WO2017090631A1

WO2017090631A1 - Fluorescent probe for detecting extracellular metabolite and screening method employing said fluorescent probe

Info

Publication number: WO2017090631A1
Application number: PCT/JP2016/084669
Authority: WO
Inventors: 泰照浦野; 小松　徹; 光一柳
Original assignee: 国立大学法人東京大学
Priority date: 2015-11-24
Filing date: 2016-11-22
Publication date: 2017-06-01

Abstract

[Problem] The present invention addresses the problem of developing a novel fluorescent probe that allows high-sensitivity detection of lactic acid, which is a substance released outside cells as the final product of the glycolytic system, and providing a screening method with which it is possible, by using such an evaluation system, to search for compounds that exhibit inhibitory effects on the glycolytic system and that are candidate anti-cancer agents. [Solution] Provided is a fluorescent probe for detecting NADH containing that contains a compound represented by formula (I) or a salt thereof [in the formula, R¹ represents the same or different types of 1-5 water-soluble substituents; R² represents the same or different types of 1-5 electron-accepting substituents; each of R³, R⁴, R⁵, and R⁶ independently represents a hydrogen atom or an alkyl group; each of R⁷ and R⁸ independently represents a hydrogen atom or the same or different types of 1-3 substituents that are independently selected from a group including a hydroxy group, a halogen atom, alkyl groups each of which may include substitution, a sulfo group, a carboxy group, an ester group, an amide group, and an azido group; and each of R^a and R^b independently represents a hydrogen atom or an alkyl group, wherein, in the case in which R³ or R⁴ is an alkyl group, an annular structure may be formed together with R⁷, which includes nitrogen atoms with which these groups are bonded, and, in the case in which R⁵ or R⁶ is an alkyl group, an annular structure may be formed together with R⁸, which includes nitrogen atoms with which these groups are bonded.].

Description

Fluorescent probe for detecting extracellular metabolites and screening method using the fluorescent probe

The present invention relates to a fluorescent probe for detecting an extracellular metabolite such as lactic acid (L-lactic acid), and a method for screening a glycolytic inhibitor using the fluorescent probe.

Over thousands of metabolic reactions are constantly progressing in the living body, and the homeostasis of the living body is maintained by various metabolic processes. Therefore, understanding and evaluating metabolic processes is thought to lead to elucidation of disease mechanisms and development of therapeutic drugs. In particular, in many cancer types, it is known that the metabolic pathway of the energy production system exhibits abnormal activity. For example, it has been found that cancer cells prioritize metabolism in the glycolytic system over oxidative phosphorylation in mitochondria even under aerobic conditions, which is called the Warburg effect. In fact, some glycolytic inhibitors exhibit anticancer activity, and 2-deoxyglucose that inhibits the glycolytic system is used as an anticancer agent. Therefore, paying attention to such metabolic pathways may lead to the development of useful therapeutic agents effective for many cancer types.

In general, metabolic pathways are complexly configured in cells and are controlled by intracellular signals and metabolites. Therefore, in order to correctly evaluate the metabolic activity of energy production systems, it is necessary to evaluate them in living cells. is there. However, it cannot be said that a useful technique for evaluating the metabolic enzyme activity for small molecule metabolites such as lactic acid, sugar and amino acid involved in the metabolic pathway of the energy production system has been established.

As existing metabolic activity evaluation methods, a method of incorporating a fluorescent probe into a substrate (Non-patent Document 1) and an in-vitro evaluation method using a purified enzyme (Non-patent Document 2) are known. However, although the former can be used in living cells, there is a problem of enzyme recognition, and it has been particularly difficult to incorporate a fluorophore into a small molecule metabolite to function as a substrate analog. In the latter case, since a single enzyme activity is evaluated in vitro, it does not reflect the complex enzyme control mechanism in the cell, and the enzyme activity in vivo cannot be detected accurately. It was.

Therefore, an object of the present invention is to construct a new technique for evaluating glycolytic activity in living cells by focusing on the glycolytic pathway in the metabolic pathway of the energy production system. More specifically, an object of the present invention is to develop a novel fluorescent probe capable of detecting lactic acid, which is a substance released extracellularly as a glycolytic end product, with high sensitivity. Another object of the present invention is to provide a screening method capable of searching for anticancer drug candidate compounds having a glycolytic inhibitory effect by using such an evaluation system.

As a result of intensive studies to solve the above problems, the present inventors have converted lactic acid into pyruvic acid by an enzyme, and selectively react with NADH (nicotinamide adenine dinucleotide) produced at that time. We have developed a new fluorescent probe that exhibits a fluorescence response of the fluorescence type, and found that it is possible to detect lactic acid. Further, it has been found that substances having a glycolytic inhibitory effect can be efficiently searched for by a screening method using such a fluorescent probe. Based on these findings, the present invention has been completed.

The fluorescent probe of the present invention has a tetrazolium moiety in the molecule and a silicon-substituted xanthene fluorophore, so that the reaction with NADH can be detected as an ON / OFF-type fluorescence response using photoinduced electron transfer (PET). Moreover, since it has water solubility, it can function in an extracellular fluid.

That is, the present invention in one aspect,
(1) A fluorescent probe for detection of NADH comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
R ¹ represents 1 to 5 identical or different water-soluble substituents;
R ² represents 1 to 5 identical or different electron withdrawing substituents;
R ³ , R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom or an alkyl group;
R ⁷ and R ⁸ are each independently a hydrogen atom, a hydroxy group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxy group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents selected from; and
R ^a and R ^b each independently represent a hydrogen atom or an alkyl group,
Here, when R ³ or R ⁴ is an alkyl group, together with R ⁷ , a ring structure containing a nitrogen atom to which they are bonded may be formed,
When R ⁵ or R ⁶ is an alkyl group, it may be combined with R ⁸ to form a ring structure containing the nitrogen atom to which they are attached. ];
(2) The above ( ¹ ), wherein R ¹ represents 1 to 5 identical or different substituents independently selected from the group consisting of a sulfo group, a sulfonamido group, a carboxy group, an ester group, and an amido group. Fluorescent probes of
(3) In the above (1) or (2), R ² represents 1 to 5 identical or different substituents independently selected from the group consisting of a nitro group, a sulfo group, a sulfonamido group, and a cyano group The described fluorescent probe;
(4) The fluorescent probe according to any one of the above (1) to (3), wherein two R ¹ are a sulfo group and R ² is a nitro group;
(5) The fluorescent probe according to any one of (1) to (4) above, wherein R ³ , R ⁴ , R ⁵ and R ⁶ are all methyl groups;
(6) The fluorescent probe according to any one of (1) to (5), wherein R ⁷ and R ⁸ are both hydrogen atoms; and (7) the compound represented by formula (I) is as follows: The fluorescent probe according to (1), which is a compound of

Is to provide.

In another aspect, the present invention provides:
(8) A method for detecting NADH using the fluorescent probe according to any one of (1) to (7) above;
(9) The detection method according to (8) above, wherein the presence of NADH is detected by observing a fluorescence response due to a reaction between NADH and the fluorescent probe;
(10) The detection method according to (9), wherein the fluorescence response is a fluorescence change caused by light-induced electron transfer (PET);
(11) Use of the fluorescent probe according to any one of (1) to (7) above for detecting lactic acid;
(12) A method for detecting lactic acid,
Step by adding lactate dehydrogenase (LDH) in the test object, is converted to pyruvate and NADH comprising lactic acid and NAD ^+,
Adding the fluorescent probe according to any one of the above (1) to (7) to the obtained test object, and reacting NADH produced in the step with the fluorescent probe;
Observing a fluorescence response generated by the reaction, and detecting the presence of NADH from the fluorescence response, thereby detecting lactic acid present in the test sample,
The detection method is provided.

Furthermore, in another aspect, the present invention provides:
(13) A method for screening a glycolytic inhibitor using the fluorescent probe according to any one of (1) to (7) above;
(14) The screening method according to (13) above, wherein the glycolytic inhibitor is an anticancer agent.

ADVANTAGE OF THE INVENTION According to this invention, the novel NADH detection fluorescent probe which becomes fluorescent by reacting with NADH can be provided, By using the said fluorescent probe, the lactic acid released outside a cell via a glycolysis system can be provided. It becomes possible to detect. Since the fluorescence probe of the present invention controls fluorescence by light-induced electron transfer (PET), it can be detected as an ON / OFF type fluorescence response. Furthermore, since the fluorescent probe of the present invention has water solubility, it can function in the extracellular fluid. In addition, the screening method using the fluorescent probe of the present invention enables efficient screening of substances having a glycolytic inhibitory effect, and is extremely useful in searching for novel anticancer drug candidate compounds.

The conventional NADH detection method using water-soluble tetrazolium has a problem in that it is detected by absorbance and thus has low sensitivity and is affected by impurities. However, according to the fluorescent probe of the present invention, NADH is fluorescent. Since it can be detected as a response, such a problem can be solved.

FIG. 1 shows the absorption spectrum (C) and fluorescence spectrum (D) of compound 20 which is a fluorescent probe of the present invention, and the absorption spectrum (A) and fluorescence spectrum (B) of compound 19 which is a reduced form of the compound. It is a thing. FIG. 2 is a graph showing the NADH concentration dependence of the fluorescence intensity of Compound 20 which is the fluorescent probe of the present invention. FIG. 3 is a graph showing the lactic acid concentration dependence of the fluorescence intensity of Compound 20, which is the fluorescent probe of the present invention. FIG. 4 is a graph showing changes in fluorescence intensity of compound 20, which is the fluorescent probe of the present invention, depending on the presence or absence of a glycolytic inhibitor. FIG. 5 is a schematic diagram showing the layout of the plate reader used in Example 5. FIG. 6 is a graph showing the results of glycolysis inhibitor screening performed using the fluorescent probe of the present invention. FIG. 7 is a schematic diagram showing the layout of the plate reader used in Example 6.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Definitions In this specification, “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present specification, “alkyl” may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited. For example, the number of carbon atoms is 1 to 20 (C _1-20 ), the number of carbons is 3 to 15 (C _{3 to 15} ), and the number of carbons is 5 to 10 (C _{5 to 10).} ). When the number of carbons is specified, it means “alkyl” having the number of carbons within the range. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part of other substituents containing an alkyl part (for example, an alkoxy group, an arylalkyl group, etc.).

In the present specification, when a functional group is defined as “may be substituted”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxy group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.

In the present specification, “aryl” may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom Etc.) may be an aromatic heterocyclic ring. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether aryl is a single ring or a fused ring, it can be attached at all possible positions. Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl Group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazole-3 -Yl group and the like. Non-limiting examples of fused polycyclic aryl include 1-naphthyl group, 2-naphthyl group, 1-indenyl group, 2-indenyl group, 2,3-dihydroinden-1-yl group, 2,3 -Dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, Isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 , 3-dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, thioxanthenyl group, etc. And the like. In the present specification, an aryl group may have one or more arbitrary substituents on the ring. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing the aryl moiety (for example, an aryloxy group and an arylalkyl group).

In the present specification, the “alkoxy group” is a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group that is linear, branched, cyclic, or a combination thereof. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group, etc. are preferable Take as an example.

As used herein, “amide” includes both RNR′CO— (when R = alkyl, alkaminocarbonyl-) and RCONR′- (where R = alkyl, alkylcarbonylamino-).

As used herein, “ester” includes both ROCO— (in the case of R = alkyl, alkoxycarbonyl-) and RCOO— (in the case of R = alkyl, alkylcarbonyloxy-).

As used herein, the term “ring structure”, when formed by a combination of two substituents, means a heterocyclic or carbocyclic group, such group being saturated, unsaturated, or aromatic. Can be. Accordingly, it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperidinyl, imidazolyl, pyrrolidinyl, pyridyl and the like. In the present specification, a substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will recognize a specific substitution, such as bonding to hydrogen. Can be understood. Therefore, when it is described that specific substituents together form a ring structure, those skilled in the art can understand that the ring structure can be formed by an ordinary chemical reaction and can be easily generated. Both such ring structures and their process of formation are within the purview of those skilled in the art.

In the present specification, “NADH” represents a reduced form of nicotinamide adenine dinucleotide, and “NAD ⁺ ” represents an oxidized form of nicotinamide adenine dinucleotide.

2. Fluorescent probe of the present invention The fluorescent probe of the present invention has a tetrazolium moiety and a fluorophore in the molecule. The tetrazolium moiety is reduced to formazan by the reaction with NADH, and is characterized in that fluorescence control by photoinduced electron transfer (PET) is performed utilizing the difference in electron density between the tetrazolium and formazan.

In order to perform such fluorescence control, the fluorophore is characterized by having an absorption band on the longer wavelength side than the absorption wavelength of formazan (near 400 to 500 nm). A silicon-substituted xanthene skeleton was employed as such a fluorophore. Furthermore, the fluorescent probe of the present invention has a water-soluble functional group so that it can be used as a fluorescent probe even outside a cell solution.

The fluorescent probe of the present invention includes a compound having a structure represented by the following general formula (I).

In the general formula (I), R ¹ represents 1 to 5 identical or different water-soluble substituents, preferably from the group consisting of a sulfo group, a sulfonamide group, a carboxy group, an ester group, and an amide group. 1 to 5 independently selected substituents which are the same or different. R ¹ is preferably a sulfo group. More preferably, there are two sulfo groups, and even more preferably, the two sulfo groups are in the meta position relative to each other. R ¹ is for adding water solubility to the fluorescent probe molecule.

R ² represents 1 to 5 identical or different electron-withdrawing substituents. Preferably, R ² represents 1 to 5 identical or different substituents independently selected from the group consisting of a nitro group, a sulfo group, a sulfonamido group, and a cyano group. More preferably, R ² is a nitro group.

R ³ , R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom or an alkyl group. The alkyl group is preferably a linear or branched alkyl group having 1 to 10 carbon atoms, and more preferably a methyl group. R ³ , R ⁴ , R ⁵ and R ⁶ are preferably all methyl groups.

Here, when either R ³ or R ⁴ is an alkyl group, it may be combined with R ⁷ to form a ring structure containing a nitrogen atom to which they are bonded. In that case, only one of R ³ and R ⁷ or a combination of R ⁴ and R ⁷ may form a ring structure, or both may form a ring structure. The ring structure may contain further hetero atoms other than the nitrogen atom.

Similarly, when either R ⁵ or R ⁶ is an alkyl group, it may be combined with R ⁸ to form a ring structure containing the nitrogen atom to which they are attached. In that case, only one of R ⁵ and R ⁸ or a combination of R ⁶ and R ⁸ may form a ring structure, or both may form a ring structure. The ring structure may contain further hetero atoms other than the nitrogen atom.

R ⁷ and R ⁸ are each independently a hydrogen atom, a hydroxy group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxy group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents. Preferably, R ⁷ and R ⁸ are both hydrogen atoms.

R ^a and R ^b each independently represent a hydrogen atom or an alkyl group. The alkyl group is preferably a linear or branched alkyl group having 1 to 10 carbon atoms, and more preferably a methyl group. Furthermore, R ^a and R ^b are, when an alkyl group, they may have one or more substituents, and examples of the substituent include an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, a carboxyl It may have one or more groups, amino groups, sulfo groups and the like. R ^a and R ^b are preferably both methyl groups.

In some cases, R ^a and R ^b may be bonded to each other to form a ring structure. For example, when R ^a and R ^b are both alkyl groups, R ^a and R ^b can be bonded to each other to form a spirocarbocycle. The ring formed is preferably about 5 to 8 membered ring, for example.

Specific examples of the compound represented by the formula (I) that are representative of the fluorescent probe of the present invention include the following compounds. However, it is not limited to this.

Since the compound represented by the above formula (I) has a monovalent positive charge at each of the tetrazolium moiety and the N atom to which R ⁶ and R ⁷ are linked, it usually exists as a salt. Examples of such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. Examples include mineral acids such as hydrochlorides, sulfates and nitrates, carboxylates such as trifluoroacetates, organic acids such as methanesulfonate, paratoluenesulfonate, citrate and oxalate. Mention may be made of salts. Examples of amino acid salts include glycine salts. However, it is not limited to these salts.

The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

The compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.

The above-described fluorescent probe may be used as a composition by blending an additive for use in a physiological environment, if necessary. As such an additive, for example, additives such as a solubilizing agent, a pH adjusting agent, a buffering agent, and an isotonic agent can be used, and the amount of these can be appropriately selected by those skilled in the art. These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, or a liquid.

In the examples of the present specification, production methods for representative compounds included in the compounds of the present invention represented by the formula (I) are specifically shown. Any compound included in the formula (I) can be easily produced by referring to, and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.

3. Detection method of NADH and lactic acid using the fluorescent probe of the present invention In the detection method of the present invention, as shown in the following scheme, an enzyme is added to lactic acid (L-lactic acid) released extracellularly via a glycolysis system. Then, the NADH (nicotinamide adenine dinucleotide) produced at that time is brought into contact with a fluorescent probe, and the fluorescence response is observed to detect the presence of lactic acid through the fluorescence response of the NADH. Is. Thereby, the metabolic activity of glycolysis can be detected and evaluated extracellularly.

More specifically, the method for detecting lactic acid in the present invention includes the following steps.
(A) A step of adding lactate dehydrogenase (LDH) to a test subject containing lactic acid and NAD ⁺ and converting it into pyruvic acid and NADH,
(B) adding the fluorescent probe of the present invention to the obtained test object, and reacting the NADH produced by the step with the fluorescent probe;
(C) a step of observing a fluorescence response generated by the reaction, and (d) a step of detecting the presence of NADH from the fluorescence response, thereby detecting lactic acid present in the test sample.

Steps (b) and (c) are steps in which NADH is detected as a fluorescence response with the fluorescent probe of the present invention using photoinduced electron transfer (PET). The control mechanism of the fluorescence response is shown in the schematic diagram below. Here, photoinduced electron transfer (PET) is a phenomenon in which electrons move between a photoexcited molecule and a molecule in the vicinity thereof, as is generally known in the art.

In the state of the tetrazolium site shown on the left of the figure, the fluorescent probe molecule is non-fluorescent (weakly fluorescent) by photoinduced electron transfer (PET). By bringing the fluorescent probe of the present invention into contact with NADH, when the tetrazolium moiety in the fluorescent probe is reduced and converted to formazan, the LUMO energy level rises. As a result, electron transfer is suppressed and fluorescence emission is suppressed. Arise. Thereby, the presence of NADH can be detected as an ON / OFF type fluorescence response.

Such fluorescence control utilizes a difference in electron density between tetrazolium and formazan. Therefore, in the present invention, as a fluorophore having an absorption band on the longer wavelength side than the absorption wavelength of formazan (near 400 to 500 nm), A silicon-substituted xanthene skeleton is used.

In this specification, the term “detection” should be interpreted in the broadest sense including measurement for various purposes such as quantification and qualitative.

As a means for observing the fluorescence response, a fluorometer having a wide measurement wavelength can be used, but it is also possible to visualize the fluorescence response using a fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image. As such a fluorimeter and a fluorescence imaging apparatus, an apparatus known in the technical field can be used.

As a means for bringing a sample containing NADH to be measured into contact with a fluorescent probe, typically, a sample containing a solution containing a fluorescent probe can be added, applied or sprayed. It is possible to select appropriately according to, for example.

The application concentration of the fluorescent probe of the present invention is not particularly limited, but for example, a solution having a concentration of about 0.1 to 50 μM can be applied.

As the fluorescent probe of the present invention, the compound represented by the above formula (I) or a salt thereof may be used as it is, but if necessary, an additive usually used for the preparation of a reagent is blended as a composition. It may be used. For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible. These compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It is sufficient to dissolve and apply to.

4). The screening method using the fluorescent probe of the present invention The present invention is also effective in detecting a substance having a glycolytic inhibitory activity by detecting extracellular metabolic activity of the glycolytic system using the fluorescent probe. It also relates to a screening method. In particular, by targeting metabolic activity in cancer cells, it can be suitably used for searching for novel anticancer drug candidate compounds.

Specifically, by adding glucose to cancer-derived cells and assessing the amount of lactic acid produced by adding various candidate compounds for the amount of lactic acid released outside the cell via the glycolysis, A compound that suppresses the production amount can be searched. By using the fluorescent probe of the present invention, the amount of lactic acid produced can be observed as an ON / OFF type fluorescence response, so that high-throughput screening is possible.

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

<Synthesis of probe molecules>
According to the following scheme, Compound 20, which is a fluorescent probe of the present invention, was synthesized.

[Synthesis of Compound 14]
2-Br-4-nitrobenzoic acid 2.54 g (10 mmol) is dissolved in THF 30 mL, (Boc) ₂ O 5.70 g (26 mmol, 2.6 eq) and DMAP 336 mg (2.75 mmol, 0.275 eq) are added, The mixture was stirred at 75 ° C. for 23 hours. Formation of the target product was confirmed with a TLC plate (n-hexane / CH ₂ Cl ₂ = 1/1), and the solvent was distilled off under reduced pressure. The resulting liquid was purified by column chromatography (silica, n-hexane / CH ₂ Cl ₂ = 100/0 → 50/50 in 9 min) to obtain 2.00 g (6.6 mmol, y. 64%) of compound 7. It was.
¹ H NMR (300 MHz, CDCl ₃ ): δ 1.60 (s. 9H), 7.81 (d, 1H, J = 8.7 Hz), 8.17 (dd, 1H, J = 2.4 Hz, 8.7 Hz), 8.41 (d, 1H, J = 2.1 Hz), ¹³ C NMR (75 MHz, CDCl ₃ ): δ27.6, 83.8, 120.9, 121,8, 128.5, 130.9, 139.8, 148.5, 163.8.

[Synthesis of Compound 15]
Compound 7 (2.00 g, 6.6 mmol) was dissolved in EtOH (32 mL), SnCl ₂ .2H ₂ O (7.94 g, 35 mmol, 5.3 eq) was added, and the mixture was stirred at 70 ° C. for 2.5 hours. After confirming the formation of the desired product with a TLC plate (silica, CH ₂ Cl ₂ ), the reaction solution was cooled to room temperature, AcOEt was added, and the mixture was washed with saturated NaHCO ₃ aq and brine. After dehydration with Na ₂ SO ₄ , the solvent was distilled off under reduced pressure to obtain 1.31 g (4.8 mmol, y. 73%) of Compound 8.
¹ H NMR (300 MHz, CDCl ₃ ): δ 1.56 (s. 9H), 4.26 (s, 2H), 6.51 (dd, 1H, J = 2.4, 7.5 Hz), 6.85 (d, 1H, J = 2.4 Hz ), 7.63 (d, 1H, J = 8.1 Hz), ¹³ C NMR (75 MHz, CDCl ₃ ): δ28.0, 81.0, 112.5, 119,2, 121.0, 123.0, 133.1, 150.3, 165.0.

[Synthesis of Compound 16]

Compound

15 1.31 g (4.8 mmol) was dissolved in MeCN 40 mL, K ₂ CO ₃ 1.63 g (12 mmol, 2.5 eq) and Allyl bromide 1.5 mL (17.3 mmol, 3.6 eq) were added, and the mixture was heated at 80 ° C. under reflux. Stir for days. After confirming the formation of the target product with a TLC plate (silica, n-hexane / CH ₂ Cl ₂ = 1/1), the reaction solution was filtered. The filtrate was collected and the solvent was distilled off under reduced pressure. The resulting liquid was purified by silica gel column chromatography (silica, n-hexane / CH ₂ Cl ₂ = 100/0 → 50/50 in 12 min) to obtain compound 9 (1.20 g, 3.4 mmol, y. 71%) Obtained.
¹ H NMR (300 MHz, CDCl ₃ ): δ 1.57 (s. 9H), 3.90 (d, 4H, J = 3.0 Hz), 5.08-5.19 (m, 4H), 5.73-5.85 (m, 2H), 6.54 . (dd, 1H, J = 3.0, 9.6 Hz), 6.86 (d, 1H, J = 2.4 Hz), 7.72 (d, 1H, J = 9.0 Hz) 13 C NMR (75 MHz, CDCl 3): δ28. 4, 52.4, 110.0, 116.4, 116.9, 119.3, 123.6, 132.2, 133.0, 151.1, 164.8, HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ 352.09122 Found 352.09595 (4.73 mmu)

[Synthesis of Compound 17]
360 mg (1.03 mmol, 8.0 eq) of Compound 16 was placed in a well-dried flask, dissolved in dehydrated THF, and stirred at −78 ° C. under Ar for 20 min. To this was added 1 M sec-BuLi 1.1 mL (1.1 mmol, 8.5 eq) little by little, and the mixture was stirred at −78 ° C. under Ar for 15 min. Further, 41.8 mg (0.129 mmol) of Si-xanthone dissolved in THF was added little by little to the reaction solution, followed by stirring at room temperature under Ar for 2 hours. 2.5 mL of TFA was added to the reaction mixture, and the mixture was stirred at room temperature for 20 min. After confirming the formation of the target product by ESI-MS, the solvent was distilled off under reduced pressure. ESI-MS The resulting liquid was purified by HPLC (A / B = 95/5 → 0/100 in 25 min, A: 0.1% TFA / H ₂ O, B: 0.1% TFA / 80% MeCN / 20% 62 mg of a crude product of compound ₂ was obtained.
HRMS (ESI ⁺ ): Calcd. For [M] ⁺ 524.27333 Found 524.27215 (-1.18 mmu)

[Synthesis of Compound 18]
62 mg of the crude product of Compound 17 was dissolved in 9 mL of CH ₂ Cl ₂ , and Pd (PPh ₃ ) ₄ 31.3 mg (0.027 mmol) and 1,3-dimethylbarbituric acid 69 mg (0.44 mmol) were added. The mixture was stirred at 35 ° C. for 14 hours under Ar. After confirming the formation of the desired product by ESI-MS, the solvent was distilled off under reduced pressure. Purification by HPLC (A / B = 95/5 → 0/100 in 25 min, A: 0.1% TFA / H ₂ O, B: 0.1% TFA / 80% MeCN / 20% H ₂ O) 23 mg (0.041 mmol, y. 32%) was obtained.
¹ H NMR (300 MHz, CDCl ₃ ): δ 0.60 (s. 3H), 0.65 (s. 3H), 3.04 (s, 12H), 4.20 (s, 2H), 6.31 (s, 1H), 6.72 (dd , 1H, J = 1.5, 8.1 Hz), 6,86 (dd, 2H, J = 3.0, 9.0 Hz), 7.07 (d, 2H, J = 8.7 Hz), 7.23 (d, 2H, J = 3.0 Hz) , 7.72 (d, 1H, J = 8.1 Hz).
HRMS (ESI ⁺ ): Calcd. For [M] ⁺ 444.21073 Found 444.20619 (-4.54 mmu)

[Synthesis of Compound 19]
Compound 18 (23 mg, 0.041 mmol) was dissolved in hydrochloric acid solution (266 μL of 12N hydrochloric acid + 1.84 mL of H ₂ O), and the mixture was stirred at 0 ° C. for 20 min. A solution prepared by dissolving 2.9 mg (0.042 mmol, 1.0 eq) of NaNO _{2 in} 69 μL of H ₂ O was added little by little, and the mixture was stirred at 0 ° C. for 30 min. To the reaction mixture, 33 mg (0.074 mmol, 1.8 eq) of compound 1 dissolved in 920 μL of H ₂ O was added little by little, and then KOHaq (KOH 266 mg + H ₂ O 920 μL) was slowly added dropwise at 0 ° C. For 1.5 hours. The production of the target product was confirmed by ESI-MS, and the solvent was distilled off under reduced pressure. The resulting solid was purified by HPLC (A / B = 95/5 → 0/100 in 25 min, A: 0.1% TEAA / H ₂ O, B: 0.1% TEAA / 80% MeCN / 20% H ₂ O ), 14.4 mg (0.017 mmol, y. 41%) of the desired product was obtained.
¹ H NMR (300 MHz, CD ₃ OD): δ 0.61 (s. 3H), 0.71 (s. 3H), 2.96 (s, 12H), 6.64 (d, 2H, J = 8.7 Hz), 6.77-6.89 ( m, 3H), 7.04 (s, 2H), 7.20 (d, 1H, J = 8.1 Hz), 7.52 (d, 2H), 7.83 (d, 1H), 7.98 (d, 2H, J = 7.5 Hz), 8.16 (d, 1H, J = 9.0 Hz), 8.27 (d, 1H, J = 8.7 Hz), 8.58 (s, 1H) HRMS (ESI -):.. Calcd For [M-2H] - 854.17343 Found 854.17682 ( -4.54 mmu)

[Synthesis of Compound 20]

Compound

19 13 mg (0.015 mmol) was dissolved in MeOH 2.6 mL, and DDQ 22.6 mg (0.104 mmol, 6.9 eq) was added. The mixture was stirred at room temperature for 1 hour in the dark, and after confirming the formation of the desired product by ESI-MS, the solvent was distilled off under reduced pressure. The resulting solid was purified by HPLC (A / B = 95/5 → 0/100 in 25 min, A: 0.1% TEAA / H ₂ O, B: 0.1% TEAA / 80% MeCN / 20% H ₂ O ) And compound 13 (1.6 mg, 0.0019 mmol, y. 12%) was obtained.
^{HRMS (ESI -):. Calcd} For [M-3H] - 852.15778 Found 852.15576 (-2.02 mmu)

<Optical properties of probe molecules>
Optical properties of Compound 20 which is the fluorescent probe molecule of the present invention obtained in Example 1 and Compound 19 which is a reduced form thereof and has a formazan moiety were measured. When the tetrazolium site of compound 20 is reduced by NADH, it becomes compound 19, and by evaluating these optical properties, the fluorescence response change when NADH is detected can be found.

Absorption spectra and fluorescence spectra of

compounds

19 and 20 in a PBS solution containing 0.1% DMSO were measured. The results are shown in FIG. The obtained absorption maximum wavelength, fluorescence maximum wavelength and quantum yield are shown in Table 1 below.

The fluorescence intensity at 661 nm has a difference of about 145 times, indicating that the S / N ratio is very good.

<Measurement of fluorescence intensity using a plate reader>
Using the fluorescent probe molecule of the present invention, the fluorescence response change accompanying the addition of NADH and lactic acid was confirmed.

A dilution series of a fluorescent probe solution and NADH was prepared according to Table 2, and after mixing, the fluorescence intensity was measured with a plate reader (fluorescence intensity was 180 minutes after the start of measurement). As the fluorescent probe, the compound 20 synthesized in Example 1 was used.

* Reaction buffer: phosphate buffer pH 7.4, 1 mM CaCl ₂ , 1 mM MgCl ₂ (including 0.1% CHAPS)

The result is shown in FIG. It can be seen that NADH on the order of several tens of μM can be detected. It can also be seen that the fluorescence intensity and NADH concentration are kept linear up to about 100 μM, and quantification is possible. This demonstrated that NADH can be detected by the fluorescent probe of the compound.

According to Table 3, a lactic acid dilution series and a fluorescent probe solution were prepared, and after mixing, the fluorescence intensity was measured with a plate reader (fluorescence intensity was 180 minutes after the start of measurement). As the fluorescent probe, the compound 20 synthesized in Example 1 was used.

The result is shown in FIG. It can be seen that lactic acid on the order of several tens of μM can be detected. It can also be seen that the fluorescence intensity and the lactic acid concentration remain linear up to about 100 μM and can be quantified. From this, it was demonstrated that lactic acid can be detected by the fluorescent probe of compound 6.

<Fluorescence assay using living cells>
We examined whether lactic acid released into the extracellular fluid using live cells could be detected with a fluorescent probe. A solution was prepared according to Table 4, and HL60 cells (3.0 × 10 ⁵ cells / mL) were incubated at 37 ° C. for 6 hours with this solution. Thereafter, the fluorescent probe solution and the cell solution were mixed according to Table 5, and the fluorescence intensity was measured with a plate reader (fluorescence intensity was 120 minutes after the start of measurement).

The result is shown in FIG. There was a sufficient difference in fluorescence intensity between glucose (2) and glucose not added (1). In addition, the fluorescence intensity was suppressed in (3) and (4) with the addition of a known glycolytic inhibitor, and the fluorescence intensity did not increase in (5) without addition of lactate dehydrogenase (LDH). From this, it was found that lactic acid metabolized by the glycolytic system and released to the outside of the cell could be detected, and the activity of the glycolytic system was captured as a fluorescent signal.

<Execution of glycolytic inhibitor screening (fluorescence assay 1)>
According to FIG. 5, human leukemia-derived cells (HL60 cells) are suspended in a phosphate buffer solution in which glucose is dissolved to 10 mM (4.5 × 10 ⁵ cells / mL) and placed in a 384 well plate containing the compound. Seeded and incubated at 37 ° C. for 2 hours. As a compound library, LOPAC (registered trademark) 1280 (Sigma-Aldrich) was used. Then, according to Table 6, after preparing the reaction solution and incubating at room temperature for 1 hour, the fluorescence intensity was measured with the plate reader. As the fluorescent probe, the compound 20 synthesized in Example 1 was used.

The result is shown in FIG. The assay was carried out twice, and in both cases, the compound with an inhibition rate exceeding 30% was regarded as a hit compound. This screening yielded 60 hit compounds.
The inhibition rate (InH) was calculated with a Mean _{Max response} of 1 and a Mean _{Minimum response} of 0.
・ InH = 1－ (Valuesample－Mean _{Minimum response} ) / (Mean _{Max respons} －Mean _{Minimum response} )

<Execution of glycolytic inhibitor screening (fluorescence assay 2)>
The 60 hit compounds obtained in Example 5 were confirmed to confirm that they did not inhibit the confirmation assay and the coupled assay. According to FIG. 7, HL60 cells are suspended in a phosphate buffer solution in which glucose is dissolved to 10 mM (4.5 × 10 ⁵ cells / mL), and a phosphate buffer solution in which 10 mM glucose and 200 μM lactic acid are dissolved is added. It seed | inoculated to 384 well plate and incubated at 37 degreeC for 2 hours. Then, according to Table 7, a reaction solution was prepared and seeded on a 384 well plate, incubated for 1 hour at room temperature, and then the fluorescence intensity was measured with a plate reader.

As a result, 28 compounds showing inhibition only in the plate seeded with the cell solution (that is, inhibiting glycolysis without inhibiting the coupled assay) were used as hit compounds.

<Execution of glycolytic inhibitor screening (detection by LC-MS)>
The 28 hit compounds obtained in Example 6 were confirmed to inhibit the glycolytic system by detecting lactic acid by LC-MS (liquid chromatography mass spectrometry). In addition, the assay concentration of the compound was assayed at 1, 10, and 50 μM, and the inhibition ability was examined.

Was suspended 15 mM HL60 cells glucose phosphate buffer solution prepared by dissolving ^{(6.75 × 10 5 cells / mL} ), the solution was created in accordance with Table 8. This was incubated at 37 ° C. for 2 hours and then centrifuged to collect the supernatant. An equal amount of 10% formic acid solution was added to the supernatant, and lactic acid was detected by LC-MS. Cells were incubated with 10 mM glucose and IAA 0.1 mM as Min.response, and cells were incubated with 10 mM glucose alone as Max.response.

* Buffer: Phosphate buffer pH 7.4, 1 mM CaCl ₂ , 1 mM MgCl ₂

As a result, it was confirmed by LC-MS that a compound capable of reducing lactic acid was obtained as a hit compound. This compound contained iodoacetamide, an analog of IAA, a known glycolytic inhibitor, and its inhibition rate was 0.648 ± 0.058 (n = 2) at 10 μM. It was shown that glycolytic inhibitors can be obtained by screening using the fluorescent probe of the present invention.
The inhibition rate (InH) was calculated with a Mean _{Max response} of 1 and a Mean _{Minimum response} of 0.
・ InH = 1－ (Valuesample－Mean _{Minimum response} ) / (Mean _{Max respons} －Mean _{Minimum response} )

The above results demonstrate that the fluorescent probe of the present invention can detect lactic acid produced from living cells and can screen for glycolytic inhibitors using the fluorescent probe with high throughput. It is.

Claims

A fluorescent probe for detection of NADH comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
R 1 represents one to five identical or different water-soluble substituent;
R 2 represents 1 to 5 identical or different electron withdrawing substituents;
R 3 , R 4 , R 5 and R 6 each independently represent a hydrogen atom or an alkyl group;
R 7 and R 8 are each independently a hydrogen atom, a hydroxy group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxy group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents selected from; and
R a and R b each independently represent a hydrogen atom or an alkyl group,
Here, when R 3 or R 4 is an alkyl group, together with R 7 , a ring structure containing a nitrogen atom to which they are bonded may be formed,
When R 5 or R 6 is an alkyl group, it may be combined with R 8 to form a ring structure containing the nitrogen atom to which they are attached. ].
The fluorescent probe according to claim 1, wherein R 1 represents 1 to 5 identical or different substituents independently selected from the group consisting of a sulfo group, a sulfonamide group, a carboxy group, an ester group, and an amide group. .
The fluorescent probe according to claim 1 or 2, wherein R 2 represents 1 to 5 identical or different substituents independently selected from the group consisting of a nitro group, a sulfo group, a sulfonamido group, and a cyano group.
The fluorescent probe according to any one of claims 1 to 3, wherein two R 1 are a sulfo group and R 2 is a nitro group.
The fluorescent probe according to any one of claims 1 to 4, wherein R 3 , R 4 , R 5 and R 6 are all methyl groups.
The fluorescent probe according to any one of claims 1 to 5, wherein R 7 and R 8 are both hydrogen atoms.
The fluorescent probe according to claim 1, wherein the compound represented by the formula (I) is the following compound.
A method for detecting NADH using the fluorescent probe according to any one of claims 1 to 7.
9. The detection method according to claim 8, wherein the presence of NADH is detected by observing a fluorescence response due to a reaction between NADH and the fluorescent probe.
The detection method according to claim 9, wherein the fluorescence response is a fluorescence change caused by light-induced electron transfer (PET).
Use of the fluorescent probe according to any one of claims 1 to 7 for detecting lactic acid.
A method for detecting lactic acid,
A step of adding lactate dehydrogenase (LDH) to a test subject containing lactic acid and NAD + to convert it into pyruvic acid and NADH;
A step of adding the fluorescent probe according to any one of claims 1 to 7 to the obtained test object, and reacting the NADH generated by the step with the fluorescent probe,
Observing a fluorescence response generated by the reaction, and detecting the presence of NADH from the fluorescence response, thereby detecting lactic acid present in the test sample,
The detection method comprising:
A method for screening a glycolytic inhibitor using the fluorescent probe according to any one of claims 1 to 7.
The screening method according to claim 13, wherein the glycolytic inhibitor is an anticancer agent.