JP2006321724A

JP2006321724A - Antitumor agent

Info

Publication number: JP2006321724A
Application number: JP2005144012A
Authority: JP
Inventors: Takeo Konakahara; 猛雄小中原; Asako Kato; 亜沙子加藤; Haruaki Kurasaki; 晴彰倉崎; Norio Sakai; 教郎坂井
Original assignee: Tokyo University of Science
Current assignee: Tokyo University of Science
Priority date: 2005-05-17
Filing date: 2005-05-17
Publication date: 2006-11-30

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA target type antitumor agent containing an ellipticine derivative as an active ingredient by searching a derivative having antitumor action from the ellipticine derivatives. <P>SOLUTION: The antitumor agent comprises a compound represented by general formula (1) (wherein X<SP>-</SP>is a monovalent anion) as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、２位置換エリプチシン誘導体を有効成分とする抗腫瘍剤に関する。 The present invention relates to an antitumor agent comprising a 2-position substituted ellipticine derivative as an active ingredient.

エリプチシンは、以下に示す化学構造を有する化合物であり、 Ellipticin is a compound having the chemical structure shown below,

キョウチクトウ等の植物に含まれるアルカロイドの一種である。
エリプチシンあるいはその誘導体は、古くから抗腫瘍作用を有することが知られ、例えば、エリプチシン及び９−メトキシエリプチシンは、マウス白血病細胞Ｌ−１２１０やマウス肉腫細胞Sarcome180に対し有効であり（非特許文献１参照）、また、９−ヒドロキシエリプチシンは、上記９−メトキシエリプチシンより、マウス白血病細胞Ｌ−１２１０に対してさらに高い活性を有することも報告されている（特許文献１、２参照））。
さらに、９−ヒドロキシエリプチシンの誘導体としては、その１位にジアミノアルキルアミノカルボニル基を有するもの（特許文献３，４参照）等が知られている。また、エリプチシン２位に置換基を有するものとしては、エリプチシン誘導体の２位に、カルボキシエチル基を導入したもの（非特許文献２参照、あるいはα−Ｌ−アラビノピラノシル基を導入したもの（特許文献５参照）等を挙げることができる。
一方、エリプチシン及びその誘導体は、ＤＮＡ塩基対間にインターカレートする能力を有し、これを利用して、さらにアルキル化剤等の抗腫瘍作用を有する化合物をエリプチシンに結合せしめ、ガン細胞に対する特異性をより高めたＤＮＡ標的型の抗腫瘍剤の研究も行われている。
しかし、これらのエリプチシン誘導体の研究においては、未だ満足する抗腫瘍作用を有する誘導体は得られていない。

It is a kind of alkaloid contained in plants such as oleander.
Ellipticin or its derivatives have long been known to have an antitumor effect. For example, ellipticine and 9-methoxyellipticine are effective against mouse leukemia cell L-1210 and mouse sarcoma cell Sarcome180 (Non-patent Document). 1), and 9-hydroxy ellipticine has also been reported to have higher activity against mouse leukemia cell L-1210 than 9-methoxy ellipticine (see Patent Documents 1 and 2). )).
Furthermore, as derivatives of 9-hydroxyellipticine, those having a diaminoalkylaminocarbonyl group at the 1-position (see Patent Documents 3 and 4) are known. Moreover, as for what has a substituent in ellipticine 2nd-position, what introduce | transduced the carboxyethyl group into 2nd-position of an ellipticine derivative (refer nonpatent literature 2 or the thing which introduce | transduced (alpha) -L-arabinopyranosyl group) (See Patent Document 5).
Ellipticin and its derivatives, on the other hand, have the ability to intercalate between DNA base pairs, and by using this, compounds having an antitumor action such as alkylating agents are bound to ellipticine and specific for cancer cells. Studies on DNA-targeted antitumor agents with higher sex are also being conducted.
However, in the study of these ellipticine derivatives, there are still no satisfactory antitumor derivatives.

J.Medicinal Chemistry 18(7) P.755-760 (1975）J. Medicinal Chemistry 18 (7) P.755-760 (1975) 「Bull.Cancer（Paris)Vol.68,P.437-441(1981)"Bull.Cancer (Paris) Vol.68, P.437-441 (1981) 特公昭５８−３５１９６号公報、Japanese Patent Publication No. 58-35196, 英国特許第1436080号明細書British Patent No. 1436080 特開平１０−１９５０７３号公報Japanese Patent Laid-Open No. 10-195073 特開平６−２１１８５２号公報JP-A-6-211182 特開平２−１９１２９７号公報Japanese Patent Laid-Open No. 2-191297

本発明の課題は、上記従来技術の現状に鑑み、エリプチシン誘導体として、顕著な抗腫瘍作用を有する誘導体を見いだし、そのインターカレーターとしての能力を利用して、腫瘍細胞の増殖を効率的に抑制しうる、ＤＮＡ標的型の抗腫瘍剤を新たに提供しようとするものである。 In view of the current state of the prior art, the object of the present invention is to find a derivative having a remarkable antitumor action as an ellipticine derivative, and to efficiently suppress the growth of tumor cells by utilizing its ability as an intercalator. It is intended to provide a new DNA-targeted antitumor agent.

本発明者らは、インターカレーターとしての機能を有するエリプチシン誘導体に着目し、これを用いてＤＮＡ塩基対周辺にアルキル化剤を運搬して、腫瘍細胞のＤＮＡの合成を阻害し、効率的にガン細胞の増殖を抑制する手段について研究してきた。このため、種々のエリプチシン誘導体を合成し、これらエリプチシン誘導体にメチルニトロソユリア等のアルキル化剤を結合せしめ、その腫瘍細胞に対する毒性を調べたが、その研究過程において、特定のエリプチシン誘導体が、極めて顕著な抗腫瘍活性を有するという知見を得て、本発明を完成させた。 The present inventors paid attention to ellipticine derivatives having a function as intercalators, and used them to transport alkylating agents around DNA base pairs to inhibit the synthesis of tumor cell DNA, thereby efficiently cancerous. We have been researching ways to suppress cell growth. For this reason, various ellipticine derivatives were synthesized, and alkylating agents such as methylnitrosourea were conjugated to these ellipticine derivatives, and their toxicity to tumor cells was examined. The present invention was completed by obtaining the knowledge that it has an antitumor activity.

すなわち本発明は以下のとおりである。
１．以下の一般式（１）又は（２）で表される化合物を有効成分として含有する抗腫瘍剤。 That is, the present invention is as follows.
1. The antitumor agent which contains the compound represented by the following general formula (1) or (2) as an active ingredient.

２．以下の一般式（１）で表される化合物。

2. The compound represented by the following general formula (1).

３．以下の一般式（２）で表される化合物。

3. The compound represented by the following general formula (2).

４．以下の一般式（３）で表される化合物。

4). The compound represented by the following general formula (3).

本発明の一般式（１）で表されるエリプチシン誘導体は特に、Sarcoma180細胞に対して顕著な細胞毒性を有し、また、一般式（２）で表されるエリプチシン誘導体は、Sarcoma180細胞に加え、ヒト子宮頸ガン由来のHeLa S-3細胞にも顕著な細胞毒性を示す。さらに、一般式（３）で表されるエリプチシン誘導体は、一般式（２）で表されるエリプチシン誘導体よりも顕著な細胞毒性を示す(Sarcoma 180)ことに加え、水溶性が良好である。また、本発明の一般式（１）および（２）の化合物は、それ自体インターカレーターであり、ＤＮＡ標的型の抗腫瘍剤として極めて有用なものである。 The ellipticine derivative represented by the general formula (1) of the present invention has particularly remarkable cytotoxicity against Sarcoma180 cells, and the ellipticine derivative represented by the general formula (2) is added to the Sarcoma180 cells, HeLa S-3 cells derived from human cervical cancer also show significant cytotoxicity. In addition, the ellipticine derivative represented by the general formula (3) exhibits remarkable cytotoxicity (Sarcoma 180) and has better water solubility than the ellipticine derivative represented by the general formula (2). The compounds of the general formulas (1) and (2) of the present invention are themselves intercalators and are extremely useful as DNA-targeted antitumor agents.

本発明において抗腫瘍剤として用いる化合物は、上記一般式（１）〜（３）で表され、いずれも、エリプチシンの２位に、窒素含有置換基を有するものである。
一般式（１）の化合物は、エリプチシン５位にメトキシカルボニル基を有し、２位の窒素原子に２−アミノエチル基が置換された４級アンモニウム化合物である点に構造上の特徴を有し、該化合物は腫瘍細胞、特にSarcoma180細胞に対して顕著な細胞毒性を有する。
これに対して、２位に２−アミノエチル基が置換されたものであっても５位に置換基を有しないもの、あるいは５位に分子鎖長が長いリンカーを有するものは、細胞毒性が低下する。また、上記２−アミノエチル基のアミノ基がさらに置換されているもの、あるいはアルキル鎖がエチル鎖よりも長い場合も顕著な細胞毒性を示さず、２位の置換基がアルキル基あるいはアルケニル基である場合も活性が低下する。
一方、陰イオンのイオン種は、その活性にあまり影響を与えないことが実験により確かめられている。該イオン種を具体的に挙げれば、塩素イオンあるいは酢酸イオン等である。
また、一般式（２）の化合物は、エリプチシンの２位に２−アミノエチルカルバモイル基を有するか、あるいは該置換基の２−アミノ基をＮ−メチルウレイド基又はＮ−メチル−Ｎ−ニトロソウレイド基に変換した置換基を有するものである。
これら一般式（２）の化合物は、Sarcoma180細胞に加え、HeLaS-3細胞にも顕著な細胞毒性を有する他、Ｌ−１２１０細胞にもある程度の細胞毒性を有し、広い範囲の腫瘍細胞に対して活性を有する。
一般式（３）の化合物は、一般式（２）の化合物の９位が、水酸基で置換された化合物であり、一般式（２）で表されるエリプチシン誘導体よりも顕著な細胞毒性を示す(Sarcoma 180)ほか、良好な水溶性を有する。
本発明の上記化合物は、以下の合成法により得られる。 The compounds used as antitumor agents in the present invention are represented by the above general formulas (1) to (3), and all have a nitrogen-containing substituent at the 2-position of ellipticine.
The compound of the general formula (1) has a structural feature in that it is a quaternary ammonium compound having a methoxycarbonyl group at the 5-position of ellipticine and a 2-aminoethyl group substituted at the 2-position nitrogen atom. The compound has significant cytotoxicity against tumor cells, especially Sarcoma 180 cells.
On the other hand, those having a 2-aminoethyl group substituted at the 2-position but not having a substituent at the 5-position, or having a linker having a long molecular chain length at the 5-position have cytotoxicity. descend. Further, when the amino group of the 2-aminoethyl group is further substituted, or when the alkyl chain is longer than the ethyl chain, no significant cytotoxicity is exhibited, and the substituent at the 2-position is an alkyl group or an alkenyl group. In some cases the activity is also reduced.
On the other hand, it has been confirmed by experiments that the ionic species of anions do not significantly affect the activity. Specific examples of the ion species include chlorine ions and acetate ions.
The compound of the general formula (2) has a 2-aminoethylcarbamoyl group at the 2-position of ellipticine, or the 2-amino group of the substituent is an N-methylureido group or N-methyl-N-nitrosoureido. It has a substituent converted into a group.
These compounds of the general formula (2) have not only Sarcoma 180 cells but also significant cytotoxicity to HeLaS-3 cells, as well as some cytotoxicity to L-1210 cells, and are effective against a wide range of tumor cells. Active.
The compound of the general formula (3) is a compound in which the 9-position of the compound of the general formula (2) is substituted with a hydroxyl group, and exhibits a marked cytotoxicity than the ellipticine derivative represented by the general formula (2) ( Sarcoma 180) Besides, it has good water solubility.
The above compound of the present invention can be obtained by the following synthesis method.

〔一般式（１）の化合物の合成〕
一般式（１）の化合物の合成法について、図１に基づき以下に説明する。
インドール(4)を出発原料として、例えば、ベンゼンスルホニルのような保護基を用いてインドールの窒素の保護を行い、その後インドールの3位をリチオ化後、熱転位により2位リチオ体とし、グリオキシレート鎖を導入することで（6）を生成させる。続いて、加水分解により（7）とした後、カルボニル基のWolff-Kishner還元、トリメチルシリルジアゾメタンによるエステル化により（9）を合成し、3-アセチルピリジンとのカップリング反応により（10）を生成させ、クロロエチルアミンの窒素原子をAc、Boc、Nsなどで保護したリンカーを用いて、ピリジンの窒素の4級化を行い、閉環反応、脱水素反応を経て（11）とする。保護基はAc基のような嵩高くないものを用いたほうが最良の結果を与える。その後、塩酸／メタノール溶液で、脱保護を行い(1a)を得る。(1a)は、例えば、酢酸銀をもちいることによりカウンターアニオンをクロライドからアセテート(1b)に変換できる。カウンターアニオンの種類は、使用する塩の種類により、適宜変更できる。 [Synthesis of Compound of General Formula (1)]
A method for synthesizing the compound of the general formula (1) will be described below with reference to FIG.
Using indole (4) as a starting material, for example, protecting the nitrogen of the indole using a protecting group such as benzenesulfonyl, and then lithiating the 3-position of the indole, followed by thermal rearrangement to give the 2-position lithiated, glyoxy (6) is generated by introducing a rate chain. Subsequently, after hydrolyzing to (7), (9) was synthesized by Wolff-Kishner reduction of the carbonyl group and esterification with trimethylsilyldiazomethane, and (10) was produced by a coupling reaction with 3-acetylpyridine. Using a linker in which the nitrogen atom of chloroethylamine is protected with Ac, Boc, Ns, etc., quaternization of nitrogen of pyridine is carried out to obtain (11) through a ring closure reaction and a dehydrogenation reaction. The best results are obtained when the protecting group is not bulky, such as the Ac group. Thereafter, deprotection is performed with a hydrochloric acid / methanol solution to obtain (1a). In (1a), for example, the counter anion can be converted from chloride to acetate (1b) by using silver acetate. The type of counter anion can be appropriately changed depending on the type of salt used.

〔一般式（２）の化合物の合成〕
一般式（２）の化合物の合成法について、図２に基づき以下に説明する。
インドール(4)を出発原料として、酸性条件下2,5-ヘキサジオンによりcarbazole環12を構築し、Vilsmeier条件による、3位へのホルミル基の導入を行い13を生成させる。続いてイミンの生成、還元、NsCl（４−ニトロベンゼンスルホン酸クロリド）によるアルキル化を行い、14を合成後、酸性条件下での閉環反応を行う。閉環反応後、直ちにNs基が脱離し、目的のエリプチシン15が得られる。つづいてアシルクロライドによるピリジン環窒素の4級化、還元反応により1,2-ジヒドロ体16とし、エチレンジアミンによる求核置換反応により(2a)を合成する。(2a)は酸性条件で容易に脱アシル化が起きることから、アミノシリカゲルクロマトグラフィーにより単離した。その後、p-ニトロフェニル-N-メチルカルバメート、またはサクシンイミジル-N-メチルニトロソカルバメートで処理することで(2b),(2c)を得ることができる。 [Synthesis of Compound of General Formula (2)]
A method for synthesizing the compound of the general formula (2) will be described below with reference to FIG.
Using indole (4) as a starting material, carbazole ring 12 is constructed with 2,5-hexadione under acidic conditions, and formyl group is introduced into the 3-position under Vilsmeier conditions to form 13. Subsequently, imine is generated, reduced, and alkylated with NsCl (4-nitrobenzenesulfonic acid chloride). After 14 is synthesized, a ring-closing reaction under acidic conditions is performed. Immediately after the ring closure reaction, the Ns group is eliminated, and the desired ellipticine 15 is obtained. Subsequently, the pyridine ring nitrogen is quaternized with acyl chloride and reduced to the 1,2-dihydro form 16, and (2a) is synthesized by nucleophilic substitution with ethylenediamine. Since (2a) easily deacylates under acidic conditions, it was isolated by amino silica gel chromatography. Thereafter, (2b) and (2c) can be obtained by treatment with p-nitrophenyl-N-methylcarbamate or succinimidyl-N-methylnitrosocarbamate.

〔一般式（３）の化合物の合成〕
一般式（３）の化合物の合成法について、図３に基づき以下に説明する。
上記エリプチシン(15)を出発原料として、Vilsmeier型反応により9位にホルミル基を導入し17を合成後、ホルミル基をBaeyer-Villiger酸化によりギ酸エステルとし、これを加水分解することにより、9-ヒドロキシエリプチシン(18)を合成する。その後アルコールのTBS保護、リンカー導入反応、還元を行い20を合成する。つづいて、TBSの脱保護、エチレンジアミンによる求核置換反応により(3a)を生成させ、さらに、所定のウレイド基を導入することで(3b), (3c)とすることができる。
本発明の一般式（１）〜（３）の化合物は、細胞膜及び細胞核膜への透過性が良好であり、このことが抗腫瘍活性の向上に寄与していると考えられる。 [Synthesis of Compound of General Formula (3)]
A method for synthesizing the compound of the general formula (3) will be described below with reference to FIG.
Using the above ellipticine (15) as a starting material, a formyl group was introduced at the 9-position by a Vilsmeier-type reaction to synthesize 17, and then the formyl group was converted to a formate ester by Baeyer-Villiger oxidation. Synthesize ellipticine (18). Thereafter, TBS protection of alcohol, linker introduction reaction, and reduction are performed to synthesize 20. Subsequently, (3a) can be generated by deprotection of TBS, nucleophilic substitution reaction with ethylenediamine, and further, a predetermined ureido group can be introduced to obtain (3b) and (3c).
The compounds of the general formulas (1) to (3) of the present invention have good permeability to the cell membrane and cell nuclear membrane, which is considered to contribute to the improvement of antitumor activity.

以下に、本発明の実施例を示すが、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〕
一般式（１）の化合物の合成（図１参照）
１）Ｎ−ベンゼンスルホニルインドール(Ｎ-benzensulfonylindole )(5)
NaOH (6.75g, 0.17mol)、tetra-n-butylammonium hydrogensulfate (0.47g, 0.0014mol)、CH₂Cl₂ 60mlを加え0℃に冷却し、インドール（indole） (4) (6.06g, 0.051mol)を滴下した。この温度を保ったまま、Benzenesulfonyl chloride (6.08g, 0.050mol)のCH₂Cl₂ (40ml)溶液を20 minかけて滴下し、3 h撹拌させた。反応混合物を吸引ろ過し、ろ液を濃縮後、得られた固体をMeOHにて洗浄し、Ｎ−ベンゼンスルホニルインドール（5） (11.4g, 0.045mol)を得た（収率： 89 %）。
＜Ｎ−ベンゼンスルホニルインドールの物性データ＞
mp 77.8-80.5 ℃; IR (KBr) ν/ cm^-1 3137(m), 1446 (s), 1376 (s), 1171 (s), 1126 (s); ¹H-NMR (500MHz, CDCl₃) δ 6.64 (1H, d, J = 4Hz, indolyl 3-position ), 7.21-8.01 (10H, m, indolylphenyl -H); MS (FAB⁺) m/z 257 (M⁺). Examples of the present invention are shown below, but the present invention is not limited to these examples.
[Example 1]
Synthesis of compound of general formula (1) (see FIG. 1)
1) N-Benzensulfonylindole (5)
NaOH (6.75g, 0.17mol), tetra -n-butylammonium hydrogensulfate (0.47g, 0.0014mol), CH 2 Cl 2 60ml and cooled to 0 ℃ added, indole (indole) (4) (6.06g , 0.051mol) Was dripped. While maintaining this temperature, a CH ₂ Cl ₂ (40 ml) solution of Benzenesulfonyl chloride (6.08 g, 0.050 mol) was added dropwise over 20 min and stirred for 3 h. The reaction mixture was suction filtered, and the filtrate was concentrated. The obtained solid was washed with MeOH to obtain N-benzenesulfonylindole (5) (11.4 g, 0.045 mol) (yield: 89%).
<Property data of N-benzenesulfonylindole>
mp 77.8-80.5 ° C; IR (KBr) ν / cm ^-1 3137 (m), 1446 (s), 1376 (s), 1171 (s), 1126 (s); ¹ H-NMR (500 MHz, CDCl ₃ ) δ 6.64 (1H, d, J = 4Hz, indolyl 3-position), 7.21-8.01 (10H, m, indolylphenyl -H); MS (FAB ⁺ ) m / z 257 (M ⁺ ).

２）Ｎ−ベンゼンスルホニル−２−メトキサリルインドール（N-benzensulfonyl-2-methoxalylindole） (6)
ジイソプロピルアミン(2.67g, 26.3mmol)、THF 50mlを加え、-75℃に冷却した後、15%n-BuLi / Hexane (8.97g, 21.0mmol)をゆっくりと加えた。その温度を保ったまま30min撹拌し、Ｎ−ベンゼンスルホニルインドール（5） (5.15g, 20.0mmol)を55mlのTHFに溶かした溶液を滴下した。さらに、-70℃で1.5 h撹拌を続けた後、1 h以上かけて15℃に暖め、再び-50℃まで冷却した。次に、あらかじめシュウ酸ジメチル (9.73g, 82.4mmol)とTHF 25mlを加えて10℃に保っていたフラスコに、1 h以上の時間をかけて前述の求核体を含む反応混合物をテフロン（登録商標）チューブを介し、Arガス圧を用いて滴下した。その後ゆっくりと室温にし、さらに2 h撹拌し、H₂Oで反応をクエンチした。得られた反応混合物をEtOAcで抽出し、有機層をMgSO₄上で乾燥し、ろ過、濃縮後、MeOHで結晶を洗浄して純粋なＮ−ベンゼンスルホニル−２− メトキサリルインドール（6） (4.26g, 12.4mmol)を得た（収率： 62%）。
＜Ｎ−ベンゼンスルホニル−２−メトキサリルインドールの物性データ＞
mp 108.5-109.4 ℃; IR (KBr) ν/ cm^-1 1740 (s, ester C=O), 1686 (s, keto C=O); ¹H-NMR (500MHz, CDCl₃) δ 3.92 (3H, s, CH₃), 7.19-8.00 (10H, m, indolyl,phenyl); MS (FAB⁺) m/z 344 (M+1). 2) N-Benzensulfonyl-2-methoxalylindole (6)
Diisopropylamine (2.67 g, 26.3 mmol) and 50 ml of THF were added and cooled to −75 ° C., and then 15% n-BuLi / Hexane (8.97 g, 21.0 mmol) was slowly added. The mixture was stirred for 30 minutes while maintaining the temperature, and a solution of N-benzenesulfonylindole (5) (5.15 g, 20.0 mmol) dissolved in 55 ml of THF was added dropwise. Further, stirring was continued at −70 ° C. for 1.5 h, and then the mixture was warmed to 15 ° C. over 1 h and cooled to -50 ° C. Next, dimethyl oxalate (9.73 g, 82.4 mmol) and 25 ml of THF were added in advance to a flask kept at 10 ° C., and the reaction mixture containing the nucleophile was added to Teflon (registered) over 1 h. (Trademark) Tube was added dropwise using Ar gas pressure. Then slowly brought to room temperature, stirred for a further 2 h, and quenched with H ₂ O. The resulting reaction mixture was extracted with EtOAc, the organic layer was dried over MgSO ₄ , filtered, concentrated, and the crystals were washed with MeOH to give pure N-benzenesulfonyl-2-methoxalylindole (6) (4.26 g, 12.4 mmol) was obtained (yield: 62%).
<Property data of N-benzenesulfonyl-2-methoxalylindole>
mp 108.5-109.4 ° C; IR (KBr) ν / cm ^-1 1740 (s, ester C = O), 1686 (s, keto C = O); ¹ H-NMR (500 MHz, CDCl ₃ ) δ 3.92 (3H, s, CH ₃ ), 7.19-8.00 (10H, m, indolyl, phenyl); MS (FAB ⁺ ) m / z 344 (M + 1).

３）インドール−２−グリオキシル酸（ Indole-2-glyoxylic acid ）(7)
アリーン冷却器を取り付けた丸底フラスコにＮ−ベンゼンスルホニル−２− メトキサリルインドール（6） (2.00g, 5.83mmol)と、あらかじめ調製しておいたK₂CO₃ 8gのMeOH 200ml、H₂O 66ml溶液を加え、8h還流した。その後、減圧下濃縮して、MeOHを完全に取り除いたあと、少量の水を加え、それを10% HClaq.で中和した。(pH=2〜3でインドール−２−グリオキシル酸（7）の結晶が析出)。EtOAcで抽出をし、有機層をMgSO₄上で乾燥し、濃縮後、CH₂Cl₂/cyclohexane混合溶媒から再結晶し、インドール−２−グリオキシル酸（7） (9.70g, 5.13mmol)を得た（収率： 88%）。
＜インドール−２−グリオキシル酸の物性データ＞
mp 162.1-164.0 ℃; IR (KBr) ν/ cm^-1 3343 (s, NH), 3500-2899 (br, dimer OH), 1715 (s, carboxyl C=O), 1642 (s, keto C=O), 1618 (s), 1422 (m), 1281 (m), 1226 (m), 1159 (m), 1140 (m); ¹H-NMR (500MHz, CDCl₃) δ 7.17-7.22 (3H, m, indolyl 3, 5, 6-H), 7.45 (1H, d, J = 4Hz, indolyl 4-H), 7.77 (1H, d, J = 8Hz, indolyl 7-H), 8.10 (1H, br, NH), 9.46 (1H, br, OH); MS (FAB⁺) m/z 190 (M+1). 3) Indole-2-glyoxylic acid (7)
N-benzenesulfonyl-2-methoxalylindole (6) (2.00 g, 5.83 mmol) in a round-bottomed flask equipped with an Allen condenser and 200 g of H ₂ O with 8 g of K ₂ CO ₃ prepared in advance 66ml solution was added and refluxed for 8h. Then, after concentration under reduced pressure to completely remove MeOH, a small amount of water was added and it was neutralized with 10% HClaq. (Indole-2-glyoxylic acid (7) crystals were precipitated at pH = 2-3). Extraction with EtOAc, the organic layer was dried over MgSO ₄ , concentrated and recrystallized from a mixed solvent of CH ₂ Cl ₂ / cyclohexane to obtain indole-2-glyoxylic acid (7) (9.70 g, 5.13 mmol). (Yield: 88%).
<Physical property data of indole-2-glyoxylic acid>
mp 162.1-164.0 ℃; IR (KBr) ν / cm ^-1 3343 (s, NH), 3500-2899 (br, dimer OH), 1715 (s, carboxyl C = O), 1642 (s, keto C = O ), 1618 (s), 1422 (m), 1281 (m), 1226 (m), 1159 (m), 1140 (m); ¹ H-NMR (500 MHz, CDCl ₃ ) δ 7.17-7.22 (3H, m , indolyl 3, 5, 6-H), 7.45 (1H, d, J = 4Hz, indolyl 4-H), 7.77 (1H, d, J = 8Hz, indolyl 7-H), 8.10 (1H, br, NH ), 9.46 (1H, br, OH); MS (FAB ⁺ ) m / z 190 (M + 1).

４）（１Ｈ−インドール−２−イル）−酢酸メチルエステル（ (1H-Indol-2-yl)-acetic acid methyl ester） (9)
アリーン冷却器とリービッヒ冷却器を取り付けた3つ口フラスコ内に、インドール−２−グリオキシル酸（7） (0.30g, 1.59mmol)、80%Hydrazine hydrate 1.49ml、KOH 1.65g、EtOH 32mlを加え、5 h還流した。その後、アリーン冷却器の冷却水を止め、溶媒を蒸留して取り除いた後、反応温度を150℃にして45 min撹拌した。反応混合物を室温まで冷やした後、水にとかし、-5℃程度の低温を保ったまま、5% HClaq.で中和した(pH=2〜3で２−インドール酢酸の結晶が析出)。反応混合物をEt₂Oで抽出し、MgSO₄上で乾燥した後、15℃以下で濃縮し溶媒を取り除き、これを次のエステル化反応に単離することなく用いた。２−インドール酢酸の粗生成物の入った丸底フラスコに、MeOH 3.3ml、Benzene 12mlを加えた後、室温で2.0M TMSCHN₂ の Hexane溶液 (1.00ml, 2.60mmol)を滴下して、30 min撹拌した。反応を酢酸を加えることでクエンチした後、Et₂Oで抽出、有機層をNa₂CO₃aq.で洗浄後MgSO₄上で乾燥し、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/EtOAc:Hexane=6:4)で精製し、（１Ｈ−インドール−２−イル）−酢酸メチルエステル（9） (0.23g, 1.21mmol)を得た（合計収率(7)→(9)：76%）。
＜（１Ｈ−インドール−２−イル）−酢酸メチルエステルの物性データ＞
mp 71.0-72.2 ℃; IR (KBr) ν/ cm^-1 3352 (s, NH), 2954 (m), 1720 (s, C=O), 1456(m), 1432 (m) 1429 (m), 1267 (m); ¹H-NMR (500MHz, CDCl₃) δ 3.74 (3H, s, Me), 3.82 (2H, s, CH₂), 6.34 (s, indolyl 3-H), 7.05-7.12 (2H, indolyl 5, 6-H), 7.32 (1H, d, J = 8Hz, indolyl 4-H), 7.54 (1H, d, J = 7Hz, indolyl 7-H), 8.79 (br, NH); MS (FAB⁺) m/z 189 (M⁺). 4) (1H-Indol-2-yl) -acetic acid methyl ester (9)
Indole-2-glyoxylic acid (7) (0.30 g, 1.59 mmol), 80% Hydrazine hydrate 1.49 ml, KOH 1.65 g, EtOH 32 ml were added to a three-necked flask equipped with an Aline cooler and a Liebig cooler. Refluxed for 5 h. Thereafter, the cooling water of the Allen cooler was stopped, the solvent was distilled off, and the reaction temperature was then raised to 150 ° C. and stirred for 45 min. The reaction mixture was cooled to room temperature, dissolved in water, and neutralized with 5% HClaq. While maintaining a low temperature of about −5 ° C. (crystals of 2-indoleacetic acid were precipitated at pH = 2 to 3). The reaction mixture was extracted with Et ₂ O, dried over MgSO ₄ and then concentrated below 15 ° C. to remove the solvent, which was used without isolation in the next esterification reaction. After adding 3.3 ml of MeOH and 12 ml of Benzene to a round bottom flask containing a crude product of 2-indoleacetic acid, 2.0M TMSCHN ₂ Hexane solution (1.00 ml, 2.60 mmol) was added dropwise at room temperature for 30 min. Stir. The reaction was quenched by adding acetic acid and extracted with Et ₂ O. The organic layer was washed with Na ₂ CO ₃ aq., Dried over MgSO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / EtOAc: Hexane = 6: 4) to obtain (1H-indol-2-yl) -acetic acid methyl ester (9) (0.23 g, 1.21 mmol). Obtained (total yield (7) → (9): 76%).
<Physical property data of (1H-indol-2-yl) -acetic acid methyl ester>
mp 71.0-72.2 ° C; IR (KBr) ν / cm ^-1 3352 (s, NH), 2954 (m), 1720 (s, C = O), 1456 (m), 1432 (m) 1429 (m), 1267 (m); ¹ H-NMR (500 MHz, CDCl ₃ ) δ 3.74 (3H, s, Me), 3.82 (2H, s, CH ₂ ), 6.34 (s, indolyl 3-H), 7.05-7.12 (2H , indolyl 5, 6-H), 7.32 (1H, d, J = 8Hz, indolyl 4-H), 7.54 (1H, d, J = 7Hz, indolyl 7-H), 8.79 (br, NH); MS ( FAB ⁺ ) m / z 189 (M ⁺ ).

５）２−インドリル−３−ピリジルエテン誘導体（2-Indolyl-3-pyridyl-ethene derivative（10）
アリーン冷却器を取り付けた3つ口フラスコにH₂SO₄を入れ、（１Ｈ−インドール−２−イル）−酢酸メチルエステル（9） (0.60g, 3.17mmol)、MeOH 30ml、3-acetyl pyridine(1.09g, 9.00mmol)を加え撹拌した。酸性下で6.5 h還流し、室温に戻した後MeOHを除去し、Na₂CO₃aq.を用いて反応混合物を中和した。抽出はEt₂Oで行い、有機層をMgSO₄上で乾燥し、ろ過、濃縮して得られた反応混合物を、シリカゲルカラムクロマトグラフィー(展開溶媒/EtOAc)で精製し、２−インドリル−３−ピリジルエテン誘導体（10） (0.61g, 2.09mmol)を得た（収率： 66%）。
＜２−インドリル−３−ピリジルエテン誘導体の物性データ＞
mp 159.6-160.6 ℃; IR (KBr) ν/ cm^-1 3138 (m, NH), 1741 (s, C=O), 1457(m), 1324 (m)1201 (m), ¹H-NMR (500MHz, CDCl₃) δ 3.72(3H, s, CH₃), 3.76 (2H, s, CH₂), 5.57, 5.83 (2H, s, ethylene CH₂), 7.00(1H, dd,indolyl 5-H), 7.12-7.26 (3H, m, indolyl 6-H, pyridyl 5, 6-H), 7.30 (1H, d, J = 8Hz, indolyl 4-H), 7.61 (1H, d, J = 8Hz, indolyl 7-H), 8.54 (1H, d, J = 4Hz, pyridyl 4-H), 8.70 (1H, s, pyridyl 2-H), 9.02 (1H, br, NH); MS (FAB⁺) m/z 293 (M+1). 5) 2-Indolyl-3-pyridyl-ethene derivative (10)
H ₂ SO ₄ was placed in a three-necked flask equipped with an Allen condenser, and (1H-indol-2-yl) -acetic acid methyl ester (9) (0.60 g, 3.17 mmol), MeOH 30 ml, 3-acetyl pyridine ( 1.09 g, 9.00 mmol) was added and stirred. The mixture was refluxed for 6.5 h under acidic conditions, returned to room temperature, MeOH was removed, and the reaction mixture was neutralized with Na ₂ CO ₃ aq. Extraction was performed with Et ₂ O, the organic layer was dried over MgSO ₄ , filtered, and concentrated. The reaction mixture obtained was purified by silica gel column chromatography (developing solvent / EtOAc), and 2-indolyl-3- A pyridylethene derivative (10) (0.61 g, 2.09 mmol) was obtained (yield: 66%).
<Physical property data of 2-indolyl-3-pyridylethene derivative>
mp 159.6-160.6 ° C; IR (KBr) ν / cm ^-1 3138 (m, NH), 1741 (s, C = O), 1457 (m), 1324 (m) 1201 (m), ¹ H-NMR ( 500MHz, CDCl ₃ ) δ 3.72 (3H, s, CH ₃ ), 3.76 (2H, s, CH ₂ ), 5.57, 5.83 (2H, s, ethylene CH ₂ ), 7.00 (1H, dd, indolyl 5-H) , 7.12-7.26 (3H, m, indolyl 6-H, pyridyl 5, 6-H), 7.30 (1H, d, J = 8Hz, indolyl 4-H), 7.61 (1H, d, J = 8Hz, indolyl 7 -H), 8.54 (1H, d, J = 4Hz, pyridyl 4-H), 8.70 (1H, s, pyridyl 2-H), 9.02 (1H, br, NH); MS (FAB ⁺ ) m / z 293 (M + 1).

６）２−（２−アセチルアミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド（2-(2-Acetylaminoethyl)-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium chloride） (11)
アリーン冷却器を取り付けた3つ口フラスコに、インドールピリジルエテン誘導体（10） (0.100g, 0.342mmol) 、DMF 1ml、N-アセチルクロロエチルアミン (0.410g, 3.42mmol)を加え、110℃で5時間撹拌後、得られたピリジニウム塩をMeOH 5mlに溶かし、さらにCH₃ONa (0.028g, 0.513mmol) /MeOH (5ml)溶液を滴下し、室温で1 h撹拌した。次に、3-エトキシカルボニル-1-メチルピリジニウムクロリド(0.103g,0.513mmol)/MeOH (5ml)溶液を滴下し、さらに室温で24 h撹拌した。得られた反応混合物を、ODSカラムクロマトグラフィー(展開溶媒/MeOH:H2O=20:80)で精製し、２−（２−アセチルアミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド（11） (0.100g, 0.243mmol)を得た（（７）からの合計収率： 71 %）。
＜２−（２−アセチルアミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライドの物性データ＞
decomp. 220 ℃; ¹H-NMR (500 MHz, D₂O) δ 1.78 (3H, s, COMe), 1.97 (3H, s, Me), 3.56 (2H, t, J = 5.5 Hz, N⁺CH₂CH₂), 3.63 (3H, s, OMe), 4.28 (2H, t, J = 5.5 Hz, N⁺CH₂CH₂), 6.55 (1H, d, J = 7 Hz, 10-H), 6.72 (1H, t, J = 7 Hz, 9-H), 6.97 (1H, t, J = 7 Hz, 8-H), 7.10 (1H, d, J = 7 Hz, 7-H), 7.81 (1H, d, J = 7 Hz, 4-H), 8.37 (1H, d, J = 7 Hz, 3-H), 9.51 (1H, d, J = 7 Hz, 1-H); MS (FAB⁺) m/z 376 (M⁺ - Cl^-); HRMS calcd for M⁺ - Cl^- (C₂₂H₂₂N₃O₃) 376.1661 found 376.1665 6) 2- (2-Acetylaminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium chloride (2- (2-Acetylaminoethyl) -5-methoxycarbonyl-11 -methyl-6H-pyrido [4,3-b] carbazol-2-ium chloride) (11)
Indolepyridylethene derivative (10) (0.100 g, 0.342 mmol), DMF 1 ml, N-acetylchloroethylamine (0.410 g, 3.42 mmol) was added to a three-necked flask equipped with an Allen condenser, and the mixture was heated at 110 ° C. for 5 hours. After stirring, the obtained pyridinium salt was dissolved in 5 ml of MeOH, and a CH ₃ ONa (0.028 g, 0.513 mmol) / MeOH (5 ml) solution was added dropwise, followed by stirring at room temperature for 1 h. Next, a solution of 3-ethoxycarbonyl-1-methylpyridinium chloride (0.103 g, 0.513 mmol) / MeOH (5 ml) was added dropwise, and the mixture was further stirred at room temperature for 24 h. The obtained reaction mixture was purified by ODS column chromatography (developing solvent / MeOH: H 2 O = 20: 80), and 2- (2-acetylaminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [ 4,3b] carbazole-2-ium chloride (11) (0.100 g, 0.243 mmol) was obtained (total yield from (7): 71%).
<Physical data of 2- (2-acetylaminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium chloride>
decomp. 220 ° C; ¹ H-NMR (500 MHz, D ₂ O) δ 1.78 (3H, s, COMe), 1.97 (3H, s, Me), 3.56 (2H, t, J = 5.5 Hz, N ⁺ CH ₂ CH ₂ ), 3.63 (3H, s, OMe), 4.28 (2H, t, J = 5.5 Hz, N ⁺ CH ₂ CH ₂ ), 6.55 (1H, d, J = 7 Hz, 10-H), 6.72 (1H, t, J = 7 Hz, 9-H), 6.97 (1H, t, J = 7 Hz, 8-H), 7.10 (1H, d, J = 7 Hz, 7-H), 7.81 (1H , d, J = 7 Hz, 4-H), 8.37 (1H, d, J = 7 Hz, 3-H), 9.51 (1H, d, J = 7 Hz, 1-H); MS (FAB ⁺ ) ^{m / z 376 (M + -} Cl -); HRMS calcd for M + - Cl - (C 22 H 22 N 3 O 3) 376.1661 found 376.1665

７）２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド（2-(2-Aminoethyl)-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium chloride） (1a)
アリーン冷却管をつけた丸底フラスコに２−（２−アセチルアミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド（11） (20.0mg, 0.053mmol) 、1.2M HCl / MeOH(10ml)を加え、72時間還流した。得られた反応混合物を濃縮し、ODSカラムクロマトグラフィー(展開溶媒/MeOH:H₂O=10: 90)で精製し２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド(1a) (18.5mg, 0.050mmol)を得た（収率： 95 %）。
＜２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライドの物性データ＞
decomp. 210 ℃; ¹H-NMR (500 MHz, D₂O) δ 1.91 (3H, s, CH₃), 3.50 (2H, t, J = 5.2 Hz, N⁺CH₂CH₂), 3.61 (3H, s, OCH₃), 4.54 (2H, t, J = 7.5 Hz, N⁺CH₂CH₂), 6.39 (1H, t, J = 7.5 Hz, H₉), 6.55 (1H, t, J = 7.5 Hz, H₈), 7.85 (1H, d, J = 7.5 Hz, H₁₀), 7.85 (1H, d, J = 7.5 Hz, H₇), 8.30 (1H, d, J = 7.5 Hz, H₄), 8.30 (1H, d, J = 7.5 Hz, H₃), 8.70 (1H, s, H₁); MS (FAB⁺) m/z 334 (M⁺ - Cl^-); HRMS calcd for M⁺-Cl^- (C₂₀H₂₀N₃O₂) 334.1556, found 334.1546. 7) 2- (2-Aminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium chloride (2- (2-Aminoethyl) -5-methoxycarbonyl-11- methyl-6H-pyrido [4,3-b] carbazol-2-ium chloride) (1a)
2- (2-acetylaminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazol-2-ium chloride (11) (20.0 mg) in a round bottom flask equipped with an Allen condenser. , 0.053 mmol), 1.2 M HCl / MeOH (10 ml) was added and refluxed for 72 hours. The obtained reaction mixture was concentrated, purified by ODS column chromatography (developing solvent / MeOH: H ₂ O = 10: 90) and purified by 2- (2-aminoethyl) -5-methoxycarbonyl-11-methyl-6H— Pyrido [4,3b] carbazole-2-ium chloride (1a) (18.5 mg, 0.050 mmol) was obtained (yield: 95%).
<Property data of 2- (2-aminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium chloride>
decomp. 210 ° C; ¹ H-NMR (500 MHz, D ₂ O) δ 1.91 (3H, s, CH ₃ ), 3.50 (2H, t, J = 5.2 Hz, N ⁺ CH ₂ CH ₂ ), 3.61 (3H , s, OCH ₃ ), 4.54 (2H, t, J = 7.5 Hz, N ⁺ CH ₂ CH ₂ ), 6.39 (1H, t, J = 7.5 Hz, H ₉ ), 6.55 (1H, t, J = 7.5 Hz, H ₈ ), 7.85 (1H, d, J = 7.5 Hz, H ₁₀ ), 7.85 (1H, d, J = 7.5 Hz, H ₇ ), 8.30 (1H, d, J = 7.5 Hz, H ₄ ) , 8.30 (1H, d, J = 7.5 Hz, H 3), 8.70 (1H, s, H 1); MS (FAB +) m / z 334 (M + - Cl -); HRMS calcd for M + -Cl ^- (C ₂₀ H ₂₀ N ₃ O ₂ ) 334.1556, found 334.1546.

８）２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムアセテート（2-(2-Aminoethyl)-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium acetate） (1b)
丸底フラスコに、２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムクロライド(1a) (35.8mg, 0.098mmol) 、酢酸銀(33.72mg, 2.02mmol)、H₂O (3ml)を入れ5分還流した。得られた反応混合物セライトを用いて脱塩し、ODSカラムクロマトグラフィー(展開溶媒/MeOH:H₂O=5:95)で精製し、２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムアセテート(1b) (130mg, 0.076mmol)を得た（収率： 78 %）。
＜２−（２−アミノエチル）−５−メトキシカルボニル−１１−メチル−６Ｈ−ピリド［４，３ｂ］カルバゾール−２−イウムアセテートの物性データ＞
¹H-NMR (500 MHz, D₂O) δ 1.76 (3H, s, MeCO), 2.26 (3H, s, Me), 3.12 (2H, t, J = 5.3 Hz, N⁺CH₂CH₂), 3.75 (3H, s, OMe), 4.32 (2H, t, J = 5.3 Hz, N⁺CH₂CH₂), 6.77 (1H, dd, J = 7.6, 7.9 Hz, 9-H), 6.92 (1H, dd, J = 7.6, 8.1 Hz, 8-H), 7.15 (1H, d, J = 7.9 Hz, 10-H), 7.41 (1H, d, J = 8.1 Hz, 7-H), 7.92 (1H, d, J = 7.7 Hz, 4-H), 8.54 (1H, d, J = 7.7 Hz, 3-H), 8.79 (1H, s, 1-H); MS (FAB⁺) m/z 334 (M⁺ - AcO^-); HRMS calcd for M⁺ - Cl^- (C₂₀H₂₀N₃O₂) 334.1556, found 334.1546. 8) 2- (2-Aminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium acetate (2- (2-Aminoethyl) -5-methoxycarbonyl-11- methyl-6H-pyrido [4,3-b] carbazol-2-ium acetate) (1b)
To a round bottom flask was added 2- (2-aminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazole-2-ium chloride (1a) (35.8 mg, 0.098 mmol), acetic acid. Silver (33.72 mg, 2.02 mmol) and H ₂ O (3 ml) were added and refluxed for 5 minutes. The obtained reaction mixture was desalted using Celite and purified by ODS column chromatography (developing solvent / MeOH: H ₂ O = 5: 95) to give 2- (2-aminoethyl) -5-methoxycarbonyl-11. -Methyl-6H-pyrido [4,3b] carbazole-2-ium acetate (1b) (130 mg, 0.076 mmol) was obtained (yield: 78%).
<Property data of 2- (2-aminoethyl) -5-methoxycarbonyl-11-methyl-6H-pyrido [4,3b] carbazol-2-ium acetate>
¹ H-NMR (500 MHz, D ₂ O) δ 1.76 (3H, s, MeCO), 2.26 (3H, s, Me), 3.12 (2H, t, J = 5.3 Hz, N ⁺ CH ₂ CH ₂ ), 3.75 (3H, s, OMe), 4.32 (2H, t, J = 5.3 Hz, N ⁺ CH ₂ CH ₂ ), 6.77 (1H, dd, J = 7.6, 7.9 Hz, 9-H), 6.92 (1H, dd, J = 7.6, 8.1 Hz, 8-H), 7.15 (1H, d, J = 7.9 Hz, 10-H), 7.41 (1H, d, J = 8.1 Hz, 7-H), 7.92 (1H, d, J = 7.7 Hz, 4-H), 8.54 (1H, d, J = 7.7 Hz, 3-H), 8.79 (1H, s, 1-H); MS (FAB ⁺ ) m / z 334 (M ^{^{+ - AcO -); HRMS calcd}} for M + - Cl - (C 20 H 20 N 3 O 2) 334.1556, found 334.1546.

〔実施例２〕
一般式（２）の化合物の合成（図２参照）
１）１，４−ジメチル−９Ｈ−カルバゾール（1,4-Dimethyl-9H-carbazole） (12)
反応容器内に、インドール (4) (0.50g, 4.27mmol)、p-トルエンスルホン酸一水和物(0.45g, 2.61mmol)、2,5-ヘキサジオン(0.49g, 4.30mmol)、EtOH (30ml)を加えた後、1.5 h 還流させた。反応混合物を濃縮後、Et₂Oにて抽出、有機層をMgSO₄上で乾燥した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/EtoAc:Hexane=2:8)で精製し、１，４−ジメチル−９Ｈ−カルバゾール（12） (0.40g, 2.09mmol)を得た（収率： 49 %）。
＜１，４−ジメチル−９Ｈ−カルバゾールの物性データ＞
mp 97.1-97.8 ℃; ¹H-NMR (500MHz, CDCl₃) δ 8.10 (1H, d, J = 8.5Hz, 5-H ), 7.91 (1H, s, NH ), 7.40 (1H, d, J = 8.5Hz, 8-H ), 7.33 (1H, t, J = 8.5Hz, 7-H ), 7.17 (1H, t, J = 8.5Hz, 6-H ), 7.06 (1H, d, J = 7Hz, 2-H), 6.86 (1H, d, J = 7Hz, 3-H ), 2.78 (3H, s, 4-Me), 2.64 (3H, s, 1-Me); MS (FAB⁺) m/z 196 (M+1). [Example 2]
Synthesis of compound of general formula (2) (see FIG. 2)
1) 1,4-Dimethyl-9H-carbazole (12)
In a reaction vessel, indole (4) (0.50 g, 4.27 mmol), p-toluenesulfonic acid monohydrate (0.45 g, 2.61 mmol), 2,5-hexadione (0.49 g, 4.30 mmol), EtOH (30 ml) ) And then refluxed for 1.5 h. The reaction mixture was concentrated, extracted with Et ₂ O, and the organic layer was dried over MgSO ₄ . The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / EtoAc: Hexane = 2: 8) to obtain 1,4-dimethyl-9H-carbazole (12) (0.40 g, 2.09 mmol) (yield). Rate: 49%).
<Physical property data of 1,4-dimethyl-9H-carbazole>
mp 97.1-97.8 ° C; ¹ H-NMR (500 MHz, CDCl ₃ ) δ 8.10 (1H, d, J = 8.5 Hz, 5-H), 7.91 (1H, s, NH), 7.40 (1H, d, J = 8.5Hz, 8-H), 7.33 (1H, t, J = 8.5Hz, 7-H), 7.17 (1H, t, J = 8.5Hz, 6-H), 7.06 (1H, d, J = 7Hz, 2-H), 6.86 (1H, d, J = 7Hz, 3-H), 2.78 (3H, s, 4-Me), 2.64 (3H, s, 1-Me); MS (FAB ⁺ ) m / z 196 (M + 1).

２）１，４−ジメチル−９Ｈ−カルバゾール−３−カルボアルデヒド（1,4-Dimethyl-9H-carbazole-3-carbaldehyde ）(13)
反応容器に１，４−ジメチル−９Ｈ−カルバゾール（ 12） (0.50g, 2.56mmol)、N-methylformanilide(0.48g, 3.55mmol)、POCl₃(0.47g, 3.06mmol)、o-dichlorobenzene 5mlを加えた後、100℃で4 h 撹拌した。反応混合物を0℃に冷却後、H₂O、benzeneを加え抽出した。有機層をMgSO₄上で乾燥し、濃縮後、得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃)で精製し１，４−ジメチル−９Ｈ−カルバゾール−３−カルボアルデヒド（13） (0.44g, 2.00mmol)を得た（収率： 78 %）
＜１，４−ジメチル−９Ｈ−カルバゾール−３−カルボアルデヒドの物性データ＞
mp 214-215 ℃; ¹H-NMR (500MHz, CDCl₃) δ 10.39 (1H, s, CHO ), 8.22 (1H, s, NH ), 8.21 (1H, d, J = 7.5Hz, H₅), 7.70 (1H, s, H₂), 7.46 (1H, d, J = 7.5Hz, H₈), 7.41 (1H, t, J = 7.5Hz, H₇), 7.25 (1H, t, J = 7.5Hz, H₆), 3.18 (3H, s, 1-Me), 2.51 (3H, s, 4-Me); MS (FAB⁺) m/z 224 (M+1). 2) 1,4-Dimethyl-9H-carbazole-3-carbaldehyde (13)
Add 1,4-dimethyl-9H-carbazole (12) (0.50 g, 2.56 mmol), N-methylformanilide (0.48 g, 3.55 mmol), POCl ₃ (0.47 g, 3.06 mmol), and 5 ml of o-dichlorobenzene to the reaction vessel. After that, the mixture was stirred at 100 ° C. for 4 hours. The reaction mixture was cooled to 0 ° C., and extracted by adding H ₂ O and benzene. The organic layer was dried over MgSO ₄ and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ ) and purified by 1,4-dimethyl-9H-carbazole-3-carbaldehyde (13). (0.44 g, 2.00 mmol) was obtained (yield: 78%)
<Physical property data of 1,4-dimethyl-9H-carbazole-3-carbaldehyde>
mp 214-215 ° C; ¹ H-NMR (500 MHz, CDCl ₃ ) δ 10.39 (1H, s, CHO), 8.22 (1H, s, NH), 8.21 (1H, d, J = 7.5 Hz, H ₅ ), 7.70 (1H, s, H ₂ ), 7.46 (1H, d, J = 7.5Hz, H ₈ ), 7.41 (1H, t, J = 7.5Hz, H ₇ ), 7.25 (1H, t, J = 7.5Hz , H ₆ ), 3.18 (3H, s, 1-Me), 2.51 (3H, s, 4-Me); MS (FAB ⁺ ) m / z 224 (M + 1).

３）Ｎ−（２，２−ジエトキシエチル）−Ｎ−（１，４−ジメチル−９Ｈ−カルバゾール−３−イルメチル）−４−ニトロベンゼンスルホンアミド（ N-(2,2-Diethoxyethyl)-N-(1,4-dimethyl-9H-carbazol-3-ylmethyl)-4-nitrobenzenesulfonamide） (14)
Dean-stark管を取り付けた3つ口フラスコ内に、１，４−ジメチル−９Ｈ−カルバゾール−３−カルボアルデヒド（13） (0.05g, 0.22mmol)、aminoacetaldehydediethoxyacetal (0.033g, 0.24mmol)、C₆H₆ (20ml)を加え、系内の水を除去しつつ、2 h還流した。系内を室温まで冷却し、濃縮後、Ar雰囲気中、反応混合物をMeOH 5mlに溶かし、NaBH₄ (0.08g, 2.11mmol)をゆっくり加え、室温で2 h撹拌した。その後、濃縮し、H₂O 20mlを加え、C₆H₆にて抽出した。得られた有機層をMgSO₄上で乾燥し、ろ過、濃縮した。次に、反応混合物をCH₃CNに溶かし、NsCl (0.15g, 0.677mmol)、pyridine (0.03ml)を加え、室温で終夜撹拌した。得られた反応混合物を濃縮し、シリカゲルカラムクロマトグラフィー(展開溶媒/ EtoAc:Hexane=3:7)で精製し、Ｎ−（２，２−ジエトキシエチル）−Ｎ−（１，４−ジメチル−９Ｈ−カルバゾール−３−イルメチル）−４−ニトロベンゼンスルホンアミド（14） (0.10g, 0.19mmol)を得た（収率： 85 %）。
＜Ｎ−（２，２−ジエトキシエチル）−Ｎ−（１，４−ジメチル−９Ｈ−カルバゾール−３−イルメチル）−４−ニトロベンゼンスルホンアミドの物性データ＞
decomp. 175.0-177.1 ℃; ¹H-NMR (500MHz, CDCl₃) δ 8.08-6.99 (9H, m, PhH), 8.00 (1H, s, NH), 4.80 (2H, s, ArCH ₂N), 4.49 (1H, t, J = 5.5Hz, CH(OEt)₂), 3.55 (2H, d, J = 5.5Hz, CH ₂CH(OEt)₂), 3.38 (4H, q, J = 7Hz, O(CH ₂CH₃)₂), 2.66 (3H, s, 1-Me), 2.33 (3H, s, 4-Me), 1.07 (6H, t, J = 7Hz, O(CH₂CH ₃)₂); MS (FAB⁺) m/z 525 (M⁺); HRMS calcd for M⁺ (C₂₇H₃₁N₃O₆S) 525.1933 found 525.1921. 3) N- (2,2-Diethoxyethyl) -N- (1,4-dimethyl-9H-carbazol-3-ylmethyl) -4-nitrobenzenesulfonamide (N- (2,2-Diethoxyethyl) -N- (1,4-dimethyl-9H-carbazol-3-ylmethyl) -4-nitrobenzenesulfonamide) (14)
In a three-necked flask equipped with a Dean-stark tube, 1,4-dimethyl-9H-carbazole-3-carbaldehyde (13) (0.05 g, 0.22 mmol), aminoacetaldehydediethoxyacetal (0.033 g, 0.24 mmol), C ₆ H ₆ (20 ml) was added and refluxed for 2 h while removing water in the system. The system was cooled to room temperature, concentrated, and the reaction mixture was dissolved in 5 ml of MeOH in an Ar atmosphere. NaBH ₄ (0.08 g, 2.11 mmol) was slowly added, and the mixture was stirred at room temperature for 2 h. Thereafter, the mixture was concentrated, 20 ml of H ₂ O was added, and the mixture was extracted with C ₆ H ₆ . The resulting organic layer was dried over MgSO ₄ , filtered and concentrated. Next, the reaction mixture was dissolved in CH ₃ CN, NsCl (0.15 g, 0.677 mmol) and pyridine (0.03 ml) were added, and the mixture was stirred at room temperature overnight. The obtained reaction mixture was concentrated and purified by silica gel column chromatography (developing solvent / EtoAc: Hexane = 3: 7), and N- (2,2-diethoxyethyl) -N- (1,4-dimethyl- 9H-carbazol-3-ylmethyl) -4-nitrobenzenesulfonamide (14) (0.10 g, 0.19 mmol) was obtained (yield: 85%).
<Property data of N- (2,2-diethoxyethyl) -N- (1,4-dimethyl-9H-carbazol-3-ylmethyl) -4-nitrobenzenesulfonamide>
decomp. 175.0-177.1 ℃; ¹ H-NMR (500MHz, CDCl ₃ ) δ 8.08-6.99 (9H, m, Ph H ), 8.00 (1H, s, NH), 4.80 (2H, s, ArC H ₂ N) , 4.49 (1H, t, J = 5.5Hz, C H (OEt) ₂ ), 3.55 (2H, d, J = 5.5Hz, C H ₂ CH (OEt) ₂ ), 3.38 (4H, q, J = 7Hz , O (C H ₂ CH ₃ ) ₂ ), 2.66 (3H, s, 1-Me), 2.33 (3H, s, 4-Me), 1.07 (6H, t, J = 7Hz, O (CH ₂ C H ₃ ) ₂ ); MS (FAB ⁺ ) m / z 525 (M ⁺ ); HRMS calcd for M ⁺ (C ₂₇ H ₃₁ N ₃ O ₆ S) 525.1933 found 525.1921.

４）エリプチシン（Ellipticine） (15)
丸底フラスコに、Ｎ−（２，２−ジエトキシエチル）−Ｎ−（１，４−ジメチル−９Ｈ−カルバゾール−３−イルメチル）−４−ニトロベンゼンスルホンアミド（14） (100mg, 0.19mmol)、6M HCl / dioxane (1:4) (20ml)を入れ、3 h還流した。室温まで冷却、NaHCO₃.にて中和した後、CHCl₃で抽出、有機層をNa₂SO₄上で乾燥し、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃)で精製し、エリプチシン（15 ）(42,6mg, 0.17mmol)を得た（収率： 91%）。
＜エリプチシンの物性データ＞
decomp. 309-311 ℃; ¹H-NMR (500MHz, CD₃OD) δ 9.41 (1H, s, H₁), 8.19-8.18 (2H, m), 7.84 (1H, d, J = 6.5Hz), 7.41 (2H, m), 7.15 (1H, d, J = 6.5Hz), 3.06 (3H, s, 11-Me), 2.62 (3H, s, 5-Me); MS (FAB⁺) m/z 247 (M+1). 4) Ellipticine (15)
In a round bottom flask, N- (2,2-diethoxyethyl) -N- (1,4-dimethyl-9H-carbazol-3-ylmethyl) -4-nitrobenzenesulfonamide (14) (100 mg, 0.19 mmol), 6M HCl / dioxane (1: 4) (20 ml) was added and refluxed for 3 h. The mixture was cooled to room temperature, neutralized with NaHCO ₃ , extracted with CHCl ₃ , the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ ) to obtain ellipticine (15) (42,6 mg, 0.17 mmol) (yield: 91%).
<Physical properties data of ellipticine>
decomp. 309-311 ° C; ¹ H-NMR (500 MHz, CD ₃ OD) δ 9.41 (1H, s, H ₁ ), 8.19-8.18 (2H, m), 7.84 (1H, d, J = 6.5 Hz), 7.41 (2H, m), 7.15 (1H, d, J = 6.5Hz), 3.06 (3H, s, 11-Me), 2.62 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 247 (M + 1).

５）２−（ｐ−ニトロフェノキシカルボニル）−１，２−デヒドロエリプチシン（2-(p-nitrophenoxycarbonyl)-1,2-dihydroellipticine) (16)
エリプチシン（15）(50.0mg, 0.203mmol)、THF (30ml)を入れ-80℃まで冷却した。p-nitrophenylchloroformate(120.0mg, 0.60mmol)のTHF溶液をゆっくり滴下し、30 min撹拌した。次にNaBH₃CN (60.0mg, 0.95mmol)のTHF溶液をゆっくり滴下し、30 min撹拌した。その後H₂O 10mlを入れ、系内を室温にもどした。反応混合物にCHCl₃を100ml加え、有機層を1M HCl、Na₂CO₃aq.、H₂Oで洗浄、有機層をNa₂SO₄上で乾燥し、ろ過、濃縮した。得られた反応混合物をMeOHにて再結晶し、２−（ｐ−ニトロフェノキシカルボニル）−１，２−デヒドロエリプチシン（16） (75.5mg, 0.183mmol)を得た（収率： 90%）。
＜２−（ｐ−ニトロフェノキシカルボニル）−１，２−デヒドロエリプチシンの物性データ＞
dec. 234-235 ℃; ¹H-NMR (500 MHz, DMSO-d₆) δ 11.9 (1H, s, NH), 8.33 (2H, d, J = 9 Hz, p-NO₂Ph-H), 8.14 (1H, dd, J = 7.5 Hz, H₁₀), 7.63, 7.58 (2H, d, J = 9 Hz, p-NO₂Ph-H), 7.51 (1H, d, J = 7.5 Hz, H₇), 7.37 (1H, dd, J = 7.5 Hz, H₉), 7.15 (1H, dd, J = 7.5 Hz, H₈), 6.95 (1H, d, J = 9 Hz, H₃), 6.36 (1H, d, J = 9 Hz, H₄), 5.22, 5.02 (2H, s, H₁), 2.71 (3H, s, 11-Me), 2.51 (3H, s, 5-Me); MS (FAB⁺) m/z 413 (M⁺); HRMS calcd for M⁺ (C₂₄H₁₉N₃O₄) 413.1376 found 413.1371 5) 2- (p-nitrophenoxycarbonyl) -1,2-dehydroellipticine (16)
Ellipticin (15) (50.0 mg, 0.203 mmol) and THF (30 ml) were added and cooled to -80 ° C. A THF solution of p-nitrophenylchloroformate (120.0 mg, 0.60 mmol) was slowly added dropwise and stirred for 30 min. Next, a THF solution of NaBH ₃ CN (60.0 mg, 0.95 mmol) was slowly added dropwise and stirred for 30 min. Thereafter, 10 ml of H ₂ O was added, and the system was returned to room temperature. 100 ml of CHCl ₃ was added to the reaction mixture, the organic layer was washed with 1M HCl, Na ₂ CO ₃ aq., H ₂ O, the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The obtained reaction mixture was recrystallized from MeOH to obtain 2- (p-nitrophenoxycarbonyl) -1,2-dehydroellipticine (16) (75.5 mg, 0.183 mmol) (yield: 90% ).
<Property data of 2- (p-nitrophenoxycarbonyl) -1,2-dehydroellipticine>
dec. 234-235 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 11.9 (1H, s, NH), 8.33 (2H, d, J = 9 Hz, p-NO ₂ Ph-H), 8.14 (1H, dd, J = 7.5 Hz, H ₁₀ ), 7.63, 7.58 (2H, d, J = 9 Hz, p-NO ₂ Ph-H), 7.51 (1H, d, J = 7.5 Hz, H ₇ ), 7.37 (1H, dd, J = 7.5 Hz, H ₉ ), 7.15 (1H, dd, J = 7.5 Hz, H ₈ ), 6.95 (1H, d, J = 9 Hz, H ₃ ), 6.36 (1H , d, J = 9 Hz, H ₄ ), 5.22, 5.02 (2H, s, H ₁ ), 2.71 (3H, s, 11-Me), 2.51 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 413 (M ⁺ ); HRMS calcd for M ⁺ (C ₂₄ H ₁₉ N ₃ O ₄ ) 413.1376 found 413.1371

６）２−（２−アミノエチル）カルバモイル−１，２−デヒドロエリプチシン（2-(2-aminoethyl)carbamoyl-1,2-dihydroellipticine) (2a)
Ar置換した丸底フラスコに、２−（ｐ−ニトロフェノキシカルボニル）−１，２−デヒドロエリプチシン16 (30.0mg, 0.073mmol)、DMF (1ml)を入れ、EDA (20.0mg, 0.33mmol)をゆっくり滴下し、3 h撹拌した。その後反応混合物をアミノシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 9 : 1)にて精製し２−（２−アミノエチル）カルバモイル−１，２−デヒドロエリプチシン(2a) (13.2mg, 0.04mmol)を得た（収率： 53%）。
＜２−（２−アミノエチル）カルバモイル−１，２−デヒドロエリプチシンの物性データ＞
decomp. 192-194 ℃; ¹H-NMR (500 MHz, CD₃OD) δ 8.05 (1H, d, J = 8 Hz, H₁₀), 7.43 (1H, d, J = 8 Hz, H₇), 7.30 (1H, t, J = 8 Hz, H₉), 7.10 (1H, t, J = 8 Hz, H₈), 6.91 (1H, d, J = 7.5 Hz, H₃), 6.12 (1H, d, J = 7.5 Hz, H₄), 4.91 (2H, s, H₁), 3.35 (3H, t, J = 6 Hz, NHCH₂), 2.80 (3H, t, J = 6 Hz, NH₂CH₂), 2.72 (3H, s, 11-Me), 2.45 (3H, s, 5-Me); MS (FAB⁺) m/z 335 (M+1); HRMS calcd for M+H (C₂₀H₂₃N₄O) 335.1872, found 335.1877. 6) 2- (2-aminoethyl) carbamoyl-1,2-dehydroellipticine (2a)
To an Ar-substituted round bottom flask, 2- (p-nitrophenoxycarbonyl) -1,2-dehydroellipticine 16 (30.0 mg, 0.073 mmol) and DMF (1 ml) were placed, and EDA (20.0 mg, 0.33 mmol) Was slowly added dropwise and stirred for 3 h. The reaction mixture was then purified by amino silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 9: 1) to give 2- (2-aminoethyl) carbamoyl-1,2-dehydroellipticine (2a) (13.2 mg 0.04 mmol) (yield: 53%).
<Property data of 2- (2-aminoethyl) carbamoyl-1,2-dehydroellipticine>
decomp. 192-194 ° C; ¹ H-NMR (500 MHz, CD ₃ OD) δ 8.05 (1H, d, J = 8 Hz, H ₁₀ ), 7.43 (1H, d, J = 8 Hz, H ₇ ), 7.30 (1H, t, J = 8 Hz, H ₉ ), 7.10 (1H, t, J = 8 Hz, H ₈ ), 6.91 (1H, d, J = 7.5 Hz, H ₃ ), 6.12 (1H, d , J = 7.5 Hz, H ₄ ), 4.91 (2H, s, H ₁ ), 3.35 (3H, t, J = 6 Hz, NHCH ₂ ), 2.80 (3H, t, J = 6 Hz, NH ₂ CH ₂ ), 2.72 (3H, s, 11-Me), 2.45 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 335 (M + 1); HRMS calcd for M + H (C ₂₀ H ₂₃ N ₄ O) 335.1872, found 335.1877.

７）１，２−ジヒドロエリプチシン−メチルウレア（1,2-dihydroellipticine-methylurea ）(2b)
Ar置換した丸底フラスコに、２−（２−アミノエチル）カルバモイル−１，２−デヒドロエリプチシン(2a) (10.0mg, 0.030mmol)、DMF (1ml)を入れ、 p-Nitrophenyl-N-methylcarbamate (7.0mg, 0.036mmol)のDMF溶液をゆっくり滴下し、室温で1 h撹拌した。その後反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 20 : 1)にて精製後、MeOH、Et₂Oより再結晶し、１，２−ジヒドロエリプチシン−メチルウレア(2b) (6.0mg, 0.015mmol)を得た（収率: 50%）。
＜１，２−ジヒドロエリプチシン−メチルウレアの物性データ＞
decomp. 148-150 ℃; ¹H-NMR (500 MHz, DMSO-d₆) δ 11.04 (1H, s, NH), 8.10 (1H, d, J = 7.5 Hz, H₁₀), 7.45 (1H, d, J = 7.5 Hz, H₇), 7.33 (1H, s, NH), 7.31 (1H, t, J = 7.5 Hz, H₉), 7.10 (1H, t, J = 7.5 Hz, H₈), 7.02 (1H, d, J = 7.5 Hz, H₃), 6.08 (1H, s, NH), 5.95 (1H, d, J = 7.5 Hz, H₄), 5.82 (1H, s, NH), 4.88 (2H, s, H1), 3.15 (4H, m, CH₂CH₂), 2.70 (3H, s, 11-Me), 2.55 (3H, s, 5-Me), 2.49 (3H, s, NHMe); MS (FAB⁺) m/z 392 (M+1); HRMS calcd for M+H (C₂₂H₂₆N₅O₂) 392.2086, found 392.2099. 7) 1,2-dihydroellipticine-methylurea (2b)
In an Ar-substituted round bottom flask, 2- (2-aminoethyl) carbamoyl-1,2-dehydroellipticine (2a) (10.0 mg, 0.030 mmol) and DMF (1 ml) were placed, and p-Nitrophenyl-N- A DMF solution of methylcarbamate (7.0 mg, 0.036 mmol) was slowly added dropwise and stirred at room temperature for 1 h. Thereafter, the reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 20: 1), recrystallized from MeOH and Et ₂ O, and 1,2-dihydroellipticine-methylurea (2b) ( 6.0 mg, 0.015 mmol) was obtained (yield: 50%).
<Physical property data of 1,2-dihydroellipticine-methylurea>
decomp. 148-150 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 11.04 (1H, s, NH), 8.10 (1H, d, J = 7.5 Hz, H ₁₀ ), 7.45 (1H, d , J = 7.5 Hz, H ₇ ), 7.33 (1H, s, NH), 7.31 (1H, t, J = 7.5 Hz, H ₉ ), 7.10 (1H, t, J = 7.5 Hz, H ₈ ), 7.02 (1H, d, J = 7.5 Hz, H ₃ ), 6.08 (1H, s, NH), 5.95 (1H, d, J = 7.5 Hz, H ₄ ), 5.82 (1H, s, NH), 4.88 (2H , s, H1), 3.15 (4H, m, CH ₂ CH ₂ ), 2.70 (3H, s, 11-Me), 2.55 (3H, s, 5-Me), 2.49 (3H, s, NHMe); MS (FAB ⁺ ) m / z 392 (M + 1); HRMS calcd for M + H (C ₂₂ H ₂₆ N ₅ O ₂ ) 392.2086, found 392.2099.

８）１，２−ジヒドロエリプチシン−メチルニトロソウレア（1,2-dihydroellipticine-methylnitrosourea (2c)
Ar置換した丸底フラスコに、２−（２−アミノエチル）アミド−１，２−デヒドロエリプチシン(2a) (10.0mg, 0.030mmol)、DMF (1ml)を入れ、Succinimidyl-N-methylnitrsocarbamate (7.0mg, 0.035mmol)のDMF溶液をゆっくり滴下し、室温で1 h撹拌した。その後反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 10 : 1)にて精製後、MeOH、Et₂Oより再結晶し、１，２−ジヒドロエリプチシン−メチルニトロソウレア(2c) (6.3mg, 0.015mmol)を得た（収率： 50%）。
＜１，２−ジヒドロエリプチシン−メチルニトロソウレアの物性データ＞
decomp. 148-150 ℃; ¹H-NMR (500 MHz, CD₃OD) δ 8.11 (1H, d, J = 7.5 Hz, H₁₀), 7.44 (1H, d, J = 7.5 Hz, H₇), 7.30 (1H, t, J = 7.5 Hz, H₉), 7.11 (1H, t, J = 7.5 Hz, H₈), 6.90 (1H, d, J = 7.5 Hz, H₃), 6.14 (1H, d, J = 7.5 Hz, H₄), 4.92 (2H, s, H₁), 3.58 (2H, t, CH₂CH₂), 3.51 (2H, t, CH₂CH₂), 3.11 (3H, s, 11-Me), 2.75 (3H, s, 5-Me), 2.45 (3H, s, NHMe); MS (FAB⁺) m/z 421 (M+1); HRMS calcd for M+H (C₂₂H₂₅N₆O₃) 421.1989, found 421.2013. 8) 1,2-dihydroellipticine-methylnitrosourea (2c)
Ar-substituted round bottom flask was charged with 2- (2-aminoethyl) amido-1,2-dehydroellipticine (2a) (10.0 mg, 0.030 mmol) and DMF (1 ml), and Succinimidyl-N-methylnitrsocarbamate ( 7.0 mg, 0.035 mmol) of DMF was slowly added dropwise and stirred at room temperature for 1 h. Thereafter, the reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 10: 1), recrystallized from MeOH and Et ₂ O, and 1,2-dihydroellipticine-methylnitrosourea (2c ) (6.3 mg, 0.015 mmol) was obtained (yield: 50%).
<Physical properties data of 1,2-dihydroellipticine-methylnitrosourea>
decomp. 148-150 ° C; ¹ H-NMR (500 MHz, CD ₃ OD) δ 8.11 (1H, d, J = 7.5 Hz, H ₁₀ ), 7.44 (1H, d, J = 7.5 Hz, H ₇ ), 7.30 (1H, t, J = 7.5 Hz, H ₉ ), 7.11 (1H, t, J = 7.5 Hz, H ₈ ), 6.90 (1H, d, J = 7.5 Hz, H ₃ ), 6.14 (1H, d , J = 7.5 Hz, H ₄ ), 4.92 (2H, s, H ₁ ), 3.58 (2H, t, CH ₂ CH ₂ ), 3.51 (2H, t, CH ₂ CH ₂ ), 3.11 (3H, s, 11-Me), 2.75 (3H, s, 5-Me), 2.45 (3H, s, NHMe); MS (FAB ⁺ ) m / z 421 (M + 1); HRMS calcd for M + H (C ₂₂ H ₂₅ N ₆ O ₃ ) 421.1989, found 421.2013.

〔実施例３〕
一般式（３）の化合物の合成（図３参照）
１） 9-ホルミルエリプチシン (17)
エリプチシン（15） (30.0mg, 0.0122mmol)、ヘキサメチレンテトラミン(200mg, 1.42mmol)、トリフルオロ酢酸 (5ml)を入れ懸濁液を20 min還流した。その後、反応液を約4分の1まで濃縮し、少量の水を加え、NaHCO₃を用いて中和した。反応混合物をCHCl₃/ MeOH 9:1で抽出し有機層を飽和Na₂CO₃水溶液、飽和食塩水で洗浄、有機層をNa₂SO₄上で乾燥し、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 9 : 1)にて精製後、MeOHにて再結晶し、9-ホルミルエリプチシン（17） (30.7mg, 0.0112mmol)を得た（収率： 92 %）。
＜9-ホルミルエリプチシンの物性データ＞
decomp. 360 ℃; ¹H-NMR (500 MHz, CD₃CD) δ 10.6 (1H, s, CHO), 9.66 (1H, s, H₁), 8.89 (1H, s, H₁₀), 8.40 (1H, d, J = 8.4 Hz, H₃), 8.06 (2H, dd, H₄, H₈), 7.65 (1H, d, J = 8.4 Hz, H₇), 2.85 (3H, s, Me); MS (FAB⁺) m/z 275 (M+1). Example 3
Synthesis of compound of general formula (3) (see FIG. 3)
1) 9-Formyl ellipticine (17)
Ellipticin (15) (30.0 mg, 0.0122 mmol), hexamethylenetetramine (200 mg, 1.42 mmol) and trifluoroacetic acid (5 ml) were added and the suspension was refluxed for 20 min. Thereafter, the reaction solution was concentrated to about 1/4, a small amount of water was added, and neutralized with NaHCO ₃ . The reaction mixture was extracted with CHCl ₃ / MeOH 9: 1, the organic layer was washed with saturated aqueous Na ₂ CO ₃ solution and saturated brine, and the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 9: 1) and recrystallized from MeOH to give 9-formyl ellipticine (17) (30.7 mg, 0.0112 mmol (Yield: 92%).
<Property data of 9-formyl ellipticine>
decomp. 360 ° C; ¹ H-NMR (500 MHz, CD ₃ CD) δ 10.6 (1H, s, CHO), 9.66 (1H, s, H ₁ ), 8.89 (1H, s, H ₁₀ ), 8.40 (1H , d, J = 8.4 Hz, H ₃ ), 8.06 (2H, dd, H ₄ , H ₈ ), 7.65 (1H, d, J = 8.4 Hz, H ₇ ), 2.85 (3H, s, Me); MS (FAB ⁺ ) m / z 275 (M + 1).

２） 9-ヒドロキシエリプチシン (18)
9−ホルミルエリプチシン（17） (100.0mg, 0.365mmol)、mCPBA(190mg, 1.09mmol)、TsOH(180mg, 1.09mmol)、THF (30ml)を入れその懸濁液を48 h撹拌した。反応混合物を濃縮し、飽和Na₂CO₃水溶液を10ml加え、CHCl₃で抽出、Na₂SO₄上で乾燥後、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 8 : 2)にて精製し、 9-ヒドロキシエリプチシン（18） (61.2mg, 0.234mmol)を得た（収率： 64 %）。
＜9-ヒドロキシエリプチシンの物性データ＞
decomp. 355 ℃; ¹H-NMR (500 MHz, DMSO-d₆) δ 11.04 (1H, s, NH), 9.64 (1H, s, H₁), 9.09 (1H, s, OH), 8.37 (1H, d, J = 6.0 Hz, H₃), 7.86 (1H, d, J = 6.0 Hz, H₄), 7.75 (1H, s, H₁₀), 7.36 (1H, d, J = 8.5 Hz, H₇), 7.00 (1H, d, J = 8.5 Hz, H₈), 3.19 (3H, s, 11-Me), 2.73 (3H, s, 5-Me); MS (FAB⁺) m/z 263 (M+1). 2) 9-Hydroxy ellipticine (18)
9-Formyl ellipticine (17) (100.0 mg, 0.365 mmol), mCPBA (190 mg, 1.09 mmol), TsOH (180 mg, 1.09 mmol), and THF (30 ml) were added and the suspension was stirred for 48 h. The reaction mixture was concentrated, 10 ml of saturated aqueous Na ₂ CO ₃ solution was added, extracted with CHCl ₃ , dried over Na ₂ SO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 8: 2) to obtain 9-hydroxyellipticine (18) (61.2 mg, 0.234 mmol) (yield) : 64%).
<Property data of 9-hydroxy ellipticine>
decomp. 355 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 11.04 (1H, s, NH), 9.64 (1H, s, H ₁ ), 9.09 (1H, s, OH), 8.37 (1H , d, J = 6.0 Hz, H ₃ ), 7.86 (1H, d, J = 6.0 Hz, H ₄ ), 7.75 (1H, s, H ₁₀ ), 7.36 (1H, d, J = 8.5 Hz, H ₇ ), 7.00 (1H, d, J = 8.5 Hz, H ₈ ), 3.19 (3H, s, 11-Me), 2.73 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 263 (M +1).

３）９−（tert−ブチルジメチルシラニルオキシ）エリプチシン（9-(tert-butyldimethylsilanyloxy)ellipticine）（19）
9-ヒドロキシエリプチシン(18) (30.0mg, 0.114mmol)、TBSCl(50.0mg, 0.342mmol)、イミダゾール(15mg, 0.228mmol)、DMF 1.5ml入れ3 h撹拌した。飽和NaHCO₃水溶液を5ml加え、CHCl₃で抽出、Na₂SO₄上で乾燥後、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 9 : 1)にて精製し９−（tert−ブチルジメチルシラニルオキシ）エリプチシン（19） (40.6mg, 0.108mmol)を得た（収率: 95 %）。
＜９−（tert−ブチルジメチルシラニルオキシ）エリプチシンの物性データ＞
decomp. 245 ℃; ¹H-NMR (500 MHz, CDCl₃)δ 9.42 (1H, s, NH), 8.22 (1H, d, J = 5.0 Hz, H₃), 7.88 (1H, s, H₁), 7.55 (2H, m, H₁₀, H₄), 7.06 (1H, d, J = 8.5 Hz, H₇), 6.79 (1H, d, J = 8.5 Hz, H₈), 2.97 (3H, s, 11-Me), 2.46 (3H, s, 5-Me), 0.78 (9H, s, t-Bu), 0.01 (6H, s, (CH₃)₂); MS (FAB⁺) m/z 377 (M+1); HRMS calcd for M+H (C₂₃H₂₉N₂OSi) 377.2049, found 377.2048. 3) 9- (tert-butyldimethylsilanyloxy) ellipticine (19)
9-hydroxy ellipticine (18) (30.0 mg, 0.114 mmol), TBSCl (50.0 mg, 0.342 mmol), imidazole (15 mg, 0.228 mmol), 1.5 ml of DMF were added and stirred for 3 h. 5 ml of a saturated aqueous NaHCO ₃ solution was added, extracted with CHCl ₃ , dried over Na ₂ SO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 9: 1) to obtain 9- (tert-butyldimethylsilanyloxy) ellipticine (19) (40.6 mg, 0.108 mmol). Obtained (yield: 95%).
<Physical property data of 9- (tert-butyldimethylsilanyloxy) ellipticine>
decomp. 245 ° C; ¹ H-NMR (500 MHz, CDCl ₃ ) δ 9.42 (1H, s, NH), 8.22 (1H, d, J = 5.0 Hz, H ₃ ), 7.88 (1H, s, H ₁ ) , 7.55 (2H, m, H ₁₀ , H ₄ ), 7.06 (1H, d, J = 8.5 Hz, H ₇ ), 6.79 (1H, d, J = 8.5 Hz, H ₈ ), 2.97 (3H, s, 11-Me), 2.46 (3H, s, 5-Me), 0.78 (9H, s, t-Bu), 0.01 (6H, s, (CH ₃ ) ₂ ); MS (FAB ⁺ ) m / z 377 ( M + 1); HRMS calcd for M + H (C ₂₃ H ₂₉ N ₂ OSi) 377.2049, found 377.2048.

４）９−（tert−ブチルジメチルシラニルオキシ）−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（9-(tert-butyldimethylsilanyloxy)-2-(p-nitrophenoxycarbonyl)-1,2-dihydroellipticine）（20）
９−（tert−ブチルジメチルシラニルオキシ）エリプチシン（19） (50.0mg, 0.132mmol)、THF 30mlを入れ-80℃まで冷却した。p-nitrophenylchloroformate(110.0mg, 0.526mmol)のTHF溶液をゆっくり滴下し、2 h撹拌した。次にNaBH₃CN (40.0mg, 0.660mmol)のTHF溶液をゆっくり滴下し、2 h撹拌した。その後NaHCO₃aq. 10mlを入れ、系内を室温にもどした。反応混合物にCHCl₃を100ml加え、有機層を1M HCl、Na₂CO₃aq.、H₂Oで洗浄、有機層をNa₂SO₄上で乾燥し、ろ過、濃縮した。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 9 : 1)で精製後、Et₂Oにて洗浄し９−（tert−ブチルジメチルシラニルオキシ）−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（20）(68.1mg, 0.125mmol)を得た（収率 : 95 %）。
＜９−（tert−ブチルジメチルシラニルオキシ）−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシンの物性データ＞
decomp. 235 ℃; ¹H-NMR (500 MHz, DMSO-d₆) δ 10.79 (1H, s, NH), 8.10 (2H, d, J = 8 Hz, p-NO₂Ph-H), 7.40, 7.36 (2H, d, J = 8 Hz, p-NO₂Ph-H), 7.32 (1H, s, H₁₀), 7.15 (1H, d, J = 8 Hz, H₇), 6.94 (1H, d, J = 8 Hz, H₈), 6.71 (1H, d, J = 9 Hz, H₃), 6.12 (1H, d, J = 9 Hz, H₄), 4.98, 4.78 (2H, s, H₁), 3.11 (3H, s, 11-Me), 2.43 (3H, s, 5-Me), 0.77 (9H, s, t-Bu), 0.00 (6H, s, (CH₃)₂); MS (FAB+) m/z 543 (M⁺); HRMS calcd for M⁺ (C₃₀H₃₃N₃O₅Si) 543.2189, found 543.2189. 4) 9- (tert-butyldimethylsilanyloxy) -2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine (9- (tert-butyldimethylsilanyloxy) -2- (p-nitrophenoxycarbonyl) -1 , 2-dihydroellipticine) (20)
9- (tert-butyldimethylsilanyloxy) ellipticine (19) (50.0 mg, 0.132 mmol) and 30 ml of THF were added and cooled to -80 ° C. A THF solution of p-nitrophenylchloroformate (110.0 mg, 0.526 mmol) was slowly added dropwise and stirred for 2 h. Next, a THF solution of NaBH ₃ CN (40.0 mg, 0.660 mmol) was slowly added dropwise and stirred for 2 h. Thereafter, 10 ml of NaHCO ₃ aq. Was added, and the system was returned to room temperature. 100 ml of CHCl ₃ was added to the reaction mixture, the organic layer was washed with 1M HCl, Na ₂ CO ₃ aq., H ₂ O, the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The resulting reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 9: 1), washed with Et ₂ O and washed with 9- (tert-butyldimethylsilanyloxy) -2- (p -Nitrophenoxycarbonyl) -1,2-dihydroellipticine (20) (68.1 mg, 0.125 mmol) was obtained (yield: 95%).
<Property data of 9- (tert-butyldimethylsilanyloxy) -2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine>
decomp. 235 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 10.79 (1H, s, NH), 8.10 (2H, d, J = 8 Hz, p-NO ₂ Ph-H), 7.40, 7.36 (2H, d, J = 8 Hz, p-NO ₂ Ph-H), 7.32 (1H, s, H ₁₀ ), 7.15 (1H, d, J = 8 Hz, H ₇ ), 6.94 (1H, d , J = 8 Hz, H ₈ ), 6.71 (1H, d, J = 9 Hz, H ₃ ), 6.12 (1H, d, J = 9 Hz, H ₄ ), 4.98, 4.78 (2H, s, H ₁ ), 3.11 (3H, s, 11-Me), 2.43 (3H, s, 5-Me), 0.77 (9H, s, t-Bu), 0.00 (6H, s, (CH ₃ ) ₂ ); MS ( FAB +) m / z 543 (M ⁺ ); HRMS calcd for M ⁺ (C ₃₀ H ₃₃ N ₃ O ₅ Si) 543.2189, found 543.2189.

５）９−ヒドロキシ−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（9-hydroxy-2-(p-nitrophenoxycarbonyl)- 1,2-dihydroellipticine）（21）
９−（tert−ブチルジメチルシラニルオキシ）−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（20）(20.0mg, 0.036mmol)、THF 5mlを入れ、AcOH(11.0mg, 0.183mmol)、滴下後、 TBAF (48.0mg, 0.183mmol)のTHF溶液をゆっくり滴下し、2 h撹拌した。。得られた反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 9 : 1)で精製後、CHCl₃、Et₂Oで再結晶し、９−ヒドロキシ−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（21） (11.7mg, 0.027mmol)を得た（収率： 76%）。
＜９−ヒドロキシ−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシンの物性データ＞
decomp. 210 ℃; ¹H-NMR (500 MHz, DMSO-d₆) δ 10.82 (1H, s, NH), 8.88 (1H, s, OH), 8.30 (2H, d, J = 8 Hz, p-NO₂Ph-H), 7.60, 7.55 (2H, d, J = 8 Hz, p-NO₂Ph-H), 7.52 (1H, s, H₁₀), 7.29 (1H, d, J = 8.5 Hz, H₇), 7.12 (1H, d, J = 8.5 Hz, H₈), 6.86 (1H, d, J = 8 Hz, H₃), 6.32 (1H, d, J = 8 Hz, H₄), 5.17, 4.98 (2H, s, H₁), 2.65 (3H, s, 11-Me), 2.46 (3H, s, 5-Me); MS (FAB⁺) m/z 429 (M⁺); HRMS calcd for M⁺ (C₂₄H₁₉N₃O₅) 429.1325, found 429.1332. 5) 9-hydroxy-2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine (21)
9- (tert-butyldimethylsilanyloxy) -2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine (20) (20.0 mg, 0.036 mmol), THF 5 ml were added, and AcOH (11.0 mg , 0.183 mmol), and then dropwise added a THF solution of TBAF (48.0 mg, 0.183 mmol) dropwise and stirred for 2 h. . The obtained reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 9: 1), recrystallized from CHCl ₃ and Et ₂ O, and 9-hydroxy-2- (p-nitrophenoxycarbonyl). ) -1,2-dihydroellipticine (21) (11.7 mg, 0.027 mmol) was obtained (yield: 76%).
<Property data of 9-hydroxy-2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine>
decomp. 210 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 10.82 (1H, s, NH), 8.88 (1H, s, OH), 8.30 (2H, d, J = 8 Hz, p- NO ₂ Ph-H), 7.60, 7.55 (2H, d, J = 8 Hz, p-NO ₂ Ph-H), 7.52 (1H, s, H ₁₀ ), 7.29 (1H, d, J = 8.5 Hz, H ₇ ), 7.12 (1H, d, J = 8.5 Hz, H ₈ ), 6.86 (1H, d, J = 8 Hz, H ₃ ), 6.32 (1H, d, J = 8 Hz, H ₄ ), 5.17 , 4.98 (2H, s, H ₁ ), 2.65 (3H, s, 11-Me), 2.46 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 429 (M ⁺ ); HRMS calcd for M ⁺ (C ₂₄ H ₁₉ N ₃ O ₅ ) 429.1325, found 429.1332.

６）２−（２−アミノエチル）カルバモイル−９−ヒドロキシ−１，２−ジヒドロエリプチシン（2-(2-aminoethyl)carbamoyl-9-hydroxy-1,2-dihydroellipticine）（3a）
９−ヒドロキシ−２−（ｐ−ニトロフェノキシカルボニル）−１，２−ジヒドロエリプチシン（21） (10.0mg, 0.023mmol)、DMF 1mlを入れ、EDA (7.0mg, 0.116mmol)をゆっくり滴下し、3 h撹拌した。その後反応混合物をアミノシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 8 : 2)にて精製し２−（２−アミノエチル）アミド−９−ヒドロキシ−１，２−ジヒドロエリプチシン(3a) (5.6mg, 0.016mmol)を得た（収率: 70%）。
＜２−（２−アミノエチル）カルバモイル−９−ヒドロキシ−１，２−ジヒドロエリプチシンの物性データ＞
decomp. 192-194 ℃; ¹H-NMR (500 MHz, CD₃OD) δ 7.55 (1H, d, J = 2 Hz, H₁₀), 7.27 (1H, d, J = 9 Hz, H₇), 6.90 (1H, d, J = 8 Hz, H₃), 6.87 (1H, dd, J = 9,2 Hz, H₈), 6.13 (1H, d, J = 8 Hz, H₄), 4.90 (2H, s, H₁), 3.38 (3H, t, J = 6 Hz, NHCH₂), 2.86 (3H, t, J = 6 Hz, NH₂CH₂), 2.70 (3H, s, 11-Me), 2.43 (3H, s, 5-Me); MS (FAB⁺) m/z 351 (M+H); HRMS calcd for M+H (C₂₀H₂₃N₄O₂) 351.1821, found 351.1742. 6) 2- (2-aminoethyl) carbamoyl-9-hydroxy-1,2-dihydroellipticine (3a)
9-Hydroxy-2- (p-nitrophenoxycarbonyl) -1,2-dihydroellipticine (21) (10.0 mg, 0.023 mmol) and 1 ml of DMF were added, and EDA (7.0 mg, 0.116 mmol) was slowly added dropwise. And stirred for 3 h. Thereafter, the reaction mixture was purified by amino silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 8: 2) to give 2- (2-aminoethyl) amido-9-hydroxy-1,2-dihydroellipticine (3a ) (5.6 mg, 0.016 mmol) was obtained (yield: 70%).
<Property data of 2- (2-aminoethyl) carbamoyl-9-hydroxy-1,2-dihydroellipticine>
decomp. 192-194 ° C; ¹ H-NMR (500 MHz, CD ₃ OD) δ 7.55 (1H, d, J = 2 Hz, H ₁₀ ), 7.27 (1H, d, J = 9 Hz, H ₇ ), 6.90 (1H, d, J = 8 Hz, H ₃ ), 6.87 (1H, dd, J = 9,2 Hz, H ₈ ), 6.13 (1H, d, J = 8 Hz, H ₄ ), 4.90 (2H , s, H ₁ ), 3.38 (3H, t, J = 6 Hz, NHCH ₂ ), 2.86 (3H, t, J = 6 Hz, NH ₂ CH ₂ ), 2.70 (3H, s, 11-Me), 2.43 (3H, s, 5-Me); MS (FAB ⁺ ) m / z 351 (M + H); HRMS calcd for M + H (C ₂₀ H ₂₃ N ₄ O ₂ ) 351.1821, found 351.1742.

７）９−ヒドロキシ−２−（２−（Ｎ’−メチルウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシン（9-hydroxy-2-(2-(N'-methylureido)ethyl)carbamoyl-1,2-dihydroellipticine） (3b)
２−（２−アミノエチル）アミド−９−ヒドロキシ−１，２−ジヒドロエリプチシン(3a) (10.0mg, 0.028mmol)、DMF (1ml)を入れ、p-Nitrophenyl-N-methylnitrosocarbamate (7.0mg, 0.035mmol)のDMF溶液をゆっくり滴下し、室温で1 h撹拌した。その後反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 7 : 3)にて精製後、MeOH、Et₂Oより再結晶し、９−ヒドロキシ−２−（２−（Ｎ’−メチルウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシン(3b) (6.3mg, 0.015mmol)を得た（収率： 55%）。
＜９−ヒドロキシ−２−（２−（Ｎ’−メチルウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシンの物性データ＞
decomp. 165 ℃; ¹H-NMR (500 MHz, DMSO-d₆ ) δ 10.68 (1H, s, NH), 8.80 (1H, s, OH), 7.48 (1H, s, H₁₀), 7.26 (1H, d, J = 8.5 Hz, H₇), 7.00 (1H, d, J = 8 Hz, H₃), 6.82 (1H, d, J = 8.5 Hz, H₈), 6.08 (1H, s, NH), 5.93 (1H, d, J = 8 Hz, H₄), 5.81 (1H, s, NH), 4.84 (2H, s, H₁), 3.14 (4H, m, CH₂CH₂), 2.65 (3H, s, 11-Me), 2.55 (3H, s, 5-Me), 2.39 (3H, s, NHMe); MS (FAB⁺) m/z 407 (M⁺); HRMS calcd for M⁺ (C₂₂H₂₅N₃O₅) 407.1957, found 407.1951. 7) 9-hydroxy-2- (2- (N'-methylureido) ethyl) carbamoyl-1,2-dihydroellipticine (9-hydroxy-2- (2- (N'-methylureido) ethyl) carbamoyl- 1,2-dihydroellipticine) (3b)
2- (2-aminoethyl) amido-9-hydroxy-1,2-dihydroellipticine (3a) (10.0 mg, 0.028 mmol) and DMF (1 ml) were added, and p-Nitrophenyl-N-methylnitrosocarbamate (7.0 mg , 0.035 mmol) in DMF was slowly added dropwise and stirred at room temperature for 1 h. Thereafter, the reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 7: 3), recrystallized from MeOH and Et ₂ O, and 9-hydroxy-2- (2- (N′-methyl). Ureido) ethyl) carbamoyl-1,2-dihydroellipticine (3b) (6.3 mg, 0.015 mmol) was obtained (yield: 55%).
<Property data of 9-hydroxy-2- (2- (N′-methylureido) ethyl) carbamoyl-1,2-dihydroellipticine>
decomp. 165 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 10.68 (1H, s, NH), 8.80 (1H, s, OH), 7.48 (1H, s, H ₁₀ ), 7.26 (1H , d, J = 8.5 Hz, H ₇ ), 7.00 (1H, d, J = 8 Hz, H ₃ ), 6.82 (1H, d, J = 8.5 Hz, H ₈ ), 6.08 (1H, s, NH) , 5.93 (1H, d, J = 8 Hz, H ₄ ), 5.81 (1H, s, NH), 4.84 (2H, s, H ₁ ), 3.14 (4H, m, CH ₂ CH ₂ ), 2.65 (3H , s, 11-Me), 2.55 (3H, s, 5-Me), 2.39 (3H, s, NHMe); MS (FAB ⁺ ) m / z 407 (M ⁺ ); HRMS calcd for M ⁺ (C ₂₂ H ₂₅ N ₃ O ₅ ) 407.1957, found 407.1951.

８）９−ヒドロキシ−２−（２−（Ｎ’−メチル−Ｎ’−ニトロソウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシン（9-hydroxy-2-(2-(N'-methyl-N'-nitrosoureido)ethyl)carbamoyl-1,2-dihydroellipticine） (3c)
２−（２−アミノエチル）カルバモイル−９−ヒドロキシ−１，２−ジヒドロエリプチシン(3a) (10.0mg, 0.028mmol)、DMF 0.3mlを入れ、Succinimidyl-N-methylnitrsocarbamate (7.0mg, 0.034mmol)のDMF溶液をゆっくり滴下し、室温で1 h撹拌した。その後反応混合物をシリカゲルカラムクロマトグラフィー(展開溶媒/ CHCl₃ : MeOH = 10 : 1)にて精製後、H₂Oより再結晶し、９−ヒドロキシ−２−（２−（Ｎ’−メチル−Ｎ’−ニトロソウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシン(3c) (6.6mg, 0.015mmol)を得た（収率； 54 %）。
＜９−ヒドロキシ−２−（２−（Ｎ’−メチル−Ｎ’−ニトロソウレイド）エチル）カルバモイル−１，２−ジヒドロエリプチシンの物性データ＞
decomp. 200 ℃; ¹H-NMR (500 MHz, CD₃OD) δ 7.46 (1H, s, H₁₀), 7.18 (1H, d, J = 9 Hz, H₇), 6.78 (1H, d, J = 9 Hz, H₃), 6.76 (1H, d, J =9 Hz, H₈), 6.03 (1H, d, J =9 Hz, H₄), 4.80 (2H, s, H₁), 3.48 (2H, t, J = 5.5 Hz, CH₂CH₂), 3.41 (2H, t, J = 5.5 Hz, CH₂CH₂), 3.03 (3H, s, 11-Me), 2.60 (3H, s, 5-Me), 2.34 (3H, s, NHMe); MS (FAB⁺) m/z 436 (M⁺); HRMS calcd for M⁺ (C₂₂H₂₄N₆O₄) 436.1859, found 436.1867. 8) 9-hydroxy-2- (2- (N′-methyl-N′-nitrosoleido) ethyl) carbamoyl-1,2-dihydroellipticine (9-hydroxy-2- (2- (N′-methyl) -N'-nitrosoureido) ethyl) carbamoyl-1,2-dihydroellipticine) (3c)
2- (2-aminoethyl) carbamoyl-9-hydroxy-1,2-dihydroellipticine (3a) (10.0 mg, 0.028 mmol) and DMF 0.3 ml were added, and Succinimidyl-N-methylnitrsocarbamate (7.0 mg, 0.034 mmol). ) Was slowly added dropwise and stirred at room temperature for 1 h. Thereafter, the reaction mixture was purified by silica gel column chromatography (developing solvent / CHCl ₃ : MeOH = 10: 1), recrystallized from H ₂ O, and 9-hydroxy-2- (2- (N′-methyl-N '-Nitrosoureido) ethyl) carbamoyl-1,2-dihydroellipticine (3c) (6.6 mg, 0.015 mmol) was obtained (yield; 54%).
<Property data of 9-hydroxy-2- (2- (N′-methyl-N′-nitrosoleido) ethyl) carbamoyl-1,2-dihydroellipticine>
decomp. 200 ° C; ¹ H-NMR (500 MHz, CD ₃ OD) δ 7.46 (1H, s, H ₁₀ ), 7.18 (1H, d, J = 9 Hz, H ₇ ), 6.78 (1H, d, J = 9 Hz, H ₃ ), 6.76 (1H, d, J = 9 Hz, H ₈ ), 6.03 (1H, d, J = 9 Hz, H ₄ ), 4.80 (2H, s, H ₁ ), 3.48 ( 2H, t, J = 5.5 Hz, CH ₂ CH ₂ ), 3.41 (2H, t, J = 5.5 Hz, CH ₂ CH ₂ ), 3.03 (3H, s, 11-Me), 2.60 (3H, s, 5 -Me), 2.34 (3H, s, NHMe); MS (FAB ⁺ ) m / z 436 (M ⁺ ); HRMS calcd for M ⁺ (C ₂₂ H ₂₄ N ₆ O ₄ ) 436.1859, found 436.1867.

〔実施例３〕
細胞毒性試験
Sarcoma-180（マウス肉腫細胞）、HelaＳ-3（ヒト子宮頸癌細胞）、L1210（マウス白血病細胞）がそれぞれ培養されている培養容器から、培地を除去後、PBS (-) で細胞を洗浄し、Trypsin-EDTAで細胞を剥離した。次いで、培地を加えてTrypsinの作用を止め、遠心分離した。上清を除去後、新しい培地を加えてから、色素排除法により細胞数をカウントした。細胞濃度を調製 ( sarcoma-180, HeLa S-3 : 2.0 x 10⁴cells / ml, L1210 : 4.0 x 10⁴cells / ml) し、37℃のCO₂インキュベーター内で3時間インキュベートした。次いで、以下の表２に示す抗腫瘍剤 (処理濃度 : 12.5 ~ 100μg/ml)を各々添加し、37℃のCO₂インキュベーター内で72時間インキュベートした。細胞死の定量にはMTT法を用いた。
この後、培養液中にMTT(0.2μg/μl)を加え、さらに、37℃のCO₂インキュベーター内で4時間インキュベートした。反応を停止させる為にDMSOを培養液と等量加えた。マイクロプレートリーダー (580nmのfilter使用) でMTT formazanの生成量を測定することにより抗腫瘍剤毎に各細胞の生存率を求め、IC₅₀を算出した。
本発明のエリプチシン誘導体（化合物Ｎｏ．１〜５，２３〜２５）、エリプチシン、及び他のエリプチシン誘導体のSarcoma-180（マウス肉腫細胞）、HelaＳ-3（ヒト子宮頸癌細胞）、L1210（マウス白血病細胞）に対するＩＣ_５０を以下の表１に示す。 Example 3
Cytotoxicity test
Remove the medium from the culture vessels in which Sarcoma-180 (mouse sarcoma cells), HelaS-3 (human cervical cancer cells), and L1210 (mouse leukemia cells) are cultured, and then wash the cells with PBS (-). The cells were detached with Trypsin-EDTA. Then, the medium was added to stop the action of Trypsin and centrifuged. After removing the supernatant, a new medium was added, and the number of cells was counted by the dye exclusion method. The cell concentration was adjusted (sarcoma-180, HeLa S-3: 2.0 × 10 ⁴ cells / ml, L1210: 4.0 × 10 ⁴ cells / ml) and incubated in a CO ₂ incubator at 37 ° C. for 3 hours. Subsequently, each of the antitumor agents shown in Table 2 below (treatment concentration: 12.5 to 100 μg / ml) was added and incubated in a CO ₂ incubator at 37 ° C. for 72 hours. MTT method was used for quantification of cell death.
Thereafter, MTT (0.2 μg / μl) was added to the culture solution, and further incubated in a CO ₂ incubator at 37 ° C. for 4 hours. In order to stop the reaction, DMSO was added in an amount equivalent to the culture solution. The viability of each cell was determined for each antitumor agent by measuring the amount of MTT formazan produced with a microplate reader (using a 580 nm filter), and the IC ₅₀ was calculated.
Ellipticin derivatives of the present invention (Compound Nos. 1-5, 23-25), ellipticine, and other ellipticin derivatives Sarcoma-180 (mouse sarcoma cells), HelaS-3 (human cervical cancer cells), L1210 (mouse leukemia) IC ₅₀ for cells) is shown in Table 1 below.

これによれば、本発明の一般式（１）あるいは（２）の化合物は、いずれも腫瘍細胞に対して良好な活性を有することが明らかである。

According to this, it is clear that the compound of the general formula (1) or (2) of the present invention has a good activity against tumor cells.

〔実施例４〕
蛍光顕微鏡による観察
Hela Ｓ-3（ヒト子宮頸癌細胞）が培養されている培養容器から、培地を除去後、PBS (-) で細胞を洗浄し、Trypsin-EDTAで細胞を剥離した。培地を加えてTrypsinの作用を止め、遠心分離した。上清を除去後、新しい培地を加えてから、色素排除法により細胞数をカウントした。細胞濃度を調製 ( 1.5x 10⁶cells / ml) 後、上記表中Ｎｏ．３の化合物及び同Ｎｏ．１の化合物及び比較として同Ｎｏ．１９の化合物剤をそれぞれ最終濃度150μMになるように添加し、37℃のCO₂インキュベーター内で24時間インキュベートした。蛍光顕微鏡U領域で観察した。結果を図４に示す。なお、図中、上段は透過光を撮影したものであり、下段は蛍光を撮影したものである。これによれば、ＩＣ_５０が小さい値を示すものほど、核内蛍光強度が強く、これは、使用化合物の膜透過性の違いが活性発現に大きく貢献していることを示す。また、５位の置換基は長いと膜透過性を阻害する。 Example 4
Observation with a fluorescence microscope
After removing the medium from the culture vessel in which Hela S-3 (human cervical cancer cells) was cultured, the cells were washed with PBS (−) and detached with Trypsin-EDTA. The medium was added to stop the action of Trypsin and centrifuged. After removing the supernatant, a new medium was added, and the number of cells was counted by the dye exclusion method. After preparing the cell concentration (1.5 × 10 ⁶ cells / ml), No. in the above table was used. 3 and No. 3 No. 1 and the same No. 1 as a comparison. Nineteen compound agents were added to a final concentration of 150 μM and incubated in a CO ₂ incubator at 37 ° C. for 24 hours. Observation was performed in the U region of the fluorescence microscope. The results are shown in FIG. In the figure, the upper part is a photograph of transmitted light, and the lower part is a photograph of fluorescence. According to this, the smaller the IC ₅₀ value, the stronger the fluorescence intensity in the nucleus, which indicates that the difference in membrane permeability of the compound used greatly contributes to the expression of activity. Further, if the substituent at the 5-position is long, the membrane permeability is inhibited.

本発明の一般式（１）の化合物の合成過程の概略を示す図である。It is a figure which shows the outline of the synthetic | combination process of the compound of General formula (1) of this invention. 本発明の一般式（２）の化合物の合成過程の概略を示す図である。It is a figure which shows the outline of the synthetic | combination process of the compound of General formula (2) of this invention. 本発明の一般式（３）の化合物の合成過程の概略を示す図である。It is a figure which shows the outline of the synthetic | combination process of the compound of General formula (3) of this invention. 実施例４における化合物添加後のHelaＳ-3を蛍光顕微鏡で撮影した写真である。It is the photograph which image | photographed HelaS-3 after the compound addition in Example 4 with the fluorescence microscope.

Claims

The antitumor agent which contains any 1 or more types among the compounds represented by the following general formula (1)-(3) as an active ingredient.

The compound represented by the following general formula (1).

The compound represented by the following general formula (2).

The compound represented by the following general formula (3).