Fluorescent compound with aggregation-induced emission characteristic as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a fluorescent compound with aggregation-induced emission characteristic and a preparation method and application thereof. The novel terpyridyl fluorescent compound with the aggregation-induced emission characteristic is synthesized, and the terpyridyl fluorescent compound has the advantages of being easy to prepare, high in fluorescence intensity, low in fluorescence background, high in biological safety and the like. The compound prepared by the invention can be used as a bacterial membrane and DNA dual-targeting fluorescent dye and a photosensitizer, has the advantages of good light stability, good biocompatibility, short time consumption and simplicity and convenience in operation in dyeing imaging effect, and can be used for imaging bacteria and distinguishing gram-positive bacteria and gram-negative bacteria. The compound disclosed by the invention can also be used for antibacterial treatment of drug-resistant bacteria, can realize an efficient bacterium killing effect mainly by damaging a bacterial membrane and a DNA (Deoxyribonucleic Acid) structure through targeted binding, and has broad spectrum.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a class of fluorescent compounds with aggregation-induced emission characteristics, a preparation method thereof, and an application thereof. Background Art

[0002] Many serious diseases caused by bacterial infections have attracted the attention of the medical community and the public worldwide. The abuse and misuse of antibiotics have led to the emergence of drug-resistant bacteria. Therefore, solving the problem of drug-resistant bacteria is a huge challenge faced globally today. At present, the single-target mechanism of traditional antibiotics easily leads to bacterial drug resistance. Therefore, it is of great significance to develop new antibacterial drugs with a multi-target mechanism.

[0003] Photodynamic therapy (PDT) is a relatively novel non-invasive treatment method that relies on the irradiation of a specific wavelength light source to activate a photosensitizer to generate reactive oxygen species (ROS). The highly active ROS can destroy the external and internal structures of microorganisms, so it is difficult for microorganisms to develop resistance. However, traditional photosensitizers have problems such as low ROS generation efficiency, poor stability, and fluorescence quenching in the aggregated state, which limit their application in biomedicine.

[0004] In recent years, aggregation-induced emission (AIE) materials have become a research hotspot for new photosensitizers due to their high-efficiency luminescence and ROS generation ability in the aggregated state. However, most of the existing AIE photosensitizers only target the bacterial membrane or DNA, and it is difficult to achieve a dual-target antibacterial effect simultaneously. Therefore, developing an AIE photosensitizer that can target both the bacterial membrane and DNA simultaneously has important clinical significance. Summary of the Invention

[0005] The purpose of the first aspect of the present invention is to provide a class of terpyridine compounds.

[0006] The purpose of the second aspect of the present invention is to provide a preparation method of the terpyridine compounds of the first aspect of the present invention.

[0007] The purpose of the third aspect of the present invention is to provide an application of the terpyridine compounds of the first aspect of the present invention.

[0008] The purpose of the fourth aspect of the present invention is to provide a product.

[0009] In order to achieve the above purposes of the present invention, the technical solutions adopted by the present invention are as follows:

[0010] In the first aspect of the present invention, there is provided a class of terpyridine compounds, the structural formula of which is shown in formula (VIII):

[0011]

[0012] In some embodiments of the present invention, X is C or N.

[0013] In some embodiments of the present invention, R1 is R2Y, and Y is absent or selected from a halide anion.

[0014] In some embodiments of the present invention, the halogen anion is I - or Br - .

[0015] In some embodiments of the present invention, R2 is selected from alkyl, One of them.

[0016] In some embodiments of the present invention, the alkyl group is selected from one of a methyl group and an ethyl group.

[0017] In some embodiments of the present invention, when X is N, the structural formula of the terpyridine compound is any one of formulas (I) to (IV):

[0018]

[0019] In some embodiments of the present invention, when X is C, the structural formula of the terpyridine compound is as shown in any one of formulas (V) to (VII):

[0020]

[0021] The second aspect of the present invention provides a method for preparing the terpyridine compound of the first aspect of the present invention.

[0022] In some embodiments of the present invention, compound 1d is reacted with reaction material 1, or compound 5d is reacted with reaction material 2 to obtain a compound of the structure represented by formula (VIII);

[0023] The structural formula of the skeleton compound 1d is shown in formula (XI):

[0024]

[0025] The structural formula of the skeleton compound 5d is shown in formula (XV):

[0026]

[0027] In some embodiments of the present invention, the reaction raw material 1 includes at least one of an alkyl halide, a quaternary ammonium halide, and a sulfonating agent.

[0028] In some embodiments of the present invention, the reaction raw material 2 includes an alkyl halide and a tertiary amine.

[0029] In some embodiments of the present invention, in reaction raw material 1, the haloalkane is selected from one of methyl iodide and iodopropane; the quaternary ammonium halide is (3-bromopropyl) trimethyl ammonium bromide; the sulfonating agent is 1,3-propane sultone.

[0030] In some embodiments of the present invention, in reaction raw material 2, the haloalkane is selected from one of 1,2-dibromoethane, 1,3-dibromopropane, and 1,4-dibromobutane; the tertiary amine is trimethylamine.

[0031] In some embodiments of the present invention, the heating temperature is 60-100.

[0032] In some embodiments of the present invention, the reaction time is  12-48 h.

[0033] The third aspect of the present invention provides the application of the terpyridine compounds described in the first aspect of the present invention in the preparation of products.

[0034] In some embodiments of the present invention, the products include at least one of fluorescent dyes, photosensitizers, bacterial dyes, and antibacterial agents.

[0035] The fourth aspect of the present invention provides a product, which includes the terpyridine compounds described in the first aspect of the present invention.

[0036] In some embodiments of the present invention, the products include at least one of fluorescent dyes, photosensitizers, bacterial dyes, and antibacterial agents.

[0037] In some embodiments of the present invention, when the product is a fluorescent dye, the fluorescent dye includes the terpyridine compounds described in the first aspect of the present invention.

[0038] In some embodiments of the present invention, when the product is a photosensitizer, the photosensitizer includes one of the terpyridine compounds represented by structural formulas (1)-(4), (6), and (7).

[0039] In some embodiments of the present invention, when the product is a bacterial dye, the bacterial dye includes the terpyridine compounds described in the first aspect of the present invention.

[0040] In some embodiments of the present invention, when the product is an antibacterial agent, the antibacterial agent includes one of the terpyridine compounds represented by structural formulas (1) and (2).

[0041] Specifically, compound 1-2 is a fluorescent dye and photosensitizer with dual targeting to bacterial membranes and DNA; the staining and imaging effect has the advantages of good photostability, good biocompatibility, short time consumption, and simple operation.

[0042] Compound 3 is a fluorescent dye and photosensitizer that targets and binds to bacterial membranes, and can be used for bacterial imaging and antibacterial therapy.

[0043] Compounds 4-7 are Gram-positive bacteria membrane-binding dyes and can be used to distinguish Gram-positive bacteria from Gram-negative bacteria.

[0044] In some embodiments of the present invention, the product further comprises a pharmaceutically acceptable excipient.

[0045] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0046] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and are inactive ingredients of pharmaceuticals. Compilations of pharmaceutically acceptable excipients can be found in "Handbook of Pharmaceutical Excipients" (2nd Edition, edited by A.Wade and P.J.Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "List of Medicinal Excipients in Chinese Pharmacopoeia" and other reference books.

[0047] The present invention further provides the use of compounds 1 and 2 as active ingredients in related drugs for the treatment of bacterial infections.

[0048] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a parenteral dosage form.

[0049] In some embodiments of the present invention, the gastrointestinal dosage forms include at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets;

[0050] In some embodiments of the present invention, the parenteral dosage forms include at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0051] The carriers selected in the preparation of oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearate, etc. The later preparation processes and equipment of each preparation belong to the conventional technologies in the pharmaceutical field, and the present invention does not limit them.

[0052] The beneficial effects of the present invention are as follows:

[0053] The present invention synthesizes a new class of terpyridine-based fluorescent compounds with aggregation-induced emission (AIE) properties. The molecular design of the present invention is based on the TPA-TPy (triphenylamine-terpyridine) skeleton, which has a donor-acceptor (D-A) structure and can achieve long-wavelength emission and efficient ROS generation; introducing positive charges (such as pyridine groups, quaternary ammonium salts, etc.) at the molecular termini significantly enhances the electrostatic interaction between the molecule and the negatively charged bacterial membrane, improving the targeting and binding ability of the molecule to bacteria; by adjusting the hydrophilic-hydrophobic balance of the molecule (such as replacing the pyridine ring with an oxygen atom), the interaction between the molecule and the bacterial membrane can be further optimized, enhancing the insertion ability of the molecule into the bacterial membrane, thereby improving the antibacterial effect; by systematically adjusting the length of the alkyl chain, the binding affinity of the molecule to the bacterial membrane and DNA can be optimized. A longer alkyl chain enhances the hydrophobic interaction and promotes the insertion of the molecule into the bacterial membrane, while a shorter chain is more suitable for binding to bacterial DNA; this design not only considers the antibacterial activity of the molecule but also, by introducing AIE (aggregation-induced emission) properties, enables the molecule to exhibit efficient fluorescence emission and ROS generation ability in the aggregated state, which is applicable to bacterial imaging and photodynamic therapy (PDT); it can be optimized for different types of bacteria (such as Gram-positive bacteria and Gram-negative bacteria), has broad-spectrum antibacterial potential, and especially shows significant antibacterial effects against multi-drug resistant bacteria (such as MRSA, MDRAB, etc.); in addition, it significantly reduces the possibility of bacteria developing drug resistance, providing a new solution to deal with multi-drug resistant bacterial infections. Description of the Drawings

[0054] Figure 1 、 2 are the proton nuclear magnetic resonance ( 1 H NMR) spectrum (400 MHz, DMSO-d6) and carbon-13 nuclear magnetic resonance ( 13 C NMR) spectrum (400 MHz, DMSO-d6) of compound 1d, respectively.

[0055] Figure 3 、 4 are the proton nuclear magnetic resonance ( 1 H NMR) spectrum (DMSO-d6) and high-resolution mass spectrometry (HRMS)

[0056] Figure 5 、6 and 7 are the proton nuclear magnetic resonance ( 1 H NMR) spectrum (400 MHz, DMSO-d6), carbon-13 nuclear magnetic resonance ( 13 C NMR) spectrum (400 MHz, DMSO-d6), and high-resolution mass spectrometry (HRMS) of Compound 2, Figure 7 The lower middle figure is the enlarged result of the boxed area in the upper figure.

[0057] Figure 8 , 9 and 10 are the proton nuclear magnetic resonance ( 1 H NMR) spectrum (DMSO-d6), carbon-13 nuclear magnetic resonance ( 13 C NMR) spectrum, and high-resolution mass spectrometry (HRMS) of Compound 3.

[0058] Figure 11 , 12 and 13 are the proton nuclear magnetic resonance ( 1 H NMR) spectrum (DMSO-d6), carbon-13 nuclear magnetic resonance ( 13 C NMR) spectrum (DMSO-d6), and high-resolution mass spectrometry (HRMS) of Compound 4, Figure 13 The lower middle figure is the enlarged result of the boxed area in the upper figure.

[0059] Figure 14 , 15 and are the proton nuclear magnetic resonance ( 1 H NMR) spectrum and high-resolution mass spectrometry of Compound 5, Figure 15 The lower middle figure is the enlarged result of the boxed area in the upper figure.

[0060] Figure 16 , 17 and are the proton nuclear magnetic resonance ( 1 H NMR) spectrum and high-resolution mass spectrometry of Compound 6, Figure 17 The lower middle figure is the enlarged result of the boxed area in the upper figure.

[0061] Figure 18 , 19 and are the proton nuclear magnetic resonance ( 1 H NMR) spectrum and high-resolution mass spectrometry of Compound 7, Figure 19 The lower middle figure is the enlarged result of the boxed area in the upper figure.

[0062] Figure 20In (B), it is the UV-visible absorption spectra of Compound 2 under the conditions of DMSO / toluene mixed solution with 0% and 99% toluene content respectively. In (A, C-G), they are the UV-visible absorption spectra of Compounds 1, 3-7 under the conditions of water / THF mixed solution with 0% and 99% THF content respectively.

[0063] Figure 21 In (B), it is the fluorescence spectrum of Compound 2 under the conditions of DMSO / toluene mixed solution. In (A, C-G), they are the fluorescence spectra of Compounds 1, 3-7 under the conditions of water / THF mixed solution.

[0064] Figure 22 In (A), it is the ROS generation ability of the compound. In (B), it is the ability of the compound to generate singlet oxygen.

[0065] Figure 23 It is the confocal laser imaging effect of Compounds 1-7 on the standard strains of Staphylococcus aureus and Escherichia coli.

[0066] Figure 24 It is the confocal laser imaging effect of Compound 2 on clinical drug-resistant strains (MRSA, MDR-AB) (A), the antibacterial effect on the plate (B, D, E), and the effect on inhibiting biofilm formation (C, F, G).

[0067] Figure 25 It is the surface morphological changes (scanning electron microscopy) and internal structural changes (transmission electron microscopy) of Compound 2 after treating MRSA and MDR-AB.

[0068] Figure 26 It is the co-localization effect after Compound 2 co-stains bacteria (methicillin-resistant Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii) and cells (L929) with commercial membrane dye CellMask and nuclear dye Hoechst 33342.

[0069] Figure 27 It is the transcriptome analysis result after Compound 2 treats MRSA (Figure A is the volcano plot of differential genes, Figure B is the GO enrichment analysis).

[0070] Figure 28 It is the verification of Compound 2 binding to bacterial DNA: In A-C, it is Compound 2 displacing commercial dye Hoechst 33342. In D, it is the change in the UV absorption spectrum after Compound 2 is mixed with exogenous DNA. In E, it is the change in the fluorescence spectrum after Compound 2 is mixed with exogenous DNA at different concentrations. In F-G, the addition of exogenous DNA inhibits the antibacterial effect of Compound 2 on MRSA and MDR-AB. In H-I, it is the molecular docking simulation calculation result of Compound 2 and DNA molecular fragment 4U8A.

[0071] Figure 29 The change in bacterial membrane potential after treating MRSA and MDR-AB with Compound 2 (indicated by the fluorescence change of Disc3(5)).

[0072] Figure 30 Drug resistance monitoring of Compound 2 (change in minimum inhibitory concentration after continuously treating standard strains of Staphylococcus aureus and Acinetobacter baumannii for two weeks).

[0073] Figure 31 Biocompatibility evaluation of Compound 2. The cell viability was detected by the MTT method after treating cells 3T3 and L929 with Compound 2.

[0074] Figure 32 In vivo antibacterial effect of Compound 2. A mouse intraperitoneal infection model was used: A is the blood biocompatibility of Compound 2, B is the white blood cell level in all infected mice after treatment with Compound 2, and C is the change in body weight of all mice during the treatment with Compound 2.

[0075] Figure 33 Therapeutic effect of Compound 2 on severe intraperitoneal infection: A is the survival rate result of infected mice in each treatment group, and B - F are the levels of organ bacterial counts after the end of treatment.

[0076] Figure 34 Reaction synthesis route diagram of various substances of the present invention. Detailed implementation manners

[0077] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0078] The synthesis reaction routes of various compounds of the present invention are as Figure 34 shown.

[0079] Example 1 Preparation of Compound 1

[0080] 1. Synthesis of Compound 1b

[0081] The structural formula of Compound 1b is shown in Formula (IX):

[0082]

[0083] The synthesis procedure of 1b was similar to the previously reported one [DOI: 10.1039 / C4PY01793E]. At 0 °C, phosphorus oxychloride (POCl3, 0.84 mL, 9.00 mmol) was added dropwise to dry N,N-dimethylformamide (DMF, 0.83 mL, 10.80 mmol). The mixture was stirred at room temperature for 20 minutes. Then, 1a (2.59 g, 8.00 mmol) was added, and the mixture was heated to 90 °C. The reaction progress was monitored by thin-layer chromatography (TLC) until 1a was completely consumed. After the reaction was completed, the mixture was cooled to room temperature, 50 mL of ice water was added, and then it was neutralized with saturated sodium bicarbonate (NaHCO3) solution. The mixture was extracted with dichloromethane (DCM) and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate (Na2SO4). The solvent was removed by rotary evaporation, and the crude product was further purified by column chromatography to obtain the desired product as a yellow solid (yield: 70%).

[0084] The nuclear magnetic resonance (NMR) spectrum was consistent with the previously reported data. 1H NMR (600 MHz, CDCl3) δ 9.64 (s, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 8.6 Hz, 4H), 6.87 - 6.80 (m, 6H).

[0085] 2. Synthesis of Compound 1c

[0086] The structural formula of Compound 1c is shown in Formula (Ⅹ):

[0087]

[0088] Compound 1c was prepared by the Suzuki [DOI: 10.1016 / j.dyepig.2016.08.052] coupling reaction: 1b (1.05 g, 2.00 mmol), pyridine-4-boronic acid (0.74 g, 6.00 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 0.23 g, 0.20 mmol), and potassium carbonate (K2CO3, 1.10 g, 8.00 mmol) were added to a 100 mL two-necked flask, and it was evacuated again for 30 minutes. Then the mixture was placed in a mixed solvent of tetrahydrofuran (THF, 20 mL) and water (4 mL), refluxed at 80 °C under a nitrogen atmosphere, and the completion of the reaction was monitored by thin-layer chromatography (TLC). After cooling, the reaction mixture was extracted with ethyl acetate (EA) as the organic solvent. The organic layer was separated, dried over anhydrous sodium sulfate (Na2SO4), and then filtered. The filtrate was concentrated by rotary evaporation under reduced pressure. Finally, the desired product was purified by silica gel column chromatography to obtain a yellow solid (yield: 50%).

[0089] 3. Synthesis of Compound 1d

[0090] The structural formula of Compound 1d is shown in Formula (Ⅺ):

[0091]

[0092] Dissolve 1c (2.10 g, 5.00 mmol) in 10 mL of ethanol. Add 2-acetylpyridine (1.12 mL, 10.00 mmol) to this mixture, then add sodium hydroxide (NaOH, 0.53 g, 13.00 mmol) and water (3 mL). Subsequently, stir the mixture for 30 minutes, and then slowly add ammonia water (NH4OH, 15 mL). Let the mixture stand overnight to precipitate. Filter the orange precipitate and wash it with cold ethanol to obtain a yellow solid (1.58 g, yield: 51%).

[0093] The characterization results of Compound 1d are as Figure 1 , 2 shown. 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 4.4 Hz, 2H), 8.73 (s, 2H), 8.68 (d, J = 8.0 Hz, 2H), 8.62 (d, J = 6.0 Hz, 4H), 8.04 (td, J = 7.8, 1.8 Hz, 2H), 7.96 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.6 Hz, 4H), 7.71 (d, J = 6.0 Hz, 4H), 7.56 - 7.51 (m, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.6 Hz, 4H). 13 C NMR (100 MHz, CDCl3) δ 156.3, 156.0, 150.3, 149.5, 149.2, 148.0, 147.7, 147.4, 136.9, 132.7, 128.6, 128.1, 124.8, 124.5, 123.9, 121.4, 121.1, 118.5.

[0094] 4. Synthesis of Compound 1:

[0095] The structural formula of Compound 1 is shown in Formula (Ⅰ):

[0096]

[0097] 1d (0.20 mmol, 0.13 g) was dissolved in tetrahydrofuran, and then methyl iodide (2.00 mmol, 0.28 g) was slowly added. The mixture was stirred at room temperature for 24 h to obtain a large amount of orange solid. The solid was filtered and washed with tetrahydrofuran and dichloromethane. The solid obtained by filtration was recrystallized with a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane. The resulting precipitate was filtered, washed with acetone and dichloromethane, and then dried under vacuum to obtain red solid 1 (0.13 g, yield 70%).

[0098] The characterization results of compound 1 are as Figure 3 , 4 shown. 1H NMR (400 MHz, DMSO-d6) δ 8.96 (d, J = 6.0 Hz, 4H), 8.77 (s, 4H), 8.70 (d, J = 7.8 Hz, 2H), 8.47 (d, J = 6.0 Hz, 4H), 8.14 (d, J = 8.4 Hz, 4H), 8.07 (d, J = 6.6 Hz, 4H), 7.59 - 7.52 (m, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 4H), 4.32 (s, 6H). HRMS: m / z: 1 / 2[M - 2I]2+ calcd for C45H36N62+: 330.1495; found: 330.1500.

[0099] Preparation of compound 2 in Example 2

[0100] The structural formula of compound 2 is shown in formula (Ⅱ):

[0101]

[0102] 1d (0.20 mmol, 0.13 g) was dissolved in tetrahydrofuran (THF), and then 1-iodopropane (2.00 mmol, 0.39 g) was slowly added. Then, the mixture was stirred at room temperature for 24 h to form a large amount of red solid. The solid was filtered and washed with tetrahydrofuran and dichloromethane (DCM). The crude product was recrystallized with a mixed solvent of dimethyl sulfoxide (DMSO) / acetone / dichloromethane. The obtained precipitate was filtered, washed with acetone and dichloromethane, and dried under vacuum to obtain red solid 2 (0.16 g, yield 80%).

[0103] The characterization results of compound 2 are as Figure 5 , 6 , 7 shown. 11H NMR (400 MHz, DMSO-d6) δ 9.05 (d, J = 5.0 Hz, 4H), 8.77 (s, 4H), 8.70 (d, J = 8.0 Hz, 2H), 8.49 (d, J = 5.0 Hz, 4H), 8.15 (d, J = 8.0 Hz, 4H), 8.08 (d, J = 7.8 Hz, 4H), 7.55 (d, J = 6.2 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 4H), 4.53 (d, J = 7.8 Hz, 4H), 1.97 (q, J = 7.6 Hz, 4H), 0.93 (t, J = 7.6 Hz, 6H). 13 13C NMR (100 MHz, DMSO-d6) δ 156.3, 155.4, 153.9, 150.0, 149.9, 149.0, 146.9, 145.0, 138.1, 134.7, 130.4, 129.3, 128.4, 127.1 125.1, 124.4, 123.9, 121.5 118.1, 61.5, 24.6 10.8 HRMS: m / z: 1 / 2[M - 2I] 2+ calcd for C 49 H 44 N6 2+ : 358.1808; found: 358.1816。

[0104] Preparation of Compound 3 in Example 3

[0105] The structural formula of Compound 3 is shown in Formula (Ⅲ):

[0106]

[0107] Synthesis of Compound 3: Dissolve 1d (0.20 mmol, 0.13 g) in acetonitrile (2 mL), and then slowly add (3-bromopropyl)trimethylammonium bromide (0.40 mmol, 0.10 g). Then, stir the mixture under reflux for 24 hours to form a large amount of red solid. Filter the solid and wash it with acetonitrile and dichloromethane. Recrystallize the filtered solid with a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane. Filter the resulting precipitate, wash it with acetone and dichloromethane, and then dry it under vacuum to obtain red solid 3 (0.15 g, yield 68%).

[0108] The characterization results of Compound 3 are as Figure 8 、 9 、shown in 10. 11H NMR (400 MHz, MeOH-d4) δ 9.01 (d, J = 6.2 Hz, 4H), 8.77 - 8.65 (m, 6H), 8.45 (d, J = 6.4 Hz, 4H), 8.13 - 8.01 (m, 8H), 7.51 (t, J = 6.4 Hz, 2H), 7.43 (dd, J = 12.8, 8.4 Hz, 6H), 4.72 (t, J = 7.8 Hz, 4H), 3.64 - 3.58 (m, 4H), 3.23 (s, 18H), 2.68 - 2.59 (m, 4H). 13 13C NMR (100 MHz, DMSO-d6) δ 156.0, 155.1, 154.2, 154.0, 149.7, 149.0, 146.9, 145.2, 138.3, 134.6, 130.5, 129.3, 128.3, 127.1, 125.2, 124.3, 124.0, 121.6, 118.2, 62.2, 53.0, 31.2, 24.8. HRMS: m / z: [M - 4Br] + calcd for C 55 H 60 N8 4+ : 832.4919; found: 832.2483。

[0109] Preparation of Compound 4 in Example 4

[0110] The structural formula of Compound 4 is shown in Formula (IV):

[0111]

[0112] Synthesis of Compound 4: Dissolve 1d (0.20 mmol, 0.13 g) in acetonitrile (2 mL), then slowly add 1,3 - propane sultone (0.40 mmol, 0.05 g). Subsequently, stir the mixture at room temperature for 24 hours to form a large amount of orange solid. Filter the solid and wash it with acetonitrile and dichloromethane. The filtered solid is recrystallized with a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane. The obtained precipitate is filtered, washed with acetone and dichloromethane, and then dried under vacuum to obtain red solid 4 (0.13 g, yield 73%).

[0113] The characterization results of Compound 4 are as shown in Figure 11 、 12 、13. 11H NMR (400 MHz, DMSO-d6) δ 9.06 (d, J = 6.6 Hz, 4H), 8.87 - 8.83 (m, 6H), 8.48 (d, J = 6.6 Hz, 4H), 8.26 - 8.15 (m, 2H), 8.13 (dd, J = 14.4, 8.6 Hz, 6H), 7.72 - 7.69 (m, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.6 Hz, 4H), 4.70 (t, J = 7.0 Hz, 4H), 3.42 (t, J = 6.4 Hz, 4H), 2.26 (t, J = 7.0 Hz, 4H). 13 13C NMR (100 MHz, DMSO-d6) δ 156.2, 155.3, 153.9, 149.9, 148.2, 147.0, 145.2, 138.1, 130.4, 129.3, 128.4, 125.1, 124.8, 124.4, 124.0, 122.0, 121.5, 118.6, 118.1, 59.0, 47.5, 27.8. HRMS: m / z: [M+Na] + calcd for C 49 H 42 N6O6NaS2: 897.2505; found: 897.2578.

[0114] Preparation of Compound 5 in Example 5

[0115] 1. Synthesis of Compound 5a

[0116] The structural formula of Compound 5a is shown in Formula (Ⅻ):

[0117]

[0118] Compound 5a was synthesized according to a previous report [DOI: 10.1039 / C8TA01773E]. Aniline (0.47 g, 5.00 mmol), 4-bromobenzyl ether (1.87 g, 10.00 mmol), palladium acetate (Pd(OAc)2, 0.06 g, 0.25 mmol), tri-tert-butylphosphine (P(t-Bu)3, 0.40 g, 0.20 mmol), and potassium tert-butoxide (t-BuOK, 1.40 g, 12.50 mmol) were added to a 100 mL flask, and toluene was added to the reaction system. The mixture was then heated to 80°C under a nitrogen atmosphere, and the reaction progress was monitored by thin-layer chromatography (TLC). After cooling, the reaction mixture was extracted with ethyl acetate (EA) to obtain an organic layer. The organic layer was separated, dried over anhydrous sodium sulfate (Na2SO4), and filtered. The filtrate was concentrated by rotary evaporation under reduced pressure, and the target product was purified by silica gel column chromatography to obtain a white solid 5a (yield 50%). 1 H NMR (600MHz, CDCl3) δ7.16 (t, J = 7.6 Hz, 2H), 7.04 (d, J = 8.8 Hz, 4H), 6.93 (d, J = 8.4 Hz, 2H), 6.86 (t, J = 7.2 Hz, 1H), 6.81 (d, J = 8.8 Hz, 4H), 3.79 (s, 6H).

[0119] 2. Synthesis of compound 5b

[0120] The structural formula of compound 5b is shown in formula (XIII):

[0121]

[0122] Compound 5b was synthesized according to a previous report [DOI: 10.1021 / acsaem.8b02100]. Phosphorus oxychloride (POCl3, 0.84 mL, 9.00 mmol) was added dropwise to dry N,N-dimethylformamide (DMF, 0.83 mL, 10.80 mmol) at 0°C, and the mixture was stirred at room temperature for 20 minutes. 5a (2.44 g, 8.00 mmol) was then added, and the mixture was heated to 90°C. The reaction was monitored by thin-layer chromatography (TLC) until 5a was completely consumed. After cooling, 50 mL of ice water was added, followed by neutralization with saturated aqueous sodium bicarbonate (NaHCO3(aq)). The mixture was extracted with dichloromethane (DCM) / water (3 x 50 mL). The organic layer was separated, dried over anhydrous sodium sulfate (Na2SO4), and filtered. The filtrate was concentrated by rotary evaporation under reduced pressure, and the target product was purified by silica gel column chromatography to obtain a yellow solid 5b (yield 70%). 11H NMR (600 MHz, CDCl3) δ 9.76 (s, 1H), 7.61 (d, J = 8.8 Hz, 4H), 7.14 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.8 Hz, 4H), 3.80 (s, 6H).

[0123] 3. Synthesis of Compound 5c

[0124] The structural formula of Compound 5c is shown in Formula (XIV):

[0125]

[0126] Compound 5c was synthesized according to the previous literature report [DOI: 10.1016 / j.dyepig.2018.05.051]. 5b (3.33 g, 5.00 mmol) was dissolved in 100 mL of ethanol, and then the resulting solution was added to a 250 mL reaction flask together with 2-acetylpyridine (3.00 g, 25.00 mmol). Under magnetic stirring, an aqueous potassium hydroxide solution (containing 0.22 g, 4.00 mmol of potassium hydroxide) dissolved in 10 mL of water was slowly added dropwise to the above mixed solution at room temperature. After 30 minutes, 25 mL of aqueous ammonia solution was added, and then the mixture was heated to 65 °C. After reacting for 3 hours, a yellow precipitate was formed, and then the precipitate was filtered. The precipitate was washed several times with ethanol and recrystallized to finally obtain yellow crystalline Compound 5c with a yield of 85%. 1 1H NMR (400 MHz, CDCl3) δ 8.72 (d, J = 4.8 Hz, 2H), 8.68 (s, 2H), 8.66 (d, J = 7.8 Hz, 2H), 7.87 (t, J = 7.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.37 - 7.30 (m, 2H), 7.11 (d, J = 8.8 Hz, 4H), 7.02 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.8 Hz, 4H), 3.82 (s, 6H).

[0127] 4. Synthesis of Compound 5d

[0128] The structural formula of Compound 5d is shown in Formula (XV):

[0129]

[0130] Under the condition of -20 °C, 5c (2.68 g, 5.00 mmol) was dissolved in dichloromethane (DCM), and then boron tribromide (BBr3, 16 mL) was slowly added. Subsequently, the mixture was stirred at -20 °C for 1 hour, and then allowed to warm to room temperature and stirred overnight. After the reaction was completed, methanol was slowly added, followed by dilute hydrochloric acid. Anhydrous sodium sulfate (Na2SO4) was added to the mixture. The solution was filtered and washed repeatedly with dichloromethane and ether to precipitate a green solid 5d (2.46 g, yield: 97%). The molecular weight of this solid was determined by gas chromatography-mass spectrometry (GC-MS) analysis, confirming it as the target product 5d.

[0131] 5. Synthesis of compound 5e:

[0132] The structural formula of compound 5e is shown in formula (XVI):

[0133]

[0134] 5d (0.4 mmol, 0.20 g), potassium hydroxide (KOH, 1.0 mmol, 0.06 g) and tetrabutylammonium bromide (0.04 mmol, 0.01 g) were added to a 50 mL flask. Subsequently, 20 mL of tetrahydrofuran (THF) was added to the mixture, and 1,2-dibromoethane (0.84 mmol, 0.16 g) was slowly added dropwise to the mixture. The reaction mixture was heated to 90 °C and stirred for 24 hours until the reaction was complete. After cooling, the mixture was extracted with ethyl acetate (EA) / water. The organic layer was separated, dried over anhydrous sodium sulfate, and then filtered. The resulting crude product was used directly in the next reaction without purification.

[0135] 6. Synthesis of compound 5:

[0136] The structural formula of compound 5 is shown in formula (V):

[0137]

[0138] 5e (0.20 mmol, 0.14 g) was added to a 50 mL flask. Subsequently, 20 mL of acetonitrile and trimethylamine (1.00 mmol, 0.06 g) were slowly added to the mixture. The mixture was heated to 70 °C and stirred for 24 hours. After cooling, a precipitate formed, and the precipitate was filtered. The solid was washed with acetonitrile and dichloromethane. The solid was recrystallized from a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane. The product was washed with acetone and dichloromethane, and then dried under vacuum to obtain a brown solid 5 (0.14 g, yield 81%).

[0139] The characterization results of compound 5 are as Figure 14 、 15as shown 1 H NMR(400MHz,DMSO-d6)δ8.76 - 8.75(m,2H),8.67(d,J=8.6Hz,4H),8.05(t,J=7.6Hz,2H),7.82(t,J=7.6Hz,2H),7.54(dd,J=7.8,4.6Hz,2H),7.17(d,J=8.2Hz,4H),7.06(d,J=8.6Hz,4H),6.94(d,J=8.2Hz,2H),4.48(s,4H),3.81(t,J=4.6Hz,4H),3.21(s,18H).HRMS:m / z:1 / 2[M - 2Br] 2+ calcd for C 43 H 48 N6O2 2+ :340.1914;found:340.1914。

[0140] Preparation of Compound 6 in Example 6

[0141] 1. Synthesis of Compound 6a

[0142] The structural formula of Compound 6a is as shown in Formula (XVII):

[0143]

[0144] Add 5d(0.40 mmol, 0.20 g), KOH(1.00 mmol, 0.06 g) and tetrabutylammonium bromide(0.04 mmol, 0.01 g) into a 50 mL flask, then slowly add THF(20 mL) and 1,3 - dibromopropane(0.84 mmol, 0.17 g) to the mixture. Heat the mixture to 90 °C and stir for 24 h. After cooling, extract the mixture with EA / water, separate the organic layer, dry it with anhydrous Na2SO4, filter, and use the obtained crude product directly in the next reaction without purification.

[0145] 2. Synthesis of Compound 6

[0146] The structural formula of Compound 6 is as shown in Formula (VI):

[0147]

[0148] 6a (0.20 mmol, 0.15 g) was added to a 50 mL flask, and then acetonitrile (20 mL) and trimethylamine (1.00 mmol, 0.06 g) were slowly added to the reaction flask. The mixture was heated to 70 °C and stirred for 24 h, during which a precipitate formed. Subsequently, the mixture was filtered, and the solid was washed with acetonitrile and dichloromethane. The solid was recrystallized with a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane, and then filtered. The obtained solid was washed with acetone and dichloromethane and dried in a vacuum oven at 50 °C to obtain a brown solid 6 (0.14 g, yield 78%).

[0149] The characterization data results of compound 6 are as Figure 16 , 17 shown. 1 H NMR (400 MHz, DMSO-d6) δ 8.84 - 8.65 (m, 4H), 8.41 - 8.29 (m, 2H), 8.10 - 8.00 (m, 2H), 7.90 - 7.81 (m, 2H), 7.56 (q, J = 6.1 Hz, 2H), 7.13 (q, J = 12.2, 10.4 Hz, 4H), 7.00 (d, J = 8.2 Hz, 4H), 6.90 (t, J = 9.4 Hz, 2H), 4.06 (d, J = 6.2 Hz, 4H), 3.13 (s, 18H), 2.98 (s, 4H), 2.19 (d, J = 13.4 Hz, 4H). HRMS: m / z: 1 / 2[M - 2Br] 2+ calcd for C 45 H 52 N6O2 2+ : 354.2071; found: 354.2071.

[0150] Preparation of Compound 7 in Example 7

[0151] 1. Synthesis of Compound 7a

[0152] The structural formula of compound 7a is shown in formula (XVIII):

[0153]

[0154] 5d (0.40 mmol, 0.20 g), potassium hydroxide (KOH, 1.00 mmol, 0.06 g) and tetrabutylammonium bromide (0.04 mmol, 0.01 g) were added to a 50 mL flask. Subsequently, tetrahydrofuran (THF, 20 mL) and 1,4-dibromobutane (0.84 mmol, 0.18 g) were slowly added to the reaction flask. The reaction mixture was heated to 90 °C and stirred for 24 h. After cooling, the mixture was extracted with ethyl acetate (EA) and water. The organic layer was separated, dried over anhydrous sodium sulfate (Na2SO4), and then filtered. The solution was concentrated by rotary evaporation. The crude product was used directly in the next reaction without purification.

[0155] 2. Synthesis of Compound 7

[0156] The structural formula of Compound 7 is shown in Formula (VII):

[0157]

[0158] 7a (0.20 mmol, 0.16 g) was added to a 50 mL flask. Then, acetonitrile (20 mL) and trimethylamine (1.00 mmol, 0.06 g) were slowly added to the reaction flask. The reaction mixture was heated to 70 °C and stirred for 24 h, during which a precipitate formed. Subsequently, the precipitate was filtered and washed with acetonitrile and dichloromethane. The washed precipitate was recrystallized with a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane, then filtered, washed again with acetone and dichloromethane, and dried in a vacuum oven at 50 °C. Finally, a brown solid 7 (0.13 g, yield 72%) was obtained.

[0159] The characterization data results of Compound 7 are as Figure 18 , 19 shown. 1 H NMR (400 MHz, DMSO-d6) δ 8.80 (t, J = 7.2 Hz, 4H), 8.73 (s, 1H), 8.70 (s, 1H), 8.16 (t, J = 8.0 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.69 - 7.54 (m, 2H), 7.15 (d, J = 8.6 Hz, 4H), 7.00 (d, J = 8.6 Hz, 4H), 6.90 (d, J = 8.4 Hz, 2H), 4.12 - 3.96 (m, 4H), 3.54 (s, 18H), 3.40 (d, J = 9.0 Hz, 4H), 1.90 (d, J = 16.2 Hz, 4H), 1.81 - 1.66 (m, 4H). HRMS: m / z: 1 / 2[M - 2Br] 2+ calcd for C 47 H 56 N6O22+ : 368.2227; found: 368.2230.

[0160] Characterization of the Photophysical Properties of Compounds 1 - 7 in Example 8

[0161] 1. Using water as the solvent, prepare a solution of fluorescent compounds 1 - 7 with a final concentration of 2×10 -5 mol·L -1 . Place the solution in a cuvette and use a Varian CARY 50 UV - Vis spectrometer to detect its UV - absorption spectrum. Figure 20 In (B), it is the UV - Vis absorption spectrum of Compound 2 under the conditions of DMSO / toluene mixed solution with 0% and 99% toluene content respectively. Figure 20 In (A, C - G), it is the UV - Vis absorption spectrum of Compounds 1, 3 - 7 under the conditions of water / THF mixed solution with 0% and 99% THF content respectively.

[0162] 2. Based on their absorption spectra, fluorescent compounds 1 - 7 are excited with light at 468, 436, 468, 435, 435, 380, 380 nm respectively. Figure 21 In (B), it is the fluorescence spectrum of Compound 2 under the conditions of DMSO / toluene mixed solution. Figure 21 In (A, C - G), it is the fluorescence spectrum of Compounds 1, 3 - 7 under the conditions of water / THF mixed solution. Figure 3 It shows that with the addition of the poor solvent THF, the luminescence of the resulting system gradually increases and reaches the maximum value when the THF content is 99, 99, 99, 70, 99, 99, 99 vol% respectively.

[0163] 3. DCFH - DA (2',7'-Dichlorodihydrofluorescein diacetate) is used as a ROS (reactive oxygen species) detection probe to detect the ROS generation of the synthesized fluorescent compounds 1 - 7 in solution (aggregated state). In the experiment, 10 μL of DCFH - DA activation solution (1.0 mM, activated by adding NaOH to the ethanol solution of DCF - DA) is added to 2 mL of the solution of fluorescent compounds 1 - 7 (10 μM) respectively, and white light (0.06 mW / cm 2 ) is used as the light source. Record the absorption values of DCFH - DA at 525 nm at different irradiation time intervals. The results are as shown in Figure 22 A. When exposed to white light, except for Compound 5, the other fluorescent compounds can rapidly and effectively generate a large amount of ROS, and their generation ability is significantly better than that of the commercial photosensitizer Rose Bengal (RB) under the same conditions.

[0164] 4. Singlet oxygen detection (ABDA method of 9,10-anthracene-diyl-bis(methylene)dimalonic acid): ABDA (9,10-anthracene-diyl-bis(methylene)dimalonic acid) is used as a singlet oxygen detection probe. In the experiment, 10 μL of the ABDA stock solution (7.5 mM) was added to 2 mL of the sample suspensions (10 μM) of fluorescent compounds 1-7 respectively, and white light (0.06 mW / cm 2 ) was used as the light source. The absorbance values of ABDA at 378 nm were recorded at different irradiation time periods to obtain its attenuation rate during the photosensitization process. The results are as shown in Figure 22 B. Under white light irradiation, except for compound 5, the ability of the other fluorescent compounds to generate 1 O2 (singlet oxygen) is stronger than that of RB.

[0165] Example 9 Fluorescent imaging effects of compounds 1-5 on bacteria (standard strains, drug-resistant strains)

[0166] (1) Bacterial culture: Bacterial strains: Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 35218), and clinical drug-resistant strains (MRSA, CRE, MDR-AB). All the above strains were provided by Nanfang Hospital, Southern Medical University. Single colonies were isolated from the LB agar plates stored in a 4°C refrigerator, added to fresh LB broth, and shaken at 160 rpm on a shaker at 37°C for 8-10 hours to ensure that the bacteria were in the logarithmic growth phase before the experiment.

[0167] (2) Bacterial imaging: The bacterial suspensions in the logarithmic growth phase were centrifuged, washed 3 times with PBS, the McFarland turbidity was measured, and then diluted to 6×10 8 CFU / mL. The compound (5 μM) was added to 500 μL of the bacterial suspension, and the mixture was cultured at 37°C for 30 min. After centrifugation and concentration, the suspension was placed on a glass slide and covered with a coverslip, and imaged with a laser confocal microscope (Carl Zeiss, LSM 880 with airscan). Excitation wavelength: 405 nm; emission band: 500-700 nm. The results are as shown in Figure 23 . Compounds 1-7 can all light up Staphylococcus aureus, and compounds 1-3 can light up Escherichia coli. According to the detailed enlarged images, compounds 1-2 can penetrate the bacterial membrane and enter the interior to light up the nuclear structure and at the same time light up the membrane structure. At the same time, compound 2 can light up the membrane structure and nuclear structure of clinical drug-resistant bacteria (MRSA, MDR-AB) ( Figure 24 A). The present invention can provide a new dual-target fluorescent detection method for bacterial detection in clinical practice.

[0168] Example 10 Bacteriostatic effects of compounds 1-7 on standard strains

[0169] The minimum inhibitory concentration of the compound against bacteria (Staphylococcus aureus and Escherichia coli) was determined by the microbroth dilution method. The bacterial suspension in the logarithmic growth phase was diluted to 3×10 8 CFU / mL, and then mixed with the compound at different concentrations on a 96-well plate. The group without the compound was used as the control group. Then the mixtures were respectively exposed to white light (0.06W / cm 2 ) or stored in the dark for 20 minutes. After incubation overnight at 37°C, the bacterial growth was monitored to evaluate the antibacterial effect. The results are shown in Table 1. Compounds 1-2 showed extremely low minimum inhibitory concentrations against Staphylococcus aureus and Escherichia coli, and the inhibitory concentrations were even lower after illumination, demonstrating the antibacterial effect of Compounds 1-2 itself and the photodynamic antibacterial pathway.

[0170] Table 1 Minimum inhibitory concentration of the compound (μM)

[0171]

[0172] Example 11 Bactericidal effect of Compound 2 on drug-resistant strains

[0173] The bacterial suspension (MRSA, MDR-AB) in the logarithmic growth phase was centrifuged, washed 2-3 times with PBS, and after measuring the McFarland turbidity, it was diluted to 3×10 8 CFU / mL. Compound 2 (5 μM) was mixed with 500 μL of the bacterial solution and cultured at 37°C for 30 min. The untreated group without the compound was used as the control group, and the illumination group was irradiated with white light at 0.06W / cm 2 for 30 min. After dilution, 20 μL of the bacterial solution was spread on an LB nutrient agar plate. After culturing at 37°C for 18-24 h, the colony growth was observed and counted. The results are shown in Figure 24 B, D-E. After treatment with Compound 2 and illumination, no colonies grew on the plate, indicating that Compound 2 showed a bactericidal effect on the clinical drug-resistant bacteria (MRSA, MDR-AB) of Gram-positive and Gram-negative bacteria, and could provide a new treatment plan for the treatment of clinical bacterial infections.

[0174] Example 12 Inhibitory effect of Compound 2 on the formation of drug-resistant strain biofilms

[0175] The bacterial suspension (MRSA, MDRAB) in the logarithmic growth phase was centrifuged, washed 2-3 times with PBS, and after measuring the McFarland turbidity, it was diluted to 3×10 8 CFU / mL, and incubated with 5 μM Compound 2 at 37°C for 30 minutes. Then the mixture was placed under white light (0.06W / cm 2) Store at room temperature or in the dark for 20 minutes. Subsequently, transfer 100 μL of the suspension onto a 96-well plate and incubate continuously for 3 days, replacing the LB medium every day. After 72 hours of incubation, remove the planktonic bacteria and wash three times with PBS. Stain the biofilm with 0.1% crystal violet solution for 30 minutes, then wash three times with PBS and air dry. Finally, add 200 μL of absolute ethanol to each well to dissolve the crystal violet, and measure the OD value at 560 nm using a microplate reader. The results are as Figure 24 shown in C, F - G in the figure. After treatment with compound 2 and light treatment, the amount of biofilm formation is less than that of the control group, and the effect is more obvious after light treatment. It is proved that compound 2 can significantly inhibit the formation of biofilms by clinical strains.

[0176] Example 13 The effect of compound 2 on the structural damage of drug-resistant strains

[0177] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM): MRSA and MDR-AB bacterial suspensions (3×10 8 CFU / mL) were treated with PBS, compound 2 (5 μM), PBS + white light (0.06 W / cm 2 ) or compound 2 (5 μM) + white light (0.06 W / cm 2 ) respectively and incubated at 37 °C for 30 minutes. Subsequently, centrifuge the bacteria at 4000 rpm for 10 minutes to collect them, and fix them overnight at 4 °C with 2.5% glutaraldehyde, and then fix them with 1% osmium tetroxide for 1 - 2 hours. The samples were dehydrated successively with different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%), and each concentration was maintained for 15 minutes. Then, treat with a 1:1 mixture of ethanol and isopentanol for 30 minutes, and then treat with pure isopentanol for 1 hour. Finally, perform critical point drying and gold plating, and observe the samples using a scanning electron microscope (SEM, HITACHI SU8010). For transmission electron microscopy (TEM) samples, after dehydration with an ethanol gradient, treat with pure acetone for 20 minutes, then treat with a 1:1 (v / v) mixture of embedding agent and acetone for 1 hour, and then treat with a 3:1 (v / v) mixture of embedding agent and acetone for 3 hours. Then, treat with pure embedding agent and heat at 70 °C overnight. Finally, use an ultramicrotome (LEICA EM UC7) to prepare ultra-thin sections of 70 - 90 nm. After staining and drying the sections, observe them using a transmission electron microscope (TEM, HITACHI H-7650). As Figure 25 shown, the SEM images show that the surfaces of MRSA and MDR-AB are ruptured and collapsed, and the TEM images show that the bacterial content and membrane structure are damaged, and the structural degradation is obvious. These observation results indicate that compound 2 induces bacterial death by destroying the internal and membrane structures, further confirming the bactericidal effect of the compound, and can provide a new treatment plan for the treatment of clinical bacterial infections.

[0178] Example 14 Verification of the targeting of Compound 2 to the bacterial membrane and the bacterial nucleus: Co-localization effect of staining with commercial dyes (CellMask / Hoechst 33342) on bacteria and cells

[0179] Separate bacterial system: The MRSA and MDR-AB bacterial suspensions in the logarithmic growth phase were diluted to 3×10 8 CFU / mL with PBS and incubated with CellMask (10 μg / mL) at 37 °C for 30 minutes. Subsequently, the bacteria were collected by centrifugation, washed three times with PBS, and then mixed with Hoechst (10 μg / mL) and Compound 2 (5 μM) for staining for 30 minutes. After staining, the bacteria were collected by centrifugation and resuspended in 10 μL of PBS. 1 μL of the stained bacterial suspension was dropped onto a glass slide and covered with a coverslip. Imaging analysis was performed using a confocal laser scanning microscope. Cell system: 5×10 4 L929 cells were seeded in a 35 mm confocal culture dish and cultured for 24 hours. After washing three times with PBS, the cells were treated with CellMask (10 μg / mL), Hoechst (10 μg / mL), and Compound 2 (5 μM) for 30 minutes. After washing three times with PBS, imaging analysis was performed using a confocal laser scanning microscope. Bacteria-cell co-culture experiment: 5×10 4 L929 cells were seeded in a 35 mm confocal culture dish and cultured for 24 hours. After washing three times with PBS, the cells were treated with 10 μg / mL CellMask and 10 μg / mL Hoechst for 30 minutes each. After washing with PBS, 3×10 8 CFU / mL of MRSA bacterial suspension and Compound 2 (5 μM) were added, and incubation was continued for 30 minutes. Finally, after washing three times with PBS, imaging analysis was performed using a confocal laser scanning microscope (CellMask: excitation wavelength 568 nm, emission wavelength 600 - 700 nm; Hoechst: excitation wavelength 405 nm, emission wavelength 410 - 500 nm; Compound 2: excitation wavelength 405 nm, emission wavelength 550 - 700 nm). As Figure 26 shown in the imaging results in A, the staining distribution of Compound 2 highly coincided with the distributions of the nuclear staining dye Hoechst 33342 (blue) and the membrane staining dye CellMask (red). Co-localization analysis of the confocal images was performed using ImageJ, and a two-dimensional fluorescence intensity histogram was generated and the Pearson correlation coefficient was calculated ( Figure 26In B-C). The histogram shows that after merging the Hoechst / CellMask channel with the compound 2 channel, strong co-localization and a high correlation coefficient were exhibited on both MRSA and MDR-AB. These results indicate that compound 2 is significantly enriched in both the bacterial membrane and the nuclear region, suggesting that its antibacterial mechanism may target both the bacterial membrane and DNA simultaneously. In contrast, extremely low fluorescence intensity was observed in L929 cells ( Figure 26 In A), indicating that compound 2 has a greater tendency to bind to bacteria rather than mammalian cells. To further verify this selectivity, a MRSA / L929 co-culture experiment was conducted in this example. The green fluorescence signal was mainly associated with MRSA, and very little co-localization was observed in the co-culture, further supporting the selective targeting of compound 2 to bacteria. The above results confirm that compound 2 binds to bacteria through dual targeting of the nucleus and membrane, and compared with cells, compound 2 has a greater tendency to bind to bacteria, showing targeting to bacteria.

[0180] Example 15 Verification of the targeting of compound 2 to the bacterial membrane and the bacterial nucleus: Transcriptomic analysis

[0181] MRSA bacterial solution (concentration of 2×10 8 CFU / mL) was treated with PBS, compound 2 (5 μM), PBS + white light (0.06 W / cm 2 ) or compound 2 (5 μM) + white light (0.06 W / cm 2 ) and incubated at 37 °C for 30 minutes. Subsequently, it was centrifuged at 4000 rpm for 10 minutes, the bacterial cells were collected and resuspended in 75% ethanol, and stored at -80 °C overnight. Finally, the frozen bacterial cells were transported to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. under dry ice conditions for sequencing analysis. As Figure 27 shown, Gene Ontology (GO) enrichment analysis indicated that most differentially expressed genes (DEGs) were related to processes associated with membrane transport and DNA / RNA synthesis. Significantly enriched GO terms included "import across the plasma membrane", "carbohydrate transmembrane transport", "carbohydrate transport", "carbohydrate import across the plasma membrane", as well as "DNA-directed 5'-3' RNA polymerase activity", "5'-3' RNA polymerase activity", and "RNA polymerase activity". These results indicate that compound 2 disrupted the bacterial membrane and DNA, affecting key biological processes such as membrane transport and DNA replication / transcription, demonstrating the targeting and disruption of compound 2 to the bacterial membrane and nucleus at the gene level.

[0182] Example 16 Verification of the binding of compound 2 to bacterial DNA: UV absorption determination and fluorescence spectroscopy determination after compound 2 binds to DNA, and inhibition of the antibacterial effect of compound 2 by addition of exogenous DNA

[0183] Extract DNA from MRSA bacterial solution using a bacterial DNA extraction kit (BIOG DNA Fungi&Bacteria Kit) according to the operating procedure in the instruction manual, and measure the DNA concentration using a NanoDrop spectrophotometer. After co-incubating the DNA solution (20 μg / mL) with compound 2 (10 μM) for 30 minutes, measure the ultraviolet-visible absorption spectrum of the mixture. At the same time, dilute the DNA to different concentrations with PBS, co-incubate the DNA solutions at different concentrations with 10 μM compound 2 for 30 minutes, and finally measure the fluorescence intensity of the mixture (excitation wavelength 405 nm, emission wavelength 450 - 800 nm). Dilute the MRSA and MDR-AB bacterial solutions in the logarithmic growth phase with LB medium to 1×10 6 CFU / mL. Mix 5 μM compound 2 with different concentrations of DNA and incubate at 37 °C for 30 minutes. Subsequently, add the bacterial suspension to the mixture to make the final bacterial solution concentration 10 6 CFU / mL, and transfer it to a 96-well plate. After incubating for 24 hours, measure the OD value at 600 nm using a microplate reader.

[0184] As Figure 28 shown in D, the absorption spectrum of the DNA-compound 2 mixture showed a significant enhancement and red shift compared to that of compound 2 alone, indicating the formation of a new complex. The fluorescence spectrum showed that with the increase in DNA concentration, the fluorescence intensity of compound 2 gradually increased ( Figure 28 shown in E), which indicated that the binding to DNA restricted the intramolecular movement of compound 2, thus promoting its luminescence. At the same time, as Figure 28 shown in F - G, DNA competitively reduced the antibacterial activity of compound 2 against MRSA and MDR-AB in a dose-dependent manner. This reduction in antibacterial activity can be attributed to the binding of exogenous DNA to compound 2, reducing the chance of compound 2 interacting with the DNA inside the bacteria. As the concentration of exogenous DNA increases, the amount of compound 2 available for binding to bacterial DNA decreases, thus weakening its antibacterial effect. The above results demonstrated the binding of compound 2 to DNA, forming a new conjugate complex, leading to changes in the photophysical properties of compound 2 and simultaneously weakening its antibacterial effect.

[0185] Example 17 Verification of the binding of compound 2 to bacterial DNA: Compound 2 competes for the binding position of the nuclear dye Hoechst 33342

[0186] Dilute the MRSA and MDR-AB bacterial solutions in the logarithmic growth phase with PBS to 2×10 8CFU / mL and incubated with Hoechst (10 μg / mL) for 30 minutes. Subsequently, different concentrations of Compound 2 were added and staining continued for 30 minutes. After staining was completed, the bacterial suspension was centrifuged and washed three times with PBS, and finally resuspended in 10 μL of PBS. 1 μL of the stained bacterial suspension was dropped onto a glass slide and covered with a coverslip. Imaging observations were performed using a confocal laser scanning microscope (Hoechst: excitation wavelength 405 nm, emission wavelength 410 - 500 nm; Compound 2: excitation wavelength 405 nm, emission wavelength 550 - 700 nm). Hoechst 33342 is a nuclear staining dye known to be able to intercalate into the minor groove of DNA. As Figure 28 shown in A - C, as the concentration of Compound 2 increased, the fluorescence intensity of Hoechst 33342 gradually decreased, while the green fluorescence signal of Compound 2 was significantly enhanced in MRSA and MDR - AB. This dose - dependent fluorescence change indicates that Compound 2 can effectively displace Hoechst 33342 on DNA. The results suggest that Compound 2 binds to DNA through a groove - binding mechanism similar to that of Hoechst 33342.

[0187] Example 18 Verification of Compound 2 Binding to Bacterial DNA: Docking Simulation Calculation of Compound 2 with the Structure of DNA Fragment 4U8A

[0188] To study the binding region and interaction mode of compound 2 with DNA, we used molecular docking to dock compound 2 with (CGCGAATTCGCG CGCGAATTCGCG, SEQ ID NO: 1) based on the X-ray crystal structure (4U8A). First, the initial structure of 4U8A was processed using AutoDock Tools 1.5.6, retaining the original charges of the bases and generating a pdbqt file for docking. Subsequently, the molecular structure of compound 2 was optimized using the MOPAC program, and the PM3 atomic charges were calculated for subsequent molecular docking. The ligand structure was also processed through AutoDock Tools 1.5.6 to generate a pdbqt file for docking. Molecular docking was achieved using the AutoDock 4.2.6 software package. The potential binding site of 4U8A is located in the base cavity region rich in A and T in the double helix structure. The central coordinates of the docking box were set to (17.169, 9.364, -23.817), the number of grid points in the XYZ directions was set to 80×80×80, and the number of docking runs was set to 50 times. The results of molecular docking showed that the binding energy of compound 2 with 4U8A was -7.748 kcal / mol, indicating a strong interaction between the two. Further analysis of the interaction mode revealed that compound 2 formed a hydrogen bond with the A17 base on the B strand of 4U8A. In addition, compound 2 also had hydrophobic interactions with the G2, C3, G4, A5, and A6 bases on the A strand and the G16, A18, T19, and T20 bases on the B strand ( Figure 28 in H-I). These interactions indicate that compound 2 can effectively bind to the circular cavity of the DNA structure, interact with the grooves and bases of DNA, confirming the strong DNA binding ability of compound 2, enabling it to interact with the grooves of bacterial DNA and exert antibacterial effects.

[0189] Example 19 Verification of the Binding of Compound 2 to the Bacterial Membrane Structure: Compound 2 Disrupts the Bacterial Membrane Potential

[0190] The MRSA and MDR-AB bacterial suspensions in the logarithmic growth phase were diluted to 6×10 8 CFU / mL with PBS. After adding 5 μM DiSC3(5), the mixture was incubated at room temperature for ı hour to allow DiSC3(5) to be fully quenched. Subsequently, 100 μL of the mixture was added to a 96-well microplate, and different concentrations of compound 2 were added to each well. The fluorescence intensity of each well was monitored for 30 minutes using a microplate reader (excitation wavelength 620 nm, emission wavelength 670 nm). The results are as Figure 29 shown. The fluorescence intensity increased significantly after treatment with compound 2, indicating that compound 2 disrupted the bacterial membrane potential, leading to membrane collapse and causing bacterial damage.

[0191] Example 20 Detection of antibacterial drug resistance of Compound 2

[0192] After treating Staphylococcus aureus and Acinetobacter baumannii with Compound 2 for 14 consecutive days and subculturing them (the operation is the same as the MIC method determination in Example 10), the minimum inhibitory concentration (MIC) values basically remained unchanged. In contrast, the MIC values of the clinical antibiotics methicillin and imipenem increased by 24 times and 128 times respectively ( Figure 30 ), indicating that these traditional antibiotics have developed drug resistance. This confirms the potential of Compound 2 as an antibacterial agent with multi-target effects and low drug resistance risk.

[0193] Example 21 Biocompatibility detection of Compound 2

[0194] MTT assay for cell viability: NIH3T3 cells and L929 cells were used for in vitro cytotoxicity evaluation by the MTT method. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). Compound 2 was added to a 96-well plate seeded with the two types of cells (4000 cells per well) and co-cultured for 24 hours. Subsequently, MTT solution was added to the 96-well plate and reacted for 2 - 4 hours. After the reaction ended, the culture was terminated, the medium in the wells was carefully aspirated, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken on a low-speed shaker for 5 minutes to fully dissolve the crystals. The absorbance value (OD560nm) of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 560 nm. As Figure 31 The results showed that the effect of Compound 2 on the viability of normal cells was negligible, especially at antibacterial active concentrations. It was demonstrated that Compound 2 had significant biocompatibility, further supporting its potential as a selective antibacterial agent.

[0195] Example 22 Treatment of drug-resistant bacterial peritoneal infection by Compound 2 in vivo and its effect on reducing bacterial load in organs

[0196] A mild MRSA-infected peritonitis model was established by intraperitoneal injection of MRSA bacterial solution (3×10 8 CFU / mL). Three hours after infection, the mice were randomly divided into four groups, and each group received the following treatment by intraperitoneal injection (5 mice per group): (1) PBS (100 μL); (2) vancomycin (0.5 mM, 100 μL); (3) Compound 2 (0.5 mM, 100 μL); (4) Compound 2 + light (0.5 mM, 0.06 W / cm 2, 100 μL, with abdominal illumination for 15 minutes). The above treatment was performed once every 24 hours for 5 consecutive times. During the entire treatment period, the body weight and survival status of the mice were monitored daily. After 5 days, the mice were sacrificed and blood and major organs were collected for blood white blood cell count analysis and hematoxylin-eosin (H&E) staining. In addition, to further explore the therapeutic effect of Compound 2 in severe infections, a severe MRSA-infected peritonitis model was established. Similarly, 3 hours after intraperitoneal injection of MRSA bacterial suspension (3×10 9 CFU / mL), the same treatment was performed according to the aforementioned method, once every 24 hours for 6 consecutive times. After 6 days, the mice were sacrificed and major organs (including heart, liver, spleen, lung, kidney) were collected, and they were homogenized to release residual bacteria. The bacterial suspension was diluted and spread on LB agar plates. After incubation at 37 °C for 24 hours, the colony-forming units (CFUs) were quantified by counting the number of colonies. As shown in Figure 32 B, compared with the normal reference range (0.8 - 6.8×10 9 / L), the WBC levels in the "PBS" group and the "vancomycin" group were significantly increased, indicating the presence of persistent inflammation and infection. In contrast, the WBC levels in the "Compound 2" group and the "Compound 2 + illumination" group remained within the normal range, indicating that Compound 2 has effective antibacterial and anti-inflammatory properties. In addition, the red blood cell (RBC) hemolysis experiment showed that even at high concentrations, the hemolytic activity of Compound 2 was negligible, indicating its excellent biosafety ( Figure 32 A). As shown in Figure 33 A, two days after severe infection, all the mice in the PBS group died, while the survival rates of the "Compound 2" group and the "Compound 2 + illumination" group were significantly higher than those of the vancomycin group. After the treatment was completed, the major organs were homogenized and the bacterial colonies were counted. The results showed ( Figure 33 B - F) that the bacterial load in the organs of the "PBS" group was high, and the bacterial loads in the "Compound 2" group and the "Compound 2 + illumination" group were significantly lower than those in the "PBS" group and the "vancomycin" group, confirming the antibacterial effect of Compound 2.

Claims

1. A class of terpyridine compounds, characterized in that: The structural formula of the said compound is shown as formula (Ⅷ): Wherein, X is C or N; R1 is R2Y, Y does not exist or is selected from halogen anions; R2 is selected from an alkyl group, and one of the following.

2. The terpyridine compound according to claim 1, characterized in that: The R2 is selected from one of methyl, ethyl, and the like; The halogen anion is selected from I - , Br - and one of them.

3. The terpyridine compound according to claim 1, characterized in that: When X is N, the structural formula of the terpyridine compound is shown as any one of formulas (I) to (IV): When X is C, the structural formula of the said terpyridine compound is shown as any one of formulas (Ⅴ) - (Ⅶ):

4. The preparation method of the terpyridine compound according to any one of claims 1 - 3, characterized in that: React compound 1d with reaction raw material 1, or react compound 5d with reaction raw material 2 to obtain the compound with the structure shown in formula (Ⅷ); The structural formula of the said skeleton compound 1d is shown as formula (Ⅺ): The structural formula of the said skeleton compound 5d is shown as formula (ⅩⅤ): The said reaction raw material 1 includes at least one of alkyl halides, quaternary ammonium halides, and sulfonating agents; The said reaction raw material 2 includes alkyl halides and tertiary amines.

5. The preparation method according to claim 4, characterized in that: In reaction raw material 1, the said alkyl halide is selected from one of methyl iodide and propyl iodide; The said quaternary ammonium halide is (3 - bromopropyl) trimethylammonium bromide; The said sulfonating agent is 1,3 - propane sultone.

6. The preparation method of the preparation method according to claim 4, characterized in that: In reaction raw material 2, the said alkyl halide is selected from one of 1,2 - dibromoethane, 1,3 - dibromopropane, and 1,4 - dibromobutane; The said tertiary amine is trimethylamine.

7. The application of the terpyridine compound according to any one of claims 1 - 3 in the preparation of products; The said products include at least one of fluorescent dyes, photosensitizers, bacterial dyes, and antibacterial agents.

8. A product, characterized in that: It includes the terpyridine compound according to any one of claims 1 - 3; The said product includes at least one of fluorescent dyes, photosensitizers, bacterial dyes, and antibacterial agents.

9. The product according to claim 8, characterized in that: When the said product is a fluorescent dye, the said fluorescent dye includes the terpyridine compound according to any one of claims 1 - 3; or When the said product is a photosensitizer, the said photosensitizer includes one of the terpyridine compounds with the structural formulas shown as formulas (Ⅰ) - (Ⅳ), (Ⅵ), and (Ⅶ) according to claim 3; or When the said product is a bacterial dye, the said bacterial dye includes the terpyridine compound according to any one of claims 1 - 3; or When the said product is an antibacterial agent, the said antibacterial agent includes one of the terpyridine compounds with the structural formulas shown as formulas (Ⅰ) and (Ⅱ) according to claim 3.

10. The product according to claim 9, characterized in that: The said product also includes pharmaceutically acceptable excipients.

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