Compound or its salt, and fluorescent probe

Abstract

To provide a low molecular compound that shows a shift in fluorescence wavelength in response to a change in pH.SOLUTION: The present invention relates to a compound represented by formula (1) or a salt thereof. R1 is a group represented by formula (2a), (2b) or (2c), R2 and R4 independently represent a substituted or unsubstituted aryl group, R3 and R5 independently represent a substituted or unsubstituted open-chain hydrocarbon group, or a substituted or unsubstituted styryl group, X1 and X2 independently represent a fluorine atom or a group represented by -O-R, and R is an alcohol residue or a sugar residue.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound or a salt thereof, and a fluorescent probe. [Background technology]

[0002] For measurements requiring spatial resolution, such as imaging, ratiometric measurement is desirable to avoid the influence of shading due to distribution. In ratiometric measurement, for example, a fluorescent dye whose fluorescence wavelength shifts with changes in pH, etc. is used, and the fluorescence intensity at two wavelengths is measured, and the pH, etc. is calculated from the ratio of these fluorescence intensities.

[0003] Polymer-type fluorescent dyes are known as fluorescent dyes that cause a fluorescence wavelength shift. However, polymeric fluorescent dyes do not have sufficient spatial resolution, so low-molecular-weight fluorescent dyes that produce a fluorescence wavelength shift are desirable.

[0004] Boron dipyrromethene (BODIPY) dyes, which have a core structure of BODIPY, are known as small molecule fluorescent dyes. BODIPY dyes are stable and have high fluorescence quantum yields. By introducing a substituent that turns fluorescence on and off depending on pH, etc., into the meso position (8-position) of BODIPY, many useful sensors have been developed.

[0005] Patent Document 1 describes a compound in which a phenyl group substituted with an amino group (which may be substituted with an alkyl group) is introduced at the meso position of BODIPY. Patent Document 2 describes a compound in which an N-alkylpyridinium group is introduced at the meso position of BODIPY. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2008 / 059910 [Patent Document 2] Chinese Patent Application Publication No. 102993763 Summary of the Invention [Problem to be solved by the invention]

[0007] However, although the compounds described in Patent Documents 1 and 2 change in fluorescence intensity depending on the pH, it is difficult to induce a fluorescence wavelength shift, and ratiometric measurement has not been possible. An object of the present invention is to provide a low molecular weight compound or a salt thereof that exhibits a fluorescence wavelength shift in response to a change in pH, and a fluorescent probe using the same. [Means for solving the problem]

[0008] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by introducing a pyridine structure into the meso-position of BODIPY and aromatic structures into the 1- and 7-positions, thereby constructing a tetrad-type molecule in which BODIPY is the sole chromophore.

[0009] The present invention has the following aspects. [1] A compound represented by the following formula (1) or a salt thereof:

[0010] [ka]

[0011] However, R 1 represents a group represented by the following formula (2a), (2b) or (2c): R 2 and R 4 each independently represents a substituted or unsubstituted aryl group, R 3 and R 5 each independently represents a substituted or unsubstituted chain hydrocarbon group or a substituted or unsubstituted styryl group, X 1 and X 2each independently represents a fluorine atom or a group represented by -OR, and R represents an alcohol residue or a sugar residue.

[0012] [ka]

[0013] However, R 6 ~R 9 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 6 and R 7 or R 7 and R 8 may be taken together to form a ring structure with the carbon atoms to which they are attached, R 10 ~R 13 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 10 and R 11 or R 11 and R 12 may be taken together to form a ring structure with the carbon atoms to which they are attached, R 14 ~R 17 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 14 and R 15 may be taken together to form a ring with the carbon atoms to which they are attached, and R 16 and R 17 may be taken together to form a ring structure with the carbon atoms to which they are attached. [2] R in the formula (1) 2 and R 4 are each independently a substituted or unsubstituted phenyl group, or a salt thereof. [3] R in the formula (1) 1 represents an alkyl-substituted or unsubstituted 2-, 3-, or 4-pyridyl group, or an alkyl-substituted or unsubstituted 2-, 3-, or 4-quinolyl group, or a salt thereof. [4] A fluorescent probe comprising any one of the compounds [1] to [3] above or a salt thereof. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a low molecular weight compound or a salt thereof that undergoes a shift in fluorescence wavelength in response to a change in pH, and a fluorescent probe using the same. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the results of evaluation of the change in UV absorption spectrum of the compound (2quBOD) of Example 1 due to a change in pH. [Figure 2] 1 shows the results of evaluating the change in fluorescence spectrum of the compound (2quBOD) in Example 1 due to changes in pH. [Figure 3] 1 shows the results of evaluating the change in the fluorescence intensity ratio of the compound (2quBOD) of Example 1 due to changes in pH. [Figure 4] 1 shows the results of evaluation of the change in UV absorption spectrum of the compound (2pyBOD) of Example 2 due to changes in pH. [Figure 5] 1 shows the results of evaluating the change in fluorescence spectrum of the compound (2pyBOD) of Example 2 due to changes in pH. [Figure 6] 1 shows the results of evaluating the change in the fluorescence intensity ratio of the compound (2pyBOD) of Example 2 due to changes in pH. [Figure 7] 1 shows the results of evaluation of the compound of Example 2 (2pyBOD) by repeated switching of pH. [Figure 8] 1 shows the results of evaluating the change in fluorescence spectrum of the compound (2pyBOD) of Example 2 depending on the concentration of trifluoroacetic acid. [Figure 9] 1 shows the results of evaluating the change in the fluorescence intensity ratio of the compound (2pyBOD) of Example 2 depending on the concentration of trifluoroacetic acid. [Figure 10] 1 shows the results of evaluating the change in fluorescence spectrum of the compound (6Me2pyBOD) of Example 3 due to changes in pH. [Figure 11] 1 shows the results of evaluating the change in the fluorescence intensity ratio due to changes in pH of the compound (6Me2pyBOD) of Example 3. [Figure 12]1 shows the results of evaluation of the compound of Example 3 (6Me2pyBOD) by repeated switching of pH. [Figure 13] 1 shows the results of evaluating the change in fluorescence spectrum of the compound (6Me2pyBOD) of Example 3 depending on the concentration of trifluoroacetic acid. [Figure 14] 1 shows the results of evaluating the change in the fluorescence intensity ratio of the compound (6Me2pyBOD) of Example 3 depending on the concentration of trifluoroacetic acid. [Figure 15] 1 shows the results of evaluating the change in the fluorescence intensity ratio due to changes in pH of the compound of Example 4 (4pyBOD). [Figure 16] Frontier orbitals of the ground state of 2pyBOD and protonated 2pyBOD. [Figure 17] Energy profiles of protonated 2pyBOD and protonated Comparative Example 1 compound (compound 5). DETAILED DESCRIPTION OF THE INVENTION

[0016] A compound according to one aspect of the present invention is represented by the following formula (1). Hereinafter, the compound represented by formula (1) will also be referred to as "compound (1)." Compounds represented by other formulas will also be referred to in the same manner.

[0017] [ka]

[0018] However, R 1 represents a group represented by the following formula (2a), (2b) or (2c): R 2 and R 4 each independently represents a substituted or unsubstituted aryl group, R 3 and R 5 each independently represents a substituted or unsubstituted chain hydrocarbon group or a substituted or unsubstituted styryl group, X 1 and X 2each independently represents a fluorine atom or a group represented by -OR (hereinafter also simply referred to as "OR"), and R represents an alcohol residue or a sugar residue.

[0019] [ka]

[0020] However, R 6 ~R 9 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 6 and R 7 or R 7 and R 8 may be taken together to form a ring structure with the carbon atoms to which they are attached, R 10 ~R 13 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 10 and R 11 or R 11 and R 12 may be taken together to form a ring structure with the carbon atoms to which they are attached, R 14 ~R 17 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 14 and R 15 may be taken together to form a ring structure with the carbon atoms to which they are attached, and R 16 and R 17 may be taken together to form a ring structure with the carbon atoms to which they are attached. In formulae (2a), (2b) and (2c), the wavy single lines represent bonds.

[0021] R 6 ~R 17 The unsubstituted alkyl group in may be linear or branched. The unsubstituted alkyl group has, for example, 1 to 3 carbon atoms. An example of the substituent in the substituted alkyl group is an alkoxy group. The number of carbon atoms in the alkoxy group is, for example, 1 to 3. The substituted alkyl group may have one or more substituents. Specific examples of the substituted or unsubstituted alkyl group include a methyl group, an ethyl group, and a 2-propyl group.

[0022] R 6 and R 7 or R 7 and R 8 , R 10 and R 11 or R 11 and R 12 , R 14 and R 15 , R 16 and R 17 Examples of the ring structure formed by combining these groups together with the carbon atoms to which they are bonded include a benzene ring and a naphthalene ring. Among these, a benzene ring is preferred from the viewpoint of solubility in aqueous solvents. The ring structure may have a substituent. Examples of the substituent include a substituted or unsubstituted alkyl group and an alkoxy group. Examples of the substituted or unsubstituted alkyl group include the same as those described above.

[0023] Specific examples of the group represented by formula (2a) (hereinafter also referred to as "group (2a)") include substituted or unsubstituted 2-pyridyl groups such as a 2-pyridyl group, a 6-methyl-2-pyridyl group, and a 3-methyl-2-pyridyl group; substituted or unsubstituted 2-quinolyl groups such as a 2-quinolyl group and a 3-methyl-2-quinolyl group; substituted or unsubstituted 2-isoquinolyl groups such as a 2-isoquinolyl group and a 3-methyl-2-isoquinolyl group; and a 2-(1,10-phenanthronyl) group. The substituted 2-pyridyl group is a 2-pyridyl group in which at least some of the hydrogen atoms bonded to the carbon atoms have been substituted with a substituent. The same applies to groups other than the 2-pyridyl group.

[0024] The substituted 2-pyridyl group, substituted 2-quinolyl group, and substituted 2-isoquinolyl group each preferably have an unsubstituted alkyl group as a substituent (an alkyl-substituted 2-pyridyl group, an alkyl-substituted 2-quinolyl group, and an alkyl-substituted 2-isoquinolyl group). The number of alkyl groups in each of the alkyl-substituted 2-pyridyl group, the alkyl-substituted 2-quinolyl group, and the alkyl-substituted 2-isoquinolyl group is, for example, 1 to 3, and one is preferred.

[0025] Specific examples of the group represented by formula (2b) (hereinafter also referred to as "group (2b)") include alkyl-substituted or unsubstituted 3-pyridyl groups such as a 3-pyridyl group and a 4-methyl-3-pyridyl group; alkyl-substituted or unsubstituted 3-quinolyl groups such as a 3-quinolyl group and a 2-methyl-3-quinolyl group; alkyl-substituted or unsubstituted 3-isoquinolyl groups such as a 3-isoquinolyl group and a 2-methyl-3-isoquinolyl group; and a 3-(1,10-phenanthronyl) group.

[0026] The substituted 3-pyridyl group, substituted 3-quinolyl group, and substituted 3-isoquinolyl group each preferably have an unsubstituted alkyl group as a substituent (an alkyl-substituted 3-pyridyl group, an alkyl-substituted 3-quinolyl group, and an alkyl-substituted 3-isoquinolyl group). The number of alkyl groups in each of the alkyl-substituted 3-pyridyl group, the alkyl-substituted 3-quinolyl group, and the alkyl-substituted 3-isoquinolyl group is, for example, 1 to 3, and 1 is preferred.

[0027] Specific examples of the group represented by formula (2c) (hereinafter also referred to as "group (2c)") include alkyl-substituted or unsubstituted 4-pyridyl groups such as a 4-pyridyl group, a 3,5-dimethyl-4-pyridyl group, and a 2,5-dimethyl-4-pyridyl group; alkyl-substituted or unsubstituted 4-quinolyl groups such as a 4-quinolyl group and a 3-methyl-4-quinolyl group; and a 4-(1,10-phenanthronyl) group.

[0028] The substituted 4-pyridyl group and the substituted 4-quinolyl group each preferably have an unsubstituted alkyl group as a substituent (an alkyl-substituted 4-pyridyl group, an alkyl-substituted 4-quinolyl group). The number of alkyl groups in each of the alkyl-substituted 4-pyridyl group and the alkyl-substituted 4-quinolyl group is, for example, 1 to 3, and preferably 1 or 2.

[0029] In one preferred embodiment, R 1 R is an alkyl-substituted or unsubstituted 2-, 3-, or 4-pyridyl group, or an alkyl-substituted or unsubstituted 2-, 3-, or 4-quinolyl group. 1 When is an alkyl-substituted or unsubstituted 2-, 3-, or 4-pyridyl group, or an alkyl-substituted or unsubstituted 2-, 3-, or 4-quinolyl group, the fluorescence quantum yield in an aqueous solvent system tends to be excellent.

[0030] In another preferred embodiment, R 1 is group (2a) or group (2c). 1 is group (2a) or group (2c), then R 1 In comparison with the case where the group (2b) is the group (2a), the change in fluorescence wavelength with respect to the change in pH tends to be greater. In this embodiment, group (2a) is preferably an alkyl-substituted or unsubstituted 2-pyridyl group or an alkyl-substituted or unsubstituted 2-quinolyl group, and group (2c) is preferably an alkyl-substituted or unsubstituted 4-pyridyl group or an alkyl-substituted or unsubstituted 4-quinolyl group.

[0031] R 2 and R 4 In the above formula, the unsubstituted aryl group may be monocyclic or polycyclic. The unsubstituted aryl group has, for example, 6 to 14 carbon atoms. Examples of the substituent in the substituted aryl group include a substituted or unsubstituted alkyl group, -NR 21 R 22 a group represented by -N + R 23 R 24 R 25 A group represented by -(OCH2CH2)n Examples of such groups include a group represented by OH (hereinafter also referred to as a "polyethylene glycol group") and a sulfone group. 21 and R 22 R each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group. 23 , R 24 and R 25 each independently represents a substituted or unsubstituted alkyl group. Examples of the substituted or unsubstituted alkyl group include the same as those described above. n represents an integer of 2 or more. The upper limit of n is, for example, 24. The substituted aryl group may have one or more substituents. Specific examples of the substituted or unsubstituted aryl group include a phenyl group, a naphthyl group, a trimethylammonium-substituted phenyl group, and a polyethylene glycol-substituted phenyl group. R 2 and R 4 is preferably a substituted or unsubstituted phenyl group from the viewpoint of solubility in aqueous solvent systems.

[0032] R 3 and R 5 In the formula (I), the unsubstituted chain hydrocarbon group may be linear or branched, and may be saturated or unsaturated. Examples of the unsubstituted chain hydrocarbon group include an unsubstituted alkyl group, an unsubstituted alkenyl group, and an unsubstituted alkynyl group. The number of carbon atoms in the unsubstituted alkyl group is, for example, 1 to 12. The number of carbon atoms in each of the unsubstituted alkenyl group and the unsubstituted alkynyl group is, for example, 2 to 12. An example of the substituent in the substituted chain hydrocarbon group is an alkoxy group. The number of carbon atoms in the alkoxy group is, for example, 1 to 3. The substituted chain hydrocarbon group may have one or more substituents. Specific examples of the substituted or unsubstituted chain hydrocarbon group include a methyl group, an ethyl group, a vinyl group, and an ethynyl group. Examples of the substituent in the substituted styryl group include a substituted or unsubstituted alkyl group and a dialkylamino group (such as a dimethylamino group). The substituted styryl group may have one or more substituents. R 3 and R5 are each independently preferably a methyl group or a styryl group, and particularly preferably a methyl group, from the viewpoint of fluorescence quantum yield.

[0033] X 1 and X 2 In the formula, R in OR represents an alcohol residue or a sugar residue. The alcohol residue is a group obtained by removing one hydroxyl group from an alcohol. The alcohol may be a monohydric alcohol or a polyhydric alcohol. The alcohol may be linear or branched. The number of carbon atoms in the alcohol is, for example, 1 to 12. A sugar residue is a group in which one hydroxyl group has been removed from a sugar. The sugar has, for example, 5 to 18 carbon atoms. Specific examples of alcohols or sugars include methanol, ethanol, 2-propanol, ethylene glycol, glycerol, glucose, lactose, and the like. X 1 and X 2 In terms of chemical stability, a fluorine atom is preferred. X 1 and X 2 In terms of increasing water solubility and promoting biological metabolism, OR is preferred.

[0034] Specific examples of compound (1) (R 1 ~R 5 , X 1 and X 2 In the following, F represents a fluorine atom. Compound (1-1a):R 1 = 2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-1b):R 1 = 2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X1 =F, X 2 =OR. Compound (1-1c):R 1 = 2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-2a):R 1 = 3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-2b):R 1 = 3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-2c):R 1 = 3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-3a):R 1 = 4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-3b):R 1 = 4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X2 =OR. Compound (1-3c):R 1 = 4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-4a):R 1 = 6-methyl-2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-4b):R 1 = 6-methyl-2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-4c):R 1 = 6-methyl-2-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-5a):R 1 = 6-methyl-3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-5b):R 1 = 6-methyl-3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1=F, X 2 =OR. Compound (1-5c):R 1 = 6-methyl-3-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-6a):R 1 = 6-methyl-4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-6b):R 1 = 6-methyl-4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-6c):R 1 = 6-methyl-4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-7a):R 1 = 2-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-7b):R 1 = 2-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1=F, X 2 =OR. Compound (1-7c):R 1 = 2-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-8a):R 1 = 3-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-8b):R 1 = 3-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-8c):R 1 = 3-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-9a):R 1 = 4-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-9b):R 1 = 4-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-9c):R 1 = 4-quinolyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-10a):R 1 = 3,5-dimethyl-4-pyridyl group, R 2 = phenyl group, R 3 = styryl group, R 4 = phenyl group, R 5 = styryl group, X 1 =F, X 2 =F. Compound (1-10b):R 1 = 6-methyl-4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-10c):R 1 = 6-methyl-4-pyridyl group, R 2 = phenyl group, R 3 = methyl group, R 4 = phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR. Compound (1-11a):R 1 = 3,5-dimethyl-4-pyridyl group, R 2 = 2-trimethylammonium substituted phenyl group, R 3 = methyl group, R 4 = 2-trimethylammonium substituted phenyl group, R 5 = methyl group, X 1 =F, X 2 =F. Compound (1-11b):R 1 = 3,5-dimethyl-4-pyridyl group, R 2 = 2-trimethylammonium substituted phenyl group, R 3 = methyl group, R4 = 2-trimethylammonium substituted phenyl group, R 5 = methyl group, X 1 =F, X 2 =OR. Compound (1-11c):R 1 = 3,5-dimethyl-4-pyridyl group, R 2 = 2-trimethylammonium substituted phenyl group, R 3 = methyl group, R 4 = 2-trimethylammonium substituted phenyl group, R 5 = methyl group, X 1 =OR, X 2 =OR.

[0035] The molecular weight of compound (1) is preferably less than 600, more preferably not more than 500. When the molecular weight is less than 600, excellent spatial resolution is easily obtained in measurements that require spatial resolution, such as imaging. The lower limit of the molecular weight of compound (1) is not particularly limited as long as it is within the range that satisfies the above formula (1), and is, for example, 450.

[0036] Compound (1) can be produced, for example, by reacting compound (3) with compound (4), reacting the resulting compound (5) with compound (6) in the presence of phosphoryl chloride (POCl3), and then reacting the resulting compound (7) with boron trifluoride ether complex in the presence of a tertiary amine. 1 and X 2 is a fluorine atom, The resulting compound is then reacted with an alcohol or sugar (ROH), as needed, to replace one or both of the two fluorine atoms with OR, resulting in X 1 and X 2 Compound (1) in which one of X is a fluorine atom and the other is OR, or X 1 and X 2 A compound in which is OR is obtained.

[0037] [ka]

[0038] R 1 ~R 5 has the same meaning as above. The compounds (3), (4), and (6) may be commercially available products or may be synthesized by known methods. An example of a boron trifluoride etherate is boron trifluoride diethyl etherate (BF3·OEt2), where Et is an ethyl group. Tertiary amines include, for example, triethylamine and diisopropylethylamine.

[0039] The reaction of compound (3) with compound (4) can be carried out, for example, by adding compound (3) and compound (4) to a solvent and reacting them in an inert atmosphere (e.g., N2 atmosphere) at 15 to 30°C for 12 to 72 hours. Examples of the solvent include dichloromethane, chloroform, and toluene.

[0040] The reaction of compound (5) with compound (6) can be carried out, for example, by adding compound (5) and compound (6) to a solution of phosphoryl chloride dissolved in a solvent, and reacting at 15 to 30°C for 12 to 72 hours. The amount of phosphoryl chloride used is, for example, 200 to 300 parts by mass per 100 parts by mass of compound (5). After the reaction, washing, extraction, etc. may be carried out as necessary.

[0041] The reaction of compound (7) with boron trifluoride etherate can be carried out, for example, by adding a tertiary amine and boron trifluoride etherate in that order to a solution of compound (7) dissolved in a solvent, and reacting the mixture at 15 to 30°C for 12 to 72 hours. After the reaction, the reaction product may be purified, if necessary. Examples of tertiary amines include triethylamine and diisopropylethylamine. The amount of the tertiary amine used is, for example, 500 to 1000 parts by mass per 100 parts by mass of the compound (7). After the reaction, purification may be carried out as necessary.

[0042] X 1 and X 2 As the alcohol or sugar to be reacted with compound (1) in which is a fluorine atom, one corresponding to OR is used. X 1 and X 2 Compound (1), in which is a fluorine atom, can be reacted with an alcohol or a sugar under relatively mild conditions (for example, at room temperature and atmospheric pressure). After the reaction, purification may be carried out as necessary.

[0043] However, the method for producing compound (1) is not limited to the above method, and appropriate modifications can be made by referring to known methods for synthesizing BODIPY dyes and the like.

[0044] Compound (1) may be in the form of a salt. Examples of salts include trifluoroacetate and borofluoride.

[0045] Because the compound (1) described above has the above structure, a fluorescence wavelength shift occurs in response to a change in pH. For example, when the pH of a solution of compound (1) is changed stepwise and each solution is irradiated with excitation light, the color tone of the fluorescence from the solution changes in response to the change in pH of the solution. For another example, when the pH of a solution of compound (1) is changed stepwise and each solution is irradiated with excitation light, the fluorescence spectrum from the solution is measured, and the ratio of the fluorescence intensities at two different wavelengths is calculated, the ratio of the fluorescence intensities changes in response to the change in pH of the solution. R of compound (1) 1 The pyridine ring nitrogen in the compound (1) is reversibly protonated, and as the pH changes, the ratio of compound (1) with a non-protonated pyridine ring nitrogen to compound (1) with a protonated pyridine ring nitrogen changes. Specifically, as the pH decreases (increases), the ratio of protonated compound (1) increases (decreases). R, a meso-position substituent of BPDIPY 1When the structure of (1) changes, the fluorescence wavelength also changes. It is thought that the ratio of compound (1) with an unprotonated pyridine ring nitrogen to compound (1) with a protonated pyridine ring nitrogen changes with changes in pH, resulting in a shift in the fluorescence wavelength and a change in the overall color tone and fluorescence intensity ratio.

[0046] The aforementioned Patent Document 2 describes a compound in which an N-alkylpyridinium group is introduced at the meso position (8th position) of BODIPY and methyl groups are introduced at the 1st and 7th positions of BODIPY. When methyl groups are introduced at the 1st and 7th positions, rotation of the meso-positioned substituents is completely inhibited, and only the ON-OFF type can be obtained. On the other hand, the absence of methyl groups at the 1st and 7th positions results in fluorescence quenching. In this embodiment, a pyridine moiety is introduced at the meso-position of BODIPY, and an aromatic structure is introduced at the 1,7-position to construct a tetrad-type molecule containing BODIPY as the sole chromophore. This allows the degree of conjugation between BODIPY and the meso-position substituent to be controlled, resulting in a fluorescence wavelength shift. Furthermore, the hydrophobic packing formed by the meso-position substituent and the 1,7-position aromatic structure prevents friction with the solvent that dissolves compound (1), which is thought to cause non-radiative deactivation, thereby maintaining fluorescence intensity.

[0047] Because the fluorescence wavelength shifts in response to changes in pH, compound (1) can be used as a fluorescent probe to enable ratiometric measurement of pH. Furthermore, compound (1) is a low-molecular-weight compound, providing excellent spatial resolution and making it useful for measurements requiring spatial resolution, such as imaging. For example, compound (1) can be introduced into cells, enabling imaging of intracellular pH. Fluorescent probes containing compound (1) or its salts can be used in the same manner as conventional fluorescent probes. The above-mentioned color tone and fluorescence intensity ratio change significantly near the acid dissociation constant pKa of compound (1). Therefore, in pH measurement, it is desirable to use compound (1) with a pKa suitable for the pH range to be measured. In compound (1), R 1By selecting the structure, the pKa of compound (1) can be controlled within the range of, for example, 1.2 to 3.7, making it applicable to a variety of measurement targets, including strongly acidic samples such as gastric acid. The pKa is determined from the inflection point of the sigmoid curve obtained from the pH response. [Example]

[0048] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. The equipment used for compound identification and evaluation is shown below. Fluorescence spectrum: Shimadzu RF-1500 spectrofluorometer. Absolute quantum yield: Hamamatsu Photonics "C9920-02G". UV absorption spectrum: Shimadzu UV-3100PC ultraviolet-visible absorption spectrophotometer. Infrared absorption spectrum (IR): "Nicolet Nexus 670NT FT-IR" manufactured by Thermo Fisher Scientific. Nuclear magnetic resonance spectrum (NMR): Agilent Technologies "NMR System 500PS SN". High-resolution mass spectrometry (HRMS): "JMS-700N" manufactured by JEOL.

[0049] (Synthesis Example 1: Synthesis of 2-methyl-4-phenylpyrrole) 5 mL of aqueous NaNO3 (402 mg, 5.83 mmol in terms of NaNO3) was added to a solution of 1 mL (5.83 mmol) of ethyl benzoylacetate in 10 mL of acetic acid at 0 °C, and the mixture was stirred at room temperature for 2 h. Subsequently, the resulting solution was added to a mixture of 762 mg (11.65 mmol) of zinc and 0.736 mL (5.83 mmol) of ethyl acetoacetate in 4 mL of acetic acid, and the mixture was refluxed at 120 °C overnight. The resulting reaction mixture was then washed with water and extracted with ethyl acetate. The extracted organic phase was dried over MgSO4, and the residue was purified by column chromatography to give diethyl 5-methyl-3-phenyl-1H-pyrrole-2,4-dicarboxylate (1.11 g, 63% yield). Next, 714 mg (2.37 mmol) of diethyl 1H-pyrrole-2,4-dicarboxylate and 652 mg (11.62 mmol) of KOH were mixed with ethylene glycol, and the resulting mixture was stirred at 170 °C for 3 hours. It was then washed with water and extracted with dichloromethane. The extracted organic phase was dried over MgSO4, and the residue was purified by column chromatography to obtain 2-methyl-4-phenylpyrrole (297 mg, 80% yield).

[0050] [ka]

[0051] Example 1 <2quBOD production> R in formula (1) 1 is a 2-quinolyl group, R 2 and R 4 is a phenyl group, R 3 and R 5 A compound in which is a methyl group (hereinafter also referred to as "2quBOD") (molecular weight 499.36) was produced by the following procedure. Quinoline-2-carbonyl chloride hydrochloride (210 mg, 0.92 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of 2-methyl-4-phenylpyrrole (145 mg, 0.92 mmol). The resulting mixture was stirred overnight at room temperature under a N atmosphere. The resulting crude ketone (165 mg) and 2-methyl-4-phenylpyrrole (146 mg, 0.93 mmol) were dissolved in dichloromethane (15 mL) containing POCl (1.95 mmol) at 0 °C. The resulting mixture was stirred at 30 °C for 48 h, washed with water, and extracted with dichloromethane. The extracted organic phase was dried and redissolved in dichloromethane (15 mL) without further purification. Triethylamine (5 mmol) and boron trifluoride diethyl etherate (7 mmol) were added to this solution, sequentially, and the reaction was allowed to proceed overnight. The resulting crude product was purified by column chromatography to give the desired compound (67 mg, yield 14.5%). The identification data of the obtained compound is shown below: From these results, it was confirmed that the obtained compound has the structure shown below. 1 H-NMR(500MHz,CDCl3,TMS,rt) δ(ppm) 2.70(6H,s),6.20(2H,s),6.44(6H,br,s),6.62(4H,m),6.82(1H,d,J=8.2Hz),7.21(1H,d,J=8.2Hz),7.38(2H,m),7.47(1H,m),7.55(1H,m); 13 C-NMR(75MHz,CDCl3,TMS,rt) δ(ppm) 14.87,121.48,123.98,125.67,126.62,126.74,126.82,128.18,128.74, 128.98,131.23,133.99,135.00,146.17,147.41,150.93,156.63;IR(ATR) 696.2,759.8,974.8,1080.9,1110.8,1164.8,1203.4,1487.8,1541.8cm -1 ;HRMS:calcd for C 32 H 24 BF2N3[M], 499.2031, Found 499.2031; Melting point 236-241℃.

[0052] [ka]

[0053] [ka]

[0054] 2quBOD was dissolved in a solvent and excited at an excitation wavelength of λ Abs , fluorescence wavelength λ em , molar extinction coefficient, fluorescence quantum yield φ f The results are shown in Table 1. ACN stands for acetonitrile.

[0055] [Table 1]

[0056] <Evaluation of UV absorption spectrum change due to pH change> 2quBOD was dissolved in an aqueous solution of 50% acetonitrile so that the final concentration became 5.0 μM, and the pH was gradually changed from 3.6 to 0.2 with hydrochloric acid. The UV absorption spectrum was measured for each solution. The results are shown in Fig. 1. As shown in Fig. 1, when the pH was 3.6, an absorption peak was observed around a wavelength of 520 nm. As the pH decreased, the absorption around a wavelength of 520 nm decreased, the peak shifted to the longer wavelength side, and when the pH was 0.2, a peak was observed around a wavelength of 528 nm. It is considered that non-protonated 2quBOD mainly existed around pH 3.6, and protonated 2quBOD (2quBOD-H) mainly existed around pH 0.2.

[0057] <Evaluation of fluorescence spectrum change due to pH change> 2quBOD was dissolved in an aqueous solution of 50% acetonitrile so that the final concentration became 5.0 μM, and the pH was gradually changed from 2.8 to 0.2 with hydrochloric acid. The fluorescence spectrum was measured for each solution. The results are shown in Fig. 2. As shown in Fig. 2, when the pH was 2.8, a peak of fluorescence intensity was observed around a wavelength of 545 nm. As the pH decreased, the fluorescence intensity around a wavelength of 545 nm decreased, and the peak shifted to the longer wavelength side. When the pH was 0.2, a peak was observed around a wavelength of 582 nm. Also, the color tone of the fluorescence of each solution was green when the pH was 2.8, and changed to yellow-green, yellow, light orange as the pH decreased, and became dark orange at pH 0.2.

[0058] <Evaluation of fluorescence intensity ratio change due to pH change> 2quBOD was dissolved in an aqueous solution of 50% acetonitrile so that the final concentration became 5.0 μM, and the pH was gradually changed from 8 to 0.2 with hydrochloric acid or sodium hydroxide. The fluorescence spectrum was measured for each solution, and the fluorescence intensity (F at a wavelength of 545 nm 545) versus the fluorescence intensity at 582 nm (F 582 ) ratio (F 582 / F 545 The results are shown in Figure 3. As shown in Figure 3, the fluorescence intensity ratio (F 582 / F 545 ) changed. The inflection point was pH 1.1. Therefore, the pKa of 2quBOD was 1.1.

[0059] Example 2 <2pyBOD production> The same procedure as in Example 1 was repeated except that pyridine-2-carbonyl chloride hydrochloride (164 mg, 0.92 mmol) was used instead of quinoline-2-carbonyl chloride hydrochloride, to obtain R 1 is a 2-pyridyl group, R 2 and R 4 is a phenyl group, R 3 and R 5 A compound in which R is a methyl group (hereinafter also referred to as "2pyBOD") (molecular weight) was produced. The yield was 26%. The identification data of the obtained compound is shown below: From these results, it was confirmed that the obtained compound has the structure shown below. 1 H-NMR(500MHz,CDCl3,TMS,rt) δ(ppm) 2.68(6H,s),6.20(2H,s),6.51(2H,t,J=6.0Hz),6.69(4H,d,J=7.6Hz),6.82(9H,m),7.71(H,d,J=4.4Hz); 13 C-NMR(125MHz,CDCl3,TMS,rt) δ(ppm) IR(KBr) 698,754,973,1081,1111,1165,1206,1500cm -1 ;HRMS:calcd for C 28 H 23 BF2N3[M+H] +,450.1953,Found 450.1963; Decomposed>.

[0060]

Chem.

[0061] 2pyBOD was dissolved in a solvent, and the excitation wavelength λ Abs , fluorescence wavelength λ em , molar absorption coefficient, and fluorescence quantum yield φ f were determined. The results are shown in Table 2.

[0062]

Table 2

[0063] <Evaluation of UV absorption spectrum change due to pH change> For 2pyBOD, except that the pH was changed from 4.0 to 0.9, the change in the UV absorption spectrum due to the pH change was evaluated in the same manner as in Example 1. The results are shown in Figure 4. As shown in Figure 4, when pH was 4.0, an absorption peak was observed around a wavelength of 520 nm. As the pH decreased, the absorption around a wavelength of 520 nm became smaller, the peak shifted to the longer wavelength side, and when pH was 0.9, a peak was observed around a wavelength of 535 nm. It is considered that around pH 4.0, unprotonated 2pyBOD mainly existed, and around pH 0.9, protonated 2pyBOD (2pyBOD-H) mainly existed.

[0064] <Evaluation of fluorescence spectrum change due to pH change> For 2pyBOD, except that the pH was changed from 4.0 to 0.9, the change in the fluorescence spectrum due to the pH change was evaluated in the same manner as in Example 1. The results are shown in Figure 5. As shown in Fig. 5, when pH was 4.0, a peak in fluorescence intensity was observed around a wavelength of 541 nm. As the pH decreased, the fluorescence intensity around a wavelength of 541 nm decreased and the peak shifted to the long wavelength side. When pH was 0.9, a peak was observed around a wavelength of 563 nm.

[0065] <Evaluation of change in fluorescence intensity ratio due to pH change> For 2pyBOD, fluorescence spectra were measured in the same manner as in Example 1 except that the pH was changed from 4.0 to 0.9, and the fluorescence intensity (F 541 ) at a wavelength of 563 nm with respect to the fluorescence intensity (F 563 ) at a wavelength of 541 nm was determined. The results are shown in Fig. 6. 563 / F 541 ) was determined. The results are shown in Fig. 6. As shown in Fig. 6, the fluorescence intensity ratio (F 563 / F 541 ) changed in response to the change in pH. Also, the pH at the inflection point was 1.7. Therefore, the pKa of 2pyBOD was 1.7.

[0066] <Evaluation of repeated switching of pH> Dissolve 2pyBOD in a 50% by mass acetonitrile aqueous solution so that the final concentration is 5.0 μM, and repeatedly switch the pH between pH 8.3 and pH 1.2 with hydrochloric acid or sodium hydroxide. For each solution, fluorescence spectra were measured in the same manner as above, and the fluorescence intensity ratio (F 563 / F 541 ) was determined. The results are shown in Fig. 7. As shown in Fig. 7, even when the pH was repeatedly switched, the values of the fluorescence intensity ratio at each pH were equivalent.

[0067] <Evaluation of change in fluorescence spectrum and fluorescence intensity ratio due to change in trifluoroacetic acid concentration> Dissolve 2pyBOD in acetonitrile so that the final concentration is 5.0 μM, add trifluoroacetic acid, and prepare solutions with trifluoroacetic acid concentrations of 10 μM to 4 mM. Fluorescence spectra were measured for each solution, and the fluorescence intensity (F 563 ) at a wavelength of 541 nm with respect to the fluorescence intensity (F541 ) ratio (F 541 / F 563 The fluorescence spectrum is shown in Figure 8, and the fluorescence intensity ratio (F 541 / F 563 ) is shown in Figure 9. As shown in Figure 8, when the trifluoroacetic acid concentration was 10 μM, an absorption peak was observed around 541 nm. As the trifluoroacetic acid concentration increased (the pH decreased), the absorption around 541 nm decreased and the peak shifted to longer wavelengths, and when the trifluoroacetic acid concentration was 4 mM, a peak was observed around 566 nm. As shown in Figure 9, the fluorescence intensity ratio (F 541 / F 563 ) was larger. In particular, a good correlation was obtained when the trifluoroacetic acid concentration was in the range of 5 to 750 μM.

[0068] Example 3 <Production of 6Me2pyBOD> The compound represented by the formula (1) R 1 is a 6-methyl-2-pyridyl group, R 2 and R 4 is a phenyl group, R 3 and R 5 is a methyl group (hereinafter also referred to as "6Me2pyBOD") (molecular weight = 463.33). The yield was 42.5%. The identification data of the obtained compound is shown below: From these results, it was confirmed that the obtained compound has the structure shown below. 1 H-NMR(500MHz,CDCl3,TMS,rt) δ(ppm) 1.98(3H,s),2.67(6H,s),6.19(2H,s),6.37(1H,d,J=7.1Hz),6.65(2H,m),6.72 (4H,d,J=7.1Hz),6.87(6H,m); 1313C-NMR (125 MHz, CDCl3, TMS, r.t.) δ (ppm) 14.85, 23.50, 115.17, 121.66, 122.65, 124.54, 126.22, 127.00, 128.28, 134.57, 135.64, 147.29, 149.48, 156.34, 156.81; IR (ATR) 695.2, 753.1, 970.0, 1067.4, 1111.8, 1161.9, 1202.4, 1360.5, 1487.8, 1541.8 cm -1 ; HRMS: calculated for C 29 H 24 BF2N3 [M], 463.2031, Found 463.2031; melting point 248 - 253 °C.

[0069]

Chem.

[0070] 6Me2pyBOD was dissolved in a solvent, and the excitation wavelength λ Abs , fluorescence wavelength λ em , molar absorption coefficient, fluorescence quantum yield φ f were determined. The results are shown in Table 3.

[0071]

Table 3

[0072] <Evaluation of Fluorescence Spectrum Changes due to pH Changes> For 6Me2pyBOD, except that the pH was changed from 7.3 to 1.2, the fluorescence spectrum changes due to pH changes were evaluated in the same manner as in Example 1. The results are shown in Figure 10. As shown in Figure 10, when pH was 7.3, a peak in fluorescence intensity was observed around a wavelength of 540 nm. As the pH decreased, the fluorescence intensity around a wavelength of 540 nm decreased and the peak shifted to the long wavelength side. When pH was 1.2, a peak was observed around a wavelength of 562 nm. Near pH 7.3, mainly non-protonated 6Me2pyBOD was present, and near pH 1.2, mainly protonated 6Me2pyBOD (6Me2pyBOD-H) was considered to be present.

[0073] <Evaluation of change in fluorescence intensity ratio due to pH change> For 6Me2pyBOD, fluorescence spectra were measured in the same manner as in Example 1 except that the pH was changed from 7.3 to 1.2, and the fluorescence intensity (F 540 ) at wavelength 562 nm with respect to the fluorescence intensity (F 562 ) at wavelength 540 nm was determined. The results are shown in Figure 11. 562 / F 540 ) was determined. The results are shown in Figure 11. As shown in Figure 11, the fluorescence intensity ratio (F 562 / F 540 ) changed in response to the change in pH. Also, the pH at the inflection point was 2.3. Therefore, the pKa of 6Me2pyBOD was 2.3.

[0074] <Evaluation of repeated switching of pH> 6Me2pyBOD was dissolved in a 50% by mass acetonitrile aqueous solution so that the final concentration was 5.0 μM, and the pH was repeatedly switched between pH 8.5 and pH 1.8 with hydrochloric acid or sodium hydroxide. For each solution, fluorescence spectra were measured in the same manner as above, and the fluorescence intensity ratio (F 562 / F 540 ) was determined. The results are shown in Figure 12. As shown in Figure 12, even when the pH was repeatedly switched, the values of the fluorescence intensity ratio at each pH were equivalent.

[0075] <Evaluation of change in fluorescence spectrum and fluorescence intensity ratio due to change in trifluoroacetic acid concentration> 6Me2pyBOD was dissolved in acetonitrile so that the final concentration was 5.0 μM, trifluoroacetic acid was added, and solutions with trifluoroacetic acid concentrations of 5 μM to 1 mM were prepared. For each solution, fluorescence spectra were measured, and the ratio of the fluorescence intensity (F 564 ) at wavelength 54l nm to the fluorescence intensity (F 541 ) at wavelength 564 nm (F541 / F 564 The fluorescence spectrum is shown in Figure 13, and the fluorescence intensity ratio (F 541 / F 564 ) is shown in Figure 14. As shown in Figure 13, when the trifluoroacetic acid concentration was 5 μM, an absorption peak was observed around 541 nm. As the trifluoroacetic acid concentration increased (the pH decreased), the absorption around 541 nm decreased and the peak shifted to longer wavelengths, and when the trifluoroacetic acid concentration was 1 mM, a peak was observed around 562 nm. As shown in Figure 14, the fluorescence intensity ratio (F 541 / F 564 ) had become larger.

[0076] Example 4 <Production of 4pyBOD> The same procedure as in Example 1 was repeated except that pyridine-4-carbonyl chloride hydrochloride (164 mg, 0.92 mmol) was used instead of quinoline-2-carbonyl chloride hydrochloride, to obtain R 1 is a 4-pyridyl group, R 2 and R 4 is a phenyl group, R 3 and R 5 A compound in which R is a methyl group (hereinafter also referred to as "4pyBOD") (molecular weight = 449.30) was produced in a yield of 24.1%. The identification data of the obtained compound is shown below: From these results, it was confirmed that the obtained compound has the structure shown below. 1 H-NMR(300MHz,CDCl3,TMS,rt) δ(ppm) 2.70(6H,s),6.22(2H,s),6.66(6H,m),6.85(4H,t,J=7.3Hz),6.97(2H,m),7.67(2H,d,J=5.9Hz); 1313C-NMR (125 MHz, CDCl3, TMS, r.t.) δ (ppm) 14.89, 122.18, 126.20, 127.21, 127.32, 128.51, 134.87, 147.36, 156.79; IR (KBr) 1075, 1112, 1171, 1206, 1502, 1552 cm -1 ; HRMS: calculated for C 28 H 23 BF2N3 [M + H]<0000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ A compound represented by the following formula 5 (hereinafter also referred to as "compound 5") (molecular weight 372.22) was produced by the following procedure. The target compound 5 (yield 6.3%) was obtained in the same manner as in Example 1, except that dimethylformamide (67 mg, 0.92 mmol) and POCl 3 (1.95 mmol) were used instead of quinoline-2-carbonyl chloride hydrochloride. The identification data of the obtained compound is shown below: From these results, it was confirmed that the obtained compound has the structure shown below. 1 H-NMR(500MHz,CDCl3,TMS,rt) δ(ppm) 2.66(6H,s),6.39(2H,s),7.28(1H,s),7.41(10H,m); 13 C-NMR(125MHz,CDCl3,TMS,rt) δ(ppm) 14.98,117.84,125.89,128.50,128.60,128.97,132.79,133.54,145.46,156.72;IR(ATR) 699.1,763.7,970.0,1018.2,1064.5,1155.2,1201.4,1486.8,1513.8,1585.2cm -1 ;HRMS:calcd for C 23 H 19 BF2N2[M], 372.1609, Found 372.1610; melting point 199-204℃.

[0082] [ka]

[0083] Compound 5 was dissolved in a solvent and excited at an excitation wavelength λ Abs , fluorescence wavelength λ em , molar extinction coefficient, fluorescence quantum yield φ f The results are shown in Table 5.

[0084] [Table 5]

[0085] <Evaluation of the change in fluorescence intensity ratio due to pH change> For Compound 5, when the fluorescence spectrum was measured in the same manner as in Example 1 except that the pH was changed from 7.0 to 1.3, almost no wavelength shift was observed (wavelength 538 nm at pH 7.0 and wavelength 539 nm at pH 1.3). As a result, an effective fluorescence intensity ratio could not be measured.

[0086] (Comparative Example 2) 2-Pyridinecarboxaldehyde (99 mg, 0.92 mmol) was dissolved in dichloromethane (10 mL), and then 2-methyl-pyrrole (150 mg, 1.84 mmol) was added. Trifluoroacetic acid (7.7 μL, 0.1 mmol) was added to the resulting mixture, and the mixture was stirred at room temperature overnight under a nitrogen atmosphere. The resulting mixture was poured into aqueous sodium hydrogen carbonate solution and extracted three times with 50 mL of dichloromethane. The dichloromethane extract was dried over 2 g of magnesium sulfate. The filtrate from which magnesium sulfate was removed by filtration was concentrated under reduced pressure. The obtained residue was dissolved in 10 mL of a dichloromethane-water mixed solvent (dichloromethane: water = 1:1), and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (209 mg, 0.92 mmol) was added to this solution and stirred at room temperature for 30 minutes. After the reaction, the mixture was poured into aqueous sodium hydrogen carbonate solution and extracted three times with 50 mL of dichloromethane. The dichloromethane extract was dried over 2 g of magnesium sulfate. The filtrate from which magnesium sulfate was removed by filtration was concentrated under reduced pressure. The obtained residue was dissolved in 10 mL of dichloromethane, and triethylamine (10 mmol) and boron trifluoride diethyl ether complex (7 mmol) were added to this solution in sequence, and the reaction was carried out overnight. The obtained crude product was purified by column chromatography to produce a compound represented by the following formula 6 (hereinafter, also referred to as "Compound ⑥"). The yield was 17.4%. The identification data of the obtained compound are shown below. From these results, it was confirmed that the obtained compound had the structure shown below. 1H-NMR (300 MHz, CDCl3, TMS, r.t.) δ (ppm) 2.65 (6H, s), 6.28 (2H, d, J = 3.5 Hz), 6.79 (2H, d, J = 4.1 Hz), 7.44 (1H, dd, J = 7.33, 5.0 Hz), 7.57 (1H, d, J = 8.2 Hz), 7.84 (1H, t, J = 7.6 Hz), 8.78 (1H, d, J = 4.7 Hz); 13 C-NMR (125 MHz, CDCl3, TMS, r.t.) δ (ppm) 14.95, 119.81, 124.25, 126.03, 130.45, 134.29, 136.26, 139.27, 149.88, 152.94, 158.32; IR (KBr) 720, 986, 1009, 1080, 1150, 1275, 1493, 1552 cm -1 ; HRMS: calcd for C 16 H 15 BF2N3 [M + H] + , 298.1327, Found 298.1330; melting point 141 - 145 °C.

[0087]

Chem.

[0091] (molecular orbital calculation) In the UV absorption and fluorescence spectra of the compounds of Examples 1 to 4, the peaks shifted to longer wavelengths by about 22 to 38 nm as the pH decreased. In this case, the molecular orbitals related to fluorescence must be reorganized by protonation. The ground-state frontier orbitals for 2pyBOD and 2pyBOD-H in acetonitrile are shown in Figure 16. Ab-initio calculations were performed using Gaussian 16 with the density functional CAM-B3LYP and 6-31G(d) basis set. The dihedral angles between the meso-substituents and BODIPY were calculated to be 67.48° for 2pyBOD and 66.29° for 2pyBOD-H in the ground state in acetonitrile, as shown in Figure 16. The HOMO shapes of these compounds are similar, but the LUMO of 2pyBOD-H is mainly extended by the meso-substituents, resulting in a lower energy.

[0092] Figure 17 shows the energy profiles of 2pyBOD-H and protonated compound 5 in acetonitrile depending on the dihedral angle (40 to 140°) between BODIPY and the meso-substituent. As shown in Figure 17, the energy profile of 2pyBOD-H depending on the dihedral angle (40–140°) showed a small barrier (7.3 kJ / mol) at 90°, unlike that of protonated compound 5. This is attributed to the 1,7-diphenyl group rotating in concert with the rotation of the meso-substituent.

[0093] This calculation result can explain the above evaluation results. In other words, the 1,7-diphenyl group controls the dihedral angle between the meso substituent and BODIPY at 60-70°. This allows partial coupling with the LUMO, but not with the HOMO. When the meso substituent is protonated, the LUMO energy is lowered, and the LUMO spreads over the meso substituent, causing a fluorescence shift. [Industrial Applicability]

[0094] Although the compound or salt thereof of the present invention is a low molecular weight compound, it exhibits a fluorescence wavelength shift in response to changes in pH. Therefore, its use as a fluorescent probe enables ratiometric measurements. Furthermore, it provides excellent spatial resolution in measurements requiring spatial resolution, such as imaging.

Claims

1. A compound represented by the following formula (1) or a salt thereof: 【Chemistry 1】 However, R １ represents a group represented by the following formula (2a), (2b) or (2c): R ２ and R ４ each independently represents a substituted or unsubstituted aryl group, R ３ and R ５ each independently represents a substituted or unsubstituted chain hydrocarbon group, or a substituted or unsubstituted styryl group, X １ and X ２ each independently represents a fluorine atom or a group represented by -O-R, where R represents an alcohol residue or a sugar residue. 【Chemistry 2】 However, R ６ ~R ９ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group; R ６ and R ７ Or R ７ and R ８ may be taken together to form a ring structure with the carbon atom to which they are attached, R １０ ~R １３ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group; R １０ and R １１ Or R １１ and R １２ may be taken together to form a ring structure with the carbon atom to which they are attached, R １４ ~R １７ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group; R １４ and R １５ may be taken together to form a ring structure with the carbon atom to which they are attached, R １６ and R １７ may be joined together to form a ring with the carbon atom to which they are attached.

2. R in the formula (1) ２ and R ４ The compound or salt thereof according to claim 1 , wherein each independently represents a substituted or unsubstituted phenyl group.

3. R in the formula (1) １ The compound or salt thereof according to claim 1 or 2, wherein represents an alkyl-substituted or unsubstituted 2-, 3-, or 4-pyridyl group, or an alkyl-substituted or unsubstituted 2-, 3-, or 4-quinolyl group.

4. A fluorescent probe comprising the compound according to any one of claims 1 to 3 or a salt thereof.

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