A ketamine small molecule probe and its synthesis method and application

Abstract

The present invention discloses a ketamine small molecule probe, a synthesis method, and an application thereof. Two small molecule probes are designed and synthesized, filling a gap in the prior art. Target fishing experiments are performed using these two probes on total proteins in cell lysates, yielding 61 and 70 target proteins, respectively. Furthermore, more than three technical repetitions have confirmed that both probes can stably bind to the target proteins. The SIRT2 protein obtained by screening is used as the target protein of ketamine, providing a reference for the synthesis and preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and in particular relates to a ketamine small molecule probe, a synthesis method and an application thereof. Background Art

[0002] Once a drug enters the human body, it acts on a specific molecular target, thereby modulating the disease phenotype to achieve the desired therapeutic effect. Therefore, target discovery and validation are crucial steps in the drug development process. Discovering and confirming the targets of small molecule drugs is currently a major task for drug developers. Over 95% of currently marketed drugs target proteins. Proteomics is the study of the protein composition and its dynamics within cells, tissues, or organisms, focusing on the proteome. Chemical proteomics technology boasts systematic, high-throughput, and high-precision capabilities. Combining multiple disciplines such as cell biology, synthetic chemistry, and biomass spectrometry, it provides a new platform for drug target screening, making it a popular choice among researchers in drug target discovery.

[0003] Activity-based protein profiling (ABPP) is a widely used chemical proteomics technique. The design and synthesis of activity probes is particularly critical. The probes must not interfere with the binding of the drug to the target protein, while effectively screening the target protein. Furthermore, the introduction of click chemistry and photoaffinity labeling has further expanded the scope of ABPP, but this technique has not yet been applied to ketamine.

[0004] Ketamine is a potent analgesic and dissociative anesthetic that has been widely used since its synthesis in 1962. S-ketamine nasal spray can relieve symptoms in patients with depression accompanied by acute suicidal thoughts or behaviors. In addition, recent studies have also found that ketamine has neuroprotective and anti-inflammatory effects. Animal and clinical trials have shown that low-dose ketamine can reduce inflammatory responses, improve the plasticity of neuronal dendritic spines, thereby improving mood and cognitive function, and reducing the occurrence of postoperative neurocognitive disorders. However, the targets and molecular mechanisms of ketamine's anti-inflammatory effects are not yet clear and require further research to determine. Therefore, this patent designs and synthesizes a ketamine small molecule probe, and uses this probe to discover new targets for ketamine. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a ketamine small molecule probe.

[0008] To solve the above technical problems, the present invention provides the following technical solution: the probe uses 6-hydroxyketamine as a substrate, as shown in the structural formula (III), and connects a propargyl group to the hydroxyl group or amino group of 6-hydroxyketamine to obtain a ketamine small molecule probe;

[0009]

[0010] As a preferred embodiment of the method for synthesizing the ketamine small molecule probe of the present invention, the ketamine small molecule probe comprises a ketamine small molecule probe having a structural formula as shown in formula (I) or formula (II);

[0011]

[0012] As a preferred embodiment of the synthesis method of the ketamine small molecule probe of the present invention, the ketamine small molecule probe having the structural formula shown in formula (I) is connected to the hydroxyl group of 6-hydroxylated ketamine by a propargyl group; the ketamine small molecule probe having the structural formula shown in formula (II) is connected to the amino group of 6-hydroxyketamine by a propargyl group.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for synthesizing a ketamine small molecule probe.

[0014] As a preferred embodiment of the synthesis method of the ketamine small molecule probe of the present invention, the synthesis method of 6-hydroxyketamine represented by the structural formula (III) comprises:

[0015] Ketamine is dissolved in anhydrous DMF, cooled to -80 to -70°C under nitrogen protection, and a tetrahydrofuran solution of potassium hexamethyldisilazide is added dropwise. After reacting for 30 minutes, the nitrogen is replaced with an oxygen system, and trimethyl phosphite is added. The reaction is carried out at low temperature for 2 to 4 hours. After the reaction is completed, the reaction is quenched and post-processed to obtain 6-hydroxyketamine as shown in formula (III);

[0016] The ratio of ketamine, potassium hexamethyldisilazide and trimethyl phosphite is 1:1.2-3:1.5-3.

[0017] As a preferred embodiment of the synthesis method of the ketamine small molecule probe of the present invention, the synthesis method of the ketamine small molecule probe having the structural formula shown in formula (I) comprises:

[0018] 6-Hydroxyketamine is dissolved in anhydrous DMF, and 3-bromopropyne, potassium tert-butoxide, and sodium iodide are added under nitrogen protection. The reaction is stirred until the reaction is complete, then quenched and post-treated to obtain a ketamine small molecule probe having a structural formula shown in formula (I);

[0019] The ratio of 6-hydroxylated ketamine, 3-bromopropyne, potassium tert-butoxide and sodium iodide is 1:1.2-1.5:2-2.5:2-2.5.

[0020] As a preferred embodiment of the method for synthesizing the ketamine small molecule probe of the present invention, the stirring reaction temperature is 25 to 35° C., and the reaction time is 2 to 4 hours.

[0021] As a preferred embodiment of the synthesis method of the ketamine small molecule probe of the present invention, the synthesis method of the ketamine small molecule probe having the structural formula shown in formula (II) comprises:

[0022] 6-Hydroxyketamine is dissolved in anhydrous DMF, and propidium bromide, cesium carbonate, and sodium iodide are added under nitrogen protection, and the reaction is stirred. After the reaction is completed, the reaction is quenched and post-processed to obtain a ketamine small molecule probe with a structural formula shown in formula (II);

[0023] The ratio of 6-hydroxylated ketamine, propyne bromide, cesium carbonate and sodium iodide is 1:1.2-1.5:2-2.5:2-2.5.

[0024] As a preferred embodiment of the method for synthesizing the ketamine small molecule probe of the present invention, the reaction temperature of the stirring reaction is 20-30° C., and the reaction time is 65-67 h.

[0025] Another object of the present invention is to overcome the deficiencies in the prior art and provide a ketamine small molecule probe for use in obtaining the target protein of ketamine by target fishing.

[0026] As a preferred embodiment of the application of the ketamine small molecule probe of the present invention, the ketamine small molecule probe with the structural formula shown in formula (I) is used to target 61 target proteins, and the ketamine small molecule probe with the structural formula shown in formula (II) is used to target 70 target proteins.

[0027] Beneficial effects of the present invention:

[0028] The present invention designed and synthesized two ketamine small molecule probes, and used these two probes to conduct a target fishing experiment on total proteins in mouse brain tissue lysates, screening 61 and 70 target proteins, respectively. After more than three technical repetitions, it was verified that both probes could stably bind to the target proteins, providing a reference for the study of the targets and molecular mechanisms of ketamine's anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0030] Figure 1 This is the hydrogen spectrum of hydroxylated ketamine prepared in Example 1 of the present invention.

[0031] Figure 2 This is the carbon spectrum of hydroxylated ketamine prepared in Example 1 of the present invention.

[0032] Figure 3 This is the hydrogen spectrum of probe 1 prepared in Example 2 of the present invention.

[0033] Figure 4 This is the carbon spectrum of probe 1 prepared in Example 2 of the present invention.

[0034] Figure 5 This is the hydrogen spectrum of probe 2 prepared in Example 3 of the present invention.

[0035] Figure 6 This is the carbon spectrum of probe 2 prepared in Example 3 of the present invention.

[0036] Figure 7 This is a flow chart of the target fishing experiment in Example 4 of the present invention.

[0037] Figure 8 This is a diagram showing the in vitro binding verification results of potential targets in Example 5 of the present invention.

[0038] Figure 9 This is a calculation simulation diagram of Example 6 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0042] The ketamine synthesis method in the present invention refers to the literature "Transition metal-free synthesis of α-aryl ketones via oxyallyl cation capture with arylboronic acids" and "Copper-Assisted Direct Nitration of Cyclic Ketones with Ceric AmmoniumNitrate for the Synthesis of Tertiaryα-Nitro,α-Substituted Scaffolds".

[0043] Unless otherwise specified, other raw materials used are commonly available in the market.

[0044] Example 1

[0045] This embodiment provides a method for synthesizing a ketamine small molecule probe having a structural formula as shown in formula (III), and a reaction formula as shown in formula (III-1), specifically:

[0046]

[0047] Ketamine (237.7 mg, 1 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), purged with nitrogen, and cooled to -78°C. A solution of potassium hexamethyldisilazide in tetrahydrofuran (2 M, 1.5 mL, 3 mmol) was slowly added dropwise. After completion of the addition, the reaction was allowed to proceed for 30 min. The nitrogen atmosphere was replaced with oxygen, and trimethyl phosphite (355 μL, 3 mmol) was slowly added. The reaction was maintained at low temperature for 2 h. TLC indicated that the reaction was complete, and the reaction was terminated.

[0048] Saturated ammonium chloride solution (10 mL) was added to the reaction system to quench the reaction. The above system was extracted with ethyl acetate (3×20 mL), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. After purification by column chromatography, 201 mg of hydroxylated ketamine was obtained as a white solid in a yield of 79.4%.

[0049] The hydrogen and carbon spectra of the product hydroxylated ketamine are as follows Figure 1 、 Figure 2As shown, the characterization data are:

[0050] 1 H NMR(300MHz, CDCl3)δ7.49(dd,J=7.8,1.4Hz,1H),7.42-7.27(m,2H),7.30(dd,1H,J=7.5,1.7Hz,), 4.19(dd,J=11.4,6.7Hz,1H),3.08-3.00(m,1H),2.39-2.30(m,1H),2.08(s,3H),1.79-1.41(m,4H).

[0051] 13 C NMR (75MHz, CDCl3) δ212.46,135.08,134.38,131.44,129.68,129.39,126.78,73.55,70.62,39.84,38.67,28.64,19.15.MS(ESI)m / z calcd for C 13 H 16 ClNNaO2 + [M+Na] + 276.08, found 275.99.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that the reaction conditions are adjusted, as shown in Table 1:

[0054] Table 1

[0055]

[0056] Example 2

[0057] This embodiment provides a method for synthesizing a ketamine small molecule probe having a structural formula as shown in formula (I), and a reaction formula as shown in formula (I-1), specifically:

[0058]

[0059] 2) Preparation of a ketamine small molecule probe having the structural formula (I):

[0060] Hydroxylated ketamine (50 mg, 0.2 mmol) was dissolved in anhydrous DMF (5 mL). After nitrogen protection, propargyl bromide (20 μL, 0.24 mmol), potassium tert-butoxide (44 mg, 0.4 mmol), and sodium iodide (60 mg, 0.4 mmol) were added respectively. The temperature was raised to 30° C. and stirred for 3 h. TLC showed that the reaction was complete, and the reaction was terminated. Saturated ammonium chloride solution (5 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (3×20 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. After purification by column chromatography, 26 mg of the ketamine small molecule probe represented by formula (I) was obtained in a yield of 44.8%.

[0061] The hydrogen and carbon spectra of the product are as follows Figure 3 、 Figure 4 As shown, the characterization data are:

[0062] 1 H NMR (300MHz, CDCl3) δ7.55(dd,J=7.7,1.8Hz,1H),7.35(dd,J=7.6,1.6Hz,1H),7.30-7.16(m,2H),6.03(dd,J=5.5,2.8Hz,1H),4.59(t, J=2.5Hz,1H),2.93-2.81(m,1H),2.68-2.54(m,1H),2.50(t,J=2.4Hz,1H),2.43-2.38(m,1H),2.37-2.31(m,1H),2.27(s,3H),2.11(br s,1H),1.94-1.86(m,1H).

[0063] 13 C NMR (75MHz, CDCl3) δ189.30,147.89,138.45,132.80,131.20,129.12,128.60,12 6.64,118.12,78.17,75.92,67.20,56.12,33.80,30.25,20.63.MS(ESI)m / zcalcd forC 16 H 17 ClNO2 + [M+H] + 290.09,found 290.01.

[0064] The difference between this comparative example and Example 2 is that the reaction conditions are adjusted, as shown in Table 2:

[0065] Table 2

[0066]

[0067] Example 3

[0068] This embodiment provides a method for synthesizing a ketamine small molecule probe having a structural formula as shown in formula (II), and a reaction formula as shown in formula (II-1), specifically:

[0069]

[0070] Hydroxylated ketamine (44 mg, 0.17 mmol) was dissolved in anhydrous DMF (5 mL). After nitrogen protection, propargyl bromide (17.5 μL, 0.21 mmol), cesium carbonate (113 mg, 0.334 mmol), and sodium iodide (52 mg, 0.34 mmol) were added, respectively. The reaction was stirred at 25°C for 66 h. TLC indicated the reaction was complete, and the reaction was terminated. Saturated ammonium chloride solution (5 mL) was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. After purification by column chromatography, 35 mg of the target ketamine small molecule probe with the structural formula shown in Formula (II) was obtained in a 70% yield.

[0071] The hydrogen and carbon spectra of the product are as follows Figure 5 、 Figure 6 As shown, the characterization data are:

[0072] 1 H NMR (300MHz, CDCl3) δ7.48(dd,J=7.8,1.4Hz,1H),7.43-7.34(m,2H),7.32(dd,J=7.4,1.7Hz,1H),4.31-4.22(m,1H),3.79(dd,J=16.9,2.3Hz,1H),3.72(br s,1H),3.15-3.03(m,2H),2.42(s,3H),2.32-2.25(m,1H),2.02(t,J=2.4Hz,1H),1.90-1.78(m,1H),1.74-1.67(m,1H),1.48-1.39(m,2H)

[0073] 13 C NMR (75MHz, CDCl3) δ211.44,135.26,132.52,131.71,130.18,129.82,127.02 ,80.82,74.65,74.33,71.87,41.28,39.51,37.99,35.03,19.00.MS(ESI)m / z calcd forC16 H 18 ClNNaO2 + [M+Na] + 314.09,found 314.03.

[0074] The difference between this comparative example and Example 3 is that the reaction conditions are adjusted, as shown in Table 3:

[0075] Table 3

[0076]

[0077] Example 4 Target fishing experiment

[0078] This example uses the ketamine probes prepared in Examples 2 and 3 to conduct a target fishing experiment, specifically:

[0079] The protein sample (1 mg / mL) was divided into two groups: a. blank control group and b. probe group, each with 1 mL. The probe was added to group b with a final concentration of 10 μM, and the same volume of DMSO was added to group a.

[0080] Incubate slowly on a shaker for 2 hours. Then, add 50 μL of n-butanol, 1 μL of 1 M tris(2-carboxyethyl)phosphine, 1 μL of 1 M copper sulfate, and 1 μL of 100 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine to each group in sequence and incubate for 1 hour to allow the system to undergo a click reaction. Then, add streptavidin magnetic beads washed with PBS (20 μL per group) and incubate with the above system for 1.5-2 hours. Finally, elute the protein on the magnetic beads with PBST, cook the sample, separate it by SDS-PAGE, and obtain protein bands by silver staining.

[0081] Reference Figure 7 The process is to incubate the probe with the total protein in the cell lysate to allow the protein to fully bind to the probe, then couple it to biotin through a click chemistry reaction, and then use streptavidin magnetic beads to separate and purify the target protein. Finally, all target protein information is obtained through silver staining and mass spectrometry analysis. Among them, probe 1 targeted 61 target proteins and probe 2 targeted 70 target proteins. The results are shown in Tables 4 and 5. There are 20 targets shared by the two, indicating that the probes synthesized by the present invention can effectively screen target proteins. SIRT2 was selected as the target protein in the following experiments.

[0082] Table 4

[0083]

[0084] Table 5

[0085]

[0086] Example 5 Potential target binding verification in vitro

[0087] Pulldown experiment Figure 8 A, B):

[0088] The target protein acquisition process of the pull-down experiment is the same as the target fishing in Example 4. Finally, the target protein band is detected by western blot experiment, as shown in Figure 8 A and B correspond to ketamine probes 1 and 2 prepared in Examples 2 and 3, respectively;

[0089] The SIRT2 protein in the probe group was much higher than that in the control group, and S-ketamine competed for binding with most of the SIRT2 protein, indicating that SIRT2 can bind well to S-ketamine, but R-ketamine cannot or rarely compete with the probe, so R-ketamine cannot bind to SIRT2.

[0090] A. Fluorescence titration experiment ( Figure 8 C):

[0091] Figure 8 C is the fluorescence titration of SIRT2 using S-ketamine and R-ketamine on a fluorescence spectrometer. First, the purified SIRT2 protein is diluted to 0.5μM (1mL) with PBS and the fluorescence intensity at this time is measured. Then, S-ketamine and R-ketamine are added respectively, and the fluorescence intensity of the system is measured when the ketamine concentration is 0.5-5μM. After each addition, the system is allowed to stand for 3 minutes to allow the drug to bind to the protein.

[0092] Data processing uses the ratio of fluorescence intensity of blank protein group and each drug-added group to plot, so the larger the slope, the greater the decrease in fluorescence intensity. Figure 8 As shown in C, with the increase of ketamine concentration, S-ketamine significantly reduced the fluorescence intensity of SIRT2 protein, while R-ketamine did not reduce it significantly, which also indicates that S-ketamine has a better binding effect with SIRT2.

[0093] B. Thermal denaturation experiment ( Figure 8 D, E):

[0094] The thermal denaturation experiment process is as follows: the cell lysate was divided into three groups, namely a. blank control group, b. S-ketamine group and c. R-ketamine group;

[0095] Groups b and c were added with S-ketamine and R-ketamine at a final concentration of 500 μM, respectively. After the three groups of samples were slowly incubated at room temperature for 2 h, each group of samples was divided into 8 200 μL centrifuge tubes, with 100 μL in each tube, corresponding to Figure 8The samples were heated at 8 temperatures as shown in D, and finally heated at 37, 42, 47, 52, 57, 62, 67, and 72 °C for 5 min to denature the proteins to varying degrees. The supernatant was collected and the samples were boiled. The target protein bands were detected by western blot. Figure 8 As shown in D, Figure 8 E is a line graph corresponding to the strips. Compared with the control group, the addition of S-ketamine reduced the stability of SIRT2 protein, indicating that S-ketamine can bind to SIRT2 protein, but R-ketamine does not bind to SIRT2 protein.

[0096] Example 6 Simulation verification of target protein

[0097] Molecular dynamics simulation was used to obtain the key residues and representative conformations in the interaction between S-ketamine and SIRT2, and a series of binding sites were obtained ( Figure 9 A) demonstrated that S-ketamine stably binds to SIRT2 protein and found a hydrogen bond between the ketone carbonyl of S-ketamine in the binding pocket and the side chain amide N atom of SIRT2 Q167 residue ( Figure 9 A). Due to hydrophobic interactions, hydrophobic residues tend to be buried inside proteins, thereby increasing protein folding and stability. S-ketamine binding reduces the percentage of hydrophobic regions in SASA buried by SIRT2, demonstrating that S-ketamine destabilizes SIRT2 ( Figure 9 B).

[0098] The root mean square deviation (RMSD) analysis of the backbone atoms of apo-SIRT2 and SIRT2 bound to S-ketamine showed that both systems quickly reached a stable state during the simulation ( Figure 9 C). To investigate the changes in protein stability caused by S-ketamine, root mean square fluctuation (RMSF) analysis was performed on the last 200 ns of the equilibrium trajectory. S-ketamine binding made most regions of SIRT2 more flexible, indicating that SIRT2 stability decreased after binding to S-KET ( Figure 9 D), which is consistent with the results of thermal denaturation experiments.

[0099] The above results show that the ketamine molecular probe synthesized by the present invention neither affects the binding with the target protein nor effectively screens out the target protein. Among them, the SIRT2 protein obtained by screening is the target of S-ketamine, and the binding effect of the two is excellent. In addition, SIRT2 protein is a target for anti-inflammatory effects, providing a reference basis for the synthesis and preparation of anti-inflammatory-related drugs.

[0100] In summary, to discover new ketamine targets from total proteins, the present invention designed and synthesized two small molecule probes, filling a gap in the existing technology. Using these two probes in a target fishing experiment on total proteins in cell lysates, 61 and 70 target proteins were identified, respectively. More than three replicates confirmed that both probes stably bound to their targets. Notably, more than ten of the targets identified in the fishing experiments were related to energy metabolism, worthy of further investigation.

[0101] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A ketamine small molecule probe, characterized in that: The probe uses 6-hydroxyketamine as a substrate, and connects a propargyl group to the hydroxyl group or amino group of 6-hydroxyketamine, thereby obtaining a ketamine small molecule probe with a structural formula as shown in formula (I) or formula (II). The ketamine small molecule probe with the structural formula shown in formula (I) is connected to the hydroxyl group of 6-hydroxylated ketamine with a propargyl group; the ketamine small molecule probe with the structural formula shown in formula (II) is connected to the amino group of 6-hydroxyketamine with a propargyl group.

2. The method for preparing a ketamine small molecule probe according to claim 1, wherein: The method for synthesizing the ketamine small molecule probe having the structural formula shown in formula (I) comprises: 6-Hydroxyketamine is dissolved in anhydrous DMF, and 3-bromopropyne, potassium tert-butoxide, and sodium iodide are added under nitrogen protection. The reaction is stirred until the reaction is complete, then quenched and post-treated to obtain a ketamine small molecule probe having a structural formula shown in formula (I); The ratio of 6-hydroxylated ketamine, 3-bromopropyne, potassium tert-butoxide and sodium iodide is 1:1.2-1.5:2-2.5:2-2.

5.

3. The method for preparing a ketamine small molecule probe according to claim 2, wherein: The stirring reaction temperature is 25-35° C., and the reaction time is 2-4 hours.

4. The method for preparing a ketamine small molecule probe according to claim 1, wherein: The method for synthesizing the ketamine small molecule probe having the structural formula shown in formula (II) comprises: 6-Hydroxyketamine is dissolved in anhydrous DMF, and propidium bromide, cesium carbonate, and sodium iodide are added under nitrogen protection, and the reaction is stirred. After the reaction is completed, the reaction is quenched and post-processed to obtain a ketamine small molecule probe with a structural formula shown in formula (II); The ratio of 6-hydroxylated ketamine, propyne bromide, cesium carbonate and sodium iodide is 1:1.2-1.5:2-2.5:2-2.

5.

5. The method for preparing a ketamine small molecule probe according to claim 4, wherein: The reaction temperature of the stirring reaction is 20-30° C., and the reaction time is 65-67 h.

6. The method for preparing a ketamine small molecule probe according to any one of claims 2 or 4, characterized in that: The synthesis method of 6-hydroxyketamine comprises: Ketamine is dissolved in anhydrous DMF, cooled to -80 to -70°C under nitrogen protection, and a tetrahydrofuran solution of potassium hexamethyldisilazide is added dropwise. After reacting for 30 minutes, the nitrogen is replaced with an oxygen system, and trimethyl phosphite is added. The reaction is carried out at low temperature for 2 to 4 hours. After the reaction is completed, the reaction is quenched and post-processed to obtain 6-hydroxyketamine as shown in formula (III); The ratio of ketamine, potassium hexamethyldisilazide and trimethyl phosphite is 1:1.2-3:1.5-3.

7. Use of the ketamine small molecule probe according to claim 1 in discovering the target protein of ketamine by target fishing.

8. Use of the ketamine small molecule probe according to claim 7 in discovering the target protein of ketamine by target fishing, characterized in that: The ketamine small molecule probe with the structural formula shown in formula (I) was used to target 61 target proteins, and the ketamine small molecule probe with the structural formula shown in formula (II) was used to target 70 target proteins.

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