Berberine derivative with antibacterial activity and aggregation-induced emission property as well as preparation method and application of berberine derivative

Abstract

The invention belongs to the field of design and synthesis of small organic molecules, and discloses a berberine derivative with antibacterial activity and aggregation-induced emission property as well as a preparation method and application of the berberine derivative. According to the invention, a triphenylamine group or a tetraphenylethylene group is respectively introduced to the 12th site of berberine, so that berberine derivatives TPA-BBR and TPE-BBR are synthesized. The berberine derivative provided by the invention shows an aggregation-induced emission (AIE) property, shows good photodynamic performance, and can be rapidly induced to generate a large amount of active oxygen under illumination. An in-vitro antibacterial activity experiment proves that the TPA-BBR and the TPE-BBR can be used for efficiently killing gram-positive bacteria. Besides, when the TPA-BBR is co-cultured with bacteria, the TPA-BBR can stain gram-positive bacteria and gram-negative bacteria, and the broad-spectrum staining performance of the TPA-BBR is shown.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of design and synthesis of small organic molecules, and particularly relates to a berberine derivative with antibacterial activity and aggregation-induced emission property, and a preparation method and application thereof. BACKGROUND

[0002] Antibacterial drugs and treatment methods that are not prone to drug resistance are of great significance for reducing the possibility of bacterial drug resistance and coping with frequent "super bacteria", among which membrane-targeting drugs and light-activated damage strategies are one of the most important solutions.

[0003] Antibacterial photodynamic therapy (aPDT) is a therapy that uses photosensitizers (PSs), light and oxygen to produce reactive oxygen species (ROS) to kill bacteria. It has attracted more and more attention due to its non-invasiveness, low drug resistance and minimal systemic toxicity. However, traditional PSs, such as phthalocyanine and porphyrin, tend to aggregate in aqueous media, which greatly reduces their bactericidal effect. Because the π-π stacking of the aggregate state leads to fluorescence quenching, the ability to generate ROS is reduced. On the contrary, the aggregation restricts the intramolecular motion of aggregation-induced emission photosensitizers (AIE-PSs), thereby inhibiting their non-radiative decay, promoting fluorescence and ROS production. Nowadays, AIE-PSs have been widely studied as a new type of antibacterial drug. For example, Liu's group developed an AIE-PSs that can be used for membrane-targeting antibacterial therapy, which showed good activity. However, most of the existing AIE-PSs are still mainly artificially synthesized, mostly involving chemical synthesis and complex separation and purification processes, and their structural diversity and physicochemical properties are limited by existing synthetic chemistry means. In addition, the high economic cost, environmental pollution, low biocompatibility and poor biodegradability faced also limit their practical application.

[0004] In contrast, nature has created many natural products with unique structures, producing some intrinsic properties that accompany unique structures: such as renewability, non-toxic / low toxicity, biodegradability, biocompatibility, and pharmacological activity. For example, Tang's group found a natural isoquinoline alkaloid, berberine chloride, isolated from Chinese herbal plants, which has an AIE effect and certain phototoxicity to gram-positive bacteria. However, the research and development of pure natural biological sources are limited by the types of natural biological sources. Among them, the biological sources obtained by chemical modification of natural products may be a good substitute for pure natural sources.

[0005] Therefore, on the basis of the research on the existing natural products, it is still necessary to further design and develop an aggregate-induced emission photosensitizer with high efficient antibacterial activity. SUMMARY

[0006] To this end, the purpose of the present application is to provide a berberine derivative with high efficient antibacterial activity and aggregate-induced emission property; another purpose is to provide a preparation method and application thereof.

[0007] To achieve the purpose of the present application, the technical solution of the present application is as follows: the structure formula of the berberine derivative antibacterial agent is as follows:

[0008]

[0009] The preparation method comprises the following steps:

[0010]

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

[0012] Step 2: A mixture of Br-BBR, 4-boronic acid triphenylamine or [1-(4-boronic acid ester phenyl)-1,2,2-triphenyl] ethylene, palladium acetate and sodium carbonate is stirred in anhydrous ethanol at room temperature. After the reaction is completed, the reaction mixture is extracted, washed, dried, and concentrated under reduced pressure to obtain a crude product, which is separated and purified by column chromatography to prepare the target compounds TPA-BBR or TPE-BBR.

[0013] The innovation of the present application lies in that the berberine derivatives TPA-BBR and TPE-BBR are designed and synthesized by modifying the natural product berberine, and the antibacterial application thereof is discussed. The berberine derivatives TPA-BBR and TPE-BBR both exhibit the property of aggregate-induced emission (AIE), can quickly generate a large amount of reactive oxygen species (ROS) under white light irradiation, and exhibit excellent antibacterial activity against gram-positive bacteria. In addition, TPA-BBR can realize bacterial imaging due to its own fluorescent property.

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

[0015] 1. The present application is based on the modification of natural product berberine, by introducing triphenylamine or tetraphenylethylene group to the 12 position of berberine, synthesizing small molecule compounds TPA-BBR and TPE-BBR, which have antibacterial activity and aggregation-induced emission properties, and are a novel structure of aggregation-induced emission photosensitizer.

[0016] 2. The synthesis process of the small molecule compounds TPA-BBR and TPE-BBR constructed by the present application is simple, the cost is low, and the synthesis yield is high, reaching more than 70%.

[0017] 3. The small molecule compounds TPA-BBR and TPE-BBR constructed by the present application are found to have excellent antibacterial activity against gram-positive bacteria through in vitro antibacterial experiment test.

[0018] 4. The compound TPA-BBR can stain gram-positive bacteria and gram-negative bacteria, showing its broad-spectrum staining performance, and has good development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the compound TPA-BBR of the present application.

[0020] Figure 2 It is the nuclear magnetic resonance carbon spectrum of the compound TPA-BBR of the present application.

[0021] Figure 3 It is the high resolution mass spectrum of the compound TPA-BBR of the present application.

[0022] Figure 4 It is the nuclear magnetic resonance hydrogen spectrum of the compound TPE-BBR of the present application.

[0023] Figure 5 It is the nuclear magnetic resonance carbon spectrum of the compound TPE-BBR of the present application.

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

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

[0026] Figure 8 It is the fluorescence spectrum of the compound TPA-BBR in 1,4-dioxane / MeOH mixed solution with different 1,4-dioxane contents of the present application.

[0027] Figure 9Plot of the maximum fluorescence emission wavelength of the compound TPA-BBR of the present invention versus the 1,4-dioxane content in 1,4-dioxane / MeOH mixed solutions.

[0028] Figure 10 Normalized UV absorption spectrum of the compound TPE-BBR of the present invention in MeOH solution.

[0029] Figure 11 Normalized fluorescence emission spectrum of the compound TPE-BBR of the present invention in MeOH solution.

[0030] Figure 12 Fluorescence spectrum of the compound TPE-BBR of the present invention in 1,4-dioxane / DMSO mixed solutions with different 1,4-dioxane content.

[0031] Figure 13 Plot of the maximum fluorescence emission wavelength of the compound TPE-BBR of the present invention versus the 1,4-dioxane content in 1,4-dioxane / DMSO mixed solutions.

[0032] Figure 14 Plot of the total reactive oxygen species generation test results of the compound TPA-BBR of the present invention under white light (50 mW / cm 2 ) irradiation.

[0033] Figure 15 Plot of the hydroxyl radical generation test results of the compound TPA-BBR of the present invention under white light (50 mW / cm 2 ) irradiation.

[0034] Figure 16 Plot of the superoxide anion radical generation test results of the compound TPA-BBR of the present invention under white light (50 mW / cm 2 ) irradiation.

[0035] Figure 17 Plot of the singlet oxygen generation test results of the compound TPA-BBR of the present invention under white light (50 mW / cm 2 ) irradiation.

[0036] Figure 18 Plot of the bacterial survival rate of Staphylococcus aureus under different concentrations of TPA-BBR of the present invention with / without white light (50 mW / cm 2 ) irradiation.

[0037] Figure 19 Photograph of the agar plate bacterial colonies of Staphylococcus aureus under different concentrations of TPA-BBR of the present invention with / without white light (50 mW / cm 2 ) irradiation.

[0038] Figure 20 The survival rate of Staphylococcus aureus under the treatment of different concentrations of TPE-BBR with / without white light irradiation (50 mW / cm 2 ) is shown in the following graph.

[0039] Figure 21 The agar plate colony photos of Staphylococcus aureus under the treatment of different concentrations of TPE-BBR with / without white light irradiation (50 mW / cm 2 ) are shown in the following photos.

[0040] Figure 22 The scanning electron microscope photos of Staphylococcus aureus under the treatment of TPA-BBR are shown in the following photos.

[0041] Figure 23 The experimental results of the permeability of the compound TPA-BBR of the present application to the cell membrane of Staphylococcus aureus are shown in the following graph.

[0042] Figure 24 The influence of the compound TPA-BBR of the present application on the cell morphology of Bacillus pumilus is shown in the following photos.

[0043] Figure 25 The laser confocal scanning photos of Staphylococcus aureus incubated with the compound TPA-BBR of the present application for 20 minutes are shown in the following photos.

[0044] Figure 26 The laser confocal scanning photos of Escherichia coli incubated with the compound TPA-BBR of the present application for 20 minutes are shown in the following photos. DETAILED DESCRIPTION

[0045] The present application will be further described below in connection with specific examples. These examples are only used to illustrate the present application and are not used to limit the scope of the present application. The percentages mentioned below are all mass percentages.

[0046] The instruments used for the characterization of the synthesized compounds are as follows: NMR spectrum is determined by using a superconducting nuclear magnetic resonance instrument of DPX-400 type of Bruker, Sweden; high resolution mass spectrum is determined by using a Q-Tof mass spectrometer of Waters-Micromass Company.

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

[0048] In a round bottom flask (100 mL), compound 12-bromoberberine (0.20 g, 0.40 mmol, 1.0 eq.), 4-boronic acid triphenylamine (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, stirred for 24 hours at room temperature, after the reaction was completed, extracted with dichloromethane (30 mL), combined organic phase and washed with water 3 times, washed with saturated sodium chloride (10 mL) 1-3 times, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 2:1, V:V), 0.18 g of orange yellow powder compound was obtained, the yield was 70%. The nuclear magnetic and mass spectrometry characterization are as follows: 1 H NMR (400 MHz, CDCl3): δ 10.71 (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 = 6.0 Hz, 2H), 4.36 (s, 3H), 4.09 (s, 3H), 3.32 (t, J = 6.0 Hz, 2H). 13 C NMR (100 MHz, CDCl3): δ 150.6, 150.5, 148.7, 148.3, 147.8, 147.1, 145.1, 137.6, 135.9, 131.4, 130.9, 130.5, 129.6, 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. for C 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), compound 12-bromoberberine (0.20 g, 0.40 mmol, 1.0 eq.), [1-(4-boronic acid ester phenyl)-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, stirred for 24 hours at room temperature, after the reaction was completed, extracted with dichloromethane (30 mL), combined organic phase and washed with water 3 times, washed with saturated sodium chloride (10 mL) 1-3 times, dried with 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 obtain 0.22 g of orange yellow powder compound with a yield of 70%. The nuclear magnetic characterization is as follows: 1 H NMR (400 MHz, CDC13): δ 10.68 (s, 1H), 7.80 (s, 1H), 7.68 (s, 1H), 7.27-7.22 (m, 5H), 7.20-7.12 (m, 12H), 7.10-7.06 (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 (100 MHz, CDC13): δ 150.7, 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, 126.4, 122.7, 120.5, 117.7, 108.9, 104.9, 102.2, 63.3, 57.2, 55.7, 27.7.

[0051] Performance test: 1. Test of photophysical properties of compound TPA-BBR and TPE-BBR

[0052] The fluorescence spectrum shown in the following formula: Figures 6-13 It can be seen from the fluorescence spectrum shown in the following formula that when the berberine derivative is dissolved in a mixed solvent of methanol and 1,4-dioxane (1,4-dioxane is a poor solvent), the proportion of the poor solvent is adjusted, and as the proportion of the poor solvent increases, the fluorescence intensity continuously increases. It can be seen that as the solubility of the berberine derivative decreases, the particles continuously aggregate, and the fluorescence intensity continuously increases, which confirms that the berberine derivative has AIE properties.

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

[0054] As shown in Figures 14-17 , the production of reactive oxygen species in TPA-BBR solution under irradiation was detected by specific fluorescent probes DCFH, DHR123, HPF and ABDA. It was found that the fluorescence increased rapidly within 5 minutes, indicating that the molecule could rapidly produce a large amount of reactive oxygen species. It was also confirmed that TPA-BBR could induce the production of singlet oxygen, as well as hydroxyl radicals and superoxide anion radicals.

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

[0056] The antibacterial activity of TPA-BBR and TPE-BBR against S. aureus was evaluated by traditional plate culture and colony counting method. Single colonies on MHA culture dishes were picked up with a inoculation loop in MHB medium, and after incubation at 37°C in a shaker at 220 r / min for 4 h, the culture medium was removed by centrifugation, washed with PBS three times, resuspended with PBS and diluted to a final concentration of ~10 7 CFU / mL. Different concentrations of TPA-BBR or TPE-BBR were first incubated with the bacterial suspension in the dark for 30 min, and then the bacterial suspension was exposed to white light (50 mW / cm 2 ) irradiation for 30 min for phototoxicity test or further incubated in the dark for 30 min to evaluate the photodynamic activity. After incubation, the bacterial suspension was diluted by 10 3 times and plated on MHA agar solid medium, and after incubation at 37°C for 16 h, colony counting was performed and the survival rate was calculated.

[0057] Figures 18-21 The killing effect of different concentrations of compound TPA-BBR and TPE-BBR on S. aureus was shown. Under irradiation, the survival rate of S. aureus decreased significantly with the increase of the concentration of the compound, showing excellent antibacterial activity.

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

[0059] (1) Electron microscopy experiment: Single colonies of S. aureus on MHA culture dishes were picked up with a inoculation loop in MHB medium, and after incubation at 37°C in a shaker at 220 r / min for 4 h, the culture medium was removed by centrifugation (7100 rpm, 3 min), washed with PBS three times, resuspended with PBS and diluted to a final concentration of ~10 9CFU / mL. After adding the compound at the desired concentration, continue incubation in a constant temperature shaker (37°C, 220 rpm) for 30 minutes. After incubation, centrifuge (4°C, 3500 rpm, 5 minutes), resuspend three times in 1× PBS, resuspend in 2.5% glutaraldehyde solution in the dark, freeze at -20°C overnight, and send to Beijing Zhongke Company for testing.

[0060] The scanning electron microscopy results of compound TPA-BBR incubated with S. aureus for 20 minutes are shown in Figure 2. Figure 22 Scanning electron microscopy images show that untreated S. aureus exhibits intact, smooth, and well-defined membrane boundaries. 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 test: A single colony of S. aureus on the MHA culture dish was picked up with an inoculation loop and placed in MHB medium. After incubation at 37°C and 220 rpm for 4 h, the culture medium was removed by centrifugation (7100 rpm, 3 min), the culture medium was washed three times with PBS, and the bacteria were resuspended and diluted with PBS to a final concentration of 10 9 CFU / mL. Take 150μL of bacterial solution and add it to a 96-well black culture plate. Then add 40μL of fluorescent dye PI (10μM) in the dark and incubate in a 37°C constant temperature incubator for 30 minutes. Use a microplate reader to measure the change in fluorescence value at an excitation wavelength of 535nm and a cutoff wavelength of 617nm. The test frequency is once per minute. After 8 minutes of continuous testing, take 10μL of diluted compound and PBS (blank control) and add them. Measure the fluorescence value for the next 30 minutes.

[0062] The results are as follows Figure 23 This indicates that the compound TPA-BBR can quickly penetrate the bacterial membrane, destroying its integrity and inducing bacterial lysis and death. The membrane integrity experiment suggests 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 the bacterial suspension was diluted with MH broth to an OD of 620 The bacterial solution contained 0.5× the MIC concentration of TPA-BBR and was grown at 37°C for 18 hours. For morphological studies, cells were collected and resuspended in 0.5 mL of PBS buffer, and 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 phase-contrast light microscopy.

[0064] Cell morphological changes such as Figure 24 The results show that TPA-BBR significantly increased the length of B. subtilis compared with the control group. These cell elongation phenomena are similar to other known FtsZ inhibitors, which preliminarily indicate the inhibitory effect of TPA-BBR on FtsZ.

[0065] Performance test: 5. Bacterial imaging experiment of compound TPA-BBR

[0066] S. aureus single colony on the MHA culture dish was picked up with a inoculating loop in MHB medium, and after 4h culture at 37℃ in a shaker at 220r / min, centrifugation (7100rpm, 3min) was performed to remove the culture medium, and the bacteria were washed with PBS for three times, resuspended with PBS and diluted to a final concentration of ~10 9 CFU / mL. After 20min incubation with the compound, centrifugation (7100rpm, 3min) was performed to remove the compound, PBS was added for resuspension, and fluorescence imaging was performed by laser confocal.

[0067] The results are shown in Figures 25-26 TPA-BBR can stain gram-positive bacteria S. aureus and gram-negative bacteria E. coli, respectively. The above results show that TPA-BBR can achieve broad-spectrum bacterial imaging.

[0068] The above only describes the embodiments of the present application, and the common knowledge of specific structures and / or characteristics in the scheme is not described in detail. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be regarded as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. A berberine derivative, characterized in that Their structural formulas are as follows:

2. The method for preparing the berberine derivative according to claim 1, characterized in that: This is achieved by following these steps: Step 1: Suspending the compound BBR in 1,4-dioxane, adding the bromine-containing 1,4-dioxane solution dropwise to the mixture at room temperature, and stirring the reaction mixture at room temperature for reaction; after the reaction, filtering out the precipitate, washing, drying, and concentrating under reduced pressure to obtain a crude product, which is separated and purified by column chromatography to obtain the compound Br-BBR; Step 2: A mixture of Br-BBR, 4-boronic acid triphenylamine or [1-(4-boronic acid ester phenyl)-1,2,2-triphenyl]ethylene, palladium acetate and sodium carbonate is stirred in anhydrous ethanol for reaction at room temperature; after the reaction is completed, the reaction mixture is extracted, washed, dried and concentrated under reduced pressure to obtain a crude product, which is separated and purified by column chromatography to obtain the target compound TPA-BBR or TPE-BBR.

3. The use of the berberine derivative in the preparation of a drug according to claim 1, wherein It is used as an active ingredient in the preparation of antibacterial drugs.

4. The use of the berberine derivative in the preparation of a drug according to claim 3, wherein The antibacterial drug is an antibacterial drug that kills Gram-positive bacteria.

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