Naphthalimide-based cyanine near-infrared fluorescent probe and application

Abstract

The invention belongs to the field of biological medicine, and relates to a naphthalimide-based cyanine near-infrared fluorescent probe and application thereof. The fluorescent probe disclosed by the invention has a structure as shown in a formula I, and in the formula I, R1-R9, m and n are described in the specification. The absorption wave band and the emission wave band of the intermediate of the fluorescent probe are both in a near-infrared region, near-infrared fluorescent probes with different functions can be obtained by changing an R5 group, and responsiveness recognition of different target molecules is achieved. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a near-infrared fluorescent probe based on naphthalimide and its application. Background Art

[0002] Fluorescent dyes are widely used in photocatalysis, optoelectronic materials, analytical testing, clinical diagnosis, etc. Small molecule dyes are indispensable in the field of chemical biology and are widely used in molecular tags, enzyme substrate labeling, cell staining, etc. Near-infrared fluorescent dyes generally refer to substances that have an absorption band in the near-infrared region (650-900nm) and can emit fluorescence. Compared with fluorescent dyes in the visible light region, near-infrared fluorescent dyes have the advantages of less biological molecule autofluorescence interference, deeper tissue penetration ability, weaker tissue autofluorescence interference, and lower signal-to-noise ratio in terms of developer, which is more conducive to in vivo application.

[0003] Traditional fluorescent dyes mainly include cyanine dyes, square acid dyes, BODIPY and the like. However, most traditional near-infrared fluorescent dyes generally have some defects, such as poor photostability, poor water solubility, and difficulty in optical modification. The classic dye naphthylimide has the advantages of high fluorescence quantum yield, easy modification, large Stokes shift, and good photostability, but its absorption wavelength is in the visible light region, making it difficult to carry out biological applications in deep tissues. CN114105982A discloses a series of near-infrared cyanine fluorescent dyes based on naphthylimide, which are mainly used in the fields of fluorescence imaging, photodynamic therapy, photothermal therapy, etc., but are less used in near-infrared fluorescent probes, and have room for further development. Summary of the invention

[0004] The present invention aims at the above-mentioned problems existing in the prior art, and provides a naphthaleneimide-based cyanine compound (compound of formula I) which can be used as a near-infrared fluorescent probe and its use.

[0005] In a first aspect of the present invention, there is provided a compound of formula I:

[0006]

[0007] In Formula I,

[0008] R1 and R9 are each independently selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C16 alkyl; the -[(CH2) p -O-] q H and C1-C16 alkyl are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C16 alkoxy and biological targeting groups; p is an integer selected from 1 to 6; q is an integer selected from 1 to 4;

[0009] R2 and R8 are each independently a C1-C16 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups;

[0010] R3, R4, R6 and R7 are each independently selected from: hydrogen and C1-C16 alkyl;

[0011] R5 is selected from: hydroxyl optionally substituted by substituents selected from C1-C16 acyl and C1-C16 alkyl, amino optionally substituted by substituents selected from C1-C16 acyl, C1-C16 alkyl, C6-C14 aryl and 5-14 membered heteroaryl, nitro, -N=N-C6-C14 aryl, and -N=N-5-14 membered heteroaryl; the C1-C16 acyl, the C1-C16 alkyl, the C6-C14 aryl and the 5-14 membered heteroaryl are optionally substituted by 1, 2, 3 or 4 substituents selected from the following groups: halogen, amino, C 1-C10 alkoxy, -(C1-C10 alkyl)NH, -(C1-C10 alkyl)2N, -(halo C1-C10 alkyl)NH, -(halo C1-C10 alkyl)(C1-C10 alkyl)N, -(halo C1-C10 alkyl)2N, and C6-C14 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxyl, amino and pinacol boryl; the 5-14 membered heteroaryl contains 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur;

[0012] m and n are each independently selected from integers from 0 to 6;

[0013] The biological targeting group is selected from: triphenylphosphine, morpholinyl and piperazine.

[0014] In one or more embodiments, the compound of formula I is selected from the following compound A, compound B, compound C, compound D and compound E:

[0015]

[0016]

[0017] The second aspect of the present invention provides a fluorescent dye, a dye composition, a fluorescent probe or a kit, wherein the fluorescent dye, the dye composition or the fluorescent probe contains the compound described in any embodiment of the present invention and an optional solvent; the kit contains the compound described in any embodiment of the present invention and an optional solvent, or contains the fluorescent dye, the dye composition or the fluorescent probe described in any embodiment of the present invention.

[0018] In one or more embodiments, the solvent is a buffer optionally containing an organic solvent.

[0019] In one or more embodiments, the buffer is a phosphate buffer.

[0020] In one or more embodiments, the solvent is a phosphate buffer containing ethanol.

[0021] In one or more embodiments, the fluorescent dye, dye composition, fluorescent probe or kit contains Compound A.

[0022] In one or more embodiments, the fluorescent dye, dye composition, fluorescent probe or kit contains compound B.

[0023] In one or more embodiments, the fluorescent dye, dye composition, fluorescent probe or kit contains Compound C.

[0024] In one or more embodiments, the fluorescent dye, dye composition, fluorescent probe or kit contains compound D.

[0025] In one or more embodiments, the fluorescent dye, dye composition, fluorescent probe or kit contains Compound E.

[0026] The third aspect of the present invention provides a use selected from the group consisting of:

[0027] (1) Use of the compound according to any embodiment of the present invention in fluorescence imaging, or in the preparation of a fluorescent probe;

[0028] (2) Use of compound A in the preparation of a fluorescent probe for detecting nitric oxide;

[0029] (3) Use of compound B in preparing a fluorescent probe for detecting nitric oxide;

[0030] (4) Use of compound C in the preparation of a fluorescent probe for detecting nitric oxide;

[0031] (5) Use of compound D and / or compound E in preparing a fluorescent probe for detecting hydrogen sulfide;

[0032] In one or more embodiments, the detection is a specific detection;

[0033] In one or more embodiments, the use is the use of compound A in the preparation of a fluorescent probe for quantitative detection of nitric oxide, and the concentration of nitric oxide is 10 to 30 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The absorption and emission spectra of compound A in ethanol, black is the absorption curve, and red is the emission curve.

[0035] Figure 2 The absorption and emission spectra of compound B in ethanol, black is the absorption curve, and red is the emission curve.

[0036] Figure 3 The absorption and emission spectra of compound C in ethanol, black is the absorption curve, and red is the emission curve.

[0037] Figure 4 The absorption and emission spectra of compound D in ethanol, black is the absorption curve, and red is the emission curve.

[0038] Figure 5 The absorption and emission spectra of compound E in ethanol, black is the absorption curve, and red is the emission curve.

[0039] Figure 6 This is the fluorescence spectrum of compound A reacting with different concentrations of nitric oxide.

[0040] Figure 7 This is a curve diagram showing the relationship between the fluorescence intensity of compound A and the concentration of nitric oxide.

[0041] Figure 8 This is the linear fitting diagram of the fluorescence intensity of compound A and the nitric oxide concentration.

[0042] Fig. 9 This is a diagram showing the selectivity experimental results of compound A.

[0043] Fig.10 This is the reaction kinetics experimental result diagram of compound A.

[0044] Fig.11 This is a diagram showing the cytotoxicity test results of compound A.

[0045] Fig.12 This is an in vivo imaging image of the acute dermatitis mouse model detected by compound A. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in this document. Unless otherwise specified, all technical and scientific terms used in this document have the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definitions in this specification shall prevail.

[0047] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0048] Herein, “comprising”, “including”, “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of”. For example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to be disclosed herein.

[0049] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible sub-ranges and individual values ​​within the range (including integers and fractions).

[0050] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0051] Herein, the sum of the percentages of the various components of the composition is 100%.

[0052] Herein, when describing embodiments or examples, it should be understood that they are not used to limit the present invention to these embodiments or examples. On the contrary, all substitutes, modifications and equivalents of the methods and materials described in the present invention are all within the scope of the present invention.

[0053] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0054] definition

[0055] Certain chemical groups defined herein are preceded by simplified notations to indicate the total number of carbon atoms present in the group. For example, C1-C4 alkyl refers to an alkyl group as defined below having a total of 1 to 4 carbon atoms. The total number of carbon atoms in the simplified notation does not include the number of carbon atoms that may be present in the substituents of the group.

[0056] As used herein, "halogen" refers to an element of Group 17 having an atomic number of 9 to 85. "Halogen" or "halogen atom" refers to F, Cl, Br, and I. "Halo" means substituted with an atom selected from F, Cl, Br, and I.

[0057] As used herein, "hydroxy" refers to -OH.

[0058] As used herein, "amino" refers to -NH2.

[0059] As used herein, "nitro" refers to -NO2.

[0060] As used herein, "carboxyl" refers to -COOH.

[0061] As used herein, "thiol" refers to -SH.

[0062] As used herein, "sulfonic acid" refers to -SO3H.

[0063] As used herein, "triphenylphosphino" refers to -P + Ph3.

[0064] As used herein, "alkyl" refers to a straight or branched monovalent saturated hydrocarbon group having a specified number of carbon atoms, and specific alkyl groups are those having 1 to 16 carbon atoms (C1-C16 alkyl), usually containing 1 to 10 carbon atoms (C1-C10 alkyl), preferably containing 1 to 8 carbon atoms (C1-C8 alkyl), containing 1 to 6 carbon atoms (C1-C6 alkyl), more preferably containing 1 to 4 carbon atoms (C1-C4 alkyl) or 1 to 3 carbon atoms (C1-C3 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0065] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl), wherein the fused rings may or may not be aromatic. In one variation, the aryl group contains 6 to 14 ring carbon atoms, preferably C6 to C10 aryl. An aryl group having more than one ring and at least one of which is a non-aromatic ring may be attached to the parent structure at an aromatic ring position or at a non-aromatic ring position. In one variation, an aryl group having more than one ring and at least one of which is a non-aromatic ring is attached to the parent structure at an aromatic ring position. Examples of aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene and fluorenyl.

[0066] As used herein, "alkoxy" refers to alkyl-O-, and preferred alkoxy is C1-C10 alkoxy, such as C1-C8 alkoxy, C1-C6 alkoxy, C1-C4 alkoxy, which includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy and the like.

[0067] As used herein, as a group or as part of other groups, the term "heteroaryl" or "heteroaromatic ring" refers to a conjugated ring system group or part having carbon atoms (e.g., 1 to 11 carbon atoms, 1 to 10 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and heteroatoms (e.g., 1 to 6 heteroatoms, such as 1, 2, 3, 4 or 5 heteroatoms) selected from nitrogen, oxygen and sulfur in the ring. In some embodiments, the heteroaryl group may contain 5 to 14 ring-forming atoms, 5 to 12 ring-forming atoms, 5 to 10 ring-forming carbon atoms, 5 to 8 ring-forming atoms, 5 to 7 ring-forming atoms, or 5 to 6 ring-forming atoms. Unless otherwise specifically indicated in this specification, the heteroaryl group may be a monocyclic, bicyclic, tricyclic or more ring system, and may also be fused with an aryl or heterocyclic group as defined above. Examples of heteroaryl fused with aryl include, but are not limited to, benzopyridyl, benzopyrazolyl, benzimidazolyl, benzopyrrolyl, etc. The nitrogen, carbon or sulfur atom in the heteroaryl may be optionally oxidized; the nitrogen atom may be optionally quaternized. For the purposes of the present disclosure, in some embodiments, the heteroaryl is a stable 5- to 14-membered aromatic group comprising 1 to 5 (e.g., 1, 2, 3, 4, or 5) heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a stable 5- to 10-membered aromatic group comprising 1 to 4 (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered aromatic group comprising 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically provided in this specification, the heteroaryl may be optionally substituted.

[0068] Non-limiting examples of heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolyl, isoquinolyl, naphthazinyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothiophenyl, oxatriol, oxazolyl, cinnolinyl, quinazolinyl, indolizinyl, o-phenanthroline, isoxazolyl, phenoxazinyl, phenothiazinyl, 4,5,6,7-tetrahydrobenzo[b]thienyl, naphthopyridinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[4,3-a]pyrazinyl, [1,2,4]triazolo[4,3-c]pyrimidinyl, [1,2,4]triazolo[4,3-a]pyridinyl, imidazo[1,2-a]pyridinyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, and the like.

[0069] As used herein, "acyl" refers to HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, etc., wherein the alkyl and substituted alkyl are as defined in the present application. Exemplary acyl groups include formyl, acetyl, etc. C1 acyl refers to HC(O)- (i.e., formyl). C2~C16 acyl refers to C1~C15 alkyl-C(O)-, and the definition of alkyl is as described above. Preferred C1~C16 acyl groups include C1~C10 acyl, C1~C8 alkyl acyl, C1~C6 alkyl acyl, C1~C4 alkyl acyl, C1~C3 alkyl acyl. Exemplary acyl groups include formyl, acetyl, propionyl, butyryl, etc.

[0070] As used herein, "optionally" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes both the occurrence and non-occurrence of the event or situation. For example, "optionally substituted aryl" means that the aryl is substituted or unsubstituted, and the description includes both substituted aryl and unsubstituted aryl. The "optionally" substituents described in the claims and the specification of the present invention are selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthiol, halogen, haloalkyl, haloalkenyl, haloalkynyl, hydroxyl, amino, carboxyl, thiol, nitro, sulfonic acid, pinacol borane, acyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclic.

[0071] As used herein, the term "substituted", whether or not preceded by the term "optionally" (i.e., equivalent to substituted or unsubstituted), refers to the replacement of one or more hydrogens of a specified group or part by a "suitable substituent". Herein, the number of substituents may be one or more, i.e., 1, 2, 3, 4, 5 or 6 or more, depending on the nature of the substituted group and the substituent. For example, when the substituent of an ethyl group is a halogen, the group may be replaced by 1, 2, 3, 4 or 5 substituents, such as trifluoroethyl, pentafluoroethyl, etc., depending on the structure of the substituted group. In some embodiments, the number of the substituents is 1, 2 or 3. In some embodiments, the number of the substituents is 1 or 2. In some embodiments, the number of the substituents is 1. It will be understood that "substituted" or "substituted by..." includes implicit conditions, i.e., such substitutions are carried out according to the allowed valence of the substituted atom, and the substitution produces a stable or chemically feasible compound, such as a compound that will not spontaneously transform, such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. It will be understood by those skilled in the art that the substituent itself may be substituted if appropriate. Unless specifically indicated as "unsubstituted", chemical moieties referred to herein should be understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes unsubstituted aryl groups and substituted variants.

[0072] For the entire application, the above-mentioned “suitable substituents” should be understood to include, but are not limited to, the alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, alkylthiol, thiol, sulfonic acid, pinacol borane, acyl, carboxyl, hydroxyl, amino, monoalkylamino, dialkylamino, nitro, aryl, heteroaryl, cycloalkyl (e.g., cycloalkyl, cycloalkenyl, etc.), heterocyclyl, etc. described herein; these groups as substituents, including alkyl, alkenyl, alkynyl, alkyl in haloalkyl, alkenyl in haloalkenyl, alkynyl in haloalkynyl, alkoxy, alkyl in monoalkylamino, alkyl in dialkylamino, aryl, heteroaryl, cycloalkyl and heterocyclyl themselves are also optionally substituted, for example, they may also be optionally substituted by one or more groups selected from alkyl, halogen, haloalkyl, alkoxy, hydroxyl, amino, monoalkylamino, dialkylamino, nitro, aryl, heteroaryl, cycloalkyl and heterocyclyl.

[0073] It will also be appreciated by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g. tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable amino, amidino and guanidino protecting groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable sulfhydryl protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, etc. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

[0074] Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, TW and PGM Wuts, Protective Groups in Organic Synthesis, (1999), 4th Ed., Wiley. The protecting group can also be a polymer resin.

[0075] Cyanine near-infrared fluorescent dyes

[0076] The present invention provides a compound that can be used as a cyanine near-infrared fluorescent fuel, and the compound has a structure shown in Formula I:

[0077]

[0078] In Formula I,

[0079] R1 is selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C16 alkyl; wherein -[(CH2) p -O-] q H and C1-C16 alkyl are optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C16 alkoxy (e.g., C1-C10 alkoxy) and biological targeting groups;

[0080] R2 is a C1-C16 alkyl group (e.g., a C1-C10 alkyl group) optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups;

[0081] R3 and R4 are each independently selected from: hydrogen and C1-C16 alkyl (e.g., C1-C10 alkyl);

[0082] R5 is selected from: hydroxyl optionally substituted with a substituent selected from C1-C16 acyl (e.g., C1-C10 acyl) and C1-C16 alkyl (C1-C10 alkyl), amino optionally substituted with a substituent selected from C1-C16 acyl (e.g., C1-C10 acyl), C1-C16 alkyl (e.g., C1-C10 alkyl), C6-C14 aryl and 5-14 membered heteroaryl, nitro, -N=N-C6-C14 aryl, and -N=N-5-14 membered heteroaryl; wherein C1-C16 acyl, C1-C16 alkyl, C6-C14 aryl, and 5-14 membered heteroaryl are optionally substituted with 1, 2, substituted by 3 or 4 substituents selected from the group consisting of halogen, amino, C1-C10 alkoxy, -(C1-C10 alkyl)NH, -(C1-C10 alkyl)2N, -(halogenated C1-C10 alkyl)NH, -(halogenated C1-C10 alkyl)(C1-C10 alkyl)N, -(halogenated C1-C10 alkyl)2N, and C6-C14 aryl optionally substituted by 1, 2, 3 or 4 substituents selected from nitro, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxy, amino and pinacol boryl; 5-14 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur;

[0083] R6 and R7 are each independently selected from: hydrogen and C1-C16 alkyl (e.g., C1-C10 alkyl);

[0084] R8 is a C1-C16 alkyl group (e.g., a C1-C10 alkyl group) optionally substituted with 1, 2, 3, or 4 substituents selected from a carboxyl group, a sulfonic acid group, and a biological targeting group;

[0085] R9 is selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C16 alkyl; wherein -[(CH2) p -O-] q H and C1-C16 alkyl are optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C10 alkoxy (e.g., C1-C10 alkoxy) and biological targeting groups;

[0086] m and n are each independently selected from integers from 0 to 3;

[0087] p is an integer selected from 1 to 6;

[0088] q is an integer selected from 1 to 4;

[0089] The biological targeting group is selected from the group consisting of triphenylphosphine, morpholinyl and piperazinyl.

[0090] The compounds of the present invention have the structure shown in Formula I, and are therefore referred to as Formula I compounds for short.

[0091] In some embodiments, R1 is selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C10 alkyl; wherein -[(CH2) p -O-] q H and C1-C10 alkyl are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C6 alkoxy and biological targeting groups; q is 1, 2 or 3; p is 1, 2 or 3.

[0092] In some embodiments, R1 is an optionally substituted C1-C6 alkyl group; wherein the C1-C6 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C4 alkoxy and biological targeting groups.

[0093] In some embodiments, R1 is C1 alkyl, C2 alkyl, C3 alkyl, or C4 alkyl.

[0094] In some embodiments, R2 is a C1-C10 alkyl group optionally substituted with 1 or 2 sulfonic acid groups.

[0095] In some embodiments, R2 is a C1-C6 alkyl group substituted with 1 or 2 sulfonic acid groups.

[0096] In some embodiments, R2 is a C1 alkyl, C2 alkyl, or C3 alkyl substituted with 1 sulfonic acid group.

[0097] In some embodiments, R3 and R4 are each independently selected from C1-C6 alkyl.

[0098] In some embodiments, R3 and R4 are each independently selected from C1-C4 alkyl.

[0099] In some embodiments, R3 and R4 are both methyl.

[0100] In some embodiments, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1-C10 acyl and C1-C10 alkyl, amino optionally substituted with substituents selected from C1-C10 acyl, C1-C10 alkyl, C6-C14 aryl and 5-14 membered heteroaryl, nitro, -N=N-C6-C14 aryl, and -N=N-5-14 membered heteroaryl; wherein C1-C10 acyl, C1-C10 alkyl, C6-C14 aryl, and 5-14 membered heteroaryl are The group is optionally substituted by 1, 2, 3 or 4 substituents selected from the following groups: halogen, amino, C1~C10 alkoxy, -(C1~C10 alkyl)NH, -(C1~C10 alkyl)2N, -(halogenated C1~C10 alkyl)NH, -(halogenated C1~C10 alkyl)(C1~C10 alkyl)N, -(halogenated C1~C10 alkyl)2N, and C6~C14 aryl optionally substituted by 1, 2, 3 or 4 substituents selected from nitro, halogen and pinacol boryl.

[0101] In some embodiments, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1~C6 acyl and C1~C4 alkyl, amino optionally substituted with substituents selected from C1~C6 acyl, C1~C4 alkyl and C6~C10 aryl, nitro and -N=N-C6~C10 aryl; wherein, C1~C6 acyl, C1~C4 alkyl and C6~C10 aryl are optionally substituted with 1, 2, 3 or 4 substituents selected from the following group: halogen, amino, C1~C6 alkoxy, -(C1~C6 alkyl)2N, -(haloC1~C6 alkyl)2N, and C6~C10 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen and pinacol borane.

[0102] In some embodiments, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1~C6 acyl and C1~C4 alkyl, amino optionally substituted with C6-C10 aryl, nitro and -N=N-C6~C10 aryl; wherein, C1~C6 acyl, C1~C4 alkyl and C6~C10 aryl are optionally substituted with 1 or 2 substituents selected from the following group: halogen, C1~C6 alkoxy, -(haloC1~C6 alkyl)2N, and C6~C10 aryl optionally substituted with 1 or 2 substituents selected from nitro, halogen and pinacol borane.

[0103] In some embodiments, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1~C6 acyl and C1~C4 alkyl, amino optionally substituted with C6~C10 aryl, nitro and -N=N-C6~C10 aryl; wherein, C6~C10 aryl is optionally substituted with 1 or 2 substituents selected from amino, C1~C6 alkoxy, -(C1~C6 alkyl)2N and -(halogenated C1~C6 alkyl)2N; C1~C6 acyl is optionally substituted with 1 or 2 substituents selected from Cl, Br and C6~C10 aryl optionally substituted with halogen (such as I); C1~C4 alkyl is optionally substituted with 1 or 2 of the following substituents: C6~C10 aryl optionally substituted with substituents selected from nitro and pinacol boryl.

[0104] In some embodiments, R5 is selected from:

[0105]

[0106] Wherein, * indicates the position where R5 is connected to other parts of the compound of the present invention.

[0107] In some embodiments, R6 and R7 are each independently selected from C1-C6 alkyl.

[0108] In some embodiments, R6 and R7 are each independently selected from C1-C4 alkyl.

[0109] In some embodiments, R6 and R7 are both methyl.

[0110] In some embodiments, R8 is C1-C10 alkyl optionally substituted with 1 or 2 sulfonic acid groups.

[0111] In some embodiments, R8 is C1-C6 alkyl substituted with 1 or 2 sulfonic acid groups.

[0112] In some embodiments, R8 is C1 alkyl, C2 alkyl, or C3 alkyl substituted with 1 sulfonic acid group.

[0113] In some embodiments, R9 is selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C10 alkyl; wherein -[(CH2) p -O-] q H and C1-C10 alkyl are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C6 alkoxy and biological targeting groups; q is 1, 2 or 3; p is 1, 2 or 3.

[0114] In some embodiments, R9 is an optionally substituted C1-C6 alkyl group; wherein the C1-C6 alkyl group is optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C4 alkoxy and biological targeting groups.

[0115] In some embodiments, R9 is C1 alkyl, C2 alkyl, C3 alkyl, or C4 alkyl.

[0116] In some embodiments, in Formula I,

[0117] R1 is an optionally substituted C1-C16 alkyl group; the C1-C16 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of a hydroxyl group, an amino group, a thiol group, a carboxyl group, a sulfonic acid group, a C1-C10 alkoxy group and a biological targeting group;

[0118] R2 is a C1-C10 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups;

[0119] R3 and R4 are each independently selected from C1-C6 alkyl;

[0120] R5 is selected from: hydroxyl optionally substituted with substituents selected from C1-C6 acyl and C1-C4 alkyl, amino optionally substituted with substituents selected from C1-C6 acyl, C1-C4 alkyl and C6-C10 aryl, nitro and -N=N-C6-C10 aryl; the C1-C6 acyl, C1-C4 alkyl and C6-C10 aryl are optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of halogen, amino, C1-C6 alkoxy, -(C1-C6 alkyl)NH, -(C1-C6 alkyl)2N, -(halogenated C1-C6 alkyl)NH, -(halogenated C1-C6 alkyl)(C1-C6 alkyl)N, -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen and pinacol boryl;

[0121] R6 and R7 are each independently selected from C1-C6 alkyl;

[0122] R8 is a C1-C10 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups;

[0123] R9 is an optionally substituted C1-C16 alkyl group; the C1-C16 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C10 alkoxy and biological targeting groups;

[0124] m and n are each independently an integer of 0 to 6; preferably, m and n are 0, 1 or 2, respectively.

[0125] In some embodiments, in Formula I,

[0126] R1 is a C1-C4 alkyl group (e.g., a C1 alkyl group, a C2 alkyl group, a C3 alkyl group, a C4 alkyl group);

[0127] R2 is a C1-C3 alkyl group (e.g., a C1 alkyl group, a C2 alkyl group, a C3 alkyl group) optionally substituted with one sulfonic acid group;

[0128] R3 and R4 are each independently selected from C1-C4 alkyl (e.g., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl);

[0129] R5 is selected from: hydroxyl optionally substituted by a substituent selected from C1-C6 acyl (e.g., C1 acyl, C2 acyl, C3 acyl, C4 acyl, C5 acyl, C6 acyl) and C1-C4 alkyl (e.g., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl), amino optionally substituted by C6-C10 aryl (e.g., C6 aryl, C10 aryl), nitro and -N=N-C6-C10 aryl; the C1-C6 Acyl, C1-C4 alkyl and C6-C10 aryl are optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, C1-C6 alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy), -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted with 1 or 2 substituents selected from the group consisting of nitro, halogen (e.g., I) and pinacol boryl;

[0130] R6 and R7 are each independently selected from C1-C4 alkyl (e.g., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl);

[0131] R8 is a C1-C3 alkyl group (e.g., a C1 alkyl group, a C2 alkyl group, a C3 alkyl group) optionally substituted with one sulfonic acid group;

[0132] R9 is C1-C4 alkyl (e.g., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl);

[0133] m is 1;

[0134] n is 0.

[0135] In some embodiments, the compound of Formula I is selected from:

[0136]

[0137]

[0138] Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)), for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0139] Fluorescent probes

[0140] The present invention also provides a fluorescent dye, a dye composition or a fluorescent probe, which contains a compound of formula I of the present invention and an optional solvent. Suitable solvents for the present invention include, but are not limited to, a buffer. The buffer optionally contains an organic solvent (e.g., ethanol). The buffer is preferably a phosphate buffer. The pH of the phosphate buffer is 7.2 to 7.6, preferably 7.3 to 7.5. In some embodiments, the solvent is a phosphate buffer containing ethanol. Preferably, in the phosphate buffer containing ethanol, the volume ratio of the phosphate buffer to ethanol may be (1 to 10):1, preferably (1 to 5):1.

[0141] The present invention also provides a kit for fluorescence imaging or for detecting a specific substance, the kit comprising a compound of formula I of the present invention and an optional solvent, or comprising a fluorescent dye, a dye composition or a fluorescent probe of the present invention. The fluorescent dye, the dye composition, the fluorescent probe and the solvent are as described above.

[0142] In some embodiments, the fluorescent dye, dye composition, fluorescent probe or kit of the present invention contains compound A.

[0143] In some embodiments, the fluorescent dye, dye composition, fluorescent probe or kit of the present invention contains compound B.

[0144] In some embodiments, the fluorescent dye, dye composition, fluorescent probe or kit of the present invention contains compound C.

[0145] In some embodiments, the fluorescent dye, dye composition, fluorescent probe or kit of the present invention contains compound D.

[0146] In some embodiments, the fluorescent dye, dye composition, fluorescent probe or kit of the present invention contains compound E.

[0147] application

[0148] The present invention also provides the use of the compound of formula I of the present invention in fluorescence imaging, or in the preparation of a fluorescent probe. The present invention also provides the use of compound A in the preparation of a fluorescent probe for detecting nitric oxide. When the concentration of nitric oxide is 10 to 30 μM, the detection is a quantitative detection. The present invention also provides the use of compound B and / or compound C in the preparation of a fluorescent probe for detecting nitric oxide. The present invention also provides the use of compound D and / or compound E in the preparation of a fluorescent probe for detecting hydrogen sulfide. In some embodiments, the detection is a specific detection.

[0149] Preparation method

[0150] The present invention provides a method for preparing a compound of formula I, wherein the method for preparing compound A to compound E is shown in the following formula, and each chemical reaction mentioned in the formula can be achieved by methods disclosed in the literature and conventional methods in the art. The preparation methods of other compounds of formula I can refer to the preparation methods of compound A to compound E.

[0151]

[0152]

[0153] An exemplary preparation step for preparing the intermediates of Compound A to Compound E is as follows:

[0154] (1) reacting 4-bromo-1,8-naphthoic anhydride with R1-NH2 (e.g., n-butylamine) to obtain compound ML-1;

[0155] (2) reacting compound ML-1 with hydrazine hydrate to prepare compound ML-2;

[0156] (3) reacting compound ML-2 with 3-methyl-2-butanone to undergo Fischer-Indole reaction to prepare compound ML-3;

[0157] (4) reacting compound ML-3 with 1,3-propane sultone and other raw materials to obtain compound ML-4 having a naphthylimide pyrrole structure;

[0158] (5) reacting cyclohexanone with Vilsmeie reagent to prepare compound ML-10;

[0159] (6) Compound ML-4 and compound ML-10 are reacted to prepare an intermediate.

[0160] In step (1), weigh 4-bromo-1,8-naphthalene anhydride and place it in a three-necked flask, and add an appropriate amount of ethanol to dissolve it. Measure an appropriate amount of R1-NH2 and slowly add it to the three-necked flask, and the color of the solution turns brown. Place the flask at 60-90oC (such as about 80°C), reflux with a condenser filled with water, and react for 6-18 hours (such as 10-12 hours). Cool the reaction to room temperature, and a large amount of yellow solid will precipitate in the flask. Filter it with a Buchner funnel, rinse the filter cake with ethanol, and dry it in an infrared oven to obtain compound ML-1.

[0161] In step (2), weigh ML-1 and place it in a three-necked flask, add an appropriate amount of ethylene glycol monomethyl ether to dissolve it, and then add an appropriate amount of 80% hydrazine hydrate. Place the flask under the protection of inert gas and react at 90-150°C (such as about 120°C) for 2-6 hours (such as about 4 hours). Cool the reaction to room temperature, and after a large amount of orange solid precipitates, filter it with a Buchner funnel, rinse the filter cake with ethanol, and dry it in an infrared oven to obtain compound ML-2.

[0162] In step (3), weigh ML-2 and place it in an eggplant-shaped bottle, add an appropriate amount of acetic acid to dissolve it, and then add an appropriate amount of 3-methyl-2-butanone. Place the reaction device under inert gas protection, heat and reflux at 90-150°C (such as about 120°C) for 6-18 hours (such as 12 hours) to stop the reaction. After removing the solvent under reduced pressure, an orange crude product is obtained. Further purification is performed by column chromatography to obtain compound ML-3.

[0163] In step (4), ML-3 is weighed and placed in a single-necked flask, and an appropriate amount of 1,2-dichlorobenzene is added to dissolve it, and then an appropriate amount of raw materials (such as 1,3-propane sultone, methyl iodide, ethyl iodide, 1,4-butane sultone, bromoacetic acid, 3-bromopropionic acid, 4-bromobutyric acid, 6-bromohexanoic acid) are added, and the reaction is protected by inert gas and then placed at 90-150° C. (such as about 120° C.) and heated to reflux for 12-20 hours (such as 18 hours). After the reaction is completed, column chromatography is performed to purify and obtain a white solid ML-4.

[0164] In step (5), POCl3 and anhydrous DMF are slowly added to a dry two-necked flask under ice bath conditions, and then an appropriate amount of anhydrous DCM and anhydrous DMF are drawn into the flask with a syringe. An appropriate amount of cyclohexanone is drawn into the flask with a syringe. The reaction is heated to reflux at 20-60°C (such as about 45°C) under inert gas protection for 2-6 hours (such as about 4 hours). After the reaction is completed, it is cooled to room temperature, the reaction solution is poured into crushed ice, and placed in a refrigerator at 4°C overnight. After a large amount of yellow solid is precipitated, it is filtered with a Buchner funnel, and the filter cake is rinsed with ice water to obtain compound ML-10.

[0165] In step (6), ML-4 and ML-10 are weighed and placed in a single-necked flask, anhydrous sodium acetate is added, and dissolved with a mixed solvent (such as acetic acid / acetic anhydride). The reaction is heated to reflux at 90-150° C. (such as about 120° C.) under the protection of an inert gas, and the reaction is terminated after 3-8 hours (such as about 5 hours), cooled to room temperature, and the solvent is removed under reduced pressure to obtain a brown crude product. The product is further separated and purified by column chromatography to obtain an intermediate.

[0166] In the above formulae, ML-1, ML-2, ML-3, ML-4 and intermediates, R1 to R9, m and n are as described in any embodiment of the present invention.

[0167] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods, reagents and materials used in the examples and comparative examples are, unless otherwise stated, conventional methods, reagents and materials in the art. The raw material compounds in the examples and comparative examples can all be purchased through commercial routes.

[0168] Preparation Example: Preparation of Intermediates

[0169]

[0170] The specific synthesis steps of the intermediate are as follows:

[0171] (1) Weigh 4-bromo-1,8-naphthalene anhydride (8.00 g, 28.88 mmol) into a three-necked flask and add 50 mL of ethanol to dissolve. Measure 6 mL of n-butylamine and slowly add it into the three-necked flask. The color of the solution turns brown. Place the flask at 80°C and reflux with a condenser filled with water for 12 hours. Cool the reaction to room temperature. A large amount of yellow solid precipitates in the flask. Filter with a Buchner funnel, rinse the filter cake with ethanol, and dry it in an infrared oven to obtain compound ML-1.

[0172] (2) Weigh ML-1 (2.00 g, 6.02 mmol) and place it in a three-necked flask. Add 30 mL of ethylene glycol monomethyl ether to dissolve it, and then add 5 mL of 80% hydrazine hydrate. Place the flask under argon protection and react at 120° C. for 4 hours. Cool the reaction to room temperature. After a large amount of orange solid precipitates, filter it with a Buchner funnel, rinse the filter cake with ethanol, and dry it in an infrared oven to obtain compound ML-2.

[0173] (3) Weigh ML-2 (6.00 g, 21.18 mmol) and place it in an eggplant-shaped bottle. Add 50 mL of acetic acid to dissolve it, and then add 5 mL of 3-methyl-2-butanone. Place the reaction apparatus under argon protection, heat at 120°C and reflux for 12 hours to stop the reaction. Remove the solvent under reduced pressure to obtain an orange crude product. Further purify it by column chromatography to obtain compound ML-3.

[0174] (4) ML-3 (500 mg, 1.49 mmol) was weighed and placed in a single-necked flask, 5 mL of 1,2-dichlorobenzene was added to dissolve it, and then 1 mL of 1,3-propane sultone was added. The flask was protected by argon and heated to reflux at 120°C for 18 hours. After the reaction was completed, column chromatography was performed to purify the mixture to obtain a white solid ML-4.

[0175] (5) Under ice bath conditions, 20 mL of POCl3 and 20 mL of anhydrous DMF were slowly added to a dry two-necked flask, and then 40 mL of anhydrous DCM and 40 mL of anhydrous DMF were drawn into the flask using a syringe. 10 g of cyclohexanone was drawn into the flask using a syringe. The reaction was heated to reflux at 45°C for 4 hours under argon protection. After the reaction was completed, the reaction solution was cooled to room temperature, poured into crushed ice, and placed in a refrigerator at 4°C overnight. After a large amount of yellow solid was precipitated, it was filtered using a Buchner funnel, and the filter cake was rinsed with ice water to obtain compound ML-10.

[0176] (6) ML-4 (185 mg, 407.7 μmol) and ML-10 (35.5 mg, 204.2 μmol) were weighed and placed in a single-necked flask, anhydrous sodium acetate (335 mg, 407.7 μmol) was added, and dissolved with 5 mL of acetic acid / acetic anhydride (Ac2O: AcOH = 3:2, v / v) mixed solvent. The reaction was heated to reflux at 120°C under argon protection, and the reaction was terminated after 5 hours. The reaction was cooled to room temperature and the solvent was removed under reduced pressure to obtain a brown crude product. The intermediate was further separated and purified by column chromatography. 1 H NMR (600MHz, DMSO-d6): δ9.09 (d, J=8.0Hz 2H),8.80(s,2H),8.43(d,J=13.0Hz,2H),8.01(t,J=7.0Hz,2H),6.90(d,J=13.0Hz,2H),4.90(s,4H),4.09(s,4H),2. 92(s,4H),2.82(s,4H),2.51(s,4H),2.30(s,4H),1.92(s,12H),1.34(dd,J=14.0,7.0Hz,6H),0.96(t,J=7.0Hz,6H).

[0177] Example 1: Synthesis and characterization of compound A

[0178]

[0179] The specific synthesis steps are as follows:

[0180] Weigh the intermediate (209.7 mg, 200 μmol) and p-anisidine (24.6 mg, 200 μmol) and add them to a 25 mL dry two-necked flask, then purge the device with argon to protect it with inert gas. Use a syringe to draw 5 mL of anhydrous DMF and triethylamine (20 mg, 200 μmol) respectively and add them to the flask to dissolve the solid. The solution is purple-red. Place the reaction device in a 55 ° C oil bath and heat it under reflux for 10 hours. Monitor the reaction by TLC. The solution is dark blue after the reaction. Stop the reaction, wait for the solution to cool to room temperature, and then remove the solvent by rotary evaporation to obtain a black crude product. Further separation and purification is carried out by silica gel column chromatography (DCM: MeOH = 6: 1), collect the product and spin dry the solvent to obtain 146.5 mg of compound A (yield: 64.57%). 1 H NMR (600MHz, DMSO) δ10.21(s,1H),8.98(d,J=8.4Hz,2H),8.55(t,J=6.5Hz,2H),8.49(s,2H),8.01(d,J= 12.1Hz,2H),7.87(t,J=7.4Hz,2H),7.32(d,J=8.8Hz,2H),7.03(d,J=8.3Hz,2H),6.47(dd,J=17.8,11.0 Hz,2H),4.70(s,4H),4.06(t,J=6.7Hz,5H),3.72(s,3H),2.76(dd,J=13.3,6.5Hz,9H),2.20(s,4H),1.8 8–1.83(m,2H),1.61(dd,J=14.6,7.4Hz,4H),1.45(s,12H),1.33(d,J=5.9Hz,4H),0.92(t,J=7.3Hz,8H).

[0181] Example 2: Synthesis and characterization of compound B

[0182]

[0183] The specific synthesis steps are as follows:

[0184] Weigh the intermediate (209.7 mg, 200 μmol) and o-anisidine (24.6 mg, 200 μmol) and add them to a 25 mL dry two-necked flask, then purge the device with argon to protect it with inert gas. Use a syringe to draw 5 mL of anhydrous DMF and triethylamine (20 mg, 200 μmol) respectively and add them to the flask to dissolve the solid. The solution is purple-red. Place the reaction device in a 55°C oil bath and heat it under reflux for 10 hours. Monitor the reaction by TLC. The solution is dark blue after the reaction. Stop the reaction, wait for the solution to cool to room temperature, and then remove the solvent by rotary evaporation to obtain a black crude product. Further separation and purification is carried out by silica gel column chromatography (DCM: MeOH = 6: 1), collect the product and spin dry the solvent to obtain 118.7 mg of compound B (yield: 52.32%). 1H NMR (400MHz, DMSO) δ9.72(s,1H),9.01(d,J=8.5Hz,2H),8.58(d,J=7.2Hz,2H),8.54(s,2H),7.96(d,J=13 .1Hz,2H),7.89(t,J=7.9Hz,2H),7.23(d,J=7.4Hz,1H),7.18(s,1H),7.10(t,J=7.6Hz,1H),7.02(t,J=7. 9Hz,1H),6.49(d,J=13.2Hz,2H),4.72(s,4H),4.08(s,4H),4.02(s,3H),2.78(s,8H),2.21(d,J=7.0Hz,4 H),2.00(d,J=7.8Hz,2H),1.67–1.60(m,4H),1.47(s,12H),1.35(d,J=6.2Hz,4H),0.94(t,J=7.3Hz,6H).

[0185] Example 3: Synthesis and characterization of compound C

[0186]

[0187] The specific synthesis steps are as follows:

[0188] Weigh the intermediate (209.7 mg, 200 μmol) and m-anisidine (24.6 mg, 200 μmol) and add them to a 25 mL dry two-necked flask, then purge the device with argon to protect it with inert gas. Use a syringe to draw 5 mL of anhydrous DMF and triethylamine (20 mg, 200 μmol) respectively and add them to the flask to dissolve the solid, and the solution is purple-red. Place the reaction device in a 55 ° C oil bath and heat it under reflux for 10 hours. Monitor the reaction by TLC. The solution is dark blue after the reaction. Stop the reaction, wait for the solution to cool to room temperature, and then remove the solvent by rotary evaporation to obtain a black crude product. Further separation and purification is carried out by silica gel column chromatography (DCM: MeOH = 6: 1), collect the product and spin dry the solvent to obtain a dark green solid compound C104.7 mg with a metallic luster (yield: 46.16%). 1 H NMR (400MHz, DMSO) δ9.56 (s, 1H), 9.04 (d, J = 8.3Hz, 2H), 8.60 (d, J = 7.3Hz, 2H), 8.55 (s, 2H), 8.10 (d, J = 13.1 Hz,2H),7.92(t,J=7.7Hz,2H),7.27(t,J=8.3Hz,1H),6.78(s,1H),6.74(d,J=8.6Hz,1H),6.61(d,J=13.5Hz ,2H),6.52(d,J=7.6Hz,1H),4.79(s,4H),4.08(s,4H),3.77(d,J=16.9Hz,3H),2.80(d,J=5.8Hz,8H),2.24( s,4H),2.02(d,J=7.5Hz,2H),1.63(d,J=7.5Hz,4H),1.49(s,12H),1.39–1.33(m,5H),0.94(t,J=7.3Hz,6H).

[0189] Example 4: Synthesis and characterization of compound D

[0190]

[0191] The specific synthesis steps are as follows:

[0192] Weigh the intermediate (209.7 mg, 200 μmol) and add it to a 25 mL dry two-necked flask, then add anhydrous sodium acetate (32.81 mg, 400 μmol). After purging the device with argon, draw 5 mL of anhydrous DMF with a syringe and add it to the flask to dissolve the solid. Place the flask in an 80 ° C oil bath, heat to reflux, stir under argon protection for 16 hours, and the solution color is purple-red. Monitor the reaction progress with TLC. After the raw material point disappears, cool to room temperature, and remove the solvent by rotary evaporation to obtain a dark purple crude product. Further purify using silica gel column chromatography (DCM: MeOH = 6: 1), collect the product and spin dry the solvent to obtain compound D134.4 mg (yield: 65.34%). 1 H NMR(400MHz,DMF)δ9.39(d,J=8.5Hz,2H),8.95(d,J=7.2Hz,2H),8.88(s,2H),8 .44(d,J=12.6Hz,2H),8.21(t,J=7.9Hz,2H),6.55(d,J=13.2Hz,2H),4.94(s,4 H),4.51–4.46(m,5H),3.18(t,J=6.5Hz,8H),2.61(s,4H),2.18(s,2H),2.12(s ,12H),2.04(dd,J=14.7,7.5Hz,5H),1.83–1.74(m,6H),1.36(t,J=7.3Hz,8H).

[0193] Example 5: Synthesis and characterization of compound E. The structural formula of the compound is as follows:

[0194]

[0195] The specific synthesis steps are as follows:

[0196] Weigh compound D (205.7 mg, 200 μmol) and add it to a 25 mL dry two-necked flask, then purge the device with argon. Use a syringe to draw 5 mL of DCM and triethylamine (20 mg, 200 μmol) and add them to the flask to dissolve the solid, and the solution is purple-red. After argon protection, place the flask in an ice-water bath. Weigh o-iodobenzoyl chloride (66.5 mg, 250 μmol) and dissolve it in 2 mL of anhydrous DCM. After reacting in an ice-water bath for 30 minutes, remove the ice-water bath and react at room temperature for 12 hours. After drying the solvent, further purify it by silica gel column chromatography (DCM: MeOH = 5: 1), collect the product and dry the solvent, and obtain 114.9 mg of reddish-brown solid compound E with a metallic luster (yield: 45.63%). 1H NMR (600MHz, DMSO) δ9.07(d,J=9.0Hz,2H),8.60(t,J=3.6Hz,4H),8.37(d,J=7.9Hz,1H) ,7.96–7.92(m,2H),7.84(d,J=6.3Hz,2H),7.62(t,J=7.7Hz,1H),7.19(d,J=13.6Hz,2H) ,6.87(d,J=14.0Hz,2H),4.90(s,4H),4.07–4.04(m,4H),2.92(s,4H),2.80(t,J=6.3Hz ,4H),1.61(d,J=7.2Hz,4H),1.53(s,12H),1.45(d,J=6.8Hz,4H),0.85(d,J=6.3Hz,6H).

[0197] Example 6: Testing of absorption and emission wavelengths of near-infrared fluorescent probes based on naphthalene imide

[0198] Use different solvents to prepare dye test solutions with a concentration of 1mM, and use UV spectrophotometer and fluorescence photometer to test. The test results are as follows:

[0199] (1) The photophysical properties of compound A in methanol, ethanol, acetic acid, acetic anhydride, chloroform, and acetone are shown in Table 1. The maximum absorption wavelength (λ abs ) range is 720-745nm, the maximum emission wavelength (λ ex ) range is 846-870nm, reaching the near-infrared band, the Stokes shift range is 97-126nm, and the molar extinction coefficient (ε) range is 38002-141545M -1 cm -1 , the fluorescence quantum yield (φ) ranges from 0.1 to 2.81%. Figure 1 The absorption and emission spectra of compound A in ethanol are shown, with the black color representing the absorption curve and the red color representing the emission curve.

[0200] (2) The photophysical properties of compound B in methanol, ethanol, acetic acid, acetic anhydride, chloroform and acetone are shown in Table 2. The maximum absorption wavelength range is 725-818 nm, the maximum emission wavelength range is 801-892 nm, reaching the near-infrared band range, the Stokes shift range is 50-126 nm, and the molar extinction coefficient range is 35631-62457 M -1 cm -1 , the fluorescence quantum yield ranged from 0.31-27.53%. Figure 2 The absorption and emission spectra of compound B in ethanol are shown, with the black color representing the absorption curve and the red color representing the emission curve.

[0201] (3) The photophysical properties of compound C in phosphate buffer, methanol, ethanol, acetic acid, chloroform and acetic anhydride are shown in Table 3. The maximum absorption wavelength range is 768-800 nm, the maximum emission wavelength range is 860-938 nm, reaching the near-infrared band, the Stokes shift range is 91-138 nm, and the molar extinction coefficient range is 30919-82544 M -1 cm -1 , the fluorescence quantum yield ranged from 0.27-46.37%. Figure 3 The absorption and emission spectra of compound C in ethanol are shown, with the black color representing the absorption curve and the red color representing the emission curve.

[0202] (4) The photophysical properties of compound D in phosphate buffer, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, ethanol, and chloroform are shown in Table 4. The maximum absorption wavelength range is 564-580 nm, the maximum emission wavelength range is 652-669 nm, the Stokes shift range is 79-105 nm, and the molar extinction coefficient range is 41944-51414 M -1 cm -1 , the fluorescence quantum yield ranged from 1.05-5.94%. Figure 4 The absorption and emission spectra of compound D in ethanol are shown, with the black color representing the absorption curve and the red color representing the emission curve.

[0203] (5) The photophysical properties of compound E in acetonitrile, N,N-dimethylformamide, methanol, ethanol, acetic acid, and acetic anhydride are shown in Table 5. The maximum absorption wavelength range is 858-870 nm, the maximum emission wavelength range is 878-895 nm, reaching the near-infrared band, the Stokes shift range is 20-33 nm, and the molar extinction coefficient range is 21304-395607 M -1 cm -1 , the fluorescence quantum yield ranged from 8.55-36.14%. Figure 5 The absorption and emission spectra of compound E in ethanol are shown, with the black color representing the absorption curve and the red color representing the emission curve.

[0204] Table 1: Photophysical properties of compound A in different solvents

[0205]

[0206] Table 2: Photophysical properties of compound B in different solvents

[0207]

[0208] Table 3: Photophysical properties of compound C in different solvents

[0209]

[0210] Table 4: Photophysical properties of compound D in different solvents

[0211]

[0212] Table 5: Photophysical properties of compound E in different solvents

[0213]

[0214] Example 7: Performance test of compound A

[0215] Weigh compound A and dissolve it in analytically pure dimethyl sulfoxide (DMSO) to obtain a 1 mM mother solution of compound A. Use MAHMA NONOate as the NO donor and phosphate buffer (PBS) as the solvent to prepare a NO donor mother solution (1 mM), and use phosphate buffer (PBS) and analytically pure ethanol (EtOH) at a volume ratio of 5:1 to prepare a test blank solvent.

[0216] (1) Determination of fluorescence emission spectra of compound A reacting with NO solutions of different concentrations

[0217] Take 20 μL dye mother solution (1 mM) and different volumes of NO donor mother solution (1 mM), add them to a quartz cuvette with four sides transparent to light, dilute with blank solvent to 2 mL, so that the probe concentration is 10 μM, and the NO concentration is 0, 5, 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300 μM. Add a magnetic particle to the cuvette, stir at 37 ° C for 5 minutes, and then test the absorbance and fluorescence intensity. The results are as follows Figure 6 As shown. With the increase of NO concentration, in the range of 10-30μM, NO concentration is proportional to fluorescence intensity. After linear fitting, the detection limit formula LOD = 3σ / k can be used to calculate the detection limit of compound A to be 0.09μM. The results are shown Figure 7 , 8 shown.

[0218] (2) Selectivity experiment of compound A

[0219] Use a pipette to measure 10 μL of Compound A stock solution and add it to a quartz cuvette with four sides transparent to light. Add 1 μL of Na + , Cl - , Fe 3+ 、NO2 - 、SO3 2- , Ca 2+ , Cu 2+ 、CO3 2- 、Zn2+ , GSH, H2S, K + , Mn 2+ 、Cys、AcO - 、Hcy、ClO - Br - , NO stock solution. Dilute to 2 mL with blank solvent, keeping the probe concentration at 5 μM. Stir at room temperature for 1 minute, and plot the fluorescence intensity of the test solution at 910 nm in a bar graph. The experimental results are shown in Fig. 9 shown.

[0220] (3) Reaction kinetics of compound A

[0221] Use a pipette to measure 10μ of compound A mother solution and add it to a quartz cuvette with four sides transparent to light. Dilute it to 2mL with blank solvent to make the probe concentration 5μM. Use a microinjector to draw 50μL of NO donor mother solution (1mM) and add it to the cuvette under light-proof conditions. The excitation wavelength is 880nm, and its fluorescence intensity is scanned at 910nm, with a time interval of 1s and a scan time of 40s. The experimental results are shown in Fig.10 shown.

[0222] (4) Cytotoxicity test of compound A

[0223] Prepare 2 mL of HeLa cells with appropriate density and good growth status into a cell suspension. Count the cells under a microscope. After calculating the cell concentration, dilute it with complete culture medium to a suitable cell suspension and add it to a 96-well plate, so that each well has 100 μL of suspension and 10 4 cells. Set up 6 concentration gradients of compound A: 0, 3.125, 6.25, 12.5, 25, and 50 μM, with 5 replicate wells for each gradient, and incubate in an incubator for 24 hours. After 24 hours, prepare a 10 mM dye stock solution. First, take an appropriate amount of the stock solution and dilute it with complete culture medium to make 2 mL of a 50 μM solution. Then perform gradient dilutions to prepare 25, 12.5, 6.25, 3.125, and 0 μM solutions. Take the 96-well plate out of the incubator, remove the old culture medium with a pipette, add the culture medium containing the dye to each well, and incubate in the incubator for 24 hours. After the incubation, replace the medium with a culture medium containing 10% CCK-8, and set up a separate cell-free group containing CCK-8 culture medium. Place the well plate in the incubator and incubate for 30 minutes, then take it out, detect the OD value at 450 nm with an enzyme reader, and calculate the cell survival rate. The experimental results are as follows Fig.11 As shown, compound A has no obvious cytotoxicity within 50 μM and has certain biocompatibility.

[0224] (5) In vivo imaging experiment of compound A

[0225] Compound A of the present invention has the ability to detect in vivo imaging of acute dermatitis mouse models. After lipopolysaccharide (LPS) modeling, compound A was subcutaneously injected at 6 hours, 12 hours, and 24 hours, and in vivo imaging images of normal group and lipopolysaccharide model mice were taken once every 5 minutes in the in vivo imaging device. The results are shown in FIG. Fig.12 As shown, after the probe was injected into the mice in the acute dermatitis group, the fluorescence intensity gradually became stronger as time went on, while the fluorescence intensity of the mice in the normal group did not change significantly, indicating that the probe compound A has the ability to distinguish between dermatitis mice and normal mice.

Claims

1. Compounds of formula I: In Formula I, R1 and R9 are each independently selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C16 alkyl; the -[(CH2) p -O-] q H and C1-C16 alkyl are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C16 alkoxy and biological targeting groups; p is an integer selected from 1 to 6; q is an integer selected from 1 to 4; R2 and R8 are each independently a C1-C16 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups; R3, R4, R6 and R7 are each independently selected from: hydrogen and C1-C16 alkyl; R5 is selected from: hydroxyl optionally substituted by substituents selected from C1-C16 acyl and C1-C16 alkyl, amino optionally substituted by substituents selected from C1-C16 acyl, C1-C16 alkyl, C6-C14 aryl and 5-14 membered heteroaryl, nitro, -N=N-C6-C14 aryl, and -N=N-5-14 membered heteroaryl; the C1-C16 acyl, the C1-C16 alkyl, the C6-C14 aryl and the 5-14 membered heteroaryl are optionally substituted by 1, 2, 3 or 4 substituents selected from the following groups: halogen, amino, C 1-C10 alkoxy, -(C1-C10 alkyl)NH, -(C1-C10 alkyl)2N, -(halo C1-C10 alkyl)NH, -(halo C1-C10 alkyl)(C1-C10 alkyl)N, -(halo C1-C10 alkyl)2N, and C6-C14 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen, C1-C10 alkyl, C1-C10 alkoxy, hydroxyl, amino and pinacol boryl; the 5-14 membered heteroaryl contains 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur; m and n are each independently selected from integers from 0 to 6; The biological targeting group is selected from: triphenylphosphine, morpholinyl and piperazine.

2. The compound of formula (I) according to claim 1, characterized in that R1 and R9 are each independently selected from: optionally substituted -[(CH2) p -O-] q H and optionally substituted C1-C10 alkyl; the -[(CH2) p -O-] q H and C1-C10 alkyl are optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C6 alkoxy and biological targeting groups; q is 1, 2 or 3; p is 1, 2 or 3; Preferably, R1 and R9 are each independently an optionally substituted C1-C6 alkyl group; the C1-C6 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C4 alkoxy and biological targeting group; Preferably, R1 and R9 are each independently C1-C4 alkyl.

3. The compound of formula I according to claim 1, characterized in that R2 and R8 are each independently a C1-C10 alkyl group optionally substituted with 1 or 2 sulfonic acid groups; Preferably, R2 and R8 are each independently a C1-C6 alkyl group substituted with 1 or 2 sulfonic acid groups; Preferably, R2 and R8 are each independently a C1-C3 alkyl group substituted with one sulfonic acid group.

4. The compound of formula I as claimed in claim 1, characterized in that R3, R4, R6 and R7 are each independently selected from C1-C6 alkyl; Preferably, R3, R4, R6 and R7 are each independently selected from C1-C4 alkyl; Preferably, R3, R4, R6 and R7 are all methyl.

5. The compound of formula I according to claim 1, characterized in that R5 is selected from: hydroxyl optionally substituted by a substituent selected from C1-C10 acyl and C1-C10 alkyl, amino optionally substituted by a substituent selected from C1-C10 acyl, C1-C10 alkyl, C6-C14 aryl and 5-14 membered heteroaryl, nitro, -N=N-C6-C14 aryl, and -N=N-5-14 membered heteroaryl; any of the C1-C10 acyl, the C1-C10 alkyl, the C6-C14 aryl and the 5-14 membered heteroaryl C6-C14 aryl optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of halogen, amino, C1-C10 alkoxy, -(C1-C10 alkyl)NH, -(C1-C10 alkyl)2N, -(halogenated C1-C10 alkyl)NH, -(halogenated C1-C10 alkyl)(C1-C10 alkyl)N, -(halogenated C1-C10 alkyl)2N, and C6-C14 aryl optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of nitro, halogen and pinacol boryl; Preferably, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1-C6 acyl and C1-C4 alkyl, amino optionally substituted with substituents selected from C1-C6 acyl, C1-C4 alkyl and C6-C10 aryl, nitro and -N=N-C6-C10 aryl; the C1-C6 acyl, the C1-C4 alkyl and the C6-C10 aryl are optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of halogen, amino, C1-C6 alkoxy, -(C1-C6 alkyl)2N, -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen and pinacol borane; Preferably, R5 is selected from: hydroxyl optionally substituted with a substituent selected from C1-C6 acyl and C1-C4 alkyl, amino optionally substituted with C6-C10 aryl, nitro and -N=N-C6-C10 aryl; the C1-C6 acyl, the C1-C4 alkyl and the C6-C10 aryl are optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, C1-C6 alkoxy, -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted with 1 or 2 substituents selected from nitro, halogen and pinacol boryl; Preferably, R5 is selected from: hydroxyl optionally substituted with substituents selected from C1~C6 acyl and C1~C4 alkyl, amino optionally substituted with C6~C10 aryl, nitro and -N=N-C6~C10 aryl; the C6~C10 aryl is optionally substituted with 1 or 2 substituents selected from amino, C1~C6 alkoxy, -(C1~C6 alkyl)2N and -(halogenated C1~C6 alkyl)2N; the C1~C6 acyl is optionally substituted with 1 or 2 substituents selected from Cl, Br and C6~C10 aryl optionally substituted with halogen; the C1~C4 alkyl is optionally substituted with 1 or 2 substituents: C6~C10 aryl optionally substituted with substituents selected from nitro and pinacol boryl.

6. The compound of formula I according to claim 1, characterized in that R5 is selected from: Wherein, * indicates the position where R5 is connected to the other parts of the compound of formula I.

7. The compound of formula I according to claim 1, characterized in that In Formula I, R1 is an optionally substituted C1-C16 alkyl group; the C1-C16 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of a hydroxyl group, an amino group, a thiol group, a carboxyl group, a sulfonic acid group, a C1-C10 alkoxy group and a biological targeting group; R2 is a C1-C10 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups; R3 and R4 are each independently selected from C1-C6 alkyl; R5 is selected from: hydroxyl optionally substituted with substituents selected from C1-C6 acyl and C1-C4 alkyl, amino optionally substituted with substituents selected from C1-C6 acyl, C1-C4 alkyl and C6-C10 aryl, nitro and -N=N-C6-C10 aryl; the C1-C6 acyl, the C1-C4 alkyl and the C6-C10 aryl are optionally substituted with 1, 2, 3 or 4 substituents selected from the group consisting of halogen, amino, C1-C6 alkoxy, -(C1-C6 alkyl)NH, -(C1-C6 alkyl)2N, -(halogenated C1-C6 alkyl)NH, -(halogenated C1-C6 alkyl)(C1-C6 alkyl)N, -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted with 1, 2, 3 or 4 substituents selected from nitro, halogen and pinacol boryl; R6 and R7 are each independently selected from C1-C6 alkyl; R8 is a C1-C10 alkyl group optionally substituted with 1, 2, 3 or 4 substituents selected from carboxyl groups, sulfonic acid groups and biological targeting groups; R9 is an optionally substituted C1-C16 alkyl group; the C1-C16 alkyl group is optionally substituted by 1, 2, 3 or 4 substituents selected from the group consisting of hydroxyl, amino, thiol, carboxyl, sulfonic acid, C1-C10 alkoxy and biological targeting groups; m and n are each independently an integer from 0 to 6; Preferably, in Formula I, R1 is a C1-C4 alkyl group; R2 is a C1-C3 alkyl group optionally substituted with one sulfonic acid group; R3 and R4 are each independently selected from C1-C4 alkyl; R5 is selected from: hydroxyl optionally substituted by a substituent selected from C1-C6 acyl and C1-C4 alkyl, amino optionally substituted by C6-C10 aryl, nitro and -N=N-C6-C10 aryl; the C1-C6 acyl, the C1-C4 alkyl and the C6-C10 aryl are optionally substituted by 1 or 2 substituents selected from the group consisting of halogen, C1-C6 alkoxy, -(halogenated C1-C6 alkyl)2N, and C6-C10 aryl optionally substituted by 1 or 2 substituents selected from nitro, halogen and pinacol boryl; R6 and R7 are each independently selected from C1-C4 alkyl; R8 is a C1-C3 alkyl group optionally substituted with one sulfonic acid group; R9 is a C1-C4 alkyl group; m is 1; n is 0.

8. The compound of formula I according to claim 1, characterized in that The compound of formula I is selected from:

9. A fluorescent dye, a dye composition, a fluorescent probe or a kit, characterized in that: The fluorescent dye, dye composition or fluorescent probe contains the compound of formula I according to any one of claims 1 to 8 and an optional solvent; the kit contains the compound of formula I according to any one of claims 1 to 8 and an optional solvent, or contains the fluorescent dye, dye composition or fluorescent probe; Preferably, the solvent is a buffer solution optionally containing an organic solvent; preferably, the buffer solution is a phosphate buffer solution; Preferably, the solvent is a phosphate buffer containing ethanol; Preferably, the fluorescent dye, dye composition, fluorescent probe or kit contains the compound A; Preferably, the fluorescent dye, dye composition, fluorescent probe or kit contains the compound B; Preferably, the fluorescent dye, dye composition, fluorescent probe or kit contains the compound C; Preferably, the fluorescent dye, dye composition, fluorescent probe or kit contains the compound D; Preferably, the fluorescent dye, dye composition, fluorescent probe or kit contains the compound E.

10. A use selected from the group consisting of: (1) Use of the compound of formula I according to any one of claims 1 to 8 in fluorescence imaging, or in the preparation of a fluorescent probe; (2) Use of the compound A according to claim 8 in the preparation of a fluorescent probe for detecting nitric oxide; (3) Use of the compound B according to claim 8 in the preparation of a fluorescent probe for detecting nitric oxide; (4) Use of the compound C according to claim 8 in the preparation of a fluorescent probe for detecting nitric oxide; (5) Use of the compound D and / or compound E according to claim 8 in the preparation of a fluorescent probe for detecting hydrogen sulfide; Preferably, the detection is a specific detection; Preferably, the use is the use of compound A in the preparation of a fluorescent probe for quantitative detection of nitric oxide, and the concentration of nitric oxide is 10 to 30 μM.

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