Preparation methods and applications of cannabidiol epoxides

Abstract

This application relates to a method for preparing cannabidiol epoxide and its application. The method includes obtaining cannabidiol, adding it to a buffer solution containing a surfactant, treating it under preset conditions to obtain a crude product, and purifying the crude product by silica gel column gradient chromatography to obtain the finished cannabidiol epoxide. Compared with the traditional method of oxidation using peroxide agents, the process of preparing cannabidiol epoxide in this invention can utilize natural oxidation in air in one step, which is greener, more environmentally friendly, and safer. It also ensures ease of operation while reducing production costs. Furthermore, using cannabidiol (CBD) as a raw material, which has a relatively mature extraction process, makes it easier to obtain compared to other naturally occurring cannabinoids with extremely low content.

Description

Technical Field

[0001] This application relates to the field of cannabinoid compound preparation technology, and in particular to a method for preparing cannabidiol epoxide and its application. Background Technology

[0002] Industrial hemp, also known as hemp, is an annual herbaceous plant belonging to the Cannabis genus of the Cannabaceae family. Internationally, varieties with a THC (tetrahydrocannabinol) content of less than 0.3% are classified as industrial hemp. It should be noted that industrial hemp contains over 400 cannabinoid and non-cannabinoid compounds, which have significant applications in the medical and health fields. Among them, CBD (cannabidiol) is the most representative, possessing anti-inflammatory, antibacterial, analgesic, anti-anxiety, antioxidant, and memory-improving properties. It can also treat multiple arteriosclerosis and Parkinson's disease, prevent myocardial infarction, inhibit glioma cell metastasis, and suppress sex hormone secretion.

[0003] Transient receptor potential (TRP) channels are a superfamily of non-selective cations. These channels primarily regulate cellular function by allowing the permeation of cations such as sodium, potassium, and calcium in response to changes in the intracellular and extracellular environment. Among them, TRPA1, TRPM8, and TRPV1 are currently the focus of research.

[0004] The opening and closing of TRPA1 ion channels are closely related to the body's maintenance of normal physiological functions. When this ion channel is activated, the body exhibits discomfort such as pain, inflammation, or itching. Conversely, when the ion channel is closed, these symptoms are significantly relieved or completely eliminated. TRPA1 channel-specific antagonists have been discovered because TRPA1 is activated under certain pathological conditions. Therefore, TRPA1 can serve as an effective target for treating pain, inflammation, respiratory diseases, and other ailments. The search for activators and antagonists of TRPA1 ion channels has become a hot topic in drug development in recent years.

[0005] The TRPV1 channel was first cloned and discovered in rat dorsal root ganglion neurons in 1997, demonstrating that both nociceptive thermal stimuli and capsaicin-induced sensory pain are mediated by the TRPV1 channel, thus promoting research into the molecular mechanisms of nociceptive thermal and chemical stimuli transmission in sensory neurons. Various mechanisms for activating or upregulating the TRPV1 channel have been studied and reported in detail, with capsaicin, acid, temperature, and voltage being the main mechanisms for TRPV1 channel activation.

[0006] TRPM8, located in primary sensory neurons and known as the cold / menthol-sensitive receptor, is a voltage-gated channel activated by cell membrane depolarization. It is distributed in tissues and organs such as the prostate, breast, colon, lung, pancreas, brain, skin, and liver. It not only participates in the regulation of pain and cold sensation but also regulates cell growth. Numerous studies have reported targeting TRPV1 and TRPM8 channels, demonstrating therapeutic potential in prostate cancer, breast cancer, colon cancer, lung cancer, and skin cancer.

[0007] CN101678059A discloses plant extracts containing cannabinoids such as THC, CBD, CBG, CBC, THCV, THCA, CBDV, and CBDA, which can alleviate diseases such as neuropathic pain, cancer, or inflammation by blocking one or more TRP channels during prevention and treatment. Specifically, the cannabinoid-containing plant extracts refer to one or more plant extracts from Cannabis plants, which also contain one or more non-cannabinoid components extracted from the plant material along with the cannabinoids.

[0008] Currently, besides extraction from cannabinoid-containing plants, only a few studies report on the preparation of cannabidiol epoxides. This primarily involves protecting the phenolic hydroxyl groups of cannabidiol with acetyl groups, then oxidizing it in organic solvents such as acetone using a peroxidizing agent, followed by deacetylation. In other words, the preparation method requires three steps and the use of peroxidizing agents, which pose an explosion risk. Summary of the Invention

[0009] In view of this, this application proposes a method for preparing cannabidiol epoxide and its application to solve the above problems.

[0010] According to one aspect of this application, a method for preparing cannabidiol epoxide is provided, comprising the following preparation steps:

[0011] Cannabidiol was obtained, added to a buffer solution containing a surfactant, and treated under preset treatment conditions to obtain a crude product.

[0012] The crude product was purified by silica gel column gradient chromatography to obtain cannabidiol epoxide as the final product.

[0013] The chemical structural formula of the cannabidiol epoxide is as follows:

[0014]

[0015] As an optional embodiment of this application, cannabidiol is optionally obtained by adding it to an aqueous solution containing a buffer salt and a surfactant, and treating it under preset treatment conditions to obtain a crude product, comprising:

[0016] Obtaining cannabidiol;

[0017] A buffer solution containing surfactants is pre-prepared;

[0018] The cannabidiol is added to the aqueous solution and converted under preset conversion reaction conditions to obtain a first mixture.

[0019] The first mixture was extracted using an organic solvent, and the extracted product was dried to obtain a crude product.

[0020] As an optional embodiment of this application, the surfactant may optionally include any one of Tween 20, Tween 40, Tween 60, Tween 80, or sodium dodecyl sulfate.

[0021] As an optional embodiment of this application, the surfactant may optionally include Tween 80.

[0022] As an optional embodiment of this application, the buffer solution may optionally include either a pH 4.0 acetic acid-acetate buffer solution or a pH 6.8 phosphate buffer solution.

[0023] As an optional embodiment of this application, the buffer solution may optionally include: a pH 6.8 phosphate buffer solution.

[0024] As an optional embodiment of this application, the crude product may be purified by silica gel column gradient chromatography to obtain cannabidiol epoxide product, comprising:

[0025] Obtain the crude product;

[0026] Preset gradient conditions;

[0027] The crude product was purified by silica gel column chromatography under the gradient conditions to obtain the cannabidiol epoxide product.

[0028] As an optional implementation of this application, the gradient condition may optionally include:

[0029] n-Hexane:ethyl acetate = 80:1-10:1.

[0030] According to another aspect of this application, an application of cannabidiol epoxide is provided, wherein the cannabidiol epoxide prepared by any of the above-described methods is used to activate the TRPA1 channel.

[0031] According to another aspect of this application, an application of cannabidiol epoxide is provided, wherein the cannabidiol epoxide prepared by any of the above-described methods is used to block the TRPM8 channel.

[0032] The beneficial effects of this application are:

[0033] 1. Compared to traditional methods using peroxide oxidation, the process for preparing cannabidiol epoxides in this invention utilizes natural oxidation in air in a single step, making it greener, more environmentally friendly, and safer. It also ensures ease of operation while reducing production costs. Furthermore, by using cannabidiol (CBD) as a raw material, which has a relatively mature extraction process, it is easier to obtain compared to other naturally occurring cannabinoids with extremely low content.

[0034] 2. The effect of the 2,3-epoxy CBD of the present invention on the TRP channel is reported for the first time. The 2,3-epoxy CBD can be applied to diseases that can alleviate the activation of the TRPA1 channel and diseases that can be alleviated by blocking the TRPM8 channel.

[0035] Other features and aspects of this application will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0036] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this application together with the specification and serve to explain the principles of this application.

[0037] Figure 1 A flowchart illustrating a method for preparing cannabidiol epoxide according to an embodiment of this application;

[0038] Figure 2 This illustration shows a schematic diagram of the chemical structure of the cannabidiol epoxide prepared using cannabidiol according to an embodiment of this application.

[0039] Figure 3 The mass spectra of 2,3-epoxy CBD according to an embodiment of this application are shown;

[0040] Figure 4 The 1H NMR spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown.

[0041] Figure 5 The nuclear magnetic resonance HH COSY spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown.

[0042] Figure 6 The nuclear magnetic resonance HSQC spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown;

[0043] Figure 7 The carbon NMR spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown. Detailed Implementation

[0044] Various exemplary embodiments, features, and aspects of this application will now be described in detail with reference to the accompanying drawings. The same reference numerals in the drawings denote elements that have the same or similar functions. Although various aspects of the embodiments are shown in the drawings, they are not necessarily drawn to scale unless specifically indicated otherwise.

[0045] It should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this application or to simplify the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0046] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0047] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0048] Furthermore, to better illustrate this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented without certain specific details. In some instances, methods, means, components, and circuits well-known to those skilled in the art have not been described in detail in order to highlight the main points of this application.

[0049] Figure 1 A flowchart illustrating a method for preparing cannabidiol epoxide according to an embodiment of this application; Figure 2 This illustration shows a schematic diagram of the chemical structure of the cannabidiol epoxide prepared using cannabidiol according to an embodiment of this application. Figure 3 The mass spectra of 2,3-epoxy CBD according to embodiments of this application are shown; as follows: Figure 3As shown, the molecular ion peak of 2,3-epoxy CBD is 331.10, and the relative molecular mass is 331.10. Figure 4 The 1H NMR spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown. Figure 5 The nuclear magnetic resonance HH COSY spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown. Figure 6 The nuclear magnetic resonance HSQC spectrum of 2,3-epoxyCBD according to an embodiment of this application is shown; Figure 7 The carbon NMR spectrum of 2,3-epoxy CBD according to an embodiment of this application is shown.

[0050] like Figure 1 As shown, in a first aspect, this application provides a method for preparing cannabidiol epoxide, comprising the following preparation steps:

[0051] S100. Obtain cannabidiol, add it to a buffer solution containing a surfactant, and treat it under preset treatment conditions to obtain crude product;

[0052] S200. The crude product is purified by silica gel column gradient chromatography to obtain cannabidiol epoxide product;

[0053] Among them, the cannabidiol epoxide (C 21 H 30 The chemical structural formula of O3 is:

[0054]

[0055] In this embodiment, cannabidiol epoxide is prepared in one step by naturally oxidizing cannabidiol (CBD) in an aqueous solution containing a surfactant. This increases the availability of cannabinoids and provides a new option for their pharmaceutical development. Since the structure of cannabidiol epoxide is very similar to that of CBD, it is expected to possess pharmacological activities related to CBD and other cannabinoid compounds.

[0056] Specifically, in step S100, cannabidiol (CBD) is obtained and added to a buffer solution containing a surfactant. The mixture is then treated under preset conditions to obtain a crude product. It should be noted that this invention uses CBD as a raw material, a material with a relatively mature extraction process. By naturally oxidizing CBD under specific conditions, CBD epoxide (2,3-epoxy CBD) can be prepared, making it easier to obtain compared to other naturally occurring cannabinoids with extremely low content. Furthermore, compared to traditional methods using peroxides, the entire preparation process utilizes natural oxidation in air in a single step, making it greener, more environmentally friendly, and safer. While ensuring ease of operation, it also results in lower production costs.

[0057] As an optional embodiment of this application, cannabidiol is obtained, added to an aqueous solution containing a buffer salt and a surfactant, and treated under preset treatment conditions to obtain a crude product, comprising: obtaining cannabidiol; pre-preparing a buffer solution containing a surfactant; adding the cannabidiol to the aqueous solution and converting it under preset conversion reaction conditions to obtain a first mixture; extracting the first mixture with an organic solvent and drying the extracted product to obtain a crude product.

[0058] As an optional embodiment of this application, the surfactant may optionally include any one of Tween 20, Tween 40, Tween 60, Tween 80, or sodium dodecyl sulfate.

[0059] As an optional embodiment of this application, the surfactant may optionally include Tween 80.

[0060] In this embodiment, after obtaining a predetermined amount of cannabidiol, it is added to a buffer solution containing a surfactant, and the pH is controlled between 4.0 and 7.0 for the conversion reaction. The conversion reaction temperature is 25-45℃, preferably 30-40℃, under which the purity of the product obtained is higher. The conversion reaction time is 24-72 hours, preferably 48 hours. After the conversion reaction, a first mixture is obtained. Subsequently, the first mixture is extracted with an organic solvent, the extracted product is dried, and the solvent is concentrated to dryness under reduced pressure to obtain the corresponding crude product. The preferred organic solvent is dichloromethane. The product is extracted twice with 500 mL of dichloromethane, the organic phases are combined, dried with anhydrous sodium sulfate, and the solvent is concentrated to dryness under reduced pressure. The crude product is then purified by gradient elution using a silica gel column.

[0061] It should be noted that the surfactant used in this invention is preferably any one of Tween 20, Tween 40, Tween 60, Tween 80, or sodium dodecyl sulfate. More preferably, the surfactant is Tween 80, and the concentration of Tween 80 is preferably 1.0%-3.0%, more preferably 2.0%. Under the same conditions, adding 2.0% Tween 80 to a buffer solution to prepare a buffer solution containing the surfactant, and then reacting it with cannabidiol, results in a higher yield of the final product, cannabidiol epoxide.

[0062] As an optional embodiment of this application, the buffer solution may optionally include either a pH 4.0 acetic acid-acetate buffer solution or a pH 6.8 phosphate buffer solution.

[0063] As an optional embodiment of this application, the buffer solution may optionally include: a pH 6.8 phosphate buffer solution.

[0064] That is, the buffer solution and corresponding pH in this invention are preferably either a pH 4.0 acetate-acetate buffer solution or a pH 6.8 phosphate buffer solution, more preferably a pH 6.8 phosphate buffer solution. Under the same conditions, using a pH 6.8 phosphate buffer solution will result in a higher yield of the final product, cannabidiol epoxide.

[0065] After obtaining crude cannabidiol epoxide using cannabidiol and a buffer solvent containing a surfactant, the crude product is further purified by silica gel column gradient chromatography in step S200 to obtain the finished cannabidiol epoxide product.

[0066] As an optional embodiment of this application, the crude product is optionally purified by silica gel column gradient chromatography to obtain cannabidiol epoxide product, including: obtaining the crude product; setting gradient conditions; purifying the crude product by silica gel column chromatography according to the gradient conditions to obtain cannabidiol epoxide product.

[0067] As an optional embodiment of this application, the gradient conditions may optionally include: n-hexane:ethyl acetate = 80:1-10:1.

[0068] In this embodiment, the crude product is purified by silica gel column gradient chromatography to obtain cannabidiol epoxide as the final product. The eluent for silica gel column chromatography is a mixture of n-hexane and ethyl acetate; more preferably, the ratio of n-hexane to ethyl acetate is 80:1-10:1, resulting in a product with higher purity.

[0069] The following will provide further explanation in conjunction with Examples 1-17.

[0070] Example 1

[0071] like Figure 2 As shown, 10g of cannabidiol (CBD) was dissolved in 1000ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35°C for 48 hours. The reaction solution was extracted twice with 500ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution using a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. Finally, 8.8g of product, namely 2,3-epoxy CBD, with a purity of 98.2%, was obtained.

[0072] Ms = 330[M+1 = 331].

[0073] Among them, the mass spectra of 2,3-epoxy CBD, i.e., cannabidiol epoxide, are as follows: Figure 3 As shown, the proton NMR spectrum is as follows: Figure 4 As shown,1 H-NMR (400 Hz, DMSO-d6) δ7.26 (s, 1H), δ6.31 (s, 1H), δ6.27 (s, 1H), δ5.41 (s, 1H), δ5.04 (s, 2H), δ4.27 (d, J = 6.4 Hz, 1H), δ3.27 (m, 1H), δ2.49 (m, 3H), δ2.10 (m, 2H), δ1.83 (s, 3H), δ1.78 (m, 2H), δ1.57 (m, 2H), δ1.38 (s, 3H), δ1.2–1.4 (m, 4H), δ0.86 (t, 3H). The HH COSY NMR spectrum is as follows: Figure 5 As shown, the nuclear magnetic resonance HSQC spectrum is as follows: Figure 6 As shown, the carbon NMR spectrum is as follows: Figure 7 As shown, 13 C-NMR (400Hz, DMSO-d6)159.5,152.7,151.8,145.0,116.6,111.7,109.8,103. 2,89.0,70.2,47.7,43.5,36.0,35.1,31.5,31.4,28.9,25.5,22.5,22.4,14.0.

[0074] Example 2

[0075] 10g of cannabidiol (CBD) was dissolved in 1000ml of a buffer solution containing 1.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35°C for 48 hours. The reaction solution was extracted twice with 500ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 6.8g of the final product, 2,3-epoxy CBD, with a purity of 98.4%, was obtained.

[0076] Example 3

[0077] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 3.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.1 g of product, 2,3-epoxy CBD, with a purity of 98.2%, was finally obtained.

[0078] Example 4

[0079] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% SDS at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 6.1 g of the product, 2,3-epoxy CBD, with a purity of 98.1%, was finally obtained.

[0080] Example 5

[0081] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 3.0% SDS at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.1 g of the product, 2,3-epoxy CBD, with a purity of 98.0%, was finally obtained.

[0082] Example 6

[0083] 10g of cannabidiol (CBD) was dissolved in 1000ml of dilute hydrochloric acid solution containing 2.0% Tween 80 at pH 1.0–1.2. The mixture was stirred and heated to approximately 35°C for 48 hours. The reaction solution was extracted twice with 500ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column under the gradient conditions of hexane:ethyl acetate = 80:1–10:1, finally yielding 4.6g of product, namely 2,3-epoxy CBD, with a purity of 98.4%.

[0084] Example 7

[0085] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a pH 4.0 acetate-acetate buffer solution containing 2.0% Tween 80. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.0 g of product, 2,3-epoxy CBD, with a purity of 98.6%, was finally obtained.

[0086] Example 8

[0087] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 30 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.2 g of the product, 2,3-epoxy CBD, with a purity of 98.6%, was finally obtained.

[0088] Example 9

[0089] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 40 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.4 g of product, 2,3-epoxy CBD, with a purity of 98.1%, was finally obtained.

[0090] Example 10

[0091] 10g of cannabidiol (CBD) was dissolved in 1000ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 45°C for 48 hours. The reaction solution was extracted twice with 500ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.3g of the final product, 2,3-epoxy CBD, with a purity of 98.0%, was obtained.

[0092] Example 11

[0093] 10g of cannabidiol (CBD) was dissolved in 1000ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35°C for 24 hours. The reaction solution was extracted twice with 500ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.5g of the final product, 2,3-epoxy CBD, with a purity of 98.3%, was obtained.

[0094] Example 12

[0095] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 36 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 8.0 g of product, 2,3-epoxy CBD, with a purity of 98.1%, was finally obtained.

[0096] Example 13

[0097] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 60 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.1 g of the product, 2,3-epoxy CBD, with a purity of 98.0%, was finally obtained.

[0098] Example 14

[0099] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 80 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 72 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.4 g of the product, 2,3-epoxy CBD, with a purity of 98.6%, was finally obtained.

[0100] Example 15

[0101] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 20 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 6.9 g of the product, 2,3-epoxy CBD, with a purity of 98.5%, was finally obtained.

[0102] Example 16

[0103] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 40 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.2 g of the product, 2,3-epoxy CBD, with a purity of 98.1%, was finally obtained.

[0104] Example 17

[0105] 10 g of cannabidiol (CBD) was dissolved in 1000 ml of a buffer solution containing 2.0% Tween 60 at pH 6.8, consisting of sodium hydrogen phosphate and sodium dihydrogen phosphate. The mixture was stirred and heated to approximately 35 °C for 48 h. The reaction solution was extracted twice with 500 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was concentrated to dryness under reduced pressure. The residue was purified by gradient elution on a silica gel column with a gradient of n-hexane:ethyl acetate ratio of 80:1 to 10:1. 7.4 g of the product, 2,3-epoxy CBD, with a purity of 98.3%, was finally obtained.

[0106] The yields and purities of cannabidiol epoxides under Examples 1-17 are shown in the following tables. Table 1 shows the yields and purities of cannabidiol epoxides at different concentrations of Tween 80; Table 2 shows the yields and purities of cannabidiol epoxides corresponding to Tween 80 and SDS; Table 3 shows the yields and purities of cannabidiol epoxides under different buffer solutions; Table 4 shows the yields and purities of cannabidiol epoxides at different heating temperatures; Table 5 shows the yields and purities of cannabidiol epoxides at different heating times; and Table 6 shows the yields and purities of cannabidiol epoxides corresponding to Tween 20, Tween 40, Tween 60, and Tween 80.

[0107]

[0108]

[0109] Table 1. Yield and purity of cannabidiol epoxide at different concentrations of Tween 80

[0110] As shown in Table 1, Examples 1-3 selected different concentrations of Tween 80 for comparison. Among them, when Tween 80 was selected as the surfactant and the concentration was controlled at 2.0%, the overall yield and purity of the finished product were optimal.

[0111] Example Types of surfactants Surfactant concentration (%) Yield (g) purity(%) 1 Twain 80 2.0 8.8 98.2 4 SDS 2.0 6.1 98.1 5 SDS 3.0 7.1 98.0

[0112] Table 2. Yield and purity of cannabidiol epoxides corresponding to Tween 80 and SDS

[0113] As shown in Table 2, Examples 1 and 4 and 5 respectively selected 2.0% Tween 80, 2.0% SDS and 3.0% SDS for comparison. At the same concentration, the yield and purity of the finished product corresponding to Tween 80 were better than those of SDS. After increasing the concentration of SDS, the yield of the finished product increased, but the yield and purity of 2% Tween 80 were still better.

[0114] Example Buffer solvents and pH Yield (g) purity(%) 1 Sodium hydrogen phosphate and sodium dihydrogen phosphate salts with a pH of 6.8 8.8 98.2 6 dilute hydrochloric acid with a pH of 1.0 to 1.2 4.6 81.4 7 pH 4.0 Acetic acid-acetate 8.0 98.6

[0115] Table 3. Yield and purity of cannabidiol epoxide in different buffer solutions

[0116] As shown in Table 3, Examples 1, 6, and 7 respectively selected sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8, dilute hydrochloric acid at pH 1.0–1.2, and acetic acid-acetate at pH 4.0 for comparison of different buffer solutions. Among them, sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 were selected as the buffer solvent, and the overall yield and purity of the finished product were optimal.

[0117]

[0118]

[0119] Table 4. Yield and purity of cannabidiol epoxide at different heating temperatures

[0120] As shown in Table 4, heating temperatures of 35℃, 30℃, 40℃, and 45℃ were selected for Examples 1, 8, 9, and 10, respectively. Among them, the yield and purity of the finished product were optimal when the heating temperature was controlled at 35℃.

[0121] Example Heating time (h) Yield (g) purity(%) 1 48 8.8 98.2 11 24 8.5 98.3 12 36 8.0 98.1 13 60 7.1 98.0 14 72 7.4 98.6

[0122] Table 5. Yield and purity of cannabidiol epoxide at different heating times.

[0123] As shown in Table 5, the heating times selected for Examples 1 and Examples 11, 12, 13 and 14 were 48h, 24h, 36h, 60h and 72h, respectively. Among them, when the heating time was controlled at 48h, the yield and purity of the finished product were optimal.

[0124] Example Types of surfactants Yield (g) purity(%) 1 Twain 80 8.8 98.2 15 Twain 20 6.9 98.5 16 Twain 40 7.2 98.1 17 Twain 60 7.4 98.3

[0125] Table 6. Cannabidiol epoxide yield and purity at Tween 20, Tween 40, Tween 60 and Tween 80

[0126] As shown in Table 6, the surfactants selected in Examples 1, 15, 16, and 17 were Tween 80, Tween 20, Tween 40, and Tween 60, respectively. Among them, when the surfactant was Tween 80, the overall yield and purity of the finished product were optimal.

[0127] Therefore, in the preparation of cannabidiol epoxide, 2% Tween 80 is added to a pH 6.8 phosphate buffer solution, and the mixture is heated at 35°C for 48 hours. After extraction twice with 500 mL of dichloromethane, the organic phases are combined and dried with anhydrous sodium sulfate. The solvent is concentrated to dryness under reduced pressure. The crude product is then purified by gradient elution using a silica gel column. The eluent for silica gel column chromatography is a mixture of n-hexane and ethyl acetate, with a preferred ratio of 80:1 to 10:1. This method yields a product with superior yield and purity. This invention...

[0128] According to another aspect of this application, an application of cannabidiol epoxide is provided, wherein the cannabidiol epoxide prepared by any of the above-described methods is used to activate the TRPA1 channel.

[0129] It should be noted that pharmacological studies have shown the use of 2,3-epoxy CBD in the preparation of drugs for the treatment of diseases that benefit from TRPA1 channel activation, including any one or more of obesity, hypertension, hyperglycemia, and aging.

[0130] According to another aspect of this application, an application of cannabidiol epoxide is provided, wherein the cannabidiol epoxide prepared by any of the above-described methods is used to block the TRPM8 channel.

[0131] It should also be noted that pharmacological studies have shown that 2,3-epoxy CBD is promising for the treatment of diseases that benefit from the relief provided by blocking the TRPM8 channel, particularly for chronic pain and migraines, irritable bowel syndrome, dysphagia (OD), chronic cough, and various cancers such as prostate cancer, breast cancer, colon cancer, lung cancer, and skin cancer.

[0132] The following three sets of experiments will provide further explanation.

[0133] Test materials

[0134] Test sample: Cannabidiol epoxide, prepared by the above-mentioned method for preparing cannabidiol epoxide, with a purity of 98.2%;

[0135] Positive controls: cinnamaldehyde (98% purity), ruthenium red (approx. 100% purity), capsaicin (approx. 100% purity), capsicum (99.3% purity), menthol (97% purity), 2-APB (approx. 100% purity).

[0136] Patch clamp assays of the effects of 1S and 2R-epoxy-CBD on TRPV1, TRPA1, and TRPM8: Experiment 1: TRPA1

[0137] The voltage stimulation protocol for whole-cell patch-clamp recording of TRPA1 currents was as follows: After whole-cell occlusion, the cell membrane voltage was clamped at -80 mV. The recording voltage was stepped from 0 mV to -100 mV and held for 1 ms, followed by a 300 ms ramp stimulation to 100 mV. Data was collected every 5 s to observe the effect of the drug on TRPA1 currents. Experimental data were acquired using an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0138] Experiment 2: TRPV1

[0139] The voltage stimulation protocol for whole-cell patch-clamp recording of TRPV1 currents was as follows: After whole-cell occlusion, the cell membrane voltage was clamped at 0 mV. The recording voltage was stepped from 0 mV to -100 mV and held for 1 ms, followed by a 300 ms ramp stimulation to 100 mV. Data was collected every 5 s to observe the effect of the drug on TRPV1 currents. Experimental data were acquired using an EPC-10 amplifier (HEKAElectronics) and stored in PatchMaster (HEKAElectronics) software.

[0140] Experiment 3: TRPM8

[0141] The voltage stimulation protocol for whole-cell patch-clamp recording of TRPM8 currents was as follows: After whole-cell occlusion, the cell membrane voltage was clamped at -80 mV. The recording voltage was stepped from 0 mV to -100 mV, followed by a 500 ms ramp stimulation to 100 mV. Data was collected repeatedly every 5 s to observe the effect of the drug on TRPM8 currents. Experimental data were acquired using an EPC-10 amplifier (HEKA Electronics) and stored in PatchMaster (HEKA Electronics) software.

[0142] A capillary glass tube was drawn into a recording electrode using a microelectrode drawing instrument. Under an inverted microscope, the microelectrode manipulator was used to bring the recording electrode into contact with the cell, and negative pressure was applied to aspirate and form a GΩ seal. After forming the GΩ seal, rapid capacitance compensation was performed, and then negative pressure was continued to rupture the cell membrane, establishing a whole-cell recording mode. Slow capacitance compensation was then performed, and the membrane capacitance and series resistance were recorded. No leakage compensation was applied.

[0143] A coverslip containing cells was placed in the recording bath of an inverted microscope. The working solution containing the test compound and the external solution without the compound were perfused through the recording bath by gravity to act on the cells. A vacuum pump was used for fluid exchange during recording. Three cells were independently and repeatedly tested. All electrophysiological experiments were performed at room temperature.

[0144] The agonistic and inhibitory effects of 2,3-epoxy CBD on TRPA1, TRPV1 and TRPM8 channels were investigated in three independent replicate experiments.

[0145] The experimental results are summarized as follows:

[0146] Table 7 shows the inhibition results of 2,3-epoxy CBD on TRPV1 current and the positive control ruthenium red on TRPV1 current; Table 8 shows the inhibition results of 2,3-epoxy CBD on TRPV1 current and the positive control capsazepine on TRPV1 current; Table 9 shows the inhibition results of 2,3-epoxy CBD on TRPM8 current and the positive control 2-APB on TRPM8 current.

[0147]

[0148] Table 7. Inhibition results of 2,3-epoxy CBD on TRPV1 current and ruthenium red (positive control) on TRPV1 current.

[0149] As shown in Table 7, the results show that 2,3-epoxy CBD has an excitatory effect on TRPA1 current and no significant inhibitory effect.

[0150]

[0151] Table 8. Results of the inhibitory effects of 2,3-epoxy CBD on TRPV1 current and the positive control capsazepine on TRPV1 current.

[0152] As shown in Table 8, the results showed that 2,3-epoxy CBD did not show any inhibitory effect compared with the positive control.

[0153]

[0154] Table 9. Inhibitory effect of 2,3-epoxy CBD on TRPM8 current and the inhibitory effect of positive control 2-APB on TRPM8 current.

[0155] As shown in Table 9, the results showed that 2,3-epoxy CBD had a similar inhibitory effect compared with the positive control.

[0156] In summary, the present invention achieves the following effects:

[0157] 1. Compared to traditional methods using peroxide oxidation, the process for preparing cannabidiol epoxides in this invention utilizes natural oxidation in air in a single step, making it greener, more environmentally friendly, and safer. It also ensures ease of operation while reducing production costs. Furthermore, by using cannabidiol (CBD) as a raw material, which has a relatively mature extraction process, it is easier to obtain compared to other naturally occurring cannabinoids with extremely low content.

[0158] 2. The effect of the 2,3-epoxy CBD of the present invention on the TRP channel is reported for the first time. The 2,3-epoxy CBD can be applied to diseases that can alleviate the activation of the TRPA1 channel and diseases that can be alleviated by blocking the TRPM8 channel.

[0159] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method of preparing cannabidiol epoxide, characterized by, The preparation method comprises the following steps: Obtain cannabidiol, add a buffer solution containing a surfactant, and process under preset processing conditions to obtain a crude product; The crude product is separated and purified by gradient chromatography on a silica gel column to obtain a cannabidiol epoxide finished product; The chemical structural formula of the cannabidiol epoxide is: ； Obtain cannabidiol, add an aqueous solution containing a buffer salt and a surfactant, and process under preset processing conditions to obtain a crude product, comprising: Obtain cannabidiol; Pre-set a buffer solution containing a surfactant; Add the cannabidiol to the aqueous solution and convert under preset conversion reaction conditions to obtain a first mixture; Extract the first mixture with an organic solvent, dry the extracted product, and obtain a crude product; The surfactant is any one of Tween 20, Tween 40, Tween 60, and Tween 80; The buffer solution is any one of a pH 4.0 acetic acid-acetate buffer solution and a pH 6.8 phosphate buffer solution; The temperature of the conversion reaction is 25-45°C, and the time of the conversion reaction is 24-72h.

2. The method of preparing cannabidiol epoxide according to claim 1, characterized in that, The crude product is separated and purified by gradient chromatography on a silica gel column to obtain a cannabidiol epoxide finished product, comprising: Obtain the crude product; Pre-set gradient conditions; Purify the crude product on a silica gel column according to the gradient conditions to obtain a cannabidiol epoxide finished product.

3. The method of preparing cannabidiol epoxide according to claim 1, wherein, The gradient conditions comprise: n-Hexane: ethyl acetate = 80:1-10:1.

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