Bacteria-targeting photosensitive compound and application thereof in photodynamic therapy

Abstract

The photosensitive compound comprises a beta-lactam ring as shown in a formula I and a Ce6 photosensitizer, and does not contain a quenching group. When the compound is in contact with MRSA and other gram positive bacteria, the beta-lactam ring structure in the compound is combined with MRSA and other gram positive bacteria cell wall synthesis key enzyme-transpeptidase to provide targeting for the photosensitizer Ce6, so that the targeting effect on the bacteria is realized, the specificity is strong, a large amount of active oxygen is generated under illumination to kill the bacteria, and the compound can be used as a photosensitizer Ce6. Meanwhile, damage to normal tissues is avoided, photodynamic therapy on bacterial infection is facilitated, and the photodynamic therapy has high efficiency and low tissue toxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound, in particular to a compound with targeting effect on bacteria, which achieves killing of gram-positive bacteria through photodynamic action, and its application in wound treatment. BACKGROUND

[0002] The continuous growth of multi-drug resistant (MDR) microorganisms has become one of the major threats to public health today. Overuse, misuse and misuse of antibiotics often lead to escalating bacterial resistance, resulting in a sharp rise in patient mortality. Since MRSA was first reported in 1961, MRSA infections have been found in various parts of the world, and MRSA infections have shown a clear upward trend, especially in burn wards, with a MRSA infection isolation rate of more than 60% in patients with wounds. MRSA has become multi-drug resistant, with an increasingly wide range of drug resistance and increasingly severe drug resistance. The number of antibiotics available for clinical treatment is decreasing, while the development of new treatment drugs is far slower than the growth of MRSA drug resistance, so the clinical treatment of MRSA infection is facing severe challenges.

[0003] In recent years, research has found that photodynamic therapy (PDT) for MRSA infection is one of the most promising treatment methods. Under the irradiation of light of appropriate wavelength, photosensitizers are excited from low-energy ground state to high-energy triplet state. Triplet-state photosensitizers react directly with surrounding biomolecules to produce free radicals (type I reaction), or react with molecular oxygen to produce highly active singlet oxygen (type II reaction). A large number of free radicals and singlet oxygen produced by the target microorganisms are toxic, which can cause damage to the cell membrane, leading to increased permeability, and damage to various important components such as proteins, enzymes, and nucleic acids in the cell, thus ultimately inactivating the microorganisms. However, traditional photosensitizers have poor water solubility and dispersibility, and no targeting between normal tissue cells and bacteria, so the ROS produced during treatment inevitably affects the tissue, limiting its clinical use. SUMMARY

[0004] One object of the present application is to provide a photosensitizing compound that achieves targeting of bacteria (such as gram-positive bacteria), thereby killing bacteria without causing damage to the tissue.

[0005] Another object of the present application is to provide a photosensitizing compound that enhances the effect of killing bacteria, which is beneficial for the development of photodynamic therapy.

[0006] Still another object of the present application is to provide the use of a photosensitizing compound in the preparation of an antibacterial drug.

[0007] Another object of the present application is to provide an application of the photosensitive compound in the preparation of antibacterial medical devices.

[0008] A photosensitive compound comprises a β-lactam ring and a Ce6 photosensitizer as shown in Formula I, and does not contain a quenching group.

[0009]

[0010] Another photosensitive compound comprises a β-lactam ring and a Ce6 photosensitizer as shown in Formula I, and a group R covalently connected to the β-lactam ring and the Ce6 photosensitizer respectively, the group R is an organic substance, such as an ester group, an ether group, an amide group, a C1-C10 hydrocarbon group, or a polymer, such as a polyester or a polyether.

[0011] Another photosensitive compound is shown in Formula II.

[0012]

[0013] The photosensitive compound provided by the present application integrates a bacterial targeting β-lactam ring on the basis of the Ce6 photosensitizer. When in contact with gram-positive bacteria such as MRSA, the β-lactam ring structure in the compound binds to the transpeptidase, a key enzyme in the synthesis of the cell wall of gram-positive bacteria such as MRSA, to provide the photosensitizer Ce6 with targeting, thereby achieving targeted action on bacteria, and having strong specificity. A large amount of reactive oxygen species (ROS) is generated under light to kill bacteria, while avoiding damage to normal tissues, and is more conducive to the implementation of photodynamic therapy on bacterial infections, so that the photodynamic therapy has high efficiency and low tissue toxicity.

[0014] The photosensitive compound of the present application is used to make drugs and drug-containing medical devices, and is applied to various types of photodynamic antibacterial therapy of wound infections, osteomyelitis, and the like, to achieve high-efficiency and low-tissue-toxicity bactericidal action. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The synthesis path diagram for preparing an embodiment of the photosensitive compound of the present application is shown in the figure;

[0016] Figure 2 The nuclear magnetic resonance hydrogen spectrum 1H NMR diagram of an embodiment of the photosensitive compound prepared by the present application is shown in the figure;

[0017] Figure 3 The bactericidal effect diagram of the photosensitive compound prepared by an embodiment of the present application is shown in the figure;

[0018] Figure 4 The confocal microscope photograph of the bacterial targeting distribution of the photosensitive compound of an embodiment of the present application is shown in the figure;

[0019] Figure 5 Figure 2 is a graph showing the change in absorption spectrum of a photosensitive compound according to an embodiment of the present application before and after treatment with beta lactamase;

[0020] Figure 6 Figure 4 is a graph showing the results of promoting wound healing using a photosensitive compound according to an embodiment of the present application;

[0021] Figure 7 Figure 6 is a graph showing the results of TUNEL staining to detect the apoptosis effect of a photosensitive compound according to an embodiment of the present application on skin tissue;

[0022] Figure 8 Figure 8 is a graph showing the comparison of the bactericidal effect of a photosensitive compound according to an embodiment of the present application and a control compound. DETAILED DESCRIPTION

[0023] The technical solutions of the present application are described in detail below. The embodiments of the present application are only used to illustrate the technical solutions of the present application and not to limit the same. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

[0024] In the following examples, all temperatures are in degrees Celsius (°C) unless otherwise indicated. Unless otherwise indicated, all starting materials and reagents are available through commercial channels such as Sigma-Aldrich, Thermo Fisher Scientific. The obtained starting materials and reagents are used directly without further purification.

[0025] Glassware was dried in an oven and / or heated dry. The reactions were followed on glass silica gel-60F254 plates (0.25 mm) (TLC). Analytical thin layer chromatography was developed with appropriate solvent ratios (v / v).

[0026] 1 H NMR spectra were determined using a Bruker instrument (400 MHz) and chemical shifts are expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. 1 H NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, dd = doublet of doublets. If coupling constants are provided, they are in Hz.

[0027] The preparation methods provided by the present application should be understood as examples for the necessity of full disclosure, rather than limiting the scope of protection required by the present application. Those skilled in the art can prepare various compounds provided by the present application according to the guidance of textbooks, experimental manuals or the examples listed in the present application. The preparation of these compounds also belongs to the general skills that should be possessed by those skilled in the art, and can be completed under the guidance of the prior art. Specifically, the preparation methods of some compounds provided by the present application are as follows Figure 1

[0028] Preparation of photosensitive compound

[0029] Ce6 and cephalosporin prodrug ACLE, and condensation reagent HATU were mixed in anhydrous dimethylformamide in a molar ratio of 1:1:1, stirred and reacted for 1 h, then diisopropylethylamine was added dropwise into the reaction solution, and the reaction was continued for 2 h. After the reaction was completed, the organic solvent dimethylformamide was removed by rotary evaporation, the reaction mixture was washed with saturated physiological saline to remove organic matter, and then the product was dried with sodium thiosulfate and further purified by chromatography, with the eluent being 10% methanol in dichloromethane.

[0030] The product was then added to a mixture of trifluoroacetic acid, anisole and dichloromethane 3 ml (in a ratio of 1:1:5), and stirred in an ice water bath for 1 h. The organic solvent was removed by rotary evaporation to obtain the product ACLE-Ce6, which removes the ester bond on the ACLE molecule that can hinder the recognition of β-lactamase.

[0031] The obtained compound was identified by NMR (see Figure 2 ) and MS, and the specific data are as follows:

[0032] ​1H NMR (400 MHz, DMSO) δ 9.82 (s, 1H), 9.72 (s, 1H), 9.14 (s, 1H), 8.30 (s, 1H), 7.27 (s, 1H), 7.15 (s, 1H), 7.02 (s, 1H), 6.87 (s, 2H), 6.86 (s, 1H), 6.83 (s, 1H), 6.46 (s, 1H), 6.42 (s, 1H), 6.19 (s, 1H), 6.16 (s, 1H), 5.38 (s, 1H), 5.35 (m, 1H), 4.61 (s, 1H), 4.46 (s, 1H), 4.43 (s, 1H), 4.41 (s, 1H), 4.29 (s, 1H), 3.80 (s, 3H), 3.76 (s, 4H), 3.74 (s, 2H), 3.72 (s, 3H), 3.71 (s, 1H), 3.69 (s, 6H), 3.58 (s, 6H), 3.51 (s, 1H), 3.46 (s, 1H), 3.35 (s, 8H), 3.33 (s, 1H), 3.29 (s, 1H), 3.09 (s, 1H), 3.08 (s, 1H), 3.07 (s, 1H), 3.06 (s, 1H), 2.59 (s, 3H), 2.50 (s, 9H), 2.18 (s, 4H), 1.73 (s, 6H), 1.70 (s, 7H), 1.68 (s, 1H), 1.66 (s, 1H), 1.65 (s, 1H), 1.52 (s, 1H), 1.36 (s, 10H), 1.30 (s, 5H), 1.29 (s, 1H), 1.26 (s, 6H), 1.25 (s, 1H), 1.23 (s, 1H), 1.20 (s, 1H), 1.18 (s, 1H), 1.17 (s, 1H), 1.16 (s, 1H), 1.14 (s, 1H), 1.11 (s, 1H), 1.08 (s, 1H), 1.06 (s, 4H), 1.03 (s, 1H), 1.00 (s, 1H). ES-MS (m / z): 838.23 (MH + )

[0033] Example 2 Experiment of photodynamic antibiosis

[0034] The specific scheme is as follows:

[0035] (1) The MRSA bacterial solution is diluted to a concentration of 10^7 / mL with a phosphate buffer at pH 7.4, and the ACLE-Ce6 photosensitizer is added to make the final concentration 1-5 μM.

[0036] (2) The bacterial solution is placed in a hole plate, and after irradiation for a certain length of time (5-10 min), the bacterial solution is gradiently diluted, and the amount of bacteria survival is calculated by plating on agar plates, such as Figure 3As shown, after 5 min blue light irradiation, MRSA has been completely killed.

[0037] Example 3 Verification of bacterial targeting of photosensitizing compound (ACLE-Ce6)

[0038] MRSA and cells (e.g. human umbilical vein vascular endothelial cells) were placed in the same culture dish, and Ce6 photosensitizer or ACLE-Ce6 was added, and incubated for 20 min;

[0039] The distribution of photosensitizer in bacteria and cells was then photographed using a confocal microscope, and it was found that the concentration of photosensitizer in bacteria was significantly higher than that in cells, and the results are shown in Figure 4 .

[0040] Example 4 Verification of photosensitivity of photosensitizing compound (ACLE-Ce6) after treatment with β-lactamase

[0041] ACLE-Ce6 was first dissolved in DMSO solution, and its absorption spectrum was measured;

[0042] Metal β-lactamase was then added, and after 1 h of treatment, the absorption spectrum of the solution was detected to compare whether there was any change in photosensitivity. The results are shown in Figure 5 , and it was found that there was no significant change in the absorption spectrum characteristics before and after treatment, indicating that ACLE-Ce6 still maintained its original photosensitivity after treatment with metal β-lactamase.

[0043] Example 5 Photodynamic antibacterial treatment of wound infection, osteomyelitis, etc.

[0044] Mice were anesthetized with isoflurane, and a circular skin cutter was used to make a circular wound with a diameter of 7 mm on the back of the mouse, and 50 μL of MRSA with a concentration of 1 x 10 9 CFU / mL was applied to the wound to induce wound infection. At the same time, the mice were divided into two groups, namely the control group and the blue light + ACLE-Ce6 group, and after 2 days of infection, 50 μL of ACLE-Ce6 solution with a concentration of 5 μM was added to the wound of the blue light + ACLE-Ce6 group, and after incubation for 20 min, 405 nm blue light with an energy density of 20 mW / cm 2 was used to irradiate for 5 min, and then the amount of residual bacteria in the wound exudate of the two groups of mice was evaluated. As shown in Figure 6 , the amount of residual bacteria in the wound of the blue light + ACLE-Ce6 group of mice was significantly less than that of the control group, and the wound healing speed was significantly accelerated.

[0045] Example 6 Verification of low tissue toxicity of photosensitizing compound (ACLE-Ce6)

[0046] The mice were anesthetized with isoflurane, and 50 μL of an ACLE-Ce6 solution with a concentration of 5 μM was applied to the skin of the mice after the fur was removed. Subsequently, the mice were irradiated with 405 nm blue light with an energy density of 20 mW / cm 2 for 5 minutes. The mice were sacrificed, and the skin tissue after irradiation was fixed and subjected to TUNEL staining to detect cell apoptosis. As shown in Figure 7 , no obvious apoptotic cells were observed in the skin tissue after irradiation, which was similar to the control group.

[0047] Example 7 Bactericidal Control Experiment

[0048] Compared with the compound recorded in the patent application with the application number 201210099069.5 (the 2012 application compound), the antibacterial effect of the photosensitizing compound ACLE-Ce6 in the present application is more excellent. As shown in Figure 8 , when the same dose of blue light irradiation was performed, the number of residual surviving bacteria in the ACLE-Ce6 group was significantly lower than that in the 2012 application compound.

Claims

1. A photosensitive compound, characterized by comprises a β-lactam ring as shown in Formula I and a Ce6 photosensitizer, and does not contain a quenching group.

2. The photosensitive compound according to claim 1, characterized in that It also comprises a group R, which is covalently linked to the β-lactam ring and the Ce6 photosensitizer, respectively.

3. The photosensitive compound according to claim 2, characterized in that The group R is an organic group selected from one or more of an ester group, an ether group, an amide group, a C1-C10 hydrocarbon group, or a polymer.

4. The photosensitive compound according to claim 2, characterized in that as shown in Formula II.

5. Use of the photosensitizing compound according to claim 1 in the preparation of an antibacterial medicament.

6. Use according to claim 5, characterized in that in a medicament for gram-positive bacteria.

7. Use of the photosensitizing compound according to claim 1 in the preparation of an antibacterial medical device.

8. A medicament for the practice of photodynamic therapy, characterized in that comprises the photosensitizing compound according to any one of claims 1-4.

9. A medical device, comprising comprises the photosensitizing compound according to any one of claims 1-4.

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