JP4138773B2 - Novel ferroceneated polycyclic hydrocarbon derivative, method for producing the same, and method for detecting target nucleic acid - Google Patents

Description

  The present invention relates to a novel compound useful as an intercalator for a double-stranded nucleic acid.

  In order to detect a nucleic acid such as DNA or RNA having a specific base sequence in biological or medical research or clinical settings, there is a method using a nucleic acid having a base sequence complementary to the base sequence as a probe. It is used. Such a method is based on the specificity that a double strand is formed by hydrogen bonding only when two nucleic acids have base sequences complementary to each other. When such a method is used, a means for confirming whether or not the target nucleic acid and the probe nucleic acid have hybridized is required for detection of the target nucleic acid.

  As a means for confirming the presence or absence of hybridization, a method of labeling a target nucleic acid with a fluorescent substance or the like is well known. For example, in order to analyze gene expression in a cell, the RNA contained in the cell is fluorescently labeled and hybridized with a probe cDNA immobilized on a solid phase carrier under appropriate conditions, and then on the solid phase carrier. Methods for detecting fluorescence are widely used. In the method using a fluorescent substance, it is necessary to chemically bind the fluorescent substance to the target nucleic acid in advance. Although the presence or absence of hybridization can be detected, the formation of double strands cannot be quantitatively measured.

  In order to solve such a problem, a method using an intercalator that selectively binds to a double-stranded nucleic acid has been proposed. An intercalator refers to a molecule having a property of intercalating so as to be sewn between nucleic acid bases, and examples thereof include dyes such as ethidium bromide. Since the intercalator is regularly sewn into the double-stranded nucleic acid, a correlation is obtained between the amount of intercalator that binds to the amount of double-stranded strands, and hybridization can be measured quantitatively. Further, the intercalator may be added after hybridization, and it is not necessary to bind to the target nucleic acid in advance.

や、下記式（９）

で表されるフェロセン化ナフタレンジイミド誘導体等が利用されている。
特開平９−２８８０８０号公報 ＷＯ０２／０５３５７１ There has also been proposed a method of electrochemically detecting an intercalator bound to a double-stranded nucleic acid by modifying the intercalator with conductive ferrocene or the like (see, for example, Patent Documents 1 and 2). According to the electrochemical method, it is possible to easily perform more accurate quantitative measurement in real time without fading like a fluorescent dye. As such an intercalator, the following formula (8)

Or the following formula (9)

The ferrocene naphthalene diimide derivative etc. which are represented by these are utilized.
JP-A-9-288080 WO02 / 053571

  The piperazine ring-containing ferrocene-naphthalenediimide derivative represented by the above formula (8) that has been used as an intercalator until now has a complicated separation operation and purification operation after the synthesis reaction, and the yield is also around 10%. And low. In addition, double-stranded selectivity is insufficient, and there is a tendency that a sufficient difference is not easily obtained from the amount of binding to a single-stranded nucleic acid due to electrostatic interaction. In addition, the double-stranded nucleic acid-intercalator complex is also unstable and the dissociation reaction proceeds rapidly.

  Moreover, although the yield of the ferrocene naphthalene diimide derivative represented by the above formula (9) is relatively high, an intercalator excellent in stability and double-stranded nucleic acid selectivity is required.

  Therefore, the object of the present invention is to provide an interface that forms a complex complex with a double-stranded nucleic acid that is easy to separate and purify after the synthesis reaction, is obtained in a high yield, has a high double-stranded nucleic acid selectivity, and the like. The object is to provide a compound useful as a calator.

（式中、Ａは多環式炭化水素若しくはその誘導体を示し；Ｘは置換基を有していてもよ
い炭素数１〜６のアルキレン基、−（ＣＨ₂）x−Ｂ−（ＣＨ₂）y−［ここでＢは窒素原子を１若しくは２個含む５員若しくは６員脂肪族複素環を示し；ｘおよびｙはそれぞれ独立に０〜３の整数を示す］、または−（ＣＨ₂）_a−（ＮＲ⁵）−（ＣＨ₂）_b−（ＮＲ⁶）−（ＣＨ₂）_c−［ここでａ、ｂおよびｃはそれぞれ独立に０〜３の整数を示し、Ｒ⁵およびＲ⁶はそれぞれ独立に水素またはメチル基を示す］のいずれかを示し；Ｒ¹は水素、炭素数１〜３のアルキル基、アルケニル基若しくはアルキニル基、炭素数７〜９のアリールアルキル基若しくはアリールアルケニル基、または炭素数２若しくは３のアルキルカルボニル基を示し；Ｒ²およびＲ³はそれぞれ独立に水溶液中で電子供与基または電子求引基となる基を示し；ｍおよびｎはそれぞれ独立に０〜２の整数を示す。１分子中の二つの同一表示基は互いに同一でも異なっていてもよい。）、および下記式（３）

（式中、Ａは多環式炭化水素若しくはその誘導体を示し；Ｙは窒素原子を１または２個含む５員または６員脂肪族複素環であって、カルボニル基に結合する基が窒素原子である環を示し；Ｒ⁴は炭素数１〜６のアルキレン基を示し；Ｒ²およびＲ³はそれぞれ独立に水溶液中で電子供与基または電子求引基となる基を示し；ｍおよびｎはそれぞれ独立に０〜２の整数を示す。１分子中の二つの同一表示基は互いに同一でも異なっていてもよい。）で表される化合物またはその塩若しくはその水和物、中でも特に、Ａがナフタレンジイミド基である化合物は、二本鎖に対する結合定数および選択性が高く、電流応答の再現性も良く、合成時に高収率で得られることを見出し、本発明を完成した。 In view of the above problems, the present inventors have intensively studied for an excellent intercalator, and as a result, the following formula (1)

(In the formula, A represents a polycyclic hydrocarbon or a derivative thereof; X represents an optionally substituted alkylene group having 1 to 6 carbon atoms, — (CH ₂ ) x —B— (CH ₂ ). y—where B represents a 5- or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms; x and y each independently represents an integer of 0 to 3, or — (CH ₂ ) _a — (NR ⁵ ) — (CH ₂ ) _b — (NR ⁶ ) — (CH ₂ ) _c — wherein a, b and c each independently represent an integer of 0 to 3, and R ⁵ and R ⁶ are each Independently represents hydrogen or a methyl group]; R ¹ represents hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkenyl group or an alkynyl group, an arylalkyl group or arylalkenyl group having 7 to 9 carbon atoms, or Represents an alkylcarbonyl group having 2 or 3 carbon atoms; R ² and R ^{2 3} independently represents a group which becomes an electron donating group or an electron withdrawing group in an aqueous solution; and m and n each independently represent an integer of 0 to 2. Two identical display groups in one molecule may be the same as each other. May be different), and the following formula (3)

Wherein A represents a polycyclic hydrocarbon or a derivative thereof; Y represents a 5-membered or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms, and the group bonded to the carbonyl group is a nitrogen atom. R ⁴ represents an alkylene group having 1 to 6 carbon atoms; R ² and R ³ each independently represents a group that becomes an electron donating group or an electron withdrawing group in an aqueous solution; Independently represents an integer of 0 to 2. Two identical display groups in one molecule may be the same or different from each other.) Or a salt or hydrate thereof, in particular, A is naphthalene. It has been found that a compound that is a diimide group has a high binding constant and selectivity for double strands, good reproducibility of current response, and can be obtained in a high yield at the time of synthesis.

（式中、Ａは多環式炭化水素若しくはその誘導体を示し；Ｘは置換基を有していてもよ
い炭素数１〜６のアルキレン基、または窒素原子を１または２個含む５員または６員脂肪族複素環を間に挟む２つの炭素数１〜３のアルキレン基を示し；Ｒ¹は水素、炭素数１〜３のアルキル基、アルケニル基若しくはアルキニル基、炭素数７〜９のアリールアルキル基若しくはアリールアルケニル基、または炭素数２若しくは３のアルキルカルボニル基を示し；Ｒ²およびＲ³はそれぞれ独立に水溶液中で電子供与基または電子求引基となる基を示し；ｍおよびｎはそれぞれ独立に０〜２の整数を示す。１分子中の二つの同一表示基は互いに同一でも異なっていてもよい。）で表される化合物またはその塩若しくはその水和物；
［２］Ｘが下記式（２）

である、前記［１］に記載の化合物またはその塩若しくはその水和物；
［３］Ｘが、−Ｃ₃Ｈ₆Ｎ（ＣＨ₃）Ｃ₃Ｈ₆−である、前記［１］に記載の化合物またはその塩若しくはその水和物；
［４］下記式（３）

（式中、Ａは多環式炭化水素若しくはその誘導体を示し；Ｙは窒素原子を１または２個含む５員または６員脂肪族複素環であって、カルボニル基に結合する基が窒素原子である環を示し；Ｒ⁴は炭素数１〜６のアルキレン基を示し；Ｒ²およびＲ³はそれぞれ独立に水溶液中で電子供与基または電子求引基となる基を示し；ｍおよびｎはそれぞれ独立に０〜２の整数を示す。１分子中の二つの同一表示基は互いに同一でも異なっていてもよい。）で表される化合物またはその塩若しくはその水和物；
［５］Ｙがピペリジル基またはピペラジル基である前記［４］に記載の化合物またはその塩若しくはその水和物；
［６］Ｒ⁴がメチレン基であり、Ｙがピペリジル基であってその窒素原子がカルボニル基の炭素原子に結合している、前記［４］に記載の化合物またはその塩若しくはその水和物；
［７］Ａがナフタレンジイミド基である前記［１］から［６］のいずれか１項に記載の化合物またはその塩若しくはその水和物；
［８］ｍ＝ｎ＝０である前記［７］に記載の化合物またはその塩若しくはその水和物；
［９］Ａがナフタレンジイミド基である請求項１に記載の化合物またはその塩若しくはその水和物を製造する方法であって、下記反応式（Ｉ）に示すように、１，４，５，８−ナフタレンテトラカルボン酸二無水物と式（４）で表される化合物とを反応させて、式（５）で表される化合物を得る第一工程と、下記反応式（ＩＩ）に示すように、式（５）で表される化合物とフェロセンカルボン酸スクシンイミドエステルとを反応させて、目的物を得る第二工程と、を含む製造方法；

［１０］Ａがナフタレンジイミド基である前記［３］に記載の化合物またはその塩若しくはその水和物を製造する方法であって、下記反応式（ＩＩＩ）に示すように、１，４，５，８−ナフタレンテトラカルボン酸二無水物と式（６）で表される化合物とを反応させて、式（７）で表される化合物を得る第一工程と、下記反応式（ＩＶ）に示すように、式（７）で表される化合物とフェロセンカルボン酸スクシンイミドエステルとを反応させて、目的物を得る第二工程と、を含む製造方法；

［１１］標的核酸と相補的な塩基配列を有するプローブ核酸と、被検試料とをハイブリダイゼーションが起こる条件下で接触させる工程と、
前記［１］から［８］のいずれか１項に記載の化合物またはその塩若しくはその水和物を添加する工程と、
二本鎖核酸に結合した前記化合物の電気化学応答を測定する工程と、
を含む、被検試料中の標的核酸を検出する方法；
［１２］標的核酸と相補的な塩基配列を有するプローブ核酸が固定された電極と、前記［１］から［８］のいずれか１項に記載の化合物またはその塩若しくはその水和物とを含む、標的核酸検出用キット、に関する。 That is, the present invention

[1] The following formula (1)

(In the formula, A represents a polycyclic hydrocarbon or a derivative thereof; X represents an optionally substituted alkylene group having 1 to 6 carbon atoms, or 5-membered or 6 containing 1 or 2 nitrogen atoms.) R ¹ represents hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkenyl group or an alkynyl group, and an arylalkyl having 7 to 9 carbon atoms. A group or an arylalkenyl group, or an alkylcarbonyl group having 2 or 3 carbon atoms; R ² and R ³ each independently represent a group which becomes an electron donating group or an electron withdrawing group in an aqueous solution; Each independently represents an integer of 0 to 2. Two identical display groups in one molecule may be the same or different from each other) or a salt or hydrate thereof;
[2] X is the following formula (2)

Or a salt or hydrate thereof according to the above [1];
[3] X _{is, -C 3 H 6 N (CH} 3) C 3 H 6 - is a compound or a salt or a hydrate thereof according to [1];
[4] The following formula (3)

Wherein A represents a polycyclic hydrocarbon or a derivative thereof; Y represents a 5-membered or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms, and the group bonded to the carbonyl group is a nitrogen atom. R ⁴ represents an alkylene group having 1 to 6 carbon atoms; R ² and R ³ each independently represents a group that becomes an electron donating group or an electron withdrawing group in an aqueous solution; Each independently represents an integer of 0 to 2. Two identical display groups in one molecule may be the same or different from each other) or a salt or hydrate thereof;
[5] The compound according to the above [4], wherein Y is a piperidyl group or a piperazyl group, or a salt or hydrate thereof;
[6] The compound according to [4], a salt thereof or a hydrate thereof, wherein R ⁴ is a methylene group, Y is a piperidyl group, and the nitrogen atom is bonded to the carbon atom of the carbonyl group;
[7] The compound according to any one of [1] to [6], wherein A is a naphthalenediimide group, or a salt or hydrate thereof;
[8] The compound according to [7], wherein m = n = 0, or a salt or hydrate thereof;
[9] A method for producing a compound according to claim 1, wherein A is a naphthalenediimide group, or a salt or hydrate thereof, as shown in the following reaction formula (I): 1, 4, 5, As shown in the following reaction formula (II), a first step of reacting 8-naphthalenetetracarboxylic dianhydride with a compound represented by formula (4) to obtain a compound represented by formula (5) And a second step of reacting the compound represented by formula (5) with ferrocenecarboxylic acid succinimide ester to obtain a target product;

[10] A process for producing the compound according to [3] above, wherein A is a naphthalenediimide group, or a salt or hydrate thereof, as shown in the following reaction formula (III): 1, 4, 5 , 8-Naphthalenetetracarboxylic dianhydride and the compound represented by the formula (6) are reacted to obtain a compound represented by the formula (7), and shown in the following reaction formula (IV) And a second step of reacting the compound represented by formula (7) with ferrocenecarboxylic acid succinimide ester to obtain a target product;

[11] contacting a probe nucleic acid having a base sequence complementary to the target nucleic acid with a test sample under conditions where hybridization occurs;
Adding the compound according to any one of [1] to [8] above or a salt or hydrate thereof;
Measuring the electrochemical response of the compound bound to a double-stranded nucleic acid;
A method for detecting a target nucleic acid in a test sample, comprising:
[12] An electrode to which a probe nucleic acid having a base sequence complementary to a target nucleic acid is immobilized, and the compound according to any one of [1] to [8] above, a salt thereof, or a hydrate thereof And a target nucleic acid detection kit.

  According to the present invention, a novel ferrocene polycyclic hydrocarbon derivative that is easy to separate and purify after the synthesis reaction, is obtained in high yield, and is useful as an intercalator having high selectivity for double-stranded nucleic acid, and Novel ferroceneated naphthalenediimide derivatives are provided. Since the ferrocened polycyclic hydrocarbon derivative and the ferrocened naphthalene diimide derivative according to the present invention form a stable complex with the double-stranded nucleic acid after intercalation with the double-stranded nucleic acid, it is detected. If easy.

  If the novel compound according to the present invention is used, nucleic acids such as DNA and RNA having a specific sequence can be detected in real time with high sensitivity and high throughput.

  Hereinafter, although the compound concerning this invention is demonstrated in detail based on the preferable embodiment, these do not limit the scope of the present invention at all.

で表される新規なフェロセン化ナフタレンジイミド誘導体（化合物（１’））を提供する。かかるフェロセン化ナフタレンジイミドは、特に、二本鎖核酸に選択的に結合するインターカレータとして有用なものである。 As an embodiment of the compound according to the present invention, the following formula (1 ′)

A novel ferroceneated naphthalenediimide derivative (compound (1 ′)) represented by the formula: Such ferrocene naphthalenediimide is particularly useful as an intercalator that selectively binds to a double-stranded nucleic acid.

  As the “salt” of the compound (1 ′) in the present invention, a salt with an inorganic base, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid Although not particularly limited, hydrochloride is most preferable.

  The compound (1 ') according to the present invention may be a hydrate or non-hydrate.

As shown in the formula (1 ′), the compound of the present invention has a naphthalenediimide part, a ferrocene part, and a linker part that connects both parts, “—X—NR ¹ —CO—C ₂ H ₄ —”. It consists of and. The naphthalene diimide portion functions as a stitched intercalator portion having an aromatic plate-like molecular structure inserted in parallel between adjacent base pairs such as double strands such as DNA. On the other hand, since the ferrocene part has redox activity, its presence can be detected electrochemically. Thereby, compound 1 'can function as an electrochemically active stitched intercalator. In the compound of the present invention, since the linker moiety has the structure shown in the formula (1 ′), the separation and purification operations after the synthesis reaction are easy, the yield is high, and the double-stranded selectivity is particularly high. It is a high intercalator.

であるもの、およびＸが−Ｃ₃Ｈ₆Ｎ（ＣＨ₃）Ｃ₃Ｈ₆−であるものは、インターカレータとして用いた場合に、二本鎖核酸への会合反応が速く進む一方で解離反応が抑制され、安定した二本鎖核酸−インターカレータ複合体を形成することができる。 In the formula (1 ′), the following formula (2) in which X contains a piperazyl group

In a one, and X is _{-C 3 H 6 N (CH 3} ) C 3 H 6 - and is intended, when used as intercalator, dissociation While association reaction to double-stranded nucleic acid proceeds faster Is suppressed, and a stable double-stranded nucleic acid-intercalator complex can be formed.

In the formula (1 ′), when m and n are 1 or 2 (not 0), the group that becomes an electron donating group or an electron withdrawing group in the aqueous solution represented by R ² and R ³ is as follows: Methyl group, methoxy group, methoxycarbonyl group, nitro group, carboxyl group, dimethylaminomethyl group, dimethylaminoethylaminocarbonyl group and the like. For example, in the case of a dimethylaminomethyl group, it is protonated in an aqueous solution and functions as a dimethylammoniummethyl group which is an electron withdrawing group. In particular, when the compound of the present invention is used as an intercalator in an aqueous solution, a compound containing an unsubstituted ferrocenyl group is also preferable, but as R ² and R ³ , an electron donating group or an electron withdrawing group in the above aqueous solution. A compound containing a group is preferable because the potential at the time of measuring the amount of current is changed by an electron donating group or an electron withdrawing group in an aqueous solution and its utility value is high.

In the formula (1 ′), examples of the alkyl group represented by R ¹ include a methyl group, an ethyl group, and a propyl group. Examples of the alkenyl group include a 2-propenyl group. As the alkynyl group, Include an ethynyl group and a 2-propynyl group; examples of the arylalkyl group include a benzyl group and a methylbenzyl group; examples of the arylalkenyl group include a 3-phenyl-2-propenyl group; Examples of the alkylcarbonyl group include an acetyl group. In particular, when the compound of the present invention is used as an intercalator, R ¹ is a group that is not sterically bulky, for example, a hydrogen, a methyl group, or the like in that it can avoid an obstacle during binding to a nucleic acid. Is preferred.

および、下記式（１０’）

で表される化合物は、高収率で得られ、二本鎖核酸選択性が高く、核酸と安定な複合体を形成するインターカレータであり、特に好適である。 In particular, among the compounds of the present invention, X represents the above formula (2), R ¹ is hydrogen, and m = n = 0 (without substituents R ² and R ³ and unsubstituted; ² and R ³ are hydrogen atoms), that is, the following formula (10)

And the following formula (10 ′)

The compound represented by is an intercalator that is obtained in high yield, has high selectivity for double-stranded nucleic acid, and forms a stable complex with nucleic acid, and is particularly suitable.

ここで、上記反応式（Ｉ）に係る反応においては、例えばテトラヒドロフラン（ＴＨＦ）等の溶媒中で反応物を加熱還流し、その後、生成物を再結晶等により分離・精製する等通常の合成法で行われる操作を必要に応じて行う。 The production method of the compound (1 ′) according to the present invention is not particularly limited. However, as shown in the following reaction formula (I), 1,4,5,8-naphthalenetetracarboxylic dianhydride A first step of obtaining a compound represented by the formula (5) by reacting the product and the compound represented by the formula (4), and the formula (5) as shown in the following reaction formula (II): The production method including the second step of obtaining the desired product by reacting the compound obtained with ferrocenecarboxylic acid succinimide ester is preferable in terms of yield.

Here, in the reaction according to the above reaction formula (I), the reaction product is heated and refluxed in a solvent such as tetrahydrofuran (THF), and then the product is separated and purified by recrystallization or the like. If necessary, perform the operations performed in.

  In the reaction according to the above reaction formula (II), for example, the compound (5) is first stirred in a solvent such as trifluoroacetic acid (TFA) and the amino group protected with a butoxycarbonyl group (Boc) is deprotected. Subsequently, the reaction is stirred in a solvent such as chloroform or triethylamine, and an operation performed by a usual synthesis method, such as separation and purification by removing the solvent under reduced pressure or recrystallization, is performed as necessary.

で表される新規なフェロセン化ナフタレンジイミド誘導体（化合物（３’））を提供する。かかるフェロセン化ナフタレンジイミドは、特に、二本鎖核酸に選択的に結合するインターカレータとして有用なものである。 Moreover, the compound which concerns on this invention is the following formula (3 ') as one Embodiment.

A novel ferroceneated naphthalenediimide derivative represented by (Compound (3 ′)) is provided. Such ferrocene naphthalenediimide is particularly useful as an intercalator that selectively binds to a double-stranded nucleic acid.

  As the “salt” of the compound (3 ′) in the present invention, a salt with an inorganic base, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid Although not particularly limited, hydrochloride is most preferable. In addition, the compound (3 ′) according to the present invention may be a hydrate or a non-hydrate.

As shown in the formula (3 ′), the compound of the present invention has a naphthalenediimide moiety, a ferrocene moiety, and a linker moiety that connects both moieties “—R ⁴ —Y—CO—C ₂ H ₄ —”. It consists of and. The naphthalene diimide portion functions as a stitched intercalator portion having an aromatic plate-like molecular structure inserted in parallel between adjacent base pairs such as double strands such as DNA. On the other hand, since the ferrocene part has redox activity, its presence can be detected electrochemically. Thereby, the compound (3 ′) can function as an electrochemically active threaded intercalator. In the compound of the present invention, since the linker moiety has the structure shown in the formula (3 ′), separation operation and purification operation after the synthesis reaction are easy, the yield is high, and the duplex selectivity is high. It is an intercalator.

  In the formula (3 ′), when Y is a 5-membered or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms, when used as an intercalator, the association reaction with a double-stranded nucleic acid proceeds rapidly. On the other hand, the dissociation reaction is suppressed, and a stable double-stranded nucleic acid-intercalator complex can be formed. In this respect, Y is particularly preferably a piperidyl group or a piperazyl group.

In the formula (3 ′), R ⁴ is a methylene group, Y is a piperidyl group, and the nitrogen atom is bonded to the carbon atom of the carbonyl group. It is particularly excellent in that it forms a stable complex with a single-stranded nucleic acid.

In the formula (3 ′), the definitions of m, n, R ² and R ³ are the same as in the compound (1 ′).

ここで、上記反応式（ＩＩＩ）および（ＩＶ）に示される反応は、上記反応式（Ｉ）および（ＩＩ）にそれぞれ準じた操作によって行なうことができる。 The production method of the compound (3 ′) is not particularly limited, but as shown in the following production method, ie, the following reaction formula (III), 1,4,5,8-naphthalenetetracarboxylic dianhydride and the formula ( A first step of reacting a compound represented by 6) to obtain a compound represented by formula (7), and a compound represented by formula (7) as shown in the following reaction formula (IV): A production method including a second step of reacting with ferrocenecarboxylic acid succinimide ester to obtain a target product is preferable in terms of yield.

Here, the reactions shown in the above reaction formulas (III) and (IV) can be carried out by operations according to the above reaction formulas (I) and (II), respectively.

  The compounds (1 ′) and (3 ′) according to the present invention are in comparison with the conventionally known ferrocene naphthalene diimide derivatives, in particular, compared with the piperazine ring-containing ferrocene naphthalene diimide derivatives that have been conventionally used as intercalators. The separation operation and the purification operation after the synthesis reaction are easy and can be obtained in a high yield.

  The use of the compound of the present invention is not particularly limited and can be applied to various uses, but as described above, it is excellent as an intercalator that regularly inserts and binds to a double-stranded nucleic acid.

  The function of the compound according to the present invention as an intercalator for the double-stranded nucleic acid is, for example, the change in the UV-visible absorption spectrum accompanying the concentration change of the double-stranded nucleic acid in the mixture of the compound of the present invention and the double-stranded nucleic acid. It can be evaluated by measuring. In general, when an intercalator is inserted and bonded to a double-stranded nucleic acid, a light color effect of small long wavelength shift and maximum absorption is observed in the UV-visible absorption spectrum. Based on the binding rate calculated from the change in the spectrum, the binding constant and the number of loci can be calculated by scatchard plot (Scatchard Plot), whereby the strength of the binding ability can be quantified and evaluated.

  Here, the double-stranded nucleic acid refers to a state in which two single-stranded nucleic acids having complementary base sequences form a relatively stable double strand by hydrogen bonding between bases. Examples of the nucleic acid include, but are not limited to, DNA and RNA. The nucleic acid may be extracted from cells or artificially synthesized.

  When the compound according to the present invention is used as an intercalator, a target nucleic acid having a specific base sequence can be detected. Such detection includes, for example, a step of bringing a probe nucleic acid having a base sequence complementary to a target nucleic acid into contact with a test sample under conditions where hybridization occurs, a step of adding a compound according to the present invention, and two steps. Measuring the electrochemical response of the compound bound to the strand nucleic acid. In addition, by immobilizing a probe nucleic acid having a base sequence complementary to the target nucleic acid to the electrode, the amount of the compound according to the present invention inserted and bound to the double-stranded nucleic acid is electrochemically determined by cyclic voltammetry or differential pulse voltammetry. Can be measured automatically. This measurement method makes it possible to quantitatively measure the formation of double strands, and can be used to quantitatively measure the target nucleic acid in a test sample in real time with high sensitivity and high throughput. .

  The present invention also provides a target nucleic acid detection kit comprising an electrode on which a probe nucleic acid having a base sequence complementary to the target nucleic acid is immobilized, and the compound according to the present invention. By forming the electrode in a chip shape that can be directly measured by voltammetry, the target nucleic acid can be detected more easily and with high throughput.

  The target nucleic acid detection method using the compound of the present invention can be applied to gene expression analysis, detection of whether a specific gene is present in the genome, detection of mutations and polymorphisms, detection of viruses and bacteria. It is widely used in fields such as biology, medicine, and forensic medicine.

  As mentioned above, although preferable embodiment of this invention was explained in full detail, this invention is not limited to this embodiment, A various change form is employable. Specifically, N, N′-disubstituted naphthalenediimide in the formula (1 ′) [corresponding to A in the formula (1)] is replaced with other various polycyclic hydrocarbons or derivatives thereof such as aromatics. Compounds having a plurality of rings such as genus hydrocarbons, aliphatic heterocycles, aromatic heterocycles, alicyclic hydrocarbons or the like, specifically 1,5-, 2,6-, 9,10-di It can be changed to substituted anthracene, 1,5-, 2,6-disubstituted anthraquinone, 1,5-, 2,6-, 4,9-disubstituted acridine derivatives and the like.

このフェロセン化アントラセン誘導体は、例えば、下記に示す合成例のように、フェロセンプロピオン酸スクシンイミドエステルを用いて得ることができる。

前記フェロセン化アントラセン誘導体も、前述した前記式（１’）および式（３’）で表されるフェロセン化ナフタレンジイミド誘導体と同様に、合成反応後の分離操作および精製操作等が容易で、しかも単離・精製収率が３０％と高く、特にインターカレータとして有用な化合物として提供できる。尚、このフェロセン化アントラセン誘導体に関して特に詳述しない点については、前述のフェロセン化ナフタレンジイミド誘導体と同様である。従って、該フェロセン化アントラセン誘導体における各置換基の例や、用途等については、前述のフェロセン化ナフタレンジイミド誘導体について説明した事項が適宜適用される。 A novel ferroceneated anthracene derivative in which A in the formula (1) is 9,10-disubstituted anthracene is also preferable, and as such a compound, for example, X is —CH ₂ —NH—C ₃ H ₆ —N ( The following formula (11), which is CH ₃ ) —C ₃ H ₆ —, may be mentioned.

This ferrocene anthracene derivative can be obtained using, for example, ferrocenepropionic acid succinimide ester as in the following synthesis example.

The ferrocene anthracene derivative is also easy to separate and purify after the synthesis reaction in the same manner as the ferrocene naphthalene diimide derivative represented by the above formulas (1 ′) and (3 ′). The separation / purification yield is as high as 30%, and can be provided as a compound particularly useful as an intercalator. The ferrocene anthracene derivative is not particularly detailed in the same manner as the above-described ferrocene naphthalenediimide derivative. Therefore, as for examples of each substituent in the ferrocene anthracene derivative, uses, and the like, the matters described for the ferrocene naphthalene diimide derivative are appropriately applied.

  In order to show the advantageous effects of the compounds according to the present invention, examples and test examples will be shown below, but these are illustrative and the present invention is in any case limited to the following specific examples. is not.

１，４−ビス（３−アミノプロピル）ピペラジン５１．６ｍｌ（０．２５ｍｏｌ）を１，４−ジオキサン約２０ｍｌに溶解させ、これにS-tert-butyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine１２．０ｇ（０．０５ｍｏｌ）を１，４−ジオキサン約８０ｍｌに溶解させた溶液を室温で約５時間かけて滴下した。滴下後しばらく撹拌していると溶液の粘性が高くなったのでさらに１，４−ジオキサンを約２００ｍｌ加え、その後一晩撹拌を続けた。
生成した白色沈殿を吸引ろ過で取り除き、ろ液を減圧留去した。減圧留去後純水約１５０ｍｌを加え、それにＮａＣｌを加えて塩析を行った後、酢酸エチルで抽出を行った(１００ｍｌ×４)。有機層を炭酸カリウムで乾燥後、溶媒を減圧留去して黄色油状物質を得た。
得られた化合物の¹Ｈ−ＮＭＲ測定を行った。¹Ｈ−ＮＭＲスペクトルデータを表１に示す。

以上の結果より、得られた黄色油状物質は、下記式で表される化合物（１２）であると同定された。収量は１２．３ｇ、収率は８２％であった。

上記構造式中のａ〜ｆは、表１中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。
なお、¹Ｈ−ＮＭＲ測定結果に酢酸エチルのピークが認められたため、真空ポンプでよく乾燥させた後、次の反応に進んだ。 Synthesis of NFcpro (i) Single Boc Protection of Linker Part As an example of the compound according to the present invention, to synthesize a novel ferrocene naphthalenediimide (hereinafter sometimes referred to as “NFcpro”) represented by the above formula (10) First, one-Boc protection of 1,4-bis (3-aminopropyl) piperazine serving as a linker portion was performed according to the following scheme.

51.6 ml (0.25 mol) of 1,4-bis (3-aminopropyl) piperazine was dissolved in about 20 ml of 1,4-dioxane, and S-tert-butyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine 12. A solution prepared by dissolving 0 g (0.05 mol) in about 80 ml of 1,4-dioxane was added dropwise at room temperature over about 5 hours. When the mixture was stirred for a while after the dropwise addition, the viscosity of the solution became high, so about 200 ml of 1,4-dioxane was further added, and the stirring was continued overnight.
The produced white precipitate was removed by suction filtration, and the filtrate was distilled off under reduced pressure. After distilling off under reduced pressure, about 150 ml of pure water was added, and NaCl was added thereto for salting out, followed by extraction with ethyl acetate (100 ml × 4). The organic layer was dried over potassium carbonate, and then the solvent was distilled off under reduced pressure to obtain a yellow oily substance.
¹ H-NMR measurement of the obtained compound was performed. ¹ H-NMR spectral data are shown in Table 1.

From the above results, the obtained yellow oily substance was identified as the compound (12) represented by the following formula. The yield was 12.3 g and the yield was 82%.

A to f in the above structural formulas indicate the positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 1.
In addition, since the peak of ethyl acetate was recognized by the < ¹ > H-NMR measurement result, after drying well with the vacuum pump, it advanced to the next reaction.

１，４，５，８−ナフタレンテトラカルボン酸二無水物４．６４ｇ（０．０１７ｍｏｌ）と化合物（１２）１２．０ｇ（０．０４０ ｍｏｌ）をＴＨＦ１５０ｍｌ中で加熱還流した。約２３時間後、溶媒を減圧留去した。得られた茶褐色固体をクロロホルムに溶解させ、不溶成分を吸引ろ過によって取り除き、ろ液を減圧留去して茶褐色固体を得た。これをメタノール約２００ｍｌに溶解させ、不溶成分(肌色固体)を吸引ろ過でろ別し、ろ液を水１０００ｍｌで再沈殿した。一晩静置したのち結晶を取り出してデシケーターで乾燥させて黄土色固体を得た。
得られた化合物の¹Ｈ−ＮＭＲ測定を行った。¹Ｈ−ＮＭＲスペクトルデータを表２に示す。

以上の結果より、得られた黄土色固体は、下記式で表される化合物（１３）であると同定された。収量は２．４ｇ、収率は１７％であった。

上記構造式中のａ〜ｋは、表２中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。
なお、¹Ｈ−ＮＭＲ測定結果で、ｈの積分比があっていないのは、メタノールのヒドロキシル基のピークと水のピークが重なっているためであると考えられる。 (Ii) Binding of compound (12) to naphthalenetetracarboxylic dianhydride Subsequently, as a reaction shown in the above reaction formula (I), a compound that becomes a linker part to naphthalenetetracarboxylic dianhydride according to the following scheme (12) was bound.

1.64 g (0.017 mol) of 1,4,5,8-naphthalenetetracarboxylic dianhydride and 12.0 g (0.040 mol) of compound (12) were heated to reflux in 150 ml of THF. After about 23 hours, the solvent was distilled off under reduced pressure. The obtained brown solid was dissolved in chloroform, insoluble components were removed by suction filtration, and the filtrate was distilled off under reduced pressure to obtain a brown solid. This was dissolved in about 200 ml of methanol, insoluble components (skin color solid) were filtered off by suction filtration, and the filtrate was reprecipitated with 1000 ml of water. After standing overnight, the crystals were taken out and dried with a desiccator to obtain an ocherous solid.
¹ H-NMR measurement of the obtained compound was performed. ^{The 1} H-NMR spectrum data is shown in Table 2.

From the above results, the obtained ocherous solid was identified as the compound (13) represented by the following formula. The yield was 2.4 g, and the yield was 17%.

In the above structural formula, a to k indicate positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 2.
In addition, it is thought that it is because the peak of the hydroxyl group of methanol and the peak of water overlap that h does not have the integration ratio of h by the < ¹ > H-NMR measurement result.

化合物（１３）２．４１ｇ（２．８９ｍｍｏｌ）にＴＦＡ７ｍｌを加え、室温で約７時間撹拌した。撹拌停止後溶媒を減圧留去し、赤色固体を得た。これをメタノールに溶解させた後、クロロホルムで再沈殿を行い薄紫色固体を得た。
得られた固体の¹Ｈ−ＮＭＲ測定および元素分析を行った。¹Ｈ−ＮＭＲスペクトルデータを表３に、元素分析の結果を表４に示す。

元素分析の結果より、得られた化合物は６ヶ所がＴＦＡの塩になっていることが確認された。¹Ｈ−ＮＭＲの結果とあわせて、上記薄紫色固体は、下記式で表される化合物（１４）であると認められた。収量は３．２ｇ、収率は８３％であった。

上記構造式中のａ〜ｊは、表３中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。 (Iii) Deprotection of amino group protected with butoxycarbonyl group (Boc) Next, according to the following scheme, de-Boc reaction of compound (13) was performed to obtain compound (14).

To 2.41 g (2.89 mmol) of compound (13) was added 7 ml of TFA, and the mixture was stirred at room temperature for about 7 hours. After the stirring was stopped, the solvent was distilled off under reduced pressure to obtain a red solid. This was dissolved in methanol and then reprecipitated with chloroform to obtain a light purple solid.
The obtained solid was subjected to ¹ H-NMR measurement and elemental analysis. ¹ H-NMR spectral data are shown in Table 3, and the results of elemental analysis are shown in Table 4.

From the results of elemental analysis, it was confirmed that six of the obtained compounds were TFA salts. Together with the result of ¹ H-NMR, the pale purple solid was recognized as the compound (14) represented by the following formula. The yield was 3.2 g and the yield was 83%.

A to j in the above structural formula indicate the positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 3.

１００ｍｌナス型フラスコに化合物（１４）０．５３ｇ（０．４ｍｍｏｌ）、クロロホルム２０ｍｌ、トリエチルアミン０．３５ｍｌ（２．４ｍｍｏｌ）を加え完全に溶解させた後、フェロセンプロピオン酸スクシンイミドエステル ０．２９ｇ（１．２ｍｍｏｌ）を加え室温で撹拌した。シリカゲルＴＬＣで反応追跡を行い、１８時間後反応を終了し溶媒を減圧留去し、茶色の油状物質を得た。これをクロロホルム１００ｍｌに溶かし、純水１００ｍｌで洗浄、抽出し、クロロホルム相を減圧留去した。得られた固体物を、シリカゲルクロマトグラフィーを用いて(展開溶媒 クロロホルム：メタノール：ジエチルアミン=９５：５：０．５)Ｒｆ値０．１５の成分を分取し、溶媒を減圧留去した。さらに１２時間減圧乾固して、融点が３００℃以上である橙色粉末を得た。
得られた化合物を¹Ｈ−ＮＭＲ、ＭＡＬＤＩ−ＴＯＦ−ＭＡＳＳを用いて同定し、およびＨＰＬＣにより純度チェックを行った。
¹Ｈ−ＮＭＲスペクトルデータを表５に示す。

以上の結果より、得られた橙色粉末は、下記式で表される化合物（１０）であると認められた。

上記構造式中のａ〜ｎは、表５中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。
また、ＭＡＬＤＩ−ＴＯＦ−ＭＡＳＳによる測定結果を図１に示す。マトリックスとしてα-cyano-ＩＶ-hydroxycinnamic acid（ＣＨＣＡ）を用い、レーザ強度は１２５０
とした。最も大きなピークのｍ／ｚ＝１１１５．８から、得られた化合物が上記化合物（１０）であることがさらに確認された。なお、ｍ／ｚ＝１０４９．７、ｍ／ｚ＝９８２．２のピークは、それぞれフェロセン部位のシクロペンタジエンが一つずつ外れたものである。
ＨＰＬＣの純度測定結果を図２に示す。検出波長は３８５ｎｍとし、カラムはＩｎｅｒｔｓｉｌＯＤＳ−３を用い、試料注入溶媒はメタノール、溶離液Ａは０．１％ＴＦＡ水溶液、溶離液Ｂは０．１％ＴＦＡメタノール溶液とした。溶離液のグラジエント条件を表６に示す。

また、リテンションタイムが２０分の時のＵＶ−ｖｉｓスペクトルを図３に示す。
上記に示したＨＰＬＣによる純度チェックにおいても、目的物由来のピークのみが観測された。この結果から、シリカゲルクロマトグラフィーにより、２つのリンカーの両方にフェロセンが導入された化合物（目的物であるＮＦｃｐｒｏ）が、２つのリンカーのうち１つだけにフェロセンが導入された化合物から効率よく分離され、純度の高いＮＦｃｐｒｏを高収率で得られたことが確認された。 (Iv) Synthesis of NFcpro Finally, according to the following scheme, compound (14) was reacted with ferrocenepropionic acid succinimide ester (compound (15)) to obtain the target compound, NFcpro.

In a 100 ml eggplant type flask, 0.53 g (0.4 mmol) of compound (14), 20 ml of chloroform and 0.35 ml (2.4 mmol) of triethylamine were added and completely dissolved, and then 0.29 g (1. ferrocenepropionic acid succinimide ester). 2 mmol) was added and stirred at room temperature. The reaction was monitored by silica gel TLC. After 18 hours, the reaction was completed, and the solvent was distilled off under reduced pressure to obtain a brown oily substance. This was dissolved in 100 ml of chloroform, washed and extracted with 100 ml of pure water, and the chloroform phase was distilled off under reduced pressure. The obtained solid substance was subjected to silica gel chromatography (developing solvent: chloroform: methanol: diethylamine = 95: 5: 0.5) to fractionate a component having an Rf value of 0.15, and the solvent was distilled off under reduced pressure. The mixture was further dried under reduced pressure for 12 hours to obtain an orange powder having a melting point of 300 ° C. or higher.
Resulting compound ¹ H-NMR, it was identified using MALDI-TOF-MASS, and was pure checked by HPLC.
^{The 1} H-NMR spectrum data is shown in Table 5.

From the above results, it was recognized that the obtained orange powder was a compound (10) represented by the following formula.

“A” to “n” in the above structural formula indicate positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 5.
Moreover, the measurement result by MALDI-TOF-MASS is shown in FIG. Α-cyano-IV-hydroxycinnamic acid (CHCA) is used as the matrix, and the laser intensity is 1250.
It was. From the largest peak m / z = 1115.8, it was further confirmed that the obtained compound was the compound (10). The peaks at m / z = 1049.7 and m / z = 982.2 are obtained by removing one cyclopentadiene from the ferrocene site.
The HPLC purity measurement results are shown in FIG. The detection wavelength was 385 nm, the column was Inertsil ODS-3, the sample injection solvent was methanol, eluent A was 0.1% TFA aqueous solution, and eluent B was 0.1% TFA methanol solution. Table 6 shows the gradient conditions of the eluent.

Further, FIG. 3 shows a UV-vis spectrum when the retention time is 20 minutes.
In the purity check by HPLC shown above, only the peak derived from the target product was observed. From this result, the compound in which ferrocene was introduced into both of the two linkers (NFCpro which is the target product) was efficiently separated from the compound in which ferrocene was introduced into only one of the two linkers by silica gel chromatography. It was confirmed that high-purity NFcpro was obtained in high yield.

化合物（Ａ）０．２９ｇ（０．３ｍｍｏｌ）とフェロセンプロピオン酸スクシンイミドエステル０．３１ｇ（０．９ｍｍｏｌ）をＣＨＣｌ₃ １５ｍｌ／ＴＥＡ０．２６ｍｌ（１．８ｍｍｏｌ）混合溶液に溶解させ、室温で撹拌した。４２時間後反応を終了させ、反応溶媒を減圧留去した。溶液にクロロホルム１００ｍｌを加え、飽和食塩水２００ｍｌ（５０ｍｌ×４回）で洗浄した。クロロホルムを減圧留去し、シリカゲルカラムクロマトグラフィー（展開溶媒ＣＨＣｌ₃：ＭｅＯＨ：ジエチルアミン＝１０：０．２：０．５）にて、Ｒｆ値０．２の茶褐色成分を分取した。溶媒を減圧留去した後、ＭｅＯＨを加えて吸引ろ過したところ、橙黄色粉末が得られた。
得られた固体を¹Ｈ−ＮＭＲ、ＭＡＬＤＩ−ＴＯＦ−ＭＡＳＳを用いて同定し、およびＨＰＬＣにより純度チェックを行った。¹Ｈ−ＮＭＲの結果を表７に示す。

¹Ｈ−ＮＭＲの結果から、上記橙黄色固体は、下記式に示す化合物（１０’）であると認められた。収量は０．２１ｇ、収率は７０％であった。

上記構造式中のａ〜ｎは、表７中のケミカルシフト（δ値）に対応する水素（プロトン）それぞれの位置を示す。
また、ＭＡＬＤＩ−ＴＯＦ−ＭＡＳＳによる測定結果を図４に示す。マトリックスとしてα-cyano-ＩＶ-hydroxycinnamic acid（ＣＨＣＡ）を用い、レーザ強度は２．２×
１０⁴とした。最も大きなピークのｍ／ｚ＝１００５．０２から、得られた化合物が上記ＦＮＤ２（１０’）であることがさらに確認された。
ＨＰＬＣの純度測定結果を図５に示す。検出波長は３８４ｎｍとし、カラムはＩｎｅｒｔｓｉｌＯＤＳ−３を用い、試料注入溶媒は０．１％ＴＦＡ水溶液、溶離液Ａは０．１％ＴＦＡ水溶液、溶離液Ｂは７０％アセトニトリル／３０％−０．１％ＴＦＡ溶液とした。溶離液のグラジエント条件を表８に示す。

また、リテンションタイムが１７．２分の時のＵＶ−ｖｉｓスペクトルを図６に示す。
上記に示したＨＰＬＣによる純度チェックにおいても、目的物由来のピークのみが観測された。この結果から、純度の高いＦＮＤ２を高収率で得られたことが確認された。 Synthesis of FND2 Next, as an example of the compound according to the present invention, a novel ferroceneated naphthalenediimide (hereinafter sometimes referred to as “FND2”) represented by the above formula (10 ′) was synthesized according to the following scheme. .

Compound (A) 0.29 g (0.3 mmol) and ferrocenepropionic acid succinimide ester 0.31 g (0.9 mmol) were dissolved in CHCl ₃ 15 ml / TEA 0.26 ml (1.8 mmol) mixed solution and stirred at room temperature. After 42 hours, the reaction was terminated, and the reaction solvent was distilled off under reduced pressure. To the solution, 100 ml of chloroform was added, and washed with 200 ml of saturated saline (50 ml × 4 times). Chloroform was distilled off under reduced pressure, and the brown component having an Rf value of 0.2 was fractionated by silica gel column chromatography (developing solvent CHCl ₃ : MeOH: diethylamine = 10: 0.2: 0.5). After the solvent was distilled off under reduced pressure, MeOH was added and suction filtered to obtain an orange-yellow powder.
The obtained solid was identified using ¹ H-NMR, MALDI-TOF-MASS, and purity was checked by HPLC. The results of ¹ H-NMR are shown in Table 7.

^From the result of ¹ H-NMR, it was recognized that the orange-yellow solid was a compound (10 ′) represented by the following formula. The yield was 0.21 g and the yield was 70%.

“A” to “n” in the above structural formula indicate positions of hydrogen (protons) corresponding to chemical shifts (δ values) in Table 7.
Moreover, the measurement result by MALDI-TOF-MASS is shown in FIG. Α-cyano-IV-hydroxycinnamic acid (CHCA) is used as the matrix, and the laser intensity is 2.2 ×
10 ⁴ . From the largest peak, m / z = 1005.02, it was further confirmed that the obtained compound was the above FND2 (10 ′).
The HPLC purity measurement results are shown in FIG. The detection wavelength was 384 nm, the column was Inertsil ODS-3, the sample injection solvent was 0.1% TFA aqueous solution, eluent A was 0.1% TFA aqueous solution, and eluent B was 70% acetonitrile / 30% -0.1. % TFA solution. Table 8 shows the gradient conditions of the eluent.

Further, FIG. 6 shows a UV-vis spectrum when the retention time is 17.2 minutes.
In the purity check by HPLC shown above, only the peak derived from the target product was observed. From this result, it was confirmed that high purity FND2 was obtained in high yield.

Evaluation of performance of NFcpro as an intercalator In order to evaluate the performance of NFcpro synthesized in Example 1 (the above formula (10)) as a stitched intercalator, accompanying the addition of Calf thymus DNA (CT-DNA) The change in the UV-visible absorption spectrum of NFcpro was measured, and Scatchard analysis was performed. In general, when a compound binds to a double-stranded nucleic acid as an intercalator, the ultraviolet-visible absorption spectrum of the compound exhibits a small long wavelength shift and a large hypochromic effect. In addition, when the concentration of the intercalator is kept constant and the DNA is changed variously, if the UV-visible absorption spectrum of the intercalator changes between two states passing through the isosbestic point, the Scatterchart analysis can determine the binding constant and the binding. The number of sites can be determined.
Under the following conditions, a small amount (about 3 μL) of the DNA solution was dropped into a solution containing NFcpro, and the UV-visible absorption of NFcpro was measured each time. A total of 300 μL of the DNA solution was dropped, and it took 6 minutes for all measurements. After completion of the dropwise addition, the mixture was stirred for 4 minutes and further left for 1 minute. The measurement temperature was 25 ° C., and NFcpro was used as a hydrochloride dissolved in water.
-Buffer; 2-morpholino-ethane-sulfonic acid (MES) 10 mM, EDTA (pH 6.25) 1 mM, NaCl 0.1 M
・ Stock solution; [NFcpro] = 1.2mM, [Calf thymus DNA] = 3mM (bp)
・ Concentration in cell; [NFcpro] = 2.4 μM
-Cell used: 10 cm cell The results are shown in FIG. A light-colored effect of maximum absorption was observed, suggesting that it was intercalated for CT-DNA.
Further, FIG. 8 shows the binding rate calculated from the change in the ultraviolet-visible absorption spectrum and the Scatchard Plot based on this binding rate. The coupling constant K and the number of seats n were calculated by optimizing the experimental data and theoretical data based on FIG. The wavelength was 382 nm.
r / L = K (1-nr) [(1-nr) / {1- (n-1) r}] ^n-1 (α)
It was calculated that the coupling constant K = 57 × 10 ⁵ [M ⁻¹ ] and the number of locus n = 1.4. As can be seen from the comparative examples described later, it was found that NFcpro has a binding constant that is 10 to 50 times stronger than that of conventionally used intercalators.

ＮＦｃｃａおよびＮＦｃａｃも塩酸塩として水に溶かして用い、bufferおよびstock solutionはＮＦｃｐｒｏと同様の条件で、セル中濃度は[ＮＦｃｃａ]＝[ＮＦｃａｃ]＝２２μＭ、使用セルは１ｃｍセルとした。
ＮＦｃａｃの紫外可視吸収スペクトル変化を図９に、Ｓｃａｔｃｈａｒｄ Ｐｌｏｔを図１０に示す。図９においては、極大吸収の淡色効果が見られ、ＣＴ−ＤＮＡに対してインターカレーションしていることが示唆された。
結合定数Ｋ＝６．８×１０⁵[Ｍ^-1]、座位数ｎ＝２．１と算出された。座位数２．１という数字から、ＤＮＡ塩基対一つおきにインターカレータが挿入結合していることが示唆された。 As a comparative example, UV-visible absorption spectrum measurement and Scatchard Plot were performed for NFcca represented by the following formula (8) and NFfac represented by formula (15), which have been used as an intercalator.

NFcca and NFcac were also dissolved in water as hydrochlorides, the buffer and stock solution were used under the same conditions as NFcpro, the concentration in the cell was [NFcca] = [NFFcac] = 22 μM, and the cell used was a 1 cm cell.
FIG. 9 shows changes in the UV-visible absorption spectrum of NFac, and FIG. 10 shows the Scatchard Plot. In FIG. 9, the light absorption effect of the maximum absorption was observed, suggesting that the CT-DNA was intercalated.
The coupling constant K was calculated as 6.8 × 10 ⁵ [M ⁻¹ ] and the number of loci n = 2.1. The number of loci 2.1 indicates that every other DNA base pair has an intercalator.

  Table 9 shows the binding constants and the number of loci determined by Scatchard Plot for NFcpro, which is a compound according to the present invention, and NFfac and NFcca, which are comparative examples.

ＮＦｃｐｒｏは、ＮＦｃｃａと比較して約５０倍、ＮＦｃａｃに対しても１０倍近い結合定数を有することがわかった。ＮＦｃｐｒｏは、長いリンカー部が有するため、ＤＮＡからの解離反応が抑制され、より安定なＤＮＡ−インターカレータ複合体を形成したものと考えられる。

NFcpro was found to have a binding constant approximately 50 times that of NFcca and nearly 10 times that of NFcac. Since NFcpro has a long linker part, it is considered that the dissociation reaction from DNA was suppressed and a more stable DNA-intercalator complex was formed.

Evaluation of double-strand selectivity of NFcpro using differential pulse voltammetry (DPV) Using an electrode with single-stranded DNA immobilized on the surface, an intercalator that binds to this single-strand by electrostatic interaction The amount and the amount of intercalator that intercalates into the double strand generated when the target DNA having a sequence complementary to this single strand hybridizes were each measured electrochemically.
As an intercalator, NFcpro according to the present invention and NFcca and NFcac as a comparative example were synthesized and used as hydrochlorides.

(I) Fixation of probe DNA to electrode 1 μL of 10 pmol / μl DNA solution was dropped onto the gold electrode surface, capped so as not to dry, and left at 25 ° C. for 2 hours. Thereafter, 1 μL of 1 mM 2-mercaptoethanol was dropped onto the gold electrode surface and allowed to stand at 25 ° C. for 2 hours to obtain a single-stranded immobilized electrode. Single-stranded DNA is fixed to the gold electrode via a sulfur atom.
In this example, a wildtype G71R partial sequence (24mer) described in SEQ ID NO: 1 was used as a target DNA, and an oligonucleotide having a complementary base sequence was used as a probe DNA. The base sequence of the probe DNA is shown in SEQ ID NO: 2. The preparation of these oligonucleotides was outsourced to Hokkaido System Science Co., Ltd. The G71R sequence is one of risk factors that are believed to cause side effects of the anticancer drug irinotecan.

(Ii) Measurement of binding amount of intercalator to probe DNA The above-mentioned single-strand immobilized electrode is composed of 0.1 M acetate buffer (pH 5.6), 0.1 M KCl aqueous solution and 50 μM intercalator solution. It was immersed in a solution (hereinafter referred to as “electrolytic solution”) for 3 minutes, and DPV measurement was performed. For the DPV measurement, ALS Model 610 was used and the sweep rate was 100 mV / sec. In the DPV measurement, the amount of intercalator bound to DNA is measured by detecting ferrocene introduced at its end as a signal.

  The measurement results for NFcpro, NFcac, and NFcca are shown as “ssDNA” in FIGS.

  Intercalator binding to single stranded DNA is thought to be due to electrostatic interactions.

(Iii) Hybridization of probe DNA and target DNA On the surface of the single-stranded immobilized electrode obtained in (i), a Wildtype G71R solution (10
1 μL of pmol / μl) solution was added dropwise. The cap was capped so that it did not dry, and it was left at 25 ° C. for 30 minutes to cause hybridization between the probe DNA and the target DNA.

(Iv) Measurement of binding amount of intercalator to double-stranded DNA This electrode was immersed in an electrolytic solution for 3 minutes, and DPV measurement was performed.
The measurement results for NFcpro, NFcac, and NFcca are indicated by “dsDNA” in FIGS.
In any intercalator, the amount of binding to double-stranded DNA exceeded the amount of binding to single-stranded DNA. Each peak potential corresponds to an oxidation potential obtained by cyclic voltammetry.
The results of DPV measurement obtained from double-stranded DNA include signals from intercalators intercalated with double strands and signals from intercalators bound by electrostatic interaction. Is considered to be included. Therefore, the above (ii
), The peak response current value in the single-stranded state obtained in step i) is i ₀ , and the peak response current value after formation of the double strand obtained in step (iv) is i. Δi was determined, and the ability to bind to double-stranded DNA was evaluated using this value.
Δi = [(i−i ₀ ) / i ₀ ] × 100 (%)
The respective peak current values and Δi values are shown in FIG. The Δi value was the largest for NFcpro, 182%, NFcca 123%, and NFcac 88%. This indicates that NFcpro has the highest selectivity for double strands. NFcpro has small variations in peak current values and a stable baseline.
In this example, the Δi value indicating the ability to bind to double-stranded DNA differs from the value obtained by Scatchard analysis in Example 2 in the double-stranded DNA in solution and on the solid support. This is caused by the difference in binding ability to DNA and the concentration of intercalator per DNA molecule.

Evaluation of binding ability by competitive intercalation Two intercalators were allowed to coexist in the electrolytic solution and competitively intercalated to compare the binding ability of both intercalators.
In the same manner as in Example 3, a single-stranded immobilized electrode was prepared using the oligonucleotide having the base sequence described in SEQ ID NO: 2. An electrolytic solution containing Wildtype G71R (target DNA) having the base sequence described in SEQ ID NO: 1 was dropped on the electrode surface to allow hybridization.
This electrode was immersed in an electrolytic solution containing NFcpro and NFcca for 3 minutes, and DPV measurement was performed. The measurement results are shown in FIG. A peak current value corresponding to each intercalator is observed, and an increase in peak current value due to intercalation into double-stranded DNA is also observed. Due to competitive intercalation, there was a difference in the rate of increase of each peak current value.
FIG. 16 shows the comparison of peak current values and the value of Δi obtained by the same method as in Example 3. The NFcpro Δi was 98%, indicating an approximately 4-fold increase compared to NFcca (Δi = 25%). This coincided with the result of the evaluation of the binding ability by Scatchard analysis, and the high binding ability of NFcpro was confirmed.

The measurement result by MALDI-TOF-MASS of the novel compound NFcpro according to the present invention is shown. The HPLC chromatogram at the time of NFcpro synthesis is shown. In the chromatogram shown in FIG. 2, a UV-vis spectrum of a fraction having a retention time of 20 minutes is shown. The measurement result by MALDI-TOF-MASS of the novel compound FND2 which concerns on this invention is shown. The HPLC chromatogram at the time of FND2 synthesis | combination is shown. In the chromatogram shown in FIG. 5, a UV-vis spectrum of a fraction having a retention time of 17.2 minutes is shown. The change of the UV-visible absorption spectrum of NFcpro accompanying DNA addition is shown. A Scatchard plot for NFcpro is shown. The change of the ultraviolet visible absorption spectrum of NFcac accompanying DNA addition is shown. A Scatchard plot for NFcac is shown. The DPV voltammogram when NFcpro is used as an intercalator is shown. The DPV voltammogram when NFfac is used as an intercalator is shown. The DPV voltammogram when NFcca is used as an intercalator is shown. The peak current values of NFcpro, NFcac, and NFcca are shown. Figure 4 shows DPV voltammograms in competitive intercalation using NFcpro and NFcca. The peak current value in the competitive intercalation shown in FIG. 15 is shown.

Claims (12)

Following formula (1)

(In the formula, A represents a polycyclic hydrocarbon or a derivative thereof; X represents an optionally substituted alkylene group having 1 to 6 carbon atoms, — (CH ₂ ) _x —B— (CH ₂ ). _y- [wherein B represents a 5- or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms; x and y each independently represents an integer of 0 to 3], or-(CH ₂ ) _a — (NR ⁵ ) — (CH ₂ ) _b — (NR ⁶ ) — (CH ₂ ) _c — wherein a, b and c each independently represent an integer of 0 to 3, and R ⁵ and R ⁶ are each Independently represents hydrogen or a methyl group]; R ¹ represents hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkenyl group or an alkynyl group, an arylalkyl group or arylalkenyl group having 7 to 9 carbon atoms, or an alkyl group having 2 or 3 carbon atoms; R ² and R ³ Each independently represents an electron donating group or an electron withdrawing group in an aqueous solution; m and n each independently represents an integer of 0 to 2. Two identical display groups in one molecule are the same or different from each other. Or a salt or hydrate thereof. X is the following formula (2)

The compound according to claim 1 or a salt or hydrate thereof. X _{is, -C 3 H 6 N (CH} 3) C 3 H 6 - is a compound or a salt or a hydrate thereof according to claim 1. Following formula (3)

Wherein A represents a polycyclic hydrocarbon or a derivative thereof; Y represents a 5-membered or 6-membered aliphatic heterocyclic ring containing 1 or 2 nitrogen atoms, and the group bonded to the carbonyl group is a nitrogen atom. R ⁴ represents an alkylene group having 1 to 6 carbon atoms; R ² and R ³ each independently represents a group that becomes an electron donating group or an electron withdrawing group in an aqueous solution; And independently represents an integer of 0 to 2. Two identical display groups in one molecule may be the same or different from each other) or a salt or hydrate thereof.   The compound according to claim 4, or a salt or hydrate thereof, wherein Y is a piperidyl group or a piperazyl group. The compound according to claim 4, a salt thereof or a hydrate thereof, wherein R ⁴ is a methylene group, Y is a piperidyl group, and the nitrogen atom is bonded to the carbon atom of the carbonyl group.   A is a naphthalene diimide group, The compound of any one of Claim 1 to 6, its salt, or its hydrate.   The compound according to claim 7, or a salt or hydrate thereof, wherein m = n = 0. The method for producing a compound according to claim 1, wherein A is a naphthalenediimide group, or a salt or hydrate thereof, as shown in the following reaction formula (I): 1,4,5,8-naphthalene A first step of reacting a tetracarboxylic dianhydride with a compound represented by the formula (4) to obtain a compound represented by the formula (5), as shown in the following reaction formula (II), A second step of obtaining a target product by reacting the compound represented by (5) with ferrocenecarboxylic acid succinimide ester.

4. A method for producing a compound according to claim 3, wherein A is a naphthalene diimide group, or a salt or hydrate thereof, as shown in the following reaction formula (III): 1,4,5,8-naphthalene A first step of reacting a tetracarboxylic dianhydride with a compound represented by the formula (6) to obtain a compound represented by the formula (7), as shown in the following reaction formula (IV), A second step of obtaining a target product by reacting the compound represented by (7) with ferrocenecarboxylic acid succinimide ester.

Contacting a probe nucleic acid having a base sequence complementary to a target nucleic acid with a test sample under conditions where hybridization occurs;
Adding the compound according to any one of claims 1 to 8, or a salt or hydrate thereof;
Measuring the electrochemical response of the compound bound to a double-stranded nucleic acid;
A method for detecting a target nucleic acid in a test sample, comprising: A kit for detecting a target nucleic acid, comprising: an electrode on which a probe nucleic acid having a base sequence complementary to the target nucleic acid is immobilized; and the compound according to any one of claims 1 to 8, or a salt or hydrate thereof. .

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