Preparation Method and Application of a Novel Fluorescent Macrocyclic Compound

Abstract

The present invention relates to the technical field of the preparation of macrocyclic compounds, specifically a preparation method and application of a novel fluorescent macrocyclic compound. The preparation method synthesizes a dialdehyde-substituted intermediate through a Suzuki coupling reaction; the obtained intermediate is reduced to benzyl alcohol and then converted into benzyl chloride; benzyl chloride and 1,4-bis(bromomethyl)benzene are subjected to a Friedel-Crafts reaction to obtain a monomer; under the catalysis of boron trifluoride diethyl ether, the monomer is cyclized to obtain a bromine-chain-substituted macrocyclic compound; the bromine-chain-substituted macrocyclic compound reacts with trimethylamine in ethanol to obtain a water-soluble fluorescent macrocyclic compound. By designing and synthesizing the novel fluorescent macrocyclic compound, the present invention can generate singlet oxygen in vitro, effectively encapsulate antibacterial drugs, and perform bacterial imaging at the same time. Through the synergistic effect, the macrocyclic compound enhances the efficacy of antibacterial drugs, overcomes the problems of drug resistance and reduced drug efficacy of traditional antibacterial agents, and provides a new strategy for antibacterial treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of macrocyclic compounds, and specifically to a preparation method and application of a novel fluorescent macrocyclic compound. Background Art

[0002] The effectiveness of traditional antibacterial drugs is restricted by bacterial drug resistance, and the research and development of new antibacterial strategies have become an important topic in the current medical field. In recent years, researchers have begun to explore enhancing antibacterial effects through non-traditional means. One promising strategy is to utilize reactive oxygen species (ROS) and their synergistic effects with antibacterial drugs. Singlet oxygen ( 1 O2) is a highly reactive oxygen molecule, usually generated by chemical reactions or photocatalytic reactions. It has strong oxidizing ability and can destroy the cell membranes, DNA, and other important cellular components of bacteria, thereby inhibiting or killing microorganisms. 1 The widespread application of 1 O2 is also limited by its instability and low production efficiency. Therefore, how to efficiently generate 1 O2 in a specific environment and synergistically act with antibacterial drugs to achieve stronger antibacterial effects has become a current research hotspot. Existing research mainly focuses on the combined application of photosensitive materials and antibacterial drugs, while the synergistic mechanism and efficiency between 1 O2 and drugs still remain an issue that needs to be deeply explored.

[0003] Traditional antibacterial drugs usually can only achieve treatment by directly killing or inhibiting the growth of bacteria, lacking an interaction mechanism with bacteria, resulting in limited effects, especially in the treatment of complex infections and multi-drug resistant bacterial infections. Existing antibacterial drugs often need to be used at high doses during treatment, which easily causes toxic and side effects. Moreover, existing 1 O2 generation methods have problems such as low efficiency, poor stability, and difficulty in local generation, restricting their widespread application in actual antibacterial treatment. Therefore, in view of the above current situation, there is an urgent need to develop a preparation method and application of a novel fluorescent macrocyclic compound to overcome the deficiencies in current practical applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a novel fluorescent macrocyclic compound to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A novel fluorescent macrocyclic compound, wherein the macrocyclic compound is [2]TpB-NExP6 having the following structure:

[0007] ;

[0008] Among them, the macrocyclic compound can generate singlet oxygen under light illumination and has a fluorescence imaging function.

[0009] As a further solution of the present invention: the fluorescence emission peak of the macrocyclic compound is located at 525 nm, presenting green fluorescence, which is suitable for bacterial imaging.

[0010] A preparation method of the novel fluorescent macrocyclic compound according to the above, comprising the following steps:

[0011] Step 1: Using 4,7-dibromo-2,1,3-benzothiadiazole and 4-formylphenylboronic acid as raw materials, a dialdehyde-substituted intermediate is synthesized through Suzuki coupling reaction.

[0012] Step 2: The intermediate obtained in Step 1 is reduced to benzyl alcohol, and then subjected to chlorination reaction to obtain benzyl chloride.

[0013] Step 3: Benzyl chloride and 1,4-bis(bromomethyl)benzene are subjected to Friedel-Crafts reaction under the catalysis of aluminum trichloride to obtain a monomer.

[0014] Step 4: Under the catalysis of boron trifluoride diethyl ether, the monomer is cyclized to obtain a bromine-chain-substituted macrocyclic compound.

[0015] Step 5: The bromine-chain-substituted macrocyclic compound reacts with trimethylamine in ethanol to obtain a water-soluble fluorescent macrocyclic compound.

[0016] As a further solution of the present invention: in Step 1, the solvent for the Suzuki coupling reaction is a mixed solution of tetrahydrofuran and water, the reaction temperature is 85 °C, and the reaction time is 48 hours.

[0017] As a further solution of the present invention: in Step 5, the reaction temperature is 90 °C, and the reaction time is 24 hours.

[0018] An application of the novel fluorescent macrocyclic compound according to the above in an antibacterial drug carrier, the macrocyclic compound can encapsulate antibacterial drugs through host-guest interaction and release singlet oxygen under light illumination to achieve a synergistic antibacterial effect.

[0019] As a further solution of the present invention: the antibacterial drug is cefotaxime sodium, and the complexation ratio of the macrocyclic compound to cefotaxime sodium is 1:1.

[0020] An application of the novel fluorescent macrocyclic compound according to the above in bacterial imaging, the macrocyclic compound can target Gram-positive bacteria and Gram-negative bacteria and achieve bacterial imaging through green fluorescence.

[0021] An antibacterial composition comprising the novel fluorescent macrocyclic compound described above, and the composition can significantly reduce the minimum inhibitory concentration of bacteria under light illumination conditions.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] In the present invention, the fluorescence emission peak of the water-soluble macrocycle (such as the macrocycle containing benzothiadiazole) is located at 525 nm, presenting green fluorescence, and can be used for bacterial imaging.

[0024] Under normal temperature and pressure, the macrocycle can be excited by an artificial light source to generate 1 O2.

[0025] In the present invention, compared with the monomer, the macrocyclic compound has a stronger ability to generate 1 O2.

[0026] The macrocyclic compound has good biocompatibility and can undergo host-guest interactions with drug molecules, meeting the principles of green chemistry and laying a foundation for the application of a series of compounds in this field. Brief Description of the Drawings

[0027] Figure 1 It is a synthetic route diagram of the macrocyclic compound in the embodiment of the present invention.

[0028] Figure 2 It is the nuclear magnetic resonance of the bromine-chain substituted macrocyclic compound in the embodiment of the present invention.

[0029] Figure 3 It is the nuclear magnetic resonance of the water-soluble macrocyclic compound in the embodiment of the present invention.

[0030] Figure 4 It is a schematic diagram for the study of the aggregation-induced emission property of [2]TpB-NExP6 in the embodiment of the present invention;

[0031] Among them, on the left is the fluorescence spectrum diagram at different acetone volumes in the mixed system of acetone and chloroform, and on the right is the broken line diagram of the fluorescence intensity change trend at 523 nm at different acetone volumes.

[0032] Figure 5 It is a schematic diagram for the study of the property of generating singlet oxygen by M-N and [2]TpB-NExP6 in the embodiment of the present invention;

[0033] Among them, on the left is the change of the ultraviolet absorption peak of ABDA with time under light illumination and in the presence of [2]TpB-NExP6, and on the right is the broken line diagram of the change trend of the absorption peak of ABDA alone and ABDA in the presence of the macrocycle [2]TpB-NExP6 and the monomer M-N with time.

[0034] Figure 6Schematic diagram of the JobPlot experiment of [2]TpB-NExP6 and cefotaxime sodium in the embodiments of the present invention;

[0035] Among them, (a) is the fluorescence emission spectrum diagram under different host-guest ratios, and (b) is the JobPlot curve diagram drawn according to the fluorescence intensity.

[0036] Figure 7 Schematic diagram of the MIC value test of the macrocyclic host-guest against three bacteria in the light and dark in the embodiments of the present invention;

[0037] Among them, Escherichia coli is on the left, Staphylococcus aureus is in the middle, and methicillin-resistant Staphylococcus aureus is on the right. Detailed implementation manners

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0040] Please refer to Figures 1 - 7 , a novel fluorescent macrocyclic compound provided by the embodiments of the present invention, and the macrocyclic compound is [2]TpB-NExP6 having the following structure:

[0041] ;

[0042] Among them, the macrocyclic compound can generate singlet oxygen under light conditions and has a fluorescence imaging function.

[0043] The fluorescence emission peak of the macrocyclic compound is located at 525 nm, presenting green fluorescence, which is suitable for bacterial imaging.

[0044] In an embodiment of the present invention, a preparation method of the novel fluorescent macrocyclic compound according to the above, includes the following steps:

[0045] Step 1: Using 4,7-dibromo-2,1,3-benzothiadiazole and 4-formylphenylboronic acid as raw materials, synthesize an intermediate substituted with dialdehyde through Suzuki coupling reaction;

[0046] Step 2: Reduce the intermediate obtained in Step 1 to benzyl alcohol, and then perform chlorination reaction to obtain benzyl chloride;

[0047] Step 3: Perform a Friedel-Crafts reaction on benzyl chloride and 1,4-bis(bromomethyl)benzene under the catalysis of aluminum trichloride to obtain a monomer;

[0048] Step 4: Cyclize the monomer under the catalysis of boron trifluoride diethyl ether to obtain a bromine-chain-substituted macrocyclic compound;

[0049] Step 5: React the bromine-chain-substituted macrocyclic compound with trimethylamine in ethanol to obtain a water-soluble fluorescent macrocyclic compound.

[0050] In Step 1, the solvent for the Suzuki coupling reaction is a mixed solution of tetrahydrofuran and water, the reaction temperature is 85 °C, and the reaction time is 48 hours.

[0051] In Step 5, the reaction temperature is 90 °C and the reaction time is 24 hours.

[0052] In an embodiment of the present invention, there is provided an application of a novel fluorescent macrocyclic compound as described above in an antibacterial drug carrier. The macrocyclic compound can encapsulate an antibacterial drug through host-guest interaction and release singlet oxygen under light irradiation to achieve a synergistic antibacterial effect.

[0053] The antibacterial drug is cefotaxime sodium, and the complexation ratio of the macrocyclic compound to cefotaxime sodium is 1:1.

[0054] In an embodiment of the present invention, there is provided an application of a novel fluorescent macrocyclic compound as described above in bacterial imaging. The macrocyclic compound can target Gram-positive bacteria and Gram-negative bacteria and achieve bacterial imaging through green fluorescence.

[0055] In an embodiment of the present invention, there is provided an antibacterial composition comprising the novel fluorescent macrocyclic compound as described above. The composition can significantly reduce the minimum inhibitory concentration (MIC) of bacteria under light irradiation.

[0056] Through the characterization of the basic physicochemical properties of the synthesized macrocyclic compound, the macrocycle with a fluorescent group is more suitable for subsequent antibacterial experiments. Therefore, a water-soluble macrocycle was obtained through post-modification to meet the requirement of generating 1 O2 under light. In this test, a light source, PBS (phosphate buffer, pH = 7.4), and 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA), etc. need to be prepared.

[0057] First, dissolve the macrocyclic compound in PBS (phosphate buffer, pH = 7.4) to prepare a 0.1 mM solution. Add the ABDA (9,10-anthracenediyl-bis(methylene)dicarboxylic acid) probe (0.01 mM), and then place the solution under visible light irradiation. Monitor the change in absorbance with a UV spectrum at regular intervals. Since 1O2 can react with ABDA and photobleach to form the corresponding endoperoxide. By measuring the absorbance value of the reaction solution at a specific wavelength (such as 400 nm) and according to its change trend, the ability of the macrocyclic compound to generate 1 O2 can be tested. During the whole experimental process, the light intensity and the concentration of the macrocyclic solution need to be well controlled to ensure the reliability of the data.

[0058] The host-guest interaction between the macrocyclic compound and the antibacterial drug cefotaxime sodium (n / n = 1:1) was proved by NMR characterization. By testing the fluorescence spectra of the macrocyclic compound and cefotaxime sodium at different ratios, a scatter plot of their fluorescence intensity and ratio was drawn to obtain the JobPlot curve, and the complexation ratio was calculated to be 1:1. Then fluorescence titration was carried out to obtain the complexation constant to prove the interaction between the macrocyclic compound and the guest molecule.

[0059] Therefore, the synthesis of novel macrocyclic compounds can effectively generate 1 O2 and encapsulate antibacterial drugs, thus achieving a synergistic antibacterial effect. Through this novel treatment strategy, the dosage of traditional antibacterial drugs can be reduced, thereby reducing side effects. By designing photosensitive macrocyclic compounds, they can efficiently generate 1 O2 under specific light conditions, thus overcoming the problems of low 1 O2 generation efficiency and poor stability in the prior art. The structural design of the macrocyclic compound endows it with good drug-loading capacity, which can effectively encapsulate antibacterial drugs and release 1 O2 in the local area, thereby improving the antibacterial effect and providing an innovative and multi-dimensional strategy for antibacterial treatment.

[0060] The present invention synthesizes the macrocyclic compound shown as Figure 1 by a five-step synthesis method. The bromine-chain substituted macrocycle can be further reacted with trimethylamine in ethanol to obtain a water-soluble fluorescent macrocycle (such as [2]TpB-NExP6 containing benzothiadiazole). We also synthesized water-soluble monomers (such as M-N) for the purpose of comparing with the macrocycle.

[0061] Example 1: The macrocyclic compound [2]TpB-NExP6 constructed with benzothiadiazole (a macrocyclic compound containing benzothiadiazole. By carrying out quaternary ammonium salt modification on it, the preparation of a water-soluble macrocycle is realized. The specific structural formula is as Figure 1For example: Weigh 5 g of 4,7-dibromo-2,1,3-benzothiadiazole, 7.6 g of 4-formylphenylboronic acid, 0.5 g of Pd(PPh3)4, and 13 g of Na2CO3. React them through Suzuki coupling in a solution of tetrahydrofuran / water (40 mL / 10 mL) at 85 °C for 48 h to obtain a dialdehyde-substituted compound with a yield of 96%. Further reduce it with sodium borohydride to obtain benzyl alcohol, and react it with thionyl chloride in a solvent at 65 °C for 24 h to obtain benzyl chloride. Both of these two steps are equimolar reactions. Then react it with 1,4-bis(bromomethyl)benzene and aluminum trichloride in dichloromethane through Friedel-Crafts reaction to obtain a monomer with a yield of 26%. Finally, catalyze it with boron trifluoride diethyl ether to obtain a macrocyclic compound with a yield of 30%. Figure 2 ). Subsequently, disperse the bromine-chain macrocyclic compound in ethanol, add trimethylamine, and react at 90 °C for 24 h to obtain the target compound with a yield of 96%. Figure 3 ). Test its fluorescence. By changing the content of acetone solvent (poor solvent) in the aqueous solution of the macrocyclic compound, it is proved that the target compound has green fluorescence and aggregation-induced emission properties. Figure 4 ). And by processing the fluorescence intensity data at different host-guest ratios, the host-guest Job Plot curve is obtained, which proves the 1:1 complexation mode between the host and the guest. Figure 6 ).

[0062] Example 2: Based on [2]TpB-NExP6 1 O2 generation ability test: First, mix the prepared aqueous solution of the macrocyclic compound with the ABDA probe evenly and transfer it to a fluorescence cuvette. Introduce a visible light source, control the system temperature to remain at room temperature, and perform UV tests at regular intervals. Under the same experimental conditions, conduct a control experiment with the monomer for the ability to generate 1 O2. It is found that the macrocycle has certain advantages. Figure 5 ).

[0063] Example 3: Antibacterial experiments were carried out on Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus. First, the minimum inhibitory concentration test (MIC) was carried out on the host-guest complex under the conditions of light and darkness. Place the bacteria in a 96-well plate and conduct experiments on the three bacteria in two groups of light treatment and dark treatment. The experimental results show that the minimum inhibitory concentration (MIC) values obtained under the condition of light are all smaller than those in the group without light. For example, under the condition of light, the MIC value of the macrocycle host-guest complex against Escherichia coli E. coli is 1.5 μM, and the MIC in the dark environment is 6.25 μM. The MIC values of Staphylococcus aureus S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) under light and dark conditions are 31.25 μM and 250 μM respectively. Figure 7). It is proved that light illumination generates 1 The important role of O2 in the antibacterial process. In addition, the quaternary ammonium salt macrocycle can target Escherichia coli and Staphylococcus aureus, and as a fluorescent probe, it can produce bright green fluorescence after binding to bacteria, realizing the fluorescence imaging of Gram-negative and Gram-positive bacteria.

[0064] In summary, by designing a novel photosensitive fluorescent macrocyclic compound, the present invention can generate 1 O2 under certain conditions and effectively encapsulate antibacterial drugs therein. This macrocyclic compound can not only bind to antibacterial drugs through host-guest interaction, but also further enhance the antibacterial effect of the drugs by generating 1 O2. 1 The high oxidizing property of O2 can directly damage biological macromolecules such as the cell membrane and DNA of bacteria, thereby achieving the effect of rapid sterilization. Compared with traditional single antibacterial drug treatment, this synergistic effect can effectively improve the antibacterial effect and reduce the generation of drug-resistant bacteria, especially having important application prospects in the treatment of complex infections and drug-resistant bacteria infections. It provides a theoretical basis for the development of novel antibacterial strategies and a new direction for the innovative design of future antibacterial systems.

[0065] It should be noted that in the present invention, it should be understood that although this specification is described according to embodiments, not each embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A novel fluorescent macrocyclic compound, characterized in that, The macrocyclic compound is [2]TpB-NExP6 with the following structure: ； Among them, the macrocyclic compound can generate singlet oxygen under light irradiation conditions and has a fluorescence imaging function.

2. A method for preparing the novel fluorescent macrocyclic compound according to claim 1, characterized in that, It includes the following steps: Step 1: Using 4,7-dibromo-2,1,3-benzothiadiazole and 4-formylphenylboronic acid as raw materials, synthesize an intermediate substituted with dialdehyde through Suzuki coupling reaction; Step 2: Reduce the intermediate obtained in Step 1 to benzyl alcohol, and then carry out chlorination reaction to obtain benzyl chloride; Step 3: Carry out Friedel-Crafts reaction on benzyl chloride and 1,4-bis(2-bromoethoxy)benzene under the catalysis of aluminum trichloride to obtain a monomer; Step 4: Under the catalysis of boron trifluoride diethyl ether, cyclize the monomer to obtain a macrocyclic compound substituted with bromine chains; Step 5: React the macrocyclic compound substituted with bromine chains with trimethylamine in ethanol to obtain a water-soluble fluorescent macrocyclic compound.

3. The preparation method of the novel fluorescent macrocyclic compound according to claim 2, wherein In Step 1, the solvent for the Suzuki coupling reaction is a mixed solution of tetrahydrofuran and water, the reaction temperature is 85 °C, and the reaction time is 48 hours.

4. The preparation method of the novel fluorescent macrocyclic compound according to claim 2, wherein In Step 5, the reaction temperature is 90 °C, and the reaction time is 24 hours.

5. An antibacterial composition, characterized in that, It contains the novel fluorescent macrocyclic compound described in Claim 1, and the composition can significantly reduce the minimum inhibitory concentration of bacteria under light irradiation conditions.

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