Cannabidiol-2-2-(3-(but-3-yn-1-yl)-3h-diazirine-3-yl)ethyl ester as a cannabidiol probe molecule and uses thereof

Abstract

The application discloses a cannabinoid derivative, a preparation method of cannabinoid-2-2-(3-(but-3-yn-1-yl)-3H-diazirine-3-yl) ethyl ester (referred to as a2) and application of the cannabinoid derivative as a cannabinoid probe molecule. Specifically, the application relates to a compound-a2 shown in formula I, the cannabinoid derivative is produced by reaction of cannabinoid and 2-(3-(but-3-yn-1-yl)-3H-diazirine-3-yl) acetic acid, the cannabinoid derivative is basically consistent with the function of the cannabinoid, and can be used as a probe molecule of the cannabinoid. After click chemistry reaction of a2 and 5-carboxy tetramethyl rhodamine azide, the location of the cannabinoid in cells can be determined, after click chemistry reaction of a2 and azido biotin, mass spectrometry analysis can be carried out, target proteins of the cannabinoid can be detected, and the finding of the target point of the cannabinoid has certain significance. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, specifically relating to a novel cannabidiol derivative, a method for preparing cannabidiol-2-2-(3-(but-3-yn-1-yl)-3H-bisacrididin-3-yl)ethyl ester (hereinafter referred to as a2), and its use in tracing the localization of cannabidiol in cells and the study of protein targets of cannabidiol action. Background Technology

[0002] Cannabidiol (CBD) is a major non-psychotropic drug extracted from the cannabis plant. Preclinical cell and rodent studies have suggested that CBD may have neuroprotective, cardioprotective, and anti-inflammatory effects.

[0003] High-purity CBD is a white or pale yellow crystal with a melting point of 66-67℃. It is almost insoluble in water but readily soluble in organic solvents such as ethanol, methanol, ether, benzene, chloroform, and petroleum ether. This ester-soluble characteristic of CBD significantly limits its application in drug development and the identification of precise drug targets. While the mechanism of action and targets of cannabidiol have been studied in various disease models, due to the very weak binding affinity of CBD to its receptors, many studies on CBD function have only described its effectiveness without identifying a clear target.

[0004] Designing and synthesizing CBD derivatives based on the structure of CBD is an important way to obtain compounds with higher activity, and it is of great significance for finding the target of CBD and preparing novel drugs.

[0005] Click chemistry, a synthetic concept proposed by chemist Barry Sharpless in 2001, aims to rapidly synthesize chemicals by assembling small units. Copper-catalyzed azido-alkynyl cycloaddition reactions are representative of click chemistry. Click chemistry is playing an increasingly important role in fields such as drug development and biomedical materials. Summary of the Invention

[0006] This invention provides a novel cannabidiol derivative a2 and its preparation method. This invention verifies that this compound has the same functional characteristics as cannabidiol. This invention utilizes click chemistry to provide a method for tracing the subcellular localization of cannabidiol and identifying the protein targets of cannabidiol action using cannabidiol derivative a2.

[0007] The compounds of the present invention have the structure shown in Formula I:

[0008] Formula I

[0009] The method for preparing cannabidiol derivatives provided by the present invention includes the following steps: reacting cannabidiol of the structure shown in Formula II with 2-(3-(but-3-yn-1-yl)-3H-bisacryl-3-yl)acetic acid of the structure shown in Formula III in the presence of a solvent and a catalyst to obtain a compound of the structure shown in Formula I.

[0010]

[0011] Formula II Formula III Formula I

[0012] In some embodiments of the present invention, the solvent is dichloromethane.

[0013] In some embodiments of the present invention, the molar ratio of 2-(3-(but-3-yn-1-yl)-3H-bisacryl-3-yl)acetic acid to cannabidiol is 1.2:1.

[0014] In some embodiments of the present invention, the preparation method specifically involves reacting the reaction mixture at 0°C in the dark for 20 min, then reacting it at room temperature in the dark for 16 hours, followed by rotary evaporation for concentration, and finally separation and purification using a plate preparation method.

[0015] The drug is formulated as an oil.

[0016] This invention provides a study on the functional similarity between compound of formula I and cannabidiol.

[0017] This invention provides compounds of Formula I for tracing the subcellular localization of cannabidiol in cells and for identifying the protein targets of cannabidiol's action. Attached Figure Description

[0018] Figure 1 Chemical structural formulas of cannabidiol and its derivative a2; Figure 2 Schematic diagram of the synthesis of cannabidiol derivative a2; Figure 3 Cannabidiol and its derivative α2 affect MIN6 cells' oxidative stress-related genes. Nrf2 The results of mRNA expression; Figure 4 Cannabidiol and its derivative α2 affect MIN6 cells' oxidative stress-related genes. Homx1 The results of mRNA expression; Figure 5 Cannabidiol and its derivative α2 affect MIN6 cells' oxidative stress-related genes. Nqo1 The results of mRNA expression; Figure 6 Cannabidiol and its derivative α2 affect MIN6 cell inflammation-related genes. Il6The results of mRNA expression; Figure 7 Cannabidiol and its derivative α2 affect MIN6 cell inflammation-related genes. Tnfα The results of mRNA expression; Figure 8 Localization results of cannabidiol derivative a2 in MIN6 cells; Figure 9 Mitochondrial proteins bound to cannabidiol derivative a2, selected based on mass spectrometry scoring; Figure 10 Western blot analysis results verifying the binding of cannabidiol derivative a2 to the mitochondrial protein VDAC1; Figure 11 Results of co-localization of cannabidiol derivative a2 and VDAC1 protein in cells. Detailed Implementation

[0019] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the materials described are all available from publicly available commercial sources.

[0020] Example 1: Preparation of cannabidiol derivative a2

[0021] The synthesis route is shown in the following formula:

[0022] Formula II Formula III Formula I

[0023] In a 25 mL round-bottom flask, 2-(3-(but-3-yn-1-yl)-3H-bisacrididin-3-yl)acetic acid (45.64 mg, 0.3 mmol, 1.2 equiv) and cannabidiol (80 mg, 0.25 mmol), along with a magnetic stir bar, were added. The starting materials were dissolved using 2 mL of anhydrous DCM. Then, under nitrogen protection, DCC (56.74 mg, 0.275 mmol, 1.1 equiv) and DMAP (15.27 mg, 0.125 mmol, 0.5 equiv) were added. During the addition of DCC and DMAP, the round-bottom flask was kept at 0 °C in the dark, and the mixture was stirred magnetically. After 20 min, the reaction mixture was removed from the 0 °C environment and allowed to continue at room temperature in the dark. The reaction was allowed to proceed for 16 hours. After the reaction was complete, the organic phase was concentrated by rotary evaporation to obtain the initial product, which was then separated and collected using a preparative plate. The collected products were dried by rotary evaporation and then sent to nuclear magnetic resonance (NMR) to confirm their structure.

[0024] The product was characterized by 1H NMR spectroscopy as follows: 1 H NMR (500 MHz, CDCl3) δ 6.57 (s, 1H), 6.43 (d, J = 1.7 Hz, 1H), 6.01(s, 1H), 5.54 (s, 1H), 4.60 (q, J = 1.9 Hz, 1H), 4.44 (s, 1H), 3.48 (s, 1H), 2.55 – 2.40 (m, 5H), 2.23 (s, 1H), 2.10 (td, J = 7.4, 2.6 Hz, 3H), 2.01 (q, J = 2.8 Hz, 1H), 1.85 (t, J = 7.4 Hz, 2H), 1.82 – 1.68 (m, 5H), 1.64 – 1.48 (m,7H), 1.37 – 1.25 (m, 5H), 0.88 (t, J = 7.0 Hz, 3H). The above-mentioned 1H NMR spectrum results demonstrate that the preparation method provided by this invention can effectively prepare a2.

[0025] Example 2: Functional comparison between cannabidiol derivative a2 and cannabidiol.

[0026] Study on the effect of cannabidiol derivative a2 on the mRNA expression of oxidative stress-related genes in MIN6 cells.

[0027] MIN6 cells were divided into 2.5 × 10 5 Cells were seeded at a density of 10 cells / mL in 24-well cell culture plates, with six groups (control group, high glucose treatment group, CBD treatment group, high glucose plus CBD treatment group, a2 treatment group, and high glucose plus a2 treatment group), with three replicates per group. After cell adhesion, cells were processed into different groups. The control group received 0.5 mL of complete culture medium per well; the high glucose treatment group received 0.5 mL of complete culture medium containing 33.3 mM glucose per well; the CBD treatment group received 0.5 mL of complete culture medium containing 10 mM CBD per well; the high glucose plus CBD treatment group received 0.5 mL of complete culture medium containing 33.3 mM glucose and 10 mM CBD per well; and the high glucose plus a2 treatment group received 0.5 mL of complete culture medium containing 33.3 mM glucose and 30 mM a2 per well. The medium was changed every 24 hours, and the treatment lasted for 48 hours. Cells were harvested, total RNA was extracted, reverse-engineered into cDNA, and quantitative real-time PCR was used to detect oxidative stress-related genes. Nrf2 , Hmox1,Nqo1 The expression of mRNA.

[0028] Experimental results are as follows Figure 3 , 4 As shown in Figure 5.

[0029] Figure 3 , 4 Figures 5 and 6 show the results of detecting the mRNA expression of genes related to oxidative stress in MIN6 cells by cannabidiol derivative a2. The results indicate that treatment of MIN6 cells with CBD and a2 for 48 h significantly improved the increased oxidative stress level in MIN6 cells induced by high glucose treatment.

[0030] Study on the effect of cannabidiol derivative a2 on the mRNA expression of inflammation-related genes in MIN6 cells.

[0031] MIN6 cells were divided into 2.5 × 10 5 Cells were seeded at a density of 10 cells / mL in 24-well cell culture plates, with six groups (control group, high glucose treatment group, CBD treatment group, high glucose plus CBD treatment group, a2 treatment group, and high glucose plus a2 treatment group), with three replicates per group. After cell adhesion, cells were processed into different groups. 0.5 mL of complete culture medium was added to each well in the control group; 0.5 mL of complete culture medium containing 33.3 mM glucose was added to each well in the high glucose treatment group; 0.5 mL of complete culture medium containing 10 mM CBD was added to each well in the CBD treatment group; 0.5 mL of complete culture medium containing 33.3 mM glucose and 10 mM CBD was added to each well in the high glucose plus CBD treatment group; and 0.5 mL of complete culture medium containing 33.3 mM glucose and 30 mM a2 was added to each well in the high glucose plus a2 treatment group. The medium was changed every 24 hours, and the treatment lasted for 48 hours. Cells were harvested, total RNA was extracted, reverse-engineered into cDNA, and then used for quantitative real-time PCR to detect inflammation-related genes. Il6,Tnfα The expression of mRNA.

[0032] Experimental results are as follows Figure 6 , 7 As shown.

[0033] Figure 6 , 7 This study examined the mRNA expression of inflammation-related genes in MIN6 cells by detecting the effect of a2. The results showed that treatment of MIN6 cells with CBD and a2 for 48 hours significantly improved the increased inflammation levels in MIN6 cells induced by high glucose treatment.

[0034] The functional comparison results of cannabidiol and its derivative a2 show that the functional results of cannabidiol derivative a2 are consistent with those of cannabidiol, and it can be used as a probe molecule for cannabidiol.

[0035] Example 3: Study on the localization of cannabidiol derivative a2 as a probe molecule of cannabidiol in cells.

[0036] 24-well cell slides were seeded onto 24-well plates, and MIN6 cells were seeded at a density of 2.5 × 10⁶ cells / well. 4 Cells / mL were seeded into 24-well plates containing cell spreaders. Two groups were established: a control group and an a2 treatment group. The a2 treatment group was treated with 10 μM a2 at 37°C for 30 min. Each group was then treated with 100 nM mitochondrial membrane potential dye at 37°C for 15 min. Afterward, the cell culture medium was discarded, and 500 μL of 4% paraformaldehyde was added to each well for fixation at 4°C for 30 min. After fixation, the cells were washed three times with PBS, and the PBS was discarded. The cell culture plates were then placed on ice and irradiated with UV light at 365 nm for 10 min. After UV irradiation, 500 μL of 0.5% Triton X100 was added to each well for treatment at room temperature for 15 min. After treatment, the cells were washed three times with PBS, and the PBS was discarded. Add 50 μL of click reaction mixture (0.25 μL 5 mM 5-carboxytetramethylrhodamine azide, 1 μL 50 mM copper sulfate, 3 μL 0.9 mg / ml TBTA, 1 μL 50 mM TCEP, 44.75 μL PBS) to each slide and react at room temperature for 1 h. After the reaction, wash three times with PBS and mount with mounting medium. Observe using a confocal microscope.

[0037] Experimental results are as follows Figure 8 As shown.

[0038] Figure 8 This is the result of detecting the localization of cannabidiol derivative a2 in MIN6 cells. Green indicates the localization staining of cannabidiol derivative a2 in cells, and red indicates the staining of mitochondria in cells. The results show that cannabidiol derivative a2 is co-localized with mitochondria in cells.

[0039] Example 4: Study on the use of cannabidiol derivative a2 as a probe molecule for cannabidiol to identify cannabidiol target proteins.

[0040] One day in advance, MIN6 cells were seeded in 10cm cell culture dishes. The next day, when the cells adhered and reached a density of over 80%, cell treatment began. Cells were treated with 10μM cannabidiol derivative a2 (control plus an equal volume of DMSO solution) in a 37℃ cell culture incubator for 30 min. The culture medium for the DMSO and a2 groups was discarded, and the cell culture plates were placed on ice and irradiated with 365nm UV light for 10 min. Cells were scraped off using ice-cold HEPES lysis buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1% (v / v) NP40 with a protease inhibitor cocktail), collected in centrifuge tubes, and disrupted using a cell sonicator. The cells were then centrifuged at 3000 rpm for 5 min at 4℃. The supernatant was collected for protein quantification.

[0041] Each group consists of four sample tubes, each containing 500 μL of 2 mg / ml total protein. Add 10 μL of 5 mM azide biotin, 10 μL of 50 mM TCEP, 30 μL of 0.9 mg / ml TBTA, and 10 μL of 50 mM copper sulfate to each tube; react at room temperature for 1 hour, vortexing every 15 minutes. Combine two tubes and centrifuge at 6500g for 4 minutes, discarding the supernatant; add 500 μL of ice-cold methanol to each tube, sonicate with a probe until the precipitate is completely dissolved in the solution, then combine the remaining two tubes from each group into a 2 ml centrifuge tube; continue centrifuging at 6500g, 4℃ for 4 minutes. Repeat the above two steps. Add 1 mL of 1.2% SDS / PBS to each tube, sonicate the probe, then boil in a metal bath at 98°C for 5 min, centrifuge at 6500g for 5 min, and collect the supernatant. Add the obtained sample and 5 mL of PBS to a 15 mL centrifuge tube, along with 170 μl of PBS-washed streptomycin beads, and incubate overnight at 4°C.

[0042] The next day, after rotating at room temperature for 1 hour, centrifuge at 4000 rpm for 5 minutes, discarding the supernatant; add 2 mL PBS, rotate for 10 minutes, then centrifuge at 1400g for 3 minutes, repeating twice. Finally, send the obtained streptomycin-affinity beads to the mass spectrometry laboratory for mass spectrometry analysis or add protein denaturation buffer for protein denaturation, which will then be used for Western blotting experiments. Because the localization results showed that cannabidiol is mainly located on mitochondria, we prioritized mitochondrial proteins with high scores.

[0043] The screening results and validation results are as follows Figure 9 , 10 As shown in Figure 11.

[0044] Figure 9 The mitochondrial protein bound to cannabidiol derivative a2 was screened based on mass spectrometry scores. The results indicate that the target protein of cannabidiol is the voltage-dependent anion channel protein VDAC1. Figure 10 The results of mass spectrometry were verified by Western blotting, which further showed that the target protein of cannabidiol is VDAC1. Figure 11 The results show the co-localization of cannabidiol derivative a2 and VDAC1 protein in cells, indicating that cannabidiol and VDAC1 protein are co-localized in cells. In summary, using cannabidiol derivative a2 as a probe molecule for cannabidiol can confirm that the target protein of cannabidiol is VDAC1.

Claims

1. A cannabidiol derivative, characterized in that, The derivative is cannabidiol-2-2-(3-(but-3-yn-1-yl)-3H-bisacrididin-3-yl)ethyl ester, with the specific chemical structural formula shown in Formula I: Formula I.

2. A method for preparing the cannabidiol derivative of claim 1, wherein, The method includes the following reaction pathway: The method includes the following steps: reacting cannabidiol of the structure shown in Formula II with 2-(3-(but-3-yn-1-yl)-3H-bisacrididin-3-yl)acetic acid of the structure shown in Formula III in the presence of a solvent and a catalyst to obtain a compound of the structure shown in Formula I, wherein the catalysts are DCC and DMAP.

3. The preparation method according to claim 2, characterized in that, The solvent is anhydrous dichloromethane.

4. The preparation method according to claim 2, characterized in that, The molar ratio of cannabidiol to 2-(3-(but-3-yn-1-yl)-3H-bisacryl-3-yl)acetic acid is 1:1.

2.

5. The preparation method according to claim 2, characterized in that, The reaction was first carried out in a light-protected environment at 0°C with magnetic stirring. After 20 minutes, the reaction was removed from the 0°C environment and allowed to continue at room temperature in the dark for 16 hours. After the reaction was completed, the organic phase was concentrated by rotary evaporation to obtain the initial product, which was then separated, purified and collected by preparative plate.

6. The application of the cannabidiol derivative of claim 1 in the preparation of probe molecules for finding cannabidiol targets.

7. The use of the cannabidiol derivative of claim 1 in the preparation of products for tracing the localization of cannabidiol in cells.

8. The use of the cannabidiol derivative of claim 1 in the preparation of products for which the target of cannabidiol is identified.

Images