Compounds, cellular vesicle staining agents, and fluorescent staining methods for cellular vesicles

Abstract

The subject of this invention is to provide novel compounds that specifically bind to intracellular and / or extracellular vesicles, cellular vesicle staining agents, and methods for fluorescent staining cellular vesicles. The subject matter can be solved by compounds represented by the following formula (1). In formula (1), R1 represents H, C1-C6 alkyl, hydroxyl, amino, or carboxyl groups; R2 represents H, C1-C6 alkyl, hydroxyl, amino, or carboxyl groups. 18 Alkyl, C1-C 18 The alkoxy group, halogen, NO2 or N(CH3)2; R3 and R4 each independently represent H or CH3; wherein, R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2; l represents 1 or 2, m represents 0 or 1, n represents 1, 2 or 3; Z represents a fluorescent group.

Description

Technical Field

[0001] The disclosure of this application relates to compounds, cellular vesicle staining agents, and methods for fluorescent staining cellular vesicles. Background Technology

[0002] In recent years, endocytosis, known as a mechanism for the uptake of extracellular molecules into cells, has been extensively studied and is associated with cellular physiological functions and diseases. The cell surface indentation caused by endocytosis subsequently translocates into the cell to form endosomes. Therefore, extracellular substances taken into the cell via endocytosis accumulate in endosomes. Furthermore, endosomes function as important intracellular organelles that determine the subsequent destination of molecules taken in from the extracellular environment.

[0003] Furthermore, exosomes are formed from endosomes created through endocytosis and released extracellularly. Since exosomes contain cell membrane components on their surface and intracellular substances inside, they reflect the characteristics of the original cell from which they were released and play an important role in intercellular communication. Moreover, exosomes released extracellularly are also present in body fluids (blood, medullary fluid, urine, etc.) and circulate throughout the body. Therefore, it is hoped that exosomes can be used as diagnostic markers for diseases. Additionally, melanosomes are known as vesicles that, like exosomes, originate from endosomes. Melanosomes are generated, released, and transported to keratinocytes in melanocytes, where they are taken up by keratinocytes. Based on this mechanism, the accumulation of melanosomes in keratinocytes can be measured for the evaluation of pharmaceuticals, cosmetics, etc.

[0004] Therefore, the detection and analysis of intracellular vesicles derived from endosomes and those released into the extracellular space are important for understanding the mechanisms of cellular uptake and release of external substances. Patent Document 1 discloses the analysis of exosomes from multiple abnormal cell sources. Furthermore, Patent Document 2 discloses a method in which fluorescently labeled keratinocytes and unlabeled melanocytes are co-cultured, and after a fluorescently labeled melanosome-specific antibody binds to the cells, the cells are analyzed using flow cytometry.

[0005] Prior art literature

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2020-204604;

[0008] Patent Document 2: Japanese Patent Application Publication No. 2005-249391. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] Patent documents 1 and 2 disclose the detection of vesicles using fluorescent labeling with fluorescent dyes. Fluorescent labeling is a simple method that involves simply recovering the vesicles and mixing them with the fluorescent dye, and is therefore widely used. However, problems exist such as sample loss due to the removal of unreacted fluorescent dye after labeling, and micelle formation due to the aggregation of fluorescent dyes. Therefore, compounds of novel staining agents capable of specifically staining vesicles are desired.

[0011] Therefore, the subject matter disclosed in this application is to provide novel compounds, cellular vesicle staining agents, and fluorescent staining methods for cellular vesicles that specifically bind to intracellular and / or extracellular vesicles. Other optional effects disclosed in this application will become apparent in the manner in which the invention is carried out.

[0012] Problem-solving methods

[0013] [1] A compound represented by the following formula (1), wherein,

[0014] [Chemical Formula 1]

[0015]

[0016] In formula (1), R1 represents H, C1-C6 alkyl, hydroxyl, amino, or carboxyl groups; R2 represents H, C1-C6 alkyl, hydroxyl, amino, or carboxyl groups. 18 Alkyl, C1-C 18 The alkoxy group, halogen, NO2 or N(CH3)2; R3 and R4 each independently represent H or CH3; wherein, R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2; l represents 1 or 2, m represents 0 or 1, n represents 1, 2 or 3; Z represents a fluorescent group.

[0017] [2] According to the compound described in [1] above, wherein the compound represented by formula (1) is the compound represented by formula (2) below.

[0018] [Chemical Formula 2]

[0019]

[0020] In formula (2), R1 represents H or CH3; R2 represents H, CH3, OCH3 or N(CH3)2; R3 and R4 each independently represent H or CH3; R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2; R5 represents NO2, SO2NH2 or SO2N(CH3)2; l represents 1 or 2, m represents 0 or 1, and n represents 1, 2 or 3.

[0021] [3] According to the compound described in [1] above, wherein the compound represented by formula (1) is the compound represented by formula (3) below.

[0022] [Chemical Formula 3]

[0023]

[0024] In formula (3), R1 represents H or CH3; R2 represents H, CH3, OCH3 or N(CH3)2; R3 and R4 each independently represent H or CH3; R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2; l represents 1 or 2, m represents 0 or 1, and n represents 1, 2 or 3.

[0025] [4] A cellular vesicle staining agent comprising any one of the compounds described in [1] to [3] above.

[0026] [5] The cellular vesicle staining agent described in [4] above also contains other fluorescent compounds.

[0027] [6] A fluorescent staining method for cellular vesicles, comprising:

[0028] The staining process involves selecting any one of the compounds described in [1] to [3] above, as well as the cellular vesicle staining agents described in [4] and [5] above, to stain the cellular vesicles; and

[0029] The testing process involves detecting stained cellular vesicles in the sample.

[0030] [7] According to the fluorescent staining method for cellular vesicles described in [6] above, wherein,

[0031] Following the testing process, there is an evaluation process for evaluating the test samples.

[0032] [8] According to the fluorescent staining method for cellular vesicles described in [7] above, wherein,

[0033] The samples included cosmetics.

[0034] The evaluation of cosmetics is based on the measurement of intracellular cellular vesicles.

[0035] [9] According to the fluorescent staining method for cellular vesicles described in [7] above, wherein,

[0036] The samples included pharmaceuticals.

[0037] Evaluation of pharmaceuticals is achieved by measuring intracellular uptake of cellular vesicles.

[0038]

[10] According to the fluorescent staining method for cellular vesicles described above [7], wherein,

[0039] The sample includes cells.

[0040] Cellular vesicles are measured within cells to evaluate cell differentiation and quality.

[0041]

[11] According to the fluorescent staining method for cellular vesicles described above [7], wherein,

[0042] The samples included food.

[0043] Food is evaluated by measuring cellular vesicles within it.

[0044]

[12] According to the fluorescent staining method for cellular vesicles described above [7], wherein,

[0045] The sample contains biological tissue collected from a living organism.

[0046] The biological tissue is evaluated by measuring cellular vesicles in the collected biological tissue.

[0047] Invention Effects

[0048] No unreacted pigments need to be removed after labeling, thus minimizing sample loss. Attached Figure Description

[0049] Figure 1 The photograph is a substitute for the accompanying drawing showing the results of Example 13. Figure 1 A shows a fluorescence microscope image of the entire cell. Figure 1 B shows that it will be by Figure 1 An enlarged view of the area enclosed by the box A.

[0050] Figure 2 The photograph is a substitute for the accompanying drawing showing the results of Example 14. Figure 2 A shows a fluorescence microscopy image of compound 1 before the addition of the PIKfyve inhibitor. Figure 2 B shows a phase-difference microscopy image before the addition of the PIKfyve inhibitor. Figure 2 C shows a fluorescence microscopy image of compound 1 after the addition of the PIKfyve inhibitor. Figure 2 D shows a phase-difference microscopy image after the addition of the PIKfyve inhibitor. Figure 2 E is a fluorescence microscopy image of the fluorescence emitted by compound 1 after cells were cultured for 3 hours following the removal of PIKfyve. Figure 2 F is a phase-contrast microscopy image taken 3 hours after removing PIKfyve cells and culturing them.

[0051] Figure 3 This is a photograph that serves as an alternative illustration to show the results of fluorescent staining of human cell vesicles using compound 1. Figure 3 A shows a fluorescence microscope image of Example 15. Figure 3 B shows a fluorescence microscope image of Example 16.

[0052] Figure 4 The photograph is a substitute for the accompanying drawing showing the results of Example 17. Figure 4 A shows a fluorescence microscope image of the fluorescence emitted by compound 1. Figure 4 B shows a fluorescence microscope image of the fluorescence emitted by the mitochondrial staining reagent. Figure 4 C shows a fluorescence microscope image of DAPI stained with a nucleus, observing its fluorescence. Figure 4 D shows a phase-contrast microscope image.

[0053] Figure 5 The photograph is an alternative illustration showing an example of the high specificity of compound 1 in cellular vesicles. Figure 5 A is a graph that overlays fluorescence microscopy images of Example 18 and Comparative Example 1. Figure 5 B is a fluorescence microscope image of Example 18. Figure 5 C is a fluorescence microscope image of Comparative Example 1.

[0054] Figure 6 The accompanying photograph is an alternative illustration showing other examples of the high specificity of compound 1 in cellular vesicles. Figure 6 A shows a fluorescence microscope image of Example 19. Figure 6 B shows a phase-contrast microscope image of Example 19. Figure 6 C shows a fluorescence microscope image of Comparative Example 2. Figure 6 D shows a phase-difference microscope image of Comparative Example 2.

[0055] Figure 7 This is a photograph that shows the results of fluorescent staining of exosomes, instead of the accompanying image.

[0056] Figure 8 This is a graph showing the standard curve in exosome detection.

[0057] Figure 9 This is a graph showing the results of exosome assays in food.

[0058] Figure 10 This is a photograph showing the uptake of labeled exosomes into cells, replacing the accompanying illustration. Figure 10 A shows a fluorescence microscopy image of the superimposed fluorescence emitted by compound 1 and ExoSparkler Mem Dye-Red. Figure 10 B shows a fluorescence microscope image of the fluorescence emitted by compound 1. Figure 10C shows a fluorescence microscope image of the fluorescence emitted by ExoSparkler Mem Dye-Red.

[0059] Figure 11 This is a diagram showing the results of inhibited cellular uptake of labeled melanosomes. Figure 11 A shows a fluorescence microscope image of Example 29 and a phase difference microscope image of Comparative Example 4. Figure 11 B shows the results of Example 29. Figure 11 C shows the results of Comparative Example 4.

[0060] Figure 12 This is a photograph that shows the results of fluorescent staining of cellular vesicles. Figure 12 A shows a fluorescence microscope image of Example 30. Figure 12 B shows a phase difference microscope image of Example 30. Figure 12 C shows a fluorescence microscope image of Example 31. Figure 12 D shows a fluorescence microscope image of Example 32. Figure 12 E shows a fluorescence microscope image of Example 33. Figure 12 F shows a fluorescence microscope image of Example 34. Figure 12 G shows a fluorescence microscope image of Example 35. Figure 12 H shows a fluorescence microscope image of Example 36. Figure 12 I shows a fluorescence microscope image of Example 37. Figure 12 J shows a fluorescence microscope image of Example 38. Figure 12 K shows a fluorescence microscope image of Example 39.

[0061] Figure 13 This is a graph showing the results of fluorescence intensity measurements of live and dead cells based on compounds 1 to 7.

[0062] Figure 14 This is a diagram showing the results of Example 47. Detailed Implementation

[0063] (Implementation of the compound)

[0064] The compounds involved in the embodiments will be described below.

[0065] The compounds involved in the embodiments are characterized as compounds represented by the following formula (1). The compounds represented by formula (1) specifically bind to cellular vesicles and emit fluorescence. In this specification, "cellular vesicles" includes "intracellular vesicles," exosomes, and other "extracellular vesicles."

[0066] [Chemical Formula 4]

[0067]

[0068] In formula (1), R1 represents H, a C1-C6 alkyl group, a hydroxyl group, an amino group, or a carboxyl group. The C1-C6 alkyl group can be straight-chain, branched, or cyclic. Examples of C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0069] R2 represents H, Cl-C 18 Alkyl, C1-C 18 Alkyl groups, halogens, NO2, or N(CH3)2. C1-C 18 The alkyl group can be straight-chain, branched, or cyclic. As a C1-C... 18 Alkyl groups, for example, include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, sec-heptyl, n-octyl, isooctyl, sec-octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, cyclotridecyl, cyclotetradecyl, cyclopentadecanyl, cyclohexadecyl, cycloheptadecyl, cyclooctadecyl, etc. C1-C 18 The alkoxy group can be saturated or unsaturated, and can also have an aromatic ring. Additionally, C1-C... 18 The alkoxy group can be straight-chain, branched, or cyclic. As a C1-C 18Alkyl groups, for example, include methoxy, ethoxy, propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, octoxy, nonoxy, decoxy, undecoxy, dodecoxy, tridecoxy, tetradecoxy, pentadecoxy, hexadecoxy, heptadecanoxy, octadecoxy, vinyloxy, allyloxy, 1-propenoxy, isopropenoxy, 1-butenoxy, 2-butenoxy, 3-butenoxy, 1,3-butadieneoxy, 1-pentenoxy, 2-pentenoxy, 3-pentenoxy, 4-pentenoxy, hexenoxy, hexadieneoxy, heptenoxy, heptenyloxy, octenoxy, octadieneoxy, nonenoxy, and nonyloxy. Dienoxy, decenoxy, decadienoxy, undecenoxy, undecadienoxy, dodecenoxy, dodecadienoxy, tridecenoxy, tridecadienoxy, tetradecenoxy, tetradecadienoxy, pentadecenoxy, pentadecadienoxy, hexadecenoxy, hexadecadienoxy, heptadecenoxy, heptadecenoxy, octadecenoxy, octadecadienoxy, phenoxy, naphthoxy, anthraceneoxy, methylphenoxy, dimethylphenoxy, trimethylphenoxy, ethylphenoxy, diethylphenoxy, triethylphenoxy, propylphenoxy, butylphenoxy, methylnaphthoxy, dimethylnaphthoxy, trimethylnaphthoxy, methylanthraoxy, ethylanthraoxy, benzyloxy, phenethyloxy, naphthylmethoxy, fluorenylmethoxy, etc. Examples of halogen elements include fluorine, chlorine, bromine, and iodine.

[0070] R3 and R4 can each independently represent H or CH3. R3 and R4 can be the same or different. In addition, R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2.

[0071] l represents 1 or 2, m represents 0 or 1, and n represents 1, 2, or 3.

[0072] Z represents a fluorescent group. The fluorescent group of the compound shown in formula (1) is not particularly restricted as long as it is a substance that emits fluorescence. Examples of fluorescent groups include NBD (nitrobenzanol), ABD (aminosulfonylbenzofuran), DBD (dimethylaminesulfonylbenzofuran), dimethylaminobenzylrhodanine, FAM, FITC, ROX, TAMRA, Alexa Fluor (registered trademark) 405, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, AMCA, AMCA-X, APC (allophycocyanin), APC-XL, ATTO 390, ATTO 465, ATTO 488, ATTO 490LS, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO 590, ATTO 594, and ATTO. 633, ATTO647N, ATTO 647, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 740, C-PC (C-phycocyanin,C-Phycocyanin), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, DY-405, DY-415, DY-475XL, DY-480XL, DY-481XL, DY-485XL, DY-490, DY-495, DY-495-X5, DY-500XL, DY-505-X5, DY-510XL, DY-520XL, DY-521XL, DY-547, DY-548, DY-549, DY-554, DY-555, DY-556, DY-560, DY-590, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647, DY-648, DY-649, DY-650, DY-651, DY-652, DY-675, DY-676, DY-677, DY-678, DY-680, DY-681, DY-682, DY-700, DY-701, DY-730, DY-731, DY-732, DY-734, DY-749, DY-750, DY-751, DY-752, DY-776, DY-777, DY-780, DY-781, DY-782, DyLight 405, DyLight 488, DyLight547, DyLight 549, DyLight 594, DyLight 633, DyLight 647, DyLight 649, DyLight 680, DyLight 750, DyLight 800, ECD, Fluorescein, HiLyte Flour 488, HiLyte Fluor 555, HiLyte Fluor 647, HiLyteFluor 680, HiLyte Fluor 750, HiLyte Fluor TR, HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, IRDye700DX, IRDye800, IRDye800CW, MFP488, MFP555, MFP590, MFP631, NorthernLights 493, NorthernLights 557, NorthernLights 637, Oyster 500, Oyster550, Oyster 556, Oyster 645, Oyster 650, Oyster 656, Pacific Blue, PE (phycoerythrin,Phycoerythrin), B-PE, R-PE, PerCP, Phycocyanin, PREX710, Quantum Red, Rhodamine, ROX (X-Rhodamine, Rhodamine Red X), Royal Blue, Spectrum Green, Spectrum Orange, Spectrum Red, Texas Red, Tri-Color, TRITC, etc. Additionally, the fluorescent group Z can be bound in any manner as long as it fluoresces when the compound shown in formula (1) stains cellular vesicles.

[0073] As more specific examples of the compounds shown in formula (1), compounds shown in formula (2) and formula (3) can be cited below.

[0074] [Chemical Formula 5]

[0075]

[0076] In equation (2), R1 represents H or CH3. R2 represents H, CH3, OCH3, or N(CH3)2. R3 and R4 each independently represent H or CH3. R3 and R4 can also bond to each other to form a ring, in which case R3 and R4 are CH2. R5 represents NO2, SO2NH2, or SO2N(CH3)2. l represents 1 or 2, m represents 0 or 1, and n represents 1, 2, or 3.

[0077] [Chemical Formula 6]

[0078]

[0079] In equation (3), R1 represents H or CH3. R2 represents H, CH3, OCH3, or N(CH3)2. R3 and R4 each independently represent H or CH3. R3 and R4 can also bond with each other to form a ring, in which case R3 and R4 are CH2. l represents 1 or 2, m represents 0 or 1, and n represents 1, 2, or 3.

[0080] The following are specific examples of compounds represented by formula (2) and formula (3), but are not limited to these exemplified compounds.

[0081] [Chemical Formula 7]

[0082]

[0083]

[0084] The compound shown in formula (1) binds to cellular vesicles derived from endosomes. Therefore, the compound according to the embodiments can fluorescently stain intracellular endosomes, melanosomes, and exosomes released extracellularly. Furthermore, when staining intracellular vesicles, the compound according to the embodiments can specifically fluorescently stain intracellular vesicles of living cells. Moreover, the compound according to the embodiments fluoresces upon binding to cellular vesicles, while compounds not bound to cellular vesicles do not fluoresce. Therefore, the process of removing compounds not bound to cellular vesicles is eliminated, and sample loss can be suppressed.

[0085] (Implementation method of cellular vesicle staining agent)

[0086] The compounds described in the above embodiments can also be dissolved in solvents and used as staining agents for cellular vesicles. The solvent is not particularly limited as long as it can dissolve the compound shown in formula (1). Examples of solvents include acetone, DMSO (dimethyl sulfoxide), ethanol, and aqueous solutions thereof.

[0087] In addition to the compound shown in formula (1), the cellular vesicle staining agent involved in the embodiments may optionally contain other fluorescent compounds, such as those that fluoresce to elements different from cellular vesicles, such as mitochondria and cell membranes. By using the compound shown in formula (1) and the fluorescent compound, multiple elements can be stained simultaneously. Furthermore, through the interaction between the compound shown in formula (1) and the fluorescent compound, the compound shown in formula (1) can be sensitized and its specificity for cellular vesicles can be enhanced.

[0088] (Implementation method of fluorescent staining for cellular vesicles)

[0089] The staining method for cellular vesicles involved in the embodiments includes at least: a staining step, which stains cellular vesicles using a compound or cellular vesicle staining agent represented by formula (1); and a detection step, which detects the stained cellular vesicles in the sample, and optionally includes an evaluation step that evaluates the sample after the detection step.

[0090] The staining process utilizes the compound shown in formula (1) to fluorescently stain cellular vesicles. For example, when staining intracellular vesicles, the compound shown in formula (1) can be added during cell culture. Alternatively, cultured cells can be recovered, the compound shown in formula (1) can be added to the recovered cells, and the cells can be placed on a culture dish or similar surface. When staining extracellular vesicles such as exosomes, the compound shown in formula (1) can be added to the solution containing the extracellular vesicles.

[0091] In the dyeing process, the amount of compound added as shown in formula (1) can be in the range of 0.01 μM to 10 μM, preferably in the range of 0.1 μM to 1 μM.

[0092] In the detection process, excitation light corresponding to the fluorophore of the compound shown in formula (1) is irradiated, and fluorescence emitted from the compound shown in formula (1) bound to cellular vesicles in the sample is detected. For excitation light irradiation, the same irradiation method as for general fluorescence detection can be used; for example, a laser source provided with a fluorescence microscope can be used, selecting a specified wavelength as needed. Fluorescence detection can be performed from the microscope tube, or images captured by a camera or similar device mounted on the fluorescence microscope can be displayed on a monitor or similar display unit. Based on the fluorophore of the compound shown in formula (1), even if fluorescence cannot be fully observed visually from the microscope tube, it can sometimes be observed by capturing images using a camera or similar device. A filter that selectively allows a specified wavelength to pass through can also be used as needed.

[0093] In addition, the fluorescent staining method for cellular vesicles may optionally include an evaluation step for the sample. The evaluation step evaluates the sample based on the cellular vesicles detected by the compound shown in formula (1). The evaluation items vary depending on the sample; for example, it can be used to evaluate cosmetics, pharmaceuticals, food, etc. Furthermore, it can also be used to evaluate the degree of cell differentiation by detecting intracellular vesicles, and to evaluate diseases by detecting exosomes in body fluids, etc.

[0094] For example, in evaluating cosmetics and pharmaceuticals, cellular vesicles can be pre-labeled using compounds represented by formula (1). Based on the presence of the cosmetics or pharmaceuticals, the uptake of these labeled cellular vesicles into cells can be detected and observed, thereby evaluating the cosmetics and pharmaceuticals. For example, pigmentation and associated skin diseases can be evaluated based on the uptake of melanosomes (vesicles that store melanin) into keratinocytes. Furthermore, food can also be evaluated by measuring the cellular vesicles contained in food. For example, the uptake efficiency of miRNAs within vesicles that affect intestinal function can be used to predict food function.

[0095] Furthermore, the degree of cell differentiation can also be evaluated by observing the distribution of cellular vesicles within the cells. For example, melanocytes with a high melanosome content and those with a low melanosome content exhibit different degrees of differentiation. Therefore, by detecting melanosomes in melanocytes, the degree of cell differentiation and quality can also be evaluated.

[0096] Furthermore, exosomes reflect the characteristics of the released protocells. Moreover, cellular vesicles such as exosomes released extracellularly are contained within biological tissues. Therefore, exosomes in biological tissues can also be used as diagnostic markers for diseases, etc. Therefore, by collecting biological tissues from an organism and measuring the cellular vesicles of the collected biological tissues using the compound shown in formula (1), diseases, etc., can be evaluated. Therefore, the compound shown in formula (1) can also be used in the development of clinical examination instruments and reagents, and can also be used in kits for measuring cellular vesicles. Additionally, "biological tissues" in this specification includes body fluids, cells, and tissues within an organism.

[0097] The compounds, cellular vesicle staining agents, and fluorescent staining methods for cellular vesicles involved in the embodiments achieve the following effects.

[0098] (1) The compound shown in formula (1) specifically binds to cellular vesicles. Furthermore, the compound shown in formula (1) that does not bind to cellular vesicles does not emit fluorescence. Therefore, since it is not necessary to remove the unbound compound shown in formula (1) after specific fluorescent staining of cellular vesicles, sample loss can be suppressed.

[0099] (2) There is no need to remove the unbonded compound shown in formula (1). Cellular vesicles can be easily stained with fluorescence simply by adding the compound shown in formula (1).

[0100] (3) When the cellular vesicle staining agent contains a fluorescent compound in addition to the compound shown in formula (1), multiple elements can be stained simultaneously. In addition, through the interaction between the compound shown in formula (1) and the fluorescent compound, the compound shown in formula (1) can be sensitized and its specificity for cellular vesicles can be improved.

[0101] (4) By using the compound shown in formula (1) to detect cellular vesicles, cosmetics, pharmaceuticals, food, cell differentiation, diseases, etc. can be evaluated.

[0102] The following examples illustrate the implementation methods disclosed in this application, but these examples are for illustrative purposes only and do not limit or restrict the scope of the invention disclosed in this application.

[0103] Example

[0104] [Synthesis of Compound 1]

[0105] <Example 1>

[0106] Compound 1 was synthesized according to the steps described below.

[0107] [Chemical Formula 8]

[0108]

[0109] 5-(2-bromoethoxy)-2,3-dihydro-1H-inden-1-one

[0110] 5-Hydroxy-1-indanone (745 mg, 5.03 mmol) was dissolved in ethyl acetate (15 mL), followed by the addition of potassium carbonate (2.45 g, 17.7 mmol), 1,2-dibromoethane (3.27 mL, 38.0 mmol), and benzyltriethylammonium chloride (BTEAC) (121 mg, 531 μmol) suspended in ethyl acetate (15 mL). The mixture was heated under reflux for 24 hours. The reaction solution was cooled to room temperature and then injected into ice water. The solution was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue after concentration under reduced pressure was purified by column chromatography (silica gel, hexane / ethyl acetate = 1 / 1) to give 5-(2-bromoethoxy)-2,3-dihydro-1H-indanone (1.06 g, 4.16 mmol, 83%) as a white solid.

[0111] 1 H NMR (400MHz, CDCl3) δ: 2.67 (m, 2H, COCH2), 3.08 (br t, J = 6Hz, 2H, ArCH2), 3.66 (t, J = 6.4Hz, 2H, BrCH2), 4.35 (t, J = 6.4Hz, 2H, OCH2), 6.89-6.93 (br m,2H,ArH),7.69(d,J=9.2Hz,1H,ArH).

[0112] 5-(2-azidoethoxy)-2,3-dihydro-1H-inden-1-one

[0113] 5-(2-bromoethoxy)-2,3-dihydro-1H-indene-1-one (410 mg, 1.61 mmol) was dissolved in N,N-dimethylformamide (5.0 mL), and sodium azide (129 mg, 1.98 mmol) was added. The mixture was heated under reflux for 35 minutes. The reaction solution was cooled to room temperature and then injected into water. The solution was extracted with diethyl ether, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure to give a reddish-brown solid of 5-(2-azidoethoxy)-2,3-dihydro-1H-indene-1-one (346 mg, 1.59 mmol, 99%).

[0114] 1 H NMR(400MHz, CDCl3)δ:2.66-2.7(m,2H,COCH2),3.09(br t,J=6Hz,2H,ArCH2),3.63(t,J=5Hz,2H,N3CH2),4.21(t,J=5Hz,2H,OCH2),6.90-6.94(br m,2H,ArH),7.70(d,J=9.2Hz,1H,ArH).

[0115] [Chemical Formula 9]

[0116]

[0117] 3-[4-(dimethylamino)phenyl]acrylonitrile

[0118] Under an argon atmosphere, potassium hydroxide (890 mg) was dissolved in acetonitrile (5.0 mL), and the mixture was heated to reflux for 10 minutes. Then, p-dimethylaminobenzaldehyde (2.03 g, 13.6 mmol) dissolved in acetonitrile (10.0 mL) was added, and the mixture was heated to reflux for 3 hours. After cooling to room temperature, water was added, and the mixture was concentrated under reduced pressure using a rotary evaporator. The residue was extracted with dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure until it did not solidify, and ethyl acetate was added. The mixture was then concentrated twice under reduced pressure to give 3-[4-(dimethylamino)phenyl]acrylonitrile (2.40 g). This compound was used directly in the next reaction without purification.

[0119] 3-[4-(dimethylamino)phenyl]propanenitrile

[0120] 3-[4-(dimethylamino)phenyl]acrylonitrile (2.40 g) was dissolved in ethyl acetate (40.0 mL), and Pd / C (10%, 240 mg) was added. The reaction was carried out at room temperature for 5 hours and 30 minutes under a hydrogen atmosphere at balloon pressure. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / ethyl acetate = 3 / 1) to give pale yellow crystalline 3-[4-(dimethylamino)phenyl]propionitrile (1.40 g, 8.03 mmol, 59% yield in two steps).

[0121] 1H NMR (400MHz, CDCl3) δ: 2.55 (t, J = 7.4Hz, 2H, CH2CN), 2.86 (t, J = 7.4Hz, 2H, ArCH2), 2.92 (s, 6H, N (CH3) 2), 6.69 (d, J = 8.7Hz, 2H, ArH), 7.09 (d, J = 8.7Hz, 2H, ArH).

[0122] 4-(3-aminopropyl)-N,N-dimethylaniline

[0123] Under an argon atmosphere, 3-[4-(dimethylamino)phenyl]propionitrile (601 mg, 3.45 mmol) was dissolved in diethyl ether (3.5 mL). Lithium aluminum hydride (391 mg, 10.3 mmol) was added in an ice-water bath, and the reaction was allowed to proceed for 3 hours. Diethyl ether was then added to the reaction mixture, followed by the sequential addition of water (391 μL), 15% sodium hydroxide aqueous solution (391 μL), and water (1.17 mL) while stirring. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily 4-(3-aminopropyl)-N,N-dimethylaniline (507 mg).

[0124] [Chemical Formula 10]

[0125]

[0126] 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-inden-1-amine

[0127] 4-(3-aminopropyl)-N,N-dimethylaniline (67.6 mg, 379 μmol) was dissolved in methanol (600 μL), and 5-(2-azidoethoxy)-2,3-dihydro-1H-inden-1-one (41.2 mg, 190 μmol), sodium cyanoborohydride (21.2 mg, 337 μmol), and acetic acid (30 μL) were added sequentially. The mixture was heated under reflux for 15 hours and 10 minutes. After cooling the reaction solution to room temperature, excess hydride was quenched by adding 1M hydrochloric acid. Then, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to obtain a pale yellow oily 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (36.3 mg, 95.6 μmol, 50%).

[0128] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.75-1.86 (complex, 3H, ArCH₂C) H 2), 2.35(m, 1H, ArCH2C H 2),2.57(dt,J=3.2 and 8.0Hz,2H,ArCH2),2.71(t,J=7.1,2H,ArCH2),2.7-2.8(m,1H), 2.90(s,6H,N(CH3)2),2.90-2.99(m,1H),3.56(t,J=5Hz,2H,N3CH2),4.12(t,J=5Hz, 2H,OCH2),4.15(t,J=6.4Hz,1H,ArCHNH),6.68(d,J=8.4Hz,2H,ArH),6.74(d,J=8.2H z,1H,ArH),6.77(s,1H,ArH),7.05(d,J=8.4Hz,2H,ArH),7.20(d,J=8.2Hz,1H,ArH).

[0129] 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine

[0130] 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (10.1 mg, 26.6 μmol) was dissolved in methanol (150 μL), followed by the sequential addition of paraformaldehyde (4.9 mg) and sodium cyanoborohydride (19.6 mg, 312 μmol), and the reaction was carried out at room temperature for 18 hours. Excess hydride was quenched by adding 1 M hydrochloric acid to the reaction solution. Next, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to obtain a pale yellow oily 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (5.4 mg, 14 μmol, 53%).

[0131] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.72-1.84 (complex, 2H, ArCH₂C) H 2), 1.98-2.05(m,2H,ArCH2C) H 2),2.15(s,3H,NCH3),2.38-2.45(m,1H),2.44-2.62(composite,2H),2.7-2.8(m, 1H),2.89(br,8H),3.57(t,J=5.0Hz,2H,N3CH2),4.13(t,J=5.0Hz,2H,OCH 2),4.35(t,J=7.4Hz,1H,ArCHN),6.68(d,J=8.7Hz,2H,ArH),6.75(s,1H,ArH),6.75-6.78(1H,ArH),7.05(d,J=8.7Hz,2H,ArH),7.25-7.26(1H,ArH).

[0132] 5-(2-aminoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine

[0133] 5-(2-Azide-ethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (22.4 mg, 57 μmol) was dissolved in methanol (500 μL), and Pd / C (10%, 4.2 mg) was added. The reaction mixture was reacted at room temperature for 6 hours and 20 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily 5-(2-aminoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (17.2 mg).

[0134] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.7-1.85 (complex, 2H, ArCH₂C) H 2), 1.97-2.06(m,2H,ArCH2C) H 2),2.16(s,3H,NCH3),2.37-2.62(composite,4H),2.7-2.9(composite,2H),2.89(s,6H),3.06(t,J=5.0Hz,2H,H2NCH2),3.96(t,J=5.0Hz,2H,OCH2),4.3 1-4.4(br,1H,ArCHN),6.68(d,J=8.7Hz,2H,ArH),6.74(s,1H,ArH),6.74-6.77(1H,ArH),7.05(d,J=8.7Hz,2H,ArH),7.25-7.26(1H,ArH).

[0135] [Chemical Formula 11]

[0136]

[0137] Compound 1

[0138] 5-(2-aminoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine (17.2 mg, 46.8 μmol) was dissolved in acetonitrile (500 μL). Then, NBD-F (9.0 mg, 49 μmol) dissolved in acetonitrile (500 μL) was added, and the reaction was carried out at room temperature for 5 hours and 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (silica gel, acetone) to give compound 1 (19.7 mg, 37 μmol, 79%), a reddish-brown oil.

[0139] 1 ¹H NMR (400MHz, CDCl₃): δ: 1.72-1.83 (complex, 2H, ArCH₂C) H 2), 1.98-2.07(m,2H,ArCH2C) H 2), 2.15(s,3H,NCH3), 2.38-2.62(complex,3H,NCH2 and ArCH2), 2.71-2.94(complex,3H,ArCH2), 2.89(s,6H,N(CH3)2), 3.86-3.92(m,2H,HNC) H 2),4.30(t,J=5Hz,2H,OCH2),4.36(t,J=6.7Hz,1H,ArCHN),6.28(d,J=8.5Hz,1H,ArH),6.52(br,1H,ArH),6.67(d ,J=8.5Hz,2H,ArH),6.76(s,1H,ArH),6.75-6.78(1H,ArH),7.05(d,J=8.5Hz,2H,ArH),8.51(d,J=8.5Hz,1H,ArH).

[0140] [Synthesis of Compound 2]

[0141] <Example 2>

[0142] Compound 2 was synthesized according to the procedures described below.

[0143] [Chemical Formula 12]

[0144]

[0145] 5-(2-azidoethoxy)-N-(3-phenylpropyl)-2,3-dihydro-1H-inden-1-amine

[0146] 3-Phenylacetamine (398 mg, 2.94 mmol) was dissolved in methanol (6.0 mL), and 5-(2-azidoethoxy)-2,3-dihydro-1H-indene-1-one (320 mg, 1.47 mmol), sodium cyanoborohydride (111 mg, 1.77 mmol), and acetic acid (400 μL) were added sequentially. The mixture was heated under reflux for 18 hours. After cooling the reaction solution to room temperature, excess hydride was quenched by adding 1 M hydrochloric acid. Then, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give a pale yellow oily 5-(2-azidoethoxy)-N-(3-phenylpropyl)-2,3-dihydro-1H-indene-1-amine (141 mg, 419 μmol, 29%).

[0147] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.76-1.88 (complex, 3H, ArCH₂C) H 2), 2.31-2.43(m, 1H, ArCH2C) H 2), 2.6-2.8 (complex, 4H), 2.91-3.0 (complex, 2H), 3.56 (t, J = 5Hz, 2H, N3CH2), 4.12 (t, J = 5Hz, 2H, OCH2), 4.15 (t, J = 6Hz, 1H, ArCHN), 6.74 (dd, J = 8.2 and 2Hz, 2H, ArH), 7.15-7.22 (complex, 4H, ArH), 7.26 (t, J = 8.4Hz, 2H, ArH).

[0148] 5-(2-azidoethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-inden-1-amine

[0149] 5-(2-Azideoxyethoxy)-N-(3-phenylpropyl)-2,3-dihydro-1H-indene-1-amine (79.0 mg, 235 μmol) was dissolved in methanol (1.0 mL), and paraformaldehyde (54.7 mg) and sodium cyanoborohydride (20.8 mg, 331 μmol) were added sequentially. The reaction was carried out at room temperature for 18 hours. Excess hydride was quenched by adding 1 M hydrochloric acid to the reaction solution. Then, the reaction solution was made weakly alkaline by adding aqueous sodium hydroxide solution, and the solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give a pale yellow oily 5-(2-azidoethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-indene-1-amine (51 mg, 146 μmol, 62%).

[0150] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.76-1.86 (complex, 2H, ArCH₂C) H 2), 1.98-2.05 (complex, 2H, ArCH2C) H 2), 2.16(s,3H,NCH3), 2.35-2.48(complex,2H,NCH2), 2.545-2.91(complex,4H,ArCH2), 3.57(t,J=5Hz,2H,N3CH2), 4.13(t,J=5Hz,2H,OCH2), 4.35(t,J=7Hz,1H,ArCHN), 6.74-6.78(br,2H,ArH), 7.13-7.19(complex,3H,ArH), 7.21-7.28(complex,3H,ArH).

[0151] 5-(2-aminoethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-inden-1-amine

[0152] 5-(2-Azide-ethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-indene-1-amine (27.4 mg, 78 μmol) was dissolved in methanol (1.0 mL), and Pd / C (10%, 2.8 mg) was added. The reaction mixture was reacted at room temperature for 7 hours and 30 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily 5-(2-aminoethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-indene-1-amine (25.3 mg).

[0153] Compound 2

[0154] 5-(2-aminoethoxy)-N-methyl-N-(3-phenylpropyl)-2,3-dihydro-1H-inden-1-amine (25.3 mg, 78 μmol) was dissolved in acetonitrile (500 μL), followed by the addition of NBD-F (14.5 mg, 79 μmol) dissolved in acetonitrile (500 μL). The reaction was carried out at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give compound 2 (24.3 mg, 50 μmol, 64%).

[0155] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.77–1.86 (complex, 2H, ArCH₂C) H 2), 1.98-2.07 (complex, 2H, ArCH2C) H 2), 2.16(s,3H,NCH3), 2.43(br t,J=7Hz,2H,NCH2), 2.54-2.92(complex,4H,ArCH2), 3.89(br,2H,HNCH2), 4.30(t,J=5Hz,2H,OCH2), 4.36(t,1H,J=7Hz,ArCHN), 6.28(d,J=8.7Hz,1H,ArH), 6.75-6.78(br,2H,ArH), 7.13-7.18(complex,4H,ArH), 7.23-7.28(complex,1H,ArH), 8.51(d,J=8.7Hz,1H,ArH).

[0156] [Synthesis of Compound 3]

[0157] <Example 3>

[0158] Compound 3 was synthesized according to the procedures described below.

[0159] [Chemical Formula 13]

[0160]

[0161] Compound 3

[0162] 5-(2-aminoethoxy)-N-(3-(4-(dimethylamino)phenyl)propyl)-N-methyl-2,3-dihydro-1H-inden-1-amine (25.0 mg, 68 μmol) was dissolved in acetonitrile (500 μL). Then, ABD-F (15.5 mg, 71 μmol) dissolved in acetonitrile (500 μL) was added, and the reaction was carried out at room temperature for 4 hours and 10 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give compound 3 (32.6 mg, 58 μmol, 85%), a reddish-brown oil.

[0163] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.75-1.84 (complex, 2H, ArCH₂C) H 2), 2.0-2.08 (complex, 2H, ArCH2C) H 2), 2.16(s, 3H, NCH3), 2.41-2.6(complex, 3H, NCH2 and ArCH2), 2.71-2.92(complex, 3H, ArCH2), 2.89(s, 6H, N(CH3)2), 3.81(t, J = 5Hz, 2H, HNC) H 2),4.27(t,J=5Hz,2H,OCH2),4.34-4.41(br,1H,ArCHN),6.01(br t,J=8.2Hz,1H,ArH),6.20(d,J=8Hz,1H,ArH),6.67(d,J=8.7Hz,2H,ArH),6.76(s, 1H, ArH), 6.75-6.79 (1H, ArH), 7.04 (d, J = 8.7Hz, 2H, ArH), 7.95 (d, J = 8Hz, 1H, ArH).

[0164] [Synthesis of Compound 4]

[0165] <Example 4>

[0166] Compound 4 was synthesized according to the procedures described below.

[0167] [Chemical Formula 14]

[0168]

[0169] 5-[2-(2-bromoethoxy)ethoxy]-2,3-dihydro-1H-inden-1-one

[0170] 5-Hydroxy-1-indanone (435 mg, 2.94 mmol) was dissolved in ethyl acetate (10 mL), followed by the addition of potassium carbonate (1.47 g, 10.6 mmol), bis(2-bromoethyl) ether (1.82 mL, 14.7 mmol), and benzyltriethylammonium chloride (76 mg, 334 μmol) suspended in ethyl acetate (10 mL). The mixture was heated under reflux for 24 hours. The reaction solution was cooled to room temperature and then injected into ice water. It was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (silica gel, hexane / ethyl acetate = 1 / 1) to give 5-[2-(2-bromoethoxy)ethoxy]-2,3-dihydro-1H-indanone (654 mg, 2.19 mmol, 74%) as a white solid.

[0171] 1 H NMR(400MHz, CDCl3)δ:2.66(m,2H,COCH2),3.07(br t,J=6Hz,2H,ArCH2),3.49(t,J=6.2Hz,2H,BrCH2),3.88(t,J=6.4Hz,2H,OCH2),3.90(t,J=5Hz,2H,OCH2),4.21(t,J=5Hz,2H,ArOCH2),6.89-6.93(br m,2H,ArH),7.68(d,J=9.2Hz,1H,ArH).

[0172] 5-[2-(2-azidoethoxy)ethoxy]-2,3-dihydro-1H-inden-1-on

[0173] 5-[2-(2-bromoethoxy)ethoxy]-2,3-dihydro-1H-indone (403 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (5.0 mL), and sodium azide (105 mg, 1.61 mmol) was added. The mixture was heated under reflux for 1 hour and 30 minutes. The reaction solution was cooled to room temperature and then injected into water. The solution was extracted with diethyl ether, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure to give a reddish-brown solid of 5-[2-(2-azidoethoxy)ethoxy]-2,3-dihydro-1H-indone (347 mg, 1.33 mmol, 99%).

[0174] 1 H NMR(400MHz, CDCl3)δ:2.64-2.68(m,2H,COCH2),3.07(br t,J=6Hz,2H,ArCH2),3.41(t,J=5Hz,2H,N3CH2),3.75(t,J=5Hz,2H,OCH2),3.89(t,J=4.6Hz,2H,OCH2),4.21(t,J=4.6Hz,2H,ArOCH2),6.89-6.93(br m,2H,ArH),7.67(d,J=9.2Hz,1H,ArH).

[0175] [Chemical Formula 15]

[0176]

[0177] 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-inden-1-amine

[0178] 4-(3-aminopropyl)-N,N-dimethylaniline (120 mg, 673 μmol) was dissolved in methanol (3.0 mL), and 5-[2-(2-azidoethoxy)ethoxy]-2,3-dihydro-1H-inden-1-one (101 mg, 386 μmol), sodium cyanoborohydride (49.2 mg, 783 μmol), and acetic acid (100 μL) were added sequentially. The mixture was heated under reflux for 38 hours. After cooling the reaction solution to room temperature, excess hydride was quenched by adding 1 M hydrochloric acid. Then, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give a pale yellow oily 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (105 mg, 248 μmol, 64%).

[0179] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.75-1.85 (complex, 3H, ArCH₂C) H 2), 2.31-2.4(m, 1H, ArCH2C) H2), 2.57(dt, J = 3 and 8 Hz, 2H, ArCH2), 2.71(t, J = 7 Hz, 2H, ArCH2), 2.7-2.8(m, 1H), 2.90(s, 6H, N(CH3)2), 2.89-3.0(m, 1H), 3.40(t, J = 5 Hz, 2H, N3CH2), 3.74(t, J = 5 Hz, 2H, OCH2), 3.84(t, J = 5 Hz, 2H, OCH2), 4.12(t, J = 5 Hz, 2H, OCH2), 4.09-4.18(complex, 3H, ArOCH2 and ArC H NH), 6.68 (d, J = 8.7Hz, 2H, ArH), 6.74 (dd, J = 2 and 8.2Hz, 1H, ArH), 6.77 (s, 1H, ArH), 7.05 (d, J = 8.7Hz, 2H, ArH), 7.18 (d, J = 8.2Hz, 1H, ArH).

[0180] 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine

[0181] 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (13 mg, 31 μmol) was dissolved in methanol (300 μL), and paraformaldehyde (10.7 mg) and sodium cyanoborohydride (9.7 mg, 154 μmol) were added sequentially. The reaction mixture was allowed to react for 17 hours at room temperature. Excess hydride was quenched by adding 1 M hydrochloric acid to the reaction solution. Then, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to obtain a pale yellow oily 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (9.8 mg, 22 μmol, 71%).

[0182] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.7-1.82 (complex, 2H, ArCH₂C) H 2), 1.96-2.05(m,2H,ArCH2C) H2),2.14(s,3H,NCH3),2.37-2.62(composite,4H),2.7-2.8(m,1H),2.89(br,8H),3.4(br,2H,N3CH2),3.74(br,2H,OCH2),3.85(br,2H, OCH2),4.12(br,2H,ArOCH2),4.34(m,1H,ArCHN),6.67(br,2H,ArH),6.75(br,2H,ArH),6.75-6.78(1H,ArH),7.05(br,2H,ArH).

[0183] 5-[2-(2-aminoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine

[0184] 5-[2-(2-azidoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (9.8 mg, 22 μmol) was dissolved in methanol (1 mL), and Pd / C (10%, 3.7 mg) was added. The reaction mixture was reacted at room temperature for 6 hours and 20 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily 5-[2-(2-aminoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-indene-1-amine (8.8 mg).

[0185] [Chemical Formula 16]

[0186]

[0187] Compound 4

[0188] 5-[2-(2-aminoethoxy)ethoxy]-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-2,3-dihydro-1H-inden-1-amine (8.8 mg, 21 μmol) was dissolved in acetonitrile (500 μL). Then, NBD-F (4.2 mg, 23 μmol) dissolved in acetonitrile (500 μL) was added, and the reaction was carried out at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (silica gel, acetone) to give compound 4 (4.3 mg, 7.5 μmol, 36%), a reddish-brown oil.

[0189] 1 H NMR(400MHz,CDCl3):δ:1.74-1.85(br,2H,ArCH2C H 2), 1.98-2.08(m,2H,ArCH2C) H 2), 2.16(s, 3H, NCH3), 2.41-2.6(complex, 4H, NCH2 and ArCH2), 2.7-2.86(complex, 2H, ArCH2), 2.89(s, 6H, N(CH3)2), 3.62-3.81(m, 2H, HNC) H 2),3.88-3.95(composite,4H),4.13(t,J=5Hz,2H,OCH2),4.39(br,1H,ArCHN),6.18(d,J=8.5Hz,1H,ArH),6.61(br,1H,ArH),6 .67(d,J=8.4Hz,2H,ArH),6.72(s,1H,ArH),6.71-6.76(1H,ArH),7.04(d,J=8.4Hz,2H,ArH),8.44(d,J=8.5Hz,1H,ArH).

[0190] [Synthesis of Compound 5]

[0191] <Example 5>

[0192] Compound 5 was synthesized according to the procedures described below.

[0193] [Chemical Formula 17]

[0194]

[0195] 5-(2-azidoethoxy)-N-[4-(dimethylamino)benzyl]-2,3-dihydro-1H-inden-1-amine

[0196] 4-(dimethylamino)benzylamine dihydrochloride (155 mg, 695 μmol) was dissolved in methanol (3.0 mL), and triethylamine (216 μL), 5-(2-azidoethoxy)-2,3-dihydro-1H-inden-1-one (100 mg, 460 μmol), sodium cyanoborohydride (48.5 mg, 772 μmol), and acetic acid (100 μL) were added sequentially. The mixture was heated under reflux for 42 hours. After cooling the reaction solution to room temperature, excess hydride was quenched by adding 1 M hydrochloric acid. Then, an aqueous solution of sodium hydroxide was added to the reaction solution to make it weakly alkaline, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give a pale yellow oily 5-(2-azidoethoxy)-N-[4-(dimethylamino)benzyl]-2,3-dihydro-1H-indene-1-amine (130 mg, 370 μmol, 80%).

[0197] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.88–2.0 and 2.35–2.45 (complex, each ¹H, ArCH₂C) H 2), 2.78 (dt, J = 7.8 and 15.8Hz, 1H, ArCH2), 2.91 (s, 6H, N (CH3)2), 3.0 (ddd, J = 5, 8.5, and 15.8Hz, 1H, ArCH2), 3.56 (t, J = 5Hz, 2H, N3CH2), 3.77 and 3.81 (d ,J=12.8Hz,2H,ArCH2N),4.11(t,J=5Hz,2H,OCH2),4.26(t,J=6.4Hz,1H,ArCHN),6.70(d,J=8.7Hz,2H,ArH),6.76(d,J=8.3Hz,1H,ArH),6.78(br s,1H,ArH),7.22-7.25(2H,ArH),7.27(d,J=8.3Hz,1H,ArH).

[0198] 5-(2-azidoethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-inden-1-amine

[0199] 5-(2-azidoethoxy)-N-[4-(dimethylamino)benzyl]-2,3-dihydro-1H-indene-1-amine (57.5 mg, 164 μmol) was dissolved in methanol (0.8 mL), and paraformaldehyde (27.1 mg) and sodium cyanoborohydride (16.0 mg, 255 μmol) were added sequentially. The reaction was carried out at room temperature for 7 hours. Excess hydrides were quenched by adding 1 M hydrochloric acid to the reaction solution. Then, the reaction solution was made weakly alkaline by adding an aqueous sodium hydroxide solution, and the solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give a pale yellow oily 5-(2-azidoethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-indene-1-amine (58 mg, 159 μmol, 97%).

[0200] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.6–1.8 and 2.0–2.11 (complex, each ¹H, ArCH₂C) H 2), 2.12(s, 3H, NCH3), 2.71-2.83 and 2.86-2.95 (complex, each 1H, ArCH2), 2.91(s, 6H, N(CH3)2), 3.32(d, J = 12.8 Hz, 1H, ArCH2), 3.50(d, J = 12.8 Hz, 1H, ArCH2), 3.57(t, J = 5 Hz, 2H, N3CH2), 4.1 3(t,J=5Hz,2H,OCH2),4.40(t,J=7.1Hz,1H,ArCHN),6.70(d,J=8.7Hz,2H,ArH),6.76(s,1 H, ArH), 6.78 (d, J = 8.2Hz, 1H, ArH), 7.21 (d, J = 8.7Hz, 2H, ArH), 7.33 (d, J = 8.2Hz, 1H, ArH).

[0201] 5-(2-aminoethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-inden-1-amine

[0202] 5-(2-Azide-ethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-indene-1-amine (28.7 mg, 79 μmol) was dissolved in methanol (1.5 mL), and Pd / C (10%, 6.5 mg) was added. The reaction mixture was reacted at room temperature for 6 hours and 50 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound, 5-(2-aminoethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-indene-1-amine (23.5 mg). This compound was used directly in the next reaction without purification.

[0203] [Chemical Formula 18]

[0204]

[0205] Compound 5

[0206] 5-(2-aminoethoxy)-N-[4-(dimethylamino)benzyl]-N-methyl-2,3-dihydro-1H-inden-1-amine (23.0 mg, 68 μmol) was dissolved in acetonitrile (500 μL). Then, NBD-F (14.4 mg, 79 μmol) dissolved in acetonitrile (500 μL) was added, and the reaction was carried out at room temperature for 1 hour and 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give compound 5 (16.1 mg, 32 μmol, 47%) as a reddish-brown solid.

[0207] 1 ¹H NMR (400MHz, CDCl₃) δ: 2.0-2.2 (complex, 2H, ArCH₂C) H2),2.12(s,3H,NCH3),2.71-2.93(composite,2H,ArCH2),2.91(s,6H,N(CH3)2),3.31(d,J=12.8Hz,1H,ArCH2N),3.49(d,J=12.8Hz,1H,ArCH2N),3.89(br,2H,HNC H 2),4.30(t,J=5Hz,2H,OCH2),4.40(br,1H,ArCHN),6.28(d,J=7.8Hz,1H,ArH),6.69(d,J=7.8Hz,2H,ArH),6. 76(s,1H,ArH),6.74-6.81(1H,ArH),7.20(d,J=7.8Hz,2H,ArH),7.35(d,J=8.2Hz,1H),8.51(d,J=7.8Hz,1H).

[0208] [Synthesis of Compound 6]

[0209] <Example 6>

[0210] Compound 6 was synthesized according to the procedures described below.

[0211] [Chemical Formula 19]

[0212]

[0213] 4-(2-bromoethoxy)benzaldehyde

[0214] 4-Hydroxybenzaldehyde (403 mg, 3.30 mmol) was dissolved in acetonitrile (25 mL), followed by the addition of 1,2-dibromoethane (2.8 mL, 33.0 mmol) and potassium carbonate (825 mg, 5.97 mmol) sequentially. The mixture was heated under reflux for 13 hours. The reaction solution was cooled to room temperature and then injected into ice water. It was extracted with diethyl ether, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue after concentration under reduced pressure was purified by column chromatography (silica gel, hexane / ethyl acetate = 3 / 1) to give 4-(2-bromoethoxy)benzaldehyde (559 mg, 2.44 mmol, 74%) as a pale pink solid.

[0215] 1 H NMR (400MHz, CDCl3) δ: 3.66 (t, J = 6.2Hz, 2H, BrCH2), 4.37 (t, J = 6.2Hz, 2H, OCH2), 7.01 (d, J = 8.7Hz, 2H, ArH), 7.84 (d, J = 8.7Hz, 2H, ArH), 9.89 (s, 1H, ArCHO).

[0216] 4-(2-azidoethoxy)benzaldehyde

[0217] 4-(2-bromoethoxy)benzaldehyde (402 mg, 1.75 mmol) was dissolved in N,N-dimethylformamide (5.0 mL), and sodium azide (157 mg, 2.42 mmol) was added. The mixture was heated under reflux for 20 minutes. The reaction solution was cooled to room temperature and then injected into water. The solution was extracted with diethyl ether, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure to give a pale yellow oily 4-(2-azidoethoxy)benzaldehyde (325 mg, 1.70 mmol, 97%).

[0218] 1 H NMR (400MHz, CDCl3) δ: 3.64 (t, J = 4.9Hz, 2H, N3CH2), 4.22 (t, J = 4.9Hz, 2H, OCH2), 7.02 (d, J = 8.9Hz, 2H, ArH), 7.85 (d, J = 8.9Hz, 1H, ArH), 9.89 (s, 1H, CHO).

[0219] [Chemical Formula 20]

[0220]

[0221] 4-(3-{[4-(2-azidoethoxy)benzyl]amino}propyl)-N,N-dimethylaniline

[0222] 4-(3-aminopropyl)-N,N-dimethylaniline (143 mg, 802 μmol) was dissolved in methanol (4 mL), and 4-(2-azidoethoxy)benzaldehyde (100 mg, 523 μmol), sodium cyanoborohydride (63.3 mg, 1.01 mmol), and acetic acid (200 μL) were added sequentially. The mixture was heated under reflux for 375 hours. After cooling the reaction solution to room temperature, excess hydride was quenched by adding 1 M hydrochloric acid. Next, an aqueous sodium hydroxide solution was added to the reaction solution to make it weakly alkaline, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give a pale yellow oily compound 4-(3-{[4-(2-azidoethoxy)benzyl]amino}propyl)-N,N-dimethylaniline (105 mg, 297 μmol, 57%). This compound was used directly in the next reaction without purification.

[0223] 4-(3-{[4-(2-azidoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline

[0224] 4-(3-{[4-(2-azidoethoxy)benzyl]amino}propyl)-N,N-dimethylaniline (16.2 mg, 46 μmol) was dissolved in methanol (500 μL), and paraformaldehyde (10 mg) and sodium cyanoborohydride (4.7 mg, 75 μmol) were added sequentially. The reaction was carried out at room temperature for 16 hours. Excess hydride was quenched by adding 1 M hydrochloric acid to the reaction solution. Then, sodium hydroxide aqueous solution was added to the reaction solution to make it weakly alkaline, and the solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give a pale yellow oily 4-(3-{[4-(2-azidoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline (7.8 mg, 21 μmol, 46%).

[0225] 1 H NMR (400MHz, CDCl3) δ: 1.79 (quin, J = 7.5Hz, 2H, CH2C H 2CH2),2.16(s,3H,NCH3),2.39(t,J=7.5Hz,2H,NC H 2CH2Ar),2.53(t,J=7.5Hz,2H,NCH2C H 2Ar),2.90(s,6H,N(CH3)2),3.42(s,2H,ArCH2N),3.58(t,J=5.0Hz,2H,N3CH2),4.14(t,J=5Hz,2H,ArOCH2), 6.68(d,J=8.7Hz,2H,ArH), 6.86(d,J=8.7Hz,2H,ArH), 7.05(d,J=8.7Hz,2H,ArH), 7.22(d,J=8.7Hz,2H,ArH).

[0226] 4-(3-{[4-(2-aminoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline

[0227] 4-(3-{[4-(2-Azide-ethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline (7.2 mg, 20 μmol) was dissolved in methanol (1 mL), and Pd / C (10%, 4.7 mg) was added. The reaction mixture was reacted at room temperature for 3 hours and 40 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound of 4-(3-{[4-(2-aminoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline (6.1 mg). This compound was used directly in the next reaction without purification.

[0228] 1 H NMR (400MHz, CDCl3) δ: 1.78 (quin, J = 7.7Hz, 2H, CH2C H 2CH2),2.15(s,3H,NCH3),2.38(t,J=7.7Hz,2H,NC H 2CH2CH2),2.53(t,J=7.7Hz,2H,CH2CH2C H 2Ar),2.90(s,6H,N(CH3)2),3.07(t,J=5Hz,2H,H2NC H 2),3.40(s,2H,ArCH2N),3.97(t,J=5Hz,2H,ArOCH2),6.68(d,J=8.7Hz,2H,ArH), 6.85(d,J=8.7Hz,2H,ArH),7.05(d,J=8.7Hz,2H,ArH),7.20(d,J=8.7Hz,2H,ArH).

[0229] [Chemical Formula 21]

[0230]

[0231] Compound 6

[0232] 4-(3-{[4-(2-aminoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline (5.9 mg, 17 μmol) was dissolved in acetonitrile (300 μL), followed by the addition of NBD-F (3.5 mg, 19 μmol) dissolved in acetonitrile (300 μL), and the reaction was carried out at room temperature for 13 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give compound 6 (6.6 mg, 13 μmol, 76%) as a reddish-brown solid.

[0233] 1 H NMR (400MHz, CDCl3) δ: 1.78 (quin, J = 7.6Hz, 2H, CH2C H 2CH2),2.15(s,3H,NCH3),2.39(t,J=7.6Hz,2H,NC H 2CH2CH2),2.53(t,J=7.6Hz,2H,CH2CH2C H 2Ar),2.89(s,6H,N(CH3)2),3.41(s,2H,ArCH2N),3.86-3.92(m,2H,HNC H 2CH2O), 4.31 (t, J = 5Hz, 2H, ArOCH2), 6.28 (d, J = 8.7Hz, 1H, ArH), 6.68 (d, J = 8.7Hz, 2H, A rH), 6.87 (d, J=8.7Hz, 2H, ArH), 7.04 (d, J=8.7Hz, 2H, ArH), 8.51 (d, J=8.7Hz, 2H, ArH).

[0234] [Synthesis of Compound 7]

[0235] <Example 7>

[0236] Compound 7 was synthesized according to the procedures described below.

[0237] [Chemical Formula 22]

[0238]

[0239] Compound 7

[0240] 5-(2-aminoethoxy)-N-(3-(4-(dimethylamino)phenyl)propyl)-N-methyl-2,3-dihydro-1H-inden-1-amine (16.2 mg, 44 μmol) was dissolved in acetonitrile (500 μL), followed by the addition of DBD-F (11.7 mg, 48 μmol) dissolved in acetonitrile (500 μL). The reaction was carried out at room temperature for 15 hours and 20 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to give compound 7 (20.7 mg, 35 μmol, 80%), which was a reddish-brown oil.

[0241] 1 H NMR(400MHz,CDCl3)δ:1.72-1.84(br m,2H,ArCH2C H 2), 1.98-2.07 (complex, 2H, ArCH2C) H 2), 2.16(s, 3H, NCH3), 2.36-2.93(complex, 6H, NCH2 and ArCH2), 2.86(s, 6H, N(CH3)2), 2.89(s, 6H, N(CH3)2), 3.80(m, 2H, HNC) H 2),4.27(t,J=5Hz,2H,OCH2),4.3-4.4(br,1H,ArCHN),5.99(br,1H,ArH),6.23(d,J=7.8Hz,1H,ArH),6. 68(d,J=8.3Hz,2H,ArH),6.73-6.8(composite,2H,ArH),7.05(d,J=8.7Hz,2H,ArH),7.91(d,J=7.8Hz,1H,ArH).

[0242] [Synthesis of Compound 8]

[0243] <Example 8>

[0244] Compound 8 was synthesized according to the procedures described below.

[0245] [Chemical Formula 23]

[0246]

[0247] 5-(2-aminoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-inden-1-amine

[0248] 5-(2-Azide-ethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (33.4 mg, 88 μmol) was dissolved in methanol (1.5 mL), and Pd / C (10%, 13.4 mg) was added. The reaction mixture was reacted at room temperature for 6 hours and 20 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound, 5-(2-aminoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-2,3-dihydro-1H-indene-1-amine (30.3 mg). This compound was used directly in the next reaction without purification.

[0249] 1 H NMR(400MHz,CDCl3)δ:1.76-1.88(1H,ArCH2C H 2),1.80(quin,J=7.3Hz,2H,ArCH2C H 2), 2.31-2.41 (complex, 1H, ArCH2C) H 2),2.51-2.62(m,2H),2.72(t,J=7.1Hz,2H),2.72-2.81(m,1H),2.90(s,6H,N(CH3)2),2.9-2.99(br m,1H),3.05(t,J=5.0Hz,2H,H2NCH2),3.96(t,J=5.0Hz,2H,OCH2),4.12-4.17(m,1H,ArCHN),6.68(d,J=8.2 Hz,2H,ArH),6.73(d,J=8Hz,1H,ArH),6.77(s,1H,ArH),7.06(d,J=8.2Hz,2H,ArH),7.19(d,J=8Hz,1H,ArH).

[0250] Compound 8

[0251] 5-(2-aminoethoxy)-N-(3-(4-(dimethylamino)phenyl)propyl)-2,3-dihydro-1H-inden-1-amine (28.2 mg, 80 μmol) was dissolved in acetonitrile (500 μL), followed by the addition of NBD-F (14.6 mg, 80 μmol) dissolved in acetonitrile (500 μL), and the reaction was carried out at room temperature for 30 min. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by thin-layer chromatography (silica gel, acetone) to give compound 8 (24.3 mg, 47 μmol, 59%) as a reddish-brown oil.

[0252] 1 H NMR(400MHz,CDCl3):δ:1.7-1.91(br,1H,ArCH2C H 2),1.82(quin,J=7.4Hz,2H,ArCH2C H 2), 2.32-2.41(m, 1H, ArCH2C H 2), 2.52-2.6 (complex, 2H, NCH2 and ArCH2), 2.72 (t, J = 7.4 Hz, 2H, ArCH2), 2.74-2.81 (complex, 1H, ArCH2C) H 2),2.88-3.0(1H),2.89(s,6H,N(CH3)2),3.86-3.92(t,J=5Hz,2H,HNC H 2CH2O),4.19(t,J=6.4Hz,1H,ArCHN),4.28(t,J=5Hz,2H,OCH2),6.28(d,J=8.7Hz,1H,ArH),6.67(d,J=8.7Hz, 2H, ArH), 6.74 (d, J = 8.7Hz, 2H, ArH), 6.77 (s, 1H, ArH), 7.04 (d, J = 8.7Hz, 2H, ArH), 8.50 (d, J = 8.7Hz, 1H, ArH).

[0253] [Synthesis of Compound 9]

[0254] <Example 9>

[0255] Compound 9 was synthesized according to the procedures described below.

[0256] [Chemical Formula 24]

[0257]

[0258] (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}amino)ethyl]phenol)

[0259] Tyramine (698 mg, 5.09 mmol) and 4-(dimethylamino)cinnamaldehyde (910 mg, 5.19 mmol) were dissolved in methanol (20 mL), and then added... Molecular sieve (969 mg) was added, and the mixture was stirred at room temperature for 22 hours. Next, the reaction vessel was cooled to 0°C, and sodium borohydride (392 mg, 10.4 mmol) was added. Methanol (26 mL) was then added to the reaction solution, and the mixture was stirred at room temperature for 42 hours. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}amino)ethyl]phenol (1.44 g) as a yellow solid. This compound was used directly in the next reaction without purification.

[0260] (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}(methyl)amino)ethyl]phenol)

[0261] (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}amino)ethyl]phenol (1.43 g) was dissolved in methanol (35 mL), and paraformaldehyde (930 mg) and sodium cyanoborohydride (389 mg, 6.19 mmol) were added sequentially. The reaction was carried out at room temperature for 17 hours. Excess hydride was quenched by adding 1 M hydrochloric acid to the reaction solution. Then, sodium hydroxide aqueous solution was added to the reaction solution to make it weakly alkaline. The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone) to give a pale yellow oil (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}(methyl)amino)ethyl]phenol (796 mg, 2.56 mmol, 50% yield in two steps).

[0262] 1H NMR (400MHz, CDCl3) δ: 2.33 (s, 3H, NCH3), 2.62 (dd, J = 6.2 and 9.6Hz, 2H, NCH2C H 2Ar), 2.74(dd, J = 6.2 and 9.6 Hz, 2H, NC H 2CH2Ar),2.94(s,6H,N(CH3)2),3.19(d,J=6.9Hz,2H,NC H 2CH=CH), 6.05(dt, J=6.9 and 16Hz, 1H, ArCH=C H CH2), 6.41 (d, J = 16 Hz, 1H, ArC H =CH), 6.67 (d, J = 8.7Hz, 2H, ArH), 6.71 (d, J = 8.3Hz, 2H, ArH), 7.04 (d, J = 8.3Hz, 2H, ArH), 7.26 (2H, ArH).

[0263] (E)-4-(3-{[4-(2-bromoethoxy)phenethyl](methyl)amino}prop-1-en-1-yl)-N,N-dimethylaniline)

[0264] (E)-4-[2-({3-[4-(dimethylamino)phenyl]allyl}(methyl)amino)ethyl]phenol (390 mg, 1.26 mmol) was dissolved in ethyl acetate (5.0 mL), followed by the addition of potassium carbonate (653 mg, 4.73 mmol), 1,2-dibromoethane (698 μL, 8.10 mmol), and benzyltriethylammonium chloride (BTEAC) (33.1 mg, 145 μmol) suspended in ethyl acetate (12 mL). The mixture was heated under reflux for 17 hours. After cooling to room temperature, the reaction solution was injected into ice water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue after concentration of the extract under reduced pressure was purified by column chromatography (silica gel, acetone / ethyl acetate / triethylamine = 100 / 100 / 3) to give (E)-4-(3-{[4-(2-bromoethoxy)phenethyl](methyl)amino}prop-1-en-1-yl)-N,N-dimethylaniline (361 mg, 865 μmol, 69%) as a yellow solid.

[0265] 1 H NMR(400MHz, CDCl3)δ:2.33(s,3H,NCH3),2.58-2.64(m,2H,NC H2CH2Ar), 2.71-2.78 (m, 2H, NCH2C) H 2Ar),2.94(s,6H,N(CH3)2),3.19(d,J=6.9Hz,2H,NC H 2CH=CH), 3.61 (t, J=6.2Hz, 2H, BrCH2), 4.25 (t, J=6.2Hz, 2H, ArOCH2), 6.05 (dt, J=6.9 and 16Hz, 1H, ArCH=C H CH2), 6.41 (d, J = 16 Hz, 1H, ArC H =CH), 6.67 (d, J = 8.7Hz, 2H, ArH), 6.73 (d, J = 8.2Hz, 2H, ArH), 7.05 (d, J = 8.2Hz, 2H, ArH), 7.25 (2H, ArH).

[0266] (E)-4-(3-{[4-(2-azidoethoxy)phenethyl](methyl)amino}prop-1-en-1-yl)-N,N-dimethylaniline)

[0267] (E)-4-(3-{[4-(2-bromoethoxy)phenethyl](methyl)amino}prop-1-en-1-yl)-N,N-dimethylaniline (290 mg, 695 μmol) was dissolved in N,N-dimethylformamide (3.0 mL), and sodium azide (71.7 mg, 1.10 mmol) was added. The reaction mixture was reacted at 90 °C for 1 hour. After cooling to room temperature, the reaction mixture was injected into water, extracted with diethyl ether, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue after concentration under reduced pressure was purified by column chromatography (silica gel, hexane / ethyl acetate = 1 / 1) to give (E)-4-(3-{[4-(2-azidoethoxy)phenethyl](methyl)amino}prop-1-en-1-yl)-N,N-dimethylaniline (206 mg, 543 μmol, 78%) as a yellow solid.

[0268] 1 H NMR(400MHz, CDCl3)δ:2.33(s,3H,NCH3),2.56-2.66(br,2H,NC H 2CH2Ar), 2.7-2.8(br,2H,NC) H 2CH2Ar),2.94(s,6H,N(CH3)2),3.19(d,J=6.9Hz,2H,NC H2CH=CH), 3.57 (t, J=4.8Hz, 2H, N3CH2), 4.11 (t, J=4.8Hz, 2H, ArOCH2), 6.05 (dt, J=6.9 and 15.6Hz, 1H, ArCH=C H CH2), 6.41 (d, J = 15.6 Hz, 1H, ArC H =CH), 6.67 (d, J = 8.5Hz, 2H, ArH), 6.83 (d, J = 8.7Hz, 2H, ArH), 7.11 (d, J = 8.5Hz, 2H, ArH), 7.25 (2H, ArH).

[0269] 4-(3-{[4-(2-aminoethoxy)phenethyl](methyl)amino}propyl)-N,N-dimethylaniline

[0270] (E)-4-(3-{[4-(2-azideoxyethoxy)phenethyl](methyl)amino}propyl-1-en-1-yl)-N,N-dimethylaniline (20.5 mg, 54 μmol) was dissolved in methanol (1.0 mL), and Pd / C (10%, 5.8 mg) was added. The reaction mixture was reacted at room temperature for 15 hours and 50 minutes under a hydrogen atmosphere at balloon pressure. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound 4-(3-{[4-(2-aminoethoxy)phenethyl](methyl)amino}propyl)-N,N-dimethylaniline (17.8 mg, 93%). This compound was used directly in the next reaction without purification.

[0271] [Chemical Formula 25]

[0272]

[0273] Compound 9

[0274] 4-(3-{[4-(2-aminoethoxy)phenethyl](methyl)amino}propyl)-N,N-dimethylaniline (17.4 mg, 49 μmol) was dissolved in acetonitrile (500 μL). Then, NBD-F (9.9 mg, 55 μmol) dissolved in acetonitrile (500 μL) was added, and the reaction was carried out at room temperature for 1 hour. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, acetone) to give compound 9 (17.3 mg, 33 μmol, 67%) as a reddish-brown oil.

[0275] 1 ¹H NMR (400MHz, CDCl₃) δ: 1.69–1.8 (complex, 2H, CH₂C) H 2CH2), 2.16(s, 3H, NCH3), 2.25-2.44(complex, 4H, ArCH2 and NCH2), 2.50(t, J = 7.8 Hz, 2H, ArCH2), 2.67-2.74(m, 2H), 2.89(s, 6H, N(CH3)2), 3.85-3.91(br m, 2H, HNC) H 2CH2O),4.28(t,J=5Hz,2H,ArOCH2),6.27(d,J=8.5Hz,1H,ArH),6.67(d,J=8.7Hz,2H,ArH),6.84(d, J=8.7Hz,2H,ArH),7.03(d,J=8.7Hz,2H,ArH),7.12(d,J=8.7Hz,2H,ArH),8.50(d,J=8.5Hz,2H,ArH).

[0276] [Synthesis of Compound 10]

[0277] <Example 10>

[0278] Compound 10 was synthesized according to the procedures described below.

[0279] The ATTO 565 (NHS ester) was prepared from 5-(2-aminoethoxy)-N-(3-(4-(dimethylamino)phenyl)propyl)-N-methyl-2,3-dihydro-1H-inden-1-amine using the ATTO 565 (NHS ester) Conjugation Kit. 1 mg of ATTO 565 (NHS ester) was mixed with an equimolar amount of 4-(3-{[4-(2-aminoethoxy)benzyl]methylamino}propyl)-N,N-dimethylaniline in 0.1 mL of acetonitrile and reacted for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase PLC (ODS, water / acetonitrile = 1 / 50) to obtain compound 10 as a reddish-brown solid.

[0280] [Synthesis of Compound 11]

[0281] <Example 11>

[0282] Compound 11 was synthesized according to the procedures described below.

[0283] Compound 11 was synthesized using 3-(4-methylphenyl)propan-1-amine by the same method as that used in the synthesis of compound 1.

[0284] 1 H NMR(400MHz,CDCl3):δ:1.75-1.85(br,2H,ArCH2C H 2), 1.96-2.1(m,2H,ArCH2C H 2), 2.16(s,3H,NCH3), 2.30(s,3H,ArCH3), 2.37-2.68(complex,4H,NCH2 andArCH2), 2.71-2.93(complex,2H,ArCH2), 3.86-3.93(br,2H,HNC) H2),4.30(t,J=5Hz,2H,OCH2),4.34-4.42(br,1H,ArCHN),6.29(d,J=8.7Hz,1H,ArH),6.76 (s,1H,ArH),6.76-6.79(m,1H,ArH),7.02-7.09(composite,5H,ArH),8.51(d,J=8.7Hz,1H,ArH).

[0285] [Synthesis of Compound 12]

[0286] <Example 12>

[0287] Compound 12 was synthesized according to the procedures described below.

[0288] Compound 12 was synthesized using 3-(4-methoxyphenyl)propan-1-amine by the same method as that used in the synthesis of compound 1.

[0289] 1 ¹H NMR (400MHz, CDCl₃): δ: 1.7-1.9 (complex, 2H, ArCH₂C) H 2), 2.0-2.1(m,2H,ArCH2C) H 2), 2.21 (s, 3H, NCH3), 2.4-2.67 (complex, 4H, NCH2 and ArCH2), 2.73-2.94 (complex, 2H, ArCH2), 3.77 (s, 3H, OCH3), 3.88-3.93 (br, 2H, HNC) H 2),4.30(t,J=5Hz,2H,OCH2),4.37-4.48(m,1H,ArCHN),6.29(d,J=8.7Hz,1H,ArH),6.76(s,1H,ArH),6.77-6.83(m ,1H,ArH),6.8(d,J=8.5Hz,2H,ArH),7.04-7.1(composite,1H,ArH),7.07(d,J=8.5Hz,2H,ArH),8.51(d,J=8.7Hz,1H,ArH).

[0290] [Fluorescent staining of intracellular vesicles using compound 1 and anti-CD63 antibody]

[0291] <Example 13>

[0292] Intracellular vesicles were fluorescently stained using compound 1 synthesized in Example 1 and anti-CD63 antibody. The procedure is shown below.

[0293] (1) 3 × 10⁻⁶ mouse malignant melanoma cells B16F10 (obtained from Riken Cell Bank) 4 Each sample was placed on a glass petri dish (Songlang) and incubated for 24 hours. The culture medium used was a high-glucose (D6046 (Merck)) medium of Dubec Modified Eagle Medium (DMEM) supplemented with 1× penicillin-streptomycin (26252-94 (Nacalai Tesque)) and 10% fetal bovine serum (Nichirei). Incubation was carried out at 37°C and 5% CO2.

[0294] (2) Add compound 1 to the culture medium to make the final concentration 0.3 μM.

[0295] (3) Five minutes after adding compound 1, the culture medium was removed by aspiration, and 1 mL of 4% paraformaldehyde / phosphate buffer (09154-14 (Nacalai Tesque)) was added. The mixture was fixed at room temperature for 15 minutes.

[0296] (4) After aspirating to remove 4% paraformaldehyde / phosphate buffer, add 1 mL of phosphate buffer.

[0297] (5) Add 1 μL of DAPI (Cellstain DAPI solution (Tongren Chemical Research Institute)), let stand for 15 minutes, and perform fluorescent staining on the nucleus.

[0298] (6) Aspirate to remove phosphate buffer and perform three cleaning operations using phosphate buffer.

[0299] (7) After washing, add 1 mL of the reaction solution prepared by diluting the anti-CD63 antibody (D263-3(MBL)) at 1 / 1000 to 10% BlockingOne(Nacalai Tesque) / phosphate buffer and let it stand at room temperature for 1 hour.

[0300] (8) Aspirate to remove phosphate buffer and perform three cleaning operations using phosphate buffer.

[0301] (9) After washing, add 1 mL of the reaction solution containing anti-mouse IgG H&L (Alexa Fluor 594) (ab150116 (Abcam)) diluted to 1 / 1000 to 10% BlockingOne / phosphate buffer and let stand at room temperature for 1 hour.

[0302] (10) Aspirate to remove phosphate buffer and perform 4 washes using phosphate buffer.

[0303] (11) Measurements were performed using a fluorescence microscope (BZ-9000 (Keyence)). Compound 1 was excited at 470 nm, and the antibody bound to CD63 was excited at 540 nm. DAPI was excited at 360 nm.

[0304] The results are shown in Figure 1 . Figure 1 A is a fluorescence microscope image of the entire cell. Figure 1 B is to be from Figure 1 A magnified view of the area enclosed by box A. Compound 1 emits green fluorescence, while the antibody binding to CD63 emits red fluorescence. Figure 1 Fluorescence was observed from compound 1 and the antibody binding to CD63. Furthermore, according to... Figure 1 A and Figure 1 B, the fluorescence from compound 1 overlaps with the fluorescence from the antibody binding to CD63. CD63 is a marker of cellular vesicles derived from endosomes. Therefore, Figure 1 The results showed that compound 1 could perform fluorescent staining on cellular vesicles.

[0305] [Detection of autophagosomes based on compound 1]

[0306] <Example 14>

[0307] Autophagosomes were detected using an endosome-derived intracellular vesicle formation inhibitor (PIKfyve inhibitor). The steps are as follows.

[0308] (1) 3 × 10 B16F10 cells 4 Place each on a glass petri dish and incubate for 24 hours.

[0309] (2) Add compound 1 to make the final concentration 3 μM.

[0310] (3) Measurements were performed using a fluorescence microscope. Additionally, measurements were taken under excitation at 470 nm.

[0311] (4) Then, add the PIKfyve inhibitor (HY-13228 (Med Chem Express)) to the culture medium to make the final concentration 10 μM and incubate for 1 hour.

[0312] (5) Measurements were performed using a fluorescence microscope.

[0313] (6) Remove the medium containing PIKfyve inhibitor by aspiration, wash twice with fresh medium, and then incubate for 3 hours with fresh medium.

[0314] (7) Measurements were performed using a fluorescence microscope.

[0315] The results are shown in Figure 2 . Figure 2 A is a fluorescence microscopy image of compound 1 before the addition of the PIKfyve inhibitor. Figure 2 B is a phase-contrast microscopy image before the addition of the PIKfyve inhibitor. Figure 2 C is a fluorescence microscopy image of compound 1 after the addition of the PIKfyve inhibitor. Figure 2 D is a phase-difference microscopy image after the addition of the PIKfyve inhibitor. Figure 2 E is a fluorescence microscopy image of the fluorescence emitted by compound 1 after cells were cultured for 3 hours following the removal of PIKfyve. Figure 2 F is a phase-contrast microscopy image taken 3 hours after PIKfyve removal from cell culture. Figure 2 A indicates the presence of cellular vesicles within the cell, which... Figure 2 B indicates the presence of melanosomes within the cell. When adding a PIKfyve inhibitor, such as... Figure 2 C and Figure 2 As shown in Figure D, a decrease in fluorescence intensity and the destruction and foaming of melanosomes were observed. This indicates that compound 1 can also produce fluorescent staining for PIKfyve-dependent cellular vesicles. Then, from... Figure 2 E and Figure 2 In cells F after removal of the PIKfyve inhibitor, the autophagosome membrane surrounding the melanosomes was fluorescently stained by compound 1. The autophagosome membrane originates from endosomes. This demonstrates that compound 1 can fluorescently stain endosome-derived cellular vesicles even with the use of the PIKfyve inhibitor.

[0316] [Fluorescent staining of cellular vesicles in human cells]

[0317] Examples 13 and 14 demonstrate the fluorescent staining of cellular vesicles from mouse-derived cells by compound 1. Therefore, fluorescent staining was also performed on human-derived cells.

[0318] <Example 15>

[0319] Compound 1 was used to fluorescently stain cellular vesicles of HEK293 cells. The steps are shown below.

[0320] (1) HEK293A cells (Thermo Fisher) were placed on glass dishes and cultured for 24 hours. The culture medium used was DMEM low glucose (D6046 (Merck)) supplemented with 10% FCS and 1× penicillin streptomycin.

[0321] (2) Add compound 1 to make the final concentration 0.3 μM.

[0322] (3) Measurements were performed using a fluorescence microscope. Additionally, measurements were taken under excitation at 470 nm.

[0323] <Example 16>

[0324] Fluorescent staining of cellular vesicles of keratinocytes (PSVK1) was performed using compound 1. The steps are shown below.

[0325] (1) PSVK1 cells (obtained from the Cell Bank of the Institute of Pharmaceuticals, Health and Nutrition) were placed on a glass dish and cultured for 24 hours. The culture medium used was the Keratinocyte Growth Medium 2 Kit (C-20111 (Takara Bio)).

[0326] (2) Add compound 1 to make the final concentration 0.3 μM.

[0327] (3) Measurements were performed using a fluorescence microscope. Additionally, measurements were taken under excitation at 470 nm.

[0328] The results are shown in Figure 3 . Figure 3 A is a fluorescence microscope image of Example 15. Figure 3 B is a fluorescence microscope image of Example 16. Figure 3 A and Figure 3 B shows that compound 1 can fluorescently stain cellular vesicles of human HEK293 cells and PSVK1 cells.

[0329] [Specificity of Compound 1 to Cellular Vesicles 1]

[0330] <Example 17>

[0331] Fluorescent staining of cellular vesicles in live cells was performed using compound 1. The steps are shown below.

[0332] (1) 3 × 10 B16F10 cells 4 Place each sample on a collagen-coated glass dish (Songlang) and incubate for 24 hours.

[0333] (2) Add mitochondrial staining reagent (Mito Tracker Red) to the culture medium to make the final concentration 1 μM and perform fluorescent staining on mitochondria.

[0334] (3) After 5 minutes, aspirate to remove the culture medium, add 4% paraformaldehyde / phosphate buffer, and fix at room temperature for 15 minutes.

[0335] (4) Aspirate to remove 4% paraformaldehyde / phosphate buffer and add 1 mL of phosphate buffer.

[0336] (5) Add 1 μL of DAPI, let stand for 15 minutes, and then perform fluorescent staining on the nucleus.

[0337] (6) Aspirate to remove phosphate buffer and perform 5 washes using phosphate buffer.

[0338] (7) After cleaning, add 2 mL of DMEM medium.

[0339] (8) Replace 3 × 10 B16F10 cells 4 Each, trained for 24 hours.

[0340] (9) Add compound 1 to make the final concentration 0.3 μM.

[0341] (10) The determination was performed using a fluorescence microscope. In addition, during the determination, compound 1 was excited at 470 nm, mitochondrial staining reagent was excited at 540 nm, and DAPI was excited at 360 nm.

[0342] The results are shown in Figure 4 . Figure 4 A is a fluorescence microscope image of the fluorescence emitted by compound 1. Figure 4 B is a fluorescence microscope image showing the fluorescence emitted by the mitochondrial staining reagent. Figure 4 C is a fluorescence microscope image showing the fluorescence emitted by DAPI in the stained nucleus. Figure 4 D is a phase-contrast microscope image. (From...) Figure 4 As can be seen from D, although cells are present throughout the entire microscope image, Figure 4 In A, only a subset of the cells' cellular vesicles were stained with fluorescence. For example... Figure 4 B and Figure 4 As indicated by the arrow in C, Figure 4 Cells in group A that are not stained by the fluorescence of compound 1 are dead cells. This demonstrates that compound 1 fluoresces the cellular vesicles of living cells.

[0343] [Specificity of Compound 1 to Cellular Vesicles 2]

[0344] Cellular vesicles were fluorescently stained using Compound 1 and other cellular vesicle staining agents, and the results were compared.

[0345] <Example 18>

[0346] Intracellular vesicles were fluorescently stained using compound 1. The steps are shown below.

[0347] (1) 3 × 10 B16F10 cells 4 Place each on a glass petri dish and incubate for 24 hours.

[0348] (2) Add compound 1 to the culture medium to make the final concentration 3 μM.

[0349] (3) Measurements were performed using a fluorescence microscope. Additionally, measurements were taken under excitation at 470 nm.

[0350] <Comparative Example 1>

[0351] Instead of compound 1, 1 μL / mL of ExoSparkler Mem Dye-Red (340-09671 (Dongjin Chemical Research Institute)) was added to the culture medium. After adding ExoSparkler Mem Dye-Red, the culture medium was removed by aspiration, and fresh culture medium was added for washing. Otherwise, it was the same as in Example 18. In addition, the excitation was measured at 540 nm.

[0352] The results are shown in Figure 5 . Figure 5 A is a graph that overlays fluorescence microscopy images of Example 18 and Comparative Example 1. Figure 5 B is a fluorescence microscope image of Example 18. Figure 5 C is a fluorescence microscope image of Comparative Example 1. Figure 5 The fluorescent staining of cellular vesicles is shown in Example 18 and Comparative Example 1. However, based on Figure 5 A and Figure 5 B, Example 18 only performed fluorescent staining on cellular vesicles, in contrast, as Figure 5 As indicated by arrow A, in Comparative Example 1, although washing was performed after staining, debris other than cellular vesicles was also fluorescently stained. This demonstrates that compound 1 exhibits high specificity for cellular vesicles.

[0353] [Specificity of Compound 1 to Cellular Vesicles 3]

[0354] In the presence of impurities, cellular vesicles were fluorescently stained using Compound 1 and other cellular vesicle staining agents, and the results were compared.

[0355] <Example 19>

[0356] Fluorescent staining of intracellular vesicles was performed using compound 1 in the presence of impurities. The steps are shown below.

[0357] (1) Collect approximately 100 mg of mouse feces and suspend the collected feces in 10 times (1 mL) of phosphate buffer.

[0358] (2) B16F10 cells were dissected and recovered using trypsin-EDTA (Nacalai Tesque), and 1×10 4One cell was suspended in 100 μL of phosphate buffer and mixed with 10 μL of mouse fecal suspension (1).

[0359] (3) Spread the suspension of cells and feces on a glass plate and add 300 μM of compound 1 to make the final concentration 0.3 μM.

[0360] (4) Measurements were performed using a fluorescence microscope. Additionally, measurements were taken under excitation at 470 nm.

[0361] <Comparative Example 2>

[0362] Except for the addition of 1 μL of CellMask reagent (C10045 (Thermo Fisher Scientific)) to the cell and fecal suspension instead of compound 1, the procedure was the same as in Example 19. Additionally, the measurements were performed at 540 nm excitation.

[0363] The results are shown in Figure 6 . Figure 6 A is a fluorescence microscope image of Example 19. Figure 6 B is a phase-contrast microscope image of Example 19. Figure 6 C is a fluorescence microscope image of Comparative Example 2. Figure 6 D is a phase-contrast microscope image of Comparative Example 2. Figure 6 A and Figure 6 As shown in Figure B, even in the presence of impurities, Example 19 only fluoresced cellular vesicles. Furthermore, according to... Figure 6 C and Figure 6 D, Comparative Example 2, shows fluorescent staining of inclusions other than cellular vesicles. This demonstrates that mouse feces contain abundant live intestinal bacteria and food digests, but compound 1 does not react with intestinal bacteria or food digests and can specifically fluorescently stain cellular vesicles.

[0364] Fluorescent staining of exosomes

[0365] <Example 20>

[0366] Perform fluorescent staining of exosomes, which are extracellular vesicles. The steps are as follows.

[0367] (1) Dissolve compound 1 dissolved in DMSO in phosphate buffer to make a final concentration of 0.3 μM.

[0368] (2) Add 10 ng of milk exosomes to the phosphate buffer solution containing compound 1.

[0369] (3) Measurements were performed using the ChemiDoc (Bio-Rad) image analysis device. Furthermore, measurements were taken under excitation at 302 nm.

[0370] <Comparative Example 3>

[0371] The same as in Example 20, except that no milk exosomes are added.

[0372] The results are shown in Figure 7 .like Figure 7 As shown, Example 20, in which milk exosomes were added, emitted fluorescence. On the other hand, Comparative Example 3 did not emit fluorescence. This demonstrates that compound 1 fluoresces by binding to exosomes, which are cellular vesicles.

[0373] [Construction of Standard Curve for Exosome Detection]

[0374] <Example 21>

[0375] Create a standard curve for exosome detection. The steps are shown below.

[0376] (1) Inject a solution of 50 μL fetal bovine serum and 50 μL phosphate buffer into the wells of a 96-well fluorescent microplate (3-3321-03(AS ONE)).

[0377] (2) Add milk exosomes to each well in the range of 0.1 to 1 ng.

[0378] (3) Autofluorescence was measured using a fluorescence microplate reader (Gro Max Multi (Promega)) (excitation 488 nm).

[0379] (4) Add compound 1 to each well to make the final concentration 0.3 μM and measure the fluorescence (excitation 488 nm). At this time, the value obtained by subtracting the fluorescence intensity from the fluorescence intensity obtained is used as the measured value.

[0380] Fluorescence intensity was measured at various exosome concentrations, and a standard curve was constructed based on the results. Figure 8 Although fetal bovine serum also contains exosomes, the fluorescence intensity showed an increase dependent on the amount of externally added milk exosomes. Therefore, according to Figure 8 The standard curve can detect 1 ng of exosomes even in the presence of impurities.

[0381] [Determination of exosomes in food]

[0382] The standard curve obtained in Example 21 was used to determine exosomes in food.

[0383] <Example 22>

[0384] Determine the exosome content in black coffee. The steps are shown below.

[0385] (1) Inject a solution containing 50 μL of black coffee (UCC) and 50 μL of phosphate buffer into the wells of a 96-well microplate for fluorescence.

[0386] (2) Measure autofluorescence using a fluorescence microplate reader (excitation at 488 nm).

[0387] (3) Add compound 1 to the well to make the final concentration 0.3 μM and measure the fluorescence (excitation 488 nm). At this time, the value obtained by subtracting the fluorescence intensity from the fluorescence intensity obtained is used as the measured value.

[0388] <Example 23>

[0389] The process is the same as in Example 22, except that milk-containing coffee (Asahi) is used instead of black coffee.

[0390] <Example 24>

[0391] The process is the same as in Example 22, except that a lactic acid bacteria beverage (Yakult) is used instead of black coffee.

[0392] <Example 25>

[0393] The process is the same as in Example 22, except that 1% skim milk (Fujifilm-Wako) is used instead of black coffee.

[0394] <Example 26>

[0395] The process is the same as in Example 22, except that milk (Meiji) is used instead of black coffee.

[0396] <Example 27>

[0397] The process is the same as in Example 22, except that vegetable juice (Kagome) is used instead of black coffee.

[0398] The results are shown in Figure 9 . Figure 9 This demonstrates the ability to measure exosomes in food. No exosomes were detected in black coffee. Additionally, exosomes are shown to be present in vegetable juice.

[0399] [Identification of exosome uptake into cells using compound 1 and exosome staining reagent]

[0400] <Example 28>

[0401] The uptake of labeled exosomes, fluorescently stained with compound 1 and an exosome staining reagent, into cells was measured. The procedure is as follows.

[0402] (1) Milk exosomes (EXO-AB-01 (Cosmo Bio)) were stained with compound 1 (final concentration 0.3 μM) and ExoSparkler Mem Dye-Red (340-09671 (Dongjin Chemical Research Institute) (1 μL) for 5 minutes, and then purified using the column attached to ExoSparkler Mem Dye-Red to prepare labeled milk exosomes.

[0403] (2) Culture HEK293A cells and add labeled milk exosomes.

[0404] (3) The samples were analyzed by fluorescence microscopy 12 hours after the addition of labeled milk exosomes. In addition, compound 1 was excited at 470 nm and ExoSparkler Mem Dye-Red was excited at 540 nm.

[0405] The results are shown in Figure 10 . Figure 10 A is a fluorescence microscopy image showing the superimposed fluorescence emitted by compound 1 and ExoSparkler Mem Dye-Red. Figure 10 B is a fluorescence microscope image of the fluorescence emitted by compound 1. Figure 10 C is a fluorescence microscopy image observing the fluorescence emitted by ExoSparkler Mem Dye-Red. ExoSparkler Mem Dye-Red is a fluorescent reagent that binds to exosomes. According to... Figure 10 A. The fluorescence of compound 1 overlaps with that of ExoSparkler Mem Dye-Red, thus demonstrating that compound 1 also specifically binds to exosomes. Additionally, it is shown that exosomes labeled with compound 1 are taken up into cells.

[0406] [Inhibition of cellular uptake by labeled melanosomes]

[0407] <Example 29>

[0408] Melanosome uptake into cells was measured in the presence of melanosome uptake inhibitors. The procedure is shown below.

[0409] (1) Culture B16F10 cells in 10 mL of culture medium.

[0410] (2) Add Forskolin (F0855 (Tokyo Chemical Industry)) to the culture medium to make the final concentration 20 μM, and culture for 48 hours to induce melanin synthesis.

[0411] (3) After removing the culture medium by aspiration, replace it with fresh culture medium containing saliva and culture for another 24 hours.

[0412] (4) After recovering the culture medium into a 15mL centrifuge tube, centrifuge at 3000rpm for 5 minutes to remove the mixed cell precipitate and recover the supernatant.

[0413] (5) Transfer the recovered supernatant to another 15mL centrifuge tube and centrifuge at 8000rpm for 10 minutes.

[0414] (6) Resuspend the precipitate in 1 mL of phosphate buffer and transfer it to a 2 mL centrifuge tube.

[0415] (7) Centrifuge at 12000 rpm for 5 minutes, discard the supernatant, and wash the precipitate twice with 1 mL of phosphate buffer.

[0416] (8) The washed precipitate was suspended in 100 μL of phosphate buffer, and compound 1 was added to make the final concentration 3 μM. The precipitate was stained for 5 minutes.

[0417] (9) Centrifuge at 12000 rpm for 5 minutes, discard the supernatant, and wash the precipitate twice with 1 mL of phosphate buffer.

[0418] (10) The washed precipitate was suspended in 100 μL of phosphate buffer to prepare labeled melanocytes.

[0419] (11) 5×10 4 One keratinocyte was placed in each well of a 96-well microplate.

[0420] (12) Labeled melanocytes (10 μL) and a melanosome uptake inhibitor (final concentration 10 μM) were added to the wells. 6-Shogaol (192-16161 (Fujifilm and light)) was used as the melanosome uptake inhibitor.

[0421] (13) After 12 hours, the cells were washed twice with 100 μL of phosphate buffer.

[0422] (14) Labeled melanosomes were measured using a microplate reader (Gro Max Multi (Promega)). The measurement was performed at 488 nm excitation.

[0423] <Comparative Example 4>

[0424] Except for the absorbance at 450 nm measured using an ELISA reader, it was the same as in Example 29.

[0425] The results are shown in Figure 11 . Figure 11 A is a fluorescence microscope image of Example 29 and a phase difference microscope image of Comparative Example 4. Figure 11 B shows the results of Example 29. Figure 11 C shows the results of Comparative Example 4. (From...) Figure 11 A and Figure 11 As shown in B, if no melanosome uptake inhibitor is present, labeled melanosomes are taken up by keratinocytes; however, if a melanosome uptake inhibitor is present, the fluorescence intensity decreases, thus inhibiting the uptake of labeled melanosomes by keratinocytes. On the other hand, in the absorbance measurement in Comparative Example 4, the uptake of melanosomes by keratinocytes could not be evaluated. Therefore, it is shown that by using compound 1, it is possible to evaluate the uptake of melanosomes by keratinocytes using a melanosome uptake inhibitor.

[0426] [Fluorescent staining of cellular vesicles using compounds 2–4 and 6–12]

[0427] <Example 30>

[0428] Except for the use of compound 2 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0429] <Example 31>

[0430] Except for the use of compound 3 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0431] <Example 32>

[0432] Except for the use of compound 4 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0433] <Example 33>

[0434] Except for the use of compound 6 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0435] <Example 34>

[0436] Except for the use of compound 7 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0437] <Example 35>

[0438] Except for the use of compound 8 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0439] <Example 36>

[0440] Except for the use of compound 9 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0441] <Example 37>

[0442] Instead of compound 1, compound 10 was used, and the excitation light was set to 540 nm during the measurement. Otherwise, it was the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0443] <Example 38>

[0444] Except for the use of compound 11 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0445] <Example 39>

[0446] Except for the use of compound 12 instead of compound 1, it is the same as in Example 14 without the addition of the PIKfyve inhibitor.

[0447] The results are shown in Figure 12 . Figure 12 A is a fluorescence microscope image of Example 30. Figure 12 B is a phase-contrast microscope image of Example 30. Figure 12 C is a fluorescence microscope image of Example 31. Figure 12 D is a fluorescence microscope image of Example 32. Figure 12 E is a fluorescence microscope image of Example 33. Figure 12 F is a fluorescence microscope image of Example 34. Figure 12 G is a fluorescence microscope image of Example 35. Figure 12 H is a fluorescence microscope image of Example 36. Figure 12 I is a fluorescence microscope image of Example 37. Figure 12 J is a fluorescence microscope image of Example 38. Figure 12 K is a fluorescence microscope image of Example 39. Figure 12 A, Figure 12 C~ Figure 12 K showed that compounds 2–4 and compounds 6–12 could perform fluorescent staining of cellular vesicles in the same way as compound 1. Furthermore, by measuring… Figure 12 The fluorescence intensity of A and Figure 12 The amount of melanin in cells B can be used to determine the correlation between fluorescence intensity and melanin content. This enables the assessment of cell quality.

[0448] [Determination of fluorescence intensity in live and dead cells]

[0449] The fluorescence intensity of compounds 1 through 7 was measured for both living and dead cells. The procedure is shown below.

[0450] <Example 40>

[0451] (1) Dissociate B16F10 cells with trypsin-EDTA and suspend them in phosphate buffer. At this time, add 30% ethanol to the dead cell preparation.

[0452] (2) Centrifuge the live cell suspension and the dead cell suspension at 2000 rpm for 3 minutes to separate the cells.

[0453] (3) Wash twice with phosphate buffer and count the cells. At this point, the viability of the live cells obtained from the live cell suspension is 70%.

[0454] (4) In cells 5×10 4 Compound 1 was added to 100 μL to make a final concentration of 1 μL, and fluorescence was measured (excitation at 488 nm). The measured value was then obtained by subtracting the fluorescence intensity of the compound itself from the obtained fluorescence intensity and dividing the result by the fluorescence intensity of the phosphate buffer alone.

[0455] <Example 41>

[0456] The same as in Example 40, except that compound 2 is used instead of compound 1.

[0457] <Example 42>

[0458] The same as in Example 40, except that compound 3 is used instead of compound 1.

[0459] <Example 43>

[0460] The same as in Example 40, except that compound 4 is used instead of compound 1.

[0461] <Example 44>

[0462] The same as in Example 40, except that compound 5 is used instead of compound 1.

[0463] <Example 45>

[0464] The same as in Example 40, except that compound 6 is used instead of compound 1.

[0465] <Example 46>

[0466] The same as in Example 40, except that compound 7 is used instead of compound 1.

[0467] <Comparative Example 5>

[0468] The same as in Example 40, except that a fluorescent compound of the following formula is used instead of compound 1.

[0469] [Chemical Formula 26]

[0470]

[0471] The results are shown in Figure 13 .based on Figure 13 In Examples 40-46, the fluorescence intensity of live cells was high, and the difference between the fluorescence intensity of live cells and dead cells was also large. On the other hand, in Comparative Example 5, the difference between the fluorescence intensity of live cells and dead cells was small. Therefore, it is shown that compounds 1-7 are specific to cellular vesicles of live cells.

[0472] [Measurement of exosomes in serum of tumor-transplanted mice]

[0473] <Example 47>

[0474] Serum was collected from mice that had received melanoma cell transplants, and the exosomes in the serum were measured. The steps are shown below.

[0475] (1) Subcutaneous transplantation of 5×10⁶ cells into mice (C57BL / 6 (8-week male)) 4 Two weeks later, the tumor weight was measured using B16F10 cells.

[0476] (2) Collect the serum of the mice and mix 20 μL of serum with 80 μL of phosphate buffer.

[0477] (3) Add compound 1 to the mixture of serum and phosphate buffer to make the final concentration 0.3 μM.

[0478] (4) The results were measured using a fluorescence microscope 15 minutes after the addition of compound 1. In addition, the measurements were performed under excitation at 488 nm.

[0479] The results are shown in Figure 14 .Depend on Figure 14 It can be seen that the fluorescence intensity increases with increasing tumor weight. This indicates that the number of exosomes in the serum increases due to increased tumor weight.

[0480] As demonstrated by the above embodiments, the compounds disclosed in this application exhibit high specificity for cellular vesicles, and do not fluoresce in compounds that do not react with cellular vesicles. Therefore, it is shown that the removal of unreacted compounds is unnecessary after labeling, thus suppressing sample loss.

[0481] Industrial application

[0482] It is useful in the field of fluorescent staining of cellular vesicles.

Claims

1. A compound represented by the following formula (2), [Chemical Formula 2] In equation (2), R1 represents H or CH3; R2 represents H, CH3, OCH3, or N(CH3)2; R3 and R4 each independently represent H or CH3; among them, R R3 and R4 can also bond with each other to form a ring. In this case, R3 and R4 are CH2; R5 represents NO2, SO2NH2 or SO2N(CH3)2; l represents 1 or 2, m represents 0 or 1, and n represents 1, 2 or 3.

2. A compound represented by the following formula (3), [Chemical Formula 3] In equation (3), R1 represents H or CH3; R2 represents H, CH3, OCH3, or N(CH3)2; R3 and R4 each independently represent H or CH3; among them, R R3 and R4 can also bond with each other to form a ring. In this case, R3 and R4 are CH2; l represents 1 or 2, m represents 0 or 1, and n represents 1, 2 or 3.

3. A cellular vesicle staining agent comprising the compound of claim 1 or 2.

4. The cellular vesicle staining agent according to claim 3 further comprises other fluorescent compounds.

5. A fluorescent staining method for cellular vesicles for non-disease diagnostic purposes, comprising: The staining process involves selecting any one of the compounds described in claim 1 or 2 and the cellular vesicle staining agents described in claims 3 and 4 to stain the cellular vesicles. as well as The testing process involves detecting stained cellular vesicles in the sample.

6. The fluorescent staining method for cellular vesicles according to claim 5, wherein, Following the testing process, there is an evaluation process for evaluating the test samples.

7. The fluorescent staining method for cellular vesicles according to claim 6, wherein, The samples included cosmetics. The evaluation of cosmetics is based on the measurement of intracellular cellular vesicles.

8. The fluorescent staining method for cellular vesicles according to claim 6, wherein, The samples included pharmaceuticals. Evaluation of pharmaceuticals is achieved by measuring intracellular uptake of cellular vesicles.

9. The fluorescent staining method for cellular vesicles according to claim 6, wherein, The sample includes cells. Cellular vesicles are measured within cells to evaluate cell differentiation and quality.

10. The fluorescent staining method for cellular vesicles according to claim 6, wherein, The samples included food. Food is evaluated by measuring cellular vesicles within it.

11. The fluorescent staining method for cellular vesicles according to claim 6, wherein, The sample contains biological tissue collected from a living organism. The biological tissue is evaluated by measuring cellular vesicles in the collected biological tissue.

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