Compound containing quinolinone skeleton and use thereof

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 a quinolinone skeleton and its application Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a compound containing a quinolinone skeleton and application thereof. Background Art

[0002] The endocannabinoid system (ECS) consists of cannabinoid receptors (CB1, CB2), endocannabinoid ligands (arachidonic acid ethanolamine AEA, 2-arachidonic acid glyceride 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, which is activated by phospholipid precursors through Ca 2+ Dependent mechanism "on demand" synthesis: Ca 2+ The influx activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol (PI) into diacylglycerol (DAG). 2-AG is then generated from DAG by diacylglycerol lipase (DAGL). When 2-AG activates CB receptors on target cells, it is taken up and degraded, terminating the signaling pathway. Monoacylglycerol esterase (MAGL) is responsible for 85% of 2-AG hydrolysis, generating arachidonic acid (AA) and glycerol. AA is then metabolized by downstream hydrolases to produce inflammatory mediators such as prostaglandins.

[0003] MAGL is responsible for hydrolyzing 2-AG to produce AA and glycerol and is involved in signal transduction of the endocannabinoid system. MAGL is highly expressed in the brain, adipose tissue, liver, and intestine. Within the brain, MAGL is expressed in the hippocampus, amygdala, and cerebellum. Endocannabinoid signaling plays an important role in these tissues. Therefore, inhibiting MAGL is a promising target for central nervous system disorders, pain, or liver diseases.

[0004] Summary of the Invention

[0005] The object of the present invention is to provide compounds having MAGL inhibitory activity.

[0006] The compound with MAGL inhibitory activity provided by the present invention has a structure shown in formula (I):

[0007] wherein R1 is selected from aryl or heteroaryl;

[0008] R2 is selected from the group consisting of substituted or replaced by any R 2A substituted aryl or heteroaryl;

[0009] R 2ASelected from -OH, -SH, -CN, halogen, nitro, carboxyl, C 1-8 Alkyl, C 1-8 Alkoxy, C 1-4 Halogenated alkyl.

[0010] In some embodiments, in R1 and R2, the aryl group is independently C6-C 10 Aryl groups are specifically and independently phenyl and naphthyl, more specifically phenyl.

[0011] In some embodiments, R1 is heteroaryl, R2 is substituted or replaced by any of R 2A Substituted aryl.

[0012] In some embodiments, in R1 and R2, 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".

[0013] In some embodiments, in R1 and R2, 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.

[0014] In some embodiments, in R1 and R2, 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 heteroatoms is 1, 2 or 3.

[0015] In some embodiments, in R1 and R2, 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 heteroatoms is 1 or 2.

[0016] In some embodiments, in R1 and R2, the heteroaryl group is a 5-6 membered heteroaryl group wherein the heteroatom is selected from N and S, and the number of the heteroatoms is 1 or 2.

[0017] In some embodiments, in R1 and R2, 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.

[0018] In some embodiments, in R1 and R2, 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, pyridyl, pyrimidinyl, pyridazinyl, oxadenyl, indolyl, quinolyl, isoquinolyl, indazolyl, benzoxazolyl, benzothiazolyl, purinyl, or oxazolopyridinyl.

[0019] In some embodiments, in R1 and R2, heteroaryl is pyridyl.

[0020] In some embodiments, R1 is pyridyl, R2 is substituted or replaced by any R 2A Substituted phenyl.

[0021] 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 Each is independently selected from -OH, -SH, -CN, cyano, halogen, nitro, carboxyl, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, -CF3, CHF2 or CH2F.

[0022] The present disclosure further provides an isotope-substituted compound of the above-mentioned compound or a pharmaceutically acceptable salt thereof. In some embodiments, the isotope-substituted compound is a deuterated compound.

[0023] In some specific examples, the present invention provides specific compounds having MAGL inhibitory activity, the structures of which are as follows:

[0024] The present disclosure provides a method for preparing a compound represented by formula (I), wherein commercially available 4-Boc-1-(5-bromo-2-pyridyl)piperazine is used as a raw material, and compound 3 is obtained by subjecting it to a Suzuki coupling reaction with a substituted phenylboronic acid. Compound 3 is then deprotected to prepare compound 4, and compound 4 is then subjected to a condensation reaction to prepare the compound of formula (I).

[0025] The present disclosure also provides a pharmaceutical composition, which includes the compound represented by formula (I) or a pharmaceutically acceptable salt or the above-mentioned isotope substitution, and a pharmaceutically acceptable excipient.

[0026] In some embodiments, the compound represented by formula (I) or its pharmaceutically acceptable salt or the above-mentioned isotopic substitution is in a therapeutically effective amount.

[0027] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotopic substitution based on the total weight of the composition. 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 isotopic 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 isotopic substitution. In some embodiments, the pharmaceutical composition contains 1%-99% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotopic substitution. In some embodiments, the pharmaceutical composition contains 2%-98% of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned isotopic substitution.

[0028] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable excipient.

[0029] The present disclosure also provides a use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the isotope-substituted product thereof or the pharmaceutical composition thereof in the preparation of a MAGL inhibitor.

[0030] The present disclosure also provides a use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the isotope substitution thereof or the pharmaceutical composition thereof in the preparation of a drug for preventing and / or treating MAGL-related diseases.

[0031] 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.

[0032] Definition of terms

[0033] On the other hand, where the present disclosure does not limit a particular configuration, the compounds of the present disclosure 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 enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0034] Additionally, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are encompassed 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.

[0035] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0036] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the 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 et al.

[0037] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the compound has a natural abundance greater than deuterium that can be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The disclosure also includes various deuterated forms of the compound 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 the compound of formula I with reference to the relevant literature. In preparing deuterated forms of compounds of formula I, commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0038] 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, and the like.

[0039] 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.

[0040] 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 pi electron system, preferably 6- to 12-membered, such as phenyl and naphthyl.

[0041] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 12-membered, more preferably 5-membered or 6-membered. For example, non-limiting examples include imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, etc.

[0042] The term "hydroxy" refers to -OH.

[0043] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0044] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0045] The term "haloalkoxy" refers to an alkoxy group substituted with a halogen, wherein alkoxy is as defined above.

[0046] The term "cyano" refers to -CN.

[0047] The term "nitro" refers to -NO2.

[0048] The term "amino" refers to -NH2.

[0049] The term "carboxy" refers to -C(O)OH.

[0050] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort.

[0051] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents.

[0052] The term "each independently selected from" means that the groups selected from the list may be the same as or different from each other.

[0053] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the above structural formulas are drawn as certain isomers for the sake of 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.

[0054] In the present disclosure, the terms "comprising" and "including" can be replaced with "consisting of".

[0055] The term "composition" refers to a mixture of one or more compounds described herein, or their physiologically acceptable salts or precursors, with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0056] 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 that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0057] Unless otherwise specified, the compounds of the present disclosure may exist independently in the form of salts, mixed salts or non-salts (e.g., free acids or free bases). When present in the form of salts or mixed salts, they may be pharmaceutically acceptable salts or pharmaceutically usable salts.

[0058] The terms "pharmaceutically acceptable salts" and "pharmaceutically usable salts" are used interchangeably and are meant to include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0059] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. These salts can be prepared by methods known in the art.

[0060] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. These salts can be prepared by methods known in the art.

[0061] 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

[0062] 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.

[0063] FIG2 shows the residence time of mice in the rotarod in the blank, model, JZL184, and IV-23 (10 mg / kg) groups.

[0064] Figure 3 shows the results of ALT and ALP content determination in mice of the Control, DDC, and IV-26 groups.

[0065] FIG4 shows the HE staining results of mice in the DDC and IV-26 groups. DETAILED DESCRIPTION

[0066] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the raw material or commercial manufacturer. Reagents whose sources are not specified can be obtained from any molecular biology reagent supplier of a quality / purity suitable for molecular biology applications.

[0067] Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0068] In the following production methods, the starting compounds and reagents used in each step and the obtained compounds may each be in the form of a salt, and examples of such salts include salts similar to the salts of the compounds of the present invention and the like.

[0069] The compound obtained in each step can be used directly in the next reaction as a reaction mixture or a crude product. Alternatively, the compound obtained in each step can be isolated from the reaction mixture and purified according to known methods, such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractionation, column chromatography, etc. When the starting compounds and reagents used in each step are commercially available, they can also be used directly.

[0070] 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.

[0071] In the reaction of each step, although the reaction temperature varies depending on the kinds of the reagent and solvent to be used, it is usually 0°C to 300°C, preferably 78°C to 150°C, unless otherwise specified.

[0072] Example 1 Preparation of intermediate

[0073] 1. tert-Butyl 4-(5-(4-(trifluoromethyl)phenyl)pyridin-2-yl)piperazine-1-carboxylate (19b)

[0074] 4-Boc-1-(5-bromo-2-pyridyl)piperazine (205 mg), 4-trifluoromethylphenylboronic acid (137 mg), Pd(OAc)2 (3 mg), and Na2CO3 (165 mg) were added to a sealed tube. H2O and DMF were added under N2, and the reaction was refluxed for 8 h. After the reaction, heating was stopped, the mixture was diluted with water, extracted three times with ethyl acetate, and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate. 19b was isolated and purified by column chromatography (petroleum ether:ethyl acetate = 16:1) to obtain 19b as a white solid (190 mg, yield 77%).

[0075] 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).

[0076] 2. tert-Butyl 4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19c)

[0077] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19c as a white solid in 83% yield.

[0078] 1 H NMR (300MHz, DMSO-d6) 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).

[0079] 3, tert-Butyl 4-(5-(3,4-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19d)

[0080] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19d as a white solid in a yield of 73%.

[0081] 1 H NMR (300MHz, DMSO-d6) 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).

[0082] 4, tert-Butyl 4-(5-(3-chloro-4-fluorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19e)

[0083] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19e as a white solid in 70% yield.

[0084] 1H 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).

[0085] 5. tert-Butyl 4-(5-(4-fluorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19f)

[0086] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19f as a pale yellow solid in 84% yield.

[0087] 1 H NMR (300MHz, DMSO-d6) 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).

[0088] 6. tert-Butyl 4-(5-(3-hydroxyphenyl)pyridin-2-yl)piperazine-1-carboxylate (19 g)

[0089] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19g as a white solid in a yield of 73%.

[0090] 1 H NMR (300MHz, DMSO-d6) 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).

[0091] 7. tert-Butyl 4-(5-(4-chloro-2-fluorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19h)

[0092] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19h as a white solid in 80% yield.

[0093] 1 H NMR (300MHz, DMSO-d6) 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), 7.3 5(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).

[0094] 8. tert-Butyl 4-(5-(3,5-difluorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19j)

[0095] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19j as a white solid in 60% yield.

[0096] 1 H NMR (300MHz, DMSO-d6) 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).

[0097] 9. tert-Butyl 4-(5-(3,5-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19k)

[0098] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19k as a white solid in a yield of 52%.

[0099] 1H NMR (300MHz, DMSO-d6) 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).

[0100] 10. tert-Butyl 4-(5-(3-fluoro-5-chloro-dichlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (191)

[0101] The synthesis method was similar to that of intermediate 19a to obtain intermediate 191 as a white solid in a yield of 52%. 1 H NMR (300MHz, DMSO-d6) 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,2H),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).

[0102] 11. tert-Butyl 4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (19n)

[0103] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19n as a white solid in 83% yield.

[0104] 1H NMR (300MHz, DMSO-d6) 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,1 H), 6.58 (dd, J = 9.6, 1.7Hz, 1H), 3.73 (d, J = 7.5Hz, 4H), 3.59 (s, 2H), 3.46 (s, 2H).

[0105] 12. tert-Butyl 4-(5-(4-nitrophenyl)pyridin-2-yl)piperazine-1-carboxylate (19o)

[0106] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19o as a white solid in 76% yield.

[0107] 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).

[0108] 13. tert-Butyl 4-(5-(3-nitrophenyl)pyridin-2-yl)piperazine-1-carboxylate (19p)

[0109] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19p as a white solid in 70% yield.

[0110] 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).

[0111] 14. tert-Butyl 4-(5-(4-methoxyphenyl)pyridin-2-yl)piperazine-1-carboxylate (19q)

[0112] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19q as a white solid in 60% yield.

[0113] 1 H NMR (300MHz, DMSO-d6) 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).

[0114] 15. tert-Butyl 4-(5-phenylpyridin-2-yl)piperazine-1-carboxylate (19r)

[0115] The synthesis method was similar to that of intermediate 19a to obtain intermediate 19r as a white solid in 83% yield.

[0116] 1 H NMR (300MHz, DMSO-d6) 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).

[0117] Example 2 7-(4-(5-(4-(trifluoromethyl)phenyl)pyridin-2-yl)piperazine-1-carbonyl)quinoline-2(1H)-1(IV-21)

[0118] The starting material, 2-oxo-1,2-dihydroquinoline-7-carboxylic acid (38 mg), was dissolved in 2 mL of DMF. HATU (91 mg) and DIPEA (104 μL) were added. After 10 minutes, intermediate 20b (82 mg, prepared from 19b by deprotection using conventional methods) was added and stirred at room temperature for 1 hour. After TLC monitoring, the reaction was stopped, diluted with water, extracted three times with ethyl acetate, and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate. Column chromatography (dichloromethane:methanol = 30:1) afforded the desired product IV-21 as a white solid (34 mg, yield 31%).

[0119] 1 H NMR (300MHz, DMSO-d6) 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, 3.0H z,3H),7.35(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).

[0120] Example 3 7-(4-(5-(4-chlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-23)

[0121] 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 in a yield of 35%.

[0122] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 11.90 (s, 1H), 8.49 (d, J = 2.4Hz, 1H), 7.96 (d, J = 9. 6Hz,1H),7.91(dd,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).

[0123] Example 4 7-(4-(5-(3,4-dichlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-22)

[0124] 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 done according to conventional methods) to obtain the target product IV-22 as a white solid with a yield of 20%.

[0125] 1 H NMR (300MHz, DMSO-d6) 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 = 8.7 Hz,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).

[0126] Example 5 7-(4-(5-(3-chloro-4-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-27)

[0127] The synthesis method refers to compound IV-21, and intermediate 20e is added (20e is prepared from 19e by deprotection, and the deprotection can be carried out according to conventional methods) to obtain the target product IV-27 as a white solid in a yield of 58%.

[0128] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 11.90 (s, 1H), 8.50 (d, J = 2.5Hz, 1H), 8.00-7.91 (m, 2 H),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=9.0 Hz, 1H), 6.58 (dd, J = 9.5, 1.7Hz, 1H), 3.72 (d, J = 20.1Hz, 4H), 3.53 (d, J = 39.9Hz, 4H).

[0129] Example 6 7-(4-(5-(4-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-24)

[0130] 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 as a yellow-brown solid with a yield of 41%.

[0131] 1 H NMR (300MHz, DMSO-d6) 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), 7.6 9-7.63(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).

[0132] Example 7 7-(4-(5-(3-hydroxyphenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-25)

[0133] The synthesis method refers to compound IV-21, and 20g of the intermediate is added (20g is prepared from 19g by deprotection, and the deprotection can be done according to conventional methods) to obtain the target product IV-25 as an off-white solid with a yield of 36%.

[0134] 1 H NMR (300MHz, DMSO-d6) 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),7.27-7.1 7(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 ,1.6Hz,1H),6.58(dd,J=9.5,1.5Hz,1H),3.71(d,J=26.3Hz,4H),3.52(d,J=31.3Hz,4H).

[0135] Example 8 7-(4-(5-(4-chloro-2-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-26)

[0136] 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 done according to conventional methods) to obtain the target product IV-26 as a white solid with a yield of 33%.

[0137] 1 H NMR (300MHz, DMSO-d6) 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).

[0138] Example 9 7-(4-(5-(3,5-difluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-14)

[0139] 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 as an off-white solid with a yield of 20%.

[0140] 1 H NMR (300MHz, DMSO-d6) 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).

[0141] Example 10 7-(4-(5-(3,5-dichlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-15)

[0142] 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 done according to conventional methods) to obtain the target product IV-15 as an off-white solid in a yield of 35%.

[0143] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 11.90 (s, 1H), 8.20 (d, J = 2.3Hz, 1H), 7.97 (d, J = 9.6Hz, 1 H),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 =8.3,2.0Hz,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).

[0144] Example 11 7-(4-(5-(3-chloro-5-fluorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-16)

[0145] 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 as an off-white solid with a yield of 30%.

[0146] 1 H NMR (300MHz, DMSO-d6) 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,2H),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).

[0147] Example 12 7-(4-(5-(3-chlorophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-19)

[0148] 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 as an off-white solid with a yield of 38%.

[0149] 1 H NMR (300MHz, DMSO-d6) 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,1 H), 6.58 (dd, J = 9.6, 1.7Hz, 1H), 3.73 (d, J = 7.5Hz, 4H), 3.59 (s, 2H), 3.46 (s, 2H).

[0150] Example 13 7-(4-(4-(4-(4-nitrophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-41)

[0151] 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 done according to conventional methods) to obtain the target product IV-41 as a yellow solid in a yield of 46%.

[0152] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.88 (s, 1H), 8.64 (d, J = 2.5Hz, 1H), 8.29-8.2 3(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).

[0153] Example 14 7-(4-(4-(4-(3-nitrophenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-42)

[0154] 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 done according to conventional methods) to obtain the target product IV-42 as a yellow solid with a yield of 40%.

[0155] 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).

[0156] Example 15 7-(4-(4-(4-(4-methoxyphenyl)pyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-43)

[0157] 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 done according to conventional methods) to obtain the target product IV-43 as an off-white solid in a yield of 42%.

[0158] 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).

[0159] Example 16 7-(4-(4-(4-phenylpyridin-2-yl)piperazine-1-carbonyl)quinolin-2(1H)-one (IV-44)

[0160] 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 as an off-white solid with a yield of 46%.

[0161] 1 H NMR (300MHz, DMSO-d6) 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 = 7.5 Hz,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).

[0162] Biological activity testing of compounds

[0163] Example 17 Determination of MAGL inhibitory activity

[0164] 1. Test Principle: Cayman's MAGL Inhibitor Screening Kit provides a simple and efficient method for screening compounds for inhibitory activity against human MAGL. MAGL hydrolyzes an alternative substrate (ethyl 4-nitrophenylacetate in place of 2-AG) to produce a yellow product (4-nitrophenol), which has a maximum absorption at 405-412 nm. The absorbance at this wavelength is measured to quantify the amount of 4-nitrophenol in the hydrolysis product, thereby characterizing enzyme activity.

[0165] 2. Preparation of reagents

[0166] (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. It was used for analysis and dilution of MAGL and the positive drug JZL195 and was stored at -20°C until use.

[0167] (2) Preparation of human recombinant MAGL: Take 30 μL of MAGL protein, add 570 μL of buffer (1×), and store for later use.

[0168] (3) Preparation of MAGL substrate: Take 150 μL substrate, add 450 μL buffer (1×), and store for later use.

[0169] (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.

[0170] 3. Experimental operation

[0171] (1) Background well: add 160 μL buffer (1×), 10 μL solvent

[0172] (2) 100% initial enzyme activity well: add 150 μL buffer (1×), 10 μL enzyme, and 10 μL solvent

[0173] (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.

[0174] Table 1

[0175] (4) Mix the contents of the wells and incubate at room temperature for 15 minutes.

[0176] (5) 10 μL of substrate was added to each well, the 96-well plate was shaken thoroughly for 10 s, and incubated at room temperature for 10 min. Readings were taken at 405 nm using a Multiscan GO (Thermo) microplate reader. The inhibitory rate was calculated according to the following formula. Nonlinear regression analysis was performed using GraphPad Prism software. The experimental data were analyzed using second-order polynomial regression analysis and mixed model inhibition fitting.

[0177] The results are shown in Table 2. As shown in Table 2, the compounds of the present invention have MAGL inhibitory activity.

[0178] Referring to the method similar to this example, the IC of the compound of the present application was determined. 50 , the specific method is as follows:

[0179] According to the above method, the inhibition rate of the applied compound at concentrations of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM was determined. The experimental data were analyzed using second-order polynomial regression analysis and mixed model inhibition fitting to obtain the IC value of the compound for MAGL inhibition. 50 data.

[0180] Example 18 Determination of 2-AG concentration in the brain

[0181] ICR male mice (3 / group) were used. The test compound IV-23 was dissolved in 10% DMSO, 10% Tween 80, and 80% normal saline to prepare a dosing solution. The dose of the test compound was prepared at 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 brain was separated at 60 min, 120 min, 240 min, and 480 min, and the cerebral hemispheres were extracted (the control was separated at 0 min). The obtained cerebral hemispheres were frozen on dry ice, and the weight of the frozen tissue was measured. About 100 mg of brain tissue was weighed and homogenized with 9 times the volume of normal saline. After homogenization, the mixture was centrifuged at 15,000 rpm for 10 minutes. The supernatant was divided into aliquots. Each tube was about 50 microliters. The 2-AG concentration in the brain was determined by the Elisa method.

[0182] The results of brain 2-AG concentrations are shown in Table 3, with the unit being ng / g.

[0183] Table 3

[0184] 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.

[0185] Example 19 Pharmacokinetics Test

[0186] 1. Experimental Materials

[0187] (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 minutes.

[0188] (2) Preparation of stock solution: Accurately weigh 2.0 mg of compound IV-23 and dissolve it in water to make a stock solution of 200.0 μg / mL in 10 mL. Accurately weigh 2.0 mg of imipramine hydrochloride and dissolve it in water to make a stock solution of 200.0 μg / mL in 10 mL. Dilute it to 3.000 μg / mL with methanol as the internal standard working solution and store it in a refrigerator at 4°C until use.

[0189] (3) Preparation of standard curve samples: Dilute the IV-23 stock solution with methanol to a series of standard solutions of 30,000, 15,000, 7,500, 3,000, 1,500, 300.0, and 30.00 ng / mL. Accurately pipette 10 μL of the above series of standard solutions, add 50 μL of blank SD rat plasma, add 10 μL of internal standard working solution, mix well, and add 230 μL of methanol. The final volume is 300 μL, and the final concentrations of compound IV-23 are 1,000, 500.0, 250.0, 100.0, 50.00, 10.00, and 1.000 ng / mL. After vortex mixing for 5 minutes, centrifuge at 12,000 r / min for 10 minutes. Transfer the supernatant to an autosampler vial for LC-MS / MS analysis to prepare for the preparation of the standard curve.

[0190] (4) LC / MS conditions: The chromatographic column was Waters 5C18-MS-Ⅱ (2.0×250 mm, 5 μm); the mobile phase was 0.5% formic acid in water (A)-methanol (B); isocratic elution was used, the organic phase ratio was 90%, the flow rate was 0.3 mL / min, the column temperature was 40°C, and the injection volume was 10 μL.

[0191] (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 and 0.25 mL Tween-80, then add 4.2 mL normal saline injection. After dissolution, sonicate and shake until the compound is clear. Prepare a 0.1 mg / mL preparation. Prepare and use 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 and 0.25 mL Tween-80, then add 4.625 mL normal saline injection. After dissolution, sonicate and shake until the compound is clear. Prepare a 1 mg / mL PO preparation and a 0.2 mg / mL IV preparation. The administration volume is 10 mL / kg, and the dosage is 10 mg / kg and 2 mg / kg, respectively. Prepare and use on the day of use.

[0192] The pharmacokinetic parameters of IV-23 are shown in Table 4:

[0193] a T max ,peak time; b C max , maximum blood concentration; c AUC, area under the drug-dose curve; d Vd, apparent distribution solvent; e t 1 / 2 , drug half-life; f CL, clearance rate; g MRT, mean residence time.

[0194] 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 gavage, indicating that compound IV-23 has good pharmacokinetic properties.

[0195] Example 20 Determination of the antidepressant effect of the compound on depressed mice

[0196] ICR male mice were used. After 1 week of adaptation, 12 mice were randomly divided into blank controls and raised normally; the remaining 70 mice were placed in 50 ml centrifuge tubes (with a hole at the end of the tube for the mice to breathe) and restrained for 4 to 8 hours per day, increasing from 4 hours per day to 8.5 hours per day, for 30 consecutive days.

[0197] Model determination:

[0198] 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 mouse's tail was fixed with tape and hung on a stand with the head 15 cm above the ground. The mice were videotaped after 2 minutes of adaptation, and the immobility time of the mice within 4 minutes was counted.

[0199] Grouping and drug administration:

[0200] After model establishment, mice were divided into a model group, a positive drug group (fluoxetine, 8 mg / kg), a low-dose compound IV-23 group (4 mg / kg), and a high-dose compound IV-23 group (8 mg / kg), based on the immobility time during the tail suspension test. Each group consisted of 6-8 mice. The compound was formulated with 10% DMSO, 40% PEG 400, 5% Tween 80, and 45% saline. The administration volume was 10 ml / kg. Dosage was continued for 7 consecutive days. After 7 days, behavioral tests including the tail suspension test, open field test, forced swim test, and sugar preference test were performed.

[0201] 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.

[0202] Table 5

[0203] Example 21 Determination of the in vitro anti-nonalcoholic fatty liver effect of the compound

[0204] 1. Instruments and Materials

[0205] (1) Cells and reagents: Human hepatocellular carcinoma cell line HepG2 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; fetal bovine serum was purchased from Solebo Biotechnology Co., Ltd.; DMEM high glucose medium, 0.25% trypsin solution, and PBS solution were purchased from Nanjing Key Biotechnology Development Co., Ltd.; DMSO, oleic acid, palmitic acid, and Oil Red O were purchased from Guangdong Xilong Chemical Co., Ltd.

[0206] (2) Main instruments: MULTISKAN MK3 fully automatic microplate reader (Thermo Scientific), vortex shaker (QL-902); centrifuge, BT224 electronic balance (Sartouris, Germany); HB-202 constant temperature water bath (Zhongxi Yuanda, Beijing); BX51 upright microscope (Olympus, Japan); other equipment and instruments include centrifuge tubes, pipettes, straws, etc.

[0207] 2. Experimental methods:

[0208] (1) Cell culture method

[0209] HepG2 cells were seeded in DMEM high-glucose medium supplemented with 10% fetal bovine serum and allowed to adhere to the culture medium. Cultured in an incubator at 37°C with 5% CO₂ saturated humidity. Depending on cell growth, cells were digested with 0.25% trypsin every 1-2 days for subculture.

[0210] (2) Effect of compound IV-23 on free fatty acid-induced NASH model in HepG2 cells

[0211] HepG2 cells were cultured in 96-well plates. After cell attachment reached 60-70%, they were co-stimulated with free fatty acids (palmitic acid:oleic acid at a molar ratio of 1:2) and varying concentrations of the test drug (Compound IV-23) 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, while the drug-treated groups were treated with DMEM at concentrations of 5μM and 10μM, respectively.

[0212] (3) Oil Red O staining to observe intracellular lipid droplets

[0213] Oil Red O staining procedures are as follows: cells were washed three times with PBS, fixed with 10% neutral formaldehyde for 30 minutes, washed twice with PBS, stained with Oil Red O at 37°C, incubated for 1 hour at 37°C, discarded the stain, and washed twice with PBS. Under a microscope, neutral lipids within cells are specifically stained red, allowing observation of lipid accumulation in cells.

[0214] 3. Experimental results: Oil red O staining to observe the effect of compound IV-23 on NASH cell model

[0215] 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. After treatment with compound IV-23, the aggregation of lipid droplets in HepG2 cells was significantly inhibited. This shows that the compound IV-23 prepared by the present invention can significantly inhibit the lipid accumulation induced by free fatty acids in a dose-dependent manner at 5μM and 10μM, and has the effect of treating non-alcoholic steatohepatitis.

[0216] Example 22 Determination of the Anti-Parkinson's Disease Effect of Compounds in Vivo

[0217] 1. Experimental Methods: Mice were acclimated for 1 week. Five animals were randomly selected from the normal group, not treated, and housed normally. Mice in the remaining four groups were intraperitoneally injected with the solvent or drug once on the first day and with MPTP (15 mg / kg, once every 2 hours, for a total of 4 injections) on the second day. The solvent or drug was injected 1 hour after the second MPTP injection. On the third day, the solvent or drug was injected intraperitoneally once. Behavioral testing was performed 2 hours after drug administration.

[0218] 2. Behavioral Experiment - Rotarod Test: The apparatus was placed 50 cm above the ground. A digital timer was located at the bottom of each partition; when an animal fell, the time was recorded. Mice were trained on the apparatus for a fixed period of time. The rotarod rotated at a speed of 20 rpm. The animals' grip strength was assessed using a rotarod. Mice were trained for two consecutive days before dosing. Behavioral testing used a 7-cm diameter rotating rod at a speed of 20 rpm, with a cutoff time of 180 s.

[0219] 3. Experimental Results: As shown in Figure 2, the average time for the blank mice on the rotarod was 59.26 seconds, while that for the model group was 24.1 seconds, showing significant differences from the blank group and indicating successful model development. The average time for the positive drug JZL184 group was 46.75 seconds, demonstrating some therapeutic effect, but not significantly different from the model group. The average time for the IV-23 (10 mg / kg) group was 53.82 seconds, also significantly different from the model group, indicating that IV-23 (10 mg / kg) significantly improved the motor function of Parkinson's mice.

[0220] Example 23 Determination of the Effect of Compounds on Cholestatic Liver Injury

[0221] 1. Experimental methods:

[0222] (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-treated 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 treated group were intraperitoneally injected with IV-26 (12 mg / kg, 10 mL / kg) every day for one week, and the control group and the 0.1% DDC group were injected with solvent (10 mL / kg) in the same way for one week. One hour after the last administration, the mice were bled from their orbits, sacrificed, and their livers were dissected. After washing with physiological saline, the surface moisture was absorbed, and the liver lobules were carefully separated and fixed in formalin fixative. The remaining liver was stored at -80 degrees.

[0223] (2) After the blood sample was allowed to stand at room temperature for 30 minutes, it was centrifuged at 3000 rpm / min for 10 minutes at 4 degrees Celsius. The upper serum sample was collected and the serum alanine aminotransferase (ALT / GPT) and serum alkaline phosphatase (ALP) levels were detected according to the kit instructions.

[0224] (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 5 μm thick sections. The prepared sections were stained with hematoxylin and eosin dyes. The sections after HE staining were dehydrated, transparentized, and mounted for observation under a microscope to record the liver tissue pathology.

[0225] 2. Experimental results:

[0226] (1) Effects of IV-26 on serum biochemical parameters in mice with DDC-induced cholestatic liver injury

[0227] Serum ALT and ALP levels are important indicators of liver function and can directly reflect the extent of liver damage. The experimental results, shown in Figure 3, show that compared with the control group, serum ALT and ALP levels in the DDC group were significantly increased, indicating that the livers of mice in the DDC group were severely damaged and that a cholestatic liver injury model was successfully established. Compared with the DDC group, serum ALT and ALP levels in the IV-26-treated group were both reduced. These results demonstrate that the test drug IV-26 significantly ameliorates DDC-induced cholestatic liver injury.

[0228] (2) Effects of IV-26 on liver histopathological changes in mice with DDC-induced cholestatic liver injury

[0229] Pathological changes in the mouse livers were observed microscopically after HE staining. The results are shown in Figure 4. The liver cells in the control group were intact and tightly arranged, with normal structures. The DDC group showed significant inflammatory cell infiltration, hepatocellular necrosis, and morphological changes. Compared with the DDC group, the IV-26-treated group improved liver damage, reduced inflammatory cell infiltration, and alleviated hepatocellular necrosis. These results demonstrate that IV-26 ameliorates the histopathological changes in DDC-induced cholestatic liver injury.

Claims

1. A compound having a structure represented by formula (I) or a pharmaceutically acceptable salt or isotope-substituted product thereof: in, R1 is selected from aryl or heteroaryl; R2 is selected from substituted or replaced by any R 2A substituted aryl or heteroaryl; R 2A Selected from -OH, -SH, -CN, halogen, nitro, carboxyl, C 1-8 Alkyl, C 1-8 Alkoxy, C 1-4 Halogenated alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt or isotope-substituted product thereof, characterized in that: The aryl groups in R1 and R2 are each independently C6-C 10 Aryl, preferably aryl is independently phenyl or naphthyl, specifically preferably, in R1 and R2, aryl is phenyl; in R1 and R2, heteroaryl is a 5-10 membered heteroaryl with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2, 3 or 4"; preferably, heteroaryl is a 5-10 membered heteroaryl with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3"; more preferably, heteroaryl is a 5-6 membered heteroaryl with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3"; further preferably, heteroaryl is a 5-6 membered heteroaryl with "heteroatoms selected from N, O and S, and the number of heteroatoms is 1 or 2"; more preferably, heteroaryl is a 5-6 membered heteroaryl with "heteroatoms selected from N and S, and the number of heteroatoms is 1 The number of heteroaryl atoms is 1 or 2. The 5-6 membered heteroaryl atoms are further preferably "a 5-6 membered heteroaryl atoms having a heteroatom selected from N and S and a heteroatom number of 1". More preferably, in R1 and R2, the heteroaryl atoms are furyl, 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, oxazolinyl, indolyl, quinolyl, isoquinolyl, indazolyl, benzoxazolyl, benzothiazolyl, purinyl, oxazolopyridinyl. Specifically preferably, in R1 and R2, the heteroaryl atoms are pyridyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt or isotope-substituted product thereof, characterized in that: R1 is a heteroaryl group, and R2 is substituted or replaced by any R 2A Preferably, R1 is pyridyl, R2 is substituted or replaced by any R 2A Substituted phenyl.

4. The compound according to claim 1 or a pharmaceutically acceptable salt or isotope-substituted product thereof, characterized in that: R 2A The number is 1, 2, 3 or 4, preferably 1 or 2, more preferably, R 2A Each is independently selected from -OH, -SH, -CN, cyano, halogen, nitro, carboxyl, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, -CF3, CHF2 or CH2F.

5. A compound having the following structure or a pharmaceutically acceptable salt or isotope thereof:

6. A method for preparing the compound of formula (I) according to claim 1:

7. A pharmaceutical composition, characterized in that It comprises the compound as claimed in any one of claims 1 to 5 or its pharmaceutically acceptable salt or isotope substitution, 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 its pharmaceutically acceptable salt or isotope substitution.

8. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or isotope substituted product thereof in the preparation of a MAGL inhibitor.

9. Use of the compound according to 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.

10. The use according to claim 9, 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.

Images