WO2025108106A1 - Compound containing quinolinone skeleton and use thereof - Google Patents

Abstract

Disclosed in the present invention are a compound containing a quinolinone skeleton and the use thereof. The compound containing a quinolinone skeleton has a structure as shown in a formula (I), and can be used in the preparation of a MAGL inhibitor and a drug for preventing and/or treating MAGL-related diseases.

Description

A compound containing quinolinone skeleton and its application Technical Field

The invention relates to the field of medicinal chemistry, and in particular to a compound containing a quinolinone skeleton and application thereof.

Background Art

The endocannabinoid system (ECS) consists of cannabinoid receptors (CB1, CB2), endocannabinoid ligands (arachidonylethanolamine AEA, 2-arachidonylglycerol 2-AG), and cannabinoid ligand hydrolases. CB receptors are the main targets of tetrahydrocannabinol (THC) in the plant cannabis, and have the functions of inhibiting adenylate cyclase activity, activating potassium ion channels, and inhibiting voltage-gated calcium ion channels. The endocannabinoid 2-AG is a full agonist of CB1 and CB2 receptors, and is synthesized "on demand" from phospholipid precursors through a Ca ²⁺ -dependent mechanism: Ca ²⁺ influx activates phospholipase C (PLC), hydrolyzes phosphatidylinositol (PI) into diacylglycerol (DAG), and then generates 2-AG from DAG through diacylglycerol lipase (DAGL). When 2-AG activates CB receptors on target cells, it is absorbed and degraded, thereby terminating the signal transduction pathway. Monoacylglycerol esterase (MAGL) is responsible for 85% of 2-AG hydrolysis to produce arachidonic acid (AA) and glycerol. Downstream AA is metabolized by downstream hydrolases to produce inflammatory mediators such as prostaglandins.

MAGL is responsible for hydrolyzing 2-AG to generate AA and glycerol, and is involved in the signal transduction of the endocannabinoid system. MAGL is highly expressed in the brain, adipose tissue, liver, and intestine. In the brain, MAGL is expressed in the hippocampus, amygdala, and cerebellum. Endocannabinoid signaling plays an important role in these tissues. Therefore, inhibiting MAGL is promising as a target for central nervous system diseases, pain, or liver diseases.

Summary of the invention

The object of the present invention is to provide compounds having MAGL inhibitory activity.

The compound with MAGL inhibitory activity provided by the present invention has a structure shown in formula (I):

Wherein, R ₁ is selected from aryl or heteroaryl;

R ₂ is selected from aryl or heteroaryl substituted or substituted by any R _2A ;

R _2A is selected from -OH, -SH, -CN, halogen, nitro, carboxyl, C _1-8 alkyl, C _1-8 alkoxy, C _1-4 haloalkyl.

In some embodiments, in R ₁ and R ₂ , the aryl group is independently a C ₆ -C ₁₀ aryl group, and specifically independently Phenyl, naphthyl, more specifically phenyl.

In some embodiments, R ₁ is heteroaryl and R ₂ is aryl substituted or substituted by any R _2A .

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-10 membered heteroaryl group "wherein the heteroatom is selected from N, O and S, and the number of the heteroatoms is 1, 2, 3 or 4".

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-10 membered heteroaryl group wherein the heteroatom is selected from N, O and S, and the number of the heteroatoms is 1, 2 or 3.

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-6 membered heteroaryl group wherein the heteroatom is selected from N, O and S, and the number of the heteroatom is 1, 2 or 3.

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-6 membered heteroaryl group "wherein the heteroatom is selected from N, O and S, and the number of the heteroatom is 1 or 2".

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-6 membered heteroaryl group wherein the heteroatom is selected from N and S, and the number of the heteroatom is 1 or 2.

In some embodiments, in R ₁ and R ₂ , the heteroaryl group is a 5-6 membered heteroaryl group wherein the heteroatom is selected from N and S, and the number of the heteroatom is 1.

In some embodiments, in R ₁ and R ₂ , heteroaryl is furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, triazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, oxaquinolyl, indolyl, quinolyl, isoquinolyl, indazolyl, benzoxazolyl, benzothiazolyl, purinyl, oxazolopyridinyl.

In some embodiments, in R ₁ and R ₂ , heteroaryl is pyridinyl.

In some embodiments, R ₁ is pyridinyl and R ₂ is phenyl substituted or substituted by any R _2A .

In some embodiments, R _2A is one or more, for example, 1, 2, 3 or 4, more specifically 1 or 2. In some examples, R _2A is independently selected from -OH, -SH, -CN, cyano, halogen, nitro, carboxyl, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, -CF ₃ , CHF ₂ or CH ₂ F.

The present disclosure further provides an isotope substitution of the above compound or a pharmaceutically acceptable salt thereof. In some embodiments, the isotope substitution is a deuterated substance.

In some specific examples, the present invention provides specific compounds having MAGL inhibitory activity, the structures of which are as follows:

The present disclosure provides a method for preparing a compound as shown in formula (I), using commercially available 4-Boc-1-(5-bromo-2-pyridyl)piperazine as a raw material, performing a Suzuki coupling reaction between the raw material and a substituted phenylboronic acid to obtain a compound 3, then performing deprotection to prepare a compound 4, and then performing a condensation reaction on the compound 4 to prepare the compound of formula (I).

The present disclosure also provides a pharmaceutical composition, which includes the compound represented by formula (I) or a pharmaceutically acceptable salt or the above isotope substitution thereof, and a pharmaceutically acceptable excipient.

In some embodiments, the compound represented by formula (I) or its pharmaceutically acceptable salt or the above-mentioned isotopic substitution is a therapeutically effective amount.

In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotope substitution. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotope substitution. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotope substitution. In some embodiments, the pharmaceutical composition contains 1%-99% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotope substitution. In some embodiments, the pharmaceutical composition contains 2%-98% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotope substitution.

In some embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable excipient based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable excipient.

The present disclosure also provides a use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the isotope substitution product or the pharmaceutical composition in the preparation of a MAGL inhibitor.

The present disclosure also provides a use of the above-mentioned compound as shown in formula (I) or its pharmaceutically acceptable salt, the above-mentioned isotope substitution or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and/or treating MAGL-related diseases.

In some embodiments, the MAGL-related diseases are central nervous system diseases, metabolic disorders and inflammatory diseases; in some instances, the MAGL-related diseases are depression, anxiety, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, neuropathic pain, inflammatory pain, cancer pain, epilepsy, cancer, fatty liver, non-alcoholic steatohepatitis, liver fibrosis, cholestasis, and inflammatory bowel disease.

Definition of terms

On the other hand, in the case where the present disclosure does not limit a specific configuration, the disclosed compounds may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present disclosure.

Additionally, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure.The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

The disclosed compounds may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The disclosed compounds containing asymmetric carbon atoms may be isolated in optically pure forms or in racemic forms. Optically pure forms may be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, and ³⁶ Cl, respectively.

Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10% deuterium incorporation) greater than the natural abundance of deuterium (which is 0.015%) by at least 1000 times. Examples of compounds having an abundance of deuterium greater than the natural abundance of deuterium may be at least 1000 times an abundance of deuterium, at least 2000 times an abundance of deuterium, at least 3000 times an abundance of deuterium, at least 4000 times an abundance of deuterium, at least 5000 times an abundance of deuterium, at least 6000 times an abundance of deuterium, at least 7000 times an abundance of deuterium, at least 8000 times an abundance of deuterium, at least 9000 times an abundance of deuterium, at least 1 ... Deuterium or deuterium with an abundance of 6000 times or more. The present disclosure also includes various deuterated forms of compounds of formula I. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula I with reference to relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds of formula I, or conventional techniques can be used to synthesize deuterated reagents, which include but are not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 8 carbon atoms, preferably an alkyl group of 1 to 6 carbon atoms, and more preferably an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.

The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings which share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 12-membered, such as phenyl and naphthyl.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 12 yuan, more preferably 5 yuan or 6 yuan. For example. Non-limiting examples thereof include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, etc.

The term "hydroxy" refers to -OH.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

The term "haloalkoxy" refers to an alkoxy group substituted with a halogen, wherein alkoxy is as defined above.

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "amino" refers to _-NH2 .

The term "carboxy" refers to -C(O)OH.

The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort.

"Substituted by one or more..." means that the group may be substituted by a single or multiple substituents. When substituted, they may be plural identical substituents or a combination of one or plural different substituents.

The term "each independently selected from" means that the groups selected from the list may be the same or different from each other.

In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both Although all of the above structural formulas are drawn as certain isomers for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in the present disclosure, the bonds No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

In the present disclosure, the terms "comprising" and "including" may be replaced with "consisting of".

The term "composition" refers to a mixture of a drug and other chemical components containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or precursors, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or domestic animals.

Unless otherwise specified, the "compounds" of the present disclosure may exist independently in the form of salts, mixed salts or non-salts (such as free acids or free bases). When they exist in the form of salts or mixed salts, they may be pharmaceutically acceptable salts or pharmaceutically usable salts.

The terms "pharmaceutically acceptable salts" and "pharmaceutically acceptable salts" are used interchangeably and are meant to include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic acid or an organic acid that retains the biological effectiveness of the free base without other side effects. These salts can be prepared by methods known in the art.

"Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can retain the biological effectiveness of the free acid without other side effects. These salts can be prepared by methods known in the art.

The compound of the present invention has good inhibitory activity on MAGL and has good therapeutic effect on the central nervous system or pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the Oil Red O staining results of cells in the blank (C), model (M), IV-23-5 μM, and IV-23-10 μM groups.

Figure 2 shows the residence time of mice in the rotarod in the blank, model, JZL184, and IV-23 (10 mg/kg) groups.

Figure 3 shows the results of ALT and ALP content determination in mice of the Control, DDC, and IV-26 groups.

FIG4 is the HE staining results of mice in the DDC and IV-26 groups.

DETAILED DESCRIPTION

The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are generally performed under conventional conditions or under conditions recommended by raw material or commodity manufacturers. Reagents that do not specify specific sources can be obtained from any supplier of molecular biology reagents with quality/purity for molecular biology applications.

Unless otherwise specified, the reagents used in the following examples are commercially available products.

In the following production methods, the starting compound and reagent used in each step and the obtained compound may each be in the form of a salt, and examples of such salt include salts similar to the salts of the compound of the present invention and the like.

The compound obtained in each step can be directly used in the next reaction as a reaction mixture or a crude product. Alternatively, the compound obtained in each step can be separated from the reaction mixture and purified according to a known method, such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractionation, column chromatography, etc. When the raw material compounds and reagents used in each step are commercially available, the commercial products can also be used directly.

In the reaction of each step, while the reaction time varies depending on the kinds of the reagent and solvent to be used, it is generally 1 minute to 48 hours, preferably 10 minutes to 12 hours, unless otherwise specified.

In the reaction of each step, while the reaction temperature varies depending on the kinds of the reagent and solvent to be used, it is generally 0°C to 300°C, preferably 78°C to 150°C, unless otherwise specified.

Example 1 Preparation of intermediate

1. tert-Butyl 4-(5-(4-(trifluoromethyl)phenyl)pyridin-2-yl)piperazine-1-carboxylate (19b)

4-Boc-1-(5-bromo-2-pyridyl)piperazine (205 mg), 4-trifluoromethylphenylboronic acid (137 mg), Pd(OAc) ₂ (3 mg), and Na ₂ CO ₃ (165 mg) were added to a sealed tube, and H ₂ O and DMF were added under N _2. The reaction was refluxed for 8 h. After the reaction was completed, the heating was stopped, and the product was diluted with water. The product was extracted with ethyl acetate three times, washed with saturated brine three times, and the organic phase was dried over anhydrous sodium sulfate. Column chromatography was used for separation and purification (petroleum ether: ethyl acetate = 16:1) to obtain 19b as a white solid (190 mg) with a yield of 77%.

¹ H NMR (300MHz, DMSO-d6) δ (ppm): 8.55 (d, J = 2.4Hz, 1H), 7.97 (dd, J = 8.9, 2.6Hz, 1H ),7.86(d,J＝8.2Hz,2H),7.76(d,J＝8.4Hz,2H),6.97(d,J＝8.9Hz,1H),3.58(dd,J ＝6.5,3.7Hz,4H),3.44(dd,J＝6.1,3.7Hz,4H),1.43(s,9H).

2. 4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19c)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19c as a white solid in a yield of 83%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.47(d,J＝2.4Hz,1H),7.89(dd,J＝8.9,2.6Hz,1H),7.66(d,J＝8.6Hz,2H),7.47(d,J＝ 8.6Hz,2H),6.93(d,J＝8.9Hz,1H),3.55(dd,J＝6.4,3.4Hz,4H),3.45-3.42(m,4H),1.43(s,9H).

3. 4-(5-(3,4-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19d)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19d as a white solid in a yield of 73%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.52(d,J＝2.1Hz,1H),7.99-7.88(m,2H),7.66(s,2H),6.94(d,J＝8.9Hz,1H),3.57(d,J＝5.2Hz,4H),3.44(s,4H),1.44(s,9H).

4. 4-(5-(3-chloro-4-fluorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19e)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19e as a white solid in a yield of 70%.

¹ H NMR (300MHz, DMSO-d6) δ (ppm): 8.48 (d, J＝2.5Hz, 1H), 7.91 (dd, J＝8.9, 2.6Hz, 1H), 7.85 (dd, J＝7.1, 2.3Hz, 1H), 7.64 (ddd, J＝8.6 ,4.7,2.4Hz,1H),7.46(t,J＝9.0Hz,1H),6.93(d,J＝8.9Hz,1H),3.55(dd,J＝6.5,3.5Hz,4H),3.44(d,J＝6.3Hz,4H),1.43(s,9H).

5. 4-(5-(4-Fluorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19f)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19f as a pale yellow solid in a yield of 84%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.44(d,J＝2.3Hz,1H),7.86(dd,J＝8.9,2.5Hz,1H),7.70-7.61(m,2H),7.26(t, J＝8.9Hz,2H),6.93(d,J＝8.9Hz,1H),3.58-3.50(m,4H),3.47-3.40(m,4H),1.43(s,9H).

6. tert-Butyl 4-(5-(3-hydroxyphenyl)pyridin-2-yl)piperazine-1-carboxylate (19 g)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19g as a white solid in a yield of 73%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):9.51(s,1H),8.39(d,J＝2.4Hz,1H),7.80(dd,J＝8.9,2.5Hz,1H),7.23(td,J＝7.8,3.9Hz,1H),7.02(d,J＝7.8Hz,1H ),6.98-6.95(m,1H),6.91(d,J＝8.9Hz,1H),6.71(dd,J＝8.0,1.6Hz,1H),3.57-3.49(m,4H),3.47-3.41(m,4H),1.43(s,9H).

7. 4-(5-(4-chloro-2-fluorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19h)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19h as a white solid in 80% yield.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.30(s,1H),7.74(d,J＝8.9Hz,1H),7.57(d,J＝8.5Hz,1H),7.53-7.47(m,1H),7.35(dd,J＝8. 3,1.7Hz,1H),6.94(d,J＝8.9Hz,1H),3.55(dd,J＝6.0,3.5Hz,4H),3.43(d,J＝5.1Hz,4H),1.42(s,9H).

8. tert-Butyl 4-(5-(3,5-difluorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19j)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19j as a white solid in a yield of 60%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.57-8.54(m,1H),7.97(dd,J＝9.0,2.6Hz,1H),7.47-7.39(m,2H),7.13(tt,J＝9 .4,2.2Hz,1H),6.93(d,J=9.0Hz,1H),3.59-3.55(m,4H),3.47-3.41(m,4H),1.43(s,9H).

9. 4-(5-(3,5-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19k)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19k as a white solid in a yield of 52%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.25(d,J＝2.4Hz,1H),7.78(d,J＝2.0Hz,1H),7.73(dd,J＝8.8,2.5Hz,1H),7.56(dd,J＝8.3,2.1Hz,1H),7 .50(d,J＝8.3Hz,1H), 6.99(d,J＝8.9Hz,1H), 3.62(dd,J＝6.6,3.5Hz,4H), 3.50(dd,J＝6.1,3.4Hz,4H), 1.49(s,9H).

10. 4-(5-(3-fluoro-5-chloro-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (191)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 191, a white solid, in a yield of 52%. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ (ppm): 11.90 (s, 1H), 8.56 (d, J = 2.6 Hz, 1H), 8.04-7.91 (m, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.64-7.47 (m, 2H), 7.38-7.30 (m, 2H), 7.24 (dd, J = 7.9, 1.0 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.58 (dd, J = 9.6, 1.6 Hz, 1H), 3.73 (s, 4H), 3.62 (s, 2H), 3.47 (s, 2H).

11. 4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19n)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19n as a white solid in a yield of 83%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.52(d,J＝2.4Hz,1H),8.00-7.92(m,2H),7.76(d,J ＝8.0Hz,1H),7.71(t,J＝1.8Hz,1H),7.66-7.58(m,1H),7.45(t,J＝7.8Hz,1H ),7.39-7.33(m,2H),7.24(dd,J＝8.0,1.4Hz,1H),6.96(d,J＝9.0Hz,1H),6. 58(dd,J＝9.6,1.7Hz,1H),3.73(d,J＝7.5Hz,4H),3.59(s,2H),3.46(s,2H).

12. 4-(5-(4-nitrophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19o)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19o as a white solid in a yield of 76%.

¹ H NMR (300MHz, DMSO-d6) δ (ppm): 8.65-8.60 (m, 1H), 8.29-8.22 (m, 2H), 8.03 (dd, J = 9.0, 2.6Hz, 1H), 7.97-7 .90(m,2H),6.98(d,J＝9.0Hz,1H),3.61(dd,J＝6.5,3.8Hz,4H),3.44(dd,J＝6.2,3.9Hz,4H),1.43(s,9H).

13. 4-(5-(3-nitrophenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19p)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19p as a white solid in a yield of 70%.

¹ H NMR (300MHz, Chloroform-d) δ (ppm): 8.48 (d, J = 2.3Hz, 1H), 8.37 (t, J = 1.9Hz, 1H), 8.19-8.11 (m, 1H), 7.88-7.82 (m, 1H) ),7.80-7.74(m,1H),7.59(t,J＝8.0Hz,1H),6.75(d,J＝8.9Hz,1H),3.66-3.60(m,4H),3.60-3.54(m,4H),1.50(s,9H).

14. 4-(5-(4-methoxyphenyl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19q)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19q as a white solid in a yield of 60%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.41(d,J＝2.4Hz,1H),7.82(dd,J＝8.8,2.6Hz,1H),7.59-7.50(m,2H),7.04-6.96(m,2H) ,6.91(d,J＝8.9Hz,1H),3.78(s,3H),3.51(dd,J＝6.7,3.2Hz,4H),3.47-3.39(m,4H),1.43(s,9H).

15. 4-(5-phenylpyridin-2-yl)piperazine-1-carboxylic acid tert-butyl (19r)

The synthesis method was similar to that of intermediate 19a to obtain intermediate 19r as a white solid in a yield of 83%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):8.46(d,J＝2.4Hz,1H),7.88(dd,J＝8.9,2.6Hz,1H),7.66-7.58(m,2H),7.43(t,J＝7.6Hz,2H),7.34 -7.27(m,1H),6.94(d,J＝8.9Hz,1H),3.54(dd,J＝6.6,3.3Hz,4H),3.44(dd,J＝6.0,2.7Hz,4H),1.43(s,9H).

Example 2 7-(4-(5-(4-(trifluoromethyl)phenyl)pyridin-2-yl)piperazine-1-carbonyl)quinoline-2(1H)-1(IV-21)

The raw material 2-oxo-1,2-dihydroquinoline-7-carboxylic acid (38 mg) was dissolved in 2 ml DMF, HATU (91 mg) and DIPEA (104 μL) were added, and after 10 min, the intermediate 20b (82 mg, 20b was prepared from 19b by deprotection, and the deprotection was carried out according to conventional methods) was added, and stirred at room temperature for 1 h. After TLC monitoring, the reaction was stopped, diluted with water, extracted with ethyl acetate three times, washed with saturated brine three times, and the organic phase was dried over anhydrous sodium sulfate. Column chromatography separation and purification (dichloromethane: methanol = 30: 1) gave the target product IV-21 as a white solid 34 mg with a yield of 31%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.57(d,J＝2.3Hz,1H),7.98(t,J＝8.1Hz,2H),7.87(d,J＝8.2Hz,2H),7.76(dd,J＝8.2,3.0Hz,3H),7.3 5(s,1H),7.25(d,J＝8.0Hz,1H),6.99(d,J＝8.9Hz,1H),6.58(d,J＝9.5Hz,1H),3.74(d,J＝6.6Hz,4H),3.55(d,J＝41.5Hz,4H).

Example 3 7-(4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-23)

The synthesis method refers to compound IV-21, and intermediate 20c is added (20c is prepared by deprotection of 19c, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-23 as a white solid with a yield of 35%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.49(d,J＝2.4Hz,1H),7.96(d,J＝9.6Hz,1H),7.91(d d,J＝8.9,2.5Hz,1H),7.75(d,J＝8.0Hz,1H),7.67(d,J＝8.6Hz,2H),7.48(d,J ＝8.6Hz,2H),7.35(s,1H),7.24(dd,J＝8.0,1.2Hz,1H),6.96(d,J＝8.9Hz,1H) ,6.58(dd,J＝9.6,1.3Hz,1H),3.72(d,J＝17.5Hz,4H),3.53(d,J＝35.5Hz,4H).

Example 4 7-(4-(5-(3,4-dichlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-22)

The synthesis method refers to compound IV-21, and intermediate 20d is added (20d is prepared by deprotection of 19d, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-22 as a white solid with a yield of 20%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.53(d,J＝2.4Hz,1H),7.98(s,1H),7.96-7.90(m,2H),7.76(d,J＝8.0Hz,1H),7.66(t,J＝8.7Hz,2H),7. 35(s,1H),7.24(dd,J＝8.0,1.3Hz,1H),6.96(d,J＝9.0Hz,1H),6.58(d,J＝10.0Hz,1H),3.73(d,J＝7.7Hz,4H),3.65-3.41(m,4H).

Example 5 7-(4-(5-(3-chloro-4-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-27)

The synthesis method refers to compound IV-21, and intermediate 20e is added (20e is prepared by deprotection of 19e, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-27 as a white solid with a yield of 58%.

¹ H NMR (300 MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.50(d,J＝2.5Hz,1H),8.00-7.91(m,2H),7.87(dd,J＝7 .1,2.3Hz,1H),7.76(d,J＝8.0Hz,1H),7.66(ddd,J＝8.7,4.6,2.3Hz,1H),7.47( t,J＝9.0Hz,1H),7.35(s,1H),7.24(dd,J＝8.0,1.4Hz,1H),6.95(d,J＝9.0Hz,1H ), 6.58 (dd, J＝9.5, 1.7Hz, 1H), 3.72 (d, J＝20.1Hz, 4H), 3.53 (d, J＝39.9Hz, 4H).

Example 6 7-(4-(5-(4-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-24)

The synthesis method refers to compound IV-21, and intermediate 20f is added (20f is prepared by deprotection of 19f, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-24, a yellow-brown solid, with a yield of 41%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.89(s,1H),8.45(d,J＝2.4Hz,1H),7.96(d,J＝9.6Hz,1H),7.88(dd,J＝8.9,2.6Hz,1H),7.75(d,J＝8.0Hz,1H),7.69-7.63(m, 2H),7.34(s,1H),7.30-7.21(m,3H),6.95(d,J＝8.9Hz,1H),6.57(dd,J＝9.5,1.7Hz,1H),3.71(d,J＝21.9Hz,4H),3.52(d,J＝30.8Hz,4H).

Example 7 7-(4-(5-(3-hydroxyphenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-25)

The synthesis method refers to compound IV-21, and 20 g of the intermediate is added (20 g is prepared from 19 g by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-25, an off-white solid, with a yield of 36%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),9.51(s,1H),8.40(d,J＝2.3Hz,1H),7.96(d,J＝9.6Hz,1H),7 .82(dd,J＝8.9,2.5Hz,1H),7.76(d,J＝8.0Hz,1H),7.34(s,1H),7.27-7.17(m,2H),7 .03(d,J＝7.9Hz,1H),6.99-6.96(m,1H),6.94(d,J＝9.0Hz,1H),6.71(dd,J＝8.0,1.6 Hz,1H),6.58(dd,J＝9.5,1.5Hz,1H),3.71(d,J＝26.3Hz,4H),3.52(d,J＝31.3Hz,4H).

Example 8 7-(4-(5-(4-chloro-2-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-26)

The synthesis method refers to compound IV-21, and intermediate 20h is added (20h is prepared by deprotection of 19h, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-26 as a white solid with a yield of 33%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.33(s,1H),7.96(d,J＝9.6Hz,1H),7.77(t,J＝7.4Hz,2H),7.62-7.49(m,2H),7.37(d,J＝10.7Hz, 2H), 7.24 (d, J = 8.0Hz, 1H), 6.97 (d, J = 9.0Hz, 1H), 6.58 (d, J = 8.8Hz, 1H), 3.73 (d, J = 11.9Hz, 4H), 3.53 (d, J = 40.8Hz, 4H).

Example 9 7-(4-(5-(3,5-difluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-14)

The synthesis method refers to compound IV-21, and intermediate 20j is added (20j is prepared by deprotection of 19j, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-14, an off-white solid, with a yield of 20%.

¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 11.90 (s, 1H), 8.57 (d, J = 2.5Hz, 1H), 8.03-7.92 (m,2H),7.76(d,J＝8.0Hz,1H),7.49-7.39(m,2H),7.35(s,1H),7.24(dd,J＝8.0,1.4Hz,1H),7.14(tt,J＝ 9.3, 2.3Hz, 1H), 6.95 (d, J = 9.0Hz, 1H), 6.58 (dd, J = 9.6, 1.6Hz, 1H), 3.73 (s, 4H), 3.54 (d, J = 44.8Hz, 4H).

Example 10 7-(4-(5-(3,5-dichlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-15)

The synthesis method refers to compound IV-21, and intermediate 20k is added (20k is prepared from 19k by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-15, an off-white solid, with a yield of 35%.

¹ H NMR (300 MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.20(d,J＝2.3Hz,1H),7.97(d,J＝9.6Hz,1H),7.76(d,J＝8. 1Hz,1H),7.73(d,J＝2.0Hz,1H),7.69(dd,J＝8.9,2.5Hz,1H),7.50(dd,J＝8.3,2.0H z,1H),7.45(d,J＝8.3Hz,1H),7.35(s,1H),7.24(dd,J＝8.0,1.4Hz,1H),6.95(d,J＝ 8.9Hz,1H),6.58(dd,J＝9.6,1.7Hz,1H),3.73(d,J＝13.2Hz,4H),3.63-3.43(m,4H).

Example 11 7-(4-(5-(3-chloro-5-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-16)

The synthesis method refers to compound IV-21, and intermediate 201 is added (201 is prepared from 191 by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-16, an off-white solid, with a yield of 30%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.56(d,J＝2.6Hz,1H),8.04-7.91(m,2H),7.76(d,J＝8.0Hz,1H),7.64-7.47(m,2H),7.38-7.30(m,2 H),7.24(dd,J＝7.9,1.0Hz,1H),6.95(d,J＝9.0Hz,1H),6.58(dd,J＝9.6,1.6Hz,1H),3.73(s,4H),3.62(s,2H),3.47(s,2H).

Example 12 7-(4-(5-(3-chlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-19)

The synthesis method refers to compound IV-21, and intermediate 20n is added (20n is prepared from 19n by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-19, an off-white solid, with a yield of 38%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.52(d,J＝2.4Hz,1H),8.00-7.92(m,2H),7.76(d,J ＝8.0Hz,1H),7.71(t,J＝1.8Hz,1H),7.66-7.58(m,1H),7.45(t,J＝7.8Hz,1H ),7.39-7.33(m,2H),7.24(dd,J＝8.0,1.4Hz,1H),6.96(d,J＝9.0Hz,1H),6. 58(dd,J＝9.6,1.7Hz,1H),3.73(d,J＝7.5Hz,4H),3.59(s,2H),3.46(s,2H).

Example 13 7-(4-(4-(4-(4-nitrophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-41)

The synthesis method refers to compound IV-21, and intermediate 20o is added (20o is prepared from 19o by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-41 as a yellow solid with a yield of 46%.

¹ H NMR (400MHz, DMSO-d ₆ )δ(ppm):11.88(s,1H),8.64(d,J＝2.5Hz,1H),8.29-8.23(m,2H),8.05(dd,J＝9.0,2.6Hz,1H),7.95(ddd,J＝9.5,5.1,2.7Hz,3H),7.75(d,J＝8.0Hz ,1H),7.35(d,J＝1.2Hz,1H),7.24(dd,J＝8.0,1.5Hz,1H),7.00(d,J＝9.0Hz,1H),6.58(dd,J＝9.6,1.8Hz,1H),3.75(s,4H),3.56(d,J＝70.6Hz,4H).

Example 14 7-(4-(4-(4-(3-nitrophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-42)

The synthesis method refers to compound IV-21, and intermediate 20p is added (20p is prepared from 19p by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-42 as a yellow solid with a yield of 40%.

¹ H NMR (300MHz, Chloroform-d) δ (ppm): 12.04 (s, 1H), 8.49 (d, J = 2.4Hz, 1H), 8.38 (t,J＝1.9Hz,1H),8.17(ddd,J＝8.2,2.1,0.9Hz,1H),7.88-7.77(m,3H),7.65(d ,J＝8.1Hz,1H),7.60(t,J＝8.0Hz,1H),7.52(s,1H),7.30(d,J＝1.3Hz,1H),6.79 (d,J＝5.7Hz,1H),6.76(d,J＝6.4Hz,1H),4.02-3.73(m,4H),3.72-3.52(m,4H).

Example 15 7-(4-(4-(4-(4-methoxyphenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-43)

The synthesis method refers to compound IV-21, and intermediate 20q is added (20q is prepared from 19q by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-43, an off-white solid, with a yield of 42%.

¹ H NMR (300MHz, DMSO-d6) δ (ppm): 11.89 (s, 1H), 8.42 (d, J = 2.4Hz, 1H), 7.96 (d, J = 9.6 Hz,1H),7.84(dd,J＝8.9,2.5Hz,1H),7.75(d,J＝8.0Hz,1H),7.55(d,J＝8.8Hz,2H), 7.35(s,1H),7.24(dd,J＝8.0,1.3Hz,1H),7.00(d,J＝8.8Hz,2H),6.93(d,J ＝8.9Hz,1H),6.58(dd,J＝9.6,1.4Hz,1H),3.78(s,3H),3.77-3.39(m,8H).

Example 16 7-(4-(4-(4-phenylpyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-44)

The synthesis method refers to compound IV-21, and intermediate 20r is added (20r is prepared from 19r by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-44, an off-white solid, with a yield of 46%.

¹ H NMR (300MHz, DMSO-d ₆ )δ(ppm):11.90(s,1H),8.54-8.43(m,1H),7.96(d,J＝9.5Hz,1H),7.93-7.86(m,1H),7.76(d,J＝8.0Hz,1H),7.63(d,J＝7.5Hz,2H),7. 44(t,J＝7.4Hz,2H),7.38-7.19(m,3H),6.96(d,J＝8.8Hz,1H),6.58(d,J＝9.5Hz,1H),3.73(d,J＝25.4Hz,4H),3.54(d,J＝35.4Hz,4H).

Biological activity testing of compounds

Example 17 Determination of MAGL inhibitory activity

1. Test principle: Cayman's MAGL inhibitor screening kit provides a simple and efficient method for screening compounds for their inhibitory activity against human MAGL. The principle is that MAGL hydrolyzes a replacement substrate (4-nitrophenyl ethyl acetate instead of 2-AG) to generate a yellow product (4-nitrophenol), which has a maximum absorption at 405-412nm. The enzyme activity is characterized by measuring the absorbance at this wavelength and quantifying the content of the hydrolysis product 4-nitrophenol.

2. Preparation of reagents

(1) Preparation of buffer: 3 mL of concentrated buffer (10×) was diluted with 27 mL of pure water. The diluted buffer (1×) contained 10 mM Tris-HCl, pH 7.2, and 1 mM EDTA, which was used for the analysis and dilution of MAGL and positive drug JZL195 and was stored at -20°C before use.

(2) Preparation of human recombinant MAGL: Take 30 μL of MAGL protein, add 570 μL of buffer (1×), and store for later use.

(3) Preparation of MAGL substrate: Take 150 μL substrate, add 450 μL buffer (1×), and store for later use.

(4) Preparation of positive drugs and compounds: JZL195 was dissolved in DMSO and buffer at a ratio of 1:1 to prepare compound solutions and JZL195 solutions with concentrations of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM, respectively, and stored for later use.

3. Experimental operation

(1) Background well: add 160 μL buffer (1×), 10 μL solvent

(2) 100% initial enzyme activity well: add 150 μL buffer (1×), 10 μL enzyme, 10 μL solvent

(3) Positive control and drug wells: Add 150 μL buffer (1×), 10 μL enzyme, and 10 μL positive drug or inhibitor. Repeat the experiment three times for each well, as shown in Table 1.

Table 1

(4) Mix the contents of the wells and incubate at room temperature for 15 min.

(5) 10 μL of substrate was added to each well, the 96-well plate was shaken for 10 seconds, and incubated at room temperature for 10 minutes. The readings were taken at 405 nm using a Multiscan GO (Thermo) microplate reader, and the inhibitor rate was calculated according to the following formula. Nonlinear regression analysis was performed using GraphPad Prism software, and the experimental data were analyzed using second-order polynomial regression analysis and by applying mixed model inhibition fitting.

The results are shown in Table 2. As can be seen from Table 2, the compounds of the present invention have MAGL inhibitory activity.

Referring to the method similar to that in this example, the IC ₅₀ of the compound of the present application was determined. The specific method is as follows:

According to the above method, the inhibition rate of the applied compound at concentrations of 0.001μM, 0.01μM, 0.1μM, 1μM, 10μM, and 100μM was determined, and the experimental data were analyzed using second-order polynomial regression analysis and by applying mixed model inhibition fitting to obtain _IC50 data of the compound's inhibition of MAGL.

Example 18 Determination of 2-AG concentration in the brain

ICR male mice (3 mice/group) were used. The test compound IV-23 was dissolved in 10% DMSO, 10% Tween 80, and 80% saline to prepare a dosing solution. The dose of the test compound was prepared to be 10 mg/kg, and the dosing volume was 10 ml/kg. The test compound was administered by gavage. After administration of the test compound, the mice were given 60 minutes, 120 minutes, The brain was separated at 240min and 480min, and the cerebral hemispheres were extracted (the control was separated at 0min). The obtained cerebral hemispheres were frozen on dry ice, and the weight of the frozen tissue was measured. About 100mg of brain tissue was weighed and homogenized with 9 times the volume of normal saline. After homogenization, the mixture was centrifuged at 15000rpm for 10 minutes. The supernatant was divided into about 50 microliters per tube. The concentration of 2-AG in the brain was determined by Elisa method.

The results of brain 2-AG concentrations are shown in Table 3, in ng/g.

Table 3

As can be seen from Table 3, compound IV-23 was transferred to the brain by oral administration to ICR male mice. Compared with the control group, these compounds significantly increased the concentration of 2-AG in the brain, and showed significant differences at 60min, 120min, and 240min (p<0.05), indicating that IV-23 acts on the MAGL target, causing an increase in the level of 2-AG in the brain.

Example 19 Pharmacokinetics Test

1. Experimental Materials

(1) Mobile phase solution: Take 500 mL of Wahaha water, add 2.5 mL of formic acid to it to prepare an aqueous solution containing 0.5% formic acid, mix well, and degas by ultrasonication for 20 min.

(2) Preparation of stock solution: Accurately weigh 2.0 mg of compound IV-23 into a 10 mL volume, add water to dissolve and make a stock solution of 200.0 ug/mL. Accurately weigh 2.0 mg of imipramine hydrochloride into a 10 mL volume, add water to dissolve and make a stock solution of 200.0 ug/mL. Add methanol to dilute to 3.000 ug/mL as the internal standard working solution and store in a 4°C refrigerator for future use.

(3) Preparation of standard curve samples: Dilute the IV-23 stock solution with methanol to a series of standard solutions of 30000, 15000, 7500, 3000, 1500, 300.0, and 30.00 ng/mL. Accurately pipette 10uL of the above series of standard solutions, add 50ul of blank SD rat plasma, add 10μL of the internal standard working solution, mix well, and add 230μL of methanol. The final volume is 300μL, and the final concentration of compound IV-23 is 1000, 500.0, 250.0, 100.0, 50.00, 10.00, and 1.000 ng/mL. After vortex mixing for 5 minutes, centrifuge at 12000r/min for 10 minutes, and transfer the supernatant to an autosampler vial for LC-MS/MS analysis to prepare for the drawing of the standard curve.

(4) HPLC conditions: The chromatographic column was Waters 5C18-MS-Ⅱ (2.0×250 mm, 5 μm); the mobile phase was 0.5% formic acid water (A)-methanol (B); isocratic elution was used, the organic phase ratio was 90%, the flow rate was 0.3 mL/min, and the column Temperature: 40°C, injection volume: 10 μL.

(5) Preparation of test sample: Weigh 0.5 mg of test sample IV-23 and place it in a 10 mL EP tube, add 0.125 ml DMSO, 0.25 ml Tween-80, and then add 4.2 mL of normal saline injection. After dissolution, sonicate and oscillate to mix until the compound is clear, and prepare a 0.1 mg/mL preparation; prepare and use it on the day of use. Weigh 5 mg of test sample IV-23 and place it in a 10 mL EP tube, add 0.125 ml DMSO, 0.25 ml Tween-80, and then add 4.625 mL of normal saline injection. After dissolution, sonicate and oscillate to mix until the compound is clear, and prepare a 1 mg/mL PO preparation and a 0.2 mg/mL IV preparation. The dosage volume is 10 ml/kg, and the dosage is 10 mg/kg and 2 mg/kg, respectively; prepare and use it on the day of use.

The pharmacokinetic parameters of IV-23 are shown in Table 4:

^a T _max , peak time; ^b C _max , maximum blood drug concentration; ^c AUC, area under the concentration-time curve; ^d Vd, apparent distribution solvent; ^e t _1/2 , drug half-life; ^f CL, clearance rate; ^g MRT, mean residence time.

As can be seen from Table 4, compound IV-23 has a higher plasma exposure and a longer half-life under different administration methods of intravenous injection and oral gavage, indicating that compound IV-23 has good pharmacokinetic properties.

Example 20 Determination of the antidepressant effect of the compound on depressed mice

ICR male mice were used. After one week of adaptation, 12 mice were randomly divided as blank controls and fed normally; the remaining 70 mice were placed in 50 ml centrifuge tubes (with holes at the end of the tubes for the mice to breathe), restrained for 4 to 8 hours a day, increasing from 4 hours a day to 8.5 hours a day, and restrained for 30 consecutive days.

Model determination:

The tail suspension test was used to determine whether the mice had adapted to the model (to evaluate the despair behavior of the mice): the posterior 1/3 of the tail of the mouse was fixed with tape and hung on a bracket with the head 15 cm above the ground. The mice were filmed after 2 minutes of adaptation, and the immobility time of the mice within 4 minutes was counted.

Grouping and dosing:

After modeling, the mice were divided into model group, positive drug group (fluoxetine, 8 mg/kg), compound IV-23-low-dose group (4 mg/kg), compound IV-23 high-dose group (8 mg/kg) according to the immobility time data in the tail suspension test, with 6-8 mice in each group. Compound preparation: 10% DMSO, 40% PEG 400, 5% Tween 80, 45% saline. Administration volume: 10 ml/kg. Administration was continued for 7 days. After 7 days, behavioral tests such as tail suspension test, open field test, forced swimming test and sugar water preference test were performed.

The results are shown in Table 5. As shown in Table 5, intraperitoneal injection of compound IV-23 for 7 consecutive days can significantly improve the depressive-like behavior of ICR depressed mice caused by chronic restraint.

Table 5

Example 21 Determination of the in vitro anti-nonalcoholic fatty liver effect of the compound

1. Instruments and Materials

(1) Cells and reagents: HepG2 cells, a human hepatocellular carcinoma cell line, were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Fetal bovine serum was purchased from Solebol Biotech Co., Ltd. DMEM high glucose medium, 0.25% trypsin solution, and PBS solution were purchased from Nanjing Keygene Biotechnology Development Co., Ltd. DMSO, oleic acid, palmitic acid, and Oil Red O were purchased from Guangdong Xilong Chemical Co., Ltd.

(2) Main instruments: MULTISKAN MK3 fully automatic microplate reader (Thermo Scientific), vortex oscillator (QL-902); centrifuge, BT224 electronic balance (Sartouris, Germany); HB-202 constant temperature water bath (Beijing Zhongxi Yuanda); BX51 upright microscope (Olympus, Japan); other equipment and instruments include centrifuge tubes, pipettes, and straws.

2. Experimental methods:

(1) Cell culture method

HepG2 cells were inoculated in DMEM high-glucose culture medium containing 10% fetal bovine serum to allow the cells to adhere to the wall and grow. The cells were cultured in an incubator at 37°C and 5% CO ₂ saturated humidity. Depending on the cell growth, the cells were digested with 0.25% trypsin every 1-2 days for subculture.

(2) Effect of compound IV-23 on free fatty acid-induced NASH model in HepG2 cells

HepG2 cells were cultured in 96-well plates. After the cells adhered to the wall and grew to 60-70%, free fatty acids (palmitic acid: oleic acid at a molar ratio of 1:2) and different concentrations of the test drug (compound IV-23) were co-stimulated for 24 hours. Three parallel wells were set up, divided into a blank group, a model group, and a drug-treated group. The blank group was replaced with blank DMEM, and the drug-treated groups were added with DMEM at concentrations of 5μM and 10μM compound IV-23, respectively.

(3) Oil Red O staining to observe intracellular lipid droplets

The steps of Oil Red O staining are as follows: wash the cells 3 times with PBS solution, fix them with 10% neutral formaldehyde for 30 minutes, wash them twice with PBS solution, stain them with Oil Red O at 37°C, incubate them at 37°C for 1 hour, discard the staining solution and wash them twice with PBS. Under the microscope, it can be seen that the neutral fat in the cells can be specifically stained red, and the lipid accumulation in the cells can be observed.

3. Experimental results: Oil red O staining to observe the effect of compound IV-23 on NASH cell model

The Oil Red O staining results of compound IV-23 are shown in Figure 1. It can be seen that compared with untreated cells, HepG2 cells exposed to free fatty acids (model group) showed higher intracellular lipid accumulation, and after intervention treatment with compound IV-23, lipid droplet aggregation in HepG2 cells was significantly inhibited; indicating that compound IV-23 prepared by the present invention can significantly inhibit free fatty acid-induced lipid accumulation in a dose-dependent manner at 5μM and 10μM, and has the effect of treating non-alcoholic fatty liver disease.

Example 22 Determination of the Anti-Parkinson's Disease Effect of Compounds in Vivo

1. Experimental methods: The mice were adapted for 1 week; 5 animals were randomly selected from the normal group, without any treatment and kept normally; the other 4 groups of mice were intraperitoneally injected with solvent or drug once on the first day, and intraperitoneally injected with MPTP (15 mg/kg, once every 2 hours, for a total of 4 injections) on the second day, and the solvent or drug was injected 1 hour after the second MPTP injection. On the third day, the solvent or drug was intraperitoneally injected once, and behavioral tests were performed 2 hours after drug administration.

2. Behavioral experiment - Rotarod test: The instrument was placed 50 cm above the ground. There was a digital time recorder at the bottom of each partition, and the time was recorded when the animal fell. The mice were trained with the instrument for a fixed time, and the rotation speed of the rotarod was 20 rpm. The grip strength of the animals was evaluated using a rotator. The mice were trained for 2 consecutive days before drug administration. A 7 cm diameter rotating rod was used for behavioral testing, and the grip strength of the animals was tested at a speed of 20 rpm, with a cut-off time of 180 s.

3. Experimental results: As shown in Figure 2, the average time of blank mice on the rotating rod was 59.26s, and that of the model group was 24.1s, which showed significant differences from the blank group, indicating that the model was successful. The average time of the positive drug JZL184 group was 46.75s, showing a certain therapeutic effect, but there was no significant difference from the model group. The average time of the IV-23 (10 mg/kg) group was 53.82s, which was significantly different from the model group, indicating that IV-23 (10 mg/kg) can significantly improve the movement of Parkinson's mice. Dynamic ability.

Example 23 Determination of the effect of compounds on cholestatic liver injury

1. Experimental methods:

(1) Eighteen C57BL/6J male mice were randomly divided into three groups (n=6) according to body weight after one week of adaptive feeding: control group, model group (0.1% DDC), and IV-26 administration group (12 mg/kg + 0.1% DDC). The control group was fed with normal feed, and the other groups were fed with feed containing 0.1% DDC for 2 weeks. After feeding with feed containing 0.1% DDC for one week, the mice in the administration group were intraperitoneally injected with IV-26 (12 mg/kg, 10 mL/kg) every day for one week, and the control group and 0.1% DDC group were injected with solvent (10 mL/kg) for 1 week in the same way. One hour after the last administration of the mice, blood was collected from the eye sockets, the mice were killed, and the livers were dissected and washed with physiological saline. The surface moisture was absorbed, and the liver lobules were carefully separated and fixed in formalin fixative. The rest of the liver was stored at -80 degrees.

(2) After the blood samples were allowed to stand at room temperature for 30 min, they were centrifuged at 3000 rpm/min for 10 min at 4°C. The upper serum samples were collected and the serum alanine aminotransferase (ALT/GPT) and serum alkaline phosphatase (ALP) levels were detected according to the kit instructions.

(3) Histopathological examination of mouse liver: The fixed mouse liver tissue was fixed with formalin solution for 24 hours, dehydrated with alcohol, embedded in paraffin and cut into slices with a thickness of 5 μm. The prepared slices were stained with hematoxylin and eosin dyes respectively. The slices after HE staining were dehydrated, transparent and sealed, and observed under a microscope to record the pathological morphology of liver tissue.

2. Experimental results:

(1) Effects of IV-26 on serum biochemical parameters in mice with DDC-induced cholestatic liver injury

The levels of ALT and ALP in serum are important indicators of liver function and can directly reflect the damage to the liver. The experimental results are shown in Figure 3. Compared with the Control group, the levels of ALT and ALP in the serum of the DDC group increased significantly, indicating that the liver damage of mice in the DDC group was severe and the cholestatic liver injury model was successfully established. Compared with the DDC group, the serum ALT and ALP in the IV-26 administration group were both reduced. The results show that the test drug IV-26 can significantly improve DDC-induced cholestatic liver injury.

(2) Effect of IV-26 on pathological changes of liver tissue in mice with DDC-induced cholestatic liver injury

The pathological changes of mouse liver were observed under the microscope after HE staining. The results are shown in Figure 4. The liver cells of mice in the Control group were intact and tightly arranged, and the structures were normal. The DDC group had obvious inflammatory cell infiltration, hepatocyte necrosis and morphological changes. Compared with the DDC group, the IV-26 administration group could improve liver damage, reduce inflammatory cell infiltration, and alleviate hepatocyte necrosis. The results showed that the test drug IV-26 could improve the pathological histological changes of DDC-induced cholestatic liver injury.

Claims (10)

A compound having a structure represented by formula (I) or a pharmaceutically acceptable salt or isotope thereof:
Wherein, R ₁ is selected from aryl or heteroaryl; R ₂ is selected from aryl or heteroaryl substituted or substituted by any R _2A ; R _2A is selected from -OH, -SH, -CN, halogen, nitro, carboxyl, C _1-8 alkyl, C _1-8 alkoxy, C _1-4 haloalkyl. The compound according to claim 1 or a pharmaceutically acceptable salt or isotope thereof, characterized in that the aryl groups in R ₁ and R ₂ are independently C ₆ -C ₁₀ aryl groups, preferably phenyl or naphthyl groups, and more preferably, in R _{1 and} R ₂ , the aryl groups are phenyl groups; ₂ , the heteroaryl group is a 5-10 membered heteroaryl group with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2, 3 or 4"; preferably, the heteroaryl group is a 5-10 membered heteroaryl group with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3"; more preferably, the heteroaryl group is a 5-6 membered heteroaryl group with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3"; further preferably, the heteroaryl group is a 5-6 membered heteroaryl group with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1 or 2"; more preferably, the heteroaryl group is a 5-6 membered heteroaryl group with "heteroatoms selected from N and S, and the number of heteroatoms is 1"; further preferably, R ₁ , R In _{R 2} , the heteroaryl group is furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, triazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, oxazolyl, indolyl, quinolyl, isoquinolyl, indazolyl, benzoxazolyl, benzothiazolyl, purinyl, oxazolopyridinyl; specifically, preferably, in R ₁ and R ₂ , the heteroaryl group is pyridyl. The compound according to claim 1 or a pharmaceutically acceptable salt or isotope thereof, characterized in that R ₁ is a heteroaryl group, and R ₂ is an aryl group substituted or substituted by any R _2A ; preferably, R ₁ is a pyridyl group, and R ₂ is a phenyl group substituted or substituted by any R _2A . The compound according to claim 1 or a pharmaceutically acceptable salt or isotope thereof, characterized in that the number of R _2A is 1, 2, 3 or 4, preferably 1 or 2, and more preferably, R _2A is independently selected from -OH, -SH, -CN, cyano, halogen, nitro, carboxyl, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, -CF ₃ , CHF ₂ or CH ₂ F. A compound having the following structure or a pharmaceutically acceptable salt or isotope thereof:
The method for preparing the compound represented by formula (I) according to claim 1:
A pharmaceutical composition, characterized in that it comprises the compound as claimed in any one of claims 1 to 5 or a pharmaceutically acceptable salt or isotope substitute thereof, and a pharmaceutically acceptable excipient; preferably, the pharmaceutical composition contains 0.01-99.99% of the compound as claimed in any one of claims 1 to 7 or a pharmaceutically acceptable salt or isotope substitute thereof. Use of the compound as claimed in any one of claims 1 to 5 or its pharmaceutically acceptable salt or isotope-substituted product in the preparation of a MAGL inhibitor. Use of the compound as claimed in any one of claims 1 to 5 or a pharmaceutically acceptable salt or isotope substitute thereof in the preparation of a medicament for preventing and/or treating a disease associated with MAGL. The use according to claim 9 is characterized in that the MAGL-related diseases are central nervous system diseases, metabolic disorders and inflammatory diseases; preferably, the MAGL-related diseases are depression, anxiety, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, neuralgia, inflammatory pain, cancer pain, epilepsy, cancer, fatty liver, non-alcoholic steatohepatitis, liver fibrosis, cholestasis, and inflammatory bowel disease.