Water-soluble organic conjugated molecules with different rigid-flexible structures and application of water-soluble organic conjugated molecules

Abstract

The invention provides a novel organic conjugated molecule, the long conjugated D-pi-A structure enables the organic conjugated molecule to form nanoparticles with high positive charge density more easily, and meanwhile, a large amount of active oxygen generated under the irradiation of white light can efficiently split bacterial cell membranes and slow down the generation of bacterial drug resistance. The organic conjugated molecule as shown in the formula (I) has a strong photodynamic killing effect on gram-negative bacteria and gram-positive bacteria, and is low in hemolysis rate and good in biocompatibility. The invention provides guidance for developing a high-efficiency and low-toxicity photodynamic antibacterial agent.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and specifically relates to a class of water-soluble organic conjugated molecules with different rigid and flexible structures and their application in antibacterial. Background Art

[0002] Currently, diseases caused by pathogens pose a major threat to public health, especially the emergence of multidrug-resistant bacteria (MDR), which makes conventional antibiotic treatment difficult. Photodynamic therapy (PDT) uses photosensitizers (Ps) to absorb light to trigger photochemical reactions (type I and type II) to produce a variety of reactive oxygen species to kill bacteria. Since PDI is a non-invasive antibacterial method, it is not easy to cause bacterial resistance and side effects, and it has the advantages of flexibility, controllability, high efficiency, and low cost. It has now become a new antibacterial technology. However, the photosensitization performance and stability of organic photosensitizers are limited, which restricts the practical application of organic photosensitizers in PDI. In order to improve the photosensitization activity of organic photosensitizers with conjugated skeletons, researchers have proposed various strategies, such as designing organic-inorganic or organic-organic heterojunctions, constructing donor-acceptor (DA) or donor-π-acceptor (D-π-A) structures, enhancing hydrophilicity and expanding the light absorption range. Among these strategies, the unique molecular structure and optoelectronic properties of the D-π-A structure give it an advantage in PDT. First, in the D-π-A structure, the intrinsic electron push-pull effect between the donor unit and the acceptor unit can promote photoinduced charge separation, thereby improving the photosensitization activity of the photosensitizer. And the photosensitizer with D-π-A structure can effectively separate the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), thereby reducing the singlet-triplet energy gap (ΔEST), enhancing the ISC process, and thus improving the photosensitization effect. In addition, the photosensitizer with D-π-A structure has extremely high photostability and excellent near-infrared absorption ability. Adjusting the conjugation degree (composition) of the donor part of the D-π-A structure can significantly affect the rigidity and flexibility, photophysical properties, energy level structure and photochemical activity of the molecule. In addition, the rigidity and flexibility of the photosensitizer molecule will affect the generation of reactive oxygen species (ROS) produced by the photosensitizer sensitizing oxygen, and affect the binding mode of the photosensitizer with cells, thereby affecting the transmission of ROS, and ultimately leading to different bactericidal effects. Therefore, it is of great significance to develop photosensitizers with excellent photophysical properties, efficient ROS generation ability and good affinity with bacteria through rational molecular design. Summary of the invention

[0003] The purpose of the present invention is to provide a class of water-soluble organic conjugated molecules with different rigid and flexible structures and their preparation methods and applications. The organic conjugated molecules have good ROS generation ability and strong ability to bind to bacteria, showing excellent antibacterial effect, and provide guidance for the development of efficient photodynamic antibacterial agents.

[0004] The present invention provides an organic conjugated molecule compound, the structural formula of which is shown in formula (I):

[0005]

[0006] Wherein, n is an integer selected from 2-12;

[0007] X is a halogen (for example, fluorine F, chlorine Cl, bromine Br, iodine I);

[0008] is selected from C6-20 aryl, 5-20 membered heteroaryl;

[0009] is selected from 5-20 membered heteroaryl groups containing a nitrogen atom.

[0010] According to an embodiment of the present invention, n is any integer selected from 3-10, for example, 3, 4, 5, 6, 7, 8, 9, or 10.

[0011] According to an embodiment of the present invention, X is Br.

[0012] According to an embodiment of the present invention, is selected from C6-16 aryl, 5-16 membered heteroaryl; preferably, it is selected from C6-10 aryl, 5-10 membered heteroaryl. According to an embodiment of the present invention, is selected from: phenyl, naphthyl, thienyl, furyl, exemplified by phenyl and thienyl. According to an embodiment of the present invention, Selected from Indicates the attachment site of a group.

[0013] According to an embodiment of the present invention, is selected from 5-16 membered heteroaryl groups containing N atoms; preferably, the N atoms are connected to phenyl groups; preferably, it is selected from carbazole and diphenylamine. According to an embodiment of the present invention, Selected from Indicates the attachment site of a group.

[0014] According to an embodiment of the present invention, the compound is selected from any one of the following formulas (I-A), (I-B) and (I-C):

[0015]

[0016] The present invention also provides a method for preparing the compound represented by the above formula (I), which comprises the following steps:

[0017] (1) 4-methylpyridine and the compound represented by formula (II) undergo a substitution reaction to obtain a compound represented by formula (III);

[0018]

[0019] Wherein, X and n have the above definitions;

[0020] (2) the compounds represented by formula (IV) and formula (V) undergo coupling reaction to obtain the compound represented by formula (VI);

[0021]

[0022] in, X has the above definition;

[0023] (3) The compounds represented by formula (III) and formula (VI) undergo addition reaction to obtain the compound represented by formula (I).

[0024] According to an embodiment of the present invention, in step (1), the molar ratio of the compound represented by formula (II) to 4-methylpyridine may be 1:(2-8), specifically 1:5;

[0025] And / or, the temperature of the substitution reaction may be 60 to 90°C, specifically 85°C;

[0026] And / or, the reaction time may be 6 to 12 hours, specifically 10 hours;

[0027] And / or, the substitution reaction may be carried out in a solvent; preferably, the solvent may be any one of methanol, ethanol, and propanol, preferably ethanol.

[0028] According to an embodiment of the present invention, in step (2), the molar ratio of the compound represented by formula (IV) to the compound represented by formula (V) may be 1:(1-1.5), specifically 1:1;

[0029] And / or, the temperature of the coupling reaction may be 60 to 90°C, specifically 80°C;

[0030] And / or, the reaction time may be 4 to 12 hours, specifically 8 hours;

[0031] and / or, the coupling reaction is carried out under an inert atmosphere, such as a nitrogen atmosphere;

[0032] and / or, the coupling reaction is carried out in the presence of a catalyst, such as tetrakistriphenylphosphine palladium;

[0033] and / or, the coupling reaction is carried out in the presence of a base, such as potassium carbonate;

[0034] And / or, the coupling reaction is carried out in a solvent; preferably, the solvent may be a mixed solvent of tetrahydrofuran and water; the volume ratio of tetrahydrofuran to water is preferably (6-3):1, preferably 4:1.

[0035] According to an embodiment of the present invention, in step (3), the molar ratio of the compound represented by formula (III) to the compound represented by formula (VI) may be 1:(2-2.3), specifically 1:2;

[0036] And / or, the temperature of the addition reaction may be 60 to 90° C., specifically 80° C.;

[0037] And / or, the reaction time may be 4 to 12 hours, specifically 7 hours;

[0038] and / or, the addition reaction is carried out under an inert atmosphere, such as a nitrogen atmosphere;

[0039] and / or, the addition reaction requires the addition of a basic catalyst, such as piperidine;

[0040] And / or, the addition reaction is carried out in a solvent; preferably, the solvent may be a mixed solvent of dichloromethane and anhydrous ethanol, and the volume ratio of dichloromethane to anhydrous ethanol is preferably (0-4):50.

[0041] The present invention also provides a nanoparticle, wherein the nanoparticle is assembled from the compound represented by the above formula (I).

[0042] According to an embodiment of the present invention, the size of the nanoparticles may be nanometer-scale. Preferably, the size of the nanoparticles is 10-20 nm.

[0043] According to an embodiment of the present invention, the nanoparticles are prepared by the following steps: preparing a solution of the compound represented by formula (I) to obtain the nanoparticles; preferably, in the solution, the concentration of the compound represented by formula (I) may be 0.5 μM to 100 μM, such as 1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 100 μM. Preferably, in the solution, the solvent may be any one, two or more of water and phosphate buffer. According to an embodiment of the present invention, the hydration kinetic diameter of the nanoparticles is 30 nm-200 nm, preferably 50 nm-150 nm, such as 60 nm, 70 nm, 75.8 nm, 80 nm, 88.8 nm, 90 nm, 98.6 nm, 100 nm.

[0044] The present invention also provides the use of the compound represented by the above formula (I) or the above nanoparticles in the preparation of antibacterial agents.

[0045] According to an embodiment of the present invention, the antimicrobial agent is an antibacterial agent or preparation.

[0046] The present invention also provides the use of the compound represented by the above formula (I) or the above nanoparticles in the preparation of a cell membrane destroying agent.

[0047] According to an embodiment of the present invention, the cell membrane disrupting agent is a bacterial cell membrane disrupting agent.

[0048] According to an embodiment of the present invention, the bacteria may be Gram-positive bacteria or Gram-negative bacteria. Preferably, the Gram-positive bacteria may be Staphylococcus aureus; preferably, the Gram-negative bacteria may be Escherichia coli.

[0049] The present invention also provides an antibacterial agent or a cell membrane destroying agent, the active ingredient of which is the compound represented by the above formula (I) or the above nanoparticles.

[0050] According to an embodiment of the present invention, the antimicrobial agent is a photodynamic antimicrobial agent. According to an embodiment of the present invention, the antimicrobial agent is an antibacterial agent or preparation.

[0051] According to an embodiment of the present invention, the cell membrane disrupting agent is a bacterial cell membrane disrupting agent.

[0052] According to an embodiment of the present invention, the bacteria may be Gram-positive bacteria or Gram-negative bacteria. Preferably, the Gram-positive bacteria may be Staphylococcus aureus; preferably, the Gram-negative bacteria may be Escherichia coli.

[0053] Beneficial Effects

[0054] The D-π-A structure of the organic conjugated molecule shown in formula (I) provided by the present invention makes it have a strong photosensitization effect, and can efficiently generate active oxygen under white light irradiation, causing oxidative damage to bacteria, thereby destroying the membrane structure of bacteria and killing bacteria. In addition, by adjusting the rigidity and flexibility of the conjugated unit of the receptor part, its photosensitization ability to generate active oxygen and the binding strength with bacteria can be regulated, thereby affecting the killing effect on bacteria.

[0055] The present invention provides a novel organic conjugated molecule, whose long conjugated D-π-A structure makes it easier for the organic conjugated molecule to form nanoparticles with high positive charge density. At the same time, a large amount of reactive oxygen generated under white light irradiation can efficiently lyse bacterial cell membranes and slow down the development of bacterial resistance. The organic conjugated molecule shown in formula (I) has a strong photodynamic killing effect on both Gram-negative bacteria and Gram-positive bacteria, and has a low hemolysis rate and good biocompatibility. The present invention provides guidance for the development of high-efficiency and low-toxic photodynamic antibacterial agents.

[0056] Definition and explanation of terms

[0057] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and combined with each other. The definitions of groups and compound structures after such combinations and combinations should be understood to be within the scope of the specification and / or claims of this application.

[0058] The term "C6-20 aryl" is understood to mean a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms, preferably a "C6-16 aryl". The term "C6-16 aryl" is to be understood as preferably meaning a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms, in particular a ring having 6 carbon atoms ("C6 aryl"), for example phenyl; or biphenyl, or a ring having 9 carbon atoms ("C9 aryl"), for example indanyl or indenyl, or a ring having 10 carbon atoms ("C10 aryl"), for example tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C13 aryl"), for example fluorenyl, or a ring having 14 carbon atoms ("C14 aryl"), for example anthracenyl.

[0059] The term "5-20 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems, including aromatic or partially aromatic, having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S, for example "5-16 membered heteroaryl". The term "5-16 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S and, in addition, in each case may be benzo-fused. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl and the like; or azinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The synthetic route of the organic conjugated molecule represented by formula (Ⅰ-A) in Example 1;

[0061] Figure 2 The synthetic route of the organic conjugated molecule represented by formula (I-B) in Example 1;

[0062] Figure 3 The synthetic route of the organic conjugated molecule represented by formula (I-C) in Example 1;

[0063] Figure 4 This is a distribution diagram of the hydration kinetic diameter of nanoparticles formed by the assembly of organic conjugated molecules in Example 2;

[0064] Figure 5 The zeta potential data of the nanoparticles formed by the organic conjugated molecules in Example 2;

[0065] Figure 6 This is a TEM image of nanoparticles assembled by organic conjugated molecules in Example 2;

[0066] Figure 7 is the UV-visible absorption spectrum of the organic conjugated molecule solution in Example 2;

[0067] Figure 8 is the fluorescence emission spectrum of the organic conjugated molecule solution in Example 2;

[0068] Fig. 9 is the total amount of active oxygen generated by the organic conjugated molecule solution in Example 3 under white light irradiation;

[0069] Fig.10 is the active oxygen species generated by the organic conjugated molecule solution in Example 3 under white light irradiation;

[0070] Fig.11 The bactericidal effect of the organic conjugated molecules on Escherichia coli in Example 4;

[0071] Fig.12 The effect of the organic conjugated molecules on the surface charge of Escherichia coli in Example 5;

[0072] Fig.13 The effect of the organic conjugated molecules on the surface morphology of Escherichia coli in Example 5;

[0073] Fig.14 The bactericidal effect of the organic conjugated molecules in Example 6 on Staphylococcus aureus;

[0074] Fig.15 This is the hemolytic activity of the organic conjugated molecule in Example 7. DETAILED DESCRIPTION

[0075] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0077] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0078] PBS was purchased from Hyclone with the product number SH30256.01.

[0079] Escherichia coli (E. coli TOP10) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0080] Staphylococcus aureus ATCC 6538 was purchased from China General Microbiological Culture Collection Center (CGMCC).

[0081] Electron microscopy imaging was performed on a scanning electron microscope (S-4800, purchased from Hitachi).

[0082] The size and charge of the nanoparticles were measured using a Malven Zetasizer Nano-ZS instrument (ZEN 3600, Malven Instruments, Worcestershire, UK).

[0083] The remaining chemical and biological reagents were purchased commercially.

[0084] Example 1: Synthesis of organic conjugated molecules

[0085] 1. Synthesis of the organic conjugated molecule represented by formula Ⅰ-A (abbreviated as Cz-Ph-Ph-Hcy)

[0086]

[0087] according to Figure 1 The organic conjugated molecule shown in formula Ⅰ-A is synthesized according to the route shown in the figure. The specific steps are as follows:

[0088] (1) After adding 50 ml of anhydrous ethanol to a single-necked flask, take 4-methylpyridine (9.80 mL, 0.100 mol) and 1,4-dibromobutane (4.3620 g, 0.0202 mol) and add them to the reaction bottle. Heat and reflux at 85°C for 10 hours. Extraction, drying with anhydrous magnesium sulfate, filtration, rotary evaporation, dissolution, recrystallization, drying and other operations are carried out in sequence to obtain 1,1-(butane-1,4-diyl)bis(4-methylpyridine) bromide. The structural verification data are as follows: 1 H NMR (400MHz): δ1.97 (t, 4H), δ2.45 (s, 6H), δ5.01 (t, 4H), δ7.67 (d, 4H), δ8.90 (d, 4H).

[0089] (2) Under nitrogen protection, 9-(4-bromophenyl)carbazole (0.4004 g, 1.243 mmol), 4-formylphenylboronic acid (0.1863 g, 1.243 mmol), tetrakistriphenylphosphine palladium (0.1145 g, 0.0991 mmol), potassium carbonate (0.1718 g, 1.243 mmol) were added to the reaction bottle. Then 20 mL of tetrahydrofuran and 5 mL of deionized water were added to a 50 mL single-necked bottle and bubbled for deoxygenation for half an hour. Then the solvent was transferred to a 50 mL double-necked bottle, sealed in a nitrogen-protected device, refluxed at 80 ° C, and the reaction progress was tracked by TLC. After 8 hours, the reaction was completed. After cooling to room temperature, the reaction solution was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography after rotary evaporation to obtain biphenylaldehyde containing carbazole moiety. The structural verification data are as follows: 1 H NMR (400MHz): δ7.16-7.20(m, 2H), δ7.35(t, 1H), δ7.50(t, 1H), δ7.58(d, 1H), δ7.83( d, 1H), δ7.90-8.00 (m, 5H), δ8.04 (d, 2H), δ8.19 (d, 2H), δ8.55 (d, 1H), δ9.89 (d, 1H).

[0090] (3) Under nitrogen protection, add biphenylaldehyde (0.2500g, 0.7199mmol) containing carbazole moiety, then measure 50mL of anhydrous ethanol and add it directly to a double-necked flask, heat to 80°C, add 4mL of dichloromethane, then add 1-(butane-1,4-diyl)bis(4-methylpyridine) bromide (0.1424g, 0.3542mmol), 15 drops of piperidine, and then seal the reaction device. After half an hour, the solution was found to be completely dissolved and dark red. After continuing the reaction for 20 minutes, orange-red solid began to precipitate. After 7 hours, the reaction was terminated and obvious solid-liquid stratification was found. Filter to obtain the lower layer of yellow solid, recrystallize from anhydrous ethanol, and obtain the organic conjugated molecule shown in formula Ⅰ-A (abbreviated as Cz-Ph-Ph-Hcy). The structural verification data are as follows: 1 H NMR (400MHz): δ1.99 (t, 4H), δ4.59 (t, 4H), δ7.32 (m, 4H), δ7.47 (m, 8H), δ7.64 (m, 2H), δ7 .79 (m, 2H), δ7.88-7.98 (m, 10H), δ8.00-8.15 (m, 6H), δ8.27-8.32 (m, 8H), δ9.00 (m, 4H).

[0091] 2. Synthesis of the organic conjugated molecule represented by formula Ⅰ-B (abbreviated as Cz-Ph-T-Hcy)

[0092]

[0093] according to Figure 2 The organic conjugated molecule shown in formula I-B is synthesized according to the route shown in the figure. The specific steps are as follows:

[0094] (1) 4-methylpyridine and 1,4-dibromobutane generate 1,1-(butane-1,4-diyl)bis(4-methylpyridine) bromide, and the specific steps are the same as (1).

[0095] (2) Under nitrogen protection, 9-(4-bromophenyl)carbazole (0.4005 g, 1.243 mmol), 5-formyl-2-thiopheneboronic acid (0.1937 g, 1.242 mmol), tetrakistriphenylphosphine palladium (0.1156 g, 0.1000 mmol), and potassium carbonate (0.1715 g, 1.241 mmol) were added to the reaction flask. Then 20 mL of tetrahydrofuran and 5 mL of deionized water were added to a 50 mL single-necked bottle and bubbled for deoxygenation for half an hour. The solvent was then transferred to a 50 mL double-necked bottle and refluxed at 80°C. The reaction progress was tracked by TLC. After 8 hours, the reaction was completed. After cooling to room temperature, the reaction solution was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography after rotary evaporation to obtain thiophene aldehyde containing carbazole moiety. The structural verification data are as follows: 1H NMR (400MHz): δ7.10-7.20 (m, 2H), δ7.35 (t, 1H), δ7.50 (t, 1H), δ7.58 (d, 1H), δ7.80 (d, 1H), δ7.90-8.00 (m, 6H), δ8.19 (d, 1H), δ8.55 (d, 1H), δ9.84 (d, 1H).

[0096] (3) Under nitrogen protection, thiophene aldehyde containing carbazole moiety (0.2502g, 0.7082mmol) was added, and then 50mL of anhydrous ethanol was directly added to a two-necked flask, the temperature was raised to 80°C, 2mL of dichloromethane was added, and then 1-(butane-1,4-diyl)bis(4-methylpyridine) bromide (0.1424g, 0.3542mmol) and 15 drops of piperidine were added in sequence, and then the reaction device was sealed. After half an hour, the solution was found to be completely dissolved and dark red. After continuing the reaction for 20 minutes, an orange-red solid began to precipitate. After 7 hours, the reaction was terminated and obvious solid-liquid stratification was found. The orange-red solid in the lower layer was filtered and recrystallized with anhydrous ethanol to obtain the organic conjugated molecule shown in formula I-B (abbreviated as Cz-Ph-T-Hcy). The structural verification data are as follows: 1 H NMR (400MHz): δ1.97 (t, 4H), δ4.57 (t, 4H), δ7.25 (m, 4H), δ7.34 (m, 6H), δ7.46 (m, 2H), δ7. 48 (m, 2H), δ7.62 (m, 2H), δ7.80 (m, 6H), δ8.00 (m, 4H), δ8.26-8.32 (m, 10H), δ8.95 (m, 4H).

[0097] 3. Synthesis of the organic conjugated molecule represented by formula Ⅰ-C (abbreviated as DPA-Ph-T-Hcy)

[0098]

[0099] according to Figure 3 The organic conjugated molecule shown in formula Ⅰ-C is synthesized according to the route shown in the figure. The specific steps are as follows:

[0100] (1) 4-methylpyridine and 1,4-dibromobutane generate 1,1-(butane-1,4-diyl)bis(4-methylpyridine) bromide, and the specific steps are the same as (1).

[0101] (2) Under nitrogen protection, 4-bromotriphenylamine (1.2212 g, 3.769 mmol), 5-formyl-2-thiopheneboronic acid (0.4901 g, 3.141 mmol), tetrakistriphenylphosphine palladium (0.1815 g, 0.1571 mmol), and potassium carbonate (1.3024 g, 9.424 mmol) were added to the reaction bottle. Then 40 mL of tetrahydrofuran and 10 mL of deionized water were added, and nitrogen was used for deoxygenation for half an hour. After that, the mixture was sealed and stirred and heated under reflux at 80° C. in an oil bath for 5 hours. The mixture was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography after rotary evaporation to obtain thiophene aldehyde containing diphenylamine motif. The structural verification data are as follows: 1 H NMR (400MHz): δ7.16-7.20 (m, 2H), δ7.34 (t, 1H), δ7.48 (t, 1H), δ7.58 (d, 1H), δ7.80 (d, 1H), δ7.90-8.00 (m, 7H), δ8.19 (d, 1H), δ8.55 (d, 1H), δ9.84 (d, 1H).

[0102] (3) Under nitrogen protection, thiophene aldehyde containing diphenylamine moiety (0.3553 g, 1.001 mmol) was added, and then 25 mL of anhydrous ethanol was directly added to a double-necked flask, and the temperature was raised to 80°C. Subsequently, 1-(butane-1,4-diyl)bis(4-methylpyridine) bromide (0.2011 g, 0.5002 mmol) and 1 mL of piperidine were added in sequence, and then the reaction apparatus was sealed and refluxed for 7 hours. Obvious solid-liquid stratification was found. The red solid in the lower layer was obtained by filtration, and recrystallization was performed with anhydrous ethanol to obtain the organic conjugated molecule shown in Formula I-C (abbreviated as DPA-Ph-T-Hcy). The structural verification data are as follows: 1 H NMR (400MHz): δ2.00 (t, 4H), δ4.57 (t, 4H), δ7.32 (m, 12H), δ7.47 (m, 8H), δ7 .64 (m, 2H), δ7.78 (m, 8H), δ7.80 (m, 4H), δ8.00-8.15 (m, 6H), δ9.00 (m, 4H).

[0103] Example 2: Preparation of organic conjugated molecular nanoparticles and characterization of their photophysical properties

[0104] (1) Preparation of organic conjugated molecular nanoparticles

[0105] The solid powders of Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy prepared in Example 1 were dissolved in DMSO to prepare a 5 mM stock solution, which was stored in a 4 °C refrigerator away from light for later use. Before use, it was diluted to 25 μM with ultrapure water. The three organic conjugated molecules are amphiphilic and can form nanoparticles by self-assembly in aqueous solution.

[0106] (2) Nanoparticle size and surface charge

[0107] The particle size distribution and surface charge of nanoparticles were measured using a Malvern nanometer. In the experiment, a 4mW He-Ne laser (λ = 632.8nm) was used as the light source: the Contin method was used to fit the backscattered light intensity data, and the obtained nanoparticle size distribution was as follows: Figure 4 As shown; collect electrophoretic light scattering data, calculate the zeta potential of the nanoparticles through the Helmholtz-Smoluchowski relationship, and calculate the average of the three results as the zeta potential of the nanoparticles. Figure 5 At the same time, 5 μL of 25 μM organic conjugated molecule aqueous solution was dropped onto the ultra-thin carbon mesh, and after natural drying, the morphology of the nanoparticles was observed using a JEOL 2100F transmission electron microscope. The results are shown in Figure 6 shown.

[0108] Depend on Figure 4 It can be seen that the hydration kinetic diameters of the nanoparticles formed by Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy in aqueous solution are 88.8, 75.8 and 98.6 nm, respectively. In other words, as the flexibility of the conjugated structure increases, the size of the nanoparticles first decreases and then increases. Figure 5 It can be determined that the surface charge of the nanoparticles formed by the three organic conjugated molecules is greater than 70mV, which provides a guarantee for the subsequent electrostatic binding of bacteria. Figure 6 ) It can be seen that the three organic conjugated molecules form spherical nanoparticles with a size of 10 to 20 nm.

[0109] (3) Characterization of photophysical properties

[0110] Subsequently, the UV-visible absorption spectra and fluorescence emission spectra of the three organic conjugated molecules were characterized by using a UV-visible absorption spectrometer and a fluorescence spectrophotometer, respectively. The organic conjugated molecules were scanned for absorption at 200-800 nm, and the wavelength corresponding to the maximum absorption peak was selected as the excitation wavelength of the fluorescence spectrum to scan the fluorescence emission spectrum of the organic conjugated molecules.

[0111] Depend on Figure 7It can be seen that the three organic conjugated molecules have strong light absorption capabilities in the near-ultraviolet and visible light regions (320-650nm), which provides a basis for the subsequent selection of white light as a light source to sensitize the production of reactive oxygen species. The maximum ultraviolet absorption wavelengths of Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy are located at 385nm, 435nm and 470nm, respectively. Figure 8 As shown, the three organic conjugated molecules all have good fluorescence emission capabilities, and the maximum fluorescence emission wavelengths are 575nm, 605nm and 634nm, respectively. Since the three organic conjugated molecules all have large Stokes shifts (>160nm), they can be used for fluorescence imaging to characterize the binding of the three organic conjugated molecules to bacteria.

[0112] Example 3: Evaluation of the photodynamic activity of the compound represented by Formula I

[0113] Under visible light irradiation, the ability of the three organic conjugated molecules to generate reactive oxygen species was detected using a 2,7-dichlorofluorescein diacetate (DCFH-DA) probe. A DCFH-DA solution with a mother liquor concentration of 10 mM was prepared and placed in a -20°C refrigerator for use. Before the experiment, ethanol and 0.01 M NaOH aqueous solution were added to the 10 mM DCFH-DA stock solution, and activated at room temperature in the dark for 30 minutes to obtain an activated solution. The activated solution was diluted with 1×PBS to finally obtain a DCFH working solution with a concentration of 40 μM. It was placed on ice in the dark for use. The amount of ROS generated was determined by testing the fluorescence emission intensity at 525 nm ( Fig. 9 Then, singlet oxygen green fluorescence probe (SOSG), dihydroethidium (DHE), and hydroxyphenylfluorescein (HPF) were used as indicators to monitor the generation of three organic conjugated molecules under visible light irradiation. 1 O2、O2 - and ·OH. 0.5 μM organic conjugated molecule solution was mixed with SOSG, DHE and HPF respectively and irradiated under visible light for 10 min. The probe solution without organic conjugated molecule was used as blank group, and the fluorescence intensity was measured with the illumination time to determine 1 O2、O2 - The amount of OH generated ( Fig.10 ).

[0114] like Fig. 9 As shown, at low light intensity (5mW·cm -2), the fluorescence signal intensity of DCFH in the blank group remained basically unchanged with time, while the fluorescence intensity of the three organic conjugated molecules Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy at 525nm increased significantly with the increase of radiation time, which indicates that the designed and synthesized organic conjugated molecules have excellent photodynamic properties and can produce a large amount of ROS in a short time to kill bacteria.

[0115] Depend on Fig.10 It can be seen that the reactive oxygen species generated by the three organic conjugated molecules under white light irradiation can oxidize the OH probe to emit a fluorescent signal, and the fluorescent signal gradually increases with time. However, the generated reactive oxygen species cannot oxidize the O2 - The probe makes it fluoresce and only oxidizes a small amount of 1 The O2 probe caused it to emit fluorescence. These results indicate that the three organic conjugated molecules mainly produce reactive oxygen species through the hypoxia-dependent Type-I pathway, and a small amount of ROS is generated through the aerobic Type-II pathway.

[0116] Example 4: Photodynamic killing effect of the compound of formula I on Escherichia coli

[0117] The agar plate counting method was used to characterize the killing effect of organic conjugate molecules on Escherichia coli: 100 μL OD 600 =1.0Amp r E. coli bacterial solution was added to 400 μL of organic conjugate molecule PBS solution. The concentrations of organic conjugate molecules in the solution were 0, 0.5, 5, 10, and 25 μM, respectively. The mixed solution was then placed in a 37°C incubator and incubated in the dark for 30 min. The subsequent antibacterial experiment was set up in two groups: light and dark. The light group was exposed to white light (65 mW·cm -2 ) for 10 min, and the dark group was wrapped in tin foil and stored for 10 min. The treated bacterial solution was diluted with 1× PBS to 1×10 4 times, take 100 μL and evenly spread it on LB medium, culture it at 37℃ for 16-18h, and record the number of colonies formed. Use the formula IR=(C-C0) / C0×100% to calculate the affinity of organic conjugated molecules to Amp r The inhibition rate IR of E.coli, where C is the colony forming unit (CFU) of the experimental group and C0 is the colony forming unit (CFU) of the control group. Each group of experiments was repeated three times and the average value was taken.

[0118] Depend on Fig.11 It can be seen that for Gram-negative bacteria (Amp rE. coli), the three organic conjugated molecules all showed a certain degree of dark toxicity, and their dark toxicity gradually increased with the increase of concentration. Under visible light irradiation, Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy had a certain degree of dark toxicity against Amp r The antibacterial activity of E.coli was significantly enhanced. The three organic conjugate molecules at 25 μM had an antibacterial effect on Amp r The sterilization rates of E. coli were 100%, 100% and 95.57% respectively. These results showed that the three organic conjugated molecules had excellent photodynamic antibacterial effects on Gram-negative bacteria.

[0119] Example 5: Mechanism of action of the compound of formula I on Escherichia coli

[0120] First, the change of surface Zeta potential after incubation of organic conjugated molecules with Escherichia coli was measured using a nanoparticle size potentiometer. The specific steps are as follows: 100 μL of bacterial solution (OD600 = 1.0), organic conjugated molecules Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and TPA-T-Hcy stock solutions were mixed with an appropriate amount of 1×PBS to a total volume of 500 μL. The final concentrations of organic conjugated molecules were 5, 10, 15, and 20 μM, respectively. Incubate at 37°C in a dark place for 30 minutes. After incubation, centrifuge to remove the supernatant, add 1 ml of sterile water to mix, and place on ice for testing. The measurement results are as follows: Fig.12 As shown. SEM was then used to characterize the changes in the surface morphology of Escherichia coli after treatment with organic conjugated molecules. According to the steps of the "Flat Antibacterial Experiment", the organic conjugated molecules were incubated with Escherichia coli and treated in the dark or light. The treated bacterial solution was centrifuged at 7100rpm for 10 minutes, and 2.5% glutaraldehyde was added after removing the supernatant, and fixed in a 4°C refrigerator overnight. Centrifuge to remove glutaraldehyde, and wash the bacterial solution twice with 1×PBS. Finally, take 5μL of the bacterial solution onto a clean silicon wafer and then air-dry it naturally in an ultra-clean bench. After the sample is dry, use 40%, 70%, 90%, and 100% ethanol to perform gradient dehydration on the sample, each time for 6 minutes. Wait until the sample is naturally dry at room temperature before spraying gold and photographing with SEM. The changes in the surface morphology of Escherichia coli are shown in Fig.13 shown.

[0121] Depend on Fig.12 It can be seen that organic conjugated molecules can electrostatically bind to the negatively charged bacterial surface. Fig.13The effect of organic conjugated molecules on Escherichia coli was directly reflected. Under dark conditions, the Escherichia coli in the blank group had a full morphology, complete structure, and relatively uniform size. Under light conditions, the surface of the bacteria treated with organic conjugated molecules showed severe shrinkage, collapse, and overflow of contents. Combining the zeta potential and SEM results, the possible antibacterial mechanism of organic conjugated molecules was analyzed as first binding to the bacterial surface through electrostatic action, and then producing ROS under light conditions, destroying the components of bacteria such as DNA, proteins, and lipids, thereby severely damaging the integrity of the bacterial membrane, and the contents continued to overflow, eventually leading to bacterial death.

[0122] Example 6: Photodynamic killing effect of the compound of formula I on Staphylococcus aureus

[0123] The same agar plate counting method was used to characterize the killing effect of organic conjugate molecules on Staphylococcus aureus: 100 μL O 600 =1.0 was added to 400 μL of organic conjugate molecule PBS solution, and the concentration of organic conjugate molecule in the action solution was 0, 1, 2, 5, 10, 15 μM, respectively, and then placed in a 37°C incubator to incubate for 30 min in the dark. The subsequent antibacterial experiment was set up in two groups: light and dark. The light group was exposed to white light (65 mW·cm -2 ) for 10 min, and the dark group was wrapped in tin foil and stored for 10 min. The treated bacterial solution was diluted with 1× PBS to 1×10 4 times, take 100 μL and evenly spread it on NB medium, culture at 37°C for 16-18h, and record the number of colonies formed. The antibacterial rate IR of organic conjugated molecules against S. aureus was calculated using the formula IR = (C-C0) / C0×100%, where C is the colony forming unit number (CFU) of the experimental group and C0 is the colony forming unit number of the control group. Each group of experiments was repeated three times and the average value was taken.

[0124] Depend on Fig.14 It can be seen that for Gram-positive bacteria (S. aureus), the three organic conjugated molecules all have high dark toxicity, and the dark toxicity gradually increases with the increase of the concentration of organic conjugated molecules. The bactericidal rates of 15μM Cz-Ph-Ph-Hcy, Cz-Ph-T-Hcy and DPA-Ph-T-Hcy against S. aureus were 79.8%, 82.2% and 58.7%. After the introduction of white light irradiation, the antibacterial activity of the three organic conjugated molecules against S. aureus reached 100%. These results show that the three organic conjugated molecules are mainly antibacterial to Gram-positive bacteria through cationic membrane disruption, and have excellent photodynamic antibacterial effects.

[0125] Example 7: Good biocompatibility of the compound represented by formula I

[0126] The biosafety of organic conjugated molecules was characterized by measuring their hemolysis rate. The specific steps are as follows: Take an equal amount of fresh rabbit blood and centrifuge to remove the supernatant to obtain sufficient red blood cells. Incubate the three organic conjugated molecules with different concentrations with the diluted red blood cells at 37°C for 3 hours. Finally, centrifuge and take the supernatant to measure the absorbance and calculate the hemolysis rate. The red blood cells incubated with TritonX100 are the positive control group PC, and the red blood cells incubated with normal saline are the negative control group NC.

[0127] In the hemolysis experiment ( Fig.15 ), compared with the positive control group (TritonX100), even if the concentration of the three organic conjugated molecules was as high as 100 μM, the hemolysis rate of red blood cells was still far below 5%. The low hemolytic activity of the three organic conjugated molecules indicates that they have good biosafety.

[0128] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An organic conjugated molecule compound, the structural formula of which is shown in formula (I), in, n is an integer selected from 2 to 12; X is a halogen; is selected from C6-20 aryl, 5-20 membered heteroaryl; is selected from 5-20 membered heteroaryl groups containing a nitrogen atom.

2. The compound according to claim 1, characterized in that n is any integer selected from 3 to 10; Preferably, X is Br.

3. The compound according to claim 1 or 2, characterized in that is selected from C6-16 aryl, 5-16 membered heteroaryl; preferably, is selected from C6-10 aryl, 5-10 membered heteroaryl; Preferably, Selected from: phenyl, naphthyl, thienyl, furanyl; Preferably, Selected from Indicates the attachment site of a group.

4. The compound according to any one of claims 1 to 3, characterized in that is selected from 5-16 membered heteroaryl groups containing a N atom; Preferably, the N atom is attached to a phenyl group; Preferably, Selected from carbazole, diphenylamine; Preferably, Selected from Indicates the attachment site of a group.

5. The compound according to any one of claims 1 to 3, characterized in that The compound is selected from any one of the following formulas (I-A), (I-B) and (I-C):

6. A method for preparing the compound of formula (I) according to any one of claims 1 to 5, comprising the following steps: (1) 4-methylpyridine and the compound represented by formula (II) undergo a substitution reaction to obtain a compound represented by formula (III); in, X and n have the meanings as set forth in any one of claims 1 to 5; (2) the compounds represented by formula (IV) and formula (V) undergo coupling reaction to obtain the compound represented by formula (VI); in, X has the meaning as defined in any one of claims 1 to 5; (3) The compounds represented by formula (III) and formula (VI) undergo addition reaction to obtain the compound represented by formula (I).

7. A nanoparticle, wherein the nanoparticle is assembled from the compound represented by formula (I) according to any one of claims 1 to 5; Preferably, the size of the nanoparticles is 10-20 nm.

8. Any of the following uses of the compound of formula (I) according to any one of claims 1 to 5 or the nanoparticles according to claim 7: (i) Use in the preparation of an antibacterial agent; preferably, the antibacterial agent is an antibacterial agent or preparation; (ii) Use in the preparation of a cell membrane disrupting agent; preferably, the cell membrane disrupting agent is a bacterial cell membrane disrupting agent; Preferably, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

9. An antibacterial agent or a cell membrane disrupting agent, wherein the active ingredient is the compound represented by formula (I) according to any one of claims 1 to 5, or the nanoparticles according to claim 7; Preferably, the antibacterial agent is a photodynamic antibacterial agent; Preferably, the antimicrobial agent is an antibacterial agent or preparation; Preferably, the cell membrane disrupting agent is a bacterial cell membrane disrupting agent; Preferably, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

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