature in the dark with 25 μl of a solution containing streptavidin. The cells were rinsed with PBS, counterstained with 2 μg / ml propidium iodide for 30 minutes and analyzed by flow cytometry (FACScan; Becton Dickinson. NJ).
Toxicity evaluation of GUT-70 against normal hematopoietic progenitor cells The sensitivity of normal hematopoietic progenitor cells to GUT-70 was examined by a standard methylcellulose culture assay containing GM-CSF, IL-3, and G-SCF. As normal hematopoietic progenitor cells, bone marrow cells from healthy volunteers with informed consent were used. The cells (1 × 10 ⁵ ) were cultured in methylcellulose at 37 ° C. for 12 days in a 5% CO ₂ incubator at a GUT-70 concentration of 0, 20 or 50 μM. The number of CFU-GM and BFU-E colonies was counted with a microscope. Three tissues were used for each group of tests and the average was determined.
Statistical analysis The Student's t test was used for statistics. P values less than 0.05 were considered statistically significant and were derived from two-sided tests.
Cell growth inhibition evaluation 15 natural extracts from Brasiliensis, namely 6 Xanthones (1-6) and 9 4-phenylcoumarins, which are two tricyclic coumarin compounds (7,8) 6 tetracyclic coumarins (9-14) and 1 dimethylcyclopropyl (15)). Compound 8 is a compound previously identified as Calophyllide (Spino) or Oblongulide (Dharmarata). For these, the anti-leukemic activity of 5 leukemia cells was examined using MTT assay. Growth of BV173 cells was inhibited by 4-phenylcoumarin compounds (compounds 7 (GUT-70) and 8) containing a tricyclic coumarin structure. This growth inhibitory effect was concentration-dependent at concentrations ranging from 0 μM to 30 μM. On the other hand, the other 13 compounds did not show growth inhibitory action.
Both Compound 7 and Compound 8 showed an inhibitory effect on the growth of BV173 cells, but Compound 7 (GUT-70) having a propyl group showed a more prominent effect (FIG. 2). Further, _{IC 50} values of compounds 7 and 8 (half-maximal inhibitory concentrations) were respectively 3μM and 9 [mu] M. Furthermore, Compound 7 also exhibited a concentration-dependent and time-dependent cell growth inhibitory action for five other leukemia cell lines (K562, HL60, SEM, NALM6, SUPB15) (FIG. 3). GUT-70 steadily suppressed the growth of human small cell lung cancer cell lines SBC-1, SBC-3, LUT134B, and H82 (IC ₅₀ value 5 μM to 8 μM, FIG. 9).
As is clear from the above results, the compound having a tricyclic coumarin structure extracted from the stem of Calophyllum Brasiliensis collected in Brazil has a remarkable cytostatic effect on leukemia cells and small cell lung cancer cells. It became clear.
Tricyclic coumarin compound (I) containing compounds 7 and 8 inhibited cell growth in a concentration-dependent manner. On the other hand, 13 compounds of other structures did not inhibit the growth. Of these, some tetracyclic coumarin compounds have been reported to have a carcinogenesis-preventing action, but did not show a growth-inhibiting action. It was also found that the action of compound 7 was more remarkable than that of compound 8.
These findings indicate that the tricyclic coumarin structure is an essential structure for the growth inhibitory action, and that the propyl group in the side chain increases the anti-leukemic action. It is presumed that the compound 7 having a propyl group in the side chain exhibits a more effective action because it has a stronger affinity for the membrane. Knowledge obtained about the importance of this side chain is expected to lead to improvement in the action of 4-phenylcoumarin compounds including tricyclic coumarins.
Inhibition of cell proliferation by induction of apoptosis Next, the relationship between the inhibitory effect of compound 7 (GUT-70) on the proliferation of leukemia cells and apoptosis was examined. NALM-6 cells were exposed for 24 hours with varying concentrations of Compound 7, and apoptosis induction was measured by morphological techniques and flow cytometry. After 24 hours exposure to 5 μM concentration of Compound 7, significant cell apoptosis was observed morphologically (FIG. 4). Compound 7 induced a significant number of TUNEL positive (ie apoptotic) cells and reduced the proportion of cells in the G ₂ / M phase. By TUNEL assay using propidium iodide double staining, compound 7 was accumulated in cells in the G ₁ phase, and apoptotic cells appeared mainly near the G ₁ / S phase (FIG. 5).
As described above, cell apoptosis was evaluated using several different methods for the action of Compound 7 that showed growth inhibitory action on five different human leukemia cells in vitro. As a result, it became clear that leukemia cells treated with compound 7 induce DNA cleavage and induce apoptosis, and also induce apoptosis mainly at the G ₁ / S boundary.
Effect of Compound 7 (GUT-70) on Cell Cycle Related Protein The time course of p21 ^{WAF1 / CIPL} , p27 ^Kip1 , p53 and p57 was examined in NALM6 cells and BV173 cells. Compound 7 induced tumor suppressor gene product p53 and cyclin-dependent kinase inhibitors p27 and p57, but not p21 in NALM6 cells. In BV173 cells, p27 and p57 were induced, but p21 and p53 were not induced (FIG. 6). Further, as described above, Compound 7 also showed a growth inhibitory action on the human leukemia cell line HL60 lacking wild-type p53. From these results, it was found that Compound 7 is p53-independent, that is, the cell growth inhibitory action of Compound 7 is not an action due to the expression of p21 through p53 induction.
Moreover, in all the cells, the expression of p27 ^Kip1 was increased, whereas the expression of p53 was increased only in NALM6. Therefore, it was found that the main signal transduction pathway after treatment with compound 7 is not the p53-p21 pathway but the p27 pathway that blocks the G1 / S transition.
Action of Compound 7 (GUT-70) on P-glycoprotein high-expressing cell K562 / D1-9 Daunorubicin (DNR), which is generally used as an anticancer agent, is a cell in which P-glycoprotein is highly expressed, K562 / D1- 9 was approximately 75 times more resistant than the parent strain K562 in which no P-glycoprotein was expressed. On the other hand, the sensitivity of Compound 7 was not decreased with respect to K562 / D1-9 in which P-glycoprotein was highly expressed as compared to parent strain K562 in which P-glycoprotein was not expressed (FIG. 7). .
These findings indicate that compound 7 (GUT-70) is not involved in the P-glycoprotein-related MDR system. Compound 7 (GUT-70) appears to be a suitable candidate component to overcome the induction of MDR. Relapsed patients have MDR that induces P-glycoprotein, which is an efflux pump for anticancer drugs, and often show multidrug resistance. Clinical trials of Compound 7 as an anticancer drug are initially applied to patients with relapsed or refractory disease. It is thought that.
Safety evaluation of compound 7 against normal hematopoietic progenitor cells Normal hematopoietic progenitor cells were treated with 20 μM and 50 μM compound 7 (GUT-70). As a result, the number of CFU-GM colonies was 75% ± 10%, % ± 2% was found. In addition, the number of BFU-E colonies was found to be 80% ± 10% and 3% ± 1% of the control, respectively (FIG. 8). From these results, it was revealed that up to a concentration of 20 μM, compound 7 (GUT-70) has an extremely low growth inhibitory effect on normal hematopoietic progenitor cells. From this, it was found that Compound 7 (GUT-70) is a compound having a large safety range.
As shown in the above results, the pharmaceutical composition of the present invention has a remarkable cancer growth inhibitory action, and can exert excellent effects on cancer treatment, particularly treatment of leukemia and small cell lung cancer. .
The anticancer agent of the present invention has a mechanism of action different from the conventional one and suppresses cancer cell proliferation in a p53-independent manner. Therefore, the present invention can be expected to be effective even for cancers that could not be expected with conventional anticancer agents. Further, by using in combination with drugs having different mechanisms, it becomes possible to treat a wider variety of cancers and reduce side effects.
Furthermore, the pharmaceutical composition of the present invention maintains excellent effects against multidrug resistant cancer cells in which P-glycoprotein has been induced. Therefore, it can be effectively used also for patients who have become ineffective for other anticancer agents and patients who need multi-drug combination therapy.
Moreover, the present invention has a large safety margin and is suitable for clinical use.
As described above, the present invention has various requirements that are eagerly desired in cancer treatment, and provides an excellent means for treating these diseases.

Claims (11)

The following general formula (1)

(Wherein R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, a cycloalkyl group or an optionally substituted aryl group, or CR ¹ R ² represents C═O;
R ³ and R ⁴ each independently represents a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, or an alkoxycarbonyl group. Represents an amino group, a monoalkylamino group, a dialkylamino group or an acylamino group;
R ⁵ , R ⁸ and R ⁹ are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, Alkoxycarbonyl group, aryloxy group which may be substituted, thiol group, alkylthio group, arylthio group which may be substituted, alkylsulfonyl group, arylsulfonyl group which may be substituted, alkylaminocarbonyl group, substituted Optionally substituted arylaminocarbonyl group, alkylaminosulfonyl group, optionally substituted arylaminosulfonyl group, alkoxycarbonyl group, optionally substituted aryloxycarbonyl group, amino group, monoalkylamino group, dialkylamino Group, acylamino Represents an alkylsulfonylamino group or an optionally substituted arylsulfonylamino group;
R ⁶ represents an alkyl group, a cycloalkyl group, an optionally substituted aryl group, an alkoxy group, an acyl group, a carboxyl group, an alkoxycarbonyl group, or the following general formula (2)

Represents the structure represented by
R ⁷ represents O, NH or NR ¹³ ;
R ¹⁰ represents a hydrogen atom, an alkyl group, a cycloalkyl group or an aryl group which may be substituted;
R ¹¹ and R ¹² each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, or an optionally substituted aryl group;
R ¹³ represents an alkyl group, a cycloalkyl group, or an optionally substituted aryl group)
Or a pharmaceutically acceptable salt thereof as an active ingredient. The anticancer agent according to claim 1, wherein R ⁷ is an oxygen atom. The anticancer agent according to claim 1, wherein R ⁵ is a methoxy group. The anticancer agent according to claim 1, wherein R ⁹ is an alkyl group or an optionally substituted aryl group. The anticancer agent according to claim 1, wherein R ⁹ is an n-propyl group. R ⁶ represents the following general formula (2)

(Wherein R ¹⁰ , R ¹¹ and R ¹² are as defined above).
The anticancer agent according to claim 1, which is a group represented by the formula: The following general formula (3)

The anticancer agent of Claim 1 represented by these. A method for treating cancer, comprising administering an effective amount of the compound according to claim 1. Cancer is acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, choriocarcinoma, multiple myeloma, soft tissue tumor, small cell lung cancer, chronic myelogenous leukemia, medullary thyroid cancer, osteosarcoma, head and neck cancer, esophagus The method according to claim 8, wherein the method is selected from the group consisting of cancer, non-small cell lung cancer, colon cancer, gastric cancer, biliary tract cancer, brain tumor, malignant melanoma, kidney cancer, pancreatic cancer, and liver cancer. The method according to claim 9, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, malignant lymphoma, chronic myelogenous leukemia, and small cell lung cancer. 9. The method of claim 8, wherein the cancer is multidrug resistant (MDR) cancer.

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