D-A-D type fluorescent probe material and synthesis and application thereof

Abstract

The invention provides a novel D-A-D type fluorescent probe material as well as a preparation method and application thereof. The fluorescent probe material has good stability, the preparation process is simple, and the synthesis condition is mild. Moreover, the D-A-D type fluorescent probe material has good solubility and stability, and a complex of the D-A-D type fluorescent probe material and zinc ions can identify pyrophosphate ions with high selectivity and high sensitivity. The interference of phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, fluorine ions, chloride ions, nitrate ions, acetate ions, sulfate ions and carbonate ions is avoided; after the zinc ion complex of the D-A-D type fluorescent probe material is combined with PPi, the fluorescence intensity is remarkably enhanced, the D-A-D type fluorescent probe material has a higher signal-to-noise ratio, in-situ imaging detection of a target object in living cells or tissues is realized, the defects of a traditional molecule and ion detection method are overcome, and the D-A-D type fluorescent probe material is suitable for being widely applied to the field of biological imaging. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a D-A-D type fluorescent probe material and its synthesis and application. Background Art

[0002] Fluorescent molecular probes are a new method for detecting molecules and ions that has emerged in recent years. They are the most attention-grabbing research hotspots in supramolecular science. Fluorescent molecular probes have characteristics such as high selectivity, high sensitivity, fast and convenient operation, enabling real-time and on-site detection. Moreover, the detection process is visualized through changes in output signals such as fluorescence, ultraviolet light, and color. More importantly, the combination of fluorescent molecular probes with imaging technology enables in-situ imaging detection of target substances in living cells or tissues, overcoming the deficiencies of traditional molecular and ion detection methods, representing a leap in detection technology, and being widely used in fields such as molecular and ion recognition, detection of bioactive substances and bioimaging, near-infrared fluorescence, and time-resolved assays.

[0003] Anion recognition is one of the important contents of current supramolecular chemistry research. With the continuous in-depth study of anion recognition, more and more anion recognition fluorescent probe materials have been designed and synthesized. The system using metal complexes as the main body to recognize anions has attracted great interest from researchers. Phosphate ions, hydrogen phosphate ions, and dihydrogen phosphate ions are important components that make up genes and genetic materials in the living system and play important roles in information transfer and energy storage; Pyrophosphate (PPI) plays a very crucial role in many biological energy transduction, DNA replication, and metabolic processes of cancer cell dysregulation in the life science process. As a product of the hydrolysis of adenosine triphosphate in the cell body, pyrophosphate is an important target molecule in the living body.

[0004] However, there are very few fluorescent probe materials that can specifically recognize and detect pyrophosphate ions without being interfered by phosphate ions, dihydrogen phosphate ions, and hydrogen phosphate ions. Summary of the Invention

[0005] In order to improve the above technical problems, the present invention provides a D-A-D type fluorescent probe material shown in formula (I) and its synthesis and application. The complex of the material with zinc ions can detect and recognize pyrophosphate (PPi) with high selectivity and high sensitivity.

[0006] The present invention provides a compound shown in formula (I):

[0007]

[0008] Wherein, R0 is selected from a C6-20 aryl group or a 5-20 membered heteroaryl group substituted by one, two, or more R1 groups;

[0009] R1 is selected from -NR2R3, C1-10 alkoxy, 3-20 membered heterocyclic group;

[0010] R2 and R3 are the same or different and are independently selected from C1-10 alkyl, C1-10 alkoxy.

[0011] According to an embodiment of the present invention, R0 is selected from C6-14 aryl, 5-14 membered heteroaryl substituted by one, two or more R1; preferably, R0 is selected from C6-14 aryl, 5-10 membered heteroaryl substituted by one, two or more R1; more preferably, R0 is selected from phenyl, anthryl, naphthyl, thienyl, furyl substituted by one, two or more R1.

[0012] According to an embodiment of the present invention, R1 is selected from -NR2R3, C1-6 alkoxy, 3-10 membered heterocyclic group; preferably, R1 is selected from -NR2R3, C1-3 alkoxy, 3-6 membered heterocyclic group.

[0013] According to an embodiment of the present invention, R2 and R3 are the same or different and are independently selected from C1-6 alkyl, C1-6 alkoxy; preferably, R2 and R3 are the same or different and are independently selected from C1-3 alkyl, C1-3 alkoxy.

[0014] According to an embodiment of the present invention, R0 is selected from the following groups:

[0015]

[0016] Indicates the connection site of the group.

[0017] According to an embodiment of the present invention, R1 is selected from -N-(CH3)2, -O-CH3, Indicates the connection site of the group.

[0018] According to a preferred embodiment of the present invention, the R0 group may be selected from the following structures:

[0019]

[0020] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following specific compounds:

[0021]

[0022] The present invention also provides a compound represented by formula (II),

[0023]

[0024] wherein X is selected from halogen;

[0025] R a Selected from one, two or more R b Substituted C6-20 aryl, 5-20 membered heteroaryl;

[0026] R b Selected from -NR c R d , C1-10 alkoxy, 3-20 membered heterocyclic group;

[0027] R c , R d The same or different, are independently selected from C1-10 alkyl and C1-10 alkoxy.

[0028] According to an embodiment of the present invention, the halogen is selected from fluorine, chlorine, bromine, iodine; preferably chlorine.

[0029] According to an embodiment of the present invention, R a Selected from one, two or more R b Substituted C6-14 aryl, 5-14 membered heteroaryl; preferably, R a Selected from one, two or more R b substituted C6-14 aryl, 5-10 membered heteroaryl; further preferably, R a Selected from one, two or more R b Substituted phenyl, anthracenyl, naphthyl, thienyl, furanyl.

[0030] According to an embodiment of the present invention, R b Selected from -NR c R d , C1-6 alkoxy, 3-10 membered heterocyclic group; preferably, R b Selected from -NR2R3, C1-3 alkoxy, 3-6 membered heterocyclic group.

[0031] According to an embodiment of the present invention, R c , R d are the same or different and are independently selected from C1-6 alkyl, C1-6 alkoxy; preferably, R c , R d The same or different, are independently selected from C1-3 alkyl, C1-3 alkoxy.

[0032] According to an embodiment of the present invention, R a Selected from the following groups:

[0033]

[0034] Indicates the attachment site of a group.

[0035] According to an embodiment of the present invention, R b is selected from -N-(CH3)2, -O-CH3, representing the connection site of the group.

[0036] According to a preferred embodiment of the present invention, the R a group may be selected from the following structures:

[0037]

[0038] According to an embodiment of the present invention, the compound represented by formula (II) is selected from the following specific compounds:

[0039]

[0040]

[0041] The present invention also provides a preparation method of the compound represented by formula (I) above. The preparation method includes the following steps: reacting the compound represented by formula (III) with the compound represented by formula (IV) to obtain the compound represented by formula (I); the reaction formula is as follows:

[0042]

[0043] R0 has the foregoing definition;

[0044] X1 is selected from halogen.

[0045] According to an embodiment of the present invention, X1 is selected from iodine.

[0046] According to an embodiment of the present invention, the reaction is carried out under the conditions of ammonium acetate and glacial acetic acid.

[0047] The present invention also provides a preparation method of the compound represented by formula (II) above. The preparation method includes the following steps: reacting the compound represented by formula (I) with zinc halide to obtain the compound represented by formula (II).

[0048] According to an embodiment of the present invention, the zinc halide is selected from zinc chloride.

[0049] The present invention also provides the application of the compound represented by formula (I) above and the compound represented by formula (II) above in the preparation of a fluorescent probe for detecting, identifying, and tracing pyrophosphate ions.

[0050] The present invention also provides the application of the compound represented by formula (I) above and the compound represented by formula (II) above in detecting, identifying, and tracing pyrophosphate ions.

[0051] The present invention also provides the use of the compound represented by the above formula (I) and the compound represented by formula (II) in the preparation of products or reagents for detecting, identifying, and tracing pyrophosphate ions.

[0052] Beneficial effects

[0053] The present invention provides a novel D-A-D type fluorescent probe material represented by formula (I). The fluorescent probe material has good stability, a simple preparation process, and mild synthesis conditions. Moreover, the complex of the fluorescent probe material with zinc ions can detect and identify pyrophosphate ions (PPi) with high selectivity and high sensitivity; after the zinc ion complex of the fluorescent probe material binds to PPi, the fluorescence intensity is significantly enhanced, with a higher signal-to-noise ratio, realizing in-situ imaging detection of the target in living cells or tissues, overcoming the deficiencies of traditional molecular and ion detection methods, and being suitable for wide application in the field of bioimaging.

[0054] Term definitions and explanations

[0055] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. The group definitions and compound structures after such combination and combination should be understood to be within the scope recorded in the specification and / or claims of this application.

[0056] Unless otherwise specified, the numerical ranges recorded in this specification and claims are equivalent to recording at least each specific integer value therein. For example, the numerical range "1-40" is equivalent to recording each integer value in the numerical range "1-10", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and each integer value in the numerical range "11-40", namely 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. It should be understood that when using one, two or more in the description of substituents, "more" should refer to an integer of ≥3, such as 3, 4, 5, 6, 7, 8, 9 or 10.

[0057] The term "halogen" or "hal" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0058] In general, the term "substituted" means that one or more hydrogen atoms in the given structure are replaced by specific substituents. Further, when the group is substituted by more than one of the said substituents, the substituents are independent of each other, that is, the more than one substituents can be different from each other or the same. Unless otherwise indicated, a substituent group can substitute at each substitutable position of the group to be substituted. When there is more than one position in the given structural formula that can be substituted by one or more substituents selected from specific groups, the substituents can substitute at each position either identically or differently. The substituents can be, but are not limited to, =O, hydrogen, deuterium, cyano, nitro, halogen, alkyl, haloalkyl, alkoxy, carboxyl, cycloalkyl, cycloalkyloxy, heterocyclic group, heterocyclic alkyl, aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, heteroaryloxy, etc.

[0059] In addition, it should be noted that unless otherwise clearly indicated, the description mode “… independently selected from” adopted in the present invention should be understood in a broad sense, which means that the described individuals are independent of each other and can be independently selected from the same or different specific groups. More specifically, the description mode “… independently selected from” can either mean that among different groups, the specific options expressed between the same symbols do not affect each other; or it can mean that within the same group, the specific options expressed between the same symbols do not affect each other.

[0060] In each part of this specification, the substituents of the compounds disclosed in the present invention are disclosed according to the group type or range. It should be specifically noted that the present invention includes each independent secondary combination of each member of these group types and ranges. For example, the term "C" 1-6 alkyl" specifically refers to C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl independently disclosed.

[0061] In each part of the present invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for this group should be understood as linking groups. For example, if the structure requires a linking group and the Markush group definition for this variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents a linked alkylene group or arylene group respectively.

[0062] The term "C" 1-10 alkyl" means a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1 to 10 carbon atoms. For example, "C" 1-6"Alkyl" means straight-chain and branched-chain alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms. Among them, the alkyl groups may optionally be substituted with one or more substituents described in the present invention. In some embodiments, the alkyl group contains 1-12 carbon atoms; in other embodiments, the alkyl group contains 1-6 carbon atoms; in still other embodiments, the alkyl group contains 1-4 carbon atoms. Examples of the alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc., or their isomers.

[0063] The term "3-20 membered heterocyclic group" should be understood to mean a saturated monovalent monocyclic, bicyclic hydrocarbon ring or bridged cycloalkane ring having a total ring atom number of 3-20 (such as 3, 4, 5, 6, 7, 8, 9, 10, etc.) containing 1-5 heteroatoms independently selected from N, O, and S, preferably a "3-10 membered heterocyclic group". The term "3-10 membered heterocyclic group" means a saturated monovalent monocyclic, bicyclic hydrocarbon ring or bridged cycloalkane ring containing 1-5, preferably 1-3 heteroatoms independently selected from N, O, and S, such as 1, 2, or 3 heteroatoms independently selected from N, O, and S. The heterocyclic group can be linked to the rest of the molecule through any one of the carbon atoms or a nitrogen atom (if present). In particular, the heterocyclic group can include, but is not limited to: a 4-membered ring, such as azetidinyl, oxetanyl; a 5-membered ring, such as tetrahydrofuranyl, dioxolanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrroline; or a 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl; or a 7-membered ring, such as diazepanyl. Optionally, the heterocyclic group can be benzo-fused. The heterocyclic group can be bicyclic, for example, but not limited to, a 5,5 bicyclic ring, such as hexahydrocyclopenta[c]pyrrol-2(1H)-yl ring, or a 5,6 bicyclic ring, such as hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring. The ring containing a nitrogen atom can be partially unsaturated, i.e., it can contain one or more double bonds, such as, but not limited to, 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or it can be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. According to the present invention, the heterocyclic group is non-aromatic. When the 3-20 membered heterocyclic group is linked to other groups to form a compound of the present invention, it can be linked to other groups through a carbon atom on the 3-20 membered heterocyclic group or through a heteroatom on the 3-20 membered heterocyclic ring. For example, when the 3-20 membered heterocyclic group is selected from piperazinyl, it can be linked to other groups through a nitrogen atom on piperazinyl. Or when the 3-20 membered heterocyclic group is selected from piperidinyl, it can be linked to other groups through a nitrogen atom and a carbon atom at the para position on the piperidine ring.

[0064] The term "C 6-20 aryl" should be understood to mean a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring having 6-20 carbon atoms, preferably "C 6-14 aryl". The term "C 6-14 aryl" should be understood to preferably mean a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms ("C 6-14"Aryl"), particularly a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or biphenyl, or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C 13 aryl"), such as fluorenyl, or a ring having 14 carbon atoms ("C 14 aryl"), such as anthryl. When the C 6-20 aryl is substituted, it may be mono-substituted or multi-substituted. And there is no restriction on the substitution site, for example, it may be ortho-substituted, para-substituted or meta-substituted.

[0065] The term "5-20 membered heteroaryl" should be understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems, including aromatic or partially aromatic ones, which have 5 to 20 ring atoms and contain 1-5 heteroatoms independently selected from N, O and S, such as "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" should be understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, particularly 5 or 6 or 9 or 10 carbon atoms, and containing 1-5, preferably 1-3 heteroatoms independently selected from N, O and S and, additionally, may be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thiazolopyrazolyl, etc. and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc. and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc. When the 5-20 membered heteroaryl is connected to other groups to form the compounds of the present invention, it may be that the carbon atoms on the 5-20 membered heteroaryl ring are connected to other groups, or the heteroatoms on the 5-20 membered heteroaryl ring are connected to other groups. When the 5-20 membered heteroaryl is substituted, it may be mono-substituted or multi-substituted. And there is no restriction on the substitution site, for example, the hydrogen connected to the carbon atom on the heteroaryl ring may be substituted, or the hydrogen connected to the heteroatom on the heteroaryl ring may be substituted.

[0066] Unless otherwise specified, a heterocyclic group, heteroaryl group or heteroarylene group includes all possible isomeric forms thereof, such as positional isomers. Thus, for some illustrative non-limiting examples, forms substituted or bonded to other groups at one, two or more positions among their 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12- positions etc. (if present) may be included, including pyridin-2-yl, pyridin-2-ylidene, pyridin-3-yl, pyridin-3-ylidene, pyridin-4-yl and pyridin-4-ylidene; thienyl or thienylene includes thien-2-yl, thien-2-ylidene, thien-3-yl and thien-3-ylidene; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, pyrazol-5-yl.

[0067] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0068] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention. Description of the Drawings

[0069] Figure 1 To observe the fluorescence changes of each solution under ultraviolet light at 365 nm, from left to right are No. 1, No. 2, No. 3, No. 4, No. 5, and No. 6.

[0070] Figure 2 For F - , Cl - , NO 3- , CO3 2- , SO4 2- , AcO - Effect of ion pairs ZnCl2-L-1 on the fluorescence recognition of PPi.

[0071] Figure 3 It is the fluorescence intensity diagram of 10 mM ZnCl2-L-1 solution at different PPi concentrations (excitation wavelength is 435 nm). Detailed Embodiments

[0072] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0073] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0074] Example 1 Synthesis of Compound 4,4″-bis(4-dimethylaminophenyl)-2,2′:6′,2″-terpyridine (L-1)

[0075] 1.1 Synthesis of 2,6-bis(pyridineacetyl)pyridine diiodide

[0076]

[0077] 16.32 g (0.1 mol) of 2,6-diacetyl-pyridine, 50.76 g (0.2 mol) of iodine and 250 mL of anhydrous pyridine were added to a 500 mL round-bottom flask, heated to 110 °C and stirred for 6 hours. After natural cooling to room temperature, filtration was carried out. The filter cake was washed with 200 mL of ethanol, and after filtration, the filter cake was recrystallized with 300 mL of ethanol to obtain a yellow-green solid. It was dried in vacuo at 65 °C for 10 hours to obtain 52.16 g of product, with a yield of 91% and a melting point of 210 - 212 °C; IR (KBr pellet, cm -1 ): 3456.44, 3414, 3041.74, 1734.01, 1716.65, 1631.78, 1579.7, 1483.26, 1408.04, 1346.31, 1305.81, 1217.08, 1209.37, 1033.85, 1020.34, 993.34, 960.55, 869.9, 817.82, 777.31; 1 1H NMR (400 MHz, DMSO-D6) δ (ppm): 9.13 - 9.15 (d, 4H), 8.78 - 8.82 (m, 2H), 8.42 - 8.46 (m, 3H), 8.34 - 8.37 (m, 4H), 6.74 (s, 4H).

[0078] 1.2 Synthesis of 4,4″-bis(4-dimethylaminophenyl)-2,2′:6′,2″-terpyridine (L-1)

[0079]

[0080] 5.73 g (10 mmol) of 2,6-bis(pyridineacetyl)pyridine diiodide, 3.51 g (20 mmol) of 4-dimethylaminocinnamaldehyde, 16 g (20 mmol) of ammonium acetate, and 60 mL of glacial acetic acid were added to a 100 mL round-bottom flask. After heating under reflux for 10 h, it was naturally cooled to room temperature, the pH was adjusted to weakly alkaline with ammonia water, and then filtered by suction. The filter cake was washed with 20 mL of distilled water to obtain a dark red solid, which was dried in vacuo at 65 °C for 10 h. Column chromatography was carried out using chloroform as the mobile phase and alumina as the stationary phase to obtain 3.86 g of a yellow solid with a yield of 81.85%. Melting point 298 - 300 °C; IR (KBr pellet, cm -1 ): 3421.72, 2924.09, 1600.92, 1577.77, 1560.41, 1527.62, 1458.18, 1444.68, 1363.67, 1228.66, 1203.58, 1168.86, 1126.43, 1064.71, 1043.49, 947.05, 810.1; 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.90 (d, 2H), 8.66 - 8.68 (d, 2H), 8.45 - 8.47 (d, 2H), 7.98 (t, 1H), 7.76 - 7.78 (d, 4H), 7.53 - 7.55 (m, 2H), 6.84 - 6.86 (d, 4H), 3.05 (s, 12H). 13 13C NMR (100 MHz, CDCl3) δ (ppm): 156.59, 155.58, 151.06, 149.43, 148.78, 137.82, 127.78, 125.54, 120.99, 120.27, 117.89, 112.46, 40.30.

[0081] 1.3 Synthesis of ZnCl2-L-1

[0082]

[0083] 0.545 g (4 mmol) of anhydrous zinc chloride and 130 mL of methanol were added to a 500 mL round-bottom flask and stirred until dissolved. 1.6 g (3.4 mmol) of L-1 was dissolved in 80 mL of dichloromethane and slowly added dropwise to the round-bottom flask. After heating at 50 °C for 3 h, it was naturally cooled to room temperature and then filtered by suction. The filter cake was washed with 10 mL of distilled water and 10 mL of methanol, and dried in vacuo at 65 °C for 10 h to obtain 0.2 g of a dark yellow solid with a yield of 96.77%. Melting point > 300 °C; IR (KBr pellet, cm -1): 2922.16, 2852.72, 1595.13, 1575.84, 1533.41, 1458.18, 1367.53, 1325.1, 1209.37, 1170.79, 1055.06, 1010.7, 947.05, 812.03, 704.02, 661.58, 565.14; 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.99 (d, 2H), 8.83 (d, 2H), 8.67 (d, 2H), 7.98 - 8.05 (d, 6H), 6.84 (m, 5H), 3.00 (s, 12H).

[0084] Synthesis of Compound 4,4″-bis(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (L-2) in Example 2

[0085]

[0086] 5.73 g (10 mmol) of 2,6-bis(pyridylacetyl)pyridine diiodide (prepared according to Part 1.1 of Example 1), 3.24 g (20 mmol) of 4-methoxycinnamaldehyde, 16 g (20 mmol) of ammonium acetate, and 60 mL of glacial acetic acid were added to a 100 mL round-bottom flask. After heating under reflux for 12 h, it was naturally cooled to room temperature, and then the pH was adjusted to weakly alkaline with ammonia water, followed by suction filtration. The filter cake was washed with 20 mL of distilled water to obtain a brownish-black solid, which was dried in vacuo at 65 °C for 10 hours. Column chromatography was performed using chloroform as the mobile phase and alumina as the stationary phase to obtain 3.68 g of a dark brown solid product with a yield of 82.65%. Melting point: 193 - 195 °C; IR (KBr pellet, cm -1 ): 2962.66, 2835.36, 1606.7, 1575.84, 1541.12, 1516.05, 1458.18, 1288.45, 1261.45, 1182.36, 1099.43, 1024.2, 815.89, 802.39, 574.79; 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.84 (d, 2H), 8.68 - 8.71 (d, 2H), 8.43 - 8.45 (d, 2H), 7.88 (t, 1H), 7.73 - 7.74 (d, 4H), 7.06 - 7.11 (m, 6H), 3.82 (s, 6H).

[0087] Synthesis of L-3 to L-6 in Example 3

[0088] Refer to the synthesis methods of Example 1 and Example 2 to synthesize the following specific compounds L-3 to L-6, and their structures are as follows in the table:

[0089] Table 1

[0090]

[0091] Application of the Fluorescent Probe Material in Example 4

[0092] Fluorescent recognition detection of PPi by the fluorescent probe material: Under normal temperature conditions, prepare a DMSO solution of L-1 (10 mmol / L) and a solution of the complex of L-1 and zinc chloride ZnCl2-L-1 (10 mmol / L, with DMSO as the solvent).

[0093] No. 1: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of L-1 (10 mmol / L).

[0094] No. 2: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of ZnCl2-L-1 (10 mmol / L).

[0095] No. 3: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of ZnCl2-L-1 (10 mmol / L), and then add 10 μL of the aqueous solution of sodium pyrophosphate (10 mmol / L).

[0096] No. 4: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of ZnCl2-L-1 (10 mmol / L), and then add 10 μL of the aqueous solution of disodium hydrogen phosphate (10 mmol / L).

[0097] No. 5: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of ZnCl2-L-1 (10 mmol / L), and then add 10 μL of the aqueous solution of ammonium dihydrogen phosphate (10 mmol / L).

[0098] No. 6: 1 mL of HEPES solution (10 mmol / L, pH = 7.4), add 10 μL of the DMSO solution of ZnCl2-L-1 (10 mmol / L), and then add 10 μL of the aqueous solution of sodium phosphate (10 mmol / L).

[0099] Observe the fluorescence changes of each solution under ultraviolet light at 365 nm.

[0100] The result shows that, as Figure 1As shown, only the addition of pyrophosphate ions (No. 3) can cause a significant enhancement in the fluorescence of the probe, while the addition of phosphate ions (No. 6), hydrogen phosphate ions (No. 4), and dihydrogen phosphate ions (No. 5) cannot enhance the fluorescence of the probe material.

[0101] The same experimental method was used to test F - , Cl - , NO3 - , AcO - , SO4 2- , CO3 2- anions on the fluorescence intensity of the fluorescent probe. The fluorescence changes of each solution were observed under ultraviolet light at 365 nm. The results are as Figure 2 shown. The addition of fluoride ions, chloride ions, nitrate ions, acetate ions, sulfate ions, and carbonate ions cannot enhance the fluorescence of the probe material.

[0102] Meanwhile, the titration experiment shows that the lowest detection limit of the fluorescent probe material ZnCl2-L-1 for PPi can reach 1×10 -6 mol / L ( Figure 3 ), and the lowest detection limits of ZnCl2-L-2, ZnCl2-L-3, ZnCl2-L-4, ZnCl2-L-5, and ZnCl2-L-6 for PPi are all lower than 1 μM.

[0103] Above, the embodiments of the present invention have been described. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A compound of formula (I): Among them, R0 is selected from C6-20 aryl or 5-20 membered heteroaryl substituted by one, two or more R1; R1 is selected from -NR2R3, C1-10 alkoxy, 3-20 membered heterocyclic group; R2 and R3 are the same or different and independently selected from C1-10 alkyl, C1-10 alkoxy.

2. The compound according to claim 1, wherein R0 is selected from C6-14 aryl or 5-14 membered heteroaryl substituted by one, two or more R1; preferably, R0 is selected from C6-14 aryl or 5-10 membered heteroaryl substituted by one, two or more R1; more preferably, R0 is selected from phenyl, anthracenyl, naphthyl, thienyl, furyl substituted by one, two or more R1; Preferably, R1 is selected from -NR2R3, C1-6 alkoxy, 3-10 membered heterocyclic group; preferably, R1 is selected from -NR2R3, C1-3 alkoxy, 3-6 membered heterocyclic group; Preferably, R2 and R3 are the same or different and independently selected from C1-6 alkyl, C1-6 alkoxy; preferably, R2 and R3 are the same or different and independently selected from C1-3 alkyl, C1-3 alkoxy; Preferably, R0 is selected from the following groups: Indicates the attachment site of the group; Preferably, R1 is selected from -N-(CH3)2, -O-CH3, indicating the attachment site of the group; Preferably, the R0 group is selected from the following structures:

3. The compound according to claim 1 or 2, characterized in that, The compound of formula (I) is selected from the following specific compounds:

4. A compound of formula (II), Among them, X is selected from halogen; R a selected from C6-20 aryl, 5-20 membered heteroaryl substituted by one, two or more R b groups; R b selected from -NR c R d , C1-10 alkoxy, 3-20 membered heterocyclic group; R c 、R d are the same or different and are each independently selected from C1-10 alkyl and C1-10 alkoxy.

5. The compound according to claim 4, characterized in that, Halogen is selected from fluorine, chlorine, bromine, iodine; preferably chlorine; Preferably, R a is selected from C6-14 aryl, 5-14 membered heteroaryl substituted by one, two or more R b ; preferably, R a is selected from C6-14 aryl, 5-10 membered heteroaryl substituted by one, two or more R b ; more preferably, R a is selected from phenyl, anthryl, naphthyl, thienyl, furyl substituted by one, two or more R b ; Preferably, R b is selected from -NR c R d , C1-6 alkoxy, 3-10 membered heterocyclic group; preferably, R b is selected from -NR2R3, C1-3 alkoxy, 3-6 membered heterocyclic group; Preferably, R c , R d are the same or different and are each independently selected from C1-6 alkyl, C1-6 alkoxy; preferably, R c , R d are the same or different and are each independently selected from C1-3 alkyl, C1-3 alkoxy; Preferably, R a is selected from the following groups: Indicates the attachment site of the group; Preferably, R b is selected from -N-(CH3)2, -O-CH3, indicating the connection site of the group; Preferably, the R a group may be selected from the following structures:

6. The compound according to claim 4 or 5, characterized in that, The compound of formula (II) is selected from the following specific compounds:

7. A method for preparing the compound of formula (I) according to any one of claims 1-3, the preparation method comprising the following steps: Reacting the compound of formula (III) with the compound of formula (IV) to obtain the compound of formula (I); The reaction formula is as follows: R0 has the definition as described in any one of claims 1-3; X1 is selected from halogen.

8. A method for preparing the compound of formula (II) according to any one of claims 4-6, the preparation method comprising the following steps: Reacting the compound of formula (I) according to any one of claims 1-3 with zinc halide to obtain the compound of formula (II).

9. Use of the compound of formula (I) according to any one of claims 1-3 and the compound of formula (II) according to any one of claims 4-6 in the preparation of a fluorescent probe for detecting, identifying and tracing pyrophosphate ions.

10. Use of the compound of formula (I) according to any one of claims 1-3 and the compound of formula (II) according to any one of claims 4-6 in detecting, identifying and tracing pyrophosphate ions.

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