A substituted cinnamic acid compound for antidepressant and anti-anxiety purposes, its pharmaceutical composition, preparation method and application

Abstract

This application relates to an antidepressant and anxiolytic substituted cinnamic acid compound, namely compound M2. This application also provides pharmaceutical compositions containing compound M2 or its solvates or pharmaceutically acceptable salts thereof, and applications as an antidepressant, anxiolytic, or both.

Description

[Technical Field]

[0001] Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202110606431.2, filed on May 28, 2021, entitled “A Novel Compound for Antidepressant and Anti-anxiety”, the entire contents of which are incorporated herein by reference.

[0003] This application relates to, but is not limited to, a pharmaceutical compound, and particularly to a novel substituted cinnamic acid compound for antidepressant and anti-anxiety purposes. [Previous Technology]

[0004] Depression and anxiety are major diseases that endanger human mental health, and with the accelerating pace of modern life, the number of people suffering from these conditions is increasing year by year. Recently, statistics show that there are 26 million people suffering from various types of depression in China, with a large proportion being teenagers. Therefore, the development of antidepressant and anti-anxiety drugs has significant economic and social benefits.

[0005] Numerous studies suggest that changes in central monoamine neurotransmitters, dopamine, cholinergic neurotransmitters, and corresponding receptor functions, as well as neuroendocrine dysfunction, may play an important role in the development and progression of this disease. Therefore, the treatment principle should focus on adjusting the levels of hypothalamic monoamine neurotransmitters and their receptor functions, and restoring normal neuroendocrine function.

[0006] Currently, the main treatment for depression and anxiety is still medication. Clinically available antidepressants and anti-anxiety drugs mainly include tricyclic antidepressants, benzodiazepines, serotonin reuptake inhibitors (SRIs), and monoamine oxidase inhibitors (MAOIs). While these drugs have some therapeutic effect on depression and anxiety, they also have significant side effects. MAOIs, due to their selectivity and irreversible inhibition of the enzyme, can cause toxic liver damage and have certain toxic side effects. Commonly used tricyclic antidepressants include doxepin, amitepine, and clomipramine. Although these drugs are effective for intrinsic depression, especially for low mood, loss of interest, and pessimism (achieving over 80% efficacy), they also have significant cardiotoxicity and numerous adverse reactions. Selective serotonin reuptake inhibitors (SSRIs) are a new type of antidepressant and anti-anxiety drug that emerged in the late 1980s. Because they significantly reduce the adverse reactions of other receptors while maintaining classic antidepressant and anti-anxiety effects, they have become commonly used first-line drugs in Europe and the United States. Commonly used medications include fluoxetine, paroxetine, sertraline, citalopram, and fluvoxamine. Because these medications are absorbed through the gastrointestinal tract and metabolized by the liver, they still cause gastrointestinal disturbances, and in some cases, sexual dysfunction, which to some extent affects the long-term use of treatment. Furthermore, traditional antidepressants and anti-anxiety medications have a slow onset of action (6-8 weeks or more) and are effective only in about 30% of patients. Therefore, in the treatment of major depressive disorder, there is an urgent medical need for drugs with faster onset of action (especially in patients with suicidal tendencies), better efficacy and fewer toxic side effects, and novel mechanisms of action.

[0007] Chinese patents CN102850317A (application number 201210123842.7, hereinafter referred to as Patent A) and CN 103687850A (application number 201280020049.2, hereinafter referred to as Patent B) disclose a substituted cinnamylamine derivative, its preparation method, and its application in drugs for treating and preventing depressive psychosis. Thirteen specific compounds, I-1 to I-13, are also disclosed. In this patent, using mouse tail-suspension "acquired despair" depression model experiments, reserpine-induced ptosis depression model experiments, and mouse forced swimming experiments, it was confirmed that compounds I-5, I-9, I-10, I-11, I-12, and I-13, administered at a dose of 10 mg / kg for 7 days, significantly shortened the immobility time of mice under tail suspension. Compounds I-4, I-5, I-10, I-11, I-12, and I-13, administered continuously at a dose of 10 mg / kg for 7 days, significantly antagonized reserpine-induced hypothermia, immobility, and improved eye-closing ability in mice, indicating a certain regulatory effect on the reuptake of 5-HT, NE, and DA. Compounds I-5, I-10, and I-13 all significantly shortened the immobility time of mice under forced swimming, with I-5 showing a dose-dependent effect. Among these, I-5, with the best effect, is currently in the clinical research stage.

[0008] Chinese Patent CN107011313A (Application No. 201710038281.3, hereinafter referred to as Patent C) discloses the application of a substituted cinnamylamine derivative in anxiolytics. A total of 17 specific 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, an elevated cross maze experiment in mice demonstrated that administration of 17 compounds at a dose of 10 mg / kg for 7 days could increase the number of times mice entered the open arm of the elevated cross maze and prolong the time mice remained in the open arm to varying degrees. A water conflict experiment in rats demonstrated that administration of 17 compounds at a dose of 5 mg / kg for 10 days could increase the number of times rats drank water during the punishment period to varying degrees. [Summary of the Invention]

[0009] This application has studied many of the compounds in the above-mentioned patents and found that although most of the compounds have good therapeutic effects, their bioavailability is low and their onset of action is slow.

[0010] In a first aspect, this application provides a substituted cinnamic acid compound, or a solvate thereof, or a pharmaceutically acceptable salt thereof, which has a faster onset of action and higher bioavailability for antidepressant and anti-anxiety purposes, and the structural formula of the substituted cinnamic acid compound is shown below: M2.

[0011] In a second aspect, this application provides a pharmaceutical composition comprising the above-mentioned substituted cinnamic acid compound or its solvate, or its pharmaceutically acceptable salt.

[0012] In a third aspect, this application provides a method for preparing the above-mentioned substituted cinnamic acid compound.

[0013] In a fourth aspect, this application provides the use of the above-mentioned substituted cinnamic acid compounds in the preparation of antidepressant, anti-anxiety, or antidepressant and anti-anxiety medicaments.

[0014] In a fifth aspect, this application provides the use of the above-mentioned compound in combination with one or more other antidepressants or anxiolytics in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic medicament.

[0015] In a sixth aspect, this application provides a method for preventing or treating mental illness, the method comprising administering the above-mentioned compound to a patient in need.

Implementation Method

[0017] Detailed Explanation

[0018] In an embodiment of the first aspect, this application provides a substituted cinnamic acid compound with the following structural formula: M2.

[0019] Furthermore, the substituted cinnamic acid compound of this application may also exist in the form of a solvate.

[0020] Furthermore, the substituted cinnamic acid compounds of this application may also exist in pharmaceutically acceptable salt forms.

[0021] In an embodiment of the second aspect, this application provides a pharmaceutical composition containing a substituted cinnamic acid compound of the application or a solvate thereof, or a pharmaceutically acceptable salt.

[0022] The pharmaceutical composition of this application may be any oral pharmaceutical form, such as tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, or patches.

[0023] The pharmaceutical composition of this application is preferably in the form of a unit dose pharmaceutical preparation.

[0024] When the pharmaceutical composition of this application is prepared into a pharmaceutical preparation, each unit dose of the preparation may contain 0.1-1000 mg of the active pharmaceutical ingredient of this application, with the remainder being a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be 0.01-99.99% by weight of the total weight of the preparation.

[0025] When using the pharmaceutical composition of this application, the dosage and administration method shall be determined according to the patient's condition, such as 1-3 times a day, or 1-10 tablets each time.

[0026] Preferably, the pharmaceutical composition of this application is an oral preparation or an injection.

[0027] The oral preparation is selected from one of the following: capsules, tablets, drops, granules, concentrated pills, oral liquids, and mixtures.

[0028] The injection is selected from one of the following: injection solution, lyophilized powder injection, and water injection.

[0029] The pharmaceutical composition of this application, in its oral administration formulation, may contain commonly used excipients, such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents or humectants, and the tablets may be coated if necessary.

[0030] Suitable fillers include cellulose, mannitol, lactose, or other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone, or starch derivatives, preferably sodium glycolate starch. Suitable lubricants are magnesium stearate. Suitable wetting agents are sodium dodecyl sulfate.

[0031] The pharmaceutical composition of this application can be prepared into a solid oral composition by commonly used methods such as mixing, filling, and tableting. Repeated mixing can distribute the active substance throughout the composition using a large amount of filler.

[0032] The oral liquid formulation may be in the form of an aqueous or oily suspension, solution, emulsion, syrup, or elixir, or it may be a dried product that can be reconstituted with water or other suitable carriers before use. Such liquid formulations may contain conventional additives, such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifiers, such as lecithin, dehydrated sorbitan monooleate, or gum arabic; non-aqueous carriers (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerides, propylene glycol, or ethanol; preservatives, such as parabens, propylparaben, or sorbic acid, and, if desired, conventional flavorings or colorings.

[0033] For injectable formulations, the prepared liquid unit dosage form contains the active substance of this application and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solution preparation typically involves dissolving the active substance in a carrier, filtering and sterilizing it before filling it into a suitable vial or ampoule, and then sealing it. Excipients such as a local anesthetic, preservative, and buffer can also be dissolved in this carrier. To improve its stability, the composition can be frozen after filling into the vial, and water can be removed under vacuum.

[0034] The pharmaceutical composition of this application may selectively incorporate a suitable pharmaceutically acceptable carrier during the preparation of the pharmaceutical agent. The pharmaceutically acceptable carrier is selected from one or more of the following: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, 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, alginate, gelatin, polyvinylpyrrolidone, glycerol, Twenty-80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipids, kaolin, talc, calcium stearate, magnesium stearate, etc.

[0035] The pharmaceutical composition described in this application, in addition to containing the substituted cinnamic acid compound of this application, may also include one or more of the following drugs for the prevention and treatment of mental illness: such as naphazoline, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, lexapro, fluvoxamine, reboxetine, venlafaxine, trifluthixeol, tetramethylanthrafenamide, and loratadine, etc.

[0036] In an embodiment of the third aspect, this application provides a method for preparing the substituted cinnamic acid compound of this application, the substituted cinnamic acid compound also referred to as compound M2, the preparation method being: (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) reacts with NH3 to obtain compound M2.

[0037] In some embodiments of the third aspect, the preparation method provided in this application, wherein NH3 can be ammonia water (NH3-H2O).

[0038] In some embodiments of the third aspect, the preparation method provided in this application is wherein the reaction is carried out in an organic solvent, including dichloromethane (DCM).

[0039] In some embodiments of the third aspect, the preparation method provided in this application, wherein (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) can be activated with a chlorinating agent such as oxalic acid or dichlorosulfite, and then reacted with NH3 to obtain compound M2.

[0040] In some embodiments of the third aspect, the preparation method provided in this application can be carried out via the following reaction route: wherein the reactant is (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid, which can be purchased commercially or prepared according to methods in prior art literature. The substituted cinnamic acid compound of this application can be prepared by the following method:

[0041] (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (intermediate A) was dissolved in dichloromethane, and a catalytic amount of N,N-dimethylformamide was added. Oxalic acid was added dropwise under an ice bath, and the mixture was stirred at room temperature until the reactants reacted completely. After the reaction solution was concentrated and dried, dichloromethane was added to dissolve the reactants, and ammonia was added dropwise under an ice bath. The reaction was carried out at room temperature until the reaction was completed. After the solvent was removed by vacuum evaporation, dilute hydrochloric acid was added to adjust the pH to acidity. The precipitated solid was filtered and washed with water to obtain the crude product, which was then purified by silica gel column chromatography to obtain compound M2.

[0042] In an embodiment of the fourth aspect, this application provides the use of compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof, in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic medicament.

[0043] In some embodiments of the fourth aspect, this application provides compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof, for use as a medicament.

[0044] In an embodiment of the fifth aspect, this application provides that compound M2 or its solvate or its pharmaceutically acceptable salt can be used in combination with other antidepressants or anxiolytics, or that compound M2 can be combined with one or more other antidepressants or anxiolytics in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic drug; wherein, the other antidepressant or anxiolytic, or other antidepressant or anxiolytic, may be selected from one or more of the following drugs for the prevention and treatment of mental illness: such as naphazoline, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, lexapro, fluvoxamine, reboxetine, venlafaxine, trifluthixeol, tetramethylammonium benzoate, and levofloxacin, etc.

[0045] In an embodiment of the sixth aspect, this application provides a method for preventing or treating mental illness, the method comprising administering to a patient in need of the compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound M2 or a solvate thereof or a pharmaceutically acceptable salt thereof.

[0046] In some implementations of the sixth aspect, the prevention or treatment of mental illness is an antidepressant, an anti-anxiety agent, or both an antidepressant and an anti-anxiety agent.

[0047] In some embodiments of the sixth aspect, the application may be oral, injectable or transdermal.

[0048] In some embodiments of the sixth aspect, the method of preventing or treating mental illness further includes combining (or combining with) one or more other antidepressants or anxiolytics; wherein the other antidepressants or anxiolytics may be selected from one or more of the following drugs for preventing and treating mental illness: such as naphazoline, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, guanethidine, fluoxetine, paroxetine, sertraline, citalopram, lexapro, fluvoxamine, reboxetine, venlafaxine, trifluthoxane, tetramethylammonium benzoate, and levofloxacin, etc.

[0049] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of the present invention will be described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other. Embodiment 1

[0050] (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (15 g, 0.058 mol) was dissolved in 200 mL of dichloromethane, and 0.1 mL of N,N-dimethylformamide catalyst was added. The mixture was stirred in an ice bath. Oxalic acid chloride (18.3 g, 0.144 mol) was dissolved in 50 mL of dichloromethane and slowly added dropwise to the system. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature until the reactants reacted completely. The reaction solution was concentrated and dried to obtain 16.5 g of crude product. 10 g of the crude product (36 mmol) was dissolved in 50 mL of dichloromethane, and ammonia water (12.6 g, 360 mmol) was slowly added dropwise with stirring in an ice bath. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature until the reaction was complete.

[0051] Concentrated hydrochloric acid was added to the system to adjust the pH to acidic. Dichloromethane was removed under reduced pressure, and the mixture was filtered and dried to obtain 8.67 g of a white crude product. Recrystallization from ethanol-water (volume ratio 1:2) yielded 6.4 g of compound M2. Example 1: Study on the efficacy and effects on brain region function of compound M2.

[0052] The objective of this study is to elucidate the antidepressant effects and brain region functional mechanisms of compound M2 through the following two experiments: 1) Forced swimming experiment: also known as the despair experiment, is usually used to test depressive behaviors in mice. Experimental animals: C57 BL / 6J male mice (18-20g). Group Dosage (mg / kg) Dosage volume (ml / kg) Animal numbers (Only) Administration method Forced swimming test time Solvent control (Vel) - 10 10 IP Detection 24 hours after single administration Esketamine (Esketa) 10 10 10 IP Fluoxetine (Flx) 10 10 10 IP Low dose M2 3 10 10 IP Medium dose M2 10 10 10 IP High dose M2 30 10 10 IP 2) Brain slice electrophysiological experiment: used to detect the effect of compound M2 on excitatory synaptic transmission in mPFC. ● Animals: Newborn litter rats C57 BL / 6J (20-30 days old). ● Instruments: Vibrating microtome (Leica), brain slice electrophysiological system (Axon). ● Brain slice electrophysiological experiment method: ● sEPSC experiment method: After anesthetizing newborn C57 litter rats, artificial cerebrospinal fluid (ACSF) was used for perfusion. Brain tissue was quickly harvested after decapitation and sliced ​​using a vibrating microtome with a thickness of 300 μm. After incubating the brain slices at 28℃ for 1 h, whole-cell recording of mPFC pyramidal neurons was performed using a voltage clamp (-70 mV) with the brain slice electrophysiological system. The baseline recording time was 6 min, after which the test drug (dissolved in ACSF) was added through the perfusion system, and the recording time after drug addition was 6-10 min. The recorded data were analyzed using Minianalysis software, and the effects of the test drugs on the frequency and amplitude of sEPSCs in the medial prefrontal pyramidal neurons of C57 mice were compared. ● Data Statistics

[0053] All data analysis was performed using SPSS data processing software, and the data are expressed as Mean ± sem. One-way ANOVA was used to analyze the effect of compound M2 on sEPSC current under the action of dopamine receptor antagonists; paired t-tests were used to analyze the sEPSC experimental results. p < 0.05 was marked with *. 3) Western blot: used to detect the effect of compound M2 on changes in mPFC protein content ● Animals: C57 BL / 6J male mice (18-20g) were divided into four groups: control group, fluoxetine group, compound M2 (30mg / kg) group, and esketamine group. ● Primary antibody used in the experiment Primary antibody brand Item number p-mTOR Cst 2971s mTOR Cst 2983s p-TrkB Cst 4621s TrkB Cst 4603s PSD95 Cst 3450s Synapsin 1 Cst 5297s β-tubulin Cst 2128s ●Western blot experimental method:

[0054] After C57 mice were administered the compound M2 for 0.5 h and 24 h, brain tissue samples were taken from the prefrontal cortex. After homogenization, the tissue samples were lysed and protein was quantified using the BCA method. The changes in protein content in the prefrontal cortex after intraperitoneal administration of compound M2 for half an hour and 24 hours were detected by steps including SDS-PAGE gel preparation, sample loading and electrophoresis, transfer, blocking, primary antibody incubation, secondary antibody incubation, and protein detection (development and fixing). 3) Experimental results 1. Compound M2 has an antidepressant-like effect

[0055] 24 hours after administration, compound M2 significantly reduced immobility time in mice, exhibiting a clear dose-response effect, with a significant effect observed at a dose of 30 mg / kg, as shown in Figure 1. Compound M2 showed a rapid onset of antidepressant action: Two positive compounds were used as controls in the experiment. Fluoxetine, a first-line clinical drug, had a slower onset of action, while esketamine had a faster onset. The results of this experiment showed that fluoxetine had no antidepressant-like effect 24 hours after a single administration, while both compound M2 and esketamine showed antidepressant-like effects, suggesting that compound M2 has the potential for rapid antidepressant action. 2. Compound M2 significantly enhanced excitatory synaptic transmission in the medial prefrontal cortex (mPFC).

[0056] During the experiment, 10 µM Bicuculine was added to the solution to block GABA receptors, thereby recording the spontaneous excitatory postsynaptic potentials (sEPSCs) of pyramidal neurons. The experiment investigated the effect of different concentrations of compound M2 on the spontaneous excitatory postsynaptic potentials (sEPSCs) of pyramidal neurons in the PrL prefrontal cortex, as shown in Figure 2. The results showed that M2 increased the firing frequency of sEPSCs in the PrL prefrontal cortex in a significant concentration-dependent manner; however, it had no significant effect on the firing amplitude of sEPSCs, as shown in Figure 3.

[0057] The increased frequency of sEPSC release suggests that compound M2 can enhance the release of excitatory glutamate neurotransmitters at prefrontal 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 projected from the midbrain to the prefrontal cortex, or it may be caused by direct action on prefrontal monoamine receptors. Excitatory synaptic transmission in the prefrontal cortex is closely related to antidepressant effects; novel antidepressants such as ketamine can enhance excitatory synaptic transmission in the prefrontal cortex. 3. Compound M2 increases excitatory synaptic transmission in the prefrontal cortex via dopamine D2R receptors 1) Dopamine D2R receptor blocking the enhancing effect of compound M2 on prefrontal sEPSC

[0058] This experiment investigated whether dopamine receptors in the prefrontal cortex affect the effects of compound M2 on the prefrontal cortex. The experiment used Sulpride, an antagonist of dopamine receptor D2R, to block D2R receptors. The results showed that blocking dopamine D2R receptors significantly blocked the enhancing effect of compound M2 on the firing frequency of sEPSCs, suggesting that D2R receptors in the prefrontal cortex may mediate M2-induced excitatory synaptic transmission (Figure 4). 2) Dopamine D1R receptors did not affect the enhancing effect of compound M2 on excitatory synaptic transmission in the prefrontal cortex.

[0059] The application of the dopamine D1R receptor antagonist SCH23390 showed that blocking dopamine D1R receptors did not block the enhancing effect of compound M2 on the firing frequency of sEPSCs, suggesting that D1R receptors in the prefrontal cortex may not be involved in the excitatory synaptic transmission induced by compound M2 (as shown in Figure 5). 4. Compound M2 rapidly initiates prefrontal synaptic generation-related signaling pathways.

[0060] This study investigated the possible intracellular signaling pathways by which compound M2 exerts its antidepressant effect. Since the activation of the mTOR signaling pathway plays a crucial role in rapid depressive response, this study examined changes in upstream and downstream proteins related to the mTOR signaling pathway. The results showed that half an hour after administration of compound M2 (Figure 6.a), the levels of p-mTOR and p-TrkB in the medial prefrontal cortex increased, while the total levels remained unchanged. Literature indicates that the mTOR signaling pathway is associated with the mechanism of rapid depressive disorder, and the rapid antidepressant effect of ketamine may be related to increased phosphorylated mTOR levels 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.

[0061] After the mTOR signaling pathway is activated, the expression level of synaptic-related proteins is further increased. Figure 6.b shows that 24 h after a single dose, compound M2 significantly increased the content of PSD95, a protein related to synapse formation in the medial prefrontal cortex. This result is consistent with the findings of electrophysiological experiments that compound M2 enhances the prefrontal synaptic transmission function. PSD95 is a postsynaptic density protein that is associated with neural plasticity. A literature study (Shinohara R, Aghajanian GK, Abdallah CG. Neurobiology of the Rapid Acting Antidepressant Effects of Ketamine: Impact and Opportunities[J]. Biological Psychiatry, 2020.) has shown that ketamine's antidepressant and anti-anxiety effects are associated with increased expression levels of PSD95 protein.

[0062] Previous studies (Pizzagalli DA, Roberts AC. Correction: Prefrontal cortex and depression[J]. Neuropsychopharmacology, 2021:1-1.) have shown that clinically depressed patients may exhibit morphological features of prefrontal cortex atrophy. Furthermore, in basic experimental studies, mice modeled under chronic social stress showed decreased density of neuronal spikes in the fifth layer of the prefrontal cortex, decreased frequency of excitatory postsynaptic currents (EPSCs), and reduced synaptic transmission of excitatory neurotransmitters in the prefrontal cortex. Excitatory synaptic transmission in the prefrontal cortex is closely related to antidepressant and anti-anxiety effects; drugs like ketamine and fluoxetine can enhance excitatory synaptic transmission in the prefrontal cortex. Therefore, this experiment, based on the pharmacological characteristics of compound M2, focuses on investigating the antidepressant and anti-anxiety effects of compound M2, and studies its influence on the glutamatergic system, an excitatory neurotransmitter in the brain. Using prefrontal lobe brain slices that more closely resemble physiological conditions, the results showed that compound M2 could promote the release of glutamate neurotransmitter and enhance the function of glutamate receptors, suggesting that compound M2 enhances the excitability of the prefrontal lobe in a concentration-dependent manner.

[0063] Based on the above results, it is indicated that compound M2 mainly exerts its rapid antidepressant effect by acting on the glutamatergic neurotransmitter system, thereby enhancing the excitability of the prefrontal cortex and increasing glutamatergic synaptic transmission. Furthermore, compound M2 rapidly activates the prefrontal mTOR signaling pathway. These results suggest that compound M2 possesses a novel antidepressant mechanism with rapid antidepressant effects and enhanced synaptic transmission and brain excitability, demonstrating antidepressant potential superior to clinical monoamine drugs. Example 2: Inhibitory effects of compounds I-5 and M2 on monoamine reuptake in rat synaptosomes.

[0064] It is currently believed that absolute or relative deficiency of central monoamine neurotransmitters serotonin (5-HT), norepinephrine (NA), and dopamine (DA) is closely related to depression and anxiety. This experiment aims to evaluate the inhibitory effects of two samples, compound I-5 and compound M2, on the reuptake of 5-HT, NA, and DA in rat brain synaptosomes using an in vitro monoamine reuptake method. The IC50 values ​​of the two test samples inhibiting the reuptake of DA, 5-HT, and NA were studied using isolated SD rat brain synaptosomes and isotope labeling methods to explore the possible antidepressant and anti-anxiety mechanisms of compounds I-5 and M2. I. Experimental Materials 1. Experimental Animals: Male SD rats, weight: 200-220g 2. Experimental Samples:

[0065] According to the experimental descriptions in Patents A and B, among all compounds, compound I-5 showed the best results in all experiments. Therefore, compound I-5 was selected as a control to compare the inhibitory effect of compound M2 on monoamine reuptake in rat brain synaptosomes. Test sample 1 name: Compound I-5 batch number: PS01068-6-CS The structure is as follows: Sample 2 name: Compound M2 batch number: 20171208 Positive control 1 name: 6-Hydroxydopamine hydrobromide batch number: 0000051379 Positive control: 100 µM can 100% inhibit synaptosome stimulation. 3 H-DA reuptake Positive control 2 name: Fluoxetine hydrochloride batch number: WXBC7489V Positive control: 100 µM can 100% inhibit synaptosome stimulation. 3 H-5-HT reuptake Positive control 3 name: Desipramine hydrochloride batch number: MKCH5352 Positive control: 100 µM can 100% inhibit synaptosome stimulation. 3 H-NA reuptake 3. Preparation of test samples and positive control samples, etc. 3.1 Preparation of test samples

[0066] Weigh the test sample (with a weighing error of ±1%), dissolve it in DMSO, prepare a 10 mM stock solution, and store it at -20℃. Test sample Concentration (mM) Weigh out (mg) Add solvent (mL) Compound I-5 10 5.63 1.79* Compound M2 10 4.48 1.73*

[0067] Before the experiment, the working solution was diluted 100 times with Kreb's solution to the concentration to be tested (1 nM-100 µM) using the serial dilution method. Conversion formulas: Preparation is as follows: Detection concentration (100 µM) — Stock solution 10 mM: 10 μl * Detection concentration (10 µM) — Working solution concentration 1 mM: 100 μl * Kreb's → Final volume to 1000 μl ① Detection concentration (1 µM) — Working solution concentration 100 µM: 100 μl ① Kreb's → Final volume to 1000 μl ② Detection concentration (100 nM) — Working solution concentration 10 µM: 100 μl ② Kreb's → Final volume to 1000 μl ③ Detection concentration (10 nM) — Working solution concentration 1 nM: 100 μl ③ Kreb's → Final volume to 1000 μl ④ Detection concentration (1 nM) — Working solution concentration 100 nM: 100 μl ④ Kreb's → Final volume to 1000 μl ⑤ Note: Vortex mixing is required at each step of the preparation process, and the solution should be protected from light. Prepare the working solution fresh for each experiment. 3.2 Positive control preparation.

[0068] Weigh the reference standard (with a weighing error of ±1%), dissolve it in double-distilled water, prepare a 10 mM stock solution, and store it at -20℃. Positive control Monoamine receptor control concentration (mM) Weighing amount (mg) Add solvent (mL) 6-hydroxydopamine DA 10 2.25 0.899 fluoxetine 5-HT 10 3.66 1.06 desipramine NA 10 3.25 1.07 II. Experimental Methods 1. Preparation of Brain Synapses

[0069] Brain synaptosomes were isolated and prepared using methods described in the literature. SD rats were rapidly decapitated using a small animal decapitation device. After decapitation, the brain was quickly removed and pre-cooled in an ice-water mixture to remove the pia mater and vascular tissue. The brain tissue was then extracted. Ten volumes (ml / g) of 10mM Tris HCl buffer (containing 0.32 M sucrose, pH 7.4) were added, and the mixture was homogenized using an ultrasonic cell disruptor (keeping the temperature low). The homogenate was centrifuged for 10 minutes (4℃, 1,000g). After equilibration centrifugation at 4℃ (1500g, 10min), the precipitate was discarded, and the supernatant was then centrifuged again (20000g) for 30 minutes. The supernatant was discarded, and the precipitate, i.e., the crude extract of synaptosomes, was retained. The precipitate was resuspended in 0.32M cold sucrose solution and carefully spread onto a gradient of 1.2M and 0.8M cold sucrose solutions (10 ml each) layered sequentially from the bottom of the tube. The mixture was then centrifuged at 4°C (38000g) for 60 min. The suspension band at the 0.8–1.2M sucrose interface was carefully collected using a puncture needle, placed in 10 ml of 0.32M cold sucrose solution, mixed, and centrifuged at 4°C (20000g) for 30 min. The precipitate was the purified brain synaptosome. The precipitate was resuspended 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 using the BCA method, following the manufacturer's instructions. 2. Monoamine reuptake

[0070] The methods described in the references have been optimized by the inventors' laboratory. The experimental method is briefly described as follows: Add 950 µl of pre-cooled Kreb's buffer to the reaction tube, followed by 30 µl of synaptosome suspension, and then 10 µl of the test compound (operated on ice). Vortex to mix, and incubate at 37°C for 5 min. Remove the reaction tube and place it on ice, then add 10 µl of substrate (3H-DA, 3H-5HT, or 3H-NA; final reaction concentration: 10 nM). Vortex to mix, and incubate at 37°C for 5 min. The reaction tube was then removed and quickly placed on ice. 3 ml of pre-chilled Kreb's buffer was added to terminate the reaction. Cells were collected using a Millipore cell sample collector, rapidly filtered through a GF / C glass fiber membrane, and elute with 3 ml of 50 mM Tris-HCl (pH 7.4) three times. The membrane was removed, dried in a microwave oven for 5–6 min, and transferred to a 1.5 ml centrifuge tube. 500 µl of lipid-soluble scintillation solution was added. The tube was incubated in the dark for at least 30 min, and the radioactivity intensity (cpm value) was measured.

[0071] Calculate the percentage inhibition rate of each compound on isotopic ligand binding using the following formula: Inhibition rate (I%) = (Total binding tube cpm - Compound cpm) / (Total binding tube cpm - Non-specific binding tube cpm) × 100% For each compound, perform three replicates in triplicate. III. Experimental Results 1. Calculation of IC50

[0072] 1) Let the concentration of the detected compound be X, and the inhibition rate be Y.

[0073] 2) Take the logarithm of X, X'=Log(X), to form new data.

[0074] 3) Perform nonlinear curve fitting on the new data, and then input the fitted data into the following equation to calculate the IC50 value. The above steps are performed using the software GraphpadPrism for fitting.

[0075] 4) Equation Equation: Sigmoidal dose-response (variable slope) Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)) : X is the logarithm of concentration. Y is the response : Y starts at Bottom and goes to Top with a sigmoid shape : This is identical to the “four parameter logistic equation” change combine thing DA 5-HT NA Inhibition rate % IC 50 (nM) Inhibition rate % IC 50 (nM) Inhibition rate % IC 50 (nM) I-5 26.02±1.33 18.52±2.47 17.21±1.59 M2 37.28±1.97 98.87±2.42 23.54±3.56 2. Experiment Summary

[0076] Monoamine reuptake inhibition is one of the main targets of antidepressant and anxiolytic drugs. The test compounds I-5 and M2 both exhibited varying degrees of inhibitory effects on the reuptake of DA, 5-HT, and NA in rat brain synaptosomes. Furthermore, the inhibitory effects of compound I-5 and compound M2 were comparable, suggesting that compounds I-5 and M2 exert their antidepressant and anxiolytic effects by inhibiting the reuptake of DA, 5-HT, and NA.

[0077] The IC50 values ​​of compound I-5 on the reuptake of DA, 5-HT and NA by rat brain synaptosomes were 26.02±1.33 nM, 18.52±2.47 nM and 17.21±1.59 nM, respectively.

[0078] The IC50 values ​​of compound M2 on the reuptake of DA, 5-HT, and NA in rat brain synaptosomes were 37.28±1.97 nM, 98.87±2.42 nM, and 23.54±3.56 nM, respectively. Example 3: Study on the antidepressant effects of compounds I-5 and M2.

[0079] The "behavioral despair" model was developed by Porsolt et al. in 1977 and includes rat and mouse forced swimming models and mouse tail-hanging models, belonging to acute stress models. The mouse tail-hanging test is a simple and easy-to-perform experimental method for evaluating antidepressants, introduced by Stern et al. in 1985. Its principle is the same as the forced swimming "immobility" test. The mice, hanging upside down, struggle to overcome the abnormal position, but after a certain period of activity, the animals exhibit intermittent immobility due to "disappointment".

[0080] This experiment observes whether the test samples, compounds I-5 and M2, affect the tail-hanging behavior of mice in the "behavioral despair" experiment, whether they shorten the time the mice remain still, and compares the rapid onset of action of compounds I-5 and M2. I. Experimental Materials

[0081] 1. Test Sample 1 Name or Code: Compound I-5 Test Sample Number: YLS-2021-CMI1203-002 Batch Number: C16101006-C17001M Preparation Method: The solvent is Tween 80 + water. Emulsify with 1.5% Tween 80 to the final volume, add water to the final volume, and store under the following conditions after preparation: 4℃ refrigerator.

[0082] 2. Test Sample 2 Name or Code: Compound M2 Test Sample Number: YLS-2021-CMI1203-01-001 Batch Number: 20201029 Preparation Method: The solvent is Tween 80 + water. Emulsify thoroughly with 1.5% Tween 80 to the final volume, then add water to the final volume. Temporary storage conditions after preparation: 4℃ refrigerator. II. Experimental Animals

[0083] C57 BL / 6 mice, 40 SPF grade, male, 4-5 weeks old. III. Experimental Methods 1. Animal grouping and dosage design basis

[0084] Forty mice were randomly divided into four groups of ten each using an Excel randomization method. Preliminary experimental results indicated that, 24 hours after administration, compound M2 dose-dependently reduced the immobility time of mice in the forced swimming test, with a significant effect at a dose of 30 mg / kg. Therefore, this study used 30 mg / kg as the low dose of compound M2 and 60 mg / kg as the high dose of compound M2 for comparison with compound I-5 at 60 mg / kg. Grouping and administration details are shown in Table 1. Table 1 Animal Grouping and Administration Group dose Number of animals Administration method Blank control group (normal animals) Purified water + Tween80 10 oral gavage (ig) Group I-5 of compounds 60mg / kg 10 High-dose group of compound M2 60mg / kg 10 low-dose group of compound M2 30mg / kg 10 2. Route of administration and timing of administration

[0085] Each experimental group was administered the drug via oral gavage (ig), at a dose of 0.2 ml / 10 g body weight. 3. Indicator Detection

[0086] A tail suspension test was performed on mice 30 minutes after a single oral administration. The tail suspension duration was 6 minutes. The time the mice remained still within 6 minutes was recorded. 4. Statistical Methods

[0087] Data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis of data differences was performed using one-way ANOVA or non-parametric tests. Differences between groups were considered statistically significant with P < 0.05. IV. Experimental Results

[0088] The results of the single-dose test in mice showed that a single dose of 60 mg / kg compound M2, administered 30 min after administration, significantly shortened the immobility time of mouse tail suspension (p < 0.05 compared to the control group). Although a single dose of 30 mg / kg compound M2 showed a certain trend in reducing the immobility time of mouse tail suspension after 30 min, the difference was not statistically significant. A single dose of 60 mg / kg compound I-5, administered 30 min after administration, had no significant effect on the immobility time of mouse tail suspension (see Table 2 for details). These results suggest that compound M2 has a rapid onset of antidepressant effect and possesses significant antidepressant activity, with a rapid onset of action, significantly superior to compound I-5 in terms of rapid effect. Table 2. Effects of single-dose administration of compounds I-5 and M2 on the tail suspension test in C57 mice (n = 10, ± s) Group Drug dosage Time spent stationary within 6 minutes (s) Blank control group Purified water + Tween80 199.3±29.2 Group I-5 of compounds 60mg / kg 218.0±35.3 High-dose group of compound M2 60mg / kg 171.8±21.7**### low-dose group of compound M2 30mg / kg 183.9±43.5 Note: Compared with the blank control group, ** P < 0.05; compared with compound I-5 group, ### P < 0.01. Experimental Example 4: Absolute bioavailability study of compound I-5 and compound M2 in rats. 1. Experimental Objective

[0089] An LC-MS / MS method for determining compounds I-5 and M2 in rat plasma was established experimentally to investigate the pharmacokinetic characteristics and absolute bioavailability of the compounds in rats. 2. Experimental Materials 2.1 Experimental Animals

[0090] Twenty SPF-grade male SD rats (weighing 220 ± 20 g) were purchased from Beijing Vital River Company. 2.2 Test Sample

[0091] Compound I-5 API, batch number C16101006-C(20170814)M, content: 99.6%, provided by Tasly Pharmaceutical Group Co., Ltd.

[0092] Compound M2 active pharmaceutical ingredient, batch number: 20201029, purity: 99.8%, provided by Tasly Pharmaceutical Group Co., Ltd. 3. Experimental Methods 3.1 Preparation and Dosage of Test Sample PK experiment Group Daily dose (mg / kg / d) Gavage volume (mL / kg / dose) Animal number Configuration method A Compound M2 single-dose gavage administration group 12.5 10 5 Preparation method: Accurately measure 25 mg of the test sample, then add 20 mL of 1.5% Tween diluted water and mix thoroughly. B single intravenous administration group of compound M2 2.5 2 5 Preparation method: Accurately measure 5 mg of the test sample, then add 4 mL of purified water (containing 10% ethanol) and mix well. C Compound I-5 single-dose gavage administration group 12.5 10 5 Preparation method: Accurately measure 25 mg of the test sample, then add 20 mL of 1.5% Tween diluted water and mix thoroughly. D Compound I-5 single intravenous administration group 2.5 2 5 Preparation method: Accurately measure 5 mg of the test sample, then add 4 mL of purified water (containing 10% ethanol) and mix well. 3.2 Determination Methods 3.2.1 Liquid Chromatography-Mass Spectroscopy (LC-MS) Conditions for Compound M2 3.2.1.1 Liquid Chromatography Conditions

[0093] Chromatographic column: Waters BEH C18 1.7μm; 2.1×100mm Column; Internal standard: DI-5 (deuterated compound I-5, structural formula shown below); Mobile phase: 0.1% formic acid aqueous solution (A), acetonitrile (B); isocratic elution (A / B: 58:42 v / v); Flow rate: 0.4ml / min; Column temperature: 40℃; Injection volume: 4 μL; 3.2.1.2 Mass spectrometry conditions

[0094] Ion detection method: Selected reaction monitoring (MRM) Ionization method: Electrospray ionization (ESI) Ion polarity: Positive ion mass spectrometry parameters Target compound ESI mode Q1 Q3 CE Compound I-5 ESI+ 316.00 243.00 -15V Compound M2 ESI+ 260.00 243.10 -15V DI-5 ESI+ 318.00 157.05 -42V 3.3 Pharmacokinetic Experiments

[0095] Twenty SD rats were randomly divided into four groups of five each. Rats were fasted for 10 hours before administration, but had free access to water. 0.3 mL of blood was collected from the orbital vein 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 samples were placed in EDTA-K2 anticoagulant tubes, gently shaken to ensure thorough mixing with the anticoagulant, placed on moist ice, and centrifuged within 0.5 hours.

[0096] Centrifugation: After centrifugation at 8000 rpm for 5 min, separate the plasma.

[0097] Storage: Frozen at -20℃. 3.4 Plasma Sample Processing Methods

[0098] Take 50 μL of plasma, add 50 μL of DI-5 (dissolved in 20 ng / mL acetonitrile), vortex for 30 s, then add 100 μL of acetonitrile; vortex thoroughly for 2 min, centrifuge at 12000 r / min for 3 min at 4℃, take 50 μL of supernatant, dilute with 50 μL of aqueous solution, vortex for 1 min, and inject 4 μL. 4. Pharmacokinetic Results Analysis

[0099] The mean plasma concentration-time curves after oral gavage and intravenous administration of compound M2 to rats are shown in Figures 8 and 9, respectively. Data processing was performed using DAS3.0 pharmacokinetic software (Chinese Society for Mathematical Pharmacology, Shanghai, China), and the statistical moment parameters of the non-compartmental model were calculated. Cmax and Tmax are the measured values. Detailed plasma concentration data and pharmacokinetic parameters are shown in the table below. Table 3: Plasma concentrations (ng / mL) of compound M2 (12.5 mg / kg) in rats after a single oral gavage administration. Time (h) Rat1 Rat2 Rat3 Rat4 Rat5 Mean Sd 0 BLOQ BLOQ BLOQ BLOQ BLOQ / / 0.08 78.15 127.05 78.40 240.77 433.00 191.47 150.45 0.25 213.75 487.46 212.80 494.43 582.67 398.22 172.95 0.50 270.87 411.67 291.90 416.38 542.56 386.67 109.77 1 144.95 184.82 165.92 270.26 288.12 210.81 64.30 2 90.66 71.40 131.47 231.27 133.28 131.62 61.71 3 175.71 60.26 194.74 114.91 122.55 133.63 53.29 4 268.66 107.99 188.58 66.64 188.12 164.00 78.66 6 57.55 48.74 11.84 15.99 299.46 86.72 120.58 8 BLOG 5.38 BLOG BLOG 3.64 2.07 2.38 BLOQ: Blood drug concentration (ng / mL) of compound M2 (2.5 mg / kg) after a single intravenous bolus injection in rats, below the detection limit. (Table 4) Time (h) Rat1 Rat2 Rat3 Rat4 Rat5 Mean Sd 0 BLOQ BLOQ BLOQ BLOQ BLOQ / / 0.08 1088.43 1221.94 1589.64 991.98 1170.61 1212.52 228.08 0.25 574.71 742.62 807.81 497.57 574.42 639.42 129.93 0.50 281.81 435.1 406.94 201.98 248.89 314.96 101.40 1 74.30 130.96 92.59 39.74 57.32 78.98 35.06 2 4.62 12.27 2.23 BLOQ 2.72 5.46 4.65 3 2.035 4.403 BLOQ BLOQ BLOQ 3.21 1.67 4 BLOQ BLOQ BLOQ BLOQ BLOQ 第34行 / / 6 BLOQ BLOQ BLOQ BLOQ BLOQ / / 8 BLOQ BLOQ BLOQ 第61行 BLOQ BLOQ / / Plasma concentration (ng / mL) of compound I-5 after single oral administration of 12.5 mg / kg to rats Time (h) Rat1 Rat2 Rat3 Rat4 Rat5 Mean Sd 0 BQL BQL BQL BQL BQL / / 0.08 140 3.59 70.1 214 164 118.3 82.5 0.25 706 7.11 371 389 361 366.8 247.5 0.50 735 4.71 416 268 246 333.9 268.4 1 450 BQL 391 92.3 132 266.3 180.4 2 224 BQL 134 35.8 43.0 109.2 88.6 3 84.5 BQL 58.4 11.3 23.6 44.4 33.3 4 27.3 BQL 17.3 10.7 6.16 15.3 9.2 6 5.64 BQL 3.20 5.66 12.2 6.6 3.9 8 BQL BQL BQL BQL BQL / / BLOQ: Below the detection limit. Table 6: Plasma concentration (ng / mL) of compound I-5 in rats after a single intravenous bolus injection of 2.5 mg / kg. Time (h) Rat1 Rat2 Rat3 Rat4 Rat5 Mean Sd 0 BQL BQL BQL BQL BQL / / 0.08 1750 1150 659 865 1350 1154.8 424.9 0.25 628 247 540 260 291 393.2 177.7 0.50 243 75.7 225 95.9 120 151.9 76.8 1 103 24.9 80.2 52.0 41.6 60.3 31.2 2 25.1 6.66 19.3 15.2 17.7 16.8 6.7 3 8.95 2.49 9.76 5.45 7.89 6.9 3.0 4 3.01 BQL 3.38 2.60 3.08 3.0 0.3 6 BQL BQL BQL BQL BQL 8 BQL BQL BQL BQL BQL Table 7. Pharmacokinetic parameters (Mean ± SD) of compound M2 administered by gavage and intravenous in rats (n=5) Parameters unit Intravenous (2.5 mg / kg) Gavage (12.5 mg / kg) M2 M2 t 1 / 2 h 0.24±0.03 1.03±0.34 C 0 / C max ng / mL 1641.93±319.23 425.47±136.97 t max h - 0.35±0.14 AUC 0-t ng.h / mL 529.41±129.86 1080.11±373.43 AUC 0-∞ ng.h / mL 532.41±126.82 1122.48±366.77 MRT 0-∞ h 0.31±0.06 2.52±0.61 F (Bioavailability, %) - 40.80 Table 8. Pharmacokinetic parameters (Mean ± SD) of compound I-5 after gavage and intravenous administration in rats (n=5) parameter unit Intravenous (2.5 mg / kg) Gavage (12.5 mg / kg) I-5 I-5 t 1 / 2 h 0.66±0.07 0.93±0.38 C 0 / C max ng / mL 2046.5±906.60 361.1±258.2 t max h / 0.35±0.14 AUC 0-t ng.h / mL 435.6±138.4 523.3±433.3 AUC 0-∞ ng.h / mL 438.3±138.8 658.9±370.6 MRT 0-∞ h 0.37±0.11 1.16±0.12 F (Bioavailability, %) - 24.05 5. Discussion

[0100] Pharmacokinetic studies of rats after gavage and intravenous administration of compounds M2 and I-5 showed that both compounds M2 and I-5 reached peak absorption relatively quickly after gavage administration, with a peak time (tmax) of 0.35 h. The peak concentrations (Cmax) of compound M2 were 425.4 ± 136.9 ng / mL, and those of compound I-5 were 361.1 ± 258.2 ng / mL, indicating that compound M2 had a higher plasma concentration. The AUC0-t of compound M2 was 1080.1 ± 373.4 ng·h / mL, and that of compound I-5 was 523.3 ± 433.3 ng·h / mL. At the same gavage dose, compound M2 showed a higher exposure in rats than compound I-5, and also had higher absolute bioavailability, at 40.8% and 24.05%, respectively. Example 5: Study on the anti-anxiety effect of M2 and its structural analogues. I. Experimental Materials 1. Test Samples

[0101] According to the results of two experiments (mouse elevated cross maze test and rat drinking conflict test) in the specification of Patent C, the compounds with better effects in both groups of experiments are II-3, II-4, II-5 and II-10. Therefore, these four compounds were selected as controls to evaluate the anti-anxiety effect of compound M2 in this application.

[0102] ① Name or code: Compound M2 Batch number: 20201029 Test sample number: YLS-2021-CMI1203-01-001 Source: Chemical Drug Development Center, Tasly Research Institute

[0103] ② Name or code: Compound II-3 (i.e., Compound I-3 in patents A and B) Batch number: 20180521 Test sample number: YSL-2021-CMI1203-01-023 Source: Chemical Drug Development Center, Tasly Research Institute

[0104] ③ Name or code: Compound II-4 (i.e., Compound I-4 in patents A and B) Batch number: 20211217 Test sample number: YSL-2022-CMI1203-01-001 Source: Chemical Drug Development Center, Tasly Research Institute

[0105] ④ Name or Code: Compound II-5 (i.e., Compound I-5 in Patents A and B) Batch Number: C16101006-C17001M Test Sample Number: YSL-2021-CMI1203-01-002 Source: Chemical Drug Development Center, Tasly Research Institute

[0106] ⑤ Name or code: Compound II-10 (i.e., Compound I-10 in patents A and B) Batch number: 20170601 Test sample number: YSL-2021-CMI1203-01-023 Source: Chemical Drug Development Center, Tasly Research Institute 2 Positive control

[0107] Name or Code: Estazolam Tablets Batch Number: 211007 Test Sample Number: YSL-2021-CMI1203-01-022 Source: Shandong Xinyi Pharmaceutical Co., Ltd. 3 Main Instruments Instrument Name Instrument Model Manufacturer Inspection items Balance T-1000 Changshu Shuangjie Testing Instrument Factory Weigh yourself Electronic balance MS204S Mettler Toledo Instruments Shanghai Co., Ltd. Weigh the test sample elevated cross maze NA Nanjing Calvin Anti-anxiety test Water-based electric shock anxiety testing system LE100-25 Panlab Anti-anxiety test II. Laboratory Animals 2.1 Laboratory Animals

[0108] ① Species: ICR mice (used for elevated plus maze experiment), Quantity: 120, Grade: SPF level, Gender: male, Body weight: 18 - 20 g, Animal qualification certificate number: 110011211113772653, 110011221101333628, Source: Beijing Vital River Laboratory Animal Technology Co., Ltd., Production license number: SCXK(Beijing)2021 - 0006

[0109] ② Species: SD rats (used for water conflict experiment), Quantity: 75, Grade: SPF level, Gender: male, Body weight: 180 - 200 g, Animal qualification certificate number: 110011221102490324, Source: Beijing Vital River Laboratory Animal Technology Co., Ltd., Production license number: SCXK(Beijing)2021 - 0011 2.2 Animal facilities

[0110] Feeding facilities: Inside the barrier environment of the animal facilities of Tianjin Tasly Holding Group Co., Ltd., Facility address: Inside the factory area at the intersection of Huaihe Road and Tingjiang West Road in Beichen Science and Technology Park, Tianjin, Experimental animal use license: SYXK(Tianjin)2017-0007, Issuing unit: Tianjin Science and Technology Commission 2.3 Animal feeding and management

[0111] Feeding environment: Barrier environment. The environmental conditions of this facility comply with the relevant standards of the national standard of China, "Laboratory Animal Environment and Facilities" (GB14925 - 2001) for barrier animal experiment facilities. Animal feeding management and animal experiment operations comply with the requirements of regulations such as the "Tianjin Regulations on Laboratory Animal Management". 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 prepared by a 1T / h type multi - microporous membrane filtration system (four - stage filtration and ultraviolet sterilization).

[0112] Animal management is the responsibility of the Animal Protection Department. Except for fasting, sufficient feed and drinking water are provided to animals every day. The drinking water bottles are changed once a day. The animal feeding bedding is changed twice a week, and is changed at any time under special circumstances. The feeding cages are changed once a week; Animal feed: Purchased from Beijing Keao Xieli Feed Co., Ltd., Production license number: SCXK(Beijing)2019 - 0003. 2.4 Animal reception and quarantine

[0113] Upon arrival of the laboratory animals, the laboratory personnel, veterinarians, and animal welfare department jointly receive them. Upon receipt, the first step is to check whether the transport vehicle meets the requirements. Then, the animal qualification certificate provided by the animal supplier is reviewed, and it is confirmed that the certificate information matches the species, grade, quantity, and sex of the animals requested for purchase. Next, the outer packaging is checked to ensure it meets the requirements and that the animal packaging is undamaged. The animal packaging is transferred to the quarantine room via the first transfer cabinet. Laboratory equipment and experimental records are transferred via the second transfer cabinet.

[0114] In the quarantine room, open the animal packaging and verify that the sex and quantity of the animals match the information on the animal certificate. Conduct a physical examination on each animal (including sex, weight, head, torso, tail, limbs, fur, mental state, and activity level), and complete the "Laboratory Animal Receipt Record" and "Laboratory Animal Quarantine Record." After inspection, place the animals in animal cages, hang quarantine period labels on the cages, and then place them in the quarantine room for an acclimatization period.

[0115] The animal's adaptation period is 2-3 days. Regularly observe the animals, including weight, head, trunk, tail, limbs, fur, mental state, activity, etc. III. Experimental methods 3.1 Single dose exploration study of compound M2 (elevated cross maze experiment) (1) Animal grouping and administration

[0116] Fifty male ICR mice were randomly divided into five groups (solvent control group, estazolam group, high-dose M2 group, medium-dose M2 group, and low-dose M2 group), with ten mice in each group. The high-, medium-, and low-dose groups of compound M2 were administered via gavage at doses of 20 mg / kg, 10 mg / kg, and 5 mg / kg, respectively; the positive control group was administered via gavage at a dose of 2.5 mg / kg; and the solvent control group was administered an equal volume of solvent via gavage. Behavioral tests were performed on all groups 30 minutes after each administration. The experiment was conducted between 8:00 AM and 2:00 PM, and all animals were brought to the testing laboratory the day before.

[0117] Dosage Design Basis: Previous anti-anxiety experiment results (Patent C) showed that the effective dose of structural analogs of compound M2, such as compounds II-3, II-4, and II-5, in the elevated cross maze test in mice was 10 mg / kg. This experiment intends to use 10 mg / kg as the intermediate dose of compound M2, exploring a lower dose of 5 mg / kg and a higher dose of 20 mg / kg. The clinical dosage of the positive control drug estazolam for anti-anxiety is 6 mg / person / day, which translates to a clinically equivalent dose of 6 mg / 60 kg * 12.3 = 1.23 mg / kg in mice, and twice the clinical dose is 2.5 mg / kg; the clinically equivalent dose in rats is 6 mg / 60 kg * 6.2 = 0.62 mg / kg, and twice the clinical dose is 1.24 mg / kg. Table 9. Single-dose Dosage Exploration of Compound M2—Groups and Dosages Group dose Number of animals per group Administration method Solvent control group --- 10 oral gavage single estazolam group 2.5 mg / kg 10 M2 high-dose group 20mg / kg 10 M2 medium-dose group 10mg / kg 10 M2 low-dose group 5mg / kg 10 (2) Behavioral testing

[0118] The laboratory is dimly lit (the lowest brightness at which the subtle movements of mice can be distinguished at a distance of 1.5m) and kept constantly lit. The room temperature is about 20℃ and the room is kept quiet. Before the maze test, each mouse is placed in a 35cm*10cm*5cm plastic box and allowed to explore freely for 5 minutes. Then, it is 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 are recorded. Each mouse is tested for 5 minutes. The observers observe and record the activities of the animals at a distance of 1.5m. The maze is wiped with a damp cloth to remove feces. After wiping it with a dry cloth, the next mouse is tested. (3) Behavioral observation indicators

[0119] ① Number of times the mouse enters the open arm (OE): The number of times the mouse enters any open arm, which is determined by the fact that all four paws of the mouse enter the arm. The entry activity is considered complete when one paw completely withdraws from the arm. ② Open arm time (OT): The time to enter the open arm, in seconds. ③ Number of times the mouse enters the closed arm (CE): The number of times the mouse enters any closed arm, which is determined by the fact that all four paws of the mouse enter the arm. ④ Close arm time (CT): The time to enter the closed arm, in seconds. ⑤ Percentage of open arm time: OT%=OT / (OT+CT)*100% ⑥ Percentage of open arm times: OE%=OE / (OE+CE)*100% 3.2 Comparative study of the efficacy of compound M2 and its structural analogs after a single dose (elevated cross maze experiment) (1) Animal grouping and administration

[0120] 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 ten mice in each group. The dosage of M2 and its four structural analogs was set at 20 mg / kg based on the experimental results in 3.1; the positive control drug estazolam was administered by gavage at 2.5 mg / kg; and the solvent control group received an equal volume of solvent by gavage. Behavioral tests were performed on all groups 30 minutes after a single administration. The experiment was conducted between 8:00 AM and 2:00 PM, and all animals were brought to the testing laboratory the day before. Table 10. Elevated Cross Maze Single-Dose Test for Compound M2 and its Structural Analogs—Groups and Doses Group dose Number of animals per group Administration method Solvent control group --- 10 oral gavage single estazolam group 2.5 mg / kg 10 Group M2 of compounds 20mg / kg 10 Group II-3 of Compounds 20mg / kg 10 Group II-4 of compounds 20mg / kg 10 Group II-5 of Compounds 20mg / kg 10 Group II-10 of compounds 20mg / kg 10 The behavioral testing methods and behavioral observation indicators are the same as in 3.1. 3.3 Comparative study of the efficacy of compound M2 and its structural analogues after a single dose (water conflict experiment) (1) Animal grouping and administration

[0121] Seventy-five male SD rats, weighing 180-220g, were acclimatized for one week and then deprived of water for 24 hours for a training period test. Qualified animals 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 eight rats in each group. The dosage of compound M2 and its four structurally similar compounds was determined to be 10 mg / kg based on the experimental results in 3.1; the positive control drug estazolam was administered by gavage at 1.24 mg / kg; and the solvent control group was administered an equal volume of solvent by gavage. The grouped animals were deprived of water for another 24 hours before a single administration, and a punishment test was conducted 30 minutes after the single administration. Table 11. Water conflict of single administration of compound M2 and its structural analogs—groups and dosages. Group dose Number of animals per group Administration method Solvent control group --- 8 oral gavage single estazolam group 1.24 mg / kg 8 Group M2 of compounds 10mg / kg 8 Group II-3 of Compounds 10mg / kg 8 Group II-4 of compounds 10mg / kg 8 Group II-5 of Compounds 10mg / kg 8 Group II-10 of compounds 10mg / kg 8

[0122] The experiment was conducted in two phases. The first phase was the training period. After being deprived of water for 24 hours, the animals were placed alone in the operating box to explore until they found the bottle mouth and began licking the water (electric shock intensity 0 mA). The counter automatically recorded the number of times the animals licked the water within 3 minutes. Animals that licked the water less than 300 times were eliminated. The second phase was the punishment period. Animals that were not eliminated were deprived of water for another 24 hours before being given medication. They were then placed alone in the operating box again. The instrument automatically started timing and gave an electric shock after the animals licked the water 20 times (the ratio of licking to electric shock was 20:1). The electric shock intensity was generally 0.3 mA and lasted for 2 seconds, but the animals could remove the electric shock by removing themselves from the bottle mouth. The number of times the animals licked the water and the number of electric shocks were recorded over 3 minutes.

[0123] Observation index: Number of times the rat licked water during the punishment period. 3.4 Statistical methods

[0124] Data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis of data differences was performed using one-way ANOVA or non-parametric tests. Differences between groups were considered to be p < 0.05. IV. Experimental Results 4.1 Exploratory Study of Single-Dose Administration of Compound M2 (Elevated Cross Maze Experiment)

[0125] The results of the single-dose dose exploration study of compound M2 showed that single gavage administration of compound M2 at high (20 mg / kg), medium (10 mg / kg), and low (5 mg / kg) doses could increase the number of times mice entered the open arm in the elevated cross maze experiment and prolong the time mice stayed in the open arm. Among them, the high-dose group of compound M2 was significantly different from the solvent control group (P < 0.05, P < 0.01).

[0126] The above results indicate that compound M2 has significant anxiolytic activity when administered as a single dose, and the effective dose for a single dose is 20 mg / kg. Table 12. Effects of different doses of compound M2 on the number of times mice enter the open arm and the duration of stay in the open arm. Group dose OE% OT% Solvent control group --- 29.36±10.96 26.53±13.42 estazolam group 2.5 mg / kg 42.78±15.27 * 50.12±12.76 ** High-dose group of compound M2 20mg / kg 38.47±7.13 * 44.60±9.33 ** Medium dose group of compound M2 10mg / kg 36.13±14.12 35.92±13.11 low-dose group of compound M2 5mg / kg 35.46±10.13 38.52±12.19 Note: Compared with the solvent control group, *P<0.05, **P<0.01 4.2 Comparative Study of the Efficacy of Compound M2 and its Structural Analogs after a Single Dosage (Elevated Cross Maze Experiment)

[0127] The results of the elevated cross maze experiment showed that a single oral administration of compound M2 and its structural analogs II-3, II-4, II-5, and II-10 at 20 mg / kg could increase the number of times mice entered the open arm in the elevated cross maze experiment and prolong the time mice stayed in the open arm. Among them, the groups of compounds M2, II-4, II-5, and II-10 showed significant differences compared with the solvent control group (P < 0.05, P < 0.01), indicating that compounds M2 and their structural analogs II-4, II-5, and II-10 have significant anxieties in this model, and compound M2 has the best anxieties compared with II-3, II-4, II-5, and II-10. Table 13. Effects of compound M2 and its structural analogs on the number of times mice entered the open arm and the time they stayed in the open arm. Group dose OE% OT% Solvent control group --- 33.03±9.45 36.52±11.47 estazolam group 2.5 mg / kg 69.60±15.12 ** 73.46±20.29 ** Group M2 of compounds 20mg / kg 57.94±11.72** 57.36±8.77 ** Group II-3 of Compounds 20mg / kg 41.04±9.52 ## 42.99±11.82 ## Group II-4 of compounds 20mg / kg 46.79±9.31 **# 53.45±8.33 ** Group II-5 of Compounds 20mg / kg 47.01±6.30 **# 46.70±9.98 *# Group II-10 of compounds 20mg / kg 50.00±10.23 ** 52.08±12.25 ** Note: Compared with the solvent control group, *P<0.05, **P<0.01; compared with M2, #P<0.05, ##P<0.01. 4.3 Comparative study of the efficacy of compound M2 and its structural analogues after a single dose (water conflict experiment).

[0128] The results of the drinking conflict experiment showed that a single oral administration of compound M2 and its structural analogs II-3, II-4, II-5, and II-10 at 20 mg / kg could increase the number of times rats drank water in the drinking conflict experiment to varying degrees. Among them, the groups of compounds M2, II-5, and II-10 showed significant differences compared with the solvent control group (P < 0.05), indicating that compounds M2 and their structural analogs II-5 and II-10 have significant anti-anxiety activity in this model, and compound M2 has the best anti-anxiety activity compared with II-3, II-4, II-5, and II-10. Table 14. Effects of compound M2 and its structural analogs on the number of times rats drank water in the Vogel drinking conflict model. Group dose Number of animals per group Number of times to drink water Solvent control group --- 8 306.94±203.83 estazolam group 1.24 mg / kg 8 540.41 / kg 2.25* Group M2 of compounds 10mg / kg 8 527.26±143.66* Group II-3 of Compounds 10mg / kg 8 363.96±145.26# Group II-4 of compounds 10mg / kg 8 387.07±57.31 # Group II-5 of Compounds 10mg / kg 8 478.02±106.32* Group II-10 of compounds 10mg / kg 8 499.61±133.93* Note: Compared with the solvent control group, *P<0.05; compared with M2, #P<0.05. V. Experimental Conclusions

[0129] In summary, compound M2 has significant anxiolytic activity, with an effective dose of 20 mg / kg (mice) after a single administration. Moreover, compound M2 exhibits the best anxiolytic activity compared to its structural analogs, compounds II-3, II-4, II-5, and II-10. [Simplified Explanation of the Diagram]

[0016] Figure 1 shows that compound M2 has an antidepressant-like effect: 24 h after administration, compound M2 significantly reduced immobility time in mice, showing a significant dose-response effect, and the effect was significant at a dose of 30 mg / kg. Figure 2 shows the effect of compound M2 on excitatory synaptic transmission in the prefrontal cortex: the figure shows the effect of compound M2 concentrations of 10 nM, 10 µM, and 150 µM on sEPSCs of pyramidal neurons in the PrL region of the prefrontal cortex. The upper figure is a schematic diagram of sEPSC waveforms, and the lower figure is a bar chart after data statistics. Figure 3 shows that compound M2 enhances excitatory synaptic transmission in the prefrontal cortex: at various concentrations, compound M2 increased the firing frequency of sEPSCs in the PrL region of the prefrontal cortex, showing a significant concentration-dependent effect; however, it did not significantly affect the firing amplitude of sEPSCs. Among them, 150 µM compound M2 significantly enhanced the firing frequency of sEPSCs. Figure 4 illustrates the enhancing effect of the dopamine D2R receptor antagonist Sulpride on prefrontal sEPSCs by blocking compound M2: Sulpride, at a working concentration of 10 μM, blocks D2R receptors, indicating that blocking D2R can block the enhancing effect of 150 µM compound M2 on sEPSC firing frequency. Figure 5 illustrates the enhancing effect of the dopamine D1R receptor antagonist SCH23390 on prefrontal sEPSCs by blocking compound M2: SCH23390, at a working concentration of 10 μM, blocks D1R receptors, indicating that blocking D1R can block the enhancing effect of 150 µM compound M2 on sEPSC firing frequency. Figure 6 illustrates the activation of mTOR-related signaling pathways by compound M2: a shows the changes in prefrontal-related signaling proteins 30 min after a single dose; b shows the changes in prefrontal-related signaling proteins 24 h after a single dose. Figure 7 shows the inhibitory effects (IC50) of compounds I-5 and M2 on the uptake of DA, 5-HT, and NA by rat brain synaptosomes. Figure 8 shows the mean drug-time curves after oral and intravenous administration of compound M2 to rats. Figure 9 shows the mean drug-time curves after oral and intravenous administration of compound I-5 to rats.

Claims

1. A substituted cinnamic acid compound with the following structural formula: .

2. The compound as claimed in claim 1, wherein, The compound can also exist in pharmaceutically acceptable salt forms.

3. A pharmaceutical composition comprising a compound as described in claim 1 or claim 2.

4. The pharmaceutical composition as claimed in claim 3 may be any oral pharmaceutical form; optionally, the pharmaceutical form is selected from: tablets, capsules, oral liquids, lozenges, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, and patches; the tablets are optionally sugar-coated tablets, film-coated tablets, or enteric-coated tablets; the capsules are optionally hard capsules or soft capsules; the injections are optionally one of injection solutions, lyophilized powder for injection, and aqueous injections.

5. The pharmaceutical composition as described in claim 3 may be used in combination with other antidepressants or anti-anxiety medications, or the pharmaceutical composition may further include other antidepressants or anti-anxiety medications selected from: naphazoline, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, lexapro, fluvoxamine, reboxetine, venlafaxine, trifluthixeol, tetramethylammonium chloride, and loratadine.

6. A method for preparing the compound as claimed in claim 1, wherein the method comprises reacting (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)-acrylic acid (i.e., intermediate A) with NH3 to give compound M2.

7. A method for preparing the compound as described in claim 6, comprising the following reaction route: .

8. The preparation method as described in claim 7, comprising the following steps: (E)-3-(3',4'-methylenedioxy-5'-trifluoromethyl-phenyl)acrylic acid (intermediate A) is dissolved in dichloromethane, and an amount of N,N-dimethylformamide is added as a catalyst. Oxalic acid is added dropwise under an ice bath, and the mixture is stirred at room temperature until the reaction is complete. After the reaction solution is concentrated and dried, dichloromethane is added to dissolve the solution, and ammonia is added dropwise under an ice bath. The reaction is carried out at room temperature until the reaction is complete. After the solvent is removed by vacuum evaporation, dilute hydrochloric acid is added to adjust the pH to acidity. The precipitated solid is filtered and washed with water to obtain the crude product, which is then purified by silica gel column chromatography to obtain M2.

9. The compound as claimed in claim 1, for use as a medicine.

10. The pharmaceutical composition as described in any one of claims 3 to 5, for use as a medicament.

11. Use of a compound or pharmaceutical composition in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic medicament, wherein the compound is any one of claims 1 to 2, or the pharmaceutical composition is any one of claims 3 to 5 in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic medicament.

12. Use of a compound in combination with one or more other antidepressants or anxiolytics in the preparation of an antidepressant, anxiolytic, or antidepressant and anxiolytic medicament, wherein the compound is the compound as described in any one of claims 1 to 2; wherein the other antidepressant or anxiolytic may be selected from one or more of the following medications for the prevention and treatment of mental illness: naphazoline, sulpiride, alprazolam, lorazepam, buspirone, tandospirone, methylphenidate, fluoxetine, paroxetine, sertraline, citalopram, lexapro, fluvoxamine, reboxetine, venlafaxine, trifluthixeol, tetramethylammonium phosphate, and levofloxacin.

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