Organic fluorescent compound as well as synthesis method and application thereof

Abstract

The invention discloses an organic fluorescent compound and a synthesis method and application thereof.The synthesis method includes the steps that reactants, first alkali and pyridine-4-boric acid are mixed, tetrakis (triphenylphosphine) palladium serving as a catalyst is added, then a 1, 4-dioxane aqueous solution is added, stirring is conducted for 10-15 h at the temperature of 90-105 DEG C under the protection of nitrogen or inert gas, the mixture is poured into ethyl acetate after being cooled to the room temperature, and the organic fluorescent compound is obtained. Extracting with water, drying the obtained organic layer, performing reduced pressure evaporation, and purifying to obtain a TPA2Py skeleton; the preparation method comprises the following steps: mixing a TPA2Py skeleton and a first solvent, adding iodomethane under the protection of nitrogen or inert gas, stirring at 60-80 DEG C for 10-15 hours, reducing pressure to remove the solvent to obtain a first crude product, uniformly distributing the first crude product in MeOH, and adding diethyl ether to obtain the organic fluorescent compound. The organic fluorescent compound can be effectively loaded in lipid droplets of mammalian cells, but does not generate toxicity to the mammalian cells, so that the sepsis caused by bacteria can be treated by adoptive transfer of macrophages through mammalian cell loading.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemistry and biomolecular technology, and particularly relates to an organic fluorescent compound, a synthesis method thereof, and an application thereof. Background Art

[0002] Sepsis is an immune response disorder to infection, which usually leads to organ dysfunction and death. The mortality rate of sepsis is alarmingly high and has been regarded as a major challenge in global healthcare, imposing a heavy burden on patients, caregivers, and the healthcare system. Clinically, antibiotic treatment is the standard treatment method in sepsis guidelines. However, due to the overuse and long-term abuse of antibiotics, antibiotic resistance is widespread, as indicated by the high clinical mortality rate caused by persistent infection during sepsis. Therefore, there is an urgent need for new therapeutic drugs that can eliminate pathogens without generating drug resistance and improve the prognosis of sepsis.

[0003] Clinical data show that more than 60% of sepsis patients initially survive the inflammatory storm but then progress to a long-term immunosuppressed state. The paralysis and death of immune cells lead to impaired ability to clear invading pathogens. Therefore, a great deal of effort has been made to identify potential therapeutic targets for inhibiting the immune disorders caused by infection, such as removing reactive oxygen species (ROS), such as H 2 O 2 、O 2- 、OH - etc., or some immune response glycoproteins, such as the anaphylatoxin C5a. Recently, strategies aimed at restoring immune function have been developed and tested in sepsis patients. An immunotherapy strategy that has attracted wide attention is adoptive cell transfer (ACT), which can potentially enhance pathogen clearance, reduce excessive inflammation, and restore immune balance. Various immune cells, such as T cells, macrophages, NK cells, and neutrophils, have been studied in ACT sepsis treatment, and promising results have been obtained in preclinical and early clinical studies. Among them, macrophages play a crucial role as effective pathogen scavengers during the infection process. Therefore, restoring or enhancing the immune function of macrophages during ACT would be an encouraging approach to promoting the eradication of infection. Currently, the nanodrugs used for adoptive transfer therapy are mainly loaded in the lysosomes of cells. However, many bacteria, such as Staphylococcus aureus and Escherichia coli, have evolved immune escape mechanisms to prevent lysosomal killing, resulting in intracellular survival and recurrent infection. Therefore, choosing other organelles to load nanodrugs is promising to provide better effects for adoptive cell transfer therapy of in vivo bacterial infections. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide an organic fluorescent compound, which can be efficiently loaded into macrophages and treat sepsis caused by bacteria through adoptive cell transfer therapy.

[0005] Another object of the present invention is to provide a synthesis method of the above-mentioned organic fluorescent compound. By modifying the TPA2Py skeleton with rigid hydrophobic groups (such as benzene, biphenyl, cumene, etc.), an organic fluorescent compound is obtained, and the organic fluorescent compound has high membrane penetration and strong bacterial affinity.

[0006] The object of the present invention is achieved by the following technical solutions.

[0007] An organic fluorescent compound, which is TPA2PyProp, TPA2PyBiPh or TPA2PyPh. The structural formula of TPA2PyBiPh is as follows:

[0008]

[0009] The structural formula of TPA2PyProp is as follows:

[0010]

[0011] The structural formula of TPA2PyPh is as follows:

[0012]

[0013] Wherein, in the structural formula of the organic fluorescent compound, R are each independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy and one or more groups capable of conjugating with one or more fluorescent substances.

[0014] A synthesis method of an organic fluorescent compound, comprising:

[0015] S1, mixing a reactant, a first base and pyridine-4-boronic acid, adding tetrakis(triphenylphosphine)palladium as a catalyst, then adding 1,4-dioxane aqueous solution, and stirring at 90-105 °C for 10-15 h under the protection of nitrogen or inert gas. After cooling to room temperature, pour it into ethyl acetate, extract with water, dry the obtained organic layer and evaporate under reduced pressure, and purify to obtain a yellow solid as the TPA2Py skeleton. Among them, in terms of the number of moles, the ratio of the reactant to pyridine-4-boronic acid is 2:(4-6), and in terms of the number of moles, the ratio of pyridine-4-boronic acid to the first base is 1:(1-1.5);

[0016] The structural formula of the reactant is: Each R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, and one or more groups capable of conjugating with one or more fluorescent substances. R' is

[0017] In S1, the first base is K 2 CO 3 , Na 2 CO 3 , potassium acetate, cesium carbonate, or sodium acetate.

[0018] In S1, based on the number of moles, the ratio of the reactant to tetrakis(triphenylphosphine)palladium is 2:(0.1 - 0.2).

[0019] In S1, the ratio of the number of moles of the reactant to the volume of the 1,4 - dioxane aqueous solution is less than or equal to 2:20. The unit of the number of moles is mmol, and the unit of the volume is mL.

[0020] In S1, the 1,4 - dioxane aqueous solution is a mixture of 1,4 - dioxane and water. Based on the volume, the ratio of 1,4 - dioxane to water in the 1,4 - dioxane aqueous solution is (4 - 5):1.

[0021] In S1, the ratio of the number of moles of the reactant to the volume of the ethyl acetate is less than or equal to 2:40. The unit of the number of moles is mmol, and the unit of the volume is mL.

[0022] In S1, purification is carried out by silica gel chromatography, using a mixture of ethyl acetate and n - hexane as the eluent. Based on the volume, the ratio of ethyl acetate to n - hexane is (3 - 5):1.

[0023] In the above technical solution, the organic layer in S1 is dried with NaSO 4 dry.

[0024] In the above technical solution, when the structural formula of the reactant is , the method for preparing the reactant is as follows: Add 1 mole fraction of compound b2, 2 - 5 mole fractions of potassium carbonate, and 1 - 1.2 mole fractions of benzyl bromide to 15 - 30 volume fractions of acetone, stir at 55 - 65 °C for 10 - 15 h. After the reaction is completed, filter under reduced pressure to remove the solid, obtain the filter cake, wash the filter cake with acetone, collect the organic phase and evaporate to dryness to obtain a crude white solid product. Purify the crude white solid product by silica gel chromatography to obtain the white solid as the reactant. Among them, the unit of the number of moles is mmol, the unit of the volume is mL, and the structural formula of benzyl bromide is R' is The structural formula of compound b2 is as follows:

[0025]

[0026] S2. Mix the TPA2Py skeleton and the first solvent, and under the protection of nitrogen or inert gas, add methyl iodide, stir at 60 - 80 °C for 10 - 15 h, remove the solvent under reduced pressure to obtain the first crude product as an orange solid. Distribute the first crude product evenly in MeOH, and then add diethyl ether to obtain an organic fluorescent compound. Among them, by the number of moles, the ratio of the TPA2Py skeleton to methyl iodide is 1:(2 - 2.5).

[0027] In S2, the first solvent is one of acetonitrile, tetrahydrofuran, and N,N - dimethylformamide.

[0028] In S2, the mass of MeOH is the minimum amount capable of dissolving the first crude product.

[0029] In S2, the ratio of the number of moles of the TPA2Py skeleton to the volume fraction of the first solvent is 1:

[0030] (50 - 150), the unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0031] In S2, by volume fraction, the ratio of the first solvent to diethyl ether is 1:(1 - 5).

[0032] The use of the organic fluorescent compound in the drug for treating sepsis caused by bacteria.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] In the present invention, the TPA2Py skeleton can significantly enhance the penetration of the organic fluorescent compound into the bacterial membrane through the modification of rigid hydrophobic groups. After penetrating the bacterial membrane, the TPA2Py skeleton can bind to the DNA in the bacterial nucleoid and induce the aggregation of DNA to achieve antibacterial effects. The organic fluorescent compound has significant inhibitory effects on both Gram - positive bacteria and Gram - negative bacteria. The MIC for Gram - positive bacteria S. aureus is 1 μM, and the MIC for Gram - negative bacteria E. coli is 5 μM. At the same time, the organic fluorescent compound can be effectively loaded in the lipid droplets of mammalian cells without being toxic to mammalian cells. Therefore, macrophage adoptive transfer can be used to treat sepsis caused by bacteria through mammalian cell loading. Brief Description of the Drawings

[0035] Figure 1: A is the normalized ultraviolet-visible absorption and emission spectra of the PBS solution of TPA2PyPh, B is the normalized ultraviolet-visible absorption and emission spectra of the PBS solution of TPA2PyBiPh, and C is the normalized ultraviolet-visible absorption and emission spectra of the PBS solution of TPA2PyProp;

[0036] Figure 2 : A is the antibacterial activity of TPA2PyPh against Staphylococcus aureus, B is the antibacterial activity of TPA2PyBiPh against Staphylococcus aureus, C is the antibacterial activity of TPA2PyProp against Staphylococcus aureus, D is the antibacterial activity of TPA2PyPh against Escherichia coli, E is the antibacterial activity of TPA2PyBiPh against Escherichia coli, and F is the antibacterial activity of TPA2PyProp against Escherichia coli; G is the cytotoxicity of TPA2PyPh against Raw 264.7 cells, H is the cytotoxicity of TPA2PyBiPh against Raw 264.7 cells, and I is the cytotoxicity of TPA2PyProp against Raw 264.7 cells;

[0037] Figure 3 : A is the confocal fluorescence image of Raw 264.7 cells incubated with TPA2PyPh and infected with GFP-S. aureus for 2 hours, B is the number of colonies of Raw 264.7 cells incubated in media with different concentrations of TPA2PyPh, and C is the survival rate of bacteria inside Raw 264.7 cells incubated with different concentrations of TPA2PyPh, with the cell / bacteria ratio being 1:20;

[0038] Figure 4 : A is the survival rate of mice in the PBS injection group, the untreated Raw 264.7 cell injection group, and the Raw 264.7 cell injection group incubated with TPA2PyPh, and B is the change in mouse weight within 10 days after injection of the liquid in the Raw 264.7 cell injection group incubated with TPA2PyPh. Detailed implementation mode

[0039] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0040] The synthesis route of TPA2PyPh in the following embodiments is as follows:

[0041]

[0042] The synthesis route of TPA2PyBiPh / TPA2PyProp in the following embodiments is as follows:

[0043]

[0044] Purity and source of purchase of drugs in the following examples:

[0045] Diphenylamine, 97%, Sigma Aldrich;

[0046] 4-Methoxytriphenylamine, 97%, BLDpharm;

[0047] N-Bromosuccinimide, 99%, Sigma Aldrich;

[0048] Boron tribromide, 1.0M in DCM, Sigma Aldrich;

[0049] K 2 CO 3 , 99%, Sigma Aldrich;

[0050] Pyridine-4-boronic acid, 90%, Sigma Aldrich;

[0051] Tetrakis(triphenylphosphine)palladium, 99%, Sigma Aldrich;

[0052] 4-(Bromomethyl)biphenyl, 98%, BLDpharm;

[0053] p-Isopropylbenzyl bromide, 98%, BLDpharm;

[0054] Methyl iodide, 99%, Sigma Aldrich;

[0055] Raw 264.7 cells were purchased from ATCC, TIB-71;

[0056] S. aureus was purchased from ATCC (Strain designation: NCTC 8532);

[0057] E. coli was purchased from ATCC (Strain designation: HB101);

[0058] Gentamicin: Purchased from sigma aldrich;

[0059] MRSA bacteria: Purchased from ATCC (Strain designation: F-182).

[0060] In the following examples, the temperature of the saturated saline solution was 21 - 25 °C.

[0061] The pH of PBS was 7.4.

[0062] Example 1

[0063] Synthesis of TPA2PyPh, including the following steps:

[0064] S1, Add compound a2 (954 mg, 2 mmol) (i.e., the reactant), K 2 CO 3 (i.e., the first base) (828 mg, 6 mmol) and pyridine-4-boronic acid (541.2 mg, 4.4 mmol) into a 100 mL round-bottom flask, then add tetrakis(triphenylphosphine)palladium (115.6 mg, 0.1 mmol) as a catalyst, and finally add 25 mL of 1,4-dioxane aqueous solution. The 1,4-dioxane aqueous solution is a mixture of 1,4-dioxane and water. By volume, the ratio of 1,4-dioxane to water in the 1,4-dioxane aqueous solution is 4:1. Seal the round-bottom flask and stir at 90 °C for 12 h under nitrogen protection. After cooling to room temperature, pour it into 100 mL of ethyl acetate, extract with water, and dry the extracted organic layer with NaSO 4 and evaporate under reduced pressure to obtain a crude product. Purify the crude product by silica gel chromatography. The eluent used for purification is a mixture of ethyl acetate and n-hexane (by volume, the ratio of ethyl acetate to n-hexane is 4:1) to obtain a yellow solid as the TPA2Py skeleton (compound a3,

[0065] N,N-bis(4-(pyridin-4-yl)phenyl)-[1,1'-biphenyl]-4-amine), with a yield of 683 mg and a yield of 71.9%. The structural formula of compound a2 is:

[0066] S2, Dissolve the TPA2Py skeleton (compound a3) (475 mg, 1 mmol) in 50 mL of the first solvent (acetonitrile), place it in a 100 mL round-bottom flask, seal the round-bottom flask, protect it with nitrogen, add methyl iodide (0.137 mL, 2.2 mmol) to the round-bottom flask under nitrogen, stir at 70 °C for 12 h, remove the solvent under reduced pressure to obtain a first crude product as an orange solid. Distribute the first crude product evenly in MeOH (the mass of MeOH is the minimum amount capable of dissolving the first crude product), and then add 100 mL of diethyl ether to obtain TPA2PyPh (orange solid). The yield of TPA2PyPh is 676 mg and the yield is 95%.

[0067] The NMR data of TPA2PyPh are as follows: 1 H NMR(400MHz,Methanol-d 4)δ8.77(d,J=6.3Hz,4H),8.32(d,J=6.4Hz,4H),8.00–7.96(m,4H),7.38(d,J=7.9Hz, 3H),7.30–7.26(m,7H),7.19–7.17(m,2H),7.12–7.09(m,2H),4.34(d,J=1.8Hz,6H).

[0068] Preparation method of compound a2: Compound a1 (1.6 g, 5 mmol) and 30 ml of DCM were added to a 100 ml round-bottom flask, cooled to 0°C with an ice bath, N-bromosuccinimide (1.72 g, 10 mmol) was dissolved in 30 mL of DCM and added dropwise to the round-bottom flask through a dropping funnel, stirred in an ice bath at 0°C in the dark for 1 hour, then warmed to room temperature and stirred in the dark for 12 hours, the reaction was quenched with water, extracted with DCM, the extracted organic layer was washed with water and heated in NaSO 4 The crude product was purified by silica gel chromatography. The eluent used for purification was a mixture of DCM and n-hexane (Hex). The ratio of DCM to n-hexane (Hex) was 1 / 10 to 1 / 4 by volume (gradient elution). A white solid was obtained as compound a2.

[0069] (N,N-bis(4-bromophenyl)-[1,1'-biphenyl]-4-amine), the yield of compound a2 is 2.23 g, and the yield is 94%.

[0070] Preparation method of compound a1: diphenylamine (1.69 g, 10 mmol), 4-iodobiphenyl (2.8 g, 10 mmol), bistriphenylphosphine palladium dichloride (112 mg, 0.5 mmol), potassium tert-butoxide (t-BuOK, 20 mmol) and tri-tert-butylphosphine tetrafluoroborate (290 mg, 1 mmol) were mixed in a 100 mL round-bottom flask, and 50 mL of toluene was added to dissolve it. The round-bottom flask was sealed and protected with nitrogen, and heated in an oil bath at 90°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature and poured into 100 mL of ethyl acetate, and the organic phase was extracted with saturated brine. The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain a crude product. The crude product was purified by silica gel column chromatography. The eluent used for purification was a mixture of DCM and n-hexane. The ratio of DCM to n-hexane was 1:10 by volume, and a white solid was obtained as compound a1.

[0071] (N,N-diphenyl-[1,1'-biphenyl]-4-amine), the yield of compound a1 was 1.8 g, and the yield was 56.8%.

[0072] Example 2

[0073] Synthesis of TPA2PyBiPh / TPA2PyProp includes the following steps:

[0074] S1, Add 0.5 mmol of compound b3 (i.e., the reactant), 1.5 mmol of K 2 CO 3 (i.e., the first base) and 1.1 mmol of pyridine-4-boronic acid into a 100 mL round-bottom flask, then add tetrakis(triphenylphosphine)palladium (0.025 mmol) as a catalyst, and finally add 25 mL of 1,4-dioxane aqueous solution. The 1,4-dioxane aqueous solution is a mixture of 1,4-dioxane and water. By volume, the ratio of 1,4-dioxane to water in the 1,4-dioxane aqueous solution is 4:1. Seal the round-bottom flask and stir at 90 °C for 12 h under nitrogen protection. After cooling to room temperature, pour it into 100 mL of ethyl acetate, extract with water, and dry the extracted organic layer with NaSO 4 and evaporate under reduced pressure. The crude product is purified by silica gel chromatography. The eluent used for purification is a mixture of ethyl acetate and n-hexane (by volume, the ratio of ethyl acetate to n-hexane is 4:1) to obtain a yellow solid of TPA2Py skeleton (compound b4). The structural formula of compound b3 is: When R’ is , the yield of compound b4 is 73%. When R’ is , the yield of compound b4 is 68%. S2, Dissolve 0.2 mmol of TPA2Py skeleton (compound b4) in 50 mL of the first solvent (acetonitrile), place it in a 100 mL round-bottom flask, seal the round-bottom flask, protect it with nitrogen, add 0.44 mmol of iodomethane to the round-bottom flask under nitrogen, stir at 70 °C for 12 h, remove the solvent under reduced pressure to obtain the first crude product as an orange solid. Distribute the first crude product evenly in MeOH (the mass of MeOH is the minimum amount capable of dissolving the first crude product), and then add 100 mL of diethyl ether to obtain an organic fluorescent compound (orange solid). When R’ is , the obtained organic fluorescent compound is TPA2PyBiPh, and the yield of TPA2PyBiPh is 95%. When R’ is , the obtained organic fluorescent compound is TPA2PyProp, and the yield of TPA2PyProp is 91%.

[0075] TPA2PyBiPh: 1 H NMR (600 MHz, Methanol-d 4 ) δ8.77 (d, J = 6.6 Hz, 4H), 8.32–8.29

[0076] (m, 4H), 7.99–7.96 (m, 4H), 7.66–7.60 (m, 4H), 7.54 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.36–7.32 (m, 1H), 7.29–7.26 (m, 4H), 7.20–7.17 (m, 2H), 7.14–7.12 (m, 2H), 5.18 (s, 2H), 4.33 (s, 6H).

[0077] TPA2PyProp: 1 H NMR (600 MHz, Methanol-d 4 ) δ 8.76 (d, J = 6.6 Hz, 4H), 8.32–8.29

[0078] (m, 4H), 7.99–7.96 (m, 4H), 7.37 (d, J = 7.9 Hz, 2H), 7.27 (t, J = 8.6 Hz, 6H), 7.19–7.16 (m, 2H), 7.10 (d, J = 9.0 Hz, 2H), 5.09 (s, 2H), 4.33 (s, 6H), 2.91 (m, 1H), 1.25 (d, J = 7.0 Hz, 6H).

[0079] Preparation method of compound b3: Add 1 mmol of compound b2, 3 mmol of potassium carbonate, and 1.1 mmol of benzyl bromide into a round-bottom flask containing 20 mL of acetone, stir at 60 °C for 12 h. After the reaction is completed, filter off the solid under reduced pressure to obtain a filter cake, wash the filter cake with acetone (10 mL, 3 times), collect the organic phase and rotary evaporate to dryness to obtain a crude white solid product. Purify the crude white solid product by silica gel chromatography, and the eluent used for purification is a mixture of DCM and n-hexane (Hex) (by volume, the ratio of DCM to n-hexane (Hex) is 1:10) to obtain a white solid as compound b3. The structural formula of benzyl bromide is R' is When R' is , the yield of compound b3 is 91%, and when R' is , the yield of compound b3 is 94%.

[0080] Preparation method of compound b2: Add compound b1 (866 mg, 2 mmol) into a 50 mL round-bottom flask, and add 20 mL of ultradry DCM to dissolve compound b1. After cooling to 0 °C in an ice bath, add 3 mmol of boron tribromide dropwise. After the addition is complete, stir in the ice bath for 30 min first, then raise the temperature to room temperature and continue stirring for 12 h. After the reaction is completed, slowly add methanol dropwise to the solution to quench the reaction, add water to extract the reaction solution, collect the organic phase and concentrate to obtain a gray crude product. Purify the gray crude product by silica gel chromatography. The eluent used for purification is a mixture of DCM and n-hexane (Hex) (by volume, the ratio of DCM to n-hexane (Hex) is 1:1), and the gray solid obtained is compound b2

[0081] (4-(bis(4-bromophenyl)amino)phenol), with a yield of 544 mg and a yield rate of 64.8%.

[0082] Preparation method of compound b1: Add 4-methoxytriphenylamine (1.38 g, 5 mmol) and 30 mL of DCM into a 100 mL round-bottom flask, cool to 0 °C in an ice bath, dissolve N-bromosuccinimide (1.72 g, 10 mmol) in 30 mL of DCM, and add it dropwise to the round-bottom flask through a dropping funnel. Stir in the ice bath at 0 °C in the dark for 1 h, raise the temperature to room temperature and stir in the dark for 12 h, quench the reaction with water, extract with DCM, wash the organic layer with water and dry it over NaSO 4 Dry, evaporate the solvent under reduced pressure, purify the crude product by silica gel chromatography. The eluent used for purification is a mixture of DCM and n-hexane (Hex). By volume, the ratio of DCM to n-hexane (Hex) is 1 / 10 - 1 / 4 (gradient elution), and the white solid obtained is compound b1 (4-bromo-N-(4-bromophenyl)-N-(4-methoxyphenyl)aniline). The yield of compound b1 is 1.96 g and the yield rate is 91%. Mix the organic fluorescent compound and DMSO to prepare a DMSO stock solution with a concentration of 1 mM of the organic fluorescent compound. The organic fluorescent compound is one of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp. Then, take 10 μL of the DMSO stock solution and add it to 0.99 mL of PBS to prepare a PBS solution with a concentration of 10 μM of the organic fluorescent compound to test the photophysical properties of the organic fluorescent compound. As Figure 1 shown in A - C of Figure 1Among A to B, Abs. is the normalized ultraviolet-visible spectral absorption and PL is the emission spectrum. TPA2PyPh, TPA2PyBiPh, and TPA2PyProp can dissolve well in PBS solution and exhibit a broad absorption spectrum from 350 nm to 500 nm. TPA2PyPh, TPA2PyBiPh, and TPA2PyProp all have a broad emission spectrum in PBS solution, with the emission coverage ranging from 500 nm to 750 nm and the peak around 620 nm.

[0083] To evaluate the antibacterial effects of organic fluorescent compounds (TPA2PyPh, TPA2PyBiPh, and TPA2PyProp), the minimum inhibitory concentration (MIC) of the organic fluorescent compounds against Gram-negative bacteria and Gram-positive bacteria was measured. MIC was defined as the lowest concentration of the organic fluorescent compounds at which no obvious bacterial growth was observed after culturing at 37 °C for 14 hours. 10 5 CFU of Staphylococcus aureus or Escherichia coli was inoculated into 100 μL of LB medium containing different concentrations of the organic fluorescent compounds, which were TPA2PyPh, TPA2PyBiPh, or TPA2PyProp, and then the absorbance (OD 600 ) of the LB medium at 600 nm was measured after incubation at 37 °C for 14 hours at a rotation speed of 220 rpm. As Figure 2 shown in A to C (S.aureus in the figure is Staphylococcus aureus), TPA2PyPh, TPA2PyBiPh, and TPA2PyProp showed a general inhibitory effect on the growth of Staphylococcus aureus, and the MICs of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp were all 1 μM. On the other hand, as Figure 2 shown in D to F (E.coli in the figure is Escherichia coli), TPA2PyPh, TPA2PyBiPh, and TPA2PyProp could significantly inhibit the growth of the Gram-negative bacterium Escherichia coli, and the MICs of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp were all 5 μM. This indicates that the antibacterial activities of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp are not specific to Gram-negative / positive bacteria.

[0084] Raw 264.7 cells were incubated in DMEM medium containing different concentrations of organic fluorescent compounds as dyes in a 37 °C, 5% carbon dioxide incubator for 48 h. The organic fluorescent compound was one of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp. The incubated Raw 264.7 cells were placed in different concentrations of the dye, and the dye concentrations were 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM. The growth of Raw 264.7 cells incubated within the range of dye concentration below 20 μM was not significantly inhibited( Figure 2 G-I), indicating the low cytotoxicity of TPA2PyPh, TPA2PyBiPh, and TPA2PyProp.

[0085] To verify that the organic fluorescent compounds (TPA2PyPh, TPA2PyBiPh, or TPA2PyProp) still have the ability to target bacteria after being loaded in macrophages, taking TPA2PyPh as an example, a fluorescence colocalization experiment was carried out by confocal microscopy. Raw 264.7 cells (500,000 - 600,000 cells / well) were inoculated in a confocal dish with DMEM medium and incubated in a 37 °C, 5% carbon dioxide cell incubator for 10 h. The DMEM medium was discarded and DMEM medium containing 5 μM TPA2PyPh was added, and incubation was continued under the same conditions for 3 h to allow TPA2PyPh to be fully loaded in Raw 264.7 cells. After the 3-h incubation, the DMEM medium was discarded, washed three times with PBS, and DMEM medium containing 5 μM TPA2PyPh was added. Staphylococcus aureus labeled with green fluorescent protein (GFP-S.aureus) was added at a multiplicity of infection of ~20. After 2 h of bacterial infection, the DMEM medium containing 5 μM TPA2PyPh was discarded and washed three times with PBS. Finally, the nuclei were labeled with DMEM medium containing Hoechst (1 μM). The intracellular bacterial imaging is as shown in Figure 3 A of (Hoechst, GFP–SA, TPA2PyPh, merged, Bright field). The green fluorescence of green fluorescent protein (GFP) and the red fluorescence of TPA2PyPh could overlap well, indicating that TPA2PyPh loaded in Raw 264.7 cells could still bind well to bacteria.

[0086] Furthermore, to prove that TPA2PyPh loaded in macrophages still retains the ability to kill bacteria, Raw 264.7 cells (500,000 - 600,000 cells / well) were first seeded in a 6-well plate and incubated with DMEM medium containing different concentrations of TPA2PyPh (0, 0.5, 1, 2, 5, 10 μM) for 3 h. Raw 264.7 cells were infected with Staphylococcus aureus at a multiplicity of infection of ~20. Two hours after bacterial infection, the cells were washed three times with PBS and then incubated with DMEM medium containing 50 μg / mL gentamicin for 1 h to remove residual bacteria outside Raw 264.7 cells. After washing three times with PBS, the cells were lysed with PBS containing 10% Triton X-100 for 10 min and the number of bacteria inside Raw 264.7 cells was counted by plate counting method. As Figure 3 shown in B - C of Figure 3 , as the concentration of TPA2PyPh increased, the colony number of macrophages incubated with TPA2PyPh (TPP-RAW) decreased significantly, and the survival rate of bacteria inside Raw 264.7 cells decreased significantly, indicating that after macrophages were loaded with TPA2PyPh, macrophages could more effectively kill the phagocytosed bacteria. These results suggest that TPA2PyPh still has the ability to bind to bacteria and antibacterial effect after being loaded in macrophages.

[0087] To prove that macrophages incubated with TPA2PyPh can treat septic mice, a mouse sepsis model was constructed. The incubation method of TPA2PyPh was as follows: Raw 264.7 cells (500,000 - 600,000 cells / well) were seeded in a plastic culture dish with DMEM medium and incubated in a 37 °C, 5% carbon dioxide cell culture incubator until 80 - 90% of the bottom of the culture dish was covered with cells. The DMEM medium was discarded and DMEM medium containing 10 μM TPA2PyPh was added, and incubation was continued under the same conditions for 3 h to allow TPA2PyPh to be fully loaded in Raw 264.7 cells, obtaining Raw 264.7 cells incubated with TPA2PyPh.

[0088] The mice were divided into three groups: PBS injection group (PBS), untreated Raw 264.7 cell injection group (PBS-RAW), and Raw 264.7 cell injection group incubated with TPA2PyPh (TPP-Raw). Cyclophosphamide (dose: 100 mg / kg, frequency: once a day) was injected into the three groups of mice continuously for three days from Day 1 to Day 3 to make the mice reach an immunosuppressed state. On Day 4, MRSA bacteria (0.1 mL, 5X10 7CFU) induced sepsis in mice. Subsequently, the same dose of 0.1 mL of liquid was injected intraperitoneally and via the tail vein into three groups of mice. The liquids injected into the PBS injection group (PBS), the untreated Raw 264.7 cell injection group (PBS-RAW), and the Raw 264.7 cell injection group incubated with TPA2PyPh (TPP-Raw) were: PBS, PBS containing 10 6 Raw 264.7 cells, and PBS containing 10 6 Raw 264.7 cells incubated with TPA2PyPh. As Figure 4 shown in A of Figure 4 , the survival rate of mice treated in TPP-Raw after 10 days was significantly higher than that of mice treated in the PBS injection group and PBS-RAW. In addition, as

[0089] shown in B of

[0090] , the body weight of mice treated with TPP-Raw increased significantly after the third day and gradually returned to the normal level, indicating that macrophages loaded with TPA2PyPh could effectively relieve the sepsis state of mice and significantly improve the survival rate of septic mice. Based on these findings, TPA2PyPh can be loaded into the lipid droplets of macrophages to enhance the immune function of macrophages during adoptive transfer therapy for sepsis caused by bacteria, thereby overcoming immune escape and drug resistance. TPA2PyPh was designed as a cationic compound with a biphenyl unit of high hydrophobicity, which helps TPA2PyPh effectively penetrate cell membranes and be loaded into the lipid droplets of macrophages. After macrophages phagocytize bacteria, TPA2PyPh will be absorbed and further inserted into the nucleic acids of bacteria, resulting in DNA aggregation and then inducing bacterial death.

[0090] The above has made an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification, or equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. An organic fluorescent compound, characterized in that it is TPA2PyProp, TPA2PyBiPh or TPA2PyPh, and the structural formula of TPA2PyBiPh is as follows: The structural formula of TPA2PyProp is as follows: The structural formula of TPA2PyPh is as follows: Wherein, in the structural formula of the organic fluorescent compound, each R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy and one or more groups capable of conjugating with one or more fluorescent substances.

2. The synthesis method of the organic fluorescent compound according to claim 1, characterized in that it includes: S1, Mix the reactant, the first base and pyridine-4-boronic acid, add tetrakis(triphenylphosphine)palladium as a catalyst, then after adding 1,4-dioxane aqueous solution, under the protection of nitrogen or inert gas, stir at 90-105 °C for 10-15 h, cool to room temperature and pour into ethyl acetate, extract with water, dry the obtained organic layer and evaporate under reduced pressure, purify to obtain a yellow solid as the TPA2Py skeleton, wherein, by the number of moles, the ratio of the reactant to pyridine-4-boronic acid is 2:(4-6), and by the number of moles, the ratio of pyridine-4-boronic acid to the first base is 1:(1-1.5); The structural formula of the reactant is: Each R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, and one or more groups capable of conjugating with one or more fluorescent substances. R' is S2, Mix the TPA2Py skeleton and the first solvent, under the protection of nitrogen or inert gas, add methyl iodide, stir at 60-80 °C for 10-15 h, remove the solvent under reduced pressure to obtain the first crude product as an orange solid, uniformly distribute the first crude product in MeOH, and then add diethyl ether to obtain the organic fluorescent compound, wherein, by the number of moles, the ratio of the TPA2Py skeleton to methyl iodide is 1:(2-2.5).

3. The synthesis method of the organic fluorescent compound according to claim 2, characterized in that When the structural formula of the reactant is The method for preparing the reactant is as follows: Add 1 molar part of compound b2, 2 - 5 molar parts of potassium carbonate, and 1 - 1.2 molar parts of benzyl bromide into 15 - 30 volume parts of acetone, stir at 55 - 65 °C for 10 - 15 h. After the reaction is completed, filter under reduced pressure to remove the solid, obtain the filter cake, wash the filter cake with acetone, collect the organic phase and rotary evaporate to dryness to obtain a crude white solid product. Purify the crude white solid product by silica gel chromatography to obtain a white solid as the reactant, wherein the unit of the molar part is mmol, the unit of the volume part is mL, and the structural formula of the benzyl bromide is R’ is The structural formula of compound b2 is as follows:

4. The synthesis method of the organic fluorescent compound according to claim 2 or 3, characterized in that In S1, by the number of moles, the ratio of the reactant to tetrakis(triphenylphosphine)palladium is 2:(0.1-0.2).

5. The synthesis method of the organic fluorescent compound according to claim 2 or 3, characterized in that In S1, the first base is K 2 CO 3 , Na 2 CO 3 , potassium acetate, cesium carbonate or sodium acetate; in S2, the first solvent is one of acetonitrile, tetrahydrofuran and N,N-dimethylformamide.

6. The synthesis method of the organic fluorescent compound according to claim 2 or 3, characterized in that In S2, the ratio of the number of moles of the TPA2Py skeleton to the volume fraction of the first solvent is 1:(50-150), the unit of the number of moles is mmol, and the unit of the volume fraction is mL.

7. The synthesis method of the organic fluorescent compound according to claim 2 or 3, characterized in that In S2, the mass of the MeOH is the minimum amount capable of dissolving the first crude product; in S2, by volume fraction, the ratio of the first solvent to diethyl ether is 1:(1-5).

8. The synthesis method of the organic fluorescent compound according to claim 2 or 3, characterized in that In S1, the ratio of the number of moles of the reactant to the volume fraction of the 1,4-dioxane aqueous solution is less than or equal to 2:

20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

9. The method for synthesizing an organic fluorescent compound according to claim 2 or 3, characterized in that in S1, the ratio of the number of moles of the reactant to the volume fraction of the ethyl acetate is less than or equal to 2:

40. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

10. Use of the organic fluorescent compound according to claim 1 in the medicine for treating sepsis caused by bacteria.

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