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WO2020095946A1 - Nouvelle sonde fluorescente - Google Patents

Nouvelle sonde fluorescente Download PDF

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Publication number
WO2020095946A1
WO2020095946A1 PCT/JP2019/043494 JP2019043494W WO2020095946A1 WO 2020095946 A1 WO2020095946 A1 WO 2020095946A1 JP 2019043494 W JP2019043494 W JP 2019043494W WO 2020095946 A1 WO2020095946 A1 WO 2020095946A1
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fluorescent probe
groups
psa
hydrogen atom
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Japanese (ja)
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泰照 浦野
真子 神谷
輝 小笠原
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University of Tokyo NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a novel fluorescent probe for detecting puromycin-sensitive aminopeptidase (PSA), a detection method using the fluorescent probe, and a detection kit containing the probe.
  • PSA puromycin-sensitive aminopeptidase
  • imaging technologies such as X-ray CT, magnetic resonance imaging (MRI), and positron emission tomography (PET) are extremely useful in cancer diagnosis. It has become an important technology. Above all, fluorescence imaging is expected as a simple and safe method having high sensitivity and excellent temporal / spatial resolution.
  • the inventors of the present invention have developed a fluorescent probe for ⁇ -glutamyl transpeptidase (GGT), which has been enhanced in breast cancer cells, and can detect a minute tumor with high sensitivity and specificity in a short time. This has been demonstrated (for example, Non-Patent Documents 1 and 2).
  • GGT fluorescent probe of Non-Patent Document 1 uses a fluorescent response in the green region. Therefore, if a probe showing a fluorescent response in a red region or the like different from this can be developed, a plurality of fluorescent probes should be used in combination. Therefore, it is expected that breast cancer can be detected with higher sensitivity and specificity.
  • the present invention provides a novel fluorescent probe that can detect a biomarker enzyme activity having high specificity for breast cancer with high sensitivity and high selectivity, and can detect it as a response of red fluorescence having a longer wavelength. This is an issue.
  • the present inventors have found puromycin-sensitive aminopeptidase (PSA) as a new biomarker enzyme for detecting breast cancer cells, and have characteristics different from conventional green probes ( Breast cancer cells can be detected as a response to red fluorescence by using a compound in which a fluorophore having wavelength, charge, and lipophilicity) is used as a nucleus and an amino acid residue that is specifically cleaved by PSA is introduced into the side chain.
  • PSA puromycin-sensitive aminopeptidase
  • a fluorescent probe for detecting puromycin-sensitive aminopeptidase which comprises a compound represented by the following formula (I) or a salt thereof: [In the formula, A is an alanine residue, B is an amino acid residue, wherein A is connected to an adjacent N atom by forming an amide bond and B is connected to A by forming an amide bond.
  • X is Si (R a ) (R b ), Ge (R a ) (R b ), Sn (R a ) (R b ), C (R a ) (R b ), P ( ⁇ O) (R a ) or O
  • R a and R b each independently represent a hydrogen atom, an alkyl group, or an aryl group
  • R 1 is a hydrogen atom, or 1 to 4 same or different independently selected from the group consisting of an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azido group.
  • R 2 represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, aryl, or heteroaryl
  • R 3 and R 4 are each independently, independently of the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group and an azido group.
  • R 5 , R 6 and R 7 each independently represent a hydrogen atom or an alkyl group, Here, R 6 or R 7 may together with R 4 form a ring structure containing a nitrogen atom to which they are bonded.
  • B is an asparagine residue or a lysine residue
  • R 3 is an optionally substituted alkyl group having 1 to 6 carbon atoms or an optionally substituted alkoxy group having 1 to 6 carbon atoms.
  • a kit for detecting a mycin-sensitive aminopeptidase (PSA) is provided.
  • the present invention provides ⁇ 9> A step of bringing the fluorescent probe according to any one of the above ⁇ 1> to ⁇ 5> into contact with a sample to be measured, and puromycin-sensitive aminopeptidase (PSA) contained in the sample and the fluorescent probe.
  • a method for detecting PSA which comprises a step of observing a fluorescence response or a change in absorbance due to the reaction of ⁇ 10> The detection method according to ⁇ 9> above, wherein the fluorescence response is visualized using a fluorescence imaging means.
  • ⁇ 11> The method according to ⁇ 9> or ⁇ 10>, wherein the sample to be measured is a cell expressing puromycin-sensitive aminopeptidase (PSA); ⁇ 12> The method according to ⁇ 11> above, wherein the cells are cancer cells; ⁇ 13> The method according to ⁇ 12> above, wherein the cancer cells are breast cancer cells.
  • PSA puromycin-sensitive aminopeptidase
  • puromycin-sensitive aminopeptidase which is expressed at a high level in breast cancer tissue, can be detected by a fluorescence response, and thereby the presence of breast cancer can be identified accurately, quickly, and with high sensitivity. It also has an excellent effect that imaging can be performed.
  • the fluorescent probe of the present invention detects the activity of PSA as a fluorescent response as a new breast cancer biomarker enzyme different from the conventionally known ⁇ -glutamyl transpeptidase (GGT), the conventional GGT is used. It is also possible to identify breast cancer specimens that could not be detected by the detection fluorescent probe.
  • GGT ⁇ -glutamyl transpeptidase
  • the fluorescent probe of the present invention can detect PSA activity as a fluorescent response in the red region, by using it in combination with a conventional green fluorescent probe, it is possible to detect breast cancer cells with higher specificity as compared with individual use of individual probes. In addition to enabling detection, multicolor imaging using a plurality of fluorescent response regions is also possible. Thereby, cancer cells such as breast cancer can be visualized and detected more precisely and with high sensitivity.
  • the fluorescent probe of the present invention is expected to be used not only for medical applications such as detecting or diagnosing cancer cells such as breast cancer and normal cells in real time, but also as a basic research tool for elucidating disease states.
  • FIG. 1 shows fluorescence imaging images (a and b) of a tumor sample and a normal sample treated with the fluorescent probe 1, and a temporal change (c) of fluorescence intensity.
  • FIG. 2 is a graph showing the time change of the T / N ratio with the addition of the fluorescent probe of the present invention to the tumor site.
  • FIG. 3 is a graph showing the fluorescence intensity change due to the reaction between the fluorescent probe 1 and the tumor site in the DEG assay (a) and PSA.
  • FIG. 4 is an absorption spectrum (left) and a fluorescence spectrum (right) of the fluorescent probes 1 and 2.
  • FIG. 5 is a table showing the fluorescence quantum yields of the fluorescent probes 1 and 2.
  • FIG. 6 is a graph showing changes in fluorescence intensity due to the reaction of the fluorescent probe 2 with PSA and GGT.
  • FIG. 7 is a multicolor imaging image using the fluorescent probe 2 and gGlu-HMRG.
  • the “amino acid” can be any compound as long as it is a compound having both an amino group and a carboxyl group, and includes natural and unnatural ones. It may be a neutral amino acid, a basic amino acid, or an acidic amino acid, and in addition to an amino acid which itself functions as a transmitter such as a neurotransmitter, a physiologically active peptide (dipeptide, tripeptide, tetrapeptide, Amino acids that are constituents of polypeptide compounds (including oligopeptides) and proteins can be used, and may be, for example, ⁇ -amino acids, ⁇ -amino acids, ⁇ -amino acids and the like.
  • amino acid residue refers to a structure corresponding to the remaining partial structure (acyl group) obtained by removing a hydroxyl group from a carboxyl group of an amino acid.
  • halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
  • alkyl may be any of linear, branched, cyclic, or a combination thereof, which is an aliphatic hydrocarbon group.
  • the number of carbon atoms of the alkyl group is not particularly limited, and examples thereof include 1 to 6 carbon atoms (C 1 to 6 ), 1 to 10 carbon atoms (C 1 to 10 ), and 1 to 15 carbon atoms (C 1 to 15). ) And having 1 to 20 carbon atoms (C 1 to 20 ). When the number of carbons is specified, it means “alkyl” having the number of carbons in the range.
  • C 1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, It includes n-heptyl, n-octyl and the like.
  • the alkyl group may have one or more optional substituents.
  • substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl.
  • alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (eg, alkoxy group, arylalkyl group, etc.).
  • a functional group when a functional group is defined as “optionally substituted”, the type of the substituent, the substitution position, and the number of the substituents are not particularly limited, and the substitution of two or more substituents is not limited. If they have groups, they may be identical or different.
  • the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group.
  • Substituents may be further present in these substituents. Examples of such a group include, but are not limited to, a halogenated alkyl group and a dialkylamino group.
  • alkenyl refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond.
  • non-limiting examples thereof are vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl.
  • the double bond may be in either the cis or trans conformation.
  • alkynyl refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond.
  • non-limiting examples include ethynyl, propynyl, 2-butynyl, 3-methylbutynyl and the like.
  • cycloalkyl refers to a monocyclic or polycyclic non-aromatic ring system consisting of the above alkyl.
  • the cycloalkyl may be unsubstituted or substituted by one or more substituents which may be the same or different and non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl. And cycloheptyl and the like, non-limiting examples of polycyclic cycloalkyl include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl and the like.
  • the cycloalkyl may be a heterocycloalkyl containing at least one hetero atom (eg, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom.
  • Any —NH in the heterocycloalkyl ring may be protected, such as, for example, as a —N (Boc), —N (CBz) and —N (Tos) group, a nitrogen atom in the ring or
  • the sulfur atom may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.
  • non-limiting examples of monocyclic heterocycloalkyl include diazapanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, lactam and Examples include lactones.
  • cycloalkenyl refers to a monocyclic or polycyclic non-aromatic ring system containing at least one carbon-carbon double bond.
  • the cycloalkenyl may be unsubstituted or substituted by one or more substituents which may be the same or different and non-limiting examples of monocyclic cycloalkenyl include cyclopentenyl, cyclohexenyl. And cyclohepta-1,3-dienyl and the like, and non-limiting examples of polycyclic cycloalkenyl include norbornylenyl and the like.
  • the cycloalkyl may be a heterocycloalkenyl containing one or more hetero atoms (eg, oxygen atom, nitrogen atom, or sulfur atom) as a ring-constituting atom. Atoms may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.
  • hetero atoms eg, oxygen atom, nitrogen atom, or sulfur atom
  • aryl may be either a monocyclic or a condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. And the like) may be included in the aromatic heterocycle. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether the aryl is a single ring or a fused ring, it may be attached at all possible positions.
  • Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl group.
  • Non-limiting examples of the fused polycyclic aryl include 1-naphthyl group, 2-naphthyl group, 1-indenyl group, 2-indenyl group, 2,3-dihydroinden-1-yl group, 2,3 -Dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, Isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 , 3-dihydrobenzothiophen-2-yl group, benzothiazolyl group
  • the aryl group may have one or more optional substituents on its ring.
  • substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl.
  • the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing an aryl moiety (for example, an aryloxy group and an arylalkyl group).
  • arylalkyl represents alkyl substituted with the above aryl.
  • the arylalkyl may have one or more optional substituents.
  • substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group.
  • the acyl group has two or more substituents, they may be the same or different.
  • Non-limiting examples of arylalkyl include benzyl group, 2-thienylmethyl group, 3-thienylmethyl group, 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group, 2-furylmethyl group, 3-furylmethyl group, 2-thiazolylmethyl group, 4-thiazolylmethyl group, 5-thiazolylmethyl group, 2-oxazolylmethyl group, 4-oxazolylmethyl group, 5-oxazolylmethyl group, 1-pyrazolylmethyl group , 3-pyrazolylmethyl group, 4-pyrazolylmethyl group, 2-pyrazinylmethyl group, 2-pyrimidinylmethyl group, 4-pyrimidinylmethyl group, 5-pyrimidinylmethyl group, 1-pyrrolylmethyl group, 2-pyrrolylmethyl group, 3-pyrrolylmethyl group , 1-imidazolylmethyl group, 2-imidazolylmethyl group, 4-imidazolylmethyl group , 3-pyridazinylmethyl group, 4-pyri
  • arylalkenyl refers to an alkenyl substituted with the above aryl.
  • the “alkoxy group” has a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group having a linear, branched, or cyclic structure or a combination thereof.
  • alkylene is a linear or branched saturated hydrocarbon divalent group such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, and the like.
  • alkenylene is a divalent group consisting of an unsaturated hydrocarbon having at least one linear or branched carbon-carbon double bond, and examples thereof include ethenylene. , 1-methylethenylene, 1-ethylethenylene, 1,2-dimethylethenylene, 1,2-diethylethenylene, 1-ethyl-2-methylethenylene, propenylene, 1-methyl-2-propenylene, 2-methyl -2-propenylene, 1,1-dimethyl-2-propenylene, 1,2-dimethyl-2-propenylene, 1-ethyl-2-propenylene, 2-ethyl-2-propenylene, 1,1-diethyl-2-propenylene , 1,2-diethyl-2-propenylene, 1-butenylene, 2-butenylene, 1-methyl-2-butenylene, 2-methyl-2-butenylene 1,1-dimethyl-2-butenylene, 1,2-dimethyl
  • arylene and arylalkylene mean a divalent group based on the above “aryl” and “arylalkyl”, respectively.
  • oxyalkylene and aryleneoxy mean a divalent group based on the above “alkoxy” and “aryloxy”, respectively.
  • ring structure when formed by the combination of two substituents, means a heterocyclic or carbocyclic group, such group being saturated, unsaturated, or aromatic.
  • it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperdinyl, imidazolyl, pyrrolidinyl, pyridyl and the like.
  • a substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art can perform a specific substitution, for example, bonding to hydrogen.
  • the fluorescent probe of the present invention contains a compound having a structure represented by the following formula (I).
  • a and B have a structure in which amino acid residues are linked, and are sites that can be selectively hydrolyzed by puromycin-sensitive aminopeptidase (PSA). More specifically, A represents an alanine residue and B represents an amino acid residue.
  • A is linked with N (R 5 ) in the formula by forming an amide bond, that is, with the N atom of N (R 5 ) with the carbonyl moiety (acyl group) of the alanine residue A.
  • A can be linked to amino acid residue B like a normal peptide chain, so that B forms an amide bond with A and links them. Therefore, B has a structure similar to that of a so-called N-terminal residue, and the intermediate amino acid residue A can be linked to B in the same manner as a normal peptide chain.
  • puromycin-sensitive aminopeptidase is a cytoplasmic alanylaminopeptidase that uses Ala-AMC as a standard substrate, and it has been reported that it is highly expressed particularly in the central nervous system. It is also used in clinical medicine as a biomarker for estimating renal function, and its function is known to be inhibited by puromycin.
  • a and B are substrate peptides of PSA and are combinations of amino acid residues that are easily hydrolyzed selectively. preferable. More specifically, as described above, A is an alanine residue (“Ala” or “A”) and B is an asparagine residue (“Asn” or “N”) or a lysine residue (“ Lys "or” K ”) is preferred. More preferably, A is an alanine residue and B is an asparagine residue.
  • R a and R b each independently represent a hydrogen atom, an alkyl group, or an aryl group.
  • R a and R b are alkyl groups, they can have one or more substituents, and examples of such substituents include an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, You may have 1 or 2 or more amino groups, a sulfo group, etc.
  • Each of R a and R b may be an optionally substituted alkyl group having 1 to 6 carbon atoms, and both are preferably methyl groups. Further, in some cases, R a and R b may be bonded to each other to form a ring structure.
  • R a and R b can combine with each other to form a spiro carbocycle.
  • the ring formed is preferably a 5- to 8-membered ring, for example.
  • X is preferably Si (R a ) (R b ), and more preferably Si (CH 3 ) 2 .
  • R 1 represents a hydrogen atom or 1 to 4 substituents bonded to the benzene ring.
  • R 1 is a hydrogen atom, or 1 to 4 independently selected from the group consisting of an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azido group. The same or different substituents are represented.
  • R 1 is other than a hydrogen atom, its position on the benzene ring is not particularly limited, but it is preferably meta to R 2 .
  • the benzene ring has two or more substituents, they may be the same or different.
  • R 1 is a hydrogen atom.
  • R 2 represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, aryl, or heteroaryl.
  • it is a hydroxyl group, a cyano group, an optionally substituted C 1 -C 4 alkyl group, a C 1 -C 4 alkoxy group, or a phenyl group. More preferably, it is a methyl group or a methoxy group.
  • R 3 and R 4 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group and an azido group. 1 to 3 of the same or different substituents. Preferably, both R 3 and R 4 are hydrogen atoms. Further, similar to R 1 described above, R 3 or R 4 may have a fluorophore that serves as a donor of fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • R 5 , R 6 and R 7 each independently represent a hydrogen atom or an alkyl group.
  • R 5 , R 6 and R 7 both represent an alkyl group, they may be the same or different.
  • R 5 , R 6 , and R 7 can each independently be a methyl group or an ethyl group.
  • R 5 , R 6 and R 7 are all hydrogen atoms.
  • R 6 or R 7 may together with R 4 form a ring structure containing a nitrogen atom to which they are bonded.
  • the ring structure is a 5-8 membered heterocyclic structure.
  • the ring structure may further include a heteroatom other than the nitrogen atom to which R 6 and R 7 are bonded.
  • R 1 , R 3 , R 4 , R 5 , R 6 , and R 7 are all hydrogen atoms.
  • the compound represented by the above formula (I) has a monovalent positive charge at the N atom to which R 6 and R 7 are linked, it usually exists as a salt.
  • salts include base addition salts, acid addition salts, amino acid salts and the like.
  • base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, and organic amine salts such as triethylamine salt, piperidine salt, and morpholine salt.
  • mineral acid salts such as hydrochlorides, sulfates, nitrates, etc.
  • organic acid salts such as carboxylates, trifluoroacetates, methanesulfonates, paratoluenesulfonates, citrates, oxalates, etc.
  • amino acid salts include glycine salts. However, it is not limited to these salts.
  • the compound represented by the formula (I) may have one or two or more asymmetric carbon atoms depending on the kind of the substituent, and stereoisomers such as optical isomers or diastereoisomers exist. There are cases. Any stereoisomers in pure form, any mixture of stereoisomers, racemates and the like are included within the scope of the present invention.
  • the compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and all of these substances are included in the scope of the present invention.
  • the type of solvent that forms the solvate is not particularly limited, and examples thereof include solvents such as ethanol, acetone, and isopropanol.
  • the above-mentioned fluorescent probe may be used as a composition by adding an additive usually used for the preparation of reagents, if necessary.
  • an additive such as a solubilizing agent, a pH adjusting agent, a buffering agent, an isotonicity agent, etc. can be used, and the blending amount thereof can be appropriately selected by those skilled in the art. is there.
  • These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, and a liquid.
  • PSA enzyme activity is detected by fluorescence control using photoinduced electron transfer (PET: Photoinduced electron transfer). More specifically, as shown in the following scheme, when the side chain at the 3-position of the xanthene ring is linked to the amino acid residue “AB” (peptide chain), it can be irradiated with excitation light. Since the xanthene ring portion has a high electron accepting property, it is in a state of being quenched by electron transfer from the benzene ring portion. On the other hand, when the fluorescent probe molecule reacts with PSA, the side chain “AB” peptide chain is cleaved by hydrolysis and the 3-position is changed to an amino group. As a result, fluorescence emission is observed.
  • PET Photoinduced electron transfer
  • fluorescence is quenched by PET from the benzene ring site before the reaction with PSA, but when the peptide chain is cleaved by PSA, the electron density of the xanthene ring site is increased. Changes and the fluorescence quenching by PET is suppressed, so that the activity of PSA can be detected as a fluorescence response.
  • the fluorescence emission due to the “AB” cleavage can be fluorescence in the red region with a fluorescence wavelength of 600 nm or more. This makes it possible to detect the presence of cancer cells and the like as a red fluorescence response.
  • visible light having a wavelength of about 400 to 600 nm may be irradiated as excitation light.
  • the fluorescence wavelength to be observed is usually in the range of about 580 to 700 nm.
  • the presence of PSA is specifically detected or visualized by observing the fluorescence response or the change in absorbance due to the reaction between PSA and the fluorescent probe.
  • detection should be construed in the broadest sense including measurement for various purposes such as quantification and qualification.
  • A) a step of bringing the fluorescent probe of the present invention into contact with a sample to be measured; and B) Only the target cells expressing PSA are specifically fluorescent by including a step of observing a fluorescence response or a change in absorbance due to a reaction between puromycin-sensitive aminopeptidase (PSA) contained in the sample and the fluorescent probe. It can be detected or visualized in response.
  • PSA puromycin-sensitive aminopeptidase
  • the method of the present invention can further include observing the fluorescence response using a fluorescence imaging means.
  • a fluorescence photometer having a wide measurement wavelength can be used, but the fluorescence response can also be visualized using a fluorescence imaging means capable of displaying a two-dimensional image.
  • the fluorescence imaging means By using the fluorescence imaging means, the fluorescence response can be visualized in two dimensions, and the PSA can be instantly visually recognized.
  • a device known in the art can be used as the fluorescence imaging device.
  • the reaction between the sample to be measured and the fluorescent probe can be detected by a change in the UV-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).
  • a solution containing the fluorescent probe is added to the sample, applied, or sprayed. It can be appropriately selected according to the measurement environment and the like. Such contacting is preferably done in an in vitro environment.
  • the fluorescent probe of the present invention is applied to diagnosis or diagnosis assistance in animal individuals, or detection of specific cells or tissues, as a means for bringing the fluorescent probe into contact with the target cells or tissues, without being limited, for example, intravenous administration and the like, which is a general administration means in the art, can be used.
  • the application concentration of the fluorescent probe of the present invention is not particularly limited, but for example, a solution having a concentration of about 0.1 to 10 ⁇ M can be applied.
  • the light irradiation performed on the target cells can be performed by irradiating the cells with light directly or via a waveguide (optical fiber etc.).
  • a waveguide optical fiber etc.
  • any light source can be used as long as it can irradiate the light having the absorption wavelength of the fluorescent probe of the present invention, and it can be used in the environment for carrying out the method of the present invention. It can be selected accordingly.
  • the compound represented by the above formula (I) or a salt thereof may be used as it is, but if necessary, an additive usually used for the preparation of a reagent may be blended to obtain a composition. You may use.
  • an additive for using the reagent in a physiological environment an additive such as a solubilizing agent, a pH adjusting agent, a buffering agent, an isotonicity agent, etc. can be used, and the blending amount thereof can be appropriately selected by those skilled in the art. It is possible.
  • compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, a solution, etc., but distilled water for injection or an appropriate buffer solution at the time of use. It is possible to apply by dissolving in.
  • the sample to be measured in the above step A) can be cells expressing puromycin-sensitive aminopeptidase (PSA), but when such cells are cancer cells or cancer tissues expressing PSA
  • PSA puromycin-sensitive aminopeptidase
  • cancer cells and cancer tissues can be detected or visualized by the detection method of the present invention. That is, the fluorescent probe of the present invention, the composition containing the fluorescent probe, and the detection method of the present invention can also be used for cancer diagnosis.
  • cancer tissue means any tissue containing cancer cells.
  • tissue should be interpreted in its broadest sense including a part or whole of an organ, and should not be limitedly interpreted in any sense. Since the fluorescent probe or the composition for detecting cancer cells containing the fluorescent probe of the present invention has an action of detecting PSA which is specifically and strongly expressed in cancer tissue, PSA is regarded as cancer tissue. A tissue that highly expresses is preferred. Further, in this specification, the term “diagnosis” should be interpreted in its broadest sense, including the confirmation of the presence of cancerous tissue at any part of a living body visually or under a microscope.
  • the sample to be measured in the above step A) is preferably a cancer cell, more preferably a breast cancer cell.
  • a fluorescent probe for ⁇ -glutamyl transpeptidase can be used in combination with the fluorescent probe of the present invention.
  • GGT ⁇ -glutamyl transpeptidase
  • the fluorescent probe of the present invention is usually prepared as a solution, but for example, it is provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, and a liquid, and at the time of use. It can also be applied by dissolving in distilled water for injection or an appropriate buffer solution.
  • the kit may optionally contain other reagents and the like as necessary.
  • a solubilizing agent, a pH adjusting agent, a buffering agent, an isotonicity agent and the like can be used, and the amount of these can be appropriately selected by those skilled in the art.
  • a green fluorescent probe for GGT detection can be included in addition to the fluorescent probe of the present invention.
  • the reaction solution was filtered off. 900 ⁇ L of DMF was added to the resin, shaken for 1 minute, and DMF was removed by filtration. This washing operation was repeated twice more.
  • 800 ⁇ L of 40% piperidine in DMF was added, shaken for 3 minutes, and the reaction solution was removed by filtration.
  • 400 ⁇ L of 40% piperidine in DMF was added, shaken for 12 minutes and the reaction solution was removed by filtration.
  • 900 ⁇ L of DMF was added to the resin, shaken for 1 minute, and DMF was removed by filtration. This washing operation was repeated 5 more times.
  • a / B 70/30 to 0/100 in 40 minutes (eluent A: H 2 containing 0.1% TFA. O, eluent B: 0.1% TFA in 80% acetonitrile and 20% H 2 O) to give compound 6 of sufficient purity.
  • Fluorescent Probe 2 (NA-2-OMe SiR600) of the Present Invention
  • the fluorescent probe 2 of the present invention (NA-2-OMe SiR600) having Asn-Ala (NA) in the side chain was synthesized as follows.
  • Fluorescent probe 2 differs from fluorescent probe 1 (NA-2-Me SiR600) in that the substituent on the benzene ring corresponding to R 2 in formula (I) is methoxy.
  • N, N, N ', N'-Tetraallyldiamino-Si-xanthone was synthesized according to the literature (Kushida, Y. et al., Bioorganic & medicinal chemistry letters 2012, 22 (12), 3908-3911.).
  • N, N, N ', N'-Tetraallyldiamino-Si-xanthone (1.28 g, 2.97 mmol) dissolved in anhydrous THF (10 mL) was added slowly and the mixture was warmed to room temperature. It was then stirred for 1 hour. The reaction was quenched by addition of 2N HCl and the mixture was stirred at room temperature for 5 minutes. Saturated NaHCO 3 was added and the whole was extracted with CH 2 Cl 2 . The organic layer was washed with brine, dried over Na 2 SO 4 and evaporated. The residue was dissolved in MeOH, stirred at 0 ° C. and NaBH 4 (340 mg, 8.99 mmol) was added.
  • Example 2 Screening Using Human Breast Cancer Surgical Specimens Using the library of fluorescent probes synthesized in Example 1, screening was performed on human breast cancer surgical specimens. First, a lysate is prepared from the surgical specimen by dividing it into a tumor site and a normal site, and each probe with a different peptide chain at the AB site is reacted with each lysate to measure the change in fluorescence intensity. Thirty-six fluorescent probes showing a sufficient increase in fluorescence and a high T / N ratio (Tumor to Normal ratio) were selected as candidate probes.
  • T / N ratio Tumor to Normal ratio
  • the probe solution was dropped on the excised specimen and fluorescence imaging was performed to measure the change in fluorescence intensity.
  • fluorescence imaging was performed to measure the change in fluorescence intensity.
  • a large number of 3-5 mm square pieces were cut from a human breast cancer specimen with a scalpel and placed in each well of an 8-well chamber slide (Thermo Fisher 155411), and each fluorescent probe solution (50 ⁇ M) was placed there.
  • each fluorescent probe solution 50 ⁇ M
  • the fluorescent probe 1 (compound 8: NA-2-MeSiR600) in which the AB site in formula (I) is “Asn-Ala” (NA) shows a rapid increase in fluorescence at the tumor site. , It was found that the normal site showed almost no increase.
  • the fluorescent probes with AB sites of “Lys-Ala” (KA) and “Met-Ala” (MA) also showed high sensitivity.
  • FIG. 1 shows fluorescence imaging images (a and b) of a tumor sample and a normal sample treated with the fluorescent probe 1, and a temporal change (c) of fluorescence intensity.
  • the time change of T / N ratio with addition to the tumor site is shown in FIG.
  • the T / N ratio at each time was higher for fluorescent probe 1 (NA) except after 1 minute.
  • a lysate prepared from the tumor site of the surgical specimen was used to perform a DEG (diced electrophoresis gel) assay, and a single highly active spot was observed (Fig. 3 ( a)). Focusing on this spot, PMF (Peptide Mass Fingerprinting) analysis was performed. As a result, puromycin-sensitive aminopeptidase (PSA) was identified as a metabolic enzyme of fluorescent probe 1. When the identified PSA was reacted with fluorescent probe 1 using a purified enzyme, a rapid increase in fluorescence was shown (FIG. 3 (b)). From these results, it was found that the fluorescent probe 1 is effective as a breast cancer detection fluorescent probe using PSA as a biomarker enzyme.
  • DEG cold electrophoresis gel
  • Fluorescence Assay for PSA The absorption and fluorescence spectra of the fluorescent probe 1 (Compound 8: NA-2-Me SiR600) and the fluorescent probe 2 (Compound 11: NA-2-OMe SiR600) synthesized in Example 1 were measured, and , The quantum yield was calculated. In each case, the fluorescent probe concentration was 1 ⁇ M, and a 0.1 M Na phosphate buffer (pH 7.4, 0.1% DMSO) solution was used.
  • the fluorescent probe 2 showed a rapid increase in fluorescence with the addition of PSA. This is probably because "Asn-Ala" at the AB site in the formula (I) was cleaved by the reaction with PSA, whereby PET was suppressed and the fluorescence emission of the xanthene ring was restored. ..
  • GGT was added, no increase in fluorescence was observed, indicating that fluorescent probe 2 was not cleaved at the AB site by GGT. This result demonstrates that the fluorescent probe 2 can specifically detect the presence of PSA.
  • gGlu-HMRG breast cancer detection green fluorescent probe
  • a tumor site and a normal site were separately cut out in a size of about 5 mm square from a breast cancer sample, and a mixed solution of fluorescent probe 2 and gGlu-HMRG was dropped on each. Merged images were created by acquiring green and red fluorescence images 30 minutes after dropping 1, 3, 5, 10, 15, 20, and 30 minutes, and superimposing them. A total of 14 cases were imaged by the same method. An image of one of these cases is shown in FIG. 7 as a representative example. In FIG. 7, both of the fluorescent probe 2 and gGlu-HMRG showed an increase in fluorescence only at the tumor site, but did not show an increase in fluorescence at the normal site. The tumor site could be detected in 13 of 14 cases (93%). This demonstrates that the combined use of fluorescent probe 2 for PSA and gGlu-HMRG for GGT can detect a tumor site of breast cancer with higher specificity.
  • the fluorescent probe of the present invention can selectively detect PSA and is a highly practical fluorescent probe for breast cancer.

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Abstract

Le problème décrit par la présente invention consiste à fournir une nouvelle sonde fluorescente capable de détecter l'activité enzymatique d'un biomarqueur hautement spécifique du cancer du sein avec une sensibilité et une sélectivité élevées, en utilisant la fluorescence rouge présentant une longueur d'onde supérieure en tant que réponse. La solution selon l'invention porte sur une sonde fluorescente pour la détection de l'aminopeptidase sensible à la puromycine (PSA) et comprend un composé représenté par la formule (I) et un sel de celui-ci [dans la formule : A représente un résidu d'alanine; B représente un résidu d'acide aminé; A est lié à un atome de N adjacent par formation d'une liaison amide avec celui-ci; B est lié à A par formation d'une liaison amide avec celui-ci; X représente Si(Ra)(Rb), Ge(Ra)(Rb), Sn(Ra)(Rb), C(Ra)(Rb), P(=O)(Ra), ou O (ici, Ra et Rb représentent chacun indépendamment un atome d'hydrogène, un groupe alkyle ou un groupe aryle); R1 représente un atome d'hydrogène ou 1 à 4 groupes substituants identiques ou différents choisis indépendamment dans le groupe constitué par les groupes alkyle, les groupes carboxyle, les groupes ester, les groupes alcoxy, les groupes amide et les groupes azido, qui sont tous éventuellement substitués; R2 représente un atome d'hydrogène, un groupe hydroxyle, un groupe cyano, ou un groupe alcoxy, un aryle, un hétéroaryle ou un groupe alkyle éventuellement substitué; R3 et R4 représentent chacun indépendamment 1 à 3 groupes substituants identiques ou différents choisis indépendamment dans le groupe constitué par un atome d'hydrogène, un groupe hydroxyle, des atomes d'halogène, et des groupes sulfo, des groupes carboxyle, des groupes ester, des groupes amide, des groupes azido et des groupes alkyle éventuellement substitués; et R5, R6 et R7 représentent chacun indépendamment un atome d'hydrogène ou un groupe alkyle, R6 ou R7 formant éventuellement conjointement avec R4 une structure cyclique comprenant des atomes d'azote correspondants liés à celle-ci].
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WO2021070906A1 (fr) * 2019-10-09 2021-04-15 国立大学法人 東京大学 Sonde fluorescente pour détecter une aminopeptidase sensible à la puromycine ou une bléomycine hydrolase
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RU234589U1 (ru) * 2025-03-11 2025-06-03 федеральное государственное автономное образовательное учреждение высшего образования "Московский физико-технический институт (национальный исследовательский университет)" Устройство для детектирования одиночных флуоресцентных молекул

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