Bisphosphoryl crosslinked stilbene compound

Abstract

To provide a compound that can stain intracellular organelle using a long wavelength excitation wavelength capable of reducing phototoxicity to intracellular organelle and that has even better light resistance as compared with MitoPB Yellow.SOLUTION: A bisphosphoryl cross-linked stilbene compound, for example, the compound trans-PO-BphoxM having the following structural formula is shown.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to bisphosphoryl crosslinked stilbene compounds.

Background Art

[0002] Fluorescent organic compounds having a high fluorescence quantum yield are important as luminescent materials for organic EL elements or fluorescent dyes for biological fluorescence imaging. There are innumerable reported examples in both basic research and applications.

[0003] However, many of the conventionally known fluorescent organic compounds gradually decompose and fade when continuous light irradiation is performed. For example, as fluorescent probes, as a group of dyes with enhanced light resistance, Alexa Fluor dyes, ATTO dyes, etc. are well-known, but even when these are used, it is difficult to repeatedly observe with a super-resolution microscope such as stimulated emission depletion (STED) imaging. For this reason, at present, the observation targets of the most advanced fluorescence microscopy techniques are limited, and improvement of the light resistance of fluorescent dyes is required.

[0004] Among such prior arts, as a fluorescent dye having excellent light resistance, a phosphole compound "MitoPB Yellow" having a specific structure is also known (see, for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, in super-resolution microscopes such as stimulated emission depletion (STED) imaging, in the case of MitoPB Yellow, since a STED laser with a wavelength of 660 nm was used as the excitation wavelength, phototoxicity to intracellular organelles such as mitochondria was observed. Therefore, it has been required to increase the excitation wavelength to a longer wavelength and reduce the phototoxicity to intracellular organelles. In addition, although MitoPB Yellow exhibited overwhelming light resistance superior to other existing fluorescent dyes, it was found that the required characteristics for light resistance did not stop there, and it was required that even with strong light irradiation that causes MitoPB Yellow to photobleach, photobleaching can be reduced.

[0007] The present invention aims to solve the above-described conventional problems, and an object thereof is to provide a compound that can stain intracellular organelles using an excitation wavelength of a long wavelength capable of reducing phototoxicity to intracellular organelles and that is even more excellent in light resistance than MitoPB Yellow.

Means for Solving the Problems

[0008] As a result of intensive studies in view of the above problems, the present inventors have found that a compound having a specific bisphosphoryl cross-linked stilbene skeleton can stain intracellular organelles, is excellent in light resistance, and even when irradiated with strong laser light used in super-resolution microscopes such as stimulated emission depletion (STED) imaging, the absorption intensity hardly decreases. Based on such findings, the present inventors have further conducted studies and completed the present invention. That is, the present invention includes the following configurations.

[0009] Item 1. General formula (1):

[0010]

Chemical formula

[0011]

Chemical formula

[0012] Item 2. The bisphosphoryl-bridged stilbene compound according to Item 1, wherein 3 R is a substituted or unsubstituted alkyl group.

[0013] Item 3. The bisphosphoryl-bridged stilbene compound according to Item 1 or 2, wherein 1 Ar is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0014] Item 4. A fluorescent dye containing the bisphosphoryl-bridged stilbene compound according to any one of Items 1 to 3.

[0015] Item 5. An organelle stain containing the bisphosphoryl-bridged stilbene compound according to any one of Items 1 to 3.

[0016] Item 6. A method for stimulated emission depletion (STED) imaging of intracellular organelles, which uses the bisphosphoryl crosslinked stilbene compound according to any one of Items 1 to 3, the fluorescent dye according to Item 4, or the intracellular organelle stain according to Item 5.

Advantages of the Invention

[0017] The bisphosphoryl crosslinked stilbene compound of the present invention has an absorption peak wavelength that is longer than that of MitoPB Yellow, and for example, intracellular organelles can be stained using a long wavelength STED laser such as 775 nm. Therefore, the phototoxicity to intracellular organelles can be reduced, and it is also useful as a light-resistant electronic material for which a longer absorption peak wavelength is required.

[0018] In addition, the bisphosphoryl crosslinked stilbene compound of the present invention is extremely excellent in light resistance and hardly fades even under strong light irradiation that causes MitoPB Yellow to fade.

[0019] Therefore, the bisphosphoryl crosslinked stilbene compound of the present invention is a fluorescent dye suitable for repeatedly observing intracellular organelles with a super-resolution microscope such as stimulated emission depletion (STED) imaging.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0021] As used herein, "comprise" is a concept that also includes "consist essentially of" and "consist of".

[0022] In this specification, when a range is expressed as "A to B", it means A or more and B or less, unless otherwise specified.

[0023] 1. Bisphosphoryl cross-linked stilbene compound The bisphosphoryl cross-linked stilbene compound of the present invention has the general formula (1):

[0024]

Chemical formula

[0025]

Chemical formula

[0026] This bisphosphoryl cross-linked stilbene compound has an oxygen atom bonded to a phosphorus atom and a ring Ar 3 Depending on the positions of

[0027]

Chemical formula

[0028] Both the trans-bisphosphoryl-bridged stilbene compound represented by the general formula (1A) and the cis-bisphosphoryl-bridged stilbene compound represented by the general formula (1B) have an absorption peak wavelength that is longer than that of MitoPB Yellow. For example, since they can stain intracellular organelles using a long-wavelength STED laser such as 775 nm, they can reduce phototoxicity to intracellular organelles and are also useful as light-resistant electronic materials that require a longer absorption peak wavelength. Furthermore, they are extremely excellent in light resistance and hardly fade even under strong light irradiation that causes MitoPB Yellow to fade.

[0029] Thus, in the bisphosphoryl-bridged stilbene compound of the present invention, since the -OR 3 group, which is a functional group, is bonded to the ring Ar 3 it is possible to control the organelle localization and dispersibility, bind to various intracellular organelles, and stain various intracellular organelles. At this time, as also shown in the examples described later, since the bisphosphoryl-bridged stilbene compound selectively exists at a desired site of the intracellular organelle, it is useful for grasping the external shape of the intracellular organelle. For example, when R 3 is a substituted alkyl group containing a phosphonium group, it can be used as a stain for the mitochondrial membrane, and when R 3 is an alkyl group such as a methyl group, it can be used as a stain for lipid droplets. Therefore, since it can be used as an intracellular organelle stain, the bisphosphoryl-bridged stilbene compound of the present invention is a compound suitable for repeated observation with a super-resolution microscope such as stimulated emission depletion (STED) imaging in vivo.

[0030] In addition, the bisphosphoryl-bridged stilbene compound of the present invention has an amino group or a substituted amino group, which is an electron-donating group, thereby imparting environmental responsiveness and achieving a longer absorption peak wavelength, and reducing phototoxicity to intracellular organelles.

[0031] Furthermore, due to having a bisphosphoryl cross-linked stilbene skeleton, the bisphosphoryl cross-linked stilbene compound of the present invention can impart light resistance such that the absorption intensity hardly decreases even under strong light irradiation that causes photo-bleaching of MitoPB Yellow.

[0032] In General Formulas (1), (1A) and (1B), Ar 1 As the aromatic hydrocarbon ring represented by, either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted. For example, a benzene ring can be mentioned as the monocyclic aromatic hydrocarbon ring, and a naphthalene ring, anthracene ring, phenanthrene ring, fluorene ring, pyrene ring, triphenylene ring, etc. can be mentioned as the polycyclic aromatic hydrocarbon ring.

[0033] Ar 1 The aromatic hydrocarbon ring represented by may have a substituent. Examples of the substituent include an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, etc., which will be described later. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0034] In General Formulas (1), (1A) and (1B), Ar 1 Examples of the heteroaromatic ring represented by include, as a monocyclic heteroaromatic ring, a pyridine ring, a pyrazine ring, etc., and as a polycyclic heteroaromatic ring, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, etc.

[0035] Ar 1The complex aromatic ring represented by may have a substituent. Examples of the substituent include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

[0036] Ar 1 From the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc., a substituted or unsubstituted aromatic hydrocarbon ring is preferred, and a polycyclic aromatic hydrocarbon ring is particularly preferred from the viewpoint of light resistance.

[0037] In addition, when Ar 1 is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring, the bisphosphoryl cross-linked stilbene compound of the present invention has the general formulas (1C) and (1D):

[0038]

Chemical formula

[0039] In the bisphosphoryl cross-linked stilbene compound represented by the general formula (1C), depending on the oxygen atom bonded to the phosphorus atom and the position of the ring Ar 3 , the general formulas (1C1) and (1C2):

[0040]

Chemical formula

[0041] Also, in the bisphosphoryl - bridged stilbene compound represented by the general formula (1D), depending on the oxygen atom bonded to the phosphorus atom and the position of the ring Ar 3 , the general formulas (1D1) and (1D2):

[0042] [Chemical formula] [In the formula, Ar 2 , Ar 3a , Ar 3b , Ar 4 , R 1 , R 2 , R 3a , R 3b , Y 3a and Y 3b are the same as described above.] All bisphosphoryl - bridged stilbene compounds represented by are included.

[0043] In the general formulas (1C), (1C1), (1C2), (1D), (1D1) and (1D2), as the aromatic hydrocarbon ring represented by Ar 4 , either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted. For example, as the monocyclic aromatic hydrocarbon ring, a benzene ring can be mentioned, and as the polycyclic aromatic hydrocarbon ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, etc. can be mentioned. Among these, from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc., the benzene ring is particularly preferred.

[0044] Ar 4The aromatic hydrocarbon ring represented by may have a substituent. Examples of the substituent include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, etc. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0045] In General Formulas (1C), (1C1), (1C2), (1D), (1D1), and (1D2), Ar 4 Examples of the heteroaromatic ring represented by include, as a monocyclic heteroaromatic ring, a pyridine ring, a pyrazine ring, etc., and as a polycyclic heteroaromatic ring, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, etc.

[0046] Ar 4 The heteroaromatic ring represented by may have a substituent. Examples of the substituent include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), etc. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0047] Note that, compared with the case where Ar 1 is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compound represented by General Formula (1C), (1C1), or (1C2) has ε in absorption and Φ F which are further excellent, and can emit light more strongly and clearly in the target intracellular organelle.

[0048] Also, Ar 1When compared with the case where it is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compound represented by the general formula (1D), (1D1) or (1D2) can have absorption peak wavelength and fluorescence peak wavelength at longer wavelengths.

[0049] From the above, Ar 1 Depending on the structure, it is possible to impart diversity to the optical properties, and it is preferable to appropriately adjust the structure of Ar 1 according to the required physical properties.

[0050] In the general formulas (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), Ar 2 , Ar 3 , Ar 3a and Ar 3b As the aromatic hydrocarbon ring represented by, either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted. For example, a benzene ring can be mentioned as the monocyclic aromatic hydrocarbon ring, and a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, etc. can be mentioned as the polycyclic aromatic hydrocarbon ring. Among these, a benzene ring is particularly preferable from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc.

[0051] Ar 2 The aromatic hydrocarbon ring represented by may have a substituent. Examples of the substituent include an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, etc. The number of substituents in the case of having a substituent is preferably, for example, 1 to 6, more preferably 1 to 3.

[0052] In the general formulas (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), Ar 2 , Ar 3 , Ar 3a and Ar3b Examples of the heteroaromatic ring represented by 3b include, as monocyclic heteroaromatic rings, a pyridine ring, a pyrazine ring, etc., and as polycyclic heteroaromatic rings, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, etc.

[0053] Ar 3 Ar 3a and Ar 3b The heteroaromatic rings represented by 3 , 3a and 3b may have substituents. Examples of the substituents include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (a vinyl group, a propenyl group, etc.), an alkynyl group (an ethynyl group, a 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.). When having substituents, the number of the substituents is preferably, for example, 1 to 6, more preferably 1 to 3.

[0054] Ar 3 Ar 3a and Ar 3b From the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc., a substituted or unsubstituted aromatic hydrocarbon ring is preferable, and a substituted or unsubstituted monocyclic aromatic hydrocarbon ring is more preferable.

[0055] In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), as the alkyl group represented by R 1 and R 2 , either a linear alkyl group or a branched alkyl group can be adopted. Examples include C1-10 alkyl groups (particularly C1-6 alkyl groups) such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group.

[0056] R 1 and R 2 ​​​​The alkyl group represented by [alkyl group] may have a substituent. Examples of the substituent include a halogen atom, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, a cyano group, a nitro group, and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, more preferably 1 to 3.

[0057] In General Formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), and (1D2), R 1 and R 2 Examples of the cycloalkyl group represented by [cycloalkyl group] include C3-10 cycloalkyl groups (particularly C4-8 cycloalkyl groups) such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group.

[0058] R 1 and R 2 The cycloalkyl group represented by [cycloalkyl group] may have a substituent. Examples of the substituent include a halogen atom, the above alkyl group, an aryl group described below, a heteroaryl group described below, a cyano group, a nitro group, and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, more preferably 1 to 3.

[0059] In General Formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), and (1D2), R 1 and R 2 As the aryl group represented by [aryl group], any of a monocyclic aryl group, a condensed-ring aryl group, and a polycyclic aryl group can be adopted. For example, the monocyclic aryl group includes a phenyl group, the condensed-ring aryl group includes a naphthyl group, an anthracenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, a triphenylenyl group, etc., and the polycyclic aryl group includes C6-18 aryl groups (particularly C6-14 aryl groups) such as a biphenyl group and a terphenyl group.

[0060] R 1 and R 2The aryl group represented by [description] may have a substituent. Examples of the substituent include a halogen atom, the above alkyl group, the above aryl group, a heteroaryl group described later, a cyano group, a nitro group, and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0061] In general formula (1), R 1 and R 2 As the heteroaryl group represented by [description], either a monocyclic heteroaryl group or a condensed heteroaryl group can be adopted. For example, as the monocyclic heteroaryl group, a pyrrolyl group, a thienyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazyl group, etc. can be mentioned, and as the condensed heteroaryl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, a quinoxalyl group, etc. can be mentioned.

[0062] R 1 and R 2 The heteroaryl group represented by [description] may have a substituent. Examples of the substituent include the above halogen atom, the above alkyl group, the above aryl group, the above heteroaryl group, a cyano group, a nitro group, and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0063] Among them, from the viewpoint of easily imparting environmental responsiveness and easily shifting the absorption peak wavelength to a longer wavelength, a substituted or unsubstituted (hetero)aryl group is preferable as R 1 and R 2 , a substituted or unsubstituted aryl group is more preferable, and an unsubstituted aryl group is even more preferable.

[0064] In addition, R 1 and R 2 may together form a ring with the adjacent nitrogen atom. That is, the group represented by -NR 1 R 2 is

[0065] [Chemical formula] It may also be a group represented by etc.

[0066] Also, R 1 and / or R 2 may combine with Ar 2 to form a ring together with the adjacent nitrogen atom. That is, the structure represented by Ar 2 -NR 1 R 2 is

[0067] [Chemical formula] etc. may be used.

[0068] In this case, in Ar 2 , the substitution position where the group represented by -NR 1 R 2 is bonded is not particularly limited.

[0069] In general formula (2), the organic group represented by R 3 is not particularly limited, and examples include an alkyl group, a polyethylene glycol group or a derivative group thereof (-(C2H4O) m R 7 ), etc.

[0070] In general formula (2), as the alkyl group represented by R 3 , either a linear alkyl group or a branched alkyl group can be adopted, but it is preferable to select an appropriate group depending on the dyeing target.

[0071] The alkyl group represented by R 3 may have a substituent. As the substituent, it is preferably a functional group so that the organelle localization and dispersibility can be easily controlled. For example, an epoxy group, a phosphorus-containing group, a carboxy group, an alkoxycarbonyl group (-COOR 8 ), an amide group or a derivative group thereof (-CONHR 9 ), etc. may be mentioned.

[0072] R 3 As the phosphorus-containing group as a substituent of the alkyl group represented by, there is no particular limitation, and from the viewpoint of easily controlling the organelle localization and dispersibility, the general formula (3): -P + R 4 n X 1 (4-n) - (3) [In the formula, R 4 represents the same or different, substituted or unsubstituted (hetero)aryl group. X 1 represents the same or different halogen atoms. n represents an integer of 1 to 3.] The group represented by is preferred.

[0073] In the general formula (3), as the aryl group represented by R 4 , for example, any of a monocyclic aryl group, a condensed aryl group, and a polycyclic aryl group can be adopted. For example, the monocyclic aryl group includes a phenyl group, and the condensed aryl group includes a naphthyl group, an anthracenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, a triphenylenyl group, etc. The polycyclic aryl group includes a C6-18 aryl group (especially a C6-14 aryl group) such as a biphenyl group and a terphenyl group.

[0074] R 4 The aryl group represented by may have a substituent. Examples of the substituent include a halogen atom, the above alkyl group, the above aryl group, the heteroaryl group described later, a cyano group, a nitro group, etc. The number of substituents when having a substituent is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0075] In the general formula (3), R 4As the heteroaryl group represented by, either a monocyclic heteroaryl group or a condensed heteroaryl group can be adopted. For example, as the monocyclic heteroaryl group, a pyrrolyl group, a thienyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazyl group, etc. can be mentioned, and as the condensed heteroaryl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, a quinoxalyl group, etc. can be mentioned.

[0076] R 4 The heteroaryl group represented by may have a substituent. Examples of the substituent include the above-mentioned halogen atom, the above-mentioned alkyl group, the above-mentioned aryl group, the above-mentioned heteroaryl group, a cyano group, a nitro group, etc. When having a substituent, the number of substituents is preferably, for example, 1 to 6, more preferably 1 to 3.

[0077] Among them, R 4 is preferably a substituted or unsubstituted aryl group, more preferably an unsubstituted aryl group, from the viewpoint of easily controlling the organelle localization and dispersibility.

[0078] In the general formula (3), as the halogen atom represented by X 1 , a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. can be mentioned. From the viewpoint of easily controlling the organelle localization and dispersibility, a chlorine atom, a bromine atom, an iodine atom, etc. are preferable, a chlorine atom, a bromine atom, etc. are more preferable, and a bromine atom is further preferable.

[0079] In the general formula (3), n is preferably an integer of 1 to 3, more preferably 2 or 3, and further preferably 3, from the viewpoint of easily controlling the organelle localization and dispersibility.

[0080] R 3 As the alkoxycarbonyl group (-COOR 8 ) as a substituent of the alkyl group represented by, R 8It can be used without particular limitation as long as it contains an alkyl group (such as methyl group, ethyl group, n-propyl group, isopropyl group, etc.). For example, methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, etc. can be mentioned.

[0081] R 3 As a substituent of the alkyl group represented by, an amide group or its derivative group (-CONHR 9 ), as long as R 8 contains a hydrogen atom or an alkyl group (such as methyl group, ethyl group, n-propyl group, isopropyl group, etc.), it can be used without particular limitation. For example, amide group, N-methylamide group, N-ethylamide group, N-n-propylamide group, N-isopropylamide group, etc. can be mentioned.

[0082] In general formula (2), as the polyethylene glycol group or its derivative group (-(C2H4O) 3 represented by R m R 7 ), as long as R 7 contains a hydrogen atom or an alkyl group (such as methyl group, ethyl group, n-propyl group, isopropyl group, etc.), and m adopts an integer of 1 to 100 for example, it can be used without particular limitation. For example, -(C2H4O) m CH3 can be preferably used.

[0083] As the organic group represented by R 3 satisfying the above conditions, specifically,

[0084] [Chemical formula] [In the formula, R 8 and R 9 are the same as above. Ph represents a phenyl group. k represents an integer of 5 to 15. m represents an integer of 2 to 100.] etc. can be mentioned.

[0085] Examples of the bisphosphoryl crosslinked stilbene compound represented by the general formula (1) that satisfies the above conditions include, for example,

[0086]

Chem.

[0087]

Chem.

[0088]

Chem.

[0089]

Chem.

[0090]

Chem.

[0091] 2. Method for producing bisphosphoryl cross-linked stilbene compound The method for producing the phosphole compound of the present invention is not particularly limited. For example, the general formula (4A) or (4B):

[0092]

Chem.

[0093] The bisphosphoryl - bridged stilbene compound represented by the above general formula (4A) or (4B) can be synthesized, for example, according to the previously reported (Chem. Asian J., 13, 1616 - 1624 (2018)).

[0094] In general formula (5), examples of the halogen atom represented by X 2 include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. A chlorine atom, a bromine atom, an iodine atom, etc. are preferred, a chlorine atom, a bromine atom, etc. are more preferred, and a bromine atom is even more preferred.

[0095] For the reaction, a base can be used if necessary. Examples of the base include potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate, etc. In this step, from the viewpoints of yield and ease of synthesis, potassium carbonate is preferred. When using a base, the amount used is preferably 2 - 30 moles, more preferably 10 - 20 moles, per 1 mole of compound (4A) or compound (4B) from the viewpoints of ease of synthesis, yield, etc.

[0096] The reaction can usually be carried out in the presence of a reaction solvent. Examples of the reaction solvents that can be used include aromatic hydrocarbon solvents such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbon solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents such as acetonitrile; amide solvents such as dimethylformamide, etc. From the viewpoints of ease of synthesis, yield, etc., amide solvents are preferred, and dimethylformamide is more preferred. These reaction solvents can be used alone or in combination of two or more.

[0097] The reaction atmosphere can usually adopt an inert gas atmosphere (such as argon gas atmosphere, nitrogen gas atmosphere, etc.). The reaction temperature can be carried out under heating, at room temperature, or under cooling, and usually, 0 to 100 °C is preferred, and 10 to 70 °C is more preferred. The reaction time is not particularly limited, and it is preferably the time for the reaction to proceed sufficiently.

[0098] After the reaction is completed, the bisphosphoryl cross-linked stilbene compound of the present invention can be obtained by performing purification treatment according to a conventional method as necessary.

[0099] In the above, an example of a synthesis method of one embodiment of the bisphosphoryl cross-linked stilbene compound of the present invention has been described, but it is not limited to this production method, and it can be synthesized by various synthesis methods.

[0100] 3. Fluorescent dye and organelle stain The fluorescent dye of the present invention contains the above-described bisphosphoryl cross-linked stilbene compound of the present invention.

[0101] The fluorescent dye of the present invention has excellent light resistance due to having a bisphosphoryl cross-linked stilbene skeleton, and 3It is possible to introduce various reactive groups. Therefore, it can be used as an organelle stain (especially lipid droplet stain, mitochondrial stain, etc.) for staining organelles in cells (especially lipid droplets, mitochondria, lysosomes, endoplasmic reticulum, cell membrane, etc.). The fluorescent dye of the present invention is suitable for repeated observation with a super-resolution microscope (especially stimulated emission depletion (STED) microscope) such as stimulated emission depletion (STED) imaging in vivo.

[0102] When the bisphosphoryl cross-linked stilbene compound of the present invention is used as an organelle stain (especially lipid droplet stain, mitochondrial stain, lysosome stain, endoplasmic reticulum stain, cell membrane stain, etc.), the organelle stain (especially lipid droplet stain, mitochondrial stain, lysosome stain, endoplasmic reticulum stain, cell membrane stain, etc.) of the present invention contains the bisphosphoryl cross-linked stilbene compound of the present invention. However, it is preferably dissolved in a culture solution (Dulbecco's modified Eagle's medium; DMEM, etc.) to form a solution. From the viewpoint of improving the light resistance and staining organelles (especially lipid droplets, mitochondria, lysosomes, endoplasmic reticulum, cell membrane, etc.) more strongly, the content of the bisphosphoryl cross-linked stilbene compound of the present invention is preferably 1 to 10000 nmol / L, and more preferably 10 to 1000 nmol / L.

[0103] As described above, the organelle stain (especially lipid droplet stain, mitochondrial stain, lysosome stain, endoplasmic reticulum stain, cell membrane stain, etc.) of the present invention preferably has a solution form. However, when observing in vivo, the pH is preferably about 5 to 11, and more preferably about 6.5 to 7.5. In order to adjust the pH, a buffer (HEPES buffer, Tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) can also be used in combination.

Examples

[0104] The present invention will be specifically described based on the examples, but the present invention is not limited thereto.

[0105] Unless otherwise specified, all reactions were carried out under a nitrogen atmosphere. Unless otherwise specified, commercially available solvents and reagents were used without purification. However, anhydrous toluene, anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), and anhydrous CH2Cl2 were purchased from Kanto Chemical Co., Inc. and purified by Glass Contour Solvent Systems.

[0106] Also, chloro(N,N-diethylamino)(4-methoxyphenyl)phosphine and the following compounds (trans-PO-B1, cis-PO-B1, trans-PO-Na1, and cis-PO-Na1):

[0107]

Chemical formula

[0108] 2-Bromo-3-iodonaphthalene was synthesized according to the previous report (Synthesis, 2005, 5, 798 - 803).

[0109] 1,2-Epoxy-6-bromohexane was synthesized according to the previous report (Angew. Chem. Int. Ed. 2019, 58, 1727 - 1731).

[0110] (4-Bromo-n-butyl)triphenylphosphonium bromide was synthesized according to the previous report (J. Am. Chem. Soc. 2015, 137, 6837 - 6843).

[0111] (10-Bromo-n-decyl)triphenylphosphonium bromide was synthesized according to the previous report (International Publication No. WO2016 / 155679).

[0112] Synthesis Example 1: 1,2-Epoxy-9-bromononane

[0113]

Chemical formula

[0114] While stirring a solution of 9-bromonon-1-ene (1.0 g, 4.87 mmol) in dichloromethane (8 mL), a solution of meta-chloroperbenzoic acid (purity 70%, 1.8 g, 10.4 mmol) in dichloromethane (8 mL) was added dropwise. Then, the mixture was stirred at 25 °C for 16 hours to precipitate meta-chloroperbenzoic acid. The reaction product was filtered to remove the white precipitate, extracted with dichloromethane (DCM; 20 mL × 2), washed with saturated NaHCO3 (20 mL), water (20 mL), and brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 10) to obtain 1,2-epoxy-9-bromononane as a colorless oil (900 mg, yield 83%). 1 H NMR (400 MHz, CDCl3) δ 3.40 (t, J = 6.9 Hz, 2H), 2.93 - 2.87 (m, 1H), 2.77 - 2.72 (m, 1H), 2.46 (dd, J = 5.0, 2.7 Hz, 1H), 1.91 - 1.78 (m, 2H), 1.55 - 1.30 (m, 10H).

[0115] Synthesis Example 2: 2-Bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene

[0116]

Chemical formula

[0117] Into a nitrogen-purged flask, 2-bromo-3-iodonaphthalene (5.4 g, 16.2 mmol), bis(triphenylphosphine)palladium dichloride (PdCl2(PPh3)2; 231 mg, 0.33 mmol), CuI (62 mg, 0.33 mmol), and triethylamine (Et3N; 70 mL) were added and rigorously degassed by the freeze-pump-thaw method. Trimethylsilylacetylene (2.47 mL) was added via syringe and the mixture was stirred at room temperature overnight. The mixture was filtered to remove insoluble compounds and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane) to give 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene as a yellow liquid (4.3 g, 86% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 19.9 Hz, 2H), 7.77 - 7.70 (m, 2H), 7.51 - 7.48 (m, 2H), 0.31 (s, 7H).

[0118] To a solution of the obtained 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene (4.2 g, 14.0 mmol) in CH3OH / tetrahydrofuran (1 / 1, v / v, 140 mL) was added K2CO3 (5.8 g, 42.0 mmol) and the mixture was stirred at room temperature for 8 hours. The product was extracted with CH2Cl2 (40 mL), washed with 1M HCl (100 mL), and then dried over Na2SO4. The solvent was removed under reduced pressure to give 2-bromo-3-ethynylnaphthalene as a brown solid (3.1 g, 97% yield). 1 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.6 Hz, 2H), 7.80 - 7.71 (m, 2H), 7.56 - 7.48 (m, 2H), 3.40 (s, 1H).

[0119] A suspension of 2-bromo-4-chloro-1-iodobenzene (3.98 g, 12.5 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4; 1.45 g, 13.0 mmol), and CuI (47.7 mg, 0.25 mmol) in triethylamine (150 mL) was degassed, and 2-bromo-3-ethynylnaphthalene (2.9 g, 12.5 mmol) obtained above was added thereto. After stirring overnight at room temperature, the mixture was diluted with toluene (150 mL) and filtered to remove insoluble compounds. The resulting filtrate was concentrated under reduced pressure and then recrystallized from ethanol for purification to obtain 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (Compound 1) as a white solid (4.4 g, yield 85%). 1 H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 5.5 Hz, 2H), 7.84-7.77 (m, 1H), 7.76-7.72 (m, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.54-7.49 (m, 2H), 7.31 (dd, J = 8.3, 2.0 Hz, 1H). 13 C NMR (125 MHz, CDCl3) δ 134.98, 134.23, 133.97, 133.76, 132.47, 131.75, 131.24, 127.97, 127.80, 127.65, 127.08, 126.99, 126.03, 123.93, 122.17, 121.63, 93.61, 91.03。

[0120] Synthesis Example 3: trans-PO-Na2 and cis-PO-Na2

[0121]

Chemical Structure

[0122] To a solution of 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (Compound 1; 1.0 g, 2.38 mmol) obtained in Synthesis Example 2 in anhydrous tetrahydrofuran (anhydrous THF; 20 mL), a solution of tert-butyllithium (tBuLi) in n-pentane (1.6 M, 6.1 mL, 9.7 mmol) was added dropwise at -78 °C over 1.5 hours. The resulting mixture was stirred at -78 °C for 0.5 hour and then kept at 0 °C for 30 minutes. Then, after cooling again to -78 °C, PCl3 (1.3 mL, 15 mmol) was added and the mixture was warmed to room temperature. After stirring at room temperature for 17 hours as it was, water (1 mL) was added to the mixture. Then, an aqueous solution of H2O2 (30%, 0.8 mL) was added at 0 °C and stirred for 30 minutes, and then an aqueous solution of Na2SO3 (10%, 50 mL) was added at 0 °C. The reaction mixture was extracted with ethyl acetate (EtOAc; 200 mL), the organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, and then filtered. The resulting filtrate was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography (CH2Cl2 / acetone = 1 / 10 to 1 / 1) to obtain 469 mg (yield 34%) of trans-PO-Na2 as a green-yellow solid and 540 mg (39%) of cis-PO-Na2 as a green-yellow solid. trans-PO-Na2: 1 H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 11.5, 1.8 Hz, 1H), 7.84-7.72 (m, 7H), 7.60 (dt, J = 11.3, 1.4 Hz, 1H), 7.56-7.45 (m, 2H), 7.42-7.34 (m, 2H), 7.02-6.92 (m, 4H), 3.80 (d, J = 0.8 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 32.54, 32.32, 31.04, 30.82. HRMS (ESI): m / z calcd. For C 32 H 24 ClO4P2: 569.0833 ([M+H] + ); found: 569.0826。 Cis-PO-Na2: 1 1H NMR (400 MHz, CDCl3) δ 8.14 (dd, J = 11.5, 1.7 Hz, 1H), 7.87 (d, J = 3.3 Hz, 1H), 7.80 (t, J = 8.2 Hz, 2H), 7.69 - 7.59 (m, 5H), 7.59 - 7.48 (m, 2H), 7.48 - 7.41 (m, 2H), 6.97 - 6.91 (m, 4H), 3.82 (d, J = 1.3 Hz, 6H). 31 31P NMR (162 MHz, CDCl3) δ 31.00 (d, J = 37.0 Hz), 29.78 (d, J = 37.0 Hz). HRMS (ESI): m / z calcd. For C 32 H 24 ClO4P2: 569.0833 ([M+H] + ); found: 569.0827。

[0123] Example 1: trans-PO-BphoxM

[0124]

Chem.

[0125] Trans-PO-B1 (0.5 g, 0.96 mmol), diphenylamine (0.82 g, 4.86 mmol), palladium acetate (Pd(OAc)2; 13 mg, 0.058 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos; 50 mg, 0.105 mmol), and K2CO3 (801 mg, 5.8 mmol) were added to anhydrous toluene (60 mL). The mixture was stirred at 80 °C for 20 h under a nitrogen atmosphere. Then, it was cooled to room temperature, and the insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (acetone / CH2Cl2 = 1 / 15 - 1 / 10) to give trans-PO-BphoxM (520 mg, yield 83%) as a red solid. 1 1H NMR (400 MHz, CDCl3) δ 7.80 - 7.68 (m, 4H), 7.64 - 7.57 (m, 1H), 7.45 - 7.35 (m, 2H), 7.33 - 7.30 (m, 2H), 7.29 - 7.21 (m, 5H), 7.09 - 7.03 (m, 6H), 7.00 - 6.95 (m, 5H), 3.83 (s, 6H). 31 31P NMR (162 MHz, CDCl3) δ 32.97. HRMS (ESI): m / z calcd. For C 40 H 32 NO4P2: 652.1801 ([M + H] + ); found: 652.1793。

[0126] Example 2: trans-PO-BphoxA1 / A2

[0127]

Chem.

[0128] To a solution of trans-PO-BphoxM (270 mg, 0.40 mmol) in anhydrous CH2Cl2 (21 mL) obtained in Example 1, BBr3 (0.85 mL, 8.3 mmol) was slowly added dropwise at -78 °C. After stirring at -78 °C for 1 hour, the temperature was slowly raised to room temperature over 4 hours. Water (5 mL) was added at 0 °C, and the mixture was extracted with ethyl acetate (EtOAc; 20 mL × 2). The organic layer was dried over Na2SO4, and the crude product was purified by column chromatography (CH3OH / CH2Cl2 = 1 / 20) to obtain trans-PO-B2 as a red solid (0.22 g, yield 88%). 1 1H NMR (400 MHz, CH3OH-d4) δ 7.71 - 7.53 (m, 6H), 7.47 - 7.41 (m, 1H), 7.38 (dd, J = 7.4, 2.3 Hz, 1H), 7.35 - 7.25 (m, 5H), 7.21 - 7.04 (m, 8H), 6.96 - 6.91 (m, 4H). HRMS (ESI): m / z calculated for C 38 H 27 NNaO4P2: 646.1308 ([M+Na] + ); found: 646.1301。

[0129]

Chem.

[0130] The trans-PO-B2 (30 mg, 0.048 mmol) obtained above, (10-bromo-n-decyl)triphenylphosphonium bromide (27 mg, 0.048 mmol) and K2CO3 (133 mg, 0.96 mmol) were added to anhydrous dimethylformamide (anhydrous DMF; 3.6 mL), and the mixture was stirred at room temperature for 12 hours. Then, epibromohydrin (13.1 mg, 0.096 mmol) was added at room temperature, and the mixture was stirred at 50 °C for 14 hours. After cooling to room temperature, water (20 mL) was added, and the mixture was extracted with CH2Cl2 (20 mL). The organic layer was washed twice with water (20 mL), dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting solid was purified by silica gel column chromatography (CH3OH / CH2Cl2 = 1 / 30 - 1 / 20) to obtain trans-PO-BphoxA1 / A2 as a red solid (15 mg, yield 27%). HRMS (ESI): m / z calculated for C 69 H 65 NO5P3: 1080.4070 ([M-Br] + ); found: 1080.4042。

[0131] Example 3: cis-PO-BphoxM

[0132]

Chem.

[0133] As a starting compound, cis-PO-BphoxM (yield 78%) was obtained in the same manner as in Example 1, except that cis-PO-B1 was used instead of trans-PO-B1. 1 H NMR (400 MHz, CDCl3) δ 7.65 - 7.53 (m, 5H), 7.43 (d, J = 4.9 Hz, 2H), 7.35 - 7.20 (m, 7H), 7.10 - 7.02 (m, 6H), 7.00 (dd, J = 8.3, 2.2 Hz, 1H), 6.92 (d, J = 8.2 Hz, 4H), 3.82 (d, J = 0.6 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 31.69. HRMS (ESI): m / z calcd. For C 40 H 31 NNaO4P2: 674.1621 ([M+Na] + ); found: 674.1615.

[0134] Example 4: trans-PO-Naphox(1,2)M

[0135]

Chemical formula

[0136] trans-PO-Na1 (110 mg, 0.19 mmol), diphenylamine (163 mg, 0.968 mmol), palladium(II) acetate (Pd(OAc)2; 2.6 mg, 0.0116 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos; 11 mg, 0.0232 mmol), and K2CO3 (160 mg, 1.16 mmol) were added to anhydrous toluene (12 mL). The mixture was stirred at 90 °C for 24 h under a nitrogen atmosphere. Then, it was cooled to room temperature and the insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (acetone / CH2Cl2 = 1 / 20 - 1 / 10) to give trans-PO-Naphox(1,2)M (120 mg, 88% yield) as a red solid. 1 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.85 - 7.72 (m, 5H), 7.52 (dd, J = 8.3, 2.8 Hz, 1H), 7.46 - 7.38 (m, 2H), 7.35 - 7.27 (m, 2H), 7.27 - 7.21 (m, 4H), 7.11 - 7.02 (m, 6H), 7.01 - 6.98 (m, 1H), 6.98 - 6.91 (m, 4H), 3.80 (d, J = 5.5 Hz, 6H). 31 31P NMR (162 MHz, CDCl3) δ 34.14 (d, J = 40.0 Hz), 32.96 (d, J = 39.9 Hz). HRMS (ESI): m / z calcd. For C 44 H 34 NO4P2: 702.1958 ([M+H] + ); found: 702.1948.

[0137] Example 5: trans-PO-Naphox(2,3)M

[0138]

Chemical Structure

[0139] The trans-PO-Na2 (90 mg, 0.158 mmol) obtained in Synthesis Example 3, diphenylamine (133 mg, 0.79 mmol), palladium acetate (Pd(OAc)2; 2.1 mg, 0.0095 mmol), 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (Xphos; 9 mg, 0.019 mmol), and K2CO3 (131 mg, 0.948 mmol) were added to anhydrous toluene (10 mL), and the mixture was stirred at 90 °C for 24 hours under a nitrogen atmosphere. Then, it was cooled to room temperature, and the insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (acetone / CH2Cl2 = 1 / 15 - 1 / 10) to obtain trans-PO-Naphox(2,3)M (100 mg, yield 90%) as a red solid. 1 H NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 11.5, 1.9 Hz, 1H), 7.84 - 7.73 (m, 7H), 7.48 (dtd, J = 16.1, 7.0, 1.0 Hz, 2H), 7.35 (dt, J = 11.3, 2.0 Hz, 1H), 7.30 - 7.21 (m, 5H), 7.11 - 7.03 (m, 6H), 7.02 - 6.93 (m, 5H), 3.82 (d, J = 2.7 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 33.21 (d, J = 37.4 Hz), 31.35 (d, J = 37.3 Hz). HRMS (ESI): m / z calcd. For C 44 H 34 NO4P2: 702.1958 ([M+H] + ); found: 702.1955。

[0140] Example 6: trans-PO-NaphoxA1 / A2

[0141]

Chemical Structure

[0142] To a solution of trans-PO-Naphox(2,3)M (90 mg, 0.128 mmol) obtained in Example 5 in anhydrous CH2Cl2 (8 mL), BBr3 (0.28 mL, 2.56 mmol) was slowly added dropwise at -78 °C. After stirring at -78 °C for 1 hour, the temperature was slowly raised to room temperature over 4 hours. Water (5 mL) was added at 0 °C, and the mixture was extracted with ethyl acetate (EtOAc; 20 mL × 2). After drying the organic layer over Na2SO4, the crude product was purified by column chromatography (CH3OH / CH2Cl2 = 1 / 20) to obtain trans-PO-Na3 as a red solid (80 mg, yield 92%). 1 H NMR (400 MHz, CH3OH-d4) δ8.19 (dd, J = 11.7, 1.9 Hz, 1H), 7.95-7.84 (m, 2H), 7.78 (d, J = 3.4 Hz, 1H), 7.73-7.63 (m, 4H), 7.63-7.52 (m, 2H), 7.36-7.26 (m, 5H), 7.20 (dt, J = 11.4, 2.0 Hz, 1H), 7.16-7.03 (m, 7H), 6.98-6.91 (m, 4H). 31 P NMR (162 MHz, CH3OH-d4) δ 36.29 (d, J = 38.0 Hz), 34.71 (d, J = 38.4 Hz). HRMS (ESI): m / z calcd. For C 42 H 30 NO4P2: 673.1650 ([M+H] + ); found: 674.1641。

[0143]

Chemical Structure

[0144] The obtained trans-PO-Na3 (30 mg, 0.0445 mmol), (10-bromodecyl)triphenylphosphonium bromide (25 mg, 0.0445 mmol), and K2CO3 (123 mg, 0.89 mmol) were added to anhydrous dimethylformamide (anhydrous DMF; 3.6 mL), and the mixture was stirred at room temperature for 24 hours. Then, epibromohydrin (12.2 mg, 0.089 mmol) was added at room temperature, and the mixture was stirred at 50 °C for 14 hours. After cooling to room temperature, water (20 mL) was added, and the mixture was extracted with CH2Cl2 (20 mL). The organic layer was washed twice with water (20 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the obtained solid was purified by silica gel column chromatography (CH3OH / CH2Cl2 = 1 / 30 - 1 / 10) to obtain trans-PO-NaphoxA1 / A2 as an orange-red solid (13 mg, yield 24%). HRMS (ESI): m / z calcd. For C 73 H 67 NO5P3: 1130.4227 ([M-Br] + ); found: 1130.4229。

[0145] Example 7: cis-PO-Naphox(2,3)M

[0146]

Chemical formula

[0147] Using cis-PO-Na2 obtained in Synthesis Example 3 instead of trans-PO-Na2 obtained in Synthesis Example 3 as the starting compound, cis-PO-Naphox(2,3)M (yield 81%) was obtained in the same manner as in Example 5. 11H NMR (400 MHz, CDCl3) δ 8.10 (dd, J = 11.5, 1.8 Hz, 1H), 7.86 - 7.74 (m, 3H), 7.71 - 7.60 (m, 4H), 7.57 - 7.51 (m, 1H), 7.50 - 7.44 (m, 1H), 7.40 - 7.31 (m, 2H), 7.28 - 7.23 (m, 3H), 7.12 - 7.00 (m, 7H), 6.93 (ddd, J = 9.0, 2.3, 1.1 Hz, 4H), 3.83 (d, J = 3.8 Hz, 6H). 31 31P NMR (162 MHz, CDCl3) δ 31.72 (d, J = 37.5 Hz), 30.11 (d, J = 37.9 Hz). HRMS (ESI): m / z calcd. For C 44 H 33 NNaO4P2: 724.1777 ([M+Na] + ); found: 724.1765。

[0148] Example 8: trans-PS-BphoxA1 / A2

[0149]

Chem.

[0150] A solution of a mixture of trans-PO-BphoxM (40.0 mg, 0.061 mmol) and Lawesson's reagent (99 mg, 0.246 mmol) in anhydrous toluene (5 mL) obtained in Example 1 was refluxed for 20 h. The solution was cooled to room temperature, and after the solvent was removed under reduced pressure, the resulting solid was purified by silica gel column chromatography (CH2Cl2 / hexane = 1 / 2 - 1 / 1) to obtain 34 g (yield 81%) of trans-PS-BphoxM and 8 mg (yield 19%) of cis-PS-BphoxM as orange solids. trans-PS-BphoxM: 11H NMR (400 MHz, CDCl3) δ 7.86 - 7.74 (m, 4H), 7.60 (dd, J = 12.8, 7.1 Hz, 1H), 7.52 - 7.46 (m, 1H), 7.44 - 7.38 (m, 1H), 7.37 - 7.29 (m, 3H), 7.26 - 7.21 (m, 4H), 7.11 - 7.03 (m, 6H), 7.00 - 6.91 (m, 5H), 3.82 (s, 6H). 31 31P NMR (162 MHz, CDCl3) δ 37.47. HRMS (ESI): m / z calcd. For C 40 H 31 NNaO2P2S2: 706.1164 ([M+Na] + ); found: 706.1151。 cis-PS-BphoxM: 1 1H NMR (400 MHz, CDCl3) δ 7.85 - 7.75 (m, 4H), 7.62 - 7.57 (m, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.36 - 7.30 (m, 3H), 7.26 - 7.21 (m, 4H), 7.09 - 7.03 (m, 6H), 6.99 - 6.91 (m, 5H), 3.82 (s, 6H). 31 31P NMR (162 MHz, CDCl3) δ 36.91. HRMS (ESI): m / z calcd. For C 40 H 31 NNaO2P2S2: 706.1164 ([M+Na] + ); found: 706.1165。

[0151]

Chem.

[0152] To a solution of the trans-PS-BphoxM (27 mg, 0.039 mmol) obtained above in anhydrous CH2Cl2 (2 mL), BBr3 (0.08 mL, 0.79 mmol) was added dropwise at -78 °C. The solution was stirred at -78 °C for 1 hour, and then the mixture was slowly warmed to room temperature over 4 hours. At 0 °C, the reaction was quenched with water (2 mL), and extracted with ethyl acetate (20 mL × 2). The orange layer was separated and dried over Na2SO4. The crude product was purified by column chromatography (CH3OH / CH2Cl2 = 1 / 20) to obtain trans-PS-B1 as an orange-red solid (20 mg, yield 79%).

[0153] [Chemical formula]

[0154] As a starting compound, trans-PS-BphoxA1 / A2 (yield 17%) was obtained in the same manner as in Example 2, except that the trans-PS-B1 obtained above was used instead of trans-PO-B2.

[0155] Comparative Example 1: Mito-PB yellow M

[0156] [Chemical formula]

[0157] Mito-PB yellow M of Comparative Example 1 was synthesized according to a previous report (PNAS, 2019, 116, 15817-15822).

[0158] Comparative Example 2: Mito Tracker Deep Red Commercially available Mito-Tracker Deep Red was used as the compound of Comparative Example 2.

[0159] Test Example 1: Optical properties For cis-PO-BphoxM of Example 3, lance-PO-Naphox(1,2)M of Example 4, and trans-PS-BphoxC4 of Example 8, measurements were carried out on the ultraviolet-visible absorption spectrum, fluorescence spectrum, absolute fluorescence quantum yield, etc. when dissolved in toluene, dichloromethane (DCM), or acetonitrile (MeCN). Appearance photos were taken. The results of Examples 3 and 8 are shown in Figure 1, the results of Example 4 are shown in Figure 2, and each optical property data is shown in Table 1.

[0160]

Table 1

[0161] From the above, the bisphosphoryl-bridged stilbene compound of the present invention can absorb light in the visible light region (especially about 450 to 550 nm) in any of various solvents, and can emit fluorescence of various wavelengths depending on the solvent. In particular, since the fluorescence was extremely small in a polar solvent such as acetonitrile, it is suggested that there is almost no fluorescence in the cytoplasm. For this reason, it is suggested that it is possible to stain hydrophobic tissues such as phospholipid membranes and lipid droplets with high contrast.

[0162] Test Example 2: Light resistance (in acetonitrile) trans-PO-BphoxM of Example 1, cis-PO-BphoxM of Example 3, trans-PO-Naphox(1,2)M of Example 4, trans-PO-Naphox(2,3)M of Example 5, and Mito-PB yellow M of Comparative Example 1 were dissolved in acetonitrile (MeCN) at 25°C. The concentration at this time was adjusted so that the absorbance at 515 nm was about the same (0.1). An LED light (2050 W / m 2 ) equipped with a band-pass filter that transmits light of 515 ± 5 nm was used to irradiate light of wavelength 515 ± 5 nm, and the ultraviolet-visible absorption spectrum was measured after various time elapses. The results of Examples 1 and Comparative Example 1 are shown in Figures 3(a) and (c), the results of Examples 3 and 5 are shown in Figures 4(a) and (c), and the result of Example 4 is shown in Figure 5(a).

[0163] Next, for each sample, the light resistance at the maximum absorption wavelength was evaluated. Specifically, an LED light equipped with a band-pass filter that transmits light at 515 ± 5 nm (2050 W / m 2 ) was used to irradiate the sample with light at a wavelength of 515 ± 5 nm. Taking the absorbance (A0) at the maximum absorption wavelength immediately after irradiation (0 hours later) (460 nm for Example 1, 487 nm for Example 3, 513 nm for Example 4, 482 nm for Example 5, and 487 nm for Comparative Example 1) as 1.00, the retention rate (A / A0) of the absorbance (A) after a predetermined time was evaluated. The results of Example 1 and Comparative Example 1 are shown in FIGS. 3(b) and (d), the results of Examples 3 and 5 are shown in FIGS. 4(b) and (d), and the result of Example 4 is shown in FIG. 5(b).

[0164] As a result, the bisphosphoryl cross-linked stilbene compound of the present invention has significantly better light resistance compared to Mito-PB yellow, and the absorbance hardly decreased.

[0165] Test Example 3: Light resistance (in DMSO / H 2 O part 1) For trans-PO-Naphox(2,3)M of Example 5 and Mito-PB yellow M of Comparative Example 1, they were dissolved in a dimethyl sulfoxide / water mixed solvent (volume ratio 1:1) at 25°C. The concentration at this time was adjusted so that the absorbance at 515 nm was about the same (0.1). An LED light equipped with a band-pass filter that transmits light at 515 ± 5 nm was used to irradiate the sample with light at a wavelength of 515 ± 5 nm at an irradiation intensity of 2050 W / m 2 , and the ultraviolet-visible absorption spectra were measured after various time intervals. The result of Comparative Example 1 is shown in FIG. 6(a), and the result of Example 5 is shown in FIG. 6(c).

[0166] Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, an LED light equipped with a band-pass filter that transmits light at 515 ± 5 nm was used to irradiate the sample with light at a wavelength of 515 ± 5 nm at an irradiation intensity of 2050 W / m 2Irradiate with [light source], and with the absorbance (A0) at the maximum absorption wavelength immediately after irradiation (0 hours later) (506 nm for Example 5 and 473 nm for Comparative Example 1) set to 1.00, the retention rate (A / A0) of the absorbance (A) after a predetermined time elapsed was evaluated. The results of Comparative Example 1 are shown in Fig. 6(b), and the results of Example 5 are shown in Fig. 6(d).

[0167] Test Example 4: Light resistance (in DMSO / H 2 O part 2) For cis-PO-BphoxM of Example 3, trans-PO-Naphox(2,3)M of Example 5, and Mito-PB ywllow M of Comparative Example 1, they were dissolved in a dimethyl sulfoxide / water mixed solvent (volume ratio 1:1) at 25 °C. The concentration at this time was adjusted so that the absorbance at 450 nm was about the same (0.1). With an LED light equipped with a band-pass filter that transmits light at 450 ± 5 nm, light with a wavelength of 450 ± 5 nm was irradiated at an irradiation intensity of 2050 W / m 2 or at an intensity of 97% of the maximum intensity of the LED light, and the ultraviolet-visible absorption spectra were measured after various time intervals. The results of the irradiation intensity of 2050 W / m for Example 3 are shown in Fig. 7(a), the results of the intensity of 97% of the maximum intensity of the LED light for Example 3 are shown in Fig. 7(c), the results of the irradiation intensity of 2050 W / m for Example 5 are shown in Fig. 8(a), the results of the intensity of 97% of the maximum intensity of the LED light for Example 5 are shown in Fig. 8(c), the results of the irradiation intensity of 2050 W / m for Comparative Example 1 are shown in Fig. 9(a), and the results of the intensity of 97% of the maximum intensity of the LED light for Comparative Example 1 are shown in Fig. 9(c). 2 The results of [intensity value 1] are shown in Fig. 7(a), the results of the intensity of 97% of the maximum intensity of the LED light for Example 3 are shown in Fig. 7(c), the results of the irradiation intensity of [intensity value 2] for Example 5 are shown in Fig. 8(a), the results of the intensity of 97% of the maximum intensity of the LED light for Example 5 are shown in Fig. 8(c), the results of the irradiation intensity of [intensity value 3] for Comparative Example 1 are shown in Fig. 9(a), and the results of the intensity of 97% of the maximum intensity of the LED light for Comparative Example 1 are shown in Fig. 9(c). 2 The results of [intensity value 2] are shown in Fig. 8(a), the results of the intensity of 97% of the maximum intensity of the LED light for Example 5 are shown in Fig. 8(c), the results of the irradiation intensity of [intensity value 3] for Comparative Example 1 are shown in Fig. 9(a), and the results of the intensity of 97% of the maximum intensity of the LED light for Comparative Example 1 are shown in Fig. 9(c). 2 The results of [intensity value 3] are shown in Fig. 9(a), and the results of the intensity of 97% of the maximum intensity of the LED light for Comparative Example 1 are shown in Fig. 9(c).

[0168] Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, with an LED light equipped with a band-pass filter that transmits light at 450 ± 5 nm, light with a wavelength of 450 ± 5 nm was irradiated at an irradiation intensity of 2050 W / m 2 or at an intensity of 97% of the maximum intensity of the LED light, and with the absorbance (A0) at the maximum absorption wavelength immediately after irradiation (0 hours later) (509 nm for Example 3, 504 nm for Example 5, and 473 nm for Comparative Example 1) set to 1.00, the retention rate (A / A0) of the absorbance (A) after a predetermined time elapsed was evaluated. The results of the irradiation intensity of 2050 W / m for Example 3 are shown in Fig. [figure number 1], the results of the intensity of 97% of the maximum intensity of the LED light for Example 3 are shown in Fig. [figure number 2], the results of the irradiation intensity of 2050 W / m for Example 5 are shown in Fig. [figure number 3], the results of the intensity of 97% of the maximum intensity of the LED light for Example 5 are shown in Fig. [figure number 4], the results of the irradiation intensity of 2050 W / m for Comparative Example 1 are shown in Fig. [figure number 5], and the results of the intensity of 97% of the maximum intensity of the LED light for Comparative Example 1 are shown in Fig. [figure number 6]. 2 It should be noted that some specific values like [light source], [intensity value 1], [intensity value 2], [intensity value 3], [figure number 1], [figure number 2], [figure number 3], [figure number 4], [figure number 5], [figure number 6] need to be filled in according to the actual context in the original text. Since they are not clearly provided in the given text, they are left as placeholders here.The results of are shown in Fig. 7(b), the results of the intensity of 97% of the maximum intensity of the LED light in Example 3 are shown in Fig. 7(d), and the irradiation intensity of Example 5 is 2050 W / m 2 The results of are shown in Fig. 8(b), the results of the intensity of 97% of the maximum intensity of the LED light in Example 5 are shown in Fig. 8(d), and the irradiation intensity of Comparative Example 1 is 2050 W / m 2 The results of are shown in Fig. 9(b), and the results of the intensity of 97% of the maximum intensity of the LED light in Comparative Example 1 are shown in Fig. 9(d).

[0169] Test Example 5: Preparation of cells (part 1) HeLa cells (RIKEN Cell Bank, Japan) were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal agent (AA, Sigma) in a 5% CO2 incubator for 24 hours. Three days before imaging, the cells (5×10 4 ) were seeded in a glass-bottom dish.

[0170] Test Example 6: Fluorescent imaging of cells (part 1) In the staining experiment of trans-PO-BphoxA1 / A2 in Example 2, HeLa cells were cultured at 37 °C for 2 hours in a DMEM medium containing 500 nM trans-PO-BphoxA1 / A2, 1% dimethyl sulfoxide and 0.1% Pluonic F-127 in a 5% CO2 incubator. After washing the cells three times with DMEM(-), they were replaced with DMEM(-) and observed using a super-resolution microscope TCS SP8 STED (manufactured by Leica). At this time, the excitation wavelength is preferably 470 - 540 nm, optimally 488 - 515 nm, and the fluorescence signal in the range of 550 - 750 nm was detected. The results are shown in Fig. 10.

[0171] In the staining experiments of trans-PO-NaphoxA1 / A2 in Example 6 and Mito Tracker Deep Red in Comparative Example 2, the same procedure was performed. The results are shown in Figs. 10 and 11.

[0172] Test Example 7: Fluorescent imaging of cells (part 2) In the staining experiment of trans-PO-BphoxA1 / A2 of Example 2, HeLa cells were cultured in DMEM medium containing 500 nM trans-PO-BphoxA1 / A2, 1% dimethyl sulfoxide and 0.1% Pluonic F-127 at 37 °C for 2 hours in a 5% CO2 incubator. After washing the cells three times with DMEM(-), they were replaced with DMEM(-), and imaging was performed using a super-resolution microscope TCS SP8 STED (manufactured by Leica).

[0173] For confocal imaging, using the super-resolution microscope TCS SP8 STED, the excitation wavelength is preferably 470 - 540 nm, optimally 488 - 515 nm, and fluorescence signals in the range of 550 - 750 nm were detected. On the other hand, for STED imaging, in order to obtain a super-resolution image, a 775 nm STED laser (pulsed laser) was used, and signals in the range of 550 - 750 nm were detected.

[0174] The same procedure was carried out in the staining experiment of trans-PO-NaphoxA1 / A2 of Example 6.

[0175] The results of trans-PO-BphoxA1 / A2 of Example 2 are shown in Figure 12, and the results of trans-PO-NaphoxA1 / A2 of Example 6 are shown in Figure 13.

[0176] Test Example 8: Preparation of cells (part 2) HeLa cells (RIKEN Cell Bank, Japan) were treated with oleic acid (400 nM) in a 5% CO2 incubator for 24 hours, and the medium was changed. HeLa cells (RIKEN Cell Bank, Japan) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal agent (AA, Sigma) in a 5% CO2 incubator for 24 hours. Three days before imaging, the cells (5×10 4 ) were seeded in a glass-bottom dish.

[0177] Test Example 9: Fluorescent imaging of cells (part 3) In the staining experiment of trans-PO-BphoxM of Example 1, HeLa cells were cultured at 37 °C for 2 hours in a 5% CO2 incubator in DMEM medium containing 500 nM of trans-PO-BphoxM, 1% dimethyl sulfoxide, and 0.1% Pluonic F-127. After washing the cells three times with DMEM(-), they were replaced with DMEM(-) and observed using a super-resolution microscope TCS SP8 STED and FV-10 (manufactured by Olympus). At this time, the excitation wavelength is preferably 470 to 540 nm, optimally 488 to 515 nm, and a fluorescence signal in the range of 550 to 750 nm was detected.

[0178] The staining experiments of cis-PO-BphoxM of Example 3, trans-PO-Naphox(1,2)M of Example 4, trans-PO-Naphox(2,3)M of Example 5, and cis-PO-Naphox(2,3)M of Example 7 were also carried out in the same manner.

[0179] The results are shown in Figure 14.

[0180] As a result, the bisphosphoryl-bridged stilbene compound of the present invention was able to selectively stain lipid droplets and mitochondria respectively by modifying R. 3 ​

Claims

1. General formula (1): 【Chemical 1】 [In the formula, Ar １ and Ar ２ are the same or different and each represents a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R １ and R ２ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may together with the adjacent nitrogen atom form a ring. R １ and / or R ２ may together with Ar ２ and the adjacent nitrogen atom form a ring. Y １ and Y ２ are the same or different and are represented by the general formula (2): 【Chemical 2】 (Ar ３ represents a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R ３ represents a substituted or unsubstituted alkyl group, or a polyethylene glycol group or a derivative thereof. Y ３ represents an oxygen atom or a sulfur atom.) represents a group represented by.] A bisphosphoryl cross-linked stilbene compound represented by.

2. The aforementioned R ３ The bisphosphoryl-crosslinked stilbene compound according to claim 1, wherein R is a substituted or unsubstituted alkyl group.

3. Ar １ The bisphosphoryl crosslinked stilbene compound according to claim 1 or 2, wherein Ar is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

4. A fluorescent dye containing the bisphosphoryl cross-linked stilbene compound according to any one of Claims 1 to 3.

5. An organelle stain containing the bisphosphoryl cross-linked stilbene compound according to any one of Claims 1 to 3.

6. A method for suppressing the induced release (STED) imaging of organelles using the bisphosphoryl cross-linked stilbene compound according to any one of Claims 1 to 3, the fluorescent dye according to Claim 4, or the organelle stain according to Claim 5.

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