阅读说明：本技术 一种大麻二酚-2-丁酸酯及其应用 (Cannabidiol-2-butyrate and application thereof ) 是由 王凤忠 范蓓 孙玉凤 佟立涛 金诺 孙晶 卢聪 王永泉 白亚娟 李敏敏 李春梅 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种大麻二酚-2-丁酸酯及其应用,具体地,本发明涉及式I所示化合物、其立体异构体或药学上可接受的盐,该大麻二酚-2-丁酸酯对神经细胞损伤、皮肤细胞损伤具有保护作用,可诱导人乳腺癌细胞凋亡,在医药及化妆品生产中具有实际应用价值,(The invention discloses a cannabidiol-2-butyrate and application thereof, and particularly relates to a compound shown in a formula I, a stereoisomer or pharmaceutically acceptable salt thereof, wherein the cannabidiol-2-butyrate has the effect of protecting nerve cell injury and skin cell injuryHas protective effect, can induce apoptosis of human breast cancer cells, has practical application value in the production of medicines and cosmetics,)

1. The cannabidiol derivative is cannabidiol-2-butyrate, and a specific chemical structural formula is shown as a formula I, and the cannabidiol derivative comprises pharmaceutically acceptable salts:

2. a process for the preparation of the cannabidiol derivative of claim 1, wherein the process comprises the following reaction scheme:

the method comprises the following steps: carrying out reaction on cannabidiol with a structure shown in a formula II and butyryl chloride with a structure shown in a formula III in the presence of a solvent to obtain a compound with a structure shown in a formula I.

3. The method of claim 2, wherein the solvent is 20mL of anhydrous acetonitrile.

4. The method of claim 2, wherein the cannabidiol and butyryl chloride are present in a molar ratio of 1: 1.

5. The process according to claim 2, wherein the reaction mixture is heated under reflux for 4 hours, concentrated by rotary evaporation, dissolved in methylene chloride, washed with water, dried over anhydrous magnesium sulfate, rotary evaporated, and purified by column chromatography.

6. The method of claim 2, wherein the product is characterized by the following nuclear magnetic hydrogen spectrum:¹HNMR(300MHz,DMSO-d₆)δ:6.41(d,J＝24.0Hz,1H),6.18(d,J＝18.0Hz,1H),6.01(s,1H),5.05(d,J＝21.0Hz,1H),4.46(d,J＝21.0Hz,2H),3.83(d,J＝9.0Hz,1H),3.03(t,J＝9.0Hz,1H),2.66(s,1H),2.43-2.30(m,3H),2.08(s,1H),2.02-1.83(m,2H),1.67(s,2H),1.58(s,7H),1.48(d,J＝6.0Hz,2H),1.30-1.26(m,5H),0.95(t,J＝9.0Hz,2H),0.86(t,J＝9.0Hz,3H)。

7. the method of claim 2, wherein the product is characterized by the following high resolution mass spectra: HRMS [ M + H ]]⁺: theoretical value is 385.27372, found 385.27313.

8. A pharmaceutical composition comprising as an active ingredient a cannabidiol derivative as claimed in any one of claims 1 to 7 or a pharmaceutically acceptable salt or solvate of the compound, together with one or more excipients, diluents or fillers.

9. Use of the cannabidiol derivative of any one of claims 1 to 7 or a pharmaceutically acceptable salt or solvate of the compound thereof, or the pharmaceutical composition of claim 8, in the manufacture of a neurocytoprotective medicament, an anti-breast cancer medicament, and a skin cytoprotective cosmetic.

10. The use according to claim 9, wherein the nerve cell is glial cell BV2, the breast cancer cell is human breast cancer cell MDA-MB-231, and the skin cell is human immortalized epidermal cell HaCaT.

Technical Field

The invention belongs to the technical field of chemical industry, and particularly relates to a novel cannabidiol derivative, and a preparation method and application thereof.

Background

Cannabidiol (CBD) is one of the main phenolic chemical components in industrial cannabis sativa, and in vivo experiments show that CBD can not only antagonize the mental activity caused by THC agonizing cannabinoid I-type receptor (CB1R), but also has the effects of anticonvulsant, sedative-hypnotic, anxiolytic, antipsychotic, anti-inflammatory and neuroprotective, and is a natural active component with great application prospects in the fields of medicine, cosmetics and food.

In recent years, studies on the physiological activity and development of applications of CBD have been advanced. UK GW pharmaceutical company has developed Sative (a mouth mucosa spray with a THC/CBD content ratio of 1) for the treatment of Tuberous Sclerosis (TSC). In 2018, the Cannabis diol oral liquid of Giewar pharmaceutical company is approved by the United states food and drug administration for treating epilepsy caused by Dravet syndrome and Lennox-Gastaut syndrome in patients of two years old or more. In addition, CBD has also made some progress in the fields of foods, cosmetics, and the like.

The high-purity CBD is a white or light yellow crystal, has a melting point of 66-67 ℃, is hardly dissolved in water or a 10% sodium hydroxide solution, and is easily dissolved in organic solvents such as ethanol, methanol, diethyl ether, benzene, chloroform, petroleum ether and the like. This ester-soluble nature of CBD has made it a great limitation in drug development. Based on the structure of cannabidiol, the design and synthesis of cannabidiol derivatives are important ways for obtaining higher active compounds, and have important significance for preparing novel medicaments and cosmetics.

The existing cannabidiol and derivatives thereof have numerous action targets but weak action on each target, and have certain toxicity, so that the application of the cannabidiol and the derivatives thereof is limited. The development of derivatives with specific groups to improve their selectivity for specific targets or to increase their effect on multiple targets is a major research direction in the future. For example, WO2011/026144a1 discloses cannabinoid prodrug delivery systems including cannabidiol-3-hydroxypropionic acid. CN112111025A discloses the preparation of cannabidiol derivatives including cannabidiol-3-butanedioic acid, but the compound only improves the water solubility, and the biological action and the patent potential are not disclosed. WO2008/107879a1 discloses the synthesis of cannabidiol derivatives, including cannabidiol-2-hydroxyacetone, for anti-inflammatory therapy. WO2009018389a1 discloses the biosynthesis of cannabinoid prodrugs including cannabidiol-glycine, cannabidiol-2-hydroxypropionic acid, cannabidiol-3-aminopropionic acid. CN1688290A discloses the preparation of liquid formulations of cannabinoids including cannabidiol-propanol, the biological effects of which are not yet clear. CN109311838A discloses the biosynthesis of cannabinoid prodrugs including cannabidiol-2-acetate, however these prodrugs still do not meet the drug development requirements.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel cannabidiol derivative and a preparation method thereof. The invention also provides the compound, pharmaceutically acceptable salts or solvates thereof, a pharmaceutical composition containing the compound and application of the compound and the pharmaceutical composition in preparing nerve cell protection medicaments.

The compounds of the invention have the structure shown in formula I:

the preparation method of the cannabidiol derivative provided by the invention comprises the following steps: carrying out reaction on cannabidiol with a structure shown in a formula II and butyryl chloride with a structure shown in a formula III in the presence of a solvent to obtain a compound with a structure shown in a formula I.

The preparation method according to a preferred embodiment of the present invention has at least the following advantageous effects:

(1) the invention provides a novel cannabidiol derivative, which is cannabidiol-2-butyrate, has a protective effect on nerve cell injury, can be prepared into a neuroprotective medicament, and has practical application value in medicine production.

(2) According to the preparation method provided by the invention, the raw materials are cannabidiol with a structure shown in a formula II and butyryl chloride with a structure shown in a formula III, no catalyst is used, and the solvent is a common solvent, so that the raw materials are cheap and easy to obtain.

(3) The preparation method provided by the invention has the advantages of mild conditions and low energy consumption.

(4) The preparation method provided by the invention has the advantages that the yield of the main product reaches 72.4%, and the yield is higher.

In some embodiments of the invention, the method of preparation comprises the following synthetic route:

in some embodiments of the invention, the solvent is anhydrous acetonitrile.

In some embodiments of the invention, the molar ratio of cannabidiol to butyryl chloride is 1: 1.

In some embodiments of the present invention, the preparation method specifically comprises heating and refluxing the reaction mixture for 4 hours, concentrating by rotary evaporation, dissolving with dichloromethane, washing with water, drying with anhydrous magnesium sulfate, rotary evaporation, and separating and purifying by column chromatography.

The invention also provides a pharmaceutical composition, which comprises the cannabidiol-2-butyrate or the pharmaceutically acceptable salt thereof or the solvate of the compound, and one or more excipients, diluents, fillers and the like;

preferably, the pharmaceutical composition is formulated in the form of powder or granules.

The invention also provides application of the cannabidiol-2-butyrate or pharmaceutically acceptable salt thereof or solvate of the compound, or a pharmaceutical composition containing the compound in preparation of nerve cell protection drugs, breast cancer resistance drugs and skin cell protection cosmetics.

Drawings

FIG. 1 is a schematic representation of the protective effect of cannabidiol-2-butyrate and its analogs (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-carboxylate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) on glutamate-induced neuronal damage;

FIG. 2 is a schematic diagram of the protective effect of cannabidiol-2-butyrate and its analogs (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-carboxylate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) on nerve cell damage caused by corticosterone;

FIG. 3 shows the chemical structure of cannabidiol-2-butyrate analogues (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-carboxylate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate);

FIG. 4 is a schematic representation of glutamic acid induced BV2 cell damage;

FIG. 5 is a schematic representation of BV2 cell damage caused by corticosterone;

FIG. 6.5. mu.M cannabidiol-2-butyrate is a schematic representation of the protective effect on glutamate-induced neuronal cell damage;

FIG. 7.5. mu.M cannabidiol-2-butyrate is a schematic representation of the protective effect on corticosterone-induced neuronal damage;

FIG. 8 is a schematic representation of the killing effect of cannabidiol-2-butyrate and its analogs (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-carboxylate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) on human breast cancer cells MDA-MB-231;

FIG. 9 cannabidiol-2-butyrate and its analogs (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-carboxylate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) vs₂O₂Schematic diagram of the protective effect of the resulting HaCaT cell damage.

Detailed Description

The conception and the resulting technical effects of the present invention will be further described with reference to specific embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The method is a conventional method unless otherwise specified. The materials are commercially available from the open literature unless otherwise specified.

Example 1 preparation of cannabidiol-2-butanoic acid ester

The synthetic route is shown as the following formula:

adding 0.53g (5mmol) of butyryl chloride and 20mL of anhydrous acetonitrile into a 50mL three-necked bottle, stirring, adding 1.57g (5mmol) of cannabidiol and 1mL of pyridine, stirring, heating, carrying out reflux reaction for 4 hours, carrying out rotary evaporation concentration, dissolving with dichloromethane, washing with water, drying with anhydrous magnesium sulfate, carrying out rotary evaporation, and carrying out column chromatography separation and purification to obtain 1.39g of colorless liquid with the yield of 72.4%.

The nuclear magnetic hydrogen spectrum of the product is characterized as follows:

¹H NMR(300MHz，DMSO-d₆)δ:6.41(d，J＝24.0Hz，1H)，6.18(d，J＝18.0Hz，1H)，6.01(s,1H),5.05(d,J＝21.0Hz,1H),4.46(d,J＝21.0Hz,2H),3.83(d,J＝9.0Hz,1H),3.03(t,J＝9.0Hz,1H),2.66(s,1H),2.43-2.30(m,3H),2.08(s,1H),2.02-1.83(m,2H),1.67(s,2H),1.58(s,7H),1.48(d,J＝6.0Hz,2H),1.30-1.26(m,5H),0.95(t,J＝9.0Hz,2H),0.86(t,J＝9.0Hz,3H)。

the high resolution mass spectrum of the product was characterized as follows:

HRMS[M+H]⁺: theoretical value is 385.27372, found 385.27313.

The nuclear magnetic hydrogen spectrum and high-resolution mass spectrum detection results show that the preparation method provided by the invention can be used for effectively preparing the cannabidiol-2-butyrate.

Example 2 determination of the neuronal injury protection Effect of cannabidiol-2-butyrate

The cannabidiol-2-butyrate compounds obtained in example 1 and analogues thereof (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-formate, cannabidiol-2-propyl ether, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate, cannabidiol-2- (2-hydroxy) propionate) were dissolved in DMSO under aseptic conditions to prepare a 20mM mother liquor. The glial cell BV2 was inoculated into a 96-well plate, the cell injury model was constructed by stimulation with glutamic acid and corticosterone, respectively, after 12 hours of culture, cannabidiol and the compound obtained in example 1 were added to the culture medium, diluted to 5.0, 2.5, 1.0, 0.5, 0.25, 0.1. mu.M, 5 duplicate wells per group were set, culture was continued for 72 hours, 20. mu.L of MTT reagent was added to each well, and culture was continued for 0.5 hours. MTT working solution was discarded, 150. mu.l DMSO was added to each well, and the mixture was mixed by shaking gently at room temperature for 10 minutes. The experiment was carried out on a normal control group, a glutamic acid-induced BV2 cell injury group and a corticosterone-induced BV2 cell injury group. The 96-well plate was placed in an microplate reader at a wavelength of 450nm to measure the OD value, and the cell survival rate was calculated.

Cell survival (%) (OD-blank OD of each treatment group)/(OD-blank OD of control group) × 100%.

The test results are shown in tables 1 and 2.

TABLE 1 protective Effect of Compounds on glutamate induced neuronal injury

Note: in a model of BV2 cell damage caused by glutamic acid, the cell survival rate (%) of a normal control group is 100.23 +/-1.91;

the cell survival rate (%) of the BV2 cell injury group was 61.32. + -. 1.66.

TABLE 2 protective Effect of Compounds on Cortisol-induced neuronal damage

Note: in a model of BV2 cell damage caused by corticosterone, the cell survival rate (%) of a normal control group is 100.12 +/-2.02; the cell survival rate (%) of the BV2 cell injury group was 62.33. + -. 1.72.

As can be seen from the results of table 1 and fig. 1, the compound of the present invention has an antagonistic effect against the cytotoxicity of glutamic acid against BV2, and as can be seen from the results of table 2 and fig. 2, the compound of the present invention has an antagonistic effect against the cytotoxicity of corticosterone against BV2, and can improve the cell survival rate, and the activity of the compound of the present invention at the same concentration is equivalent to that of the analog cannabidiol-2-imidazole-1-carboxylate, higher than that of the analog cannabidiol, cannabidiol-2-acetate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, and cannabidiol-2-bicarbonate. The research results show that the compound has good protection effect on nerve cell injury.

Example 3 determination of anti-Breast cancer Effect of cannabidiol-2-butyrate

The cannabidiol-2-butyrate compounds obtained in example 1 and their analogues (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-formate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) were dissolved in DMSO under aseptic conditions to prepare a 20mM mother liquor. Inoculating human breast cancer cell MDA-MB-231 to a 6-well plate, setting 3 multiple wells for each group, culturing for 12 hours, adding a culture medium to dilute into 5.0, 2.5 and 1.0 mu M drug treatment groups after the cells adhere to the wall, and culturing for 24 hours. The experiment was set as a normal control group. Digesting the cells by trypsin without EDTA, washing the cells for 2 times by PBS, resuspending the cells by 100 muL of binding buffer, transferring the cells into a flow tube, then respectively adding 5 muL of Annexin V-FITC and 5 muL of PI staining solution, incubating the cells for 15 minutes in a dark place at room temperature, and finally adding 400 muL of binding buffer and mixing the cells uniformly. And (5) detecting on a computer, and processing data by using software.

The test results are shown in Table 3.

TABLE 3 apoptosis rate of Compounds on human Breast cancer cells MDA-MB-231

Note: the apoptosis rate of the control group is 2.21 +/-0.31.

As can be seen from the results in table 3, the compounds of the present invention induced apoptosis in human breast cancer cells MDA-MB-231, and at the same concentration, the activity of the compounds of the present invention was comparable to that of the analogues cannabidiol-2-imidazole-1-carboxylate, higher than that of the analogues cannabidiol, cannabidiol-2-acetate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, and cannabidiol-2-hydrocarbonate. The research results show that the compound has good breast cancer resistance.

Example 4 assay of the skin cell injury protective Effect of cannabidiol-2-butyrate

The cannabidiol-2-butyrate compounds obtained in example 1 and their analogues (cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-formate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate, cannabidiol-2-bicarbonate) were dissolved in DMSO under aseptic conditions to prepare a 20mM mother liquor. Human immortalized epidermal cells HaCaT were inoculated into 96-well plates with H₂O₂Cell injury model was constructed by stimulation, and after 24 hours of culture, the compound obtained in example 1 and its analogue were added to the culture medium, diluted to 10.0, 5.0, 2.5, 1.0, 0.5 μ M, and 5 wells per group were set, and culture was continued for 24 hours, and 16 μ L of MTT reagent was added to each well, and culture was continued for 4 hours. MTT working solution was discarded, 160. mu.L DMSO was added to each well, and the mixture was shaken gently in a shaker at room temperature for 10 minutes and mixed well. The experiment was set as a normal control group H₂O₂The resulting HaCaT cell injury group. The 96-well plate was placed in an microplate reader at a wavelength of 450nm to measure the OD value, and the cell survival rate was calculated.

Cell survival (%) (OD-blank OD of each treatment group)/(OD-blank OD of control group) × 100%.

The test results are shown in Table 4.

TABLE 4 Compound vs. H₂O₂Protective effects of HaCaT cell damage caused by HaCaT

Note: cell survival (%) 100.33 ± 1.37 of the normal control group; cell survival (%) 56.66 ± 0.58 in HaCaT cell injury group.

As can be seen from the results in Table 4, the compounds of the present invention have antagonistic H activity₂O₂The compound has higher activity than analogues of cannabidiol, cannabidiol-2-acetate, cannabidiol-2-imidazole-1-formate, cannabidiol-2-propyl ether, cannabidiol-2- (2-hydroxy) propionate, cannabidiol-2- (3-hydroxy) propionate and cannabidiol-2-bicarbonate at the same concentration. The research results show that the compound has good protective effect on skin cell damage.

Although the present invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it is not limited to the above-described embodiments, but may be modified or improved on the basis of the present invention, as will be apparent to those skilled in the art. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.