Antidepressant and anti-anxiety substituted cinnamamide compounds

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 the Chinese invention patent application filed on 28 May 2021, titled "Novel Compounds for Antidepressant and Anxiolytic Effects," with application number 202110606431.2, the entire contents of said Chinese patent application are incorporated herein by reference.

[0002] This application relates to pharmaceutical compounds, but is not limited thereto, and more particularly to novel substituted cinnamamide compounds for antidepressant and anxiolytic effects. [Background technology]

[0003] Depression and anxiety disorders are major illnesses that harm people's mental health, and the number of people suffering from them is increasing year by year as the pace of modern life accelerates. Recent statistics show that the number of people suffering from various types of depression in China has reached 26 million, with a relatively large proportion of them being adolescents. For this reason, research and development of antidepressants and anti-anxiety drugs has significant economic and social benefits.

[0004] Extensive research has shown that alterations in central monoamine neurotransmitters, dopamine, cholinergic activity, corresponding receptor function changes, and neuroendocrine dysfunction may play a significant role in the onset and progression of the disease. Therefore, the treatment principle should focus on regulating the hypothalamic monoamine neurotransmitter content and their receptor function, as well as restoring normal neuroendocrine function.

[0005] Currently, the primary treatment for depression and anxiety disorders remains medication. Traditionally, antidepressants and anxiolytics used clinically have mainly been chemical drugs such as tricyclic antidepressants, benzodiazepines, 5-hydroxytryptamine reuptake inhibitors, and monoamine oxidase inhibitors. While these drugs have some therapeutic effect on depression and anxiety disorders, they also have drawbacks, such as relatively high toxicity and side effects. Monoamine oxidase inhibitors, in particular, cause toxic liver damage due to their selectivity and irreversible inhibition of the enzyme, thus exhibiting some toxicity and side effects. Common tricyclic antidepressants include doxepin, amitriptyline, and clomipramine. These drugs are relatively effective against endogenous depression, achieving a therapeutic effect of over 80% in cases of despondency, loss of interest, and pessimism, but they have strong cardiotoxicity, resulting in a relatively high incidence of adverse reactions. Selective 5-HT reuptake inhibitors (SSRIs) are novel antidepressants and anxiolytics that emerged in the late 1980s. They maintain the antidepressant and anxiolytic effects of conventional drugs while significantly reducing adverse reactions to other receptors, and have become the standard first-line treatment in Western countries. Examples of commonly used SSRIs include fluoxetine, paroxetine, sertraline, citalopram, and fluvoxamine. Due to gastrointestinal absorption and hepatic metabolism, gastrointestinal dysfunction and sexual dysfunction can occur, affecting long-term use to some extent. Furthermore, conventional antidepressants and anxiolytics have a slow onset of action (6-8 weeks or more), and are only effective in about 30% of patients. Therefore, in the field of treating severe depression, there is an urgent need for drugs with a rapid onset of action (especially in patients with suicidal tendencies), relatively good therapeutic efficacy, relatively low toxicity and side effects, and a novel mechanism of action.

[0006] Chinese patents CN102850317A (application number 201210123842.7, hereinafter abbreviated as "Patent A") and CN103687850A (application number 201280020049.2, hereinafter abbreviated as "Patent B") disclose substituted cinnamamide derivatives, methods for producing them, and their use as therapeutic and preventive drugs for mental disorders such as depression. Thirteen specific compounds, I-1 to I-13, are disclosed. In these patents, mouse tail suspension tests, "acquired despair" depression model experiments, anti-reserpine ptosis depression model experiments, and mouse forced swimming tests have verified that administration of 10 mg / kg of compounds I-5, I-9, I-10, I-11, I-12, and I-13 for 7 days can significantly shorten the mouse tail suspension immobility time. Seven consecutive days of administration of compounds I-4, I-5, I-10, I-11, I-12, and I-13 at a dose of 10 mg / kg significantly antagonized the reserpine-induced decrease in mouse body temperature and immobility, and improved the degree of eye closure, indicating a certain modulatory effect on the re-intake of 5-HT, NE, and DA. Compounds I-5, I-10, and I-13 all clearly shortened the immobility time during forced swimming in mice, and the effect of I-5 on the immobility time during forced swimming in mice showed a certain dose-dependent effect. Among these, I-5, which showed the best effect, is currently in clinical research.

[0007] Chinese Patent CN107011313A (Application No. 201710038281.3, hereinafter abbreviated as "Patent C") discloses the anxiolytic applications of substituted cinnamamide derivatives. Specifically, a total of 17 compounds are disclosed: II-3 (compound I-3 in Patents A and B), II-4 (compound I-4 in Patents A and B), II-5 (compound I-5 in Patents A and B), II-10 (compound I-10 in Patents A and B), II-11 (compound I-11 in Patents A and B), II-12 (compound I-12 in Patents 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, a mouse elevated cruciform maze test demonstrated that administration of 17 compounds at a dose of 10 mg / kg for 7 days increased the number of times mice entered the open arm in the elevated cruciform maze test and extended the time mice spent in the open arm, to varying degrees. A rat Vogel-type conflict test demonstrated that administration of 17 compounds at a dose of 5 mg / kg for 10 days increased the number of times rats drank water during the punishment period, to varying degrees. [Overview of the project]

[0008] This application is based on research into many compounds in the above-mentioned patent, and it was discovered that while many of the compounds exhibited excellent therapeutic effects, they had low bioavailability and slow onset of action.

[0009] In the first aspect, the present application provides a faster-acting and more bioavailable substituted cinnamamide compound for depressant and anxiolytic purposes, or a solvate thereof, or a pharmaceutically acceptable salt thereof, the structural formula of the substituted cinnamamide compound is as follows. [ka]

[0010] In a second aspect, the present application provides a pharmaceutical composition containing the above-mentioned substituted cinnamamide compound, or a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0011] In a third aspect, the present application provides a method for producing the above-mentioned substituted cinnamamide compound.

[0012] In a fourth aspect, the present invention provides the above-mentioned substituted cinnamamide compound for use in the manufacture of antidepressants, anxiolytics, or antidepressants / anxiolytics.

[0013] In a fifth aspect, the present invention provides a combination of the above compound with one or more other antidepressants or anxiolytics for use in the manufacture of antidepressants, anxiolytics, or antidepressants / anxiolytics.

[0014] In the sixth aspect, the present application provides a method for preventing or treating a mental disorder, comprising administering the above-mentioned compound to a patient in need of treatment. [Brief explanation of the drawing]

[0015] [Figure 1] This study demonstrates that compound M2 has antidepressant-like effects. 24 hours after administration, compound M2 significantly reduced immobility time in mice, showing a clear dose-response effect, and exhibiting a significant effect at a dose of 30 mg / kg. [Figure 2] This study demonstrates the effects of compound M2 on excitatory synaptic transmission in the frontal lobe, specifically showing the effects of compound M2 concentrations of 10 nM, 10 μM, and 150 μM on sEPSCs in pyramidal neurons of the PrL brain area of ​​the frontal lobe. The upper graph shows a schematic representation of the sEPSC waveform, and the lower graph shows the bar graph after data statistics. [Figure 3] This study demonstrates the enhancement of excitatory synaptic transmission in the frontal lobe by compound M2. At multiple concentrations, compound M2 increased the firing frequency of sEPSCs in the PrL brain area of ​​the frontal lobe, exhibiting a clear concentration-dependent effect. However, the effect on the firing amplitude of sEPSCs was not clear, and compound M2 at 150 μM significantly enhanced the firing frequency of sEPSCs. [Figure 4]It is shown that the dopamine D2R antagonist Sulpride inhibits the enhancing effect of compound M2 on sEPSC in the prefrontal cortex. The D2R antagonist sulpride can inhibit the D2R receptor at an effective concentration of 10 μM. The results indicate that inhibition of D2R can inhibit the enhancing effect of 150 μM compound M2 on the firing frequency of sEPSC. [Figure 5] It is shown that the dopamine D1R antagonist SCH23390 inhibits the enhancing effect of compound M2 on sEPSC in the prefrontal cortex. The D1R receptor antagonist SCH23390 can inhibit the D1R receptor at an effective concentration of 10 μM. The results indicate that inhibition of D1R can inhibit the enhancing effect of 150 μM compound M2 on the firing frequency of sEPSC. [Figure 6] It is shown that compound M2 activates the mTOR-related signaling pathway. a shows the changes in prefrontal cortex-related signaling proteins 30 min after single administration, and b shows the changes in prefrontal cortex-related signaling proteins 24 h after single administration. [Figure 7] Shows the inhibitory effects (IC50) of compounds I-5 and M2 on the uptake of DA, 5-HT, and NA in rat brain synaptosomes. [Figure 8] Shows the average plasma drug concentration-time curve after administering compound M2 to rats by forced oral and intravenous routes. [Figure 9] Shows the average plasma drug concentration-time curve after administering compound I-5 to rats by forced oral and intravenous routes.

Embodiments for Carrying Out the Invention

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

Chemical formula

[0017] Furthermore, the substituted cinnamic amide compound of the present application may exist in the form of a solvate.

[0018] Furthermore, the substituted cinnamamide compounds of this application may exist in the form of pharmaceutically acceptable salts.

[0019] In a second embodiment, the present application provides a pharmaceutical composition containing the substituted cinnamamide compound of the present application, or a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0020] The pharmaceutical composition of this application may be any administerable pharmaceutical form, such as tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral solutions, buccal solutions, granules, pills, powders, ointments, erythemas, suspensions, powders, solutions, injections, suppositories, ointments, hard ointments, creams, sprays, drops, or patches.

[0021] Preferably, the pharmaceutical composition of the present application is in the form of a unit dose pharmaceutical formulation.

[0022] In the pharmaceutical composition of this application, when preparing the drug, a unit dose of the drug may contain 0.1 to 1000 mg of the pharmaceutically active substance of this application, with the remainder being a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be 0.01 to 99.99% by weight of the total weight of the preparation.

[0023] The pharmaceutical composition of this application is used according to the patient's condition, with the dosage and administration determined accordingly, for example, 1 to 3 times a day, with 1 to 10 tablets per dose.

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

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

[0026] The aforementioned injectable preparation is one selected from injectable solutions, lyophilized powder injectable preparations, and liquid injectable preparations.

[0027] In the pharmaceutical composition of the present application, orally administered formulations may contain common excipients, such as adhesives, fillers, diluents, tablets, lubricants, disintegrants, colorants, flavoring agents, or wetting agents, and the 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 composition of this application can be manufactured by conventional methods such as mixing, filling, and tableting. Repeated mixing allows the active substance to be distributed throughout the entire composition, even when a large amount of filler is used.

[0030] The oral liquid formulation may be in the form of an aqueous or oily suspension, solution, emulsion, syrup, or elixir, or a dry product that can be compounded with water or another suitable carrier before use. The liquid formulation may contain common additives, such as suspending agents like sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fat; emulsifiers like lecithin, dehydrated sorbitol monooleate, or gum arabic; a non-aqueous carrier [which may include edible oils (e.g., almond oil, fractionated coconut oil), e.g., glycerin oil esters, propylene glycol, or ethanol)]; preservatives such as p-hydroxybenzoic acid, p-hydroxybenzoate propyl, or sorbic acid; and, if necessary, common flavorings or colorings.

[0031] For injectable preparations, the liquid unit dosage form produced contains the active substance of this application and a sterile carrier. Depending on the carrier and concentration, the compound may be suspended or dissolved. For the preparation of solutions, the active substance is usually dissolved in one type of carrier, filtered and disinfected before being placed in a suitable vial or ampoule, and then sealed. Auxiliary materials (e.g., local anesthetics, preservatives, and buffers) may also be dissolved in such carriers. To enhance stability, such compositions may be frozen after being placed in vials to remove water under vacuum.

[0032] The pharmaceutical composition of this application may be selectively modified to include a suitable pharmaceutically acceptable carrier during the manufacturing process. The pharmaceutically 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, monovalent alkali metal carbonates, acetates, phosphates 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, etc.

[0033] In addition to the substituted cinnamamide compound of the present application, the pharmaceutical composition of the present application may further include one or more drugs for the prevention and treatment of mental disorders, such as nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracene, neurostane, etc.

[0034] In a third embodiment, the present application provides a method for producing the substituted cinnamamide compound, the substituted cinnamamide compound also referred to as compound M2, and the production method is as follows: [ka] The process involves reacting (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid (i.e., intermediate A) with NH3 to obtain compound M2.

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

[0036] In some embodiments of the third aspect, the manufacturing method according to the present application is carried out in an organic solvent, the organic solvent comprising dichloromethane (DCM).

[0037] In some embodiments of the third aspect, in the production method according to the present invention, (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid (i.e., intermediate A) is activated with an acyl chloride reagent, for example, oxalyl chloride or thionyl chloride, and then reacts with NH3 to obtain compound M2.

[0038] In some embodiments of the third aspect, the manufacturing method according to the present invention can be carried out by the following reaction pathway. [ka] Among these, the reaction raw material is (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid, which may be purchased commercially or manufactured by methods described in conventional technical literature.

[0039] The substituted cinnamamide compounds of this invention can be produced by the following method. (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid (intermediate A) is dissolved in dichloromethane, a catalytic amount of N,N-dimethylformamide is added, oxalyl chloride is added dropwise under an ice bath, and the mixture is stirred at room temperature until the reaction of the starting materials is complete. After concentrating and drying the reaction mixture, dichloromethane is added to dissolve it, and aqueous ammonia is added dropwise under an ice bath, and the reaction is carried out at room temperature until the reaction is complete. After removing the solvent under reduced pressure, dilute hydrochloric acid is added to adjust the acidity, the solid is precipitated, and the crude product is obtained by filtration and washing, and the crude product is purified by silica gel column chromatography to obtain compound M2.

[0040] In a fourth embodiment, the present application provides a pharmaceutical composition containing compound M2 or its solvate or a pharmaceutically acceptable salt thereof, or compound M2 or its solvate or a pharmaceutically acceptable salt thereof, for use in the manufacture of an antidepressant, an anxiolytic, or an antidepressant / anxiolytic.

[0041] In some embodiments of the fourth aspect, the present application provides drug uses for pharmaceutical compositions containing compound M2 or its solvate or a pharmaceutically acceptable salt thereof, or compound M2 or its solvate or a pharmaceutically acceptable salt thereof.

[0042] In a fifth embodiment, the present application provides a use in the manufacture of an antidepressant, anxiolytic, or antidepressant / anxiolytic, in combination of compound M2 or its solvate or a pharmaceutically acceptable salt thereof with other antidepressants or anxiolytics, or in combination of compound M2 with one or more other antidepressants or anxiolytics. Here, the other antidepressants or anxiolytics, or other antidepressants or anxiolytics, may be selected from one or more of the drugs used to prevent and treat mental disorders, such as nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracene, neurostane, etc.

[0043] In a sixth embodiment, the present application provides a method for the prevention or treatment of a mental disorder, the method comprising administering to a patient in need of treatment a pharmaceutical composition containing compound M2 or its solvate or a pharmaceutically acceptable salt thereof, or compound M2 or its solvate or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments of the sixth aspect, the prevention or treatment of the mental disorder is an antidepressant, anxiolytic, or antidepressant-anxiolytic.

[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 the mental disorder further comprises a combination (or concomitant use) with one or more other antidepressants or anxiolytics, where the other antidepressants or anxiolytics may be selected from one or more of the drugs for preventing and treating mental disorders, such as nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracene, neurostane, etc.

[0047] Specific Embodiments To further clarify the purpose, technical proposal, and advantages of this application, embodiments of the present invention will be described in detail below. The embodiments and features described herein can be combined in any way, provided they do not conflict with each other.

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

[0049] Concentrated hydrochloric acid was added to the reaction system to adjust the pH to acidic, and dichloromethane was removed by rotary distillation under reduced pressure. The mixture was then filtered by suction until dry to obtain 8.67 g of a white crude product, which was recrystallized with 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 its impact on brain region function. The 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) Compulsory swimming test (also known as the despair test, commonly used to test depressive behaviors in mice) [Table 1]

[0052] 2) Brain slice electrophysiological testing (used to measure the effect of compound M2 on excitatory synaptic transmission in the mPFC) ● Animals: Newborn mice from a litter BL / 6J (20-30 days old) ●Equipment: Vibratome (Leica), Brain Slice Electrophysiology System (Axon) ● Brain Slice Electrophysiological Testing Methods ●sEPSC Experimental Method: Neonatal C57 mice from a single litter are anesthetized and perfused with artificial cerebrospinal fluid (ACSF). After decapitation, brain tissue is immediately extracted and sliced ​​using a vibratome, with a brain slice thickness of 300 μm. After incubating the brain slices at 28°C for 1 hour, whole-cell recordings of mPFC pyramidal neurons are performed using a brain slice electrophysiology system employing voltage clamping (-70 mV). The baseline recording time is 6 min, after which the drug awaiting measurement (dissolved in ACSF) is added via a perfusion system, and the recording time after drug addition is 6-10 min. The recorded data is analyzed using Minianalysis software to compare the effects of the drug awaiting measurement on the frequency and amplitude of sEPSCs in medial frontal lobe pyramidal neurons of C57 mice. ●Data Statistics All data analysis is performed using spss data processing software, and data are presented as Mean ± SEM. One-way ANOVA is used to analyze the effect of compound M2 on sEPSC current under the action of compound M2 and dopamine receptor antagonists, and paired t-tests are used to analyze the sEPSC experimental results. An asterisk (*) is used 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 experimental method: After administering compound M2 to C57 mice, a sample of the frontal lobe brain region was taken from the brain tissue 0.5 hours / 24 hours later. The tissue sample was lysed and homogenized, and the protein was quantified using the BCA method. The changes in protein content in the frontal lobe brain region under the effect of compound M2 intraperitoneally for 0.5 hours / 24 hours were then measured through processes such as SDS-PAGE gel formulation, application and electrophoresis, transmembrane scanning, blocking, primary antibody incubation, secondary antibody incubation, and protein measurement (development and fixation).

[0054] 3) Experimental results 1. Compound M2 has antidepressant-like effects. As shown in Figure 1, 24 hours after administration, compound M2 significantly reduced the immobility time of mice, exhibiting a clear dose-response effect, and a significant effect was observed at a dose of 30 mg / kg. The antidepressant effect of compound M2 was rapid. Two positive compounds were used as controls in the experiment; of these, fluoxetine, a first-line clinical drug, had a relatively slow onset of action, while esketamine had a rapid onset. The results of this experiment showed that fluoxetine did not have an antidepressant-like effect 24 hours after a single dose, while compounds M2 and esketamine did, indicating that compound M2 has the potential for rapid antidepressant action.

[0055] 2. Compound M2 significantly enhances excitatory synaptic transmission in the medial frontal lobe (mPFC). During the experiment, 10 μM bicuculine was added to the solution to inhibit GABA receptors, and sEPSCs of pyramidal neurons were recorded. As shown in Figure 2, the experiment investigated the effect 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 and exhibits a concentration-dependent effect, 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 at frontal lobe synapses. Based on the targeting characteristics of compound M2, this increase may be due to compound M2 enhancing synaptic transmission of monoamine neurotransmitters released from the midbrain to the frontal lobe, and may also be due to a direct action 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 reinforcing effect of compound M2 on the frontal lobe sEPSC. This experiment investigates whether frontal lobe dopamine receptors affect the effects of compound M2 on the frontal lobe. In the experiment, the dopamine receptor D2R antagonist Sulpride was used to inhibit D2R receptors. The results showed that inhibition of dopamine D2R receptors significantly inhibited the enhancing effect of compound M2 on sEPSC firing frequency, suggesting that frontal lobe D2R receptors may be involved in excitatory synaptic transmission mediated by M2 (shown in Figure 4).

[0057] 2) Dopamine D1R receptors do not affect the reinforcing effect of compound M2 on frontal lobe excitatory synaptic transmission. Results using the dopamine D1R receptor antagonist SCH23390 showed that inhibition of dopamine D1R receptors could not inhibit the reinforcing effect of compound M2 on sEPSC firing frequency, suggesting 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 frontal lobe synapses and generates related signaling pathways. This experiment investigates the intracellular signaling pathway through which compound M2 exerts its antidepressant effects. Since activation of the mTOR signaling pathway plays a crucial role in rapid antidepressant activity, this study examines 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 medial frontal lobe p-mTOR and p-TrkB increased, but the total content remained unchanged. Literature has shown that the mTOR signaling pathway is associated with the rapid depressive disorder mechanism, and that the rapid antidepressant effect of ketamine may be related to increased phosphorylated mTOR content and activation of the TrkB protein (BDNF receptor). These results suggest that the antidepressant effect of compound M2 may also be related to the activation of mTOR and TrkB proteins.

[0059] Following activation of the mTOR signaling pathway, the expression levels of synapse-related proteins were further enhanced. Figure 6b shows that, 24 hours after a single dose, compound M2 significantly increased the content of the synapse-related protein PSD95 formed in medial frontal lobe synapses. This result is consistent with the function of compound M2 in enhancing frontal lobe synaptic transmission, as discovered by electrophysiological testing. PSD95 is a postsynaptic density protein involved in neuronal plasticity. A study (Shinohara R, Aghajanian GK, Abdallah CG. Neurobiology of the Rapid Acting Antidepressant Effects of Ketamine: Impact and Opportunities[J]. Biological Psychiatry, 2020.) showed that the antidepressant and anxiolytic effects of ketamine are associated with increased expression levels of the PSD95 protein.

[0060] Previous research (Pizzagalli DA, Roberts AC. Correction: Prefrontal cortex and depression[J]. Neuropsychopharmacology, 2021:1-1.) suggests that clinically, depressed patients may exhibit morphological characteristics of frontal lobe atrophy. Furthermore, basic experimental research has shown that model mice induced by chronic social stress exhibit decreased neurite density in layer 5 of the frontal lobe, reduced frequency of postsynaptic excitatory currents (EPSCs), and this reduction in EPSC frequency decreases synaptic transmission of frontal lobe excitatory neurotransmitters. Frontal lobe excitatory synaptic transmission is closely involved in antidepressant and anxiolytic effects, and substances like ketamine and fluoxetine can enhance it. Therefore, this experiment will combine the pharmacological characteristics of compound M2 to primarily investigate its antidepressant and anxiolytic effects, and to examine its impact on the brain's excitatory neurotransmitter glutamatergic system. Experiments involving frontal lobe brain slices, which closely simulated physiological conditions, revealed that all compounds M2 could promote the release of glutamate neurotransmitters and enhance the function of glutamate receptors. This demonstrated that compound M2 enhances frontal lobe excitability and exhibits a concentration-dependent effect.

[0061] The results above demonstrate that compound M2 primarily acts on the glutamatergic neurotransmitter system, enhancing excitatory glutamatergic synaptic transmission, thereby improving frontal lobe excitability and exerting a rapid antidepressant effect. Furthermore, compound M2 rapidly activates the frontal lobe mTOR signaling pathway. These results indicate that compound M2 possesses a rapid antidepressant effect and a novel antidepressant mechanism that enhances synaptic transmission and brain excitability, demonstrating superior antidepressant potential compared to clinical monoamine drugs.

[0062] Experimental Example 2: Inhibitory effect of compound I-5 and compound M2 on monoamine reuptake in rat brain synaptosomes. From a current perspective, absolute or relative deficiencies of the central monoamine neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NA), and dopamine (DA) are closely associated with depression and anxiety. This experiment evaluates the inhibitory effects of two test compounds, I-5 and M2, on the reuptake of 5-HT, NA, and DA in rat brain synaptosomes using an in vitro monoamine reuptake method. Preparation of isolated SD rat brain synaptosomes and isotope tracer analysis are performed to evaluate the IC of the two test compounds that inhibit the reuptake of monoamines DA, 5-HT, and NA, respectively. 50 This study investigates the possible antidepressant and anxiolytic mechanisms of action of compounds I-5 and M2.

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

[0064] 2. Experimental specimens: As can be seen from the experimental descriptions in the specifications of patents A and B, compound I-5 showed favorable results in all the experiments described in each section. Therefore, compound I-5 was selected as a control, and the inhibitory effect of compound M2 on rat brain synaptosome monoamine reuptake was compared. [Table 3]

[0065] 3. Combination of test substance and positive control substance, etc. 3.1 Composition of test substances The test substance is weighed (allowable weighing error ±1%), dissolved in DMSO, prepared as a 10 mM storage solution, and stored at -20°C. [Table 4] Before the experiment, dilute the Kreb's solution using the doubling dilution method to obtain a 100-fold dilution of the solution required to reach the detection concentration (1 nM to 100 μM). Conversion formula:

number

[0066] 3.2. Formulation of positive control substance Weigh the control substance (allowable weighing error ±1%), dissolve it in redistilled water to prepare a 10 mM storage solution, and store it at -20°C. [Table 5]

[0067] 2. Experimental Method 1. Preparation of brain synaptosomes Brain synaptosomes were prepared by isolating synaptosomes using a method described in the literature. The heads of SD rats were rapidly cut with a small animal decapitator, and the brains were then quickly extracted and pre-cooled in an ice water mixture to remove the pia mater and vascular tissue. Brain tissue was extracted. 10 times the volume (ml / g) of 10 mM Tris HCl buffer (containing 0.32 M sucrose, pH 7.4) was added, and the brain tissue was homogenized using a cell disruptor sonicator (maintaining a low temperature) and centrifuged for 10 minutes (4°C, 1,000 g). Equilibrium centrifugation was performed at 4°C (1500 g, 10 min), the precipitate was removed, and the supernatant was taken and centrifuged again for 30 minutes (20000 g). The supernatant was removed, and the precipitate, i.e., the crude extract of synaptic capitellum, was retained. The precipitate was further suspended in a 0.32 M cold sucrose solution, then carefully spread onto a gradient of 1.2 M and 0.8 M (10 ml each) cold sucrose solutions laid sequentially from the bottom of the tube, and centrifuged at 4°C for 60 minutes (38000 g). The suspension zone at the 0.8-1.2 M sucrose interface was carefully collected with a puncture needle, added to 10 ml of 0.32 M cold sucrose solution, mixed uniformly, and centrifuged at 4°C for 30 minutes (20000 g). The precipitate was purified brain synaptosomes. The precipitate is suspended in a small amount of Kreb's buffer (NaCl 118mM, KCl 4.7mM, CaCl 22.5mM, MgSO4 1.2mM, KH2PO4 1.2mM, NaHCO3 25mM, and Glucose 11.1mM, pH 7.2-7.4), and protein quantification is performed by the BCA method, following the instructions for the procedure.

[0068] 2. Reuptake of monoamines Based on relevant methods in the literature and optimized in the inventor's laboratory, the experimental procedure is as follows: Add 950 μl of pre-cooled Kreb's buffer to the reaction tube, then add 30 μl of synaptic head suspension, and then add 10 μl of the compound awaiting measurement (performed on ice). Mix uniformly with a turbine and bathe in water at 37°C for 5 minutes. Remove the reaction tube and place it on ice, then add 10 μl of substrate ( 3 H-DA or 3 H-5HT or 3Add H-NA (final reaction concentration: 10 nM), mix uniformly with a turbine, and bathe in water at 37°C for 5 minutes. Then, remove the reaction tube and quickly place it on ice, add 3 ml of pre-cooled Kreb's buffer to stop the reaction, collect the sample with a Millipore cell sample collector, quickly filter by suction through a GF / C glass fiber filter membrane, elute three times with 3 ml of eluate (50 mM Tris-HCl, pH 7.4), remove the filter membrane, dry in a microwave oven for 5-6 minutes, transfer the filter membrane to a 1.5 ml centrifuge tube, and add 500 μl of lipid-soluble scintillator solution. Let stand for at least 30 minutes away from light, and count to measure the radioactivity intensity (cpm value).

[0069] The inhibition rate of each compound on isotopic ligand binding is calculated using the following formula. Inhibition rate (1%) = (Total binding tubule cpm - Compound cpm) / (Total binding tubule cpm - Nonspecific binding tubule cpm) × 100% Each time, we conduct experiments with three sets of compounds and repeat the individual experiments three times.

[0070] III. Experimental Results 1. IC 50 Calculation 1) Let X be the concentration of the detected compound and Y be the inhibition rate. 2) Take the logarithm of X, so X' = Log(X), and generate new data. 3) Perform a nonlinear curve fitting on the new data, and after fitting, substitute it into the following equation and calculate IC 50 The values ​​are calculated. The above steps are fitted using the GraphpadPrism software. 4)

number

[0071] 2. Experiment Summary Monoamine reuptake inhibition is one of the main targets of antidepressant and anxiolytic drugs. The test substances, compounds I-5 and M2, exhibited different degrees of inhibitory activity against the reuptake of DA, 5-HT, and NA in rat brain synaptosomes. Furthermore, compounds I-5 and M2 showed similar levels of inhibitory activity, indicating that compounds I-5 and M2 exert their antidepressant and anxiolytic effects by inhibiting the reuptake of DA, 5-HT, and NA. IC5 for the reuptake of DA, 5-HT, and NA in rat brain synaptosomes. 50 The values ​​are 26.02±1.33 nM, 18.52±2.47 nM, and 17.21±1.59 nM, respectively. IC2 of compound M2 on the reuptake of DA, 5-HT, and NA in rat brain synaptosomes. 50 The values ​​are 37.28±1.97 nM, 98.87±2.42 nM, and 23.54±3.56 nM, respectively.

[0072] Experimental Example 3: Study of the antidepressant effects of compound I-5 and compound M2 The "behavioral despair" model, developed by Porsolt et al. in 1977, includes rat-mouse forced swimming models and mouse tail suspension models, and belongs to the category of acute stress models. 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. Suspended mice struggle to overcome the abnormal body posture, but after a certain period of movement, the animals express intermittent immobility due to "despair." In this experiment, we will observe whether compounds I-5 and M2 in samples awaiting measurement affect the tail-hanging behavior of mice in a "behavioral despair" experiment, whether they can shorten the immobility time of mice, and study the rapid onset of action of compounds I-5 and M2.

[0073] 1. Experimental materials 1. Test subject 1 Name or code: Compound I-5 Test substance number: YLS-2021-CMI1203-002 Batch number: C16101006-C17001M Preparation method: The solvent is Tween 80 + water. The mixture is fully emulsified with 1.5% Tween 80 until the final volume is reached, and water is added until the final volume is achieved. The initial storage conditions after preparation are a 4°C refrigerator.

[0074] 2. Test subject 2 Name or code: Compound M2 Test substance number: YLS-2021-CMI1203-01-001 Batch number: 20201029 Preparation method: The solvent is Tween 80 + water. The mixture is fully emulsified with 1.5% Tween 80 until the final volume is reached, and water is added until the final volume is achieved. The initial storage conditions after preparation are a 4°C refrigerator.

[0075] 2. Laboratory animals C57 BL / 6 mice, 40 individuals, SPF grade, male, 4-5 weeks old.

[0076] III. Experimental Method 1. Animal grouping and rationale for dose design Forty mice were divided into four groups of 10 mice each, according to the Excel complete random grouping method. As shown in the results of the preliminary experiment, compound M2 could reduce the immobility time of mice in the forced swimming test in a dose-dependent manner 24 hours after administration, and the effect was significant at a dose of 30 mg / kg. Therefore, in this experiment, 30 mg / kg was used as the low dose of compound M2, and 60 mg / kg was used as the high dose of compound M2, and it was studied in comparison with compound I-5 at 60 mg / kg. See Table 1 for details on grouping and administration.

[0077] [Table 7]

[0078] 2. Dosage form and administration time Each test group received the drug solution via forced oral administration (ig), with a dose of 0.2 ml / 10 g body weight.

[0079] 3. Indicator detection A single oral administration was administered to mice 30 minutes prior to the tail suspension test, which was observed to last 6 minutes. The time the mice remained stationary and immobile during this 6-minute period was recorded.

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

[0081] IV. Experimental Results As shown in the results of the single-dose mouse test, a single dose of compound M2 at 60 mg / kg 30 mins after administration significantly reduced the time of immobility in mouse tail suspension, with p<0.05 compared to the blank group. A single dose of compound M2 at 30 mg / kg 30 mins after administration showed a certain degree of reduction in the time of immobility in mouse tail suspension, but it did not show a statistically significant difference. A single dose of compound I-5 at 60 mg / kg 30 mins after administration had no significant effect on the time of immobility in mouse tail suspension (see Table 2). The above results indicate that compound M2 has a relatively rapid onset of antidepressant effect, possesses significant antidepressant activity, and is clearly superior to compound I-5 in terms of its rapid onset of action.

[0082] [Table 8]

[0083] Experimental Example 4: Study of the absolute bioavailability of compounds I-5 and M2 in rats. 1. Experimental Objectives Through experiments, we will establish an LC-MS / MS method for measuring compounds I-5 and M2 in rat plasma, and investigate the rat pharmacokinetic characteristics and absolute bioavailability of these compounds.

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

[0085] 3 Experimental Methods 3.1 Preparation and Dosage of Test Substances

Table 9

[0086] 3.2 Measurement Methods 3.2.1 LC-MS Conditions (Normal Phase) for Compound M2 3.2.1.1 Liquid Phase Conditions <000...​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0088] 3.3 Pharmacokinetic Experiments Twenty SD rats were randomly divided into four groups of five. The rats were fasted for 10 hours before administration and allowed to drink water freely. 0.3 mL of venous blood was collected from the edge of the eye at 0 hours before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 hours after administration. The blood was placed in an EDTA-K2 anticoagulant tube, gently shaken to ensure thorough and uniform mixing with the anticoagulant, placed on wet ice, and centrifugation was completed within 0.5 hours. Centrifugation: After centrifugation at 8000 rpm for 5 minutes, separate the plasma. Storage: Store frozen at -20°C.

[0089] 3.4 Processing method for plasma samples Take 50 μL of plasma, dissolve 50 μL of DI-5 (in 20 ng / mL acetonitrile), run the turbine for 30 seconds, add another 100 μL of acetonitrile, run the turbine thoroughly for 2 minutes, centrifuge at 12000 r / min at 4°C for 3 minutes, take 50 μL of supernatant, dilute with 50 μL of aqueous solution, run the turbine for 1 minute, and you may inject 4 μL.

[0090] 4. Analysis of pharmacokinetic results For the mean blood concentration-time curves after forced oral and intravenous administration to rats, refer to Figures 8 and 9. Data processing was performed using DAS3.0 pharmacokinetic software (China Mathematical Pharmacology Professional Committee, Shanghai, China), and non-compartmental model statistical moment parameters were calculated. max and T max These are measured values. For more details on blood concentration data and pharmacokinetic parameters, please refer to 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 administering compounds M2 and I-5 to rats via forced oral and intravenous administration, pharmacokinetic studies showed that the time to reach peak absorption of compounds M2 and I-5 after forced oral administration was relatively fast, as indicated by the time to reach peak absorption t. max The interval is 0.35h, and the C of compound M2 max The concentration was 425.4 ± 136.9 ng / mL, and the C of compound I-5 max The AUC of compound M2 is 361.1 ± 258.2 ng / mL, indicating that compound M2 has a relatively high blood concentration. 0-t The AUC of compound I-5 is 1080.1 ± 373.4 ng.h / mL. 0-t The concentration is 523.3 ± 433.3 ng.h / mL. At the same forced oral dose, compound M2 showed higher exposure in rats and had higher absolute bioavailability than compound I-5, at 40.8% and 24.05%, respectively.

[0098] Experimental Example 5: Study of the anxiolytic effects of M2 and its structural analogs 1. Experimental materials 1. Test specimen As can be seen from the results of the two experiments described in the specification of Patent C (mouse elevated cruciform maze test, rat Vogel-type conflict test), compounds II-3, II-4, II-5, and II-10 showed relatively good effects in both experiments. Therefore, these four compounds were 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 Research Institute Chemical 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 Research Institute Chemical 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 Research Institute Chemical 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 Research Institute Chemical 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 Research Institute Chemical 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] 2. Laboratory animals 2.1 Laboratory animals (1) Species: ICR mouse (for elevated cross maze testing) Quantity: 120 animals Grade: SPF Gender: Male Weight: 18-20g Animal identification numbers: 110011211113772653, 110011221101333628 Origin: Beijing Vital River Laboratory Animal Technology Co., Ltd. Production License Number: SCXK(Beijing)2021-0006

[0107] (2) Species: SD Rats (for Vogel Conflict Test) Quantity: 75 Grade: SPF Gender: Male Weight: 180 - 200g Animal Certificate Number: 110011221102490324 Origin: Beijing Vital River Laboratory Animal Technology Co., Ltd. Production License Number: SCXK(Beijing)2021-0011<00OO622>

[0108] 2.2 Animal Facilities Breeding Facility: Inside the barrier environment of the animal facility of Tasly Holding Group Co., LTD in Tianjin Facility Address: Inside the factory area at the intersection of Huaiao Road and Tingjiang West Road in Beichen Science and Technology Park, Tianjin Experimental Animal Use Permit: SYXK(Tianjin)2017-0007 Issuing Authority: Tianjin Science and Technology Commission

[0109] 2.3 Animal Breeding and Management Breeding Environment: Barrier environment. The environmental conditions of this facility meet the standards for barrier animal experiment facilities in the Chinese national standard "Laboratory Animal Environment and Facilities" (GB14925-2001). Animal breeding and animal experiment operations meet the regulations such as the "Tianjin Laboratory Animal Management Regulations". Temperature: 20 - 26°C, Humidity: 40% - 70%, Lighting: 12 hours light, 12 hours dark, Ventilation: ≥15 times / hour of fresh air, Animals drink sterile water (4-stage filtration, UV sterilization) prepared by a 1T / h type multi-microporous filter membrane filtration system. Animal management is the responsibility of the Animal Protection Department. Except for fasting, sufficient feed and drinking water should be provided to animals every day. The water bottle should be replaced once a day. The bedding material 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 experimenter, veterinarian and Animal Protection Department shall jointly receive them. 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 is transported to the quarantine room via the first transport cabinet. The test supplies and experimental records are 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, activities, 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 regularly (including weight, head, body, tail, limbs, fur, spirit, activities, etc.).

[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 in each group. The high, medium, and low-dose groups of compound M2 were force-administered at 20 mg / kg, 10 mg / kg, and 5 mg / kg respectively, while the positive drug estazolam was force-administered at 2.5 mg / kg. The solvent control group received a force-administered volume of solvent. Behavioral tests were performed 30 minutes after a single dose in each group. The experiment was conducted between 8:00 AM and 2:00 PM, with all animals entering the test laboratory the day before. Dosage Design Basis: As shown in the results of the previous anxiolytic experiment (Patent C), compounds II-3, II-4, II-5, etc., which are structural analogs of compound M2, have an effective anxiolytic dose of 10 mg / kg in the mouse elevated cruciform maze test. In this experiment, 10 mg / kg will be used as the intermediate dose of compound M2, and the doses will be decreased by 5 mg / kg and increased by 20 mg / kg respectively. The clinical dose of estazolam, a positive anxiolytic drug, is 6 mg / person / day. Converting this to a mouse clinically equivalent dose, it is 6 mg / 60 kg * 12.3 = 1.23 mg / kg, and the double clinical dose is 2.5 mg / kg. Converting this to a rat clinically equivalent dose, it is 6 mg / 60 kg * 6.2 = 0.62 mg / kg, and the double clinical dose is 1.24 mg / kg.

[0112] [Table 18]

[0113] (2) Behavioral Test In the laboratory, the lighting should be dim (based on the minimum brightness required to distinguish slight mouse movements at a distance of 1.5m), the brightness should be kept constant, the room temperature should be around 20°C, and the environment should be quiet. Before the maze test, each mouse should be placed in a 35cm*10cm*5cm plastic box and allowed to explore freely. After 5 minutes, it should be quickly placed on the central platform of the elevated cross maze, with its head facing one of the open arms. After release, the following indicators should be recorded. Each mouse should be tested for 5 minutes. The observer should observe and record the activity of each animal from a distance of 1.5m. Midway through, the maze should be wiped with a damp cloth to remove feces, then wiped with a dry cloth before testing the next mouse.

[0114] (3) Behavioral observation indicators (1) Open arm entry (OE): This is the number of times the mouse enters any of the open arms. The criterion is that all four of the mouse's legs enter the arm, and if one leg completely withdraws from the arm during the process, it indicates the completion of the entry activity. (2) Open arm entry time (OT): This is the time taken to enter the open arm, and is measured in seconds. (3) Number of close arm entries (CE): This is the number of times the mouse enters any of the closed arms, with the criterion being that all four of the mouse's feet enter the arm. (4) Close arm entry time (CT): This is the time taken to enter the closed arm, and is 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 its structural analog (elevated cross maze test) (1) Animal grouping and administration 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 in each group. The doses of M2 and its four structural analogs were set at 20 mg / kg based on the experimental results in 3.1, and the positive drug estazolam was administered orally at 2.5 mg / kg. The solvent control group received an equivalent volume of solvent orally. Behavioral tests were performed 30 minutes after a single dose in each group. The experiment was conducted between 8:00 AM and 2:00 PM, with all animals entering the test laboratory the day before.

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

[0117] 3.3. Comparative study of single-dose drug effects of compound M2 and its structural analog (Vogel-type conflict trial) (1) Animal grouping and administration 75 male SD rats weighing 180-220g were adaptively reared for one week, then subjected to a training period test with 24 hours of water deprivation. Qualified animals were selected and randomly divided into seven groups based on body weight (solvent control group, estazolam group, compound M2 group, compound II-3 group, compound II-4 group, compound II-5 group, compound II-10 group), with 8 animals per group. Of these, compound M2 and its four structural analogs were administered orally at a dose of 10 mg / kg based on the experimental results in 3.1, and the positive drug estazolam was administered orally at a dose of 1.24 mg / kg. The solvent control group received an equivalent volume of solvent orally. The animals in each group were then subjected to a 24-hour water deprivation, followed by a single dose, and a punishment experiment test was conducted 30 minutes after the single dose.

[0118] [Table 20]

[0119] The experiment is conducted in two stages. The first stage is the training stage, in which animals are placed individually in a control box after 24 hours of water deprivation and allowed to explore thoroughly until they find the bottle opening and begin licking the water (electric shock intensity 0mA). The number of water licks within 3 minutes is automatically recorded using a counter, and animals with fewer than 300 water licks are eliminated. The second stage is the punishment stage, in which animals that have not been eliminated are given the treatment after another 24 hours of water deprivation and again placed individually in a control box. An electric shock is administered once the animal has licked the water 20 times, with the counter automatically starting to count (the ratio of water licks to electric shocks is 20:1). The electric shock intensity is generally 0.3mA and is maintained for 2 seconds, but the electric shock can be released if the animal removes the bottle opening. The number of water licks and electric shocks of the animals over 3 minutes are recorded. Observation indicator: Number of times rats licked water during the punishment period.

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

[0121] IV. Experimental Results 4.1. Exploratory study on single-dose dosage of compound M2 (elevated cross maze test) As shown in the exploratory study results for single doses of compound M2, single forced oral administration of high (20 mg / kg), medium (10 mg / kg), and low (5 mg / kg) doses of compound M2 all increased the number of times mice entered the open arm in the elevated cruciform maze test and extended the time mice spent in the open arm, to varying degrees. Of these, the high-dose group of compound M2 showed a significant difference compared to the solvent control group (P<0.05, P<0.01). As shown in the results above, a single dose of compound M2 exhibits significant anxiolytic activity, and its effective single dose is 20 mg / kg.

[0122] [Table 21]

[0123] 4.2. Comparative study of single-dose drug effects of compound M2 and its structural analog (elevated cross maze test) As shown in the results of the elevated cruciform maze test, a single forced oral administration of 20 mg / kg of compound M2 and its structural analogs, compounds II-3, II-4, II-5, and II-10, increased the number of times mice entered the open arm in the elevated cruciform maze test and extended the time mice spent in the open arm, to varying degrees. Of these, the groups of compounds M2, II-4, II-5, and II-10 showed significant differences compared to the solvent control group (P<0.05, P<0.01). Compound M2 and its structural analogs II-4, II-5, and II-10 exhibited significant anxiolytic activity in this model, and compound M2 was shown to have optimal anxiolytic activity compared to II-3, II-4, II-5, and II-10.

[0124] [Table 22]

[0125] 4.3. Comparative study of single-dose drug effects of compound M2 and its structural analog (Vogel-type conflict trial) As shown in the results of the Vogel conflict test, a single forced oral administration of 20 mg / kg of compound M2 and its structural analogs, compounds II-3, II-4, II-5, and II-10, can increase the number of water swallows in rats in the Vogel conflict test to varying degrees. Of these, the groups of compound M2, compound II-5, and compound II-10 showed a significant difference compared to the solvent control group (P<0.05). Compound M2 and its structural analogs, compounds II-5 and II-10, exhibited significant anxiolytic activity in this model, and compound M2 was shown to have optimal anxiolytic activity compared to II-3, II-4, II-5, and II-10.

[0126] [Table 23]

[0127] V. Experimental Conclusions In summary, compound M2 exhibits significant anxiolytic activity, with an effective single dose of 20 mg / kg (in mice). Furthermore, compound M2 exhibits optimal anxiolytic activity compared to its structural analogs, compounds II-3, II-4, II-5, and II-10.

Claims

1. The following structural formula: 【Chemistry 1】 A substituted cinnamamide compound represented by [formula].

2. The following structural formula: 【Chemistry 2】 Antidepressant and anti-anxiety compounds as indicated by [this symbol].

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

4. The compound according to claim 1 or 2, characterized in that it exists in the form of a pharmaceutically acceptable salt.

5. A pharmaceutical composition containing the compound described in claim 1.

6. The pharmaceutical composition according to claim 5, which exists in any administerable pharmaceutical form, and which is selectively selected from tablets, capsules, oral solutions, buccal preparations, granules, pills, powders, ointments, erythemas, suspensions, powders, solutions, injections, suppositories, ointments, hard ointments, creams, sprays, drops, and patches, wherein the tablets are selectively sugar-coated tablets, film-coated tablets, or enteric-coated tablets, the capsules are selectively hard capsules or soft capsules, and the injections are selectively one of injection solutions, lyophilized powder injections, and liquid injections.

7. The pharmaceutical composition according to claim 5, which is used in combination with other antidepressants or anxiolytics when used, or which further comprises other antidepressants or anxiolytics, the other antidepressants or anxiolytics being selected from nefazodone, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, reboxetine, venlafaxine, flupentixol, melitracene, and neurostane. 【Request Item 8】 【Chemistry 3】 (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid (i.e., intermediate A) and NH 3 A method for producing the compound according to claim 1 or 2, comprising reacting with to obtain compound M2.

9. The reaction pathway is as follows: 【Chemistry 4】 A method for producing the compound according to claim 8, including the method described above.

10. The manufacturing method according to claim 9, comprising the steps of: dissolving (E)-3-(3',4'-methylenedioxy-5'-trifluoromethylphenyl)-acrylic acid (intermediate A) in dichloromethane, adding a catalytic amount of N,N-dimethylformamide, adding oxalyl chloride dropwise under an ice bath, stirring at room temperature until the reaction of the starting materials is complete, concentrating and drying the reaction solution, adding dichloromethane to dissolve it, adding aqueous ammonia dropwise under an ice bath, allowing the reaction to be completed at room temperature, removing the solvent under reduced pressure, adjusting the acidity by adding dilute hydrochloric acid, precipitating the solid, filtering and washing to obtain the crude product, purifying it 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, in the manufacture of an antidepressant, an anxiolytic, or an antidepressant / anxiolytic.

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