Compound emitting light under specific condition, and method for detecting biological activity using said compound

Abstract

The present invention provides a compound that responds with an aldehyde dehydrogenase, particularly ALDH1A1 and ALDH1A3 to emit light with high sensitivity, and creates a molecular probe that can be applied to detection of various cancer stem cells. The present invention relates to a compound represented by the following formula (in the formula, Ar1 and Ar2 are each an aromatic ring and may be the same or different, X is an oxygen atom, a nitrogen atom, a sulfur atom, or a selenium atom, R1 and R2 are each an alkylene having one or more carbon atoms or an arylene which may include a hetero atom, the total number of carbon atoms of R1 and R2 is 2-10, Y is -NHCO-, -COO-, -COS-, -O-, -S-, -NR3- (R3 is an alkyl group having 1-5 carbon atoms), or -C=N-, p is an integer of 2-4, q is an integer of 0-4, and r is 0 or 1).

Description

Compound that emits light under specific conditions and method for detecting biological activity using said compound

[0001] The present invention relates to a compound that emits light under specific conditions and a method for detecting biological activity using said compound.

[0002] Cancer cells are known to contain cancer stem cells (CSCs), which exhibit resistance to anticancer drugs and metastatic potential. Luminescent agents that enable visualization of cancer stem cells are effective for real-time detection of cancer stem cells using a microscope, for sorting cancer stem cells, and for detecting malignant tumors containing many cancer stem cells (in clinical settings). Aldehyde dehydrogenase 1A1 (ALDH1A1) and 1A3 (ALDH1A3) are known biomarkers expressed in cells exhibiting stemness. Molecular probes for detecting ALDH1A1, such as the commercially available drug ALDEFLUOR (Non-Patent Document 1), have been developed (Non-Patent Documents 2-5), and have been shown to be able to identify cancer stem cells in cancer cells. However, in addition to ALDH1A1, the expression of its isoform, ALDH1A3, has also been reported in cancer stem cells. Furthermore, some cancer cell types are known to express higher levels of ALDH1A3 than ALDH1A1. If a molecular probe that responds with high sensitivity to both ALDH1A1 and ALDH1A3 and emits light can be developed, it could be said to be more suitable for detecting a wider variety of cancer stem cells than the above-mentioned luminescent probes.

[0003] Furthermore, when metastatic cancer is discovered, the rate of diagnosis of cancer of unknown primary origin is only a few percent. Currently, there are limitations to the interpretation of comprehensive whole-body imaging results, and when a diagnosis of unknown primary tumor is made, doctors have no choice but to make predictions based on surrounding information and provide treatment. If it were possible to predict the organ where the primary tumor is located, intensive diagnosis would dramatically improve the accuracy of detection, and the probability of remission would increase.

[0004] Cancer metastasis occurs primarily through cancer stem cells, which exhibit high metastatic potential, and metastatic cancers exhibit the properties of cancer stem cells from the primary tumor. Meanwhile, as mentioned above, it is known that the expression levels of ALDH1A1 and ALDH1A3 vary depending on the cancer cell type. Furthermore, since metastatic cancers inherit the properties of the primary tumor, it is expected that the expression ratio of ALDH1A1 and ALDH1A3 will not change even after metastasis. Quantifying the expression ratio of ALDH1A1 and ALDH1A3 in unknown cells can be useful for predicting the primary tumor of metastatic cancer.

[0005]

[0006] Known methods for predicting the primary site of metastatic cancer include investigating the mRNA contained in metastatic cancer, and methods such as antigen testing and cytokine evaluation. While the mRNA investigation method is highly accurate, it requires a long and multi-stage diagnosis. Antigen testing and cytokine evaluation methods are considered to be less accurate. Therefore, the development of simpler methods is desirable.

[0007] R. W. Storms, et al. Proc. Natl. Acad. Sci. USA 1999, 96, 9118. S. Maity, et al. Chem. Sci. 2017, 8, 7143. DOI: 10.1039 / c7sc03017gC. Anorma, et al. ACS Cent. Sci. 2018, 4, 1045. DOI: 10.1021 / acscentsci.8b00313; T. E. Bearrood, et al. Bioconjugate Chem. 2020, 31, 224. DOI: 10.1021 / acs.bioconjchem.9b00723M. Oe, et al. ACS Sens. 2021, 6, 3320. DOI: 10.1021 / acssensors.1c01136Q. W. Wang, et al. Anal. Chem. 2022, 94, 49, 17328−17333.

[0008] The present invention aims to provide a compound that emits light with high sensitivity to aldehyde dehydratase, particularly ALDH1A1 and ALDH1A3, and to create a molecular probe that can be applied to the detection of various cancer stem cells.

[0009] That is, the present invention (1) is a compound represented by the following formula: (In the formula, Ar １ and Ar ２ are each an aromatic ring and may be the same or different. X is an oxygen atom, a nitrogen atom, a sulfur atom or a selenium atom. R １ and R ２ are each alkylene having one or more carbon atoms or arylene which may contain a heteroatom, and R １ and R ２ The total number of carbon atoms is 2 to 10. Y is —NHCO—, —COO—, —COS—, —O—, —S—, or —NR ３ - (R ３ is an alkyl group having 1 to 5 carbon atoms. ) or —C═N—. p is an integer of 2 to 4, q is an integer of 0 to 4, and r is 0 or 1.

[0010] The present invention (2) is the above-mentioned Ar １ and Ar ２ are expressed as follows: ,or, (wherein Z is a halogeno group, -SO ３ Na, -SO ３ H or -COOR ４ and R ４ is H or an alkyl group having 1 to 6 carbon atoms.

[0011] The present invention (3) is a compound represented by the following formula: (where n is an integer of 2 to 10).

[0012] The present invention (4) is a fluorescent reagent containing the compound according to any one of the present inventions (1) to (3).

[0013] The present invention (5) is a fluorescent reagent according to the present invention (4) for detecting cellular ALDH1A1 activity and ALDH1A3 activity.

[0014] The present invention (6) is a method for detecting ALDH1A1 activity or ALDH1A3 activity in cells, comprising the steps of administering the fluorescent reagent according to the present invention (4) to cells, irradiating the cells with light having a wavelength of 300 to 1300 nm, and detecting the fluorescence of the fluorescent reagent.

[0015] The present invention (7) is the detection method according to the present invention (6), in which the cells are detected in vivo, in vitro or ex vivo.

[0016] The compounds of the present invention emit light in response to aldehyde dehydratase, particularly both ALDH1A1 and ALDH1A3, with high sensitivity, and therefore can easily detect a variety of cancer stem cells.

[0017] (a) Absorption spectrum and (b) fluorescence spectrum of C5NC5-A synthesized in Example 1 in buffer solutions of various pH values. (c) Fluorescence spectrum of C5NC4-A synthesized in Example 2 and (d) fluorescence spectrum of C5NC6-A synthesized in Example 3 in buffer solutions of various pH values. (a) ALDH1A1 or (b) ALDH1A3 was reacted with C5NC4-A synthesized in Example 2. (c) ALDH1A1 or (d) ALDH1A3 was reacted with C5NC5-A synthesized in Example 1. (a) Absorption spectrum and (b) fluorescence spectrum of C5NC5-A synthesized in Example 1 in buffer solutions of various pH values. (b) Fluorescence spectrum of C5NC4-A synthesized in Example 2 and (d) fluorescence spectrum of C5NC6-A synthesized in Example 3. (c) ALDH1A1 or (d) ALDH1A3 was reacted with C5NC5-A synthesized in Example 1. (c) ALDH1A1 or (d) ALDH1A3 was reacted with C5NC5-A synthesized in Example 1. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC4-A synthesized in Example 2 and (d) ALDH1A3. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC5-A synthesized in Example 1. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC5-A synthesized in Example 1. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC5-A synthesized in Example 2 and (d) ALDH1A3. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC5-A synthesized in Example 1 and (d) ALDH1A1 and ALDH1A3. (c) ALDH1A1 and ALDH1A3 were reacted with C5NC5-A synthesized (a) Confocal laser microscope images of SUIT-2 cells treated with C5NC5-A. The inhibitor was disulfiram. (b) Confocal laser microscope images of SUIT-2 cells treated with ALDEFLUOR and C5NC5-A. The scale bar is 50 μm. Confocal laser microscope images of SUIT-2, KATO-III, and MKN1 cells treated with C3S-A and C5NC5-A are shown. Changes in fluorescence intensity when ALDH1A3 was allowed to respond to the compounds of the present invention synthesized in each Example for 1 hour are shown. Changes in fluorescence intensity when ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH3A1, or ALDH7A1 was allowed to respond to C5NC5-A synthesized in Example 1 are shown. The figure shows the results of a cytotoxicity test when C5NC5-A was applied to SUIT-2 cells for 6 hours. The figure shows confocal laser microscope images of MKN1 cells when (a) C5NC5-A or (b) C5S-A was applied to MKN1 cells. (c) The fluorescence intensity (F ) of luminescent and non-luminescent cells stained with C5NC5-A or C5S-A was measured. ＣＳＣ and F ＮＣＣ ) are shown. Confocal laser microscope images of SUIT-2 treated with C5NC5-A at various concentrations ((a) 10, (b) 5, (c) 1, (d) 0.5, (e) 0.1, (f) 0.05, (g) 0.01 μM). (h) Fluorescence intensity (F ＣＳＣ and F ＮＣＣ) are shown. The results of flow cytometric analysis of SUIT-2 cells stained with C5NC5-A synthesized in Example 1 are shown. These are confocal laser microscope images obtained when 1 μM C5NC5-A was allowed to act on SUIT-2 for (a) 5 minutes, (b) 20 minutes, and (c) 60 minutes, respectively. (d) The fluorescence intensity (F ＣＳＣ and F ＮＣＣ ) is shown.

[0018] The present inventors have previously developed three ALDH1A1-responsive molecular probes: C3S-A (K. Miki, D. Yamanaka, H. Mu, K. Miki, K. Ohe, The 103rd CSJ Annual Meeting, D1441-1am-07), C5S-A (M. Oe, K. Miki, Y. Ueda, Y. Mori, A. Okamoto, Y. Funakoshi, H. Minami, K. Ohe, ACS Sens. 2021, 6, 3320), and C7S-A (M. Oe, K. Suzuki, K. Miki, H. Mu, K. Ohe, Chem Plus Chem 2022, 87, e220200319). These molecular probes feature a nucleophilic mercapto group and an enzyme-responsive ω-formylalkyl group bound to the same indole moiety. We reasoned that changing the position of the enzyme-responsive site was necessary to allow the probe to respond to a different isoform of ALDH1A3. We synthesized a molecular probe, C5NBn-A, bearing a 4-formylphenylmethyl group at the indole site, which differs from indoles that previously possess a nucleophilic mercapto group, and evaluated its ALDH1A3 responsiveness. However, while C5NBn-A exhibited responsiveness with ALDH1A1, its responsiveness with ALDH1A3 was not very high. Given that the enzyme active site of ALDH1A3 is located more recessed than that of ALDH1A1, we designed three types of molecular probes, C5NCn-A (n = 4), C5NC5-A (n = 5), and C5NC6-A (n = 6)), which possess an ω-formylalkyl group instead of the 4-formylphenylmethyl group. Docking simulations (AutoDock) of C5NC4-A, C5NC5-A, C5NC6-A, and C5NBn-A with ALDH1A3 estimated the distances from the active site of ALDH1A3 to be 5.0 Å, 4.0 Å, 4.5 Å, and 5.6 Å, respectively. This suggests that all three types of C5NCn-A are more easily accessible to the enzyme active site of ALDH1A3 than C5NBn-A. Based on these guidelines, the compounds of the present invention were discovered.

[0019]

[0020] The compound of the present invention is represented by the following formula (1): (In the formula, Ar １ and Ar ２ are each an aromatic ring and may be the same or different. X is an oxygen atom, a nitrogen atom, a sulfur atom or a selenium atom. R １ and R ２ are each alkylene having one or more carbon atoms or arylene which may contain a heteroatom, and R １ and R ２ The total number of carbon atoms is 2 to 10. Y is —NHCO—, —COO—, —COS—, —O—, —S—, or —NR ３ - (R ３ is an alkyl group having 1 to 5 carbon atoms. ) or -C=N-. p is an integer of 2 to 4, q is an integer of 0 to 4, and r is 0 or 1.

[0021] The compound of the present invention emits light under specific conditions. The mechanism of this light emission will be explained based on C5NC5-A, which has the following formula: Aldehyde dehydrogenase converts a formyl group to a carboxy group. As the formyl group is converted to a carboxy group, the heterocycle containing N and S dissociates (the bond between S and C dissociates), forming an iminium, which then conjugates with two benzene rings via vinylene and emits light when irradiated with light of 300 to 1300 nm. This mechanism makes it possible to detect aldehyde dehydratase, making it possible to detect cancer stem cells in which aldehyde dehydratase is present.

[0022]

[0023] In the chemical formula (1), the formyl group is oxidized to a carboxy group by aldehyde dehydrogenase, thereby reducing the cell membrane permeability of the compound from cancer stem cells to the outside of the cells. １ -(Y) ｒ -R ２ It is bonded to the nitrogen atom of the light-emitting site via a -.

[0024] In the chemical formula (1), Ar １ and Ar ２ are each an aromatic ring, and may be the same or different. Specific examples of aromatic rings include Here, Z is a halogeno group, -SO ３ Na, -SO ３ H or -COOR ４ It is. ４ is H or an alkyl group having 1 to 6 carbon atoms. １ , Ar ２ The combination of the aryliminium formed by aldehyde dehydrogenase and the vinylene that binds them constitutes the light-emitting moiety. １ and Ar ２ When the heterocycle forms an indole ring, the indole ring structure can be synthesized by a known method (M. Oe, et al. ACS Sens. 2021, 6, 3320. DOI: 10.1021 / acssensors.1c01136).

[0025] X is an oxygen atom, nitrogen atom, sulfur atom, or selenium atom. Of these, a sulfur atom is preferred. The ring structure containing X and N has the function of turning the light-emitting portion into a non-light-emitting state. The -XH group bonds to the carbon-nitrogen double bond to form a single bond, severing the conjugated system and thereby changing the light-emitting state into a non-light-emitting state.

[0026] R １ and R ２ R is an alkylene having one or more carbon atoms or an arylene which may contain a heteroatom. Examples of the alkylene having one or more carbon atoms include methylene, ethylene, propylene, butylene, pentene, pentylene, and hexylene. Examples of the arylene which may contain a heteroatom include phenylene, thienylene, pyrrolylene, and triazolylene. １ and R ２ The total number of carbon atoms in the group is 2 to 10, preferably 3 to 7, and more preferably 4 to 6. Y is —NHCO—, —COO—, —COS—, —O—, —S—, or —NR ３ - (R ３ is an alkyl group having 1 to 5 carbon atoms, or —C═N—, and r is 0 or 1.

[0027] q is an integer of 0 to 4, preferably an integer of 1 to 3.

[0028] Aldehyde dehydrogenase is an enzyme that can convert aldehyde group to carboxy group, such as ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH16A1, ALDH18A1, etc. Among them, ALDH1A1 and ALDH1A3 are preferred because they are known to be highly expressed in cancer stem cells.Since these aldehyde dehydrogenases are contained in cancer stem cells, cancer stem cells can be detected by the compound of the present invention.

[0029] The compound of the present invention is preferably a compound represented by the following chemical formula: Here, n is an integer of 2 to 10, preferably an integer of 2 to 6.

[0030] More specific examples of the compound include compounds represented by the following chemical formulas.

[0031] The fluorescent reagent of the present invention is characterized by containing the compound of the present invention. The fluorescent reagent can also contain solvents, buffers, pH adjusters, stabilizers, serum, antibacterial agents, nutrients, and the like that are commonly added to detection reagents. Examples of solvents include water, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). When used as a fluorescent reagent, the concentration of the compound of the present invention is preferably 0.1 to 50 μM, and more preferably 10 to 20 μM.

[0032] The fluorescent reagent of the present invention can be used as a cancer stem cell detection reagent and can also be used as a component of a cancer stem cell detection kit, which may include, in addition to the cancer stem cell detection reagent of the present invention, a buffer, a test tube, a Petri dish, a positive control sample, a negative control sample, etc.

[0033] The method for detecting ALDH1A1 activity or ALDH1A3 activity of the present invention is characterized by comprising the steps of administering the fluorescent reagent of the present invention to cells, and irradiating the cells with light having a wavelength of 300 to 1300 nm and detecting the fluorescence of the compound.

[0034] The cells to be administered are not particularly limited and may be human cells or cells from non-human animals. Examples of non-human animals include primates, rodents (mice, rats, etc.), and rabbits. The tissue from which the cells are derived is not particularly limited and examples include the prostate, uterus, ovaries, lungs, skin, stomach, intestines, liver, pancreas, gallbladder, blood cells, and brain. The cells to be administered may be cultured cells or cells contained in tissue. Examples of tissues containing cells include cultured tissues, biological tissues obtained from test individuals, and tissue slices.

[0035] The method of administering the fluorescent reagent to cells is not particularly limited, and may be in vitro administration, in vivo administration to cells present in a test individual, or ex vivo administration. In vitro administration methods include immersing cells or tissues containing cells in a medium or buffer containing the fluorescent reagent, or applying or dripping the fluorescent reagent onto tissues containing cells. In vivo administration methods include application, injection, oral administration, enteral administration, vaginal administration, and inhalation. Ex vivo administration methods include immersing biological tissues or tissue sections obtained from a test individual in a medium or buffer containing the fluorescent reagent, or applying or dripping a medium or buffer containing the fluorescent reagent.

[0036] The method for detecting cellular ALDH1A1 activity and ALDH1A3 activity is not particularly limited. After administering a fluorescent reagent to the cells, the cells are allowed to stand for preferably 5 to 180 minutes, more preferably 30 to 60 minutes, and then irradiated with light having a wavelength of 300 to 1300 nm. The wavelength is preferably 350 to 750 nm, more preferably 550 to 650 nm. The luminescence generated upon irradiating the cells with light is observed, and it can be determined that cells having ALDH1A1 activity and ALDH1A3 activity, such as cancer stem cells, are present at sites where luminescence is observed. The luminescence wavelength is preferably 450 to 800 nm, more preferably 650 to 800 nm.

[0037] When a fluorescent reagent is administered to cells in vitro, in vitro detection can be performed by irradiating cells present in a container such as a petri dish with red to near-infrared light and observing the luminescence. When a fluorescent reagent is administered to cells in vivo, in vivo detection can be performed by irradiating near-infrared light at the location of the cells in the test subject and observing the luminescence. In vivo detection allows non-invasive observation of cells present inside the body by adjusting the wavelength of the irradiated near-infrared light and the detection wavelength.

[0038] It is known that the expression levels of ALDH1A1 and ALDH1A3 vary depending on the cancer cell type. Furthermore, since metastatic cancers that are likely to be diagnosed as cancer of unknown primary origin inherit the characteristics of the primary tumor, it is expected that the expression ratio of ALDH1A1 and ALDH1A3 will not change even after metastasis. Quantifying the expression ratio of ALDH1A1 and ALDH1A3 in unknown cells is effective in predicting the primary tumor of metastatic cancer.

[0039] The compounds of the present invention can be used in screening for cancer therapeutic drugs. Examples of methods for screening cancer therapeutic drugs include the following: (i) administering the compounds of the present invention to cancer stem cells in the presence or absence of a test substance, (ii) irradiating the cancer stem cells with light (preferably near-infrared light) having a wavelength of 300 to 1300 nm and detecting luminescence from the compound, and (iii) selecting the test substance as a cancer therapeutic drug when the amount of luminescence from the compound is reduced in the presence of the test substance compared to its absence.

[0040] The test substance used in step (i) is not particularly limited as long as it is a candidate substance for a cancer therapeutic drug. The cancer stem cells may be cells that have been confirmed to be cancer stem cells, and the animal or organ from which they are derived is not particularly limited. In steps (i) to (ii), the same conditions as those for the in vitro administration method and detection method described in relation to the method for detecting cancer stem cells can be used.

[0041] In step (iii), the luminescence intensity of the compound is compared in the presence and absence of the test substance. A decrease in the luminescence intensity in the presence of the test substance compared to its absence indicates that the test substance has inactivated or eliminated cancer stem cells, and the test substance can be selected as a cancer therapeutic agent. To select a test substance as a cancer therapeutic agent, the decrease in the luminescence intensity of the compound in the presence of the test substance compared to its absence is preferably 50% or more, more preferably 80% or more. High-throughput screening is possible by performing steps (i) to (iii) in parallel for tens to thousands of test substances.

[0042] Unless otherwise specified, solvents and reagents used were special grade or first grade reagents from Nacalai Tesque, Inc., Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Kishida Chemical Co., Ltd., or Aldrich.

[0043] Example 1 (Synthesis of C5NC5-A (Compound 11, n=5)) (The synthesis method of Compound 1 is known from a paper.)

[0044]

[0045] (Synthesis of Compound 2) 1,6-hexanediol (5.1 g, 43 mmol), Et ３ N (9.2 mL, 66 mmol) was dissolved in CH ２ Cl ２ (80 mL). The mixture was cooled to 0°C, and TBSCl (t-butyldimethylsilyl chloride) (6.5 g, 43 mmol) was added in small portions. The reaction was allowed to proceed at room temperature for 2.5 hours. The reaction solution was washed with water (20 mL x 2), and the aqueous layer was extracted with CH ２ Cl ２ (10 mL x 2). The combined organic layers were extracted with MgSO ４ The organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (hexane: EtOAc = 4:1) to obtain compound 2 (4.6 g, 20 mmol, 46%, rf = 0.3).

[0046] (Synthesis of Compound 3) Compound 2 (1.5 g, 6.5 mmol), CH ２ Cl ２ (35 mL), PPh ３ (1.9 g, 7.2 mmol) and imidazole (0.89 g, 13 mmol) were added. The mixture was cooled to 0°C, and iodine (1.8 g, 7.2 mmol) was added. The mixture was allowed to react at room temperature for 3 hours, and the solvent was evaporated under reduced pressure. Hexane was added to the residue, and the mixture was stirred. The organic layer of the supernatant was separated, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane only) to obtain compound 3 (1.8 g, 5.4 mmol, 83%, rf≧0.9 (hexane: EtOAc = 4:1)).

[0047] Compound 3 １ H NMR (400MHz, CDCl ３ , 25℃) δ0.05 (s, 6H), 0.89 (s, 9H), 1.32-1.42 (m, 4H), 1.48-1.55 (m, 2H), 1 .84 (tt, J=7.2, 7.2Hz, 2H), 3.19 (t, J=7.1Hz, 2H), 3.61 (t, J=6.6Hz, 2H).

[0048]

[0049] (Synthesis of Compound 4) In a nitrogen atmosphere, 2,3,3-trimethylindolenine (0.70 g, 4.4 mmol), Compound 1 (1.4 g, 5.8 mmol), and MeCN (9 mL) were placed in a 50 mL two-necked flask and refluxed for 12 hours. The solvent was removed from the reaction solution by distillation under reduced pressure. A small amount of CH ２ Cl ２ Transfer to a centrifuge tube using Et ２ The solid was precipitated by adding 200 ml of CH. The mixture was centrifuged and the supernatant was removed. ２ Cl ２ Dissolved in Et ２ O was added to precipitate a solid. The solid was centrifuged, the supernatant was removed, and the mixture was dried under reduced pressure to obtain Compound 4 (1.5 g, 3.7 mmol, 84%).

[0050] (Synthesis of Compound 5) Under a nitrogen atmosphere, compound 4 (1.5 g, 3.7 mmol), acetic anhydride (12 mL), and acetic acid (8 mL) were placed in a 100 mL two-neck flask. N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline hydrochloride (CAS: 58467-94-0, 0.96 g, 3.7 mmol) was added thereto, and the mixture was reacted at 125°C for 2 hours. Et ２ The reaction solution was poured into 10 ml of ethanol with stirring. The mixed solution was stored in a freezer to precipitate a solid. The obtained solid was ２ Repeated washing with O gave compound 5 (0.62 g, 1.1 mmol, 29%).

[0051]

[0052] (Synthesis of Compound 6) Under a nitrogen atmosphere, 2,3,3-trimethylindolenine (0.51 g, 3.2 mmol), Compound 3 (1.1 g, 3.2 mmol), and MeCN (35 mL) were placed in a 100 mL two-neck flask and refluxed for 3 days. Purification was performed using a centrifuge tube in the same manner as in the purification of Compound 4, but no solid precipitated and the compound remained in an oily state. The supernatant was removed, leaving the oil, and the resulting mixture was diluted with Et ２ The extract was washed with O. The extract was dried under reduced pressure to obtain Compound 6 (0.91 g, 1.9 mmol, 58%).

[0053] (Synthesis of Compound 7) Compound 6 (0.63 g, 1.3 mmol) was dissolved in MeOH (20 mL), and p-toluenesulfonic acid monohydrate (58 mg, 0.30 mmol) was added thereto. The mixture was stirred at room temperature for 24 hours, and the solvent was evaporated under reduced pressure. The residue was dissolved in CH ２ Cl ２ The organic layer was washed with water and then with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ :MeOH=15:1) to obtain Compound 7 (0.27 g, 0.70 mmol, 53%).

[0054] (Synthesis of Compound 8) Under a nitrogen atmosphere, Compound 7 (0.27 g, 0.70 mmol) was dissolved in dry pyridine (10 mL). To this was added a solution of Compound 5 (0.52 g, 0.91 mmol) dissolved in dry pyridine (5 mL) at room temperature. The reaction was carried out at 40°C for 2.5 hours. The solvent was distilled off under reduced pressure in the dark. The residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a 1:1 mixture of HCl, HCl, and MeOH (15:1 to 10:1) to obtain Compound 8 (0.34 g, 0.50 mmol, 72%, blue solid).

[0055] (Synthesis of Compound 9) Compound 8 (88 mg, 0.13 mmol) and sodium hexafluorophosphate (0.17 g, 1.0 mmol) were dissolved in MeOH (2 mL) and water (0.5 mL), and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with water and added to CH ２ Cl ２ The organic layer was extracted with MgSO ４ The solvent was evaporated under reduced pressure to quantitatively obtain Compound 9 as a crude product.

[0056] (Synthesis of Compound 10) Compound 9 (0.12 g, 0.16 mmol) was dissolved in CH ２ Cl ２ (3 mL). Dess-Martin periodinane (DMP, 86 mg, 0.24 mmol) was added at room temperature in the dark, and the reaction was allowed to proceed. After 30 minutes, DMP (17 mg, 0.057 mmol) was added, and the reaction was continued. After 30 minutes, water was added to stop the reaction, and the aqueous layer was separated using CH ２ Cl ２ The organic layer was extracted with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ :MeOH=30:1) to obtain Compound 10 (15 mg, 0.021 mmol, 13%).

[0057] Compound 10 １ H NMR (400MHz, CDCl ３, 25℃) δ1.49-1.55 (m, 2H), 1.71 (s, 12H), 1.78-1.87 (m, 4H), 2.09 (tt, J=7.4, 7.4Hz, 2H), 2.36 (s, 3H), 2.54 (d t, J=1.4, 6.9Hz, 2H), 3.00 (t, J=7.1Hz, 2H), 4.02 (t, J=7.8Hz, 2H), 4.07 (t, J=7.8Hz, 2H), 6.24 (d, J=13.5Hz, 1H), 6.25 (d, J = 13.5Hz, 1H), 6.79 (dd, J = 12.8, 12.8Hz, 1H), 7.08 (d, J = 7.8Hz, 1H), 7.09 (d, J = 7.8Hz, 1H), 7.2 0-7.26 (m, 2H), 7.35-7.40 (m, 4H), 7.89 (dd, J=13.213.2Hz, 1H), 7.90 (dd, J=13.2, 13.2Hz, 1H), 9.78 (s, 1H). HRMS (ESI) calcd for C ３６ H ４５ N ２ O ２ S ([M] ＋ )569.3196, found569.3196.

[0058] (Synthesis of Compound 11) Compound 10 (6.1 mg, 8.6 μmol) was dissolved in MeOH (1 mL), and the solution was heated at room temperature under light shielding. ２ CO ３ (2.7 mg, 20 μmol) was added. After 20 minutes, the reaction mixture was ２ Cl ２ The organic layer was diluted with MgSO and washed twice with saturated aqueous sodium bicarbonate. ４ The solvent was evaporated under reduced pressure to give Compound 11 (3.7 mg, 7.0 μmol, 82%).

[0059] Compound 11 HRMS (ESI) calcd for C ３４ H ４３ N ２ OS ([M ＋ H] ＋ )527.3091, found527.3096.

[0060] Example 2 (Synthesis of C5NC4-A)

[0061]

[0062] C5NC4-A was synthesized in the same manner as in Example 1, except that 1,5-pentanediol was used instead of 1,6-hexanediol.

[0063] Compound 12 １ H NMR (400MHz, CDCl ３ , 25℃) δ0.04 (s, 6H), 0.89 (s, 9H), 1.36-1.43 (m, 2H), 1.47-1.54 (m, 2H), 1 .82 (tt, J=7.0, 7.0Hz, 2H), 3.18 (t, J=7.1Hz, 2H), 3.59 (t, J=6.6Hz, 2H).

[0064] Compound 13 １ H NMR (400MHz, CDCl ３ , 25℃) δ1.37-1.45 (m, 4H), 1.74 (s, 12H), 2.08-2.11 (m, 2H), 2.36 (s, 3H), 2.63 (t, J = 5.3 Hz, 2H), 3.06 (t, J = 7.1Hz, 2H), 4.13-4.22 (m, 4H), 6.52 (d, J = 13.7Hz, 1H), 6.54 (d, J = 13. 7Hz, 1H), 6.95 (dd, J = 12.8, 12.8Hz, 1H), 7.09 (d, J = 8.2Hz, 2H), 7.19-7.25 (m, 2H), 7.33- 7.37 (m, 4H), 8.06 (dd, J=13.3, 13.3Hz, 1H), 8.07 (dd, J=13.3, 13.3Hz, 1H), 9.81 (s, 1H). HRMS (ESI) calcd for C ３５ H ４３ N ２ O ２ S ([M] ＋ )555.3040, found555.3044.

[0065] Example 3 (Synthesis of C5NC6-A)

[0066]

[0067] C5NC6-A was synthesized in the same manner as in Example 1, except that 1,7-heptanediol was used instead of 1,6-hexanediol.

[0068] Compound 14 １ H NMR (400 MHz, CDCl ３ , 25 °C) δ 0.04 (s, 6H), 0.89 (s, 9H), 1.30 - 1.34 (m, 4H), 1.36 - 1.43 (m, 2H), 1.47 - 1.54 (m, 2H), 1.82 (tt, J = 7.0, 7.0 Hz, 2H), 3.18 (t, J = 7.1 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H).

[0069] Compound 15 １ H NMR (400 MHz, CDCl ３ , 25 °C) δ 1.40 - 1,49 (m, 4H), 1.64 - 1.68 (m, 2H), 1.72 (s, 12H), 1.78 - 1.82 (m, 2H), 2.07 - 2.10 (m, 2H), 2.36 (s, 3H), 2.50 (t, J = 5.7 Hz, 2H), 3.00 (t, J = 7.1 Hz, 2H), 4.01 (t, J = 7.5 Hz, 2H), 4.07 (t, J = 7.5 Hz, 2H), 6.25 (d, J = 13.7 Hz, 2H), 6.77 (dd, J = 12.6, 12.6 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 7.20 - 7.24 (m, 2H), 7.35 - 7.40 (m, 4H), 7.89 (dd, J = 13.2, 13.2 Hz, 1H), 7.90 (dd, J = 13.2, 13.2 Hz, 1H), 9.78 (s, 1H), HRMS (ESI) calcd for C ３７ H ４７ N ２ O ２ S ([M] ＋ ) 583.3367, found 583.3353.

[0070] Example 4 (Synthesis of C5NBn - A)

[0071]

[0072] (Synthesis of Compound 19) Under a nitrogen atmosphere, Compound 16 (0.29 g, 1.7 mmol), 4-(bromomethyl)benzenemethanol (0.34 g, 1.7 mmol), and MeCN (8 mL) were placed in a Schlenk tube and refluxed overnight. The solvent was distilled off under reduced pressure, and the residue was transferred to a centrifuge tube. A small amount of CH２ Cl ２ Dissolved in Et ２ The solid was precipitated by adding 200 ml of CH. The mixture was centrifuged and the supernatant was removed. ２ Cl ２ Dissolved in Et ２ The solid was precipitated by adding Et. The mixture was centrifuged and the supernatant was removed. The solid was left behind and the supernatant was removed. ２ The extract was washed with O. The extract was dried under reduced pressure to give compound 17 (0.44 g, crude yield 73%).

[0073] Under a nitrogen atmosphere, compound 17 (0.16 g) was placed in a 100 mL two-neck flask and dissolved in dry pyridine (7 mL). To this was added a solution of compound 18 (0.22 g, 0.52 mmol) dissolved in dry pyridine (5.5 mL) at room temperature. The reaction was carried out at 40°C for 2.5 hours. The solvent was distilled off under reduced pressure in the dark. The residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a hexanes / MeOH mixture (MeOH = 30:1) to obtain Compound 19 (44 mg, 61 μmol, yield 14%, blue solid).

[0074] Compound 19 a blue solid (yield 14%) １ H NMR (400MHz, CDCl ３ , 25°C) δ1.73 (s, 6H), 1.79 (s, 6H), 2.02-2.10 (m, 2H), 2.30 (s, 3H), 3.02 (t, J=7. 3Hz, 2H), 4.19 (t, J = 7.6Hz, 2H), 4.64 (s, 2H), 5.23 (s, 2H), 6.18 (d, J = 13.8Hz, 1H ), 6.44 (d, J = 13.3Hz, 1H), 6.73 (t, J = 12.2Hz, 1H), 7.02 (d, J = 8.2Hz, 1H), 7.14- 7.23 (m, 6H), 7.34-7.37 (m, 5H), 8.09 (t, J=12.8Hz, 1H), 8.11 (t, J=13.3Hz, 1H). HRMS (ESI) calcd for C ３８ H ４３ N ２ O ２ S ([M] ＋ )591.3040; found591.3033.

[0075] (Synthesis of Compound 20) Compound 19 (44 mg, 61 μmol) and sodium hexafluorophosphate (95 mg, 0.57 mmol) were dissolved in MeOH (10 mL) and water (2.5 mL) in a sample tube, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with water and added to CH ２ Cl ２ The organic layer was extracted with MgSO ４ The solvent was evaporated under reduced pressure to give a blue solid, which was used as it was in the next reaction.

[0076] A blue solid (52 mg, 71 μmol) was added to a sample tube. ２ Cl ２ (8 mL). Dess-Martin periodinane (DMP, 52 mg, 0.12 mmol) was added at room temperature in the dark, and the mixture was stirred for 1 hour. The reaction solution was washed once each with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium thiosulfate, and then once with saturated brine. The organic layer was washed with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a hexanes / MeOH mixture (MeOH = 30:1) to obtain Compound 20 (15 mg, 21 μmol, yield 29%, blue solid).

[0077] Compound 20 a blue solid (yield 29%); １ H NMR (400MHz, CDCl ３ , 25℃) δ1.70 (s, 6H), 1.76 (s, 6H), 2.02-2.10 (m, 2H), 2.33 (s, 3H), 2.97 (t, J = 7.1Hz , 2H), 4.09 (t, J = 7.5Hz, 2H), 5.26 (s, 2H), 6.08 (d, J = 13.3Hz, 1H), 6.25 (d, J = 13.7H z, 1H), 6.62 (t, J = 12.8Hz, 1H), 7.01 (d, J = 8.2Hz, 1H), 7.14 (d, J = 8.2Hz, 1H), 7.25- 7.21 (m, 1H), 7.28-7.55 (m, 2H), 7.37-7.42 (m, 5H), 7.85-7.95 (m, 4H), 9.98 (s, 1H). HRMS (ESI) calcd for C ３８ H４１ N ２ O ２ S ([M] ＋ ) 589.2883; found 589.2874.

[0078] (Synthesis of C5NBn-A) Compound 20 (2.5 mg, 3.4 μmol) was dissolved in MeOH (2.5 mL) in a sample tube, and the solution was heated at room temperature under light shielding. ２ CO ３ (0.47 mg, 3.4 μmol) was added. After 1 hour, the reaction mixture was ２ Cl ２ The organic layer was diluted with MgSO and washed twice with saturated aqueous sodium bicarbonate. ４ The solvent was evaporated under reduced pressure to obtain compound C5NBn-A (1.5 mg, 2.7 μmol, yield 80%).

[0079] C5NBn-A a greenish blue solid (yield 80%); HRMS (ESI) calcd for C ３３ H ３９ N ２ OS ([M+H] ＋ ) 547.2278; found 547.2782.

[0080] Example 5 (Synthesis of C5Namide-A)

[0081] (Synthesis of Compound 24) Compound 21 (0.56 g, 3.6 mmol), 3-bromopropionic acid (0.66 g, 4.3 mmol), and MeCN (5 mL) were placed in a Schlenk tube under a nitrogen atmosphere and refluxed overnight. The solvent was distilled off under reduced pressure, and the residue was transferred to a centrifuge tube. ２ Cl ２ Dissolved in Et ２ The solid was precipitated by adding 200 ml of CH. The mixture was centrifuged and the supernatant was removed. ２ Cl ２ Dissolved in Et ２ The solid was precipitated by adding Et. The mixture was centrifuged and the supernatant was removed. The solid was left behind and the supernatant was removed. ２ The extract was washed with O. The extract was dried under reduced pressure to give Compound 22 (0.59 g, crude yield 53%).

[0082] Under a nitrogen atmosphere, compound 22 (0.21 g) was placed in a 100 mL two-neck flask and dissolved in dry pyridine (10 mL). To this was added a solution of compound 23 (0.44 g, 0.77 mmol) dissolved in dry pyridine (10 mL) at room temperature. The reaction was carried out at 40°C for 2.5 hours. The solvent was distilled off under reduced pressure in the dark. The residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a hexanes / MeOH mixture (30:1 to 5:1) to obtain compound 24 (0.21 g, 0.34 mmol, 50%, blue solid).

[0083] (Synthesis of Compound 25) Under a nitrogen atmosphere, compound 24 (78 mg, 0.13 mmol), tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 48 mg, 0.16 mmol), and N,N-diisopropylethylamine (DIEA, 41 mg, 0.32 mmol) were placed in a 100 mL two-neck flask and cooled with dry CH ２ Cl ２ (25 mL) and stirred at room temperature for 2 hours. Amino acetaldehyde dimethyl acetal (28 mg, 0.27 mmol) and DIEA (49 mg, 38 μmol) were added to the solution and stirred for 1 hour. The organic layer was washed with water. The aqueous layer was washed with CH ２ Cl ２ (10 mL × 2), and the combined organic layer was extracted with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a hexanes / MeOH mixture (MeOH = 30:1) to obtain Compound 25 (47 mg, 66 μmol, yield 52%, blue solid).

[0084] Compound 25 a blue solid (yield 52%); １ H NMR (400MHz, CDCl ３, 25℃) δ1.68 (s, 6H), 1.69 (s, 6H), 2.03-2.11 (m, 2H), 2.37 (s, 3H), 2.78 (t, J = 7.3Hz, 2H), 2.98 (t, J = 7.1Hz, 2H), 3.34 (s, 6H), 3.36-3.39 (m, 2H), 4.03 (t, J = 7.3Hz, 2H), 4.38 (t, J = 6.2Hz, 2H), 4.45 (t , J=5.5Hz, 1H), 6.17 (d, J=13.3Hz, 1H), 6.46 (d, J=13.7Hz, 1H), 6.81 (t, J=12.6Hz, 1H), 7.07 (d, J= 8.2Hz, 2H), 7.18-7.24 (m, 2H), 7.32-7.40 (m, 4H), 7.85 (t, J=13.1Hz, 1H), 7.90 (t, J=13.1Hz, 1H). HRMS (ESI) calcd for C ３７ H ４８ N ３ O ４ S ([M] ＋ )630.3360;found 630.3367.

[0085] (Synthesis of Compound 26) Compound 25 (38 mg, 54 μmol) and p-toluenesulfonic acid monohydrate (0.52 g, 2.8 mmol) were added to a sample tube, and the mixture was dissolved in acetone (5 mL) and water (5 mL) and stirred at room temperature for 4.5 hours. After washing once with a saturated aqueous solution of sodium bicarbonate, the organic layer was washed once with saturated saline. The organic layer was then washed with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ The residue was purified with a 20:1 mixture of hexane and MeOH (21 mg, 31 μmol, yield 58%, blue solid) to give Compound 26.

[0086] Compound 26 a blue solid (yield 58%); １ H NMR (400MHz, CDCl ３, 25℃) δ1.68 (s, 6H), 1.69 (s, 6H), 2.03-2.11 (m, 2H), 2.37 (s, 3H), 3.03-2.96 (m , 4H), 3.93 (d, J = 4.9Hz, 2H), 4.01 (t, J = 7.6Hz, 2H), 4.53 (t, J = 8.5Hz, 2H), 6.15 ( d, J = 12.5Hz, 1H), 6.81-6.84 (m, 2H), 7.05 (d, J = 8.0Hz, 1H), 7.18-7.25 (m, 2H), 7.33-7.42 (m, 5H), 7.80 (t, J=12.8Hz, 1H), 7.89 (t, J=12.8Hz, 1H), 9.54 (s, 1H). HRMS (ESI) calcd for C ３５ H ４２ N ３ O ３ S ([M] ＋ ) 584.2941; found 584.2938.

[0087] (Synthesis of Compound C5Namide-A) Compound 26 (1.8 mg, 2.7 μmol) was dissolved in MeOH (4 mL) in a sample tube, and the solution was heated at room temperature under light shielding. ２ CO ３ (0.90 mg, 6.5 μmol) was added. After 3 hours, the reaction mixture was ２ Cl ２ The organic layer was diluted with MgSO and washed twice with saturated aqueous sodium bicarbonate. ４ The solvent was evaporated under reduced pressure to obtain compound C5Namide-A (1.0 mg, 1.9 μmol, yield 69%).

[0088] Compound C5Namide-A was a greenish blue solid (yield 69%); HRMS (ESI) calculation for C ３３ H ４０ N ３ O ２ S ([M+H] ＋ ) 547.2836; found 542.2834.

[0089] Example 6 (Synthesis of C7NC5-A)

[0090] (Synthesis of Compound 27) Compound 4 (1.9 g, 4.7 mmol), glutaconaldehyde hydrochloride (0.43 g, 2.8 mmol), and Ac were placed in a 100 mL flask. ２ 0 (20 mL) was added and the reaction was carried out at 100°C for 2.5 hours. The reaction solution was cooled to 0°C with Et ２ The reaction vessel was poured into 200 mL of HCl (500 mL). ２ Cl ２ and washed with ice-cold Et ２ The solution was cooled to -20°C overnight, filtered through a Kiriyama funnel, and ２ The extract was washed with O. The extract was dried under reduced pressure to give Compound 27 (2.0 g, crude yield 70%).

[0091] (Synthesis of Compound 28) In a nitrogen atmosphere, compound 27 (0.13 g) was dissolved in dry pyridine (10 mL) in a 100 mL two-necked flask. Compound 7 (0.24 g, 0.40 mmol) was added thereto at room temperature. The reaction was carried out at 40°C for 2 hours. The solvent was distilled off under reduced pressure in the dark. The residue was purified by silica gel chromatography (CH ２ Cl ２ :MeOH=20:1) to obtain Compound 28 (0.16 g, 0.23 mmol, 69%, greenish blue solid).

[0092] Compound 28 A greenish blue solid (yield 69%); １ H NMR (400MHz, CDCl ３, 25℃) δ1.51-1.52 (m, 4H), 1.56-1.63 (m, 2H), 1.68 (s, 12H), 1.78-1.86 (m, 2H), 2.04-2.11 (m , 2H), 2.35 (s, 3H), 3.04 (t, J = 7.1Hz, 2H), 3.67 (t, J = 6.2Hz, 2H), 4.07-4.09 (m, 4H), 6.31 (d, J = 13.7Hz, 1H), 6.39 (d, J = 15.1Hz, 1H), 6.72-6.83 (m, 2H), 7.06 (d, J = 7.8Hz, 1H), 7.10 (d, J = 8.2Hz, 1H), 7.14-7.23 (m, 2H), 7.30-7.38 (m, 4H), 7.60 (t, J=14.0Hz, 1H), 7.73-7.85 (m, 2H). HRMS (ESI) calcd for C ３８ H ４９ N ２ O ２ S ([M] ＋ ) 597.3509, found 597.3510.

[0093] (Synthesis of Compound C7NC5-A) Under a nitrogen atmosphere, N,N'-dicyclohexylcarbodiimide (DCC, 36 mg, 0.17 mmol), trifluoroacetic acid (TFA, 2.9 μL, 26 μmol), and dry DMSO (3 mL) were placed in a 50 mL two-neck flask and stirred at room temperature for 1 hour. Compound 28 (38 mg, 53 μmol) was added to dry CH ２ Cl ２ (10 mL) was added and reacted for 21 hours. After washing once with saturated sodium bicarbonate aqueous solution, the organic layer was washed once with saturated brine. The organic layer was washed with MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (CH ２ Cl ２ Impurities were removed with HCl (MeOH = 20:1) to give compound 29.

[0094] Compound 29 A greenish blue solid; HRMS (ESI) calculation for C ３８ H ４７ N ２ O ２ S ([M] ＋) 595.3353, found 595.3356.

[0095] Compound 29 was dissolved in MeOH (10 mL) in a sample tube, and K ２ CO ３ After 50 minutes, the reaction mixture was ２ Cl ２ The organic layer was diluted with MgSO 4 and washed once with water, and then washed once with saturated brine. ４ The solvent was evaporated under reduced pressure to obtain compound C7NC5-A.

[0096] Compound C7NC5-A A green solid; HRMS (ESI) calcd for C ３４ H ４３ N ２ OS ([M+H] ＋ ) 553.3247, found 553.3249.

[0097] The compounds synthesized in Examples 1 to 5 were evaluated as follows.

[0098] <Ultraviolet-visible absorption spectrum, fluorescence spectrum> The ultraviolet-visible absorption spectrum was measured using a UH-5300 spectrophotometer (Hitachi, Ltd.), and the fluorescence emission spectrum was measured using an RF-6000 (Shimadzu Corporation).

[0099] The compounds synthesized in Examples 1, 2, and 3 (C5NC5-A, C5NC4-A, C5NC6-A) were weighed using a precision balance and dissolved in DMSO to prepare a dye solution (0.5 mM). This dye solution (20 μL) was dissolved in a buffer solution (1980 μL) to prepare a sample solution (5.0 μM). The absorption and fluorescence spectra were measured by varying the pH. The results are shown in Figures 1-1(a) and 1-1(b) and 1-2(c) and 1-2(d).

[0100] C5NC5-A has absorption peaks at 300-400 nm and 500-700 nm. The former is a closed-ring compound in which the nucleophilic mercapto group is intramolecularly cyclized, while the latter is a ring-open compound in which the nucleophilic mercapto group is eliminated, resulting in a luminescent pi-conjugated structure (see the formula below).

[0101]

[0102] The absorption peak between 300 and 400 nm increased with increasing pH, while the peak between 500 and 700 nm decreased with increasing pH. The fluorescence spectrum correlated with the absorption spectrum between 500 and 700 nm, and the fluorescence intensity (emission maximum at 662 nm) decreased with increasing pH.

[0103] The proportion of open ring forms was evaluated at pH 7.6, which is close to physiological conditions, using fluorescence spectroscopy. ｏ is defined by the following formula: ｏ =F ｐＨ７．６ / F ｍａｘ Here, F ｐＨ７．６ is the luminescence intensity at pH 7.6, F ｍａｘ is the maximum fluorescence intensity obtained under low pH conditions. ｏ were 0.088, 0.037, and 0.081, respectively. ｏ was 0.16 (M. Oe, K. Suzuki, K. Miki, H. Mu, K. Ohe, ChemPlusChem 2022, 87, e220200319.), indicating that C5NC4-A, C5NC5-A, and C5NC6-A have the advantage of weaker background luminescence than C5S-A (n=2) represented by the above formula.

[0104]

[0105] Next, C5NC4-C and C5NC5-C, which are the expected products of the enzymatic reaction of C5NC4-A and C5NC5-A, were synthesized, and R ｏ The R of C5NC4-C and C5NC5-C was evaluated from the fluorescence spectrum intensity. ｏ were calculated to be 0.96 and 0.81, respectively. These results suggest that C5NC4-A and C5NC5-A are mainly composed of closed ring structures before the enzyme response and show almost no fluorescence, whereas C5NC4-C and C5NC5-C, which are produced after the enzyme response, are mostly open ring structures and show strong fluorescence.

[0106] <Enzyme responsiveness experiment> ALDH1A1 was dissolved in 0.1 M phosphate buffer (pH 7.6) to prepare an ALDH1A1 solution. ＋ A DMSO solution of C5NC4-A or C5NC5-A (20 μL, 0.5 mM) was mixed with phosphate buffer (1780 μL) containing ALDH1A1. After stirring the mixture at 37°C for 15 minutes, ALDH1A1 solution (200 μL, 500 nM) was added and the change in fluorescence intensity over time was measured. The experimental results are shown in Figure 2-1(a) and Figure 2-2(c).

[0107] ALDH1A3 was dissolved in 0.1 M phosphate buffer (pH 7.6) to prepare an ALDH1A3 solution. ＋ A DMSO solution of C5NC4-A or C5NC5-A (20 μL, 0.5 mM) was mixed with phosphate buffer (1780 μL) containing ALDH1A3. After stirring the mixture at 37°C for 15 minutes, ALDH1A3 solution (200 μL, 500 nM) was added and the change in fluorescence intensity over time was measured. The experimental results are shown in Figure 2-1(b) and Figure 2-2(d).

[0108] Potassium chloride, DTT, NAD ＋ A DMSO solution (20 μL, 0.5 mM) of C5NC4-A, C5NC5-A, C5NC6-A, C5Namide-A, or known C5S-A or C5NBn-A was mixed with phosphate buffer (1780 μL) containing ALDH1A3. After stirring for 15 minutes at 37°C, an ALDH1A3 solution (200 μL, 500 nM) was added and the mixture was incubated at 37°C for 1 hour, after which the fluorescence intensity was measured. The experimental results are shown in Figure 6. All probes showed responsiveness to ALDH1A3, but C5NC5-A was found to respond most sensitively to ALDH1A3.

[0109] Potassium chloride, DTT, NAD ＋ or nicotinamide adenine dinucleotide phosphate (NADP ＋A DMSO solution of C5NC5-A (20 μL, 0.5 mM) was mixed with phosphate buffer (1780 μL) containing potassium chloride, DTT, and NAD. After stirring this mixture for 15 minutes at 37°C, ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH3A1, or ALDH7A1 solution (200 μL, 500 nM) was added and incubated at 37°C for 1 hour, after which the fluorescence intensity was measured. ＋ or NADP ＋ The final concentrations of ALDH, ALDH, and C5NC5-A were 100 mM, 2 mM, 1 mM, 50 nM, and 5.0 μM, respectively. ＋ In the case of ALDH3A1, NADP ＋ The experimental results are shown in Figure 7.

[0110] To compare the ALDH1A1 and ALDH1A3 responsiveness of C5NC4-A and C5NC5-A, the enzymes were applied and the time course of luminescence was monitored (Figures 2-1 and 2-2). The fluorescence intensity ratios were calculated based on the fluorescence intensity at 662 nm before enzyme application (Figure 3). When ALDH1A1 and ALDH1A3 were applied to C5NC4-A, the fluorescence intensity ratios increased 10-fold and 9-fold, respectively, after 3 hours. Without ALDH application, the fluorescence intensity ratio was 0.73, demonstrating that C5NC4-A functions as a turn-on fluorescent probe responsive to both ALDH1A1 and ALDH1A3. When ALDH1A1 and ALDH1A3 were applied to C5NC5-A, the fluorescence intensity ratios increased 24-fold and 18-fold, respectively, after 3 hours. When ALDH was not applied, the fluorescence intensity ratio was 0.79, indicating that C5NC5-A functions as a turn-on fluorescent probe similar to C5NC4-A. However, since the fluorescence intensity ratio was higher for C5NC5-A, it is believed that C5NC5-A can detect enzyme activity with better contrast. Furthermore, when C5NBn-A was applied to ALDH1A1 and ALDH1A3, the fluorescence intensity ratios were 2.9 and 1.2 times, respectively, after 3 hours. This indicates that both C5NC4-A and C5NC5-A can distinguish the enzyme activity of both ALDH1A1 and ALDH1A3 with good detection sensitivity.

[0111] <Cytotoxicity experiment> Pancreatic cancer cell SUIT-2 was placed in a 96-well plate at 100 μL (5.0 × 10 ５ C5NC5-A was dissolved in DMSO and diluted to 1, 10, 20, or 50 μM (DMSO content 1%) with RPMI-1640 medium containing phenol red and FBS. The medium was removed, and 100 μL of C5NC5-A solution (1, 10, 20, or 50 μM) was added to the cells. The cells were incubated in 5% CO ２The cells were cultured for 6 hours at 37°C under air containing phenol red. The C5NC5-A solution was then removed, and the cells were washed three times with PBS (100 μL). 100 μL of Cell Counting Kit-8 (Dojindo Laboratories) diluted 10-fold with RPMI-1640 medium containing phenol red and FBS was added to the cells, and the cells were cultured for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability under each condition was calculated. The experimental results are shown in Figure 8.

[0112] <Staining experiment of gastric cancer cell MNK1> A staining test was performed on gastric cancer cell MKN1, which has low expression of ALDH1A1 and high expression of ALDH1A3. MKN1 cells (300 μL, 5.0 × 10 ５ C5S-A or C5NC5-A was dissolved in DMSO and diluted to 1 μM (DMSO content 0.1%) with RPMI-1640 medium containing no phenol red or FBS. 150 μL of C5S-A or C5NC5-A solution was applied to the cells, and the cells were incubated in an incubator (5% CO ２ The cells were cultured at 4°C (37°C) for 20 minutes. Then, the cells were observed using a confocal laser microscope. The laser excitation wavelength was 633 nm, and the detection wavelength was 647-759 nm. The experimental results using C5NC5-A are shown in Figure 9(a), and the experimental results using C5S-A are shown in Figure 9(b) (left: fluorescent image, right: superimposition of bright-field image and fluorescent image).

[0113] To evaluate the fluorescence intensity of CSCs in confocal laser microscope images, the fluorescence intensity F per any luminescent cell was calculated. ＣＳＣ and the fluorescence intensity F per any non-luminescent cell ＮＣＣ was measured using ZEN 2011 software (Carl Zeiss). The experimental results are shown in Figure 9(c). The ratio of FCSC to FLNCC (F ＣＳＣ / F ＮＣＣ) was calculated to be 8.59±3.3 for C5S-A and 16.0±4.6 for C5NC5-A, and it can be said that C5NC5-A is more suitable for detecting cancer stem cells with high ALDH1A3 expression levels than C5S-A, which is suitable for detecting ALDH1A1.

[0114] <Staining experiment of pancreatic cancer cells SUIT-2> Pancreatic cancer cells SUIT-2, which have been confirmed to contain cancer stem cells with high ALDH1A1 activity by staining tests using C5S-A and ALDEFLUOR, were selected and stained with C5NC5-A (Figure 4(a)). Confocal laser microscope observation revealed strong red luminescence from some cells. When C5NC5-A was applied to SUIT-2 that had been treated with disulfiram, an ALDH inhibitor, for 2 hours, no luminescence was observed from the cells. This suggests that C5NC5-A detects intracellular ALDH activity. Next, SUIT-2 was stained with C5NC5-A and ALDEFLUOR (Figure 4(b)). It was confirmed that the green luminescence of ALDEFLUOR and the red luminescence of C5NC5-A overlapped. This confirmed that C5NC5-A functions as a molecular probe capable of detecting cancer stem cells in cancer cells.

[0115] When SUIT-2 cells were stained with C5S-A, the fluorescence intensity ratio (F ＣＳＣ / F ＮＣＣ , F ＣＳＣ and F ＮＣＣ The F (luminescence intensity per unit area) of cancer stem cells and other cancer cells was 3.7 times higher (M. Oe, K. Suzuki, K. Miki, H. Mu, K. Ohe, Chem Plus Chem 2022, 87, e220200319.). ＣＳＣ / F ＮＣＣ was 10.4 times higher, indicating that C5NC5-A is a molecular probe capable of detecting cancer stem cells with better contrast than C5S-A (n=2).

[0116] <Evaluation of ALDH expression levels> In addition to SUIT-2, gastric cancer cells KATO-III and MKN1 were stained with C3S-A and C5NC5-A, respectively, as shown in the formula below, and observed under a confocal laser microscope (Figure 5). In all cells, the green emission of C3S-A and the red emission of C5NC5-A showed good overlap. This demonstrated that C5NC5-A can be applied to cell groups other than SUIT-2.

[0117] SUIT-2 cells (300 μL, 9.0 × 10 cells / well) were added to an 8-well dish (μ-Slide 8 Well ibiTreat, Ibidi, Germany). ５ C5NC5-A was dissolved in DMSO and diluted to 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μM (DMSO content: 0.1%) with RPMI-1640 medium containing no phenol red or FBS. 150 μL of C5NC5-A solution at each concentration was applied to the cells, and the cells were incubated in an incubator (5% CO ２ The cells were cultured at 47°C (37°C) for 1 hour. After that, they were observed using a confocal laser microscope. The laser excitation wavelength was 633 nm, and the detection wavelength was 647 to 759 nm. The experimental results are shown in Figures 10(a) to 10(g).

[0118] To evaluate the fluorescence intensity of CSCs in confocal laser microscope images, the fluorescence intensity F per any luminescent cell was calculated. ＣＳＣ and the fluorescence intensity F per any non-luminescent cell ＮＣＣ was measured using ZEN 2011 software (Carl Zeiss). The experimental results are shown in Figure 10(h). ＣＳＣ / F ＮＣＣ The ratio was more than 10 times at 0.05-0.5 μM. When C5S-A was applied at 1 μM, F ＣＳＣ / F ＮＣＣ The ratio is about four times, and the contrast is greatly improved. It can also be said that C5NC5-A provides an image with good contrast even at lower densities.

[0119]

[0120] Fluorescence intensity F in cancer stem cells ＣＳＣis defined by the following formula: ＣＳＣ = F-F ｂｌａｎｋ where F and F ｂｌａｎｋ is the amount of luminescence of any luminescent cell and any non-luminescent cell, and is the green luminescence F obtained from the observation results of a confocal laser microscope. ＣＳＣ，ｇｒｅｅｎ and red emitting F ＣＳＣ，ｒｅｄ were 1749 and 2939 in SUIT-2, 8222 and 7843 in KATO-III, and 1738 and 5972 in MKN1.

[0121] The luminescence intensity in cells is considered to be proportional to the "expression level of ALDH" and the "response of the probe to ALDH." We considered that the "response of the probe to ALDH" could be evaluated by the increase in fluorescence intensity when the enzyme was allowed to act. When ALDH1A1 and ALDH1A3 were allowed to act on C5NC5-A, the fluorescence intensity increased by 13.9-fold and 11.0-fold, respectively, so the "response of the probe to ALDH1A1 and ALDH1A3" was determined to be 13.9 and 11.0, respectively. Since the expression levels of ALDH1A1 and ALDH1A3 in KATO-III (Cancer Cell Line Encyclopedia) were 6.607 and 0.824, the F observed in KATO-III was 13.9 and 11.0, respectively. ＣＳＣ，ｒｅｄ The breakdown of 7843 is considered to be: Amount of luminescence due to ALDH1A1: 7843 x (6.607 x 13.9) / (6.607 x 13.9 + 0.824 x 11.0) = 7145 Amount of luminescence due to ALDH1A3: 7843 x (0.824 x 11.0) / (6.607 x 13.9 + 0.824 x 11.0) = 698. Since C3S-A responds only to ALDH1A1, the green luminescence can be said to be luminescence due to ALDH1A1. Since the ratio of green luminescence to red luminescence due to ALDH1A1 is thought to be independent of cell type, the ratio (8222 / 7145) in KATO-III is also considered to be valid in SUIT-2. From this, the red luminescence caused by ALDH1A1 in SUIT-2 is calculated to be 1519, and the red luminescence caused by ALDH1A3 is found to be 1420 (=2929-1519).

[0122] Therefore, the expression ratio of ALDH1A1 to ALDH1A3 in SUIT-2 was calculated as (1519 / 13.9) / (1420 / 11.0) = 0.84. This ratio in SUIT-2 was 0.394 (Cancer Cell Line Encyclopedia), which showed relatively good agreement with the literature value. This indicates the possibility that co-staining with C5NC5-A and C3S-A may enable evaluation of the expression ratio of ALDH1A1 and ALDH1A3 in unknown cancer cells based on the luminescence intensity of cancer cells with known ALDH1A1 and ALDH1A3 expression levels. Furthermore, the expression ratio of ALDH1A1 to ALDH1A3 in MKN1 was calculated based on the luminescence intensity of KATO-III and was found to be 0.265. Since the literature value was 0.074 (Cancer Cell Line Encyclopedia), this also showed good agreement. Although it cannot be said to be an accurate evaluation of the expression ratio, it is possible to evaluate the amount, and therefore it is thought to be effective as a means for evaluating the expression ratio of ALDH1A1 to ALDH1A3 in unknown cancer cells.

[0123] <Flow cytometer analysis> SUIT-2 cells were placed in a 24-well plate in 1 mL (5.0 × 10 ５ The cells were seeded at 1000 x 1000 cells / mL and left to stand for 24 hours. The inhibitor disulfiram was dissolved in DMSO and diluted with RPMI-1640 medium containing phenol red and FBS to prepare a solution (0.30 mM, DMSO content 1%). The medium was removed from the wells, and the prepared disulfiram solution (1 mL) was added to the wells. The cells were incubated in a 5% CO atmosphere. ２ The cells were cultured for 2 hours at 37°C under an atmosphere containing 0.1% CO. The disulfiram solution was removed, and the cells were washed once with PBS (500 μL). When no inhibitor was used, a medium containing no disulfiram was used instead of the disulfiram solution. C5NC5-A was dissolved in DMSO and diluted with RPMI-1640 medium containing no phenol red or FBS to prepare a solution (1 μM, DMSO content 0.1%). The medium was removed, and the prepared solution (500 μM) was applied, followed by incubation at 37°C under 5% CO. ２The cells were cultured at 37°C for 20 minutes in air containing ethanol. The C5NC5-A solution was then removed, and the cells were washed once with PBS (500 μL). Trypsin-1 mM EDTA (200 μL) was added, and the cells were incubated at 37°C for 3 minutes. The cells were then suspended in RPMI-1640 medium (800 μL) containing phenol red and FBS. The cells were precipitated by centrifugation at 4°C, 3000 rpm, for 5 minutes, the supernatant was removed, and PBS solution (1000 μL) was added and suspended. The cells were again precipitated by centrifugation at 4°C, 3000 rpm, for 5 minutes, the supernatant was removed, and PBS solution (1000 μL) was added and resuspended to prepare the sample. A flow cytometer (BD Accuri™ C6 Plus, Becton Dickinson Japan) was used to analyze the samples. A red laser (wavelength 640 nm) was used as excitation light, and the detection wavelength was 675±12.5 nm. 10,000 events were analyzed for each sample. The experimental results are shown in Figure 11. In the figure, gate % indicates the percentage of cells showing a fluorescence intensity stronger than a given fluorescence intensity.

[0124] To evaluate the gate %, which indicates the percentage of cells showing a fluorescence intensity higher than a given fluorescence intensity, the gate % was set to 1.7% for the sample treated with the inhibitor, and the gate % for the sample not treated with the inhibitor was calculated to be 20.1%. This indicates that the inhibitor significantly suppresses the luminescence intensity. These results indicate that cell sorting by fluorescence-activated cell sorting using a flow cytometer is possible.

[0125] <Short-term staining test> 300 μL (5.0 × 10) of SUIT-2 cells were added to an 8-well dish (μ-Slide 8 Well ibiTreat, Ibidi, Germany). ５ C5NC5-A was dissolved in DMSO and diluted to 1 μM (DMSO content 0.1%) with RPMI-1640 medium containing no phenol red or FBS. 150 μL of C5NC5-A solution was applied to the cells, and the cells were incubated in an incubator (5% CO ２The cells were cultured at 47°C (37°C) for 5, 20, and 60 minutes. Then, observations were made using a confocal laser microscope. The laser excitation wavelength was 633 nm, and the detection wavelength was 647-759 nm. The experimental results for a 5-minute culture time are shown in Figure 12(a), 20-minute culture time in (b), and 60-minute culture time in (c) (left: fluorescent image, right: superimposed fluorescent and bright-field images).

[0126] To evaluate the fluorescence intensity of CSCs in confocal laser microscope images, the fluorescence intensity F per any luminescent cell was calculated. ＣＳＣ and the fluorescence intensity F per any non-luminescent cell ＮＣＣ was measured using ZEN 2011 software (Carl Zeiss). The experimental results are shown in Figure 12(d). ＣＳＣ / F ＮＣＣ The ratio was approximately 5 to 8 times. This suggests that C5NC5-A is suitable for high-contrast visualization of CSCs even with a staining time of 5 minutes, and is a drug that can contribute to rapid diagnosis.

[0127] The compounds of the present invention enable the preparation of molecular probes that emit light with high sensitivity to aldehyde dehydratase, particularly ALDH1A1 and ALDH1A3, and can easily detect cancer stem cells, which is expected to be highly effective in the medical field of cancer diagnosis and treatment.

Claims

1. The following formula (In the formula, Ar １ and Ar ２ are each an aromatic ring and may be the same or different. X is an oxygen atom, a nitrogen atom, a sulfur atom or a selenium atom. R １ and R ２ are each alkylene having one or more carbon atoms or arylene which may contain a heteroatom, and R １ and R ２ The total number of carbon atoms is 2 to 10. Y is —NHCO—, —COO—, —COS—, —O—, —S—, or —NR ３ - (R ３ is an alkyl group having 1 to 5 carbon atoms, or —C═N—, p is an integer of 2 to 4, q is an integer of 0 to 4, and r is 0 or 1.

2. The Ar １ and Ar ２ are expressed as follows: ,or, (wherein Z is a halogeno group, -SO ３ Na, -SO ３ H or -COOR ４ and R ４ is H or an alkyl group having 1 to 6 carbon atoms.

3. The following formula (wherein n is an integer of 2 to 10).

4. A fluorescent reagent comprising the compound according to claim 1 or 2.

5. The fluorescent reagent according to claim 4, which is used for detecting cellular ALDH1A1 activity and ALDH1A3 activity.

6. A method for detecting ALDH1A1 activity or ALDH1A3 activity in cells, comprising the steps of: administering the fluorescent reagent according to claim 4 to cells; and irradiating the cells with light having a wavelength of 300 to 1300 nm and detecting the fluorescence of the fluorescent reagent.

7. The detection method according to claim 6, wherein the cells are detected in vivo, in vitro or ex vivo.

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