JP6116842B2 - Target cell specific photosensitizing compound. - Google Patents

Description

The present invention relates to a novel photosensitizing compound capable of acting specifically on a target cell, and a method for specifically inducing cell death of a target cell expressing a specific enzyme using the compound. More specifically, the present invention relates to a photosensitizing compound that selectively induces cell death in cells expressing a reporter enzyme such as β-galactosidase.

A photosensitizer is a dye compound that can generate active oxygen species such as singlet oxygen ( ¹ O ₂ ) by light irradiation and can load oxidative stress on the surrounding environment. Expected as a tool to conditionally induce cell death and analyze its function because it is relatively toxic in the dark and can be loaded with oxidative stress at any time by controlling light irradiation Is growing.

As typical photosensitizers, photofurin and porphyrin are known in the art. However, in order to induce cell death in a specific cell group, it is necessary to exhibit phototoxicity only in those target cells. However, conventional photosensitizers are always activated by irradiation with light after being administered in vivo. In order to generate seeds, there has been a problem that non-specific damage caused by the sensitizer occurs outside the cells and tissues to which oxidative stress is to be applied. For example, when a specific cell death is aimed at in vivo using a photosensitizer, nonspecific cell death caused by a photosensitizer distributed outside the target cell is expressed, and high spatial resolution is achieved. Realizing selective cell death is difficult. If oxidative stress is applied to only certain types of cells among various cells and cell death can be realized, it is expected to be used as an innovative biological tool.

Similar to green fluorescent protein (GFP), luciferase, β-galactosidase, alkaline phosphatase and the like, β-galactosidase is widely used as a reporter enzyme for verifying transcriptional control and evaluating transfection efficiency ( Non-Patent Documents 1 and 2). Until now, development of a fluorescent substrate for visualizing the activity of β-galactosidase in living cells has been attempted, but there are problems such as insufficient selectivity and low cell permeability ( Non-patent document 3). In addition, in order to load β-galactosidase-expressing cells with oxidative stress and verify their functions, it is necessary to exhibit phototoxicity only in those target cells, but conventional photosensitizers have sufficient selectivity. At present, there is no such thing that has not been widely used.

J. et al. Alam et al. Anal. Biochem. 1990, 188, 245 D. J. et al. Supergel et al. Prog. Neurobiol. , 2001, 63, 673 V. A. Rakhmanova et al. Anal. Biochem. 1998, 257, 234

The problem to be solved by the present invention is to provide a photosensitizing compound capable of selectively applying oxidative stress to a target cell, particularly a reporter enzyme-expressing cell such as β-galactosidase.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have used a compound having a serenrodol skeleton containing a group cleaved by a reporter enzyme such as β-galactosidase to thereby produce a spiro ring by an enzymatic reaction. It has been found that by controlling the ring opening of the compound to change the visible light absorption of the compound, a photosensitizing action that selectively causes cell death of the enzyme-expressing cells becomes possible. Based on this finding, the present invention has been completed.

That is, this invention provides the compound for selective photosensitization containing the compound or its salt represented by the following formula | equation (I) in one aspect | mode.

In the formula, A represents a monovalent group cleaved by an enzyme; R ¹ represents 1 to 4 identical or different substituents bonded to a hydrogen atom or a benzene ring; R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group; X is C ₁ —C ₃ represents an alkylene group. Preferably, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are hydrogen, R ⁸ and R ⁹ are ethyl groups, and X is a methylene group.

A is preferably a group cleaved by a reporter enzyme, wherein the reporter enzyme is preferably β-galactosidase, β-lactamase, alkaline phosphatase, luciferase, or peroxidase.

In a more preferred embodiment, the reporter enzyme is β-galactosidase and A is a galactopyranosyl group. Specifically, it is a compound for selective photosensitization including a compound represented by the following formula (II) or a salt thereof (wherein Et represents an ethyl group).

式中、Ｒ^１〜Ｒ^９は、上記式（I）における定義と同じである。当該式（III）の光増感剤は、式（I）の化合物と酵素との反応によって基Ａが切断され、含セレンロドール骨格におけるスピロ環部位が開環し構造変化したものである。当該化合物に光照射することによって、空気中の酸素から一重項酸素（^１Ｏ_２）などの活性酸素種が生成され、それによって、標的細胞に酸化ストレスを与え細胞死を誘発することができる。すなわち、上記の一般式（I）で表される本発明の化合物はそれ自身は光照射に伴ない活性酸素種を生成する光増感能を有しないが、酵素との接触による基Ａの切断によって上記の一般式(III)で表される強い光増感能を有する化合物を与える性質を有する。 In another embodiment, the present invention relates to a photosensitizer represented by the following formula (III).

In the formula, R ^{1 to} R ⁹ are the same as defined in the above formula (I). In the photosensitizer of the formula (III), the group A is cleaved by the reaction of the compound of the formula (I) with an enzyme, and the spiro ring site in the serenrodol skeleton is opened to change the structure. By irradiating the compound with light, reactive oxygen species such as singlet oxygen ( ¹ O ₂ ) are generated from oxygen in the air, thereby applying oxidative stress to the target cell and inducing cell death. That is, the compound of the present invention represented by the above general formula (I) does not itself have a photosensitizing ability to generate reactive oxygen species upon light irradiation, but the group A is cleaved by contact with an enzyme. To give a compound having a strong photosensitizing ability represented by the above general formula (III).

In another aspect, the present invention provides a method for specifically inducing cell death of a target cell in which a specific enzyme is expressed, using the photosensitizing compound of the above formula (I). . The method comprises a step of bringing a compound for photosensitization of formula (I) into contact with an enzyme that is specifically expressed in the target cell to produce a compound represented by formula (III) above, and irradiation with excitation light And a step of generating reactive oxygen species with the compound represented by the formula (III) and loading the target cells with oxidative stress. Preferably, the target cell is a β-galactosidase expressing cell.

The present invention also relates to the use of the photosensitizing compound of formula (I) for specifically inducing cell death in target cells expressing a specific enzyme. As above, the target cell is preferably a β-galactosidase expressing cell.

  According to the present invention, by using a compound having a selenrodol skeleton into which a group cleaved by a reporter enzyme such as β-galactosidase is introduced, the opening of the spiro ring by the enzymatic reaction is controlled, and the visible light absorption of the compound is achieved. It is possible to bring about a photosensitizing effect that selectively causes cell death of the enzyme-expressing cells.

More specifically, the compound of formula (I) has no visible light absorption before the reaction with the enzyme, so that the phototoxicity is suppressed to a very low level. Due to the structural change in which the spiro ring site is opened, visible light absorption is greatly recovered, and high phototoxicity is obtained. By using such a technique, only target cells transfected with a specific enzyme lead to cell death depending on the concentration of the photosensitizer. Therefore, selective phototoxicity that could not be achieved with conventional photosensitizers. It has an excellent effect that control becomes possible.

Further, since a rhodol skeleton having a high intracellular retention property is used as the basic skeleton of the photosensitizer, the compound of the present invention can be retained in the cell and exhibit sufficient phototoxicity, and is close to the living body. There is also an effect of having excellent absorption characteristics that can be used as a photosensitizer in a pH environment.

In particular, the photosensitizer of formula (III) used in the present invention can be excited by the green light of a general-purpose lamp, and a system that leads to the death of transfected cells at any timing without special equipment is established. Therefore, it is very useful when targeting cells that express β-galactosidase, which is widely used as a reporter enzyme. It can be easily combined with a technique for expressing β-galactosidase at a specific site or tissue using an animal with established genetics such as Drosophila or mouse, and can be applied to any cell in vivo at any timing. It is also excellent in terms of practicality in that it can be subjected to oxidative stress to investigate its function.

FIG. 1 shows changes in the absorption spectrum (FIG. 1a) and fluorescence spectrum of SeRhodol, a photosensitizer produced by an enzymatic reaction between SeRhodol-gal, which is a photosensitizing compound of the present invention, and β-galactosidase. (FIG. 1 b) and the emission of excited singlet oxygen ( ¹ O ₂ ) (FIG. 1 c). FIG. 2 is a diagram showing the pH dependence of the absorption spectrum change of SeRhodol-gal, which is a photosensitizer produced by an enzymatic reaction between SeRhodol-gal, which is a photosensitizing compound of the present invention, and β-galactosidase. It is. FIG. 3 is a graph showing the result of CCK assay after HEK293 cells introduced with β-galactosidase (LacZ) are subjected to photosensitization treatment with the compound of the present invention as compared with the case where luciferase (Luc) is introduced. It is.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

In this specification, the alkyl group may be any of an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof. Although carbon number of an alkyl group is not specifically limited, For example, it is about C1-C6, Preferably it is C1-C4. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of the substituent include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. A group, an aryl group, and the like can be mentioned, but are not limited thereto. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (for example, an alkyloxy group or an aralkyl group).

In the present specification, the aryl group may be either a monocyclic aryl group or a condensed polycyclic aryl group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom 1 or more may be included. In the present specification, an aryl group may have one or more arbitrary substituents on the ring. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing an aryl moiety (for example, an aryloxy group and an aralkyl group).

(1) Compound for selective photosensitization The compound for selective photosensitization of the present invention is a compound having a structure represented by the following general formula (I) in one aspect.

In the above general formula (I), R ¹ represents a hydrogen atom or 1 to 4 substituents bonded to a benzene ring. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When having two or more substituents on the benzene ring, they may be the same or different. R ¹ is more preferably a hydrogen atom, a lower alkyl group or a lower alkoxy group. A hydrogen atom is particularly preferred.

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom. R ² and R ⁷ are preferably hydrogen atoms. It is also preferred that R ³ , R ⁴ , R ⁵ , R ⁶ are hydrogen atoms. More preferably, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are all hydrogen atoms.

R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group. When R ⁸ and R ⁹ both represent an alkyl group, they may be the same or different. For example, R ⁸ and R ⁹ are each independently preferably a methyl group or an ethyl group, and more preferably R ⁸ and R ⁹ are both ethyl groups.

X represents a ^C 1 -C ³ alkylene group. The alkylene group may be a linear alkylene group or a branched alkylene group. For example, in addition to a methylene group (—CH ₂ —), an ethylene group (—CH ₂ —CH ₂ —), a propylene group (—CH ₂ —CH ₂ —CH ₂ —), a branched alkylene group such as —CH ( CH ₃ ) —, —CH ₂ —CH (CH ₃ ) —, —CH (CH ₂ CH ₃ ) — and the like can also be used. Among these, a methylene group or an ethylene group is preferable, and a methylene group is more preferable.

The group A represents a monovalent group that can be cleaved by an enzyme. Examples of the enzyme for cleaving A include a reductase, an oxidase, a hydrolase, and the like. More specifically, Examples include, but are not limited to, β-galactosidase, β-lactamase, alkaline phosphatase, luciferase, peroxidase cytochrome P450 oxidase, β-glucosidase, β-glucuronidase, β-hexosaminidase, lactase, etc. It will never be done. Preferred are enzymes that can be used as reporters such as β-galactosidase, β-lactamase, alkaline phosphatase, luciferase, or peroxidase. Most preferred is β-galactosidase.

The compound represented by the above formula (I) (including the case of the embodiment of formula (II). The same applies to the following description) may exist as a salt. Examples of the salt include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. Examples thereof include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate. Examples of amino acid salts include glycine salts. However, the salt of the compound of the present invention is not limited to these.

The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

The compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.

  In the examples of the present specification, production methods for typical compounds included in the compounds of the present invention represented by the general formula (I) are specifically shown. Any compound included in the general formula (I) can be easily produced by referring to the disclosure and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.

(2) Mechanism of photosensitizing action in the present invention When the selective photosensitizing compound represented by the formula (I) provided by the present invention is incorporated into cells, the group represented by A is cleaved. In a cell expressing a possible enzyme, the group represented by A is cleaved in the cell to produce a compound of formula (III) having a photosensitizing ability.

For example, in the case of a photosensitizing compound of the formula (II), cleavage of the group A and opening of the spiro ring are caused by β-galactosidase as follows, and a photosensitizer corresponding to the formula (III) is obtained. Generate.

When irradiated with excitation light in this state, active oxygen species such as singlet oxygen ( ¹ O ₂ ) are generated from the compound represented by the above formula (II) existing in the cell, and oxidative stress is loaded on the cell. As a result, cell death is induced. On the other hand, if the cell incorporating the selective photosensitizing compound represented by formula (I) does not express an enzyme or reactive oxygen species capable of cleaving the group represented by A, formula (III) The represented compound is not generated, and reactive oxygen species such as singlet oxygen are not generated in the cells by light irradiation. Since the compound represented by the formula (I) itself does not have photosensitizing ability, it is not loaded with oxidative stress even by light irradiation. As described above, by using the selective photosensitizing compound represented by the formula (I), only cells expressing an enzyme or reactive oxygen species capable of cleaving the group represented by A are selectively selected. It can lead to death.

Therefore, the compound represented by the formula (I) of the present invention can be used as a tool for cell biological research in a cell line in which a reporter enzyme is expressed. In cell biology research, known gene transfer methods can be used, and the function and role of the gene-transferred cells can be analyzed by comparing the response to external stimuli before and after light irradiation. it can.

The compound for selective photosensitization represented by the formula (I) of the present invention, and the photosensitizer represented by the formula (III), which is a photosensitizer generated by an enzymatic reaction between the compound and a target enzyme, Typically, the acid dissociation equilibrium is shown as follows, and as shown in FIG. 2, the absorbance difference in the vicinity of neutral pH, which is the in vivo environment, is very large. Can be provided. Therefore, it is very useful in the use of the tool for cell biological research as described above.

(3) Method for inducing selective cell death by the photosensitizing compound of the present invention In order to show the above-described mechanism of photosensitizing action, the photosensitizing compound of the present invention is expressed as a target expressed by a specific enzyme. It can be used in a method for specifically inducing cell death of cells. Specifically, the step of contacting the photosensitizing compound of formula (I) with an enzyme such as β-galactosidase that is specifically expressed in the target cell to produce a compound represented by formula (III), Only target cells such as β-galactosidase-expressing cells by performing a step of irradiating excitation light to generate reactive oxygen species with the compound represented by the formula (III) and loading the target cells with oxidative stress. Can specifically lead to cell death.

As a means for bringing the photosensitizing compound of the present invention into contact with an enzyme that is specifically expressed in a target cell, typically, a sample containing a photosensitizing compound may be added, applied, or sprayed. Although it is mentioned, it is possible to select appropriately according to the use.

Moreover, the light irradiation performed to a target cell can irradiate the said cell with light directly or via a waveguide (optical fiber etc.). As the light source, any light source can be used as long as it can irradiate light including the absorption wavelength of the photosensitizer represented by the formula (III). Depending on the environment in which the method of the present invention is performed, etc. It can be appropriately selected.

As the photosensitizing compound of the present invention, the compound represented by the above general formula (I) or a salt thereof may be used as it is, but if necessary, an additive usually used for the preparation of a reagent is blended. And may be used as a composition. For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible. These compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It is sufficient to dissolve and apply to.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.

Compound 5 (SeRhodol-gal), which is a selective photosensitizing compound of the present invention, was synthesized according to the following scheme.

４−ヒドロキシ安息香酸（３．０ｇ、２１．７ｍｍｏｌ）を１５ｍＬのＳＯＣｌ_２中に懸濁させ、２時間還流した後、反応溶媒を減圧除去した。残渣をジクロロメタン５０ｍＬに溶解し、ジエチルアミン１５ｍＬを滴下した。混合物を室温で２時間攪拌し、反応溶媒を減圧除去した。再度、ジエチルアミン２０ｍＬを添加し、１８時間還流した。反応溶媒を減圧除去し、粗生成物をシリカゲルクロマトグラフィーで精製して（ジクロロメタン、５％メタノール）、白色固体の目的化合物を得た（３．２８ｇ、収率７８％）。 (A) Synthesis of Compound 1 (N, N-diethyl-4-hydroxybenzamide)

4-Hydroxybenzoic acid (3.0 g, 21.7 mmol) was suspended in 15 mL of SOCl ₂ and refluxed for 2 hours, after which the reaction solvent was removed under reduced pressure. The residue was dissolved in 50 mL of dichloromethane, and 15 mL of diethylamine was added dropwise. The mixture was stirred at room temperature for 2 hours and the reaction solvent was removed under reduced pressure. Again, 20 mL of diethylamine was added and refluxed for 18 hours. The reaction solvent was removed under reduced pressure, and the crude product was purified by silica gel chromatography (dichloromethane, 5% methanol) to obtain the target compound as a white solid (3.28 g, yield 78%).

Ｎ，Ｎ−ジエチル−４−ヒドロキシベンズアミド（１．２ｇ、６．２５ｍｍｏｌ）及びＫ_２ＣＯ_３（２．０ｇ、１４．５ｍｍｏｌ）をジメチルホルムアミド（ＤＭＦ）２０ｍＬに懸濁し、臭化アリル２０ｍＬ（２３．７ｍｍｏｌ）を滴下した。混合物を８０℃まで加熱し、攪拌した。室温に冷却後、反応溶液に水５０ｍＬを添加し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥した。反応溶媒を減圧除去し、シリカゲルクロマトグラフィーで精製して（ジクロロメタン）、無色油状の目的化合物を得た（１．３６ｇ、収率７５％）。 (B) Synthesis of compound 2 (4- (allyloxy) -N, N-diethylbenzamide)

N, N-diethyl-4-hydroxybenzamide (1.2 g, 6.25 mmol) and K ₂ CO ₃ (2.0 g, 14.5 mmol) were suspended in 20 mL of dimethylformamide (DMF), and 20 mL of allyl bromide (23 0.7 mmol) was added dropwise. The mixture was heated to 80 ° C. and stirred. After cooling to room temperature, 50 mL of water was added to the reaction solution, extracted with ethyl acetate, washed with brine, and dried over Na ₂ SO ₄ . The reaction solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane) to obtain the target compound as a colorless oil (1.36 g, yield 75%).

¹ H NMR (300 MHz, CDCl ₃ ): 1.19 (br s, 6H), 3.43 (br s, 4H), 4.58 (d, 2H, J = 6.0 Hz), 5.45-5.48 (m, 2H), 6.03-6.11 (m, 1H), 6.93 (d, 2H, J = 8.8 Hz), 7.35 (d, 2H, J = 8.8 Hz)
¹³ C NMR (75 MHz, CDCl ₃ ): 168.8, 101.6, 114.4, 117.9, 128.2, 132.9, 171.2, 188.8
LRMS (ESI ⁺ ): [M + H] ⁺ 234, Found 234

３−ブロモアニリン（６．３ｇ、３６．７ｍｍｏｌ）をリン酸トリエチル６．２ｍｌ（３６．２ｍｍｏｌ）に加え、２１０℃まで加熱し、３時間攪拌した。室温まで冷却した後、反応溶液に食塩水を添加してＣＨ_２Ｃｌ_２で抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４によって乾燥した。続いて、反応溶媒を減圧除去し、粗生成物をシリカゲルクロマトグラフィーで精製して（ジクロロメタン／ヘキサン＝２／８）、無色油状の目的化合物（５．７４ｇ、収率６９％）を得た。 (C) Synthesis of 3,3′-diselanediylbis (N, N-diethylaniline) (c-1) Synthesis of N, N-diethyl-3-bromoaniline:

3-Bromoaniline (6.3 g, 36.7 mmol) was added to 6.2 ml (36.2 mmol) of triethyl phosphate, heated to 210 ° C., and stirred for 3 hours. After cooling to room temperature, brine was added to the reaction solution, extracted with CH ₂ Cl ₂ , washed with brine, and dried over Na ₂ SO ₄ . Subsequently, the reaction solvent was removed under reduced pressure, and the crude product was purified by silica gel chromatography (dichloromethane / hexane = 2/8) to obtain a colorless oily target compound (5.74 g, yield 69%).

¹ H NMR (300 MHz, CDCl ₃ ): 1.14 (t, 6H J = 7.3 Hz), 3.31 (q, 4H, J = 7.3 Hz), 6.56 (dd, 1H J = 2.2, 8.8 Hz), 6.72-6.76 ( m, 2H), 7.03 (t, 1H, J = 8.8 Hz)
¹³ C NMR (75 MHz, CDCl ₃ ): 12.4, 44.3, 110.2, 114.2, 117.9, 123.6, 130.4, 148.9

Ｍｇ（１．２３ｇ、５０ｍｍｏｌ）をアルゴン雰囲気下で乾燥テトラヒドロフラン（ＴＨＦ）５０ｍＬに添加した。溶液が暗灰色になるまでＮ，Ｎ−ジエチル−３−ブロモアニリン（２．８９ｇ、１２．７ｍｍｏｌ）の乾燥ＴＦＨ溶液５０ｍＬを添加し、５０℃に加熱した。混合物を室温に冷却し、セレン粉末（１．０ｇ、１２．７ｍｍｏｌ）を添加した。続いて、混合物を３時間攪拌し、飽和ＮａＨＣＯ_３溶液３０ｍＬを添加し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥した。残渣をシリカゲルクロマトグラフィーで精製して（ジクロロメタン／ヘキサン＝３／７）、黄色油状のジセレニドを得た（１．７８ｇ、収率６２％）。 (C-2) Synthesis of 3,3′-diselanediylbis (N, N-diethylaniline):

Mg (1.23 g, 50 mmol) was added to 50 mL of dry tetrahydrofuran (THF) under an argon atmosphere. 50 mL of a dry TFH solution of N, N-diethyl-3-bromoaniline (2.89 g, 12.7 mmol) was added and heated to 50 ° C. until the solution turned dark gray. The mixture was cooled to room temperature and selenium powder (1.0 g, 12.7 mmol) was added. Subsequently, the mixture was stirred for 3 hours, 30 mL of saturated NaHCO ₃ solution was added, extracted with ethyl acetate, washed with brine and dried over Na ₂ SO ₄ . The residue was purified by silica gel chromatography (dichloromethane / hexane = 3/7) to give a yellow oily diselenide (1.78 g, 62% yield).

４−（アリルオキシ）−Ｎ，Ｎ−ジエチルベンズアミド（６６１ｍｇ、２．３ｍｍｏｌ）をアルゴン雰囲気下にて乾燥ＴＨＦ１０ｍＬに溶解させた。溶液を−７８℃に冷却し、１Ｍのｓ−ＢｕＬｉ（２．３ｍＬ）をゆっくり添加した。溶液を１０分間攪拌し続け、ジセレニド（８５０ｍｇ、１．６８ｍｍｏｌ）のＴＦＨ溶液１０ｍＬを添加した。混合物を室温の戻し、２時間攪拌した。反応溶液にＮａＨ_２ＰＯ_４溶液を添加し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥した。反応溶媒を減圧除去し、シリカゲルクロマトグラフィーで粗精製して（酢酸エチル／ヘキサン＝１／９）、淡黄色油状の中間体を得た（２６１ｍｇ）。続いて、当該中間体及び１ｍＬのジエチルベンズアミドをアセトニトリル５ｍＬに溶解し、１ｍＬのＰＯＣｌ_３を添加した。混合物をアルゴン雰囲気下で１時間加熱還流した。室温に冷却後、２ＮのＮａＯＨを１５ｍＬ添加し、３時間攪拌した。溶液に水を添加し、酢酸エチルで抽出し、食塩水で洗浄後、減圧除去し、Ｎａ_２ＳＯ_４で乾燥した。残渣をシリカゲルクロマトグラフィーで精製して（酢酸エチル／ヘキサン＝２／８）、淡黄色固体のセレノキサンテンを得た（１１２ｍｇ、２ステップで収率１７％）。 (D) Synthesis of compound 3 (selenoxanthene)

4- (Allyloxy) -N, N-diethylbenzamide (661 mg, 2.3 mmol) was dissolved in 10 mL of dry THF under an argon atmosphere. The solution was cooled to −78 ° C. and 1M s-BuLi (2.3 mL) was added slowly. The solution was kept stirring for 10 minutes and 10 mL of a TFH solution of diselenide (850 mg, 1.68 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 2 hours. A NaH ₂ PO ₄ solution was added to the reaction solution, extracted with ethyl acetate, washed with brine, and dried over Na ₂ SO ₄ . The reaction solvent was removed under reduced pressure, and the residue was roughly purified by silica gel chromatography (ethyl acetate / hexane = 1/9) to obtain a pale yellow oily intermediate (261 mg). Subsequently, the intermediate and 1 mL of diethylbenzamide were dissolved in 5 mL of acetonitrile and 1 mL of POCl ₃ was added. The mixture was heated to reflux under an argon atmosphere for 1 hour. After cooling to room temperature, 15 mL of 2N NaOH was added and stirred for 3 hours. Water was added to the solution, extracted with ethyl acetate, washed with brine, removed under reduced pressure, and dried over Na ₂ SO ₄ . The residue was purified by silica gel chromatography (ethyl acetate / hexane = 2/8) to give selenoxanthene as a pale yellow solid (112 mg, 17% yield over 2 steps).

¹ H NMR (300 MHz, CDCl ₃ ): 1.19 (t, 6H, J = 7.2 Hz), 3.39 (q, 4H, J = 7.2 Hz), 4.58 (d, 2H, J = 5.9 Hz), 5.29-5.45 ( m, 2H), 6.01-6.04 (m, 1H), 6.60 (d, 1H, J = 2.6 Hz), 6.72 (dd, 1H, J = 2.6, 9.2 Hz), 6.95 (dd, 1H, J = 2.2, 8.8 Hz), 6.98 (d, 1H, J = 2.6 Hz), 8.45 (d, 1H, J = 8.8 Hz), 8.55 (d, 1H, J = 8.8 Hz)
¹³ C NMR (75 MHz, CDCl ₃ ): 12.5, 44.4, 68.9, 106.9, 111.3, 111.7, 114.4, 118.1, 119.2, 125.0, 132.3, 136.2, 137.0, 149.7, 160.6, 179.6
LRMS (ESI ⁺ ): [M + H] ⁺ 388, Found 388

１−ブロモ−２−（ｔｅｒｔ−ブトキシメチル）ベンゼン（１４２ｍｇ、０．５８ｍｍｏｌ）をアルゴン雰囲気下にて乾燥ＴＨＦ５ｍＬに溶解させた。当該溶液を−７８℃に維持し、１Ｍのｓ−ＢｕＬｉ（２．３ｍＬ）を滴下した。溶液を１０分間攪拌し続け、セレノキサンテン（３１ｍｇ、０．０８０ｍｍｏｌ）のＴＦＨ溶液５ｍＬを添加した。混合物を室温の戻し、３時間攪拌した。反応溶液に２ＮのＨＣｌを添加して酸性化し、数分間攪拌した。反応溶液を飽和ＮａＨＣＯ_３で中和し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥し、減圧除去した。残渣をジクロロメタン２ｍＬに溶解し、ＴＦＡ２ｍＬを添加し、４０℃で１時間攪拌した。続いて、混合物に２ＮのＮａＯＨを１０ｍＬ添加し、さらに３０分間攪拌し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥し、減圧除去した。残渣をシリカゲルクロマトグラフィーで粗精製した。 (E) Synthesis of Compound 4 (SeRhodol)

1-Bromo-2- (tert-butoxymethyl) benzene (142 mg, 0.58 mmol) was dissolved in 5 mL of dry THF under an argon atmosphere. The solution was maintained at −78 ° C. and 1M s-BuLi (2.3 mL) was added dropwise. The solution was kept stirring for 10 minutes and 5 mL of a TFH solution of selenoxanthene (31 mg, 0.080 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction solution was acidified by adding 2N HCl and stirred for several minutes. The reaction solution was neutralized with saturated NaHCO ₃ , extracted with ethyl acetate, washed with brine, dried over Na ₂ SO ₄ and removed under reduced pressure. The residue was dissolved in 2 mL of dichloromethane, 2 mL of TFA was added, and the mixture was stirred at 40 ° C. for 1 hour. Subsequently, 10 mL of 2N NaOH was added to the mixture, and the mixture was further stirred for 30 minutes, extracted with ethyl acetate, washed with brine, dried over Na ₂ SO ₄ and removed under reduced pressure. The residue was roughly purified by silica gel chromatography.

To the crude product, 3 mL of a methanol solution of K ₂ CO ₃ (13.0 mg, 0.094 mmol) and Pd (PPh ₃ ) ₄ (40.6 mg, 0.0048 mmol) was added and stirred at room temperature for 3 hours under an argon atmosphere. did. 10 mL no water was added, the mixture was extracted with ethyl acetate, washed with brine, dried over Na ₂ SO ₄ and removed under reduced pressure. Purification by preparative HPLC gave pink powder SeRhodol (6.8 mg, 20% yield over 3 steps).

¹ H NMR (300 MHz, CD ₃ OH): 1.33 (t, 6H, J = 7.1 Hz), 3.74 (q, 4H, J = 7.1 Hz), 4.23 (s, 2H), 6.97 (dd, 1H, J = 8.8, 2.2 Hz), 7.18 (dd, 1H, J = 2.6, 9.5 Hz), 7.23 (d, 1H, J = 7.3 Hz), 7.46 (d, 1H, J = 9.5 Hz), 7.53-7.57 (m, 3H), 7.67 (t, 1H, J = 7.3 Hz), 7.76-7.78 (m, 2H)
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 438.09723, Found, 438.09640 (-0.83mmu)

ＳｅＲｈｏｄｏｌ（４．２ｍｇ、０．００９５ｍｍｏｌ）及びＣｓ_２Ｃｏ_３（１５．８ｍｇ、０．０４４ｍｍｏｌ）を２ｍＬのＤＭＦ中に懸濁させ、０．０５３ｍＬの２，３，４，６−テトラ−Ｏ−アセチル−α−Ｄ−ガラクト−ピラノシル ブロミドのＤＭＦ溶液（１Ｍ）を添加した。アルゴン雰囲気下、混合物を室温で一晩攪拌した。反応混合物に水を添加し、酢酸エチルで抽出し、食塩水で洗浄後、Ｎａ_２ＳＯ_４で乾燥し、減圧除去した。残渣を１ｍＬのメタノールに溶解し、２８％ナトリウムメトキシドのメタノール溶液を２００μＬ添加し、４時間攪拌した。混合物をアンバーライト（登録商標）で中和し、ろ過後、減圧除去した。粗生成物をＨＰＬＣで精製し、ピンク色粉末のＳｅＲｈｏｄｏｌ−ｇａｌを得た（４．９ｍｇ、収率８６％）。 (F) Synthesis of Compound 5 (SeRhodol-gal)

SeRhodol (4.2 mg, 0.0095 mmol) and Cs ₂ Co ₃ (15.8 mg, 0.044 mmol) were suspended in 2 mL DMF and 0.053 mL 2,3,4,6-tetra-O—. A DMF solution of acetyl-α-D-galacto-pyranosyl bromide (1M) was added. The mixture was stirred overnight at room temperature under an argon atmosphere. Water was added to the reaction mixture, extracted with ethyl acetate, washed with brine, dried over Na ₂ SO ₄ and removed under reduced pressure. The residue was dissolved in 1 mL of methanol, and 200 μL of 28% sodium methoxide in methanol was added and stirred for 4 hours. The mixture was neutralized with Amberlite (registered trademark), filtered and removed under reduced pressure. The crude product was purified by HPLC to obtain pink powder SeRhodol-gal (4.9 mg, 86% yield).

¹ H NMR (300 MHz, acetone-d ₆ ): 1.01 (t, 6H, J = 7.1 Hz), 3.26 (q, 4H, J = 7.1 Hz), 3.49-3.52 (m, 1H), 3.65-3.70 (m , 4H), 3.84 (d, 1H, J = 3.7 Hz), 4.79 (m, 1H), 5.40 (d, 2H, J = 6.6 Hz), 6.44-6.47 (m, 1H), 6.74 (d, 1H, J = 2.9 Hz), 6.78-6.80 (m, 1H), 6.97-7.03 (m, 1H), 7.10-7.23 (m, 3H), 7.27 (d, 1H, J = 8.1 Hz), 7.36 (d, 1H , J = 8.1 Hz), 7.66 (m, 1H)
LRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 600.15005, Found, 600.14729 (-2.76 mmu)

Spectral changes of Compound 4 (SeRhodol) and Compound 5 (SeRhodol-gal) By an enzymatic reaction between Compound 5 (SeRhodol-gal), which is a photosensitizing compound of the present invention, and β-galactosidase. Changes in absorption spectrum and fluorescence spectrum (excitation wavelength: 550 nm) of compound 4 (SeRhodol), which is the resulting photosensitizer, are shown in FIGS. 1a and 1b, respectively. The measurement was performed in the presence of 100 mM sodium phosphate buffer (pH 7.4).

From FIG. 1a, compound 5 showed no absorption, whereas compound 4 showed absorption with a peak at around 570 nm. From FIG. 1b, since compound 4 exhibits fluorescence emission at around 590 nm, the photosensitizing compound of the present invention can have a function as a fluorescent probe for enzyme reaction with β-galactosidase. Is also suggested.

FIG. 1c shows the emission of excited singlet oxygen ( ¹ O ₂ ) when compounds 4 and 5 are each excited at 532 nm. In Compound 4, since a spectrum is observed in the vicinity of 1270 nm in the near infrared, which is an emission wavelength when singlet oxygen returns to ground state oxygen (triplet), singlet oxygen is generated by Compound 4, and Compound 4 Has been shown to function as a photosensitizer. On the other hand, compound 5 did not emit light due to singlet oxygen.

Using compound 5 having a galactopyranosyl group cleaved by enzymatic reaction with β-galactosidase selectively loaded with β-galactosidase on β-galactosidase expressing cells using compound 5 (SeRhodol-gal) We examined whether changes in oxidative stress load (cell death) associated with light irradiation were observed depending on the presence or absence of expression. The test was performed using HEK293 cells expressing β-galactosidase (LacZ) as test cells and HEK293 cells expressing luciferase (Luc) as comparative examples. Each HEK293 cell was cultured in a PLL-coated 96-well plastic bottom plate, and a cell density of 2 to 5 × 10 ⁵ cells / mL was used.

Each test cell was irradiated with light (510-550 nm, 50 mW / cm ² , 180 seconds) 4 hours after compound 5 was added, and 24 hours later, a CCK assay was performed to confirm viable cells. The obtained results are shown in FIG. From FIG. 3, it was found that LacZ-expressing cells can be selectively killed by using Compound 5, and the degree thereof depends on the concentration of Compound 5. On the other hand, in the Luc-expressing cells, even when the concentration of Compound 5 was increased, almost no change was observed in the proportion of viable cells. The above results demonstrate that by using Compound 5 which is a photosensitizing compound of the present invention, only lactress-expressing cells can be selectively loaded with oxidative stress by light irradiation and induced to cell death. is there.

Claims (7)

A compound represented by the following formula (I) or a salt thereof :

Wherein A is a galactopyranosyl group; R ¹ represents 1 to 4 identical or different substituents bonded to a hydrogen atom or a benzene ring; R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ each independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group; X is C ₁ -C ₃ Represents an alkylene group). 2. The method according to claim ¹ , wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are hydrogen, R ⁸ and R ⁹ are ethyl groups, and X is a methylene group . Compound. A compound represented by the following formula (II) or a salt thereof :

(In the formula, Et represents an ethyl group). Photosensitizer represented by the following formula (III):

Wherein R ¹ represents 1 to 4 identical or different substituents bonded to a hydrogen atom or a benzene ring; R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently Represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group; X represents a C ₁ -C ₃ alkylene group). Using the compound according to any one of claims 1 to 3, specific cell death of a target cell in which a specific enzyme is expressed (except when the target cell is present in the human body) The target cell is a β-galactosidase-expressing cell. A step of bringing the compound according to any one of claims 1 to 3 into contact with an enzyme that is specifically expressed in the target cell to produce a compound represented by the following formula (III), and excitation light: 6. The method according to claim 5, comprising the step of performing irradiation to generate reactive oxygen species by the compound represented by the formula (III) and loading the target cells with oxidative stress.

Wherein R ¹ represents 1 to 4 identical or different substituents bonded to a hydrogen atom or a benzene ring; R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently Represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group; X represents a C ₁ -C ₃ alkylene group). 4. The method according to any one of claims 1 to 3, which specifically induces cell death of a target cell in which a specific enzyme is expressed (except when the target cell is present in the human body). use of a compound, said target cell is a β- galactosidase expressing cells, said use.

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