WO2018159810A1 - Fluorescent probe for detecting alkaline phosphatase, and use for same - Google Patents

Abstract

Provided is a fluorescent probe for detecting alkaline phosphatase. The fluorescent probe is suitable for measurements that require high sensitivity and quantitativity. A fluorescent probe for detecting alkaline phosphatase, the fluorescent probe including a compound that has an anionic functional group and a phosphate group.

Description

Fluorescent probe for alkaline phosphatase detection and use thereof

The present invention relates to a fluorescent probe for detecting alkaline phosphatase and its use. Specifically, the present invention relates to a compound, a fluorescent probe for detecting alkaline phosphatase, a microdevice, and a method for detecting the enzymatic activity of alkaline phosphatase in a biological sample. This application claims priority on March 3, 2017 based on Japanese Patent Application No. 2017-041149 filed in Japan, the contents of which are incorporated herein by reference.

The method for detecting the enzyme activity of a single enzyme using a microdevice is to clarify individual biochemical parameters of the enzyme, to detect the presence of a specific protein with high sensitivity using the enzyme as a reporter protein, etc. It is widely used for the purpose. In recent years, high-sensitivity observation of enzyme activity in biological samples showing abnormal values in connection with specific diseases is expected to be applied to diagnosis of pathological conditions.

As a typical research example of a method for detecting enzyme activity using a microdevice, there is a research on alkaline phosphatase (ALP). ALP has so far been used as a reporter protein for detecting a specific protein with high sensitivity using a microdevice. Further, it is known that abnormalities of ALP activity in vivo, particularly in blood, serve as diagnostic markers for many diseases. Therefore, it is expected to establish a new method for diagnosing a disease state by detecting ALP activity in a biological sample with high sensitivity.

The present inventors have so far developed a fluorescent probe for detecting ALP shown in Patent Document 1.

International Publication No. 2006/093067

Conventional fluorescent probes for detecting ALP, such as the fluorescent probe described in Patent Document 1, have high fat solubility. Therefore, in the method for detecting enzyme activity using a multi-well chamber type micro device, when encapsulating the device, the fluorescent probe leaks from the aqueous solution in the well of the device into the organic solvent used for sealing outside the device. This is a problem that is not suitable for measurement requiring high quantitativeness and sensitivity.

The present invention has been made in view of the above circumstances, and provides a fluorescent probe for ALP detection suitable for measurement requiring high quantitativeness and sensitivity.

As a result of intensive studies to achieve the above-mentioned object, the present inventors leaked the fluorescent probe out of the microdevice by adding an anionic functional group to the compound used in the conventional fluorescent probe for ALP detection. It was found that a fluorescent probe for ALP detection suitable for measurement requiring high quantitativeness and sensitivity was obtained, and the present invention was completed.

That is, the present invention includes the following aspects.
The fluorescent probe for detecting alkaline phosphatase according to the first aspect of the present invention includes a compound having an anionic functional group and a phosphate group.
In the fluorescent probe for detecting alkaline phosphatase according to the first aspect, the anionic functional group may be a carboxy group, a sulfonic acid group, or a phosphoric acid group.
In the fluorescent probe for detecting alkaline phosphatase according to the first aspect, the compound may be a compound represented by the following general formula (1), (2), (3) or (4).

In the general formula (1), R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, or a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Any one of R ¹¹ , R ¹² and R ¹³ is a group having an anionic functional group at the terminal. R ¹⁴ and R ¹⁵ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms. Y ¹¹ represents a single _{bond, -O- (CH 2) n11 -} , - O- (CH 2) n12 -Ar 11 -, - NH- (CH 2) n13 -, or, -NH- _{(CH 2) n14} - Ar ¹² - is. n11, n12, n13 and n14 are each independently an integer of 1 to 10. Ar ¹¹ and Ar ¹² are each independently a substituted or unsubstituted arylene group.

In the general formula (2), R ²¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ²¹ may be the same as or different from each other. R ²² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ²² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. X ²¹ is an oxygen atom or N ⁺ HR ′. R ′ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²¹ represents a single _{bond, -O- (CH 2) n21 -} , - O- (CH 2) n22 -Ar 21 -, - NH- (CH 2) n23 -, or, -NH- _{(CH 2) n24} - Ar ²² - is. n21, n22, n23 and n24 are each independently an integer of 1 to 10. Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.

In the general formula (3), R ³¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ³¹ may be the same as or different from each other. R ³² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ³² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. X ³² is an oxygen atom or N ⁺ HR ″. R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ³¹ represents a single _{bond, -O- (CH 2) n31 -} , - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, or, -NH- _{(CH 2) n34} - Ar ³² —. n31, n32, n33 and n34 are each independently an integer of 1 to 10. Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.

In the general formula (4), R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ⁴¹ may be the same as or different from each other. R ⁴² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ⁴² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁵ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴¹ represents a single _{bond, -O- (CH 2) n41 -} , - O- (CH 2) n42 -Ar 41 -, - NH- (CH 2) n43 -, or, -NH- _{(CH 2) n44} - Ar ⁴² —. n41, n42, n43 and n44 are each independently an integer of 1 to 10. Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group.

In the fluorescent probe for detecting alkaline phosphatase according to the first aspect, the compound is represented by the following general formulas (1-1), (1-2), (2-1), (2-2), (3-1), It may be a compound represented by (3-2), (4-1) or (4-2).

(In the general formula (1-1), R ¹¹¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, where n111 and n112 are each independently an integer of 1 to 10 Ar ¹¹¹ is a substituted or unsubstituted arylene group.
In the general formula (1-2), R ¹²¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —. n121 and n122 are each independently an integer of 1 to 10. Ar ¹²¹ is a substituted or unsubstituted arylene group. )

(In General Formula (2-1), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ²¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or a carbon number of Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, wherein n ²¹¹ and n 212 are each independently 1 to ^{.Ar 211} is an integer of 10 is a substituted or unsubstituted arylene group.
In general formula (2-2), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. R ²²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. )

(In the general formula (3-1), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ³¹³ is a group having an anionic functional group at its terminal. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms X ³¹ is a silicon atom, phosphorus atom, germanium atom or tin it is an atomic ^{.Y 311} is a single _{bond, -O- (CH 2) n311 -} , ^{_{or, -O- (CH 2) n312 -Ar}} 311 - a is .n311 and N312 are each independently ^{.Ar 311} is an integer of 1-10 is a substituted or unsubstituted arylene group.
In general formula (3-2), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. R ³²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. )

(In General Formula (4-1), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are Each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. The .n411 and n412 is the ^{.Ar 411} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. )

In the fluorescent probe for detecting alkaline phosphatase according to the first aspect, the compound is represented by the following general formulas (1-1-1), (1-1-2), (1-1-3), (1-2-1). ), (1-2-2), (1-2-3), (2-1-1), (2-1-2), (2-1-3), (2-2-1), (2-2-2), (2-2-3), (3-1-1), (3-1-2), (3-1-3), (3-2-1), (3 -2-2), (3-2-2), (4-1-1), (4-1-2), (4-1-3), (4-2-1), (4-2 -2) or (4-2-3).

(In the general formulas (1-1-1) to (1-2-3), Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —. N111 and n112 are each independently an integer of 1 to 10. Ar ¹¹¹ is a substituted or unsubstituted arylene group, Y ¹²¹ is —NH— (CH ₂ ) _n121 —, or —NH— ( CH ₂ ) _n122 —Ar ¹²¹ —, where n121 and n122 are each independently an integer of 1 to 10. Ar ¹²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (2-1-1) to (2-2-3), Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —. the .n211 and n212 is the ^{.Ar 211} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group ^{.Y 221} is a single _{bond, -NH- (CH 2) n221 -} , or - NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (3-1-1) to (3-2-1), Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —. N311 and n312 are each independently an integer of 1 to 10. Ar ³¹¹ is a substituted or unsubstituted arylene group, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or — NH— (CH ₂ ) _n322 —Ar ³²¹ —, wherein n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (4-1-1) to (4-2-3), Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. N411 and n412 are each independently an integer of 1 to 10. Ar ⁴¹¹ is a substituted or unsubstituted arylene group, Y ⁴²¹ is —NH— (CH ₂ ) _n421 —, or —NH— ( CH ₂ ) _n422 —Ar ⁴²¹ —, where n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group.

The alkaline phosphatase detection fluorescent probe according to the first aspect may be for a micro device.

A microdevice according to a second aspect of the present invention includes the fluorescent probe for detecting alkaline phosphatase according to the first aspect.
The microdevice according to the second aspect may include one kind of the fluorescent probe for detecting alkaline phosphatase in one well of the microdevice.
The microdevice according to the second aspect may include two or more fluorescent probes for detecting alkaline phosphatase having different reaction points in one well of the microdevice and having different fluorescence wavelengths.

The method for detecting the enzymatic activity of alkaline phosphatase according to the third aspect of the present invention is a method using the microdevice according to the second aspect.

The compound according to the fourth aspect of the present invention is a compound represented by the following general formula (2).

(In the general formula (2), R ²¹ represents 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ²¹ may be the same as each other. , good .R ²² be different a 1-2 monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ²² there are a plurality of mutually identical The anionic functional group may be any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ²³ , R ²⁴ , R ^25, and R ²⁶ is independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, X ²¹ is an oxygen atom or N ⁺ HR ′, and R ′ is a hydrogen atom or carbon atom having 1 to 10 carbon atoms. Y ²¹ is a single bond, —O— ( _{CH 2) n21 -, - O-} (CH 2) n22 -Ar 21 -, - NH- (CH 2) n23 -, ^{_{or, -NH- (CH 2) n24 -Ar}} 22 - are as .n21, n22, n23 and n24 are each independently an integer of 1 to 10. Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.)

The compound according to the fourth aspect may be a compound represented by the following general formula (2-1) or (2-2).

(In General Formula (2-1), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ²¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, wherein n ²¹¹ and n 212 are each independently 1 to ^{.Ar 211} is an integer of 10 is a substituted or unsubstituted arylene group.
In general formula (2-2), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. R ²²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. )

The compound according to the fourth aspect includes the following general formulas (2-1-1), (2-1-2), (2-1-3), (2-2-1), (2-2-2) Or a compound represented by (2-2-3).

(In the general formulas (2-1-1) to (2-2-3), Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —. the .n211 and n212 is the ^{.Ar 211} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group ^{.Y 221} is a single _{bond, -NH- (CH 2) n221 -} , or - NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group.

The compound according to the fifth aspect of the present invention is a compound represented by the following general formula (3).

(In the general formula (3), R ³¹ is 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ³¹ may be the same as each other. may .R ³² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ³² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ³³ , R ³⁴ , R ³⁷ and R ³⁸ is each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom , Phosphorus atom, germanium atom or tin atom. X ³² is an oxygen atom or N ⁺ HR ″. R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ³¹ is a single bond, —O _{- (CH 2) n31 -,} - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, ^{_{or, -NH- (CH 2) n34 -Ar}} 32 - are as .N31, n32, n33 and n34 are each independently an integer of 1 to 10. Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.)

The compound according to the fifth aspect may be a compound represented by the following general formula (3-1) or (3-2).

(In the general formula (3-1), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ³¹³ is a group having an anionic functional group at its terminal. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms X ³¹ is a silicon atom, phosphorus atom, germanium atom or tin it is an atomic ^{.Y 311} is a single _{bond, -O- (CH 2) n311 -} , ^{_{or, -O- (CH 2) n312 -Ar}} 311 - a is .n311 and N312 are each independently ^{.Ar 311} is an integer of 1-10 is a substituted or unsubstituted arylene group.
In general formula (3-2), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. R ³²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. )

In the compound according to the fifth aspect, the X ³¹ may be a silicon atom.
The compound according to the fifth aspect includes the following general formulas (3-1-1), (3-1-2), (3-1-3), (3-2-1), (3-2-2) Alternatively, it may be a compound represented by (3-2-3).

(In the general formulas (3-1-1) to (3-2-1), Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —. N311 and n312 are each independently an integer of 1 to 10. Ar ³¹¹ is a substituted or unsubstituted arylene group, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or — NH— (CH ₂ ) _n322 —Ar ³²¹ —, wherein n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group.

The compound according to the sixth aspect of the present invention is a compound represented by the following general formula (4).

(In the general formula (4), R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ⁴¹ may be the same as each other. may .R ⁴² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ⁴² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, and R ⁴³ is a hydrogen atom or a carbon number of 1. R ⁴⁴ , R ⁴⁵ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently a carbon number. 1 to 10 alkyl groups or carbon 6-10 aryl group ^{.X 41} silicon atom, a phosphorus atom, a germanium atom or a tin atom ^{.Y 41} is a single _{bond, -O- (CH 2) n41 -} , - O- (CH 2) n42 —Ar ⁴¹ —, —NH— (CH ₂ ) _n43 —, or —NH— (CH ₂ ) _n44 —Ar ⁴² —, wherein n41, n42, n43 and n44 are each independently an integer of 1 to 10. Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group.)

The compound according to the sixth aspect may be a compound represented by the following general formula (4-1) or (4-2).

(In General Formula (4-1), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are Each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. The .n411 and n412 is the ^{.Ar 411} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. )

In the compound according to the sixth aspect, X ⁴¹ may be a silicon atom.
The compound according to the sixth aspect includes the following general formulas (4-1-1), (4-1-2), (4-1-3), (4-2-1), (4-2-2) Or a compound represented by (4-2-3).

(In the general formulas (4-1-1) to (4-2-3), Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. N411 and n412 are each independently an integer of 1 to 10. Ar ⁴¹¹ is a substituted or unsubstituted arylene group, Y ⁴²¹ is —NH— (CH ₂ ) _n421 —, or —NH— ( CH ₂ ) _n422 —Ar ⁴²¹ —, where n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group.

The ALP detection fluorescent probe of the above aspect is suitable for measurement requiring high quantitativeness and sensitivity.

1 is a perspective view schematically showing a microdevice according to an embodiment of the present invention. It is sectional drawing which compared the microdevice which concerns on one Embodiment of this invention, and the microdevice provided with the conventional fluorescence probe. It is the graph (conceptual figure) which compared the result of having detected the enzyme activity of the alkaline phosphatase in the biological sample derived from the healthy subject and the patient who has a specific disease using the microdevice which concerns on one Embodiment of this invention. 2 is a graph showing optical characteristics (absorbance) of Compound (1-1-1a) -1 in Production Example 1. 6 is a graph showing optical characteristics (fluorescence intensity) of Compound (2-1-2a) -1 in Production Example 2. It is the image which image | photographed the microdevice 22 minutes after the fluorescence measurement start by the alkaline phosphatase fluorescence assay using the compound (1-1-1a) or the compound (10) in Test Example 1, and 132 minutes after with the fluorescence microscope. . 2 is a graph showing the fluorescence intensity 132 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (1-1-1a) or the compound (10) in Test Example 1. FIG. 2 is an image obtained by photographing a microdevice after 22 minutes and 132 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using Compound (2-1-2a) or Compound (20) in Test Example 1. . 3 is a graph showing the fluorescence intensity 132 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (2-1-2a) or the compound (20) in Test Example 1. 6 is a graph showing optical characteristics (absorbance) of compound (3-1-2a) -1 in Production Example 5. 6 is a graph showing optical characteristics (fluorescence intensity) of compound (3-1-2a) -1 in Production Example 5. It is a graph which shows the fluorescence intensity 5 minutes after the fluorescence measurement start by the alkaline phosphatase fluorescence assay using the compound (3-1-2a) in Test Example 2. 10 is a graph showing the fluorescence intensity 10 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (1-1-1b) in Test Example 3. 6 is a graph showing the fluorescence intensity 10 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (2-1-2b) in Test Example 3. 6 is a graph showing the fluorescence intensity 10 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (3-1-2b) in Test Example 3. 10 is a graph showing the fluorescence intensity 10 minutes after the start of fluorescence measurement in an alkaline phosphatase fluorescence assay using the compound (2-1-2c) in Test Example 4.

Hereinafter, the present invention will be described in detail.
In the present specification, the unit of concentration “M” means “mol / L”.

<< Compound (1) >>
The compound according to the first embodiment of the present invention is represented by the following general formula (1) (in this specification, it may be referred to as “compound (1)”).

(In the general formula (1), R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, or a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, and any one of R ¹¹ , R ¹² and R ¹³ is an anionic functional group at the terminal. R ¹⁴ and R ¹⁵ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms Y ¹¹ is a single bond, —O— (CH ₂ ) _n11 — ^{_{, -O- (CH 2) n12 -Ar}} 11 -, - NH- (CH 2) n13 -, ^{_{or, -NH- (CH 2) n14 -Ar}} 12 - are as .n11, n12, n13 and n14 it The And each independently represents an integer of 1 to 10. Ar ¹¹ and Ar ¹² are each independently a substituted or unsubstituted arylene group.

Compound (1) is a coumarin derivative, and is a compound having a phosphate group that is hydrolyzed by alkaline phosphatase (ALP) under alkaline conditions.
Compound (1) is a fluorescent compound that emits fluorescence when a phosphate group is eliminated by hydrolysis with ALP.

In the present specification, the “derivative” means that one or more hydrogen atoms of the original compound are substituted with a group other than a hydrogen atom (substituent), or one or more carbons of the original compound. This means that an atom is substituted alone or together with a hydrogen atom bonded to this carbon atom, is substituted with another group (substituent).

<R ¹¹ , R ¹² and R ¹³ >
In the general formula (1), R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, or a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.
Any one of R ¹¹ , R ¹² and R ¹³ is a group having an anionic functional group at the terminal. In particular, the maximum absorption wavelength of compound (1) (non-dissociation type (neutral type)) and compound (1) -1 (dissociation type (anion type)) after elimination of a phosphate group, which will be described later, greatly deviate. Therefore, R ¹³ is preferably a group having an anionic functional group at the terminal.

In addition, the “group having an anionic functional group at the end” in the present specification may consist of, for example, a group consisting only of an anionic functional group, or an anionic functional group bonded to the linker Y ′. It may be a group. Examples of the linker Y ′ include an alkylene group having 1 to 10 carbon atoms which may contain —O— or —NH—.
Among them, the “group having an anionic functional group at the terminal” in the general formula (1) is preferably a group consisting of only an anionic functional group because it is easy to synthesize.

As said halogen atom in R <^11> , R < ¹² > and R < ¹³ >, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom etc. are mentioned, for example. Especially, it is preferable that the said halogen atom in R < ¹¹ >, R < ¹² > and R < ¹³ > is a chlorine atom, a bromine atom, or an iodine atom.

The alkyl group having 1 to 10 carbon atoms in R ¹¹ , R ¹² and R ¹³ may be linear or branched. Specific examples of the alkyl group having 1 to 10 carbon atoms include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n -Pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethyl Butyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3,3-dimethylpentyl Group, 3-ethylpentyl group, 2,2,3-trimethylbutyl group, n-octyl group, isooctyl group, 2-ethylhexyl group, nonyl group, Sill group, and the like. Among them, the alkyl group having 1 to 10 carbon atoms in R ¹¹ , R ¹² and R ¹³ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in general formula (1), R ¹³ is preferably a group having an anionic functional group at the terminal.
Among them, in general formula (1), R ¹¹ and R ¹² are preferably the same because they are easily synthesized, and R ¹¹ and R ¹² are more preferably hydrogen atoms.

^{<R 14} and ^{R 15>}
Examples of the halogen atom in R ¹⁴ and R ¹⁵ include the same ones as exemplified in the above-mentioned “<R ¹¹ , R ¹² , and R ¹³ >”. Among them, it is preferable that the halogen atom in R ¹⁴ and R ¹⁵ is chlorine atom, bromine atom or iodine atom.

Examples of the alkyl group having 1 to 10 carbon atoms for R ¹⁴ and R ¹⁵ include the same groups as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ¹⁴ and R ¹⁵ is preferably a linear group, and more preferably a methyl group or an ethyl group.

Among them, in general formula (1), R ¹⁴ and R ¹⁵ are preferably the same because they are easily synthesized, and R ¹¹ and R ¹² are more preferably hydrogen atoms.

<Y ¹¹ >
In the general formula (1), Y ¹¹ is a single bond, —O— (CH ₂ ) _n11 —, —O— (CH ₂ ) _n12 —Ar ¹¹ —, —NH— (CH ₂ ) _n13 —, or —NH — (CH ₂ ) _n14 —Ar ¹² —. In Y ¹¹ , the bond opposite to the alkylene group of —O— or —NH— is bonded to the carbon atom constituting the coumarin ring in the general formula (1). In addition, a bond opposite to the oxygen atom (O), amino group (NH), or alkylene group of — (CH ₂ ) _n11 —, —Ar ¹¹ —, — (CH ₂ ) _n13 —, or —Ar ¹² — It is bonded to the phosphate group in formula (1).
n11, n12, n13 and n14 are each independently an integer of 1 to 10.
Ar ¹¹ and Ar ¹² are each independently a substituted or unsubstituted arylene group.

n11, n12, n13 and n14 are the number of repetitions of the alkylene group for each ^{Y 11.} n11, n12, n13 and n14 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .

Ar ¹¹ and Ar ¹² are each independently a substituted or unsubstituted arylene group.
The unsubstituted arylene group in Ar ¹¹ and Ar ¹² is preferably one having 6 to 14 carbon atoms, and specific examples include a phenylene group and a naphthylene group. Among them, the unsubstituted arylene group in Ar ¹¹ and Ar ¹² is preferably a phenylene group.
Examples of the substituent that the arylene group has include a halogen atom and an alkyl group having 1 to 10 carbon atoms.
Examples of the halogen atom are the same as those exemplified above in “<R ¹¹ , R ¹² , and R ¹³ >”. Among these, the halogen atom is preferably a chlorine atom, a bromine atom or an iodine atom.
Examples of the alkyl group having 1 to 10 carbon atoms include the same groups as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among these, the alkyl group having 1 to 10 carbon atoms is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in the general formula (1), Y ¹¹ is a single bond, —O—CH ₂ —, —O— (CH ₂ ) ₂ —, —O—CH ₂ —Ph—, —NH—CH ₂ —, —NH It is preferably — (CH ₂ ) ₂ — or —NH—CH ₂ —Ph—. Here, “Ph” represents a substituted or unsubstituted phenylene group.

Preferred examples of the compound (1) include a compound represented by the following general formula (1-1) (hereinafter sometimes abbreviated as “compound (1-1)”), or a compound represented by the following general formula ( 1-2) (hereinafter sometimes abbreviated as “compound (1-2)”) and the like.
In addition, these compounds are only examples of a preferable compound (1), and a preferable compound (1) is not limited to these.

(In the general formula (1-1), R ¹¹¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, where n111 and n112 are each independently an integer of 1 to 10 Ar ¹¹¹ is a substituted or unsubstituted arylene group.
In the general formula (1-2), R ¹²¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —. n121 and n122 are each independently an integer of 1 to 10. Ar ¹²¹ is a substituted or unsubstituted arylene group. )

<R ¹¹¹ and R ¹²¹ >
R ¹¹¹ and R ¹²¹ are each independently an anionic functional group selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.

< ^Y111 and ^Y121 >
Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —. Is a substituted or unsubstituted arylene group.
Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —. Ar ¹²¹ is a substituted or unsubstituted arylene group.
n111, n112, n121 and n122 are the number of repetitions of the alkylene group for ^{Y 111} and ^{Y 121,} respectively. n111, n112, n121 and n122 are each preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .
Ar ¹¹¹ and Ar ¹²¹ are each independently a substituted or unsubstituted arylene group. Examples of the substituted or unsubstituted arylene group include the same groups as those exemplified above for “<Y ¹¹ >”.

Preferred examples of the compound (1-1) include, for example, R ¹¹¹ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O ^{_{- (CH 2) n112 -Ar 111}} - a is an integer of 1 ~ 8 n111 and n112 are each ^{independently, Ar 111} can be cited, such as those substituted or unsubstituted phenylene group.
More preferable examples of the compound (1-1) include, for example, R ¹¹¹ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or — O— (CH ₂ ) _n112 —Ar ¹¹¹ —, wherein n111 and n112 are each independently an integer of 1 to 6, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.

Preferred examples of the compound (1-2) include, for example, R ¹²¹ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH _{2) n122} -Ar ¹²¹ - a is an integer of 1 ~ 8 n121 and n122 are each ^{independently, Ar 121} can be cited, such as those substituted or unsubstituted phenylene group.
More preferable examples of the compound (1-2) include, for example, R ¹²¹ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— ( CH ₂ ) _n122 —Ar ¹²¹ —, wherein n121 and n122 are each independently an integer of 1 to 6, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.

Among the compounds (1), preferred as the compound (1-1) are, for example, compounds represented by the following general formula (1-1-1) (hereinafter referred to as “compound (1-1-1)”). May be abbreviated), a compound represented by the following general formula (1-1-2) (hereinafter may be abbreviated as “compound (1-1-2)”), or a compound represented by the following general formula (1 -1-3) (hereinafter sometimes abbreviated as “compound (1-1-3)”) and the like.
Among the compounds (1), preferred as the compound (1-2) are, for example, compounds represented by the following general formula (1-2-1) (hereinafter referred to as “compound (1-2-1)”). A compound represented by the following general formula (1-2-2) (hereinafter referred to as “compound (1-2-2)”, or represented by the following general formula (1-2-3)). And the like (hereinafter sometimes abbreviated as “compound (1-2-3)”).
In addition, these compounds are only examples of a preferable compound (1), and a preferable compound (1) is not limited to these.

(In general formulas (1-1-1) to (1-2-3), Y ¹¹¹ and Y ¹²¹ are the same as described above.)

As a preferable compound (1-1-1), for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, and n111 And n112 is each independently an integer of 1 to 8, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.
More preferable compounds (1-1-1) include, for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, n111 and n112 are each independently an integer of 1 to 6, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (1-1-2), for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, and n111 And n112 is each independently an integer of 1 to 8, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (1-1-2) is, for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, n111 and n112 are each independently an integer of 1 to 6, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.

Preferred examples of the compound (1-1-3) include, for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, and n111 And n112 is each independently an integer of 1 to 8, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (1-1-3) is, for example, Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, n111 and n112 are each independently an integer of 1 to 6, and Ar ¹¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (1-2-1), for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 are Examples thereof are each independently an integer of 1 to 8, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (1-2-1) is, for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 Are each independently an integer of 1 to 6, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (1-2-2), for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 are Examples thereof are each independently an integer of 1 to 8, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.
As a more preferable compound (1-2-2), for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 Are each independently an integer of 1 to 6, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (1-2-3), for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 are Examples thereof are each independently an integer of 1 to 8, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (1-2-3) is, for example, Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —, and n121 and n122 Are each independently an integer of 1 to 6, and Ar ¹²¹ is a substituted or unsubstituted phenylene group.

Among the compounds (1), preferred as the compound (1-1-1) are, for example, compounds represented by the following formula (1-1-1a) (hereinafter referred to as “compound (1-1-1a)” Or a compound represented by the following formula (1-1-1b) (hereinafter sometimes abbreviated as “compound (1-1-1b)”), or a compound represented by the following formula (1- 1-1c) (hereinafter sometimes abbreviated as “compound (1-1-1c)”) and the like.
Among the compounds (1), preferred as the compound (1-1-2) are, for example, compounds represented by the following formula (1-1-2a) (hereinafter referred to as “compound (1-1-2a)”. Or a compound represented by the following formula (1-1-2b) (hereinafter sometimes abbreviated as “compound (1-1-2b)”), or a compound represented by the following formula (1- 1-2c) (hereinafter, may be abbreviated as “compound (1-1-2c)”) and the like.
Among the compounds (1), preferred as the compound (1-1-3) are, for example, compounds represented by the following formula (1-1-3a) (hereinafter referred to as “compound (1-1-3a)” Or a compound represented by the following formula (1-1-3b) (hereinafter sometimes abbreviated as “compound (1-1-3b)”), or a compound represented by the following formula (1- 1-3c) (hereinafter sometimes abbreviated as “compound (1-1-3c)”) and the like.
Among the compounds (1), preferred as the compound (1-2-1) are, for example, compounds represented by the following formula (1-2-1a) (hereinafter referred to as “compound (1-2-1a)”) Or a compound represented by the following formula (1-2-1b) (hereinafter sometimes abbreviated as “compound (1-2-1b)”), or a compound represented by the following formula (1- 2-1c) (hereinafter sometimes abbreviated as “compound (1-2-1c)”) and the like.
Among the compounds (1), preferred as the compound (1-2-2) are, for example, compounds represented by the following formula (1-2-2a) (hereinafter referred to as “compound (1-2-2a)”. Or a compound represented by the following formula (1-2-2b) (hereinafter sometimes abbreviated as “compound (1-2-2b)”), or a compound represented by the following formula (1- 2-2c) (hereinafter sometimes abbreviated as “compound (1-2-2c)”) and the like.
Among the compounds (1), preferred as the compound (1-2-3) are, for example, compounds represented by the following formula (1-2-3a) (hereinafter referred to as “compound (1-2-3a)”. Or a compound represented by the following formula (1-2-3b) (hereinafter sometimes abbreviated as “compound (1-2-3b)”), or a compound represented by the following formula (1- 2-3c) (hereinafter sometimes abbreviated as “compound (1-2-3c)”) and the like.
In addition, these compounds are only examples of a preferable compound (1), and a preferable compound (1) is not limited to these.

The compound (1) is hydrolyzed by ALP, the phosphate group is eliminated, and the compound (1) is changed into the compound (1) -1, thereby generating blue (fluorescence wavelength: about 350 nm to less than about 450 nm) fluorescence.

The compound (1) of the present embodiment exists as a substantially non-dissociation type (neutral type) compound, but the dissociation type (anion type) is obtained by the elimination of the phosphate group by an enzymatic reaction with ALP. ) Compound (1) -1. Therefore, the compound (1) of the present embodiment can be used as a fluorescent probe for measuring ALP with high sensitivity because the maximum absorption wavelength greatly changes before and after the enzyme reaction with ALP.

<< Method for Producing Compound (1) >>
Compound (1) can be produced, for example, by forming a coumarin skeleton by performing a known reaction according to the types of Y ¹¹ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ . More specifically, it is as follows.

<Method for Producing Compound (1-1)>
Among the compounds (1), the compound (1-1) is, for example, a compound represented by the following general formula (1-1a) (hereinafter sometimes abbreviated as “compound (1-1a)”); A step of reacting 2,4-dihydroxybenzaldehyde to obtain a compound represented by the following general formula (1-1b) (hereinafter sometimes abbreviated as “compound (1-1b)”) (hereinafter referred to as “compound (1-1b)”) (Sometimes abbreviated as “compound (1-1b) production process”), compound (1-1b), and a compound represented by the following general formula (1-1c) (hereinafter referred to as “compound (1-1c)”) To obtain a compound represented by the following general formula (1-1d) (hereinafter sometimes abbreviated as “compound (1-1d)”) (hereinafter referred to as “compound (1-1d)”). , “Compound (1-1d) production process”), and compound (1- Compound from d) (1-1) the step of obtaining (hereinafter, "Compound (1-1) production step" by a production method having sometimes abbreviated as), can be produced.
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group. R ¹¹² has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and (It is an alkylene group having 1 to 10 carbon atoms which may contain at least one of an oxygen atom and an arylene group. Y ¹¹¹ and R ¹¹¹ are both the same as above.)

[Compound (1-1b) Production Process]
In the production step of the compound (1-1b), the compound (1-1a) and 2,4-dihydroxybenzaldehyde are reacted to obtain the compound (1-1b).

(Compound (1-1a))
Compound (1-1a) is a known compound.
In the compound (1-1a), R ¹¹¹ is a group having an anionic functional group at the terminal.
Among them, in the compound (1-1a), R ¹¹¹ is preferably a group consisting only of an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. .

(Compound (1-1b))
Compound (1-1b) is a known compound when R ¹¹¹ is a carboxy group. Further, when R ¹¹¹ is a sulfonic acid group or a phosphoric acid group, it is a novel compound.
In compound (1-1b), R ¹¹¹ is the same as R ¹¹¹ in compound (1-1a).

(Reaction conditions)
In the production process of compound (1-1b), it is preferable to carry out the reaction using a deprotecting agent.
Examples of the deprotecting agent include piperidine.
It is preferable that the usage-amount of the said deprotecting agent is 0.01 times mole amount or more and 0.10 times mole amount or less of the usage-amount of a compound (1a), for example.

In the production process of compound (1-1b), the amount of 2,4-dihydroxybenzaldehyde used is preferably 0.5 to 2.0 times the molar amount of compound (1-1a). .

In the production process of compound (1-1b), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production step of compound (1-1b), the reaction time is preferably 5 hours or more and 25 hours or less, and more preferably 10 hours or more and 20 hours or less.

In the production process of compound (1-1b), after completion of the reaction, the compound (1-1b) may be removed by performing post-treatment as necessary by a known method. That is, as needed, post-treatment operations such as filtration, washing, extraction, pH adjustment, dehydration, concentration, etc. are performed alone or in combination of two or more, and concentration, crystallization, reprecipitation, column chromatography are performed. The compound (1-1b) may be taken out by, for example. In addition, the extracted compound (1-1b) can be used alone or in combination of two or more kinds of operations such as crystallization, reprecipitation, column chromatography, extraction, and stirring and washing of the crystals with a solvent, if necessary. May be purified once or more.
In the production step of compound (1-1b), after completion of the reaction, compound (1-1b) may be used in the next step without taking out, but the yield of the target compound (1-1) is improved. Therefore, it is preferable to take out the compound (1-1b) by the above-mentioned method.

[Compound (1-1d) Production Process]
In the production step of compound (1-1d), compound (1-1b) and compound (1-1c) are reacted to obtain compound (1-1d).

(Compound (1-1c))
Compound (1-1c) is a known compound.
In the compound (1-1c), R ¹¹² has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.

Among these, in the compound (1-1c), R ¹¹² is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (1-1d))
Compound (1-1d) is a novel compound.
In the compound (1-1d), Y ¹¹¹ is the same as that exemplified for “<Y ¹¹¹ and Y ¹²¹ >” in the above “<< Compound (1) >>”, and R ¹¹¹ represents the compound (1-1a). ) ^{Is the} same as R ¹¹¹ in FIG.

(Reaction conditions)
In the production step of compound (1-1d), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamine such as triethylamine and N, N-diisopropylethylamine (DIEA).
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (1-1b).

In the production step of compound (1-1d), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include N, N-dimethyl-4-aminopyridine (DMAP).
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 mol amount or less of the amount of compound (1-1b) used.

In the compound (1-1d) production process, an aprotic solvent is preferably used as a reaction solvent.
The aprotic solvent is not particularly limited. For example, perfluorohexane, α, α, α-trifluorotoluene, pentane, hexane, cyclohexane, methylcyclohexane, decahydronaphthalene, carbon tetrachloride, dioxane, fluorotrichloromethane, benzene , Toluene, triethylamine, carbon disulfide, diisopropyl ether, diethyl ether, t-butyl methyl ether, chloroform, ethyl acetate, 1,2-dimethoxyethane, 2-methoxyethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, methylene chloride Pyridine, 2-butanone, acetone, hexamethylphosphoramide, N-methylpyrrolidinone, nitromethane, dimethylformamide, acetonitrile, sulfolane, dimethyl sulfoxide, di Isopropyl ethyl amine, isopropyl acetate, dichloromethane, dimethylamine, N, N- dimethylformamide, propylene, and the like carbonates.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (1-1b).

In the production process of the compound (1-1d), it is preferable to carry out the reaction in an inert gas atmosphere.
The inert gas is not particularly limited, and examples thereof include nitrogen, helium, neon, argon, krypton, and xenon.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the compound (1-1d) production process, the amount of compound (1-1c) used is preferably 1 to 2 times the amount of compound (1-1b).

In the step of producing compound (1-1d), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (1-1d), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, and more preferably 1 hour or longer and 5 hours or shorter.

Compound (1-1d) can be taken out in the same manner as in the above-mentioned compound (1-1b) production step, and the taken out compound (1-1d) may be further purified in the same manner. Further, the obtained compound (1-1d) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the point that the yield of the target compound (1-1) is improved. Is preferred.

[Compound (1-1) Production Process]
In the production process of the compound (1-1), the compound (1-1) is obtained from the compound (1-1d).
The method for obtaining the compound (1-1) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Even when a protective group is bonded to R ¹¹¹ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (1-1), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (1-1), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (1-1), the reaction time is preferably 30 minutes or longer and 5 hours or shorter, more preferably 1 hour or longer and 3 hours or shorter.

In the production process of compound (1-1), after completion of the reaction, compound (1-1) can be taken out by the same method as in the production process of compound (1-1b), and the taken out compound (1-1 ) May be further purified by the same method.

Each compound such as the compound (1-1), the compound (1-1a), the compound (1-1b), the compound (1-1c), the compound (1-1d), and the like is, for example, nuclear magnetic resonance (NMR) spectroscopy The structure can be confirmed by a known method such as mass spectrometry (MS) or infrared spectroscopy (IR).

<Method for Producing Compound (1-2)>
Among the compounds (1), the compound (1-2) is, for example, a compound represented by the following general formula (1-2a) (hereinafter sometimes abbreviated as “compound (1-2a)”), A step of reacting 2-hydroxy-4-amino-benzaldehyde to obtain a compound represented by the following general formula (1-2b) (hereinafter sometimes abbreviated as “compound (1-2b)”) (Hereinafter may be abbreviated as “compound (1-2b) production process”), compound (1-2b) and a compound represented by the following general formula (1-2c) (hereinafter referred to as “compound (1- 2c) ”) to obtain a compound represented by the following general formula (1-2d) (hereinafter sometimes abbreviated as“ compound (1-2d) ”). Process (hereinafter may be abbreviated as “compound (1-2d) production process”), and compound Compound from (1-2d) (1-2) the step of obtaining (hereinafter, "Compound (1-2) Manufacturing process" may be abbreviated as) by a production method having a can be prepared.
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group. R ¹²² has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and (It is an alkylene group having 1 to 10 carbon atoms that may contain at least one of an oxygen atom and an arylene group. Y ¹²¹ and R ¹²¹ are the same as above.)

[Compound (1-2b) Production Process]
In the step of producing compound (1-2b), compound (1-2a) is reacted with 2,4-dihydroxybenzaldehyde to obtain compound (1-2b).

(Compound (1-2a))
Compound (1-2a) is a known compound.
In the compound (1-2a), R ¹²¹ is an anionic functional group selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.

Compound (1-2b) is a known compound.
In the compound (1-2b), R ¹²¹ is the same as R ¹²¹ in the compound (1-2a).

(Reaction conditions)
In the production process of compound (1-2b), it is preferable to carry out the reaction using a deprotecting agent.
Examples of the deprotecting agent include piperidine.
The amount of the deprotecting agent used is preferably 0.01 to 0.10 times the amount of the compound (1-2a) used.

In the production process of compound (1-2b), the amount of 2-hydroxy-4-amino-benzaldehyde used is 0.5 to 2.0 times the amount of compound (1-2a) used. It is preferable.

In the production step of compound (1-2b), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production step of compound (1-2b), the reaction time is preferably 5 hours or more and 25 hours or less, more preferably 10 hours or more and 20 hours or less.

In the compound (1-2b) production process, after completion of the reaction, the compound (1-2b) can be removed in the same manner as in the above-mentioned compound (1-1b) production process. You may refine by. In addition, the obtained compound (1-2b) may be used in the next step without being removed after the completion of the reaction. However, it should be removed from the viewpoint of improving the yield of the target compound (1-2). Is preferred.

[Compound (1-2d) Production Process]
In the step of producing compound (1-2d), compound (1-2b) and compound (1-2c) are reacted to obtain compound (1-2d).

(Compound (1-2c))
Compound (1-2c) is a known compound.
In the compound (1-2c), R ¹²² has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.

Among these, in the compound (1-2c), R ¹¹² is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (1-2d))
Compound (1-2d) is a novel compound.
In the compound (1-2d), Y ¹²¹ is the same as those exemplified for “<Y ¹¹¹ and Y ¹²¹ >” in the above “<< Compound (1) >>”, and R ¹²¹ represents the compound (1-2a). ) ^{Is the} same as R ¹²¹ in FIG.

(Reaction conditions)
In the production step of compound (1-2d), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (1-2b) used.

In the production step of compound (1-2d), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of compound (1-2b) used.

In the production process of compound (1-2d), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (1-2b).

In the production process of the compound (1-2d), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (1-2d), the amount of compound (1-2c) used is preferably 1 to 2 times the amount of compound (1-2b).

In the production process of compound (1-2d), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (1-2d), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, and more preferably 1 hour or longer and 5 hours or shorter.

Compound (1-2d) can be taken out in the same manner as in the above-mentioned compound (1-1b) production step, and the taken out compound (1-2d) may be further purified in the same manner. Further, the obtained compound (1-2d) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the point that the yield of the target compound (1-2) is improved. Is preferred.

[Compound (1-2) Production Process]
In the production step of the compound (1-2), the compound (1-2) is obtained from the compound (1-2d).
The method for obtaining the compound (1-2) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Even when a protective group is bonded to R ¹²¹ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (1-2), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (1-2), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (1-2), the reaction time is preferably 30 minutes or longer and 5 hours or shorter, more preferably 1 hour or longer and 3 hours or shorter.

In the production process of compound (1-2), after completion of the reaction, compound (1-2) can be taken out in the same manner as in the production process of compound (1-1b), and the taken out compound (1-2) ) May be further purified by the same method.

Each compound such as the compound (1-2), the compound (1-2a), the compound (1-2b), the compound (1-2c), the compound (1-2d) and the like can be obtained by, for example, nuclear magnetic resonance (NMR) spectroscopy. The structure can be confirmed by a known method such as mass spectrometry (MS) or infrared spectroscopy (IR).

≪Compound (2) ≫
The compound according to the second embodiment of the present invention is represented by the following general formula (2) (in this specification, sometimes referred to as “compound (2)”).

(In the general formula (2), R ²¹ represents 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ²¹ may be the same as each other. , good .R ²² be different a 1-2 monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ²² there are a plurality of mutually identical The anionic functional group may be any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ²³ , R ²⁴ , R ^25, and R ²⁶ is independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, X ²¹ is an oxygen atom or N ⁺ HR ′, and R ′ is a hydrogen atom or carbon atom having 1 to 10 carbon atoms. Y ²¹ is a single bond, —O— ( _{CH 2) n21 -, - O-} (CH 2) n22 -Ar 21 -, - NH- (CH 2) n23 -, ^{_{or, -NH- (CH 2) n24 -Ar}} 22 - are as .n21, n22, n23 and n24 are each independently an integer of 1 to 10. Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.)

Compound (2) is a fluorescein derivative or a rhodamine derivative, and is a compound having a phosphate group that is hydrolyzed by ALP under alkaline conditions.
Compound (2) is a fluorescent compound that emits fluorescence when a phosphate group is eliminated by hydrolysis with ALP.

<R ²¹ >
R ²¹ is 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group.
The “electron donating group” in the present specification may be any substituent that can donate electrons to the benzene ring. Specific examples include, but are not limited to, a hydroxyl group, an alkoxy group having 1 to 10 carbon atoms, an amino group, an alkylamino group having 1 to 10 carbon atoms, and the like.
The number of R ²¹ is 1 or 2, and is preferably 2. When two R ²¹ are present, they may be the same as or different from each other. Among these, when there are two R ^{21 s} , it is preferable that they are the same as each other because they are easily synthesized.

The alkoxy group having 1 to 10 carbon atoms in R ²¹ may have a structure in which a linear or branched alkyl group having 1 to 10 carbon atoms is bonded to an oxygen atom. Specific examples of the alkoxy group having 1 to 10 carbon atoms include, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentoxy group, isopentoxy group, neopentoxy group, tert-pentoxy group, 1-methylbutoxy group, n-hexoxy group, 2-methylpentoxy group, 3-methylpentoxy group, 2,2-dimethylbutoxy group, 2 , 3-dimethylbutoxy group, n-heptoxy group, 2-methylhexoxy group, 3-methylhexoxy group, 2,2-dimethylpentoxy group, 2,3-dimethylpentoxy group, 2,4-dimethylpentoxy group, 3 , 3-dimethylpentoxy group, 3-ethylpentoxy group, 2,2,3-trimethylbutoxy group, n-octyl Alkoxy group, Isookutokishi group, 2-Echiruhekitokishi group, Noninokishi group, decyloxy group and the like. Among these, the alkoxy group having 1 to 10 carbon atoms in R ²¹ is preferably a straight chain, and more preferably a methoxy group or an ethoxy group.

The alkylamino group having 1 to 10 carbon atoms in R ²¹ may have a structure in which a linear or branched alkyl group having 1 to 10 carbon atoms is bonded to an amino group. Specific examples of the alkylamino group having 1 to 10 carbon atoms include a methylamino group, an ethylamino group, an isopropylamino group, a dimethylamino group, a diethylamino group, and a diisopropylamino group. Among them, the alkylamino group having 1 to 10 carbon atoms in R ²¹ is preferably a straight chain, and more preferably a methylamino group or an ethylamino group.

Among these, in general formula (2), R ²¹ is preferably a straight-chain alkoxy group having 1 to 10 carbon atoms or a straight-chain alkylamino group having 1 to 10 carbon atoms because it is highly hydrophilic. A group, an ethylamino group, a methoxy group, or an ethoxy group is more preferable.

Further, in the general formula ^(2), it is preferred ^{that R 21} is two. The two R ²¹ positions in the benzene ring are preferably arranged at positions that are ortho positions relative to each other.

<R ²² >
R ²² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. Among them, the “group having an anionic functional group at the terminal” in the general formula (2) is preferably a group consisting of only an anionic functional group because it is easy to synthesize.
The number of R ²² is 1 or 2, and is preferably 1. When there are two R ^{22 s} , they may be the same or different. Among these, when there are two R ^{22 s} , they are preferably the same as each other because they are easily synthesized.

In particular, the maximum absorption wavelength of compound (2) (non-dissociation type (neutral type)) and compound (2) -1 (dissociation type (anion type)) after elimination of a phosphate group, which will be described later, greatly deviate. Therefore, in the general formula (2), in the benzene ring having two R ²¹ and one R ²² , the xanthene skeleton is in the 1st position, one R ²¹ is in the 4th position, and the other R ²¹ is in the 6th position. In this case, R ²² is preferably in the 3rd or 5th position, more preferably in the 3rd position.

<R ²³ , R ²⁴ , R ²⁵ and R ²⁶ >
Examples of the halogen atom in R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are the same as those exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among ^them, it is preferable that the halogen atom in R ^23, R ^{24, R 25} and ^{R 26} is a chlorine atom, a bromine atom, or iodine atom.
Examples of the alkyl group having 1 to 10 carbon atoms in R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are the same as those exemplified above in “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ²³ , R ²⁴ , R ²⁵ and R ²⁶ is preferably a linear group, and more preferably a methyl group or an ethyl group.

Among them, in the general formula (2), R ²³ and R ^26, since the easy synthesis, preferably the same, and more preferably a hydrogen atom.
Of these, in general formula (2), R ²⁴ and R ²⁵ are preferably the same, and more preferably a hydrogen atom or a halogen atom, because they are easily synthesized.

<X ²¹ >
X ²¹ is an oxygen atom or N ⁺ HR ′.
When X ²¹ is an oxygen atom, Y ²¹ is preferably a single bond, —O— (CH ₂ ) _n21 —, or —O— (CH ₂ ) _n22 —Ar ²¹ —, and X ²¹ is N ^+. when a HR ^{', Y 21} is a single _{bond, -NH- (CH 2) n23 -} , ^{_{or, -NH- (CH 2) n24 -Ar}} 22 - it is preferably.

R ′ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Examples of the alkyl group having 1 to 10 carbon atoms for R ′ include the same ones as exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ′ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in the general formula ^(2), it is preferred that ^{X 21} is an oxygen atom or ^N + _{H 2.}

<Y ²¹ >
In the general formula (2), Y ²¹ represents a single bond, —O— (CH ₂ ) _n21 —, —O— (CH ₂ ) _n22 —Ar ²¹ —, —NH— (CH ₂ ) _n23 —, or —NH — (CH ₂ ) _n24 —Ar ²² —. In Y ²¹ , the bond opposite to the alkylene group of —O— or —NH— is bonded to the carbon atom constituting the xanthene ring in the general formula (2). In addition, a bond opposite to the oxygen atom (O), amino group (NH), or alkylene group of — (CH ₂ ) _n21 —, —Ar ²¹ —, — (CH ₂ ) _n23 —, or —Ar ²² — It is bonded to the phosphate group in formula (2).
n21, n22, n23 and n24 are each independently an integer of 1 to 10.
Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.

n21, n22, n23 and n24 are the number of repetitions of the alkylene group for each ^{Y 21.} n21, n22, n23 and n24 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .

As the substituted or unsubstituted arylene group for Y ^21, the same groups as those exemplified above for “<Y ¹¹ >” can be mentioned.

Among them, in the general formula (2), Y ²¹ represents a single bond, —O—CH ₂ —, —O— (CH ₂ ) ₂ —, —O—CH ₂ —Ph—, —NH—CH ₂ —, —NH It is preferably — (CH ₂ ) ₂ — or —NH—CH ₂ —Ph—. Here, “Ph” represents a substituted or unsubstituted phenylene group.

Preferred examples of the compound (2) include, for example, a compound represented by the following general formula (2-1) (hereinafter sometimes abbreviated as “compound (2-1)”), or a compound represented by the following general formula ( 2-2) (hereinafter sometimes abbreviated as “compound (2-2)”) and the like.
In addition, these compounds are only examples of a preferable compound (2), and a preferable compound (2) is not limited to these.

(In General Formula (2-1), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ²¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or a carbon number of Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, wherein n ²¹¹ and n 212 are each independently 1 to ^{.Ar 211} is an integer of 10 is a substituted or unsubstituted arylene group.
In general formula (2-2), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. R ²²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. )

<R ²¹¹ , R ²²² , R ²²³ and R ²²⁴ >
Examples of the alkyl group having 1 to 10 carbon atoms in R ²¹¹ , R ²²² , R ²²³ and R ²²⁴ include the same as those exemplified above in “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ²¹¹ , R ²²² , R ²²³ and R ²²⁴ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among these, in general formula (2-1), R ²¹¹ and R ²¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.
In general formula (2-2), R ²²¹ and R ²²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.

< ^R213 and ^R223 >
R ²¹³ and R ²²³ are each independently a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.
Among them, the “group having an anionic functional group at the terminal” in the general formulas (2-1) and (2-2) is preferably a group consisting only of an anionic functional group because it is easy to synthesize.

< ^R224 >
R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Examples of the alkyl group having 1 to 10 carbon atoms for R ²²⁴ include the same groups as those exemplified above in “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ²²⁴ is preferably a straight chain, and more preferably a methyl group or an ethyl group.
In the general formula (2-2), R ²²⁴ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom, a methyl group or an ethyl group.

< ^Y211 and ^Y221 >
Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —. Ar ²¹¹ is a substituted or unsubstituted arylene group.
Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. Ar ²²¹ is a substituted or unsubstituted arylene group.
n211, n212, n221 and n222 are the number of repetitions of the alkylene group for ^{Y 211} and ^{Y 221,} respectively. n211, n212, n221, and n222 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .
Ar ²¹¹ and Ar ²²¹ are each independently a substituted or unsubstituted arylene group. Examples of the substituted or unsubstituted arylene group include the same groups as those exemplified above for “<Y ¹¹ >”.

Preferred examples of the compound (2-1) include, for example, R ²¹¹ and R ²¹² are straight-chain alkyl groups having 1 to 10 carbon atoms, R ²¹³ is a group consisting of only an anionic functional group, and R ²³ and R ²⁶ are a hydrogen atom, a methyl group or an ethyl group, R ²⁴ and R ²⁵ are a hydrogen atom, a halogen atom, a methyl group or an ethyl group, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, Or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, wherein n211 and n212 are each independently an integer of 1 to 8, and Ar ²¹¹ is a substituted or unsubstituted phenylene group. .
More preferable examples of the compound (2-1) include, for example, R ²¹¹ and R ²¹² are a methyl group or an ethyl group, R ²¹³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ²³ and R ²⁶ Is a hydrogen atom, R ²⁴ and R ²⁵ are a hydrogen atom or a halogen atom, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —. , and the integers of 1 ~ 6 N211 and n212 are each ^{independently, Ar 211} can be cited, such as those substituted or unsubstituted phenylene group.

Preferred examples of the compound (2-2) include, for example, R ²²¹ and R ²²² are linear alkyl groups having 1 to 10 carbon atoms, R ²²³ is a group consisting of only an anionic functional group, ²²⁴ is a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, R ²³ and R ²⁶ are a hydrogen atom, a methyl group or an ethyl group, and R ²⁴ and R ²⁵ are a hydrogen atom, a halogen atom, methyl Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently 1 to 8 And Ar ²²¹ is a substituted or unsubstituted phenylene group.
More preferable compounds (2-2) include, for example, R ²²¹ and R ²²² are a methyl group or an ethyl group, R ²²³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ²²⁴ is a hydrogen atom. , A methyl group or an ethyl group, R ²³ and R ²⁶ are hydrogen atoms, R ²⁴ and R ²⁵ are hydrogen atoms or halogen atoms, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, Or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently an integer of 1 to 6, and Ar ²²¹ is a substituted or unsubstituted phenylene group.

Among the compounds (2), preferred as the compound (2-1) are, for example, compounds represented by the following general formula (2-1-1) (hereinafter referred to as “compound (2-1-1)”). May be abbreviated), a compound represented by the following general formula (2-1-2) (hereinafter sometimes abbreviated as “compound (2-1-2)”), or a compound represented by the following general formula (2 -1-3) (hereinafter sometimes abbreviated as “compound (2-1-3)”) and the like.
Of the compounds (2), preferred as the compound (2-2) are, for example, compounds represented by the following general formula (2-2-1) (hereinafter referred to as “compound (2-2-1)”). Or a compound represented by the following general formula (2-2-2) (hereinafter referred to as “compound (2-2-2)”, or represented by the following general formula (2-2-3)). And the like (hereinafter sometimes abbreviated as “compound (2-2-3)”).
In addition, these compounds are only examples of a preferable compound (2), and a preferable compound (2) is not limited to these.

(In General Formulas (2-1-1) to (2-2-3), Y ²¹¹ and Y ²²¹ are the same as described above.)

As a preferable compound (2-1-1), for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, and n211 And n212 each independently represents an integer of 1 to 8, and Ar ²¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-1-1) is, for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, n211 and n212 are each independently a 1-6 ^{integer, Ar 211} can be cited, such as those substituted or unsubstituted phenylene group.

As a preferable compound (2-1-2), for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, and n211 And n212 each independently represents an integer of 1 to 8, and Ar ²¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-1-2) is, for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, n211 and n212 are each independently a 1-6 ^{integer, Ar 211} can be cited, such as those substituted or unsubstituted phenylene group.

Preferred examples of the compound (2-1-3) include, for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, and n ²¹¹ And n212 each independently represents an integer of 1 to 8, and Ar ²¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-1-3) is, for example, Y ²¹¹ is a single bond, —O— (CH ₂ ) _n211 —, or —O— (CH ₂ ) _n212 —Ar ²¹¹ —, n211 and n212 are each independently a 1-6 ^{integer, Ar 211} can be cited, such as those substituted or unsubstituted phenylene group.

As a preferable compound (2-2-1), for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, and n221 And n222 are each independently an integer of 1 to 8, and Ar ²²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-2-1) is, for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, n221 and n222 are each independently an integer of 1 to 6, and Ar ²²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (2-2-2), for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, and n221 And n222 are each independently an integer of 1 to 8, and Ar ²²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-2-2) is, for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, n221 and n222 are each independently an integer of 1 to 6, and Ar ²²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (2-2-3), for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, and n221 And n222 are each independently an integer of 1 to 8, and Ar ²²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (2-2-3) is, for example, Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —, n221 and n222 are each independently an integer of 1 to 6, and Ar ²²¹ is a substituted or unsubstituted phenylene group.

Among the compounds (2), preferred as the compound (2-1-1) are, for example, compounds represented by the following formula (2-1-1a) (hereinafter referred to as “compound (2-1-1a)”) Or a compound represented by the following formula (2-1-1b) (hereinafter sometimes abbreviated as “compound (2-1-1b)”), or a compound represented by the following formula (2- 1-1c) (hereinafter sometimes abbreviated as “compound (2-1-1c)”) and the like.
Among the compounds (2), preferred as the compound (2-1-2) are, for example, compounds represented by the following formula (2-1-2a) (hereinafter referred to as “compound (2-1-2a)”) Or a compound represented by the following formula (2-1-2b) (hereinafter sometimes abbreviated as “compound (2-1-2b)”), or a compound represented by the following formula (2- 1-2c) (hereinafter sometimes abbreviated as “compound (2-1-2c)”) and the like.
Among the compounds (2), preferred as the compound (2-1-3) are, for example, compounds represented by the following formula (2-1-3a) (hereinafter referred to as “compound (2-1-3a)”) Or a compound represented by the following formula (2-1-3b) (hereinafter sometimes abbreviated as “compound (2-1-3b)”), or a compound represented by the following formula (2- 1-3c) (hereinafter sometimes abbreviated as “compound (2-1-3c)”) and the like.
Among the compounds (2), preferred as the compound (2-2-1) are, for example, compounds represented by the following formula (2-2-1a) (hereinafter referred to as “compound (2-2-1a)”. Or a compound represented by the following formula (2-2-1b) (hereinafter sometimes abbreviated as “compound (2-2-1b)”), or a compound represented by the following formula (2- 2-1c) (hereinafter sometimes abbreviated as “compound (2-2-1c)”) and the like.
Among the compounds (2), preferred as the compound (2-2-2) are, for example, compounds represented by the following formula (2-2-2a) (hereinafter referred to as “compound (2-2-2a)”) Or a compound represented by the following formula (2-2-2b) (hereinafter sometimes abbreviated as “compound (2-2-2b)”), or a compound represented by the following formula (2- 2-2c) (hereinafter sometimes abbreviated as “compound (2-2-2c)”) and the like.
Among the compounds (2), preferred as the compound (2-2-3) are, for example, compounds represented by the following formula (2-2-3a) (hereinafter referred to as “compound (2-2-3a)”. Or a compound represented by the following formula (2-2-3b) (hereinafter sometimes abbreviated as “compound (2-2-3b)”), or a compound represented by the following formula (2- 2-3c) (hereinafter sometimes abbreviated as “compound (2-2-3c)”) and the like.
In addition, these compounds are only examples of a preferable compound (2), and a preferable compound (2) is not limited to these.

Compound (2) is hydrolyzed by ALP, the phosphate group is eliminated, and the compound (2) -1 is changed to compound (2) -1, thereby generating green (fluorescence wavelength: about 450 nm or more and less than about 550 nm) fluorescence.

Although the compound (2) of this embodiment exists as a substantially non-dissociation type (neutral type) compound, the dissociation type (anion type) is obtained by the elimination of the phosphate group by an enzymatic reaction with ALP. ) Compound (2) -1. Therefore, the compound (2) of the present embodiment can be used as a fluorescent probe for measuring ALP with high sensitivity because the maximum absorption wavelength greatly changes before and after the enzyme reaction with ALP.

<< Method for Producing Compound (2) >>
Compound (2) can be produced, for example, by reacting a compound having a benzene derivative and a phosphate group on the xanthene skeleton using a known reaction according to the types of Y ²¹ , R ²¹ and R ²² . More specifically, it is as follows.

<Method for Producing Compound (2-1)>
Among the compounds (2), the compound (2-1) is, for example, a compound represented by the following general formula (2-1a) (hereinafter sometimes abbreviated as “compound (2-1a)”), A compound represented by the following general formula (2-1b) (hereinafter sometimes abbreviated as “compound (2-1b)”) is reacted with the compound to represent the following general formula (2-1c). A step of obtaining a compound (hereinafter sometimes abbreviated as “compound (2-1c)”) (hereinafter abbreviated as “a compound (2-1c) production step”), a compound (2-1c) and And a compound represented by the following general formula (2-1d) (hereinafter, may be abbreviated as “compound (2-1d)”) to give a compound represented by the following general formula (2-1e). (Hereinafter referred to as “compound (2-1e)”) (hereinafter referred to as “compound (2-1e)”). ) A production step ”and a step of obtaining compound (2-1) from compound (2-1e) (hereinafter, sometimes abbreviated as“ compound (2-1) production step ”). It can manufacture with the manufacturing method which has.
Hereinafter, each step will be described in detail.

(Wherein Bzl is a benzyl group and TBS is a tert-butyldimethylsilyl group. R ²¹⁴ is a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a terminal group) And an alkylene group having 1 to 10 carbon atoms which may contain at least one of an oxygen atom and an arylene group, Y ²¹¹ , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²¹¹ , R ²¹² , and R ²¹³ are all the same as above.)

[Compound (2-1c) Production Process]
In the production step of the compound (2-1c), the compound (2-1a) and the compound (2-1b) are reacted to obtain the compound (2-1c).

(Compound (2-1a))
Compound (2-1a) is a known compound.
In the compound (2-1a), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (2-1a), R ²¹¹ and R ²¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (2-1a), R ²¹³ is a group having an anionic functional group at the terminal.
Among them, in the compound (2-1a), R ²¹³ is preferably a group consisting of only an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group, because it is easy to synthesize. preferable.

(Compound (2-1b))
Compound (2-1b) is a known compound.
In the compound (2-1b), R ²³ , R ²⁴ , R ²⁵ , and R ²⁶ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms.
Among them, in the compound (2-1b), R ²³ and R ²⁶ are preferably the same and more preferably a hydrogen atom because they are easily synthesized. R ²⁴ and R ²⁵ are preferably the same, and more preferably a hydrogen atom or a halogen atom, because they are easy to synthesize.

(Reaction conditions)
In the production step of compound (2-1c), it is preferred that a strong base is previously mixed with compound (2-1a) and then reacted with compound (2-1b).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (2-1a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (2-1c), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (2-1c), the amount of compound (2-1b) used is preferably 0.1 to 0.3 times the amount of compound (2-1a) used. .

In the production step of compound (2-1c), it is preferable to react compound (2-1a), compound (2-1b), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1M or more and 5M or less.

In the production step of compound (2-1c), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production step of compound (2-1c), the reaction time is preferably 5 hours or more and 25 hours or less, more preferably 10 hours or more and 20 hours or less.

In the compound (2-1c) production process, after completion of the reaction, the compound (2-1c) can be taken out in the same manner as in the above-mentioned compound (1-1b) production process, and the taken out compound (2-1c) is further treated in the same way. You may refine by. Further, the obtained compound (2-1c) may be used in the next step without being removed after completion of the reaction, but it should be removed from the viewpoint of improving the yield of the target compound (2-1). Is preferred.

[Compound (2-1e) Production Process]
In the step of producing compound (2-1e), compound (2-1c) and compound (2-1d) are reacted to obtain compound (2-1e).

(Compound (2-1c))
Compound (2-1c) is a known compound.
In the compound ^{(2-1c), R 211, R} 212 and ^{R 213} are the same as ^{R 211, R 212} and ^{R 213} in the compound ^{(2-1a), R 23, R} 24, R 25 and ^{R 26} The same as R ²³ , R ²⁴ , R ²⁵ and R ²⁶ in the compound (2-1b).

(Compound (2-1d))
Compound (2-1d) is a known compound.
In the compound (2-1d), R ²¹⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among these, in the compound (2-1d), R ²¹⁴ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (2-1e))
Compound (2-1e) is a novel compound.
In the compound (2-1e), Y ²¹¹ is the same as those exemplified for “<Y ²¹¹ and Y ²²¹ >” in the above “<< Compound (2) >>”.
Further, in the compound ^{(2-1e), R 211, R} 212 and ^{R 213} are the same as ^{R 211, R 212} and ^{R 213} in the compound ^{(2-1a), R 23, R} 24, R 25 and R ²⁶ is the same as R ²³ , R ²⁴ , R ²⁵ and R ²⁶ in the compound (2-1b).

(Reaction conditions)
In the production step of compound (2-1e), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (2-1c).

In the production process of compound (2-1e), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of compound (2-1c) used.

In the production process of compound (2-1e), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in [Compound (1-1d) Production Process].
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (2-1c).

In the production process of the compound (2-1e), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the compound (2-1e) production process, the amount of compound (2-1d) used is preferably 1 to 2 times the amount of compound (2-1c).

In the production process of compound (2-1e), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production process of compound (2-1e), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

The compound (2-1e) can be taken out by the same method as in the above-mentioned compound (1-1b) production step, and the taken out compound (2-1e) may be further purified by the same method. Further, the obtained compound (2-1e) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the point that the yield of the target compound (2-1) is improved. Is preferred.

[Compound (2-1) Production Process]
In the production step of the compound (2-1), the compound (2-1) is obtained from the compound (2-1e).
The method for obtaining the compound (2-1) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Note that when a protective group is bonded to ^R213 , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (2-1), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of the compound (2-1), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, and more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (2-1), the reaction time is preferably 30 minutes or longer and 5 hours or shorter, more preferably 1 hour or longer and 3 hours or shorter.

In the compound (2-1) production process, after completion of the reaction, the compound (2-1) can be removed in the same manner as in the compound (1-1b) production process, and the removed compound (2-1 ) May be further purified by the same method.

Each compound such as compound (2-1), compound (2-1a), compound (2-1b), compound (2-1c), compound (2-1d), compound (2-1e), etc. is, for example, a nucleus The structure can be confirmed by a known method such as magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

<Method for Producing Compound (2-2)>
Among the compounds (2), the compound (2-2) is, for example, a compound represented by the following general formula (2-2a) (hereinafter sometimes abbreviated as “compound (2-2a)”); A compound represented by the following general formula (2-2b) (hereinafter sometimes abbreviated as “compound (2-2b)”) is reacted with the compound to represent the following general formula (2-2c). A step of obtaining a compound (hereinafter sometimes abbreviated as “compound (2-2c)”) (hereinafter abbreviated as “a compound (2-2c) production step”), a compound (2-2c) and And a compound represented by the following general formula (2-2d) (hereinafter, may be abbreviated as “compound (2-2d)”) to give a compound represented by the following general formula (2-2e). (Hereinafter referred to as “compound (2-2e)”) (hereinafter referred to as “compound (2-2e)”). ) A production step ”and a step of obtaining compound (2-2) from compound (2-2e) (hereinafter, abbreviated as“ compound (2-2) production step ”). It can manufacture with the manufacturing method which has.
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group and Ph is an unsubstituted phenyl group. R ²²⁵ is a hydrogen atom, a hydroxyl group, a leaving group (for example, a halogen atom), or a leaving group at the end (for example, And an alkylene group having 1 to 10 carbon atoms, which may contain at least one of an oxygen atom and an arylene group, and includes Y ²²¹ , R ²³ , R ²⁴ , R ^25. , R ²⁶ , R ²²¹ , R ²²² , R ²²³ and R ²²⁴ are all the same as above.)

[Compound (2-2c) Production Process]
In the production step of the compound (2-2c), the compound (2-2a) and the compound (2-2b) are reacted to obtain the compound (2-2c).

(Compound (2-2a))
Compound (2-2a) is a known compound.
In the compound (2-2a), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (2-2a), R ²²¹ and R ²²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (2-2a), R ²²³ is a group having an anionic functional group at the terminal.
Among these, in the compound (2-2a), R ²²³ is preferably a group consisting only of an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. preferable.

(Compound (2-2b))
Compound (2-2b) is a known compound.
In the compound (2-2b), R ²³ , R ²⁴ , R ²⁵ , and R ²⁶ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms.
Among them, in the compound (2-2b), R ²³ and R ²⁶ are preferably the same and more preferably a hydrogen atom because they are easily synthesized. R ²⁴ and R ²⁵ are preferably the same, and more preferably a hydrogen atom or a halogen atom, because they are easy to synthesize.

In the compound (2-2b), R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Among these, in the compound (2-2b), R ²²⁴ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom, a methyl group, or an ethyl group. preferable.

(Reaction conditions)
In the production step of compound (2-2c), it is preferable that a strong base is mixed with compound (2-2a) in advance and then reacted with compound (2-2b).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (2-2a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (2-2c), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (2-2c), the amount of compound (2-2b) used is preferably 0.1 to 0.3 times the amount of compound (2-2a) used. .

In the production step of compound (2-2c), it is preferable to react compound (2-2a), compound (2-2b), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the production step of compound (2-2c), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, and more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production step of compound (2-2c), the reaction time is preferably 5 hours or more and 25 hours or less, more preferably 10 hours or more and 20 hours or less.

In the compound (2-2c) production process, after completion of the reaction, the compound (2-2c) can be removed in the same manner as in the above-mentioned compound (1-1b) production process. You may refine by. The obtained compound (2-2c) may be used in the next step without being removed after the reaction is completed, but it should be removed from the viewpoint that the yield of the target compound (2-2) is improved. Is preferred.

[Compound (2-2e) Production Process]
In the production step of the compound (2-2e), the compound (2-2c) and the compound (2-2d) are reacted to obtain the compound (2-2e).

(Compound (2-2c))
Compound (2-2c) is a known compound.
In the compound ^{(2-2c), R 221, R} 222 and ^{R 223} are the same as ^{R 221, R 222} and ^{R 223} in the compound ^{(2-2a), R 23, R} 24, R 25, R 26 and R ²²⁴ is the same as ^{R 23, R 24, R 25} , R 26 and ^{R 224} in the compound (2-2b).

(Compound (2-2d))
Compound (2-2d) is a known compound.
In the compound (2-2d), R ²²⁵ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among these, in the compound (2-2d), R ²²⁵ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (2-2e))
Compound (2-1e) is a novel compound.
In the compound (2-1e), the same as those exemplified for “<Y ²¹¹ and Y ²²¹ >” in the above “<< Compound (2) >>”.
Further, in the compound ^{(2-2e), R 221, R} 222 and ^{R 223} are the same as ^{R 221, R 222} and ^{R 223} in the compound ^{(2-2a), R 23, R} 24, R 25, R ²⁶ and ^{R 224} are the same as ^{R 23, R 24, R 25} , R 26 and ^{R 224} in the compound (2-2b).

(Reaction conditions)
In the production step of compound (2-2e), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (2-2c).

In the production process of compound (2-2e), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (2-2c) used.

In the production process of compound (2-2e), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (2-2c).

In the production process of the compound (2-2e), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (2-2e), the amount of compound (2-2d) used is preferably 1 to 2 times the amount of compound (2-2c).

In the production process of compound (2-2e), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production process of compound (2-2e), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (2-2e) can be extracted in the same manner as in the above-mentioned “[Compound (1-1b) production step]”, and the extracted compound (2-2e) is further purified by the same method. May be. Further, the obtained compound (2-2e) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the viewpoint of improving the yield of the target compound (2-2). Is preferred.

[Compound (2-2) Production Process]
In the production step of the compound (2-2), the compound (2-2) is obtained from the compound (2-2e).
The method for obtaining the compound (2-2) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. In addition, also when a protective group has couple ^| bonded with ^R223 , a protective group is similarly removed and a carboxy group, a sulfonic acid group, or a phosphoric acid group is formed. Further, when a protective group is bonded to R ²²⁴ , the protective group is similarly removed.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (2-2), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (2-2), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, and more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (2-2), the reaction time is preferably 30 minutes or longer and 5 hours or shorter, more preferably 1 hour or longer and 3 hours or shorter.

In the production step of compound (2-2), after completion of the reaction, compound (2-2) can be taken out in the same manner as in the case of “[Compound (1-1b) production step]”. (2-2) may be further purified by the same method.

Each compound such as compound (2-2), compound (2-2a), compound (2-2b), compound (2-2c), compound (2-2d), compound (2-2e) is, for example, a nucleus The structure can be confirmed by a known method such as magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

<< Compound (3) >>
The compound according to the third embodiment of the present invention is represented by the following general formula (3) (sometimes referred to as “compound (3)” in the present specification).

(In the general formula (3), R ³¹ is 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ³¹ may be the same as each other. may .R ³² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ³² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ³³ , R ³⁴ , R ³⁷ and R ³⁸ is each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom , Phosphorus atom, germanium atom or tin atom. X ³² is an oxygen atom or N ⁺ HR ″. R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ³¹ is a single bond, —O _{- (CH 2) n31 -,} - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, ^{_{or, -NH- (CH 2) n34 -Ar}} 32 - are as .N31, n32, n33 and n34 are each independently an integer of 1 to 10. Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.)

Compound (3) is a fluorescein analog or rhodamine analog, and has a phosphate group that is hydrolyzed by ALP under alkaline conditions.
Compound (3) is a fluorescent compound that emits fluorescence when a phosphate group is eliminated by hydrolysis with ALP.

<R ³¹ >
R ³¹ is 1-2 monovalent substituents present on the benzene ring and is an electron donating group.
The electron donating group in R ³¹ is the same as those exemplified in the above “<R ²¹ >”.
The number of R ³¹ is 1 or 2, and is preferably 2. When there are two R ^{31 s} , they may be the same or different. Among these, when there are two R ^{31 s} , it is preferable that they are the same as each other because they are easily synthesized.

Among these, in general formula (3), R ³¹ is highly hydrophilic, and is preferably a straight-chain alkoxy group having 1 to 10 carbon atoms or a straight-chain alkylamino group having 1 to 10 carbon atoms. A group, an ethylamino group, a methoxy group, or an ethoxy group is more preferable.

Further, in the general formula ^(3), it is preferred ^{that R 31} is two. The two R ²¹ positions in the benzene ring are preferably arranged at positions that are ortho positions relative to each other.

<R ³² >
R ³² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. Among them, the “group having an anionic functional group at the terminal” in the general formula (3) is preferably a group consisting of only an anionic functional group because it is easy to synthesize.
The number of R ³² is one or two, and preferably one. When two R ³² are present, they may be the same as or different from each other. Among these, when there are two R ^{32 s} , it is preferable that they are the same as each other because they are easily synthesized.

In particular, the maximum absorption wavelength of compound (3) (non-dissociation type (neutral type)) and compound (3) -1 (dissociation type (anion type)) after elimination of a phosphate group, which will be described later, greatly deviate. Therefore, in general formula (3), in the benzene ring having two R ³¹ and one R ³² , the xanthene skeleton is in the 1st position, one R ³¹ is in the 4th position, and the other R ³¹ is in the 6th position. In this case, R ³² is preferably located at the 3rd or 5th position, more preferably at the 3rd position.

<R ³³ , R ³⁴ , R ³⁷ and R ³⁸ >
Examples of the halogen atom in R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are the same as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Especially, it is preferable that the said halogen atom in R ^{< 33} >, R ^{< 34>} , R ^<37> and R ^<38 > is a chlorine atom, a bromine atom, or an iodine atom.
Examples of the alkyl group having 1 to 10 carbon atoms in R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are the same as those exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ³³ , R ³⁴ , R ³⁷ and R ³⁸ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Especially, in general formula (3), since ^R33 and ^R38 are easy to synthesize | combine, it is preferable that it is the same, and it is more preferable that they are a hydrogen atom.
Of these, in general formula (3), R ³⁴ and R ³⁷ are preferably the same, more preferably a hydrogen atom or a halogen atom, because they are easily synthesized.

< ^R35 and ^R36 >
Examples of the alkyl group having 1 to 10 carbon atoms for R ³⁵ and R ³⁶ are the same as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ³⁵ and R ³⁶ is preferably a linear group, and more preferably a methyl group or an ethyl group.
Examples of the aryl group having 6 to 10 carbon atoms in R ³⁵ and R ³⁶ include phenyl group, benzyl group, tolyl group, o-xylyl group, m-xylyl group, p-xylyl group and the like. Not. Among them, the aryl group having 6 to 10 carbon atoms in R ³⁵ and R ³⁶ is preferably a phenyl group or a benzyl group, and more preferably a phenyl group.

Among these, in general formula (3), R ³⁵ and R ³⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, More preferably, it is an ethyl group.

<X ³¹ >
X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom.
Among them, in the general formula ^{(3), X 31} is preferable a silicon atom.

< ^X32 >
X ³² is an oxygen atom or N ⁺ HR ″.
When X ³² is an oxygen ^{atom, Y 31} represents a single _{bond, -O- (CH 2) n31 -} , ^{_{or, -O- (CH 2) n32 -Ar}} 31 - is ^{preferably, X 32} is ^{N +} When HR ″, Y ³¹ is preferably a single bond, NH— (CH ₂ ) _n33 —, or —NH— (CH ₂ ) _n34 —Ar ³² —.

R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Examples of the alkyl group having 1 to 10 carbon atoms for R ″ include the same ones as exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ″ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in the general formula ^{(3), X 32} is preferably is an oxygen atom or ^N + _{H 2.}

<Y ³¹ >
In the general formula ^{(3), Y 31} represents a single _{bond, -O- (CH 2) n31 -} , - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, or, -NH — (CH ₂ ) _n34 —Ar ³² —. In Y ³¹ , the bond opposite to the alkylene group of —O— or —NH— is bonded to the carbon atom constituting the hetero three-membered ring in the general formula (3). ^{_{Also, - (CH 2) n31 -}} , - Ar 31 -, - (CH 2) n33 - or ^{-Ar 32} - oxygen atoms (O), amino group (NH) or opposite bond is the general alkylene group It is bonded to the phosphate group in formula (3).
n31, n32, n33 and n34 are each independently an integer of 1 to 10.
Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.

n31, n32, n33 and n34 are the number of repetitions of the alkylene group for each ^{Y 31.} n31, n32, n33 and n34 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .

As the substituted or unsubstituted arylene group for Y ^31, the same groups as those exemplified above for “<Y ¹¹ >” can be mentioned.

Among them, in the general formula (3), Y ³¹ is a single bond, —O—CH ₂ —, —O— (CH ₂ ) ₂ —, —O—CH ₂ —Ph—, —NH—CH ₂ —, —NH It is preferably — (CH ₂ ) ₂ — or —NH—CH ₂ —Ph—. Here, “Ph” represents a substituted or unsubstituted phenylene group.

Preferred examples of the compound (3) include a compound represented by the following general formula (3-1) (hereinafter sometimes abbreviated as “compound (3-1)”), or a compound represented by the following general formula ( 3-2) (hereinafter sometimes abbreviated as “compound (3-2)”) and the like.
In addition, these compounds are only examples of a preferable compound (3), and a preferable compound (3) is not limited to these.

(In the general formula (3-1), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ³¹³ is a group having an anionic functional group at its terminal. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms X ³¹ is a silicon atom, phosphorus atom, germanium atom or tin it is an atomic ^{.Y 311} is a single _{bond, -O- (CH 2) n311 -} , ^{_{or, -O- (CH 2) n312 -Ar}} 311 - a is .n311 and N312 are each independently ^{.Ar 311} is an integer of 1-10 is a substituted or unsubstituted arylene group.
In general formula (3-2), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. R ³²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. )

<R ³¹¹ , R ³²² , R ³²¹ and R ³²² >
Examples of the alkyl group having 1 to 10 carbon atoms in R ³¹¹ , R ³²² , R ³²¹ and R ³²² are the same as those exemplified above in “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ³¹¹ , R ³²² , R ³²¹ and R ³²² is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in general formula (3-1), R ³¹¹ and R ³¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.
In general formula (3-2), R ³²¹ and R ³²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.

< ^R313 and ^R323 >
R ³¹³ and R ³²³ are each independently a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.
Among them, the “group having an anionic functional group at the terminal” in the general formulas (3-1) and (3-2) is preferably a group consisting only of an anionic functional group because it is easy to synthesize.

< ^R324 >
R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Examples of the alkyl group having 1 to 10 carbon atoms for R ³²⁴ include the same groups as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among these, the alkyl group having 1 to 10 carbon atoms in R ³²⁴ is preferably a linear group, and more preferably a methyl group or an ethyl group.
In General Formula (3-2), R ³²⁴ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom, a methyl group, or an ethyl group.

<Y ³¹¹ and Y ³²¹ >
Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —. Ar ³¹¹ is a substituted or unsubstituted arylene group.
Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. Ar ³²¹ is a substituted or unsubstituted arylene group.
n311, n312, n321 and n322 are the number of repetitions of the alkylene group for ^{Y 311} and ^{Y 321,} respectively. n311, n312, n321 and n322 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .
Ar ³¹¹ and Ar ³²¹ are each independently a substituted or unsubstituted arylene group. Examples of the substituted or unsubstituted arylene group include the same groups as those exemplified above for “<Y ¹¹ >”.

Preferred examples of the compound (3-1) include, for example, R ³¹¹ and R ³¹² are linear alkyl groups having 1 to 10 carbon atoms, R ³¹³ is a group consisting of only an anionic functional group, ³³ and R ³⁸ are a hydrogen atom, a methyl group or an ethyl group, R ³⁴ and R ³⁷ are a hydrogen atom, a halogen atom, a methyl group or an ethyl group, and R ³⁵ and R ³⁶ are a methyl group, an ethyl group or a phenyl group. Or a benzyl group, X ³¹ is a silicon atom, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, and n311 and n312 Are each independently an integer of 1 to 8, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.
More preferable examples of the compound (3-1) include, for example, R ³¹¹ and R ³¹² are a methyl group or an ethyl group, R ³¹³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ³³ and R ³⁸ Is a hydrogen atom, R ³⁴ and R ³⁷ are a hydrogen atom or a halogen atom, R ³⁵ and R ³⁶ are a methyl group or an ethyl group, X ³¹ is a silicon atom, Y ³¹¹ is a single bond, —O — (CH ₂ ) _n311 — or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, wherein n311 and n312 are each independently an integer of 1 to 6, and Ar ³¹¹ is a substituted or unsubstituted phenylene group And the like.

Preferred examples of the compound (3-2) include, for example, R ³²¹ and R ³²² are linear alkyl groups having 1 to 10 carbon atoms, R ³²³ is a group consisting of only an anionic functional group, ³²⁴ is a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, R ³³ and R ³⁸ are a hydrogen atom, a methyl group or an ethyl group, and R ³⁴ and R ³⁷ are a hydrogen atom, a halogen atom, methyl A group or an ethyl group, R ³⁵ and R ³⁶ are a methyl group, an ethyl group, a phenyl group or a benzyl group, X ³¹ is a silicon atom, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 — ^{_{or, -NH- (CH 2) n322 -Ar}} 321 - a is an integer of 1 ~ 8 N321 and n322 are each ^{independently} also ^{Ar 321} is a substituted or unsubstituted phenylene group Etc. The.
More preferable examples of the compound (3-2) include, for example, R ³²¹ and R ³²² are a methyl group or an ethyl group, R ³²³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ³²⁴ is a hydrogen atom. , Methyl group or ethyl group, R ³³ and R ³⁸ are hydrogen atoms, R ³⁴ and R ³⁷ are hydrogen atoms or halogen atoms, R ³⁵ and R ³⁶ are methyl groups or ethyl groups, and X ³¹ Is a silicon atom, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, wherein n321 and n322 are each independently 1 to 6 Examples thereof include an integer and Ar ³²¹ is a substituted or unsubstituted phenylene group.

Of the compounds (3), preferred as the compound (3-1) are, for example, compounds represented by the following general formula (3-1-1) (hereinafter referred to as “compound (3-1-1)”). May be abbreviated), a compound represented by the following general formula (3-1-2) (hereinafter sometimes abbreviated as “compound (3-1-2)”), or a compound represented by the following general formula (3 -1-3) (hereinafter sometimes abbreviated as “compound (3-1-3)”) and the like.
Among the compounds (3), preferred as the compound (3-2) are, for example, compounds represented by the following general formula (3-2-1) (hereinafter referred to as “compound (3-2-1)”). May be abbreviated), a compound represented by the following general formula (3-2-2) (hereinafter referred to as “compound (3-2-2)”, or represented by the following general formula (3-2-3) (Hereinafter sometimes abbreviated as “compound (3-2-3)”) and the like.
In addition, these compounds are only examples of a preferable compound (3), and a preferable compound (3) is not limited to these.

(In the general formula, Y ³¹¹ and Y ³²¹ are the same as above.)

As a preferable compound (3-1-1), for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, and n311 And n312 are each independently an integer of 1 to 8, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-1-1) is, for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, n311 and n312 are each independently an integer of 1 to 6, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (3-1-2), for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, and n311 And n312 are each independently an integer of 1 to 8, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-1-2) is, for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, n311 and n312 are each independently an integer of 1 to 6, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (3-1-3), for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, and n311 And n312 are each independently an integer of 1 to 8, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-1-3) is, for example, Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —, n311 and n312 are each independently an integer of 1 to 6, and Ar ³¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (3-2-1), for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, and n321 And n322 each independently represents an integer of 1 to 8, and Ar ³²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-2-1) is, for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, n321 and n322 are each independently an integer of 1 to 6, and Ar ³²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (3-2-2), for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, and n321 And n322 each independently represents an integer of 1 to 8, and Ar ³²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-2-2) is, for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, n321 and n322 are each independently an integer of 1 to 6, and Ar ³²¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (3-2-3), for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, and n321 And n322 each independently represents an integer of 1 to 8, and Ar ³²¹ is a substituted or unsubstituted phenylene group.
More preferable compound (3-2-3) is, for example, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —, n321 and n322 are each independently an integer of 1 to 6, and Ar ³²¹ is a substituted or unsubstituted phenylene group.

Among the compounds (3), preferred as the compound (3-1-1) are, for example, compounds represented by the following formula (3-1-1a) (hereinafter referred to as “compound (3-1-1a)”. Or a compound represented by the following formula (3-1-1b) (hereinafter sometimes abbreviated as “compound (3-1-1b)”), or a compound represented by the following formula (3- 1-1c) (hereinafter sometimes abbreviated as “compound (3-1-1c)”) and the like.
Among the compounds (3), preferred as the compound (3-1-2) are, for example, compounds represented by the following formula (3-1-2a) (hereinafter referred to as “compound (3-1-2a)”. Or a compound represented by the following formula (3-1-2b) (hereinafter sometimes abbreviated as “compound (3-1-2b)”), or a compound represented by the following formula (3- 1-2c) (hereinafter sometimes abbreviated as “compound (3-1-2c)”) and the like.
Among the compounds (3), preferred as the compound (3-1-3) are, for example, compounds represented by the following formula (3-1-3a) (hereinafter referred to as “compound (3-1-3a)”) Or a compound represented by the following formula (3-1-3b) (hereinafter sometimes abbreviated as “compound (3-1-3b)”), or a compound represented by the following formula (3- 1-3c) (hereinafter sometimes abbreviated as “compound (3-1-3c)”) and the like.
Among the compounds (3), preferred as the compound (3-2-1) are, for example, compounds represented by the following formula (3-2-1a) (hereinafter referred to as “compound (3-2-1a)”. Or a compound represented by the following formula (3-2-1b) (hereinafter sometimes abbreviated as “compound (3-2-1b)”), or a compound represented by the following formula (3- 2-1c) (hereinafter sometimes abbreviated as “compound (3-2-1c)”) and the like.
Of the compounds (3), preferred as the compound (3-2-2) are, for example, compounds represented by the following formula (3-2-2a) (hereinafter referred to as “compound (3-2-2a)”) Or a compound represented by the following formula (3-2-2b) (hereinafter sometimes abbreviated as “compound (3-2-2b)”), or a compound represented by the following formula (3- 2-2c) (hereinafter sometimes abbreviated as “compound (3-2-2c)”) and the like.
Among the compounds (3), preferred as the compound (3-2-3) are, for example, compounds represented by the following formula (3-2-3a) (hereinafter referred to as “compound (3-2-3a)”) Or a compound represented by the following formula (3-2-3b) (hereinafter sometimes abbreviated as “compound (3-2-3b)”), or a compound represented by the following formula (3- 2-3c) (hereinafter sometimes abbreviated as “compound (3-2-3c)”) and the like.
In addition, these compounds are only examples of a preferable compound (3), and a preferable compound (3) is not limited to these.

The compound (3) is hydrolyzed by ALP, the phosphate group is eliminated, and the compound (3) is converted into the compound (3) -1, thereby generating red (fluorescence wavelength: about 550 nm or more and less than about 650 nm) fluorescence.

The compound (3) of the present embodiment exists as a substantially non-dissociation type (neutral type) compound, but the dissociation type (anion type) is obtained by the elimination of the phosphate group by an enzymatic reaction with ALP. ) Compound (3) -1. Therefore, the compound (3) of the present embodiment can be used as a fluorescent probe for measuring ALP with high sensitivity because the maximum absorption wavelength largely changes before and after the enzyme reaction with ALP.

<< Method for Producing Compound (3) >>
Compound (3) can be obtained by reacting a compound having a benzene derivative and a phosphate group in the xanthene skeleton using a known reaction according to the types of Y ³¹ , R ³¹ , R ³² and R ³³ , for example. Can be manufactured. More specifically, it is as follows.

<Method for Producing Compound (3-1)>
Among the compounds (3), the compound (3-1) is, for example, a compound represented by the following general formula (3-1a) (hereinafter sometimes abbreviated as “compound (3-1a)”); A compound represented by the following general formula (3-1b) (hereinafter sometimes abbreviated as “compound (3-1b)”) is reacted with the compound to represent the following general formula (3-1c). A step of obtaining a compound (hereinafter sometimes abbreviated as “compound (3-1c)”) (hereinafter abbreviated as “a compound (3-1c) production step”), a compound (3-1c) and And a compound represented by the following general formula (3-1d) (hereinafter, may be abbreviated as “compound (3-1d)”) to give a compound represented by the following general formula (3-1e). (Hereinafter referred to as “compound (3-1e)”) (hereinafter referred to as “compound (3-1e)”. ) A production step ”and a step of obtaining compound (3-1) from compound (3-1e) (hereinafter, abbreviated as“ compound (3-1) production step ”). It can manufacture with the manufacturing method which has.
Hereinafter, each step will be described in detail.

(Wherein Bzl is a benzyl group and TBS is a tert-butyldimethylsilyl group. R ³¹⁴ is a hydrogen atom, a hydroxyl group, a leaving group (for example, a halogen atom), or a leaving group (for example, a terminal group) And an alkylene group having 1 to 10 carbon atoms, which may contain at least one of an oxygen atom and an arylene group, X ³¹ , Y ³¹¹ , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³¹¹ , R ³¹² and R ³¹³ are all the same as above.)

[Compound (3-1c) Production Process]
In the step of producing compound (3-1c), compound (3-1a) is reacted with compound (3-1b) to obtain compound (3-1c).

(Compound (3-1a))
Compound (3-1a) is a known compound.
In the compound (3-1a), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (3-1a), R ³¹¹ and R ³¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (3-1a), R ³¹³ is a group having an anionic functional group at the terminal.
Among them, in the compound (3-1a), R ³¹³ is preferably a group consisting of only an anionic functional group, more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. .

(Compound (3-1b))
Compound (3-1b) is a known compound.
In the compound (3-1b), R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms.
Among them, the compound ^{(3-1b), R 33} and ^{R 38,} since the easy synthesis, preferably the same, and more preferably a hydrogen atom. R ³⁴ and R ³⁷ are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.

In the compound ^{(3-1b), R 35} and ^{R 36} are each independently an aryl group an alkyl group or having 6 to 10 carbon atoms having 1 to 10 carbon atoms.
Among them, in the compound (3-1b), R ³⁵ and R ³⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group. Or it is more preferable that it is an ethyl group.

In the compound (3-1b), X ³¹ is a silicon atom, a germanium atom or a tin atom.
Among them, the compound ^{(3-1b), X 31} is preferable a silicon atom.

(Reaction conditions)
In the production step of compound (3-1c), it is preferred that a strong base is mixed with compound (3-1a) in advance and then reacted with compound (3-1b).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (3-1a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (3-1c), an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (3-1c), the amount of compound (3-1b) used is preferably 0.1 to 0.3 times the amount of compound (3-1a) used. .

In the production step of compound (3-1c), it is preferable to react compound (3-1a), compound (3-1b), and strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the production step of compound (3-1c), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, and more preferably 80 ° C. or higher and 130 ° C. or lower.
In the step of producing compound (3-1c), the reaction time is preferably 5 hours or more and 25 hours or less, more preferably 10 hours or more and 20 hours or less.

In the compound (3-1c) production process, after completion of the reaction, the compound (3-1c) can be taken out in the same manner as in the above-mentioned compound (1-1b) production process, and the taken out compound (3-1c) is further treated in the same way. You may refine by. Further, the obtained compound (3-1c) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the viewpoint that the yield of the target compound (3-1) is improved. Is preferred.

[Compound (3-1e) Production Process]
In the production step of the compound (3-1e), the compound (3-1c) and the compound (3-1d) are reacted to obtain the compound (3-1e).

(Compound (3-1c))
Compound (3-1c) is a known compound.
In the compound ^{(3-1c), R 311, R} 312 and ^{R 313} are ^each the same as ^{R 311, R 312} and ^{R 313} in the compound ^{(3-1a), R 33, R} 34, R 35, R 36, R ^{37, R 38} and ^{X 31} are the same as ^{R 33, R 34, R 35} , R 36, R 37, R 38 and ^{X 31} in the compound (3-1b).

(Compound (3-1d))
Compound (3-1d) is a known compound.
R ³¹⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and at least one of an oxygen atom and an arylene group. And an alkylene group having 1 to 10 carbon atoms which may contain one of them.
Among them, the compound ^{(3-1d), R 314} is a hydrogen atom, a halogen atom, _-O-CH 2 -X, or, preferably a _-O-Ph-CH 2 -X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (3-1e))
Compound (3-1e) is a novel compound.
In the compound (3-1e), the same as those exemplified for “<Y ³¹¹ and Y ³²¹ >” in the above “<< Compound (3) >>”.
Further, in the compound ^{(3-1e), R 311, R} 312 and ^{R 313} are ^each the same as ^{R 311, R 312} and ^{R 313} in the compound ^{(3-1a), R 33, R} 34, R 35, R ^{36, R 37, R 38} and ^{X 31} are the same as ^{R 33, R 34, R 35} , R 36, R 37, R 38 and ^{X 31} in the compound (3-1b).

(Reaction conditions)
In the production step of compound (3-1e), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (3-1c).

In the production process of compound (3-1e), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (3-1c) used.

In the production process of compound (3-1e), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (3-1c).

In the production process of the compound (3-1e), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (3-1e), the amount of compound (3-1d) used is preferably 1 to 2 times the amount of compound (3-1c).

In the production step of compound (3-1e), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the step of producing compound (3-1e), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (3-1e) can be removed in the same manner as in the above-mentioned “[Compound (1-1b) production step]”, and the removed compound (3-1e) is further purified by the same method. Also good. Further, the obtained compound (3-1e) may be used in the next step without being removed after the completion of the reaction, but it should be removed from the viewpoint that the yield of the target compound (3-1) is improved. Is preferred.

[Compound (3-1) Production Process]
In the production step of compound (3-1), compound (3-1) is obtained from compound (3-1e).
The method for obtaining the compound (3-1) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Note that when a protective group is bonded to R ³¹³ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (3-1), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (3-1), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production step of compound (3-1), the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.

In the compound (3-1) production process, after completion of the reaction, the compound (3-1) can be taken out in the same manner as in the compound (1-1b) production process, and the taken out compound (3-1 ) May be further purified by the same method.

Each compound such as compound (3-1), compound (3-1a), compound (3-1b), compound (3-1c), compound (3-1d), compound (3-1e) is, for example, a nucleus The structure can be confirmed by a known method such as magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

<Method for Producing Compound (3-2)>
Among the compounds (3), the compound (3-2) is, for example, a compound represented by the following general formula (3-2a) (hereinafter sometimes abbreviated as “compound (3-2a)”); A compound represented by the following general formula (3-2b) (hereinafter sometimes abbreviated as “compound (3-2b)”) is reacted to be represented by the following general formula (3-2c). A step of obtaining a compound (hereinafter sometimes abbreviated as “compound (3-2c)”) (hereinafter abbreviated as a “compound (3-2c) production step”), a compound (3-2c) and And a compound represented by the following general formula (3-2d) (hereinafter, may be abbreviated as “compound (3-2d)”). (Hereinafter referred to as “compound (3-2e)”) (hereinafter referred to as “compound (3-2e)”). ) A production process ”) and a process for obtaining compound (3-2) from compound (3-2e) (hereinafter sometimes abbreviated as“ compound (3-2) production process ”). It can manufacture with the manufacturing method which has.
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group and Ph is a phenyl group. R ³²⁵ is a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal. And an alkylene group having 1 to 10 carbon atoms which may contain at least one of an oxygen atom and an arylene group, X ³¹ , Y ³²¹ , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³²¹ , R ³²² , R ³²³ and R ³²⁴ are all the same as above.)

[Compound (3-2c) Production Process]
In the production step of compound (3-2c), compound (3-2a) and compound (3-2b) are reacted to obtain compound (3-2c).

(Compound (3-2a))
Compound (3-2a) is a known compound.
In the compound (3-2a), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (3-2a), R ³²¹ and R ³²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (3-2a), R ³²³ is a group having an anionic functional group at the terminal.
Among them, the compound (3-2a), R ^323, since the easy synthesis, is preferably a group consisting of only the anionic functional group, a carboxy group, and more preferably a sulfonic acid group or phosphoric acid group .

(Compound (3-2b))
Compound (3-2b) is a known compound.
In the compound (3-2b), R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms.
Among these, in compound (3-2b), R ³³ and R ³⁸ are preferably the same, and more preferably a hydrogen atom, because they are easily synthesized. R ³⁴ and R ³⁷ are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.

In the compound (3-2b), R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms.
Among them, the compound (3-1b), R ³⁵ and R ^36, since the easy synthesis, preferably the same, a methyl group, an ethyl group, preferably a phenyl group or a benzyl group, a methyl group or More preferably, it is an ethyl group.

In the compound (3-2b), R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
Among these, in the compound (3-2b), R ³²⁴ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom, a methyl group, or an ethyl group.

In the compound (3-2b), X ³¹ is a silicon atom, a germanium atom or a tin atom.
Among these, in the compound (3-2b), X ³¹ is preferably a silicon atom.

(Reaction conditions)
In the production step of compound (3-2c), it is preferred that a strong base is mixed with compound (3-2a) in advance and then reacted with compound (3-2b).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (3-2a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (3-2c), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (3-2c), the amount of compound (3-2b) used is preferably 0.1 to 0.3 times the amount of compound (3-2a) used. .

In the production step of compound (3-2c), it is preferable to react compound (3-2a), compound (3-2b), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the production step of compound (3-2c), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production step of compound (3-2c), the reaction time is preferably 5 hours or more and 25 hours or less, and more preferably 10 hours or more and 20 hours or less.

In the production step of compound (3-2c), after completion of the reaction, it can be taken out by the same method as in the production step of compound (1-1b) described above. You may refine by. In addition, the obtained compound (3-2c) may be used in the next step without being taken out after completion of the reaction, but is taken out from the point that the yield of the target compound (3-2) is improved. Is preferred.

[Compound (3-2e) Production Process]
In the production step of compound (3-2e), compound (3-2c) and compound (3-2d) are reacted to obtain compound (3-2e).

(Compound (3-2c))
Compound (3-2c) is a known compound.
In the compound ^{(3-2c), R 321, R} 322 and ^{R 323} are the same as ^{R 321, R 322} and ^{R 323} in the compound ^{(3-2a), R 33, R} 34, R 35, R 36, ^{R 37,} R 38, R ³²⁴ and ^{X 31} are the same as ^{R 33, R 34, R 35} , R 36, R 37, R 38, R 324 and ^{X 31} in the compound (3-2b).

(Compound (3-2d))
Compound (3-2d) is a known compound.
R ³²⁵ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and at least one of an oxygen atom and an arylene group. And an alkylene group having 1 to 10 carbon atoms which may contain one of them.
Among these, in the compound (3-2d), R ³²⁵ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (3-2e))
Compound (3-2e) is a novel compound.
In the compound (3-2e), the same as those exemplified for “<Y ³¹¹ and Y ³²¹ >” in the above “<< Compound (3) >>”.
Further, in the compound ^{(3-2e), R 321, R} 322 and ^{R 323} are the same as ^{R 321, R 322} and ^{R 323} in the compound ^{(3-1a), R 33, R} 34, R 35, R ^{36, R 37, R 38,} R 324 and ^{X 31} are the same as ^{R 33, R 34, R 35} , R 36, R 37, R 38, R 324 and ^{X 31} in the compound (3-1b).

(Reaction conditions)
In the production step of compound (3-2e), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (3-2c).

In the production step of compound (3-2e), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (3-2c) used.

In the production process of compound (3-2e), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (3-2c).

In the production process of the compound (3-2e), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (3-2e), the amount of compound (3-2d) used is preferably 1 to 2 times the amount of compound (3-2c).

In the production process of compound (3-2e), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (3-2e), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (3-2e) can be taken out by the same method as in the above-mentioned compound (1-1b) production step, and the taken out compound (3-2e) may be further purified by the same method. Further, the obtained compound (3-2e) may be used in the next step without being removed after completion of the reaction, but it should be removed from the viewpoint that the yield of the target compound (3-2) is improved. Is preferred.

[Compound (3-2) Production Process]
In the production step of compound (3-2), compound (3-2) is obtained from compound (3-2e).
The method for obtaining the compound (3-2) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Incidentally, if even a protective group R ³²³ are attached, similarly protecting group is removed, a carboxyl group, a sulfonic acid group or phosphoric acid group is formed. Further, when a protecting group is bonded to R ³²⁴ , the protecting group is similarly removed.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (3-2), an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (3-2), the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production step of compound (3-2), the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.

In the production step of compound (3-2), after completion of the reaction, compound (3-2) can be taken out by the same method as in the production step of compound (1-1b), and the taken out compound (3-2) ) May be further purified by the same method.

Each compound such as the compound (3-2), the compound (3-2a), the compound (3-2b), the compound (3-2c), the compound (3-2d), the compound (3-2e), The structure can be confirmed by a known method such as magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

<< Compound (4) >>
The compound according to the fourth embodiment of the present invention is represented by the following general formula (4) (in this specification, sometimes referred to as “compound (4)”).

(In the general formula (4), R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ⁴¹ may be the same as each other. may .R ⁴² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ⁴² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, and R ⁴³ is a hydrogen atom or a carbon number of 1. R ⁴⁴ , R ⁴⁵ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently a carbon number. 1 to 10 alkyl groups or carbon 6-10 aryl group ^{.X 41} silicon atom, a phosphorus atom, a germanium atom or a tin atom ^{.Y 41} is a single _{bond, -O- (CH 2) n41 -} , - O- (CH 2) n42 —Ar ⁴¹ —, —NH— (CH ₂ ) _n43 —, or —NH— (CH ₂ ) _n44 —Ar ⁴² —, wherein n41, n42, n43 and n44 are each independently an integer of 1 to 10. Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group.)

Compound (4) is a fluorescein analog or rhodamine analog, and has a phosphate group that is hydrolyzed by ALP under alkaline conditions.
Compound (4) is a fluorescent compound that emits fluorescence when a phosphate group is eliminated by hydrolysis with ALP.

<R ⁴¹ >
R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group.
The electron donating group in R ⁴¹ is the same as those exemplified in the above “<R ²¹ >”.
The number of R ⁴¹ is 1 or 2, and is preferably 2. When there are two R ^{41 s} , they may be the same or different. Among these, when there are two R ^{41 s} , it is preferable that they are the same as each other because they are easily synthesized.

Among these, in general formula (4), R ⁴¹ is preferably a straight-chain alkoxy group having 1 to 10 carbon atoms or a straight-chain alkylamino group having 1 to 10 carbon atoms because it has high hydrophilicity. A group, an ethylamino group, a methoxy group or an ethoxy group is more preferable.

Further, in the general formula ^(4), it is preferred ^{that R 41} is two. The two R ²¹ positions in the benzene ring are preferably arranged at positions that are ortho positions relative to each other.

< ^R42 >
R ⁴² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. Among them, the “group having an anionic functional group at the terminal” in the general formula (4) is preferably a group consisting of only an anionic functional group because it is easy to synthesize.
The number of R ⁴² is 1 or 2, is preferably one. When there are two R ^{42 s} , they may be the same as or different from each other. Above all, if R ⁴² is two, since the easy synthesis, it is preferably the same to each other.

In particular, the maximum absorption wavelength of compound (4) (non-dissociation type (neutral type)) and compound (4) -1 (dissociation type (anion type)) after elimination of a phosphate group, which will be described later, greatly deviate. From the general formula (4), in the benzene ring having two R ⁴¹ and one R ⁴² , the xanthene skeleton is in the 1st position, one R ⁴¹ is in the 4th position, and the other R ⁴¹ is in the 6th position. When it does, it is preferable to have ^R42 in 3rd-position or 5th-position, and it is more preferable to have in 3rd-position.

<R ⁴³ >
Examples of the alkyl group having 1 to 10 carbon atoms for R ⁴³ include the same groups as those exemplified above in “<R ¹¹ , R ¹² and R ¹³ >”. Among these, in general formula (4), R ⁴³ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom methyl group or an ethyl group.

<R ⁴⁴ , R ⁴⁷ and R ⁴⁸ >
Examples of the halogen atom in R ⁴⁴ , R ⁴⁷ and R ⁴⁸ include the same halogen atoms as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among ^them, it is preferable that the halogen atom in R ^{44, R 47} and ^{R 48} is a chlorine atom, a bromine atom, or iodine atom.
Examples of the alkyl group having 1 to 10 carbon atoms for R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are the same as those exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ⁴⁴ , R ⁴⁷ and R ⁴⁸ is preferably a straight chain, and more preferably a methyl group or an ethyl group.

Among them, in the general formula (4), R ⁴⁸ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, and more preferably a hydrogen atom, a methyl group or an ethyl group. .
Among them, in the general formula (4), R ⁴⁴ and R ⁴⁷ are preferably the same, more preferably a hydrogen atom or a halogen atom, because they are easily synthesized.

< ^R45 and ^R46 >
Examples of the alkyl group having 1 to 10 carbon atoms for R ⁴⁵ and R ⁴⁶ are the same as those exemplified above for “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ⁴⁵ and R ⁴⁶ is preferably a linear group, and more preferably a methyl group or an ethyl group.
Examples of the aryl group having 6 to 10 carbon atoms in R ⁴⁵ and R ⁴⁶ include phenyl group, benzyl group, tolyl group, o-xylyl group, m-xylyl group, p-xylyl group and the like. Not. Among these, the aryl group having 6 to 10 carbon atoms in R ⁴⁵ and R ⁴⁶ is preferably a phenyl group or a benzyl group, and more preferably a phenyl group.

Among these, in general formula (4), R ⁴⁵ and R ⁴⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, More preferably, it is an ethyl group.

< ^X41 >
X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom.
Among these, in the general formula (4), X ⁴¹ is preferably a silicon atom.

<Y ⁴¹ >
In the general formula (4), Y ⁴¹ is a single bond, —O— (CH ₂ ) _n41 —, —O— (CH ₂ ) _n42 —Ar ⁴¹ —, —NH— (CH ₂ ) _n343 —, or —NH — (CH ₂ ) _n44 —Ar ⁴² —. In Y ⁴¹ , the bond opposite to the alkylene group of —O— or —NH— is bonded to the carbon atom constituting the hetero four-membered ring in the general formula (3). ^{_{Also, - (CH 2) n41 -}} , - Ar 41 -, - (CH 2) n43 - or ^{-Ar 42} - oxygen atoms (O), amino group (NH) or opposite bond is the general alkylene group It is bonded to the phosphate group in formula (4).
n41, n42, n43 and n44 are each independently an integer of 1 to 10.
Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group.

n41, n42, n43 and n44 are the number of repetitions of the alkylene group for each ^{Y 41.} n41, n42, n43 and n44 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .

As the substituted or unsubstituted arylene group for Y ^41, the same groups as those exemplified above for “<Y ¹¹ >” can be mentioned.

Among them, in the general formula (4), Y ⁴¹ is a single bond, —O—CH ₂ —, —O— (CH ₂ ) ₂ —, —O—CH ₂ —Ph—, —NH—CH ₂ —, —NH It is preferably — (CH ₂ ) ₂ — or —NH—CH ₂ —Ph—. Here, “Ph” represents a substituted or unsubstituted phenylene group.

Preferred examples of the compound (4) include, for example, a compound represented by the following general formula (4-1) (hereinafter sometimes abbreviated as “compound (4-1)”), or a compound represented by the following general formula ( 4-2) (hereinafter sometimes abbreviated as “compound (4-2)”) and the like.
In addition, these compounds are only examples of a preferable compound (4), and a preferable compound (4) is not limited to these.

(In General Formula (4-1), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are Each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. The .n411 and n412 is the ^{.Ar 411} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. )

< ^R411 , ^R422 , ^R421 and ^R422 >
Examples of the alkyl group having 1 to 10 carbon atoms in R ⁴¹¹ , R ⁴²² , R ⁴²¹ and R ⁴²² are the same as those exemplified in the above “<R ¹¹ , R ¹² and R ¹³ >”. Among them, the alkyl group having 1 to 10 carbon atoms in R ⁴¹¹ , R ⁴²² , R ⁴²¹ and R ⁴²² is preferably a linear group, and more preferably a methyl group or an ethyl group.

Among them, in general formula (4-1), R ⁴¹¹ and R ⁴¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or More preferred is an ethyl group.

< ^R413 and ^R423 >
R ⁴¹³ and R ⁴²³ are each independently a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group.
Among them, the “group having an anionic functional group at the terminal” in the general formulas (4-1) and (4-2) is preferably a group consisting only of an anionic functional group because it is easy to synthesize.

< ^Y411 and ^Y421 >
Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. Ar ⁴¹¹ is a substituted or unsubstituted arylene group.
Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. Ar ⁴²¹ is a substituted or unsubstituted arylene group.
n411, n412, n421 and n422 are the number of repetitions of the alkylene group for ^{Y 411} and ^{Y 421,} respectively. n411, n412, n421, and n422 are preferably an integer of 1 to 8, more preferably an integer of 1 to 6, further preferably an integer of 1 to 4, and particularly preferably an integer of 1 to 2 because of high hydrophilicity. .
Ar ⁴¹¹ and Ar ⁴²¹ are each independently a substituted or unsubstituted arylene group. Examples of the substituted or unsubstituted arylene group include the same groups as those exemplified above for “<Y ¹¹ >”.

Preferred examples of the compound (4-1) include, for example, R ⁴¹¹ and R ⁴¹² are linear alkyl groups having 1 to 10 carbon atoms, R ⁴¹³ is a group composed of only an anionic functional group, ⁴³ and R ⁴⁸ are a hydrogen atom, a methyl group or an ethyl group, R ⁴⁴ and R ⁴⁷ are a hydrogen atom, a halogen atom, a methyl group or an ethyl group, and R ⁴⁵ and R ⁴⁶ are a methyl group, an ethyl group or a phenyl group. Or a benzyl group, X ⁴¹ is a silicon atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, and n411 and n412 Are each independently an integer of 1 to 8, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.
More preferable examples of the compound (4-1) include, for example, R ⁴¹¹ and R ⁴¹² are a methyl group or an ethyl group, R ⁴¹³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ⁴³ and R ⁴⁸ Is a hydrogen atom, R ⁴⁴ and R ⁴⁷ are a hydrogen atom or a halogen atom, R ⁴⁵ and R ⁴⁶ are a methyl group or an ethyl group, X ⁴¹ is a silicon atom, Y ⁴¹¹ is a single bond, —O — (CH ₂ ) _n411 — or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, wherein n411 and n412 are each independently an integer of 1 to 6, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group And the like.

Preferred examples of the compound (4-2) include, for example, R ⁴²¹ and R ⁴²² are linear alkyl groups having 1 to 10 carbon atoms, R ⁴²³ is a group consisting of only an anionic functional group, ⁴³ and R ⁴⁸ are a hydrogen atom, a methyl group or an ethyl group, R ⁴⁴ and R ⁴⁷ are a hydrogen atom, a halogen atom, a methyl group or an ethyl group, and R ⁴⁵ and R ⁴⁶ are a methyl group, an ethyl group or a phenyl group. Or a benzyl group, X ⁴¹ is a silicon atom, Y ⁴²¹ is —NH— (CH ₂ ) _n421 —, or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 are independent of each other. And an integer of 1 to 8 and Ar ^421- substituted or unsubstituted phenylene group.
More preferable examples of the compound (4-2) include, for example, R ⁴²¹ and R ⁴²² are a methyl group or an ethyl group, R ⁴²³ is a carboxy group, a sulfonic acid group, or a phosphoric acid group, and R ⁴³ and R ⁴⁸ Is a hydrogen atom, R ⁴⁴ and R ⁴⁷ are a hydrogen atom or a halogen atom, R ⁴⁶ and R ⁴⁷ are a methyl group or an ethyl group, X ⁴¹ is a silicon atom, and Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, wherein n421 and n422 are each independently an integer of 1 to 6, and Ar ^{421 is a} substituted or unsubstituted phenylene group, etc. Is mentioned.

Among the compounds (4), preferred as the compound (4-1) are, for example, compounds represented by the following general formula (4-1-1) (hereinafter referred to as “compound (4-1-1)”). May be abbreviated), a compound represented by the following general formula (4-1-2) (hereinafter sometimes abbreviated as “compound (4-1-2)”), or a compound represented by the following general formula (4 -1-3) (hereinafter sometimes abbreviated as “compound (4-1-3)”) and the like.
Among the compounds (4), preferred as the compound (4-2) are, for example, compounds represented by the following general formula (4-2-1) (hereinafter referred to as “compound (4-2-1)”). May be abbreviated), a compound represented by the following general formula (4-2-2) (hereinafter referred to as “compound (4-2-2)”, or represented by the following general formula (4-2-3): (Hereinafter sometimes abbreviated as “compound (4-2-3)”) and the like.
In addition, these compounds are only examples of a preferable compound (4), and a preferable compound (4) is not limited to these.

(In the general formula, Y ⁴¹¹ and Y ⁴²¹ are the same as described above.)

As a preferable compound (4-1-1), for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, and n411 And n412 each independently represents an integer of 1 to 8, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (4-1-1) is, for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, n411 and n412 are each independently an integer of 1 to 6, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (4-1-2), for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, and n411 And n412 each independently represents an integer of 1 to 8, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (4-1-2) is, for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, n411 and n412 are each independently an integer of 1 to 6, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (4-1-3), for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, and n411 And n412 each independently represents an integer of 1 to 8, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.
More preferable compound (4-1-3) is, for example, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —, n411 and n412 are each independently an integer of 1 to 6, and Ar ⁴¹¹ is a substituted or unsubstituted phenylene group.

As a preferable compound (4-2-1), for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 are Examples thereof are each independently an integer of 1 to 8, and Ar ⁴²¹ substituted or unsubstituted phenylene group.
More preferable compound (4-2-1) is, for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 Are each independently an integer of 1 to 6, and Ar ^421- substituted or unsubstituted phenylene group.

As a preferable compound (4-2-2), for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 are Examples thereof are each independently an integer of 1 to 8, and Ar ⁴²¹ substituted or unsubstituted phenylene group.
More preferable compound (4-2-2) is, for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 Are each independently an integer of 1 to 6, and Ar ^421- substituted or unsubstituted phenylene group.

As a preferable compound (4-2-3), for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 are Examples thereof are each independently an integer of 1 to 8, and Ar ⁴²¹ substituted or unsubstituted phenylene group.
More preferable compound (4-2-3) is, for example, Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —, and n421 and n422 Are each independently an integer of 1 to 6, and Ar ^421- substituted or unsubstituted phenylene group.

Among the compounds (4), preferred as the compound (4-1-1) are, for example, compounds represented by the following formula (4-1-1a) (hereinafter referred to as “compound (4-1-1a)”) Or a compound represented by the following formula (4-1-1b) (hereinafter sometimes abbreviated as “compound (4-1-1b)”), or a compound represented by the following formula (4- 1-1c) (hereinafter, may be abbreviated as “compound (4-1-1c)”) and the like.
Among the compounds (4), preferred as the compound (4-1-2) are, for example, compounds represented by the following formula (4-1-2a) (hereinafter referred to as “compound (4-1-2a)”) Or a compound represented by the following formula (4-1-2b) (hereinafter sometimes abbreviated as “compound (4-1-2b)”), or a compound represented by the following formula (4- 1-2c) (hereinafter sometimes abbreviated as “compound (4-1-2c)”) and the like.
Of the compounds (4), preferred as the compound (4-1-3) are, for example, compounds represented by the following formula (4-1-3a) (hereinafter referred to as “compound (4-1-3a)”. Or a compound represented by the following formula (4-1-3b) (hereinafter sometimes abbreviated as “compound (4-1-3b)”), or a compound represented by the following formula (4- 1-3c) (hereinafter sometimes abbreviated as “compound (4-1-3c)”) and the like.
Among the compounds (4), preferred as the compound (4-2-1) are, for example, compounds represented by the following formula (4-2-1a) (hereinafter referred to as “compound (4-2-1a)”. Or a compound represented by the following formula (4-2-1b) (hereinafter sometimes abbreviated as “compound (4-2-1b)”), or a compound represented by the following formula (4- 2-1c) (hereinafter sometimes abbreviated as “compound (4-2-1c)”) and the like.
Of the compounds (4), preferred as the compound (4-2-2) are, for example, compounds represented by the following formula (4-2-2a) (hereinafter referred to as “compound (4-2-2a)”. Or a compound represented by the following formula (4-2-2b) (hereinafter sometimes abbreviated as “compound (4-2-2b)”), or a compound represented by the following formula (4- 2-2c) (hereinafter sometimes abbreviated as “compound (4-2-2c)”) and the like.
Among the compounds (4), preferred as the compound (4-2-3) are, for example, compounds represented by the following formula (4-2-3a) (hereinafter referred to as “compound (4-2-3a)”. Or a compound represented by the following formula (4-2-3b) (hereinafter sometimes abbreviated as “compound (4-2-3b)”), or a compound represented by the following formula (4- 2-3c) (hereinafter sometimes abbreviated as “compound (4-2-3c)”) and the like.
In addition, these compounds are only examples of a preferable compound (4), and a preferable compound (4) is not limited to these.

The compound (4) is hydrolyzed by ALP, the phosphate group is eliminated, and the compound (4) -1 is changed to the compound (4) -1, thereby generating near-infrared (fluorescence wavelength: about 650 nm or more) fluorescence.

Although the compound (4) of this embodiment exists as a substantially non-dissociation type (neutral type) compound, the dissociation type (anion type) is caused by the elimination of the phosphate group by an enzymatic reaction with ALP. ) Compound (4) -1. Therefore, the compound (4) of the present embodiment can be used as a fluorescent probe for measuring ALP with high sensitivity because the maximum absorption wavelength greatly changes before and after the enzyme reaction with ALP.

<< Method for Producing Compound (4) >>
Compound (4) can be produced, for example, by reacting a compound having a xanthene skeleton with a benzene derivative and a phosphate group using a known reaction according to the types of Y ⁴¹ , R ⁴¹ and R ⁴² . More specifically, it is as follows.

<Method for Producing Compound (4-1)>
R ⁴³ : hydrogen atom Among the compounds (4), the compound (4-1) is, for example, a compound represented by the following general formula (4-1a) when R ⁴³ is a hydrogen atom (hereinafter referred to as “compound (4-1a) ”and a compound represented by the following general formula (4-1b1) (hereinafter sometimes abbreviated as“ compound (4-1b1) ”) To obtain a compound represented by the following general formula (4-1c1 ′) (hereinafter sometimes abbreviated as “compound (4-1c1 ′)”) (hereinafter referred to as “compound (4-1c1 ′)”). Production process ”), a compound represented by the following general formula (4-1c1) from the compound (4-1c1 ′) (hereinafter sometimes abbreviated as“ compound (4-1c1) ”) (Hereinafter sometimes abbreviated as “compound (4-1c1) production process”), The compound (4-1c1) and a compound represented by the following general formula (4-1d1) (hereinafter sometimes abbreviated as “compound (4-1d1)”) are reacted to give the following general formula: (4-1) may be abbreviated as a “compound (4-1e1) production process” (hereinafter, abbreviated as “compound (4-1e1)”). ) And a step of obtaining compound (4-1) -1 from compound (4-1e1) (hereinafter sometimes abbreviated as “compound (4-1) -1 production step”). it can.
Hereinafter, each step will be described in detail.

(Wherein Bzl is a benzyl group and TBS is a tert-butyldimethylsilyl group. R ⁴¹⁴ is a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a terminal) X ⁴¹ , Y ⁴¹¹ , R ⁴⁴ , R 1, R ⁴ , R 4, R ⁴ , R 4, R ⁴ , R 4, R ⁴ , and R 4, ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴¹¹ , R ⁴¹² and R ⁴¹³ are all the same as above.)

[Production Process of Compound (4-1c1 ′)]
In the production step of the compound (4-1c1 ′), the compound (4-1a) and the compound (4-1b1) are reacted to obtain the compound (4-1c1 ′).

(Compound (4-1a))
Compound (4-1a) is a known compound.
In the compound (4-1a), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (4-1a), R ⁴¹¹ and R ⁴¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.

In the compound (4-1a), R ⁴¹³ is a group having an anionic functional group at the terminal.
Among them, in the compound (4-1a), R ⁴¹³ is preferably a group consisting only of an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group, because it is easy to synthesize. preferable.

(Compound (4-1b1))
Compound (4-1b1) is a known compound.
In the compound (4-1b1), R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms.
Among them, in the compound (4-1b1), R ⁴⁴ and R ⁴⁷ are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.
R ⁴⁸ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group.

In the compound (4-1b1), R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms.
Among them, in the compound (4-1b1), R ⁴⁵ and R ⁴⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group. Or it is more preferable that it is an ethyl group.

In the compound (4-1b1), X ⁴¹ is a silicon atom, a germanium atom or a tin atom.
Among these, in the compound (4-1b1), X ⁴¹ is preferably a silicon atom.

(Compound (4-1c1 ′))
The compound (4-1c1 ′) is a known compound.
In the compound ^{(4-1c1 '), R 411,} R 412 and ^{R 413} are the same as ^{R 411, R 412} and ^{R 413} in the compound ^{(4-1a), R 44, R} 45, R 46, R 47 , ^{R 48} and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-1b1).

(Reaction conditions)
In the production step of compound (4-1c1 ′), it is preferred that a strong base is mixed with compound (4-1a) in advance and then reacted with compound (4-1b1).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (4-1a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (4-1c1 ′), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-1c1 ′), the amount of compound (4-1b1) used is 0.1 to 0.3 times the amount of compound (4-1a) used. preferable.

In the production step of compound (4-1c1 ′), it is preferable to react compound (4-1a), compound (4-1b), and strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the step of producing compound (4-1c1 ′), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production process of compound (4-1c1 ′), the reaction time is preferably 5 hours or longer and 25 hours or shorter, and more preferably 10 hours or longer and 20 hours or shorter.

In the production process of compound (4-1c1 ′), after completion of the reaction, the compound (4-1c1 ′) can be taken out in the same manner as in the production process of compound (1-1b) described above. You may refine | purify by the method of. Further, the obtained compound (4-1c1 ′) may be used in the next step without being taken out after completion of the reaction. However, from the viewpoint of improving the yield of the target compound (4-1) -1. It is preferable to take out.

[Production process of compound (4-1c1)]
In the production step of the compound (4-1c1), the compound (4-1c1) is obtained from the compound (4-1c1 ′).

(Compound (4-1c1))
Compound (4-1c1) is a known compound.
In the compound ^{(4-1c1), R 411, R} 412 and ^{R 413} are the same as ^{R 411, R 412} and ^{R 413} in the compound ^{(4-1a), R 44, R} 45, R 46, R 47, R ⁴⁸ and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-1b1).

(Reaction conditions)
The production process of compound (4-1c1) is preferably carried out using a catalyst such as tetrakis (triphenylphosphine) palladium and 1,3-dimethylbarbituric acid.

In the production process of the compound (4-1c1), it is preferable to carry out the reaction in an inert gas atmosphere.
The inert gas is not particularly limited, and examples thereof include nitrogen, helium, neon, argon, krypton, and xenon.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-1c1), the reaction temperature is preferably 20 ° C. or higher and 50 ° C. or lower, more preferably 25 ° C. or higher and 45 ° C. or lower.
In the production process of compound (4-1c1), the reaction time is preferably 5 hours or longer and 25 hours or shorter, more preferably 8 hours or longer and 16 hours or shorter.

In the compound (4-1c1) production process, after completion of the reaction, the compound (4-1c1) can be removed in the same manner as in the above-mentioned compound (1-1b) production process. You may refine by. Further, the obtained compound (4-1c1) may be used in the next step without being taken out after completion of the reaction. However, since the yield of the target compound (4-1) -1 is improved, It is preferable to take out.

[Production process of compound (4-1e1)]
In the step of producing compound (4-1e), compound (4-1c1) and compound (4-1d1) are reacted to obtain compound (4-1e1).

(Compound (4-1d1))
Compound (4-1d1) is a known compound.
In the compound (4-1d1), R ⁴¹⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among these, in the compound (4-1d1), R ⁴¹⁴ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (4-1e1))
Compound (4-1e1) is a novel compound.
In the compound (4-1e1), Y ⁴¹¹ is the same as those exemplified for “<Y ⁴¹¹ and Y ⁴²¹ >” in the above “<< Compound (4) >>”.
Further, in the compound ^{(4-1e1), R 411, R} 412 and ^{R 413} are the same as ^{R 411, R 412} and ^{R 413} in the compound ^{(4-1a), R 44, R} 45, R 46, R ^{47, R 48} and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-1b1).

(Reaction conditions)
In the production step of compound (4-1e1), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (4-1c1) used.

In the production step of compound (4-1e1), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (4-1c1) used.

In the production process of compound (4-1e1), an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (4-1c1).

In the production process of the compound (4-1e1), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-1e1), the amount of compound (4-1d1) used is preferably 1 to 2 times the amount of compound (4-1c1).

In the production process of compound (4-1e1), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production process of compound (4-1e1), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (4-1e1) can be taken out in the same manner as in the above-described compound (1-1b) production step, and the taken out compound (4-1e1) may be further purified in the same manner. Further, the obtained compound (4-1e1) may be used in the next step without removal after the completion of the reaction. However, since the yield of the target compound (4-1) -1 is improved, It is preferable to take out.

[Compound (4-1) -1 Production Process]
In the production step of compound (4-1) -1, compound (4-1) -1 is obtained from compound (4-1e1).
The method for obtaining the compound (4-1) -1 is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Note that when a protective group is bonded to R ⁴¹³ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (4-1) -1, an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-1) -1, the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (4-1) -1, the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.

In the production process of compound (4-1) -1, after completion of the reaction, compound (4-1) -1 can be taken out in the same manner as in the production process of compound (1-1b). (4-1) may be further purified by the same method.

Compound (4-1) -1, Compound (4-1a), Compound (4-1b1), Compound (4-1c1 ′), Compound (4-1c1), Compound (4-1d1), Compound (4-1e1) The structure of each compound such as) can be confirmed by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

R ⁴³ : other than a hydrogen atom Among the compounds (4), the compound (4-1) is, for example, a compound represented by the following general formula (4-1a) when R ⁴³ is other than a hydrogen atom (hereinafter, “Sometimes abbreviated as“ compound (4-1a) ”), a compound represented by the following general formula (4-1b2) (hereinafter sometimes abbreviated as“ compound (4-1b2) ”), To obtain a compound represented by the following general formula (4-1c2) (hereinafter sometimes abbreviated as “compound (4-1c2)”) (hereinafter referred to as “compound (4-1c2) production”). Step ”), compound (4-1c2), compound represented by general formula (4-1d2) below (hereinafter sometimes abbreviated as“ compound (4-1d2) ”), , To give a compound represented by the following general formula (4-1e2) Compound (4-1e2) ”(hereinafter sometimes abbreviated as“ compound (4-1e2) production step ”), and compound (4-1e2) to compound (4 -1) -2 (hereinafter may be abbreviated as “production step of compound (4-1) -2”).
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group and TBS is a tert-butyldimethylsilyl group. X ⁴¹ , Y ⁴¹¹ , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴¹¹ , R ⁴¹² , R ⁴¹⁴ and R ⁴¹⁴ are all the same as above.)

[Production process of compound (4-1c2)]
In the step of producing compound (4-1c2), compound (4-1a) and compound (4-1b2) are reacted to obtain compound (4-1c2).

(Compound (4-1a))
Compound (4-1a) is a known compound.
In the compound (4-1a), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (4-1a), R ⁴¹¹ and R ⁴¹² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (4-1a), R ⁴¹³ is a group having an anionic functional group at the terminal.
Among them, in the compound (4-1a), R ⁴¹³ is preferably a group consisting only of an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. .

(Compound (4-1b2))
Compound (4-1b2) is a known compound.
In the compound ^{(4-1b2), R 43} is an alkyl group having 1 to 10 carbon atoms linear. Among these, in the compound (4-1b2), R ⁴³ is preferably a methyl group or an ethyl group.
In the compound (4-1b2), R ⁴⁴ , R ⁴⁷ , and R ⁴⁸ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms.
Among these, in the compound (4-1b2), R ⁴⁴ and R ⁴⁷ are preferably the same, and more preferably a hydrogen atom or a halogen atom, because they are easily synthesized.
R ⁴⁸ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group.

In the compound (4-1b2), R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms.
Among them, in the compound (4-1b2), R ⁴⁵ and R ⁴⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, More preferably, it is an ethyl group.

In the compound (4-1b2), X ⁴¹ is a silicon atom, a germanium atom or a tin atom.
Among these, in the compound (4-1b2), X ⁴¹ is preferably a silicon atom.

(Reaction conditions)
In the production step of compound (4-1c2), it is preferable that a strong base is mixed with compound (4-1a) in advance and then reacted with compound (4-1b2).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (4-1a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (4-1c2), an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (4-1c2), the amount of compound (4-1b2) used is preferably 0.1 to 0.3 times the amount of compound (4-1a) used. .

In the production step of compound (4-1c2), it is preferable to react compound (4-1a), compound (4-1b2), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the step of producing compound (4-1c2), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, more preferably 80 ° C. or higher and 130 ° C. or lower.
In the step of producing compound (4-1c2), the reaction time is preferably 5 hours or longer and 25 hours or shorter, more preferably 10 hours or longer and 20 hours or shorter.

In the compound (4-1c2) production process, after completion of the reaction, the compound (4-1c2) can be removed in the same manner as in the above-described compound (1-1b) production process. You may refine by. Further, the obtained compound (4-1c2) may be used in the next step without being taken out after completion of the reaction. However, since the yield of the target compound (4-1) -2 is improved, It is preferable to take out.

[Compound (4-1e) Production Process]
In the production step of the compound (4-1e2), the compound (4-1c2) and the compound (4-1d2) are reacted to obtain the compound (4-1e2).

(Compound (4-1c2))
Compound (4-1c2) is a known compound.
In the compound ^{(4-1c2), R 411, R} 412 and ^{R 413} are the same as ^{R 411, R 412} and ^{R 413} in the compound ^{(4-1a), R 43, R} 44, R 45, R 46, R ^{47, R 48} and ^{X 41} are the same as ^{R 43, R 44, R 45} , R 46, R 47, R 48 and ^{X 41} in the compound (4-1b2).

(Compound (4-1d2))
The compound (4-1d2) is a known compound.
In the compound (4-1d2), R ⁴¹⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among these, in the compound (4-1d2), R ⁴¹⁴ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (4-1e2))
Compound (4-1e2) is a novel compound.
In compound (4-1e2), Y ⁴¹¹ is the same as that exemplified for “<Y ⁴¹¹ and Y ⁴²¹ >” in “<< Compound (4) >>” above.
Further, in the compound ^{(4-1e2), R 411, R} 412 and ^{R 413} are the same as ^{R 411, R 412} and ^{R 413} in the compound ^{(4-1a), R 43, R} 44, R 45, R ^{46, R 47, R 48} and ^{X 41} are the same as ^{R 43, R 44, R 45} , R 46, R 47, R 48 and ^{X 41} in the compound (4-1b2).

(Reaction conditions)
In the production step of compound (4-1e2), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (4-1c2) used.

In the production step of compound (4-1e2), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (4-1c2) used.

In the production process of compound (4-1e2), an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (4-1c2).

In the production process of compound (4-1e2), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (4-1e2), the amount of compound (4-1d2) used is preferably 1 to 2 times the amount of compound (4-1c2).

In the production step of compound (4-1e2), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (4-1e2), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (4-1e2) can be taken out by the same method as in the above-mentioned compound (1-1b) production step, and the taken out compound (4-1e2) may be further purified by the same method. Further, the obtained compound (4-1e2) may be used in the next step without being taken out after completion of the reaction. However, since the yield of the target compound (4-1) -2 is improved, It is preferable to take out.

[Production Process of Compound (4-1) -2]
In the production step of compound (4-1) -2, compound (4-1) -2 is obtained from compound (4-1e2).
The method for obtaining the compound (4-1) -2 is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. In addition, also when a protective group has couple ^| bonded with ^R413 and ^R43 , a protective group is similarly removed and a carboxy group, a sulfonic acid group, or a phosphoric acid group is formed.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (4-1) -2, an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-1) -2, the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (4-1) -2, the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.

In the production process of compound (4-1) -2, after completion of the reaction, compound (4-1) -2 can be taken out in the same manner as in the production process of compound (1-1b). (4-1) -2 may be further purified by the same method.

Each compound such as compound (4-1) -2, compound (4-1a), compound (4-1b2), compound (4-1c2), compound (4-1d2), compound (4-1e2), etc. The structure can be confirmed by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

<Method for Producing Compound (4-2)>
R ⁴³ : hydrogen atom Of the compound (4), the compound (4-2) is, for example, a compound represented by the following general formula (4-2a) when R ⁴³ is a hydrogen atom (hereinafter referred to as “compound (4-2a) ”and a compound represented by the following general formula (4-2b1) (hereinafter, sometimes abbreviated as“ compound (4-2b1) ”) To obtain a compound represented by the following general formula (4-2c1 ′) (hereinafter sometimes abbreviated as “compound (4-2c1 ′)”) (hereinafter referred to as “compound (4-2c1 ′)”). Production process ”), a compound represented by the following general formula (4-2c1) from the compound (4-2c1 ′) (hereinafter sometimes abbreviated as“ compound (4-2c1) ”) (Hereinafter sometimes abbreviated as “compound (4-2c1) production process”), The compound (4-2c1) is reacted with a compound represented by the following general formula (4-2d1) (hereinafter sometimes abbreviated as “compound (4-2d1)”) to give the following general formula: (4-2-2) may be abbreviated as “compound (4-2e1) production step” (hereinafter, referred to as “compound (4-2e1) production step”). ) And a step of obtaining compound (4-2) -1 from compound (4-2e1) (hereinafter sometimes abbreviated as “compound (4-2) -1 production step”). it can.
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group and Ph is an unsubstituted phenyl group. R ⁴²⁴ is a hydrogen atom, a hydroxyl group, a leaving group (for example, a halogen atom), or a leaving group (for example, a terminal group) And an alkylene group having 1 to 10 carbon atoms, which may contain at least one of an oxygen atom and an arylene group, X ⁴¹ , Y ⁴²¹ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴²¹ , R ⁴²² and R ⁴²³ are all the same as above.)

[Production Process of Compound (4-2c1 ′)]
In the production step of the compound (4-2c1 ′), the compound (4-2a) and the compound (4-2b1) are reacted to obtain the compound (4-2c1 ′).

(Compound (4-2a))
Compound (4-2a) is a known compound.
In the compound (4-2a), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (4-2a), R ⁴²¹ and R ⁴²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (4-2a), R ⁴²³ is a group having an anionic functional group at the terminal.
Among these, in the compound (4-2a), R ⁴²³ is preferably a group consisting only of an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. preferable.

(Compound (4-2b1))
Compound (4-2b1) is a known compound.
In the compound (4-2b1), R ⁴⁴ , R ⁴⁷ , and R ⁴⁸ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms.
Among these, in the compound (4-2b1), R ⁴⁴ and R ⁴⁷ are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.
R ⁴⁸ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group.

In the compound (4-2b1), R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms.
Among them, in the compound (4-2b1), R ⁴⁵ and R ⁴⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, More preferably, it is an ethyl group.

In the compound (4-2b1), X ⁴¹ is a silicon atom, a germanium atom or a tin atom.
Among these, in the compound (4-2b1), X ⁴¹ is preferably a silicon atom.

(Compound (4-2c1 ′))
The compound (4-2c1 ′) is a known compound.
In the compound ^{(4-2c1 '), R 421,} R 422 and ^{R 423} are the same as ^{R 421, R 422,} and ^{R 423} in the compound ^{(4-2a), R 44, R} 45, R 46, R ^{47, R 48} and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-2b1).

(Reaction conditions)
In the production step of compound (4-2c1 ′), it is preferred that a strong base is previously mixed with compound (4-2a) and then reacted with compound (4-2b1).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (4-2a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (4-2c1 ′), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-2c1 ′), the amount of compound (4-2b1) used is 0.1 to 0.3 times the amount of compound (4-2a) used. preferable.

In the production step of compound (4-2c1 ′), it is preferable to react compound (4-2a), compound (4-2b1), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the production process of compound (4-2c1 ′), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, and more preferably 80 ° C. or higher and 130 ° C. or lower.
In the production process of compound (4-2c1 ′), the reaction time is preferably 5 hours or longer and 25 hours or shorter, more preferably 10 hours or longer and 20 hours or shorter.

In the production process of the compound (4-2c1 ′), after completion of the reaction, the compound (4-2c1 ′) can be taken out in the same manner as in the production process of the compound (1-1b) described above. You may refine | purify by the method of. The obtained compound (4-2c1 ′) may be used in the next step without being taken out after completion of the reaction. However, the yield of the target compound (4-2) -1 is improved. It is preferable to take out.

[Production process of compound (4-2c1)]
In the production step of the compound (4-2c1), the compound (4-2c1) is obtained from the compound (4-2c1 ′).

(Compound (4-2c1))
Compound (4-2c1) is a known compound.
In the compound ^{(4-2c1), R 421, R} 422 and ^{R 413} are the same as ^{R 421, R 422} and ^{R 413} in the compound ^{(4-2a), R 44, R} 45, R 46, R 47, R ⁴⁸ and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-2b1).

(Reaction conditions)
The production process of the compound (4-2c1) is preferably carried out using a catalyst such as tetrakis (triphenylphosphine) palladium and 1,3-dimethylbarbituric acid.

In the production process of compound (4-2c1), it is preferable to carry out the reaction in an inert gas atmosphere.
The inert gas is not particularly limited, and examples thereof include nitrogen, helium, neon, argon, krypton, and xenon.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (4-2c1), the reaction temperature is preferably 20 ° C. or higher and 50 ° C. or lower, more preferably 25 ° C. or higher and 45 ° C. or lower.
In the production step of compound (4-2c1), the reaction time is preferably 5 hours or longer and 25 hours or shorter, and more preferably 8 hours or longer and 16 hours or shorter.

In the production step of compound (4-2c1), after completion of the reaction, it can be taken out in the same manner as in the production step of compound (1-1b) described above, and the taken out compound (4-2c1) is further treated in the same manner. You may refine by. Further, the obtained compound (4-2c1) may be used in the next step without being taken out after completion of the reaction. However, since the yield of the target compound (4-2) -1 is improved, It is preferable to take out.

[Production process of compound (4-2e1)]
In the production step of the compound (4-2e1), the compound (4-2c1) and the compound (4-2d1) are reacted to obtain the compound (4-2e1).

(Compound (4-2d1))
The compound (4-2d1) is a known compound.
In the compound (4-2d1), R ⁴²⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among these, in the compound (4-2d1), R ⁴²⁴ is preferably a hydrogen atom, a halogen atom, —O—CH ₂ —X, or —O—Ph—CH ₂ —X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (4-2e1))
Compound (4-2e1) is a novel compound.
In the compound (4-2e1), Y ⁴²¹ is the same as those exemplified for “<Y ⁴¹¹ and Y ⁴²¹ >” in the above “<< Compound (4) >>”.
Further, in the compound ^{(4-2e1), R 421, R} 422 and ^{R 423} are the same as ^{R 421, R 422} and ^{R 423} in the compound ^{(4-2a), R 44, R} 45, R 46, R ^{47, R 48} and ^{X 41} are the same as ^{R 44, R 45, R 46} , R 47, R 48 and ^{X 41} in the compound (4-2b1).

(Reaction conditions)
In the production step of compound (4-2e1), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (4-2c1).

In the production step of compound (4-2e1), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (4-2c1) used.

In the production process of compound (4-2e1), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (4-2c1).

In the production process of the compound (4-2e1), the reaction is preferably performed in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (4-2e1), the amount of compound (4-2d1) used is preferably 1 to 2 times the amount of compound (4-2c1).

In the production step of compound (4-2e1), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (4-2e1), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

Compound (4-2e1) can be taken out in the same manner as in the above-described compound (1-1b) production step, and the taken out compound (4-2e1) may be further purified in the same manner. Further, the obtained compound (4-2e1) may be used in the next step without removal after the completion of the reaction. However, since the yield of the target compound (4-2) -1 is improved, It is preferable to take out.

[Compound (4-2) -1 Production Process]
In the production step of compound (4-2) -1, compound (4-2) -1 is obtained from compound (4-2e1).
The method for obtaining the compound (4-2) is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Even when a protective group is bonded to R ⁴²³ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (4-2) -1, an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-2) -1, the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (4-2) -1, the reaction time is preferably 30 minutes or longer and 5 hours or shorter, more preferably 1 hour or longer and 3 hours or shorter.

In the production process of compound (4-2) -1, after completion of the reaction, compound (4-2) -1 can be taken out in the same manner as in the production process of compound (1-1b). (4-2) -1 may be further purified by the same method.

Each compound such as compound (4-2) -1, compound (4-2a), compound (4-2b1), compound (4-2c1), compound (4-2d1), compound (4-2e1) The structure can be confirmed by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

R ⁴³ : other than a hydrogen atom Among the compounds (4), the compound (4-2) is, for example, a compound represented by the following general formula (4-2a) (wherein R ⁴³ is other than a hydrogen atom) “Sometimes abbreviated as“ compound (4-2a) ”), a compound represented by the following general formula (4-2b2) (hereinafter sometimes abbreviated as“ compound (4-2b2) ”), To obtain a compound represented by the following general formula (4-2c2) (hereinafter sometimes abbreviated as “compound (4-2c2)”) (hereinafter referred to as “compound (4-2c2) production). Step (sometimes abbreviated as “step”), compound (4-2c2), compound represented by the following general formula (4-2d2) (hereinafter sometimes abbreviated as “compound (4-2d2)”), , To give a compound represented by the following general formula (4-2e2) Compound (4-2e2) ”(hereinafter sometimes abbreviated as“ compound (4-2e2) production step ”), and compound (4-2e2) to compound (4 -2) It can be produced by a production method having a step of obtaining -2 (hereinafter sometimes abbreviated as "compound (4-2) -2 production step").
Hereinafter, each step will be described in detail.

(In the formula, Bzl is a benzyl group and Ph is an unsubstituted phenyl group. X ⁴¹ , Y ⁴²¹ , R ⁴³ , R ⁴⁴ , R ⁴⁵ , R ⁴⁶ , R ⁴⁷ , R ⁴⁸ , R ⁴²¹ , R ⁴²² , R ⁴²³ and R ⁴²⁴ are the same as above.)

[Production process of compound (4-2c2)]
In the step of producing compound (4-2c2), compound (4-2a) and compound (4-2b2) are reacted to obtain compound (4-2c2).

(Compound (4-2a))
Compound (4-2a) is a known compound.
In the compound (4-2a), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. Among them, in the compound (4-2a), R ⁴²¹ and R ⁴²² are preferably the same because they are easy to synthesize, more preferably a linear alkyl group having 1 to 10 carbon atoms, a methyl group or an ethyl group Is more preferable.
In the compound (4-2a), R ⁴²³ is a group having an anionic functional group at the terminal.
Among these, in the compound (4-2a), R ⁴²³ is preferably a group consisting of only an anionic functional group, and more preferably a carboxy group, a sulfonic acid group, or a phosphoric acid group because it is easy to synthesize. .

(Compound (4-2b2))
The compound (4-2b2) is a known compound.
In the compound (4-2b2), R ⁴³ is a linear alkyl group having 1 to 10 carbon atoms. Among these, in the compound (4-2b2), R ⁴³ is preferably a methyl group or an ethyl group.
In the compound (4-2b2), R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms.
Among them, in the compound (4-2b2), R ⁴⁴ and R ⁴⁷ are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.
R ⁴⁸ is preferably a hydrogen atom or a linear alkyl group having 1 to 10 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group.

In the compound (4-2b2), R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms.
Among them, in the compound (4-2b2), R ⁴⁵ and R ⁴⁶ are preferably the same because they are easily synthesized, and are preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, More preferably, it is an ethyl group.

In the compound (4-2b2), X ⁴¹ is a silicon atom, a germanium atom or a tin atom.
Among these, in the compound (4-2b2), X ⁴¹ is preferably a silicon atom.

(Reaction conditions)
In the production step of compound (4-2c2), it is preferable that a strong base is mixed with compound (4-2a) in advance and then reacted with compound (4-2b2).
Examples of the strong base include sec-butyllithium.
The amount of the strong base used is preferably 0.5 to 1.0 times the amount of the compound (4-2a) used.
The temperature when mixing with a strong base in advance is preferably −90 ° C. or higher and −60 ° C. or lower.
The time for mixing with a strong base in advance is preferably from 10 minutes to 1 hour.

In the production process of compound (4-2c2), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-2c2), the amount of compound (4-2b2) used is preferably 0.1 to 0.3 times the amount of compound (4-2a) used. .

In the production step of compound (4-2c2), it is preferable to react compound (4-2a), compound (4-2b2), and a strong base under acidic conditions.
Examples of the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
In the reaction, for example, the amount of acid used is preferably 1 M or more and 5 M or less, for example.

In the production step of compound (4-2c2), the reaction temperature is preferably 70 ° C. or higher and 150 ° C. or lower, and more preferably 80 ° C. or higher and 130 ° C. or lower.
In the step of producing compound (4-2c2), the reaction time is preferably 5 hours or longer and 25 hours or shorter, more preferably 10 hours or longer and 20 hours or shorter.

In the production step of compound (4-2c2), after completion of the reaction, it can be taken out in the same manner as in the production step of compound (1-1b) described above, and the taken out compound (4-2c2) is further treated in the same manner. You may refine by. Further, the obtained compound (4-2c2) may be used in the next step without being taken out after the completion of the reaction. However, since the yield of the target compound (4-2) -2 is improved, It is preferable to take out.

[Production process of compound (4-2e2)]
In the production step of the compound (4-2e2), the compound (4-2c2) and the compound (4-2d2) are reacted to obtain the compound (4-2e2).

(Compound (4-2c2))
Compound (4-2c2) is a known compound.
In the compound ^{(4-2c2), R 421, R} 422 and ^{R 423} are the same as ^{R 421, R 422} and ^{R 423} in the compound ^{(4-2a), R 43, R} 44, R 45, R 46, R ^{47, R 48} and ^{X 41} are the same as ^{R 43, R 44, R 45} , R 46, R 47, R 48 and ^{X 41} in the compound (4-2b2).

(Compound (4-2d2))
The compound (4-2d2) is a known compound.
In the compound (4-2d2), R ⁴²⁴ has a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a halogen atom) at the terminal, and an oxygen atom and An alkylene group having 1 to 10 carbon atoms which may contain at least one of arylene groups.
Among them, the compound ^{(4-2d2), R 424} represents a hydrogen atom, a halogen atom, _-O-CH 2 -X, or, preferably a _-O-Ph-CH 2 -X. Here, “X” represents a halogen atom, and “Ph” represents a substituted or unsubstituted phenylene group.

(Compound (4-2e2))
Compound (4-2e2) is a novel compound.
In the compound (4-2e2), Y ⁴²¹ is the same as that exemplified for “<Y ⁴¹¹ and Y ⁴²¹ >” in the above “<< Compound (4) >>”.
Further, in the compound ^{(4-2e2), R 421, R} 422 and ^{R 423} are the same as ^{R 421, R 422} and ^{R 423} in the compound ^{(4-2a), R 43, R} 44, R 45, R ^{46, R 47, R 48} and ^{X 41} are the same as ^{R 43, R 44, R 45} , R 46, R 47, R 48 and ^{X 41} in the compound (4-2b2).

(Reaction conditions)
In the production step of compound (4-2e2), it is preferable to carry out the reaction using a base.
The base is not particularly limited, and examples thereof include trialkylamines such as triethylamine and DIEA.
The bases may be used alone or in combination of two or more, and when two or more are used in combination, their combination and ratio can be arbitrarily selected.
The amount of the base used is preferably 1 to 3 times the amount of the compound (4-2c2).

In the production step of compound (4-2e2), it is preferable to carry out the reaction using a condensing agent.
The condensing agent is not particularly limited, and examples thereof include DMAP.
The said condensing agent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the condensing agent used is preferably 0.05 mol amount or more and 0.2 times mol amount or less of the amount of the compound (4-2c2) used.

In the production process of compound (4-2e2), it is preferable to use an aprotic solvent as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.
The amount of the solvent used is preferably 1 to 5 times the amount of the compound (4-2c2).

In the production process of the compound (4-2e2), it is preferable to carry out the reaction in an inert gas atmosphere.
Examples of the inert gas include those similar to those exemplified in “[Compound (1-1d) Production Process]”.
The said inert gas may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production step of compound (4-2e2), the amount of compound (4-2d2) used is preferably 1 to 2 times the amount of compound (4-2c2).

In the production process of compound (4-2e2), the reaction temperature is preferably −50 ° C. or higher and 0 ° C. or lower, and more preferably −30 ° C. or higher and −5 ° C. or lower.
In the production step of compound (4-2e2), the reaction time is preferably 30 minutes or longer and 10 hours or shorter, more preferably 1 hour or longer and 5 hours or shorter.

The compound (4-2e2) can be extracted by the same method as in the above-described compound (1-1b) production process, and the extracted compound (4-2e2) may be further purified by the same method. Further, the obtained compound (4-2e2) may be used in the next step without being taken out after the completion of the reaction. However, since the yield of the target compound (4-2) -2 is improved, It is preferable to take out.

[Production process of compound (4-2) -2]
In the production step of compound (4-2) -2, compound (4-2) -2 is obtained from compound (4-2e2).
The method for obtaining the compound (4-2) -2 is a known deprotection reaction. That is, in this step, the benzyl group is removed and a hydroxyl group is formed. Even when a protective group is bonded to R ⁴²³ , the protective group is similarly removed to form a carboxy group, a sulfonic acid group, or a phosphoric acid group. Further, when a protective group is bonded to R ⁴³ , the protective group is similarly removed.
The deprotection reaction can be performed, for example, under reducing conditions.

Examples of what is used for reducing conditions include a method using a palladium carbon catalyst in a hydrogen atmosphere, a birch reduction method using sodium / liquid ammonia, and the like.

In the production process of compound (4-2) -2, an aprotic solvent is preferably used as a reaction solvent.
Examples of the aprotic solvent include the same solvents as those exemplified in “[Compound (1-1d) Production Process]”.
The said solvent may be used individually by 1 type, may use 2 or more types together, and when using 2 or more types together, those combinations and ratios can be selected arbitrarily.

In the production process of compound (4-2) -2, the reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
In the production process of compound (4-2) -2, the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.

In the production process of compound (4-2) -2, after completion of the reaction, compound (4-2) -2 can be taken out in the same manner as in the production process of compound (1-1b). (4-2) -2 may be further purified by the same method.

Each compound such as compound (4-2) -2, compound (4-2a), compound (4-2b2), compound (4-2c), compound (4-2d2), compound (4-2e2) is, for example, The structure can be confirmed by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).

≪Fluorescent probe for ALP detection≫
A fluorescent probe for detecting ALP according to an embodiment of the present invention includes a compound having an anionic functional group and a phosphate group.

According to the fluorescent probe for ALP detection of the present embodiment, the enzymatic activity of ALP in a biological sample can be measured with high quantitativeness and sensitivity.

<Compound>
The compound in this embodiment has an anionic functional group and a phosphate group.
In the present specification, “anionic functional group” means a functional group that ionically dissociates with a negative charge in an aqueous medium, and “cationic functional group” means a positive charge in an aqueous medium. It means a functional group that has ionic dissociation.
Examples of the anionic functional group include a hydroxyl group, a thiol group, a carboxy group, a dithiocarboxy group, a hydroxythioxomethyl group, a mercaptocarbonyl group, a sulfonic acid group, a phosphoric acid group, a cyano group, and salts thereof. Can be mentioned. Especially, as said anionic functional group, a carboxy group, a sulfonic acid group, or a phosphoric acid group is preferable.

The compound in this embodiment does not emit fluorescence as it is, but emits fluorescence for the first time when the phosphate group is eliminated by hydrolysis with ALP.

More specifically, examples of the compound in the present embodiment include the above-mentioned compound (1), the above-mentioned compound (2), the above-mentioned compound (3), the above-mentioned compound (4), and the like.
Preferred examples of the compound (1) include the above compound (1-1) and the above compound (1-2).
Preferred examples of the compound (2) include the above compound (2-1) and the above compound (2-2).
Preferred examples of the compound (3) include the above compound (3-1) and the above compound (3-2).
Preferred examples of the compound (4) include the above compound (4-1) and the above compound (4-2).
These compounds are only examples of preferred compounds (1), (2), (3) and (4), and preferred compounds (1), (2), (3) and (4) are limited to these. Not.

Among the compounds (1), preferable examples of the compound (1-1) include, for example, the above compound (1-1-1), the above compound (1-1-2), and the above compound (1-1). -3).
Among the compounds (1), preferable examples of the compound (1-2) include, for example, the above compound (1-2-1), the above compound (1-2-2), and the above compound (1-2). -3).

Among the compounds (2), preferable examples of the compound (2-1) include, for example, the above compound (2-1-1), the above compound (2-1-2), and the above compound (2-1). -3).
Among the compounds (2), preferable examples of the compound (2-2) include, for example, the above compound (2-2-1), the above compound (2-2-2), and the above compound (2-2). -3).

Among the compounds (3), preferred as the compound (3-1) are, for example, the above compound (3-1-1), the above compound (3-1-2), and the above compound (3-1). -3).
Among the compounds (3), preferred as the compound (3-2) are, for example, the above compound (3-2-1), the above compound (3-2-2), and the above compound (3-2). -3).

Among the compounds (4), preferred as the compound (4-1) are, for example, the above compound (4-1-1), the above compound (4-1-2), and the above compound (4-1). -3).
Among the compounds (4), preferable examples of the compound (4-2) include, for example, the above compound (4-2-1), the above compound (4-2-2), and the above compound (4-2). -3).
These compounds are only examples of preferred compounds (1), (2), (3) and (4), and preferred compounds (1), (2), (3) and (4) are limited to these. Not.

Among the compounds (1), preferable examples of the compound (1-1-1) include, for example, the above compound (1-1-1a), the above compound (1-1-1b), or the above compound (1-1-1c) and the like.
Among the compounds (1), preferred as the compound (1-1-2) are, for example, the above compound (1-1-2a), the above compound (1-1-2b), or the above compound (1-1-2c) and the like.
Among the compounds (1), preferred as the compound (1-1-3) are, for example, the above compound (1-1-3a), the above compound (1-1-3b), or the above compound (1-1-3c) and the like.
Among the compounds (1), preferable examples of the compound (1-2-1) include, for example, the above compound (1-2-1a), the above compound (1-2-1b), or the above compound (1-2-1c) and the like.
Among the compounds (1), preferred as the compound (1-2-2) are, for example, the above compound (1-2-2a), the above compound (1-2-2b), or the above compound (1-2-2c) and the like.
Among the compounds (1), preferable examples of the compound (1-2-3) include, for example, the above compound (1-2-3a), the above compound (1-2-3b), or the above compound (1-2-3c) and the like.

Among the compounds (2), preferable examples of the compound (2-1-1) include, for example, the above compound (2-1-1a), the above compound (2-1-1b), or the above compound (2-1-1c) and the like.
Among the compounds (2), preferable examples of the compound (2-1-2) include, for example, the above compound (2-1-2a), the above compound (2-1-2b), or the above compound (2-1-2c) and the like.
Among the compounds (2), preferred as the compound (2-1-3) are, for example, the above compound (2-1-3a), the above compound (2-1-3b), or the above compound (2-1-3c) and the like.
Among the compounds (2), preferable examples of the compound (2-2-1) include, for example, the above compound (2-2-1a), the above compound (2-2-1b), or the above compound (2-2-1c).
Among the compounds (2), preferable examples of the compound (2-2-2) include, for example, the above compound (2-2-2a), the above compound (2-2-2b), or the above compound (2-2-2c).
Among the compounds (2), preferred as the compound (2-2-3) are, for example, the above compound (2-2-3a), the above compound (2-2-3b), or the above compound (2-2-3c) and the like.

Among the compounds (3), preferred as the compound (3-1-1) are, for example, the above compound (3-1-1a), the above compound (3-1-1b), or the above compound (3-1-1c).
Among the compounds (3), preferred as the compound (3-1-2) are, for example, the above compound (3-1-2a), the above compound (3-1-2b)), or the above compound (3-1-2). Examples thereof include compound (3-1-2c).
Among the compounds (3), preferred as the compound (3-1-3) are, for example, the above compound (3-1-3a), the above compound (3-1-3b)), or the above compound (3-1-3). Examples thereof include compound (3-1-3c).
Among the compounds (3), preferred as the compound (3-2-1) are, for example, the above compound (3-2-1a), the above compound (3-2-1b)), or the above compound (3-2-1). Examples thereof include compound (3-2-1c).
Among the compounds (3), preferred as the compound (3-2-2) are, for example, the above compound (3-2-2a), the above compound (3-2-2b), or the above compound (3-2-2c) and the like.
Among the compounds (3), preferred as the compound (3-2-3) are, for example, the above compound (3-2-3a), the above compound (3-2-3b), or the above compound (3-2-3c) and the like.

Among the compounds (4), preferred as the compound (4-1-1) are, for example, the above compound (4-1-1a), the above compound (4-1-1b), or the above compound (4-1-1c) and the like.
Among the compounds (4), preferable examples of the compound (4-1-2) include, for example, the above compound (4-1-2a), the above compound (4-1-2b), or the above compound (4-1-2c) and the like.
Among the compounds (4), preferable examples of the compound (4-1-3) include, for example, the above compound (4-1-3a), the above compound (4-1-3b), or the above compound (4-1-3c).
Among the compounds (4), preferable examples of the compound (4-2-1) include, for example, the above compound (4-2-1a), the above compound (4-2-1b), or the above compound (4-2-1c) and the like.
Among the compounds (4), preferable examples of the compound (4-2-2) include, for example, the above compound (4-2-2a), the above compound (4-2-2b), or the above compound (4-2-2c) and the like.
Among the compounds (4), preferable examples of the compound (4-2-3) include, for example, the above compound (4-2-3a), the above compound (4-2-3b), or the above compound (4-2-3c) and the like.
These compounds are only examples of preferred compounds (1), (2), (3) and (4), and preferred compounds (1), (2), (3) and (4) are limited to these. Not.

It is preferable to add the fluorescent probe for ALP detection of the present embodiment to about 1 nM to 1 mM, particularly about 1 μM to 50 μM to a biological sample containing ALP.
The biological sample is not particularly limited as long as it contains ALP, for example, a body fluid sample collected from a test animal, a cell extract collected from a test animal, a culture cell extract, etc. But are not limited to these.

In this embodiment, the test animal is not particularly limited, and examples thereof include, but are not limited to, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and the like.

More specifically, as a body fluid sample collected from the test animal, for example, blood, serum, plasma, urine, puffy coat, saliva, semen, chest exudate, cerebrospinal fluid, tears, sputum, mucus, Examples include, but are not limited to, lymph, ascites, pleural effusion, amniotic fluid, bladder lavage fluid, and bronchoalveolar lavage fluid.

More specifically, examples of the types of cells or cultured cells collected from test animals include cells derived from tissues such as liver, kidney, osteoblast, placenta, and small intestine, but are not limited thereto.

<Application>
The fluorescent probe for detecting ALP of the present embodiment reacts, for example, a target substance (for example, an antigen or the like) fixed on a blot membrane with a specific binding substance (for example, an antibody against the antigen) for the target substance, Furthermore, it can be used in a blotting method such as a method of detecting with a secondary binding substance (for example, an ALP-labeled secondary antibody) specific to the specific binding substance labeled with ALP, a method of modifying or modifying the method.
Examples of the target substance include biological substances such as proteins, nucleic acids, lipids, and saccharides.
Specific examples of the blotting method include blotting methods widely used in the industry such as Western blotting, Southern blotting, Northern blotting, dot / slot blotting, and colony blotting. Furthermore, immunochromatography and the like are also included in the blotting method.

Alternatively, the ALP detection fluorescent probe of the present embodiment can be used in a method for detecting the enzymatic activity of ALP in a biological sample by providing it in a well of a microdevice described later.

≪Micro device≫
A microdevice according to an embodiment of the present invention includes the above-described fluorescent probe for ALP detection.

According to the microdevice of this embodiment, the enzymatic activity of ALP in a biological sample can be detected with high quantitativeness and sensitivity.

The material of the microdevice is not particularly limited, and examples thereof include a glass material, silicon, a plastic including a dendritic polymer, or a copolymer. Examples of the glass material include soda lime glass, Pyrex (registered trademark) glass, Vycor (registered trademark) glass, and quartz glass. Examples of the resin polymer include poly (vinyl chloride), poly (vinyl alcohol), poly (methyl methacrylate), poly (vinyl acetate-co-maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cyclic olefin polymer, Fluorocarbon polymer, polystyrene, polypropylene, polyethyleneimine and the like can be mentioned. Examples of the copolymer include poly (vinyl acetate-co-maleic anhydride), poly (styrene-co-maleic anhydride), poly (ethylene-co-acrylic acid), and derivatives thereof.
Examples of the shape of the microdevice include a multi-well plate in which an arbitrary number of wells are arranged as shown in FIG. 1A. Examples of the number of wells include 1 to 1 million, for example, 10 to 500,000, for example, about 100,000 per plate.

The pore diameter of the well of the microdevice of the present embodiment may be, for example, 10 nm or more and 10 μm or less, for example, 100 nm or more and 10 μm or less, for example, 1 μm or more and 10 μm or less.
Further, the depth of the well of the microdevice of the present embodiment may be, for example, 10 nm or more and 1 μm or less, for example, 100 nm or more and 800 μm or less, for example, 200 nm or more and 700 nm or less.
When the pore diameter and depth are within the above ranges, one molecule of ALP can be captured in the well, and the enzyme activity of each ALP molecule in the biological sample can be detected.

The microdevice of this embodiment may include one type of the above-described fluorescent probe for ALP detection per well.
Thereby, with respect to ALP1 molecule in a biological sample, the fluorescence intensity of one type of fluorescent probe for ALP detection can be detected, and the enzyme activities of ALP1 molecules can be compared.

In addition, the microdevice of the present embodiment may include two or more kinds of the above-described fluorescent probes for ALP detection having different reaction points in one well and having different fluorescence wavelengths.
In the present specification, “having different reaction points” means that the cleavage position by ALP is different, that is, a phosphate group hydrolyzed by ALP from the mother nucleus (for example, coumarin skeleton) of a fluorescent compound. It means that the length is different. Specifically, as two types of ALP detection fluorescent probes having different reaction points and different fluorescence wavelengths, for example, the following compound (1-1-1a) and compound (2-2-1b) Is mentioned.

It is known that ALP has subtypes such as ALP1, ALP2, ALP3, ALP4, ALP5, ALP6, and ALPI. The characteristics of each subtype are as follows.
ALP1: high molecular ALP. The bile duct obstruction increases the internal pressure of the bile duct, and the bile component appears in the blood in a state of flowing back into the sinusoids. When ALP1 appears, it is accompanied by an increase in ALP2.
ALP2: Hepatic ALP. Consists of adult serum ALP. If the bile duct is damaged in some way, synthesis in the liver increases and blood levels increase.
ALP3: Bone ALP. The main component of pediatric serum ALP. It increases with bone renewal.
ALP4: placental ALP. Detected as ALP derived from placenta in serum in late pregnancy. In addition, it is detected as ALP produced by tumor cells in the serum of malignant tumor patients with low frequency.
ALP5: small intestinal ALP. This ALP enters the large circulatory system through the thoracic lymph from the small intestinal mucosa with the absorption of fat. However, in humans with blood types B and O, even if they are normal, they appear in the blood, and in liver cirrhosis, chronic renal failure, diabetes, etc., the degree is enhanced. This is thought to be due to a decrease in processing in the liver. There is familial hyperalpemia due to increased small bowel ALP.
ALP6: immunoglobulin-bound ALP. It often appears at the extreme stage of ulcerative colitis.
ALPI: isozyme detected by polyacrylamide gel electrophoresis. Liver cancer ALP (variant ALP) produced by hepatocellular carcinoma. It is a tumor marker for hepatocellular carcinoma.

By providing two or more kinds of the above-described fluorescent probes for ALP detection having different reaction points in one well and different fluorescent wavelengths, ALP subtypes can be classified from the fluorescence intensity pattern.
Furthermore, a biological sample derived from a healthy subject and a subject having a disease using a microdevice having two or more kinds of the above-described fluorescent probes for detecting ALP having different reaction points in one well. Thus, it can be applied to the discovery of a subtype of ALP that is found in a disease-specific manner and a method for diagnosing the disease (see FIG. 2).

In addition, the amount of the fluorescent probe for ALP detection contained in one well of the microdevice of the present embodiment may be, for example, 100 nM or more and 100 μM or less, for example, 1 μM or more and 100 μM or less, for example, 10 μM or more and 100 μM or less. I just need it.

As a method of using the microdevice of this embodiment, first, a solution containing a biological sample is added to the microdevice. Next, sealing oil is dropped to encapsulate ALP in the biological sample in the well of the microdevice. At this time, the phosphate group is eliminated due to hydrolysis by ALP and emits fluorescence. However, since the conventional compound (for example, compound (10)) has high fat solubility, there existed a problem which melt | dissolves in sealing oil and leaks out of a microdevice (refer FIG. 1B).
On the other hand, in the microdevice of this embodiment, the compound (eg, compound (1-1-1a)) contained in the fluorescent probe has an anionic functional group (eg, carboxy group), and thus does not dissolve in the sealing oil. Does not leak out of the microdevice (see FIG. 1B). Thereby, ALP can be detected with high quantitativeness and sensitivity.

<< Method for detecting enzyme activity of ALP in biological sample >>
The method for detecting the enzymatic activity of alkaline phosphatase in a biological sample according to an embodiment of the present invention is a method using the above-described microdevice.

According to the detection method of this embodiment, the enzymatic activity of ALP in a biological sample can be detected with high quantitativeness and sensitivity.
Details of the detection method of this embodiment will be described below.

[Step 1]
First, a solution containing a biological sample is added to a microdevice including the above-described ALP detection fluorescent probe. Examples of the biological sample include those similar to those exemplified in the above-mentioned “<< ALP detection fluorescent probe >>”.
The pH of the solution containing the biological sample may be a value close to that in the living body, and specifically, for example, may be 6.0 or more and 8.0 or less.
The protein concentration of the biological sample may be, for example, from 1 pM to 100 pM, for example, from 10 pM to 100 pM.
Examples of the method for measuring the protein concentration of a biological sample include a method using an antibody antigen reaction (for example, ELISA method), a colorimetric method using a reaction between a protein and a reagent (for example, a bicinchoninic acid (BCA) method, Bradford method, Raleigh method, Biuret method, etc.).
The biological sample may be diluted with various aqueous solvents or the like so as to have the above concentration. Examples of the aqueous solvent include water, physiological saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline, and the like. However, it is not limited to these.

[Step 2]
Next, sealing oil is dropped to encapsulate the ALP in the biological sample in the well of the microdevice provided with the above-described fluorescent probe for ALP detection. Any known sealing oil may be used as long as it is generally used for enclosing a sample in a microdevice, and examples thereof include fluorinated oil (FC-40, etc.).

[Step 3]
Next, the fluorescence in the well of the microdevice is detected using a fluorescence scanner or the like. Enzyme activity can be evaluated from the detected fluorescence intensity.
In addition, in the case of using a microdevice having two or more kinds of the above-described fluorescent probes for detecting ALP having different reaction points in one well, a living body derived from a healthy subject and a subject having a disease By comparing the enzyme activity of ALP in a sample, it can be applied to the discovery of ALP subtypes that are found in a disease-specific manner and a method for diagnosing the disease (see FIG. 2).

EXAMPLES Hereinafter, although an Example and a comparative example etc. are given and this invention is further explained in full detail, this invention is not limited to these Examples.
In the following, the name of the compound was determined using the symbols attached to each compound, for example, “the compound represented by the general formula (1)” is referred to as “compound (1)”. .

[Production Example 1] Production of Compound (1-1-1a) Compound (1-1-1a) was produced as Compound (1) by the route shown below.

[Production of Compound (1-1b-1)]
To a dried flask was added 6.18 g (21.7 mmol, 1 eq) of dibenzyl malonate, 3.00 g (21.7 mmol, 1 eq) of 2,4-dihydroxybenzaldehyde, and pipette. Lysine (Piperidine) 92.5 mg (1.09 mmol, 0.05 eq) was added, and the mixture was heated to reflux at 110 ° C. for 14 hours. Subsequently, the disappearance of the raw material was confirmed by thin-layer chromatography (TLC). Subsequently, the obtained crude product was purified using column chromatography (silica; ethyl acetate / hexane = 1/1, containing 0.5% acetic acid) to obtain 3.89 g of compound (1-1b-1). (13.1 mmol, 60% yield).

The analysis results of the obtained compound (1-1b-1) by ¹ H-NMR, ¹³ C-NMR, and high-resolution mass spectrometry (HR-MS) are shown below.
^{1 H-NMR (DMSO-d} 6, 400MHz) δ5.36 (s, 2H), 6.74 (d, 1H, J = 2.4 Hz), 6.85 (dd, 1H, J = 8.5, 2.2 Hz), 7.36-7.45 (m, 5H), 7.78 (d, 1H, J = 8.3Hz), 8.73 (s, 1H), 11.13 (s, 1H).
¹³ C-NMR (DMSO-d ₆ , 100 MHz) δ 66.1, 101.8, 110.4, 111.7, 114.0, 127.8, 128.4, 132.3, 136.0, 149.8, 156.4, 157.2, 162.8, 164.2.
HR-MS (ESI ⁺ ): Calcd for [M + Na] ⁺ , 319.05824, Found, 319.05829 (+0.05 mmu).

[Production of Compound (1-1d-1)]
In a dried flask, 900 mg (3.04 mmol, 1 eq) of compound (1-1b-1) was placed and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Further, carbon tetrachloride (1169 mg, 7.6 mmol, 2.5 eq), N, N-diisopropylethylamine (DIEA) (824 mg, 6.38 mmol, 2.1 eq) dissolved in acetonitrile (dehydrated) was added. ) And 38 mg (0.31 mmol, 0.1 eq) of N, N-dimethyl-4-aminopyridine (DMAP) were added, and the mixture was cooled to −10 ° C. Furthermore, 1156 mg (4.41 mmol, 1.45 eq) of dibenzyl phosphonate dissolved in acetonitrile (dehydrated) was added little by little to the reaction solution, and the mixture was stirred at −10 ° C. for 2 hours under an argon atmosphere. Subsequently, formation of the compound (1-1d-1) was confirmed by a liquid chromatography-mass spectrometry (LC-MS) meter. Subsequently, pH 8.0 Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) was added, and acetonitrile was distilled off under reduced pressure. The obtained solid was purified using column chromatography (silica; ethyl acetate / hexane = 22/78 → 43/57) to obtain 612 mg (1.01 mmol, yield 36%) of compound (1-1d-1). Obtained.

The results of analysis of the obtained compound (1-1d-1) using an HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + Na] ⁺ , 579.11847, Found, 579.11535 (-3.12 mmu).

[Production of Compound (1-1-1a)]
In a dry flask, 407 mg (0.73 mmol) of the compound (1-1d-1) was added and dissolved in acetonitrile (dehydrated). A 10% palladium carbon catalyst was added to a spatula, and the mixture was stirred for 2 hours at room temperature in a hydrogen atmosphere. Subsequently, formation of the compound (1-1-1a) was confirmed with an LC-MS meter. Next, a small amount of Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) having a pH of 7.0 was added, and the palladium carbon catalyst was removed by suction filtration. Subsequently, acetonitrile was distilled off under reduced pressure. Subsequently, the obtained solution was subjected to high performance liquid chromatography (HPLC) (A / B = 99/1 → 0/100 in 30 minutes, eluted at 13 minutes, A: 0.1% triethylamine-acetic acid Purification was performed using a buffer solution (Trithylamonium acetate; TEAA), B: 0.1% TEAA / 80% acetonitrile / 20% water), and 34 mg (0.12 mmol, 16% yield) of the compound (1-1-1a) was obtained. )Obtained.

The analysis results of the obtained compound (1-1-1a) by ¹ H-NMR, ¹³ C-NMR, and HR-MS meter are shown below.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ 7.16 (d, 1H.J = 8.3 Hz), 7.21 (s, 1H), 7.78 (d, 1H, J = 8.8 Hz), 8.68 (s, 1H ).
¹³ C-NMR (DMSO-d ₆ , 100 MHz) δ 106.2, 112.3, 114.7, 117.3, 130.9, 148.6, 155.9, 157.3, 159.7, 164.3.
HR-MS (ESI ⁺ ): Calcd for [M + Na] ⁺ , 308.97762, Found, 308.97724 (-0.38 mmu).

Further, in the obtained compound (1-1-1a), the optical properties of the compound (1-1-1a) -1 after the phosphate group is eliminated by an enzymatic reaction with ALP are shown in Table 1 and FIG. Shown in “Neutral Form” was measured by dissolving compound (1-1-1a) -1 in a sodium phosphate solution (pH 3.0), and “Anion Form” was measured by adding compound (1- 1-1a) -1 was dissolved and measured.

[Production Example 2] Production of Compound (2-1-2a) Compound (2-1-2a) was produced as Compound (2) by the route shown below.

[Production of 1-bromo-2,4-dimethoxy-5-sulfobenzene]
To a dry flask, 0.925 mL (17.40 mmol, 1 eq) of concentrated sulfuric acid and 5.26 mL (36.54 mmol, 2.1 eq) of trifluoroacetic anhydride (TFAA) were added at 0 ° C. under an argon atmosphere. Slowly added. The mixture was then returned to room temperature and stirred for 3 hours. Then, when the solution became uniform, 3775 mg (17.40 mmol, 1 eq) of 1-bromo-2,4-dimethoxybenzene was added little by little at 0 ° C., and then water was added. added. Subsequently, TFAA and water were distilled off under reduced pressure. The resulting solid was then redissolved in water and filtered three times. Next, water was distilled off from the filtrate under reduced pressure to obtain 4429 mg (14.91 mmol, yield 86%) of 1-bromo-2,4-dimethoxy-5-sulfobenzene.

The analysis results of the obtained 1-bromo-2,4-dimethoxy-5-sulfobenzene by ¹ H-NMR, ¹³ C-NMR, and HR-MS are shown below.
¹ H-NMR (D ₂ O, 300 MHz) δ 3.78, 3.79 (s, 3H), 6.59 (s, 1H), 7.75 (s, 1H).
¹³ C-NMR (CD ₃ OD, 75 MHz) δ 56.7, 57.1, 98.4, 101.1, 126.4, 133.6, 159.1, 160.4.
^{HR-MS (ESI -):} Calcd for [MH] -, 294.92758, Found, 294.92760 (+0.01 mmu).

[Production of Compound (2-1a-1)]
To the dried flask was added 1011 mg (3.40 mmol) of 1-bromo-2,4-dimethoxy-5-sulfobenzene dissolved in 30 mL of 2-propanol and 6 mL of triisopropyl orthoformate at 55 ° C. Stir for 3 hours. Then, production of compound (2b-1) was confirmed using TLC. Subsequently, the solvent was distilled off under reduced pressure. Subsequently, the obtained solid was purified by column chromatography (silica; ethyl acetate / hexane = 18/82 → 39/61), and 782 mg (2.31 mmol, yield 68%) of compound (2-1a-1) was obtained. Obtained.

The analysis results by ¹ H-NMR and ¹³ C-NMR of the obtained compound (2-1a-1) are shown below.
¹ H-NMR (CDCl ₃ , 300 MHz) δ1.31 (d, 6H, J = 5.9 Hz), 3.98, 3.99 (s, 3H), 4.76 (m, 1H), 6.52 (s, 1H), 8.07 (s , 1H).
¹³ C-NMR (CDCl ₃ , 75 MHz) δ 22.7, 56.5, 56.6, 77.5, 96.6, 101.4, 117.9, 134.9, 158.3, 161.1.

[Production of Compound (2-1c-1)]
In a dried flask, 202 mg (0.60 mmol, 1 eq) of compound (2-1a-1) was added, dissolved in dehydrated tetrahydrofuran (THF), and stirred at −78 ° C. for 30 minutes under an argon atmosphere. Next, 0.60 mL (0.60 mmol, 1 eq) of 1M secondary butyllithium (sec-butyllithium; sec-BuLi) was added little by little, and the mixture was stirred at −78 ° C. for 30 minutes in an argon atmosphere. Next, after further adding 54.8 mg (0.12 mmol, 0.2 eq) of diTBS Xanthone dissolved in anhydrous THF to the reaction solution little by little, the mixture was stirred at room temperature for 2 hours under an argon atmosphere. Next, 2N HCl solution was added to the reaction solution, and the mixture was heated to reflux at 110 ° C. for 13 hours. Then, formation of compound (2-1c-1) was confirmed by LC-MS. Subsequently, toluene was added to the solution, and the solvent was distilled off under reduced pressure. Subsequently, the obtained solid was subjected to HPLC (A / B in 99 minutes = 99/1 → 0/100, eluted at 15 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1 % TFA / 80% acetonitrile / 20% water) to obtain 5 mg (0.012 mmol, yield 10%) of the compound (2-1c-1).

The analysis results of the obtained compound (2-1c-1) by ¹ H-NMR, ¹³ C-NMR, and HR-MS meter are shown below.
¹ H-NMR (D ₂ O / KOD, 400 MHz) δ3.51 (s, 3H, b), 4.15 (s, 3H, a), 6.28 (m, 4H), 6.77 (s, 1H), 6.83 (d , 2H, J = 9.6 Hz), 7.94 (s, 1H).
¹³ C-NMR (D ₂ O / KOD, 100 MHz) δ 56.6, 57.1, 97.7, 104.3, 112.5, 113.3, 123.6, 131.2, 131.7, 152.6, 158.7, 159.8, 160.9, 169.2, 181.3.
HR-MS (ESI ⁺ ): Calcd for [M + Na] ⁺ , 451.04636, Found, 451.05134 (+4.99 mmu).

[Production of Compound (2-1e-1)]
120 mg (0.28 mmol, 1 eq) of the compound (2-1c-1) was put in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 215 mg (1.4 mmol, 5 eq) of carbon tetrachloride dissolved in acetonitrile (dehydrated), 76 mg (0.59 mmol, 2.1 eq) of DIEA, and 3.7 mg (0.03 mmol, 0.1 eq) of DMAP were added thereto. And cooled to -10 ° C. Further, 114 mg (0.41 mmol, 1.45 eq) of dibenzyl phosphonate dissolved in acetonitrile (dehydrated) was added little by little to the reaction solution, and the mixture was stirred at −10 ° C. for 2 hours under an argon atmosphere. Then, formation of compound (2-1e-1) was confirmed by LC-MS. Subsequently, Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) at pH 8.0 was added, and acetonitrile was distilled off under reduced pressure. Subsequently, extraction with dichloromethane was performed, and after dehydration with sodium sulfate, the solvent was distilled off under reduced pressure. Subsequently, the obtained solid was subjected to HPLC (A / B in 30 minutes = 60/40 → 0/100, eluted at 15 minutes, A: 0.1% TFA / water, B: 0.1% TFA / 80% Purification with acetonitrile / 20% water) yielded 13 mg (0.019 mmol, yield 7%) of compound (2-1e-1).

The results of analysis of the obtained compound (2-1e-1) using an HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 689.12464, Found, 689.12239 (-2.26 mmu).

[Production of Compound (2-1-2a)]
10 mg (0.015 mmol) of the compound (2-1e-1) was put in a dried flask and dissolved in methanol and dichloromethane. Next, a palladium carbon catalyst was added to the spatula, and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. Next, disappearance of the raw materials was confirmed by LC-MS. Subsequently, the palladium carbon catalyst was removed by suction filtration, PBS was added, and the organic solvent was distilled off under reduced pressure. Thereafter, chloranil dissolved in dichloromethane was added to the solution, and the mixture was stirred at room temperature for 1 hour. Then, formation of compound (2-1-2a) was confirmed by LC-MS. It was then extracted with PBS and dichloromethane. The amount of PBS solution was then reduced under reduced pressure. The solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1% TFAA / water, B: 0.1% TFAA / 80% acetonitrile / 20 % Water) to obtain a trace amount of compound (2-1-2a).

The analysis results of the obtained compound (2-1-2a) by ¹ H-NMR and HR-MS are shown below.
¹ H-NMR (D ₂ O, 300 MHz) δ3.63 (s, 3H), 3.95 (s, 3H), 6.43 (m, 2H), 6.82 (s, 1H), 7.04 (d, 1H, J = 8.8 Hz), 7.23 (m, 3H), 7.55 (s, 1H).
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 509.03074, Found, 509.02780 (-2.95 mmu).

Further, in the obtained compound (2-1-2a), the optical properties of the compound (2-1-2a) -1 after the phosphate group is eliminated by an enzymatic reaction with ALP are shown in Table 2 and FIG. Shown in “Neutral Form” was measured by dissolving compound (2-1-2a) -1 in a sodium phosphate solution (pH 3.0), and “Anion Form” was measured by adding compound (2- 1-2a) -1 was dissolved and measured.

[Production Example 3] Production of Compound (10) Compound (10) was produced as a conventional fluorescent probe compound by the route shown below.

[Production Example 4] Production of Compound (20) As a conventional fluorescent probe compound, a known method (reference document: International Publication No. 2006/093067, “Urano Y et al.,“ Evolution of Fluorescein as a platform for finely tunable fluorescence probes ", Journal of the American Chemical Society, vol. 127, no. 13, p4888-4894, 2005.

[Test Example 1] In vitro alkaline phosphatase fluorescence assay Dilution of fluorescent probe First, the compound (1-1-1a), the compound (2-2-1a), the compound (10), and the compound (20) obtained in Production Examples 1 to 4 are each 10 μmol / L. Dilute with assay buffer as follows. The composition of the assay buffer is 0.05 mol / L Tris-HCl buffer (pH 7.0), 1.0 mmol / L magnesium chloride.

2. Alkaline phosphatase fluorescence assay using a microdevice 3 mL of the diluted solution diluted in “1. Dilution of fluorescent probe” was added to each well of a microdevice consisting of a multiwell plate. Next, alkaline phosphatase (ALP) (molecular weight of about 120,000 to 150,000, EC 3.1.3.1, manufactured by Sigma-Aldrich) was added, and fluorescence measurement was started. FIGS. 5A and 6A show images obtained by photographing a microdevice to which each diluent is added 22 minutes and 132 minutes after the start of fluorescence measurement with a fluorescence microscope. In addition, FIG. 5B and FIG. 6B show the fluorescence intensity in the microdevice to which each diluted solution 132 minutes after the start of fluorescence measurement was added.

From FIG. 5A and FIG. 5B, in the ALP fluorescence assay using the compound (10) that emits blue fluorescence by the enzymatic reaction with ALP, the increase in fluorescence is completely observed and detected due to leakage of the compound (10) to the outside of the device. There wasn't. On the other hand, in the ALP fluorescence assay using the compound (1-1-1a) that emits blue fluorescence by the enzymatic reaction with ALP, an increase in fluorescence was observed and detected only in the wells in which ALP was encapsulated.

Also, from FIGS. 6A and 6B, in the ALP fluorescence assay using the compound (2-2-1a) and the compound (20) that emit green fluorescence by the enzymatic reaction with ALP, both are only in wells encapsulating ALP. An increase in fluorescence was observed and detected. However, in the ALP fluorescence assay using compound (20), the fluorescence intensity was low, suggesting that leakage outside the device occurred.

[Production Example 5] Production of Compound (3-1-2a) Compound (3-1-2a) was produced as Compound (3) by the route shown below.

[Production of Compound (3-1-2a) -1]
In a dry flask, 249 mg (0.73 mmol, 1 eq) of compound (2-1a-1) was added, dissolved in dehydrated tetrahydrofuran (THF), and stirred at −78 ° C. for 30 minutes under an argon atmosphere. Next, 0.73 mL (0.73 mmol, 1 eq) of 1M secondary butyllithium (sec-butyllithium; sec-BuLi) was added little by little, and the mixture was stirred at −78 ° C. for 30 minutes in an argon atmosphere. Next, 53 mg (0.15 mmol, 0.20 eq) of compound (3-1b-1) dissolved in anhydrous THF was added little by little to the reaction solution, and the mixture was stirred at room temperature for 5 hours in an argon atmosphere. Next, 2N HCl solution was added to the reaction solution, and the mixture was heated to reflux at 110 ° C. for 13 hours. Subsequently, formation of compound (3-1-2a) -1 was confirmed by LC-MS. Subsequently, toluene was added to the solution, and the solvent was distilled off under reduced pressure. The resulting solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 15 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1 % TFA / 80% acetonitrile / 20% water) to obtain 32 mg (0.068 mmol, yield 45%) of compound (3-1-2a) -1.

[Production of Compound (3-1e-1)]
714 mg (1.52 mmol) of the compound (3-1-2a) -1 was put in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 18 mg (0.15 mmol) of DMAP, 557 μL (3.19 mmol) of DIEA, and 735 μL (7.6 mmol) of carbon tetrachloride dissolved in acetonitrile (dehydrated) were added thereto, and the mixture was heated to −10 ° C. Cooled down. Further, 514 μL (2.20 mmol) of dibenzyl phosphonate dissolved in acetonitrile (dehydrated) was added little by little to the reaction solution, followed by stirring at −10 ° C. for 2 hours under an argon atmosphere. Then, formation of compound (3-1e-1) was confirmed by LC-MS. Subsequently, Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) at pH 8.0 was added, and acetonitrile was distilled off under reduced pressure. Then HPLC (A / B = 30/30 in 30 minutes → 0/100, elution at 15 minutes, A: 0.1 M TEAA, B: 0.1 M TEAA / 80% acetonitrile / 20% water) Purification gave 99 mg (0.14 mmol, 9.2% yield) of an orange solid compound (3-1e-1).

The results of analysis of the obtained compound (3-1e-1) using an HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 731.15360, Found, 731.14971 (-3.89 mmu).

[Production of Compound (3-1-2a)]
91 mg (0.13 mmol) of compound (3-1e-1) was placed in a dry flask and dissolved in 2 mL of methanol and 1 mL of dichloromethane. Next, a 10% palladium carbon catalyst was added to the spatula, and the mixture was stirred at room temperature for 2 hours in a hydrogen atmosphere. Next, disappearance of the raw materials was confirmed by LC-MS. Subsequently, the palladium carbon catalyst was removed by suction filtration, PBS was added, and the organic solvent was distilled off under reduced pressure. Thereafter, dichloromethane and 44 mg (0.20 mmol) of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) were added to the solution, followed by stirring at room temperature for 1 hour. Then, formation of compound (2-1-2a) was confirmed by LC-MS. Subsequently, PBS was added and the organic solvent was distilled off under reduced pressure. The solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile. / 20% water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Subsequently, freeze-drying gave 1415 mg (0.03 mmol, 23% yield) of a red solid compound (3-1-2a).

The analysis results of the obtained compound (3-1-2a) by ¹ H-NMR and HR-MS are shown below.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 6.99 (s, 1H), 6.59 (dd, 1H, J = 8.8 Hz, 2.0 Hz), 6.50 (d, 1H, J = 10.0 Hz), 6.40 ( d, 1H, J = 8.8 Hz), 6.25 (d, 1H, J = 2.4 Hz), 6.19 (d, 1H, J = 2.0 Hz), 5.62 (d, 1H, J = 10.0 Hz), 5.59 (d, 1H, J = 2.4 Hz), 3.37 (s, 3H), 3.27 (s, 3H), 0.00 (s, 3H), -0.11 (s, 3H).
¹³ C (100 MHz, DMSO-d ₆ ) δ 183.6, 161.4, 160.2, 159.6, 155.5, 148.3, 143.3, 139.8, 139.1, 136.7, 135.7, 135.5, 129.6, 127.2, 126.7, 125.6, 121.6, 121.5, 106.6, 99.7, 56.5, 56.2, 0.0, -1.7.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 551.05970, Found, 551.05808 (-1.62 mmu).

Further, in the obtained compound (3-1-2a), the optical characteristics of the compound (3-1-2a) -1 after the phosphate group is eliminated by an enzymatic reaction with ALP are shown in FIGS. 7A and 7B. . “Phenolform” was measured by dissolving compound (3-1-2a) -1 in 100 mM sodium phosphate solution (pH 3.4), and “Phenoxide Form” was 0.1N sodium hydroxide aqueous solution (pH 13). Compound (3-1-2a) -1 was dissolved in and measured.

[Test Example 2] In vitro alkaline phosphatase fluorescence assay Dilution of fluorescent probe First, the compound (3-1-2a) obtained in Production Example 5 was diluted with an assay buffer so as to be 10 μmol / L. The composition of the assay buffer is 1.0 mM magnesium chloride-containing DEA buffer (pH 9.3).

2. Alkaline phosphatase fluorescence assay using a microdevice 3 mL of the diluted solution diluted in “1. Dilution of fluorescent probe” was added to each well of a microdevice consisting of a multiwell plate. Subsequently, ALP (recombinant protein using methanol-utilizing yeast Pichia pastoris) was added, and fluorescence measurement was started. FIG. 8 shows the fluorescence intensity in the microdevice to which the diluted solution before addition of ALP (ALP (−)) and 5 minutes after addition of ALP (ALP (+)) was added. The fluorescence measurement of the compound (3-1-2a) was performed under excitation at 590 nm.

As shown in FIG. 8, the fluorescence spectrum before and after the enzyme reaction with ALP using the compound (3-1-2a) was measured, and as a result, the fluorescence increased about 84 times.

[Production Example 6] Production of Compound (1-1-1b) Compound (1-1-1b) was produced as Compound (1) by the route shown below.

[Production of Compound (1-1d-2)]
300 mg (1.01 mmol) of the compound (1-1-1a) -1 was placed in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 687 μL (4.04 mmol) of DIEA and 394 mg (1.21 mmol) of dibenzyl chloromethyl phosphate dissolved in acetonitrile (dehydrated) were added little by little, followed by stirring at 50 ° C. for 2 hours under an argon atmosphere. did. Subsequently, acetonitrile was distilled off under reduced pressure. Subsequently, the residue was purified by column chromatography (ethyl acetate / hexane = 30/70 → 80/20 over 20 minutes) to obtain 83 mg (0.14 mmol, 14% yield) of the compound (1-1d-2).

[Production of Compound (1-1-1b)]
In a dry flask, 83 mg (0.14 mmol) of the compound (1-1d-2) was added and dissolved in acetonitrile (dehydrated). A 10% palladium carbon catalyst was added to a spatula, and the mixture was stirred for 2 hours at room temperature in a hydrogen atmosphere. Subsequently, formation of the compound (1-1-1b) was confirmed with an LC-MS meter. Next, a small amount of Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) having a pH of 7.0 was added, and the palladium carbon catalyst was removed by suction filtration. Subsequently, acetonitrile was distilled off under reduced pressure. Then, the resulting solution was subjected to HPLC (A / B = 30/1 → 0/100 in 30 minutes, eluted at 13 minutes, A: 0.1% triethylamine-acetate buffer (TEAA), B: Purification using 0.1% TEAA / 80% acetonitrile / 20% water) gave 12 mg (0.038 mmol, 27% yield) of compound (1-1-1b).

The results of analysis of the obtained compound (1-1-1b) using an HR-MS meter are shown below.
^{HR-MS (ESI -):} Calcd for [MH] -, 314.99059, Found, 314.98632 (-4.27 mmu).

[Production Example 7] Production of Compound (2-1-2b) Compound (2-1-2b) was produced as Compound (2) by the route shown below.

[Production of Compound (2-1e-2)]
100 mg (0.23 mmol) of the compound (2-1-2a) -1 was put in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 150 mg (0.46 mmol) of cesium carbonate dissolved in acetonitrile (dehydrated) was added thereto and mixed at room temperature under an argon atmosphere. Further, 150 mg (0.46 mmol) of dibenzyl chloromethyl phosphate dissolved in acetonitrile (dehydrated) was added little by little to the reaction solution, and the mixture was stirred at 60 ° C. for 2 hours in an argon atmosphere. Then, formation of compound (2-1e-2) was confirmed by LC-MS. Subsequently, PBS was added and acetonitrile was distilled off under reduced pressure. Next, HPLC (A / B in 30 minutes = 99/1 → 0/100, elution at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile / 20 % Water) to obtain 16 mg (0.022 mmol, 9.6% yield) of the compound (2-1e-2).

[Production of Compound (2-1-2b)]
5 mg (0.007 mmol) of compound (2-1e-2) was placed in a dry flask and dissolved in 2 mL of methanol and 1 mL of dichloromethane. Next, a 10% palladium carbon catalyst was added to the spatula, and the mixture was stirred at room temperature for 2 hours in a hydrogen atmosphere. Next, disappearance of the raw materials was confirmed by LC-MS. Subsequently, the palladium carbon catalyst was removed by suction filtration, PBS was added, and the organic solvent was distilled off under reduced pressure. Thereafter, dichloromethane and 2 mg (0.009 mmol) of chloranil were added to the solution and stirred at room temperature for 1 hour. Then, formation of compound (2-1-2b) was confirmed by LC-MS. Subsequently, PBS was added and the organic solvent was distilled off under reduced pressure. The solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile. / 20% water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Subsequently, lyophilization was carried out to obtain a trace amount of compound (2-1-2b).

The results of analysis of the obtained compound (2-1-2b) by HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 539.04131, Found, 539.03653 　　　 (-4.78 mmu).

[Production Example 8] Production of compound (3-1-2b) Compound (3-1-2b) was produced as compound (3) by the route shown below.

[Production of Compound (3-1e-2)]
1134 mg (2.41 mmol) of compound (3-1-2a) -1 was placed in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 3141 mg (9.64 mmol) of cesium carbonate, dibenzyl chloromethyl phosphate 1030 mg (3.15 mmol) and 5 mL of DMSO dissolved in 15 mL of acetonitrile (dehydrated) were added little by little, and then under an argon atmosphere. Stir at 60 ° C. for 2 hours. Then, formation of compound (3-1e-2) was confirmed by LC-MS. Subsequently, PBS was added and acetonitrile was distilled off under reduced pressure. Next, HPLC (A / B in 30 minutes = 99/1 → 0/100, elution at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile / 20 % Water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Next, freeze-drying gave 45 mg (0.059 mmol, yield 2.4%) of a red solid compound (3-1e-2).

The results of analysis of the obtained compound (3-1e-2) using an HR-MS meter are shown below.
^{HR-MS (ESI -):} Calcd for [MH] -, 759.14907, Found, 759.14793 (-1.14 mmu).

[Production of Compound (3-1-2b)]
45 mg (0.059 mmol) of the compound (3-1e-2) was placed in a dry flask and dissolved in 2 mL of methanol and 1 mL of dichloromethane. Next, a 10% palladium carbon catalyst was added to the spatula, and the mixture was stirred at room temperature for 2 hours in a hydrogen atmosphere. Next, disappearance of the raw materials was confirmed by LC-MS. Subsequently, the palladium carbon catalyst was removed by suction filtration, PBS was added, and the organic solvent was distilled off under reduced pressure. Thereafter, dichloromethane and 16 mg (0.059 mmol) of chloranil were added to the solution, and the mixture was stirred at room temperature for 1 hour. Then, formation of compound (2-1-2b) was confirmed by LC-MS. Subsequently, PBS was added and the organic solvent was distilled off under reduced pressure. The solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile. / 20% water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Subsequently, lyophilization was performed to obtain a trace amount of red solid compound (3-1-2b).

The results of analysis of the obtained compound (3-1-2b) using an HR-MS meter are shown below.
^{HR-MS (ESI -):} Calcd for [MH] -, 579.05462, Found, 579.05270 (-1.91 mmu).

[Test Example 3] In vitro alkaline phosphatase fluorescence assay Dilution of fluorescent probe First, the compound (1-1-1b), the compound (2-1-2b) and the compound (3-1-2b) obtained in Production Examples 6 to 8 were each adjusted to 10 μmol / L. Dilute with assay buffer. The composition of the assay buffer is 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 mM magnesium chloride.

2. Alkaline phosphatase fluorescence assay using a microdevice 3 mL of the diluted solution diluted in “1. Dilution of fluorescent probe” was added to each well of a microdevice consisting of a multiwell plate. Subsequently, ALP (recombinant protein using methanol-utilizing yeast Pichia pastoris) was added, and fluorescence measurement was started. FIG. 9 (compound (1-1-1b)) shows the fluorescence intensity in the microdevice to which the diluted solution was added 10 minutes after the addition of ALP (ALP (−)) and 10 minutes after the addition of ALP (ALP (+)). Shown in FIG. 10 (compound (2-1-2b)) and FIG. 11 (compound (3-1-2b)). The fluorescence measurement of the compound (1-1-1b) was conducted under excitation of 400 nm, the fluorescence measurement of the compound (2-1-2b) was conducted under excitation of 490 nm, and the fluorescence measurement of the compound (3-1-2b) was measured. Performed under 590 nm excitation.

As shown in FIGS. 9 to 11, fluorescence spectra before and after the enzymatic reaction with ALP using the compound (1-1-1b), the compound (2-1-2b) and the compound (3-1-2b) were measured. As a result, a significant increase in fluorescence was detected.

[Production Example 9] Production of Compound (2-1-2c) Compound (2-1-2c) was produced as Compound (2) by the route shown below.

[Production of Compound (21)]
In a dried flask, 248 mg (2.0 mmol) of 4-hydroxybenzyl alcohol was added and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 96 μL (10 mmol) of carbon tetrachloride dissolved in acetonitrile (dehydrated), 733 μL (4.2 mmol) of DIEA, and 24 mg (0.2 mmol) of DMAP were added thereto, and the mixture was cooled to −10 ° C. Further, 639 μL (2.9 mmol) of dibenzyl phosphonate dissolved in acetonitrile (dehydrated) was added little by little to the reaction solution, followed by stirring at −10 ° C. for 2 hours in an argon atmosphere. Then, formation of compound (21) was confirmed by LC-MS. Subsequently, Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) at pH 8.0 was added, and acetonitrile was distilled off under reduced pressure. Subsequently, extraction with dichloromethane was performed, and after dehydration with sodium sulfate, the solvent was distilled off under reduced pressure. Subsequently, the obtained solid was subjected to HPLC (A / B in 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile / 20% water) was roughly purified to obtain 233 mg (including impurities) of compound (21).

[Production of Compound (22)]
In a dry flask, 233 mg (including impurities) of compound (21) was added and dissolved in dichloromethane. Next, 477 mg (1.44 mmol) of carbon tetrabromide dissolved in dichloromethane and 226 mg (0.86 mmol) of triphenylphosphine were added thereto, and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. Then, formation of compound (22) was confirmed by LC-MS. Subsequently, Tris-HCl buffer (containing 50 mM and 1 mM magnesium chloride) at pH 8.0 was added, and acetonitrile was distilled off under reduced pressure. Subsequently, extraction with dichloromethane was performed, and after dehydration with sodium sulfate, the solvent was distilled off under reduced pressure. The resulting solution was then HPLC (A / B = 30/40 → 0/100 in 30 minutes, eluted at 15 minutes, A: 0.1% TFA / water, B: 0.1% TFA / 80% Crude purification was performed with acetonitrile / 20% water) to obtain 753 mg (including impurities) of Compound (22).

[Production of Compound (2-1e-3)]
Next, 51 mg (0.12 mmol) of the compound (2-1-2a) -1 was placed in a dried flask and dissolved in acetonitrile (dehydrated) under an argon atmosphere. Next, 156 mg (0.48 mmol) of cesium carbonate dissolved in acetonitrile (dehydrated), 753 mg (including impurities) of compound (22), and 5 mL of DMSO were added little by little, and then at room temperature under an argon atmosphere. For 2 hours. Then, formation of compound (2-1e-3) was confirmed by LC-MS. Subsequently, PBS was added and acetonitrile was distilled off under reduced pressure. Next, HPLC (A / B in 30 minutes = 99/1 → 0/100, elution at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile / 20 % Water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Then, it was freeze-dried to obtain 32 mg (0.04 mmol) of the compound (2-1e-3).

The results of analysis of the obtained compound (2-1e-3) by HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 795.16651, Found, 795.16473 (-1.77 mmu).

[Production of Compound (2-1-2c)]
32 mg (0.04 mmol) of the compound (2-1e-3) was placed in a dry flask and dissolved in dichloromethane. Next, 25 μL (0.18 mmol) of trimethylsilyl iodide dissolved in dichloromethane and 37 μL (0.46 mmol) of pyridine were added thereto, followed by stirring at 0 ° C. for 2 hours in a hydrogen atmosphere. Then, formation of compound (2-1-2c) was confirmed by LC-MS. Subsequently, PBS was added and the organic solvent was distilled off under reduced pressure. The solution was then HPLC (A / B for 30 minutes = 99/1 → 0/100, eluted at 17 minutes, A: 0.1 M TEAA / 100% water, B: 0.1 M TEAA / 80% acetonitrile. / 20% water). The resulting solution was then desalted and the water was evaporated under reduced pressure. Then, it was freeze-dried to obtain a trace amount of compound (2-1-2c).

The results of analysis of the obtained compound (2-1-2c) by HR-MS meter are shown below.
HR-MS (ESI ⁺ ): Calcd for [M + H] ⁺ , 613.05696, Found, 613.05837 (+1.41 mmu).

Further, the obtained compound (2-1-2c) has a self-cleaving linker, and as shown in the following formula, an elimination reaction occurs in two steps by an enzymatic reaction with ALP, and a fluorophore is released. Is done.

[Test Example 3] In vitro alkaline phosphatase fluorescence assay Dilution of fluorescent probe First, the compound (2-1-2c) obtained in Production Example 9 was diluted with an assay buffer so as to be 10 μmol / L. The composition of the assay buffer is 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 mM magnesium chloride.

2. Alkaline phosphatase fluorescence assay using a microdevice 3 mL of the diluted solution diluted in “1. Dilution of fluorescent probe” was added to each well of a microdevice consisting of a multiwell plate. Subsequently, ALP (recombinant protein using methanol-utilizing yeast Pichia pastoris) was added, and fluorescence measurement was started. FIG. 12 shows the fluorescence intensity in the microdevice to which the diluted solution was added before the addition of ALP (ALP (−)) and after 10 minutes from the addition of ALP (ALP (+)). The fluorescence measurement of the compound (2-1-2c) was performed under excitation at 490 nm.

As shown in FIG. 12, as a result of measuring the fluorescence spectrum before and after the enzymatic reaction with ALP using the compound (2-1-2c), a remarkable increase in fluorescence was detected.

From the above, the fluorescent probe for ALP detection of the present embodiment does not leak out of the microdevice, and can measure the enzymatic activity of ALP with high accuracy.

According to the fluorescent probe for ALP detection of this embodiment, the enzymatic activity of ALP in a biological sample can be measured with high quantitativeness and sensitivity.

Claims (21)

A fluorescent probe for detecting alkaline phosphatase comprising a compound having an anionic functional group and a phosphate group. The fluorescent probe for detecting alkaline phosphatase according to claim 1, wherein the anionic functional group is a carboxy group, a sulfonic acid group or a phosphoric acid group. The fluorescent probe for detecting alkaline phosphatase according to claim 1 or 2, wherein the compound is a compound represented by the following general formula (1), (2), (3) or (4).

(In the general formula (1), R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, or a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, and any one of R ¹¹ , R ¹² and R ¹³ is an anionic functional group at the terminal. R ¹⁴ and R ¹⁵ are each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms Y ¹¹ is a single bond, —O— (CH ₂ ) _n11 — ^{_{, -O- (CH 2) n12 -Ar}} 11 -, - NH- (CH 2) n13 -, ^{_{or, -NH- (CH 2) n14 -Ar}} 12 - are as .n11, n12, n13 and n14 it The Each independently represents an integer of 1 to 10. Ar ¹¹ and Ar ¹² are each independently a substituted or unsubstituted arylene group.
In the general formula (2), R ²¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ²¹ may be the same as or different from each other. R ²² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ²² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. X ²¹ is an oxygen atom or N ⁺ HR ′. R ′ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²¹ represents a single _{bond, -O- (CH 2) n21 -} , - O- (CH 2) n22 -Ar 21 -, - NH- (CH 2) n23 -, or, -NH- _{(CH 2) n24} - Ar ²² - is. n21, n22, n23 and n24 are each independently an integer of 1 to 10. Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.
In the general formula (3), R ³¹ is 1-2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ³¹ may be the same as or different from each other. R ³² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ³² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. X ³² is an oxygen atom or N ⁺ HR ″. R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ³¹ represents a single _{bond, -O- (CH 2) n31 -} , - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, or, -NH- _{(CH 2) n34} - Ar ³² —. n31, n32, n33 and n34 are each independently an integer of 1 to 10. Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.
In the general formula (4), R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ⁴¹ may be the same as or different from each other. R ⁴² is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal. A plurality of R ⁴² may be the same as or different from each other. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁵ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴¹ represents a single _{bond, -O- (CH 2) n41 -} , - O- (CH 2) n42 -Ar 41 -, - NH- (CH 2) n43 -, or, -NH- _{(CH 2) n44} - Ar ⁴² —. n41, n42, n43 and n44 are each independently an integer of 1 to 10. Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group. The compound is represented by the following general formula (1-1), (1-2), (2-1), (2-2), (3-1), (3-2), (4-1) or (4 The fluorescent probe for detecting alkaline phosphatase according to claim 3, which is a compound represented by -2).

(In the general formula (1-1), R ¹¹¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —, where n111 and n112 are each independently an integer of 1 to 10 Ar ¹¹¹ is a substituted or unsubstituted arylene group.
In the general formula (1-2), R ¹²¹ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. Y ¹²¹ is —NH— (CH ₂ ) _n121 — or —NH— (CH ₂ ) _n122 —Ar ¹²¹ —. n121 and n122 are each independently an integer of 1 to 10. Ar ¹²¹ is a substituted or unsubstituted arylene group. )

(In General Formula (2-1), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ²¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or a carbon number of Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, wherein n ²¹¹ and n 212 are each independently 1 to ^{.Ar 211} is an integer of 10 is a substituted or unsubstituted arylene group.
In general formula (2-2), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. R ²²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. )

(In the general formula (3-1), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ³¹³ is a group having an anionic functional group at its terminal. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms X ³¹ is a silicon atom, phosphorus atom, germanium atom or tin it is an atomic ^{.Y 311} is a single _{bond, -O- (CH 2) n311 -} , ^{_{or, -O- (CH 2) n312 -Ar}} 311 - a is .n311 and N312 are each independently ^{.Ar 311} is an integer of 1-10 is a substituted or unsubstituted arylene group.
In general formula (3-2), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. R ³²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. )

(In General Formula (4-1), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are Each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. The .n411 and n412 is the ^{.Ar 411} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. ) The compound is represented by the following general formulas (1-1-1), (1-1-2), (1-1-3), (1-2-1), (1-2-2), (1-2 -3), (2-1-1), (2-1-2), (2-1-3), (2-2-1), (2-2-2), (2-2-3 ), (3-1-1), (3-1-2), (3-1-3), (3-2-1), (3-2-2), (3-2-3), (4-1-1), (4-1-2), (4-1-3), (4-2-1), (4-2-2) or (4-2-3) The fluorescent probe for detecting alkaline phosphatase according to claim 4, wherein the fluorescent probe is a compound.

(In the general formulas (1-1-1) to (1-2-3), Y ¹¹¹ is a single bond, —O— (CH ₂ ) _n111 —, or —O— (CH ₂ ) _n112 —Ar ¹¹¹ —. N111 and n112 are each independently an integer of 1 to 10. Ar ¹¹¹ is a substituted or unsubstituted arylene group, Y ¹²¹ is —NH— (CH ₂ ) _n121 —, or —NH— ( CH ₂ ) _n122 —Ar ¹²¹ —, where n121 and n122 are each independently an integer of 1 to 10. Ar ¹²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (2-1-1) to (2-2-3), Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —. the .n211 and n212 is the ^{.Ar 211} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group ^{.Y 221} is a single _{bond, -NH- (CH 2) n221 -} , or - NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (3-1-1) to (3-2-1), Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —. N311 and n312 are each independently an integer of 1 to 10. Ar ³¹¹ is a substituted or unsubstituted arylene group, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or — NH— (CH ₂ ) _n322 —Ar ³²¹ —, wherein n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group.

(In the general formulas (4-1-1) to (4-2-3), Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. N411 and n412 are each independently an integer of 1 to 10. Ar ⁴¹¹ is a substituted or unsubstituted arylene group, Y ⁴²¹ is —NH— (CH ₂ ) _n421 —, or —NH— ( CH ₂ ) _n422 —Ar ⁴²¹ —, where n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. The fluorescent probe for detecting alkaline phosphatase according to any one of claims 1 to 5, which is used for a micro device. A microdevice comprising the alkaline phosphatase detection fluorescent probe according to any one of claims 1 to 6. The microdevice according to claim 7, wherein one well of the microdevice is provided with one type of fluorescent probe for detecting alkaline phosphatase. The microdevice according to claim 7, comprising two or more fluorescent probes for detecting alkaline phosphatase having different reaction points in one well of the microdevice and having different fluorescence wavelengths. A method for detecting the enzymatic activity of alkaline phosphatase in a biological sample using the microdevice according to any one of claims 7 to 9. A compound represented by the following general formula (2).

(In the general formula (2), R ²¹ represents 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ²¹ may be the same as each other. , good .R ²² be different a 1-2 monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ²² there are a plurality of mutually identical The anionic functional group may be any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ²³ , R ²⁴ , R ^25, and R ²⁶ is independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, X ²¹ is an oxygen atom or N ⁺ HR ′, and R ′ is a hydrogen atom or carbon atom having 1 to 10 carbon atoms. Y ²¹ is a single bond, —O— ( _{CH 2) n21 -, - O-} (CH 2) n22 -Ar 21 -, - NH- (CH 2) n23 -, ^{_{or, -NH- (CH 2) n24 -Ar}} 22 - are as .n21, n22, n23 and n24 are each independently an integer of 1 to 10. Ar ²¹ and Ar ²² are each independently a substituted or unsubstituted arylene group.) The compound according to claim 11, wherein the compound is a compound represented by the following general formula (2-1) or (2-2).

(In General Formula (2-1), R ²¹¹ and R ²¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ²¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or a carbon number of Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —, wherein n ²¹¹ and n 212 are each independently 1 to ^{.Ar 211} is an integer of 10 is a substituted or unsubstituted arylene group.
In general formula (2-2), R ²²¹ and R ²²² are each independently an alkyl group having 1 to 10 carbon atoms. R ²²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ²²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ²³ , R ²⁴ , R ²⁵ and R ²⁶ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. Y ²²¹ is a single bond, —NH— (CH ₂ ) _n221 —, or —NH— (CH ₂ ) _n222 —Ar ²²¹ —. n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. ) The compound is represented by the following general formula (2-1-1), (2-1-2), (2-1-3), (2-2-1), (2-2-2) or (2-2 The compound according to claim 12, which is a compound represented by -3).

(In the general formulas (2-1-1) to (2-2-3), Y ²¹¹ is a single bond, —O— (CH ₂ ) _{n 211} —, or —O— (CH ₂ ) _{n 212} —Ar ²¹¹ —. the .n211 and n212 is the ^{.Ar 211} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group ^{.Y 221} is a single _{bond, -NH- (CH 2) n221 -} , or - NH— (CH ₂ ) _n222 —Ar ²²¹ —, wherein n221 and n222 are each independently an integer of 1 to 10. Ar ²²¹ is a substituted or unsubstituted arylene group. A compound represented by the following general formula (3).

(In the general formula (3), R ³¹ is 1 to 2 monovalent substituents present on the benzene ring and is an electron donating group. A plurality of R ³¹ may be the same as each other. may .R ³² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ³² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, R ³³ , R ³⁴ , R ³⁷ and R ³⁸ is each independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom , Phosphorus atom, germanium atom or tin atom. X ³² is an oxygen atom or N ⁺ HR ″. R ″ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Y ³¹ is a single bond, —O _{- (CH 2) n31 -,} - O- (CH 2) n32 -Ar 31 -, - NH- (CH 2) n33 -, ^{_{or, -NH- (CH 2) n34 -Ar}} 32 - are as .N31, n32, n33 and n34 are each independently an integer of 1 to 10. Ar ³¹ and Ar ³² are each independently a substituted or unsubstituted arylene group.) The compound according to claim 14, wherein the compound is a compound represented by the following general formula (3-1) or (3-2).

(In the general formula (3-1), R ³¹¹ and R ³¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ³¹³ is a group having an anionic functional group at its terminal. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, and R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or a carbon number of 1 R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms X ³¹ is a silicon atom, phosphorus atom, germanium atom or tin it is an atomic ^{.Y 311} is a single _{bond, -O- (CH 2) n311 -} , ^{_{or, -O- (CH 2) n312 -Ar}} 311 - a is .n311 and N312 are each independently ^{.Ar 311} is an integer of 1-10 is a substituted or unsubstituted arylene group.
In general formula (3-2), R ³²¹ and R ³²² are each independently an alkyl group having 1 to 10 carbon atoms. R ³²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ³²⁴ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ³³ , R ³⁴ , R ³⁷ and R ³⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ³⁵ and R ³⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ³¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or —NH— (CH ₂ ) _n322 —Ar ³²¹ —. n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. ) The compound according to claim 14 or 15, wherein X ³¹ is a silicon atom. The compound is represented by the following general formula (3-1-1), (3-1-2), (3-1-3), (3-2-1), (3-2-2) or (3-2) The compound according to claim 16, which is a compound represented by -3).

(In the general formulas (3-1-1) to (3-2-1), Y ³¹¹ is a single bond, —O— (CH ₂ ) _n311 —, or —O— (CH ₂ ) _n312 —Ar ³¹¹ —. N311 and n312 are each independently an integer of 1 to 10. Ar ³¹¹ is a substituted or unsubstituted arylene group, Y ³²¹ is a single bond, —NH— (CH ₂ ) _n321 —, or — NH— (CH ₂ ) _n322 —Ar ³²¹ —, wherein n321 and n322 are each independently an integer of 1 to 10. Ar ³²¹ is a substituted or unsubstituted arylene group. A compound represented by the following general formula (4).

(In the general formula (4), R ⁴¹ is 1 to 2 monovalent substituents present on the benzene ring, and is an electron donating group. A plurality of R ⁴¹ may be the same as each other. may .R ⁴² be different from a one to two monovalent substituents present on the benzene ring, the terminal is a group having an anionic functional group. R ⁴² there are a plurality of mutually identical The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group, and R ⁴³ is a hydrogen atom or a carbon number of 1. R ⁴⁴ , R ⁴⁵ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently a carbon number. 1 to 10 alkyl groups or carbon 6-10 aryl group ^{.X 41} silicon atom, a phosphorus atom, a germanium atom or a tin atom ^{.Y 41} is a single _{bond, -O- (CH 2) n41 -} , - O- (CH 2) n42 —Ar ⁴¹ —, —NH— (CH ₂ ) _n43 —, or —NH— (CH ₂ ) _n44 —Ar ⁴² —, wherein n41, n42, n43 and n44 are each independently an integer of 1 to 10. Ar ⁴¹ and Ar ⁴² are each independently a substituted or unsubstituted arylene group.) The compound according to claim 18, wherein the compound is a compound represented by the following general formula (4-1) or (4-2).

(In General Formula (4-1), R ⁴¹¹ and R ⁴¹² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴¹³ is a group having an anionic functional group at its end. Is any one selected from the group consisting of a carboxy group, a sulfonic acid group and a phosphoric acid group, R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are Each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 10 carbon atoms, and R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom, Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. The .n411 and n412 is the ^{.Ar 411} are each independently an integer of 1 to 10 is a substituted or unsubstituted arylene group.
In general formula (4-2), R ⁴²¹ and R ⁴²² are each independently an alkyl group having 1 to 10 carbon atoms. R ⁴²³ is a group having an anionic functional group at the terminal. The anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphoric acid group. R ⁴³ is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁴ , R ⁴⁷ and R ⁴⁸ are each independently a hydrogen atom, a halogen atom or an alkyl group having 1 to 10 carbon atoms. R ⁴⁵ and R ⁴⁶ are each independently an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 10 carbon atoms. X ⁴¹ is a silicon atom, a phosphorus atom, a germanium atom or a tin atom. Y ⁴²¹ is —NH— (CH ₂ ) _n421 — or —NH— (CH ₂ ) _n422 —Ar ⁴²¹ —. n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group. ) 20. The compound according to claim 18 or 19, wherein ^X41 is a silicon atom. The compound is represented by the following general formula (4-1-1), (4-1-2), (4-1-3), (4-2-1), (4-2-2) or (4-2) The compound according to claim 20, which is a compound represented by -3).

(In the general formulas (4-1-1) to (4-2-3), Y ⁴¹¹ is a single bond, —O— (CH ₂ ) _n411 —, or —O— (CH ₂ ) _n412 —Ar ⁴¹¹ —. N411 and n412 are each independently an integer of 1 to 10. Ar ⁴¹¹ is a substituted or unsubstituted arylene group, Y ⁴²¹ is —NH— (CH ₂ ) _n421 —, or —NH— ( CH ₂ ) _n422 —Ar ⁴²¹ —, where n421 and n422 are each independently an integer of 1 to 10. Ar ⁴²¹ is a substituted or unsubstituted arylene group.

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