A cannabidiol small molecule probe and its synthesis method and application

Abstract

The present invention discloses a cannabidiol small molecule probe and a synthesis method and application thereof, belonging to the technical field of acyclic or carbocyclic compounds. Cannabidiol with a structural formula as shown in formula (1) is used as a substrate, and a compound CP2 with a structural formula as shown in formula (2) is connected. Formula (1); Formula (2); The hydroxyl group in the structural formula of formula (1) is connected with the Br group in the structural formula of formula (2) to obtain a cannabidiol small molecule probe CP1 with a structural formula as shown in formula (3); Formula (3); The small molecule probe CP1 is used to perform a target fishing experiment on the total protein in the BV2 cell lysate, and the binding of the CBD small molecule probe to the target protein is verified by two biological repetitions, and finally 51 common target proteins are screened, which provides a reference for the study of the target and molecular mechanism of CBD anti-addiction.

Description

Technical Field

[0001] The present invention belongs to the field of acyclic or carbocyclic compounds, and specifically relates to a cannabidiol small molecule probe and a synthesis method and application thereof. Background Art

[0002] After entering the human body, drugs act on specific molecular targets to regulate disease phenotypes, thereby achieving therapeutic effects. Therefore, target discovery and verification play a key role in drug development. Among the drugs currently on the market, more than 95% of drug targets are proteins. Proteomics research focuses on the composition and changes of proteins in cells, tissues or organisms. With the advantages of systematicity, high throughput and high precision, chemical proteomics combines multiple disciplines such as cell biology, synthetic chemistry and biomass spectrometry, becoming a new platform for drug target screening and is deeply favored by researchers.

[0003] Activity-Based Protein Profiling (ABPP) is a widely used chemical proteomics technology, in which the design and synthesis of activity probes are particularly important. The probe must be able to effectively screen the target protein without affecting the binding of the drug to the target protein. The introduction of click chemistry and photoaffinity labeling technology has further expanded the application scope of ABPP technology. However, this technology has not yet been applied in CBD.

[0004] CBD is the main active compound in cannabis, and its potential therapeutic properties have been explored in several neuropsychiatric conditions, including substance use disorders caused by various psychostimulants. In recent years, CBD has attracted much attention for its potential therapeutic effects on methamphetamine (METH) addiction. Studies have shown that CBD can reduce METH-induced hyperactivity and conditioned place preference (CPP), suggesting that it may reduce METH craving behavior and relapse risk. Recent studies have revealed the effects of CBD on ATP, which is essential in the regulation of cellular energy metabolism and mitochondrial function. CBD effectively restores mitochondrial function and reduces oxidative stress, thereby treating pulmonary arterial hypertension (PAH). In Alzheimer's disease, CBD activates TRPV2 channels, enhances the clearance of amyloid-β by microglia, and increases ATP production to reduce neuroinflammation. In addition, CBD protects astrocytes from oxidative stress and inflammation by optimizing their mitochondrial function, suggesting its potential in treating metabolic and neuroinflammatory diseases. However, the targets and molecular mechanisms of CBD's biological effects have not yet been clarified and further studies are needed to determine them. Currently, there is no CBD probe for ABPP technology. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot 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 cannabidiol small molecule probe.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0009] Using cannabidiol of the structural formula (1) as a substrate, a compound CP2 of the structural formula (2) is connected, wherein the molar ratio of cannabidiol to CP2 is 1-10 mol:1-10 mol;

[0010] Formula (1);

[0011] Formula (2);

[0012] The hydroxyl group in the structural formula (1) is reacted with the Br group in the structural formula (2) to obtain a cannabidiol small molecule probe having a structural formula as shown in formula (3);

[0013] Formula (3).

[0014] Another object of the present invention is to provide a cannabidiol small molecule probe for use in screening target proteins of cannabidiol by target fishing.

[0015] Another object of the present invention is to provide an application of a cannabidiol small molecule probe in the preparation of anti-addiction drugs. Specifically, the application method includes obtaining the anti-addiction target of cannabidiol by target fishing using the cannabidiol small molecule probe as the target for preparing anti-addiction drugs.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a cannabidiol small molecule probe.

[0017] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0018] 1-10 mmol of compound 1 and 1-10 mmol of 3-bromopropyne are dissolved in 10-30 mL of acetone aqueous solution, and 3-30 mmol of powdered potassium carbonate is added thereto to obtain a mixture I;

[0019] The mixture I is stirred and heated to react at 40-80° C. After the reaction is completed, the reaction mixture is quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction is washed with saturated brine and dried with sodium sulfate to obtain a crude product 1. The crude product 1 is purified by flash column chromatography to obtain compound 2;

[0020] Wherein, the structural formula of the compound 1 is shown in formula (4), and the structural formula of the compound 2 is shown in formula (5);

[0021] Formula (4);

[0022] Formula (5);

[0023] 2~20 mmol of compound 2 and 4~40 mmol of 1,3-dibromopropane are dissolved in 15~150 mL of acetone solution, and 2~20 mmol of ground potassium carbonate is added thereto to obtain mixture II.

[0024] The mixture II was stirred and heated to react at 40-80°C. After the reaction was completed, the reaction mixture was quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 2.

[0025] The crude product 2 was purified by flash column chromatography to obtain a compound CP2 having a structural formula as shown in formula (2);

[0026] 1~10 mol CP2 and 1~10 mol cannabidiol are dissolved in 10~100 mL acetone solution, and 1~10 mmol of ground potassium carbonate is added thereto to obtain a mixture III;

[0027] The mixture III was stirred and heated to react at 40-80°C. After the reaction was completed, the reaction mixture was quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 3.

[0028] The crude product 3 was purified by preparative thin layer chromatography to obtain a cannabidiol small molecule probe CP1 having a structural formula as shown in formula (3).

[0029] As a preferred embodiment of the method for preparing the cannabidiol small molecule probe of the present invention, the acetone aqueous solution is a mixture of H2O and acetone in a volume ratio of 1:9 to 1:1.

[0030] As a preferred embodiment of the method for preparing the cannabidiol small molecule probe of the present invention, ethyl acetate is used as the solvent for extraction in the synthesis method.

[0031] As a preferred embodiment of the method for preparing the cannabidiol small molecule probe of the present invention, the purification in the synthesis method is eluted using a mixed solvent of petroleum ether and ethyl acetate.

[0032] As a preferred embodiment of the method for preparing the cannabidiol small molecule probe of the present invention, the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 40:1 to 10:1.

[0033] As a preferred embodiment of the method for preparing the cannabidiol small molecule probe of the present invention, the heating reaction time in the synthesis method is 12 to 48 hours.

[0034] Beneficial effects of the present invention:

[0035] The present invention designs and synthesizes a CBD small molecule probe CP1, uses the CBD small molecule probe CP1 to carry out a target fishing experiment on the total protein in the BV2 cell lysate, and verifies the binding of the CBD small molecule probe to the target protein through two biological repetitions, and finally screens out 51 common target proteins, which provides a reference for the study of the target and molecular mechanism of CBD anti-addiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0037] Figure 1 Schematic diagram of the process of synthesizing cannabidiol small molecule probe in Example 1 of the present invention

[0038] Figure 2 This is the hydrogen spectrum of compound 2 prepared in Example 1 of the present invention.

[0039] Figure 3 This is the carbon spectrum of compound 2 prepared in Example 1 of the present invention.

[0040] Figure 4 This is the hydrogen spectrum of CP2 prepared in Example 1 of the present invention.

[0041] Figure 5 This is the carbon spectrum of CP2 prepared in Example 1 of the present invention.

[0042] Figure 6 This is the hydrogen spectrum of CP1 prepared in Example 1 of the present invention.

[0043] Figure 7 This is the carbon spectrum of CP1 prepared in Example 1 of the present invention.

[0044] Figure 8 This is a target fishing result diagram of Example 2 of the present invention.

[0045] Fig. 9 This is a diagram showing the in vitro binding verification results of potential targets in Example 3 of the present invention.

[0046] Fig.10 This is the result diagram showing that ATP5A1 is a new anti-addiction target in Example 4 of the present invention. DETAILED DESCRIPTION

[0047] 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.

[0048] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and 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.

[0049] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0050] Unless otherwise specified, the raw materials used in the present invention are all commercially available in the art.

[0051] Example 1, reference Figure 1 This embodiment provides a method for synthesizing a cannabidiol small molecule probe, specifically:

[0052] 1) Compound 1 (3 mmol, 642.18 mg) and 3-bromopropyne (3 mmol, 353.82 mg, 1 equivalent) were dissolved in 20 mL of acetone aqueous solution (the volume ratio of acetone to water was 9:1), and powdered potassium carbonate (3 mmol, 414.63 mg, 1 equivalent) was added thereto to obtain a mixture I;

[0053] The mixture I was stirred at room temperature and heated under reflux at 60° C. for 12 h. After the reaction was completed, the reaction mixture was quenched with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 1.

[0054] The crude product 1 was purified by flash column chromatography with petroleum ether / ethyl acetate (40:1) to obtain compound 2;

[0055] Figure 2 is the hydrogen spectrum of compound 2, Figure 3 For its carbon spectrum, the characterization data are as follows;

[0056] 1 H NMR (300 MHz, MeOD) δ = 7.81 – 7.63 (m, 4H), 7.16 – 7.04 (m, 2H), 6.94 – 6.82 (m, 2H), 4.85 (s, 2H), 3.03 (t, J =2.3, 1H).

[0057] 13 C NMR (75 MHz, MeOD) δ = 195.4, 162.0, 161.1, 132.3, 131.7, 131.1,128.9, 114.7, 114.1, 100.0, 77.8, 76.0, 55.3. MS (ESI - ) calcd for: 251.08 [M - H] - ; found: 251.15.

[0058] 2) Compound 2 (2 mmol, 504.16 mg) and 4 mmol 1,3-dibromopropane (4 mmol, 807.56 mg, 2 equivalents) were dissolved in 15 mL acetone, and powdered potassium carbonate (2 mmol, 276.42 mg, 1 equivalent) was added thereto to obtain a mixture II;

[0059] The mixture II was stirred at room temperature and heated under reflux at 60°C for 12 h. After the reaction was completed, the reaction mixture was quenched with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 2.

[0060] The crude product 2 was purified by flash column chromatography with petroleum ether / ethyl acetate (40:1) to obtain compound CP2;

[0061] Figure 4 is the hydrogen spectrum of CP2, Figure 5 For its carbon spectrum, the characterization data are as follows;

[0062] 1 H NMR (300 MHz, CDCl3) δ = 7.88 – 7.73 (m, 4H), 7.05 (d, J =5.8, 2H),6.96 (d, J =5.2, 2H), 4.77 (d, J =2.4, 2H), 4.19 (t, J =5.8, 2H), 3.62 (t, J =6.4, 2H), 2.59 (t, J =2.4, 1H), 2.35 (p, J =6.1, 2H).

[0063] 13 C NMR (75 MHz, CDCl3) δ = 194.3, 162.0, 160.6, 132.3, 132.1, 131.5,130.7, 114.4, 114.0, 77.9, 76.2, 65.5, 55.9, 32.1, 29.9. MS (ESI - ) calcd for:371.04 [M - H] - ; found: 371.06.

[0064] 3) CP2 (1 mmol, 252.08 mg) and cannabidiol (1 mmol, 314.22 mg, 1 equivalent) were dissolved in 10 mL of acetone, and powdered potassium carbonate (1 mmol, 138.21 mg, 1 equivalent) was added thereto to obtain a mixture III;

[0065] The mixture III was stirred at room temperature and heated under reflux at 60°C for 12 h. After the reaction was completed, the reaction mixture was quenched with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 3.

[0066] The crude product 3 was purified by preparative thin layer chromatography using petroleum ether / ethyl acetate (40:1) to obtain the cannabidiol small molecule probe CP1.

[0067] Figure 6 is the hydrogen spectrum of CP1, Figure 7 For its carbon spectrum, the characterization data are as follows;

[0068] 1 H NMR (300 MHz, CDCl3) δ = 7.81 (d, J =8.5, 4H), 7.06 (d, J =8.7, 2H),6.98 (d, J =8.7, 2H), 6.33 (s, 1H), 6.28 (s, 1H), 6.06 (s, 1H), 5.57 (s, 1H), 4.79 (d, J =2.3, 2H), 4.54 (s, 1H), 4.41 (s, 1H), 4.24 (t, J =6.1, 2H), 4.10(t, J =5.5, 2H), 3.99 (s, 1H), 2.59 (t, J =2.2, 1H), 2.54 – 2.44 (m, 3H), 2.33– 2.17 (m, 3H), 2.15 – 2.00 (m, 1H), 1.76 (s, 4H), 1.67 – 1.53 (m, 5H), 1.38– 1.23 (m, 6H), 0.90 (t, J =6.7, 3H).

[0069] 13 C NMR (75 MHz, CDCl3) δ = 194.4, 162.3, 160.6, 132.3, 132.1, 131.6,130.6, 114.3, 114.0, 77.9, 77.3, 76.1, 64.9, 55.9, 36.0, 31.6, 30.9, 29.4,23.7, 22.6, 14.1. MS (ESI - ) calcd for: 605.33 [M - H] - ; found: 605.36.

[0070] Comparative Example 1: This comparative example differs from Example 1 in that the acetone aqueous solution in step 1) is adjusted to a pure acetone solution, and the remaining steps and processes are all based on Example 1 to obtain the cannabidiol small molecule probe CP1 of this comparative example, but the product yield is significantly reduced.

[0071] Comparative Example 2. The difference between this comparative example and Example 1 is that the volume ratio of the acetone aqueous solution in step 1) is adjusted to 5:5 between water and acetone, and the remaining steps are processed with reference to Example 1 to obtain the cannabidiol small molecule probe CP1 of this comparative example, but the product yield is significantly reduced.

[0072] Comparative Example 3

[0073] The difference between this comparative example and Example 1 is that the mixture I in step 1) is stirred and refluxed at 20° C. for 12 seconds, and the remaining steps are all based on Example 1 to obtain the cannabidiol small molecule probe CP1 of this comparative example, but the product yield is significantly reduced.

[0074] Comparative Example 4: This comparative example is different from Example 1 in that the mixture I in step 1) is stirred and refluxed at -20°C for 12 seconds, and the remaining steps are all based on Example 1. As a result, the reaction cannot proceed.

[0075] Comparative Example 5. The difference between this comparative example and Example 1 is that the molar ratio of CP2 to cannabidiol in step 3) is adjusted to 2:1, and the remaining steps and processes are all based on Example 1 to obtain the cannabidiol small molecule probe CP1 of this comparative example, but the product yield is reduced.

[0076] Comparative Example 6: This comparative example differs from Example 1 in that the molar ratio of CP2 to cannabidiol in step 3) is adjusted to 5:1, and the remaining steps are carried out according to Example 1 to obtain the cannabidiol small molecule probe CP1 of this comparative example, but the product yield is significantly reduced.

[0077] Comparative Example 7: This comparative example is different from Example 1 in that the mixture III in step 1) is stirred and refluxed at 20° C. for 12 seconds, and the remaining steps are all based on Example 1. As a result, the product yield is significantly reduced.

[0078] Comparative Example 8: This comparative example is different from Example 1 in that the mixture III in step 1) is stirred and refluxed at -20°C for 12 seconds, and the remaining steps are all based on Example 1. As a result, the reaction cannot proceed.

[0079] The synthesis condition variables and results of Example 1 and Comparative Examples 1 to 8 are shown in Table 1.

[0080] Table 1

[0081] ,

[0082] As can be seen from Table 1, each reaction condition has a significant effect on the synthesis of CP1.

[0083] Example 2, reference Figure 8In this example, the cannabidiol small molecule probe CP1 obtained in Example 1 was used to carry out a target fishing experiment, and CP2 was used as a control probe of CP1. Specifically:

[0084] 1) Divide BV2 cells into five groups:

[0085] Blank control group, b. CP1 probe-non-click reaction group, c. CP2 probe-click reaction group, d. CP1 probe-click reaction-CBD competitive binding group, e. CP1 probe-click reaction group, 1 ml for each group;

[0086] Among them, group b was added with CP1 probe at a final concentration of 20 μM, group c was added with CP2 probe at a final concentration of 20 μM, group d was added with CP1 probe at a final concentration of 10 μM, the CBD concentration was 60 μM, group e was added with CP1 probe at a final concentration of 10 μM, and group a was added with an equal volume of DMSO.

[0087] Reference Figure 8 The cells were placed on ice and irradiated with 365 nm UV light for 30 min. After washing twice with 3 mL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.4), the cells were lysed with a buffer containing 25 mM HEPES (pH = 8.0), 150 mM KCl, 5 mM EDTA, 0.5% NP-40, and 1% protease inhibitor cocktail.

[0088] The resulting lysate was centrifuged at 12,000 g for 10 min at 4 °C, and the protein concentration was adjusted to 1 mg / mL using a BCA kit (YEASEN, 20201ES86, CHN);

[0089] Then the click reaction was carried out at room temperature for 1 h using 80 μM biotin-N3, 100 μM TBTA, 1 mM TCEP, 1 mM CuSO4, and 5% tert-butanol;

[0090] Proteins were captured by 20 μL streptavidin magnetic beads (Invitrogen, 11206D, USA) and mixed with 50 μL 2× Laemmli buffer, then incubated at 100°C for 8 min. The samples were separated by SDS-PAGE and silver stained (Beyotime, P0017S, CHN) ( Figure 8 The band was cut out and then analyzed by LC-MS / MS to identify the protein ( Figure 8 The results are shown in Table 2.

[0091] Table 2

[0092] ,

[0093] ,

[0094] ,

[0095] As can be seen from Table 2, the two probes have 51 common targets, indicating that the probes synthesized by the present invention can effectively screen target proteins.

[0096] Example 3, based on Example 1, this example selects ATP5A1 as the target protein to perform in vitro binding verification of potential targets, specifically:

[0097] 1. Pull down experiment:

[0098] The target protein acquisition process of the pull-down experiment is the same as the target fishing in Example 2. Finally, the target protein band is detected by western blot experiment. The results are as follows Fig. 9 As shown in A, they respectively correspond to the CBD probes prepared in Example 1;

[0099] It can be seen that the ATP5A1 protein in the probe group is much higher than that in the control group, and CBD competes to bind to most of the ATP5A1 protein, indicating that ATP5A1 can bind to CBD well.

[0100] 2. Thermal denaturation experiment:

[0101] The cell lysate was divided into two groups: a. blank control group and b. CBD group;

[0102] Group b was added with CBD at a final concentration of 500 μM. After the two 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 Fig. 9 The samples were heated at 7 temperatures as shown in B, and finally heated at 48, 50, 52, 54, 56, 58, and 60 for 5 min to denature the proteins to different degrees. The supernatant was collected, the samples were boiled, and the target protein bands were detected by western blot. Fig. 9 As shown in B, the line graph is a statistical change graph of the optical density of the protein bands. Compared with the control group, the addition of CBD increased the stability of the ATP5A1 protein, indicating that CBD can bind to the ATP5A1 protein.

[0103] 3. Fluorescence titration experiment:

[0104] CBD was used to perform fluorescence titration of ATP5A1 on a fluorescence spectrometer. First, the purified ATP5A1 protein was diluted to 0.5 μM (1 mL) with PBS and the fluorescence intensity at this time was measured. Then CBD was added separately and the fluorescence intensity of the system was measured when the CBD concentration was 0.5-80 μM. After each addition of the drug, the system was allowed to stand for 3 min to allow the drug to bind to the protein.

[0105] Data processing uses the ratio of the fluorescence intensity of the blank reagent group to that of each drug-added group to plot the graph. Therefore, the larger the slope, the greater the decrease in fluorescence intensity. Fig. 9 It can be seen from the C that with the increase of CBD concentration, CBD significantly reduced the fluorescence intensity of ATP5A1 protein, which also indicates a better binding effect between CBD and ATP5A1.

[0106] 4. Mitochondrial complex V activity detection experiment:

[0107] The Mi-toTox™ Complex V OXPHOS Activity Assay Kit (Abcam, ab109907, UK) was used to detect the effect of CBD on the activity of mitochondrial complex V, in order to further determine the effect of CBD on the complex composition and function after binding to ATP5A1.

[0108] Different concentrations of CBD (0.001 to 200 μM) were incubated with 96-well plates pre-coated with bovine heart mitochondria at 37°C for 45 minutes. After incubation, complex V activity buffer was added and the absorbance at 340 nm was measured every minute for 60 minutes. The ATP synthase activity was then calculated using the following formula: Complex V activity = sample reaction rate – background reaction rate; reaction rate (OD / minute) = (OD2- OD1) / (T2-T1). OD represents the absorbance at 340 nm and T represents the test time. The experimental results show ( Fig. 9 D), CBD can effectively enhance the activity of mitochondrial complex V.

[0109] Simulation verification of target protein:

[0110] Molecular dynamics simulation was used to obtain the key residues and representative conformations in the interaction between CBD and ATP5A1, and a series of binding sites were obtained ( Fig. 9 E- Fig. 9 F), demonstrating that CBD is stably bound to ATP5A1 protein, and that hydrogen bonds exist between CBD and the side chains of ATP5A1 Q255 and D333 residues in the binding pocket ( Fig. 9 F).

[0111] The above results indicate that the CBD molecular probe synthesized by the present invention does not affect the binding with the target protein, and can effectively screen out the target protein. Among them, the screened ATP5A1 protein is the target of CBD, and the combination of the two has an excellent effect.

[0112] Example 4: This example is used to verify that ATP5A1 obtained by screening in the present invention is a new anti-addiction target, specifically:

[0113] 1. ATP5A1 silencing experiment:

[0114] AAV9-shATP5A1-CMV-EGFP-WPRE and AAV9-CMV-EGFP-WPRE vectors were provided by Shanghai Gene Chemical Technology Co., Ltd. (OBiO Technology). 500 nL AAV9 (250 nL per brain side) was injected into the ventral tegmental area (VTA) ( Fig.10 A~ Fig.10 (B) (anteroposterior coordinate: -3.16 mm, left-right coordinate: ±0.50 mm, superior-inferior coordinate: -4.50 mm). Injection was performed by a microinjection pump and controller (R480, RWD, China) at a rate of 200 nL / min, and the injection needle remained in place for an additional 10 min. Subsequently, the interference efficiency was verified by western blotting ( Fig.10 C). The experimental results showed that ATP5A1 gene silencing can be achieved in specific brain regions, effectively reducing ATP5A1 protein expression.

[0115] Addiction behavior experiment in mice after ATP5A1 silencing:

[0116] (A) Behavioral sensitization experiment: In the experiment, mice were placed in an open field (40 × 40 × 40 cm^3) for 1 hour to adapt to the equipment in the first 3 days. The behavioral sensitization test was performed for 7 consecutive days. After daily injection of METH (1 mg / kg, intraperitoneal injection), CBD (intraperitoneal injection), or a combination of METH (1 mg / kg) and CBD, the mice were placed in the open field for 1 hour, and their movement trajectories were recorded. After a 7-day withdrawal period, the mice were given METH (0.5 mg / kg, intraperitoneal injection) to assess behavioral sensitization. Their movement trajectories were subsequently recorded to evaluate the continued therapeutic effect of CBD. The experimental results are shown in the figure ( Fig.10 D~ Fig.10 The results showed that METH can increase the distance of mice's movement, and CBD can effectively shorten the extension of the distance of mice's movement caused by METH. After silencing ATP5A1, the effect of CBD was significantly reduced. The above results indicate that ATP5A1 may be the target of CBD's anti-addiction effect.

[0117] (B) Conditioned place preference procedure: Conditioned place preference (CPP) experiments were conducted in a 12-day protocol. On the first two days, mice were placed in the CPP apparatus for 15 minutes each to adapt to the equipment. On the third day (pre-conditioning), mice were allowed to freely explore the apparatus for 15 minutes, and their movement trajectories were recorded. The time the mice stayed in each compartment was analyzed to determine their preference. The compartment where the mice stayed less was the METH-treated paired compartment. The mice were divided into six groups: control group, CBD (50 mg / kg) group, METH (2 mg / kg) group, METH (2 mg / kg) + CBD (2 mg / kg) group, METH (2 mg / kg) + CBD (20 mg / kg) group, and METH (2 mg / kg) + CBD (50 mg / kg) group. CPP training lasted for 8 days, with a total of 4 cycles. After daily treatment, the mice were isolated in the compartments for 30 minutes each. On days 3, 5, 7, and 9, mice received METH (2 mg / kg, intraperitoneal injection) and on days 4, 6, 8, and 10, they received vehicle (5 mL / kg saline, intraperitoneal injection). The specified doses of CBD were administered daily with METH or vehicle. CPP testing was performed 24 hours after the last training. Mice were allowed to explore the apparatus freely, and their movement trajectories were recorded for 15 minutes. The CPP score (seconds) was calculated by subtracting the time spent in the METH-paired compartment from the time spent in the control vehicle-paired compartment. The experimental results are shown in the figure ( Fig.10 F). The experimental results showed that mice treated with METH had obvious conditioned place preference behavior. After CBD treatment, the conditioned place preference behavior of mice treated with METH was significantly weakened. After ATP5A1 gene silencing, the effect of CBD was significantly reduced.

[0118] In summary, the present invention designed and synthesized a CBD small molecule probe CP1, used the CBD small molecule probe CP1 to carry out a target fishing experiment on the total protein in the BV2 cell lysate, and verified the binding of the CBD small molecule probe to the target protein after two biological repetitions. Finally, 51 common target proteins were screened, which provided a reference for the study of the targets and molecular mechanisms of CBD anti-addiction.

[0119] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. 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 cannabidiol small molecule probe, characterized in that: The structural formula is shown in formula (3); Formula (3).

2. Use of the cannabidiol small molecule probe as described in claim 1 in the preparation of a reagent for screening the target protein of cannabidiol by target fishing.

3. The use of the cannabidiol small molecule probe as claimed in claim 1 in the preparation of anti-addiction drugs, characterized in that: The application method includes obtaining the anti-addiction target of cannabidiol through target fishing using a cannabidiol small molecule probe as the action target for preparing anti-addiction drugs.

4. A method for synthesizing a cannabidiol small molecule probe, characterized in that: include, 1-10 mmol of compound 1 and 1-10 mmol of 3-bromopropyne are dissolved in 10-30 mL of acetone aqueous solution, and 3-30 mmol of powdered potassium carbonate is added thereto to obtain a mixture I; The mixture I is stirred and heated to react at 40-80° C. After the reaction is completed, the reaction mixture is quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction is washed with saturated brine and dried with sodium sulfate to obtain a crude product 1. The crude product 1 is purified by flash column chromatography to obtain compound 2; Wherein, the structural formula of the compound 1 is shown in formula (4), and the structural formula of the compound 2 is shown in formula (5); Formula (4); Formula (5); 2~20 mmol of compound 2 and 4~40 mmol of 1,3-dibromopropane are dissolved in 15~150 mL of acetone solution, and 2~20 mmol of ground potassium carbonate is added thereto to obtain mixture II. The mixture II was stirred and heated to react at 40-80°C. After the reaction was completed, the reaction mixture was quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 2. The crude product 2 was purified by flash column chromatography to obtain a compound CP2 having a structural formula as shown in formula (2); Formula (2); 1~10 mol CP2 and 1~10 mol cannabidiol are dissolved in 10~100 mL acetone solution, and 1~10 mmol ground potassium carbonate is added thereto to obtain mixture III; Wherein, the structural formula of cannabidiol is shown as formula (I); Formula (I); The mixture III was stirred and heated to react at 40-80°C. After the reaction was completed, the reaction mixture was quenched with 1-5 M hydrochloric acid and extracted. The organic phase obtained by extraction was washed with saturated brine and dried over sodium sulfate to obtain a crude product 3. The crude product 3 was purified by preparative thin layer chromatography to obtain a cannabidiol small molecule probe CP1 having a structural formula as shown in formula (3).

5. The method for synthesizing the cannabidiol small molecule probe according to claim 4, characterized in that: The acetone aqueous solution is a mixture of H2O and acetone in a volume ratio of 1:9 to 1:

1.

6. The method for synthesizing the cannabidiol small molecule probe according to claim 4, characterized in that: The extraction in the synthesis method uses ethyl acetate as solvent.

7. The method for synthesizing the cannabidiol small molecule probe according to claim 4, characterized in that: The purification in the synthesis method is eluted using a mixed solvent of petroleum ether and ethyl acetate.

8. The method for synthesizing the cannabidiol small molecule probe according to claim 7, characterized in that: The volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 40:1-10:

1.

9. The method for synthesizing the cannabidiol small molecule probe according to claim 4, characterized in that: The heating reaction time in the synthesis method is 12 to 48 hours.

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