Compound and reagent for detecting sialidase

Abstract

To provide a detection reagent hardly causing noise due to generation of nonspecific fluorescence, and capable of detecting sialidase with higher accuracy.SOLUTION: The present invention relates to a compound represented by formula (I) or a salt thereof, or a solvate thereof. In the formula (I), R1 represents a halogen atom, and R2 represents an alkyl group having 5 to 11 carbon atoms or a hydrocarbyl group having 5 to 11 carbon atoms and having one or more unsaturated bonds. ] SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel compound represented by formula (I) and a reagent containing this compound for detecting sialidase or viruses having sialidase. [Background technology]

[0002] The present inventors have developed "BTP3-Neu5Ac" and "mC9-yne-BTP-Neu5Ac (BTP9-Neu5Ac)," shown in the chemical formulas below, as detection reagents capable of fluorescently detecting sialidase, a hydrolase that releases sialic acid from sugar chains (see Non-Patent Document 1 and Patent Document 1). When sialic acid (Neu5Ac) in the substrate structure of these detection reagents is cleaved by sialidase activity, an insoluble fluorescent benzothiazolylphenol (BTP) derivative is generated, which emits fluorescence and deposits in the surrounding area. Therefore, these detection reagents are used for fluorescent imaging of virus-infected cells that exhibit sialidase activity, as well as for measuring sialidase activity and detecting viruses.

[0003] [ka]

[0004] Of these, "BTP3-Neu5Ac" tends to diffuse the fluorescent staining area due to BTP3 when used for fluorescent imaging of virus-infected cells, resulting in unclear fluorescent staining images and poor localized staining performance. Therefore, "BTP9-Neu5Ac" was developed as a detection reagent that can obtain clear fluorescent staining images in fluorescent imaging.

[0005] In human lung cancer-derived A549 cells infected with influenza A virus, newly synthesized neuraminidase (NA) from the viral gene undergoes glycosylation, resulting in transient localization of neuraminidase in the Golgi apparatus 7 hours after infection. Using "BTP9-Neu5Ac," which produces clear fluorescent staining images, the enzymatic activity of sialidase localized in the Golgi apparatus within infected cells was visualized for the first time in the world (see Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6493964 [Non-patent literature]

[0007] [Non-Patent Document 1] Takahashi, Tadanori, Kurebayashi, Yuki, Otsubo, Tadamune, Ikeda, Kiyoshi, Minami, Akira, and Suzuki, Takashi, "Fluorescence imaging of virus-infected cells using sialidase," Analytical Chemistry, 2016, Vol. 65, No. 12, pp. 689-701 [Non-patent document 2] Journal of Virological Methods, January 2024, Vol. 323, 114838 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the present inventors noticed that when performing fluorescence imaging of virus-infected cells using "BTP9-Neu5Ac," multiple granular fluorescent staining images sometimes appeared. Because these granular fluorescent staining images appeared in locations that did not match the immunostaining images obtained with anti-sialidase antibodies, they were presumed to be nonspecific fluorescent images (noise) unrelated to sialidase activity. Therefore, it was sometimes difficult to determine the localization of sialidase within virus-infected cells based solely on the results of fluorescence imaging.

[0009] Therefore, the present invention has been made in consideration of the above points, and its object is to provide a detection reagent that is less likely to generate noise due to the generation of nonspecific fluorescence and can detect sialidase with higher accuracy.

[0010] Another object of the present invention is to provide a reagent for detecting sialidase-containing viruses or sialidase, which, when used in fluorescence imaging, is less likely to produce nonspecific fluorescent staining images (noise) and can provide clear fluorescent staining images. [Means for solving the problem]

[0011] The present inventors focused on the structure of benzothiazolylphenol (BTP) derivatives that function as fluorescent substances and discovered a novel BTP derivative-sialic acid (Neu5Ac) compound that does not produce nonspecific granular fluorescent staining images during fluorescence imaging, but rather produces specific, clear fluorescent staining images. Based on this finding, the present invention was completed.

[0012] The present invention, which solves the above-mentioned problems, is a compound represented by the following formula (I), or a salt thereof, or a solvate thereof, wherein in formula (I), R 1 is a halogen atom, R 2 represents an alkyl group having 5 to 11 carbon atoms or a hydrocarbon group having 5 to 11 carbon atoms and one or more unsaturated bonds.

[0013] [ka]

[0014] The compound represented by formula (I) is hydrolyzed by sialidase activity into a benzothiazolylphenol (BTP) derivative and Neu5Ac (sialic acid), generating an insoluble fluorescent BTP derivative, which emits fluorescence and accumulates at the site. The compound represented by formula (I) is less likely to generate nonspecific fluorescence, reducing noise due to nonspecific fluorescence, enabling highly accurate detection of sialidase-containing viruses or sialidase. Furthermore, the BTP derivative generated by sialidase activity in a sample remains at the site without diffusing, enabling the generation of clear fluorescent stained images when used in fluorescence imaging.

[0015] In addition, in the compound according to the present invention, R 1 is a chlorine atom or a bromine atom, and R in formula (I) 2 is also preferably a linear alkyl group having 6 to 10 carbon atoms. This makes it possible to obtain a compound that constitutes a detection reagent that is less likely to generate noise due to the generation of nonspecific fluorescence and that can detect sialidase-containing viruses or sialidase with higher accuracy. Furthermore, since this compound is less likely to generate nonspecific fluorescent staining images (noise) and can obtain clear fluorescent staining images, it can be suitably used as a fluorescent imaging probe.

[0016] In order to solve the above problems, the detection reagent of the present invention is a reagent for detecting a virus having a sialidase or a sialidase, and contains a compound represented by the following formula (I) or a salt thereof, or a solvate thereof, wherein in formula (I), R 1 is a halogen atom, R 2 represents an alkyl group having 5 to 11 carbon atoms or a hydrocarbon group having 5 to 11 carbon atoms and one or more unsaturated bonds.

[0017] [ka]

[0018] The above-described compound contained in the detection reagent of the present invention is hydrolyzed by sialidase activity into a benzothiazolylphenol (BTP) derivative and Neu5Ac (sialic acid), thereby generating an insoluble fluorescent BTP derivative, which emits fluorescence and accumulates at the site. The compound represented by formula (I) is less likely to generate nonspecific fluorescence and reduces noise due to nonspecific fluorescence, allowing for highly accurate detection of sialidase-containing viruses or sialidase. Therefore, a detection reagent is obtained that is less likely to generate noise due to nonspecific fluorescence and allows for highly accurate detection of sialidase. Furthermore, the BTP derivative generated by sialidase activity in a sample remains at the site without diffusing, allowing for clear fluorescent staining images to be obtained when used in fluorescence imaging.

[0019] In addition, in the detection reagent according to the present invention, R 1 is a chlorine atom or a bromine atom, and R in formula (I) 2 is also preferably a linear alkyl group having 6 to 10 carbon atoms. This makes it possible to obtain a detection reagent that is less likely to generate noise due to nonspecific fluorescence and that can detect sialidase-containing viruses or sialidase with higher accuracy. Furthermore, when this detection reagent is used for fluorescent imaging of virus-infected cells, etc., it is possible to obtain a clear fluorescent stained image with less nonspecific fluorescent stained images (noise).

[0020] Furthermore, in the detection reagent of the present invention, it is also preferable that the virus to be detected is influenza A virus, and the sialidase to be detected is sialidase of influenza A virus. This allows the selection of a preferred virus species or type of sialidase to be detected.

[0021] The detection reagent of the present invention is also preferably a fluorescent imaging probe for detecting a virus having sialidase or a sialidase, which is less likely to produce nonspecific fluorescent staining images (noise) and can provide clear fluorescent staining images, and therefore can be suitably used as a fluorescent imaging probe.

[0022] Furthermore, the detection method of the present invention is a method for detecting sialidase in virus-infected cells, which is a method for fluorescent imaging of virus-infected cells using the above-described detection reagent. The above-described detection reagent of the present invention can be easily transferred into virus-infected cells, and a free BTP derivative is generated by sialidase activity in the infected cells. The free BTP derivative emits fluorescence, and this fluorescence can be used as a signal to detect sialidase. Furthermore, the detection reagent of the present invention is less likely to produce nonspecific fluorescent staining images, and a clear fluorescent staining image can be obtained, making it possible to detect the localization of sialidase in virus-infected cells.

[0023] Furthermore, the detection method of the present invention is a method for detecting viruses having sialidase, which is a method for fluorescent imaging of virus-infected cells using the above-mentioned detection reagent. The above-mentioned detection reagent of the present invention generates a free BTP derivative by sialidase activity inside virus-infected cells and / or on the surface of virus cells. Since the free BTP derivative emits fluorescence, viruses possessing sialidase can be detected using this fluorescence as a signal. Furthermore, the detection reagent of the present invention is less likely to produce nonspecific fluorescent staining images and can provide clear fluorescent staining images, allowing for highly accurate detection of sialidase-containing viruses.

[0024] The detection method of the present invention is a method for detecting sialidase, which comprises the step of adding the above-described detection reagent to a sample and determining the presence or absence of sialidase based on the fluorescent substance generated by this step. The above-described detection reagent of the present invention generates a free BTP derivative by the sialidase activity in the sample. The free BTP derivative emits fluorescence, and this fluorescence can be used as a signal to determine the presence or absence of sialidase. Furthermore, the detection reagent of the present invention is less likely to generate nonspecific fluorescence, and noise due to nonspecific fluorescence is reduced, allowing for highly accurate detection of sialidase. [Effects of the Invention]

[0025] According to the present invention, it is possible to provide a virus-containing sialidase or sialidase detection reagent that has the following excellent effects. (1) Because non-specific fluorescence is reduced, local staining is excellent, and sialidase-containing viruses or sialidase can be detected with high accuracy. (2) Since non-specific fluorescent staining images are unlikely to occur and clear fluorescent staining images can be obtained, it can be used as a fluorescent imaging probe for fluorescent imaging of sialidase activity. (3) Because it is possible to detect the localization of sialidase in virus-infected cells, it can be used to search for sialidase inhibitors (antiviral agents) that target sialidase in virus-infected cells. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a photograph showing a fluorescent image obtained when BTP9-Neu5Ac, Compound 21-188b, and Compound 21-189b were used as fluorescent imaging probes in Example 1. [Figure 2] 10 is a photograph showing the results of fluorescent multiplex immunostaining in Example 2 using (a) compound 21-189b and (b) BTP9-Neu5Ac as fluorescent imaging probes. [Figure 3]1 is a photograph showing a fluorescent image obtained when BTP9-Neu5Ac, compound 21-049a, compound 21-049b, and compound 21-049c were used as fluorescent imaging probes in Example 3. [Figure 4] 10 is a photograph showing the results of fluorescent multiplex immunostaining using compound 21-049a as a fluorescent imaging probe in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0027] The novel compound, the reagent for detecting a virus having sialidase or a sialidase, and the detection method using the same according to the present invention will be described in detail below.

[0028] The reagent for detecting sialidase-containing viruses or sialidase according to the present invention contains a compound represented by formula (I): The compound according to the present invention may be a salt, preferably a pharmacologically acceptable salt. The pharmacologically acceptable salt of this compound is not particularly limited as long as it is a salt formed with an acid or a base. Furthermore, this compound or its salt may be a solvate, including, but not limited to, a hydrate or a solvate with an organic solvent such as ethanol.

[0029] [ka]

[0030] In formula (I) representing the above-mentioned compound, R 1 The atom or molecule represented by is a halogen atom. Having a halogen atom at the para-position to sialic acid contributes to reducing non-specific fluorescence and improving the clarity of fluorescent stained images (improving local staining). Examples of halogen atoms include fluorine, chlorine, bromine, and iodine atoms, with chlorine and bromine atoms being preferred from the viewpoint of excellent detection accuracy.

[0031] Next, R 2The atom or molecule represented by is an alkyl group having 5 to 11 carbon atoms or a hydrocarbon group having 5 to 11 carbon atoms and one or more unsaturated bonds. The presence of an ether bond with the alkyl group, i.e., an alkoxy group, at the meta position relative to the sialic acid contributes to reducing nonspecific fluorescence and improving the clarity of fluorescent stained images (improving local staining). Of these, the alkyl group having 5 to 11 carbon atoms may be either a linear or branched alkyl group, and from the viewpoint of reducing nonspecific fluorescence, a linear alkyl group having 5 to 11 carbon atoms is preferred. Specific examples of the linear alkyl group having 5 to 11 carbon atoms include an n-pentyl group (C5), an n-hexyl group (C6), an n-heptyl group (C7), an n-octyl group (C8), an n-nonyl group (C9), an n-decyl group (C10), and an n-undecyl group (C11). Of these, from the viewpoint of excellent detection accuracy, a linear alkyl group having 6 to 10 carbon atoms is more preferred, and a linear alkyl group having 7 to 9 carbon atoms (an n-heptyl group, an n-octyl group, an n-nonyl group) is particularly preferred.

[0032] On the other hand, examples of hydrocarbon groups having 5 to 11 carbon atoms and one or more unsaturated bonds include linear or branched alkenyl groups having 5 to 11 carbon atoms and linear or branched alkynyl groups having 5 to 11 carbon atoms. Of these, linear alkenyl or alkynyl groups are preferred, linear alkenyl or alkynyl groups having 6 to 10 carbon atoms are more preferred, and linear alkenyl or alkynyl groups having 7 to 9 carbon atoms are particularly preferred.

[0033] Specific examples of the compound represented by the above formula (I) include compounds (1) and (2) represented by the following formulae: Formula (1): In formula (I), R 1 = bromine atom, R 2 = n-octyl group N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-bromo-5-octyloxyphenyl]-α-D-neuraminic acid. Formula (2): R in formula (I) 1 = chlorine atom, R 2 = n-octyl group N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-chloro-5-octyloxyphenyl]-α-D-neuraminic acid.

[0034] [ka]

[0035] [ka]

[0036] In addition to the compounds represented by the above formulas (1) and (2), the following compounds can be mentioned as examples: 1 = bromine atom, R 2 As a compound having an n-heptyl group, N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-bromo-5-heptyloxyphenyl]-α-D-neuraminic acid, R in formula (I) 1 = chlorine atom, R 2 As a compound having an n-heptyl group, N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-chloro-5-heptyloxyphenyl]-α-D-neuraminic acid, R in formula (I) 1 = bromine atom, R 2 As a compound having an n-nonyl group, N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-bromo-5-nonyloxyphenyl]-α-D-neuraminic acid, R in formula (I) 1 = chlorine atom, R 2 = As a compound with an n-nonyl group, N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-chloro-5-nonyloxyphenyl]-α-D-neuraminic acid.

[0037] Furthermore, among the compounds represented by formula (I), compounds that are particularly preferred from the standpoint of detection accuracy include N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-bromo-5-octyloxyphenyl]-α-D-neuraminic acid shown in formula (1) and N-acetyl-[2-(benzo[d]thiazol-2-yl)-4-chloro-5-octyloxyphenyl]-α-D-neuraminic acid shown in formula (2).In the examples described below, it has been shown that these compounds are less likely to produce nonspecific fluorescence and produce clear fluorescent staining images.

[0038] The compound represented by formula (I) can be synthesized, for example, by the synthetic route shown in the following scheme. As an example, the 4-position hydroxyl group of known compound a (2,4-dihydroxybenzaldehyde) is protected by allylation and then reacted with 2-aminobenzenethiol to obtain compound c (benzothiazolylphenol derivative). Subsequently, the hydroxyl group of compound c is protected with a methoxymethyl group, and the allyl group is deprotected and the hydroxyl group is alkylated to obtain compound f. Subsequently, the 3-position of compound f is halogenated and the methoxymethyl group is deprotected to synthesize compound g. The compound g obtained is coupled with compound h derived from sialic acid methyl ester to obtain compound i, which is then hydrolyzed to obtain the compound represented by formula (I).

[0039] [ka]

[0040] The detection reagent of the present invention is a sialidase-containing virus or a sialidase detection reagent. Viruses containing sialidase to be detected include influenza virus, human parainfluenza virus, mumps virus, Newcastle disease virus, Sendai virus, etc., and among these, influenza virus is preferred from the viewpoint of excellent detectability, with influenza A virus being particularly accurately detected. The detection reagent of the present invention is capable of histochemical fluorescent imaging of the location of a virus in a virus-infected cell. Therefore, the detection reagent of the present invention can be used not only as a simple fluorescence measurement reagent but also as a fluorescent imaging probe.

[0041] Furthermore, the sialidase to be detected preferably includes viral sialidases of viruses containing sialidase, such as influenza virus, human parainfluenza virus, mumps virus, Newcastle disease virus, and Sendai virus, as described above, as well as animal (mammalian) sialidases and bacterial sialidases (e.g., sialidases derived from Arthrobacter ureafaciens and Salmonella typhimurium). As shown in the Examples below, the detection reagent of the present invention is suitable for detecting viral sialidases, and can particularly detect influenza A virus sialidase with high accuracy. The detection reagent of the present invention enables histochemical fluorescent imaging of the localization of sialidase activity in tissues and cells. Therefore, the detection reagent of the present invention can be used not only as a simple fluorescence measurement reagent but also as a fluorescent imaging probe.

[0042] The detection reagent of the present invention may contain other components in addition to those described above, provided that the effects of the present invention are not impaired. Examples of such components include solvents, pH adjusters, pH buffers, and inorganic salts. The detection reagent of the present invention can be used in a variety of situations, including as a research reagent, clinical diagnostic reagent, and clinical analytical reagent.

[0043] A method for detecting sialidase in virus-infected cells by fluorescence imaging using a detection reagent containing the compound represented by formula (I) according to the present invention is described below. First, a detection reagent containing the compound represented by formula (I) is used as the detection reagent. When this detection reagent is added to a sample, the compound represented by formula (I) is translocated into the virus-infected cells. The sample also includes immobilized virus-infected cells. When sialidase newly synthesized from the viral gene is present in the virus-infected cells, the compound represented by formula (I) is hydrolyzed into "Neu5Ac (sialic acid)" and "free BTP derivative" by the sialidase activity. The free BTP derivative emits fluorescence and deposits at the site of hydrolysis, allowing sialidase in the virus-infected cells to be detected by fluorescence imaging using this fluorescence as a signal. Furthermore, the detection reagent of the present invention is less likely to produce nonspecific fluorescent staining images and can provide clear fluorescent staining images, making it possible to detect the localization of sialidase in virus-infected cells. As shown in the Examples below, sialidase localized in the Golgi apparatus in virus-infected cells can be detected. The reaction concentration of the compound represented by formula (I) varies depending on the sample and the mode of use, but is preferably 5 μM to 100 mM, and more preferably 10 μM to 10 mM. The reaction time depends on the concentration of the compound and the state of the sample to be added, but, for example, when added to a culture medium, it is preferably several minutes to about 1 hour, and more preferably about 5 to 30 minutes. When added to a sample such as an agar medium, it is preferably several hours to about 1 day, and more preferably about 3 to 12 hours.

[0044] This section also describes a method for detecting viruses by fluorescence imaging using a detection reagent containing a compound represented by formula (I) according to the present invention. When a detection reagent containing a compound represented by formula (I) is added to a sample, the compound represented by formula (I) is hydrolyzed into "Neu5Ac (sialic acid)" and a "free BTP derivative" by the sialidase activity inside virus-infected cells and / or on the surface of the virus cells. The free BTP derivative emits fluorescence, and this fluorescence can be used as a signal to detect viruses containing sialidase. Furthermore, the detection reagent according to the present invention is less likely to produce nonspecific fluorescent staining images, and produces clear fluorescent staining images, allowing for highly accurate detection of sialidase-containing viruses. The reaction concentration of the compound represented by formula (I) varies depending on the sample and mode of use, but is preferably 5 μM to 100 mM, and more preferably 10 μM to 10 mM. The reaction time depends on the concentration of the compound and the state of the sample to be added. For example, when the compound is added to a culture medium, the reaction time is preferably several minutes to 1 hour, and more preferably 5 to 30 minutes. When added to a specimen such as an agar medium, the incubation time is preferably from several hours to about one day, and more preferably from about 3 hours to 12 hours.

[0045] A method for detecting sialidase using a detection reagent containing the compound represented by formula (I) according to the present invention will now be described. When a detection reagent containing the compound represented by formula (I) is added to a sample, if sialidase is present in the sample, the compound represented by formula (I) is hydrolyzed into "Neu5Ac (sialic acid)" and a "free BTP derivative" due to the sialidase activity. The free BTP derivative emits fluorescence, and this fluorescence can be used as a signal to detect the presence or absence and activity level of sialidase in the sample. The detection reagent according to the present invention is less likely to generate nonspecific fluorescence, reducing noise due to nonspecific fluorescence and enabling highly accurate detection of sialidase. The reaction concentration of the compound represented by formula (I) varies depending on the sample and mode of use, but is preferably 5 μM to 100 mM, more preferably 10 μM to 10 mM. The reaction time depends on the concentration of the compound and the state of the sample to be added. For example, when the compound is added to a suspension or culture medium, it is preferably several minutes to 1 hour, more preferably 5 to 30 minutes. When added to a specimen such as an agar medium, the incubation time is preferably from several hours to about one day, and more preferably from about 3 hours to 12 hours.

[0046] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0047] [Example 1] 1. Fluorescence imaging of the sialidase activity of influenza A virus neuraminidase (NA) localized in the Golgi apparatus (1) In this example, fluorescent imaging of influenza A virus-infected cells was performed using the following three compounds.

[0048] [ka]

[0049] Compounds 21-189b and 21-188b were synthesized based on the following formula. The compounds (including intermediates) obtained by the synthesis were confirmed to be the target compounds by high-resolution mass spectrometry (HRMS). High-resolution mass spectrometry was performed using an AccuTOF (JMS-T100LC) manufactured by JEOL Ltd., connected to an electrospray ion source (ES) and measured in positive mode.

[0050] [ka]

[0051] [Synthesis of compound 21-189b] Compound 21-189b was synthesized based on the formula shown above, where R 1 = bromine atom, R 2 = n-octyl group. First, sodium bicarbonate (84 g, 1.0 mol) and allyl bromide (48 mL, 0.55 mol) were added to a solution of compound a (2,4-dihydroxybenzaldehyde; 69 g, 0.5 mol) in acetonitrile (600 mL) and refluxed. The reaction was monitored every 18 to 29 hours, and finally 25 mL of allyl bromide and 84 g of sodium bicarbonate were added. The reaction solution was filtered, the salt was washed with acetone, and the combined filtrate was evaporated under reduced pressure. Acetic acid (120 mL) and 2-aminobenzenethiol (53 mL, 0.5 mol) were added to the resulting compound b, and the reaction was continued at 120 °C for 5 hours, followed by cooling. The resulting solid was dispersed in methanol, filtered, and the resulting solid was washed with methanol to obtain compound c (57.9 g) in 41% yield. Compound c HRMS (ESI-TOF): m / z Calcd. for C 16 H 14 NO2S [M+H] + 284.07452; Found. 284.07673.

[0052] Subsequently, a tetrahydrofuran / N,N-dimethylformamide (50 mL / 50 mL) mixture was added to sodium hydride (4.0 g, 2 equivalents, 60% liquid paraffin dispersion), and compound c (14.2 g, 50 mmol) was added portionwise. Subsequently, chloromethyl methyl ether (5.7 mL, 1.5 equivalents) was added, and the mixture was reacted at 70°C for 19 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue (18.94 g) containing compound d was used in the next reaction without purification. Compound d HRMS (ESI-TOF): m / z Calcd. for C 18 H 17 NNaO3S [M+Na] + 350.08268; Found. 350.08445, Calcd. for C 18 H 18 NO3S [M+H] + 328.10074; Found. 328.10237.

[0053] Crude compound d (6.49 g) was added to tetrahydrofuran (70 mL), sodium borohydride (760 mg), and bis(triphenylphosphine)palladium(II) dichloride (140 mg) and reacted at 75°C for 20 hours. The reaction mixture was cooled to room temperature, and methanol was added to remove unreacted sodium borohydride. The mixture was then diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and dried over sodium sulfate. The drying agent was filtered off, and the solvent was evaporated under reduced pressure to give a residue containing compound e (5.84 g), which was used unpurified in the next reaction. Compound e HRMS (ESI-TOF): m / z Calcd. for C 15 H 13 NNaO3S [M+Na] + 310.05138; Found. 310.05048, Calcd. for C 15 H 14 NO3S [M+H] +288.06944; Found. 288.0701.

[0054] Crude compound e (3.3 g, 11.5 mmol) was added to N,N-dimethylformamide (30 mL), octyl 4-methylbenzenesulfonate (4.08 g, 1.25 equivalents), and potassium carbonate (4.75 g, 3.0 equivalents) and reacted at 120 °C for 20 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound f1 (4.07 g) in 89% yield. HRMS (ESI-TOF): m / z Calcd. for C 23 H 29 NNaO3S [M+Na] + 422.17658; Found. 422.17885, Calcd. for C 23 H 30 NO3S [M+H] + 400.19464; Found. 400.19186.

[0055] Compound f1 (1.5 g, 3.75 mmol) was added with N,N-dimethylformamide (20 mL) and N-bromosuccinimide (768 mg, 1.15 equivalents) and reacted at 80°C for 18 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (10 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the mixture was reacted at room temperature for 19 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (10 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the mixture was reacted at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate, neutralized with aqueous potassium carbonate, and then separated. The organic layer was washed with water (three times) and saturated brine (one time) in that order, and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure to give compound g1 (1.48 g) in 91% yield. HRMS (ESI-TOF): m / z Calcd. for C 21 H 25 81 BrNO2S [M+H] + 436.07689; Found. 436.07887, Calcd. for C 21 H 25 79 BrNO2S [M+H] + 434.07894; Found. 434.07948.

[0056] Compound h, which was to be coupled with compound g1, was prepared as shown in the following formula. Specifically, methyl N-acetylneuraminate (1 g) was dissolved in a mixture of acetyl chloride and acetic acid (10 mL / 10 mL) and left overnight. After the solvent was removed by distillation under reduced pressure, toluene was added and the mixture was azeotroped three times, followed by drying under reduced pressure to obtain compound h as a foam. The resulting compound h was used in the subsequent reaction without purification.

[0057] [ka]

[0058] Sodium hydride (80 mg, 2 mmol, 60% liquid paraffin dispersion) was added to a tetrahydrofuran / N,N-dimethylformamide (5 mL / 5 mL) mixture of compound h obtained from methyl N-acetylneuraminate (0.5 g equivalent) and compound g1 (434 mg, 1 mmol), and the mixture was allowed to react at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound i1 (469 mg) in 52% yield. HRMS (ESI-TOF): m / z Calcd. for C 41 H 51 81 BrN2NaO 14 S [M+Na] + 931.21216; Found. 931.21163, Calcd. for C 41 H 51 79 BrN2NaO 14 S [M+Na] + 929.21421; Found. 929.21128.

[0059] Compound i1 (150 mg, 165 μmol) was added to dry methanol (1 mL) and sodium methoxide methanol solution (100 μL, 5.0 mol / L) and reacted at room temperature for 10 minutes. Sodium hydroxide solution (200 μL, 2.5 mol / L) was then added and reacted at room temperature for an additional 12 hours. Acetic acid (70 μL) was added to the reaction mixture, and the mixture was purified using high-performance liquid chromatography to obtain the desired compound 21-189b (65.6 mg) in 54% yield. HRMS (ESI-TOF): m / z Calcd. for C 32 H 41 81 BrN2NaO 10 S [M+Na] + 749.15425; Found. 749.15611, Calcd. for C 32H 41 79 BrN2NaO 10 S [M+Na] + 747.1563; Found. 747.15166, Calcd. for C 32 H 42 81 BrNO 10 S [M+H] + 727.17231; Found. 727.17077, Calcd. for C 32 H 42 79 BrNO 10 S [M+H] + 725.17435; Found. 725.17432.

[0060] [Synthesis of compound 21-188b] Compound 21-188b was synthesized as follows in the same manner as in the synthesis of compound 21-189b described above. 1 = hydrogen atom, R 2 = n-octyl group. In the synthesis route from compound f1 in the above scheme, tetrahydrofuran (10 mL) and concentrated hydrochloric acid (5 mL) were added sequentially to compound f1 (1.0 g, 2.5 mmol) and the reaction was carried out at room temperature for 19 hours. The reaction solution was diluted with ethyl acetate, neutralized with aqueous potassium carbonate solution, and then separated. The organic layer was washed sequentially with water (twice) and saturated brine (once) and dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure to quantitatively obtain compound g2. HRMS (ESI-TOF): m / z Calcd. for C 21 H 26 NO2S [M+H] + 356.16842; Found. 356.16882.

[0061] Sodium hydride (80 mg, 2 mmol, 60% liquid paraffin dispersion) was added to a tetrahydrofuran / N,N-dimethylformamide (5 mL / 5 mL) mixture of compound h obtained from methyl N-acetylneuraminate (0.5 g equivalent) and compound g2 (356 mg, 1 mmol), and the mixture was allowed to react at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound i2 (490 mg) in 59% yield.

[0062] Compound i2 (83 mg, 100 μmol) was added to dry methanol (1 mL) and sodium methoxide methanol solution (100 μL, 5.0 mol / L) and reacted at room temperature for 30 minutes. Sodium hydroxide solution (200 μL, 2.5 mol / L) was then added and reacted at room temperature for an additional 17 hours. Acetic acid (60 μL) was added to the reaction mixture, and the mixture was purified using high-performance liquid chromatography to give the desired compound 21-188b (35.7 mg) in 40% yield.

[0063] [Synthesis of compound BTP9-Neu5Ac] In addition, the compound BTP9-Neu5Ac was synthesized based on the following formula.

[0064] [ka]

[0065] Compound k (2-(benzo[d]thiazol-2-yl)-5-bromophenol; 947 mg) was added portionwise to a suspension of sodium hydride (154 mg, 60% dispersion in liquid paraffin) in tetrahydrofuran (10 mL) and stirred at room temperature for 10 minutes. To the reaction mixture was slowly added an N,N-dimethylformamide solution (10 mL) of compound h obtained from methyl N-acetylneuraminate (1 g) at room temperature, and the mixture was further reacted overnight. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once) and dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound L (1.44 g) in 60% yield. 1 H-NMR (600MHZ, CDCl3) δ 8.34 (d, J = 8.5 Hz, 1 H), 8.09 (d, J = 8.2 Hz, 1 H), 7.94 (d, J = 7.9 Hz, 1 H), 7.71 (d, J = 1.8 Hz, 1 H), 7.51 (ddd, J = 8.2, 7.2, 1.1 Hz, 1 H), 7.43-7.40 (m, 2 H), 5.55 (d, J = 10.0 Hz, 1 H), 5.40 (td, J = 6.5, 3.0 Hz, 1 H), 5.35 (dd, J = 7.0, 1.8 Hz, 1 H), 5.07 (ddd, J = 11.6, 10.4, 4.7 Hz, 1 H), 4.40 (dd, J = 12.4, 3.1 Hz, 1 H), 4.38 (dd, J = 10.7, 1.8 Hz, 1 H), 4.28 (dd, J = 12.5, 6.0 Hz, 1 H), 4.14 (q, J = 10.3 Hz, 1 H), 3.70 (s, 3 H), 2.89 (dd, J = 13.0, 4.7 Hz, 1 H), 2.46 (t, J = 12.4 Hz, 1 H), 2.14 (s, 3 H), 2.11 (s, 3 H), 2.07 (s, 3 H), 2.04 (s, 3 H), 1.92 (s, 3 H). 13C-NMR (CDCl3, 150 MHz) δ 171.1, 170.8, 170.5, 170.3, 170.2, 167.4, 161.8, 152.2, 152.0, 136.1, 131.2, 128.2, 126.5, 125.4, 125.1, 124.8, 123.4, 123.3, 121.5, 101.5, 74.0, 69.8, 68.6, 67.5, 62.0, 53.5, 49.5, 37.5, 23.3, 21.1, 21.0, 21.0, 20.9.

[0066] Compound L (721 mg, 925 mmol), non-1-yne (302 μL, 2 equiv.), bis(triphenylphosphine)palladium(II) dichloride (129 mg, 0.2 equiv.), and copper(I) iodide (35 mg, 0.2 equiv.) were added to a triethylamine / acetonitrile (2 mL / 20 mL) mixture and reacted at 80°C for 16 hours under an argon atmosphere. The reaction solution was diluted with ethyl acetate, and the organic layer was washed sequentially with water (three times) and saturated brine (once) and dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound m (427 mg) in 56% yield. HRMS calculation for C 42 H 50 N2NaO 13 S [M+Na] + 845.29313; found 845.29578, calcd for C 42 H 51 N2O 13 S [M+H] + 823.31118; found 823.31235.

[0067] Compound m (82 mg, 100 μmol) was added to dry methanol (1 mL) and sodium methoxide methanol solution (50 μL, 5 mol / L) and reacted for 10 minutes. After that, aqueous sodium hydroxide solution (400 μL, 2.5 mol / L) was added and reacted for an additional 14 hours. The reaction mixture was neutralized with acetic acid and purified using high-performance liquid chromatography to obtain compound BTP9-Neu5Ac (44.1 mg) in 67% yield. 1 H-NMR (CD3OD, 600 MHz) δ 8.29 (d, J = 8.2 Hz, 1 H), 8.01-7.99 (m, 2 H), 7.86 (d, J = 1.3 Hz, 1 H), 7.50 (ddd, J = 8.2, 7.2, 1.1 Hz, 1 H), 7.40 (ddd, J = 8.0, 7.2, 0.9 Hz, 1 H), 7.13 (dd, J = 8.2, 1.4 Hz, 1 H), 4.00 (dd, J = 10.1, 1.7 Hz, 1 H), 3.97 (ddd, J = 9.3, 4.7, 2.8 Hz, 1 H), 3.89-3.83 (m, 3 H), 3.74 (dd, J = 11.4, 4.6 Hz, 1 H), 3.68 (dd, J = 9.1, 1.6 Hz, 1 H), 3.26 (dd, J = 12.3, 4.2 Hz, 1 H), 2.43 (t, J = 7.2 Hz, 2 H), 2.23 (t, J = 11.7 Hz, 1 H), 2.04 (s, 3 H), 1.62 (quin, J = 7.4 Hz, 2 H), 1.50-1.45 (m, 2 H), 1.40-1.32 (m, 6 H), 0.92 (t, J = 6.9 Hz, 3 H) HRMS calculation for C 33 H 39 N2Na2O9S [M+Na] + 685.21716; found 685.21270, calcd for C 33 H 40 N2NaO9S [M+H] + 663.23522; found 663.23504.

[0068] [Fluorescence imaging of influenza A virus-infected cells] Using the three compounds obtained as described above, fluorescent imaging of influenza A virus-infected cells was performed. In this example, influenza A virus was the A / Memphis / 1 / 1971 H3N2 strain, and human lung cancer-derived A549 cells (ATCC-CCL-185) were used as host cells to prepare virus-infected cells. Cells were cultured in 5% FBS-containing DMEM medium at 5% CO2 and 37°C.

[0069] A549 cells were plated at 2 × 10 in each well of a 10-well cell imaging slide (CELLview glass-bottom slide, Greiner Japan). 3 The cells were seeded at 8 × 10 cells / well and cultured overnight at 37°C in 5% CO2 until the cells reached confluence. The virus was then added to the wells at a titer of infection (MOI: number of virus particles / number of cells) of 2. 5A549 cells were infected with influenza A virus by adding 50 μL of serum-free medium (Hybridoma-SFM Complete DPM, Thermo Fisher Scientific, hereafter referred to as SFM) containing focus-forming units (hereafter referred to as FFU) of virus particles to each well, incubating on ice for 30 minutes, and then incubating at 37°C for 1 hour. The cells were washed with phosphate buffer (131 mM NaCl, 14 mM NaHPO, 1.5 mM KHPO, 2.7 mM KCl, pH 7.2, hereafter referred to as PBS), then the medium was replaced with 50 μL / well of SFM and cultured at 37°C in 5% CO for 6 hours. After 6 hours of incubation (7 hours postinfection), infected cells were washed with PBS and immersion-fixed in 4% paraformaldehyde / PBS solution for 15 minutes at room temperature. Then, they were permeabilized by immersion in 0.1% Triton® X-100 / PBS solution for 15 minutes at room temperature. At 7 hours postinfection, newly synthesized neuraminidase (NA) from the influenza A virus genome infecting A549 cells was localized to the Golgi apparatus for glycosylation. The sialidase activity of this neuraminidase was visualized by fluorescence imaging using the compounds mentioned above (21-189b, 21-188b, and BTP9-Neu5Ac).

[0070] Each compound (21-189b, 21-188b, and BTP9-Neu5Ac) was diluted to 50 μM in SFM and centrifuged (4°C, 10,000 × g, 10 min). The supernatant was collected and nonspecifically cleaved fluorescent material was isolated. Fixed and permeabilized infected cells were washed with PBS, and SFM supernatant containing each compound at 50 μM was added. The cells were then incubated on ice for 30 min and then incubated at 37°C for 15 min. After washing each cell with PBS, fluorescent images were observed using a 20x objective lens with a custom-made BTP filter (excitation wavelength: 360 / 40 nm, absorption wavelength: 525 / 50 nm, dichroic mirror wavelength: 400 nm) under a fluorescence microscope (model number: BZ-X700, Keyence Corporation). The results are shown in Figure 1.

[0071] According to these results, compound 21-189b produced a very clear fluorescent staining image in the fluorescent imaging of the sialidase activity of neuraminidase localized in the Golgi apparatus of influenza A virus-infected cells. On the other hand, compound 21-188b produced a poorly defined fluorescent staining image, making it unsuitable for use as a fluorescent imaging probe. Therefore, in the next study, compound 21-189b was selected as a candidate compound for a fluorescent imaging probe, and we investigated whether there was any nonspecific fluorescent image (noise) in the fluorescent staining image obtained by fluorescent imaging with compound 21-189b.

[0072] [Example 2] 2. Study of non-specific fluorescence images (noise) in fluorescent staining images in fluorescence imaging (1) Fluorescence imaging of the sialidase activity of neuraminidase localized in the Golgi apparatus of influenza A virus-infected cells was performed using compound 21-189b and BTP9-Neu5Ac using the same method as in Example 1 described above. After observing the fluorescent stained images, immunostaining was performed as follows. First, the cells were fixed with methanol for 1 minute and then blocked with a blocking agent for immunostaining (Blocking One Histo, Nacalai Tesque, Inc.) for 15 minutes. After washing with PBS, the cells were immunostained with rabbit anti-TGN46 antibody (Golgi marker antibody) and mouse anti-N2NA antibody (NA antibody) for 1 hour at room temperature. Subsequently, the cells were stained with secondary antibodies Alexa Fluor® 488-goat anti-rabbit IgG antibody and Alexa Fluor® 555-goat anti-mouse IgG antibody, as well as the nuclear stain DAPI, for 1 hour at room temperature. After washing the cells with PBS, Z-stack fluorescent images were obtained using a fluorescence microscope. The results for compound 21-189b are shown in Figure 2(a), and the results for the comparative example, BTP9-Neu5Ac, are shown in Figure 2(b). The merged images in Figure 2 are a combination of the compounds (21-189b or BTP9-Neu5Ac) used as fluorescent imaging probes, neuraminidase (NA) antibody, Golgi marker antibody, and DAPI.

[0073] As shown in Figure 2(b), when BTP9-Neu5Ac was used as a fluorescent imaging probe, granular fluorescent staining was detected in areas not corresponding to the localization of neuraminidase (sialidase) (indicated by the white arrow in the merged image), confirming nonspecific fluorescent image noise. In contrast, compound 21-189b did not exhibit the granular fluorescent staining observed with BTP9-Neu5Ac, and fluorescent staining was detected only in areas consistent with the immunostaining image with the NA antibody, i.e., the localization of sialidase. These results demonstrate that compound 21-189b is an excellent fluorescent imaging probe, capable of producing clear, localized fluorescent images without nonspecific fluorescent staining (noise).

[0074] [Example 3] 3. Fluorescence imaging of the sialidase activity of influenza A virus neuraminidase (NA) localized in the Golgi apparatus (2) The above-mentioned Examples 1 and 2 demonstrated that compound 21-189b is a fluorescent imaging probe that produces clear fluorescent stained images without producing nonspecific fluorescent stained images (noise). Therefore, in this example, several derivatives of compound 21-189b were selected in which the number of carbon atoms in the para-position halogen atom and meta-position alkoxy group in the BTP structure was varied, and their detection performance was examined. In this example, the following four compounds were used to perform fluorescent imaging of influenza A virus-infected cells.

[0075] [ka]

[0076] Each compound (21-049a, 21-049b, and 21-049c) was synthesized as follows. The compounds (including intermediates) obtained by the synthesis were confirmed to be the target compounds by high-resolution mass spectrometry (HRMS). High-resolution mass spectrometry was performed using an AccuTOF (JMS-T100LC) manufactured by JEOL Ltd., connected to an electrospray ion source (ES), and measurements were performed in positive mode. The BTP9-Neu5Ac synthesized in Example 1 was used.

[0077] [Synthesis of compound 21-049a] Compound 21-049a was synthesized as follows by the same method as the synthetic route of compound 21-189b in Example 1. In the formula showing the synthetic route shown in Example 1, compound 21-049a is R 1 = chlorine atom, R 2 = n-octyl group. In the synthesis route from compound f1, compound f1 (800 mg, 2.0 mmol) was added to N,N-dimethylformamide (10 mL) and N-chlorosuccinimide (294 mg, 1.1 equivalents) and reacted at 80°C for 17 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the residue obtained, tetrahydrofuran (10 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the reaction was continued at room temperature for 19 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the residue obtained, tetrahydrofuran (5 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the reaction was continued at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, neutralized with aqueous potassium carbonate, and then separated. The organic layer was washed with water (twice) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound g3 (713 mg) in 91% yield. HRMS (ESI-TOF): m / z Calcd. for C 21 H 25 37ClNO2S [M+H] + 392.1265; Found. 392.12676, Calcd. for C 21 H 25 35 ClNO2S [M+H] + 390.12945; Found. 390.13216.

[0078] Sodium hydride (80 mg, 2 mmol, 60% liquid paraffin dispersion) was added to a tetrahydrofuran / N,N-dimethylformamide (5 mL / 5 mL) mixture of compound h, obtained from methyl N-acetylneuraminate (0.5 g equivalent), and compound g3 (500 mg, 1.28 mmol), and the mixture was allowed to react at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound i3 (416 mg) in 38% yield. HRMS (ESI-TOF): m / z Calcd. for C 41 H 51 37 ClN2NaO 14 S [M+Na] + 887.26177; Found. 887.26128, Calcd. for C 41 H 51 35 ClN2NaO 14 S [M+Na] + 885.26472; Found. 885.26278.

[0079] Compound i3 (150 mg, 174 μmol) was added to dry methanol (1 mL) and sodium methoxide methanol solution (100 μL, 5.0 mol / L) and reacted at room temperature for 10 minutes. Sodium hydroxide solution (200 μL, 2.5 mol / L) was then added and reacted at room temperature for an additional 12 hours. Acetic acid (70 μL) was added to the reaction mixture, and the mixture was purified using high-performance liquid chromatography to give compound 21-049a (58.1 mg) in 49% yield. HRMS (ESI-TOF): m / z Calcd. for C 32 H 41 37 ClN2NaO 10 S [M+Na] + 705.20386; Found. 705.20679, Calcd. for C 32 H 41 35 ClN2NaO 10 S [M+Na] + 703.20681; Found. 703.20380, Calcd. for C 32 H 42 37 ClNO 10 S [M+H] + 683.22192; Found. 683.22296, Calcd. for C 32 H 42 35 ClNO 10 S [M+H] + 681.22487; Found. 681.22293.

[0080] [Synthesis of compound 21-049b] Compound 21-049b was synthesized as follows by the same method as the synthetic route of compound 21-189b in Example 1. In the formula showing the synthetic route shown in Example 1, compound 21-049b is R 1 = chlorine atom, R 2 = n-dodecyl group. In the synthesis route from compound e, unpurified compound e (287 mg, 1.0 mmol) was added to N,N-dimethylformamide (5 mL), 4-methylbenzenesulfonate dodecyl (426 mg, 1.25 equivalents), and potassium carbonate (414 mg, 3.0 equivalents) and reacted at 120 °C for 16 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound f2 (326 mg) in 73% yield. HRMS (ESI-TOF): m / z Calcd. for C 27 H 37 NNaO3S [M+Na] + 478.23918; Found. 478.23930, Calcd. for C 27 H 38 NO3S [M+H] + 456.25724; Found. 456.25852.

[0081] Compound f2 (223 mg, 0.5 mmol) was added to a mixture of tetrahydrofuran / N,N-dimethylformamide (1 mL / 3 mL) and N-chlorosuccinimide (76 mg, 1.15 equivalents), and the mixture was reacted at 80°C for 21 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (3 mL) and concentrated hydrochloric acid (3 mL) were added successively, and the mixture was reacted at room temperature for 15 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (5 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the mixture was reacted at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, neutralized with aqueous potassium carbonate, and then separated. The organic layer was washed with water (twice) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure, and the resulting residue was purified by flash column chromatography to give compound g4 (186 mg) in 83% yield. HRMS (ESI-TOF): m / z Calcd. for C 25 H 33 37 ClNO2S [M+H] + 448.1891; Found. 448.19306, Calcd. for C 25 H 33 35 ClNO2S [M+H] + 446.19205; Found. 446.19662.

[0082] Compound g4 (100 mg, 224 μmol) was added to a tetrahydrofuran suspension (1 mL) of sodium hydride (30 mg, 0.75 mmol, 60% liquid paraffin dispersion). This was followed by an N,N-dimethylformamide solution (1 mL) of compound h, obtained from methyl N-acetylneuraminate (0.25 g). The mixture was allowed to react at room temperature for 21 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed sequentially with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound i4 (157 mg) in 76% yield. HRMS (ESI-TOF): m / z Calcd. for C 45 H 59 37 ClN2NaO 14 S [M+Na] + 943.32437; Found. 943.32804, Calcd. for C 45 H 59 35 ClN2NaO 14 S [M+Na] + 941.32732; Found. 941.32266, Calcd. for C 45 H 60 37 ClNO 14 S [M+H] + 921.34243; Found. 921.34609, Calcd. for C 45 H 60 35 ClNO 14 S [M+H] + 919.34538; Found. 919.34460.

[0083] Compound i4 (30 mg, 32.6 μmol) was added to dry methanol (0.5 mL) and sodium methoxide methanol solution (100 μL, 5.0 mol / L) and reacted at room temperature for 30 minutes. Sodium hydroxide solution (200 μL, 2.5 mol / L) was then added and reacted at room temperature for an additional 16 hours. Acetic acid (60 μL) was added to the reaction mixture, and the mixture was purified using high-performance liquid chromatography to give compound 21-049b (16.8 mg) in 70% yield. HRMS (ESI-TOF): m / z Calcd. for C 36 H 50 37 ClNO 10 S [M+2Na-H] + 783.24841; Found. 783.25126, Calcd. for C 36 H 50 35 ClNO 10 S [M+2Na-H] + 781.25136; Found. 781.25202, Calcd. for C 36 H 49 37 ClN2NaO 10 S [M+Na] + 761.26646; Found. 761.26876, Calcd. for C 36 H 49 35 ClN2NaO 10 S [M+Na] + 759.26941; Found. 759.26831, Calcd. for C 36 H 50 37 ClNO 10 S [M+H] + 739.28452; Found. 739.28720, Calcd. for C 36 H 50 35 ClNO 10 S [M+H] + 737.28747; Found. 737.28604.

[0084] [Synthesis of compound 21-049c] Compound 21-049c was synthesized as follows by the same method as the synthetic route of compound 21-189b in Example 1. In the formula showing the synthetic route shown in Example 1, compound 21-049c is R 1 = chlorine atom, R 2 = n-octadecyl group. In the synthesis route from compound e, unpurified compound e (287 mg, 1.0 mmol) was added to N,N-dimethylformamide (5 mL), octadecyl 4-methylbenzenesulfonate (531 mg, 1.25 equivalents), and potassium carbonate (414 mg, 3.0 equivalents) and reacted at 120 °C for 16 hours. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound f3 (453 mg) in 84% yield. HRMS (ESI-TOF): m / z Calcd. for C 33 H 49 NNaO3S [M+Na] + 562.33308; Found. 562.33766, Calcd. for C 33 H 50 NO3S [M+H] + 540.35114; Found. 540.35440.

[0085] Compound f3 (270 mg, 0.5 mmol) was added to a mixture of tetrahydrofuran / N,N-dimethylformamide (1 mL / 3 mL) and N-chlorosuccinimide (76 mg, 1.15 equivalents), and the mixture was reacted at 80°C for 21 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (3 mL) and concentrated hydrochloric acid (3 mL) were added successively, and the mixture was reacted at room temperature for 15 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. To the resulting residue, tetrahydrofuran (5 mL) and concentrated hydrochloric acid (5 mL) were added successively, and the mixture was reacted at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, neutralized with aqueous potassium carbonate, and then separated. The organic layer was washed with water (twice) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure, and the resulting residue was purified by flash column chromatography to give compound g5 (239 mg) in 90% yield. HRMS (ESI-TOF): m / z Calcd. for C 31 H 45 37 ClNO2S [M+H] + 532.283; Found. 532.28101, Calcd. for C 31 H 45 35 ClNO2S [M+H] + 530.28595; Found. 530.28829.

[0086] Compound g5 (100 mg, 189 μmol) was added to a tetrahydrofuran suspension (1 mL) of sodium hydride (30 mg, 0.75 mmol, 60% liquid paraffin dispersion). This was followed by an N,N-dimethylformamide solution (1 mL) of compound h, obtained from methyl N-acetylneuraminate (0.25 g). The mixture was allowed to react at room temperature for 21 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed sequentially with water (three times) and saturated brine (once), and then dried over sodium sulfate. After filtering off the desiccant, the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography to give compound i5 (91 mg) in 48% yield. HRMS (ESI-TOF): m / z Calcd. for C 51 H 72 37 ClNO 14 S [M+H] + 1005.43633; Found. 1005.43896, Calcd. for C 51 H 72 35 ClNO 14 S [M+H] + 1003.43928; Found. 1003.43861.

[0087] Compound i5 (30 mg, 29.9 μmol) was added to dry methanol (0.5 mL) and sodium methoxide methanol solution (100 μL, 5.0 mol / L) and reacted at room temperature for 30 minutes. Sodium hydroxide solution (200 μL, 2.5 mol / L) was then added and reacted at room temperature for an additional 16 hours. Acetic acid (60 μL) was added to the reaction mixture, and the mixture was purified using high-performance liquid chromatography to give compound 21-049c (9.6 mg) in 39% yield. HRMS (ESI-TOF): m / z Calcd. for C 42 H 62 37 ClNO 10 S [M+2Na-H] + 867.34231; Found. 867.34581, Calcd. for C 42 H 62 35ClNO 10 S [M+2Na-H] + 865.34526; Found. 865.34615, Calcd. for C 42 H 61 37 ClN2NaO 10 S [M+Na] + 845.36034; Found. 845.36228, Calcd. for C 42 H 61 35 ClN2NaO 10 S [M+Na] + 843.36331; Found. 843.36531.

[0088] [Fluorescence imaging of influenza A virus-infected cells] Fluorescence imaging of influenza A virus-infected cells was performed using the three compounds obtained as described above and the control BTP9-Neu5Ac. Fluorescence imaging was performed in the same manner as in Example 1. The results are shown in Figure 3.

[0089] These results showed that compound 21-049a produced very clear fluorescent staining images in the fluorescent imaging of the sialidase activity of neuraminidase localized in the Golgi apparatus of influenza A virus-infected cells. On the other hand, compounds 21-049b and 21-049c produced unclear fluorescent staining images, making them unsuitable for use as fluorescent imaging probes. Therefore, in the next study, compound 21-049a was selected as a candidate compound for a fluorescent imaging probe, and we investigated whether there was any nonspecific fluorescent image (noise) in the fluorescent staining images obtained by fluorescent imaging with compound 21-049a.

[0090] [Example 4] 4. Investigation of non-specific fluorescence images (noise) in fluorescent staining images in fluorescence imaging (2) Using compound 21-049a, fluorescent imaging of the sialidase activity of neuraminidase localized in the Golgi apparatus of influenza A virus-infected cells and immunostaining using a neuraminidase (NA) antibody and a Golgi marker antibody were performed using the same method as in Example 2 described above. The results are shown in Figure 4. The merge (overlay) in Figure 4 is a combined image of compound 21-049a used as a fluorescent imaging probe, an NA (neuraminidase) antibody, a Golgi marker antibody, and DAPI.

[0091] As shown in Figure 4, compound 21-049a did not produce granular fluorescent staining like the BTP9-Neu5Ac staining in Figure 3(b), and fluorescent staining was detected only in the area consistent with the immunostaining image using the NA antibody, i.e., the area consistent with the localization of sialidase. This indicates that compound 21-049a is an excellent fluorescent imaging probe that does not produce nonspecific fluorescent staining (noise), has excellent local staining ability, and can produce clear fluorescent staining images.

[0092] [summary] The results of Examples 1 to 4 above revealed that in order to obtain a BTP derivative-sialic acid (Neu5Ac) compound that reduces nonspecific fluorescent staining images (noise), has excellent local staining properties, and provides clear fluorescent staining images, it is important to add a halogen to the para-position relative to the sialic acid in the BTP derivative structure, and to add an ether bond (alkoxy group) to an alkyl group at the meta-position relative to the sialic acid, with the alkyl group having fewer than 12 carbon atoms. Furthermore, it was shown that compounds in which the alkyl group has 8 carbon atoms are particularly preferred.

[0093] The present invention is not limited to the above-described embodiments or examples, and its technical scope also includes various modified designs within the scope that does not deviate from the gist of the invention described in the claims. [Industrial Applicability]

[0094] The present invention provides a reagent for detecting sialidase-containing viruses or viral sialidases and a detection method using the same, which will be widely useful in industries such as virus research, including antiviral drug research, virus diagnosis, and medical care.

Claims

1. A compound represented by the following formula (I), a salt thereof, or a solvate thereof: 【Chemistry 1】 [In formula (I), R １ is a halogen atom, R ２ represents an alkyl group having 5 to 11 carbon atoms or a hydrocarbon group having 5 to 11 carbon atoms and one or more unsaturated bonds.]

2. In the formula (I), the R １ is a chlorine atom or a bromine atom, and the R ２ The compound or salt thereof, or a solvate thereof according to claim 1, wherein is a linear alkyl group having 6 to 10 carbon atoms.

3. A reagent for detecting a virus having sialidase or a sialidase, comprising: A detection reagent comprising a compound represented by the following formula (I) or a salt thereof, or a solvate thereof: 【Chemistry 2】 [In formula (I), R １ is a halogen atom, R ２ represents an alkyl group having 5 to 11 carbon atoms or a hydrocarbon group having 5 to 11 carbon atoms and one or more unsaturated bonds.]

4. In the formula (I), the R １ is a chlorine atom or a bromine atom, and the R ２ The detection reagent according to claim 3, wherein is a linear alkyl group having 6 to 10 carbon atoms.

5. the virus is an influenza A virus, 5. The detection reagent according to claim 3, wherein the sialidase is an influenza A virus sialidase.

6. 5. The detection reagent according to claim 3, which is a fluorescent imaging probe for detecting a virus having sialidase or a sialidase.

7. A method for detecting sialidase in virus-infected cells, comprising: A detection method comprising fluorescent imaging of the virus-infected cells using the detection reagent according to claim 3 or 4.

8. A method for detecting a virus having sialidase, comprising: A detection method comprising fluorescent imaging of cells infected with the virus using the detection reagent according to claim 3 or 4.

9. A method for detecting sialidase, comprising: adding the detection reagent according to claim 3 or 4 to a specimen; and A detection method characterized by determining the presence or absence of the sialidase based on the fluorescent substance produced by the above step.

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