Preparation method and application of cannabidiol epoxide

Abstract

The invention relates to a preparation method and application of cannabidiol epoxide, and the method comprises the following steps: obtaining cannabidiol, adding the cannabidiol into a buffer solution containing a surfactant, and treating under a preset treatment condition to obtain a crude product; and separating and purifying the crude product through silica gel column gradient chromatography to obtain a cannabidiol epoxide finished product. Compared with a traditional oxidation method adopting a peroxide agent, the process for preparing the cannabidiol epoxide can be obtained in one step through air natural oxidation, the cannabidiol epoxide is greener, more environmentally friendly and safer, and the production cost is lower while it is guaranteed that operation is easy. Meanwhile, the cannabidiol CBD which is relatively mature in extraction process is adopted as a raw material, and compared with other cannabinoids which are extremely low in natural content, the cannabidiol CBD is easier to obtain.

Description

Technical Field

[0001] The present application relates to the technical field of preparation of cannabinoid compounds, and in particular to a preparation method and application of cannabidiol epoxide. Background Art

[0002] Industrial hemp, also known as hemp, is an annual herbaceous plant belonging to the genus Cannabis of the family Cannabaceae. Internationally, varieties with a THC (Tetrahydrocanabinol) content of less than 0.3% are called industrial hemp. It should be noted that there are more than 400 cannabinoid and non-cannabinoid compounds in industrial hemp, which have important application value in the field of medical and health care. Among them, CBD (Cannabidiol) is the most representative. It can not only resist inflammation, sterilize, relieve pain, relieve anxiety, resist oxidation and improve memory, but also treat multiple arteriosclerosis and Parkinson's disease, prevent myocardial infarction, inhibit the metastasis of glioma cells and inhibit the secretion of sex hormones.

[0003] Transient receptor potential ion channels, or TRP channels, are a superfamily of non-selective cations that are mainly permeable to cations such as sodium, potassium, and calcium according to changes in the intracellular and extracellular environments, thereby regulating cell functions. Among them, TRPA1, TRPM8, and TRPV1 are currently hot topics of research.

[0004] The opening or closing of TRPA1 ion channels is closely related to the body's maintenance of normal physiological activities. When the ion channel is activated, the body will show discomfort reactions such as pain, inflammation, or itching. When the ion channel is closed, the above discomfort symptoms will be significantly alleviated or completely eliminated. When the TRPA1 channel-specific antagonist is discovered, TRPA1 is activated in certain pathological conditions. Therefore, TRPA1 can be used as an effective target for the treatment of pain, inflammation, respiratory diseases and other diseases. The search for activators and antagonists of TRPA1 ion channels has become a hot spot in drug research and development in recent years.

[0005] TRPV1 channel was first cloned and discovered in rat dorsal root ganglion neurons in 1997, and it was proved that noxious heat stimulation and capsaicin-induced pain were both mediated by TRPV1 channel, which promoted the study of the molecular mechanism of noxious heat stimulation and chemical stimulation in sensory neurons. There have been many detailed studies and reports on the mechanisms of stimulating or upregulating TRPV1 channels, among which capsaicin, acid, temperature and voltage are the main ways to stimulate TRPV1 channels.

[0006] TRPM8 exists in primary sensory neurons and is known as the cold / menthol sensitive receptor. It is a voltage-gated channel activated by cell membrane depolarization and is distributed in tissues and organs such as the prostate, breast, colon, lung, pancreas, brain, skin, and liver. It is not only involved in the regulation of pain and cold sensation, but also can regulate cell growth. There are also many research reports targeting TRPV1 and TRPM8 channels, which show therapeutic prospects 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 relieve diseases such as neuropathic pain, cancer or inflammation by blocking one or more TRP channels in the prevention and treatment process. The plant extract containing cannabinoids refers to one or more plant extracts from plants of the genus Cannabis, and also contains one or more non-cannabinoid components extracted from plant materials together with cannabinoids.

[0008] At present, how to obtain cannabinoid substances and use them to provide new options for the development of the pharmaceutical field, in addition to the extraction from the plants containing cannabinoids mentioned above, there are only a few literature reports on the preparation of cannabidiol epoxide, which mainly involves first protecting the phenolic hydroxyl group of cannabidiol with an acetyl group, then oxidizing it with a peroxidation agent in an organic solvent such as acetone, and then removing the acetyl group. That is, the preparation method requires a total of three steps of reaction, and a peroxidation agent with an explosion risk must be used. Summary of the invention

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

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

[0011] Obtaining cannabidiol, adding it to a buffer solution containing a surfactant, and treating it under preset treatment conditions to obtain a crude product;

[0012] The crude product is separated and purified by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product;

[0013] Wherein, the chemical structural formula of the cannabidiol epoxide is:

[0014]

[0015] As an optional embodiment of the present application, cannabidiol is optionally obtained, added to an aqueous solution containing a buffer salt and a surfactant, and treated under preset treatment conditions to obtain a crude product, including:

[0016] Get cannabidiol;

[0017] Pre-prepare a buffer solution containing a surfactant;

[0018] Adding the cannabidiol to the aqueous solution and converting it under preset conversion reaction conditions to obtain a first mixed material;

[0019] The first mixed material is extracted with an organic solvent, and the extracted product is dried to obtain a crude product.

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

[0021] As an optional embodiment of the present application, optionally, the surfactant includes: Tween 80.

[0022] As an optional embodiment of the present application, optionally, the buffer solution includes: any one of a pH 4.0 acetic acid-acetate buffer solution and a pH 6.8 phosphate buffer solution.

[0023] As an optional embodiment of the present application, optionally, the buffer solution includes: pH 6.8 phosphate buffer solution.

[0024] As an optional embodiment of the present application, optionally, the crude product is separated and purified by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product, including:

[0025] obtaining the crude product;

[0026] Preset gradient conditions;

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

[0028] As an optional embodiment of the present application, optionally, the gradient condition comprises:

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

[0030] According to another aspect of the present application, there is provided an application of cannabidiol epoxide, and the application of cannabidiol epoxide prepared by any of the preparation methods of cannabidiol epoxide described above in stimulating TRPA1 channels.

[0031] According to another aspect of the present application, there is provided an application of cannabidiol epoxide, and the application of cannabidiol epoxide prepared by any of the preparation methods of cannabidiol epoxide described above in blocking TRPM8 channels.

[0032] Beneficial effects of this application:

[0033] 1. Compared with the traditional method of oxidation by peroxide, the process of preparing cannabidiol epoxide in the present invention can be obtained by natural oxidation in air in one step, which is greener, more environmentally friendly and safer. While ensuring easy operation, the production cost is lower. At the same time, cannabidiol CBD with a relatively mature extraction process is used as a raw material, which is easier to obtain than other cannabinoids with extremely low natural content.

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

[0035] Other features and aspects of the present application will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the present application and, together with the description, serve to explain the principles of the present application.

[0037] Figure 1 A flow chart showing the method for preparing cannabidiol epoxide according to an embodiment of the present application;

[0038] Figure 2 A schematic diagram showing the chemical structure corresponding to the preparation of cannabidiol epoxide using cannabidiol in an embodiment of the present application;

[0039] Figure 3 The mass spectrum of 2,3-epoxy CBD of the present application example is shown;

[0040] Figure 4 The nuclear magnetic resonance hydrogen spectrum of 2,3-epoxy CBD of the present application example is shown;

[0041] Figure 5 The nuclear magnetic resonance HH COSY spectrum of 2,3-epoxy CBD of the present application example is shown;

[0042] Figure 6 The NMR HSQC spectrum of 2,3-epoxy CBD of the present application example is shown;

[0043] Figure 7 The carbon nuclear magnetic resonance spectrum of 2,3-epoxy CBD of the example of the present application is shown. DETAILED DESCRIPTION

[0044] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0045] Among them, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application or simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0046] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0047] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0048] In addition, in order to better illustrate the present application, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0049] Figure 1 A flow chart showing the method for preparing cannabidiol epoxide according to an embodiment of the present application; Figure 2 A schematic diagram showing the chemical structure corresponding to the preparation of cannabidiol epoxide using cannabidiol in an embodiment of the present application; Figure 3 The mass spectrum of 2,3-epoxy CBD of the present application example is shown; 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 nuclear magnetic resonance hydrogen spectrum of 2,3-epoxy CBD of the present application example is shown; Figure 5 The nuclear magnetic resonance HH COSY spectrum of 2,3-epoxy CBD of the present application example is shown; Figure 6 The NMR HSQC spectrum of 2,3-epoxyCBD of the present application example is shown; Figure 7 The carbon nuclear magnetic resonance spectrum of 2,3-epoxy CBD of the example of the present application is shown.

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

[0051] S100, obtaining cannabidiol, adding it to a buffer solution containing a surfactant, and treating it under preset treatment conditions to obtain a crude product;

[0052] S200, separating and purifying the crude product by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product;

[0053] Wherein, the cannabidiol epoxide (C 21 H 30 O3) is as follows:

[0054]

[0055] In this embodiment, cannabidiol epoxide can be prepared in one step by suspending cannabidiol in an aqueous solution containing a surfactant and using air for natural oxidation. This can not only increase the availability of cannabinoids, but also provide a new option for the development of cannabinoids in the pharmaceutical field. Since the structure of cannabidiol epoxide is very similar to cannabidiol CBD, it is expected to have pharmacological activities related to cannabinoid compounds such as CBD.

[0056] Specifically, through step S100, cannabidiol is obtained, added to a buffer solution containing a surfactant, and processed under preset processing conditions to obtain a crude product. Here, it should be noted that the present invention uses cannabidiol CBD with a relatively mature extraction process as a raw material, and naturally oxidizes cannabidiol under specific conditions to achieve the preparation of cannabidiol epoxide, i.e. 2,3-epoxy CBD, which is easier to obtain than other cannabinoids with extremely low natural content. At the same time, compared with the traditional method of oxidation using peroxide agents, the entire preparation process can be obtained by natural oxidation in air in one step, which is more green, environmentally friendly, and safer. While ensuring easy operation, the production cost is lower.

[0057] As an optional embodiment of the present application, cannabidiol is optionally obtained, added to an aqueous solution containing a buffer salt and a surfactant, and treated under preset treatment conditions to obtain a crude product, including: obtaining cannabidiol; pre-setting 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 mixed material; extracting the first mixed material using an organic solvent, and drying the extracted product to obtain a crude product.

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

[0059] As an optional embodiment of the present application, optionally, the surfactant includes: Tween 80.

[0060] In this embodiment, after obtaining a preset amount of cannabidiol, it is added to a buffer containing a surfactant, and the pH is controlled to be between 4.0-7.0 for conversion reaction. Wherein, the conversion reaction temperature is: 25-45°C, preferably 30-40°C, and the finished product obtained under this temperature condition has a higher purity. At the same time, the conversion reaction time is: 24-72h, preferably 48h. After the conversion reaction, a first mixed material is obtained. Subsequently, the first mixed material is extracted with an organic solvent, the extracted product is dried, and the solvent is concentrated to dryness under reduced pressure to obtain a corresponding crude product. Wherein, the organic solvent is preferably dichloromethane, and 500mL of dichloromethane is used for extraction twice. After the organic phase is combined, it is dried with anhydrous sodium sulfate, and the solvent is concentrated to dryness under reduced pressure. The crude product is then separated and purified by gradient elution on a silica gel column.

[0061] It should be noted that the surfactant in the present 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, 2.0% Tween 80 is added to the buffer solution to prepare a buffer solution containing the surfactant, and the conversion reaction is carried out with cannabidiol, and the final product cannabidiol epoxide has a higher yield.

[0062] As an optional embodiment of the present application, optionally, the buffer solution includes: any one of a pH 4.0 acetic acid-acetate buffer solution and a pH 6.8 phosphate buffer solution.

[0063] As an optional embodiment of the present application, optionally, the buffer solution includes: pH 6.8 phosphate buffer solution.

[0064] That is, the buffer solution and the corresponding pH in the present invention are preferably any one of pH 4.0 acetic acid-acetate buffer solution or pH 6.8 phosphate buffer solution, and more preferably pH 6.8 phosphate buffer solution. Under the same conditions, pH 6.8 phosphate is used as the buffer solution, and the yield of the final product cannabidiol epoxide is higher.

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

[0066] As an optional embodiment of the present application, the crude product is optionally separated and purified by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product, including: obtaining the crude product; presetting gradient conditions; purifying the crude product by silica gel column chromatography corresponding to the gradient conditions to obtain a finished cannabidiol epoxide product.

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

[0068] In this embodiment, the crude product is separated and purified by silica gel column gradient chromatography to obtain the finished cannabidiol epoxide product. The eluent of the silica gel column chromatography is a mixture of n-hexane and ethyl acetate. Furthermore, the ratio of n-hexane to ethyl acetate is preferably 80:1-10:1, so that the finished product prepared in this way has higher purity.

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

[0070] Example 1

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

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

[0073] Among them, the mass spectrum of 2,3-epoxy CBD, i.e. cannabidiol epoxide, is as follows Figure 3 As shown, the H NMR spectrum is 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), HH COSY spectrum as shown Figure 5 As shown, the NMR HSQC spectrum is Figure 6 As shown, the carbon NMR spectrum is 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] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 1.0% Tween 80, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 6.8 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.4%.

[0076] Example 3

[0077] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 3.0% Tween 80, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 8.1 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.2%.

[0078] Example 4

[0079] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% SDS, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 6.1 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.1%.

[0080] Example 5

[0081] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 3.0% SDS, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.1 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.0%.

[0082] Example 6

[0083] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a dilute hydrochloric acid solution with a pH of 1.0 to 1.2 containing 2.0% Tween 80, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 4.6 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.4%.

[0084] Example 7

[0085] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of pH 4.0 acetic acid-acetate buffer solution containing 2.0% Tween 80, start stirring, heat to about 35°C, maintain for 48 hours, extract the reaction solution twice with 500 ml of dichloromethane, combine the organic phases and dry them with anhydrous sodium sulfate, concentrate the solvent to dryness under reduced pressure, and separate and purify the residue by gradient elution on a silica gel column, the gradient condition is n-hexane: ethyl acetate = 80:1 to 10:1, and finally obtain 8.0 g of product, i.e., 2,3-epoxy CBD, with a purity of 98.6%.

[0086] Example 8

[0087] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 30°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.2 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.6%.

[0088] Example 9

[0089] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 40°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 8.4 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.1%.

[0090] Example 10

[0091] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 45°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 8.3 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.0%.

[0092] Embodiment 11

[0093] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 35°C, and maintain for 24 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 8.5 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.3%.

[0094] Example 12

[0095] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 35°C, and maintain for 36 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 8.0 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.1%.

[0096] Embodiment 13

[0097] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 35°C, and maintain for 60 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.1 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.0%.

[0098] Embodiment 14

[0099] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 80, start stirring, heat to about 35°C, and maintain for 72 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.4 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.6%.

[0100] Embodiment 15

[0101] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 20, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 6.9 g of the product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.5%.

[0102] Example 16

[0103] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 40, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.2 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.1%.

[0104] Embodiment 17

[0105] Dissolve 10 g of cannabidiol (CBD) in 1000 ml of a buffer solution of sodium hydrogen phosphate and sodium dihydrogen phosphate at pH 6.8 containing 2.0% Tween 60, start stirring, heat to about 35°C, and maintain for 48 hours. The reaction solution is extracted twice with 500 ml of dichloromethane, the organic phases are combined and dried over anhydrous sodium sulfate, the solvent is concentrated to dryness under reduced pressure, and the residue is separated and purified by gradient elution on a silica gel column, the gradient condition being n-hexane: ethyl acetate = 80:1 to 10:1, and finally 7.4 g of product, i.e., 2,3-epoxy CBD, is obtained with a purity of 98.3%.

[0106] The yield and purity of cannabidiol epoxide under Examples 1 to 17 are shown in the following table. Among them, Table 1 shows the yield and purity of cannabidiol epoxide under different concentrations of Tween 80; Table 2 shows the yield and purity of cannabidiol epoxide corresponding to Tween 80 and SDS; Table 3 shows the yield and purity of cannabidiol epoxide under different buffer solutions; Table 4 shows the yield and purity of cannabidiol epoxide under different heating temperatures; Table 5 shows the yield and purity of cannabidiol epoxide under different heating times; Table 6 shows the yield and purity of cannabidiol epoxide corresponding to Tween 20, Tween 40, Tween 60 and Tween 80.

[0107]

[0108]

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

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

[0111] Example Surfactant Type 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 epoxide corresponding to Tween 80 and SDS

[0113] As shown in Table 2, Example 1 and Example 4 and Example 5 respectively selected 2.0% Tween 80 and 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, and after increasing the SDS concentration, the finished product increased, but the yield and purity of 2% Tween 80 were still better.

[0114] Example Buffer solvent and pH Yield (g) purity(%) 1 pH 6.8 Sodium hydrogen phosphate and sodium dihydrogen phosphate salt 8.8 98.2 6 pH 1.0~1.2 dilute hydrochloric acid 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 and Examples 6 and 7 respectively selected pH 6.8 sodium hydrogen phosphate and sodium dihydrogen phosphate salt, pH 1.0-1.2 dilute hydrochloric acid and pH 4.0 acetic acid-acetate for comparison of different buffer solutions, wherein pH 6.8 sodium hydrogen phosphate and sodium dihydrogen phosphate salt were selected as buffer solvents, and the 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, Example 1 and Examples 8, 9, and 10 respectively selected heating temperatures of 35°C, 30°C, 40°C, and 45°C, among which when the heating temperature was controlled at 35°C, the yield and purity of the finished product were optimal.

[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 of Example 1 and Examples 11, 12, 13, and 14 are 48 h, 24 h, 36 h, 60 h, and 72 h, respectively. When the heating time is controlled at 48 h, the yield and purity of the finished product are optimal.

[0124] Example Surfactant Type 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 purity under Tween 20, Tween 40, Tween 60 and Tween 80

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

[0127] It can be seen that in the process of preparing cannabidiol epoxide, 2% Tween 80 is selected and added to a pH 6.8 phosphate buffer solution, and after heating at 35°C for 48 hours, it is extracted twice with 500 mL of dichloromethane, the organic phases are combined and dried with anhydrous sodium sulfate, and the solvent is concentrated to dryness under reduced pressure. The crude product is then separated and purified by gradient elution on a silica gel column, wherein the eluent for silica gel column chromatography is a mixture of n-hexane and ethyl acetate, and the ratio of n-hexane to ethyl acetate is preferably 80:1-10:1, so that the yield and purity of the finished product prepared in this way are better.

[0128] According to another aspect of the present application, there is provided an application of cannabidiol epoxide, and the application of cannabidiol epoxide prepared by any of the preparation methods of cannabidiol epoxide described above in stimulating TRPA1 channels.

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

[0130] According to another aspect of the present application, there is provided an application of cannabidiol epoxide, and the application of cannabidiol epoxide prepared by any of the preparation methods of cannabidiol epoxide described above in blocking TRPM8 channels.

[0131] It should also be noted that pharmacological experiments have found that 2,3-epoxy CBD is useful in the preparation of drugs for the treatment of diseases that benefit from blocking the TRPM8 channel, especially in chronic pain and migraines, irritable bowel syndrome, oropharyngeal dysphagia (OD), chronic cough, and various cancers, such as prostate cancer, breast cancer, colon cancer, lung cancer, and skin cancer.

[0132] Three groups of experiments are provided below for further explanation.

[0133] Test materials

[0134] Test sample: cannabidiol epoxide, prepared by the above cannabidiol epoxide preparation method, purity 98.2%;

[0135] Positive control substances: cinnamaldehyde (purity 98%), ruthenium red (purity about 100%), capsaicin (purity about 100%), capsaicin (purity 99.3%), menthol (purity 97%), 2-APB (purity about 100%).

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

[0137] The voltage stimulation scheme for whole-cell patch clamp recording of whole-cell TRPA1 current is as follows: After the whole-cell seal is formed, the cell membrane voltage is clamped at -80mV. The recording voltage is stepped from 0mV to -100mV for 1ms, and then a 300ms ramp stimulation is given to 100mV. Data is collected repeatedly every 5s to observe the effect of drugs on TRPA1 current. The experimental data are collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0138] Trial 2: TRPV1

[0139] The voltage stimulation scheme for whole-cell patch clamp recording of whole-cell TRPV1 current is as follows: After the whole-cell seal is formed, the cell membrane voltage is clamped at 0mV. The recording voltage is stepped from 0mV to -100mV for 1ms, and then a 300ms ramp stimulation is given to 100mV. Data is collected repeatedly every 5s to observe the effect of drugs on TRPV1 current. The experimental data are collected by EPC-10 amplifier (HEKAElectronics) and stored in PatchMaster (HEKAElectronics) software.

[0140] Trial 3: TRPM8

[0141] The voltage stimulation scheme for whole-cell patch clamp recording of whole-cell TRPM8 current is as follows: After the whole-cell seal is formed, the cell membrane voltage is clamped at -80mV. The recording voltage is stepped from 0mV to -100mV, and a 500ms ramp stimulation is given to 100mV. Data is collected repeatedly every 5s to observe the effect of the drug on TRPM8 current. The experimental data are collected by an EPC-10 amplifier (HEKA Electronics) and stored in PatchMaster (HEKA Electronics) software.

[0142] Use a microelectrode puller to pull a capillary glass tube into a recording electrode. Under an inverted microscope, use the microelectrode manipulator to bring the recording electrode into contact with the cell, apply negative pressure and suction to form a GΩ seal. After the GΩ seal is formed, perform fast capacitance compensation, and then continue to apply negative pressure to break the cell membrane and form a whole-cell recording mode. Then perform slow capacitance compensation and record the membrane capacitance and series resistance. No leakage compensation is given.

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

[0144] The agonist and inhibitory effects of 2,3-epoxy CBD on TRPA1, TRPV1, and TRPM8 channels were tested in three independent replicates.

[0145] The test results are summarized as follows:

[0146] Table 7 shows the inhibitory effects of 2,3-epoxy CBD on TRPA1 current and the positive control ruthenium red on TRPV1 current; Table 8 shows the inhibitory effects of 2,3-epoxy CBD on TRPV1 current and the positive control capsazepine on TRPV1 current; Table 9 shows the inhibitory effects 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 TRPA1 current and positive control ruthenium red on TRPV1 current

[0149] As shown in Table 7, the results showed that 2,3-epoxy CBD had an agonistic effect on TRPA1 currents but had no significant inhibitory effect.

[0150]

[0151] Table 8 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 showed no 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 showed a similar inhibitory effect compared with the positive control.

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

[0157] 1. Compared with the traditional method of oxidation by peroxide, the process of preparing cannabidiol epoxide in the present invention can be obtained by natural oxidation in air in one step, which is greener, more environmentally friendly and safer. While ensuring easy operation, the production cost is lower. At the same time, cannabidiol CBD with a relatively mature extraction process is used as a raw material, which is easier to obtain than other cannabinoids with extremely low natural content.

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

[0159] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing cannabidiol epoxide, characterized in that: The method comprises the following preparation steps: Obtaining cannabidiol, adding it to a buffer solution containing a surfactant, and treating it under preset treatment conditions to obtain a crude product; The crude product is separated and purified by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product; Wherein, the chemical structural formula of the cannabidiol epoxide is:

2. The method for preparing cannabidiol epoxide according to claim 1, characterized in that: Obtain cannabidiol, add it to an aqueous solution containing a buffer salt and a surfactant, and treat it under preset treatment conditions to obtain a crude product, including: Get cannabidiol; Pre-prepare 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 mixed material; The first mixed material is extracted with an organic solvent, and the extracted product is dried to obtain a crude product.

3. The method for preparing cannabidiol epoxide according to claim 1, characterized in that: The surfactant includes any one of Tween 20, Tween 40, Tween 60, Tween 80 or sodium dodecyl sulfate.

4. The method for preparing cannabidiol epoxide according to claim 3, characterized in that: The surfactant includes Tween 80.

5. The method for preparing cannabidiol epoxide according to claim 1, characterized in that: The buffer solution includes: any one of a pH 4.0 acetic acid-acetate buffer solution and a pH 6.8 phosphate buffer solution.

6. The method for preparing cannabidiol epoxide according to claim 5, characterized in that: The buffer solution includes: pH 6.8 phosphate buffer solution.

7. The method for preparing cannabidiol epoxide according to claim 1, characterized in that: The crude product is separated and purified by silica gel column gradient chromatography to obtain a finished cannabidiol epoxide product, including: obtaining the crude product; Preset gradient conditions; The crude product is purified by silica gel column chromatography according to the gradient conditions to obtain the finished cannabidiol epoxide product.

8. The method for preparing cannabidiol epoxide according to claim 7, characterized in that: The gradient conditions include: n-Hexane:ethyl acetate=80:1-10:

1.

9. An application of cannabidiol epoxide, characterized in that: Application of cannabidiol epoxide prepared by the method for preparing cannabidiol epoxide according to any one of claims 1 to 8 in stimulating TRPA1 channels.

10. An application of cannabidiol epoxide, characterized in that: Application of cannabidiol epoxide prepared by the method for preparing cannabidiol epoxide according to any one of claims 1 to 8 in blocking TRPM8 channels.

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