WO2014142320A1 - Novel compound, and reagent for detecting lipid droplets and/or adipose tissue containing said compound - Google Patents
Novel compound, and reagent for detecting lipid droplets and/or adipose tissue containing said compound Download PDFInfo
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- WO2014142320A1 WO2014142320A1 PCT/JP2014/056973 JP2014056973W WO2014142320A1 WO 2014142320 A1 WO2014142320 A1 WO 2014142320A1 JP 2014056973 W JP2014056973 W JP 2014056973W WO 2014142320 A1 WO2014142320 A1 WO 2014142320A1
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- compound
- fluorescence
- lipid
- cells
- alkyl group
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0028—Oxazine dyes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Definitions
- the main storage site for mammalian fat is the adipocyte cytoplasm.
- fat droplets When fat cells take in fatty acids and cholesterol and produce enzymes for fat synthesis, they start to accumulate fat droplets (fat droplets). The fat droplets gradually merge and become large, and the inside of the fat cells is almost filled with one large fat droplet (Non-patent Document 1).
- the lipid droplets are monolayer droplets composed of neutral fats such as triacylglycerol synthesized from fatty acids by enzymes of the endoplasmic reticulum membrane and cholesterol esters synthesized from cholesterol.
- Fat droplets play an important role in energy balance at the individual level through accumulation, decomposition, and release of fat (Non-patent Document 2).
- the degradation of lipid droplets occurs when triacylglycerol is hydrolyzed by lipase. Fatty acids released by hydrolysis of triacylglycerol are mobilized for energy production by ⁇ -oxidation, membrane formation, lipoprotein synthesis, and the like.
- Nile Red is uncertain because the absorption wavelength region varies greatly from 450 to 560 nm due to strong solvatochromism (a phenomenon in which the maximum absorption wavelength varies depending on the polarity of the solvent). Also, in order to observe the dynamics such as the formation of lipid droplets in detail, it is often necessary to use multiple staining of lipid droplet membranes and cell nuclei in addition to lipid droplet nuclei, but Nile Red has a wide fluorescence wavelength (green, yellow , Orange, red), such multiple staining observation is difficult.
- Nile red is an off / on fluorescent reagent that has a low fluorescence intensity in an aqueous solution and a high fluorescence intensity in a hydrophobic environment, but also emits background fluorescence in cells.
- BODIPY493 / 503 is not environmentally responsive and always provides stable fluorescence around 500 nm. Further, due to its hydrophobic structure, it tends to be distributed in hydrophobic parts such as lipid droplets in the cell. Therefore, even if there is no off / on function, it can function as a lipid droplet detection reagent.
- BODIPY493 / 503 needs to perform a washing operation after fixing cells or tissues for staining. Therefore, it cannot be applied to imaging of living individuals. Therefore, there is a great demand for compounds that can specifically detect lipid droplets and / or fat cells than conventional compounds.
- the present invention has been made in view of the above points, and an object thereof is to provide a novel compound capable of specifically detecting fat droplets and / or adipose tissue.
- the present inventor can detect fat droplets and / or fat cells more specifically than the conventional fluorescent reagent for detecting lipid droplets by using the compound represented by the general formula (I).
- the present invention has been completed by finding out what can be done. That is, the gist of the present invention is as follows.
- X is a group represented by the following formula (III): (Where R 6 represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least a part of a hydrogen atom is substituted with a halogen atom, —C ( ⁇ O) —C 1-2 alkyl group, or CN; R 7 represents a hydrogen atom or NO 2 , Provided that both R 6 and R 7 are not NO 2 ).
- R 1 and R 2 each independently represent a C 1-4 alkyl group, and R 3 and R 4 each represent a hydrogen atom.
- the novel compound which can detect specifically the lipid droplet contained in a cell can be provided.
- the compound can also specifically detect adipose tissue in an individual organism. Further, since the compound maintains a fluorescence signal for a long time, the effect of the lipid droplet formation inhibitor can be observed over time, and can be used for screening for a lipid droplet formation inhibitor.
- FIG. 6 is a frequency distribution diagram of fluorescence intensity in HeLa cells (fat droplets (+)) having lipid droplets added with each compound or HeLa cells having no lipid droplets (fat droplets ( ⁇ )).
- a C 1-4 alkyl group means an alkyl group having 1 to 4 carbon atoms.
- a halogen atom is a concept including a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
- R 1 represents a C 1-4 alkyl group, or R 1 is a nitrogen atom adjacent to R 1 , R 3 , and a benzene ring bonded to the nitrogen atom and R 3 . Together with these two carbon atoms may form a 5- to 7-membered (preferably 5- to 6-membered) ring.
- R 1 is preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group, and even more preferably an ethyl group.
- R 1 3 is a hydrogen atom.
- R 3 is preferably a hydrogen atom.
- R 4 is a hydrogen atom.
- R 4 is preferably a hydrogen atom.
- X is a group represented by the following formula (II).
- Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C ( ⁇ O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5; Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is in the range of ⁇ 0.240 to ⁇ 0.190.
- each R 5 independently represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C ( ⁇ O) —C 1- It is preferably a 2 alkyl group or CN, more preferably a NO 2 , halogen atom, CF 3 , —C ( ⁇ O) —CH 3 group, or CN.
- R 5 is preferably bonded to the 2-position and / or 4-position of the benzene ring of formula (II), and more preferably bonded to the 4-position.
- n is preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
- R 6 is NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C ( ⁇ O) —C 1-2 alkyl group, Or CN, and R 7 represents a hydrogen atom or NO 2 .
- R 6 and R 7 are not both NO 2 .
- R 6 is preferably NO 2 , a halogen atom, CF 3 , C ( ⁇ O) CH 3 , or CN, more preferably NO 2 , a halogen atom, or CF 3 , and most preferably NO 2 .
- R 7 is preferably a hydrogen atom.
- X is more preferably a group selected from the group consisting of:
- X is more preferably a group selected from the group consisting of the following.
- X is even more preferably a group selected from the group consisting of:
- X is most preferably the following group.
- the second aspect of the present invention is a reagent for detecting lipid droplets and / or adipose tissue (probe) comprising the compound of the first aspect.
- the detection reagent may be composed of only the compound of the first aspect, or may be in the form of a mixture with a compound other than the compound of the present invention that can be used as a reagent for detecting lipid droplets and / or adipose tissue. Good.
- the compound other than the compound of the present invention include Nile Red, BODIPY493 / 503, LD450, Lipid Green, and SF44.
- a third aspect of the present invention is a method for detecting intracellular lipid droplets, which comprises the step of adding the compound of the first aspect to cells containing lipid droplets.
- the compound of the present invention can specifically detect lipid droplets present in cells. Therefore, it is very useful as a reagent for detecting fat droplets.
- the method for detecting lipid droplets present in cells in the present invention comprises the step of adding the compound of the first aspect to cells containing lipid droplets. Thereafter, lipid droplets contained in the cells can be detected by observing the fluorescence signal of the compound of the present invention with a fluorescence microscope or the like.
- the addition amount of the compound of the present invention or a salt thereof may vary depending on the cells to be used, the ratio of lipid droplets, and the like. can do.
- a solvent for example, dimethyl sulfoxide (DMSO) can be used as the solvent.
- DMSO dimethyl sulfoxide
- the cells to which the compound of the present invention is added are not particularly limited as long as they contain lipid droplets, and examples thereof include 3T3-L1 cells and isolated adipocytes. Moreover, you may use the cell which formed the lipid droplet artificially in the cell which does not contain a lipid droplet, or a cell with little content of a lipid droplet.
- a fourth aspect of the present invention is a method for detecting adipose tissue present in an individual living organism comprising the step of administering the compound of the first aspect into the individual living organism.
- the compound of the present invention can specifically detect adipose tissue in living organisms (in vivo). Therefore, it is very useful as a reagent for detecting adipose tissue in a living body.
- adipose tissue in a living body include subcutaneous fat, visceral fat, ectopic fat (for example, fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney).
- the method for detecting adipose tissue in a living organism individual comprises the step of administering the compound of the present invention to the individual organism. Then, by observing the fluorescence signal of the compound of the present invention using a biomolecular imaging technique using an inverted confocal microscope, the adipose tissue in the living body is detected in a living state without immobilizing the individual organism. be able to.
- Examples of the dosage form of the compound of the present invention include intravenous administration, subcutaneous administration and intramuscular administration.
- the dose of the compound of the present invention varies depending on the animal to be administered and the administration form, but is, for example, 0.01 to 1.0 ⁇ M / kg body weight, preferably 0.05 to 0.5 ⁇ M / kg body weight.
- a range of the compounds can be administered.
- DMSO can be used as the solvent.
- the individual organism to be administered is not particularly limited, and examples thereof include vertebrates and invertebrates including mammals (mouse, human, pig, dog, rabbit, etc.). Further, the administration subject may or may not include a human.
- the fifth aspect of the present invention includes a step of adding a lipid droplet formation inhibitor candidate compound to a cell containing lipid droplets, and a step of adding the compound of the present invention described in the first aspect to the cell.
- the compound of the present invention can specifically detect fat droplets and can maintain a fluorescent signal for a long time. Therefore, it can be suitably used to search for compounds and / or compositions that inhibit lipid droplet formation (ie, lipid droplet formation inhibitors).
- a lipid droplet formation inhibitor can be searched.
- the candidate compounds for lipid droplet formation inhibitors and compounds that are expected to have lipid droplet formation inhibitory activity from other experimental results may be used, and it is unknown whether they have lipid droplet formation inhibitory activity.
- Such compounds may be used.
- a novel lipid droplet formation inhibitor can be searched using various compounds contained in a commercially available compound library.
- Ts-NB having the following structural formula as a blue solid (12 mg, 0.025 mmol, 21%).
- Example 1 and Comparative Examples 1 and 2 obtained by Synthesis Examples 1 to 3 above, and commercially available compounds such as Nile Blue (NB) (Sigma Aldrich), Nile Red (Sigma Aldrich), BODIPY493 having the following structure Various tests were performed using / 503 (Invitrogen).
- the test method in each test example is as follows.
- DMEM-low glucose medium hereinafter referred to as mesenchymal stem cell expansion medium
- mesenchymal stem cell expansion medium L-glutamine containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (1 ⁇ concentration
- FBS fetal bovine serum
- penicillin / streptomycin 1 ⁇ concentration
- UEET-12 cells were cultured at 37 ° C. and 5% CO 2 concentration. Before differentiation into adipose tissue, cells were seeded at 2 ⁇ 10 4 cells / well in a 24-well plate and cultured until the cells became confluent.
- DMEM low containing 10% FBS containing adipogenesis induction medium (1 ⁇ M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 10 ⁇ M insulin).
- the medium was replaced with a glucose medium) and cultured for 7 days while changing the medium every two days.
- the lipogenesis-introducing medium was replaced with a lipogenesis maintenance medium (DMEM low glucose medium containing 10% FBS containing 10 ⁇ M insulin), and further cultured for 2 days.
- the medium was again replaced with an adipogenic medium and cultured for 6 days (medium was changed every 2 days). Thereafter, the medium was replaced with an adipogenesis maintenance medium, and further cultured for 2 days.
- NB, MNs-NB, Nile Red, and BODIPY493 / 503 reagents As a negative control, undifferentiated cells were used.
- mice Male C57BL / 6J mice were obtained from Charles River Japan. All mice were raised under a 12 hour light-dark cycle and were ready to eat. Mice were fed a standard chow diet (6% fat, Oriental Yeast) or a high fat diet (D12492, 60 Kcal% fat, Research Diets). All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly followed the University of Tokyo animal experiment guidelines.
- mice were anesthetized by injection of urethane (1.5 g / kg). After a moderate depth of anesthesia was obtained, a small incision was made and moistened with sarin. The window was then covered with wrap. In vivo imaging was performed through a small window (3 mm or less) without taking the tissue out of the body. A heating pad was used to maintain a body temperature of 37 ° C.
- FITC-dextran 5 mg / kg body weight, molecular weight 150,000, Sigma was injected into mice via the tail vein to visualize blood cells.
- Hoechst 33342 (1 mM / kg body weight, Invitrogen) was injected to visualize the nuclei in vivo. Imaging was performed 15 minutes after injection.
- MNs-NB dissolved in DMSO was injected (0.1 uM / kg body weight). Image with high time and spatial resolution (160 nm / pixel) using an inverted microscope (Ti, Nikon) provided for resonance-scanning confocal microscopy (Nikon A1R) Got.
- Stromal blood vessel (SV) cells have been isolated using a slightly modified method described in the past (Am J Physiol Cell Physiol December 2006 vol. 291 no. 6 C1232-C1239). After systemic heparinization, mice were sacrificed under general anesthesia conditions. Thereafter, epididymis and subcutaneous adipose tissue were removed, chopped into small pieces, and cultured in collagenase solution (2 mg / ml collagenase type 2 [Worthington] in Tyrode buffer) for 20 minutes with gentle agitation.
- collagenase solution 2 mg / ml collagenase type 2 [Worthington] in Tyrode buffer
- the digested tissue was centrifuged, and the resulting pellet containing the SV fraction was resuspended in PBS and then filtered through a 70 ⁇ m mesh filter. Thereafter, the collected cells were washed twice with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The cells were cultured in a standard adipogenic mixture containing dexamethasone, IBMX (3-isobutyl-1-methylxanthine) and insulin. In this state, in order to observe accumulation of fat droplets, observation was performed using a confocal microscope.
- DMEM Dulbecco's modified Eagle's medium
- the lipid reagent for detecting lipid droplets enhances fluorescence with chloroform having a low dielectric constant, and exhibits high specificity when it does not enhance fluorescence with other solvents such as methanol having a high dielectric constant. This was used as a guideline when evaluating the off / on function of the reagent.
- logP of the lipid droplet detection reagent reported so far was calculated (the left column of Table 2). As a result, LD450 was the highest, showing 4.38, and LipidGreen was the lowest, showing 2.92.
- logP of Nile Blue (NB), DNs-NB, MNs-NB and Ts-NB was calculated, Nile Blue showed 3.2, whereas derivatives DNs-NB, MNs- NB and Ts-NB were 5.95, 5.99, 5.13 and higher values than the conventional reagents, respectively (Table 2 right column).
- the calculation result of the log P value supported that these NB derivatives have high distribution specificity to lipid droplets.
- the molar extinction coefficient of the maximum absorption wavelength of each reagent was calculated (Table 3).
- MNs-NB showed the lowest value.
- the value of the reagent under chloroform solvent was higher than that of methanol. This is expected to be due to the presence of a plurality of equilibrium structures in the ground state of each reagent in the hydrophilic solution. In highly polar methanol, it is advantageous to dissolve the cationic equilibrium structure.
- Each reagent shows a blue shift in a highly hydrophobic chloroform solution, and is particularly remarkable in MNs-NB and Ts-NB (FIG. 2).
- the blue shift of the hydrophobic solvent indicates that the dipole transition moment of the excited state structure is greater than the ground state structure. This is expected because NB is in a ⁇ - ⁇ excited state.
- FIG. 4 outlines the relationship between absorption, emission and energy in fluorescence emission.
- the molecule (S 0 ) in the ground state undergoes an electronic transition due to light absorption (hv), and changes to an excited state (S 1 ) in a time of about 10 ⁇ 15 seconds. Thereafter, fluorescence emission occurs when the molecule in the excited state returns to the ground state.
- the fluorescence intensity per unit concentration obtained in the fluorescence spectrum is proportional to the molar extinction coefficient ( ⁇ ) and the fluorescence quantum yield ( ⁇ f ) (fluorescence intensity ⁇ molar extinction coefficient ( ⁇ ) ⁇ fluorescence quantum yield ( ⁇ f ) ). This fluorescence intensity corresponds to the sensitivity of a so-called fluorescent compound.
- the fluorescence quantum yield ( ⁇ f ) is expressed in the relational expression (1) between the radiation relaxation rate constant (k r ) and the non-radiation relaxation rate constant (k nr ). That is, the fluorescence quantum yield increases as the radiation relaxation rate constant increases and as the non-radiation relaxation rate constant decreases. Furthermore, the fluorescence lifetime ( ⁇ f ) that can be measured in an experiment has a relationship of the radiation relaxation rate constant (k f ), the non-radiation relaxation rate constant (k nr ), and the equation (2).
- the fluorescence quantum yield of MNs-NB decreases with chloroform and methanol to 0.21 and 0.03, while its radiation relaxation rate constant also decreases to 0.055 and 0.030.
- the non-radiative relaxation rate constant increases to 0.203 and 0.960.
- MNs-NB has a dominant route of non-radiative relaxation in methanol. Since DNs-NB could not measure the fluorescence lifetime ( ⁇ f ) in all organic solvents, k r and k nr could not be calculated. However, the molar extinction coefficient indicates high excitation efficiency. Therefore, it is clear that DNs-NB returns to the ground state through the non-radiative relaxation process with high probability from the excited state.
- an NB reagent that gives a long-wavelength fluorescence signal is more suitable for biological imaging.
- the ratio of luminance in chloroform to water was defined as the S / B (signal / background) ratio. This allows numerical evaluation of the off / on function that enhances fluorescence in a hydrophobic environment.
- the luminance value was calculated by integrating the molar extinction coefficient ( ⁇ ) value and the fluorescence quantum yield ( ⁇ f ) value.
- the flow cytometer does not provide localization information, but enables high-speed cell analysis of tens of thousands or more, and gives fluorescence intensity for each cell unit as a frequency distribution. Analysis by a flow cytometer gives all signals in the cell as a single fluorescence intensity, and therefore requires a highly specific detection reagent.
- HeLa cells fat droplets (+)
- normal HeLa cells without lipid droplets fat droplets (-)
- lipid droplet detection reagent As the lipid droplet detection reagent, NB, MNs-NB, Ts-NB, Nile Red and BODIPY493 / 503 were used. After adding 0.5 ⁇ M reagent to HeLa cells and incubating at 37 ° C. for 15 minutes, the fluorescence intensity of each reagent was measured with a flow cytometer. FIG. 7 shows the frequency distribution obtained as a result of the analysis.
- MNs-NB When comparing the distribution of lipid droplet present cells and non-existing cells, the most dominant difference in fluorescence intensity distribution was observed in MNs-NB. In addition, almost no significant distribution difference was observed between NB and Ts-NB. On the other hand, a significant distribution difference was observed in BODIPY493 / 503, but no significant difference was observed in Nile Red.
- the median fluorescence intensity was calculated from the frequency distribution (Table 8). The ratio of the median values in the presence and absence of lipid droplets (specificity: lipid droplet (+) / lipid droplet ( ⁇ )) is an indicator of the specificity of the reagent. Among the reagents, MNs-NB gave the highest specificity 1.94. As described above, from the results of imaging analysis using a fluorescence microscope and relative quantity analysis using a flow cytometer, MNs-NB was confirmed to be the reagent with the highest lipid droplet specificity.
- the bone marrow-derived human mesenchymal cell line UEET-12 cell line is known to differentiate into various cells. Treatment of the cells with dexamethasone and insulin differentiates into adipocytes. In this case, when it differentiates into fat cells, lipid droplets are generated inside. Therefore, it is possible to evaluate the process of UEET-12 cell line differentiation into adipocytes by imaging lipid droplets. Thus, it was verified using a fluorescence microscope whether differentiation of UEET-12 into adipocytes could be identified using the MNs-NB reagent.
- UEET-12 was cultured for 14 days (subculture every 2 days) using DMEM (10% FBS) supplemented with dexamethasone and insulin to induce differentiation into adipocytes (UEET-12 (+)) .
- DMEM 10% FBS
- UEET-12 (+) UEET-12 cultured in DMEM (10% FBS) to which nothing was added was also prepared (UEET-12 ( ⁇ )).
- Various reagents of 5 ⁇ M were added to UEET-12 (+) and incubated at 37 ° C. for 15 minutes, and then fluorescence signals were observed with a fluorescence microscope (FIG. 8).
- NB did not give a specific signal in the comparison of UEET-12 (+) and UEET-12 (-).
- Nile red and BODIPY493 / 503 also showed a slight non-specific fluorescence signal in UEET-12 (+) and UEET-12 ( ⁇ ).
- UEET-12 (+) the fluorescence signals from other than lipid droplets are observed in any of the reagents, so it can be said that the specificity is lower than that of MNs-NB. From the above results, it was revealed that MNs-NB is an excellent reagent for detecting lipid droplets.
- Triaxin C (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the inhibitor. Triaccin C is a fungal metabolite and has a mechanism of inhibiting lipid droplet formation by inhibiting the synthesis process of triacylglycerol.
- fluorescence imaging was analyzed over time (FIG. 11). As a result, it was confirmed that the fluorescence signal in the cell was attenuated every time after 4 hours and after 24 hours. From this basic experiment, it was confirmed that high-throughput screening (HTS) of inhibitors at the cellular level was possible by using MNs-NB.
- HTS high-throughput screening
- Adipose tissue imaging of living mouse individuals is the first example in the world.
- lean mice and obese mice with a high fat diet were prepared.
- Each mouse's vein was injected with MNs-NB (DMSO solution) in an amount of 0.1 ⁇ M / kg body weight.
- MNs-NB DMSO solution
- FIG. 12 shows a skeletal muscle tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed.
- FIG. 12 shows a liver tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice.
- MNs-NB succeeded in the world's first adipose tissue imaging in living mice. It was revealed that MNs-NB is a practical reagent that exhibits tissue permeability and membrane permeability even in animal individuals and can be stained and imaged specifically for adipose tissue.
- FIG. 13 shows a visceral fat image. In obese mice, fat cell hypertrophy occurred.
- FIG. 13 shows a heart image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice. From this, by using MNs-NB, the deposition of ectopic fat in the heart, which is not seen in the lean type, was visualized by obese mice.
- a novel compound capable of detecting fat droplets and / or adipose tissue can be provided. Therefore, the compound of this invention is very useful industrially.
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Abstract
Provided is a compound represented by general formula (I) and capable of detecting lipid droplets and/or adipose tissue. (In formula (I), R1 represents a C1-4 alkyl group, R2 represents a C1-4 alkyl group, and X is a group represented by formula (II), where R5's each independently represent NO2, a halogen atom, a C1-4 alkyl group in which at least one hydrogen atom has been substituted by a halogen atom, a -C(=O)-C1-4 alkyl group, or CN, and n represents an integer of 1-5, with the proviso that the electron density of carbon atoms at position 1 of the benzene ring in formula (II) is within the -0.240 to -0.190 range)).
Description
本発明は、新規化合物並びに該化合物を含む脂肪滴及び／又は脂肪組織検出用試薬に関する。
The present invention relates to a novel compound and a reagent for detecting lipid droplets and / or adipose tissue containing the compound.
脂肪は、生命活動に必要なエネルギー源になると同時に、体を構築する材料として重要な役割を果たしている。哺乳動物の脂肪の主な貯蔵部位は、脂肪細胞の細胞質である。脂肪細胞は、脂肪酸やコレステロールを取り込み、脂肪合成のための酵素類を生産するようになると、脂肪滴（脂肪の液滴）を蓄積し始める。脂肪滴は、徐々に融合して大きくなり、脂肪細胞の内部は、一個の大きな脂肪滴でほとんど埋め尽くされている（非特許文献１）。脂肪滴は、小胞体膜の酵素によって脂肪酸から合成されるトリアシルグリセロールや、コレステロールから合成されるコレステロールエステルなど中性脂肪から成る単分層の小滴である。
Fats play an important role as a material for building the body as well as an energy source necessary for life activities. The main storage site for mammalian fat is the adipocyte cytoplasm. When fat cells take in fatty acids and cholesterol and produce enzymes for fat synthesis, they start to accumulate fat droplets (fat droplets). The fat droplets gradually merge and become large, and the inside of the fat cells is almost filled with one large fat droplet (Non-patent Document 1). The lipid droplets are monolayer droplets composed of neutral fats such as triacylglycerol synthesized from fatty acids by enzymes of the endoplasmic reticulum membrane and cholesterol esters synthesized from cholesterol.
脂肪滴は、脂肪の蓄積・分解・放出を通じて個体レベルでのエネルギーバランスに重要な役割を担っている（非特許文献２）。脂肪滴の分解は、トリアシルグリセロールがリパーゼにより加水分解を受けることによって起こる。トリアシルグリセロールの加水分解で遊離した脂肪酸は、β酸化によるエネルギー生産、膜新生、リポタンパク質合成などに動員される。
Fat droplets play an important role in energy balance at the individual level through accumulation, decomposition, and release of fat (Non-patent Document 2). The degradation of lipid droplets occurs when triacylglycerol is hydrolyzed by lipase. Fatty acids released by hydrolysis of triacylglycerol are mobilized for energy production by β-oxidation, membrane formation, lipoprotein synthesis, and the like.
自然環境に生息する動物は、得られる食べ物が多様で予測ができない状態と折り合いをつけなければならない。したがって、脂肪細胞は、環境変化によって脂肪の貯蓄・放出をする。脂肪組織に求められるのは、一生を通じ、栄養分の供給量に応じて組織の量を加減する機能である。この機能は我々の祖先にとっては、極めて重要であった。しかしながら、現在の食事事情に恵まれた先進国では、肥満の原因として問題になっている。肥満はメタボリックシンドロームの最大の危険因子である。肥満の二次的な結果として、あるいはそれ以外の原因で、脂肪組織以外の組織に脂肪が異常蓄積すると、それらの組織の機能不全を招くとともに、糖尿病や動脈硬化などの発症につながる。また、脂肪滴は、Ｃ型肝炎ウイルスなどの複製、クラミジアなどの増殖に関与している。脂肪滴の形成・成長・分解のメカニズムが詳細に解明されることによって、これらの疾患の診断・治療に寄与する新たな研究展開が期待されることから、現在も、細胞レベルから個体レベルにわたって脂肪滴研究が、精力的に展開されている。そのため、生きた細胞内や個体内の脂肪滴の動態を観察できる分子プローブの開発は重要である。
動物 Animals that inhabit the natural environment must come to terms with a variety of foods that cannot be predicted. Therefore, fat cells store and release fat according to environmental changes. What is required of adipose tissue is the function of adjusting the amount of tissue according to the supply of nutrients throughout life. This function was extremely important for our ancestors. However, it is a problem as a cause of obesity in developed countries that are blessed with current dietary conditions. Obesity is the greatest risk factor for metabolic syndrome. As a secondary result of obesity or for other reasons, abnormal accumulation of fat in tissues other than adipose tissue leads to dysfunction of those tissues and leads to the development of diabetes, arteriosclerosis and the like. In addition, lipid droplets are involved in replication such as hepatitis C virus and proliferation such as chlamydia. As the mechanism of lipid droplet formation / growth / degradation is elucidated in detail, new research development that contributes to the diagnosis and treatment of these diseases is expected. Drop studies are being vigorously developed. Therefore, it is important to develop molecular probes that can observe the dynamics of lipid droplets in living cells and individuals.
蛍光イメージング法は、生きた細胞内や個体内の脂肪滴の動態を観察するために、広く用いられる重要なツールとなっている。脂肪滴は機能的および形態学的に多様性のある亜集団を形成するので、すべての脂肪滴を染色するための単一のマーカーはない。それゆえ、脂肪滴を視覚化するための信頼できる唯一の方法は脂肪親和性の強い分子で脂肪滴の核部分（脂質エステル）を染色することである。現在汎用されている脂肪滴検出用試薬（脂肪滴検出用プローブ）としては、ナイルレッドおよびＢＯＤＩＰＹ４９３／５０３等が挙げられる（非特許文献３～６）。
Fluorescence imaging is an important tool that is widely used to observe the dynamics of lipid droplets in living cells and individuals. Since lipid droplets form functionally and morphologically diverse subpopulations, there is no single marker for staining all lipid droplets. Therefore, the only reliable way to visualize lipid droplets is to stain the lipid droplet core (lipid ester) with a lipophilic molecule. Examples of lipid droplet detection reagents (fat droplet detection probes) that are currently widely used include Nile Red and BODIPY493 / 503 (Non-Patent Documents 3 to 6).
しかしながら、ナイルレッドは、強いソルバトクロミズム（溶媒の極性に応じて最大吸収波長が異なる現象）により吸収波長領域が４５０～５６０ｎｍと大きく変化するため不確定になる。また、脂肪滴の形成等、動態を詳細に観察するために、しばしば脂肪滴核以外にも脂肪滴膜や細胞核等の多重染色を用いる必要があるが、ナイルレッドは広い蛍光波長（グリーン、イエロー、オレンジ、レッド）をもつため、このような多重染色観察が難しい。また、ナイルレッドは、水溶液下において蛍光強度が低く、疎水性環境下において高い蛍光強度を示すオフ／オン型蛍光試薬であるが、細胞内でバックグラウンド蛍光を発することも問題となる。
一方、ＢＯＤＩＰＹ４９３／５０３は、ナイルレッドと異なり、環境応答性ではなく、常に約５００ｎｍ付近に安定した蛍光を与える。また、その疎水性構造のため細胞内において脂肪滴などの疎水性部分に分布しやすい。そのため、オフ／オン機能がなくても脂肪滴検出試薬としての機能を果たすことができる。しかしながら、一般的に、ＢＯＤＩＰＹ４９３／５０３は、その染色のために、細胞或いは組織を固定し洗浄操作を行う必要がある。そのため、生きた個体のイメージング等に応用できない。
従って、従来の化合物よりも脂肪滴及び／又は脂肪細胞を特異的に検出することのできる化合物に対する大きな需要が存在する。 However, Nile Red is uncertain because the absorption wavelength region varies greatly from 450 to 560 nm due to strong solvatochromism (a phenomenon in which the maximum absorption wavelength varies depending on the polarity of the solvent). Also, in order to observe the dynamics such as the formation of lipid droplets in detail, it is often necessary to use multiple staining of lipid droplet membranes and cell nuclei in addition to lipid droplet nuclei, but Nile Red has a wide fluorescence wavelength (green, yellow , Orange, red), such multiple staining observation is difficult. Nile red is an off / on fluorescent reagent that has a low fluorescence intensity in an aqueous solution and a high fluorescence intensity in a hydrophobic environment, but also emits background fluorescence in cells.
On the other hand, unlike Nile Red, BODIPY493 / 503 is not environmentally responsive and always provides stable fluorescence around 500 nm. Further, due to its hydrophobic structure, it tends to be distributed in hydrophobic parts such as lipid droplets in the cell. Therefore, even if there is no off / on function, it can function as a lipid droplet detection reagent. However, in general, BODIPY493 / 503 needs to perform a washing operation after fixing cells or tissues for staining. Therefore, it cannot be applied to imaging of living individuals.
Therefore, there is a great demand for compounds that can specifically detect lipid droplets and / or fat cells than conventional compounds.
一方、ＢＯＤＩＰＹ４９３／５０３は、ナイルレッドと異なり、環境応答性ではなく、常に約５００ｎｍ付近に安定した蛍光を与える。また、その疎水性構造のため細胞内において脂肪滴などの疎水性部分に分布しやすい。そのため、オフ／オン機能がなくても脂肪滴検出試薬としての機能を果たすことができる。しかしながら、一般的に、ＢＯＤＩＰＹ４９３／５０３は、その染色のために、細胞或いは組織を固定し洗浄操作を行う必要がある。そのため、生きた個体のイメージング等に応用できない。
従って、従来の化合物よりも脂肪滴及び／又は脂肪細胞を特異的に検出することのできる化合物に対する大きな需要が存在する。 However, Nile Red is uncertain because the absorption wavelength region varies greatly from 450 to 560 nm due to strong solvatochromism (a phenomenon in which the maximum absorption wavelength varies depending on the polarity of the solvent). Also, in order to observe the dynamics such as the formation of lipid droplets in detail, it is often necessary to use multiple staining of lipid droplet membranes and cell nuclei in addition to lipid droplet nuclei, but Nile Red has a wide fluorescence wavelength (green, yellow , Orange, red), such multiple staining observation is difficult. Nile red is an off / on fluorescent reagent that has a low fluorescence intensity in an aqueous solution and a high fluorescence intensity in a hydrophobic environment, but also emits background fluorescence in cells.
On the other hand, unlike Nile Red, BODIPY493 / 503 is not environmentally responsive and always provides stable fluorescence around 500 nm. Further, due to its hydrophobic structure, it tends to be distributed in hydrophobic parts such as lipid droplets in the cell. Therefore, even if there is no off / on function, it can function as a lipid droplet detection reagent. However, in general, BODIPY493 / 503 needs to perform a washing operation after fixing cells or tissues for staining. Therefore, it cannot be applied to imaging of living individuals.
Therefore, there is a great demand for compounds that can specifically detect lipid droplets and / or fat cells than conventional compounds.
本発明は、上記の点に鑑みてなされたものであり、脂肪滴及び／又は脂肪組織を特異的に検出することのできる新規化合物を提供することを目的とする。
The present invention has been made in view of the above points, and an object thereof is to provide a novel compound capable of specifically detecting fat droplets and / or adipose tissue.
本発明者は、鋭意検討の結果、一般式（Ｉ）で表される化合物を用いることにより、従来の脂肪滴検出用蛍光試薬よりも脂肪滴及び／又は脂肪細胞を特異的に検出することができることを見出し、本発明を完成させた。
すなわち、本発明の要旨は以下のとおりである。 As a result of intensive studies, the present inventor can detect fat droplets and / or fat cells more specifically than the conventional fluorescent reagent for detecting lipid droplets by using the compound represented by the general formula (I). The present invention has been completed by finding out what can be done.
That is, the gist of the present invention is as follows.
すなわち、本発明の要旨は以下のとおりである。 As a result of intensive studies, the present inventor can detect fat droplets and / or fat cells more specifically than the conventional fluorescent reagent for detecting lipid droplets by using the compound represented by the general formula (I). The present invention has been completed by finding out what can be done.
That is, the gist of the present invention is as follows.
＜１＞ 下記一般式（Ｉ）で表される化合物：
式中、
Ｒ1は、Ｃ1-4アルキル基を表し、あるいは
Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ2は、Ｃ1-4アルキル基を表し、あるいは
Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ3は、Ｒ1が５～７員の環を形成していない場合には水素原子であり；
Ｒ4は、Ｒ2が５～７員の環を形成していない場合には水素原子であり；且つ
Ｘは下記式（ＩＩ）で表される基である：
式中、
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 <1> Compound represented by the following general formula (I):
Where
R 1 represents a C 1-4 alkyl group, or R 1 together with a nitrogen atom adjacent to R 1 , R 3 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 , May form a 5- to 7-membered ring;
R 2 represents a C 1-4 alkyl group, or R 2 together with a nitrogen atom adjacent to R 2 , R 4 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 4 , May form a 5- to 7-membered ring;
R 3 is a hydrogen atom when R 1 does not form a 5- to 7-membered ring;
R 4 is a hydrogen atom when R 2 does not form a 5- to 7-membered ring; and X is a group represented by the following formula (II):
Where
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is in the range of −0.240 to −0.190.
Ｒ1は、Ｃ1-4アルキル基を表し、あるいは
Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ2は、Ｃ1-4アルキル基を表し、あるいは
Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ3は、Ｒ1が５～７員の環を形成していない場合には水素原子であり；
Ｒ4は、Ｒ2が５～７員の環を形成していない場合には水素原子であり；且つ
Ｘは下記式（ＩＩ）で表される基である：
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 <1> Compound represented by the following general formula (I):
R 1 represents a C 1-4 alkyl group, or R 1 together with a nitrogen atom adjacent to R 1 , R 3 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 , May form a 5- to 7-membered ring;
R 2 represents a C 1-4 alkyl group, or R 2 together with a nitrogen atom adjacent to R 2 , R 4 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 4 , May form a 5- to 7-membered ring;
R 3 is a hydrogen atom when R 1 does not form a 5- to 7-membered ring;
R 4 is a hydrogen atom when R 2 does not form a 5- to 7-membered ring; and X is a group represented by the following formula (II):
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is in the range of −0.240 to −0.190.
＜２＞ Ｘが下記式（ＩＩＩ）で表される基である、上記＜１＞に記載の化合物：
（式中、
Ｒ6は、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-2アルキル基、－Ｃ（＝Ｏ）－Ｃ1-2アルキル基、又はＣＮを表し、
Ｒ7は、水素原子又はＮＯ2を表し、
但し、Ｒ6及びＲ7の両方が共にＮＯ2ではないことを条件とする）。
＜３＞ Ｒ1及びＲ2がそれぞれ独立して、Ｃ1-4アルキル基を表し、Ｒ3及びＲ4が水素原子を表す、上記＜１＞又は＜２＞に記載の化合物。
＜４＞ Ｘが下記からなる群から選択される基である、上記＜１＞～＜３＞のいずれかに記載の化合物。
＜５＞ Ｘが下記の基である、上記＜４＞に記載の化合物。
＜６＞ 上記＜１＞～＜５＞のいずれかに記載の化合物を含む、脂肪滴及び／又は脂肪組織の検出用試薬。
＜７＞ 上記＜１＞～＜５＞のいずれかに記載の化合物を、脂肪滴を含む細胞に添加する工程を含む、細胞内の脂肪滴を検出する方法。
＜８＞ 上記＜１＞～＜５＞のいずれかに記載の化合物を、生きている生物個体内（ヒトを除く）に投与する工程を含む、生物個体内に存在する脂肪組織を検出する方法。
＜９＞ 脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び
上記＜１＞～＜５＞のいずれかに記載の化合物を、該細胞に添加する工程、
を含む、脂肪滴形成阻害剤を探索する方法。 <2> The compound according to <1>, wherein X is a group represented by the following formula (III):
(Where
R 6 represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least a part of a hydrogen atom is substituted with a halogen atom, —C (═O) —C 1-2 alkyl group, or CN;
R 7 represents a hydrogen atom or NO 2 ,
Provided that both R 6 and R 7 are not NO 2 ).
<3> The compound according to <1> or <2>, wherein R 1 and R 2 each independently represent a C 1-4 alkyl group, and R 3 and R 4 each represent a hydrogen atom.
<4> The compound according to any one of <1> to <3>, wherein X is a group selected from the group consisting of:
<5> The compound according to <4>, wherein X is the following group.
<6> A reagent for detecting lipid droplets and / or adipose tissue, comprising the compound according to any one of <1> to <5> above.
<7> A method for detecting intracellular lipid droplets, comprising a step of adding the compound according to any one of <1> to <5> above to cells containing lipid droplets.
<8> A method for detecting adipose tissue present in an individual living organism, comprising a step of administering the compound according to any one of <1> to <5> above into a living organism individual (excluding a human). .
<9> a step of adding a lipid droplet formation inhibitor candidate compound to cells containing lipid droplets, and a step of adding the compound according to any one of <1> to <5> to the cells,
A method for searching for a lipid droplet formation inhibitor.
Ｒ6は、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-2アルキル基、－Ｃ（＝Ｏ）－Ｃ1-2アルキル基、又はＣＮを表し、
Ｒ7は、水素原子又はＮＯ2を表し、
但し、Ｒ6及びＲ7の両方が共にＮＯ2ではないことを条件とする）。
＜３＞ Ｒ1及びＲ2がそれぞれ独立して、Ｃ1-4アルキル基を表し、Ｒ3及びＲ4が水素原子を表す、上記＜１＞又は＜２＞に記載の化合物。
＜４＞ Ｘが下記からなる群から選択される基である、上記＜１＞～＜３＞のいずれかに記載の化合物。
＜７＞ 上記＜１＞～＜５＞のいずれかに記載の化合物を、脂肪滴を含む細胞に添加する工程を含む、細胞内の脂肪滴を検出する方法。
＜８＞ 上記＜１＞～＜５＞のいずれかに記載の化合物を、生きている生物個体内（ヒトを除く）に投与する工程を含む、生物個体内に存在する脂肪組織を検出する方法。
＜９＞ 脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び
上記＜１＞～＜５＞のいずれかに記載の化合物を、該細胞に添加する工程、
を含む、脂肪滴形成阻害剤を探索する方法。 <2> The compound according to <1>, wherein X is a group represented by the following formula (III):
R 6 represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least a part of a hydrogen atom is substituted with a halogen atom, —C (═O) —C 1-2 alkyl group, or CN;
R 7 represents a hydrogen atom or NO 2 ,
Provided that both R 6 and R 7 are not NO 2 ).
<3> The compound according to <1> or <2>, wherein R 1 and R 2 each independently represent a C 1-4 alkyl group, and R 3 and R 4 each represent a hydrogen atom.
<4> The compound according to any one of <1> to <3>, wherein X is a group selected from the group consisting of:
<7> A method for detecting intracellular lipid droplets, comprising a step of adding the compound according to any one of <1> to <5> above to cells containing lipid droplets.
<8> A method for detecting adipose tissue present in an individual living organism, comprising a step of administering the compound according to any one of <1> to <5> above into a living organism individual (excluding a human). .
<9> a step of adding a lipid droplet formation inhibitor candidate compound to cells containing lipid droplets, and a step of adding the compound according to any one of <1> to <5> to the cells,
A method for searching for a lipid droplet formation inhibitor.
本発明によれば、細胞内に含まれる脂肪滴を特異的に検出することのできる新規化合物を提供することができる。
また、該化合物は、生物個体中での脂肪組織をも特異的に検出することができる。
さらには、該化合物は、長時間蛍光シグナルが維持されるため、脂肪滴形成阻害剤の効果を継時的に観察することもでき、脂肪滴形成阻害剤のスクリーニングに利用することができる。 ADVANTAGE OF THE INVENTION According to this invention, the novel compound which can detect specifically the lipid droplet contained in a cell can be provided.
The compound can also specifically detect adipose tissue in an individual organism.
Further, since the compound maintains a fluorescence signal for a long time, the effect of the lipid droplet formation inhibitor can be observed over time, and can be used for screening for a lipid droplet formation inhibitor.
また、該化合物は、生物個体中での脂肪組織をも特異的に検出することができる。
さらには、該化合物は、長時間蛍光シグナルが維持されるため、脂肪滴形成阻害剤の効果を継時的に観察することもでき、脂肪滴形成阻害剤のスクリーニングに利用することができる。 ADVANTAGE OF THE INVENTION According to this invention, the novel compound which can detect specifically the lipid droplet contained in a cell can be provided.
The compound can also specifically detect adipose tissue in an individual organism.
Further, since the compound maintains a fluorescence signal for a long time, the effect of the lipid droplet formation inhibitor can be observed over time, and can be used for screening for a lipid droplet formation inhibitor.
以下、本発明を詳細に説明する。
＜＜定義＞＞
本明細書及び特許請求の範囲において、脂肪滴とは、脂肪を蓄積した涙滴状の細胞内構造物をする（図１参照）。
本明細書及び特許請求の範囲において、脂肪細胞とは、細胞質内に脂肪滴を含む細胞を意味する。
本明細書及び特許請求の範囲において、脂肪滴形成阻害剤とは、脂肪滴の形成を阻害することのできる物質を意味する。例えば、脂肪滴の構成成分であるトリグリセライドの形成を阻害する物質、細胞への脂肪酸の吸収を阻害する物質、及び脂肪酸の合成を阻害する物質などが挙げられる。
本明細書及び特許請求の範囲において、アルキル基は直鎖状であっても分岐鎖状であってもよい。また、例えば、Ｃ1-4アルキル基とは、炭素数１～４のアルキル基を意味する。
本明細書及び特許請求の範囲において、ハロゲン原子とは、フッ素原子、塩素原子、臭素原子及びヨウ素原子を含む概念である。 Hereinafter, the present invention will be described in detail.
<< Definition >>
In the present specification and claims, a lipid droplet is a teardrop-like intracellular structure in which fat is accumulated (see FIG. 1).
In the present specification and claims, an adipocyte means a cell containing lipid droplets in the cytoplasm.
In the present specification and claims, the lipid droplet formation inhibitor means a substance capable of inhibiting the formation of lipid droplets. Examples include substances that inhibit the formation of triglyceride, which is a constituent of lipid droplets, substances that inhibit the absorption of fatty acids into cells, and substances that inhibit the synthesis of fatty acids.
In the present specification and claims, the alkyl group may be linear or branched. For example, a C 1-4 alkyl group means an alkyl group having 1 to 4 carbon atoms.
In the present specification and claims, a halogen atom is a concept including a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
＜＜定義＞＞
本明細書及び特許請求の範囲において、脂肪滴とは、脂肪を蓄積した涙滴状の細胞内構造物をする（図１参照）。
本明細書及び特許請求の範囲において、脂肪細胞とは、細胞質内に脂肪滴を含む細胞を意味する。
本明細書及び特許請求の範囲において、脂肪滴形成阻害剤とは、脂肪滴の形成を阻害することのできる物質を意味する。例えば、脂肪滴の構成成分であるトリグリセライドの形成を阻害する物質、細胞への脂肪酸の吸収を阻害する物質、及び脂肪酸の合成を阻害する物質などが挙げられる。
本明細書及び特許請求の範囲において、アルキル基は直鎖状であっても分岐鎖状であってもよい。また、例えば、Ｃ1-4アルキル基とは、炭素数１～４のアルキル基を意味する。
本明細書及び特許請求の範囲において、ハロゲン原子とは、フッ素原子、塩素原子、臭素原子及びヨウ素原子を含む概念である。 Hereinafter, the present invention will be described in detail.
<< Definition >>
In the present specification and claims, a lipid droplet is a teardrop-like intracellular structure in which fat is accumulated (see FIG. 1).
In the present specification and claims, an adipocyte means a cell containing lipid droplets in the cytoplasm.
In the present specification and claims, the lipid droplet formation inhibitor means a substance capable of inhibiting the formation of lipid droplets. Examples include substances that inhibit the formation of triglyceride, which is a constituent of lipid droplets, substances that inhibit the absorption of fatty acids into cells, and substances that inhibit the synthesis of fatty acids.
In the present specification and claims, the alkyl group may be linear or branched. For example, a C 1-4 alkyl group means an alkyl group having 1 to 4 carbon atoms.
In the present specification and claims, a halogen atom is a concept including a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
＜＜化合物＞＞
本発明の第１の態様は、下記一般式（Ｉ）で表される化合物である。後に詳述するように、該化合物は、脂肪滴及び／又は脂肪組織を検出する試薬として非常に有用である。 << Compound >>
A first aspect of the present invention is a compound represented by the following general formula (I). As described later in detail, the compound is very useful as a reagent for detecting lipid droplets and / or adipose tissue.
本発明の第１の態様は、下記一般式（Ｉ）で表される化合物である。後に詳述するように、該化合物は、脂肪滴及び／又は脂肪組織を検出する試薬として非常に有用である。 << Compound >>
A first aspect of the present invention is a compound represented by the following general formula (I). As described later in detail, the compound is very useful as a reagent for detecting lipid droplets and / or adipose tissue.
式中、
Ｒ1は、Ｃ1-4アルキル基を表し、あるいは
Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ2は、Ｃ1-4アルキル基を表し、あるいは
Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ3は、Ｒ1が５～７員の環を形成していない場合には水素原子であり；
Ｒ4は、Ｒ2が５～７員の環を形成していない場合には水素原子であり；且つ
Ｘは下記式（ＩＩ）で表される基である： Where
R 1 represents a C 1-4 alkyl group, or R 1 together with a nitrogen atom adjacent to R 1 , R 3 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 , May form a 5- to 7-membered ring;
R 2 represents a C 1-4 alkyl group, or R 2 together with a nitrogen atom adjacent to R 2 , R 4 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 4 , May form a 5- to 7-membered ring;
R 3 is a hydrogen atom when R 1 does not form a 5- to 7-membered ring;
R 4 is a hydrogen atom when R 2 does not form a 5- to 7-membered ring; and X is a group represented by the following formula (II):
Ｒ1は、Ｃ1-4アルキル基を表し、あるいは
Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ2は、Ｃ1-4アルキル基を表し、あるいは
Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ3は、Ｒ1が５～７員の環を形成していない場合には水素原子であり；
Ｒ4は、Ｒ2が５～７員の環を形成していない場合には水素原子であり；且つ
Ｘは下記式（ＩＩ）で表される基である： Where
R 1 represents a C 1-4 alkyl group, or R 1 together with a nitrogen atom adjacent to R 1 , R 3 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 , May form a 5- to 7-membered ring;
R 2 represents a C 1-4 alkyl group, or R 2 together with a nitrogen atom adjacent to R 2 , R 4 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 4 , May form a 5- to 7-membered ring;
R 3 is a hydrogen atom when R 1 does not form a 5- to 7-membered ring;
R 4 is a hydrogen atom when R 2 does not form a 5- to 7-membered ring; and X is a group represented by the following formula (II):
式中、
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子（一般式（Ｉ）の－ＳＯ2基と結合する炭素原子）の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 Where
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
However, the electron density of the carbon atom at the 1-position of the benzene ring in the formula (II) (carbon atom bonded to the —SO 2 group in the general formula (I)) is within the range of −0.240 to −0.190. Subject to being.
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子（一般式（Ｉ）の－ＳＯ2基と結合する炭素原子）の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 Where
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
However, the electron density of the carbon atom at the 1-position of the benzene ring in the formula (II) (carbon atom bonded to the —SO 2 group in the general formula (I)) is within the range of −0.240 to −0.190. Subject to being.
一般式（Ｉ）において、Ｒ1は、Ｃ1-4アルキル基を表し、あるいは、Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員（好ましくは５～６員）の環を形成していてもよい。
これらのなかでも、Ｒ1は、Ｃ1-4アルキル基であることが好ましく、Ｃ1-2アルキル基であることがより好ましく、エチル基であることがさらにより好ましい。 In the general formula (I), R 1 represents a C 1-4 alkyl group, or R 1 is a nitrogen atom adjacent to R 1 , R 3 , and a benzene ring bonded to the nitrogen atom and R 3 . Together with these two carbon atoms may form a 5- to 7-membered (preferably 5- to 6-membered) ring.
Among these, R 1 is preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group, and even more preferably an ethyl group.
これらのなかでも、Ｒ1は、Ｃ1-4アルキル基であることが好ましく、Ｃ1-2アルキル基であることがより好ましく、エチル基であることがさらにより好ましい。 In the general formula (I), R 1 represents a C 1-4 alkyl group, or R 1 is a nitrogen atom adjacent to R 1 , R 3 , and a benzene ring bonded to the nitrogen atom and R 3 . Together with these two carbon atoms may form a 5- to 7-membered (preferably 5- to 6-membered) ring.
Among these, R 1 is preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group, and even more preferably an ethyl group.
一般式（Ｉ）において、Ｒ2は、Ｃ1-4アルキル基を表し、あるいは、Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよい。
これらのなかでも、Ｒ2は、Ｃ1-4アルキル基であることが好ましく、Ｃ1-2アルキル基であることがより好ましく、エチル基であることがさらにより好ましい。
Ｒ1が、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していないとき、Ｒ3は水素原子である。Ｒ3は水素原子であることが好ましい。
Ｒ2が、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していないとき、Ｒ4は水素原子である。Ｒ4は水素原子であることが好ましい。 In the general formula (I), R 2 represents a C 1-4 alkyl group, or R 2 represents a nitrogen atom adjacent to R 2 , R 4 , and a benzene ring bonded to the nitrogen atom and R 4 . And 5 carbon atoms may form a 5- to 7-membered ring.
Among these, R 2 is preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group, and even more preferably an ethyl group.
When R 1 does not form a 5- to 7-membered ring with the nitrogen atom adjacent to R 1 , R 3 , and the two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 ,R 1 3 is a hydrogen atom. R 3 is preferably a hydrogen atom.
When R 2 is a nitrogen atom adjacent to R 2, R 4, and with two carbon atoms on the benzene ring bonded with the nitrogen atom and R 4, it does not form a ring of 5 to 7-membered, R 4 is a hydrogen atom. R 4 is preferably a hydrogen atom.
これらのなかでも、Ｒ2は、Ｃ1-4アルキル基であることが好ましく、Ｃ1-2アルキル基であることがより好ましく、エチル基であることがさらにより好ましい。
Ｒ1が、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していないとき、Ｒ3は水素原子である。Ｒ3は水素原子であることが好ましい。
Ｒ2が、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していないとき、Ｒ4は水素原子である。Ｒ4は水素原子であることが好ましい。 In the general formula (I), R 2 represents a C 1-4 alkyl group, or R 2 represents a nitrogen atom adjacent to R 2 , R 4 , and a benzene ring bonded to the nitrogen atom and R 4 . And 5 carbon atoms may form a 5- to 7-membered ring.
Among these, R 2 is preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group, and even more preferably an ethyl group.
When R 1 does not form a 5- to 7-membered ring with the nitrogen atom adjacent to R 1 , R 3 , and the two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 ,
When R 2 is a nitrogen atom adjacent to R 2, R 4, and with two carbon atoms on the benzene ring bonded with the nitrogen atom and R 4, it does not form a ring of 5 to 7-membered, R 4 is a hydrogen atom. R 4 is preferably a hydrogen atom.
一般式（Ｉ）において、Ｘは下記式（ＩＩ）で表される基である。
In general formula (I), X is a group represented by the following formula (II).
式中、
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 Where
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is in the range of −0.240 to −0.190.
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする。 Where
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is in the range of −0.240 to −0.190.
式（ＩＩ）中、Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-2アルキル基、－Ｃ（＝Ｏ）－Ｃ1-2アルキル基、又はＣＮであることが好ましく、ＮＯ2、ハロゲン原子、ＣＦ3、－Ｃ（＝Ｏ）－ＣＨ3基、又はＣＮであることがより好ましい。
また、Ｒ5は、式（ＩＩ）のベンゼン環の２位及び／又は４位に結合していることが好ましく、４位に結合していることがより好ましい。
式（ＩＩ）中、ｎは、１～３が好ましく、１～２がより好ましく、１が最も好ましい。 In formula (II), each R 5 independently represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C (═O) —C 1- It is preferably a 2 alkyl group or CN, more preferably a NO 2 , halogen atom, CF 3 , —C (═O) —CH 3 group, or CN.
R 5 is preferably bonded to the 2-position and / or 4-position of the benzene ring of formula (II), and more preferably bonded to the 4-position.
In the formula (II), n is preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
また、Ｒ5は、式（ＩＩ）のベンゼン環の２位及び／又は４位に結合していることが好ましく、４位に結合していることがより好ましい。
式（ＩＩ）中、ｎは、１～３が好ましく、１～２がより好ましく、１が最も好ましい。 In formula (II), each R 5 independently represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C (═O) —C 1- It is preferably a 2 alkyl group or CN, more preferably a NO 2 , halogen atom, CF 3 , —C (═O) —CH 3 group, or CN.
R 5 is preferably bonded to the 2-position and / or 4-position of the benzene ring of formula (II), and more preferably bonded to the 4-position.
In the formula (II), n is preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度は、－０．２４０～－０．１９０である。なかでも、－０．２２０～－０．１９５が好ましく、－０．２１５～－０．１９５がより好ましく、－０．２１０～－０．２００が特に好ましい。
式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、上記範囲内になるようにＲ5の各置換基の種類及び結合位置を選択することにより、得られる化合物は、脂肪滴及び／又は脂肪細胞に対して特異的に蛍光を発しやすくなり、脂肪滴及び／又は脂肪細胞の特異的に検出することができる。
また、Ｘは、下記式（ＩＩＩ）で表される基であることが好ましい。 The electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is -0.240 to -0.190. Among these, −0.220 to −0.195 is preferable, −0.215 to −0.195 is more preferable, and −0.210 to −0.200 is particularly preferable.
By selecting the type and bonding position of each substituent of R 5 so that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is within the above range, the resulting compound is It becomes easy to fluoresce specifically with respect to a fat cell, and can detect a lipid droplet and / or a fat cell specifically.
X is preferably a group represented by the following formula (III).
式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、上記範囲内になるようにＲ5の各置換基の種類及び結合位置を選択することにより、得られる化合物は、脂肪滴及び／又は脂肪細胞に対して特異的に蛍光を発しやすくなり、脂肪滴及び／又は脂肪細胞の特異的に検出することができる。
また、Ｘは、下記式（ＩＩＩ）で表される基であることが好ましい。 The electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is -0.240 to -0.190. Among these, −0.220 to −0.195 is preferable, −0.215 to −0.195 is more preferable, and −0.210 to −0.200 is particularly preferable.
By selecting the type and bonding position of each substituent of R 5 so that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is within the above range, the resulting compound is It becomes easy to fluoresce specifically with respect to a fat cell, and can detect a lipid droplet and / or a fat cell specifically.
X is preferably a group represented by the following formula (III).
式（ＩＩＩ）中、Ｒ6は、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-2アルキル基、－Ｃ（＝Ｏ）－Ｃ1-2アルキル基、又はＣＮを表し、Ｒ7は、水素原子又はＮＯ2を表す。但し、Ｒ6及びＲ7の両方が共にＮＯ2ではないことを条件とする。
Ｒ6は、好ましくはＮＯ2、ハロゲン原子、ＣＦ3、Ｃ（＝Ｏ）ＣＨ3、又はＣＮであり、より好ましくはＮＯ2、ハロゲン原子、又はＣＦ3であり、最も好ましくはＮＯ2である。
Ｒ7は、好ましくは水素原子である。
Ｘは、より好ましくは下記からなる群から選択される基である。 In the formula (III), R 6 is NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C (═O) —C 1-2 alkyl group, Or CN, and R 7 represents a hydrogen atom or NO 2 . Provided that R 6 and R 7 are not both NO 2 .
R 6 is preferably NO 2 , a halogen atom, CF 3 , C (═O) CH 3 , or CN, more preferably NO 2 , a halogen atom, or CF 3 , and most preferably NO 2 . .
R 7 is preferably a hydrogen atom.
X is more preferably a group selected from the group consisting of:
Ｒ6は、好ましくはＮＯ2、ハロゲン原子、ＣＦ3、Ｃ（＝Ｏ）ＣＨ3、又はＣＮであり、より好ましくはＮＯ2、ハロゲン原子、又はＣＦ3であり、最も好ましくはＮＯ2である。
Ｒ7は、好ましくは水素原子である。
Ｘは、より好ましくは下記からなる群から選択される基である。 In the formula (III), R 6 is NO 2 , a halogen atom, a C 1-2 alkyl group in which at least one hydrogen atom is substituted with a halogen atom, —C (═O) —C 1-2 alkyl group, Or CN, and R 7 represents a hydrogen atom or NO 2 . Provided that R 6 and R 7 are not both NO 2 .
R 6 is preferably NO 2 , a halogen atom, CF 3 , C (═O) CH 3 , or CN, more preferably NO 2 , a halogen atom, or CF 3 , and most preferably NO 2 . .
R 7 is preferably a hydrogen atom.
X is more preferably a group selected from the group consisting of:
これらの中でも、Ｘは、下記からなる群から選択される基であることがより好ましい。
Among these, X is more preferably a group selected from the group consisting of the following.
Ｘは、さらにより好ましくは、下記からなる群から選択される基である。
X is even more preferably a group selected from the group consisting of:
＜＜脂肪滴及び又は脂肪組織検出用試薬＞＞
本発明の第２の態様は、上記第１の態様の化合物を含む、脂肪滴及び／又は脂肪組織の検出用試薬（プローブ）である。
該検出用試薬は、第１の態様の化合物のみから構成されていてもよく、脂肪滴及び又は脂肪組織検出用試薬として用いられ得る本発明の化合物以外の化合物との混合物の形態であってもよい。
本発明の化合物以外の化合物としては、ナイルレッド、ＢＯＤＩＰＹ４９３／５０３、ＬＤ４５０、リピッドグリーン（Ｌｉｐｉｄ Ｇｒｅｅｎ）、ＳＦ４４などが挙げられる。 << Reagent for detecting lipid droplets and / or adipose tissue >>
The second aspect of the present invention is a reagent for detecting lipid droplets and / or adipose tissue (probe) comprising the compound of the first aspect.
The detection reagent may be composed of only the compound of the first aspect, or may be in the form of a mixture with a compound other than the compound of the present invention that can be used as a reagent for detecting lipid droplets and / or adipose tissue. Good.
Examples of the compound other than the compound of the present invention include Nile Red, BODIPY493 / 503, LD450, Lipid Green, and SF44.
本発明の第２の態様は、上記第１の態様の化合物を含む、脂肪滴及び／又は脂肪組織の検出用試薬（プローブ）である。
該検出用試薬は、第１の態様の化合物のみから構成されていてもよく、脂肪滴及び又は脂肪組織検出用試薬として用いられ得る本発明の化合物以外の化合物との混合物の形態であってもよい。
本発明の化合物以外の化合物としては、ナイルレッド、ＢＯＤＩＰＹ４９３／５０３、ＬＤ４５０、リピッドグリーン（Ｌｉｐｉｄ Ｇｒｅｅｎ）、ＳＦ４４などが挙げられる。 << Reagent for detecting lipid droplets and / or adipose tissue >>
The second aspect of the present invention is a reagent for detecting lipid droplets and / or adipose tissue (probe) comprising the compound of the first aspect.
The detection reagent may be composed of only the compound of the first aspect, or may be in the form of a mixture with a compound other than the compound of the present invention that can be used as a reagent for detecting lipid droplets and / or adipose tissue. Good.
Examples of the compound other than the compound of the present invention include Nile Red, BODIPY493 / 503, LD450, Lipid Green, and SF44.
＜＜脂肪滴の検出方法＞＞
本発明の第３の態様は、上記第１の態様の化合物を、脂肪滴を含む細胞に添加する工程を含む、細胞内の脂肪滴を検出する方法である。
本発明の化合物は、細胞内に存在する脂肪滴を特異的に検出することが出来る。したがって、脂肪滴を検出するための試薬として非常に有用である。
本発明における細胞内に存在する脂肪滴を検出する方法は、第１の態様の化合物を、脂肪滴を含む細胞に添加する工程を含む。その後、本発明の化合物の蛍光シグナルを蛍光顕微鏡等により観測することにより、細胞内に含まれる脂肪滴を検出することができる。
本発明の化合物又はその塩の添加量は、使用する細胞や脂肪滴の割合などによっても変化し得るが、例えば、０．０１～１００μＭ、好ましくは０．１～１０μＭの終濃度で細胞に添加することができる。
本発明の化合物を溶媒に溶解させてから細胞に添加する場合、該溶媒として、例えば、ジメチルスルホキシド（ＤＭＳＯ）用いることが出来る。
本発明の化合物を添加する細胞としては、脂肪滴を含む細胞であれば特に制限はなく、例えば３Ｔ３－Ｌ１細胞、単離脂肪細胞が挙げられる。また、脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞中に人為的に脂肪滴を形成させた細胞を用いてもよい。脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞としては、ＨｅＬａ細胞、ＵＥＥＴ－１２細胞、ＮＩＨ３Ｔ３細胞などが挙げられる。また、脂肪滴を形成させる方法としては、例えば、オレイン酸を細胞に添加する方法、インスリン、ＩＢＭＸ，ＤＥＸのカクテルを細胞に添加することにより脂肪滴を誘導する方法が挙げられる。 << Fat droplet detection method >>
A third aspect of the present invention is a method for detecting intracellular lipid droplets, which comprises the step of adding the compound of the first aspect to cells containing lipid droplets.
The compound of the present invention can specifically detect lipid droplets present in cells. Therefore, it is very useful as a reagent for detecting fat droplets.
The method for detecting lipid droplets present in cells in the present invention comprises the step of adding the compound of the first aspect to cells containing lipid droplets. Thereafter, lipid droplets contained in the cells can be detected by observing the fluorescence signal of the compound of the present invention with a fluorescence microscope or the like.
The addition amount of the compound of the present invention or a salt thereof may vary depending on the cells to be used, the ratio of lipid droplets, and the like. can do.
When the compound of the present invention is dissolved in a solvent and then added to cells, for example, dimethyl sulfoxide (DMSO) can be used as the solvent.
The cells to which the compound of the present invention is added are not particularly limited as long as they contain lipid droplets, and examples thereof include 3T3-L1 cells and isolated adipocytes. Moreover, you may use the cell which formed the lipid droplet artificially in the cell which does not contain a lipid droplet, or a cell with little content of a lipid droplet. Examples of cells not containing lipid droplets or cells containing a small amount of lipid droplets include HeLa cells, UEET-12 cells, NIH3T3 cells, and the like. Examples of the method of forming lipid droplets include a method of adding oleic acid to cells, and a method of inducing lipid droplets by adding a cocktail of insulin, IBMX, and DEX to cells.
本発明の第３の態様は、上記第１の態様の化合物を、脂肪滴を含む細胞に添加する工程を含む、細胞内の脂肪滴を検出する方法である。
本発明の化合物は、細胞内に存在する脂肪滴を特異的に検出することが出来る。したがって、脂肪滴を検出するための試薬として非常に有用である。
本発明における細胞内に存在する脂肪滴を検出する方法は、第１の態様の化合物を、脂肪滴を含む細胞に添加する工程を含む。その後、本発明の化合物の蛍光シグナルを蛍光顕微鏡等により観測することにより、細胞内に含まれる脂肪滴を検出することができる。
本発明の化合物又はその塩の添加量は、使用する細胞や脂肪滴の割合などによっても変化し得るが、例えば、０．０１～１００μＭ、好ましくは０．１～１０μＭの終濃度で細胞に添加することができる。
本発明の化合物を溶媒に溶解させてから細胞に添加する場合、該溶媒として、例えば、ジメチルスルホキシド（ＤＭＳＯ）用いることが出来る。
本発明の化合物を添加する細胞としては、脂肪滴を含む細胞であれば特に制限はなく、例えば３Ｔ３－Ｌ１細胞、単離脂肪細胞が挙げられる。また、脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞中に人為的に脂肪滴を形成させた細胞を用いてもよい。脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞としては、ＨｅＬａ細胞、ＵＥＥＴ－１２細胞、ＮＩＨ３Ｔ３細胞などが挙げられる。また、脂肪滴を形成させる方法としては、例えば、オレイン酸を細胞に添加する方法、インスリン、ＩＢＭＸ，ＤＥＸのカクテルを細胞に添加することにより脂肪滴を誘導する方法が挙げられる。 << Fat droplet detection method >>
A third aspect of the present invention is a method for detecting intracellular lipid droplets, which comprises the step of adding the compound of the first aspect to cells containing lipid droplets.
The compound of the present invention can specifically detect lipid droplets present in cells. Therefore, it is very useful as a reagent for detecting fat droplets.
The method for detecting lipid droplets present in cells in the present invention comprises the step of adding the compound of the first aspect to cells containing lipid droplets. Thereafter, lipid droplets contained in the cells can be detected by observing the fluorescence signal of the compound of the present invention with a fluorescence microscope or the like.
The addition amount of the compound of the present invention or a salt thereof may vary depending on the cells to be used, the ratio of lipid droplets, and the like. can do.
When the compound of the present invention is dissolved in a solvent and then added to cells, for example, dimethyl sulfoxide (DMSO) can be used as the solvent.
The cells to which the compound of the present invention is added are not particularly limited as long as they contain lipid droplets, and examples thereof include 3T3-L1 cells and isolated adipocytes. Moreover, you may use the cell which formed the lipid droplet artificially in the cell which does not contain a lipid droplet, or a cell with little content of a lipid droplet. Examples of cells not containing lipid droplets or cells containing a small amount of lipid droplets include HeLa cells, UEET-12 cells, NIH3T3 cells, and the like. Examples of the method of forming lipid droplets include a method of adding oleic acid to cells, and a method of inducing lipid droplets by adding a cocktail of insulin, IBMX, and DEX to cells.
＜＜脂肪組織の検出方法＞＞
本発明の第４の態様は、上記第１の態様の化合物を、生きている生物個体内に投与する工程を含む、生きている生物個体に存在する脂肪組織を検出する方法である。
本発明の化合物は、生きている生物個体内（生体内）の脂肪組織をも特異的に検出することができる。したがって、生体内の脂肪組織を検出するための試薬として非常に有用である。生体内の脂肪組織としては、皮下脂肪、内臓脂肪、異所性脂肪（例えば、筋肉、肝臓、心臓、膵臓、腎臓などの臓器に蓄積する脂肪）などが挙げられる。
本発明における生きている生物個体中の脂肪組織を検出する方法は、本発明の化合物を、該生物個体に投与する工程を含む。その後、倒立共焦点顕微鏡を用いた生体分子イメージング手法を用いて本発明の化合物の蛍光シグナルを観測することにより、生物個体を固定化することなく生きた状態で、生体内の脂肪組織を検出することができる。
本発明の化合物の投与形態としては、例えば、静脈内投与、皮下投与、筋肉内投与が挙げられる。
また、本発明の化合物の投与量は、投与対象となる動物、投与形態によっても異なるが、例えば、０．０１～１．０μＭ／ｋｇ体重、好ましくは０．０５～０．５μＭ／ｋｇ体重の範囲で該化合物を投与することができる。
本発明の化合物を溶媒に溶解させてから生体内に投与する場合、該溶媒としては、例えば、ＤＭＳＯなどを用いることが出来る。
投与対象となる生物個体としては、特に限定されず、例えば、哺乳動物（マウス、ヒト、ブタ、イヌ、ウサギなど）を含む脊椎動物や無脊椎動物が挙げられる。また、投与対象にはヒトが含まれていても含まれていなくてもよい。 << Adipose tissue detection method >>
A fourth aspect of the present invention is a method for detecting adipose tissue present in an individual living organism comprising the step of administering the compound of the first aspect into the individual living organism.
The compound of the present invention can specifically detect adipose tissue in living organisms (in vivo). Therefore, it is very useful as a reagent for detecting adipose tissue in a living body. Examples of adipose tissue in a living body include subcutaneous fat, visceral fat, ectopic fat (for example, fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney).
The method for detecting adipose tissue in a living organism individual according to the present invention comprises the step of administering the compound of the present invention to the individual organism. Then, by observing the fluorescence signal of the compound of the present invention using a biomolecular imaging technique using an inverted confocal microscope, the adipose tissue in the living body is detected in a living state without immobilizing the individual organism. be able to.
Examples of the dosage form of the compound of the present invention include intravenous administration, subcutaneous administration and intramuscular administration.
The dose of the compound of the present invention varies depending on the animal to be administered and the administration form, but is, for example, 0.01 to 1.0 μM / kg body weight, preferably 0.05 to 0.5 μM / kg body weight. A range of the compounds can be administered.
When the compound of the present invention is dissolved in a solvent and then administered into a living body, for example, DMSO can be used as the solvent.
The individual organism to be administered is not particularly limited, and examples thereof include vertebrates and invertebrates including mammals (mouse, human, pig, dog, rabbit, etc.). Further, the administration subject may or may not include a human.
本発明の第４の態様は、上記第１の態様の化合物を、生きている生物個体内に投与する工程を含む、生きている生物個体に存在する脂肪組織を検出する方法である。
本発明の化合物は、生きている生物個体内（生体内）の脂肪組織をも特異的に検出することができる。したがって、生体内の脂肪組織を検出するための試薬として非常に有用である。生体内の脂肪組織としては、皮下脂肪、内臓脂肪、異所性脂肪（例えば、筋肉、肝臓、心臓、膵臓、腎臓などの臓器に蓄積する脂肪）などが挙げられる。
本発明における生きている生物個体中の脂肪組織を検出する方法は、本発明の化合物を、該生物個体に投与する工程を含む。その後、倒立共焦点顕微鏡を用いた生体分子イメージング手法を用いて本発明の化合物の蛍光シグナルを観測することにより、生物個体を固定化することなく生きた状態で、生体内の脂肪組織を検出することができる。
本発明の化合物の投与形態としては、例えば、静脈内投与、皮下投与、筋肉内投与が挙げられる。
また、本発明の化合物の投与量は、投与対象となる動物、投与形態によっても異なるが、例えば、０．０１～１．０μＭ／ｋｇ体重、好ましくは０．０５～０．５μＭ／ｋｇ体重の範囲で該化合物を投与することができる。
本発明の化合物を溶媒に溶解させてから生体内に投与する場合、該溶媒としては、例えば、ＤＭＳＯなどを用いることが出来る。
投与対象となる生物個体としては、特に限定されず、例えば、哺乳動物（マウス、ヒト、ブタ、イヌ、ウサギなど）を含む脊椎動物や無脊椎動物が挙げられる。また、投与対象にはヒトが含まれていても含まれていなくてもよい。 << Adipose tissue detection method >>
A fourth aspect of the present invention is a method for detecting adipose tissue present in an individual living organism comprising the step of administering the compound of the first aspect into the individual living organism.
The compound of the present invention can specifically detect adipose tissue in living organisms (in vivo). Therefore, it is very useful as a reagent for detecting adipose tissue in a living body. Examples of adipose tissue in a living body include subcutaneous fat, visceral fat, ectopic fat (for example, fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney).
The method for detecting adipose tissue in a living organism individual according to the present invention comprises the step of administering the compound of the present invention to the individual organism. Then, by observing the fluorescence signal of the compound of the present invention using a biomolecular imaging technique using an inverted confocal microscope, the adipose tissue in the living body is detected in a living state without immobilizing the individual organism. be able to.
Examples of the dosage form of the compound of the present invention include intravenous administration, subcutaneous administration and intramuscular administration.
The dose of the compound of the present invention varies depending on the animal to be administered and the administration form, but is, for example, 0.01 to 1.0 μM / kg body weight, preferably 0.05 to 0.5 μM / kg body weight. A range of the compounds can be administered.
When the compound of the present invention is dissolved in a solvent and then administered into a living body, for example, DMSO can be used as the solvent.
The individual organism to be administered is not particularly limited, and examples thereof include vertebrates and invertebrates including mammals (mouse, human, pig, dog, rabbit, etc.). Further, the administration subject may or may not include a human.
＜＜脂肪滴形成阻害剤の候補化合物の探索方法＞＞
本発明の第５の態様は、脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び第１の態様に記載の本発明の化合物を、該細胞に添加する工程を含む、脂肪滴形成阻害剤を探索する方法である。
本発明の化合物は、脂肪滴を特異的に検出することができ、且つ、長時間にわたって蛍光シグナルを維持することができる。したがって、脂肪滴の形成を阻害する化合物及び／又は組成物（すなわち、脂肪滴形成阻害剤）を探索するために好適に用いることができる。
本発明の探索方法は、脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び第１の態様の化合物を、該細胞に添加する工程を含む。その後、蛍光顕微鏡等を用いて、本発明の化合物の蛍光シグナルを観測する。脂肪滴形成阻害能のある化合物を添加した細胞では、脂肪滴が形成されにくくなるため、第１の態様の化合物は添加するが脂肪滴形成阻害剤の候補化合物は添加しない場合（コントロール）よりも、本発明の化合物による脂肪滴部分からの蛍光シグナルが低減される。したがって、候補化合物を添加していないコントロールの細胞の蛍光シグナルに対して、候補化合物を添加した細胞の蛍光シグナルがどの程度低減したかを指標として、脂肪滴形成阻害剤の候補化合物群の中から脂肪滴形成阻害剤を探索することができる。
脂肪滴形成阻害剤の候補化合物としては、特に制限はなく、他の実験結果から脂肪滴形成阻害能を有すると予想される化合物を用いてもよく、脂肪滴形成阻害能を有するか否か不明な化合物を用いてもよい。例えば、市場で入手可能な化合物ライブラリーに含まれる種々の化合物を用いて、新規脂肪滴形成阻害剤を探索することができる。 << Method for Searching Candidate Compound for Lipid Formation Inhibitor >>
The fifth aspect of the present invention includes a step of adding a lipid droplet formation inhibitor candidate compound to a cell containing lipid droplets, and a step of adding the compound of the present invention described in the first aspect to the cell. A method for searching for a lipid droplet formation inhibitor.
The compound of the present invention can specifically detect fat droplets and can maintain a fluorescent signal for a long time. Therefore, it can be suitably used to search for compounds and / or compositions that inhibit lipid droplet formation (ie, lipid droplet formation inhibitors).
The search method of the present invention includes a step of adding a candidate compound for a lipid droplet formation inhibitor to a cell containing a lipid droplet, and a step of adding the compound of the first aspect to the cell. Thereafter, the fluorescence signal of the compound of the present invention is observed using a fluorescence microscope or the like. In cells to which a compound capable of inhibiting lipid droplet formation is added, lipid droplets are less likely to be formed, so that the compound of the first aspect is added but the candidate compound for lipid droplet formation inhibitor is not added (control). The fluorescence signal from the lipid droplet portion by the compound of the present invention is reduced. Therefore, using the index of how much the fluorescence signal of the cells to which the candidate compound was added decreased compared to the fluorescence signal of the control cells to which the candidate compound was not added, from among the candidate compound group of lipid droplet formation inhibitors A lipid droplet formation inhibitor can be searched.
There are no particular limitations on the candidate compounds for lipid droplet formation inhibitors, and compounds that are expected to have lipid droplet formation inhibitory activity from other experimental results may be used, and it is unknown whether they have lipid droplet formation inhibitory activity. Such compounds may be used. For example, a novel lipid droplet formation inhibitor can be searched using various compounds contained in a commercially available compound library.
本発明の第５の態様は、脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び第１の態様に記載の本発明の化合物を、該細胞に添加する工程を含む、脂肪滴形成阻害剤を探索する方法である。
本発明の化合物は、脂肪滴を特異的に検出することができ、且つ、長時間にわたって蛍光シグナルを維持することができる。したがって、脂肪滴の形成を阻害する化合物及び／又は組成物（すなわち、脂肪滴形成阻害剤）を探索するために好適に用いることができる。
本発明の探索方法は、脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び第１の態様の化合物を、該細胞に添加する工程を含む。その後、蛍光顕微鏡等を用いて、本発明の化合物の蛍光シグナルを観測する。脂肪滴形成阻害能のある化合物を添加した細胞では、脂肪滴が形成されにくくなるため、第１の態様の化合物は添加するが脂肪滴形成阻害剤の候補化合物は添加しない場合（コントロール）よりも、本発明の化合物による脂肪滴部分からの蛍光シグナルが低減される。したがって、候補化合物を添加していないコントロールの細胞の蛍光シグナルに対して、候補化合物を添加した細胞の蛍光シグナルがどの程度低減したかを指標として、脂肪滴形成阻害剤の候補化合物群の中から脂肪滴形成阻害剤を探索することができる。
脂肪滴形成阻害剤の候補化合物としては、特に制限はなく、他の実験結果から脂肪滴形成阻害能を有すると予想される化合物を用いてもよく、脂肪滴形成阻害能を有するか否か不明な化合物を用いてもよい。例えば、市場で入手可能な化合物ライブラリーに含まれる種々の化合物を用いて、新規脂肪滴形成阻害剤を探索することができる。 << Method for Searching Candidate Compound for Lipid Formation Inhibitor >>
The fifth aspect of the present invention includes a step of adding a lipid droplet formation inhibitor candidate compound to a cell containing lipid droplets, and a step of adding the compound of the present invention described in the first aspect to the cell. A method for searching for a lipid droplet formation inhibitor.
The compound of the present invention can specifically detect fat droplets and can maintain a fluorescent signal for a long time. Therefore, it can be suitably used to search for compounds and / or compositions that inhibit lipid droplet formation (ie, lipid droplet formation inhibitors).
The search method of the present invention includes a step of adding a candidate compound for a lipid droplet formation inhibitor to a cell containing a lipid droplet, and a step of adding the compound of the first aspect to the cell. Thereafter, the fluorescence signal of the compound of the present invention is observed using a fluorescence microscope or the like. In cells to which a compound capable of inhibiting lipid droplet formation is added, lipid droplets are less likely to be formed, so that the compound of the first aspect is added but the candidate compound for lipid droplet formation inhibitor is not added (control). The fluorescence signal from the lipid droplet portion by the compound of the present invention is reduced. Therefore, using the index of how much the fluorescence signal of the cells to which the candidate compound was added decreased compared to the fluorescence signal of the control cells to which the candidate compound was not added, from among the candidate compound group of lipid droplet formation inhibitors A lipid droplet formation inhibitor can be searched.
There are no particular limitations on the candidate compounds for lipid droplet formation inhibitors, and compounds that are expected to have lipid droplet formation inhibitory activity from other experimental results may be used, and it is unknown whether they have lipid droplet formation inhibitory activity. Such compounds may be used. For example, a novel lipid droplet formation inhibitor can be searched using various compounds contained in a commercially available compound library.
＜＜製造方法＞＞
本発明の化合物は、例えば、市販あるいは自ら合成した下記構造のナイルブルーを出発物質として、ジイソピルエチルアミン存在下、本発明のＲ5の置換基を有する芳香族スルホニル塩化物で処理することにより製造することができる。 << Manufacturing method >>
The compound of the present invention is treated, for example, with an aromatic sulfonyl chloride having a substituent of R 5 of the present invention in the presence of diisopropylmethylamine, starting from commercially available or self-synthesized Nile blue having the following structure. Can be manufactured.
本発明の化合物は、例えば、市販あるいは自ら合成した下記構造のナイルブルーを出発物質として、ジイソピルエチルアミン存在下、本発明のＲ5の置換基を有する芳香族スルホニル塩化物で処理することにより製造することができる。 << Manufacturing method >>
The compound of the present invention is treated, for example, with an aromatic sulfonyl chloride having a substituent of R 5 of the present invention in the presence of diisopropylmethylamine, starting from commercially available or self-synthesized Nile blue having the following structure. Can be manufactured.
次に、実施例により、本発明を具体的に説明する。しかしながら、本発明はこれらの例によって限定されるものではない。
Next, the present invention will be specifically described with reference to examples. However, the present invention is not limited by these examples.
ナイルブルー（ＮＢ）の５位のアミノ基に求電子性が段階的に低くなる芳香族スルホニル制御ユニットとして、それぞれジニトロベンゼンスルホニル基（ＤＮｓ基）、モノニトロベンゼンスルホニル基（ＭＮｓ基）およびトシル基（Ｔｓ基）を導入した化合物（ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢ）をそれぞれ合成した（合成例１～３）。
［合成例１］
＜ＤＮｓ－ＮＢ（比較例１の化合物）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ、０．１８ｍｍｏｌ）及び２，４－ジニトロベンゼンスルホニルクロライド（３８ｍｇ、０．１４ｍｍｏｌ）を添加した。２０分後、該溶液を７０℃の周囲温度に温め、該温度で２時間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、水で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＤＮｓ－ＮＢを得た（９．５ｍｇ、０．０１７ｍｍｏｌ、１４％）。融点>400℃;1H NMR (500 MHz, ピリジン-d5): δ 9.13 (1H, s) 8.87-8.85 (1H, d, J = 8.5 Hz), 8.59-8.76 (1H, d, J = 9.0 Hz), 8.39-8.40 (1H, d, J = 8.5 Hz), 8.31-8.29 (1H, d, J = 9.5 Hz), 7.90 (1H, s), 7.90-7.89 (1H, d, J = 8.8 Hz), 7.79-7.76 (1H, dd, J = 8.5, 8.0 Hz), 7.70-7.67 (1H, dd, J = 8.0, 7.5 Hz), 7.00-6.98 (1H, d, J = 9.0 Hz), 6.69 (1H, s), 3.46-3.3.41 (4H, q, J = 7.0 Hz), 1.18-1.15 (6H, t, J = 7.0 Hz). 13C NMR (100 MHz, C5D5N-d5): δ 164.17, 153.28, 151.18, 147.90, 132.56, 132.24, 131.73, 131.44, 130.70, 129.77, 129.32, 129.19, 126.61, 120.73, 114.08, 101.28, 96.34, 79,79, 55.05, 45.74, 29.98, 28.81, 12.58. HRMS (ESI) m/z: 計算値C26H21N5O7SNa+ ([M+Na]+) 570.1083, 測定値570.1081. As an aromatic sulfonyl control unit in which the electrophilicity gradually decreases to the 5-position amino group of Nile Blue (NB), a dinitrobenzenesulfonyl group (DNs group), a mononitrobenzenesulfonyl group (MNs group) and a tosyl group ( Compounds (DNs-NB, MNs-NB and Ts-NB) into which Ts group was introduced were synthesized (Synthesis Examples 1 to 3).
[Synthesis Example 1]
<Synthesis of DNs-NB (Compound of Comparative Example 1)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 2, under an argon atmosphere. 4-Dinitrobenzenesulfonyl chloride (38 mg, 0.14 mmol) was added. After 20 minutes, the solution was warmed to an ambient temperature of 70 ° C. and stirred at that temperature for 2 hours. The reaction mixture was diluted with EtOAc and washed with water. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain DNs-NB having the following structural formula as a blue solid (9.5 mg, 0.017 mmol, 14%). Melting point> 400 ° C; 1 H NMR (500 MHz, pyridine-d5): δ 9.13 (1H, s) 8.87-8.85 (1H, d, J = 8.5 Hz), 8.59-8.76 (1H, d, J = 9.0 Hz ), 8.39-8.40 (1H, d, J = 8.5 Hz), 8.31-8.29 (1H, d, J = 9.5 Hz), 7.90 (1H, s), 7.90-7.89 (1H, d, J = 8.8 Hz) , 7.79-7.76 (1H, dd, J = 8.5, 8.0 Hz), 7.70-7.67 (1H, dd, J = 8.0, 7.5 Hz), 7.00-6.98 (1H, d, J = 9.0 Hz), 6.69 (1H , s), 3.46-3.3.41 (4H, q, J = 7.0 Hz), 1.18-1.15 (6H, t, J = 7.0 Hz) 13 C NMR (100 MHz, C 5 D 5 Nd 5):. δ 164.17, 153.28, 151.18, 147.90, 132.56, 132.24, 131.73, 131.44, 130.70, 129.77, 129.32, 129.19, 126.61, 120.73, 114.08, 101.28, 96.34, 79,79, 55.05, 45.74, 29.98, 28.81, 12.58.MS ESI) m / z: Calculated value C 26 H 21 N 5 O 7 SNa + ([M + Na] + ) 570.1083, measured value 570.1081.
［合成例１］
＜ＤＮｓ－ＮＢ（比較例１の化合物）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ、０．１８ｍｍｏｌ）及び２，４－ジニトロベンゼンスルホニルクロライド（３８ｍｇ、０．１４ｍｍｏｌ）を添加した。２０分後、該溶液を７０℃の周囲温度に温め、該温度で２時間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、水で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＤＮｓ－ＮＢを得た（９．５ｍｇ、０．０１７ｍｍｏｌ、１４％）。融点>400℃;1H NMR (500 MHz, ピリジン-d5): δ 9.13 (1H, s) 8.87-8.85 (1H, d, J = 8.5 Hz), 8.59-8.76 (1H, d, J = 9.0 Hz), 8.39-8.40 (1H, d, J = 8.5 Hz), 8.31-8.29 (1H, d, J = 9.5 Hz), 7.90 (1H, s), 7.90-7.89 (1H, d, J = 8.8 Hz), 7.79-7.76 (1H, dd, J = 8.5, 8.0 Hz), 7.70-7.67 (1H, dd, J = 8.0, 7.5 Hz), 7.00-6.98 (1H, d, J = 9.0 Hz), 6.69 (1H, s), 3.46-3.3.41 (4H, q, J = 7.0 Hz), 1.18-1.15 (6H, t, J = 7.0 Hz). 13C NMR (100 MHz, C5D5N-d5): δ 164.17, 153.28, 151.18, 147.90, 132.56, 132.24, 131.73, 131.44, 130.70, 129.77, 129.32, 129.19, 126.61, 120.73, 114.08, 101.28, 96.34, 79,79, 55.05, 45.74, 29.98, 28.81, 12.58. HRMS (ESI) m/z: 計算値C26H21N5O7SNa+ ([M+Na]+) 570.1083, 測定値570.1081. As an aromatic sulfonyl control unit in which the electrophilicity gradually decreases to the 5-position amino group of Nile Blue (NB), a dinitrobenzenesulfonyl group (DNs group), a mononitrobenzenesulfonyl group (MNs group) and a tosyl group ( Compounds (DNs-NB, MNs-NB and Ts-NB) into which Ts group was introduced were synthesized (Synthesis Examples 1 to 3).
[Synthesis Example 1]
<Synthesis of DNs-NB (Compound of Comparative Example 1)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 2, under an argon atmosphere. 4-Dinitrobenzenesulfonyl chloride (38 mg, 0.14 mmol) was added. After 20 minutes, the solution was warmed to an ambient temperature of 70 ° C. and stirred at that temperature for 2 hours. The reaction mixture was diluted with EtOAc and washed with water. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain DNs-NB having the following structural formula as a blue solid (9.5 mg, 0.017 mmol, 14%). Melting point> 400 ° C; 1 H NMR (500 MHz, pyridine-d5): δ 9.13 (1H, s) 8.87-8.85 (1H, d, J = 8.5 Hz), 8.59-8.76 (1H, d, J = 9.0 Hz ), 8.39-8.40 (1H, d, J = 8.5 Hz), 8.31-8.29 (1H, d, J = 9.5 Hz), 7.90 (1H, s), 7.90-7.89 (1H, d, J = 8.8 Hz) , 7.79-7.76 (1H, dd, J = 8.5, 8.0 Hz), 7.70-7.67 (1H, dd, J = 8.0, 7.5 Hz), 7.00-6.98 (1H, d, J = 9.0 Hz), 6.69 (1H , s), 3.46-3.3.41 (4H, q, J = 7.0 Hz), 1.18-1.15 (6H, t, J = 7.0 Hz) 13 C NMR (100 MHz, C 5 D 5 Nd 5):. δ 164.17, 153.28, 151.18, 147.90, 132.56, 132.24, 131.73, 131.44, 130.70, 129.77, 129.32, 129.19, 126.61, 120.73, 114.08, 101.28, 96.34, 79,79, 55.05, 45.74, 29.98, 28.81, 12.58.MS ESI) m / z: Calculated value C 26 H 21 N 5 O 7 SNa + ([M + Na] + ) 570.1083, measured value 570.1081.
［合成例２］
＜ＭＮｓ－ＮＢ（実施例１の化合物）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ、０．１８ｍｍｏｌ）及び４－ニトロベンゼンスルホニルクロライド（３２ｍｇ、０．１４ｍｍｏｌ）を添加した。５分後、該溶液を周囲温度に温め、該温度で１０分間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、飽和炭酸水素ナトリウム水溶液で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＭＮｓ－ＮＢを得た（９ｍｇ、０．０２ｍｍｏｌ、１５％）。融点（Ｍｐ）：272 ℃; 1H NMR (500 MHz, DMSO-d6) : δ 8.688.66 (1H, d, J = 8 Hz), 8.40-8.39 (1H, d, J = 8 Hz), 8.308.29 (2H, d, J = 8.5 Hz), 8.248.22 (2H, d, J = 8.5 Hz), 7.70 (1H, s), 7.697.67 (1H, d, J = 7.5 Hz), 7.587.55 (2H, dd, J = 8 Hz, 8 Hz), 6.82-6.81 (1H, d, J = 7.5 Hz), 6.55 (1H, s), 3.503.46 (4H, q, J = 6.5 Hz), 1.261.24 (6H, t, 6.5 Hz). 13C NMR (125 MHz, DMSO-d6): δ 164.23, 150.67, 149.56, 148.83, 147.25, 148.29, 132.01, 131.56, 131.52, 130.99, 130.76, 129.56, 128.07, 126.21, 123.99, 123.83, 112.87, 101.31, 96.92, 65.66, 46.08. HRMS (ESI) m/z: 計算値 C26H23N4O5S+ ([M+H]+) 503.1389, 測定値 503.1404. [Synthesis Example 2]
<Synthesis of MNs-NB (Compound of Example 1)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 4- Nitrobenzenesulfonyl chloride (32 mg, 0.14 mmol) was added. After 5 minutes, the solution was warmed to ambient temperature and stirred at that temperature for 10 minutes. The reaction mixture was diluted with EtOAc and washed with saturated aqueous sodium bicarbonate. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain MNs-NB having the following structural formula as a blue solid (9 mg, 0.02 mmol, 15%). Melting point (Mp): 272 ° C .; 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.688.66 (1H, d, J = 8 Hz), 8.40-8.39 (1H, d, J = 8 Hz), 8.308.29 (2H, d, J = 8.5 Hz), 8.248.22 (2H, d, J = 8.5 Hz), 7.70 (1H, s), 7.697.67 (1H, d, J = 7.5 Hz), 7.587 .55 (2H, dd, J = 8 Hz, 8 Hz), 6.82-6.81 (1H, d, J = 7.5 Hz), 6.55 (1H, s), 3.503.46 (4H, q, J = 6.5 Hz) , 1.261.24 (6H, t, 6.5 Hz) 13 C NMR (125 MHz, DMSO-d 6):. δ 164.23, 150.67, 149.56, 148.83, 147.25, 148.29, 132.01, 131.56, 131.52, 130.99, 130.76, 129.56 , 128.07, 126.21, 123.99, 123.83, 112.87, 101.31, 96.92, 65.66, 46.08.HRMS (ESI) m / z: Calculated value C 26 H 23 N 4 O 5 S + ([M + H] + ) 503.1389, measured Value 503.1404.
＜ＭＮｓ－ＮＢ（実施例１の化合物）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ、０．１８ｍｍｏｌ）及び４－ニトロベンゼンスルホニルクロライド（３２ｍｇ、０．１４ｍｍｏｌ）を添加した。５分後、該溶液を周囲温度に温め、該温度で１０分間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、飽和炭酸水素ナトリウム水溶液で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＭＮｓ－ＮＢを得た（９ｍｇ、０．０２ｍｍｏｌ、１５％）。融点（Ｍｐ）：272 ℃; 1H NMR (500 MHz, DMSO-d6) : δ 8.688.66 (1H, d, J = 8 Hz), 8.40-8.39 (1H, d, J = 8 Hz), 8.308.29 (2H, d, J = 8.5 Hz), 8.248.22 (2H, d, J = 8.5 Hz), 7.70 (1H, s), 7.697.67 (1H, d, J = 7.5 Hz), 7.587.55 (2H, dd, J = 8 Hz, 8 Hz), 6.82-6.81 (1H, d, J = 7.5 Hz), 6.55 (1H, s), 3.503.46 (4H, q, J = 6.5 Hz), 1.261.24 (6H, t, 6.5 Hz). 13C NMR (125 MHz, DMSO-d6): δ 164.23, 150.67, 149.56, 148.83, 147.25, 148.29, 132.01, 131.56, 131.52, 130.99, 130.76, 129.56, 128.07, 126.21, 123.99, 123.83, 112.87, 101.31, 96.92, 65.66, 46.08. HRMS (ESI) m/z: 計算値 C26H23N4O5S+ ([M+H]+) 503.1389, 測定値 503.1404. [Synthesis Example 2]
<Synthesis of MNs-NB (Compound of Example 1)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 4- Nitrobenzenesulfonyl chloride (32 mg, 0.14 mmol) was added. After 5 minutes, the solution was warmed to ambient temperature and stirred at that temperature for 10 minutes. The reaction mixture was diluted with EtOAc and washed with saturated aqueous sodium bicarbonate. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain MNs-NB having the following structural formula as a blue solid (9 mg, 0.02 mmol, 15%). Melting point (Mp): 272 ° C .; 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.688.66 (1H, d, J = 8 Hz), 8.40-8.39 (1H, d, J = 8 Hz), 8.308.29 (2H, d, J = 8.5 Hz), 8.248.22 (2H, d, J = 8.5 Hz), 7.70 (1H, s), 7.697.67 (1H, d, J = 7.5 Hz), 7.587 .55 (2H, dd, J = 8 Hz, 8 Hz), 6.82-6.81 (1H, d, J = 7.5 Hz), 6.55 (1H, s), 3.503.46 (4H, q, J = 6.5 Hz) , 1.261.24 (6H, t, 6.5 Hz) 13 C NMR (125 MHz, DMSO-d 6):. δ 164.23, 150.67, 149.56, 148.83, 147.25, 148.29, 132.01, 131.56, 131.52, 130.99, 130.76, 129.56 , 128.07, 126.21, 123.99, 123.83, 112.87, 101.31, 96.92, 65.66, 46.08.HRMS (ESI) m / z: Calculated value C 26 H 23 N 4 O 5 S + ([M + H] + ) 503.1389, measured Value 503.1404.
［合成例３］
＜Ｔｓ－ＮＢ（比較例２）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ，０．１８ｍｍｏｌ）及び４－メチルベンゼンスルホニルクロライド（３４ｍｇ，０．１８ｍｍｏｌ）を添加した。１５分後、該溶液を８０℃の周囲温度に温め、該温度で３０分間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、飽和炭酸水素ナトリウム水溶液で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＴｓ－ＮＢを得た（12 mg, 0.025 mmol, 21%）。融点（Ｍｐ）：246 ℃; 1H NMR (300 MHz, DMSO-d6) : δ 8.618.59(1H, d, J = 8.1 Hz), 8.448.41 (1H, d, J = 8.4 Hz), 7.957.92 (2H, d, J = 8.1 Hz), 7.267.23 (2H, d, J = 7.8 Hz), 7.667.49 (4H, m), 6.736.70 (1H, d, J = 8.7 Hz), 6.46 (1H, s), 3.473.30 (4H, q, J = 7.2 Hz), 2.35 (3H, s), 1.241.19 (6H, t, J = 6.9 Hz). 13C NMR (125 MHz, CDCl3) : δ 163.52, 150.33, 147.16, 142.58, 140.24, 138.82, 131.76, 131.61, 131.29, 131.14, 131.11, 129.87, 129.38, 126.96, 126.35, 123.76, 112.29, 96.71, 45.91, 29.86, 21.59, 12.75. HRMS (ESI) m/z: 計算値 C27H26N3O3S+ ([M+H]+) 471.1171, 測定値 471.1695. [Synthesis Example 3]
<Synthesis of Ts-NB (Comparative Example 2)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 4- Methylbenzenesulfonyl chloride (34 mg, 0.18 mmol) was added. After 15 minutes, the solution was warmed to an ambient temperature of 80 ° C. and stirred at that temperature for 30 minutes. The reaction mixture was diluted with EtOAc and washed with saturated aqueous sodium bicarbonate. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain Ts-NB having the following structural formula as a blue solid (12 mg, 0.025 mmol, 21%). Melting point (Mp): 246 ° C .; 1 H NMR (300 MHz, DMSO-d 6 ): δ 8.618.59 (1H, d, J = 8.1 Hz), 8.448.41 (1H, d, J = 8.4 Hz), 7.957.92 (2H, d, J = 8.1 Hz), 7.267.23 (2H, d, J = 7.8 Hz), 7.667.49 (4H, m), 6.736.70 (1H, d, J = 8.7 Hz) , 6.46 (1H, s), 3.473.30 (4H, q, J = 7.2 Hz), 2.35 (3H, s), 1.241.19 (6H, t, J = 6.9 Hz). 13 C NMR (125 MHz, CDCl 3 ): δ 163.52, 150.33, 147.16, 142.58, 140.24, 138.82, 131.76, 131.61, 131.29, 131.14, 131.11, 129.87, 129.38, 126.96, 126.35, 123.76, 112.29, 96.71, 45.91, 29.86, 21.59, 12.75. (ESI) m / z: Calculated value C 27 H 26 N 3 O 3 S + ([M + H] + ) 471.1171, measured value 471.1695.
＜Ｔｓ－ＮＢ（比較例２）の合成＞
無水ＤＭＦ（１．２ｍＬ）中で０℃に冷却されたナイルブルーＡ（５０ｍｇ、０．１２ｍｍｏｌ）の攪拌溶液に、アルゴン雰囲気下でＮ，Ｎ－ジイソプロピルエチルアミン（３４μＬ，０．１８ｍｍｏｌ）及び４－メチルベンゼンスルホニルクロライド（３４ｍｇ，０．１８ｍｍｏｌ）を添加した。１５分後、該溶液を８０℃の周囲温度に温め、該温度で３０分間攪拌した。この反応混合液をＥｔＯＡｃで希釈し、飽和炭酸水素ナトリウム水溶液で洗浄した。有機相をＮａ2ＳＯ4で乾燥し、ｉｎ ｖａｃｕｏエバポレート（真空内蒸発）させた。フラッシュカラムクロマトグラフィーで残渣を精製し、青色の固体として下記構造式のＴｓ－ＮＢを得た（12 mg, 0.025 mmol, 21%）。融点（Ｍｐ）：246 ℃; 1H NMR (300 MHz, DMSO-d6) : δ 8.618.59(1H, d, J = 8.1 Hz), 8.448.41 (1H, d, J = 8.4 Hz), 7.957.92 (2H, d, J = 8.1 Hz), 7.267.23 (2H, d, J = 7.8 Hz), 7.667.49 (4H, m), 6.736.70 (1H, d, J = 8.7 Hz), 6.46 (1H, s), 3.473.30 (4H, q, J = 7.2 Hz), 2.35 (3H, s), 1.241.19 (6H, t, J = 6.9 Hz). 13C NMR (125 MHz, CDCl3) : δ 163.52, 150.33, 147.16, 142.58, 140.24, 138.82, 131.76, 131.61, 131.29, 131.14, 131.11, 129.87, 129.38, 126.96, 126.35, 123.76, 112.29, 96.71, 45.91, 29.86, 21.59, 12.75. HRMS (ESI) m/z: 計算値 C27H26N3O3S+ ([M+H]+) 471.1171, 測定値 471.1695. [Synthesis Example 3]
<Synthesis of Ts-NB (Comparative Example 2)>
To a stirred solution of Nile Blue A (50 mg, 0.12 mmol) cooled to 0 ° C. in anhydrous DMF (1.2 mL) was added N, N-diisopropylethylamine (34 μL, 0.18 mmol) and 4- Methylbenzenesulfonyl chloride (34 mg, 0.18 mmol) was added. After 15 minutes, the solution was warmed to an ambient temperature of 80 ° C. and stirred at that temperature for 30 minutes. The reaction mixture was diluted with EtOAc and washed with saturated aqueous sodium bicarbonate. The organic phase was dried over Na 2 SO 4 and evaporated in vacuo (evaporation in vacuo). The residue was purified by flash column chromatography to obtain Ts-NB having the following structural formula as a blue solid (12 mg, 0.025 mmol, 21%). Melting point (Mp): 246 ° C .; 1 H NMR (300 MHz, DMSO-d 6 ): δ 8.618.59 (1H, d, J = 8.1 Hz), 8.448.41 (1H, d, J = 8.4 Hz), 7.957.92 (2H, d, J = 8.1 Hz), 7.267.23 (2H, d, J = 7.8 Hz), 7.667.49 (4H, m), 6.736.70 (1H, d, J = 8.7 Hz) , 6.46 (1H, s), 3.473.30 (4H, q, J = 7.2 Hz), 2.35 (3H, s), 1.241.19 (6H, t, J = 6.9 Hz). 13 C NMR (125 MHz, CDCl 3 ): δ 163.52, 150.33, 147.16, 142.58, 140.24, 138.82, 131.76, 131.61, 131.29, 131.14, 131.11, 129.87, 129.38, 126.96, 126.35, 123.76, 112.29, 96.71, 45.91, 29.86, 21.59, 12.75. (ESI) m / z: Calculated value C 27 H 26 N 3 O 3 S + ([M + H] + ) 471.1171, measured value 471.1695.
上記合成例１～３により得られた実施例１及び比較例１～２の化合物、並びに市販の化合物としてナイルブルー（ＮＢ）（シグマアルドリッチ社）、ナイルレッド（シグマアルドリッチ社）、下記構造のＢＯＤＩＰＹ４９３／５０３（インビトロジェン社）を用いて、種々の試験を行った。
なお、各試験例における試験方法は下記の通りである。 The compounds of Example 1 and Comparative Examples 1 and 2 obtained by Synthesis Examples 1 to 3 above, and commercially available compounds such as Nile Blue (NB) (Sigma Aldrich), Nile Red (Sigma Aldrich), BODIPY493 having the following structure Various tests were performed using / 503 (Invitrogen).
In addition, the test method in each test example is as follows.
なお、各試験例における試験方法は下記の通りである。 The compounds of Example 1 and Comparative Examples 1 and 2 obtained by Synthesis Examples 1 to 3 above, and commercially available compounds such as Nile Blue (NB) (Sigma Aldrich), Nile Red (Sigma Aldrich), BODIPY493 having the following structure Various tests were performed using / 503 (Invitrogen).
In addition, the test method in each test example is as follows.
［試験方法］
＜量子収量の測定＞
ＮＢ，ＤＮｓ－ＮＢ（比較例１）、ＭＮｓ－ＮＢ（実施例１）及びＴｓ－ＮＢ（比較例２）の１ｍＭ・ＤＭＳＯ（ジメチルスルホキシド）ストック溶液を調製した。１ｍＭ・ＤＭＳＯストック溶液の適切な希釈により調節された、所望の濃度における各化合物の各溶液の吸収スペクトルを得た（６１０ｎｍにおける吸光度＜０．０２）。蛍光の量子効率(φfl)を決定するために、蛍光スタンダードとして、メタノール（ＭｅＯＨ）中のクレシル・バイオレットを用いた(φ = 0.66)。以下の方程式から、蛍光の量子効率を得た（式中、Ｆは、各波長における蛍光強度を表し、Σ [F]は、蛍光強度の合計により求められる。）
φfl サンプル= φfl スタンダード Abs スタンダード Σ [F サンプル] / Abs サンプル Σ [F スタンダード] [Test method]
<Measurement of quantum yield>
A 1 mM DMSO (dimethyl sulfoxide) stock solution of NB, DNs-NB (Comparative Example 1), MNs-NB (Example 1) and Ts-NB (Comparative Example 2) was prepared. Absorption spectra of each solution of each compound at the desired concentration, adjusted by appropriate dilution of a 1 mM DMSO stock solution, were obtained (absorbance at 610 nm <0.02). In order to determine the quantum efficiency (φ fl ) of fluorescence, cresyl violet in methanol (MeOH) was used as the fluorescence standard (φ = 0.66). The quantum efficiency of fluorescence was obtained from the following equation (where F represents the fluorescence intensity at each wavelength, and Σ [F] is determined by the total fluorescence intensity).
φ fl sample = φ fl standard Abs standard Σ [F sample ] / Abs sample Σ [F standard ]
＜量子収量の測定＞
ＮＢ，ＤＮｓ－ＮＢ（比較例１）、ＭＮｓ－ＮＢ（実施例１）及びＴｓ－ＮＢ（比較例２）の１ｍＭ・ＤＭＳＯ（ジメチルスルホキシド）ストック溶液を調製した。１ｍＭ・ＤＭＳＯストック溶液の適切な希釈により調節された、所望の濃度における各化合物の各溶液の吸収スペクトルを得た（６１０ｎｍにおける吸光度＜０．０２）。蛍光の量子効率(φfl)を決定するために、蛍光スタンダードとして、メタノール（ＭｅＯＨ）中のクレシル・バイオレットを用いた(φ = 0.66)。以下の方程式から、蛍光の量子効率を得た（式中、Ｆは、各波長における蛍光強度を表し、Σ [F]は、蛍光強度の合計により求められる。）
φfl サンプル= φfl スタンダード Abs スタンダード Σ [F サンプル] / Abs サンプル Σ [F スタンダード] [Test method]
<Measurement of quantum yield>
A 1 mM DMSO (dimethyl sulfoxide) stock solution of NB, DNs-NB (Comparative Example 1), MNs-NB (Example 1) and Ts-NB (Comparative Example 2) was prepared. Absorption spectra of each solution of each compound at the desired concentration, adjusted by appropriate dilution of a 1 mM DMSO stock solution, were obtained (absorbance at 610 nm <0.02). In order to determine the quantum efficiency (φ fl ) of fluorescence, cresyl violet in methanol (MeOH) was used as the fluorescence standard (φ = 0.66). The quantum efficiency of fluorescence was obtained from the following equation (where F represents the fluorescence intensity at each wavelength, and Σ [F] is determined by the total fluorescence intensity).
φ fl sample = φ fl standard Abs standard Σ [F sample ] / Abs sample Σ [F standard ]
＜吸光度測定＞
１０μＭの試薬を含む各溶液において反応を行った。ＵＶ／ＶＩＳ分光法により吸光度スペクトルを観察した（V-550; JASCO）。４５０～７５０ｎｍのスキャン範囲で吸光度を得た。 <Absorbance measurement>
Reactions were performed in each solution containing 10 μM reagent. Absorbance spectra were observed by UV / VIS spectroscopy (V-550; JASCO). Absorbance was obtained in the scan range of 450-750 nm.
１０μＭの試薬を含む各溶液において反応を行った。ＵＶ／ＶＩＳ分光法により吸光度スペクトルを観察した（V-550; JASCO）。４５０～７５０ｎｍのスキャン範囲で吸光度を得た。 <Absorbance measurement>
Reactions were performed in each solution containing 10 μM reagent. Absorbance spectra were observed by UV / VIS spectroscopy (V-550; JASCO). Absorbance was obtained in the scan range of 450-750 nm.
＜蛍光測定＞
１μＭの試薬を含む各溶液において反応を行った。蛍光分光法により蛍光スペクトルを観察した（FP-6500; JASCO）。６２０～７２０ｎｍのスキャン範囲で、６１０ｎｍの励起を有する蛍光を得た（励起バンド幅（excitation band）：３ｎｍ、蛍光バンド幅（emission band）：３ｎｍ、感度：中間（medium））。 <Fluorescence measurement>
Reactions were performed in each solution containing 1 μM reagent. The fluorescence spectrum was observed by fluorescence spectroscopy (FP-6500; JASCO). Fluorescence with 610 nm excitation was obtained in the scan range of 620-720 nm (excitation band: 3 nm, emission band: 3 nm, sensitivity: medium).
１μＭの試薬を含む各溶液において反応を行った。蛍光分光法により蛍光スペクトルを観察した（FP-6500; JASCO）。６２０～７２０ｎｍのスキャン範囲で、６１０ｎｍの励起を有する蛍光を得た（励起バンド幅（excitation band）：３ｎｍ、蛍光バンド幅（emission band）：３ｎｍ、感度：中間（medium））。 <Fluorescence measurement>
Reactions were performed in each solution containing 1 μM reagent. The fluorescence spectrum was observed by fluorescence spectroscopy (FP-6500; JASCO). Fluorescence with 610 nm excitation was obtained in the scan range of 620-720 nm (excitation band: 3 nm, emission band: 3 nm, sensitivity: medium).
＜蛍光寿命測定＞
室温で、ＮＢ，ＤＮｓ－ＮＢ，ＭＮｓ－ＮＢ又はＴｓ－ＮＢを含む各溶液において、寿命測定を行った（吸収強度（absorbance intensity）< 0.02）（標準物質はLUDOXを使用した）。蛍光寿命測定装置（HORIBA TemPro-01, Nano LED-561）により蛍光寿命を観察した。 <Fluorescence lifetime measurement>
Lifetime measurements were performed in each solution containing NB, DNs-NB, MNs-NB or Ts-NB at room temperature (absorbance intensity <0.02) (LUDOX was used as the standard substance). The fluorescence lifetime was observed with a fluorescence lifetime measuring device (HORIBA TemPro-01, Nano LED-561).
室温で、ＮＢ，ＤＮｓ－ＮＢ，ＭＮｓ－ＮＢ又はＴｓ－ＮＢを含む各溶液において、寿命測定を行った（吸収強度（absorbance intensity）< 0.02）（標準物質はLUDOXを使用した）。蛍光寿命測定装置（HORIBA TemPro-01, Nano LED-561）により蛍光寿命を観察した。 <Fluorescence lifetime measurement>
Lifetime measurements were performed in each solution containing NB, DNs-NB, MNs-NB or Ts-NB at room temperature (absorbance intensity <0.02) (LUDOX was used as the standard substance). The fluorescence lifetime was observed with a fluorescence lifetime measuring device (HORIBA TemPro-01, Nano LED-561).
＜ＨｅＬａ細胞における脂肪滴の導入＞
（オレイン酸－ＢＳＡコンジュゲート）
ＨｅＬａ細胞に脂肪滴形成を誘導するために、オレイン酸－ＢＳＡ溶液の４ｍＭのストック溶液を調製した。０．１Ｍ・Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）溶液１０ｍＬに、１．４ｇのＢＳＡを添加した。清潔なスクリューキャップのついたチューブに、オレイン酸を加え、これに１０ｍＬのＢＳＡ溶液を添加し、回転式振とう器を用いて混合した。ＢＳＡ溶液を添加すると曇りが生じたが、複合体形成が完了すると曇りは消失した。得られた複合体（コンジュゲート）を０．２２μｍのフィルターユニットに通し、４℃で保存した。
（脂肪滴の導入）
１０％ＦＢＳ、０．１ｍＭ非必須アミノ酸（ＮＥＡＡ）、１ｍＭピルビン酸ナトリウム及びペニシリン／ストレプトマイシン（１×濃度）を含むＤＭＥＭ－低グルコース培地中でＨｅＬａ細胞を維持した。脂肪滴の導入のために、２４時間、３７℃、５％の二酸化炭素条件下で、２４ウェルプレートにＨｅＬａ細胞を２×１０4細胞の濃度で播いた。その後、２００μＭのオレイン酸－ＢＳＡコンジュゲートを該細胞に添加し、２４時間培養した。ネガティブコントロールの細胞は、オレイン酸－ＢＳＡコンジュゲートで処理していないＨｅＬａ細胞であった。指し示された時間後、ＮＢ、ＭＮｓ、ＭＮｓ、ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３などの試薬で細胞を染色した。 <Introduction of lipid droplets in HeLa cells>
(Oleic acid-BSA conjugate)
A 4 mM stock solution of oleic acid-BSA solution was prepared to induce lipid droplet formation in HeLa cells. 1.4 g of BSA was added to 10 mL of 0.1 M Tris-HCl (pH 8.0) solution. To a tube with a clean screw cap, oleic acid was added, 10 mL of BSA solution was added thereto and mixed using a rotary shaker. When the BSA solution was added, cloudiness occurred, but when the complex formation was completed, the cloudiness disappeared. The resulting complex (conjugate) was passed through a 0.22 μm filter unit and stored at 4 ° C.
(Introduction of fat droplets)
HeLa cells were maintained in DMEM-low glucose medium containing 10% FBS, 0.1 mM non-essential amino acid (NEAA), 1 mM sodium pyruvate and penicillin / streptomycin (1 × concentration). For the introduction of lipid droplets, HeLa cells were seeded at a concentration of 2 × 10 4 cells in 24-well plates under conditions of 5% carbon dioxide at 37 ° C. for 24 hours. Thereafter, 200 μM oleic acid-BSA conjugate was added to the cells and cultured for 24 hours. Negative control cells were HeLa cells not treated with oleic acid-BSA conjugate. After the indicated times, cells were stained with reagents such as NB, MNs, MNs, NB, Nile Red and BODIPY493 / 503.
（オレイン酸－ＢＳＡコンジュゲート）
ＨｅＬａ細胞に脂肪滴形成を誘導するために、オレイン酸－ＢＳＡ溶液の４ｍＭのストック溶液を調製した。０．１Ｍ・Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）溶液１０ｍＬに、１．４ｇのＢＳＡを添加した。清潔なスクリューキャップのついたチューブに、オレイン酸を加え、これに１０ｍＬのＢＳＡ溶液を添加し、回転式振とう器を用いて混合した。ＢＳＡ溶液を添加すると曇りが生じたが、複合体形成が完了すると曇りは消失した。得られた複合体（コンジュゲート）を０．２２μｍのフィルターユニットに通し、４℃で保存した。
（脂肪滴の導入）
１０％ＦＢＳ、０．１ｍＭ非必須アミノ酸（ＮＥＡＡ）、１ｍＭピルビン酸ナトリウム及びペニシリン／ストレプトマイシン（１×濃度）を含むＤＭＥＭ－低グルコース培地中でＨｅＬａ細胞を維持した。脂肪滴の導入のために、２４時間、３７℃、５％の二酸化炭素条件下で、２４ウェルプレートにＨｅＬａ細胞を２×１０4細胞の濃度で播いた。その後、２００μＭのオレイン酸－ＢＳＡコンジュゲートを該細胞に添加し、２４時間培養した。ネガティブコントロールの細胞は、オレイン酸－ＢＳＡコンジュゲートで処理していないＨｅＬａ細胞であった。指し示された時間後、ＮＢ、ＭＮｓ、ＭＮｓ、ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３などの試薬で細胞を染色した。 <Introduction of lipid droplets in HeLa cells>
(Oleic acid-BSA conjugate)
A 4 mM stock solution of oleic acid-BSA solution was prepared to induce lipid droplet formation in HeLa cells. 1.4 g of BSA was added to 10 mL of 0.1 M Tris-HCl (pH 8.0) solution. To a tube with a clean screw cap, oleic acid was added, 10 mL of BSA solution was added thereto and mixed using a rotary shaker. When the BSA solution was added, cloudiness occurred, but when the complex formation was completed, the cloudiness disappeared. The resulting complex (conjugate) was passed through a 0.22 μm filter unit and stored at 4 ° C.
(Introduction of fat droplets)
HeLa cells were maintained in DMEM-low glucose medium containing 10% FBS, 0.1 mM non-essential amino acid (NEAA), 1 mM sodium pyruvate and penicillin / streptomycin (1 × concentration). For the introduction of lipid droplets, HeLa cells were seeded at a concentration of 2 × 10 4 cells in 24-well plates under conditions of 5% carbon dioxide at 37 ° C. for 24 hours. Thereafter, 200 μM oleic acid-BSA conjugate was added to the cells and cultured for 24 hours. Negative control cells were HeLa cells not treated with oleic acid-BSA conjugate. After the indicated times, cells were stained with reagents such as NB, MNs, MNs, NB, Nile Red and BODIPY493 / 503.
＜フローサイトメトリー＞
サイトミックスＬＳＲ装置（Cytomics LSR instrument）(Beckman Coulter)を用いて、洗浄工程を経ることなく生ＨｅＬａ細胞懸濁液を直接分析した。以下の条件で蛍光シグナルを観察した。ＢＤ／ＬＳＲによる励起；アルゴン４８８ｎｍ／５３０／２８ＢＰ（BODIPY及びナイルレッド）、ヘリウム－ネオン６３３ｎｍ／６６０・１６ＢＰ（ＮＢ試薬）。
前方散乱（forward angle light scatter）（ＦＳＣ）、側方散乱（side angle light scatter）（ＳＳＣ）及び蛍光データを記録した。各測定について、１００００イベントよりも多く保存した。（脂肪滴を用いてあるいは用いずに）０．５μＭの試薬を用いて、１５分間３７℃でＨｅＬａ細胞の中で反応を行った。FLOWJOソフトウェア(FLOWJO)を用いてデータを分析した。 <Flow cytometry>
Using a Cytomics LSR instrument (Beckman Coulter), the raw HeLa cell suspension was directly analyzed without going through a washing step. The fluorescence signal was observed under the following conditions. Excitation with BD / LSR; Argon 488 nm / 530/28 BP (BODIPY and Nile Red), Helium-Neon 633 nm / 660 · 16 BP (NB reagent).
Forward angle light scatter (FSC), side angle light scatter (SSC) and fluorescence data were recorded. More than 10,000 events were stored for each measurement. Reactions were performed in HeLa cells for 15 minutes at 37 ° C. with 0.5 μM reagent (with or without fat droplets). Data was analyzed using FLOWJO software (FLOWJO).
サイトミックスＬＳＲ装置（Cytomics LSR instrument）(Beckman Coulter)を用いて、洗浄工程を経ることなく生ＨｅＬａ細胞懸濁液を直接分析した。以下の条件で蛍光シグナルを観察した。ＢＤ／ＬＳＲによる励起；アルゴン４８８ｎｍ／５３０／２８ＢＰ（BODIPY及びナイルレッド）、ヘリウム－ネオン６３３ｎｍ／６６０・１６ＢＰ（ＮＢ試薬）。
前方散乱（forward angle light scatter）（ＦＳＣ）、側方散乱（side angle light scatter）（ＳＳＣ）及び蛍光データを記録した。各測定について、１００００イベントよりも多く保存した。（脂肪滴を用いてあるいは用いずに）０．５μＭの試薬を用いて、１５分間３７℃でＨｅＬａ細胞の中で反応を行った。FLOWJOソフトウェア(FLOWJO)を用いてデータを分析した。 <Flow cytometry>
Using a Cytomics LSR instrument (Beckman Coulter), the raw HeLa cell suspension was directly analyzed without going through a washing step. The fluorescence signal was observed under the following conditions. Excitation with BD / LSR; Argon 488 nm / 530/28 BP (BODIPY and Nile Red), Helium-Neon 633 nm / 660 · 16 BP (NB reagent).
Forward angle light scatter (FSC), side angle light scatter (SSC) and fluorescence data were recorded. More than 10,000 events were stored for each measurement. Reactions were performed in HeLa cells for 15 minutes at 37 ° C. with 0.5 μM reagent (with or without fat droplets). Data was analyzed using FLOWJO software (FLOWJO).
＜ＵＥＥＴ－１２細胞の脂肪組織への分化＞
１０％のウシ胎仔血清（ＦＢＳ）及びペニシリン／ストレプトマイシン（１×濃度）を含むＬ－グルタミンを含むＤＭＥＭ－低グルコース培地（以下、間葉性幹細胞拡大培地（mesenchymal stem cell expansion medium）と呼ぶことがある）中で、３７℃、５％ＣＯ２濃度でＵＥＥＴ－１２細胞を培養した。脂肪組織に分化する前に、２４ウェルプレートに細胞を２×１０4cell/wellで播き、細胞がコンフルエントになるまで培養した。細胞がコンフルエントに達した後、脂質生成導入培地（adipogenesis induction medium）（１μＭデキサメタゾン、０．５ｍＭの３－イソブチル-１-メチルキサンチン（ＩＢＭＸ）及び１０μＭのインスリン）を含む１０％ＦＢＳを含むＤＭＥＭ低グルコース培地）に置き換え、２日毎に培地交換しながら７日間培養した。７日後、脂質生成導入培地を脂質生成維持培地（１０μＭのインスリンを含む１０％ＦＢＳを含むＤＭＥＭ低グルコース培地）に置き換え、さらに２日間培養した。再度脂質生成導入培地に置き換え、６日間培養した（２日毎に培地交換を行った）。その後、脂質生成維持培地に置き換え、さらに２日間培養した。脂肪滴を検出するために、ＮＢ，ＭＮｓ－ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３の各試薬を用いて、細胞を染色した。ネガティブコントロールは、未分化の細胞を用いた。 <Differentiation of UEET-12 cells into adipose tissue>
DMEM-low glucose medium (hereinafter referred to as mesenchymal stem cell expansion medium) containing L-glutamine containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (1 × concentration) In certain cases, UEET-12 cells were cultured at 37 ° C. and 5% CO 2 concentration. Before differentiation into adipose tissue, cells were seeded at 2 × 10 4 cells / well in a 24-well plate and cultured until the cells became confluent. After the cells reach confluence, DMEM low containing 10% FBS containing adipogenesis induction medium (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 10 μM insulin). The medium was replaced with a glucose medium) and cultured for 7 days while changing the medium every two days. Seven days later, the lipogenesis-introducing medium was replaced with a lipogenesis maintenance medium (DMEM low glucose medium containing 10% FBS containing 10 μM insulin), and further cultured for 2 days. The medium was again replaced with an adipogenic medium and cultured for 6 days (medium was changed every 2 days). Thereafter, the medium was replaced with an adipogenesis maintenance medium, and further cultured for 2 days. To detect lipid droplets, cells were stained with NB, MNs-NB, Nile Red, and BODIPY493 / 503 reagents. As a negative control, undifferentiated cells were used.
１０％のウシ胎仔血清（ＦＢＳ）及びペニシリン／ストレプトマイシン（１×濃度）を含むＬ－グルタミンを含むＤＭＥＭ－低グルコース培地（以下、間葉性幹細胞拡大培地（mesenchymal stem cell expansion medium）と呼ぶことがある）中で、３７℃、５％ＣＯ２濃度でＵＥＥＴ－１２細胞を培養した。脂肪組織に分化する前に、２４ウェルプレートに細胞を２×１０4cell/wellで播き、細胞がコンフルエントになるまで培養した。細胞がコンフルエントに達した後、脂質生成導入培地（adipogenesis induction medium）（１μＭデキサメタゾン、０．５ｍＭの３－イソブチル-１-メチルキサンチン（ＩＢＭＸ）及び１０μＭのインスリン）を含む１０％ＦＢＳを含むＤＭＥＭ低グルコース培地）に置き換え、２日毎に培地交換しながら７日間培養した。７日後、脂質生成導入培地を脂質生成維持培地（１０μＭのインスリンを含む１０％ＦＢＳを含むＤＭＥＭ低グルコース培地）に置き換え、さらに２日間培養した。再度脂質生成導入培地に置き換え、６日間培養した（２日毎に培地交換を行った）。その後、脂質生成維持培地に置き換え、さらに２日間培養した。脂肪滴を検出するために、ＮＢ，ＭＮｓ－ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３の各試薬を用いて、細胞を染色した。ネガティブコントロールは、未分化の細胞を用いた。 <Differentiation of UEET-12 cells into adipose tissue>
DMEM-low glucose medium (hereinafter referred to as mesenchymal stem cell expansion medium) containing L-glutamine containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (1 × concentration) In certain cases, UEET-12 cells were cultured at 37 ° C. and 5
＜脂肪滴の生細胞染色＞
ＮＢ，ＭＮｓ－ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３を用いてＨｅＬａ細胞及びＵＥＥＦ－１２細胞を染色した。３７℃、５％ＣＯ2条件下、試薬（５μＭ）を細胞に添加し、で１５分間、インキュベートした。ＰＢＳ（ｐＨ７．４）で１回洗浄した後、蛍光顕微鏡（Carl Zeiss, Gottingen, Germany）により細胞内脂肪滴を観察した。 <Live cell staining of lipid droplets>
HeLa cells and UEEF-12 cells were stained with NB, MNs-NB, Nile Red and BODIPY493 / 503. Reagent (5 μM) was added to the cells at 37 ° C., 5% CO 2 and incubated for 15 minutes. After washing once with PBS (pH 7.4), intracellular lipid droplets were observed with a fluorescence microscope (Carl Zeiss, Gottingen, Germany).
ＮＢ，ＭＮｓ－ＮＢ，ナイルレッド及びＢＯＤＩＰＹ４９３／５０３を用いてＨｅＬａ細胞及びＵＥＥＦ－１２細胞を染色した。３７℃、５％ＣＯ2条件下、試薬（５μＭ）を細胞に添加し、で１５分間、インキュベートした。ＰＢＳ（ｐＨ７．４）で１回洗浄した後、蛍光顕微鏡（Carl Zeiss, Gottingen, Germany）により細胞内脂肪滴を観察した。 <Live cell staining of lipid droplets>
HeLa cells and UEEF-12 cells were stained with NB, MNs-NB, Nile Red and BODIPY493 / 503. Reagent (5 μM) was added to the cells at 37 ° C., 5% CO 2 and incubated for 15 minutes. After washing once with PBS (pH 7.4), intracellular lipid droplets were observed with a fluorescence microscope (Carl Zeiss, Gottingen, Germany).
＜動物モデル＞
Charles River JapanからオスのC57BL/6Jマウスを得た。全てのマウスは１２時間の光と闇のサイクル下で育てられ、いつでも餌を食べることが出来る状態であった。マウスには、標準的な固形飼料（chow diet）（6%脂肪, Oriental Yeast）又は高脂肪食（D12492, 60 Kcal%脂肪, Research Diets）を与えた。全ての実験は東京大学動物実験倫理委員会（University of Tokyo Ethics Committee for Animal Experiments）により承認され、東京大学の動物実験ガイドラインに厳密に従った。 <Animal model>
Male C57BL / 6J mice were obtained from Charles River Japan. All mice were raised under a 12 hour light-dark cycle and were ready to eat. Mice were fed a standard chow diet (6% fat, Oriental Yeast) or a high fat diet (D12492, 60 Kcal% fat, Research Diets). All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly followed the University of Tokyo animal experiment guidelines.
Charles River JapanからオスのC57BL/6Jマウスを得た。全てのマウスは１２時間の光と闇のサイクル下で育てられ、いつでも餌を食べることが出来る状態であった。マウスには、標準的な固形飼料（chow diet）（6%脂肪, Oriental Yeast）又は高脂肪食（D12492, 60 Kcal%脂肪, Research Diets）を与えた。全ての実験は東京大学動物実験倫理委員会（University of Tokyo Ethics Committee for Animal Experiments）により承認され、東京大学の動物実験ガイドラインに厳密に従った。 <Animal model>
Male C57BL / 6J mice were obtained from Charles River Japan. All mice were raised under a 12 hour light-dark cycle and were ready to eat. Mice were fed a standard chow diet (6% fat, Oriental Yeast) or a high fat diet (D12492, 60 Kcal% fat, Research Diets). All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly followed the University of Tokyo animal experiment guidelines.
＜生肝臓／筋肉／脂肪組織イメージング＞
生肝臓、大腿四頭筋骨格筋（quadriceps skeletal muscles）及び精巣上体脂肪組織のイメージング方法のために、２５週齢の高脂肪食負荷肥満マウス（diet-induced-obese）及び同じ年齢の通常食痩せ型マウス（lean control mice）を用いた。
ｉｎ ｖｉｖｏの細胞動力学を視覚化するために、ウレタン（１．５ｇ／ｋｇ）を注射することによりマウスに麻酔をかけた。適度な深さの麻酔が得られた後、小さな切り込みを入れ、サリンで湿らせた。その後、この窓をラップで覆った。組織を体外に出すことなく、小さな窓（３ｍｍ以下）を介して生体内イメージングを行った。３７℃の体温を維持するために加熱パッドを用いた。
血液の流れ及び血球を視覚化するために、尾静脈を介してＦＩＴＣ－デキストラン（5 mg/kg体重、分子量150,000, Sigma）をマウスに注入し血球を視覚化した。さらに、核をｉｎ ｖｉｖｏで視覚化するため、Hoechst 33342 (1mM/kg体重, Invitrogen)を注入した。注入から１５分後、イメージングを行った。脂肪滴をｉｎ ｖｉｖｏで視覚化するために、ＤＭＳＯに溶かしたＭＮｓ－ＮＢを注入した(0.1 uM/kg体重)。レゾナンス共焦点スキャナー（resonance-scanning confocal microscopy (Nikon A1R)）用に備わっている倒立顕微鏡（Ti, Nikon）を用いて、高時空間解像度（high time and spatialresolution（160 nm/pixel））を有するイメージを得た。
各イメージにつき１秒あたり３０フレームで全ての連続イメージを得ている。使用したシステムは、405, 488, 561, 又は640nmの励起波長用の４種のレーザーラインを備えており、同時４波長励起、同時４波長測光により観察を行なった。 <Live liver / muscle / adipose tissue imaging>
For imaging methods of live liver, quadriceps skeletal muscles and epididymal adipose tissue, 25-week-old high-fat diet-induced obese mice and normal diets of the same age Lean control mice were used.
To visualize in vivo cell kinetics, mice were anesthetized by injection of urethane (1.5 g / kg). After a moderate depth of anesthesia was obtained, a small incision was made and moistened with sarin. The window was then covered with wrap. In vivo imaging was performed through a small window (3 mm or less) without taking the tissue out of the body. A heating pad was used to maintain a body temperature of 37 ° C.
To visualize blood flow and blood cells, FITC-dextran (5 mg / kg body weight, molecular weight 150,000, Sigma) was injected into mice via the tail vein to visualize blood cells. In addition, Hoechst 33342 (1 mM / kg body weight, Invitrogen) was injected to visualize the nuclei in vivo. Imaging was performed 15 minutes after injection. In order to visualize the lipid droplets in vivo, MNs-NB dissolved in DMSO was injected (0.1 uM / kg body weight). Image with high time and spatial resolution (160 nm / pixel) using an inverted microscope (Ti, Nikon) provided for resonance-scanning confocal microscopy (Nikon A1R) Got.
All consecutive images are acquired at 30 frames per second for each image. The system used was equipped with four types of laser lines for excitation wavelengths of 405, 488, 561, or 640 nm, and observation was performed by simultaneous 4-wavelength excitation and simultaneous 4-wavelength photometry.
生肝臓、大腿四頭筋骨格筋（quadriceps skeletal muscles）及び精巣上体脂肪組織のイメージング方法のために、２５週齢の高脂肪食負荷肥満マウス（diet-induced-obese）及び同じ年齢の通常食痩せ型マウス（lean control mice）を用いた。
ｉｎ ｖｉｖｏの細胞動力学を視覚化するために、ウレタン（１．５ｇ／ｋｇ）を注射することによりマウスに麻酔をかけた。適度な深さの麻酔が得られた後、小さな切り込みを入れ、サリンで湿らせた。その後、この窓をラップで覆った。組織を体外に出すことなく、小さな窓（３ｍｍ以下）を介して生体内イメージングを行った。３７℃の体温を維持するために加熱パッドを用いた。
血液の流れ及び血球を視覚化するために、尾静脈を介してＦＩＴＣ－デキストラン（5 mg/kg体重、分子量150,000, Sigma）をマウスに注入し血球を視覚化した。さらに、核をｉｎ ｖｉｖｏで視覚化するため、Hoechst 33342 (1mM/kg体重, Invitrogen)を注入した。注入から１５分後、イメージングを行った。脂肪滴をｉｎ ｖｉｖｏで視覚化するために、ＤＭＳＯに溶かしたＭＮｓ－ＮＢを注入した(0.1 uM/kg体重)。レゾナンス共焦点スキャナー（resonance-scanning confocal microscopy (Nikon A1R)）用に備わっている倒立顕微鏡（Ti, Nikon）を用いて、高時空間解像度（high time and spatialresolution（160 nm/pixel））を有するイメージを得た。
各イメージにつき１秒あたり３０フレームで全ての連続イメージを得ている。使用したシステムは、405, 488, 561, 又は640nmの励起波長用の４種のレーザーラインを備えており、同時４波長励起、同時４波長測光により観察を行なった。 <Live liver / muscle / adipose tissue imaging>
For imaging methods of live liver, quadriceps skeletal muscles and epididymal adipose tissue, 25-week-old high-fat diet-induced obese mice and normal diets of the same age Lean control mice were used.
To visualize in vivo cell kinetics, mice were anesthetized by injection of urethane (1.5 g / kg). After a moderate depth of anesthesia was obtained, a small incision was made and moistened with sarin. The window was then covered with wrap. In vivo imaging was performed through a small window (3 mm or less) without taking the tissue out of the body. A heating pad was used to maintain a body temperature of 37 ° C.
To visualize blood flow and blood cells, FITC-dextran (5 mg / kg body weight, molecular weight 150,000, Sigma) was injected into mice via the tail vein to visualize blood cells. In addition, Hoechst 33342 (1 mM / kg body weight, Invitrogen) was injected to visualize the nuclei in vivo. Imaging was performed 15 minutes after injection. In order to visualize the lipid droplets in vivo, MNs-NB dissolved in DMSO was injected (0.1 uM / kg body weight). Image with high time and spatial resolution (160 nm / pixel) using an inverted microscope (Ti, Nikon) provided for resonance-scanning confocal microscopy (Nikon A1R) Got.
All consecutive images are acquired at 30 frames per second for each image. The system used was equipped with four types of laser lines for excitation wavelengths of 405, 488, 561, or 640 nm, and observation was performed by simultaneous 4-wavelength excitation and simultaneous 4-wavelength photometry.
＜脂肪組織の間質血管フラクション（stromal vascular fraction）の培養＞
精巣上体の脂肪組織由来の間質血管（ＳＶ）フラクションを集め、コンフルエントになるまで培養した。過去に記載された方法（Am J Physiol Cell Physiol December 2006 vol. 291 no. 6 C1232-C1239）を若干修正した上で用いて、間質血管（ＳＶ）細胞を単離している。全身ヘパリン化後、一般的な麻酔条件下でマウスを犠牲にした。その後、精巣上体及び皮下の脂肪組織を取り出し、小さな断片に切り刻み、穏やかに攪拌しながら、コラゲナーゼ溶液（Tyrodeバッファー中の2 mg/mlコラゲナーゼtype 2 [Worthington]）中で２０分間培養した。消化処理された組織を遠心分離し、得られたＳＶフラクションを含むペレットをＰＢＳ中に再懸濁した後、７０μｍメッシュのフィルターでろ過した。その後、回収された細胞を、１０％ＦＢＳを加えたダルベッコ変法イーグル培地（Dulbecco's modified Eagle's medium（DMEM））で２回洗浄した。デキサメタゾン、ＩＢＭＸ（３－イソブチル－１－メチルキサンチン）及びインスリンを含む標準的な脂肪生成混合液中で該細胞を培養した。その状態で、脂肪滴の蓄積を観察するために、共焦点顕微鏡を用いて観察を行なった。 <Culture of stromal vascular fraction of adipose tissue>
Stromal blood vessel (SV) fractions from epididymal adipose tissue were collected and cultured until confluent. Stromal blood vessel (SV) cells have been isolated using a slightly modified method described in the past (Am J Physiol Cell Physiol December 2006 vol. 291 no. 6 C1232-C1239). After systemic heparinization, mice were sacrificed under general anesthesia conditions. Thereafter, epididymis and subcutaneous adipose tissue were removed, chopped into small pieces, and cultured in collagenase solution (2 mg / ml collagenase type 2 [Worthington] in Tyrode buffer) for 20 minutes with gentle agitation. The digested tissue was centrifuged, and the resulting pellet containing the SV fraction was resuspended in PBS and then filtered through a 70 μm mesh filter. Thereafter, the collected cells were washed twice with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The cells were cultured in a standard adipogenic mixture containing dexamethasone, IBMX (3-isobutyl-1-methylxanthine) and insulin. In this state, in order to observe accumulation of fat droplets, observation was performed using a confocal microscope.
精巣上体の脂肪組織由来の間質血管（ＳＶ）フラクションを集め、コンフルエントになるまで培養した。過去に記載された方法（Am J Physiol Cell Physiol December 2006 vol. 291 no. 6 C1232-C1239）を若干修正した上で用いて、間質血管（ＳＶ）細胞を単離している。全身ヘパリン化後、一般的な麻酔条件下でマウスを犠牲にした。その後、精巣上体及び皮下の脂肪組織を取り出し、小さな断片に切り刻み、穏やかに攪拌しながら、コラゲナーゼ溶液（Tyrodeバッファー中の2 mg/mlコラゲナーゼtype 2 [Worthington]）中で２０分間培養した。消化処理された組織を遠心分離し、得られたＳＶフラクションを含むペレットをＰＢＳ中に再懸濁した後、７０μｍメッシュのフィルターでろ過した。その後、回収された細胞を、１０％ＦＢＳを加えたダルベッコ変法イーグル培地（Dulbecco's modified Eagle's medium（DMEM））で２回洗浄した。デキサメタゾン、ＩＢＭＸ（３－イソブチル－１－メチルキサンチン）及びインスリンを含む標準的な脂肪生成混合液中で該細胞を培養した。その状態で、脂肪滴の蓄積を観察するために、共焦点顕微鏡を用いて観察を行なった。 <Culture of stromal vascular fraction of adipose tissue>
Stromal blood vessel (SV) fractions from epididymal adipose tissue were collected and cultured until confluent. Stromal blood vessel (SV) cells have been isolated using a slightly modified method described in the past (Am J Physiol Cell Physiol December 2006 vol. 291 no. 6 C1232-C1239). After systemic heparinization, mice were sacrificed under general anesthesia conditions. Thereafter, epididymis and subcutaneous adipose tissue were removed, chopped into small pieces, and cultured in collagenase solution (2 mg / ml collagenase type 2 [Worthington] in Tyrode buffer) for 20 minutes with gentle agitation. The digested tissue was centrifuged, and the resulting pellet containing the SV fraction was resuspended in PBS and then filtered through a 70 μm mesh filter. Thereafter, the collected cells were washed twice with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The cells were cultured in a standard adipogenic mixture containing dexamethasone, IBMX (3-isobutyl-1-methylxanthine) and insulin. In this state, in order to observe accumulation of fat droplets, observation was performed using a confocal microscope.
［試験例１］
これまでの報告された脂肪滴検出試薬の機能は、疎水性環境下でのオフ／オン機能を有するものか（ナイルレッドなど）、或いは生体中・細胞内において疎水性部分への分布が優位に起こる機能をもつもの（ＢＯＤＩＰＹ４９３／５０３など）に大別できる。この２つの機能を同時に併せ持つ試薬は、極めて有用であることが予想される。脂肪滴は、トリアシルグリセロールなどの疎水性の高い生体分子が集合した液滴である。そのため、試薬は、疎水性環境下でのみ蛍光が増強する性質が有効である。表１に示すように、各種トリアシルグリセロールが形成する脂肪滴の誘電率が報告されている。誘電率が低いほどその媒質の疎水性が高いことになる。各種トリアシルグリセロールの誘電率は、５以下の値を示している。一方、各種有機溶媒及び水の誘電率については表１に示す通りであり、クロロホルム、メタノール、エチレングリコール、アセトニトリルおよび水の誘電率は、それぞれ、４．８９、３２．３５、３１，６９、３６．００及び８０．１０である。つまり、脂肪滴に近い誘電率を示す溶媒は、クロロホルムとなる。そこで、脂肪滴検出蛍光試薬は、誘電率が低いクロロホルムで蛍光を増強し、他の誘電率が高いメタノールなどの溶媒で蛍光を増強しないものが高い特異性を示すと考えた。試薬のオフ／オン型機能を評価する際の一つの指針とした。 [Test Example 1]
The function of the lipid droplet detection reagent reported so far has an off / on function in a hydrophobic environment (such as Nile Red), or the distribution to the hydrophobic part is dominant in the body and in the cell It can be roughly divided into those having functions that occur (BODIPY493 / 503, etc.). A reagent having both of these functions at the same time is expected to be extremely useful. A lipid droplet is a droplet in which highly hydrophobic biomolecules such as triacylglycerol are assembled. Therefore, the reagent is effective in the property that fluorescence is enhanced only in a hydrophobic environment. As shown in Table 1, the dielectric constant of lipid droplets formed by various triacylglycerols has been reported. The lower the dielectric constant, the higher the hydrophobicity of the medium. The dielectric constants of various triacylglycerols have a value of 5 or less. On the other hand, the dielectric constants of various organic solvents and water are as shown in Table 1. The dielectric constants of chloroform, methanol, ethylene glycol, acetonitrile, and water are 4.89, 32.35, 31, 69, and 36, respectively. 0.00 and 80.10. That is, the solvent showing a dielectric constant close to that of fat droplets is chloroform. Therefore, it was considered that the lipid reagent for detecting lipid droplets enhances fluorescence with chloroform having a low dielectric constant, and exhibits high specificity when it does not enhance fluorescence with other solvents such as methanol having a high dielectric constant. This was used as a guideline when evaluating the off / on function of the reagent.
これまでの報告された脂肪滴検出試薬の機能は、疎水性環境下でのオフ／オン機能を有するものか（ナイルレッドなど）、或いは生体中・細胞内において疎水性部分への分布が優位に起こる機能をもつもの（ＢＯＤＩＰＹ４９３／５０３など）に大別できる。この２つの機能を同時に併せ持つ試薬は、極めて有用であることが予想される。脂肪滴は、トリアシルグリセロールなどの疎水性の高い生体分子が集合した液滴である。そのため、試薬は、疎水性環境下でのみ蛍光が増強する性質が有効である。表１に示すように、各種トリアシルグリセロールが形成する脂肪滴の誘電率が報告されている。誘電率が低いほどその媒質の疎水性が高いことになる。各種トリアシルグリセロールの誘電率は、５以下の値を示している。一方、各種有機溶媒及び水の誘電率については表１に示す通りであり、クロロホルム、メタノール、エチレングリコール、アセトニトリルおよび水の誘電率は、それぞれ、４．８９、３２．３５、３１，６９、３６．００及び８０．１０である。つまり、脂肪滴に近い誘電率を示す溶媒は、クロロホルムとなる。そこで、脂肪滴検出蛍光試薬は、誘電率が低いクロロホルムで蛍光を増強し、他の誘電率が高いメタノールなどの溶媒で蛍光を増強しないものが高い特異性を示すと考えた。試薬のオフ／オン型機能を評価する際の一つの指針とした。 [Test Example 1]
The function of the lipid droplet detection reagent reported so far has an off / on function in a hydrophobic environment (such as Nile Red), or the distribution to the hydrophobic part is dominant in the body and in the cell It can be roughly divided into those having functions that occur (BODIPY493 / 503, etc.). A reagent having both of these functions at the same time is expected to be extremely useful. A lipid droplet is a droplet in which highly hydrophobic biomolecules such as triacylglycerol are assembled. Therefore, the reagent is effective in the property that fluorescence is enhanced only in a hydrophobic environment. As shown in Table 1, the dielectric constant of lipid droplets formed by various triacylglycerols has been reported. The lower the dielectric constant, the higher the hydrophobicity of the medium. The dielectric constants of various triacylglycerols have a value of 5 or less. On the other hand, the dielectric constants of various organic solvents and water are as shown in Table 1. The dielectric constants of chloroform, methanol, ethylene glycol, acetonitrile, and water are 4.89, 32.35, 31, 69, and 36, respectively. 0.00 and 80.10. That is, the solvent showing a dielectric constant close to that of fat droplets is chloroform. Therefore, it was considered that the lipid reagent for detecting lipid droplets enhances fluorescence with chloroform having a low dielectric constant, and exhibits high specificity when it does not enhance fluorescence with other solvents such as methanol having a high dielectric constant. This was used as a guideline when evaluating the off / on function of the reagent.
［試験例２］
＜脂肪検出試薬の評価（分布特性）＞
次に、脂肪滴への選択的な分布特性を評価した。化合物の疎水性が高くなるほど脂肪滴への分布が有利になる。疎水性のパラメータとして、オクタノール・水分配計数ｌｏｇＰを用いることができる。ｌｏｇＰは、Ｖｉｓｗａｎａｄｈａｎのフラグメーション法を用いてＣｈｅｍＤｒａｗなどのソフトを用いて計算できる。ｌｏｇＰが高いほど脂肪滴への分布選択性が上がることが予想される。 [Test Example 2]
<Evaluation of fat detection reagent (distribution characteristics)>
Next, selective distribution characteristics to fat droplets were evaluated. The higher the hydrophobicity of the compound, the more advantageous the distribution to the lipid droplets. As a hydrophobic parameter, octanol / water partition count logP can be used. logP can be calculated using software such as ChemDraw using the Viswanadhan fragmentation method. It is expected that the distribution selectivity to fat droplets increases as log P increases.
＜脂肪検出試薬の評価（分布特性）＞
次に、脂肪滴への選択的な分布特性を評価した。化合物の疎水性が高くなるほど脂肪滴への分布が有利になる。疎水性のパラメータとして、オクタノール・水分配計数ｌｏｇＰを用いることができる。ｌｏｇＰは、Ｖｉｓｗａｎａｄｈａｎのフラグメーション法を用いてＣｈｅｍＤｒａｗなどのソフトを用いて計算できる。ｌｏｇＰが高いほど脂肪滴への分布選択性が上がることが予想される。 [Test Example 2]
<Evaluation of fat detection reagent (distribution characteristics)>
Next, selective distribution characteristics to fat droplets were evaluated. The higher the hydrophobicity of the compound, the more advantageous the distribution to the lipid droplets. As a hydrophobic parameter, octanol / water partition count logP can be used. logP can be calculated using software such as ChemDraw using the Viswanadhan fragmentation method. It is expected that the distribution selectivity to fat droplets increases as log P increases.
まず、これまでに報告されている脂肪滴検出試薬のｌｏｇＰを計算した（表２左欄）。
その結果、ＬＤ４５０が最も高く、４．３８を示し、ＬｉｐｉｄＧｒｅｅｎは最も低く２．９２を示した。次に、ナイルブルー（ＮＢ）、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢおよびＴｓ－ＮＢのｌｏｇＰを計算したところ、ナイルブルーが３．２を示したのに対して、誘導体であるＤＮｓ－ＮＢ、ＭＮｓ－ＮＢおよびＴｓ－ＮＢは、それぞれ５．９５、５．９９、５．１３とこれまでの試薬よりも高い値を示した（表２右欄）。ｌｏｇＰ値の計算結果は、これらＮＢ誘導体が脂肪滴への高い分布特異性を有することを支持した。 First, logP of the lipid droplet detection reagent reported so far was calculated (the left column of Table 2).
As a result, LD450 was the highest, showing 4.38, and LipidGreen was the lowest, showing 2.92. Next, when logP of Nile Blue (NB), DNs-NB, MNs-NB and Ts-NB was calculated, Nile Blue showed 3.2, whereas derivatives DNs-NB, MNs- NB and Ts-NB were 5.95, 5.99, 5.13 and higher values than the conventional reagents, respectively (Table 2 right column). The calculation result of the log P value supported that these NB derivatives have high distribution specificity to lipid droplets.
その結果、ＬＤ４５０が最も高く、４．３８を示し、ＬｉｐｉｄＧｒｅｅｎは最も低く２．９２を示した。次に、ナイルブルー（ＮＢ）、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢおよびＴｓ－ＮＢのｌｏｇＰを計算したところ、ナイルブルーが３．２を示したのに対して、誘導体であるＤＮｓ－ＮＢ、ＭＮｓ－ＮＢおよびＴｓ－ＮＢは、それぞれ５．９５、５．９９、５．１３とこれまでの試薬よりも高い値を示した（表２右欄）。ｌｏｇＰ値の計算結果は、これらＮＢ誘導体が脂肪滴への高い分布特異性を有することを支持した。 First, logP of the lipid droplet detection reagent reported so far was calculated (the left column of Table 2).
As a result, LD450 was the highest, showing 4.38, and LipidGreen was the lowest, showing 2.92. Next, when logP of Nile Blue (NB), DNs-NB, MNs-NB and Ts-NB was calculated, Nile Blue showed 3.2, whereas derivatives DNs-NB, MNs- NB and Ts-NB were 5.95, 5.99, 5.13 and higher values than the conventional reagents, respectively (Table 2 right column). The calculation result of the log P value supported that these NB derivatives have high distribution specificity to lipid droplets.
［試験例３］
＜ＮＢ誘導体を用いた光化学特性の解析＞
合成した各ＮＢ誘導体の光化学特性を解析した。第一に、誘電率の異なる各種溶媒中におけるＮＢ試薬（ＮＢ，ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ、Ｔｓ－ＮＢ）の吸収スペクトルを測定した。溶媒は、メタノール、エチレングリコール、アセトニトリル及びクロロホルムを用いた。図２に示すように、ＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢは、クロロホルム溶液下を除いて、どれもほとんど同じスペクトルの形状を示した。 [Test Example 3]
<Analysis of photochemical properties using NB derivatives>
The photochemical characteristics of each synthesized NB derivative were analyzed. First, absorption spectra of NB reagents (NB, DNs-NB, MNs-NB, Ts-NB) in various solvents having different dielectric constants were measured. As the solvent, methanol, ethylene glycol, acetonitrile, and chloroform were used. As shown in FIG. 2, NB, DNs-NB, MNs-NB and Ts-NB all showed almost the same spectral shape except under chloroform solution.
＜ＮＢ誘導体を用いた光化学特性の解析＞
合成した各ＮＢ誘導体の光化学特性を解析した。第一に、誘電率の異なる各種溶媒中におけるＮＢ試薬（ＮＢ，ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ、Ｔｓ－ＮＢ）の吸収スペクトルを測定した。溶媒は、メタノール、エチレングリコール、アセトニトリル及びクロロホルムを用いた。図２に示すように、ＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢは、クロロホルム溶液下を除いて、どれもほとんど同じスペクトルの形状を示した。 [Test Example 3]
<Analysis of photochemical properties using NB derivatives>
The photochemical characteristics of each synthesized NB derivative were analyzed. First, absorption spectra of NB reagents (NB, DNs-NB, MNs-NB, Ts-NB) in various solvents having different dielectric constants were measured. As the solvent, methanol, ethylene glycol, acetonitrile, and chloroform were used. As shown in FIG. 2, NB, DNs-NB, MNs-NB and Ts-NB all showed almost the same spectral shape except under chloroform solution.
各試薬の最大吸光波長のモル吸光係数を算出した（表３）。各試薬の値を比較したところ、ＭＮｓ－ＮＢが最も低い値を示した。また、クロロホルム溶媒下の試薬の値は、メタノールより高い値を示した。これは、親水性溶液中では、各試薬の基底状態において複数の平衡構造が存在するためであると予想される。極性の高いメタノール中では、カチオン性の平衡構造が溶解するのに有利である。一方、クロロホルム中では電荷のない分子構造のみが存在すると考えられる。いずれの試薬も疎水性の高いクロロホルム溶液中で青色シフトを示しており、特にＭＮｓ－ＮＢとＴｓ－ＮＢにおいて顕著である（図２）。疎水性溶媒の青色シフトは、基底状態構造よりも励起状態構造の双極子遷移モーメントが大きいことを示している。これは、ＮＢがπ－π励起状態を取っているためであると予想される。
The molar extinction coefficient of the maximum absorption wavelength of each reagent was calculated (Table 3). When the value of each reagent was compared, MNs-NB showed the lowest value. Moreover, the value of the reagent under chloroform solvent was higher than that of methanol. This is expected to be due to the presence of a plurality of equilibrium structures in the ground state of each reagent in the hydrophilic solution. In highly polar methanol, it is advantageous to dissolve the cationic equilibrium structure. On the other hand, only an uncharged molecular structure exists in chloroform. Each reagent shows a blue shift in a highly hydrophobic chloroform solution, and is particularly remarkable in MNs-NB and Ts-NB (FIG. 2). The blue shift of the hydrophobic solvent indicates that the dipole transition moment of the excited state structure is greater than the ground state structure. This is expected because NB is in a π-π excited state.
次に、ナイルブルー（ＮＢ）、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢおよびＴｓ－ＮＢの各試薬の蛍光スペクトルを解析した（図３）。溶媒は、水（ＰＢＳ、ｐＨ７．４）、メタノール、アセトニトリル、エチレングリコール及びクロロホルムを用いた。水中においてはＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢのどの試薬もその蛍光強度は低かった。最も極性の低いクロロホルム溶液中においては、ＤＮｓ－ＮＢの蛍光は極めて弱いが、他のＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢの蛍光は強かった。さらに、誘電率３０付近のメタノール、アセトニトリル、エチレングリコール溶液中においては、ＮＢとＴｓ－ＮＢは蛍光を与えるが、ＤＮｓ－ＮＢ及びＭＮｓ－ＮＢはほとんど蛍光を与えなかった。
試験例１の結果から、脂肪滴検出試薬の設計指針として、誘電率の低いクロロホルム溶液でのみ蛍光発光が強くなり、それよりも誘電率の高い溶媒では蛍光発光が弱くなる光化学特性が望まれる。したがって、蛍光スペクトル解析の結果からは、ＭＮｓ－ＮＢがこの設計指針に合致する試薬であるといえる。 Next, the fluorescence spectra of Nile Blue (NB), DNs-NB, MNs-NB, and Ts-NB reagents were analyzed (FIG. 3). As the solvent, water (PBS, pH 7.4), methanol, acetonitrile, ethylene glycol and chloroform were used. In water, the fluorescence intensity of all reagents NB, DNs-NB, MNs-NB and Ts-NB was low. In the least polar chloroform solution, the fluorescence of DNs-NB was very weak, while the fluorescence of other NB, MNs-NB and Ts-NB was strong. Furthermore, in methanol, acetonitrile, and ethylene glycol solutions with a dielectric constant of around 30, NB and Ts-NB gave fluorescence, but DNs-NB and MNs-NB gave little fluorescence.
From the results of Test Example 1, as a design guideline for the lipid droplet detection reagent, photochemical characteristics that enhance fluorescence emission only with a chloroform solution having a low dielectric constant and weaken fluorescence emission with a solvent having a higher dielectric constant are desired. Therefore, from the result of fluorescence spectrum analysis, it can be said that MNs-NB is a reagent that matches this design guideline.
試験例１の結果から、脂肪滴検出試薬の設計指針として、誘電率の低いクロロホルム溶液でのみ蛍光発光が強くなり、それよりも誘電率の高い溶媒では蛍光発光が弱くなる光化学特性が望まれる。したがって、蛍光スペクトル解析の結果からは、ＭＮｓ－ＮＢがこの設計指針に合致する試薬であるといえる。 Next, the fluorescence spectra of Nile Blue (NB), DNs-NB, MNs-NB, and Ts-NB reagents were analyzed (FIG. 3). As the solvent, water (PBS, pH 7.4), methanol, acetonitrile, ethylene glycol and chloroform were used. In water, the fluorescence intensity of all reagents NB, DNs-NB, MNs-NB and Ts-NB was low. In the least polar chloroform solution, the fluorescence of DNs-NB was very weak, while the fluorescence of other NB, MNs-NB and Ts-NB was strong. Furthermore, in methanol, acetonitrile, and ethylene glycol solutions with a dielectric constant of around 30, NB and Ts-NB gave fluorescence, but DNs-NB and MNs-NB gave little fluorescence.
From the results of Test Example 1, as a design guideline for the lipid droplet detection reagent, photochemical characteristics that enhance fluorescence emission only with a chloroform solution having a low dielectric constant and weaken fluorescence emission with a solvent having a higher dielectric constant are desired. Therefore, from the result of fluorescence spectrum analysis, it can be said that MNs-NB is a reagent that matches this design guideline.
［試験例４］
＜ＮＢ誘導体の蛍光発光特性の解析＞
次に、蛍光発光特性を明らかにするために詳細なスペクトル解析を行った。各試薬の各溶媒における蛍光量子収率（φf）及び蛍光寿命（τf）を測定した（表４）。 [Test Example 4]
<Analysis of fluorescence emission characteristics of NB derivative>
Next, detailed spectral analysis was performed to clarify the fluorescence emission characteristics. The fluorescence quantum yield (φ f ) and fluorescence lifetime (τ f ) of each reagent in each solvent were measured (Table 4).
＜ＮＢ誘導体の蛍光発光特性の解析＞
次に、蛍光発光特性を明らかにするために詳細なスペクトル解析を行った。各試薬の各溶媒における蛍光量子収率（φf）及び蛍光寿命（τf）を測定した（表４）。 [Test Example 4]
<Analysis of fluorescence emission characteristics of NB derivative>
Next, detailed spectral analysis was performed to clarify the fluorescence emission characteristics. The fluorescence quantum yield (φ f ) and fluorescence lifetime (τ f ) of each reagent in each solvent were measured (Table 4).
図４に蛍光発光における吸収、発光とエネルギーの関係を概略した。基底状態にある分子（Ｓ0）は、光吸収（ｈｖ）によって電子遷移が起こり、１０-15秒程度の時間で励起状態（Ｓ1）に変化する。その後、励起状態にある分子が、基底状態に戻るときに蛍光発光が起こる。蛍光スペクトルで得られる単位濃度当たりの蛍光強度は、モル吸光係数（ε）と蛍光量子収率（φf）に比例する（蛍光強度∝モル吸光係数（ε）×蛍光量子収率（φf））。この蛍光強度がいわゆる蛍光化合物の感度に当たるものである。蛍光量子収率（φf）は、放射緩和速度定数（ｋr）と無放射緩和速度定数（ｋnr）の関係式（１）において表される。すなわち、放射緩和速度定数が大きくなるほど、また無放射緩和速度定数が小さくなるほど蛍光量子収率は高くなる。さらに、実験で測定できる蛍光寿命（τf）は、放射緩和速度定数（ｋf）と無放射緩和速度定数（ｋnr）と式（２）の関係にある。
蛍光量子収率と蛍光寿命の実験値を用いて、式（１）と式（２）から放射緩和速度定数（ｋr）と無放射緩和速度定数（ｋnr）の値を求めることができる。その結果を表５に示す。 FIG. 4 outlines the relationship between absorption, emission and energy in fluorescence emission. The molecule (S 0 ) in the ground state undergoes an electronic transition due to light absorption (hv), and changes to an excited state (S 1 ) in a time of about 10 −15 seconds. Thereafter, fluorescence emission occurs when the molecule in the excited state returns to the ground state. The fluorescence intensity per unit concentration obtained in the fluorescence spectrum is proportional to the molar extinction coefficient (ε) and the fluorescence quantum yield (φ f ) (fluorescence intensity ∝molar extinction coefficient (ε) × fluorescence quantum yield (φ f ) ). This fluorescence intensity corresponds to the sensitivity of a so-called fluorescent compound. The fluorescence quantum yield (φ f ) is expressed in the relational expression (1) between the radiation relaxation rate constant (k r ) and the non-radiation relaxation rate constant (k nr ). That is, the fluorescence quantum yield increases as the radiation relaxation rate constant increases and as the non-radiation relaxation rate constant decreases. Furthermore, the fluorescence lifetime (τ f ) that can be measured in an experiment has a relationship of the radiation relaxation rate constant (k f ), the non-radiation relaxation rate constant (k nr ), and the equation (2).
Using the experimental values of the fluorescence quantum yield and the fluorescence lifetime, the values of the radiation relaxation rate constant (k r ) and the non-radiation relaxation rate constant (k nr ) can be obtained from the equations (1) and (2). The results are shown in Table 5.
蛍光量子収率と蛍光寿命の実験値を用いて、式（１）と式（２）から放射緩和速度定数（ｋr）と無放射緩和速度定数（ｋnr）の値を求めることができる。その結果を表５に示す。 FIG. 4 outlines the relationship between absorption, emission and energy in fluorescence emission. The molecule (S 0 ) in the ground state undergoes an electronic transition due to light absorption (hv), and changes to an excited state (S 1 ) in a time of about 10 −15 seconds. Thereafter, fluorescence emission occurs when the molecule in the excited state returns to the ground state. The fluorescence intensity per unit concentration obtained in the fluorescence spectrum is proportional to the molar extinction coefficient (ε) and the fluorescence quantum yield (φ f ) (fluorescence intensity ∝molar extinction coefficient (ε) × fluorescence quantum yield (φ f ) ). This fluorescence intensity corresponds to the sensitivity of a so-called fluorescent compound. The fluorescence quantum yield (φ f ) is expressed in the relational expression (1) between the radiation relaxation rate constant (k r ) and the non-radiation relaxation rate constant (k nr ). That is, the fluorescence quantum yield increases as the radiation relaxation rate constant increases and as the non-radiation relaxation rate constant decreases. Furthermore, the fluorescence lifetime (τ f ) that can be measured in an experiment has a relationship of the radiation relaxation rate constant (k f ), the non-radiation relaxation rate constant (k nr ), and the equation (2).
Using the experimental values of the fluorescence quantum yield and the fluorescence lifetime, the values of the radiation relaxation rate constant (k r ) and the non-radiation relaxation rate constant (k nr ) can be obtained from the equations (1) and (2). The results are shown in Table 5.
メタノール中において、各ＮＢ試薬の蛍光強度がクロロホルム中と比べて低い値となっている（図３）。この結果は、蛍光寿命が短いことと、蛍光量子収率が低いことの両方に起因する（表４）。一方、各種有機溶媒において、各ＮＢ試薬の蛍光強度は変化しているが、モル吸光係数はほぼ同じ値を示している。これは、各有機溶媒中において、励起効率はほぼ同じであるが、蛍光量子収率が変化することから起こる。蛍光量子収率の変化は、各有機溶媒において放射緩和速度と無放射緩和速度の割合が変化することから起こっている。例えば、ＭＮｓ－ＮＢの蛍光量子収率はクロロホルム、メタノールで０．２１、０．０３と低下するのに対して、その放射緩和速度定数も０．０５５、０．０３０と低下する。一方、その無放射緩和速度定数は、０．２０３、０．９６０と増加する。特に、ＭＮｓ－ＮＢはメタノール中において無放射緩和の経路が極めて優勢であることがわかる。ＤＮｓ－ＮＢはすべての有機溶媒中で蛍光寿命（τf）が測定不可能であったため、ｋrおよびｋnrの算出はできなかった。しかしながら、モル吸光係数は励起効率が高いことを示している。そのため、ＤＮｓ－ＮＢは、励起状態から高い確率で無放射緩和過程を通じて基底状態に戻ることが明らかである。表５から、まず、より極性の高い溶媒中において、励起状態分子が無放射緩和過程を通じて基底状態に戻る確率が高くなっていることが判る。さらに、芳香族スルホニル制御ユニット上の置換基が求電子性であるほど、無放射緩和過程の確立が高くなっていることが判る。
In methanol, the fluorescence intensity of each NB reagent is lower than that in chloroform (FIG. 3). This result is due to both the short fluorescence lifetime and the low fluorescence quantum yield (Table 4). On the other hand, in various organic solvents, the fluorescence intensity of each NB reagent varies, but the molar extinction coefficient shows almost the same value. This occurs because the excitation efficiency is almost the same in each organic solvent, but the fluorescence quantum yield changes. The change in the fluorescence quantum yield occurs because the ratio between the radiation relaxation rate and the non-radiation relaxation rate changes in each organic solvent. For example, the fluorescence quantum yield of MNs-NB decreases with chloroform and methanol to 0.21 and 0.03, while its radiation relaxation rate constant also decreases to 0.055 and 0.030. On the other hand, the non-radiative relaxation rate constant increases to 0.203 and 0.960. In particular, it can be seen that MNs-NB has a dominant route of non-radiative relaxation in methanol. Since DNs-NB could not measure the fluorescence lifetime (τ f ) in all organic solvents, k r and k nr could not be calculated. However, the molar extinction coefficient indicates high excitation efficiency. Therefore, it is clear that DNs-NB returns to the ground state through the non-radiative relaxation process with high probability from the excited state. From Table 5, it can be seen that first, in a more polar solvent, the probability that the excited state molecule returns to the ground state through the non-radiative relaxation process is high. Furthermore, it can be seen that the more electrophilic the substituent on the aromatic sulfonyl control unit, the higher the establishment of a non-radiative relaxation process.
次に、各種ＮＢ試薬（ＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢ）の光学特性及び物理化学特性を、既存の脂肪滴検出試薬（ナイルレッド及びＢＯＤＩＰＹ４９３／５０３）と比較検討した。励起波長６１０ｎｍで、クロロホルム溶液中の蛍光スペクトルを解析した結果、ＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢの最大蛍光波長は、それぞれ、６４４ｎｍ、６５８ｎｍ、６５１ｎｍ及び６４２ｎｍを示した（図３）。一方、ナイルレッド及びＢＯＤＩＰＹ４９３／５０３の最大蛍光波長は、５９０ｎｍ及び５０３ｎｍを示すことから（図５）、長波長の蛍光シグナルを与えるＮＢ試薬がより生体イメージングには適している。脂肪滴検出試薬のための評価基準として、クロロホルムと水中における輝度の比をＳ／Ｂ（シグナル／バックグラウンド）比とした。これによって、疎水性環境で蛍光を増強するオフ／オン機能を数値で評価できる。輝度の値は、モル吸光係数（ε）の値と蛍光量子収率（Φf）の値の積算により算出した。ＮＢ、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢの値は、それぞれ表６に示した。その結果、ＭＮｓ－ＮＢが９．１と最も高いＳ／Ｂ比を示した。一方、既存のナイルレッドおよびＢＯＤＩＰＹ４９３／５０３のＳ／Ｂ比は、４．７及び１．０を示した。
以上の結果から、ＭＮｓ－ＮＢは、ｌｏｇＰの値だけでなくＳ／Ｂ比の値も高く、オフ／オン機能及び脂肪滴への高い分布特性のいずれをも兼ね備えていることが判った。したがって、ＭＮｓ－ＮＢは、オフ／オン機能又は疎水性環境に分布しやすいかオフ／オン機能を有するかのいずれか１つを有する従来の化合物よりも、脂肪滴を特異的に検出することができ、脂肪滴検出試薬として優れているといえる。 Next, the optical properties and physicochemical properties of various NB reagents (NB, DNs-NB, MNs-NB and Ts-NB) were compared with those of existing lipid droplet detection reagents (Nile Red and BODIPY493 / 503). As a result of analyzing the fluorescence spectrum in the chloroform solution at an excitation wavelength of 610 nm, the maximum fluorescence wavelengths of NB, DNs-NB, MNs-NB and Ts-NB were 644 nm, 658 nm, 651 nm and 642 nm, respectively (FIG. 3). ). On the other hand, since the maximum fluorescence wavelengths of Nile Red and BODIPY493 / 503 are 590 nm and 503 nm (FIG. 5), an NB reagent that gives a long-wavelength fluorescence signal is more suitable for biological imaging. As an evaluation standard for the lipid droplet detection reagent, the ratio of luminance in chloroform to water was defined as the S / B (signal / background) ratio. This allows numerical evaluation of the off / on function that enhances fluorescence in a hydrophobic environment. The luminance value was calculated by integrating the molar extinction coefficient (ε) value and the fluorescence quantum yield (Φ f ) value. The values of NB, DNs-NB, MNs-NB and Ts-NB are shown in Table 6, respectively. As a result, MNs-NB showed the highest S / B ratio of 9.1. On the other hand, the S / B ratios of existing Nile Red and BODIPY493 / 503 were 4.7 and 1.0.
From the above results, it was found that MNs-NB has not only a logP value but also a high S / B ratio value, and has both an off / on function and a high distribution characteristic to fat droplets. Therefore, MNs-NB can detect lipid droplets more specifically than conventional compounds having either one of off / on function or easily distributed in a hydrophobic environment or having off / on function. It can be said that it is excellent as a lipid droplet detection reagent.
以上の結果から、ＭＮｓ－ＮＢは、ｌｏｇＰの値だけでなくＳ／Ｂ比の値も高く、オフ／オン機能及び脂肪滴への高い分布特性のいずれをも兼ね備えていることが判った。したがって、ＭＮｓ－ＮＢは、オフ／オン機能又は疎水性環境に分布しやすいかオフ／オン機能を有するかのいずれか１つを有する従来の化合物よりも、脂肪滴を特異的に検出することができ、脂肪滴検出試薬として優れているといえる。 Next, the optical properties and physicochemical properties of various NB reagents (NB, DNs-NB, MNs-NB and Ts-NB) were compared with those of existing lipid droplet detection reagents (Nile Red and BODIPY493 / 503). As a result of analyzing the fluorescence spectrum in the chloroform solution at an excitation wavelength of 610 nm, the maximum fluorescence wavelengths of NB, DNs-NB, MNs-NB and Ts-NB were 644 nm, 658 nm, 651 nm and 642 nm, respectively (FIG. 3). ). On the other hand, since the maximum fluorescence wavelengths of Nile Red and BODIPY493 / 503 are 590 nm and 503 nm (FIG. 5), an NB reagent that gives a long-wavelength fluorescence signal is more suitable for biological imaging. As an evaluation standard for the lipid droplet detection reagent, the ratio of luminance in chloroform to water was defined as the S / B (signal / background) ratio. This allows numerical evaluation of the off / on function that enhances fluorescence in a hydrophobic environment. The luminance value was calculated by integrating the molar extinction coefficient (ε) value and the fluorescence quantum yield (Φ f ) value. The values of NB, DNs-NB, MNs-NB and Ts-NB are shown in Table 6, respectively. As a result, MNs-NB showed the highest S / B ratio of 9.1. On the other hand, the S / B ratios of existing Nile Red and BODIPY493 / 503 were 4.7 and 1.0.
From the above results, it was found that MNs-NB has not only a logP value but also a high S / B ratio value, and has both an off / on function and a high distribution characteristic to fat droplets. Therefore, MNs-NB can detect lipid droplets more specifically than conventional compounds having either one of off / on function or easily distributed in a hydrophobic environment or having off / on function. It can be said that it is excellent as a lipid droplet detection reagent.
＜各置換基の求電子性評価＞
試験例３、４から、蛍光発光特性は芳香族スルホニル制御ユニット（すなわち、一般式（Ｉ）の「－ＳＯ2－Ｘ」基部分）の置換基の求電子性に応じて変化し得ること、並びに、求電子性の低いＤＮｓ－ＮＢ（２，４－ジニトロベンゼン）は各溶媒下において無蛍光を示し、求電子性の高いＴｓ－ＮＢ（４－メチルベンゼン）は逆に全ての溶媒下において高い蛍光強度を示すのに対し、求電子性が両化合物の間にあるＭＮｓ－ＮＢはクロロホルム溶媒にのみ高い蛍光スペクトルを示すことが判った。すなわち、芳香族スルホニル制御ユニットの置換基の電子密度を一定の範囲内にすることが、脂肪滴を特異的に検出する化合物を得るのに重要であると判った。
そこで、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢがそれぞれ有する２，４－ジニトロベンジル基、４－ニトロベンジル基、４－メチルベンジル基を含む、種々のベンゼン誘導体の電子密度を、計算化学的手法を用いて算出した（表７）。なお、ベンゼンのＣ１の電子密度の値は、密度汎関数法のＢ３ＬＹＰ／６－３１Ｇ＊の計算レベルを用いて算出した。計算ソフトウェアはスパルタン’０８を使用した。
表７から判るように、ベンゼン環の１位の炭素原子の電子密度が低い程、ベンゼン誘導体は求電子性である。例えば、２，４－ジニトロベンゼンの電子密度は、－０．１８６（エレクトロン）と最も低い絶対値を示すことから、最も求電子性であるといえる。一方、４－メチルベンゼンの電子密度は最も高い絶対値を示し、最も求電子性が低いこととなる。他の８種類のベンゼン誘導体の求電子性は、２，４－ジニトロベンゼンと４－メチルベンゼンの間にある。
したがって、本発明の化合物は、２，４－ジニトロベンジル基の電子密度（－０．１８６）よりも求電子性が高く、４－メチルベンジル基の電子密度（－０．２４３）よりも求電子性が高い基を、ＮＢの５位のアミノ基にスルホニル基を介して結合させることが重要であり、これによりクロロホルム溶媒で顕著に高い蛍光スペクトルを示し、脂肪滴の検出に好適に用いることができる化合物を得ることができると理解できる。 <Electrophilic evaluation of each substituent>
From Test Examples 3 and 4, the fluorescence emission characteristics can be changed according to the electrophilicity of the substituent of the aromatic sulfonyl control unit (that is, the “—SO 2 —X” group moiety of the general formula (I)), In addition, DNs-NB (2,4-dinitrobenzene) with low electrophilicity shows no fluorescence under each solvent, and Ts-NB (4-methylbenzene) with high electrophilicity is conversely under all solvents. In contrast to the high fluorescence intensity, MNs-NB, which has electrophilicity between both compounds, was found to show a high fluorescence spectrum only in the chloroform solvent. That is, it has been found that it is important to obtain a compound that specifically detects lipid droplets by setting the electron density of the substituent of the aromatic sulfonyl control unit within a certain range.
Therefore, the electron density of various benzene derivatives containing 2,4-dinitrobenzyl group, 4-nitrobenzyl group, and 4-methylbenzyl group respectively possessed by DNs-NB, MNs-NB and Ts-NB are calculated by chemical calculation. Calculations were made using the method (Table 7). In addition, the value of the electron density of C1 of benzene was calculated using a calculation level of B3LYP / 6-31G * of the density functional method. The calculation software used was Spartan '08.
As can be seen from Table 7, the lower the electron density of the carbon atom at the 1-position of the benzene ring, the more electrophilic the benzene derivative. For example, the electron density of 2,4-dinitrobenzene has the lowest absolute value of -0.186 (electrons), so it can be said to be the most electrophilic. On the other hand, the electron density of 4-methylbenzene has the highest absolute value and the lowest electrophilicity. The electrophilicity of the other eight benzene derivatives is between 2,4-dinitrobenzene and 4-methylbenzene.
Therefore, the compound of the present invention has higher electrophilicity than the electron density of 2,4-dinitrobenzyl group (−0.186), and more electrophilic than the electron density of 4-methylbenzyl group (−0.243). It is important to bond a highly functional group to the amino group at the 5-position of NB via a sulfonyl group, and as a result, it shows a significantly high fluorescence spectrum in a chloroform solvent and can be suitably used for detection of lipid droplets. It can be understood that a compound that can be obtained can be obtained.
試験例３、４から、蛍光発光特性は芳香族スルホニル制御ユニット（すなわち、一般式（Ｉ）の「－ＳＯ2－Ｘ」基部分）の置換基の求電子性に応じて変化し得ること、並びに、求電子性の低いＤＮｓ－ＮＢ（２，４－ジニトロベンゼン）は各溶媒下において無蛍光を示し、求電子性の高いＴｓ－ＮＢ（４－メチルベンゼン）は逆に全ての溶媒下において高い蛍光強度を示すのに対し、求電子性が両化合物の間にあるＭＮｓ－ＮＢはクロロホルム溶媒にのみ高い蛍光スペクトルを示すことが判った。すなわち、芳香族スルホニル制御ユニットの置換基の電子密度を一定の範囲内にすることが、脂肪滴を特異的に検出する化合物を得るのに重要であると判った。
そこで、ＤＮｓ－ＮＢ、ＭＮｓ－ＮＢ及びＴｓ－ＮＢがそれぞれ有する２，４－ジニトロベンジル基、４－ニトロベンジル基、４－メチルベンジル基を含む、種々のベンゼン誘導体の電子密度を、計算化学的手法を用いて算出した（表７）。なお、ベンゼンのＣ１の電子密度の値は、密度汎関数法のＢ３ＬＹＰ／６－３１Ｇ＊の計算レベルを用いて算出した。計算ソフトウェアはスパルタン’０８を使用した。
表７から判るように、ベンゼン環の１位の炭素原子の電子密度が低い程、ベンゼン誘導体は求電子性である。例えば、２，４－ジニトロベンゼンの電子密度は、－０．１８６（エレクトロン）と最も低い絶対値を示すことから、最も求電子性であるといえる。一方、４－メチルベンゼンの電子密度は最も高い絶対値を示し、最も求電子性が低いこととなる。他の８種類のベンゼン誘導体の求電子性は、２，４－ジニトロベンゼンと４－メチルベンゼンの間にある。
したがって、本発明の化合物は、２，４－ジニトロベンジル基の電子密度（－０．１８６）よりも求電子性が高く、４－メチルベンジル基の電子密度（－０．２４３）よりも求電子性が高い基を、ＮＢの５位のアミノ基にスルホニル基を介して結合させることが重要であり、これによりクロロホルム溶媒で顕著に高い蛍光スペクトルを示し、脂肪滴の検出に好適に用いることができる化合物を得ることができると理解できる。 <Electrophilic evaluation of each substituent>
From Test Examples 3 and 4, the fluorescence emission characteristics can be changed according to the electrophilicity of the substituent of the aromatic sulfonyl control unit (that is, the “—SO 2 —X” group moiety of the general formula (I)), In addition, DNs-NB (2,4-dinitrobenzene) with low electrophilicity shows no fluorescence under each solvent, and Ts-NB (4-methylbenzene) with high electrophilicity is conversely under all solvents. In contrast to the high fluorescence intensity, MNs-NB, which has electrophilicity between both compounds, was found to show a high fluorescence spectrum only in the chloroform solvent. That is, it has been found that it is important to obtain a compound that specifically detects lipid droplets by setting the electron density of the substituent of the aromatic sulfonyl control unit within a certain range.
Therefore, the electron density of various benzene derivatives containing 2,4-dinitrobenzyl group, 4-nitrobenzyl group, and 4-methylbenzyl group respectively possessed by DNs-NB, MNs-NB and Ts-NB are calculated by chemical calculation. Calculations were made using the method (Table 7). In addition, the value of the electron density of C1 of benzene was calculated using a calculation level of B3LYP / 6-31G * of the density functional method. The calculation software used was Spartan '08.
As can be seen from Table 7, the lower the electron density of the carbon atom at the 1-position of the benzene ring, the more electrophilic the benzene derivative. For example, the electron density of 2,4-dinitrobenzene has the lowest absolute value of -0.186 (electrons), so it can be said to be the most electrophilic. On the other hand, the electron density of 4-methylbenzene has the highest absolute value and the lowest electrophilicity. The electrophilicity of the other eight benzene derivatives is between 2,4-dinitrobenzene and 4-methylbenzene.
Therefore, the compound of the present invention has higher electrophilicity than the electron density of 2,4-dinitrobenzyl group (−0.186), and more electrophilic than the electron density of 4-methylbenzyl group (−0.243). It is important to bond a highly functional group to the amino group at the 5-position of NB via a sulfonyl group, and as a result, it shows a significantly high fluorescence spectrum in a chloroform solvent and can be suitably used for detection of lipid droplets. It can be understood that a compound that can be obtained can be obtained.
［試験例５］
＜脂肪滴形成細胞での検討＞
今回合成したＭＮｓ－ＮＢ及びＴｓ－ＮＢを用いて細胞内脂肪滴のイメージングモデルを実験系で検討した。ＤＮｓ－ＮＢは細胞内でＧＳＨなどのチオールと反応するため、細胞実験には用いなかった。比較としてＮＢを用いて検討した。ＨｅＬａ細胞にオレイン酸（０．２ｍＭ）を添加し、３７℃、２４時間インキュベートすることで、細胞内に脂肪滴を有するモデル細胞を作成した。脂肪滴を内部に形成しているＨｅＬａ細胞（脂肪滴（＋））に、１μＭの各種試薬を添加し、３７℃、１５分間インキュベートした後に、蛍光顕微鏡を用いてイメージング像を得た（図６）。いずれの試薬も、脂肪滴部分からの蛍光シグナルを観測できたが、そのシグナル・バックグラウンドに違いがあった。ＭＮｓ－ＮＢは脂肪滴部分のみから顕著な蛍光シグナルを与えた。一方で、ＮＢ及びＴｓ－ＮＢでは、脂肪滴以外の細胞質全体からも非特異的な蛍光シグナルが観測された。 [Test Example 5]
<Examination with lipid droplet-forming cells>
An imaging model of intracellular lipid droplets was examined in an experimental system using MNs-NB and Ts-NB synthesized this time. Since DNs-NB reacts with thiols such as GSH in cells, it was not used in cell experiments. NB was used for comparison. By adding oleic acid (0.2 mM) to HeLa cells and incubating at 37 ° C. for 24 hours, model cells having lipid droplets in the cells were prepared. 1 μM various reagents were added to HeLa cells (fat droplets (+)) in which lipid droplets were formed, and after incubation at 37 ° C. for 15 minutes, an imaging image was obtained using a fluorescence microscope (FIG. 6). ). Each reagent was able to observe a fluorescent signal from the lipid droplet portion, but there was a difference in the signal background. MNs-NB gave a significant fluorescent signal only from the lipid droplet portion. On the other hand, in NB and Ts-NB, non-specific fluorescence signals were also observed from the whole cytoplasm other than lipid droplets.
＜脂肪滴形成細胞での検討＞
今回合成したＭＮｓ－ＮＢ及びＴｓ－ＮＢを用いて細胞内脂肪滴のイメージングモデルを実験系で検討した。ＤＮｓ－ＮＢは細胞内でＧＳＨなどのチオールと反応するため、細胞実験には用いなかった。比較としてＮＢを用いて検討した。ＨｅＬａ細胞にオレイン酸（０．２ｍＭ）を添加し、３７℃、２４時間インキュベートすることで、細胞内に脂肪滴を有するモデル細胞を作成した。脂肪滴を内部に形成しているＨｅＬａ細胞（脂肪滴（＋））に、１μＭの各種試薬を添加し、３７℃、１５分間インキュベートした後に、蛍光顕微鏡を用いてイメージング像を得た（図６）。いずれの試薬も、脂肪滴部分からの蛍光シグナルを観測できたが、そのシグナル・バックグラウンドに違いがあった。ＭＮｓ－ＮＢは脂肪滴部分のみから顕著な蛍光シグナルを与えた。一方で、ＮＢ及びＴｓ－ＮＢでは、脂肪滴以外の細胞質全体からも非特異的な蛍光シグナルが観測された。 [Test Example 5]
<Examination with lipid droplet-forming cells>
An imaging model of intracellular lipid droplets was examined in an experimental system using MNs-NB and Ts-NB synthesized this time. Since DNs-NB reacts with thiols such as GSH in cells, it was not used in cell experiments. NB was used for comparison. By adding oleic acid (0.2 mM) to HeLa cells and incubating at 37 ° C. for 24 hours, model cells having lipid droplets in the cells were prepared. 1 μM various reagents were added to HeLa cells (fat droplets (+)) in which lipid droplets were formed, and after incubation at 37 ° C. for 15 minutes, an imaging image was obtained using a fluorescence microscope (FIG. 6). ). Each reagent was able to observe a fluorescent signal from the lipid droplet portion, but there was a difference in the signal background. MNs-NB gave a significant fluorescent signal only from the lipid droplet portion. On the other hand, in NB and Ts-NB, non-specific fluorescence signals were also observed from the whole cytoplasm other than lipid droplets.
さらに、脂肪滴を形成していないＨｅＬａ細胞（脂肪滴（－））において、同様のイメージング実験を行ったところ、ＭＮｓ－ＮＢは全く非特異的な蛍光を示さないのに対して、ＮＢ及びＴｓ－ＮＢは細胞質全体に非特異的な蛍光シグナルが観測された（図６）。続いて、市販の試薬のナイルレッド及びＢＯＤＩＰＹ４９３／５０３を用いて同様に検討した。ナイルレッドは脂肪滴特異性の高い蛍光画像を与えるのに対して、ＢＯＤＩＰＹ４９３／５０３は比較的特異性の低い蛍光画像を与えた。脂肪滴を形成していないＨｅＬａ細胞において、ナイルレッドとＢＯＤＩＰＹ４９３／５０３はいずれも非特異的なシグナルを細胞質全体に与えた。これらの結果は、ＭＮｓ－ＮＢの脂肪滴検出における高い特異性を示している。
Furthermore, when a similar imaging experiment was performed on HeLa cells (lipid droplets (−)) that did not form lipid droplets, MNs-NB did not show any nonspecific fluorescence, whereas NB and Ts For -NB, a non-specific fluorescence signal was observed throughout the cytoplasm (FIG. 6). Subsequently, the same examination was performed using commercially available reagents Nile Red and BODIPY493 / 503. Nile red gave a fluorescent image with high lipid droplet specificity, whereas BODIPY493 / 503 gave a fluorescent image with relatively low specificity. In HeLa cells that did not form lipid droplets, both Nile Red and BODIPY493 / 503 gave a non-specific signal throughout the cytoplasm. These results indicate a high specificity in detecting lipid droplets of MNs-NB.
続いて、フローサイトメトリーを用いて脂肪滴の相対的量解析を検討した。フローサイトメーターは、蛍光顕微鏡を用いたイメージング解析と異なり局在化情報は得られないが、数万個以上の高速細胞解析を可能とし、一細胞単位ごとの蛍光強度を頻度分布として与える。フローサイトメーターによる解析は、細胞内のすべてのシグナルを一つの蛍光強度として与えるため、特異性の高い検出試薬が必要になる。先ほど同様に、脂肪滴を形成させたＨｅＬａ細胞（脂肪滴（＋））と脂肪滴がない通常のＨｅＬａ細胞（脂肪滴（－））を調整した。脂肪滴検出用試薬は、ＮＢ、ＭＮｓ－ＮＢ、Ｔｓ－ＮＢ、ナイルレッド及びＢＯＤＩＰＹ４９３／５０３を用いた。ＨｅＬａ細胞に０．５μＭ試薬を添加し、３７℃、１５分間インキュベートした後、フローサイトメーターによりそれぞれの試薬の蛍光強度を測定した。図７に解析の結果得られた頻度分布を示した。
Subsequently, the relative amount analysis of fat droplets was examined using flow cytometry. Unlike imaging analysis using a fluorescence microscope, the flow cytometer does not provide localization information, but enables high-speed cell analysis of tens of thousands or more, and gives fluorescence intensity for each cell unit as a frequency distribution. Analysis by a flow cytometer gives all signals in the cell as a single fluorescence intensity, and therefore requires a highly specific detection reagent. Similarly, HeLa cells (fat droplets (+)) in which lipid droplets were formed and normal HeLa cells without lipid droplets (fat droplets (-)) were prepared. As the lipid droplet detection reagent, NB, MNs-NB, Ts-NB, Nile Red and BODIPY493 / 503 were used. After adding 0.5 μM reagent to HeLa cells and incubating at 37 ° C. for 15 minutes, the fluorescence intensity of each reagent was measured with a flow cytometer. FIG. 7 shows the frequency distribution obtained as a result of the analysis.
脂肪滴存在細胞及び非存在細胞において分布を比較すると、ＭＮｓ－ＮＢにおいて最も優位な蛍光強度の分布差が観察された。また、ＮＢとＴｓ－ＮＢにおいては、ほとんど有意な分布差は観測されなかった。一方、ＢＯＤＩＰＹ４９３／５０３においては有意な分布差が観察されたが、ナイルレッドは有意な差は観測されなかった。頻度分布から蛍光強度の中央値を算出した（表８）。脂肪滴存在細胞及び非存在細胞における中央値の比（特異度：脂肪滴（＋）／脂肪滴（－））は、試薬の特異性を示す指標になる。試薬の中では、ＭＮｓ－ＮＢが最も高い特異度１．９４を与えた。以上、蛍光顕微鏡によるイメージング解析とフローサイトメーターによる相対的量解析の結果から、ＭＮｓ－ＮＢが最も脂肪滴特異性の高い試薬と確認した。
When comparing the distribution of lipid droplet present cells and non-existing cells, the most dominant difference in fluorescence intensity distribution was observed in MNs-NB. In addition, almost no significant distribution difference was observed between NB and Ts-NB. On the other hand, a significant distribution difference was observed in BODIPY493 / 503, but no significant difference was observed in Nile Red. The median fluorescence intensity was calculated from the frequency distribution (Table 8). The ratio of the median values in the presence and absence of lipid droplets (specificity: lipid droplet (+) / lipid droplet (−)) is an indicator of the specificity of the reagent. Among the reagents, MNs-NB gave the highest specificity 1.94. As described above, from the results of imaging analysis using a fluorescence microscope and relative quantity analysis using a flow cytometer, MNs-NB was confirmed to be the reagent with the highest lipid droplet specificity.
［試験例６］
＜脂肪細胞内脂肪滴のイメージング＞
骨髄由来のヒト間葉系細胞株ＵＥＥＴ－１２細胞株は、様々な細胞に分化することが知られている。デキサメタゾン及びインスリンでこの細胞を処理すると、脂肪細胞に分化する。この場合、脂肪細胞に分化すると内部に脂肪滴を生じる。そのため、脂肪滴をイメージングすることでＵＥＥＴ－１２細胞株が脂肪細胞に分化する過程を評価することができる。そこで、ＭＮｓ－ＮＢ試薬を用いてＵＥＥＴ－１２の脂肪細胞への分化を識別できるかを、蛍光顕微鏡を用いて検証した。デキサメタゾン及びインスリンを添加したＤＭＥＭ（１０％ＦＢＳ）を用いてＵＥＥＴ－１２を１４日間培養（２日おきに継代）することで、脂肪細胞への分化を誘導した（ＵＥＥＴ－１２（＋））。コントロールとして、何も添加してないＤＭＥＭ（１０％ＦＢＳ）で培養したＵＥＥＴ－１２も調整した（ＵＥＥＴ－１２（－））。ＵＥＥＴ－１２（＋）に５μＭの各種試薬を添加し、３７℃、１５分間インキュベートした後、蛍光顕微鏡にて蛍光シグナルを観測した（図８）。ＮＢは、ＵＥＥＴ－１２（＋）とＵＥＥＴ－１２（－）の比較において、特異的なシグナルを与えなかった。また、ナイルレッド及びＢＯＤＩＰＹ４９３／５０３は、ＵＥＥＴ－１２（＋）とＵＥＥＴ－１２（－）においてもわずかな非特異蛍光シグナルを示した。また、ＵＥＥＴ－１２（＋）において、いずれの試薬も脂肪滴以外からの蛍光シグナルが観測されていることから、ＭＮｓ－ＮＢと比較し特異性が低いといえる。以上の結果から、ＭＮｓ－ＮＢは、脂肪滴を検出するための優れた試薬であることが明らかとなった。 [Test Example 6]
<Imaging of lipid droplets in fat cells>
The bone marrow-derived human mesenchymal cell line UEET-12 cell line is known to differentiate into various cells. Treatment of the cells with dexamethasone and insulin differentiates into adipocytes. In this case, when it differentiates into fat cells, lipid droplets are generated inside. Therefore, it is possible to evaluate the process of UEET-12 cell line differentiation into adipocytes by imaging lipid droplets. Thus, it was verified using a fluorescence microscope whether differentiation of UEET-12 into adipocytes could be identified using the MNs-NB reagent. UEET-12 was cultured for 14 days (subculture every 2 days) using DMEM (10% FBS) supplemented with dexamethasone and insulin to induce differentiation into adipocytes (UEET-12 (+)) . As a control, UEET-12 cultured in DMEM (10% FBS) to which nothing was added was also prepared (UEET-12 (−)). Various reagents of 5 μM were added to UEET-12 (+) and incubated at 37 ° C. for 15 minutes, and then fluorescence signals were observed with a fluorescence microscope (FIG. 8). NB did not give a specific signal in the comparison of UEET-12 (+) and UEET-12 (-). Nile red and BODIPY493 / 503 also showed a slight non-specific fluorescence signal in UEET-12 (+) and UEET-12 (−). In addition, in UEET-12 (+), the fluorescence signals from other than lipid droplets are observed in any of the reagents, so it can be said that the specificity is lower than that of MNs-NB. From the above results, it was revealed that MNs-NB is an excellent reagent for detecting lipid droplets.
＜脂肪細胞内脂肪滴のイメージング＞
骨髄由来のヒト間葉系細胞株ＵＥＥＴ－１２細胞株は、様々な細胞に分化することが知られている。デキサメタゾン及びインスリンでこの細胞を処理すると、脂肪細胞に分化する。この場合、脂肪細胞に分化すると内部に脂肪滴を生じる。そのため、脂肪滴をイメージングすることでＵＥＥＴ－１２細胞株が脂肪細胞に分化する過程を評価することができる。そこで、ＭＮｓ－ＮＢ試薬を用いてＵＥＥＴ－１２の脂肪細胞への分化を識別できるかを、蛍光顕微鏡を用いて検証した。デキサメタゾン及びインスリンを添加したＤＭＥＭ（１０％ＦＢＳ）を用いてＵＥＥＴ－１２を１４日間培養（２日おきに継代）することで、脂肪細胞への分化を誘導した（ＵＥＥＴ－１２（＋））。コントロールとして、何も添加してないＤＭＥＭ（１０％ＦＢＳ）で培養したＵＥＥＴ－１２も調整した（ＵＥＥＴ－１２（－））。ＵＥＥＴ－１２（＋）に５μＭの各種試薬を添加し、３７℃、１５分間インキュベートした後、蛍光顕微鏡にて蛍光シグナルを観測した（図８）。ＮＢは、ＵＥＥＴ－１２（＋）とＵＥＥＴ－１２（－）の比較において、特異的なシグナルを与えなかった。また、ナイルレッド及びＢＯＤＩＰＹ４９３／５０３は、ＵＥＥＴ－１２（＋）とＵＥＥＴ－１２（－）においてもわずかな非特異蛍光シグナルを示した。また、ＵＥＥＴ－１２（＋）において、いずれの試薬も脂肪滴以外からの蛍光シグナルが観測されていることから、ＭＮｓ－ＮＢと比較し特異性が低いといえる。以上の結果から、ＭＮｓ－ＮＢは、脂肪滴を検出するための優れた試薬であることが明らかとなった。 [Test Example 6]
<Imaging of lipid droplets in fat cells>
The bone marrow-derived human mesenchymal cell line UEET-12 cell line is known to differentiate into various cells. Treatment of the cells with dexamethasone and insulin differentiates into adipocytes. In this case, when it differentiates into fat cells, lipid droplets are generated inside. Therefore, it is possible to evaluate the process of UEET-12 cell line differentiation into adipocytes by imaging lipid droplets. Thus, it was verified using a fluorescence microscope whether differentiation of UEET-12 into adipocytes could be identified using the MNs-NB reagent. UEET-12 was cultured for 14 days (subculture every 2 days) using DMEM (10% FBS) supplemented with dexamethasone and insulin to induce differentiation into adipocytes (UEET-12 (+)) . As a control, UEET-12 cultured in DMEM (10% FBS) to which nothing was added was also prepared (UEET-12 (−)). Various reagents of 5 μM were added to UEET-12 (+) and incubated at 37 ° C. for 15 minutes, and then fluorescence signals were observed with a fluorescence microscope (FIG. 8). NB did not give a specific signal in the comparison of UEET-12 (+) and UEET-12 (-). Nile red and BODIPY493 / 503 also showed a slight non-specific fluorescence signal in UEET-12 (+) and UEET-12 (−). In addition, in UEET-12 (+), the fluorescence signals from other than lipid droplets are observed in any of the reagents, so it can be said that the specificity is lower than that of MNs-NB. From the above results, it was revealed that MNs-NB is an excellent reagent for detecting lipid droplets.
［試験例７］
＜イメージングを基礎とした創薬スクリーニング法＞
細胞内における脂肪滴形成を阻害できる化合物は、高脂血症治療薬や成人病関連の治療薬として期待できる。今回開発したＭＮｓ－ＮＢは優れた細胞内脂肪滴イメージング試薬であることから、イメージングを基盤とした脂肪滴形成阻害剤のハイスループットスクリーニング（ＨＴＳ）に応用できると考えた（図９）。そこで、脂肪滴を形成したＮＩＨ３Ｔ３細胞株を用いて、長期的な脂肪滴イメージングが可能か、また、脂肪滴形成阻害剤の効果が蛍光シグナルで相対的量的に観測できるかを検証した。ＮＩＨ３Ｔ３細胞にオレイン酸を添加し、１２時間後、細胞内に脂肪滴形成を顕微鏡明視野にて確認した。その後、オレイン酸を培養液から除去し、ＭＮｓ－ＮＢを添加して１５分後の細胞の蛍光イメージング像を示した（図１０）。細胞株からは、脂肪滴由来の顕著な蛍光シグナルが観測された。さらに、４６時間経過した細胞においてもほぼ同じ強度の蛍光シグナルが維持されていた。このことから、少なくとも４６時間後でも試薬は安定な蛍光シグナルを与えることが確認できた。 [Test Example 7]
<Drug discovery screening method based on imaging>
A compound capable of inhibiting lipid droplet formation in cells can be expected as a therapeutic drug for hyperlipidemia and a therapeutic drug for adult diseases. Since MNs-NB developed this time is an excellent intracellular lipid droplet imaging reagent, we thought that it could be applied to high-throughput screening (HTS) of lipid droplet formation inhibitors based on imaging (FIG. 9). Therefore, using the NIH3T3 cell line in which lipid droplets were formed, it was verified whether long-term lipid droplet imaging is possible and whether the effect of the lipid droplet formation inhibitor can be observed in a relative quantitative manner with a fluorescence signal. Oleic acid was added to NIH3T3 cells, and after 12 hours, lipid droplet formation in the cells was confirmed in a bright light microscope. Thereafter, oleic acid was removed from the culture, and MNs-NB was added, and a fluorescence imaging image of the cells 15 minutes after was shown (FIG. 10). From the cell line, a remarkable fluorescent signal derived from lipid droplets was observed. Further, the fluorescence signal having almost the same intensity was maintained in the cells after 46 hours. From this, it was confirmed that the reagent gave a stable fluorescent signal even after at least 46 hours.
＜イメージングを基礎とした創薬スクリーニング法＞
細胞内における脂肪滴形成を阻害できる化合物は、高脂血症治療薬や成人病関連の治療薬として期待できる。今回開発したＭＮｓ－ＮＢは優れた細胞内脂肪滴イメージング試薬であることから、イメージングを基盤とした脂肪滴形成阻害剤のハイスループットスクリーニング（ＨＴＳ）に応用できると考えた（図９）。そこで、脂肪滴を形成したＮＩＨ３Ｔ３細胞株を用いて、長期的な脂肪滴イメージングが可能か、また、脂肪滴形成阻害剤の効果が蛍光シグナルで相対的量的に観測できるかを検証した。ＮＩＨ３Ｔ３細胞にオレイン酸を添加し、１２時間後、細胞内に脂肪滴形成を顕微鏡明視野にて確認した。その後、オレイン酸を培養液から除去し、ＭＮｓ－ＮＢを添加して１５分後の細胞の蛍光イメージング像を示した（図１０）。細胞株からは、脂肪滴由来の顕著な蛍光シグナルが観測された。さらに、４６時間経過した細胞においてもほぼ同じ強度の蛍光シグナルが維持されていた。このことから、少なくとも４６時間後でも試薬は安定な蛍光シグナルを与えることが確認できた。 [Test Example 7]
<Drug discovery screening method based on imaging>
A compound capable of inhibiting lipid droplet formation in cells can be expected as a therapeutic drug for hyperlipidemia and a therapeutic drug for adult diseases. Since MNs-NB developed this time is an excellent intracellular lipid droplet imaging reagent, we thought that it could be applied to high-throughput screening (HTS) of lipid droplet formation inhibitors based on imaging (FIG. 9). Therefore, using the NIH3T3 cell line in which lipid droplets were formed, it was verified whether long-term lipid droplet imaging is possible and whether the effect of the lipid droplet formation inhibitor can be observed in a relative quantitative manner with a fluorescence signal. Oleic acid was added to NIH3T3 cells, and after 12 hours, lipid droplet formation in the cells was confirmed in a bright light microscope. Thereafter, oleic acid was removed from the culture, and MNs-NB was added, and a fluorescence imaging image of the cells 15 minutes after was shown (FIG. 10). From the cell line, a remarkable fluorescent signal derived from lipid droplets was observed. Further, the fluorescence signal having almost the same intensity was maintained in the cells after 46 hours. From this, it was confirmed that the reagent gave a stable fluorescent signal even after at least 46 hours.
次に、細胞レベルで脂肪滴形成阻害剤の効果を観測できるか検証した。阻害剤はトリアクシンＣ（和光純薬工業株式会社製）を用いた。トリアクシンＣは、真菌の代謝産物で、トリアシルグリセロールの合成過程を阻害することで、脂肪滴の形成を阻害するメカニズムを有する。脂肪滴を形成させたＮＩＨ３Ｔ３細胞に対して、０．８μＭトリアクシンＣを添加した後、経時的に蛍光イメージングを解析した（図１１）。その結果、４時間後、２４時間後と時間を経るごとに、細胞内の蛍光シグナルが減弱するのを確認できた。本基礎実験から、ＭＮｓ－ＮＢを用いることで細胞レベルでの阻害剤のハイスループットスクリーニング（ＨＴＳ）が可能であることを確認できた。
Next, it was verified whether the effect of the lipid droplet formation inhibitor could be observed at the cellular level. Triaxin C (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the inhibitor. Triaccin C is a fungal metabolite and has a mechanism of inhibiting lipid droplet formation by inhibiting the synthesis process of triacylglycerol. After adding 0.8 μM triaxin C to NIH3T3 cells in which lipid droplets were formed, fluorescence imaging was analyzed over time (FIG. 11). As a result, it was confirmed that the fluorescence signal in the cell was attenuated every time after 4 hours and after 24 hours. From this basic experiment, it was confirmed that high-throughput screening (HTS) of inhibitors at the cellular level was possible by using MNs-NB.
［試験例８］
＜生きている生物個体での脂肪組織のイメージング（１）＞
これまでに、蛍光試薬を用いてゼブラフィッシュや線虫における脂肪組織イメージングが報告されている。しかしながら、そのプロトコルは固定化・洗浄を伴うため、生きている生物個体中の脂肪組織のイメージング例は殆ど報告されていない。また、マウスなどの哺乳動物の生体内における脂肪組織イメージングは全く報告されていない。これまでの細胞実験から、ＭＮｓ－ＮＢは脂肪組織への高い分布特性と脂肪組織での特異的蛍光増強が期待できる。そのため、洗浄操作をできないマウス個体深部の脂肪組織イメージングが可能であると考えた。生きたマウス個体の脂肪組織イメージングは世界で初めての例である。本実験のために、痩せ型マウスと高脂肪食負荷の肥満型マウスを調製した。それぞれのマウスの静脈に、ＭＮｓ－ＮＢ（ＤＭＳＯ溶液）を０．１μＭ／ｋｇ体重の量で注射した。この際、フルオレイン修飾デキストランとＨｏｅｃｈｓｔ３３３４２を同時に注射することで、マウス組織中の血管を緑色蛍光で染色し、細胞核を青色蛍光で染色した。１５分後、それぞれのマウスにおける骨格筋、肝臓を蛍光顕微鏡で観測した。
図１２に骨格筋組織像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。これは骨格筋内における異所性脂肪（皮下脂肪や内臓脂肪に蓄積されずに、内臓や骨格筋に沈着する脂肪）の存在を示している。一方で、痩せ型マウスでは、ＭＮｓ－ＮＢ由来の蛍光シグナルは観察されなかった。図１２に肝臓組織画像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。一方で、痩せ型マウスでは、蛍光シグナルが観察されなかった。以上、ＭＮｓ－ＮＢを用いることで、世界で初めての生きているマウスでの脂肪組織イメージングに成功した。ＭＮｓ－ＮＢは、動物個体においても組織浸透性かつ膜透過性を示し、脂肪組織特異的に染色・イメージングできる実用的な試薬であることが明らかとなった。 [Test Example 8]
<Imaging of adipose tissue in living organisms (1)>
So far, imaging of adipose tissue in zebrafish and nematodes using fluorescent reagents has been reported. However, since the protocol involves immobilization / washing, few examples of imaging of adipose tissue in living organisms have been reported. In addition, imaging of adipose tissue in vivo in mammals such as mice has never been reported. From previous cell experiments, MNs-NB can be expected to have high distribution characteristics in adipose tissue and specific fluorescence enhancement in adipose tissue. Therefore, we thought that it was possible to image adipose tissue in the deep part of a mouse individual that cannot be washed. Adipose tissue imaging of living mouse individuals is the first example in the world. For this experiment, lean mice and obese mice with a high fat diet were prepared. Each mouse's vein was injected with MNs-NB (DMSO solution) in an amount of 0.1 μM / kg body weight. At this time, by simultaneously injecting fluorescein-modified dextran and Hoechst 33342, blood vessels in mouse tissues were stained with green fluorescence, and cell nuclei were stained with blue fluorescence. After 15 minutes, skeletal muscle and liver in each mouse were observed with a fluorescence microscope.
FIG. 12 shows a skeletal muscle tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. This indicates the presence of ectopic fat in the skeletal muscle (the fat deposited in the viscera and skeletal muscle without being accumulated in the subcutaneous fat and visceral fat). On the other hand, no fluorescent signal derived from MNs-NB was observed in lean mice. FIG. 12 shows a liver tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice. As described above, the use of MNs-NB succeeded in the world's first adipose tissue imaging in living mice. It was revealed that MNs-NB is a practical reagent that exhibits tissue permeability and membrane permeability even in animal individuals and can be stained and imaged specifically for adipose tissue.
＜生きている生物個体での脂肪組織のイメージング（１）＞
これまでに、蛍光試薬を用いてゼブラフィッシュや線虫における脂肪組織イメージングが報告されている。しかしながら、そのプロトコルは固定化・洗浄を伴うため、生きている生物個体中の脂肪組織のイメージング例は殆ど報告されていない。また、マウスなどの哺乳動物の生体内における脂肪組織イメージングは全く報告されていない。これまでの細胞実験から、ＭＮｓ－ＮＢは脂肪組織への高い分布特性と脂肪組織での特異的蛍光増強が期待できる。そのため、洗浄操作をできないマウス個体深部の脂肪組織イメージングが可能であると考えた。生きたマウス個体の脂肪組織イメージングは世界で初めての例である。本実験のために、痩せ型マウスと高脂肪食負荷の肥満型マウスを調製した。それぞれのマウスの静脈に、ＭＮｓ－ＮＢ（ＤＭＳＯ溶液）を０．１μＭ／ｋｇ体重の量で注射した。この際、フルオレイン修飾デキストランとＨｏｅｃｈｓｔ３３３４２を同時に注射することで、マウス組織中の血管を緑色蛍光で染色し、細胞核を青色蛍光で染色した。１５分後、それぞれのマウスにおける骨格筋、肝臓を蛍光顕微鏡で観測した。
図１２に骨格筋組織像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。これは骨格筋内における異所性脂肪（皮下脂肪や内臓脂肪に蓄積されずに、内臓や骨格筋に沈着する脂肪）の存在を示している。一方で、痩せ型マウスでは、ＭＮｓ－ＮＢ由来の蛍光シグナルは観察されなかった。図１２に肝臓組織画像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。一方で、痩せ型マウスでは、蛍光シグナルが観察されなかった。以上、ＭＮｓ－ＮＢを用いることで、世界で初めての生きているマウスでの脂肪組織イメージングに成功した。ＭＮｓ－ＮＢは、動物個体においても組織浸透性かつ膜透過性を示し、脂肪組織特異的に染色・イメージングできる実用的な試薬であることが明らかとなった。 [Test Example 8]
<Imaging of adipose tissue in living organisms (1)>
So far, imaging of adipose tissue in zebrafish and nematodes using fluorescent reagents has been reported. However, since the protocol involves immobilization / washing, few examples of imaging of adipose tissue in living organisms have been reported. In addition, imaging of adipose tissue in vivo in mammals such as mice has never been reported. From previous cell experiments, MNs-NB can be expected to have high distribution characteristics in adipose tissue and specific fluorescence enhancement in adipose tissue. Therefore, we thought that it was possible to image adipose tissue in the deep part of a mouse individual that cannot be washed. Adipose tissue imaging of living mouse individuals is the first example in the world. For this experiment, lean mice and obese mice with a high fat diet were prepared. Each mouse's vein was injected with MNs-NB (DMSO solution) in an amount of 0.1 μM / kg body weight. At this time, by simultaneously injecting fluorescein-modified dextran and Hoechst 33342, blood vessels in mouse tissues were stained with green fluorescence, and cell nuclei were stained with blue fluorescence. After 15 minutes, skeletal muscle and liver in each mouse were observed with a fluorescence microscope.
FIG. 12 shows a skeletal muscle tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. This indicates the presence of ectopic fat in the skeletal muscle (the fat deposited in the viscera and skeletal muscle without being accumulated in the subcutaneous fat and visceral fat). On the other hand, no fluorescent signal derived from MNs-NB was observed in lean mice. FIG. 12 shows a liver tissue image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice. As described above, the use of MNs-NB succeeded in the world's first adipose tissue imaging in living mice. It was revealed that MNs-NB is a practical reagent that exhibits tissue permeability and membrane permeability even in animal individuals and can be stained and imaged specifically for adipose tissue.
［試験例９］
＜生きている生物個体での脂肪組織のイメージング（２）＞
生きたマウス個体の内臓脂肪及び心臓における脂肪組織のイメージングを試みた。本実験のため、痩せ型マウスと高脂肪食負荷の肥満型マウスを調製した。それぞれのマウスの尾静脈に、ＭＮｓ－ＮＢ（ＤＭＳＯ溶液）を０．１μＭ／ｋｇ体重の量で注射した。この際、Ａｌｅｘａ４８８標識レクチンとＨｏｅｃｈｓｔ３３３４２を同時に注射することで、マウス組織中の血管を緑色蛍光で染色し、細胞核を青色蛍光で染色した。３０分後、それぞれのマウスにおける内臓脂肪（精巣上体脂肪）および心臓を摘出し、臓器を倒立共焦点顕微鏡により観察した。
図１３に内臓脂肪画像を示した。肥満型マウスでは、脂肪細胞の肥大化が生じていた。図１３に心臓画像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。一方で、痩せ型マウスでは、蛍光シグナルが観察されなかった。このことから、ＭＮｓ－ＮＢを用いることで、痩せ型では見られない、異所性脂肪の心臓での沈着が肥満型マウスによって可視化された。 [Test Example 9]
<Imaging of adipose tissue in living organisms (2)>
We attempted to image visceral fat and adipose tissue in the heart of living mice. For this experiment, lean mice and obese mice with a high fat diet were prepared. The tail vein of each mouse was injected with MNs-NB (DMSO solution) in an amount of 0.1 μM / kg body weight. At this time, Alexa 488-labeled lectin and Hoechst 33342 were simultaneously injected to stain blood vessels in mouse tissue with green fluorescence and cell nuclei with blue fluorescence. After 30 minutes, the visceral fat (epididymal fat) and heart in each mouse were removed, and the organs were observed with an inverted confocal microscope.
FIG. 13 shows a visceral fat image. In obese mice, fat cell hypertrophy occurred. FIG. 13 shows a heart image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice. From this, by using MNs-NB, the deposition of ectopic fat in the heart, which is not seen in the lean type, was visualized by obese mice.
＜生きている生物個体での脂肪組織のイメージング（２）＞
生きたマウス個体の内臓脂肪及び心臓における脂肪組織のイメージングを試みた。本実験のため、痩せ型マウスと高脂肪食負荷の肥満型マウスを調製した。それぞれのマウスの尾静脈に、ＭＮｓ－ＮＢ（ＤＭＳＯ溶液）を０．１μＭ／ｋｇ体重の量で注射した。この際、Ａｌｅｘａ４８８標識レクチンとＨｏｅｃｈｓｔ３３３４２を同時に注射することで、マウス組織中の血管を緑色蛍光で染色し、細胞核を青色蛍光で染色した。３０分後、それぞれのマウスにおける内臓脂肪（精巣上体脂肪）および心臓を摘出し、臓器を倒立共焦点顕微鏡により観察した。
図１３に内臓脂肪画像を示した。肥満型マウスでは、脂肪細胞の肥大化が生じていた。図１３に心臓画像を示した。肥満型マウスでは、ＭＮｓ－ＮＢ由来の強い蛍光シグナルが観察された。一方で、痩せ型マウスでは、蛍光シグナルが観察されなかった。このことから、ＭＮｓ－ＮＢを用いることで、痩せ型では見られない、異所性脂肪の心臓での沈着が肥満型マウスによって可視化された。 [Test Example 9]
<Imaging of adipose tissue in living organisms (2)>
We attempted to image visceral fat and adipose tissue in the heart of living mice. For this experiment, lean mice and obese mice with a high fat diet were prepared. The tail vein of each mouse was injected with MNs-NB (DMSO solution) in an amount of 0.1 μM / kg body weight. At this time, Alexa 488-labeled lectin and Hoechst 33342 were simultaneously injected to stain blood vessels in mouse tissue with green fluorescence and cell nuclei with blue fluorescence. After 30 minutes, the visceral fat (epididymal fat) and heart in each mouse were removed, and the organs were observed with an inverted confocal microscope.
FIG. 13 shows a visceral fat image. In obese mice, fat cell hypertrophy occurred. FIG. 13 shows a heart image. In obese mice, a strong fluorescent signal derived from MNs-NB was observed. On the other hand, no fluorescence signal was observed in lean mice. From this, by using MNs-NB, the deposition of ectopic fat in the heart, which is not seen in the lean type, was visualized by obese mice.
本発明によれば、脂肪滴及び／又は脂肪組織を検出できる新規化合物を提供することができる。したがって、本発明の化合物は産業上極めて有用である。
According to the present invention, a novel compound capable of detecting fat droplets and / or adipose tissue can be provided. Therefore, the compound of this invention is very useful industrially.
Claims (9)
- 下記一般式（Ｉ）で表される化合物：
Ｒ1は、Ｃ1-4アルキル基を表し、あるいは
Ｒ1は、Ｒ1に隣接する窒素原子、Ｒ3、並びに該窒素原子及びＲ3と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ2は、Ｃ1-4アルキル基を表し、あるいは
Ｒ2は、Ｒ2に隣接する窒素原子、Ｒ4、並びに該窒素原子及びＲ4と結合するベンゼン環上の２個の炭素原子と共に、５～７員の環を形成していてもよく；
Ｒ3は、Ｒ1が５～７員の環を形成していない場合には水素原子であり；
Ｒ4は、Ｒ2が５～７員の環を形成していない場合には水素原子であり；且つ
Ｘは下記式（ＩＩ）で表される基である：
Ｒ5はそれぞれ独立して、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-4アルキル基、－Ｃ（＝Ｏ）－Ｃ1-4アルキル基、又はＣＮを表し； ｎは、１～５の整数を表し；
但し、式（ＩＩ）におけるベンゼン環の１位の炭素原子の電子密度が、－０．２４０～－０．１９０の範囲内にあることを条件とする））。 Compound represented by the following general formula (I):
R 1 represents a C 1-4 alkyl group, or R 1 together with a nitrogen atom adjacent to R 1 , R 3 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 3 , May form a 5- to 7-membered ring;
R 2 represents a C 1-4 alkyl group, or R 2 together with a nitrogen atom adjacent to R 2 , R 4 , and two carbon atoms on the benzene ring bonded to the nitrogen atom and R 4 , May form a 5- to 7-membered ring;
R 3 is a hydrogen atom when R 1 does not form a 5- to 7-membered ring;
R 4 is a hydrogen atom when R 2 does not form a 5- to 7-membered ring; and X is a group represented by the following formula (II):
Each R 5 independently represents NO 2 , a halogen atom, a C 1-4 alkyl group in which at least a part of the hydrogen atom is substituted with a halogen atom, a —C (═O) —C 1-4 alkyl group, or CN N represents an integer of 1 to 5;
Provided that the electron density of the carbon atom at the 1-position of the benzene ring in formula (II) is within the range of -0.240 to -0.190)). - Ｘが下記式（ＩＩＩ）で表される基である、請求項１に記載の化合物：
Ｒ6は、ＮＯ2、ハロゲン原子、少なくとも水素原子の１部がハロゲン原子に置換されたＣ1-2アルキル基、－Ｃ（＝Ｏ）－Ｃ1-2アルキル基、又はＣＮを表し、
Ｒ7は、水素原子又はＮＯ2を表し、
但し、Ｒ6及びＲ7の両方が共にＮＯ2ではないことを条件とする）。 The compound according to claim 1, wherein X is a group represented by the following formula (III):
R 6 represents NO 2 , a halogen atom, a C 1-2 alkyl group in which at least a part of a hydrogen atom is substituted with a halogen atom, —C (═O) —C 1-2 alkyl group, or CN;
R 7 represents a hydrogen atom or NO 2 ,
Provided that both R 6 and R 7 are not NO 2 ). - Ｒ1及びＲ2がそれぞれ独立して、Ｃ1-4アルキル基を表し、Ｒ3及びＲ4が水素原子を表す、請求項１又は２に記載の化合物。 The compound according to claim 1 or 2, wherein R 1 and R 2 each independently represents a C 1-4 alkyl group, and R 3 and R 4 represent a hydrogen atom.
- Ｘが下記からなる群から選択される基である、請求項１～３のいずれか１項に記載の化合物。
- Ｘが下記の基である、請求項４に記載の化合物。
- 請求項１～５のいずれか１項に記載の化合物を含む、脂肪滴及び／又は脂肪組織の検出用試薬。 A reagent for detecting lipid droplets and / or adipose tissue, comprising the compound according to any one of claims 1 to 5.
- 請求項１～５のいずれか１項に記載の化合物を、脂肪滴を含む細胞に添加する工程を含む、細胞内の脂肪滴を検出する方法。 A method for detecting intracellular lipid droplets, comprising a step of adding the compound according to any one of claims 1 to 5 to cells containing lipid droplets.
- 請求項１～５のいずれか１項に記載の化合物を、生きている生物個体内（ヒトを除く）に投与する工程を含む、生物個体内に存在する脂肪組織を検出する方法。 A method for detecting adipose tissue present in an organism individual, comprising the step of administering the compound according to any one of claims 1 to 5 into an organism organism (excluding humans).
- 脂肪滴形成阻害剤の候補化合物を、脂肪滴を含む細胞に添加する工程、及び
請求項１～５のいずれか１項に記載の化合物を、該細胞に添加する工程、
を含む、脂肪滴形成阻害剤を探索する方法。 Adding a lipid droplet formation inhibitor candidate compound to cells containing lipid droplets, and adding the compound according to any one of claims 1 to 5 to the cells;
A method for searching for a lipid droplet formation inhibitor.
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| WO2020189721A1 (en) * | 2019-03-19 | 2020-09-24 | 国立大学法人群馬大学 | Reagent for fluorescence imaging of lipid droplets in cell and tissue |
| JP2020186192A (en) * | 2019-05-13 | 2020-11-19 | 学校法人昭和薬科大学 | Novel compound and reagent for lipid droplet detection containing the same |
| WO2023074778A1 (en) * | 2021-10-27 | 2023-05-04 | 国立大学法人群馬大学 | Fluorescence imaging reagent for lipid droplets in tissues and cells |
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| JP2021191807A (en) * | 2018-08-28 | 2021-12-16 | 株式会社同仁化学研究所 | Novel fluorescent dye, and composition for lipid droplet staining and method for imaging intracellular lipid droplets using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008523379A (en) * | 2004-12-11 | 2008-07-03 | ザ サイエンス アンド テクノロジー ファシリティーズ カウンシル | Analysis of lipoproteins using phosphor K-37 |
-
2014
- 2014-03-14 JP JP2015505607A patent/JP6241014B2/en not_active Expired - Fee Related
- 2014-03-14 WO PCT/JP2014/056973 patent/WO2014142320A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008523379A (en) * | 2004-12-11 | 2008-07-03 | ザ サイエンス アンド テクノロジー ファシリティーズ カウンシル | Analysis of lipoproteins using phosphor K-37 |
Non-Patent Citations (5)
| Title |
|---|
| BONILLA E ET AL.: "Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle", THE JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, vol. 35, no. 5, 1987, pages 619 - 621 * |
| JIE ZHANG ET AL.: "Synthesis and Characterization of a Series of Highly FluorogenicSubstrates for Glutathione Transferases, a General Strategy", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 133, no. 35, 2011, pages 14109 - 14119, XP055062761, DOI: doi:10.1021/ja205500y * |
| JINEY JOSE ET AL.: "Benzophenoxazine-based fluorescent dyes for labeling biomolecules", TETRAHEDRON, vol. 62, no. 48, 2006, pages 11021 - 11037 * |
| P GREENSPAN ET AL.: "Spectrofluorometric studies of the lipid probe, nile red.", THE JOURNAL OF LIPID RESEARCH, vol. 26, no. 7, 1985, pages 781 - 789, XP002459744 * |
| WU LIU ET AL.: "Reversible Near-Infrared pH Probes Based on Benzo[a]phenoxazine", ANALYTICAL CHEMISTRY, vol. 85, no. 15, pages 7419 - 7425 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020189721A1 (en) * | 2019-03-19 | 2020-09-24 | 国立大学法人群馬大学 | Reagent for fluorescence imaging of lipid droplets in cell and tissue |
| JP7369468B2 (en) | 2019-03-19 | 2023-10-26 | 国立大学法人群馬大学 | Fluorescent imaging reagents for lipid droplets in cells and tissues |
| JP2020186192A (en) * | 2019-05-13 | 2020-11-19 | 学校法人昭和薬科大学 | Novel compound and reagent for lipid droplet detection containing the same |
| JP7217460B2 (en) | 2019-05-13 | 2023-02-03 | 学校法人昭和薬科大学 | A novel compound and a reagent for detecting lipid droplets containing the compound |
| WO2023074778A1 (en) * | 2021-10-27 | 2023-05-04 | 国立大学法人群馬大学 | Fluorescence imaging reagent for lipid droplets in tissues and cells |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6241014B2 (en) | 2017-12-06 |
| JPWO2014142320A1 (en) | 2017-02-16 |
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