WO2018159810A1 - Sonde fluorescente pour la détection de phosphatase alcaline et son utilisation - Google Patents

Sonde fluorescente pour la détection de phosphatase alcaline et son utilisation Download PDF

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WO2018159810A1
WO2018159810A1 PCT/JP2018/007993 JP2018007993W WO2018159810A1 WO 2018159810 A1 WO2018159810 A1 WO 2018159810A1 JP 2018007993 W JP2018007993 W JP 2018007993W WO 2018159810 A1 WO2018159810 A1 WO 2018159810A1
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group
compound
independently
carbon atoms
atom
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泰照 浦野
小松　徹
眞伍 坂本
博行 野地
力也 渡邉
翼 張
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/26—Triarylmethane dyes in which at least one of the aromatic nuclei is heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • 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.
  • ALP alkaline phosphatase
  • 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.
  • 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.
  • 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.
  • the anionic functional group may be a carboxy group, a sulfonic acid group, or a phosphoric acid group.
  • the compound may be a compound represented by the following general formula (1), (2), (3) or (4).
  • 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.
  • 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.
  • 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.
  • 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 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).
  • 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.
  • 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 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.
  • 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 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
  • 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 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
  • 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.
  • 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), (3-2-2), (4-1-1), (4-1-2), (4-1-3), (4-2-1), (4-2 -2) or (4-2-3).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 ′
  • 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).
  • 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.
  • 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).
  • 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).
  • 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
  • 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).
  • 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
  • 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 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • FIG. 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.
  • . 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.
  • the unit of concentration “M” means “mol / L”.
  • 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.
  • 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 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 13 is preferably a group having an anionic functional group at the terminal.
  • 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.
  • the linker Y ′ include an alkylene group having 1 to 10 carbon atoms which may contain —O— or —NH—.
  • 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.
  • 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.
  • 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
  • R 13 is preferably a group having an anionic functional group at the terminal.
  • 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.
  • halogen atom in R 14 and R 15 examples 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.
  • 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 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 —.
  • 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).
  • 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.
  • 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 >”.
  • the halogen atom is preferably a chlorine atom, a bromine atom or an iodine atom.
  • 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.
  • 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—.
  • “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.
  • compound (1-1) a compound represented by the following general formula (1-1)
  • compound 1-2 a compound represented by the following general formula (1-2)
  • these compounds are only examples of a preferable compound (1), and a preferable compound (1) is not limited to these.
  • 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.
  • 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 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 is a single bond, —O— (CH 2 ) n111 —, or —O— (CH 2 ) n112 —Ar 111 —.
  • 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.
  • R 111 is a carboxy group, a sulfonic acid group, or a phosphoric acid group
  • 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.
  • R 121 is a carboxy group, a sulfonic acid group, or a phosphoric acid group
  • 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.
  • the compound (1-1) 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.
  • preferred as the compound (1-2) 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)”).
  • these compounds are only examples of a preferable compound (1), and a preferable compound (1) is not limited to these.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound (1-1-1) 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.
  • 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)”.
  • compound (1-1-2b) a compound represented by the following formula (1-1-2b)
  • compound (1-1-2c) a compound represented by the following formula (1-1-2c)
  • compound (1-1-2c) a compound represented by the following formula (1-1-2c)
  • compound (1-1-3a) compounds represented by the following formula (1-1-3a)
  • compound (1-1-3b) a compound represented by the following formula (1-1-3b)
  • compound (1-1-3c) a compound represented by the following formula (1-1-3c)
  • the compound (1-2-1) 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.
  • 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)”.
  • compound (1-2-2b) a compound represented by the following formula (1-2-2b)
  • compound (1-2-2c) a compound represented by the following formula (1-2-2c)
  • compound (1-2-2c) a compound represented by the following formula (1-2-2c)
  • 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)”.
  • a compound represented by the following formula (1-2-3b) hereinafter sometimes abbreviated as “compound (1-2-3b)
  • compound (1-2-3c) a compound represented by the following formula (1- 2-3c)
  • 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.
  • 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.
  • 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)”).
  • 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 (1-1a) is a known compound.
  • R 111 is a group having an anionic functional group at the terminal.
  • 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) 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.
  • 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.
  • the amount of 2,4-dihydroxybenzaldehyde used is preferably 0.5 to 2.0 times the molar amount of compound (1-1a).
  • 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.
  • 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.
  • 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.
  • 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.
  • compound (1-1b) 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 (1-1c) is a known compound.
  • 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.
  • R 112 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (1-1d) is a novel compound.
  • 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).
  • R 111 represents the compound (1-1a).
  • 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).
  • DIEA N, N-diisopropylethylamine
  • 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).
  • the condensing agent is not particularly limited, and examples thereof include N, N-dimethyl-4-aminopyridine (DMAP).
  • DMAP N, N-dimethyl-4-aminopyridine
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • the aprotic solvent is not particularly limited.
  • 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.
  • the amount of compound (1-1c) used is preferably 1 to 2 times the amount of compound (1-1b).
  • 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.
  • 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.
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • 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.
  • compound (1-1) 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
  • NMR nuclear magnetic resonance
  • the structure can be confirmed by a known method such as mass spectrometry (MS) or infrared spectroscopy (IR).
  • 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) ”).
  • 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.
  • 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.
  • each step will be described in detail.
  • 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 (1-2a) is a known compound.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound (1-2b) 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 (1-2c) is a known compound.
  • 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.
  • R 112 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (1-2d) is a novel compound.
  • 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).
  • R 121 represents the compound (1-2a).
  • 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.
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (1-2c) used is preferably 1 to 2 times the amount of compound (1-2b).
  • 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.
  • 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.
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • 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.
  • compound (1-2) 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.
  • NMR nuclear magnetic resonance
  • the structure can be confirmed by a known method such as mass spectrometry (MS) or infrared spectroscopy (IR).
  • 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.
  • 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 ′
  • 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 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-dimethylp
  • 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.
  • 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.
  • 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.
  • 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 is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal.
  • 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.
  • 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.
  • R 23 and R 26 since the easy synthesis, preferably the same, and more preferably a hydrogen atom.
  • 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 is an oxygen atom or N + HR ′.
  • Y 21 is preferably a single bond, —O— (CH 2 ) n21 —, or —O— (CH 2 ) n22 —Ar 21 —, and X 21 is N +.
  • 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 >”.
  • 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.
  • X 21 is an oxygen atom or N + H 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 —.
  • 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).
  • 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. .
  • 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—.
  • “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.
  • compound (2-1) a compound represented by the following general formula (2-1)
  • compound (2-2) a compound represented by the following general formula (2-2)
  • these compounds are only examples of a preferable compound (2), and a preferable compound (2) is not limited to these.
  • 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.
  • 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 >”.
  • 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.
  • 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.
  • 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.
  • R 213 and R223 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.
  • 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.
  • 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.
  • 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.
  • 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 >”.
  • 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
  • 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.
  • 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.
  • compound (2-1) 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.
  • compound (2-2) 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)”).
  • compound (2-2-2) a compound represented by the following general formula (2-2-2)
  • compound (2-2-3) a compound represented by the following general formula (2-2-3)
  • compound (2-2-3) a compound represented by the following general formula (2-2-3)
  • compound (2-2-3) a compound represented by the following general formula (2-2-3)
  • these compounds are only examples of a preferable compound (2), and a preferable compound (2) is not limited to these.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compound (2-1-1) 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.
  • compound (2-1-1a) compounds represented by the following formula (2-1-1a)
  • compound (2-1-1b) hereinafter sometimes abbreviated as “compound (2-1-1b)
  • compound (2-1-1c) a compound represented by the following formula (2- 1-1c)
  • compound (2-1-2a) compounds represented by the following formula (2-1-2a)
  • compound (2-1-2b) a compound represented by the following formula (2-1-2b)
  • compound (2-1-2c) a compound represented by the following formula (2-1-2c)
  • compound (2-1-3) 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.
  • 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)”.
  • compound (2-2-1b) a compound represented by the following formula (2-2-1b)
  • compound (2-2-1c) a compound represented by the following formula (2-2-1c)
  • compound (2-2-1c) a compound represented by the following formula (2-2-1c)
  • compound (2-2-2c) a compound represented by the following formula (2-2-2c)
  • compound (2-2-2c) a compound represented by the following formula (2-2-2c)
  • compound (2-2-3) 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • R 214 is a hydrogen atom, a hydroxyl group, a leaving group (eg, a halogen atom), or a leaving group (eg, a terminal group)
  • 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 (2-1a) is a known compound.
  • 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.
  • R 213 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (2-1b) used is preferably 0.1 to 0.3 times the amount of compound (2-1a) used. .
  • compound (2-1c) it is preferable to react compound (2-1a), compound (2-1b), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1M or more and 5M or less.
  • 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.
  • 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.
  • the compound (2-1c) 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 (2-1c) is a known compound.
  • 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) is a known compound.
  • 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.
  • R 214 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (2-1e) is a novel compound.
  • Y 211 is the same as those exemplified for “ ⁇ Y 211 and Y 221 >” in the above “ ⁇ Compound (2) >>”.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (2-1d) used is preferably 1 to 2 times the amount of compound (2-1c).
  • 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.
  • 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.
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 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.
  • 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.
  • the compound (2-1) 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).
  • NMR magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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).
  • 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.
  • 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 (2-2a) is a known compound.
  • R 221 and R 222 are each independently an alkyl group having 1 to 10 carbon atoms.
  • 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.
  • R 223 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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.
  • 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.
  • R 224 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (2-2b) used is preferably 0.1 to 0.3 times the amount of compound (2-2a) used. .
  • compound (2-2c) it is preferable to react compound (2-2a), compound (2-2b), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • the compound (2-2c) 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 (2-2c) is a known compound.
  • 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) is a known compound.
  • 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.
  • R 225 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (2-1e) is a novel compound.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (2-2d) used is preferably 1 to 2 times the amount of compound (2-2c).
  • 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.
  • 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.
  • 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.
  • a protective group has couple
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 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.
  • 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.
  • compound (2-2) 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).
  • NMR magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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
  • 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 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.
  • R 31 s 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.
  • 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.
  • 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 is a group having 1 to 2 monovalent substituents present on the benzene ring and having an anionic functional group at the terminal.
  • 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.
  • 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.
  • 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.
  • R33 and R38 are easy to synthesize
  • 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.
  • 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 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.
  • X 32 is an oxygen atom or N + HR ′′.
  • Y 31 represents a single bond, -O- (CH 2) n31 - , or, -O- (CH 2) n32 -Ar 31 - is preferably, X 32 is N +
  • 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 >”.
  • 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.
  • X 32 is preferably is an oxygen atom or N + H 2.
  • 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 —.
  • 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).
  • 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. .
  • 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—.
  • “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.
  • compound (3-1) a compound represented by the following general formula (3-1)
  • compound (3-2) a compound represented by the following general formula (3-2)
  • these compounds are only examples of a preferable compound (3), and a preferable compound (3) is not limited to these.
  • 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
  • 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 >”.
  • 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.
  • 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.
  • 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.
  • R 313 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.
  • 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.
  • 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 >”.
  • 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.
  • 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 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.
  • X 31 is a silicon atom
  • Y 311 is a single bond
  • n311 and n312 Are each independently an integer of 1 to 8
  • 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.
  • 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
  • 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
  • n322 -Ar 321 - a is an integer of 1 ⁇ 8 N321 and n322 are each independently also Ar 321
  • 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.
  • 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
  • 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.
  • compound (3-1) 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.
  • 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)”).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)”.
  • compound (3-1-2b) a compound represented by the following formula (3-1-2b)
  • compound (3-1-2c) a compound represented by the following formula (3-1-2c)
  • compound (3-1-2c) a compound represented by the following formula (3-1-2c)
  • 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.
  • compound (3-2-1a) compounds represented by the following formula (3-2-1a)
  • compound (3-2-1b) compounds represented by the following formula (3-2-1b)
  • compound (3-2-1c) compounds represented by the following formula (3-2-1c)
  • compound (3-2-2a) compounds represented by the following formula (3-2-2a)
  • compound (3-2-2b) a compound represented by the following formula (3-2-2b)
  • compound (3-2-2c) a compound represented by the following formula (3-2-2c)
  • compound (3-2-3) 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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)
  • 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 (3-1a) is a known compound.
  • 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.
  • R 313 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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 34 and R 37 are preferably the same because of easy synthesis, and more preferably a hydrogen atom or a halogen atom.
  • 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.
  • 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.
  • 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).
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 compound (3-1b) used is preferably 0.1 to 0.3 times the amount of compound (3-1a) used. .
  • compound (3-1c) it is preferable to react compound (3-1a), compound (3-1b), and strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • the compound (3-1c) 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 (3-1c) is a known compound.
  • 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) 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.
  • 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.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (3-1e) is a novel compound.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (3-1d) used is preferably 1 to 2 times the amount of compound (3-1c).
  • 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.
  • 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) 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.
  • an aprotic solvent as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.
  • the compound (3-1) 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).
  • NMR magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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)”).
  • 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.
  • 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.
  • 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 (3-2a) is a known compound.
  • 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.
  • R 323 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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 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.
  • 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.
  • 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.
  • R 324 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
  • 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.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (3-2b) used is preferably 0.1 to 0.3 times the amount of compound (3-2a) used. .
  • compound (3-2c) it is preferable to react compound (3-2a), compound (3-2b), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • Compound (3-2c) is a known compound.
  • 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).
  • 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.
  • R 325 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (3-2e) is a novel compound.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (3-2d) used is preferably 1 to 2 times the amount of compound (3-2c).
  • 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.
  • 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) 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.
  • compound (3-2) 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).
  • NMR magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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.
  • 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.
  • 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 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.
  • R 41 s 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the xanthene skeleton in the 1st position, one R 41 is in the 4th position, and the other R 41 is in the 6th 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.
  • halogen atom in R 44 , R 47 and R 48 examples 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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 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 —.
  • 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).
  • 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. .
  • 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—.
  • “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.
  • these compounds are only examples of a preferable compound (4), and a preferable compound (4) is not limited to these.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • R 413 and R423 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.
  • 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.
  • 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.
  • X 41 is a silicon atom
  • Y 411 is a single bond
  • n411 and n412 Are each independently an integer of 1 to 8
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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)”).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compound (4-1-1) 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.
  • compound (4-1-2a) compounds represented by the following formula (4-1-2a)
  • compound (4-1-2b) a compound represented by the following formula (4-1-2b)
  • compound (4-1-2c) a compound represented by the following formula (4-1-2c)
  • compound (4-1-2c) a compound represented by the following formula (4-1-2c”
  • compound (4-1-3a) compounds represented by the following formula (4-1-3a)
  • compound (4-1-3a) compounds represented by the following formula (4-1-3a)
  • compound (4-1-3b) a compound represented by the following formula (4-1-3b)
  • compound (4-1-3c) a compound represented by the following formula (4-1-3c)
  • compound (4-2-1c) a compound represented by the following formula (4-2-1c)
  • compound (4-2-1c) a compound represented by the following formula (4-2-1c)
  • compound (4-2-2a) compounds represented by the following formula (4-2-2a)
  • compound (4-2-2b) compounds represented by the following formula (4-2-2b)
  • compound (4-2-2c) compounds represented by the following formula (4-2-2c)
  • compound (4-2-3a) compounds represented by the following formula (4-2-3a)
  • compound (4-2-3a) compounds represented by the following formula (4-2-3a)
  • compound (4-2-3b) a compound represented by the following formula (4-2-3b)
  • compound (4- 2-3c) a compound represented by the following formula (4-2-3c)
  • compound (4-2-3c) a compound represented by the following formula (4-2-3c)
  • 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.
  • 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.
  • 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.
  • 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) ”)
  • compound (4-1c1 ′) a compound represented by the following general formula (4-1c1 ′)
  • compound (4-1c1 ′) hereinafter sometimes abbreviated as “compound (4-1c1 ′)”.
  • 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.
  • Compound (4-1a) is a known compound.
  • 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.
  • R 413 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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 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.
  • 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.
  • 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.
  • the compound (4-1c1 ′) is a known compound.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (4-1b1) used is 0.1 to 0.3 times the amount of compound (4-1a) used. preferable.
  • compound (4-1c1 ′) it is preferable to react compound (4-1a), compound (4-1b), and strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • the compound (4-1c1 ′) 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
  • Compound (4-1c1) is a known compound.
  • 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.
  • a catalyst such as tetrakis (triphenylphosphine) palladium and 1,3-dimethylbarbituric acid.
  • 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.
  • 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.
  • 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.
  • the compound (4-1c1) 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.
  • Compound (4-1d1) is a known compound.
  • 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.
  • R 414 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (4-1e1) is a novel compound.
  • Y 411 is the same as those exemplified for “ ⁇ Y 411 and Y 421 >” in the above “ ⁇ Compound (4) >>”.
  • 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.
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (4-1d1) used is preferably 1 to 2 times the amount of compound (4-1c1).
  • 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.
  • 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
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.
  • compound (4-1) -1 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.
  • 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) ”), ,
  • compound (4-1e2) compound represented by general formula (4-1e2)
  • production step compound (4-1e2)
  • production step of compound (4-1) -2 compound (4-1) -2
  • 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.
  • Compound (4-1a) is a known compound.
  • 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.
  • R 413 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 compound (4-1b2) used is preferably 0.1 to 0.3 times the amount of compound (4-1a) used. .
  • compound (4-1c2) it is preferable to react compound (4-1a), compound (4-1b2), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • the compound (4-1c2) 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 (4-1c2) is a known compound.
  • 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).
  • the compound (4-1d2) is a known compound.
  • 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.
  • R 414 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (4-1e2) is a novel compound.
  • Y 411 is the same as that exemplified for “ ⁇ Y 411 and Y 421 >” in “ ⁇ Compound (4) >>” above.
  • 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.
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (4-1d2) used is preferably 1 to 2 times the amount of compound (4-1c2).
  • 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.
  • 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.
  • 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.
  • a protective group has couple
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.
  • compound (4-1) -2 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.
  • the structure can be confirmed by a known method such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared spectroscopy (IR).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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) ”)
  • compound (4-2c1 ′) a compound represented by the following general formula (4-2c1 ′)
  • compound (4-2c1 ′) hereinafter sometimes abbreviated as “compound (4-2c1 ′)”.
  • 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)
  • 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.
  • Compound (4-2a) is a known compound.
  • 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.
  • R 423 is a group having an anionic functional group at the terminal.
  • 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) is a known compound.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the compound (4-2c1 ′) is a known compound.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (4-2b1) used is 0.1 to 0.3 times the amount of compound (4-2a) used. preferable.
  • compound (4-2c1 ′) it is preferable to react compound (4-2a), compound (4-2b1), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • the compound (4-2c1 ′) 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
  • Compound (4-2c1) is a known compound.
  • 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).
  • 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.
  • a catalyst such as tetrakis (triphenylphosphine) palladium and 1,3-dimethylbarbituric acid.
  • 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.
  • 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.
  • 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.
  • the compound (4-2d1) is a known compound.
  • 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.
  • R 424 is preferably a hydrogen atom, a halogen atom, —O—CH 2 —X, or —O—Ph—CH 2 —X.
  • X represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (4-2e1) is a novel compound.
  • Y 421 is the same as those exemplified for “ ⁇ Y 411 and Y 421 >” in the above “ ⁇ Compound (4) >>”.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction is preferably performed in an inert gas atmosphere.
  • 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.
  • the amount of compound (4-2d1) used is preferably 1 to 2 times the amount of compound (4-2c1).
  • 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.
  • 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
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • 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.
  • compound (4-2) -1 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).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • 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).
  • compound (4-2e2) compound represented by the following general formula (4-2e2)
  • compound (4-2e2) production step compound represented by the following general formula (4-2e2)
  • 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.
  • Compound (4-2a) is a known compound.
  • 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.
  • R 423 is a group having an anionic functional group at the terminal.
  • 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. .
  • the compound (4-2b2) is a known compound.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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 compound (4-2b2) used is preferably 0.1 to 0.3 times the amount of compound (4-2a) used. .
  • compound (4-2c2) it is preferable to react compound (4-2a), compound (4-2b2), and a strong base under acidic conditions.
  • the acid include inorganic acids such as hydrochloric acid; organic acids such as acetic acid and p-toluenesulfonic acid.
  • the amount of acid used is preferably 1 M or more and 5 M or less, for example.
  • 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.
  • 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.
  • Compound (4-2c2) is a known compound.
  • 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).
  • the compound (4-2d2) is a known compound.
  • 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.
  • 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.
  • “X” represents a halogen atom
  • Ph represents a substituted or unsubstituted phenylene group.
  • Compound (4-2e2) is a novel compound.
  • Y 421 is the same as that exemplified for “ ⁇ Y 411 and Y 421 >” in the above “ ⁇ Compound (4) >>”.
  • 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).
  • 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.
  • an aprotic solvent as a reaction solvent.
  • 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).
  • the reaction in an inert gas atmosphere.
  • 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.
  • the amount of compound (4-2d2) used is preferably 1 to 2 times the amount of compound (4-2c2).
  • 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.
  • 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.
  • 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.
  • an aprotic solvent is preferably used as a reaction solvent.
  • 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 reaction temperature is preferably 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 30 ° C. or lower.
  • the reaction time is preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours.
  • compound (4-2) -2 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).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • IR infrared spectroscopy
  • a fluorescent probe for detecting ALP includes a compound having an anionic functional group and a phosphate group.
  • the enzymatic activity of ALP in a biological sample can be measured with high quantitativeness and sensitivity.
  • the compound in this embodiment has an anionic functional group and a phosphate group.
  • anionic functional group means a functional group that ionically dissociates with a negative charge in an aqueous medium
  • cationic functional group means a positive charge in an aqueous medium. It means a functional group that has ionic dissociation.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • the compound (4-1-1) 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the fluorescent probe for ALP detection of the present embodiment 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 ⁇ M to 50 ⁇ M 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • a target substance include biological substances such as proteins, nucleic acids, lipids, and saccharides.
  • blotting methods 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.
  • 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.
  • a microdevice includes the above-described fluorescent probe for ALP detection.
  • 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.
  • the glass material include soda lime glass, Pyrex (registered trademark) glass, Vycor (registered trademark) glass, and quartz glass.
  • 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 ⁇ m or less, for example, 100 nm or more and 10 ⁇ m or less, for example, 1 ⁇ m or more and 10 ⁇ m or less.
  • the depth of the well of the microdevice of the present embodiment may be, for example, 10 nm or more and 1 ⁇ m or less, for example, 100 nm or more and 800 ⁇ m or less, for example, 200 nm or more and 700 nm or less.
  • 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.
  • 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.
  • “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.
  • 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.
  • ALP has subtypes such as ALP1, ALP2, ALP3, ALP4, ALP5, ALP6, and ALPI.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ M or less, for example, 1 ⁇ M or more and 100 ⁇ M or less, for example, 10 ⁇ M or more and 100 ⁇ M or less. I just need it.
  • the microdevice of this embodiment 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.
  • the conventional compound for example, compound (10)
  • 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.
  • the method for detecting the enzymatic activity of alkaline phosphatase in a biological sample is a method using the above-described microdevice.
  • 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.
  • a solution containing a biological sample is added to a microdevice including the above-described ALP detection fluorescent probe.
  • 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.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGS. 7A and 7B 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.
  • 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.
  • 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.
  • FIG. 9 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.
  • 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.
  • 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.
  • 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.
  • the enzymatic activity of ALP in a biological sample can be measured with high quantitativeness and sensitivity.

Abstract

L'invention concerne une sonde fluorescente pour détecter une phosphatase alcaline. La sonde fluorescente est appropriée pour des mesures qui nécessitent une sensibilité et une quantitativité élevées. La sonde fluorescente pour détecter une phosphatase alcaline comprend un composé qui possède un groupe fonctionnel anionique et un groupe phosphate.
PCT/JP2018/007993 2017-03-03 2018-03-02 Sonde fluorescente pour la détection de phosphatase alcaline et son utilisation WO2018159810A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2020246616A1 (fr) 2019-06-07 2020-12-10 国立大学法人 東京大学 Sonde fluorescente pour la détection de l'activité de l'enpp

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Publication number Priority date Publication date Assignee Title
WO2020246616A1 (fr) 2019-06-07 2020-12-10 国立大学法人 東京大学 Sonde fluorescente pour la détection de l'activité de l'enpp
EP3981778A4 (fr) * 2019-06-07 2023-07-19 The University of Tokyo Sonde fluorescente pour la détection de l'activité de l'enpp

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