Polysubstituted tetrahydropyrimidine compound and application thereof in preparation of medicine for treating HsClpP-mediated diseases

Abstract

The invention relates to a polysubstituted tetrahydropyrimidine compound and application of the polysubstituted tetrahydropyrimidine compound in preparation of a medicine for treating HsClpP mediated diseases. The polysubstituted tetrahydropyrimidine compound or pharmaceutically acceptable salt, hydrate or crystal form thereof plays a role in treatment of human casein lyase (HsClpP) mediated diseases, and belongs to the field of chemical medicines. The technical problem to be solved by the invention is to provide a polysubstituted tetrahydropyrimidine compound. The compound is characterized in that the compound has a framework structure of ectoine, nitrogen atoms on a pyrimidine ring are linked with other substituent groups, and double bond parts of the compound are substituted by cyclic aromatic. The compounds have obvious activity of regulating and controlling HsClpP, and can be used for treating HsClpP-mediated related diseases.

Description

Technical Field

[0001] This invention relates to the use of a class of ternary topological HsClpP agonists or their pharmaceutically acceptable salts, hydrates or crystal forms in antitumor and anti-inflammatory effects, belonging to the field of chemical medicine. Background Technology

[0002] HsClpP is an ATP-dependent unfolded peptidase-protein complex found in the mitochondrial matrix. HsClpP maintains organelle homeostasis, controls protein quality, regulates mitochondrial metabolism, and plays a crucial role in the integrity of mitochondrial unfolded protein responses and oxidative phosphorylation. Abnormal expression or functional mutations of HsClpP can lead to mitochondrial dysfunction and induce various diseases. These include neurological diseases such as Perrault syndrome (PRLTS), Parkinson's disease (PD), and Alzheimer's disease (AD), as well as tumors and metabolic syndromes such as obesity and diabetes, which have been extensively studied. The mechanisms of action of HsClpP in human diseases include regulating ROS levels and ATP production, affecting the activity of mitochondrial respiratory chain complex enzymes, and Ca2+ signaling. Current preclinical data support restoring normal HsClpP activity as a novel treatment for mitochondrial dysfunction. This treatment provides a new strategy for treating HsClpP-related diseases. In recent years, several small molecules have been developed to regulate the proteolytic activity of HsClpP. This includes HsClpP inhibitors (such as β-lactones, phenyl esters, and borate peptides) and HsClpP agonists (such as ADEP, D9, and imipridone compounds). Among these, the imipridone compound ONC201 is currently in the new drug approval stage for clinical trials in glioma treatment. However, there are currently no reports of HsClpP agonists or inhibitors being used to treat small cell lung cancer or other tumors such as leukemia, liver cancer, and inflammatory diseases. Summary of the Invention

[0003] HsClpP is an ATP-dependent unfolded peptidase-protein complex found in the mitochondrial matrix. HsClpP maintains organelle homeostasis, controls protein quality, regulates mitochondrial metabolism, and plays a crucial role in the integrity of mitochondrial unfolded protein responses and oxidative phosphorylation. Abnormal expression or functional mutations of HsClpP can lead to mitochondrial dysfunction and induce various diseases. These include neurological diseases such as Perrault syndrome (PRLTS), Parkinson's disease (PD), and Alzheimer's disease (AD), as well as tumors and metabolic syndromes such as obesity and diabetes, which have been extensively studied. The mechanisms of action of HsClpP in human diseases include regulating ROS levels and ATP production, affecting the activity of mitochondrial respiratory chain complex enzymes, and Ca2+ signaling. Current preclinical data support restoring normal HsClpP activity as a novel treatment for mitochondrial dysfunction. This treatment approach provides a new strategy for treating HsClpP-related diseases.

[0004] In recent years, several small molecules have been developed to regulate the hydrolytic activity of HsClpP proteins. These include HsClpP inhibitors (such as β-lactones, phenyl esters, and borate peptides) and HsClpP agonists (such as ADEP, D9, and imipridone compounds). Among these, the imipridone compound ONC201 is currently in clinical trials for glioma treatment and has entered the new drug approval stage. However, there are currently no reports of HsClpP agonists or inhibitors being used to treat small cell lung cancer and other tumors such as leukemia, liver cancer, and inflammatory diseases.

[0005] Specifically, the technical solution and content of the present invention involve the following three aspects.

[0006] In a first aspect, the present invention relates to a class of compounds with a Z1-L-Z2 ternary topological structure, characterized in that the compound is as shown in Formula I: Z1-L-Z2 Formula I Z1 is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkylacyl, arylacyl, and aralkylacyl; Z2 is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyalkyl, alkylamino, cycloalkylamino, heterocycloalkylamino, aralkyloxy, and aralkylthio; the connecting arm L is independently selected from the following structures: R1 to R7 are independently selected from hydrogen, halogen, nitro, hydroxyl, alkoxy, and alkyl.

[0007] Furthermore, the Z1-L-Z2 ternary topological structure compound is characterized in that the compound is as shown in Formula III. Ar1 and Ar2 are independently selected from 0-5 phenyl groups substituted with R14 or R15, where R14 and R15 are selected from halogens, cyano groups, C1-C6 alkyl groups, C3-C9 substituted cycloalkyl groups, C1-C6 haloalkyl groups, -CF3, -NH2, -NO2, -SH, -SR16, -OH, C1-C6 substituted alkoxy groups, -NR16, R17, (C3-C9)cycloalkyl, (C2-C6)alkynyl, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, substituted aryl, substituted heterocyclic aryl, -COOH, -COOR18, -OCOOR18, C2-C6 alkynyl, C2-C8 alkenyl, -SO2OR18, SO2NR18R19, -SO2R18, -NR18SO2R19, -CONR18R19, -COR18, -NR18. COR19; R1 to R8 are independently selected from hydrogen, halogen, C3-C6 cycloalkyl, and C1-C6 substituted alkyl; R10 to R13 are independently selected from hydrogen, halogen, C3-C6 cycloalkyl, C1-C6 substituted alkyl, and heteroatoms; R16 to R17 are independently selected from hydrogen, halogen, and C1-C3 substituted alkyl; R18 to R19 are independently selected from hydrogen, C3-C6 cycloalkyl, and C1-C6 substituted alkyl.

[0008] The Z1-L-Z2 ternary topological structure compound according to claim 3 is characterized in that the compound is as shown in Formula IV. R12 and R13 are independently selected from hydrogen, halogen, cyano, C1-C6 alkyl, C3-C9 substituted cycloalkyl, C1-C6 haloalkyl, and CF3, while R7 is independently selected from hydrogen and halogen.

[0009] Furthermore, the Z1-L-Z2 ternary topological structure compound has the following structural formula:

[0010] Furthermore, the salts, hydrates, or crystal forms of the Z1-L-Z2 ternary topological structure compounds and the isotopically substituted compounds of any one atom, wherein the salts are preferably pharmaceutically acceptable salts that can be formed by the compounds of the present invention with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, or aspartic acid.

[0011] This invention also provides the use of the above-mentioned compounds in the treatment of HsClpP-mediated inflammatory diseases and tumor-related diseases. The inflammatory diseases include inflammatory bowel disease, etc., and the tumors include central nervous system tumors, brain tumors, peripheral nervous system tumors, pheochromocytomas, paragangliomas, neuroendocrine tumors, liver cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, prostate cancer, endometrial cancer, hematologic malignancies, and lymphatic system tumors.

[0012] Through extensive experimental screening, this invention has identified a highly active HsClpP agonist, providing a better option for the clinical treatment of HsClpP-mediated diseases.

[0013] The pharmaceutically acceptable adjuvant component possesses certain physiological activities, but its addition does not alter the dominant role of the pharmaceutical composition in the disease treatment process; rather, it merely exerts an adjuvant effect. These adjuvant effects are simply the utilization of the known activities of the component, and are a commonly used adjuvant therapy method in the pharmaceutical field. If the aforementioned adjuvant component is used in combination with the pharmaceutical composition of the present invention, it still falls within the scope of protection of this invention.

[0014] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular, or subcutaneous) and topical administration.

[0015] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient or carrier, such as sodium citrate or dicalcium phosphate, or with: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as premethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0016] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0017] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0018] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, and fragrances.

[0019] In addition to active compounds, suspensions may contain suspending agents such as mixtures of substances such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar.

[0020] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0021] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0022] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable mixtures.

[0023] The isotope-labeled compounds described in this invention refer to compounds identical to those listed herein, but in which one or more atoms are replaced by another atom whose atomic mass or mass number differs from that of naturally occurring atoms. Isotopes that can be introduced into the compounds include hydrogen, carbon, nitrogen, oxygen, and sulfur, i.e., 2H, 3H, 13C, 14C, 15N, 17O, 18O, 35S, etc. Compounds containing the above-mentioned isotopes and / or other atomic isotopes, their stereoisomers, and pharmaceutically usable salts of such compounds and stereoisomers should all be included within the scope of this invention.

[0024] Secondly, the method for preparing the compound described in this invention includes the following synthetic route: Reaction conditions: (a) Borates, Pd(Ph3)4, K2CO3, ACN, 90℃, 2hr-overnight; (b) TMSOK, ACN / H2O, 100℃ 50-90%; (c) HATU, DIPEA, p-(Trifluoromethyl)benzylamine, DMF, rt, 46-88% yield; (d) 1) TFA, DCM; 2) 3-(bromomethyl) benzonitrile, K2CO3, DMF, rt, 45-80% yield. Route 2 is: Reaction conditions: (a) Borates, Pd(Ph3)4, K2CO3, ACN, 90℃, 2hr-overnight; (b) TMSOK, ACN / H2O, 100℃ 50-90%; (c) HATU, DIPEA, p-(Trifluoromethyl)benzylamine, DMF, rt, 46-88% yield; (d) 1) TFA, DCM; 2) 3-(bromomethyl) benzonitrile, K2CO3, DMF, rt, 45-80% yield.

[0025] Thirdly, the technical solution and content of this invention relate to the use of the compound of Formula I for anti-tumor drugs and the treatment of inflammatory diseases. The compound of Formula I can be used as an HsClpP agonist, which can regulate mitochondrial protein homeostasis, thereby affecting cell proliferation, apoptosis, etc. This enriches the types of drugs targeting mitochondria and provides new probe molecules for the treatment and mechanistic research of tumor drugs and inflammatory bowel diseases. Attached Figure Description

[0026] Figure 1The present invention provides experiments on the degradation of α-casein by HsClpP protein using preferred compounds.

[0027] Figure 2 The preferred compounds of this invention affect the thermodynamic stability of intracellular HsClpP protein.

[0028] Figure 3 The preferred compound of this invention is used to inhibit the formation of tumor cell clones in vitro.

[0029] Figure 4 Cell scratch assay of the preferred compounds of this invention.

[0030] Figure 5 The preferred compound of this invention demonstrates its therapeutic effect on a mouse model of small cell lung cancer. Detailed Implementation

[0031] Example 1: 1-(3-cyanobenzyl)-4-phenyl-N-(4-(trifluoromethyl)benzyl)-1,2,5,6-tetrahydropyridine-3-carboxamide (5a) Reference 1 for the synthesis of raw material 1. Starting material 1 (0.5 g, 1.2 mmol, 1.00 equivalent) and 2-(2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoboronyl pentane (0.35 g, 1.5 mmol, 1.2 equivalent) were dissolved in 6 mL of 1,2-dimethoxyethane solution, and K₂CO₃ (0.26 g, 1.86 mmol, 1.5 equivalent) was added. The mixture was degassed with N₂ for 15 min, and then refluxed at 90 °C for 6 h. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the solid fraction was washed with ethyl acetate (EA, 10 mL). The filtrate was concentrated under reduced pressure and purified by Biotage Isolera One rapid column chromatography system (using a 200-300 mesh silica gel column, eluting with a petroleum ether (PE):ethyl acetate (EA) = 0-10%), finally yielding an oily liquid 2 in 76% yield. Compound 2 (0.5 g, 1.5 mmol) was added to an acetonitrile / water (ACN / H2O, 4 mL / 1 mL) solution with potassium trimethylsilanolate (TMSOK) (967.8 mg, 7.25 mmol, 5 equivalents), and the mixture was refluxed at 100 °C for 6 hours. After the reaction was complete, the mixture was acidified to pH 6.0 with acetic acid and quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate (EA, 8 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate (Na2SO4). After filtration, the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (elution gradient: methanol / dichloromethane (CH3OH / CH2Cl2) = 0:100 to 10:100) to give compound 3 (247.2 mg) as a pale yellow oil, in 54% yield. Compound 3 (0.2 g, 0.66 mmol, 1.0 equivalent) was dissolved in anhydrous N,N-dimethylformamide (5 mL), followed by the addition of p-trifluoromethylbenzylamine (0.79 mmol, 0.12 mL, 1.2 equivalent) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.99 mmol, 0.38 g, 1.5 equivalent). After stirring for 20 minutes, N,N-diisopropylethylamine (1.32 mmol, 0.21 mL, 2 equivalent) was added to the reaction mixture, and the reaction was continued with stirring for 1 hour. After the reaction was complete, the solution was quenched with saturated ammonium chloride solution (5 mL) and extracted with ethyl acetate (5 mL × 3). The organic phase was washed with water (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by Biotage Isolera One rapid column chromatography system (200-300 mesh silica column, elution gradient: petroleum ether / ethyl acetate = 0-50%) to give compound 4 (167 mg) in 55% yield. Compound 4 (0.2 g; 0.43 mmol; 1.00 equivalent) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added dropwise at 0 °C. The mixture was heated to room temperature and the reaction was continued for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure and used directly in the next reaction without further purification. The crude product was dissolved in dimethylformamide (4 mL) under nitrogen protection, followed by the addition of potassium carbonate (90 mg; 0.65 mmol; 1.5 equivalents) and 3-cyanobenzyl bromide (0.1 g; 0.52 mmol; 1.2 equivalents). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was quenched with saturated ammonium chloride solution (5 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with water and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by a Biotage Isolera One rapid column chromatography system equipped with a 200-300 mesh silica gel column to give a white solid product 5a (157 mg), in 76% yield. 1 H NMR (400MHz, chloroform-d): δ7.72(s,1H),7.63(d,J=8.0Hz,1H),7.57(d,J=8 .0Hz,1H),7.45(t,J=8.0Hz,1H),7.41(d,J=8.0Hz,2H),7.28-7.25(m,3H),7.22 -7.19(m,2H),6.88(d,J=8.0Hz,2H),5.43(t,J=4.0Hz,1H),4.21(d,J=4.0Hz,2 H),3.71(s,2H),3.4(t,J=4.0Hz,1H),2.69(t,J=8.0Hz,1H),2.55-2.51(m,2H). 13 C NMR (101MHz, chloroform-d): δ171.1,168.5,141.5,140.4,139.5,138.5,133.4,132.5,131.0,130.0,129.6,129.3,129.2,128 .9(2C),128.0,127.8(2C),127.2(2C),125.3(q,J=4.04Hz),122.7,118.8,112.5,77.2,61.3,53.5,49.4,43.0,32.2.HRMS(ESI + ):m / z calcdfor C 28 H 25 F3N3O[M+H] + ,476.1944; found 476.1967.

[0032] Example 2: 1-(3-cyanobenzyl)-4-(2-methoxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5b) Synthesis of compound 5b: 5a. 1H NMR (400MHz, chloroform-d): δ 7.57 (s, 1H), 7.47 (d, J = 4.0Hz, 1H), 7.40 (d, J = 8.0Hz, 1H), 7.29 (d, J = 8.0Hz, 1H), 7.23 (d, J = 8.0Hz, 2H), 7.12–7.06 (m, 1H), 6.94 (d, J = 8.0Hz, 1H), 6.76 (t, J = 8.0Hz, 2H). .0Hz,1H),6.66(d,J=8.0Hz,2H),6.59(d,J=8.0Hz,1H),5.79(t,J=8.0Hz,1H),4.06-3.94(m,2 H),3.55(s,2H),3.52(s,3H),3.34-3.17(m,2H),2.50(t,J=8.0Hz,2H),2.28-2.21(m,2H).13C NMR (101MHz, chloroform-d): δ168.1,155.4,141.9,139.8,136.2,133.3,132.3,130.8,130.5,129.7,12 9.3,129.1,129,127.6,121.3,118.9,112.4,110.9,61.3,55.2,53.1,49.3,42.7,31.7.HRMS(ESI+):m / z calcd for C29H27F3N3O2[M+H]+,506.2050; found,506.2057.

[0033] Example 3: 1-(3-cyanobenzyl)-4-(2-fluorophenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5c) The synthesis of compound 5c is as follows: 5a. 1 H NMR (400MHz, chloroform-d): δ7.7(s,1H),7.62(d,J=8.0Hz,1H),7.55(d,J=8.0H z,1H),7.44(d,J=8.0Hz,1H),7.39(d,J=8.0Hz,2H),7.02(t,J=8.0Hz,1H),6.94( t,J=8.0Hz,1H),6.88(d,J=8.0Hz,2H),5.57(t,J=8.0Hz,1H),4.23(d,J=8.0Hz,2 H),3.69(s,2H),3.40(t,J=4.0Hz,2H),2.68(t,J=8.0Hz,2H),2.48-2.46(m,2H). 13C NMR (101MHz, chloroform-d): δ167.9,160.2,157.8,141.6,139.6,133.3,132.9 ,132.4(d,J=3.03Hz),131.0,129.9(d,J=4.04Hz),129.7(d,J=8.08Hz),129.6,1 29.3,129.2,127.9,127.7,127.6,125.3(q,J=4.04Hz),124.6(d,J=3.03Hz),12 2.7,118.9,115.8(d,J=22.02Hz),112.5.61.2,53.3,49.1,42.8,31.5.HRMS(ESI + ):m / z calcdfor C 28 H 23 F3N3NaO[M+Na] + ,532.1374; found,532.1389.

[0034] Example 4: 4-(2-chlorophenyl)-1-(3-cyanobenzyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5d) The synthesis of compound 5d is as follows: 5a. 1 H NMR (400MHz, chloroform-d): δ7.72(s,1H),7.63(d,J=8.0Hz,1H),7.57(d,J=8.0Hz,1H),7.46 -7.43(m,1H),7.39(d,J=12.0Hz,1H),7.27-7.24(m,1H),7.20-7.17(m,3H),6.84(d,J=8.0Hz, 1H),5.68(t,J=8.0Hz,1H),4.40-4.36(m,1H),4.09-4.05(m,1H),3.72(d,J=8.0Hz,2H),3.64( d,J=16.0Hz,1H),3.2(d,J=16.0Hz,1H),2.78(brs,1H),2.60-2.51(m,2H),2.34-2.29(m,1H). 13C NMR(101MHz,chloroform-d): δ167.5(2C),141.6,139.4(d,J=28.28Hz),136.3,133.2(2C),132.2(2C),131.5(d,J=5.05Hz),130.8,129.5(d,J= 5.05Hz),129.2,129(d,J=4.04Hz),128.9,127.4(3C),125.3,125.2(q,J=4.04Hz),122.6,118.8,112.2,61.0,52.8,48.9,42.5,31.2.HRMS(ESI + ):m / z calcd for C 28 H 23 ClF3N3NaO[M+Na] + ,532.1374; found,532.1389.

[0035] Example 5: 1-(3-cyanobenzyl)-4-(o-tolyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5e) The synthesis of compound 5e is as follows: 5a. 1 H NMR (400MHz, chloroform-d): δ7.73(s,1H),7.63(d,J=8.0Hz,1H),7.56(d,J=8.0Hz,1H),7.45(d ,J=8.0Hz,1H),7.41(d,J=8.0Hz,2H),7.17-7.06(m,4H),6.84(d,J=8.0Hz,2H),5.41(d,J=8.0Hz ,1H),4.28(dd,J1=4.0Hz,J2=8.0Hz,1H),4.08(dd,J1=4.0Hz,J2=8.0Hz,1H),3.72(s,2H),3.44( q,J=16.0Hz,2H),2.74-2.70(m,1H),2.65-2.59(m,1H),2.48-2.33(m,2H),2.16(s,3H).HRMS(ESI + ):m / zcalcd for C 29 H 26 F3N3NaO[M+Na] + ,512.1920; found,512.1946.

[0036] Example 6: 1-(3-cyanobenzyl)-4-(2-isopropylphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5f) The synthesis of compound 5f is as follows: 5a. 1 H NMR (400MHz, chloroform-d): δ7.73(s,1H),7.63(d,J=8.0Hz,1H),7.56(d,J=8.0Hz,1H),7.44(t,J=8.0Hz, 1H),7.38(d,J=8.0Hz,2H),7.31-7.28(m,1H),7.16-7.12(m,1H),7.05(d,J=8.0Hz,1H),6.79(d,J=8.0Hz,1H ),5.44(t,J=4.0Hz,1H),4.16(d,J=4.0Hz,2H),3.72(s,2H),3.51-3.39(m,1H),2.98-2.89(m,1H),2.73-2.6 8(m,1H),2.65-2.59(m,1H),2.52-2.43(m,1H),2.39-2.31(m,1H).1.28-1.23(m,1H),1.18(s,3H),1(s,3H). 13 C NMR (101MHz, chloroform-d): δ167.0,145.8,141.4,140.0,139.1,133.4,132.5,131.0,129.4,129.1,128.5,127.7(2 C),127.2,126.8,126.6,125.3(q,J=4.04Hz),118.9,112.5,61.5,53.1,49.3,43.0,34.3,29.8,24.8,23.8.HRMS(ESI + ):m / z calcd for C 31 H 30 F3N3NaO[M+Na] + ,540.2233;found,540.2235.

[0037] Example 7: 1-(3-cyanobenzyl)-4-(2-nitrophenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5g) The synthesis of compound 5g is shown in 5a. 1H NMR (400MHz, chloroform-d): δ7.77 (dd, J1=4.0Hz, J2=12.0Hz, 1Hz), 7.69 (s, 1H), 7. 61(d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.48-7.41(m,2H),7.36-7.33(m,3H),7.26 (dd, J1=4.0Hz, J2=8.0Hz, 1Hz), 6.82 (d, J=8.0Hz, 2H), 4.47 (brs, 1H), 3.96 (brs, 1H) ,3.7(s,3H),3.11-3.07(m,1H),2.86-2.81(m,1H),2.52(brs,1H),2.32-2.14(m,2H). 13 C NMR (101MHz, chloroform-d): δ167.8,147.4,141.6,139.5,136.0,134.8,134.0,133.3,132.3,131.9,131.0,129.9,129.5 ,129.2,128.8,127.6(2C),125.4(q,J=4.04Hz),124.3,122.6,119.9,118.8,112.5,61.0,52.9,49.2,42.6,31.4.HRMS(ESI + ):m / z calcd for C 28 H 24 F3N4O3[M+H] + ,521.1795; found,521.1865.

[0038] Example 8: 1-(3-cyanobenzyl)-4-(3-methoxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5h) The synthesis of compound 5h is as follows: 5a. 1H NMR (400MHz, chloroform-d): δ7.71(s,1H),7.61(d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.45-7.40(m,3H),7.18(t,J=8.0Hz,1H),6.9(d,J=8.0Hz,2H),6 .8-6.76(m,2H),6.7(s,1H),5.48(t,J=8.0Hz,1H),4.2(d,J=4.0Hz,2H),3.7 2(s,2H),3.68(s,3H),3.37(s,2H),2.66(t,J=8.0Hz,2H),2.51-2.47(m,2H). 13 C NMR (101MHz, chloroform-d): δ168.3,159.9,142.0,141.5,139.6,138.7,133.4,132.4,131.0,130.0,129.9,129.5,129.3,12 9.2,127.8(2C),125.3(q,J=4.04Hz),122.7,119.3,118.9,113.6,112.6,112.5,61.4,55.1,53.5,49.4,43.0,32.4.HRMS(ESI + ):m / z calcd forC 26 H 26 F3N3NaO2[M+Na] + ,528.1869;found,528.1892.

[0039] Example 9: 1-(3-cyanobenzyl)-4-(3-methanesulfonamide phenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5i) The synthesis of compound 5i is as follows: 5a. 1H NMR (400MHz, MeOH-d4): δ7.69-7.68(m,1H),7.61-7.54(m,3H),7.45-7.40( m,3H),7.23(d,J=8.0Hz,2H),7.16-7.14(m,2H),7.03-7.01(m,1H),6.90(d ,J=8.0Hz,2H),5.6(d,J=8.0Hz,1H),4.24(d,J=4.0Hz,2H),3.68(s,2H),3. 36(t,J=4.0Hz,2H),2.88(s,3H),2.67(t,J=8.0Hz,2H),2.51-2.48(m,2H). 13 C NMR (101MHz, chloroform-d): δ168.8,142.0,141.5,139.5,137.7,137.2,133.4,132.4,131.0,130.1,129.4,129.2,127.9,127 .7,125.5(d,J=4.04Hz),125.4,125.3,123.8,122.6,119.8,119.4,118.9,112.5,61.3,53.6,49.3,43.0,39.6,31.8.HRMS(ESI + ):m / z calcd forC 29 H 27 F3N4NaO3S[M+Na] + ,591.1648; found,591.1643.

[0040] Example 10: 1-(3-cyanobenzyl)-4-(3-fluorophenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5j) The synthesis of compound 5j is as follows: 5a. 1 H NMR (400MHz, chloroform-d): δ7.69(s,1H),7.61(d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.45-7.41(m,3H),7.20-7.14(m,1H) ,6.97-6.89(m,5H),5.43(brs,1H),4.22(d,J=8.0Hz,1H),3.88(s,2H),3.36(s,2H),2.67(t,J=8.0Hz,2H),2.50-2.47(m,2H). 13C NMR (101MHz, chloroform-d): δ168.2,164.0,161.5,142.4(d,J=7.07Hz),141.3,139.4,136.7,133.4(2C),132.4(2C),131.1,130.4(d,J=8Hz ),129.2,127.8(3C),125.4(q,J=3.03Hz),123.0,118.8,114.8(d,J=21Hz),114.3(d,J=22Hz),112.5,61.3,53.5,49.3,43.0,31.8.HRMS(ESI + ):m / z calcd forC 28 H 23 F4N3NaO[M+Na] + ,516.1669; found,516.1703.

[0041] Example 11: 1-(3-cyanobenzyl)-4-(3-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5k) The synthesis of compound 5k is shown in 5a. 1 H NMR (400MHz, chloroform-d): δ7.68-7.67(m,1H),7.61-7.59(m,1H),7.55-7.52(m,1H),7.43-7.40(m,3H),6.94-6.90(m,3H),6.50- 6.45(m,2H),5.87(t,J=8.0Hz,1H),4.26(d,J=8.0Hz,1H),3.69(s,2H),3.39-3.37(m,2H),2.68(t,J=8.0Hz,1H),2.48-2.44(m,2H). 13 C NMR (101MHz, chloroform-d): δ189.2,157.4,141.4,141.1,139.3,139.0,133.6,132.6,131.0,130.2,129.6,129.2,12 9.1,127.6(2C),125.4(q,J=4.04Hz),122.7,118.7,118.1,115.6,114.4,112.3,53.2,49.2,43.2,31.9,29.6.HRMS(ESI + ):m / z calcd for C 28 H 24F3N3NaO2[M+Na] + ,514.1713;found,514.1732.

[0042] Example 12: 1-(3-cyanobenzyl)-4-(3-nitrophenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5l) The synthesis of compound 5l is as described in 5a. 1 H NMR (400MHz, chloroform-d): δ8.05(t,J=4.0Hz,1H),8.02-7.99(m,1H),7.69(s,1H),7.61( d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.50(t,J1=4.0Hz,J2=8.0Hz,1H),7.44(t,J=8.0Hz, 1H),7.39(d,J=8.0Hz,2H),7.30(t,J=8.0Hz,1H),6.97(d,J=8.0Hz,2H),5.66(t,J=8.0Hz,1 H), 4.21 (d, J = 4.0Hz, 2H), 3.7 (s, 2H), 3.37 (s, 2H), 2.72 (t, J = 8.0Hz, 2H), 2.54-2.52 (m, 2H). 13 C NMR (101MHz, chloroform-d): δ168.0,148.2,141.7,141.2,139.3,135.1,133.4(d,J=19.19Hz),132.7,132.4,131.1,129.9 ,129.6,129.3,128.0(3C),125.4(q,J=4.04Hz),125.2,122.6,122.0,118.8,112.5,61.2,53.6,49.2,42.9,31.3.HRMS(ESI + ):m / z calcd for C 28 H 23 F3N4NaO3[M+Na] + ,543.1614; found,543.1670.

[0043] Example 13: 1'-(3-cyanobenzyl)-N-[4-(trifluoromethyl)benzyl]-1',2',5',6'-tetrahydro-[3,4'-bipyridine]-3'-carboxamide (5m) The synthesis of compound 5m is as shown in 5a.1 H NMR (400MHz, chloroform-d): δ8.41(d,J=8.0Hz,1H),7.71(s,1H),7.63(d,J=8.0Hz,1H),7.57(d ,J=8.0Hz,1H),7.49(td,J1=4.0Hz,J2=8.0Hz,1H),7.44(t,J=8.0Hz,1H),7.21(d,J=8.0Hz,1H), 7.09(ddd,J1=4.0Hz,J2=8.0Hz,J3=12.0Hz,1H),6.99(d,J=8.0Hz,1H),6.2(t,J=8.0Hz,1H),4.2 5(d,J=8.0Hz,1H),3.71(s,3H),3.41(t,J=4.0Hz,1H),2.71(t,J=8.0Hz,1H),2.61-2.58(m,2H). 13 C NMR(101MHz,chloroform-d): δ168.4,158.4,149.0(2C),141.6,139.7,137.2,136.8(2C),133.3,132.4(2C),131. 0,129.2,128(2C),125.4(q,J=4.04Hz),123.1,122.7,122.6,116.9,112.5,61.2,53.5,49.4,43.0,30.5.HRMS(ESI + ):m / z calcd for C 27 H 23 F3N4NaO[M+Na] + ,499.1716; found,499.1746.

[0044] Example 14: 1-(3-cyanobenzyl)-4-(4-methoxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5n) The synthesis of compound 5n is as follows: 5a. 1H NMR (400MHz, chloroform-d): δ7.7(d,J=8.0Hz,2H),7.49-7.44(m,3H),7.33(t,J=8.0 1Hz),7.16(d,J=8.0Hz,1H),7.08(d,J=8.0Hz,2H),7.03(s,1H),6.82(d,J=8.0Hz,2H),3.87(d,J=1 2.0Hz,2H),3.8(s,3H),3.7(s,2H),3.37(t,J=4.0Hz,3H),2.68(t,J=8.0Hz,2H),2.57-2.53(m,2H). 13 C NMR (101MHz, chloroform-d): δ167.3,159.2,147.0,139.7,137.0,133.9,133.3,132. 6,132.4,132.0,131.7,131.4,131.0(q,J=4.04Hz),130.9,130.1,129.2(d,J=5.05Hz) ,129.1,128.9,128.4,128.0,125.4(q,J=4.04Hz),124.2,118.8(d,J=8.08Hz),118.4 ,113.6(2C),112.5(d,J=3.03Hz),112.4,64.7,61.3,55.2,53.3,49.3,33.5.HRMS(ESI + ):m / z calcd forC 29 H 26 F3N3NaO2[M+Na] + ,528.1869; found,528.1901.

[0045] Example 15: 1-(3-cyanobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5o) The synthesis of compound 5o is as follows: 5a. 1H NMR (400MHz, chloroform-d): δ7.78(s,1H),7.72(d,J=8.0Hz,1H),7.65(d,J=8.0Hz,1H),7.53(t,J=8.0Hz,1H),7.44(d,J=8.0Hz,1H),7.1(d,J=8 .0Hz,1H),6.99(d,J=8.0Hz,1H),6.71(d,J=8.0Hz,1H),4.24(s,2H),3.7 4(s,2H),3.26(t,J=4.0Hz,1H),2.75(t,J=8.0Hz,1H),2.53-2.49(m,2H). 13 C NMR (101MHz, chloroform-d): δ172.1,158.2,143.3,140.5,138.5,135,133.7,132.6,132.1,130.4,130.1,129.8(2 C),128.5(3C),125.9(q,J=4.04Hz),124.0,121.4,119.6,116.2(2C),113.2,61.9,54.3,50.7,43.3,32.1.HRMS(ESI + ):m / z calcd for C 28 H 24 F3N3NaO2[M+H] + ,514.1713;found,514.1762.

[0046] Example 16: 1-(3-cyanobenzyl)-4-[4-(hydroxymethyl)phenyl]-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5p) The synthesis of compound 5p is as follows: 5a. 1H NMR (400MHz, chloroform-d): δ7.71(s,1H),7.62(d,J=8.0Hz,1H),7.57(d,J=8.0Hz,1H) ,7.45(d,J=4.0Hz,1H),7.43(d,J=4.0Hz,2H),7.27(s,1H),7.25(d,J=8.0Hz,1H),7.20( s,1H),7.18(d,J=4.0Hz,1H),6.93(d,J=8.0Hz,2H),5.37(brs,1H),4.66(s,2H),4.21(d ,J=4.0Hz,2H),3.7(s,2H),3.39-3.36(m,2H),2.68(t,J=8.0Hz,1H),2.53-2.49(m,2H). 13 C NMR (101MHz, chloroform-d): δ168.6,141.5,141.0,139.4,139.3,138.2,133.5,132.5,131.0,129.9,129.6,129.3,129.2,1 27.9(2C),127.3(d,J=7.07Hz),125.3(q,J=4.04Hz),122.7,118.8,112.4,64.3,61.3,53.5,50.6,49.4,43.0,32.0.HRMS(ESI + ):m / z calcd for C 29 H 26 F3N3NaO2[M+Na] + ,528.1869; found,528.1882.

[0047] Example 17: 1-(3-cyanobenzyl)-4-[4-(methylcarbamoyl)phenyl]-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5q) The synthesis of compound 5q is as follows: 5a. 1H NMR (400MHz, chloroform-d): δ7.81 (s, 1H), 7.75-7.67 (m, 4H), 7.56 (t, J = 8.0Hz, 1H), 7.39 (d, J = 8.0Hz, 1H), 7.33 (d, J = 8.0Hz, 1H),6.88(d,J=8.0Hz,1H),4.22(s,2H),3.78(s,2H),3.34-3.31(m,2H),2.95(s,3H),2.80(t,J=8.0Hz,1H),2.59-2.55(m,2H). 13 C NMR (101MHz, chloroform-d): δ178.3,178.2,175.7,175.6,153.3,152.8,149.4,143.3,143.2,142.8,141.8,141.3,140.4,139,136.8( d,J=8.08Hz),136.7,136.5(2C),136.4,135.1,134.2(q,J=4.04Hz),132.4,128.3,120.8,69.5,62.7,58.6,50.8,39.7,35.6.HRMS(ESI + ):m / z calcd for C 30 H 27 F3N4NaO2[M+H] + ,555.1978;found,555.2030,C 28 H 23 F3N4NaO3[M+H] + ,543.1614;found,543.1622.

[0048] Example 18: 1-(3-cyanobenzyl)-4-(4-nitrophenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5r) The synthesis of compound 5r is as shown in 5a. 1H NMR (400MHz, chloroform-d): δ8.03(d,J=8.0Hz,2H),7.70(s,1H),7.61(d,J=8.0Hz ,1H),7.57(d,J=8.0Hz,1H),7.46(d,J=8.0Hz,1H),7.41(d,J=8.0Hz,2H),7.36(d,J= 8.0Hz,2H),7.0(d,J=8.0Hz,2H),6.54(t,J=8.0Hz,1H),5.54(t,J=8.0Hz,1H),4.21( d,J=4.0Hz,2H),3.71(s,2H),3.39(s,2H),2.72(t,J=8.0Hz,2H),2.55-2.52(m,2H). 13 C NMR (101MHz, chloroform-d): δ171.1,167.9,147.2,146.7,141.0,139.1,135.4,133.4,132.9,132.4,131.1,130.1,129.8,129.3,128 .7(d,J=13.13Hz),128.0,125.4(q,J=4.04Hz),125.1,123.5,122.4,118.7,112.6,61.2,60.4,53.6,49.2,31.1,21.0,14.1.HRMS(ESI + ):m / z calcd for C 28 H 23 F3N4NaO3[M+H] + ,543.1614;found,543.1622.

[0049] Example 19: 1-(3-cyanobenzyl)-4-(2,6-dimethylphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5s) The synthesis of compound 5S is as shown in 5a. 1H NMR (400MHz, chloroform-d): δ7.73(s,1H),7.64(d,J=8.0Hz,2H),7.46-7.32(m,3H),7.09-7.05(m,1H),7.0(d,J=4.0Hz,2H ),6.86(d,J=8.0Hz,2H),5.51(brs,1H),4.18(d,J=8.0Hz,2H),3.75(s,2H),3.49(s,2H),2.69(t,J=8.0Hz,2H),2.18(s,6H). 13 C NMR (101MHz, chloroform-d): δ166.1,141.9,139.9,139.3,134.5(2C),133.4,132.5,131.0,129.6,129.3,129.2,129.1,128. 5(2C),128.0,127.6(2C),125.5,125.4(q,J=4.04Hz),122.8,119.0,112.6,61.4,52.9,49.3,43.0,31.8,19.4(2C).HRMS(ESI + ):m / z calcd for C 30 H 29 F3N3O[M+H] + ,504.2257,found 504.2259.

[0050] Example 20: 1-(3-cyanobenzyl)-4-(2-fluoro-4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5t) The synthesis of compound 5t is as shown in 5a. 1 H NMR (400MHz, chloroform-d): δ7.67(s,1H),7.6(d,J=8.0Hz,1H),7.54(d,J=8.0Hz,1H),7.43-7.40(m,3H),6.94-6.90(m,3H),6.5 0-6.45(m,3H),5.89-5.85(m,1H),4.26(d,J=8.0Hz,2H),3.7(s,2H),3.39-3.37(m,2H),2.68(t,J=8.0Hz,2H),2.48-2.44(m,2H). 13C NMR (101MHz, chloroform-d): δ169.0,160.9,158.5(d,J=6.06Hz),158.4,141.3,139.2,133.8,133.6,132.5,131.3,131.1,130.2(d,J=4.04Hz), 129.6,129.3,129.2,127.7,125.4(q,J=4.04Hz),118.8,118.7,118.5,1 12.4,112,103.7(d,J=24.24Hz),61.1,53.2,49.2,43.0,31.4.HRMS(ESI + ):m / z calcd for C 28 H 23 F4N3NaO2[M+H + ,532.1619; found,532.1635.

[0051] Example 21: 1-(3-cyanobenzyl)-4-(1-methyl-1H-pyrazole-4-yl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (5u) The synthesis of compound 5u is as follows: 5a. 1 H NMR (400MHz, MeOH-d4): δ7.77(s,1H),7.71(d,J=8.0Hz,1H),7.65(d,J=8.0Hz,1H),7.58(d,J=8.0Hz,1H),7.53(t,J=8.0Hz,1H) ,7.43(s,2H),7.31(d,J=8.0Hz,2H),4.39(s,2H),3.72(s,5H),3.23(t,J=4.0Hz,2H),2.73(t,J=8.0Hz,2H),2.52-2.49(m,2H). 13 C NMR (101MHz, MeOH-d4): δ172.7,146.3,143.8,140.8,138.4,136.1,135.0,133.7,132.2,130.5,130.2,1 29.3(2C),129.1,128.4,127.3,126.3,121.8,119.7,113.3,61.8,54.6,50.4,43.6,38.8,30.5.HRMS(ESI + ):m / z calcd forC 26 H 24 F3N5NaO[M+H]+ ,502.1825; found,502.1835.

[0052] Example 22: 1-(2-cyanobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6a) Starting material 1 (0.5 g, 1.2 mmol, 1.00 equivalent) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenol (0.33 g, 1.5 mmol, 1.2 equivalent) were dissolved in 1,2-dimethoxyethane (6 mL) solution, and K₂CO₃ (0.26 g, 1.86 mmol, 1.5 equivalent) was added. The mixture was degassed with N₂ for 15 min, and then refluxed at 90 °C for 6 h. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the solid fraction was washed with ethyl acetate (EA, 10 mL). The filtrate was concentrated under reduced pressure and purified by Biotage Isolera One rapid column chromatography system (using a 200-300 mesh silica gel column, eluting with a petroleum ether (PE):ethyl acetate (EA) = 0-10%) to finally obtain 2a in 79% yield. Compound 2a (0.5 g, 1.5 mmol) was reacted with potassium trimethylsilanolate (TMSOK) (967.8 mg, 7.25 mmol, 5 equivalents) in an acetonitrile / water (ACN / H2O, 4 mL / 1 mL) solution. The mixture was refluxed at 100 °C for 6 hours. After the reaction was complete, the mixture was acidified to pH 6.0 with acetic acid and quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate (EA, 8 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate (Na2SO4). After filtration, the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (elution gradient: methanol / dichloromethane (CH3OH / CH2Cl2) = 0:100 to 10:100) to give a pale yellow oily intermediate 3a (247.2 mg), in 54% yield. Compound 3a (0.2 g, 0.42 mmol, 1.00 equivalent) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added dropwise at 0 °C. The mixture was heated to room temperature and the reaction was continued for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure, and the crude product was used directly in the next reaction without further purification. The crude product was dissolved in dimethylformamide (4 mL) under nitrogen protection, followed by the sequential addition of 3-cyanobenzyl bromide (98 mg, 0.5 mmol, 1.2 equivalents) and potassium carbonate (86.5 mg, 0.63 mmol, 1.5 equivalents). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was quenched with saturated ammonium chloride solution (5 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed sequentially with water (20 mL) and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure and purified by Biotage Isolera One rapid column chromatography system (200-300 mesh silica gel column) to give a white solid product 6a in 82% yield. 1 H NMR (400MHz, chloroform-d): δ7.64-7.52(m,3H),7.42(d,J=8.0Hz,2H),7.36(t,J=8.0Hz,1H),7.0(d,J=8.0Hz,2H),6.89(d,J=8.0 Hz,2H),6.71(d,J=8.0Hz,2H),5.64(brs,1H),4.21(d,J=4.0Hz,2H),3.85(s,2H),3.4(s,2H),2.77-2.74(m,2H),2.49-2.45(m,2H). 13 C NMR(101MHz,chloroform-d): δ169.5,156.7,141.6(d,J=43.4Hz),138.8,132.9(d,J=13.1Hz),131.9,130.3,129.7,129.3, 129.1,128.6,127.8,127.7,125.3(q,J=4.04Hz),122.7,117.8,115.8,112.9,60.5,59.6,53.2,49.7,43.1,31.6.HRMS(ESI + ):m / z calcd for C 28 H 24 F3N3NaO2[M+Na] + ,514.1713;found,514.1722.

[0053] Example 23: 1-(2-chlorobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6b) The synthesis of 6b is similar to that of compound 6a. 1H NMR (400MHz, chloroform-d): δ7.49 (dd, J1=4.0Hz, J2=8.0Hz, 1H), 7.41 (d, J=8.0Hz, 2H), 7.34 (dd, J1=4.0Hz, J2=8.0Hz, 1H), 7.22-7.18 (m, 2H), 7.0 (d, J=8.0 Hz,2H),6.88(d,J=8.0Hz,2H),6.71(d,J=8.0Hz,2H),5.65(brs,1H),4.21(d,J =8.0Hz,2H),3.8(s,2H),3.47(s,2H),2.75(t,J=4.0Hz,2H),2.49-2.45(m,2H). 13 CNMR (101MHz, chloroform-d): δ169.6,156.8,141.4,138.8,134.9,134.5,131.7,131.0,129.9,129.6,129.5,129.3, 129.1,129,128.5(2C),127.7(2C),126.8,125.3(q,J=4.04Hz),122.7,115.9,58.3,53.5,49.4,43.1,31.9.HRMS(ESI + ):m / z calcd forC 27 H 24 ClF3N2NaO2[M+Na] + ,523.1371; found,523.1383.

[0054] Example 24: 1-(3-nitrobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6c) The synthesis of 6c is similar to that of compound 6a. 1H NMR (400MHz, chloroform-d): δ8.23(s,1H),8.1(d,J=8.0Hz,1H),7.71(d,J=8.0Hz ,2H),7.48(td,J1=4.0Hz,J2=8.0Hz),7.41(d,J=8.0Hz,2H),7.01(d,J=8.0Hz,2H) ,6.99(d,J=8.0Hz,2H),6.72(d,J=8.0Hz,2H),6.66-6.64(m,1H),4.21(d,J=4.0Hz ,1H),3.75(s,2H),3.39(t,J=4.0Hz,2H),2.72(d,J=8.0Hz,2H),2.50-2.47(m,2H). 13 C NMR (101MHz, chloroform-d): δ169.3,156.7,148.4,141.4,139.9,138.9,135.2,131.7,129.6,129.4,12 9.3,129,128.6,127.8,125.3(q,J=4.04Hz),123.9,122.5,115.8,77.2,61.2,53.5,43.1,32.0.HRMS(ESI + ):m / z calcd for C 27 H 24 F3N3NaO4[M+H] + ,534.1611; found,534.1619.

[0055] Example 25: 1-(3-methoxybenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6d) The synthesis of 6d is similar to that of compound 6a. 1 H NMR (400MHz, chloroform-d): δ7.43(d,J=8.0Hz,2H),7.27-7.23(m,2H),7.0-6.91(m,6H),6.84(d,J=8.0Hz,1H),6.66 (d,J=8.0Hz,1H),5.70-5.68(m,1H),4.21(d,J=4.0Hz,2H),3.8(s,2H),3.72(s,2H),2.74-2.72(m,2H),2.49(brs,2H). 13C NMR (101MHz, chloroform-d): δ168.9,168.2,166.2,159.8,159.2,150.0,141.8,138.7,137.3,136.0,133.0,131.5,129. 5(2C),128.6(2C),127.9,125.3(q,J=4.04Hz),121.9,115.8,115.0,113.5,62.0,55.3,53.4,49.1,43.0,29.7.HRMS(ESI + ):m / z calcdfor C 28 H 27 F3N2NaO3[M+Na] + ,519.1866; found,519.1865.

[0056] Example 26: 1-(4-nitrobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6e) The synthesis of 6e is as shown in compound 6a. 1 H NMR (400MHz, chloroform-d): δ8.11(d,J=8.0Hz,2H),7.53(d,J=8.0Hz,2H),7.4(d,J=8.0Hz,2H),7.02(d,J=8.0Hz,2H),6.87(d,J=8.0Hz ,2H),6.73(d,J=8.0Hz,2H),5.68-5.64(m,1H),4.22(d,J=4.0Hz,1H),3.74(s,2H),3.37(s,2H),2.69(t,J=8.0Hz,2H),2.50-2.47(m,2H). 13 C NMR (101MHz, chloroform-d): δ171.5,169.4,156.8,147.2,145.6,141.3,138.9,131.6,129.6(2C),129.3 ,129.0,128.6(2C),127.8(2C),125.3(q,J=4.04Hz),123.6,115.9,61.3,53.5,49.6,43.1,32.0.HRMS(ESI + ):m / z calcd for C 27 H 24 F3N3NaO4[M+H] +,534.1611;found,534.1604.

[0057] Example 27: 1-(4-cyanobenzyl)-4-(4-hydroxyphenyl)-N-[4-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6f) The synthesis of 6f is as shown in compound 6a. 1 H NMR (400MHz, chloroform-d): δ7.61(d,J=8.0Hz,2H),7.50(d,J=8.0Hz,2H),7.42(d,J=8.0Hz,2H),7.23(d,J=8.0Hz,2H),7.17(d,J=8.0Hz,2H),6. 9(d,J=8.0Hz,2H),5.44(brs,1H),4.63(s,2H),4.19(d,J=8.0Hz,2H),3.7 2(s,2H),3.39(t,J=4.0Hz,2H),2.68(t,J=8.0Hz,2H),2.52-2.48(m,2H). 13 C NMR(101MHz,chloroform-d): δ168.5(2C),162.6,143.4,141.5,141.0,139.5,138.2,132.2(3C),130.0,129.6(2C),129.4,1 27.9(2C),127.3(d,J=8.08Hz),125.3(q,J=4.04Hz),122.7(2C),118.8,111.2,64.5,61.7,53.6,49.5,43.0,31.4.HRMS(ESI + ):m / z calcd forC 28 H 24 F3N3NaO2[M+Na] + ,514.1713;found,514.1734.

[0058] Example 28: 1-(4-cyanobenzyl)-4-(4-hydroxyphenyl)-N-[2-(trifluoromethyl)benzyl]-1,2,5,6-tetrahydropyridine-3-carboxamide (6g) The synthesis of 6g is as shown in compound 6a. 1H NMR (400MHz, chloroform-d): δ7.69(s,1H),7.6(d,J=8.0Hz,2H),7.55(d,J=8.0H z,1H),7.47(d,J=8.0Hz,1H),7.44-7.39(m,2H),7.32(t,J=8.0Hz,1H),7.21(d,J =8.0Hz,1H),7.04(s,4H),5.38(t,J=8.0Hz,1H),4.56(s,2H),4.31(d,J=4.0Hz,1 H),3.67(s,2H),3.36(t,J=4.0Hz,1H),2.65(t,J=8.0Hz,2H),2.48-2.45(m,2H). 13 C NMR (101MHz, chloroform-d): δ168.3,140.7,139.6,139.1,138.2,135.8,133.4,132.4,132.1,131.4,131.0,130.1,129.1, 128.1,127.8,127.4,127.1,126.9,125.5(q,J=4.04Hz),125.4,122.7,118.8,112.4,64.5,53.5,49.4,39.7,32.0.HRMS(ESI + ):m / zcalcd for C 28 H 25 F3N3O2[M+H] + ,514.1713; found,514.1729. The beneficial effects of the compounds of the present invention are illustrated below through experimental examples.

[0059] Example 29: Experiment on the hydrolysis of short peptide substrates by the compound stimulating HsClpP enzyme.

[0060] This experiment investigated the effect of compounds on the hydrolysis of AC-WLA-AMC substrate by HsClpP protein, evaluating the regulatory activity of the compounds on HsClpP. The test system consisted of a 100 μL test volume, with a final HsClpP protein concentration of 0.5 μM and a final AC-WLA-AMC substrate concentration of 200 μM. The preferred compound stock solution was diluted to 1 μM. The small molecule and HsClpP protein solution were first added to flat-bottomed black 96-well plates, with three replicates per group. After incubation at room temperature for 10 min, the AC-WLA-AMC substrate was added. The fluorescence intensity in the wells was immediately detected using a fluorescence microplate reader (excitation: 360 nm, emission: 440 nm), with readings taken every 5 min, shaking for 5 s before each reading, for a total of 30 min. DMSO was used as the negative control group instead of the small molecule. The fold increase in the readings for the DMSO group at different concentrations was recorded as the hydrolysis activity evaluation index. Graphpad Prism was used to plot the obtained fold increases, and EC50 was calculated. 50 Values. The results are shown in Table 1. At a single concentration of 1 μM, the invented compounds all showed certain regulatory performance against HsClpP. -: No agonistic activity

[0061] Example 30: In vitro antitumor proliferation experiment of the compound. The purpose of this experiment was to detect the inhibitory activity of the invented compound on the proliferation of tumor cells in vitro using CCK-8 assay. Main reagents: RPMI-1640, DMED high-glucose medium, fetal bovine serum, trypsin, etc., were purchased from Gibco BRL. For in vitro experiments, the test compound was prepared as a 10mM stock solution with DMSO and stored at -20°C protected from light. Before use, it was diluted to the required concentration with complete culture medium. Experimental method: When cells showed good growth during culture, they were digested, centrifuged, and collected. The previous culture medium was discarded, and the cells were resuspended in fresh culture medium. Cell counts were then performed. The cell plating concentration was determined based on the different cell growth rates, generally 3000-5000 cells / well. After determining the plating concentration, the cell suspension was diluted to the required concentration with fresh culture medium and then added to 96-well plates at 100 μL per well. 200 μL of PBS was added to the side wells to prevent evaporation of the culture medium. On the second day, drug treatment was performed. First, the compound was diluted into a series of gradients using culture medium. Then, the drug solutions were added to 96-well plates, with three parallel replicates for each gradient. A blank control group and ONC201 cells were included in each plate as a positive control group. After 72 hours of drug treatment, cell growth in the 96-well plates was observed visually. Then, CCK-8 solution was added to each well, and the plates were incubated for 1-2 hours. Finally, absorbance was measured at 450 nm. The inhibition rate at each drug concentration was calculated as: Cell inhibition rate = (OD450 of blank control group - OD450 of experimental group) / OD450 of blank control group × 100%. The IC50 was then calculated using Graphpad Prism software. The results are shown in Table 2. Some preferred compounds exhibit antiproliferative activity against MKN45 (human gastric cancer cells) and human melanoma cells (A375). Some compounds exhibit antitumor proliferation activity against human small cell lung cancer cells (SBC-2), human myeloid monocytic leukemia cells (MV4-11), and human liver cancer cells (HepG2). +++:<1μM; +++:1-5μM; +:5-10μM

[0062] Example 31: α-Casein degradation experiment. This experiment verified the effect of the selected compound in activating HsClpP and enhancing its hydrolytic activity by detecting its activation of HsClpP, thereby enhancing its degradation of the protein substrate α-Casein. The specific procedure was as follows: 2 μM HsClpP was added to the detection buffer (25 mM HEPES, pH 7.6, 5 mM MgCl2, 200 mM NaCl, 0.5 mM ATP, 10% glycerol, and 1 mM DTT), followed by the addition of 1 μM of the compound or DMSO. Then, 4.8 mg / mL FITC-casein was added to initiate the reaction. Fluorescence values ​​were then read every 5 minutes at 488 / 525 nm at 30 °C for 1.5 hours. Finally, the EC50 of the protease activity based on fluorescence intensity was calculated using GraphPad Prism 9 software. 50 value.

[0063] Example 32: Differential Scanning Fluorescence (DSF) Experiment. This experiment verified the interaction between the invented compound and HsClpP by detecting the effect of the preferred compound 5p on the stability of HsClpP protein at different temperatures. 10 μM HsClpP, 5×SSYPRO Orange, and 100 μM compound solution were added to eight-tube RT-PCR apparatus, with at least two replicates per well, and incubated at room temperature for 30 min. The reaction system consisted of K₂HPO₄ / KH₂PO₄ 50 mM pH 7.6, KCl 100 mM, and 5% glycerol. Detection: Fluorescence detection was performed using an RT-PCR instrument. A melting curve was selected, and the temperature was increased from 25℃ to 99℃ within 40 min. The SSYPRO Orange channel was selected for fluorescence detection, and the data were recorded. The results are as follows: Figure 2 As shown, with DMSO as a blank control, the above compounds shifted the Tm value of HsClpP protein to the right, indicating a significant effect on the thermal stability of HsClpP. At the same concentration of 100 μM, compound 5p caused the largest ΔTm value, making it an excellent HsClpP regulator.

[0064] Example 33: In vitro anti-tumor cell clonogenic assay using preferred compounds. Besides being closely related to tumor cell proliferation, HsClpP regulation also induces cell cycle arrest and clonogenic formation. This experiment used non-SMC cells for monoclonal cell experiments. Crystal violet staining was used, and the results are as follows: Figure 3 As shown. The preferred compound 5p significantly inhibits the formation of non-SMC monoclonal antibodies at lower concentrations.

[0065] Example 34: Cell scratch assay. Non-SMC cells were cultured at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1,000 cells / well in six-well plates. When the cells reached 85% confluence, a scratch assay was performed. Three transverse scratches were then made in each well using a 200 μL pipette tip, and the cells were washed with PBS buffer to remove any detached cells. The designated drug was then added, and the scratch width was immediately observed and photographed using an optical microscope at time 0. The scratch width was measured again at 24 and 48 hours post-drug administration.

[0066] Example 35: Evaluation of the therapeutic effect of the selected compound in a mouse small cell lung cancer model. This experiment verified the therapeutic effect of HsClpP against small cell lung cancer by observing the in vivo therapeutic effect of the selected compound on a mouse model of small cell lung cancer. Male BALB / c nude mice (6-8 weeks old, 18-22g) provided by Beijing Huafukang Biotechnology Co., Ltd. were used in the experiment. After acclimatization for 1 week, 10 mg of HsClpP was injected subcutaneously. 6 Non-smooth muscle cells (suspended in a mixture of 0.05 mL antibiotic-free culture medium and 0.05 mL basement membrane matrix). Approximately 9 days later, when the tumor diameter grew to about 6 mm, the tumor-bearing mice were randomly divided into groups and administered 8o (1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg, dissolved in physiological saline) and NCA029 (5 mg / kg, dissolved in physiological saline) by gavage, respectively. A control group with an equal volume of physiological saline was also included. For 20 consecutive days, mouse body weight and tumor size, and non-smooth muscle cell tumor volume (mm³) were recorded every two days. 3 According to the formula V = major diameter (mm) × minor diameter (mm) 2 / 2 calculation.

Claims

1. The present invention provides a class of tetrahydropyrimidine compounds characterized in that The compound is shown in Formula I, Z1-L-Z2 Z1 is independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkylacyl, arylacyl, and aralkylacyl; Z2 is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkoxyalkyl, alkylamino, cycloalkylamino, heterocycloalkylamino, aralkyloxy, and aralkylthio; the connecting arm L is independently selected from the following structure: R1 to R7 are independently selected from hydrogen, halogen, C3-C6 cycloalkyl, and C1-C6 substituted alkyl; R9 to R12 are independently selected from hydrogen, halogen, and C1-C6 substituted alkyl; Q is independently selected from aryl and heterocyclic alkyl.

2. Further, the Z1-L-Z2 ternary topological structure compound is characterized in that The compound shown is the one represented by Formula III. Ar1 and Ar2 are independently selected from 0-5 phenyl groups substituted with R12 or R13, where R12 and R13 are selected from halogens, nitro groups, C1-C6 alkyl groups, C3-C9 substituted cycloalkyl groups, C1-C6 haloalkyl groups, -CF3, -NH2, -NO2, -SH, -SR16, -OH, C1-C6 substituted alkoxy groups, -NR16R17, (C3-C9)cycloalkyl, (C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenylalkyl, and substituted aryl groups.

3. The Z1-L-Z2 ternary topological structure compound according to claim 3, characterized in that... The compound shown is the one represented by formula IV. R12 and R13 are independently selected from hydrogen, halogen, cyano, C1-C6 alkyl, C3-C9 substituted cycloalkyl, C1-C6 haloalkyl, and CF3, while R7 is independently selected from hydrogen and halogen.

4. Furthermore, the Z1-L-Z2 ternary topological structure HsClpP agonist has the following structural formula:

5. The salt, hydrate, or crystal form of the Z1-L-Z2 ternary topological structure compound and any isotopically substituted compound of any atom as described in any one of claims 1 to 5, wherein the salt is preferably a pharmaceutically acceptable salt that can be formed by the compound of the present invention with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, or aspartic acid.

6. The use of the Z1-L-Z2 ternary topological structure compounds according to any one of claims 1 to 6, and their salts, hydrates or crystal forms, in the treatment of HsClpP-mediated nervous system diseases, metabolic syndromes, inflammatory diseases and tumor-related diseases.

7. The use of the Z1-L-Z2 ternary topological structure compounds and their salts, hydrates or crystal forms as described in any one of claims 1 to 6 in the treatment of HsClpP-regulated malignant tumors such as: central nervous system tumors, brain tumors, peripheral nervous system tumors, pheochromocytoma, paraganglioma, neuroendocrine tumors, liver cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, prostate cancer, endometrial cancer, hematologic malignancies and lymphatic system tumors.

8. The Z1-L-Z2 ternary topological structure compounds and their salts, hydrates or crystal forms as described in any one of claims 1 to 6, for the treatment of HsClpP-regulated inflammatory diseases, such as inflammatory bowel disease (IBD), hepatitis, etc.

9. A pharmaceutical composition for treating HsClpP-mediated diseases, characterized in that: A formulation prepared by adding pharmaceutically acceptable excipients and / or auxiliary ingredients to a compound of the Z1-L-Z2 ternary topological structure as described in any one of claims 1-6 or its crystal form.

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