WO2022050233A1 - Light-emitting system and cytochrome p450 quantification method - Google Patents

Abstract

The purpose of the present invention is to provide a cytochrome P450 light emission system which can easily be made to emit light without the need for excitation light; this purpose is met by a light emitting system characterized by containing cytochrome P450 and a heterocyclic compound represented by general formula (1) or a salt thereof [in the formula, R1 is a hydrogen or a C1-4 alkyl group, R2 is NR4 2 or OH; the R4s are independently a hydrogen or a C1-6 alkyl group, and two R4s of the NR4 2s may optionally bond together and form a ring; A is represented by general formula (2) or (3), the R3s are independently CR5 or N; R5s are independently a hydrogen, a C1-8 alkyl group or a C2-8 alkenyl group, and n is an integer 0-3].

Description

Luminous system and method for quantifying cytochrome P450

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).

Conventionally, 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. Here, the test compound that modulates the activity of cytochrome P450 is identified by its effect on the amount of fluorescent product accumulated.
On the other hand, in the luminescence assay, a luminescence reaction between firefly luciferase and firefly luciferin, which is a substrate thereof, is used (Non-Patent Document 2). Specifically, 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.

"Drug Interactions Through Cytochrome P450", Pharmacia, 31 (9), 992-996 (1995) "Screening for Cytochrome P450 Activity Using Luminescence Assay", Promega, November 19, 2006

However, the excitation light required for the fluorescence assay generates a background signal, which limits assay sensitivity.
On the other hand, since 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.

Therefore, it is an object of the present invention to solve the above-mentioned problems of the prior art and to provide a cytochrome P450 light emitting system capable of easily emitting light without requiring excitation light.
Further, it is a further object of the present invention to provide a method for quantifying cytochrome P450 easily and with high sensitivity by using the above light emitting system.

As a result of diligent studies to solve the above problems, the present inventors have found that cytochrome P450 reacts with a compound having a specific structure to emit light, and by quantifying the luminescence, cytochrome P450 can be quantified. Has been completed.
That is, the gist structure of the present invention that solves the above problems is as follows.

［式中、Ｒ^１は、水素又は炭素数１～４のアルキル基であり、
　Ｒ^２は、ＮＲ^４ _２又はＯＨであり、ここで、Ｒ^４は、それぞれ独立して水素又は炭素数１～６のアルキル基であり、また、ＮＲ^４ _２の２つのＲ^４は、互いに結合して環を形成してもよく、
　Ａは、下記一般式（２）又は（３）：

で表され、
　Ｒ^３は、それぞれ独立してＣＲ^５又はＮであり、ここで、Ｒ^５は、それぞれ独立して水素、炭素数１～８のアルキル基又は炭素数２～８のアルケニル基であり、
　ｎは、０～３の整数である］で表される複素環式化合物又はその塩と、シトクロムＰ４５０と、を含むことを特徴とする。
　かかる本発明の発光システムは、励起光を必要とせず、簡便にシトクロムＰ４５０由来の発光を得ることができる。 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.

In a preferred example of the light emitting system of the present invention, the cytochrome P450 is present in the tissue in the living body. In this case, luminescence derived from cytochrome P450 in the tissue in the living body can be obtained.

In another preferred example of the light emitting system of the present invention, the cytochrome P450 is present in mammalian liver tissue. In this case, luminescence derived from cytochrome P450 in mammalian liver tissue is obtained.

［式中、Ｒ^１は、水素又は炭素数１～４のアルキル基であり、
　Ｒ^２は、ＮＲ^４ _２又はＯＨであり、ここで、Ｒ^４は、それぞれ独立して水素又は炭素数１～６のアルキル基であり、また、ＮＲ^４ _２の２つのＲ^４は、互いに結合して環を形成してもよく、
　Ａは、下記一般式（２）又は（３）：

で表され、
　Ｒ^３は、それぞれ独立してＣＲ^５又はＮであり、ここで、Ｒ^５は、それぞれ独立して水素、炭素数１～８のアルキル基又は炭素数２～８のアルケニル基であり、
　ｎは、０～３の整数である］で表される複素環式化合物又はその塩を反応させ、生じる発光量を測定し、
　前記発光量から、前記シトクロムＰ４５０の量を測定することを特徴とする。
　かかる本発明のシトクロムＰ４５０の定量方法は、簡便に且つ高感度で、シトクロムＰ４５０を定量することができる。 The method for quantifying cytochrome P450 of the present invention is based on cytochrome P450 according to 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 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.

According to the present invention, it is possible to provide a 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.

It is a result of measuring the amount of luminescence in the system containing the arthropod extract and the compound of the structural formula (1-1) or firefly luciferin. It is the result of measuring the amount of luminescence in the system containing the cultured mammalian liver cell and the compound of the structural formula (1-1) or firefly luciferin. It is a result of measuring the amount of decrease in the amount of luminescence in the system containing the pill bug (male) extract, the cytochrome P450 inhibitor, and the compound of the structural formula (1-1). It is a result of measuring the amount of decrease in the amount of luminescence in the system containing the pill bug (female) extract, the cytochrome P450 inhibitor, and the compound of the structural formula (1-1). It is a result of measuring the amount of decrease in the amount of luminescence in the system containing the crokin fly (male) extract, the cytochrome P450 inhibitor, and the compound of the structural formula (1-1). It is a result of measuring the amount of decrease in the amount of luminescence in the system containing the crokin fly (female) extract, the cytochrome P450 inhibitor, and the compound of the structural formula (1-1). It is a result of measuring the amount of decrease in the amount of luminescence in the system containing the liver extract of mouse, the cytochrome P450 inhibitor, and the compound of structural formula (1-1). This is the result of administering a cytochrome P450 inhibitor and a compound of structural formula (1-2) to mice and measuring the amount of decrease in the amount of luminescence (in vivo). It is a result of measuring the amount of light emission in the system containing CYP1A2 and the heterocyclic compound represented by the general formula (1) or a salt thereof. It is a result of measuring the amount of light emission in the system containing CYP2A6 and the heterocyclic compound represented by the general formula (1). It is a result of measuring the amount of light emission in the system containing CYP2B6 and the heterocyclic compound represented by the general formula (1) or a salt thereof. It is a result of measuring the amount of light emission in the system containing CYP2C9 and the heterocyclic compound represented by the general formula (1) or a salt thereof. It is a result of measuring the amount of light emission in the system containing CYP2D6 and the heterocyclic compound represented by the general formula (1) or a salt thereof. It is a result of measuring the amount of light emission in the system containing CYP2E1 and the heterocyclic compound represented by the general formula (1) or a salt thereof. It is a result of measuring the amount of light emission in the system containing CYP3A4 and the heterocyclic compound represented by the general formula (1) or a salt thereof. This is the result of administering the compound (D form) of the structural formula (1-2) or the compound (L form) of the structural formula (1-8) to the mice and measuring the amount of luminescence (in vivo). It is a luminescence image taken by administering the compound of the structural formula (1-2) to the larva of the black fly larva (in vivo). It is the result of administering the compound of structural formula (1-2) to the mouse model of chronic liver disease due to carbon tetrachloride and the mouse of the control group, and measuring the amount of luminescence (in vivo). This is the result of administering the compound of the structural formula (1-2) to the non-alcoholic fatty liver disease model mouse and the control group mouse, and measuring the luminescence amount (in vivo).

Hereinafter, the light emitting system of the present invention and the method for quantifying cytochrome P450 will be exemplified in detail based on the embodiment thereof.

［式中、Ｒ^１は、水素又は炭素数１～４のアルキル基であり、
　Ｒ^２は、ＮＲ^４ _２又はＯＨであり、ここで、Ｒ^４は、それぞれ独立して水素又は炭素数１～６のアルキル基であり、また、ＮＲ^４ _２の２つのＲ^４は、互いに結合して環を形成してもよく、
　Ａは、下記一般式（２）又は（３）：

で表され、
　Ｒ^３は、それぞれ独立してＣＲ^５又はＮであり、ここで、Ｒ^５は、それぞれ独立して水素、炭素数１～８のアルキル基又は炭素数２～８のアルケニル基であり、
　ｎは、０～３の整数である］で表される複素環式化合物又はその塩と、シトクロムＰ４５０と、を含むことを特徴とする。 <Light emitting system>
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.

In the light emitting system of the present invention, the heterocyclic compound represented by the general formula (1) or a salt thereof reacts with cytochrome P450 to emit light. Upon such emission, the emission system of the present invention does not require excitation light, unlike the fluorescence assay.
Further, 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.

According to the light emitting system of the present invention, it is possible to provide a method for directly visualizing the activity of cytochrome P450. When 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. Unlike the firefly luminescence system, this luminescence does not require luciferase, so there is no need to introduce the luciferase gene into the measurement target. Further, by combining 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.

(Heterocyclic compound or salt thereof)
In the above general formula (1), R ¹ is hydrogen or an alkyl group having 1 to 4 carbon atoms. Here, 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. From the viewpoint of luminous efficiency, hydrogen is preferable as ^R1 .
Regarding the asymmetric carbon of the thiazolin ring to which -COOR ¹ is bonded, the configuration may be R or S.

で表される１－アザシクロプロピル基（三員環）、１－アザシクロブチル基（四員環）、１－アザシクロペンチル基（五員環）、１－アザシクロヘキシル基（六員環）、１－アザシクロヘプチル基（七員環）等が挙げられる。発光効率の観点から、Ｒ^４としては、メチル基が好ましい。
　Ｒ^２としては、発光効率の観点から、ＮＲ^４ _２が好ましく、Ｎ（ＣＨ_３）_２が特に好ましい。 In the above general formula (1), 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. Regarding ^R4 , 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. .. Further, 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. From the viewpoint of luminous efficiency, a methyl group is preferable as ^R4 .
As R ² , NR _{42 is preferable, and N (CH 3 ) 2} ^is _particularly preferable, from the viewpoint of luminous efficiency.

In the general formula (1), 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). ..

In the above general formulas (2) and (3), 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. Regarding R5, ^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.
From the viewpoint of ease of synthesis, R ³ is preferably CR ⁵ . Further, from the viewpoint of solubility in a buffer solution having water or a pH near neutral ^, it is preferable that one or more of R3 is N.

In the above general formula (1), n indicates the number of repetitions in vinylene unit (-CH = CH-) and is an integer of 0 to 3. The larger the number of n, the longer the emission wavelength. Therefore, from the viewpoint of visualization of the deep part of the living body, n is preferably 2 or 3, and from the viewpoint of ease of synthesis, n is preferably 2.

The 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.
Here, 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. For example, as 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. Phosphate, nitrate, phosphite, nitrite, citrate, formate, acetate, oxalate, maleate, lactate, tartrate, fumarate, benzoate, mandelate, Silica syrup, pamoate, stearate, glutamate, aspartate, methanesulfonate, ethanedisulfonate, p-toluenesulfonate, salicylate, succinate, trifluoroacetate, etc. Can be mentioned. On the other hand, 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. For example, 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.
Examples of the cytochrome P450 subfamily (molecular species) include CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5.
Among these, 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.
Further, from the viewpoint of reactivity with the heterocyclic compound represented by the general formula (1) or a salt thereof, 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.

In one preferred embodiment of the light emitting system of the present invention, the cytochrome P450 is present in the tissue in the living body. In this case, luminescence derived from cytochrome P450 in the tissue in the living body can be obtained. In order to detect the emission derived from cytochrome P450 present in the tissue in the living body, 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. ..

In another preferred embodiment of the light emitting system of the present invention, the cytochrome P450 is present in mammalian liver tissue. In this case, luminescence derived from cytochrome P450 in mammalian liver tissue is obtained. Generally, in mammals, 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. In order to detect cytochrome P450-derived luminescence present in the liver tissue of a mammal, 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.

In another preferred embodiment of the light emitting system of the present invention, the cytochrome P450 is present in a container such as a test tube. In this case, since 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. In this case, depending on the amount of cytochrome P450, 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.

(others)
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. As a precursor of the heterocyclic compound represented by the general formula (1) or a salt thereof, a compound in which sugar, ATP, a phospholipid or the like is bound to R ¹ or R ² in the general formula (1) or a salt thereof. Etc., but are not limited to this. 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. 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. Here, the pH of the light emitting system is preferably 4 to 10, more preferably 6 to 8. Examples of 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. Examples of the surfactant include cationic surfactants, anionic surfactants, nonionic surfactants, amphoteric surfactants and the like, and nonionic surfactants are preferable. Further, as 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. When 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.

<Calculation method of cytochrome P450>
In the method for quantifying cytochrome P450 of the present invention, 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.
When carried out in vivo, 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. By reacting with the cytochrome P450 of the above and measuring the amount of generated light, the cytochrome P450 in the living body can be quantified.
When carried out in vitro, for example, in vivo tissue (particularly, mammalian liver 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.

<Use of light emitting system, quantification method of cytochrome P450>
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 addition, 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.
Further, since 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.

(1) Bio-optical imaging Since 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. ..

(2) Diagnosis of disease When the activity (amount) of cytochrome P450 changes due to a disease, 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.

(3) Pharmacokinetics / safety evaluation It is known that many drugs are metabolized by cytochrome P450 and excreted from the body. Therefore, which cytochrome P450 metabolizes the new drug to what extent is an essential consideration item in the safety evaluation of the drug. Since the metabolic process can be easily detected by using the luminescence system of the present invention, the luminescence system of the present invention is useful for pharmacokinetic and safety evaluation.

(4) Agriculture, forestry and fisheries (agricultural chemicals, breeding, increased production, etc.)
In plants, 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.
As described above, in both agriculture and fisheries, in the development of antibiotics for pesticides and livestock, the light emitting system of the present invention enables easy evaluation of safety as in human drug discovery. ..

(5) Environmental field (environmental hormones, environmental pollution, etc.)
Fish may be used to detect environmental pollution, including endocrine disrupters. At that time, 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.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

で表される化合物（黒金化成株式会社製「ＴｏｋｅＯｎｉ」の脱ＨＣｌタイプ）、又は、下記構造式（１－２）：

で表される化合物（黒金化成株式会社製「ＴｏｋｅＯｎｉ」）、又は、下記構造式（１－３）：

で表される化合物（黒金化成株式会社製「ＳｅＭｐａｉ」）、又は、下記構造式（１－４）：

で表される化合物、又は、下記構造式（１－５）：

で表される化合物、又は、下記構造式（１－６）：

で表される化合物、又は、下記構造式（１－７）：

で表される化合物、又は、下記構造式（１－８）：

で表される化合物を使用した。
　なお、構造式（１－３）の化合物及び構造式（１－７）の化合物は、特許第６３５３７５１号公報に記載の方法で合成した。
　また、構造式（１－６）の化合物は、国際公開第２０１３／０２７７７０号に記載の方法で合成した。
　また、構造式（１－４）の化合物は、構造式（１－６）の化合物の合成法において、原料のナフタレン環を有する化合物を、ベンゼン環を有する化合物に置き換えて、合成した。
　また、構造式（１－５）の化合物は、特許第５４６４３１１号公報に記載の合成法において、二重結合の形成工程を繰り返して、合成した。
　また、構造式（１－８）の化合物は、特許第５４６４３１１号公報に記載の合成法において、実施例１－３の「メチルエステル１の合成」の工程で、「Ｄ－システイン－Ｓ－トリチル化合物」に代えて「Ｌ－システイン－Ｓ－トリチル化合物」を使用する以外は、同様にして、構造式（１－１）の化合物のＬ体を合成した後、特許第６０１１９７４号公報に記載の合成法に従い塩酸塩（ＨＣｌ）化して、合成した。 <Tested heterocyclic compound>
In the following experiment, 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.
In addition, the compound of structural formula (1-6) was synthesized by the method described in International Publication No. 2013/0277770.
Further, 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).
Further, 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.
Further, 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.

で表される天然のホタルルシフェリン（富士フイルム和光純薬株式会社製）を使用した。 In addition, as a comparative control, the following chemical formula (a):

Natural firefly luciferin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) represented by is used.

<Measuring equipment>
A luminometer (“Luminette Sensor AB2200” manufactured by Atto Co., Ltd.) was used for measuring the amount of light emitted. 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.

(1) Observation of luminescence by arthropod, annelid or mammalian liver cultured cells and cytochrome P450 (1-1) Arthropod and annelid extract 0.5 mL buffer of each arthropod or annelid Homogenized in solution (10 mM phosphate buffer (pH 7.4), 20% glycerol). The homogenate was centrifuged at 15,000 × g at 4 ° C. for 15 minutes, and the supernatant was used as an extract for measurement.
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 μM structural formula (1). -1) compound [Compound (1-1) in FIG. 1] or 200 μM firefly luciferin.
The results are shown in FIG.

(1-2) Mammalian liver cultured cells After collecting rat, mouse, and human liver cultured cells cultured in a 150 cm ² culture dish, 0.1 mL of buffer solution (10 mM phosphate buffer (pH 7.4), 20%). Homogenized in glycerol). The homogenate was centrifuged at 15,000 × g at 4 ° C. for 15 minutes, and the supernatant was used as an extract for measurement.
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 μM structural formula (1). -1) compound [Compound (1-1) in FIG. 2] or 200 μM firefly luciferin.
The results are shown in FIG.

From FIG. 1, it can be seen that 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.

(2) Observation of decrease in luminescence due to cytochrome P450 inhibitor (2-1) Extract of pill bug, pill bug extract 3 pill bugs or 5 pill bugs in 0.5 mL buffer (10 mM phosphate buffer (pH 7.4)) , 20% glycerol) homogenized. The homogenate was centrifuged at 15,000 × g at 4 ° C. for 15 minutes, and the supernatant was used as an extract for measurement.
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.

In the graphs shown in FIGS. 3 to 6, 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.

The relationship between each inhibitor and the target cytochrome P450 (CYP) is shown in Table 1.

(2-2) Mouse liver extract The liver was removed from C57BL6 mice after euthanasia, and 3.0 mL of buffer solution (i) (10 mM phosphate buffer (pH 7.4), 20% glycerol) per animal was removed. Was added and homogenized. The homogenate was centrifuged at 15,000 × g at 4 ° C. for 15 minutes, and the supernatant was collected and filtered using a syringe filter having a pore size of 0.45 mm.
The filtrate was then subjected to an anion exchange column (HiTrap Q FF: GE Healthcare). After equilibrating the column with the buffer solution (i), the filtrate was loaded onto the column at a flow rate of 0.6 mL / min, and the passing fraction was collected.
Next, this pass-through fraction was subjected to a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge: Bio-Rad). After equilibrating the column with the buffer solution (i), 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.

In the graph shown in FIG. 7, 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.

The relationship between each inhibitor and the target cytochrome P450 (CYP) is shown in Table 2.

From FIGS. 2 to 6, when a cytochrome P450 inhibitor was added to an extract derived from a pill bug or a pill bug, the effect of inhibiting luminescence was observed by some inhibitors.
Further, from FIG. 7, even if a cytochrome P450 inhibitor was added to the liver extract of a mouse, the effect of inhibiting luminescence was observed by some inhibitors.
From these results, it can be seen that the compound of structural formula (1-1) emits light due to the action of cytochrome P450.

(2-3) Test in mice (in vivo) 30 minutes before luminescence measurement, 100 μL of each inhibitor of 10 mM dissolved in DMSO was administered intraperitoneally to 6 to 10 week old C57BL6 mice. Next, 15 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, 5 minutes before the luminescence measurement, 20 mM was applied to the abdominal cavity of the mouse. 250 μL of the compound of the structural formula (1-2) of the above was administered. The measurement was performed with an image analyzer LS4000 (GE Healthcare Japan) for 20 minutes. The results are shown in FIG.

The relationship between each inhibitor and the target cytochrome P450 (CYP) is shown in Table 3.

From FIG. 8, even if a cytochrome P450 inhibitor was added to the living body of a mouse, the effect of inhibiting luminescence was observed.
From these results, it can be seen that the compound of structural formula (1-2) emits light due to the action of cytochrome P450.

(3) Examination of luminescence by 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 μM for the compound of the structural formula (1-1), 800 μM for the compound of the structural formula (1-2), 200 μM for the compound of the structural formula (1-3), and the structural formula (1-). The compound of 4) is 100 μM, the compound of structural formula (1-5) is 40 μM, the compound of structural formula (1-6) is 100 μM, and the compound of structural formula (1-7) is 200 μM.
The results are shown in FIGS. 9 to 15.

In the graphs shown in FIGS. 9 to 15, the value of the amount of light emitted when the compound of the structural formula (1-3) is added is taken as 100%, and is shown as a relative value.

From FIGS. 9 to 15, it can be seen that cytochrome P450 is combined with a heterocyclic compound represented by the general formula (1) or a salt thereof to emit light.

(4) Confirmation experiment using isomers Fifteen minutes before luminescence measurement, the anesthetic pentobarbital was administered intraperitoneally to 6 to 10 week old C57BL6 mice so as to be 65 mg / kg body weight, and further, luminescence measurement was performed. Five minutes before, 250 μL of 20 mM the compound (D form) of the structural formula (1-2) or the compound (L form) of the structural formula (1-8) was administered to the abdomen of the mouse. The measurement was performed with an image analyzer LS4000 (GE Healthcare Japan) for 20 minutes. The results are shown in FIG.

From FIG. 16, it can be seen that the amount of light emitted is the same regardless of whether the compound (D form) of the structural formula (1-2) is used or the compound (L form) of the structural formula (1-8) is used.

(5) Luminescence test in insects (in vivo) 15 minutes before the measurement of luminescence, 3 μL 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.

From FIG. 17, it can be seen that the larvae of Crokin fly (containing cytochrome P450) are combined with the heterocyclic compound represented by the general formula (1) or a salt thereof to emit light.

(6) Luminescence test in 7-week-old C57BL6 mice by administration of carbon tetrachloride Carbon tetrachloride was diluted 5-fold with olive oil to 3 mL / kg twice a week, 4 A mouse model of chronic liver disease was prepared by intraperitoneal administration for a week. The control group received the same amount of olive oil.
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 μL of the compound (1-2) was administered, and measurement was performed. The results are shown in FIG.

From FIG. 18, it can be seen that the amount of luminescence of the chronic liver disease model mouse is reduced as compared with the control group mouse. This indicates that drug-induced liver injury reduced cytochrome P450 in mice.

As literature on drug-induced toxic liver injury, for example, the Journal of the Japanese Society of Internal Medicine, 84, 188-193 (1995) discloses that administration of carbon tetrachloride reduces the amount of cytochrome P450 in the liver. It was confirmed that it was consistent with the above test results.

(7) 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 μL of the compound (1-2) was administered, and measurement was performed. The results are shown in FIG.

From FIG. 19, it can be seen that the amount of luminescence is increased in the non-alcoholic fatty liver disease model mouse as compared with the control group mouse. This indicates that non-alcoholic fatty liver disease increased cytochrome P450 in mice.

As literature on non-alcoholic steatohepatitis, for example, Weltman M. et al. D. et al. (1998), "Hepatological cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis", Hepatology, 27 (1): 128-33 in non-alcoholic steatohepatitis patients It was confirmed that it was consistent with the above test results.

As described above, 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.
In addition, 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.).

Claims (4)

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 a heterocyclic compound or a salt thereof.
A light emitting system comprising cytochrome P450. The light emitting system according to claim 1, wherein the cytochrome P450 is present in a tissue in a living body. The light emitting system according to claim 1 or 2, wherein the cytochrome P450 is present in the liver tissue of a mammal. Cytochrome P450 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 the heterocyclic compound or a salt thereof is reacted and the amount of light emitted is measured.
A method for quantifying cytochrome P450, which comprises measuring the amount of cytochrome P450 from the amount of light emitted.

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