Thiophene derivative as fluorescent probe

Abstract

Disclosed herein, in one embodiment, is a compound represented by formula (I) (wherein each symbol is as defined in the specification) or a salt thereof, which is useful as a fluorescent probe for imaging organelles, such as lipid droplets within cells.

Description

Thiophene derivatives as fluorescent probes

[0001] In one embodiment, the present invention provides a novel thiophene derivative that is useful, for example, as a fluorescent probe for imaging intracellular organelles (e.g., lipid droplets), and is useful, for example, in the field of pharmaceutical research and development.

[0002] Various organelles (cell organelles) exist within cells, each performing important functions and attracting considerable research attention. Among these, lipid droplets (lipid droplets) are highly conserved among cells in vivo. They are widely present in both adipocytes and non-adipocytes and consist of a hydrophobic core composed primarily of triacylglycerides (TAGs) and cholesterol esters (CEs). Due to their ability to trap lipids, lipid droplets serve as energy storage and, importantly, buffer intracellular fatty acid concentrations. The fatty acid buffering capacity provided by increased lipid droplets promotes the suppression of lipotoxicity and the unfolded protein response (ERP). Therefore, dysfunction of lipid droplet function is associated with various diseases, including cancer, mitochondrial disorders, obesity, and diabetes. Recent studies have revealed that lipid droplets are essential hubs actively involved in antitumor immunity, anti-infective immunity, and metabolic diseases. Against this background, lipid droplet staining dyes, such as BODIPY, Lipid Blue, and Nile Red, have been developed and commercialized and are actively used in lipid droplet research. Furthermore, a push-pull type fluorescent probe, DAF (Dimethyl Aniline Furaldehyde), has also been reported (Non-Patent Document 1).

[0003] Anal. Chem. 2019, 91, 1928-1935

[0004] However, while lipid droplet research is gaining importance, there remain uncertainties and questions regarding the role of lipid droplets. Therefore, there is a growing demand for more versatile dye probes for imaging lipid droplets than conventional ones. The development of compact fluorescent probes with high cell permeability while minimizing interference with the natural interactions of biomolecules has been a long-awaited goal in the field.

[0005] The present inventors conducted extensive research to solve the above-mentioned problems and created a thiophene derivative, which is a novel compound having a thiophene skeleton. As described below, this compound exhibits an absorption spectrum with a large bathochromic shift and excellent solvatochromism in various organic solvents, and was therefore found to be useful as a versatile fluorescent probe. Further research led to the completion of the present invention. Specific embodiments of the present invention include the following, but the present invention is not limited to these embodiments.

[0006] [1] Formula (I):

[0007]

[0008] (In the formula, R １ represents an electron donating group, and R ２ represents an electron-withdrawing group.) or a salt thereof. [2] R １ However, mono- or di- (C １－６ alkyl)amino group, or C １－６ an alkoxy group, and R ２ is a nitro group or C １－６ The compound or salt thereof according to the above [1], wherein R is an alkanoyl group. １ But, Gee (C １－６ alkyl)amino group, and R ２ [4] The compound or salt thereof according to the above [2], wherein the structure represented by formula (I) is the following formula (Ia):

[0009]

[0010] The compound or salt thereof according to any one of the above [1] to [3], wherein the compound has a structure represented by the following formula:

[0011] [5] A fluorescent probe comprising the compound according to any one of [1] to [4] above, or a salt thereof. [6] The fluorescent probe according to [5] above, which is for imaging intracellular organelles. [7] The fluorescent probe according to [6] above, wherein the imaging of intracellular organelles is for evaluating the responsiveness of a cell to changes in the cellular environment by monitoring fluorescence intensity during imaging. [8] The fluorescent probe according to [6] or [7] above, wherein the cell is an immune cell. [9] The fluorescent probe according to any one of [6] to [8] above, wherein the organelle is a lipid droplet.

[0012]

[10] A method for imaging intracellular organelles, comprising a step of contacting the compound or salt thereof according to any one of [1] to [4] above with organelles in a collected cell.

[11] The imaging method according to

[10] above, wherein imaging of intracellular organelles is for evaluating the responsiveness of the cells to changes in the cellular environment by monitoring fluorescence intensity during imaging.

[12] The imaging method according to

[10] or

[11] above, wherein the cells containing the organelles are cells collected from a mammal.

[13] The imaging method according to any one of

[10] to

[12] above, wherein the cells containing the organelles are immune cells.

[0013]

[14] A method for imaging intracellular organelles, comprising the step of administering the compound or salt thereof according to any one of [1] to [4] above to a mammal and contacting it with cells containing the organelles.

[15] The imaging method according to

[14] above, wherein the mammal is an animal other than a human.

[16] The imaging method according to

[14] or

[15] above, wherein imaging of intracellular organelles is for evaluating the responsiveness of the cell to changes in the cellular environment by monitoring fluorescence intensity during imaging.

[17] The imaging method according to any one of

[10] to

[16] above, wherein the organelles are lipid droplets.

[0014]

[18] Use of the compound or salt thereof according to any one of [1] to [4] above as a fluorescent probe.

[19] The use according to

[18] above, wherein the compound or salt is for imaging intracellular organelles.

[20] The use according to

[18] or

[19] above, wherein the imaging of intracellular organelles is for evaluating the responsiveness of a cell to changes in the cellular environment by monitoring fluorescence intensity during imaging.

[21] The use according to any one of

[18] to

[20] above, wherein the organelle-containing cells are cells collected from a mammal.

[22] The use according to any one of

[18] to

[21] above, wherein the organelle-containing cells are immune cells.

[23] The use according to any one of

[18] to

[22] above, wherein the organelles are lipid droplets.

[0015] According to one embodiment of the present invention, there is provided a novel thiophene derivative that is useful, for example, as a fluorescent probe for imaging intracellular organelles (for example, lipid droplets).

[0016] FIG. 1 shows the results of fluorescence microscopy of T cells, B cells, and macrophages stained with the thiophene derivative ("NiTA") synthesized in Example 1 (described later) in Test Example 1 (described later). FIG. 2 shows the results of an investigation of the wavelength of fluorescence emitted from NiTA in various solvents in Test Example 2 (described later). FIG. 3 shows the results of fluorescence microscopy of cells stained with NiTA and / or Lipi-Blue (described later) in Test Example 3 (described later). FIG. 4 shows the results of fluorescence microscopy of T cells stained with NiTA under oxidative stress conditions in Test Example 4 (described later). FIG. 5 shows the results of fluorescence microscopy of B cells stained with NiTA under oxidative stress conditions in Test Example 4 (described later). FIG. 6 shows the results of fluorescence microscopy of T cells stained with NiTA under starvation conditions in Test Example 5 (described later). Figure 7 shows the results of fluorescence microscopy of fluorescence from B cells stained with NiTA under starvation conditions in Test Example 5, which will be described later. Figure 8 shows the results of fluorescence microscopy of fluorescence from T cells stained with NiTA in the presence of a cholesterol production inhibitor in Test Example 6, which will be described later. Figure 9 shows the results of fluorescence microscopy of fluorescence from B cells stained with NiTA in the presence of a cholesterol production inhibitor in Test Example 6, which will be described later.

[0017] The present invention will be described in detail below based on the following embodiments, but the present invention is not limited thereto. Those skilled in the art may modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included within the scope of the present invention.

[0018] [Regarding Compound (I) or a Salt thereof] One embodiment of the present invention is the following novel thiophene derivative: "[A] A compound represented by the following formula (I):

[0019]

[0020] (In the formula, R １ represents an electron donating group, and R ２represents an electron-withdrawing group.) or a salt thereof (hereinafter also referred to as "compound (I) or a salt thereof"). (Embodiment A)

[0021] Regarding the above-mentioned compound (I) or a salt thereof, the following embodiments are preferred: (1) Compound (I-1) or a salt thereof R １ However, mono- or di- (C １－６ alkyl)amino group, or C １－６ an alkoxy group, and R ２ is a nitro group or C １－６ (2) Compound (I-2) or a salt thereof, R １ But, Gee (C １－６ alkyl)amino group, and R ２ is a nitro group. (3) Compound (I-3) or a salt thereof. Compound (I), compound (I-1) and compound (I-2) are compounds in which the structure represented by formula (I) is the following formula (Ia):

[0022]

[0023] (4) Compound (I-4) or a salt thereof. Compound (I-3) or a salt thereof, wherein R １ is a dimethylamino group, and R ２ is a nitro group, or a salt thereof. Herein, the free form of compound (I-4) represented by the following formula is also referred to as "NiTA" in this specification.

[0024]

[0025] Each symbol in the above compound (I) will be explained. In this specification, the term "electron-donating group" refers to a substituent that more easily donates electrons to the atom to which it is bonded, compared to a hydrogen atom. This means that the group is more likely to donate electrons as a sum of inductive effect, mesomeric effect (or resonance effect), etc. In this specification, the term "electron-withdrawing group" refers to a substituent that more easily attracts electrons from the atom to which it is bonded, compared to a hydrogen atom. This means that the group attracts electrons as a result of the substituent effect, such as inductive effect, mesomeric effect (or resonance effect). In this specification, the term "C ａ－ｂ ” (e.g., C １－６ ) indicates that the number of carbon atoms constituting the group is a to b (for example, 1 to 6). １－６ alkyl group ("C １－６ Examples of "mono- or di-(C alkyl)" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl. １－６ The "(alkyl)amino group" is a C １－６ by an alkyl group or by two identical or different C １－６ It is an amino group substituted with an alkyl group, and examples thereof include methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, and N-ethyl-N-methylamino. １－６ In the present specification, "C" is preferably a dimethylamino group, more preferably a dimethylamino group. １－６ Examples of the "alkoxy group" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy. １－６Examples of the "alkanoyl group" include acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl, hexanoyl, and heptanoyl.

[0026] (Regarding the Aspects of Compound (I)) Compound (I) can be used in either the free form or the form of a salt thereof (e.g., a pharmaceutically acceptable salt thereof). A person skilled in the art can carry out this embodiment by appropriately selecting from either form, taking into account the properties of the individual compound (I) used. Suitable salts include, for example, salts with inorganic acids such as hydrochloride, hydrobromide, sulfate, phosphate, etc.; salts with organic acids such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, tosylate, maleate, etc.; salts with amino acids such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, aspartate, etc.

[0027] (Method for Obtaining Compound (I) or a Salt Thereof) Compound (I) or a salt thereof can be obtained by synthesis using, as a starting material, a known compound or a compound that can be easily synthesized from a known compound by a person skilled in the art, by a synthesis method known in the art. For example, a person skilled in the art can synthesize compound (I) or a salt thereof by taking into consideration the synthesis method described in Example 1 below.

[0028] [Regarding the Usefulness of Compound (I) or Its Salt as a Fluorescent Probe] Another embodiment of the present invention is "a fluorescent probe containing [B] Compound (I) or its salt" (Embodiment B). That is, Compound (I) or its salt (hereinafter collectively referred to as "Compound (I) or the like") can be used as a fluorescent probe for imaging a target. In one embodiment, Compound (I) or its salt is contained in the fluorescent probe as the sole imaging component. In this embodiment, intracellular organelles are preferred targets for imaging, and intracellular lipid droplets are more preferred targets. Hereinafter, fluorescent probes containing Compound (I) or the like will be described using lipid droplets as an example of such target organelles. The fluorescent probe can be used, for example, to elucidate the function of intracellular organelles (e.g., lipid droplets) and to research and develop preventive or therapeutic drugs for diseases involving the target. For preferred embodiments of compound (I) etc. in embodiment B, reference can be made to the preferred forms of compound (I) or a salt thereof detailed in embodiment (A). The usefulness of compound (I) etc. as a fluorescent probe will be described in detail below. Where necessary, the usefulness of compound (I) etc. as a fluorescent probe may be described based on specific test results using NiTA as a representative of compound (I) etc.

[0029] (Regarding the fluorescent properties of compound (I) and the like) (1) Compound (I) and the like have a sufficient difference between the maximum excitation wavelength and the maximum fluorescence wavelength of the fluorescence emitted from the compound. In addition, the emitted fluorescence can be observed in two wavelength regions, the green region and the red region. For example, when cells containing lipid droplets imaged with NiTA are excited at a wavelength of 473 nm and observed under a fluorescence microscope, the fluorescence emitted from the stained object within the cells can be observed in both the green channel (490-540 nm) and the red channel (570-670 nm) wavelength regions (see Test Example 1, Figure 1 below). (2) Compound (I) and the like exhibit "solvatochromism," in which the spectrum of the emitted fluorescence changes depending on the polarity of the solvent. In the case of compound (I) etc., as the hydrophobicity of the solvent (cellular environment) in which the target to be stained exists increases (i.e., as the polarity decreases), the fluorescence intensity increases on the shorter wavelength side (in the case of NiTA, the green channel (490 to 540 nm) region). On the other hand, as the hydrophilicity of the solvent (cellular environment) increases (i.e., as the polarity increases), the fluorescence intensity increases on the longer wavelength side (in the case of NiTA, the red channel (570 to 670 nm) region) (see Test Example 2, Figure 2 below). (3) From the above characteristics, compound (I) etc. can easily observe the emitted fluorescence, and by observing the fluctuations in fluorescence intensity in the two wavelength regions, it is also possible to evaluate the responsiveness of intracellular organelles (e.g., lipid droplets) to changes in the cellular environment in which intracellular organelles (e.g., lipid droplets) exist. (4) Compound (I) and the like have low cytotoxicity and exhibit high retention in intracellular organelles (e.g., lipid droplets), allowing for long-term observation of the dynamics of intracellular organelles (e.g., lipid droplets). (5) Compound (I) and the like can also be used for multiplex staining of organelles (e.g., lipid droplets) that are the target of imaging, by combining them with other fluorescent dyes having different fluorescence wavelengths, if necessary.

[0030] (Form as a fluorescent probe) The form of a fluorescent probe containing compound (I) or the like (hereinafter also referred to as "the present fluorescent probe") is not limited and can be appropriately selected by a person skilled in the art depending on the intended use. For example, compound (I) or the like itself can be used as the present fluorescent probe, or compound (I) or the like can be dissolved in an appropriate solvent and used as the present fluorescent probe in the form of a solution. Examples of such solvents include chloroform and dimethyl sulfoxide (DMSO).

[0031] (Cells to which the present fluorescent probe is applied) The cells to which the present fluorescent probe is applied are not particularly limited as long as they are the target of imaging by compound (I) or the like, specifically, cells containing intracellular organelles (e.g., lipid droplets). For example, cells to which compound (I) or the like is added include immune cells (e.g., T cells, B cells, macrophages, etc.) and non-immune cells such as cancer cells (e.g., HeLa cells, etc.). Here, the mammal is not particularly limited, and examples thereof include humans, mice, pigs, dogs, rabbits, etc.

[0032] By using this fluorescent probe, it is possible to observe the fluorescence emitted from a target cell in which the target object to be imaged, more specifically, intracellular organelles (e.g., lipid droplets) have been stained with compound (I) or the like, both in vitro and in vivo. These imaging methods are further embodiments of the present invention. Specifically, they are: "[B-1] A method for imaging intracellular organelles, comprising the step of contacting compound (I) or the like with collected intracellular organelles." (Example Form B-1) "[B-2] A method for imaging intracellular organelles, comprising the step of administering compound (I) or the like to a mammal and contacting the compound (I) or the like with cells containing intracellular organelles." (Embodiment B-2)

[0033] Regarding Embodiment B-1 (In Vitro Imaging) In this embodiment, the "intracellular organelle" targeted for imaging is, for example, "intracellular lipid droplets." "Collected cells" refer to cells to be imaged that have been previously collected and isolated from a mammal or the like. "Collecting" can be performed by methods commonly used in the art. In this embodiment, the step of contacting the fluorescent probe with intracellular organelles can be performed by methods commonly used in the art, for example, by adding the fluorescent probe to cells containing the target organelle. When compound (I) or the like is dissolved in a solvent and then added to cells containing the target organelle, the solvent can be, for example, a polar solvent such as dimethyl sulfoxide (DMSO) or physiological saline, preferably dimethyl sulfoxide. The amount of compound (I) or the like added to cells can vary depending on the cells used, the proportion of target organelles, etc., but can be added to cells containing the target organelle at a final concentration of, for example, 0.05 to 50 μM, preferably 0.3 to 2.5 μM.

[0034] This embodiment may further include a step of irradiating the cells with excitation light to generate fluorescence (imaging), and may additionally include a step of measuring the fluorescence from the fluorescent probe. Cells stained with the fluorescent probe may be irradiated with excitation light that excites compound (I) or the like, and the fluorescence from the fluorescent probe may be observed by in vitro fluorescence imaging using an in vitro fluorescence imaging device such as a fluorescence microscope or flow cytometer. For example, compound (I) or the like may be excited by irradiating the cells with excitation light having a wavelength range of, for example, 300 to 800 nm, e.g., 380 to 500 nm, and the emitted fluorescence may be observed in two wavelength regions (e.g., green channel and red channel) within, for example, the range of 400 to 800 nm.

[0035] Regarding Embodiment B-2 (In Vivo Imaging) Cells containing intracellular organelles (e.g., lipid droplets) present in living organisms can also be detected. The "intracellular organelles" targeted for imaging are, for example, intracellular lipid droplets. In other words, the fluorescent probe is also useful as a reagent for detecting cells containing target organelles in vivo or tissues composed of such cells. Examples of such intracellular tissues include adipose tissues such as subcutaneous fat, visceral fat, and ectopic fat (e.g., fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney). In this embodiment, the mammal is not particularly limited as long as it can be observed by fluorescence. Examples include humans, mice, pigs, dogs, and rabbits. Furthermore, the subject of administration may or may not include humans. In this embodiment, the step of contacting the fluorescent probe with intracellular organelles (e.g., lipid droplets) can be performed by administering Compound (I) or the like to a mammal. Examples of administration routes include intravenous administration, subcutaneous administration, and intramuscular administration. The dosage of compound (I) or the like varies depending on the animal to be administered and the administration form, but can be, for example, 0.01 to 1.0 μM / kg body weight, preferably 0.05 to 0.5 μM / kg body weight. When compound (I) or the like is dissolved in a solvent and then administered to a living body, DMSO or the like can be used as the solvent. Fluorescence can be observed by, for example, observing the fluorescent signal from this fluorescent probe using a biomolecular imaging technique using an inverted confocal microscope or the like, thereby enabling detection of cells containing target organelles in a living body or tissues composed of such cells.

[0036] As described in detail above, by using this fluorescent probe, it is possible to observe the fluorescence emitted from target cells in which the imaging target, specifically the intracellular target organelle (e.g., lipid droplets) has been stained with compound (I) or the like, both in vitro and in vivo. Furthermore, as described in the above section (Regarding Fluorescence Properties), compound (I) or the like exhibits "solvatochromism," in which the spectrum of the emitted fluorescence changes depending on the polarity of the solvent. In the case of compound (I) or the like, as the hydrophobicity of the solvent (cellular environment) in which the staining target (e.g., lipid droplets) exists increases (i.e., as the polarity decreases), the fluorescence intensity at shorter wavelengths increases. On the other hand, as the hydrophilicity of the solvent (cellular environment) increases (i.e., as the polarity increases), the fluorescence intensity at longer wavelengths increases (see Test Example 2, Figure 2, below). Due to the above characteristics, by observing the fluctuations in fluorescence intensity in the two wavelength regions emitted by compound (I) and the like, it is possible to evaluate the responsiveness of cells containing target organelles (e.g., lipid droplets) to changes in the cellular environment in which the organelles are present. For example, if the intensity of fluorescence on the longer wavelength side increases due to a change in the cellular environment, this means that the polarity inside the cell containing the organelle stained with compound (I) and the like has shifted toward increasing. Such changes in the cellular environment are diverse, such as oxidative stress imposed on the cell, nutrient deficiency in the cell, and the action of drugs on the cell. By observing the change in the wavelength of the fluorescence emitted from compound (I) and the like, it is possible to explore the state of the target cell using the "change in polarity" within the cell as an indicator of the effect of such changes in the cellular environment on the internal environment of the cell. Here, the degree of wavelength change varies depending on the responsiveness of the cell to changes in the cellular environment. Therefore, by comparing the degree of change in the wavelength of the fluorescence emitted from each cell using multiple types of cells under the same conditions, it is possible to understand the differences in the responsiveness of each cell to a change in the cellular environment. Therefore, the present fluorescent probe containing compound (I) etc. is useful for evaluating the responsiveness of cells to changes in the cellular environment by monitoring the fluorescence intensity during imaging.For specific embodiments of the present fluorescent probe described above, reference can be made to the descriptions in Test Examples 4 to 6 below.

[0037] Another embodiment of the present invention is "[C] Use of compound (I) or the like as a fluorescent probe" (Embodiment C). More specific embodiments include: [C-1] the use according to [C] above, wherein the use is for imaging intracellular organelles. [C-2] the use according to [C-1] above, wherein the imaging of intracellular organelles is for evaluating the responsiveness of cells to changes in the cellular environment by monitoring fluorescence intensity during imaging. [C-3] the use according to [C-1] or [C-2] above, wherein the organelle-containing cells are cells collected from a mammal. [C-4] the use according to any one of [C-1] to [C-3] above, wherein the organelle-containing cells are immune cells. [C-5] the use according to any one of [C-1] to [C-4] above, wherein the organelles are lipid droplets. Each of the above embodiments can be easily implemented by those skilled in the art by referring to the specific aspects detailed in Embodiments A and B. In one embodiment, Compound (I) or a salt thereof is used as the sole imaging component.

[0038] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Those skilled in the art may modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also within the scope of the present invention.

[0039] Example 1: Synthesis of NiTA

[0040]

[0041] 2-Bromo-5-nitrothiophene (3.6 mmol, 1.5 eq.), 4-(dimethylamino)-phenylboronic acid (2.4 mmol, 1.0 eq.), Pd(OAc) (0.024 mmol, 0.01 eq.), and PPh (0.072 mmol, 0.03 eq.) were dissolved in dry THF (15 mL) under an Ar atmosphere. 2M aqueous NaCO (7.5 mL) was then added, and the mixture was heated under reflux (80 °C) overnight. After cooling to room temperature, the mixture was filtered through EA, and the filtrate was washed with water and brine. The organic layer was dried over NaSO, filtered, and concentrated. The crude product was washed with cold MeOH to give the target compound (compound 3 above; N,N-dimethyl-4-(5-nitrothiophen-2-yl)aniline) as a powder.

[0042] Yield: 91% (deep red powder) 1 H-NMR (600 MHz, CHLOROFORM-D) δ 7.86 (d, J = 4.1 Hz, 1H), 7.52 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 4.5 Hz, 1H), 6.71 (d, J = 8.9 Hz, 2H), 3.04 (s, 6H) 13 C NMR (150 MHz, CDCl3): δ 154.4, 151.7, 147.8, 130.6, 127.6, 119.9, 119.7, 112.3, 40.3 ESI-HRMS calculated for C 12 H 12 N2O2S [M+Na] + 271.0512, found 271.0511

[0043] In the following test examples, the results of fluorescence observation from NiTA-stained lipid droplets under various conditions are shown to demonstrate the usefulness of NiTA as a fluorescent probe.

[0044] Test Example 1: Fluorescence Observation Under NiTA Staining T cells, B cells, and macrophages were each stained using NiTA. After staining, each cell was excited with 473 nm excitation light, and the fluorescence emitted from each cell was observed under a fluorescence microscope. Figure 1 shows the observation results in both the green channel (490-540 nm) and red channel (570-670 nm) wavelength ranges. In the figure, the upper row shows the observation results for T cells, the middle row shows the observation results for B cells, and the lower row shows the observation results for macrophages. The observation results showed that in all cells, fluorescence was emitted from NiTA in both the green channel (490-540 nm) and red channel (570-670 nm) wavelength ranges.

[0045] Test Example 2: Investigation of the Fluorescence Wavelength Emitted from NiTA in Various Solvents The wavelength of fluorescence emitted from solutions of NiTA dissolved in various solvents (hexane, ethyl ether, dioxane, ethyl acetate (EA), and acetone) was observed. The results are shown in Figure 2. The graph showing the fluorescence wavelength in the figure shows, from left to right, the results of observation in hexane, ethyl ether, dioxane, ethyl acetate (EA), and acetone. The observation results showed that NiTA exhibits solvatochromism, and the wavelength of the fluorescence emitted from NiTA becomes longer as the polarity (hydrophilicity) of the solvent increases.

[0046] Test Example 3: Fluorescence observation under co-staining with NiTA and Lipi-Blue HeLa cells were co-stained with NiTA and Lipi-Blue, a dye for lipid droplets. NiTA and Lipi-Blue were also stained separately. Under confocal imaging, the fluorescence emitted from each cell was observed under a fluorescence microscope in the wavelength ranges of the blue and green channels. The observation results are shown in Figure 3. The observation results showed that the fluorescence emitted from NiTA staining and Lipi-Blue staining colocalized with each other, indicating that NiTA can selectively stain lipid droplets.

[0047] Lipid droplets play many roles, including regulating lipotoxicity in response to environmental stress and maintaining cellular lipid homeostasis. To monitor the state of lipid droplets in response to changes in the cellular environment, we performed imaging of lipid droplets using NiTA under oxidative stress and starvation conditions.

[0048] Experimental Example 4: Observation of Fluorescence from NiTA-Stained Lipid Droplets under Oxidative Stress Conditions. Hydrogen peroxide is one of the intracellular reactive oxygen species (ROS). To induce oxidative stress due to ROS, T cells and B cells were stained with 2.5 μM NiTA and then treated with 2 mM hydrogen peroxide for 1 hour. Untreated and H2O2-treated cells were imaged using two color channels, and significant changes in fluorescent signal were observed in both the green and red channels. An overall increase in fluorescent signal was observed in both T and B cells. This indicates the presence of more lipid droplets in H2O2-treated cells. Lipid droplets accumulate to protect cells by sequestering toxic lipids from peroxidation and mitigating oxidative stress. Furthermore, when the R / G fluorescence ratio was analyzed from imaging of untreated and H2O2-treated cells, the R / G fluorescence ratio of H2O2-treated cells increased compared to untreated cells (from 0.87 to 0.89 in T cells and from 0.87 to 0.92 in B cells). The results of the evaluation in T cells are shown in Figure 4, and the results of the evaluation in B cells are shown in Figure 5. (Summary) It has been reported that in H2O2-treated samples, cholesterol esters are converted to more polar products through oxidation reactions. The change in the R / G fluorescence ratio under oxidative stress conditions in this study indicates that oxidative stress conditions increase the polarity of the environment within lipid droplets.

[0049] Experimental Example 5: Observation of Fluorescence from NiTA-Stained Lipid Droplets under Starvation Conditions (1) To further explore the state of lipid droplets in T and B cells by monitoring lipid droplets under starvation conditions using NiTA, we conducted another series of experiments. To this end, cells treated with low-glucose medium without FBS were stained with NiTA to visualize lipid droplets. An increase in fluorescent signal was observed in both cell types, suggesting an increase in lipid droplets under starvation conditions. (2) Under nutrient deprivation, cells shift their metabolism from relying on glycolysis to mitochondrial fatty acid oxidation, using lipids instead of glucose as an energy source. To facilitate this process, cells increase the number of lipid droplets as lipid conduits. Furthermore, to provide energy during nutrient deprivation, lipid beta-oxidation increases, altering the polarity of the lipid droplet environment. Furthermore, previous studies have shown that cholesterol levels in adipose tissue increase during starvation. In this study, the changes in the R / G fluorescence ratio observed in each cell type during starvation were significantly different between T and B cells. In T cells, the R / G fluorescence ratio was 0.53 (initial 0.6) after 24 hours, and in B cells, the R / G fluorescence ratio was 1.36 (initial 0.70) after 24 hours. In B cells, the fluorescence shifted significantly toward the red. Figure 6 shows the results of (1) and (2) in T cells, and Figure 7 shows the results of (1) and (2) in B cells. (Summary) These results suggest that NiTA can be used to monitor lipid droplets in immune cells and may provide important information about the susceptibility of immune cells to stress conditions such as nutrient deprivation. The increase in red fluorescence is thought to suggest a change in the polarity of the LD environment due to beta-oxidation during lipid degradation. As described above, the changes in the R / G fluorescence ratio observed during starvation were significantly different between T cells and B cells. These results provide important information about the susceptibility of cells to stressful conditions such as nutrient deprivation.In other words, it is thought that T cells adapt to environmental changes even in a state of starvation, but B cells are more stressed than T cells in an environment of nutrient deficiency, which causes lipid breakdown through beta-oxidation reactions to progress, increasing the polarity of the cellular environment and causing the increase in red fluorescence to exceed the increase in green fluorescence.

[0050] Experimental Example 6: Observation of Fluorescence from NiTA-Stained Lipid Droplets in the Presence of a Cholesterol Production Inhibitor. Red fluorescence from NiTA in lipid droplets was examined in both T cells and B cells in the presence of simvastatin. Simvastatin is an HMG-CoA reductase inhibitor that reduces cholesterol production. To alter cholesterol levels in these cells, cells were treated with simvastatin. Live imaging of T cells revealed no significant difference in red fluorescence intensity over time between simvastatin-treated and untreated T cells (Figure 8). In contrast, simvastatin-treated B cells showed a significant change in fluorescence intensity over time compared to untreated B cells (Figure 9). These results demonstrate that using NiTA as a dye to measure the ratio of cholesterol moieties in lipid droplets can be a valuable tool for visualizing lipid droplet composition in live cells. (Summary) Lipid droplet imaging using NiTA revealed distinct responsiveness of T cells and B cells to the same drug treatment. This result suggests that when the same drug treatment is applied to different cells, the reactivity and responsiveness of each cell to the drug can be compared using NiTA.

[0051] (Summary) The results of various tests using the representative compound NiTA revealed the following characteristics of compound (I) and others. (1) Compound (I) and others are solvatochromic fluorescent probes that target lipid droplets and can selectively stain intracellular lipid droplets. (2) Compound (I) and others can be used to clearly image lipid droplets in various types of cells, including cancer cells and immune cells such as T cells, B cells, and macrophages. (3) Compound (I) and others can observe fluorescence emitted in two wavelength regions, the green channel and the red channel. As the polarity of the solvent increases, fluorescence is emitted at longer wavelengths. Therefore, by focusing on these characteristics, changes in the lipid droplet environment in response to external stress such as oxidative stress or starvation can be detected as polarity changes. (4) Compound (I) and others are expected to be used in a variety of applications, such as fluorescence mapping to monitor changes in the number, morphology, and polarity of lipid droplets.

[0052] In one embodiment, the present invention provides a novel thiophene derivative that is useful, for example, as a fluorescent probe for imaging intracellular organelles (e.g., lipid droplets), and is useful, for example, in the field of pharmaceutical research and development. This application is based on Japanese Patent Application No. 2024-152503 (filing date: September 4, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A compound of the following formula (I): (In the formula, R １ represents an electron donating group, and R ２ represents an electron-withdrawing group.) or a salt thereof.

2. R １ However, mono- or di- (C １－６ alkyl)amino group, or C １－６ an alkoxy group, and R ２ is a nitro group or C １－６ The compound or salt thereof according to claim 1 , wherein the aryl group is an alkanoyl group.

3. R １ But, Gee (C １－６ alkyl)amino group, and R ２ The compound or salt thereof according to claim 2, wherein is a nitro group.

4. The structure represented by formula (I) is the following formula (Ia): The compound according to claim 3 or a salt thereof, wherein the compound has a structure represented by:

5. A fluorescent probe comprising the compound or salt thereof according to any one of claims 1 to 4.

6. The fluorescent probe according to claim 5, which is for imaging intracellular organelles.

7. The fluorescent probe according to claim 6, wherein imaging of intracellular organelles is for evaluating the responsiveness of cells to changes in the cellular environment by monitoring the fluorescence intensity during imaging.

8. The fluorescent probe according to claim 6, wherein the cell is an immune cell.

9. The fluorescent probe according to any one of claims 6 to 8, wherein the organelle is a lipid droplet.

10. A method for imaging intracellular organelles, comprising the step of contacting the compound or salt thereof according to any one of claims 1 to 4 with organelles extracted from cells.

11. The imaging method according to claim 10, wherein imaging of intracellular organelles is for evaluating the responsiveness of cells to changes in the cellular environment by monitoring fluorescence intensity during imaging.

12. The imaging method according to claim 10, wherein the cells containing organelles are cells taken from a mammal.

13. The imaging method according to claim 10, wherein the cells containing organelles are immune cells.

14. The imaging method according to claim 10, wherein the organelle is a lipid droplet.

15. A method for imaging intracellular organelles, comprising the step of administering the compound or salt thereof according to any one of claims 1 to 4 to a mammal and contacting it with cells containing the organelles.

16. The imaging method of claim 15, wherein the mammal is a non-human animal.

17. The imaging method according to claim 15, wherein imaging of intracellular organelles is for evaluating the responsiveness of cells to changes in the cellular environment by monitoring fluorescence intensity during imaging.

18. The imaging method of claim 15, wherein the organelle is a lipid droplet.

19. Use of the compound according to any one of claims 1 to 4 or a salt thereof as a fluorescent probe.

20. The use according to claim 19 for imaging organelles within a cell.

21. The use according to claim 20, wherein the imaging of intracellular organelles is for evaluating the responsiveness of cells to changes in the cellular environment by monitoring fluorescence intensity during imaging.

22. The use according to claim 20, wherein the cells containing the organelles are cells obtained from a mammal.

23. The use according to claim 20, wherein the cells containing the organelles are immune cells.

24. The use according to any one of claims 20 to 23, wherein the organelle is a lipid droplet.

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