Fluorescent compound with aggregation-induced emission property and preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a kind of fluorescent compound with aggregation-induced emission characteristics and a preparation method and application thereof.A new kind of terpyridine fluorescent compound with aggregation-induced emission characteristics is synthesized, which has the characteristics of easy preparation, high fluorescence intensity, low fluorescence background, high biological safety and the like.The compound prepared by the application can be used as a fluorescent dye and photosensitizer for bacterial membrane and DNA double targeting, and has the advantages of good light stability, good biocompatibility, short time consumption and simple operation in dyeing imaging effect, and can be used for bacterial imaging, distinguishing gram-positive bacteria and gram-negative bacteria.The compound of the application can also be used for antibacterial treatment of drug-resistant bacteria, mainly by targeted binding to destroy the structure of bacterial membrane and DNA, to achieve high-efficiency bacterial killing effect, and has broad spectrum.

Description

TECHNICAL FIELD

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

[0002] Many serious diseases caused by bacterial infection have attracted the attention of the medical profession and the public worldwide. Antibiotic misuse and abuse have led to the emergence of drug-resistant bacteria. Therefore, solving the problem of drug-resistant bacteria is a great challenge facing the world today. At present, the single target mechanism of traditional antibiotics is easy to lead to bacterial drug resistance, so it is of great significance to develop new antibacterial drugs with multiple target mechanisms.

[0003] Photodynamic therapy (PDT) is a relatively novel non-invasive treatment method, which relies on a specific wavelength light source to activate photosensitizers to produce reactive oxygen species (ROS). The high activity of 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 aggregation state fluorescence quenching, 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 of luminescence and ROS generation in the aggregated state. However, existing AIE photosensitizers mostly only target bacterial membranes or DNA, making it difficult to achieve dual-targeting antibacterial effects. Therefore, developing an AIE photosensitizer that can simultaneously target bacterial membranes and DNA has important clinical significance. SUMMARY

[0005] The first aspect of the present application aims to provide a class of terpyridine compounds.

[0006] The second aspect of the present application aims to provide a preparation method of the terpyridine compound of the first aspect of the present application.

[0007] The third aspect of the present application aims to provide an application of the terpyridine compound of the first aspect of the present application.

[0008] The fourth aspect of the present application aims to provide a product.

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

[0010] The first aspect of the present application provides a class of terpyridine compounds, the structural formula of which is shown in formula (VIII):

[0011]

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

[0013] In some embodiments of the present application, R1 is R2Y, Y is absent or selected from halide anions.

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

[0015] In some embodiments of the present application, R2 is selected from one of alkyl, .

[0016] In some embodiments of the present application, the alkyl is selected from one of methyl, ethyl.

[0017] In some embodiments of the present application, when X is N, the structure of the terpyridine compound is shown in any one of formula (I)-(IV):

[0018]

[0019] In some embodiments of the present application, when X is C, the structure of the terpyridine compound is shown in any one of formula (V)-(VII):

[0020]

[0021] In a second aspect of the present application, a method for preparing the terpyridine compound of the first aspect of the present application is provided.

[0022] In some embodiments of the present application, compound 1d is reacted with reaction material 1, or compound 5d is reacted with reaction material 2, to obtain a compound with the structure shown in formula (VIII):

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

[0024]

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

[0026]

[0027] In some embodiments of the present application, the reaction material 1 comprises at least one of halogenated alkane, quaternary ammonium halide, sulfonating agent.

[0028] In some embodiments of the present application, the reaction material 2 comprises halogenated alkane and tertiary amine.

[0029] In some embodiments of the present application, in the reaction raw material 1, the halogenated alkane is selected from one of methyl iodide, propyl iodide; the quaternary ammonium halide is (3-bromopropyl) trimethylammonium bromide; and the sulfonating agent is 1,3-propane sulfone lactone.

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

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

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

[0033] In a third aspect of the present application, the terpyridine compound of the first aspect of the present application is used in the preparation of a product.

[0034] In some embodiments of the present application, the product comprises at least one of a fluorescent dye, a photosensitizer, a bacterial dye, and an antibacterial agent.

[0035] In a fourth aspect of the present application, a product comprises the terpyridine compound of the first aspect of the present application.

[0036] In some embodiments of the present application, the product comprises at least one of a fluorescent dye, a photosensitizer, a bacterial dye, and an antibacterial agent.

[0037] In some embodiments of the present application, when the product is a fluorescent dye, the fluorescent dye comprises the terpyridine compound of the first aspect of the present application.

[0038] In some embodiments of the present application, when the product is a photosensitizer, the photosensitizer comprises one of the terpyridine compounds of structural formula (1)-(4), (6), (7).

[0039] In some embodiments of the present application, when the product is a bacterial dye, the bacterial dye comprises the terpyridine compound of the first aspect of the present application.

[0040] In some embodiments of the present application, when the product is an antibacterial agent, the antibacterial agent comprises one of the terpyridine compounds of structural formula (1), (2).

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

[0042] Compound 3 is a bacterial membrane-targeting binding fluorescent dye, photosensitizer, which can be used for imaging and antibacterial treatment of bacteria.

[0043] Compounds 4-7 are gram-positive bacterial membrane-binding dyes, which can be used to distinguish gram-positive bacteria from gram-negative bacteria.

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

[0045] In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retardant, a carrier.

[0046] The above pharmaceutically acceptable excipients are generally recognized for this purpose and are non-active ingredients of a medicament. A compilation of pharmaceutically acceptable excipients can be found in Handbook of Pharmaceutical Excipients, 2ndEdition, Edited by A. Wade and P. J. Weller; Published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; and in other tool books such as Chinese Pharmacopoeia-Pharmaceutical Excipients Name List.

[0047] The present application further provides the use of compounds 1 and 2 as active ingredients for the preparation of a medicament for the treatment of bacterial infection.

[0048] In some embodiments of the present application, the dosage form of the medicament comprises a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

[0049] In some embodiments of the present application, the gastrointestinal administration dosage form comprises at least one of a powder, a tablet, a granule, a capsule, a sustained-release agent, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet.

[0050] In some embodiments of the present application, the non-gastrointestinal administration dosage form comprises at least one of an injection administration dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucosa administration dosage form, a cavity administration dosage form.

[0051] The carriers used in the preparation of oral formulations can be conventional pharmaceutical excipients such as starch, dextrin, cyclodextrin, various chemically modified cyclodextrins, sucrose, and stearates. The subsequent preparation processes and equipment for each formulation are all conventional technologies in the pharmaceutical field, and this invention does not limit them.

[0052] The beneficial effects of this invention are:

[0053] This invention synthesizes a novel class of terpyridine fluorescent compounds with aggregation-induced emission properties. The molecular design is based on the TPA-TPy (triphenylamine-terpyridine) backbone, which possesses a donor-acceptor (DA) structure, enabling long-wavelength emission and efficient ROS generation. Introducing positive charges (such as pyridine groups, quaternary ammonium salts, etc.) at the molecule's ends significantly enhances the electrostatic interaction between the molecule and the negatively charged bacterial membrane, improving the molecule's targeting and binding ability to bacteria. Adjusting the molecule's hydrophilicity-hydrophobicity balance (e.g., replacing the pyridine ring with an oxygen atom) further optimizes the interaction between the molecule and the bacterial membrane, enhancing the molecule's insertion ability into the bacterial membrane, thereby improving the antibacterial effect. Systematically adjusting the length of the alkyl chain optimizes the binding affinity of the molecule to the bacterial membrane and DNA; longer alkyl chains... The alkyl chain enhances hydrophobic interactions, facilitating molecular insertion into bacterial membranes, while the shorter chain is better suited for binding to bacterial DNA. This design not only considers the antibacterial activity of the molecule but also incorporates AIE (aggregation-induced emission) properties, enabling the molecule to exhibit efficient fluorescence emission and ROS generation in the aggregated state, making it suitable for bacterial imaging and photodynamic therapy (PDT). It can be optimized for different types of bacteria (such as Gram-positive and Gram-negative bacteria), exhibiting broad-spectrum antibacterial potential, especially showing significant antibacterial effects against multidrug-resistant bacteria (such as MRSA, MDRAB, etc.). In addition, it significantly reduces the possibility of bacteria developing drug resistance, providing a new solution for combating multidrug-resistant bacterial infections. Attached Figure Description

[0054] Figure 1 , 2 The proton nuclear magnetic resonance (NMR) values ​​of compound 1d are shown below. 1 ¹H NMR spectrum (400MHz, DMSO-d6), carbon-13 NMR (… 13 C NMR spectrum (400MHz, DMSO-d6).

[0055] Figure 3 , 4 The proton nuclear magnetic resonance (NMR) values ​​of compound 1 are shown below. 1 1H NMR spectrum (DMSO-d6), high-resolution mass spectrometry (HRMS)

[0056] Figure 5 ,6 7 and 8 are the proton nuclear magnetic resonance (NMR) values ​​of compound 2, respectively. 1 ¹H NMR spectrum (400MHz, DMSO-d6), carbon-13 NMR (… 13 C10 NMR spectrum (400 MHz, DMSO-d6), high-resolution mass spectrometry (HRMS), Figure 7 The lower image is a magnified view of the area within the box in the upper image.

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

[0058] Figure 11 , 12 13 and 13 are the proton nuclear magnetic resonance (NMR) values ​​of compound 4, respectively. 1 1H NMR spectrum (DMSO-d6), carbon-13 NMR ( 13 10⁻⁶ C NMR spectrum (DMSO-d6), high-resolution mass spectrometry (HRMS), Figure 13 The lower image is a magnified view of the area within the box in the upper image.

[0059] Figure 14 , 15 The proton nuclear magnetic resonance (NMR) values ​​of compound 5 are shown below. 1 1H NMR spectrum, high-resolution mass spectrometry Figure 15 The lower image is a magnified view of the area within the box in the upper image.

[0060] Figure 16 , 17 The proton nuclear magnetic resonance (NMR) of compound 6 are respectively 1 1H NMR spectrum, high-resolution mass spectrometry Figure 17 The lower image is a magnified view of the area within the box in the upper image.

[0061] Figure 18 , 19 The proton nuclear magnetic resonance (NMR) of compound 7 are respectively 1 1H NMR spectrum, high-resolution mass spectrometry Figure 19 The lower image is a magnified view of the area within the box in the upper image.

[0062] Figure 20(B) is the UV-Vis absorption spectrum of compound 2 in DMSO / toluene mixed solution, (A, C-G) is the UV-Vis absorption spectrum of compounds 1, 3-7 in water / THF mixed solution, respectively, in the presence of 0% and 99% THF.

[0063] Figure 21 (B) is the fluorescence spectrum of compound 2 in DMSO / toluene mixed solution; (A, C-G) is the fluorescence spectrum of compounds 1, 3-7 in water / THF mixed solution.

[0064] Figure 22 (A) is the compound ROS generation ability; (B) is the compound singlet oxygen generation ability.

[0065] Figure 23 The laser confocal imaging effect of compounds 1-7 on standard strains of Staphylococcus aureus and Escherichia coli.

[0066] Figure 24 The laser confocal imaging effect of compound 2 on clinical drug-resistant strains (MRSA, MDR-AB) (A), the effects of plate antibiosis (B, D, E), and the inhibition of biofilm formation (C, F, G).

[0067] Figure 25 The surface morphology change (scanning electron microscope) and internal structure change (transmission electron microscope) of MRSA and MDR-AB after treatment with compound 2.

[0068] Figure 26 The colocalization effect after compound 2 was used to co-stain 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 The results of transcriptome analysis after MRSA was treated with compound 2 (A is the volcano plot of differential genes, and B is the GO enrichment analysis).

[0070] Figure 28 Verification of the binding of compound 2 to bacterial DNA: A-C is the replacement of commercial dye hoechst 33342 by compound 2, D is the change in UV absorption spectrum after compound 2 was mixed with exogenous DNA, E is the change in fluorescence spectrum after compound 2 was mixed with different concentrations of exogenous DNA, F-G is that the addition of exogenous DNA inhibits the antibacterial effect of compound 2 on MRSA and MDR-AB, H-I is the molecular docking simulation result of compound 2 and DNA molecule fragment 4U8A.

[0071] Figure 29 Changes of membrane potential of MRSA and MDR-AB after treatment with compound 2 (indicated by fluorescence changes of Disc3(5)).

[0072] Figure 30 Drug resistance monitoring of compound 2 (changes of minimum inhibitory concentration after two weeks of continuous treatment of standard strains of Staphylococcus aureus and Acinetobacter baumannii).

[0073] Figure 31 Biocompatibility evaluation of compound 2, using MTT method to detect cell activity after treatment of cells 3T3 and L929 with compound 2.

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

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

[0076] Figure 34 Reaction synthesis route map of each substance of the present application. DETAILED DESCRIPTION

[0077] The concept and technical effects of the present application will be described below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0078] The synthesis reaction route of each compound of the present application is shown in Figure 34 .

[0079] Preparation of compound 1 of example 1

[0080] 1. Synthesis of compound 1b

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

[0082]

[0083] The synthesis of 1b was similar to the previous report [DOI: 10.1039 / C4PY01793E]. Phosphorus oxychloride (POCI3, 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 min. Then, 1a (2.59 g, 8.00 mmol) was added and the mixture was heated to 90 °C and the progress of the reaction was monitored by thin layer chromatography (TLC) until the complete consumption of 1a. After the completion of the reaction, the mixture was cooled to room temperature, 50 mL of ice water was added followed by neutralization with saturated sodium bicarbonate (NaHCO3) solution. The mixture was extracted with dichloromethane (DCM) and washed with saturated brine and 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] Nuclear magnetic resonance (NMR) spectra were consistent with previously reported data.1H NMR (600 MHz, CDC13) 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 structure of compound 1c is shown in Formula (X):

[0087]

[0088] Compound 1c was prepared by 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 vacuumed for 30 min again. Then, the mixture was placed in a mixed solvent of tetrahydrofuran (THF, 20 mL) and water (4 mL) and refluxed at 80 °C under 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 under reduced pressure by rotary evaporation. 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 structure of compound 1d is shown in formula (IX):

[0091]

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

[0093] The results of the characterization of compound 1d are shown in Figure 1 , 2 . 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 structure of compound 1 is shown in formula (I):

[0096]

[0097] Dissolve 1d (0.20 mmol, 0.13 g) in tetrahydrofuran, then slowly add iodomethane (2.00 mmol, 0.28 g). Stir the mixture at room temperature for 24 hours to obtain a large amount of orange solid. Filter the solid and wash with tetrahydrofuran and dichloromethane. Recrystallize the filtered solid with dimethyl sulfoxide / acetone / dichloromethane mixed solvent. Filter the obtained precipitate, wash with acetone and dichloromethane, and then dry under vacuum to obtain red solid 1 (0.13 g, yield 70%).

[0098] The characterization results of compound 1 are shown in Table 1. Figure 3 、 4 The characterization results of compound 1 are shown in Table 1.

[0099] Preparation of compound 2 of Example 2

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

[0101]

[0102] Dissolve 1d (0.20 mmol, 0.13 g) in tetrahydrofuran (THF), then slowly add 1-iodopropane (2.00 mmol, 0.39 g). Then, stir the mixture at room temperature for 24 hours to generate a large amount of red solid. Filter the solid and wash with tetrahydrofuran and dichloromethane (DCM). Recrystallize the crude product with dimethyl sulfoxide (DMSO) / acetone / dichloromethane mixed solvent. Filter the obtained precipitate, wash with acetone and dichloromethane, and dry under vacuum to obtain red solid 2 (0.16 g, yield 80%).

[0103] The characterization results of compound 2 are shown in Table 2. Figure 5 、 6 1 ​H 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 C 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 of example 3

[0105] The structural formula of compound 3 is shown as formula (III):

[0106]

[0107] Synthesis of compound 3: Id (0.20 mmol, 0.13 g) was dissolved in acetonitrile (2 mL), then (3-bromopropyl)trimethylammonium bromide (0.40 mmol, 0.10 g) was added slowly. Next, the mixture was stirred under reflux for 24 hours, generating a large amount of red solid. The solid was filtered and washed with acetonitrile and dichloromethane. The obtained solid was recrystallized with dimethyl sulfoxide / acetone / dichloromethane mixed solvent. The obtained precipitate was filtered, washed with acetone and dichloromethane, and then dried under vacuum to obtain red solid 3 (0.15 g, yield 68%).

[0108] The characterization results of compound 3 are shown in Figure 8 、 9 , 10. 1H 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 C 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 of example 4

[0110] The structural formula of compound 4 is shown as formula (IV):

[0111]

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

[0113] The characterization results of compound 4 are shown in Table Figure 11 、 12 , 13. 1H 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 C 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 of example 5

[0115] 1. Synthesis of compound 5a

[0116] The structural formula of compound 5a is shown as formula (X):

[0117]

[0118] Compound 5a was synthesized according to previous reports [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. Then, the mixture was 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 with anhydrous sodium sulfate (Na2SO4), and then filtered. The filtrate was concentrated by rotary evaporation under reduced pressure, and the target product was purified by silica gel column chromatography to obtain white solid 5a (yield 50%). 1 H NMR (600 MHz, CDC13) δ 7.16 (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 as formula (XIII):

[0121]

[0122] Compound 5b was synthesized according to previous reports [DOI: 10.1021 / acsaem.8b02100]. At 0 °C, phosphorus oxychloride (POCI3, 0.84 mL, 9.00 mmol) was added dropwise to dry N,N-dimethylformamide (DMF, 0.83 mL, 10.80 mmol), and then the mixture was stirred at room temperature for 20 minutes. Next, 5a (2.44 g, 8.00 mmol) was added, and the mixture was heated to 90 °C, and 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 sodium bicarbonate aqueous solution (NaHCO3(aq)). The mixture was extracted with dichloromethane (DCM) / water (3 x 50 mL). The organic layer was separated, dried with anhydrous sodium sulfate (Na2SO4), and then filtered. The filtrate was concentrated by rotary evaporation under reduced pressure, and the target product was purified by silica gel column chromatography to obtain yellow solid 5b (yield 70%). 1H NMR (400 MHz, CDC13) 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).

[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). A potassium hydroxide solution (containing 0.22 g, 4.00 mmol of potassium hydroxide) dissolved in 10 mL of water was slowly added to the above mixture under magnetic stirring at room temperature. After 30 minutes, 25 mL of an ammonia solution was added, and then the mixture was heated to 65°C. After 3 hours of reaction, a yellow precipitate was generated, and then the precipitate was filtered. The precipitate was washed with ethanol several times and recrystallized, and finally yellow crystalline compound 5c was obtained with a yield of 85%. 1 H NMR (400 MHz, CDC13) 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] 5c (2.68 g, 5.00 mmol) was dissolved in dichloromethane (DCM) at -20 °C, followed by the slow addition of boron tribromide (BBr3, 16 mL). The mixture was then stirred at -20 °C for 1 hour, followed by warming to room temperature and stirring overnight. After the reaction was complete, 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 diethyl ether to precipitate a green solid 5d (2.46 g, yield: 97%). The molecular weight of the solid was determined by gas chromatography-mass spectrometry (GC-MS), 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] 5 d (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. Next, 20 mL of tetrahydrofuran (THF) was added to the mixture, and 1,2-dibromoethane (0.84 mmol, 0.16 g) was slowly added dropwise. 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. Then, 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, which was filtered. The solid was washed with acetonitrile and dichloromethane. The solid was recrystallized from the solid using a mixed solvent of dimethyl sulfoxide / acetone / dichloromethane. The product was washed with acetone and dichloromethane and then dried under vacuum to give brown solid 5 (0.14 g, 81% yield).

[0139] The characterization results of compound 5 are as follows: Figure 14 , 15shown. 1 H NMR (400 MHz, DMSO-d6) δ 8.76-8.75 (m, 2H), 8.67 (d, J = 8.6 Hz, 4H), 8.05 (t, J = 7.6 Hz, 2H), 7.82 (t, J = 7.6 Hz, 2H), 7.54 (dd, J = 7.8, 4.6 Hz, 2H), 7.17 (d, J = 8.2 Hz, 4H), 7.06 (d, J = 8.6 Hz, 4H), 6.94 (d, J = 8.2 Hz, 2H), 4.48 (s, 4H), 3.81 (t, J = 4.6 Hz, 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 of Example 6

[0141] 1. Synthesis of compound 6a

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

[0143]

[0144] 5d (0.40 mmol, 0.20 g), KOH (1.00 mmol, 0.06 g) and tetra-n-butylammonium bromide (0.04 mmol, 0.01 g) were added into a 50 mL flask, then THF (20 mL) and 1,3-dibromopropane (0.84 mmol, 0.17 g) were slowly added into the mixture, the mixture was heated to 90°C and stirred for 24 h, after cooling, the mixture was extracted with EA / water, the organic layer was separated, dried over anhydrous Na2SO4, filtered, and the obtained crude product was directly used in the next reaction without purification.

[0145] 2. Synthesis of compound 6

[0146] The structural formula of compound 6 is shown as formula (VI):

[0147]

[0148] To a 50 mL flask was added 6a (0.20 mmol, 0.15 g), followed by the slow addition of acetonitrile (20 mL) and trimethylamine (1.00 mmol, 0.06 g) to the reaction flask. The mixture was heated to 70 °C and stirred for 24 h, during which time a precipitate formed. The mixture was then filtered and the solid washed with acetonitrile and dichloromethane. The solid was recrystallized from dimethylsulfoxide / acetone / dichloromethane and filtered. The resulting solid was washed with acetone and dichloromethane and dried in a vacuum oven at 50 °C to yield brown solid 6 (0.14 g, 78% yield).

[0149] The results of the characterization data for compound 6 are shown in Figure 16 、 17 . 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 of example 7

[0151] 1. Synthesis of compound 7a

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

[0153]

[0154] Into a 50 mL flask was placed 5d (0.40 mmol, 0.20 g), potassium hydroxide (KOH, 1.00 mmol, 0.06 g), and tetra-n-butylammonium bromide (0.04 mmol, 0.01 g). To the reaction flask was then added tetrahydrofuran (THF, 20 mL) and 1,4-dibromobutane (0.84 mmol, 0.18 g) slowly. 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 (Na2S04), and then filtered. The solution was concentrated by rotary evaporation. The crude product was used directly for the next reaction without purification.

[0155] 2. Synthesis of compound 7

[0156] The structure of compound 7 is shown in formula (VII):

[0157]

[0158] Into a 50 mL flask was placed 5d (0.40 mmol, 0.20 g), potassium hydroxide (KOH, 1.00 mmol, 0.06 g), and tetra-n-butylammonium bromide (0.04 mmol, 0.01 g). To the reaction flask was then added tetrahydrofuran (THF, 20 mL) and 1,4-dibromobutane (0.84 mmol, 0.18 g) slowly. 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 (Na2S04), and then filtered. The solution was concentrated by rotary evaporation. The crude product was used directly for the next reaction without purification.

[0159] The characterization data results of compound 7 are shown in Table 1. Figure 18 , 19 Table 1 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] Photophysical property characterization of compounds 1-7

[0161] 1. Prepare 2 x 10 -5 mol L -1 solution of fluorescent compounds 1-7 in water as solvent, and detect their UV- absorption spectra in a cuvette using Varian CARY 50 UV-Vis spectrometer. Figure 20 (A, C-G) are the UV-Vis absorption spectra of compounds 1, 3-7 in water / THF mixed solution with the THF content of 0% and 99% respectively. Figure 20 (A, C-G) are the UV-Vis absorption spectra of compounds 1, 3-7 in water / THF mixed solution with the THF content of 0% and 99% respectively.

[0162] 2. Based on their absorption spectra, fluorescent compounds 1-7 are excited by 468, 436, 468, 435, 435, 380, 380 nm light respectively. Figure 21 (A, C-G) are the fluorescence spectra of compounds 1, 3-7 in water / THF mixed solution. Figure 21 (A, C-G) are the fluorescence spectra of compounds 1, 3-7 in water / THF mixed solution. Figure 3 It is shown that the luminescence of the resulting system gradually increases with the addition of a poor solvent THF, and reaches a maximum 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 active oxygen detection probe to detect the ROS production of fluorescent compounds 1-7 in solution (aggregated state) respectively. In the experiment, 10 μL of DCFH-DA activation solution (1.0 mM, activated by NaOH in DCF-DA ethanol solution) is added to 2 mL of fluorescent compound 1-7 (10 μM) solution respectively, and white light (0.06 mW / cm 2 ) is used as the light source. The absorption value of DCFH-DA at 525 nm is recorded at different irradiation time periods. The results are shown in Figure 22 A, when exposed to white light, the remaining fluorescent compounds can quickly and effectively produce a large amount of ROS, and its production ability is significantly better than the commercial photosensitizer Rose Bengal (RB) under the same conditions.

[0164] 4. Singlet oxygen detection (dimethylglycolic anthracene-ABDA method): ABDA (9,10-anthracene-diyl-bis(methylene)dimalonic acid) was used as a singlet oxygen detection probe. In the experiment, 10 μL of ABDA stock solution (7.5 mM) was added to 2 mL of fluorescent compound 1-7 sample suspension (10 μM) respectively, and white light (0.06 mW / cm 2 ) was used as the light source. The absorption value of ABDA at 378 nm was recorded at different irradiation time periods to obtain the decay rate of photosensitization process. The results are shown in Figure 22 B, under white light irradiation, the remaining fluorescent compounds have stronger ability to generate 1 O2 (singlet oxygen) than RB.

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

[0166] (1) Bacterial culture: bacterial strains: Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 35218) and clinical drug-resistant strains (MRSA, CRE, MDR-AB). The above strains were provided by Nanfang Hospital of Southern Medical University. Single colonies were isolated from LB agar plates stored in a 4°C refrigerator, added to fresh LB broth, and shaken at 160 rpm 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 logarithmic growth phase bacterial suspension was centrifuged, washed with PBS 3 times, the McFarland turbidity was measured, and then diluted to 6 x 10 8 CFU / mL. The compound (5 μM) was added to 500 μL of bacterial suspension, and mixed and cultured at 37°C for 30 min. After centrifugal concentration, the suspension was placed on a glass slide and covered with a cover glass, 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 shown in Figure 23 , compounds 1-7 can light up Staphylococcus aureus, and compounds 1-3 can light up Escherichia coli. According to the detail magnification, compounds 1-2 can penetrate the bacterial membrane to light up the internal nuclear structure, while lighting up the membrane structure. At the same time, compound 2 can light up the membrane structure and nuclear structure of the clinical drug-resistant bacteria (MRSA, MDR-AB) (A in Figure 24 ). The present application can provide a new bacterial double-targeted fluorescent detection method for clinical bacterial detection.

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

[0169] The minimum inhibitory concentration (MIC) of the compound against bacteria (Staphylococcus aureus and Escherichia coli) was determined using the micro-broth dilution method. The bacterial suspension in the logarithmic growth phase was diluted to 3 × 10⁻⁶. 8 CFU / mL was then mixed with different concentrations of the compound in a 96-well plate. The group without the compound served as a control. The mixture was then exposed to white light (0.06 W / cm²). 2 Alternatively, store in the dark for 20 minutes. After incubation at 37°C overnight, monitor bacterial growth to assess the antibacterial effect. The results are shown in Table 1. Compounds 1-2 exhibited extremely low minimum inhibitory concentrations (MICs) against Staphylococcus aureus and Escherichia coli, with even lower MICs after light exposure, demonstrating the antibacterial effect of compounds 1-2 themselves and the photodynamic antibacterial pathway.

[0170] Table 1. Minimum inhibitory concentration (μM) of compounds

[0171]

[0172] Example 11: Killing effect of compound 2 on drug-resistant strains

[0173] Centrifuge the suspension of bacteria (MRSA, MDR-AB) in the logarithmic growth phase, wash 2-3 times with PBS, measure McFarland turbidity, and then dilute to 3×10⁻⁶. 8 CFU / mL, compound 2 (5 μM) was mixed with 500 μL of bacterial culture and incubated at 37°C for 30 min. The control group (without compound treatment) and the light group (at 0.06 W / cm²) were treated with the compound. 2 Irradiate the bacterial culture under white light for 30 min. Dilute the culture and spread 20 μL onto LB nutrient agar plates. Incubate at 37℃ for 18-24 h, then observe and count the colony growth. Results are as follows: Figure 24 As shown in Figures B and D, after treatment with compound 2 and light, no colonies grew on the plates, indicating that compound 2 exhibits a killing effect on both clinically resistant Gram-positive and Gram-negative bacteria (MRSA, MDR-AB), which can provide a new treatment option for clinical bacterial infection treatment.

[0174] Example 12: Inhibitory effect of compound 2 on biofilm formation of drug-resistant strains

[0175] Logarithmic growth phase bacterial suspension (MRSA, MDRAB) centrifuged, washed 2-3 times with PBS, and diluted to 3×10⁻⁶ after measuring McFarland turbidity. 8 CFU / mL, incubated with 5 μM compound 2 at 37°C for 30 minutes. The mixture was then placed under white light (0.06 W / cm²). 2) or in the dark for 20 min. Subsequently, 100 μL of the suspension was transferred to a 96-well plate and incubated continuously for 3 days, with LB medium being replaced every day. After 72 h of incubation, planktonic bacteria were removed and washed with PBS three times. The biofilm was stained with 0.1% crystal violet solution for 30 min, then washed with PBS three times and air-dried. Finally, 200 μL of absolute ethanol was added to each well to dissolve the crystal violet, and the OD value at 560 nm was determined using a microplate reader. The results are shown in Figure 24 As shown in Figs. C, F-G, the amount of biofilm formation after treatment with compound 2 and light treatment was less than that of the control group, and the effect after light treatment was more obvious. It is proved that compound 2 can significantly inhibit the formation of biofilm of clinical strains.

[0176] Example 13 Effect of compound 2 on structural damage to drug-resistant strains

[0177] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM): MRSA and MDR-AB bacterial liquid (3 x 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 min. Then, the bacteria were collected by centrifugation at 4000 rpm for 10 min, and fixed with 2.5% glutaraldehyde at 4°C overnight, and then with 1% osmium tetroxide for 1-2 hours. The samples were dehydrated with different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15 min each. Then, they were treated with a mixture of ethanol and isoamyl alcohol 1:1 for 30 min, and then with pure isoamyl alcohol for 1 hour. Finally, critical point drying and gold plating were performed, and the samples were observed using a scanning electron microscope (SEM, HITACHI SU8010). For transmission electron microscopy (TEM) samples, after ethanol gradient dehydration, they were treated with pure acetone for 20 min, then with a mixture of embedding agent and acetone 1:1 (v / v) for 1 hour, and then with a mixture of embedding agent and acetone 3:1 (v / v) for 3 hours. Then, they were treated with pure embedding agent and heated at 70°C overnight. Finally, ultrathin sections of 70-90 nm were prepared using an ultramicrotome (LEICA EMUC7). After staining and drying, the sections were observed using a transmission electron microscope (TEM, HITACHI H-7650). As shown in Figure 25 The SEM images show that the surface of MRSA and MDR-AB is ruptured and collapsed, and the TEM images show that the bacterial content and membrane structure are damaged, with obvious structural degradation. These observations indicate that compound 2 induces bacterial death by damaging the internal and membrane structures, further confirming the bactericidal effect of the compound, which can provide a new treatment for clinical bacterial infections.

[0178] Example 14. Compound 2 targets bacterial membrane and nucleus validation: colocalization effect with commercial dyes (cell mask / hoechst 33342) on bacterial and cell staining

[0179] Single bacterial system: Log phase MRSA and MDR-AB bacterial solution was diluted with PBS to 3 x 10 8 CFU / mL and incubated with CellMask (10 pg / mL) for 30 min at 37 °C. Subsequently, the bacteria were collected by centrifugation, washed with PBS for three times, and then mixed with Hoechst (10 pg / mL) and Compound 2 (5 pM) for 30 min staining. After staining, the bacteria were collected by centrifugation and resuspended in 10 pL PBS. 1 pL 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 x 10 4 CFU / mL and incubated with CellMask (10 pg / mL) for 30 min at 37 °C. Subsequently, the bacteria were collected by centrifugation, washed with PBS for three times, and then mixed with Hoechst (10 pg / mL) and Compound 2 (5 pM) for 30 min staining. After staining, the bacteria were collected by centrifugation and resuspended in 10 pL PBS. 1 pL 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 x 10 4 CFU / mL and incubated with CellMask (10 pg / mL) for 30 min at 37 °C. Subsequently, the bacteria were collected by centrifugation, washed with PBS for three times, and then mixed with Hoechst (10 pg / mL) and Compound 2 (5 pM) for 30 min staining. After staining, the bacteria were collected by centrifugation and resuspended in 10 pL PBS. 1 pL 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 x 10 8 CFU / mL and incubated with CellMask (10 pg / mL) for 30 min at 37 °C. Subsequently, the bacteria were collected by centrifugation, washed with PBS for three times, and then mixed with Hoechst (10 pg / mL) and Compound 2 (5 pM) for 30 min staining. After staining, the bacteria were collected by centrifugation and resuspended in 10 pL PBS. 1 pL 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 x 10 Figure 26 CFU / mL and incubated with CellMask (10 pg / mL) for 30 min at 37 °C. Subsequently, the bacteria were collected by centrifugation, washed with PBS for three times, and then mixed with Hoechst (10 pg / mL) and Compound 2 (5 pM) for 30 min staining. After staining, the bacteria were collected by centrifugation and resuspended in 10 pL PBS. 1 pL 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 x 10 Figure 26Figure 10. Compound 2 binds to both bacterial membrane and nucleus. (A) The merged image of Hoechst / CellMask and compound 2 channels. (B) The merged image of Hoechst / CellMask and compound 2 channels. (C) The histogram shows that compound 2 exhibits strong co-localization and high correlation coefficient on both MRSA and MDR-AB after merging the Hoechst / CellMask channel with the compound 2 channel. These results indicate that compound 2 is significantly enriched in both bacterial membrane and nucleus, suggesting that its antibacterial mechanism may simultaneously target bacterial membrane and DNA. In contrast, the fluorescence intensity observed in L929 cells is extremely low Figure 26 Figure 11. Compound 2 preferentially binds to bacteria rather than mammalian cells. (A) The merged image of Hoechst / CellMask and compound 2 channels. (B) The merged image of Hoechst / CellMask and compound 2 channels. (C) The histogram shows that compound 2 preferentially binds to bacteria rather than mammalian cells. These results demonstrate that compound 2 preferentially binds to bacteria and exhibits targeting specificity for bacteria compared to cells.

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

[0181] MRSA bacterial solution (concentration of 2 x 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 ) respectively and incubated at 37 °C for 30 minutes. Subsequently, centrifugation was performed at 4000 rpm for 10 minutes, the bacterial bodies were collected and resuspended in 75% ethanol, and stored at -80 °C overnight. Finally, the frozen bacterial bodies were transported to Shanghai Meiji Biotech Co., Ltd. on dry ice for sequencing analysis. As shown in Figure 27 GO enrichment analysis showed that most of the differentially expressed genes (DEGs) were related to processes associated with membrane transport and DNA / RNA synthesis. The significantly enriched GO entries included “transmembrane import,” “carbohydrate transmembrane transport,” “carbohydrate transport,” “carbohydrate transmembrane import,” 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 disrupts the bacterial membrane and DNA, affecting key biological processes such as membrane transport and DNA replication / transcription, and prove at the gene level that compound 2 targets and disrupts the bacterial membrane and nucleus.

[0182] Example 16 Verification of compound 2 binding to bacterial DNA: UV absorption assay and fluorescence spectrum assay after compound 2 binds to DNA, and exogenous DNA addition inhibits the antibacterial effect of compound 2

[0183] DNA was extracted from MRSA bacterial culture using a BIOG DNA Fungi & Bacteria Kit following the manufacturer's instructions. DNA concentration was determined using a NanoDrop spectrophotometer. The DNA solution (20 μg / mL) was incubated with compound 2 (10 μM) for 30 minutes, and the UV-Vis absorption spectrum of the mixture was measured. Simultaneously, DNA was diluted to different concentrations with PBS, and each concentration of DNA solution was incubated with 10 μM compound 2 for 30 minutes. Finally, the fluorescence intensity of the mixture was measured (excitation wavelength 405 nm, emission wavelength 450-800 nm). MRSA and MDR-AB bacterial cultures in the logarithmic growth phase were diluted to 1×10⁻⁶ in LB medium. 6 CFU / mL. 5 μM compound 2 was mixed with DNA of varying concentrations and incubated at 37°C for 30 minutes. Subsequently, bacterial suspension was added to the mixture to bring the final bacterial concentration to 10. 6 The concentration of CFU / mL was transferred to a 96-well plate. After incubation for 24 hours, the OD value at 600 nm was measured using a microplate reader.

[0184] like Figure 28 As shown in Figure D, the absorption spectrum of the DNA-compound 2 mixture exhibits a significant enhancement and red shift compared to compound 2 alone, indicating the formation of a new complex. Fluorescence spectroscopy shows that the fluorescence intensity of compound 2 gradually increases with increasing DNA concentration. Figure 28 (E), indicating that binding to DNA restricts the intramolecular movement of compound 2, thereby promoting its luminescence. Meanwhile, as... Figure 28 As shown in Figure FG, 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, which reduces the opportunity for compound 2 to interact with bacterial endoDNA. With increasing exogenous DNA concentration, the amount of compound 2 available for binding to bacterial DNA decreases, thereby weakening its antibacterial effect. These results demonstrate that the binding of compound 2 to DNA forms a new conjugated complex, leading to changes in the photophysical properties of compound 2 and simultaneously weakening its antibacterial effect.

[0185] Example 17: Validation of Compound 2's binding to bacterial DNA: Binding site of Compound 2 competing nuclear dye hoechst33342

[0186] MRSA and MDR-AB bacterial cultures in the logarithmic growth phase were diluted with PBS to a concentration of 2 × 10⁻⁶. 8CFU / mL and incubated with Hoechst (10 pg / mL) for 30 min. Then, different concentrations of compound 2 were added and the staining was continued for another 30 min. After the completion of staining, the bacterial solution was centrifuged and washed with PBS for three times, and finally resuspended in 10 pL PBS. 1 pL of the stained bacterial suspension was dropped onto a glass slide and covered with a cover glass. Imaging was 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, which is known to be able to intercalate into the minor groove of DNA. As shown in FIG. 18A-C, the fluorescence intensity of Hoechst 33342 gradually decreased with the increase of the concentration of compound 2, while the green fluorescence signal of compound 2 was significantly enhanced in both MRSA and MDR-AB. This dose-dependent fluorescence change indicates that compound 2 is able to effectively displace Hoechst 33342 on DNA. The results suggest that compound 2 binds to DNA through a similar groove-binding mechanism as Hoechst 33342. Figure 28

[0187] Example 18 Verification of compound 2 binding to bacterial DNA: compound 2 docking simulation with DNA fragment 4U8A structure

[0188] ​To investigate the binding region and interaction mode of compound 2 with DNA, we used molecular docking method 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 charge 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 charge was calculated for subsequent molecular docking. The ligand structure was also processed by AutoDock Tools 1.5.6 to generate a pdbqt file for docking. Molecular docking was achieved by the AutoDock 4.2.6 software package. The potential binding site of 4U8A is located in the A and T-rich base cavity region of the double helix structure. The center coordinates of the docking box were set to (17.169, 9.364, -23.817), the grid point number in XYZ direction was set to 80x80x80, and the docking running number was set to 50 times. The molecular docking results 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 found 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 G2, C3, G4, A5, A6 bases on the A strand and G16, A18, T19, and T20 bases on the B strand Figure 28 of compound 2 can effectively bind to the loop cavity of the DNA structure, interact with the groove and bases of DNA, and confirm the strong DNA binding ability of compound 2, enabling it to interact with the groove of bacterial DNA and exert antibacterial effects.

[0189] Example 19 Verification of compound 2 binding to bacterial membrane structure: Compound 2 destroys bacterial membrane potential

[0190] The MRSA and MDR-AB bacterial solutions in the logarithmic growth phase were diluted with PBS to 6x10 8 CFU / mL. After adding 5 μM DiSC3(5), the mixture was incubated at room temperature for 1 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 using a microplate reader for 30 minutes (excitation wavelength 620 nm, emission wavelength 670 nm). The results are shown in Figure 29 that the fluorescence intensity significantly increased after treatment with compound 2, indicating that compound 2 destroyed the bacterial membrane potential, leading to membrane collapse and triggering bacterial damage.

[0191] Example 20 Bacteriostatic resistance test of compound 2

[0192] After continuous treatment of S. aureus and A. baumannii with compound 2 for 14 days and subculture, the minimum bacteriostatic concentration (MIC) values remained essentially unchanged (as determined according to the MIC method of Example 10). In contrast, the MIC values of the clinical antibiotics methicillin and imipenem increased by 24-fold and 128-fold, respectively Figure 30 ), indicating that these traditional antibiotics have developed resistance. This confirms the potential of compound 2 as an antibacterial agent with multi-target action and low risk of resistance.

[0193] Example 21 Biocompatibility test of compound 2

[0194] MTT assay for cell activity: In vitro cytotoxicity evaluation was performed by MTT method using NIH3T3 cells and L929 cells. Cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). Compound 2 was added to 96-well plates seeded with two types of cells (4000 cells per well), and co-cultured for 24 hours. Subsequently, MTT solution was added to the 96-well plates, and reacted for 2-4 hours. After the reaction was completed, 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 crystals were fully dissolved by shaking on a low-speed shaker for 5 minutes. The absorbance value (OD560nm) of each well was measured using a microplate reader at a wavelength of 560 nm. As Figure 31 The results show that the effect of compound 2 on normal cell activity is negligible, especially at concentrations with antibacterial activity. It is proved that compound 2 has significant biocompatibility, further supporting its potential as a selective antibacterial agent.

[0195] Example 22 Treatment of abdominal infection with resistant bacteria and reduction of bacterial load in organs in vivo by compound 2

[0196] A mild MRSA infectious peritonitis model was established by intraperitoneal injection of MRSA bacterial solution (3 x 10 8 CFU / mL). After 3 hours of 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 2100 μL of compound 2 was administered, followed by abdominal light exposure for 15 minutes. This treatment was repeated every 24 hours for a total of 5 treatments. Throughout the treatment period, the mice's weight and survival status were monitored daily. After 5 days, the mice were sacrificed, and blood and major organs were collected for white blood cell count analysis and hematoxylin-eosin (H&E) staining. Furthermore, to further investigate the therapeutic effect of compound 2 in severe infections, a severe MRSA infectious peritonitis model was established. Similarly, MRSA bacterial solution (3 × 10⁻⁶) was injected intraperitoneally. 9 Three hours after the initial treatment (CFU / mL), the mice were treated again using the same method described above, once every 24 hours, for a total of 6 treatments. Six days later, the mice were euthanized and major organs (including heart, liver, spleen, lungs, and kidneys) were collected and homogenized to release residual bacteria. The bacterial suspension was diluted and spread onto LB agar plates, incubated at 37°C for 24 hours, and the colony-forming units (CFUs) were quantified by counting the number of colonies. Figure 32 As shown in Figure B, this is different from the normal reference range (0.8-6.8×10). 9 Compared to the PBS and vancomycin groups, WBC levels were significantly elevated in the compound 2 group and the compound 2 + light exposure group, indicating persistent inflammation and infection. In contrast, WBC levels in the compound 2 group and the compound 2 + light exposure group remained within the normal range, indicating that compound 2 possesses effective antibacterial and anti-inflammatory properties. Furthermore, the erythrocyte (RBC) hemolysis assay showed that even at high concentrations, the hemolytic activity of compound 2 was negligible, indicating its excellent biocompatibility. Figure 32 (A) For example Figure 33 As shown in Figure A, two days after severe infection, all mice in the PBS group died, while the survival rates of the "Compound 2" group and the "Compound 2 + Light" group were significantly higher than those of the vancomycin group. After treatment, major organs were homogenized and bacterial colonies were counted. The results showed ( Figure 33 In the study of BF, the bacterial load in organs of the "PBS" group was higher than that of the "Compound 2" group and the "Compound 2 + Light" group, which showed significantly lower bacterial loads than the "PBS" group and the "Vancomycin" group, thus confirming the antibacterial effect of Compound 2.

Claims

1.A kind of terpyridine compound, characterized in that: The structural formula of the compound is shown as formula (VIII): Formula (VIII); Wherein, X is C or N; R1 is R2Y, Y is nothing or selected from halogen anion; R2is selected from one of methyl, ethyl, , , , , . 2.The terpyridine compound according to claim 1, characterized in that: The halogen anion is selected from one of I - , Br - . 3.A terpyridine compound, characterized in that: The structural formula of the terpyridine compound is shown as any one of formula (I)-(VII): (Formula I); or (Formula II); or (Formula III); or (Formula IV); (Formula V); or (Formula VI); or (Formula VII). 4.The preparation method of the terpyridine compound according to any one of claims 1-3, characterized in that: Compound 1d is reacted with reaction material 1, or compound 5d is reacted with reaction material 2 to obtain a compound with the structure shown in formula (VIII); The structural formula of the skeleton compound 1d is shown as formula (IX): Formula (XI); The structural formula of the skeleton compound 5d is shown as formula (XV): Formula (XV); The reaction material 1 includes at least one of alkyl halide, quaternary ammonium halide and sulfonating agent; The reaction material 2 includes alkyl halide and tertiary amine. 5.The preparation method according to claim 4, characterized in that: In the reaction material 1, the alkyl halide is selected from one of methyl iodide and propyl iodide; The quaternary ammonium halide is (3-bromopropyl) trimethylammonium bromide; The sulfonating agent is 1,3-propane sulfone lactone. 6.The preparation method according to claim 4, characterized in that: In the reaction material 2, the alkyl halide is selected from one of 1,2-dibromoethane, 1,3-dibromopropane and 1,4-dibromobutane; The tertiary amine is trimethylamine. 7.Use of the terpyridine compound according to any one of claims 1-3 in the preparation of a product; The product includes at least one of fluorescent dye, photosensitizer, bacterial dye and antibacterial agent. 8.A product, characterized in that: It includes the terpyridine compound according to any one of claims 1-3; The product includes at least one of fluorescent dye, photosensitizer, bacterial dye and antibacterial agent. 9.The product according to claim 8, characterized in that: When the product is fluorescent dye, the fluorescent dye includes the terpyridine compound according to any one of claims 1-3; or When the product is photosensitizer, the photosensitizer includes one of the terpyridine compounds with the structural formula shown as formula (I)-(IV), formula (VI) and formula (VII) according to claim 3; or When the product is bacterial dye, the bacterial dye includes the terpyridine compound according to any one of claims 1-3; or When the product is antibacterial agent, the antibacterial agent includes one of the terpyridine compounds with the structural formula shown as formula (I) and (II) according to claim 3. 10.The product according to claim 9, characterized in that: The product further includes pharmaceutically acceptable excipients.

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