Ketamine small molecule probe, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a ketamine small molecule probe, and a preparation method therefor and the use thereof. Two small molecular probes are designed and synthesized, and the blank in the prior art is filled. A target fishing experiment is performed on the total protein in a cell lysis buffer by means of using the two probes, and 61 and 70 target proteins are obtained, respectively. In addition, more than three technical repetitions verify that the two probes can stably bind to the target proteins. A screened SIRT2 protein is used as a target protein of ketamine, and a reference basis is provided for the synthesis and preparation of an anti-inflammatory related drug.

Description

A ketamine small molecule probe and its preparation method and application Technical Field

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

[0002] After entering the human body, drugs act on specific molecular targets, thereby regulating the disease phenotype to achieve the corresponding therapeutic effect. Therefore, target discovery and verification are crucial links in the drug development process. How to discover and confirm the targets of small molecule drugs is one of the main tasks of current drug developers. Among the drugs currently on the market, more than 95% of them have protein as their target molecules. Proteomics is a science that studies the protein composition and its changes in cells, tissues or organisms based on the proteome. Chemical proteomics technology has the characteristics of systematicity, high throughput and high precision. It combines multiple disciplines such as cell biology, synthetic chemistry and biological mass spectrometry, providing a new platform for drug target screening. Therefore, this technology is favored by 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.

[0005] Summary of the Invention

[0006] 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.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a ketamine small molecule probe.

[0009] To solve the above technical problems, according to one aspect of the present invention, a ketamine small molecule probe is provided, wherein the ketamine small molecule probe has a structure represented by the following formula (I) or formula (II):

[0010] According to another aspect of the present invention, a method for preparing a ketamine small molecule probe is provided, comprising: using 6-hydroxyketamine having a structure represented by the following formula (III) as a substrate, and attaching a propargyl group to the hydroxyl group or amino group of 6-hydroxyketamine to obtain the ketamine small molecule probe:

[0011] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the ketamine small molecule probe is a ketamine small molecule probe having a structure represented by formula (I) or formula (II):

[0012] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the ketamine small molecule probe having a structure represented by formula (I) has a structure in which a propargyl group is connected to the hydroxyl group of 6-hydroxyketamine; and the ketamine small molecule probe having a structure represented by formula (II) has a structure in which a propargyl group is connected to the amino group of 6-hydroxyketamine.

[0013] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the 6-hydroxyketamine having the structure represented by formula (III) is prepared by the following method:

[0014] Ketamine is dissolved in anhydrous DMF or tetrahydrofuran, 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 a low temperature for 2 to 4 hours. After the reaction is completed, the reaction is quenched and post-processed to obtain the 6-hydroxyketamine having the structure shown in formula (III).

[0015] The ratio of ketamine, potassium hexamethyldisilazide and trimethyl phosphite is 1:1.2-3:1.5-3.

[0016] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the ketamine small molecule probe having the structure represented by formula (I) is prepared by the following method:

[0017] 6-Hydroxyketamine was dissolved in anhydrous DMF, and propidium bromide, potassium tert-butoxide and sodium iodide were added under nitrogen protection. After stirring until the reaction was completed, the reaction was quenched and post-processed to obtain a ketamine small molecule probe having a structure shown in formula (I).

[0018] The ratio of 6-hydroxyketamine, propynyl bromide, potassium tert-butoxide and sodium iodide is 1:1.2-1.5:2-2.5:2-2.5.

[0019] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the reaction temperature is 25 to 35° C., and the reaction time is 2 to 4 hours.

[0020] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the ketamine small molecule probe having the structure represented by formula (II) is prepared by the following method:

[0021] 6-Hydroxyketamine was dissolved in anhydrous DMF, and propidium bromide, cesium carbonate and sodium iodide were added under nitrogen protection. After stirring until the reaction was completed, the reaction was quenched and post-processed to obtain a ketamine small molecule probe having a structure shown in formula (II).

[0022] Wherein, the ratio of 6-hydroxyketamine, propidium bromide, cesium carbonate and sodium iodide is 1:1.2-1.5:2-2.5:2-2.5.

[0023] As a preferred embodiment of the method for preparing the ketamine small molecule probe of the present invention, the reaction temperature is 20-30° C., and the reaction time is 65-67 h.

[0024] 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.

[0025] As a preferred embodiment of the application of the ketamine small molecule probe of the present invention, the ketamine small molecule probe having the structure shown in formula (I) targets 61 target proteins, and the ketamine small molecule probe having the structure shown in formula (II) targets 70 target proteins.

[0026] Beneficial effects of the present invention:

[0027] 1. The present invention designs and synthesizes two ketamine small molecule probes, filling the gap in ketamine target discovery in the prior art.

[0028] 2. The present invention 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.

[0029] 3. The screened SIRT2 protein is used as the target protein of ketamine, providing a reference for the synthesis and preparation of anti-inflammatory drugs.

[0030] 4. The probe design and application of the present invention provide new tools for drug developers, helping to discover and confirm the targets of small molecule drugs and promoting the drug development process. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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:

[0032] FIG1 is a hydrogen spectrum of 6-hydroxyketamine prepared in Example 1 of the present invention.

[0033] FIG2 is a carbon spectrum of 6-hydroxyketamine prepared in Example 1 of the present invention.

[0034] FIG3 is a hydrogen spectrum of the probe 1 prepared in Example 2 of the present invention.

[0035] FIG4 is a carbon spectrum of the probe 1 prepared in Example 2 of the present invention.

[0036] FIG5 is a hydrogen spectrum of the probe 2 prepared in Example 3 of the present invention.

[0037] FIG6 is a carbon spectrum of the probe 2 prepared in Example 3 of the present invention.

[0038] FIG7 is a flow chart of a target fishing experiment according to Example 4 of the present invention.

[0039] FIG8 is a diagram showing the in vitro binding verification results of potential targets according to Example 5 of the present invention.

[0040] FIG9 is a calculation simulation diagram according to Example 6 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 Ammonium Nitrate for the Synthesis of Tertiary α-Nitro, α-Substituted Scaffolds".

[0045] Unless otherwise specified, other raw materials used are commonly available in the market.

[0046] Example 1

[0047] This embodiment provides a method for preparing a ketamine small molecule probe having a structure shown in formula (III). The reaction formula is shown in formula (III-1), specifically:

[0048] Ketamine (237.7 mg, 1 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), purged with nitrogen, and cooled to -78°C. A tetrahydrofuran solution of potassium hexamethyldisilazide (2 M, 1.5 mL, 3 mmol) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 30 min. The nitrogen atmosphere was replaced with an oxygen system, and trimethyl phosphite (355 μL, 3 mmol) was slowly added. The reaction was maintained at low temperature for 2 h. TLC showed that the reaction was complete, and the reaction was terminated.

[0049] 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, which was purified by column chromatography to give 201 mg of 6-hydroxyketamine as a white solid in a yield of 79.4%.

[0050] The hydrogen spectrum and carbon spectrum of the product 6-hydroxyketamine are shown in Figures 1 and 2, and the characterization data are:

[0051] 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).

[0052] 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.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 1 is that the reaction conditions are adjusted, as shown in Table 1:

[0055] Table 1

[0056] Example 2

[0057] This embodiment provides a method for preparing a ketamine small molecule probe having a structure shown in formula (I). The reaction formula is shown in formula (I-1), specifically:

[0058] 2) Preparation of a ketamine small molecule probe having the structural formula (I):

[0059] 6-Hydroxyketamine (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%.

[0060] The hydrogen spectrum and carbon spectrum of the product are shown in Figures 3 and 4, and the characterization data are:

[0061] 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).

[0062] 13 C NMR (75MHz, CDCl3) δ189.30, 147.89, 138.45, 132.80, 131.20, 129.12, 128.60, 126.64, 118.12, 78.17, 75.92, 67.20, 56.12, 33.80, 30.25, 20.63.MS(ESI)m / z calcd for C 16 H 17 ClNO2 + [M+H] + 290.09, found 290.01.

[0063] The difference between this comparative example and Example 2 is that the reaction conditions are adjusted, as shown in Table 2:

[0064] Table 2

[0065] Example 3

[0066] This embodiment provides a method for preparing a ketamine small molecule probe having a structure represented by formula (II). The reaction formula is shown in formula (II-1), specifically:

[0067] 6-Hydroxyketamine (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 a 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.

[0068] The hydrogen spectrum and carbon spectrum of the product are shown in Figures 5 and 6, and the characterization data are:

[0069] 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)

[0070] 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 for C 16 H 18 ClNNaO2 + [M+Na] + 314.09, found 314.03.

[0071] The difference between this comparative example and Example 3 is that the reaction conditions are adjusted, as shown in Table 3:

[0072] Table 3

[0073] Example 4 Target fishing experiment

[0074] This example uses the ketamine probes prepared in Examples 2 and 3 to conduct a target fishing experiment, specifically:

[0075] 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.

[0076] 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.

[0077] Referring to the process in Figure 7, the probe was incubated with the total protein in the cell lysate to allow the protein to fully bind to the probe. The biotin was coupled through a click chemistry reaction, and then the target protein was separated and purified using streptavidin magnetic beads. Finally, all target protein information was obtained through silver staining and mass spectrometry analysis. Among them, 61 target proteins were obtained by targeting probe one, and 70 target proteins were obtained by targeting probe two. 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.

[0078] Table 4

[0079] Table 5

[0080] Example 5 Potential target binding verification in vitro

[0081] Pull-down experiment (Figure 8A, B):

[0082] The target protein acquisition process of the pull-down experiment was the same as that of the target fishing in Example 4. Finally, the target protein bands were detected by western blot experiment, as shown in Figures 8A and C, which correspond to ketamine probes 1 and 2 prepared in Examples 2 and 3, respectively;

[0083] 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.

[0084] A. Fluorescence titration experiment (Figure 8C):

[0085] Figure 8E shows the fluorescence titration of SIRT2 using S-ketamine and R-ketamine on a fluorescence spectrometer. First, the purified SIRT2 protein was diluted with PBS to 0.5 μM (1 mL) and the fluorescence intensity at this time was measured. Then, S-ketamine and R-ketamine were added respectively, and the fluorescence intensity of the system was measured when the ketamine concentration was 0.5-5 μM. After each addition, the system was allowed to stand for 3 minutes to allow the drug to bind to the protein.

[0086] Data processing was performed by plotting the ratio of fluorescence intensity between the blank protein group and each drug-treated group. Therefore, the larger the slope, the greater the decrease in fluorescence intensity. As can be seen from Figure 8E, with the increase in ketamine concentration, S-ketamine significantly reduced the fluorescence intensity of SIRT2 protein, while R-ketamine did not significantly reduce it, which also indicates that S-ketamine has a better binding effect with SIRT2.

[0087] B. Thermal denaturation experiment (Figure 8D, E):

[0088] 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;

[0089] S-ketamine and R-ketamine were added to groups b and c, respectively, with a final concentration of 500 μM. 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 the 8 temperatures shown in Figure 5B. Finally, the samples were 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 experiment as shown in Figure 8B. Figure 8D is the line graph corresponding to the bands. 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.

[0090] Example 6 Simulation verification of target protein

[0091] Molecular dynamics simulations were used to identify key residues and representative conformations in the interaction between S-ketamine and SIRT2, yielding a series of binding sites (Figure 9A), demonstrating that S-ketamine stably binds to the SIRT2 protein. A hydrogen bond was found between the ketocarbonyl group of S-ketamine and the side chain amide N atom of the SIRT2 Q167 residue in the binding pocket (Figure 9A). Due to hydrophobic interactions, hydrophobic residues tend to be buried within the protein, thereby increasing its folding and stability. The binding of S-ketamine reduced the percentage of hydrophobic regions in the SASA buried by SIRT2, demonstrating that S-ketamine destabilizes SIRT2 (Figure 9B).

[0092] 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 9C). To investigate the changes in protein stability caused by S-ketamine, root mean square fluctuation (RMSF) analysis was performed on the final 200 ns equilibrium trajectory. S-ketamine binding made most regions of SIRT2 more flexible, indicating that SIRT2 stability decreased after binding to S-KET (Figure 9D), a result consistent with the results of the thermal denaturation experiment.

[0093] 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.

[0094] 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.

[0095] 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, wherein the ketamine small molecule probe has a structure represented by the following formula (I) or formula (II):

2. A method for preparing a ketamine small molecule probe, the method comprising: The ketamine small molecule probe is obtained by using 6-hydroxyketamine having a structure shown in the following formula (III) as a substrate and connecting a propargyl group to the hydroxyl group or amino group of 6-hydroxyketamine:

3. The method for preparing a ketamine small molecule probe according to claim 2, wherein the ketamine small molecule probe is a ketamine small molecule probe having a structure represented by formula (I) or formula (II):

4. The method for preparing a ketamine small molecule probe according to claim 3, wherein the ketamine small molecule probe having a structure represented by formula (I) has a structure in which a propargyl group is connected to the hydroxyl group of 6-hydroxyketamine; and the ketamine small molecule probe having a structure represented by formula (II) has a structure in which a propargyl group is connected to the amino group of 6-hydroxyketamine.

5. The method for preparing a ketamine small molecule probe according to claim 2, wherein the 6-hydroxyketamine having a structure represented by formula (III) is prepared by the following method: Ketamine is dissolved in anhydrous DMF or tetrahydrofuran, cooled to -80 to -70°C under nitrogen protection, a tetrahydrofuran solution of potassium hexamethyldisilazide is added dropwise, the nitrogen is replaced with an oxygen system after reacting for 30 minutes, trimethyl phosphite is added, and 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 the 6-hydroxyketamine having the structure shown in formula (III). in, The ratio of ketamine, potassium hexamethyldisilazide and trimethyl phosphite is 1:1.2-3:1.5-3.

6. The method for preparing a ketamine small molecule probe according to claim 3, wherein the ketamine small molecule probe having a structure represented by formula (I) is prepared by the following method: 6-Hydroxyketamine is dissolved in anhydrous DMF, and propidium bromide, potassium tert-butoxide and sodium iodide are added under nitrogen protection. After stirring until the reaction is completed, the reaction is quenched and post-treated to obtain a ketamine small molecule probe having a structure shown in formula (I). in, The ratio of 6-hydroxyketamine, propinyl bromide, potassium tert-butoxide and sodium iodide is 1:1.2-1.5:2-2.5:2-2.

5.

7. The method for preparing a ketamine small molecule probe according to claim 6, wherein the reaction temperature is 25 to 35°C and the reaction time is 2 to 4 hours.

8. The method for preparing a ketamine small molecule probe according to claim 3, wherein the ketamine small molecule probe having a structure represented by formula (II) is prepared by the following method: 6-Hydroxyketamine is dissolved in anhydrous DMF, and propyne bromide, cesium carbonate and sodium iodide are added under nitrogen protection. After stirring until the reaction is completed, the reaction is quenched and post-treated to obtain a ketamine small molecule probe having a structure shown in formula (II). in, The ratio of 6-hydroxyketamine, propargyl bromide, cesium carbonate and sodium iodide is 1:1.2-1.5:2-2.5:2-2.

5.

9. The method for preparing a ketamine small molecule probe according to claim 8, wherein the reaction temperature is 20 to 30°C and the reaction time is 65 to 67 hours.

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

11. Use of the ketamine small molecule probe according to claim 10 in discovering target proteins of ketamine by target fishing, wherein the ketamine small molecule probe having a structure shown in formula (I) obtains 61 target proteins by target fishing, and the ketamine small molecule probe having a structure shown in formula (II) obtains 70 target proteins by target fishing.

Images