CN117430522A - GPR139 receptor agonist and preparation method thereof - Google Patents

Abstract

The invention provides a compound as shown in formula (I)A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ And R is ₆ Etc. have the definitions given in the description. In addition, the invention also provides a preparation method of the compound and pharmaceutically acceptable salts thereof and application of the compound in preparation of medicines for treating GPR139 receptor related diseases.

Description

A GPR139 receptor agonist and preparation method thereof

Technical Field

The present invention relates to the field of medicinal chemistry. Specifically, the present invention relates to a class of ligand molecules of G protein-coupled receptor 139 (GPR139 for short).

Background Art

G protein-coupled receptor 139 (GPR139) was first discovered by Gloriam et al. in 2005 (Biochim Biophys Acta 2005; 1722: 235-246), also known as GPCR12, PGR3, KOR3L, GPRg1. GPR139 is mainly expressed in the habenula, striatum, thalamus, hypothalamus and pituitary in the central nervous system (CNS). Its amino acid sequence is highly conserved in different species. For example, the homology of human GPR139 protein sequence with mouse, chicken and zebrafish is 96%, 92% and 70% respectively. When GPR139 receptor is expressed on the cell surface, it is composed of N-terminal fragment, 7 transmembrane fragments and C-terminal fragment, of which the N-terminal fragment is composed of 26 amino acids and contains a cysteine. Wang et al. reported that GPR139 receptors and dopamine receptor D2 are co-expressed in different tissues of the mouse brain, and in vitro cell experiments confirmed that GPR139 can promote the agonist of dopamine receptor D2 to produce calcium flow signals, and the calcium flow signals can be inhibited by both dopamine receptor D2 antagonists and GPR139 antagonists (Frontiers in Neuroscience, March 2019, Volume 13, Article 281). In addition, Nepomuceno et al. confirmed that GPR139 can synergize with melanocortin receptor 3 or 5 (MC3/MC5). These results indicate that GPR139 can form heterodimers with other receptors on the cell surface to exert physiological effects. In addition, our experiments have confirmed that the GPR139 receptor can form homodimers through the N-terminal cysteine.

Different research teams were looking for ligands of GPR139, including agonists and antagonists. At the same time, Liu et al. reported that L-phenylalanine and L-tryptophan were endogenous agonists of GPR139 (Mol Pharmacol 2015; 88:911-925).

Existing studies have shown that the GPR139 receptor has important physiological functions and is a potential drug target for anti-anxiety, anti-depression, anti-schizophrenia, treatment of Parkinson's syndrome, treatment of drug and alcohol abuse, and treatment of metabolic-related diseases. The relevant GPR139 structure, expression, pharmacology, and physiological function information provide a solid foundation for the design and synthesis of new GPR139 agonists.

Summary of the invention

In the first aspect of the present invention, there is provided a compound as shown in formula (I), or a pharmaceutically acceptable salt thereof

in,

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, halogen, hydroxy, carboxyl, C _1-6 acyl, C _1-3 alkyl-carbonyl-C _1-3 alkyl, C _1-6 alkyl-aldehyde, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy;

R7 and R8 are each independently selected from the following group: hydrogen, C1-4 alkyl.

In some embodiments, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, hydroxyl, carboxyl, C _1-6 acyl, C _1-2 alkyl-carbonyl-C _1-2 alkyl, C _1-3 alkyl-aldehyde, C _1-3 alkyl, C _1-6 haloalkyl, C _1-3 haloalkoxy.

In some embodiments, R ₁ , R ₂ and R ₃ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, hydroxy, carboxyl, C _1-6 acyl, C _1-3 alkyl-carbonyl-C _1-3 alkyl, C _1-6 alkyl-aldehyde, methyl, trifluoromethyl, trifluoromethoxy.

In some embodiments, R ₄ , R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, hydroxy, carboxyl, methyl, trifluoromethyl, trifluoromethoxy.

In some embodiments, _R7 and _R8 are each independently selected from the group consisting of hydrogen and methyl.

In some embodiments, the compound is selected from the group consisting of:

In the second aspect of the present invention, there is provided a method for preparing the compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, comprising the steps of:

or

wherein R', R", and R'" are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, keto, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, and C _1-6 haloalkoxy;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are as defined above.

In the third aspect of the present invention, a pharmaceutical composition is provided, characterized in that it comprises the compound as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In the fourth aspect of the present invention, a method for preparing the pharmaceutical composition as described in the third aspect of the present invention is provided, characterized in that it comprises the steps of: mixing the compound as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier to form a drug mixture.

In the fifth aspect of the present invention, there is provided a use of the compound as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in the third aspect of the present invention, characterized in that it is used for preparing a drug for treating GPR139 receptor-related diseases.

In another preferred embodiment, the GPR139 receptor-related diseases include anxiety, depression, Parkinson's syndrome, drug and alcohol abuse.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventor unexpectedly found that the compound of the present invention has excellent GPR139 receptor agonist activity, and thus conducted a series of synthesis and biological activity tests, on which basis the present invention was completed.

The experimental methods in the following examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

the term

The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

In the present invention, the term "multiple" refers to two or more than two. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the associated objects are in an "or" relationship.

In the compounds described herein, when any variable occurs more than once in any constituent, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations render the compound stable. Lines drawn from substituents into the ring system indicate that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds are attached only to any suitable carbon atom of an adjacent ring. It is to be understood that one of ordinary skill in the art may select substituents and substitution patterns for the compounds of the invention to provide chemically stable compounds that can be readily synthesized from readily available raw materials by techniques of the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

As used herein, the term "alkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C ₁ -C ₈ " in "C ₁ -C ₈ alkyl" includes groups having 1, 2, 3, 4, 5, or 8 carbon atoms in a straight or branched arrangement. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, etc.

The term "alkoxy" as used herein represents an alkyl-oxy group wherein alkyl is as defined above.

As will be appreciated by those skilled in the art, "halogen" as used herein is meant to include chlorine, fluorine, bromine and iodine.

The term "haloalkyl" as used herein represents an alkyl group wherein one or more hydrogen atoms are replaced by halogen, wherein alkyl is as defined above.

The term "haloalkoxy" as used herein represents an alkyl-oxy group having one or more hydrogen atoms replaced by a halogen, wherein alkyl is as defined above.

Active ingredients

In the present invention, an active ingredient that can effectively excite the GPR139 receptor is provided. The active ingredient is a compound represented by general formula (I), and the active ingredient can effectively prevent, treat and/or alleviate GPR139-related diseases.

Experiments have shown that the active ingredients of the present invention can effectively stimulate the GPR139 receptor, thereby preventing, treating and/or alleviating GPR139-related diseases.

It should be understood that the active ingredients of the present invention include compounds represented by general formula (I), or pharmaceutically acceptable salts thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, and deuterated compounds of the compounds of general formula (I).

The term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

Preparation method

The preparation method of the compound of formula (I) of the present invention is described in more detail below, but these specific methods do not constitute any restriction to the present invention. The compounds of the present invention can also be optionally combined with various synthetic methods described in this specification or known in the art and easily prepared, and such combination can be easily carried out by those skilled in the art to which the present invention belongs.

Usually, in the preparation process, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (such as 0°C to 80°C, preferably 0°C to 50°C). The reaction time is usually 0.1 hour to 60 hours, preferably 0.5 to 48 hours.

The following general preparation route can be used to synthesize the compounds of formula (I) of the present invention.

or

wherein R', R", R'", _R1 , _R2 , _R3 , _R4 , _R5 and _R6 are as defined above.

Pharmaceutical compositions and methods of administration

Since the compounds of the present invention have excellent GPR139 receptor agonist activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases related to GPR139 receptors or abnormal expression.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-3000 (active dose range 3-30 mg/kg) mg of the compound of the present invention/dose, more preferably, 10-2000 mg of the compound of the present invention/dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 6 to 600 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

The main advantages of the present invention include:

(a) The compounds of the present invention can effectively stimulate the GPR139 receptor, and the EC50 values of some compounds are less than 0.1 μM.

(b) The compound of the present invention is easy to synthesize and has a high yield.

(c) The compounds of the present invention have low toxicity and side effects and good drugability.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The starting materials used in the present invention are commercially purchased without special instructions.

Example

Example 1

Preparation of compound NRB025

Under nitrogen protection, S-phenylethylamine (171 mg, 1.42 mmol) and diisopropylethylamine (183 mg, 1.42 mmol) were dissolved in 2 ml of dichloromethane and cooled to about 5 degrees Celsius with an ice-water bath. Malonyl chloride (100 mg, 0.71 mmol) was slowly added dropwise to the above reaction solution. It took about 2 minutes to drip off. After the addition, the reaction was continued to stir for 15 minutes. The completion of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 ml of saturated ammonium chloride solution and extracted with dichloromethane (10 ml x 2). The organic phase was dried over sodium sulfate and concentrated after filtration. Purification was performed by column chromatography to obtain 37.5 mg of the product. The analytical data are as follows:

LCMS(ESI+): m/z 311(M+H); 1H-NMR(300MHz, CDCl3)δ7.66-7.26(m,8H),7.08(brs,2H),5.09(m,2H),3.14( s, 2H), 1.50 (d, J = 6.9Hz, 6H).

Embodiment 2-5

Preparation of compounds NRB037, NRB038, NRB040, and NRB064

The method of Example 1 is adopted, except that a different substrate is used to replace S-phenylethylamine, thereby preparing compound 2-5.

Example 6

Preparation of compound NRB035

Step 1: Preparation of Intermediate 1

Under nitrogen protection, S-phenylethylamine (870 mg, 7.32 mmol) and diisopropylethylamine (1.72 g, 13.3 mmol) were dissolved in 20 ml of dichloromethane and cooled to about 5 degrees Celsius with an ice-water bath. Then ethyl chloroformyl acetate (1 g, 6.67 mmol) was slowly added dropwise to the above reaction solution. It took about 5 minutes to drip off. After the addition, the reaction was continued to stir for 15 minutes. The completion of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 50 ml of saturated ammonium chloride solution and extracted with dichloromethane (20 ml x 2). The organic phase was dried over sodium sulfate and concentrated after filtration. 1.35 grams of oily liquid was obtained. Analytical data: LCMS (ESI+): m/z236 (M+H).

Step 2: Preparation of intermediate 2

Intermediate 1 (1.25 g, 5.32 mmol) was dissolved in tetrahydrofuran (10 ml) and water (10 ml), and lithium hydroxide monohydrate (447 mg, 10.64 mmol) was added at room temperature in one go. The reaction was stirred at room temperature for 3 hours. After the reaction was completed, the pH value was adjusted to 3 with dilute hydrochloric acid, and extracted with ethyl acetate (50 ml x 2). The organic phase was dried over sodium sulfate, filtered, and concentrated to obtain 1.34 g of an oily substance. The oily substance slowly solidified at room temperature. Analytical data: LCMS (ESI-): m/z206 (M-H).

Step 3: Preparation of compound NRB035

Under nitrogen protection, intermediate 2 (30 mg, 0.14 mmol) and S-4-chlorophenylethylamine (33 mg, 0.17 mmol) were dissolved in dichloromethane (2 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (54 mg, 0.28 mmol), 1-hydroxybenzotriazole (38 mg, 0.28 mmol) and diisopropylethylamine (36 mg, 0.28 mmol) were added in sequence. The reaction was stirred overnight at room temperature. The completion of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into a saturated aqueous ammonium chloride solution (10 ml) and extracted with dichloromethane (10 ml x 2). The organic phase was dried, filtered and concentrated, and purified by preparative TLC to obtain compound NRB035 as a white solid (14 mg). The analytical data are as follows:

LCMS(ESI+):m/z 345,347(M+H); 1H-NMR(300MHz,CD3OD)(m,9H),5.05-4-90(m,2H),3.07(s,2H),1.44(m ,6H).

Example 7-25

Preparation of compounds NRB036, NRB039, NRB041, NRB042, NRB043, NRB044, NRB045, NRB046, NRB047, NRB048, NRB026, NRB032, NRB056, NRB057, NRB058, NRB061, NRB063, NRB065, NRB066

The method of Example 1 was adopted, except that different substrates were used to replace S-phenylethylamine and S-4-chlorophenylethylamine, thereby preparing compound 6-14.

Embodiment 26

Preparation of compound NRB060

Step 1: Preparation of intermediate 2

S-1-phenylethylamine (100 mg, 0.83 mmol) and potassium cyanate (335 mg, 4.13 mmol) were dissolved in 0.85 ml of 1N HCl and 0.5 ml of water and reacted in a microwave at 80 °C for 1 hour.

The reaction was completed by monitoring by TLC. There was a solid in the reaction, which was filtered and washed three times with n-hexane (3x5 ml). The solid was dried in vacuo to obtain 120 mg of crude product. Analytical data: LCMS (ESI+): m/z 165 (M+H).

Step 2: Preparation of intermediate 3

Intermediate 2 (120 mg, 0.73 mmol) and pyridine (173 mg, 2.19 mmol) were dissolved in 2 ml of dichloromethane, phenyl chloroformate (137 mg, 0.88 mmol) was added, and stirred at room temperature overnight. The reaction was completed by monitoring by TLC. 3 ml of water was added to the reaction solution, extracted with dichloromethane (3x3 ml), dried over anhydrous sodium sulfate, and dried over a column (ethyl acetate: petroleum ether = 1:10) to obtain 55 mg of product. Analytical data: LCMS (ESI+): m/z 285 (M+H).

Step 3: Preparation of compound NRB060

Intermediate 3 (45 mg, 0.16 mmol) and raw material A (33 mg, 0.19 mmol) were dissolved in 1 ml of acetonitrile and potassium carbonate (43.7 mg, 0.32 mmol) was added. The reaction was stirred at 50 degrees for 5 hours. The reaction was monitored by TLC and completed. The reaction solution was spin-dried and purified by preparative TLC to obtain 20 mg of white oily product. Analytical data: LCMS (ESI+): m/z 326 (M+H).

¹ H-NMR (300MHz, CDCl ₃ ) δ7.66-7.26 (m, 8H), 7.08 (br s, 2H), 5.09 (m, 2H), 3.14 (s, 2H), 1.50 (d, J = 6.9 Hz,6H).

¹ H-NMR (300MHz, CD ₃ OD) δ7.34-7.12 (m, 9H), 5.11 (q, J = 6.9Hz 1H), 5.01-4.95 (m, 1H), 2.36 (s, 3H), 1.45 (d,J＝7.5Hz,3H),1.42(d,J＝7.2Hz,3H).

Embodiment 27

Preparation of compound NRB062

Step 1: Preparation of intermediate 2

S-1-phenylethylamine (200 mg, 1.65 mmol) and S1 (161 mg, 1.10 mmol), EDCl (423 mg, 2.2 mmol), HOBT (297 mg, 2.2 mmol), diisopropylethylamine (284 mg, 2.2 mmol) were dissolved in 5 ml of dichloromethane and stirred at room temperature overnight.

The reaction was completed by monitoring by TLC. 1N dilute hydrochloric acid was added to quench, extracted with dichloromethane (3x5 ml), dried over anhydrous sodium sulfate, and purified by preparative TLC to obtain 240 mg of a white oily product. Analytical data:

LCMS (ESI+): m/z 250 (M+H).

Step 2: Preparation of intermediate 3

Intermediate 2 (240 mg, 0.96 mmol) was dissolved in 3 ml of tetrahydrofuran and 3 ml of water, lithium hydroxide monohydrate (122 mg, 2.9 mmol) was added, and stirred at room temperature overnight. The reaction was completed by monitoring by TLC. The reaction solution was adjusted to pH 3 to 4 by adding 1N dilute hydrochloric acid, extracted with ethyl acetate (3x10 ml), dried over anhydrous sodium sulfate, and spin-dried to obtain 200 mg of white solid product. Analytical data: LCMS (ESI+): m/z 222 (M+H).

Step 3: Preparation of compound NRB062

Intermediate 3 (60 mg, 0.27 mmol) and S2 (35 mg, 0.2 mmol), EDCl (77 mg,

0.4 mmol), HOBT (54 mg, 0.4 mmol), diisopropylethylamine (52 mg, 0.4 mmol) were dissolved in 3 ml of dichloromethane and stirred at room temperature for 3 hours. The reaction was completed by monitoring by TLC. 5 ml of water was added, extracted with dichloromethane (3x5 ml), dried over anhydrous sodium sulfate, and dried by spin drying. Purification by preparative TLC (dichloromethane: methanol = 10: 1) gave 17 mg of white solid product.

Analytical data: LCMS (ESI+): m/z 339 (M+H).

¹ H-NMR (300 MHz, DMSO-d ₆ )δ

8.20-8.07(m,2H),7.31-7.13(m,9H),5.04-4.83(m,2H),3.21(q,J＝7.2Hz,1H),2.28(d,J＝7.2Hz,3H) ,1.38-1.23(m,6H),1.14(d,J=6.9Hz,3H).

Embodiment 28

GPR139 receptor activity assay

1. Experimental Methods

HEK293 cells were grown in 10 cm culture dishes containing DMEM complete medium (containing glutamine, sodium pyruvate, penicillin/streptomycin, and 10% fetal bovine serum) and cultured in a 37°C, 5% CO ₂ incubator to 90% confluence. Then subculture, subculture twice a week at 1:5. When subculturing cells, first completely aspirate the culture medium, wash once with 4 mL of DPBS, add 3 mL of trypsin digestion solution, and shake gently. When the cells split, aspirate the trypsin digestion solution and add 5 mL of DMEM complete medium. After dispersing the cells with a pipette, add 1 mL of dispersed cell solution to each of 5 new 10 cm culture dishes, and then add 7 mL of DMEM complete medium to each, mix well, and culture in a 37°C, 5% CO ₂ incubator.

On the day of transfection, digest the cells at an appropriate density into a 10 cm dish, add 6 mL of DMEM/F12 medium (containing glutamine, sodium pyruvate, HEPES, and fetal bovine serum), and culture in a 37°C, 5% CO ₂ incubator for 5 hours. Take a 50 mL sterile centrifuge tube A, add 1 mL of serum-free DMEM/F12 medium (containing glutamine, sodium pyruvate, and HEPES), add 10 μg of GPR139 DNA, and mix well. Take another 50 mL sterile centrifuge tube B, add 1 mL of serum-free DMEM medium (containing glutamine, sodium pyruvate, and HEPES), add 60 μL of Fugene HD transfection reagent, and mix well. Take 1 mL of Fugene HD transfection reagent from centrifuge tube B, add it to centrifuge tube A, mix well, and leave at room temperature for 20 minutes. Add 2 mL of DMEM/F12 medium (containing glutamine, sodium pyruvate, HEPES, and fetal bovine serum) to centrifuge tube A and mix well. Then take out the mixture (4 ml) in centrifuge tube A and add it to the cells in the above 10 cm culture dish (a total of 10 mL of liquid). After mixing well, culture in a 37°C, 5% _CO2 incubator for 20-24 hours.

The next day, the transfected cells were digested with trypsin, and appropriate DMEM/F12 medium (containing glutamine, sodium pyruvate, HEPES, penicillin/streptomycin, and fetal bovine serum) was added. The cells were seeded in a Matrigel-coated 384-well plate at a cell density of 6.67×106 cells/mL, 30 μL/well. The 384-well plate was placed in a 37°C, 5% CO ₂ incubator for culture.

On the third day, add 10 μL/well FLIPR dye (diluted in HEPES solution containing Pluronic and Probenicid) to the 384-well cell plate and incubate for 40 minutes at 37°C in a 5% CO ₂ incubator. Add the diluted compound to the cell plate and read the calcium flux signal in the FLIPR plate reader.

XL-fit software was used for data analysis and the _EC50 value of each compound was obtained.

2. Experimental results

Table 1. EC50 values of compounds for GPR139 FLIPR cell calcium flux signal detection

EC50 value of GPR139 FLIPR cell calcium flow signal detection compound (measured data, μM)

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

1. A compound represented by formula (I), or a pharmaceutically acceptable salt thereof in, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, carboxyl, C _1-6 acyl, C _1-3 alkyl-carbonyl-C _{1 -3} alkyl, C _1-6 alkyl-aldehyde group, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy; R7 and R8 are each independently selected from the group consisting of hydrogen and C1-4 alkyl. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, Fluorine, chlorine, hydroxyl, carboxyl, C _1-6 acyl, C _1-2 alkyl-carbonyl-C _1-2 alkyl, C _1-3 alkyl-aldehyde, C _1-3 alkyl, C _{1- 6} haloalkyl, C _1-3 haloalkoxy. 3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₁ , R ₂ and R ₃ are each independently selected from the following group: hydrogen, fluorine, chlorine, hydroxyl, carboxyl, C _1-6 acyl, C _1-3 alkyl-carbonyl-C _1-3 alkyl, C _1-6 alkyl-aldehyde, methyl, trifluoromethyl, trifluoromethoxy. 4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₄ , R ₅ and R ₆ are each independently selected from the following group: hydrogen, fluorine, chlorine, hydroxyl, carboxyl, methane base, trifluoromethyl, trifluoromethoxy. 5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₇ and R ₈ are each independently selected from the group consisting of hydrogen and methyl. 6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 7. The preparation method of the compound as claimed in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, comprising the steps: or Wherein, R', R", R"' are each independently selected from the following group: hydrogen, halogen, hydroxyl, carboxyl, ketone, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy , C _1-6 haloalkoxy; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are as defined in claim 1 . 8. A pharmaceutical composition, characterized by comprising the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. 9. The preparation method of pharmaceutical composition according to claim 8, characterized in that it includes the step of: combining the compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof. The received carriers are mixed to form a drug mixture. 10. The compound as claimed in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or the use of the pharmaceutical composition as claimed in claim 8, characterized in that it is used to prepare the treatment of GPR139 receptors Drugs for related diseases. In another preferred embodiment, the GPR139 receptor-related diseases include anxiety, depression, Parkinson's disease, drug and alcohol abuse.