JPWO2007013201A1 - Zinc fluorescent probe - Google Patents

Abstract

The present invention relates to a zinc fluorescent probe excellent in water solubility and capable of observing a change in intracellular zinc ion concentration with high sensitivity in a ratiometric manner using a single excitation wavelength, a method for producing the same, and the zinc fluorescent probe Provided is a method for measuring zinc ions using As a zinc fluorescent probe, general formula (I): [Chemical Formula 1] (In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, and n is 2 or 3, R1 and R2 are the same or different and may have a substituent, an alkyl group which may have a substituent, an aryl group which may have a substituent or a heteroaryl group which may have a substituent, Z is a single bond or a formula : A group represented by [Chemical Formula 2], A represents a benzene ring which may have a substituent, Y represents a group represented by —O— or —S—, or a compound thereof Provide salt.

Description

  The present invention relates to a novel zinc fluorescent probe for measuring zinc ions in living cells and living tissues with high sensitivity under physiological conditions.

Zinc is an essential trace metal element present in the human body, second only to iron. However, free zinc ions (Zn ²⁺ ) in cells are usually below the nmol / l level, and most of the intracellular Zn ²⁺ is thought to be mainly tightly bound to proteins. It is done. Its function is important and includes the maintenance of protein structure and the control of enzyme activity.

In addition, DNA binding proteins such as transcription factors have Zn ²⁺ binding motifs such as zinc finger and LIM motif, and are said to bind to DNA via them. Zn ²⁺ deficiency affects DNA transcription and is suggested to be involved in carcinogenesis.

In recent years, focusing on the relationship with cell death, there have been many studies that relate the localization and dynamics of label Zn ²⁺ . For example, various studies have been conducted, such as selective cell death by exogenous Zn ^2+, regulation of enzyme activity closely related to cell death, influence of oxidative stress on enzyme activity center, etc. Knowledge about the workings of people is being accumulated. However, there are many unexplained parts about their dynamics and localization.

  In the research as described above, in order to measure the zinc ion in the tissue, a compound that specifically captures the zinc ion to form a complex and emits fluorescence along with the complex formation, that is, “ "Zinc fluorescent probe".

Various zinc probes have been proposed so far. For example, Patent Document 1 proposes a compound having a polyamine as a substituent. Although this compound can measure zinc in vivo with high sensitivity, the fluorescence intensity detected is affected by the difference in the thickness of the introduced cells, etc., and the Zn ²⁺ ion concentration is accurately and quantitatively analyzed from the fluorescence intensity. There was a problem that it was difficult to do.

  In order to solve such problems, Patent Document 2 has proposed a ratiometric fluorescent probe. This is an analysis method that utilizes a shift in excitation wavelength when the probe binds to zinc ions. In this method, since the ratio of the two excitation wavelengths is taken, an accurate quantitative analysis can be performed. However, since light having two excitation wavelengths is used, a large-scale and complicated apparatus must be used for the light source and the detection system (fluorescence microscope, flow cytometry, etc.), and there is a problem that versatility is poor.

In view of such problems, the present inventors have developed a zinc probe that can observe a change in fluorescence wavelength due to the concentration of zinc ions using a single excitation wavelength (Non-patent Document 1). However, this compound has a problem that it is not sufficiently water-soluble and sensitive to zinc ions. Furthermore, since the excitation wavelength is in the ultraviolet region, there is a problem that it is not preferable for cells.
JP 2000-239272 A International Publication No. 02/102795 Pamphlet J. Am. Chem. Soc. Communications, 2004,126, p712-713

  The present invention relates to a zinc fluorescent probe that is excellent in water solubility and capable of observing a change in intracellular zinc ion concentration with high sensitivity using a single wavelength of excitation light, a method for producing the same, and the zinc fluorescence An object of the present invention is to provide a method for measuring zinc ions using a probe.

  As a result of intensive studies in view of the above problems, the present invention has found that the above problems can be improved or solved when a compound represented by the general formula (I) described later is used as a zinc probe. As a result of further research based on this knowledge, the present invention has been completed.

  That is, the present invention provides the following compounds, a zinc fluorescent probe, a method for producing the same, a method for measuring zinc ions using the zinc fluorescent probe, and the like.

  Item 1. Formula (I):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, Z is a single bond or a formula:

A represents a benzene ring which may have a substituent, and Y represents a group represented by —O— or —S—. )
Or a salt thereof.

  Item 2. In the general formula (I), Ar is an alkyl group, alkoxy group, hydroxyl group, amino group, mono- or dialkylamino group, mono- or di (hydroxyalkyl) amino group, carboxyl group, alkoxycarbonyl group, carboxyalkyl group, alkoxycarbonylalkyl. Item 2. The compound or a salt thereof according to Item 1, which is an aryl group substituted with at least one group selected from the group consisting of a group, a carboxyalkoxy group and an alkoxycarbonylalkoxy group.

  Item 3. In the general formula (I), Ar is one group selected from the group consisting of a phenyl group, a toluyl group, a naphthyl group, a carboxyalkoxy group-substituted phenyl group and a 5- (dimethylamino) -1-naphthyl group. Or a salt thereof.

  Item 4. Item 4. The compound or a salt thereof according to any one of Items 1 to 3, wherein in General Formula (I), X and Y are groups represented by -O-, n is 2, and A is a benzene ring.

Item 5. In the general formula (I), R ¹ and R ² are the same or different, and at least one selected from the group consisting of a nitrogen-containing heteroaryl group, a carboxyl group, a hydroxyl group, an amino group, a mono- or dialkylamino group and an alkoxy group Item 5. The compound or a salt thereof according to any one of Items 1 to 4, which is an alkyl group substituted with a group.

  Item 6. General formula (II):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. The benzene ring, R ¹ and R ² may be the same or different and each may have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a heteroaryl group which may have a substituent; Show.)
Or a salt thereof.

  Item 7. Formula (III):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An alkyl group that may have, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent.
Or a salt thereof.

  Item 8. General formula (II):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. The benzene ring, R ¹ and R ² may be the same or different and each may have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a heteroaryl group which may have a substituent; Show.)
Wherein the compound represented by the general formula (16):

(In the formula, R ³ represents an alkyl group, and Ar, X, Y, n, A, R ¹ and R ² are the same as above.)
The ester compound represented by this is hydrolyzed, The manufacturing method characterized by the above-mentioned.

  Item 9. Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
The ester compound represented by these.

Item 10. Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A method for producing an ester compound represented by general formula (14):

(In the formula, R ³ represents an alkyl group, and Ar, X, Y, n and A are the same as above.)
An aldehyde compound represented by the general formula (15):
HN (R ¹ ) (R ² ) (15)
(In the formula, R ¹ and R ² are the same or different and each represents an alkyl group which may have a substituent.)
The manufacturing method characterized by making the amine compound represented by these react.

Item 11. General formula (14):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ³ represents an alkyl group.)
An aldehyde compound represented by

  Item 12. General formula (6):

Wherein X and Y are the same or different and are represented by —O— or —S—, n is 2 or 3, A is a benzene ring which may have a substituent, and m is 1 or 2. )
A compound represented by

Item 13. Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A method for producing an ester compound represented by the general formula (23):

(In the formula, Ar, X, Y, n, A, R ¹ , R ² and R ³ are the same as above.)
And a trialkyl phosphite.

Item 14. Formula (23):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A compound represented by

  Item 15. General formula (19):

(Wherein X and Y are the same or different and are represented by —O— or —S—, n is 2 or 3, and A represents a benzene ring which may have a substituent)
A compound represented by

Item 16. General formula (III):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An alkyl group that may have, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent.
Or a salt thereof, which is represented by the general formula (34):

(In the formula, R ³ represents an alkyl group, and Ar, X, n, R ¹ and R ² are the same as above.)
The ester compound represented by this is hydrolyzed, The manufacturing method characterized by the above-mentioned.

  Item 17. The zinc fluorescent probe containing the compound or its salt in any one of the said Claims 1-7.

  Item 18. A reagent for measuring zinc ions, comprising the compound or salt thereof according to any one of claims 1 to 7.

  Item 19. The zinc complex containing the compound and zinc ion in any one of the said Claims 1-7.

Item 20. A method for measuring zinc ions,
(A) a step of reacting the compound according to any one of claims 1 to 7 or a salt thereof with a zinc ion to form a zinc complex; and (b) measuring the fluorescence intensity of the zinc complex formed in (a) above. A measuring method including a step of performing.

  Item 21. In the step (b), the light of a single excitation wavelength is irradiated, and the fluorescence intensity of each of the compound according to any one of claims 1 to 7 or a salt thereof and the zinc complex formed in the above (a) is measured. The measurement method according to claim 20.

  Item 22. The measurement method according to claim 20, wherein the excitation wavelength is 340 to 400 nm.

  Item 23. In the step (b), the difference between the peak wavelength of the fluorescence spectrum of the compound or salt thereof according to any one of claims 1 to 7 and the peak wavelength of the fluorescence spectrum of the zinc complex formed in the above (a) is 20 nm. It is the above, The measuring method of Claim 20, 21 or 22.

Item 24. A method for measuring intracellular zinc ions,
(A) a step of incorporating the compound or salt thereof according to any one of claims 1 to 7 into a cell; and (b) the compound or salt thereof incorporated into the cell in (a) above and an intracellular zinc ion. Measuring method including the process of measuring each fluorescence intensity of the zinc complex produced | generated from.

The present invention is described in detail below.
I. Zinc Fluorescent Probe A zinc fluorescent probe is a compound that exhibits a fluorescence response when complexed with zinc, and is used for imaging free zinc ions in a living body.

The zinc fluorescent probe of the present invention has the general formula (I):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, Z is a single bond or a formula:

A represents a benzene ring which may have a substituent, and Y represents a group represented by —O— or —S—. )
Or a salt thereof.

As an aryl group of the aryl group which may have a substituent represented by Ar, for example, a monocyclic or bicyclic C ₆ such as a phenyl group, (o-, m- or p-) toluyl group, naphthyl group and the like. _-10 aryl groups. Of these, a phenyl group or a p-toluyl group is preferred.

  Examples of the substituent on the aryl group include an alkyl group, an alkoxy group, a hydroxyl group, an amino group, a mono- or dialkylamino group, a mono- or di (hydroxyalkyl) amino group, a carboxyl group, an alkoxycarbonyl group, a carboxyalkyl group, Examples include an alkoxycarbonylalkyl group, a carboxyalkoxy group, an alkoxycarbonylalkoxy group, and the like, and 1 to 3 (preferably 1) groups selected from these may be substituted on the aryl group.

Examples of the alkyl group include ethyl group, a propyl group, an alkyl group of C _1-6 and butyl group.

Examples of the alkoxy group include a methoxy group, an ethoxy group, an alkoxy group of C _1-6, such as a propoxy group.

Examples of the mono- or dialkylamino group include mono- or di-C _1-6 alkylamino groups such as a methylamino group, an ethylamino group, a propylamino group, a dimethylamino group, a diethylamino group, and a dipropylamino group.

Examples of the mono or di (hydroxyalkyl) amino group include mono or di (hydroxy) such as 2-hydroxyethylamino group, hydroxypropylamino group, di (2-hydroxyethyl) amino group, and di (hydroxypropyl) amino group. C _1-6 alkyl) amino group.

Examples of the alkoxycarbonyl group include C _1-6 alkoxycarbonyl groups such as a methoxycarbonyl group and an ethoxycarbonyl group.

Examples of the carboxyalkyl group include carboxy C _1-6 alkyl groups such as carboxymethyl group and carboxyethyl group.

Examples of the alkoxycarbonylalkyl group include C _1-6 alkoxycarbonyl C _1-6 alkyl groups such as ethoxycarbonylmethyl group and methoxycarbonylmethyl group.

Examples of the carboxyalkoxy group include carboxy C _1-6 alkyl groups such as a carboxymethoxy group and a carboxyethoxy group.

The alkoxycarbonyl alkoxy group, for example, methoxycarbonyl methoxy group, and a C _1-6 alkoxycarbonyl C _1-6 alkoxy group such as ethoxycarbonyl methoxy group.

  Among these, as Ar, a phenyl group, a toluyl group, a naphthyl group, a carboxymethoxy group-substituted phenyl group, a 5- (dimethylamino) -1-naphthyl group (dansyl group) and the like are preferable.

  X is preferably a group represented by -O-, and n is preferably 2.

Z is the formula:

(In the formula, A and Y are the same as above.)
In the case where A has a substituent, examples of the substituent include an alkyl group, an alkoxy group, and a halogen atom. A is preferably a benzene ring. A group in which Y is represented by -O- is preferred.

As the alkyl group of the alkyl group which may have a substituent represented by R ¹ or R ² , for example, a C _1-3 alkyl group such as a methyl group, an ethyl group, or a propyl group is preferable.

Examples of the substituent on the alkyl group include a nitrogen-containing heteroaryl group, a carboxyl group, a hydroxyl group, an amino group, a mono- or dialkylamino group, an alkoxy group (for example, a C _1-3 alkoxy group), and the like. As a nitrogen-containing heteroaryl group, nitrogen-containing six-membered heteroaryl groups, such as a pyridyl group (especially 2-pyridyl group), a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, are mentioned, for example. Examples of the mono- or dialkylamino group include mono- or di-C _1-6 alkylamino groups such as a methylamino group and a dimethylamino group. One or two (preferably one) of these may be substituted on the alkyl group.

The alkyl group which may have a substituent represented by R ¹ or R ² is preferably a 2-pyridylmethyl group, 2-pyridylethyl group, hydroxymethyl group, 2-hydroxyethyl group, carboxymethyl group, 2- A carboxyethyl group, a dimethylamino group, etc. are mentioned.

Examples of the aryl group of the aryl group which may have a substituent represented by R ¹ or R ² include monocyclic or bicyclic C _6-10 aryl groups such as a phenyl group and a naphthyl group.

Examples of the substituent on the aryl group include a nitrogen-containing heteroaryl group, a carboxyl group, a hydroxyl group, an amino group, a mono- or dialkylamino group, and an alkoxy group (for example, a C _1-3 alkoxy group). As a nitrogen-containing heteroaryl group, nitrogen-containing six-membered heteroaryl groups, such as a pyridyl group (especially 2-pyridyl group), a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, are mentioned, for example. Examples of the mono- or dialkylamino group include mono- or di-C _1-6 alkylamino groups such as a methylamino group and a dimethylamino group. One or two (preferably one) of these may be substituted on the aryl group.

Examples of the heteroaryl group of the heteroaryl group which may have a substituent represented by R ¹ or R ² include C ₆ -containing at least one heteroatom selected from the group consisting of N, O and S. ₁₀ heteroaryl groups are mentioned. Specific examples of the heteroaryl group include pyridyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, imidazolyl group, pyrazolyl group, oxazolyl group, isoxazolyl group, thienyl group, and furyl group.

Examples of the substituent on the heteroaryl group include a nitrogen-containing heteroaryl group, a carboxyl group, a hydroxyl group, an amino group, a mono- or dialkylamino group, and an alkoxy group (for example, a C _1-3 alkoxy group). . As a nitrogen-containing heteroaryl group, nitrogen-containing six-membered heteroaryl groups, such as a pyridyl group (especially 2-pyridyl group), a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, are mentioned, for example. Examples of the mono- or dialkylamino group include mono- or di-C _1-6 alkylamino groups such as a methylamino group and a dimethylamino group. One or two (preferably one) of these may be substituted on the heteroaryl group.

  Examples of the salt of the compound represented by the general formula (I) include alkali metal salts such as lithium salt, sodium salt and potassium salt.

  Of the compounds represented by the general formula (I) or salts thereof, typically, the general formula (II):

(In the formula, Ar, X, Y, n, A, R ¹ and R ² are the same as above.)
Or a salt thereof, or a general formula (III):

(In the formula, Ar, X, Y, n, R ¹ and R ² are the same as above.)
Or a salt thereof.

  Of the compounds represented by the general formula (II) or salts thereof, as specific examples suitable as a zinc fluorescent probe, the general formula (IIa):

(In the formula, Ar, X, Y, n, R ¹ and R ² are the same as above.)
Or a salt thereof.

  As a more preferred specific example, the general formula (IIb):

(In the formula, Ar, X, Y and n are the same as above.)
Or a salt thereof, or a general formula (IIc):

(In the formula, Ar, X, Y and n are the same as above.)
Or a salt thereof.

  Of the compounds represented by the general formula (III) or a salt thereof, specific examples suitable as a zinc fluorescent probe include the general formula (IIIa):

(In the formula, Ar, X, n, R ¹ and R ² are the same as above.)
Or a salt thereof.

  As a more preferred specific example, the general formula (IIIb):

(In the formula, Ar, X and n are the same as above.)
Or a salt thereof, or a general formula (IIIc):

(In the formula, Ar, X, Y and n are the same as above.)
Or a salt thereof.

The compound represented by the general formula (I) is suitably used as a fluorescent probe for zinc ion measurement. The compound has the following characteristics.
(1) Since it is a ratiometric type fluorescent probe, fluctuations in measured values due to the number of cells, excitation light, substrate concentration, etc. are small, and the concentration of zinc ions can be quantitatively analyzed more accurately. The ratiometric means a method of observing a change in intensity ratio at two different wavelengths. Here, the change in the peak intensity ratio of each fluorescence wavelength is observed for the free zinc fluorescent probe and its zinc complex.
(2) Since a change in fluorescence wavelength (visible light region) is observed using a single excitation light wavelength, both the excitation system and the detection system can be simplified. Furthermore, since the excitation light wavelength is single, it is easy to use a confocal laser microscope.
(3) Since the excitation light wavelength is longer than the ultraviolet region (around 340 to 370 nm), the influence on cells can be reduced as much as possible.
(4) Good sensitivity and selectivity for zinc ions. That is, zinc ions of nM to μM order can be captured.
(5) It is excellent in water solubility and has sufficient solubility even in an appropriate aqueous medium containing cells and tissues.

II. Production of Zinc Fluorescent Probe A method for producing a compound represented by the general formula (I) or a salt thereof (compounds represented by the general formulas (II) and (III) or a salt thereof) contained in the zinc fluorescent probe of the present invention is described. To do.

Manufacturing method A
The compound represented by the general formula (II) can be produced, for example, as follows.

(In the formula, R ³ represents an alkyl group, m represents 1 or 2, and Ar, X, Y, n, A, R ¹ and R ² are the same as above.)
Examples of the alkyl group represented by R ³ include C _1-3 alkyl groups such as a methyl group, an ethyl group, and a propyl group. In particular, a methyl group and an ethyl group are preferable.

  The compound represented by the general formula (1) is prepared in the presence of a base (for example, potassium carbonate) according to the description of J. Am. Chem. Soc. Communications, 2002, 124 (5), p776-778. A dibromo compound represented by the formula (2) is reacted to obtain a compound represented by the general formula (3).

  The compound represented by the general formula (3) is reacted with the diol represented by the general formula (3) in the presence of an acid catalyst (for example, p-toluenesulfonic acid (pTsOH), pyridinium p-toluenesulfonate (PPTS), etc.). To obtain an acetal (4), which is reacted with an aldehyde compound represented by the general formula (5) in the presence of a base (for example, sodium hydride, sodium hydroxide, potassium carbonate, etc.) to give a general formula (6) To obtain a compound represented by:

The compound represented by the general formula (6) is reduced to formyl group by reducing the formyl group with a reducing agent such as NaBH ₄ , and this is reacted with acetic anhydride (Ac ₂ O) and protected with an acetyl group. The compound represented by (7) is obtained.

The compound represented by the general formula (7) is represented by the general formula (8) by reducing a nitro group to an amino group in the presence of a hydrogenation catalyst (PtO ₂ , Pd-carbon, etc.) in a hydrogen atmosphere. To obtain a compound.

  The compound represented by the general formula (8) is reacted with the sulfonic acid chloride represented by the general formula (9) in the presence of a base (for example, pyridine or the like) to form a sulfonic acid amide. By treatment with an acid such as an acid, the acetal protecting group is removed to obtain a formyl compound represented by the general formula (10).

The formyl compound represented by the general formula (10) is reacted with the Wittig reagent represented by the general formula (11) to obtain the olefin compound represented by the general formula (12). This is reductively closed using P (OEt) ₃ to obtain an indole compound represented by the general formula (13).

The acetyl group of the indole compound represented by the general formula (13) is removed with EtONa and oxidized with an oxidizing agent such as MnO ₂ to obtain the aldehyde compound represented by the general formula (14). This is reacted with a secondary amine represented by the general formula (15) (reductive amination reaction) in the presence of a reducing agent (for example, sodium triacetoxyborohydride) to give a general formula (16) To obtain a compound represented by:

The compound of the present invention represented by the general formula (II) is obtained by hydrolyzing the carboxylic acid ester of the compound represented by the general formula (16) using an alkali metal hydroxide such as LiOH. In addition, when Ar, R ¹ and / or R ² contain a carboxylic acid ester, the carboxylic acid ester is usually hydrolyzed to a carboxylic acid in this hydrolysis step.

Manufacturing method B
Or the compound represented by general formula (II) can also be manufactured as follows, for example.

(In the formula, Ar, X, Y, n, A, R ¹ , R ² and R ³ are the same as above.)
The compound represented by the general formula (17) is synthesized as described in Journal of Medicinal Chemical Society, 2003, 46, pp. 691-701. A compound represented by the general formula (17) is reacted with a compound represented by the general formula (18) in the presence of a base (for example, potassium carbonate) to give a formyl compound represented by the general formula (19). obtain.

  The formyl compound represented by the general formula (19) is reacted with the secondary amine represented by the general formula (15) in the presence of a reducing agent (for example, sodium triacetoxyborohydride) (reductive amination). Reaction) to obtain a compound represented by the general formula (20).

The compound represented by the general formula (20) is reduced to an amino group in the presence of a hydrogenation catalyst (PtO ₂ , Pd-carbon, etc.) in a hydrogen atmosphere, and then a base (for example, pyridine, etc.) is obtained. ) Is reacted with a sulfonic acid chloride represented by the general formula (9) to give a sulfonic acid amide represented by the general formula (21).

The sulfonic acid amide represented by the general formula (21) is oxidized with an oxidizing agent such as MnO ₂ to obtain a compound represented by the general formula (22). This is reacted with a Wittig reagent represented by general formula (11) to obtain an olefin compound represented by general formula (23). This is reductively closed using a trialkyl phosphite such as P (OEt) ₃ to obtain an indole compound represented by the general formula (16).

  The ester of the indole compound represented by the general formula (16) is hydrolyzed in the same manner as in Production Method A to obtain the compound of the present invention represented by the general formula (II).

Manufacturing method C
The compound represented by the general formula (III) can be produced, for example, as follows.

Wherein W ¹ and W ² represent a leaving group, and Ar, X, n, R ¹ , R ² and R ³ are the same as above.

  The compound represented by the general formula (17) is reacted with the compound represented by the general formula (24) in the presence of a base (for example, potassium carbonate) to obtain a compound represented by the general formula (25). .

The compound represented by the general formula (25) is represented by the general formula (26) by reducing a nitro group to an amino group in the presence of a hydrogenation catalyst (PtO ₂ , Pd-carbon, etc.) in a hydrogen atmosphere. A compound is obtained.

  A compound represented by the general formula (26) is reacted with a sulfonic acid chloride represented by the general formula (9) in the presence of a base (for example, pyridine and the like) to obtain a sulfone represented by the general formula (27). The acid amide is obtained.

The sulfonic acid amide represented by the general formula (27) is oxidized with an oxidizing agent such as MnO ₂ to obtain an aldehyde compound represented by the general formula (28). This compound is reacted with a Wittig reagent represented by the general formula (11) to obtain an olefin compound represented by the general formula (29), and this is reductively cyclized using P (OEt) ₃ in general. An indole compound represented by the formula (30) is obtained.

  The indole compound represented by the general formula (30) is treated with an acid (for example, hydrochloric acid, trifluoroacetic acid, etc.) to remove the Boc group to obtain the compound represented by the general formula (31).

The compound represented by the general formula (31) is reacted with the compound represented by the general formula (32) and (33) in the presence of an activator such as a base (for example, potassium carbonate). The compound represented by (34) is obtained. Note that W ^{1 of the} compound represented by the general formula (32) and W ² of the compound represented by the general formula (33) may be the same or different. Examples of the leaving group represented by W ¹ and W ² include halogen atoms such as a chlorine atom, a bromine atom, and an iodine atom, TfO—, MsO—, and TsO—.

  Furthermore, instead of the compounds represented by the general formulas (32) and (33), a predetermined aldehyde compound is used, and in the presence of a reducing agent (for example, sodium triacetoxyborohydride), the general formula (31 The compound represented by the general formula (34) can also be produced by subjecting the compound represented by) to a reductive amination reaction.

  The ester of the indole compound represented by the general formula (34) is hydrolyzed in the same manner as in Production Method A to obtain the compound of the present invention represented by the general formula (III).

In addition, the compound represented by general formula (II) and (III) can also be converted into a salt as needed. Examples of acceptable salts include alkali metal salts such as lithium salts, sodium salts, and potassium salts. Moreover, depending on the case, it can also obtain as solvates, such as water and alcohol.

III. Method for Measuring Zinc Ion Using Zinc Fluorescent Probe The compound of the present invention represented by the above general formula (I) or a salt thereof (compound represented by the general formulas (II) and (III) or a salt thereof) is zinc It is useful as a fluorescent probe and a reagent for measuring zinc ions.

  Even if the compound (I) or a salt thereof captures zinc ions to form a zinc complex, the shift of the peak wavelength of the absorption spectrum is small. For this reason, the wavelength of the excitation light to be irradiated may be single, and the excitation device and the detection device can be simplified. The peak of the absorption light wavelength varies depending on the chemical structure of the compound (I) or a salt thereof, but is usually about 340 to 400 nm, preferably about 340 to 370 nm, and the wavelength of the excitation light can be appropriately selected from this range. it can.

  In addition, when the compound (I) or a salt thereof captures zinc ions to form a zinc complex, the peak of the fluorescence spectrum causes a remarkable wavelength shift. The wavelength shift of the fluorescence spectrum peak is usually about 20 nm or more, further about 25 to 50 nm, particularly about 25 to 45 nm.

  That is, by using the compound of the present invention as a zinc fluorescent probe, irradiating excitation light of an appropriate single wavelength, and measuring the fluorescence intensity ratio between the compound (I) and the zinc complex at that time, zinc in the sample Ions can be measured by the ratio method.

  Regarding the ratio method, Mason W. et al. T.A. (Mason WT in Fluorescent and Luminescent Probes for Biological Activity, Second Edition, Edited by Mason WT, Academic Press) and the like.

  In addition, the compound (I) or a salt thereof of the present invention has a feature that it can specifically capture zinc ions and forms a complex very rapidly. For example, compound (I) or a salt thereof hardly affects even if other metal ions (for example, sodium ion, calcium ion, potassium ion, magnesium ion, etc.) coexist in the sample other than zinc ion. A complex is formed specifically with respect to zinc ions without being received.

  From the above characteristics, the compound (I) or a salt thereof of the present invention is extremely useful as a zinc fluorescent probe for measuring zinc ions in living cells and living tissues under physiological conditions. The term “measurement” used in this specification is interpreted in the broadest sense including quantitative and qualitative.

  The specific example is shown below about the measuring method of the zinc ion in the sample using the zinc fluorescent probe of this invention. The zinc ion measurement method of the present invention comprises (a) a step of reacting a compound represented by the above general formula (I) or a salt thereof with a zinc ion to form a zinc complex, and (b) the above (a). And a step of measuring the fluorescence intensity of the zinc complex formed in (1).

  Specifically, in an aqueous medium such as physiological saline or a buffer, or a mixed medium of a water-compatible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, dimethylformamide and water, the compound (I) or Dissolve the salt. This solution may be added to an appropriate buffer containing cells and tissues, irradiated with excitation light having a single wavelength, and the respective fluorescence intensity ratios of compound (I) and its zinc complex may be measured. In order to enhance the fat solubility in consideration of the permeability of the cell membrane, a hydrophilic group such as a carboxyl group of compound (I) may be converted into a predetermined ester group. The ester is not particularly limited as long as it can be hydrolyzed with esterase after entering the cell.

  When the zinc concentration is measured using the compound (I) of the present invention as a zinc fluorescent probe, the concentration of the compound (I) may be, for example, about 0.05 to 100 μM, preferably about 0.1 to 10 μM. The concentration of zinc ions can be measured, for example, from about 0.05 nM to 200 μM, preferably from about 0.1 nM to 100 μM.

  As a specific example, the compound (IIc) has an absorption wavelength of 342 nm and a fluorescence wavelength of 427 nm, and can be used as a zinc fluorescent probe at a concentration of about 0.1 to 10 μM. The concentration of zinc ions in the detectable living cells or living tissues may be about 0.1 nM to 100 μM. Compound (IIc) captures zinc ions to form a complex, and the peak of the fluorescence spectrum is shifted by a long wavelength by about 30 to 50 nm. Therefore, when this compound (IIc) is used as a zinc probe, for example, if excitation light with an excitation wavelength of 342 nm is used and the fluorescence intensity of compound (IIc) and its zinc complex at the excitation wavelength is obtained, the ratio is calculated. Good. From this fluorescence intensity ratio, the concentration of zinc ions present in cells and tissues can be determined.

  The measurement of zinc ions using the zinc fluorescent probe of the present invention can be carried out using a fluorescence microscope conventionally used for measuring the ratio of calcium ions. Since the method of the present invention is two-wavelength photometry, it is advantageous in that it can be used even in an apparatus that can use only single excitation light.

  The zinc fluorescent probe of the present invention may be used in the form of a composition in combination with an appropriate additive. For example, it can be combined with additives such as a buffer, a solubilizing agent and a pH adjuster.

  The compound of the present invention is extremely useful as a ratiometric type fluorescent probe for measuring zinc.

In particular, even when the compound of the present invention forms a complex with zinc ions, the wavelength shift hardly occurs in the peak of the excitation spectrum, and when the complex with zinc ions is formed, the peak of the fluorescence spectrum shifts by a long wavelength. Therefore, it is possible to irradiate excitation light having a single wavelength and detect the zinc ion concentration in the cell with high sensitivity in a ratiometric manner. Further, since an excitation light source having a single wavelength may be used, there is an advantage that the excitation system and the detection system can be simplified.

In addition, since the excitation light wavelength of the compound of the present invention is longer than that in the ultraviolet region (about 340 to 370 nm), the influence on cells is small. And it has the characteristic that the sensitivity and selectivity with respect to zinc ion are favorable.

The complex formation schematic diagram of a compound (IIc) and zinc ion is shown. The absorption spectrum when compound (IIc) and zinc are added is shown. The fluorescence spectrum when compound (IIc) and zinc are added is shown. It is a graph which shows the metal ion selectivity of a compound (IIc).

  The present invention will be described in more detail with reference to examples, but is not limited thereto.

Example 1 (Production method A)
(1) 3- (2- (2-Formylphenoxy) -1-ethoxy) -4-nitrobenzaldehyde dioxolane (6)
3- (2-Bromoethoxy) -4-nitrobenzaldehyde dioxolane was synthesized according to the method described in Journal of the American Chemical Society, 2002, 124, pp. 776-778. To a solution of 4.8 g (15.1 mmol) of the compound and 1.9 g (15.1 mmol) of salicylaldehyde in 50 ml of dimethylformamide, 2.76 g (20.0 mmol) of potassium carbonate was added and stirred at 100 ° C. for 3 hours. The reaction solution was added to 300 ml of ice water with stirring, and the precipitated solid was collected by filtration to obtain 4.3 g of the title compound (6) (yield 80%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 4.03-4.15 (4H, m), 4.50-4.59 (4H, m), 5.84 (1H, s), 7.04-7.10 (2H, m), 7.19 (1H , dd, J = 8.4, 1.4 Hz), 7.32 (1H, d, J = 1.4 Hz), 7.53 (1H, m), 7.82-7.85 (2H, m).

(2) 3- (2- (2-Acetoxymethylphenoxy) -1-ethoxy) -4-nitrobenzaldehyde dioxolane (7)
To a solution of compound (6) 3.6 g (10.0 mmol) suspended in 100 ml of methanol, sodium tetrahydroborate NaBH ₄ 0.38 g (10.0 mmol) was added little by little at 0 ° C. and stirred overnight at room temperature. After distilling off methanol under reduced pressure, the residue was washed with water and recrystallized with ethanol / water to obtain 3.6 g of an alcohol form (yield quantitative).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 4.03-4.14 (4H, m), 4.41-4.46 (2H, m), 4.51-4.55 (2H, m), 4.64 (2H, s), 5.84 (1H , s), 6.93 (1H, d, J = 8.1 Hz), 6.98 (1H, t, J = 7.0 Hz), 7.17 (1H, dd, J = 8.1, 1.4 Hz), 7.25-7.31 (3H, m) , 7.84 (1H, d, J = 8.1 Hz).
To a solution obtained by dissolving 2.9 g (8.0 mmol) of the above compound in 30 ml of pyridine, 1.02 g (10.0 mmol) of acetic anhydride was added at 0 ° C. and stirred overnight at room temperature. 10 ml of ethanol was added to the reaction solution, and pyridine / ethanol was distilled off under reduced pressure. The residue was dissolved in 50 ml of ethyl acetate, washed 3 times with saturated brine, and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain 2.90 g of the title compound (7) (yield 90%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 2.06 (3H, s), 4.05-4.11 (4H, m), 4.39-4.43 (2H, m), 4.48-4.55 (2H, m), 5.15 (2H , s), 5.85 (1H, s), 6.94-7.01 (2H, m), 7.17 (1H, dd, J = 8.4, 1.4 Hz), 7.28-7.34 (3H, m), 7.83 (1H, d, J = 8.1 Hz).

(3) 3- (2- (2-Acetoxymethylphenoxy) -1-ethoxy) -4-aminobenzaldehyde dioxolane (8)
To a solution of compound (7) 2.0 g (5.0 mmol) dissolved in 40 ml of tetrahydrofuran, 114 mg (0.5 mmol) of platinum dioxide was added and stirred overnight at room temperature under 2 atmospheres of hydrogen gas. The platinum dioxide was filtered off through celite, and the filtrate was concentrated under reduced pressure to obtain 1.9 g of the title compound (8). (Yield quantitative).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 2.05 (3H, s), 3.99-4.02 (2H, m), 4.11-4.14 (2H, m), 4.38 (4H, s), 5.18 (2H, s ), 5.70 (1H, s), 6.69 (1H, d, J = 8.0 Hz), 6.93 (1H, dd, J = 8.0, 1.5 Hz), 6.95 (1H, d, J = 8.0 Hz), 6.97-7.00 (2H, m), 7.31 (1H, td, J = 7.5, 1.5 Hz), 7.34 (1H, dd, J = 7.5, 1.5 Hz).

(4) 3- (2- (2-Acetoxymethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) benzaldehyde (10)
To a solution of 1.9 g (5.0 mmol) of compound (8) in 20 ml of pyridine, 1.14 g (6.0 mmol) of p-toluenesulfonyl chloride was added at 0 ° C. and stirred overnight at room temperature. The reaction solution was added to 300 ml of ice water with stirring, and stirring was continued at room temperature for 30 minutes. The precipitated solid was collected by filtration to obtain 2.4 g of a sulfonamide (yield quantitative).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 2.07 (3H, s), 2.31 (3H, s), 3.97-4.14 (4H, m), 4.22 (4H, s), 5.17 (2H, s), 5.69 (1H, s), 6.90 (1H, d, J = 7.8 Hz), 6.95 (1H, d, J = 1.6 Hz), 7.00-7.05 (2H, m), 7.12 (2H, d, J = 8.1 Hz ), 7.23 (1H, s), 7.32-7.38 (2H, m), 7.54 (1H, d, J = 7.8 Hz), 7.61 (2H, d, J = 8.1 Hz).
To a solution of 2.4 g (5.0 mmol) of the above compound dissolved in 70 ml of acetone, 87 mg (0.5 mmol) of toluenesulfonic acid monohydrate was added and stirred overnight at room temperature. To the reaction solution was added 10 ml of saturated brine, and acetone was distilled off under reduced pressure, followed by extraction with dichloromethane. The dichloromethane layer was washed with saturated brine and dried over magnesium sulfate. Dichloromethane was distilled off under reduced pressure, and the residue was purified by a silica gel column to obtain 2.3 g of Compound (10) (yield 97%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 2.06 (3H, s), 2.34 (3H, s), 4.31-4.40 (4H, m), 5.15 (2H, s), 6.92 (1H, d, J = 8.4 Hz), 7.04 (1H, t, J = 7.6 Hz), 7.17 (2H, d, J = 8.1 Hz), 7.31-7.42 (4H, m), 7.64-7.71 (4H, m).

(5) 3-Nitro-4- [2- [3- (2- (2-acetoxymethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -trans-vinyl] benzoic acid Ethyl ester (12)
Compound (10) 0.43 g (1.0 mmol) in 6 ml dimethylformamide was dissolved in (4-ethoxycarbonyl-2-nitrobenzyl) triphenylphosphonium bromide (11) 0.83 g (1.5 mmol), 0.69 g (5.0 mmol) of potassium was added, and the mixture was stirred at 90 ° C. overnight. The reaction solution was added to 100 ml of water and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure, and the residue was purified by a silica gel column to obtain 0.49 g of Compound (12) (yield 73%).
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.42 (3H, t, J = 7.0 Hz), 2.05 (3H, s), 2.32 (3H, s), 4.27 (4H, s), 4.43 (2H, q, J = 7.0 Hz), 5.17 (2H, s), 6.92 (1H, d, J = 7.5 Hz), 7.02 (1H, d, J = 1.5 Hz), 7.04 (1H, d, J = 8.0 Hz) , 7.08-7.11 (2H, m), 7.15 (2H, d, J = 8.5 Hz), 7.34-7.38 (3H, m), 7.46 (1H, d, J = 14.0 Hz), 7.56 (1H, d, J = 8.5 Hz), 7.64 (2H, d, J = 8.5 Hz), 7.81 (1H, d, J = 8.0 Hz), 8.21 (1H, dd, J = 8.0, 1.5 Hz), 8.57 (1H, d, J = 1.5 Hz).

(6) 2- [3- (2- (2-Acetoxymethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid ethyl ester (13)
Compound (12) (0.32 g, 0.48 mmol) was dissolved in 3 ml of triethyl phosphite and stirred at 125 ° C. for 4 hours. Triethyl phosphite was distilled off under reduced pressure and purified by a silica gel column to obtain 0.23 g of Compound (13) (yield 74%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.42 (3H, t, J = 7.3 Hz), 2.03 (3H, s), 2.30 (3H, s), 4.22-4.28 (4H, m), 4.40 ( 2H, q, J = 7.3 Hz), 5.17 (2H, s), 6.76 (1H, d, J = 1.4 Hz), 6.90 (1H, d, J = 8.6 Hz), 7.03 (1H, t, J = 7.3 Hz), 7.14 (2H, d, J = 8.4 Hz), 7.22 (1H, dd, J = 1.8 Hz), 7.30 (1H, dd, J = 8.4, 1.8 Hz), 7.32-7.38 (2H, m), 7.59 (1H, d, J = 8.4 Hz), 7.61 (1H, d, J = 8.4 Hz), 7.65 (2H, d, J = 8.4 Hz), 7.80 (1H, dd, J = 8.4, 1.1 Hz), 8.15 (1H, s), 9.19 (1H, brs).

(7) 2- [3- (2- (2-Formylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid ethyl ester (14)
Sodium ethoxide 0.39 g (5.7 mmol) was added to a solution of compound (13) 0.18 g (0.29 mmol) in 20 ml of ethanol, and the mixture was stirred overnight at room temperature. Acetic acid 0.34 g (5.7 mmol) was added to the reaction solution, and ethanol was distilled off under reduced pressure. To the residue was added 25 ml of saturated brine, extracted 3 times with ethyl acetate, and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure and purified by a silica gel column to obtain 0.12 g of alcohol (yield 69%).
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.41 (3H, t, J = 7.0 Hz), 2.26 (3H, s), 4.21 (2H, t, J = 3.5 Hz), 4.30 (2H, t, J = 3.5 Hz), 4.39 (2H, q, J = 7.0 Hz), 4.67 (2H, s), 6.74 (1H, d, J = 1.0 Hz), 6.89 (1H, d, J = 8.0 Hz), 7.01 (1H, t, J = 7.5 Hz), 7.09 (2H, d, J = 8.5 Hz), 7.25 (1H, s), 7.29-7.34 (3H, m), 7.47 (1H, s), 7.58 (1H, d, J = 8.3 Hz), 7.59 (1H, d, J = 8.3 Hz), 7.61 (2H, d, J = 8.5 Hz), 7.74 (1H, dd, J = 8.3, 1.0 Hz), 8.13 (1H, s), 9.63 (1H, s).
To a solution of 0.12 g (0.2 mmol) of the above compound in 15 ml of dichloromethane, 0.17 g (2.0 mmol) of manganese dioxide was added and stirred overnight at room temperature. The insoluble material was filtered off through celite, and the filtrate was evaporated under reduced pressure to give compound (14).
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.34 (3H, t, J = 7.0 Hz), 2.28 (3H, s), 4.31 (2H, q, J = 7.0 Hz), 4.37-4.43 (4H, m), 6.99 (1H, s), 7.13 (1H, t, J = 7.5 Hz), 7.21 (1H, d, J = 8.0 Hz), 7.31 (2H, d, J = 7.5 Hz), 7.39-7.44 ( 2H, m), 7.53 (1H, s), 7.58-7.63 (4H, m), 7.72-7.73 (2H, m), 8.03 (1H, s), 9.55 (1H, brs), 10.31 (1H, s) , 11.83 (1H, s).

(8) 2- [3- (2- (2-Bis (2-pyridylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6- Carboxylic acid ethyl ester (16b)
After adding 36 mg (0.18 mmol) of di- (2-picolyl) amine to a solution of compound (14) 53 mg (0.09 mmol) in 10 ml of 1,2-dichloroethane, sodium triacetoxyboro Hydride 38 mg (0.18 mmol) was added, and the mixture was stirred at room temperature overnight. 20 ml of water was added to the reaction solution, extracted three times with chloroform and then dried over magnesium sulfate. Chloroform was distilled off under reduced pressure and purified by a silica gel column to obtain 52 mg of Compound (16b) (yield 75%).
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.37 (3H, t, J = 7.0 Hz), 2.26 (3H, s), 3.60 (2H, s), 3.75 (4H, s), 3.97-3.99 ( 2H, m), 4.11-4.14 (2H, m), 4.36 (2H, q, J = 7.0 Hz), 4.37-4.43 (4H, m), 6.71 (1H, d, J = 7.5 Hz), 6.76 (1H , dd, J = 2.0, 1.0 Hz), 6.95 (1H, td, J = 7.5, 1.0 Hz), 7.04-7.07 (4H, m), 7.19 (1H, td, J = 7.5, 1.5 Hz), 7.25 ( 1H, d, J = 2.0 Hz), 7.29 (1H, dd, J = 8.5, 2.0 Hz), 7.41 (1H, dd, J = 7.5, 1.5 Hz), 7.47 (1H, d, J = 7.5 Hz), 7.52 (1H, td, J = 7.5, 1.5 Hz), 7.55 (1H, d, J = 8.5 Hz), 7.59 (1H, d, J = 8.5 Hz), 7.61 (1H, d, J = 8.0 Hz), 7.78 (1H, dd, J = 5.5, 1.5 Hz), 8.04 (1H, d, J = 1.5 Hz), 8.46 (2H, ddd, J = 5.0, 2.0, 1.0 Hz), 10.39 (1H, s).

(9) 2- [3- (2- (2-Bis (2-pyridylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6- Carboxylic acid (IIb)
To a solution of 12 mg (15 mmol) of compound (16b) in 10 ml of methanol was added 42 mg (1.0 mmol) of lithium hydroxide monohydrate, and the mixture was stirred overnight at room temperature. Methanol was distilled off under reduced pressure and neutralized with dilute hydrochloric acid, and insoluble matter was collected by filtration to obtain 7.3 mg of compound (IIb) (yield 81%).

Example 2 (Production method A)
(1) 2- [3- (2- (2-Bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carvone Acid ethyl ester (16c)
Compound (16c) was obtained in the same manner as in Example (8) except that diethyliminodiacetate was used in place of di- (2-picolyl) amine in Example 1 (8). 68%).
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.17 (3H, t, J = 7.0 Hz), 1.40 (3H, t, J = 7.0 Hz), 2.30 (3H, s), 3.53 (4H, s) , 3.93 (2H, s), 4.07 (4H, q, J = 7.0 Hz), 4.14-4.16 (2H, m), 4.25-4.27 (2H, m), 4.39 (2H, q, J = 7.0 Hz), 6.77 (1H, s), 6.84 (1H, d, J = 8.0 Hz), 6.98 (1H, t, J = 7.5 Hz), 7.13 (2H, d, J = 8.5 Hz), 7.24-7.28 (2H, m ), 7.31 (1H, dd, J = 8.0, 1.5 Hz), 7.38 (1H, dd, J = 7.5, 1.5 Hz), 7.48 (1H, brs), 7.58 (1H, d, J = 8.0 Hz), 7.59 (1H, d, J = 8.0 Hz), 7.66 (2H, d, J = 8.5 Hz), 7.89 (1H, dd, J = 8.0, 1.5 Hz), 8.14 (1H, s), 9.59 (1H, s) .

(2) 2- [3- (2- (2-Bis (carboxymethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid (IIc)
Lithium hydroxide monohydrate 42 mg (1.0 mmol) was added to a solution of compound (16c) 40 mg (52 mmol) in 10 ml of methanol, and the mixture was stirred overnight at room temperature. Methanol was distilled off under reduced pressure, 1 ml of 3N hydrochloric acid was added, and the insoluble material was collected by filtration. This was suspended in 1 ml of distilled water, and diluted hydrochloric acid was added until pH8 was reached. Purification by column chromatography using G-25 Sephadex gave 4.1 mg of compound (IIc) (yield 10%).

Example 3 (Production method A)
(1) 2- [3- (2- (2-Bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (4- (ethoxycarbonylmethoxy) benzenesulfonylamino) -phenyl] -1H- Indole-6-carboxylic acid ethyl ester (16d)
In Example 1 (4) above, by reacting in the same manner as in Examples 1 (1) to (8) except that ethyl (4-chlorosulfonylphenoxy) acetate was reacted instead of p-toluenesulfonyl chloride. Compound (16d) was obtained.
¹ H-NMR (CDCl ₃ , 500 MHz): δ 1.27 (3H, t, J = 7.0 Hz), 1.42 (3H, t, J = 7.0 Hz), 3.61 (2H, s), 3.75 (4H, s) , 4.19-4.24 (6H, m), 4.40 (2H, q, J = 7.0 Hz), 4.56 (2H, s), 6.77 (1H, s), 6.80 (1H, d, J = 8.5 Hz), 6.91 ( 1H, d, J = 7.5 Hz), 7.02-7.07 (3H, m), 7.20-7.22 (2H, m), 7.32-7.38 (3H, m), 7.47 (2H, d, J = 7.5 Hz), 7.51 (2H, td, J = 7.5, 2.0 Hz), 7.58-7.61 (2H, m), 7.67 (2H, d, J = 8.5 Hz), 7.80 (1H, d, J = 8.0 Hz), 8.16 (1H, s), 8.46 (2H, dd, J = 5.0, 2.0 Hz), 9.79 (1H, s).

(2) 2- [3- (2- (2-Bis (carboxymethyl) aminomethylphenoxy) -1-ethoxy) -4- (4- (carboxymethoxy) benzenesulfonylamino) -phenyl] -1H-indole- 6-carboxylic acid (IId)
To a solution of 12 mg (15 μmol) of the compound (16d) in 10 ml of methanol, 42 mg (1.0 mmol) of lithium hydroxide monohydrate was added and stirred overnight at room temperature. Methanol was distilled off under reduced pressure and neutralized with dilute hydrochloric acid, and insoluble matter was collected by filtration to obtain 7.3 mg of compound (IId) (yield 81%).

Example 4 (Production method B)
(1) 3- (2- (2-Formylphenoxy) -1-ethoxy) -4-nitrobenzyl alcohol (19)
2-nitoro-5-hidroxymethyl-phenol (17) was synthesized according to the method described in Journal of Medicinal Chemical Society, 2003, 46, pp. 691-701. To a solution obtained by dissolving 3.4 g (20.0 mmol) of the compound (17) and 4.6 g (20.0 mmol) of 2- (2-bromoethoxy) benzaldehyde in 100 ml of dimethylformamide, 6.9 g (50.0 mmol) of potassium carbonate was added. Stir at 5 ° C. for 5 hours. The reaction solution was added to 300 ml of ice water with stirring, and the precipitated solid was collected by filtration to obtain 4.7 g of the title compound (19) (yield 74%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 4.03-4.15 (4H, m), 4.50-4.59 (4H, m), 5.84 (1H, s), 7.04-7.10 (2H, m), 7.19 (1H , dd, J = 8.4, 1.4 Hz), 7.32 (1H, d, J = 1.4 Hz), 7.53 (1H, m), 7.82-7.85 (2H, m).

(2) 3- (2- (2-Bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4-nitrobenzyl alcohol (20)
To a solution of compound (19) 2.78 g (8.76 mmol) in 50 ml of 1,2-dichloroethane was added diethyliminodiacetate 6.24 g (33.0 mmol), and then sodium triacetoxyborohydride 2.12 g (10.0 mol) ) And 0.60 g (10 mmol) of acetic acid were added, and the mixture was stirred overnight at room temperature. 100 ml of water was added to the reaction solution, extracted three times with chloroform and then dried over magnesium sulfate. Chloroform was distilled off under reduced pressure and purified by a silica gel column to obtain 3.07 g of compound (20) (yield 71%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 4.03-4.14 (4H, m), 4.41-4.46 (2H, m), 4.51-4.55 (2H, m), 4.64 (2H, s), 5.84 (1H , s), 6.93 (1H, d, J = 8.1 Hz), 6.98 (1H, t, J = 7.0 Hz), 7.17 (1H, dd, J = 8.1, 1.4 Hz), 7.25-7.31 (3H, m) , 7.84 (1H, d, J = 8.1 Hz).

(3) 3- (2- (2-Bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) benzyl alcohol (21)
To a solution of compound (20) 0.71 g (1.45 mmol) in 20 ml of tetrahydrofuran was added 5% palladium / carbon 70 mg, and the mixture was stirred overnight at room temperature under 2 atmospheres of hydrogen gas. The catalyst was filtered off through celite, and the filtrate was concentrated under reduced pressure to obtain 0.67 g of a reduced amino compound. (Yield quantitative)
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.22 (6H, t, J = 7.2 Hz), 3.55 (4H, s), 3.95 (2H, s), 4.10 (4H, q, J = 7.2 Hz) , 4.32-4.39 (4H, m), 4.56 (2H, s), 6.67 (1H, d, J = 7.8 Hz), 6.79 (1H, dd, J = 7.8, 1.9 Hz), 6.89-6.92 (3H, m ), 6.96 (1H, td, J = 7.3, 1.9 Hz), 7.23 (1H, td, J = 7.3, 1.6 Hz), 7.43 (1H, dd, J = 7.3, 1.6 Hz).
To a solution of 0.67 g (1.45 mmol) of the above compound dissolved in 10 ml of chloroform, 150 ml of pyridine was added, and to this solution was added a solution of toluenesulfonyl chloride 0.28 g (1.50 mmol) in 2 ml of chloroform at 0 ° C. And stirred overnight. 100 ml of water was added to the reaction solution, extracted three times with chloroform and then dried over magnesium sulfate. Chloroform was distilled off under reduced pressure and purified by a silica gel column to obtain 3.07 g of compound (21) (yield 71%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.21 (6H, t, J = 7.0 Hz), 2.31 (3H, s), 3.56 (4H, s), 3.89 (2H, s), 4.05-4.23 ( 8H, m), 4.61 (2H, s), 6.85 (2H, m), 6.92 (1H, d, J = 1.9 Hz), 7.00 (1H, td, J = 7.6, 1.1 Hz), 7.10 (2H, d , J = 7.6 Hz), 7.23-7.30 (2H, m), 7.43 (1H, dd, J = 7.6, 1.9 Hz), 7.49 (1H, d, J = 8.4 Hz), 7.60 (2H, d, J = (7.6 Hz).

(4) 3- (2- (2-bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) benzaldehyde (21)
To a solution of compound (21) 0.60 g (0.98 mmol) in 15 ml of dichloromethane was added manganese dioxide 0.34 g (4.0 mmol), and the mixture was stirred overnight at room temperature. The insoluble material was filtered off through celite, and the filtrate was evaporated under reduced pressure to give 0.54 g of compound (21) (yield 89%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.21 (6H, t, J = 7.0 Hz), 2.26 (3H, s), 3.49 (4H, s), 3.87 (2H, s), 4.00 (4H, q, J = 7.0 Hz), 4.19-4.30 (4H, m), 6.79 (1H, d, J = 7.8 Hz), 6.91 (1H, t, J = 7.8 Hz), 7.09 (2H, d, J = 8.2 Hz), 7.19 (1H, t, J = 7.8 Hz), 7.28-7.35 (3H, m), 7.59 (1H, d, J = 7.8 Hz), 7.64 (2H, d, J = 8.2 Hz).

(5) 3-Nitro-4- [2- [3- (2- (2-bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl]- Trans-Vinyl] benzoic acid ethyl ester (23)
Compound (21) 0.54 g (0.87 mmol) in 5 ml dimethylformamide was dissolved in (4-ethoxycarbonyl-2-nitrobenzyl) triphenylphosphonium bromide 0.72 g (1.3 mmol), potassium carbonate 0.60 g (4.4 mmol) was added and stirred at 90 ° C. overnight. The reaction mixture was added to 50 ml of water and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure and purified by a silica gel column to obtain 0.17 g of compound (23) (yield 24%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.20 (6H, t, J = 7.3 Hz), 1.42 (3H, t, J = 7.3 Hz), 2.34 (3H, s), 3.57 (4H, s) , 3.96 (2H, s), 4.08 (4H, q, J = 7.3 Hz), 4.20-4.29 (4H, m), 4.43 (2H, q, J = 7.3 Hz), 6.88 (1H, d, J = xx Hz), 6.94-7.16 (5H, m), 7.28-7.34 (2H, m), 7.43-7.49 (2H, m), 7.55-7.67 (4H, m), 7.80 (1H, d, J = 8.6 Hz) , 8.21 (1H, dd, J = 8.1, 1.6 Hz), 8.56 (1H, d, J = 1.6 Hz).

(6) 2- [3- (2- (2-Bis (ethoxycarbonylmethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carvone Acid ethyl ester (16c)
Compound (23) 0.17 g (0.21 mmol) was dissolved in 2 ml of triethyl phosphite and stirred at 125 ° C. for 4 hours. Triethyl phosphite was distilled off under reduced pressure and purified by a silica gel column to obtain 40 mg of Compound (16c) (yield 25%).

(7) 2- [3- (2- (2-Bis (carboxymethyl) aminomethylphenoxy) -1-ethoxy) -4- (p-toluenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid (IIe)
Compound (16c) was treated in the same manner as in Example 1 (9) to give compound (IIe).

Example 5 (Production method C)
(1) (2- (5-hydroxymethyl-2-nitrophenoxy) ethyl) carbamic acid-tert-butyl ester (25)
Compound (17) 2.57 g (15.2 mmol) and (2-bromoethyl) carbamic acid-tert-butyl ester 3.40 g (15.2 mmol) in 50 ml dimethylformamide were dissolved in 3.15 g (22.8 mmol) potassium carbonate. The mixture was further stirred at 100 ° C. for 22 hours. 150 ml of water was added to the reaction solution, extracted three times with ethyl acetate, and then dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure and purified by a silica gel column to obtain 0.85 g of compound (25) (yield 18%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.44 (9H, s), 3.55-3.61 (2H, m), 4.18 (2H, t, J = 5.1 Hz), 4.77 (2H, d, J = 5.7 Hz), 5.18 (1H, bs), 6.99 (1H, d, J = 8.4 Hz), 7.13 (1H, s), 7.88 (1H, d, J = 8.4 Hz).

(2) (2- (5-hydroxymethyl-2- (4- (ethoxycarbonylmethoxy) phenylsulfonylamino) ethyl) carbamic acid-tert-butyl ester (27)
To a solution of 0.85 g (2.73 mmol) of Compound (25) in 30 ml of ethanol, 70% of 5% palladium / carbon was added, and the mixture was stirred overnight at room temperature under 2 atmospheres of hydrogen gas. The catalyst was filtered off through celite, and the filtrate was concentrated under reduced pressure to obtain 0.77 g of a reduced amino compound (26). (Yield 85%)
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.45 (9H, s), 3.54-3.61 (2H, m), 4.07 (2H, t, J = 5.1 Hz), 4.55 (2H, s), 4.98 ( 1H, bs), 6.69 (1H, d, J = 7.8 Hz), 6.77-6.81 (2H, m).
2 ml of pyridine was added to a solution of the above compound (26) 0.65 g (2.31 mmol) in 20 ml of dichloromethane, and 2 ml of ethyl (4-chlorosulfonylphenoxy) acetate 0.64 g (2.31 mmol) was added to this solution. Chloroform solution was added at 0 ° C. and stirred overnight at room temperature. 50 ml of water was added to the reaction solution, extracted three times with ethyl acetate, and then dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure and purified by silica gel column to obtain 1.13 g of compound (27) (yield 94%).
¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.28 (3H, t, J = 7.0 Hz), 1.43 (9H, s), 3.25-3.29 (2H, m), 3.65-3.69 (2H, m), 4.24 (2H, q, J = 7.0 Hz), 4.57 (2H, s), 4.63 (2H, s), 5.44 (1H, bs), 6.70 (1H, s), 6.80-6.85 (3H, m), 7.44 (1H, d, J = 8.4 Hz), 7.60 (1H, bs), 7.65 (2H, d, J = 8.6 Hz).

(3) (2- (5-Formyl-2- (4- (ethoxycarbonylmethoxy) phenylsulfonylamino) ethyl) carbamic acid-tert-butyl ester (28)
To a solution of compound (27) 1.13 g (2.16 mmol) in 30 ml dichloromethane was added manganese dioxide 1.88 g (21.6 mmol), and the mixture was stirred overnight at room temperature. The insoluble material was filtered off through celite, and the filtrate was evaporated under reduced pressure to give compound (28).

(4) 3-Nitro-4- [2- [3- (2- (tert-butoxycarbonylamino) -1-ethoxy) -4- (4- (ethoxycarbonylmethoxy) benzenesulfonylamino) -phenyl] -trans -Vinyl] benzoic acid ethyl ester (29)
Compound (29) is obtained by subjecting compound (28) to a Wittig reaction in the same manner as in Example 1 (5).

(5) 2- [3- (2- (tert-butoxycarbonylamino) -1-ethoxy) -4- (4- (ethoxycarbonylmethoxy) benzenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid Ethyl ester (30)
Compound (30) is obtained by reacting compound (29) in the same manner as in Example 1 (6).

(6) 2- [3- (2-Amino-1-ethoxy) -4- (4- (ethoxycarbonylmethoxy) benzenesulfonylamino) -phenyl] -1H-indole-6-carboxylic acid ethyl ester (31)
Compound (30) is added to a mixed solvent of trifluoroacetic acid / dichloromethane 1: 1 at 0 degree and stirred for 30 minutes. The solvent is distilled off under reduced pressure to obtain compound (31).

(7) 2- [3- (2-Bis (2-pyridylmethyl) amino-1-ethoxy) -4- (4- (ethoxycarbonylmethoxy) benzenesulfonylamino) -phenyl] -1H-indole-6-carvone Acid ethyl ester (34)
2-Pyridinecarboxaldehyde is added to a 1,2-dichloroethane solution of compound (31), sodium triacetoxyborohydride is added, and the mixture is stirred overnight at room temperature. Add 20 ml of water to the reaction mixture, extract 3 times with chloroform, and dry over magnesium sulfate. Chloroform was distilled off under reduced pressure and purified by silica gel column to obtain compound (34).

(8) 2- [3- (2-Bis (2-pyridylmethyl) amino-1-ethoxy) -4- (4- (carboxymethoxybenzene) sulfonylamino) -phenyl] -1H-indole-6-carboxylic acid (IIIb)
Compound (34) is treated in the same manner as in Example 1 (9) to give compound (IIIb).

Test Example 1 (Fluorescence characteristics of compound (IIc))
The fluorescence characteristic of the compound (IIc) obtained in Example 1 was examined. Compound (IIc) was dissolved in 100 mM HEPES buffer (pH 7.2, containing 5 μM EDTA) so as to have a concentration of 10 μM, and absorption and fluorescence spectra were measured.
<Measurement conditions>
JASCO Corporation spectrofluorometer FP-750DS
Slit: Ex, Em 2.5nm
Scan speed: 240 nm / min
Measurement temperature: 25 ° C
The fluorescence characteristics of the compound (IIc) before the addition of zinc ion (20 μM ZnCl ₂ ) and after the addition of zinc ion are shown in Table 1 below.

  In compound (IIc), the absorption wavelength peak due to coordination with zinc ions was shifted to the wavelength side of 8 nm, and the fluorescence wavelength peak was shifted to the longer wavelength side of 37 nm. This is considered to be because the pKa value of the sulfonamide group of the compound (IIc) was reduced by the interaction with the zinc ion, so that the conjugated system was expanded by deprotonation even under neutral conditions.

  A schematic diagram of the equilibrium state of compound (IIc) and zinc ions after addition of zinc ions is shown in FIG. Moreover, the change of each absorption spectrum is shown in FIG. 2, and the change of a fluorescence spectrum is shown in FIG. The excitation wavelength in FIG. 3 is 342 nm, which is the intersection of the two absorption spectra in FIG.

According to this, it was found that in compound (IIc) and its zinc complex, the shift of the absorption wavelength is small and the peak of the fluorescence wavelength is large. Therefore, when compound (IIc) is used as a zinc fluorescent probe, single-wavelength excitation light can be used, and the fluorescence wavelength peaks of compound (IIc) and its zinc complex are greatly different and the fluorescence intensity ratio is accurately determined. Since it can detect, the zinc concentration in a sample (cell) can be measured correctly.

Test Example 2 (Zinc ion selectivity of compound (IIc))
Manganese divalent ions, iron trivalent ions, or zinc ions are added to 100 mM HEPES buffer (containing pH 7.2, 5 μM EDTA) containing 10 μM compound (IIc) until the concentration reaches 50 μM, respectively, and the excitation wavelength is set to 350 nm. The fluorescence intensity (I ₄₁₀ ) at a fluorescence wavelength of 410 nm of the compound (IIc) and the fluorescence intensity (I ₄₈₅ ) at a fluorescence wavelength of 485 nm of the metal complex of the compound (IIc) were measured, and the fluorescence intensity ratio (I ₄₈₅ / I ₄₁₀ ) was calculated.

Similarly, sodium ion, potassium ion, calcium ion, or magnesium ion is added to 100 mM HEPES buffer solution (pH 7.2, containing 5 μM EDTA) containing 10 μM compound (IIc) until the concentration reaches 5 mM, respectively. The fluorescence intensity (I ₄₁₀ ) at a fluorescence wavelength of 410 nm of the compound (IIc) and the fluorescence intensity (I ₄₈₅ ) at a fluorescence wavelength of 485 nm of the metal complex of the compound (IIc) were measured, and the fluorescence intensity ratio (I ₄₈₅ / _I410 ).

The results are shown in FIG. 4 (without Zn ²⁺ ).

  Compound (IIc) shows a high ratio change (fluorescence intensity ratio) when zinc ion is added, but no change in the ratio is observed when other ions present in the living body are added, or Fluorescence quenching was observed. From this, it was found that the compound (IIc) shows high selectivity for zinc ions.

Next, zinc ions were added to each solution prepared above so as to be 50 μM, and the spectrum was measured under the same measurement conditions. The results are shown in FIG. 4 (with Zn ²⁺ ). According to this, by adding zinc ions to each solution, the same fluorescence intensity ratio of the zinc complex as when only the above zinc ions were added was obtained. From this, it was found that when compound (IIc) was used, the ratio of zinc ions could be measured without being influenced by other metal ions.

Claims (24)

Formula (I):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, Z is a single bond or a formula:

A represents a benzene ring which may have a substituent, and Y represents a group represented by -O- or -S-. )
Or a salt thereof.   In the general formula (I), Ar is an alkyl group, alkoxy group, hydroxyl group, amino group, mono- or dialkylamino group, mono- or di (hydroxyalkyl) amino group, carboxyl group, alkoxycarbonyl group, carboxyalkyl group, alkoxycarbonylalkyl. The compound or a salt thereof according to claim 1, which is an aryl group substituted with at least one group selected from the group consisting of a group, a carboxyalkoxy group and an alkoxycarbonylalkoxy group.   In the general formula (I), Ar is one group selected from the group consisting of a phenyl group, a toluyl group, a naphthyl group, a carboxyalkoxy group-substituted phenyl group and a 5- (dimethylamino) -1-naphthyl group. 2. The compound according to 2, or a salt thereof.   In general formula (I), X and Y are groups shown by -O-, n is 2, and A is a benzene ring, The compound or salt in any one of Claims 1-3. In the general formula (I), R ¹ and R ² are the same or different, and at least one selected from the group consisting of a nitrogen-containing heteroaryl group, a carboxyl group, a hydroxyl group, an amino group, a mono- or dialkylamino group and an alkoxy group The compound or a salt thereof according to any one of claims 1 to 4, which is an alkyl group substituted with a group. General formula (II):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. The benzene ring, R ¹ and R ² may be the same or different and each may have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a heteroaryl group which may have a substituent; Show.)
Or a salt thereof. General formula (III):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An alkyl group that may have, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent.
Or a salt thereof. General formula (II):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. The benzene ring, R ¹ and R ² may be the same or different and each may have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a heteroaryl group which may have a substituent; Show.)
Wherein the compound represented by the general formula (16):

(In the formula, R ³ represents an alkyl group, and Ar, X, Y, n, A, R ¹ and R ² are the same as above.)
The ester compound represented by this is hydrolyzed, The manufacturing method characterized by the above-mentioned. Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
The ester compound represented by these. Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A method for producing an ester compound represented by general formula (14):

(In the formula, R ³ represents an alkyl group, and Ar, X, Y, n and A are the same as above.)
An aldehyde compound represented by the general formula (15):
HN (R ¹ ) (R ² ) (15)
(In the formula, R ¹ and R ² are the same or different and each represents an alkyl group which may have a substituent.)
The manufacturing method characterized by making the amine compound represented by these react. General formula (14):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ³ represents an alkyl group.)
An aldehyde compound represented by General formula (6):

Wherein X and Y are the same or different and are represented by —O— or —S—, n is 2 or 3, A is a benzene ring which may have a substituent, and m is 1 or 2. )
A compound represented by Formula (16):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A method for producing an ester compound represented by the general formula (23):

(In the formula, Ar, X, Y, n, A, R ¹ , R ² and R ³ are the same as above.)
And a trialkyl phosphite. Formula (23):

(In the formula, Ar is an aryl group which may have a substituent, X and Y are the same or different and are represented by -O- or -S-, n is 2 or 3, and A has a substituent. An optionally substituted benzene ring, R ¹ and R ² may be the same or different and may have a substituent, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent, R ³ represents an alkyl group.)
A compound represented by General formula (19):

(Wherein X and Y are the same or different and are represented by —O— or —S—, n is 2 or 3, and A represents a benzene ring which may have a substituent)
A compound represented by General formula (III):

(In the formula, Ar is an aryl group which may have a substituent, X is a group represented by —O— or —S—, n is 2 or 3, and R ¹ and R ² are the same or different and each represents a substituent. An alkyl group that may have, an aryl group that may have a substituent, or a heteroaryl group that may have a substituent.
Or a salt thereof, which is represented by the general formula (34):

(In the formula, R ³ represents an alkyl group, and Ar, X, n, R ¹ and R ² are the same as above.)
The ester compound represented by this is hydrolyzed, The manufacturing method characterized by the above-mentioned.   The zinc fluorescent probe containing the compound or its salt in any one of the said Claims 1-7.   A reagent for measuring zinc ions, comprising the compound or salt thereof according to any one of claims 1 to 7.   The zinc complex containing the compound and zinc ion in any one of the said Claims 1-7. A method for measuring zinc ions,
(A) a step of reacting the compound according to any one of claims 1 to 7 or a salt thereof with a zinc ion to form a zinc complex; and (b) measuring the fluorescence intensity of the zinc complex formed in (a) above. A measuring method including a step of performing.   In the step (b), the light of a single excitation wavelength is irradiated, and the fluorescence intensity of each of the compound according to any one of claims 1 to 7 or a salt thereof and the zinc complex formed in the above (a) is measured. The measurement method according to claim 20.   The measurement method according to claim 20, wherein the excitation wavelength is 340 to 400 nm.   In the step (b), the difference between the peak wavelength of the fluorescence spectrum of the compound or salt thereof according to any one of claims 1 to 7 and the peak wavelength of the fluorescence spectrum of the zinc complex formed in the above (a) is 20 nm. It is the above, The measuring method of Claim 20, 21 or 22. A method for measuring intracellular zinc ions,
(A) a step of incorporating the compound or salt thereof according to any one of claims 1 to 7 into a cell; and (b) the compound or salt thereof incorporated into the cell in (a) above and an intracellular zinc ion. Measuring method including the process of measuring each fluorescence intensity of the zinc complex produced | generated from.

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