Nitric oxide-activated near-infrared fluorescent-photoacoustic compounds, their preparation methods and applications

Abstract

This application discloses a nitric oxide-activated near-infrared fluorescent-photoacoustic compound, its preparation method, and its applications. The fluorescent-photoacoustic compound has the structure shown below, where R includes groups containing oxanthracene or indole structures, etc. Compared to existing technologies, the fluorescent-photoacoustic compound provided in this application has a longer excitation / emission wavelength, exhibiting not only a highly specific response to nitric oxide but also deeper tissue penetration, effectively avoiding interference from biofluorescence during bioimaging. It can be used for near-infrared fluorescence imaging and photoacoustic imaging, enabling real-time monitoring of changes in nitric oxide concentration at the in vivo level.

Description

Technical Field

[0001] This application relates to a nitric oxide (NO) probe, specifically to a nitric oxide-activated near-infrared fluorescent-photoacoustic compound, its preparation method, and its application as a nitric oxide probe, belonging to the field of fluorescence detection technology. Background Technology

[0002] Tumor-associated macrophages (TAMs) comprise 30%–50% of the tumor stroma and are a crucial component of the tumor microenvironment. They assist tumor growth and metastasis, promote immunosuppression, and hinder the efficacy of immune checkpoint blockade. TAMs are complex and dynamic populations composed of various cell types. Within the tumor microenvironment, activated macrophages can be classified into M1 and M2 types based on their characteristics and properties. M1-TAMs release large amounts of pro-inflammatory cytokines, promoting inflammation, and produce reactive oxygen species (ROS) such as H2O2, NO, and ONOO- to promote cell apoptosis or necrosis; therefore, M1-TAMs are also known as pro-inflammatory macrophages. M2-TAMs, on the other hand, are the majority of TAMs and have a negative impact on tumor treatment.

[0003] Nitric oxide functions as a biosignaling agent in the body, promoting muscle relaxation, vasodilation, lowering blood pressure, and synaptic signal transduction. In the tumor microenvironment, the large amounts of nitric oxide produced by M1 macrophages act as reactive oxygen species, inhibiting tumor growth. Detecting nitric oxide levels in tumor microenvironments (TMMs) can reflect patient prognosis and the efficacy of immunosuppressive drugs. Since the number of M1 macrophages is positively correlated with the nitric oxide content at the tumor site, detecting nitric oxide levels can effectively reflect M1 macrophage information.

[0004] Near-infrared fluorescence imaging and photoacoustic imaging technologies offer numerous advantages, including high tissue penetration, high resolution, high sensitivity, and real-time imaging. Utilizing near-infrared fluorescent-photoacoustic compounds for non-invasive in vivo imaging of nitric oxide offers advantages such as simple operation and high spatiotemporal resolution, making it significant for deep tumor imaging and macrophage immunotherapy drug evaluation. Commercially available nitric oxide probes such as DAF-FM (515nm) and DAN (406nm) exist, but their short wavelengths and low tissue penetration depth limit their use to in vitro and cellular-level NO content assessment. Summary of the Invention

[0005] The main objective of this application is to provide a nitric oxide-activated near-infrared fluorescent-photoacoustic compound, its preparation method, and its application, in order to overcome the shortcomings of existing nitric oxide probes, such as short wavelength and low tissue penetration depth.

[0006] To achieve the aforementioned objectives, the technical solution adopted in this application includes:

[0007] The first aspect of this application provides a class of nitric oxide-activated near-infrared fluorescent-photoacoustic compounds having the structure shown below:

[0008]

[0009] Wherein, R has at least one of the following structures:

[0010]

[0011] Where m = 0 to 2, h = 0 to 1.

[0012] The second aspect of this application provides a class of nitric oxide detection probes comprising the aforementioned nitric oxide-activated near-infrared fluorescent-photoacoustic compound.

[0013] A third aspect of this application provides a method for detecting nitric oxide, comprising: applying the nitric oxide-activated near-infrared fluorescent-photoacoustic compound to a test object containing nitric oxide or a nitric oxide donor, and detecting the absorption spectrum and / or emission spectrum and / or photoacoustic signal of the test object, thereby realizing the detection of nitric oxide in the test object.

[0014] A fourth aspect of this application provides a method for preparing the nitric oxide-activated near-infrared fluorescent-photoacoustic compound, comprising:

[0015] At least the half containing the R group reacts with malondialdehyde diphenylimine salt and rhodamine half containing carboxyl functional groups to form an intermediate with an asymmetric structure.

[0016] The intermediate is reacted with o-phenylenediamine at least to obtain the nitric oxide-activated near-infrared fluorescent-photoacoustic compound.

[0017] Compared with the prior art, the nitric oxide-activated near-infrared fluorescent-photoacoustic compound provided in this application has a longer fluorescence emission wavelength. When used as a nitric oxide detection probe, it not only has a high specific response to nitric oxide, but can also penetrate tissues more deeply and effectively avoid interference from biofluorescence during bioimaging. It can be used for near-infrared fluorescence imaging and photoacoustic tomography to achieve real-time monitoring of changes in in vivo nitric oxide concentration. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a diagram illustrating the NO response mechanism of a nitric oxide detection probe in an embodiment of this application.

[0020] Figure 2 The product RhNO-3 in Example 1 of this application 1 H NMR spectrum;

[0021] Figure 3 This is the absorption spectrum of product RhNO-3 in Example 1 of this application;

[0022] Figure 4 The emission spectrum of product RhNO-3 in Example 1 of this application;

[0023] Figure 5 The absorption spectra of product RhNO-4 in Example 4 of this application before and after NO response;

[0024] Figure 6 The emission spectra of product RhNO-4 in Example 4 of this application before and after NO response;

[0025] Figure 7 This is a linear correlation graph of product RhNO-4 in Example 4 of this application after reacting with NO;

[0026] Figure 8 This is a graph showing the NO selectivity test of product RhNO-4 in Example 4 of this application;

[0027] Figure 9 This is a high-resolution mass spectrum of product RhNO-10 in Example 10 of this application;

[0028] Figure 10 This is the absorption spectrum of product RhNO-10 in Example 10 of this application;

[0029] Figure 11 The emission spectrum of product RhNO-10 in Example 10 of this application;

[0030] Figure 12 The image shows the 1H NMR spectrum of product RhNO-14 in Example 14 of this application.

[0031] Figure 13This is the absorption spectrum of product RhNO-14 in Example 14 of this application;

[0032] Figure 14 The emission spectrum of product RhNO-14 in Example 14 of this application;

[0033] Figure 15 These are photoacoustic tomographic images of product RhNO-4 in Example 4 of this application before and after NO response;

[0034] Figure 16 This is a photoacoustic tomography image of the product RhNO-4 applied to the mouse brain in Example 4 of this application;

[0035] Figure 17 This is a photoacoustic tomography image of the product RhNO-4 in Example 4 of this application applied to a mouse body. Detailed Implementation

[0036] As mentioned above, nitric oxide has many important functions in the body, especially for cancer patients, where timely detection of changes in nitric oxide levels is essential. However, existing nitric oxide detection probes are limited by their short wavelengths, making it impossible to meet the need for timely and accurate assessment of nitric oxide levels in the body.

[0037] In view of this, the applicant, through long-term research and extensive practice, has proposed the technical solution of this application, which firstly provides a class of nitric oxide-activated near-infrared fluorescent-photoacoustic compounds, having the structure shown in the following formula:

[0038]

[0039] Wherein, R has at least one of the following structures:

[0040]

[0041] Where m = 0 to 2, h = 0 to 1.

[0042] Some embodiments of this application also provide the use of the nitric oxide-activated near-infrared fluorescent-photoacoustic compound in the preparation of nitric oxide detection probes.

[0043] In one embodiment, the nitric oxide detection probe has near-infrared fluorescence imaging and / or photoacoustic imaging capabilities, and the use includes: using the nitric oxide detection probe to detect changes in nitric oxide content in cells or biological tissues via near-infrared fluorescence imaging and / or photoacoustic imaging (e.g., photoacoustic tomography).

[0044] In one embodiment, the biological tissue includes living biological tissue, such as tumor tissue.

[0045] The nitric oxide detection probe described in this application has a highly specific response to nitric oxide, enabling real-time monitoring of changes in in vivo nitric oxide concentration. Furthermore, its fluorescence emission wavelength can reach 1040 nm, exhibiting high tissue penetration capability and effectively avoiding background fluorescence interference during bioimaging. It can be used for near-infrared fluorescence imaging and photoacoustic imaging.

[0046] Some embodiments of this application also provide the use of the nitric oxide-activated near-infrared fluorescent-photoacoustic compounds in the preparation of macrophage immunotherapies. Exemplarily, the fluorescent-photoacoustic compounds of this application can be used to evaluate macrophage immunotherapies.

[0047] Some embodiments of this application also provide the use of the nitric oxide-activated near-infrared fluorescent-photoacoustic compound in the preparation of nitric oxide detection probes, near-infrared fluorescent imaging reagents, photoacoustic imaging reagents, or kits.

[0048] For example, this application provides a near-infrared fluorescence imaging and photoacoustic tomography method, which includes administering the fluorescent-photoacoustic compound to a subject and then performing near-infrared fluorescence imaging and photoacoustic tomography.

[0049] Some embodiments of this application also provide a method for detecting nitric oxide, comprising: applying the aforementioned nitric oxide-activated near-infrared fluorescent-photoacoustic compound to a test object containing nitric oxide or a nitric oxide donor, and detecting the absorption spectrum and / or emission spectrum and / or photoacoustic signal of the test object to achieve the detection of nitric oxide in the test object. The detection limit of this method is as low as 25 nM, and the detectable NO concentration range is between 0.002 and 1.5 equivalents of the probe.

[0050] The objects to be detected include, but are not limited to, wastewater containing NO or its donors, animal body fluids, biological cells, isolated biological tissues, living biological tissues, and living organisms.

[0051] Some embodiments of this application also provide a method for preparing the nitric oxide-activated near-infrared fluorescent-photoacoustic compound, comprising:

[0052] At least the half containing the R group reacts with malondialdehyde diphenylimine salt and rhodamine half containing carboxyl functional groups to form an intermediate with an asymmetric structure.

[0053] The intermediate is reacted with o-phenylenediamine at least to obtain the fluorescent-photoacoustic compound.

[0054] This application describes a near-infrared dye obtained by condensing rhodamine dye with a corresponding half-body. By changing the half-body, probes that meet different needs can be obtained. Furthermore, by adding an o-phenylenediamine triggering group to the spirocyclic structure of rhodamine, the fluorescent-photoacoustic compound that can serve as a highly sensitive nitric oxide-responsive probe can be obtained. The synthesis process is simple.

[0055] In one embodiment, the preparation method specifically includes the following steps:

[0056] S1. Dissolve the R-group-containing half-body and malondialdehyde diphenylimine salt in a first solvent, heat to 90-120°C under a protective atmosphere and react for 3-5 hours, then cool to room temperature, add the Rhodamine half-body solution dropwise, then add excess acetate, continue stirring at room temperature for 3-5 hours, and then separate the intermediate from the reaction mixture.

[0057] S2. Dissolve the intermediate in a second solvent, and add phosphorus oxychloride dropwise under ice bath conditions. After thorough mixing, heat to reflux under a protective atmosphere and react for 3-5 hours. Then remove the second solvent from the reaction mixture and add a third solvent. Next, add anhydrous acetonitrile solution containing o-phenylenediamine and a basic catalyst dropwise and react at room temperature for more than 12 hours to obtain the fluorescent-photoacoustic compound.

[0058] In one embodiment, the rhodamine half containing a carboxyl functional group has the structure shown in the following formula:

[0059]

[0060] Where R' is ethyl, M - Including Cl - ,Br - I - ClO4 - CH3SO3 - Or CH3COO, etc., and not limited to these.

[0061] In one embodiment, the molar ratio of the half containing the R group, malondialdehyde bisphenylimine monohydrochloride, and the rhodamine half in step S1 is 1:1.1-1.5:1.1-1.5.

[0062] In one embodiment, the molar ratio of the intermediate, phosphorus oxychloride, o-phenylenediamine and alkaline catalyst in step S2 is 1:1.5-3:1.5-5:1-2:2-4, and is preferably 1:1.5-3:1.5-5:1.5:3.

[0063] In one embodiment, the malondialdehyde diphenylimine salt includes at least one of malondialdehyde diphenylimine monohydrochloride, malondialdehyde diphenylimine acetate, and malondialdehyde diphenylimine sulfate, but is not limited thereto.

[0064] In one embodiment, the first solvent includes one or more of acetic anhydride, methanol, toluene, and n-butanol, but is not limited thereto.

[0065] In one embodiment, the second solvent comprises, but is not limited to, 1,2-dichloroethane.

[0066] In one embodiment, the solvent of the rhodamine half-solution includes, but is not limited to, anhydrous pyridine.

[0067] In one embodiment, the acetate includes sodium acetate or potassium acetate, but is not limited thereto.

[0068] In one embodiment, the alkaline catalyst is selected from organic bases, such as triethylamine, but is not limited thereto.

[0069] In one embodiment, the third solvent is selected from aprotic polar solvents, including, but not limited to, anhydrous acetonitrile or DMF.

[0070] In a typical embodiment, the preparation method includes: firstly, synthesizing a rhodamine half containing a carboxyl functional group, condensing it with the corresponding xanthene half or indole half (i.e., half containing an R group) to obtain an intermediate with an asymmetric structure, and then introducing an o-phenylenediamine triggering group to make it specifically responsive to nitric oxide to obtain the fluorescent-photoacoustic compound.

[0071] For example, the synthetic route of the fluorescent-photoacoustic compound is as follows:

[0072] (I)

[0073] (II)

[0074] Where R represents a half containing an R group, the structure of which is described above.

[0075] For example, a method for preparing the fluorescent-photoacoustic compound specifically includes the following steps:

[0076] S1. Dissolve the R-group-containing half-body and malondialdehyde bisphenylimine monohydrochloride in acetic anhydride, and heat to 90-120℃ under nitrogen protection for 3-5 hours. After the reaction cools to room temperature, dissolve the rhodamine half-body in anhydrous pyridine and add it dropwise to the above acetic anhydride mixture. Add excess sodium acetate and continue stirring at room temperature for 3-5 hours. Remove pyridine by rotary evaporation, followed by extraction. Purify the intermediate (which can be defined as asymmetric near-infrared probe A) by column chromatography after rotary evaporation.

[0077] S2. Dissolve the asymmetric near-infrared probe A in 1,2-dichloroethane. Add phosphorus oxychloride dropwise under ice bath conditions, stirring for 10 min. Then, heat to reflux under nitrogen protection and react for 3-5 h. Remove the 1,2-dichloroethane by rotary evaporation and add anhydrous acetonitrile for later use. Dissolve o-phenylenediamine and triethylamine in anhydrous acetonitrile and add them dropwise to the aforementioned prepared solution, stirring overnight at room temperature. After the reaction is complete, remove the acetonitrile by rotary evaporation. Obtain the final product, the fluorescent-photoacoustic compound, using silica gel column chromatography or HPLC.

[0078] The fluorescent-photoacoustic compound described in this application, by extending the conjugated chain based on rhodamine dye, extends the absorption and emission wavelengths to the near-infrared region. When used as a nitric oxide-activated probe, it effectively reduces the influence of in vivo autofluorescence, allowing for deeper tissue penetration and broadening the application scenarios of fluorescence imaging, such as cell imaging and biofluorescence labeling. Moreover, it can non-invasively and visually assess nitric oxide content. Compared with invasive tissue biopsy methods, it reduces patient suffering while enabling doctors to treat the condition more promptly and accurately.

[0079] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0080] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0081] Example 1

[0082]

[0083] Rhodamine hemipolymer R3 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-3. Intermediate Rh-3 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO₃. HRMS(M⁺) = 809.07. Figure 2 The product RhNO-3 is shown. 1 H NMR spectrum. Figure 3 , Figure 4 The absorption and emission spectra of the product RhNO-3 are shown respectively.

[0084] Example 2

[0085]

[0086] Rhodamine hemipolymer R2 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-2. Intermediate Rh-2 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO₂. HRMS(M⁺) = 877.04.

[0087] Example 3

[0088]

[0089] Half-body R1 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, half-body Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-1. Intermediate Rh-1 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO₃⁻. HRMS(M⁺) = 775.01.

[0090] Example 4

[0091]

[0092] Rhodamine hemipolymer R4 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-4. Intermediate Rh-4 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and evaporated to dryness to give a black powdery final product, RhNO₄⁻. HRMS(M⁺) = 764.97.

[0093] Figure 5 , Figure 6 The absorption and emission spectra of RhNO-4 before and after the NO response are shown respectively.

[0094] Example 5

[0095]

[0096] Rh-5 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL) and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-5. Intermediate Rh-5 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO₅. HRMS(M⁺) = 789.97.

[0097] Example 6

[0098]

[0099] Rhodamine hemipolymer R6 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-6. Intermediate Rh-6 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-6. HRMS(M+) = 879.11.

[0100] Example 7

[0101]

[0102] Rh-7 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL) and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-7. Intermediate Rh-7 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-7. HRMS(M+) = 893.14.

[0103] Example 8

[0104]

[0105] Rhodamine hemipolymer R8 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-8. Intermediate Rh-8 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and evaporated to dryness to give a black powdery final product, RhNO₃⁻⁸. HRMS(M⁺) = 907.17.

[0106] Example 9

[0107]

[0108] Rh-9 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL) and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-9. Intermediate Rh-9 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-9. HRMS(M+) = 759.01.

[0109] Example 10

[0110]

[0111] Rhodamine hemipolymer R10 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-10. Intermediate Rh-10 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-10. HRMS(M+) = 773.03.

[0112] Figure 9 The high-resolution mass spectrum of RhNO-10 is shown. Figure 10 , Figure 11 The absorption and emission spectra of RhNO-10 are shown respectively.

[0113] Example 11

[0114]

[0115] Rh-11 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL) and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-11. Intermediate Rh-11 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-11. HRMS(M+) = 787.06.

[0116] Example 12

[0117]

[0118] Rhodamine hemipolymer R12 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate product Rh-12. Intermediate Rh-12 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and evaporated to dryness to give a black powdery final product, RhNO-12. HRMS(M+) = 858.11.

[0119] Example 13

[0120]

[0121] Rhodamine hemipolymer R13 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL), and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, rhodamine hemipolymer A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-13. Intermediate Rh-13 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powdery final product, RhNO-13. HRMS(M+) = 872.14.

[0122] Example 14

[0123]

[0124] Rh-14 (0.2 mmol) and malondialdehyde bisphenylimine monohydrochloride (0.21 mmol) were dissolved in acetic anhydride (20 mL) and the mixture was heated to 110 °C under nitrogen protection for 4 h. After the reaction cooled to room temperature, Rhodamine A (0.2 mmol) was dissolved in anhydrous pyridine and added dropwise to the above acetic anhydride mixture, and the mixture was stirred for 3-5 h. The pyridine was removed by rotary evaporation, followed by extraction, drying, and purification by column chromatography to obtain the black intermediate Rh-14. Intermediate Rh-14 (0.1 mmol) was dissolved in 1,2-dichloroethane (20 mL), and phosphorus oxychloride (1 mL) was added dropwise under ice bath conditions. After stirring for 10 min, the mixture was heated to 80 °C under nitrogen protection for 4 h. The 1,2-dichloroethane was removed by rotary evaporation, and anhydrous acetonitrile (20 mL) was added for later use. o-Phenylenediamine (0.2 mmol) and triethylamine (1 mL) were dissolved in anhydrous acetonitrile (5 mL), and the solution was added dropwise to the prepared solution. The mixture was stirred overnight at room temperature. After the reaction was complete, the acetonitrile was removed by rotary evaporation, purified by silica gel column chromatography, and dried to obtain a black powder, RhNO-14. HRMS(M+) = 696.90.

[0125] Figure 12 RhNO-14 is shown 1 H NMR spectrum. Figure 13 , Figure 14The absorption and emission spectra of RhNO-14 are shown respectively.

[0126] This application also tested the NO selectivity when RhNO-1 to RhNO-14 were used as NO probes, and the test method is as follows:

[0127] Solutions of NaCl, MnCl2, MgSO4, ZnCl2, NiCl2, CuSO4, Na2S, Na2CO3, NaNO2, N2NO3, NaHCO3, L-Cys, L-Arg, L-Lys, L-Leu, H2O2, ONOO-, ·OH, and NO were prepared at a concentration of 100 μM as test solutions. RhNO-1 to RhNO-14 were prepared as 20 μM NO probe solutions.

[0128] Mix 1 mL of the test solution and 1 mL of the NO probe solution thoroughly. Set up 3 parallel samples for each group. Wait for the reaction to stabilize for 30 seconds, and then test the absorption spectrum and emission spectrum. Take the highest peak value in the emission spectrum to make a bar chart.

[0129] A typical example is the NO selectivity test result of RhNO-4, as shown below. Figure 8 As shown. The NO selectivity test results for RhNO-1 to RhNO-3 and RhNO-5 to RhNO-14 are similar to those for RhNO-4.

[0130] This application also utilizes RhNO-1 to RhNO-14 as NO probes for in vitro fluorescence detection of NO, the specific method of which is as follows:

[0131] DEANONO ATE (NO donor reagent) was prepared into solutions with concentrations of 0.4 μM, 1 μM, 2 μM, 4 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 100 μM, and 150 μM using PBS in an ice bath. 1 mL of each solution was placed in a cuvette and preheated to 37°C for 2 min to allow for complete NO release. Each NO probe was dissolved in acetonitrile to prepare a 20 μM solution. Then, 1 mL of the probe solution was added to the preheated cuvette, ensuring a 1:1 ratio of organic to aqueous phase. The reaction was allowed to stabilize for 30 s before the absorption and emission spectra were measured. Fitting the spectral data yielded the NO linearity correlation and detection limit of the probe.

[0132] A typical linear correlation spectrum of the reaction between RhNO-4 and NO is as follows: Figure 7As shown in the figure. The linear correlation test results of RhNO-1 to RhNO-3 and RhNO-5 to RhNO-14 with NO are similar to those of RhNO-4. This indicates that RhNO-1 to RhNO-15 all possess good NO responsiveness.

[0133] This application also utilizes RhNO-1 to RhNO-14 as NO probes for in vitro photoacoustic detection of NO, and the specific method is as follows:

[0134] DEANONO ATE (NO donor reagent) was prepared into solutions with concentrations of 0.4 μM, 1 μM, 2 μM, 4 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 100 μM, and 150 μM using PBS in an ice bath. 10 μL of each solution was placed in a centrifuge tube and preheated to 37°C for 2 min to allow for complete NO release. NO probes were prepared into micelles and dissolved in water to prepare solutions with concentrations of 0.1 mg / mL and 0.01 mg / mL. Then, 0.5 mL of the NO probe solution (maintaining a NO probe to NO molar ratio of less than 1:2) was added to the preheated centrifuge tubes. After the reaction stabilized for 30 s, 808 nm laser excitation was performed to obtain a photoacoustic signal.

[0135] A typical example is the photoacoustic tomography image of RhNO-4 before and after the NO response, as shown below. Figure 15 As shown. The photoacoustic tomography results of RhNO-1 to RhNO-3 and RhNO-5 to RhNO-14 before and after NO response are similar to the test results of RhNO-4.

[0136] This application also utilizes RhNO-1 to RhNO-14 as NO probes for in vivo photoacoustic detection of NO, and the specific method is as follows:

[0137] Each NO probe was dissolved in physiological saline containing 10% DMSO to prepare a 50 μM solution. These solutions were injected into mice via the tail vein at a dose of 3 mg / kg. Mice were continuously anesthetized with isoflurane immediately before and 30 minutes after injection, and placed in a prone position on an animal support for imaging. The imaging chamber temperature was set to 36°C, and the animals were allowed 10 minutes to equilibrate to the temperature before imaging. Photoacoustic images were acquired in the brain or chest region of the mice using the optimal absorption wavelengths before and after each probe response. Images were reconstructed using a LOIS-3D device, with all scanning parameters kept consistent.

[0138] Typical examples include photoacoustic tomography images of the mouse brain or chest region obtained using RhNO-4, such as... Figure 16 , Figure 17As shown. The photoacoustic tomography results of mouse brain or chest regions obtained using RhNO-1 to RhNO-3 and RhNO-5 to RhNO-14 were also similar to the results obtained using RhNO-4.

[0139] Therefore, RhNO-1 to RhNO-14 can be used for non-invasive in vivo imaging and real-time monitoring of changes in in vivo nitric oxide concentration. They can effectively reduce the influence of autofluorescence in vivo and penetrate deeper into tissues.

[0140] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0141] It should be understood that the technical solution of this application is not limited to the specific implementation examples mentioned above. Any technical modifications made to the technical solution of this application without departing from the spirit and scope of protection of the claims shall fall within the scope of protection of this application.

Claims

1. A class of nitric oxide-activated near-infrared fluorescent-photoacoustic compounds, characterized in that, The fluorescent-photoacoustic compound has any of the following structures: , 。 2. The use of the nitric oxide-activated near-infrared fluorescent-photoacoustic compound of claim 1 in the preparation of a nitric oxide detection probe.

3. The use according to claim 2, characterized in that, The nitric oxide detection probe has near-infrared fluorescence imaging and / or photoacoustic imaging capabilities.

Images