JPWO2010126075A1 - Fluorescent probe for NPP detection - Google Patents

Abstract

Compounds of formula (I) useful as fluorescent probes capable of detecting NPP6 with high sensitivity and specificity [R1 and R2 are hydrogen atoms, alkyl groups, or alkoxy groups; R3 and R4 are hydrogen atoms or halogen atoms; m Is 0 or 1; L is an alkylene group having 2 to 5 carbon atoms; R5 is -N + (R6) (R7) (R8) or -N (R9) (R10) (R6 to R10 are alkyl groups).

Description

  The present invention relates to a fluorescent probe capable of specifically detecting NPP2 or NPP6 among NPP (Nucleotide Pyrophosphatase / Phosphodiesterase) subtypes.

  NPP is a general term for an ecto-type phosphodiesterase having an enzyme active site outside the cell, and seven subtypes from NPP1 to NPP7 are known to date. These enzymes act on phosphodiester and pyrophosphate linkages present in various molecules and are involved in phospholipid degradation (NPP2, NPP6, and NPP7) and nucleic acid degradation (NPP1, NPP3, NPP4, and NPP5). ing.

  Among these, NPP2 (Autotaxin) has been elucidated to be a factor involved in angiogenesis in cancer, and an inhibitor has been developed. NPP6 is highly expressed in the brain and kidney, and has a feature that it recognizes choline of glycerophosphocholine (GPC) and lysophosphatidylcholine (LPC) and exhibits phospholipase C (LPC) activity. However, there are still many unclear points regarding the functions of NPP6.

  As a means to detect NPP6 activity, pNPPC (J. Biochem., 63, 678, 1968), which is a light-absorbing substrate, is known. Has the problem of not being able to. Further, although FS-2,3 is known as a fluorescent substrate (Org. Lett., 8, 2023, 2006), this compound has low specificity and has a problem that NPP2 and NPP6 cannot be distinguished. ing. In order to further elucidate the functions of NPP2 and NPP6, it is desired to use highly sensitive fluorescent probes specific to these subtypes, but fluorescent probes having such properties have been provided in the past. There wasn't.

J. Biochem., 63, 678, 1968 Org. Lett., 8, 2023, 2006

  An object of the present invention is to provide a fluorescent probe that can detect NPP2 or NPP6 with high sensitivity and specificity.

  As a result of intensive studies to solve the above problems, the present inventors have found that the compounds represented by the following general formula (I) and general formula (II) are probes for specifically detecting NPP6 and NPP2, respectively. It was found to be extremely useful. The present invention has been completed based on the above findings.

〔式(I)中、R¹及びR²はそれぞれ独立に水素原子、C_1-6アルキル基、又はC_1-6アルコキシ基を示し、ただしR¹及びR²が同時に水素原子となることはなく、R¹及びR²の組み合わせはR¹及びR²が結合するベンゼン環の酸化電位が1.40V〜1.70Vの範囲となるように選択され；R³及びR⁴はそれぞれ独立に水素原子又はハロゲン原子を示し；mは0又は1を示し；Lは炭素数2〜5のアルキレン基を示し；R⁵は-N⁺(R⁶)(R⁷)(R⁸)又は-N(R⁹)(R¹⁰)（R⁶、R⁷、R⁸、R⁹、及びR¹⁰はそれぞれ独立にC_1-18アルキル基を示し、R⁶、R⁷、及びR⁸から選ばれる2個又は3個の基は互いに結合して環構造を形成していてもよく、R⁹及びR¹⁰は互いに結合して環構造を形成してもよい）を示し；式(II)中、R¹¹及びR¹²はそれぞれ独立に水素原子、C_1-6アルキル基、又はC_1-6アルコキシ基を示し、ただしR¹¹及びR¹²が同時に水素原子を示すことはなく、R¹¹及びR¹²の組み合わせはR¹¹及びR¹²が結合するベンゼン環の酸化電位が1.40V〜1.70Vの範囲となるように選択され；R¹³及びR¹⁴はそれぞれ独立に水素原子又はハロゲン原子を示し；nは0又は1を示し；R¹⁵はカルボキシル基で置換されていてもよいC_1-18アルキル基を示す〕で表される化合物又はその塩が提供される。That is, according to the present invention, the following general formula (I) or general formula (II):

[In the formula (I), R ¹ and R ² each independently represent a hydrogen atom, a C _1-6 alkyl group, or a C _1-6 alkoxy group, provided that R ¹ and R ² can simultaneously be hydrogen atoms. without the combination of R ¹ and R ² are selected such that the oxidation potential of the benzene ring to which R ¹ and R ² are attached is in the range of 1.40V~1.70V; R ³ and R ⁴ are each independently a hydrogen atom or M represents 0 or 1; L represents an alkylene group having 2 to 5 carbon atoms; R ⁵ represents —N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ) or —N (R ⁹ ) (R ¹⁰ ) (R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ each independently represent a C _1-18 alkyl group, and two or three selected from R ⁶ , R ⁷ , and R ⁸ ) Each group may be bonded to each other to form a ring structure, and R ⁹ and R ¹⁰ may be bonded to each other to form a ring structure); in formula (II), R ¹¹ and R ¹² each independently represent a hydrogen atom, C _1-6 alkyl group, or a C _1-6 alkoxy group, However R ¹¹ and R ¹² are never represents a hydrogen atom at the same time, selecting the combination of R ¹¹ and R ¹² are as oxidation potential of the benzene ring which R ¹¹ and R ¹² are attached is in the range of 1.40V~1.70V R ¹³ and R ¹⁴ each independently represent a hydrogen atom or a halogen atom; n represents 0 or 1; R ¹⁵ represents a C _1-18 alkyl group which may be substituted with a carboxyl group]. Or a salt thereof.

According to a preferred embodiment of the present invention, a compound represented by the above general formula (I) or a salt thereof wherein R ³ and R ⁴ are hydrogen atoms; a compound represented by the above general formula (I) wherein m is 1 Or a salt thereof; a compound represented by the above general formula (I) or a salt thereof wherein L is a propylene group or a butylene group; the above general formula wherein R ⁵ is —N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ) A compound represented by the formula (I) or a salt thereof; a compound represented by the above general formula (I) or a salt thereof, wherein R ⁶ , R ⁷ and R ⁸ are each independently a C _1-6 alkyl group; ⁶ , a compound represented by the above general formula (I) wherein R ⁷ and R ⁸ are methyl groups or a salt thereof; a compound represented by the above general formula (II) wherein R ¹³ and R ¹⁴ are hydrogen atoms; A salt thereof; and a compound represented by the above general formula (II) wherein n is 1, or a salt thereof.

According to a further preferred embodiment, the phenyl group having a combination of R ¹ and R ² or a combination of R ¹¹ and R ¹² is the following group: 2,4-diC _1-6 alkoxyphenyl group, 2-C _{1- 6} alkoxy-5-C _1-6 alkylphenyl group, 2-C _1-6 alkyl-4-C _1-6 alkoxyphenyl group, or 2-C _1-6 alkoxyphenyl group (wherein the benzene ring is xanthene A compound represented by the above general formula (I) or (II) or a salt thereof is provided, and a particularly preferred embodiment is a combination of R ¹ and R ² , or R ¹¹ And a phenyl group having a combination of R ¹² is the following group: 2,4-dimethoxyphenyl group, 2-methoxy-5-methylphenyl group, 2-methyl-4-methoxyphenyl group, or 2-methoxyphenyl group A compound represented by the above general formula (I) or (II) or a salt thereof is provided. In a most preferred embodiment, the phenyl group having a combination of R ¹ and R ² or a combination of R ¹¹ and R ¹² is represented by the above general formula (I) or (II), which is a 2-methyl-4-methoxyphenyl group. Or a salt thereof.

  From another aspect, according to the present invention, an NPP6-specific fluorescent probe containing a compound represented by the above general formula (I) or a salt thereof, and an NPP2 comprising a compound represented by the above general formula (II) or a salt thereof Specific fluorescent probes are provided.

  Further, according to the present invention, there is provided a method for measuring NPP6, the following steps: (A) reacting NPP6 with an NPP6-specific fluorescent probe containing a compound represented by the above general formula (I) or a salt thereof, and (B) a method comprising measuring the fluorescence of the probe-derived degradation product produced in the step (A); and a method for measuring NPP2, comprising the following steps: (A) in the general formula (II) A method comprising reacting NPP2 with an NPP2-specific fluorescent probe containing a compound represented by the formula or a salt thereof, and (B) measuring the fluorescence of a degradation product derived from the probe generated in the step (A). Provided.

  A method for screening an inhibitor against NPP6, comprising a step of measuring NPP6 in the presence of a test compound using the compound represented by the general formula (I) or a salt thereof; an inhibitor against NPP2 And a method comprising the step of measuring NPP2 in the presence of a test compound using the compound represented by the above general formula (II) or a salt thereof.

  Further, a method for screening for a specific inhibitor against NPP6, wherein (A) measuring NPP6 in the presence of a test compound using a compound represented by the above general formula (I) or a salt thereof, (B ) A step of measuring NPP2 in the presence of a test compound using the compound represented by the general formula (II) or a salt thereof, and (C) suppressing an increase in fluorescence intensity caused by NPP6 in the step (A). And a method for selecting a test substance that does not suppress an increase in fluorescence intensity caused by NPP2 in the step (B), and a screening method for a specific inhibitor against NPP2, comprising (A) the above general formula (I ) A step of measuring NPP6 in the presence of a test compound using a compound represented by the formula (II) or a salt thereof, (B) in the presence of a test compound using a compound represented by the above general formula (II) or a salt thereof The step of measuring NPP2 in (C) and the above step The present invention provides a method comprising the step of selecting a test substance that suppresses the increase in fluorescence intensity caused by NPP2 in (B) and does not suppress the increase in fluorescence intensity caused by NPP6 in the above step (A).

  The compounds of the present invention represented by the above general formulas (I) and (II) or salts thereof are substantially non-fluorescent or weakly fluorescent per se, and after reacting specifically with NPP6 and NPP2, respectively. It has the property of giving a strong fluorescent degradation product. Therefore, the compounds of the present invention represented by the above general formulas (I) and (II) or salts thereof are useful as specific high-sensitivity fluorescent probes for NPP6 and NPP2, respectively.

It is the figure which showed reaction of the fluorescent probe of this invention, and NPPs. (A) shows the result of TG-mPC, and (B) shows the result of TG-mPhosPr. It is the figure which showed reaction of the fluorescent probe of this invention, and NPP2 or NPP6. (A) shows the reaction with NPP6, (B) shows the reaction with NPP2, and (C) shows the reactivity ratio of NPP6 / NPP2.

  In this specification, the term alkyl group includes an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof. The same applies to a substituent having an alkyl moiety (such as an alkoxy group). Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

R ¹ and R ² each independently represent a hydrogen atom, a C _1-6 alkyl group, or a C _1-6 alkoxy group, but R ¹ and R ² are not simultaneously hydrogen atoms. Examples of the C _1-6 alkyl group represented by R ¹ and R ² include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Examples of the C _1-6 alkoxy group represented by R ¹ and R ² include, but are not limited to, a methoxy group, an ethoxy group, an n-propoxy group, and an isopropoxy group. A particularly preferred C _1-6 alkyl group is a methyl group, and a particularly preferred C _1-6 alkoxy group is a methoxy group. The same applies to R ¹¹ and R ¹² .

The combination of R ¹ and R ² are selected such oxidation potential of the benzene ring to which R ¹ and R ² are attached is in the range of 1.40V～1.70V. By selecting an oxidation potential within this range, the compound represented by the general formula (I) becomes substantially non-fluorescent or weakly fluorescent, and the degradation product after undergoing side chain hydrolysis by NPP6 Due to the strong fluorescence, the compound represented by the general formula (I) can be used as a highly sensitive fluorescent probe for NPP6. The above concept is disclosed in International Publication No. WO2005 / 24049. For example, by referring to FIG. 1 of the above International Publication, a person skilled in the art can easily understand a mother nucleus structure that can be used as a fluorescent probe. is there. All of the above international disclosures are incorporated herein by reference. The oxidation potential of the benzene ring can be easily obtained, for example, by calculating according to a quantum chemical method.

For example, a phenyl group having a combination of R ¹ and R ² is the following group: 2,4-diC _1-6 alkoxyphenyl group, 2-C _1-6 alkoxy-5-C _1-6 alkylphenyl group, 2 -C _1-6 alkyl-4-C _1-6 alkoxyphenyl group or 2-C _1-6 alkoxyphenyl group (in the above definition, the position where the benzene ring is bonded to the 9-position of the xanthene ring is the 1-position) Although it is preferable, it is not limited to these combinations. A particularly preferred combination is when the phenyl group having a combination of R ¹ and R ² is a 2-C _1-6 alkyl-4-C _1-6 alkoxyphenyl group. In these combinations, the C _1-6 alkyl is preferably a methyl group and / or the alkoxy group is preferably a methoxy group. The same applies to R ¹¹ and R ¹² .

R ³ and R ⁴ each independently represent a hydrogen atom or a halogen atom, but R ³ and R ⁴ are preferably hydrogen atoms. The same applies to R ¹³ and R ¹⁴ . m represents 0 or 1. When m is 0, it means that —O—CH ₂ — is not present. The same applies to n. m and n are preferably 1. L represents an alkylene group having 2 to 5 carbon atoms. As the alkylene group, a linear or branched alkylene group can be used, and for example, a propylene group or a butylene group is preferable.

R ⁵ represents -N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ) or -N (R ⁹ ) (R ¹⁰ ), wherein R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ each independently represent a C _1-18 alkyl group, R ^6, R ^7, and 2 or 3 groups selected from R ⁸ may be bonded to form a ring structure, R ⁹ and is R ¹⁰ may be bonded to each other to form a ring structure. The C _1-18 alkyl group represented by R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ is preferably a C _1-10 alkyl group, and more preferably a C _1-6 alkyl group. The C _1-18 alkyl group represented by R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ may have, for example, one or more substituents such as a halogen atom, a hydroxyl group, and an amino group. Good. R ⁵ is preferably —N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ).

Two or three groups selected from R ⁶ , R ⁷ and R ⁸ may be bonded to each other to form a ring structure. Examples of the ring structure include 5- to 7-membered rings, but are not limited thereto. R ⁶ , R ⁷ , and R ⁸ may be bonded to form a bicyclo ring, in which case the quaternary nitrogen atom to which R ⁶ , R ⁷ , and R ⁸ are bonded becomes a bridgehead nitrogen atom. It may be. R ⁹ and R ¹⁰ may be bonded to each other to form a ring structure. Examples of the ring structure include 5- to 7-membered rings, but are not limited thereto. The ring structure can contain one or more ring-constituting heteroatoms such as an oxygen atom, a nitrogen atom, and a sulfur atom as necessary. R ⁶ , R ⁷ , and R ⁸ are preferably the same or different C _1-4 alkyl groups, and R ⁶ , R ⁷ , and R ⁸ are preferably methyl groups. R ¹⁵ represents a C _1-18 alkyl group, and the C _1-18 alkyl group is preferably a C _1-10 alkyl group, more preferably a C _1-6 alkyl group. The C _1-18 alkyl group represented by R ¹⁵ may have a carboxyl group, and may preferably have a carboxyl group at the terminal. In addition to the carboxyl group, the C _1-18 alkyl group represented by R ¹⁵ may have one or more substituents such as a halogen atom, a hydroxyl group, and an amino group.

  The compound represented by the general formula (I) may exist in the form of an intramolecular counter ion with the phosphate moiety, or has an appropriate counter ion so as to form a quaternary ammonium salt. It may be. Examples of the counter ion include halogen ions, but are not limited thereto. The compound of the present invention represented by the above general formula (II) can also exist in the form of a base addition salt. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salts, and organic amine salts such as triethylamine salt. In addition to these, a salt with an amino acid such as glycine may be formed. The compound of the present invention represented by the above general formula (I) or (II) 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. The

  The compound of the present invention represented by the above general formula (I) or (II) may have one or two or more asymmetric carbons depending on the type of substituent, but one or two or more asymmetric carbons may be present. In addition to stereoisomers such as optically active substances based on asymmetric carbons and diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates, etc. are all within the scope of the present invention. Is included.

  The production methods of representative compounds of the compounds of the present invention represented by the above general formula (I) or (II) are shown in detail and specifically in the examples of the present specification. A person skilled in the art appropriately selects reaction raw materials, reaction conditions, reaction reagents, and the like based on the description of this example, and modifies and modifies these methods as necessary, thereby adding the above general formula (I). Alternatively, any of the compounds of the present invention represented by (II) can be produced. A method for producing a 9-substituted phenylxanthene compound used as a raw material compound (for example, Compound 3 in the Examples) is disclosed in, for example, International Publication WO2005 / 24049, so that those skilled in the art can easily obtain the raw material compound. Can do.

  As used herein, the term “measurement” should be interpreted in the broadest sense, including measurement, examination, detection, etc. performed for purposes such as quantification, qualitative or diagnostic. The method for measuring NPP6 according to the present invention generally comprises (A) a step of reacting the compound of the present invention represented by the above general formula (I) with NPP6, and (B) the above step (A). The method for measuring NPP2 according to the present invention generally comprises (A) the compound of the present invention represented by the above general formula (II) and NPP2. A step of reacting, and (B) a step of measuring fluorescence derived from the decomposition product generated in the step (A). The compound represented by the above general formula (I) or (II) is itself non-fluorescent or weakly fluorescent, but when it comes into contact with NPP6 and NPP2, respectively, the side chain is hydrolyzed and strongly fluorescent 9- A substituted phenylxanthene compound is provided.

  The fluorescence measurement means using the fluorescent probe of the present invention is not particularly limited, and a method of measuring a fluorescence spectrum in vitro, a method of measuring a fluorescence spectrum in vivo using a bioimaging technique, or the like is adopted. be able to. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method. If the fluorescent probe of the present invention is incorporated into cells by a microinjection method or the like, NPP2 or NPP6 localized in individual cells can be measured with high sensitivity in real time by a bioimaging technique. In addition, for example, a specific inhibitor for NPP2 or NPP6 can be screened using the fluorescent probe of the present invention.

  For example, measuring NPP2 or NPP6 in vitro in the presence of a test substance, and selecting a test substance that suppresses the increase in fluorescence intensity of the fluorescent probe caused by NPP2 or NPP6 in the absence of the test substance Thus, a candidate compound having an inhibitory action on NPP2 or NPP6 can be selected. Further, in this screening method, an inhibitory substance that specifically acts on NPP2 or NPP6 can be selected by selecting a test substance that exhibits inhibitory activity on either NPP2 or NPP6. Specific inhibitors for NPP2 or NPP6 selected by the above screening method of the present invention are also included in the scope of the present invention.

  The reagent of this invention may mix | blend the additive normally used for preparation of a reagent as needed, and may use it as a composition. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible. These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example. In the following examples, Me represents a methyl group.
Example 1
The compounds of the present invention were prepared according to the following scheme.

(a) Compound 2
Compound 1 (556 mg, 2.0 mmol), NaHCO ₃ (673 mg, 8.0 mmol), and tetrabutylammonium hydrogen sulfate (69 mg, 0.20 mmol) were dissolved in water and stirred at 0 ° C. for 10 minutes. After adding 10 mL of methylene chloride and stirring, chloromethyl chlorosulfate (263 μL, 2.6 mmol) dissolved in 7 mL of methylene chloride was slowly added dropwise. After vigorous stirring at 0 ° C. for 30 minutes, the mixture was returned to room temperature and stirred vigorously for 20 hours. The methylene chloride layer was dried over saturated brine and anhydrous Na ₂ SO ₄ and filtered, and then the solvent was distilled off. The residue was purified by silica gel column chromatography to obtain compound 2 (435 mg).

(b) Compound 4
2-Me-4-OMe TG (50 mg, 0.15 mmol) and Cs ₂ CO ₃ (195 mg, 0.6 mmol) were dissolved in 5 mL of dimethylformamide (DMF), and compound 2 ( 102 mg, 0.3 mmol) was slowly added dropwise. After stirring at room temperature for 13 hours, the solvent was distilled off, and the residue was dissolved in dichloromethane and washed three times with phosphate buffer (pH 9.2). The extract was washed with saturated brine, dried over anhydrous Na ₂ SO ₄ and filtered, and then the solvent was distilled off. The residue was purified by silica gel column chromatography to obtain compound 4 (40 mg).

Compound 5 (TG-mPhos)
Compound 4 (40.2 mg, 0.064 mmol) was dissolved in dichloromethane / methanol (3 mL / 7 mL), 10% palladium on carbon was added, and the mixture was vigorously stirred in a hydrogen atmosphere for 1 hour. After palladium carbon was removed by filtration, 5 mL of TEAA buffer was added, and the solvent other than water was distilled off. 2 mL of dichloromethane and chloranil (excess amount) were added to the resulting solution and stirred at room temperature for 30 minutes. The solution obtained after distilling off dichloromethane was purified by preparative HPLC and desalted to obtain Compound 5 (TG-mPhos) (6.9 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 1.30 (t, 9H, J = 7.3 Hz), 2.03 (s, 3H), 3.19 (q, 6H, J = 7.3 Hz), 3.90 (s, 3H ), 5.70 (d, 2H, J = 10.2 Hz), 6.50 (d, 1H, J = 2.2 Hz), 6.63 (dd, 1H, J = 1.5, 9.5 Hz), 6.96-7.07 (m, 2H), 7.09 -7.23 (m, 4H), 7.43 (d, 1H, J = 2.1 Hz)
¹³ C-NMR (100 MHz, CD ₃ OD): δ 187.5, 164.5, 162.4, 161.6, 156.2, 155.3, 139.1, 133.0, 131.6, 131.3, 129.6, 125.4, 119.5, 117.1, 116.8, 116.5, 112.8, 105.5, 104.3, 88.9, 88.9, 55.9, 47.7, 20.0, 9.2
LRMS (ESI ^-): 441

Compound 6 (TG-mPC)
TG-mPhos (3.0 mg, 0.0068 mmol), bromocholine bromide (3.3 mg, 0.014 mmol), and DIEA (3.5 μL, 0.0020 mmol) were added to 1 mL of DMF, then at 50 ° C under argon atmosphere for 24 hours Stir. The residue after evaporation of the solvent was purified by preparative HPLC, and desalted to obtain Compound 6 (TG-mPC) (0.6 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 2.01 (s, 3H), 3.17 (s, 9H), 3.55-3.61 (m, 2H), 4.18-4.27 (m, 2H), 5.74 (d, 2H, J = 13.2 Hz), 6.49 (d, 1H, J = 1.5 Hz), 6.63 (dd, 1H, J = 1.5, 9.5 Hz), 6.97-7.08 (m, 2H), 7.09-7.25 (m, 4H ), 7.43 (d, 1H, J = 2.2 Hz)
¹³ C-NMR (100 MHz, CD ₃ OD): δ 187.4, 164.0, 162.4, 161.5, 156.2, 155.1, 139.1, 133.1, 131.6, 131.6, 129.8, 125.3, 119.8, 117.2, 117.1, 116.2, 112.9, 105.6, 104.2, 89.2, 89.1, 67.3, 60.6, 60.6, 55.9, 54.6, 54.6, 20.0
HRMS (ESI ⁺ ): calcd for [M] ⁺ , 528.17873; found, 528.18319.

Compound 7 (TG-mPC ₃ C)
TG-mPhos (4.0 mg, 0.009 mmol), (3-bromopropyl) trimethylammonium bromide (10 mg, 0.038 mmol), and DIEA (16 μL, 0.16 mmol) were added to 1 mL of DMF, followed by 40 ° C. For 48 hours. The residue after the solvent was distilled off was purified by preparative HPLC and desalted to obtain Compound 7 (TG-mPC ₃ C) (3.6 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 2.03 (s, 3H), 2.05-2.10 (m, 2H), 3.10 (s, 9H), 3.39-3.47 (m, 2H), 3.87-3.95 ( m, 5H), 5.70 (d, 2H, J = 12.5 Hz), 6.48 (d, 1H, J = 2.0 Hz), 6.61 (dd, 1H, J = 2.0, 9.5 Hz), 7.00 (dd, 1H, J = 2.6, 8.3 Hz), 7.05 (d, 1H, J = 2.6 Hz), 7.08-7.15 (m, 2H), 7.16-7.23 (m, 2H), 7.41 (d, 1H, J = 2.4 Hz)
HRMS (ESI ⁺ ): calcd for [M] ⁺ , 542.19438; found, 542.19645.

Compound 8 (TG-mPC ₅ C)
TG-mPhos (3.1 mg, 0.007 mmol), (5-bromopentyl) triethylammonium bromide (10 mg, 0.035 mmol), and DIEA (10 μL, 0.10 mmol) were added to 1 mL of DMF, followed by 40 ° C. For 48 hours. The residue after the solvent was distilled off was purified by preparative HPLC and desalted to obtain Compound 8 (TG-mPC ₅ C) (1.2 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 1.40-1.52 (m, 2H), 1.58-1.69 (m, 2H), 1.75-1.87 (m, 2H), 2.03 (s, 3H), 3.10 ( s, 9H), 3.80-3.90 (m, 5H), 5.69 (d, 2H, J = 12.3 Hz), 6.48 (d, 1H, J = 2.0 Hz), 6.62 (dd, 1H, J = 2.0, 9.7 Hz ), 7.01 (dd, 1H), 7.05 (d, 1H), 7.08-7.15 (m, 2H), 7.16-7.22 (m, 2H), 7.41 (d, 1H, J = 2.2 Hz)
HRMS (ESI ⁺ ): calcd for [M] ⁺ , 570.22568; found, 570.22651.

Compound 9 (TG-mPdiEtNethyl)
TG-mPhos (2.5 mg, 0.0056 mmol), 2- (diethylamino) ethyl bromide hydrobromide (10 mg, 0.038 mmol), and DIEA (10 μL, 0.10 mmol) were added to 1 mL of DMF, followed by 45 Stir at 48 ° C. for 48 hours. The residue after the solvent was distilled off was purified by preparative HPLC and desalted to obtain Compound 8 (TG-mPC ₅ C) (1.2 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 1.28 (t, 6H, J = 7.1 Hz), 2.04 (s, 3H), 3.14-3.25 (m, 4H), 3.90 (s, 3H), 4.04 -4.12 (m, 2H), 5.73 (d, 2H, J = 13.0 Hz), 6.48 (d, 1H, J = 2.0 Hz), 6.61 (dd, 1H, J = 2.0, 6.7 Hz), 7.00 (dd, 1H, J = 2.4, 7.9 Hz), 7.05 (d, 1H, J = 2.4 Hz), 7.08-7.15 (m, 2H), 7.16-7.23 (m, 2H), 7.41 (d, 1H, J = 2.4 Hz )
HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 542.19438; found, 542.19047.

Compound 10 (TG-mPhosMe)
TG-mPhos (4.6 mg, 0.010 mmol), methyl iodide (0.6 μL, 0.010 mmol), and Cs ₂ CO ₃ (8.1 mg, 0.025 mmol) were added to 1 mL of DMF and then stirred at room temperature for 48 hours. . The residue after evaporation of the solvent was purified by preparative HPLC, and desalted to obtain Compound 10 (TG-mPhosMe) (0.7 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 2.03 (s, 3H), 3.51 (d, 3H, J = 11.0 Hz), 3.89 (s, 3H), 5.69 (d, 2H, J = 12.1 Hz ), 6.49 (d, 1H, J = 2.0 Hz), 6.61 (dd, 1H, J = 2.0, 9.7 Hz), 6.97-7.23 (m, 6H), 7.41 (d, 1H, J = 2.4 Hz)
^{HRMS (ESI -): calcd for} [MH] -, 455.08958; found, 455.09138.

Compound 11 (TG-mPhosPr)
TG-mPhos (3.0 mg, 0.007 mmol), propyl iodide (0.6 μL, 0.009 mmol), and Cs ₂ CO ₃ (8.1 mg, 0.025 mmol) were added to 1 mL of DMF and then stirred at room temperature for 24 hours. . The residue after evaporation of the solvent was purified by preparative HPLC, and desalted to obtain Compound 10 (TG-mPhosPr) (1.0 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 0.86 (t, 3H, J = 5.4 Hz), 1.31 (t, 9H, J = 7.3 Hz) 1.49-1.62 (m, 2H), 2.04 (s, 3H), 3.20 (q, 6H, J = 7.3 Hz), 3.73-3.78 (m, 2H), 3.90 (s, 3H), 5.71 (d, 2H, J = 9.0 Hz), 6.54 (d, 1H, J = 1.6 Hz), 6.65 (dd, 1H, J = 1.6, 7.2 Hz), 7.01 (dd, 1H, J = 1.6, 6.3 Hz), 7.06 (d, 1H, J = 1.6 Hz), 7.10-7.26 (m , 4H), 7.44 (d, 1H, J = 1.8 Hz)
¹³ C-NMR (100 MHz, CD ₃ OD): δ 186.9, 164.6, 162.5, 161.6, 156.5, 156.0, 139.1, 133.2, 131.7, 131.6, 129.4, 125.4, 119.6, 117.2, 117.1, 116.7, 112.9, 105.5, 104.2, 89.0, 88.9, 68.5, 68.5, 55.9, 47.9, 25.0, 24.9, 20.0, 10.6, 9.2
^{LRMS (ESI -): calcd for} [MH] -, 483.12088; found, 483.11941.

Compound 12
TG-mPhos (4.7 mg, 0.011 mmol), benzyl 2-bromoacetate (3.9 mg, 0.017 mmol), and Cs ₂ CO ₃ (11.4 mg, 0.035 mmol) were added to 1 mL of DMF, followed by 48 hours at room temperature. Stir. The residue after evaporation of the solvent was purified by preparative HPLC, and desalted to obtain Compound 12 (4.0 mg).

Compound 13 (TG-mPhosAcOH)
Compound 12 (4.0 mg, 0.0068 mmol) was dissolved in dichloromethane / methanol (1 mL / 1 mL), 10% palladium carbon was added, and the mixture was vigorously stirred under a hydrogen atmosphere for 3 hours. After palladium carbon was removed by filtration, 4 mL of TEAA buffer was added, and the solvent other than water was distilled off. 1 mL of dichloromethane and chloranil (excess amount) were added to the resulting solution and stirred at room temperature for 30 minutes. The residue obtained after distilling off dichloromethane was purified by preparative HPLC and desalted to obtain Compound 13 (TG-mPhosAcOH) (3.0 mg).
¹ H-NMR (300 MHz, CD ₃ OD): δ 2.03 (s, 3H), 3.89 (s, 3H), 4.25 (d, 2H, J = 8.8 Hz), 5.75 (d, 2H, J = 12.1 Hz ), 6.49 (d, 1H, J = 2.0 Hz), 6.61 (dd, 1H, J = 2.0, 9.7 Hz), 6.99 (dd, 1H, J = 2.4, 8.3 Hz), 7.04 (d, 1H, J = 2.4 Hz), 7.10-7.15 (m, 2H), 7.16-7.22 (m, 2H), 7.41 (d, 1H, J = 2.2 Hz)
^{HRMS (ESI -): calcd for} [M] -, 499.07941; found, 499.07742.

Example 2
TG-mPC is quenched by the photo-induced electron transfer (PeT) mechanism from the benzene ring perpendicular to the 9-position of the xanthene ring to the xanthene ring, but the phosphorylcholine structure that is the substrate sequence of NPP6 is It is cut out, followed by formaldehyde elimination to give highly fluorescent 2-Me-4-OMe TG.

The reactivity with NPPs was investigated using TG-mPC and TG-mPhosPr. For TG-mPC, measurement was performed 3 minutes after the start of the enzyme reaction, and for TG-mPhosPr, 27 minutes after the start of the enzyme reaction, and the ratio to the background fluorescence was obtained to obtain the value of fluorescence enhancement. The reaction was performed at 37 ° C. in 100 mM Tris-HCl buffer (pH 9.0) containing 5 mM MgCl ₂ , 500 mM NaCl, and 0.05% Triton X-100. TG-mPC showed an increase in fluorescence intensity only when reacted with NPP6, and almost no increase in fluorescence was observed with other NPPs (FIG. 1 (A)). From this result, it was shown that TG-mPC is a very specific probe for NPP6. Furthermore, TG-mPhosPr showed an increase in fluorescence intensity only when reacted with NPP2, indicating that it is a probe specific to NPP2 (FIG. 1 (B)).

Other probes were similarly reacted with NPPs, but only NPP2 and NPP6 showed reactivity. FIG. 2 shows the reactivity with NPP2 and NPP6, and the specificity for NPP6. The reaction with NPP6 (FIG. 2 (a)) and NPP2 (FIG. 2 (b)) was determined as specificity for the reactivity ratio of NPP6 / NPP2 in 3 minutes (FIG. 2 (C)). From these results, TG-mPC, TG-mPC ₃ C, TG-mPC ₅ C, and TG-mPhosdiEtNethyl are fluorescent probes specific for NPP6, and TG-mPhosPr is a fluorescent probe specific for NPP2. It is believed that there is.

Claims (12)

The following general formula (I) or general formula (II):

[In the formula (I), R ¹ and R ² each independently represent a hydrogen atom, a C _1-6 alkyl group, or a C _1-6 alkoxy group, provided that R ¹ and R ² can simultaneously be hydrogen atoms. without the combination of R ¹ and R ² are selected such that the oxidation potential of the benzene ring to which R ¹ and R ² are attached is in the range of 1.40V~1.70V; R ³ and R ⁴ are each independently a hydrogen atom or M represents 0 or 1; L represents an alkylene group having 2 to 5 carbon atoms; R ⁵ represents —N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ) or —N (R ⁹ ) (R ¹⁰ ) (R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ each independently represent a C _1-18 alkyl group, and two or three selected from R ⁶ , R ⁷ , and R ⁸ ) Each group may be bonded to each other to form a ring structure, and R ⁹ and R ¹⁰ may be bonded to each other to form a ring structure); in formula (II), R ¹¹ and R ¹² each independently represent a hydrogen atom, C _1-6 alkyl group, or a C _1-6 alkoxy group, However R ¹¹ and R ¹² are never represents a hydrogen atom at the same time, selecting the combination of R ¹¹ and R ¹² are as oxidation potential of the benzene ring which R ¹¹ and R ¹² are attached is in the range of 1.40V~1.70V R ¹³ and R ¹⁴ each independently represent a hydrogen atom or a halogen atom; n represents 0 or 1; R ¹⁵ represents a C _1-18 alkyl group which may be substituted with a carboxyl group]. Or a salt thereof. The compound represented by the above general formula (I) or a salt thereof according to claim 1, wherein R ³ and R ⁴ are hydrogen atoms, m is 1, and L is a propylene group or a butylene group. The general formula according to claim 1, wherein R ⁵ is -N ⁺ (R ⁶ ) (R ⁷ ) (R ⁸ ), and R ⁶ , R ⁷ and R ⁸ are each independently a C _1-6 alkyl group. A compound represented by the formula (I) or a salt thereof. The compound represented by the above general formula (II) or a salt thereof according to claim 1, wherein R ¹³ and R ¹⁴ are a hydrogen atom and n is 1. A phenyl group having a combination of R ¹ and R ² or a combination of R ¹¹ and R ¹² is the following group: 2,4-diC _1-6 alkoxyphenyl group, 2-C _1-6 alkoxy-5-C _{1 -6} alkylphenyl group, 2-C _1-6 alkyl-4-C _1-6 alkoxyphenyl group, or 2-C _1-6 alkoxyphenyl group (in the above definition, the position where the benzene ring is bonded to the xanthene ring is 1 The compound represented by the general formula (I) or (II) according to any one of claims 1 to 4 or a salt thereof. The general formula (I) according to any one of claims 1 to 4, wherein the phenyl group having a combination of R ¹ and R ² or a combination of R ¹¹ and R ¹² is a 2-methyl-4-methoxyphenyl group. Or a compound represented by (II) or a salt thereof. An NPP6-specific fluorescent probe comprising the compound represented by the general formula (I) according to claim 1 or a salt thereof. An NPP2-specific fluorescent probe comprising the compound represented by the general formula (II) according to claim 1 or a salt thereof. A method for measuring NPP6, comprising the following steps: (A) reacting NPP6 with an NPP6-specific fluorescent probe comprising a compound represented by the general formula (I) according to claim 1 or a salt thereof; and (B) A method comprising a step of measuring the fluorescence of the degradation product derived from the probe generated in the step (A). A method for measuring NPP2, comprising the following steps: (A) reacting NPP2 with an NPP2-specific fluorescent probe comprising a compound represented by the general formula (II) according to claim 1 or a salt thereof; and (B) A method comprising a step of measuring the fluorescence of the degradation product derived from the probe generated in the step (A). A method for screening an inhibitor against NPP6, comprising the step of measuring NPP6 in the presence of a test compound using the compound represented by the general formula (I) according to claim 1 or a salt thereof. A method for screening an inhibitor against NPP2, comprising the step of measuring NPP2 in the presence of a test compound using the compound represented by the general formula (II) according to claim 1 or a salt thereof.

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