Selective nucleus ATP (adenosine triphosphate) small-molecule probe as well as preparation method and application thereof

Abstract

The invention relates to a selective cell nucleus ATP (adenosine triphosphate) micromolecular probe and a preparation method and application thereof, 2, 7-dihydroxynaphthalene is used as a starting material, and the selective cell nucleus ATP micromolecular probe is obtained through key steps such as amination, protection / deprotection operation, aldehyde group introduction, benzopyrone cyclization, benzyl bromination and functional DPA group introduction and other multi-step functionalization reactions. And finally, high-efficiency synthesis of the nucleus ATP fluorescent probe molecule is realized. The probe molecule can be selectively positioned in a cell nucleus in a cell and can generate an enhanced fluorescence signal through specific binding with ATP, so that high-sensitivity detection of ATP is realized.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis and biological detection, and particularly relates to a selective cell nucleus ATP small molecule probe and a preparation method and application thereof. Background Art

[0002] Adenosine 5′-triphosphate (ATP) is an indispensable biological macromolecule in living organisms. It is the main energy source for cell life activities and plays a key role in cell respiration, enzyme catalysis, energy and signal transmission. Mitochondria are the main place for the production of ATP. Changes in ATP content will inevitably have irreversible effects on mitochondrial function. Scientific research shows that mitochondrial dysfunction is closely related to cardiovascular disease, malignant tumors, Parkinson's disease and other diseases. Therefore, the precise detection of intracellular ATP is of great significance for understanding cell biological events and studying the pathogenesis of diseases related to mitochondrial damage.

[0003] Fluorescent probes have obvious advantages in intracellular and in vivo ATP imaging due to their high sensitivity, good selectivity, real-time imaging, and good biocompatibility. Many fluorescent probes for detecting ATP have been developed. Fluorescent probes based on metal complexes (such as zinc complex fluorescent probes) have been widely studied due to their strong affinity for phosphate anions, good selectivity, high sensitivity, and simple preparation. At present, a variety of zinc complex fluorescent probes with selectivity for different organelles have been developed. Recently, the Ahn group prepared the first nuclear-selective zinc complex fluorescent probe I, which detects changes in ATP in the nucleus through reversible binding and has excellent nuclear selectivity. At physiological pH 7.4, the fluorescence intensity of I increased by 25 times. Using this probe, it can be observed that the nuclear ATP levels in cancer cells and tumor tissues are 2.1-3.3 times and 3.9-7.8 times higher than those in normal cells, respectively. This probe can be used to study the life processes related to the nucleus and distinguish tumor tissues from normal tissues.

[0004] The literature does not provide a specific synthetic route, only similar synthetic methods, but slight differences in structure require major changes in the synthetic route.

[0005] The route reported in the literature is shown in Scheme 1:

[0006]

[0007] Solution 1

[0008] Compound 3 was obtained by reductive amination of aldehyde 2 and dipyridylamine (DPA), and then complexed with a zinc reagent to obtain the target product NucATP. However, the conversion yield of compound 1 to compound 2 was very low (<5%) and the reproducibility was very poor. Summary of the invention

[0009] In view of the technical problems of the route reported in the literature, the purpose of the present invention is to provide a selective nuclear ATP small molecule probe and its preparation method and application, using 2,7-dihydroxynaphthalene as the starting material, through key steps such as amination, protection / deprotection operation, aldehyde group introduction, benzopyrone cyclization, benzyl bromination and functional DPA group introduction and other multi-step functionalization reactions, finally achieving efficient synthesis of nuclear ATP fluorescent probe molecules. The probe molecule can selectively locate in the cell nucleus in the cell, and generate enhanced fluorescence signals by specifically binding to ATP, thereby achieving high-sensitivity detection of ATP.

[0010] In one aspect, the present invention provides a method for preparing a selective nuclear ATP small molecule probe, which is suitable for preparing a small molecule probe having a substituted benzocoumarin structure, comprising the following steps:

[0011] Initial functional group modification: Using 2,7-dihydroxynaphthalene as the starting material, the hydroxyl group is first subjected to an amination reaction to form an amino-containing intermediate; then, the hydroxyl group is protected;

[0012] Introduction of aldehyde group and deprotection of hydroxyl group: Under the shielding of protecting group, aldehyde functional group is introduced at the unprotected site, and then the hydroxyl protecting group is selectively removed to release the activity of the site;

[0013] Ring structure construction: The benzopyrone ring skeleton is generated by anhydride cyclization reaction;

[0014] Benzyl bromination and DPA substitution: Bromination at the benzyl position followed by introduction of a dipyridylamine group;

[0015] Final deprotection and metal complexation: The amino protecting group is removed by acid catalysis to restore the activity of the amino group; followed by metal complexation to complete the synthesis of the target probe.

[0016] As a preferred embodiment, the structural formula of the small molecule probe corresponds to Formula I,

[0017]

[0018] Wherein, R1, R3 represent C1-C 25 A hydrocarbon chain which may be substituted by one or more groups, which may be the same or different.

[0019] As a preferred embodiment, the preparation method uses 2,7-dihydroxynaphthalene as a starting material, undergoes hydroxyl amination, hydroxyl protection, aldehyde addition, hydroxyl deprotection, ring closure, temporary deprotection, amino protection, benzyl bromination, benzyl alkylation, deamino protection, and finally complexation with a zinc reagent to obtain the target product.

[0020] As a preferred embodiment, the specific steps of the preparation method are as follows:

[0021] S1. Using 2,7-dihydroxynaphthalene as the starting material, react with amine under the action of sodium pyrosulfite to obtain compound II-2:

[0022]

[0023] S2. Use a strong base to introduce a protecting group R4 into compound II-2 to generate compound II-3:

[0024]

[0025] S3. Under the action of a strong base, compound II-3 removes the ortho-hydrogen, reacts with DMF to introduce an aldehyde group, and removes the protecting group R4 to obtain compound II-5:

[0026]

[0027] S4, compound II-5 undergoes condensation reaction with anhydride under the catalysis of carboxylate to obtain key intermediate II-6:

[0028]

[0029] S5, removing the protecting group R2 by metal palladium catalysis, replacing the new protecting group R5, to obtain compound II-8:

[0030]

[0031] S6. Compound II-8 is brominated using NBS under free radical bromination conditions to obtain bromide II-9:

[0032]

[0033] S7, bromide II-9 and dipyridylamine undergo alkylation reaction to obtain compound II-10:

[0034]

[0035] S8, removing the protecting group R5 under the action of a strong acid to generate compound II-11:

[0036]

[0037] S9. The methanol solution of compound II-11 is reacted with an aqueous solution of zinc perchlorate to generate a zinc complex I of the target probe.

[0038] As a preferred embodiment, R1, R2, and R3 represent C1-C 25A hydrocarbon chain which may be substituted by one or more groups, which may be the same or different.

[0039] As a preferred embodiment, R4 is selected from any one of MOMcl, Ac, Boc and Cbz; and / or,

[0040] R5 is selected from any one of Ac, Boc and Cbz;

[0041] The structural formula of the protecting group is shown in Formula III.

[0042]

[0043]

[0044] As a preferred embodiment, the strong acid is any one of hydrochloric acid, sulfuric acid, methanesulfonic acid and trifluoromethanesulfonic acid.

[0045] On the other hand, the present invention further provides a selective nuclear ATP small molecule probe, which is prepared by the above-mentioned preparation method, and its structural features include DPA fragments introduced by bromination and alkylation, and ATP selectivity achieved by zinc ion complexation.

[0046] In yet another aspect, the present invention further provides the use of the above-mentioned selective nuclear ATP small molecule probe for detecting the ATP level in the nucleus to distinguish tumor tissue from normal tissue.

[0047] As a preferred embodiment, the detection method includes using the probe to perform fluorescence imaging on target cells to detect changes in ATP levels in the cell nucleus.

[0048] The present invention adopts the above technical solution to have at least the following beneficial effects:

[0049] 1. The present invention provides a selective nuclear ATP small molecule probe and its preparation method and application, using 2,7-dihydroxynaphthalene as the starting material, through key steps such as amination, protection / deprotection operation, aldehyde group introduction, benzopyrone cyclization, benzyl bromination and functional DPA group introduction and other multi-step functionalization reactions, and finally realizes the efficient synthesis of nuclear ATP fluorescent probe molecules. The probe molecule can selectively locate in the cell nucleus in the cell, and generate an enhanced fluorescent signal by specifically binding to ATP, thereby realizing high-sensitivity detection of ATP;

[0050] 2. The present invention stabilizes the molecular structure and ensures the selectivity of the reaction site by amino and hydroxyl protection; accurately introduces aldehyde groups and removes the protecting groups under acidic conditions to release the reactive sites, and then forms a benzopyrone skeleton by cyclization; bromination and introduction of DPA (bispyridylamine) groups at the benzyl position enable the probe to selectively bind to nuclear ATP; and finally, zinc complexation is used to complete the probe structure and enhance the fluorescence signal when binding to ATP. This step-by-step construction and optimized design ensures the high selectivity and high sensitivity of the probe molecule to nuclear ATP, providing a reliable means for the dynamic detection of ATP;

[0051] 3. The present invention uses propionic anhydride to obtain a benzopyrone structure in a moderate yield (50%) under the condition of sodium propionate, which is greatly improved compared with the yield reported in the literature (<5%);

[0052] 4. The raw materials in the preparation method of the present invention are simple and easy to obtain, the reaction conditions are mild, the operation is simple, there is no selectivity problem, the yield is high, and it is universal for this series of compounds;

[0053] 5. The probe molecules of the present invention can be used for cell metabolism research and disease mechanism analysis, especially in the detection of ATP level differences between tumors and normal tissues, which is helpful for the early diagnosis and monitoring of cancer. DETAILED DESCRIPTION

[0054] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0055] In the examples of the present application, NMR: All NMR spectra were detected by Bruker AV 400 MHz NMR instrument, with TMS as internal standard or without internal standard. MS: All mass spectra were detected by Shimadzu LCMS2020.

[0056] Unless otherwise defined, all terms (including technical and scientific terms) used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal meaning unless explicitly defined in the present invention.

[0057] As used herein, a hyphen ("-") not between two letters or symbols is used to indicate the position of the point of attachment of a substituent. For example: -C1-C 10 Alkyl-NH2 through C1-C 10 Alkyl group connected.

[0058] As used herein, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes instances where the event or condition occurs and instances where it does not occur. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically unrealistic, synthetically unfeasible, and / or inherently unstable.

[0059] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, which is generally 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl but have two points of attachment.

[0060] As used herein, "alkene group" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group. The group can be a cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues identical to alkenyl but with two points of attachment.

[0061] As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to a residue that is identical to alkynyl but has two points of attachment.

[0062] As used herein, "haloalkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms is replaced by one or more, up to the maximum permitted number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.

[0063] Various hydroxyl protecting groups can be used in the present disclosure. In general, protecting groups make chemical functional groups insensitive to specific reaction conditions, and can be attached and removed from the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entirety.

[0064] This application adopts a synthesis strategy different from Scheme 1, using bromide and dipyridine methylamine, alkylation to obtain compound I-3. The specific reaction is shown in Scheme 2: The initial synthesis route uses 2,7-dihydroxynaphthalene as the starting material, and is substituted, MOM protected, aldehyde group added, MOM protection removed, ring closed, deallyl protection, Boc protection group added, benzyl bromination, substitution, Boc protection removed, and finally complexed with a zinc reagent to obtain the target product.

[0065] The advantages of this synthetic strategy are: 1. Using propionic anhydride under sodium propionate conditions, the benzopyrone structure is ring-closed in a moderate yield (50%), which is a great improvement over the yield reported in the literature (<5%). 2. Bromine is introduced at the benzyl position by NBS bromination, and then the dipyridylamine (DPA) fragment is introduced by alkylation. The protection of the left methylamino group using Boc is proved to be necessary for bromination. The bromination of substrates without Boc protection requires a very complicated reaction system.

[0066]

[0067] Solution 2

[0068] In order to fully understand the above solution and clarify its application value, this application also gives the following specific examples.

[0069] Step A: Synthesis of intermediate 2, namely: Synthesis of 7-(allyl(methyl)amino)naphthalene-2-ol

[0070] In a sealed tube with a volume of 150mL, naphthalene-2,7-diol (14g, 87.407mmol) and sodium metabisulfite (33.56g, 174.814mmol) were dissolved in 30mL of water. After fully dissolved, N-methyl-2-propylene-1-amine (24.87g, 349.629mmol) was added. After cooling, the whole mixture was placed in an oil bath at 120°C and stirred for 5 hours. After the reaction was completed, it was cooled to room temperature, diluted with water (60mL), and extracted with ethyl acetate (30mL*3). The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (EtOAc PE from 1% to 5%) to obtain 7-(allyl (methyl) amino) naphthalene-2-ol (10g, 46.887mmol, 53.64%) as a light yellow oil.

[0071] 1 HNMR(400MHz,DMSO-d6)δ9.40(s,1H),7.60–7.46(m,2H),6.98–

[0072] 6.83(m,2H),6.76–6.67(m,2H),5.90–5.77(m,1H),5.17–5.07(m,2H),4.04–4.00(m,2H),2.96(s,3H).

[0073] Step B: Synthesis of intermediate 3, i.e., synthesis of N-allyl-7-(methoxymethoxy)-N-methylnaphthalene-2-amine

[0074] At 0°C, 60% sodium hydride (2.25 g, 56.26 mmol) suspended in mineral oil was added portionwise to a solution of 7-(allyl(methyl)amino)naphthalene-2-ol (10 g, 46.887 mmol) in dry DMF (80 mL), and the mixture was stirred for 10 minutes under the protection of nitrogen. Chloromethyl methyl ether (4.27 mL, 56.26 mmol) was added to the above mixture, and the mixture was stirred for 2 hours. After the reaction was completed, saturated ammonium chloride solution (20 mL) was slowly added dropwise to the reaction, diluted with water (300 mL), and extracted with ethyl acetate (120 mL*3). The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (EtOAc in PE from 1% to 5%) to give N-allyl-7-(methoxymethoxy)-N-methylnaphthalen-2-amine (8 g, 31.09 mmol, 66.31%) as a light yellow oil.

[0075] LC-MS(ESI):258.1[M+H] + .

[0076] Step C: Synthesis of intermediate 4, i.e., 3-[(methoxymethyl)oxy]-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde

[0077] To a THF solution of {7-[(methoxymethyl)oxy]-2-naphthyl}(methyl)(prop-2-enyl)amine (10 g, 38.86 mmol), t-BuLi (29.89 mL, 38.86 mmol) was carefully added dropwise at -40°C and the reaction was kept at this temperature for 30 minutes. DMF (9.38 mL, 116.58 mmol) was then added dropwise to the reaction mixture, stirred for another 2 h, and then quenched with a saturated NH4Cl solution. The mixture was extracted with EA, the organic layer was dried over anhydrous Na2SO4, concentrated, and flash column chromatography (PE / EA=10 / 1) was used to give 3-[(methoxymethyl)oxy]-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde (2.1 g, 7.36 mmol, 18.94%) as a yellow solid.

[0078] LC-MS(ESI):256.1[M+H] + .

[0079] Step D: Synthesis of intermediate 5, i.e., synthesis of 3-hydroxy-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde

[0080] To a solution of 3-[(methoxymethyl)oxy]-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde (2.1 g, 7.36 mmol) in acetonitrile (30 mL) was added NbCl5 (1.99 g, 7.36 mmol) at 0°C and the reaction was stirred at room temperature for 2 h. The reaction mixture was then quenched with a saturated NaHCO3 solution and extracted with EA. The organic layer was washed with brine, dried over anhydrous Na2SO4, and then purified by flash chromatography (0-5% PE / EA) to give 3-hydroxy-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde (920 mg, 3.81 mmol, 51.81%) as a yellow oil.

[0081] LC-MS(ESI):242.1[M+H] + .

[0082] Step E: Synthesis of intermediate 6, i.e., 3-methyl-8-[methyl(prop-2-enyl)amino]-2H-benzo[g]benzopyran-2-one

[0083] 3-Hydroxy-6-[methyl(prop-2-enyl)amino]naphthalene-2-carboxaldehyde (920 mg, 3.81 mmol), propionic anhydride (10 mL), sodium propionate (732.52 mg, 7.63 mmol) and TEA (0.53 mL, 3.81 mmol) were added to a microwave tube and then stirred in a microwave at 170° C. for 50 minutes. The reaction was then cooled, extracted with saturated aqueous NaHCO3, and the organic layer was washed with brine, dried over anhydrous Na2SO4, then filtered, concentrated under reduced pressure, and purified by flash chromatography (0-5% PE / EA) to give 3-methyl-8-[methyl(prop-2-enyl)amino]-2H-benzo[g]benzopyran-2-one (530 mg, 1.90 mmol, 49.76%) as a yellow oil.

[0084] LC-MS(ESI):280.1[M+H] + .

[0085] Step F: Synthesis of intermediate 7, i.e., 3-methyl-8-(methylamino)-2H-benzo[g]benzopyran-2-one

[0086] Pd(PPh3)4 (219.25 mg, 0.190 mmol) and 1,3-dimethyl-2-oxohexanehydropyrimidine-4,6-dione (888.75 mg, 5.692 mmol) were added to 3-methyl-8-[methyl(prop-2-enyl)amino]-2H-benzo[g]benzo[g]benzopyran-2-one (530 mg, 1.897 mmol) in DCM (20 mL), and the reaction was stirred at 50°C for 2 hours, and then concentrated under reduced pressure to obtain a residue. Purification by flash chromatography (80% PE / DCM) gave 3-methyl-8-(methylamino)-2H-benzo[g]benzopyran-2-one (330 mg, 1.379 mmol, 72.69%) as a yellow oil.

[0087] LC-MS(ESI):240.1[M+H] + .

[0088] Step G: Synthesis of intermediate 8, i.e., N-methyl-N-(3-methyl-2-oxobenzo[g]benzopyran-8-yl)acetamide

[0089] To a solution of 3-methyl-8-(methylamino)-2H-benzo[g]benzo[g]benzopyran-2-one (330 mg, 1.38 mmol) in THF (20 mL) was added Ac2O (422.40 mg, 4.14 mmol), Na2CO3 (438.54 mg, 4.14 mmol), and the reaction was stirred at room temperature for 16 h, then water was added and extracted three times with EA (20 mL*3). The combined organic layer was washed with brine (40 mL*3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give N-methyl-N-(3-methyl-2-oxobenzo[g]benzopyran-8-yl)acetamide (240 mg, 0.85 mmol, 61.86%) as a yellow oil.

[0090] LC-MS(ESI):282.1[M+H] + .

[0091] Step H: Synthesis of intermediate 9, i.e., N-[3-(bromomethyl)-2-oxobenzo[g]benzopyran-8-yl]-N-methylacetamide

[0092] To a solution of N-methyl-N-(3-methyl-2-oxobenzo[g]benzo[g]benzopyran-8-yl)acetamide (240 mg, 0.85 mmol) in carbon tetrachloride were added NBS (455.56 mg, 2.56 mmol) and AIBN (420.29 mg, 2.56 mmol), and the reaction was stirred at 70°C for 6 hours, and then concentrated under reduced pressure to give N-[3-(bromomethyl)-2-oxobenzo[g]benzopyran-8-yl]-N-methylacetamide (170 mg, 0.33 mmol, 38.72%) as a yellow oil.

[0093] LC-MS(ESI):360.1[M+H] + .

[0094] Step I: Synthesis of intermediate 10, i.e., synthesis of N-(3-{[bis(pyridin-2-ylmethyl)amino]methyl}-2-oxobenzo[g]benzopyran-8-yl)-N-methylacetamide

[0095] To a solution of N-[3-(bromomethyl)-2-oxobenzo[g]benzo[g]chromen-8-yl]-N-methylacetamide (170 mg, 0.47 mmol) in acetonitrile (10 mL) were added 2-{[(pyridin-2-ylmethyl)amino]methyl}pyridine (94.04 mg, 0.47 mmol) and K2CO3 (195.67 mg, 1.42 mmol). The reaction was stirred at room temperature for 1 h. The reaction mixture was quenched with water and extracted with EA (20 mL*3). The combined organic layers were washed with brine (40 mL*3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and purified by chromatographic column (DCM / MeOH=20 / 1) to give N-(3-{[bis(pyridin-2-ylmethyl)amino]methyl}-2-oxobenzo[g]chromen-8-yl)-N-methylacetamide (160 mg, 0.33 mmol, 70.84%) as a yellow oil.

[0096] LC-MS(ESI):479.2[M+H] + .

[0097] Step J: Synthesis of intermediate 11, i.e., 3-{[bis(pyridin-2-ylmethyl)amino]methyl}-8-(methylamino)-2H-benzo[g]benzopyran-2-one

[0098] To a solution of N-(3-{[bis(pyridin-2-ylmethyl)amino]methyl}-2-oxobenzo[g]benzo[g]benzopyran-8-yl)-N-methylacetamide (150 mg, 0.31 mmol) in MeOH (10 mL) was added SOCl2 (111.86 mg, 0.94 mmol), and the reaction was stirred at 70°C for 6 h. A saturated aqueous NaHCO3 solution was added and extracted with EA (40 mL*3). The combined organic layer was washed with brine (40*3 mL), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by Pre-HPLC to give 3-{[bis(pyridin-2-ylmethyl)amino]methyl}-8-(methylamino)-2H-benzo[g]benzopyran-2-one (30 mg, 0.07 mmol, 21.93%) as a yellow solid.

[0099] LC-MS(ESI):437.2[M+H] + .

[0100] 1 HNMR(400MHz,DMSO-d6)δ8.50–8.48(m,2H),8.13(s,1H),8.00(s,1H),7.79–7.77(m,2H),7.71(d,J=8.0Hz,1H),7.62(d,J=8.0Hz,2H),7.4 3(s,1H),7.25–7.21(m,2H),6.97–6.95(m,1H),6.66(d,J=2.0Hz,1H),6.47–6.44(m,1H),3.85(s,4H),3.60(s,2H),2.80(d,J=4.0Hz,3H).

[0101] Stepk: Synthesis of NucATP

[0102] To a solution of 3-{[bis(pyridin-2-ylmethyl)amino]methyl}-8-(methylamino)-2H-benzo[g]benzo[g]chromen-2-one (10 mg, 0.02 mmol) in MeOH (5 mL) was added bis(trioxohypochlorite) hexahydrate zinc (8.53 mg, 0.02 mmol), and the reaction was stirred at room temperature for 2 h, then concentrated under reduced pressure to give NucATP (6.7 mg, 0.01 mmol, 58.49%) as a red solid.

[0103] 1HNMR(400MHz,DMSO-d6)δ8.65(d,J=4.8Hz,2H),8.22(s,1H),8.06–7.97(m,3H),7.77(d,J=8.0Hz,1H),7.64–7.51(m,4H),7 .45(s,1H),7.04–6.96(m,1H),6.68(s,2H),4.39(d,J=16.0Hz,2H),4.15–4.10(m,2H),3.77(s,2H),2.81(d,J=4.8Hz,3H).

[0104] 13 CNMR(100MHz,DMSO-d6)δ154.96,150.82,150.66,148.00,147.91,141.14,138.34,130.11,12 9.76,125.20,124.96,124.05,119.70,117.11,114.10,109.01,100.35,57.87,49.07,29.82.

[0105] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the conceptual scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a selective nuclear ATP small molecule probe, characterized in that: The method is applicable to the preparation of a small molecule probe having a substituted benzocoumarin structure, comprising the following steps: Initial functional group modification: Using 2,7-dihydroxynaphthalene as the starting material, the hydroxyl group is first subjected to an amination reaction to form an amino-containing intermediate; then, the hydroxyl group is protected; Introduction of aldehyde group and deprotection of hydroxyl group: Under the shielding of protecting group, aldehyde functional group is introduced at the unprotected site, and then the hydroxyl protecting group is selectively removed to release the activity of the site; Ring structure construction: The benzopyrone ring skeleton is generated by anhydride cyclization reaction; Benzyl bromination and DPA substitution: Bromination at the benzyl position followed by introduction of a dipyridylamine group; Final deprotection and metal complexation: The amino protecting group is removed by acid catalysis to restore the activity of the amino group; followed by metal complexation to complete the synthesis of the target probe.

2. The preparation method according to claim 1, characterized in that: The structural formula of the small molecule probe corresponds to Formula I, Wherein, R1, R3 represent C1-C 25 A hydrocarbon chain which may be substituted by one or more groups, which may be the same or different.

3. The preparation method according to claim 2, characterized in that: The preparation method uses 2,7-dihydroxynaphthalene as a starting material, undergoes hydroxyl amination, hydroxyl protection, aldehyde group addition, hydroxyl group removal, ring closure, temporary protection removal, amino group addition, benzyl bromination, benzyl alkylation, deamination protection, and finally complexation with a zinc reagent to obtain the target product.

4. The preparation method according to claim 2 or 3, characterized in that: The specific steps of the preparation method are as follows: S1. Using 2,7-dihydroxynaphthalene as the starting material, react with amine under the action of sodium pyrosulfite to obtain compound II-2: S2. Use a strong base to introduce a protecting group R4 into compound II-2 to generate compound II-3: S3. Under the action of a strong base, compound II-3 removes the ortho-hydrogen, reacts with DMF to introduce an aldehyde group, and removes the protecting group R4 to obtain compound II-5: S4, compound II-5 undergoes condensation reaction with anhydride under the catalysis of carboxylate to obtain key intermediate II-6: S5, removing the protecting group R2 by metal palladium catalysis, replacing the new protecting group R5, to obtain compound II-8: S6. Compound II-8 is brominated using NBS under free radical bromination conditions to obtain bromide II-9: S7, bromide II-9 and dipyridylamine undergo alkylation reaction to obtain compound II-10: S8, removing the protecting group R5 under the action of a strong acid to generate compound II-11: S9. The methanol solution of compound II-11 is reacted with an aqueous solution of zinc perchlorate to generate a zinc complex I of the target probe.

5. The preparation method according to claim 4, characterized in that: R1, R2, R3 represent C1-C 25 A hydrocarbon chain which may be substituted by one or more groups, which may be the same or different.

6. The preparation method according to claim 4, characterized in that: R4 is selected from any one of MOMCl, Ac, Boc and Cbz; and / or, R5 is selected from any one of Ac, Boc and Cbz.

7. The preparation method according to claim 4, characterized in that: The strong acid is any one of hydrochloric acid, sulfuric acid, methanesulfonic acid and trifluoromethanesulfonic acid.

8. A selective nuclear ATP small molecule probe, characterized in that: The preparation method according to any one of claims 1 to 7 is adopted for preparation, and its structural features include DPA fragments introduced by bromination and alkylation, and ATP selectivity achieved by zinc ion complexation.

9. The use of the selective nuclear ATP small molecule probe as claimed in claim 8, characterized in that: Used to detect the ATP level in the cell nucleus to distinguish tumor tissue from normal tissue.

10. The use according to claim 9, characterized in that: The detection method comprises using the probe to perform fluorescence imaging on target cells to detect changes in ATP levels in the cell nucleus.

Images