JP2018145423A - Azapyrene compound or salt thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent dye that allow lipid metabolism into a cell and can distinction between a fatty acid metabolized to a cell membrane and a fatty acid metabolized to a lipid droplet.SOLUTION: Provided is an azapyrene compound represented by general formula (1) or a salt thereof. [in the formula, Rrepresents a hydrogen atom, a substituted or unsubstituted alkyl group, or a cyano group, Rrepresents a divalent hydrocarbon group having six or more carbon atoms, Yrepresents an oxygen atom or a sulfur atom, and Yrepresents a single bond, an oxygen atom, or a group expressed by -NH-].SELECTED DRAWING: None

Description

  The present invention relates to an azapyrene compound or a salt thereof.

  Solvatochromic fluorescent dyes are widely used for fluorescent probes because they can distinguish the polarity of the surrounding environment. However, solvatochromic fluorescent dyes that have been conceived in the past are fluorescent dyes whose absorption maximum wavelength and fluorescence maximum wavelength change depending on the polarity of the solvent, but in a solvent with a very large polarity, such as water, The yield is extremely small.

  Solvatochromic fluorescent dyes are classified into fluorescent dyes having a positive solvent effect and fluorescent dyes having a negative solvent effect. Most solvatochromic fluorescent dyes have a positive solvent effect, and as shown in the left diagram of FIG. 1, the absorption maximum wavelength and the fluorescence maximum wavelength are shifted by a longer wavelength as the polarity of the solvent increases. Conversely, a solvatochromic fluorescent dye having a negative solvent effect typically shifts the absorption maximum wavelength and the fluorescence maximum wavelength by a short wavelength as the polarity of the solvent increases, as shown in the right diagram of FIG.

  Lipid metabolism is a typical example where the molecule starts with an aqueous medium and ends with a very non-polar medium. During metabolism of long-chain fatty acid analogs by viable cells, as shown in FIG. 2, first, the uptake of exogenous long-chain fatty acids resulting from the immediate conversion of free fatty acids to the active coenzyme A (CoA) form. start from. Acyl-CoA converted from these long chain fatty acids is easily metabolized to diacyl glycerides and triacyl glycerides (DAG and TAG), phospholipids, or cholesteryl esters. Here, in TAG, ie in cells where it is preferred to preserve adipocytes, exogenous fatty acids are rapidly converted to TAG and stored in lipid droplets. For this reason, it is expected to visualize each metabolic process in the metabolism of long-chain fatty acids by fat cells using solvatochromic fluorescent dyes.

  To date, various fluorescent fatty acid analogs have been reported, some of which have been commercialized. Fluorescent dyes should be small molecules and non-polar structures because they should not disrupt the biological distribution and processing of fatty acids. For this reason, commonly used bright fluorescent dyes such as fluorescein compounds, rhodamine compounds, cyanine compounds and the like cannot be used because of cytotoxicity. Under such restrictions, BODIPY compounds and NBD compounds, which are generally neutral molecules resulting from zwitterionic structures, are known.

  BODIPY compounds to which long chain fatty acids are added (BODIPY C12 etc.) are the most widely used fluorescent dyes for visualizing lipid metabolism, and the technology has been established as a research tool. However, although BODIPY compounds (such as BODIPY C12) are taken into cells as long-chain fatty acid analogs and metabolized into cell membranes and lipid droplets, BODIPY compounds (such as BODIPY C12) are absorbed at the maximum wavelength and fluorescence depending on the polarity of the solvent. Because the maximum wavelength does not change, it is possible to distinguish between fatty acids that have been converted to phospholipids by the lipid-metabolizing enzyme system and moved to the cell membrane, and fatty acids that have formed intracellular lipid droplets after being converted to triglycerides by the lipid-metabolizing enzyme system. Can not. On the other hand, NBD compounds change the absorption maximum wavelength and the fluorescence maximum wavelength according to the polarity of the solvent, but in polar solvents such as aqueous media, the fluorescence intensity is remarkably weak and hardly fluoresces, so detecting fatty acids in the cytoplasm itself. Can not. In addition, NBD compounds are regarded as a problem that they are slow-metabolizing compounds.

  As described above, fluorescence that can distinguish between fatty acids that have been converted to phospholipids by the lipid-metabolizing enzyme system and transferred to the cell membrane, and fatty acids that have formed intracellular lipid droplets after being converted to triglycerides by the lipid-metabolizing enzyme system. There is currently no pigment. Therefore, the present invention can transfer fatty acids from the outside of the cells to the inside of the cells, or convert fatty acids into lipids such as phospholipids, diacylglycerides, and triglycerides by a lipid metabolizing enzyme system existing in the cells. Yes, it is possible to distinguish between fatty acids that have been converted to phospholipids by the lipid-metabolizing enzyme system and moved to the cell membrane, and fatty acids that have formed intracellular lipid droplets after being converted to triglycerides by the lipid-metabolizing enzyme system. The object is to provide a dye.

  As a result of intensive studies in view of the above object, the present inventors have found that a compound in which a long-chain fatty acid skeleton is introduced into a compound having a specific azapyrene skeleton is a fluorescent dye having a negative solvent effect, and is a polar substance such as an aqueous solvent. It was found that it was strongly fluorescent even in the solvent. Moreover, this compound can carry out the conversion of fatty acids from lipids into phospholipids, diacylglycerides, triglycerides, etc. by the transport of fatty acids from the outside of the cells into the cells and the lipid metabolism enzyme system present in the cells, In addition, it is possible to distinguish between fatty acids that have been converted to phospholipids by the lipid metabolizing enzyme system and transferred to the cell membrane, and fatty acids that have formed intracellular lipid droplets after being converted to triglycerides by the lipid metabolizing enzyme system. I also found it. The present invention has been completed by further research based on such knowledge. That is, the present invention includes the following configurations.

  Item 1. General formula (1):

[Wherein, R ¹ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a cyano group. R ² represents a divalent hydrocarbon group having 6 or more carbon atoms. Y ¹ represents an oxygen atom or a sulfur atom. Y ² represents a single bond, an oxygen atom or a group represented by —NH—. ]
Or an azapyrene compound represented by the formula:

Item 2. Item 2. The azapyrene compound or a salt thereof according to Item 1, wherein R ¹ is a hydrogen atom.

Item 3. Item 3. The azapyrene compound or a salt thereof according to Item 1 or 2, wherein Y ¹ is an oxygen atom.

Item 4. Item 4. The azapyrene compound or a salt thereof according to any one of Items 1 to 3, wherein Y ² is an oxygen atom.

Item 5. Item 5. The azapyrene compound or a salt thereof according to any one of Items 1 to 4, wherein R ² is a divalent chain hydrocarbon group having no branched chain.

  Item 6. Item 6. A fluorescent dye containing the azapyrene compound or a salt thereof according to any one of Items 1 to 5.

  Item 7. Item 6. A solvatochromic fluorescent dye containing the azapyrene compound or a salt thereof according to any one of Items 1 to 5.

  Item 8. Item 6. A cell stain containing the azapyrene compound or a salt thereof according to any one of Items 1 to 5.

  Item 9. Item 9. The cell stain according to Item 8, which is at least one stain selected from the group consisting of lipid droplets, cell membranes and cytoplasm.

  Item 10. Item 6. An assay kit containing the azapyrene compound or a salt thereof according to any one of Items 1 to 5.

Item 11. Comparing the fluorescence of the azapyrene compound or the salt thereof according to any one of Items 1 to 5 in the presence and absence of the test compound, and determining the presence or absence of DGAT inhibitory activity of the test compound, Screening method for anti-fat drugs.

  Since the azapyrene compound or a salt thereof of the present invention has a long-chain fatty acid skeleton, it is converted from fatty acid to phospholipid, diacyl as a fatty acid analog by transport of fatty acid from the outside of the cell to the inside of the cell or a lipid metabolizing enzyme system existing in the cell. Conversion to lipids such as glycerides and triglycerides is possible.

  Further, the azapyrene compound of the present invention or a salt thereof has a negative solvent effect and is a compound in which the polarity of the solvent is increased and the absorption maximum wavelength and the fluorescence maximum wavelength are shifted by a short wavelength, and a polar solvent such as an aqueous solvent Above all, it is strongly fluorescent. At this time, it is possible to fluoresce desired lipid droplets, cell membranes, and cytoplasm by adjusting the wavelength of the light to be irradiated. For this reason, it is possible to distinguish lipid droplets, cell membranes and cytoplasm.

It is the schematic explaining the positive solvent effect and the negative solvent effect. It is the schematic explaining the lipid metabolism in a cell. It is a schematic diagram of the pathway inhibited by Wortmannin (PI-3 inhibitor) or 2-bromooctanoic acid (DGAT inhibitor). Wortmannin (PI-3 inhibitor) inhibits fatty acid uptake itself, whereas 2-bromooctanoic acid (DGAT inhibitor) inhibits the conversion of acyl-CoA to TAG by blocking DGAT. 2 is an ultraviolet-visible absorption spectrum and a fluorescence spectrum of compound 1a in various solvents. It is a plot of the absorption maximum wavelength with respect to the orientation polarizability (Δf) of the solvent. The plot was measured in the presence (right figure) and absence (left figure) of a solvent having a hydrogen bond. Compounds showing a light resistance results in CH ₂ Cl ₂ for 1 a to 1 c. The sample was irradiated with a 300 W Xe lamp equipped with a 460 nm bandpass filter. It is the excitation spectrum (dashed line) and the fluorescence spectrum (solid line) of the compound 1a in different pH. Excitation maximum (lower side) and fluorescence maximum (upper side) of compound 1a at different pHs. It is the excitation spectrum (left figure) and fluorescence spectrum (right figure) of compound 1-C12 in aqueous buffer (phosphate, pH7.4), LUV, and soybean oil. It is the excitation spectrum (left figure) and the fluorescence spectrum (right figure) of the compound 1a, the compound 1C-6, and the compound 1-C12 in an aqueous buffer. It is the result of the light resistance test of compound 1a, BODIPY493 / 503 (left figure), and NBD-C6 (right figure). The sample was irradiated with a 300 W Xe lamp equipped with a bandpass filter of 480 nm (left figure) or 460 nm (right figure). It is a confocal microscope picture of HepG2 cells. Cells were stained with 5 μM compound 1-C12 for 1 hour. Arrows indicate lipid droplets (fat droplets). A) λ _ex = 473 nm; λ _em = 490-590 nm. B) λ _ex = 559 nm; λ _em = 570-670 nm. C) Composite image. D) Bright field. It is a confocal microscope picture of HepG2 cells. Cells were stained with 5 μM compound 1-C12 for 4 hours and then with Lipidye for 15 minutes. Arrows indicate lipid droplets (fat droplets). A) λ _ex = 473 nm; λ _em = 490-590 nm. B) λ _ex = 559 nm; λ _em = 570-670 nm. C) Bright field. D) LipiDye. λ _ex = 405 nm; λ _em = 430-530 nm. E) A composite image of A and B. It is a confocal microscope picture of the cell (EH) which performed the 30-minute pretreatment for 200 micromol phloretin of HepG2 cell, and the cell which is not performed (AD). Cells were stained with 5 μM compound 1-C12 for 1 hour. A, E) Bright field. Scale bar = 10 μm. B, F) λ _ex = 473 nm; λ _em = 490-590 nm. C, G) λ _ex = 559 nm; λ _em = 570-670 nm. D, H) Composite image. It is the result of inhibition of fatty acid metabolism of HepG2 cells by phloretin, measured by comparing the fluorescence of compound 1-C12. Images were acquired using a confocal microscope, and the fluorescence intensity of lipid droplets (fat droplets; η _ex = 559 nm) was compared. Error bars represent standard deviation (n = 20). Statistical analysis was performed using two-sided Student's t-test with unequal variance: P = 2.7 × 10 ⁻¹⁰ . It is the result of the cell viability assay using MTT reagent. HeLa cells were incubated with a predetermined concentration of compound 1-C12 for 24 hours. Error bar: Standard deviation (n = 4) It is the result of the cell viability assay using MTT reagent. HeLa cells were incubated with a predetermined concentration of Compound 1a for 24 hours. Error bar: Standard deviation (n = 4) It is a confocal microscope picture of HepG2 cells. Cells were stained with 5 μM compound 1-C6 for 1 hour. A) λ _ex = 473 nm; λ _em = 490-590 nm. B) λ _ex = 559 nm; λ _em = 570-670 nm. C) Bright field. It is a confocal microscope picture of a HepG2 cell (AD) and a HeLa cell (EH). Cells were stained with 5 μM compound 1-C12 for 2 hours and then with ER tracker red for 15 minutes. A, E) λ _ex = 473 nm; λ _em = 490-590 nm. B, F) λ _ex = 559 nm; λ _em = 570-620 nm. C, G) Composite image. D, H) Bright field. It is a result of the photobleaching property test in a living cell. It is the confocal microscope picture which dye | stained HepG2 cell with 5 micromol compound 1-C12 (AD) or BODIPY-C12 (EH). Images were taken 30 times. A, E) First time. B, F) 10th time. C, G) 30th time. D, H) Bright field. Scale bar = 10 μm. λ _ex = 473 nm; λ _em = 490-590 nm. It is a result of the comparison of the intracellular intensity | strength of FIG. The fluorescence intensity for the first image is shown. Error bars are standard deviation (n = 10). It is a confocal microscope picture of 3T3-L1 cell. The cells were stained with 5 μM compound 1-C12 and photographed at predetermined times. A) λ _ex = 473 nm; λ _em = 490-590 nm. B) λ _ex = 559 nm; λ _em = 570-670 nm. C) Composite image. It is a confocal microscope picture of 3T3-L1 cell when not treating (A to B) and not (C to D) when treated with 4 μM Wortmannin for 20 minutes. Cells were stained with 5 μM compound 1-C12 for 1 hour. A, C) λ _ex = 473 nm; λ _em = 490-590 nm. B, D) λ _ex = 559 nm; λ _em = 570-670 nm. E) Comparison of the fluorescence intensity of lipid droplets (fat droplets) (η _ex = 559 nm). Error bars are standard deviation (n = 20). Statistical analysis was performed using two-sided Student's t-test with non-uniform variance: P <2 × 10 ^-10 It is a confocal microscope picture of 3T3-L1 cells treated with 1.2 mM 2-bromooctanoic acid for 30 minutes. In the presence of 2-bromooctanoic acid, the cells were stained with 5 μM compound 1-C12 for a predetermined time. A) λ _ex = 473 nm; λ _em = 490-590 nm. B) λ _ex = 559 nm; λ _em = 570-670 nm. C) Composite image.

  In the present specification, “containing” is a concept including any of “comprise”, “consist essentially of”, and “consist of”. In the present specification, when the numerical range is represented by A to B, A to B is shown.

1． Azapyrene compounds or salts thereof The azapyrene compounds or salts thereof of the present invention are represented by the general formula (1):

[Wherein, R ¹ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a cyano group. R ² represents a divalent hydrocarbon group having 6 or more carbon atoms. Y ¹ represents an oxygen atom or a sulfur atom. Y ² represents a single bond, an oxygen atom or a group represented by —NH—. ]
Or a salt thereof. The azapyrene compound represented by the general formula (1) or a salt thereof is a novel compound not described in any literature.

In the general formula (1), as the alkyl group represented by R ¹ , both a linear alkyl group and a branched alkyl group can be employed. As the linear alkyl group, for example, a linear alkyl group having 1 to 6 (particularly 1 to 4) carbon atoms is preferable, and specifically, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, n -Pentyl group, n-hexyl group and the like. As the branched alkyl group, for example, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5 carbon atoms) is preferable. Specifically, an isopropyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, or neopentyl. Group, isohexyl group, 3-methylpentyl group and the like.

The alkyl group represented by R ¹ may have a substituent. The substituent that the alkyl group may have is not particularly limited, and examples thereof include a hydroxyl group and a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). The number of substituents in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

Among them, as R ^1, as the polarity of the solvent increases, the absorption maximum wavelength and the fluorescence maximum wavelength shift shorter, and the fluorescence of the lipid droplet, cell membrane and cytoplasm is desired more strongly in a polar solvent such as an aqueous solvent. From the viewpoint of making it possible to more strongly fluoresce these, a hydrogen atom, a halogenated alkyl group (such as a trifluoromethyl group), a cyano group, and the like are preferable, and a hydrogen atom is more preferable.

In the general formula (1), as the divalent hydrocarbon group represented by R ² , as the polarity of the solvent increases, the absorption maximum wavelength and the fluorescence maximum wavelength shift shorter, and even in polar solvents such as aqueous solvents From the viewpoint that it can fluoresce more strongly, and the desired lipid droplets, cell membranes and cytoplasm can be more strongly fluoresced, a hydrocarbon group having no branched chain (especially a divalent chain having no branched chain) Hydrocarbon group) is preferable, and examples thereof include an alkylene group, an alkenylene group, and an alkynylene group.

Examples of the alkylene group include-(CH ₂ ) ₆ -,-(CH ₂ ) ₇ -,-(CH ₂ ) ₈ -,-(CH ₂ ) ₉ -,-(CH ₂ ) ₁₀ -,-(CH ₂ ) ₁₁ -,-(CH ₂ ) ₁₂ -,-(CH ₂ ) ₁₃ -,-(CH ₂ ) ₁₄ -,-(CH ₂ ) ₁₅ -,-(CH ₂ ) ₁₆ -,-(CH ₂ ) _{17 -, - (CH 2)} 18 - carbon atoms, such as 6 to 40, and particularly alkylene groups having 8 to 30. As the alkenylene group, _{e.g., -CH = CH (CH 2)} 4 -, - CH = CH (CH 2) 5 -, - CH = CH (CH 2) 6 -, - CH = CH (CH 2) 7 - _{, -CH = CH (CH 2)} 8 -, - CH = CH (CH 2) 9 -, - CH = CH (CH 2) 10 -, - CH = CH (CH 2) 11 -, - CH = CH ( _{CH 2) 12 -, - CH} = CH (CH 2) 13 -, - CH = CH (CH 2) 14 -, - CH = CH (CH 2) 15 -, - CH = CH (CH 2) 16 - etc. And alkenylene groups having 6 to 40 carbon atoms, particularly 8 to 30 carbon atoms. The alkynylene group, for _{example, -C≡C (CH 2) 4 -} , - C≡C (CH 2) 5 -, - C≡C (CH 2) 6 -, - C≡C (CH 2) 7 - _{, -C≡C (CH 2) 8 -} , - C≡C (CH 2) 9 -, - C≡C (CH 2) 10 -, - C≡C (CH 2) 11 -, - C≡C ( _{CH 2) 12 -, - C≡C} (CH 2) 13 -, - C≡C (CH 2) 14 -, - C≡C (CH 2) 15 -, - C≡C (CH 2) 16 - etc. And an alkynylene group having 6 to 40 carbon atoms, particularly 8 to 30 carbon atoms. These divalent hydrocarbon groups may have a substituent. The substituent that the divalent hydrocarbon group may have is not particularly limited, and examples thereof include a hydroxyl group and a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). The number of substituents in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

Among these, as the divalent hydrocarbon group represented by R ² , the polarity of the solvent increases, the absorption maximum wavelength and the fluorescence maximum wavelength shift shorter, and the fluorescence is stronger in polar solvents such as aqueous solvents. An alkylene group having no branched chain is preferred from the viewpoint that the desired lipid droplet, cell membrane and cytoplasm can be more strongly fluorescent.

As Y ¹ , the polarity of the solvent increases, the absorption maximum wavelength and the fluorescence maximum wavelength shift shorter, and the fluorescence is more intense in polar solvents such as aqueous solvents, and the desired lipid droplets, cell membranes and cytoplasm are obtained. From the viewpoint of enabling more intense fluorescence, an oxygen atom is preferable.

As Y ² , the polarity of the solvent increases and the absorption maximum wavelength and the fluorescence maximum wavelength shift shorter, and the fluorescence is more intense in polar solvents such as aqueous solvents, and the desired lipid droplets, cell membranes and cytoplasm are obtained. From the viewpoint of enabling more intense fluorescence, an oxygen atom is preferable.

  As an azapyrene compound of the present invention that satisfies such conditions, for example,

Etc.

  The azapyrene compound of the present invention can also exist in the form of a salt. Examples of such salts include, for example, the general formula (1 ′):

[Wherein R ¹ , R ² , Y ¹ and Y ² are the same as defined above. ]
The salt represented by these is mentioned.

  The azapyrene compound of the present invention may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.

  Such an azapyrene compound or a salt thereof of the present invention has a long-chain fatty acid skeleton, and therefore, as a fatty acid analog, the fatty acid is transferred from the fatty acid to the cell by the transport of the fatty acid from the outside of the cell to the inside of the cell or the lipid metabolic enzyme system existing in the cell. Conversion to lipids such as lipids, diacylglycerides and triglycerides can be performed. Further, the azapyrene compound of the present invention or a salt thereof has a negative solvent effect and is a compound in which the polarity of the solvent is increased and the absorption maximum wavelength and the fluorescence maximum wavelength are shifted by a short wavelength, and a polar solvent such as an aqueous solvent Above all, it is strongly fluorescent. At this time, it is possible to fluoresce desired lipid droplets, cell membranes, and cytoplasm by adjusting the wavelength of the light to be irradiated. For this reason, it is possible to distinguish lipid droplets, cell membranes and cytoplasm.

2． Method for Producing Azapyrene Compound or its Salt The azapyrene compound or its salt of the present invention is not particularly limited. For example, an azapyrene compound (1A) in which Y ¹ and Y ² are oxygen atoms is represented by the following reaction formula 1:

[Wherein, R ¹ and R ² are the same as defined above. R ³ represents a protecting group. ]
Can be synthesized according to

In the formula, examples of the protecting group represented by R ³ include a substituted or unsubstituted alkyl group. As the alkyl group, those described above can be adopted. The types and number of substituents that the alkyl group may have are the same.

An azapyrene compound in which Y ¹ is a sulfur atom is an appropriate substrate instead of the compound represented by the general formula (3):

The azapyrene compound in which Y ² is a single bond can be synthesized by using a suitable substrate instead of the compound represented by the general formula (3):

Etc. can be synthesized.

(2-1) Compounds (2) and (3) → Compound (4)
In this step, for example, compound (2) and compound (3) can be reacted in the presence of a cobalt catalyst, a silver compound and a base in an organic solvent.

  As the compounds (2) and (3), known or commercially available compounds can be used or synthesized. Compound (2) can be synthesized according to the previous report. In the case of synthesizing compound (3), for example, reaction formula 2:

[Wherein, R ² and R ³ are the same as defined above. X ¹ and X ² are the same or different and each represents a halogen atom. ]
Can be synthesized according to For example, after reacting compound (5) and compound (6) in an organic solvent (pyridine, dichloromethane, etc.) to obtain compound (7), presence of a base (triethylamine, etc.) in organic solvent (chloroform, etc.) It can be synthesized by reacting the compound (7) with an azide compound (p-ABSA; 4-acetamidobenzenesulfonyl azide etc.) below. The amount of compound (6) used is preferably 1.5 to 3 moles per mole of compound (5), and the amount of azide compound used is preferably 0.5 to 2 moles per mole of compound (7). The amount used is preferably 1 to 2 moles relative to 1 mole of compound (7).

  In the above reaction formula 1, the amount of the compound (3) used is preferably 0.5 to 3 mol, more preferably 1 to 2 mol, relative to 1 mol of the compound (2), from the viewpoint of ease of synthesis, yield, and the like. preferable.

Cobalt catalysts include cobalt metal, halogen (chlorine, bromine, iodine, etc.), hydroxide ions, carbon monoxide (CO), 1,5-cyclooctadiene, dibenzylideneacetone, bipyridine, phenanthroline, benzonitrile. Further, cobalt catalysts in which ligands such as isocyanide, acetylacetonato, cyclopentadienyl (Cp *), and ethylenediamine are coordinated are also exemplified. In the present invention, Cp * Co (CO) I ₂ is preferable from the viewpoint of ease of synthesis, yield, and the like. These cobalt catalysts can be used alone or in combination of two or more.

  In the above reaction formula 1, the amount of cobalt catalyst used is preferably 0.01 to 0.5 mol, more preferably 0.02 to 0.2 mol, relative to 1 mol of compound (2), from the viewpoint of ease of synthesis, yield, and the like.

The silver compound is not particularly limited, and is an organic silver compound such as silver acetate, silver pivalate (AgOPiv), silver trifluoromethanesulfonate (AgOTf), silver benzoate (AgOCOPh); silver nitrate, silver fluoride, silver chloride, Inorganic silver such as silver bromide, silver iodide, silver sulfate, silver oxide, silver sulfide, silver tetrafluoroborate (AgBF ₄ ), silver hexafluorophosphate (AgPF ₆ ), silver hexafluoroantimonate (AgSbF ₆ ) Inorganic silver compounds are preferred from the viewpoint of ease of synthesis, yield, etc., and silver tetrafluoroborate (AgBF ₄ ), silver hexafluorophosphate (AgPF ₆ ), silver hexafluoroantimonate (AgSbF ₆ ) and the like are more preferable, and silver hexafluoroantimonate (AgSbF ₆ ) is more preferable. These silver compounds can be used alone or in combination of two or more.

  In the above reaction formula 1, the amount of the silver compound used is preferably 0.02 to 1 mol, more preferably 0.05 to 0.5 mol with respect to 1 mol of the compound (2), from the viewpoints of ease of synthesis, yield, and the like.

  Examples of the base include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, etc. In this step, from the viewpoint of yield and ease of synthesis, Potassium acetate is preferred. These bases can be used alone or in combination of two or more.

  In the above reaction formula 1, the amount of the base used is preferably 0.05 to 1 mol, more preferably 0.1 to 0.5 mol, relative to 1 mol of the compound (2), from the viewpoint of ease of synthesis, yield and the like.

  As the organic solvent that can be used in this step, a known one can be employed. In this step, for example, 2-fluoroethanol, 2,2′-difluoroethanol, 2,2 ′, 2 ″ -tril Fluoroalcohols such as fluoroethanol, 2,2,3,3,3-pentafluoro-1-propanol, 2,2,3,3,4,4-hexafluoro-1-butanol, 2,2,3,3 , 4,4,4-heptafluoro-1-butanol, 2,2,3,3,4,4,5,5-octafluoro-1-pentanol, 1,1,1,3,3,3- Fluorinated alcohols such as hexafluoro-2-propanol and 1,1,1,3,3,3-hexafluoro-2-phenyl-2-propanol; 2,2,3,3,4,4-hexafluoro- Fluorine-containing diols such as 1,5-pentanediol and 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol; halogenated hydrocarbons such as dichloroethane and chloroform It is done. These organic solvents can be used alone or in combination of two or more.

  The reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction atmosphere is preferably an inert gas atmosphere (nitrogen gas atmosphere, argon gas atmosphere, etc.), and the reaction temperature is 0 to 150 ° C., particularly 50 to 100. The reaction time is preferably 5 minutes to 36 hours, particularly 10 minutes to 24 hours.

  After completion of the reaction, purification may be performed according to a conventional method as necessary. Moreover, the following process can also be performed without performing a refinement | purification process.

(2-2) Oxidation reaction (compound (4) → compound (1A))
In this step, for example, an oxidation reaction can be caused to compound (4) in the above reaction formula 1 in an organic solvent.

  For the oxidation reaction, use acids such as hydrogen chloride (hydrochloric acid), sulfuric acid, formic acid, acetic acid, trifluoroacetic acid (TFA), trifluoroacetic anhydride, boron trifluoride diethyl ether complex, trifluoromethanesulfonic acid, etc. Can do. These acids can be used alone or in combination of two or more.

  In the above reaction scheme 1, the acid is preferably used in an excess amount (solvent amount) from the viewpoint of ease of synthesis, yield, and the like.

  As the organic solvent that can be used in this step, known ones can be employed. In this step, for example, halogenated hydrocarbons such as dichloromethane and chloroform; cyclic ether compounds such as tetrahydrofuran and dioxane; dimethyl sulfoxide (DMSO ) And the like. These organic solvents can be used alone or in combination of two or more.

  The reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction atmosphere is preferably an inert gas atmosphere (nitrogen gas atmosphere, argon gas atmosphere, etc.), and the reaction temperature is −50 to 100 ° C., particularly 0 to 50 ° C. is preferred, and the reaction time is preferably 5 minutes to 36 hours, particularly preferably 10 minutes to 24 hours.

  In this way, the azapyrene compound represented by the general formula (1) can be obtained, and can be used through ordinary isolation and purification steps as necessary.

  Note that, although an example of the synthesis method of one embodiment of the azapyrene compound of the present invention has been described above, the synthesis method is not limited to this production method, and the synthesis can be performed by various synthesis methods.

3． Fluorescent dye and cell staining agent The fluorescent dye of the present invention contains the azapyrene compound of the present invention or a salt thereof.

  The fluorescent dye of the present invention has a negative solvent effect in which the absorption maximum wavelength and the fluorescence maximum wavelength are shifted by a short wavelength as the polarity of the solvent increases. At this time, the fluorescence quantum yield does not change much depending on the polarity of the solvent. Therefore, while the polarity of the solvent is increased, the absorption maximum wavelength and the fluorescence maximum wavelength are shifted by a short wavelength, and it is strongly fluorescent even in a very polar solvent such as an aqueous solvent. be able to. For this reason, the fluorescent dye of the present invention is useful as a solvatochromic fluorescent dye having a negative solvent effect.

  In addition, since the fluorescent dye of the present invention has a long-chain fatty acid skeleton, the fatty acid analog can be used to transport fatty acids from the outside of the cell to the inside of the cell, or from lipids to phospholipids and diacylglycerides by a lipid metabolizing enzyme system existing in the cells. It is possible to cause conversion to lipids such as triglycerides. Thereby, it is possible to stain the lipid droplets, the cell membrane and the cytoplasm in the cells as necessary. As described above, the azapyrene compound of the present invention or a salt thereof is a compound in which the absorption maximum wavelength and the fluorescence maximum wavelength change depending on the surrounding environment. The desired fluorescence can be obtained. For example, when the light having a wavelength of 473 nm is irradiated, the azapyrene compound of the present invention or the salt thereof in the lipid droplet is hardly excited and therefore does not fluoresce. From this, when light with a wavelength of 473 nm is irradiated, it is possible to fluoresce cytoplasm and cell membrane. On the other hand, when the light having a wavelength of 559 nm is irradiated, the azapyrene compound of the present invention in the cytoplasm or a salt thereof is hardly excited and is not fluorescent, whereas the azapyrene compound of the present invention in a lipid droplet or a salt thereof is strongly fluorescent. . For this reason, when light having a wavelength of 559 nm is irradiated, fat droplets can be strongly fluorescent and cell membranes can be fluorescent. In particular, in lipid metabolism in a lipid-metabolizing enzyme system, the azapyrene compound of the present invention or a salt thereof is recognized as a fatty acid, so that it tends to be present inside the lipid droplet, and the fluorescence of the lipid droplet tends to be stronger. Thus, when the azapyrene compound of the present invention or a salt thereof is used, the lipid droplets, cytoplasm, and cell membrane in the cells can be stained and dyed as necessary. Therefore, by using the azapyrene compound or a salt thereof of the present invention, it is possible to analyze the behavior when fat cells are formed in the cells and how the fat cells change. In addition, when using a BODIPY compound (BODIPY C12, etc.) to which a long-chain fatty acid that has been used in the past is used, the absorption maximum wavelength and the fluorescence maximum wavelength do not change depending on the surrounding environment. In addition, the cell membrane cannot be dyed separately, and the above analysis cannot be performed.

Four. Assay Kit and Screening Method The assay kit of the present invention (particularly, lipid droplet, cytoplasm and cell membrane fluorescent staining kit) contains the azapyrene compound of the present invention or a salt thereof. In addition, if necessary, a plate having many (6, 24, 96, 384, etc.) wells (holes), a washing solution, a fixing solution, an instruction manual, etc. can be contained.

  As described above, the azapyrene compound of the present invention or a salt thereof can fluoresce lipid droplets strongly and fluoresce cell membranes when irradiated with light having a wavelength of 559 nm, for example. As shown in FIG. 3, the azapyrene compound of the present invention or a salt thereof can be converted to triacylglyceride (TAG) and can be prevented from being unevenly distributed inside the lipid droplets. It is possible to suppress fluorescence. For this reason, when the azapyrene compound of the present invention or a salt thereof and a DGAT inhibitor are used in combination, fluorescence derived from lipid droplets (excitation at 559 nm) is not observed, while fluorescence derived from fat (excitation at 473 nm) can be observed. . On the other hand, when a fatty acid uptake inhibitor, an acyl CoA synthesis inhibitor, or the like is used, neither cytoplasm-derived fluorescence (473 nm excitation) nor lipid droplet-derived fluorescence (559 nm excitation) is observed. By utilizing this difference, it is possible to determine whether or not the test compound has DGAT inhibitory activity.

  Therefore, if the assay kit of the present invention is used, it can be analyzed whether the test compound has DGAT inhibitory activity. Specifically, by comparing the fluorescence of the azapyrene compound of the present invention or a salt thereof in the presence and absence of the test compound and detecting the difference in the fluorescence, the presence or absence of the DGAT inhibitory activity of the test compound is determined. Can be determined. At this time, when the fluorescence derived from the lipid droplet is observed in the presence of the test compound, while the fluorescence derived from the cytoplasm can be observed, it can be determined to have DGAT inhibitory activity.

  By using this determination method, it is possible to determine whether the test compound has DGAT inhibitory activity, that is, whether it is converted to triacylglyceride (TAG) and unevenly distributed in the lipid droplets. Therefore, it is possible to screen for anti-fat drugs using this fact.

  The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

¹ H NMR spectrum and ¹³ C { ¹ H} NMR spectrum were measured using JEOL AL-400 spectrometer ( ¹ H: 400 MHz, ¹³ C: 100 MHz) or JEOL ECA 500 II spectrometer ( ¹³ C: 125 MHz) as a solvent. Measured in CDCl ₃ or DMSO-d ₆ . The chemical shift of the ¹ H NMR spectrum is expressed in δ ppm using the residual proton of the solvent as the internal standard (CHCl ₃ δ 7.26, DMSO δ 2.50), and the chemical shift of the ¹³ C NMR spectrum is expressed as the internal standard (CDCl ₃ δ 77.16). DMSO-d ₆ δ 73.78) using the solvent signal. The mass spectrum was measured by an electrospray ionization (ESI) method using a Thermo Fisher Scientific Exactive spectrometer. Thin layer chromatography (TLC) was performed on plates coated with silica gel 60F ₂₅₄ (Merck) at a thickness of 0.25 mm and visualized by UV (254 nm) or basic permanganate staining. Column chromatography was performed using neutral silica gel PSQ100B (Fuji Silysia Chemical Ltd.). Recycle preparative gel permeation chromatography (GPC) is LC-918 (Nippon Analytical Industrial Co., Ltd.) equipped with polystyrene gel columns (JAIGEL 1H and 2H, Nippon Analytical Industrial Co., Ltd.) using CHCl ₃ as the eluent. ). Anhydrous CH ₂ Cl ₂ was purchased from Kanto Chemical Co., Inc. and purified with Glass Contour Solvent Systems. Compound 1a (Angew. Chem. Int. Ed. 2015, 54, 4508.), Compound 1b (Chem. Asian J. 2015, 10, 553.), Compound 1c (Angew. Chem. Int. Ed. 2015, 54, 10975.), 6-hydroxy-tert-butylhexanoate (J. Org. Chem. 1984, 49, 2144.), 11-hydroxy-tert-butyldodecanoate (Tetrahedron 1995, 51, 10531.), and Cyclopentadienylcarbonylcobalt diiodide ([Cp * Co (CO) I ₂ ]; Adv. Synth. Catal. 2014, 356, 1491.) was synthesized according to a previous report. AgSbF ₆ and potassium acetate (KOAc) were purchased from a reagent company and stored in a glove box. Dry pyridine was purchased from Wako Pure Chemical Industries, Ltd. and stored on 4 cm of Linde molecular sieves. All other reagents were purchased from reagent companies and used as they were. Unless otherwise limited, all reactions were performed under an argon or nitrogen atmosphere.

  The 3a-azapyren-4-one skeleton was easily synthesized by a previously reported CH alkylation / cyclization cascade using a CoIII catalyst (Angew. Chem. Int. Ed. 2015, 54, 4508.) . In summary, commercially available benzo [h] quinoline and the corresponding diazomalonate tert-butyl protected long chain omega carboxylic acids (3a and 3b) are subjected to a CH activation reaction to give 3a-azapyrene-4 A -one derivative was obtained. Subsequent release of the protecting group by treatment with excess trifluoroacetic acid gave the desired products (1-C6 and 1-C12). The diazomalonate used for C—H activation was prepared in two steps from the corresponding alcohol and malonyl chloride, followed by diazotization of malonic acid. Details of each synthesis are shown below.

Reference Example 1: Compound 1a

  The compound described in a previous report (Angew. Chem. Int. Ed. 2015, 54, 4508.) was defined as Compound 1a.

Reference Example 2: Compound 1b

  The compound described in the previous report (Chem. Asian J. 2015, 10, 553.) was designated as Compound 1b.

Reference Example 3: Compound 1c

  The compound described in a previous report (Angew. Chem. Int. Ed. 2015, 54, 10975.) was defined as Compound 1c.

Synthesis Example 1 Synthesis of Compound 2a To a solution of 6-hydroxy-tert-butylhexanoate (3.10 g, 16.5 mmol) and anhydrous pyridine (1.60 mL, 19.8 mmol) in anhydrous CH ₂ Cl ₂ (50.0 mL) Malonyl chloride (730 μL, 1.06 g, 7.51 mmol) was added. The mixture was stirred at room temperature for 19 hours and quenched with water. The aqueous layer was extracted with CH ₂ Cl ₂ and the combined organic extracts were washed with saturated NH ₄ Cl solution, water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate = 10: 1) gave compound 2a as a colorless oil (1.63 g, 3.68 mmol, 49%).

Synthesis Example 2: Synthesis of Compound 2b To a solution of 12-hydroxy-tert-butyldodecanoate (4.55 g, 16.7 mmol) and anhydrous pyridine (1.45 mL, 18.0 mmol) in anhydrous CH ₂ Cl ₂ (20.0 mL) A solution of malonyl chloride (730 μL, 1.06 g, 7.51 mmol) in anhydrous CH ₂ Cl ₂ (20.0 mL) was added over 15 minutes at room temperature. The mixture was stirred at room temperature for 16 hours and quenched with water. The aqueous layer was extracted with CH ₂ Cl ₂ and the combined organic extracts were washed with water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate = 10: 1) gave compound 2b as a colorless oil (3.65 g, 5.95 mmol, 79%).

Synthesis Example 3 Synthesis of Compound 3a Suspension of Compound 2a (1.41 g, 3.18 mmol) obtained in Synthesis Example 1 and 4-acetamidobenzenesulfonyl azide (p-ABSA; 965 mg, 4.02 mmol) in acetonitrile (15.0 mL) To the solution was added triethylamine (630 μL, 4.52 mmol). The mixture was stirred at room temperature for 3 days. CH ₂ Cl ₂ was added to this suspension and filtered through a plug of celite. The filtrate was concentrated under reduced pressure, dissolved in CH ₂ Cl ₂ and washed with saturated aqueous NH ₄ Cl, water and brine. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (CH ₂ Cl ₂ ) followed by GPC (CHCl ₃ ) gave compound 3a as a yellow oil (1.40 g, 2.97 mmol, 93%). This reaction was performed in the atmosphere.

Synthesis Example 4: Synthesis of Compound 3b Suspension of Compound 2b (3.09 g, 5.04 mmol) and 4-acetamidobenzenesulfonyl azide (p-ABSA; 1.44 g, 5.60 mmol) obtained in Synthesis Example 2 in acetonitrile (15.0 mL) To the suspension was added triethylamine (1.05 mL, 7.53 mmol). The mixture was stirred at room temperature for 40 hours. To this suspension was added additional triethylamine (500 μL, 3.59 mmol) and the mixture was stirred for another 2 days. CH ₂ Cl ₂ was added to this suspension and filtered through a plug of celite. The filtrate was concentrated under reduced pressure, dissolved in CH ₂ Cl ₂ and washed with water and brine. The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate = 9: 1) gave compound 3b as a yellow oil (2.64 g, 4.13 mmol, 82%). This reaction was performed in the atmosphere.

Synthesis Example 5 Synthesis of Compound 1d AgSbF ₆ (20.6 mg, 60 μmol) and potassium acetate (KOAc; 11.8 mg, 120 μmol) were added to an oven-dried Schlenk flask under an argon atmosphere. Then, here, cyclopentadienylcarbonylcobalt diiodide ([Cp * Co (CO) I ₂ ]; 14.3 mg, 30 μmol) in trifluoroethanol (TFE; 6.0 mL), benzo [h] quinoline (109 mg, 0.608 mmol) and a solution of Compound 3a (419 mg, 0.890 mmol) obtained in Synthesis Example 3 were added. The resulting suspension was stirred at 80 ° C. for 16 hours. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ / acetone = 10: 1, then 4: 1). It was then further purified by preparative GPC (CHCl ₃ ) to give compound 1d as a red solid (97.9 mg, 0.226 mmol, 38%).

Synthesis Example 6 Synthesis of Compound 1e AgSbF ₆ (35.0 mg, 0.102 mmol) and potassium acetate (KOAc; 20.3 mg, 0.207 mmol) were added to an oven-dried Schlenk flask under an argon atmosphere. Then, here, cyclopentadienylcarbonylcobalt diiodide ([Cp * Co (CO) I ₂ ]; 23.8 mg, 50 μmol), benzo [h] quinoline (91.6 mg) in trifluoroethanol (TFE; 5.0 mL) , 0.511 mmol) and a solution of Compound 3b (478 mg, 0.749 mmol) obtained in Synthesis Example 4 were added. The resulting suspension was stirred at 80 ° C. for 16 hours. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ / acetone = 20: 1, then 4: 1). It was then further purified by preparative GPC (CHCl ₃ ) to give compound 1e as a red solid (80.1 mg, 0.155 mmol, 30%).

Comparative Example 1: Synthesis of Compound 1-C6 A solution of Compound 1d (76.2 mg, 0.176 mmol) obtained in Synthesis Example 5 in trifluoroacetic acid (TFA; 5.0 mL) and CH ₂ Cl ₂ (5.0 mL) at room temperature. Stir for 12 hours. Water was added to this solution and the organic layer was separated. The aqueous layer was extracted with CH ₂ Cl ₂ and saturated aqueous NaHCO ₃ was added to the combined organic extracts. The aqueous layer was washed with CH ₂ Cl ₂ and hydrochloric acid was added to make the aqueous layer acidic. CH ₂ Cl ₂ was added to dissolve the precipitate, and the aqueous layer was washed with CH ₂ Cl ₂ . The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting crude product was suspended in a small amount of CHCl ₃ and filtered to give compound 1-C6 as a red solid (12.3 mg, 32.6 μmol). Separately, the filtrate was concentrated under reduced pressure and suspended in a small amount of CH ₂ Cl ₂ . The suspension was filtered, and the solid was collected to obtain Compound 1-C6 separately as a red solid (22.7 mg, 60.1 μmol; total yield 53%).

Example 1: Synthesis of Compound 1-C12 A solution of Compound 1e (54.6 mg, 0.106 mmol) obtained in Synthesis Example 6 in trifluoroacetic acid (TFA; 2.0 mL) and CH ₂ Cl ₂ (2.0 mL) at room temperature. Stir for 12 hours. Water was added to this solution and the organic layer was separated. The aqueous layer was extracted with CH ₂ Cl ₂ and the combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting crude product was suspended in a small amount of CH ₂ Cl ₂ and filtered to give a red solid. The obtained solid was dried in an oven (100 ° C.) to obtain Compound 1-C12 as a red solid (24.9 mg, 53.9 μmol, 51%).

Photophysical property measurement UV-visible absorption spectra were recorded on a Shimadzu UV-3510 spectrometer with a resolution of 0.5 nm. The steady state fluorescence spectrum of the sample solution was recorded on a Hitachi F-4500 spectrometer with a resolution of 0.5 nm. A sample solution having a concentration of 10 ⁻⁵ M was placed in a 1 cm square quartz cuvette. For fluorescence measurement, the sample solution was excited at the absorption maximum wavelength of each compound. Absolute fluorescence quantum yields were measured with a Hamamatsu C9920-02 calibrated integrating sphere system equipped with a multichannel spectrometer (PMA-11).

Light resistance measurement Light resistance is light stability due to change in absorption intensity in aqueous buffer (HEPES, pH7.4, 1% DMSO co-solvent) or soybean oil (1% by Wako Pure Chemical Industries, Ltd.) Was monitored. A solution in a 1 cm square quartz cuvette was irradiated with a 300 W Xe lamp (Asahi spectrum, MAX-302) equipped with a 460 nm or 480 nm bandpass filter. The irradiation intensity was 300 Wm ^-2 . The absorbance at 460 nm (Compounds 1a to 1c and Compound NBD-C6) or 480 nm (Compound 1a and BODIPY) of each sample was adjusted to the same range.

Test Example 1: Evaluation of negative solvent effect FIG. 4 and Table 1 show the results of measurement for Compound 1a of Reference Example 1. The absorption maximum wavelength was in the order of toluene, CH ₂ Cl ₂ , CHCl ₃ , DMSO, acetonitrile, methanol, water, and the fluorescence maximum wavelength was in order of toluene, DMSO, CH ₂ Cl ₂ , CHCl ₃ , acetonitrile, methanol, water. . From this result, thorough studies of photophysical properties revealed that compound 1a exhibits a significant negative solvent effect while maintaining high fluorescence quantum yields in all solvents. The absorption maximum wavelength shifted abruptly depending on the solvent, and the fluorescence maximum wavelength was significant but more gradual change was observed. Fluorescence quantum yields and lifetimes were similar for all solvents, indicating that the lowest excited state properties were similar regardless of the solvent polarity.

  To further insight into the origin of the negative solvent effect, a series of experiments was conducted to clarify that the degree of short wavelength shift is significantly affected by both solvent polarity and hydrogen bonding. The present inventors plotted the absorption maximum in terms of wave number against the orientation polarizability (Δf) of the solvent (Principles of Fluorescence Spectroscopy, 3rd Ed. Springer, 2006.). Since Δf only considers physical parameters, ie, refractive index and dielectric constant, Δf can be regarded as the polarity of the solvent without considering chemical interaction. The results are shown in FIG. Interestingly, a good linear correlation was observed in the case of solvents without significant hydrogen bond donating ability. In the presence of a solvent having hydrogen bonds such as methanol and water, the linear correlation deviates greatly, suggesting the influence of hydrogen bonds. This suggests that depending on the solvent having hydrogen bonds such as water and methanol, the absorption maximum is further increased not only by the polarity of the solvent but also by the presence of hydrogen bonds.

Since different substituents can be introduced into the carbon atom adjacent to the carbonyl group of the azapyrene compound, the photophysical properties of Compound 1a were compared with those already reported for Compound 1b and Compound 1c. The results are shown in Table 2 and FIG. For both CH ₂ Cl ₂ and aqueous buffer, only subtle differences in emission maximum and absorption maximum wavelengths and fluorescence quantum yields were observed, so that the azapyrene compounds of the present invention or their salts likewise have negative solvent effects. It is suggested to have On the other hand, a remarkable difference was observed in the photobleaching property. Obviously, the presence of an electron-withdrawing ester group or a similar group has been suggested to greatly improve the photostability, suggesting that the azapyrene compound of the present invention or a salt thereof is also excellent in photostability. Is done.

Test example 2: PH dependence

  Since compound 1a can be represented as two canonical structures, we evaluated whether compound 1a is protonated at pH in the acidic region. When both the excitation maxima and fluorescence maxima at various physiologically relevant pHs were monitored, the pH dependence of compound 1a was negligible. The results are shown in FIGS. This result also shows that compound 1a can be represented as a zwitterionic canonical structure, but the basicity of the carbonyl group is low enough that no protonation event occurs at least within physiological pH. Such lack of pH dependence makes Compound 1a an excellent candidate for a polar probe.

Preparation of large monolayer vesicles (LUV) Large monolayer vesicles were prepared according to the procedure described previously (Biochim. Biophys. Acta 1985, 812, 55.). A dried lipid film was prepared from a chloroform solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and compound 1-C12 obtained in Example 1 in a molar ratio of 200: 1. The film was hydrated with phosphate buffer (pH 7.4) to a final lipid concentration of 1 mM and multilamellar vesicles were prepared using sonication. The obtained vesicle solution was extruded 10 times through a polycarbonate filter having a pore size of 100 nm. The LUV solution was characterized by dynamic light scattering (DLS) measurements.

Test Example 3: In order to evaluate the photophysical property polarity in the model system and the environmental response due to hydrogen bonding, the present inventors analyzed free dispersion in an aqueous medium (corresponding to the cytoplasm), and water molecules in the fluorescent dye. It was confirmed whether a membrane-bound state (corresponding to a cell membrane) that was partially accessible and an oily environment (corresponding to fat droplets) in which no water molecule was present could be distinguished. Specifically, as long-chain fatty acid analogs, Compound 1a, Compound 1-C6 and Compound 1-C12 in an aqueous buffer; large single-layer vesicles (LUV); and soybean oil fluorescence and excitation spectra were measured. The results are shown in FIGS. Interestingly, the excitation maxima of the three model systems are very different, indicating that compound 1-C12 can distinguish between cytoplasm, endoplasmic reticulum and related membranes (cell membranes), and lipid droplets (fat droplets). Since compound 1a, compound 1-C6, and compound 1-C12 had almost the same fluorescence spectrum and excitation spectrum, it was suggested that micelle aggregates were not formed. A noticeable difference was observed between the aqueous buffer and LUV, indicating that compound 1-C12 is probably incorporated into the lipid bilayer, but the fluorescent dye was surfaced because it showed a significant difference between LUV and soybean oil. May be in the vicinity. In particular, considering that the excitation maximum of LUV is similar to that of pure methanol (Principles of Fluorescence Spectroscopy, 3rd Ed. Springer, 2006.), water probably approaches the fluorescent dye, forms hydrogen bonds, and is excited. It is suggested to cause a significant short wavelength shift of the maximum wavelength. Considering all these results, it is suggested that compound 1-C12 can distinguish three different environments, the cell membrane, cytoplasm, and lipid droplets that are metabolized into cells.

  In order to ensure the applicability of the 3a-azapyren-4-one skeleton to cell imaging, the photobleaching properties of compound 1a are used as fluorescent dyes commonly used for lipid drop staining and lipid analog staining. BODIPY493 / 503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a, 4a-diaza-s-indacene) and NBD-C6 (6- (7-nitrobenzo) (Furazan-4-ylamino) hexanoic acid). The results are shown in FIG. In soybean oil, compound 1a showed excellent photostability compared to both fluorescent dyes. From the above results, it is suggested that the azapyrene compound or a salt thereof of the present invention maintains the fluorescence similarly to the compound 1a.

Cell culture
HeLa cells (RIKEN Cell Bank, Japan), HepG2 cells (RIKEN Cell Bank, Japan), and 3T3-L1 cells (JCRB Cell Bank, Japan), 10% fetal bovine serum (FBS, Gibco), antibiotics / presence The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing a mitotic inhibitor (AA; penicillin, streptomycin and amphotericin B; Wako chemical) in a 37 ° C, 5% CO ₂ /95% air incubator. Two days after the cells reached confluence, the medium was added to DMEM (10% FBS, 1% AA, 10 μg / mL insulin, 2.5 μM dexamethasone, 0.5 mM 3-isobutyl-methyl to initiate 3T3-L1 adipocyte differentiation. Xanthine) and incubated for 3 days. The culture medium was then changed to DMEM (10% FBS, 1% AA, 10 μg / mL insulin) and the medium was changed every 2 days. For other cells, the culture medium was changed every 2 or 3 days.

Cell viability To assess the cytotoxicity of compound 1-C12 on live cells, an MTT assay was performed. HeLa cells are seeded in 96-well plates and CO in DMEM containing various concentrations of Compound 1-C12 or Compound 1a (0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 15 μM, or 20 μM) for 24 hours at 37 ° C. Incubated in ₂ incubators. MTT reagent (final concentration = 0.5 mg / mL) was then added to each well and the plates were incubated for an additional hour in a CO ₂ incubator. After DMSO was added to each well to dissolve formazan, the absorbance of each well was measured with SpectraMaxi3 (Molecular Devices) at a wavelength of 600 nm (wavelength was selected to avoid interference from compound 1-C12).

Cell staining and fluorescence imaging
HepG2 cells were cultured in a single well glass bottom dish (Matsunami Glass). Prior to fluorescence imaging, HepG2 cells were treated with DMEM (10% FBS, 1%) containing 1 mM fatty acid (oleic acid / palmitate 2: 1) and 2% bovine serum albumin (essentially free of fatty acids). AA) and incubated in a CO ₂ incubator for 24 hours. The cells were rinsed with PBS (0.5% BSA) and PBS (−). Cells were then incubated with DMEM (phenol red free, 1% AA) containing 5 μM BODIPY-C12, compound 1-C6, or compound 1-C12. For inhibition experiments, cells were treated with 200 mM phloretin in medium for 30 minutes and then incubated with 5 μM compound 1-C12 in the presence of phloretin. For co-staining experiments, cells stained with Compound 1-C12 were further incubated with 1 μM Lipidye or 1 μM ER Tracker Red for 15-30 minutes.

  HeLa cells were cultured in a single well glass bottom dish (Matsunami Glass). Cells were incubated for 2 hours in DMEM (phenol red free, 1% AA) containing 5 μM compound 1-C12. The medium was rinsed with Hank's balanced salt solution without calcium and magnesium (HBSS, Wako Chemical) and the cells were incubated with 1 μM ER Tracker Red in HBSS for an additional 15 minutes.

  Fully differentiated 3T3-L1 cells (8-11 days) were cultured in single well glass bottom dishes (Matsunami Glass). Cells were incubated with DMEM (phenol red free, 1% AA) containing 5 μM compound 1-C12. For inhibition experiments, cells were treated with 1.2 mM 2-bromooctanoic acid or 4 mM Wortmannin in medium for 30 minutes and then incubated with 5 μM compound 1-C12 in the presence of inhibitors.

Test Example 4: Cell Staining Experiment In order to examine whether Compound 1-C12 acts as a fluorescent long chain fatty acid analog, Compound 1a was established as a negative solvatochromic fluorescent dye that responds significantly to the surrounding environment. Since HepG2 cells do not naturally contain lipid droplets (fat droplets) (Hepatology 2008, 47, 1905.), 1mM fatty acid (oleic acid) is used to promote the growth of lipid droplets (fat droplets). Art: Palmitate 2: 1, mixed with bovine serum albumin) for 24 hours. Screening of several conditions showed that a good signal was obtained when incubated with 5 μM compound 1-C12. When cells incubated with compound 1-C12 for 1 hour were excited with 473 nm and 559 nm lasers, different staining patterns were observed. The results are shown in FIG. When excited with a 473 nm laser, a membrane-like structure (cell membrane) was observed (no fluorescence of lipid droplets), whereas with a 559 nm laser, fluorescence from lipid droplets (lipid droplets) was found along with the membrane-like structure (cell membrane). Was also observed. Prolonging the incubation time increases the signal from the lipid droplet (fat droplet), suggesting that compound 1-C12 is slowly metabolized by HepG2 cells into the lipid droplet (fat droplet). The result is shown in FIG. Furthermore, when phloretin-treated cells were stained, the fluorescence intensity decreased significantly, suggesting that compound 1-C12 was metabolically treated. The results are shown in FIGS. This result shows that a short wavelength shift of the excitation maximum wavelength occurred when entering a more hydrophobic medium from the aqueous solution as in the above model experiment. The fact that membrane-like structures (cell membranes) can be excited by both lasers and lipid droplets (fat droplets) can only be excited by a 559 nm laser is also consistent with previous observations.

  Importantly, despite compound 1a being highly cytotoxic, compound 1-C12 was less cytotoxic. The results are shown in FIGS. The cell viability of Compound 1-C12 and Compound 1a determined by MTT assay using HeLa cells was almost non-toxic up to 20 μM for Compound 1-C12, whereas Compound 1a was significantly cytotoxic at a concentration of 1 μM Met.

  In contrast to Compound 1-C12, Compound 1-C6 did not produce appreciable intracellular fluorescence. The results are shown in FIG. When incubated with 5 μM compound 1-C6 for 1 hour, staining of HepG2 cells was negligible. This staining difference is probably due to the fact that short chain fatty acid analogs are less likely to be incorporated as natural fatty acid analogs. In fact, it is also known by comparison of fatty acid uptake kinetics with BODIPY derivatives that short acyl chains result in significantly reduced or negligible cellular uptake (J. Biol. Chem. 1983, 258, 2034. and Methods Enzymol. 2014, 538, 107.). Thus, when the hydrophilicity of compound 1-C6 is greatly enhanced due to the short alkyl chain, compound 1-C6 can be inhibited from passing through the plasma membrane.

  A series of co-staining experiments demonstrated that compound 1-C12 localizes to both the endoplasmic reticulum (ER; cell membrane) and lipid droplets (fat droplets). First, in order to confirm that the membrane-like signal observed by both excitation wavelengths was due to ER staining, a colocalization experiment with ER tracker red was performed. To minimize complications due to signals from lipid droplet-like structures, HepG2 cells without pretreatment with fatty acids were used. When HepG2 cells were incubated with compound 1-C12 for 1 hour and then incubated with ER Tracker Red for 30 minutes, good colocalization was observed. The results are shown in FIG. The laser intensity and detection wavelength were adjusted so that no significant fluorescent signal was observed from the 559 nm excitation channel in the absence of ER tracker red.

  Since HepG2 cells have highly developed ER membranes, essentially most cell compartments appear to be stained. In order to further confirm the localization of ER, co-staining experiments were performed using HeLa cells. The results are shown in FIG. As expected, compound 1-C12 in HeLa cells also showed good colocalization. From these experiments it can be concluded that an overlap of red and green signals from compound 1-C12 results from the ER.

  As mentioned above, in the case of HepG2 cells treated with fatty acids, compound 1-C12 stained a lipid droplet-like structure (fat droplet), which was only observed with excitation at 559 nm. From the result of simultaneous staining with the lipid droplet marker LipiDye, it was confirmed that a red signal was actually generated from the lipid droplet (fat droplet) (FIG. 13). Combined with the above results, it was established that Compound 1-C12 stains ER excited by 473 nm and 559 nm lasers and lipid droplets (fat droplets) excited only by 559 nm laser.

  Compared with BODIPY 500 / 510-C12 (hereinafter referred to as “BODIPY-C12”) to confirm the localization pattern of compound 1-C12 and to confirm whether compound 1-C12 is a really useful fluorescent probe Compound 1-C12 was confirmed to have good photobleaching resistance. HepG2 cells were incubated with BODIPY-C12 or compound 1-C12, and multiple images were taken with strong laser intensity (20%). The results are shown in FIGS. Comparison of intracellular fluorescence from 30 consecutive images showed that compound 1-C12 retained 76 ± 8% of the initial fluorescence and BODIPY-C12 retained only 59 ± 6% of the initial value. It was revealed. This result means that compound 1-C12 has sufficient photostability to follow fatty acid metabolism.

  To further evaluate the potential of compound 1-C12 as a fluorescent fatty acid analog, a series of experiments were performed using 3T3-L1 adipocyte-like cells, which is a simpler system compared to HepG2 cells. is there. We began by following the cellular uptake of compound 1-C12 by 3T3-L1 cells. Fully differentiated 3T3-L1 cells were treated with 5 μM compound 1-C12 and immediate uptake into membrane-like structures (presumed to be ER) was observed. The results are shown in FIG. Further incubation resulted in rapid accumulation of fluorescent signal in lipid droplets (fat droplets), suggesting that compound 1-C12 was mechanically rapidly processed into TAG in the cell. Importantly, the fluorescence signal from lipid droplets (fat droplets) is only observed upon excitation with a 559 nm laser, and compound 1-C12 is present in a very non-polar environment, with a dominant locality at the lipid droplet surface. It was shown that the presence was excluded.

Test Example 5: Compound 1-C12 as a natural lipid analog
Free fatty acid uptake by 3T3-L1 cells is known to be reduced by inhibition of PI-3 kinase (PLOS One 2015, 10, e0120289.), And similar behavior was observed for compound 1-C12. 1-C12 was confirmed to be taken up at least partially by 3T3-L1 cells via biological transport pathways. Fully differentiated 3T3-L1 cells were treated with 4 μM Wortmannin, a known PI-3 kinase inhibitor, and then incubated with compound 1-C12 for 60 minutes. The results are shown in FIG. From the results of comparison of intracellular fluorescence intensity with control cells, it is clear that treatment with Wortmannin actually decreases the uptake of compound 1-C12. In particular, the signals of both channels decrease when treated with Wortmannin, suggesting that fatty acid uptake is inhibited without metabolism. Since a similar phenomenon is observed with BODIPY-C12, the results suggest that compound 1-C12 behaves as a natural fatty acid analog.

  Different results when 3T3-L1 cells were treated with 2-bromooctanoic acid (J. Biol. Chem. 1985, 260, 6528. and Traffic 2008, 9, 338.), a TAG synthesis inhibitor (DGAT inhibitor) Was observed. Conversion of diacylglyceride (DAG) to triacylglyceride (TAG) is catalyzed by the enzyme DGAT (diacylglycerol acyltransferase), which is expressed at high levels in 3T3-L1 cells (FIG. 3). Thus, although 3T3-L1 cells rapidly convert exogenously incorporated fatty acids to TAG, inhibiting this pathway should leave a significant amount of exogenous fatty acids. In line with these expectations, incubation of cells treated with 1.2 mM 2-bromooctanoic acid significantly reduced the accumulation of compound 1-C12 in lipid droplets (fat droplets). Unlike Wortmannin, a strong signal from whole cells other than lipid droplets (fat droplets) is obtained by excitation at 473 nm, indicating that compound 1-C12 is taken up by cells and remains in a very polar medium. Show. The results are shown in FIG. Since 2-bromooctanoic acid inhibits the conversion of compound 1-C12 to the corresponding TAG, but not uptake, the observed fluorescence is probably in the cytoplasm, probably as an active coenzyme A (CoA) form There is a high possibility that the fluorescence is derived from the compound 1-C12. It can also be understood from the fact that prolonged incubation reduces the cytoplasmic signal and increases the lipid droplet signal. This is because Compound 1-C12 distinguishes between fatty acid uptake inhibitors (DGAT inhibitors) and fatty acid metabolism inhibitors, as has been observed in hexadecapentenoic acid (Traffic 2008, 9, 338.). Demonstrate what you can do.

  In conclusion, the azapyrene compounds or salts thereof of the present invention are negative solvatochromic fluorescent dyes for distinguishing local lipid environments at different excitation wavelengths. Maintaining the significant short wavelength shift and fluorescence quantum yield observed when entering a polar solvent from a non-polar solvent was essential to this achievement. The low toxicity and good photostability of the azapyrene compounds or salts thereof of the present invention can make this molecule a practical fluorescent dye. Furthermore, the azapyrene compounds or salts thereof of the present invention had the ability to distinguish cytoplasm, cell membranes and lipid droplets at different excitation wavelengths. This unique ability to distinguish the lipid environment is useful, for example, in finding and distinguishing new inhibitors for fatty acid uptake and metabolism. Finally, to the best of our knowledge, this is the first example of a fluorescent dye that takes advantage of the negative solvent effect.

Claims (11)

General formula (1):
[Wherein, R ¹ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a cyano group. R ² represents a divalent hydrocarbon group having 6 or more carbon atoms. Y ¹ represents an oxygen atom or a sulfur atom. Y ² represents a single bond, an oxygen atom or a group represented by —NH—. ]
Or an azapyrene compound represented by the formula: The azapyrene compound or a salt thereof according to claim 1, wherein R ¹ is a hydrogen atom. The azapyrene compound or a salt thereof according to claim 1 or 2, wherein Y ¹ is an oxygen atom. The azapyrene compound or a salt thereof according to claim 1, wherein Y ² is an oxygen atom. The azapyrene compound or a salt thereof according to any one of claims 1 to 4, wherein R ² is a divalent chain hydrocarbon group having no branched chain. The fluorescent pigment | dye containing the azapyrene compound or its salt in any one of Claims 1-5. The solvatochromic fluorescent pigment | dye containing the azapyrene compound or its salt in any one of Claims 1-5. A cell stain containing the azapyrene compound or a salt thereof according to any one of claims 1 to 5. The cell stain according to claim 8, which is at least one stain selected from the group consisting of lipid droplets, cell membranes and cytoplasm. The assay kit containing the azapyrene compound or its salt in any one of Claims 1-5. A step of comparing the fluorescence of the azapyrene compound or a salt thereof according to any one of claims 1 to 5 in the presence and absence of the test compound and determining the presence or absence of DGAT inhibitory activity of the test compound. , Screening method for anti-fat drugs.