A targeted MASH diagnosis and treatment integrated probe based on azoreductase response and a synthesis method and application thereof

Abstract

The application discloses a kind of targeted MASH diagnosis and treatment integrated probe based on azoreductase response and its synthesis method and application, the probe of the present application integrates the following functional units: 1) near-infrared fluorescent Cy skeleton;2) Gd-DO3A magnetic resonance imaging contrast agent;3) H2S release unit.Under azoreductase trigger, nitrogen-nitrogen double bond is reduced, probe occurs 1,6-self-elimination and intramolecular charge transfer reaction, releases carbonyl sulfide, then rapidly converted into H2S under the action of carbonic anhydrase, simultaneously generating quinone methide intermediate, covalently bound with adjacent protein.This double mechanism makes near-infrared fluorescence enhance by 50 times, r1relaxation rate is increased from 8.9mM ‑1 s ‑1 to 14.1 mM ‑1 s ‑1 The probe of the present application has good biological safety, good water dispersibility, is suitable for azoreductase magnetic resonance and photoacoustic dual-mode imaging, and has great application potential in the monitoring of MASH.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging technology, specifically relating to a targeted MASH diagnostic and therapeutic probe based on azoreductase response, its synthesis method, and its application. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive subtype of metabolic-associated fatty liver disease (MASLD). Its histopathological features mainly include hepatocellular steatosis, ballooning degeneration, and lobular inflammation. As the disease progresses, MASH can further develop into liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). With the global prevalence of obesity and metabolic syndrome, MASH has become one of the most common chronic liver diseases today, posing a heavy public health burden.

[0003] Currently, liver biopsy remains the gold standard for diagnosing MASH. However, as an invasive procedure, liver biopsy suffers from significant sampling errors, bleeding risks, and low patient acceptance, making it unsuitable for dynamic disease monitoring or large-scale population screening. Therefore, there is an urgent clinical need to develop a non-invasive, sensitive, and integrated imaging method that can simultaneously and accurately assess disease activity and treatment response.

[0004] Unfortunately, current non-invasive imaging techniques for assessing MASH each have significant limitations. Conventional ultrasound heavily relies on operator experience and has low sensitivity for mild steatosis. Computed tomography (CT) can quantify liver fat content, but it inevitably carries the risk of ionizing radiation. Magnetic resonance imaging proton density fat fraction (MRI-PDFF) is currently the most accurate non-invasive method for quantifying fat, but it only reflects the degree of triglyceride accumulation in the liver and cannot effectively distinguish between simple non-fatty liver fibrosis (NAFL) and MASH with inflammation and ballooning degeneration. Clinical studies have shown that approximately 30%-50% of MASH patients do not have significantly elevated liver fat content, while some NAFL patients show high fat content, resulting in PDFF having a specificity of less than 60% in the diagnosis of MASH. On the other hand, while magnetic resonance elastography (MRE) can assess the degree of liver fibrosis, it is not sensitive to the inflammatory activity in early MASH. Overall, existing imaging methods overemphasize "structural changes" such as fat accumulation or fibrosis, and severely lack the ability of "functional molecular imaging" to directly detect MASH-related enzyme activity or inflammatory signals.

[0005] Liver inflammation and metabolic stress are characterized by local hypoxia, altered redox homeostasis, immune cell infiltration, and dysregulation of reductase activity. Azoreductase reaction motifs are widely used as hypoxia / redox-sensitive triggers because azo bonds can be selectively reduced under reductive enzymatic conditions. Therefore, azoreductase activity (a reductase activity capable of cleaving azo bonds under hypoxia / reduction conditions) can serve as a functional alternative to the hypoxic and inflammatory microenvironment associated with active lipids.

[0006] Meanwhile, drug treatment for MASH also faces significant challenges. In exploring novel therapies for MASH, gaseous neurotransmitter therapy has shown great potential. Hydrogen sulfide (H2S) is the third endogenous gaseous signaling molecule in mammals, following nitric oxide (NO) and carbon monoxide (CO), playing a crucial regulatory role in liver physiology and chronic liver disease. Recent studies have shown that exogenous H2S or its donors can significantly improve liver function, reduce hepatic lipid deposition, alleviate oxidative stress, and inhibit the expression of inflammatory cytokines in MASH and liver fibrosis models. However, traditional H2S donors lack tissue specificity, and their uncontrolled release in vivo not only greatly limits the precision of treatment but also raises safety concerns regarding non-targeted tissues and organs. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a targeted MASH diagnostic and therapeutic probe based on azoreductase response, its synthesis method, and its application. The probe of this invention generates near-infrared fluorescence and T1-weighted MR dual-modal signal enhancement upon AzoR activation, while simultaneously releasing therapeutic gas molecules H2S in situ. This not only enables early (4-week) diagnosis of MASH but also allows for dynamic monitoring of treatment response and proactive relief of inflammation, overcoming the fundamental limitation of existing imaging technologies that can only provide structural information.

[0008] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows: A targeted MASH diagnostic and therapeutic probe based on azoreductase response has the following structural formula: .

[0009] A method for synthesizing a targeted MASH therapeutic probe based on azoreductase response includes the following steps: S1. Under alkaline conditions, 2,4-dihydroxybenzyl alcohol reacts with heptamethrin in a Knovengel reaction to produce compound (1), the reaction formula of which is as follows: ; S2. In the presence of acid, ethyl 4-nitrosobenzoate undergoes an addition reaction with 3,5-difluoroaniline to produce compound (2), the reaction formula of which is as follows: ; S3. In the presence of a reducing agent, compound (2) undergoes a reduction reaction to produce compound (3), and the reaction formula is as follows: ; S4. In the presence of a catalyst, compound (3) undergoes a bromination reaction with a brominating reagent to produce compound (4), the reaction formula of which is as follows: ; S5. Under alkaline conditions, compound (1) undergoes a substitution reaction with compound (4) to produce compound (5), and the reaction equation is as follows: ; S6. Under alkaline conditions, compound (5) undergoes a substitution reaction with phenyl thiochloroformate to produce compound (6), the reaction formula of which is as follows: ; S7. Under the presence of a base and an activator, 2,2,2-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester and azidoacetic acid undergo an amide condensation reaction to produce compound (7), the reaction formula of which is as follows: ; S8. Under acidic conditions, the compound of formula (7) first undergoes a hydrolysis reaction, and then undergoes a coordination reaction with the trivalent gadolinium salt. The reaction formula for compound (8) is as follows: ; S9. In the presence of a catalyst and a reducing agent, compounds of formula (6) and (8) undergo a click chemistry reaction to generate the azo reductase-based targeted MASH diagnostic and therapeutic probe, the reaction formula of which is as follows:

[0010] Furthermore, in step S1, the alkali is anhydrous potassium carbonate, the solvent is anhydrous N,N-dimethylformamide, the Knovengel reaction temperature is 70~80 ℃, the time is 4-8 h, and the molar ratio of 2,4-dihydroxybenzyl alcohol, heptamethrin and alkali is 1.0:0.5~2.0:1.0~2.5.

[0011] Furthermore, in step S1, the alkali is anhydrous potassium carbonate, the solvent is anhydrous N,N-dimethylformamide, the Knovengel reaction temperature is 70~80 ℃, the time is 4-8 h, and the molar ratio of 2,4-dihydroxybenzyl alcohol, heptamethrin and alkali is 1.0:0.5~2.0:1.0~2.5.

[0012] Furthermore, in step S2, the acid is glacial acetic acid, the solvent is glacial acetic acid, the condensation reaction temperature is room temperature, the time is 10~12 h, and the molar ratio of ethyl 4-nitrosobenzoate to 3,5-difluoroaniline is 1.0:1.0~3.0.

[0013] Furthermore, in step S3, the reducing agent is lithium aluminum hydride, the solvent is tetrahydrofuran, the temperature of the reduction reaction is -40~-20℃, the time is 1~2 h, and the molar ratio of compound (2) and lithium aluminum hydride is 1.0:1.0~3.0.

[0014] Furthermore, in step S5, the base is anhydrous potassium carbonate, the solvent is acetonitrile, the temperature of the substitution reaction is 70~80℃, the time is 3~6 h, and the molar ratio of compound (1), compound (4) and base is 1.0:1.5~7.5:2.5~12.5.

[0015] Furthermore, in step S6, the base is triethylamine, the solvent is anhydrous dichloromethane, the temperature of the substitution reaction is 20~35 ℃, the time is 6-12 h, and the molar ratio of compound (5), phenyl thiocarbamate and base is 1.0:5.0~20.0:5.0~10.0.

[0016] Further, in step S7, the base is N,N-diisopropylethylamine, the activator is 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate, the solvent is N,N-dimethylformamide or triethylamine, the amide condensation reaction temperature is 20~35 °C, the time is 4-10 h, and the molar ratio of 2,2',2''-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester, azidoacetic acid, activator and base is 1.0:1.3~5:1.3~5:1.6~6.

[0017] Furthermore, in step S8, the acidic reagent used is ethyl hydrochloric acid solution, the hydrolysis reaction temperature is 20~35 ℃, and the time is 4-6 h; The trivalent gadolinium salt is gadolinium chloride. The coordination reaction temperature is 20~35 ℃, the time is 3~8 h, the solvent is ultrapure water, and the molar ratio of compound (7) and trivalent gadolinium salt is 1.0:2.0~4.0.

[0018] Furthermore, in step S9, the catalyst is anhydrous copper sulfate, the reducing agent is ascorbic acid, the solvent is a mixed solvent of N,N-dimethylformamide, dimethyl sulfoxide and ultrapure water, the temperature of the click chemical reaction is 20~35 ℃, the reaction time is 12~24 h, and the molar ratio of compound (6), compound (8), catalyst and reducing agent is 1.0:1.5~4.0:0.3~3.0:1.2~5.0.

[0019] Application of an azoreductase-responsive targeted MASH diagnostic and therapeutic probe in the preparation of targeted liver multimodal imaging contrast agents and drugs for treating MASH.

[0020] Furthermore, the multimodal imaging contrast agent is a fluorescence imaging contrast agent and a T1-weighted MRI contrast agent.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The probe of this invention integrates the following functional units: 1) a near-infrared fluorescent Cy backbone; 2) a Gd-DO3A magnetic resonance imaging contrast agent; and 3) an H2S release unit. Triggered by azoreductase, the nitrogen-nitrogen double bond is reduced, and the probe undergoes a 1,6-self-elimination and intramolecular charge transfer reaction, releasing carbonyl sulfide (COS, which is then rapidly converted to H2S by carbonic anhydrase), while simultaneously generating a quinone methylation intermediate that covalently binds to a neighboring protein. This dual mechanism enhances near-infrared fluorescence by up to 50-fold, and the r1 relaxation rate increases from 8.9 mM. -1 s -1 Increased to 14.1 mM -1 s -1 (Increases by approximately 1.6 times).

[0022] 2. This invention introduces fluorine substituents into the responsive scaffold to modulate the electronic properties of the self-eliminating linker, thereby inhibiting premature H2S release while maintaining enzymatic responsiveness. Upon activation by azoreductase, the probe undergoes a 1,6-self-elimination reaction to generate a methylquinone intermediate, which can covalently anchor to a neighboring protein, achieving sustained retention and enhancement of fluorescence and magnetic resonance signals. Simultaneously, the released carbonyl sulfide is converted into H2S by endogenous carbonic anhydride, thereby enabling the delivery of local gaseous messengers in the hypoxic liver microenvironment. This strategy provides a molecular-level integrated approach for dual-mode visualization and image-guided H2S therapy in MASH.

[0023] 3. The probe of the present invention has good biocompatibility and good water dispersibility, and is suitable for in vivo magnetic resonance and fluorescence dual-modal imaging of azoreductase, and has great application potential in the monitoring of MASH.

[0024] 4. In a MASH mouse model induced by methionine / choline deficiency, the probe AzoR-Cy-Gd of this invention was used to detect MASH mice 8 weeks after injection. Eight hours later, the liver fluorescence intensity of the MASH mouse model was 2.8 times that of the normal group, and the magnetic resonance signal enhancement reached 58%. Furthermore, sustained H2S release significantly reduced liver function indicators (ALT, AST) and pro-inflammatory cytokines (IL-6, TNF-α). This work realizes a diagnostic and therapeutic platform integrating diagnosis and treatment through a single enzyme-triggered event. Attached Figure Description

[0025] Figure 1 The image shows the HPLC-MS / MS analysis results of AzoR-Cy-Gd prepared in Example 1. Figure 1 a is the liquid chromatogram of AzoR-Cy-Gd. Figure 1 b is the high-resolution mass spectrum of AzoR-Cy-Gd.

[0026] Figure 2 The fluorescence emission spectra of AzoR-Cy-Gd prepared in Example 1 under the action of different concentrations of azo reductase AzoR are shown.

[0027] Figure 3 The graph shows a comparison of the longitudinal molar relaxation rate of AzoR-Cy-Gd prepared in Example 1 with and without azo reductase.

[0028] Figure 4 The H2S release curve of AzoR-Cy-Gd prepared in Example 1 under the presence or absence of carbonic anhydrase.

[0029] Figure 5 The graph shows the change in uptake of AzoR-Cy-Gd prepared in Example 1 over time in 4T1 hypoxic cells.

[0030] Figure 6 The image shows the in vivo fluorescence imaging of MASH model mice with AzoR-Cy-Gd prepared in Example 1.

[0031] Figure 7 The in vivo effects of AzoR-Cy-Gd prepared in Example 1 on MASH model mice 1 HMRI image.

[0032] Figure 8 The image shows the therapeutic effect of AzoR-Cy-Gd prepared in Example 1 on MASH model mice. Figure 8 a is a graph showing the content of ALT and AST in different treatment groups. Figure 8 b is a graph showing the levels of IL-6 and TNF-α in different treatment groups.

[0033] Figure 9 The fluorescence intensity of compound AzoR-H / F / CF3 under the action of azoreductase AzoR is shown as a function of time. Figure 9 a represents the fluorescence emission spectra of AzoR-H at different times. Figure 9 b shows the fluorescence emission spectra of AzoR-F at different times. Figure 9 c shows the fluorescence emission spectra of AzoR-CF3 at different times. Figure 9 d represents the linear relationship between the fluorescence intensity of AzoR-H / F / CF3 and time. Detailed Implementation

[0034] The present invention will now be described in detail with reference to specific embodiments, but these embodiments are not to be construed as limiting the scope of protection of the present invention in any sense.

[0035] Example 1

[0036] 1. Synthesis of compound (1): Anhydrous N,N-dimethylformamide (10 mL) was added to 2,4-dihydroxybenzyl alcohol (2 mmol, CAS No.: 33617-60-4), followed by the addition of heptamethrin (1 mmol) and anhydrous potassium carbonate (2.5 mmol). The mixture was stirred at 80°C for 4 hours. After the reaction was completed, the mixture was extracted with ethyl acetate and water. The organic phase was collected and dried with anhydrous sodium sulfate. The lyophilized organic phase was concentrated under vacuum. The residue was purified by rapid column chromatography (mobile phase: dichloromethane / methanol = 100:5, v / v), lyophilized, and yielded a blue solid of compound (1) (210 mg, yield 35.4%).

[0037] 1 H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 10.0 Hz, 1H), 7.34 – 7.23 (m,3H), 7.12 (s, 1H), 7.04 (t, J = 10.0 Hz, 1H), 6.87 (d, J = 10.0 Hz, 1H), 6.51(s, 1H), 5.72 (d, J = 10.0 Hz, 1H), 4.70 (s, 2H), 3.93 (t, J = 5.0 Hz, 2H), 2.68 (t, J = 5.0 Hz, 2H), 2.63 (t, J= 5.0 Hz, 2H), 2.34 (t, J = 5.0 Hz, 2H),2.13 (s, 1H), 1.98 (t, J = 5.0 Hz, 2H), 1.90 (p, J = 5.0 Hz, 2H), 1.69 (s, 6H).

[0038] HRMS (ESI) Calculation for [C 31 H 32 INO3, MI] ＋ : 466.2377, Found: 466.2377.

[0039] 2. Synthesis of compound (2): Ethyl 4-nitrosobenzoate (1 mmol: CAS No.: 7476-79-1) was dissolved in glacial acetic acid (10 mL), followed by the addition of 3,5-difluoroaniline (1.5 mmol, CAS No.: 372-39-4). The mixture was stirred at room temperature for 10 hours. After the reaction was complete, the insoluble matter was removed by filtration. Water (150 mL) was added to the filtrate, and a precipitate was formed. After the precipitation was complete, the precipitate was filtered. The precipitate was dissolved in dichloromethane, and anhydrous sodium sulfate was added to dry the solution to remove any small amount of water. The organic solution was concentrated under reduced pressure, and the residue was recrystallized from methanol. The residue was filtered, and the filter cake was washed with cold methanol and dried to obtain a yellow solid compound of formula (2) (120 mg, yield 41.4%).

[0040] 1 H NMR (500 MHz, CDCl3) δ 8.23 ​​(d, J = 10.0 Hz, 2H), 7.97 (d, J = 5.0Hz, 2H), 7.52 (d, J = 5.0 Hz, 2H), 6.98 (td, J = 10.0, 5.0 Hz, 1H), 4.45 (q, J = 5.0 Hz, 2H), 1.45 (t, J = 5.0 Hz, 3H).

[0041] 13C NMR (126 MHz, CDCl3) δ 164.29, 164.19, 162.30, 162.20, 154.37,154.30, 154.23, 133.06, 130.65, 130.59, 122.99, 106.66, 106.50, 106.45,106.33, 106.28, 106.24, 61.41, 61.37, 14.33.

[0042] 3. Synthesis of compound (3): The compound of formula (2) (1.00 mmol) was dissolved in anhydrous tetrahydrofuran (2.5 mL). The resulting solution was added dropwise to a suspension of lithium aluminum hydride (1.20 mmol, CAS No.: 16853-85-3) in anhydrous tetrahydrofuran (4 mL) pre-cooled to -20 °C. After the addition was complete, the mixture was stirred at -20 °C for 1 hour. After the reaction was complete, 10% NaOH solution (0.5 mL) was added to quench the reaction, and a precipitate was formed. After the precipitation was complete, the resulting mixture was filtered through diatomaceous earth to remove the precipitate and washed with ethyl acetate. The filtrate from the filtration and washing steps was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 100:3, v / v), lyophilized, and the resulting yellow solid compound of formula (3) (200 mg, yield 80.6%) was obtained.

[0043] 1 H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 10.0 Hz, 2H), 7.54 (d, J = 10.0Hz, 2H), 7.48 (d, J = 10.0 Hz, 2H), 6.95 (tt, J = 10.0, 5.0 Hz, 1H), 4.81 (s, 2H).

[0044] 13 C NMR (126 MHz, CDCl3) δ 164.29, 164.19, 162.31, 162.20, 154.60, 154.52, 154.45, 151.51, 144.94, 127.46, 123.50, 106.21, 106.16, 106.05, 106.00, 105.80, 105.59, 64.77. 4. Synthesis of compound (4): At 0 °C, triphenylphosphine (1.5 mmol, CAS No.: 603-35-0) and N-bromosuccinimide (1.5 mmol, CAS No.: 128-08-5) were added sequentially to an anhydrous tetrahydrofuran (11 mL) solution of compound (3) (1 mmol), and then the mixture was brought to room temperature and stirred for 1 hour. After the reaction was completed, the resulting mixture was filtered through diatomaceous earth, the filtrate was collected and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate = 80:20, v / v), lyophilized, and the resulting yellow solid compound (4) (150 mg, 48.9%) was obtained.

[0045] 1 H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 10.0 Hz, 2H), 7.58 (d, J = 5.0Hz, 2H), 7.49 (d, J = 5.0 Hz, 2H), 6.96 (tt, J = 10.0, 5.0 Hz, 1H), 4.57 (s, 2H).

[0046] 13 C NMR (126 MHz, CDCl3) δ 164.29, 164.19, 162.30, 162.20, 154.50,154.43, 154.36, 151.71, 141.63, 130.00, 123.68, 106.30, 106.25, 106.23,106.14, 106.09, 106.02, 105.81, 32.46.

[0047] 5. Synthesis of compound (5): Compound (1 mmol) and anhydrous potassium carbonate (2.5 mmol) were dissolved in acetonitrile (10 mL), stirred at room temperature for 30 minutes, then heated to 70 °C, and compound (4) (1.5 mmol) was added. The mixture was stirred at 70 °C for 4 hours. After the reaction was complete, the solvent in the resulting mixed solution was removed by rotary evaporation under vacuum. The residue was extracted with dichloromethane (150 mL), followed by extraction with saturated ammonium chloride (100 mL). The aqueous phase was extracted with dichloromethane (3 × 50 mL). The organic phases were combined and collected, dried with anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 100:3, v / v), lyophilized, and yielded compound (5) (365 mg, yield 44.4%) as a blue solid.

[0048] 1 H NMR (500 MHz, MeOD) δ 8.76 (d, J = 14.8 Hz, 1H), 8.07 (d, J = 10.0Hz, 2H), 7.78 (d, J = 10.0 Hz, 2H), 7.66 (s, 2H), 7.60 – 7.51 (m, 5H), 7.46(td, J = 10.0, 5.0 Hz, 1H), 7.20 – 7.14 (m, 2H), 6.57 (d, J = 15.0 Hz, 1H),5.50 (s, 2H), 4.80 (s, 2H), 4.46 (t, J = 5.0 Hz, 2H), 2.83 (t, J = 5.0 Hz, 2H), 2.75 (t, J = 5.0 Hz, 2H), 2.57 (t, J = 5.0 Hz, 1H), 2.43 (td, J = 10.0, 5.0 Hz, 2H), 2.11 (p, J = 5.0 Hz, 2H), 1.96 (t, J = 5.0 Hz, 2H), 1.83 (s, 6H).

[0049] 13C NMR (126 MHz, DMSO) δ 177.42, 170.82, 164.29, 164.18, 162.32,162.21, 161.73, 158.65, 154.45, 154.38, 153.44, 151.53, 145.06, 142.33,141.98, 141.61, 134.95, 130.43, 129.33, 128.92, 128.87, 127.65, 127.22,126.06, 123.80, 123.22, 115.67, 114.58, 113.20, 107.02, 106.82, 106.58,106.37, 103.92, 100.13, 93.34, 83.89, 72.87, 70.10, 60.23, 58.05, 55.39,50.68, 44.16, 28.86, 28.16, 26.69, 24.24, 21.23, 20.35, 15.76, 14.56. HRMS(ESI) Calcd for [C 44 H 40 F2IN3O3, MI] ＋ : 696.3032, Found: 696.3034.

[0050] 6. Synthesis of compound (6): Compound (5) (1 mmol), phenyl thiochloroformate (20 mmol, CAS 1005-56-7), and triethylamine (5 mmol) were dissolved in anhydrous dichloromethane (15 mL) and stirred at room temperature for 6 h. After the reaction was completed, the solvent in the resulting mixed solution was removed by rotary evaporation under vacuum. The residue was purified by reversed-phase high-performance liquid chromatography on a C18 column (mobile phase: CH3CN / H2O = 95:5, v / v), and lyophilized to obtain compound (6) (290 mg, 30.2%) as a blue solid.

[0051] 1 H NMR (500 MHz, CDCl3) δ 8.68 (d, J = 14.6 Hz, 1H), 8.03 (d, J = 10.0Hz, 2H), 7.74 (d, J = 5.0 Hz, 2H), 7.57 – 7.47 (m, 5H), 7.45 – 7.34 (m, 4H), 7.27 (d,J = 10.0 Hz, 1H), 7.22 (s, 1H), 7.13 (t, J = 10.0 Hz, 3H), 6.97 (td, J = 10.0, 5.0 Hz, 1H), 6.44 (d, J = 15.0 Hz, 1H), 5.44 (s, 2H), 4.40 (t, J =10.0 Hz, 2H), 4.23 (s, 2H), 2.75 (t, J = 5.0 Hz, 2H), 2.68 (t, J = 5.0 Hz, 2H), 2.41 (t, J = 5.0 Hz, 2H), 2.19 (s, 1H), 2.11 (t, J = 5.0 Hz, 2H), 1.93(t, J = 5.0 Hz, 2H), 1.81 (s, 6H). 13 C NMR (126 MHz, MeOD) δ 177.41, 164.48,164.37, 162.51, 162.40, 162.29, 159.28, 154.57, 153.91, 151.74, 145.55,141.91, 141.57, 140.99, 134.66, 129.54, 128.81, 127.77, 127.55, 126.76,126.29, 123.29, 122.35, 115.70, 114.60, 112.16, 105.70, 105.65, 105.57, 105.48, 102.78, 99.38, 82.32, 70.33, 69.99, 58.32, 56.08, 53.40, 50.45, 48.22, 43.39, 28.63, 27.11, 26.04, 23.91, 20.14, 15.13.

[0052] HRMS (ESI) Calculation for [C 51 H 44 F2IN3O4S, MI] ＋ : 832.3015, Found:832.3628.

[0053] 7. Synthesis of compound (7): Weigh 1.3 mmol of azidoacetic acid (CAS No. 18523-48-3) and 1.3 mmol of 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (CAS No. 148893-10-1) into a 100 mL round-bottom flask, add 15 mL of N,N-dimethylformamide, and stir at room temperature for 10 min. Then add a solution of 2,2',2''-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (CAS No. 173308-19-5) and N,N-diisopropylethylamine (1.6 mmol) in N,N-dimethylformamide, and stir at room temperature for 4 h. After the reaction was completed, the N,N-dimethylformamide was removed by vacuum distillation of the resulting mixed solution. The residue was extracted with a mixed solvent of dichloromethane / water, separated and the aqueous phase was retained. The aqueous phase was washed three times with dichloromethane. The organic phases from the three washing steps and the extraction step were combined. The organic phase was dried with anhydrous sodium sulfate for 30 min, and then the solvent was removed by vacuum distillation. The residue was purified and separated by silica gel column chromatography (mobile phase: dichloromethane / methanol = 96:4) to obtain an oily compound of formula (7) (320 mg, yield 47.0%).

[0054] 1 H NMR (500 MHz, CDCl3): δ 3.89 (s, 2H), 3.36 (s, 8H), 2.80 (s, 8H), 2.66 -2.58 (d, J = 40.0 Hz, 4H), 2.23 - 2.20 (m, 6H), 1.66 (s, 2H), 1.46-1.45 (d, J = 6.1 Hz, 27H).

[0055] 8. Synthesis of compound (8): Weigh 0.43 mmol of compound (7) into a 100 mL round-bottom flask, add 60 mL of 4 M hydrogen chloride-ethyl acetate solution, and react at room temperature for 4 h. A white solid precipitates. After the reaction is complete, the resulting mixed solution... Hydrogen chloride-ethyl acetate solution was removed by rotary evaporation under reduced pressure. The residue was dissolved in 40 mL of ultrapure water and gadolinium chloride (0.86 mmol) aqueous solution was added. The pH of the system was then adjusted to maintain at 5.8–6.2. After the reaction was completed, half of the solvent in the resulting mixed solution was removed by rotary evaporation under vacuum. The residual solution was filtered through a 0.22 μm nylon filter. The filter cake was purified by reverse column chromatography (mobile phase: CH3CN / H2O = 95:5, v / v). Finally, it was lyophilized under vacuum at -80 °C to obtain a white solid compound of formula (8) (215.4 mg, yield 73.1%).

[0056] HRMS (ESI) Calculation for [C 20 H 32 GdN9O8, M] ＋ : 684.1615, Found: 685.1619.

[0057] 9. Synthesis of therapeutic probes: Compound (6) (21.9 μmol), compound (8) (32.1 μmol), anhydrous copper sulfate (6.4 μmol), ascorbic acid (25.7 μmol), N,N-dimethylformamide (0.2 mL), dimethyl sulfoxide (0.1 mL), and ultrapure water (0.25 mL) were added to a round-bottom flask and stirred at room temperature for 12 h. After the reaction was completed, the resulting mixed solution was concentrated under vacuum, and the residue was purified by preparative RP-HPLC on a C18 column (mobile phase: CH3CN / H2O = 95:5, v / v) to obtain a blue solid azoreductase-responsive targeted MASH therapeutic probe (15 mg, 41.7%), named AzoR-Cy-Gd.

[0058] The AzoR-Cy-Gd prepared in Example 1 was analyzed by liquid chromatography (C18 reversed-phase column)-mass spectrometry (LC-MS). The obtained liquid chromatograms and mass spectra are shown below. Figure 1 a, Figure 1 As shown in b, by Figure 1 As can be seen from a, the elution time of AzoR-Cy-Gd is 32 min, indicating that AzoR-Cy-Gd has strong hydrophobicity.

[0059] Figure 1 The mass spectrometry of b showed that HRMS (ESI) Calcd for [C 71 H 76 F2GdIN12O 12 S, MI] ＋:1516.4630, Found: 1516.4805.

[0060] Experiment 1: Fluorescence spectroscopy test of AzoR-Cy-Gd of the present invention under the action of azo reductase Experimental methods: 1. Take 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL and 11 μL of azoreductase AzoR (1000 U / mL) stock solution, add PBS buffer (pH 7.4) and adjust the volume to 200 μL to obtain azoreductase AzoR solutions with concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 U / mL, respectively.

[0061] 2. Weigh 8.86 mg of NADH powder and add it to 5 mL of PBS buffer (pH 7.4) to prepare a NADH solution with a concentration of 2.5 mM.

[0062] 3. Prepare a 5 mM AzoR-Cy-Gd stock solution using DMSO with the AzoR-Cy-Gd prepared in Example 1. Then, take 1 μL of the AzoR-Cy-Gd stock solution and add it to 599 μL of PBS buffer (pH 7.4) to obtain a 600 μL AzoR-Cy-Gd solution.

[0063] 4. Mix 200 μL of 1 U / mL azoreductase AzoR solution, 200 μL of NADH solution, and 600 μL of AzoR-Cy-Gd solution to obtain a mixed solution. The final concentrations of AzoR, NADH, and AzoR-Cy-Gd in the mixed solution are 1 U / mL, 500 μM, and 5 μM, respectively. Then, incubate the mixed solution at 37 ℃ for different times (0, 30, 60, 90, 120, 150, 180, 210, and 240 min). After each incubation, record the fluorescence emission spectrum on a fluorescence spectrometer with the excitation wavelength set to 685 nm.

[0064] 5. Perform the tests with azoreductase AzoR solutions of 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 U / mL respectively, following the method in step 4.

[0065] 6. Set up a control experiment without adding azoreductase AzoR solution. In the control experiment, replace the azoreductase AzoR solution with an equal volume of PBS buffer (pH 7.4).

[0066] Experimental results: Fluorescence emission spectra of AzoR-Cy-Gd solution under the action of different concentrations of azo reductase AzoR solution are as follows: Figure 2 As shown, after the probe AzoR-Cy-Gd was activated by azoreductase, the fluorescence intensity increased from 15 × 10⁻⁶ to 10⁻⁶. 7 au increased to 760×10 7 The au value increased by approximately 50 times. This indicates that the AzoR-Cy-Gd probe exhibits good solution imaging performance.

[0067] Experiment 2: Relaxation rate test of AzoR-Cy-Gd under the action of azo reductase according to the present invention Experimental methods: 1. The AzoR-Cy-Gd prepared in Example 1 was prepared into a 5 mM AzoR-Cy-Gd stock solution using DMSO. Subsequently, AzoR-Cy-Gd solutions with concentrations of 50 μM, 100 μM, 150 μM and 200 μM were prepared using PBS buffer (pH 7.4).

[0068] 2. Add 200 μL of azo reductase (1000 U / mL) and 200 μL of NADH (25 mM) to 600 μL of 50 μM AzoR-Cy-Gd solution to prepare a mixed solution with an AzoR-Cy-Gd concentration of 50 μM.

[0069] 3. Prepare mixed solutions of AzoR-Cy-Gd with concentrations of 100 μM, 150 μM and 200 μM according to the method in step 2.

[0070] 4. The AzoR-Cy-Gd mixed solution with a concentration of 50 μM was incubated at 37 ℃ for 240 min. After incubation, the T1 value of the reaction solution was acquired using a 20 MHz magnetic resonance scanner (Bruker, Germany) through a series of inversion recovery fast spin echo sequences.

[0071] 5. Perform the tests on the mixed solutions of AzoR-Cy-Gd with concentrations of 100 μM, 150 μM and 200 μM respectively, according to step 4.

[0072] 6. Plot the concentration of AzoR-Cy-Gd solution on the x-axis and the relaxation rate r1 (i.e., 1 / T1) of AzoR-Cy-Gd solution at the corresponding concentration on the y-axis. Graph the collected data, fit the plot to obtain a standard curve, and calculate the longitudinal molar relaxation rate (r1, unit: mM) based on the slope of the standard curve. -1 s -1 ).

[0073] 7. Perform a control experiment without adding azoreductase AzoR solution, following the steps 1-6.

[0074] Experimental results: The longitudinal relaxation rate of AzoR-Cy-Gd solution with and without azoreductase activity is shown in the figure below. Figure 3 As shown, by Figure 3 It can be seen that, without the action of azoreductase, the longitudinal molar relaxation rate r1 of AzoR-Cy-Gd prepared in Example 1 is 8.9 mM. -1 s -1 Under the action of azoreductase, the longitudinal molar relaxation rate r1 of AzoR-Cy-Gd prepared in Example 1 was 14.10 mM. -1 s -1 The longitudinal molar relaxation rate increased by 1.6 times. This indicates that the probe AzoR-Cy-Gd exhibits good T1-weighted magnetic resonance imaging performance under the action of azo reductase.

[0075] Experiment 3: H2S release test of AzoR-Cy-Gd of the present invention Test method (methylene blue (MB) method): 1. Add the AzoR-Cy-Gd, NADH, carbonic anhydrase CA and azo reductase AzoR prepared in Example 1 to 0.01M PBS and mix well to obtain a mixed solution with final concentrations of 100 μM, 200 U / mL, 5 mM and 25 μg / mL for AzoR-Cy-Gd, AzoR, NADH and CA, respectively.

[0076] 2. Incubate the mixed solution at 37 °C. At different incubation times (0, 50, 100, 150, 200, 250, 300 min), add 100 μL of the solution to the wells of a 96-well plate. Then add 25 μL of 30 mM FeCl3 (dissolved in 1.2 M HCl solution) and 25 μL of 20 mM N,N-dimethyl-1,4-phenylenediamine sulfate (dissolved in 7.2 M HCl solution) to the wells of the 96-well plate. Incubate the 96-well plate in the dark for 60 min. Finally, measure the absorbance of the reaction solution in the wells of the 96-well plate at 750 nm using a microplate reader.

[0077] 3. Conduct a control experiment without adding carbonic anhydrase (CA) following the steps in steps 1-2.

[0078] Experimental results: The H2S release curve of AzoR-Cy-Gd solution under the action of azoreductase AzoR in the presence or absence of carbonic anhydrase (CA) is shown below. Figure 4As shown, by Figure 4 It is known that the carbonyl sulfide compound produced by the cleavage of the probe AzoR-Cy-Gd by the azo reductase AzoR can only be hydrolyzed in the presence of carbonic anhydrase to generate H2S.

[0079] Experiment 4: Optimal uptake time of AzoR-Cy-Gd in hypoxic cells according to the present invention. Test method: 1. Prepare a 5 mM stock solution of AzoR-Cy-Gd using DMSO. Then, add 20 μL of the stock solution to 9980 μL of fetal bovine serum-free (FBS) DMEM to prepare a 10 μM AzoR-Cy-Gd culture medium.

[0080] 2. Spread 4T1 cells at a rate of 1 × 10⁻⁶ cells per dish. 5 4T1 cells were seeded at a density of 1000 cells / h in confocal culture dishes and cultured overnight until adherence. The adhered 4T1 cells were then added to culture medium containing AzoR-Cy-Gd (10 μM) and incubated at 37°C for 10 h under a 5% O2 atmosphere. After incubation, the culture medium was discarded, and the hypoxic 4T1 cells were washed three times with PBS. Fresh PBS was then added to cover the hypoxic 4T1 cells, and cell images were taken at different time points (1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h) using a fluorescence microscope (Nikon, Japan) in the Cy5.5 channel (excitation / emission).

[0081] Experimental results: The graph shows the change in AzoR-Cy-Gd uptake by hypoxic 4T1 cells over time. Figure 5 As shown, by Figure 5 It was observed that the fluorescence / NMR signal intensity of AzoR-Cy-Gd in hypoxic 4T1 cells gradually increased with increasing co-incubation time, indicating that the uptake of this probe in hypoxic cells continued to accumulate over time. The uptake of AzoR-Cy-Gd by hypoxic 4T1 cells reached a plateau at 6 hours, at which point the cells exhibited good imaging capabilities.

[0082] Experiment 5: In vivo fluorescence imaging and T1-MRI in vivo imaging experiments of the present invention using AzoR-Cy-Gd. Test method: 1. In vivo fluorescence imaging: 1.1 Prepare a 5 mM stock solution of AzoR-Cy-Gd prepared in Example 1 using DMSO. Then, take 20 μL of the stock solution and add it to 980 μL of PBS buffer (pH 7.4) to prepare a 100 μM AzoR-Cy-Gd solution.

[0083] 1.2. Eight-week-old C57BL / 6 wild-type male mice (three mice per group) were fed MCD diet (methionine-choline deficient diet) for 8 weeks to establish the MASH mouse model. AzoR-C-Gd solution (0.0005 mmol / kg Gd) was injected into the MASH model mice via tail vein injection. 3+ Whole-body fluorescence images of MASH model mice were acquired using the IVIS Lumina XRMS Series III imaging system (excitation wavelength 710 nm, emission wavelength 760 nm) before injection and at 1, 2, 4, 8, 12, 24, and 48 hours after injection. Regions of interest (ROIs) were measured using Living Image software (PerkinElmer, USA), and fluorescence intensity was quantitatively analyzed.

[0084] 1.3. Eight-week-old wild-type male C57BL / 6 mice fed a normal diet were used as a control.

[0085] 2. In vivo T1-MRI imaging: 2.1 The AzoR-Cy-Gd prepared in Example 1 was prepared into a stock solution with a concentration of 50 mM using DMSO. Then, 60 μL of the stock solution was added to 940 μL of PBS buffer (pH 7.4) to prepare a 3 mM AzoR-Cy-Gd solution.

[0086] 2.2. Eight-week-old C57BL / 6 wild-type male mice (three mice per group) were fed MCD diet (methionine-choline deficient diet) for 8 weeks to establish the MASH mouse model. AzoR-Cy-Gd solution (0.015 mmol / kg Gd) was injected into the MASH model mice via tail vein injection. 3+Magnetic resonance imaging (MRI) was performed on mice before injection and at 1, 2, 4, 8, 12, 24, and 48 hours after injection. All MRI experiments were performed on a 4.7 T MRI scanner (Bruker, Germany), with mice scanned tail-first in a prone position. Images were acquired using the T1-RARE imaging sequence with the following parameters: repetition time / echo time (TR / TE) = 500 / 8 ms; image matrix 128 × 128; slice thickness 1 mm; number of slices 20; field of view 35 mm × 35 mm; and acquisition time for each MRI scan was 8 minutes and 32 seconds. The acquired MRI data were imported into RadiAnt DICOM Viewer in DICOM image format for quantitative image analysis. The percentage of signal enhancement (%SE) at each time point was calculated, using the tumor signal intensity (SI) in the plain scan (pre-injection) image as a baseline: %SE(t) = (SI(t) - SI(t=0)) / SI(t=0), calculated separately for each mouse and each scan.

[0087] 2.3. Eight-week-old wild-type male C57BL / 6 mice fed a normal diet were used as a control.

[0088] Experimental results: 1. In vivo fluorescence imaging images of MASH model mice at different time points after injection of AzoR-Cy-Gd solution are shown below. Figure 6 As shown, by Figure 6 It was found that fluorescence in the MASH model mice was mainly concentrated in the liver, reaching its peak brightness at 8 hours, and the brightness could last up to 48 hours. Eight hours after injection of AzoR-Cy-Gd solution, the fluorescence intensity in the liver of the MASH model mice was 2.8 times that of the control group. This indicates that the probe AzoR-Cy-Gd utilizes the highly expressed AzoR enzyme in the MASH liver as a "key" to activate imaging, and "locks" itself onto the lesion protein through a chemical reaction, thereby enabling long-term imaging.

[0089] 2. In vivo T1-MRI images of MASH model mice at different time points after injection of AzoR-Cy-Gd solution, as shown below. Figure 7 As shown, AzoR-Cy-Gd generated T1-MRI signals in the liver, illuminating the liver of MASH model mice and enabling precise imaging of the liver of MASH model mice. Eight hours after injection of AzoR-Cy-Gd solution, the T1-MRI signal intensity of the liver of MASH model mice increased by 58% compared with the control group mice.

[0090] The above results indicate that AzoR-Cy-Gd exhibits good dual-modal imaging performance, including both fluorescence imaging and T1-MRI.

[0091] Experiment 6: Therapeutic efficacy trial of the present invention AzoR-Cy-Gd against MASH Test method: 1. Eight-week-old C57BL / 6 wild-type male mice (three mice per group) were fed MCD diet (methionine-choline deficient diet) for 8 weeks to establish the MASH mouse model. AzoR-Cy-Gd solution (0.0005 mmol / kg Gd3+) was injected into the MASH model mice via tail vein injection every two days for three weeks.

[0092] 2. Blood was collected from the mouse eyeballs and placed in an EP tube. After standing, the supernatant was collected. The supernatant was then sent for testing of blood biochemical indicators such as ALT and AST. The mice were then sacrificed, and fresh livers were collected for testing of pro-inflammatory cytokine cytokines IL-6 and TNF-α.

[0093] 3. A control experiment was conducted by injecting AzoR-Cy-Gd solution into 8-week-old wild-type male C57BL / 6 mice fed a normal diet.

[0094] 4. A control experiment was conducted by injecting MASH model mice with an equal volume of PBS buffer (pH 7.4) to AzoR-Cy-Gd solution.

[0095] Experimental results: For ease of representation, the experimental group treated using the method in steps 1-2 is labeled AzoR-Cy-Gd, the control group treated using the method in step 3 is labeled MASH, and the control group treated using the method in step 4 is labeled control.

[0096] After three weeks of treatment with AzoR-Cy-Gd solution, the biochemical indicators of MASH model mice were as follows: Figure 8 As shown, Figure 8 The results showed that, compared with the MASH group mice, the AzoR-Cy-Gd group mice treated with AzoR-Cy-Gd solution for three weeks had significantly lower levels of blood biochemical indicators ALT and AST. Figure 8 a), while inflammatory factors IL-6 and TNF-α were also significantly reduced ( Figure 8 b). The above results indicate that AzoR-Cy-Gd can effectively alleviate MASH.

[0097] Preparation of compound AzoR-H Hemicyanine (1 mmol) and anhydrous potassium carbonate (2.5 mmol) were dissolved in acetonitrile (10 mL), stirred at room temperature for 30 minutes, then heated to 70 °C, and (E)-4-(bromomethyl)-azobenzene (1.5 mmol, CAS No.: 57340-21-3) was added. The reaction was continued at 70 °C for 4 hours with stirring. After the reaction was completed, the solvent in the resulting mixed solution was removed by rotary evaporation under vacuum. The residue was extracted with dichloromethane (150 mL), followed by extraction with saturated ammonium chloride (100 mL). The aqueous phase was extracted with dichloromethane (3 × 50 mL). The organic phases from the three washing and extraction steps were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 100:3, v / v), lyophilized, and yielded a blue solid compound AzoR-H (300 mg, yield 39.6%).

[0098] 1 H NMR (500 MHz, CDCl3) δ 8.64 (d, J = 15.0 Hz, 1H), 7.98 (d, J = 5.0Hz, 2H), 7.92 (d, J = 5.0 Hz, 2H), 7.66 (d, J = 10.0 Hz, 2H), 7.59 (d, J =10.0 Hz, 1H), 7.56 - 7.42 (m, 6H), 7.37 (t, J = 5.0 Hz, 1H), 7.31 (s, 1H),7.03 - 6.96 (m, 2H), 6.70 (d, J = 15.0 Hz, 1H), 5.35 (s, 2H), 4.70 (t, J =5.0 Hz, 2H), 2.83 (t, J = 5.0 Hz, 2H), 2.75 (t, J = 5.0 Hz, 2H), 2.54 (t, J =5.0 Hz, 2H), 2.19 - 2.07 (m, 3H), 1.93 (p, J = 5.0 Hz, 2H), 1.81 (s, 6H).

[0099] 13C NMR (126 MHz, CDCl3) δ 177.38, 161.94, 161.75, 154.38, 152.55,152.46, 146.03, 141.68, 141.46, 138.82, 133.93, 131.27, 129.33, 129.16,129.01, 128.20, 127.68, 127.25, 123.24, 122.91, 122.49, 116.20, 115.52,113.81, 112.94, 104.36, 102.18, 83.14, 70.56, 70.20, 53.47, 50.60, 44.60, 31.92, 31.66, 29.69, 29.66, 29.24, 28.46, 26.57, 24.68, 22.32, 20.34, 16.18, 1.02.

[0100] HRMS (ESI) Calculation for [C 43 H 40 IN3O2, MI] ＋ : 630.3111, Found: 630.3115.

[0101]

[0102] Preparation of compound AzoR-F The preparation method is basically the same as that of compound AzoR-H, except that (E)-4-(bromomethyl)-azobenzene is replaced with compound (4), and finally a blue solid compound AzoR-F (285 mg, 35.9%) is generated.

[0103] 1H NMR (500 MHz, CDCl3) δ 8.67 (d, J = 14.8 Hz, 1H), 7.99 (d, J = 10.0Hz, 2H), 7.70 (d, J = 10.0 Hz, 2H), 7.59 (d, J = 5.0 Hz, 1H), 7.53 (d, J =5.0 Hz, 1H), 7.50 - 7.47 (m, 3H), 7.44 (d, J = 10.0 Hz, 1H), 7.41 (d, J = 5.0Hz, 1H), 7.30 (s, 1H), 7.04 - 7.00 (m, 2H), 6.95 (t, J = 5.0 Hz, 2H), 6.72(d, J = 15.0 Hz, 1H), 5.38 (s, 2H), 4.71 (t, J = 5.0 Hz, 2H), 2.84 (t, J =5.0 Hz, 2H), 2.76 (t, J = 5.0 Hz, 2H), 2.55 (t, J = 5.0 Hz, 2H), 2.16 (t, J =5.0 Hz, 2H), 2.13 (t, J = 5.0 Hz, 1H), 1.95 (p, J = 5.0 Hz, 2H),1.86 (s, 6H)。

[0104] 13C NMR (126 MHz, CDCl3) δ 177.45, 164.28, 164.17, 162.29, 162.19,161.84, 161.70, 154.46, 154.41, 154.38, 154.31, 152.29, 151.82, 146.08,142.36, 141.73, 141.46, 139.94, 135.01, 133.81, 129.33, 128.96, 128.31,128.12, 127.74, 127.50, 127.28, 123.83, 123.63, 123.45, 122.52, 116.23, 116.09, 115.56, 113.69, 112.89, 106.29, 106.24, 106.12, 106.07, 106.03, 105.82, 104.45, 102.21, 83.12, 70.40, 70.22, 50.65, 44.61, 34.73, 34.23, 31.94, 31.65, 30.31, 29.71, 29.66, 29.38, 29.27, 28.48, 26.56, 24.72, 22.71, 20.34, 18.46, 16.18, 14.15.

[0105] HRMS (ESI) Calculation for [C 43 H 38 F2IN3O2, MI] ＋ : 666.2927, Found: 666.2969.

[0106]

[0107] Preparation of compound AzoR-CF3 The preparation method is basically the same as that of compound AzoR-H, except that (E)-4-(bromomethyl)-azobenzene is replaced with compound (9). The preparation method of compound (9) is basically the same as that of compound (4), except that 3,5-difluoroaniline is replaced with 3,5-bis(trifluoromethyl)aniline (CAS No.: 328-74-5). Finally, a blue solid compound AzoR-CF3 (300 mg, 33.6%) is generated.

[0108]

[0109] 1H NMR (500 MHz, CDCl3) δ 8.68 (d, J = 15.0 Hz, 1H), 8.37 (s, 2H),8.03 (d, J = 10.0 Hz, 2H), 7.98 (s, 1H), 7.73 (d, J = 5.0 Hz, 2H), 7.59 -7.52 (m, 2H), 7.52 - 7.43 (m, 2H), 7.42 - 7.35 (m, 1H), 7.32 (s, 1H), 7.08(d, J = 5.0 Hz, 1H), 7.02 (dd, J = 10.0, 5.0 Hz, 1H), 6.67 (t, J = 15.0 Hz,1H), 5.40 (s, 2H), 4.66 (t, J = 5.0 Hz, 2H), 2.81 (t, J = 5.0 Hz, 2H), 2.76(t, J = 5.0 Hz, 2H), 2.53 (t, J = 5.0 Hz, 2H), 2.18 - 2.11 (m, 3H), 1.93 (t,J = 5.0 Hz, 4H), 1.84 (s, 6H)。

[0110] 13 C NMR (126 MHz, CDCl3) δ 177.49, 161.88, 161.79, 154.47, 152.81,151.80, 146.10, 141.77, 141.44, 140.56, 133.92, 133.15, 132.88, 132.61,132.34, 129.29, 128.97, 128.38, 127.71, 127.28, 126.31, 124.14, 123.93,123.77, 123.01, 122.98, 122.57, 121.97, 119.80, 116.25, 115.51, 113.75,112.79, 104.30, 102.22, 83.05, 70.35, 70.25, 50.69, 44.56, 29.26, 28.48,26.53, 24.68, 20.35, 16.17。

[0111] HRMS (ESI) Calcd for [C 45 H 38 F6IN3O2, M-I] ＋: 766.2863, Found: 766.2840.

[0112] Experiment 7: Screening Experiment of Fluorescent Groups of the Invention Experimental methods: 1. Weigh 8.86 mg of NADH powder and add it to 5 mL of PBS buffer (pH 7.4) to prepare a NADH solution with a concentration of 2.5 mM.

[0113] 2. Prepare a 5 mM AzoR-H stock solution using DMSO. Then, add 1 μL of the AzoR-H stock solution to 599 μL of PBS buffer (pH 7.4) to obtain a 600 μL AzoR-H solution.

[0114] 3. Prepare 600 μL AzoR-F solution and AzoR-CF3 solution respectively according to the method in step 2.

[0115] 4. Mix 11 μL of azoreductase AzoR (1000 U / mL), 189 μL of PBS buffer (pH 7.4), 200 μL of NADH solution, and 600 μL of AzoR-H solution thoroughly to obtain a mixed solution. The final concentrations of AzoR, NADH, and AzoR-Cy-Gd in the mixed solution are 11 U / mL, 500 μM, and 5 μM, respectively. Then, incubate the mixed solution at 37 °C for different times (0, 30, 60, 90, 120, 150, 180, 210, and 240 min). After each incubation, record the fluorescence emission spectrum on a fluorescence spectrometer with an excitation wavelength of 685 nm.

[0116] 5. Perform the tests on the AzoR-F solution and AzoR-CF3 solution according to the method in step 4.

[0117] Experimental results: The fluorescence emission spectrum of AzoR-H solution under the action of azoreductase AzoR solution is as follows: Figure 9 As shown in figures a and 9d, after the probe AzoR-H was activated by azoreductase, the fluorescence intensity reached its peak at 120 min. The fluorescence emission spectrum of the AzoR-F solution under the action of azoreductase AzoR solution is shown in the figure below. Figure 9 As shown in b and 9d, after AzoR-F was activated by azoreductase, the fluorescence intensity reached its peak at 210 min. The fluorescence emission spectrum of AzoR-CF3 solution under the action of azoreductase AzoR solution is shown in the figure. Figure 9As shown in c and 9d, the fluorescence intensity of AzoR-CF3 remained almost unchanged after AzoR was activated by azoreductase. This indicates that the fluorine atom attached to azobenzene can significantly reduce the sensitivity to the nitrogen-nitrogen double bond while ensuring a complete response by azoreductase, thus enabling a slow and controlled release of H2S and avoiding excessively rapid H2S release, which could lead to excessively high local H2S concentrations and damage to cells.

Claims

1. A targeted MASH diagnostic and therapeutic probe based on azoreductase response, characterized in that... Its structural formula is as follows: 。 2. A method for synthesizing the azo reductase-responsive targeted MASH diagnostic and therapeutic probe as described in claim 1, characterized in that... Includes the following steps: S1. Under alkaline conditions, 2,4-dihydroxybenzyl alcohol reacts with heptamethrin in a Knovengel reaction to produce compound (1), the reaction formula of which is as follows: ； S2. In the presence of acid, ethyl 4-nitrosobenzoate undergoes a condensation reaction with 3,5-difluoroaniline to produce compound (2), the reaction formula of which is as follows: ； S3. In the presence of a reducing agent, compound (2) undergoes a reduction reaction to produce compound (3), and the reaction formula is as follows: ； S4. In the presence of a catalyst, compound (3) undergoes a bromination reaction with a brominating reagent to produce compound (4), the reaction formula of which is as follows: ； S5. Under alkaline conditions, compound (1) undergoes a substitution reaction with compound (4) to produce compound (5), and the reaction equation is as follows: ； S6. Under alkaline conditions, compound (5) undergoes a substitution reaction with phenyl thiochloroformate to produce compound (6), the reaction formula of which is as follows: ； S7. Under the presence of a base and an activator, 2,2,2-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester and azidoacetic acid undergo an amide condensation reaction to produce compound (7), the reaction formula of which is as follows: ； S8. Under acidic conditions, the compound of formula (7) first undergoes a hydrolysis reaction, and then undergoes a coordination reaction with the trivalent gadolinium salt. The reaction formula for compound (8) is as follows: ； S9. In the presence of a catalyst and a reducing agent, compounds of formula (6) and (8) undergo a click chemistry reaction to generate the azo reductase-based targeted MASH diagnostic and therapeutic probe, the reaction formula of which is as follows: 。 3. The method for preparing the azo reductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S1, the alkali is anhydrous potassium carbonate, the solvent is anhydrous N,N-dimethylformamide, the Knovengel reaction temperature is 70-80 °C, the time is 4-8 h, and the molar ratio of 2,4-dihydroxybenzyl alcohol, heptamethrin and alkali is 1.0:0.5-2.0:1.0-2.

5.

4. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S2, the acid is glacial acetic acid, the solvent is glacial acetic acid, the condensation reaction temperature is room temperature, the time is 10~12 h, and the molar ratio of ethyl 4-nitrosobenzoate to 3,5-difluoroaniline is 1.0:1.0~3.

0.

5. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S3, the reducing agent is lithium aluminum hydride, the solvent is tetrahydrofuran, the temperature of the reduction reaction is -40~-20℃, the time is 1~2 h, and the molar ratio of compound (2) and lithium aluminum hydride is 1.0:1.0~3.

0.

6. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S4, the catalyst is triphenylphosphine, the brominating agent is N-bromosuccinimide, the solvent is anhydrous tetrahydrofuran, the bromination reaction temperature is 20~35℃, the time is 0.5~1.5 h, and the molar ratio of compound (3), triphenylphosphine, and N-bromosuccinimide is 1.0:1.0~2.0:1.0~4.

0.

7. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S5, the base is anhydrous potassium carbonate, the solvent is acetonitrile, the temperature of the substitution reaction is 70~80℃, the time is 3~6 h, and the molar ratio of compound (1), compound (4) and base is 1.0:1.5~7.5:2.5~12.

5.

8. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S6, the base is triethylamine, the solvent is anhydrous dichloromethane, the temperature of the substitution reaction is 20~35 ℃, the time is 6-12 h, and the molar ratio of compound (5), phenyl thiocarbamate and base is 1.0:5.0~20.0:5.0~10.

0.

9. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S7, the base is N,N-diisopropylethylamine, the activator is 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate, the solvent is N,N-dimethylformamide or triethylamine, the amide condensation reaction temperature is 20~35 °C, the time is 4-10 h, and the molar ratio of 2,2',2''-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester, azidoacetic acid, activator and base is 1.0:1.3~5:1.3~5:1.6~6.

10. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S8, the acidic reagent used is ethyl acetate hydrochloric acid solution, the hydrolysis reaction temperature is 20~35 ℃, and the time is 4-6 h; The trivalent gadolinium salt is gadolinium chloride. The coordination reaction temperature is 20~35 ℃, the time is 3~8 h, the solvent is ultrapure water, and the molar ratio of compound (7) and trivalent gadolinium salt is 1.0:2.0~4.

0.

11. The method for preparing the azoreductase-responsive targeted MASH diagnostic and therapeutic probe according to claim 2, characterized in that: In step S9, the catalyst is anhydrous copper sulfate, the reducing agent is ascorbic acid, the solvent is a mixed solvent of N,N-dimethylformamide, dimethyl sulfoxide and ultrapure water, the temperature of the click chemical reaction is 20~35 ℃, the reaction time is 12~24 h, and the molar ratio of compound (6), compound (8), catalyst and reducing agent is 1.0:1.5~4.0:0.3~3.0:1.2~5.

0.

12. The application of the azoreductase-responsive targeted MASH diagnostic and therapeutic probe of claim 1 in the preparation of a targeted liver multimodal imaging contrast agent and a drug for treating MASH, wherein the multimodal imaging contrast agent is a fluorescence imaging contrast agent and a T1-weighted MRI contrast agent.

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