WO2022050233A1 - 発光システム及びシトクロムｐ４５０の定量方法 - Google Patents

発光システム及びシトクロムｐ４５０の定量方法 Download PDF

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WO2022050233A1
WO2022050233A1 PCT/JP2021/031785 JP2021031785W WO2022050233A1 WO 2022050233 A1 WO2022050233 A1 WO 2022050233A1 JP 2021031785 W JP2021031785 W JP 2021031785W WO 2022050233 A1 WO2022050233 A1 WO 2022050233A1
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cytochrome
luminescence
general formula
salt
amount
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French (fr)
Japanese (ja)
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厚志 仲村
昌次郎 牧
亮平 森屋
昇雄 北田
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黒金化成株式会社
国立大学法人電気通信大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Definitions

  • the present invention relates to a light emitting system and a method for quantifying cytochrome P450.
  • Cytochrome P450 is a metabolic enzyme present in almost all living organisms from bacteria to plants and mammals, and is responsible for drug / toxic metabolism, hormone biosynthesis, fatty acid metabolism, and plant secondary metabolism. ing. In drug discovery, clarifying the process by which a drug is metabolized by cytochrome P450 is extremely important in examining side effects and drug swallowing (Non-Patent Document 1).
  • a fluorescence assay and a luminescence assay are known as methods for detecting the activity of cytochrome P450.
  • the fluorescence assay uses a substrate that produces a fluorescent product after metabolism by cytochrome P450.
  • the test compound that modulates the activity of cytochrome P450 is identified by its effect on the amount of fluorescent product accumulated.
  • a luminescence reaction between firefly luciferase and firefly luciferin, which is a substrate thereof, is used (Non-Patent Document 2).
  • a derivative of firefly luciferin which is not a substrate of firefly luciferase, is prepared in advance, and cytochrome P450 converts the derivative of firefly luciferin into firefly luciferin in the first reaction. Then, in the second reaction, the produced firefly luciferin and firefly luciferase react with each other to emit light, and the amount of the emitted light is measured to quantify cytochrome P450.
  • the excitation light required for the fluorescence assay generates a background signal, which limits assay sensitivity.
  • the luminescence assay does not require excitation light, the background is suppressed to a low level and the assay sensitivity is high, but it is necessary to introduce a gene for the expression of firefly luciferase into the object. Therefore, in vitro (in vitro experiment), it is necessary to add firefly luciferase, and in vivo (in vivo experiment), it is necessary to prepare a genetically modified organism into which the firefly luciferase gene has been introduced in advance. Therefore, it is not possible to easily emit light.
  • cytochrome P450 reacts with a compound having a specific structure to emit light, and by quantifying the luminescence, cytochrome P450 can be quantified.
  • the gist structure of the present invention that solves the above problems is as follows.
  • the light emitting system of the present invention has the following general formula (1): [In the formula, R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms. R 2 is NR 4 2 or OH, where R 4 is an independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and the two R 4s of NR 4 2 are bonded to each other. May form a ring, A is the following general formula (2) or (3): Represented by R 3 is independently CR 5 or N, where R 5 is independently hydrogen, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms, respectively.
  • n is an integer of 0 to 3], and is characterized by containing a heterocyclic compound or a salt thereof, and cytochrome P450.
  • the light emitting system of the present invention does not require excitation light and can easily obtain light emission derived from cytochrome P450.
  • the cytochrome P450 is present in the tissue in the living body.
  • luminescence derived from cytochrome P450 in the tissue in the living body can be obtained.
  • the cytochrome P450 is present in mammalian liver tissue.
  • luminescence derived from cytochrome P450 in mammalian liver tissue is obtained.
  • the method for quantifying cytochrome P450 of the present invention is based on cytochrome P450 according to the following general formula (1):
  • R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms.
  • R 2 is NR 4 2 or OH, where R 4 is an independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and the two R 4s of NR 4 2 are bonded to each other. May form a ring
  • A is the following general formula (2) or (3): Represented by R 3 is independently CR 5 or N, where R 5 is independently hydrogen, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms, respectively.
  • n is an integer of 0 to 3]
  • the heterocyclic compound or a salt thereof is reacted and the amount of light emitted is measured. It is characterized in that the amount of the cytochrome P450 is measured from the amount of light emitted.
  • the method for quantifying cytochrome P450 of the present invention can easily and highly sensitively quantify cytochrome P450.
  • cytochrome P450 light emitting system that does not require excitation light and can easily emit light. Further, according to the present invention, it is possible to provide a method for quantifying cytochrome P450 easily and with high sensitivity.
  • the light emitting system of the present invention has the following general formula (1): [In the formula, R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms. R 2 is NR 4 2 or OH, where R 4 is an independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and the two R 4s of NR 4 2 are bonded to each other. May form a ring, A is the following general formula (2) or (3): Represented by R 3 is independently CR 5 or N, where R 5 is independently hydrogen, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms, respectively. n is an integer of 0 to 3], and is characterized by containing a heterocyclic compound or a salt thereof, and cytochrome P450.
  • the heterocyclic compound represented by the general formula (1) or a salt thereof reacts with cytochrome P450 to emit light.
  • the emission system of the present invention does not require excitation light, unlike the fluorescence assay.
  • the light emitting system of the present invention does not require firefly luciferase, unlike the light emitting system using firefly luciferin. Therefore, even in the case of in vivo (in vivo), it is not necessary to prepare a genetically modified organism into which the firefly luciferase gene has been introduced in advance. Therefore, the light emitting system of the present invention does not require excitation light and can easily emit light.
  • the light emitting system of the present invention it is possible to provide a method for directly visualizing the activity of cytochrome P450.
  • a heterocyclic compound represented by the above general formula (1) or a salt thereof that reacts with the cytochrome P450 is allowed to act on the cytochrome P450, it emits light according to the activity of the cytochrome P450.
  • this luminescence does not require luciferase, so there is no need to introduce the luciferase gene into the measurement target.
  • a specific cytochrome P450 with a heterocyclic compound represented by the general formula (1) or a salt thereof that interacts with the specific cytochrome P450, it is possible to sense cytochrome P450 that is widely present in a living body.
  • R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms.
  • examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group and the like.
  • hydrogen is preferable as R1 .
  • the configuration may be R or S.
  • R 2 is NR 42 or OH, where R 4 is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and is also of NR 42 .
  • the two R4s may combine with each other to form a ring.
  • examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group and the like. ..
  • the group of the cyclic structure formed by combining the two R 4s of NR 4 2 together with N is as follows.
  • 1-azacyclopropyl group (three-membered ring), 1-azacyclobutyl group (four-membered ring), 1-azacyclopentyl group (five-membered ring), 1-azacyclohexyl group (six-membered ring), represented by 1-Azacycloheptyl group (seven-membered ring) and the like can be mentioned.
  • a methyl group is preferable as R4 .
  • R 2 NR 42 is preferable, and N (CH 3 ) 2 is particularly preferable, from the viewpoint of luminous efficiency.
  • A is represented by the general formula (2) or (3). From the viewpoint of luminous efficiency, A is preferably represented by the general formula (2) or (3), R 3 is preferably CR 5 , and more preferably represented by the general formula (2). ..
  • R 3 is independently CR 5 or N, respectively, where R 5 is independently hydrogen, an alkyl group having 1 to 8 carbon atoms or carbon. It is an alkenyl group of the number 2-8.
  • R5 is independently hydrogen, an alkyl group having 1 to 8 carbon atoms or carbon. It is an alkenyl group of the number 2-8.
  • examples of the alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group and a pentyl group.
  • Examples include a hexyl group, a heptyl group, an octyl group and the like, and examples of the alkenyl group having 2 to 8 carbon atoms include a vinyl group, an allyl group, a 1-propenyl group, an isopropenyl group, a 1-butenyl group, a 2-butenyl group and 3 -Butenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group and the like can be mentioned.
  • R 3 is preferably CR 5 .
  • it is preferable that one or more of R3 is N.
  • heterocyclic compound represented by the general formula (1) or a salt thereof is described in, for example, Japanese Patent No. 5464311, Japanese Patent No. 60111974, Japanese Patent No. 6353751, and International Publication No. 2013/0277770. It can be synthesized according to the above, and a commercially available product can also be used.
  • the heterocyclic compound represented by the general formula (1) can also be a salt.
  • the salt of the heterocyclic compound represented by the general formula (1) can also react with cytochrome P450 to emit light.
  • the salt of the heterocyclic compound represented by the general formula (1) may be an addition salt with an acid or an addition salt with a base.
  • the acid in the addition salt of the heterocyclic compound of the general formula (1) and the acid hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphite, sub-acid.
  • Nitrate citric acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, pamoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanedisulfonic acid.
  • P-Toluenesulfonic acid, salicylic acid, succinic acid, trifluoroacetic acid and the like, and examples of the acid addition salt include hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, etc.
  • examples of the base in the addition salt of the heterocyclic compound of the general formula (1) and the base include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, ethanolamine, and meglumin.
  • examples of the base addition salt include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, ethanolamine salt, meglumin salt and the like.
  • the salt of the heterocyclic compound represented by the above general formula (1) has excellent solubility in water or a buffer solution having a pH near neutral. Therefore, the salt of the heterocyclic compound represented by the general formula (1) can be dissolved in water or a buffer solution having a pH near neutral at a high concentration, and the emission brightness can be improved.
  • Cytochrome P450 The cytochrome P450 (CYP) is a group of reduced protoheme-containing proteins having monooxygenase activity, and when carbon monoxide is aerated through the protein in a reduced state, the absorption spectrum changes and the difference spectrum (CO) having a maximum at 450 nm. The difference spectrum) appears.
  • the cytochrome P450 gene is known to be present in most organisms except some bacteria such as Escherichia coli. Cytochrome P450 is known to be involved in various reactions such as hydroxylation reaction, epoxidation reaction, and demethylation reaction, and its role in the living body is secondary metabolism, steroid hormone biosynthesis, and foreign body metabolism. , Assimilation of hydrocarbons, etc.
  • cytochrome P450 is involved in the metabolism of hydrophobic drugs, carcinogens, and other potentially toxic compounds and metabolites that circulate in the blood.
  • the liver is a major organ for xenobiotic metabolism, containing high levels of the most important CYP mixed functional oxidases. It is also said that cytochrome P450 is involved in about 80% of drug metabolism reactions, and by measuring the activity of cytochrome P450, it is possible to evaluate side effects caused by the drug.
  • the cytochrome P450 is classified based on the identity of the amino acid sequence. As a general rule, if the amino acid sequences match 40% or more, they are classified into the same family, and if they match 55% or more, they are classified into the same subfamily, and a unique classification symbol is given.
  • the classification symbol includes a CYP representing cytochrome P450, a family number, a subfamily number, and a gene number in this order, and the gene numbers are given in the order of discovery.
  • cytochrome P450 subfamily examples include CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5.
  • CYP3A4, CYP2D6, CYP2C9, CYP2C8, and CYP1A2 are preferable, and CYP3A4, CYP2D6, and CYP2C9 are more preferable from the viewpoint of the contribution rate to the drug metabolism reaction.
  • These cytochrome P450 molecular species have a high contribution rate of drug metabolism and are particularly effective in evaluating drug metabolism.
  • CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5 are preferable, and CYP2A6, CYP2 CYP2C9 and CYP3A4 are more preferable.
  • These cytochrome P450 molecular species have a high amount of light emission due to a reaction with the heterocyclic compound represented by the general formula (1) or a salt thereof, and can be detected with high sensitivity.
  • the cytochrome P450 is present in the tissue in the living body.
  • luminescence derived from cytochrome P450 in the tissue in the living body can be obtained.
  • the heterocyclic compound represented by the above general formula (1) or a salt thereof is administered to the living body, and the light generated by the reaction is emitted. It may be detected, or a living body may be collected and brought into contact with a heterocyclic compound represented by the general formula (1) or a salt thereof in a container such as a test tube to detect the generated light. ..
  • the cytochrome P450 is present in mammalian liver tissue.
  • luminescence derived from cytochrome P450 in mammalian liver tissue is obtained.
  • cytochrome P450 is present in high concentration in liver tissue. Therefore, by detecting luminescence derived from cytochrome P450 in liver tissue, drug metabolism ability and side effects of the target mammal can be detected with high accuracy. Can be evaluated.
  • the heterocyclic compound represented by the above general formula (1) or a salt thereof is administered to the mammal and delivered to the liver.
  • the light generated by the reaction with cytochrome P450 present in the liver tissue may be detected, or the liver tissue may be collected from a mammal and placed in a container such as a test tube in a heterocycle represented by the general formula (1).
  • the generated light may be detected by contacting with the formula compound or a salt thereof.
  • the cytochrome P450 is present in a container such as a test tube.
  • a container such as a test tube.
  • the emission derived from cytochrome P450 can be detected outside the living body, the influence on the living body can be reduced, and the absorption and scattering of light by the components in the living body (for example, hemoglobin, oxidized hemoglobin, water) can be reduced. It is easy to detect the generated light. Further, by performing a purification treatment on the collected biological tissue as needed, it is possible to detect the luminescence derived from cytochrome P450 with higher accuracy.
  • the heterocyclic compound represented by the general formula (1) or a salt thereof is preferably used in an amount of 1 mol to 1 ⁇ 10 5 mol with respect to 1 mol of the cytochrome P450, and 1 ⁇ 10 1 mol to 1 ⁇ 10 4 It is more preferable to use mol.
  • the amount of luminescence due to the reaction between cytochrome P450 and the heterocyclic compound represented by the general formula (1) or a salt thereof increases, and the amount of luminescence of cytochrome P450 is quantified. Accuracy is improved.
  • the light emitting system of the present invention may contain the heterocyclic compound represented by the above general formula (1) or a salt thereof and cytochrome P450 as constituent elements, but the amount of light emitted may be increased or the light emission may be stabilized. Other components may be further contained for the purpose of making it into a substance. Further, the heterocyclic compound represented by the general formula (1) or a salt thereof may be present when reacting with cytochrome P450. For example, when it is administered in a container such as a test tube or in vivo, it is generally used. It may be a precursor of a heterocyclic compound represented by the formula (1) or a salt thereof.
  • These precursors also react with cytochrome P450 by deviating from R 1 and R 2 such as sugar, ATP, and phospholipid, and changing to a heterocyclic compound represented by the general formula (1) or a salt thereof. , Luminous.
  • the light emitting system of the present invention preferably further contains a buffer.
  • a buffer When a buffer is included, it is easy to adjust the pH of the light emitting system and it is easy to obtain stable light emission.
  • the pH of the light emitting system is preferably 4 to 10, more preferably 6 to 8.
  • the buffer include potassium phosphate, tris-hydrochloric acid (Tris / HCl), glycine, HEPES and the like.
  • the light emitting system of the present invention preferably further contains a surfactant.
  • the surfactant include cationic surfactants, anionic surfactants, nonionic surfactants, amphoteric surfactants and the like, and nonionic surfactants are preferable.
  • the nonionic surfactant poly (oxyethylene) octylphenyl ether (Triton X-100 or the like) or the like is preferable.
  • the content of the surfactant in the light emitting system is preferably in the range of 0.1 to 3% by mass, more preferably in the range of 0.5 to 2% by volume.
  • the light emitting system of the present invention preferably does not contain firefly luciferase.
  • the light emitting system does not contain firefly luciferase, it is possible to prevent light emission due to the reaction between the firefly luciferase and the heterocyclic compound represented by the general formula (1) or a salt thereof, and the cytochrome P450 is represented by the general formula (1). Only light emission due to the reaction with the heterocyclic compound or a salt thereof can be observed, and the sensitivity to cytochrome P450 is improved.
  • cytochrome P450 is reacted with a heterocyclic compound represented by the general formula (1) or a salt thereof, the amount of luminescence generated is measured, and the amount of luminescence is measured from the amount of luminescence. It is characterized by measuring the amount of.
  • the method for quantifying cytochrome P450 of the present invention can easily and highly sensitively quantify cytochrome P450.
  • the method for quantifying cytochrome P450 of the present invention may be carried out in vivo or in vitro.
  • the heterocyclic compound represented by the general formula (1) or a salt thereof is administered to the living body, and the administered heterocyclic compound of the general formula (1) or a salt thereof is in vivo.
  • the cytochrome P450 in the living body can be quantified.
  • in vitro tissue particularly, mammalian liver tissue
  • in vitro tissue is collected from the living body, stored in a container, and represented by the above general formula (1) in the container.
  • the heterocyclic compound or a salt thereof is added, and the added heterocyclic compound of the general formula (1) or a salt thereof reacts with cytochrome P450 in the in vivo tissue in the container, and the amount of light generated is measured. By doing so, cytochrome P450 in the tissue in the living body can be quantified.
  • the luminescence (amount) generated by the reaction between the cytochrome P450 and the heterocyclic compound represented by the general formula (1) or a salt thereof is a luminometer, an image analyzer, a scintillation counter, a photomultiplier tube photometer, and a photosensitive emulsion. It can be measured using a film or the like.
  • the luminescent system of the present invention directly evaluates the activity of cytochrome P450 present in almost all organisms including bacteria, plants, and mammals, and thereby, drug metabolism in those organisms, biosynthesis of hormones, and fatty acids. Photosensing of metabolism and secondary metabolism of plants can be performed.
  • in-situ observation (in) in vivo which was difficult in the past, is achieved by non-invasive, deep visualization of the living body, which is a feature of bioluminescence imaging using the heterocyclic compound represented by the general formula (1) or a salt thereof. Vivo evaluation) is possible.
  • the luminescence system of the present invention does not require firefly luciferase and does not require the introduction of a luciferase gene, it is easy to evaluate the activity of cytochrome P450, and it is suitable for diagnosing diseases and health conditions of humans as well as experimental animals. Can also be applied. Hereinafter, the use of this will be described in more detail.
  • the luminescence system of the present invention is a luminescence system that does not require the introduction of genes such as firefly luciferase, it can be applied not only to experimental animals but also to human disease and health diagnosis. ..
  • the disease can be diagnosed by measuring the change in the amount of luminescence derived from cytochrome P450 by using the luminescence system of the present invention. Will be.
  • cytochrome P450 is heavily involved in secondary metabolism.
  • the luminescence system of the present invention is useful in agriculture and forestry because screening can be facilitated if the activity of cytochrome P450 can be easily visualized when breeding to a food containing a specific nutrient.
  • Cytochrome P450 is also greatly involved in secondary metabolism in marine products, and the light emitting system of the present invention can be utilized in the fishery industry as well as in the cultivation of marine products containing a large amount of specific nutrients.
  • the light emitting system of the present invention enables easy evaluation of safety as in human drug discovery. ..
  • Environmental field environmental hormones, environmental pollution, etc.
  • Fish may be used to detect environmental pollution, including endocrine disrupters.
  • the action of fish cytochrome P450 may be used as an index. Therefore, by using the light emitting system of the present invention for detecting the action of cytochrome P450 in fish, the light emitting system of the present invention can also be effectively used for detecting environmental pollution.
  • the heterocyclic compound represented by the general formula (1) or a salt thereof is used as the following structural formula (1-1): (DeHCl type of "TokeOni” manufactured by Kurokin Kasei Co., Ltd.) or the following structural formula (1-2): Compound represented by (“TokeOni” manufactured by Kurokin Kasei Co., Ltd.) or the following structural formula (1-3): Compound represented by (“SeMPai” manufactured by Kurokin Kasei Co., Ltd.) or the following structural formula (1-4): The compound represented by, or the following structural formula (1-5): The compound represented by, or the following structural formula (1-6): The compound represented by, or the following structural formula (1-7): The compound represented by, or the following structural formula (1-8): The compound represented by is used.
  • the compound of structural formula (1-3) and the compound of structural formula (1-7) were synthesized by the method described in Japanese Patent No. 6353751.
  • the compound of structural formula (1-6) was synthesized by the method described in International Publication No. 2013/0277770.
  • the compound of the structural formula (1-4) was synthesized by replacing the compound having a naphthalene ring as a raw material with the compound having a benzene ring in the method for synthesizing the compound of the structural formula (1-6).
  • the compound of the structural formula (1-5) was synthesized by repeating the step of forming a double bond in the synthesis method described in Japanese Patent No. 5464311.
  • the compound of the structural formula (1-8) is described in the synthetic method described in Japanese Patent No. 5464311, in the step of "synthesis of methyl ester 1" of Example 1-3, "D-cysteine-S-trityl". Except for the use of "L-cysteine-S-trityl compound” instead of “compound”, the L-form of the compound of structural formula (1-1) is synthesized in the same manner, and then described in Japanese Patent No. 6011974. It was synthesized by hydrochloride (HCl) according to the synthesis method.
  • HCl hydrochloride
  • Luminette Sensor AB2200 manufactured by Atto Co., Ltd.
  • the measurement was carried out by recording the integrated value of luminescence for 30 seconds after adding the heterocyclic compound represented by the general formula (1) or a salt thereof, or firefly luciferin.
  • the solution composition at the time of luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl 2 , 6% (v / v) glycerol, 1% (v / v) TritonX-100, and 200 ⁇ M structural formula (1).
  • the solution composition at the time of luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl 2 , 6% (v / v) glycerol, 1% (v / v) TritonX-100, and 200 ⁇ M structural formula (1).
  • luminescence can be induced by adding the compound of structural formula (1-1) to the extracts of arthropods and annelids. Further, from FIG. 2, it can be seen that luminescence can be induced even by adding the compound of structural formula (1-1) to the extracts of cultured human, rat, and mouse liver cells. On the other hand, it can be seen that luminescence cannot be detected by administration of firefly luciferin.
  • the solution composition at the time of luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl 2 , 6% (v / v) glycerol, 1% (v / v) Triton X-100, 200 mM structural formula (1-1). ) Is a compound.
  • the results are shown in FIGS. 3 to 6.
  • the filtrate was loaded onto the column at a flow rate of 0.6 mL / min, and the passing fraction was collected.
  • this pass-through fraction was subjected to a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge: Bio-Rad).
  • the mixture was loaded onto the column at a flow rate of 0.6 mL / min, and the passing fraction was collected.
  • the hydroxyapatite column was washed with 15 mL of buffer (i) and then eluted with buffer (ii) (150 mM phosphate buffer (pH 7.4), 20% glycerol).
  • a mixture of the pass-through fraction and the eluate fraction was used for the luminescence measurement.
  • the solution composition at the time of luminescence measurement was 50 mM Tris / HCl, 65 mM PB, 30 mM KCl, 8 mM MgCl 2 , 6% (v / v) glycerol, 1% (v / v) Triton X-100, 200 mM structural formula (1-1). ) Is a compound. The results are shown in FIG.
  • the luminescence value when DMSO as a solvent is added is taken as 100% and is shown as a relative value. Inhibitor concentrations are all 2 mM. The amount of DMSO added is 2% in all cases.
  • cytochrome P450 As cytochrome P450 (CYP), a human P450 enzyme manufactured by CORNING was used. As a control, a membrane fraction of insect cells sold by CORNING for a control experiment was used. The value obtained by subtracting the measured value in the control from the measured value in CYP was taken as the amount of light emitted by the light emitting system composed of CYP and each substrate.
  • the CYP concentration at the time of luminescence measurement is 100 pmol / mL.
  • Other compositions of the solution at the time of luminescence measurement were 46 mM Tris / HCl, 17 mM phosphate buffer, 24 mM KCl, 6.4 mM MgCl 2 , 17 mM NaCl, 2% (v / v) Triton X-100, and a luminescent substrate ( Variable).
  • the luminescent substrate concentrations were 200 ⁇ M for the compound of the structural formula (1-1), 800 ⁇ M for the compound of the structural formula (1-2), 200 ⁇ M for the compound of the structural formula (1-3), and the structural formula (1-).
  • the compound of 4) is 100 ⁇ M
  • the compound of structural formula (1-5) is 40 ⁇ M
  • the compound of structural formula (1-6) is 100 ⁇ M
  • the compound of structural formula (1-7) is 200 ⁇ M.
  • the results are shown in FIGS. 9 to 15.
  • cytochrome P450 is combined with a heterocyclic compound represented by the general formula (1) or a salt thereof to emit light.
  • Luminescence test in insects 15 minutes before the measurement of luminescence, 3 ⁇ L of a compound having a structural formula (1-2) of 20 mM was administered to larvae of black flies 5 to 6 days after hatching. The measurement was performed with an image analyzer LS4000 (GE Healthcare Japan) for 5 minutes, and an image was acquired. The results are shown in FIG. In FIG. 17, a white portion indicates a light emitting portion.
  • Luminescence test in non-alcoholic fatty liver disease model mice 7-week-old C57BL6 mice were allowed to freely ingest a choline-deficient CDAHFD high-fat diet for 8 weeks. The control group was allowed to freely ingest the same amount of normal diet. Fifteen minutes before the luminescence measurement, the anesthetic pentobarbital was administered to the abdominal cavity of the mouse so as to be 65 mg / kg of body weight, and further, five minutes before the luminescence measurement, the structural formula of 20 mM was applied to the abdominal cavity of the mouse. 250 ⁇ L of the compound (1-2) was administered, and measurement was performed. The results are shown in FIG.
  • the light emitting system of the present invention has biophotoacoustic imaging, disease diagnosis, pharmacokinetic / safety evaluation, agriculture, forestry and fisheries fields (pesticides, breeding, production increase, etc.), environmental fields (environmental hormones, environmental pollution, etc.). Can be used for.
  • the luminescence system of the present invention includes a glucose monitoring kit, a heart marker, an infectious disease test kit, a pregnancy and infertility test kit, a blood glucose level, a blood gas and electrolyte test kit, a tumor / cancer marker, a urine test kit, and a cholesterol test. It can also be used for various kits such as kits, immunological test kits, pharmacokinetic / safety evaluation kits, food toxicity evaluation kits, and the like. Further, the light emitting system of the present invention can be used in the fields of medical research, evolution / life research, space biology (International Space Station (ISS), microgravity science experiment using lunar orbit platform gateway, etc.).
  • ISS International Space Station

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