A fluorescent compound, and a preparation method and application thereof

Abstract

The application provides a fluorescent compound, a preparation method and application of the fluorescent compound, and the compound is shown in formula (I). The compound can be used as a fluorescent probe for Gram-positive bacteria targeted imaging, and can directly kill bacteria, so that dual effects of fluorescent identification and bacterial killing on Gram-positive bacteria are realized.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a fluorescent compound, its preparation method, and its application. Background Technology

[0002] Multidrug-resistant Gram-positive bacterial infections are one of the major threats to global public health security in the 21st century. The World Health Organization (WHO) lists methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and drug-resistant Streptococcus pneumoniae as "high-priority pathogens," with mortality rates of 20%-40% for bloodstream infections, pneumonia, and skin and soft tissue infections caused by these pathogens. Although last-line antibiotics such as vancomycin, daptomycin, and linezolid are widely used in clinical practice, the frequent emergence of drug-resistant mutant strains in recent years and the severe lack of new antibiotic development highlight the urgency of developing novel antibacterial agents that break through traditional mechanisms of action.

[0003] The field of antimicrobial therapy still faces challenges: firstly, while bacterial culture is the gold standard for diagnosis, it is time-consuming; secondly, treatment often faces problems such as antibiotic resistance, biofilm barriers, relatively singular antimicrobial strategies, and relapses due to neglect of microenvironment regulation. This disconnect between diagnosis and treatment has prompted researchers to turn their attention to the construction of integrated diagnostic and therapeutic systems, namely, developing novel probes that combine real-time detection of pathogens with highly effective killing capabilities to achieve a precise antimicrobial model of "detection equals treatment."

[0004] Recent studies have revealed that the reactive oxygen species (ROS)-mediated oxidative damage mechanism provides a new approach to overcoming bacterial resistance barriers. Photodynamic therapy has unique advantages, such as broad-spectrum antibacterial activity, rapid action, and low susceptibility to inducing resistance. Developing photosensitizers that can specifically target Gram-positive bacteria and achieve effective antibacterial action could provide new theoretical basis and treatment options for overcoming the challenge of multidrug-resistant bacteria. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent compound, its preparation method, and its applications. This compound can not only target and image Gram-positive bacteria, but also directly kill bacteria, achieving the dual effects of fluorescent identification and bacterial killing of Gram-positive bacteria.

[0006] In some embodiments, the present invention provides a fluorescent compound, which is a compound represented by formula (I).

[0007]

[0008] In the formula,

[0009] Ar1 and Ar2 are selected from substituted or unsubstituted aromatic ring groups;

[0010] m is an integer not less than 2.

[0011] n is an integer not less than 3.

[0012] Preferably, in the fluorescent compound of the present invention, the Ar1 is selected from one of the following groups:

[0013]

[0014] In the formula,

[0015] R1 is a hydrogen atom, or a C1-C6 alkyl or alkenyl group.

[0016] R2 is selected from C1-C6 alkyl or cycloalkyl;

[0017] R3 and R4 may be the same or different, and each can be independently selected from hydrogen atoms, C1-C6 alkyl groups and cycloalkyl groups;

[0018] Preferably, in the fluorescent compound of the present invention, Ar2 is selected from one of the following groups:

[0019]

[0020] In the formula, R5 and R6 may be the same or different, and each is independently selected from hydrogen atoms, C1-C6 alkyl groups and C1-C6 alkoxy groups.

[0021] In some embodiments, the fluorescent compound of the present invention, wherein the Ar1 group is

[0022] In the formula, R1 is H.

[0023] The group Ar2 is

[0024]

[0025] Preferably, in the fluorescent compound of the present invention, m is 2 and n is 4.

[0026] In a preferred embodiment, the fluorescent compound of the present invention is a compound of formula TPV-CyS or its pharmaceutical salt and isomers.

[0027]

[0028] In the above preferred embodiments, the isomers of the fluorescent compound TPV-CyS compound of the present invention are selected from the following compounds:

[0029]

[0030] In another embodiment, the present invention provides a method for preparing a TPV-CyS compound, comprising the following steps:

[0031] 1) Compound 1 and compound 2 were reacted in a solvent in the presence of catalyst Pd(PPh3)4 and potassium carbonate to generate compound 3;

[0032] 2) Compounds 3 and 4 were refluxed in an organic solvent to prepare compound TPV-CyS.

[0033] The reaction formula is as follows:

[0034]

[0035] In some embodiments, in the method of the present invention described above, the solvent in step 1) is a toluene / ethanol / water mixture, preferably in a volume ratio of 8:1:1. In step 2), the organic solvent is selected from at least one of anhydrous ethanol, methanol, acetonitrile, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, and dimethyl sulfoxide, preferably anhydrous ethanol.

[0036] In some embodiments, the present invention provides the use of the compound of formula (I) of the present invention in the preparation of fluorescent probes for targeted imaging of Gram-positive bacteria, that is, in the manufacture of diagnostic reagents for targeted imaging of Gram-positive bacteria.

[0037] In some embodiments, the present invention provides the use of the compound of formula (I) of the present invention in the manufacture of an anti-Gram-positive bacteria drug.

[0038] In some embodiments, the application or use of the compound shown in (I) is preferably that the compound is a TVP-CyS compound or an isomer thereof.

[0039] In some embodiments, the Gram-positive bacteria used in the application of the present invention described above are selected from at least one of the following: Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), agalactococcus (also known as group B streptococci, abbreviated GBS), Streptococcus mutans, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae (KPN), Enterobacter cloacae (ECL), and Pseudomonas aeruginosa (PA).

[0040] The fluorescent compounds of the present invention have excellent aggregation-induced emission (AIE) properties, that is, they emit weak light in solution, but the emission is significantly enhanced in the aggregated state.

[0041] The fluorescent compound of the present invention can selectively "light up" live Gram-positive bacteria (emitting bright fluorescence) and has a good fluorescence co-localization effect with the bacterial membrane. It can quickly distinguish between Gram-positive and Gram-negative bacteria through fluorescence, and has the characteristics of high fluorescence intensity and high reactive oxygen species (ROS) generation rate.

[0042] The fluorescent compounds of this invention can enter dead Gram-positive bacteria and exhibit good fluorescent co-localization effects with bacterial nucleic acids.

[0043] The fluorescent compounds of this invention are applicable to imaging Gram-positive bacteria under different pH conditions (acidic, neutral, alkaline).

[0044] Under light source irradiation, the fluorescent compound of the present invention can induce the production of a large amount of ROS in Gram-positive bacteria, triggering lipid peroxidation and rupture of the bacterial membrane, destroying the bacterial cell membrane structure, and exhibiting excellent photodynamic antibacterial effect. In particular, it has excellent photodynamic antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA), and the compound has good biocompatibility and low toxicity.

[0045] The fluorescent compounds of this invention are effective at killing Gram-positive bacteria under different pH conditions (acidic, neutral, and alkaline).

[0046] The fluorescent compounds of the present invention can be used as photosensitizers and antibacterial agents, and can specifically kill Gram-positive bacteria by disrupting cell membrane structure and photodynamic effects, thereby promoting wound repair. Attached Figure Description

[0047] Figure 1 The image shows the 1H NMR spectrum of the intermediate (compound 3) from Example 1.

[0048] Figure 2 The image shows the carbon NMR spectrum of the intermediate (compound 3) from Example 1.

[0049] Figure 3 This is a high-resolution mass spectrum of the intermediate (compound 3) of Example 1.

[0050] Figure 4 The image shows the 1H NMR spectrum of the target product TPV-CyS from Example 1.

[0051] Figure 5 The image shows the carbon NMR spectrum of the target product TPV-CyS in Example 1.

[0052] Figure 6 The image shows a high-resolution mass spectrum of the target product TPV-CyS from Example 1.

[0053] Figure 7 The image shows the 1H NMR spectra of the product TPV-CyS from Example 1 under acidic and alkaline conditions.

[0054] Figure 8 The image shows the carbon NMR spectra of the product TPV-CyS from Example 1 under acidic and alkaline conditions.

[0055] Figure 9 The image shows the photophysical property detection results of Example 2, where A is the UV-Vis absorption spectrum and B is the spectrum of different toluene contents (f). T The fluorescence emission spectrum of TPV-CyS measured in a DMSO / toluene mixed solution, where C represents the relative fluorescence intensity (I / I0) versus different toluene contents (f). T The relationship diagram; DMSO is dimethyl sulfoxide, Wavelength represents wavelength, Molar absorptivity represents molar absorptivity, PL intensity represents fluorescence intensity, I / I0 represents the ratio of the fluorescence intensity measured in the DMSO / toluene mixture with and without toluene, f T This indicates the volume fraction of toluene.

[0056] Figure 10 The images shown are the bacterial imaging results from 3.1 and 3.2 in Example 3. A is a confocal image of live Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli (E. coli) after co-incubation with TPV-CyS, with a scale bar of 10 μm; B is a confocal image of dead S. aureus and E. coli after co-incubation with TPV-CyS, with a scale bar of 10 μm; C is an image of live S. aureus, MRSA, TPV-CyS, and Cell Mask (a commercially available cell membrane dye) after co-staining, with a scale bar of 10 μm; and D is an image of dead S. aureus and E. coli after co-staining with TPV-CyS and DAPI, with a scale bar of 10 μm.

[0057] Figure 11 The images shown are the bacterial imaging results under different pH conditions in Example 3.3. A is a confocal image of methicillin-resistant Staphylococcus aureus (MRSA) after co-incubation with TPV-CyS, with a scale bar of 10 μm; B is an imaging result of co-staining MRSA, TPV-CyS and Cell Mask (a commercial cell membrane dye), with a scale bar of 10 μm.

[0058] Figure 12The image shows the detection results of reactive oxygen species (ROS) in Example 4. In the image, A represents 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), B represents dihydrorhodamine 123 (DHR 123), and C represents the change in fluorescence intensity (PL intensity) of hydroxyphenylfluorescein (HPF) at 523 nm. These were used to detect the total reactive oxygen species (ROS) and superoxide anion (O2) in TPV-CyS, respectively. -· ) and hydroxyl radicals (·OH), D represents the change in absorbance of TPV-CyS at 380 nm under white light irradiation with or without 9,10-anthratridimyl-bis(methylene)dicarboxylic acid (ABDA), used to detect singlet oxygen ( ). 1 O2); A / A0 is the relative absorbance.

[0059] Figure 13 The image shows the results of the selective killing test of Gram-positive and Gram-negative bacteria in Example 5.1. In this image, A is an agar plate photograph of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli treated with different concentrations of TPV-CyS under light or dark conditions. B shows the corresponding bacterial viability. +L represents light conditions, and -L represents dark conditions.

[0060] Figure 14 The image shows the results of the bacterial killing test under different pH conditions in Example 5.2. In the image, A is an agar plate photograph of methicillin-resistant Staphylococcus aureus (MRSA) treated with different concentrations of TPV-CyS under light or dark conditions, B is the corresponding bacterial activity, +L represents light conditions, and -L represents dark conditions.

[0061] Figure 15 The following figures show the results of the TPV-CyS antibacterial mechanism study in Example 6. A shows the SYTO 9 / PI double staining results of MRSA treated with TPV-CyS, where L represents the light conditions. SYTO 9 is a commercially available green fluorescent nucleic acid dye, and PI (propidium iodide) is a commercially available DNA-binding dye. The scale bar is 10 μm. B shows the ROS generation results in MRSA treated with TPV-CyS, with a scale bar of 10 μm. C shows the lipid peroxidation results of the bacterial membrane in MRSA treated with TPV-CyS, with a scale bar of 10 μm. D shows the FITC-Dextran staining results of MRSA treated with TPV-CyS, where L represents the light conditions. FITC-Dextran is a commercially available green fluorescent membrane integrity detection probe, with a scale bar of 10 μm. E shows the morphology of MRSA treated under different conditions, with a scale bar of 1 μm.

[0062] Figure 16The following is a diagram showing the in vivo antibacterial test results of Example 7. In this diagram, A shows the wound healing status of mice in different treatment groups, with a scale bar of 10 mm; B shows the relative infection area of ​​the wounds of mice in different treatment groups; and C shows the results of hematoxylin-eosin (H&E) staining and Masson staining of the wounds of mice in different treatment groups on day 15, with a scale bar of 500 μm. Detailed Implementation

[0063] The following embodiments are provided to describe the present invention in more detail. However, these embodiments are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0064] Example 1: Preparation of TPV-CyS compound

[0065] The reaction route for preparing TPV-CyS compounds is as follows:

[0066]

[0067] Synthesis of intermediate (compound 3): Compound 1 (1.38 g, 3.6 mmol), compound 2 (633.2 mg, 3 mmol), Pd(PPh3)4 (173 mg, 0.15 mmol), and potassium carbonate (829 mg, 6 mmol) were reacted in a toluene / ethanol / water mixture (8 / 1 / 1, v / v / v, 50 mL) under a nitrogen atmosphere and refluxed for 10 h. After the reaction, the mixture was cooled to room temperature and then poured into water, and extracted several times with dichloromethane. The combined organic layers were washed with saturated brine and water. The resulting organic solution was dried over anhydrous magnesium sulfate, filtered, and then the solvent was removed by rotary evaporation. The crude product was further purified by silica gel column chromatography using n-hexane / dichloromethane as the eluent. The obtained intermediate (compound 3) was a yellow solid, 1.06 g, yield 91%.

[0068] Synthesis of the target product TPV-CyS: Compound 3 (212 mg, 0.5 mmol) and compound 4 (148 mg, 0.5 mmol) were dissolved in anhydrous ethanol (5 mL). The reaction solution was refluxed overnight under a nitrogen atmosphere. The solvent was removed by rotary evaporation, and the crude product was further purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain the target compound TPV-CyS. The target product was a red solid, 140 mg, with a yield of 42%.

[0069] The molecular structure of the target compound TPV-CyS undergoes corresponding transformations under acidic or alkaline conditions, as detailed below:

[0070]

[0071] The target compound TPV-CyS was converted into the isomer TPV-CyS-H under acidic conditions (e.g., with the addition of hydrochloric acid).

[0072] The target compound TPV-CyS was converted into the isomer TPV-CyS-OH under alkaline conditions (e.g., with the addition of sodium hydroxide solution).

[0073] Molecular structure characterization:

[0074] Nuclear magnetic resonance spectrometer (Bruker Avance 600MHz) and high-resolution mass spectrometer (ThermoScientific) were used. TM Orbitrap Fusion TM Tribrid TM The structures of the intermediate (compound 3) and the target product TPV-CyS were characterized, and the results are as follows: Figures 1-6 As shown.

[0075] The structure of the target product TPV-CyS was characterized using nuclear magnetic resonance spectroscopy (Bruker Avance 600MHz) under acidic (TPV-CyS-H) and basic (TPV-CyS-OH) conditions. The results are as follows: Figure 7 and Figure 8 As shown.

[0076] like Figure 1 As shown, the 1H NMR characterization data of the intermediate (compound 3) are as follows: 1 H NMR(600MHz, CDCl3) δ9.66(d,J=7.7Hz,1H),7.37(d,J=15.9Hz,1H),7.30(d,J=8 .2Hz,2H),7.13–7.08(m,11H),7.04–7.01(m,6H),6.63(dd,J=15.9,7.7Hz,1H).

[0077] like Figure 2 As shown, the carbon NMR characterization data of the intermediate (compound 3) are as follows: 13 C NMR (150MHz, CDCl3) δ193.69,152.56,147.32,143.27,143.26,143.14,142.44,139.93,132.0 7,131.97,131.33,131.32,131.27,128.17,127.97,127.90,127.88,127.71,126.90,126.77.

[0078] like Figure 3 As shown, the high-resolution mass spectrometry characterization data of the intermediate (compound 3) are: HRMS (ESI): m / z 387.17523 ([M+H] + C 29 H 23 O, calcd 387.17434).

[0079] like Figure 4 As shown, the 1H NMR characterization data of the target product TPV-CyS are as follows: 1 H NMR (600MHz, CD3OD) δ8.27 (dd, J=15.2, 10.7Hz, 1H), 7.83 (dd, J=7.5, 1.5Hz, 1H) ,7.72–7.71(m,1H),7.63–7.57(m,2H),7.53(d,J=15.3Hz,1H),7.48–7.40(m,3H) ,7.26(d,J=15.2Hz,1H),7.14–7.08(m,11H),7.05–6.99(m,6H),4.55(t,J=7.9H z,2H),2.91(t,J=7.0Hz,2H),2.15–2.09(m,2H),2.00–1.95(m,2H),1.79(s,6H).

[0080] like Figure 5 As shown, the carbon NMR characterization data of the target product TPV-CyS are as follows: 13 C NMR (150MHz, CD3OD) δ182.58,156.95,150.19,148.30,144.56,144.36,144. 28,144.12,143.60,141.85,141.22,134.78,132.86,132.00,131.98,131.9 0,130.24,130.19,129.38,128.92,128.56,128.55,128.36,127.61,127.45,127.42,123.59,115.80,115.56,54.38,53.12,50.75,27.58,26.18,22.90.

[0081] like Figure 6 As shown, the high-resolution mass spectrometry characterization data of the target product TPV-CyS are: HRMS (ESI): m / z 664.29083 ([M+H] + C 44 H 42 NO3S, calcd 664.28799).

[0082] like Figure 7 and Figure 8 As shown, the 1H and 1C NMR characterization data of the target product TPV-CyS under acidic (TPV-CyS-H) and alkaline (TPV-CyS-OH) conditions are as follows:

[0083] TPV-CyS: 1 H NMR (600MHz, DMSO-d6 / D2O) δ8.28(dd,J=15.2,10.8Hz,1H),7.91(d,J=7.2Hz,1H),7.82(d ,J=8.3Hz,1H),7.63–7.59(m,3H),7.50(d,J=8.3Hz,2H),7.45(dd,J=15.3,10.8Hz,1H),7 .29(d,J=15.3Hz,1H),7.20–7.13(m,9H),7.07(d,J=8.2Hz,2H),7.02–6.98(m,6H),4.48( t,J=7.6Hz,2H),2.59(t,J=7.3Hz,2H),1.97–1.92(m,2H),1.80–1.76(m,2H),1.73(s,6H). 13 C NMR (150MHz, DMSO-d6 / D2O) δ180.91,154.94,147.93,146.14,143.43,142.82,142.79,142.58,141.82,140.62,139.87,133.52,131.48,13 0.63,130.58,130.51,128.99,127.98,127.82,126.94,126.82,126.7 8,122.88,115.56,115.00,51.75,50.15,46.22,26.86,25.60,22.00.

[0084] TPV-CyS-OH (add sodium hydroxide to TPV-CyS): 11H NMR (600 MHz, DMSO-d6 / D2O) δ 7.22 (d, J = 8.1 Hz, 2H), 7.17–7.10 (m, 9H), 6.99–6.93 (m, 7H), 6.90–6.84 (m, 4H), 6.58–6.52 (m, 2H), 6.47 (d, J = 15.6 Hz, 1H), 6.31 (d, J = 7.8 Hz, 1H), 5.90 (d, J = 15.1 Hz, 1H), 2.93–2.85 (m, 2H), ~2.56 (overlapped with DMSO-d6, 2H), 1.68–1.57 (m, 4H), 1.11 (s, 3H), 0.99 (s, 3H). 13 13C NMR (150 MHz, DMSO-d6 / D2O) δ 148.80, 143.13, 143.11, 142.97, 142.01, 140.52, 140.29, 138.38, 130.87, 130.56, 130.50, 127.86, 127.82, 127.78, 126.64, 126.61, 125.51, 121.12, 115.99, 109.66, 105.02, 51.14, 47.62, 42.60, 28.10, 25.29, 22.46, 21.83.

[0085] TPV-CyS-H (Adding hydrochloric acid to TPV-CyS-OH): 1 1H NMR (600 MHz, DMSO-d6 / D2O) δ 8.27 (dd, J = 14.9, 11.0 Hz, 1H), 7.86 (d, J = 6.42 Hz, 1H), 7.80 (d, J = 6.6 Hz, 1H), 7.65–7.58 (m, 3H), 7.53 (d, J = 8.2 Hz, 2H), 7.44 (dd, J = 15.1, 11.0 Hz, 1H), 7.25 (d, J = 15.3 Hz, 1H), 7.20–7.14 (m, 9H), 7.08 (d, J = 8.2 Hz, 2H), 7.03–7.00 (m, 6H), 4.49 (t, J = 7.2 Hz, 2H), 2.68 (t, J = 7.2 Hz, 2H), 1.99–1.94 (m, 2H), 1.81–1.76 (m, 2H), 1.73 (s, 6H). 13C NMR(150MHz,DMSO-d6 / D2O)δ181.04,155.03,148.02,146.20,143.38,142 .79,142.76,142.53,141.82,140.44,139.88,133.46,131.41,130.56,13 0.51,130.44,129.36,129.27,128.87,128.09,128.00,127.86,127.00,1 26.89,122.87,115.33,114.84,51.77,50.08,46.11,26.75,25.63,21.71.

[0086] Example 2: Photophysical property testing

[0087] The TPV-CyS compound obtained in Example 1 was detected by UV-Vis absorption spectroscopy using a UV-Vis spectrometer.

[0088] The specific steps are as follows:

[0089] The product was prepared to a concentration of 1.0 × 10⁻⁶ using dimethyl sulfoxide (DMSO) as a solvent. -5 A mol / L TPV-CyS solution was prepared, and the solution was placed in a cuvette. The UV-Vis absorption spectrum was detected using a UV-Vis spectrometer. The results are as follows: Figure 9 As shown in Figure A.

[0090] The fluorescence emission spectrum of the TPV-CyS compound obtained in Example 1 was detected using a fluorescence spectrometer. The specific steps are as follows:

[0091] Dimethyl sulfoxide and toluene were mixed in different proportions to obtain liquids with toluene volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively. The products were then prepared to a concentration of 1.0 × 10⁻⁶ using these liquids. -5 The fluorescence emission spectra of each TPV-CyS solution (mol / L) were detected using a fluorescence spectrometer. The results are as follows: Figure 9 B and Figure 9 As shown in C.

[0092] Depend on Figure 9 It can be seen that the maximum absorption peak of TPV-CyS in dimethyl sulfoxide is located at a wavelength of 473 nm. Figure 9 A); TPV-CyS exhibits weak fluorescence in dimethyl sulfoxide, but the fluorescence intensity significantly increases with increasing water content in the system. Figure 9 B and Figure 9(c) The reason is that as the water content in the system increases, the solubility of TPV-CyS gradually decreases, leading to the formation of aggregates. This restricts intramolecular movement, thereby activating the aggregation-induced emission (AIE) effect. This result demonstrates that the compounds of this invention possess excellent AIE properties, exhibiting weak fluorescence in solution but significantly enhanced luminescence in the aggregated state.

[0093] Example 3 Bacterial Imaging Test

[0094] 3.1 Selective Imaging Test for Planktonic Gram-Positive and Gram-Negative Bacteria

[0095] The specific steps are as follows:

[0096] (1) Gram-positive bacteria were represented by Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria were represented by Escherichia coli. The diluted bacterial solution (1×10⁻⁶) was... 8 CFU / mL was mixed with TPV-CyS (final concentration 5 μM), stained for 60 min, centrifuged (3500 rpm, 2 min) to remove the supernatant, washed 3 times with PBS buffer, centrifuged at 3500 rpm for 2 min, discarded the supernatant, resuspended in 20 μL PBS, and then 10 μL was dropped onto a glass slide and sealed with a coverslip. Observation and imaging were performed using a laser confocal scanning microscope FV3000; imaging conditions: excitation wavelength 488 nm, emission wavelength range 600-700 nm; results are as follows. Figure 10 As shown in Figure A.

[0097] Depend on Figure 10 As can be seen from Figure A, after bacteria were co-incubated with TPV-CyS (5 μM) for 1 h at room temperature, Gram-positive S. aureus and MRSA emitted strong red fluorescence, while Gram-negative E. coli showed no obvious staining, indicating that TPV-CyS has good imaging capabilities for Gram-positive bacteria (S. aureus and MRSA).

[0098] (2) Dead bacterial cell suspension: Gram-positive bacteria were represented by *S. aureus* and MRSA, and Gram-negative bacteria were represented by *E. coli*. Take 1 mL of bacterial suspension diluted with PBS (1×10⁻⁶). 8 Add CFU / mL to a sterile 50mL centrifuge tube, add 20mL of 75% ethanol to the tube, mix well, and incubate at room temperature for 1h, mixing once every 15min; after 1h, centrifuge at 7000rpm for 8min, remove the supernatant, wash twice with PBS, and resuspend in 1mL in an EP tube for later use.

[0099] Staining and imaging of dead bacteria: Diluted bacterial suspension (1×10⁻⁶)8 CFU / mL was mixed with TPV-CyS (final concentration 5 μM), stained for 60 min, centrifuged (3500 rpm, 2 min) to remove the supernatant, washed 3 times with PBS buffer, centrifuged at 3500 rpm for 2 min, discarded the supernatant, resuspended in 20 μL PBS, and then 10 μL was dropped onto a glass slide and sealed with a coverslip. Observation and imaging were performed using a laser confocal scanning microscope FV3000. Imaging conditions: excitation wavelength 488 nm, emission wavelength range 600-700 nm; results are as follows. Figure 10 As shown in B.

[0100] Depend on Figure 10 As shown in Figure B, after incubating dead bacteria with TPV-CyS (5 μM) for 1 h at room temperature, the dead Gram-positive S. aureus and MRSA bacteria emitted strong red fluorescence, and the dead Gram-negative E. coli also emitted strong red fluorescence, indicating that TPV-CyS has good imaging capabilities for both dead Gram-positive bacteria (S. aureus and MRSA) and Gram-negative bacteria (E. coli).

[0101] 3.2 Bacterial Co-localization Imaging Assay

[0102] To further explore the imaging and localization capabilities of TPV-CyS for Gram-positive bacteria (S. aureus and MRSA). The specific steps are as follows:

[0103] Dilute the bacterial suspension with PBS to 1 mL (1×10⁻⁶). 8 CFU / mL), stained with TPV-CyS (final concentration 5 μM) for 1 h, then 1 μL of commercial membrane dye (Cell Mask, final concentration 5 μg / mL) or 1 μL of commercial nuclear dye (DAPI, final concentration 10 μM) was added for co-staining for 15 min. The supernatant was discarded by centrifugation, and the cells were washed three times with PBS buffer, resuspended in 20 μL of PBS, and then 10 μL of the bacterial culture was dropped onto a glass slide and observed under a laser confocal microscope. Imaging conditions: excitation wavelength: DAPI 405 nm, TPV-CyS 488 nm, Cell Mask 633 nm; emission wavelength range: DAPI 410-460 nm, TPV-CyS 600-700 nm, Cell Mask 640-750 nm; results are as follows. Figure 10 C and Figure 10 As shown in D.

[0104] Depend on Figure 10As can be seen from Figure C, the red fluorescence signal of *S. aureus* and MRSA overlaps with the green fluorescence signal of the Cell Mask, indicating that TPV-CyS has good targeting ability for Gram-positive bacteria and is mainly localized on the bacterial membrane. Figure 10 As can be seen from D, the red fluorescence signal of the dead S. aureus and MRSA groups overlaps with the blue fluorescence signal of DAPI, indicating that TPV-CyS has good targeting ability for dead bacteria and is mainly located on the nucleus.

[0105] 3.3 Bacterial Imaging Tests under Different pH Conditions

[0106] (1) Bacterial imaging under different pH conditions: pH values ​​of 5, 7, and 9 represent acidic, neutral, and alkaline conditions, respectively. MRSA is used as an example. The specific steps are as follows:

[0107] TPV-CyS (5 μM) and MRSA (concentration 1 × 10⁻⁶) in 1 mL of solutions with different pH values ​​(pH 5, 7, and 9). 8 Incubate with CFU / mL for 1 h, centrifuge (3500 rpm, 2 min), and discard the supernatant. Wash the precipitate three times with PBS, resuspend in 20 μL of PBS, then drop 10 μL onto a glass slide, cover with a coverslip, and observe and image using a laser confocal scanning microscope FV3000. Imaging conditions: excitation wavelength 488 nm, emission wavelength range 600-700 nm; results are shown below. Figure 11 As shown in Figure A.

[0108] Depend on Figure 11 As can be seen from A, after co-incubation with TPV-CyS (5 μM) for 1 h, MRSA in different pH solutions (pH 5, 7, and 9) emitted strong red fluorescence, indicating that TPV-CyS has good imaging ability for MRSA under different pH conditions.

[0109] (2) Bacterial colocalization imaging assay under different pH conditions

[0110] The specific steps are as follows:

[0111] TPV-CyS (5 μM) and MRSA (1 × 10⁻⁶) in 1 mL of solutions with different pH values ​​(pH 5, 7, and 9) 8Incubate with TPV-CyS for 1 h using CFU / mL cell membrane staining agent (Cell Mask, 10 μM) for 15 min. After incubation, centrifuge to remove supernatant, wash three times with PBS buffer, resuspend in 20 μL PBS, then drop 10 μL onto a glass slide, cover with a coverslip, and observe and image using a laser confocal scanning microscope FV3000. Imaging conditions: excitation wavelength: 488 nm for TPV-CyS, 633 nm for Cell Mask; emission wavelength range: 600-700 nm for TPV-CyS, 640-750 nm for Cell Mask; results are as follows. Figure 11 As shown in B.

[0112] Depend on Figure 11 As can be seen from B, the red fluorescence signal of MRSA in different pH solutions (pH 5, 7, and 9) overlaps with the green fluorescence signal of CellMask, indicating that TPV-CyS has good targeting ability for MRSA under different pH conditions and is mainly located on the bacterial film.

[0113] Example 4: Detection of Reactive Oxygen Species (ROS)

[0114] The ROS generation capacity of compound TPV-CyS after white light irradiation was evaluated using ROS indicators, and the specific results are shown below.

[0115] 4.1 Total ROS Detection

[0116] The specific steps are as follows:

[0117] DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was used as an indicator. A DCFH-DA solution (final concentration 5 μM) was mixed with a TPV-CyS solution (final concentration 5 μM), and the mixture was irradiated under white light. Changes in fluorescence intensity at 523 nm were monitored. Another group used a commercially available photosensitizer, dihydroporphyrin e6 (Ce6, 5 μM), instead of TPV-CyS, and was detected using the same experimental method. The group containing only DCFH-DA served as a control group. Results are as follows: Figure 12 As shown in Figure A.

[0118] Depend on Figure 12 As can be seen from A, the DCFH-DA+TPV-CyS group and the DCFH-DA+Ce6 group showed obvious fluorescence enhancement, while the control group (DCFH-DA) showed no obvious fluorescence change, proving that both TPV-CyS and Ce6 have good total ROS generation capacity; at the same time, TPV-CyS showed better total ROS generation capacity than Ce6.

[0119] 4.2 Superoxide anion (O2) -· ) detection

[0120] The specific steps are as follows:

[0121] DHR123 (dihydrorhodamine 123) was used as an indicator. A DHR123 solution (final concentration 5 μM) was mixed with a TPV-CyS solution (final concentration 5 μM), irradiated under white light, and the change in fluorescence intensity at 523 nm was monitored. Another group used a commercially available photosensitizer, Ce6 (final concentration 5 μM), instead of TPV-CyS, and was detected using the same experimental method; the group containing only DHR123 served as a control group. Results are as follows... Figure 12 As shown in B.

[0122] Depend on Figure 12 As shown in Figure B, the DHR123+TPV-CyS group and the DHR123+Ce6 group exhibited significant fluorescence enhancement, while the control group (DHR123) showed no significant fluorescence change, demonstrating that both TPV-CyS and Ce6 possess good O2... -· Generative capacity; meanwhile, TPV-CyS exhibits superior O2 production compared to Ce6. -· Generation capability.

[0123] 4.3 Detection of hydroxyl radicals (·OH)

[0124] The specific steps are as follows:

[0125] HPF (hydroxyphenyl fluorescein) was used as an indicator. HPF solution (final concentration 5 μM) was mixed with TPV-CyS solution (final concentration 5 μM), and the mixture was irradiated under white light. Changes in fluorescence intensity at 523 nm were monitored. Another group used commercially available photosensitizer Ce6 (final concentration 5 μM) instead of TPV-CyS, and the same experimental method was used. The group containing only HPF served as a control group. Results are as follows: Figure 12 As shown in C.

[0126] Depend on Figure 12 As can be seen from C, the HPF+TPV-CyS group showed obvious fluorescence enhancement, while the HPF+Ce6 group and the control group (HPF) showed no obvious fluorescence change, proving that TPV-CyS has good ·OH generation ability.

[0127] 4.4 Singlet oxygen ( 1 O2) detection

[0128] The specific steps are as follows:

[0129] ABDA (9,10-anthratridiyl-bis(methylene)dicarboxylic acid) was used as an indicator. An ABDA solution (final concentration 5 μM) was mixed with a TPV-CyS solution (final concentration 5 μM), and the mixture was irradiated under white light. The absorbance change of ABDA at 380 nm was monitored. Another group used a commercially available photosensitizer, Ce6 (final concentration 5 μM), instead of TPV-CyS, and the same experimental method was used for detection; the group containing only ABDA served as a control group. Results are as follows: Figure 12 As shown in D.

[0130] Depend on Figure 12 As can be seen from D, the ABDA+TPV-CyS group and the ABDA+Ce6 group showed a significant decreasing trend in absorbance, while the absorbance of the control group (HPF) did not change significantly, proving that both TPV-CyS and Ce6 have good absorbance properties. 1 O2 generation capacity; meanwhile, TPV-CyS exhibits slightly lower O2 generation capacity than Ce6. 1 O2 generation capability.

[0131] In summary, TPV-CyS exhibits excellent ROS generation capabilities: it possesses both good I-type photosensitizer (·OH and O2) properties. -· It has the ability to generate and also possesses good type II photosensitizer ( 1 O2) generation capacity.

[0132] Example 5 In vitro antibacterial test

[0133] 5.1 Selective killing test for Gram-positive and Gram-negative bacteria

[0134] The specific steps are as follows:

[0135] Different concentrations of TPV-CyS (0, 1, 2.5, 5, 10, 20 μM) were used to treat 1 mL of bacterial suspension (1 × 10⁻⁶ μM). 4 Staining was performed using CFU / mL (temperature 37℃, staining time 1h). 100μL of bacterial suspension was spread onto agar plates under both dark and light (30min) conditions, and incubated at 37℃ for 24h. Afterwards, these plates were placed on white light plates, photographed, and the bacterial colony count was recorded. Bacterial viability was calculated. Results are shown below. Figure 13 As shown.

[0136] Depend on Figure 13It can be seen that Gram-positive bacteria (S. aureus and MRSA) maintained good viability under both dark and light conditions without TPV-CyS treatment. After TPV-CyS treatment, antibacterial effects were observed regardless of light exposure, but the antibacterial effect was more significant under light conditions—a low concentration of TPV-CyS (2.5 μM) showed a significant antibacterial effect. However, for Gram-negative bacteria (E. coli), the antibacterial effect was not obvious regardless of light exposure or the use of different concentrations of TPV-CyS. This indicates that TPV-CyS can selectively kill Gram-positive bacteria (S. aureus and MRSA).

[0137] 5.2 Bactericidal test under different pH conditions

[0138] The specific steps are as follows:

[0139] MRSA bacterial suspensions (1×10⁻⁶) in solutions with different pH values ​​(pH 5, 7, and 9, respectively) 4 CFU / mL of TPV-CyS at different concentrations (0, 1, 2.5, 5, 10, and 20 μM) were treated at 37°C for 1 h, followed by treatment under white light or darkness for 30 min. Each suspension (100 μL) was inoculated onto agar plates and incubated at 37°C for 24 h. These plates were then placed on white light plates, photographed, and bacterial colony counts were recorded. Bacterial viability was calculated, and the results are shown below. Figure 14 As shown.

[0140] Depend on Figure 14 It can be seen that TPV-CyS treatment of MRSA in different pH solutions (pH 5, 7, and 9) showed good antibacterial effects regardless of whether there was light exposure. Furthermore, the antibacterial effect under acidic and alkaline conditions was better than that under neutral conditions, indicating that TPV-CyS can effectively kill MRSA in different pH microenvironments.

[0141] Example 6: Study on antibacterial mechanism

[0142] 6.1 SYTO 9 / PI live / dead bacteria double staining test

[0143] SYTO 9 is a commercially available green fluorescent nucleic acid dye, and PI (propidium iodide) is a commercially available DNA-binding dye that can stain dead cells.

[0144] The specific steps are as follows: Mix TPV-CyS (final concentration 5 μM) with MRSA bacterial culture (1 × 10⁻⁶ μM). 8The bacteria were treated with a mixture of CFU / mL for 1 h, followed by 30 min under both dark and light conditions. Then, they were co-incubated with SYTO 9 / PI at 37 °C for 30 min. The supernatant was removed by centrifugation (3500 rpm, 2 min). The bacteria were washed three times with PBS, then resuspended in 20 μL of PBS. The stained bacteria were then imaged using an FV3000 laser confocal microscope with excitation and emission wavelengths of 561 / 558-700 nm and 488 / 503 nm, respectively. The results are as follows: Figure 15 As shown in Figure A.

[0145] Depend on Figure 15 As can be seen from A, even a small amount of MRSA can be stained red by PI under dark conditions, while after light treatment, almost all MRSA is stained red by PI, indicating that TPV-CyS can cause the death of some bacteria under dark conditions, but the effect is more significant after light exposure.

[0146] 6.2 Detection of ROS in bacteria

[0147] The specific steps are as follows:

[0148] MRSA suspension (1×10) 8 CFU / mL) was mixed with PBS or TPV-CyS (final concentration 5 μM) and incubated for 1 h. The light group was treated with white light for 30 min, while the dark group was incubated in the dark for 30 min under the same conditions. After incubation, the mixture was centrifuged (3500 rpm, 2 min), the supernatant was removed, and the mixture was washed once with PBS. Then, it was incubated with DCFH-DA (final concentration 10 μM) for 30 min, washed again, and confocal imaging was performed. The excitation wavelength of DCFH-DA was 488 nm, and the emission wavelength was 525 nm. The results are as follows: Figure 15 As shown in B.

[0149] Depend on Figure 15 As shown in Figure B, compared with the control group (Control) and the blank light-illuminated group (L), green fluorescence signals were observed in both the TPV-CyS group and the TPV-CyS+L group, indicating the production of ROS. Furthermore, the TPV-CyS+L group exhibited a stronger green fluorescence signal than the TPV-CyS group, indicating that more ROS was produced after light treatment. In other words, TPV-CyS treatment can promote the production of ROS within bacteria, but TPV-CyS+L treatment further increases the level of ROS production within bacteria, achieving a more significant photodynamic antibacterial effect.

[0150] 6.3 Detection of lipid peroxidation

[0151] As mentioned earlier, TPV-CyS can stain Gram-positive bacteria (MRSA) and locate them on the bacterial membrane. The main component of the membrane is lipid, and TPV-CyS will produce a large amount of ROS after being exposed to light, causing lipid peroxidation of the bacterial membrane.

[0152] The specific steps are as follows:

[0153] MRSA suspension (1×10) 8 The samples were incubated with PBS or TPV-CyS (final concentration 5 μM) for 1 h. The light-treated group was exposed to white light for 30 min, while the dark-treated group was incubated in darkness for 30 min under the same conditions. After incubation, the samples were centrifuged (3500 rpm, 2 min), the supernatant was removed, and the samples were washed once with PBS. Both groups of samples were then stained with a lipid peroxidation fluorescent probe (BODIPY581 / 591C11, commercially available, concentration 10 μM) for 30 min. After washing, confocal imaging was performed. BODIPY581 / 591C11 showed red fluorescence in the reduced state (Ex = 581 nm, Em = 591 nm) and green fluorescence (Ex = 500 nm, Em = 510 nm) when interacting with lipids in the oxidized state. The results are as follows: Figure 15 As shown in C.

[0154] Depend on Figure 15 As can be seen from C, green fluorescence signals (oxidized) can be observed in both the TPV-CyS group and the TPV-CyS+L group, indicating that both have lipid peroxidation of the bacterial film. However, the TPV-CyS+L group showed a stronger green fluorescence signal than the TPV-CyS group, indicating that lipid peroxidation of the bacterial film was more significant after TPV-CyS+L treatment.

[0155] 6.4 Detection of bacterial film integrity

[0156] The specific steps are as follows:

[0157] MRSA suspension (1×10) 8 CFU / mL) was mixed with PBS or TPV-CyS (final concentration 5 μM) and incubated for 1 h. The light group was treated with white light for 30 min, while the dark group was incubated in the dark for 30 min under the same conditions. After incubation, the mixture was centrifuged (3500 rpm, 2 min), the supernatant was removed, and the mixture was washed once with PBS. Then, it was incubated with Dextran-FITC (final concentration 10 μM) for 30 min, washed again, and confocal imaging was performed. The excitation wavelength of Dextran-FITC was 488 nm, and the emission wavelength was 525 nm. The results are as follows: Figure 15 As shown in D.

[0158] Depend on Figure 15As can be seen from D, compared with the control group (Control) and the blank light group (L), green fluorescence signals can be observed in both the TPV-CyS group and the TPV-CyS+L group, proving that the integrity of the bacterial membrane is destroyed. At the same time, the TPV-CyS+L group showed a stronger green fluorescence signal than the TPV-CyS group, indicating that TPV-CyS destroys the integrity of the bacterial membrane, thereby exerting an antibacterial effect, and the antibacterial effect is more significant after light exposure.

[0159] 6.5 Bacterial Morphology Observation

[0160] Bacterial morphology was observed using a scanning electron microscope (SEM, model SU8010, HITACHI). The specific steps were as follows:

[0161] MRSA suspension (1×10) 8 The bacterial culture was mixed with PBS or TPV-CyS (final concentration 5 μM) and incubated for 1 h. The light group was treated with white light for 30 min, while the dark group was incubated in the dark for 30 min under the same conditions. After incubation, the culture was centrifuged (3500 rpm, 2 min), the supernatant was removed, and the culture was washed three times with PBS, then resuspended in PBS (1 mL) containing 2.5% glutaraldehyde. The bacterial culture was stored at 4 °C for 12 h, then subjected to a gradient wash with ethanol solution (100 μL of PBS buffer was mixed with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, respectively), and then mixed with 10 μL of ethanol solution. Finally, 1 μL of the bacterial culture was dropped onto the surface of a silicon wafer. After the ethanol solvent evaporated and dried, the silicon wafer was attached to the SEM platform using a black conductive tap, and the morphology of the bacteria was observed using SEM. The results are as follows. Figure 15 As shown in E.

[0162] Depend on Figure 15 As can be seen from E, the bacteria in the control group (Control) and the light-only group (L) had intact morphology, while the TPV-CyS group and TPV-CyS+L showed different degrees of bacterial morphological changes. In particular, the bacterial membrane in the TPV-CyS+L group showed obvious shrinkage and even rupture, which indicates that TPV-CyS can destroy the integrity of the bacterial membrane, thereby exerting an antibacterial effect, and the antibacterial effect is more significant after light exposure.

[0163] Example 7 In vivo antibacterial test

[0164] Eight-week-old female BALB / c mice purchased commercially were selected. Before the experiment, all mice were acclimatized to the animal facility for one week under pathogen-free conditions, and then housed at 25°C and 55% humidity. The specific steps are as follows:

[0165] Before the experiment, mice were divided into four groups: control group, light-only group (L), TPV-CyS group, and TPV-CyS+L (light-only) group. In the control group, wounds infected with MRSA were treated with PBS buffer (20 μL); in the light-only group (L), wounds infected with MRSA were treated with PBS buffer (20 μL) followed by light exposure for 30 min; in the TPV-CyS group, wounds infected with MRSA were treated with TPV-CyS (5 μM, 20 μL) for 1 h; and in the TPV-CyS+L group, wounds infected with MRSA were treated with TPV-CyS (5 μM, 20 μL) for 1 h and then light-exposed for 30 min.

[0166] On day 1 of the experiment, the mice were anesthetized after shaving their backs. A full-thickness skin wound with a diameter of approximately 10 mm was created on the back, and then 20 μL of MRSA bacterial solution (10 8 CFU / mL was applied to the wound for infection, and an anaerobic environment was created using a transparent dressing. On the second day of the experiment, mice were divided into a light group and a dark group, and then treated with PBS or TPV-CyS (final concentration 5 μM) for 1 hour, respectively. The light group was exposed to white light for 30 minutes, while the dark group was treated in darkness for 30 minutes under the same conditions. The wound was then measured and photographed on days 1, 3, 9, 11, and 15 to record the wound healing process. The results are as follows: Figure 16 A and Figure 16 As shown in B.

[0167] Furthermore, on day 15 of the experiment, the entire wound bed of each mouse was harvested for histological analysis. The wounds were fixed with 4% paraformaldehyde, dehydrated with a series of gradient ethanol solutions, embedded in paraffin, and cut into 4 μm thick longitudinal sections. Staining was then performed: hematoxylin-eosin (H&E) staining was used to analyze re-epithelialization rate, and Masson staining was used to assess collagen accumulation. The results are as follows: Figure 16 As shown in C.

[0168] Depend on Figure 16 A and Figure 16 As can be seen from B, the TPV-CyS+L group exhibited the best wound healing effect; by Figure 16 As can be seen from Figure C, the epidermis in the TPV-CyS+L group recovered well, with obvious hair follicle cells visible, further demonstrating the ability of TPV-CyS+L treatment to significantly repair wounds.

[0169] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A fluorescent compound, which is a compound of the formula TPV-CyS or a pharmaceutical salt thereof and its isomers. 。 2. A fluorescent compound selected from compounds of the formula TPV-CyS-H and TPV-CyS-OH: 、 。 3. A method for preparing the fluorescent compound of claim 1, comprising the following steps: 1) Compound 1 and compound 2 were reacted in a solvent in the presence of catalyst Pd(PPh3)4 and potassium carbonate to generate compound 3; 2) Compounds 3 and 4 were refluxed in an organic solvent to prepare compound TPV-CyS. 。 4. In the method of claim 3, in step 1), the solvent is a toluene / ethanol / water mixture.

5. The method as described in claim 4, in step 1), the volume ratio of toluene / ethanol / water in the mixed solution is 8:1:

1.

6. The method of claim 3, wherein in step 2), the organic solvent is selected from at least one of anhydrous ethanol, methanol, acetonitrile, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, and dimethyl sulfoxide.

7. The method of claim 6, wherein in step 2), the organic solvent is anhydrous ethanol.

8. Use of the fluorescent compound of claim 1 or 2 as a fluorescent probe for targeted imaging of Gram-positive bacteria.

9. The use of the fluorescent compound of claim 1 or 2 in the manufacture of an anti-Gram-positive bacteria drug.

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