A photosensitizer containing a no donor structure, its preparation method and antibacterial application

Abstract

The application discloses a photosensitizer with a NO donor structure with a general formula I structure and a preparation method and antibacterial application thereof. A rhodamine analogue with a photodynamic effect is selected as a mother nucleus, and N-nitrosophenylamine is used as a NO controlled-release segment to construct a green light (532 nm) or near-infrared light (660 nm) light-controlled NO donor and a photodynamic integrated compound. The application not only provides a new idea for solving the problem of precise control of NO, but also realizes integrated release of NO and ROS under the same light excitation through the introduction of the photosensitizer, and the NO can react with the superoxide anion in the ROS component to generate ONOO ‑ , which can realize a more efficient synergistic antibacterial effect. Therefore, the photosensitizer can realize precise and efficient treatment of drug-resistant bacterial infection and can be used for preparing antibacterial drugs, especially drugs for treating drug-resistant bacterial infection diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a photosensitizer containing an NO donor structure, a preparation method thereof and antibacterial application. BACKGROUND

[0002] Bacterial infection is a major problem in the fields of public health, environment and food safety. In clinic, the main treatment drug for inflammation caused by bacteria is broad-spectrum antibiotics, such as levofloxacin and moxifloxacin. However, due to the abuse of antibiotics, the clinical and public health burden caused by antibiotic resistance has become a major problem faced by the world.

[0003] The emergence of drug-resistant bacteria makes anti-infection treatment twice as difficult. Methicillin-resistant Staphylococcus aureus (MRSA) is a common drug-resistant bacteria that can cause skin infections, blood infections and pneumonia, and other life-threatening diseases. Nitric oxide (NO) as an endogenous diatomic free radical can affect various physiological processes in the body, and is also used to kill antibiotic-resistant bacteria, and the bactericidal process does not cause the generation of bacterial resistance.

[0004] At present, although the developed NO prodrugs (such as sodium nitroprusside and furacillin) have shown good effects in biofilm removal, but due to the instability of these NO prodrugs in complex biological environment and the inability to achieve targeted delivery, it limits its further clinical application. And the single NO prodrug developed at present often has unsatisfactory antibacterial effect, and needs to be combined with antibiotics or other antibacterial agents to achieve better efficacy, which still inevitably exists the problem of bacterial resistance.

[0005] Photodynamic therapy (PDT) is an effective treatment method for killing bacteria by light-induced photosensitizer to generate highly active ROS to induce bacterial cell wall damage and DNA breakage, and it also has the ability to kill drug-resistant bacteria. It has the characteristics of non-invasiveness and high spatiotemporal precision treatment. However, the clinical application of PDT is limited due to its dependence on oxygen. Because when inflammation occurs, various pathogenic bacteria will cause a large amount of inflammatory exudate, even tissue fragments, to appear in the inflammatory tissue site, resulting in an obstacle to the utilization of oxygen, causing local hypoxia. Therefore, single photodynamic antibacterial also has certain limitations. The combination of NO donor antibacterial therapy and photodynamic therapy will be a more effective means for treating bacterial infection.

[0006] At present, although the photosensitizers containing NO donor structure developed have both photodynamic antibacterial and NO antibacterial functions, most of these photosensitizers containing NO donor structure are induced to release NO and ROS by short-wavelength ultraviolet light or blue light excitation, and the short-wavelength light has high phototoxicity, which limits its application in deep diseases. In view of this, it is of great significance to design photosensitizers containing NO donor structure with low phototoxicity and high tissue penetration for long-wavelength light-controlled release of NO and ROS for the treatment of drug-resistant bacterial infections.

[0007] The PDT and NO synergistic antibacterial design strategy can not only make up for the shortcoming that the defense system of the bacterial biofilm established by the single photosensitizer can consume part of ROS and is not enough to completely kill the mature biofilm, but also can generate more active and oxidative peroxynitrite (ONOO - ) by reacting with the superoxide anion generated by PDT, thereby enhancing the antibacterial effect. Therefore, by combining the advantages of photodynamic therapy such as good targeting, small side effects and high antibacterial efficacy, and the advantages of NO such as the ability to remove bacterial biofilm, the design of long-wavelength light-controlled NO release and photodynamic therapy in one prodrug provides a useful tool for clinical application in the field of drug-resistant bacteria. SUMMARY

[0008] The purpose of the present application is to provide a photosensitizer containing NO donor structure and its preparation method and antibacterial application, which solves the problems of the prior art. The photosensitizer containing NO donor structure provided by the present application can generate NO and ROS, including superoxide anion and singlet oxygen, under light conditions. The preparation method of the present application is simple and has good light stability.

[0009] Technical scheme: The purpose of the present application is achieved by the following technical scheme:

[0010] The present application provides a photosensitizer containing NO donor structure with the general formula I:

[0011]

[0012] Among them,

[0013] R is selected from any one of the following structures:

[0014]

[0015] In a preferred embodiment of the present application, the substitution position of R on the benzene ring is para or ortho.

[0016] The compound of general formula I of the present application is preferably the following compound:

[0017]

[0018] The application also provides a preparation method of the photosensitizer containing the NO donor structure, which comprises the following steps: subjecting a rhodamine analogue mother nucleus to an aldehyde amine condensation, a sodium borohydride reduction and a nitrosation reaction, respectively, to obtain a symmetrical photosensitizer containing the NO donor structure excited by green light of 532 nm and an asymmetrical photosensitizer containing the NO donor structure excited by near-infrared light of 660 nm.

[0019] Rhodamine is an organic fluorescent dye and has the characteristics of a photosensitizer. The preparation method of the application selects a rhodamine analogue with a photodynamic effect as a mother nucleus and constructs a green light (532 nm) or near-infrared light (660 nm) light-controlled NO donor and a photodynamic integrated compound by using N-nitrosophenol as an NO controlled-release segment. The N-nitrosophenol transfers electrons to the photosensitizer rhodamine analogue mother nucleus, generates a short-lived phenoxy radical, and forms a relatively stable quinone by releasing NO, so as to finally realize the controlled release of NO.

[0020] The application not only provides a new idea for solving the problem of precise control of NO, but also realizes the integrated release of NO and ROS under the same light excitation by introducing the photosensitizer, and the NO can react with the superoxide anion in the ROS component to generate ONOO-, which is more toxic, so as to realize more efficient synergistic antibacterial effect, thus realizing precise and efficient treatment of drug-resistant bacterial infection, and further promoting the biological application of the compound of the application in the field of drug-resistant bacteria.

[0021] The application provides a preparation method of a photosensitizer containing a NO donor structure, which comprises the following steps:

[0022] (1) reacting compound 1 with p-aminophenol at room temperature to obtain compound 2;

[0023]

[0024] (2) reacting compound 2 with sodium borohydride at room temperature to obtain compound 3;

[0025]

[0026] (3) reacting compound 3 with sodium nitrite at room temperature to obtain compound 4;

[0027]

[0028] wherein R is as defined in the general formula I.

[0029] The compounds of the present application of general formula I can be prepared by the above or similar methods. Depending on the different substituents and the positions of the substituents, the corresponding starting materials can be selected. Those skilled in the art should recognize that the above routes are helpful for understanding the present application, but do not limit the present application, and the variables are defined as mentioned in general formula I, unless otherwise specified.

[0030] The present application also provides a pharmaceutical composition comprising the NO-donor-containing photosensitizer having the structure of general formula I and a pharmaceutically acceptable carrier or excipient.

[0031] The compounds of the present application can be combined with pharmaceutically acceptable carriers or excipients to form formulations for administration. For example, solvents, diluents, sprays, etc. The various dosage forms of the pharmaceutical compositions of the present application can be prepared according to methods well known in the pharmaceutical art. These pharmaceutical formulations can contain, in combination with the carrier, for example, 0.05% to 90% by weight of the active ingredient, more commonly, about 15% to 60% by weight of the active ingredient. The dosage of the compounds of the present application can be 0.005 to 5000 mg / kg / day, and doses outside this range can also be used depending on the severity of the disease or the dosage form.

[0032] The present application also provides the use of the NO-donor-containing photosensitizer in the preparation of an antibacterial drug. The drug has antibacterial activity.

[0033] The photosensitizer is a green light 532 nm or near-infrared light 660 nm light-controlled NO donor and a photodynamic integrated compound.

[0034] The green light or near-infrared light excited NO-donor-containing photosensitizer of the present application can effectively release NO after laser irradiation.

[0035] The green light or near-infrared light excited NO-donor-containing photosensitizer of the present application can generate superoxide anion and singlet oxygen after laser irradiation.

[0036] The green light or near-infrared light excited NO-donor-containing photosensitizer of the present application can effectively kill drug-resistant bacteria under laser irradiation conditions.

[0037] The present application also provides the use of the NO-donor-containing photosensitizer in the preparation of a drug for treating drug-resistant bacterial infection diseases.

[0038] Preferably, the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus (MRSA).

[0039] Beneficial effects:

[0040] The present application utilizes the PET principle, selects a rhodamine analogue with a photodynamic effect as a mother nucleus, and N-nitroso aminophenol as an NO controlled-release fragment, and constructs a photosensitizer containing an NO donor structure excited by green light or near-infrared light. On the one hand, under the condition of light excitation, N-nitroso aminophenol transfers electrons to the photosensitizer to produce a short-lived phenoxy radical, and forms a relatively stable quinone by releasing NO, so as to realize the controlled release of NO; on the other hand, the rhodamine analogue can produce ROS molecules with antibacterial activity under the action of light. Through the study of the antibacterial effect on drug-resistant bacteria MRSA, it is found that the compound has obvious antibacterial effect on drug-resistant bacteria MRSA, and in addition, the compound has little damage to normal cells at a certain concentration, so it is expected to be applied to the treatment of drug-resistant bacterial infection diseases such as skin abscess, diabetes, foot ulcer, osteomyelitis, and can further promote the biological application of the compound in the field of drug-resistant bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the compound RhB-NH-1 of Example 1.

[0042] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the photosensitizer RhB-NO-1 containing an NO donor structure excited by green light of Example 1.

[0043] Figure 3 The mass spectrum of the photosensitizer RhB-NO-1 containing an NO donor structure excited by green light of Example 1.

[0044] Figure 4 The nuclear magnetic resonance hydrogen spectrum of the compound RhB-NH-2 of Example 2.

[0045] Figure 5 The nuclear magnetic resonance hydrogen spectrum of the photosensitizer RhB-NO-2 containing an NO donor structure excited by near-infrared light of Example 2.

[0046] Figure 6 The mass spectrum of the photosensitizer RhB-NO-2 containing an NO donor structure excited by near-infrared light of Example 2.

[0047] Figure 7 The NO release capacity spectrum of the compounds RhB-NO-1 and RhB-NO-2 of the present application is detected by DAN fluorescence kit detection method, electron paramagnetic resonance spectroscopy and electrochemical method, respectively.

[0048] Figure 8 The superoxide anion generation capacity spectrum of the compounds RhB-NO-1 and RhB-NO-2 of the present application is detected by DHR123 fluorescence kit detection method and electron paramagnetic resonance method, respectively.

[0049] Figure 9 The singlet oxygen generation ability spectrum of the compounds RhB-NO-1 and RhB-NO-2 of the present application was detected by ABDA kit detection method and electron paramagnetic resonance method, respectively.

[0050] Figure 10 The antibacterial experiment results of the compounds RhB-NO-1 and RhB-NO-2 of the present application under light irradiation. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be described in detail below through specific examples, but the protection scope of the present application is not limited to the examples.

[0052] The specific techniques or conditions not mentioned in the examples were carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer were all conventional products that can be purchased through regular channels.

[0053] The experimental methods in the following examples were all conventional methods unless otherwise specified. The test materials used in the following examples were all commercially available products unless otherwise specified.

[0054] Example 1 Synthesis of photosensitizer RhB-NO-1 containing NO donor structure excited by green light

[0055] (1) Synthesis of compound RhB-CHO-1

[0056]

[0057] Compound 3-hydroxy-N,N-diethyl aniline (1a, 5.13 g, 31.05 mmol) and p-benzaldehyde (2a, 2.08 g, 15.52 mmol) were added to a 100 mL two-necked flask, 25 mL of acetic acid was added, and p-toluenesulfonic acid (502 mg, 3 mmol) was slowly added. After stirring under nitrogen protection in an oil bath at 75°C, the reaction was refluxed overnight. After the reaction was completed, the solution was purple red, and the reaction liquid was pressurized to remove acetic acid at 50°C. A deep purple oil was obtained, saturated sodium bicarbonate was added, and a large amount of bubbles was observed. Extraction was carried out with dichloromethane and water, the organic phase was collected, and after drying on a rotary evaporator, a deep purple solid compound RhB-1 was obtained.

[0058] Dark purple solid compound RhB-1 (2 g, 4.66 mmol) and chloranil (573.7 mg, 2.33 mmol) were added to a 100 mL round bottom flask, and 25 mL of dichloromethane was added, and the reaction was stirred at room temperature for 3 hours. After the reaction was completed, the color of the reaction solution was further deepened, and after drying on a rotary evaporator, the crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 30:1, v / v) to obtain 500 mg of compound RhB-CHO-1 with a yield of 40%.

[0059] ESI-MS (m / z): 427.21 [M+H] + .

[0060] 1H NMR (400 MHz, DMSO-d6) d (ppm): 10.20 (s, 1H), 8.20 (d, J = 7.6 Hz, 2H), 7.85-7.68 (m, 2H), 7.50-7.29 (m, 1H), 7.26-7.11 (m, 3H), 6.99 (s, 2H), 3.80-3.56 (m, 6H), 3.38 (s, 2H), 1.12 (d, J = 84.6 Hz, 12H).

[0061] (2) Synthesis of compound RhB-NH-1

[0062]

[0063] Compound RhB-CHO-1 (427 mg, 1 mmol) and p-aminophenol (545 mg, 5 mmol) were weighed into a 50 mL round bottom flask, and 20 mL of dichloromethane and 2 mL of acetic acid were added, and the reaction was stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (635.82 mg, 3 mmol) was added, and stirring was continued for 10 min. After the reaction was completed, saturated sodium bicarbonate was added, and a large amount of gas bubbles were observed, and extraction was performed with dichloromethane and water, and the organic phase was collected, dried with anhydrous sodium sulfate, and dried on a rotary evaporator. The crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 30:1, v / v) to obtain 500 mg of compound RhB-NH-1 with a yield of 40%.

[0064] The nuclear magnetic resonance hydrogen spectrum of compound RhB-NH-1 is shown in Figure 1 .

[0065] ESI-MS (m / z): 520.30 [M+H] + .

[0066] 1H NMR (400 MHz, DMSO-d6) d (ppm): 8.51 (s, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.46 (d, J = 7.9 Hz, 2H), 7.26 (d, J = 9.6 Hz, 2H), 7.16 (d, J = 9.9 Hz, 2H), 6.96 (s, 2H), 6.67-6.47 (m, 4H), 5.79 (s, 1H), 4.35 (s, 2H), 3.65 (q, J = 7.2 Hz, 8H), 1.22 (q, J = 9.1, 7.1 Hz, 12H).

[0067] (3) Synthesis of NO-donor photosensitizer RhB-NO-1

[0068]

[0069] Compound RhB-NH-1 (52 mg, 0.1 mmol) was weighed into a 50 mL round bottom flask, 6 mL acetic acid was added to dissolve it completely, then 6 mL aqueous sodium nitrite solution (6.9 mg, 0.1 mmol) was added and stirred at room temperature for 10 min. Then diluted with ethyl acetate and extracted, and washed with 10% citric acid. The organic layer was evaporated in vacuum. Then saturated sodium bicarbonate and dichloromethane were added to extract. The organic layer was washed with brine, dried over sodium sulfate and spin dried on a rotary evaporator. The crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 30:1, v / v) to obtain 32 mg of photosensitizer RhB-NO-1 (N-(6-(diethylamino)-9-(((4-hydroxyphenyl)(nitroso)amino)methyl)phenyl)-3H-xanthen-3-ylidene)-N- ethylethanaminium), with a yield of 59%.

[0070] The proton nuclear magnetic resonance spectrum of photosensitizer RhB-NO-1 is shown in Figure 2 , and the mass spectrum is shown in Figure 3 .

[0071] ESI-MS (m / z): 549.29 [M+H] + .

[0072] 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.87 (s, 1H), 7.52-7.47 (m, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.38-7.33 (m, 2H), 7.19 (d, J = 9.5 Hz, 2H), 7.13 (dd, J = 9.6, 2.4 Hz, 2H), 6.96 (d, J = 2.4 Hz, 2H), 6.94-6.90 (m, 2H), 5.43 (s, 2H), 3.63 (t, J = 7.1 Hz, 8H), 1.21 (d, J = 6.9 Hz, 12H).

[0073] Synthesis of NIR light-excited photosensitizer with NO donor structure RhB-NO-2

[0074] (1) Synthesis of compound RN-1

[0075]

[0076] Compound RN (559 mg, 1 mmol) was weighed into a 50 mL round-bottom flask, and 20 mL of dichloromethane was added to dissolve it completely, followed by the addition of N-methylmorpholine (110 μL, 1 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (230 mg, 1.2 mmol) and N-methyl-N-methoxyamine hydrochloride (107 mg, 1.1 mmol). The mixture was stirred at room temperature for 4.5 hours, dried by a rotary evaporator, and then the crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 30:1, v / v) to obtain 350 mg of compound RN-1, with a yield of 58%.

[0077] ESI-MS (m / z): 602.34 [M+H] + .

[0078] 1H NMR (300 MHz, DMSO-d6) d (ppm): 8.55 (d, J = 14.4 Hz, 1H), 7.76-7.57 (m, 4H), 7.55-7.27 (m, 4H), 6.89-6.62 (m, 3H), 6.32 (d, J = 14.5 Hz, 1H), 4.03 (q, J = 7.3 Hz, 1H), 3.75 (s, 3H), 3.53 (dd, J = 13.5, 6.2 Hz, 8H), 3.03 (s, 3H), 2.69 (t, J = 19.3 Hz, 2H), 2.33 (s, 1H), 1.99 (s, 1H), 1.75 (d, J = 4.3 Hz, 6H), 1.18 (t, J = 7.0 Hz, 6H).

[0079] (2) Synthesis of compound RhB-CHO-2

[0080]

[0081] Compound RN-1 (301 mg, 0.5 mmol) was weighed into a 50 mL round-bottom flask, and 20 mL of anhydrous THF was added to dissolve it completely, and then hydrochloride dichlorobis (zirconocene) (155 mg, 0.6 mmol) was added. The mixture was stirred at room temperature for 5 min, and then dried by a rotary evaporator, extracted with water and dichloromethane, and the organic layer was dried with anhydrous sodium sulfate, and dried by a rotary evaporator, and the crude product was purified by silica gel column chromatography (eluent: PE: DCM = 1:3, v / v) to obtain 350 mg of compound RhB-CHO-2, with a yield of 64.4%.

[0082] ESI-MS (m / z): 543.30 [M+H]+.

[0083] 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.90 (s, 1H), 8.58 (d, J = 14.5 Hz, 1H), 8.13 (dd, J = 7.9, 1.4 Hz, 1H), 7.91 (td, J = 7.5, 1.4 Hz, 1H), 7.80 (td, J = 7.6, 1.2 Hz, 1H), 7.72-7.68 (m, 1H), 7.55-7.39 (m, 3H), 7.35 (td, J = 7.4, 1.2 Hz, 1H), 6.80-6.74 (m, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.57 (d, J = 9.1 Hz, 1H), 6.35 (d, J = 14.5 Hz, 1H), 3.76 (s, 3H), 3.52 (q, J = 7.0 Hz, 5H), 2.65 (t, J = 6.4 Hz, 2H), 2.22 (ddt, J = 47.3, 15.7, 6.2 Hz, 2H), 1.77 (d, J = 1.4 Hz, 6H), 1.17 (t, J = 7.0 Hz, 6H).

[0084] (3) Synthesis of compound RhB-NH-2

[0085]

[0086] Referring to the synthesis method of compound RhB-NH-1 in Example 1, compound RhB-CHO-2 was replaced with compound RhB-CHO-2 in the method to obtain 330 mg of compound RhB-NH-2, with a yield of 51.8%.

[0087] The proton nuclear magnetic resonance spectrum of compound RhB-NH-2 is shown in Figure 4 .

[0088] ESI-MS (m / z): 636.36 [M+H]+.

[0089] 1H NMR (400 MHz, DMSO-d6) d (ppm): 8.54 (d, J = 14.2 Hz, 1H), 8.37 (s, 1H), 7.68 (dd, J = 7.6, 4.8 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.53 - 7.40 (m, 4H), 7.32 (td, J = 7.1, 1.7 Hz, 1H), 7.17 (dd, J = 7.5, 1.5 Hz, 1H), 6.82 (dd, J = 9.2, 2.5 Hz, 1H), 6.71 - 6.62 (m, 2H), 6.45 - 6.38 (m, 2H), 6.28 (d, J = 14.2 Hz, 1H), 6.23 - 6.17 (m, 2H), 5.50 (s, 1H), 3.93 (q, J = 14.9, 14.0 Hz, 2H), 3.74 (d, J = 6.4 Hz, 3H), 3.53 (q, J = 7.0 Hz, 5H), 2.64 (dd, J = 14.0, 7.1 Hz, 3H), 2.38 - 2.21 (m, 2H), 1.75 (d, J = 3.9 Hz, 6H), 1.19 (t, J = 7.0 Hz, 6H).

[0090] (4) Synthesis of photosensitizer RhB-NO-2 containing NO donor

[0091]

[0092] Referring to the synthesis method of photosensitizer RhB-NO-1 containing NO donor in Example 1, compound RhB-NH-2 was used instead of compound RhB-NO-2 in the method, and finally 40 mg of photosensitizer RhB-NO-2 ((Z)-6-(diethylamino)-9-(2-((4-hydroxyphenyl)(nitro)amino) methyl) phenyl)-4-(2-((E)-1,3,3-trimethylindolin-2-ylidene) ethylidene)-1,2,3,4-tetrahydroxanthene) containing NO donor was obtained, with a yield of 63%.

[0093] The proton nuclear magnetic resonance spectrum of photosensitizer RhB-NO-2 is shown in Figure 5 , and the mass spectrum of photosensitizer RhB-NO-2 is shown in Figure 6 .

[0094] ESI-MS (m / z): 665.40 [M+H]+.

[0095] 1H NMR (400 MHz, DMSO-d6) d (ppm): 8.55 (d, J = 14.0 Hz, 1H), 8.11 (d, J = 9.9 Hz, 1H), 7.66 (dd, J = 26.3, 7.4 Hz, 1H), 7.53 - 7.44 (m, 4H), 7.34 (t, J = 7.4 Hz, 1H), 7.30 - 7.22 (m, 2H), 7.18 (dt, J = 9.0, 2.6 Hz, 3H), 6.79 - 6.64 (m, 3H), 6.53 (d, J = 9.1 Hz, 1H), 6.34 (d, J = 14.7 Hz, 1H), 4.99 (d, J = 8.3 Hz, 2H), 3.79 - 3.46 (m, 8H), 2.66 (qd, J = 16.0, 14.0, 7.2 Hz, 2H), 2.28 - 2.14 (m, 2H), 1.78 - 1.74 (m, 4H), 1.72 (d, J = 4.4 Hz, 2H), 1.25 (t, J = 6.9 Hz, 2H), 1.20 (t, J = 7.0 Hz, 4H).

[0096] NO release test of the compounds RhB-NO-1 and RhB-NO-2 of the present application

[0097] The NO release test of the compounds RhB-NO-1 and RhB-NO-2 of the present application was carried out in a 10% DMSO solution in PBS, and the compounds were prepared into a test solution with a final concentration of 20 mM. The NO release capacity of RhB-NO-1 and RhB-NO-2 under light irradiation was verified by NO detection kit, electron paramagnetic resonance spectroscopy (EPR) and electrochemical method.

[0098] Among them, the NO detection mechanism of DAN fluorescence kit (2,3-diaminonaphthalene, CAS No.: 771-97-1, purchased from Shanghai Maieryi Biochemical Technology) is that under acidic conditions, the commercial probe reacts with NO 2- to produce fluorescent naphthol triazole, and the change of the fluorescence intensity of naphthol triazole at 410 nm can be observed to judge the NO release of RhB-NO-1 and RhB-NO-2.

[0099] The NO detection mechanism of EPR method is to capture the generated NO free radical in situ by using spin trapping agent 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) to form 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI), and the signal of PTI is detected by electron paramagnetic resonance spectrometer to judge the NO release capacity.

[0100] The mechanism of NO detection by electrochemical method is that the released NO can pass through the NO sensor, and finally react to form an electrical signal, which is measured by an amplifier recorder to measure the NO release level.

[0101] Figure 7 The NO release ability spectrum of the compounds RhB-NO-1 and RhB-NO-2 of the present application is detected by DAN fluorescence kit detection method, electron paramagnetic resonance spectroscopy and electrochemical method respectively.

[0102] The results of DAN fluorescence kit detection method show that after irradiation, the characteristic fluorescence emission peak of the compound RhB-NO-1 (A) and RhB-NO-2 (D) and the buffer solution of DAN at 405 nm increases with the increase of irradiation time. Figure 7 Figure 7 The results of EPR method show that under laser irradiation, the characteristic EPR signal of PTI is obviously observed for RhB-NO-1 (B) and RhB-NO-2 (E).

[0103] The results of electrochemical method also show that under laser irradiation, RhB-NO-1 (C) and RhB-NO-2 (F) can successfully release NO, and there is no premature leakage of NO. Figure 7 Figure 7 Since the photosensitizer provided by the present application with the structure of general formula I also contains the N-nitrosophenol NO controlled release fragment, it also has the ability of light-controlled release of NO.

[0104] The photodynamic property test of the compounds RhB-NO-1 and RhB-NO-2 of the present application Figure 7 Figure 7 The photodynamic property test of the compounds RhB-NO-1 and RhB-NO-2 of the present application is carried out in 10% DMSO pure water solution, and the compounds of the present application are prepared into detection solution with a final concentration of 10 μM.

[0105] The photodynamic property test of the compounds RhB-NO-1 and RhB-NO-2 of the present application is carried out in 10% DMSO pure water solution, and the compounds of the present application are prepared into detection solution with a final concentration of 10 μM.

[0106] The photodynamic property test of the compounds RhB-NO-1 and RhB-NO-2 of the present application is carried out in 10% DMSO pure water solution, and the compounds of the present application are prepared into detection solution with a final concentration of 10 μM.

[0107] The photodynamic property of RhB-NO-1 and RhB-NO-2 is verified by using DHR123 fluorescence kit (superoxide anion detection probe, CAS number: 109244-58-8, Shanghai Qiyuan Technology), ABDA kit (singlet oxygen indicator, 9,10-anthracenediyl-bis(methylene)dipropionic acid, CAS number: 307554-62-7, Shanghai Mokang Biological Technology) and electron paramagnetic resonance spectroscopy.

[0108] The photodynamic property of RhB-NO-1 and RhB-NO-2 is verified by using DHR123 fluorescence kit (superoxide anion detection probe, CAS number: 109244-58-8, Shanghai Qiyuan Technology), ABDA kit (singlet oxygen indicator, 9,10-anthracenediyl-bis(methylene)dipropionic acid, CAS number: 307554-62-7, Shanghai Mokang Biological Technology) and electron paramagnetic resonance spectroscopy.

[0109] ​​​DHR123 emits strong fluorescence after being oxidized by superoxide anion to the highly fluorescent product Rhodamine 123, with a fluorescence emission peak at 535 nm.

[0110] The ABDA kit reacts with singlet oxygen to generate an endogenous oxidation product, which leads to a decrease in the characteristic UV absorption peak of ABDA, specifically a decrease in the absorption peak at 378 nm in the absorption spectrum.

[0111] The EPR method for detecting superoxide anions uses 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO) as a trapping agent. Superoxide anions react with DMPO to form a complex, and the superoxide anion generating capacity of RhB-NO-1 and RhB-NO-2 is determined by detecting the signal of the complex using electron paramagnetic resonance spectroscopy.

[0112] The EPR method for detecting singlet oxygen uses 2,2,6,6,6-tetramethyl-4-piperidinol (TMP) as a trapping agent. Singlet oxygen can react with TMP to generate TMPN radicals. The singlet oxygen generation capacity of RhB-NO-1 and RhB-NO-2 is determined by detecting the TMPN signal using electron paramagnetic resonance spectroscopy.

[0113] Figure 8 The superoxide anion generating capacity spectra of compounds RhB-NO-1 and RhB-NO-2 of the present invention were determined by the DHR123 fluorescence reagent kit and the electron paramagnetic resonance method, respectively.

[0114] Figure 9 The singlet oxygen generation capacity spectra of compounds RhB-NO-1 and RhB-NO-2 of the present invention were determined by the ABDA kit detection method and the electron paramagnetic resonance method, respectively.

[0115] The results showed that after irradiating the solution of RhB-NO-1 or a mixture of RhB-NO-2 and DHR123 with a laser, the fluorescence intensity at 536 nm increased significantly, indicating that the compound RhB-NO-1 of this invention ( Figure 8 A) and RhB-NO-2( Figure 8 C) Both compounds can effectively generate superoxide anions under light irradiation. After irradiating a solution of RhB-NO-1 or a mixture of RhB-NO-2 and ABDA with a laser, the ultraviolet absorption around 410 nm was significantly reduced, indicating that the compound RhB-NO-1 (…) of this invention… Figure 9 A) and RhB-NO-2( Figure 9 C) It can effectively produce singlet oxygen under light conditions.

[0116] EPR results showed that under laser irradiation conditions, both RhB-NO-1 and RhB-NO-2 exhibited trapping of superoxide anions.Figure 8 B, D) and singlet oxygen Figure 9 B, D) and singlet oxygen

[0117] Since the photosensitizer with NO donor structure provided by the present application has the rhodamine analogue mother nucleus with general structure I, it also has the photodynamic property.

[0118] Example 5 Anti-bacterial activity test of the compounds RhB-NO-1 and RhB-NO-2 of the present application

[0119] MRSA (from the Medical Examination Center of Zhongda Hospital Affiliated to Southeast University) was selected as the research object. The scanning electron microscope method was used to determine the damage degree of the bacterial membrane under the light control condition of RhB-NO-1 or RhB-NO-2. Four groups (PBS+light, RhB-NO-1 / RhB-NO-2+light, RhB-NO-1 / RhB-NO-2+darkness, levofloxacin control group) were set in parallel. The bacterial suspension with OD600 value of 0.5-0.8 was taken, RhB-NO-1 or RhB-NO-2 (10 μM) was added, and after treatment under different experimental conditions: PBS+light, RhB-NO-1 / RhB-NO-2+light, RhB-NO-1 / RhB-NO-2+darkness, levofloxacin, centrifugation, discarding the supernatant, collecting the bacterial precipitate, washing 3 times (0.9% physiological saline), fixing (2% glutaraldehyde), dehydrating, scanning electron microscope shooting, and characterizing the morphology of the bacterial membrane.

[0120] The anti-bacterial experiment results of the compounds RhB-NO-1 and RhB-NO-2 under light condition are shown in Figure 10 .

[0121] The results show that under the condition of laser light, the compounds RhB-NO-1 Figure 10 A) and RhB-NO-2 Figure 10 B) provided by the present application both have the effect of destroying the bacterial cell membrane.

[0122] Since the photosensitizer with NO donor structure provided by the present application has the photodynamic and ROS generating properties, it also has the effect of destroying the bacterial cell membrane, and has the anti-bacterial activity.

[0123] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is not to be construed as a limitation on the present application itself. Various changes in form and details can be made therein without departing from the spirit and scope of the present application defined in the appended claims.

Claims

1. A photosensitizer containing a NO donor structure, characterized in that... Selected from:

2. A method for preparing a photosensitizer containing a NO donor structure as described in claim 1, characterized in that, The rhodamine analog nucleus was subjected to aldehyde-amine condensation, sodium borohydride acetate reduction, and nitrosation to obtain a symmetrical photosensitizer with a NO donor structure excited by green light at 532 nm and an asymmetric photosensitizer with a NO donor structure excited by near-infrared light at 660 nm. The preparation method includes the following steps: (1) Compound 1 was reacted with p-aminophenol at room temperature to obtain compound 2; (2) Compound 2 was reacted with sodium borohydride acetate at room temperature to obtain compound 3; (3) Compound 3 was reacted with sodium nitrite at room temperature to obtain compound 4; Wherein, R is selected from:

3. A pharmaceutical composition, characterized in that, This includes photosensitizers containing NO donor structures as described in claim 1, as well as pharmaceutically acceptable excipients.

4. The use of the photosensitizer containing the NO donor structure as described in claim 1 in the preparation of antibacterial drugs.

5. The application according to claim 4, characterized in that, The photosensitizer is a photosensitive NO donor and photodynamic compound that integrates green light 532nm or near-infrared light 660nm.

6. The use of the photosensitizer containing the NO donor structure as described in claim 1 in the preparation of a medicament for treating drug-resistant bacterial infections.

Images