Responsive antibacterial hemicyanine photosensitizer with bacterial division protein FtsZ inhibition capability as well as synthesis method and application of responsive antibacterial hemicyanine photosensitizer

Abstract

The invention discloses a responsive antibacterial hemicyanine photosensitizer with bacterial division protein FtsZ inhibition capability, and a synthesis method and application thereof. The structure of the responsive antibacterial hemicyanine photosensitizer is shown as a general formula I, the test result of the IHcy-PbE synthesized in the embodiment shows that the IHcy-PbE can inhibit the growth of bacteria under the condition of no light, and can generate active oxygen to kill the bacteria under the light and after being activated by hydrogen peroxide, and the bacterial colony number is obviously reduced. Scanning electron microscope observation shows that bacteria treated by IHcy-PbE and hydrogen peroxide have an obvious splitting blocking condition relative to a control group, and the phenomena of membrane surface collapse and rupture occur in addition to splitting blocking after illumination. The dye not only can realize long-acting bacteriostasis, but also can perform rapid sterilization through photodynamic after responding to a bacterial infection environment, and has a wide application prospect in the fields of bacterial imaging and antibacterial treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of fine chemical organic dyes, and in particular to a responsive antibacterial hemicyanine photosensitizer having the ability to inhibit bacterial fission proteins, a synthesis method thereof, and an application thereof. Background Art

[0002] As a new type of antibacterial therapy, Photodynamic Antimicrobial Therapy (PDAT) has received more and more research in recent years because it can rapidly kill bacteria by generating reactive oxygen species and hardly produces drug resistance itself. Although much progress has been made in the use of photosensitizing dyes for antibacterial treatment, many problems still restrict their practical applications. Existing traditional photoantibacterial agents completely stop working after illumination, have no killing and inhibitory ability for a small number of bacteria that have not been completely inactivated, and are prone to rapid recurrence of bacterial infections, thus rendering the treatment ineffective. Moreover, the phototoxicity to non-treatment target areas will also cause phototoxic side effects, which limits the application of photoantibacterial therapy. Existing traditional photosensitizers cannot achieve infection microenvironment response, cannot report the infection situation of bacteria, and are also difficult to achieve precise and intelligent treatment.

[0003] Among the proteins related to bacterial fission, filamentous temperature-sensitive protein FtsZ is a protein with rich content and stable structure in bacteria, and is almost present in all pathogenic bacteria. A series of inhibitors of FtsZ protein have been proven to be able to effectively inhibit bacterial fission and kill bacteria, thereby treating bacterial infections. Although filamentous temperature-sensitive protein FtsZ is a key protein for bacterial fission, its mechanism for treating bacterial infections is mainly through inhibiting bacterial fission, and it is difficult to achieve rapid sterilization of bacteria that are not in the process of fission, which reduces the ability of the inhibitor to clear bacterial infections.

[0004] In summary, the existing technology is difficult to combine the rapid bactericidal ability and long-acting bacteriostatic ability of antibacterial agents, and at the same time lacks the ability to respond to and report the infection microenvironment, has strong phototoxicity to non-target areas, has many deficiencies, and seriously restricts its practical applications. A multifunctional and intelligent antibacterial photosensitizing dye that can simultaneously inhibit bacteria for a long time and be activated in the infection microenvironment is a more ideal choice. Therefore, there is an urgent need to propose a new type of antibacterial hemicyanine photosensitizer having the ability to inhibit bacterial fission protein FtsZ and the ability to respond to the infection microenvironment, which is of great significance for solving the key problems in the treatment of bacterial infections. Summary of the Invention

[0005] In view of the above problems of the prior art, the present invention provides a method for synthesizing a responsive antibacterial hemicyanine photosensitizer with the ability to inhibit bacterial division protein FtsZ and its application, which can be used for fluorescence imaging, can effectively inhibit and kill Gram-positive bacteria, and has important research significance for the diagnosis and treatment of bacterial infections.

[0006] To achieve the above object, the first aspect of the present invention discloses a responsive antibacterial hemicyanine photosensitizer with the ability to inhibit bacterial division protein FtsZ, and its structure is shown in general formula I:

[0007]

[0008] In general formula I,

[0009] X is selected from one of oxygen, sulfur, selenium, tellurium, and dimethylsilicon. Further preferably, in the embodiments of the present invention, X is selected as oxygen;

[0010] Y - is selected from one of halogen ions. Further preferably, in the embodiments of the present invention, Y - is selected as iodide ion;

[0011] R1 is selected from one of H, C1-C12 alkyl, phenyl, benzyl, and further preferably, in the embodiments of the present invention, R1 is selected as ethyl;

[0012] R2 is selected from one of carbon, oxygen, sulfur, selenium, and carbon dimethyl. Further preferably, in the embodiments of the present invention, R2 is selected as carbon dimethyl;

[0013] R3 and R4 are each independently selected from one of H, phenyl, Cl, Br, and I groups. Further preferably, in the embodiments of the present invention, R3 is selected as iodine and R4 is selected as H;

[0014] R5 is selected from any one of the groups represented by the following structural formulas M-1 or M-2:

[0015]

[0016] Further preferably, in the embodiments of the present invention, R5 is selected as M-1.

[0017] The second aspect of the present invention lies in disclosing a method for synthesizing a responsive antibacterial hemicyanine photosensitizer with the ability to inhibit bacterial division protein FtsZ;

[0018]

[0019] Its synthesis method includes the following steps:

[0020] (1) In organic solvent 1, add N,N-dimethylformamide (DMF), slowly dropwise add phosphorus tribromide under low-temperature conditions and stir for a period of time, then slowly dropwise add cyclohexanone. After full reaction, pour it into ice water and adjust the pH to 6 - 8 using inorganic base 1, and extract with organic solvent 1. The obtained organic phase is concentrated under reduced pressure, and then add the raw material containing the R4 substituent and inorganic base 2, add organic solvent 2 as the reaction solvent. After full reaction, it is concentrated, purified under reduced pressure to obtain the compound shown in formula Z-1;

[0021] (2) In organic solvent 3, add the compound shown in formula Z-1, slowly add the demethylation reagent under low-temperature conditions, fully react, and concentrate, purify under reduced pressure to obtain the compound shown in formula Z-2;

[0022] (3) In organic solvent 4, add the compound shown in formula Z-2, the raw material containing the R5 substituent and inorganic base 3, fully react, and then concentrate, purify under reduced pressure to obtain the compound shown in formula Z-3;

[0023] (4) At 50 - 150 °C, in organic solvent 5, add the compound shown in formula Z-3 and the raw material containing the R1, R2, R3 substituents, fully react, and then concentrate, purify under reduced pressure to obtain the compound shown in formula Z-3.

[0024] Further preferably, in the step (1), the molar ratio of the raw material containing the R4 substituent to DMF, phosphorus tribromide, cyclohexanone, and inorganic base 2 is 1:2 - 10:2 - 10:1 - 2:1 - 5, and the preferred ratio is 1:5 - 10:5 - 10:1.5 - 2:1.5 - 3.

[0025] Further preferably, in the above step (1), the organic solvent 1 is selected from one or a combination of chloroform, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, ethyl acetate, petroleum ether, toluene, xylene; the inorganic base 1 and inorganic base 2 are each independently selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, sodium ethoxide; the organic solvent 2 is selected from one or a combination of DMF, DMSO, ethyl acetate, ethylene glycol dimethyl ether, acetonitrile.

[0026] Further preferably, in the above step (2), the molar ratio of the compound shown in Z-1 to the demethylation reagent is 1:1 - 20, and the preferred ratio is 1:5 - 10.

[0027] Further preferably, in the above step (2), the organic solvent 3 is selected from one or a combination of chloroform, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, petroleum ether, toluene, xylene, acetonitrile; the demethylation reagent is selected from one or a combination of boron tribromide, aluminum trichloride, trimethylsilyl iodide.

[0028] Further preferably, in the above step (3), the molar ratio of the compound shown in formula Z-2, the raw material containing the R5 substituent and the inorganic base 3 is 1:1-3:1-5, and the preferred ratio is 1:1-1.5:1-2.

[0029] Further preferably, in the above step (3), the organic solvent 4 is selected from one or a combination of petroleum ether, acetone, ethyl acetate, dichloromethane, DMF, DMSO; the inorganic base 3 is selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, sodium ethoxide.

[0030] Further preferably, in the above step (4), the molar ratio of the compound shown in formula Z-3 and the raw material containing the R1, R2, R3 substituents is 1:0.5-5, and the preferred ratio is 1:1-1.5.

[0031] Further preferably, in the above step (4), the organic solvent 5 is selected from one or a combination of methanol, ethanol, isopropanol, petroleum ether, acetone, ethyl acetate, acetonitrile, tetrahydrofuran, chloroform, dichloromethane, DMF, DMSO.

[0032] Another aspect of the present invention is to protect the application of the class of responsive antibacterial hemicyanine photosensitizers with the ability to inhibit bacterial fission protein FtsZ, including:

[0033] Bacterial fission inhibition: By targeting the bacterial FtsZ protein, interfering with its polymerization process, inhibiting bacterial fission and proliferation.

[0034] Fluorescence imaging technology for bacterial infection: Utilizing the fluorescence characteristics of the dye to achieve highly sensitive and highly selective imaging of the bacterial infection site.

[0035] Photodynamic therapy technology: Preparing a preparation for photodynamic therapy, generating singlet oxygen under light irradiation conditions, and having a highly efficient killing effect on bacteria.

[0036] Antibacterial material development: Used for developing antibacterial coatings, medical devices, medical dressings, etc., to prevent and treat bacterial infections.

[0037] Environment and public health: Applied to environmental water treatment, food preservation and agricultural disease control, reducing the risk of bacterial transmission and pollution.

[0038] The present invention particularly focuses on the targeted imaging and interference polymerization of bacterial FtsZ protein, the practical applications of bacterial fluorescence imaging technology, and antibacterial treatment technology, and has broad application prospects and social and economic value.

[0039] In summary, the present invention has the following beneficial effects:

[0040] 1. The responsive antibacterial hemicyanine photosensitizer designed with a hemicyanine dye as the backbone and having the ability to inhibit bacterial fission protein FtsZ retains the photodynamic performance of traditional substituted hemicyanine dyes while increasing the ability to inhibit bacterial fission and respond to overexpressed hydrogen peroxide in the bacterial infection area. Test Example 1 of the present invention shows that IHcy-PbE synthesized in the examples exhibits excellent absorption in PBS, dichloromethane, and methanol, facilitating practical applications. Test Example 2 of the present invention shows that the fluorescence of IHcy-PbE is enhanced in an aqueous solution containing hydrogen peroxide, indicating its ability to respond to hydrogen peroxide and enabling activation at the infection site. This responsiveness enables IHcy-PbE to be specifically activated at the bacterial infection site, which can not only report the bacterial infection situation through fluorescence but also help reduce damage to normal tissues and improve the accuracy and safety of treatment.

[0041] 2. Through experimental tests, the responsive antibacterial hemicyanine photosensitizer with the ability to inhibit bacterial fission protein FtsZ described in the present application can effectively inhibit Gram-positive bacteria represented by Staphylococcus aureus and has the ability to kill positive bacteria under light conditions after activation. Test Example 3 of the present invention shows that IHcy-PbE has the ability to bind to the recombinant FtsZ protein of Staphylococcus aureus, and the dissociation equilibrium constant K D value is 5.58 ± 0.15 μM. Test Examples 4 and 5 further confirm that IHcy-PbE can effectively inhibit Gram-positive bacteria represented by Staphylococcus aureus. Under the conditions of hydrogen peroxide and light, the number of bacterial colonies CFU of methicillin-resistant Staphylococcus aureus treated with IHcy-PbE is significantly reduced. IHcy-PbE can effectively inhibit and kill bacteria at low concentrations, showing its great potential in the treatment of bacterial infections.

[0042] 3. The responsive antibacterial hemicyanine photosensitizer with the ability to inhibit bacterial division protein FtsZ described in this application has long-lasting antibacterial ability, good response performance at the infection site, and excellent photoantibacterial ability, and has good application prospects in the imaging and treatment of bacterial infections. In Test Example 6 of the present invention, it was found by scanning electron microscopy that when the photosensitizer was added but not irradiated, it was obvious that the division of bacteria was inhibited, and the size and volume of the bacteria increased. After adding the photosensitizer, hydrogen peroxide and irradiating, in addition to the change in size and morphology of the bacteria, obvious collapse, wrinkling and rupture also appeared on the membrane surface, further verifying that the IHcy-PbE photosensitizer synthesized in the examples of the present invention has effectively inhibited the bacterial FtsZ protein and can cause photodynamic damage to bacteria under the activation of hydrogen peroxide and irradiation conditions. This morphological damage further confirms the killing mechanism of IHcy-PbE on bacteria and provides strong support for its effectiveness in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0044] Figure 1 Absorption and fluorescence spectra of IHcy-PbE in the examples of the present invention, IHcy-OH in Comparative Example 1, and Hcy-OH in Comparative Example 2 in different reagents.

[0045] Figure 2 Fluorescence spectra of IHcy-PbE in the examples of the present invention and IHcy-OH in Comparative Example 1 under the activation of hydrogen peroxide at different concentrations.

[0046] Figure 3 UV absorption decay curves of singlet oxygen quencher DPBF of IHcy-PbE in the examples of the present invention, IHcy-OH in Comparative Example 1, and Hcy-OH in Comparative Example 2 in methanol.

[0047] Figure 4 Determination of the binding ability of IHcy-PbE in the examples of the present invention, IHcy-OH in Comparative Example 1, and Hcy-OH in Comparative Example 2 to FtsZ protein.

[0048] Figure 5 Inhibition experiments of IHcy-PbE in the examples of the present invention, IHcy-OH in Comparative Example 1, and Hcy-OH in Comparative Example 2 on different bacteria.

[0049] Figure 6Colony counting of bacteria killed by IHcy-PbE in the embodiments of the present invention, IHcy-OH in Comparative Example 1, and Hcy-OH in Comparative Example 2.

[0050] Figure 7 Observation of morphological damage to different bacteria by IHcy-PbE in the embodiments of the present invention under a scanning electron microscope. Detailed Description of the Invention

[0051] The following combines the accompanying drawings and embodiments to further describe in detail the specific implementation manners of the present invention. The following details the hemicyanine dyes shown in General Formula I in combination with embodiments, but does not limit the scope of the present invention.

[0052] In the specific implementation process of the present invention, in addition to the key steps described in detail, a variety of conventional chemical reagents and standard operating procedures are used. For example, all solvents such as dichloromethane, N,N-dimethylformamide (DMF), methanol, etc. are purchased from commercial suppliers and their purity is ensured. In addition, common chemical reaction conditions such as reaction systems under nitrogen protection, rotary evaporation under reduced pressure, silica gel column chromatography, etc. are all standard operations in the field of organic synthesis.

[0053] The selection of these conventional steps and reagents is based on their wide applicability and reliability, and does not constitute a key impact on understanding the core technology of the present invention, so they are not described in detail.

[0054] Unless otherwise specified, the terms used herein have the following meanings.

[0055] The term "halogen" used herein includes fluorine, chlorine, bromine, and iodine.

[0056] The term "alkyl" used in the present invention includes straight-chain alkyls and branched-chain alkyls.

[0057] Example 1

[0058] The hemicyanine dye IHcy-PbE has the following structural formula:

[0059]

[0060] Its synthesis method includes the following steps:

[0061]

[0062] (1) Synthesis of Compound 1

[0063]

[0064] Take 11.2 mL of N,N-dimethylformamide (DMF), add it to 50 mL of chloroform for dissolution. While cooling in an ice-water bath, slowly drip 12.4 mL of phosphorus tribromide. After stirring for 45 min, slowly drip 5 mL of cyclohexanone, and stir at room temperature for 16 h. Pour the reaction solution into ice water for quenching, slowly add solid sodium bicarbonate to adjust the pH to neutral, then extract three times with dichloromethane. Take the organic phase, add anhydrous sodium sulfate for drying, filter, and rotary evaporate. Add the obtained liquid to 20 mL of DMF. Weigh 2-hydroxy-4-methoxybenzaldehyde (3.05 g, 20 mmol) and cesium carbonate (13.03 g, 40 mmol), and slowly add them to the reaction system. Stir the reaction at room temperature for 24 h under nitrogen protection. After the reaction is completed, remove the solvent by distillation under reduced pressure, and use column chromatography for separation and purification (V 二氯甲烷 :V 石油醚 =1:1) to obtain a yellow solid with a yield of 1.41 g and a yield of 29%.

[0065] (2) Synthesis of Compound 2

[0066]

[0067] Weigh Compound 1 (0.29 g, 1.2 mmol) and dissolve it in 10 mL of anhydrous dichloromethane. While cooling in an ice-water bath, slowly drip boron tribromide (3.01 g, 10.0 mmol). React at room temperature for 24 h under nitrogen protection. After the reaction is completed, pour the reaction solution into ice water for quenching, slowly add solid sodium bicarbonate to adjust the pH to 6 - 8, then extract three times with dichloromethane. Take the organic phase and remove the solvent by distillation under reduced pressure, and use column chromatography for separation and purification (V 二氯甲烷 :V 甲醇 =100:2) to obtain a yellow solid with a yield of 0.21 g and a yield of 75%.

[0068] (3) Synthesis of Compound 3

[0069]

[0070] Weigh Compound 2 (184 mg, 0.8 mmol), 4-bromomethylphenylboronic acid pinacol ester (184 mg, 0.88 mmol), and potassium carbonate (138 mg, 1.0 mmol), add them to 10 mL of acetone, and reflux for 12 h under nitrogen protection. After the reaction is completed, pour the reaction solution into water, extract three times with dichloromethane. Take the organic phase and remove the solvent by distillation under reduced pressure, and use column chromatography for separation and purification (V 二氯甲烷 :V 甲醇 =100:2) to obtain a yellow solid with a yield of 263 mg and a yield of 74%.

[0071] (4) Synthesis of the target compound IHcy-PbE

[0072]

[0073] Weigh 3 (223 mg, 0.50 mmol) and 1-ethyl-5-iodo-2,3,3-trimethyl-3H-indolium iodide (243 mg, 0.55 mmol), add them to 10 mL of ethanol, and reflux for 4 h under nitrogen protection. After the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, and purify by column chromatography (V 二氯甲烷 :V 甲醇 = 100:5) to obtain a dark blue solid with a yield of 0.059 g and a yield of 89%.

[0074] 1 H NMR (400 MHz, MeOD) δ 8.71 (d, J = 14.8 Hz, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.87 (dd, J = 8.4, 1.7 Hz, 1H), 7.85–7.74 (m, 2H), 7.51 (dd, J = 8.1, 1.5 Hz, 3H), 7.47 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 6.44 (d, J = 14.7 Hz, 1H), 4.33 (q, J = 7.3 Hz, 2H), 3.49 (q, J = 7.0 Hz, 1H), 2.75 (dt, J = 27.5, 6.0 Hz, 4H), 1.93 (p, J = 6.0 Hz, 2H), 1.79 (s, 6H), 1.44 (t, J = 7.2 Hz, 3H), 1.34 (s, 12H), 1.17 (t, J = 7.0 Hz, 2H).

[0075] 13 C NMR (101 MHz, MeOD) δ 176.21, 162.53, 154.50, 145.88, 144.24, 141.12, 139.77, 137.95, 134.82, 134.75, 131.72, 128.86, 127.38, 126.34, 116.12, 114.93, 114.49, 113.88, 102.49, 101.34, 90.43, 83.86, 70.46, 65.51, 50.35, 39.98, 28.58, 26.90, 23.85, 23.65, 20.20, 14.04, 11.31, -1.45.

[0076] Comparative Example 1

[0077] The comparative example IHcy-OH of the hemicyanine dye with a heavy atom modification but without a hydrogen peroxide-responsive linker was synthesized using a method similar to that of Example IHcy-PbE, and the structure and synthetic route are as follows:

[0078]

[0079] 1 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.56 (d, J = 14.7 Hz, 1H), 8.16 (d, J = 1.7 Hz, 1H), 7.87 (dd, J = 8.3, 1.7 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.88 (dd, J = 8.5, 2.3 Hz, 1H), 6.45 (d, J = 14.7 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H), 1.86–1.80 (m, 2H), 1.73 (s, 6H), 1.33 (t, J = 7.1 Hz, 3H).

[0080] 13 13C NMR (101 MHz, DMSO-d6) δ 175.74, 162.44, 162.21, 154.78, 145.09, 144.60, 141.57, 137.92, 135.86, 131.95, 129.93, 126.41, 115.49, 115.09, 114.83, 103.26, 102.46, 92.02, 50.53, 28.74, 27.75, 24.13, 20.45, 12.87.

[0081] Comparative Example 2

[0082] The comparative example Hcy-OH of the hemicyanine dye without a hydrogen peroxide-responsive linker and without heavy atom modification was synthesized using a method similar to that of Example I Hcy-PbE, and the structure and synthetic route are as follows:

[0083]

[0084] 11H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.59 (d, J = 14.9 Hz, 1H), 7.76 (dd, J = 7.5, 1.2 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.57–7.40 (m, 4H), 6.92 (d, J = 2.3 Hz, 1H), 6.85 (dd, J = 8.5, 2.3 Hz, 1H), 6.52 (d, J = 14.8 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 2.71 (dt, J = 16.9, 6.0 Hz, 4H), 1.86–1.80 (m, 2H), 1.75 (s, 5H), 1.37 (t, J = 7.1 Hz, 3H).

[0085] 13 13C NMR (101 MHz, DMSO-d6) δ 176.90, 162.10, 161.49, 154.62, 145.22, 142.47, 141.61, 134.79, 129.75, 129.37, 127.24, 126.36, 123.30, 115.07, 114.89, 114.24, 113.25, 103.81, 102.45, 50.68, 28.80, 27.88, 24.08, 20.47, 13.05.

[0086] Test Example

[0087] In order to prove that the hemicyanine dye IHcy-PbE prepared in the examples can target the FtsZ protein, prevent bacterial division, and can perform photodynamic antibacterial therapy on Gram-positive bacteria under light conditions after being activated in response to the bacterial infection microenvironment, which has important research significance for the treatment of bacterial infections, the following tests were carried out on the hemicyanine dye synthesized in the above examples:

[0088] Test Example 1 Determination of Ultraviolet-Visible Absorption Spectrum and Hydrogen Peroxide Activation Ability

[0089] The test solutions with a concentration of 5 μM of IHcy-PbE in the examples, IHcy-OH and Hcy-OH in the comparative examples were prepared, and their absorption spectra were tested in different solvents. The test results are shown in Figure 1 . The test solutions with a concentration of 10 μM of IHcy-PbE in the examples and IHcy-OH in the comparative examples were prepared and incubated in phosphate buffered saline (PBS) containing different concentrations of hydrogen peroxide for 30 min, and the fluorescence changes before and after treatment were observed. The test results are shown in Figure 2 .

[0090] Figure 1(a) Absorption curves of IHcy-PbE in methanol, dichloromethane and PBS; Figure 1 (b) Absorption curves of IHcy-OH in methanol, dichloromethane and PBS; Figure 1 (c) Absorption curves of Hcy-OH in methanol, dichloromethane and PBS.

[0091] Figure 2 (a) Fluorescence changes of IHcy-PbE in hydrogen peroxide solutions with different concentrations; Figure 2 (b) Fluorescence changes of IHcy-OH in hydrogen peroxide solutions with different concentrations.

[0092] From Figure 1 it can be seen that in the systems of dichloromethane, methanol and PBS, IHcy-PbE, IHcy-OH and Hcy-OH all exhibit excellent optical absorption at 600 nm - 700 nm. And, from Figure 2 it can be seen that IHcy-PbE has low fluorescence in the solution without hydrogen peroxide, while in the solution with hydrogen peroxide, its fluorescence increases with the increase of hydrogen peroxide concentration, which is beneficial for the diagnosis and treatment of in vivo infections in practical applications. However, IHcy-OH as a comparative example has strong fluorescence in both the solution with and without hydrogen peroxide, and cannot achieve the response to bacterial-infected tissues.

[0093] Test Example 2 Test for singlet oxygen generation ability

[0094] The samples to be tested were respectively added to a quartz cell containing 3 mL of methanol, and DPBF was added to make the absorbance at 415 nm approximately equal to 1. The quartz cell was irradiated with a xenon light source through a near-infrared filter (700 nm, 10 mW / cm 2 ), and the ultraviolet absorption was measured every 60 s. The test results are shown in Figure 3 .

[0095] Figure 3 (a) DPBF absorption curves of IHcy-PbE with irradiation time; Figure 3 (b) DPBF absorption curves of IHcy-OH with irradiation time; Figure 3 (c) DPBF absorption curves of Hcy-OH with irradiation time; Figure 3 (d) Comparative diagram of the absorption changes at 415 nm of DPBF and pure DPBF irradiated with dyes IHcy-PbE, IHcy-OH, and Hcy-OH respectively.

[0096] From Figure 3It can be seen that under the irradiation of 700 nm light, the absorbance of the DPBF solution containing IHcy-PbE at 415 nm decreased slowly, indicating that capping can inhibit the ability to generate singlet oxygen. The absorbance of the DPBF solution containing IHcy-OH at 415 nm decreased rapidly, and the activated structure IHcy-OH had a good ability to generate singlet oxygen, indicating that IHcy-PbE could be used for subsequent photoantibacterial therapy after activation.

[0097] Test Example 3: Test for the binding ability of bacterial FtsZ protein

[0098] Using the OCTET-R8 biolayer interferometry (BLI) technology of Sartorius Company, the binding ability of the recombinant FtsZ protein with six histidine tags (His) immobilized on the Ni-NTA sensor was tested with the test dyes at different concentrations (0.16 μM, 0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, 5 μM). The collected data was automatically fitted by the supporting software of the instrument after double background subtraction to obtain the fitting curve and report the dissociation equilibrium constant K D value.

[0099] Figure 4 (a) is the fitting curve of the binding thickness of the example IHcy-PbE on the Ni-NTA sensor immobilizing the recombinant FtsZ protein and the K D value; Figure 4 (b) is the fitting curve of the binding thickness of IHcy-OH on the Ni-NTA sensor of the recombinant FtsZ protein and the K D value; Figure 4 (c) is the fitting curve of the binding thickness of Hcy-OH on the Ni-NTA sensor of the recombinant FtsZ protein and the K D value.

[0100] Figure 4 The arrow indicates the change of the binding thickness curve on the sensor surface when the concentration of the test dye increases from low to high.

[0101] From Figure 4 it can be seen that the example IHcy-PbE, Comparative Example 1 IHcy-OH, and Comparative Example 2 Hcy-OH all had the binding ability to the recombinant FtsZ protein, and the dissociation equilibrium constant K D values were 5.58 ± 0.15 μM, 5.68 ± 0.34 μM, and 6.12 ± 0.41 μM, respectively.

[0102] Test Example 4: Bacterial inhibition experiment

[0103] To evaluate the inhibitory ability of the dye on bacteria, a 96-well plate was used with 100 μL of bacterial suspension (about 2×106 100 μL of sample solutions of Test Example IHcy-PbE, Comparative Example 1 IHcy-OH, and Comparative Example 2 Hcy-OH with different concentrations were added to 100 μL of Staphylococcus aureus suspensions (about 2×10

[0104] Figure 5 (a) shows the growth of Staphylococcus aureus treated with IHcy-PbE, IHcy-OH, and Hcy-OH; Figure 5 (b) shows the growth of Bacillus subtilis treated with IHcy-PbE, IHcy-OH, and Hcy-OH; Figure 5 (c) shows the growth of Enterococcus faecalis treated with IHcy-PbE, IHcy-OH, and Hcy-OH; Figure 5 (d) shows the growth of methicillin-resistant Staphylococcus aureus treated with IHcy-PbE, IHcy-OH, and Hcy-OH.

[0105] Figure 5 The lowest concentration at which bacterial growth is inhibited, i.e., the minimum inhibitory concentration (MIC), is marked with arrows and circles.

[0106] From Figure 5 it can be seen that compared with Comparative Example 1 IHcy-OH and Comparative Example 2 Hcy-OH, Test Example IHcy-PbE has a lower inhibitory concentration for bacteria. The minimum inhibitory concentrations of IHcy-PbE against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus are 0.63 μM, 0.63 μM, 0.63 μM, and 1.25 μM, respectively.

[0107] Test Example 5 Bacterial Killing Experiment

[0108] To evaluate the phototoxicity and dark toxicity of the dye to bacteria, 100 μL of sample solutions of Test Example IHcy-PbE, Comparative Example 1 IHcy-OH, and Comparative Example 2 Hcy-OH with different concentrations were added to 100 μL of methicillin-resistant Staphylococcus aureus bacterial suspensions (about 2×10 5 CFU / mL). After incubation in a light-proof incubator for 2 hours, the light treatment group was irradiated (680 - 690 nm LED, 30 mW cm -2 , 5 min). The concentration of hydrogen peroxide was 100 μM. Subsequently, 100 μL of each treated sample was taken, serially diluted 10-fold, and 100 μL of the diluted solution was spread on an LB solid plate and incubated overnight.

[0109] Figure 6(a) Bacterial CFU plate images of different concentrations of IHcy-PbE under dark conditions and under the activation of hydrogen peroxide with additional light illumination. Figure 6 (b) Bacterial CFU plate images of different concentrations of IHcy-OH under light and dark conditions. Figure 6 (c) Bacterial CFU plate images of different concentrations of Hcy-OH under light and dark conditions. Figure 6 (d) Bacterial activities after treatment with different concentrations of IHcy-PbE, IHcy-OH, and Hcy-OH under different conditions.

[0110] As Figure 6 shown, under dark conditions, after treatment with IHcy-PbE, the number of bacterial colonies CFU was less than that of the control group, indicating that the dye has the ability to kill bacteria in the dark. After activation with hydrogen peroxide and additional light illumination, the number of bacterial colonies CFU further decreased, indicating that the activated dye has efficient photodynamic antibacterial ability under light conditions. In the comparative examples, IHcy-OH and Hcy-OH also have the ability to kill bacteria under dark and light conditions, but the effect is relatively worse than that of the test example IHcy-PbE.

[0111] Test Example 6 Observation of bacterial damage under scanning electron microscopy and transmission electron microscopy under different treatment conditions

[0112] The bacterial suspensions of 15 mL of overnight-cultured Staphylococcus aureus, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus were evenly divided into three groups and centrifuged. Among them, the first group was resuspended with normal saline, the second group was resuspended with normal saline containing 2 μM Hcy-PbE and incubated at 37 °C for 4 h (L-group), and the third group was resuspended with normal saline containing 2 μM Hcy-PbE and 20 μM hydrogen peroxide and incubated at 37 °C for 4 h followed by light illumination (L+ group). After treatment, centrifugation was performed, and the samples were fixed with glutaraldehyde solution for 4 h, gradient eluted with ethanol solutions of different contents, and then dropped onto silicon wafers and ultra-thin carbon films to prepare scanning electron microscopy and transmission electron microscopy samples respectively, and observed after drying.

[0113] Figure 7 (a) Scanning electron microscopy images of bacteria under different treatment conditions; Figure 7 (b) Transmission electron microscopy images of bacteria under different treatment conditions.

[0114] Figure 7 The scale bar is 1 μm. Figure 7 The positions indicated by the arrows are the positions where the bacteria have morphological changes.

[0115] The change in bacterial morphology, such as the elongation of bacilli or the swelling of cocci, is an important phenotype for the action of FtsZ inhibitors. As Figure 7As shown, the bacterial morphology in the normal saline treatment group was intact and normal. When the photosensitizer was added but without light irradiation, it was obvious that the bacterial division was inhibited and the size and volume of the bacteria increased. After adding the photosensitizer, hydrogen peroxide and light irradiation, in addition to the changes in size and morphology, obvious collapse, wrinkling and rupture appeared on the membrane surface of the bacteria, indicating that the photosensitizer had effectively inhibited the bacterial FtsZ protein and could cause photodynamic damage to the bacteria under the activation of hydrogen peroxide and light irradiation conditions.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A responsive antibacterial hemicyanine photosensitizer having the ability to inhibit bacterial fission protein FtsZ, characterized in that: Its structure is shown in general formula I: In the general formula I, X is selected from one of oxygen, sulfur, selenium, tellurium and dimethyl silicon; Y - One selected from the group consisting of halogen ions; R1 is selected from H, C1-C12 alkyl, phenyl, benzyl, One of; R2 is selected from one of X selected from carbon, oxygen, sulfur, selenium, and carbon dimethyl; R3 and R4 are each independently selected from one of H, phenyl, Cl, Br, and I groups; R5 is selected from any one of the groups described in the following structural formula M-1 or M-2:

2. The method for synthesizing a hemicyanine photosensitizer according to claim 1, characterized in that: The process is as follows: Its synthesis method comprises the following steps: (1) Add N,N-dimethylformamide (DMF) to an organic solvent 1, slowly drop phosphorus tribromide under low temperature and stir for a period of time, then slowly drop cyclohexanone, pour into ice water after sufficient reaction, adjust the pH to 6-8 with inorganic base 1, and extract with organic solvent 1; after the obtained organic phase is decompressed and concentrated, add a raw material containing an R4 substituent and inorganic base 2, add organic solvent 2 as a reaction solvent, and after sufficient reaction, decompress, concentrate, and purify to obtain a compound represented by formula Z-1; (2) Adding the compound represented by formula Z-1 to the organic solvent 3, slowly adding the demethylation agent under low temperature conditions, reacting sufficiently, and obtaining the compound represented by formula Z-2 by decompression, concentration, and purification; (3) Adding the compound represented by formula Z-2, a raw material containing an R5 substituent and an inorganic base 3 to an organic solvent 4, and after sufficient reaction, reducing pressure, concentrating and purifying to obtain a compound represented by formula Z-3; (4) At 50-150°C, a raw material containing R1, R2, and R3 substituents of the compound represented by formula Z-3 is added to an organic solvent 5. After sufficient reaction, the compound represented by formula Z-3 is obtained by decompression, concentration, and purification.

3. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (1), the molar ratio of the raw material containing the R4 substituent to DMF, phosphorus tribromide, cyclohexanone and inorganic base 2 is 1:2-10:2-10:1-2:1-5.

4. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (1), the organic solvent 1 is selected from one or a combination of chloroform, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, ethyl acetate, petroleum ether, toluene, and xylene; in step (1), the inorganic base 1 and the inorganic base 2 are independently selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; in step (1), the organic solvent 2 is selected from one or a combination of DMF, DMSO, ethyl acetate, ethylene glycol dimethyl ether, and acetonitrile.

5. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (2), the molar ratio of the compound represented by Z-1 to the demethylation agent is 1:1-20.

6. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (2), the organic solvent 3 is selected from one or a combination of chloroform, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, petroleum ether, toluene, xylene, acetonitrile; in step (2), the demethylation agent is selected from one or a combination of boron tribromide, aluminum trichloride, and trimethylsilane iodide.

7. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (3), the molar ratio of the compound represented by formula Z-2 to the raw material containing the R5 substituent and the inorganic base 3 is 1:1-3:1-5.

8. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (3), the organic solvent 4 is selected from one or a combination of petroleum ether, acetone, ethyl acetate, dichloromethane, DMF, and DMSO; in step (3), the inorganic base 3 is selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; in step (4), the organic solvent 5 is selected from one or a combination of methanol, ethanol, isopropanol, petroleum ether, acetone, ethyl acetate, acetonitrile, tetrahydrofuran, chloroform, dichloromethane, DMF, and DMSO.

9. The method for synthesizing a type of hemicyanine photosensitizer according to claim 2, characterized in that: In step (4), the molar ratio of the raw materials containing R1, R2, and R3 substituents in the compound represented by formula Z-3 is 1:0.5-5.

10. The use of a hemicyanine photosensitizer as claimed in claim 1, characterized in that: The applications include: bacterial division inhibition, fluorescent imaging technology of bacterial infection, preparation of photodynamic therapy preparations, development of antibacterial materials, and environmental and public health technology fields.

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