JPH1129500A - Fluorescent photodissociating protective group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject new protective group enabling the tracing of a physiologicaly active substance, etc., by protecting an active point of a physiologically active substance, diffusing the protected substance to the desired part in the living body, separating from the physiologically active substance by photo-reaction and detecting the extremely intense fluorescent light emitted by a carbostyryl fluorescent group. SOLUTION: This protective group has (E) 3-(2-amino-4,5-dimethoxyphenyl)2- methylacryloyl group expressed by formula I. The protective group can be produced e.g. by reacting a salicylaldehyde derivative with a Wittig reagent to form a cinnamic acid skeleton and converting the nitro group to amino group by reduction reaction to form a trans-isomer of a cinnamate. The detection of e.g. the development of physiologically active substance can be carried out by introducing an active group into the product for bonding with a physiologically active substance to protect the active substance, deprotecting the protective group by irradiation with light after diffusing the substance into the living body and determining the fluorescence of the carbostyryl derivative of the formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel fluorescent photolabile protecting group.

[0002]

2. Description of the Related Art In order to investigate the mechanism of action of a substance in a living body, it is necessary to quantitatively measure the fate of the substance in a biological system in a short time. It is also important to observe the various changes that follow the material.

On the other hand, biological reactions are often extremely fast, and a plurality of complicatedly related reactions usually proceed simultaneously. Therefore, when the substance is added from the outside, the process of diffusing in the system becomes rather rate-determining, and the subsequent reaction to be actually measured often cannot be clearly grasped. In order to solve the problem, various methods for quickly adding the target substance have been proposed, but none of them is satisfactory at present.

As one of them, a technique based on light irradiation has recently been reported. It is a protecting group known as a so-called Caged reagent, a light blocking agent and the like. In general, those in which the active site of a physiologically active substance to be tracked is protected (blocked or caded) with the protective group are introduced into a biological system, and it is confirmed that the substance has sufficiently diffused to the action point. After that, the protecting group is deprotected based on light irradiation. Therefore, it is possible to trace the reaction by the substance having recovered the released physiological activity. The feature of this protecting group is that it can be deprotected only by light irradiation, that it can be deprotected very quickly, and that it is possible to squeeze light irradiation only to a specific part if necessary. . Here, as the biologically active substance protected by the protecting group, various biological substances, for example, amino acids, proteins, enzymes,
Nucleotide-3-phosphate, cAMP, nucleic acid and the like are conceivable, and various attempts have been made.

[0005] However, although the quantification of the reaction in which the biologically active substance protected by the above-mentioned protecting group is deprotected by light irradiation in a biological system is extremely important, an established means has not yet been established. Not reported.

SUMMARY OF THE INVENTION In view of the above points, the present inventor has studied diligently to protect the active site of a physiologically active substance and, after being diffused to a desired site in a biological system, to apply this protective group to light. A photo-dissociable protecting group that can be deprotected by irradiating the compound with a protecting group (fluorescent photo-dissociating protecting group) capable of quantitatively measuring the deprotecting reaction of the protecting group. ) Was successfully found, and the present invention was completed.

[0006]

That is, the protecting group according to the present invention is a compound which expresses an extremely strong fluorescence based on the carbostyril fluorophore after being cleaved from the physiologically active substance by light irradiation. . At this time, the amount of the bioactive substance to be deprotected can be quantified based on the measured fluorescence intensity based on the carbostyril skeleton.

[0007] More specifically, the present invention relates to a protecting group represented by the following formula 1.

[0008]

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Further, the present invention relates to a method for detecting the above protecting group by measuring the fluorescence of a carbostyril derivative represented by the following formula 2, which is formed based on a deprotection reaction based on a photoreaction. .

[0010]

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Further, the present invention provides an ethyl (E) 3-
(2-amino-4,5-dimethoxyphenyl) -2-methylpropenate and (E) 3- (2-amino-
4,5-dimethoxyphenyl) -2-methylpropenoic acid.

The fluorescent photodissociation protecting group according to the present invention basically has an o-aminocinnamic acid skeleton as described above, and therefore has an ester (including thioester) bond, an amide (thioamide) These compounds can be widely used as protecting groups for physiologically active substances having hydroxyl groups (including phenolic and alcoholic hydroxyl groups), thiol groups and amino groups as active sites based on bonds such as bonds.

[0013]

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Here, X is O, S (ester or thioester, etc.) or NHR (R is H or alkyl group, etc.).

Furthermore, the fluorescent photo-dissociation protecting group according to the present invention is one that deprotects upon irradiation with light.
The trans-oriented double bond is cis-isomerized, causing an intramolecular transamidation reaction with the aniline amino group of the benzene ring, and as a result, a deprotected bioactive substance and
It is considered to be a carbostyril derivative (FIG. 1).

The resulting carbostyril derivative is a substance having strong fluorescence and can be quantified by ordinary fluorescence measurement means.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below.

Synthesis Method The method for synthesizing the cadide compound according to the present invention is not particularly limited, and generally known organic chemical reactions can be preferably used. Specifically, a cinnamic acid skeleton is formed by reacting a salicylaldehyde derivative with a Wittig reagent according to the synthetic route illustrated in FIG. 2, and a trans isomer is converted into a nitro group into an amino group by a reduction reaction. It is possible to obtain

Further, after the reaction with the above Wittig reagent, the compound is converted into a cinnamic acid derivative by hydrolysis, and thereafter, an appropriate physiologically active substance is condensed by an amide bond, and then the nitro group is converted into an amino group by a reduction reaction. It is possible to

Further, according to the synthetic route illustrated in FIG. 2, the obtained cinnamate is an alkyl ester (eg, ethyl ester), and this ester bond is necessary for performing a labeling reaction to another physiologically active substance. Is considered too stable. Therefore, it is convenient to store the reagent, but it is necessary to further introduce an active group in order to carry out the labeling reaction. For this purpose, conventionally used active groups such as carboxylic acid groups, acid halides, acid anhydrides, active ester groups, active imide groups and the like are conceivable. The above carboxylic acid can be easily carried out by a usual hydrolysis reaction, and the carboxylic acid can be isolated.
The resulting carboxylic acid can be further derivatized into various active groups by a usual synthesis method. Specifically, the active group includes acid halides (bromine, chlorine, iodine), imidazolyl group, succinimide group, cyano group, butyroyl group, alkyloyl group such as bivaloyl group, 4-amidinophenyloxy group, p-amido group and the like. It is a phenoxy group such as a nitrophenyloxy group.

By reacting the protecting group according to the present invention having these activating groups with various physiologically active substances, the physiologically active substance can be protected. For example, when the physiologically active substance is an amino acid, an oligopeptide, a protein, or the like, and has an amino group, a phenolic hydroxyl group, a thiol group, an alcoholic hydroxyl group, and the like, it can be labeled with an amide, ester, or thioamide bond. Become.

By the photoreaction of the labeled physiologically active substance
That eliminated by light irradiation protecting group according to the deprotection ized present invention, the carbostyril derivatives and the light reaction conditions to release the bioactive substance is not particularly limited, conventional light sources can be used. If necessary, the irradiation light can be adjusted under a microscope to partially release the protected bioactive substance present at a specific position in a cell at a specific time.

Regardless of the location of the protected physiologically active substance in a tissue, it is possible to cause a deprotection reaction only at a specific position by using a microscope or the like (FIG. 1).

Physiologically active substance when a cadide reagent is used
Quantification of expression level of quality Using a physiologically active substance for expression purpose as a protecting group according to the present invention, the amount of expression of a target compound (bioactive substance) can be controlled by controlling light irradiation in the system.

As shown in FIG. 3, when the ethyl ester derivative having a protecting group according to the present invention is irradiated with a specific light, a desired fluorescent phenomenon by carbostyril occurs.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0027]

【Example】

(Synthesis of ethyl ester derivative) (E) 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid ethyl ester (1) ((E) 3- (3,4-Dimethoxy) -6-nitrophenyl-2-methyl
Synthesis of 2-propenic acid ethyl ester).

10 g of 6-nitroveratraldehyde (6-N
Itoroveratlaldehyde (Aldrich) and 18.1 g (50 mmol) of Wittig reagent (carboethoxy ethylidene triphenylphosphorane, Aldrich) were reacted by stirring in benzene at room temperature for about 18 hours. At this time, the reaction was performed in a dark room. After completion of the reaction, benzene was removed under reduced pressure to obtain white crystals. This was recrystallized from ethyl acetate to obtain 28.5 g of the target compound (1) (yield: 76%).

The structure of the compound was confirmed by infrared absorption spectrum (IR), ¹ H-NMR and TOF-MS. IR: 1700 cm ^-1 (ester). TOF-MS: 296
(M / C). ¹ H-NMR (chloroform, δ ppm): 7.92 (s,
1H, methine group proton of aromatic ring or double bond), 7.74 (s,
1H, methine group proton of aromatic ring or double bond), 6.72 (s,
1H, aromatic ring or double bond methine group proton), 4.29 (q,
2H, methylene proton of ethyl group), 3.99 (methyl proton of s, 3H, methoxy group), 3.96 (methyl proton of s, 3H, methoxy group), 3.57 (amino proton of s, 2H, aniline) , 1.92 (s, 3H, 2-position methyl group proton), 1.36
(t, 3H, methyl group proton of ethyl group).

(E) Ethyl 3- (2-amino-4,5-dimethoxyphenyl) -2-methyl-2-propenoate ((E) 3- (2-Amino-4,5-Dimethoxyphenyl-2-) methyl
Synthesis of 2-propenic acid ethyl ester).

19.0 g of (1) obtained above was mixed with 250 ml of glacial acetic acid.
And 15.0 g of iron powder and 20 ml of ion-exchanged water were added thereto, and the mixture was heated under reflux for 40 minutes. Thereafter, the reaction solution was filtered to remove iron powder, the obtained filtrate was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, and then ethyl acetate was successively added with water, 5% aqueous sodium bicarbonate, and a saturated aqueous sodium chloride solution. The layer was washed and dried over anhydrous sodium sulfate overnight. Ethyl acetate was removed under reduced pressure, and the residue was recrystallized from hexane / ethanol to obtain 12.5 g of the desired product (78% yield).

The structure of the compound was determined by infrared absorption spectrum (IR), ¹ H-NMR, and TOF-MS. IR: 1689 cm ^-1 (ester). TOF-MS: 265
(M / C). ¹ H-NMR (chloroform, δ): 7.57 (s, 1H,
Methine group proton of an aromatic ring or a double bond), 6.68 (s, 1H,
Methine group proton of an aromatic ring or a double bond), 6.29 (s, 1H,
Methine group proton of aromatic ring or double bond), 4.25 (q, 2H,
Methylene group proton of ethyl group), 3.84 (methyl proton of s, 3H, methoxy group), 3.79 (methyl proton of s, 3H, methoxy group), 3.57 (s, 2H, amino proton of aniline), 2.03 (s, 3H, 2-position methyl group proton), 1.33 (t, 3H,
(Ethyl methyl proton) (Synthesis of phenethylamide derivative) (E) 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid ((E) 3- (3,4 -Dimeth
Synthesis of oxy-6-nitrophenyl-2-methyl-2-propenic acid).

The above-obtained (E) 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid ethyl ester (1) (11.3 g) was dissolved in methanol (250 ml). 45 ml of a normal aqueous sodium hydroxide solution was added, and the mixture was reacted at 40 ° C. for 4 hours. After completion of the reaction, the solution was adjusted to pH 4 using 1 N hydrochloric acid, cooled, and the precipitated crystals were separated by filtration to obtain 10.1 g of the target compound (yield 9).
9%).

The structure of the compound was determined by infrared absorption spectrum (IR), ¹ H-NMR, and TOF-MS. IR: 1685 cm ^-1 (carboxyl group). TOF-M
S: 268 (M / C). ¹ H-NMR (heavy DMSO, δppm): 7.76
(s, 1H, aromatic ring or double bond methine group proton), 7.72
(s, 1H, methine group proton of aromatic ring or double bond), 6.97
(s, 1H, methine group proton of aromatic ring or double bond), 3.90
(s, 6H, methyl group proton of methoxy group), 3.57 (s, 2H, amino group proton of aniline), 1.84 (s, 3H, 2-position methyl group proton).

(E) 3- (3,4-Dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid phenethylamide ((E) 3- (3,4-Dimethoxy-6-nitorphenyl-2- meth
Synthesis of yl-2-propenic acid phenethylamide).

The obtained (E) 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid and 2-phenethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were mixed with 100 ml of dichloromethane. And cooled to 0 ° C,
EDC-HCl (1- (3-Dimethylaminopropyl) -3-ethylcarbodii
mide hydrochloride (manufactured by Aldrich) was added and stirred overnight to react. The solvent was removed under reduced pressure, and ethyl acetate and water were added to the residue. After washing the organic solvent layer, it was dried over sodium sulfate. The solvent was removed under reduced pressure, and the resulting residue was subjected to silica gel chromatography (developing solvent: chloroform / methanol = 50/1) to obtain 14.1 g of the target compound (quantitative yield).

The structure of the compound was determined by infrared absorption spectrum (IR), ¹ H-NMR, and TOF-MS. IR: 3280, 1627 cm ^-1 (amide group). TOF-M
S: 370 (M / C). ¹ H-NMR (chloroform, δ ppm):
7.73-7.22 (m, 7H, aromatic proton or double bond proton), 6.70 (s, 1H, aromatic proton or double bond proton), 6.01 (s, 1H, amide bond NH), 3.97 (s, 3H , Methoxy group methyl group proton), 3.94 (s, 3H, methoxy group methyl group proton), 3.63 (dt, 2H, phenethyl group 1 'methylene group proton), 2.91 (t, 2H, phenethyl group 2 'Position methylene group proton), 1.86 (s, 3H, methyl group proton).

(E) 3- (2-Amino-4,5-dimethoxyphenyl) -2-methyl-2-propenoic acid phenethylamide ((E) 3- (2-Amino-4,5-Dimethoxyphenyl) -2- meth
Synthesis of yl-2-propionic acid Phenethyl amide).

The above-obtained (E) 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid phenethylamide (14.1 g) was dissolved in glacial acetic acid (164 ml), and iron powder 9.8 was added thereto. g and 13 ml of ion-exchanged water.
The reaction was carried out for 40 minutes. Thereafter, the reaction solution was filtered to remove iron powder, the filtrate was concentrated under reduced pressure, and the obtained residue was dissolved in ethyl acetate. The ethyl acetate layer was successively separated with water, 5% aqueous sodium hydrogen carbonate and saturated aqueous sodium chloride. Washed and dried over anhydrous sodium sulfate overnight. The ethyl acetate was removed under reduced pressure, and the residue was recrystallized from hexane / ethanol to obtain 8.9 g of the desired product (69% yield).

The structure of this compound was determined by infrared absorption spectrum (IR), ¹ H-NMR, and TOF-MS. IR: 3326, 3207, 1602 cm ^-1 (amide group). TOF
-MS: 340 (M / C). ¹ H-NMR (chloroform,
δ): 7.35 to 7.21 (m, 6H, aromatic ring proton), 6.60 (s, 1H,
Aromatic ring proton), 5.93 (s, 1H, amide proton), 3.84
(s, 3H, methoxy group methyl proton), 3.78 (s, 3H, methoxy methyl proton), 3.64 (dt, 2H, 1 'methylene proton of phenethyl group), 3.54 (s, 2H, Aniline group amino proton), 2.90 (t, 2H, phenethyl group 2'-position methylene group proton), 1.96 (s, 3H, 2-position methyl group proton).

(Synthesis of Fluorescent Carbostyril Derivative)
6,7-dimethoxy-3-methylcarbostyril (6,7-D
imethoxy-3-methylcarbostyril) (E) Dissolve 0.5 g of 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid ethyl ester (1) in 30 ml of ethyl alcohol, and add 36 g of
Ultraviolet rays of 5 nm were irradiated for about 20 hours, the precipitated crystals were separated by filtration, and the obtained crystals were recrystallized from ethanol to obtain 0.38 g of the target compound (yield 92.1%).

The structure of the compound was determined by infrared absorption spectrum (IR), ¹ H-NMR, and TOF-MS. IR: 1654 cm ^-1 (lactam). TOF-MS: 219
(M / C). ¹ H-NMR (chloroform, δ): 12.3 (s, 1H,
Amide proton), 7.56 (s, 1H, 4-position proton), 6.90 (s, 1
H, 5th or 8th proton), 6.88 (s, 1H, 5th or 8th proton), 3.99 (s, 3H, methyl of methoxy group),
3.92 (s, 3H, methyl of methoxy group), 2.28 (s, 3H, 3-position methyl).

(Deprotection grouping reaction by light irradiation) (E) 3
-(3,4-dimethoxy-6-nitrophenyl) -2-
The solution containing methyl-2-propenoic acid ethyl ester (1) was irradiated with light to monitor the deprotection reaction by HPLC (340 nm).

HPLC conditions: column, ODS φ4.6 mm × 150 mm
(SHISEIDO CacellPakC18); moving bed, acetonitrile /
Water = 30/70 to 60/40 (0 to 20 minutes) with a flow rate of 1.0 ml / min.

Before light irradiation, a peak of the above compound was observed at a retention time of 7.7 minutes. A new peak was observed at a retention time of 3.7 minutes by light irradiation. This 3.7 minute peak coincided with the carbostyril peak.

Similarly, a solution containing (E) 3- (2-amino-4,5-dimethoxyphenyl) -2-methyl-2-propenoic acid phenethylamide was irradiated with light to monitor the deprotection reaction by HPLC (260 nm). )did.

HPLC conditions: column, ODS φ4.6 mm × 150 mm
(SHISEIDO CacellPakC18); moving bed, acetonitrile /
Water = 30/70 to 60/40 (0 to 20 minutes) with a flow rate of 1.0 ml / min.

Before the light irradiation, the peak of the phenethylamide compound was observed at a retention time of 6.9 minutes. A new peak was observed at a retention time of 3.8 minutes by light irradiation. This peak at 3.8 minutes coincided with the peak of carbostyril.

Further, (E) 3-
The deprotection reaction was monitored (260 nm) by irradiating the solution containing (2-amino-4,5-dimethoxyphenyl) -2-methyl-2-propenoic acid phenethylamide with light.

HPLC conditions: column, ODS φ4.6 mm × 150 mm
(SHISEIDO CacellPakC18); moving bed, acetonitrile /
Water = 20/80 to 60/40 (5 to 20 minutes) at a flow rate of 1.0 ml / min.

Before the light irradiation, the peak of the phenethylamide compound was observed at a retention time of 19.2 minutes. New peaks were observed at the retention time of 10.0 minutes and 6.6 minutes by light irradiation. The peak at 10.0 minutes coincided with the peak of carbostyril, and the peak at 6.6 minutes coincided with the peak of phenethylamine.

[Brief description of the drawings]

FIG. 1 is a diagram showing that a biologically active substance protected using a protecting group according to the present invention is deprotected by light irradiation to release a biologically active substance.

FIG. 2 is a diagram illustrating a method for preparing a physiologically active substance protected by a protecting group according to the present invention.

FIG. 3 (E) is a diagram showing a deprotection grouping reaction of a solution containing 3- (3,4-dimethoxy-6-nitrophenyl) -2-methyl-2-propenoic acid ethyl ester by light irradiation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI // C07D 215/22 C07D 215/22

Claims (4)

[Claims] (E) 3- (2) represented by the following formula 1.
-Amino-4,5-dimethoxyphenyl) -2-methylacryloyl group having a novel fluorescent photolabile protecting group. Embedded image 2. A method for detecting a deprotection reaction based on a photoreaction of a protecting group according to claim 1 by measuring fluorescence of a carbostyril derivative represented by the following formula 2 to be formed. Embedded image 3. Ethyl (E) 3- (2-amino-
4,5-Dimethoxyphenyl) -2-methylpropenate. (E) 3- (2-amino-4,5-dimethoxyphenyl) -2-methylpropenoic acid.