D-pi-a type organic antibacterial photosensitizer and synthesis method and application thereof

Abstract

The application provides a D-pi-A type organic antibacterial photosensitizer and a synthesis method and application thereof, and belongs to the field of photosensitizers and antibacterial photodynamic therapy; the application takes methoxy-substituted triphenylamine as an electron donor, pyridine salt as an electron acceptor, and thiophene as an electron pi bridge; through a series of simple organic reactions, an AIE type photosensitizer with bacterial cell selectivity is synthesized, and the photosensitizer can be applied to antibacterial photodynamic therapy; the photosensitizer can realize bacterial imaging and selectively exert photodynamic effect on bacteria by connecting a hydrophilic group, has good therapeutic effect and biological safety, and has strong application prospect in antibacterial photodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of photosensitizer technology, specifically to a D-π-A type organic antibacterial photosensitizer, its synthesis method, and its application. Background Technology

[0002] Photodynamic therapy, as a novel antibacterial therapy, is characterized by its non-invasiveness, lack of drug resistance, low cytotoxicity, selective targeting, spatiotemporal precision, and synergistic effects.

[0003] Photodynamic therapy is a novel method for treating bacterial infections using photosensitizing drugs and laser activation. Irradiating the bacterial infection site with light of a specific wavelength activates photosensitizing drugs that selectively enter the bacteria, triggering a photochemical reaction that produces reactive oxygen species (ROS). These ROS can then kill the bacteria. When the photosensitizer absorbs light, it transforms into a triplet excited state. 3 PS*, then with adjacent photosensitive molecules 1 Hydrogen atom or electron transfer occurs between PS to form ionic free radicals (PS). -· PS +· ); Ionic free radicals (PS) -· PS +· Through a series of reactions, hydroxyl radicals (OH·) are eventually formed. These radicals can interfere with the biological functions of nucleic acids, fatty acids, and amino acids, thereby inducing bacterial death. This process is called type I photodynamic therapy; energy is directly transferred from the triplet excited state. 3 PS* is transferred to ground-state molecular oxygen ( 3 O2), 3 O2 absorbs energy and transforms into singlet oxygen. 1 O2). Singlet oxygen can oxidize biologically active macromolecules, inducing oxidative stress in bacteria, thereby inducing bacterial death. This process is known as type II photodynamic therapy.

[0004] Many reported antimicrobial photosensitizers, upon entering bacteria, readily accumulate in living mammalian cells, exhibiting a significant degree of invasiveness towards these cells. Due to the differences in surface composition and structure between bacterial and mammalian cells, the amphiphilicity of drugs greatly influences their hydrophobic interactions with organisms and biological components. Therefore, eliminating the invasiveness of antimicrobial drugs to living mammalian cells is a crucial task in meeting biosafety and accuracy requirements. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology, and to provide a D-π-A type organic antibacterial photosensitizer, its synthesis method, and its application. The technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides a D-π-A type organic antibacterial photosensitizer, the structural formula of which is:

[0007]

[0008] Wherein: the electron-donating group is a methoxy-substituted triphenylamine group; the electron-withdrawing group is a pyridinium salt ion.

[0009] A second aspect of the present invention provides a method for synthesizing the D-π-A type organic antibacterial photosensitizer as described above, the chemical formula of which is as follows:

[0010]

[0011] The method includes the following steps:

[0012] (1) Using N,N-dimethylformamide as a solvent, compound 1, valeric acid, cesium carbonate, and 5 moles of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were added. After the reactants were mixed evenly, the mixture was stirred at room temperature. After the reaction was completed, deionized water was added to the reaction solution, and the mixture was extracted multiple times with dichloromethane. The organic phases obtained from the extraction were combined, dried with anhydrous magnesium sulfate, filtered, and rotary evaporated to remove the organic solvent, yielding a crude product. The crude product was purified by gradient elution using column chromatography to obtain an organic antibacterial photosensitizer named OTTPy-Va. The structural formula of this photosensitizer is:

[0013]

[0014] Thirdly, the present invention also provides the application of the aforementioned D-π-A type organic antibacterial photosensitizer in the preparation of photodynamic antibacterial therapeutic drugs, and the application of the aforementioned D-π-A type organic antibacterial photosensitizer in selectively killing bacteria in bacteria and normal cells, wherein the bacteria are Staphylococcus aureus, and the normal cells are human umbilical vein endothelial cells and epidermal cells; in application, the present D-π-A type organic antibacterial photosensitizer can enter the bacteria and kill the bacteria by white light irradiation, and can be used in bacterial imaging and photodynamic therapy; the present photosensitizer has good biocompatibility.

[0015] The beneficial effects of this invention are as follows:

[0016] (1) The present invention uses a methoxy-substituted triphenylamine group as an electron donor and connects it to a pyridine salt group with strong electron absorption capacity through a thiophene group as a π bridge. This can accelerate intramolecular electron transfer, accelerate intersystem crossing process, and promote the occurrence of photodynamic reaction. This organic antibacterial photosensitizer is synthesized for the first time in the present invention. The raw materials are readily available, the process is simple, it can be industrialized, and it has strong commercial value.

[0017] (2) The D-π-A type organic antibacterial photosensitizer provided by the present invention has a large Stokes shift and excellent AIE performance, and has the potential for biofluorescence imaging.

[0018] (3) The D-π-A type organic antibacterial photosensitizer provided by the present invention can be activated to generate a large amount of active oxygen under white light irradiation, and the generation rate is faster than that of commercial dye RB.

[0019] (4) The D-π-A type organic antibacterial photosensitizer provided by the present invention can enter bacteria and emit red fluorescence.

[0020] (5) The amide group on the right side of the pyridine salt and the methoxy group on the triphenylamine of the D-π-A type organic antibacterial photosensitizer provided by the present invention play a role in regulating the water solubility of the molecule, so that it can selectively produce strong phototoxicity to bacteria, and has excellent therapeutic effect and good biological safety.

[0021] In summary, this invention provides a D-π-A type organic antibacterial photosensitizer. This organic antibacterial photosensitizer is simple to prepare and can selectively exert photodynamic effects within bacteria. Compared with commercial photosensitizers, the organic antibacterial photosensitizer provided by this invention has a larger Stokes shift, which can be used for intrabacterial fluorescence imaging; it has a higher and faster reactive oxygen species generation capacity; and it can selectively enter bacteria from normal cells and bacteria to exert photodynamic therapeutic effects, greatly improving the treatment effect of bacterial infections. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0023] Figure 1 This is the synthesis route diagram for Example 1;

[0024] Figure 2 In (a), the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 is in DMF / Toluene (v / v = 1 / 99) (concentration: 1 × 10⁻⁶). -5 The UV-Vis absorption spectrum of M). Figure 2 (b) shows the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 in DMF / Toluene (v / v = 1 / 99) (concentration: 1 × 10⁻⁶). -5 Fluorescence emission spectrum in M);

[0025] Figure 3In (a), it is the fluorescence emission diagram of the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 in DMSO / Toluene solvent (concentration: 1×10 -5 M) at different volume ratios of DMSO / Toluene, Figure 3 In (b), it is Figure 3 the quantitative dot line diagram of the fluorescence intensity in (a);

[0026] Figure 4 In (a), it is the diagram showing the enhanced fluorescence intensity of the ROS indicator DCFH by the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 under white light illumination, Figure 4 in (b), it is the diagram showing the enhanced fluorescence intensity of DCFH by the commercial dye Rose Bengal under the same conditions; Figure 4 in (c), it is the fluorescence intensity diagram of the ROS indicator DCFH under white light illumination; Figure 4 in (d), it is Figure 4 the quantitative dot line diagram of the fluorescence intensity at 525 nm in (a-c);

[0027] Figure 5 It is the confocal fluorescence imaging diagram of the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 in Staphylococcus aureus; it includes the blue fluorescence diagram of the commercial nuclear dye DAPI; the red fluorescence diagram of the synthesized organic antibacterial photosensitizer OTTPy-Va; the bright field diagram of the bacteria; the overlay diagram of the three; scale bar: 5 μm.

[0028] Figure 6 The organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 was subjected to cell imaging experiments, and normal cells Hacat cells (human immortalized epidermal cells) and normal cells HUVEC cells (human umbilical vein endothelial cells) were selected respectively. The first row is the confocal fluorescence images taken after the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 was incubated with HUVEC cells for 15, 30, 60, 120 min; the second row is the confocal fluorescence images taken after the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 was incubated with Hacat cells for 15, 30, 60, 120 min; scale bar: 100 μm.

[0029] Figure 7 The antibacterial experiment of the organic antibacterial photosensitizer OTTPy-Va of Example 1 against Staphylococcus aureus was carried out; among them Figure 7 in (a), it is the plate coating diagram of different drug concentrations against Staphylococcus aureus; Figure 7 in (b), it is the quantitative diagram of the plate coating experiment. Detailed implementation mode

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and Embodiment 1 and Test Examples 1-7.

[0031] Example 1

[0032] A D-π-A type organic antibacterial photosensitizer is used in the synthesis of antibacterial photodynamic therapy and the selective bactericidal drug OTTPy-Va.

[0033] (1) Synthesis of photosensitizer OTTPy-Va:

[0034]

[0035] Take a 50 mL round-bottom flask, add 20 mL of DMF as solvent, then add compound 1 (70 mg, 0.1091 mmol), valeric acid (22.3 mg, 0.2184 mmol), and cesium carbonate (106 mg).

[0036] mg (0.3273 mmol) and HATU (207 mg, 0.5455 mmol). The reaction solution was mixed evenly and reacted at room temperature for 24 h. After the reaction was completed, 30 mL of deionized water was added to the reaction solution, and the mixture was extracted multiple times with dichloromethane. The organic phases obtained from the extraction were combined, dried with anhydrous magnesium sulfate, filtered, and rotary evaporated to remove the organic solvent to obtain the crude product. The crude product was purified by neutral alumina column chromatography using (dichloromethane / methanol, v / v = 25 / 1) as the eluent to obtain a deep purple compound in 60% yield. The organic dye OTTPy-Va was prepared. NMR: 1 H NMR (400MHz, DMSO-d6) δ8.86 (d, J=7.0

[0037] Hz,2H),8.24–8.14(m,3H),7.82(t,J=5.6Hz,1H),7.56–7.41(m,4H),7.09

[0038] (d,J=8.9Hz,5H),6.95(d,J=8.9Hz,4H),6.77(d,J=8.8Hz,2H),4.46(t,J=

[0039] 7.3Hz, 2H), 3.76(s, 6H), 3.07(q, J = 7.0Hz, 2H), 2.04(t, J = 7.4Hz, 2H), 1.87(t, J = 7.0Hz, 2H), 1.49–1.37(m, 4H), 1.26(d, J = 7.6Hz, 2H), 0.85(t, J = 7.3Hz, 3H). The structural formula of the organic dye OTTPy-Va is:

[0040]

[0041] Test Example 1

[0042] like Figure 2 As shown, the UV-Vis absorption and fluorescence emission spectra of the organic antibacterial photosensitizer OTTPy-Va from Example 1 were tested. The UV-Vis absorption spectrum of the organic antibacterial photosensitizer OTTPy-Va in DMF / toluene (v / v = 1:99) is shown below. Figure 2 As shown in (a), the organic antibacterial photosensitizer OTTPy-Va has a maximum absorption wavelength of 526 nm, and its absorption range covers most of the visible light region. Figure 2 (b) shows the fluorescence emission spectrum of the organic antibacterial photosensitizer OTTPy-Va in DMF / toluene (v / v = 1:99). The maximum fluorescence emission peak of the photosensitizer is around 670 nm, which shows good potential for biofluorescence imaging.

[0043] Test Example 2

[0044] like Figure 3 As shown, the AIE performance of the organic antibacterial photosensitizer OTTPy-Va from Example 1 was tested. Figure 3 As shown in (a), the AIE performance of the organic antibacterial photosensitizer OTTPy-Va was tested in DMSO / Toluene. From Figure 3 As can be seen in (a), the fluorescence intensity of the solution gradually increases with the increase of the proportion of toluene in the system. Figure 3 (b) is Figure 3 (a) I / I0 quantification dot plot of fluorescence. Where I represents the fluorescence intensity of the organic antibacterial photosensitizer OTTPy-Va at 670 nm in different DMSO / Toluene systems, and I0 represents the initial fluorescence intensity of the organic antibacterial photosensitizer OTTPy-Va at 670 nm. Figure 3 As can be seen in (b), the fluorescence intensity of OTTPy-Va was enhanced by about 75 times, demonstrating excellent AIE performance.

[0045] Test Example 3

[0046] like Figure 4 As shown, the in vitro ROS generation capacity of the organic antibacterial photosensitizer OTTPy-Va from Example 1 was tested. A commercially available photosensitizer with good biocompatibility and clinical applicability, 2-7-dichlorodihydrofluorescein (DCFH-DA), was used as the ROS indicator. DCFH-DA is activated to form DCFH for use. Figure 4 As shown in (a), a mixture of the organic antibacterial photosensitizer OTTPy-Va and DCFH under white light (25 mW / cm²) 2Under irradiation, fluorescence emission gradually increased at a wavelength of 525 nm, reaching its maximum fluorescence at approximately 60 seconds, indicating that the organic antibacterial photosensitizer OTTPy-Va has the ability to generate reactive oxygen species in vitro. Figure 4 As shown in (b), the commercially available dye Rose Bengal was used as a positive control, and its fluorescence emission under white light was detected after mixing with DCFH. Figure 4 As shown in (c), when the DCFH solution is placed under the same light conditions, almost no increase in fluorescence emission is detected, which eliminates the possible influence of the indicator on the experimental results and indirectly shows that the ROS indicator DCFH has good stability. Figure 4 (d) is Figure 4 A dot plot of I / I0 quantification of mid-ac fluorescence. Where I represents the fluorescence of the organic dye OTTPy-Va and DCFH mixture at 525 nm after different illumination times, and I0 represents the fluorescence of the organic antibacterial photosensitizer OTTPy-Va and DCFH mixture at 525 nm without illumination. Figure 4 As shown in (d), the ROS generation efficiency of the organic antibacterial photosensitizer OTTPy-Va is higher than that of the commercial dye Rose Bengal, indicating that the organic antibacterial photosensitizer OTTPy-Va exhibits excellent ROS generation performance.

[0047] Test Example 4

[0048] like Figure 5 As shown, bacterial fluorescence imaging experiments were performed on the organic antibacterial photosensitizer OTTPy-Va from Example 1. Staphylococcus aureus was incubated with 5 μM of OTTPy-Va for 15, 30, 60, and 120 minutes, and the location of the photosensitizer was confirmed by comparing the fluorescence. The commercially available bacterial nuclear dye DAPI was used to indicate the bacterial location, exhibiting blue fluorescence; the organic antibacterial photosensitizer OTTPy-Va penetrated the bacteria, exhibiting red fluorescence; the bright field image shows the bacteria observed under white light; the Merge plot is an image of the three channels overlapping; when the incubation time reached 15 minutes, the drug could penetrate the bacteria, showing obvious red fluorescence.

[0049] Test Example 5

[0050] like Figure 6As shown, cell imaging experiments were performed on the organic antibacterial photosensitizer OTTPy-Va from Example 1. Hacat cells (human immortalized epidermal cells) and normal HUVEC cells (human umbilical vein endothelial cells) were used for testing. Normal cells were incubated with 5 μM of the organic antibacterial photosensitizer OTTPy-Va for 15, 30, 60, and 120 minutes, and then images were taken using a fluorescence microscope. When the drug was incubated with both types of cells for 120 minutes, no reverse red fluorescence was observed, indicating that OTTPy-Va had almost not entered the cells.

[0051] Test Example 6

[0052] like Figure 7 As shown, a photodynamic antibacterial experiment was conducted on the organic antibacterial photosensitizer OTTPy-Va from Example 1. The survival rate of Staphylococcus aureus incubated with different concentrations of the organic dye OTTPy-Va shown in the figure under light and dark conditions was detected using the plate coating method. This was to investigate the photodynamic therapeutic effect of OTTPy-Va on bacteria. Bacteria were plated in 96-well plates and divided into two groups: one group under white light (25 mW / cm²). 2 One group was exposed to light for 10 minutes, and the other group received no light. Figure 7 Image (a) shows a plate coating photograph of the phototoxicity and dark toxicity tests of the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 against Staphylococcus aureus. Figure 7 Figure (b) shows the quantitative graph of bacterial survival rate. Under dark conditions, the bacterial survival rate remained around 100% with increasing drug concentration, indicating that the organic antibacterial photosensitizer OTTPy-Va has extremely low dark toxicity and good biocompatibility. Under white light irradiation, the bacterial survival rate gradually decreased with increasing OTTPy-Va drug concentration, demonstrating good photodynamic therapy and excellent antibacterial effect.

[0053] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A D-π-A type organic antibacterial photosensitizer, characterized in that, The structural formula of this organic antibacterial photosensitizer is:

2. The method for synthesizing the D-π-A type organic antibacterial photosensitizer as described in claim 1, characterized in that, Its chemical reaction formula is as follows: The method includes the following steps: Compound 1,2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) was dissolved in N,N-dimethylformamide (DMF), then cesium carbonate was added, and finally valeric acid was added. The reactants were mixed thoroughly, and the reaction solution was stirred at room temperature. After the reaction was completed, deionized water was added, and the mixture was extracted with dichloromethane. The resulting organic phase was dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by gradient elution using column chromatography to obtain the organic dye named OTTPy-Va, which is the D-π-A type organic antibacterial photosensitizer.

3. The application of the D-π-A type organic antibacterial photosensitizer as described in claim 1 in the preparation of antibacterial photodynamic therapy drugs.

4. The application of the D-π-A type organic antibacterial photosensitizer as described in claim 1 in the preparation of reagents for selective imaging or selective killing of bacteria in bacteria and normal cells.

5. The application according to claim 4, characterized in that: The bacteria mentioned include Staphylococcus aureus, and the normal cells are human umbilical vein endothelial cells and epithelial cells.

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