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EP-3805236-B1 - FLUORESCENT COMPOUND AND FLUORESCENT LABELED BIOLOGICAL SUBSTANCE USING THE SAME

EP3805236B1EP 3805236 B1EP3805236 B1EP 3805236B1EP-3805236-B1

Inventors

  • NAKATA, HIYOKU
  • WATANABE, KOUSUKE
  • ASAKURA, Akihiro
  • MAKITA, KEIKO
  • KANAZAWA, YOSHINORI
  • KANEKO, KAZUHEI

Dates

Publication Date
20260506
Application Date
20190531

Claims (15)

  1. A fluorescent compound represented by Formula (1) or Formula (4), in the formula, X represents CR 5 or N, R 1 to R 6 represent a halogen atom, a cyano group, or a group represented by Formula (A), R 7 represents an alkyl group, a cycloalkyl group, an aliphatic heterocyclic group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a hydroxyl group, a mercapto group, an alkoxy group, an alkylthio group, an aryloxy group, an arylthio group, an aryl group, a heteroaryl group, or a halogen atom, where R 6 and R 7 do not bond to each other to form a ring, Q 1 and Q 2 represent a group represented by any one of Formulae (1-1) to (1-3), L 1 and L 2 represent an alkylene group, an alkenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, a cycloalkenylene group, a divalent aliphatic heterocyclic group, or a group represented by any one of Formulae (1-1) to (1-8), or a linking group formed by combining two to four of these groups, and a ring β 1 and a ring β 2 are a 5- to 8-membered ring, provided that at least one of R 1 to R 7 , L 1 , L 2 , Q 1 , or Q 2 has at least one of a carboxy group or a salt thereof, a sulfo group or a salt thereof, a phosphono group or a salt thereof, an onio group, or a polyamino acid residue, the polyamino acid residue being a group in which one of the hydrogen atoms of -NH 2 , -CO 2 H, -OH, and -SH contained in the amino acids constituting the polyamino acid is replaced with a bond, in a case where X is CR 5 , where R 5 is a group represented by Formula (A), the ring β 1 and the ring β 2 are a 6-membered ring, and L 1 and L 2 are an arylene group, (a) there is no case in which L 3 is a single bond and R 111 is an aryl group having a linear alkyl group having 18 or more carbon atoms, as a substituent, and (b) there is no case in which L 3 is arylene group and R 111 is a linear alkyl group having 18 or more carbon atoms, and in a case where X is CR 5 , where R 5 is a group represented by Formula (A), the ring β 1 and the ring β 2 are a 6-membered ring, L 1 and L 2 are an arylene group, and R 111 has a substituent having a dipyrromethene boron complex structure, the dipyrromethene boron complex structure has a structure in which a dipyrromethene skeleton is coordinately bonded to a boron atom in a tridentate or tetradentate coordination, in the formula, L 3 represents a single bond or a linking group formed by combining one or two or more of an alkylene group, an arylene group, and a group represented by any one of Formulae (1-1) to (1-8), R 111 represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group, and * represents a bonding portion, in the formula, R 11 and R 12 represent a hydrogen atom or a substituent, and * represents a bonding portion.
  2. The fluorescent compound according to claim 1, wherein both the Q 1 and the Q 2 are a group represented by Formula (1-1).
  3. The fluorescent compound according to claim 1 or 2, wherein the X is CR 5 , where R 5 is represented by Formula (A), and L 3 and R 111 in Formula (A) satisfy the following; the L 3 is a single bond, the R 111 is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group, or the L 3 is a group formed by combining one or two or more of an alkylene group, an arylene group, and a group represented by any one of Formulae (1-1) to (1-8), and the R 111 is a hydrogen atom.
  4. The fluorescent compound according to any one of claims 1 to 3, wherein at least one of the R 3 or the R 4 is a group containing a carboxy group or a salt thereof, a group containing a sulfo group or a salt thereof, a group containing a phosphono group or a salt thereof, a group containing an onio group, or a group containing a polyamino acid residue.
  5. The fluorescent compound according to any one of claims 1 to 4, wherein at least one of the R 3 or the R 4 is a carboxy group or a salt thereof, a sulfo group or a salt thereof, or a phosphono group or a salt thereof.
  6. The fluorescent compound according to any one of claims 1 to 5, wherein at least one of the R 3 or the R 4 is a sulfo group or a salt thereof.
  7. The fluorescent compound according to any one of claims 1 to 6, wherein the L 1 and the L 2 are an alkylene group, an ethenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, or a divalent aliphatic heterocyclic group, or a linking group formed by combining two of these groups, and the ring β 1 and the ring β 2 are a 6- to 8-membered ring.
  8. The fluorescent compound according to any one of claims 1 to 7, wherein the fluorescent compound is represented by Formula (2) or Formula (5), in the formula, X represents CR 5 or N, R 1 to R 7 and Q respectively have the same meanings as R 1 to R 7 and Q 1 in Formula (1) or (4), a ring A represents a hydrocarbon ring or a heterocycle, and L 11 represents a single bond or an alkylene group, an alkenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, a divalent aliphatic heterocyclic group, or a group represented by any one of Formulae (1-1) to (1-8), or a linking group formed by combining two of these groups, where a ring B 11 and a ring β 21 are a 6- to 8-membered ring.
  9. The fluorescent compound according to any one of claims 1 to 7, wherein the fluorescent compound is represented by Formula (3) or Formula (6), in the formula, X represents CR 5 or N, R 1 to R 7 and Q respectively have the same meanings as R 1 to R 7 and Q 1 in Formula (1) or (4), and L 11 has the same meaning as L 11 in Formula (2) or (5), and R 8 represents a substituent, and n is an integer of 0 to 4, where a ring β 11 and a ring β 21 are a 6- to 8-membered ring.
  10. The fluorescent compound according to any one of claims 1 to 6, wherein the fluorescent compound is represented by Formula (7) or Formula (8), in the formula, X represents CR 5 or N, R 1 to R 7 and Q respectively have the same meanings as R 1 to R 7 and Q 1 in Formula (1) or (4), L 12 represents a single bond or an alkylene group, an alkenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, a divalent aliphatic heterocyclic group, or a group represented by any one of Formulae (1-1) to (1-8), or a linking group formed by combining two of these groups, and L 13 represents a methylene group or a group represented by any one of Formulae (1-1) to (1-8), where a ring β 12 and a ring β 22 are a 5- to 8-membered ring.
  11. The fluorescent compound according to any one of claims 1 to 6, wherein the fluorescent compound is represented by Formula (9), in the formula, X represents CR 5 or N, R 1 to R 5 and Q respectively have the same meanings as R 1 to R 5 and Q 1 in Formula (1) or (4), L 11 and L 12 represent a single bond or an alkylene group, an alkenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, a divalent aliphatic heterocyclic group, or a group represented by any one of Formulae (1-1) to (1-8), or a linking group formed by combining two of these groups, L 13 represents a methylene group or a group represented by any one of Formulae (1-1) to (1-8), and a ring A represents a hydrocarbon ring or a heterocycle, and where a ring β 12 is a 5- to 8-membered ring, and a ring β 21 is a 6- to 8-membered ring.
  12. The fluorescent compound according to any one of claims 1 to 11, wherein at least one of R 1 to R 8 , L 1 , L 2 , L 11 to L 13 , Q, Q 1 , or Q 2 has a moiety bondable to a biological substance.
  13. A fluorescent labeled biological substance, which is obtained through bonding between the fluorescent compound according to claim 12 and a biological substance.
  14. The fluorescent labeled biological substance according to claim 13, wherein the biological substance is any one of a protein, a peptide, an amino acid, a nucleic acid, a sugar chain, or a lipid.
  15. The fluorescent labeled biological substance according to claim 13 or 14, wherein the bonding between the fluorescent compound and the biological substance is formed by any one of the followings i) to v), i) non-covalent or covalent bond between peptides, ii) Van der Waals interaction between a long-chain alkyl group in a fluorescent compound and a lipid bilayer or lipid in a biological substance, iii) an amide bond formed by reacting an N-hydroxysuccinimide ester in a fluorescent compound with an amino group in a biological substance, iv) a thioether bond formed by reacting a maleimide group in a fluorescent compound with a sulfanyl group in a biological substance, and v) a bond with a formation of a triazole ring, which is formed by Click reaction between an azido group in a fluorescent compound and an acetylene group in a biological substance, or between an acetylene group in a fluorescent compound and an azido group in a biological substance.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent compound and a fluorescent labeled biological substance using the fluorescent compound. 2. Description of the Related Art Bioimaging technology for analyzing the dynamics and functions of living body molecules, cells, tissues, and the like in the living body has been utilized for the diagnosis of various diseases. In recent years, in vivo fluorescence imaging, which visualizes and observes a specific portion of a living body with a fluorescent dye, is expected as a new technique for living body observation. In this in vivo fluorescence imaging, an organic fluorescent dye is generally used. However, the organic fluorescent dye has low light resistance and deteriorates by irradiation with excitation light, and thus the observation of the target living body may not be sufficiently performed. A dipyrromethene boron complex is known as a fluorescent dye that has a high quantum yield and exhibits sharp emission characteristics and thus is used in various fields. For example, JP2016-193907A discloses a fluorescent lipid ether compound that is expected to be used for tumor diagnosis and discloses a dipyrromethene boron complex as an example of a fluorophore in the fluorescent lipid ether compound. In addition, US2009/0192298A discloses an energy transfer cassette through a chemical bond and discloses a dipyrromethene boron complex as an example of a donor or an acceptor of this cassette. Clarke et al ChemPhotoChem 2017, 1, 513-517 describe a helically chiral dipyrromethene (BODIPY) with a N,N,O,C-boron-chelation motif, synthesized by means of a one-pot boron metathesis, nucleophilic aromatic substitution, Suzuki coupling, boron chelation, cascade reaction. SUMMARY OF THE INVENTION However, the dipyrromethene boron complex is generally poor water solubility and has low light resistance. Accordingly, in order to apply the dipyrromethene boron complex to a tool for living body observation, such as in vivo fluorescence imaging, it is necessary to impart both water solubility and light resistance to the dipyrromethene boron complex at a high level. From this viewpoint, the inventors of the present invention performed studies and found that although dipyrromethene boron complexes having high water solubility have been reported, such dipyrromethene boron complexes having high water solubility are easily discolored and still have poor light resistance. An object of the present invention is to provide a fluorescent compound having a dipyrromethene boron complex structure and realizing both sufficient hydrophilicity required for in vivo fluorescence imaging and excellent light resistance. In addition, another object of the present invention is to provide a fluorescent labeled biological substance obtained by bonding the fluorescent compound to a biological substance. The inventors of the present invention speculated that the discoloring of the existing dipyrromethene boron complexes having water solubility is mainly due to photolysis involving the boron atom by reactive oxygen species. Under this speculation, the inventors of the present invention further introduced a specific hydrophilic group into a compound having a specific structure in which a compound having a dipyrromethene skeleton into which a substituent had been introduced was coordinated to a boron atom as a tridentate ligand or a tetradentate ligand and have found that discoloring is successfully reduced and a fluorescent dye having excellent hydrophilicity can be obtained. The present invention has been completed through further studies based on these findings. That is, the above objects of the present invention have been achieved by the following means. <1> A fluorescent compound represented by Formula (1) or Formula (4). In the formula, X represents CR5 or N. R1 to R6 represent a halogen atom, a cyano group, or a group represented by Formula (A).R7 represents an alkyl group, a cycloalkyl group, an aliphatic heterocyclic group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a hydroxyl group, a mercapto group, an alkoxy group, an alkylthio group, an aryloxy group, an arylthio group, an aryl group, a heteroaryl group, or a halogen atom. Here, R6 and R7 do not bond to each other to form a ring.Q1 and Q2 represent a group represented by any one of Formulae (1-1) to (1-3).L1 and L2 represent an alkylene group, an alkenylene group, an arylene group, a heteroarylene group, a cycloalkylene group, a cycloalkenylene group, a divalent aliphatic heterocyclic group, a group represented by any one of Formulae (1-1) to (1-8), or a linking group formed by combining two to four of these groups.A ring β1 and a ring β2 are a 5- to 8-membered ring. Here, at least one of R1 to R7, L1, L2, Q1, or Q2 has at least one of a carboxy group or a salt thereof, a sulfo group or a salt thereof, a phosphono group or a salt thereof, an onio group, or a polyamino acid residue. In a c