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WO2010026743A1 - Réactif pour mesurer un environnement hypoxique - Google Patents

Réactif pour mesurer un environnement hypoxique Download PDF

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Publication number
WO2010026743A1
WO2010026743A1 PCT/JP2009/004313 JP2009004313W WO2010026743A1 WO 2010026743 A1 WO2010026743 A1 WO 2010026743A1 JP 2009004313 W JP2009004313 W JP 2009004313W WO 2010026743 A1 WO2010026743 A1 WO 2010026743A1
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group
compound
hypoxic environment
compound according
measuring
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Japanese (ja)
Inventor
長野哲雄
清瀬一貴
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University of Tokyo NUC
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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
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9

Definitions

  • the present invention relates to a compound useful as a low oxygen environment measurement reagent or a salt thereof.
  • the present invention also relates to a low oxygen environment measuring reagent containing the above compound or a salt thereof.
  • hypoxic microenvironment in vivo has been suggested to be associated with various diseases, so detecting tissues and cells in hypoxic conditions is useful for early diagnosis of diseases and determination of treatment strategies.
  • detecting tissues and cells in hypoxic conditions is useful for early diagnosis of diseases and determination of treatment strategies.
  • several proposals have been made as methods capable of measuring a hypoxic environment. For example, PET tracers and immunostaining have been proposed, but these methods have problems in terms of safety and simplicity.
  • a method for measuring a trace component in a living body or a specific environment using a fluorescent probe is simple and high safety can be expected. Therefore, development of a method for measuring a hypoxic microenvironment using a fluorescent probe is required.
  • hitherto few fluorescent probes capable of measuring a hypoxic microenvironment have been provided, and there have been few proposals for fluorescent probes using small molecules.
  • Some of the reported fluorescent probes have many problems in terms of specificity and excitation wavelength (for example, JP 2007-77036 A, ChemBioChem., 9, pp.426-432, 2008). .
  • An object of the present invention is to provide a compound useful for measurement of a hypoxic environment. More specifically, it is an object of the present invention to provide a compound capable of imaging a tissue in a living body or a hypoxic environment of a cell with fluorescence safely and easily.
  • the present inventors have reduced the substantially non-fluorescent compound represented by the following general formula (I) in a low oxygen environment, and emits strong fluorescence. And by using these compounds, it has been found that a hypoxic microenvironment in cells and tissues in a living body can be efficiently imaged.
  • the present invention has been completed based on these findings.
  • Y 1 represents an alkylene group having 1 to 6 carbon atoms
  • X 1 represents a single bond, —CO—, or —SO 2 —
  • X 2 represents —OY 2 —N (R 14 ) —
  • Y 2 represents an alkylene group having 1 to 6 carbon atoms
  • R 14 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • m represents 0 or 1
  • pC 6 H 4 - represents a p- phenylene group
  • Ar represents an aryldiyl group
  • R 13 represents a group represented by showing a monoalkylamino group or a dialkylamino group
  • R 2 is an optionally substituted group good C 1-12 alkyl group, which may have a substituent C 1-12 alkoxy group, a carboxy group, a sulfo group, or an optionally substituted C 1-12 alkoxycarbonyl group
  • the above compound wherein R 2 is a C 1-12 alkyl group or a carboxy group; the above compound wherein R 2 is a methyl group or a carboxy group; R 3 , R 4 , R 5 ,
  • R 6 , R 7 and R 8 are hydrogen atoms; the above compound wherein Ar—R 13 is a p-dimethylaminophenyl group; the above compound wherein m is 0; X 1 is —SO 2
  • Y 1 is an ethylene group; the above compound wherein R 11 and R 12 are bonded to each other to form an ethylene group; and Y 1 is an ethylene group, and R 11 and There is provided the above compound wherein R 12 is bonded to each other to form an ethylene group.
  • a reagent for measuring a low oxygen environment comprising the compound represented by the above general formula (I) is provided by the present invention, and preferably in the presence of an enzyme capable of reductively cleaving an azo bond.
  • an enzyme capable of reductively cleaving an azo bond In the presence of NADPH-cytochrome) P450 reductase in the presence of NADPH-cytochrome P450 reductase. ) Is provided.
  • an enzyme substrate having the ability to reductively cleave an azo bond comprising a compound represented by the general formula (I), particularly NADPH-cytochrome ⁇ ⁇ ⁇ P450 reductase substrate, is provided by the present invention.
  • a method for measuring a low oxygen environment comprising the following steps: (A) introducing the compound represented by the general formula (I) into the low oxygen environment; and (B) the above steps ( fluorescent compound formed by a) or a group bound to X 1 and an azo group cleavable in (the general formula (I) - (X 2) m - group bonded is p- aminophenyl to X 1 through a
  • a method comprising the step of measuring the fluorescence of the underlying compound).
  • the step (A) is performed in the presence of an enzyme having the ability to reductively cleave the azo bond in a hypoxic environment, such as NADPH-cytochrome P450 reductase, azoreductase, DT diaphorase and the like.
  • an enzyme having the ability to reductively cleave the azo bond in a hypoxic environment such as NADPH-cytochrome P450 reductase, azoreductase, DT diaphorase and the like.
  • the compound of the present invention is useful as a reagent for the measurement of a hypoxic environment, particularly a hypoxic microenvironment.
  • a hypoxic environment particularly a hypoxic microenvironment.
  • tissues and cells in a hypoxic environment such as solid tumors on the surface of mucous membranes, skin or organs can be safely and simply Can be imaged.
  • FIG. 3 shows the results of a reaction between DBCF-PIP (3 ⁇ M) and rat liver microsomes in the presence of NADPH or NADH under normal and hypoxic conditions.
  • ⁇ indicates the results in a hypoxic environment
  • ⁇ indicates the results in a normal oxygen environment
  • (A) indicates the results of 50 ⁇ M NADPH
  • (B) indicates the results of 250 ⁇ M NADH.
  • FIG. 2 is a diagram showing an absorption spectrum (A) of DBCF-PIP and a fluorescence spectrum (B) when rat liver microsomes are reacted with DBCF-PIP under the respective conditions of DBCF-PIP, hypoxic environment and normal oxygen environment.
  • DBCF-PIP is the fluorescence spectrum of DBCF-PIP
  • MS-aerobic is the fluorescence spectrum of rat liver microsomes and DBCF-PIP in a normal oxygen environment
  • MS-hypoxic is the rat in a hypoxic environment.
  • the fluorescence spectrum at the time of making liver microsome and DBCF-PIP react is shown. It is the figure which showed the result of having added the NADPH-cytochrome P450 reductase inhibitor in the hypoxic environment and reacting DBCF-PIP with the rat liver microsome.
  • FIG. 3 (A) shows the results of changes over time
  • FIG. 3 (B) shows the measurement results after 1 hour of reaction.
  • FIG. 4 is a graph showing the results of reaction with rat liver microsomes in a hypoxic environment for the four compounds obtained in Examples 1 to 4.
  • an “alkyl group” or an alkyl part of a substituent containing an alkyl part means an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof.
  • the “alkylene group” may be linear or branched, and may include a cyclic structure. For example, ethylene group, propylene group, cyclopentane-1,2-diyl group, cyclohexane-1,2-diyl group, cyclohexane-1,3-diyl group, cyclohexane-1,4-diyl group, etc. Also good.
  • the “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • C 1-12 alkyl group represented by R 2 C 1-12 alkoxy group, or the C 1-12 alkoxycarbonyl group has a substituent
  • the kind of substituents, number, and substitution position is not particularly limited, for example, ,
  • a halogen atom, a hydroxyl group, an amino group, a carboxy group, an alkoxycarbonyl group, a sulfo group, an alkyl sulfonate group, or the like may be used as a substituent.
  • Examples of the aryldiyl group represented by Ar include aromatic hydrocarbon diyl groups such as a phenylene group, a naphthalenediyl group, an anthracenediyl group, and a phenanthrenediyl group, as well as a pyridinediyl group, a quinolinediyl group, a furandiyl group, and a thiophenediyl group. Or a heteroaromatic diyl group.
  • R 11 and R 12 are preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom. It is also preferred that R 11 and R 12 are combined to form an ethylene group.
  • Y 1 is preferably a methylene group, an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group.
  • R 11 and R 12 are combined to form an ethylene group, Y 1 is preferably an ethylene group.
  • X 1 is preferably —CO— or —SO 2 —, more preferably —SO 2 —.
  • the X 2 Y 2 is a methylene group, an ethylene group, or propylene group, it is preferred that R 14 is a hydrogen atom or a methyl group.
  • m represents 0 or 1, but is preferably 0. In this case, it means that X 2 does not exist and becomes a single bond.
  • the aryldiyl group represented by Ar is preferably a phenylene group, more preferably an o-phenylene group or a p-phenylene group, and particularly preferably a p-phenylene group.
  • R 13 is preferably a monomethylamino group or a dimethylamino group, and more preferably a dimethylamino group.
  • R 2 is preferably a C 1-12 alkyl group or a carboxy group, more preferably a C 1-6 alkyl group or a carboxy group, and even more preferably a methyl group or a carboxy group.
  • the C 1-12 alkyl group optionally having a substituent represented by R 2 in the above general formula (I), substituted The water-solubility of the compound of the present invention by appropriately selecting a C 1-12 alkoxy group which may have a group or a substituent of a C 1-12 alkoxycarbonyl group which may have a substituent, It can be used as a cell membrane permeation type and non-membrane permeation type probe.
  • the compound of the present invention having one or two, preferably three or more, sulfo groups and carboxy groups as the substituents is highly water-soluble and exhibits non-membrane permeability and cannot be taken into cells. It can be suitably used for detection of extracellular hypoxic environment.
  • the compound of the present invention in which R 2 is an acetoxymethoxycarbonyl group has high lipid solubility and exhibits cell membrane permeability and is taken into cells (the acetoxymethoxycarbonyl group is hydrolyzed by esterase present in the cells).
  • R 2 in the above general formula (I) may be converted to a compound represented by a carboxy group, resulting in a decrease in fat solubility and intracellular retention). In some cases, it can be suitably used.
  • the 6-position hydroxyl group of the xanthene ring moiety of the above general formula (I) can be acetylated and converted to an acetoxy group to enhance the lipid solubility of the compound of the present invention and impart cell membrane permeability (the acetoxy group).
  • the group is hydrolyzed by the esterase present in the cell, it is converted into the compound represented by the above general formula (I)).
  • the compound of the present invention is a fluorescein-like compound in which R 2 in the above general formula (I) is a carboxy group
  • the fluorescein-like compound takes a lactone form and the carbonyl group at the 3-position of the xanthene ring moiety is a hydroxyl group
  • one or both of the hydroxyl groups at the 3rd and 6th positions of the xanthene ring moiety are acetylated to convert them into acetoxy groups, thereby increasing the fat solubility of the compound of the present invention.
  • Cell membrane permeability can be imparted.
  • R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are preferably hydrogen atoms.
  • Preferred compounds as the compound represented by the general formula (I) are: (a) Ar—R 13 is a p-dimethylaminophenyl group; (b) m is 0; (c) X 1 is —SO 2 —; (d) Y 1 is an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group; or (e) a case where R 11 and R 12 bonded to each other is an ethylene group, more preferably a case where two or more of the above (a) to (e) are included, and particularly preferably the above (a) to (e ).
  • the compound represented by the general formula (I) may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.
  • the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.
  • the compound represented by the general formula (I) may exist in the form of a salt, and the scope of the present invention includes a salt form. Examples of the salt include acid addition salts and base addition salts.
  • mineral salts such as hydrochloride and sulfate
  • organic acid salts such as maleate and p-toluenesulfonate
  • sodium salt and potassium in addition to metal salts such as salts, organic amine salts such as ammonium salts and triethylamine salts, salts of amino acids such as glycine salts may be used.
  • the compound represented by the general 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 of the present invention represented by the general formula (I) is generally an amine compound represented by the general formula (I) in which the terminal carbonyl group is replaced with a hydrogen atom in the group represented by the general formula (A). It can be easily produced by reacting a compound represented by R 1 with a carboxy group. Since the production method of a representative compound of the compound represented by the general formula (I) of the present invention is specifically shown in the examples of the present specification, those skilled in the art will be based on the specific description of the examples. The compound of the present invention can be easily produced by appropriately selecting starting materials and reaction reagents, and appropriately changing or modifying the reaction conditions and steps as necessary.
  • the target product can be efficiently produced by carrying out the reaction while protecting specific functional groups as necessary in the reaction step.
  • Organic synthesis Protective Groups, Organic Synthesis, TWGreene, John Wiley & Sons, Inc., 1981
  • Organic synthesis and the like can be selected by those skilled in the art.
  • isolation and purification of the product in the above production method can be performed by appropriately combining methods used in ordinary organic synthesis, for example, filtration, extraction, washing, drying, concentration, crystallization, various chromatography and the like.
  • the production intermediate in the above step can be subjected to the next reaction without particular purification.
  • the salt of the compound of the present invention when the salt of each compound is obtained in the above production method, it may be purified as it is.
  • the free form compound is obtained, the free form compound is obtained. May be dissolved or suspended in a suitable solvent, acid or base may be added to form a salt, and purification may be performed as necessary.
  • the compound of the present invention represented by the general formula (I) When the compound of the present invention represented by the general formula (I) is placed in a hypoxic environment under mild conditions, for example, physiological conditions, the azo group in the general formula (A) is cleaved, and the general formula (A group or bound to X 1 in I) - (X 2) m - is a group bound to X 1 via a has the property of giving the compound a p- aminophenyl group.
  • the compound represented by the general formula (I) is substantially non-fluorescent, while the above compound produced by cleavage of the azo group has a property of emitting high intensity fluorescence.
  • hypoxic environment examples include a hypoxic environment of about 0% to 5% (38 mmHg) in solid cancer. It is not limited to this state.
  • examples of the hypoxic environment include cancer tissue, cancer cells, or ischemic tissue.
  • cancer tissue for example, a solid cancer generated on the mucosal surface, skin, or organ surface is preferable. It is.
  • a tissue in a hypoxic environment can be identified using a reagent of the present invention by means such as endoscopy, and the presence of cancer can be detected at an early stage.
  • resistance to radiation increases when the intratumoral oxygen partial pressure pO 2 is less than 10 mmHg, and the reagent of the present invention is also useful for identifying tumors having such radiation resistance.
  • the compound (reagent) of the present invention When the compound (reagent) of the present invention is introduced (applied) into a hypoxic environment in a biological sample (tissue or cell), it has the ability to reductively cleave the azo bond in the hypoxic environment present in the tissue or cell.
  • a compound in which the azo group is cleaved is generated by the enzyme having it, and generates fluorescence.
  • Enzymes that have the ability to reductively cleave the azo bond in such a hypoxic environment include NADPH-cytochrome P450 reductase (present in various tissue microsomes), cytochrome-b 5 reductase (present in various tissue microsomes).
  • DT diaphorase present in the cytosol of various tissues
  • azoreductase derived from microorganisms
  • NADPH-cytochrome P450 reductase is preferable because it exists in a wide range of tissues and cells.
  • the enzyme may be derived from a sample depending on the sample for measuring the hypoxic environment or the test conditions, or may be added from the outside. When added from the outside, it may be obtained by a genetic recombination technique.
  • the method for measuring a hypoxic environment of the present invention generally comprises (A) a step of introducing the compound represented by the above general formula (I) into a low oxygen environment, and (B) the step (A).
  • a step of measuring fluorescence A step of measuring fluorescence.
  • Measurement of the fluorescence of a compound with an azo group cleaved can be performed by a normal method, such as a method of measuring a fluorescence spectrum in vitro or a method of measuring a fluorescence spectrum in vivo using a bioimaging method. Can do. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method.
  • DBTG-PIP has the property of being taken up into cells, and other probes can be easily converted into a structure that can be taken up into cells by protecting the hydroxyl group of the xanthene skeleton with an acetyl group. Is possible. Therefore, a hypoxic environment localized in individual cells and tissues can be measured with high sensitivity by a bioimaging technique.
  • the compound represented by the above general formula (I) may be used as it is, but if necessary, it is composed of additives usually used for the preparation of the reagent. You may use as a thing.
  • additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible.
  • These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.
  • the compound of the present invention can be the main component of the diagnostic imaging composition by preparing it as a pharmacologically or pharmaceutically acceptable formulation (formulation) as necessary.
  • a diagnostic imaging composition based on the compound of the present invention is provided.
  • applications of the above reagents and compositions to diagnostic imaging are also included.
  • Example 1 DBCF-PIP synthesis (1) Compound 1
  • N-Boc-piperazine (366 mg, 2 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 334 ⁇ L of triethylamine and dabsyl chloride (332 mg, 1 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, the residue was purified on silica gel to obtain 327 mg of the target compound 1 (yield 69%, orange powder).
  • N-Boc-ethylenediamine (345.8 mg, 2.16 mmol) was dissolved in 40 mL of anhydrous dichloromethane, 334 ⁇ L of triethylamine and dabsyl chloride (330.4 mg, 1.02 mmol) were added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by silica gel column chromatography to obtain 392.5 mg of the target compound 2 (yield 85.8%, dark red solid).
  • N-Boc-hexanediamine (216 mg, 1 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 167 ⁇ L of triethylamine and dabsyl chloride (241 mg, 0.74 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere.
  • Example 5 Reaction between DBCF-PIP and rat liver microsomes
  • reaction between DBCF-PIP (3 ⁇ M) and rat liver microsomes was performed under normal or hypoxic conditions. It was. Measurement was performed at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent. A hypoxic environment was created by bubbling 100% argon for 30 minutes. The results are shown in Figure 1.
  • DMSO dimethyl sulfoxide
  • (1) shows the results in a hypoxic environment
  • ⁇ shows the results in a normal oxygen environment
  • (A) shows the results in the presence of 50 ⁇ M NADPH
  • (B) shows the results in the presence of 250 ⁇ M NADH.
  • Liver microsomes contain cytochrome reductases, and those using NADPH as an electron donor are NADPH-cytochrome P450 reductase, and those using NADH as an electron donor are cytochrome-b 5 reductases.
  • the compounds of the present invention have been shown to have excellent properties as substrates for cytochrome reductases in liver microsomes, particularly NADPH-cytochrome P450 reductase, under anaerobic (hypoxic) conditions.
  • Fig. 2 (A) shows the absorption spectrum (3 ⁇ M) of DBCF-PIP
  • Fig. 2 (B) shows the fluorescence spectrum when reacted with rat liver microsomes under the conditions of DBCF-PIP and hypoxic / normal oxygen environment.
  • the sample was prepared with potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and reacted with rat liver microsomes in the presence of 250 ⁇ M NADH for 3 hours, measured at an excitation wavelength of 490 nm. Went.
  • DMSO dimethyl sulfoxide
  • the absorption spectrum of DBCF-PIP showed a spectrum shape in which dabsyl and fluorescein were superimposed, and the fluorescence intensity was very small (FIG. 2B, fluorescence quantum yield 0.01). A slight increase in fluorescence was observed even in a normal oxygen environment, but a very large increase in fluorescence of 10 times or more that in a normal oxygen environment was observed in a low oxygen environment.
  • Diphenyliodonium chloride an inhibitor of NADPH-cytochrome-P450-reductase
  • a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm).
  • FIG. 3 (A) shows the results of changes over time
  • FIG. 3 (B) shows the measurement results after 1 hour of reaction. In a hypoxic environment, the increase in fluorescence was suppressed depending on the concentration of the inhibitor (FIG. 3 (A)).
  • FIG. 4 shows the results of reaction with DBCF-PIP (3 ⁇ M) using purified human NADPH-cytochrome-P450-reductase (5 ⁇ g).
  • a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm). While a large increase in fluorescence was observed in the hypoxic environment, no substantial increase in fluorescence was observed in the normal oxygen environment.
  • DMSO dimethyl sulfoxide
  • Example 6 The four compounds (3 ⁇ M) obtained in Examples 1 to 4 were reacted with rat liver microsomes (50 ⁇ L, 5-fold dilution) in a hypoxic environment in the same manner as in Example 5.
  • a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and measurement was performed in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm). It was confirmed that all four compounds of the present invention obtained in Examples 1 to 4 fluoresce in a hypoxic environment.
  • DMSO dimethyl sulfoxide

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Abstract

L'invention concerne un composé représenté par la formule (I), qui peut être utilisé pour mesurer par fluorescence de manière pratique et avec une grande sensibilité un environnement hypoxique (dans la formule (I), R1 représente -CO-N(R11)-Y1-N(R12)-X1-(X2)m-p-C6H4-N=N-Ar-R13 [R11 et R12 représentant indépendamment H ou un alkyle (y compris un alkylène formé par la liaison entre R11 et R12); Y1 représentant un alkylène; X1 représentant une simple liaison, -CO- ou -SO2-; X2 représentant -O-Y2-N(R14)- (Y2 représentant un alkylène; et R14 représentant H ou un alkyle); m représentant un nombre de 0 ou 1; p-C6H4- représentant un p-phénylène; Ar représentant un aryldiyle; et R13 représentant un alkylamino]; R2 représente un alkyle, un alcoxy, un carboxy, un sulfo ou un alcoxycarbonyle; et R3 à R8 représentent indépendamment H ou un halogène).
PCT/JP2009/004313 2008-09-03 2009-09-02 Réactif pour mesurer un environnement hypoxique Ceased WO2010026743A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111818A1 (fr) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Sonde fluorescente
WO2012111817A1 (fr) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Sonde fluorescente

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62120378A (ja) * 1985-11-19 1987-06-01 ザ　ジヨ−ンズ　ホプキンズ　ユニバ−シテイ− タンパク質ラベル及び医薬物質放出システム
JP2007077036A (ja) * 2005-09-12 2007-03-29 Kyoto Univ 標的部位で選択的に蛍光強度が強くなる新規化合物および画像診断用組成物
WO2007076379A2 (fr) * 2005-12-20 2007-07-05 Gilead Colorado, Inc. Composes de 4,7-dihydrothieno[2,3-b]pyridine et compositions pharmaceutiques
WO2007092963A1 (fr) * 2006-02-09 2007-08-16 Spectrum Pharmaceuticals, Inc. Traitement du cancer de la vessie et procedes correspondants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62120378A (ja) * 1985-11-19 1987-06-01 ザ　ジヨ−ンズ　ホプキンズ　ユニバ−シテイ− タンパク質ラベル及び医薬物質放出システム
JP2007077036A (ja) * 2005-09-12 2007-03-29 Kyoto Univ 標的部位で選択的に蛍光強度が強くなる新規化合物および画像診断用組成物
WO2007076379A2 (fr) * 2005-12-20 2007-07-05 Gilead Colorado, Inc. Composes de 4,7-dihydrothieno[2,3-b]pyridine et compositions pharmaceutiques
WO2007092963A1 (fr) * 2006-02-09 2007-08-16 Spectrum Pharmaceuticals, Inc. Traitement du cancer de la vessie et procedes correspondants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHEN, H. ET AL.: "Poly(ethylene glycol) carbodiimide coupling reagents for the biological and chemical functionalization of water-soluble nanoparticles", ACSNANO, vol. 3, no. 4, 2009, pages 915 - 923 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111818A1 (fr) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Sonde fluorescente
WO2012111817A1 (fr) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Sonde fluorescente
US9170266B2 (en) 2011-02-18 2015-10-27 The University Of Tokyo Fluorescent probe
JP2016033134A (ja) * 2011-02-18 2016-03-10 国立大学法人 東京大学 蛍光プローブ
US9329184B2 (en) 2011-02-18 2016-05-03 The University Of Tokyo Fluorescent probe

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