Substituted cinnamic amide compounds for antidepressant and antianxiety

Abstract

The present application relates to an antidepressant / anti-anxiety substituted cinnamic amide compound, namely compound M2. The present application further provides a pharmaceutical composition containing compound M2 or a solvate or a pharma- ceutically acceptable salt thereof, and its use for antidepressant, anti-anxiety, or antidepressant / anti-anxiety. TIFF2024521282000041.tif34149

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application for invention filed on May 28, 2021, for the invention titled "Novel antidepressant and anti-anxiety compound" and bearing application number 202110606431.2, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to pharmaceutical compounds, but is not limited thereto, and in particular to novel compounds of substituted cinnamic amides having antidepressant and anxiolytic properties. [Background technology]

[0003] Depression and anxiety are the main diseases that harm people's mental health, and with the accelerating rhythm of modern life, the number of people suffering from these diseases is increasing year by year.Recent statistics show that the number of people suffering from various types of depression in China has reached 26 million, of which adolescents account for a relatively large proportion.Therefore, the research and development of antidepressant and antianxiety drugs has important economic and social benefits.

[0004] A large amount of research results show that the changes in central monoamine neurotransmitters, dopamine, cholinergic, the corresponding receptor function changes, and neuroendocrine dysfunction may have important effects on the development and progression of this disease.Here, the treatment principle should focus on adjusting the content of hypothalamic monoamine neurotransmitters and their receptor function, and restoring normal neuroendocrine.

[0005] At present, the main treatment for depression and anxiety is still taking drugs. Traditionally, antidepressants and antianxiety drugs in clinical practice mainly include tricyclics, benzodiazepines, 5-hydroxytryptamine reuptake inhibitors, monoamine oxidase inhibitors and other chemical drugs, which have a certain therapeutic effect on depression and anxiety, but also have the disadvantage of relatively high toxicity and side effects. Monoamine oxidase inhibitors cause toxic liver damage due to selectivity and irreversible inhibition of enzymes, and have certain toxicity and side effects. Common tricyclic drugs include doxepin, amitriptyline, clomipramine, etc., which are relatively effective against endogenous depression, especially for depression, loss of hobbies, and pessimism, and can achieve a treatment effect of more than 80%, but due to strong toxicity to the heart, there are relatively many adverse reactions. Selective 5-HT reuptake inhibitors (SSRIs) are new antidepressants and antianxiety drugs that appeared in the late 1980s. They maintain the traditional antidepressant and antianxiety properties while significantly reducing the adverse reactions that occur in other receptors, and are now the usual first-choice drugs in Europe and the United States. Typical drugs include fluoxetine, paroxetine, sertraline, citalopram, and fluvoxamine. Due to gastrointestinal absorption and liver metabolism, some of them have gastrointestinal dysfunction and sexual dysfunction, which has affected long-term use in treatment to some extent. Furthermore, traditional antidepressants and antianxiety drugs are slow to take effect (6 to 8 weeks or more), and only work in about 30% of patients. Therefore, in the field of treatment of severe depression, there is an urgent need for drugs that have a fast onset (especially in patients with suicidal tendencies), relatively good therapeutic effects, relatively low toxicity and side effects, and a new mechanism of action.

[0006] Chinese Patent CN102850317A (Application No. 201210123842.7, hereinafter referred to as "Patent A") and CN103687850A (Application No. 201280020049.2, hereinafter referred to as "Patent B") disclose substituted cinnamamide derivatives, their preparation methods, and their use in drugs for treating and preventing mental disorders of depression. Thirteen specific compounds I-1 to I-13 are disclosed. In the patent, it was verified by the mouse tail suspension test, the "acquired despair" depression model experiment, the anti-reserpine ptosis depression model experiment, and the mouse forced swimming test that the administration of I-5, I-9, I-10, I-11, I-12, and I-13 at a dose of 10 mg / kg for 7 days can significantly shorten the tail suspension immobility time of mice. Compounds I-4, I-5, I-10, I-11, I-12 and I-13 were administered at a dose of 10mg / kg for 7 days in succession, and could significantly antagonize the hypothermia and immobility caused by reserpine in mice, and improve the degree of eye closure, which shows that they have a certain regulating effect on the re-uptake of 5-HT, NE and DA. Compounds I-5, I-10 and I-13 can all significantly shorten the forced swimming immobility time of mice, and the effect of I-5 on the forced swimming immobility time of mice is dose-dependent. Among them, I-5, which has the best effect, is currently in clinical research stage.

[0007] Chinese Patent CN107011313A (application number 201710038281.3, hereinafter referred to as "Patent C") disclosed the use of substituted cinnamamide derivatives in anti-anxiety. Specifically, a total of 17 compounds were disclosed, including II-3 (compound I-3 in Patent A and B), II-4 (compound I-4 in Patent A and B), II-5 (compound I-5 in Patent A and B), II-10 (compound I-10 in Patent A and B), II-11 (compound I-11 in Patent A and B), II-12 (compound I-12 in Patent A and B), II-13, II-14, II-15, II-16, III-2, III-4, III-7, III-9, III-10, III-11, and III-13. In this patent, it is proved that 17 kinds of compounds are administered at a dose of 10mg / kg for 7 days by mouse elevated plus maze test, and can increase the number of mouse's entering into the open arm in elevated plus maze test to different extents, and can extend the time spent in the open arm by mouse.In rat Vogel type conflict test, it is proved that 17 kinds of compounds are administered at a dose of 5mg / kg for 10 days, and can increase the number of rat's drinking water in punishment period to different extents. Summary of the Invention

[0008] The present applicant has studied many of the compounds in the above patents and found that many of the compounds have excellent therapeutic effects, but have low bioavailability and slow onset of action.

[0009] In a first aspect, the present application provides an antidepressant / anti-anxiety substituted cinnamic amide compound, or a solvate or pharma- ceutically acceptable salt thereof, having a faster onset of action and higher bioavailability, the structural formula of the substituted cinnamic amide compound being: [ka]

[0010] In a second aspect, the present application provides a pharmaceutical composition comprising the above-mentioned substituted cinnamic amide compound, or a solvate thereof, or a pharma- ceutically acceptable salt thereof.

[0011] In a third aspect, the present application provides a method for preparing the above-mentioned substituted cinnamic amide compounds.

[0012] In a fourth aspect, the present application provides a use of the above-mentioned substituted cinnamic amide compound in the manufacture of an antidepressant, an anxiolytic, or an antidepressant / anxiolytic.

[0013] In a fifth aspect, the present application provides the use of a compound as defined above in combination with one or more other antidepressants or anxiolytics in the manufacture of an antidepressant, an anxiolytic, or an antidepressant / anxiolytic medicament.

[0014] In a sixth aspect, the present application provides a method for the prevention or treatment of a psychiatric disorder comprising administering to a patient in need thereof a compound as described above. [Brief description of the drawings]

[0015] [Figure 1] It shows that compound M2 has antidepressant-like effect. 24 hours after administration, compound M2 significantly reduces the immobility time of mice, and has obvious dose effect, and when the dose is 30mg / kg, it has significant effect. [Diagram 2] The effect of compound M2 on excitatory synaptic transmission in the frontal lobe is shown, specifically, the effect of compound M2 at concentrations of 10 nM, 10 μM, and 150 μM on sEPSCs of pyramidal neurons in the PrL brain area of ​​the frontal lobe. The upper part shows a schematic diagram of sEPSC waveforms, and the lower part shows a columnar graph after data statistics. [Diagram 3] Compound M2 enhances excitatory synaptic transmission in the frontal lobe. At multiple concentrations, compound M2 increases the firing frequency of sEPSCs in the PrL brain area of ​​the frontal lobe, and exhibits obvious concentration-dependence, but the effect on the amplitude of sEPSC firing is unclear. Compound M2 at 150 μM can significantly enhance the firing frequency of sEPSCs. [Figure 4]This shows that the dopamine D2R receptor antagonist Sulpride inhibits the enhancing effect of compound M2 on sEPSCs in the frontal lobe. The D2R antagonist Sulpride can inhibit D2R receptors at an effective concentration of 10μM. The results show that inhibition of D2R can inhibit the enhancing effect of 150μM compound M2 on sEPSC firing frequency. [Diagram 5] This shows that the dopamine D1R receptor antagonist SCH23390 inhibits the enhancing effect of compound M2 on sEPSCs in the frontal lobe. The D1R receptor antagonist SCH23390 can inhibit D1R receptors at an effective concentration of 10 μM. The results show that inhibition of D1R can inhibit the enhancing effect of 150 μM compound M2 on sEPSC firing frequency. [Figure 6] It shows that compound M2 activates the mTOR-related signal pathway. a shows the change in frontal lobe-related signal protein 30 min after a single administration, and b shows the change in frontal lobe-related signal protein 24 h after a single administration. [Figure 7] The inhibitory effects (IC50) of compounds I-5 and M2 on the uptake of DA, 5-HT, and NA by rat brain synaptosomes are shown. [Figure 8] 1 shows the mean drug blood concentration time curves after oral gavage and intravenous administration of compound M2 to rats. [Figure 9] 1 shows the mean drug blood concentration-time curves after administration of compound I-5 to rats by oral gavage and intravenous route. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In an embodiment of the first aspect, the present application provides a substituted cinnamic amide compound, the structural formula of which is as follows: [ka]

[0017] Additionally, the substituted cinnamic amide compounds of the present application may exist in the form of solvates.

[0018] Additionally, the substituted cinnamic amide compounds of the present application can exist in the form of pharma- ceutically acceptable salts.

[0019] In an embodiment of the second aspect, the present application provides a pharmaceutical composition comprising a substituted cinnamic amide compound of the present application, or a solvate or a pharma- ceutically acceptable salt thereof.

[0020] The pharmaceutical composition of the present application may be in any administrable pharmaceutical form, such as a tablet, a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a capsule, a hard capsule, a soft capsule, an oral liquid, a buccal tablet, a granule, a pill, a powder, an ointment, an elixir, a suspension, a powder, a solution, an injection, a suppository, an ointment, a plaster, a cream, a spray, a drop, or a patch.

[0021] Preferably, the pharmaceutical compositions of the present application are in the form of unit dose pharmaceutical preparations.

[0022] When the pharmaceutical composition of the present application is prepared into a drug, the unit dose of the drug may contain 0.1 to 1000 mg of the pharmacologic active substance of the present application, and the remainder is a pharmaceutically acceptable carrier, which may be 0.01 to 99.99% by weight of the total weight of the formulation.

[0023] When using the pharmaceutical composition of the present application, the method and dosage are determined according to the condition of the patient, for example, 1 to 3 times a day, 1 to 10 tablets at a time, etc.

[0024] Preferably, the pharmaceutical composition of the present application is an oral formulation or an injectable formulation.

[0025] The oral preparation is one selected from capsules, tablets, drop pills, granules, concentrated pills, oral liquids and combination preparations.

[0026] The injection is one selected from an injection solution, a freeze-dried powder injection, and a liquid injection.

[0027] In the pharmaceutical composition of the present application, the formulation for oral administration may contain conventional excipients such as adhesives, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents or wetting agents, and tablets may be coated if necessary.

[0028] Suitable fillers include cellulose, mannitol, lactose or other similar fillers.Suitable disintegrants include starch, polyvinylpyrrolidone or starch derivatives, preferably sodium starch glycolate.Suitable lubricant is magnesium stearate.Suitable wetting agent is sodium lauryl sulfate.

[0029] The pharmaceutical compositions of the present application may be prepared into solid oral compositions by conventional methods of blending, filling, tableting, etc. Repeated blending may distribute the active agent throughout compositions in which large amounts of filler are used.

[0030] Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be dry products that can be mixed with water or other suitable carriers before use. These liquid preparations may contain conventional additives, such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents such as lecithin, dehydrated sorbitol monooleate or gum arabic, non-aqueous carriers [edible oils (e.g., almond oil, fractionated coconut oil), e.g., oil esters of glycerin, propylene glycol or ethanol)], preservatives such as p-hydroxybenzoic acid, p-hydroxybenzoic acid propyl or sorbic acid, and may contain conventional flavorings or colorings, if necessary.

[0031] For injections, the liquid unit dosage forms prepared contain the active substance of the present application and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. For preparing a solution, the active substance is usually dissolved in a carrier, and then filtered and sterilized before being placed in a suitable vial or ampoule, and then sealed. Auxiliary materials (e.g., local anesthetics, preservatives and buffers) can also be dissolved in such a carrier. To enhance stability, such compositions can be frozen after being placed in a vial, and water can be removed under vacuum.

[0032] When the pharmaceutical composition of the present application is prepared into a drug, a suitable pharma- ceutically acceptable carrier can be optionally added. The pharma- ceutically acceptable carrier is one or more selected from mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginates, gelatin, polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.

[0033] The pharmaceutical compositions of the present application may further comprise, in addition to the substituted cinnamic amide compounds of the present application, one or more of the following drugs for the prevention or treatment of mental disorders, such as, for example, nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracen, neurostan, and the like.

[0034] In an embodiment of the third aspect, the present application provides a method for preparing a substituted cinnamic amide compound of the present application, the substituted cinnamic amide compound is also referred to as compound M2, the method comprising: [ka] (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) is reacted with NH3 to obtain compound M2.

[0035] In some embodiments of the third aspect, in the process according to the present application, NH3 may be aqueous ammonia (NH3-H2O).

[0036] In some embodiments of the third aspect, in the process according to the present application, the reaction is carried out in an organic solvent, and the organic solvent comprises dichloromethane (DCM).

[0037] In some embodiments of the third aspect, in the process according to the present application, (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) is activated with an acyl chloride reagent, such as oxalyl chloride or thionyl chloride, and then reacted with NH3 to give compound M2.

[0038] In some embodiments of the third aspect, the process according to the present application can be carried out according to the following reaction pathway: [ka] The raw material for the reaction is (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid, which can be purchased commercially or prepared according to the methods in the prior art literature.

[0039] The substituted cinnamic amide compounds of the present invention can be prepared by the following method. (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (intermediate A) is dissolved in dichloromethane, a catalytic amount of N,N-dimethylformamide is added, oxalyl chloride is added dropwise in an ice bath, and the mixture is stirred at room temperature until the reaction of the raw materials is complete. The reaction solution is concentrated and dried, and then dichloromethane is added to dissolve the mixture, and ammonia water is added dropwise in an ice bath, and the mixture is reacted at room temperature until the reaction is complete. After the solvent is distilled off under reduced pressure, dilute hydrochloric acid is added to adjust the mixture to acidity, and the solid is precipitated. After filtration and washing, a crude product is obtained, which is purified by silica gel column chromatography to obtain compound M2.

[0040] In an embodiment of the fourth aspect, the present application provides the use of compound M2, or a solvate thereof, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition containing compound M2, or a solvate thereof, or a pharma- ceutically acceptable salt thereof, in the manufacture of an antidepressant, an anxiolytic, or an antidepressant-anxiolytic drug.

[0041] In some embodiments of the fourth aspect, the present application provides the use of compound M2 or a solvate or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition containing compound M2 or a solvate or a pharma- ceutically acceptable salt thereof, for a drug.

[0042] In a fifth embodiment, the present application provides the use of compound M2 or a solvate or a pharma- ceutically acceptable salt thereof in combination with another antidepressant or anxiolytic, or a combination of compound M2 with one or more other antidepressants or anxiolytics, in the manufacture of an antidepressant, anxiolytic, or an antidepressant-anxiolytic medicament, wherein the other antidepressant or anxiolytic, or another antidepressant or anxiolytic, may be selected from one or more of the following drugs for the prophylaxis or treatment of mental disorders: nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracen, neurostan, and the like.

[0043] In an embodiment of the sixth aspect, the present application provides a method for preventing or treating a psychiatric disorder, the method comprising administering to a patient in need thereof a compound M2 or a solvate thereof or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition containing compound M2 or a solvate thereof or a pharma- ceutically acceptable salt thereof.

[0044] In some embodiments of the sixth aspect, the prevention or treatment of a psychiatric disorder is antidepressant, anti-anxiety, or both antidepressant and anti-anxiety.

[0045] In some embodiments of the sixth aspect, the administration may be oral, by injection, or transdermal.

[0046] In some embodiments of the sixth aspect, the method for preventing or treating a psychiatric disorder further comprises combination with one or more additional antidepressants or anti-anxiety drugs, which may be selected from one or more of the following drugs for preventing or treating a psychiatric disorder: nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracen, neurostan, and the like.

[0047] Specific embodiments In order to make the objectives, technical solutions and advantages of the present application clearer, the embodiments of the present invention are described in detail below, whereby the embodiments and features in the embodiments of the present application can be arbitrarily combined with each other unless there is a conflict.

[0048] Example 1 (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (15g, 0.058mol) was dissolved in 200mL of dichloromethane, 0.1mL of catalyst N,N-dimethylformamide was added, and the mixture was stirred under an ice bath. Oxalyl chloride (18.3g, 0.144mol) was dissolved in 50mL of dichloromethane, and slowly added dropwise to the reaction system. After the addition was completed, the ice bath was turned off, and the mixture was stirred at room temperature until the reaction of the raw materials was complete. The reaction solution was concentrated and dried to obtain 16.5g of crude product. The crude product (10g, 36mmol) was taken and dissolved in 50mL of dichloromethane, and ammonia water (12.6g, 360mmol) was slowly added dropwise while stirring under an ice bath. After the addition was completed, the ice bath was turned off, and the mixture was reacted at room temperature until the reaction was complete.

[0049] Concentrated hydrochloric acid was added to the reaction system to adjust the pH to acidic, dichloromethane was removed by rotary distillation under reduced pressure, and the mixture was suction filtered to dryness to obtain 8.67 g of a white crude product, which was recrystallized from ethanol-water (volume ratio 1:2) to obtain 6.4 g of compound M2.

[0050] Experimental Example 1: Study on the effects of compound M2 and brain region functions The research objective of this study is to explain the mechanism of the antidepressant effect and brain region function of compound M2 through the following two experiments.

[0051] 1) Forced swimming test (also called the despair test, commonly used to test depressive behavior in mice) [Table 1]

[0052] 2) Brain slice electrophysiological study (used to measure the effect of compound M2 on excitatory synaptic transmission in the mPFC) Animals: Human litter of newborn BL / 6J mice (20-30 days old) Equipment: Vibratome (Leica), brain slice electrophysiology system (Axon) Brain slice electrophysiological testing method ●sEPSC experiment method: After anesthetizing newborn mice C57 from a litter, they were perfused with artificial cerebrospinal fluid (ACSF). After decapitation, the brain tissue was immediately extracted and sliced ​​with a vibratome, with the thickness of the brain slice being 300 μm. After incubating the brain slice at 28°C for 1 h, whole-cell recording was performed on mPFC pyramidal neurons using a brain slice electrophysiological system with voltage clamp (-70 mV). The baseline recording time was 6 min, after which the perfusion system was used to add the drug to be measured (the drug was dissolved in ACSF), and the recording time after drug addition was 6 to 10 min. The recorded data was analyzed using Minianalysis software, and the effects of the drug to be measured on the frequency and amplitude of sEPSCs in medial frontal lobe pyramidal neurons of C57 mice were compared. ●Data Statistics All data analysis is performed by spss data processing software, and data is shown as Mean±sem. One-way ANOVA is used to analyze the effect of compound M2 and compound M2 under the action of dopamine receptor antagonist on sEPSC current, and paired t-test is used to analyze the sEPSC experimental results. * is indicated when p<0.05.

[0053] 3) Western Blot (used to measure the effect of compound M2 on changes in mPFC protein content) ●Animals: C57 BL / 6J male mice (18-20g). Divided into four groups: control group, fluoxetine group, compound M2 (30mg / kg) group, and esketamine group. ● Primary antibodies used in the experiment [Table 2] Western Blot Experiment Method: 0.5h / 24h after administration to C57 mice, the frontal lobe brain region was sampled from the brain tissue, the tissue sample was dissolved and homogenized, and the protein was quantified by the BCA method. The changes in protein content in the frontal lobe brain region under the action of intraperitoneal administration of compound M2 for 0.5h / 24h were measured through the processes of SDS-PAGE gel preparation, application / electrophoresis, membrane penetration, blocking, primary antibody incubation, secondary antibody incubation, protein measurement (development / fixation), etc.

[0054] 3) Experimental results 1. Compound M2 has antidepressant-like effects As shown in FIG. 1, after 24 h administration, compound M2 significantly reduced the immobility time of mice, exhibiting obvious dose effect, and has significant effect when administered at a dose of 30 mg / kg. Compound M2 has a fast onset of antidepressant effect. Two positive compounds were used as controls in the experiment, of which fluoxetine, the first-choice clinical drug, has a relatively slow onset of effect, while esketamine has a fast onset of effect. The results of this experiment showed that after 24 h of single administration, fluoxetine had no antidepressant-like effect, while compound M2 and esketamine had antidepressant-like effect, indicating that compound M2 has the potential for fast antidepressant effect.

[0055] 2. Compound M2 significantly enhances excitatory synaptic transmission in the medial prefrontal cortex (mPFC) During the experiment, 10 μM Bicuculline was added to the solution to block GABA receptors, and sEPSCs of pyramidal neurons were recorded. As shown in Figure 2, the experiment examines the effects of different concentrations of compound M2 on spontaneous excitatory postsynaptic potentials (sEPSCs) of pyramidal neurons in the PrL brain region of the frontal lobe. As shown in Figure 3, the results show that M2 increases the firing frequency of sEPSCs in the PrL brain region of the frontal lobe in a concentration-dependent manner, but the effect on the firing amplitude of sEPSCs is not clear. The increase in sEPSC firing frequency indicates that compound M2 can enhance the release of excitatory glutamate neurotransmitters in frontal lobe synapses. Based on the target action characteristics of compound M2, this increase may be due to compound M2 enhancing the synaptic transmission of monoamine neurotransmitters released from the midbrain to the frontal lobe, or it may be due to a direct effect on frontal lobe monoamine receptors. Frontal lobe excitatory synaptic transmission is closely involved in antidepressant effects, and novel antidepressants such as ketamine can enhance frontal lobe excitatory synaptic transmission.

[0056] 3. Compound M2 enhances excitatory synaptic transmission in the frontal lobe via dopamine D2R receptors 1) Dopamine D2R receptors inhibit the enhancing effect of compound M2 on frontal sEPSCs In this experiment, we investigate whether frontal lobe dopamine receptors affect the action of compound M2 on the frontal lobe. In the experiment, we use the dopamine receptor D2R antagonist Sulpride to inhibit D2R receptors. The results show that inhibition of dopamine D2R receptors can significantly inhibit the enhancing effect of compound M2 on sEPSC firing frequency, indicating that D2R receptors in the frontal lobe may mediate excitatory synaptic transmission by M2 (shown in Figure 4).

[0057] 2) Dopamine D1R receptors do not affect the enhancing effect of compound M2 on excitatory synaptic transmission in the frontal lobe Using the dopamine D1R receptor antagonist SCH23390, it was shown that inhibition of dopamine D1R receptors could not inhibit the enhancing effect of compound M2 on sEPSC firing frequency, indicating that D1R receptors in the frontal lobe may not be involved in excitatory synaptic transmission by compound M2 (shown in Figure 5).

[0058] 4. Compound M2 rapidly activates prefrontal synapses and generates related signal pathways In this experiment, we investigated the possible intracellular signaling pathway through which compound M2 exerts its antidepressant effect. Because activation of the mTOR signaling pathway plays an important role in fast antidepressant action, this study investigated the changes in related proteins upstream and downstream of the mTOR signaling pathway. The results showed that 0.5 hours after administration of compound M2 (Figure 6a), the content of p-mTOR and p-TrkB in the medial frontal lobe could be increased, but the total content did not change. Literature has shown that the mTOR signaling pathway is related to the mechanism of fast depressive disease, and the fast antidepressant effect of ketamine may be related to the increase in phosphorylated mTOR content and the activation of TrkB protein (BDNF receptor). The results showed that the antidepressant effect of compound M2 may also be related to the activation of mTOR and TrkB proteins.

[0059] After the mTOR signal pathway was activated, the expression level of synapse-related proteins was further increased, and Figure 6b showed that after a single administration for 24 hours, compound M2 significantly increased the content of the related protein PSD95 formed in the medial frontal lobe synapse. This result is consistent with the function of compound M2 in enhancing frontal lobe synaptic transmission found by electrophysiological tests. PSD95 is a postsynaptic density protein and is involved in neuroplasticity. Research literature (Shinohara R, Aghajanian GK, Abdallah CG. Neurobiology of the Rapid Acting Antidepressant Effects of Ketamine: Impact and Opportunities[J]. Biological Psychiatry, 2020.) showed that ketamine's antidepressant and anxiolytic effects are associated with an increase in the expression level of PSD95 protein.

[0060] Previous research (Pizzagalli DA, Roberts AC. Correction: Prefrontal cortex and depression [J]. Neuropsychopharmacology, 2021:1-1.) has shown that morphological characteristics of frontal lobe atrophy may appear in clinical depression patients, and basic experimental research has shown that chronic social stress model mice have a decreased neuronal projection density in the fifth layer of the frontal lobe, a decreased frequency of neuronal excitatory postsynaptic currents (EPSCs), and a decrease in the frequency of EPSCs reduces the synaptic transmission of frontal lobe excitatory neurotransmitters. Frontal lobe excitatory synaptic transmission is closely related to antidepressant and anxiolytic effects, and ketamine, fluoxetine, and other drugs can enhance frontal lobe excitatory synaptic transmission. Therefore, in this experiment, we combine the pharmacological properties of compound M2 to mainly investigate the antidepressant and anxiolytic effects of compound M2, and investigate the effects of the compound on the brain excitatory neurotransmitter glutamatergic system. As a result of conducting frontal lobe brain slice experiments similar to physiological conditions, it was found that compound M2 can promote the release of glutamate neurotransmitter and enhance the function of glutamate receptors, and it was shown that compound M2 enhances the excitability of the frontal lobe and exhibits a concentration effect.

[0061] The above results show that compound M2 mainly acts on the glutamatergic neurotransmitter system, and enhances excitatory glutamatergic synaptic transmission, thereby improving the excitability of the frontal lobe and exerting a fast antidepressant drug effect. Compound M2 also quickly activates the frontal lobe mTOR signal pathway. The results show that compound M2 has a fast antidepressant effect and a novel antidepressant mechanism of enhancing synaptic transmission and brain excitability, and exhibits better antidepressant potential than clinical monoamine drugs.

[0062] Experimental Example 2: Inhibitory effect of compound I-5 and compound M2 on monoamine reuptake in rat brain synaptosomes From the current viewpoint, absolute or relative deficiency of central monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NA) and dopamine (DA) are closely related to depression and anxiety. In this experiment, the inhibitory effects of two test compounds, test compound I-5 and compound M2, on the reuptake of 5-HT, NA and DA in rat brain synaptosomes were evaluated by in vitro monoamine reuptake method. The IC values ​​of the two test compounds, which inhibit the reuptake of monoamines DA, 5-HT and NA, respectively, were determined by preparing isolated SD rat brain synaptosomes and using the isotope tracer method. 50 To investigate the possible antidepressant and anxiolytic mechanisms of action of compound I-5 and compound M2.

[0063] 1. Experimental materials 1. Experimental animals: SD rats, male, weight 200-220g

[0064] 2. Test specimens: As can be seen from the experimental sections of the specifications of Patents A and B, among all the compounds, compound I-5 has good results in all the experiments in each section, so compound I-5 is selected as a control to compare the inhibitory effect of compound M2 on rat brain synaptosomal monoamine reuptake. [Table 3]

[0065] 3. Mixing of test substance and positive control substance, etc. 3.1. Test substance composition Test substances are weighed (allowable weighing error ±1%), dissolved in DMSO, prepared into 10 mM stock solutions and stored at -20°C. [Table 4] Before the experiment, dilute the solution with Kreb's solution by doubling dilution method to obtain a 100-fold working solution required for the detection concentration (1 nM to 100 μM). Conversion formula:

number

[0066] 3.2. Positive control substance combination The control substance is weighed (allowable weighing error ±1%), dissolved in double distilled water, prepared as a 10 mM stock solution and stored at -20°C. [Table 5]

[0067] II. Experimental Method 1. Preparation of Brain Synaptosomes Synaptosomes are isolated by adopting the method described in the literature to prepare brain synaptosomes. The head of SD rats is rapidly cut with a small animal decapitator, then the brain is rapidly removed and pre-cooled in ice-water mixture, and the pia mater and vascular tissue are removed. The brain tissue is removed. 10 volumes (ml / g) of 10 mM Tris HCl buffer (containing 0.32 M sucrose, pH 7.4) are added, and the brain tissue is homogenized with a Cell Disruptor ultrasonic homogenizer (maintaining a low temperature), and the brain tissue is homogenized and centrifuged for 10 minutes (4°C, 1,000g). Equilibrium centrifugation is performed at 4°C (1500g, 10min), the precipitate is removed, and then the supernatant is taken and centrifuged again for 30 minutes (20000g). The supernatant is removed, and the precipitate, i.e., the crude extract of synaptic heads, is retained. The precipitate was further suspended in 0.32M cold sucrose solution, then carefully placed on 1.2M and 0.8M (10ml each) cold sucrose gradient solutions laid in order from the bottom of the tube, and centrifuged at equilibrium for 60 minutes at 4°C (38000g). The suspension zone at the 0.8-1.2M sucrose interface was carefully collected with a puncture needle, added to 10ml of 0.32M cold sucrose solution, mixed uniformly, and centrifuged at equilibrium for 30 minutes at 4°C (20000g). The precipitate was purified brain synaptosomes. The precipitate was suspended in a small amount of Kreb's buffer (NaCl 118 mM, KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, and glucose 11.1 mM, pH 7.2-7.4), and protein quantification was performed by the BCA method, following the operating steps in the manufacturer's instructions.

[0068] 2. Monoamine reuptake With reference to the relevant methods in the literature, the inventor's laboratory optimized the experimental method, which is as follows: Add 950 μl of pre-cooled Kreb's buffer to the reaction tube, then add 30 μl of the suspension of synaptic heads, and then add 10 μl of the compound to be measured (operated on ice), mix uniformly with a turbine, and perform a water bath at 37°C for 5 minutes. Take out the reaction tube and place it on ice, add 10 μl of substrate ( 3 H-DA or 3 H-5HT or 3H-NA, final reaction concentration: 10 nM) is added, mixed homogeneously with a turbine, and a water bath is performed at 37 °C for 5 min. Then, the reaction tube is removed and quickly placed on ice, and 3 ml of pre-cooled Kreb's buffer is added to stop the reaction, collected with a Millipore cell sample collector, quickly filtered by suction through a GF / C glass fiber filter membrane, eluted three times with 3 ml of elution solution (50 mM Tris-HCl, PH 7.4), removed the filter membrane, dried in a microwave oven for 5 to 6 min, transferred the filter membrane to a 1.5 ml centrifuge tube, and 500 μl of lipid-soluble scintillator solution is added. The mixture is left to stand for 30 min or more away from light, and counted to measure the radioactivity (cpm value).

[0069] The inhibition rate of each compound to isotopic ligand binding is calculated using the following formula. Inhibition rate (I%) = (total binding cpm - compound cpm) / (total binding cpm - non-specific binding cpm) x 100% Experiments are performed with triplicate compounds each time, with three separate replicates.

[0070] 3. Experimental results 1. IC 50 Calculation of 1) The detection compound concentration is X and the inhibition rate is Y. 2) Take the logarithm of X, X' = Log(X), to generate new data. 3) Perform nonlinear curve fitting on the new data, and after fitting, substitute the following equation to obtain IC 50 The values ​​are calculated. The above steps are fitted using the software Graphpad Prism. 4)

number

[0071] 2. Summary of the experiment Monoamine reuptake inhibition is one of the main targets of antidepressant and anxiolytic drugs. The test substances, Compound I-5 and Compound M2, had different inhibitory effects on the reuptake of DA, 5-HT, and NA in rat brain synaptosomes, while Compound I-5 and Compound M2 had similar inhibitory effects, indicating that Compound I-5 and Compound M2 exert their antidepressant and anxiolytic effects by inhibiting the reuptake of DA, 5-HT, and NA. IC of compound I-5 on the reuptake of DA, 5-HT and NA in rat brain synaptosomes 50 The values ​​are 26.02±1.33nM, 18.52±2.47nM, and 17.21±1.59nM, respectively. IC of compound M2 on the reuptake of DA, 5-HT and NA in rat brain synaptosomes 50 The values ​​are 37.28±1.97nM, 98.87±2.42nM, and 23.54±3.56nM, respectively.

[0072] Experimental Example 3: Study of the antidepressant effect of compound I-5 and compound M2 The "behavioral despair" model was developed by Porsolt et al. in 1977 and includes the rat and mouse forced swimming model and the mouse tail suspension model, which belong to the acute stress model. The mouse tail suspension test is a simple experimental method for evaluating antidepressants introduced by Stern et al. in 1985, and its principle is the same as the forced swimming "immobility" experiment. The suspended mouse struggles to overcome the abnormal body posture, but after a certain period of movement, the animal expresses intermittent immobility due to "despair." In this experiment, we observed whether the pending samples, compound I-5 and compound M2, affected the tail suspension behavior of mice in the "behavioral despair" experiment, whether they could shorten the immobility time of mice, and also compared the fast onset effects of compound I-5 and compound M2.

[0073] 1. Experimental materials 1. Test specimen 1 Name or code: Compound I-5 Test substance number: YLS-2021-CMI1203-002 Batch number: C16101006-C17001M Blending method: The solvent is Tween 80 + water. The final volume is thoroughly emulsified with 1.5% Tween 80, and water is added until the final volume is reached. After blending, the temporary storage conditions are a 4°C refrigerator.

[0074] 2. Test specimen 2 Name or code: Compound M2 Test substance number: YLS-2021-CMI1203-01-001 Batch number: 20201029 Blending method: The solvent is Tween 80 + water. The final volume is thoroughly emulsified with 1.5% Tween 80, and water is added until the final volume is reached. After blending, the temporary storage conditions are a 4°C refrigerator.

[0075] II. Experimental animals C57 BL / 6 mice, 40 mice, SPF grade, male, 4-5 weeks old.

[0076] III. Experimental Method 1. Animal grouping and dosage design basis 40 mice were divided into 4 groups according to Excel complete random grouping method, each group had 10 mice. As shown in the results of the preliminary experiment, after 24 h administration, compound M2 can dose-dependently reduce the immobility time of mice in the forced swimming test, and has a significant effect when administered at a dose of 30 mg / kg. Therefore, in this experiment, 30 mg / kg is the low dose of compound M2, and 60 mg / kg is the high dose of compound M2, and the study is conducted in comparison with 60 mg / kg of compound I-5. For details on grouping and administration, please refer to Table 1.

[0077] [Table 7]

[0078] 2. Dosage form and administration time Each test group was administered the drug solution by oral gavage (ig) at a dose of 0.2 ml / 10 g body weight.

[0079] 3. Indicator detection Thirty minutes after the single oral administration, a tail suspension test is performed on the mice, and the duration of the tail suspension is 6 minutes. The time during which the mice remain motionless for 6 minutes is recorded.

[0080] 4, statistical methods Data are shown as mean ± SD. Data difference statistics were performed using one-way analysis of variance (ANOVA) or nonparametric tests, and differences between groups were judged at P<0.05.

[0081] 4. Experimental results As shown in the results of single-dose test in mice, after 30 min of single administration of 60mg / kg compound M2, the time of immobility of mouse tail suspension can be significantly shortened, p<0.05 compared with blank group. After 30 min of single administration of 30mg / kg compound M2, the time of immobility of mouse tail suspension showed a certain tendency to decrease, but no statistically significant difference was shown. After 30 min of single administration of 60mg / kg compound I-5, the time of immobility of mouse tail suspension had no obvious effect (see Table 2). The above results show that compound M2 has a relatively fast onset of antidepressant effect, compound M2 has a significant antidepressant effect, and the onset of antidepressant effect is relatively fast, and is obviously superior to compound I-5 in terms of fast onset.

[0082] [Table 8]

[0083] Experimental Example 4: Absolute bioavailability study of Compound I-5 and Compound M2 in rats 1. Purpose of the experiment Through the experiments, an LC-MS / MS method was established to measure compound I-5 and compound M2 in rat plasma, and the rat pharmacokinetic characteristics and absolute bioavailability of the compounds were investigated.

[0084] 2. Experimental materials 2.1. Experimental animals Twenty SPF SD male rats (body weight 220±20 g) were purchased from Beijing Vital River. 2.2 Test substance Compound I-5 drug substance, batch number C16101006-C(20170814)M, content 99.6%, provided by TIANJIN TASLY PHARMACEUTICAL CO.,LTD. Compound M2 drug substance, batch number 20201029, content 99.8%, provided by TIANJIN TASLY PHARMACEUTICAL CO.,LTD.

[0085] 3. Experimental method 3.1. Test substance composition and dosage [Table 9]

[0086] 3.2, Measurement method 3.2.1. LC-MS conditions for compound M2 (normal phase) 3.2.1.1 Liquid-phase conditions Column: Waters BEH C 18 1.7μm; 2.1×100mm Column Internal standard: DI-5 (deuterium compound I-5, structural formula is as follows) [ka] Mobile phase: 0.1% formic acid (A), acetonitrile (B), isocratic elution (A / B: 58:42 v / v) Flow rate: 0.4 ml / min, column temperature: 40°C, injection volume: 4 μL

[0087] 3.2.1.2 Mass spectrum conditions Ion detection method: Selected reaction monitoring (MRM) Ionization method: Electrospray ionization (ESI) Ion polarity: positive ion [Table 10]

[0088] 3.3 Pharmacokinetic experiments Twenty SD rats were randomly divided into four groups, five in each group. They were fasted for 10 h before administration and allowed to drink water ad libitum. 0.3 mL of blood was collected from the eye vein at 0 h and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h after administration. The blood was placed in an EDTA-K2 anticoagulant tube, gently shaken to mix thoroughly and homogeneously with the anticoagulant, placed on wet ice, and centrifuged within 0.5 h. Centrifugation: After centrifugation at 8000 rpm for 5 minutes, plasma is separated. Storage: Store frozen at -20℃.

[0089] 3.4. Plasma sample processing method Take 50μL of plasma, add 50μL of DI-5 (dissolved in 20ng / mL acetonitrile), spin the tube for 30 seconds, add 100μL of acetonitrile, spin the tube thoroughly for 2 minutes, centrifuge at 4℃ 12000r / min for 3 minutes, take 50μL of the supernatant, add 50uL of aqueous solution to dilute, spin the tube for 1 minute, and then inject 4μL.

[0090] 4. Pharmacokinetic analysis The mean blood concentration-time curves after oral gavage and intravenous administration to rats, respectively, are shown in Figures 8 and 9. The DAS3.0 pharmacokinetic software (China Professional Committee of Mathematical Pharmacology, Shanghai, China) was used to process the data and calculate the non-compartment model statistical moment parameters. C max and T max are measured values. For details on blood concentration data and pharmacokinetic parameters, see the table below.

[0091] [Table 11]

[0092] [Table 12]

[0093] [Table 13]

[0094] [Table 14]

[0095] [Table 15]

[0096] [Table 16]

[0097] 5. Consideration After oral and intravenous administration of compound M2 and compound I-5 to rats, the pharmacokinetic study showed that the absorption peak of compound M2 and compound I-5 was relatively fast after oral gavage, with the peak absorption time t max is 0.35h, and C of compound M2 max was 425.4 ± 136.9 ng / mL, and the C max The AUC of Compound M2 was 361.1±258.2 ng / mL, and Compound M2 had a relatively high blood concentration. 0-t was 1080.1 ± 373.4 ng.h / mL, and the AUC 0-t is 523.3±433.3ng.h / mL. At the same gavage dose, compound M2 exhibits higher exposure and has higher absolute bioavailability in rats compared to compound I-5, 40.8% and 24.05%, respectively.

[0098] Experimental Example 5: Study of the anxiolytic effects of M2 and its structural analogues 1. Experimental materials 1. Test specimen As can be seen from the results of two experiments (elevated plus maze test in mice and Vogel-type conflict test in rats) in the specification of Patent C, the compounds with relatively good effects in the two experiments are II-3, II-4, II-5, and II-10, so these four compounds are selected as controls to evaluate the anxiolytic effect of compound M2 of the present application.

[0099] (1) Name or code: Compound M2 [ka] Batch number: 20201029 Test substance number: YLS-2021-CMI1203-01-001 Origin: TASLY Institute Chemical Drug Development Center

[0100] (2) Name or code: Compound II-3 (i.e., Compound I-3 in Patents A and B) [ka] Batch number: 20180521 Test substance number: YSL-2021-CMI1203-01-023 Origin: TASLY Institute Chemical Drug Development Center

[0101] (3) Name or code: Compound II-4 (i.e., Compound I-4 in Patents A and B) [ka] Batch number: 20211217 Test substance number: YSL-2022-CMI1203-01-001 Origin: TASLY Institute Chemical Drug Development Center

[0102] (4) Name or code: Compound II-5 (i.e., Compound I-5 in Patents A and B) [ka] Batch number: C16101006-C17001M Test substance number: YSL-2021-CMI1203-01-002 Origin: TASLY Institute Chemical Drug Development Center

[0103] (5) Name or code: Compound II-10 (i.e., Compound I-10 in Patents A and B) [ka] Batch number: 20170601 Test substance number: YSL-2021-CMI1203-01-023 Origin: TASLY Institute Chemical Drug Development Center

[0104] 2. Positive control substance Name or code: Estazolam tablets Batch number: 211007 Test substance number: YSL-2021-CMI1203-01-022 Origin: Shandong Xinyi Pharmaceutical Co., Ltd.

[0105] 3. Main equipment [Table 17]

[0106] II. Experimental animals 2.1. Experimental animals (1) Species: ICR mice (for elevated plus maze tests) Quantity: 120 animals Grade: SPF grade Gender: Male Weight: 18~20g Animal certificate number: 110011211113772653, 110011221101333628 Origin: Beijing Vital River Laboratory Animal Technology Co., Ltd. Production license number: SCXK(Kyoto)2021-0006

[0107] (2) Species: SD rats (for Vogel-type conflict tests) Quantity: 75 animals Grade: SPF grade Gender: Male Weight: 180-200g Animal certificate number: 110011221102490324 Origin: Beijing Vital River Laboratory Animal Technology Co., Ltd. Production license number: SCXK(Kyoto)2021-0011

[0108] 2.2 Animal facilities Breeding facility: Tasly Holding Group Co. LTD Tianjin Animal Facility, inside a barrier environment Facility address: Tianjin Beichen Science and Technology Park, Huaihe Road and Tingjiang West Road intersection, factory area Experimental animal use permit: SYXK(Tsu)2017-0007 Issued by: Tianjin Municipal Science and Technology Commission

[0109] 2.3 Animal care and management Breeding environment: Barrier environment. The environmental conditions of this facility meet the standards for barrier animal experimental facilities in the Chinese national standard "Experimental Animal Environment and Facilities" (GB14925-2001). Animal breeding and animal experimental procedures meet the regulations of the "Tianjin City Experimental Animal Management Regulations" and other regulations. Temperature: 20-26°C, humidity: 40%-70%, lighting: 12 hours light, 12 hours dark, ventilation: total fresh air ≥15 times / hour, animals drink sterile water (grade 4 filtration, UV sterilization) prepared by a 1T / h type multi-micropore filter membrane filtration system. Animal management is the responsibility of the Animal Protection Department. In addition to fasting, sufficient feed and drinking water should be provided to animals every day. The water bottle should be replaced once a day. The bedding for animal breeding should be replaced twice a week and replaced as needed in special cases. The breeding cage should be replaced once a week. Animal feed is purchased from Beijing Keao Xieli Feed Co., Ltd., and the production license number is SCXK(Beijing)2019-0003.

[0110] 2.4, Receiving and Quarantining of Animals After the experimental animals arrive, the experimenters, veterinarians and the Animal Protection Department will receive them jointly. When receiving, first check whether the transportation tool meets the regulations, and then check the animal certificate provided by the animal supplier to confirm the consistency between the content of the certificate and the species, grade, quantity, and gender of the animals applied for purchase. Then, check whether the package meets the regulations and whether there is any damage to the animal package. The animal package will be transported to the quarantine room via the first transport cabinet. The test supplies and experimental records will be transported via the second transport cabinet. Open the animal package in the quarantine room and check whether the gender and quantity of the animals match the items described in the animal certificate. Check the appearance of the animals (including gender, weight, head, body, tail, limbs, fur, spirit, activity, etc.) one by one, and fill in the "Experimental Animal Receiving Record" and "Experimental Animal Quarantine Record". After checking the animals, place them in the animal breeding cage, hang the quarantine period label in the cage, and then conduct adaptive breeding in the quarantine room. The adaptive breeding period of the animals is 2-3 days. Observe the animals (including weight, head, body, tail, limbs, fur, spirit, activity, etc.) regularly.

[0111] III. Experimental Methods 3.1, Exploratory Study on the Single-Dose Administration of Compound M2 (Elevated Plus Maze Test) (1) Animal Grouping and Administration Fifty male ICR mice were randomly divided into five groups (solvent control group, estazolam group, M2 high dose group, M2 medium dose group, and M2 low dose group), with 10 mice per group. Compound M2 was administered orally at 20 mg / kg, 10 mg / kg, and 5 mg / kg in the high, medium, and low dose groups, respectively, and the positive drug estazolam was administered orally at 2.5 mg / kg, while the solvent control group was administered an equivalent volume of solvent orally. Each group was subjected to behavioral testing 30 min after a single administration. The experiment was conducted between 8:00 and 14:00 in the morning, and all animals were placed in the testing laboratory the day before. Dose design basis: As shown in the results of the early anti-anxiety experiment (patent C), the structural analogues of compound M2, such as compounds II-3, II-4, and II-5, have an effective anti-anxiety dose of 10mg / kg in the mouse elevated plus maze test. In this experiment, 10mg / kg is the medium dose of compound M2, and the dose is lowered by 5mg / kg and raised by 20mg / kg, respectively. The clinical dose of estazolam, an anti-anxiety positive drug, is 6mg / person / day, which is converted into a mouse clinical effect equivalent dose of 6mg / 60kg*12.3=1.23mg / kg, and the double clinical dose is 2.5mg / kg, and the rat clinical effect equivalent dose is 6mg / 60kg*6.2=0.62mg / kg, and the double clinical dose is 1.24mg / kg.

[0112] [Table 18]

[0113] (2) Conduct test In the laboratory, the light is dim (based on the minimum brightness that can distinguish the mouse's slight movements at a distance of 1.5m), the light is constant, the room temperature is about 20℃, and quiet is maintained. Before the maze test, each mouse is placed in a 35cm*10cm*5cm plastic box and allowed to explore freely. After 5min, it is quickly placed on the central platform of the elevated plus maze, its head is directed toward one of the open arms, and the following indicators are recorded after it is released. Each mouse is tested for 5min. The observer observes and records the activity of the animals at a distance of 1.5m. In the middle, the maze is wiped with a wet cloth, feces is removed, and then it is wiped with a dry cloth before the next mouse is tested.

[0114] (3) Behavioral Observation Indicators (1) Open arm entry (OE): The number of entries into any of the open arms. The entry of all four paws of the mouse into the arm was defined as the criterion. Complete withdrawal of one paw from the arm during the course of the entry activity indicated the completion of this session of entry activity. (2) Open arm time (OT): Time required to enter an open arm, measured in seconds. (3) Number of entries into closed arms (CE): The number of entries into any of the closed arms, defined as the number of entries into the arm with all four paws of the mouse. (4) Closed arm time (CT): Time to enter a closed arm, measured in seconds. (5) Percentage of open arm time: OT% = OT / (OT+CT)*100% (6) Percentage of open arm counts: OE% = OE / (OE+CE)*100%

[0115] 3.2. Comparative study of single-dose drug effects of compound M2 and structural analogs (elevated plus maze test) (1) Animal grouping and dosing Seventy male ICR mice were randomly divided into seven groups (solvent control group, estazolam group, compound M2 group, compound II-3 group, compound II-4 group, compound II-5 group, and compound II-10 group), with 10 mice per group. Among them, the dose of M2 and its four structural analogs was set to 20 mg / kg based on the experimental results of 3.1, the positive drug estazolam was administered orally at 2.5 mg / kg, and an equivalent volume of solvent was administered orally forcibly in the solvent control group. In each group, a behavioral test was performed 30 min after a single administration. The experiment was performed between 8:00 and 14:00 in the morning, and all animals entered the testing laboratory the day before.

[0116] [Table 19] The behavioral test method and behavioral observation index are the same as in 3.1.

[0117] 3.3. Comparative study of single-dose drug effects of compound M2 and structural analogs (Vogel-type conflict test) (1) Animal grouping and dosing 75 male SD rats weighing 180-220g were adaptively raised for one week, and then subjected to a training test with 24h water deprivation. Eligible animals were selected and randomly divided into seven groups (solvent control group, estazolam group, compound M2 group, compound II-3 group, compound II-4 group, compound II-5 group, compound II-10 group) according to weight, with eight animals per group. Among them, the dose of compound M2 and its four structural analogs was set to 10mg / kg based on the experimental results of 3.1, and the positive drug estazolam was administered orally at 1.24mg / kg, and the solvent control group was administered an equivalent volume of solvent orally. The animals divided into groups were then deprived of water for 24h, administered a single dose, and subjected to a punishment experiment test 30min after the single dose.

[0118] [Table 20]

[0119] The experiment is carried out in two stages. The first stage is the training stage, in which the animals are individually placed in the operation box after 24 hours of water deprivation, and are allowed to explore until they find the mouth of the bottle and begin to lick the water (electric shock intensity 0 mA). The number of times the animals lick the water within 3 minutes is automatically recorded by a counter, and animals that lick the water less than 300 times are eliminated. The second stage is the punishment stage, in which the animals that have not been eliminated are administered after 24 hours of water deprivation, and are placed in the operation box again individually. The counter automatically starts counting from the animal's 20th water lick and delivers one electric shock (the ratio of water licks to electric shocks is 20:1). The electric shock intensity is generally 0.3 mA and is maintained for 2 s, but the shock can be released by the animal's removal from the mouth of the bottle. The number of times the animals lick the water and the number of electric shocks are recorded for 3 minutes. Observation index: number of times the rat licked water during the punishment period.

[0120] 3.4, Statistical methods Data are shown as mean ± SD. Data difference statistics were performed using one-way analysis of variance (ANOVA) or nonparametric tests, and differences between groups were judged at P < 0.05.

[0121] 4. Experimental results 4.1. Exploratory study of single dose of compound M2 (elevated plus maze test) As shown in the results of the single dose exploratory study of compound M2, single oral gavage administration of high (20mg / kg), medium (10mg / kg), and low doses (5mg / kg) of compound M2 can increase the number of mouse entries into the open arms and extend the time spent in the open arms in the elevated plus maze test to different degrees, with the high dose group of compound M2 showing significant differences compared to the vehicle control group (P<0.05, P<0.01). As shown by the above results, a single administration of compound M2 has significant anxiolytic activity, and the effective single administration dose is 20 mg / kg.

[0122] [Table 21]

[0123] 4.2. Comparative study of single-dose drug effects of compound M2 and structural analogs (elevated plus maze test) As shown by the research results of elevated plus maze test, compound M2 and its structural analogues compounds II-3, II-4, II-5, II-10 can be administered by single forced oral administration at 20mg / kg to different extents to increase the number of mouse's entry into open arms in elevated plus maze test, and extend the time spent in open arms.Compound M2, compound II-4, compound II-5, compound II-10 group have significant difference compared with solvent control group (P<0.05, P<0.01).Compound M2 and its structural analogues II-4, II-5, II-10 have significant anxiolytic activity in this model, and compound M2 has the best anxiolytic activity compared with II-3, II-4, II-5, II-10.

[0124] [Table 22]

[0125] 4.3. Comparative study of single-dose drug effects of compound M2 and structural analogs (Vogel-type conflict test) As shown by the results of Vogel-type conflict test, compound M2 and its structural analogues II-3, II-4, II-5, II-10 can be administered by single forced oral administration at 20mg / kg to different extents to increase the number of rats drinking water in Vogel-type conflict test.Compound M2, compound II-5, compound II-10 group have significant difference compared with solvent control group (P<0.05).Compound M2 and its structural analogues II-5, II-10 have significant anxiolytic activity in this model, and compound M2 has the best anxiolytic activity compared with II-3, II-4, II-5, II-10.

[0126] [Table 23]

[0127] 5. Experimental Conclusion In summary, compound M2 has significant anxiolytic activity, with a single effective dose of 20 mg / kg (in mice), and its anxiolytic activity is optimal compared to its structural analogues, compounds II-3, II-4, II-5, and II-10.

Claims

1. The structural formula: 【Chemistry 1】 A substituted cinnamic amide compound represented by the formula:

2. The structural formula: 【Chemistry 2】 An antidepressant and anti-anxiety compound represented by the formula:

3. A compound according to claim 1 or 2, characterized in that it exists in the form of a solvate.

4. A compound according to claim 1 or 2, characterized in that it exists in the form of a pharma- ceutically acceptable salt.

5. A pharmaceutical composition comprising the compound of claim 1.

6. The pharmaceutical composition according to claim 5, which is in any administrable pharmaceutical form, optionally selected from the group consisting of tablets, capsules, oral liquids, buccal tablets, granules, pills, powders, plasters, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops and patches, wherein the tablets are optionally sugar-coated tablets, film-coated tablets or enteric-coated tablets, the capsules are optionally hard capsules or soft capsules, and the injections are optionally one of injections, freeze-dried powder injections and liquid injections.

7. The pharmaceutical composition of claim 5, which is used in combination with another antidepressant or anti-anxiety drug, or which further comprises another antidepressant or anti-anxiety drug, and which is selected from nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracen, and neurostan. 【Request 8】 【Chemical 3】 (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) and NH 3 and reacting the compound M2 with the compound M1 to obtain a compound M2.

9. The reaction pathway below: 【Chemistry 4】 A method for producing the compound of claim 8, comprising:

10. The process according to claim 9, which is a process of dissolving (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (intermediate A) in dichloromethane, adding catalytic amount of N,N-dimethylformamide, adding oxalyl chloride dropwise under ice bath, stirring at room temperature until the reaction of the raw material is complete, concentrating and drying the reaction solution, adding dichloromethane to dissolve, adding ammonia water dropwise under ice bath, reacting at room temperature until the reaction is completed, distilling off the solvent under reduced pressure, adding dilute hydrochloric acid to adjust the acidity, precipitating a solid, filtering and washing to obtain a crude product, and purifying the product by silica gel column chromatography to obtain M2.

11. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 5 to 7 for a drug.

12. Use of the compound according to claim 1 or 2, or the pharmaceutical composition according to any one of claims 5 to 7, in the manufacture of an antidepressant, an anxiolytic, or an antidepressant / anxiolytic drug.

13. 8. A combination of a compound according to claim 1 or 2 or a pharmaceutical composition according to any one of claims 5 to 7 with another antidepressant or anxiolytic, or a combination of a compound according to claim 1 or 2 with one or more other antidepressants or anxiolytics in the manufacture of an antidepressant, anxiolytic, or an antidepressant-anxiolytic, wherein the other antidepressant or anxiolytic, or the other antidepressant or anxiolytic may be selected from one or more of the following drugs for the prevention and treatment of mental disorders: nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracen, neurostan.

14. A method for the prevention or treatment of a psychiatric disorder comprising administering to a patient in need thereof a compound according to claim 1 or 2, or a pharmaceutical composition according to any one of claims 5 to 7.

15. 15. The method of claim 14, wherein the prevention or treatment of a psychiatric disorder is antidepressant, anti-anxiety, or antidepressant-anti-anxiety.

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