Novel rhodamine derivative

Abstract

A compound represented by general formula (I) or (II), or a salt thereof. (In the formulas, R1, R2, R3, R4, R5, R6, R7, R10, R11, X, Y, and Z are as defined in the specification)

Description

Novel rhodamine derivatives

[0001] This disclosure relates to novel rhodamine derivatives.

[0002] Rhodamines are a general term for dyes in which nitrogen atoms are bonded to the 3rd and 6th positions of a xanthene ring. They have been widely used in fluorescence imaging as dyes that possess high fluorescence quantum yield, strong photobleaching resistance, and water solubility. On the other hand, some rhodamines exhibit nonfluorescence due to some quenching mechanism. Such "nonfluorescent rhodamines" are not only used as quenchers that quench the fluorescence of donor molecules as acceptors in FRET, but by elucidating the mechanism of their nonfluorescence and designing appropriate molecules that can release the nonfluorescence using specific biological phenomena as a switch, it will be possible to develop fluorescent probes based on new fluorescence control principles.

[0003] The QSY series, representative non-fluorescent rhodamines, are dyes in which a phenyl molecule is bonded to the N atom on the xanthene ring of rhodamine (hereinafter also referred to as "N-phenylrhodamines," see Figure 1). N-phenylrhodamines such as QSY7 and QSY21 exhibit strong quenching, and our prior research has suggested that this quenching is caused by the generation of a p-TICT (phenyl-induced twisted intramolecular charge transfer) state in the excited state (Non-Patent Literature 1). TICT is a phenomenon in which a charge imbalance occurs within a molecule and simultaneously causes a twist in the molecular structure in the excited state. Non-Patent Literature 1 discloses Halo rhodamine 4 as an example of a Halo tag probe based on phenyl-induced TICT.

[0004]

[0005] Hanaoka et. al. J. Am. Chem. Soc. 2022. 144, 19778

[0006] Incidentally, in compounds in which an aryl group is bonded to the N atom on the xanthene ring, such as N-phenylrhodamines, water solubility may decrease due to the introduction of an aromatic ring. When water solubility decreases, the permeability of the cell membrane also decreases, and it may become difficult for the compound to reach the target inside the cell. An object of the present invention is to provide a novel rhodamine derivative that is quenched by the p-TICT mechanism and has high water solubility.

[0007] The present inventors have completed the present invention by introducing a monovalent heterocyclic group which may have a substituent and contains at least one hetero atom as a polar functional group to the N atom on the xanthene ring.

[0008] Item 1. A compound represented by the following general formula (I) or (II) or a salt thereof. (In formula (I), R １ represents a hydrogen atom or one to three identical or different monovalent substituents present on the benzene ring, and R ２ and R ３ each independently represent a hydrogen atom or a monovalent substituent present on the benzene ring, and R ４ and R ５ each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a carboxyl group, an ester group, an amide group or a halogen atom, and R ６ and R ７ each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a carboxyl group, an ester group, an amide group or a halogen atom, and R １０ is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, R １１ is a monovalent heterocyclic group which may have a substituent and contains at least one hetero atom, R １０ and R １１ together may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R １０ and R １１ are bonded, and X is an oxygen atom, Si(R ａ )(R ｂ ), C(R ａ )(R ｂ ), Ge(Rａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Y is -NR ８ R ９ Or -OH, where R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, and (i) Y is -NR ８ R ９ In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1 to 6 carbon atoms, (ii) When Y is -OH, R １０ is a group other than a hydrogen atom, and R ５ and R ７ (One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.) (In formula (II), R １R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Z is And (i) Z is In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R８ or R ９ may form a 5- to 7-membered heterocyclyl containing a nitrogen atom to which it is attached, and further the heterocyclyl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, or an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms, R ８ and R ９ may together form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R ８ and R ９ are attached, and further the heterocyclyl may be substituted with an alkyl group having 1 to 6 carbon atoms, (ii) when Z is R １０ is a group other than a hydrogen atom, and at least one of R ５ and R ７ is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms or a halogen atom.) [[ID=--]]

[0009] Item 2. A compound represented by the general formula (I) or (II) or a salt thereof, R ８ and R ４ may together form a 5- to 7-membered heterocyclyl containing a nitrogen atom to which R ８ or R ９ is attached, R ８ and R ９ may together form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R ８ and R ９ are attached, R ９ and R ６ may together form a 5- to 7-membered heterocyclyl containing a nitrogen atom to which R ８ or R ９ is attached, the compound or a salt thereof according to Item 1.

[0010] Item 3. The compound or a salt thereof according to Item 1 or 2, wherein in the formula (I) or (II), R １１ is a monocyclic heteroaryl group containing at least one heteroatom selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom. Item 4. In the formula (I) or (II), R１１ However, the compound or salt thereof described in any one of items 1 to 3, which is a heterocyclic group of a five-membered ring or a six-membered ring containing a nitrogen atom. Item 5. In formula (I) or (II), R １１ However, the compound or salt thereof described in item 1, which is a monocyclic heteroaryl group containing a sulfur atom. Item 6. A compound or salt thereof represented by formula (I), wherein Y is -NR ８ R ９ And R ８ and R ９ The compound or salt thereof according to any one of claims 1 to 4, wherein is the same or different substituted or unsubstituted alkyl group having 1 to 14 carbon atoms.

[0011] Item 7. A fluorescent probe comprising a compound or a salt thereof as described in any one of Items 1 to 6. Item 8. A method for detecting reactive oxygen species, comprising the steps of reacting a compound or a salt thereof as described in any one of Items 1 to 6 or a fluorescent probe as described in Item 7 with reactive oxygen species, and measuring the fluorescence emitted by the reaction.

[0012] Item 9. A cell detection agent containing a compound or salt thereof described in any one of items 1 to 6, or a fluorescent probe described in item 7. Item 10. A method for bioimaging cells using a compound or salt thereof described in any one of items 1 to 6, a fluorescent probe described in item 7, or a cell detection agent described in item 9.

[0013] Item 11. A method for detecting cells is provided, comprising contacting a cell with a compound or salt thereof described in any one of items 1 to 6, a fluorescent probe described in item 7, or a cell detection agent described in item 9, and measuring the fluorescence emitted from the reaction product of the compound or salt thereof described in any one of items 1 to 6, the fluorescent probe described in item 7, or the cell detection agent described in item 9.

[0014] Item 12. A method for reacting a compound or salt thereof described in any one of Items 1 to 6 or a fluorescent probe described in Item 7 with a target molecule, wherein the reaction releases the quenching of the compound or salt thereof described in any one of Items 1 to 6 or the fluorescent probe described in Item 7, and restores its fluorescence (or increases its fluorescence intensity). Item 13. A method for detecting a target molecule, further comprising the steps of reacting a compound or salt thereof described in any one of Items 1 to 6 or a fluorescent probe described in Item 7 with a target molecule, wherein the reaction releases the quenching of the compound or salt thereof described in any one of Items 1 to 6 or the fluorescent probe described in Item 7, and restores its fluorescence or increases its fluorescence intensity, and measuring the fluorescence emitted by the release of quenching as described in Item 12.

[0015] The present invention makes it possible to provide a novel rhodamine dye that is quenched by a p-TICT mechanism and has high water solubility.

[0016] Chemical structures of various rhodamine dyes. Optical properties of N-Imidazole Rho1, N-Imidazole Rho2, and diEt Rho are shown. Fluorescence intensity of solutions in which N-Imidazole Rho1, N-Imidazole Rho2, and N-Thiazole Rho are dissolved in various solvents is shown. Reactivity of various ROS with N-Imidazole Rho1 and N-Imidazole Rho2 is shown. N-Imidazole Rho1 is dissolved in hypochlorous acid (HOCl) and singlet oxygen ( 1 The fluorescence intensity when N-Imidazole Rho2 is reacted with hypochlorous acid (HOCl) and singlet oxygen (O2) is shown. 1 The fluorescence intensity when reacted with each of the following (O2): Fluorescence images of living cells stained with N-Imidazole Rho1 (top left), N-Imidazole Rho2 (top right), and N-Thiazole Rho (bottom left) are shown. The scale in the bottom right of each image is 20 μm. Comparison of fluorescence quantum yield. Results of viscosity sensitivity test. Results of dielectric constant sensitivity test. Switching of donor and acceptor by diethylamino group-mediated TICT and heterocyclic structure-mediated TICT in PBS. 1Fluorescence spectra of each probe (1 μM) when reacted with O2 (excitation 500 nm). Singlet oxygen in PBS ( 1 Fluorescence spectra of each probe (1 μM) when reacted with O2 (excitation 500 nm). Fluorescence imaging of HeLa cells after addition of Imidazole-THQR (5 μM) and 5-ALA-inducing PpIX (Ex. 514 nm, Em. 530–600 nm). HeLa cells. 1 Box plot of fluorescence intensity before and after O2 reaction. *p < 0.0001 Two-sided Welch's t-test. Fluorescence intensity of HeLa cells with various reagents added. *p < 0.0001 Two-sided Welch's t-test. Measurement of the HOMO energy level of Thiophen-THQR. Calculation of the energy change of the S1 state with respect to the dihedral angle φ between the Xanthene ring and the N atom. Measurement of absorbance (left) and fluorescence intensity (right) of each compound.

[0017] In this specification, "alkyl group" or the alkyl group of a substituent containing an alkyl group (e.g., an alkoxy group) means, unless otherwise specified, an alkyl group consisting of a linear, branched, cyclic, or combination thereof, having, for example, 1 to 14 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. If a number of carbon atoms is specified, it means an alkyl group having a number of carbon atoms within that range. More specifically, examples of alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropylmethyl group, n-pentyl group, n-hexyl group, and the like.

[0018] In this specification, "halogen atom" refers to any of fluorine, chlorine, bromine, or iodine atoms, preferably fluorine, chlorine, or bromine atoms. In this specification, "heteroaryl group" refers to an optionally substituted heteroaryl group which comprises 5 to 20 (e.g., 5 to 12 or 5 to 10) skeletal ring-forming atoms, of which at least one (e.g., 1 to 4, 1 to 3, or 1 to 2) ring-forming atoms is a heteroatom, and the heteroatom is a heteroatom independently selected from oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, tin, and tellurium, but is not limited to these. In this specification, "heterocyclyl" refers to a monovalent group derived from a cyclic compound containing at least one heteroatom (such as oxygen, nitrogen, or sulfur), and includes heterocyclylalkyl groups, heteroalkenyl groups, and heteroaryl groups. In this specification, “heterocyclic” means a non-aromatic ring in which one or more ring-forming groups are heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms). In this specification, “monovalent heterocyclic group” means the group remaining after removing one hydrogen atom from a heterocyclic compound that is directly bonded to a carbon atom or heteroatom constituting the ring.

[0019] 1. Compounds represented by general formula (I) or (II) or salts thereof This disclosure provides compounds represented by the following general formulas (I) or (II) or salts thereof.

[0020] (In formula (I), R １ R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Y is -NR ８ R ９ Or -OH, where R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, and (i) Y is -NR ８ R ９ In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９R may form a 5-7 membered heterocycline containing a nitrogen atom to which it is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1 to 6 carbon atoms, (ii) When Y is -OH, R １０ is a group other than a hydrogen atom, and R ５ and R ７ (One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.) (In formula (II), R １ R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Cautionｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Z is And (i) Z is In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which it is bonded, and (ii) Z is In the case of R １０ is a group other than a hydrogen atom, and R ５ and R ７ (One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.)

[0021] In some embodiments, the present disclosure relates to a compound represented by the above general formula (I) or a salt thereof, wherein R １ , R ２ , R ３ , R ４ , R５ , R ６ , R ７ , R １０ , R １１ X and Y are defined above with respect to the compound represented by general formula (I), and R ８ and R ４ Together, R ８ or R ９ It may form a 5-7 member heterocycline containing a nitrogen atom to which it is bonded, R ８ and R ９ Together, R ８ and R ９ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which it is bonded, R ９ and R ６ Together with R ８ or R ９ The present invention provides a compound or a salt thereof that may form a 5- to 7-membered heterocycline containing a nitrogen atom to which is bonded.

[0022] In some embodiments, the disclosure is a compound represented by the above general formula (II) or a salt thereof, where R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R １０ , R １１ X and Y are defined above with respect to equation (II).

[0023] In some embodiments, the disclosure provides compounds or salts thereof represented by the following general formulas (Ia) or (IIa).

[0024] (In formula (Ia), R １ R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ It may form a 4-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Cautionｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ (This is an alkyl group having 1 to 6 carbon atoms or a substituted phenyl group.)

[0025] (In formula (IIa), R １ R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９It may form a 4-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ (This is an alkyl group having 1 to 6 carbon atoms or a substituted phenyl group.)

[0026] In each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R １ This represents either a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring.

[0027] R １ The type of monovalent substituent represented is not particularly limited, but it is preferable to select from the group consisting of, for example, an alkyl group having 1 to 14 carbon atoms (preferably 1 to 12, more preferably 1 to 6), an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 14 carbon atoms (preferably 1 to 12, more preferably 1 to 6), a hydroxyl group, a carboxyl group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, an amino group, an amide group, and an alkylamide group.

[0028] These monovalent substituents may further have one or more substituents. For example, R １ The alkyl group represented by may contain one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., for example R １ The alkyl group indicated may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group, etc.

[0029] Also, for example, R １ The amino group shown may have one or two alkyl groups present, R １ The amino group indicated by may be a monoalkylamino group or a dialkylamino group. Furthermore, R １ Examples of cases where the alkoxy group shown has substituents include carboxylated alkoxy groups or alkoxycarbonyl substituted alkoxy groups, and more specifically, 4-carboxybutoxy groups or 4-acetoxymethyloxycarbonylbutoxy groups. Also, for example, R １ The amide group, alkylamide group, sulfonyl group, and alkoxycarbonyl group shown may have one or two alkyl groups present.

[0030] In one preferred embodiment of the present disclosure, R １ is one carboxyl group, sulfonyl group, hydroxyl group, amino group, or alkoxy group. In one preferred embodiment of the present disclosure, R １ They are all hydrogen atoms.

[0031] In each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R ２ and R ３ Each of these independently represents a monovalent substituent present on a hydrogen atom or a benzene ring. ２ and R ３ The monovalent substituent is preferably selected from an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a carboxyl group, or an ester group. ２ and R ３The monovalent substituent may further have one or more substituents. Examples of such substituents include halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, and alkoxy groups. For example, R ２ and R ３ However, the alkyl group may have one or more further substituents such as halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., for example R ２ or R ３ If the substituted alkyl group is a substituted alkyl group, such substituted alkyl group may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group, etc. ２ or R ３ If the substituted alkoxy group has a substituent, then such substituted alkoxy group is a halogenated alkyl group (e.g., -OCF ３ ) is also acceptable.

[0032] In each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group (-COOH), an ester group (COOR), an amide group (CONR), or a halogen atom (where R is an alkyl group). ４ or R ５ Examples of substituents on the alkyl group include halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, and alkoxy groups, and these may be present in one or more quantities. ４ or R ５ Examples of substituted alkyl groups include halogenated alkyl groups, hydroxyalkyl groups, and carboxyalkyl groups.

[0033] In each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R ６ and R ７Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group (-COOH), an ester group (COOR), an amide group (CONR), or a halogen atom (where R is an alkyl group). ６ and R ７ For more details, see R ４ and R ５ This is similar to the explanation given earlier.

[0034] In one preferred embodiment of the present disclosure, R ４ , R ５ , R ６ and R ７ In all cases, R ４ , R ５ , R ６ and R ７ At least one of these groups is a group other than a hydrogen atom. Although not intended to be theoretically bound, in this disclosure, it is believed that by introducing a heterocycle bonded to the amino group on the xanthene ring, intramolecular charge transfer (ICT) occurs, promoting the formation of a TICT state in the excited state, and each of the compounds represented by formulas (I), (II), (Ia), and (IIa) exhibits non-fluorescence.

[0035] R １０ However, the alkyl group is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of substituents on the alkyl group include halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, and alkoxy groups. Furthermore, part or all of the hydrocarbon chain of the alkyl group may be substituted with an ether.

[0036] R １１ However, it is a monovalent heterocyclic group which may have substituents and contain at least one heteroatom. The heterocyclic group may have 3 to 14 ring-forming atoms (for example, 3 to 10, 3 to 8, 4 to 7, or 5 to 6). １０ and R １１ Together, R １０ and R １１It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which it is bonded.

[0037] R １１ The heterocyclic groups of the heterocyclic group include heterocyclic rings containing a nitrogen atom as a heteroatom (for example, five-membered rings such as pyrrole rings, pyrrolidine rings, pyrazole rings, imidazole rings, triazole rings, and tetrazole rings; six-membered rings such as isocyanuric rings, pyridine rings, pyridazine rings, pyrimidine rings, pyrazine rings, piperidine rings, and piperazine rings; and condensed rings such as indole rings, indoline rings, quinoline rings, acridine rings, naphthyridine rings, quinazoline rings, and purine rings), heteroatoms Heterocyclic rings containing an oxygen atom as a child (e.g., three-membered rings such as oxirane rings; four-membered rings such as oxetane rings; five-membered rings such as furan rings, tetrahydrofuran rings, oxazole rings, isoxazole rings, and γ-butyrolactone rings; six-membered rings such as 4-oxo-4H-pyran rings, tetrahydropyran rings, and morpholine rings; condensed rings such as benzofuran rings, isobenzofuran rings, 4-oxo-4H-chromene rings, chroman rings, and isochroman rings; and 3-oxatricyclo[4.3.1.1 4,8 ]Undecane-2-one ring, and 3-oxatricyclo[4.2.1.0 4,8 Examples include, but are not limited to, bridging rings such as nonane-2-one rings, and heterocycles containing a sulfur atom as a heteroatom (e.g., five-membered rings such as thiophene rings, thiazole rings, isothiazole rings, and thiadiazole rings; six-membered rings such as 4-oxo-4H-thiopyran rings; and fused rings such as benzothiophene rings), and heterocycles containing a selenium atom as a heteroatom (e.g., five-membered rings such as selenofen rings, fused rings such as benzo[b]selenophene rings and seleno[3,2-b]thiophene rings).

[0038] Examples of substituents that a monovalent heterocyclic group containing at least one heteroatom may optionally have include, but are not limited to, C1-C4 alkyl groups, C1-C4 alkynyl groups, C1-C4 alkoxy groups, C1-C8 amino groups, C1-C4 sulfide groups, C1-C4 fluorinated alkyl groups such as trifluoromethyl groups, cyano groups, nitro groups, halogen atoms, azide groups, carboxyl groups, ester groups, amide groups, sulfonyl groups, sulfonamide groups, phosphone groups, and hydroxyl groups.

[0039] In this disclosure, in each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R １１ By designing molecules that incorporate a monovalent heterocyclic group containing at least one heteroatom and potentially having substituents, intramolecular charge transfer occurs, promoting the formation of a TICT state in the excited state. It is believed that each of the compounds represented by formulas (I), (II), (Ia), and (IIa) are strongly quenched by the p-TICT quenching mechanism.

[0040] In one preferred embodiment of the present disclosure, in each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R １０ is hydrogen, R １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom.

[0041] In one preferred embodiment of the present disclosure, R １１ R is a monocyclic heteroaryl group comprising at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur atoms. In one preferred embodiment of the present disclosure, R １１ is a monocyclic heteroaryl group containing an oxygen atom, a nitrogen atom, or both. In one preferred embodiment of the present disclosure, R １１ is a five-membered or six-membered heterocyclic group containing a nitrogen atom. In one preferred embodiment of the present disclosure, R １１ is a five-membered or six-membered heteroaryl group containing a nitrogen atom. In one preferred embodiment of the present disclosure, R １１It is a monocyclic heteroaryl group containing a sulfur atom.

[0042] In another preferred embodiment of the present disclosure, in each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), R ８ and R ９ All of these are identical or different substituted or unsubstituted alkyl groups having 1 to 6 carbon atoms, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom.

[0043] In each of the compounds represented by general formula (I), formula (II), formula (Ia), and formula (IIa), X is an oxygen atom, Si(R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te(=O). In one preferred embodiment of the present disclosure, X is an oxygen atom or Si(R ａ ) (Caution ｂ )

[0044] R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group. ａ and R ｂ Each of these is preferably an alkyl group having 1 to 3 carbon atoms, R ａ and R ｂ It is more preferable that both are methyl groups. ａ and R ｂ The alkyl group represented by may contain one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., for example R ａ and R ｂ The alkyl group indicated may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like.

[0045] R ａ and R ｂ When is an aryl group, the aryl group may be either a monocyclic aromatic group or a fused aromatic group, and the aryl ring may contain one or more ring-forming heteroatoms (e.g., nitrogen, oxygen, or sulfur atoms). A phenyl group is preferred as the aryl group. One or more substituents may be present on the aryl ring. Examples of substituents may include one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc.

[0046] R ｃ R is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group. Substituents for the phenyl group include methyl, hydroxyl, and methoxy groups. From the standpoint of ease of introduction in synthesis, ｃ The group is preferably a methyl group or a phenyl group. Also, R ｃ A methyl group is preferable because it results in higher water solubility.

[0047] In the compound represented by general formula (I), Y is -NR ８ R ９ or -OH, R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of substituents on the alkyl group include halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, and alkoxy groups. (i) Y is -NR ８ R ９ In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ A 4- to 7-membered (preferably 5-membered) heterocycline containing a nitrogen atom to which is bonded may be formed, and the heterocycline may further be substituted with an alkyl group having 1 to 6 carbon atoms. (ii) When Y is -OH, R １０ is a group other than a hydrogen atom, and R ５ and R ７ One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.

[0048] In one preferred embodiment of this disclosure, Y is -NR ８ R ９ And R ８ and R ９ These are all groups other than hydrogen atoms. In one preferred embodiment of this disclosure, Y is -NR ８ R ９ And R ８ and R ９ These are identical or different substituted or unsubstituted alkyl groups having 1 to 14 carbon atoms, preferably alkyl groups having 1 to 6 carbon atoms.

[0049] In the compound represented by general formula (II), Z is And R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of substituents on the alkyl group include halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, and alkoxy groups. (i) Z is In the case of R ８ and R ９At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which it is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ (ii) Z is a 4- to 7-membered (preferably 5-membered) heterocycline containing a nitrogen atom to which is bonded, and the heterocycline may be further substituted with an alkyl group having 1 to 6 carbon atoms. In the case of R １０ is a group other than a hydrogen atom, and R ５ and R ７ One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.

[0050] In one preferred embodiment of the present disclosure, Z is And R ８ and R ９ These are all groups other than hydrogen atoms.

[0051] In one preferred embodiment of the present disclosure, Z is And R ８ and R ９ The alkyl groups are identical or different substituted or unsubstituted groups having 1 to 14 carbon atoms, preferably 1 to 6 carbon atoms.

[0052] Each of the compounds represented by general formulas (I), (II), (Ia), and (IIa) of this disclosure may exist as an acid addition salt or a base addition salt. Examples of acid addition salts include mineral salts such as hydrochloride, sulfate, and nitrate, or organic salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Examples of base addition salts include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt. In addition, they may also form salts with amino acids such as glycine. The compound of general formula (I) of this disclosure or its salts may also exist as hydrates or solvates, and these substances are also within the scope of this disclosure.

[0053] Each of the compounds represented by the general formulas (I), (II), (Ia), and (IIa) of this disclosure may have one or more chiral carbons depending on the type of substituent. However, stereoisomers such as optically active compounds based on one or more chiral carbons and diastereoisomers based on two or more chiral carbons, as well as any mixture of stereoisomers and racemates, are all included within the scope of this disclosure.

[0054] Methods for producing representative compounds among those represented by general formulas (I), (II), (Ia), and (IIa) of this disclosure are specifically shown in the examples of this specification. Accordingly, those skilled in the art can produce each of the compounds represented by general formulas (I), (II), (Ia), and (IIa) by appropriately selecting reaction materials, reaction conditions, reaction reagents, etc., and modifying or altering these methods as necessary, based on these descriptions.

[0055] Non-limiting examples of the compounds or salts thereof of this disclosure are shown below.

[0056]

[0057] 2. Fluorescent Probes Another embodiment of the present disclosure is a fluorescent probe comprising a compound of general formula (I) or (II) or a salt thereof.

[0058] The compounds of general formula (I) or (II) of this disclosure exhibit strong fluorescence quenching due to the introduction of a potentially substituted monovalent heterocyclic group containing at least one heteroatom to the alkyl group on the N atom. Furthermore, because a potentially substituted monovalent heterocyclic group containing at least one heteroatom is introduced to the alkyl group on the N atom, these compounds have higher water solubility compared to compounds containing a phenyl group at the position of the potentially substituted monovalent heterocyclic group containing at least one heteroatom. Since the potentially substituted monovalent heterocyclic group containing at least one heteroatom is polar, the compounds of general formula (I) also exhibit higher cell membrane permeability compared to compounds containing a phenyl group at the position of the potentially substituted monovalent heterocyclic group containing at least one heteroatom. It is believed that when the potentially substituted monovalent heterocyclic group containing at least one heteroatom is oxidized by reaction with a target molecule, the TICT-induced quenching is released and fluorescence is restored.

[0059] The fluorescent probes of this disclosure can be applied to the detection of target molecules. Examples of target molecules include, but are not limited to, reactive oxygen species (ROS).

[0060] This disclosure further provides a method for detecting reactive oxygen species, comprising the steps of reacting a compound of general formula (I) or (II) or a fluorescent probe with reactive oxygen species, and measuring the fluorescence emitted by the reaction.

[0061] The method of use of the fluorescent probes of this disclosure is not particularly limited and can be used in the same way as conventionally known fluorescent probes. Typically, the compound represented by general formula (I) or a salt thereof is dissolved in an aqueous medium such as physiological saline or buffer, or in a mixture of an aqueous medium and a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide, and this solution is added to a suitable buffer containing cells or tissues, and the fluorescence spectrum is measured. The fluorescent probes of this disclosure may also be used in the form of a composition in combination with appropriate additives. For example, they can be combined with additives such as buffers, solubilizers, and pH adjusters. The fluorescent probes of this disclosure have high permeability into cells and, by introducing desired substituents, can be selectively localized in desired cells (e.g., cancer cells such as HeLa cells) in vivo.

[0062] Therefore, this disclosure provides a cell detection agent containing a compound represented by the above general formula (I) or (II) or a salt thereof, or the above fluorescent probe. The cell detection agent may contain a solvent (e.g., an organic solvent), a buffer for adjusting the pH (e.g., HEPES buffer, Tris buffer, Trisine-sodium hydroxide buffer, phosphate buffer, phosphate-buffered saline, etc.). The cell detection agent is preferably a cancer cell detection agent.

[0063] This disclosure allows the compound represented by the above general formula (I) or (II) or a salt thereof, the fluorescent probe, or the cell detection agent to be used in a cell bioimaging method. This disclosure provides a method for detecting cells, comprising contacting a cell with the compound represented by the above general formula (I) or (II) or a salt thereof, the fluorescent probe, or the cell detection agent, and measuring the fluorescence produced by the contact. The compound represented by the general formula (I) or (II) or a salt thereof taken up into the cell is R １１ When the monovalent heterocyclic group is oxidized, fluorescence is restored, and cells (e.g., cancer cells) can be detected by measuring the fluorescence emitted by the compound represented by the above general formula (I) or its salt, the above fluorescent probe, or the reaction product of the cell detection agent.

[0064] This disclosure provides a method for reacting a compound represented by the general formula (I) or (II) or a salt thereof, or the fluorescent probe, with a target molecule, wherein the reaction releases the quenching of the compound represented by the general formula (I) or (II) or a salt thereof, or the fluorescent probe, thereby restoring its fluorescence or increasing its fluorescence intensity.

[0065] The Disclosure further provides a method for detecting a target molecule, comprising reacting a compound or salt thereof represented by the general formula (I) or (II), or the fluorescent probe, with the target molecule, wherein the reaction releases the quenching of the compound or salt thereof represented by the general formula (I) or (II), or the fluorescent probe, thereby restoring its fluorescence or increasing its fluorescence intensity, and measuring the fluorescence emitted as a result of the release of quenching.

[0066] The present invention will be described below with reference to examples, but the present invention is not limited thereto.

[0067] Synthesis Example 1: Synthesis of Imidazole Rho1 Synthesis of Compound 1 p-toluenesulfonic acid monohydrate (228 mg, 1.20 mmol, 1.2 eq.) was mixed with anhydrous toluene (2 mL), and under an argon atmosphere, 2-bromo-1-methyl-1H-imidazole (0.15 mL, 1.5 mmol, 1.5 eq.) and m-anisidine (0.11 mL, 1.0 mmol, 1 eq.) were added. The mixture was stirred at 120°C for 22 hours. The solution was cooled to room temperature, and the reaction was stopped with saturated sodium bicarbonate aqueous solution. The solvent was evaporated to dryness, and the residue was extracted by DCM. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated to dryness, and the resulting residue was purified by column chromatography (aminosilica, DCM / MeOH) to obtain Compound 1 (71.2 mg, yield 35%). 1H NMR (500 MHz, CDCl3): δ 7.11 (brs, 1H), 7.06 (t, J = 8.4 Hz, 1H), 6.89 (d, J = 1.5 Hz, 1H), 6.71 (d, J = 1.5 Hz, 1H), 6.39-6.37 (m, 1H), 6.35-6.33 (m, 2H), 3.71 (s, 3H), 3.40 (s, 3H); 13 HRMS (ESI + ): Calcd. for [M+H] + , 204.1137; Found, 204.1146 (+0.9 mmu).

[0068]

[0069] Synthesis of N-Imidazole Rho1: 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (20.4 mg, 0.0651 mmol, 1 eq.) and compound 1 (13.2 mg, 0.0651 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The solution was cooled to room temperature and neutralized with sodium hydroxide. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 80 / 20 to 50 / 50 (50 min); flow rate = 5.0 mL / min) to obtain N-Imidazole Rho1 (20.0 mg, yield 53%). 1H NMR (500 MHz, CD3OD) : δ 8.37 (dd, J = 7.8 Hz, 1.2 Hz, 1H), 7.89 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.84 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.48 (d, J = 2.2 Hz, 1H), 7.46 (dd, J = 7.5 Hz, 1.0 Hz 1H), 7.40 (d, J = 2.3 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.31-7.24 (m,3H), 7.22 (dd, J = 9.0 Hz, 2.2 Hz 1H), 7.13 (d, J = 2.3 Hz, 1H), 3.79 (q, J = 7.2 Hz, 4H), 3.79 (s, 3H), 1.35 (t, J = 7.2 Hz, 6H); 13 C NMR (125 MHz, CD3OD) : δ 168.1, 160.3, 158.9, 157.1, 149.6, 141.3, 135.8, 134.2, 133.5, 132.4, 132.3, 131.9, 131.8, 131.1, 122.2, 118.5, 118.3, 117.7, 117.5, 117.4, 104.6, 97.8, 47.7, 34.0, 12.9; HRMS (ESI + ): Calcd. for [M] + , 467.2083; Found, 467.2106 (+2.3 mmu).

[0070]

[0071] Synthesis Example 2: Synthesis of N-Imidazole Rho2 Synthesis of Compound 2 4-bromo-1H-imidazole (147 mg, 1.00 mmol, 1 eq.) and tBuBrettPhos Pd G3 (26.8 mg, 0.03 mmol, 0.03 eq.) were mixed with lithium bis(trimethylsilyl)amide (1.0 M THF solution) (2.2 mL, 2.2 mmol, 2.2 eq.), and m-anisidine (0.13 mL, 1.2 mmol, 1.2 eq.) was added under an argon atmosphere. The mixture was stirred at 50°C for 6 hours. The solution was cooled to room temperature and evaporated to dryness. The resulting residue was purified by column chromatography (aminosilica, AcOEt / MeOH) to obtain Compound 2 (108 mg, yield 57%). 1 H NMR (500 MHz, CD2Cl2): δ 7.42 (d, J = 1.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.77 (d, J = 1.0 Hz, 1H), 6.50-6.46 (m, 2H), 6.61 (ddd, J = 8.0 Hz, 2.4 Hz, 0.7 Hz, 1H), 3.75 (s, 3H); 13 C NMR (125 MHz, CD2Cl2): δ 161.2, 146.4, 141.3, 132.5, 130.3, 107.6, 104.5, 102.4, 100.7, 55.5; HRMS (ESI + ): Calcd. for [M+H] + , 190.0980; Found, 190.0975 (-0.5 mmu).

[0072]

[0073] Synthesis of N-Imidazole Rho2: 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (20.8 mg, 0.0664 mmol, 1 eq.) and compound 2 (12.6 mg, 0.0664 mmol, 1 eq.) were mixed with TFA (2 mL). The mixture was stirred at 150°C for 4 hours. The solution was cooled to room temperature and evaporated to dryness. The resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 80 / 20 to 50 / 50 (50 min); flow rate = 5.0 mL / min) to obtain N-Imidazole Rho2 (15.0 mg, yield 40%). 1 H NMR (500 MHz, CD3OD): δ 8.49 (d, J = 1.3 Hz, 1H), 8.36 (dd, J = 7.8, 1.3 Hz, 1H), 7.87 (td, J = 7.5 Hz, 1.4 Hz, 1H), 7.82 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.45 (d, J = 1.4 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1.4 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.22 (s, 1H), 7.20 (d, J = 9.1 Hz, 1H), 7.16 (dd, J = 9.6 Hz, 2.5 Hz, 1H), 7.06-7.04 (m, 2H), 3.73 (q, J = 7.2 Hz, 4H), 1.33 (t, J = 7.2 Hz, 6H); 13 C NMR (125 MHz, CD3OD): δ 168.0, 162.2, 160.1, 158.7, 158.3, 155.6, 135.2, 134.3, 134.3, 134.0, 133.1, 132.6, 132.3, 132.2, HRMS (ESI + ): Calcd. for [M] +, 453.1927; Found, 453.1950 (+2.3 mmu).

[0074]

[0075] Synthesis Example 3: Synthesis of N-Thiazole Rho Synthesis of Compound 3 p-toluenesulfonic acid monohydrate (190 mg, 1.00 mmol, 0.5 eq.) was mixed with i-PrOH (2 mL), and 2-bromothiazole (0.18 mL, 2.00 mmol, 1 eq.) and m-anisidine (0.448 mL, 4.00 mmol, 2 eq.) were added. The mixture was stirred at 80°C for 72 hours. The solution was cooled to room temperature, and the reaction was stopped with saturated sodium bicarbonate aqueous solution. The solvent was evaporated to dryness, and the residue was extracted by DCM. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. The resulting residue was purified by column chromatography (aminosilica, AcOEt / MeOH) to obtain Compound 3 (199 mg, yield 48%). 1 H NMR (500 MHz, CDCl3): δ 9.90 (s, 1H), 7.33 (d, J = 3.7 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H), 6.95 (t, J = 2.2 Hz, 1H), 6.92 (ddd, J = 8.0 Hz, 2.2 Hz, 0.7 Hz, 1H), 6.62 (d, J = 3.7 Hz, 1H), 6.61 (ddd, J = 8.1 Hz, 2.2 Hz, 0.7 Hz, 1H), 3.81 (s, 3H); 13 C NMR (125 MHz, CDCl3): δ 166.4, 160.7, 142.2, 138.2, 130.4, 110.5, 108.3, 107.2, 104.0, 55.4; + ): Calcd. for [M+H] + , 207.0592; Found, 207.0610 (+1.8 mmu).

[0076]

[0077] Synthesis of N-Thiazole Rho: 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (20.1 mg, 0.0641 mmol, 1 eq.) and compound 3 (12.1 mg, 0.0641 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The mixture was cooled to room temperature and neutralized with NaOH. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Thiazole Rho (16.1 mg, yield 36%). 1 H NMR (500 MHz, CD3OD): δ 8.56 (d, J = 2.1 Hz, 1H), 8.37 (dd, J = 7.8 Hz, 1.2 Hz, 1H), 7.88 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.83 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.47-7.45 (m, 2H), 7.32 (dd, J = 9.0 Hz, 2.1 Hz, 1H), 7.25 (d, J = 9.7 Hz, 1H) 7.21 (d, J = 9.0 Hz, 1H), 7.17 (dd, J = 9.7 Hz, 2.4Hz, 1H), 7.09 (d, J = 3.6 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 3.74 (q, J = 7.2 Hz, 4H), 1.33 (t, J = 7.2 Hz, 6H); 13 C NMR (125 MHz, CD3OD): δ 168.1, 163.4, 161.7, 160.3, 158.5, 158.3, 150.9, 140.6, 135.2, 134.0, 133.1, 132.6, 132.3, 131.7, HRMS (ESI + ): Calcd. for [M]+ , 470.1538; Found, 470.1557 (+1.9 mmu).

[0078]

[0079] Synthesis Example 4: Synthesis of N-Imidazole Rho3 228 mg, 1.20 mmol, 1.2 eq. p-toluenesulfonic acid monohydrate was mixed with anhydrous toluene (2 mL) and, under an argon atmosphere, 2-bromo-1H-imidazole (221 mg, 1.50 mmol, 1.5 eq.) and m-anisidine (0.11 mL, 1.0 mmol, 1 eq.) were added. The mixture was stirred at 120°C for 144 hours. The solution was cooled to room temperature and the reaction was stopped with saturated sodium bicarbonate aqueous solution. The solvent was evaporated to dryness and the residue was extracted by DCM. The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated to dryness. The resulting residue was purified by column chromatography (aminosilica, DCM / MeOH) to obtain compound 4 (150 mg, yield 79%). 1 H NMR (500 MHz, CD3OD): δ 7.09 (t, J = 8.1 Hz, 1H), 7.03 (s, 1H), 6.76-6.74 (m, 3H), 6.65 (ddd, J = 8.1 Hz, 2,1 Hz, 0.8 Hz, 1H), 6.42 (ddd, J = 8.1 Hz, 2,4 Hz, 0.8 Hz, 1H), 3.74 (s, 3H); 13 C NMR (125 MHz, CD3OD): δ 162.1, 146.3, 145.6, 130.8, 119.5, 117.3, 109.5, 106.7, 102.7, 55.5; + ): Calcd. for [M+H] + , 190.0980; Found, 190.0979 (-0.1 mmu).

[0080]

[0081] 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (31.3 mg, 0.100 mmol, 1 eq.) and compound 4 (18.9 mg, 0.100 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The solution was cooled to room temperature and neutralized with NaOH. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 80 / 20 to 50 / 50 (50 min); flow rate = 5.0 mL / min) to obtain N-Imidazole3 Rho (15.3 mg, yield 27%). 1 H NMR (500 MHz, CD3OD): δ 8.34 (dd, J = 7.8 Hz, 1.1 Hz, 1H), 7.88 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.83 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.43 (dd, J = 7.6 Hz, 1.1 Hz, 1H), 7.27-7.17 (m, 6H), 7.07 (dd, J = 2.4 Hz, 1H), 3.75 (q, J = 7.2 Hz, 4H), 1.33 (t, J = 7.2 Hz, 6H); 13 C NMR (125 MHz, CD3OD): δ 168.4, 159.8, 158.3, 157.0, 148.5, 142.2, 137.4, 134.4, 133.1, 132.1, 131.9, 131.8, 131.7, 130.6, 118.3, 117.7, 117.5, 117.4, 116.6, 104.6, 97.8, 47.5, 12.9; + ): Calcd. for [M] + , 453.1927; Found, 453.1951 (+2.4 mmu).

[0082]

[0083] Synthesis Example 5: Synthesis of N-Oxazole Rho 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (313 mg, 1.00 mmol, 1 eq.) and 3-iodophenol (308 mg, 1.40 mmol, 1.4 eq.) were mixed with methanesulfonic acid (10 mL). The mixture was stirred at 150°C for 2 hours. The solution was cooled to room temperature and neutralized with NaOH. The residue was extracted with DCM. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated to dryness, and the resulting residue was purified by column chromatography (silica, DCM / AcoEt) to obtain compound 5 (440 mg, yield 88%). 1 H NMR (500 MHz, CDCl3) : δ; 8.01 (dt, J = 7.5 Hz, 1.0 Hz, 1H), 7.67-7.64 (m, 2H), 7.61 (td, J = 7.5 Hz, 1.0 Hz, 1H), 7.30 (dd, J = 8.3 Hz, 1.7 Hz, 1H), 7.17 (dt, J = 7.6 Hz, 0.9 Hz, 1H), 6.57 (d, J = 8.9 Hz, 1H), 6.48 (d, J = 8.3 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.37 (dd, J = 8.9 Hz, 2.6Hz, 1H), 3.36 (q, J = 7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H); 13 C NMR (125 MHz, CDCl3): δ 169.5, 153.0, 152.5, 152.1, 149.8, 135.1, 132.4, 129.8, 129.5, 128.9, 127.0, 126.2, 125.1, 124.1, 119.5, 108.8, 104.7, 97.7, 95.1, 83.5, 44.6, 12.6; + ): Calcd. for [M] + , 498.0566; Found, 498.0540 (-2.6 mmu).

[0084]

[0085] Compound 5 (49.7 mg, 0.100 mmol, 1 eq.), 1,3-oxazole-2-amine (18.6 mg, 0.200 mmol, 2 eq.), cesium carbonate (163 mg, 0.500 mmol, 5 eq.), tris(dibenzylideneacetone)dipalladium(0) (18.3 mg, 0.0200 mmol, 0.2 eq.), and xanthophos (8.6 mg, 0.015 mmol, 0.15 eq.) were mixed with anhydrous toluene (5 mL) under an argon atmosphere. The mixture was stirred at 100°C for 2 hours. The solution was cooled to room temperature, the solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Oxazole Rho (13.5 mg, yield 24%). 1 H NMR (500 MHz, CD3OD): δ 8.40 (d, J = 2.1 Hz, 1H), 8.38 (dd, J = 7.8 Hz, 1.3 Hz, 1H), 7.89 (td, J = 7.5 Hz, 1.4 Hz, 1H), 7.84 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.61 (d, J = 1.0 Hz, 1H), 7.46 (dd, J = 7.5 Hz, 1.3 Hz, 1H), 7.37 (dd, J = 9.1 Hz, 2.1 Hz, 1H), 7.27 (d, J = 9.7 Hz, 1H), 7.24 (d, J = 9.1 Hz, 1H), 7.21 (dd, J = 9.7 Hz, 2.4 Hz, 1H), 7.12-7.08 (m, 2H), 3.76 (q, J = 7.2 Hz, 4H), 1.34 (t, J = 7.2 Hz, 6H); 13C NMR (125 MHz, CD3OD): δ 168.0, 162.5, 160.5, 158.8, 158.0, 156.7, 149.6, 135.4, 135.0, 134.0, 133.3, 132.7, 132.2, 131.7, HRMS (ESI + ): Calcd. for [M] + , 454.1767; Found, 454.1784 (+1.7 mmu).

[0086]

[0087] Synthesis Example 6: Synthesis of N-Triazole Rho p-toluenesulfonic acid monohydrate (228 mg, 1.20 mmol, 1.2 eq.) was mixed with anhydrous toluene (2 mL), and under an argon atmosphere, 3-chloro-1,2,4-tetrazole (155 mg, 1.50 mmol, 1.5 eq.) and m-anisidine (0.112 mL, 1.00 mmol, 1 eq.) were added. The mixture was stirred at 120°C for 48 hours. The solution was cooled to room temperature, and the reaction was stopped with saturated sodium bicarbonate aqueous solution. The solvent was evaporated to dryness, and the residue was extracted by DCM. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated to dryness, and the resulting residue was purified by column chromatography (aminosilica, DCM / MeOH) to obtain compound 6 (164 mg, yield 86%). 1 H NMR (500 MHz, CD3OD) : δ 8.00 (s, 1H), 7.15-7.11 (m, 2H), 6.94 (dd, J = 8.2 Hz, 1.6 Hz, 1H), 6.47 (dd, J = 8.2 Hz, 2.4 Hz, 1H), 3.76 (s, 3H); 13 C NMR (125 MHz, CD3OD): δ161.9, 159.3, 145.4, 143.8, 130.7, 110.4,107.3,103.8, 55.5; HRMS (ESI + ): Calcd. for [M+H]+ , 191.0933; Found, 191.0959 (+2.6 mmu).

[0088]

[0089] 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (31.3 mg, 0.100 mmol, 1 eq.) and compound 6 (19.0 mg, 0.100 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The mixture was cooled to room temperature and neutralized with NaOH. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Triazole Rho (8.1 mg, yield 14%). 1 H NMR (500 MHz, CD3OD): δ 8.37 (dd, J = 7.8 Hz, 1.2 Hz, 1H), 8.33 (s, 1H), 8.28 (d, J = 2.0 Hz, 1H), 7.88 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.82 (td, J = 7.6 Hz, 1.4 Hz, 1H), 7.46 (dd, J = 7.5 Hz, 1.2 Hz, 1H), 7.35 (dd, J = 9.2 Hz, 2.0 Hz, 1H), 7.23 (d, J = 9.6 Hz, 1H), 7.18 (d, J = 9.2 Hz, 1H), 7.15 (dd, J = 9.6 Hz, 2.5 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 3.72 (q, J = 7.3 Hz, 4H), 1.33 (t, J = 7.3 Hz, 6H); 13C NMR (125 MHz, CD3OD): δ 168.1, 162.5, 160.2, 159.5, 158.7, 158.3, 152.5, 144.1, 135.1, 134.0, 133.0, 132.6, 132.2, 131.6, 131.5, 131.4, 118.2, 117.2, 116.6, 116.6, 102.0,97.3, 47.3, 12.9 + ): Calcd. for [M] + , 454.1879; Found, 454.1906 (+2.7 mmu).

[0090]

[0091] Synthesis Example 7: Synthesis of N-Thiadiazole Rho 1,3,4-thiadiazole-2-amine (55.6 mg, 0.550 mmol, 1 eq.), potassium carbonate (106 mg, 0.77 mmol, 1.4 eq.), and tBuBrettPhos Pd G3 (9.4 mg, 0.011 mmol, 0.02 eq.) were mixed with anhydrous 2-butanol (8 mL). Under an argon atmosphere, in the presence of molecular sieves 4 Å (300 mg), 3-bromoanisole (0.07 mL, 0.55 mmol, 1 eq.) and acetic acid (0.001 mL, 0.0165 mmol, 0.03 eq.) were added. The mixture was stirred at 115°C for 3 hours. The solution was cooled to room temperature, filtered, and evaporated to dryness. The resulting residue was purified by column chromatography (aminosilica, DCM / AcOEt) to obtain compound 7 (108 mg, yield 95%). 1 H NMR (500 MHz, CD3OD): δ 11.13 (s, 1H), 8.51 (s, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.10 (t, J = 2.3 Hz, 1H), 7.03 (ddd, J = 8.1 Hz, 2.3 Hz, 0.5 Hz, 1H), 6.65 (ddd, J = 8.1 Hz, 2.3 Hz, 0.5 Hz, 1H), 3.85 (s, 3H); 13C NMR (125 MHz, CD3OD): δ166.8, 160.9, 141.9, 141.2, 130.6, 110.0,109.3,104.1, 55.5; HRMS (ESI + ): Calcd. for [M+H] + , 208.0545; Found, 208.0552 (+0.7 mmu).

[0092]

[0093] 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (31.3 mg, 0.100 mmol, 1 eq.) and compound 7 (20.7 mg, 0.100 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The mixture was cooled to room temperature and neutralized with NaOH. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Thiadiazole Rho (14.1 mg, yield 24%). 1 H NMR (500 MHz, CD3OD): δ 9.04 (s, 1H), 8.63 (d, J = 2.2 Hz, 1H), 8.38 (dd, J = 7.8 Hz, 1.3 Hz, 1H), 7.89 (td, J = 7.5 Hz, 1.4 Hz, 1H), 7.84 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.47 (dd, J = 7.5 Hz, 1.3 Hz, 1H), 7.39 (dd, J = 9.0 Hz, 2.2 Hz, 1H), 7.29 (d, J = 9.6 Hz, 1H), 7.28 (d, J = 9.0 Hz, 1H), 7.24 (dd, J = 9.6 Hz, 2.4 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 3.78 (q, J = 7.3 Hz, 4H), 1.36 (t, J = 7.3 Hz, 6H); 13C NMR (125 MHz, CD3OD): δ 168.0, 164.8, 160.6, 159.0, 157.8, 149.9, 148.3, 135.2, 134.1, 133.4, 132.6, 132.2, 131.8, 131.4, 118.4, 118.2, 117.8, 117.6, 104.7, 97.5, 47.6, 12.9; + ): Calcd. for [M] + , 471.1491; Found, 471.1513 (+2.2 mmu).

[0094]

[0095] Synthesis Example 8: Synthesis of N-Oxadiazole Rho Compound 5 (49.7 mg, 0.100 mmol, 1 eq.), 1,3,4-Oxadiazole-2-amine (18.6 mg, 0.200 mmol, 2 eq.), Cesium carbonate (97.7 mg, 0.300 mmol, 3 eq.), Tris(dibenzylideneacetone)dipalladium(0) (18.3 mg, 0.0200 mmol, 0.2 eq.), and Xanthophos (8.6 mg, 0.015 mmol, 0.15 eq.) were mixed with anhydrous toluene (5 mL) under an argon atmosphere. The mixture was stirred at 80°C for 4 hours. The solution was cooled to room temperature, the solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Oxadiazole Rho (9.3 mg, yield 16%).

[0096]

[0097] Synthesis Example 9: Synthesis of N-Tetrazole Rho. Compound 5 (199 mg, 0.400 mmol, 1 eq.), 1H-tetrazole-5-amine (68.1 mg, 0.800 mmol, 2 eq.), cesium carbonate (391 mg, 1.20 mmol, 3 eq.), tris(dibenzylideneacetone)dipalladium(0) (73.3 mg, 0.0800 mmol, 0.2 eq.), and xanthophos (34.7 mg, 0.060 mmol, 0.15 eq.) were mixed with anhydrous toluene (20 mL) under an argon atmosphere. The mixture was stirred at 80°C for 18 hours. The solution was cooled to room temperature, the solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA; gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain N-Oxadiazole Rho (5.7 mg, yield 2.5%).

[0098]

[0099] Synthesis Example 10: Synthesis of Imidazole-THQR 1-Methyl-1,2,3,4-tetrahydroquinoline-7-ol (163 mg, 1.00 mmol, 1 eq.) and phthalic anhydride (148 mg, 1.00 mmol, 1 eq.) were mixed with anhydrous toluene (5 mL) under an argon atmosphere. The mixture was stirred at 120°C for 24 hours. The solution was cooled to room temperature, filtered, and dried to obtain compound 8 (235 mg, yield 75%). 1H NMR (500 MHz, DMSO-d6): δ 13.05 (s, 1H), 12.65 (s, 1H), 7.95 (dd, J = 7.7, 1.0 Hz, 1H), 7.67 (td, J = 7.5, 1.3 Hz, 1H), 7.59 (td, J = 7.7, 1.3 Hz, 1H), 7.36 (dd, J = 7.5, 1.0 Hz, 1H), 6.51 (s, 1H), 6.00 (s, 1H), 3.30 (t, J = 5.8 Hz, 2H), 2.95 (s, 3H), 2.41 (t, J = 6.2 Hz, 2H), 1.78-1.73 (m, 2H); 13 C NMR (125 MHz, DMSO-d6): δ 198.1, 167.0, 163.9, 152.5, 140.1, 131.9, 131.2, 129.9, 129.8, 129.3, 127.7, 114.0, 108.9, 95.7, 50.3, 38.6, 26.4, 21.3; HRMS (ESI － ): Calcd. for [M-2H+Na] － , 332.0899; Found, 332.0888 (-1.1 mmu).

[0100]

[0101] Compound 8 (31.1 mg, 0.100 mmol, 1 eq.) and Compound 2 (18.9 mg, 0.100 mmol, 1 eq.) were mixed with TFA (2 mL). The mixture was stirred at 150°C for 4 hours. The solution was cooled to room temperature and evaporated to dryness. The resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 80 / 20 to 50 / 50 (50 min); flow rate = 5.0 mL / min) to obtain Imidazole-THQR (14.6 mg, yield 26%). 1H NMR (500 MHz, CD3OD) : δ 8.55 (s, 1H), 8.36 (dd, J = 7.8 Hz, 1.2 Hz, 1H), 7.86 (td, J = 7.5 Hz, 1.4 Hz, 1H), 7.81 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.46 (s, 1H), 7.41 (dd, J = 7.5 Hz, 1.2 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 9.1 Hz, 1H), 7.02 (dd, J = 9.1 Hz, 2.2 Hz, 1H), 6.97 (s, 1H), 6.86 (s, 1H), 3.67 (t, J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.73 (t, J = 5.8 Hz, 2H), 2.00-1.95 (m, 2H); 13 C NMR (125 MHz, CD3OD) : δ 168.0, 160.8, 159.7, 158.0, 157.9, 154.8, 135.2, 134.2, 134.0, 133.4, 132.6, 132.2, 131.9, 131.6, 131.4, 129.1, 128.9, 116.8, 116.6, 116.4, 111.7, 99.9, 96.4, 53.2, 40.7, 28.1, 21.7; HRMS (ESI + ): Calcd. for [M] + , 451.1770; Found, 451.1773 (+0.3 mmu).

[0102]

[0103] Synthesis Example 11: Synthesis of diEt Rho 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (57.3 mg, 0.183 mmol, 1 eq.) and m-anisidine (0.020 mL, 0.18 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The solution was cooled to room temperature and neutralized with sodium hydroxide. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain diEt Rho (41.2 mg, yield 45%). 1 H NMR (500 MHz, CD3OD): δ 8.33 (dd, J = 7.8 Hz, 1.1 Hz, 1H), 7.84 (td, J = 7.6 Hz, 1.3 Hz, 1H), 7.79 (td, J = 7.6 Hz, 1.3 Hz, 1H), 7.40 (dd, J = 7.6 Hz, 1.1 Hz, 1H), 7.13 (d, J = 9.5 Hz, 1H), 7.05-7.01 (m, 2H), 6.95 (d, J = 2.4 Hz, 1H), 6.81-6.78 (m, 2H), 3.66 (q, J = 7.2 Hz, 4H), 1.29 (t, J = 7.2 Hz, 6H); 13 C NMR (125 MHz, CD3OD): δ 168.1, 161.6, 161.2, 159.7, 159.3, 157.1, 135.3, 133.8, 132.8, 132.5, 132.5, 132.2, 131.4, 131.4, 117.7, 115.4, 115.1, 114.7, 98.4, 97.1, 46.8, 12.8; + ): Calcd. for [M] + , 387.1709; Found, 387.1698 (-1.1 mmu).

[0104]

[0105] Compound 8 (31.1 mg, 0.100 mmol, 1 eq.) and m-anisidine (0.011 mL, 0.10 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150 °C for 2 hours. The solution was cooled to room temperature and neutralized with sodium hydroxide. The solvent was evaporated to dryness, and the resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain THQ Rho (19.8 mg, yield 40%). 1 H NMR (500 MHz, CD3OD): δ 8.33 (dd, J = 7.8 Hz, 1.2 Hz, 1H), 7.84 (td, J = 7.5 Hz, 1.4 Hz, 1H), 7.78 (td, J = 7.8 Hz, 1.4 Hz, 1H), 7.38 (dd, J = 7.5 Hz, 1.2 Hz, 1H), 7.00 (d, J = 9.1 Hz, 1H), 6.89 (s, 1H), 6.81 (d, J = 2.1 Hz, 1H), 6.79-6.76(m, 2H) 3.60 (t, J = 5.8 Hz, 2H), 3.27 (s, 3H), 2.70 (t, J = 6.0 Hz, 2H), 1.99-1.93 (m, 2H); 13 C NMR (125 MHz, CD3OD): δ 168.1, 160.6, 160.4, 159.2, 158.9, 156.6, 135.5, 133.8, 132.5, 132.3, 132.3, 131.5, 131.3, 128.6, 127.3, 117.4, 114.9, 98.3, 96.2, 52.8, 40.2, 28.2, 21.9; HRMS (ESI + ): Calcd. for [M] + , 385.1552; Found, 385.1542 (-1.0 mmu).

[0106]

[0107] Synthesis Example 12: Synthesis of Thiophene-THQR 2-bromothiophene (0.11 mL, 1.1 mmol, 1 eq.), potassium carbonate (199 mg, 1.44 mmol, 1.4 eq.), and tBuBrettPhos Pd G3 (18.8 mg, 0.022 mmol, 0.02 eq.) were mixed with anhydrous 2-butanol (16 mL). Under an argon atmosphere and in the presence of molecular sieves 4 Å (600 mg), m-anisidine (0.13 mL, 1.1 mmol, 1 eq.) and acetic acid (0.001 mL, 0.0165 mmol, 0.15 eq.) were added. The mixture was stirred at 115°C for 5 hours. The solution was cooled to room temperature, filtered, and evaporated to dryness. The resulting residue was purified by column chromatography (aminosilica, hexane / DCM) to obtain compound 8 (196 mg, yield 87%). 1 H NMR (500 MHz, CDCl3): δ 7.19 (t, J = 8.5 Hz, 1H), 6.99 (dd, J = 5.6 Hz, 1.3 Hz, 1H), 6.94 (dd, J = 5.6 Hz, 3.6 Hz, 1H), 6.78 (dd, J = 3.6 Hz, 1.3 Hz, 1H), 6.54-6.51 (m, 2H), 6.50-6.46 (m, 1H), 5.68 (s, 1H), 3.80 (s, 3H); 13 HRMS (ESI + ): Calcd. for [M+H] + , 206.0640; Found, 206.0660 (+2.0 mmu).

[0108]

[0109] Compound 8 (31.1 mg, 0.100 mmol, 1 eq.) and Compound 9 (20.6 mg, 0.100 mmol, 1 eq.) were mixed with methanesulfonic acid (1 mL). The mixture was stirred at 150°C for 2 hours. The solution was cooled to room temperature and evaporated to dryness. The resulting residue was purified by HPLC (mobile phase, A: H2O / 0.1% TFA, B: CH3CN / 0.1% TFA, gradient: A / B = 70 / 30 to 40 / 60 (50 min); flow rate = 5.0 mL / min) to obtain Thiophene-THQR (2.9 mg, yield 5%). 1 H NMR (500 MHz, CD3OD): δ 8.34 (d, J = 7.8 Hz, 1H), 7.84 (td, J = 7.4 Hz, 1.1 Hz, 1H), 7.80 (td, J = 7.8 Hz, 1.1 Hz, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.26 (dd, J = 5.6 Hz, 1.4 Hz, 1H), 7.14-7.10 (m, 2H), 7.04 (dd, J = 5.6 Hz, 3.7 Hz, 1H), 7.00 (dd, J = 9.1 Hz, 2.2 Hz, 1H), 6.97 (dd, J = 3.7 Hz, 1.4 Hz, 1H), 6.95 (s, 1H), 6.85 (s, 1H), 3.64 (t, J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.73 (t, J = 5.8 Hz, 2H), 2.00-1.95 (m, 2H); HRMS (ESI + ): Calcd. for [M] + , 467.1429; Found, 467.1424 (-0.5 mmu).

[0110]

[0111] Example 1 Optical properties of heterocyclic rhodamines The absorption and fluorescence spectra of diEt Rho and heterocyclic N-Imidazole Rho1, N-Imidazole Rho2, and N-Thiazole Rho were evaluated (Figure 2). All of N-Imidazole Rho1, N-Imidazole Rho2, and N-Thiazole Rho exhibited absorption in the wavelength range of 500 nm to 600 nm. On the other hand, the fluorescence spectra showed that N-Imidazole Rho1, N-Imidazole Rho2, and N-Thiazole Rho all had very low fluorescence intensity compared to diEt Rho. This indicates that rhodamine can be quenched even with the introduction of heterocyclic structures.

[0112] Example 2: Investigation of Viscosity Dependence of Fluorescence Spectra To confirm the quenching principle of rhodamine derivatives with heterocycles, the viscosity dependence of the fluorescence spectra was investigated. For quenching by the p-TICT mechanism, the aniline portion needs to be twisted relative to the xanthene ring. Therefore, previous studies have shown that under conditions where intramolecular rotation is inhibited, such as in high-viscosity environments, the p-TICT mechanism partially releases quenching and increases fluorescence intensity. Accordingly, the fluorescence spectra of N-Imidazole Rho1, N-Imidazole Rho2, and N-Thidazole Rho were measured in PBS, methanol, ethylene glycol, and glycerin to investigate their viscosity dependence (Figure 3). All probes showed increased fluorescence in the high-viscosity solvents of ethylene glycol and glycerin, suggesting that quenching was due to the p-TICT mechanism.

[0113] Example 3: Investigation of Reactivity with Reactive Oxygen Species The reactivity of N-Imidazole Rho1 and N-Imidazole Rho2 with reactive oxygen species was evaluated by fluorescence spectral measurement (Figure 4). Reactive oxygen species were added to the probe solution to the respective concentrations and reacted at 37°C for 30 minutes, after which the fluorescence spectrum was measured. For singlet oxygen, methylene blue, a photosensitizer, was added and irradiated with 660 nm light, which only methylene blue absorbs, to generate it. It was found that N-Imidazole Rho1 and N-Imidazole Rho2 showed increased fluorescence upon reaction with hypochlorous acid and singlet oxygen.

[0114] Example 4: Confirmation of the reactivity of N-Imidazole Rho1 with hypochlorous acid and singlet oxygen. Fluorescence spectra were measured before and after the reaction of N-Imidazole Rho1 with hypochlorous acid and singlet oxygen (Figure 5). The fluorescence intensity around 570 nm increased when hypochlorous acid and singlet oxygen were added to an aqueous solution of N-Imidazole Rho1.

[0115] Example 5: Confirmation of the reactivity of N-Imidazole Rho2 with hypochlorous acid and singlet oxygen. Fluorescence spectra were measured before and after the reaction of N-Imidazole Rho2 with hypochlorous acid and singlet oxygen (Figure 6). The fluorescence intensity around 570 nm increased when hypochlorous acid and singlet oxygen were added to an aqueous solution of N-Imidazole Rho2.

[0116] Example 6 Evaluation of Cell Membrane Permeability HeLa cells were seeded at a density of 300,000 cells / dish in a glass-bottom dish (35 mm) and cultured for 1 day at 37°C under 5% CO2. After culturing, the dish was washed twice with medium (1 mL), and medium (2 mL) and Probe (final concentration 1 μM) were added. After standing for 30 minutes at 37°C under 5% CO2, the cells were observed with a confocal fluorescence microscope (Ex. 514 nm, Em. 530-630 nm; HV: 550 V). Staining experiments were performed on HeLa cells to confirm the cell membrane permeability of N-Imidazole Rho1, N-Imidazole Rho2, and N-Thiazole Rho (Figure 7). From the fluorescence images, it was clear that all probes were permeable to the cell membrane.

[0117] Example 7 Comparison of Fluorescence Quantum Yields The fluorescence particle yield of the compounds from Synthesis Examples 1 to 9 in PBS solution was measured by absolute quantum yield using Quantaurus-QY (Hamamatsu Photonics K.K.). The results are shown in Figure 8. All rhodamine derivatives with heterocycles showed quenching.

[0118] Example 8 Analysis of the quenching mechanism The quenching observed in Example 7 was presumed to be caused by twisting of bonds within the compound and intramolecular charge transfer (ICT).

[0119]

[0120] Therefore, we investigated the change in fluorescence due to the difference in solvents using three types of solvents (methanol, ethylene glycol, and glycerin). We also investigated the change in fluorescence due to the difference in dielectric constant of two types of solvents with different dielectric constants (acetonitrile and dichloroethane). As shown in Figure 9, as the viscosity of the solvent increased, the twisting of the CN bond was suppressed and fluorescence was restored. This indicates that the twisting of bonds within the compound is involved in quenching. As shown in Figure 10, as the dielectric constant of the solvent decreased, charge transfer was suppressed and fluorescence was restored. This indicates that intramolecular charge transfer (ICT) within the compound is involved in quenching.

[0121] Example 9 Measurement of HOMO Energy The HOMO energy levels of each compound prepared in Synthesis Examples 1-9 were calculated at the CAM-B3LYP / 6-31+G(d,p) level using Gaussian 16. As shown in Figure 11, it was suggested that switching between donor and acceptor occurs in TICT due to the diethylamino group and TICT due to the heterocyclic structure, and that the direction of the TICT mechanism is determined by the HOMO energy. In TICT where the heterocyclic structure is the donor, a correlation was observed between the electron-donating ability of the donor and the fluorescence quantum yield. On the other hand, in TICT where the diethylamino group is the donor, a correlation was observed between the electron-accepting ability of the acceptor and the fluorescence quantum yield (data not shown). From the above, it was strongly supported that quenching is due to TICT.

[0122] Example 10 Confirmation of reactivity with singlet oxygen The fluorescence spectra of each probe, Imidazole-THQR and N-Imidazole Rho2, were measured before and after reaction with singlet oxygen (Figure 12). 1 O2: Using methylene blue (0.5 equivalents), at 660 nm (25 mW / cm²). 2 It was generated by light irradiation. Imidazole-THQR showed a greater increase in fluorescence upon reaction with singlet oxygen compared to aqueous solution of N-Imidazole Rho2.

[0123] Example 11 Evaluation of reactive oxygen species selectivity The fluorescence intensity (Ex. 500 nm, Em. 560 nm) of N-Imidazole Rho2 (1 μM) was measured after reacting it with various ROS in PBS at 37°C. - :HPX / Xanthine Oxidase;H2O2:H2O2;HO・:Fenton reaction;NO:NOC 7 (0.5 equivalent);TBHP:t-BuOOH;ONOO - :ONOONa;HOCl:NaOCl; 1 O2: Using methylene blue (0.5 μM), at 660 nm (25 mW / cm²). 2 It was generated by light irradiation. As shown in Figure 13, N-Imidazole Rho2 showed a significant increase in fluorescence compared to singlet oxygen.

[0124] Example 12: Intracellular singlet oxygen imaging. Fluorescence imaging (Ex. 514 nm, Em. 530–600 nm) when Imidazole-THQR (5 μM) and 5-ALA-inducing PpIX were added to HeLa cells. 1 O2:5-ALA (1 mM) was incubated for 4 hours, followed by irradiation with 640 nm light for 60 seconds. Images were acquired using a confocal laser scanning microscope (FV3000, Olympus). HeLa cells. 1 Box plot of fluorescence intensity before and after O2 reaction. Each group n = 30 cells. Image 14, HeLa cells. 1 Figure 15 shows a box plot of fluorescence intensity before and after the O2 reaction. Light irradiation of PpIX resulted in an increase in fluorescence.

[0125] Example 13 Quantitative Intracellular Imaging Quantitative determination of fluorescence intensity (Ex. 514 nm, Em. 530–600 nm) of HeLa cells treated with Imidazole-THQR (5 μM), 5-ALA-inducible PpIX, and NaN3 (1 mM). 1 O2:5-ALA (1 mM) was incubated for 4 hours, followed by irradiation with 640 nm light. Each group consisted of n = 30 cells. As shown in Figure 16, singlet oxygen was successfully detected intracellularly by adding Imidazole-THQR.

[0126] Example 14 Measurement of HOMO Energy The HOMO energy level of Thiophen-THQR prepared in Synthesis Example 12 was calculated at the CAM-B3LYP / 6-31+G(d,p) level using Gaussian 16. As shown in Figure 17, charge separation occurs in conjunction with the twisting of the xanthene-thiophene bond after excitation, suggesting that Thiophen-THQR takes the TICT state at the HOMO energy.

[0127] Example 15 Changes in Stability in the Excited State When the energy change of the S1 state with respect to the dihedral angle φ between the Xanthene ring and the N atom was determined by TD-DFT calculation, it was shown that the TICT state was more stable in thiophene (right) than in imizole-THQR (left) (Figure 18).

[0128] Example 16 Comparison of quenching of Thiophene-THQR and Imidazole-THQR The absorbance and fluorescence intensity of PBS solutions containing each compound (using a 0.1% DMF solution containing 1 μm of each compound, Thiophene and Imidazole-THQR) were measured at an excitation wavelength of 500 nm. Thiophene-THQR showed stronger quenching than Imidazole-THQR (Figure 19).

[0129]

Claims

1. A compound represented by the following general formula (I) or (II) or a salt thereof. (In formula (I), R １ represents a hydrogen atom or one to three identical or different monovalent substituents present on the benzene ring, and R ２ and R ３ each independently represent a hydrogen atom or a monovalent substituent present on the benzene ring, and R ４ and R ５ each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a carboxyl group, an ester group, an amide group or a halogen atom, and R ６ and R ７ [ each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a carboxyl group, an ester group, an amide group or a halogen atom, and R １０ is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, and R １１ is a monovalent heterocyclic group which may have a substituent and contains at least one hetero atom, and R １０ and R １１ together may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R １０ and R １１ are attached, X is an oxygen atom, Si(R ａ )(R ｂ ), C(R ａ )(R ｂ ), Ge(R ａ )(R ｂ ), P(=O)R ｃ ), SO ２ ), Se, Te or Te(=O), where R ａ and R ｂ each independently represent an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, and R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Y is -NR ８ R ９ or -OH, where R ８ and R ９ Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms, and (i) Y is -NR ８ R ９ In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1 to 6 carbon atoms, (ii) When Y is -OH, R １０ is a group other than a hydrogen atom, and R ５ and R ７ (One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.) (In formula (II), R １ R represents a hydrogen atom or one to three identical or different monovalent substituents present on a benzene ring. ２ and R ３ Each of these independently represents a monovalent substituent located on a hydrogen atom or a benzene ring, and R ４ and R ５ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R ６ and R ７ Each of these independently represents a hydrogen atom, a substituted or unsubstituted C1-C6 alkyl group, a carboxyl group, an ester group, an amide group, or a halogen atom, R １０ However, R is a hydrogen atom, or a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms. １１ However, it is a monovalent heterocyclic group which may have substituents and contains at least one heteroatom, R １０ and R １１ Together, R １０ and R １１ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, where X is an oxygen atom, Si (R ａ ) (Caution ｂ ), C (R ａ ) (Caution ｂ ), Ge(R ａ ) (Caution ｂ ), P(=O)R ｃ SO ２ , selected from Se, Te or Te (=O), where R ａ and R ｂ Each of these is independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, R ｃ is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group, and Z is And (i) Z is In the case of R ８ and R ９ At least one of the groups constituting the set is a group other than a hydrogen atom, R ８ or R ９ R ４ or R ６ Together with R ８ or R ９ R may form a 5-7 membered heterocycline containing a nitrogen atom to which is bonded, and furthermore, the heterocycline may be substituted with an alkyl group having 1-6 carbon atoms, an alkenyl group having 2-6 carbon atoms, or an alkynyl group having 2-6 carbon atoms, an aralkyl group having 6-10 carbon atoms, or an alkyl-substituted alkenyl group having 6-10 carbon atoms. ８ and R ９ Together, R ８ and R ９ (ii) Z is In the case of R １０ is a group other than a hydrogen atom, and R ５ and R ７ (One or more of these are substituted or unsubstituted alkyl or halogen atoms having 1 to 6 carbon atoms.) 2. A compound represented by the general formula (I) or a salt thereof, wherein R ８ and R ４ may together form a 5- to 7-membered heterocyclyl containing the nitrogen atom to which R ８ or R ９ is attached; R ８ and R ９ may together form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R ８ and R ９ are attached; R ９ and R ６ may together form a 5- to 7-membered heterocyclyl containing the nitrogen atom to which R ８ or R ９ is attached, the compound or a salt thereof according to claim 1.

3. In formula (I) or (II), R １１ is a monocyclic heteroaryl group containing at least one heteroatom selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, the compound according to claim 1, or a salt thereof.

4. In equation (I) or (II), R １１ The compound or salt thereof according to claim 1, wherein the compound is a heterocyclic group of a five-membered ring or a six-membered ring containing a nitrogen atom.

5. In equation (I) or (II), R １１ The compound or salt thereof according to claim 1, wherein the compound is a monocyclic heteroaryl group containing a sulfur atom.

6. A compound represented by formula (I) or a salt thereof, wherein Y is -NR ８ R ９ And R ８ and R ９ The compound or salt thereof according to claim 1, wherein is the same or different substituted or unsubstituted alkyl group having 1 to 14 carbon atoms.

7. A fluorescent probe comprising the compound or a salt thereof as described in claim 1.

8. A method for detecting reactive oxygen species, comprising the steps of reacting reactive oxygen species with a compound or salt thereof according to any one of claims 1 to 6 or a fluorescent probe according to claim 7, and measuring the fluorescence emitted by the reaction.

9. A cell detection agent containing the compound or salt thereof described in claim 1, or the fluorescent probe described in claim 7.

10. A method for bioimaging cells using the compound or salt thereof described in claim 1, the fluorescent probe described in claim 7, or the cell detection agent described in claim 9.

11. A method for detecting cells is provided, comprising contacting a cell with the compound or salt thereof described in claim 1, the fluorescent probe described in claim 7, or the cell detection agent described in claim 9, and measuring the fluorescence emitted from the reaction product of the compound or salt thereof described in claim 1, the fluorescent probe described in claim 7, or the cell detection agent described in claim 9.

12. A method for reacting a compound or salt thereof described in claim 1 or a fluorescent probe described in claim 7 with a target molecule, wherein the reaction releases the quenching of the compound or salt thereof described in claim 1 or the fluorescent probe described in claim 7, restores its fluorescence, or increases its fluorescence intensity.

13. A method for detecting a target molecule, comprising reacting a compound or salt thereof according to claim 1 or a fluorescent probe according to claim 7 with a target molecule, wherein the reaction releases the quenching of the compound or salt thereof according to claim 1 or the fluorescent probe according to claim 7, restores its fluorescence, or increases its fluorescence intensity, and measures the fluorescence emitted as a result of the release of the quenching.

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