A berberine derivative with antibacterial activity and aggregation-induced emission properties, and a preparation method and application thereof

Abstract

The application belongs to the field of design and synthesis of organic small molecules, and discloses a berberine derivative with antibacterial activity and aggregation-induced emission property, and a preparation method and application thereof. The berberine derivative TPA-BBR and TPE-BBR are synthesized by introducing triphenylamine or tetraphenylethylene groups to the 12th position of berberine respectively. The berberine derivative provided by the application exhibits aggregation-induced emission (AIE) property, and exhibits good photodynamic performance, and can quickly induce a large amount of active oxygen under light. The in-vitro antibacterial activity experiment proves that TPA-BBR and TPE-BBR can efficiently kill gram-positive bacteria. In addition, when co-cultured with bacteria, TPA-BBR can stain gram-positive bacteria and gram-negative bacteria, and exhibits broad-spectrum staining performance.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule design and synthesis, and particularly relates to a berberine derivative with antibacterial activity and aggregation-induced emission properties, its preparation method and application. Background Technology

[0002] Antimicrobial drugs and treatments that are less likely to induce drug resistance are of great significance in reducing the possibility of bacterial resistance and dealing with the frequent occurrence of "superbugs." Among them, membrane-targeted drugs and photoactive damage strategies are among the most important solutions.

[0003] Antibacterial photodynamic therapy (aPDT) is a therapy that uses photosensitizers (PSs), light, and oxygen to generate reactive oxygen species (ROS) to kill bacteria. It has attracted increasing attention due to its non-invasiveness, low resistance, and minimal systemic toxicity. However, traditional PSs, such as phthalocyanines and porphyrins, tend to aggregate in aqueous media, significantly reducing their bactericidal efficacy. This is because the π-π stacking of aggregated states leads to fluorescence quenching, reducing the ability to generate ROS. Conversely, aggregation restricts the intramolecular movement of aggregation-induced emission photosensitizers (AIE-PSs), thereby inhibiting their non-radiative decay and promoting fluorescence and ROS generation. Currently, AIE-PSs are widely studied as novel antibacterial drugs. For example, Liu's research group developed an AIE-PSs for membrane-targeted antibacterial therapy that exhibits excellent activity. However, most existing AIE-PSs are still primarily synthesized artificially, involving chemical synthesis and complex separation and purification processes. Furthermore, their structural diversity and physicochemical properties are limited by existing synthetic chemistry methods. In addition, high economic costs, environmental pollution, low biocompatibility, and poor degradability also limit their practical application.

[0004] In contrast, nature creates many natural products with unique structures, resulting in inherent properties associated with these structures, such as renewability, non-toxicity / low toxicity, biodegradability, biocompatibility, and pharmacological activity. For example, Tang's research group discovered berberine chloride, a natural isoquinoline alkaloid isolated from traditional Chinese medicinal plants, which exhibits an AIE effect and some phototoxicity to Gram-positive bacteria. However, the research and development of purely natural biosources is limited by the variety of natural biosources available. Among these, biosources obtained through chemical modification of natural products may be a good alternative to purely natural biosources.

[0005] Therefore, based on existing research on natural products, it is necessary to further design and develop aggregation-induced emission photosensitizers with highly efficient antibacterial activity. Summary of the Invention

[0006] Therefore, the present invention aims to provide a berberine derivative with highly efficient antibacterial activity and aggregation-induced emission properties; another objective is to provide its preparation method and application.

[0007] To achieve the objective of this invention, the technical solution is as follows: The berberine derivative antibacterial agent has the following structural formulas:

[0008]

[0009] Its preparation method includes the following steps:

[0010]

[0011] Step 1: Compound BBR was suspended in 1,4-dioxane. At room temperature, a bromine-containing solution of 1,4-dioxane was added dropwise to the mixture, and the reaction mixture was stirred at room temperature. After the reaction was complete, the precipitate was filtered off, washed, dried, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound Br-BBR.

[0012] Step 2: At room temperature, a mixture of Br-BBR, triphenylamine 4-borate or [1-(4-boronylphenyl)-1,2,2-triphenyl]ethylene, palladium acetate, and sodium carbonate was stirred and reacted in anhydrous ethanol. After the reaction was completed, the reaction mixture was extracted, washed, dried, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain the target compound TPA-BBR or TPE-BBR.

[0013] The innovation of this invention lies in the design and synthesis of berberine derivatives TPA-BBR and TPE-BBR by modifying the natural product berberine, and the exploration of their antibacterial applications. Both TPA-BBR and TPE-BBR exhibit aggregation-induced emission (AIE) properties, rapidly generating large amounts of reactive oxygen species (ROS) under white light irradiation, and demonstrate excellent antibacterial activity against Gram-positive bacteria. Furthermore, due to the inherent fluorescent properties of the compounds, TPA-BBR can be used for bacterial imaging.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention is based on the modification of the natural product berberine. By introducing triphenylamine or tetraphenylethylene groups into the 12 position of berberine, small molecule compounds TPA-BBR and TPE-BBR are synthesized. These compounds have antibacterial activity and aggregation-induced emission properties, and are novel aggregation-induced emission photosensitizers.

[0016] 2. The small molecule compounds TPA-BBR and TPE-BBR constructed in this invention have simple synthesis processes, low cost, and high synthesis yields, reaching over 70%.

[0017] 3. The small molecule compounds TPA-BBR and TPE-BBR constructed in this invention exhibit excellent antibacterial activity against Gram-positive bacteria, as revealed by in vitro antibacterial experiments.

[0018] 4. The compound TPA-BBR can stain both Gram-positive and Gram-negative bacteria, demonstrating its broad-spectrum staining performance and showing great potential for development. Attached Figure Description

[0019] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the compound TPA-BBR of this invention.

[0020] Figure 2 This is the carbon NMR spectrum of the compound TPA-BBR of this invention.

[0021] Figure 3 This is a high-resolution mass spectrum of the compound TPA-BBR of this invention.

[0022] Figure 4 This is the 1H NMR spectrum of the compound TPE-BBR of this invention.

[0023] Figure 5 This is the carbon NMR spectrum of the compound TPE-BBR of this invention.

[0024] Figure 6 This is the normalized ultraviolet absorption spectrum of the compound TPA-BBR in MeOH solution.

[0025] Figure 7 This is the normalized fluorescence emission spectrum of the compound TPA-BBR in MeOH solution.

[0026] Figure 8 The fluorescence spectra of the compound TPA-BBR of this invention are shown in mixed solutions of 1,4-dioxane / MeOH with different contents of 1,4-dioxane.

[0027] Figure 9This is a graph showing the relationship between the maximum fluorescence emission wavelength of the compound TPA-BBR and the content of 1,4-dioxane in a 1,4-dioxane / MeOH mixed solution.

[0028] Figure 10 This is the normalized ultraviolet absorption spectrum of the compound TPE-BBR in MeOH solution.

[0029] Figure 11 This is the normalized fluorescence emission spectrum of the compound TPE-BBR in MeOH solution.

[0030] Figure 12 The fluorescence spectra of the compound TPE-BBR of this invention are shown in mixed solutions of 1,4-dioxane / DMSO with different contents of 1,4-dioxane.

[0031] Figure 13 The graph shows the relationship between the maximum fluorescence emission wavelength of the compound TPE-BBR and the content of 1,4-dioxane in the 1,4-dioxane / DMSO mixed solution.

[0032] Figure 14 The compound TPA-BBR of this invention is used in white light (50mW / cm²). 2 Figure showing the test results of total reactive oxygen species production under irradiation.

[0033] Figure 15 The compound TPA-BBR of this invention is used in white light (50mW / cm²). 2 Figure showing the test results of hydroxyl radical generation under irradiation.

[0034] Figure 16 The compound TPA-BBR of this invention is used in white light (50mW / cm²). 2 Figure showing the test results of superoxide anion free radical generation under irradiation.

[0035] Figure 17 The compound TPA-BBR of this invention is used in white light (50mW / cm²). 2 Figure showing the test results of singlet oxygen production under irradiation.

[0036] Figure 18 Staphylococcus aureus at different concentrations of TPA-BBR under / without white light irradiation (50mW / cm²) 2 )Statistical chart of bacterial survival rate under condition treatment.

[0037] Figure 19 Staphylococcus aureus at different concentrations of TPA-BBR under / without white light irradiation (50mW / cm²) 2 Photographs of bacterial colonies on agar plates under certain conditions.

[0038] Figure 20 Staphylococcus aureus at different concentrations of TPE-BBR under / without white light irradiation (50mW / cm²) 2 )Statistical chart of bacterial survival rate under condition treatment.

[0039] Figure 21 Staphylococcus aureus at different concentrations of TPE-BBR under / without white light irradiation (50mW / cm²) 2 Photographs of bacterial colonies on agar plates under certain conditions.

[0040] Figure 22 Scanning electron microscope images of Staphylococcus aureus with and without TPA-BBR treatment.

[0041] Figure 23 The figure shows the experimental results of the permeability of the compound TPA-BBR of this invention to the cell membrane of Staphylococcus aureus.

[0042] Figure 24 The effect of the compound TPA-BBR of this invention on the cell morphology of Bacillus pumilus.

[0043] Figure 25 This is a laser confocal scan image of the compound TPA-BBR of this invention after incubation with Staphylococcus aureus for 20 minutes.

[0044] Figure 26 This is a laser confocal scan image of the compound TPA-BBR of this invention incubated with Escherichia coli for 20 minutes. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection claimed by the present invention. Unless otherwise specified, all percentage contents mentioned below are mass percentage contents.

[0046] The instruments used for characterizing the synthesized compounds were as follows: NMR spectra were measured using a Bruker DPX-400 superconducting nuclear magnetic resonance spectrometer (Sweden); high-resolution mass spectrometry was performed using a Waters-Micromass Q-Tof mass spectrometer.

[0047] Example 1: Preparation of compound TPA-BBR

[0048] In a round-bottom flask (100 mL), 12-bromoberberine (0.20 g, 0.40 mmol, 1.0 eq.), triphenylamine 4-borate (0.18 g, 0.60 mmol, 1.5 eq.), sodium carbonate (0.08 g, 0.8 mmol, 2.0 eq.), and palladium acetate (0.90 mg, 0.004 mmol, 0.1 eq.) were dissolved in anhydrous ethanol. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL). The organic phases were combined and washed three times with water, then washed 1-3 times with saturated sodium chloride (10 mL). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 2:1, V:V) to give 0.18 g of an orange-yellow powder, with a yield of 70%. NMR and mass spectrometry characterization are as follows: 1 H NMR (400MHz, CDCl3): δ10.71(s,1H),8.24(s,1H),7.74(s,1H),7.38–7.29(m,6H),7.26–7.21(m,6H),7.15–7 .08(m,3H),6.83(s,1H),6.09(s,2H),5.41(t,J=6.0Hz,2H),4.36(s,3H),4.09(s,3H),3.32(t,J=6.0Hz,2H). 13 C NMR (100MHz, CDCl3): δ150.6,150.5,148.7,148.3,147.8,147.1,145.1,137.6,135.9,131.4,130.9,130.5,12 9.6,129.5,126.2,125.4,124.0,122.8,122.3,120.5,117.9,108.8,105.0,102.2,63.3,57.1,55.7,27.7.ESI HRMS:Calcd.forC 38 H 31 BrN2O4[M–Br] + 579.2278, found 579.2286.

[0049] Example 2: Preparation of compound TPE-BBR

[0050] In a round-bottom flask (100 mL), 12-bromoberberine (0.20 g, 0.40 mmol, 1.0 eq.), [1-(4-boronylphenyl)-1,2,2-triphenyl]ethylene (0.23 g, 0.60 mmol, 1.5 eq.), sodium carbonate (0.08 g, 0.8 mmol, 2.0 eq.), and palladium acetate (0.90 mg, 0.004 mmol, 0.1 eq.) were dissolved in anhydrous ethanol. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL). The organic phases were combined and washed three times with water, then washed 1-3 times with saturated sodium chloride (10 mL). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 2:1, V:V) to give 0.22 g of an orange-yellow powder, with a yield of 70%. NMR characterization is as follows: 1 H NMR (400MHz, CDCl3): δ10.68(s,1H),7.80(s,1H),7.68(s,1H),7.27–7.22(m,5H),7.20–7.12(m,12H),7.1 0–7.06(m,2H),6.95(s,1H),6.85(s,1H),6.15(s,2H),5.38(s,2H),4.35(s,3H),4.06(s,3H),3.31(s,2H). 13 C NMR (100MHz, CDCl3): δ150.7,150.3,148.4,147.9,145.4,144.7,143.6,143.2,143.2,142.3,140.0,137.7,135.7,134.6,132.2,131.5,131.4, 131.3,131.3,131.0,129.0,128.0,127.9,127.8,127.2,126.9,126.8,1 26.4,122.7,120.5,117.7,108.9,104.9,102.2,63.3,57.2,55.7,27.7.

[0051] Performance testing: 1. Photophysical property testing of compounds TPA-BBR and TPE-BBR

[0052] Depend on Figures 6-13 The fluorescence spectra shown indicate that when berberine derivatives are dissolved in a mixed solvent of methanol and 1,4-dioxane (1,4-dioxane is a poor solvent), and the proportion of the poor solvent is adjusted, the fluorescence intensity continuously increases with the increase of the proportion of the poor solvent. This demonstrates that as the solubility of berberine derivatives decreases, their particles continuously aggregate, and the fluorescence intensity continuously increases, thus confirming that this type of berberine derivative possesses AIE properties.

[0053] Performance testing: 2. Detection of reactive oxygen species in the TPA-BBR compound solution.

[0054] like Figures 14-17 As shown, the generation of reactive oxygen species (ROS) in TPA-BBR solution under illumination was detected using specific fluorescent probes DCFH, DHR123, HPF, and ABDA. A rapid increase in fluorescence was observed within 5 minutes, indicating that this molecule can rapidly generate a large amount of ROS. This also confirms that TPA-BBR can induce the generation of singlet oxygen, as well as hydroxyl radicals and superoxide anion radicals.

[0055] Performance testing: 3. In vitro photodynamic antibacterial activity testing of compounds TPA-BBR and TPE-BBR

[0056] The antibacterial activity of TPA-BBR and TPE-BBR against Staphylococcus aureus was evaluated using conventional plate culture and colony counting methods. Single colonies from MHA culture dishes were picked with an inoculation loop and transferred to MHB medium. After incubation at 37°C and 220 rpm for 4 hours in a shaker, the medium was removed by centrifugation, the bacteria were washed three times with PBS, and resuspended in PBS to dilute to a final bacterial concentration of ~10⁻⁶. 7 CFU / mL, different concentrations of TPA-BBR or TPE-BBR were first incubated with the bacterial suspension in the dark for 30 minutes, and then the bacterial suspension was exposed to white light (50mW / cm²). 2 Irradiate for 30 minutes to perform a phototoxicity test, or incubate in the dark for another 30 minutes to assess photodynamic activity. After incubation, serially dilute the bacterial suspension 10⁻⁶ times. 3 The cells were spread onto MHA agar solid medium and incubated at 37°C for 16 hours before colony counting and survival rate calculation.

[0057] Figures 18-21 The study investigated the antibacterial activity of different concentrations of compounds TPA-BBR and TPE-BBR against Staphylococcus aureus. Under light irradiation, the survival rate of Staphylococcus aureus decreased significantly with increasing compound concentration, demonstrating excellent antibacterial activity.

[0058] Performance Testing: 4. Study on the antibacterial mechanism of compound TPA-BBR

[0059] (1) Electron microscopy experiment: A single colony of S. aureus was picked from an MHA culture dish using an inoculation loop and placed in MHB medium. After incubation at 37℃ and 220r / min for 4h, the culture medium was removed by centrifugation (7100rpm, 3min). The bacteria were washed three times with PBS and resuspended in PBS to dilute to a final concentration of ~10. 9After adding the desired concentration of compound (CFU / mL) to a constant temperature shaker (37℃, 220 rpm) for 30 minutes, the mixture was incubated for 30 minutes. After incubation, the mixture was centrifuged (4℃, 3500 rpm, 5 min), resuspended three times in 1×PBS, and then resuspended again in 2.5% glutaraldehyde solution in the dark. The mixture was then frozen overnight at -20℃ and sent to Beijing Zhongke Company for analysis.

[0060] The scanning electron microscopy results of compound TPA-BBR after incubation with S. aureus for 20 minutes are as follows: Figure 22 As shown in the scanning electron microscope images, untreated *S. aureus* exhibits a complete, smooth, and well-defined membrane boundary. *S. aureus* treated with the compound TPA-BBR exhibits collapsed and fused morphologies, which are more pronounced under light conditions, indicating that the bacterial membrane is disrupted.

[0061] (2) Plasma membrane integrity experiment: A single colony of S. aureus was picked from an MHA culture dish using an inoculation loop and placed in MHB medium. After incubation at 37℃ and 220 r / min for 4 h in a shaker, the culture medium was removed by centrifugation (7100 rpm, 3 min). The bacteria were washed three times with PBS and resuspended in PBS to dilute to a final concentration of ~10. 9 CFU / mL. Add 150 μL of bacterial culture to a 96-well black culture plate, then add 40 μL of fluorescent dye PI (10 μM) in the dark, and incubate at 37℃ for 30 min. Measure the fluorescence value at an excitation wavelength of 535 nm and a cutoff wavelength of 617 nm using a microplate reader, measuring once per minute for 8 minutes. After this, add 10 μL of the diluted compound and PBS (blank control), and measure the fluorescence value for the next 30 minutes.

[0062] The results are as follows Figure 23 As shown, this indicates that the compound TPA-BBR can rapidly penetrate bacterial membranes, disrupting membrane integrity and inducing bacterial lysis and death. Membrane integrity experiments suggest that the cell membrane may be an important target of the compound TPA-BBR.

[0063] (3) Visualization of bacterial morphology: Bacillus pumilus CMCC63202 cells were incubated in MH broth at 37°C, and then the bacterial suspension was diluted to OD using MH broth. 620 The concentration was 0.01. The prepared bacterial solution contained 0.5 × MIC TPA-BBR and was grown at 37 °C for 18 hours. Cells used for morphological studies were collected and resuspended in 0.5 mL PBS buffer. 10 mL of the suspension mixture was transferred to a microscope slide pretreated with 0.1% (w / v) poly-L-lysine. Cell morphology was captured using a phase-contrast optical microscope.

[0064] Cell morphological changes such as Figure 24 As shown in the figure. The results indicate that TPA-BBR significantly increased the length of Bacillus pumilus compared to the control group. These cell elongation phenomena are similar to those of other known FtsZ inhibitors, preliminarily indicating their inhibitory effect on FtsZ.

[0065] Performance Testing: 5. Bacterial Imaging Experiment of Compound TPA-BBR

[0066] A single colony of *S. aureus* was picked from an MHA culture dish using an inoculation loop and placed in MHB medium. After incubation at 37°C and 220 rpm for 4 hours on a shaker, the culture medium was removed by centrifugation (7100 rpm, 3 min). The bacteria were washed three times with PBS and resuspended in PBS to a final concentration of ~10⁻⁶. 9 CFU / mL. After incubating the compound for 20 min, centrifuge (7100 rpm, 3 min) to remove the compound, resuspend in PBS, and perform fluorescence imaging using laser confocal microscopy.

[0067] The results are as follows Figures 25-26 As shown, TPA-BBR can stain both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. These results demonstrate that TPA-BBR can achieve broad-spectrum bacterial imaging.

[0068] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific structures and / or characteristics in the solutions are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A berberine derivative characterized in that, Its structural formula is as follows: 。 2. A process for preparing the berberine derivative as claimed in claim 1, characterized in that, This is achieved by the following steps: Step 1: The compound BBR was suspended in 1,4-dioxane. At room temperature, a bromine-containing solution of 1,4-dioxane was added dropwise to the above mixture. The reaction mixture was stirred at room temperature. After the reaction was completed, the precipitate was filtered off, washed, dried, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain the compound Br-BBR. Step 2: At room temperature, a mixture of Br-BBR, [1-(4-boronylphenyl)-1,2,2-triphenyl]ethylene, palladium acetate, and sodium carbonate was stirred and reacted in anhydrous ethanol. After the reaction was completed, the reaction mixture was extracted, washed, dried, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain the target compound TPE-BBR.

3. Use of berberine derivatives of the following structural formulae, respectively, in the preparation of medicaments, characterized in that, It is used as an active ingredient in the preparation of antibacterial drugs that kill Gram-positive bacteria; 。

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