PH-sensitive fluorescent probe for detecting thionitrous acid as well as preparation method and application of pH-sensitive fluorescent probe

Abstract

The invention provides a pH-sensitive fluorescent probe for detecting thionitrous acid and a preparation method and application thereof, the fluorescent probe has good stability, the structural formula is shown in the specification, the fluorescent probe has good selectivity and sensitivity (the detection limit is 415 nM) to HSNO in an acid environment, specific detection of HSNO in HCT-116 cells is achieved, and the fluorescent probe has good application prospects. And the fluorescent probe is applied to fluorescence imaging of HSNO in an HCT-116 colon cancer nude mouse transplanted tumor. Besides, after the HSNO is intravenously injected into the tail of a transplanted tumor nude mouse, the tumor growth is inhibited, and the HSNO level in the tumor tissue is increased, which indicates that the HSNO has the anti-tumor activity, and an effective detection tool is provided for researching the relationship between the HSNO and the tumor development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemistry and analytical detection technology, and particularly relates to a pH-sensitive fluorescent probe for detecting thionitrous acid and a preparation method and application thereof. BACKGROUND

[0002] Thionitrous acid (HSNO) is the smallest molecule of S-nitrosothiol, which is generated from H2S and RSNOs or H2S and NO, and is a key signal transduction molecule connecting H2S and NO signaling pathways. NO and H2S interact with each other in the regulation of blood vessels and the nervous system, and jointly regulate physiological functions. HSNO can also generate H2S n and HNO with H2S, and metabolically generate NO, NO + , and NO - , and plays a role as a signal molecule and a cellular oxidative regulator. In colon cancer, NO and H2S regulate cell proliferation through a bell-shaped concentration-response curve, and endogenous NO and H2S promote tumor cell proliferation by maintaining the cGMP / VASP pathway, while exogenous NO and H2S at higher than physiological concentrations play an anti-tumor role, and both exist in a series of cross-talk in the regulation of the cGMP / VASP signaling pathway. It can be seen that the expression levels of NO and H2S and the cross-talk relationship between them affect the development of colon cancer. HSNO is the source of NO and the cross-talk product of NO and H2S, and its level change is also closely related to the development of colon cancer.

[0003] Acidity is one of the characteristics of the tumor microenvironment, which is caused by the tumor metabolic pattern, enhanced proton pump activity, and abnormal structure and function of blood vessels. The acidic microenvironment can activate the MAPK signaling pathway to promote tumor cell proliferation, induce the secretion of MMPs to enhance the invasiveness of tumor cells, inhibit the function of immune cells, and reduce the activity of chemotherapeutic drugs to affect tumor treatment. In recent years, more and more tumor targeting probes activated by acidic microenvironment have been developed, and these fluorescent probes show high specificity and high signal-to-noise ratio in tumor imaging potential.

[0004] The traditional methods for detecting HSNO at present include high-resolution mass spectrometry, Fourier transform infrared spectroscopy, 15 Nuclear magnetic resonance method, but these methods are not suitable for real-time visual detection of biological samples. Fluorescent probes have the advantages of high sensitivity, high signal-to-noise ratio, and simple operation in real-time visual detection of biological samples, and there are two examples of HSNO fluorescent probes reported, but these two fluorescent probes cannot realize the detection of HSNO at the level of living body, and cannot specifically detect HSNO in tumors. Therefore, it is necessary to develop a fluorescent probe that can specifically detect HSNO in tumors. SUMMARY

[0005] The application aims to provide a pH-sensitive fluorescent probe for detecting thionitrite based on the prior art, which has good stability, good selectivity and sensitivity (detection limit is 415 nM) to HSNO in an acidic environment, realizes specific detection of HSNO in HCT-116 cells, and is applied to fluorescence imaging of HSNO in HCT-116 colon cancer transplanted tumors of nude mice. In addition, after the transplanted tumor nude mice are injected with HSNO through the tail vein, the tumor growth is inhibited, and the HSNO level in the tumor tissue is increased, which indicates that HSNO has anti-tumor activity, and provides an effective detection tool for studying the relationship between HSNO and tumor development.

[0006] The second object of the application is to provide a preparation method of the above-mentioned pH-sensitive fluorescent probe for detecting thionitrite.

[0007] The third object of the application is to provide application of the above-mentioned pH-sensitive fluorescent probe in detection of thionitrite.

[0008] The technical scheme of the application is as follows:

[0009] A pH-sensitive fluorescent probe for detecting thionitrite, the structural formula of the fluorescent probe is as follows:

[0010]

[0011] The application also provides a preparation method of the above-mentioned pH-sensitive fluorescent probe for detecting thionitrite, and the synthetic route is as follows:

[0012]

[0013]

[0014] The application also provides a preparation method of the above-mentioned pH-sensitive fluorescent probe for detecting thionitrite, and the synthetic route is as follows:

[0015] (1) Carrying out chemical reaction on p-carboxyphenylhydrazine and methyl isopropyl ketone to prepare compound CHC-1;

[0016] (2) Carrying out chemical reaction on compound CHC-1 and N-tert-butoxycarbonyl-bromoethylamine to prepare compound CHC-2;

[0017] (3) Carrying out chemical reaction on 4-(diethylamino)salicylaldehyde and diethyl malonate in the presence of piperidine to prepare compound CHC-3; (4) Carrying out chemical reaction on compound CHC-3 in the presence of phosphorus trichloride to prepare compound CHC-4;

[0018] (5) Compound CHC-2 and compound CHC-4 are subjected to a chemical reaction to prepare compound CHC-5;

[0019] (6) Compound CHC-5 is subjected to a chemical reaction in the presence of trifluoroacetic acid to prepare compound CHC-6;

[0020] (7) Thiosalicylic acid and 2,2'-dithiodipyridine are subjected to a chemical reaction to prepare compound S-1;

[0021] (8) Compound S-1 and p-hydroxybenzaldehyde are subjected to a chemical reaction in the presence of EDCI and DMAP to prepare compound S-2;

[0022] (9) Compound S-2 is subjected to a chemical reaction in the presence of sodium borohydride to prepare compound S-3;

[0023] (10) Compound S-3, o-phenylenediamine and triphosgene are subjected to a chemical reaction in the presence of K2CO3 and pyridine to prepare compound S-4;

[0024] (11) Compound CHC-6 and compound S-4 are subjected to a chemical reaction in the presence of HBTU and HOBT to prepare fluorescent probe CHC-HSNO.

[0025] In the present application, in step (1), the molar ratio of p-carboxyphenylhydrazine and methyl isopropyl ketone is 1:1.2-2.0, preferably 1:1.4-1.6, more preferably 1:1.5.

[0026] In the present application, in step (2), the molar ratio of compound CHC-1 and N-tert-butoxycarbonyl-bromoethylamine is 1:1.5-2.5, preferably 1:1.8.0-2.2; more preferably 1:2.0.

[0027] In the present application, in step (3), the molar ratio of 4-(diethylamino)salicylaldehyde and diethyl malonate is 1:1.5-2.5, preferably 1:1.8.0-2.2; more preferably 1:2.0.

[0028] In step (3), the molar ratio of 4-(diethylamino)salicylaldehyde and piperidine is 2.0-3.0:1, preferably 2.6-2.8:1; more preferably 2.75:1.

[0029] In the present application, in step (4), the mass-volume ratio of compound CHC-3 to phosphorus trichloride is 0.1-0.8:1 g / mL, preferably 0.3-0.5:1 g / mL, more preferably 0.44:1 g / mL.

[0030] In step (4), the reaction temperature is 40-60°C, preferably 45-55°C, more preferably 50°C.

[0031] In the present application, in step (5), the molar ratio of compound CHC-2 and compound CHC-4 is 1.5-2.5:1, preferably 1.8-2.2:1; more preferably 2.0:1.

[0032] In the present application, in step (6), the mass-volume ratio of compound CHC-5 and trifluoroacetic acid is 0.1-0.8:1 g / mL, preferably 0.2-0.4:1 g / mL, more preferably 0.3:1 g / mL.

[0033] In the present application, in step (7), the molar ratio of thiosalicylic acid and 2,2'-dithiodipyridine is 2.5-3.5:1, preferably 2.8-3.2:1; more preferably 3.0:1.

[0034] In the present application, in step (8), the molar ratio of compound S-1 and p-hydroxybenzaldehyde is 0.5-1.5:1, preferably 0.8-1.2:1; more preferably 1.0:1.

[0035] In step (8), the molar ratio of compound S-1 and EDCI is 1:1.2-2.0, preferably 1:1.4-1.6; more preferably 1:1.5.

[0036] In step (8), the molar ratio of compound S-1 and DMAP is 1:0.1-0.5, preferably 1:0.15-0.25; more preferably 1:0.2.

[0037] In the present application, in step (9), the molar ratio of compound S-2 and sodium borohydride is 1:0.8-1.5, preferably 1:1.0-1.2; more preferably 1:1.1.

[0038] In the present application, in step (10), the molar ratio of compound S-3 and o-phenylenediamine is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0.

[0039] In step (10), the molar ratio of compound S-3 and triphosgene is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0.

[0040] In step (10), the molar ratio of compound S-3 and K2CO3 is 1:3.0-4.0, preferably 1:3.8-4.2; more preferably 1:4.0.

[0041] In step (10), the molar ratio of compound S-3 and pyridine is 1:3.0-4.0, preferably 1:3.8-4.2; more preferably 1:4.0.

[0042] In this invention, in step (11), the molar ratio of compound CHC-6 and compound S-4 is 1:1.5-2.5, preferably 1:1.8.0-2.2; more preferably 1:2.0.

[0043] In step (11), the molar ratio of compound CHC-6 to HBTU is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0.

[0044] In step (11), the molar ratio of compound CHC-6 to HOBT is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0.

[0045] The advantages of using the technical solution of this invention are as follows:

[0046] This invention provides a pH-sensitive fluorescent probe for detecting thionitrite. This probe exhibits good stability and excellent selectivity and sensitivity for HSNO in acidic environments (detection limit of 415 nM), enabling specific detection of HSNO in HCT-116 cells. It has been applied to fluorescence imaging of HSNO in HCT-116 colon cancer xenografts in nude mice. Furthermore, intravenous injection of HSNO into the tail vein of xenograft mice inhibits tumor growth and increases HSNO levels in tumor tissue, indicating that HSNO possesses antitumor activity. This provides an effective detection tool for studying the relationship between HSNO and tumor development. Attached Figure Description

[0047] Figure 1 It is the fluorescent probe CHC-HSNO 1 H NMR spectrum;

[0048] Figure 2 It is the fluorescent probe CHC-HSNO 13 C NMR spectrum;

[0049] Figure 3 This is a schematic diagram illustrating the design concept of the fluorescent probe CHC-HSNO;

[0050] Figure 4 This is a schematic diagram of the mechanism by which the fluorescent probe CHC-HSNO recognizes HSNO;

[0051] Figure 5 It is the HRMS (calculated for C) of the reaction product of the fluorescent probe CHC-HSNO and HSNO. 28 H 32 N3O4[M+H] + ,474.2393; found,474.2401);

[0052] Figure 6 yes Figure 5 A magnified view of a medium-to-high resolution mass spectrum;

[0053] Figure 7 It is compound CHC-6-A 1 H NMR spectrum;

[0054] Figure 8 It is compound CHC-6-B. 1 H NMR spectrum;

[0055] Figure 9 The UV absorption spectra of probe CHC-HSNO before and after incubation with HSNO; UV absorption spectra of probe CHC-HSNO (10 μM), probe CHC-HSNO (10 μM) + HSNO (100 μM), and compound CHC-6 (10 μM) after incubation at 37°C for 30 min in PBS buffer (50 mM, pH = 4.0 or 7.4, 1% DMSO);

[0056] Figure 10 The fluorescence spectra of the probe CHC-HSNO in response to HSNO; the fluorescence spectra of probe CHC-HSNO (10 μM), probe CHC-HSNO (10 μM) + HSNO (100 μM), and compound CHC-6 (10 μM) incubated in PBS buffer (50 mM, pH = 4.0 or 7.4, 1% DMSO) at 37 °C for 30 min; (λ ex =600nm, slit:10nm);

[0057] Figure 11 The fluorescence response intensity changes of probes CHC-HSNO and HSNO (0-80 μM) after incubation in PBS buffer (50 mM, pH = 4.0, 1% DMSO) for 30 min; among which, Figure 11 Fluorescence spectra of probe CHC-HSNO (10 μM) and HSNO (0-80 μM, 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM) after incubation in PBS buffer (50 mM, pH = 4.0, 1% DMSO) at 37 °C for 30 min. Figure 11 The fluorescence intensity changes at 660 nm of the probe CHC-HSNO (10 μM) and different concentrations of HSNO (0-80 μM) in PBS buffer (50 mM, pH=4.0, 1% DMSO) after incubation at 37 °C for 30 min; Figure 11The linear relationship (λ) between the fluorescence intensity of the c-probe CHC-HSNO at 660 nm and the HSNO concentration (0-10 μM) ex =600nm, slit:10nm);

[0058] Figure 12 These are the fluorescence spectra of the probe CHC-HSNO incubated with HSNO for different times; among them, Figure 12 Fluorescence spectra of probe CHC-HSNO (10 μM) and HSNO (100 μM) in PBS buffer (50 mM, pH = 4.0, 1% DMSO) at 37 °C for different times (10, 20, 30, 40, 50, 60, 70, 80, 90 min); Figure 12 The fluorescence intensity changes at 660 nm of the b-probe CHC-HSNO (10 μM) and HSNO (100 μM) at different times (10, 20, 30, 40, 50, 60, 70, 80, 90 min) after incubation at 37 °C in PBS buffer (50 mM, pH = 4.0, 1% DMSO); (λ ex =600nm, slit:10nm);

[0059] Figure 13 These are the fluorescence spectra of the probes CHC-HSNO and HSNO reacting at different pH values; among them, Figure 13 Fluorescence spectra of probe CHC-HSNO (10 μM) and HSNO (100 μM) after incubation at 37 °C for 30 min in PBS buffers at different pH values ​​(50 mM, pH = 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 1% DMSO); Figure 13 The fluorescence intensity changes at 660 nm of the b-probe CHC-HSNO (10 μM) and HSNO (100 μM) at different pH values ​​in PBS buffers (50 mM, pH = 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 1% DMSO) after incubation at 37 °C for 30 min; (λ ex =600nm, slit:10nm);

[0060] Figure 14 It is the selectivity of the probe CHC-HSNO for HSNO; among which, Figure 14Fluorescence spectra of probe CHC-HSNO (10 μM) and amino acids (blank; 1 mM Gly; 1 mM D-Ala; 1 mM Val; 1 mM Ile; 1 mM L-Cys; 1 mM L-Thr; 1 mM MAsp; 1 mM L-Glu; 1 mM L-Arg; 1 mM L-Lys; 1 mM L-His; 1 mM Hcy) or HSNO (100 μM) in PBS buffer (50 mM, pH = 4.0, 1% DMSO) at 37 °C for 30 min. Figure 14 The fluorescence intensity at 660 nm of the probe CHC-HSNO (10 μM) with amino acids and HSNO (100 μM) was measured after incubation in PBS buffer (50 mM, pH 4.0, 1% DMSO) at 37 °C for 30 min. The following are the fluorescence intensities: 1. blank; 2. Gly (1 mM); 3. D-Ala (1 mM); 4. Val (1 mM); 5. Ile (1 mM); 6. L-Cys (1 mM); 7. L-Thr (1 mM); 8. Asp (1 mM); 9. L-Glu (1 mM); 10. L-Arg (1 mM); 11. L-Lys (1 mM); 12. L-His (1 mM); 13. Hcy (1 mM); 14. HSNO (100 μM) (λ) ex =600nm, slit:10nm);

[0061] Figure 15 It is the selectivity of the probe CHC-HSNO for HSNO; among which, Figure 15 The probe CHC-HSNO (10 μM) and inorganic salt ions (blank; 1 mM Sn) 2+ 1mM Cd 2+ ;1mM Mn 2+ 1mM Co 2+ 1mM Cu 2+ 1mM Hg 2+ 1mM Zn 2+ ;1mMFe 2+ 1mM Fe 3+ 1mM Ca 2+ 1mM Al 3+ 1mM Ni 2+ 1mM Mg 2+ 1mM Li + 1mM Na + 1mM K + 1mMAg +Fluorescence spectra of HNSO (100 μM) or HNSO (100 μM) in PBS buffer (50 mM, pH = 4.0, 1% DMSO) incubated at 37 °C for 30 min; Figure 15 The fluorescence intensity at 660 nm of the probe CHC-HSNO (10 μM) with inorganic salt ions and HSNO (100 μM) in PBS buffer (50 mM, pH 4.0, 1% DMSO) at 37 °C for 30 min was measured. 1. blank; 2. Sn 2+ (1mM); 3.Cd 2+ (1mM); 4.Mn 2+ (1mM); 5.Co 2+ (1mM); 6.Cu 2+ (1mM); 7.Hg 2+ (1mM); 8.Zn 2+ (1mM); 9.Fe 2+ (1mM); 10.Fe 3+ (1mM); 11.Ca 2+ (1mM); 12.Al 3+ (1mM); 13.Ni 2+ (1mM); 14.Mg 2+ (1mM); 15.Li + (1mM); 16.Na + (1mM); 17.K + (1mM); 18.Ag + (1mM))19.HNSO(100μM)(λ ex =600nm, slit:10nm);

[0062] Figure 16 It is the selectivity of the probe CHC-HSNO for HSNO; among which, Figure 16 Fluorescence spectra of probe CHC-HSNO (10 μM) and RSS (blank; 100 μM Na2S; 100 μM Na2S+100 μM DEA·NONOate; 100 μM Na2S2; 100 μM Na2S2+100 μM MDEA·NONOate; 500 μM NaHS; 500 μM Na2SO3; 500 μM NaHSO3; 100 μM CH3SSSCH3; 500 μM MysSSCys; 1 mM GSSG, 500 μM S8; 10 mM GSH) or HSNO (100 μM) in PBS buffer (50 mM, pH = 4.0, 1% DMSO) incubated at 37 °C for 30 min. Figure 16The fluorescence intensity at 660 nm of the probe CHC-HSNO (10 μM) was measured after incubating it with RSS and HSNO (100 μM) in PBS buffer (50 mM, pH 4.0, 1% DMSO) at 37 °C for 30 min. 1.blank; 2Na2S (100μM); 3Na2S (100μM) + DEA·NONOate (100μM); 4Na2S2 (100μM); 5Na2S2 (100μM) + DEA·NONOate (100μM); 6NaHS (500μM); 7N a2SO3 (500μM); 8NaHSO3 (500μM); 9CH3SSSCH3 (100μM); 10CysSSCys (500μM); 11GSSG (1mM), 12S8 (500μM); 13GSH (10mM); 14HSNO (100μM) (λ ex =600nm, slit:10nm);

[0063] Figure 17 It is the selectivity of the probe CHC-HSNO for HSNO; among which, Figure 17 The probe CHC-HSNO (10 μM) and ROS (blank; 100 μM H2O2; 100 μM ClO) were used. - 100μM t BuOOH; 100μM·OH; 100μM 1 O2; 100μM O2 ·- 100μM NO3 - 100μM NO2 - 100μM ONOO - Fluorescence spectra of 100 μM HSNO or 100 μM in PBS buffer (50 mM, pH 4.0, 1% DMSO) incubated at 37 °C for 30 min; Figure 17 The fluorescence intensity at 660 nm of the probe CHC-HSNO (10 μM) was measured after incubation with ROS and HSNO (100 μM) in PBS buffer (50 mM, pH 4.0, 1% DMSO) at 37 °C for 30 min. 1. blank; 2. H2O2 (100 μM); 3. ClO - (100μM); 4. t BuOOH(100μM); 5.·OH(100μM); 6. 1 O2 (100μM); 7.O2 ·- (100μM); 8.NO3 - (100μM); 9.NO2 - (100μM); 10000 -(100μM); 11HSNO(100μM)(λ) ex =600nm, slit:10nm);

[0064] Figure 18 This relates to the effect of the probe CHC-HSNO on cell viability; among which, Figure 18 In Figure a, HCT-116 cells were co-incubated with different concentrations of the probe CHC-HSNO (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) for 48 h to improve cell viability. Figure 18 Figure b shows the effect of co-incubation of NCM-460 cells with different concentrations of the probe CHC-HSNO (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) for 48 h on cell viability; data are expressed as mean ± standard deviation (n = 3);

[0065] Figure 19 This relates to the effect of the probe CHC-HSNO on cell viability; among which, Figure 19 In Figure a, HCT-116 cells were co-incubated with the probe CHC-HSNO (20 μM) for different times (0 h, 6 h, 12 h, 24 h, 48 h) to improve cell viability. Figure 19 Figure b shows the effect of co-incubation of NCM-460 cells with the probe CHC-HSNO (20 μM) for different times (0 h, 6 h, 12 h, 24 h, 48 h) on cell viability. Data are expressed as mean ± standard deviation (n = 3).

[0066] Figure 20 It is a fluorescence imaging method for detecting HSNO using the CHC-HSNO probe; among which, Figure 20 In step a, probe CHC-HSNO (10 μM) was co-incubated with HCT-116 cells for 30 min; Figure 20 In step b, the probe CHC-HSNO (10 μM) was first co-incubated with HCT-116 cells for 30 min, and then HSNO (100 μM) was added and co-incubated for 10 min. Figure 20 In step c, the probe CHC-HSNO (10 μM) was first co-incubated with HCT-116 cells for 30 min, and then GSNO (300 μM) was added and co-incubated for 10 min. Figure 20 In the middle, the probe CHC-HSNO (10 μM) was first co-incubated with HCT-116 cells for 30 min, and then Na2S (300 μM) was added and co-incubated for 10 min. Figure 20 The middle part of the text refers to the co-incubation of NCM-460 cells with the probe CHC-HSNO (10 μM) for 30 min. Figure 20In the middle f, the probe CHC-HSNO (10μM) was first co-incubated with NCM-460 cells for 30 min, and then HSNO (100μM) was added and co-incubated for 10 min. Figure 20 g is Figure 20 Mean fluorescence intensity of cells in subcategories a, b, c, d, e, and f; data are expressed as mean ± SD (n = 3);

[0067] Figure 21 yes Figure 20 The corresponding bright-field image of the cells;

[0068] Figure 22 It is a fluorescence imaging method for detecting HSNO using the CHC-HSNO probe; among which, Figure 22 In step a, probe CHC-HSNO (10 μM) was co-incubated with HCT-116 cells for 30 min; Figure 22 b is the co-incubation of HCT-116 cells transfected with CBS-siRNA and probe CHC-HSNO (10μM) for 30 min; Figure 22 In step c, HCT-116 cells were first co-incubated with AOAA (20 μM) for 45 min, and then co-incubated with the probe CHC-HSNO (10 μM) for 30 min. Figure 22 In the middle section, d represents the co-incubation of NCM-460 cells with the probe CHC-HSNO (10 μM) for 30 min. Figure 22 The middle e is Figure 22 Mean fluorescence intensity of cells in cells a, b, c, and d; data are expressed as mean ± SD (n = 3);

[0069] Figure 23 yes Figure 22 The corresponding bright-field image of the cells;

[0070] Figure 24 The data represent the expression levels of CBS protein in HCT-116 cells and HCT-116 cells transfected with CBS siRNA; data are expressed as mean ± SD (n = 3).

[0071] Figure 25 The probe CHC-HSNO was used to detect HSNO in colon cancer xenografts in nude mice. Control group mice were injected intratumorally with probe CHC-HSNO (500 μM, 10 μL, saline: DMSO = 100:1 v / v); HSNO group mice were injected intratumorally with probe CHC-HSNO (500 μM, 10 μL, saline: DMSO = 100:1 v / v), followed by intratumoral injection of exogenous HSNO (100 μM) in PBS solution (50 mM, pH = 7.4).

[0072] Figure 26 It is a quantitative expression Figure 27Fluorescence signal intensity in two groups of mice; data are expressed as mean ± standard deviation (n=3);

[0073] Figure 27 This involves research on the antitumor activity of HSNO; among which, Figure 27 In the middle section, a represents the weight monitoring of a nude mouse xenograft model during a 15-day treatment period; Figure 28 In the middle b, tumor volume monitoring was performed on a nude mouse xenograft model during a 15-day treatment cycle. Figure 29 In the middle, c represents the size of the tumor tissue in the nude mouse xenograft model after 15 days of treatment;

[0074] Figure 29 HE staining of tumor tissue from a nude mouse xenograft model after 15 days of treatment;

[0075] Figure 29 Fluorescence imaging of tumor tissue in a nude mouse xenograft model after 15 days of treatment; among them, Figure 1 In the control group (a), 100 μL of normal saline was administered via tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. Tumor tissue was then removed, frozen sections were prepared, and the tissue was incubated with the probe CHC-HSNO (100 μM, 10 μL, normal saline: DMSO = 100:1 v / v) for 30 min before fluorescence imaging. In the HSNO group, 1.6 mg / kg of HSNO (Saline: DMSO = 99:1) was administered via tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. Tumor tissue was then removed, frozen sections were prepared, and the tissue was incubated with the probe CHC-HSNO (100 μM, 10 μL, normal saline: DMSO = 100:1 v / v) for 30 min before fluorescence imaging. Figure 2 b represents the fluorescence intensity of tumor tissues in the control group and the HSNO group; excitation wavelength is 638 nm, and emission wavelength is 650 nm-800 nm; data are expressed as mean ± standard deviation (n = 3). Detailed Implementation

[0076] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0077] I. Implementation Methods

[0078] 1. Materials

[0079] 1.1 Solution Preparation

[0080] (1) Preparation of the fluorescent probe CHC-HSNO solution: Dissolve the probe CHC-HSNO (8.49 mg, 0.01 mmol) in DMSO (1 mL) to obtain a 10.0 mM probe solution, and store it in a refrigerator at -20℃ protected from light.

[0081] (2) Preparation of S-nitrosoglutathione (GSNO) stock solution: Weigh GSNO (11.37 mg, 0.034 mmol), dissolve it in PBS buffer solution (50 mM, pH = 7.4, 33.8 mL) to prepare a 1 mM stock solution, and store it at -20℃.

[0082] (3) Preparation of sodium sulfide (Na2S) stock solution: Weigh 3.21 mg (0.041 mmol) of Na2S and dissolve it in PBS buffer solution (50 mM, pH = 7.4, 13.7 mL) to prepare a 3.0 mM stock solution. Then, dilute the stock solution to a 300 μM solution for later use.

[0083] (4) Preparation of thionitrous acid (HSNO) stock solution: Under light-protected, room temperature, and nitrogen protection conditions, freshly prepared 1.0 mM HSNO solution and 0.3 mM Na2S solution were placed in PBS buffer (50 mM, pH = 7.4) to obtain a 300 μM HSNO stock solution. Subsequently, the stock solution was diluted to a 100 μM solution and used immediately.

[0084] (5) Preparation of glutathione (GSH) stock solution: Add GSH (15.36 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then, dilute the stock solution to 1.0 mM and 10.0 μM solutions for later use.

[0085] (6) Preparation of L-cysteine ​​(L-Cys) stock solution: Add Cys (6.05 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then, dilute the stock solution to 1.0 mM and 10.0 μM solutions for later use.

[0086] (7) Preparation of DEA·NONOate stock solution (as a source of NO): DEA·NONOate (15.5 mg, 0.1 mmol) was added to 10 mL of 0.01 M sodium hydroxide solution to prepare a 10.0 mM stock solution. The stock solution was then diluted to 1.0 mM and 10.0 μM solutions for later use.

[0087] (8) Preparation of H2O2 stock solution: Add 1 mL of H2O2 (30%) to deionized water (9.0 mL), and determine the concentration of the H2O2 stock solution by measuring the absorbance at 240 nm. The molar extinction coefficient is 43.6 M. -1 cm -1 The calculation formula is c = A / (bε).

[0088] (9)ClO - Preparation of the stock solution: Add NaClO (10%) to deionized water, and then measure the absorbance at 209 nm. The molar extinction coefficient is 350 M. -1 cm -1 Determination of ClO - The concentration of the stock solution is calculated using the formula c = A / (bε).

[0089] (10) t Preparation of BuOOH stock solution: commercial t BuOOH (5mM).

[0090] (11) · Preparation of OH solution: Add ferrous sulfate (15.20 mg, 0.10 mmol) to 10 mL of hydrogen peroxide solution and mix to prepare a 10 mM solution. · OH stock solution.

[0091] (12) 1 Preparation of O2 stock solution: Methylene blue (31.98 mg, 0.10 mmol) was irradiated with an LED lamp, and 5 mL of NaClO4 (1 mM) solution and 5 mL of H2O2 (30%) solution were added to prepare a 10 mM stock solution, which was prepared and used immediately.

[0092] (13)O2 ·- Preparation of stock solution: Under nitrogen protection, add KO2 (7.10 mg, 0.10 mmol) to anhydrous DMSO (10 mL) to prepare a 10 mM stock solution, which should be prepared and used immediately.

[0093] (14) NO3 - Preparation of stock solution: Add NaNO3 (8.50 mg, 0.1 mmol) to deionized water (10 mL) to prepare a 10 mM NO3 solution. - Stock solution.

[0094] (15) NO2 - Preparation of stock solution: Add NaNO2 (6.90 mg, 0.1 mmol) to deionized water (10 mL) to prepare a 10 mM NO2 solution. - Stock solution.

[0095] (16)ONOO - Preparation of the stock solution: Add NaNO₂ solution (0.6 M, 10 mL) and H₂O₂ (0.7 M, 10 mL) to deionized water and stir vigorously. Add HCl (0.6 M, 10 mL) at 0 °C, then quickly add NaOH solution (1.5 M, 20 mL). Determine the ONOO₂ concentration by UV analysis. - The concentration of [concentration] has a molar extinction coefficient of 1670 M at 302 nm. -1 cm -1 The calculation formula is c = A / (bε).

[0096] In addition to the substances mentioned above, other stock solutions may be prepared using amino acids (Gly, D-Ala, Val, Ile, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy); inorganic salts (Sn... 2+ Cd 2+ Mn 2+ Co 2+ Cu 2+ Hg 2+ Zn 2+ Fe 2+ Fe 3 + Ca 2+ Al 3+ Ni 2+ Mg 2+ Li + Na + K + Ag + Active sulfur (Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8) were prepared by directly dissolving them in deionized water.

[0097] All the above solutions should be prepared and used immediately.

[0098] 1.2 Cells

[0099] Species and strains: HCT-116 (human colon cancer cell line), NCM-460 (human colon epithelial cell line). Source: Zhejiang Nuobo Biotechnology Co., Ltd.

[0100] 1.3 Animals

[0101] Species: Healthy male BALB / C-Nude nude mice, weighing 20-25g. Source: Changzhou Cavens Laboratory Animal Co., Ltd.

[0102] 2. Materials

[0103] 2.1 Synthetic route of probe CHC-HSNO

[0104] Compound CHC-1 was obtained by condensation reaction of p-carboxyphenylhydrazine with methyl isopropyl ketone. Compound CHC-1 was then substituted with N-tert-butoxycarbonyl-bromoethylamine to yield compound CHC-2. Compound CHC-3 was obtained by condensation reaction of 4-(diethylamino)salicylaldehyde with diethyl malonate. Compound CHC-3 was converted to compound CHC-4 via a Vilsmeier-Haack reaction. Compound CHC-5 was synthesized by condensation reaction of compound CHC-2 and compound CHC-4. Compound CHC-5 was deprotected with trifluoroacetic acid and then hydrolyzed to synthesize compound CHC-6.

[0105] Using thiosalicylic acid as a starting material, compound S-1 was first synthesized via a substitution reaction with 2,2'-dithiodipyridine. Compound S-1 was then esterified with p-hydroxybenzaldehyde to yield compound S-2. Compound S-2 was reduced with sodium borohydride to convert its aldehyde group to a hydroxyl group, yielding compound S-3. Compound S-3 was then condensed with o-phenylenediamine via triphosgene to form compound S-4.

[0106] Compound CHC-6 and compound S-4 undergo an amide condensation reaction via HOBT and HBTU, followed by reduction of the disulfide bond via triphenylphosphine to obtain the probe CHC-HSNO.

[0107] Synthesis of compound CHC-1: p-Carboxyphenylhydrazine (15.22 g, 100 mmol) and methyl isopropyl ketone (12.93 g, 150 mmol) were dissolved in 250 mL of anhydrous ethanol, and 2.5 mL of concentrated sulfuric acid was added dropwise. The resulting mixture was heated under reflux for 18 h. After the reaction was complete, the reaction solution was cooled to 20-30 °C, and 500 mL of distilled water was added. The precipitate was obtained by filtration, washed with distilled water, dried, and a yellow compound was obtained. The compound was purified by column chromatography (silica, DCM:MeOH, 20:1 v / v) to give a yellow solid compound CHC-1 in 56.1% yield. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.2.

[0108] Compound CHC-1: 1 H NMR (400MHz, Chloroform-d) δ8.17(dd,J=8.0,1.6Hz,1H),8.07(d,J=2.0Hz,1H),7.68(d,J=8.0Hz,1H),2.39(s,3H),1.38(s,6H).

[0109] Synthesis of compound CHC-2: Compound CHC-1 (203.00 mg, 1 mmol) and N-tert-butoxycarbonyl-bromoethylamine (448.00 mg, 2 mmol) were dissolved in 20 mL of acetonitrile and reacted under nitrogen protection by reflux for 16 h. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a yellow oil, which was purified by column chromatography (silica, DCM:MeOH, 200:1 v / v) to give the yellow oil compound CHC-2, with a yield of 53.1%. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.8.

[0110] Compound CHC-2: 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J=8.0Hz,1H),7.98(s,1H),7.56(d,J=8.0Hz,1H ), 4.40 (t, J = 4.0Hz, 2H), 3.56 (d, J = 8.0Hz, 2H), 2.33 (s, 3H), 1.44 (s, 9H), 1.34 (s, 6H).

[0111] Synthesis of compound CHC-3: 4-(diethylamino)salicylaldehyde (10.60 g, 55 mmol) and diethyl malonate (17.60 g, 110 mmol) were dissolved in 100 mL of anhydrous ethanol. Piperidine (2 mL, 20 mmol) was added, and the mixture was heated under reflux for 3 h. After the reaction was complete, the solvent was removed by vacuum distillation. The reactants were then dissolved in a mixed solution of 50 mL glacial acetic acid and 50 mL concentrated hydrochloric acid, and heated under reflux at 130 °C for 3 h. After the reaction was complete, the reaction solution was poured into 200 mL of ice water, and the pH was adjusted to neutral with 40% sodium hydroxide solution. A large amount of solid precipitated out. The solid was filtered, and the filter cake was washed with distilled water and dried to obtain a yellow compound. After purification by column chromatography (silica, DCM:MeOH, 100:1 v / v), the yellow solid compound CHC-3 was obtained, with a yield of 85.4%. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.6.

[0112] Compound CHC-3: 1H NMR(400MHz,Chloroform-d3)δ7.54(d,J=12.0Hz,1H),7.25(d,J=16.0Hz,1H),6.57(dd,J=8.0Hz,12.0Hz ,1H),6.50(d,J=8.0Hz,1H),6.04(d,J=9.2Hz,1H),3.41(q,J=8.0Hz,12.0Hz,4H),1.21(t,J=8.0Hz,6H).

[0113] Synthesis of compound CHC-4: 10 mL of phosphorus trichloride and 10 mL of DMF were mixed and heated at 50 °C for 30 min under nitrogen protection. Compound CHC-3 (4.40 g, 20 mmol) was dissolved in 20 mL of DMF and added dropwise to the mixture under nitrogen protection. The mixture was stirred at 50 °C for 5 h. After the reaction was complete, the reaction solution was poured into 100 mL of ice water, and the pH was adjusted to neutral with 40% sodium hydroxide solution. A large amount of solid precipitated out. The solid was filtered, washed with distilled water, and dried to obtain a yellow compound. Purification by column chromatography (silica, DCM:MeOH, 100:1 v / v) yielded a yellow solid compound CHC-4, with a yield of 67.3%. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.5.

[0114] Compound CHC-4: 1 H NMR(400MHz,Chloroform-d3)δ10.13(s,1H),8.31(s,1H),7.46-7.41(m,1H),6.66(d,J =8.0Hz,1H),6.51(s,1H),3.48(q,J=8.0Hz,16.0Hz,4H),1.26(t,J=8.0Hz,4.0Hz,6H).

[0115] Synthesis of compound CHC-5: Compounds CHC-2 (694.00 mg, 2 mmol) and CHC-4 (217.00 mg, 1 mmol) were dissolved in 20 mL of anhydrous ethanol and reacted under nitrogen protection by reflux for 18 h. After the reaction was completed, the solvent was removed by vacuum distillation to obtain an orange oily compound. This oily compound CHC-5 was purified by column chromatography (silica, DCM:MeOH, 100:1 v / v) in a yield of 40.9%. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.7.

[0116] Compound CHC-5:1 H NMR(400MHz,Chloroform-d)δ8.11(d,J=12.0Hz,2H),8.03(s,1H),7.87(s,1H),7. 71(d,J=8.0Hz,1H),7.64(d,J=16.0Hz,1H),7.37(d,J=12.0Hz,1H),6.65(d,J=8.0 Hz,1H),6.54(d,J=2.4Hz,1H),4.42(t,J=8.0Hz,4.0Hz,2H),3.58(d,J=5.6Hz,2H) ,3.49(q,J=8.0Hz,12.0Hz,4H),1.52(s,6H),1.467(s,9H),1.27(t,J=8.0Hz,6H).

[0117] Synthesis of compound CHC-6: Compound CHC-5 (574.00 mg, 1 mmol) was dissolved in 6 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added. The mixture was stirred at 20-30 °C for 6 h. After the reaction was complete, the resulting reaction solution was poured into 50 mL of 0.01 M sodium bicarbonate solution and extracted with ethyl acetate (100 mL × 3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain an orange solid compound. After purification by column chromatography (silica, DCM:MeOH, 200:1 v / v), the orange solid compound CHC-6 was obtained, with a yield of 66.6%. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.5.

[0118] Compound CHC-6: 1 H NMR (400MHz, Methanol-d4) δ8.06(d,J=8.0Hz,1H),7.99(s,1H),7.782(s,1H),7.78-7.72(m,2H),7.59-7.55(m,2H),7.33(d,J=8.0Hz,1 H),6.61(d,J=8.0Hz,1H),6.50(s,1H),4.48(m,2H),3.45(q,J=12.0,4.0Hz,4H),3.27(m,2H),1.44(s,6H),1.24(t,J=8.0Hz,4.0Hz,6H).

[0119] Compound CHC-6: 13C NMR: 400MHz, CDCl3) δ 187.09, 168.77, 160.71, 156.86, 156.31, 151.56, 146.88, 143.30, 133.81, 130.92, 130. 23,126.41,120.65,119.77,115.79,109.51,109.03,97.00,62.36,53.01,50.76,44.59,42.28,23.45,12.49.

[0120] Synthesis of compound S-1: Thiosylsalicylic acid (1.54 g, 10 mmol) was dissolved in 30 mL of chloroform, and 2,2'-dithiodipyridine (6.6 g, 30 mmol) was added. The mixture was stirred at 20-30 °C for 12 h. After the reaction was complete, the mixture was filtered, and the filter cake was washed with dichloromethane to obtain a white solid, compound S-1. No purification was required, and the mixture was directly proceeded to the next step with a yield of 81.0%. TLC (silica, DCM:MeOH, 10:1 v / v): R f =0.2.

[0121] Compound S-1: 1 H NMR (400MHz, DMSO-d6) δ8.44 (s, 1H), 7.99 (d, J = 8.0Hz, 1H), 7.77-7.71 (m, 2H), 7.5 8(d,J=8.0Hz,1H),7.45(d,J=8.0Hz,1H),7.32(t,J=8.0Hz,1H),7.23-7.20(m,1H).

[0122] Synthesis of compound S-2: Compound S-1 (263 mg, 1 mmol) was dissolved in 10 mL of dichloromethane, and EDCI (287 mg, 1.5 mmol) and DMAP (24.4 mg, 0.2 mmol) were added, followed by p-hydroxybenzaldehyde (122 mg, 1 mmol). The mixture was stirred at 20-30 °C for 6 h. After the reaction was complete, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with saturated brine (10 mL × 3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a white solid compound. After purification by column chromatography (silica, DCM:EtOAc, 200:1 v / v), white solid compound S-2 was obtained, with a yield of 52.9%. TLC (silica, DCM:EtOAc, 20:1 v / v), R f =0.7.

[0123] Compound S-2: 1H NMR (400MHz, DMSO-d6) δ10.01(s,1H),8.45(d,J=8.0Hz,1H),8.30(d,J=8.0Hz,1H),8.03(d,J=8.0Hz,2H),7.86(d,J=8.0 Hz,1H),7.77-7.69(m,2H),7.58(d,J=8.0Hz,2H),7.52(d,J=8.0Hz,1H),7.45(t,J=8.0Hz,4.0Hz,1H),7.23-7.26(m,1H).

[0124] Synthesis of compound S-3: Compound S-2 (367.00 mg, 1 mmol) was dissolved in 20 mL of anhydrous THF to obtain a mixed solution. Sodium borohydride (42 mg, 1.1 mmol) was dissolved in 10 mL of anhydrous ethanol and slowly added dropwise to the mixed solution at 0 °C. After the addition was complete, the reaction was continued at 0 °C for 1 h. After the reaction was complete, 100 mL of ethyl acetate was added to the reaction solution and washed with saturated ammonium chloride solution (100 mL × 3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a yellow oily compound. After purification by column chromatography (silica, DCM:EtOAc, 200:1 v / v), a white solid compound S-3 was obtained, with a yield of 14.6%. f =0.3.

[0125] Compound S-3: 1 H NMR (400MHz, DMSO-d6) δ8.49(d,J=4.0Hz,1H),8.33(d,J=8.0Hz,1H),8.00(d,J=8.0Hz,1H),7.623-7.55(m,3H ),7.48(d,J=8.0Hz,1H),7.36(t,J=8.0Hz,4.0Hz,1H),7.28(d,J=4.0Hz,2H),7.11-7.15(m,1H),4.76(s,2H).

[0126] Synthesis of compound S-4: K₂CO₃ (552.82 mg, 4 mmol) and triphosgene (296.75 mg, 1 mmol) were dissolved in 10 mL of anhydrous THF and reacted at 0 °C for 1 h under nitrogen protection. Then, an anhydrous THF solution (5 mL) of compound S-3 (369.04 mg, 1 mmol) was added dropwise, and the reaction was carried out at 30 °C in the dark for 12 h. After the reaction was complete, the mixture was filtered, and the filtrate was collected. The organic phase was concentrated under reduced pressure to obtain a white solid.

[0127] o-Phenylenediamine (108.06 mg, 1 mmol) and pyridine (0.32 mL, 4 mmol) were dissolved in 10 mL of anhydrous dichloromethane and reacted at 30 °C for 30 min under nitrogen protection. Then, a solution of the above white solid in anhydrous dichloromethane (5 mL) was added dropwise to the above mixture, and the reaction was carried out at 20-30 °C for 12 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure to give a pale yellow oily compound, which was purified by column chromatography (silica, DCM:EtOAc, 200:1 v / v) to give a white solid compound S-4, with a yield of 29.0%. TLC (silica, DCM:EtOAc, 20:1 v / v), R f =0.5.

[0128] Compound S-4: 1 H NMR (400MHz, DMSO-d6) δ8.49(d,J=4.0Hz,1H),8.30(d,J=4.0Hz,1H),8.00(d,J=8.0Hz,1H),7.59-7.49(m,5H), 7.38-7.27(m,4H),7.12(t,J=8.0Hz,4.0Hz,1H),7.05(t,J=8.0Hz,4.0Hz,1H),6.84-6.79(m,2H),5.23(s,2H).

[0129] Compound S-4: 13 C NMR(400MHz,Chloroform-d3)δ192.73,165.11,158.88,157.07,149.91,149.28,141.18,138.79,137.92,137.72, 134.00,132.15,130.31,128.34,126.25,126.02,125.94,122.88,122.31,121.84,121.34,120.13,115.09,65.34.

[0130] Synthesis of compound CHC-HSNO: Compounds CHC-6 (240.50 mg, 0.5 mmol), S-4 (503.09 mg, 1.0 mmol), HBTU (189.48 mg, 0.5 mmol), and HOBT (67.58 mg, 0.5 mmol) were dissolved in 10 mL of anhydrous DMF and reacted at 20–30 °C for 5 min under nitrogen protection. DIPEA (87.03 μL, 0.5 mmol) was added to the mixture, and the reaction was continued at 20–30 °C for 12 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the solution was then dissolved in THF / H₂O (10 mL / 5 mL). PPh₃ (131.16 mg, 0.5 mmol) was added at 0 °C, followed by one drop of 1 M HCl solution, and the reaction was continued at 0 °C for 1 h. After the reaction was complete, the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to give a yellow solid compound. This solid compound was purified by column chromatography (silica, DCM:MeOH, 200:1 v / v) to give a yellow solid compound CHC-HSNO in 67.3% yield. TLC (silica, DCM:MeOH, 20:1 v / v), R f =0.7.

[0131] Compound CHC-HSNO: 1H NMR (400MHz, Methanol-d4) δ 8.42 (d, J = 2.8 Hz, 1H), 8.31 (d, J = 6.4 Hz, 1H), 8.14–8.10 (m, 3H), 7.94 (d, J = 8.0 Hz, 1H), 7.78–7.71 (m, 2H), 7.63–7.54 (m, 4H), 7.49–7.45 (m, 3H), 7.41 (t, J = 8.0 Hz, 1H), 7.28–7. .22(m,3H), 6.76(dd,J=9.2,6.8Hz,1H), 6.53(d,J=2.4Hz,1H), 4.67(s,2H), 4.46(t,J=5.6Hz,5.2Hz,2H), 3.51(q,J=6.8Hz,14Hz,4H), 3.21(t,J=5.6Hz,5.2Hz,2H), 1.48(s,6H), 1.23(t,J=7.2Hz,6H). (Specific details are as follows...) Figure 3 As shown.

[0132] Compound CHC-HSNO: 13C NMR(400MHz,Methanol-d4)δ188.21,166.45,164.80,161.11,158.21,157.32,156.43,152.1 2,149.80,149.20,146.48,144.31,140.48,139.56,137.94,135.91,133.62,131.78,130.22 ,127.79,126.60,126.41,125.99,125.47,122.71,121.40,121.34,119.89,119.77,119.05,118.47,114.30,109.83,108.92,96.13,63.93,63.18,52.68,44.54,38.91,22.55,10.72. (Specific details are as follows...) Figure 4 As shown.

[0133] 2.2 Mechanism verification of CHC-HSNO probe recognizing HSNO under acidic conditions

[0134] Under light-protected conditions, the probe CHC-HSNO (50 μM) and HSNO (300 μM) were incubated in PBS buffer (pH = 7.4, 50 mM, 1 mL) at 37 °C for 30 min. Extraction was performed with ethyl acetate (1 mL × 3), and the organic phase was concentrated by vacuum distillation. After separation, the reaction product was confirmed by high-resolution mass spectrometry to verify the mechanism by which the probe CHC-HSNO recognizes HSNO.

[0135] The above product was dissolved in 1 mL of methanol, and 1 M HCl (1 mL) was added. The mixture was stirred at room temperature in the dark for 10 min. After the reaction, methanol was removed by vacuum distillation. The remaining reaction solution was extracted with ethyl acetate (2 mL × 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the acidified product. The structures of the products before and after acidification were confirmed by 1H NMR spectroscopy to verify the mechanism by which the probe CHC-HSNO recognizes HSNO under acidic conditions.

[0136] 2.3 Fluorescence Spectroscopy Determination of the Response of Probe CHC-HSNO to HSNO

[0137] Under light-protected conditions, the probe CHC-HSNO was dissolved in DMSO, and a mixture of freshly prepared 1 mM nitrosoglutathione (GSNO) and 0.3 mM Na2S was dissolved in PBS buffer (pH = 4.0 or 7.4, 50 mM) as the source of HSNO (300 μM) (hereinafter referred to as "HSNO solution"). Subsequently, the probe CHC-HSNO (10 μM) and the HSNO (300 μM) solution were mixed and co-incubated at 37°C for a period of time. The sample was then poured into a quartz cuvette, and its fluorescence intensity was measured using a fluorescence spectrophotometer. PBS buffer (pH = 4.0 or 7.4, 50 mM) was used as a blank control during the experiment. Each set of data was measured in at least three parallel measurements, and the final results are expressed as mean ± SD.

[0138] The detection conditions for the fluorescence spectrophotometer were as follows: for the reaction between the probe CHC-HSNO and HSNO, the excitation wavelength was set to 600 nm, the excitation slit width and the emission slit width were both set to 10 nm, the instrument scanning speed was set to 1200 nm / min, and the emission spectral range was set to 620-800 nm. The voltage of the photomultiplier tube was set to 650 V.

[0139] 2.4 Determination of the detection limit of HSNO by probe CHC-HSNO

[0140] The probe CHC-HSNO (10 μM) was incubated with different concentrations of HSNO solution (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 80 μM) in PBS buffer (pH = 4.0, 50 mM, 1% DMSO) for 30 min, and then the fluorescence intensity was measured. The linear equation between fluorescence intensity and HSNO concentration was calculated. The fluorescence intensity of the probe CHC-HSNO in HSNO-free PBS buffer (pH = 4.0, 50 mM, 1% DMSO) was measured 10 times, and the standard deviation of the 10 detection results was calculated. The limit of detection (LOD) was calculated as: LOD = 3σ / k, where σ is the standard deviation of the blank sample, and k is the slope of the linear equation between fluorescence intensity and HSNO concentration.

[0141] 2.5 Cell Culture Methods

[0142] HCT-116 cell culture: McCoy's 5A medium containing 10% fetal bovine serum and 100 μg / mL penicillin and streptomycin was used. Cells were cultured in a cell culture incubator at 37°C and 5% CO2. Cell passages were performed every two days, and cells were transplanted into new culture dishes at a ratio of 1:3.

[0143] NCM-460 cell culture: RPMI-1640 medium containing 10% fetal bovine serum and 100 μg / mL penicillin and streptomycin was used. Cells were cultured in a cell culture incubator at 37°C and 5% CO2. Cell passages were performed every four days, with cells transferred to new culture dishes at a 1:3 ratio.

[0144] 2.6 Cytotoxicity Detection

[0145] The cytotoxicity of the probe CHC-HSNO against HCT-116 cells was detected using the CCK8 assay. HCT-116 cells were seeded at a density of 10,000 cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. After cell adhesion and proliferation, the old culture medium was discarded, and 0, 1, 5, 10, and 20 μM of probe CHC-HSNO and culture medium mixture were added to each well, followed by 24 h of further culture. After culturing, the culture medium in the wells was aspirated, and 100 μL of CCK-8 solution (McCOY's 5A:CCK-8 = 9:1) was added to each well. The plates were then transferred to a 37°C, 5% CO2 incubator and cultured in the dark for 1 h. The OD values ​​at 450 nm in the 96-well plates were then measured using a microplate reader. The process was repeated after incubating HCT-116 cells with probe CHC-HSNO (20 μM) for different times (0, 6, 12, 24, and 48 h).

[0146] The method for detecting the cytotoxicity of the probe CHC-HSNO against NCM-460 cells is the same as above.

[0147] Cell viability calculation formula: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0148] As: Absorbance of the experimental wells (containing probes, CCK8, and cell culture medium)

[0149] Ac: Absorbance of control wells (containing CCK8 and cell culture medium, but without probe)

[0150] Ab: Blank wells (containing no cells, only the absorbance of CCK8 and cell culture medium)

[0151] 2.7 Cell-level fluorescence imaging

[0152] Preparation of cell suspension: Collect cells from T25 cell culture flasks and dilute an appropriate amount of cells to a suitable concentration. Pour the prepared cell suspension into a solution at approximately 1 × 10⁻⁶ cells / mL. 5 The cells were seeded into a confocal microscope dish and cultured for a further period of time.

[0153] 2.7.1 Fluorescence imaging of exogenous HSNO in cells

[0154] The cells were divided into the following 6 groups: (1) HCT-116 cells were co-incubated with probe CHC-HSNO (10 μM) for 30 min. (2) HCT-116 cells were first co-incubated with probe CHC-HSNO (10 μM) for 30 min, and then HSNO (100 μM) was added and co-incubated for 30 min. (3) HCT-116 cells were first co-incubated with probe CHC-HSNO (10 μM), and then GSNO (300 μM) was added and co-incubated for 30 min. (4) HCT-116 cells were first co-incubated with probe CHC-HSNO (10 μM) for 30 min, and then Na2S (100 μM) was added and co-incubated for 30 min. (5) NCM460 cells were co-incubated with probe CHC-HSNO (10 μM) for 30 min. (6) NCM460 cells were first co-incubated with probe CHC-HSNO (10 μM) for 30 min, and then co-incubated with HSNO (100 μM) for 30 min. Before imaging, the confocal dish was rinsed three times with PBS buffer.

[0155] 2.7.2 Fluorescence imaging of endogenous HSNO in cells

[0156] The cells were divided into the following 4 groups: (1) HCT-116 cells were co-incubated with probe CHC-HSNO (10 μM) for 30 min. (2) HCT-116 cells transfected with CBS siRNA were co-incubated with probe CHC-HSNO (10 μM) for 30 min. (3) HCT-116 cells were first co-incubated with AOAA (20 μM) for 45 min, and then co-incubated with probe CHC-HSNO (10 μM) for 30 min. (4) NCM460 cells were co-incubated with probe CHC-HSNO (10 μM) for 30 min.

[0157] This experiment used a Leica STELLARIS 5 laser confocal microscope (63× oil immersion) for imaging. The excitation wavelength was 638 nm, and the emission wavelength range was 650 nm–800 nm. Analysis was performed using Leica software. All data are expressed as mean ± SD (n = 3).

[0158] 2.8 Animal husbandry

[0159] Healthy male Balb / C nude mice, weighing 20-25g, were used and provided by Changzhou Cavens Laboratory Animal Co., Ltd. The animal experimental protocol was approved by the Animal Protection and Use Committee of Xuzhou Medical University and complies with relevant Chinese laws. The nude mice were allowed one week to acclimatize to the environment before the experiment began. They were housed in separate cages under natural diurnal light conditions, with the ambient temperature maintained at 22±2℃ and humidity at 50±10%, and free access to food and water.

[0160] 2.8.1 Construction of HCT-116 subcutaneous xenograft model of colon cancer in nude mice

[0161] HCT-116 cells were cultured in T75 flasks until they reached the logarithmic growth phase, then digested with trypsin and transferred to serum-free McCoy's 5A medium, resulting in a cell suspension with a concentration of approximately 4 × 10⁻⁶. 7 Cells / mL. Next, select 4-6 week old, 20-25g Balb / C nude mice, and administer approximately 4 × 10⁶ cells / mL. 6 One cell (100 μL cell suspension) was inoculated subcutaneously in the middle and posterior part of the left upper limb axilla of nude mice.

[0162] 2.9 Fluorescence imaging of a nude mouse xenograft model

[0163] Select tumors with a volume exceeding 200 mm 3 Nude mice in good condition were randomly divided into two groups of three each. The groups were the saline group and the HSNO group. After anesthetizing each group of mice with isoflurane gas, (1) control group: mice were injected intratumorally with probe CHC-HSNO (500μM, 10μL, saline: dimethyl sulfoxide = 100:1v / v); (2) HSNO group: mice were injected intratumorally with probe CHC-HSNO (500μM, 10μL, saline: dimethyl sulfoxide = 100:1v / v), and then intratumorally with exogenous HSNO (100μM, 100μL) in PBS solution (50mM, pH = 7.4).

[0164] Imaging was performed using the IVIS Lumina S5 small animal live imaging system. Imaging conditions: excitation wavelength 600nm, emission wavelength 670nm. Image and data analysis were performed using Living Image software.

[0165] 2.10 Study on the antitumor activity of HSNO

[0166] Select tumors with a volume exceeding 200 mm 3 Nude mice in good condition were randomly divided into two groups of six each: a saline group and an HSNO group. (1) Saline group: 100 μL of saline was injected via the tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. (2) HSNO group: HSNO solution (1.6 mg / kg, Saline:DMSO = 99:1) was injected via the tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. Tumor volume was recorded every two days for a period of 15 days. Changes in body weight and tumor volume of each group of nude mice were recorded over the 15 days.

[0167] 2.10.1 HE staining

[0168] (1) Preparation of tissue sections: First, the tumor tissue specimen is fixed, dehydrated, cleared, impregnated with paraffin and embedded, and then cut into thin sections.

[0169] (2) Dewaxing and hydration: The slices are placed in organic solvents such as xylene to remove paraffin and then hydrated with alcohol of various concentrations.

[0170] (3) Staining:

[0171] Hematoxylin staining: Immerse the slide in hematoxylin staining solution to stain the cell nuclei.

[0172] Hydrochloric acid-ethanol differentiation: removes excess hematoxylin dye, making the cell nucleus staining clearer.

[0173] Ammonia turns blue: It makes the cell nucleus appear purple-blue.

[0174] Eosin staining: Immerse the slide in eosin staining solution to stain the cytoplasm and intercellular matrix.

[0175] (4) Dehydration and clearing: The stained sections are dehydrated by a series of alcohol concentrations and cleared with clearing agents such as xylene.

[0176] (5) Mounting: Add a neutral resin or other sealing agent to the slide and cover it with a coverslip for mounting.

[0177] 2.10.2 Frozen sections

[0178] (1) Tissue fixation: Fresh tumor tissue was fixed in 4% paraformaldehyde for 24 hours. The tissue was removed from the fixative and the target area was trimmed with a scalpel in a fume hood.

[0179] (2) Dehydration: The trimmed tissue was placed in a 15% sucrose solution and dehydrated at 4°C in a refrigerator until it settled. Then it was transferred to a 30% sucrose solution and dehydrated at 4°C in a refrigerator until it settled.

[0180] (3) OCT embedding: Take out the dehydrated tissue, use filter paper to slightly dry the surface water, and place it on the embedding stage with the cut side facing up. Drip OCT embedding agent around the tissue, place the embedding stage on the quick-freezing stage of the cryostat for quick-freezing and embedding. After the OCT turns white and hard, it can be sectioned.

[0181] (4) Sectioning: Fix the freezing stage on the microtome, first make a rough cut to smooth the tissue surface, and then start slicing. The section thickness is 10μm. Place a clean glass slide on top of the cut tissue section and then attach the tissue to the glass slide. Store at -20℃ for later use.

[0182] 2.10.3 Tumor tissue fluorescence imaging

[0183] (1) Incubation: Frozen sections of tumor were incubated with probe CHC-HSNO (100 μM, 10 μL, physiological saline: DMSO = 100: 1 v / v) for 30 min.

[0184] (2) Washing: Wash three times with PBS for 3 minutes each time.

[0185] (3) Imaging: The tissue sections were imaged using a confocal microscope.

[0186] Images were captured using a Leica STELLARIS 5 laser confocal microscope. The excitation wavelength was 638 nm, and the emission wavelength range was 650 nm–800 nm. Analysis was performed using Leica software. All data are expressed as mean ± SD (n = 3).

[0187] 2.11 Data Processing

[0188] Each experimental data set was measured at least three times. Final results were expressed as mean ± standard deviation (Mean ± SD) and statistically analyzed using SPSS 16.0 software. One-way ANOVA with a completely randomized design was used to compare differences among multiple groups. P < 0.05 was considered statistically significant.

[0189] III. Results and Discussion

[0190] 1. Probe design and recognition principle

[0191] The design concept of the pH-sensitive fluorescent probe CHC-HSNO for detecting thionitrite provided by this invention is as follows: A coumarin-hemicyanine derivative is used as the near-infrared fluorophore. Under neutral or alkaline conditions, this fluorophore undergoes an intramolecular cyclization reaction to generate an imidazoline ring, disrupting the conjugated structure and quenching the fluorescence. Under acidic conditions, the imidazoline ring opens, restoring the conjugated structure and activating the fluorescence. An o-phenylenediamine and o-mercaptobenzoic acid group are attached to the carboxyl group of indole as specific recognition sites for HSNO. When the probe reacts with HSNO under acidic conditions, the imidazoline ring opens, activating the fluorescence. This allows the probe to specifically respond to HSNO in the acidic tumor microenvironment, thus constructing the pH-sensitive fluorescent probe CHC-HSNO for the specific detection of thionitrite in tumors. Figure 5 ).

[0192] Mechanism of CHC-HSNO probe for identifying HSNO ( Figure 6The "S" in HSNO is electrophilic, undergoing a nucleophilic reaction and intramolecular cyclization with the exposed thiol group of compound CHC-HSNO to generate and release benzodithione. Subsequently, the self-elimination chain undergoes a self-elimination reaction, exposing o-phenylenediamine. The "N=O" in HSNO reacts with o-phenylenediamine to generate benzotriazole, which then undergoes hydrolysis to remove benzotriazole and release the fluorescent parent nucleus CHC-6-A. Then, under acidic conditions, the imidazoline ring opens, at which point the near-infrared fluorescence of the coumarin-hemiflorum derivative CHC-6-B is activated.

[0193] like Figure 5 As shown, the product of the reaction between the probe CHC-HSNO and HSNO was verified by high-resolution mass spectrometry. Figure 7 For is Figure 8 The high-resolution mass spectrum after local magnification shows that the probe reacts with HSNO to generate compound CHC-6-A.

[0194] Next, 1H NMR spectroscopy confirmed that compound CHC-6-A regenerates into compound CHC-6-B under acidic conditions. Under acidic conditions, the spirocyclic ring in compound CHC-6-A undergoes ring opening to form compound CHC-6-B. The nitrogen atom in the indole group becomes a nitrogen cation, and the chemical shift of the hydrogen atom in the adjacent methylene group (b) shifts to a lower field. Figure 9 and Figure 10 The 1H NMR spectroscopy results confirmed this, showing that the chemical shift of the methylene β-hydrogen changed from 3.57 to 4.58. The experimental results also demonstrated that CHC-6-A generated compound CHC-6-B under acidic conditions.

[0195] Compound CHC-6-A: 1 H NMR(400MHz, Methanol-d4)δ7.90(s,1H),7.84(d,J=12.0Hz,1H),7.72(s,1H),7.40(dd,J=4.0Hz,1H),6.81-6.63(m,4H),6 .51(s,1H),3.77(t,J=8.0Hz,2H),3.57(m,2H),3.50-3.44(m,4H),1.41(s,3H),1.19(t,J=8.0Hz,4.0Hz,6H),1.14(s,3H).

[0196] Compound CHC-6-B: 1H NMR (400MHz, Methanol-d4) δ8.49(s,1H),8.44(s,1H),8.39(d,J=16.0Hz,1H),8.27(d,J=8.0Hz,1H),7.77(d,J=12.0Hz,1H),7.62(t,J=12.0Hz,2H),6.9 2(d,J=12.0Hz,1H),6.64(s,1H),4.58(t,J=4.4Hz,2H),3.61(q,J=12.0Hz,8 .0Hz,4H),3.41(t,J=5.2Hz,2H),1.75(s,6H),1.26(t,J=4.0Hz,8.0Hz,6H).

[0197] 2. Study on the detection performance of the CHC-HSNO probe for HSNO

[0198] 2.1 UV absorption spectrum of the reaction between probe CHC-HSNO and HSNO

[0199] To investigate the detection performance of the probe CHC-HSNO for HSNO, the changes in the ultraviolet absorption spectrum before and after the reaction of CHC-HSNO with HSNO were first examined. For example... Figure 11 As shown, the probe CHC-HSNO itself exhibits no significant UV absorption peak between 500 nm and 700 nm under acidic conditions (pH 4.0 or 7.4). At pH 7.4, after reacting with HSNO, the probe CHC-HSNO also shows no significant UV absorption peak between 500 nm and 700 nm. At pH 4.0, after reacting with HSNO, the probe CHC-HSNO exhibits a maximum UV absorption peak at 600 nm, consistent with the maximum UV absorption peak of compound CHC-6 at pH 4.0. The experimental results indicate that the probe CHC-HSNO can only react with HSNO under acidic conditions to generate CHC-6-B, consistent with the expected reaction mechanism.

[0200] 2.2 Fluorescence spectrum of the reaction between probe CHC-HSNO and HSNO

[0201] The fluorescence response performance of the probe CHC-HSNO to HSNO was studied, such as... Figure 12 As shown, the probe itself showed no fluorescence emission at pH 4.0 or pH 7.4; at pH 7.4, co-incubation with HSNO also resulted in no fluorescence emission. However, at pH 4.0, co-incubation with HSNO produced significant fluorescence emission at 660 nm, consistent with the fluorescence emission peak of compound CHC-6 at pH 4.0. The experimental results indicate that fluorescence is only produced by the reaction of the probe with HSNO under acidic conditions, consistent with the expected reaction mechanism.

[0202] 2.3 Fluorescence spectrum and detection limit of the reaction between probe CHC-HSNO and HSNO

[0203] The relationship between the fluorescence intensity of the probe CHC-HSNO response and the concentration of HSNO, as well as the detection limit, were further investigated. Figure 13 As shown in Figure a, under pH 4.0 conditions, after incubating the probe CHC-HSNO with different concentrations (0-80 μM) of HSNO, the fluorescence intensity at 660 nm increased 63-fold with increasing HSNO concentration. Furthermore, the fluorescence intensity showed a good linear relationship with the HSNO concentration (0-10 μM). In addition, the detection limit of this probe was 415 nM, indicating that the probe CHC-HSNO has good detection sensitivity for HSNO.

[0204] 2.4 Reaction time of probe CHC-HSNO with HSNO

[0205] The reaction time between the probe CHC-HSNO and HSNO was investigated. Figures 14-17 As shown, after co-incubating the probe CHC-HSNO (10 μM) and HSNO (100 μM) for approximately 30 min, the fluorescence intensity reached its peak at 660 nm, and the fluorescence intensity did not decrease even after 90 min. These results indicate that the fluorescence response generated by the probe CHC-HSNO and HSNO exhibits good stability.

[0206] 2.5 Reaction of probe CHC-HSNO with HSNO under different pH conditions

[0207] The study investigated the response of the probe CHC-HSNO to HSNO under different pH conditions, such as... Figure 18 As shown, when the probe CHC-HSNO and HSNO were co-incubated in PBS buffer at pH 3.0-4.0, the strongest fluorescence signal was observed at 660 nm. Within the pH range of 4.0-7.0, the fluorescence signal gradually weakened with increasing pH, and was quenched at pH 7.0-8.0. The results indicate that the probe CHC-HSNO responds to HSNO within the pH range of 3.0-6.5, and the fluorescence signal increases with increasing acidity, suggesting that the probe is suitable for the specific detection of HSNO in the acidic tumor microenvironment. 2.6 Selectivity of the probe CHC-HSNO for HSNO detection

[0208] Considering the complex environment within organisms, high selectivity of probes for analytes is crucial for accurate detection. To verify the selectivity of the probe CHC-HSNO for HSNO, CHC-HSNO was combined with the following interfering substances: amino acids (Gly, D-Ala, Val, Ile, L-Cys, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy) and inorganic salts (Sn). 2+ Cd 2+ Mn 2+ Co 2+ Cu 2+ Hg 2+ Zn 2+ Fe 2+ Fe 3+ Ca 2+ Al 3+ Ni 2+ Mg 2+ Li + Na + K + Ag + ), reactive sulfur (Na2S, Na2S2, DEA·NONOate, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8, GSH), reactive oxygen and reactive nitrogen (H2O2, ClO) - , t BuOOH、 · OH、 1 O2, O2 ·- NO3 - NO2 - ONOO - After co-incubation for 30 minutes, the fluorescence intensity was measured. The results are as follows: Figure 19 The results showed that the probe CHC-HSNO only produced a fluorescent signal after co-incubation with HSNO, and no fluorescent signal was produced after co-incubation with other interfering substances. These experimental results indicate that the probe CHC-HSNO has good selectivity for HSNO.

[0209] 3. Cell-level experiments

[0210] 3.1 Cytotoxicity assay

[0211] Cellular experiments were conducted using human colon cancer cells HCT-116 and human colon epithelial cells NCM-460. The cytotoxicity of the probe CHC-HSNO was studied using the CCK8 assay. The cells were co-incubated with different concentrations of the probe CHC-HSNO (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) for 48 h. The results are as follows: Figure 20As shown, the cell viability rate is above 83%, indicating that the probe has good biocompatibility and can be used for intracellular HSNO imaging.

[0212] To observe the changes in cell viability over time after incubation with the probe, the cell viability of HCT-116 and NCM-460 cells was further examined after incubation with the probe CHC-HSNO (20 μM) for different times (0 h, 6 h, 12 h, 24 h, 48 h). Figure 20 As shown, after incubation with the probe for 48 hours, the survival rate of both cell types was above 85%, indicating that the probe CHC-HSNO has low toxicity to both cell types and can be used for intracellular HSNO imaging.

[0213] 3.2 Fluorescence imaging of exogenous HSNO in cells

[0214] In vitro experiments demonstrated that the probe exhibited good selectivity and sensitivity for HSNO under acidic conditions, but showed no response under neutral or alkaline conditions, suggesting that the probe is suitable for the specific detection of HSNO in the acidic environment of tumors. Next, the detection capability of the probe CHC-HSNO for HSNO in tumor cells was further investigated. First, HCT-116 cells were co-incubated with the probe CHC-HSNO for 30 min, and a weak fluorescence signal was detected (…). Figure 20 (a) Next, the addition of exogenous HSNO to the cells significantly enhanced the fluorescence signal (6-fold). Figure 20 (b) Since the added exogenous HSNO was synthesized from GSNO and Na2S, in order to eliminate interference, the probe was reacted with GSNO ( Figure 20 c) and Na2S ( Figure 20 (d) Pretreated HCT-116 cells were co-incubated by Figure 20 c and Figure 20 As can be seen from d, the fluorescence signal is related to Figure 20 There was no significant change compared to a, indicating Figure 20 The fluorescence signal generated in b is caused by exogenous HSNO. The probe CHC-HSNO can effectively detect exogenous HSNO.

[0215] Next, the specific detection capability of the probe for HSNO in tumor cells was investigated. The probe CHC-HSNO was co-incubated with NCM-460 cells, and then... Figure 21 As shown in Figure e, no fluorescence signal was observed. NCM-460 cells were incubated with the probe, followed by incubation with exogenous HSNO, but still no fluorescence was observed. Figure 24(f) This is because the probe only activates a fluorescence response after reacting with HSNO under acidic conditions, while the human colonic epithelial NCM-460 cells have a neutral environment, thus no fluorescence signal is generated. Experiments have demonstrated that the probe CHC-HSNO can specifically detect exogenous HSNO in tumor cells. Figure 22 The bright-field image of the cells shows normal cell morphology. This demonstrates that the CHC-HSNO probe can specifically detect exogenous HSNO within tumor cells.

[0216] 3.3 Fluorescence imaging of endogenous HSNO in cells

[0217] As a crosstalk product of hydrogen sulfide (H2S) and nitric oxide (NO), the level of thionitrite (HSNO) in vivo is mainly regulated by changes in H2S and NO concentrations. Studies have confirmed that when exogenous NO donors react with endogenous H2S in cells, HSNO can be generated in situ, demonstrating the crucial role of H2S in HSNO biosynthesis. Knocking out the CBS gene resulted in a significant decrease in intracellular H2S levels, which directly led to a reduction in endogenous HSNO levels. In this invention, to reduce the generation of endogenous HSNO, we transfected HCT-116 cells with CBS-siRNA to inhibit the expression of CBS protein, such as... Figure 22 As shown, CBS protein expression was reduced in HCT-116 cells transfected with CBS siRNA. Figure 22 a and Figure 22 As shown in Figure b, compared with HCT-116 cells, the fluorescence intensity in HCT-116 cells transfected with CBS siRNA was significantly reduced, indicating that the HSNO level in HCT-116 cells transfected with CBS siRNA was decreased. Studies have shown that aminooxyacetate acid (AOAA) is an inhibitor of the H2S synthase cystathionine β-synthase (CBS), and can reduce endogenous H2S levels. This invention uses AOAA to pretreat cells, such as... Figure 23 As shown in Figure c, compared with HCT-116 cells, the fluorescence intensity in HCT-116 cells treated with AOAA was also significantly reduced, indicating that the HSNO level in HCT-116 cells was reduced after AOAA treatment. Figure 25 As shown in Figure d, no fluorescence was produced after the probe CHC-HSNO was incubated with human colonic epithelial cells NCM-460. Figure 26 The bright-field image of the cells shows normal cell morphology. These results demonstrate that the probe CHC-HSNO can specifically detect endogenous HSNO in tumor cells.

[0218] 4. Animal-level experiments

[0219] This invention further investigates the imaging performance of the CHC-HSNO probe for HSNO detection at the in vivo level. Tumors with a volume exceeding 200 mm² were selected. 3 Nude mice in good condition were randomly divided into two groups of three each. The control group and the HSNO group were respectively. After anesthetizing each group of mice with isoflurane gas, (1) control group: mice were injected intratumorally with probe CHC-HSNO (500μM, 10μL, physiological saline: dimethyl sulfoxide = 100:1v / v); (2) HSNO group: mice were injected intratumorally with probe CHC-HSNO (500μM, 10μL, physiological saline: dimethyl sulfoxide = 100:1v / v), and then intratumorally with exogenous HSNO (100μM, 100μL) in PBS solution (50mM, pH = 7.4). The experimental results showed that mice that were injected intratumorally with probe CHC-HSNO alone showed almost no fluorescence signal ( Figure 27 In contrast, mice injected with the probe CHC-HSNO intratumorally followed by exogenous HSNO (100 μM) showed a gradual increase in fluorescence signal. Figure 27 The fluorescence intensity reached its peak (17-fold increase) at approximately 40 minutes and still showed a strong fluorescence signal at 60 minutes. This demonstrates that the CHC-HSNO probe exhibits good stability and can be used for the detection of HSNO in mice.

[0220] 5. Study on the antitumor activity of HSNO

[0221] Studies have shown that H2S and NO at concentrations higher than physiological levels have anti-tumor effects. In colorectal cancer, H2S and NO regulate cell proliferation in a concentration-dependent manner. Higher than physiological concentrations of H2S and NO inhibit tumor cell growth by suppressing the cGMP / VASP pathway, Akt, and p44 / 42MAPK (Erk1 / 2), respectively. HSNO, as a key signal transduction molecule connecting the H2S and NO signaling pathways, is both a source of NO and a crosstalk product of NO and H2S, possessing similar physiological functions to H2S and NO. Therefore, it is speculated that HSNO may also have anti-tumor effects. To investigate the anti-tumor activity of HSNO, xenografted nude mice were randomly divided into two groups, with tumors exceeding 200 mm² selected. 3 Nude mice in good condition, 6 mice per group. (1) Control group: 100 μL of physiological saline was injected via tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. (2) HSNO group: 1.6 mg / kg, Saline:DMSO = 99:1, was injected via tail vein on days 1, 3, 5, 7, 9, 11, 13, and 15. Tumor volume was recorded every 2 days for a 15-day treatment period. Changes in body weight and tumor volume of each group of nude mice were recorded over 15 days to study the antitumor activity of HSNO. Results are as follows:Figure 27 b and Figure 28 As shown in Figure c, the tumor volume in the control group nude mice rapidly increased to 1264 mm. 3 In the HSNO group, the tumor volume gradually decreased to 148 mm. 3 Preliminary results indicate that HSNO has an inhibitory effect on tumor growth. For example... Figure 29 As shown in Figure a, after 15 days of treatment, there was no significant difference in body weight between the control group and the HSNO group of nude mice. The mice were then euthanized, and tumor tissue from the xenografted nude mice was removed. The excised tumors were photographed for comparison, followed by pathological analysis and fluorescence imaging. The results are as follows: ​ HE staining results showed that the tumor cells in the control group mice were densely packed, with deeply stained nuclei exhibiting significant atypia, rich blood vessels within the tumors, and no necrotic foci. In the HSNO group sections, tumor cell nuclei were pyknoid, nuclear structure was indistinct, and extensive cell necrosis was observed. ​ As shown, the fluorescence intensity of tumor tissue in the HSNO group was significantly higher than that in the control group, indicating that the HSNO level was elevated in the HSNO group. This suggests a possible link between tumor growth inhibition and elevated HSNO levels, but the specific mechanism requires further investigation. The experimental results preliminarily indicate that HSNO possesses antitumor activity.

[0222] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pH-sensitive fluorescent probe for detecting thionitrous acid, the structural formula of which is shown below:

2. A method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid as described in claim 1, characterized in that, Its synthetic route is as follows:

3. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 2, characterized in that, Includes the following steps: (1) Chemical reaction of carboxyphenylhydrazine and methyl isopropyl ketone to prepare compound CHC-1; (2) Compound CHC-1 and N-tert-butoxycarbonyl-bromoethylamine were chemically reacted to prepare compound CHC-2; (3) In the presence of piperidine, 4-(diethylamino)salicylaldehyde and diethyl malonate react to prepare compound CHC-3; (4) In the presence of phosphorus trichloride, compound CHC-3 undergoes a chemical reaction to prepare compound CHC-4; (5) Compound CHC-2 and compound CHC-4 undergo a chemical reaction to prepare compound CHC-5; (6) In the presence of trifluoroacetic acid, compound CHC-5 undergoes a chemical reaction to prepare compound CHC-6; (7) Thiosyl salicylic acid and 2,2'-dithiodipyridine were chemically reacted to prepare compound S-1; (8) Compound S-1 and p-hydroxybenzaldehyde were chemically reacted in the presence of EDCI and DMAP to prepare compound S-2. (9) Compound S-2 undergoes a chemical reaction in the presence of sodium borohydride to prepare compound S-3; (10) Compound S-3, o-phenylenediamine and triphosgene reacted in the presence of K2CO3 and pyridine to prepare compound S-4; (11) In the presence of HBTU and HOBT, compound CHC-6 and compound S-4 reacted chemically to prepare the fluorescent probe CHC-HSNO.

4. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (1), the molar ratio of p-carboxyphenylhydrazine and methyl isopropyl ketone is 1:1.2-2.0, preferably 1:1.4-1.6, and more preferably 1:1.5; In step (2), the molar ratio of compound CHC-1 and N-tert-butoxycarbonyl-bromoethylamine is 1:1.5-2.5, preferably 1:1.8.0-2.2; A more preferred ratio is 1:2.

0.

5. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (3), the molar ratio of 4-(diethylamino)salicylaldehyde to diethyl malonate is 1:1.5-2.5, preferably 1:1.8.0-2.2; more preferably 1:2.0; the molar ratio of 4-(diethylamino)salicylaldehyde to piperidine is 2.0-3.0:1, preferably 2.6-2.8:1; more preferably 2.75:1; in step (4), the mass-volume ratio of compound CHC-3 to phosphorus trichloride is 0.1-0.8:1 g / mL, preferably 0.3-0.5:1 g / mL, more preferably 0.44:1 g / mL; the reaction temperature is 40-60℃, preferably 45-55℃, more preferably 50℃.

6. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (5), the molar ratio of compound CHC-2 to compound CHC-4 is 1.5-2.5:1, preferably 1.8-2.2:1; more preferably 2.0:1; in step (6), the mass-volume ratio of compound CHC-5 to trifluoroacetic acid is 0.1-0.8:1 g / mL, preferably 0.2-0.4:1 g / mL, more preferably 0.3:1 g / mL.

7. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (7), the molar ratio of thiosalicylic acid to 2,2'-dithiodipyridine is 2.5-3.5:1, preferably 2.8-3.2:1; more preferably 3.0:1; In step (8), the molar ratio of compound S-1 to p-hydroxybenzaldehyde is 0.5-1.5:1, preferably 0.8-1.2:1; more preferably 1.0:1; the molar ratio of compound S-1 to EDCI is 1:1.2-2.0, preferably 1:1.4-1.6; more preferably 1:1.5; the molar ratio of compound S-1 to DMAP is 1:0.1-0.5, preferably 1:0.15-0.25; more preferably 1:0.

2.

8. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (9), the molar ratio of compound S-2 to sodium borohydride is 1:0.8-1.5, preferably 1:1.0-1.2; more preferably 1:1.1; in step (10), the molar ratio of compound S-3 to o-phenylenediamine is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0; the molar ratio of compound S-3 to triphosgene is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0; the molar ratio of compound S-3 to K2CO3 is 1:3.0-4.0, preferably 1:3.8-4.2; more preferably 1:4.0; the molar ratio of compound S-3 to pyridine is 1:3.0-4.0, preferably 1:3.8-4.2; more preferably 1:4.

0.

9. The method for preparing a pH-sensitive fluorescent probe for detecting thionitrous acid according to claim 3, characterized in that, In step (11), the molar ratio of compound CHC-6 to compound S-4 is 1:1.5-2.5, preferably 1:1.8.0-2.2; more preferably 1:2.0; the molar ratio of compound CHC-6 to HBTU is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.0; the molar ratio of compound CHC-6 to HOBT is 1:0.5-1.5, preferably 1:0.8-1.2; more preferably 1:1.

0.

10. The application of the pH-sensitive fluorescent probe according to claim 1 in the detection of thionitrous acid.

Images