Compound for in-vivo detection of carboxylesterase 2, or salt thereof, or stereoisomer thereof, and preparation method and application thereof

Abstract

The invention provides a compound for in-vivo detection of carboxylesterase 2, or a salt thereof, or a stereoisomer thereof, and a preparation method and application thereof, and belongs to the technical field of in-vivo luminescence detection of organisms. The invention provides a bioluminescent probe as shown in a formula I which is described in the specification. The probe is high in sensitivity, good in selectivity and suitable for accurate quantitative and qualitative analysis of carboxylesterase 2. Moreover, the preparation process is simple and convenient, the preparation cost is low, the operation condition is mild, the biocompatibility is good, the application prospect is wide, and the method is suitable for medical and biological research on carboxylesterase 2 detection in a living body. # imgabs0 #

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, and a preparation method and application thereof. Background Art

[0002] Carboxylesterase is a type of Phase I drug metabolizing enzyme widely found in mammals, mainly in the liver. It can mediate and catalyze the hydrolysis of various ester bonds, amide bonds, thioester bonds, etc., participate in the detoxification and metabolism of various ester drugs, environmental toxins and carcinogens, and is closely related to the occurrence and development of various diseases. Therefore, the in vivo detection of carboxylesterase activity in living organisms is of great significance for understanding the metabolism and excretion of drugs, environmental substances and carcinogens containing ester bond structures in the body.

[0003] Although several effective methods have been established to assess carboxylesterase 2 (CES2) activity, including fluorescent probes 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 may damage cells, and tissue components also affect the intensity of fluorescence signals. In addition, the inherent defects of fluorescence imaging, such as photobleaching, autofluorescence, and light scattering, limit its application in in vivo biological detection. Autofluorescence increases background noise, and light scattering reduces the clarity and deep penetration of the signal, especially in living tissues. These problems affect its applicability for rapid detection.

[0004] Bioluminescence imaging has come into our sight because it does not require external light sources for excitation and avoids the problem of light damage in fluorescence imaging. Bioluminescence imaging is based on the principle of generating photons by chemical reactions catalyzed by enzymes in organisms. The most common bioluminescence system is the firefly luciferase-luciferin system. Its essence is that firefly luciferase catalyzes the substrate luciferin in the presence of energy (ATP) and oxygen, causing electron transitions. When the molecule returns from the excited state to the steady state, photons are generated and oxidized luciferin is released.

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

[0006] Document 1 (CN117024422A) and Document 2 (CN118026953B) respectively 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 these two probes are used to detect carboxylesterase 2 in organisms, there are problems of low detection signal-to-noise ratio and low sensitivity.

[0007] Summary of the invention

[0008] In order to solve the shortcomings of carboxylesterase 2 (CES2) optical probes, such as poor selectivity, low sensitivity and harsh detection conditions, the purpose of the present invention is to provide a compound, or a salt thereof, or a stereoisomer thereof for detecting carboxylesterase 2 in vivo, and 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, the method comprising 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] Further,

[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: dissolving compound 1 in anhydrous dichloromethane, and then adding an anhydrous dichloromethane solution containing compound 2 and a base;

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

[0023] Further,

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

[0025] And / or, in step (2), in the mixed solution of anhydrous methanol and water, the volume ratio of anhydrous methanol to 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 1:1-3;

[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] Further,

[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 in 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 solids, 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 uses cyclopropanecarboxyl as the recognition group and hydroxyl fluorescein as the bioluminescent group to synthesize a novel bioluminescent probe I for detecting carboxylesterase 2. 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 broken, thereby releasing hydroxyl fluorescein, which is activated by ATP, luciferase, and Mg in the organism. 2+ Strong bioluminescence can be produced under the action of the above factors. The luminescence intensity is linearly related to the concentration of CES2, which is 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, the sensitivity is significantly improved, and a higher detection signal-to-noise ratio can be provided.

[0044] The bioluminescent probe developed by the present invention 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. It has a low detection limit, and within a specific concentration range, the bioluminescent intensity is linearly correlated with the concentration of carboxylesterase 2 and the bioluminescent 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, especially suitable for medical and biological research on CES2 detection in vivo.

[0045] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0046] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to 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 graph 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 The mice in the control group and experimental group in Experimental Example 3 (FVB-Luc + Comparison of bioluminescence of transgenic mice. DETAILED DESCRIPTION

[0052] The raw materials and equipment used in the present invention are all known products, which 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 instrument (JEOL, Japan); LB983NightOWL II small animal living 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. Solution preparation

[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 MgCl2-containing solution. 2+ Tris-HCl buffer solution.

[0058] (2) Preparation of luciferase solution containing ATP: Luciferase was dispensed into 60 μg / tubes, and 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. Then 3.63 mg of ATP (adenosine-5'-triphosphate disodium salt hydrate, 98%, MW = 605.2) was 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: Use purchased PBS mixed salt to prepare a solvent system with a concentration of 25 mM (pH 7.2).

[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, and add PBS to dilute the probe solution to a 100 μM solution for later use. Prepare it before use and store it at low temperature and away from light.

[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, weight 20-30g. FVB-Luc + Transgenic mice can express luciferase throughout the body, making them an ideal animal model for real-time imaging of CES2 in vivo. + The transgenic mice were raised in a specific environment. The animals were adapted to the experimental environment one week before the experiment and were housed in groups under natural circadian lighting conditions. The temperature was (22±2)°C and the humidity was 50±10%. The animals 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 mice in each group. The control group was injected with 100 μL of Ⅰ (100 μM) solution through the tail vein, and the experimental group was injected with 100 μL of loperamide solution (10 mM), a specific inhibitor of carboxylesterase 2, through the tail vein to inhibit the activity of carboxylesterase 2. One hour later, 100 μL of Ⅰ (100 μM) solution was injected through the tail vein. Immediately imaged with a small animal live imager. Select the bioluminescence mode and the exposure time is 1 s.

[0067] Example 1. Preparation of the bioluminescent probe for detecting carboxylesterase 2 in vivo according to the present invention

[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 raised 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 mixture was reacted at 0°C for 3 h. The mixture was then moved to room temperature (26±2)°C for further reaction. The reaction time was 5 to 6 h. The mixture was dried by rotation. Ethyl acetate was added to dissolve the residue. Water was added to extract the organic phase. Anhydrous sodium sulfate was added to the organic phase for drying. The 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, a white solid powder probe I. 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). The yield was 67% and the purity was 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 placed 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 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 replicate wells were made for each concentration, and then images were taken under a living imager.

[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, and then ATP (2 mM, 50 μL) and Luciferase (20 μg / mL) were added for reaction, and the relative bioluminescence intensity was measured. The results are shown in Figure 2. Figure 3 As shown, the inserted graph is 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 It can be seen that 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 It can be seen that with the increase of carboxylesterase 2 concentration, the bioluminescence intensity gradually increased. In Tris-HCl buffer, the bioluminescence intensity showed a good linear relationship with carboxylesterase 2 in the concentration range of (0-20 μg / mL). The detection limit (LOD) of probe I for carboxylesterase 2 was 0.000673753 μg / mL. The above results show that probe I has good detection 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 probe I was fixed (10 μM, 50 μL), and different analytes were taken: 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 (5μg / mL); AchE (5μg / mL); Trypsin (5μg / mL); BchE (5μg / mL); chymotrypsin (5μg / mL); HSA (5μg / mL); CES1 (5μg / mL); CES2 (5μg / mL)) were prepared with buffer solution, incubated at 37°C after adding the probe for 30 minutes, and 50μL of luciferase (20μg / mL) solution containing ATP (2mM) was added / well, and the images were immediately taken under the in vivo imaging instrument. CES1 is carboxylesterase 1, and CES2 is carboxylesterase 2.

[0085] After probe I was incubated with various biologically active 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 It can be seen that among various enzymes and molecular compounds in the body, only carboxylesterase 2 caused strong bioluminescence and had strong selectivity relative to carboxylesterase 1, and other enzymes and molecular compounds in the body produced almost no obvious bioluminescence signal. It can be seen that probe I can selectively detect carboxylesterase 2 in the body without being interfered by other substances, and the probe I of the present invention has good specificity in 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 + Transgenic mice), 10 adult transgenic male mice were selected and healthy male FVB-Luc were transfected 2 h before imaging. +Transgenic mice were divided into two groups, 5 in each group, and anesthetized with isoflurane. The control group was injected with 100 μL of probe I (100 μM) solution through the tail vein; the experimental group was injected with 100 μL of loperamide (10 mM) solution through the tail vein to inhibit the activity of carboxylesterase 2. One hour later, 100 μL of probe I (100 μM) solution was injected into the tail vein. Immediately, imaging was recorded under a live imaging device, with the time recorded as 0 min, and then recorded every one minute until the bioluminescence intensity decreased. The total intensity of all luminous parts of the mouse except the tail was plotted.

[0091] 2 h before imaging, healthy male FVB-Luc + Transgenic mice were divided into two groups, 5 in each group. The control group was injected with 100 μL of Ⅰ (100 μM) solution through the tail vein, and the experimental group was injected with 100 μL of loperamide (10 mM) solution through the tail vein to inhibit the activity of carboxylesterase 2. One hour later, 100 μL of Ⅰ (100 μM) solution was injected through the tail vein, and the images were immediately taken with a small animal live imager. The bioluminescence mode was selected, and the exposure time was 1 s.

[0092] 2. Experimental results

[0093] Depend on Figure 5 It can be seen that 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 that before the probe injection, with a significant statistical difference. Compared with the control group, the bioluminescence intensity of mice injected with loperamide first and then injected with probe I 1 hour later was significantly lower than that of the control group, with a significant statistical difference.

[0094] Moreover, since the experiment was conducted under anesthesia in mice, the changes of the probe in the control group and the experimental group can also be observed, and the normal life state of the mice during and after the experiment can be observed. Strong bioluminescent signals can be observed by in vivo imaging of small animals, and the bioluminescent intensity reaches the maximum 1 minute after the probe is injected into the tail vein, and then tends to decrease. The above results show that probe I reacts quickly with carboxylesterase 2, and the bioluminescent signal generated is strong and relatively stable. Therefore, probe I can achieve 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, and a preparation method and application thereof. The probe provided by the present invention for detecting carboxylesterase 2 in vivo 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. 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 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, especially suitable for medical and biological research on CES2 detection in vivo, and 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 Ⅰ, 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, characterized in that: 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, characterized in that: 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; And / or, the order of adding materials in step (2) is: compound 3 is dissolved in a mixed solution of dichloromethane and anhydrous methanol, and then a mixed solution of anhydrous methanol and water containing a base and D-cysteine ​​hydrochloride is added.

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

1.

6. The preparation method according to claim 4, characterized in that: 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 1:1-3; 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, characterized in that: 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 in 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 solids, 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.

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