Nile blue dyes substituted with a ring and an amino group in the side chain, synthesis method and application thereof

Abstract

The application discloses a novel ring-substituted and side-chain-amino-substituted Nile blue dye, a synthesis method and application thereof, and relates to a series of dyes which can be simultaneously used for bacterial fluorescence imaging and photodynamic antibacterial treatment and are designed and synthesized based on Nile blue dye as a matrix, the structure of the dyes being shown as a general formula I. The dyes have high molar extinction coefficient, fluorescence imaging and active oxygen generation efficiency. MNBS-C2-NH2 and MNBS-C6-NH2 synthesized in the examples exhibit excellent absorption and fluorescence performance in PBS and dichloromethane, especially in the range of 640-660 nm. Test results show that the dyes have a significant killing effect on E. coli under light conditions, and the log CFU is obviously reduced, while the dark toxicity is low. Scanning electron microscope observation further proves that the bacteria after light treatment have obvious membrane surface collapse and rupture phenomenon. The dyes can penetrate through the outer membrane protein of gram-negative bacteria, thereby realizing enhanced penetration and killing ability for gram-negative bacteria, and have wide application prospects in the field of antibacterial treatment.

Description

Technical Field

[0001] This invention relates to the field of fine chemical organic dyes technology, and in particular to a class of Nile blue dyes, their preparation methods and applications. Background Technology

[0002] Bacterial infections have long been a significant threat to human health. Due to the increasing prevalence of antimicrobial resistance (AMR), the pace of new drug development cannot keep up with the rate of emergence of resistant bacteria. Furthermore, existing antibiotics commonly used to treat AMR infections, such as polymyxin and vancomycin, have significant toxic side effects, including kidney damage, hearing impairment, neurotoxicity, liver damage, and anaphylactic shock. Therefore, AMR infections have become a global public health challenge, making the exploration of new treatments for AMR infections particularly important.

[0003] Photodynamic antimicrobial therapy (PDAT), as a novel non-antibiotic antibacterial therapy, has shown great potential in addressing the growing problem of multidrug-resistant bacteria due to its ability to overcome antibiotic resistance in multidrug-resistant bacteria while exhibiting minimal resistance itself. It has received increasing attention and research in recent years. Currently, PDAT is already used clinically in the treatment of skin and oral infections, effectively replacing antibiotics and reducing their use in local infection treatment. Furthermore, it has been reported that various drug-resistant bacteria can be inactivated through PDAT. Although there has been some progress in the antibacterial therapy of photosensitized dyes, their poor water solubility, poor bacterial penetration, and low reactive oxygen species production severely limit their practical application, especially their unsatisfactory bactericidal effect against Gram-negative bacteria. Enhancing the penetration ability against Gram-negative bacteria is a key focus and challenge in antibacterial dye design. Therefore, there is an urgent need to develop a class of photosensitized dyes with excellent bacterial penetration and photosensitization capabilities to enhance the penetration and killing ability against Gram-negative bacteria and multidrug-resistant bacteria, which is of great significance for solving the diagnosis and treatment of bacterial infections. Summary of the Invention

[0004] To address the problems of poor water solubility, poor penetration into Gram-negative bacteria, and low reactive oxygen species yield of existing photosensitive dyes, this invention provides a class of cyclic-substituted and side-chain amino-substituted Nile Blue dyes. The synthesis method and application utilize a positively charged hydrophilic photosensitizer with an amino group to adsorb and bind to bacteria via electrostatic interaction. It also leverages the transport capacity of porins in the outer membrane of Gram-negative bacteria to penetrate their internal structure, thereby enhancing their imaging and killing capabilities. This dye can be used for fluorescence imaging and not only kills Gram-positive bacteria but also exhibits excellent photodynamic antibacterial effects against most Gram-negative bacteria for which photosensitive dyes are less effective. This has significant research value for the diagnosis and treatment of bacterial infections.

[0005] To achieve the above objectives, the technical solution of the present invention is: a method for synthesizing Nile Blue dyes with cyclic substitution and side-chain amino substitution, the structure of which is shown in general formula I:

[0006]

[0007] In general formula I,

[0008] X is selected from oxygen, sulfur, selenium, and tellurium. More preferably, in the embodiments of the present invention, X is selected as sulfur.

[0009] Y - Selected from halide ions, ClO4 - BF4 - PF6 - One of them, and more preferably, in the embodiment of the present invention, Y - Chloride ions were selected.

[0010] R1 is selected from One of the groups, more preferably, in the embodiments of the present invention, R1 is selected as

[0011] n is a positive integer selected from 1 to 18, which is the length of the alkyl chain of the side chain, more preferably 2 to 6, and more preferably, in the embodiments of the present invention, n is 2 or 6;

[0012] R2 and R3 are each independently selected from H, methyl, ethyl, propyl, ... One of the groups, more preferably, in the embodiments of the present invention, R2 and R3 are both selected as H.

[0013] The synthesis method of cyclic-substituted and side-chain amino-substituted Nile blue dyes is shown below:

[0014]

[0015] Its synthesis method includes the following steps:

[0016] (1) In an aqueous solution, aniline containing R1 substituent was added to an aqueous solution of aluminum sulfate. NaS2O3 and ZnCl2 were added sequentially under low temperature stirring. Oxidizing agent was added dropwise. After the reaction was complete, the precipitate was obtained by filtration and then purified by recrystallization to obtain the compound shown in formula Z-1.

[0017] Alternatively, phenol containing the R1 substituent can be added to 5-50 wt% acid; sodium nitrite can be slowly added under low temperature and stirring, allowed to react completely, allowed to stand, filtered to obtain a precipitate, and washed to obtain the compound shown in formula Z-2;

[0018] (2) At 80-150℃, 1-bromonaphthalene, an inorganic base, a catalyst and an amino compound containing R2 and R3 substituents are added to an organic solvent and reacted for 12-48h. The compound shown in formula Z-3 is obtained by depressurization, concentration and purification.

[0019] (3) At 0-80℃, the compound shown in formula Z-1 or Z-2 and the compound shown in formula Z-3 are oxidized with an oxidant or catalyzed by a base in an organic solvent to form a ring, and then subjected to a process containing Y. - The compound shown in Formula I was obtained by acid treatment, concentration, and purification.

[0020] More preferably, in step (1), the molar ratio of aniline containing the R1 substituent, aluminum sulfate, NaS2O3, ZnCl2 and oxidant is 1:1-2:1-3:1-2:0.1-2, and the preferred ratio is 1:1-1.5:2-3:1-1.5:0.3-1.

[0021] More preferably, in step (1) above, the molar ratio of phenol containing the R1 substituent to sodium nitrite is 1:1-3, and the preferred ratio is 1:1-1.5; the acid is more preferably 10-20 wt%.

[0022] More preferably, in step (1) above, the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, and potassium chlorate; and the acid is selected from at least one of hydrochloric acid, sulfuric acid, hydrobromic acid, and acetic acid.

[0023] More preferably, in step (2), the molar ratio of the 1-bromonaphthalene, catalyst, inorganic base and amino compound containing R2 and R3 substituents is 1:0.05-0.6:1-3:1-5, and the preferred ratio is 1:0.05-0.2:1-1.5:2-3.

[0024] More preferably, in step (2) above, the organic solvent is selected from one or a mixture of several of the following solvents: ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, propylene glycol methyl ether, butanediol, ethanol, acetonitrile, and ethyl acetate; the inorganic base is selected from at least one of the following: sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; and the catalyst is selected from at least one of the following: cuprous iodide, cuprous bromide, cuprous chloride, cuprous oxide, copper sulfate, copper sulfate pentahydrate, copper oxide, and copper chloride.

[0025] More preferably, in step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-3 is 1:0.5-3, and the preferred ratio is 1:1-1.5.

[0026] More preferably, in step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the oxidant or base is 1:0.5-5, and the preferred ratio is 1:1-2.

[0027] More preferably, in step (3) above, the organic solvent is selected from one or a mixture of several of methanol, ethanol, isopropanol, petroleum ether, ethyl acetate, glycerol, dichloromethane, and DMF.

[0028] More preferably, in step (3), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, potassium chlorate, and hydrogen peroxide.

[0029] More preferably, in step (3), the alkali is selected from at least one of silver carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium ethoxide, sodium hydroxide, sodium perchlorate, potassium perchlorate, sodium hexafluorophosphate, sodium fluoroborate, and triethylamine;

[0030] More preferably, in step (3), the Y-containing - The acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, perchloric acid, fluoroboric acid, and hexafluorophosphate.

[0031] Another aspect of the present invention is to protect the application of the aforementioned class of cyclic-substituted and side-chain amino-substituted Nile blue dyes, the applications of which include spectroscopic testing techniques, bacterial imaging techniques, and photodynamic therapy techniques used in non-disease diagnosis and treatment, with particular emphasis on the practical application of high-resolution fluorescence imaging techniques and antibacterial therapy techniques for bacteria.

[0032] In summary, the present invention has the following beneficial effects:

[0033] 1. This application describes a novel class of cyclic-substituted and side-chain amino-substituted Nile blue dyes designed using Nile blue dye as a backbone. While retaining the fluorescence properties of traditional Nile blue dyes, it improves the water solubility and photosensitization ability of the dyes. The MNBS-C2-NH2 and MNBS-C6-NH2 synthesized in Examples 1 and 2 of this invention exhibit excellent absorption and fluorescence properties in PBS and dichloromethane, particularly in the 640-660 nm range. Furthermore, the solubility of these dyes in water is significantly improved, facilitating practical applications.

[0034] 2. Experimental tests show that the Nile Blue dyes with ring substitution and side-chain amino substitution described in this application can effectively penetrate Gram-negative bacteria, represented by *Escherichia coli*, exhibiting good fluorescence imaging capabilities against Gram-negative bacteria and strong bactericidal activity under light conditions, while retaining the penetration and bactericidal capabilities of traditional photosensitizers against Gram-positive bacteria. Test Example 3 of this invention shows that the fluorescence signal intensity of MNBS-C2-NH2 and MNBS-C6-NH2 against *E. coli* gradually increases within 0-120 minutes, indicating their good bacterial penetration ability. Test Example 5 further confirms that under light conditions, MNBS-C2-NH2 and MNBS-C6-NH2 have a significant bactericidal effect on *E. coli*, with a significant reduction in log CFU, while exhibiting low dark toxicity.

[0035] 3. The cyclic-substituted and side-chain amino-substituted Nile blue dyes described in this application exhibit low dark toxicity, good fluorescence properties, and enhanced antibacterial activity, showing promising application prospects in bacterial imaging and the treatment of bacterial infections. In Test Example 6 of this invention, scanning electron microscopy revealed that bacteria treated with light (L+ group) showed obvious membrane surface collapse, wrinkling, and rupture, while bacteria in the light-shielded group (L- group) and the control group maintained intact morphology, further validating the highly efficient antibacterial activity of these dyes in photodynamic therapy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The absorption and fluorescence spectra of MNBS-C2-NH2 and MNBS-C6-NH2 in different reagents are presented in the embodiments of the present invention.

[0038] Figure 2The UV absorption decay curves of MNBS-C2-NH2 and MNBS-C6-NH2 in dichloromethane as reactive oxygen species scavenger DPBF are shown in the embodiments of the present invention.

[0039] Figure 3 This invention includes an uptake experiment of MNBS-C2-NH2, MNBS-C6-NH2 and Comparative Example 1MNBS-C0-NH2 in different bacteria.

[0040] Figure 4 This invention analyzes the penetration ability of MNBS-C2-NH2 and MNBS-C6-NH2 against the outer membrane proteins of Gram-negative bacteria Escherichia coli.

[0041] Figure 5 The bacterial colony counts of MNBS-C2-NH2, MNBS-C6-NH2 and Comparative Example 2 (ampicillin) against different bacteria in this invention embodiment.

[0042] Figure 6 The morphological damage of MNBS-C2-NH2 to different bacteria was observed under a scanning electron microscope in this embodiment of the invention. Detailed Implementation

[0043] The present invention will now be described in further detail.

[0044] Unless otherwise stated, the terms used herein have the following meanings.

[0045] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0046] The term "alkyl" as used in this invention includes straight-chain alkyl and branched-chain alkyl.

[0047] The following examples illustrate the cyclic-substituted and side-chain amino-substituted Nile Blue dyes of General Formula I in detail.

[0048] Example 1

[0049] Nile blue dye MNBS-C2-NH2 has the following structural formula:

[0050]

[0051] Its synthesis method includes the following steps:

[0052]

[0053] (1) Synthesis of Compound 1

[0054]

[0055] Aluminum sulfate octadecate (3.66 g, 5.5 mmol) was dissolved in 10 mL of water to prepare an aqueous solution. 4-morpholinoaniline (0.89 g, 5 mmol) was weighed and added to the aluminum sulfate solution, followed by sodium thiosulfate pentahydrate (2.85 g, 11.5 mmol) and zinc chloride (0.72 g, 5.25 mmol). The mixture was cooled in an ice bath. Potassium dichromate (0.44 g, 1.5 mmol) was weighed and prepared into a 4 mL aqueous solution, which was slowly added dropwise to the reaction system. The mixture was stirred in an ice bath for approximately 2 hours, the solid precipitate was filtered off and dissolved in 12 mL of methanol, refluxed for 10 minutes, cooled, filtered, and dried to obtain a grayish-white solid. No further purification was required; this solid was used directly in the next step. The yield was 0.60 g, with a yield of 41%.

[0056] (2) Synthesis of compound 2

[0057]

[0058] Weigh 1-bromonaphthalene (2.07 g, 10.0 mmol), ethylenediamine (1.20 g, 20.0 mmol), cuprous iodide (0.095 g, 0.5 mmol), and cesium carbonate (3.26 g, 10 mmol) and add them to 20 mL of ethylene glycol monomethyl ether. React at 125 °C for 24 h under nitrogen protection. After the reaction is complete, remove the solvent by vacuum distillation, and purify by column chromatography (V... 二氯甲烷 V 甲醇 A pale yellow oily liquid was obtained by mixing (5:1), yielding 1.12 g, with a yield of 61%.

[0059] (3) Synthesis of the target compound MNBS-C2-NH2

[0060]

[0061] Compound 1 (0.145 g, 0.50 mmol) and compound 2 (0.122 g, 0.65 mmol) were weighed and added to 3 mL of dimethyl sulfoxide. Potassium dichromate (0.162 g, 0.55 mmol) was slowly added, and the mixture was stirred at room temperature for 10 min. Then, 50 mL of methanol and 6 mL of dilute hydrochloric acid solution (1.0 mol / L) were added, and the reaction was continued at room temperature for another 40 min. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the mixture was purified by column chromatography (V...). 二氯甲烷 V 甲醇 (100:14) yielded a dark blue solid with a yield of 0.059 g and a yield of 30%.

[0062] 1H NMR(500MHz,D2O)δ7.72(d,J=6.1Hz,1H),7.30(d,J=8.2Hz,3H),6.84(d,J=8.5Hz,1H),6.61(d,J=8.2Hz ,1H),6.45(s,1H),6.11(s,1H),3.71(s,4H),3.56(t,J=7.0Hz,2H),3.20(t,J=7.0Hz,2H),3.15(s,4H). 13 C NMR(126MHz,D2O)δ152.64,152.19,140.71,136.46,134.38,132.54,132.49,131.76,131. 50,130.08,125.21,123.70,122.06,118.11,105.82,101.87,66.04,46.68,40.75,37.71.

[0063] Example 2

[0064] MNBS-C6-NH2 dye has the following structural formula:

[0065]

[0066] Its synthesis method includes the following steps:

[0067]

[0068] (1) Synthesis of Compound 1

[0069] The synthesis of intermediate compound 1 in Example 2 is the same as that of intermediate compound 1 in Example 1.

[0070] (2) Synthesis of compound 3

[0071]

[0072] Weigh 1-bromonaphthalene (2.07 g, 10.0 mmol), 1,6-hexanediamine (2.33 g, 20.0 mmol), cuprous iodide (0.095 g, 0.5 mmol), and cesium carbonate (3.26 g, 10 mmol) and add them to 20 mL of ethylene glycol monomethyl ether. React at 125 °C for 24 h under nitrogen protection. After the reaction is complete, remove the solvent by vacuum distillation, and purify by column chromatography (V... 二氯甲烷 V 甲醇 A pale yellow oily liquid was obtained by mixing (5:1), yielding 1.45g, with a yield of 60%.

[0073] (3) Synthesis of the target compound MNBS-C6-NH2

[0074]

[0075] Compound 1 (0.145 g, 0.50 mmol) and compound 3 (0.157 g, 0.65 mmol) were weighed and added to 3 mL of dimethyl sulfoxide. Potassium dichromate (0.162 g, 0.55 mmol) was slowly added, and the mixture was stirred at room temperature for 10 min. Then, 50 mL of methanol and 6 mL of dilute hydrochloric acid solution (1.0 mol / L) were added, and the reaction was continued at room temperature for another 40 min. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the mixture was purified by column chromatography (V...). 二氯甲烷 V 甲醇 The ratio of 100:12 yielded a dark blue solid with a yield of 0.078 g and a yield rate of 34%.

[0076] 1 H NMR(500MHz,D2O)δ8.05(d,J=8.0Hz,1H),7.51(t,J=7.3Hz,1H),7.44(dt,J=14.6,7.6Hz,2H),7.08(d,J=9.1Hz,1H),6.73(d,J=9.4Hz,1H) ,6.53(s,1H),6.35(s,1H),3.77(t,J=5.1Hz,4H),3.23(d,J=5.1Hz,6H),3.00–2.94(m,2H),1.65(q,J=13.0,9.9Hz,4H),1.44–1.37(m,4H). 13 C NMR(126MHz,D2O)δ152.68,151.44,140.11,135.47,134.22,131.68,131.60,131.38,129.98,129.72,12 4.96,123.31,121.76,116.57,105.52,102.63,65.93,46.14,44.12,39.45,28.11,26.79,25.93,25.55.

[0077] Comparative Example 1

[0078] The Nile blue dye MNBS-C0-NH2, which has only left-side ring substitution, was synthesized using a similar method to that used in Examples MNBS-C2-NH2 and MNBS-C6-NH2. The structure and synthetic route are as follows:

[0079]

[0080] 1H-NMR (500MHz, Methanol-d4) δ9.09(d,J=8.1Hz,1H),8.30(d,J=8.1Hz,1H),8.04(d,J=9.2Hz,1H),7.97(t,J=7.5Hz, 1H),7.87(t,J=7.5Hz,1H),7.48(d,J=9.1Hz,1H),7.37(s,1H),7.23(s,1H),3.89–3.84(m,4H),3.66(d,J=4.4Hz,4H). 13 C-NMR(126MHz,Methanol-d4)δ159.32,154.34,142.49,138.15,137.27,135.97,134.43, 133.53,131.87,131.52,126.96,125.41,124.79,118.69,107.95,107.65,67.53,48.34.

[0081] Comparative Example 2

[0082] (2S,5R,6R)-3,3-dimethyl-6-[(R)-2-amino-2-phenylacetamido]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate sodium salt (Ampicillin Sodium) is a commercially available antibiotic used in clinical practice. Its structure is shown below. This product is available from Bid Pharmaceuticals Ltd.

[0083]

[0084] Test case

[0085] To demonstrate that the Nile blue dyes prepared in Examples 1 and 2, with ring substitution and side-chain amino substitution, can effectively penetrate Gram-negative bacteria, represented by Escherichia coli, exhibit good fluorescence imaging capabilities against Gram-negative bacteria, and demonstrate strong killing ability against Gram-negative bacteria under light conditions, while retaining the penetration and killing ability of traditional photosensitizers against Gram-positive bacteria, and thus possessing important research significance for the treatment of drug-resistant bacterial infections, the Nile blue dyes synthesized in the above examples were subjected to the following tests:

[0086] Test Example 1: UV-Vis Absorption Spectroscopy and Fluorescence Quantum Yield Determination

[0087] MNBS-C2-NH2 and MNBS-C6-NH2 were prepared into 10 μM test solutions. The absorption and fluorescence spectra of MNBS-C2-NH2 and MNBS-C6-NH2 were measured in different solvents, and their maximum absorption and emission wavelengths were recorded. The test results are shown in [reference needed]. Figure 1 .

[0088] Figure 1 (a) shows the absorption curves of MNBS-C2-NH2 and MNBS-C6-NH2 in dichloromethane; Figure 1 (a) shows the absorption curves of MNBS-C2-NH2 and MNBS-C6-NH2 in phosphate buffered saline (PBS).

[0089] Table 1. Absolute fluorescence quantum yield of MNBS-C2-NH2 and MNBS-C6-NH2.

[0090] <![CDATA[MNBS-C2-NH2]]> dichloromethane 0.216 <![CDATA[MNBS-C2-NH2]]> PBS 0.020 <![CDATA[MNBS-C6-NH2]]> dichloromethane 0.377 <![CDATA[MNBS-C6-NH2]]> PBS 0.026

[0091] from Figure 1 It can be seen that in the PBS and dichloromethane system, MNBS-C2-NH2 and MNBS-C6-NH2 both exhibit excellent optical absorption in the 640nm-660nm range, and show significant fluorescence in dichloromethane and low fluorescence quantum yield in water. In practical applications, they are beneficial for in vivo imaging, effectively reducing background fluorescence interference and improving the signal-to-noise ratio and resolution of the imaging.

[0092] Test Example 2: Singlet Oxygen Generation Capacity Test

[0093] Compounds MNBS-C2-NH2 and MNBS-C6-NH2 were added to quartz dishes containing 3 mL of dichloromethane, respectively, and a certain amount of DPBF was added to make the absorbance at 415 nm approximately equal to 1. Near-infrared light (660 nm, 2 mW / cm²) was then used to measure the absorbance. 2 Irradiate the test quartz dish, and measure the ultraviolet absorption every minute. See the test results below. Figure 2 .

[0094] Figure 2 (a) shows the absorption curve of MNBS-C2-NH2 with light exposure time; Figure 2 (b) shows the absorption curve of MNBS-C6-NH2 with light exposure time.

[0095] Depend on Figure 2 It can be seen that under 660nm light irradiation, the absorbance of the DPBF solution containing the photosensitive dye decreases rapidly at 415nm, indicating that the photosensitizer has good ROS generation ability and can be used for subsequent antibacterial treatment.

[0096] Test Example 3: Bacterial Intake Test

[0097] Test samples MNBS-C2-NH2, MNBS-C6-NH2, and comparative sample MNBS-C0-NH2 were added to overnight cultured Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), respectively. The samples were then added to confocal cell culture dishes with glass bottoms and imaged using a single-photon laser scanning confocal microscope. Bright-field and fluorescence images of the bacteria were acquired at different time points to observe the bacterial uptake of the photosensitive dye. The test results are shown in [reference needed]. Figure 3 .

[0098] Figure 3 (a) Changes in fluorescence signal over time as bacteria take up MNBS-C2-NH2; Figure 3 (b) Changes in fluorescence signal of bacteria uptake of MNBS-C6-NH2 over time; Figure 3 (c) Changes in fluorescence signal of bacteria taking up MNBS-C0-NH2 over time. Figure 3 The scale bar is 10 μm.

[0099] from Figure 3 It can be seen that within 0-120 min, the fluorescence signal intensity of MNBS-C2-NH2 and MNBS-C6-NH2 gradually increased for Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, indicating good penetration ability for bacteria. In contrast, the comparative example 1 MNBS-C0-NH2 showed poor penetration ability for bacteria, especially for Gram-negative Escherichia coli, where the fluorescence intensity was low. This characteristic makes MNBS-C2-NH2 and MNBS-C6-NH2 have broad application potential in bacterial imaging and antibacterial therapy, and can be used for real-time monitoring of bacterial growth and distribution to improve diagnostic accuracy.

[0100] Test Example 4: Analysis of the penetration ability of MNBS-C2-NH2 and MNBS-C6-NH2 against the outer membrane proteins of Gram-negative bacteria *Escherichia coli*.

[0101] Using spermine as an inhibitor of Gram-negative bacterial membrane proteins, two groups of bacterial suspensions of Gram-negative Escherichia coli were prepared. One group was pre-incubated with 20 μM spermine for 30 min, and then dye was added and incubated for 2 hours. The effect of the presence or absence of an inhibitor on the dye uptake of Escherichia coli was observed.

[0102] like Figure 4 As shown, the Gram-negative bacteria in the group without inhibitors showed obvious red fluorescence signals, while the fluorescence signals decreased significantly after the addition of inhibitors, indicating that the dyes MNBS-C2-NH2 and MNBS-C6-NH2 mainly enter the Gram-negative bacteria through membrane protein channels. Figure 4The scale bar is 10 μm. This mechanism allows the dye to efficiently penetrate bacterial cells and accumulate within them, enabling precise monitoring of bacterial distribution and activity, thereby enhancing the effectiveness of fluorescence imaging and antibacterial therapy.

[0103] Test Example 5: Bacterial Phototoxicity and Dark Toxicity

[0104] To assess phototoxicity and dark toxicity to bacteria, a bacterial suspension of 100 μL (10 8 ~10 9 Add the test sample solutions MNBS-C2-NH2, MNBS-C6-NH2, Comparative Example 1 MNBS-C0-NH2, and Comparative Example 2 ampicillin sodium to the CFU / mL solution, respectively. After incubation in a light-protected incubator for 2 hours, the light-treated group was irradiated (640-660nm LED, 30mW cm⁻¹). -2 (10 minutes). Then, 100 μL of each treated sample was taken and serially diluted 10-fold. The diluted solutions were plated on LB agar plates and incubated overnight. Bacterial colony counts were recorded, and the log CFU for each sample was calculated. For the lowest dilution plate with a colony count less than or equal to 1, the log CFU was recorded as 0. All experiments were repeated three times.

[0105] Figure 5 (a) Changes in log CFU of bacteria under light and dark conditions for MNBS-C2-NH2; Figure 5 (b) Changes in log CFU of MNBS-C6-NH2 on bacteria under light and dark conditions; Figure 5 (c) Changes in log CFU of bacteria under light and dark conditions caused by MNBS-C0-NH2; Figure 5 (d) Changes in log CFU of MNBS-C6-NH2 on bacteria under light and dark conditions; Figure 5 (e) Bacterial plate images of MNBS-C2-NH2, MNBS-C6-NH2, Comparative Example 1 MNBS-C0-NH2 and Comparative Example 2 ampicillin sodium under light and dark conditions.

[0106] like Figure 5As shown, under light-protected conditions, the log CFU of bacteria treated with MNBS-C2-NH2 and MNBS-C6-NH2 was basically similar to that of the control group, indicating that these two dyes have low toxicity under dark conditions. However, after light exposure, the log CFU decreased significantly, indicating that these two dyes have highly efficient bactericidal ability under light conditions. In contrast, the log CFU of bacteria in the comparative ampicillin group decreased less, indicating that the photosensitizers MNBS-C2-NH2 and MNBS-C6-NH2 have excellent bactericidal ability under light conditions, demonstrating their great potential in photodynamic therapy and their ability to effectively kill bacteria, thereby improving treatment efficacy.

[0107] Test Example 6: Observation of bacterial damage under different treatment conditions using scanning electron microscopy.

[0108] The 15 mL overnight bacterial suspension was divided into three equal groups and centrifuged. The first group was resuspended in physiological saline, and the second group was resuspended in physiological saline containing 10 μM dye MNBS-C2-NH2 and incubated at 37°C for 30 min before being centrifuged at 660 nm (30 mW cm⁻¹). -2 The first group was irradiated with light for 10 min (L+ group), while the second group was resuspended in physiological saline containing 10 μM dye MNBS-C2-NH2 and incubated at 37℃ for 30 min, followed by 10 min in the dark (L- group). After treatment, the samples were centrifuged, fixed with glutaraldehyde solution for 4 h, eluted with gradients of ethanol solutions of different concentrations, and then dropped onto an ultrathin carbon membrane. After drying, the membrane was observed under a scanning electron microscope.

[0109] Figure 6 The scale bar is 1 μm.

[0110] like Figure 6 As shown, the bacteria in the saline group and the light-protected group had intact morphology, while the overall morphology of the negative bacteria after light exposure was significantly damaged, and the membrane surface showed obvious collapse, wrinkling and rupture, indicating that the photosensitizer had effectively damaged the bacteria.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of Nile blue dyes, characterized in that, Its structure is shown in general formula I: I In general formula I, X is selected from oxygen and sulfur; Y - Selected from halide ions, ClO4 － BF4 － PF6 － One of them; R1 is ; n is a positive integer selected from 2 to 6; R2 and R3 are each independently selected from H, methyl, ethyl, and propyl.

2. The method for synthesizing a type of Nile blue dye as described in claim 1, characterized in that, The process is as follows: Its synthesis method includes the following steps: (1) In an aqueous solution, aniline containing R1 substituent is added to an aqueous solution of aluminum sulfate. NaS2O3 and ZnCl2 are added sequentially under low temperature stirring. Oxidizing agent is added dropwise. After the reaction is complete, the precipitate is obtained by filtration and then purified by recrystallization to obtain the compound shown in formula Z-1. Alternatively, phenol containing the R1 substituent can be added to 5-50 wt% acid, and sodium nitrite can be slowly added under stirring at low temperature. After the reaction is complete and the mixture is allowed to stand, the precipitate is obtained by filtration and washing to obtain the compound shown in formula Z-2. (2) At 80-150℃, 1-bromonaphthalene, an inorganic base, a catalyst, and an amino compound containing R2 and R3 substituents are added to an organic solvent and reacted fully. The compound shown in formula Z-3 is obtained by depressurization, concentration, and purification. (3) At 0-80℃, the compound shown in formula Z-1 or Z-2 and the compound shown in formula Z-3 are oxidized with an oxidant or catalyzed by a base in an organic solvent to form a ring, and then subjected to a process containing Y. - The compound shown in Formula I is obtained by treating with acid or salt, concentrating, and purifying.

3. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (1), the molar ratio of aniline containing R1 substituent, aluminum sulfate, NaS2O3, ZnCl2 and oxidant is 1:1-2:1-3:1-2:0.1-2; in step (1), the molar ratio of phenol containing R1 substituent and sodium nitrite is 1:1-3.

4. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (1), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, and potassium chlorate; in step (1), the acid is selected from at least one of hydrochloric acid, sulfuric acid, hydrobromic acid, and acetic acid.

5. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (2), the molar ratio of the 1-bromonaphthalene, catalyst, inorganic base and amino compound containing R2 and R3 substituents is 1:0.05-0.6:1-3:1-5.

6. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (2), the organic solvent is selected from one or a mixture of several of the following: ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, propylene glycol methyl ether, butanediol, ethanol, acetonitrile, and ethyl acetate; in step (2), the inorganic base is selected from at least one of the following: sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; in step (2), the catalyst is selected from at least one of the following: cuprous iodide, cuprous bromide, cuprous chloride, cuprous oxide, copper sulfate, copper sulfate pentahydrate, copper oxide, and copper chloride.

7. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-3 is 1:0.5-3; In step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the oxidant or base is 1:0.5-5.

8. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that, In step (3), the organic solvent is selected from one or a mixture of several of the following solvents: methanol, ethanol, isopropanol, petroleum ether, ethyl acetate, glycerol, dichloromethane, and DMF. In step (3), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, potassium chlorate, and hydrogen peroxide; In step (3), the alkali is selected from at least one of silver carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium ethoxide, sodium hydroxide, sodium perchlorate, potassium perchlorate, sodium hexafluorophosphate, sodium fluoroborate, and triethylamine; In step (3), the Y-containing - The acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, perchloric acid, fluoroboric acid, and hexafluorophosphate.

9. The application of the Nile Blue dye according to claim 1, characterized in that: The applications include spectral testing techniques, bacterial imaging techniques, and photodynamic therapy techniques used in the diagnosis and treatment of non-disease conditions.

10. The application according to claim 9, characterized in that: The applications include fluorescent imaging technology for bacteria and antibacterial treatment technology.

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