A compound, composition and use

Abstract

The application discloses a compound, a composition and application, and claims the compound shown in formula (I) or a salt thereof. The compound provided by the application is an enzyme responsive fluorescent substrate specially designed for sulfotransferase SULT1A1, and precise recognition and specific catalytic response to SULT1A1 are realized from the molecular structure level. The compound has a low fluorescent background itself, the sulfonated fluorophore in the catalytic reaction product has the characteristics of near-infrared emission and large Stokes shift, can effectively avoid the background interference of excitation light on fluorescent detection, and is excellent in specificity, stability and linearity. Therefore, the compound or the salt thereof and the composition containing the compound or the salt thereof have the prospect of being developed into a reagent, a kit or a microplate for in vitro quantitative or qualitative detection of SULT1A1 activity, in situ imaging and positioning of SULT1A1 in cells or in vivo, and high-throughput screening of SULT1A1 inhibitors or activators.

Description

Technical Field

[0001] This invention belongs to the field of chemistry and relates to new compounds, specifically to a compound, a composition, and its application. Background Technology

[0002] Sulfonate transferases (SULTs) are key phase II metabolic enzymes in the human body, widely involved in core biochemical metabolic processes. These enzymes catalyze the transfer of sulfonic acid groups from the cofactor 3'-adenosine-5'-phosphosulfate (PAPS) to endogenous substances or exogenous foreign substrates containing hydroxyl or amino groups, completing the sulfonation biotransformation of substances. There are multiple subtypes of SULTs in the human body, among which SULT1A1 is the most abundant and physiologically crucial subtype. Sulfonation, as an indispensable metabolic pathway, has bidirectional and diverse functional effects. It is not only a major detoxification metabolic process for drugs and exogenous toxins, but also an important pathway for the activation of some prodrugs and the generation of toxic metabolites, directly regulating the body's metabolic homeostasis and physiological and pathological states.

[0003] SULT1A1-mediated sulfonation metabolism disorders are closely related to the occurrence and development of various diseases. Under the cascade catalysis of gut microbiota and SULT enzymes, various endogenous metabolites in the body can undergo sulfonation modification, generating various uremic toxins and other metabolites. Under normal physiological conditions, the kidneys can efficiently clear sulfonated metabolites from the gut microbiota, preventing their accumulation in the body. However, in pathological states such as acute and chronic kidney disease, renal clearance function is significantly impaired, leading to obstructed excretion of toxic sulfonated metabolites such as p-cresol sulfate and indophenol sulfate, which continuously accumulate in the blood and kidney tissues. Numerous studies have confirmed that these metabolic abnormalities can further induce or aggravate kidney damage, liver and kidney fibrosis, inflammatory bowel disease, and atherosclerosis, and are also highly correlated with the pathological process and treatment prognosis of various malignant tumors. Therefore, accurate detection and dynamic monitoring of SULT1A1 enzyme activity has significant clinical value and scientific research significance for disease mechanism research, pathological state assessment, and intervention and treatment of related diseases.

[0004] Currently, the detection and characterization methods for sulfonate transferase activity still have significant technical shortcomings, greatly limiting basic research and translational applications related to SULT1A1. The mainstream detection methods at present heavily rely on large-scale LC-MS / MS instruments. This method is cumbersome, has a long detection cycle, and is highly dependent on equipment, making it impossible to achieve rapid, real-time sample detection, let alone meet the needs of high-throughput sample screening. Furthermore, existing traditional detection technologies and reported fluorescent probe systems have inherent limitations, only enabling endpoint quantitative detection of in vitro samples, failing to achieve real-time visual evaluation of SULT1A1 activity, and not supporting in-situ imaging and activity localization characterization at the cellular or in vivo levels.

[0005] In summary, the current lack of a highly specific, widely applicable SULT1A1 detection tool that can simultaneously perform in vitro quantitative detection and in vivo in situ imaging severely restricts research on SULT1A1 enzyme function, exploration of related disease mechanisms, and targeted drug screening. Therefore, developing a novel fluorescent probe substrate capable of specifically recognizing SULT1A1 and enabling precise in vitro enzyme activity detection and in situ visualization imaging in cells and in vivo has become a pressing technical challenge in this field. Summary of the Invention

[0006] The first objective of this invention is to provide a compound or its salt that can generate a fluorescent product by specific catalysis of the SULT1A1 enzyme. The second objective is to provide a composition containing the compound or its salt. The third objective is to provide specific uses of the compound or its salt and composition for preparing reagents, kits or microplates for in vitro quantitative or qualitative detection of sulfotransferase SULT1A1 activity, in situ imaging and localization of sulfotransferase SULT1A1 in cells or in vivo, and high-throughput screening of sulfotransferase SULT1A1 inhibitors or activators.

[0007] This invention provides a compound of formula (I) or a salt thereof.

[0008]

[0009] (I)

[0010] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

[0011] The core structure of formula (I) designed in this invention is adapted to the specific recognition and catalytic reaction of sulfonyltransferase SULT1A1, and the overall molecular structure highly matches the pocket configuration of the active site of SULT1A1. This core structure balances molecular structural stability, enzyme binding specificity, and fluorescence response performance, ensuring that the compound undergoes significant fluorescence spectral changes before and after sulfonation modification catalyzed by SULT1A1, achieving the detection characteristics of "no fluorescence / weak fluorescence background, strong fluorescence output after enzyme activation," while greatly avoiding the non-specific recognition of other subtypes of human sulfonyltransferases, thus ensuring its exclusive response capability to SULT1A1 at the molecular structure level.

[0012] Preferably, the compound or its salt is a compound or its salt of formula (II).

[0013]

[0014] (II)

[0015] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

[0016] The substituents in Formula (II) are further simplified while maintaining the core structure framework of Formula (I).

[0017] More preferably, the compound or its salt is a compound or its salt represented by formula (III).

[0018]

[0019] (III)

[0020] Among them, R1, R2, R3, R4, R5, R6, R 12 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

[0021] The substituents in Formula (III) are further simplified while maintaining the original core structure of Formula (II).

[0022] More preferably, the compound or its salt is a compound or its salt represented by formula (IV).

[0023]

[0024] (IV)

[0025] Among them, R3 and R4 are each independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

[0026] The substituents in the (IV) structure are further simplified while maintaining the core structure framework of the (III) structure.

[0027] More preferably, the compound or its salt is a compound or its salt represented by formula (V).

[0028]

[0029] (V).

[0030] Equation (V) represents the optimal specific implementation structure of this invention.

[0031] This invention provides a composition comprising any one of the compounds described above or their salts, and at least one excipient. The compound or its salt is an enzyme-responsive fluorescent substrate that specifically responds to the sulfotransferase SULT1A1. It is non-fluorescent or exhibits only weak fluorescence, and can undergo a structural transformation catalyzed by SULT1A1, activating and generating a significant fluorescent signal. It can specifically recognize and respond to SULT1A1 enzyme activity. By combining it with suitable functional excipients, the solubility, stability, and enzyme-catalyzed reaction compatibility of the fluorescent substrate can be significantly improved, optimizing the sensitivity and specificity of the detection system, and adapting it to various application scenarios such as in vitro enzyme activity detection, in vivo cell imaging, and high-throughput drug screening.

[0032] Preferably, the excipients are selected from at least one of buffers, solubilizers, stabilizers, antioxidants, antibacterial preservatives, ion modifiers, surfactants, and blocking agents. Specifically, the buffer stabilizes the pH of the reaction system, maintains the optimal catalytic activity environment for SULT1A1, and ensures the stable and efficient conduction of the enzymatic reaction; the solubilizer effectively improves the solubility of the fluorescent substrate in the aqueous system, preventing substrate precipitation and aggregation, and eliminating interference in the detection system; the stabilizer protects the substrate structure and SULT1A1 enzyme activity, improving the long-term storage stability of the reagent; the antioxidant prevents substrate oxidation; the antibacterial preservative inhibits microbial growth; the ion modifier provides the metal cofactors required for enzyme catalysis; the surfactant improves substrate dispersibility and also has cell permeability properties, making it suitable for cell imaging experiments; and the blocking agent blocks non-specific binding sites on the solid-phase carrier, effectively reducing background noise and significantly improving detection accuracy.

[0033] This invention provides the application of any of the compounds or salts thereof, or any of the compositions described above, in the preparation of reagents, kits, or microplates for the in vitro quantitative or qualitative detection of sulfotransferase SULT1A1 activity. The compounds, as SULT1A1-specific fluorescent substrates, can achieve qualitative discrimination and precise quantitative detection of SULT1A1 activity in biological samples based on the principle of enzyme-catalyzed fluorescence response, with a good linear correlation between fluorescence signal intensity and SULT1A1 enzyme activity. The prepared reagents can be configured as concentrated stock solutions or ready-to-use working solutions. The kits can be equipped with standards, controls, diluents, and stop solutions for standardized detection. The pre-fabricated microplates are compatible with high-throughput automated detection equipment, eliminating reliance on traditional large-scale LC-MS / MS instruments. The methods are simple to operate, fast, and highly sensitive, making them suitable for scientific research experiments, batch sample detection, and enzyme kinetic analysis.

[0034] This invention provides the application of any of the compounds or salts thereof, or any of the compositions described above, in the preparation of reagents or kits for in situ imaging and localization of the sulfotransferase SULT1A1 in cells or in vivo. The fluorescent substrate possesses good biocompatibility and cell membrane permeability, allowing it to penetrate into living cells and tissues. It specifically catalyzes and activates fluorescence only at the SULT1A1 enzyme expression site, enabling direct and in situ visualization of the expression distribution and abundance of SULT1A1 in cells, tissues, and living animals, achieving visualized dynamic monitoring. This effectively overcomes the limitations of traditional detection methods, which only allow endpoint detection, cannot provide in situ localization, and cannot dynamically track changes in enzyme activity. It provides an intuitive and visualized detection tool for studying the physiological mechanisms and pathological evolution of SULT1A1.

[0035] This invention provides the application of any of the compounds described above, or their salts, or any of the compositions described above, in the preparation of reagents, kits, or microplates for high-throughput screening of sulfotransferase SULT1A1 inhibitors or activators. Based on the specific enzyme-responsive fluorescence characteristics of the compounds of this invention, changes in fluorescence signal intensity can directly reflect the inhibitory or activating effect of the test compound on SULT1A1 enzyme activity, enabling rapid screening of small molecule compounds that regulate SULT1A1 activity. The prepared high-throughput screening reagents are compatible with pre-fabricated microplates and automated screening platforms for batch screening of compound libraries, resulting in high screening throughput, stable results, and good reproducibility, significantly improving the screening efficiency of SULT1A1-targeted drugs.

[0036] Beneficial effects:

[0037] The compounds or their salts provided by this invention are enzyme-responsive fluorescent substrates specifically designed for sulfonyltransferase SULT1A1, achieving precise recognition and specific catalytic response to SULT1A1 at the molecular structure level. The compounds of this invention have low fluorescence background, and the sulfonated fluorophores in the catalytic reaction products exhibit near-infrared emission and a large Stokes shift, effectively avoiding background interference from excitation light on fluorescence detection. They also demonstrate excellent specificity, stability, and linearity. Therefore, the compounds or their salts provided by this invention, as well as compositions containing these compounds or their salts, have the potential to be developed into reagents, kits, or microplates for in vitro quantitative or qualitative detection of SULT1A1 activity, in situ imaging and localization of SULT1A1 in cells or in vivo, and high-throughput screening of SULT1A1 inhibitors or activators. Attached Figure Description

[0038] Figure 1 The 1H NMR spectrum and mass spectrometry detection spectrum of the target compound in Example 1 are shown below;

[0039] Figure 2The UV absorption spectrum and fluorescence spectrum of the target compound in Example 1 are shown below;

[0040] Figure 3 The UV absorption and fluorescence spectra of the target compound in Example 1 were obtained under different pH and solvent conditions;

[0041] Figure 4 The results of the specific response of the target compound to SULT1A1 in Example 1 are shown below; where A represents the response of the target compound to different isoforms of sulfonate transferase; B represents the change of the response level of the target compound to SULT1A1 over time; C represents the change of the fluorescence response of the target compound to SULT1A1 with enzyme concentration; and D represents the linear relationship between fluorescence intensity and enzyme concentration.

[0042] Figure 5 The results of the biocompatibility test of the target compound in Example 1 are shown.

[0043] Figure 6 The results of cellular fluorescence imaging of the target compound in Example 1;

[0044] Figure 7 The correlation between SULT1A1 protein expression level (A) and fluorescence intensity (B) in cells of each group;

[0045] Figure 8 The results are shown in mouse in vivo fluorescence imaging of the target compound in Example 1. Detailed Implementation

[0046] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0047] Example 1: Synthesis and structural confirmation of compounds

[0048] The target compound has the following structure and is named TMP-SIN-OH.

[0049]

[0050] The synthesis route is as follows:

[0051]

[0052] Synthesis steps:

[0053] 1. Synthesis of S-IN-OMe

[0054] 5-Methoxy-2-methyl-2H-indazole (200 mg, 1.24 mmol) was added to a 25 mL flask and dissolved in 5 mL of 1,4-dioxane. Then, 2-thiophenecarboxaldehyde (0.31 mL, 3.74 mmol), Cu(OAc)₂H₂O (375 mg, 1.86 mmol), pyridine (0.2 mL), and Pd(PPh₃)₄ (73 mg, 0.06 mmol) were added, and a magnetic stir bar was placed on the flask. The flask was fixed to a constant-temperature magnetic stirrer and heated to reflux at 120°C for 24 h under nitrogen protection. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, filtered, and the organic phase was concentrated by rotary evaporation. The crude product was purified by normal-phase silica gel column chromatography (PE / CH₂Cl₂ = 1:1, Et₃N) to give product S-IN-OMe (137.8 mg).

[0055] 2. Synthesis of TMP-SIN-OMe

[0056] S-IN-OMe (130 mg, 0.50 mmol) was added to a 50 mL round-bottom flask and dissolved in 20 mL of anhydrous ethanol. 2-(3,5,5-trimethylcyclohexyl-2-en-1-ethyl)malonadionitrile (123 mg, 0.59 mmol) and piperidine (0.5 mL) were added, and a magnetic stir bar was placed inside. The mixture was fixed to a magnetic stirrer with constant temperature heating, and the reaction was refluxed at 80°C for 3 h under nitrogen protection. After the reaction was complete, heating was stopped, and the mixture was allowed to cool naturally to room temperature. The organic phase was concentrated by rotary evaporation. The crude product was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to obtain the product TMP-SIN-OMe (149.2 mg).

[0057] 3. Synthesis of TMP-SIN-OH

[0058] TMP-SIN-OMe (145 mg, 0.33 mmol) was added to a 50 mL round-bottom flask and dissolved in 10 mL of CH2Cl2. A magnetic stir bar was placed in the flask, and the mixture was cooled to 0°C with stirring in an ice bath. BBr3 solution was added dropwise until the solution color changed from red to deep blue. The reaction temperature was slowly increased to room temperature and stirred overnight. After the reaction was complete, ice-cold distilled water was added to terminate the reaction, and the mixture was washed with a saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was purified by normal-phase silica gel column chromatography (PE / EA = 5:2) to obtain TMP-SIN-OH (68.3 mg). 1H NMR (500 MHz, DMSO-d6): δ 9.41 (s, 1H), 7.63-7.59 (m, 1H), 7.59-7.54 (m, 1H), 7.52 (d, J =4.1 Hz, 1H), 7.46-7.37 (m, 1H), 7.21 (d, J = 15.8 Hz, 1H), 7.12-7.07 (m, 1H), 6.95 (s, 1H), 4.26 (s, 3H), 2.64 (s, 2H), 2.56 (s, 2H), 1.05 (s, 6H). HRMS(ESI-MS, m / z): [M+H] + calcd for C 25 H 23 N4OS, 427.1593; found, 427.1540. Mass and proton spectra are shown below. Figure 1 As shown in Figures A and B.

[0059] Example 2: Catalytic response of the compound to sulfonate transferase

[0060] I. Experimental Materials

[0061] The test compound TMP-SIN-OH was prepared according to the method in Example 1.

[0062] Human hepatocellular carcinoma cell line HepG2; SPF-grade male C57BL / 6J mice, 6-8 weeks old, weighing 20-22 g; sulfonyltransferases SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1E1, SULT2A1, SULT2B1, SULT4A1; 3′-phosphoadenosine-5′-phosphorylsulfate (PAPS) tetralithium salt.

[0063] II. Experimental Methods

[0064] 1. Compound spectral testing

[0065] TMP-SIN-OH was prepared into a 1 mM stock solution with dimethyl sulfoxide (DMSO). Before spectral testing, it was diluted to 10 μM with DMSO and then spectrally tested using a BioTek Synergy H1 multi-functional microplate detector (λex = 480 nm, λem = 730 nm).

[0066] 2. Sulfonation reaction test

[0067] A 190 μL sulfonation metabolism reaction system was prepared in advance, including PBS buffer (pH 7.4), recombinant human sulfonate transferase (1.42 mg / mL, 0.5 μL), and TMP-SIN-OH (10 mM, 4 μL), and the operation was performed on ice. 10 μL of 2 mM PAPS was added to the above reaction system, and the system was quickly incubated in a 37 °C metal bath to initiate the reaction. After the reaction was completed, 50 μL of ice-cold methanol was added to terminate the reaction. After centrifugation at 4 °C, 14000 rpm, and 10 min, the supernatant was collected for fluorescence detection and HPLC-MS / MS analysis. The control group contained no PAPS or recombinant human sulfonate transferase to ensure that biotransformation depended on the enzyme and PAPS.

[0068] 3. Cytotoxicity test

[0069] When HepG2 cells reach the logarithmic growth phase, they can be digested and centrifuged, and the cells can be resuspended in culture medium at 5 × 10⁻⁶. 3 Cells were seeded at a density of 100 µL per well in 96-well plates. Cells were randomly assigned to a control group (containing cells, cultured in medium without TMP-SIN-OH), a TMP-SIN-OH treatment group (containing cells, cultured in medium containing TMP-SIN-OH), and a blank group (containing no cells, cultured in medium without TMP-SIN-OH). Each group had three replicates. After incubating the cells in an incubator for 6 h, 20 µL of MTT (5 mg / mL) was added to each well, and incubation continued for another 2 h. The plates were then removed, the liquid in the wells was aspirated, and 150 µL of DMSO was added to each well. The plates were then shaken at 37°C for 10 min on a microplate reader. The absorbance (OD) value was measured at 570 nm using a microplate reader.

[0070] 4. Cell fluorescence imaging

[0071] HepG2 cells were seeded in culture dishes and cultured until the cell density reached approximately 60% confluence. Then, TMP-SIN-OH was added to a final concentration of 10 µM and the cells were incubated at 37°C for 3 h. After incubation, the culture medium was discarded, and the cells were washed 2-3 times with PBS before fluorescence imaging.

[0072] 5. In vivo fluorescence imaging

[0073] Before the experiment, hair was removed from the imaging area of ​​the mice to avoid interference with fluorescence signal acquisition. The imaging system and computer software were turned on, and the signal pathway was set as follows: Ex: Fluorescence Green / Em: 720 nm. The gas anesthesia machine was turned on, the isoflurane concentration was adjusted, and the marked mice were anesthetized. After anesthesia, TMP-SIN-OH injection (100 μM, 200 μL) was injected intraperitoneally, and the mice were placed in the same position on the dark chamber platform. Fluorescence images were then acquired in the dark.

[0074] 6. Statistical Analysis

[0075] The experimental data were processed using Origin 2024 software, and the results are expressed as mean ± SD. Two-tailed unpaired t-tests were used to compare two independent samples to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.

[0076] III. Experimental Results

[0077] 1. Compound spectral test results

[0078] The TMP-SIN-OH provided by this invention has low fluorescence intensity before activation, and the UV absorption peak and fluorescence peak of the sulfonated fluorophore generated after activation are located at 480 nm and 730 nm, respectively. Figure 2 The fluorophore exhibits near-infrared emission and a large Stokes shift, effectively avoiding background interference from excitation light in fluorescence detection. It also possesses good tissue penetration, making it suitable for fluorescence imaging of deep tissues. This experiment further investigated the effects of different solvents and pH on the performance of the TMP-SIN-OH probe. Figure 3 The results showed that the probe could maintain stable low fluorescence characteristics under different physicochemical conditions when it was not activated, and had excellent specific response performance.

[0079] 2. Results of sulfonation reaction test

[0080] The specific response detection results of the TMP-SIN-OH probe to SULT1A1 are as follows: Figure 4 As shown, A represents the response of TMP-SIN-OH to different sulfonate transferase subtypes, B represents the change of TMP-SIN-OH response level to SULT1A1 over time, C represents the variation of TMP-SIN-OH fluorescence response to SULT1A1 with enzyme concentration, and D represents the linear relationship between fluorescence intensity and enzyme concentration.

[0081] To investigate the responsiveness of fluorescent probes to different isoforms of sulfonate transferases, the following isoforms were selected as test subjects: SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1E1, SULT2A1, SULT2B1, and SULT4A1. The results are as follows: Figure 4 As shown in Figure A, the fluorescence level was significantly increased in the SULT1A1 group, indicating that SULT1A1 can bind to the fluorescent probe TMP-SIN-OH and transfer the sulfonic acid group to generate a fluorophore product. Other isoforms, however, could not catalyze the sulfonation of the fluorescent probe.

[0082] The effect of incubation time on sulfonation level was investigated by setting different incubation times (0, 20, and 40 min) and detecting changes in fluorescence intensity at each time point. Figure 4 As can be seen from Figure B, the fluorescence intensity also increases with increasing incubation time (R). 2 = 0.9946), indicating that the enzymatic reaction continued during the 40 min incubation period, the sulfonation products accumulated continuously, and the reaction equilibrium was not reached or the enzyme activity did not decrease significantly.

[0083] By incubating different concentrations of SULT1A1 (0, 2.5, 5, 7.5, 10, 20 μg / mL) with the fluorescent probe, the fluorescence response of the fluorescent probe TMP-SIN-OH to SULT1A1 as a function of enzyme concentration was evaluated. Figure 4 (C). Within the concentration range of 0-20 μg / mL SULT1A1 ( Figure 4 In the middle D), there was a good linear relationship between fluorescence intensity and enzyme concentration (R). 2 =0.9917).

[0084] 3. Cytotoxicity test results

[0085] Cytotoxicity tests demonstrated that TMP-SIN-OH exhibits good cell biocompatibility. Figure 5 ).

[0086] 4. Cell fluorescence imaging results

[0087] Figure 7 To differentiate SULT1A1 gene expression between silencing (si-SULT1A1) and overexpression (OE-SULT1A1), fluorescence imaging was performed on cells in the control group and the gene silencing (siRNA) / overexpression (OE) groups. HepG2 cells were co-incubated with the TMP-SIN-OH probe in DMEM high-glucose medium at 37°C for 30 min, and the fluorescence intensity at 690 nm was detected by flow cytometry. Results are as follows: Figure 6As shown, the fluorescence signal of HepG2 cells incubated with the probe was significantly enhanced, and the intensity changes of the cell fluorescence signal were consistent with the upregulation and downregulation trends of SULT1A1 protein expression level. Figure 7 Therefore, it can be confirmed that intracellular fluorescence signals mainly depend on SULT1A1-mediated TMP-SIN-OH sulfonation.

[0088] 5. Results of in vivo fluorescence imaging

[0089] In in vivo fluorescence imaging experiments, high-intensity near-infrared fluorescence was observed in mice 10 minutes after injection of TMP-SIN-OH, peaking at approximately 30 minutes. Subsequently, the fluorescence in the mice gradually decreased over time, demonstrating a high metabolic clearance rate. Figure 8 ).

[0090] The above experimental results show that the TMP-SIN-OH probe prepared in Example 1 can specifically recognize and respond to the enzyme activity of SULT1A1. It has a low fluorescence background, and the sulfonated fluorophore in the catalytic reaction product has the characteristics of near-infrared emission and large Stokes shift, which can effectively avoid background interference of excitation light on fluorescence detection. It has excellent specificity, stability and linearity, and high biocompatibility.

[0091] Example 3: Kit for in vitro quantitative or qualitative detection of sulfotransferase SULT1A1 activity

[0092] This embodiment provides a detection kit for the in vitro quantitative or qualitative detection of sulfotransferase SULT1A1 activity. It is prepared based on TMP-SIN-OH prepared in Example 1 as an enzyme-responsive fluorescent substrate. By utilizing the characteristic that SULT1A1 can specifically catalyze the sulfonation modification of TMP-SIN-OH and generate a significant fluorescence signal enhancement, rapid, sensitive and specific detection of SULT1A1 activity in biological samples can be achieved.

[0093] The kit in this example includes fluorescent substrate working solution, reaction buffer, cofactor solution, sample dilution solution, stop solution, SULT1A1 standard, negative control solution, and concentrated washing solution.

[0094] Working principle: The active SULT1A1 in the sample can specifically recognize TMP-SIN-OH. With the participation of PAPS cofactor, it catalyzes a specific sulfonation reaction of the substrate, causing a change in the conjugated structure of the substrate molecule and generating a high-intensity fluorescence signal. The fluorescence signal intensity has a good linear relationship with the activity of SULT1A1 in the system. By detecting the fluorescence intensity at the corresponding wavelength and combining it with the standard curve plotted with the standard, the activity of SULT1A1 in the sample can be accurately and quantitatively detected. At the same time, rapid qualitative discrimination can be achieved based on the presence or absence of fluorescence.

[0095] This kit can be used to detect SULT1A1 activity in various in vitro samples such as cell lysates, tissue homogenates, and biological fluids. The detection operation is simple, does not require large-scale precision instruments such as LC-MS / MS, has a short detection cycle, high sensitivity and strong specificity, and does not have significant cross-reaction with other SULTs isoenzymes in the human body. It effectively solves the problems of high background interference, poor accuracy and inability to quickly detect in batches in existing detection technologies.

[0096] Example 4: SULT1A1 in situ imaging and localization kit for cells or in vivo

[0097] This embodiment provides a kit for in situ imaging and localization of the sulfotransferase SULT1A1 in cells or in vivo, prepared using TMP-SIN-OH as the core functional fluorescent probe prepared in Example 1. TMP-SIN-OH possesses excellent biocompatibility, cell membrane penetration ability, and high SULT1A1 enzyme-specific response characteristics. It is non-fluorescent or has only very weak background fluorescence, and its fluorescence output is activated only after being specifically catalyzed by SULT1A1 sulfonation. It can achieve in situ tracing, spatial localization, and activity visualization analysis of SULT1A1 at the cellular, tissue, and in vivo levels without damaging the physiological activity of cells or living tissues. This overcomes the technical drawbacks of traditional detection methods, which can only detect the endpoint by lysing the sample and cannot reflect the in situ distribution and dynamic changes of the enzyme.

[0098] The in situ imaging kit of this embodiment mainly includes fluorescence imaging working solution, cell incubation buffer, permeabilization enhancement solution, in vivo injection buffer, anti-fluorescence quenching mounting medium, and sterile cleaning solution.

[0099] Working Principle: The TMP-SIN-OH of this invention can freely penetrate living cell membranes or living tissue barriers. In areas within cells or tissues where SULT1A1 is not expressed, the substrate maintains its original weak fluorescence / non-fluorescence state with extremely low background. Only in subcellular regions or tissue sites where SULT1A1 is highly expressed and active, the substrate undergoes specific catalytic sulfonation modification, reconstructing the molecular conjugated system and generating a bright, specific fluorescence signal. Fluorescence signals can be acquired using fluorescence microscopy, confocal microscopy, or in vivo imaging systems, allowing for direct observation of the subcellular localization and tissue distribution characteristics of SULT1A1. Simultaneously, the activity and expression level of SULT1A1 can be semi-quantitatively analyzed based on fluorescence intensity.

[0100] This kit can be widely used for live cell in situ imaging, intracellular SULT1A1 subcellular localization analysis, in situ staining of pathological tissue sections, and dynamic visualization monitoring of SULT1A1 at the live level in model animals.

[0101] Example 5: Microplate for high-throughput screening of SULT1A1 inhibitors

[0102] This embodiment provides a prefabricated microplate for high-throughput screening of sulfotransferase SULT1A1 inhibitors. It is prepared using TMP-SIN-OH as the specific enzyme-responsive fluorescent substrate, combined with a suitable excipient system, and belongs to the category of solid-phase detection carriers for high-throughput drug screening. This invention utilizes the characteristic that TMP-SIN-OH is specifically activated by active SULT1A1, allowing for a direct reflection of the inhibitory effect of the target small molecule compound on SULT1A1 enzyme activity through changes in fluorescence signal intensity. This enables large-scale, automated, and highly reproducible screening of SULT1A1 inhibitors, overcoming the technical shortcomings of traditional screening methods, such as cumbersome operation, low throughput, poor stability, reliance on large mass spectrometry equipment, and high cost.

[0103] This embodiment is a prefabricated 96-well or 384-well high-throughput screening microplate. The substrate of the microplate is a polystyrene transparent plate, and the wells are preloaded with immobilized fluorescent substrate TMP-SIN-OH.

[0104] Working principle: A fixed amount of SULT1A1-specific fluorescent substrate is immobilized in the wells of a pre-fabricated plate. The screening system is then incubated with SULT1A1 enzyme solution, PAPS cofactor, and different candidate small molecule compounds. In the absence of inhibitors, SULT1A1 normally catalyzes the sulfonation reaction of the substrate, resulting in significant fluorescence activation. If the test compound exhibits SULT1A1 inhibitory activity, the SULT1A1 catalytic function is blocked or weakened, the substrate cannot effectively produce a fluorescence response, and the fluorescence intensity of the system is significantly reduced. The fluorescence signal values ​​of each well are quantitatively read using a microplate reader. By comparing the fluorescence difference between the blank control group and the negative control group, the enzyme inhibition rate of the test compound can be accurately calculated, allowing for rapid screening of lead compounds with SULT1A1 inhibitory activity.

[0105] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A compound of formula (I) or a salt thereof, （I） in, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

2. The compound or a salt thereof according to claim 1, wherein it is a compound or a salt thereof of formula (II), (II) in, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

3. The compound or a salt thereof according to claim 2, wherein it is a compound or a salt thereof of formula (III), (III) in, R1, R2, R3, R4, R5, R6, R 12 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

4. The compound or a salt thereof according to claim 3, wherein it is a compound or a salt thereof of formula (IV). (IV) in, R3 and R4 are each independently selected from hydrogen, halogens, and C. 1-3 Alkyl, C 1-3 Halogenated alkyl groups.

5. The compound or a salt thereof according to claim 4, wherein it is a compound or a salt thereof of formula (V). （V）。 6. A composition, characterized in that, It comprises the compound or salt thereof as described in any one of claims 1 to 5, and at least one excipient.

7. The composition according to claim 6, characterized in that, The excipients are selected from at least one of the following: buffers, solubilizers, stabilizers, antioxidants, antibacterial preservatives, ion modifiers, surfactants, and blocking agents.

8. The use of the compound of any one of claims 1 to 5 or a salt thereof, or the composition of any one of claims 6 to 7, in the preparation of reagents, kits, or microplates for the in vitro quantitative or qualitative detection of sulfotransferase SULT1A1 activity.

9. The use of the compound of any one of claims 1 to 5 or a salt thereof, or the composition of any one of claims 6 to 7, in the preparation of reagents or kits for in situ imaging and localization of sulfotransferase SULT1A1 in cells or in vivo.

10. The use of the compound of any one of claims 1 to 5 or a salt thereof or the composition of any one of claims 6 to 7 in the preparation of reagents, kits or microplates for high-throughput screening of sulfotransferase SULT1A1 inhibitors or activators.

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