WO2018159631A1 - Sonde fluorescente dans l'infrarouge proche pour la détection d'une activité peptidase - Google Patents

Sonde fluorescente dans l'infrarouge proche pour la détection d'une activité peptidase Download PDF

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WO2018159631A1
WO2018159631A1 PCT/JP2018/007327 JP2018007327W WO2018159631A1 WO 2018159631 A1 WO2018159631 A1 WO 2018159631A1 JP 2018007327 W JP2018007327 W JP 2018007327W WO 2018159631 A1 WO2018159631 A1 WO 2018159631A1
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fluorescent probe
peptidase
detecting
cancer
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PCT/JP2018/007327
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Japanese (ja)
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泰照 浦野
真子 神谷
竜 岩立
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国立大学法人 東京大学
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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
    • 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

Definitions

  • the present invention relates to a fluorescent probe for detecting peptidase activity. More specifically, the present invention relates to a novel fluorescent probe capable of detecting peptidase activity such as aminopeptidase by fluorescence in the near infrared region, a composition for cancer diagnosis containing the probe, and a detection method using the fluorescent probe. .
  • GTT ⁇ -glutamyltransferase
  • protease a peptidase (protease)
  • Non-patent Document 1 a fluorescent probe group capable of detecting the activity of ⁇ -glutamyltransferase based on a fluorescent dye exhibiting an intramolecular spirocyclization equilibrium
  • the absorption / emission wavelength of such a conventional fluorescent probe is 550 nm or less, and although it can be detected with high sensitivity to cancer cells and the like present on the tissue surface, living organisms such as lymph node metastasis can be used. There is a restriction that it cannot be applied to cancer cells existing in tissues or inside organs.
  • the near-infrared region from 650 nm to 900 nm is called “biological window”, and it is known that light absorption by water and light scattering by tissues are small.
  • autofluorescence of living tissue decreases as it is excited at a longer wavelength, it is considered that imaging with a lower background and higher contrast is possible.
  • a fluorescent probe that can detect peptidase activity by such a fluorescence response in the near-infrared region and is excellent in cell penetration has not been developed.
  • an object of the present invention is to provide a novel fluorescent probe that can detect peptidase activity highly expressed in cancer cells with a longer-wavelength fluorescence response and has excellent tissue permeability. is there.
  • Another object of the present invention is to provide a system capable of detecting and visualizing cancer cells existing in a living organization or an organ with high sensitivity in surgical extraction operations and the like.
  • the present inventors have obtained a fluorescent molecule having a structure in which a group cleaved by a peptidase is introduced into a silicon rhodamine skeleton and having optimized intramolecular spirocyclization characteristics. It was found that a fluorescent probe that is colorless and non-fluorescent before contact with the target peptidase, but exhibits a fluorescence response in the near-infrared region, can be obtained by reaction with the peptidase. In addition, as a result of applying the fluorescent probe to clinical specimens, it has been found that cancer cells and tissues expressing the target peptidase can be detected and visualized rapidly. Based on this knowledge, the present invention has been completed.
  • a fluorescent probe for detecting peptidase activity comprising a compound represented by the following formula (I) or a salt thereof: [Where, X represents Si (R a ) (R b ) (where R a and R b each independently represents a hydrogen atom or an alkyl group); Y represents a C 0 -C 3 alkylene group, and the alkylene group may be substituted with a halogen atom or haloalkyl; R 1 represents 1 to 4 identical groups independently selected from the group consisting of a hydrogen atom, a cyano group, an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group.
  • R 2 together with R 3 forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached
  • R 4 together with R 5 forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached
  • R 6 , R 7 , R 8 and R 9 each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group
  • R 10 represents an amino acid-derived acyl residue.
  • the heterocyclic structure formed by combining R 2 and R 3 is a 6-membered ring, and the heterocyclic structure formed by combining R 4 and R 5 is a 6-membered ring.
  • the fluorescent probe for detecting peptidase activity according to ⁇ 1>; ⁇ 3> The fluorescent probe for detecting a peptidase activity according to the above ⁇ 1> or ⁇ 2>, wherein R a and R b are both C 1 -C 4 alkyl groups; ⁇ 4>
  • ⁇ 5> The fluorescent probe for detecting peptidase activity according to any one of ⁇ 1> to ⁇ 4>, wherein the peptidase is ⁇ -glutamyltranspeptidase, dipeptidylpeptidase IV (DPP-IV), or calpain
  • the present invention provides: ⁇ 9> A composition for cancer diagnosis comprising the fluorescent probe for detecting peptidase activity according to any one of ⁇ 1> to ⁇ 8>above; ⁇ 10> The composition for cancer diagnosis as described in ⁇ 9> above, which is used for cancer surgical treatment or cancer examination; and ⁇ 11> the cancer surgical treatment or cancer examination is open surgery, endoscopic surgery, Or the composition for cancer diagnosis as described in said ⁇ 10> which is an endoscopy is provided.
  • the present invention also relates to a method for detecting or visualizing a target cell in which a specific peptidase is expressed, specifically, ⁇ 12>
  • the present invention also relates to an apparatus comprising means for observing a fluorescence response by the above-described fluorescent probe for detecting peptidase activity, specifically, ⁇ 16> a fluorescence imaging means for observing a fluorescence response due to a reaction between the peptidase specifically expressed in the target cell and the peptidase activity detection fluorescent probe according to any one of the above ⁇ 1> to ⁇ 8>.
  • apparatus ⁇ 17> The apparatus according to ⁇ 16>, wherein the apparatus is an endoscope or an in vivo fluorescence imaging apparatus; and ⁇ 18> the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8.
  • a kit is provided.
  • the fluorescent probe of the present invention is colorless and non-fluorescent before contact with the target peptidase, but can detect the fluorescence response in the near infrared region specifically and on / off by reaction with the peptidase.
  • to show the fluorescence response in the near-infrared region which is about 100 nm longer than conventional fluorescent probes, to visualize cancer cells that exist in the deep part of the body, such as lymph node metastasis, which was difficult in the past It has the effect of being able to.
  • the fluorescent probe of the present invention to intraoperative or preoperative imaging, various cancers can be identified accurately, rapidly, and with high sensitivity.
  • accurate diagnosis of cancer becomes possible, microscopic cancer at the time of endoscopy with relatively small invasion can be detected at an early stage, and it is useful for radical treatment with chemoradiotherapy.
  • it is suitable for evaluation of a resection stump by applying it at the time of surgical operation such as ESD or laparotomy, and it is possible to perform radical treatment without any residual. It can be said that the medical and industrial utility value and economic effect of the present invention are extremely large.
  • FIG. 1 is an absorption and fluorescence spectrum of Compound 1 (HMJSiR).
  • FIGS. 1A and 1B are an absorption spectrum and a fluorescence spectrum, respectively, and
  • FIG. 1C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 2 is an absorption and fluorescence spectrum of Compound 2 (gGlu-HMJSiR) which is a fluorescent probe of the present invention.
  • 2A and 2B are an absorption spectrum and a fluorescence spectrum, respectively, and
  • FIG. 2C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 2D is an absorption spectrum of Compound 2 normalized with respect to the absorption spectrum of Compound 1.
  • FIG. 1 is an absorption and fluorescence spectrum of Compound 1 (HMJSiR).
  • FIGS. 1A and 1B are an absorption spectrum and a fluorescence spectrum, respectively, and
  • FIG. 1C is a graph showing the pH dependence of absorbance and fluorescence
  • FIG. 3 is an absorption and fluorescence spectrum of Compound 3 (HMSiR600) of Comparative Example.
  • 3A and 3B are an absorption spectrum and a fluorescence spectrum, respectively, and
  • FIG. 3C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 4 is an absorption and fluorescence spectrum of Compound 4 (HMSiR620h) of Comparative Example.
  • 4A and 4B are an absorption spectrum and a fluorescence spectrum, respectively, and
  • FIG. 4C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 5 shows changes in fluorescence intensity with time when ⁇ -glutamyltranspeptidase (GGT) was added to Compound 2, which is the fluorescent probe of the present invention, and addition of ⁇ -glutamyltranspeptidase (GGT) and an inhibitor (GGsTop).
  • the time change of the fluorescence intensity at the time of performing is shown.
  • FIG. 6 is a fluorescence confocal imaging image of GGT activity in living cells using Compound 2, which is the fluorescent probe of the present invention.
  • FIG. 7 is a two-color imaging image showing the intracellular localization of Compound 2 in SHIN3 cells.
  • FIG. 8 is a fluorescence imaging image in the mesentery of a model mouse having peritoneal metastasis using Compound 2 which is the fluorescent probe of the present invention.
  • FIG. 9 is a fluorescence imaging image of a model mouse model of a subcutaneous tumor using Compound 2, which is the fluorescent probe of the present invention.
  • halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
  • alkyl may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof.
  • the number of carbon atoms of the alkyl group is not particularly limited.
  • the number of carbon atoms is 1 to 20 (C 1-20 )
  • the number of carbons is 1 to 15 (C 1 to 15 )
  • the number of carbon atoms is 1 to 10 (C 1 to 10). ).
  • the number of carbons it means “alkyl” having the number of carbons within 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, n-heptyl, n-octyl and the like are included.
  • the alkyl group may have one or more arbitrary substituents.
  • Examples of such a substituent 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 alkyl group has two or more substituents, they may be the same or different.
  • a functional group when a functional group is defined as “may be substituted”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different.
  • the substituent group 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. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.
  • alkenyl refers to a linear or branched hydrocarbon group having at least one carbon-carbon double bond.
  • non-limiting examples include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl 4-pentenyl, 1,3-pentanedienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1,4-hexanedienyl).
  • the double bond may be either cis or trans conformation.
  • alkynyl refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond.
  • non-limiting examples include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl.
  • cycloalkyl refers to a monocyclic or polycyclic non-aromatic ring system composed of the above alkyl.
  • the cycloalkyl can be unsubstituted or substituted by one or more substituents, which can be the same or different, and non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl
  • Non-limiting examples of polycyclic cycloalkyls include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl and the like.
  • the cycloalkyl may be a heterocycloalkyl containing one or more hetero atoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as ring-constituting atoms.
  • Any —NH in the heterocycloalkyl ring may be protected, for example as a —N (Boc) group, —N (CBz) group 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 non-limiting examples of polycyclic cycloalkenyl include norbornylenyl and the like.
  • the cycloalkyl may be a heterocycloalkenyl which may be a heterocycloalkenyl containing one or more heteroatoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. Atoms may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.
  • heteroatoms for example, an oxygen atom, a nitrogen atom, or a sulfur atom
  • alkylene is a divalent group consisting of a linear or branched saturated hydrocarbon, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1 -Methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1 -Diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrime Len, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-d
  • aryl may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom Etc.) may be an aromatic heterocyclic ring. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether aryl is a single ring or a fused ring, it can 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, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazole-3 -Yl group and the like.
  • Non-limiting examples of 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,
  • an aryl group may have one or more arbitrary substituents on the 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 the aryl moiety (for example, an aryloxy group and an arylalkyl group).
  • the “alkoxy group” is a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group that is linear, branched, cyclic, or a combination thereof.
  • methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group, etc. are preferable Take as an example.
  • ring structure when formed by a combination of two substituents, means a heterocyclic or carbocyclic ring, such ring being saturated, unsaturated, or aromatic. be able to. Accordingly, it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above.
  • heterocyclic structure is synonymous with a heterocyclic ring, and means a monocyclic heterocycle having one or more heteroatoms arbitrarily selected from O, S and N in the ring. And such rings can be saturated, unsaturated, or aromatic. Further, these monocyclic heterocycles may further include, for example, a ring (polycyclic heterocycle) in which one or two 3- to 8-membered rings are condensed.
  • the non-aromatic hetero ring include a piperidine ring, a piperazine ring, and a morpholine ring.
  • aromatic hetero ring include a pyridine ring, a pyrimidine ring, a pyrrole ring, and an imidazole ring. Other examples include julolidine and indoline.
  • a specific substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will recognize a specific substituent, for example, hydrogen. It can be understood that the bonds are formed. Therefore, when it is described that specific substituents together form a ring structure, those skilled in the art can understand that the ring structure can be formed by an ordinary chemical reaction and can be easily generated. Both such ring structures and their process of formation are within the purview of those skilled in the art. Moreover, the said heterocyclic structure may have arbitrary substituents on the ring.
  • the fluorescent probe for detecting peptidase activity of the present invention comprises a compound having a structure represented by the following formula (I).
  • X represents Si (R a ) (R b ).
  • R a and R b each independently represent a hydrogen atom or an alkyl group.
  • the alkyl group can be a C 1 -C 4 alkyl group.
  • R a and R b are alkyl groups, they can have one or more substituents, and examples of such substituents include alkyl groups, alkoxy groups, halogen atoms, hydroxyl groups, carboxyl groups, You may have 1 or 2 or more amino groups, sulfo groups, etc.
  • R a and R b are preferably each a C 1 -C 4 alkyl group, more preferably a methyl group (in which case X is Si (CH 3 ) 2 ).
  • R a and R b may be bonded to each other to form a ring structure.
  • R a and R b are both alkyl groups, R a and R b can be bonded to each other to form a spirocarbocycle.
  • the ring formed is preferably about 5 to 8 membered ring, for example.
  • Y represents a C 0 -C 3 alkylene group.
  • the alkylene group may be substituted with a halogen atom or haloalkyl. In the case of a C 0 alkylene group, Y means a direct bond.
  • the alkylene group may be a linear alkylene group or a branched alkylene group.
  • a methylene group (—CH 2 —)
  • an ethylene group (—CH 2 —CH 2 —)
  • a propylene group (—CH 2 —CH 2 —CH 2 —)
  • a branched alkylene group such as —CH ( CH 3 ) —, —CH 2 —CH (CH 3 ) —, —CH (CH 2 CH 3 ) — and the like
  • a methylene group or an ethylene group is preferable, and a methylene group is more preferable.
  • R 1 represents a hydrogen atom, a cyano group, or an optionally substituted alkyl group (for example, the “optionally substituted alkyl group” may include haloalkyl such as fluoroalkyl), a carboxyl group, 1 to 4 identical or different substituents independently selected from the group consisting of an ester group, an alkoxy group, an amide group, and an azide group. Moreover, when it has two or more substituents on the benzene ring, they may be the same or different.
  • R 1 is more preferably a hydrogen atom, a lower alkyl group or a lower alkoxy group. A hydrogen atom is particularly preferred.
  • R 2 together with R 3 forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached. That is, the heterocyclic structure forms a heterocyclic structure in which R 2 and R 3 are connected to each other, and the nitrogen atom to which R 2 is bonded and the carbon atom to which R 3 is bonded are ring member atoms. .
  • the heterocyclic structure is a 6-membered ring.
  • the hetero ring structure can further contain heteroatoms other than the nitrogen atom to which R 2 is attached, it ring member atoms of the nitrogen atom to which R 2 is attached are both carbon atoms Is preferred.
  • R 4 together with R 5 forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached. That is, the heterocyclic structure forms a heterocyclic structure in which R 4 and R 5 are connected to each other so that the nitrogen atom to which R 4 is bonded and the carbon atom to which R 5 is bonded are ring members. .
  • the heterocyclic structure is a 6-membered ring.
  • the hetero ring structure can further contain heteroatoms other than the nitrogen atom to which R 4 is bonded, that ring member atoms of the nitrogen atom to which R 4 is bonded are both carbon atoms Is preferred.
  • both the heterocyclic structure formed by combining R 2 and R 3 and the heterocyclic structure formed by combining R 4 and R 5 are both 6-membered rings.
  • R 6 , R 7 , R 8 and R 9 each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group.
  • R 6 , R 7 , R 8 and R 9 are preferably all hydrogen atoms.
  • R 10 represents an amino acid-derived acyl residue.
  • the said acyl residue means the acyl group which is the remaining partial structure which removed the hydroxyl group from the carboxyl group of the amino acid. That is, the carbonyl moiety of the acyl residue derived from an amino acid and NH of the formula (I) are linked to the silicon rhodamine skeleton by forming an amide bond.
  • 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 non-natural compounds. It may be any of neutral amino acids, basic amino acids, or acidic amino acids. In addition to amino acids that themselves function as transmitters such as neurotransmitters, bioactive peptides (in addition to dipeptides, tripeptides, tetrapeptides, An amino acid that is a constituent component of a polypeptide compound such as an oligopeptide or a protein can be used. As the amino acid, an optically active amino acid is preferably used. For example, as the ⁇ -amino acid, either D- or L-amino acid may be used, but it may be preferable to select an optically active amino acid that functions in a living body.
  • R 10 is a site cleaved by reaction with a target peptidase.
  • the target peptidase can be ⁇ -glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), or calpain. Therefore, when the target peptidase is ⁇ -glutamyl transpeptidase, R 10 is preferably a ⁇ -glutamyl group. When the target peptidase is dipeptidyl peptidase IV, R 10 is preferably an acyl group containing a proline residue.
  • R 10 can be, for example, an acyl group containing a cysteine residue, or Suc-Leu-Leu-Val-Tyr (Suc--) known in the art as a calpain substrate.
  • LLVY or AcLM can also be used.
  • the compound represented by the above formula (I) may exist as a salt.
  • such salts include base addition salts, acid addition salts, amino acid salts and the like.
  • the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt.
  • examples thereof include mineral acid salts such as hydrochloride, sulfate and nitrate, and organic acid salts such as carboxylate, methanesulfonate, paratoluenesulfonate, citrate and oxalate.
  • Examples of 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 more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all 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 any of these substances is included in the scope of the present invention.
  • solvents such as ethanol, acetone, isopropanol, can be illustrated.
  • the above-mentioned fluorescent probe may be used as a composition by blending additives usually used in the preparation of reagents as necessary.
  • additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for use in a physiological environment, and the amount of these 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, or a liquid.
  • the fluorescent probe of the present invention when detecting the peptidase activity using the fluorescent probe of the present invention, or when used for cancer diagnosis as described later, it can be used as a kit containing the fluorescent probe.
  • the fluorescent probe of the present invention is usually prepared as a solution.
  • it is provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid. It can also be applied by dissolving in distilled water for injection or an appropriate buffer.
  • the kit may contain the above-described additives as necessary.
  • Fluorescence emission mechanism of fluorescence probe of the present invention Hereinafter, the fluorescence emission mechanism in the fluorescence probe for detecting peptidase activity of the present invention will be described.
  • the compound represented by the above formula (I) has a structure in which the upper part of the silicon rhodamine skeleton having a structure in which the central atom of rhodamine is substituted from O to Si is closed to form a spiro ring.
  • the fluorescent probe itself is substantially non-absorbing and non-fluorescent (the fluorescence response is off).
  • the acyl residue derived from the amino acid of R 10 is hydrolyzed by a peptidase and cleaved from the silicon rhodamine skeleton, a compound of the formula (II) is generated.
  • the compound of the formula (II) accumulates in lysosomes (pH 4.0 to 5.0) of intracellular acidic vesicles, thereby rapidly opening the spiro ring and showing strong fluorescence.
  • the compound represented by the formula (I) hardly emits fluorescence when irradiated with excitation light of, for example, about 500 to 650 nm in an in vivo pH environment, but the cleavage caused by the reaction with peptidase occurs.
  • the ring compound (II) has a property of emitting extremely strong fluorescence under the same conditions.
  • cells which have taken up a fluorescent probe of the formula (I) is, if not expressing cleavable peptidases hydrolyze the R 10 is preferably a ring-opening compound (II) does not generate a fluorescent Although no substance is produced in the cell, when such a peptidase is present, a ring-opening compound (II) is produced and strong fluorescence is obtained. Therefore, the presence of the target peptidase is observed by the on / off change of the fluorescence intensity, thereby detecting the presence of a cancer cell or the like that expresses the peptidase.
  • a structure having a silicon atom at X which is the 10th element of the xanthene ring and having a hetero ring at one of the ring parts is used. Fluorescence emission due to the ring opening of can be made into fluorescence in the near-infrared region having a fluorescence peak wavelength of 650 nm to 900 nm. This makes it possible to visualize cancer cells and the like existing in the deep part of the living body, such as lymph node metastasis, which has been difficult in the past.
  • the ring-opening compound (II) hydrolyzed by the peptidase When the fluorescent probe of the present invention is applied to living cells, the ring-opening compound (II) hydrolyzed by the peptidase will accumulate in the lysosome of the cell, and 1) the maximum absorption wavelength Is increased by about 100 nm, 2) the fluorescence quantum yield and the molar extinction coefficient are increased, and 3) the spirocyclization equilibrium is shifted due to the low pH in the lysosome to change from the closed ring structure to the open ring structure, and the fluorescence response is changed. can get. In addition, 4) since the fluorescence of the ring-opening compound (II) decreases outside the lysosome, the background signal emitted from the probe leaked from the cell is suppressed, and high-sensitivity detection is possible.
  • a target cell expressing a specific peptidase can be specifically detected or visualized using the fluorescent probe of the present invention.
  • the term “detection” should be interpreted in the broadest sense including measurement for various purposes such as quantitative and qualitative.
  • the specific peptidase can preferably be ⁇ -glutamyl transpeptidase, dipeptidyl peptidase IV (DPP-IV), or calpain.
  • DPP-IV dipeptidyl peptidase IV
  • calpain calpain
  • the target cell is preferably a cancer cell.
  • the method of the present invention may further include observing the fluorescence response using a fluorescence imaging means.
  • a fluorometer having a wide measurement wavelength can be used.
  • the fluorescence response can be visualized by using a fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image.
  • the fluorescence imaging apparatus an apparatus known in the technical field can be used.
  • the reaction between the peptidase and the fluorescent probe can be detected by a change in the ultraviolet-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).
  • step A as means for bringing the fluorescent probe of the present invention into contact with the peptidase specifically expressed in the target cell, typically, a solution containing the fluorescent probe is added, applied or sprayed. However, it can be appropriately selected depending on the application. Further, when the fluorescent probe of the present invention is applied to diagnosis or assistance in an animal individual or detection of a specific cell or tissue, the fluorescent probe and a peptidase expressed in the target cell or tissue are brought into contact with each other. There is no particular limitation, and for example, administration means common in the art such as intravenous administration 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 100 ⁇ M can be applied.
  • the light irradiation performed on the target cell can be performed by irradiating the cell with light directly or via a waveguide (such as an optical fiber).
  • a waveguide such as an optical fiber.
  • any light source can be used as long as it can irradiate light including the absorption wavelength of the fluorescent probe of the present invention after being subjected to enzymatic cleavage. It can be selected as appropriate.
  • the compound represented by the above general formula (I) or a salt thereof may be used as it is, but if necessary, a composition containing additives usually used in the preparation of reagents. It may be used as For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible.
  • additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment.
  • These 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, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It can be dissolved and applied in
  • Cancer Diagnostic Composition Containing the Fluorescent Probe of the Present Invention As described above, cancer cells and cancer tissues expressing a specific peptidase can be detected and visualized by using the fluorescent probe of the present invention. Therefore, in another aspect, the present invention also relates to a cancer diagnostic composition comprising the above-described fluorescent probe for detecting peptidase activity.
  • cancer tissue means any tissue containing cancer cells.
  • tissue must be interpreted in the broadest sense including part or all of an organ, and should not be limitedly interpreted in any way.
  • the cancer diagnostic composition of the present invention has an action of detecting peptidase specifically expressed strongly in cancer tissue, typically ⁇ -glutamyltransferase. A tissue that highly expresses ⁇ -glutamyltransferase is preferred.
  • diagnosis should be interpreted in the broadest sense, including confirming the presence of cancer tissue at an arbitrary biological site under the naked eye or under a microscope.
  • the cancer diagnostic composition of the present invention can be used, for example, during surgery or during examination.
  • the term “surgery” means, for example, craniotomy with open wound, thoracotomy, laparotomy, or skin surgery, as well as gastroscope, colonoscope, laparoscope, thoracoscope, etc. Includes any surgery applied for the treatment of cancer, including microscopic surgery.
  • the term “examination” refers to examinations using endoscopes such as gastroscopes and colonoscopes, treatments such as excision and collection of tissues associated with examinations, and tissues separated and collected from living bodies. This includes inspections performed on
  • Cancers that can be diagnosed by the cancer diagnostic composition of the present invention are not particularly limited, and include any malignant tumor including sarcoma, but preferably used for diagnosis of solid cancer.
  • the cancer diagnostic composition of the present invention is applied to a part or the whole of a surgical field under the naked eye or under a microscope by an appropriate method such as spraying, applying, or injecting, After 10 seconds to several minutes, the application site can be irradiated with light having a wavelength of about 500 nm.
  • the tissue When cancer tissue is included in the application site, the tissue emits fluorescence. Therefore, the tissue is identified as cancer tissue and is excised together with surrounding tissues including the tissue.
  • the composition for cancer diagnosis of the present invention is applied to an examination site by an appropriate method such as spraying, applying, or injecting in a gastroscope or colonoscopy,
  • an appropriate method such as spraying, applying, or injecting in a gastroscope or colonoscopy
  • the application site is irradiated with light having a wavelength of about 500 nm after 10 seconds to several minutes and a fluorescent tissue is confirmed, it can be identified as a cancer tissue.
  • a cancer tissue can be confirmed by endoscopy, it can be subjected to examination resection or therapeutic resection.
  • the cancer diagnostic composition of the present invention may be blended with the above-mentioned additives usually used for the preparation of reagents, if necessary.
  • Apparatus for Using Fluorescent Probe of the Present Invention The present invention is for observing a fluorescence response due to a reaction between a peptidase specifically expressed in a target cell and the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8. It also relates to an apparatus comprising fluorescence imaging means.
  • the device can be an endoscope or an in vivo fluorescence imaging device.
  • NMR measurement was performed using AVANCE III-400 Nanobay (Bruker, Co. Ltd.) (400 MHz for 1 H NMR, 101 MHz for 13 C NMR). Mass spectrometry measurement was performed using MicrOTOF® (ESI-TOF, Bruker, Co. Ltd.). For high-resolution MS (HRMS) measurement, sodium formate was used as an external standard.
  • HRMS high-resolution MS
  • the reaction was quenched with 1 M dilute hydrochloric acid and neutralized with saturated aqueous sodium bicarbonate.
  • the aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure.
  • the resulting residue was purified by aminated silica gel flash column chromatography (normal hexane / dichloromethane) to obtain a colorless liquid. This was dissolved in 10 ml of acetone, cooled to ⁇ 15 ° C., and 153.0 mg (0.968 mmol, 2.0 equivalents) of potassium permanganate was added in 6 portions over 60 minutes.
  • reaction mixture was diluted with dichloromethane, filtered through celite, and purified by silica gel flash column chromatography (normal hexane / ethyl acetate) to obtain 33.0 mg of a pale yellow solid (77.0 ⁇ mol, 15.9% over 2 steps).
  • the obtained residue was dissolved in 10 ml of dichloromethane, 38.7 mg of chloranil (0.157 mmol, 3.9 equivalents) was added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction mixture was purified by short silica chromatography to remove chloranil and concentrated under reduced pressure to obtain an intermediate compound A3.
  • To the residue was added 4 ml of a 1: 1 mixture of trifluoroacetic acid and dichloromethane, and the mixture was stirred at room temperature for 9 hours.
  • the reaction mixture was diluted with dichloromethane and the solvent was removed under reduced pressure. It was then dissolved in 4 ml of a 1: 1 mixture of methanol and water.
  • Compound 2 (fluorescent probe molecule)
  • Compound 2 (gGlu-HMJSiR), which is the fluorescent probe molecule of the present invention, was synthesized from Compound 1 according to Scheme 2 below.
  • Compound 2 is a compound in which a ⁇ -glutamyl group is introduced at the site corresponding to R 10 in formula (I), but by introducing an arbitrary acyl group according to the target peptidase as described above.
  • Other fluorescent probes can be synthesized similarly.
  • reaction solvent was removed under reduced pressure, the residue was dissolved in 5 ml of a 4: 1 mixture of trifluoroacetic acid and dichloromethane, and the solution was stirred for 8 hours.
  • the solvent was removed under reduced pressure and the residue was purified by preparative HPLC to give 1.5 mg of red powder (2.6 ⁇ mol, 12.1%).
  • N, N, N ′, N′-tetraallyldiamino-Si-xanthone A4 was published in literature (T. Egawa, Y. Koide, K. Hanaoka, T. Komatsu, T. Terai, T. Nagano, Chem. Commun. 2011). , 47, 4162.). 70 ⁇ l (0.360 mmol, 5.0 eq) 1-bromo-2-[(1,1-dimethylethoxy) methyl] -benzene in 10 ml dry tetrahydrofuran was cooled to ⁇ 78 ° C. under an argon atmosphere.
  • N, N-diallyl-N ′, N′-dimethylamino-Si-xanthone (A5) was synthesized according to Example 1 of WO2014 / 106957.
  • 1000 ⁇ l (5.14 mmol, 25.7 equivalents) of 1-bromo-2-[(1,1-dimethylethoxy) methyl] -benzene in 10 ml of dehydrated tetrahydrofuran was cooled to ⁇ 78 ° C. under an argon atmosphere.
  • sec-Butyllithium 5.0 ml (0.99 M, 4.95 mmol, 24.8 equivalents) was added dropwise, and the mixture was stirred at ⁇ 78 ° C. under an argon atmosphere for 5 minutes.
  • FIGS. 1A and 1B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 1C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 2A and 2B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 2C is a graph showing the pH dependence of absorbance and fluorescence intensity.
  • FIG. 2D is an absorption spectrum of Compound 2 normalized with respect to the absorption spectrum of Compound 1.
  • Experimental conditions Excitation wavelength of fluorescence spectrum: 500 nm, excitation / fluorescence slit width: 5.0 nm, photomultiplier tube voltage: 700 V
  • Table 1 shows the absorption maximum wavelength ( ⁇ abs ), fluorescence maximum wavelength ( ⁇ fl ), quantum yield ( ⁇ fl ), and spirocyclization equilibrium constant (pK cycl) obtained for the compounds 1 to 4.
  • HMSiR (650) is a compound having the following structure, and each value is S. Uno, M. Kamiya, T. Yoshihara, K. Sugawara, K. Okabe, MC Tarhan, H. Fujita, Based on T. Funatsu, Y. Okada, S. Tobita, et al., Nat. Chem. 2014, 6, 681-9.
  • Compound 1 exhibits 100% ring-opening and thus has a large absorption and strong fluorescence.
  • Compound 3 and Compound 4 The close proximity of the two pK cycls revealed that there was no pH present in 100% ring-opened form and only relatively weak fluorescence was shown.
  • Compound 2 is present in a colorless and non-fluorescent closed ring structure near physiological pH 7.4, but in addition to that, the maximum absorption wavelength is greatly shortened, so that when excited at the maximum absorption wavelength of Compound 1, it becomes more fluorescent. Was shown to be suppressed.
  • Live cell imaging Compound 2 (gGlu-HMJSiR) was applied to 4 types of cancer cells with different ⁇ -glutamyltranspeptidase (GGT) activities, and live cell imaging was performed. Specifically, SHIN3 human ovarian cancer-derived cells, A549 human lung cancer-derived cells with high GGT activity, and H226 human lung cancer-derived cells with low GGT activity, NMuMG normal mouse mammary epithelial cells Using. The results are shown in FIG. The scale bar represents 100 ⁇ m.
  • LysoTracker Green was added to SHIN3 cells incubated for 5 minutes after application of 1 ⁇ M compound 2, and fluorescence images were acquired in two colors immediately using a confocal fluorescence microscope.
  • mice Fluorescence spectrum imaging was performed using a mouse model in which SHIN3 cells were seeded intraperitoneally.
  • SHIN3-seeded model mice were established by intraperitoneal injection of 2 ⁇ 10 6 SHIN3 cells suspended in 300 ⁇ l of PBS ( ⁇ ) into 7-8 week old female nude mice. Experiments were performed 7-10 days after injection when peritoneal seeding reached a size of about 1 mm.
  • the mice were anesthetized by intraperitoneal injection of ketamine (2 mg / animal) and xylazine (0.2 mg / animal), and the abdominal skin was incised about 1 cm. The intestine was pulled out from the incision and placed on a black rubber plate to spread the mesentery.
  • Subcutaneous tumor model mice were created by subcutaneously injecting 7 ⁇ 10 female nude mice with 5 ⁇ 10 6 A549 cells and 1.5 ⁇ 10 7 H226 cells suspended in 100 ⁇ l of PBS ( ⁇ ). .
  • A549 cells were injected into the left flank of the mice, and H226 cells were injected approximately 1 cm away from the caudal side.
  • Experiments were performed 7 days after the injection when the long sides of both tumors reached approximately 5 mm. Mice were anesthetized with an intraperitoneal injection of ketamine (2 mg / animal) and xylazine (0.2 mg / animal).
  • microprobe expressing peptidase can be detected with high sensitivity and speed as a fluorescence response in the near infrared region even in vivo by the fluorescent probe of the present invention.

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Abstract

Le problème décrit par la présente invention est de fournir une nouvelle sonde fluorescente qui présente une perméabilité tissulaire supérieure et qui est capable de détecter l'activité de peptidases hautement exprimées dans les cellules cancéreuses à l'aide d'une réponse fluorescente ayant une longueur d'onde supérieure. La solution selon l'invention porte sur une sonde fluorescente pour détecter une activité peptidase comprenant un composé représenté par la formule (I) ou un sel de celui-ci [dans la formule : X représente Si(Ra)(Rb) (dans lequel Ra et Rb représentent chacun indépendamment un atome d'hydrogène ou un groupe alkyle) ; Y représente un groupe alkylène C0-C3, et le groupe alkylène peut être substitué par un atome d'halogène ou un haloalkyle ; R1 représente 1 à 4 substituants, qui sont identiques ou différents les uns des autres, et qui sont indépendamment sélectionnés dans le groupe constitué par un atome d'hydrogène, un groupe cyano, et un groupe alkyle éventuellement substitué, un groupe carboxyle, un groupe ester, un groupe alcoxy, un groupe amide et un groupe azide ; R2 forme, conjointement avec R3, une structure hétérocyclique à 5 à 8 chaînons contenant un atome d'azote et un atome de carbone auquel R2 et R3 sont liés ; R4 forme avec R5, une structure hétérocyclique à 5 à 8 chaînons contenant un atome d'azote et un atome de carbone auquel R4 et R5 sont liés ; R6, R7, R8, et R9 représentent chacun indépendamment un atome d'hydrogène, un atome d'halogène, un groupe hydroxyle, un groupe alkyle, un groupe sulfo, un groupe carboxyle, un groupe ester, un groupe amide ou un groupe azide ; et R10 représente un résidu acyle dérivé d'acide aminé].
PCT/JP2018/007327 2017-02-28 2018-02-27 Sonde fluorescente dans l'infrarouge proche pour la détection d'une activité peptidase WO2018159631A1 (fr)

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CN113474342A (zh) * 2019-02-28 2021-10-01 国立大学法人东京大学 检测癌症的荧光探针
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CN115011141A (zh) * 2022-06-01 2022-09-06 华东理工大学 一类基于氯取代硅罗丹明类近红外染料母体、制备方法及其应用

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