Near-infrared two-region micromolecule optical diagnosis and treatment reagent based on hydrogen sulfide activation as well as preparation method and application thereof

Abstract

The invention provides a near-infrared two-region micromolecule optical diagnosis and treatment reagent based on hydrogen sulfide activation. The chemical structural formula of the reagent is # imgabs0 #. The diagnosis and treatment reagent provided by the invention can accurately identify solid tumors overexpressed by hydrogen sulfide, realizes effective distinguishing between the tumors and normal tissues, and can perform tumor excision under the guidance of NIR-II fluorescence imaging; in addition, the material has good photothermal conversion efficiency, and can successfully ablate tumors under laser irradiation. The reagent shows huge superiority in tumor recognition and treatment, and is expected to become an efficient 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 for specifically identifying hydrogen sulfide and an application thereof. Background Art

[0002] Cancer, medically known as malignant tumors, is one of the diseases that seriously threaten human health and life. It is still regarded as a difficult or even incurable disease. With the increase in the incidence and death of cancer, the diagnosis and treatment of cancer are particularly important. Early diagnosis and precise treatment are effective solutions to improve the prognosis of cancer patients. In clinical applications, HE staining is an effective method for tumor diagnosis, but this method is invasive and cannot achieve real-time monitoring. There are currently many methods for treating cancer, including surgery, chemotherapy, radiotherapy, and photothermal therapy. Although surgical resection is currently one of the most reliable methods for treating cancer patients. However, during surgery, it is difficult for clinicians to accurately distinguish cancerous tissue from healthy tissue with the naked eye. Excessive tissue removal will affect the normal function of the body, and underestimating the tumor range will cause residual tumors. For some small or residual tumor tissues that are difficult to remove surgically, chemotherapy and radiotherapy are mainly used in clinical practice. However, using these methods will bring patients greater side effects when treating tumors, such as nausea, vomiting, hair loss, and neuropathic pain. Therefore, developing an agent that can evaluate 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 diagnosis method that does not require the removal of patient tissue, thus avoiding further harm to the patient. Therefore, this diagnostic method has attracted the attention of a large number of researchers and clinicians. In addition, photothermal therapy can be combined with imaging technology for integrated tumor diagnosis and treatment research. Therefore, photothermal diagnostic and therapeutic agents have always received widespread attention. Near-infrared II (NIR-II) fluorescence imaging has the advantages of high spatial resolution, deep tissue penetration, and minimal interference from autofluorescence, which has attracted the attention of researchers. At present, some NIR-II diagnostic and therapeutic agents based on metal nanoparticles, quantum dots, rare earth nanomaterials and organic small molecules have been reported. Among them, organic small molecule NIR-II fluorescent agents are regarded as ideal materials for clinical applications because of their easy metabolism in the body, strong structural adjustability and simple synthesis steps. However, the fluorescence signals of most NIR-II organic molecule agents are always in the "turn-on" state, lacking sufficient specificity, resulting in these non-specific agents being unable to effectively distinguish between cancerous tissues and non-cancerous tissues, and also causing damage to normal tissues when treating tumors. Therefore, it is urgent to develop activatable NIR-II organic small molecule probes to improve the accuracy of cancer diagnosis and treatment.

[0004] According to literature reports, the amount of hydrogen sulfide produced increases significantly in some cancer diseases (such as melanoma, colon cancer, and prostate cancer). Therefore, changes in hydrogen sulfide content can be regarded as a biomarker to distinguish these tumors from normal tissues. The construction of hydrogen sulfide-activated NIR-II fluorescent probes combined with photothermal properties can achieve 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, which can selectively react with hydrogen sulfide overexpressed in tumor tissues. After the reaction, the fluorescence intensity is significantly enhanced. 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 purpose, the present invention adopts the following technical solution.

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

[0010] The preparation method of the above compound comprises the following steps: (1) Demethylation with boron tribromide gives ; (2) and After heating reaction, ; (3) and 2,4-dinitrobenzenesulfonyl chloride under strong alkaline conditions to obtain .

[0011] The above compounds can be used for quantitative or qualitative detection of hydrogen sulfide, HS - and S 2- .

[0012] The compound can be used for tracing cancer cells or cancer tissues and for the formulation and evaluation of disease progression and treatment plans.

[0013] The above compounds can also ablate diseased cells through laser excitation and can be used as therapeutic agents for solid tumors that accumulate hydrogen sulfide.

[0014] The mechanism of the present invention is as follows: ; The present invention utilizes the strategy of extending the conjugated structure and constructs a NIR-II fluorescence platform based on the advantages of the xanthene skeleton. At the same time, by utilizing the strategy of extending the conjugated structure, expanding the wavelength can narrow the energy gap, thereby giving the fluorescent agent photothermal therapy capabilities. Based on these principles, the present invention introduces electron-rich xanthene derivatives into the rhodamine skeleton to construct a NIR-II fluorophore. At the same time, the hydrogen sulfide-specific recognition group (2,4-dinitrobenzenesulfonyl chloride) is connected to the fluorescent group to construct a NIR-II diagnostic and therapeutic agent. The diagnostic and therapeutic agent itself has no fluorescence, but in the presence of hydrogen sulfide, the NIR-II fluorescence signal is significantly enhanced, and under the guidance of fluorescence imaging, precise photothermal therapy of tumor tissue can be achieved.

[0015] The present invention has the following advantages: The diagnostic and therapeutic reagent provided by the present invention can accurately distinguish solid tumors with overexpression of hydrogen sulfide in vivo; in addition, it has good photothermal conversion efficiency and can successfully ablate tumors under laser irradiation. The diagnostic and therapeutic reagent can not only effectively distinguish tumors from normal tissues, but also effectively kill tumors under the guidance of NIR-II fluorescence imaging. This probe shows great superiority in biological analysis and detection, and is expected to become a new therapeutic reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the mass spectrum of compound NS; Figure 2 It is the compound NS 1 H NMR spectrum; Figure 3 The fluorescence spectra of compound NS (10 µM) in PBS buffer for different concentrations of hydrogen sulfide (A) and the linear relationship between fluorescence intensity and log value of hydrogen sulfide concentration (B); excitation wavelength: 808 nm; 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; Figure 5 This is the selective fluorescence diagram of compound NS recognizing hydrogen sulfide. The excitation wavelength is 808 nm; the concentration of the probe is 10 µM; Figure 6 It is the temperature change of compounds with different concentrations under laser irradiation. Excitation wavelength: 880 nm; Figure 7 Fluorescence imaging of compounds in tumor-bearing mice. Excitation wavelength: 808 nm, probe concentration: 25 µM; Figure 8Photothermal therapy of tumor-bearing mice with compounds. Excitation wavelength: 880 nm, probe concentration: 25 µM. DETAILED DESCRIPTION

[0017] The present invention provides a compound that specifically recognizes hydrogen sulfide, referred to as NS, and has the chemical structural formula: .

[0018] The preparation method of the above compound can be carried out by Demethylation gives , and then with The salt is heated to obtain (NH), and then react with 2,4-dinitrobenzenesulfonyl chloride under strong alkaline conditions to obtain (NS).

[0019] and The preparation can be carried out by methods and steps disclosed in the prior art.

[0020] In some embodiments, NS can be prepared by the following method: (1) 3-(Diethylamino)phenol and phthalic anhydride were heated to reflux in toluene for reaction. 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 ; (2) Compound 1 and excess cyclohexanone were heated to about 90°C under the catalysis of concentrated sulfuric acid to react. 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; (3) Cyclohexanone, boron tribromide and dimethylformamide react in a solution. After the reaction is completed, the reaction system is poured into ice water, the pH value is adjusted to neutral, and extracted with dichloromethane. After the organic layer is dried, the solvent is removed to obtain an oily compound 3. ; (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 to obtain a filtrate, which was extracted with ethyl acetate. After the organic layer was freed of solvent, column chromatography was performed with dichloromethane and ethyl acetate in a volume ratio of 10:1 as the eluent to obtain compound 4. ; (5) Compound 4 was reacted under the catalysis of boron tribromide, and 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 the eluent to obtain compound 5 ; (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 ; (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. The compound NS was purified by silica gel chromatography using dichloromethane and methanol in a volume ratio of 20:1 as the eluent to obtain the compound NS .

[0021] 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.

[0022] Since hydrogen sulfide accumulates in some cancer cells compared to normal cells, the compound can be used to trace cancer cells or cancer tissues and to evaluate disease progression and treatment plans. In addition, the product of the above compound after recognizing hydrogen sulfide has photothermal conversion properties. After cancer cells or tissues are enriched, the diseased cells can be ablated by laser excitation. Therefore, it can be used as a therapeutic agent for solid tumors that accumulate hydrogen sulfide.

[0023] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0024] Example 1 Synthesis of Compound NS (1) 165 mg 3-(diethylamino)phenol (1.0 mmol) and 148 mg phthalic anhydride (1.0 mmol) were dissolved in a toluene solution, heated to 100°C for 6 hours, and then the reaction system was filtered and washed with methanol to obtain a solid of compound 1, which was used in the next step without further purification; (2) At 0°C, 110 µL of cyclohexanone (1.0 mmol) was dropped into concentrated sulfuric acid (1.2 mL). 167 mg of compound 1 (0.5 mmol) was added to the above mixture, heated at 90°C for 1.5 hours, cooled to room temperature, poured into a liquid containing 5 g of ice water and 70% perchloric acid (0.7 mL), filtered to obtain a solid, and washed with ice water to obtain the perchlorate salt of compound 2; (3) Under 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 min, 1 mL of cyclohexanone was added at room temperature and stirred at room temperature for 8 hours. The solution was then poured into ice water, the pH value was adjusted to 7 with sodium bicarbonate, extracted with dichloromethane, and the organic layer was dried and the solvent was removed to obtain oily compound 3; (4) 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 mixture was reacted at 25°C-30°C for 8 hours. The insoluble matter was filtered, and the filtrate was concentrated. The mixture 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 the mixture was purified by column chromatography using dichloromethane / ethyl acetate (10:1, v / v) as the eluent to obtain compound 4. (5) 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, and the reaction was stirred at room temperature for 16 hours; the reaction was then quenched with a 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; and the compound was purified by silica gel chromatography using dichloromethane / methanol (10:1, v / v) as the eluent to obtain compound 5; (6) 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. (7) 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, and 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, and the ethyl acetate was removed under reduced pressure, and dichloromethane / methanol (20:1, v / v) was used as the eluent to purify by silica gel chromatography to obtain compound NS; Mass spectra of compound NS and 1 The H NMR spectra are shown in Figure 1 and Figure 2 As shown, its molecular formula is C 44 H 38 N3O 11 S + , the structural formula is: , which is the same as the expected product.

[0025] Example 2 Response of compound NS to test substances 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.

[0026] In the test solution, an appropriate amount of NS stock solution was measured and fixed to volume using PBS buffer (pH = 7.4), and an appropriate amount of sodium hydrosulfide was added to one of them. The concentration of the probe in the test solution was 10 μM, and the concentrations of sodium hydrosulfide used in the test 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%.

[0027] Fluorescence detection of the test fluid (λ ex = 808 nm, λ em = 900 nm-1100 nm), and a graph was established showing the relationship between concentration and fluorescence intensity. 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 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.

[0028] Example 3 Response kinetics of compound NS to test substances Add 20 μL of NS DMSO stock solution and 380 μL of DMSO to a 2 mL volumetric flask, dilute with PBS buffer, and then add an appropriate amount of sodium hydrosulfide, where the concentration of NS is 10 μM and the concentration of sodium hydrosulfide 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.

[0029] The results are as follows Figure 4 As shown, in the presence of sodium hydrosulfide, 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 hydrosulfide.

[0030] Example 4 Selectivity of compound NS to different ions Prepare 100 mM solutions of various ions, amino acids, and reactive oxygen / nitrogen species for backup. Add 20 μL probe stock solution, 380 μL DMSO, and 50 equivalents of each ion solution or 10 equivalents of each active substance solution to a 2 mL volumetric flask, and make up the volume with PBS buffer (pH = 7.4), where 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; shake well and perform fluorescence detection (λ ex = 808 nm), and a histogram of fluorescence intensity and each ion was constructed.

[0031] The results are as follows Figure 5 As shown, 1-21 are: H2O2 (100 μM), arginine (1 mM), vitamin C (1 mM), ONOO - (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), NaHS(60 μM). As can be seen from the figure, other substances have almost no effect on the fluorescence of probe NS. Only in the presence of sodium hydrosulfide, the fluorescence signal is significantly enhanced, indicating that the probe has good specificity for sodium hydrosulfide.

[0032] Example 5 Photothermal properties of compound NH Prepare a 1 mM dimethyl sulfoxide mother solution of the intermediate product NH prepared in Example 1 for later use. Add 40 μL, 100 μL or 200 μL of NH mother solution and 360 μL, 300 μL or 200 μL of DMSO to a 2 mL volumetric flask, dilute to volume with PBS buffer, shake well, and irradiate with 808 nm laser respectively. Record the temperature change at different time points, and establish a linear graph of temperature change and time at different NH concentrations.

[0033] Results Figure 6As 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 its concentration, indicating that the probe has good photothermal properties and has application potential in laser ablation of tumors.

[0034] Example 6 Application of compound NS to imaging of tumor-bearing mice 5×10 6 B16 cells were suspended in 200 µL PBS and then implanted subcutaneously into the right hind leg of 4-week-old Kunming mice. Tumors were visible to the naked eye about 7 days later. Then, NS solution (25 µM, 50 µL) was injected in situ into the tumor area of ​​B16 tumor-bearing mice. For comparison, NS solution (25 µM, 50 µL) was injected subcutaneously into normal mice. Fluorescence imaging of anesthetized mice was performed at different times using the NIR-II in vivo imaging system.

[0035] 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.

[0036] Example 7 Imaging application of fluorescent probe in organisms 5×10 6 B16 cells were suspended in 200 µL PBS and then implanted subcutaneously into the right hind leg of 4-week-old Kunming mice. The tumor was visible to the naked eye about 7 days later. Two mice were injected intratumorally with 50 µL PBS and NS solution (25 µM, 50 µL), respectively. Then, the tumor site of the mouse was irradiated with 808 laser for 8 min, and the temperature changes of the living mice were recorded with an infrared thermal imager. The mice were photographed on days 0, 1, 7, and 10 to observe the changes in the tumor.

[0037] The results are as follows Figure 8 As shown in the figure, after laser irradiation, the temperature of the mouse tumor increased significantly. On the 10th day, the tumor of the mouse injected with the probe became smaller and showed no trend of further growth compared with the tumor mouse injected with PBS. This indicates that the probe of the present invention has good photothermal properties and can kill tumor tissue.

Claims

1. A compound that specifically recognizes hydrogen sulfide, whose chemical structural formula is: .

2. A method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Demethylation with boron tribromide gives ; (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 detection 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, and the fluorescence excitation wavelength is preferably 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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