JP2023013344A - Bisbenzimide analog, production method thereof, and use thereof - Google Patents

Abstract

To provide a bisbenzimide analog realizing selective light emission from hypoxic cells.SOLUTION: The bisbenzimide analog is represented by the general formula (1) in the figure, where -R1 represents a C1-3 alkyl group, and -X represents an azido group or azo group-containing substituent.SELECTED DRAWING: Figure 1

Description

The present invention relates to a novel bisbenzimide analog compound, a method for producing the compound, and uses of the compound.

Hypoxic cells are generated in tumors, ischemic diseases, and the like. Hypoxic cells refer to cells with low oxygen concentration. In tumor tissue, hypoxic cells (hypoxic regions) occur with very rapid cell proliferation and angiogenic imbalance of tumor cells. On the other hand, in ischemic diseases such as infarction, blood vessels become clogged, resulting in the generation of hypoxic cells in the downstream region. Since cells in a severely hypoxic environment do not occur in normal tissues, visualization of hypoxic cells is expected to lead to visualization of these diseases. Techniques for staining the nuclei of hypoxic cells have not been previously reported. In general, staining cell nuclei is a technique that is very easy to see and is widely used in microscopic imaging of animal tissues. It is expected to be applied to life science research that requires hypoxic cells.

で表されるビスベンズイミド化合物が知られ、Hoechst分子などとも呼ばれる。Hoechst分子は細胞核内に集積し、ゲノムＤＮＡと結合する結果、発光する性質をもつ。このことから、細胞核を染色する核染色として汎用されてきた。 As a nuclear stain that stains the cell nucleus, the following structural formula

A bisbenzimide compound represented by is known and is also called a Hoechst molecule. Hoechst molecules accumulate in the cell nucleus and have the property of emitting light as a result of binding with genomic DNA. For this reason, it has been widely used as a nuclear stain for staining cell nuclei.

As a separate issue from the staining of cells described above, compounds with high affinity for cell nuclei are expected to be applied to the Huisgen reaction. The Huisgen reaction is a chemical reaction that has been widely used in organic synthesis. A cycloaddition reaction occurs between an azide group and an acetylene group in the presence of a copper catalyst. Moreover, when an acetylene derivative with a large strain is used, the cycloaddition reaction proceeds even in the absence of a catalyst. This reaction has been widely used as a reaction that connects various biomolecules (proteins, nucleic acids, etc.) and functional molecules. It has also been used to modify Hoechst molecules (Patent Document 1).

International Publication No. WO2015/042438

As described above, Hoechst molecules accumulate in the cell nucleus and have the property of emitting light as a result of binding to genomic DNA. However, the Hoechst molecule has no cell-selective function and stains the nuclei of all cells. Therefore, the Hoechst molecule is unsuitable for detecting hypoxic cells. In view of such circumstances, an object of the present invention is to provide a compound that selectively causes hypoxic cells to emit light.

The above-mentioned Huisgen reaction is achieved by condensing a compound having an acetylene group on the Hoechst side and an azide group on the functional molecule side in conventional compounds. However, introduction of an azide group usually involves complicated reactions in many cases. Therefore, it is more preferable to provide a compound that can be used in the Huisgen reaction without introducing an azide group into the functional molecule.

The present inventors completed the present invention shown below as a result of intensive studies.

（式中、－Ｒ１は炭素数１～３のアルキル基を表し、－Ｘは下記（Ｘ１）～（Ｘ３）のいずれかで表される官能基を表す。）

で表される、ビスベンズイミド類縁体。
（ＩＩ）下記一般式（２）で表される化合物（式中、－Ｒ１は上記と同じ官能基を表す。）及び下記一般式（３）で表される４－ニトロベンズアルデヒド

を共存させた状態で酸性条件下で還元して、次いで環化させることで、下記一般式（４）

（式中、－Ｒ１は上記と同じ官能基を表す。）で表されるビスベンズイミド類縁体を得て、上記一般式（４）で表されるビスベンズイミド類縁体の末端アミノ基を上記官能基Ｘに置換することによる、上記（Ｉ）の一般式（１）で表される、ビスベンズイミド類縁体の製造方法。
（ＩＩＩ）被検細胞の細胞核に上記（Ｉ）のビスベンズイミド類縁体を結合させ、前記結合後の被検細胞に光を照射して、前記照射に基づく蛍光を観測する、被検細胞の酸素濃度の評価方法。
（ＩＶ）上記（Ｉ）のビスベンズイミド類縁体を含有する診断薬であって、被検細胞の細胞核に前記ビスベンズイミド類縁体を結合させ、前記結合後の被検細胞に光を照射して、前記照射に基づく蛍光を観測することにより、被検細胞の酸素濃度を評価するための、前記診断薬。
（Ｖ）上記（Ｉ）のビスベンズイミド類縁体（但し、－Ｘは下記（Ｘ１）で表される官能基である。）及び下記一般式（５）

（式中、－Ｙは、一般式（５）に表示される炭素原子とＣ－Ｃ単結合を形成する官能基を表す。）で表される化合物を反応させて、下記一般式（６）

で表される化合物を得て、得られた一般式（６）で表される化合物を細胞核に結合させる、官能基－Ｙを有する化合物を細胞核へ導入する方法。
（ＶＩ）上記（Ｉ）のビスベンズイミド類縁体（但し、－Ｘは下記（Ｘ１）で表される官能基である。）及び下記一般式（７）

（式中、－Ｚは、一般式（７）に表示される窒素原子とＮ－Ｃ単結合を形成する官能基を表す。）で表される化合物を反応させて、下記一般式（８）

で表される化合物を得て、得られた一般式（８）で表される化合物を細胞核に結合させる、官能基－Ｚを有する化合物を細胞核へ導入する方法。 (I) the following general formula (1)

(Wherein, -R1 represents an alkyl group having 1 to 3 carbon atoms, and -X represents a functional group represented by any one of the following (X1) to (X3).)

A bisbenzimide analogue represented by
(II) a compound represented by the following general formula (2) (wherein —R represents the same functional group as above) and 4-nitrobenzaldehyde represented by the following general formula (3)

in the presence of the following general formula (4) by reducing under acidic conditions and then cyclizing

(Wherein, -R1 represents the same functional group as above), and the terminal amino group of the bisbenzimide analogue represented by the general formula (4) is attached to the functional group X. A method for producing a bisbenzimide analogue represented by general formula (1) of (I) above by substitution.
(III) The bisbenzimide analog of (I) is bound to the cell nucleus of the test cell, the test cell after binding is irradiated with light, and the fluorescence based on the irradiation is observed, whereby the oxygen concentration of the test cell. evaluation method.
(IV) A diagnostic agent containing the bisbenzimide analogue of (I) above, wherein the bisbenzimide analogue is bound to the cell nucleus of a test cell, the test cell after binding is irradiated with light, and the irradiation is performed. said diagnostic agent for assessing the oxygen concentration of a test cell by observing fluorescence based on
(V) the bisbenzimide analog of (I) above (where -X is a functional group represented by (X1) below) and the following general formula (5)

(Wherein, -Y represents a functional group that forms a C—C single bond with the carbon atom shown in general formula (5).) by reacting the compound represented by the following general formula (6)

A method of introducing a compound having a functional group -Y into cell nuclei, comprising obtaining a compound represented by and binding the obtained compound represented by general formula (6) to cell nuclei.
(VI) the bisbenzimide analog of (I) above (where -X is a functional group represented by (X1) below) and the following general formula (7)

(Wherein, -Z represents a functional group that forms an NC single bond with the nitrogen atom represented by the general formula (7).) The compound represented by the following general formula (8)

A method of introducing a compound having a functional group -Z into a cell nucleus, which comprises obtaining a compound represented by and binding the obtained compound represented by the general formula (8) to the cell nucleus.

The bisbenzimide analogue of (I) provided by the present invention can selectively stain hypoxic cells when taken into the cell nucleus. Therefore, the analogue is expected to be used as a diagnostic agent for measuring oxygen concentration in cells.

Furthermore, with regard to the bisbenzimide analogue (I) ₍ where X is -N3) provided by the present invention, a functional molecule having a carbon-carbon triple bond can be incorporated into cells by the Huisgen reaction. Be expected. In this case, the functional molecule need not have an azide group, so there is an advantage that the options for the functional molecule that can be used are widened.

1 shows observation images of fluorescence intensity in the presence of the bisbenzimide analogue of the present invention under aerobic and hypoxic conditions. 1 shows the time course of relative fluorescence intensity in the presence of the bisbenzimide analogue of the present invention under aerobic and hypoxic conditions.

The novel bisbenzimide analogue provided by the present invention (hereinafter also referred to as the compound of the present invention) is represented by the following general formula (1).

In general formula (1), -R1 represents an alkyl group having 1 to 3 carbon atoms. Preferably, it is a methyl group or an ethyl group.

In general formula (1), -X represents a functional group represented by any one of the following (X1) to (X3).

In consideration of the structural similarity with the Hoechst molecule introduced in the prior art column, the following abbreviations may be used in this specification.

Hoechst-N3: A bisbenzimide analog in which -R1 is a methyl group and -X is the functional group represented by (X1) in the general formula (1).

Hoechst-Azo-NMe2: A bisbenzimide analog in which -R1 is a methyl group and -X is the functional group represented by (X2) in the general formula (1).

Hoechst-Azo-NO2: A bisbenzimide analog in which -R1 is a methyl group and -X is the functional group represented by (X3) in the general formula (1).

Hoechst-NH2: A bisbenzimide analog in which -R1 is a methyl group and -X is an amino group in the general formula (1), in other words, a compound in which -R1 is a methyl group in the general formula (4). , is a compound that can be a precursor of the compound of the present invention.

（例：Ｒ１がメチル基である場合は、Hoechst-NH2である。）を得て、前記アミノ基を所望の官能基Ｘ、すなわち、上記（Ｘ１）～（Ｘ３）に置換する合成スキームが提案される。一般式（４）で表される化合物からアミノ基を上記（Ｘ１）～（Ｘ３）に置換する具体的方法は、後述の実施例を参考にすることができる。 The method for producing the compound of the present invention is not particularly limited, and is preferably a compound in which -X is an amino group in the general formula (1), that is, a compound represented by the following general formula (4)

(Example: when R1 is a methyl group, it is Hoechst-NH2), and a synthetic scheme is proposed in which the amino group is substituted with the desired functional group X, that is, (X1) to (X3) above. be done. Specific methods for substituting (X1) to (X3) for the amino group in the compound represented by the general formula (4) can refer to Examples described later.

を共存させた状態で酸性条件下で還元して、次いで環化させることで得られ、具体的な合成例は後述の実施例を参照することができる。 The compound represented by the general formula (4) is a compound represented by the following general formula (2) and 4-nitrobenzaldehyde represented by the following general formula (3)

is coexisted, and then reduced under acidic conditions, followed by cyclization. Specific synthetic examples can be referred to the examples described later.

The compounds of the present invention are functional molecules that bind to DNA duplexes within the cell nucleus in the same way as conventional Hoechst molecules. However, unlike the conventional Hoechst molecule, it has the characteristic that the degree of fluorescence changes depending on the oxygen concentration of the cell. This mechanism is roughly inferred as follows.

The compound of the present invention has a functional group represented by any one of the above (X1) to (X3) as -X in general formula (1). These functional groups have an azide structure or an azo structure, and these structures do not emit fluorescence. Here, when the compound of the present invention is taken into cells with low oxygen concentration, it undergoes reduction by a reductase, is reduced to an amino group, and is converted to a compound represented by the above general formula (4). be done. Since the compound represented by the general formula (4) having an amino group emits fluorescence when irradiated with light of a specific wavelength, a luminescence phenomenon is observed.

As described above, the compound of the present invention can selectively stain the cell nuclei of hypoxic cells, so that it can be used as a diagnostic agent for clarifying the location of hypoxic cells and evaluating the oxygen concentration of test cells. can be used as Hypoxic cells are present in ischemic diseases, solid cancer tissues, and the like, and thus the compound of the present invention is expected to be useful for early detection of these diseases. In Examples described later, fluorescence is observed with respect to light irradiation with an excitation wavelength of 405 nm.

Next, the function of the compound of the present invention to transport functional molecules into the cell nucleus will be described. The compound of the present invention, that is, the compound represented by the general formula (1), wherein the functional group -X is -N3 _, can be chemically bonded to a compound having an acetylene structure by Huisgen reaction. Further bonding of such a chemically bonded compound to the cell nucleus corresponds to transporting the compound having the azide structure or acetylene structure into the cell nucleus.

一般式（５）において、－Ｙは、一般式（５）に表示される炭素原子とＣ－Ｃ単結合を形成する官能基を表す。一般式（５）で表される化合物及び上記一般式（１）で表される化合物のうち、官能基－Ｘが－Ｎ_３である化合物を、同触媒の存在下、Huisgen反応に供することにより、下記一般式（６）

で表される化合物を得ることができる。得られた一般式（６）で表される化合物を細胞核に結合させることは、官能基－Ｙを有する化合物を細胞核へ導入することに相当する。 An example of the compound having the above-mentioned acetylene structure is the compound represented by the following general formula (5).

In general formula (5), —Y represents a functional group forming a C—C single bond with the carbon atom shown in general formula (5). By subjecting the compound represented by the general formula (5) and the compound represented by the general formula (1), wherein the functional group -X is -N3 _, to the Huisgen reaction in the presence of the same catalyst. , the following general formula (6)

A compound represented by can be obtained. Binding the obtained compound represented by the general formula (6) to the cell nucleus corresponds to introducing the compound having the functional group -Y into the cell nucleus.

Specific examples of the functional group -Y preferably include signal-transmitting molecules such as fluorescent dyes, agents that act on genomic DNA in cell nuclei such as DNA alkylating agents, and the like.

一般式（７）において、－Ｚは、一般式（７）に表示される窒素原子とＮ－Ｃ単結合を形成する官能基を表す。一般式（７）で表される化合物及び上記一般式（１）で表される化合物のうち、官能基－Ｘが－Ｎ_３である化合物を、Huisgen反応に供することにより、下記一般式（８）

で表される化合物を得ることができる。得られた一般式（８）で表される化合物を細胞核に結合させることは、官能基－Ｚを有する化合物を細胞核へ導入することに相当する。通常は、アセチレン構造を有する化合物が一般式（８）で表されるように、比較的に複雑な構造の環式構造を有する場合には、Huisgen反応において同触媒は通常は不要である。 Another example of the compound having the acetylene structure is the compound represented by the following general formula (7).

In general formula (7), -Z represents a functional group that forms an NC single bond with the nitrogen atom shown in general formula (7). Among the compounds represented by the general formula (7) and the compounds represented by the general formula (1), the compound in which the functional group -X is -N ₃ is subjected to the Huisgen reaction to obtain the following general formula (8) )

A compound represented by can be obtained. Binding the obtained compound represented by general formula (8) to the cell nucleus corresponds to introducing a compound having a functional group -Z into the cell nucleus. Generally, when a compound having an acetylene structure has a cyclic structure with a relatively complicated structure as represented by general formula (8), the same catalyst is usually unnecessary in the Huisgen reaction.

Specific examples of the functional group -Z preferably include signal-transmitting molecules such as fluorescent dyes, agents that act on intranuclear genomic DNA such as DNA alkylating agents, and the like.

The advantage of introducing a functional molecule using the Huisgen reaction according to the present invention is that, in the above example, the compound represented by the general formula (5) or the general formula (7), that is, the functional molecule intended to be introduced into a cell , there is no need to introduce an azide structure. Since introduction of an azide structure into a compound having an arbitrary chemical structure usually requires a complicated reaction, the use of the Huisgen reaction eliminates the need to introduce an azide structure as in the present invention. can lead to further promotion of

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

The following equipment was used for the measurement of the synthesized compounds.
NMR spectra JNM-ECX500II ( ¹ H: 500 MHz, ¹³ C: 125 MHz) spectrometer (JEOL) and chemical shifts: set by residual protons of solvent
(CD3OD: δ3.30 ( ¹ H NMR)
FAB mass spectrometry: JMS-700N mass spectrometer (JEOL)
Matrix: nitrobenzylalcohol
confocal microscope
C2 confocal laser scanning microscope (Nikon)
Unless otherwise specified, reagents and the like for synthesis were manufactured by Fujifilm Wako Pure Chemical Industries, Tokyo Kasei Kogyo, Kanto Kagaku, and Sigma-Aldrich.

ニトロアニリン誘導体1 (680 mg, 1.93×10^-2 mol)、アルデヒド2 (1.46 g, 9.66×10^-3 mol)、Sn (1.29 g, 1.09×10^-2 mol)の混合物にEtOH (15 mL)と濃塩酸 (1 mL)を加え、90 ℃で3時間攪拌した。セライトを用いてろ過をした後、水酸化ナトリウム水溶液を用いてろ液を中和した。続いて、Na₂S₂O₅水溶液（388 mg, 2.04×10^-3 molを水2 mLに溶解したもの)を加えて、100 ℃で2時間攪拌した。反応終了後、溶媒を留去した後、カラムクロマトグラフィー（固定相: SiO₂, 移動相: CHCl₃-MeOH-aq.NH₃）で分離し、Hoechst-NH2（680 mg, 83%）をオレンジ色固体として得た。
なお、ニトロアニリン誘導体1は、文献J. Med. Chem. 1996, 39, 4804-4809を参考に合成し、得た。
同定：¹H NMR (CD₃OD, 500 MHz) δ8.21 (d, 1H, J = 1.0 Hz), 7.91 (dd, 1H, J = 1,0, 8.5 Hz), 7.87 (d, 1H, J = 9.0 Hz), 7.64 (d, 1H, J = 8.0 Hz), 7.50 (d, 1H, J = 8.5 Hz), 7.14 (brs, 1H), 7.04 (dd, 1H, J = 1,5, 9.0 Hz), 6.80 (d, 1H, J = 8.5 Hz), 3.22 (t, 4H, J = 4.5 Hz), 2.67 (t, 4H, J = 4.5 Hz), 2.37 (s, 3H); FABMS (NBA) m/z 424 [(M + H)⁺]; HRMS calcd. for C₂₅H₂₆N₇ [(M + H)⁺] 424.2250, found 424.2251. (1) Synthesis of Hoechst-NH2:
Hoechst-NH2 is a compound represented by the above general formula (4) (wherein R1 is a methyl group), and this compound was obtained by the following scheme.

A mixture of nitroaniline derivative 1 (680 mg, 1.93×10 ^-2 mol), aldehyde 2 (1.46 g, 9.66×10 ^-3 mol) and Sn (1.29 g, 1.09×10 ^-2 mol) was added with EtOH (15 mL). and concentrated hydrochloric acid (1 mL) were added, and the mixture was stirred at 90°C for 3 hours. After filtration using celite, the filtrate was neutralized using an aqueous sodium hydroxide solution. Subsequently, an aqueous Na ₂ S ₂ O ₅ solution (388 mg, 2.04×10 ⁻³ mol dissolved in 2 mL of water) was added, and the mixture was stirred at 100° C. for 2 hours. After completion of the reaction, the solvent was distilled off and separated by column chromatography (stationary phase: SiO ₂ , mobile phase: CHCl ₃ -MeOH-aq.NH ₃ ) to obtain Hoechst-NH2 (680 mg, 83%) as an orange Obtained as a colored solid.
Nitroaniline derivative 1 was obtained by synthesizing with reference to J. Med. Chem. 1996, 39, 4804-4809.
Identification: ¹ H NMR (CD ₃ OD, 500 MHz) δ8.21 (d, 1H, J = 1.0 Hz), 7.91 (dd, 1H, J = 1,0, 8.5 Hz), 7.87 (d, 1H, J = 9.0 Hz), 7.64 (d, 1H, J = 8.0 Hz), 7.50 (d, 1H, J = 8.5 Hz), 7.14 (brs, 1H), 7.04 (dd, 1H, J = 1,5, 9.0 Hz ), 6.80 (d, 1H, J = 8.5 Hz), 3.22 (t, 4H, J = 4.5 Hz), 2.67 (t, 4H, J = 4.5 Hz), 2.37 (s, 3H); FABMS (NBA) m /z 424 [(M + H) ⁺ ]; HRMS _calcd . for _C25H26N7 [(M + H) ⁺ ] _424.2250 , found 424.2251.

Hoechst-NH2 (18 mg, 4.25×10^-5 mol)に水（2 mL）と6N 塩酸(200 μL)を加え、0 ℃で10分攪拌した後、NaNO₂水溶液 (14 mgのNaNO₂を水1mLに溶解したもの)を加えた。続いて、0 ℃で30分攪拌した後、NaN₃水溶液（17 mgのNaN₃を水1mLに溶解したもの)を加え、0 ℃で30分攪拌した。さらに、室温に温度を上げて30分攪拌した。反応終了後、溶媒を留去した後、カラムクロマトグラフィー（固定相: SiO₂, 移動相: CHCl₃-MeOH-aq.NH₃）で分離し、Hoechst-N3（18mg, 94%）を赤色固体として得た。
同定：¹H NMR (CD₃OD, 500 MHz) δ 8.28 (d, 1H, J = 1.0 Hz), 8.14 (d, 2H, J = 9.0 Hz), 7.97 (dd, 1H, J = 1.5, 8.5 Hz), 7.71 (d, 1H, J = 8.5 Hz), 7.51 (d, 1H, J = 9.0 Hz), 7.25 (d, 2H, J = 8.5 Hz), 7.14 (d, 1H, J = 1.5 Hz), 7.05 (dd, 1H, J = 2.5, 9.5 Hz), 3.23 (t, 4H, J = 4.5 Hz), 2.68 (t, 1H, J = 4.5 Hz), 2.37 (s, 3H); ¹H NMR (CD₃OD, 125 MHz) δ 154.4, 151.5, 149.1, 143.5, 136.7, 131.3, 129.3, 126.1, 122.5, 120.8, 120.3, 118.4, 117.5, 116.6, 115.8. 115.3, 114.5, 101.6, 54.8, 54.7, 43.7; FABMS (NBA) m/z 450 [(M + H)⁺]; HRMS calcd. for C₂₅H₂₄N₉ [(M + H)⁺] 450.2155, found 450.2155. (2) Synthesis of Hoechst-N3:
Hoechst-N3 is a compound represented by the above general formula ( ₁ ) (wherein R1 is a methyl group and X is -N3), and this compound was obtained by the following scheme.

Water ( ₂ mL) and 6N hydrochloric acid (200 μL) were added to Hoechst- _NH2 (18 mg, 4.25×10 ^-5 mol) and stirred at 0 °C for 10 minutes. 1 mL) was added. Subsequently, after stirring at 0°C for 30 minutes, an aqueous NaN ₃ solution (17 mg of NaN ₃ dissolved in 1 mL of water) was added, and the mixture was stirred at 0°C for 30 minutes. Furthermore, the temperature was raised to room temperature and stirred for 30 minutes. After completion of the reaction, the solvent was distilled off and separated by column chromatography (stationary phase: SiO ₂ , mobile phase: CHCl ₃ -MeOH-aq.NH ₃ ) to give Hoechst-N3 (18mg, 94%) as a red solid. obtained as
Identification: ¹ H NMR (CD ₃ OD, 500 MHz) δ 8.28 (d, 1H, J = 1.0 Hz), 8.14 (d, 2H, J = 9.0 Hz), 7.97 (dd, 1H, J = 1.5, 8.5 Hz ), 7.71 (d, 1H, J = 8.5 Hz), 7.51 (d, 1H, J = 9.0 Hz), 7.25 (d, 2H, J = 8.5 Hz), 7.14 (d, 1H, J = 1.5 Hz), 7.05 (dd, 1H, J = 2.5, 9.5 Hz), 3.23 (t, 4H, J = 4.5 Hz), 2.68 (t, 1H, J = 4.5 Hz), 2.37 (s, ^3H ); ₃ OD, 125 MHz) δ 154.4, 151.5, 143.5, 143.5, 143.5, 136.3, 129.3, 126.1, 122.5, 120.3, 120.3, 118.4, 117.5, 116.6, 115.8. (NBA) m/z 450 [(M + H) ⁺ ]; HRMS calcd. for C ₂₅ H ₂₄ N ₉ [(M + H) ⁺ ] 450.2155, found 450.2155.

Hoechst-NH2 (86 mg, 2.01×10^-4 mol)を1N 塩酸(400 μL)に溶かし、室温で20分攪拌した。続いて、NaNO₂ (42 mg, 6.09×10^-4 mol)を添加し、30分室温で攪拌した。さらに、ジメチルアニリンの塩酸溶液(73 mg, 6.09×10^-4 molのジメチルアニリンを1N 塩酸に溶解したもの)を添加し、1時間室温で攪拌した。反応終了後、NaHCO₃飽和溶液を加えて中和し、酢酸エチルで抽出した。最後にカラムクロマトグラフィー（固定相: SiO₂, 移動相: CHCl₃-MeOH-aq.NH₃）で分離し、Hoechst-Azo-NMe2（26 mg, 23%, cis体とtrans体の混合物）をオレンジ色固体として得た。
同定：¹H NMR (CD₃OD, 500 MHz) δ 8.30 (br, 1H), 8.26-8.17 (1H), 8.24 (dd, 1H, J = 1.5, 9.0 Hz), 8.14 (m, 1H), 8.30-7.96 (1H), 7.95 (dd, 1H, J = 2.0, 9.0 Hz), 7.86 (m, 1H), 7.83-7.69 (2H), 7.61-7.46 (2H), 7.20-7.09 (1H), 7.06 (dd, 1H, J = 2.0, 9.0 Hz), 6.83 (d, 1H, J = 7.0 Hz), 3.30 (6H), 3.24 (m, 4H), 2.68 (m, 4H), 2.38 (s, 3H); FABMS (NBA) m/z 556 [(M + H)⁺]; HRMS calcd. for C₃₃H₃₄N₉ [(M + H)⁺] 556.2937, found 556.2935. (3) Synthesis of Hoechst-Azo-NMe2:
Hoechst-Azo-NMe2 is a compound represented by the above general formula (1) (wherein R1 is a methyl group and X is a functional group represented by (X2) above), and this compound was obtained by the following scheme.

Hoechst-NH2 (86 mg, 2.01×10 ^-4 mol) was dissolved in 1N hydrochloric acid (400 μL) and stirred at room temperature for 20 minutes. Subsequently, NaNO ₂ (42 mg, 6.09×10 ^-4 mol) was added and stirred for 30 minutes at room temperature. Furthermore, a hydrochloric acid solution of dimethylaniline (73 mg, 6.09×10 ⁻⁴ mol of dimethylaniline dissolved in 1N hydrochloric acid) was added and stirred at room temperature for 1 hour. After completion of the reaction, NaHCO ₃ saturated solution was added to neutralize and extracted with ethyl acetate. Finally, column chromatography (stationary phase: SiO ₂ , mobile phase: CHCl ₃ -MeOH-aq.NH ₃ ) separated Hoechst-Azo-NMe2 (26 mg, 23%, mixture of cis and trans isomers). Obtained as an orange solid.
Identification: ¹ H NMR (CD ₃ OD, 500 MHz) δ 8.30 (br, 1H), 8.26-8.17 (1H), 8.24 (dd, 1H, J = 1.5, 9.0 Hz), 8.14 (m, 1H), 8.30 -7.96 (1H), 7.95 (dd, 1H, J = 2.0, 9.0 Hz), 7.86 (m, 1H), 7.83-7.69 (2H), 7.61-7.46 (2H), 7.20-7.09 (1H), 7.06 ( dd, 1H, J = 2.0, 9.0 Hz), 6.83 (d, 1H, J = 7.0 Hz), 3.30 (6H), 3.24 (m, 4H), 2.68 (m, 4H), 2.38 (s, 3H); FABMS (NBA) m/z 556 [(M + H) ⁺ ]; HRMS calcd. for C ₃₃ H ₃₄ N ₉ [(M + H) ⁺ ] 556.2937, found 556.2935.

4-Nitroaniline 3 (21 mg, 1.52×10^-4 mol)とPotassium Peroxymonosulfate (122 mg, 1.98×10^-4 mol) の混合物にCH₂Cl₂ (0.5 mL), H₂O (0.5 mL)を加えて、室温で3時間攪拌した。反応終了後、反応終了後、水を加えた後、クロロホルムで抽出し、4（ニトロソニトロベンゼン）の粗生成物を得た。続いて、得られた粗生成物に、Hoechst-NH2 (19 mg, 4.48×10^-5 mol)、さらにCH₂Cl₂ ( 0.5 mL)、酢酸(0.5 mL)を加えて、室温で16時間攪拌した。反応終了後、NaOH水溶液で中和した。最後にカラムクロマトグラフィー（固定相: SiO₂, 移動相: CHCl₃-MeOH-aq.NH₃）で分離し、Hoechst-Azo-NO2（21 mg, 84%）をオレンジ色固体として得た。
同定：¹H NMR (CD₃OD, 500 MHz) δ 7.90-7.70 (5H), 7.68-7.58 (1H), 7.58-7.42 (5H), 7.25 (d, 2H, J = 8.0 Hz), 6.90-6.76 (2H), 3.32 (br, 4H), 2.86 (br, 4H), 2.53 (s, 3H); FABMS (NBA) m/z 558 [(M + H)⁺]; HRMS calcd. for C₃₁H₂₈N₉O₂ [(M + H)⁺] 558.2366, found 558.2366. (3) Hoechst-Azo-NO2
Hoechst-Azo-NO2 is a compound represented by the above general formula (1) (wherein R1 is a methyl group and X is a functional group represented by (X3) above), and this compound was obtained by the following scheme.

CH ₂ Cl ₂ (0.5 mL) and H ₂ O (0.5 mL) were added to a mixture of 4-Nitroaniline 3 (21 mg, 1.52×10 ^-4 mol) and Potassium Peroxymonosulfate (122 mg, 1.98×10 ^-4 mol). and stirred at room temperature for 3 hours. After completion of the reaction, water was added, and the mixture was extracted with chloroform to obtain a crude product of 4 (nitrosonitrobenzene). Subsequently, Hoechst-NH2 (19 mg, 4.48×10 ^-5 mol), CH ₂ Cl ₂ (0.5 mL) and acetic acid (0.5 mL) were added to the obtained crude product, and the mixture was stirred at room temperature for 16 hours. bottom. After completion of the reaction, it was neutralized with an aqueous NaOH solution. Finally, separation was performed by column chromatography (stationary phase: _SiO2 , mobile phase: CHCl3-MeOH _{- aq.NH3} ) to obtain Hoechst-Azo-NO2 (21 mg, 84%) as an orange solid.
Identification: ¹ H NMR (CD ₃ OD, 500 MHz) δ 7.90-7.70 (5H), 7.68-7.58 (1H), 7.58-7.42 (5H), 7.25 (d, 2H, J = 8.0 Hz), 6.90-6.76 (2H), 3.32 (br, 4H), 2.86 (br, 4H), 2.53 (s, 3H); FABMS (NBA) m/z 558 [ ₍ M + H) ⁺ ]; HRMS calcd. for _{C31H28 N9O2} [(M ₊ H) ⁺ ]558.2366, found 558.2366.

Hoechst-N3 (16 mg, 3.56×10^-5 mol)と1-ethynyl-4-nitrobenzene 5 (61 mg, 4.15×10^-5 mol)の混合物をDMF (300 mL)に溶かし、続いて、TBTA (4.7 mg, 2.94×10^-5 mol)、CuSO₄ (4.7 mg, 2.94×10^-5 mol)、アスコルビン酸(6.6 mg, 3.33×10^-5 mol)を加えて、4時間室温で攪拌した。反応終了後、溶媒を留去した後、カラムクロマトグラフィー（固定相: SiO₂, 移動相: CHCl₃-MeOH-aq.NH₃）で分離し、付加体6（8 mg, 38%）をオレンジ色固体として得た。
同定：¹H NMR (CD₃OD, 500 MHz) δ9.71 (s, 1H), 8.48 (d, 2H, J = 8.0 Hz), 8.40 (d, 2H, J = 7.5 Hz), 8.40-8.31 (1H), 8.24 (d, 2H, J = 7.5 Hz), 8.20 (d, 2H, J = 7.5 Hz), 8.06 (d, 1H, J = 8.0 Hz), 7.80-7.70 (1H), 7.69 (m, 1H), 6.93 (br, 2H), 3.12 (br, 4H), 2.26-2.20 (4H); FABMS (NBA) m/z 597 [(M + H)⁺]; HRMS calcd. for C₃₃H₂₉N₁₀O₂ [(M + H)⁺] 597.2475, found 597.2477. (4) Huisgen reaction of Hoechst-N3:
Hoechst-N3 synthesized above was subjected to Huisgen reaction according to the following scheme.

A mixture of Hoechst-N3 (16 mg, 3.56×10 ^-5 mol) and 1-ethynyl-4-nitrobenzene 5 (61 mg, 4.15×10 ^-5 mol) was dissolved in DMF (300 mL), followed by TBTA ( 4.7 mg, 2.94×10 ⁻⁵ mol), CuSO ₄ (4.7 mg, 2.94×10 ⁻⁵ mol) and ascorbic acid (6.6 mg, 3.33×10 ⁻⁵ mol) were added and stirred at room temperature for 4 hours. After completion of the reaction, the solvent was distilled off and separated by column chromatography (stationary phase: SiO ₂ , mobile phase: CHCl ₃ -MeOH-aq.NH ₃ ) to give adduct 6 (8 mg, 38%) as an orange Obtained as a colored solid.
Identification: ¹ H NMR (CD ₃ OD, 500 MHz) δ9.71 (s, 1H), 8.48 (d, 2H, J = 8.0 Hz), 8.40 (d, 2H, J = 7.5 Hz), 8.40-8.31 ( 1H), 8.24 (d, 2H, J = 7.5Hz), 8.20 (d, 2H, J = 7.5Hz), 8.06 (d, 1H, J = 8.0Hz), 7.80-7.70 (1H), 7.69 (m, 1H), 6.93 (br, 2H), 3.12 (br, 4H), 2.26-2.20 (4H); FABMS (NBA) m/z 597 [(M + H) ⁺ ]; HRMS calcd. for C ₃₃ H ₂₉ N _{10 O2} [(M + H) ⁺ ] 597.2475, found 597.2477.

By binding the adduct 6 thus obtained to a desired cell nucleus, 1-ethynyl-4-nitrobenzene 5 used as a raw material can be introduced into the cell nucleus.

(5) Hoechst-N3 cell experiments:
Human lung cancer cell A549 was plated on wells (medium DMEM) and cultured at 37° C. for 24 hours under aerobic conditions (O ₂ concentration 21%) and hypoxic conditions (O ₂ concentration 0.3%). Subsequently, after removing the medium, the medium (DMEM 100 μL) in which Hoechst-N3 (10 μM) was dissolved was immediately added to the cells and incubated at 37° C. for a predetermined time (10 minutes, 30 minutes, 60 minutes or 24 hours). cultured in Subsequently, after washing the cells with PBS, the cells were observed with a confocal microscope, irradiated with excitation light (Ex. 405 nm), and fluorescence emission was observed. FIG. 1 is the observed image. Although blue fluorescence is actually observed, it is converted to grayscale and drawn in FIG. The closer to white the drawing in FIG. 1 is, the stronger the blue light emission is. FIG. 1(A) is an observation image of cells under hypoxic conditions, and FIG. 1(B) is an observation image of cells under aerobic conditions. FIG. 2 shows the relative values of the observed fluorescence intensity on the vertical axis and the time course on the horizontal axis. Hoechst-N3 has been shown to selectively illuminate hypoxic cells.

Claims (6)

General formula (1) below

(Wherein, -R1 represents an alkyl group having 1 to 3 carbon atoms, and -X represents a functional group represented by any one of the following (X1) to (X3).)

A bisbenzimide analogue represented by A compound represented by the following general formula (2) (wherein —R represents the same functional group as above) and 4-nitrobenzaldehyde represented by the following general formula (3)

in the presence of the following general formula (4) by reducing under acidic conditions and then cyclizing

(Wherein, -R1 represents the same functional group as above) to obtain a bisbenzimide analogue represented by
By substituting the terminal amino group of the bisbenzimide analogue represented by the general formula (4) with the functional group X,
A method for producing a bisbenzimide analog represented by the general formula (1) according to claim 1. binding the bisbenzimide analog according to claim 1 to the cell nucleus of the test cell, irradiating the test cell after the binding with light, and observing fluorescence based on the irradiation;
A method for evaluating the oxygen concentration of test cells. A diagnostic agent containing the bisbenzimide analogue according to claim 1,
The bisbenzimide analog is bound to the cell nuclei of the test cells, the test cells after the binding are irradiated with light, and fluorescence based on the irradiation is observed to evaluate the oxygen concentration of the test cells. ,
Said diagnostic agent. The bisbenzimide analogue according to claim 1 (where -X is a functional group represented by the following (X1)) and the following general formula (5)

(Wherein, -Y represents a functional group that forms a C—C single bond with the carbon atom shown in general formula (5).) by reacting the compound represented by the following general formula (6)

Obtaining a compound represented by and binding the obtained compound represented by the general formula (6) to the cell nucleus,
A method for introducing a compound having a functional group -Y into a cell nucleus. The bisbenzimide analogue according to claim 1 (where -X is a functional group represented by the following (X1)) and the following general formula (7)

(Wherein, -Z represents a functional group that forms an NC single bond with the nitrogen atom represented by the general formula (7).) The compound represented by the following general formula (8)

Obtaining a compound represented by and binding the obtained compound represented by the general formula (8) to the cell nucleus,
A method for introducing a compound having a functional group -Z into a cell nucleus.

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