A compound for detecting carboxylesterase 2 in vivo, or its salt, or its stereoisomer, and its preparation method and application

Abstract

The present invention provides a compound, salt, or stereoisomer thereof for detecting carboxylesterase 2 in vivo, as well as a preparation method and application thereof, belonging to the field of bioluminescent detection technology. The present invention provides a bioluminescent probe represented by Formula I. This probe has high sensitivity and good selectivity, and is suitable for accurate quantitative and qualitative analysis of carboxylesterase 2. Furthermore, the preparation process is simple, the production cost is low, the operating conditions are mild, and the probe has good biocompatibility and broad application prospects, making it suitable for medical and biological research on the detection of carboxylesterase 2 in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioluminescence detection, and specifically relates to a compound for detecting carboxylesterase 2 in vivo, or a salt thereof, or a stereoisomer thereof, as well as a preparation method and application thereof. Background Art

[0002] Carboxylesterases are a class of Phase I drug-metabolizing enzymes widely found in mammals, primarily in the liver. They mediate and catalyze the hydrolysis of various ester, amide, and thioester bonds, participating in the detoxification and metabolism of numerous ester drugs, environmental toxicants, and carcinogens. They are also closely associated with the development and progression of numerous diseases. Therefore, in vivo detection of carboxylesterase activity is crucial for understanding the metabolism and excretion of drugs, environmental pollutants, and carcinogens containing ester structures.

[0003] Although several effective methods for assessing carboxylesterase 2 (CES2) activity have been established, including fluorescent and near-infrared fluorescent probes (doi:10.1039 / d0cc00297f; doi:10.1016 / j.bios.2016.04.075; 10.1021 / acs.analchem.9b04189; doi:10.1039 / c9sc00283a), fluorescence imaging relies on excitation light, which can damage cells, and tissue composition can also affect the intensity of the fluorescence signal. Furthermore, inherent drawbacks of fluorescence imaging, such as photobleaching, autofluorescence, and light scattering, limit its application in in vivo bioassays. Autofluorescence increases background noise, while light scattering reduces signal clarity and deep penetration, particularly in living tissues. These issues hinder its applicability for rapid detection.

[0004] Bioluminescence imaging has come into our attention because it does not require external light sources for excitation, thus avoiding the photodamage problem that exists in fluorescence imaging. Bioluminescence imaging utilizes the principle of photon generation through enzyme-catalyzed chemical reactions within organisms. The most common bioluminescence system is the firefly luciferase-luciferin system. In essence, firefly luciferase catalyzes the substrate luciferin in the presence of energy (ATP) and oxygen, causing an electronic transition. When the molecule returns from an excited state to a steady state, photons are generated and oxidized luciferin is released.

[0005] Because there is almost no endogenous luminescence in living cells and tissues of humans and rodents, the background signal of bioluminescence is negligible and interference from biological matrix absorption is strongly suppressed. Therefore, bioluminescence imaging can provide a higher detection signal-to-noise ratio, high sensitivity, and good tissue penetration, making it more advantageous in in vivo biological detection. Therefore, bioluminescence detection provides an attractive alternative for CES2 analysis.

[0006] Document 1 (CN117024422A) and Document 2 (CN118026953B) each disclose a bioluminescent probe for detecting carboxylesterase 2 in organisms. However, the detection limit of the probe in Document 1 is 0.001034495 mg / mL, and the detection limit of the probe in Document 2 is 0.00154 μg / mL. When using these two probes to detect carboxylesterase 2 in organisms, there are problems with low signal-to-noise ratio and low sensitivity.

[0007] Summary of the Invention

[0008] In order to address the shortcomings of carboxylesterase 2 (CES2) optical probes, such as poor selectivity, low sensitivity, and harsh detection conditions, the present invention aims to provide a compound, or a salt thereof, or a stereoisomer thereof, for in vivo detection of carboxylesterase 2, as well as a preparation method and application thereof. The probe is specifically targeted at the CES2 enzyme, has extremely high selectivity and sensitivity, and can perform qualitative and quantitative analysis in complex biological environments.

[0009] The present invention provides a compound, or a salt thereof, or a stereoisomer thereof, wherein the structure of the compound is shown in Formula I:

[0010]

[0011] The present invention also provides a method for preparing the above compound, which comprises the following steps:

[0012]

[0013] (1) Compound 1 and Compound 2 react in a solvent under the action of a base to obtain Compound 3;

[0014] (2) Compound 3 and D-cysteine ​​hydrochloride react in a solvent under the action of a base to obtain compound I.

[0015] Furthermore,

[0016] In step (1), the solvent is anhydrous dichloromethane;

[0017] And / or, in step (1), the base is triethylamine or potassium carbonate;

[0018] And / or, in step (2), the solvent is a mixed solution of dichloromethane, anhydrous methanol and water;

[0019] And / or, in step (2), the base is triethylamine or potassium carbonate.

[0020] Further,

[0021] The order of adding materials in step (1) is as follows: compound 1 is dissolved in anhydrous dichloromethane, and then an anhydrous dichloromethane solution containing compound 2 and a base is added;

[0022] And / or, the order of adding materials in step (2) is: dissolving compound 3 in a mixed solution of dichloromethane and anhydrous methanol, and then adding a mixed solution of anhydrous methanol and water containing a base and D-cysteine ​​hydrochloride.

[0023] Further,

[0024] In step (2), the volume ratio of dichloromethane to anhydrous methanol in the mixed solution of dichloromethane and anhydrous methanol is 1:1;

[0025] And / or, in step (2), the volume ratio of anhydrous methanol to water in the mixed solution of anhydrous methanol and water is 1:1.

[0026] Further,

[0027] In step (1), the molar ratio of compound 1, compound 2 and base is 1:1-3:1-3;

[0028] And / or, in step (1), the reaction is first carried out at 0-4°C for 1-5 hours, and then at 20-40°C for 2-8 hours;

[0029] And / or, in step (2), the molar ratio of compound 3 to D-cysteine ​​hydrochloride is 1:1 to 3;

[0030] And / or, in step (2), the mass ratio of the compound 3 to the base is 1:1 to 2.5.

[0031] Further,

[0032] In step (1), the molar ratio of compound 1, compound 2 and base is 1:2.5:2.5;

[0033] And / or, in step (1), the reaction is first carried out at 0°C for 3 hours, and then at 25-30°C for 5-6 hours;

[0034] And / or, in step (2), the molar ratio of compound 3 to D-cysteine ​​hydrochloride is 1:2;

[0035] And / or, in step (2), the mass ratio of the compound 3 to the base is 1:1.2-1.5.

[0036] Furthermore,

[0037] In step (1), after the reaction, the reaction solution is purified to obtain compound 3, and the purification step comprises distilling the reaction solution under reduced pressure, extracting, drying, filtering, distilling under reduced pressure, and separating by column chromatography, wherein the eluent used for the column chromatography separation is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 15:1;

[0038] And / or, in step (2), after the reaction, the reaction solution is purified to obtain compound I, and the purification step comprises the steps of drying the reaction solution, adding water, adjusting the pH to 1 with hydrochloric acid, precipitating a solid, and filtering.

[0039] The present invention also provides use of the above compound, or a salt thereof, or a stereoisomer thereof in preparing a bioluminescent probe.

[0040] Furthermore, the bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2 in an organism.

[0041] The present invention synthesizes a novel bioluminescent probe I for detecting carboxylesterase 2 using cyclopropanecarboxyl as the recognition group and hydroxyl fluorescein as the bioluminescent group. The preparation method of the bioluminescent probe is simple, with high yield and low cost. The probe can selectively react with CES2 in vivo, and the ester group in the probe structure is hydrolyzed and cleaved, thereby releasing hydroxyl fluorescein, which is released in the body by ATP, luciferase, and Mg. 2+ Strong bioluminescence is generated under the action of the above agents. The luminescence intensity is linearly related to the CES2 concentration, making it suitable for accurate quantitative and qualitative analysis of CES2.

[0042] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0043] The detection limit of the probe in document 1 (CN117024422A) is 0.001034495 mg / mL, the detection limit of the probe in document 2 (CN118026953B) is 0.00154 μg / mL, and the detection limit of probe I of the present invention is 0.000673753 μg / mL, which significantly improves the sensitivity and can provide a higher detection signal-to-noise ratio.

[0044] The bioluminescent probe developed in this invention not only has mild operating conditions and good biocompatibility, but also can rapidly and highly selectively detect carboxylesterase 2 activity. The probe is virtually unresponsive to other potential interfering substances and exhibits extremely high sensitivity and good response to CES2. It has a low detection limit, and within a specific concentration range, the bioluminescence intensity is linearly correlated with the concentration of carboxylesterase 2, and the bioluminescent signal is stable, making it suitable for qualitative and quantitative analysis of CES2. Furthermore, the probe is simple to prepare, has low production costs, and has broad application potential, particularly in medical and biological research for in vivo CES2 detection.

[0045] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0046] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of probe I prepared in Example 1.

[0048] Figure 2 This is the mass spectrum of probe I prepared in Example 1.

[0049] Figure 3 This is the result of the bioluminescence linear response of probe I to carboxylesterase 2 in Experimental Example 1.

[0050] Figure 4 This is the selectivity result diagram of probe I for carboxylesterase 2 in Experimental Example 2.

[0051] Figure 5 For the mice in the control group and experimental group in Experimental Example 3 (FVB-Luc + Comparison of bioluminescence in transgenic mice. DETAILED DESCRIPTION

[0052] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0053] 1. Materials and instruments

[0054] Experimental animals: healthy male FVB-Luc + Transgenic mice (luciferase-expressing mice).

[0055] Instruments: ECZ-400S nuclear magnetic resonance spectrometer (JEOL, Japan); LB983NightOWL II small animal in vivo imager (BERTHOLD, Germany); YRT-3 melting point meter (Tianjin Tianda Tianfa Technology Co., Ltd.); ABIQ-starElite high-resolution mass spectrometer (Applied Biosystems, USA); automatic double-pure water distiller (Millipore, USA); PB-21 pH meter (Sartorius, Germany); RTC basic magnetic stirrer (IKA, Germany).

[0056] 2. Preparation of solution

[0057] (1) Preparation of Tris-HCl buffer solution containing MgCl2 (10 mM): First, 1.5143 g of Tris-base (tris(hydroxymethyl)aminomethane, 99.5%, MW = 121.14) was dissolved in 250 mL of water, and the pH was adjusted to 7.2-7.4 with HCl to obtain a 50 mM Tris-HCl buffer solution; then, 0.508 g of MgCl2 (MgCl2·6H2O, MW = 203.30) was dissolved in 250 mL of Tris-HCl buffer to obtain a 10 mM Tris-HCl buffer solution. 2+ Tris-HCl buffer solution.

[0058] (2) Preparation of luciferase solution containing ATP: Luciferase was divided into 60 μg / tubes. 3 mL of Tris-HCl buffer solution (50 mM, containing MgCl2 = 10 mM, pH = 7.4) containing MgCl2 (10 mM) was added to obtain a luciferase solution with a concentration of 20 μg / mL. 3.63 mg of ATP (adenosine-5'-triphosphate disodium salt hydrate, 98%, MW = 605.2) was then added to obtain a luciferase solution (20 μg / mL) containing ATP (2 mM), which was prepared and used immediately.

[0059] (3) Preparation of PBS solution: Prepare a 25 mM solvent system (pH 7.2) using purchased PBS mixed salts.

[0060] (4) Preparation of Probe I Stock Solution: Dissolve Probe I (0.35 mg, 0.001 mmol) in DMSO (333 μL) to obtain a 3 mM probe solution. Add PBS to dilute the probe solution to a 100 μM solution for later use. Prepare immediately before use and store at low temperature in the dark.

[0061] (5) Preparation of carboxylesterase 2 solution: Carboxylesterase 2 (50 μg) was dissolved in sterile water (100 μL) to obtain a 500 μg / mL solution. Sterile water was added to dilute the solution to (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) solutions for use.

[0062] (6) Preparation of loperamide solution: Loperamide (1.3 mg, 0.0025 mmol) was dissolved in 1.265 μL of DMSO, and then 2.53 μL of PEG was added. 200 , to obtain a 0.659 mM loperamide solution, which was diluted to a 10 mM solution by adding PBS for later use.

[0063] 3. Animal husbandry

[0064] FVB-Luc + Transgenic mice, weighing 20-30 g. FVB-Luc + Transgenic mice can express luciferase throughout the body, making them an ideal animal model for real-time imaging of CES2 in vivo. + Transgenic mice were raised in a specific environment. One week before the experiment, the animals were acclimatized to the experimental environment and housed in groups under natural circadian lighting conditions. The temperature was (22±2)°C, the humidity was 50±10%, and they had free access to food and water.

[0065] 4. In vivo bioluminescence imaging

[0066] 2 h before imaging, healthy male FVB-Luc + Transgenic mice were divided into two groups, 5 in each. The control group received a tail vein injection of 100 μL of Ⅰ (100 μM) solution, while the experimental group received a tail vein injection of 100 μL of loperamide (10 mM), a specific inhibitor of carboxylesterase 2, to inhibit carboxylesterase 2 activity. One hour later, 100 μL of Ⅰ (100 μM) solution was injected into the tail vein. Immediately, images were taken using a small animal live imager. Bioluminescence mode was selected, with an exposure time of 1 s.

[0067] Example 1: Preparation of a bioluminescent probe for detecting carboxylesterase 2 in vivo

[0068] The synthesis route of the bioluminescent probe for detecting carboxylesterase 2 in an organism of the present invention is as follows:

[0069]

[0070] (1) 528 mg (3.0 mmol) of 6-hydroxybenzothiazole-2-carbonitrile was dissolved in 10 mL of anhydrous dichloromethane. Under nitrogen protection, the temperature was brought to 0°C in an ice-water bath. 148 mg (7.5 mmol) of triethylamine and 1.646 mg (7.5 mmol) of 3-bromobenzoyl chloride dissolved in 5 mL of anhydrous dichloromethane were added dropwise. The reaction was carried out at 0°C for 3 h. The mixture was then moved to room temperature (26±2)°C and the reaction was continued for 5 to 6 h. The mixture was dried by rotation. Ethyl acetate was added to dissolve the residue. The organic phase was extracted with water. Anhydrous sodium sulfate was added to the organic phase for drying. The organic phase was filtered, dried by rotation, and separated by column chromatography. The eluent was petroleum ether / ethyl acetate (15:1, v / v). A yellow oily liquid intermediate 3 was obtained with a yield of 80%.

[0071] (2) 286 mg (1.0 mmol) of intermediate 3 was dissolved in 10 mL of dichloromethane and 10 mL of anhydrous methanol. Under nitrogen protection, 370 mg of potassium carbonate and 450 mg (2.0 mmol) of D-cysteine ​​hydrochloride dissolved in 2 mL of anhydrous methanol and 2 mL of distilled water were added dropwise. The reaction was carried out at 20°C to 30°C for 1 to 2 h. The reaction was dried by spin drying, 2 mL of distilled water was added, and the pH was adjusted to 1 with 1 mM hydrochloric acid. The solid was precipitated and filtered to obtain the target product, probe I, as a white solid powder. The structure of probe I is shown below:

[0072]

[0073] 1 H NMR (400 MHz, DMSO-d6) δ 8.06 (t, J = 1.9 Hz, 1H), 8.04–7.89 (m, 2H), 7.93–7.80 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 5.41 (dd, J = 9.7, 8.2 Hz, 1H), 3.76 (dd, J = 11.2, 9.8 Hz, 1H), 3.66 (dd, J = 11.3, 8.2 Hz, 1H). Yield: 67%, purity: 93%. Figure 1 is the H NMR spectrum of probe Ⅰ; Figure 2 This is the mass spectrum of probe I.

[0074] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0075] Experimental Example 1: Linearity test of probe I response to carboxylesterase 2

[0076] 1. Experimental methods

[0077] The concentration of fixed probe I (100 μM) and different concentrations of carboxylesterase 2 (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) were in a black 96-well plate. 50 μL of probe solution (concentration of 100 μM) and 50 μL of carboxylesterase 2 solution of different concentrations were first added to each well, and the plates were incubated at 37°C for 30 min. Then, 50 μL of luciferase (20 μg / mL) solution containing ATP (2 mM) was added. Three replicates were made for each concentration, and then imaging was performed under a live imaging device.

[0078] The probe (100 μM) was incubated with carboxylesterase 2 (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) in Tris-HCl buffer (50 mM, containing MgCl2 = 10 mM, pH = 7.4) at 37°C for 30 min. ATP (2 mM, 50 μL) and Luciferase (20 μg / mL) were then added for reaction, and the relative bioluminescence intensity was measured. The results are shown in Table 1. Figure 3 As shown, the inserted graph shows the linear relationship between the bioluminescence intensity and the concentration of carboxylesterase 2 (0-20 μg / mL).

[0079] 2. Experimental results

[0080] The experimental results show that when the probe concentration is 0, the standard deviation of the three groups of values ​​is 3050754.06. Figure 3 The slope is 4528000000, and the standard deviation divided by the slope gives the limit of detection (LOD), which is 0.000673753 μg / mL.

[0081] Depend on Figure 3 As can be seen, the bioluminescence intensity gradually increased with increasing carboxylesterase 2 concentration. In Tris-HCl buffer, the bioluminescence intensity exhibited a good linear relationship with carboxylesterase 2 over the concentration range of 0 to 20 μg / mL. The limit of detection (LOD) of Probe I for carboxylesterase 2 was 0.000673753 μg / mL. These results demonstrate that Probe I has excellent sensitivity and can quantitatively detect carboxylesterase 2 at the millimolar level in biological samples.

[0082] Experimental Example 2: Study on the Selectivity of Probes and Carboxylesterase 2

[0083] 1. Experimental methods

[0084] The concentration of fixed probe I (10 μM, 50 μL) was used to analyze different analytes: Blank; NaCl (1 mM); Na2CO3 (1 mM); MgCl2 (1 mM); NaNO3 (1 mM); ZnCl2 (1 mM); CaCl2 (1 mM); CuCl2 (1 mM); KCl (1 mM); Ser (1 mM); Trp (1 mM); Ala (1 mM); Thr (1 mM); Lys (1 mM); Cys (1 mM); Asp (1 mM); Phe (1 mM) ); ALT (5 μg / mL); AchE (5 μg / mL); Trypsin (5 μg / mL); BchE (5 μg / mL); chymotrypsin (5 μg / mL); HSA (5 μg / mL); CES1 (5 μg / mL); and CES2 (5 μg / mL) were prepared in buffer. After adding the probes, the cells were incubated at 37°C for 30 minutes. 50 μL of luciferase (20 μg / mL) solution containing ATP (2 mM) was added per well and the cells were immediately imaged using an in vivo imaging system. CES1 represents carboxylesterase 1, and CES2 represents carboxylesterase 2.

[0085] After incubation of probe I with various bioactive substances (Blank; NaCl; Na2CO3; MgCl2; NaNO3; ZnCl2; CaCl2; CuCl2; KCl; Ser; Trp; Ala; Thr; Lys; Cys; Asp; Phe; ALT; AchE; Trypsin; BchE; chymotrypsin; HSA; CES1; CES2), the bioluminescence intensity was observed.

[0086] 2. Experimental results

[0087] Depend on Figure 4 As can be seen, among various enzymes and molecular compounds in vivo, only carboxylesterase 2 elicited strong bioluminescence and exhibited strong selectivity over carboxylesterase 1. Other enzymes and molecular compounds in vivo produced almost no significant bioluminescent signal. This demonstrates that Probe I can selectively detect carboxylesterase 2 in vivo without interference from other substances, demonstrating that Probe I of the present invention exhibits high specificity for detecting carboxylesterase 2.

[0088] Experimental Example 3: FVB-Luc + Transgenic mouse imaging

[0089] 1. Experimental methods

[0090] The animals used were luciferase transgenic mice (FVB-Luc + 10 adult transgenic male mice were taken and healthy male FVB-Luc were placed in the 2h before imaging. +Transgenic mice were divided into two groups, five in each, and anesthetized with isoflurane. The control group received a tail vein injection of 100 μL of Probe I (100 μM); the experimental group received a tail vein injection of 100 μL of loperamide (10 mM) to inhibit carboxylesterase 2 activity. One hour later, the tail vein was injected with 100 μL of Probe I (100 μM). Immediately, the mice were imaged using a live imaging device, with time marked as 0 min. Thereafter, the images were recorded every minute until the bioluminescence intensity decreased. The total intensity of all bioluminescent areas of the mouse, excluding the tail, was plotted.

[0091] 2 h before imaging, healthy male FVB-Luc + Transgenic mice were divided into two groups, 5 in each. The control group received a tail vein injection of 100 μL of I (100 μM) solution, while the experimental group received a tail vein injection of 100 μL of loperamide (10 mM) solution to inhibit carboxylesterase 2 activity. One hour later, the tail vein was injected with 100 μL of I (100 μM) solution. Immediately, imaging was performed using a small animal in vivo imager. Bioluminescence mode was selected, with an exposure time of 1 s.

[0092] 2. Experimental results

[0093] Depend on Figure 5 The probe can also detect the presence of carboxylesterase 2 in animals. The bioluminescence intensity of mice injected with probe I was significantly higher than before probe injection, with a statistically significant difference. Compared with the control group, the bioluminescence intensity of mice injected with loperamide first and then probe I one hour later was significantly lower than that of the control group, with a statistically significant difference.

[0094] Furthermore, because the experiments were conducted with anesthetized mice, changes in the probe's activity in both the control and experimental groups could be observed without affecting the mice's normal life during and after the experiments. In vivo imaging of small animals revealed a strong bioluminescent signal, reaching its maximum intensity one minute after probe injection into the tail vein and then declining. These results suggest that Probe I reacts rapidly with carboxylesterase 2, generating a strong and stable bioluminescent signal. Therefore, Probe I is capable of bioluminescent imaging of endogenous carboxylesterase 2 in vivo.

[0095] In summary, the present invention provides a compound, a salt thereof, or a stereoisomer thereof for detecting carboxylesterase 2 in vivo, as well as a preparation method and application thereof. The probe provided by the present invention for detecting carboxylesterase 2 in vivo not only has mild operating conditions and good biocompatibility, but also can quickly and highly selectively detect the activity of carboxylesterase 2. The probe has almost no response to other potential interferents and exhibits extremely high sensitivity and good response performance to CES2. Its detection limit is low, and within a specific concentration range, the bioluminescence intensity is linearly correlated with the concentration of carboxylesterase 2 and the bioluminescence signal is stable, which can be used for qualitative and quantitative analysis of CES2. At the same time, the probe preparation process is simple, the production cost is low, and it has a wide range of application potential. It is particularly suitable for medical and biological research on CES2 detection in organisms, as well as in vitro detection of inhibitors against CES2.

Claims

1. A compound, or a salt thereof, or a stereoisomer thereof, characterized in that: The structure of the compound is shown in Formula I:

2. A method for preparing the compound according to claim 1, characterized in that: The method comprises the following steps: (1) Compound 1 and Compound 2 react in a solvent under the action of a base to obtain Compound 3; (2) Compound 3 and D-cysteine ​​hydrochloride react in a solvent under the action of a base to obtain compound I.

3. The preparation method according to claim 2, wherein: In step (1), the solvent is anhydrous dichloromethane; And / or, in step (1), the base is triethylamine or potassium carbonate; And / or, in step (2), the solvent is a mixed solution of dichloromethane, anhydrous methanol and water; And / or, in step (2), the base is triethylamine or potassium carbonate.

4. The preparation method according to claim 3, wherein: The order of adding materials in step (1) is as follows: dissolving compound 1 in anhydrous dichloromethane, and then adding an anhydrous dichloromethane solution containing compound 2 and a base; And / or, the order of adding materials in step (2) is: dissolving compound 3 in a mixed solution of dichloromethane and anhydrous methanol, and then adding a mixed solution of anhydrous methanol and water containing a base and D-cysteine ​​hydrochloride.

5. The preparation method according to claim 4, characterized in that: In step (2), the volume ratio of dichloromethane to anhydrous methanol in the mixed solution of dichloromethane and anhydrous methanol is 1:1; And / or, in step (2), the volume ratio of anhydrous methanol to water in the mixed solution of anhydrous methanol and water is 1:

1.

6. The preparation method according to claim 4, wherein: In step (1), the molar ratio of compound 1, compound 2 and base is 1:1-3:1-3; And / or, in step (1), the reaction is first carried out at 0-4°C for 1-5 hours, and then at 20-40°C for 2-8 hours; And / or, in step (2), the molar ratio of compound 3 to D-cysteine ​​hydrochloride is 1:1 to 3; And / or, in step (2), the mass ratio of the compound 3 to the base is 1:1 to 2.

5.

7. The preparation method according to claim 6, characterized in that: In step (1), the molar ratio of compound 1, compound 2 and base is 1:2.5:2.5; And / or, in step (1), the reaction is first carried out at 0°C for 3 hours, and then at 25-30°C for 5-6 hours; And / or, in step (2), the molar ratio of compound 3 to D-cysteine ​​hydrochloride is 1:2; And / or, in step (2), the mass ratio of the compound 3 to the base is 1:1.2-1.

5.

8. The preparation method according to claim 2, wherein: In step (1), after the reaction, the reaction solution is purified to obtain compound 3, and the purification step comprises distilling the reaction solution under reduced pressure, extracting, drying, filtering, distilling under reduced pressure, and separating by column chromatography, wherein the eluent used for the column chromatography separation is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 15:1; And / or, in step (2), after the reaction, the reaction solution is purified to obtain compound I, and the purification step comprises the steps of drying the reaction solution, adding water, adjusting the pH to 1 with hydrochloric acid, precipitating a solid, and filtering.

9. Use of the compound according to claim 1, or a salt thereof, or a stereoisomer thereof in the preparation of a bioluminescent probe.

10. The use according to claim 9, characterized in that: The bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2 in an organism.

Images