Bacteriostatic light cage for light-operated rapid release of sulfadiazine as well as preparation method and application of bacteriostatic light cage

Abstract

The invention discloses a bacteriostatic light cage for light-controlled rapid release of sulfadiazine, a preparation method and application thereof, and the bacteriostatic light cage is based on a light-controlled release system of a Sanger reagent, and realizes efficient space-time release and targeted antibiosis of SD. The light-operated bacteriostatic light cage capable of rapidly releasing sulfadiazine, disclosed by the invention, has sulfadiamidine-2, 4-dinitrobenzene derivative # imgabs0 # with a structure as shown in a formula SD-Py-N < + >

Description

Technical Field

[0001] The present invention relates to an antibacterial drug-loading system, a preparation method and an application thereof, and more particularly to a bacteriostatic photocage for photocontrolled rapid release of sulfadiazine, a preparation method and an application thereof. Background Art

[0002] As a Gram-positive pathogenic bacterium, Staphylococcus aureus can cause various fatal infections ranging from skin abscesses to sepsis. Its rapidly evolving drug resistance has made it an ongoing challenge in clinical treatment. Current first-line therapeutic drugs (such as β-lactams, vancomycin) have reduced efficacy due to the widespread presence of drug-resistant genes (mecA, vanA) and the bacterial metabolic dormancy mechanism. There is an urgent need to develop new antibacterial strategies to improve efficacy and reduce side effects. Based on its strong antibacterial properties, sulfadiazine (abbreviated as SD) is widely used in the treatment of local infections such as skin abscesses, postoperative infections and osteomyelitis caused by Staphylococcus aureus. Especially when combined with silver compounds (such as silver sulfadiazine), it can synergistically enhance the anti-infection effect of burn wounds. However, systemic administration of SD has problems such as dose-dependent crystalluria, nephrotoxicity and insufficient penetration into deep tissues (such as osteomyelitis) due to poor water solubility. There is an urgent need to develop a controlled release system to enhance efficacy and reduce toxicity.

[0003] Photocontrolled prodrugs release active molecules by light triggering, which can precisely limit the activation of drugs to the lesion area, providing an innovative strategy for precision treatment. Compared with traditional prodrugs that rely on enzyme or environmental activation, the photocontrolled release system achieves precise spatio-temporal control through local activation, significantly reducing the off-target effect. Sanger reagent (i.e., 2,4-dinitrofluorobenzene), as a photodeprotecting group (PPG), has shown unique advantages in the release of amide molecules, but its application in the delivery of sulfonamide drugs has not been reported. Developing an efficient and controllable photocage for photocontrolled release of sulfadiazine, that is, a photocontrolled release system or a photo-responsive drug delivery system, is of great significance for improving the antibacterial treatment effect. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of uncontrollable release and high systemic toxicity of existing SD preparations, and provide a bacteriostatic photocage for photocontrolled rapid release of sulfadiazine. This bacteriostatic photocage is based on a photocontrolled release system of Sanger reagent, realizing the efficient spatio-temporal release of SD and targeted antibacterial.

[0005] The present invention also provides a preparation method of the bacteriostatic photocage for photocontrolled rapid release of sulfadiazine and the application of the bacteriostatic photocage for photocontrolled rapid release of sulfadiazine in the rapid dissociation of sulfadiazine and the bacteriostasis of Staphylococcus aureus.

[0006] The technical concept and principle of the present invention are as follows: The photocontrolled antibacterial photocage based on sulfadiazine-2,4-dinitrobenzene derivative can achieve the effective release of sulfadiazine under ultraviolet light irradiation at 400 nm. The present invention for the first time prepares an antibacterial photocage that can rapidly release sulfadiazine by photocontrol, namely sulfadiazine-2,4-dinitrobenzene derivative, and for the first time applies it to the rapid dissociation of sulfadiazine and the antibacterial activity against Staphylococcus aureus.

[0007] The technical solution for the present invention to solve its technical problems is as follows:

[0008] The antibacterial photocage for the rapid release of sulfadiazine by photocontrol of the present invention has a sulfadiazine-2,4-dinitrobenzene derivative with the structure shown in formula SD-Py-N + as follows:

[0009]

[0010] The preparation method of the antibacterial photocage for the rapid release of sulfadiazine by photocontrol of the present invention includes the following steps:

[0011] Dissolve 2-(2,4-dinitrophenyl)(pyridin-4-ylmethyl)amino)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamide (abbreviated as SD-Sanger) and (3-bromopropyl)trimethylammonium bromide in a solvent, stir and react for more than 24 hours under an oil bath at 80-90 °C, nitrogen protection and light avoidance conditions. After the reaction, purify and vacuum dry to obtain the sulfadiazine-2,4-dinitrobenzene derivative abbreviated as SD-Py-N + which is the antibacterial photocage for the rapid release of sulfadiazine by photocontrol.

[0012] For the preparation method of the antibacterial photocage for the rapid release of sulfadiazine by photocontrol of the present invention, a further technical solution is that the reaction temperature is preferably 80-90 °C, and the solvent is preferably ethanol. In the present invention, the most preferred reaction temperature is 85 °C.

[0013] For the preparation method of the antibacterial photocage for the rapid release of sulfadiazine by photocontrol of the present invention, a further technical solution can also be that after the reaction, purification is carried out. The purification step is: subject the crude product to vacuum filtration, and further purify the crude product on a silica gel column with dichloromethane / methanol to obtain SD-Py-N + .

[0014] Application of the above-mentioned antibacterial photocage for the rapid release of sulfadiazine by photocontrol of the present invention in the rapid dissociation of sulfadiazine and the antibacterial activity against Staphylococcus aureus.

[0015] The present invention has the following beneficial effects:

[0016] 1) The antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention can be efficiently photocleaved, and SD is completely released within 480 seconds under 400 nm light irradiation.

[0017] 2) After using the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention, the IC 50 value of the released SD against Staphylococcus aureus 29213 was measured to be 1.86 μM, which is equivalent to the activity of the raw drug (verified by the dilution plate experiment).

[0018] 3) The antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention has a strong ability to target the bacterial surface. The trimethylammonium group enhances the bacterial membrane targeting and reduces the toxicity of the system exposure.

[0019] 4) The preparation process of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention is simple and easy to implement, and is easy to scale up production.

[0020] The preparation reaction equation of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention is as follows:

[0021] Description of the Drawings

[0022] Figure 1 1H-NMR spectrum of the antibacterial photocage for photocontrolled rapid release of sulfadiazine in Example 1 of the present invention. 1 1H-NMR spectrum.

[0023] Figure 2 High performance liquid chromatography spectrum and photolysis kinetic curve of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention.

[0024] Figure 3 Study on the inhibitory effect of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention on Staphylococcus aureus (plate experiment).

[0025] Figure 4 Live / Dead fluorescence imaging results of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention.

[0026] Figure 5 IC 50 determination curve of the antibacterial photocage for photocontrolled rapid release of sulfadiazine of the present invention. Detailed Description of the Invention

[0027] The present invention will be further described in detail below with reference to the drawings and in combination with the embodiments. However, the present invention is not limited to the given examples.

[0028] Preparation of the antibacterial photocage for photocontrolled rapid release of sulfadiazine in Example 1

[0029] Dissolve 200 mg of 2-(2,4-dinitrophenyl)(pyridin-4-ylmethyl)amino)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamide and 64 mg of (3-bromopropyl)trimethylammonium bromide in 100 ml of ethanol solution; stir for 24 hours under an 85 °C oil bath, nitrogen protection and light avoidance conditions; after the reaction is completed, first concentrate the reaction solution under reduced pressure, then purify the crude product by silica gel column chromatography (dichloromethane / methanol), and finally dry it under vacuum to obtain 140 mg of an oily substance, which is the pure antibacterial photocage for photoresponsive rapid release of sulfadiazine (its 1 1H-NMR spectrum is shown in Figure 1 ).

[0030] Example 2 High-performance liquid chromatography spectrum and photolysis kinetic curve of the antibacterial photocage for photoresponsive rapid release of sulfadiazine

[0031] Accurately weigh 1.0 mg of SD-Py-N prepared in Example 1, dissolve it in 1 ml of acetonitrile to prepare a stock solution of 1.0 mg / mL. Each stock solution is diluted with a 50% methanol-water mixture to obtain a test sample of 100 μg / mL. The sample is irradiated at 400 nm, and its photoreaction process is monitored by HPLC. The results are as + shown in Figure 2 .

[0032] The above results show that:

[0033] The antibacterial photocage SD-Py-N for photoresponsive rapid release of sulfadiazine + can rapidly release SD under 400 nm light irradiation (about 480 s for complete photolysis).

[0034] Example 3 Study on the inhibitory effect of the antibacterial photocage for photoresponsive rapid release of sulfadiazine on Staphylococcus aureus

[0035] Prepare solutions of unphotolyzed SD-Py-N + , photolyzed SD-Py-N + (treated by 400 nm ultraviolet photolysis) and the control drug SD at 0, 2, 5, 10 μM respectively, and add them to the Staphylococcus aureus 29213 bacterial suspension. After incubating at 37 °C for 1 hour, the bacterial solution is diluted 1000-fold twice and evenly spread on the LB agar plate. The plate is placed in a 37 °C constant temperature incubator for 15 hours, the colony morphology is photographed using a gel imaging system, and the number of colonies on each plate is quantitatively counted by ImageJ software. The results are as Figure 3 shown in

[0036] The above results show that:

[0037] When SD-Py-N +(0 - 10 μM) was co - incubated with Staphylococcus aureus 29213, plated and cultured after 5 - minute illumination at 400 nm. The results showed that the number of colonies in the illuminated group decreased by >80% at a concentration of 10 μM, comparable to the SD control group( Figure 3 ).

[0038] Live / Dead fluorescence imaging results of the antibacterial photocage for photo - controlled rapid release of sulfadiazine in Example 4

[0039] Suspensions of Staphylococcus aureus 29213 were incubated with 10 μM of SD - Py - N + (before UV photolysis), SD - Py - N + +UV (after UV photolysis) and SD at 37 °C for 15 minutes. After incubation, the samples were stained with NucGreen / EthD - III for 10 minutes and imaged using total internal reflection fluorescence microscopy (TIRF). The green channel was excited at 488 nm with an emission range of 520 - 560 nm, while the red channel was excited at 561 nm with an emission range of 580 - 640 nm. The results obtained are as Figure 4 shown.

[0040] The above results indicate that:

[0041] Most of the Staphylococcus aureus 29213 cells treated with SD - Py - N + were viable and only visible in the green channel, indicating that SD - Py - N + had little bactericidal activity. In contrast, when co - incubated with UV - photolyzed SD - Py - N + , a significant increase in red fluorescence was detected, indicating a large number of bacteria died. These results are consistent with the observations of Staphylococcus aureus 29213 cells treated with SD alone, further confirming that the bactericidal activity of photolyzed SD - Py - N + originates from the released SD. This finding highlights the effectiveness of SD - Py - N + as a photo - masking compound for photo - controlled release of active antibacterial agents.

[0042] IC of the antibacterial photocage for photo - controlled rapid release of sulfadiazine in Example 5 50 assay curve

[0043] To evaluate the antibacterial activity of the antibacterial photocage for photo - controlled rapid release of sulfadiazine against Staphylococcus aureus 29213, the IC 50Values. In LB medium, the stock solution of the drug-loaded system (1 mM in dimethyl sulfoxide) was diluted in a 2-fold serial dilution manner to obtain final concentrations ranging from 0.1 μM to 100 μM. In a 96-well plate, 50 μL of the drug-loaded system solution and 50 μL of the bacterial suspension were mixed with 100 μL of LB medium, and the total volume per well was 200 μL. The samples were irradiated with ultraviolet light at 400 nm (200 mW / cm 2 ) for 5 minutes, while the dark controls were protected from light. The plates were incubated at 37 °C for 24 hours, and the OD 600 values were recorded using a microplate reader. The IC 50 values were calculated by non-linear regression analysis, and the results obtained are as shown in Figure 5 .

[0044] The above results indicate that:

[0045] SD-Py-N + showed strong antibacterial activity against Staphylococcus aureus 29213 after photolysis, with an IC 50 value of 1.8620 μM.

Claims

1. An antibacterial photocage for rapid release of sulfadiazine controlled by light, characterized in that, The antibacterial photocage has a sulfadiazine-2,4-dinitrobenzene derivative with the structure shown in formula SD-Py-N + as follows:

2. A method for preparing an antibacterial photocage for rapid release of sulfadiazine controlled by light as described in claim 1, characterized in that It includes the following steps: Dissolve 2-(2,4-dinitrophenyl)(pyridin-4-ylmethyl)amino)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamide and (3-bromopropyl)trimethylammonium bromide in a solvent, stir and react under an 80-90 °C oil bath, nitrogen protection and light avoidance conditions for more than 24 hours. After the reaction, purify and vacuum dry to obtain the sulfadiazine-2,4-dinitrobenzene derivative, abbreviated as SD-Py-N + That is, an antibacterial photocage for light-controlled rapid release of sulfadiazine.

3. The preparation method of the antibacterial photocage for phototriggered rapid release of sulfadiazine according to claim 2, wherein, The reaction temperature is 85 °C, and the solvent is ethanol; the mass ratio of 2-(2,4-dinitrophenyl)(pyridin-4-ylmethyl)amino)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamide to (3-bromopropyl)trimethylammonium bromide is 2.08:

1.

4. The preparation method of the antibacterial photocage for rapid release of sulfadiazine controlled by light according to claim 2, characterized in that, Purification is carried out after the reaction, and the purification steps are as follows: The crude product is filtered under reduced pressure, and the crude product is further purified on a silica gel column with dichloromethane / methanol to obtain SD-Py-N + .

5. Use of the light-controlled rapid-release sulfadiazine antibacterial photocage as claimed in claim 1 in the rapid dissociation of sulfadiazine and antibacterial of Staphylococcus aureus.

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