D-pi-A type organic antibacterial photosensitizer as well as synthesis method and application thereof

Abstract

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

Description

Technical Field

[0001] The invention relates to the technical field of photosensitizers, in particular to a D-π-A type organic antibacterial photosensitizer and a synthesis method and application thereof. Background Art

[0002] As an emerging antibacterial therapy, photodynamic antimicrobial therapy has the characteristics of non-invasiveness, no drug resistance, low cytotoxicity, selective targeting, spatiotemporal precision and synergistic effect.

[0003] Photodynamic antibacterial therapy is a new method for treating bacterial infections using photosensitive drugs and laser activation. Irradiating the bacterial infection site with light of a specific wavelength can activate the photosensitive drug that selectively enters the bacteria, triggering a photochemical reaction to produce reactive oxygen species (ROS), which can cause bacterial death. When the photosensitizer absorbs light, it will transform into a triplet excited state. 3 PS*, then interacts with adjacent photosensitive molecules 1 Hydrogen atom transfer or electron transfer occurs between PS to form ion free radicals (PS -· ,PS +· ); ion radical (PS -· ,PS +· ) through a series of reactions to finally form hydroxyl radicals (OH·), which can interfere with the biological functions of nucleic acids, fatty acids and amino acids, thereby inducing bacterial death. This process is called type I photodynamics; energy is directly transferred from the triplet excited state to the 3 PS* transfers 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, induce oxidative stress in bacteria, and thus induce bacterial death. This process is called type II photodynamic therapy.

[0004] A large number of reported antimicrobial photosensitizer drugs tend to accumulate in mammalian living cells while entering bacteria, that is, they have non-negligible invasiveness to mammalian living cells. Due to the different surface composition and structure of bacteria and mammalian cells, the amphiphilicity of the drug has a great influence on the hydrophobic interaction between the drug and the organism and biological components. Therefore, eliminating the invasiveness of antimicrobial diagnostic and therapeutic drugs to mammalian living cells is an important task to meet the requirements of biological safety and accuracy. Summary of the invention

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

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

[0007]

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

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

[0010]

[0011] The method comprises the following steps:

[0012] (1) Compound 1, valeric acid, cesium carbonate, and 5 molar parts of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were added to N,N-dimethylformamide as solvent, the reactants were mixed evenly, and stirred at room temperature for reaction. After the reaction, deionized water was added to the reaction solution, and it was extracted with dichloromethane several times. The organic phases obtained by the extraction were combined, dried with anhydrous magnesium sulfate, filtered, and rotary evaporated to remove the organic solvent to obtain a crude product, which was separated and purified by column chromatography gradient elution to obtain an organic antibacterial photosensitizer named OTTPy-Va. The photosensitizer has the structural formula:

[0013]

[0014] In a third aspect, the present invention also provides the use of the above-mentioned D-π-A type organic antibacterial photosensitizer in the preparation of photodynamic antibacterial therapeutic drugs, and the use of the 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 the application, the D-π-A type organic antibacterial photosensitizer can enter the bacteria, kill the bacteria by white light irradiation, and be used in bacterial imaging and photodynamic therapy; the photosensitizer has good biological safety.

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

[0016] (1) The present invention uses a methoxy-substituted triphenylamine group as an electron donor, and connects it to a pyridinium salt group with strong electron absorption ability through a thiophene group as a π bridge, which can accelerate intramolecular electron transfer, accelerate the intersystem crossing process, and promote the occurrence of photodynamic reaction; the organic antibacterial photosensitizer is synthesized for the first time in the present invention, the raw materials are easily 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 bioluminescence imaging.

[0018] (3) The D-π-A type organic antibacterial photosensitizer provided by the present invention can be activated under white light to produce a large amount of active oxygen, and the production rate is faster than that of the 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 pyridinium 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, the present invention provides a D-π-A type organic antibacterial photosensitizer, which is simple to prepare and can selectively exert photodynamic effects in bacteria; compared with commercial photosensitizers, the organic antibacterial photosensitizer provided by the present invention has a larger Stokes shift and can be used for fluorescence imaging in bacteria; it has a higher and faster ability to generate active oxygen; it can selectively enter bacteria among normal cells and bacteria and exert a photodynamic therapy effect, greatly improving the treatment effect of bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0023] Figure 1 The synthetic route diagram of Example 1 is shown in FIG.

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

[0025] Figure 3(a) is the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 in DMSO / Toluene solvent (concentration: 1×10 -5 M) Fluorescence emission graphs at different DMSO / Toluene volume ratios. Figure 3 (b) Yes Figure 3 (a) Fluorescence intensity quantification dotted line graph;

[0026] Figure 4 (a) is a graph showing the fluorescence intensity enhancement of the ROS indicator DCFH by the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 under white light irradiation. Figure 4 (b) is the fluorescence intensity enhancement diagram of DCFH by commercial dye Rose Bengal under the same conditions; Figure 4 (c) is the fluorescence intensity diagram of ROS indicator DCFH under white light irradiation; Figure 4 (d) Yes Figure 4 (ac) Quantified dotted line graphs of fluorescence intensity at 525 nm;

[0027] Figure 5 This is a confocal image of fluorescence imaging of the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 in Staphylococcus aureus; it includes a blue fluorescence image of the commercial cell nucleus dye DAPI; a red fluorescence image of the synthesized organic antibacterial photosensitizer OTTPy-Va; a bacterial bright field image; and a superimposed image of the three; scale: 5 μm.

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

[0029] Figure 7 The organic antibacterial photosensitizer OTTPy-Va of Example 1 was subjected to an antibacterial experiment on Staphylococcus aureus; Figure 7 (a) is a plate coating diagram of different drug concentrations on Staphylococcus aureus; Figure 7 (b) Quantification of the plate coating experiment. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings and Example 1 and Test Examples 1-7.

[0031] Example 1

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

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

[0034]

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

[0036] mg, 0.3273mmol) and HATU (207mg, 0.5455mmol). After the reaction solution was evenly mixed, the reaction was allowed to react at room temperature for 24h. After the reaction was completed, 30mL of deionized water was added to the reaction solution, and it was extracted with dichloromethane several times. The organic phases obtained by the extraction were combined, dried with anhydrous magnesium sulfate, filtered, and rotary evaporated to remove the organic solvent to obtain a crude product. The crude product was separated and purified by neutral alumina column chromatography using (dichloromethane / methanol, v / v=25 / 1) as eluent to obtain a dark purple compound with a yield of 60%. The organic dye OTTPy-Va was obtained. 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-visible absorption and fluorescence emission spectra of the organic antibacterial photosensitizer OTTPy-Va of Example 1 were tested. The UV-visible absorption spectrum of the organic antibacterial photosensitizer OTTPy-Va in DMF / toluene (v / v=1:99) is shown in FIG. Figure 2 As shown in (a), the maximum absorption wavelength of the organic antibacterial photosensitizer OTTPy-Va is 526 nm, and the absorption range covers most of the visible light region. Figure 2 (b) is 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 670nm, which has good bioluminescence imaging potential.

[0043] Test Example 2

[0044] like Figure 3 As shown, the AIE performance test of the organic antibacterial photosensitizer OTTPy-Va in Example 1 was carried out. Figure 3 As shown in (a), the AIE performance of the organic antibacterial photosensitizer OTTPy-Va was tested in DMSO / Toluene. Figure 3 It can be seen in (a) that as the proportion of toluene in the system increases, the fluorescence intensity of the solution gradually increases. Figure 3 (b) Figure 3 (a) Fluorescence I / I0 quantitative dotted line graph. I is the fluorescence intensity of the organic antibacterial photosensitizer OTTPy-Va at 670 nm in the DMSO / Toluene system with different ratios, and I0 is 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 is enhanced by about 75 times at most, showing extremely excellent AIE performance.

[0045] Test Example 3

[0046] like Figure 4 As shown, the organic antibacterial photosensitizer OTTPy-Va of Example 1 was tested for its ROS generation ability in vitro. A commercial photosensitizer 2-7-dichlorodihydrofluorescein (DCFH-DA) with good biocompatibility and clinical use was used as a ROS indicator. DCFH-DA was converted into DCFH after activation. Figure 4 As shown in (a), the mixture of organic antibacterial photosensitizer OTTPy-Va and DCFH was exposed to white light (25 mW / cm 2) irradiation, the fluorescence emission at a wavelength of 525 nm gradually increased and reached the maximum fluorescence around 60 s, indicating that the organic antibacterial photosensitizer OTTPy-Va has the ability to produce reactive oxygen species in vitro. Figure 4 As shown in (b), a commercial dye Rose Bengal was used as a positive control and mixed with DCFH to detect its fluorescence emission under white light. Figure 4 As shown in (c), when the DCFH solution was placed under the same lighting conditions, almost no increase in fluorescence emission was detected, eliminating the possible impact of the indicator on the experimental results and indirectly indicating that the ROS indicator DCFH has good stability. Figure 4 (d) Figure 4 (ac) Quantitative dotted line graph of fluorescence I / I0. I is the fluorescence of the mixture of organic dye OTTPy-Va and DCFH at 525nm after different illumination times, and I0 is the fluorescence of the mixture of organic antibacterial photosensitizer OTTPy-Va and DCFH at 525nm when no illumination is given. Figure 4 (d) It can be seen that the ROS generation efficiency of the organic antibacterial photosensitizer OTTPy-Va is higher than that of the commercial dye Rose Bengal, which indicates that the organic antibacterial photosensitizer OTTPy-Va shows excellent ROS generation performance.

[0047] Test Example 4

[0048] like Figure 5 As shown, a bacterial fluorescence imaging experiment was conducted on the organic antibacterial photosensitizer OTTPy-Va of Example 1. Staphylococcus aureus was incubated with 5 μM of the organic antibacterial photosensitizer OTTPy-Va for 15, 30, 60, and 120 minutes, and the location of the photosensitizer was confirmed by comparing the fluorescence. The commercial bacterial nuclear dye DAPI was used to indicate the location of the bacteria, showing blue fluorescence; the organic antibacterial photosensitizer OTTPy-Va entered the bacteria and showed red fluorescence; the bright field was an image of the bacteria observed under white light; the Merge image was an image of the overlapping of the above three channels; when the incubation time reached 15 minutes, the drug could enter the bacteria, and obvious red fluorescence appeared.

[0049] Test Example 5

[0050] like Figure 6As shown, the organic antibacterial photosensitizer OTTPy-Va of Example 1 was subjected to a cell imaging experiment, and Hacat cells (human immortalized epidermal cells) and normal cells HUVEC cells (human umbilical vein endothelial cells) were selected for testing. Normal cells were incubated with 5 μM organic antibacterial photosensitizer OTTPy-Va for 15, 30, 60, and 120 minutes and photographed with a fluorescence microscope. After the drug was incubated with the two cells for 120 minutes, no inverted red fluorescence was observed, indicating that OTTPy-Va hardly entered the cells.

[0051] Test Example 6

[0052] like Figure 7 As shown in the figure, the organic antibacterial photosensitizer OTTPy-Va of Example 1 was subjected to a photodynamic antibacterial experiment. 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 by the plate coating method. In order to explore the effect of OTTPy-Va on photodynamic therapy in bacteria. The bacteria were spread into a 96-well plate and divided into two groups, one group was a white light lamp (25mW / cm 2 ) for 10 minutes, and one group was not exposed to light. Figure 7 (a) is a plate coating photograph of the phototoxicity and dark toxicity test of the organic antibacterial photosensitizer OTTPy-Va synthesized in Example 1 on Staphylococcus aureus, Figure 7 (b) is a quantitative graph of bacterial survival rate. Under dark conditions, as the dosage concentration increases, the bacterial survival rate remains at around 100%, which indicates that the organic antibacterial photosensitizer OTTPy-Va has extremely low dark toxicity and good biosafety. Under white light irradiation, as the dosage concentration of OTTPy-Va increases, the bacterial survival rate gradually decreases, showing good photodynamic therapy and reflecting excellent antibacterial effect.

[0053] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to 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 the organic antibacterial photosensitizer is:

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

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

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

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

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