Infrared fluorescent dyes and their use

Abstract

The present application provides an infrared fluorescent dye and application thereof. Specifically, the present application provides a compound shown in the following formula A, wherein R1-R 17 and A ‑ are defined as defined herein. The maximum absorption wavelength of the dye molecule of the present application is distributed in the range of 740-838 nm, which is uniformly distributed in the near infrared region, and shows excellent fluorescence brightness and light stability, which is superior to other categories of small organic molecule near infrared fluorescent dyes in the field.

Description

Technical Field

[0001] This invention belongs to the field of dye chemistry, and specifically relates to an infrared fluorescent dye and its applications. Background Technology

[0002] Over the past two decades, super-resolution fluorescence microscopy has improved the resolution of dynamic microscopic imaging of live cells to the nanometer level. Among these advancements, the development of fluorescent dyes for super-resolution microscopy is at the forefront of dye chemistry research. Visible-light super-resolution fluorescence microscopy is susceptible to interference from biological background signals and exhibits phototoxicity, making it unsuitable for long-term imaging of live biological samples. Near-infrared dyes can overcome these limitations and have attracted significant attention in the field of super-resolution fluorescence microscopy. Cyanide dyes, with their high fluorescence brightness and low cytotoxicity, are widely used in near-infrared super-resolution fluorescence microscopy. However, cyanide dyes also have drawbacks. Currently, the cyanide dyes used in near-infrared super-resolution fluorescence microscopy have relatively short wavelengths, ranging from 650-800 nm, and exhibit poor photostability. Existing dyes with wavelengths greater than 800 nm generally suffer from deficiencies in photostability and fluorescence brightness. Therefore, developing super-resolution fluorescence microscopy dyes with wavelengths greater than 800 nm can achieve long-term, low-toxicity dynamic fluorescence microscopy and also enable multi-channel super-resolution imaging in conjunction with visible-light dyes. In addition, near-infrared dyes with wavelengths greater than 800 nm can also be used in fields such as in vivo fluorescence imaging, photodynamic therapy for diseases, and photothermal ablation of tumors, taking advantage of their strong tissue penetration ability and low fluorescence background. Summary of the Invention

[0003] A first aspect of the present invention provides a compound represented by formula A:

[0004]

[0005] In the formula:

[0006] R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from: hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, aryl C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, and optionally one or two selected from C 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl groups; or R1 with R2, R2 with R3, or R3 with R4 and / or R5 with R6, R6 with R7, or R7 with R8, together with their respective attached C atoms, optionally consisting of 1-3 atoms selected from halogens, C atoms ... 1-4 Alkyl and C 1-4 Alkoxy substituents in benzene rings;

[0007] R9, R 10 R 11 R 12 R 13 Each group is independently selected from: hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, aryl C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, optionally selected by 1-5 halogens, C 1-4 The C substituents of alkoxy and azido groups 1-4 Alkyl groups, optionally composed of one or two selected from C 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl group, optionally oxidized to a heterocyclic group -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkoxy groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted aryl C 1-4 Alkoxy;

[0008] R 14 and R 15 Each is independently selected from H, and C is arbitrarily substituted with halogens. 1-6 Alkyl, C 6-14 Aryl and C 2-4 alkenyl; or R 14 and R 15 Together with the nitrogen atoms bonded to them, they form optional groups of 1-5 atoms selected from C. 1-6 3-8 membered heterocyclic groups or 4-15 membered heterobridged cyclic groups substituted with alkyl, halogen, or oxo groups; or R 14 and R 15 Together with the nitrogen atoms to which they are attached, they form 3-8 membered cycloalkyl spiro-4-15 membered heterobridged cyclogroups or 3-8 membered heterocyclic spiro-4-15 membered heterobridged cyclogroups; the heterocyclic groups and heterobridged cyclogroups have 1-5 heteroatoms selected from N, O and S;

[0009] R 16 and R 17 Each is independently selected from H, and C is arbitrarily substituted with halogens. 1-4 Alkyl, C 1-4 Alkoxy and C 2-4 alkenyl; or R 16 Its connected C, R14 R 14 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents of 5-7 membered nitrogen-containing heterocycles, and / or R 17 Its connected C, R 15 R 15 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents in 5-7 membered nitrogen-containing heterocycles;

[0010] A - The acid is an acid radical ion that has been deprotonated, wherein the acid is selected from one or more of HCl, trifluoroacetic acid, methanesulfonic acid, acetic acid, and sulfuric acid;

[0011] X is a halogen;

[0012] Y is either O or S;

[0013] n is an integer selected from 1 to 500.

[0014] The present invention also provides a fluorescent dye comprising the compound described in any embodiment herein and an optional solvent.

[0015] The present invention also provides a dye composition comprising the compound described in any embodiment herein and an optional solvent.

[0016] The present invention also provides a kit containing the compound described in any embodiment herein and an optional solvent, or containing the fluorescent dye or dye composition described herein.

[0017] The present invention also provides the use of the compounds described in any embodiment herein in the preparation of reagents for near-infrared fluorescence imaging, photothermal therapy and / or photodynamic therapy. Attached Figure Description

[0018] Figure 1 This is the absorption and emission spectrum of compound 10 in dichloromethane.

[0019] Figure 2 This is the absorption and emission spectrum of compound 12 in dichloromethane.

[0020] Figure 3 This is the absorption and emission spectrum of compound 17 in dichloromethane.

[0021] Figure 4 This is the absorption and emission spectrum of compound 21 in dichloromethane.

[0022] Figure 5This is the absorption and emission spectrum of compound 24 in dichloromethane.

[0023] Figure 6 This is the absorption and emission spectrum of compound 26 in dichloromethane.

[0024] Figure 7 This is the absorption and emission spectrum of compound 28 in dichloromethane.

[0025] Figure 8 This is the absorption and emission spectrum of compound 30 in dichloromethane.

[0026] Figure 9 This is the absorption and emission spectrum of compound 32 in dichloromethane.

[0027] Figure 10 This is the absorption and emission spectrum of compound 34 in dichloromethane.

[0028] Figure 11 This is the absorption and emission spectrum of compound 46 in dichloromethane.

[0029] Figure 12 These are super-resolution imaging results of compound 34 for nanospheres. Figure A shows a comparison of wide-field imaging and structured illumination microscopy (SIM) super-resolution imaging of nanospheres with different nanoparticle sizes. Figure B shows the imaging results of 300 nm nanospheres. Figure C is a magnified view of the SIM super-resolution imaging of the area selected in Figure B. Figure D is a magnified view of the wide-field fluorescence microscopy imaging of the area selected in Figure B.

[0030] Figure 13 These are SIM super-resolution microscopy and wide-field imaging images of compound 36 used in fixed cells (U2OS cell line) mitochondria and live endosomes.

[0031] Figure 14 These are in vivo fluorescence images of mice after injecting compounds 28, 24, 34 and DSPE-mPEG2000 to encapsulate compound 34.

[0032] Figure 15 Figure A shows the photothermal heating curve of compound 17. Figure A shows the aqueous solutions of compound 17 at different concentrations at 1 W / cm². 2 Figure B shows the temperature change over time of an 808 nm laser irradiation at different power densities for an aqueous solution of compound 17 at the same concentration (10 μM). Figure C shows the temperature change over time of a 15 μM aqueous solution of compound 17 irradiated by an 808 nm laser at a power density of 1 W / cm². 2 The heating curves (0s-1200s) and cooling curves (1200s-2400s) under irradiation by an 808nm laser.

[0033] Figure 16 This is the UV absorption spectrum of a compound produced by detecting singlet oxygen using DPBF. Figure A shows the UV absorption spectrum of an acetonitrile solution with a concentration of 20 μM DPBF after passing through a 1 W / cm² solution. 2 Figure B shows the UV absorption spectra of DPBF and compound 21 after irradiation with an 808 nm laser. Figure C shows the UV absorption spectra of the DPBF and compound 21 in acetonitrile solutions and the UV absorption spectra of their mixture. Figure D shows the UV absorption spectra of the acetonitrile mixture of DPBF and compound 21 after irradiation with a 1 W / cm² laser for different durations. 2 The ultraviolet absorption spectrum after irradiation by an 808nm laser. Detailed Implementation

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0035] the term

[0036] As used herein, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (e.g., C1-C1). 20 C1-C 10 Or C1-C6, where C1-C 20 (Indicates 1-20 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0037] As used herein, "alkenyl" refers to a straight or branched group containing 2 to 20 carbon atoms (e.g., 2 to 10 carbon atoms, 2 to 6 carbon atoms), unless the carbon chain length is otherwise limited, wherein at least two carbon atoms in the chain contain a double bond. Typical alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl.

[0038] As used herein, "alkynyl" refers to a straight-chain or branched group containing 2 to 20 carbon atoms (e.g., 2 to 10 carbon atoms, 2 to 6 carbon atoms), unless the chain length is otherwise limited, wherein at least two carbon atoms in the chain are connected by a triple bond. Typical alkynyl groups include ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl, and 2-butynyl.

[0039] As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. "Halogenated" means substituted by an atom selected from F, Cl, Br, and I. "Halogen anion" refers to an anion formed when a halogen atom gains an electron, including Cl... — ,Br — I— F — .

[0040] As used in this article, "alkylene" refers to a group that is identical to an alkyl group but has a divalent oxidation state. C 1-4 Alkyl groups are divalent groups having 1 to 4 carbon atoms. Examples of alkyl groups include methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-).

[0041] As used herein, a "heterocyclic group" refers to a saturated or partially saturated 3-7 membered monocyclic group or a 7-10 membered bicyclic group, consisting of a carbon atom and 1-4 heteroatoms selected from O, N, and S. Examples of heterocyclic groups include tetrahydrofuranyl, pyranyl, piperidinyl, piperazineyl, tetrahydro-1,4-thiazinyl, tetrahydro-1,3-thiazinyl, tetrahydro-1,2-thiazinyl, 1,3-dioxolanecycloyl, 1,2-oxazinylalkyl, 1,3-oxazinylalkyl, 1,4-oxazinylalkyl, 1,4-diazaheptanyl, aziridine, oxazinyl, pyrrolidinyl, imidazolinyl, imidazolinyl, dihydroindolyl, isodihydroindolyl, morpholinyl, pyrazolylalkyl, pyrazolyl, and tetrahydroisoquinolinyl. "Nitrogen-containing heterocycle" refers to a "heterocyclic group" containing at least one nitrogen atom, and also includes a carbon atom and optionally 1-4 additional heteroatoms selected from O, N and S. Examples of "nitrogen-containing heterocycles" include, but are not limited to, piperidine, tetrahydrothiazine, and oxazine.

[0042] As used herein, “oxo” refers to a group that is substituted with an “=O” group. “Oxyheterocyclic group” refers to a heterocyclic group in which a carbon atom or a heteroatom selected from N or S is substituted with one, two, or three “=O” groups. Examples of “oxyheterocyclic groups” include, but are not limited to, oxopyrrolyl, 3-oxo-1,2,4-triazolyl, 5-oxo-1,2,4-triazolyl, dioxopyrrolyl, oxomorpholinyl, oxopiperidyl, oxopiperazinyl, and dioxothiomorpholinyl, such as 1,1-dioxothiomorpholinyl.

[0043] As used herein, "heteroaryl" refers to a group containing 5-14, preferably 5-10, ring atoms, and having 6, 10, or 14 electrons shared in the ring system. The ring atoms in a heteroaryl group are carbon atoms and 1-3 heteroatoms selected from O, N, and S. In this invention, preferred heteroaryl groups are those containing N atoms, more preferably 5- or 6-membered heteroaryl groups containing N atoms. Examples of heteroaryl groups include: triazolyl, thiophenyl, furanyl, pyranyl, pyrroleyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indazinyl, isoindolyl, indolyl, benzimidazolyl, and pyrazolopyrimidinyl, etc.

[0044] As used herein, "alkoxy" refers to an RO- group, wherein R is an alkyl group as described herein. Preferred alkoxy groups are C0. 1-4 Alkyl groups, such as methoxy, ethoxy, propoxy, etc.

[0045] As used in this article, "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 carbon atoms, i.e., C 6-14 Aryl, preferably C 6-10 Aryl groups. Examples of aryl groups include phenyl, naphthyl, phenanthryl, anthraceneyl, indyl, azulel, biphenyl, biphenylene, and fumonisinyl.

[0046] Aryl C 1-4 Alkoxy refers to C that has been substituted with an aryl group as described in this application. 1-4 Alkyl group. An exemplary aryl C 1-4 Alkoxy groups include benzyloxy groups.

[0047] As used herein, "cycloalkyl" or "carbocyclic" refers to a saturated cyclic hydrocarbon having 3 to 13 ring carbon atoms, comprising one ring such as cyclohexyl or multiple rings such as adamantyl. Cycloalkyl compounds comprising more than one ring can be fused, spirocyclic, bridged, or combinations thereof. Preferred cycloalkyl compounds are saturated cyclic hydrocarbons having 3 to 8 ring carbon atoms ("C..."). 3-8 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 4 to 6 cyclic carbon atoms (“C”). 4-6 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 7 cyclic carbon atoms (“C”). 5-7 Cycloalkyl groups ("cycloalkyl"). Examples of cycloalkyl groups include adamantyl, decahydronaphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0048] As used herein, a "hybrid bridged ring" refers to a structure formed by two or more ring structures sharing two non-adjacent ring atoms, and having 1-4 heteroatoms selected from N, S, and O. A hybrid bridged ring can have 4-15 ring atoms, preferably a 6-12 membered hybrid bridged ring, and more preferably a 7-11 membered hybrid bridged ring. Examples of hybrid bridged rings include, but are not limited to: A "hybridized cyclogroup" is a group formed by the loss of a hydrogen atom when a hybrid cyclogroup is attached to another group.

[0049] As used herein, "heterocyclic spiroheterobridged cyclic group" refers to the heterocyclic group described in this application that is spirolinked with the heterobridged cyclic group described in this application, such as a ketal obtained by reacting a carbonyl-containing bridged heterocycle with ethylene glycol.

[0050] Compounds of the present invention

[0051] This invention provides a compound represented by formula A:

[0052]

[0053] In the formula:

[0054] R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, aryl, and C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, and optionally one or two selected from C 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl groups; or R1 with R2, R2 with R3 or R3 with R4 and / or R5 with R6, R6 with R7 or R7 with R8, together with their respective attached C atoms, optionally formed by 1-3 atoms selected from halogens, C 1-4 Alkyl and C 1-4 Alkoxy substituents in benzene rings;

[0055] R9, R 10 R 11 R 12 R 13 Each group is independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, and aryl C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, 1-5 of which are selected from halogens, C 1-4 The C substituents of alkoxy and azido groups 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 alkoxy or aryl C 1-4 Alkoxy;

[0056] R 14 and R 15 Each is independently selected from H, and C is arbitrarily substituted with halogens. 1-6 Alkyl, C 6-14 Aryl and C 2-4 alkenyl; or R 14 and R 15 Together with the nitrogen atoms bonded to them, they form optional groups of 1-5 atoms selected from C. 1-63-8 membered heterocyclic groups or 4-15 membered heterobridged cyclic groups substituted with alkyl, halogen, or oxo groups; or R 14 and R 15 Together with the nitrogen atoms to which they are attached, they form 3-8 membered cycloalkyl spiro-4-15 membered heterobridged cyclogroups or 3-8 membered heterocyclic spiro-4-15 membered heterobridged cyclogroups; the heterocyclic groups and heterobridged cyclogroups have 1-5 heteroatoms selected from N, O and S;

[0057] R 16 and R 17 Each is independently selected from H, and C is arbitrarily substituted with halogens. 1-4 Alkyl, C 1-4 Alkoxy and C 2-4 alkenyl; or R 16 Its connected C, R 14 R 14 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents of 5-7 membered nitrogen-containing heterocycles, and / or R 17 Its connected C, R 15 R 15 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents in 5-7 membered nitrogen-containing heterocycles;

[0058] A - The acid is an acid radical ion that has been deprotonated, wherein the acid is selected from one or more of HCl, trifluoroacetic acid, methanesulfonic acid, acetic acid, and sulfuric acid;

[0059] X is a halogen;

[0060] Y is either O or S;

[0061] n is an integer between 1 and 500.

[0062] In some implementations, X is Cl.

[0063] In some implementations, Y is O.

[0064] In some implementation schemes, A - For Cl - F3CCOO - or CH3SO3 - .

[0065] Preferably, n is an integer selected from 1 to 400, for example, an integer selected from 10 to 350, an integer selected from 20 to 300, an integer selected from 50 to 250, or an integer selected from 80 to 200. Preferably, n is an integer selected from 100 to 150. In some embodiments, n is an integer selected from 1 to 50. In some embodiments, n is an integer selected from 1 to 20. In some embodiments, n is an integer selected from 1 to 10.

[0066] In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, aryl, and C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, and optionally one or two selected from C 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl group, X, is as described in any embodiment herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, aryl, and C. 1-4 Alkoxy, C 1-4 Alkyl, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, where X is as described in any embodiment herein. Preferably, R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogens, C. 1-4 Alkyl, -SO 3- And -SO2X, X as described in any embodiment herein.

[0067] In some implementations, R1, R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, -SO 3- C 1-4 Alkyl group. Preferably, R1, R2, R4, R5, R7, and R8 are hydrogen, and R3 is H or -SO. 3- R6 is H or -SO 3- Even better, the R3 and R6 substituents are the same.

[0068] In some implementation schemes, R9, R 10 R 11 R 12 R 13 Each group is independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, and aryl C. 1-4 Alkoxy, C 2-4 alkenyl, -SO 3--SO2X, -SO2NH2, C groups optionally substituted with 1-5 substituents selected from halogens and azide groups 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 C substituents of alkoxy, halogen, and azide groups 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 The definitions of alkoxy groups X, Y, and n are as previously stated. In some embodiments, R9, R... 10 R 11 R 12 R 13 Each is selected independently from hydrogen, -SO 3- -SO2X, -SO2NH2, C groups optionally substituted with 1-3 substituents selected from halogens and azide groups 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 C substituents of alkoxy, halogen, and azide groups 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 The definitions of alkoxy groups X, Y, and n are as previously stated. In some embodiments, R9, R... 10 R 11 R 12 R 13 Each is independently selected from: hydrogen, -SO 3- C10 groups are optionally substituted with 1-3 substituents selected from halogens and azide groups. 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 Alkoxy and halogen substituents of C 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 The definitions of alkoxy groups X, Y, and n are as described above.

[0069] In some implementation schemes, R9, R 10 R 12 R 13 Each is independently selected from hydrogen, -SO 3- and optional C1-4 alkoxy-substituted C 1-4 Alkyl; R 11 C10 groups selected from hydrogen, optionally substituted with 1-3 substituents selected from halogens and azide groups. 1-4 Alkoxy groups and optionally oxy-substituted heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkyl group.

[0070] In some embodiments, the oxoheterocyclic group is a 1-, 2-, or 3-oxoheterocyclic group. Preferably, the oxoheterocyclic group is a dioxoheterocyclic group. In some embodiments, the oxoheterocyclic group is a 2,5-dicarbonyl nitrogen-containing heterocyclic group. In this invention, the oxoheterocyclic group is a 5-7 membered heterocyclic group having 1-3 heteroatoms selected from O, S, and N, such as a succinimide group. In some implementations, the oxoheterocyclic group is a dioxo 4-6 membered heterocyclic group.

[0071] In some implementation schemes, R9, R 12 Each is independently H, -SO 3- Or be chosen by C 1-4 alkoxy-substituted C 1-4 Alkyl group. Preferably, R9, R 12 Each is independently H, -SO 3- Or be chosen by C 1-2 alkoxy-substituted C 1-2 Alkyl group. R 10 R 13 It is hydrogen. R 11 C is hydrogen, halogenated and azido-substituted. 1-4 Alkyl groups or succinimide groups -OC(=O)-C 1-2 Alkylene-O-[C 1-2 [alkylene-O] n -C 1-2 alkylene-substituted heteroaryl C 1-4 The definition of alkoxy group n is as described above.

[0072] In some implementation schemes, R 14 and R 15 Each is independently selected from H and C 1-6 Alkyl and C 6-14 aryl; or R 14 and R 15 Together with the nitrogen atoms bonded to them, they form optional groups of 1-3 atoms selected from C. 1-6 4-7 membered heterocyclic groups or 5-12 membered heterobridged cyclic groups substituted with alkyl, halogen, or oxo groups; or R14 and R 15 Together with the nitrogen atoms connected to them, they form 4-7 membered heterocyclic spirochetes and 5-12 membered heterobridged cyclic groups; said heterocyclic groups and heterobridged cyclic groups have 1-3 heteroatoms selected from N, O, and S. In some embodiments, R 14 and R 15 Each is independently selected from H and C 1-6 Alkyl and C 6-10 aryl; or R 14 and R 15 Together with the nitrogen atoms bonded to them, they form optional groups of 1-3 atoms selected from C. 1-6 4-6 membered heterocyclic groups or 6-9 membered heterobridged cyclic groups substituted with alkyl, halogen, or oxo groups; or R 14 and R 15 Together with the nitrogen atoms connected to them, they form 4-6 membered heterocyclic spirocyclic and 6-9 membered heterobridged cyclic groups; the heterocyclic groups and heterobridged cyclic groups have 1-2 heteroatoms selected from N, O and S.

[0073] In some embodiments, the heterocyclic group has one or two ions selected from oxo, fluorine, chlorine, C... 1-4 Alkyl substituents.

[0074] In some implementation schemes, R 16 and R 17 Each is independently selected from H and C 1-4 Alkyl and C 1-4 alkoxy; or R 16 Its connected C, R 14 R 14 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents of 5-7 membered nitrogen-containing heterocycles, and / or R 17 Its connected C, R 15 R 15 The connected N together form 1-4 selected from C 1-4 Alkyl and Halogenated C 1-4 Alkyl substituents substituted 5-7 membered nitrogen-containing heterocycles.

[0075] In some implementation schemes, R 16 and R 17 Each is independently selected from H and C 1-4 alkoxy; or R 16 Its connected C, R 14 R 14 The attached N atoms together form a 5-7 member nitrogen-containing heterocycle, and / or R atoms. 17 Its connected C, R 15 R 15The attached N atoms together form a 5-7 member nitrogen-containing heterocycle. In some embodiments, R... 16 and R 17 C 1-4 Alkoxy, R 16 Its connected C, R 14 R 14 The attached N atoms together form a 5-7 member nitrogen-containing heterocycle, and R 17 Its connected C, R 15 R 15 The connected N atoms together form 5-7 member nitrogen-containing heterocycles.

[0076] Preferably, the compound of formula A is selected from the following compounds:

[0077]

[0078]

[0079] The near-infrared fluorescent dyes of this invention have novel structures and are simple to synthesize. The maximum absorption wavelength of these dyes is in the spectral range of 740 nm to 850 nm, and the maximum fluorescence emission wavelength is in the spectral range of 780 nm to 875 nm. Furthermore, the fluorescence emission peak exhibits a strong tail peak in the range of 1000 nm to 1200 nm, demonstrating strong infrared absorption, fluorescence emission, and photoacoustic effects. These dyes can be used for fluorescence and photoacoustic imaging of cells, tissues, or living organisms. They can also be used for imaging-based disease diagnosis, photodynamic therapy for diseases, or photothermal ablation of tumors.

[0080] Compound A of this invention can be prepared using the corresponding raw materials according to the preparation examples provided in the embodiments herein.

[0081] Near-infrared fluorescent dyes or dye compositions

[0082] The present invention also provides a near-infrared fluorescent dye or dye composition containing a compound of formula A of the present invention. The dye or dye composition may also contain a solvent. Exemplary solvents include, but are not limited to, one or more of MeOH, EtOH, PrOH, iPrOH, BuOH, water, acetonitrile, acetone, DMF, DMSO, pyridine, DCM, chloroform, dichloroethane, benzene, toluene, p-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, THF, ethyl acetate, AcOH, and buffer solutions. In some embodiments, the solvent may be selected from water, acetonitrile, or a buffer solution. Preferably, the buffer solution may be PBS buffer.

[0083] In some embodiments, the dye composition contains nanospheres and the compound described in any of the embodiments herein. Preferably, the nanospheres are polystyrene nanospheres. The dye composition can be prepared by first preparing an aqueous solution of the compound described in any of the embodiments herein, and then mixing and shaking it with the solution / suspension containing the nanospheres.

[0084] Reagent test kit

[0085] The present invention also provides a kit for fluorescence imaging, the kit containing a compound of formula A of the present invention, or a near-infrared fluorescent dye or dye composition.

[0086] application

[0087] This invention provides the use of compound A of this invention in the preparation of reagents for near-infrared fluorescence imaging, photothermal therapy, or photodynamic therapy. Preferably, the near-infrared fluorescence imaging is nanomaterial imaging or cellular-level imaging.

[0088] In some embodiments, the nanomaterial is a nanosphere. Cell-level imaging can image living cells and their organelles or fixed cells and their organelles. Cells can be selected from one or more cell lines such as U2OS, HeLa, HepG2, RAW264.7, 4T1, A549, and PC12. In some embodiments, the compounds of the present invention are used to image mitochondria and / or endosomes. Cell fixation methods are employed using methods commonly used in the art, such as using paraformaldehyde to fix cells.

[0089] The advantages of this invention include:

[0090] (1) This invention provides a novel near-infrared fluorescent dye core structure, which is expected to be widely used in the field of fluorescent dyes.

[0091] (2) The maximum absorption wavelength of the dye molecules of the present invention is distributed in the range of 740nm-838nm, with a large range of uniform distribution in the near-infrared region, and exhibits excellent fluorescence brightness and photostability, which is superior to other types of organic small molecule near-infrared fluorescent dyes in the field.

[0092] (3) The dye molecules of the present invention have good biocompatibility and can realize super-resolution fluorescence microscopy imaging at the in vitro and cellular levels, such as material nanoparticles, fixed cell mitochondria, and living cell endosomes. It has important application potential in super-resolution fluorescence microscopy imaging technology.

[0093] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0094] Example 1: Preparation of Compound 10

[0095]

[0096] (1) Synthesis of compound 2: Weigh 30.00 g of compound 1 (147.78 mmol, 1 equiv.) into a 500 ml round-bottom flask, add a magnetic stir bar, add 18.34 g of ethylene glycol (295.55 mmol, 2 equiv.) and 1 g of p-toluenesulfonic acid as catalysts, add 200 mL of toluene to dissolve, use a water separator to separate the water and reflux for 5 hours, and separate the aqueous phase in time. Once the water level in the separator stopped increasing, heating was stopped, and the mixture was cooled. 7.38 mL of triethyl orthoformate (44.33 mmol, 1 equiv.) and 5 g of anhydrous magnesium sulfate were added. The mixture was then heated under reflux for 2 hours using a spherical condenser. After the reaction was confirmed to be complete by thin-layer chromatography, heating was stopped, and the mixture was cooled to room temperature. p-Toluenesulfonic acid was neutralized with saturated sodium bicarbonate, and 100 mL of water was added to dissolve the salt. The mixture was separated using a separatory funnel, and the aqueous phase was extracted with DCM. The solution was dried over an appropriate amount of anhydrous magnesium sulfate, filtered, and toluene was evaporated under reduced pressure. The resulting product was distilled under reduced pressure to obtain 35.78 g of compound 2 as a colorless liquid, with a yield of 98%.

[0097] 1 H NMR (400MHz, CDCl3) δ7.59 (s, 1H), 7.31 (dd, J = 5.2, 2.3Hz, 1H), 7.05 (s, 1H), 6.04 (s, 1H), 4.21–3.99 (m, 4H).

[0098] (2) Synthesis of Compound 3: 10.00 g of Compound 2 (40.48 mmol, 1 equivalent) was weighed into a 250 mL round-bottom flask, dissolved in 100 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. At -78 °C, 19.43 mL of n-butyllithium (2.5 M, 1.2 equivalent) was added, and the mixture was stirred at -78 °C for 15 minutes. Then, 3.76 mL of anhydrous DMF (48.57 mmol, 1.2 equivalent) was added, and the mixture was gradually brought to room temperature. After the reaction was confirmed to be complete by thin-layer chromatography, it was quenched with saturated ammonium chloride solution. The mixture was separated using a separatory funnel, and the aqueous phase was extracted with DCM. The aqueous phase was dried over 3 g of anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / EA, 100 / 5, v / v) yielded 6.35 g of Compound 3 as a viscous pale yellow liquid, with a yield of 80%.

[0099] 1 H NMR (400MHz, CDCl3) δ10.40 (d, J=2.5Hz, 1H), 7.71 (dd, J=8.6, 5.2Hz, 1H), 7. 62(dd,J=8.8,2.8Hz,1H),7.31–7.25(m,1H),6.30(s,1H),4.18–4.07(m,4H).

[0100] (3) Synthesis of compound 4: 10.00 g of compound 3 (50.97 mmol, 1 equiv.) and 3.98 g (25.49 mmol, 0.5 equiv.) of 1,4-cyclohexanedione monoethylene glycol ketal were weighed into a 100 mL round-bottom flask. A magnetic stir bar was added, and 10 mL of ethanol was measured to dissolve the compound. A 30% sodium hydroxide solution was slowly added dropwise at room temperature until a pale yellow solid precipitated. 50 mL of water was added to dilute the reaction solution, and the mixture was stirred for 30 min. After thin-layer chromatography confirmed that the reaction of the starting material was complete, the mixture was filtered, washed with an appropriate amount of ice-cold ethanol, and recrystallized with dichloromethane and petroleum ether to obtain 12.80 g of compound 4 as a pale yellow solid, with a yield of 98%.

[0101] 1 H NMR (400MHz, CDCl3) δ8.05 (s, 2H), 7.60 (dd, J=8.6, 5.8Hz, 2H), 7.06 (td, J=8.4, 2.6Hz, 2H), 6.93 (dd, J= 9.4, 2.5Hz, 2H), 5.84 (s, 2H), 4.17–4.09 (m, 4H), 4.06–3.99 (m, 4H), 3.87 (s, 4H), 2.88 (d, J = 1.5Hz, 4H).

[0102] (4) Synthesis of compound 5: 4.15 g of diphenyl ether (24.39 mmol, 2.5 equiv.) was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, a magnetic stir bar was added, and the mixture was pre-cooled in an ice-water bath for 10 min. Under argon protection, 9.76 mL of n-butyllithium (2.5 M, 2.5 equiv.) was added, and the mixture was reacted at 0 °C for 1 hour. Weigh 5.00 g of compound 4 (9.76 mmol, 1 equiv.) into a 250 mL round-bottom flask, add 120 mL of anhydrous tetrahydrofuran to dissolve it, add a magnetic stir bar, and pre-cool in an ice-water bath for 10 min. Transfer the previously prepared lithium diphenyl ether reagent to the reaction solution and react at 0 °C for 2 hours. Thin-layer chromatography showed that the reaction of the starting material was complete. Quench with saturated ammonium chloride solution, add 50 mL of water to dissolve the solid salt, separate the liquid phase using a separatory funnel, extract the aqueous phase with DCM, dry with an appropriate amount of anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure. The product was dissolved in 50 mL of dichloromethane in a 100 mL round-bottom flask. A magnetic stir bar was added, and 1 mL of methanesulfonic acid was slowly added dropwise. The reaction was carried out at 40 °C for 30 min. Heating was stopped, and the mixture was cooled. 10 mL of water was added, and the reaction was continued at 40 °C for 4 hours. Thin-layer chromatography confirmed that the starting material had reacted completely. Saturated sodium bicarbonate solution was added to neutralize the methanesulfonic acid, followed by the addition of 50 mL of water. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 3 / 1, v / v) yielded 1.50 g of compound 5 as a pale yellow solid, with a yield of 31%.

[0103] 1 H NMR (400MHz, CDCl3) δ9.06 (s, 2H), 8.05 (dd, J=8.8, 5.7Hz, 2H), 7.50 (s, 2H), 7.30 (ddd, J= 12.0,8.4,3.5Hz,4H),7.25–7.16(m,4H),6.82–6.75(m,2H),6.61(dd,J=7.9,1.4Hz,2H).

[0104] (5) Synthesis of compound 6: 1.0 g of compound 5 (2.01 mmol, 1 equiv.) was weighed into a 100 mL round-bottom flask, dissolved in 30 mL of dimethyl sulfoxide, and 1.45 g of 30% sodium methoxide methanol solution (8.06 mmol, 4 equiv.) was added. A magnetic stir bar was added, and the reaction was carried out at 80 °C for 5 hours. Thin-layer chromatography showed that the reaction of the starting material was complete. The methanol was evaporated under reduced pressure, and the reaction solution was poured into ice water. A solid precipitated out. The solid was filtered to obtain a yellow solid. Column chromatography (PE / DCM, 2 / 1, v / v) yielded 0.86 g of compound 6 as a yellow solid, with a yield of 82.5%.

[0105] 1H NMR (400MHz, CDCl3) δ8.98(s,2H),7.93(d,J=9.1Hz,2H),7.41(s,2H),7.30(d,J=8.3Hz,2H),7.19(t,J=7.7 Hz,2H),7.12(dd,J=9.0,1.8Hz,2H),6.88(s,2H),6.78(t,J=7.5Hz,2H),6.65(d,J=7.9Hz,2H),3.83(s,6H).

[0106] (6) Synthesis of compound 7: 500 mg of compound 6 was weighed into a 250 mL round-bottom flask, dissolved in 100 mL of 1,2-dichloroethane, pre-cooled in an ice-water bath for 10 min, and 1 mL of boron tribromide was added under argon protection. After reacting in an ice-water bath for 30 min, a spherical condenser was used, and the mixture was heated under reflux at 60 °C for 6 hours. Thin-layer chromatography showed that the starting material had reacted completely. After cooling to room temperature, 20 mL of water was slowly added dropwise from the top of the condenser to quench the reaction. The mixture was separated by a separatory funnel, and the aqueous phase was extracted with EA. The aqueous phase was dried with an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / EA / DCM, 10 / 2 / 1, v / v) yielded 435.2 mg of compound 7 as a pale yellow solid, with a yield of 92%.

[0107] 1 H NMR (400MHz, CDCl3) δ8.98(s,2H),7.95(d,J=8.9Hz,2H),7.34(s,2H),7.28(s,2H),7.17(dd,J=11.1 ,4.2Hz,2H),7.13–7.08(m,2H),6.91(s,2H),6.76(t,J=7.5Hz,2H),6.67–6.60(m,2H),5.50(s,2H).

[0108] (7) Synthesis of compound 8: 500 mg of compound 7 (1.02 mmol, 1 equiv.) was weighed into a 250 mL round-bottom flask, dissolved in 100 mL of anhydrous dichloromethane, a magnetic stir bar was added, and the flask was pre-cooled in an ice-water bath for 10 min. 0.8 mL of trifluoromethanesulfonic anhydride and 0.8 mL of pyridine were slowly added, and the mixture was reacted at 0 °C for 1 hour. The ice-water bath was removed, and the mixture was allowed to rise to room temperature and continue reacting for 1 hour. Thin-layer chromatography showed that the starting material had reacted completely. 30 mL of water was slowly added to quench the reaction. The mixture was separated by a separatory funnel, and the aqueous phase was extracted with DCM. The aqueous phase was dried with an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 3 / 1, v / v) yielded 683.6 mg of compound 8 as a white solid, with a yield of 89%.

[0109] 1H NMR (400MHz, CDCl3) δ9.10 (s, 2H), 8.16 (d, J = 9.1Hz, 2H), 7.62 (s, 2H), 7.57 (d, J = 2.3Hz, 2H), 7.38 (dd, J = 9.1 ,2.4Hz,2H),7.34(dd,J=8.3,1.1Hz,2H),7.26–7.20(m,2H),6.83–6.78(m,2H),6.57(dd,J=7.9,1.5Hz,2H).

[0110] (8) Synthesis of Compound 9: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 95.81 mg of benzophenone imine (528.65 μmol, 8 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM / TEA, 1 / 1 / 0.01, v / v / v) yielded compound 9 as a yellow solid, 28 mg, with a yield of 51.7%.

[0111] 1 H NMR (400MHz, CDCl3) δ8.87(s,2H),7.77(d,J=8.8Hz,2H),7.72(d,J=7.6Hz,4H),7.48(dd,J=9.7,5.1Hz,2H),7.39(t,J=7.5Hz,4H),7.29(s,2H) ,7.24(s,2H),7.22–7.07(m,12H),6.95(s,2H),6.82(dd,J=8.7,1.7Hz,2H),6.77(t,J=7.4Hz,2H),6.62(d,J=7.8Hz,2H).HRMS(ESI)m / z:Calcd for C 60 H 39 N2O2 + [M+H] + ,819.3006,Found,819.3013.

[0112] (9) Synthesis of Compound 10: 104.42 mg (610.52 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.24 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to proceed for 5 min at room temperature. 100 mg (126.43 μmol, 1 equiv.) of Compound 9 was weighed into a 50 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran solution of Compound 9. The reaction was allowed to proceed for 5 min at 0 °C, then allowed to proceed for 2 min at room temperature. TLC was used to detect complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in methanol, and 1 mL of 3 M methyl butyllithium was added. HCl was added and reacted for 1 min. Saturated sodium bicarbonate was added to neutralize the mixture. The mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The mixture was then purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to obtain 37 mg of green solid, with a yield of 54%.

[0113] Compound 10 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 10 in dichloromethane is shown below. Figure 1 As shown.

[0114] 1 H NMR (400MHz, MeOD) δ7.67(dd,J=7.5,1.2Hz,1H),7.64(s,2H),7.63–7.49(m,5H),7.35(dd,J=8.3,1.2Hz,2H),7.32–7.25(m,2H),7.02(s,2H) ,6.99–6.93(m,2H),6.91(dd,J=9.1,2.0Hz,2H),6.86(dd,J=7.9,1.4Hz,1H),6.80(dd,J=7.9,1.4Hz,1H),6.58(d,J=1.9Hz,2H),2.30(s,3H). 13C NMR(101MHz,MeOD)δ168.59,158.54,150.25,150.20,150.14,143.34,140.82,137.70,136.84,136.59,131.83,131.43,131.22,131.07,131.03 ,130.95,130.50,130.36,129.84,127.60,127.22,127.03,125.24,125. 15,121.12,118.28,118.16,108.85,48.67,20.04.HRMS(ESI)m / z:Calcd for C 41 H 29 N2O + [M] + ,565.2274,Found,565.2278.

[0115] Example 2: Preparation of Compound 12

[0116]

[0117] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0118] (2) Synthesis of Compound 11: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 43 mg of dimethylamine hydrochloride (528.6 μmol, 8 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve them. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, a spherical condenser was connected, and the temperature was raised to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting material was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred, and the mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Compound 11 has poor solubility, so the product was used directly in the next step of the reaction without separation and purification.

[0119] (3) Synthesis of Compound 12: 78.22 mg (457.31 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.18 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then the temperature was raised to room temperature and the reaction was allowed to continue for 5 min. 50 mg (91.46 μmol, 1 equiv.) of Compound 11 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 11. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of trifluoroacetic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added to neutralize the mixture, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 40 mg of purple solid, with a yield of 70%.

[0120] Compound 12 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 12 in dichloromethane is shown below. Figure 2 As shown.

[0121] 1 H NMR(600MHz, CDCl3)δ7.61(td,J=7.6,1.0Hz,1H),7.59–7.47(m,6H),7.39( d,J=7.2Hz,1H),7.31(d,J=8.2Hz,2H),7.26–7.22(m,2H),7.08(s,2H),7.0 5(dd,J=9.4,2.4Hz,2H),6.92–6.84(m,2H),6.77(dd,J=7.9,1.4Hz,1H),6. 74(dd,J=7.9,1.4Hz,1H),6.52(d,J=2.3Hz,2H),3.22(s,12H),2.26(s,3H); 13C NMR (151MHz, CDCl3) δ154.9,149.1,149.0,135.3,131.3,131.1,130.3,130.1,128.9,128. 9,126.4,124.4,124.3,117.5,117.5,117.5,107.4,41.3,20.2; HRMS(ESI)m / z:Calcd.for C 45 H 37 N2O + [M] + ,621.2900;Found,621.2908.

[0122] Example 3: Preparation of compounds 13, 14 and 15

[0123]

[0124] (1) The synthesis steps of compounds 2-8 are as described in Example 1, and the synthesis steps of compound 11 are as described in Example 2.

[0125] (2) Synthesis of Compound 13: 120.53 mg (457.31 μmol, 5 equiv.) of 2-bromo-4-(3-chloropropoxy)-toluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.18 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to proceed for another 5 min at room temperature. 50 mg (91.46 μmol, 1 equiv.) of Compound 11 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared phenyllithium reagent was transferred to a compound... In a tetrahydrofuran solution of compound 11, the reaction was carried out at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to obtain 46 mg of purple solid, with a yield of 70%.

[0126] 1H NMR(600MHz,Chloroform-d)δ7.64(s,2H),7.60(d,J=9.5Hz,2H),7.36–7.32(m,3H),7.31–7.28(m ,1H),7.10(d,J=8.0Hz,2H),7.06(d,J=6.8Hz,4H),6.96–6.92(m,1H),6.92–6.89(m,1H),6.80(dd, J=7.9,1.3Hz,1H),6.76(dd,J=7.9,1.3Hz,1H),6.53(d,J=2.0Hz,2H),4.35(t,J=5.7Hz,2H),3.89( t,J=6.3Hz,2H),3.57(q,J=7.1Hz,8H),2.39(p,J=6.0Hz,2H),2.25(s,3H),1.25(t,J=7.1Hz,12H). 13 C NMR(151MHz,Chloroform-d)δ166.10,159.90,152.92,148.85,148.72,148.54,141.12,139.30,135.39,130.90,130.22,129.97,129.18,128.6 4,128.61,126.64,125.80,124.23,124.17,117.27,117.17,117.12,11 6.85,112.18,106.74,64.54,47.36,45.85,41.66,32.28,20.33,13.04.

[0127] (3) Synthesis of Compound 14: 100 mg of Compound 13 (129.80 μmol, 1.0 equiv.) was weighed into a 25 mL round-bottom flask. 5 mL of 98% concentrated sulfuric acid was added to the flask under ice bath conditions, and the mixture was stirred for 8 h. The reaction was monitored by TLC until completion. The mixture was neutralized with saturated sodium bicarbonate solution, the solvent was evaporated under reduced pressure, and the solution was reconstituted with methanol. The insoluble salt was removed by filtration. Three drops of 3M hydrochloric acid solution were added, and the solution changed from yellow to purple-red. The compound was purified by reverse-phase silica gel column chromatography with an eluent ratio of H₂O:MeCN = 5:1 (v / v), yielding 95.0 mg of a purple-red solid, with a yield of 71%.

[0128] 1H NMR (400MHz, MeOD) δ7.96 (s, 0H), 7.76 (ddd, J = 8.7, 4.3, 2.1Hz, 1H), 7.69 (s ,1H),7.65(d,J=9.5Hz,1H),7.46(d,J=8.7Hz,1H),7.43–7.37(m,2H),7.18 –7.11(m,2H),6.81(s,1H),4.48(t,J=5.5Hz,2H),4.03(t,J=6.3Hz,1H),3. 58(q,J=6.9Hz,4H),2.46–2.41(m,1H),2.39(s,1H),1.19(t,J=7.0Hz,6H); 13 C NMR (101MHz, CD3OD) δ165.4,153.2,149.5,149.4,147.7,141.2,141.1,138.9,136.8,135.3,134.9,131.1,130.3,129.8,1 29.5,128.2,128.2,128.0,128.0,126.5,126.4,125.8,125.8,117.1,117.0,106.8,78.1,62.9,45.3,19.0,11.7; HRMS(ES + calcd for C 49 H 43 N2Na2O7S2[M] 2- ,503.1021;Found,503.0990.

[0129] (4) Synthesis of Compound 15: 100 mg of Compound 14 (92.89 μmol, 1.0 equiv.) was weighed into a 50 mL round-bottom flask, dissolved in 5 mL of N,N-dimethylformamide, and then 12.08 mg of sodium azide (18.58 μmol, 2.0 equiv.) and a catalytic equivalent of potassium iodide (1 mg) were added. The mixture was heated to 70 °C and stirred for 24 h. The reaction was monitored by TLC until it ended. The reaction solution was directly purified by reverse-phase silica gel column chromatography with an eluent ratio of H2O:MeCN = 4:1 (v / v) to give 66 mg of a purple-red solid, with a yield of 66%.

[0130] ESI-HRMS (m / z) [M] 2- :calcd.For C 52 H 47 N5O 11 S3 2- 506.6223, found 506.6227.

[0131] Example 4: Preparation of Compound 17

[0132]

[0133] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0134] (2) Synthesis of Compound 16: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 28 mg of N-methylaniline (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 3 / 1, v / v) yielded compound 16 as a yellow solid, 31 mg, in 70% yield.

[0135] 1 H NMR (600MHz, CDCl3) δ8.88(s,2H),7.77(d,J=9.1Hz,2H),7.36–7.33(m,4H),7.27(d,J=3.5Hz,4H),7.19–7.16(td,J=8.4,1.2Hz,2H),7.15(m ,4H),7.13(s,2H),7.05(dd,J=9.1,2.4Hz,2H),6.84(d,J=2.4Hz,2H),6.77(td,J=7.2,0.6Hz,2H),6.70(dd,J=8.4,1.8Hz,2H),3.33(s,6H). 13 C NMR (151MHz, CDCl3) δ183.8,149.2,148.7,148.0,148.0,138.1,131.3,130.2,129.7, 129.5,128.37,127.7,126.3,126.0,125.0,124.6,123.6,120.4,116.6,108.9,40.6.

[0136] (3) Synthesis of Compound 17: 63.74 mg (372.68 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.15 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to rise to room temperature for 5 min. 50 mg (74.54 μmol, 1 equiv.) of Compound 16 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 16. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 42 mg of purple solid, with a yield of 75%.

[0137] Compound 17 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 17 in dichloromethane is shown below. Figure 3 As shown.

[0138] 1 H NMR (400MHz, CDCl3) δ7.69–7.62(m,3H),7.60–7.53(m,2H),7.47(m,7H),7.38(d ,J=7.6Hz,1H),7.37(t,J=7.4Hz,1H),7.33(dd,J=8.3,1.3Hz,2H),7.31–7.27(m, 2H),7.20–7.15(m,4H),7.11(s,2H),6.98–6.88(m,4H),6.76(dd,J=7.9,1.4Hz,1 H),6.72(dd,J=7.9,1.4Hz,1H),6.63(d,J=2.2Hz,2H),3.52(s,6H),2.28(s,3H); 13C NMR (101MHz, CDCl3) δ168.7,154.9,149.2,149.1,149.1,145.3,141.7,140. 3,136.5,135.1,135.1,131.8,131.3,130.8,130.6,130.3,130.1,130.1,129 .1,129.1,129.0,128.9,128.4,127.2,127.1,126.8,126.5,124.6,124.5,12 4.5,119.4,117.8,117.7,109.2,47.8,41.9,20.3; HRMS(ESI)m / z:Calcd.for C 55 H 41 N2O + [M] + ,745.3213;Found,745.3217.

[0139] Example 5: Preparation of Compound 19

[0140]

[0141] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0142] (2) Synthesis of Compound 18: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 45 mg of diphenylamine (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 1 / 1, v / v) yielded compound 18 as a yellow solid, 33 mg, in 64% yield.

[0143] 1H NMR(400MHz, CDCl3)δ8.91(s,2H),7.84(d,J=9.0Hz,2H),7.28(s,4H),7.25–7.17(m ,8H),7.15–7.04(m,16H),7.00(s,2H),6.75(t,J=7.4Hz,2H),6.64(d,J=7.8Hz,2H). 13 C NMR (101MHz, CDCl3) δ149.05,148.00,147.86,146.96,137.61,131.06,130.54,129.96,129.51,128.92,128. 31,127.77,127.72,126.73,125.47,124.04,123.97,123.54,116.81,116.65,46.03.HRMS(EI)m / z:Calcd.for C 58 H 38 N2O2[M] + ,794.2933,Found,794.2931.

[0144] (3) Synthesis of Compound 19: 53.79 mg (314.48 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.13 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then the temperature was raised to room temperature and the reaction was allowed to continue for 5 min. 50 mg (62.90 μmol, 1 equiv.) of Compound 18 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 18. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 41 mg of purple solid, with a yield of 75%.

[0145] 1H NMR (400MHz, CDCl3) δ7.72(s,1H),7.64(s,1H),7.55(d,J=27.5Hz,1H),7.47–7.43(m,1H),7.41(d,J=7.6Hz,1H),7.34–7.29(m,1H),7.28(s ,5H),7.20(d,J=3.8Hz,1H),7.15(d,J=7.6Hz,6H),7.09–6.98(m,7H),6.91(s,5H),6.76(s,1H),6.70(dd,J=15.3,7.3Hz,4H),2.28(s,3H). 13 C NMR (151MHz, CDCl3) δ170.50,154.40,149.08,148.74,148.71,144.19,141.37,14 0.46,136.17,134.87,134.51,131.83,131.07,130.64,130.56,130.29,130.02,1 29.80,128.85,128.26,128.12,128.03,127.76,127.54,126.93,126.28,124.30, 124.24,122.02,117.59,117.49,113.89,47.51,19.99.HRMS(ESI)m / z:Calcd.forC 65 H 45 N2O + [M] + ,869.3526;Found,869.3533.

[0146] Example 6: Preparation of Compound 21

[0147]

[0148] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0149] (2) Synthesis of Compound 20: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 15 mg of cyclobutylamine (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 1 / 1, v / v) yielded compound 20 as a yellow solid, 25 mg, in 66% yield.

[0150] 1 H NMR (400MHz, CDCl3) δ8.88 (s, 2H), 7.85 (d, J = 8.8Hz, 2H), 7.26-7.24 (m, 2H), 7.22-7.13 (m, 4H) ,6.76-6.73(m,4H),6.71-6.66(m,2H),6.32(s,2H),3.94(t,J=7.2Hz,8H),2.40-2.33(m,4H); 13 C NMR(101MHz, CDCl3)δ157.6,144.5,142.2,131.3,128.8,123.6,103.4,58.5,51.9,18.4,16.8; HRMS(ESI)m / z:Calcd.for C 40 H 30 N₂O₂[M+H] + ,571.2380; found,571.2385.

[0151] (3) Synthesis of Compound 21: 74.93 mg (438.06 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.18 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to proceed for another 5 min at room temperature. 50 mg (87.61 μmol, 1 equiv.) of Compound 20 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 20. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 50 mg of purple solid, with a yield of 88%.

[0152] Compound 21 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 21 in dichloromethane is shown below. Figure 4 As shown.

[0153] 1 H NMR (600MHz, CDCl3) δ7.62(td,J=7.2,1.2Hz,1H),7.53(m,4H),7.48(d,J=9.6Hz,2H),7.40(d,J=7. 2Hz,1H),7.31(d,J=8.4Hz,2H),7.28–7.26(m,1H),7.26–7.24(m,1H),7.04(s,1H),6.92(td,J=7.2 ,1.2Hz,1H),6.90(td,J=7.2,1.2Hz,1H),6.79(dd,J=7.8,1.8Hz,1H),6.75(dd,J=7.8,1.8Hz,1H), 6.65(dd,J=9.0,1.8Hz,2H),6.19(d,J=1.2Hz,1H),4.22(t,J=7.2Hz,8H),2.51(m,4H),2.28(s,3H); 13C NMR (151MHz, CDCl3) δ154.3,149.1,149.1,140.9,139.6,135.4,131.2,130.9,130.4,130.3,130.2,129 .0,128.9,126.5,124.5,124.4,117.6,117.5,115.8,105.3,52.2,20.3,16.4; HRMS(ESI)m / z:Calcd.for C 47 H 37 N2O + [M] + ,645.2900;Found,645.2907.

[0154] Example 7: Preparation of Compound 22

[0155]

[0156] (1) The synthesis steps of compounds 2-8 are as described in Example 1, and the synthesis steps of compound 20 are as described in Example 6.

[0157] (2) Synthesis of Compound 22: 88.08 mg (438.06 μmol, 5 equiv.) of 2-methoxymethylbromobenzene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.18 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the mixture was reacted for 20 min, then allowed to rise to room temperature for 5 min. 50 mg (87.61 μmol, 1 equiv.) of Compound 20 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 20. The reaction was carried out in a hydrogen furan solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, and 1 mL of methanesulfonic acid was added. The reaction was carried out for 10 min, then neutralized with saturated sodium bicarbonate. The mixture was separated using a separatory funnel, and the aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 52 mg of a purple solid, with a yield of 88%.

[0158] 1H NMR (400MHz, CDCl3) δ7.78-7.74(m,2H),7.65(t,J=7.3Hz,1H),7.54(s,2H),7.47(t,J=8.0Hz,3H),7.34-7.23(m,4H),7.04(s,2H),6.96-6.88( m,2H),6.85-6.79(m,2H),6.66(dd,J1=9.0Hz,J2=1.9Hz,2H),6.20(s,2H),4.40(s,2H),4.23(t,J=7.5Hz,8H),3.16(s,3H),2.55-2.48(m,4H); 13 C NMR (101MHz, CDCl3) δ149.9,149.0,148.5,143.9,134.6,130.3,130.1,129.8,129.4,128.8,128.7,128 .6,126.2,124.2,124.1,117.3,117.2,115.0,103.8,72.8,58.5,52.3,51.9,16.1; HRMS(ESI)m / z:[M+H] + Calculated for C 48 H 39 N2O2 + ,675.3006;found,675.3011.

[0159] Example 8: Preparation of Compound 24

[0160]

[0161] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0162] (2) Synthesis of Compound 23: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 34 mg of 3,3-difluoroazacyclobutane hydrochloride (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction proceeds were completely reacted. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 3 / 1, v / v) yielded compound 24 as a yellow solid, 30 mg, in 71% yield.

[0163] 1 H NMR (400MHz, CDCl3) δ8.93(s,2H),7.92(d,J=8.9Hz,2H),7.29(s,3H),7.26(d,J=4.3Hz,2H),7.16( t,J=7.6Hz,2H),6.76(t,J=9.1Hz,4H),6.65(d,J=7.8Hz,2H),6.45(s,2H),4.26(t,J=11.7Hz,8H); 13 C NMR (101MHz, CDCl3) δ184.0,149.5,149.2,148.6,137.8,131.7,131.4,129.6,129.5,129.2,12 8.2,126.4,126.3,124.0,117.0,116.1,105.9,63.7,63.5,63.2,46.4; HRMS(EI)m / z:Calcd.for C 40 H 26 F4N2O2[M] + ,642.1930;Found,642.1934.

[0164] (3) Synthesis of Compound 24: 66.54 mg (389.01 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.16 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to rise to room temperature for 5 min. 50 mg (77.80 μmol, 1 equiv.) of Compound 23 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 23. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 46 mg of brown solid, with a yield of 83%.

[0165] Compound 24 was dissolved to prepare a 2.5 μM solution in dichloromethane in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 24 in dichloromethane is shown below. Figure 5 As shown.

[0166] 1 H NMR (600MHz, CDCl3) δ7.78(s,2H),7.67(t,J=7.4Hz,3H),7.57(dd,J=16.1,7.7Hz,2H),7.44(d,J=7.2Hz,1H),7.34(d,J=8.3Hz,2H),7.29(dd,J=6.8 ,5.2Hz,2H),7.19(s,2H),6.91(dt,J=12.0,7.2Hz,2H),6.77(d,J=7.0Hz, 2H),6.74–6.67(m,2H),6.37(s,2H),4.53(t,J=10.8Hz,8H),2.28(s,3H); 13CNMR(151MHz, CDCl3)δ170.6,153.1,149.3,149.0,148.9,141.5,141.3,135.9,131.1,131.0,130.5,129.9,129.7,129.6,129.0,128.9,128 .4,128.3,127.0,126.9,126.2,124.3,124.3,117.5,117.4,116.3,114.5,106.6,63.2(t,J=28.69Hz),47.6,20.0; HRMS(ESI)m / z:Calcd.for C 47 H 33 F4N2O + [M] + ,717.2524;Found,717.2527.

[0167] Example 9: Preparation of Compound 26

[0168]

[0169] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0170] (2) Synthesis of Compound 25: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 19 mg of cyclopentanamine (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 1 / 1, v / v) yielded compound 25 as an orange solid, 28 mg, in 70% yield.

[0171] 1H NMR (400MHz, CDCl3) δ8.87(s,2H),7.85(d,J=9.1Hz,2H),7.24(s,2H),7.18(s,2H),7.14(dd,J=14.8,1.6Hz,2H),6.95(d d,J=9.0,1.8Hz,2H),6.76(d,J=7.8Hz,2H),6.73–6.70(m,2H),6.42(s,2H),3.33(t,J=6.2Hz,8H),1.99(t,J=6.2Hz,8H). 13 C NMR (151MHz, CDCl3) δ183.6,149.1,147.9,147.5,138.3,131.3,131.0,129.9,128.6,128.4, 127.4,124.8,124.6,123.5,116.7,116.3,103.7,47.6,46.1,25.4.HRMS(ESI)m / z:Calcd.for C 42 H 34 N₂O₂[M+H] + ,599.2693; found,599.2698.

[0172] (3) Synthesis of Compound 26: 71.41 mg (417.54 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.17 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then the temperature was raised to room temperature and the reaction was allowed to continue for 5 min. 50 mg (77.80 μmol, 1 equiv.) of Compound 25 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 25. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 41 mg of purple solid, with a yield of 72%.

[0173] Compound 26 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 26 in dichloromethane is shown below. Figure 6 As shown.

[0174] 1 H NMR (400MHz, CDCl3) δ7.64(t,J=7.5Hz,1H),7.61–7.49(m,6H),7.43(d,J=7.4Hz,1H),7.34(d,J=8.2Hz,2H),7.31–7.25(m,3H),7.09(s ,2H),6.96(s,2H),6.93–6.88(m,2H),6.82(d,J=7.9Hz,1H),6.79(d,J=7.9Hz,1H),6.45(s,2H),3.54(s,8H),2.30(s,3H),2.08(s,8H). 13 C NMR (151MHz, CDCl3) δ152.2,148.9,148.8,148.4,140.9,139.3,135.1,130.9,130.8,130.1,130.0,129 .9,128.6,126.1,124.2,124.1,118.1,117.3,117.2,107.6,48.9,25.2,20.0.HRMS(ESI)m / z:Calcd.for C 49 H 41 N2O + [M] + ,673.3213;Found,673.3218.

[0175] Example 10: Preparation of Compound 28

[0176]

[0177] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0178] (2) Synthesis of Compound 27: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 35 mg of 7-azabicyclo[2,2,1]heptane hydrochloride (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve them. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, a spherical condenser was connected, and the temperature was raised to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting material was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred, and the mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 1 / 1, v / v) yielded compound 27 as 31 mg of orange solid, with a yield of 72%.

[0179] 1 H NMR (400MHz, CDCl3) δ8.89(s,2H),7.85(d,J=9.0Hz,2H),7.28(d,J=8.2Hz,2H),7.24(s,2H),7.16(dd,J=13.6,4.8Hz,4 H),6.79(s,2H),6.76(d,J=7.6Hz,2H),6.69(d,J=7.8Hz,2H),4.23(s,4H),1.75(d,J=7.3Hz,9H),1.42(t,J=6.4Hz,8H); 13 C NMR (101MHz, CDCl3) δ184.2,149.5,148.5,148.1,138.3,131.6,131.2,129.8,129.6,128.7, 128.0,126.7,126.2,123.9,120.3,116.8,109.6,58.1,46.4,29.1.HRMS(ESI)m / z:Calcd.for C 46 H 38 N₂O₂[M+H] + ,651.3006; found,651.3018.

[0180] (3) Synthesis of Compound 28: 65.70 mg (384.13 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.15 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then the temperature was raised to room temperature and the reaction was allowed to continue for 5 min. 50 mg (76.83 μmol, 1 equiv.) of Compound 27 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of Compound 27. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of methanesulfonic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 25:1 to 20:1) to give 42 mg of purple solid, with a yield of 75%.

[0181] Compound 28 was dissolved to prepare a 2.5 μM solution in dichloromethane in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 28 in dichloromethane is shown below. Figure 7 As shown.

[0182] 1 H NMR(600MHz, CDCl3)δ7.64(ddd,J=7.5,7.5,0.8Hz,1H),7.58–7.50(m,6H),7.40(d,J=7.2H z,1H),7.34(d,J=8.3Hz,2H),7.31–7.26(m,2H),7.09(s,2H),7.02(dd,J=9.2,1.7Hz,2H),6 .95–6.91(m,1H),6.90(m,1H),6.79(dd,J=7.9,1.4Hz,1H),6.75(dd,J=7.9,1.4Hz,1H),6. 67(d,J=1.4Hz,2H),4.48(s,4H),2.28(s,3H),1.80(d,J=7.6Hz,8H),1.60(d,J=7.3Hz,8H); 13C NMR (151MHz, CDCl3) δ165.8,151.8,149.2,149.1,148.9,141.5,139.2,13 6.5,135.9,135.2,131.3,131.2,130.4,130.3,130.2,130.0,129.2,129. 1,129.0,129.0,127.4,127.2,126.5,124.5,124.5,119.4,117.7,117.6, 109.9,77.6,77.4,77.1,58.0,29.1,29.1,20.2; HRMS(ESI)m / z:Calcd.for C 53 H 45 N2O + [M] + ,725.3526;Found,725.3530.

[0183] Example 11: Preparation of Compound 30

[0184]

[0185] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0186] (2) Synthesis of Compound 29: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 26 mg of N-methylpiperazine (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure.

[0187] 1H NMR (400MHz, CDCl3) δ8.91(s,2H),7.88(d,J=9.2Hz,2H),7.30(s,2H),7.28(d,J=8.3Hz,2H),7.21(dd,J=9.2,1.9Hz,2H),7.15 (t,J=7.6Hz,2H),6.79(s,2H),6.75(t,J=7.5Hz,2H),6.66(d,J=7.8Hz,2H),3.34–3.22(m,8H),2.57–2.49(m,8H),2.32(s,6H). 13 CNMR(151MHz, CDCl3)δ183.89,150.74,149.19,147.94,137.90,131.21,130.66,129.79,129.40,128.31, 127.78,126.41,126.20,123.66,119.58,116.61,108.57,54.80,48.31,46.05.HRMS(ESI)m / z:Calcd.for C 44 H 42 N4O2 2+ [M+2H] 2+ ,329.1648;Found,329.1667.

[0188] (3) Synthesis of compound 30: 65.10 mg (380.62 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.15 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the mixture was reacted for 20 min, then allowed to rise to room temperature and react for 5 min. 50 mg (76.12 μmol, 1 equiv.) of compound 29 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of compound 29. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of trifluoroacetic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 10:1 to 1:1) to give 29 mg of purple solid, with a yield of 52%.

[0189] Compound 30 was dissolved to prepare a 2.5 μM solution in dichloromethane in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 30 in dichloromethane is shown below. Figure 8 As shown.

[0190] 1 H NMR (600MHz, CD2Cl2) δ7.86(s,2H),7.68(s,3H),7.63–7.54(m,2H),7.48(d,J=6.5Hz,1H),7.36(d,J=7.4Hz,2H),7.30(d,J=10.4Hz,4 H),7.19(s,2H),6.91(d,J=7.7Hz,2H),6.84(s,2H),6.75(dd,J=24.0,7.3Hz,2H),3.91(s,8H),3.34(s,8H),2.84(s,6H),2.29(s,3H). 13 C NMR (151MHz, CD2Cl2) δ172.3,161.3,154.1,149.4,149.1,149.0,141.8,141.5,136.4,135.6,134.5,131.9,130.9,130.4,129.9,129.9,129.7 ,128.9,128.9,128.2,128.1,127.7,127.1,126.0,124.1,118.2,117.6,117.5,109.1,52.6,47.7,44.4,43.2,19.9.HRMS(ESI)m / z:Calcd.for C 51 H 47 N4O + [M] + ,731.3744;Found,731.3749.

[0191] Example 12: Preparation of compound 32

[0192]

[0193] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0194] (2) Synthesis of Compound 31: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask. A magnetic stir bar was added, and argon gas was introduced for 5 min. 36 mg of thiomorpholine dioxide (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction of the starting materials was complete. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure.

[0195] 1 H NMR (400MHz, CDCl3) δ8.94(s,2H),7.97(d,J=9.1Hz,2H),7.33(s,2H),7.29(d,J=8.1Hz,2H),7.22–7.16(m,4H),6.8 5(d,J=2.0Hz,2H),6.78(dd,J=11.0,3.9Hz,2H),6.64(dd,J=7.9,1.3Hz,2H),4.03–3.85(m,8H),3.15–3.01(m,8H). 13 C NMR (151MHz, CDCl3) δ183.67,149.13,148.31,137.63,131.74,131.09,129.94,129.04,128.46,128. 04,126.68,126.65,123.82,118.65,116.73,109.59,50.50,46.94,46.14.HRMS(ESI)m / z:Calcd.for C 42 H 34 N2NaO6S2 + [M+Na] + ,749.1750;Found,749.1755.

[0196] (3) Synthesis of compound 32: 58.83 mg (343.94 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.14 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to rise to room temperature for 5 min. 50 mg (68.79 μmol, 1 equiv.) of compound 31 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of compound 31. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of trifluoroacetic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 20:1 to 10:1) to give 24 mg of purple solid, with a yield of 44%.

[0197] Compound 32 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 32 in dichloromethane is shown below. Figure 9 As shown.

[0198] 1 H NMR (600MHz, CDCl3) δ7.84(s,2H),7.74(s,2H),7.68(t,J=7.1Hz,1H),7.61–7.56(m,2H),7.45(d,J=6.9Hz,1H),7.3 7–7.26(m,8H),6.95–6.87(m,2H),6.85(s,2H),6.69(dd,J=21.6,7.7Hz,2H),4.15(s,8H),3.11(s,8H),2.28(s,3H). 13C NMR (151MHz, CDCl3) δ172.6,160.1,159.8,153.0,149.5,149.0,148.9,1 42.0,141.7,136.6,136.1,134.2,132.1,131.2,130.7,130.0,129.9,129 .7,129.1,128.1,128.0,127.7,127.1,126.3,124.4,118.4,117.7,117. 6,116.5,114.6,109.4,51.9,47.7,46.5,20.3.HRMS(ESI)m / z:Calcd.for C 49 H 41 N2O5S2 + [M] + ,801.2451;Found,801.2458.

[0199] Example 13: Preparation of compound 34

[0200]

[0201] (1) The synthesis steps of compounds 2-8 are as described in Example 1.

[0202] (2) Synthesis of Compound 33: 50 mg of Compound 8 (66.08 μmol, 1 equiv.) was weighed into a 50 mL anaerobic reaction flask, a magnetic stir bar was added, and argon gas was introduced for 5 min. 45 mg of nortropinone glycol ketal (264.3 μmol, 4 equiv.) and 215.31 mg of cesium carbonate (660.81 μmol, 10 equiv.) were added and poured into the anaerobic reaction flask. Catalytic amounts of BINAP and palladium acetate were added, and 35 mL of dry toluene was added to dissolve the compounds. Argon gas was introduced for 10 min. Vacuum grease was applied to the ground glass joint, and a spherical condenser was connected. The mixture was heated to 115 °C and refluxed for 12 hours under argon protection. Thin-layer chromatography showed that the reaction proceeds were completely reacted. The mixture was cooled to room temperature, 20 mL of water was added and stirred. The mixture was separated using a separatory funnel. The aqueous phase was extracted with DCM, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Column chromatography (PE / DCM, 3 / 1, v / v) yielded compound 33 as an orange solid, 45 mg, in 85% yield.

[0203] 1H NMR(400MHz,CDCl3)δ8.87(s,2H),7.87(d,J=9.1Hz,2H),7.25(d,J=6.6Hz,3H),7.19(s,2H),7.15(t,J=7.7Hz,2H),7.10(dd,J=9.1,1.8Hz,2H),6.78(t,J=7.5Hz,2H),6.71(d,J=6.9Hz,2H),6.66(s,2H),4.31(s,4H),3.95(t,J=6.3Hz,4H),3.72(t,J=6.3Hz,4H),2.16(t,J=6.4Hz,4H),2.02(dd,J=13.9,2.6Hz,4H),1.98–1.90(m,4H),1.67(d,J=13.7Hz,4H); 13 C NMR(101MHz,CDCl3)δ184.1,149.4,148.3,146.2,138.8,131.7,130.0,129.2,128.7,127.9,125.8,125.7,124.1,118.7,116.7,108.0,107.9,64.9,63.3,54.0,46.4,37.9,27.7;HRMS(ESI)m / z:Calcd.for C 52 H 46 N2O6[M+H] + ,795.3429;Found,795.3438.

[0204] (3) Synthesis of compound 34: 53.79 mg (314.49 μmol, 5 equiv.) of o-bromotoluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.14 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min, then allowed to rise to room temperature for 5 min. 50 mg (62.90 μmol, 1 equiv.) of compound 33 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran of compound 33. The reaction was carried out in uranium solution at 0°C for 5 min, then at room temperature for 2 min. TLC was used to confirm the complete reaction of the starting material. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in dichloromethane, 1 mL of trifluoroacetic acid was added, and the reaction was carried out for 10 min. Saturated sodium bicarbonate was added for neutralization, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was purified by column chromatography (DCM / MeOH = 20:1 to 10:1) to give 47 mg of purple solid, with a yield of 85%.

[0205] Compound 34 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 34 in dichloromethane is shown below. Figure 10 As shown.

[0206] 1 H NMR (400MHz, CDCl3) δ7.67–7.62(m,1H),7.61(s,2H),7.58(s,2H),7.54(d,J=7.3Hz,1H),7.42(d, J=7.1Hz,1H),7.36–7.27(m,4H),7.26–7.23(m,1H),7.16(d,J=8.7Hz,2H),7.07(s,2H),6.92(ddd, J=16.4,8.2,1.0Hz,2H),6.80(dd,J=7.9,1.0Hz,1H),6.76(dd,J=7.8,1.1Hz,1H),6.60(s,2H),4. 55(s,4H),3.97(t,J=6.3Hz,4H),3.75(t,J=6.3Hz,4H),2.28(d,J=5.0Hz,7H),2.03–1.84(m,12H); 13C NMR (151MHz, CDCl3) δ150.7,148.8,148.7,148.6,141.4,139.1,135.8,131.0,130.1,130.1,129.9,128.7,128.7,126.4,126.2,1 24.4,124.3,124.3,123.5,118.8,117.3,117.2,108.8,106.7,64.8,63.3,55.1,31.5,30.1,26.8,20.0; HRMS(ESI)m / z:Calcd.for C 59 H 53 N2O5 + [M] + ,869.3949;Found,869.3953.

[0207] Example 14: Preparation of compounds 35 and 36

[0208]

[0209] (1) The synthesis steps of compounds 2-8 are as described in Example 1, and the synthesis steps of compound 33 are as described in Example 13.

[0210] (2) Synthesis of compound 35: 82.89 mg (314.49 μmol, 5 equiv.) of 2-bromo-4-(3-chloropropoxy)-toluene was weighed into a 100 mL round-bottom flask, dissolved in 40 mL of anhydrous tetrahydrofuran, and a magnetic stir bar was added. 0.14 mL (2.5 M, 5 equiv.) of n-butyllithium was added at -78 °C, and the reaction was allowed to proceed for 20 min. The reaction was then allowed to proceed for 5 min at room temperature. 50 mg (62.90 μmol, 1 equiv.) of compound 33 was weighed into a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and the prepared methylbenzene lithium was transferred to the tetrahydrofuran solution of compound 33. The reaction was allowed to proceed for 5 min at 0 °C, and then allowed to proceed for 2 min at room temperature. The reaction of the starting material was detected by TLC to be complete. The reaction was quenched with saturated ammonium chloride solution. Water was added, and the mixture was separated by a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in 10 mL of N,N-dimethylformamide, and 8.18 mg of sodium azide (125.79 μmol, 2 equiv.) and a catalytic equivalent of potassium iodide (1 mg) were added. The mixture was heated to 70 °C and reacted for 2 hours. The mixture was extracted with 50 mL of water and 50 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in 10 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added. The mixture was reacted for 10 min, neutralized with saturated sodium bicarbonate, and separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (DCM / MeOH = 20:1 to 10:1) to give 42 mg of a purple solid, with a yield of 69%.

[0211] 1 H NMR(400MHz, CDCl3)δ7.65(s,2H),7.63(s,2H),7.32(dd,J=9.2,4.8Hz,3H),7.29–7.26(m,1H),7.26–7.22(m,1H),7 .16(d,J=8.6Hz,2H),7.12–7.07(m,2H),7.06(s,2H),6.95–6.91(m,1H),6.89(dd,J=9.6,2.7Hz,1H),6.78(dd,J=7.9 ,1.1Hz,1H),6.73(dd,J=7.9,1.2Hz,1H),6.59(s,2H),4.54(s,4H),4.29(t,J=5.8Hz,2H),3.97(t,J=6.3Hz,4H),3.7 4(t,J=6.3Hz,4H),3.65(t,J=6.5Hz,2H),2.28(d,J=7.5Hz,4H),2.25(s,3H),2.23–2.18(m,2H),2.02–1.83(m,13H); 13C NMR (151MHz, CDCl3) δ166.8,160.2,150.9,149.2,149.0,148.9,141.7,139.7,138.4 ,136.1,131.5,131.2,130.5,130.2,129.4,129.3,129.0,128.9,127.6,127.3,126. 7,124.6,124.5,119.0,117.5,117.4,117.2,112.5,109.0,107.1,65.2,65.1,63.6, 55.3,48.6,47.7,41.0,31.9,29.2,27.1,23.0,20.7,14.4; HRMS(ESI)m / z:Calcd.for C 62 H 58 N5O6 + [M] + ,968.4382;Found,968.4388.

[0212] (3) Synthesis of compound 36: 20 mg (20.64 μmol, 1 equiv.) of compound 35 and 41.3 mg (20.64 μmol, 1 equiv.) of Alkyne-PEG-NHS were weighed and dissolved in 2 mL of anhydrous N,N-dimethylformamide in a 20 mL round-bottom flask. The flask was purged three times with argon gas and sealed with a rubber stopper. 7.69 mg (20.64 μmol, 1 equiv.) of copper tetraacetonitrile hexafluorophosphate and 2.67 mg (20.64 μmol, 1 equiv.) of DIPEA were dissolved in 2 mL of anhydrous N,N-dimethylformamide and added to the round-bottom flask using a syringe. The mixture was stirred at room temperature for 2 hours. TLC analysis confirmed the reaction of the starting material was complete. Water was added, and the mixture was separated using a separatory funnel. The aqueous phase was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The mixture was then purified by column chromatography (DCM / MeOH = 20:1 to 10:1) to obtain 19 mg of purple solid, with a yield of 32%.

[0213] MALDI-TOF m / z Expected MW:~2968, Measured MW:~2988.

[0214] Example 15: Preparation of Compound 46

[0215]

[0216] (1) Synthesis of compound 38: 20 g of 2-nitrobenzene-1,3-diol (129 mmol, 1 equiv.), 54 mL of 1-bromo-2-chloroethane (645 mmol, 5 equiv.), and 10 g (excess) of potassium carbonate were dissolved in 200 mL of acetonitrile and heated to 90 °C for 48 hours. After cooling to room temperature, the mixture was filtered. The filtrate was poured into 200 mL of petroleum ether, and a solid precipitated out. After filtration and drying, 35 g of white solid product 38 was obtained, with a yield of 97%.

[0217] 1 H NMR (400MHz, CDCl3) δ7.34(t,J=8.0Hz,1H),6.67(d,J=8.0Hz,2H),4.30(t,J=4.0Hz,4H),3.78(t,J=4.0Hz,4H); 13 C NMR (101MHz, CDCl3) δ150.5,132.8,131.3,106.5,69.5,41.0; HRMS (ES + calcd for C 10 H 11 Cl2NO4[M+Na] + ,301.9957;Found,301.9961.

[0218] (2) Synthesis of compound 39: 10 g of compound 38 (35.7 mmol, 1 equiv.) was dissolved in 200 mL of ethyl acetate in 2.74 mL of liquid bromine (1.5 equiv.) and stirred at room temperature for 24 hours. The reaction was quenched with sodium hydroxide solution, and the mixture was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give 11.7 g of yellow oily product 39, with a yield of 91%.

[0219] 1 H NMR (400MHz, CDCl3) δ7.60 (d, J=12.0Hz, 1H), 6.76 (d, J=8.0Hz, 1H), 4.36 (t, J= 8.0Hz,2H),4.30(t,J=8.0Hz,2H),3.79(t,J=8.0Hz,2H),3.78(t,J=8.0Hz,2H); 13 CNMR(101MHz, CDCl3)δ149.7,148.1,137.9,134.8,111.0,108.8,74.4,69.9,41.6,40.9; HRMS(ES + calcd for C 10 H 10 BrCl2NO4[M+Na] +,379.9062;Found,379.9070.

[0220] (3) Synthesis of compound 40: 20 g of compound 39 (55.76 mmol, 1 equiv.), 2.2 mL of acetic acid (39 mmol, 0.7 equiv.), and 12.45 g of iron powder (223 mmol, 4 equiv.) were dissolved in 100 mL of ethanol, and 1 mL of water was added. The reaction mixture was heated to 90 °C for 12 hours under argon protection. The reaction mixture was filtered, and the pH of the filtrate was adjusted to 12. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography (PE / DCM = 1 / 1, v / v) yielded 12.4 g of blue-violet solid product 40, with a yield of 87%.

[0221] 1 H NMR (400MHz, CDCl3) δ6.80(d,J=8.0Hz,1H),6.31(d,J=8.0Hz,1H),4.47(t,J=4.0Hz,2H),4.36(t,J=4.0Hz,2H),3.18(t,J=4.0Hz,2H),3.17(m,2H); 13 C NMR (101MHz, CDCl3) δ143.6,140.8,122.9,121.8,109.2,100.6,65.7,65.0,46.5,46.4; HRMS (ES + )calcdfor C 10 H 10 BrNO2[M+H] + ,255.9968;Found,255.9970.

[0222] (4) Synthesis of compound 41: 21.8 mL of phosphorus oxychloride (234.3 mmol, 2 equiv.) was added to 150 mL of N,N-dimethylformamide and reacted in an ice bath for 40 minutes. The mixture was then transferred to a solution of 30 g of compound 40 (117.1 mmol, 1 equiv.) in 1,2-dichloroethane. The reaction solution was heated to 80 °C and reacted for 6 hours. After cooling to room temperature, the mixture was poured into ice water. Filtering yielded 28.3 g of yellow solid product 41, with a yield of 85%.

[0223] 1 H NMR (400MHz, CDCl3) δ10.16(s,1H),7.10(s,1H),4.48–4.42(t,J=4.0Hz,2H),4.36–4.30(t,J=4.0Hz,2H),3.34(m,4H); 13C NMR (151MHz, CDCl3) δ196.0,142.4,139.4,128.8,123.1,109.8,107.9,64.8,64.1,46.1,46.0; HRMS (ES + calcd for C 11 H 10 BrNO[M+H] + ,283.9917;Found,283.9925.

[0224] (5) Synthesis of compound 42: 10 g of compound 41 (35.2 mmol, 1 equiv.), 3.28 g of ethylene glycol (52.8 mmol, 1.5 equiv.), 5.22 g of triethyl orthoformate (35.2 mmol, 1 equiv.), and 0.6 g of p-toluenesulfonic acid (3.52 mmol, 0.1 equiv.) were dissolved in 200 mL of toluene. The mixture was heated to 100 °C and reacted for 12 hours. After cooling to room temperature, the mixture was neutralized with saturated sodium bicarbonate, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography (PE / EA / TEA = 100 / 10 / 1, v / v / v) yielded 8.4 g of yellow solid compound 42, in 73% yield.

[0225] 1 H NMR (400MHz, CDCl3) δ6.68(s,1H),6.03(s,1H),4.48–4.40(m,2H),4.34–4.28(m,2H),4.12–4.04(m,2H),4.03–3.96(m,2H),3.20–3.12(m,4H).

[0226] (6) Synthesis of compound 43: 10 g of compound 42 (30.5 mmol, 1 equiv.) was dissolved in 200 mL of anhydrous tetrahydrofuran. The mixture was reacted with 14.6 mL of n-butyllithium (45.7 mmol, 2.5 M, 1.2 equiv.) in a syringe at -78 °C for 20 min. 3.54 mL of LDM (45.7 mmol, 1.5 equiv.) was added to the reaction solution, and the mixture was allowed to rise to room temperature for 2 h. The reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography (PE / EA / TEA = 100 / 25 / 1, v / v / v) yielded 7.3 g of yellow solid compound 43, with a yield of 89%.

[0227] 1H NMR (400MHz, CDCl3) δ10.38(s,1H),6.82(s,1H),6.53(s,1H),4.53–4.45(m,2H),4.44–4.39(m,2H),4.14–3.96(m,4H),3.29–3.13(m,4H). 13 CNMR(151MHz, CDCl3)δ189.7,149.1,148.4,131.0,115.8,107.2,99.9,65.6,65.2,46.4; 46.0; HRMS(ES + )calcdfor C 14 H 15 NO5[M+Na] + ,300.0842;Found,300.0853.

[0228] (7) Synthesis of compound 44: 10 g of compound 43 (36 mmol, 2 equiv.) and 2.82 g of 1,4-cyclohexanedione monoethylene glycol ketal (18 mmol, 1 equiv.) were dissolved in 20 mL of ethanol. 0.5 mL of 40% sodium hydroxide aqueous solution was slowly added dropwise to the reaction solution, and the mixture was stirred for 12 hours. The mixture was filtered, and the filter cake was dried to give 9.9 g of compound 44, with a yield of 81%.

[0229] 1 H NMR(600MHz, CDCl3)δ7.81(s,2H),6.66(s,2H),5.70(s,2H),4.41–4.31(m,8H),4.13–4.04 (m,4H),3.97–3.90(m,4H),3.83(s,2H),3.18–3.15(m,4H),3.15–3.12(m,4H),2.72(s,4H). 13 C NMR (151MHz, CDCl3) δ186.5,144.1,135.8,134.3,126.6,122.5,115.9,107.3,106.7,65.3,65.2,64.4,46.7; 46.4,37.4; HRMS (ES + calcd for C 36 H 38 N2O 11 [M+H] + ,675.2548;Found,675.2553.

[0230] (8) Synthesis of Compound 45: 2 g of diphenyl ether (11.9 mmol, 4 equiv.) was dissolved in 30 mL of anhydrous tetrahydrofuran. 4.7 mL of n-butyllithium (11.9 mmol, 4 equiv.) was slowly added using a syringe under an ice-water bath. After reacting at 0 °C for 40 min, the solution was transferred to 100 mL of tetrahydrofuran containing 2 g of compound 44 (2.9 mmol, 1 equiv.). The reaction was carried out at 0 °C for 2 h. The reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in 50 mL of dichloromethane, and 2 mL of methanesulfonic acid (excess) was added and reacted for 4 h. The mixture was neutralized with saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and recrystallized to give 0.88 g of compound 45, with a yield of 45%. This compound was used directly in the next step without further purification.

[0231] (9) Synthesis of Compound 46: 2.6 g of 2-bromotoluene (15.2 mmol, 10 equiv.) was dissolved in 30 mL of anhydrous tetrahydrofuran. 6.1 mL of n-butyllithium (15.2 mmol, 2.5 M, 10 equiv.) was added to the syringe at -78 °C, and the mixture was stirred for 20 minutes. The solution was then transferred to 150 mL of tetrahydrofuran containing 1 g of Compound 45 (1.52 mmol, 1 equiv.). The reaction was carried out at 0 °C for 2 hours. The reaction was quenched with water, and the mixture was separated. The aqueous phase was extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was dissolved in 50 mL of dichloromethane, and 2 mL of methanesulfonic acid (excess) was added. The mixture was stirred for 8 hours. The solution was neutralized with saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography (DCM / MeOH = 20 / 1, v / v) yielded 700 mg of compound 46, with a yield of 63%.

[0232] Compound 46 was dissolved to prepare a 2.5 μM dichloromethane solution in a 4 mL quartz cuvette. The UV-Vis absorption spectrum of the dye was acquired using a UV2600i, and the fluorescence emission spectrum was acquired using a Dutta detector. The detectors were an R928 PMT and an Inga AS. The absorption and emission spectrum of compound 46 in dichloromethane is shown below. Figure 11 As shown.

[0233] 1 H NMR(600MHz, CDCl3)δ7.60(t,J=7.4Hz,1H),7.56–7.47(m,2H),7.45–7.34(m,5H),7.30(d,J=8.2Hz,2H ),7.24(s,2H),6.87(dt,J=13.9,7.4Hz,2H),6.78–6.63(m,4H),4.33(m,8H),3.73(m,8H),2.24(s,2H);13 C NMR (151MHz, CDCl3) δ148.9,130.7,129.9,129.8,129.7,128.3,126.0,125.0, 123.8,123.8,117.3,117.2,109.9,64.1,63.8,47.3,47.2,31.5,19.9; HRMS(ES + )m / z calcd for C 49 H 37 N2O5[M] + ,733.2697;Found,733.2701.

[0234] Example 16: Super-resolution fluorescence microscopy of nanospheres

[0235] Compound 34 was prepared as a 40 μM, 0.5 mL aqueous solution and added to 30 μL of a 2.5 wt% suspension of polystyrene nanospheres (purchased from Wuxi Ruige Biotechnology Co., Ltd.). The solution was shaken for 5 minutes, then diluted 1 / 100 by volume. 5 μL of the diluted solution was pipetted onto a coverslip, allowed to evaporate to dryness, and then glycerol was added before placing it on a glass slide. Using λ... ex =808nm, λ em =830nm for structured illumination micro-imaging (SIM) and wide-field imaging.

[0236] The super-resolution imaging results of compound 34 on nanospheres are as follows: Figure 12 As shown in the figure. A represents a comparison between wide-field imaging and structured illumination microscopy (SIM) super-resolution imaging of microspheres with different nanoparticle sizes. B represents the imaging results of relatively dense 300 nm polystyrene nanospheres. C and D are magnified views of the area selected in B, where C shows the effect of SIM super-resolution imaging and D shows the effect of traditional wide-field fluorescence microscopy. Experiments show that the compounds of this invention have the potential for near-infrared (>800 nm) super-resolution fluorescence microscopy imaging.

[0237] Example 17: Cell-level super-resolution fluorescence microscopy

[0238] Compound 36 was prepared as a 1 μM PBS buffer solution and co-incubated with paraformaldehyde-fixed U2OS cells and unfixed live cells for approximately 30 minutes. Structured light microscopy (SIM) and wide-field imaging were then performed on the incubated U2OS cells using λ... ex =808nm, λ em =Shot at 830nm.

[0239] Compound 36 was used for SIM super-resolution microscopy and wide-field imaging of mitochondria and endosomes in fixed cells (U2OS cell line), such as... Figure 13 As shown in the figure. The inset in the upper right corner of the right figure is a bright-field image of a live cell. Experiments show that the compounds of this invention have the potential for near-infrared (>800 nm) cellular-level super-resolution fluorescence microscopy imaging.

[0240] Example 18: In vivo fluorescence imaging in mice

[0241] Compounds 28, 24, 34, and DSPE-mPEG2000 encapsulating compound 34 were dissolved in PBS buffer to prepare a 50 mM injection solution. The solution was injected into mice via the tail vein. Mice were anesthetized with isoflurane gas and placed on a near-infrared in vivo fluorescence imaging platform. The images were then analyzed using λ... ex Images were taken using an excitation light of 808nm and long-pass filters of different wavelengths.

[0242] In vivo fluorescence imaging of mice after injection of compounds 28, 24, 34 and DSPE-mPEG2000-encapsulated compound 34 in nanomicelles, as shown in the figure. Figure 14 As shown, different long-pass filters (LP) and exposure times (ET) were used, and imaging was performed after different injection times (PI). Near-infrared II in vivo fluorescence imaging was performed on the liver, spleen, duodenum, small intestine, saphenous vasculature, abdominal vasculature, sternum, and tibia of mice. This experiment demonstrates the potential of the compounds of this invention for near-infrared in vivo fluorescence imaging applications.

[0243] Example 19: Photothermal conversion performance

[0244] Compound 17 was prepared as a 5 mM acetonitrile stock solution, which was then diluted with water to aqueous solutions with concentrations of 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, and 80 μM. Under this system, a power density of 0.2 W / cm² was used. 2 0.4W / cm 2 0.6W / cm 2 0.8W / cm 2 1W / cm 2An 808nm laser was used for irradiation, with the light source approximately 10cm away. A high-sensitivity temperature probe was inserted into the center of the solution, and a camera was used to record the heating process of the laser irradiation for 10 minutes under different dye concentrations. The temperature of each group of solutions was taken at different time points, and the temperature change curve of the solution over time was plotted.

[0245] The photothermal heating curve of compound 17 is as follows: Figure 15 As shown, this demonstrates that the compounds of the present invention have the potential for application in near-infrared photothermal ablation of tumors.

[0246] Example 20: The compound of this application generates singlet oxygen upon excitation.

[0247] A 20 μM acetonitrile solution of singlet oxygen detector DPBF was prepared, and its UV-Vis absorption spectrum was measured. A 2 μM acetonitrile solution of compound 21 was prepared, and its absorption spectrum was measured. A mixed solution of 20 μM DPBF and 2 μM compound 21 in acetonitrile was prepared, and its UV-Vis absorption spectrum was measured. The mixed solution was then subjected to a power density of 1 W / cm². 2 The UV-Vis absorption spectra of the mixed solution were measured after irradiation with an 808 nm laser for different durations (0 s, 5 s, 10 s, 20 s, 30 s). A 20 μM DPBF solution in acetonitrile was subjected to a 1 W / cm² laser. 2 After irradiation with an 808nm laser, its ultraviolet absorption spectrum was measured.

[0248] The generation of singlet oxygen in compound 21 after irradiation with an 808 nm laser was detected using the singlet oxygen detector DPBF. The experimental results are as follows: Figure 16 As shown in Figure A, the 0s irradiation curve and the 30s irradiation curve coincide, indicating that an acetonitrile solution with a concentration of 20 μM DPBF has been subjected to irradiation at a rate of 1 W / cm². 2 After irradiation with an 808nm laser, the absorbance of DPBF remained unchanged, indicating that the absorption spectrum of the DPBF detector did not change when irradiated only in the presence of DPBF. B represents the absorption spectra of 20μM singlet oxygen detector DPBF and 2μM compound 21 in acetonitrile solution, and the absorption spectrum of their mixture, showing that the absorption peak at 400nm is almost entirely contributed by DPBF. C represents the absorption of a mixed solution of 20μM singlet oxygen detector DPBF and 2μM compound 21 in acetonitrile solution after irradiation with a 1W / cm² laser for different durations. 2The absorption spectrum changes after irradiation with an 808nm laser were observed, with the 20s and 30s irradiation curves coinciding. After irradiation for 0s, 5s, 10s, and 20s, the absorbance at the absorption wavelength of DPBF decreased sequentially. C indicates a significant decrease in the absorbance of the singlet oxygen detector DPBF at 400nm, suggesting that compound 21 generates singlet oxygen after light irradiation. These test results indicate that compound 21 has potential application in photodynamic therapy for diseases.

Claims

1. The compound represented by formula A: In the formula: R1, R2, R3, R4, R5, R6, R7, and R8 are all hydrogen; R9, R 10 R 11 R 12 R 13 Each group is independently selected from: hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, C 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, optionally selected by 1-5 halogens, C 1-4 The C substituents of alkoxy and azido groups 1-4 Alkyl groups, optionally composed of one or two selected from C 1-4 C substituents of alkoxy, halogen, azide, amino, and thiol groups 1-4 Alkyl group, optionally oxidized to a heterocyclic group -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkoxy groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted aryl C 1-4 Alkoxy; heteroaryl C 1-4 The heteroaryl group in alkoxy refers to a 5-14 membered heteroaryl group composed of a carbon atom and 1-3 heteroatoms selected from O, N, and S; the aryl C 1-4 The aryl group in the alkoxy group is C. 6-14 Aryl; among which, The oxoheterocyclic group is a succinimide group. ; R 14 and R 15 Together with the nitrogen atoms connected to them, they form 3-8 membered heterocyclic spirochetes and 4-15 membered heterobridged cyclic groups, wherein the heterocyclic group is a 1,3-dioxolane ring group, and the heterobridged cyclic group is... ; R 16 and R 17 All are H; A - The acid is an acid radical ion that has been deprotonated, wherein the acid is selected from one of HCl, trifluoroacetic acid, methanesulfonic acid, acetic acid, and sulfuric acid; X is a halogen; Y is either O or S; n is an integer selected from 1 to 500.

2. The compound according to claim 1, characterized in that, R9, R 10 R 11 R 12 R 13 Each group is independently selected from hydrogen, amino, hydroxyl, nitro, cyano, carboxyl, halogen, and C. 2-4 alkenyl, -SO 3- -SO2X, -SO2NH2, C groups optionally substituted with 1-5 substituents selected from halogens and azide groups 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 C substituents of alkoxy, halogen, and azide groups 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkyl group.

3. The compound according to claim 1, characterized in that, R9, R 10 R 11 R 12 R 13 Each is selected independently from hydrogen, -SO 3- -SO2X, -SO2NH2, C groups optionally substituted with 1-3 substituents selected from halogens and azide groups 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 C substituents of alkoxy, halogen, and azide groups 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkyl group.

4. The compound according to claim 1, characterized in that, R9, R 10 R 11 R 12 R 13 Each is independently selected from: hydrogen, -SO 3- C10 groups are optionally substituted with 1-3 substituents selected from halogens and azide groups. 1-4 Alkyl groups, optionally selected from C10 and C20, are alkoxy groups. 1-4 Alkoxy and halogen substituents of C 1-4 Alkyl groups, and optionally oxidized heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 The alkoxy groups, X, Y, and n are defined as described in claim 1.

5. The compound according to claim 1, characterized in that, n is an integer selected from 1 to 300.

6. The compound according to claim 5, characterized in that, n is an integer selected from 1 to 200.

7. The compound according to claim 1, characterized in that, R1, R2, R3, R4, R5, R6, R7, and R8 are all hydrogen; R9, R 10 R 12 R 13 Each is independently selected from hydrogen, -SO 3- and optional C 1-4 alkoxy-substituted C 1-4 alkyl; R 11 C10 groups selected from hydrogen, optionally substituted with 1-3 substituents selected from halogens and azide groups. 1-4 Alkoxy groups and optionally oxy-substituted heterocyclic groups -OC(=O)-C 1-4 Alkylene-Y-[C 1-4 [alkylene-O] n -C 1-4 alkylene-substituted heteroaryl C 1-4 Alkoxy group, where Y is O and n is an integer selected from 1 to 100; R 16 and R 17 All are H.

8. The compound according to any one of claims 1-7, characterized in that, R 11 C is hydrogen, halogenated and azido-substituted. 1-4 Alkyl groups or succinimide groups -OC(=O)-C 1-2 Alkylene-O-[C 1-2 [alkylene-O] n -C 1-2 alkylene-substituted heteroaryl C 1-4 Alkyl group.

9. The compound according to claim 1, characterized in that, The compound is selected from the following compounds: , and .

10. A fluorescent dye comprising the compound of any one of claims 1-9 and an optional solvent.

11. The fluorescent dye according to claim 10, characterized in that, The solvent is selected from one or more of MeOH, EtOH, PrOH, iPrOH, BuOH, water, acetonitrile, acetone, DMF, DMSO, pyridine, dichloromethane, chloroform, dichloroethane, benzene, toluene, p-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, THF, ethyl acetate, AcOH, and buffer solutions.

12. The fluorescent dye according to claim 11, characterized in that, The buffer solution is PBS buffer.

13. A dye composition comprising the compound of any one of claims 1-9 and an optional solvent.

14. The dye composition according to claim 13, characterized in that, The solvent is selected from one or more of MeOH, EtOH, PrOH, iPrOH, BuOH, water, acetonitrile, acetone, DMF, DMSO, pyridine, dichloromethane, chloroform, dichloroethane, benzene, toluene, p-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, THF, ethyl acetate, AcOH, and buffer solutions.

15. The dye composition according to claim 14, characterized in that, The buffer solution is PBS buffer.

16. A kit comprising the compound of any one of claims 1-9 and optionally a solvent.

17. The kit according to claim 16, characterized in that, The solvent is selected from one or more of MeOH, EtOH, PrOH, iPrOH, BuOH, water, acetonitrile, acetone, DMF, DMSO, pyridine, dichloromethane, chloroform, dichloroethane, benzene, toluene, p-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, THF, ethyl acetate, AcOH, and buffer solutions.

18. The kit according to claim 17, characterized in that, The buffer solution is PBS buffer.

19. Use of the compound of any one of claims 1-9 in the preparation of reagents for near-infrared fluorescence imaging, photothermal therapy and / or photodynamic therapy.

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