A near-infrared second-zone small molecule optical diagnostic and therapeutic reagent based on hydrogen sulfide activation, and its preparation method and application

Abstract

The present invention provides a hydrogen sulfide-activated near-infrared II small molecule optical diagnostic and therapeutic agent, whose chemical structure is: [Illegible text - likely a missing link]. This diagnostic and therapeutic agent can accurately identify solid tumors overexpressing hydrogen sulfide, effectively distinguishing tumors from normal tissue, and can be used for tumor resection under the guidance of NIR-II fluorescence imaging. Furthermore, it has excellent photothermal conversion efficiency and can successfully ablate tumors under laser irradiation. This agent demonstrates significant advantages in tumor identification and treatment and is expected to become a highly effective molecular tool for tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule optical diagnostic reagents, and in particular relates to a near-infrared second zone small molecule optical diagnostic reagent that specifically recognizes hydrogen sulfide and its application. Background Art

[0002] Cancer, medically known as malignant tumors, is a serious threat to human health and life, and is still considered difficult or even incurable. With the increasing incidence and mortality of cancer, the diagnosis and treatment of cancer have become increasingly important. Early diagnosis and precise treatment are effective strategies for improving the prognosis of cancer patients. In clinical practice, hematoxylin and eosin staining is an effective method for tumor diagnosis, but this method is invasive and cannot achieve real-time monitoring. Currently, there are multiple methods for treating cancer, including surgery, chemotherapy, radiotherapy, and photothermal therapy. While surgical resection is currently one of the most reliable treatments for cancer patients, it is difficult for clinicians to accurately distinguish cancerous from healthy tissue during surgery. Excessive tissue removal can affect normal body function, while underestimating the extent of the tumor can result in residual tumor. For small or residual tumors that are difficult to remove surgically, chemotherapy and radiotherapy are the main treatments in clinical practice. However, these treatments can cause significant side effects for patients, such as nausea, vomiting, hair loss, and neuropathic pain. Therefore, developing a reagent that can assess tumors early and ablate tiny or residual tumor tissue is of great significance for cancer diagnosis and treatment.

[0003] Fluorescence imaging is a non-invasive cancer diagnostic method that does not require tissue removal, thus avoiding further harm to the patient. Therefore, this diagnostic method has attracted widespread attention from researchers and clinicians. Furthermore, photothermal therapy can be combined with imaging techniques for integrated tumor diagnosis and treatment, leading to widespread interest in photothermal diagnostic and therapeutic agents. Near-infrared II (NIR-II) fluorescence imaging has attracted considerable attention due to its advantages, including high spatial resolution, deep tissue penetration, and minimal interference from autofluorescence. Currently, several NIR-II diagnostic and therapeutic agents based on metal nanoparticles, quantum dots, rare earth nanomaterials, and organic small molecules have been reported. Among these, organic small molecule NIR-II fluorescent agents are considered promising candidates for clinical application due to their ease of metabolism in the body, strong structural tunability, and simple synthesis steps. However, the fluorescence signals of most NIR-II organic molecule agents are always in a "turn-on" state, lacking sufficient specificity. This results in these nonspecific agents being unable to effectively distinguish between cancerous and non-cancerous tissues and can also damage normal tissue during tumor treatment. Therefore, the development of activatable NIR-II organic small molecule probes is urgently needed to improve the accuracy of cancer diagnosis and treatment.

[0004] Literature reports indicate that hydrogen sulfide production is significantly increased in some cancers, such as melanoma, colon cancer, and prostate cancer. Therefore, changes in hydrogen sulfide content can be considered a biomarker that distinguishes these tumors from normal tissue. The construction of hydrogen sulfide-activated NIR-II fluorescent probes combined with photothermal properties could enable early diagnosis and photothermal therapy of tumors. Summary of the Invention

[0005] In response to the problems in the prior art, the present invention provides a near-infrared zone II small molecule compound that specifically recognizes hydrogen sulfide. It can selectively react with hydrogen sulfide overexpressed in tumor tissue, and the fluorescence intensity is significantly enhanced after the reaction. In addition, the reagent has photothermal properties and can effectively kill tumor cells through laser irradiation.

[0006] Another object of the present invention is to provide an application of the above compound in tracing tumor tissue in vivo.

[0007] Another object of the present invention is to provide a use of the above compound in laser ablation of tumor tissue.

[0008] To achieve the above objectives, the present invention adopts the following technical solutions.

[0009] A compound that specifically recognizes hydrogen sulfide, referred to as NS, has a chemical structure as shown in formula (I):

[0010]

[0011] Formula (I).

[0012] The preparation method of the above compound comprises the following steps:

[0013] (1) Demethylation reaction with boron tribromide yields ;

[0014] (2) and After heating reaction, ;

[0015] (3) and 2,4-dinitrobenzenesulfonyl chloride under strong alkaline conditions to obtain .

[0016] The above compounds can be used for quantitative or qualitative detection of hydrogen sulfide, HS - and S 2- .

[0017] The compound can be used for tracing cancer cells or cancer tissues and for the formulation and evaluation of disease progression and treatment plans.

[0018] The above-mentioned compounds can also ablate diseased cells through laser excitation and can be used as therapeutic agents for solid tumors that accumulate hydrogen sulfide.

[0019] The mechanism of the present invention is as follows:

[0020] ;

[0021] This invention utilizes an extended conjugated structure strategy, building on the advantages of the xanthene backbone, to construct a NIR-II fluorescence platform. Furthermore, by utilizing this extended conjugated structure strategy, expanding the wavelength can narrow the energy gap, thereby endowing the fluorescent reagent with photothermal therapeutic capabilities. Based on these principles, the present invention incorporates electron-rich xanthene derivatives into the rhodamine backbone to construct a NIR-II fluorophore. Furthermore, a hydrogen sulfide-specific recognition group (2,4-dinitrobenzenesulfonyl chloride) is linked to the fluorophore to create a NIR-II diagnostic and therapeutic reagent. This diagnostic and therapeutic reagent itself is non-fluorescent, but in the presence of hydrogen sulfide, the NIR-II fluorescence signal is significantly enhanced. Under the guidance of fluorescence imaging, precise photothermal therapy of tumor tissue can be achieved.

[0022] The present invention has the following advantages:

[0023] The diagnostic and therapeutic reagent provided by this invention can accurately distinguish solid tumors that overexpress hydrogen sulfide in vivo. Furthermore, it exhibits excellent photothermal conversion efficiency, successfully ablating tumors under laser irradiation. This diagnostic and therapeutic reagent not only effectively distinguishes tumors from normal tissue but also effectively kills tumors under the guidance of NIR-II fluorescence imaging. This probe demonstrates significant advantages in biological analysis and detection, and is expected to become a new therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the mass spectrum of compound NS;

[0025] Figure 2 It is compound NS 1 H NMR spectrum;

[0026] Figure 3 Figure 1 is the fluorescence spectrum of compound NS (10 µM) in PBS buffer for different concentrations of hydrogen sulfide (A), and the linear relationship between fluorescence intensity and the log value of hydrogen sulfide concentration (B); excitation wavelength: 808 nm;

[0027] Figure 4 The kinetics of compound NS recognizing hydrogen sulfide; the excitation wavelength is 808 nm; the concentration of the probe is 10 μM; the concentration of hydrogen sulfide is 60 μM;

[0028] Figure 5This is the selective fluorescence image of compound NS recognizing hydrogen sulfide. The excitation wavelength is 808 nm and the probe concentration is 10 μM.

[0029] Figure 6 The temperature change of compounds with different concentrations under laser irradiation. Excitation wavelength: 880 nm;

[0030] Figure 7 Fluorescence imaging of the compound in tumor-bearing mice. Excitation wavelength: 808 nm, probe concentration: 25 µM;

[0031] Figure 8 This is a photothermal treatment of tumor-bearing mice using a compound. Excitation wavelength: 880 nm, probe concentration: 25 µM. DETAILED DESCRIPTION

[0032] The present invention provides a compound that specifically recognizes hydrogen sulfide, referred to as NS, whose chemical structure is:

[0033] .

[0034] The preparation method of the above compound can be carried out by Demethylation gives , then with The salt is heated to obtain (NH), and then react with 2,4-dinitrobenzenesulfonyl chloride under strong alkaline conditions to obtain (NS).

[0035] and The preparation can be carried out using methods and steps disclosed in the prior art.

[0036] In some embodiments, NS can be prepared by the following method:

[0037] (1) 3-(Diethylamino)phenol and phthalic anhydride were heated to reflux in toluene. After the reaction was completed, the reaction system was cooled to room temperature and filtered. The solid was washed with methanol to obtain compound 1. ;

[0038] (2) Compound 1 and excess cyclohexanone were heated to about 90°C under the catalysis of concentrated sulfuric acid. After the reaction was completed, the reaction system was cooled to room temperature and poured into ice water containing perchloric acid for acidification. The solid was filtered and washed with ice water to obtain compound 2. perchlorate;

[0039] (3) Cyclohexanone, boron tribromide and dimethylformamide react in a solution. After monitoring the reaction, the reaction system is poured into ice water, the pH value is adjusted to neutral, and extraction is performed with dichloromethane. The organic layer is dried and the solvent is removed to obtain an oily compound 3. ;

[0040] (4) Compound 3 and 2-hydroxy-4-methoxybenzaldehyde were dissolved in dimethylformyl solution and reacted under the catalysis of cesium carbonate. After the reaction was completed, the filtrate was filtered and extracted with ethyl acetate. The organic layer was stripped of solvent and purified by column chromatography using dichloromethane and ethyl acetate in a volume ratio of 10:1 as eluent to obtain compound 4. ;

[0041] (5) Compound 4 was reacted under the catalysis of boron tribromide. After completion, the reaction was quenched with a base and extracted with dichloromethane. The organic layer was dried and the solvent was removed. Then, the compound was purified by silica gel chromatography using dichloromethane and methanol in a volume ratio of 10:1 as eluent to obtain compound 5. ;

[0042] (6) The perchlorate of compound 2 and compound 5 were heated to react, cooled to room temperature and filtered, the solid was washed with water, and purified by silica gel chromatography using dichloromethane and methanol in a volume ratio of 20:1 as eluent to obtain compound NH ;

[0043] (7) Compound NH and 2,4-dinitrobenzenesulfonyl chloride were reacted in the presence of trimethylamine. After the reaction was completed, the reaction system was extracted with ethyl acetate. The organic layer was dried and the solvent was removed. Compound NS was purified by silica gel chromatography using dichloromethane and methanol in a volume ratio of 20:1 as eluent to obtain compound NS. .

[0044] The above compounds can be used to identify hydrogen sulfide, HS - and S 2- The fluorescence intensity of the compound is in a certain concentration range and is quantitatively related to the concentration of the above-mentioned detection substance, and the quantitative relationship can be used to quantify the concentration of the detection substance.

[0045] Because some cancer cells accumulate hydrogen sulfide compared to normal cells, this compound can be used to track cancer cells or cancerous tissues and assess disease progression and treatment options. Furthermore, the product produced by the compound after hydrogen sulfide recognition has photothermal properties. Once accumulated in cancer cells or tissues, laser excitation can ablate diseased cells. Therefore, it can be used as a therapeutic agent for solid tumors that accumulate hydrogen sulfide.

[0046] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0047] Example 1 Synthesis of Compound NS

[0048] (1)

[0049] 165 mg of 3-(diethylamino)phenol (1.0 mmol) and 148 mg of phthalic anhydride (1.0 mmol) were dissolved in toluene solution and heated to 100°C for 6 hours. The reaction system was then filtered and washed with methanol to obtain a solid of compound 1, which was used in the next reaction without further purification.

[0050] (2)

[0051] At 0°C, 110 µL of cyclohexanone (1.0 mmol) was added dropwise to concentrated sulfuric acid (1.2 mL). 167 mg of compound 1 (0.5 mmol) was added to the mixture, heated at 90°C for 1.5 hours, cooled to room temperature, and poured into a solution containing 5 g of ice water and 70% perchloric acid (0.7 mL). The solid was filtered and washed with ice water to obtain the perchlorate salt of compound 2.

[0052] (3)

[0053] With stirring at 0°C, 2.5 mL of boron tribromide was gradually added to a mixture of 2.3 mL of dimethylformamide and 10 mL of dichloromethane. After stirring for 45 minutes, 1 mL of cyclohexanone was added at room temperature, and the mixture was stirred for 8 hours. The solution was then poured into ice water, the pH adjusted to 7 with sodium bicarbonate, and extracted with dichloromethane. The organic layer was dried and the solvent removed to obtain compound 3 as an oil.

[0054] (4)

[0055] 378 mg of compound 3 (2.0 mmol) was dissolved in 2 mL of dimethylformamide, and then 152 mg of 2-hydroxy-4-methoxybenzaldehyde (1.0 mmol) and 400 mg of cesium carbonate (3.0 mmol) were added. The reaction was carried out at 25°C-30°C for 8 hours. The insoluble material was filtered, and the filtrate was concentrated. The product was extracted with ethyl acetate. After the ethyl acetate layer was dried over anhydrous sodium sulfate, the ethyl acetate was removed under reduced pressure and purified by column chromatography using dichloromethane / ethyl acetate (10:1, v / v) as the eluent to obtain compound 4.

[0056] (5)

[0057] 242 mg of compound 4 (1.0 mmol) was dissolved in 10 mL of dichloromethane, and then 1.9 mL of boron tribromide was added dropwise at -40°C. The reaction was stirred at room temperature for 16 hours. The reaction was then quenched with saturated sodium bicarbonate solution at 0°C and extracted with dichloromethane. The dichloromethane layer was dried over anhydrous sodium sulfate, and the dichloromethane was removed under reduced pressure. Compound 5 was purified by silica gel chromatography using dichloromethane / methanol (10:1, v / v) as the eluent to obtain compound 5.

[0058] (6)

[0059] 376 mg of the perchlorate salt of compound 2 (1.0 mmol) and 228 mg of compound 5 (1.0 mmol) were added to 10 mL of ethanol and stirred at 80°C for 8 hours. After cooling to room temperature, the crude product was filtered, the solid was washed with water, and purified by silica gel chromatography using dichloromethane / methanol (20:1, v / v) as the eluent to obtain compound NH.

[0060] (7)

[0061] 58 mg of compound NH (1 mmol) and 27 mg of 2,4-dinitrobenzenesulfonyl chloride (1 mmol) were dissolved in 6 mL of dichloromethane, and then 40 μL of trimethylamine was added. The reaction was stirred at room temperature for 8 hours. The reaction system was extracted with ethyl acetate, and the ethyl acetate layer was dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure, and the product was purified by silica gel chromatography using dichloromethane / methanol (20:1, v / v) as the eluent to obtain compound NS.

[0062] Mass spectra of compound NS and 1 The H NMR spectra were Figure 1 and Figure 2 As shown, its molecular formula is C 44 H 38 N3O 11 S + , the structural formula is: , the same as the expected product.

[0063] Example 2 Response of Compound NS to Detection Substances

[0064] A test stock solution of the compound NS prepared in Example 1 in dimethyl sulfoxide (DMSO) with a concentration of 1 mM was prepared for use.

[0065] In the test solution, an appropriate amount of NS stock solution was measured and brought to volume using PBS buffer (pH = 7.4). An appropriate amount of sodium bisulfide was added to one of the solutions. The concentration of the probe in the test solution was 10 μM. The sodium bisulfide concentrations used in the tests were 4 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, 40 μM, 44 μM, 48 μM, 50 μM, 55 μM, and 60 μM, respectively. The volume fraction of DMSO in the test solution was 20%.

[0066] Fluorescence detection of the test liquid (λ ex = 808 nm, λ em = 900 nm-1100 nm), and a graph showing the relationship between concentration and fluorescence intensity was established. Figure 3 As shown in A, as the concentration of sodium hydrosulfide increases, the fluorescence intensity at 925 nm gradually increases. When the concentration of sodium hydrosulfide reaches 60 μM, the fluorescence intensity of the reaction system reaches saturation. 2- , HS - , which is the same as the state of existence of hydrogen sulfide in the body; in addition, Figure 3 As shown in Figure B, there is a linear relationship between the logarithmic value of hydrogen sulfide concentration and the fluorescence intensity. Therefore, the above results prove that compound NS can react with H2S in the body and can be detected and quantified by detecting fluorescence.

[0067] Example 3 Response kinetics of compound NS to test substances

[0068] Add 20 μL of NS DMSO stock solution and 380 μL of DMSO to a 2 mL volumetric flask, dilute to volume with PBS buffer, and then add an appropriate amount of sodium bisulfide, where the concentration of NS is 10 μM and the concentration of sodium bisulfide is 60 μM; shake well and perform fluorescence detection (λ ex = 808 nm), and a graph of fluorescence intensity at 925 nm versus time was constructed.

[0069] The results are as follows Figure 4 As shown, in the presence of sodium bisulfide, the fluorescence signal of the probe increased significantly over time and reached saturation after about 600 seconds, indicating that the probe can quickly recognize sodium bisulfide.

[0070] Example 4 Selectivity of compound NS for different ions

[0071] Prepare 100 mM solutions of various ions, amino acids, and reactive oxygen / nitrogen species for later use. Add 20 μL of probe stock solution, 380 μL of DMSO, and 50 equivalents of each ion solution or 10 equivalents of each active substance solution to a 2 mL volumetric flask. Use PBS buffer (pH = 7.4) to make up the volume. The concentration of NS is 10 μM, the concentration of the test ion is 500 μM, and the concentration of reactive oxygen / nitrogen species is 200 μM. After shaking, perform fluorescence detection (λ ex = 808 nm), and a histogram was constructed to plot the fluorescence intensity against each ion.

[0072] The results are as follows Figure 5 As shown, 1-21 are: H2O2 (100 μM), arginine (1 mM), vitamin C (1 mM), ONOO - The fluorescence of the probe NS was detected in the presence of 100 μM, MgCl2 (2.5 mM), NaCl (2.5 mM), CaCl2 (2.5 mM), FeSO4 (2.5 mM), NS (10 μM), GSH (2.5 mM), ZnCl2 (2.5 mM), glycine (1 mM), NaNO3 (2.5 mM), FeCl3 (1 mM), CuCl2 (2.5 mM), AlCl3 (2.5 mM), Cys (1 mM), Hcy (1 mM), HClO (100 μM), NaHSO3 (100 μM), and NaHS (60 μM). As can be seen from the figure, other substances have little effect on the fluorescence of the probe NS. Only in the presence of sodium bisulfide does the fluorescence signal increase significantly, indicating that the probe has good specificity for sodium bisulfide.

[0073] Example 5 Photothermal properties of compound NH

[0074] Prepare a 1 mM dimethyl sulfoxide stock solution of the intermediate NH prepared in Example 1 for later use. Add 40 μL, 100 μL, or 200 μL of the NH stock solution and 360 μL, 300 μL, or 200 μL of DMSO to a 2 mL volumetric flask. Dose to volume with PBS buffer. Shake well and irradiate with an 808 nm laser. Record the temperature change at different time points and create a linear plot of temperature change versus time at different NH concentrations.

[0075] See the results Figure 6 As can be seen from the figure, after 8 minutes of laser irradiation, the temperature of the NH solution can reach 63°C, and the temperature change increases with time and the increase of its concentration, indicating that the probe has good photothermal properties and has application potential in laser ablation of tumors.

[0076] Example 6 Application of Compound NS in Imaging of Tumor-Bearing Mice

[0077] 5×10 6 B16 cells were suspended in 200 µL of PBS and implanted subcutaneously into the right hind legs of 4-week-old Kunming mice. Tumors became visible approximately 7 days later. The B16 tumor-bearing mice were then orthotopically injected with a 50 µL NS solution (25 µM) into the tumor region. For comparison, normal mice were subcutaneously injected with a 50 µL NS solution (25 µM). Fluorescence imaging of the anesthetized mice was performed at various times using the NIR-II in vivo imaging system.

[0078] The results are as follows Figure 7 As shown, it can be seen that as time goes by, the fluorescence signal of tumor mice is significantly enhanced, while the fluorescence signal of normal mice is weak, indicating that compound NS can be used for imaging of tumor tissues.

[0079] Example 7 Imaging Application of Fluorescent Probes in Organisms

[0080] 5×10 6 B16 cells were suspended in 200 µL of PBS and subcutaneously implanted into the right hind legs of 4-week-old Kunming mice. Tumors became visible approximately 7 days later. Two mice were then injected intratumorally with 50 µL of PBS and 50 µL of NS solution (25 µM). The tumor sites were then irradiated with an 808 laser for 8 minutes. Temperature changes in the mice were recorded using an infrared thermal imager. The mice were photographed on days 0, 1, 7, and 10 to observe tumor changes.

[0081] The results are as follows Figure 8 As shown in the images, after laser irradiation, the temperature of the mouse tumor increased significantly. On day 10, the tumors of the mice injected with the probe were smaller and showed no signs of further growth compared to those of the mice injected with PBS. This indicates that the probe of the present invention has excellent photothermal properties and can kill tumor tissue.

Claims

1. A compound that specifically recognizes hydrogen sulfide, having the chemical formula: 。 2. A method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Demethylation reaction with boron tribromide yields ; (2) and After heating reaction, ; (3) and 2,4-dinitrobenzenesulfonyl chloride under strong alkaline conditions to obtain 。 3. A compound as claimed in claim 1 for preparing a test solution, a cell, a tissue, or a biological body for detecting hydrogen sulfide, HS - and S 2- Application of reagents.

4. The use according to claim 3, characterized in that The detection is fluorescent qualitative or quantitative detection.

5. Use of the compound according to claim 1 as a tracer for cancer cells or cancer tissues.

6. The use according to claim 5, characterized in that The tracer is a fluorescent tracer with a fluorescence excitation wavelength of 808 nm.

7. Use of the compound according to claim 1 in preparing a therapeutic agent for solid tumors.

8. The use according to claim 7, characterized in that The solid tumor is a tumor that accumulates hydrogen sulfide; the treatment method of the therapeutic agent is laser thermal ablation of diseased cells under the guidance of fluorescent imaging.

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