Iridoid compound, preparation method thereof, pharmaceutical composition thereof and application thereof

Abstract

The present invention discloses an iridoid compound, a preparation method, a pharmaceutical composition, and an application thereof, belonging to the field of pharmaceutical chemistry technology. Based on the traditional Chinese medicine small molecule genipin, the present invention designs a GSK-3β compound containing an iridoid structure, which has a certain therapeutic effect on Alzheimer's disease and can also enhance the specificity and effectiveness of the compound, reduce toxic side effects, and prevent drug resistance. Furthermore, the compound of the present invention has good GSK-3β inhibitory activity and has important practical value in the field of anti-Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to an iridoid compound, a preparation method thereof, and a pharmaceutical composition and application thereof. Background Art

[0002] Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine / threonine kinase that is a key molecule connecting Aβ, tau protein, inflammation, synapses, and neurons. This kinase is involved in almost all pathways leading to the pathogenesis of Alzheimer's disease (AD).

[0003] GSK-3β can phosphorylate the cytoplasmic domain of amyloid precursor protein (APP), promote the production of amyloid β-protein (Aβ), and increase the toxicity of Aβ. Inhibiting the activity of GSK-3β can reduce Aβ in the brains of AD model mice. 1-40 and Aβ 1-42 Accumulation. GSK-3β is one of the main players in tau (a microtubule-associated protein) phosphorylation, microtubule stabilization, and promotion of microtubule assembly. Overactivation of GSK-3β leads to hyperphosphorylation of tau protein, which prevents tau protein from binding to microtubules, thereby destabilizing microtubules. Subsequently, free tau protein accumulates and aggregates in neurons, leading to neurofibrillary tangles (NFTs) and neuronal death. Studies have found that GSK-3β participates in the regulation of tau protein activity in an insulin-dependent manner. Akt / PKB has been shown to be the main inhibitory kinase upstream of GSK-3β. Activation of Akt / PKB signaling can promote GSK-3β phosphorylation and reduce its enzymatic activity. The insulin-stimulated PI3K signaling pathway reversely regulates GSK-3β, and activation of this enzyme can regulate the hyperphosphorylation of tau protein, which is of great significance for the formation of neurofibrillary tangles in the AD brain.

[0004] Currently, FDA-approved anti-AD drugs include: acetylcholinesterase inhibitors (donepezil, rivastigmine, tacrine, and galantamine), and NMDA inhibitors (memantine). However, these anti-AD drugs only alleviate symptoms and cannot reverse the disease progression. Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In response to the above problems, one of the objectives of the present invention is to provide an iridoid compound, wherein the compound is a compound represented by Formula III, or an isomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof; the general structural formula of the compound is as follows:

[0006]

[0007] Wherein, X, Y, and Z each independently represent CH or N; A represents O or N-R3; B represents CH or O; n1 represents 1 or 2; n2 represents 0, 1, 2, or 3; Linker represents a piperazine ring, acetamide, propionamide, butyramide, or piperazinamide;

[0008] Wherein, R1 represents a C1-C4 alkyl group or a benzyl group; R3 represents a C1 or C3 alkyl group;

[0009] Each R2 independently represents H, methyl, methoxy or acetonide;

[0010] Each bond represented by a dashed line in Formula III may be present or absent.

[0011] Furthermore, the compound is selected from any one of the structures in Formula I or II, or an isomer of the compound or a solvate thereof or a pharmaceutically acceptable salt thereof, and the general structural formula of the compound is as follows:

[0012]

[0013]

[0014] in,

[0015] R1 is selected from one of methyl, ethyl, n-propyl, n-butyl, isobutyl, tert-butyl or benzyl;

[0016] R2 is selected from pyrazine, pyrimidine or paeonol;

[0017] R3 is selected from methyl or n-propyl;

[0018] The linker is selected from piperazine, acetamide, butanamide, 2-(piperazin-1-yl)acetamide, (piperazin-1-yl)acetamide, or 3-(piperazin-1-yl)propionamide.

[0019] Furthermore, the iridoid compound is selected from any one of the following compounds or their isomers or solvates or pharmaceutically acceptable salts:

[0020]

[0021]

[0022] A second object of the present invention is to provide a method for preparing an iridoid compound as described in Formula I, comprising the following steps:

[0023] Compound 3 was prepared using compound 1 genipin as a starting material. Compound 3, K2CO3, and DMF were reacted with compound 13 or compound 14 at room temperature to obtain a compound represented by general formula I. The reaction scheme is as follows:

[0024]

[0025] Or include the following steps:

[0026] Compound 17 or 19, EDCI·HCl, HOBt and dichloromethane were stirred at room temperature and then compound 6 was added. After reaction at room temperature, the compound represented by general formula I was obtained. The reaction scheme is as follows:

[0027]

[0028] The compound 17 is selected from any one of 2-(4-((3,5,6-trimethylpiperazin-2-yl)methyl)piperazin-1-yl)acetic acid and 2-(4-((3,5,6-trimethylpiperazin-2-yl)methyl)piperazin-1-yl)propionic acid; the compound 19 is selected from any one of 2-(4-(pyrimidin-2-yl)piperazin-1-yl)acetic acid and 2-(4-(pyrimidin-2-yl)piperazin-1-yl)propionic acid;

[0029] Or include the following steps:

[0030] Compound 6, compound 22, EDCI·HCl, HOBt, triethylamine and dichloromethane are reacted at room temperature to obtain a compound represented by formula I; the reaction scheme is as follows:

[0031]

[0032] Wherein, R1 represents a C1-C4 alkyl group or a benzyl group; n1 represents 1 or 2.

[0033] Furthermore, the preparation of compound 3 using compound 1 genipin as a starting material comprises the following steps:

[0034] Compound 2 was prepared by reacting compound 1 genipin with p-toluenesulfonic acid monohydrate and reaction reagent 1 at 80°C;

[0035] The compound 2 is reacted with dichloromethane, methanesulfonyl chloride, and triethylamine in an ice bath to generate the compound 3; the reaction scheme is as follows:

[0036]

[0037] Wherein, the reaction reagent 1 is selected from any one of n-propanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol, methanol or ethanol;

[0038] The compound 6 was prepared according to the following method:

[0039] Compound 4 phthalimide, the compound 2, and triphenylphosphine were dissolved in anhydrous tetrahydrofuran under nitrogen protection, and DIAD diluted in anhydrous tetrahydrofuran was added dropwise to the dissolved solution to react at room temperature to generate compound 5;

[0040] Compound 5 is then reacted with anhydrous ethanol and 40%-50% hydrazine hydrate at room temperature to generate compound 6. The reaction scheme is as follows:

[0041]

[0042] The compound 22 was prepared according to the following steps:

[0043] Compound 20, paeonol, potassium carbonate, DMF, and reaction reagent 3 are reacted at room temperature to obtain compound 21; then, aqueous sodium hydroxide solution is added to a mixed solution of compound 21 and anhydrous methanol and reacted at room temperature to obtain compound 22. The reaction scheme is as follows:

[0044]

[0045] The reaction reagent 3 is ethyl chloroacetate or ethyl chlorobutyrate.

[0046] A third object of the present invention is to provide a method for preparing an iridoid compound as described in Formula II, comprising the following steps:

[0047] Compound 26 was prepared using compound 1 genipin as the starting material. Compound 26, K2CO3, and DMF were reacted with compound 13 and compound 14 at room temperature to obtain the compound represented by formula II. The reaction scheme is as follows:

[0048]

[0049] Alternatively, compound 28 is reacted with compound 22, EDCI·HCl, HOBt, triethylamine and dichloromethane at room temperature to obtain a compound represented by formula II. The reaction scheme is as follows:

[0050]

[0051] Wherein, R3 represents methyl or n-propyl, and n1 represents 1 or 2.

[0052] Furthermore, the preparation of compound 26 using compound 1 genipin as a starting material comprises the following steps:

[0053] Compound 1, genipin, imidazole, N,N-dimethylformamide, and tert-butyldimethylsilyl chloride were reacted in an ice bath to obtain compound 23;

[0054] The compound 23 is then reacted with dichloromethane and Dess-Martin periodinane at room temperature to obtain compound 24;

[0055] The compound 24 is reacted with a reaction reagent 2 and pyridine to obtain the corresponding compound 25; the reaction reagent 2 includes any one of methylamine ethanol or n-propylamine;

[0056] The compound 25 is reacted with triethylamine and dichloromethane to obtain the corresponding compound 26;

[0057] The reaction route is as follows:

[0058]

[0059] The compound 28 was prepared according to the following steps:

[0060] Compound 4 phthalimide, compound 25, and triphenylphosphine were dissolved in anhydrous tetrahydrofuran under nitrogen protection, and 2 mL of anhydrous tetrahydrofuran-diluted DIAD was added dropwise to react at room temperature to generate compound 27;

[0061] Compound 27 is reacted with anhydrous ethanol and 40%-50% hydrazine hydrate at room temperature to produce compound 28; the reaction scheme is as follows:

[0062]

[0063] A fourth object of the present invention is to provide a pharmaceutical composition containing the iridoid compound, wherein the pharmaceutical composition further comprises at least one or more pharmaceutically acceptable carriers or excipients.

[0064] The fifth object of the present invention is to propose the use of the iridoid-containing compound or the pharmaceutical composition in the preparation of GSK-3β inhibitor drugs or reagents.

[0065] Furthermore, the iridoid-containing compound or the pharmaceutical composition thereof can be used in the preparation of drugs for treating Alzheimer's disease.

[0066] Beneficial effects of the present invention:

[0067] This invention designs a GSK-3β compound containing an iridoid structure based on the small molecule genipin, a traditional Chinese medicine. Genipin and geniposide can combat endoplasmic reticulum stress, Aβ toxicity, inflammation, oxidative stress, and mitochondrial damage, repairing damaged neurons and promoting neuronal growth. Furthermore, geniposide significantly inhibits Aβ metabolism and tau phosphorylation in primary cultured cortical neurons and enhances the regulatory effects of insulin on Aβ metabolism and tau phosphorylation in primary cultured cortical neurons. The iridoid compound designed based on genipin in this invention has a novel structure and demonstrates promising therapeutic effects for Alzheimer's disease. This is of great significance for enhancing the compound's specificity and efficacy, reducing toxic side effects, and preventing drug resistance. Furthermore, the compound exhibits excellent GSK-3β inhibitory activity, possessing significant practical value in the field of Alzheimer's disease treatment.

[0068] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0070] Figure 1 Effects of different compounds on the viability of SH-SY5Y cells induced by TBHP (100 μM);

[0071] Figure 2 Compound JD-5 for Aβ 25-35 (80 μM)-induced viability in SH-SY5Y cells;

[0072] Figure 3 Compound JD-7 for Aβ 25-35 (80 μM)-induced viability in SH-SY5Y cells. DETAILED DESCRIPTION

[0073] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0074] The general structural formula of the iridoid compound proposed in the present invention is shown in Formula III:

[0075]

[0076] Wherein, X, Y, and Z each independently represent CH or N; A represents O or N-R3; B represents CH or O; n1 represents 1 or 2; n2 represents 0, 1, 2, or 3; Linker represents a piperazine ring, acetamide, propionamide, butyramide, or piperazinamide;

[0077] Wherein, R1 represents a C1-C4 alkyl group or a benzyl group; R3 represents a C1 or C3 alkyl group;

[0078] Each R2 independently represents H, methyl, methoxy, or acetonide;

[0079] Each bond represented by a dashed line in Formula III may be present or absent.

[0080] Furthermore, the general structural formula of the iridoid compound is as follows:

[0081]

[0082] in,

[0083] R1 is selected from one of methyl, ethyl, n-propyl, n-butyl, isobutyl, tert-butyl or benzyl;

[0084] R2 is selected from pyrazine, pyrimidine or paeonol;

[0085] R3 is selected from methyl or n-propyl;

[0086] The linker is selected from a piperazine ring, an acetamide, a propionamide, a butyramide or a piperazinamide, and further selected from piperazine, acetamide, butyramide, 2-(piperazin-1-yl)acetamide (piperazin-1-yl)acetamide or 3-(piperazin-1-yl)propionamide.

[0087] The iridoid compounds proposed in the present invention have the following two preparation methods depending on their structures. The first preparation method is:

[0088] 1) Compound 1 (400.0 mg, 1.77 mmol) was reacted with p-toluenesulfonic acid monohydrate (403.6 mg, 2.12 mmol) and 3.96 mL of reaction reagent 1 (including any one of n-propanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol, and ethanol) at 80°C for 1 h to prepare intermediate compound 2; compound 2 (200.0 mg, 745.4 μmol) was reacted with 5 mL of dichloromethane, methanesulfonyl chloride (0.057 mL, 745.4 μmol), and triethylamine (0.16 mL, 1.12 mmol) in an ice bath to produce compound 3; compound 3, K2CO3, and DMF (N,N-dimethylformamide) were reacted with intermediate compounds 13 and 14, respectively, at room temperature for 4 h to obtain target compounds A1 to A5; B1 to B5;

[0089] 2) Compound 4 (710.7 mg, 2.71 mmol), compound 2 (530.0 mg, 2.08 mmol), and triphenylphosphine (398.7 mg, 2.71 mmol) were dissolved in anhydrous tetrahydrofuran (6 mL) under nitrogen protection. The reaction flask was placed in an ice bath, and DIAD (diisopropyl azodicarboxylate 820.8 mmol, 4.17 mmol) diluted with 2 mL of anhydrous tetrahydrofuran was slowly added dropwise. The mixture was reacted at room temperature for 6 h to produce compound 5. Compound 5 was then added to the mixture. 5 (750.0 mg, 1.96 mmol) was reacted with anhydrous ethanol (10 mL) and 40%-50% hydrazine hydrate (752.3 mg, 23.47 mmol) at room temperature for 12 h to produce compound 6; compound 6 (145.6 mg, 574.8 μmol), compound 17 (200.0 mg, 718.5 μmol), EDCI·HCl (228.4 mg, 1.15 mmol), and HOBt (155.34 mg, 1.15 mmol) and dichloromethane (10 mL), reacted at room temperature for 12 h to obtain target compounds A8 and A9; compound 6 (205.2 mg, 809.9 μmol), compound 19 (225.0 mg, 1.01 mmol), EDCI·HCl (310.5 mg, 1.62 mmol), HOBt (218.9 mg, 1.62 mmol) and dichloromethane (10 mL), reacted at room temperature for 12 h to obtain target compounds B8 and B9; compound 6 (113.0 mg, 4 46.0 μmol), compound 22 (100.0 mg, 446.0 μmol), EDCI·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 128.3 mg, 669.0 μmol), HOBt (N-hydroxybenzotriazole ester 90.4 mg, 669.0 μmol), triethylamine (217.0 mmL, 1.56 mmol) and dichloromethane (5 mL) were reacted at room temperature for 12 h to obtain the target compounds JD-1 to JD-4;

[0090] 3) Compound 7, ligustrazine (4.00 g, 29.37 mmol), 30% H2O2 (5.93 mL, 58.74 mmol), reacted at 70°C for 4 h to obtain compound 8, compound 8 (2.50 g, 16.41 mmol), acetic acid (16 mL), acetic anhydride (10 ml), reacted at 110°C for 6 h to obtain compound 9, compound 9 (2.92 g, 15.03 mmol), anhydrous ethanol (15 mL), NaOH (3.61 g, 90.20 mmol) aqueous solution, reacted at 75°C for 2 h to obtain compound 10 Compound 10 (1.59 g, 10.43 mmol) and anhydrous dichloromethane (10 mL) were added with SOCl2 (1.86 g, 15.64 mmol) under ice bath and reacted at room temperature for 3 h to obtain compound 11. Anhydrous piperazine (compound 12) (2.73 g, 31.65 mmol), dichloromethane (15 mL) and 2-(chloromethyl)-3,5,6-trimethylpyrazine (compound 11) (900.0 mg, 5.27 mmol) in dichloromethane were added. After the addition was complete, the reaction was continued for 6 h under ice bath conditions to obtain compound 13.

[0091] 4) Compound 13 (300.0 mg, 1.36 mmol), compound 15 (356.3 mmL, 3.13 mmol), K2CO3 (473.9 mg, 3.4 mmol), and acetonitrile (10 mL) were reacted at room temperature for 2 h to obtain compound 16. Compound 16 (400.0 mg, 1.25 mmol), anhydrous methanol (5 mL), and aqueous sodium hydroxide solution (149.8 mg, 3.75 mmol) were dissolved in 2 mL of anhydrous methanol and heated under reflux at 70°C for 4 h to obtain compound 17;

[0092] 5) Compound 14 (300.0 mg, 1.83 mmol), compound 15 (572.2 mmL, 4.20 mmol), K2CO3 (631.2 mg, 4.57 mmol), and acetonitrile (10 mL) were reacted at room temperature for 2 h to obtain compound 18. Compound 18 (420.0 mg, 1.59 mmol), anhydrous methanol (6 mL), and aqueous sodium hydroxide solution (190.7 mg, 4.77 mmol) were dissolved in 2 mL of anhydrous methanol and heated under reflux at 70°C for 4 h to obtain compound 19;

[0093] 6) Paeonol (Compound 20) (500.0 mg, 3.01 mmol), potassium carbonate (1.66 g, 3.61 mmol), DMF (5 mL), reaction reagent 3 (including either ethyl chloroacetate or ethyl chlorobutyrate) (386.4 mmL, 3.61 mmol), reacted at room temperature for 12 h to obtain Compound 21, and Compound 21 (690.0 mg, 2.74 mmol), anhydrous methanol (5 mL), and aqueous sodium hydroxide solution (437.6 mmL, 10.94 mmol), reacted at room temperature for 12 h to obtain Compound 22;

[0094] The reaction route of the first preparation method is as follows:

[0095] The second preparation method is:

[0096] (1) Compound 1 (10.0 g, 44.2 mmol) and imidazole (6.02 g, 88.4 mmol) were dissolved in DMF (60 mL), N,N-dimethylformamide and tert-butyldimethylsilyl chloride (TBSCl: 13.32 g, 88.4 mmol) were dissolved in DMF (30 mL), and the mixture was reacted in an ice bath for 2 h to obtain compound 23; the mixture was reacted with dichloromethane (80 mL) and Dess-Martin periodinane (DMP; 34.1 g, 74.2 mmol) at room temperature for 1 h to obtain compound 24; the mixture was reacted with reaction reagent 2 (including either methylamine ethanol or n-propylamine) (30-33%, 8 mL) and pyridine (20 mL) at 65 °C for 2 h and then directly used for the next reaction. Trifluoroacetic acid (20 mL) and tetrahydrofuran (20 mL) were added to a pressure tube and reacted at 65°C for 2.5 h to obtain compound 25; the mixture was reacted with triethylamine (1.2 mL, 8.4 mmol) and dichloromethane (20 mL) to obtain compound 26; compound 26, K2CO3, and DMF were reacted with intermediate compound 13 and compound 14, respectively, at room temperature for 4 h to obtain A6, A7; B6, B7;

[0097] (2) Compound 4 (710.7 mg, 4.83 mmol) was dissolved in anhydrous tetrahydrofuran (6 mL) with compound 25 (882.4 mg, 3.72 mmol) and triphenylphosphine (709.5 mg, 2.70 mmol) under nitrogen. The reaction flask was placed in an ice bath and DIAD (948.4 mmol, 7.74 mmol) diluted with 2 mL of anhydrous tetrahydrofuran was slowly added dropwise. The mixture was reacted at room temperature for 6 h to produce compound 27. Compound 27 (750.0 mg, 2.05 mmol) was reacted with anhydrous ethanol (10 mL) and 40%-50% hydrazine hydrate (787.2 mg, 24.57 mmol) for 12 h to produce compound 28. Compound 28 was reacted with compound 22, EDCI·HCl, HOBt, triethylamine and dichloromethane at room temperature for 12 h to obtain JD-5 to JD-8.

[0098] The reaction scheme of the second preparation method is as follows:

[0099]

[0100] The above reaction scheme is exemplified below in conjunction with specific examples. The reagents or raw materials used in the embodiments of the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in this area or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the present application method. The preferred implementation methods and materials described in the present invention are for demonstration purposes only.

[0101] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) measurements using a Bruker AVANCE-300 / 500 NMR spectrometer in DMSO-d6 solvent and TMS as the internal standard.

[0102] In Examples 1-10, genipin and different reaction reagents 1 are used to first prepare the corresponding compound 2, then compound 2 is used to prepare compound 3, and finally compound 3 and 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine or 2-(piperazin-1-yl)pyrimidine are used to prepare compounds A1-A5 and B1-B5 shown in general formula I.

[0103] Example 1: The synthesis of compound methyl 1-propoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A1) is taken as an example:

[0104]

[0105] The specific preparation process is as follows:

[0106] S1: Synthesis of methyl 7-(hydroxymethyl)-1-propoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 2);

[0107] Genipin (400.0 mg, 1.77 mmol), p-toluenesulfonic acid monohydrate (403.6 mg, 2.12 mmol), and 3.96 mL of n-propanol were added sequentially to a 50 mL round-bottom flask. After reacting at 80° C. for 1 h, TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of saturated NaHCO3 aqueous solution was added to quench the reaction, and the mixture was extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow oil, which was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 4:1 as the eluent] to obtain 416.6 mg of a light yellow oil (compound 2) in a yield of 87.8%.

[0108] S2: Synthesis of methyl 7-((methylsulfonyloxy)methyl)-1-propoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 3)

[0109]

[0110] In a 50 mL round-bottom flask, methyl 7-(hydroxymethyl)-1-propoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (compound 2) (200.0 mg, 745.4 μmol), 5 mL of dichloromethane, and Et3N (0.16 mL, 1.12 mmol) were added sequentially. Methanesulfonyl chloride (0.057 mL, 745.4 μmol) was slowly added dropwise in an ice bath. After completion of the addition, the reaction was continued in an ice bath for 2 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was almost complete. The reaction was quenched with 10% dilute hydrochloric acid and extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and concentrated to give 250 mg of a light yellow oil (compound 3) in a yield of 96.9%.

[0111] S3: Synthesis of 2,3,5,6-tetramethylpyrazine 1-oxide (Compound 8)

[0112]

[0113] To a 100 mL round-bottom flask was added ligustrazine (compound 7) (4.00 g, 29.37 mmol), dissolved with 30 mL of glacial acetic acid, and then added 30% H2O2 (5.93 mL, 58.74 mmol). The reaction was carried out at 70°C for 4 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. The reaction was quenched by adding 20 mL of saturated NaHCO3 aqueous solution, and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.479 g of a white solid with a yield of 75%.

[0114] S4: Synthesis of methyl (3,5,6-trimethylpyrazin-2-yl)acetate (Compound 9)

[0115]

[0116] 2,3,5,6-ligustrazine 1-oxide (Compound 8) (2.50 g, 16.41 mmol) was added to a 100 mL round-bottom flask, and 16 mL of acetic acid was added to dissolve the mixture. Then, 10 mL of acetic anhydride was added and the mixture was reacted at 110 ° C for 6 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of saturated NaHCO3 aqueous solution was added to quench the reaction, and the mixture was extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to obtain 2.92 g of a brown oily liquid with a yield of 91.5%.

[0117] S5: Synthesis of (3,5,6-trimethylpyrazin-2-yl)methanol (Compound 10)

[0118]

[0119] Methyl (3,5,6-trimethylpyrazin-2-yl)acetate (Compound 9) (2.92 g, 15.03 mmol) was added to a 100 mL round-bottom flask and dissolved in 15 mL of anhydrous ethanol. Then, an aqueous solution of NaOH (3.61 g, 90.20 mmol) was added and reacted at 75°C for 2 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was almost complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.587 g of a yellow solid with a yield of 69.4%.

[0120] S6: Synthesis of 2-(chloromethyl)-3,5,6-trimethylpyrazine (Compound 11)

[0121]

[0122] To a 50 mL round-bottom flask was added (3,5,6-trimethylpyrazin-2-yl)methanol (Compound 10) (1.59 g, 10.43 mmol), dissolved in 10 mL of anhydrous dichloromethane, and then SOCl2 (1.86 g, 15.64 mmol) was added under ice bath. The reaction was allowed to react at room temperature for 3 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as developing solvent] showed that the reaction was almost complete. The reaction was quenched by adding 20 mL of ice water, and extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a light yellow oily liquid, which was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 6:1 as eluent] to obtain 900.0 mg of a colorless oily liquid in a yield of 50.6%.

[0123] S7: Synthesis of 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine (Compound 13)

[0124]

[0125] Anhydrous piperazine (2.73 g, 31.65 mmol) was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. A dichloromethane solution of 2-(chloromethyl)-3,5,6-trimethylpyrazine (900.0 mg, 5.27 mmol) was slowly added dropwise at 0°C. After completion of the addition, the reaction was continued in an ice bath for 6 h. TLC [V (petroleum ether):V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was essentially complete. The reaction was quenched by adding 20 mL of distilled water and extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude white solid product. The product was purified by silica gel column chromatography [V (dichloromethane):V (methanol) = 10:1 as the eluent] to obtain 432.0 mg of a white solid with a yield of 37.2%.

[0126] S8: Synthesis of methyl 1-propoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl ester)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A1)

[0127] In a 50 mL round-bottom flask, 7-((methylsulfonyloxy)methyl)-1-propoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylic acid methyl ester (Compound 3) (188.65 mg, 544.61 μmol), 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine (Compound 13) (239.60 mg, 1.09 mmol), K2CO3 (150.54 mg, 1.09 mmol) and 15 mL of The product was dissolved in DMF and reacted at room temperature for 4 h. TLC [V (petroleum ether):V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of distilled water was added to quench the reaction, and the product was extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (dichloromethane):V (methanol) = 20:1 as the eluent] to obtain 87.2 mg of a yellow oil with a yield of 34.1%. 1 H NMR (400MHz, DMSO-d6) δ7.47(d,J=1.1Hz,1H,3-H),5.67(s,1H,7-H),4.82(d,J=6.7Hz,1H,1-H),3. 63(s,3H,COOCH3),3.53(d,J=2.8Hz,2H,CH2),3.49(d,J=9.6Hz,2H,CH2),3.50–3.44(m,2H,10-CH2 ),3.09(d,J=13.9Hz,1H,9-H),3.00(t,J=7.6Hz,1H,5-H),2.94–2.85(m,2H,CH2),2.52–2.50(m,9H , CH3), 2.40 (d, J=2.7Hz, 8H, piperazine-H), 1.55 (q, J=7.0Hz, 2H, CH2), 0.88 (t, J=7.4Hz, 3H, CH3). 13 CNMR(101MHz,DMSO)δ167.44,150.72,149.93,149.70,148.00,147.87,140.86,129.32,111.02,100.84,70.76,6 1.92,57.77,53.46,53.40,51.44,46.47,46.23,38.65,34.83,22.84,21.52,21.42,20.90,10.98.ESI-HRMS:m / s calcd for C 26 H 38 N4O4[M+H] + 471.2966, found 471.2964.

[0128] Example 2 Synthesis of methyl 1-butoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl ester)-1,4a,5,7a-tetrahydrocyclopentyl[c]pyran-4-carboxylate (A2)

[0129]

[0130] Compound A2 was synthesized according to the method of Example 1, except that n-propanol in S1 of Example 1 was replaced by n-butanol. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 30.0%. 1 H NMR (400MHz, DMSO-d6) δ7.46(s,1H,3-H),5.67(d,J=6.0Hz,1H,7-H),4.82(d,J=6.6Hz,1H,1-H),3.83–3.64(m,2 H,CH2),3.63(s,3H,COOCH3),3.52(q,J=3.9,3.4Hz,4H,piperazine-H),3.13–3.00(m,2H,CH2),2.91–2.83(m,2 H,10-CH2),2.75–2.68(m,1H,9-H),2.65(d,J=8.0Hz,1H,5-H),2.59(q,J=8.8,8.0Hz,2H,CH2),2.48(s,4H,pipe razine-H),2.40(d,J=2.7Hz,9H,CH3),1.42–1.25(m,4H,CH2),0.88(t,J=7.4Hz,3H,CH3).3.83–3.64(m,1H,CH). 13 C NMR (101MHz, DMSO) δ167.42,149.93,149.69,148.00,147.86,147.60,140.82,129.29,111.03,100.78,68.80,61.93,6 1.36,58.40,57.74,53.42,53.39,52.40,51.43,38.64,31.57,31.50,21.52,21.42,20.89,19.22,14.10.ESI-HRMS:m / s calcd for C 26 H 38 N4O4[M+H] + 485.3122,found485.3122.

[0131] Example 3 Synthesis of methyl 1-isobutoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl ester)-1,4a,5,7a-tetrahydrocyclopentyl[c]pyran-4-carboxylate (A3)

[0132]

[0133] Compound A3 was synthesized according to the method of Example 1, except that n-propanol in S1 of Example 1 was replaced by isobutanol. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 22.9%. 1 H NMR (400MHz, DMSO-d6) δ7.49–7.39(m,1H,3-H),5.68(s,1H,7-H),4.80(d,J=6.8Hz,1H,1-H),3.63(d,J= 3.1Hz,3H,COOCH3),3.62–3.56(m,2H,CH2),3.53(s,2H,CH2),3.15(dd,J=9.1,6.0Hz,2H,10-CH2),3.02( d,J=7.8Hz,1H,9-H),2.88(d,J=10.8Hz,1H,5-H),2.63(d,J=8.4Hz,2H,CH2),2.48(s,4H,piperazine-H ),2.40(d,J=2.8Hz,9H,CH3),2.36–2.25(m,4H,piperazine-H),1.82(m,1H,CH),0.93–0.83(m,6H,CH3). 13 C NMR(151MHz,DMSO-d6)δ167.42,161.18,152.41,150.70,149.93,147.97,147.93,116.05,111.01,100.96,75.62,74.84, 61.68,55.38,53.74,52.56,51.46,46.42,45.25,38.66,34.92,29.88,21.54,21.43,20.90,19.62,19.58.ESI-HRMS:m / s calcd for C 27 H 40 N4O4[M+H] + 485.3122, found 485.3118.

[0134] Example 4 Synthesis of methyl 1-(tert-butoxy)-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A4)

[0135]

[0136] Compound A4 was synthesized according to the method of Example 1, except that n-propanol in S1 was replaced with tert-butanol. The remaining steps were the same, resulting in a pale yellow oily liquid with a yield of 20.5%. 1H NMR (600 MHz, DMSO-d6) δ 7.44 (s, 1H, 3-H), 5.65 (s, 1H, 7-H), 5.10 (d, J = 6.4 Hz, 1H, 1-H), 3.62 (s, 3H, COOCH3), 3.56 (d, J = 14.8 Hz, 2H, CH2), 3.26 (dd, J = 10.4, 6.1 Hz, 1H, 10-H), 3.18–3. 10(m,2H,CH2),2.99(q,J=7.5Hz,1H,10-H),2.87(d,J=13.8Hz,1H,9-H),2.65(dd,J=16.1, 8.1Hz,1H,5-H),2.48(s,9H,CH3),2.41(d,J=6.5Hz,8H,piperazine-H),1.21(s,9H,CH3). 13 C NMR(151MHz,DMSO-d6)δ167.56,153.09,149.94,149.71,147.97,147.88,141.12,129.28,110.54,95.43,76.22, 61.93,61.67,60.73,57.76,53.44,51.37,47.38,45.26,38.55,34.99,28.84,21.53,21.42,20.90.ESI-HRMS:m / s calcd for C 27 H 40 N4O4[M+H] + 485.3122, found 485.3116.

[0137] Example 5 Synthesis of methyl 1-(benzyloxy)-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl ester)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A5)

[0138]

[0139] Compound A5 was synthesized according to the method of Example 1, except that n-propanol in S1 of Example 1 was replaced by benzyl alcohol. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 29.9%. 1H NMR(400MHz,DMSO-d6)δ7.50(s,1H,3-H),7.33(q,J=13.5,10.2Hz,5H,ArH),5.77–5.63(m,1 H,7-H),5.05(d,J=6.0Hz,1H,1-H),4.87–4.61(m,2H,CH2),3.64(s,3H,COOCH3),3.51(s,2H ,CH2),3.03(dd,J=13.8,6.3Hz,2H,CH2),2.92–2.80(m,2H,CH2),2.69(dd,J=22.5,7.4Hz,2 H, CH2), 2.46 (d, J = 6.1Hz, 4H, piperazine-H), 2.40 (s, 9H, CH3), 2.32 (s, 4H, piperazine-H). 13 C NMR (151MHz, DMSO) δ167.40,149.93,149.70,148.01,147.89,147.60,140.48,137.68,128.81,128.67,128.23,127.51,127.21,111. 72,111.36,99.57,70.47,61.87,61.33,53.41,53.35,53.29,52.39,51.49,46.80,38.57,34.24,21.52,21.43,20.89.ESI-HRMS:m / s calcd forC 30 H 38 N4O4[M+H] + 519.2966,found 519.2961.

[0140] Example 6 Synthesis of methyl 1-propoxy-7-(4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (B1)

[0141]

[0142] Compound B1 was synthesized according to the method of Example 1. We replaced 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in S8 of Example 1 with 2-(piperazin-1-yl)pyrimidine (Compound 14). The remaining steps were the same. The final product was a light yellow oily liquid with a yield of 19.0%. NMR (400MHz, DMSO-d6) δ8.35(d,J=4.7Hz,2H,ArH),7.46(dd,J=29.8,1.3Hz,1H,3-H),6.62(td,J=4.7,1.2Hz,1H,ArH),5.75(s,1H ,7-H),4.98–4.77(m,1H,1-H),3.83–3.76(m,1H,10-H),3.72(s,4H,piperazine-H),3.65(d,J=2.7Hz,3H,COOCH3),3.51(dt,J=9. 7,6.7Hz,1H,10-H),3.19(d,J=13.7Hz,1H,9-H),3.05(dd,J=16.1,8.4Hz,1H,5-H),2.95(d,J=13.8Hz,1H,CH2),2.76–2.63(m,2H, 10-CH2),2.38(dq,J=31.6,5.8Hz,4H,piperazine-H),2.03(m,1H,CH2),1.57(h,J=7.1Hz,2H,CH2),0.89(t,J=7.4Hz,3H,CH3).13C NMR (151MHz, DMSO) δ167.45,161.69,158.36,152.46,140.70,129.69,111.01,110.52,100.95,70 .85,57.91,53.21,53.14,51.47,46.45,46.27,43.84,38.68,34.97,22.88,11.00.ESI-HRMS:m / s calcd for C22H30N4O4[M+H]+415.2340,found415.2334.

[0143] Example 7 Synthesis of methyl 1-butoxy-7-(4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopentyl[c]pyran-4-carboxylate (B2)

[0144]

[0145] Compound B2 was synthesized according to the method of Example 1. In Example 1, the n-propanol in S1 was replaced by n-butanol, and the 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in S8 was replaced by 2-(piperazin-1-yl)pyrimidine. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 49.5%. 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=4.8Hz,2H,ArH),7.56–7.35(m,1H,3-H),6.62(t,J=4.7Hz,1H,ArH),5.74(s,1H,7-H),4.86(d ,J=6.9Hz,1H,1-H),3.81(dt,J=9.8,6.5Hz,1H,10-H),3.72(s,4H,piperazine-H),3.65(d,J=3.0Hz,3H,COOCH3),3.55(dt,J=9. 8,6.9Hz,1H,10-H),3.18(d,J=14.0Hz,1H,9-H),3.06–2.99(m,1H,5-H),2.94(d,J=13.5Hz,1H,CH2),2.75–2.62(m,2H,10-CH2) ,2.40(ddd,J=21.6,11.2,5.6Hz,4H,piperazine-H),2.08–1.98(m,1H,CH2),1.24(d,J=4.0Hz,4H,CH2),0.93–0.85(m,3H,CH3). 13 CNMR(101MHz,DMSO)δ167.44,161.70,158.34,150.70,140.67,129.67,111.48,111.03,110.52,68.8 9,68.03,58.48,57.87,53.20,53.13,51.45,46.49,38.68,32.00,31.59,19.22,14.10.ESI-HRMS:m / s calcd for C 23 H 32 N4O4[M+H] + 429.2496, found 429.2491.

[0146] Example 8 Synthesis of methyl 1-isobutoxy-7-(4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (B3)

[0147]

[0148] Compound B3 was synthesized according to the method of Example 1. In Example 1, the n-propanol in S1 was replaced by isobutanol, and the 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in S8 was replaced by 2-(piperazin-1-yl)pyrimidine. The remaining steps were the same, and a colorless oily liquid was finally obtained with a yield of 32.9%. 1 H NMR (400MHz, DMSO-d6) δ8.35(dd,J=4.7,0.7Hz,2H,ArH),7.45(dd,J=28.9,1.3Hz,1H,3-H),6.62(t,J=4.7Hz,1H,ArH),5.74(s,1H ,7-H),4.86(d,J=6.8Hz,1H,1-H),3.81(dt,J=9.7,6.5Hz,1H,10-H),3.72(s,4H,piperazine-H),3.65(d,J=3.0Hz,3H,COOCH3),3 .60–3.51(m,1H,10-H),3.42(s,1H,CH),3.18(d,J=13.9Hz,1H,9-H),3.03(d,J=9.8Hz,1H,5-H),2.98–2.88(m,1H,CH2),2.69–2.6 3(m,1H,CH),2.38(dtd,J=32.9,10.8,5.0Hz,4H,piperazine-H),2.03(m,1H,CH2),1.55–1.50(m,1H,CH),1.44–1.16(m,6H,CH3). 13 C NMR (101MHz, DMSO) δ167.46,161.71,158.37,152.44,140.67,129.68,111.04,110.54,100.90,68 .90,57.87,53.20,53.13,51.47,46.50,43.86,38.68,34.89,31.58,19.22,14.11.ESI-HRMS:m / s calcd for C 23 H 32 N4O4[M+H] + 429.2496, found 429.2493.

[0149] Example 9 Synthesis of methyl 1-(benzyloxy)-7-(4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (B4)

[0150]

[0151] Compound B4 was synthesized according to the method of Example 1. In Example 1, the n-propanol in S1 was replaced by benzyl alcohol, and the 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in S8 was replaced by 2-(piperazin-1-yl)pyrimidine. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 20.9%. 1 H NMR(400MHz, DMSO-d6)δ8.34(dd,J=6.1,4.7Hz,2H,ArH),7.48(dd,J=33.8,1.3Hz,1H,3-H),7.39–7.2 1(m,5H,ArH),6.61(q,J=4.9Hz,1H,ArH),5.81–5.69(m,1H,7-H),5.55–5.06(m,1H,1-H),4.90–4.49( m,2H,10-CH2),3.65(s,7H,piperazine-H,COOCH3),3.11(t,J=9.2Hz,1H,9-H),2.98(s,1H,5-H),2.9 3–2.87(m,1H,CH2),2.84–2.60(m,2H,CH2),2.37–2.22(m,4H,piperazine-H),2.12–1.95(m,1H,CH2) 13 CNMR(151MHz,DMSO)δ167.40,161.67,158.34,152.24,140.35,137.69,128.80,128.73,128.28,128.24,127.86 ,127.13,111.36,110.51,99.61,70.52,57.69,53.19,53.01,51.49,46.11,43.77,38.63,34.85.ESI-HRMS:m / s calcd for C 26 H 30 N4O4[M+H] + 463.2340, found 463.2352.

[0152] Example 10 Synthesis of methyl 1-ethoxy-7-((4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-2,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (B5)

[0153]

[0154] B5 was synthesized according to the method of Example 1. A light yellow oily liquid was obtained. In Example 1, the n-propanol in S1 was replaced by ethanol, and the 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in S8 was replaced by 2-(piperazin-1-yl)pyrimidine. The yield was 42.5%. 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=4.7Hz,2H,ArH),7.49(s,1H,3-H),6.61(t,J=4.7Hz,1H,ArH),5.74(s,1H,7-H),4. 87(d,J=6.7Hz,1H,1-H),3.86(dq,J=9.7,7.0Hz,1H,10-H),3.71(d,J=6.0Hz,4H,piperazine-H),3.65(s,3H,COOCH3) ,3.62–3.55(m,1H,10-H),3.18(d,J=13.9Hz,1H,9-H),3.05(q,J=7.6Hz,1H,5-H),2.94(d,J=13.9Hz,1H,CH2),2.69( dt,J=28.0,7.8Hz,2H,10-CH2),2.46–2.30(m,4H,piperazine-H),2.12–1.95(m,1H,CH2),1.17(t,J=7.0Hz,3H,CH3). 13 C NMR(151MHz,DMSO)δ167.45,161.69,158.36,152.45,140.68,129.63,111.01,110.51, 100.77,64.90,57.90,53.15,51.46,46.49,43.84,38.67,34.87,15.49.ESI-HRMS:m / s calcd for C 21 H 28 N4O4[M+H] + 401.2183,found401.2175.

[0155] In Examples 11-14, compound 23 is first prepared using genipin, compound 24 is then prepared using compound 23, compound 25 is then prepared using compound 24, compound 26 is then prepared using compound 25, and then compounds A6, A7, B6, and B7 of formula II are prepared using compound 26.

[0156] Example 11 2.4.2 Synthesis of methyl 2-methyl-1-oxo-7-(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl]-2,4a,5,7a-tetrahydro-1H-cyclopentadienyl[c]pyridine-4-carboxylate (A6)

[0157]

[0158] Step 1) Synthesis of methyl 7-(((tert-butyldimethylsilyl)oxy)methyl)-1-hydroxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 23)

[0159]

[0160] Genipin (10.0 g, 44.2 mmol) and imidazole (6.02 g, 88.4 mmol) were added to a 250 mL round-bottom flask and dissolved in DMF (60 mL). Tert-butyldimethylsilyl chloride (TBSCl: 13.32 g, 88.4 mmol) was dissolved in DMF (30 mL) and slowly added dropwise to the reaction mixture under an ice bath. After the addition was complete, the reaction was allowed to proceed for 2 h. TLC (developing solvent: petroleum ether:ethyl acetate = 4:1) indicated near-complete reaction. The reaction was quenched by adding 20 mL of distilled water and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 6:1) afforded 12.5 g of a white solid in an 83.1% yield.

[0161] Step 2) Synthesis of methyl 7-(((tert-butyldimethylsilyl)oxy)methyl)-1-oxo-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 24)

[0162]

[0163] In a 250 mL round-bottom flask, methyl 7-(((tert-butyldimethylsilyl)oxy)methyl)-1-hydroxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (12.5 g, 36.7 mmol) and dichloromethane (80 mL) were added, and Dess-Martin periodinane (DMP; 25 g, 58.9 mmol) was added in three additions, each with an interval of 10 min. The reaction was carried out at room temperature for 1 h. TLC [V (petroleum ether): V (ethyl acetate) = 4:1 was used for the development The reaction mixture was stirred for 15 min, and the mixture was transferred to a separatory funnel for separation and extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 8:1] to obtain 10.0 g of a white solid with a yield of 80.5%.

[0164] Step 3) Synthesis of methyl 7-(hydroxymethyl)-2-methyl-1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylate (Compound 25)

[0165]

[0166] In a 100 mL pressure tube, methyl 7-(((tert-butyldimethylsilyl)oxy)methyl)-1-oxo-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (10.0 g, 29.54 mmol), ethanolic methylamine solution (30-33%, 8 mL) and pyridine (20 mL) were added. The mixture was reacted at 65°C for 2 h. TLC [developing solvent: V (petroleum ether): V (ethyl acetate) = 4:1] showed that the reaction was almost complete. N-hexane was added to the reaction solution and pyridine was evaporated under reduced pressure to obtain a dark brown oil, which was directly used in the next step. Trifluoroacetic acid (20 mL) and tetrahydrofuran (20 mL) were added to a pressure tube, and the reaction was carried out at 65° C. for 2.5 h. TLC [V (petroleum ether):V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (petroleum ether):V (ethyl acetate) = 2:1 as the eluent] to obtain 2.9 g of a yellow solid in a yield of 41.4%.

[0167] Step 4) Synthesis of methyl 2-methyl-7-(((methylsulfonyl)oxy)methyl))1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylate (Compound 26)

[0168]

[0169] In a 100 mL round-bottom flask, methyl 7-(hydroxymethyl)-2-methyl-1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylate (0.5 g, 2.1 mmol), triethylamine (1.2 mL, 8.4 mmol) and dichloromethane (20 mL) were added. Methanesulfonyl chloride (0.65 mL, 8.4 mmol) was slowly added dropwise to the reaction solution under ice bath. After the addition was complete, the reaction was continued for 1 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was almost complete. The reaction was quenched by adding 20 mL of distilled water and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a light yellow oil for use.

[0170] Step 5) Synthesis of methyl 2-methyl-1-oxo-7-(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl]-2,4a,5,7a-tetrahydro-1H-cyclopentadienyl[c]pyridine-4-carboxylate (A6)

[0171] In a 50 mL round-bottom flask, 2-methyl-7-(((methylsulfonyl)oxy)methyl))1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopentane[c]pyridine-4-carboxylic acid methyl ester (Compound 26) (83.2 mg, 310.2 μmol), 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine (Compound 13) (136.7 mg, 620.4 μmol), potassium carbonate (85.7 mg, 620.4 μmol) and DMF ( The reaction mixture was added with 4% paraformaldehyde (5 mL) and reacted at room temperature for 18 h. TLC [V (petroleum ether): V (ethyl acetate) = 1:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (dichloromethane): V (methanol) = 20:1 as the eluent] to obtain 54.0 mg of a light yellow oily liquid in a yield of 39.6%. 1 H NMR(600MHz,DMSO-d6)δ7.34(s,1H,3-H),5.65(s,1H,7-H),3.63(s,3H,COOCH3),3 .57–3.41(m,4H,piperazine-H),3.39(t,J=5.3Hz,2H,CH2),3.30(s,3H,CH2),3.1 8(m,2H,CH2),3.04(s,3H,NCH3),2.94(d,J=13.9Hz,1H,9-H),2.64(m,1H,5-H),2. 45(s,4H,piperazine-H),2.37(d,J=4.1Hz,9H,CH3),2.07(dd,J=16.3,8.6Hz,1H). 13 C NMR (151MHz, DMSO) δ169.51,166.92,149.95,149.70,148.01,147.86,140.80,140.02,128.34,108.98,72.75,67.7 3,67.66,61.91,60.72,58.16,53.40,53.35,51.66,48.57,37.68,35.20,21.53,21.42,20.90,20.45.ESI-HRMS:m / s calcd for C 24H 33 N5O3[M+H] + 440.2656, found 440.2647.

[0172] Example 12 Synthesis of methyl 1-oxo-2-propyl-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)methyl)-2,4a,5,7a-tetrahydro-1H-cyclopentyl[c]pyridine-4-carboxylate (A7)

[0173]

[0174] Compound A7 was synthesized as a light yellow oily liquid according to the method of Example 11. The methylamine ethanol solution in step 3 of Example 11 was replaced with n-propylamine. The remaining steps were the same, and the final yield was 21.4%. 1 H NMR(600MHz,DMSO-d6)δ7.35(s,1H,3-H),5.72(d,J=38.7Hz,1H,7-H),3.66(s,3H,COOCH3),3.54–3 .49(m,4H,piperazine-H),3.48(d,J=7.3Hz,2H,CH2),3.45(d,J=6.8Hz,2H,CH2),3.36(d,J=13.9H z,2H,CH2),3.34–3.28(m,2H,CH2),2.96(d,J=13.9Hz,1H,9-H),2.71–2.64(m,1H,5-H),2.47(s,4H ,piperazine-H),2.39(d,J=5.4Hz,9H,CH3),1.48(q,J=7.3Hz,2H,CH2),0.79(t,J=7.4Hz,3H,CH3). 13 C NMR (151MHz, DMSO) δ169.11,166.92,149.95,149.71,147.99,147.88,140.90,139.02,128.29,109.25,61.87,61.7 9,58.22,55.35,53.42,53.30,51.69,48.68,48.58,37.52,30.86,22.05,21.50,21.40,20.89,11.26.ESI-HRMS:m / s calcd for C 26 H 37 N5O3[M+H] + 468.2969, found 468.2965.

[0175] Example 13 Synthesis of methyl 2-methyl-1-oxo-7-((4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylate (B6)

[0176]

[0177] Compound B6 was synthesized according to the method of Example 11. We replaced 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in Step 5 of Example 11 with 2-(piperazin-1-yl)pyrimidine (Compound 14). The remaining steps were the same. The final light yellow oily liquid was obtained with a yield of 51.7%. 1 H NMR (600MHz, DMSO-d6) δ8.34(dd,J=4.7,1.4Hz,2H,ArH),7.38(d,J=1.4Hz,1H,3-H),6.61(td,J=4.7,1.3Hz,1H,A rH),5.75(s,1H,7-H),3.70(t,J=5.2Hz,4H,piperazine-H),3.68(s,3H,COOCH3),3.56(d,J=10.5Hz,1H,CH),3.4 5(d,J=13.8Hz,1H,CH),3.08(s,3H,NCH3),3.03(d,J=13.7Hz,1H,9-H),2.71(ddd,J=16.4,8.4,2.6Hz,1H,5-H),2 .50(d,J=36.2Hz,1H,CH2),2.38(ddt,J=33.8,10.8,5.1Hz,4H,piperazine-H),2.13(dd,J=16.3,8.6Hz,1H,CH2). 13 C NMR (151MHz, DMSO) δ169.55,166.94,161.70,158.34,140.71,140.03,128.61,110. 49,109.01,58.29,53.20,51.67,48.52,43.83,37.67,35.20,29.88.ESI-HRMS:m / s calcd for C 20 H 25 N5O3[M+H] + 384.2030, found 384.2023.

[0178] Example 14 Synthesis of methyl 1-oxo-2-propyl-7-((4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-2,4a,5,7a-tetrahydro-1H-cyclopentyl[c]pyridine-4-carboxylate (B7)

[0179]

[0180] Compound B7 was synthesized according to the method of Example 12, except that 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine in Example 12 was replaced by 2-(piperazin-1-yl)pyrimidine. The remaining steps were the same, and a colorless oily liquid was finally obtained with a yield of 23.5%. 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=4.7Hz,2H,ArH),7.38(s,1H,3-H),6.61(t,J=4.7Hz,1H,ArH),5.76(s, 1H,7-H),3.71(t,J=5.2Hz,4H,piperazine-H),3.68(s,3H,COOCH3),3.60–3.52(m,2H,CH2),3.50–3.43(m ,2H,CH2),3.42–3.37(m,1H,9-H),3.03(d,J=13.8Hz,1H,5-H),2.77–2.68(m,1H,CH2),2.45–2.33(m,4H, piperazine-H), 2.10 (dd, J=16.0, 8.7Hz, 1H, CH2), 1.50 (q, J=7.3Hz, 2H, CH2), 0.81 (t, J=7.4Hz, 3H, CH3). 13 C NMR (151MHz, DMSO) δ169.11,166.92,161.69,158.33,140.86,139.04,128.51,110.47,109.25,60 .37,58.37,55.37,53.23,51.69,48.68,48.53,43.81,37.54,29.87,22.07,11.27.ESI-HRMS:m / s calcd for C 22 H 29 N5O3[M+H] + 412.2343, found 412.2341.

[0181] Examples 15-18 are to prepare compound 5, which is then used to prepare compound 6, which is then used to prepare compounds A8, A9, B8, and B9 of general formula I.

[0182] Example 15 Synthesis of methyl 1-ethoxy-7-((2-(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)acetylamino)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A8)

[0183]

[0184] Step 1) Synthesis of methyl 7-((1,3-dioxoisoindolin-2-yl)methyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 5)

[0185]

[0186] In a 50 mL three-necked flask, methyl 7-(hydroxymethyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (compound 2 obtained when reaction reagent 1 is ethanol) (530.0 mg, 2.08 mmol), phthalimide (compound 4) (710.7 mg, 2.71 mmol), and triphenylphosphine (398.7 mg, 2.71 mmol) were added in sequence. The mixture was vacuumed 3 to 4 times and nitrogen was replaced in the reaction flask. Under nitrogen protection, anhydrous tetrahydrofuran (6 mL) was added to dissolve the mixture. The reaction flask was placed in an ice bath and 2 mL was slowly added dropwise. DIAD (820.8 mm L, 4.17 mmol) diluted with anhydrous tetrahydrofuran was reacted at room temperature for 6 h. TLC [V (petroleum ether):V (ethyl acetate) = 5:1 as the developing solvent] showed that the reaction was almost complete. The reaction was quenched by adding 20 mL of distilled water and extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (petroleum ether):V (ethyl acetate) = 8:1 as the eluent] to obtain 750.0 mg of a light yellow solid in a yield of 93.8%. 1 H NMR (600MHz, DMSO-d6) δ7.91–7.85(m,4H,ArH),7.52(d,J=1.2Hz,1H,3-H),5.50(t,J=2.3Hz,1H,7-H),4 .73(d,J=8.0Hz,1H,1-H),4.24(dd,J=17.3,2.6Hz,1H,10-H),4.18–4.08(m,1H,10-H),3.93(m,J=9.8,7 .1Hz,1H,CH),3.71(m,J=9.8,7.1Hz,1H,CH),3.63(d,J=4.0Hz,3H,COOCH3),3.05(m,J=8.5,1.3Hz,1H,9 -H),2.68–2.59(m,1H,5-H),2.58–2.53(m,1H,CH2),2.04–1.94(m,1H,CH2),0.88(t,J=7.5Hz,3H,CH3).

[0187] Step 2) Synthesis of methyl 7-(aminomethyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (Compound 6)

[0188]

[0189] To a 50 mL round-bottom flask was added methyl 7-((1,3-dioxoisoindolin-2-yl)methyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (Compound 5) (750.0 mg, 1.96 mmol) and dissolved in anhydrous ethanol (10 mL). 40%-50% hydrazine hydrate (752.3 mg, 23.47 mmol) was added and reacted at room temperature for 12 h. TLC [V (petroleum ether):V (ethyl acetate) = 3:1 as the developing solvent] showed that the reaction was essentially complete. The white precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a pale yellow oily crude product, which was purified by silica gel column chromatography [V (dichloromethane):V (methanol) = 10:1 as the eluent] to obtain 350.0 mg of a pale yellow oily liquid in a yield of 70.6%. 1 H NMR(600MHz,DMSO-d6)δ7.48(s,1H,3-H),5.66(s,1H,7-H),4.70(d,J=7.5Hz,1H,1-H), 3.89–3.81(m,1H,CH),3.64(s,4H,COOCH3,CH2),3.61–3.58(m,1H,CH),3.00(q,J=8.0H z,2H,CH2),2.67(dd,J=16.1,8.3Hz,1H,9-H),2.67(dd,J=16.2,8.3Hz,1H,5-H),2.54( t,J=7.7Hz,1H,CH2),2.04–1.97(m,1H,CH2),1.16(t,J=7.0Hz,3H,CH3).ESI-HRMS:m / s calcd for C 13 H 19 NO4[M+H] + 254.1387, found 254.1386.

[0190] Step 3) Synthesis of methyl 1-ethoxy-7-((2-(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)acetylamino)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A8)

[0191] In a 50 mL round-bottom flask, 2-(4-((3,5,6-trimethylpiperazin-2-yl)methyl)piperazin-1-yl)acetic acid (Compound 17) (200.0 mg, 718.5 μmol), EDCI·HCl (228.4 mg, 1.15 mmol), HOBt (155.34 mg, 1.15 mmol) and dichloromethane (10 mL) were added and stirred at room temperature for 30 min. Then, 7-(aminomethyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-yl dissolved in 2 mL of dichloromethane was added. Methyl carboxylate (Compound 6) (145.6 mg, 574.8 μmol) was reacted at room temperature for 12 h. TLC [V (dichloromethane): V (methanol) = 10:1 as the developing solvent] showed that the reaction was almost complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (dichloromethane): V (methanol) = 100:1 as the eluent] to obtain 209.6 mg of a light yellow oily liquid with a yield of 56.8%. 1 H NMR (600MHz, DMSO-d6) δ7.83(t,J=6.0Hz,1H,NH),7.49(d,J=1.2Hz,1H,3-H),5.58(s,1H,7-H),4.73(d,J=7.4Hz,1H,1-H) ,3.97(dq,J=16.6,2.4Hz,1H,CH2),3.87(dq,J=9.8,7.1Hz,1H,CH2),3.83–3.71(m,2H,CH2),3.64(s,3H,COOCH3),3.59(s, 1H,CH2),3.54(d,J=2.1Hz,3H,CH2),3.19(s,1H,9-H),3.02(qd,J=8.1,1.3Hz,1H,5-H),2.68(ddd,J=16.2,8.2,3.0Hz,1H, CH2),2.52(d,J=6.4Hz,9H,CH3),2.40(d,J=2.9Hz,8H,piperazine-H),2.04–1.99(m,1H,CH2),1.18(t,J=7.1Hz,3H,CH3). 13C NMR(151MHz,DMSO-d6)δ170.94,167.47,152.44,149.98,149.81,147.96,147.91,141.23,126.79,110.89,101.07,65 .35,61.77,61.57,53.35,53.11,52.47,51.52,46.86,38.64,38.56,35.29,21.47,21.38,20.85,15.44.ESI-HRMS:m / s calcd for C 27 H 39 N5O5[M+H] + 514.3024, found 514.3019.

[0192] Example 16 Synthesis of methyl 1-ethoxy-7-((3-(4-((3,5,6-trimethylpyrazin-2-yl)methyl)piperazin-1-yl)propionamido)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (A9)

[0193]

[0194] Compound A9 was synthesized according to the method of Example 15. We replaced the 2-(4-((3,5,6-trimethylpiperazin-2-yl)methyl)piperazin-1-yl)acetic acid in Step 3 of Example 15 with 2-(4-((3,5,6-trimethylpiperazin-2-yl)methyl)piperazin-1-yl)propionic acid. The remaining steps were the same. The final light yellow oily liquid was obtained with a yield of 44.4%. 1H NMR (600MHz, DMSO-d6) δ8.08(t,J=5.7Hz,1H,NH),7.50(d,J=1.2Hz,1H,3-H),5.70(d,J=3.0Hz,1H,7-H),4.72(d,J=7.4Hz,1H,1-H),3.9 2(dt,J=16.2,4.9Hz,1H,CH2),3.86(dq,J=9.8,7.1Hz,1H,CH2),3.70(ddd,J=16.4,5.5,2.5Hz,1H,CH2),3.65(s,5H,CH2,COOCH3),3.62 –3.57(m,1H,CH2),3.51(s,2H,CH2),3.03(qd,J=8.1,1.3Hz,1H,9-H),2.68(dt,J=15.0,5.6Hz,1H,5-H),2.49(d,J=12.1Hz,9H,CH3),2. 40(d,J=2.7Hz,8H,piperazine-H),2.24(t,J=7.0Hz,3H,CH2,CH2),2.02(ddt,J=16.1,7.8,2.2Hz,1H,CH2),1.17(t,J=7.1Hz,3H,CH3). 13 CNMR(151MHz,DMSO-d6)δ171.27,167.41,152.49,149.93,149.73,147.94,147.88,141.44,126.91,110.89,101.17,65.3 0,61.93,54.53,53.26,52.92,51.49,46.67,38.74,38.56,35.54,33.51,21.53,21.43,20.90,15.45.ESI-HRMS:m / scalcd for C 28 H 41 N5O5[M+H] + 528.3180, found 528.3169.

[0195] Example 17 Synthesis of methyl 1-ethoxy-7-((2-(4-(pyrimidin-2-yl)piperazin-1-yl)acetamido)methyl)-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (B8)

[0196]

[0197] 2-(4-(pyrimidin-2-yl)piperazin-1-yl)acetic acid (Compound 19) (225.0 mg, 1.01 mmol), EDCI·HCl (310.5 mg, 1.62 mmol), HOBt (218.9 mg, 1.62 mmol) and dichloromethane (10 mL) were added to a 50 mL round-bottom flask and stirred at room temperature for 30 min. 7-(aminomethyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylic acid methyl ester (Compound 19) dissolved in 2 mL of dichloromethane was then added. Compound 6) (205.2 mg, 809.9 μmol) was reacted at room temperature for 12 h. TLC [V (dichloromethane): V (methanol) = 10:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3×50 mL). The organic phases were combined, washed with saturated brine (3×50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V (dichloromethane): V (methanol) = 100:1 as the eluent] to obtain 360.0 mg of a light yellow oily liquid with a yield of 77.7%. 1 H NMR (600MHz, DMSO-d6) δ8.35(d,J=4.7Hz,2H,ArH),7.99(t,J=5.9Hz,1H,NH),7.50(d,J=1.2Hz,1H,3-H),6.62(t,J=4.7Hz,1 H, ArH), 5.61 (d, J = 3.1Hz, 1H, 7-H), 4.74 (d, J = 7.4Hz, 1H, 1-H), 4.00 (m, J = 16.6, 2.6Hz, 1H, CH2), 3.88 (dt, J = 9.8, 7.1Hz, 1H, CH2),3.81–3.71(m,6H,CH2,piperazine-H),3.64(s,3H,COOCH3),3.04(td,J=8.1,1.3Hz,1H,9-H),3.01(s,2H,CH2),2.72– 2.66(m,1H,5-H),2.54(t,J=7.8Hz,1H,CH2),2.50(s,4H,piperazine-H),2.06–1.99(m,1H,CH2),1.19(t,J=7.0Hz,3H,CH3). 13CNMR(151MHz,DMSO-d6)δ169.35,167.41,161.67,158.39,152.44,141.34,126.78,110.91,110. 59,101.12,65.34,61.62,53.09,51.50,46.90,43.71,38.66,38.61,35.38,15.47.ESI-HRMS:m / s calcd for C 23 H 31 N5O5[M+H] + 458.2398, found 458.2391.

[0198] Example 18 Synthesis of methyl 1-ethoxy-7-((3-(4-(pyrimidin-2-yl)piperazin-1-yl)propionamido)methyl)-1,4a,5,7a-tetrahydrocyclopentyl[c]pyran-4-carboxylate (B9)

[0199]

[0200] Compound B9 was synthesized according to the method of Example 17. We replaced the 2-(4-(pyrimidin-2-yl)piperazin-1-yl)acetic acid in Example 17 with 2-(4-(pyrimidin-2-yl)piperazin-1-yl)propionic acid. The remaining steps were the same. The final light yellow oily liquid was obtained with a yield of 59.2%. 1H NMR (600MHz, DMSO-d6) δ8.35(d,J=4.6Hz,2H,ArH),8.14(q,J=6.1Hz,1H,NH),7.48(d,J=1.2Hz,1H,3-H),6.62(t,J=4.7Hz,1H,ArH),5.70(d,J =2.9Hz,1H,7-H),4.73(d,J=7.4Hz,1H,1-H),3.97–3.90(m,1H,CH2),3.88–3.83(m,1H,CH2),3.74(dp,J=4.5,2.3Hz,1H,CH2),3.71–3.68(m,4H ,piperazine-H),3.67–3.64(m,1H,CH2),3.63(s,3H,COOCH3),3.02(qd,J=8.1,1.3Hz,1H,9-H),2.69–2.63(m,1H,5-H),2.57(t,J=7.0Hz,2H,C H2),2.54(d,J=8.4Hz,1H,CH2),2.43(t,J=5.1Hz,4H,piperazine-H),2.32(t,J=7.1Hz,2H,CH2),2.02(m,1H,CH2),1.17(t,J=7.0Hz,3H,CH3). 13 C NMR(151MHz,DMSO-d6)δ171.29,167.42,161.67,158.38,152.46,141.41,126.89,110.91,110.58,101 .12,65.30,54.66,52.75,51.49,46.71,43.73,38.76,38.56,35.47,33.54,15.44.ESI-HRMS:m / scalcd for C 24 H 33 N5O5[M+H] + 472.2554, found 472.2538.

[0201] In Examples 19-22, Compound 20 and Paeonol were first used to prepare Compound 21, Compound 21 was then used to prepare Compound 22, and then Compound 22 and Compound 6 were used to prepare JD1-JD4.

[0202] Example 19 2.4.4 Synthesis of 7-((2-(2-acetyl-5-methoxyphenoxy)acetamido)methyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylic acid methyl ester (JD-1)

[0203]

[0204] Step 1) Synthesis of ethyl 2-(2-acetyl-5-methoxyphenoxy)acetate (Compound 21)

[0205]

[0206] Paeonol (compound 20) (500.0 mg, 3.01 mmol), potassium carbonate (1.66 g, 3.61 mmol) and DMF (5 mL) were added to a 50 mL round-bottom flask. The reaction was placed in an ice bath, and ethyl chloroacetate (386.4 mm L, 3.61 mmol) was added. The reaction was allowed to react at room temperature for 12 h. TLC [V (petroleum ether): V (ethyl acetate) = 5:1 as the developing solvent] showed that the reaction was essentially complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 690.0 mg of a brown solid with a yield of 90.9%.

[0207] Step 2) Synthesis of 2-(2-acetyl-5-methoxyphenoxy)acetic acid (Compound 22)

[0208]

[0209] To a 50 mL round-bottom flask were added ethyl 2-(2-acetyl-5-methoxyphenoxy)acetate (690.0 mg, 2.74 mmol) and anhydrous methanol (5 mL). The reaction was placed in an ice bath and aqueous sodium hydroxide solution (437.6 mm L, 10.94 mmol) was added. The reaction was allowed to react at room temperature for 12 h. TLC [V (petroleum ether): V (ethyl acetate) = 3:1 as the developing solvent] showed that the reaction was essentially complete. 1 M hydrochloric acid solution was added to adjust the pH to 3-4. The precipitate was precipitated and filtered. The filter cake was dried to obtain 483.9 mg of a light yellow solid with a yield of 78.9%. 1 H NMR (600MHz, DMSO-d6) δ13.14(s,1H,OH),7.66(d,J=8.7Hz,1H,ArH),6.68–6.57(m,2H ArH),4.86(s,2H,CH2),3.81(s,3H,CH3),2.57(s,3H,CH3).

[0210] Step 3) Synthesis of methyl 7-((2-(2-acetyl-5-methoxyphenoxy)acetamido)methyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (JD-1)

[0211] In a 50 mL round-bottom flask were added 2-(2-acetyl-5-methoxyphenoxy)acetic acid (Compound 22) (100.0 mg, 446.0 μmol), 7-(aminomethyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylic acid methyl ester (Compound 6 obtained in Example 15) (113.0 mg, 446.0 μmol), EDCI·HCl (128.3 mg, 669.0 μmol), HOBt (90.4 mg, 669.0 μmol), triethylamine (21 The product was added with 4% paraformaldehyde (7.0 mm L, 1.56 mmol) and dichloromethane (5 mL), and the reaction was allowed to proceed at room temperature for 12 h. TLC [V (dichloromethane): V (methanol) = 10:1 as the developing solvent] showed that the reaction was basically complete. 20 mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The organic phases were purified by silica gel column chromatography [V (dichloromethane): V (methanol) = 200:1 as the eluent] to obtain 55.8 mg of a white solid in a yield of 27.2%. 1 H NMR (600MHz, DMSO-d6) δ8.36(t,J=5.8Hz,1H,NH),7.73(d,J=8.7Hz,1H,ArH),7.49(d,J=1.2Hz,1H,3-H),6.64(dd,J=8.7, 2.3Hz,1H,ArH),6.58(d,J=2.3Hz,1H,ArH),5.66–5.61(m,1H,7-H),4.72(d,J=7.6Hz,1H,1-H),4.70(s,2H,CH2),4.07–4.0 1(m,2H,CH2),3.86(dq,J=10.0,7.1Hz,2H,CH2),3.81(s,3H,OCH3),3.64(s,3H,COOCH3),3.02(qd,J=8.2,1.3Hz,1H,9-H), 2.70–2.63(m,1H,5-H),2.55(s,3H,COCH3),2.53(d,J=8.1Hz,1H,CH2),2.05–2.00(m,1H,CH2),1.17(d,J=1.6Hz,3H,CH3). 13C NMR(151MHz,DMSO-d6)δ197.13,167.50,167.40,164.54,159.60,152.49,141.00,132.80,127.16,120.94,110.8 5,106.90,101.23,100.11,67.92,65.34,56.11,51.49,46.69,38.71,38.61,35.59,31.65,15.42.ESI-HRMS:m / s calcd for C 24 H 29 NO8[M+H] + 460.1966,found 460.1964.

[0212] Example 20 Synthesis of methyl 7-((4-(2-acetyl-5-methoxyphenoxy)butyramido)methyl)-1-ethoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (JD-2)

[0213]

[0214] Compound JD-2 was synthesized according to the method of Example 19. In step 1) of Example 19, ethyl chloroacetate was replaced with ethyl chlorobutyrate. The remaining steps were the same, and a light yellow oily liquid was finally obtained with a yield of 36.4%. 1 H NMR (600MHz, DMSO-d6) δ8.06(t,J=5.7Hz,1H,NH),7.67(d,J=8.7Hz,1H,ArH),7.48(d,J=1.3Hz,1H,3-H),6.61(d,J=2.3Hz,1H,ArH),6 .59(dd,J=8.6,2.3Hz,1H,ArH),5.57(s,1H,7-H),4.71(d,J=7.4Hz,1H,1-H),4.10(t,J=6.2Hz,2H,CH2),3.93(m,J=15.5,4.7Hz,1H,C H2),3.87–3.81(m,5H,CH2,OCH3),3.73(m,J=16.6,5.6,2.6Hz,1H,CH2),3.64(s,3H,COOCH3),2.99(m,J=8.0,1.3Hz,1H,9-H),2.63(m ,J=15.6,9.5,4.8Hz,1H,5-H),2.52(s,3H,COCH3),2.34(t,J=7.3Hz,2H,CH2),2.04(m,J=6.9Hz,3H,CH2),1.17(t,J=7.0Hz,3H,CH3). 13C NMR(151MHz,DMSO-d6)δ196.76,171.63,167.40,164.74,160.75,152.44,141.45,132.25,126.80,120.76,110.86,106.46 ,101.12,99.25,68.32,65.30,56.04,51.48,46.70,38.76,38.54,35.46,32.41,32.22,25.21,15.41.ESI-HRMS:m / scalcd forC26H33NO8[M+H]+488.2279,found 488.2290.

[0215] Example 21 Synthesis of methyl 7-((2-(2-acetyl-5-methoxyphenoxy)acetamido)methyl)-1-methoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (JD-3)

[0216]

[0217] Compound JD-3 was synthesized according to the method of Example 19. We replaced the n-propanol in S1 in Example 1 with methanol. The remaining steps were the same. A light yellow oily liquid was finally obtained with a yield of 32.1%. 1 H NMR (600MHz, DMSO-d6) δ8.39(t,J=5.8Hz,1H,NH),7.73(d,J=8.7Hz,1H,ArH),7.49(d,J=1.2Hz,1H,3-H),6 .64(dd,J=8.7,2.3Hz,1H,ArH),6.58(d,J=2.4Hz,1H,ArH),5.63(d,J=3.0Hz,1H,7-H),4.71(s,2H,CH2),4. 69(d,J=7.0Hz,1H,1-H),3.81(s,5H,OCH3,CH2),3.64(s,3H,COOCH3),3.46(s,3H,CH3),3.05–2.99(m,1H, 9-H),2.69–2.62(m,1H,5-H),2.58(dd,J=8.5,6.3Hz,1H,CH2),2.55(s,3H,COCH3),2.05–1.99(m,1H,CH2). 13C NMR(151MHz,DMSO-d6)δ152.26,132.79,127.37,106.90,102.00,100.08,67 .88,56.95,56.11,51.51,46.77,39.85,38.55,35.14,31.69.ESI-HRMS:m / s calcd for C 23 H 27 NO8[M+H] + 446.1809, found 446.1820.

[0218] Example 22 Synthesis of methyl 7-((4-(2-acetyl-5-methoxyphenoxy)butyramido)methyl)-1-methoxy-1,4a,5,7a-tetrahydrocyclopentane[c]pyran-4-carboxylate (JD-4)

[0219]

[0220] Compound JD-4 was synthesized according to the method of Example 19. We replaced the ethyl chloroacetate in step 1) of Example 19 with ethyl chlorobutyrate, and replaced the n-propanol in S1 of Example 1 with methanol. The remaining steps were the same, and a milky white oily liquid was finally obtained with a yield of 38.2%. 1 H NMR(600MHz,DMSO-d6)δ8.06(t,J=5.7Hz,1H,NH),7.66(dd,J=8.7,3.2Hz,1H,ArH),7.47(d,J=1.2Hz,1H,3-H),6.61 (d,J=2.4Hz,1H,ArH),6.59(dd,J=8.7,2.3Hz,1H,ArH),5.57(q,J=1.9Hz,1H,7-H),4.68(d,J=6.9Hz,1H,1-H),4.09( t,J=6.2Hz,2H,CH2),3.89–3.80(m,5H,OCH3,CH2),3.64(s,3H,COOCH3),3.45(s,3H,CH3),3.03–2.97(m,1H,9-H),2. 66–2.60(m,1H,5-H),2.59–2.54(m,1H,CH2),2.52(s,3H,COCH3),2.34(t,J=7.3Hz,2H,CH2),2.06–2.00(m,3H,CH2). 13C NMR(151MHz,DMSO-d6)δ196.76,171.67,167.34,164.74,160.75,152.20,141.07,132.26,127.00,120.75,111.14, 106.45,101.92,99.25,68.30,56.92,56.04,51.49,46.79,38.61,38.48,35.02,32.41,32.21,25.21.ESI-HRMS:m / s calcd for C25H31NO8[M+H]+474.2122,found 474.2132.

[0221] Examples 23-26 used Compound 28 and Compound 22 to prepare JD-5-JD-8.

[0222] Example 23 Synthesis of methyl 7-((2-(2-acetyl-5-methoxyphenoxy)acetamido)methyl)-2-methyl-1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylate (JD-5)

[0223]

[0224] In a 50 mL round-bottom flask were added 2-(2-acetyl-5-methoxyphenoxy)acetic acid (Compound 22) (100.0 mg, 446.0 μmol), 7-(aminomethyl)-2-methyl-1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopenta[c]pyridine-4-carboxylic acid methyl ester (Compound 28) (105.4 mg, 446.0 μmol), EDCI·HCl (128.3 mg, 669.0 μmol), HOBt (90.4 mg, 669.0 μmol), triethylamine (217 0mmL, 1.56mmol) and dichloromethane (5mL), and react at room temperature for 12h. TLC [V(dichloromethane):V(methanol)=10:1 as developing solvent] showed that the reaction was basically complete. 20mL of distilled water was added to quench the reaction, and the mixture was extracted with dichloromethane (3×50mL). The organic phases were combined, washed with saturated brine (3×50mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography [V(dichloromethane):V(methanol)=200:1 as eluent] to obtain 64.5mg of a light yellow solid in a yield of 32.7%. 1H NMR (600MHz, DMSO-d6) δ8.38(t,J=5.9Hz,1H,NH),7.72(d,J=8.7Hz,1H,ArH),7.37(s,1H,3-H),6.64(d d,J=8.7,2.3Hz,1H,ArH),6.57(d,J=2.4Hz,1H,ArH),5.64–5.58(m,1H,7-H),4.71(s,2H,CH2),4.21(m, 1H,CH2),4.00(m,1H,CH2),3.81(s,3H,OCH3),3.67(s,3H,COOCH3),3.55(d,J=10.7Hz,1H,9-H),3.40–3 .35(m,1H,5-H),3.08(s,3H,NCH3),2.73–2.65(m,1H,CH2),2.55(s,3H,COCH3),2.11–2.02(m,1H,CH2). 13 C NMR(151MHz,DMSO-d6)δ197.11,169.29,167.54,166.84,164.52,159.56,140.47,139.70,132.79,126.10,12 0.94,109.25,106.85,100.07,67.91,56.10,51.73,49.08,39.93,39.23,37.47,35.15,31.69.ESI-HRMS:m / s calcd for C 23 H 26 N2O7[M+H] + 443.1818,found443.1812.

[0225] Example 24 Synthesis of methyl 7-((4-(2-acetyl-5-methoxyphenoxy)butyramido)methyl)-2-methyl-1-oxo-2,4a,5,7a-tetrahydro-1H-cyclopentadienyl[c]pyridine-4-carboxylate (JD-6)

[0226]

[0227] Compound JD-6 was synthesized according to the method of Example 23. We replaced the ethyl chloroacetate in step 1) of Example 19 with ethyl chlorobutyrate, and the obtained compound 22 was reacted with compound 28. The remaining steps were the same to finally obtain a white solid with a yield of 38.9%. 1H NMR(600MHz,DMSO-d6)δ8.09(t,J=5.7Hz,1H,NH),7.67(d,J=8.6Hz,1H,ArH),7.35(s,1H,3-H),6.62–6 .57(m,2H,ArH),5.56(m,J=3.3,1.8Hz,1H,7-H),4.15–4.07(m,3H,CH2),3.91(m,J=16.6,5.7,2.9Hz,1H ,CH2),3.82(s,3H,OCH3),3.67(s,3H,COOCH3),3.50(d,J=10.8Hz,1H,9-H),3.33(m,J=10.8,8.6Hz,1H, 5-H),3.07(s,3H,NCH3),2.70–2.63(m,1H,CH2),2.35(t,J=7.3Hz,2H,CH2),2.04(p,J=6.5Hz,3H,CH2). 13 C NMR(151MHz,DMSO-d6)δ196.77,171.68,169.25,166.84,164.72,160.74,140.95,139.65,132.24,125.74,120.71, 109.24,106.42,99.25,99.22,68.27,56.03,51.70,49.01,39.37,37.49,35.14,32.41,32.22,25.20.ESI-HRMS:m / s calcd for C 25 H 30 N2O7[M+H] + 471.2126, found 471.2121.

[0228] Example 25 Synthesis of methyl 7-((2-(2-acetyl-5-methoxyphenoxy)acetylamino)methyl)-1-oxo-2-propyl-2,4a,5,7a-tetrahydro-1H-cyclopentadienyl[c]pyridine-4-carboxylate (JD-7)

[0229]

[0230] Compound JD-7 was synthesized according to the method of Example 23. We replaced the methylamine ethanol solution in step 3) of Example 11 with n-propylamine. The remaining steps were the same, and a light yellow solid was finally obtained with a yield of 45.7%. 1H NMR (600MHz, DMSO-d6) δ8.39(t,J=5.8Hz,1H,NH),7.72(d,J=8.7Hz,1H,ArH),7.37(s,1H,3-H),6.64(dd,J=8.8,2.3Hz,1H,ArH),6. 57(d,J=2.4Hz,1H,ArH),5.62(m,J=3.4,1.9Hz,1H,7-H),4.70(s,2H,CH2),4.19(m,J=16.3,4.4Hz,1H,CH2),4.00(m,J=16.9,5.9,2 .9Hz,1H,CH2),3.81(s,3H,OCH3),3.68(s,3H,COOCH3),3.59–3.51(m,2H,CH2),3.47(m,J=13.6,7.1Hz,1H,9-H),3.37(d,J=8.7Hz, 1H,5-H),2.73–2.66(m,1H,CH2),2.55(s,3H,COCH3),2.09–2.01(m,1H,CH2),1.49(h,J=7.3Hz,2H,CH2),0.80(t,J=7.4Hz,3H,CH3). 13 C NMR (151MHz, DMSO-d6) δ138.78,132.77,106.86,100.06,67.92,56.10,51.76,49.22,48.63,39.99,39.25,37.42,31.70,22.00,11.24.ESI-HRMS: m / s calcd for C 25 H 30 N2O7[M+H] + 471.2126,found471.2116.

[0231] Example 26 Synthesis of methyl 7-((4-(2-acetyl-5-methoxyphenoxy)butyramido)methyl)-1-oxo-2-propyl-2,4a,5,7a-tetrahydro-1H-cyclopentadienyl[c]pyridine-4-carboxylate (JD-8)

[0232]

[0233] Compound JD-8 was synthesized according to the method of Example 23. We replaced the methylamine ethanol solution in step 3 of Example 11 with n-propylamine, and replaced the ethyl chloroacetate in step 1 of Example 19 with ethyl chlorobutyrate. The remaining steps were the same, and a white solid was finally obtained with a yield of 34.6%. 1H NMR (600MHz, DMSO-d6) δ8.10(t,J=5.7Hz,1H,NH),7.67(d,J=8.7Hz,1H,ArH),7.35(s,1H,3-H),6.63–6.56(m,2H,ArH),5.56( m,J=3.3,1.8Hz,1H,7-H),4.09(t,J=6.2Hz,3H,CH2),3.91(m,J=16.8,5.8,2.8Hz,1H,CH2),3.82(s,3H,OCH3),3.67(s,3H,CO OCH3),3.58–3.49(m,2H,CH2),3.45(m,J=13.6,7.1Hz,1H,9-H),3.33(m,J=10.7,8.6Hz,1H,5-H),2.69–2.63(m,1H,CH2),2.5 2(s,3H,COCH3),2.35(t,J=7.3Hz,2H,CH2),2.03(m,J=6.4Hz,3H,CH2),1.49(p,J=7.3Hz,2H,CH2),0.80(t,J=7.4Hz,3H,CH3). 13 C NMR(151MHz,DMSO-d6)δ196.73,171.64,168.87,166.83,164.72,160.74,141.11,138.77,132.24,125.60,120.71,109.42 ,106.43,99.22,68.29,56.04,51.73,49.16,48.62,39.74,39.37,37.45,32.42,32.22,25.20,22.01,11.25.ESI-HRMS:m / s calcd forC 27 H 34 N2O7[M+H]+499.2439,found 499.2439.

[0234] Example 27 In vitro enzyme inhibition activity experiment

[0235] 1. Experimental methods:

[0236] The GSK-3β inhibitory activity experiment was tested using the Kinase-Glo assay with Staurosporine as a positive control. The test compound concentrations were 1.25, 2.5, 5, 10, 20, and 40 μM. The Kinase-Glo assay was performed using a black 96-well plate in assay buffer. 10 μL of the test compound (dissolved in dimethyl sulfoxide and diluted to the desired concentration in assay buffer), 10 μL of human recombinant GSK-3β (20 ng), 10 μL of substrate (0.05 μg / μL), and 10 μL of ATP (2 μM) were added to the 96-well plate in sequence. After incubation at 37°C for 30 minutes, the enzymatic reaction was stopped with 40 μL of Kinase-Glo detection reagent, and the fluorescence intensity was measured using a multi-function microplate reader (SpectraMax i3x). The inhibition rate was calculated based on the fluorescence value, and the log (inhibitor) vs. Response-Variable slope analysis software GraphPad Prism 8 was used to fit the dose-effect curve and calculate the IC 50 Buffer preparation: 40 mM Tris, pH 7.5; 20 mM MgCl2; 0.1 mg / mL BSA; 50 μM DTT.

[0237] 2. The experimental results are shown in Table 1;

[0238]

[0239] Table 1 Inhibitory activity of target compounds on GSK-3β

[0240]

[0241]

[0242] In Table 1, the a Genipin (Bide), the leading compound; b Staurosporine (McLean), a typical ATP competitive GSK-3βkinase inhibitor, was used as a reference compound in this study.

[0243] Example 28 Cytotoxicity Experiment

[0244] 1. Experimental Methods

[0245] SH-SY5Y cells were grown in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. SH-SY5Y cells were digested, collected, and centrifuged. After resuspending and counting, the cells were counted at 1×10 4 The cell suspension was seeded into a 96-well cell culture plate at a concentration of 10 cells / well, and 100 μL of SH-SY5Y cell suspension was added to each well. After 24 hours, the target compound was added to the cells and incubated for an additional 48 hours. Then, 100 μL of culture medium containing 10% CCK-8 working solution was added to each well and incubated at 37°C for 1.5 hours. Finally, the absorbance (OD) value was measured at a wavelength of 450 nm using a multi-function microplate reader to calculate cell viability.

[0246] 2. The experimental results are shown in Table 2:

[0247] Table 2 Cytotoxicity of target compounds

[0248] Cpd. Cell survival rate (%) Cpd. Cell survival rate (%) Control 100.00 B6 87.41 Genipin 92.14 B7 85.17 Donepezil 91.55 A8 87.44 A1 67.05 A9 96.11 A2 70.90 B8 89.28 A3 73.35 B9 91.16 A4 86.10 JD-1 71.76 A5 71.70 JD-2 71.49 A6 97.23 JD-3 61.57 A7 89.43 JD-4 68.84 B1 76.86 JD-5 78.14 B2 71.07 JD-6 91.31 B3 67.86 JD-7 94.61 B4 69.58 JD-8 82.27 B5 91.15 / /

[0249] As can be seen, the CCK8 assay was used to evaluate the cytotoxicity of the target compound against the human neuroblastoma cell line SH-SY5Y, with donepezil used as a positive control. The results showed that the compound had no significant cytotoxicity against the SH-SY5Y cell line after 48 hours. These results suggest that the target compound is worthy of further study as a GSK-3β inhibitor.

[0250] Example 29 Experiment on the protective effect of the compound on SH-SY5Y cell damage induced by TBHP and Aβ25-35.

[0251] 1. Experimental Methods

[0252] 100 μL of SH-SY5Y cells in logarithmic growth phase were plated at 1×10 4 Cells were seeded into 96-well plates at a density of 10 cells / mL and cultured in an incubator containing 5% CO2 at 37°C. After 24 hours of culture, the culture medium was discarded and the cells were divided into the Blank group, negative control group, model group and drug-treated group. After 4 hours of pretreatment, the culture medium of the drug-treated group was discarded and the cells were treated with 100 μM TBHP (or 80 μM Aβ) 25-35 ) was replaced with the drug-containing medium (positive drug or target compound). After 24 hours of incubation, the medium was discarded and 100 μL of medium containing 10% CCK-8 working solution was added to each well. The cells were incubated at 37°C for 1.5 hours. Finally, the absorbance (OD) was measured at 450 nm using a multifunctional microplate reader to calculate cell viability. Each experiment was repeated three times, and data are presented as mean ± SD.

[0253] 2. Experimental Results

[0254] Oxidative stress is an important pathological indicator of neurodegenerative diseases, and cells in the nervous system are particularly susceptible to oxidative stress-mediated damage. TBHP is widely used as an inducer of oxidative stress in vitro. Therefore, the neuroprotective activity of the target compound against TBHP-induced oxidative stress was evaluated in SH-SY5Y cells. Figure 1 As shown in Figure 3, after 24 hours of incubation, the cell viability of SH-SY5Y cells exposed to 100 μM TBHP decreased to 33.89%. However, the compound group was able to significantly improve the cell viability of SH-SY5Y cells at 20 μM. Based on the above results, the compound JD-5 (GSK-3β, IC 50 =3.54) and JD-7 (GSK-3β, IC 50 =2.47) to conduct further research.

[0255] The experimental results are as follows Figure 2 and Figure 3 As shown in the results, after 24 hours of induction with 80μM Aβ25-35, the cell survival rate dropped to about 47.52%, which was significantly different from the normal control group (P<0.001); after 4 hours of protection with the target compounds JD-5 and JD-7 at different concentrations, the cell survival rate was significantly improved compared with the model group. JD-5 showed a good cell protection effect at a concentration of 10μM, and the cell protection effect was the strongest at a concentration of 20μM, with a cell survival rate of about 95.73%, which was significantly different from the Aβ 25-35 Compared with the induced group, there was a statistically significant difference (P<0.0001). Similarly, JD-7 had a strong cell protection at a concentration of 20μM, with a cell survival rate of about 94.47%, which was comparable to Aβ 25-35 There was a statistically significant difference compared with the induced group (P<0.0001).

[0256] From Examples 27-29, it can be seen that the compounds proposed in the present invention have good inhibitory activity against GSK-3β; the cytotoxicity test experiment shows that the compounds have no obvious cytotoxicity to SH-SY5Y cell line at 48h; TBHP and Aβ 25-35 In the induced SH-SY5Y cell model, the compound had a protective effect on neurons.

[0257] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An iridoid compound, characterized in that: The iridoid compound is a compound represented by the following general structural formula or a pharmaceutically acceptable salt thereof; JD-5 to JD-8 Wherein, R3 is selected from methyl or n-propyl, and n1 represents 1 or 2.

2. An iridoid compound, characterized in that: The iridoid compound is selected from any one of the following compounds or pharmaceutically acceptable salts thereof: 、 、 or .

3. A method for preparing an iridoid compound according to claim 1, characterized in that: The following steps are involved: Compound 28 was reacted with compound 22, EDCI·HCl, HOBt, triethylamine and dichloromethane at room temperature to obtain compounds as shown in JD-5 to JD-8. The reaction scheme is as follows: Wherein, R3 represents methyl or n-propyl, and n1 represents 1 or 2.

4. The method for preparing an iridoid compound according to claim 3, wherein: The compound 28 was prepared according to the following steps: Compound 4 phthalimide, compound 25, and triphenylphosphine were dissolved in anhydrous tetrahydrofuran under nitrogen protection, and 2 mL of anhydrous tetrahydrofuran-diluted DIAD was added dropwise to react at room temperature to generate compound 27; Compound 27 is reacted with anhydrous ethanol and 40%-50% hydrazine hydrate at room temperature to produce compound 28; the reaction scheme is as follows: 。 5. A pharmaceutical composition containing an iridoid compound, characterized in that: Contains the iridoid compound according to claim 1 or 2 and one or more pharmaceutically acceptable carriers or excipients.

6. Use of the iridoid compound according to claim 1 or 2 or the pharmaceutical composition containing the iridoid compound according to claim 5 in the preparation of a GSK-3β inhibitor drug or reagent.

7. Use of the iridoid compound according to claim 1 or 2 or the pharmaceutical composition containing the iridoid compound according to claim 5 in the preparation of a drug for treating Alzheimer's disease.

8. The iridoid compound according to claim 1 or 2 or the pharmaceutical composition containing the iridoid compound according to claim 5 in the preparation of a method for protecting TBHP and Aβ 25-35 The invention relates to a drug for inducing SH-SY5Y cell damage, characterized in that: The concentration of the iridoid compound is 20 μM.

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