Cannabidiol acid ester composition and application thereof

Abstract

The present application relates to cannabidiol esters, compositions comprising them and their use in the treatment of a variety of different diseases, disorders and symptoms.

Description

[0001] This application is a divisional application of the patent application for the invention with the same title "Cannabidiolic acid ester compositions and uses thereof", having the priority date of 20 June 2017, the filing date of 20 June 2018, the application number of 201880041597.0. BACKGROUND

[0002] Cannabidiolic acid (CBDA) is a major constituent of Cannabis (Cannabis sativa L.) plants. It was first isolated in 1955 Cannabis sativa and its structure was elucidated in 1965 by analysis of the physical properties of its methyl ester (Mechoulam and Gaoni, 1965). Its synthesis from cannabidiol was subsequently reported (Mechoulam and Ben-Zvi, 1969).

[0003]

[0004] CBDA Cannabidiolic acid (CBDA) is decarboxylated to cannabidiol (Mechoulam, 1973) while still in the plant, a process that is accelerated by heat. Although cannabidiol has been the subject of many publications and its biological / therapeutic properties are now well established (Mechoulam et al., 2002; Zhornitsky & Potvin, 2012; Cascio and Pertwee, 2014), our understanding of the pharmacology of cannabidiolic acid is still very limited.

[0005] The limited amount of information published about this phytocannabinoid suggests that it can have a wide range of various different effects and actions. Accordingly, it has been shown to inhibit breast cancer cell migration (Takeda et al., 2017) and to cause downregulation of cyclooxygenase-2 (COX-2) (Takeda et al., 2014). Recent evidence suggests that CBDA (i.p. at doses as low as 1 µg·kg -1 1A mediated anti-emetic effects, as it potentiates 5-HT 1A receptor activation in the ferret (Musty, 1989). CBDA has also been shown to inhibit the growth of human breast cancer cells (Takeda et al., 2017) and to inhibit the growth of human lung cancer cells (Takeda et al., 2014). Suncus murinus ​​indicated by its apparent ability to prevent both emesis in the dog (Grill & Norgren, 1978) and acute nausea-induced conditioned gaping behavior in the rat (Bolognini et al., 2013; Rock et al., 2013; 2015b). In addition to reducing acute nausea, CBDA has the potential to reduce anticipatory (conditioned) nausea, an effect experienced by chemotherapy patients upon their return to the clinic where they received the nausea-inducing treatment (Rock et al., 2014; 2015a; 2016). Once anticipatory nausea occurs in these patients, there are currently no effective selective treatment options. It is therefore noteworthy that Rock et al. (2014; 2015a; 2016) have demonstrated that CBDA also reduces situationally-induced conditioned gaping, a model of anticipatory nausea, via 5-HT 1A dependent mechanisms. Finally, similar to CBD, CBDA has also been shown to produce anxiolytic-like effects under high stress conditions at i.p. doses as low as 0.1 pg-kg -1

[0006] However, CBDA is highly unstable, particularly when heated. There is therefore a growing need to discover CBDA analogs with higher stability. The inventors of the present application have surprisingly found that ester derivatives of CBDA provide both the sought stability and similar or more potent biological properties compared to CBDA. SUMMARY

[0007] The present application therefore provides a compound having the general formula (I):

[0008] (I) wherein R1 is selected from -C(=0)0R3, -OC(=0)R4; R2 is selected from linear or branched C1-C 15 alkyl, linear or branched C2-C 15 alkenyl and linear or branched C2-C 15 alkynyl, each independently optionally substituted with at least one substituent selected from hydroxyl (-OH), halogen, amine and amide, or any combination thereof; R3 and R4 are each independently selected from linear or branched C1-C 15 alkyl, linear or branched C2-C 15 alkenyl, linear or branched C2-C 15 alkynyl (each of said alkyl, alkenyl or alkynyl is optionally substituted with at least one substituent selected from hydroxyl (-OH), halogen, amine and amide, or any combination thereof), halogen, amine and amide.

[0009] ​The present application also provides a compound having the general formula (II):

[0010] (II) wherein R2is selected from the group consisting of linear or branched C1-C 15 alkyl, linear or branched C2-C 15 alkenyl, and linear or branched C2-C 15 alkynyl, each of which is independently optionally substituted with at least one substituent selected from the group consisting of hydroxyl, halogen, amine, and amide, or any combination thereof; and R3is selected from the group consisting of linear or branched C1-C 15 alkyl, linear or branched C2-C 15 alkenyl, and linear or branched C2-C 15 alkynyl (each of the alkyl, alkenyl, or alkynyl is optionally substituted with at least one substituent selected from the group consisting of hydroxyl (-OH), halogen, amine, and amide, or any combination thereof), halogen, amine, and amide.

[0011] In certain embodiments, R2is linear or branched C1-C 15 alkyl. In other embodiments, R2is linear or branched C2-C 15 alkenyl. In yet other embodiments, R2is linear or branched C2-C 15 alkynyl.

[0012] In certain embodiments, R3is linear or branched C1-C 15 alkyl. In other embodiments, R3is linear or branched C2-C 15 alkenyl. In yet other embodiments, R3is linear or branched C2-C 15 alkynyl. In still other embodiments, R3is selected from the group consisting of halogen, amine, and amide. In certain embodiments, R3as alkyl, alkenyl, or alkynyl is optionally substituted with at least one substituent selected from the group consisting of hydroxyl (-OH), halogen, amine, and amide, or any combination thereof.

[0013] In certain embodiments, the compound of the present application is HU-580:

[0014] HU-580.

[0015] The present application also provides a compound having the general formula (III):

[0016] (III) wherein R2is selected from the group consisting of linear or branched C1-C 15 alkyl, linear or branched C2-C 15Alkenyl and straight-chain or branched C2-C 15 The alkynyl group is optionally substituted independently by at least one substituent selected from hydroxyl, halogen, amine and amide or any combination thereof; R4 is selected from straight-chain or branched C1-C. 15 Alkyl, straight-chain or branched C2-C 15 Alkenyl, straight-chain or branched C2-C 15 Alkyne (each of the alkyl, alkenyl or alkynyl groups is optionally substituted with at least one substituent selected from hydroxyl (-OH), halogen, amine and amide or any combination thereof), halogen, amine and amide.

[0017] the term" halogen "This means F, Cl, Br, or I.

[0018] When used in this document, the term " amine " refers to the "-NRR'R" group, where R, R', and R'' are each selected from H, straight-chain or branched C1-C atoms. 15 Alkyl, straight-chain or branched C2-C 15 Alkenyl and straight-chain or branched C2-C 15 Alkyne group.

[0019] When used in this document, the term " amide "" refers to the -C(=O)NRR'R" or NRC(=O)R' group, where R, R', and R'' are each selected from H, straight-chain or branched C1-C atoms. 15 Alkyl, straight-chain or branched C2-C 15 Alkenyl and straight-chain or branched C2-C 15 Alkyne group.

[0020] When used in this document, the term " C 1 -C 15 alkyl "" represents a saturated branched or straight-chain hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms connected by σ bonds.

[0021] When used in this document, the term " C 2 -C 15 alkenyl "" represents a branched or straight-chain hydrocarbon group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms, wherein at least one of the bonds connecting the carbon atoms is a double bond, and all other bonds can be any other type (single bond and / or double bond).

[0022] When used in this document, the term "C 2 -C 15 alkynyl "Represents a branched or straight-chain hydrocarbon group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms, wherein at least one of the bonds connecting the carbon atoms is a triple bond, and all other bonds can be any other type (single bond and / or double bond and / or triple bond).

[0023] When used herein, the term "optional substituent" means that the corresponding substituent may or may not be present. Therefore, the compounds of the present invention may have one, two, three, or more optional substituents at any point where the radical is defined as having such optional substitution.

[0024] It should be understood that the compounds presented herein may contain one or more chiral centers. These chiral centers may each be ( R )or( S The configuration is ( ). In the case where the compounds of the present invention contain more than one chiral center, each of these chiral centers may independently have ( ). R )or( S Configuration. Therefore, the compounds presented herein may be enantiomers pure, or stereoisomers or mixtures of diastereomers.

[0025] The present invention also provides a composition comprising at least one compound of general formulas (I) and (II) as defined above and below.

[0026] On the other hand, the present invention provides compounds of general formulas (I) and (II) as defined above and below, for the treatment of 5-HT 1A Receptor-related conditions, diseases, or symptoms.

[0027] On the other hand, the present invention provides a compound of general formulas (I) and (II) as defined above and below, for treating a condition, disease or symptom selected from nausea, vomiting, convulsions and any combination thereof.

[0028] On the other hand, the present invention provides a compound of general formulas (I) and (II) as defined above and below, for the treatment of symptoms, diseases or conditions related to depression.

[0029] On the other hand, the present invention provides a compound of general formulas (I) and (II) as defined above and below, for the treatment of a condition, disease, or symptom selected from anxiety, tension, depression, schizophrenia, panic, withdrawal syndrome, autoimmune disease, inflammation, infarct reduction, increased blood flow during stroke, obesity, metabolic syndrome, retinopathy, nausea, myocardial, hepatic, and renal ischemia / reperfusion injury, neuronal injury, Huntington's disease, Alzheimer's disease, cerebral infarction, hepatic encephalopathy, traumatic brain injury, cerebral ischemia, spinal cord injury, memory recovery effect, cancer, angiogenesis, epilepsy, spasm, neuropathic pain, airway obstruction, compulsive behaviors, cognitive impairment, impaired libido and sexual function, sleep disorders, opioid-related respiratory depression, addiction, and any combination thereof.

[0030] In another aspect, the present invention relates to a method of treating a disease characterized by improvement by cannabidiol (CBDA), the method comprising administering to a subject requiring such treatment a therapeutically effective amount of a compound of general formulas (I) and (II) as defined above and below.

[0031] Non-limiting examples of diseases, symptoms, and conditions include: anxiety and tension, depression, schizophrenia, panic and anxiety, withdrawal symptoms in marijuana and tobacco addiction, the reward-promoting effect of morphine and cocaine, any type of autoimmune disease (specific non-limiting examples being type 1 diabetes, GVHD), inflammation (Crohn's disease, colitis, pancreatitis, rheumatoid arthritis), infarct size reduction and increased blood flow during stroke, obesity (treated by reducing food intake or by reducing appetite), metabolic syndrome, diabetic retinopathy, nausea, ischemic / reperfusion injury of the myocardium, liver, and kidneys, neuronal damage (caused by neurological diseases or injuries), Parkinson's disease, Huntington's disease, Alzheimer's disease, cerebral infarction, hepatic encephalopathy, traumatic brain injury, cerebral ischemia, spinal cord injury, memory salvage effects, cancer and resistance to cancer chemotherapy, cancer cell migration (metastasis), angiogenesis, epilepsy and spasms, chronic inflammatory and neuropathic pain, airway obstruction, compulsive behaviors, and any combination thereof.

[0032] In some implementations, the disease, condition, and symptom are selected from nausea (both anticipatory and acute), vomiting, anxiety, and any type of mood disorder such as depression (including major depressive disorder, mild depressive disorder, and bipolar disorder).

[0033] This invention relates to a method for treating a disease, wherein the clinically beneficial effects are achieved through 5HT. 1A The method involves administering an effective amount of at least one compound of formula (I) or (II) to a subject requiring such treatment via receptor activation.

[0034] Non-limiting examples of these diseases and conditions include: high blood pressure, anxiety, vomiting and nausea, pain, schizophrenia, Parkinson's disease, cognitive impairment, impaired libido and sexual function, obesity (which has the effect of suppressing food intake), sleep disorders (especially REM shortness of breath), opioid-related respiratory depression, addiction, and any combination thereof.

[0035] Specifically, due to 5HT 1A Diseases that show improved clinical outcomes upon activation are nausea (both anticipatory and acute) and vomiting (antiemetic and anti-nausea), anxiety, and any type of mood disorder, primarily depression (including major depressive disorder, mild depression, and bipolar disorder), and any combination thereof.

[0036] Therefore, when referring to "in this article 5HT 1A receptor-related condition, symptom or disease "When referring to this, it should be understood that it includes high blood pressure, anxiety, vomiting and nausea, pain, schizophrenia, Parkinson's disease, cognitive impairment, impaired libido and sexual function, obesity (the effect of which is to suppress food intake), sleep disorders (especially short duration of rapid eye movement), opioid-related respiratory depression, addiction, nausea (both anticipatory and acute) and vomiting (antiemetics and anti-nausea), anxiety and any type of mood disorder, primarily depression (including major depressive disorder, mild depressive disorder and bipolar disorder) and any combination thereof.

[0037] In another aspect, the present invention relates to a method for treating a disease selected from nausea (both anticipatory and acute), anxiety, and any type of mood disorder, primarily depression (including major depressive disorder, mild depressive disorder, and bipolar disorder), the method comprising administering an effective amount of CBDA-ME to a subject requiring such treatment.

[0038] On the other hand, the present invention provides a compound as disclosed above and below for treating at least one disease, condition, symptom, or disorder associated with renal dysfunction. The present invention also provides a method for treating at least one disease, condition, symptom, or disorder associated with renal dysfunction in a patient in need, the method comprising administering at least one compound of the present invention to the patient.

[0039] When referring to "in the context of this application" renal dysfunction When referring to kidney function impairment, it should be understood that it includes any type (qualitative or quantitative) of decreased or failed kidney function, and can be acute or chronic. Such kidney dysfunction can be caused by any reason, including injury, disease, genetic characteristics, etc. Causes of acute kidney dysfunction include, but are not limited to, hypotension, urinary tract obstruction, drug therapy, muscle breakdown, hemolytic uremic syndrome, and any combination thereof. Other causes of chronic kidney dysfunction include, but are not limited to, diabetes mellitus, hypertension, nephrotic syndrome, polycystic kidney disease, and any combination thereof.

[0040] In some implementations, these diseases, conditions, symptoms, and disorders associated with renal dysfunction include, but are not limited to, diabetic nephropathy, chronic and acute kidney injury, chronic and acute kidney disease, acute-on-chronic renal failure, obesity-related kidney damage, and any combination thereof.

[0041] Conditions and symptoms associated with renal dysfunction include, but are not limited to: high urea levels in the blood, vomiting, diarrhea, nausea, weight loss, nocturia, changes in the frequency and amount of urination, hematuria, stress or difficulty urinating, phosphate buildup in the blood, itching, bone damage, nonunion of broken bones, muscle cramps, potassium buildup in the blood, abnormal heart rhythms, muscle paralysis, the kidneys' inability to clear excess fluid, swelling of the legs, ankles, feet, face, or hands, shortness of breath, polycystic kidney disease, large fluid-filled cysts on the kidneys, back or side pain, decreased erythropoietin production, decreased red blood cell production, anemia, foamy or bubbly urine, swelling of the hands, feet, abdomen, or face, loss of appetite, excessive protein in the blood and urine, seizures during administration of high doses of penicillin, and any combination thereof.

[0042] The present invention also relates to pharmaceutical compositions comprising compounds of the present invention mixable with pharmaceutically acceptable adjuvants and optionally other therapeutic agents. The adjuvants must be "acceptable" in the sense that they are compatible with the other components of the composition and harmless to their receptors.

[0043] Pharmaceutical compositions include those suitable for oral, nasal, topical (including transdermal, buccal, and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration or via implantation. The compositions may be prepared by any method known in the field of pharmacology.

[0044] These methods include the step of associating the compounds or combinations thereof used in this invention with any adjuvants. The adjuvants, also referred to as auxiliary ingredients, include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavorings, antioxidants, and wetting agents.

[0045] Pharmaceutical compositions suitable for oral administration may be available as discrete dosage units such as pills, tablets, sugar-coated pills, or capsules, or as powders or granules, or as solutions or suspensions. The active ingredient may also be available as large pills or pastes. The compositions may be further processed into suppositories or enemas for rectal administration.

[0046] The present invention also includes the pharmaceutical compositions described above, which, in combination with packaging materials and instructions for use of the compositions, are used for the purposes described above.

[0047] For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injectable solutions. These compositions can be present in single-dose or multi-dose containers such as sealed tubes and ampoules and can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water, before use. For transdermal administration, gels, patches, or sprays are considered. Suitable compositions or dosage forms for pulmonary administration, such as nasal inhalation, include fine powders or mists that can be generated using metered-dose pressurized aerosols, nebulizers, or inhalers.

[0048] The precise dosage and administration regimen of the composition necessarily depend on the therapeutic or nutritional effect to be achieved, and can vary depending on the specific formulation, route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

[0049] This invention also provides a treatment for 5-HT 1A Methods for treating receptor-related conditions, diseases, or symptoms, the methods comprising administering to a patient in need at least one compound disclosed herein or above (compounds of formula (I), (II), etc.).

[0050] The present invention also provides a method for treating a condition, disease or symptom selected from nausea, vomiting, convulsions and any combination thereof, the method comprising administering to a patient in need at least one compound disclosed herein or above (compounds of formula (I), (II) etc.).

[0051] The present invention also provides a method for treating a condition, disease or symptom associated with depression, the method comprising administering to a patient in need at least one compound disclosed herein or above (compounds of formula (I), (II) etc.).

[0052] The present invention also provides a method for treating a condition, disease, or symptom selected from anxiety, tension, depression, schizophrenia, panic, withdrawal syndrome, autoimmune disease, inflammation, infarct size reduction, increased blood flow during stroke, obesity, metabolic syndrome, retinopathy, nausea, myocardial, hepatic, and renal ischemia / reperfusion injury, neuronal injury, Huntington's disease, Alzheimer's disease, cerebral infarction, hepatic encephalopathy, traumatic brain injury, cerebral ischemia, spinal cord injury, memory recovery effect, cancer, angiogenesis, epilepsy, spasm, neuropathic pain, airway obstruction, compulsive behaviors, cognitive impairment, impaired libido and sexual function, sleep disorders, opioid-related respiratory depression, addiction, and any combination thereof, said method comprising administering to a patient in need at least one of the compounds disclosed herein or above (compounds of formula (I), (II), etc.).

[0053] The present invention also provides a method for treating at least one disease, condition, symptom or disorder associated with renal dysfunction, the method comprising administering to a patient in need at least one compound disclosed herein or above (compounds of formula (I), (II) etc.).

[0054] When used in this document, the term " treating a disease, disorder, condition or symptom "This refers to the slowing or reversal of the progression of the disease, disorder, or its symptoms. Treating a disease or disorder includes treating symptoms and / or alleviating the symptoms of the disease." Attached Figure Description

[0055] The subject matter of this invention is specifically pointed out and explicitly claimed in the concluding section of the specification. However, the organization and operation of the invention, as well as its objects, features, and advantages, can be best understood by referring to the following detailed description in conjunction with the accompanying drawings.

[0056] Figures 1A-1E The diagram shows the pairing of CBDA (0.01, 0.1, 1.0, 10, or 100 nM) with [ 35 S]-GTPγS and from stable transfection of human 5-HT 1A Effect of 8-OH-DPAT-induced stimulation on membrane binding of CHO cells to the receptor. Symbols represent mean ± SEM ( n = 6). 8-OH-DPAT determined in the presence of CBDA or only its medium (VEH) DMSO. E max and EC 50 The average values, along with their 95% confidence limits, are listed in Table 1.

[0057] Figures 2A-2F The HU-580 (0.001, 0.01, 0.1, 1.0, 10, or 100 nM) is shown to be compatible with [ 35 S]-GTPγS and from stable transfection of human 5-HT 1A Effect of 8-OH-DPAT-induced stimulation on membrane binding of CHO cells to the receptor. Symbols represent mean ± SEM ( n = 6). 8-OH-DPAT determined in HU-580 or only in the presence of its medium (VEH) DMSO. E max and EC 50 The average values, along with their 95% confidence limits, are listed in Table 2.

[0058] Figure 3The mean number of conditioned mouth openings induced by LiCl-paired saccharin solution is shown in rats pretreated with various doses of CBDA (n = 8 per group), HU-580 (n = 8 per group), or mediator alone (VEH; n = 8). The other groups were pretreated with WAY100635 (0.1 mg·kg⁻¹) 15 min prior to 0.1 mg·kg⁻¹ HU-580 (n = 6) or VEH (n = 8). Results are presented as mean ± SEM and *P < 0.05, depicting the mean response to CBDA or HU-580, which differed significantly from the mean response to VEH.

[0059] Figure 4A CBDA or HU-580 (0.01, 0.1 μg / kg) administered intraperitoneally 45 min prior to the anticipatory nausea test is shown. -1 The influence of the medium (VEH) or medium ( n = 6 animals per group). The other groups were at 0.1 mg·kg⁻¹. -1 HU-580 ( n = 8) or VEH ( n = 8) Administer WAY100635 (0.1 mg / kg) 15 minutes prior to administration. -1 Pretreatment of [the sample / sample]. The mean number of conditioned mouth-opening responses was measured during the anticipated nausea test. Each bar represents the mean ± SEM. * P <0.05, a significant difference compared to the VEH-treated control animals.

[0060] Figure 4B The mean distance traveled (cm) in the activity test performed after the anticipatory nausea test is shown. Each bar represents the mean ± SEM.

[0061] Figure 5 The mean time rats remained in the light chamber 24 h after exposure to footless electric shock (no FS) or FS is shown. Forty-five minutes prior to the 5-minute light-dark chamber stress test, all rats were treated with a medium (VEH; n =9 or 12), 0.01 μg·kg -1 CBDA ( n =8) or 0.01 μg·kg -1 HU-580 ( n =8) IP injection. The other group received VEH ( n = 7 or 8) or 0.01 μg·kg - 1 HU-580 ( n = 8) 0.1 mg·kg 15 min before -1WAY100635 injection. Each bar represents the mean ± SEM. * P <0.05 indicates a significant difference between the FS and non-FS stress groups.

[0062] It should be understood that, for the sake of simplicity and clarity, the components shown in the figures are not necessarily drawn to scale. For example, the dimensions of some components may be exaggerated relative to others for clarity. Furthermore, reference numerals may be repeated between figures where deemed appropriate to indicate corresponding or similar components. Detailed Implementation

[0063] In the following detailed description, numerous specific details are set forth in order to provide a full understanding of the invention. However, those skilled in the art will understand that the invention can be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the invention.

[0064] synthesis Synthesis of Cannabinoid Acid B (CBDA): A mixture of cannabidiol (CBD, 314 mg, 1 mmol) and magnesium monomethyl carbonate (MMC / 2M, 1.5 ml, 3 mmol) in a 2-molar solution of dimethylformamide (DMF) was heated at 130 °C for 3 hours. The reaction was then cooled to 0 °C, acidified with 10% hydrochloric acid, and extracted with diethyl ether. The organic layer was washed with brine, dried on magnesium sulfate (MgSO4) as a drying agent, and then evaporated. The crude compound was then cleaned by column chromatography (20% diethyl ether-petroleum ether).

[0065] Synthesis of Cannabinoid Acid B Methyl Ester (HU-580): To a solution of cannabidiol (CBDA) (175 mg, 0.488 mmol) in 2.5 mL of dichloromethane (CH2Cl2), 0.02 mL of methanol (CH3OH, 0.488 mmol) and 7.2 mg of 4-pyrrolidine (0.048 mmol) were added. The reaction was stirred at room temperature for 5 minutes, then the coupling agent N,N'-dicyclohexylcarbodiimide (DCC) (121 mg, 0.585 mmol) was added and stirred overnight. The solvent was then evaporated, and the crude mixture was acidified with 5% hydrochloric acid and extracted with dichloromethane (CH2Cl2). The organic layer was washed with a saturated aqueous solution of sodium bicarbonate (NaHCO3), dried on magnesium sulfate (MgSO4) as a drying agent, and then evaporated. The crude compound was then cleaned by column chromatography (2% diethyl ether-petroleum ether).

[0066] 11H-NMR spectra were obtained using a Bruker AMX 300 MHz instrument with deuterated DMSO. Thin-layer chromatography (TLC) was performed on silica gel 60F. 254 The chromatography was performed on a Merck phase. Column chromatography was performed on silica gel 60 Å (Merck). Compounds were localized using a UV lamp at 254 nm. GCMS analysis was performed on an HP GCMS instrument (GCD PLUS type) using an EI detector and a 30 m methyl silicone column.

[0067] 1 H NMR (300 MHz, ((CD3)2SO)) δ 6.18 (1H, s, Ar), 5.07 (1H, s), 4.44(1H, s), 4.41 (1H, s), 3.82 (3H, s), 3.35 (1H, m), 2.66 (1H, m), 2.49 (2H,t), 2.09 (1H, b), 1.95 (3H, s), 1.71-1.05 (12, ms), 0.86 (3H, t). GCMS=314m / z.

[0068] Biological methods External procedures CHO cells. CHO cells stably transfected with cDNA encoding the human 5-HT1A receptor (a generous gift from Dr. Keith Parker) were maintained at 37°C and 5% CO2 in a Gibco container supplied by Fisher Scientific UK Ltd. ™ Ham's F-12 nutritional blend is supplemented with 2 mM L-glutamine, 10% FBS, and 0.6% penicillin-streptomycin, all supplied by Fisher Scientific UK Ltd, and G418 disulfate [(2R,3S,4R,5R,6S)-5-amino-6-{[(1R,2S,3S,4R,6S)-4,6-diamino-3-{[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-(methylamino)oxacyclohexane-2-yl]oxy}2-hydroxycyclohexyl]oxy}-2-[(1R)-1-hydroxyethyl]oxacyclohexane-3,4-diol, 600 mg·mL, supplied by Sigma-Aldrich UK. -1 ].

[0069] [ 35 S]-GTP γ S binding assay. Each assay used human 5-HT 1ACHO cell membrane (50 μg protein per well), GTPγS binding buffer (50 mM Tris–HCl, 50 mM Tris base, 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM DTT and 0.1% BSA), 0.1 nM [ 35 [S]-GTPγS and 30 μM GDP were added in a final volume of 500 μL (Cascio et al., 2010). The binding was achieved by adding [S]-GTPγS and 30 μM GDP to the pores. 35 Initiated by [S]-GTPγS. Nonspecific binding was measured in the presence of 30 μM GTPγS. Assays were performed at 30 °C for 60 min (Cascio et al., 2010). The reaction was terminated by rapid vacuum filtration using Tris-binding buffer, as previously described by Cascio et al. (2010), and radioactivity was quantified by liquid scintillation spectroscopy. In all [ 35 In the [S]-GTPγS binding assay, 0.1 nM [ 35 [S]-GTPγS, 30 mM GDP, and protein concentration of 5 μg per well. CBDA, HU-580, 8-OH-DPAT, and WAY100635 were stored as 10 mM stock solutions dissolved in DMSO at -20°C.

[0070] In vivo program Animals.Animal procedures were performed in accordance with the Canadian Council on Animal Care, and the protocols were approved by the Institutional Animal Care Committee at the University of Guelph. Animal studies were reported according to the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). A total of 200 juvenile male Sprague-Dawley rats obtained from Charles River Laboratories (St. Constant, Quebec) were used for all in vivo studies. Rats were housed individually (for acute nausea studies) or in pairs (for anticipated nausea and light-dark stress studies) in cages (48 × 26 × 20 cm) made of opaque white plastic containing bed-o-cob bedding, brown paper towels, and Crinkl'Nest from Harlan Laboratories, Inc. (Mississauga, Ontario) and Crinkl'Nest from The Andersons, Inc. (Maumee, Ohio). ™ In addition, in the cages, rats were provided with soft, white paper containers 14 cm long and 12 cm in diameter. All rats were subjected to an ambient temperature of 21°C and a 12 / 12 h light-dark schedule (lights turned off at 07:00 h) and were kept on an ad libitum basis with food (Highland rat food

[8640] ) and water. For acute and anticipatory nausea studies, their body weight was in the range of 263 to 329 g on the day of conditioning. For light-dark stress studies, their body weight was in the range of 320 to 387 g on the day of testing.

[0071] Apparatus.For the acute nausea study (in vivo experiment 1), rats were placed in a taste responsiveness (Grill and Norgren, 1978) chamber, their cannulas attached to an infusion pump (KDS100, KD Scientific, Holliston, MA, USA) for fluid delivery. The taste responsiveness chamber was made of transparent plexiglass (22.5 × 26 × 20 cm) and placed on a table with a transparent glass top. A mirror positioned at a 45° angle below the chamber facilitated observation of the rats' ventral side to observe orofacial responses. The conditioned chamber was located in a dark room near a 25 W light source. A camera (Sony DCR-HC48, Henry's Cameras, Waterloo, ON, Canada) connected to a computer was focused onto the mirror and used to record the orofacial responses of each rat during a 2-minute taste responsiveness test. The videotapes were later scored using The Observer software (Noldus Information Technology Inc., Leesburg, VA, USA).

[0072] For in vivo experiment 2, situation-induced conditioned mouth opening (a model of anticipatory nausea) was measured using a unique conditioned chamber (22.5 × 26 × 20 cm) made of opaque black plexiglass with an opaque lid, placed on a table with a transparent glass top. A mirror positioned at a 45° angle below the chamber facilitated observation of the rat's ventral side to observe orofacial responses. The conditioned chamber was located in a dark room near a 25 W light source. A camera, wired to a computer, was focused onto the mirror to record each rat's orofacial responses over a 5-minute experiment. The videotapes were later scored using "The Observer" software. To assess activity, an activity chamber (60 × 25 × 25 cm) made of white plexiglass was used, illuminated with red light to create a different situation than the AN chamber, in a room different from the room using the situation chamber. Each rat's activity was captured by a camera and sent to the Ethovision software program (Noldus, Inc., NL) to measure the distance traveled (cm).

[0073] For in vivo experiment 3, a light-dark stress response device was used to assess anxieties-like responses. This device consisted of an opaque white rectangular box divided into two chambers: a small (25 cm wide × 20.5 cm long × 20.5 cm high) enclosed dark chamber made of opaque black plastic, with a door (8 cm wide × 10 cm high) leading to a larger (39.5 cm long × 25 cm wide) open bright chamber. The open bright chamber was illuminated by a lamp (with a 60 W bulb, 180 lux in the light chamber) located 115 cm above the center of the bright chamber. A camera was mounted on top of the light-dark chamber, and the videotape was analyzed using Ethovision software (Noldus Information Technology, Leesburg, VA, USA) to obtain the time spent in the light chamber for the 5-minute test. For the foot shock (FS) phase, rats were placed in a silenced MED Associates fear conditioning chamber (St. Albans, VT, USA). The 6-minute FS phase consists of six 0.8 mA foot shocks delivered at 1-minute intervals. As described by Bluett et al. (2014), a 30-second tone (90 dB, 5000 Hz) is played before each 0.5-second shock.

[0074] In vivo program In vivo experiment 1 : Dose-related effects of CBDA and HU-580 on acute nausea and 5-HT 1A receptor mediation of the effects of HU-580 In vivo experiment 2: Effects of CBDA and HU-580 on anticipatory nausea and 5-HT Following the procedure described by Limebeer et al. (2010), cannulas were surgically inserted into the oral cavity of all rats. On the day of surgery, rats were injected with antibiotics (Derapin: 0.00 mg / kg) 30 min before anesthesia with isoflurane (4-5% for induction, 1.5% for maintenance in O2). -1 sc; Pfizer Animal Health, Pfizer Canada Inc, Kirkland, Quebec, Canada). Induce surgical-level anesthesia prior to any surgery, as indicated by the absence of a hindlimb withdrawal reflex and as defined by the Canadian Council of Animal Care, and adjust as necessary. Once adequate anesthesia has been induced, insert an anesthesia 2 cm above the scapula at the back of the neck. 2The skin was shaved. Skin preparation was performed by washing with soap (Bactistat; Ecolab, St. Paul, MN, USA) and wiping with 70% isopropanol followed by 7% povidone-iodine solution (Purdue Products LP, Stamford, CT, USA). Each rat was then administered 5 mg / kg of the solution. -1 Injection (ip) of the anti-inflammatory / analgesic drug carbofenac (Limodi; Pfizer Canada Inc., Kirkland, Quebec, Canada). Insert a 15-gauge thin-walled stainless steel needle into the shaved area on the neck, guide it subcutaneously to near the ear, and bring it out behind the first molar in the mouth. Then, insert a 10 cm long Intra Medic PE90 tubing (Clay Adams Brand; Becton Dickinson and Co., Sparks, MD, USA) with an inner diameter of 0.86 mm and an outer diameter of 1.27 mm through the needle, and then remove the needle. Apply iodine (10%) to the puncture site and insert three flexible discs (2 cm). 2 The cannula was placed on the exposed end of the tubing and pulled towards the skin at the back of the neck to stabilize it. The cannula was held in place in the oral cavity by a 6 mm polypropylene mesh (297 μm; Small Parts Inc., Miramar, FL, USA) disc fixed behind the opening of the heated flange. The rats were then returned to their cages and monitored daily for three days. For three days post-surgery, the rats were weighed and their cannulas were rinsed with antibacterial mouthwash. During this period, the rats' activity, vocalization, dehydration, rigidity, and the presence of porphyrin staining around the eyes were also monitored. On the first day post-reoperation, the rats were also administered analgesic / anti-inflammatory injection (5 mg / kg). -1 IP).

[0075] Following postoperative monitoring, the rats underwent an adaptation test, in which they were placed in a taste-responsive chamber and each rat's cannula was attached to an infusion pump. During the adaptation period, water was administered at a rate of 1 mL / min. -1 The drugs were infused into their intraoral cannulas at a rate of 2 min. On the day following the acclimatization test, the rats underwent a conditioned reflex test, in which they were administered a mediator (VEH) (n=8), CBDA (0.01, 0.1, 1 μg·kg⁻¹), and other drugs. -1 ; n =8 per group) or HU-580 (0.01, 0.1, 1 μg·kg -1 ; nThe rats were pretreated with an injection of 0.1% saccharin solution at a rate of 1 mL / min for 45 minutes after the pretreatment injection. -1 The saccharin was infused at a rate of 2 mL / kg for 2 min. Immediately after the saccharin infusion, all rats were given 20 mL / kg of saccharin. -1 The rats were injected with 0.15M LiCl and then returned to their home cages. A drug-free test was performed on the rats 72 hours later. The rats were then injected again with 0.1% saccharin solution at 1 mL / min. -1 The rats were infused at a rate of 0.1 mg / kg orally for 2 minutes, while their oral-facial responses were video-recorded from a mirror positioned at a 45° angle below the chamber. The rats were then returned to their cages. Two additional groups were added to determine the mechanism of action. These rats were then administered WAY100635 (0.1 mg / kg). -1 ) Inject, 15 minutes later inject the medium ( n =8) or 0.1 μg·kg - 1 HU-580 ( n =6). The videotape was later used by an observer unaware of the experimental conditions to rate the mouth-opening behavior (mouth and jaw opening wide, with the lower incisors exposed) using "The Observer".

[0076] receptor mediation of the effects of HU-580 1A In vivo experiment 3: Effects of CBDA and HU-580 on anxiety-like responses and 5-HT To compare the potential of HU-580 and CBDA in alleviating anticipatory nausea, a situation-induced conditioned mouth-opening paradigm was used (e.g., Limebeer et al., 2010; Rock et al., 2014). Rats underwent four conditioned tests during which a unique situation was associated with 127 mg / kg. -1 LiCl pairing. In each experiment, rats were injected with LiCl and then immediately placed in a conditioned chamber for 30 min. This procedure was repeated four times, with a 48 h interval between conditioned experiments. For the test experiments, rats were randomly assigned to one of five treatment groups ( n = 6 animals per group): VEH, 0.1 μg·kg -1 CBDA, 0.1 μg·kg -1 HU-580, 0.01 μg·kg 1 CBDA, 0.01 μg·kg -1 HU-580. 45 min after injection pretreatment, rats were given a saline injection (20 mL / kg). -1Rats were individually placed in conditioned (contextual) chambers for 5 min, and their oral and facial responses were recorded via video. To investigate the mechanism of action of HU-580, two other groups of rats were administered 0.1 mg / kg of HU-580. -1 WAY-VEH ( n =8), 0.1 mg·kg -1 WAY-0.1 μg·kg -1 HU-580 ( n =8). VEH or WAY100635 was administered 15 min prior to HU-308 or VEH administration. Videotapes from the test experiment were used by an observer unaware of the experimental conditions to score mouth-opening behavior (wide opening of the mouth and jaws, with the lower incisors exposed) using "The Observer". Immediately after the test experiment, rats were placed in an activity chamber (white plexiglass, 60 × 25 × 25 cm, illuminated with red light) for 15 min. Spontaneous activity was captured by a camera and transmitted to a computer, where the distance traveled (cm) was measured using EthoVision software (Noldus, Inc, NL).

[0077] receptor mediation of the effects of HU-580 1A Drugs and materials used in vitro. The effects of CBDA and HU-580 on anxiety-like responses were assessed using a light-dark chamber stress test following foot shock or no-foot shock (FS) stress. Bluett et al. (2014) demonstrated that anxiety-like responses were significantly enhanced 24 hours after foot shock stress in this test. Furthermore, Rock et al. (2017) showed that CBDA (at doses as low as 0.1 μg·kg⁻¹) could significantly enhance anxiety-like responses. -1 (at the dose of ip) via 5-HT 1A The mechanism of action is dependent on the prevention of increased anxiety-like responses following foot shock. Therefore, even lower doses (0.01 μg·kg⁻¹) were compared. -1 The relative efficacy of CBDA and HU-508 (in 1 ip) was compared. Since HU-580 was found to be an anxiolytic at this low dose, 5HT was subsequently evaluated. 1A The ability of the receptor antagonist WAY100635 to reverse the inhibition of the anxiety-like response induced by HU-580.

[0078] All rats were acclimatized to the environment for 13 days prior to the experimental procedures, during which they were weighed and operated on for 8 of those days. Following this acclimatization, rats were subjected to either a single FS stress phase or a non-FS stress phase, followed by a light-dark stress test 24 h later (Bluett et al., 2014). For the FS group, rats were placed in a silenced MED Associates fear conditioning chamber (St. Albans, VT, USA). The 6-min FS phase consisted of six 0.8 mA FS shocks delivered 1 min apart. As described by Bluett et al. (2014), a 30-second tone (90 dB, 5000 Hz) was played before each 0.5-second shock. The non-FS stress group remained in their cages during this phase.

[0079] Twenty-four hours later, the rats underwent a light-dark stress test. Rats in the FS group and the non-FS group were treated with VEH at 0.01 μg / kg. -1 CBDA or 0.01 μg·kg -1 HU-580 pretreatment. After 45 minutes, they were placed in a dark chamber of a light-dark box, and their movement was tracked during a 5-minute experiment. To investigate the effects of HU-580, 5-HT... 1A To investigate the receptor-mediated possibility, another group was injected with WAY100635, followed by VEH or 0.01 μg / kg 15 min later. -1 HU-580. Measure the number of seconds spent in the illumination chamber. Groups are as follows: No FS–VEH ( n =9), FS-VEH ( n =12), no FS-0.01 μg·kg -1 CBDA ( n =8), FS-0.01 μg·kg -1 CBDA ( n = 8), no FS- 0.01 HU-580 ( n = 8), FS-0.01 HU-580 ( n = 8), no FS-0.1 μg·kg -1 WAY-VEH ( n =8), FS-0.1 μg·kg -1 WAY-VEH ( n =7), no FS-0.1 μg·kg -1 WAY-0.01 μg·kg - 1 HU580 ( n =8), FS-0.1 μg·kg -1 WAY-0.01 μg·kg -1HU-580 ( n =8).

[0080] In vitro and in vivo data analysis agonist-stimulated [ 35 The net binding value of [S]-GTPγS is calculated by subtracting the baseline binding value (obtained in the absence of an agonist) from the agonist stimulation value (obtained in the presence of an agonist) (Cascio et al., 2010). Values ​​are expressed as a mean and variation as SEM or as a 95% confidence limit. Mean EC 50 and average maximum effect (E max The values ​​and their SEM or 95% confidence limits were calculated using the equations for the S-shaped concentration response curves via nonlinear regression analysis (GraphPad Prism). P A value <0.05 was considered significant. The data and statistical analysis conformed to the recommended experimental design and analysis protocols in pharmacology (Curtis et al., 2015).

[0081] For the analysis of data from the acute nausea test (in vivo experiment 1), a one-way ANOVA was performed on the mean number of mouth openings during the 2-minute test, followed by a post-hoc test of least significant difference (LSD) to assess paired comparisons. For the analysis of data from the anticipatory nausea (AN) test (in vivo experiment 2), a one-way ANOVA was performed on the number of mouth openings during the 5-minute AN test and the total distance moved during the activity test, followed by a post-hoc test of LSD to assess paired comparisons. For the analysis of data from the anxiety-like response test (in vivo experiment 3), the amount of time spent in the lightbox during the light-dark stress test was entered into a 2 × 5 factorial ANOVA, where the factors were FS stress / no FS stress and each pretreatment and μg·kg. -1 IP Dosage Conditions (VEH, 0.01 μg·kg) -1 CBDA, 0.01 μg·kg -1 HU-580, WAY-VEH, or WAY-HU-580). This was followed by independent [further details needed]. t - Tests were conducted to explore interactions. The significance level was set at... P <0.05.

[0082] Drugs used in vivo. 8-OH-DPAT and WAY100635 are supplied by Bio-Techne (Abingdon, UK). 35 S]-GTPγS (1250 Ci mmol) -1The GTPγS, GDP, and DMSO were purchased from PerkinElmer Life Sciences, Inc. (Boston, MA, USA). CBDA and its methyl ester (HU-580) were purchased from Sigma-Aldrich UK.

[0083] CBDA and HU-580 enhance the ability of 5-HT Lithium chloride (LiCl; Sigma Aldrich) was prepared into a 0.15 M solution using sterile water and diluted at a concentration of 20 mL·kg⁻¹. -1 (127.2 mg·kg) -1 Administer via intraperitoneal (ip) at the specified volume. CBDA and its methyl ester (HU-580), both supplied by Raphael Mechoulam, were dissolved in 1 mL of ethanol in a graduated glass tube. 1 mL of Tween80 (Sigma) was added to the solution, and the ethanol was evaporated using a nitrogen stream. Then, 9 mL of saline was added (final Tween80:saline ratio = 1:9). CBDA or HU-580 was administered at doses of 0.01, 0.1, or 1.0 μg / mL, respectively. -1 The concentration of the stock solution containing one or more of these compounds is in 1 mL·kg -1 In the volume of 0.01, 0.1 or 1.0 μg·kg 1 The dose was administered intraperitoneally to rats. WAY100635 (Sigma, St Louis, MO, USA) was administered at 0.1 mg / mL. -1 The concentration was dissolved in salt water at 0.1 mg / kg. -1 (1 mL·kg) -1 The dose was administered ip to rats.

[0084] result receptor agonists to stimulate [35S]GTPyS binding to human 5-HT 1A receptors in vitro 1A Figures 1A-1E Figures 2A-2F As previously performed using rat brainstem membranes [ 35 As discovered in experiments involving the binding of 5-HT to GTPγS (Bolognini et al., 2013), CBDA enhancement is achieved through selective 5-HT. 1A 8-OH-DPAT receptor agonist-induced [ 35 Stimuli that bind to 5-GTPγS, said binding is associated with the transfer of human 5-HT from stably transfected cells. 1A Membrane binding of the receptor to CHO cells ( Figures 1A-1E(See Table 1). CBDA concentrations in the submicromolar range caused average 8-OH-DPAT at 0.1, 1.0, and 10 nM, but not at 0.01 or 100 nM. E max A significant increase. These averages E max The increase was not accompanied by an average EC50 of 8-OH-DPAT. 50 Any significant changes ( P >0.05; Table 1). CBDA methyl ester HU-580 in enhanced [ 35 S]-GTPγS and expression of human 5-HT 1A The binding of the receptor to the CHO cell membrane by 8-OH-DPAT is even more effective than CBDA in inducing stimulation. Figures 2A-2F (and Table 2). Therefore, it causes an average 8-OH-DPA not only at 0.1, 1.0, and 10 nM (similar to CBDA) but also at 0.01 nM (different from CBDA). E max Significant improvement. HU-580 does not improve the average 8-OH-DPAT at 100 nM (similar to CBDA) or 0.001 nM. E max Furthermore, it did not significantly affect the mean EC50 of 8-OH-DPAT at any of the concentrations studied. 50 (Table 2). When administered autologously, HU-580 did not exhibit 5-HT at concentrations of 0.01, 0.1, 1, 10, or 100 nM. 1A Receptor agonists or inverse agonists, as either of these concentrations affects [ 35 S]-GTPγS and from human 5-HT 1A The membrane binding of receptor-transfected CHO cells lacked detectable effects. n = 6; data not shown) indicates.

[0085] Table 1: For 8-OH-DPAT pairs [ 35 S]GTPγS and from stable transfection with human 5-HT 1A Regarding the stimulation of receptor CHO cells to bind to the membrane, various concentrations of CBDA showed an average EC50 of 8-OH-DPAT. 50 and E max The effect of the value (see also) In vivo experiment 1 : Dose-related effects of CBDA and HU-580 on acute nausea and 5-HT )

[0086] Each asterisk indicates the average 8-OH-DPAT determined in the presence of a specific concentration of CBDA.E max The value is the average of 8-OH-DPAT determined in the same experiment in the presence of a medium (DMSO) instead of CBDA, as shown in the previous row. E max Significant differences between values ​​(*) P <0.05). Significant differences are indicated by a non-overlapping 95% confidence limit.

[0087] Table 2: For 8-OH-DPAT pairs [ 35 S]GTPγS and from stable transfection with human 5-HT 1A Regarding the stimulation of receptor CHO cells to bind to the membrane, various concentrations of HU-580 showed average EC50 values ​​for 8-OH-DPAT. 50 and E max The effect of the value (see also) receptor mediation of the effects of HU-580 )

[0088] Each asterisk indicates the average 8-OH-DPAT determined in the presence of a specific concentration of HU-580. E max The value is the average of 8-OH-DPAT determined in the same experiment in the presence of a medium (DMSO) instead of HU-580, as shown in the previous row. E max Significant differences between values ​​(*) P <0.05). Significant differences are indicated by a non-overlapping 95% confidence limit.

[0089] Figure 3 1A In vivo experiment 2: Effects of CBDA and HU-580 on anticipatory nausea and 5-HT receptor mediation of the effects of HU-580 As assessed using a rat open-mouth model, at 0.1 μg·kg -1 But not at 0.01 or 1 μg·kg -1 At the recommended dosage, HU-580 was more effective than CBDA in relieving acute nausea. HU-580 (0.1 μg·kg⁻¹) -1 The inhibitory effect of WAY100635 on acute nausea was blocked. One-way ANOVA revealed a significant group effect F(8, 61) = 3.9. P <0.05. Figure 4A The average number of mouth openings observed by various pretreatment groups is presented. Subsequent post-hoc comparative tests using LSD revealed that at 1 μg·kg⁻¹… -1 At the given dosage, both compounds reduced the LiCl-induced mouth-opening response relative to the medium. P<0.05%, which repeats our previous findings (Limebeer et al., 2010; Rock and Parker, 2013). However, at 0.1- μg·kg -1 At even lower doses, i.e. below the threshold for CBDA-induced reduction in nausea-like behavior, HU-580 reduced LiCl-induced conditioned mouth-opening behavior relative to the medium. P <0.05). Use HU-580 (0.1 μg·kg -1 The pretreated rats also had significantly less mouth opening than those treated with WAY-0.1 μg·kg⁻¹. -1 HU-580 group ( P <0.05), indicating 5-HT 1A Receptor-mediated effects.

[0090] Figure 4B 1A In vivo experiment 3: Anti-anxiety effects of CBDA and HU-580 As assessed by the scenario-induced conditional mouth-opening model, at 0.01 μg·kg -1 At extremely low doses, but not below 0.1 μg·kg -1 In this study, HU-580 was more effective than CBDA in reducing anticipatory nausea. HU-580 (0.1 μg·kg⁻¹) -1 The inhibitory effect of ) was blocked by WAY100635. One-way ANOVA revealed a significant group effect F(6, 39) = 8.7; P <0.05. Figure 5 The average number of mouth openings observed was presented. Subsequent post-hoc comparisons with LSD revealed that, compared to the VEH control, at 0.1 μg·kg⁻¹ -1 At the doses, both CBDA and HU-580 reduce conditioned mouth opening ( P Value <0.05); however, at 0.01 μg·kg -1 The groups showed differences at the specified dosages, with the HU-580 group exhibiting significantly less mouth opening than the VEH control group. P <0.05) and 0.01 CBDA group ( P =0.05). Use HU-580 (0.1 μg·kg -1 The pretreated rats also had significantly less mouth opening than those treated with WAY-0.1 μg·kg⁻¹. -1 HU-580 group ( P <0.05), indicating 5-HT 1A Receptor-mediated effects. For spontaneous activity tests ( Figure 3 For example, single-factor ANOVA revealed no significant effect on travel distance. F(6, 39) = 0.9, P >0.05.

[0091] ​ ​ The mean number of seconds spent in the light chamber by rats in each of the various pretreatment groups (those receiving FS or not receiving FS) 24 h prior to the light-dark test is presented. As can be seen, FS stress greatly enhances the anxiety-like response, i.e., the time spent in the light chamber is reduced. At 0.01 μg·kg -1 At low doses, HU-580 reversed the effect of FS on anxiety-like responses (reduced time spent in the lightbox). A 2×5 ANOVA on the number of seconds spent in the lightbox revealed FS stress (F(1, 84) = 25.6). P <0.05) and the interaction between FS stress and pretreatment (F(4, 84) = 3.2; P The significant main effect was 0.05. To analyze the interaction, subsequent independent... t - The test revealed the use of VEH ( P <0.05), 0.01 μg·kg -1 CBDA ( P <0.05), WAY-VEH ( P <0.05) or WAY-0.01 μg·kg 1 HU-580 ( P Rats pretreated with 0.05 μg / kg spent less time in the light chamber after FS stress compared to rats without FS stress, but with 0.01 μg / kg -1 Rats pretreated with HU-580 did not show this anxiety-inducing response. Furthermore, subsequent one-way ANOVA of the time spent in the lightbox revealed a significant pretreatment effect between the FS groups (F(4, 38) = 4.6). P <0.05), but not among the groups without FS. Within the FS groups, subsequent Bonferroni tests revealed only 0.01 μg·kg⁻¹. -1 The HU-580 group spent significantly more time in the light box compared to the VEH group (P<0.05).

[0092] discuss The results confirmed that CBDA induces 5-HT in vitro. 1A The receptor is directly 5-HT 1A The epigenetic enhancement of 5-HT receptor agonist 8-OH-DPAT activation and its induction of both acute and anticipatory nausea in rats in vivo. 1ABoth receptor-mediated reductions showed significant efficacy.

[0093] The new in vitro data show that, firstly, CBDA can enhance 5-HT in the rat brainstem. 1A Receptor activation (Bolognini et al., 2013), and can enhance human 5-HT 1A Receptor activation, and secondly, in the rat brainstem and human 5-HT receptors. 1A Of the two receptors, CBDA induced this enhancement with a bell-shaped concentration-response curve in the submicromolar range. The in vitro data described in this article also reveal important similarities between the pharmacological effects of CBDA and its methyl ester HU-580. More specifically, these data provide compelling evidence that HU-580 enjoys the pharmacological benefits of CBDA in […]. 35 The S-GTPγS binding assay for human 5-HT 1A The ability of the receptor to be apparently enhanced by 8-OH-DPAT activation. Importantly, HU-580 induced this enhancement with higher potency and even broader bell-shaped concentration-response curves compared to CBDA. Thus, significant enhancements were induced by HU-580 at concentrations of 0.01 to 10 nM (Table 2) and CBDA at concentrations of 0.1 to 10 nM (Table 1). However, at concentrations of 1, 10, and 100 nM, HU-580 induced 8-OH-DPAT-induced 5-HT receptor activation compared to CBDA. 1A The receptor activation was slightly less enhanced, while at concentrations of 0.01 and 0.1 nM, HU-580 caused a slightly greater enhancement of this activation compared to CBDA (Tables 1 and 2).

[0094] It is worth noting that for 8-OH-DPAT [ 35 S]-GTPγS and 5-HT 1A Regarding the stimulation of receptor binding, it significantly increases the E2O2 of 8-OH-DPAT. max Neither the concentration of CBDA nor HU-580 caused EC50 in 8-OH-DPAT. 50 Any significant changes (Tables 1 and 2). This finding suggests that CBDA and HU-580 may act as positive allosteric regulators of 8-OH-DPAT activation in these receptors, as there is evidence that some positive allosteric regulators do indeed enhance E in certain receptors. max It has the value but does not improve the efficacy of the agonist (Christopoulos et al., 2014). There is a possibility that CBDA and HU-580 act as positive allosteric modulators targeting 5-HT. 1AAllosteric sites on the receptor. It is also noteworthy that the positive in vitro data for CBDA and HU-580 obtained in this study were derived using transfected human 5-HT. 1A Experiments conducted on the receptor's CHO cells.

[0095] The in vivo data revealed a similarity between the pharmacological effects of HU-580 and CBDA. Therefore, these data suggest that the ability of CBDA to reduce acute and anticipatory nausea in rats extends to HU-580. Importantly, and as we found in our in vitro experiments, HU-580 exhibited even higher potency than CBDA. More specifically, HU-580 showed even greater potency at dosages as low as 0.1 μg·kg⁻¹. -1 At an intraperitoneal dose, CBDA effectively inhibits conditioned mouth opening induced by acute nausea, and the lowest effective dose of CBDA to produce this inhibition is 1 μg / kg. -1 IP ( ​ In fact, it has been found at concentrations as low as 0.01 μg·kg⁻¹. -1 At intraperitoneal doses, HU-580, but not CBDA, inhibits situation-induced conditioned mouth opening. It has also been shown that LiCl-induced mouth opening and situation-induced conditioned mouth opening are inhibited by HU-580, which can be mitigated by 5-HT. 1A The receptor-selective antagonist WAY100635 provided complete inhibition. Finally, although CBDA has recently been found to be effective in light-dark chamber emergency tests at 0.1, 1, and 100 μg·kg⁻¹,… -1 The FS dose at ip reduced the enhancement of similar anxiety-inducing behaviors (Rock et al., 2017), but in this study it was found not to have the same effect as HU-580 in the light-dark chamber emergency test at doses as low as 0.01 μg·kg⁻¹. -1 The ability of HU-580 to reduce the enhancement of stress-induced anxiety by FS at intraperitoneal doses suggests that HU-580 may be even more effective than CBDA in reducing stress-induced anxiety. Furthermore, the ability of HU-580 to reduce the enhancement of stress-induced anxiety by FS is also shown to be due to 5-HT. 1A Receptor-mediated. The results showed that HU-580 was not only more stable than CBDA, but also more effective than CBDA (for acute and anticipatory nausea).

[0096] Ideally, pharmaceutical drugs should exhibit stability during storage for a reasonable period of time. Therefore, since CBDA undergoes significant decomposition even at 4°C, the primary objective of this project is to develop a compound that produces efficacy no less than CBDA in the assays described herein, and exhibits significantly higher stability when stored at that temperature for a reasonable duration. Consequently, it is noteworthy that the inventors have found that HU-580 is indeed more stable than CBDA when stored at 4°C for 21 days. Furthermore, the finding that HU-580 is more effective than CBDA in both in vitro and in vivo conditions supports the hypothesis that the pharmacological effects produced by HU-580 in our experiments are independent of its decomposition or metabolism into CBDA.

[0097] In summary, the evidence suggests that HU-580 is more effective than CBDA in suppressing both acute and anticipatory nausea and stress-induced anxiety in rats, and that it is more potent than CBDA in inhibiting 5-HT. 1A These effects are produced in a receptor-dependent manner.

[0098] Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, alterations, and equivalents will be possible to those skilled in the art. Therefore, it should be understood that the claims of this invention are intended to cover all such modifications and alterations falling within the true spirit of the invention.

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Claims

1. The use of the compounds shown below in the preparation of a medicament for the treatment of 5-HT 1A Receptor-related conditions, diseases, or symptoms: ， The one mentioned with 5-HT 1A Receptor-related disorders, diseases, or symptoms are selected from anxiety, tension, depression, panic, and combinations thereof.

2. The application according to claim 1, wherein the compound is administered orally, nasally, topically, parenterally, or via an implant.

3. The application according to claim 2, wherein the compound is administered orally.

4. The application according to claim 1, wherein the drug comprises an adjuvant selected from carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavorings, antioxidants, and wetting agents.

5. The application according to claim 1, for preparing a medicine for treating anxiety.

6. The application according to claim 1, for preparing a medicine for treating tension.

7. The application according to claim 1, for preparing a medicament for treating depression.

8. The application according to claim 1, for preparing a medicine for treating panic.

9. The use of a pharmaceutical composition comprising, with pharmaceutically acceptable excipients, the following compounds in the preparation of a medicament for the treatment of 5-HT 1A Receptor-related conditions, diseases, or symptoms: ， The one mentioned with 5-HT 1A Receptor-related disorders, diseases, or symptoms are selected from anxiety, tension, depression, panic, and combinations thereof.

10. The application according to claim 9, wherein the compound is administered orally, nasally, topically, parenterally, or via an implant.

11. The application according to claim 10, wherein the compound is administered orally.

12. The application according to claim 9, wherein the drug comprises an adjuvant selected from carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavorings, antioxidants, and wetting agents.

13. The application according to claim 9, for preparing a medicament for treating anxiety.

14. The application according to claim 9, for preparing a medicament for treating tension.

15. The application according to claim 9, for preparing a medicament for treating depression.

16. The application according to claim 9, for preparing a medicament for treating panic.

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