JP2018150293A - Caged compound and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a photoresponsive caged compound that can suppress biological damage, has an excellent quantum yield, and can perform efficiently spatiotemporal control of biologically active material such as amino acid, and a method for producing the photoresponsive caged compound.SOLUTION: A caged compound is represented by a formula (1) (where Ris a methoxy group or a diethylamino group, Ris a hydrogen atom or a methoxy group, and Ris a nitro group or a dimethylamino group).SELECTED DRAWING: None

Description

  The present invention relates to a photoresponsive caged compound and a method for producing the same.

  Conventionally, a functional group important for biological activity is protected (modified) with a photo-releasable protective group to inactivate it, and irradiation with light releases the protection by the photo-releasable protective group, and exhibits activity There are known caged compounds (that generate bioactive substances).

  More specifically, for example, a caged compound produced by performing an esterification reaction between a carboxylic acid and acetoxymethyl bromide in the presence of diisopropylethylamine is disclosed (for example, see Patent Document 1).

JP2015-124171A

  However, since the conventional caged compound has a benzofuran skeleton excited at a short wavelength, there is a problem in that the irradiated light becomes ultraviolet light (wavelength: 330 to 370 nm), and as a result, biological damage is caused.

  Further, the conventional caged compound has a problem in that the quantum yield is low because the benzofuran skeleton is decomposed.

  Therefore, the present invention has been made in view of the above points, and can suppress living body damage, is excellent in quantum yield, and can efficiently perform spatiotemporal control of physiologically active substances such as amino acids. An object is to provide a photoresponsive caged compound and a method for producing the same.

  In order to achieve the above object, the caged compound of the present invention is represented by the following formula (1).

(In the formula, R ₁ represents a methoxy group or a diethylamino group, R ₂ represents a hydrogen atom or a methoxy group, and R ₃ represents a nitro group or a dimethylamino group.)

  According to the caged compound of the present invention, biological damage can be suppressed, the quantum yield is excellent, and space-time control of physiologically active substances such as amino acids can be efficiently performed.

It is a ¹ HNMR spectrum data of the caged compound according to Example 1 of the present invention. It is a ¹ HNMR spectrum data of the caged compound according to Example 2 of the present invention. It is a ¹ HNMR spectrum data of the caged compound according to Example 3 of the present invention. It is a ¹ HNMR spectrum data of the caged compound according to Example 4 of the present invention. It is a ¹ HNMR spectrum data of the caged compound according to Example 5 of the present invention.

  The caged compound of the present invention has a function of freely and easily releasing amino acids such as glutamic acid by light stimulation, and is obtained by an esterification reaction of an alcohol derivative and a carboxylic acid. For example, the caged compound is represented by the following formula (2). Photoresponsive caged compounds.

  This caged compound is a coumarin derivative having an amino acid, and is a derivative compound having an electron-donating substituent introduced at both ends thereof.

  The caged compound of the present invention is inactivated by protecting (modifying) a functional group important for biological activity, for example, an amino acid such as glutamic acid with a photoreleasable protective group, and irradiating light. The protection by the protecting group is released and the activity is exhibited. In addition, the above-mentioned glutamic acid is a neurotransmitter and is an essential element indispensable for life activity.

  In the caged compound of the present invention, two-photon excitation (two photons are simultaneously absorbed by the molecule) by irradiation with near-infrared light (wavelength: 680 to 1050 nm) in the electron-donating substituents introduced at both ends thereof. Phenomenon that causes excitation).

  Accordingly, the quantum yield is dramatically improved, and the spatiotemporal control of physiologically active substances such as amino acids can be efficiently performed. Moreover, since near-infrared light having biological permeability can be used, it is possible to suppress biological damage compared to the case of using ultraviolet light.

  As this caged compound, the compound shown by following formula (3)-(6) is mentioned, for example.

  The caged compound represented by the formula (3) is a derivative compound having a methoxy group and a nitro group introduced at both ends thereof, and the caged compound represented by the formula (4) has two methoxy groups at both ends thereof, It is a derivative compound into which a nitro group has been introduced. The caged compound represented by the formula (5) is a derivative compound in which a diethylamino group and a nitro group are introduced at both ends, and the caged compound represented by the formula (6) is a diethylamino group and a dimethyl group at both ends. It is a derivative compound into which an amino group has been introduced.

Next, an outline of a method for producing a caged compound represented by the above formula (3) is shown in the following reaction scheme 1.
<Reaction scheme 1>

  To obtain a caged compound represented by the formula (3), first, an acetic anhydride mixed solution of 2′-hydroxy-4′-methoxyacetophenone of the formula (7) and 4-nitrophenylacetic acid of the formula (8) is used. Triethylamine is added dropwise and heated to reflux to obtain 7-methoxy-4-methyl-3- (4-nitrophenyl) coumarin represented by the formula (9).

  Next, benzoyl peroxide (bpo) is added to a carbon tetrachloride suspension of 7-methoxy-4-methyl-3- (4-nitrophenyl) coumarin and N-bromosuccinimide (NBS) represented by the formula (9). In addition, 7-methoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin represented by formula (10) is obtained by heating under reflux.

  Then, 7-methoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin represented by the formula (10) was dissolved in dry DMF, and potassium iodide, potassium carbonate and benzoic acid were added to the solution. After heating and stirring, the solvent is removed, and the resulting reaction mixture is purified using silica gel chromatography (GPC), whereby the caged compound represented by (3) above can be obtained.

  In silica gel chromatography using a particle packed column, purification (isolation) can be performed by using a mixed solvent (polar solvent) of dichloromethane and ethyl acetate as a mobile phase.

  The mixing ratio of dichloromethane and ethyl acetate is not particularly limited, but from the viewpoint of increasing the polarity of the mobile phase and facilitating isolation of the caged compound, the mixing ratio (volume ratio) of dichloromethane / ethyl acetate is dichloromethane. : It is preferable to set to ethyl acetate = 5: 1.

Next, an outline of a method for producing a caged compound represented by the above formula (4) is shown in the following reaction scheme 2.
<Reaction scheme 2>

  In order to obtain a caged compound represented by the formula (4), first, 2′-hydroxy-4 ′, 5′-dimethoxyacetophenone of the formula (11) and acetic anhydride of 4-nitrophenylacetic acid of the formula (8) Triethylamine is added dropwise to the mixture and heated to reflux to obtain 6,7-dimethoxy-4-methyl-3- (4-nitrophenyl) coumarin represented by the formula (12).

  Next, benzoyl peroxide (bpo) was added to a carbon tetrachloride suspension of 6,7-dimethoxy-4-methyl-3- (4-nitrophenyl) coumarin and N-bromosuccinimide (NBS) represented by formula (12). ) And heated under reflux, and purified using silica gel chromatography (dichloromethane: ethyl acetate = 4: 1) to obtain 6,7-dimethoxy-4-bromomethyl-3 represented by (13) above. -(4-Nitrophenyl) coumarin is obtained.

  Then, 6,7-dimethoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin represented by the formula (13) is dissolved in dry DMF, and potassium iodide, potassium carbonate and benzoic acid are added to the solution. After the mixture was heated and stirred, the solvent was removed and the resulting reaction mixture was purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 5: 1) to obtain the above (4 ) Can be obtained.

Next, an outline of a method for producing a caged compound represented by the above formula (5) is shown in the following reaction scheme 3.
<Reaction scheme 3>

  To obtain a caged compound represented by the formula (5), first, selenium dioxide is added to a xylene solution of 7- (diethylamino) -4-methylcoumarin represented by the formula (14), and this solution is heated to reflux. Next, ethanol and sodium borohydride were added and stirred at room temperature, the solution was neutralized with aqueous hydrochloric acid solution to remove the solvent, and the resulting reaction mixture was subjected to silica gel chromatography (dichloromethane: ethyl acetate (volume ratio)). = 4: 1) to obtain 7- (diethylamino) -4- (hydroxymethyl) coumarin represented by the above formula (15).

  Next, after adding tert-butyldimethylchlorosilane and 4-dimethylaminopyridine (DMAP) to a dichloromethane mixed solution of 7- (diethylamino) -4- (hydroxymethyl) coumarin represented by the formula (15), triethylamine is added. (TEA) is further added and stirred. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 4: 1), whereby 7- represented by formula (16) is obtained. (Diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] coumarin is obtained.

  Next, N-bromosuccinimide (NBS) was added to an acetonitrile solution of 7- (diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] coumarin represented by the formula (16). And ammonium acetate are added and stirred. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- represented by formula (17) is obtained. (Diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin is obtained.

Next, 7- (diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin represented by the formula (17) and 4-nitrophenylboronic acid Sodium carbonate and dichlorobis (triphenylphosphine) palladium (II) are added to a DMF-H ₂ O solution of pinacol ester, and the mixture is heated and stirred under a nitrogen atmosphere. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate = (volume ratio) 4: 1), whereby 7- represented by the formula (18) is expressed. (Diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin is obtained.

  Next, tetrabutylammonium fluoride was added to a THF solution of 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin represented by the formula (18). Add and stir. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 4: 1), whereby 7- represented by the formula (19) is obtained. (Diethylamino) -4- (hydroxymethyl) -3- (4-nitrophenyl) coumarin is obtained.

  Next, 7- (diethylamino) -4- (hydroxymethyl) -3- (4-nitrophenyl) coumarin represented by the formula (19) and benzoic acid in a dry methylene chloride solution were mixed with 4-dimethylaminopyridine (DMAP). Then, N, N′-dicyclohexylcarbodiimide (DCC) is further added and stirred. Then, the solvent is removed, and the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 4: 1), whereby the caged compound represented by (5) above is obtained. Can be obtained.

Next, an outline of a method for producing a caged compound represented by the above formula (6) is shown in the following reaction scheme 4.
<Reaction Scheme 4>

7- (Diethylamino) -4-[[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin and 4- (N, N′dimethylamino) phenylboron represented by the formula (17) Sodium carbonate and dichlorobis (triphenylphosphine) palladium (II) are added to a DMF-H ₂ O solution of acid pinacol ester, and the mixture is heated and stirred under a nitrogen atmosphere. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- represented by the formula (20) is expressed. (Diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin is obtained.

  Next, tetrabutylammonium fluoride is added to a THF solution of 7- (diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin represented by the formula (20) and stirred. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- represented by the formula (21) is expressed. (Diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin is obtained.

  Then, 4-dimethylaminopyridine (DMAP) is added to a dry methylene chloride solution of 7- (diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin and benzoic acid represented by formula (21). Then, N, N′-dicyclohexylcarbodiimide (DCC) is added and stirred. Then, the solvent was removed, and the resulting reaction mixture was purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby the caged compound represented by (6) above was obtained. Can be obtained.

  Furthermore, the caged compound of the present invention includes a compound represented by the following formula (22).

  The caged compound represented by the formula (22) is a derivative compound in which a double bond is introduced between the coumarin site and the dimethylaminophenyl group of the caged compound represented by the formula (6).

Next, an outline of a method for producing a caged compound represented by the above formula (22) is shown in the following reaction scheme 5.
<Reaction scheme 5>

  In order to obtain a caged compound represented by the formula (22), first, 4-bromo-N, N-dimethylbenzenamine represented by the formula (23) and 4,5,5,5-tetramethyl-2-vinyl- N, N-diisopropylethylamine is added to a dry toluene solution of 1,3,2-dioxaborolane and tri-tert-butylphosphonium tetrafluoroborate, and the mixture is heated and stirred under a nitrogen atmosphere. Then, after removing the solvent, the reaction mixture obtained is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 5: 1), whereby N, represented by formula (24). N-dimethyl-4-[(1E) -2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) ethenyl] benzenamine is obtained.

Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin represented by the formula (17) and N, N-dimethyl-4- [ (1E) -2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) ethenyl] benzeneamine in DMF-H ₂ O solution was added sodium carbonate and dichlorobis (triphenyl). Add phosphine) palladium (II) and stir under nitrogen atmosphere. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- represented by the formula (25) is expressed. (Diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin Get.

  Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3- (N-[(1E) -2- [4] represented by formula (25) -(Dimethylamino) phenyl] ethenyl]) Add tetrabutylammonium fluoride to a solution of coumarin in THF and stir. Then, after removing the solvent, the resulting reaction mixture is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- represented by formula (26) is obtained. (Diethylamino) -4- (hydroxymethyl) -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin is obtained.

  And 7- (diethylamino) -4- (hydroxymethyl) -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin and benzoic acid represented by formula (26) 4-Dimethylaminopyridine (DMAP) is added to the dry methylene chloride solution, and then N, N′-dicyclohexylcarbodiimide (DCC) is added and stirred. Then, the solvent was removed, and the resulting reaction mixture was purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby the caged compound represented by (22) above. Can be obtained.

  Hereinafter, the present invention will be described based on examples. In addition, this invention is not limited to these Examples, These Examples can be changed and changed based on the meaning of this invention, and they are excluded from the scope of the present invention. is not.

Example 1
(Synthesis of caged compounds)
A caged compound represented by the above formula (3) was synthesized. More specifically, first, 500 mg (3.0 mol) of 2′-hydroxy-4′-methoxyacetophenone of formula (7) (manufactured by Tokyo Chemical Industry Co., Ltd.) and 4-nitrophenylacetic acid of formula (8) 1 ml of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to a mixed solution in which 589 mg (3.3 mol) (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 2 ml of acetic anhydride (manufactured by Wako Pure Chemical Industries, Ltd.). . Next, the reaction mixture was heated to reflux at 90 ° C. for 24 hours, cooled to room temperature, 10 ml of ether was added, and the mixture was stirred for 2 hours. The precipitated solid was filtered and dried to obtain 7-methoxy-4-methyl-3- (4-nitrophenyl) coumarin (475 mg, yield: 51%) represented by the formula (9).

  Next, 7-methoxy-4-methyl-3- (4-nitrophenyl) coumarin (100 mg, 0.32 mmol) represented by the formula (9) and N-bromosuccinimide (manufactured by Tokyo Chemical Industry Co., Ltd.) 62.9 mg A catalytic amount of 11 mg of benzoyl peroxide (bpo) was added to a suspension of (0.35 mol) of carbon tetrachloride (2 ml), and the mixture was heated to reflux at 90 ° C. for 15 hours. The solid was then stirred in 10 ml of warm water and filtered to remove the solvent and remove succinimide. Then, 7-methoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin represented by the formula (10) was obtained by recrystallization in chloroform (50 mg, yield 40%).

  Then, 7-methoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin (129 mg, 0.33 mmol) represented by the formula (10) was dissolved in 5 ml of dry DMF, and potassium iodide (109 mg,. 66 mmol), potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd., 91 mg, 0.66 mmol) and benzoic acid (40 mg, 0.33 mmol) were added and heated and stirred at 50 ° C. for 6 hours. Then, extraction with ethyl acetate was performed, the organic layer was washed with saturated brine, the organic layer was dried over sodium sulfate, the solvent was distilled off, and the crude product was purified by silica gel column chromatography. The caged compound represented by the above (3) was obtained.

  More specifically, it was purified by gel permeation chromatography using a column (trade name: C300, manufactured by Wako Pure Chemical Industries, Ltd.) packed with high purity silica gel.

  A mixed solvent of dichloromethane (manufactured by Wako Pure Chemical Industries, Ltd.) and ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the mobile phase. The mixing ratio (volume ratio) of dichloromethane / ethyl acetate was set to dichloromethane: ethyl acetate = 5: 1. The purification conditions were a flow rate of 10 ml / min and 25 ° C., and the column retention time was 30 minutes.

  The yield of the obtained caged compound was 28 mg (0.53 mmol), and the yield was 20%.

(Light irradiation)
Next, the synthesized caged compound (1150 μg) was added to 10 ml of dimethyl sulfoxide (DMSO) solution to prepare an absorbance of 5 at a wavelength of 360 nm.

  Next, 3 ml of this solution is put into a cell together with a stirrer, and light irradiation (wavelength: 360 ± 10 nm, irradiation time: 1 minute) using a xenon lamp (trade name: BPS-500B, manufactured by Spectrometer Co., Ltd.) )

(Degauge evaluation)
Next, by the two-photon absorption of near-infrared light by the above-mentioned light irradiation, benzoic acid (a kind of carboxylic acid, which is a superordinate concept of amino acids) is generated from the synthesized caged compound, resulting in a degageation reaction. It was confirmed by ¹ HNMR spectrum data (see FIG. 1).

More specifically, the measurement of ¹ HNMR spectrum in this example uses a nuclear magnetic resonance measuring apparatus (manufactured by BRUKER, trade name: Ascend ^™ 400), uses DMSO-d ₆ as an internal reference, and sets the frequency. It was set at 400 MHz. The measurement temperature was set to 297.3 K, and the measurement was performed 8 hours, 16 hours, and 24 hours after the light irradiation.

  As shown in FIG. 1, benzoic acid-derived signals were observed in the vicinity of 7.95, 7.62, and 7.50 ppm, but the raw material-derived signals (8.30, 7.95, 7.70). , 7.49, around 7.10 ppm) was not observed over time. This can be said to indicate that benzoic acid is generated from the caged compound and a degageation reaction occurs.

(Calculation of quantum yield)
Next, the particle yield of the synthesized gauged compound was calculated. More specifically, first, 10 μl of the above-mentioned solution before light irradiation (hereinafter referred to as “raw material solution”) and 10 μl of the solution after light irradiation (hereinafter referred to as “reaction solution”) are used. Using high performance liquid chromatography (manufactured by JASCO Corporation, trade name: 880-PU), the integral value when 10 μl of the raw material solution is injected and the integral value when 10 μl of the reaction solution are injected are calculated. The reaction rate was calculated using the following formula (1). As a result, the reaction rate was 3.5%.

(Equation 1)
Reaction rate (%) = (integral value when 10 μl of raw material solution is injected−integral value when 10 μl of reaction solution is injected) ÷ integral value when 10 μl of raw material solution is injected × 100 (1)

  For the column, Inertsil ODS-3 was used, and a solvent (acetonitrile: water (volume ratio) = 8: 2) was used as a mobile phase.

Next, the quantum yield was calculated from the following formula (2) using the light quantity I (3.31 × 10 ⁻⁷ mol / min) of a xenon lamp, which is a light source used for light irradiation. The number of molecules (mol) of the caged compound represented by formula (3) reacted by 1 minute irradiation was measured by high performance liquid chromatography, and the caged compound represented by formula (3) was absorbed by 1 minute irradiation. The amount of light (mol) measured was measured by high performance liquid chromatography. As a result, the number of molecules (mol) of the caged compound represented by the formula (3) reacted by 1 minute irradiation was 0.282 × 10 ⁻⁷ (mol), and represented by the formula (3) by 1 minute irradiation. The amount of light (mol) absorbed by the caged compound was 3.31 × 10 ⁻⁷ (mol), and the quantum yield was 0.09.

(Equation 2)
Quantum yield = number of molecules of caged compound represented by formula (3) reacted by irradiation for 1 minute (mol) ÷ light quantity absorbed by caged compound represented by formula (3) by irradiation for 1 minute (mol) (2 )

(Example 2)
(Synthesis of caged compounds)
A caged compound represented by the above formula (4) was synthesized. More specifically, in order to obtain the caged compound represented by the formula (4), first, 2′-hydroxy-4 ′, 5′-dimethoxyacetophenone of the formula (11) (manufactured by Tokyo Chemical Industry Co., Ltd.) 1.0 g (5.1 mol), 4-nitrophenylacetic acid of formula (8) (Tokyo Chemical Industry Co., Ltd.) 996 mg (5.5 mol) and acetic anhydride (Wako Pure Chemical Industries, Ltd.) 3 ml 1 ml of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the mixed solution. Next, the reaction mixture is heated to reflux overnight at 90 ° C., cooled to room temperature, 10 ml of ether is added, and the mixture is stirred for 2 hours. The precipitated solid is filtered and dried to obtain the formula (12). The indicated 6,7-dimethoxy-4-methyl-3- (4-nitrophenyl) coumarin (823 mg, yield: 47%) was obtained.

  Next, 6,7-dimethoxy-4-methyl-3- (4-nitrophenyl) coumarin (398 mg, 1.16 mmol) represented by the formula (12) and N-bromosuccinimide (manufactured by Tokyo Chemical Industry Co., Ltd.) 374 mg A catalytic amount of benzoyl peroxide (60 mg) was added to a suspension of (2.10 mmol) of carbon tetrachloride (4 ml), and the mixture was heated to reflux at 90 ° C. for 24 hours. Thereafter, the solvent was distilled off and the solid was stirred and filtered in 10 ml of warm water to remove succinimide. Then, by purification using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 4: 1), the 6,7-dimethoxy-4-bromomethyl-3- (4) represented by (13) above is obtained. -Nitrophenyl) coumarin (287 mg, yield: 59%) was obtained.

  Then, 6,7-dimethoxy-4-bromomethyl-3- (4-nitrophenyl) coumarin (50 mg, 0.12 mmol) represented by the formula (13) was dissolved in 2 ml of dry DMF, and potassium iodide ( 40 mg, 0.24 mmol), potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd., 33 mg, 0.24 mmol) and benzoic acid (15 mg, 0.12 mmol) were added and heated at 50 ° C. for 3 hours. And stirred. Thereafter, the mixture was extracted with ethyl acetate, the organic layer was washed with saturated brine, the organic layer was dried over sodium sulfate, and the solvent was distilled off. And the caged compound represented by said (4) was obtained by refine | purifying a crude product using a silica gel chromatography.

  More specifically, it was purified by gel permeation chromatography using a column (trade name: C300, manufactured by Wako Pure Chemical Industries, Ltd.) packed with high purity silica gel.

  A mixed solvent of dichloromethane (manufactured by Wako Pure Chemical Industries, Ltd.) and ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the mobile phase. The mixing ratio (volume ratio) of dichloromethane / ethyl acetate was set to dichloromethane: ethyl acetate = 5: 1. The purification conditions were a flow rate of 10 ml / min and 25 ° C., and the column retention time was 30 minutes.

  The yield of the obtained caged compound was 23 mg (0.049 mmol), and the yield was 42%.

Then, in the same manner as in Example 1 described above, light irradiation and degauge evaluation were performed. As a result, benzoic acid (amino acid) was synthesized from the synthesized caged compound by two-photon absorption of near-infrared light by the light irradiation described above. It was confirmed by ¹ HNMR spectrum data (see FIG. 2) that one kind of carboxylic acid, which is a superordinate concept of 1), was generated and that a degazing reaction occurred.

  More specifically, as shown in FIG. 2, benzoic acid-derived signals were observed in the vicinity of 7.95, 7.62, and 7.50 ppm, but the raw material-derived signals (8.30, 7.88, 7.70, 7.45, 7.22 ppm)) was not observed over time. This can be said to indicate that benzoic acid is generated from the caged compound and a degageation reaction occurs.

  And when the quantum yield was computed like the above-mentioned Example 1, the quantum yield was 0.03.

(Example 3)
(Synthesis of caged compounds)
A caged compound represented by the above formula (5) was synthesized. More specifically, to obtain the caged compound represented by the formula (5), first, 570 mg of 7- (diethylamino) -4-methylcoumarin (manufactured by Tokyo Chemical Industry Co., Ltd.) represented by the formula (14) ( To a xylene solution (70 ml) of 2.46 mmol), 414 mg (2.46 mol) of selenium dioxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and this solution was heated to reflux for 24 hours. Next, after cooling to room temperature, celite filtration was performed, and the solvent of the obtained solution was distilled off with an evaporator. Next, ethanol (100 ml) and 46 mg (1.21 mmol) of sodium borohydride (Tokyo Chemical Industry Co., Ltd.) were added and stirred at room temperature for 4 hours, and then the solution was neutralized with 1M aqueous hydrochloric acid solution. Then, the mixture was extracted three times with methylene chloride, the organic layer was dried with magnesium sulfate, and the solvent was distilled off. Then, the brown residue is purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 4: 1) to obtain 7- (diethylamino) -4- represented by the above formula (15). (Hydroxymethyl) coumarin (300 mg, yield: 49%) was obtained.

  Next, tert-butyldimethylchlorosilane (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to a dichloromethane mixed solution of 7- (diethylamino) -4- (hydroxymethyl) coumarin (307 mg, 1.24 mmol) represented by the formula (15). ) 225 mg (1.49 mmol) and 4-dimethylaminopyridine (45 mg, 0.37 mmol) were added, then triethylamine (0.26 ml, 1.86 mmol) was further added, and the mixture was stirred at room temperature for 3 hours. Next, it was quenched with a saturated aqueous ammonium chloride solution, extracted with methylene chloride, the organic layer was dried over magnesium sulfate, and the solvent was distilled off. The crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 4: 1), whereby 7- (diethylamino) -4-[[ [(1,1-Dimethylethyl) dimethylsilyl] oxy] methyl] coumarin (253 mg, yield: 56%) was obtained.

  Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] coumarin (195 mg, 0.538 mmol) represented by the formula (16) was added to an acetonitrile solution. 105 mg (0.592 mmol) of N-bromosuccinimide (manufactured by Tokyo Chemical Industry Co., Ltd.) and ammonium acetate (5 mg, 0.0538 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was added to water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and evaporated. The crude product is purified using silica gel chromatography (hexane: ethyl acetate = 2: 1), whereby 7- (diethylamino) -4-[[[(1, 1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (115 mg, yield: 49%) was obtained.

Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (100 mg, 0.227 mmol) represented by formula (17) and To a solution of 4-nitrophenylboronic acid pinacol ester (manufactured by Aldrich) 57 mg (0.227 mmol) in DMF-H ₂ O (4 ml: 2 ml), sodium carbonate (manufactured by Wako Pure Chemical Industries, 63 mg, 0.59 mmol) And dichlorobis (triphenylphosphine) palladium (II) (manufactured by Tokyo Chemical Industry Co., Ltd., 3 mg, 0.00454 mmol) were added, and the mixture was heated and stirred at 90 ° C. for 24 hours in a nitrogen atmosphere. Thereafter, the mixture was cooled to room temperature, extracted with methylene chloride in addition to water, the organic layer was dried over magnesium sulfate, filtered and evaporated. Then, the crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 4: 1) to give 7- (diethylamino) -4- [ [[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (28 mg, yield: 26%) was obtained.

  Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (12.5 mg, 0.026 mmol) represented by formula (18) Tetrabutylammonium fluoride (manufactured by Tokyo Chemical Industry Co., Ltd., 1 mol / L tetrahydrofuran solution 0.052 ml, 0.052 mmol) was added to the THF solution at 0 ° C. Then, it returned to room temperature, stirred for 30 minutes, quenched with saturated ammonium chloride, extracted with ethyl acetate, the organic layer was dried with magnesium sulfate, filtered, and the solvent was distilled off. The crude product is purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 4: 1) to give 7- (diethylamino) -4- (hydroxy) represented by the formula (19). Methyl) -3- (4-nitrophenyl) coumarin (7.6 mg, yield: 80%) was obtained.

  Next, 7- (diethylamino) -4- (hydroxymethyl) -3- (4-nitrophenyl) coumarin (7.6 mg, 0.021 mmol) represented by formula (19) and benzoic acid (2.5 mg, 4-Dimethylaminopyridine (manufactured by Tokyo Chemical Industry Co., Ltd., 0.025 mg, 0.0021 mmol) was added to 1 ml of a dry methylene chloride solution of 0.021 mmol) and stirred for 10 minutes. Thereafter, N, N′-dicyclohexylcarbodiimide (4.3 mg, 0.021 mmol) was further added, and the mixture was stirred at room temperature for 17 hours. Next, water was added to the reaction solution and the mixture was extracted with methylene chloride. The organic layer was dried over magnesium sulfate, filtered and evaporated. And the caged compound represented by said (5) was obtained by refine | purifying a crude product using a silica gel chromatography.

  More specifically, it was purified by gel permeation chromatography using a column (trade name: C300, manufactured by Wako Pure Chemical Industries, Ltd.) packed with high purity silica gel.

  As a mobile phase, a mixed solvent of hexane (manufactured by Wako Pure Chemical Industries, Ltd.) and ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The mixing ratio (volume ratio) of hexane / ethyl acetate was set to hexane: ethyl acetate = 4: 1. The purification conditions were a flow rate of 10 ml / min and 25 ° C., and the column retention time was 30 minutes.

  The yield of the obtained caged compound was 8.8 mg (0.019 mmol), and the yield was 88%.

(Light irradiation)
Next, the synthesized caged compound (2590 μg) was added to 5 ml of a dimethyl sulfoxide (DMSO) solution to adjust the absorbance to 5 at a wavelength of 360 nm.

  Next, 3 ml of this solution is placed in a cell together with a stirrer, and light irradiation (wavelength: 400 ± 10 nm, irradiation time: 1 minute) using a xenon lamp (trade name: BPS-X500B, manufactured by Spectrometer Co., Ltd.) )

Then, when the degauge evaluation was performed in the same manner as in Example 1 described above, benzoic acid (carboxyl, which is a superordinate concept of amino acids), was synthesized from the synthesized caged compound by the two-photon absorption of near-infrared light by the above-described light irradiation. It was confirmed by ¹ HNMR spectral data (see FIG. 3) that one kind of acid) was generated and a degazing reaction occurred.

  More specifically, as shown in FIG. 3, benzoic acid-derived signals were observed in the vicinity of 7.95, 7.62, and 7.50 ppm, but the raw material-derived signals (8.25, 7.83, 7.72, 7.63, 7.50, 6.80, and 6.62 ppm) were not observed over time. This can be said to indicate that benzoic acid is generated from the caged compound and a degageation reaction occurs.

  And when the quantum yield was computed like the above-mentioned Example 1, the quantum yield was 0.01.

(Example 4)
(Synthesis of caged compounds)
A caged compound represented by the above formula (6) was synthesized. More specifically, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (206 mg, 0.468 mmol) represented by formula (17) And 4- (N, N′dimethylamino) phenylboronic acid pinacol ester (manufactured by Aldrich, 116 mg, 0.468 mmol) in a DMF-H ₂ O (4 ml: 2 ml) solution with sodium carbonate (Wako Pure Chemical Industries, Ltd.) 99 mg, 0.935 mmol) and dichlorobis (triphenylphosphine) palladium (II) (Tokyo Chemical Industry Co., Ltd., 7 mg, 0.00935 mmol) were added, and the mixture was heated and stirred at 90 ° C. for 24 hours in a nitrogen atmosphere. did. Then, it cooled to room temperature, added water, extracted with methylene chloride, the organic layer was dried with magnesium sulfate, filtered, and the solvent was distilled off. The crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1) to give 7- (diethylamino) -4- ( Hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin (206 mg, yield: 92%) was obtained.

  Next, tetrabutylammonium fluoride (Tokyo) was added to a THF solution of 7- (diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin (130 mg, 0.27 mmol) represented by the formula (20). A 1 mol / L tetrahydrofuran solution (0.054 ml, 0.054 mmol) manufactured by Kasei Kogyo Co., Ltd. was added at 0 ° C., and then the mixture was returned to room temperature and stirred for 30 minutes. Then, it was quenched with saturated ammonium chloride, extracted with ethyl acetate, the organic layer was dried over magnesium sulfate, filtered and evaporated. The crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1) to give 7- (diethylamino) -4- (hydroxy) represented by the formula (21). Methyl) -3- (4-dimethylaminophenyl) coumarin (40 mg, yield: 40%) was obtained.

  Next, 7- (diethylamino) -4- (hydroxymethyl) -3- (4-dimethylaminophenyl) coumarin (30 mg, 0.082 mmol) and benzoic acid (10 mg, 0.082 mmol) represented by formula (21) 4-dimethylaminopyridine (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 mg, 0.0082 mmol) was added to 4 ml of the dry methylene chloride solution, and the mixture was stirred for 10 minutes. Then, N, N′-dicyclohexylcarbodiimide (17 mg, 0.082 mmol) was added and stirred at room temperature for 17 hours. Next, water was added to the reaction solution and the mixture was extracted with methylene chloride. The organic layer was dried over magnesium sulfate, filtered and evaporated. And the caged compound represented by said (6) was obtained by refine | purifying a crude product using a silica gel chromatography.

  More specifically, it was purified by gel permeation chromatography using a column (trade name: C300, manufactured by Wako Pure Chemical Industries, Ltd.) packed with high purity silica gel.

  As a mobile phase, a mixed solvent of hexane (manufactured by Wako Pure Chemical Industries, Ltd.) and ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The mixing ratio (volume ratio) of hexane / ethyl acetate was set to hexane: ethyl acetate = 2: 1. The purification conditions were a flow rate of 10 ml / min and 25 ° C., and the column retention time was 30 minutes.

  The yield of the obtained caged compound was 33 mg (0.070 mmol), and the yield was 86%.

Then, in the same manner as in Example 3 described above, light irradiation and degauge evaluation were performed, and benzoic acid (amino acid) was synthesized from the synthesized caged compound by two-photon absorption of near-infrared light by the light irradiation described above. It was confirmed by ¹ HNMR spectrum data (see FIG. 4) that one kind of carboxylic acid, which is a superordinate concept of (1), was generated and a degageation reaction occurred.

  More specifically, as shown in FIG. 4, benzoic acid-derived signals were observed in the vicinity of 7.95, 7.62, and 7.50 ppm, but the raw material-derived signals (7.90, 7.67, 7.60, 7.57, 7.18, 6.78, and 6.58 ppm)) were not observed over time. This can be said to indicate that benzoic acid is generated from the caged compound and a degageation reaction occurs.

  And when the quantum yield was computed like the above-mentioned Example 1, the quantum yield was 0.15.

(Example 5)
(Synthesis of caged compounds)
A caged compound represented by the above formula (22) was synthesized. More specifically, first, 4-bromo-N, N-dimethylbenzenamine (Tokyo Chemical Industry Co., Ltd., 200 mg, 0.995 mmol) and tris (dibenzylideneacetone) dipalladium represented by the formula (23) ( Aldrich (45 mg, 0.050 mmol) and tri-tert-butylphosphonium tetrafluoroborate (Wako Pure Chemical Industries, 29 mg, 0.10 mmol) in a dry toluene solution was 4,5,5,5- Tetramethyl-2-vinyl-1,3,2-dioxaborolane (Tokyo Chemical Industry Co., Ltd., 0.19 ml, 1.1 mmol) and N, N-diisopropylethylamine (Tokyo Chemical Industry Co., Ltd., 0.33 ml) 2.0 mmol), and the mixture was heated and stirred at 95 ° C. for 3 hours under a nitrogen atmosphere. Thereafter, the mixture was cooled to room temperature, extracted with ethyl acetate in addition to water, the organic layer was dried over sodium sulfate, filtered and evaporated. The crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 5: 1) to obtain N, N-dimethyl-4- [ (1E) -2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) ethenyl] benzenamine (160 mg, yield: 65%) was obtained.

Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3-bromocoumarin (200 mg, 0.454 mmol) represented by formula (17) and N, N-dimethyl-4-[(1E) -2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) ethenyl] benzenamine (125 mg, 0.454 mmol) Of DMF-H ₂ O (16 ml: 4 ml) in sodium carbonate (manufactured by Wako Pure Chemical Industries, 96 mg, 0.908 mmol) and dichlorobis (triphenylphosphine) palladium (II) (Tokyo Chemical Industry Co., Ltd.) Product, 8 mg, 0.00908 mmol) was added, and the mixture was heated and stirred at 90 ° C. for 7 hours under a nitrogen atmosphere. Thereafter, the mixture was cooled to room temperature, extracted with methylene chloride in addition to water, the organic layer was dried over magnesium sulfate, filtered and evaporated. Then, the crude product is purified using silica gel chromatography (hexane: ethyl acetate (volume ratio) = 2: 1), whereby 7- (diethylamino) -4- [ [[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin (155 mg, yield: 67 %).

  Next, 7- (diethylamino) -4-[[[(1,1-dimethylethyl) dimethylsilyl] oxy] methyl] -3- (N-[(1E) -2- [ 4- (dimethylamino) phenyl] ethenyl]) coumarin (39 mg, 0.077 mmol) in a THF solution of tetrabutylammonium fluoride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.15 ml of a 1 mol / L tetrahydrofuran solution; 15 mmol) was added at 0 ° C. Then, it returned to room temperature, stirred for 30 minutes, quenched with saturated ammonium chloride, extracted with ethyl acetate, the organic layer was dried with magnesium sulfate, filtered, and the solvent was distilled off. The crude product is purified using silica gel chromatography (dichloromethane: ethyl acetate (volume ratio) = 2: 1) to give 7- (diethylamino) -4- (hydroxy) represented by the formula (26). Methyl) -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin (24 mg, yield: 79%) was obtained.

  Next, 7- (diethylamino) -4- (hydroxymethyl) -3- (N-[(1E) -2- [4- (dimethylamino) phenyl] ethenyl]) coumarin represented by the formula (26) ( 4-dimethylaminopyridine (manufactured by Tokyo Chemical Industry Co., Ltd., 0.83 mg, 0.0069 mmol) was added to 4 ml of a dry methylene chloride solution of 27 mg, 0.069 mmol) and benzoic acid (8.3 mg, 0.069 mmol). Stir for 10 minutes. Thereafter, N, N′-dicyclohexylcarbodiimide (15 mg, 0.069 mmol) was further added, and the mixture was stirred at room temperature for 12 hours. Next, water was added to the reaction solution and the mixture was extracted with methylene chloride. The organic layer was dried over magnesium sulfate, filtered and evaporated. And the caged compound represented by said (22) was obtained by refine | purifying a crude product using a silica gel chromatography.

  More specifically, it was purified by gel permeation chromatography using a column (trade name: C300, manufactured by Wako Pure Chemical Industries, Ltd.) packed with high purity silica gel.

  As a mobile phase, a mixed solvent of hexane (manufactured by Wako Pure Chemical Industries, Ltd.) and ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The mixing ratio (volume ratio) of hexane / ethyl acetate was set to hexane: ethyl acetate = 2: 1. The purification conditions were a flow rate of 10 ml / min and 25 ° C., and the column retention time was 30 minutes.

  The yield of the obtained caged compound was 30 mg (0.060 mmol), and the yield was 88%.

(Light irradiation)
Next, the synthesized caged compound (980 μg) was added to 5 ml of a dimethyl sulfoxide (DMSO) solution to adjust the absorbance to 5 at a wavelength of 458 nm.

  Next, 3 ml of this solution is placed in a cell together with a stirrer, and light irradiation (wavelength: 458 ± 10 nm, irradiation time: 1 minute) using a xenon lamp (trade name: BPS-X500B, manufactured by Spectrometer Co., Ltd.) )

(Degauge evaluation)
Next, by the two-photon absorption of near-infrared light by the above-mentioned light irradiation, benzoic acid (a kind of carboxylic acid, which is a superordinate concept of amino acids) is generated from the synthesized caged compound, resulting in a degageation reaction. It was confirmed by ¹ HNMR spectrum data (see FIG. 5). As a characteristic of two-photon absorption, the two-photon absorption cross section at 700 nm was 850 GM.

More specifically, the measurement of ¹ HNMR spectrum in this example uses a nuclear magnetic resonance measuring apparatus (manufactured by BRUKER, trade name: Ascend ^™ 400), uses DMSO-d ₆ as an internal reference, and sets the frequency. It was set at 400 MHz. The measurement temperature was set to 297.3 K, and the measurement was performed 20 minutes, 35 minutes, and 45 minutes after the light irradiation.

  As shown in FIG. 5, benzoic acid-derived signals were observed in the vicinity of 7.95, 7.62, and 7.50 ppm, but the raw material-derived signals (7.95, 7.71, 7.66, 7.51, 7.48, 7.38, 7.11, 6.77, 6.71, 6.58, and 5.70 ppm) were not observed over time. This can be said to indicate that benzoic acid is generated from the caged compound and a degageation reaction occurs.

(Calculation of quantum yield)
Next, the particle yield of the synthesized gauged compound was calculated. More specifically, first, 10 μl of the raw material solution and 10 μl of the reaction solution are used, and 10 μl of the raw material solution is injected using high performance liquid chromatography (trade name: 880-PU, manufactured by JASCO Corporation). The integrated value when the reaction solution was injected and the integrated value when 10 μl of the reaction solution was injected were calculated, and the reaction rate was calculated using Equation (1) above. As a result, the reaction rate was 7.9%.

  Inertsil ODS-3 was used for the column, and acetonitrile solvent was used as the mobile phase.

Next, the quantum yield was calculated from the following formula (3) using the light quantity I (3.34 × 10 ⁻⁶ mol / min) of a xenon lamp which is a light source used for light irradiation. The number of molecules (mol) of the caged compound represented by formula (22) reacted by irradiation for 15 seconds was measured by a high performance liquid chromatography measurement method, and the caged compound represented by formula (22) by irradiation for 15 seconds. The amount of light (mol) absorbed by was measured by a high performance liquid chromatography measurement method. As a result, the number of molecules (mol) of the caged compound represented by the formula (22) reacted by irradiation for 15 seconds is 0.938 × 10 ⁻⁷ (mol), and expressed by the formula (22) by irradiation for 15 seconds. The amount of light absorbed by the caged compound (mol) was 0.835 × 10 ⁻⁶ (mol), and the quantum yield was 0.112.

(Equation 3)
Quantum yield = number of molecules of caged compound represented by formula (22) reacted by irradiation for 15 seconds (mol) ÷ light quantity absorbed by caged compound represented by formula (22) by irradiation for 15 seconds (3) (3) )

  From the above, it can be seen that according to the caged compounds of Examples 1 to 5, the quantum yield is remarkably improved, and the spatiotemporal control of physiologically active substances such as amino acids can be efficiently performed. Moreover, since near-infrared light having biological permeability can be used, it is possible to suppress biological damage compared to the case of using ultraviolet light.

  Examples of utilization of the present invention include photoresponsive caged compounds and methods for producing the same.

Claims (7)

A caged compound represented by the following formula (1):
(In the formula, R ₁ represents a methoxy group or a diethylamino group, R ₂ represents a hydrogen atom or a methoxy group, and R ₃ represents a nitro group or a dimethylamino group.) The caged compound according to claim 1, which is represented by the following formula (2).
The caged compound according to claim 1, which is represented by the following formula (3).
The caged compound according to claim 1, which is represented by the following formula (4).
The caged compound according to claim 1, which is represented by the following formula (5).
The caged compound according to claim 1, which is represented by the following formula (6).
The caged compound manufacturing method characterized by manufacturing the caged compound represented by following formula (7) by performing esterification reaction of an alcohol derivative and carboxylic acid.
(In the formula, R ₁ represents a methoxy group or a diethylamino group, R ₂ represents a hydrogen atom or a methoxy group, and R ₃ represents a nitro group or a dimethylamino group.)