PYRROLOTRIAZINONE COMPOUND, PHARMACEUTICAL COMPOSITION COMPRISING SAME, PREPARATION METHOD THEREFOR, AND USE THEREOF

Provided are a pyrrolotriazinone compound represented by formula I or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising same, a preparation method therefor, and use thereof. The pyrrolotriazinone compound has agonistic activity on GPR139 receptor.

The present application claims the right of priority of Chinese patent application No. 202210195902X filed on Mar. 1, 2022. The contents of the above Chinese patent application are incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to a pyrrolotriazinone compound, a pharmaceutical composition comprising same, a preparation method therefor, and a use thereof.

BACKGROUND

Schizophrenia affects more than 20 million people worldwide. Its symptoms can be roughly divided into three categories based on their clinical characteristics: positive symptoms, negative symptoms, and cognitive dysfunction. Positive symptoms refer to exaggerations or distortions of normal functional behaviors, including hallucinations, delusions, confusion, etc. Negative symptoms refer to some defects in normal emotional responses or other thought processes, including flat affect, alogia, loss of motivation and pleasure, and depressive-like features such as a negative attitude and social withdrawal. Cognitive dysfunction mainly manifests as decline in attention, memory, learning ability, etc.

Based on the historical context of drug development and pharmacological properties, current anti-schizophrenia drugs can be divided into three generations. First-generation anti-schizophrenia drugs, also known as typical anti-schizophrenia drugs, are potent antagonists of dopamine D2 receptors. Currently commonly used first-generation anti-schizophrenia drugs include haloperidol, chlorpromazine, perphenazine, etc., which can effectively suppress the positive symptoms of schizophrenia, but long-term use of these drugs can induce extrapyramidal reactions similar to Parkinson's disease. Second-generation anti-schizophrenia drugs, including clozapine, risperidone, and paliperidone, exert their effects by dual blockade of dopamine D2 receptors and serotonin 2A (5-HT2A) receptors. Since inhibition of 5-HT2A receptors can indirectly promote the release of dopamine from dopaminergic neurons, the extrapyramidal side effects induced by second-generation drugs are significantly reduced. However, the risk of metabolic syndrome (excessive obesity, insulin resistance, dyslipidemia, and hypertension) is higher with second-generation drugs than with typical anti-schizophrenia drugs. The third-generation anti-schizophrenia drugs include aripiprazole, brexpiprazole, and cariprazine, which are partial agonists of dopamine D2 receptors. As a “stabilizer” for dopamine, the third-generation drugs stabilize the activity of the brain's dopamine circuit in a moderate range, greatly reducing extrapyramidal side effects and significantly improving safety. The second and third generations are also collectively known as atypical anti-schizophrenia drugs. Currently, all drugs for the treatment of schizophrenia can effectively alleviate positive symptoms, but most drugs have limited or ineffective efficacy in improving negative symptoms and cognitive function. Therefore, there is an urgent need to develop drugs with novel mechanisms of action targeting the negative symptoms and cognitive disorder associated with schizophrenia.

The orphan receptor GPR139 was identified from bioinformatics analysis of the human genome and belongs to class A G protein-coupled receptors (GPCRs). In mammals, GPR139 is mainly expressed in the central nervous system, with the highest expression sites being the striatum, pituitary, habenula, thalamus, and hypothalamus (Matsuo et al., Biochem Biophys Res Commun 2005, 331:363-369). In both rodents and humans, GPR139 is highly expressed in the medial habenula. Several studies have shown a link between the habenula and schizophrenia. Overall structural damage to the rodent habenula can lead to schizophrenia-related symptoms such as reduced social activities, cognitive impairment, and excessive responses to stressful stimuli (Wang et al., Neuroreport 2013, 24:276-280). Patients with chronic schizophrenia have a higher frequency of habenula calcification and changes in habenula volume than normal individuals (Sandyk et al., Int J Neurosci 1992, 67:19-30). Functional magnetic resonance imaging (fMRI) studies have shown that, during a matching task, the habenula is activated in normal individuals when an error occurs, but in patients with chronic schizophrenia, their habenula is not significantly activated after a matching error occurs (Shepard et al., Schizophr Bull 2006, 32:417-421). The GPR139 knockout mouse model shows behavioral characteristics associated with schizophrenia such as decreased spontaneous movement, sensorimotor gating defects, and cognitive disorder. Symptoms can be improved by administering the dopamine D2 receptor antagonist haloperidol (Dao et al., Neuropsychopharmacology 2021). Based on these results, targeting GPR139 has the potential to develop new anti-schizophrenia drugs, especially drugs targeting negative symptoms.

The selective GPR139 agonist JNJ-63533054 (EC50=16 nM) reported in 2018 can reduce self-drinking and hyperalgesia in alcohol-dependent rats (Kononoff et al., eNeuro 2018, 5), but did not show obvious behavioral modulation in in vivo studies. In 2021, Takeda Company of Japan announced TAK-041, a selective GPR139 agonist with a benzotriazinone structure (EC50=22 nM), which has been confirmed to be effective in treating negative symptoms associated with schizophrenia in a mouse model (Reichard et al., J Med Chem 2021, 64:11527-11542) and has successfully entered Phase II clinical trials.

CONTENT OF THE PRESENT INVENTION

The technical problem to be solved by the present disclosure is the defect of the existing GPR139 receptor agonists with a single structure, and the present disclosure provides a pyrrolotriazinone compound, a pharmaceutical composition comprising same, a preparation method therefor, and a use thereof. The pyrrolotriazinone compound of the present disclosure has a novel structure and has strong agonistic activity on GPR139 receptors.

The present disclosure solves the above technical problem through the following technical solutions.

The present disclosure provides a pyrrolotriazinone compound of formula I or a pharmaceutically acceptable salt thereof;

In a certain embodiment, in the pyrrolotriazinone compound of formula I or the pharmaceutically acceptable salt thereof, certain groups can be defined as described below, and other groups can be defined as described in any embodiment of the present disclosure (hereinafter referred to as “in a certain embodiment”):

In a certain embodiment,

for example,

In a certain embodiment, R1, R2, and R3 are independently H or C1-6 alkyl.

In a certain embodiment, R4 is independently H.

In a certain embodiment, R5 is H.

In a certain embodiment, R6 is H.

In a certain embodiment, R7 is C1-6 alkyl or C1-6 alkyl substituted by 1, 2, or 3 R6-1.

In a certain embodiment, R6-1 is independently —NRaRb.

In a certain embodiment, Ra and Rb are independently C1-6 alkyl. In a certain embodiment, n is 0 or 1.

In a certain embodiment, in R1-1, R1-2, and R1-3, the halogen is F, Cl, Br, or I.

In a certain embodiment, in R6 and R7, the “C1-6 alkyl” in the C1-6 alkyl and C1-6 alkyl substituted by 1, 2, or 3 R6-1 is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, for example, methyl or ethyl.

In a certain embodiment, in Ra and Rb, the C1-6 alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, for example, methyl.

In a certain embodiment, when Ra and Rb, together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocycloalkyl group or a 5- to 6-membered heterocycloalkyl group substituted by 1, 2, or 3 Ra-1, the “5- to 6-membered heterocycloalkyl group” in the 5- to 6-membered heterocycloalkyl group or 5- to 6-membered heterocycloalkyl group substituted by 1, 2, or 3 Ra-1 is independently a 5- to 6-membered heterocycloalkyl group with 1 or 2 heteroatoms independently selected from N and O; for example,

In a certain embodiment, in Q, the C6-10 aromatic ring is a benzene ring or a naphthalene ring, for example, a benzene ring.

In a certain embodiment, in Q, the 5- to 10-membered heteroaromatic ring is a 5- to 6-membered heteroaromatic ring, for example, a pyridine ring, for another example,

In a certain embodiment, in Q, the C3-6 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, for example, cyclohexyl.

In a certain embodiment, in R8, the halogen is independently F, Cl, Br, or I, for example F, Cl, or Br.

In a certain embodiment, in R8-1 and R8-2, the halogen is independently F, Cl, Br, or I, for example, F.

In a certain embodiment, when R8 and R7 together form —(CH2)m—, m is 3.

In a certain embodiment, when Q is a benzene ring, then

In a certain embodiment,

In a certain embodiment, R6 is H.

In a certain embodiment, R7 is —CH3, —CH2CH3,

In a certain embodiment,

for example,

In a certain embodiment,

In a certain embodiment, the pyrrolotriazinone compound of formula I is selected from any one of the following compounds:

The present disclosure further provides a pharmaceutical composition, and the pharmaceutical composition comprises the pyrrolotriazinone compound of formula I or the pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

The present disclosure further provides a preparation method for the pyrrolotriazinone compound of formula I and the pharmaceutically acceptable salt thereof, and the preparation method comprises a following step: carrying out a condensation reaction between compound II and compound III;

The present disclosure further provides a use of the pyrrolotriazinone compound of formula I and the pharmaceutically acceptable salt thereof or the pharmaceutical composition in the manufacture of a medicament for treating and/or preventing a GPR139 receptor-associated disease; the GPR139 receptor-associated disease may be schizophrenia, bipolar disorder, depression, cognitive disorder, autism spectrum disorder, sleep disorder, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, or fibromyalgia.

The present disclosure further provides a use of the pyrrolotriazinone compound of formula I and the pharmaceutically acceptable salt thereof or the pharmaceutical composition in the manufacture of a medicament for treating and/or preventing schizophrenia, bipolar disorder, depression, cognitive disorder, autism spectrum disorder, sleep disorder, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, or fibromyalgia.

Unless otherwise specified, the terms used in the present disclosure have the following meanings:

The term “alkyl” refers to a straight or branched chain alkyl group with a specified number of carbon atoms (e.g., C1-6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc.

The term “cycloalkyl” refers to a cyclic group, composed solely of carbon atoms, with a specified number of carbon atoms (e.g., C3-6). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

The term “heterocycloalkyl” refers to a cyclic group with a specified number of ring atoms (e.g., 5- to 6-membered), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatoms (one or more kinds of N, O, and S). Heterocycloalkyl groups include, but are not limited to, azetidinyl, tetrahydropyrrolyl, tetrahydrofuryl, morpholinyl, piperidinyl, etc.

The term “aromatic ring” refers to a cyclic group composed solely of carbon atoms, with a specified number of carbon atoms (e.g., C6-10). It can be monocyclic or polycyclic, with at least one ring being aromatic (conforming to Huckel's rule). Aromatic rings include but are not limited to benzene rings, naphthalene rings, etc.

The term “heteroaromatic ring” refers to a cyclic group with a specified number of ring atoms (e.g., 5- to 10-membered), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatoms (one or more kinds of N, O, and S). It can be monocyclic or polycyclic, with at least one ring being aromatic (conforming to Huckel's rule). Heteroaromatic rings are connected to other moieties in the molecule either through an aromatic or a non-aromatic ring. Heteroaromatic rings include, but are not limited to, furan rings, pyrrole rings, thiophene rings, pyrazole rings, imidazole rings, oxazole rings, thiazole rings, pyridine rings, pyrimidine rings, indole rings, etc.

The “-” at the end of a group indicates that the group is connected at that point to other moieties in the molecule. For example, —OCH3 refers to methoxy.

When any variable (e.g., group Ra-1) appears multiple times in the definition of a compound, their definitions are independent of each other and do not affect each other. For example, 5- to 6-membered heterocycloalkyl substituted by 3 Ra-1 means that the 5- to 6-membered heterocycloalkyl is substituted by 3 Ra-1, and the definitions of these 3 Ra-1 are independent and do not affect each other.

The term “pharmaceutically acceptable salt” refers to salts derived from the reaction of a compound with a pharmaceutically acceptable acid or base, which are relatively non-toxic, safe, and suitable for patient use. When a compound contains relatively acidic functional groups, the corresponding base addition salts can be obtained by contacting the free form of the compound with a sufficient amount of pharmaceutically acceptable base in an appropriate inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, etc. When a compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of the compound with a sufficient amount of pharmaceutically acceptable acid in an appropriate inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, methanesulfonate salts, etc. For details, refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, 2002).

The term “therapeutically effective amount” refers to an amount of a compound administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the compound, type of disease, severity of the disease, patient's age, etc., but can be adjusted by those skilled in the art as required.

The term “pharmaceutical excipient” refers to the formulating agents and additives used in the manufacture of drugs and the compounding of prescriptions. They are substances contained in a pharmaceutical formulation, apart from the active ingredients. For details, refer to the Pharmacopoeia of the People's Republic of China (2020 Edition) or the Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009).

The term “treat/treatment” refers to any of the following situations: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that initiates the disease; (3) slowing the progression of one or more biological manifestations of the disease.

The term “prevent/prevention” refers to reducing the risk of developing a disease.

The term “patient” refers to any animal who has already received or is about to receive treatment, preferably a mammal, most preferably a human. Mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans.

On the basis of not violating the common sense in the field, the preferred conditions above can be arbitrarily combined to obtain the preferred examples of the present disclosure.

The reagents and raw materials used in the present disclosure are commercially available.

The positive and progressive effect of the present disclosure is that the pyrrolotriazinone compound of the present disclosure has a novel structure and has strong agonistic activity on GPR139 receptors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present disclosure is further described below by the way of examples, but the present disclosure is not thereby limited to the scope of the described examples. In the following examples, experimental methods without specified conditions are carried out according to conventional methods and conditions, or are selected according to the product instructions.

Step 1: Preparation of pyrrolo[1,2-d][1,2,4]triazin-4(3H)-one

Step 2: Preparation of ethyl 2-(4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate

Step 3: Preparation of 2-(4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetic Acid

Ethyl 2-(4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate (0.40 g, 1.81 mmol) was dissolved in a solution of THF:H2O (1:1, 8 mL), then lithium hydroxide (0.227 g, 5.42 mmol) was added thereto at 0° C., and the reaction mixture was stirred at room temperature for 2 hours. Then, the pH of the reaction mixture was adjusted to about 2 using 1 M hydrochloric acid. The reaction mixture was evaporated to dryness under reduced pressure to remove the solvent to obtain the corresponding solid title compound (0.35 g, crude product), which was used without further purification. HRMS (ESI) calculated for C8H8N3O3+ [M+H]+: 194.0560; found: 194.0561.

The(S)-1-(4-methylphenyl)ethylamine in Example 2 was replaced with (S)-4-(1-aminoethyl) phenol, and the rest of the required raw materials, reagents, and preparation methods were the same as those in Example 2.

Step 1: Preparation of 5-methylpyrrole-2-carbaldehyde

Dichloromethane (12 mL) was added to a three-necked round-bottomed flask, and then DMF (0.854 g, 11.7 mmol) was added thereto, and the system was replaced with nitrogen. POCl3 (1.79 g, 11.7 mmol) was added dropwise thereto at 0° C. and stirred at room temperature for 15 minutes, then 2-methylpyrrole (1.0 g, 12.3 mmol) was added thereto at 0° C. and stirred at room temperature for 30 minutes. Anhydrous sodium acetate (4.6 g, 56.0 mmol) was dissolved in 13 mL of water, slowly added to a round-bottomed flask at room temperature, and stirred at 80° C. for 20 minutes. The reaction mixture was diluted with water and extracted twice with dichloromethane. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The remaining solid was purified by silica gel column chromatography (0 to 20% EA/PE) to obtain the title compound (0.600 g, yield: 44%) as a light yellow solid. 1H NMR (800 MHZ, DMSO-d6) δ 11.86 (s, 1H), 9.30 (s, 1H), 6.89-6.86 (m, 1H), 6.03-5.99 (m, 1H), 2.24 (s, 3H). HRMS (ESI) calculated for C6H8NO+ [M+H]+: 110.0600, found: 110.0599.

Step 2: Preparation of 6-methylpyrrolo[1,2-d][1,2,4]triazin-4-one

Step 3: Preparation of ethyl 2-(6-methyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate

Step 4: Preparation of 2-(6-methyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetic Acid

Ethyl 2-(6-methyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate (2.25 g, 9.56 mmol) was dissolved in a solution of THF:H2O (1:1, 20 mL), then lithium hydroxide (1.20 g, 28.69 mmol) was added thereto at 0° C., and the reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to about 2 using 1 M hydrochloric acid. The reaction mixture was evaporated to dryness under reduced pressure to remove the solvent to obtain the corresponding solid title compound (1.7 g, crude product), which was used without further purification. HRMS (ESI) calculated for C9H10N3O3+ [M+H]+: 208.0717, found: 208.0717.

Step 1: Preparation of 6,8-dimethylpyrrolo[1,2-d][1,2,4]triazin-4-one

Step 2: Preparation of ethyl 2-(6,8-dimethyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate

Step 3: Preparation of 2-(6,8-dimethyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetic Acid

Ethyl 2-(6,8-dimethyl-4-oxopyrrolo[1,2-d][1,2,4]triazin-3(4H)yl)acetate (2.10 g, 8.42 mmol) was dissolved in a solution of THF:H2O (1:1, 10 mL), then lithium hydroxide (1.06 g, 25.27 mmol) was added thereto at 0° C., and the reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to about 2 using 1 M hydrochloric acid. The reaction mixture was evaporated to dryness under reduced pressure to remove the solvent to obtain the corresponding solid title compound (1.8 g, crude product), which was used without further purification. HRMS (ESI) calculated for C10H12N3O3+ [M+H]+: 222.0873; found: 222.0874.

Bioassay Example 1: Determination of the Agonistic Activity of the Compounds of the Present Disclosure on GPR139

The agonistic activity of the compounds of the present disclosure on GPR139 was determined using a calcium flux detection assay (Molecular Devices). The specific operation method is as follows:

Results: The agonistic activity of the compounds of the present disclosure on GPR139 is shown in Table 1.

I-7
NT
NT

I-9
NT
NT

I-10
NT
NT

I-25
NT
NT

I-28
NT
NT

I-29
NT
NT

I-30
NT
NT

I-31
NT
NT

I-33
NT
NT

I-35
NT
NT

I-36
NT
NT

I-42
NT
NT

I-43
NT
NT

I-44
NT
NT

I-45
NT
NT

In the table above, Emax ± maximum effect of the compound of the present disclosure/maximum effect of TAK-041;

Bioassay Example 2: Study on the Pharmacokinetics of Compounds in Mice In Vivo after Intraperitoneal Injection Administration

After a single-dose intraperitoneal injection administration of the compound in male mice in vivo, blood samples and brain tissue were collected at various time points. The concentrations of the compound in the mouse plasma and brain tissue were determined using LC-MS/MS, and the relevant pharmacokinetic parameters were calculated to investigate the pharmacokinetic characteristics and brain distribution of the compounds in mice in vivo.

2.1 Experimental Design

Thirty-six male C57 mice were randomly divided into 4 groups according to body weight, with 9 mice in each group. They were fasted for 12 to 14 hours without water restriction the day before administration and were given food 4 hours after administration. Compound solvent: 10% DMSO+10% solutol (polyethylene glycol-12 hydroxystearate)+80% normal saline.

Administration information

Route of

Quantity
Test
dose
tration
volume
Sample
adminis-

2.2 Sample Collection

Before and after administration, blood (0.1 mL) was collected from the orbit under isoflurane anesthesia, placed in EDTA K2 centrifuge tubes, and placed on an ice bath. The samples were centrifuged at 5000 rpm for 10 minutes at 4° C., and the plasma was collected. IP plasma and brain tissue were collected at 0.5 hours, 2 hours, and 4 hours. All plasma samples were stored at −80° C. before analysis. The brain tissue was collected at 0.5 hours, 2 hours, and 4 hours. After the mice were bled and euthanized, the brain tissue was collected and cleaned, weighed accurately, and homogenized with 50% methanol in water at a ratio of 1:4. The homogenate samples were stored at −80° C. for analysis.

2.3 Data Processing

The data acquisition and control system software was Analyst 1.5.1 (Applied Biosystem). The sample peak integration mode of the spectrum was automatic integration; the ratio of the sample peak area to the internal standard peak area was used as an indicator to perform regression with the concentration of the sample. Regression mode: linear regression with a weight coefficient of 1/X2. Pharmacokinetic parameters were analyzed using non-compartmental model analysis using WinNonlin Professional v6.3 (Pharsight, USA). Cmax was the measured maximum plasma concentration, and the area under the plasma concentration-time curve (AUC(0→t)) was calculated using the trapezoidal rule, and tmax was the time at which the peak plasma concentration was reached after administration. Experimental data are expressed as “mean” (n=3).

2.4 Experimental Results

Parameters
Brain
Plasma
Brain
Plasma
Brain
Plasma
Brain
Plasma

NA means that it is unable to be calculated.

Bioassay Example 3: Behavioral Study on BALB/c Mice after Compound Administration

BALB/c mice were intraperitoneally injected with different doses of I-5, and the therapeutic effect of the developed compounds on the negative symptoms of schizophrenia was verified through social interaction experiments.

3.1 Experimental Subjects

The experimental subjects were 6-week-old male BALB/c wild-type mice, weighing approximately 22 grams, purchased from Shanghai Lingchang Biotechnology Co., Ltd. They were housed in SPF-grade breeding facilities, with 2 mice per cage, under a 12-hour light/dark cycle, with free access to food and water. All behavioral experiments in this project were completed during the light cycle. The experiment adhered to all relevant regulations and guidelines for animal welfare and ethics, and was supervised and inspected by the ethics committee and laboratory animal managers.

3.2 Behavioral System

The length, width, and height of the behavioral box used in this experiment were all 40 cm. During the experiment, 4 mice were tested in 4 behavioral boxes at the same time. In this experiment, Etho Vision XT was used for experimental setup and video recording.

3.3 Experimental Process

The mice were 5 weeks old when purchased. After one week of observation, the experiment was started when they were 6 weeks old and weighed about 22 grams. Before each experiment, the experimental mice were restricted from eating 18 hours in advance. On the day of the experiment, the mice were transferred to the behavioral laboratory to familiarize themselves with the environment 1 hour in advance. Subsequently, they were administered by intraperitoneal injection, and each mouse was returned to its original cage after injection. After 20 minutes, the experimental mice were placed in the behavioral box to acclimate for 10 minutes. Then, a stimulus mouse (from the same batch of BALB/c) was placed in a corner of the behavioral box, away from the experimental mouse. The two mice were allowed to explore freely for 10 minutes while their activity was monitored and recorded by a camera, and the data was archived.

The experimental data were analyzed. The social interaction time (including approaching, sniffing, grooming, chasing, attacking, etc.) was calculated for each experimental mouse over a 10-minute period, and the total social time of each mouse across different groups was analyzed by one-way analysis of variance. During the analysis, mice that did not engage in social interactions or had social interaction times of less than 10 seconds were excluded.

3.4 Experimental Results

The experimental results are shown in FIG. 1 and the table below:

Social time
analysis of

Test compound
Dose
(seconds)
variance

Experimental results show that compound I-5 has a significant improvement effect on social disorders in mice at a dose of 3 mg/kg.