A group of substituted benzopentagonal unsaturated heterocyclic compounds, and preparation method and application thereof

Abstract

The application discloses a group of substituted benzopentatomic unsaturated heterocyclic compounds and a preparation method and application thereof, and the structure of the substituted benzopentatomic unsaturated heterocyclic compounds is shown as formula I. The compound provided by the application can effectively reduce the expression of aging-related proteins P16, P21 and P27, and can reduce the proportion of SA-beta-gal positive cells. Therefore, the application provides a theoretical basis for further development of anti-cell aging drugs and provides a technical means for development of anti-aging products.

Description

Technical Field

[0001] This invention relates to a group of substituted benzo[a]-5-membered unsaturated heterocyclic compounds, their preparation methods, and applications. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Cellular senescence refers to the irreversible arrest of cells within a certain growth cycle. This arrest is accompanied by changes in cell morphology, metabolism, related genes, and epigenetic regulation, including increased expression of cell cycle inhibitory regulatory proteins, enhanced senescence-related β-galactosidase activity, and senescence-related chromatin aggregation. The cell cycle regulates cell differentiation and proliferation, as well as the decline in cell function and cellular senescence. Inhibiting cellular senescence may offer the potential to extend human lifespan.

[0003] Regulatory molecules such as cyclins play positive or negative roles in related molecular signaling pathways. Currently reported related proteins include P16, P21, and P27. P16's involvement in cellular senescence is mainly related to the Rb signaling pathway. P16 inhibits the phosphorylation of Rb protein by the cyclin-CDK complex, thereby inhibiting the binding of Rb protein to E2F transcription factors and suppressing the transcription of DNA replication-related genes. The Rb signaling pathway inhibits CDK activity, inducing cell cycle arrest in the G1 phase. This transcriptional process is crucial for the transition of stem cell cycles from G1 to S phase. P21 is a marker protein of senescence. Increased p21 expression induced by p53-activated transcription can significantly lead to cellular senescence; p21 is one of the important signaling molecules promoting cellular senescence. After p21 binds to its binding site on CDK, it forms a p21-cvclin-CDK-PCNA complex with proliferating cell nuclear antigen (PCNA). This complex inhibits the binding of CDK and PCNA to other molecules, thus inactivating them. The binding of PCNA and p21 also inhibits complex elongation; once the complex detaches from the template, it inhibits PCNA-dependent DNA synthesis. p21 has a dual role in inhibiting cell entry into S phase. p27 plays a negative regulatory role in the cell cycle; its expression level and distribution during the cell cycle are regulated by multiple mechanisms. p27 inhibits the activity of the CE-CDK2 complex; the binding of p27 to p21 and the CD-CDK complex prevents p27 from binding to CE-CDK2. After cell cycle arrest induced by p16, the binding of p27 to CE-CDK2 inhibits the activity of CE-CDK2 D, thereby inhibiting cell cycle progression. PTEN has a negative regulatory effect on the cell cycle, accelerating differentiation and arresting proliferation. In many senescent cells, PTEN activation leads to decreased AKT phosphorylation and upregulation of p27 expression, ultimately resulting in cell arrest at the G0 / G1 phase. p27 is a target molecule of transforming growth factor-β (TGF-β), which can significantly attenuate the effects of cyclin D-CDK, including cyclin D-CDK4. Overexpression of p27 will cause cell arrest in the G1 phase. SKP2 can induce cellular senescence, a process independent of stem cell n53 or p16-related signaling pathways. Decreased or inactivated SKP2 expression is usually accompanied by upregulation of n27 and FoxO3a expression, promoting cellular senescence. Inactivation of the senescence stimulant vonhippel lindau tumor suppressor (VHL) can cause a decrease in SKP2 expression, which in turn increases p27 expression, thereby inducing cellular senescence.The co-inactivation of the phosphatase and tensin homolog Pten protein, both deleted on chromosome 10, and SKP2 can lead to increased p27 expression, inducing cellular senescence. Inhibiting the expression of senescence-related proteins P16, P21, and P27 counteracts cellular senescence, ultimately extending lifespan.

[0004] During cellular senescence, the activity of senescence-associated β-galactosidase (SA-β-gal) increases significantly. Therefore, SA-β-gal activity is often measured to distinguish normal cells from senescent cells. Currently, SA-β-gal detection is frequently used as a marker of cellular senescence after cells have been treated with different conditions or compounds. β-galactosidase is a class of enzymes that hydrolyze β-galactosidic bonds. Enzymes from different sources have different characteristics, such as their optimal pH. Based on these characteristics, methods for detecting SA-β-gal can be designed. SA-β-gal is present in lysosomes of cells. Lysosomes are intracellular digestive organs, and in their resting state, their pH is 4.5–5.0, where β-gal can exert a catalytic effect. Senescent cells still express enzyme activity at pH 6.0, which is called SA-β-gal and is now commonly used as a biological marker of senescent cells.

[0005] IL-10 is a multifunctional negative regulator, primarily produced by Th2 cells, activated B cells, monocytes, and macrophages. It participates in the biological regulation of various cells, including immune cells, inflammatory cells, and tumor cells, playing a crucial role in autoimmune diseases, severe infectious diseases, tumors, and transplant immunology. Furthermore, IL-10 is a key pathway in combating inflammation. It combats inflammation by blocking the metabolism of immune cells and by clearing damaged mitochondria, the cell's electrical engines. The accumulation of this cellular damage can gradually develop into inflammation, making it important in the treatment of aging and other degenerative diseases. Summary of the Invention

[0006] One of the objectives of this invention is to provide a group of substituted benzo[a] pentane unsaturated heterocyclic compounds or their pharmaceutical salts.

[0007] Another object of the present invention is to provide a method for preparing the substituted benzo[a] pentane unsaturated heterocyclic compound.

[0008] Another object of the present invention is to provide compositions comprising the substituted benzo[a] pentane unsaturated heterocyclic compound or its pharmaceutical salt or ester.

[0009] Another object of the present invention is to provide a pharmaceutical composition, pharmaceutical preparation or cosmetic having the substituted benzo[a] pentane unsaturated heterocyclic compound as an active ingredient, and the use of the pharmaceutical composition in the treatment or prevention of aging diseases.

[0010] To achieve the above objectives, the present invention employs the following technical means:

[0011] First, this invention proposes a substituted benzo[a]-5-membered unsaturated heterocyclic compound having the structure shown in Formula I, or a pharmaceutical salt thereof:

[0012]

[0013] Where X is O or S;

[0014] R1 and R2 are each independently selected from the group consisting of H and C1-C6 alkoxy groups; or R1 and R2 are connected together by carbon, oxygen or nitrogen to form a five- to seven-membered ring structure;

[0015] Q is selected from -O(CH2) n NR a R b -N(CH2) n CR s COOH, -N(CH2) n NR a R b -N(CH2) m1 C(O)O(CH2) m2 NR a R b And -N(CH2) m1 C(O)N(CH2) m2 NR a R b The group consisting of;

[0016] R3 represents one or more substituents, independently selected from the group consisting of H, hydroxyl, C1-C6 alkyl, 1-3 halogenated C1-C6 alkyl and R5 substituted phenyl;

[0017] R4 represents one or more substituents, independently selected from the group consisting of H, hydroxyl, halogen and C1-C6 alkyl groups;

[0018] R5 is selected from the group consisting of H, C1-C6 alkyl groups, and benzyl groups;

[0019] R a R b Each independently represents a C1-C6 alkyl, C1-C6 alkoxy, or R a Rb The rings are linked together by carbon, oxygen, or nitrogen to form a five- to seven-membered ring structure or a five- to seven-membered ring structure with one or more substituents R6; R6 is independently selected from the group consisting of hydroxyl, halogen, and C1-C6 alkyl groups;

[0020] n is 1, 2, 3, 4, 5 or 6;

[0021] m1 is 1, 2, or 3;

[0022] m2 can be 1, 2, 3 or 4.

[0023] Preferably, the alkoxy group of C1-C6 is methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexyloxy, or isohexyloxy.

[0024] Preferably, the C1-C6 alkyl group is methyl, ethyl, isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl or isohexyl;

[0025] Preferably, R1 and R2 are connected together by oxygen to form a 1,3-dioxocyclopentane structure.

[0026] Preferably, the R a R b The resulting five- to seven-membered ring structures are piperazine rings, morpholine rings, piperidine rings, tetrahydropyrrole rings, or azaheptan rings.

[0027] Among them, the preferred one is -NR a R b is -N(CH3)2, -N(CH2 CH3)2, -N(CH2CH2CH3)2,

[0028] Preferably, the substituted benzo[a] 5-membered unsaturated heterocyclic compound is selected from the following compounds:

[0029] Compound 1: 2-(diethylamino)ethyl-6-methoxybenzofuran-2-carboxylate;

[0030] Compound 2: 2-(pyrrolidone-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate;

[0031] Compound 3: 2-(piperidin-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate;

[0032] Compound 4: 2-morpholinoethyl-6-methoxybenzofuran-2-carboxylate;

[0033] Compound 5: 2-(4-methylpiperazin-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate;

[0034] Compound 6: (6-methoxybenzofuran-2-carbonyl)glycine;

[0035] Compound 7: (6-methoxybenzofuran-2-carbonyl)-D-alanine;

[0036] Compound 8: (6-methoxybenzofuran-2-carbonyl)-D-valine;

[0037] Compound 9: (6-methoxybenzofuran-2-carbonyl)-D-leucine;

[0038] Compound 10: (6-methoxybenzofuran-2-carbonyl)-D-phenylalanine;

[0039] Compound 11: 2-(dimethylamino)ethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0040] Compound 12: N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-6-methoxybenzofuran-2-carboxamide;

[0041] Compound 13: 6-methoxy-N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)benzofuran-2-carboxamide;

[0042] Compound 14: 6-methoxy-N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)benzofuran-2-carboxamide;

[0043] Compound 15: (6-methoxybenzofuran-2-yl)(4-methylpiperazin-1-yl)methyl ketone;

[0044] Compound 16: 6-methoxy-N-(2-(pyrrolidone-1-yl)ethyl)benzofuran-2-carboxamide;

[0045] Compound 17: 6-methoxy-N-(2-(piperidin-1-yl)ethyl)benzofuran-2-carboxamide;

[0046] Compound 18: 6-Methoxy-N-(2-morpholinoethyl)benzofuran-2-carboxamide;

[0047] Compound 19: 6-Methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzofuran-2-carboxamide;

[0048] Compound 20: N-(3-(diethylamino)propyl)-6-methoxybenzofuran-2-carboxamide;

[0049] Compound 21: N-(4-(dimethylamino)butyl)-6-methoxybenzofuran-2-carboxamide;

[0050] Compound 22: N-(4-(diethylamino)butyl)-6-methoxybenzofuran-2-carboxamide;

[0051] Compound 23: 6-Methoxy-N-(2-(piperazin-1-yl)ethyl)benzofuran-2-carboxamide;

[0052] Compound 24: N-(2-(diethylamino)ethyl)-6-methoxybenzofuran-2-carboxamide;

[0053] Compound 25: (4-hydroxypiperidin-1-yl)(6-methoxybenzofuran-2-yl)methyl ketone;

[0054] Compound 26: (6-methoxybenzofuran-2-yl)(4-(trifluoromethyl)piperidin-1-yl)methyl ketone;

[0055] Compound 27: N-(3-(dimethylamino)propyl)-6-methoxybenzofuran-2-carboxamide;

[0056] Compound 28: (4-(4-fluorophenyl)piperidin-1-yl)(6-methoxybenzofuran-2-yl)methyl ketone;

[0057] Compound 29: (6-methoxybenzofuran-2-yl)(4-(pyridin-4-yl)piperidin-1-yl)methyl ketone;

[0058] Compound 30: N-(4-hydroxycyclohexyl)-6-methoxybenzofuran-2-carboxamide;

[0059] Compound 31: 2-(diethylamino)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate;

[0060] Compound 32: 2-(pyrrolidone-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate;

[0061] Compound 33: 2-(piperidin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate;

[0062] Compound 34: 2-morpholinoethyl-6-methoxybenzo[b]thiophene-2-carboxylate;

[0063] Compound 35: 2-(4-methylpiperazin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate;

[0064] Compound 36: (6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0065] Compound 37: 6-Methoxybenzo[b]thiophene-2-carbonyl)-D-alanine;

[0066] Compound 38: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-valine;

[0067] Compound 39: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-leucine;

[0068] Compound 40: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-phenylalanine;

[0069] Compound 41: 2-(dimethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0070] Compound 42: N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0071] Compound 43: 6-methoxy-N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide;

[0072] Compound 44: 6-methoxy-N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide;

[0073] Compound 45: (6-methoxybenzothiophene-2-yl)(4-methylpiperazin-1-yl) methyl ketone;

[0074] Compound 46: 6-methoxy-N-(2-(pyrrolidone-1-yl)ethyl)benzo[b]thiophene-2-carboxamide;

[0075] Compound 47: 6-methoxy-N-(2-(piperidin-1-yl)ethyl)benzo[b]thiophene-2-formamide;

[0076] Compound 48: 6-Methoxy-N-(2-morpholinoethyl)benzo[b]thiophene-2-carboxamide;

[0077] Compound 49: 6-methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide;

[0078] Compound 50: N-(3-(diethylamino)propyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0079] Compound 51: N-(4-(dimethylamino)butyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0080] Compound 52: N-(4-(diethylamino)butyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0081] Compound 53: 6-methoxy-N-(2-(piperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide;

[0082] Compound 54: N-(2-(diethylamino)ethyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0083] Compound 55: (4-hydroxypiperidin-1-yl)(6-methoxybenzothiophen-2-yl) methyl ketone;

[0084] Compound 56: (6-methoxybenzo[b]thiophen-2-yl)(4-(trifluoromethyl)piperidin-1-yl)methyl ketone;

[0085] Compound 57: N-(3-(dimethylamino)propyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0086] Compound 58: (4-(4-fluorophenyl)piperidin-1-yl)(6-methoxybenzothiophene-2-yl)methyl ketone;

[0087] Compound 59: (6-methoxybenzothiophene-2-yl)(4-(pyridin-4-yl)piperidin-1-yl)methyl ketone;

[0088] Compound 60: N-(4-hydroxycyclohexyl)-6-methoxybenzo[b]thiophene-2-carboxamide;

[0089] Compound 61: 2-(dimethylamino)ethyl 6-methoxybenzofuran-2-carboxylate;

[0090] Compound 62: 2-(piperazin-1-yl)ethyl 6-methoxybenzofuran-2-carboxylate;

[0091] Compound 63: 3-(dimethylamino)propyl6-methoxybenzofuran-2-carboxylate;

[0092] Compound 64: 3-(diethylamino)propyl 6-methoxybenzofuran-2-carboxylate;

[0093] Compound 65: 3-(pyrrolidone-1-yl)propyl6-methoxybenzofuran-2-carboxylate;

[0094] Compound 66: 3-(piperidin-1-yl)propyl 6-methoxybenzofuran-2-carboxylate;

[0095] Compound 67: 3-morpholinopropyl-6-methoxybenzofuran-2-carboxylate;

[0096] Compound 68: 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzofuran-2-carboxylate;

[0097] Compound 69: 3-(piperidin-4-yl)propyl-6-methoxybenzofuran-2-carboxylate;

[0098] Compound 70: 4-(dimethylamino)butyl-6-methoxybenzofuran-2-carboxylate;

[0099] Compound 71: 4-(diethylamino)butyl-6-methoxybenzofuran-2-carboxylate;

[0100] Compound 72: 4-(pyrrolidone-1-yl)butyl-6-methoxybenzofuran-2-carboxylate;

[0101] Compound 73: 4-(piperidin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate;

[0102] Compound 74: 4-morpholine n-butyl-6-methoxybenzofuran-2-carboxylate;

[0103] Compound 75: 4-(piperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate;

[0104] Compound 76: 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate;

[0105] Compound 77: 2-(diethylamino)ethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0106] Compound 78: 2-morpholinoethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0107] Compound 79: 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0108] Compound 80: 2-(piperidin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0109] Compound 81: 2-(piperazin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine;

[0110] Compound 82: 2-(dimethylamino)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate;

[0111] Compound 83: Ethyl 2-(piperazin-1-yl)6-methoxybenzo[b]thiophene-2-carboxylate;

[0112] Compound 84: 3-(dimethylamino)propyl6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0113] Compound 85: 3-(diethylamino)propyl6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0114] Compound 86: 3-(pyrrolidone-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0115] Compound 87: 3-(piperidin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0116] Compound 88: 3-morpholinopropyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0117] Compound 89: 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0118] Compound 90: 3-(piperidin-4-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0119] Compound 91: 4-(dimethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0120] Compound 92: 4-(diethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0121] Compound 93: 4-(pyrrolidone-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0122] Compound 94: 4-(piperidin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0123] Compound 95: 4-morpholinobutyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester;

[0124] Compound 96: 4-(piperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate;

[0125] Compound 97: 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate;

[0126] Compound 98: 2-(diethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0127] Compound 99: 2-morpholinoethyl (6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0128] Compound 100: 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0129] Compound 101: 2-(piperidin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0130] Compound 102: 2-(piperazin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine;

[0131] Compound 103: 2-(diethylamino)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxylate;

[0132] Compound 104: 2-(pyrrolidone-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxene-6-carboxylate;

[0133] Compound 105: 2-(piperidin-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxy-6-carboxylate;

[0134] Compound 106: 2-morpholinoethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxocyclo-6-carboxylate;

[0135] Compound 107: 2-(4-methylpiperazin-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxane-6-carboxylate;

[0136] Compound 108: (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxy-6-carbonyl)glycine;

[0137] Compound 109: (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-alanine;

[0138] Compound 110: (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-valine;

[0139] Compound 111: (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-leucine;

[0140] Compound 112: (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxy-6-carbonyl)-d-phenylalanine;

[0141] Compound 113: 2-(dimethylamino)ethyl(thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)glycine ester;

[0142] Compound 114: N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide;

[0143] Compound 115: N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide;

[0144] Compound 116: N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide;

[0145] Compound 117: N-(2-(pyrrolidone-1-yl)ethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxene-6-carboxamide.

[0146] Furthermore, the present invention also provides a method for preparing the substituted benzo[a]-5-membered unsaturated heterocyclic compounds. Although the compounds of the present invention can be prepared by the following method, the conditions of the method, such as reactants, solvents, acids, bases, amounts of compounds used, reaction temperatures, and reaction times, are not limited to the following description. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known to those skilled in the art, and those skilled in the art can easily perform the above combinations.

[0147] The method for preparing the substituted benzo[a]-5-membered unsaturated heterocyclic compound of the present invention is synthesized via the following route:

[0148] Route 1: Synthetic routes for compounds 1-30 and 61-81

[0149]

[0150] Reaction conditions: (a) BrCH2COOC2H5, dry DMF, anhydrous K2CO3, 0℃, 1h, 90℃, 18h; (b) 0.1NNaOH, H2O, dioxane, 40℃; (c) HOB T, EDCI, dry DMF, rt; (d) HOBT, EDCI, dry DMF, rt;

[0151] Route 2: Synthetic routes for compounds 31-60 and 82-102

[0152]

[0153] Reaction conditions: (a) 0.1N NaOH, H2O, dioxane, 40℃, 1h; (b) SOCl2, DMF, anhydrous toluene, 80℃, 3h; (c) Et3N, anhydrous toluene, 0℃, 1h;

[0154] Route 3: Synthetic route for compounds 103-117

[0155]

[0156] Reaction conditions: (a) 0.1N NaOH, H2O, dioxane, 40℃, 1h; (b) SOCl2, DMF, anhydrous toluene, 80℃, 3h; (c) Et3N, anhydrous toluene, 0℃, 1h.

[0157] Furthermore, the present invention also proposes a pharmaceutical composition comprising the substituted benzo[a]penta-unsaturated heterocyclic compound described in this invention or a pharmaceutical salt thereof, and optionally, one or more pharmaceutically acceptable carriers or excipients.

[0158] Furthermore, the present invention also proposes the use of the aforementioned substituted benzo[a]penta-unsaturated heterocyclic compounds or their pharmaceutical salts in the preparation of products for treating or preventing aging, and the use of the aforementioned substituted benzo[a]penta-unsaturated heterocyclic compounds or their pharmaceutical salts in the preparation of medicaments for clearing or reducing senescent cells.

[0159] Preferably, the products include, but are not limited to, pharmaceuticals and cosmetics.

[0160] Terminology Definition

[0161] The terminology used in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The nomenclature used herein and the laboratory procedures described herein in organic chemistry, medicinal chemistry, and biology are well-known and commonly used in the art. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0162] As used in the description of embodiments of the invention and the appended claims, the singular forms of “a,” “an,” “the,” and “its” are used to refer to the singular and plural of the article, unless the context clearly indicates otherwise. For example, a compound comprises one or more compounds.

[0163] As used in this article, “and / or” means any and all possible combinations of one or more of the related listed items.

[0164] As used herein, the term “disease” or “patient” refers to any change in physical condition or organ that interrupts or interferes with the functioning of the organ and / or causes symptoms.

[0165] As used herein, the term "treatment" aims to alleviate or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of a compound or its pharmaceutically acceptable salt, isomer, polymorph, solvate, isotopically labeled compound, metabolite, or prodrug, or a pharmaceutical composition thereof, is received and one or more indications and symptoms show an observable and / or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0166] As used herein, the term "subject" may refer to a patient or other animal that receives the compositions of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described herein, and in particular to humans and mammals.

[0167] As used herein, "halogenated" or "halogen" can be fluorine, chlorine, bromine or iodine, and is preferably F, Cl or Br.

[0168] As used herein, "pharmaceutical salt" refers to a salt that retains the desired biological activity of the target compound and exhibits minimal undesirable toxicological effects. When the substituted benzo[a]-5-membered unsaturated heterocyclic compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of the compound of the present invention with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases, wherein the inorganic base salts include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, etc. The organic non-toxic base salts include salts of primary amines, secondary amines, and tertiary amines, including substituted amines and cyclic amines. Examples include: N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; and organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, terpentinic acid, 2-hydroxyethanesulfonic acid, itaconic acid, and aminosulfonic acid. Trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, and salts formed from sodium, potassium, magnesium, lithium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine.

[0169] As used herein, a "pharmaceutical composition" comprises a therapeutically effective amount of the substituted benzo[a]pentaunsaturated heterocyclic compound or its pharmaceutical salt or ester, solvate, isomer, polymorph, isotopically labeled compound, metabolite or prodrug, and one or more pharmaceutically acceptable carriers, and is prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, tinctures, decoctions, lozenges, mixtures, suppositories, injections, inhalants or sprays, etc. Preferably, the pharmaceutical composition contains 0.1%-99.5% by weight of the substituted benzo[a]pentaunsaturated heterocyclic compound or its pharmaceutical salt of the present invention as the active ingredient, more preferably 0.5%-99.5% by weight of the active ingredient.

[0170] As used herein, “pharmaceutically acceptable carriers or excipients” include: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that some pharmaceutically acceptable excipients may be used for more than one function and for alternative functions, depending on the amount of said excipient present in the formulation and what other ingredients are present in the formulation. For example, when used orally, it can be formulated into oral preparations such as tablets, capsules, granules, and pills, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose and polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, and leucine), stabilizers (methylparaben, propylparaben, etc.), and flavoring agents (e.g., commonly used sweeteners, acidulants, and flavorings, etc.). When used parenterally, the drug can be formulated as an injection, comprising a sterile powder for injection and a solvent for injection. The carrier or excipient used may contain sterile water, Ringer's solution, and isotonic sodium chloride solution. Suitable adjuvants such as antioxidants, buffers, and antibacterial agents may also be added depending on the properties of the drug. When used for rectal administration, the drug can be formulated as suppositories, etc. When used for pulmonary administration, the drug can be formulated as an inhaler or spray, etc. Many resources are available to those skilled in the art describing pharmaceutically acceptable excipients and which can be used to select suitable pharmaceutically acceptable excipients, such as the Remington Pharmacy Encyclopedia, the Chinese Pharmaceutical Yearbook, and Pharmaceutics.

[0171] The substituted benzo[a]-5-membered unsaturated heterocyclic compounds of this invention can be administered by any suitable method known in the art, such as oral, intravenous, intraperitoneal, intramuscular, local, transdermal, ocular, nasal, inhalation, subcutaneous, intramuscular, sublingual, sublingual, or rectal administration. The compounds described above can be administered at any amount from 1 μg to 2000 mg / kg of the subject's body weight, for example, at doses of 1 μg to 1000 mg / kg of body weight / day, 50 μg to 1000 mg / kg of body weight / day, 100 μg to 1000 mg / kg of body weight / day, 1 to 500 mg / kg of body weight / day, 2 to 200 mg / kg of body weight / day, or 5 to 100 mg / kg of body weight / day. In some embodiments of this invention, the compounds described above can be administered four times daily, three times daily, twice daily, once daily, once every two days, once weekly, or at other intervals, optionally repeating the dosing regimen as appropriate weekly or monthly. In this invention, the dosage of the compound can be adjusted according to factors such as the severity of the patient's or subject's condition, age, weight, gender, administration method, and course of treatment.

[0172] The substituted benzo[a]-5-membered unsaturated heterocyclic compounds of this invention can be used alone or in combination with one or more other active ingredients for the treatment, prevention, inhibition, or improvement of diseases or symptoms, wherein the combination of drugs is safer or more effective than the use of any single drug alone. Such other drugs can be administered simultaneously or sequentially with the compounds of this invention in the usual routes and amounts. When the compounds of this invention are used simultaneously with one or more other drugs, pharmaceutical compositions containing the other drug and the compounds of this invention in a unit dosage form are preferred, particularly in combination with pharmaceutically acceptable carriers. However, combination therapy may also include treatment with the compounds of this invention and one or more other drugs administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the compounds of this invention and the other active ingredients can be used at lower doses than when used alone. Therefore, in addition to the compounds of this invention, the pharmaceutical compositions of this invention also include those compositions containing one or more other active ingredients.

[0173] Compared with the prior art, the beneficial effects of the present invention are:

[0174] The substituted benzo[a]penta-unsaturated heterocyclic compounds provided in this invention have been verified to effectively reduce the expression of aging-related proteins P16, P21, and P27, and to decrease the proportion of SA-β-gal positive cells. This invention lays a theoretical foundation for the further development of anti-cellular aging drugs and provides technical means for the development of anti-aging products. Attached Figure Description

[0175] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0176] Figure 1 A schematic diagram illustrating the expression of cell senescence-related proteins p16, p21, and p27 after compound administration;

[0177] Figure 2 Schematic diagram of β-galactosidase staining for cell senescence 48 h after compound administration;

[0178] Figure 3 This is a mouse survival cycle curve diagram of the present invention;

[0179] Figure 4 This is a graph showing the concentration of the cytokine IL-10 in mouse serum during the present invention. Detailed Implementation

[0180] The present invention is described below with reference to specific embodiments. These embodiments are not intended to limit the scope of the invention, but rather to provide guidance to those skilled in the art for the preparation and use of the compounds, compositions, and methods of the present invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0181] The chemical names of the compounds described in this application are generally derived from ChemDraw Ultra (Chambridge Soft) and are generated / or generally follow the principles of IUPAC nomenclature.

[0182] Some of the compounds in this embodiment were synthesized via the following route:

[0183] Route 1: Synthetic route for compounds 1-30 and 61-81

[0184]

[0185] Reaction conditions: (a) BrCH2COOC2H5, dry DMF, anhydrous K2CO3, 0℃, 1h, 90℃, 18h; (b) 0.1NNaOH, H2O, dioxane, 40℃; (c) HOB T, EDCI, dry DMF, rt; (d) HOBT, EDCI, dry DMF, rt.

[0186] Route 2: Synthetic routes for compounds 31-60 and 82-102

[0187]

[0188] Reaction conditions: (a) 0.1N NaOH, H2O, dioxane, 40℃, 1h; (b) SOCl2, DMF, anhydrous toluene, 80℃, 3h; (c) Et3N, anhydrous toluene, 0℃, 1h.

[0189] Route 3: Synthetic route for compounds 103-117

[0190]

[0191] Reaction conditions: (a) 0.1N NaOH, H2O, dioxane, 40℃, 1h; (b) SOCl2, DMF, anhydrous toluene, 80℃, 3h; (c) Et3N, anhydrous toluene, 0℃, 1h.

[0192] Example 1

[0193]

[0194] 2-(diethylamino)ethyl-6-methoxybenzofuran-2-carboxylate (1)

[0195] To a stirred solution of 6-methoxybenzofuran-2-carboxylic acid (0.19 g, 1.0 mmol, 1.00 equivalence) in DCM (10 mL), HOBT (0.15 g, 1.1 mmol, 1.10 equivalence) and EDCI (0.21 g, 1.1 mmol, 1.1 equivalence) were added, and the mixture was stirred at room temperature for 1 h. Then, 2-(diethylamino)ethane-1-ol (0.082 g, 1.1 mmol, 1.10 equivalence) was added, and the reaction was carried out at room temperature for 10 h. The mixture was diluted with H₂O (40 mL) and extracted with EA (20 mL × 3). The organic phase was washed with saturated NaCl solution (10 mL × 3), dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified on a silica gel column (DCM:MeOH = 10:1) to give a white powder 1 (0.21 g, 0.87 mmol, 87%), mp: 43.5–43.9 °C. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.2, 2.2 Hz, 1H), 6.98–6.90 (m, 2H), 4.71 (t, J = 5.3 Hz, 2H), 3.83 (s, 3H), 3.52 (t, J = 5.3 Hz, 2H), 2.89 (q, J = 6.9 Hz, 4H), 1.05 (t, J = 6.9 Hz, 6H).

[0196] Example 2

[0197]

[0198] 2-(pyrrolidone-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate (2)

[0199] Brown solid 2 was obtained from 2-(pyrrolidone-1-yl)ethane-1-ol according to the preparation method of compound 1, with a yield of 35% and mp: 37.2-38.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.79 (d, J=2.2Hz, 0H), 7.65 (dd, J=8.1, 2.1Hz, 0H), 6.98-6.90 (m, 1H), 4.71 (t, J=6.0Hz, 1H), 3.83 (s, 1H), 3.25-3.19 (m, 2H), 3.15 (t, J=6.0Hz, 1H), 1.96 (p, J=2.1Hz, 2H).

[0200] Example 3

[0201]

[0202] 2-(piperidin-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate (3)

[0203] Using 2-(piperidin-1-yl)ethane-1-ol as a starting material, a yellow solid 3 was obtained according to the preparation method of compound 1, with a yield of 38% and mp: 39.4-40.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.1, 2.1Hz, 0H), 6.98-6.90 (m, 1H), 4.71 (t, J =6.0Hz, 1H), 3.83 (s, 1H), 3.15 (t, J = 5.9Hz, 1H), 2.45-2.39 (m, 2H), 1.53-1.38 (m, 3H).

[0204] Example 4

[0205]

[0206] 2-Morpholinyl-6-methoxybenzofuran-2-carboxylate (4)

[0207] Using 2-morpholinoethanol-1-ol as a raw material, a pale yellow solid 4 was obtained according to the preparation method of compound 1, with a yield of 53% and mp: 58.7-59.8℃. 1H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.3, 2.2Hz, 0H), 6.98-6.90 (m, 1H), 4.71 (t, J=6.0 Hz, 1H), 3.83 (s, 1H), 3.72 (t, J=4.7Hz, 2H), 3.15 (t, J=5.9Hz, 1H), 2.54-2.42 (m, 2H).

[0208] Example 5

[0209]

[0210] 2-(4-methylpiperazin-1-yl)ethyl-6-methoxybenzofuran-2-carboxylate (5)

[0211] Using 2-(4-methylpiperazin-1-yl)ethane-1-ol as a starting material, a brown oily substance 5 was obtained according to the preparation method of compound 1, with a yield of 29%. 1 H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.3, 2.2Hz, 0H), 6.98-6.90 (m, 1H), 4.71 (t, J=6.0Hz, 1H), 3.83 (s, 1H), 3.15 (t, J=5.9Hz, 1H), 2.57-2.48 (m, 4H).

[0212] Example 6

[0213]

[0214] (6-Methoxybenzofuran-2-carbonyl)glycine(6)

[0215] Using glycine as a raw material, brown solid 6 was obtained according to the preparation method of compound 1, with a yield of 35% and mp: 67.2-68.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.2Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H ), 7.00 (t, J=6.4Hz, 1H), 6.97-6.90 (m, 2H), 3.84 (d, J=6.4Hz, 2H), 3.83 (s, 4H).

[0216] Example 7

[0217]

[0218] (6-Methoxybenzofuran-2-carbonyl)-D-alanine (7)

[0219] Using D-alanine as a raw material, a white solid 7 was obtained according to the preparation method of compound 1, with a yield of 41% and mp: 31.4-32.58℃. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H), 6.98-6 .90 (m, 2H), 6.35 (d, J=9.9Hz, 1H), 4.46 (dq, J=9.9, 6.2Hz, 1H), 3.83 (s, 3H), 1.41 (s, 2H).

[0220] Example 8

[0221]

[0222] (6-Methoxybenzofuran-2-carbonyl)-D-valine (8)

[0223] Using D-valine as a raw material, a white solid 8 was obtained according to the preparation method of compound 1, with a yield of 37% and mp: 67.6-68.4℃. 1 H NMR (500MHz, Chloroform-d) δ9.72 (s, 1H), 7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.1, 2.1Hz, 1H), 6.98-6.90 (m, 2H), 6.35 (d, J= 10.8Hz, 1H), 4.26 (ddp, J=11.1, 6.8, 1.5Hz, 1H), 3.83 (s, 2H), 2.16-2.04 (m, J=6.6Hz, 1H), 0.93 (ddd, J=6.5, 4.9, 1.5Hz, 6H).

[0224] Example 9

[0225]

[0226] (6-Methoxybenzofuran-2-carbonyl)-D-leucine (9)

[0227] Using D-leucine as a raw material, a pale yellow oil 9 was obtained according to the preparation method of compound 1, with a yield of 58%. 1H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.1, 2.1Hz, 1H), 6.98-6.90 (m, 2H), 6.35 ( d, J=9.7Hz, 1H), 4.40 (dt, J=9.7, 7.0Hz, 1H), 3.83 (s, 2H), 1.76-1.55 (m, 3H), 0.93 (dd, J=6.4, 5.0Hz, 6H).

[0228] Example 10

[0229]

[0230] (6-Methoxybenzofuran-2-carbonyl)-D-phenylalanine (10)

[0231] Using D-phenylalanine as a raw material, a white solid 10 was obtained according to the preparation method of compound 1, with a yield of 49% and mp: 67.2-68.3℃. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.2Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H), 7.31-7.17 (m, 4H), 6.98-6.90 (m, 2H), 6.35 (d, J =10.4Hz, 1H), 4.78 (dt, J = 10.4, 6.9Hz, 1H), 3.83 (s, 2H), 3.17 (ddt, J = 14.1, 6.8, 0.8Hz, 1H), 2.92 (ddt, J = 14.1, 6.8, 0.8Hz, 1H).

[0232] Example 11

[0233]

[0234] 2-(dimethylamino)ethyl(6-methoxybenzofuran-2-carbonyl)glycine (11)

[0235] To a stirred solution of 6-methoxy-2,3-dihydrobenzofuran-2-carboxylic acid (0.25 g, 1.0 mmol, 1.00 equivalence) in DCM (10 mL), HOBT (0.15 g, 1.1 mmol, 1.10 equivalence) and EDCI (0.21 g, 1.1 mol, 1.10 equivalence 1.1 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Then, 2-(dimethylamino)ethane-1-ol (0.09 g, 1.1 mmol, 1.10 equivalence) was added, and the reaction was repeated at room temperature for 10 hours. The mixture was diluted with H₂O (40 mL) and extracted with EA (20 mL × 3). The organic phase was washed with saturated NaCl solution (10 mL × 3), dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified on a silica gel column (DCM:MeOH = 20:1) to give a white oily product, 11 (0.17 g, 0.54 mmol, 54%). ¹H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.3, 2.2 Hz, 1H), 7.03–6.90 (m, 3H), 4.35 (t, J = 6.8 Hz, 2H), 4.13 (d, J = 5.9 Hz, 2H), 3.83 (s, 2H), 3.33 (t, J = 6.7 Hz, 2H).

[0236] Example 12

[0237]

[0238] N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-6-methoxybenzofuran-2-carboxamide (12)

[0239] Using N1,N1-dimethylethane-1,2-diamine as a raw material, a white solid 12 was obtained according to the preparation method of compound 11, with a yield of 65% and mp: 45.2-46.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H), 7.00 (t, J=6.0Hz, 1H), 6.98-6.90 ( m, 2H), 6.26 (t, J=4.3Hz, 1H), 3.83 (s, 2H), 3.77 (d, J=5.9Hz, 2H), 3.35 (td, J=6.0, 4.2Hz, 2H), 2.46 (t, J=6.0Hz, 2H).

[0240] Example 13

[0241]

[0242] 6-Methoxy-N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)benzofuran-2-carboxamide (13)

[0243] Using 2-(pyrrolidone-1-yl)ethane-1-amine as a starting material, a white solid 13 was obtained according to the preparation method of compound 11, with a yield of 44% and mp: 36.9-38.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.1, 2.1Hz, 1H), 7.00 (t, J=6.0Hz, 1H), 6.98-6.90 (m, 2H), 6.26 (t, J=4.2Hz , 1H), 3.83 (s, 2H), 3.77 (d, J=5.9Hz, 2H), 3.14 (td, J=5.4, 4.3Hz, 2H), 2.67 (t, J=5.4Hz, 2H), 2.60 (td, J=3.6, 1.9Hz, 4H), 1.85-1.79 (m, 2H).

[0244] Example 14

[0245]

[0246] 6-Methoxy-N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)benzofuran-2-carboxamide (14)

[0247] Using 2-(piperidin-1-yl)ethane-1-amine as a starting material, a white solid 14 was obtained according to the preparation method of compound 11, yielding 51% yield, mp: 47.8-48.8℃. 1 H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.1, 2.1Hz, 0H), 7.03-6.90 (m, 1H), 6.26 (t, J=4.2Hz, 0H), 3.83 (s, 1H), 3.77 (d, J=5.9Hz, 1H), 3.14 (td, J=5.4, 4.2Hz, 1H), 2.67 (t, J=5.4Hz, 1H), 2.43-2.37 (m, 2H), 1.61-1.53 ​​(m, 2H), 1.46-1.38 (m, 1H).

[0248] Example 15

[0249]

[0250] (6-Methoxybenzofuran-2-yl)(4-methylpiperazin-1-yl)methyl ketone (15)

[0251] Using 1-methylpiperazine as a raw material, a white solid 15 was obtained according to the preparation method of compound 1, with a yield of 65% and mp: 44.2-45.5℃. 1 H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.3, 2.2Hz, 0H), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.66 (ddd, J=11.3, 6.2, 3.5Hz, 1 H), 3.27 (ddd, J=11.3, 6.2, 3.5Hz, 1H), 3.00 (ddd, J=11.3, 6.2, 3.5Hz, 1H), 2.72 (ddd, J=11.5, 6.1, 3.5Hz, 1H), 2.56 (s, 1H).

[0252] Example 16

[0253]

[0254] 6-Methoxy-N-(2-(pyrrolidone-1-yl)ethyl)benzofuran-2-carboxamide (16)

[0255] Using N-methyl-2-(pyrrolidone-1-yl)ethane-1-amine as a starting material, a white oily substance 16 was obtained according to the preparation method of compound 1, with a yield of 51%. 1 H NMR (500MHz, Chlorofom-d) δ7.65 (dd, J=8.1, 2.1Hz, 0H), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.54 (td, J=5.3, 4.4Hz, 1H), 2.67 (t, J=5.3Hz, 1H), 2.60 (td, J=3.6, 1.9Hz, 2H), 1.85-1.79 (m, 1H).

[0256] Example 17

[0257]

[0258] 6-Methoxy-N-(2-(piperidin-1-yl)ethyl)benzofuran-2-carboxamide (17)

[0259] Using 2-(piperidin-1-yl)ethane-1-amine as a starting material, a white oily substance 17 was obtained according to the preparation method of compound 1, with a yield of 55%. 1H NMR (500MHz, Chloroform-d) δ7.65 (dd, J=8.1, 2.1Hz, 0H), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.54 (td, J=5.3, 4.4Hz, 1H), 2.67 (t, J=5.3Hz, 1H), 2.43-2.37 (m, 2H), 1.57 (ddd, J=9.5, 6.1, 4.8Hz, 2H), 1.46-1.38 (m, 1H).

[0260] Example 18

[0261]

[0262] 6-Methoxy-N-(2-morpholinoethyl)benzofuran-2-carboxamide (18)

[0263] Using 2-morpholinoethanol-1-amine as a raw material, a pale yellow oil 18 was obtained according to the preparation method of compound 1, with a yield of 42%. 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 0H), 7.65 (dd, J=8.1, 2.1Hz, 0H), 6.98-6.90 (m, 1H), 3 .83 (s, 1H), 3.76-3.70 (m, 2H), 3.54 (td, J=5.3, 4.4Hz, 1H), 2.60 (t, J=5.3Hz, 1H), 2.51-2.45 (m, 2H).

[0264] Example 19

[0265]

[0266] 6-Methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzofuran-2-carboxamide (19)

[0267] Using 2-(4-methylpiperazin-1-yl)ethane-1-amine as a starting material, a white oil 19 was obtained according to the preparation method of compound 1, with a yield of 51%. ¹H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 2.3 Hz, OH), 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98–6.90 (m, ¹H), 3.83 (s, ¹H), 3.54 (td, J = 5.3, 4.4 Hz, ¹H), 2.67 (t, J = 5.3 Hz, ¹H), 2.57–2.48 (m, 4H).

[0268] Example 20

[0269]

[0270] N-(3-(diethylamino)propyl)-6-methoxybenzofuran-2-carboxamide (20)

[0271] Using N1,N1-diethylpropane-1,3-diamine as a starting material, a brown oily substance 20 was obtained according to the preparation method of compound 1, with a yield of 72%. ¹H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.3, 2.2 Hz, 1H), 7.08 (d, J = 10.4 Hz, 0H), 6.98–6.90 (m, 2H), 3.83 (s, 2H), 3.08 (td, J = 5.9, 5.1 Hz, 2H), 3.01 (q, J = 6.9 Hz, 4H), 2.53 (t, J = 6.0 Hz, 2H), 1.88–1.80 (m, 1H), 1.02 (t, J = 6.9 Hz, 5H).

[0272] Example 21

[0273]

[0274] N-(4-(dimethylamino)butyl)-6-methoxybenzofuran-2-carboxamide (21)

[0275] Using N1,N1-dimethylbutane-1,4-diamine as a starting material, a white oil 21 was obtained according to the preparation method of compound 1, with a yield of 61%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98–6.90 (m, ¹H), 3.83 (s, ¹H), 3.48–3.42 (m, ¹H), 2.51 (t, J = 5.7 Hz, ¹H), 1.61–1.52 (m, ¹H), 1.45–1.37 (m, ¹H).

[0276] Example 22

[0277]

[0278] N-(4-(diethylamino)butyl)-6-methoxybenzofuran-2-carboxamide (22)

[0279] Using N1,N1-diethylbutane-1,4-diamine as a starting material, a white solid 22 was obtained according to the preparation method of compound 1, with a yield of 49% and mp: 48.0-49.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.48-3.42 (m, 1H), 3.05-2.97 (m, 3H), 1.74-1.64 (m, 1H), 1.41 (tt, J = 8.1, 5.4 Hz, 1H), 1.02 (t, J = 6.9 Hz, 3H).

[0280] Example 23

[0281]

[0282] 6-Methoxy-N-(2-(piperazin-1-yl)ethyl)benzofuran-2-carboxamide (23)

[0283] Using 2-(piperazin-1-yl)ethane-1-amine as a starting material, a white solid 23 was obtained according to the preparation method of compound 1, in 37% yield, mp: 48.0-49.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.54 (td, J = 5.3, 4.4 Hz, 1H), 2.92-2.86 (m, 2H), 2.67 (t, J = 5.3 Hz, 1H), 2.57-2.51 (m, 2H).

[0284] Example 24

[0285]

[0286] N-(2-(diethylamino)ethyl)-6-methoxybenzofuran-2-carboxamide (24)

[0287] Using N1,N1-diethylethane-1,2-diamine as a starting material, a white solid 24 was obtained according to the preparation method of compound 1, with a yield of 56%, mp: 88.1-89.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.2, 2.2 Hz, OH), 6.98-6.90 (m, 1H), 3.83 (s, 1H), 3.54 (q, J = 4.5 Hz, 1H), 2.67 (t, J = 4.6 Hz, 1H), 2.46 (q, J = 6.9 Hz, 2H), 1.09 (t, J = 6.9 Hz, 3H).

[0288] Example 25

[0289]

[0290] (4-Hydroxypiperidin-1-yl)(6-methoxybenzofuran-2-yl)methyl ketone (25)

[0291] Using piperidine-4-ol as a starting material, white solid 25 was obtained according to the preparation method of compound 1, with a yield of 62% and mp: 69.0-70.8℃. 1H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.4Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H), 6.98-6.90 (m, 2H), 4.07 (ddd, J=12.3, 9.1, 6.4Hz, 2H), 3.84 (h, J=5.1Hz, 1H ), 3.83 (s, 3H), 3.03 (ddd, J=12.1, 9.1, 6.4Hz, 2H), 2.60 (d, J=4.8Hz, 1H), 2.15 (dddd, J=12.8, 9.0, 6.3, 4.9Hz, 2H), 2.06 (dddd, J=13.0, 9.0, 6.3, 4.9Hz, 2H).

[0292] Example 26

[0293]

[0294] (6-Methoxybenzofuran-2-yl)(4-(trifluoromethyl)piperidin-1-yl)methyl ketone (26)

[0295] Using 4-(trifluoromethyl)piperidine as a starting material, a white solid 26 was obtained according to the preparation method of compound 1, in 48% yield, mp: 57.0-59.4℃. ¹H NMR (500MHz, Chloroform-d) δ 7.84 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.3, 2.2 Hz, 1H), 6.98-6.90 (m, 2H), 4.07 (ddd, J = 12.1, 11.3, 8.5 Hz, 2H), 3.83 (s, 2H), 3.03 (ddd, J = 12.1, 11.3, 8.5 Hz, 2H), 2.43-2.29 (m, 1H), 1.93-1.81 (m, 2H), 1.41-1.28 (m, 2H).

[0296] Example 27

[0297]

[0298] N-(3-(dimethylamino)propyl)-6-methoxybenzofuran-2-carboxamide (27)

[0299] Using N1,N1-dimethylpropane-1,3-diamine as a starting material, a pale yellow solid 27 was obtained according to the preparation method of compound 1, with a yield of 38% and mp: 39.5-40.8℃. ¹H NMR (500 MHz, Chlorofon m-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98-6.90 (m, ¹H), 3.83 (s, ¹H), 3.08 (q, J = 5.3 Hz, ¹H), 2.94 (t, J = 5.8 Hz, ¹H), 1.88-1.80 (m, ¹H).

[0300] Example 28

[0301]

[0302] (4-(4-fluorophenyl)piperidin-1-yl)(6-methoxybenzofuran-2-yl)methyl ketone (28)

[0303] Using 4-(4-fluorophenyl)piperidine as a raw material, a white solid 28 was obtained according to the preparation method of compound 1, with a yield of 39% and mp: 49.5-49.7℃. 1H NMR (500MHz, Chloroform-d) δ7.84 (d, J=2.2Hz, 1H), 7.65 (dd, J=8.3, 2.2Hz, 1H), 7.15-7.09 (m, 2H), 6.98-6.88 (m, 4H), 4.07 (ddd, J=12.1, 8.4, 5.7Hz, 2H), 3.83 (s, 2H), 3.03 (ddd, J=12.3, 8.4, 5.7Hz, 2H), 2.80 (tt, J=6.0, 5.0Hz, 1H), 1.89 (ddt, J=13.0, 8.4, 5.6Hz, 2H), 1.69-1.59 (m, 2H).

[0304] Example 29

[0305]

[0306] (6-Methoxybenzofuran-2-yl)(4-(pyridin-4-yl)piperidin-1-yl)methyl ketone (29)

[0307] Using 4-(piperidin-4-yl)pyridine as a starting material, a white solid 29 was obtained according to the preparation method of compound 1, with a yield of 37% and mp: 54.8-56.1℃. 1H NMR (500MHz, Chloroform-d) δ 8.60-8.56 (m, 2H), 7.84 (d, J=2.3Hz, 1H), 7.65 (dd, J=8.1, 2.1Hz, 1H), 7.25-7.21 (m, 2H), 6.98-6.90 (m, 2H), 4.07 (ddd, J=1 2.1, 8.4, 5.7Hz, 2H), 3.83 (s, 2H), 3.03 (ddd, J=12.3, 8.4, 5.7Hz, 2H), 2.51 ( qd, J=5.6, 4.8Hz, 1H), 1.90 (ddt, J=13.0, 8.4, 5.6Hz, 2H), 1.70-1.60 (m, 2H).

[0308] Example 30

[0309]

[0310] N-(4-hydroxycyclohexyl)-6-methoxybenzofuran-2-carboxamide (30)

[0311] Using 4-aminocyclohexane-1-ol as a starting material, a pale yellow solid 30 was obtained according to the preparation method of compound 1, with a yield of 62% and mp: 52.9-59.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 2.2 Hz, 1H), 7.65 (dd, J = 8.3, 2.2 Hz, 1H), 6.98-6.90 (m, 2H), 5.05 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 3.90-3.76 (m, 2H), 1.75-1.63 (m, 4H), 1.54 (s, 1H), 1.25-1.15 (m, 2H), 1.14-1.04 (m, 2H).

[0312] Example 31

[0313]

[0314] 2-(diethylamino)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate (31)

[0315] HOBT (0.15 g, 1.1 mmol, 1.10 equivalence) and EDCI (0.21 g, 1.1 mmol, 1.1 equivalence) were added to a stirred solution of 6-methoxybenzo[b]thiophene-2-carboxylic acid in DCM (10 mL), and the mixture was stirred at room temperature for 1 h. Then, 2-(diethylamino)ethane-1-ol (0.082 g, 1.1 mmol, 1.10 equivalence) was added, and the mixture was reacted at room temperature for 10 h. The mixture was diluted with H₂O (40 mL) and extracted with EA (20 mL × 3). The organic phase was washed with saturated NaCl solution (10 mL × 3), dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified on a silica gel column (DCM:MeOH = 10:1) to give a white powder 31 (yield: 77%), mp: 64.9–65.5 °C. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=1.9Hz, 0H), 7.74 (dd, J=7.8, 2.1Hz, 0H), 7.54 (d, J=1.9Hz, 0H), 7.01 (dd, J=7. 8, 2.1Hz, 0H), 4.43 (t, J=5.2Hz, 1H), 3.80 (s, 1H), 3.15 (t, J=5.3Hz, 1H), 2.89 (q, J=6.9Hz, 2H), 1.05 (t, J=6.9Hz, 3H).

[0316] Example 32

[0317]

[0318] 2-(pyrrolidone-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate (32)

[0319] Using 2-(pyrrolidine-1-yl)ethane-1-ol as a starting material, a pale yellow oil 32 was obtained according to the preparation method of compound 31, with a yield of 48%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.8, 2.1 Hz, 1H), 4.43 (t, J = 6.0 Hz, 2H), 3.80 (s, 2H), 3.25–3.19 (m, 3H), 3.15 (t, J = 6.0 Hz, 2H), 1.96 (p, J = 2.1 Hz, 3H).

[0320] Example 33

[0321]

[0322] 2-(piperidin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate (33)

[0323] Using 2-(piperidin-1-yl)ethane-1-ol as a starting material, a pale yellow solid 33 was obtained according to the preparation method of compound 31, with a yield of 71% and mp: 52.1-53.7℃. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=2.0Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.54 (d, J=2.0Hz, 1H), 7.01 (dd, J=7.7, 2.1 Hz, 1H), 4.43 (t, J=5.9Hz, 2H), 3.80 (s, 2H), 3.15 (t, J=5.9Hz, 2H), 2.45-2.39 (m, 4H), 1.53-1.46 (m, 3H), 1.46-1.38 (m, 2H).

[0324] Example 34

[0325]

[0326] 2-Morpholinoethyl-6-methoxybenzo[b]thiophene-2-carboxylate (34)

[0327] Using 2-morpholinoethanol-1-ol as a starting material, a white solid 34 was obtained according to the preparation method of compound 31, with a yield of 63% and mp: 57.9-59.5℃. ¹H NMR (500MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.43 (t, J = 5.9 Hz, 1H), 3.80 (s, 1H), 3.72 (t, J = 4.7 Hz, 2H), 3.15 (t, J = 5.9 Hz, 1H), 2.54-2.42 (m, 2H).

[0328] Example 35

[0329]

[0330] 2-(4-methylpiperazin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate (35)

[0331] Using 2-(4-methylpiperazin-1-yl)ethane-1-ol as a starting material, compound 35 was prepared according to the method described for compound 31, yielding a white oily product, 35, in 73% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.0 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 4.43 (t, J = 5.9 Hz, 2H), 3.80 (s, 2H), 3.15 (t, J = 5.9 Hz, 2H), 2.57–2.48 (m, 7H).

[0332] Example 36

[0333]

[0334] (6-Methoxybenzo[b]thiophene-2-carbonyl)glycine (36)

[0335] Using glycine as a starting material, compound 36 was prepared according to the method described for compound 31, yielding a brown oily substance in 59%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.63–7.57 (m, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 3.86–3.78 (m, 4H).

[0336] Example 37

[0337]

[0338] 6-Methoxybenzo[b]thiophene-2-carbonyl)-D-alanine (37)

[0339] Using D-alanine as a starting material, a pale yellow solid 37 was obtained according to the preparation method of compound 31, with a yield of 51% and mp: 52.6-54.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 4.46 (dq, J = 8.1, 6.3 Hz, 1H), 3.80 (s, 2H), 1.41 (s, 1H).

[0340] Example 38

[0341]

[0342] (6-Methoxybenzo[b]thiophene-2-carbonyl)-D-valine (38)

[0343] Using D-valine as a starting material, a pale yellow solid 38 was obtained according to the preparation method of compound 31, with a yield of 62% and mp: 52.9-59.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 9.72 (s, 1H), 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 4.30-4.22 (m, 1H), 3.80 (s, 2H), 2.16-2.04 (m, J = 6.6 Hz, 1H), 0.93 (ddd, J = 6.5, 4.9, 1.5 Hz, 6H).

[0344] Example 39

[0345]

[0346] (6-Methoxybenzo[b]thiophene-2-carbonyl)-D-leucine (39)

[0347] Brown solid 39 was obtained from D-leucine according to the preparation method of compound 31, with a yield of 38% and mp: 72.9-73.7℃. ¹H NMR (500MHz, Chloroform-d) δ 8.51 (d, J = 2.5Hz, 1H), 7.74 (dd, J = 7.9, 2.1Hz, 1H), 7.54 (d, J = 1.9Hz, 1H), 7.01 (dd, J = 7.7, 2.1Hz, 1H), 6.95 (d, J = 8.1Hz, 1H), 4.40 (dt, J = 8.1, 7.0Hz, 1H), 3.80 (s, 2H), 1.76-1.55 (m, 3H), 0.93 (dd, J = 6.4, 5.0Hz, 6H).

[0348] Example 40

[0349]

[0350] (6-methoxybenzo[b]thiophene-2-carbonyl)-D-phenylalanine

[0351] (6-Methoxybenzo[b]thiophene-2-carbonyl)-D-phenylalanine (40)

[0352] Using D-phenylalanine as a raw material, a white oily substance 40 was obtained according to the preparation method of compound 31, with a yield of 56%. 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.8, 2.1Hz, 1H), 7.54 (d, J=2.0Hz, 1H), 7.31-7.17 (m, 5H), 7.01 (dd, J=7.7, 2.1 Hz, 1H), 6.95 (d, J=8.6Hz, 1H), 4.78 (dt, J=8.6, 6.8Hz, 1H), 3.80 (s, 2H), 3.17 (ddt, J=14.1, 6.8, 0.8Hz, 1H), 2.92 (ddt, J=14.1, 6.8, 0.8Hz, 1H).

[0353] Example 41

[0354]

[0355] 2-(dimethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (41)

[0356] HOBT (0.15 g, 1.1 mmol, 1.10 equivalence) and EDCI (0.21 g, 1.1 mol, 1.10 equivalence 1.1 mmol) were added to a stirred solution of (6-methoxybenzo[b]thiophene-2-carbonyl)glycine in DCM (10 mL), and the mixture was stirred for 1 h at room temperature. Then, 2-(dimethylamino)ethane-1-ol (0.09 g, 1.1 mmol, 1.10 equivalence) was added, and the mixture was reacted again for 10 h at room temperature. The mixture was diluted with H₂O (40 mL) and extracted with EA (20 mL × 3). The organic phase was washed with saturated NaCl solution (10 mL × 3), dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified on a silica gel column (DCM:MeOH = 20:1) to give a white oily 41 (54%). 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.8, 2.1Hz, 1H), 7.60 (t, J=5.6Hz, 1H), 7.54 (d, J=2.0 Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4.35 (t, J=6.8Hz, 2H), 4.13 (d, J=5.5Hz, 2H), 3.80 (s, 2H), 3.33 (t, J=6.7Hz, 2H).

[0357] Example 42

[0358]

[0359] N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-6-methoxybenzo[b]thiophene-2-carboxamide (42)

[0360] Using N1,N1-dimethylethane-1,2-diamine as a starting material, compound 41 was prepared by the same method, yielding a brown oily substance, 42, in 39% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 6.26 (t, J = 4.3 Hz, 1H), 3.82–3.75 (m, 4H), 3.35 (td, J = 6.0, 4.2 Hz, 2H), 2.46 (t, J = 6.0 Hz, 2H).

[0361] Example 43

[0362]

[0363] 6-Methoxy-N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide (43)

[0364] Using N-methyl-2-(pyrrolidone-1-yl)ethane-1-amine as a starting material, a pale yellow oil 43 was obtained according to the preparation method of compound 41, with a yield of 76%. 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.2Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.60 (t, J=5.6Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.8, 2.1H z, 1H), 6.26 (t, J=4.2Hz, 1H), 3.82-3.75 (m, 4H), 3.14 (td, J=5.4, 4.3Hz, 2 H), 2.67 (t, J=5.4Hz, 2H), 2.60 (td, J=3.6, 1.9Hz, 4H), 1.85-1.79 (m, 2H).

[0365] Example 44

[0366]

[0367] 6-Methoxy-N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide (44)

[0368] Using 2-(piperidin-1-yl)ethane-1-amine as a starting material, a pale yellow oil 44 was obtained according to the preparation method of compound 41, with a yield of 63%. 1H NMR (500MHz, Chloroform-d) δ7.74 (dd, J=7.9, 2.1Hz, 0H), 7.60 (t, J=5.6Hz, 0H), 7.54 (d, J=2.0Hz, 0H), 7.01 (dd, J=7.7, 2.1Hz, 0H), 6.26 (t, J=4. 2Hz, 0H), 3.82-3.75 (m, 2H), 3.14 (td, J=5.4, 4.2Hz, 1H), 2.67 (t, J=5.4H z, 1H), 2.43-2.37 (m, 2H), 1.57 (tt, J=5.9, 4.8Hz, 2H), 1.46-1.38 (m, 1H).

[0369] Example 45

[0370]

[0371] (6-Methoxybenzothiophene-2-yl)(4-methylpiperazin-1-yl)methyl ketone (45)

[0372] Using 1-methylpiperazine as a raw material, a pale yellow oil 45 was obtained according to the preparation method of compound 41, with a yield of 18%. 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.8, 2.1Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 3.80 (s, 2H), 3.66 (ddd, J=11.3, 6.2, 3.5Hz, 2H), 3.27 (ddd, J=11.3, 6.2, 3.5Hz, 2H), 3.00 (dd d, J=11.3, 6.2, 3.5Hz, 2H), 2.72 (ddd, J=11.5, 6.1, 3.5Hz, 2H), 2.56 (s, 2H).

[0373] Example 46

[0374]

[0375] 6-Methoxy-N-(2-(pyrrolidone-1-yl)ethyl)benzo[b]thiophene-2-carboxamide (46)

[0376] Using 2-(pyrrolidine-1-yl)ethane-1-amine as a starting material, a pale yellow oil 46 was obtained according to the preparation method of compound 31, with a yield of 78%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.2 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.8, 2.1 Hz, 1H), 6.58 (t, J = 4.0 Hz, 1H), 3.80 (s, 2H), 3.54 (td, J = 5.3, 4.0 Hz, 2H), 2.67 (t, J = 5.3 Hz, 2H), 2.63–2.57 (m, 4H), 1.85–1.79 (m, 2H).

[0377] Example 47

[0378]

[0379] 6-Methoxy-N-(2-(piperidin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide (47)

[0380] Using 2-(piperidin-1-yl)ethane-1-amine as a starting material, a pale yellow oil 47 was obtained according to the preparation method of compound 31, with a yield of 82%. 1H NMR (500MHz, Chloroform-d) δ7.74 (dd, J=7.9, 2.1Hz, 0H), 7.54 (d, J=2.0Hz, 0H), 7.01 (dd, J=7.7, 2.1Hz, 0H), 6.58 (t, J=4.0Hz, 0H), 3. 80 (s, 1H), 3.54 (td, J=5.3, 4.0Hz, 1H), 2.67 (t, J=5.3Hz, 1H), 2.43-2.37 (m, 2H), 1.57 (ddd, J=9.5, 6.0, 4.8Hz, 2H), 1.46-1.38 (m, 1H).

[0381] Example 48

[0382]

[0383] 6-Methoxy-N-(2-morpholinoethyl)benzo[b]thiophene-2-carboxamide (48)

[0384] Using 2-morpholinoethanol-1-amine as a starting material, a white oily substance 48 was obtained according to the preparation method of compound 31, with a yield of 42%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 6.58 (t, J = 4.0 Hz, OH), 3.80 (s, 1H), 3.76–3.70 (m, 2H), 3.54 (td, J = 5.3, 4.0 Hz, 1H), 2.60 (t, J = 5.3 Hz, 1H), 2.51–2.45 (m, 2H).

[0385] Example 49

[0386]

[0387] 6-Methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide (49)

[0388] Using 2-(4-methylpiperazin-1-yl)ethane-1-amine as a starting material, a pale yellow oil 49 was obtained according to the preparation method of compound 31, with a yield of 48%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 6.58 (t, J = 4.0 Hz, OH), 3.80 (s, ¹H), 3.54 (td, J = 5.3, 4.0 Hz, ¹H), 2.67 (t, J = 5.3 Hz, ¹H), 2.57–2.48 (m, 4H).

[0389] Example 50

[0390]

[0391] N-(3-(diethylamino)propyl)-6-methoxybenzo[b]thiophene-2-carboxamide (50)

[0392] Using N1,N1-diethylpropane-1,3-diamine as a raw material, a brownish-yellow oil 50 was obtained according to the preparation method of compound 31, with a yield of 30%. 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.9, 2.0Hz, 1H), 7.68 (t, J=4.8Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.7 , 2.1Hz, 1H), 3.80 (s, 2H), 3.08 (td, J=5.9, 4.8Hz, 2H), 3.01 (q, J=6.9Hz, 4H), 2.53 (t, J=6.0Hz, 2H), 1.88-1.80 (m, 1H), 1.02 (t, J=6.9Hz, 5H).

[0393] Example 51

[0394]

[0395] N-(4-(dimethylamino)butyl)-6-methoxybenzo[b]thiophene-2-carboxamide (51)

[0396] Using N1,N1-dimethylbutane-1,4-diamine as a starting material, a brown oily substance 51 was obtained according to the preparation method of compound 31, with a yield of 55%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.68 (t, J = 4.5 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 3.80 (s, 2H), 3.45 (td, J = 5.4, 4.4 Hz, 2H), 2.51 (t, J = 5.7 Hz, 2H), 1.61–1.52 (m, 2H), 1.45–1.37 (m, 2H).

[0397] Example 52

[0398]

[0399] N-(4-(diethylamino)butyl)-6-methoxybenzo[b]thiophene-2-carboxamide (52)

[0400] Using N1,N1-diethylbutane-1,4-diamine as a raw material, a pale yellow solid 52 was obtained according to the preparation method of compound 31, with a yield of 30% and mp: 199.1-201.9℃. 1H NMR (500MHz, Chloroform-d) δ7.74 (dd, J=7.9, 2.1Hz, 0H), 7.68 (t, J=4.5Hz, 1H), 7.54 (d, J=1.9Hz, 0H), 7.01 (dd, J=7.7, 2.1Hz, 0H) , 3.80 (s, 1H), 3.45 (td, J=5.5, 4.5Hz, 1H), 3.05-2.97 (m, 3H), 1.74-1.64 (m, 1H), 1.41 (tt, J=8.1, 5.4Hz, 1H), 1.02 (t, J=6.9Hz, 3H).

[0401] Example 53

[0402]

[0403] 6-Methoxy-N-(2-(piperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide (53)

[0404] Using 2-(piperazin-1-yl)ethane-1-amine as a starting material, a pale yellow solid 53 was obtained according to the preparation method of compound 31, with a yield of 96%, mp: 73.6-75.0℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 6.58 (t, J = 4.0 Hz, OH), 3.80 (s, 1H), 3.54 (td, J = 5.3, 4.0 Hz, 1H), 2.92-2.86 (m, 2H), 2.67 (t, J = 5.3 Hz, 1H), 2.57-2.51 (m, 2H).

[0405] Example 54

[0406]

[0407] N-(2-(diethylamino)ethyl)-6-methoxybenzo[b]thiophene-2-carboxamide (54)

[0408] Using N1,N1-diethylethane-1,2-diamine as a starting material, a pale pink solid 54 was obtained according to the preparation method of compound 31, with a yield of 30% and mp: 209.1-211.9℃. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.8, 2.1 Hz, OH), 6.58 (t, J = 4.0 Hz, 1H), 3.80 (s, 1H), 3.54 (q, J = 4.5 Hz, 1H), 2.67 (t, J = 4.6 Hz, 1H), 2.46 (q, J = 6.9 Hz, 2H), 1.09 (t, J = 6.9 Hz, 3H).

[0409] Example 55

[0410]

[0411] (4-Hydroxypiperidin-1-yl)(6-methoxybenzothiophene-2-yl)methyl ketone (55)

[0412] Using piperidine-4-ol as a starting material, a pale yellow oil 55 was obtained according to the preparation method of compound 31, with a yield of 98%. 1HNMR (500MHz, Chloroform-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4.07 (d dd, J=12.3, 9.1, 6.4Hz, 2H), 3.84 (h, J=5.1Hz, 1H), 3.80 (s, 2H), 3.03 (ddd, J=12.1, 9.1, 6.4Hz, 2H), 2.60 (d, J=4.8Hz, 1H), 2.20-2.01 (m, 4H).

[0413] Example 56

[0414]

[0415] (6-Methoxybenzo[b]thiophene-2-yl)(4-(trifluoromethyl)piperidin-1-yl)methyl ketone (56)

[0416] Using 4-(trifluoromethyl)piperidine as a raw material, a pink solid 56 was obtained according to the preparation method of compound 31, with a yield of 38% and mp: 221.5-224.1℃. 1H NMR (500MHz, Chlorofonm-d) δ8.51 (d, J=2.5Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.54 (d, J=2.0Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4.07 ( ddd, J=12.1, 11.3, 8.5Hz, 2H), 3.80 (s, 2H), 3.03 (ddd, J=12.1, 11.3, 8.5Hz, 2H), 2.43-2.29 (m, 1H), 1.93-1.81 (m, 2H), 1.41-1.28 (m, 2H).

[0417] Example 57

[0418]

[0419] N-(3-(dimethylamino)propyl)-6-methoxybenzo[b]thiophene-2-carboxamide (57)

[0420] Using N1,N1-dimethylpropane-1,3-diamine as a starting material, colorless oil 57 was obtained according to the preparation method of compound 31, with a yield of 74%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.68 (t, J = 4.8 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 3.80 (s, 2H), 3.08 (q, J = 5.2 Hz, 2H), 2.94 (t, J = 5.8 Hz, 2H), 1.88–1.80 (m, 2H).

[0421] Example 58

[0422]

[0423] (4-(4-fluorophenyl)piperidin-1-yl)(6-methoxybenzothiophene-2-yl)methyl ketone (58)

[0424] Using 4-(4-fluorophenyl)piperidine as a starting material, compound 31 was prepared by the same method, yielding a pale yellow oil, 58, in 49% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.15–7.09 (m, 2H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 6.96–6.88 (m, 2H). , 4.07 (ddd, J=12.1, 8.4, 5.7Hz, 2H), 3.80 (s, 2H), 3.03 (ddd, J=12.3, 8.4, 5.7Hz, 2H) , 2.80 (tt, J=6.0, 5.0Hz, 1H), 1.89 (ddt, J=13.1, 8.4, 5.6Hz, 2H), 1.69-1.59 (m, 2H).

[0425] Example 59

[0426]

[0427] (6-Methoxybenzothiophene-2-yl)(4-(pyridin-4-yl)piperidin-1-yl)methyl ketone (59)

[0428] Using 4-(piperidin-4-yl)pyridine as a starting material, brown solid 59 was obtained according to the preparation method of compound 31, in 45% yield, mp: 110.0-112.2℃. ¹H NMR (500MHz, Chloroform-d) δ 8.60-8.56 (m, 2H), 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.25-7.21 (m, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H) , 4.07 (ddd, J=12.1, 8.4, 5.7Hz, 2H), 3.80 (s, 2H), 3.03 (ddd, J=12.3, 8.4, 5.7Hz, 2H) , 2.51 (qd, J=5.6, 4.8Hz, 1H), 1.90 (ddt, J=13.0, 8.4, 5.6Hz, 2H), 1.70-1.60 (m, 2H).

[0429] Example 60

[0430]

[0431] N-(4-hydroxycyclohexyl)-6-methoxybenzo[b]thiophene-2-carboxamide (60)

[0432] Using 4-aminocyclohexane-1-ol as a starting material, a pale yellow oil 60 was obtained according to the preparation method of compound 31, with a yield of 82%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 5.65 (d, J = 7.3 Hz, 1H), 3.90–3.76 (m, 2H), 3.80 (s, 3H), 1.75–1.63 (m, 4H), 1.54 (s, 1H), 1.25–1.15 (m, 2H), 1.14–1.04 (m, 2H).

[0433] Example 61

[0434]

[0435] 2-(dimethylamino)ethyl 6-methoxybenzofuran-2-carboxylate (61)

[0436] Using 2-(dimethylamino)ethane-1-ol as a starting material, a brown oily compound 61 was obtained according to the preparation method of compound 1, with a yield of 56%. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.3, 2.2 Hz, 1H), 6.98–6.90 (m, 2H), 4.70 (t, J = 6.6 Hz, 2H), 3.83 (s, 2H), 3.53 (t, J = 6.6 Hz, 2H).

[0437] Example 62

[0438]

[0439] 2-(piperazin-1-yl)ethyl 6-methoxybenzofuran-2-carboxylate (62)

[0440] Using 2-(piperazin-1-yl)ethane-1-ol as a starting material, a colorless oil 62 was obtained according to the preparation method of compound 1, with a yield of 26%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98–6.90 (m, ¹H), 4.71 (t, J = 6.0 Hz, ¹H), 3.83 (s, ¹H), 3.15 (t, J = 5.9 Hz, ¹H), 2.92–2.86 (m, 2H), 2.57–2.51 (m, 2H).

[0441] Example 63

[0442]

[0443] 3-(dimethylamino)propyl6-methoxybenzofuran-2-carboxylate (63)

[0444] Using 3-(dimethylamino)prop-1-ol as a starting material, a brown solid 63 was obtained according to the preparation method of compound 1, with a yield of 46%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98–6.90 (m, ¹H), 4.24 (t, J = 5.9 Hz, ¹H), 3.83 (s, ¹H), 2.50 (t, J = 6.6 Hz, ¹H), 2.07–1.99 (m, ¹H).

[0445] Example 64

[0446]

[0447] 3-(diethylamino)propyl 6-methoxybenzofuran-2-carboxylate (64)

[0448] Using 3-(diethylamino)prop-1-ol as a starting material, a colorless oil 64 was obtained according to the preparation method of compound 1, with a yield of 38%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.2, 2.2 Hz, OH), 6.98–6.90 (m, 1H), 4.24 (t, J = 6.6 Hz, 1H), 3.83 (s, 1H), 3.01 (q, J = 6.9 Hz, 2H), 2.85 (t, J = 6.7 Hz, 1H), 2.03 (p, J = 6.6 Hz, 1H), 1.02 (t, J = 6.9 Hz, 3H).

[0449] Example 65

[0450]

[0451] 3-(pyrrolidone-1-yl)propyl6-methoxybenzofuran-2-carboxylate (65)

[0452] Using 3-(pyrrolidone-1-yl)prop-1-ol as a starting material, a brown solid 65 was obtained according to the preparation method of compound 1, in 35% yield, mp: 37.2-38.5℃. ¹H NMR (500MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98-6.90 (m, 1H), 4.24 (t, J = 6.6 Hz, 1H), 3.83 (s, 1H), 3.79-3.71 (m, 1H), 2.85 (t, J = 6.7 Hz, 1H), 2.80-2.72 (m, 1H), 2.03 (p, J = 6.6 Hz, 1H), 1.88-1.80 (m, 1H).

[0453] Example 66

[0454]

[0455] 3-(piperidin-1-yl)propyl6-methoxybenzofuran-2-carboxylate (66)

[0456] Using 3-(piperidin-1-yl)prop-1-ol as a starting material, a pale yellow solid 66 was obtained according to the preparation method of compound 1, in 57% yield, mp: 57.9-58.5℃. ¹H NMR (500MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98-6.90 (m, 1H), 4.24 (t, J = 6.6 Hz, 1H), 3.83 (s, 1H), 2.85 (t, J = 6.7 Hz, 1H), 2.48-2.42 (m, 2H), 2.03 (p, J = 6.6 Hz, 1H), 1.65-1.57 (m, 2H), 1.46-1.38 (m, 1H).

[0457] Example 67

[0458]

[0459] 3-morpholinopropyl-6-methoxybenzofuran-2-carboxylate (67)

[0460] Using 3-morpholinopropane-1-ol as a starting material, a brown solid 65 was obtained according to the preparation method of compound 1, in 35% yield, mp: 37.2-38.5℃. ¹H NMR (500MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98-6.90 (m, 1H), 4.24 (t, J = 6.6 Hz, 1H), 3.83 (s, 1H), 3.77-3.71 (m, 2H), 2.85 (t, J = 6.7 Hz, 1H), 2.51-2.45 (m, 2H), 2.03 (p, J = 6.6 Hz, 1H).

[0461] Example 68

[0462]

[0463] 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzofuran-2-carboxylate (68)

[0464] Using 3-(4-methylpiperazin-1-yl)propanol as a starting material, a brown oily substance 68 was obtained according to the preparation method of compound 1, with a yield of 23%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98–6.90 (m, 1H), 4.24 (t, J = 6.6 Hz, 1H), 3.83 (s, 1H), 2.85 (t, J = 6.7 Hz, 1H), 2.57–2.49 (m, 4H), 2.03 (p, J = 6.6 Hz, 1H).

[0465] Example 69

[0466]

[0467] 3-(piperidin-4-yl)propyl-6-methoxybenzofuran-2-carboxylate (69)

[0468] Using 3-(piperidin-4-yl)prop-1-ol as a starting material, a brown solid 69 was obtained according to the preparation method of compound 1, with a yield of 46% and mp: 47.9-49.4℃. ¹H NMR (500MHz, Chloroform-d) δ 7.79 (d, J = 2.5Hz, 1H), 7.65 (dd, J = 8.3, 2.2Hz, 1H), 6.98-6.90 (m, 2H), 4.33 (t, J = 6.3Hz, 2H), 3.83 (s, 2H), 3.13 (dd, J = 13.4, 5.3, 3.9, 2). .6Hz, 2H), 2.77 (dddd, J=13.4, 5.3, 4.0, 2.6Hz, 2H), 2.00 (p, J=3.8Hz, 1H), 1.82 (tt , J=8.9, 6.3Hz, 2H), 1.77-1.68 (m, 2H), 1.40 (hept, J=6.1Hz, 1H), 1.25-1.15 (m, 4H).

[0469] Example 70

[0470]

[0471] 4-(dimethylamino)butyl-6-methoxybenzofuran-2-carboxylate (70)

[0472] Using 4-(dimethylamino)but-1-ol as a starting material, a pale yellow solid 70 was obtained according to the preparation method of compound 1, with a yield of 22%, mp: 127.2-1288.5℃. ¹H NMR (500MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98-6.90 (m, 1H), 4.33 (t, J = 6.1 Hz, 1H), 3.83 (s, 1H), 2.54-2.48 (m, 1H), 1.82 (tt, J = 7.3, 5.9 Hz, 1H), 1.58 (tt, J = 7.3, 5.9 Hz, 1H).

[0473] Example 71

[0474]

[0475] 4-(diethylamino)butyl-6-methoxybenzofuran-2-carboxylate (71)

[0476] Using 4-(diethylamino)but-1-ol as a starting material, a pale yellow oil 72 was obtained according to the preparation method of compound 1, with a yield of 39%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 6.98–6.90 (m, 1H), 4.33 (t, J = 6.1 Hz, 1H), 3.83 (s, 1H), 3.01 (q, J = 6.9 Hz, 2H), 2.38 (t, J = 6.0 Hz, 1H), 1.87–1.77 (m, 1H), 1.63–1.53 (m, 1H), 1.02 (t, J = 6.9 Hz, 3H).

[0477] Example 72

[0478]

[0479] 4-(pyrrolidone-1-yl)butyl-6-methoxybenzofuran-2-carboxylate (72)

[0480] Using 4-(pyrrolidone-1-yl)but-1-ol as a starting material, a pale yellow oil was obtained according to the preparation method of compound 1, with a yield of 75%. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.2 Hz, OH), 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98–6.90 (m, 1H), 4.33 (t, J = 6.1 Hz, 1H), 3.83 (s, 1H), 2.60 (td, J = 3.6, 1.9 Hz, 2H), 2.38 (t, J = 6.0 Hz, 1H), 1.88–1.77 (m, 2H), 1.63–1.53 (m, 1H).

[0481] Example 73

[0482]

[0483] 4-(piperidin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate (73)

[0484] Using 4-(piperidin-1-yl)but-1-ol as a starting material, a brownish-yellow oil 73 was obtained according to the preparation method of compound 1, with a yield of 26%. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.2 Hz, OH), 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98–6.90 (m, 1H), 4.33 (t, J = 6.1 Hz, 1H), 3.83 (s, 1H), 2.41–2.35 (m, 2H), 1.87–1.77 (m, 1H), 1.65–1.53 (m, 3H), 1.46–1.38 (m, 1H).

[0485] Example 74

[0486]

[0487] 4-morpholine n-butyl-6-methoxybenzofuran-2-carboxylate (74)

[0488] Using 4-morpholinobutane-1-ol as a starting material, a pale yellow solid 74 was obtained according to the preparation method of compound 1, in 10% yield, mp: 49.2-50.3℃. ¹H NMR (500MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 6.98-6.90 (m, 1H), 4.33 (t, J = 6.1 Hz, 1H), 3.83 (s, 1H), 3.77-3.71 (m, 2H), 2.51-2.45 (m, 2H), 2.38 (t, J = 6.0 Hz, 1H), 1.87-1.77 (m, 1H), 1.63-1.53 ​​(m, 1H).

[0489] Example 75

[0490]

[0491] 4-(piperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate (75)

[0492] Using 4-(piperazin-1-yl)but-1-ol as a starting material, a pale yellow oil 75 was obtained according to the preparation method of compound 1, with a yield of 35%. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.1, 2.1 Hz, 1H), 6.98–6.90 (m, 2H), 4.33 (t, J = 6.1 Hz, 2H), 3.83 (s, 2H), 2.92–2.86 (m, 4H), 2.57–2.51 (m, 4H), 2.40 (t, J = 6.0 Hz, 2H), 1.87–1.77 (m, 2H), 1.63–1.53 (m, 2H), 1.50 (p, J = 3.4 Hz, 1H).

[0493] Example 76

[0494]

[0495] 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate (76)

[0496] Using 4-(4-methylpiperazin-1-yl)but-1-ol as a starting material, a white solid 76 was obtained according to the preparation method of compound 1, with a yield of 93%, mp: 103.6–3105.4 °C. ¹H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 2.2 Hz, OH), 6.94 (d, J = 2.2 Hz, OH), 6.77 (dd, J = 8.4, 2.2 Hz, OH), 4.33 (t, J = 6.1 Hz, 1H), 2.57–2.49 (m, 4H), 2.40 (t, J = 6.0 Hz, 1H), 1.87–1.77 (m, 1H), 1.63–1.53 (m, 1H).

[0497] Example 77

[0498]

[0499] 2-(Diethylamino)ethyl(6-methoxybenzofuran-2-carbonyl)glycine (77)

[0500] Using 2-(diethylamino)ethane-1-ol as a starting material, a pale yellow oil 77 was obtained according to the preparation method of compound 1, with a yield of 82%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.2, 2.2 Hz, OH), 7.03–6.90 (m, 2H), 4.35 (t, J = 5.4 Hz, 1H), 4.13 (d, J = 5.9 Hz, 1H), 3.83 (s, 1H), 2.97 (t, J = 5.4 Hz, 1H), 2.89 (q, J = 6.9 Hz, 2H), 1.05 (t, J = 6.9 Hz, 3H).

[0501] Example 78

[0502]

[0503] 2-Morpholinylethyl(6-methoxybenzofuran-2-carbonyl)glycine (78)

[0504] Using 2-morpholinoethanol-1-ol as a starting material, a colorless oil 78 was obtained according to the preparation method of compound 1, with a yield of 42%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.3, 2.2 Hz, OH), 7.03–6.90 (m, ¹H), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.9 Hz, 1H), 3.83 (s, 1H), 3.72 (t, J = 4.7 Hz, 2H), 2.97 (t, J = 6.1 Hz, 1H), 2.54–2.42 (m, 2H).

[0505] Example 79

[0506]

[0507] 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine (79)

[0508] Using 2-(pyrrolidone-1-yl)ethane-1-ol as a starting material, a pale yellow oil 79 was obtained according to the preparation method of compound 1, with a yield of 48%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 7.03–6.90 (m, 1H), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.9 Hz, 1H), 3.83 (s, 1H), 3.25–3.19 (m, 2H), 2.97 (t, J = 6.1 Hz, 1H), 1.96 (p, J = 2.1 Hz, 2H).

[0509] Example 80

[0510]

[0511] 2-(piperidin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine (80)

[0512] Using 2-(piperidin-1-yl)ethane-1-ol as a starting material, a brownish-yellow oil 80 was obtained according to the preparation method of compound 1, in 30% yield. ¹H NMR (500 MHz, Chloroform-d) δ 7.84 (d, J = 2.4 Hz, OH), 7.65 (dd, J = 8.1, 2.1 Hz, 1H), 7.03–6.90 (m, 2H), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.9 Hz, 1H), 3.83 (s, 1H), 2.97 (t, J = 6.1 Hz, 1H), 2.45–2.39 (m, 2H), 1.53–1.38 (m, 3H).

[0513] Example 81

[0514]

[0515] 2-(piperazin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine (81)

[0516] Using 2-(piperazin-1-yl)ethane-1-ol as a starting material, a brown oily compound 81 was obtained according to the preparation method of compound 1, with a yield of 55%. ¹H NMR (500 MHz, Chloroform-d) δ 7.65 (dd, J = 8.1, 2.1 Hz, OH), 7.03–6.90 (m, 1H), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.9 Hz, 1H), 3.83 (s, 1H), 2.97 (t, J = 6.1 Hz, 1H), 2.92–2.86 (m, 2H), 2.57–2.51 (m, 2H).

[0517] Example 82

[0518]

[0519] 2-(dimethylamino)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate (82)

[0520] Using 2-(dimethylamino)ethane-1-ol as a starting material, a pale yellow oil 82 was obtained according to the preparation method of compound 31, with a yield of 78%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.0 Hz, 1H), 7.74 (dd, J = 7.9, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 4.43 (t, J = 6.6 Hz, 2H), 3.80 (s, 2H), 2.95 (t, J = 6.6 Hz, 2H).

[0521] Example 83

[0522]

[0523] 2-(piperazin-1-yl)6-methoxybenzo[b]thiophene-2-carboxylic acid ethyl ester (83)

[0524] Using 2-(piperazin-1-yl)ethane-1-ol as a starting material, a pale yellow oil 83 was obtained according to the preparation method of compound 31, with a yield of 93%. ¹H NMR (500 MHz, Chlorofon n-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.43 (t, J = 5.9 Hz, 1H), 3.80 (s, 1H), 3.15 (t, J = 5.9 Hz, 1H), 2.92–2.86 (m, 2H), 2.57–2.51 (m, 2H).

[0525] Example 84

[0526]

[0527] 3-(dimethylamino)propyl6-methoxybenzo[b]thiophene-2-carboxylic acid ester (84)

[0528] Using 3-(dimethylamino)prop-1-ol as a starting material, a pale yellow oil 84 was obtained according to the preparation method of compound 31, with a yield of 90%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 4.24 (t, J = 5.9 Hz, 2H), 3.80 (s, 2H), 2.50 (t, J = 6.6 Hz, 2H), 2.07–1.99 (m, 2H).

[0529] Example 85

[0530]

[0531] 3-(diethylamino)propyl6-methoxybenzo[b]thiophene-2-carboxylic acid ester (85)

[0532] Using 3-(dimethylamino)prop-1-ol as a starting material, a pale yellow oil 85 was obtained according to the preparation method of compound 31, with a yield of 85%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.8, 2.1 Hz, OH), 4.24 (t, J = 6.6 Hz, 1H), 3.80 (s, 1H), 3.01 (q, J = 6.9 Hz, 2H), 2.85 (t, J = 6.7 Hz, 1H), 2.03 (p, J = 6.6 Hz, 1H), 1.02 (t, J = 6.9 Hz, 3H).

[0533] Example 86

[0534]

[0535] 3-(pyrrolidone-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (86)

[0536] Using 3-(pyrrolidone-1-yl)prop-1-ol as a starting material, a pale yellow solid 86 was obtained according to the preparation method of compound 31, with a yield of 55% and mp: 94.6-96.2℃. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=1.9Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4. 24 (t, J=6.6Hz, 2H), 3.80 (s, 2H), 3.79-3.71 (m, 2H), 2.85 (t, J=6.7Hz, 2H), 2.80-2.72 (m, 2H), 2.03 (p, J=6.6Hz, 2H), 1.88-1.80 (m, 2H).

[0537] Example 87

[0538]

[0539] 3-(piperidin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (87)

[0540] Using 3-(piperidin-1-yl)prop-1-ol as a starting material, a pale yellow solid 87 was obtained according to the preparation method of compound 31, with a yield of 69% and mp: 78.4-79.3℃. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=1.9Hz, 0H), 7.74 (dd, J=7.9, 2.1Hz, 0H), 7.54 (d, J=1.9Hz, 0H), 7.01 (dd, J=7.7, 2.1Hz, 0H), 4. 24 (t, J=6.6Hz, 1H), 3.80 (s, 1H), 2.85 (t, J=6.7Hz, 1H), 2.48-2.42 (m, 2H), 2.03 (p, J=6.6Hz, 1H), 1.65-1.57 (m, 2H), 1.46-1.38 (m, 1H).

[0541] Example 88

[0542]

[0543] 3-Morpholinyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (88)

[0544] Using 3-morpholinopropane-1-ol as a starting material, a pale yellow solid 88 was obtained according to the preparation method of compound 31, with a yield of 52% and mp: 51.2-53.9℃. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.0 Hz, OH), 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.24 (t, J = 6.6 Hz, 1H), 3.80 (s, 1H), 3.77-3.71 (m, 2H), 2.85 (t, J = 6.7 Hz, 1H), 2.51-2.45 (m, 2H), 2.03 (p, J = 6.6 Hz, 1H).

[0545] Example 89

[0546]

[0547] 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (89)

[0548] Using 3-(4-methylpiperazin-1-yl)propanol as a starting material, a white oil 89 was obtained according to the preparation method of compound 31, with a yield of 75%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.0 Hz, OH), 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.24 (t, J = 6.6 Hz, 1H), 3.80 (s, 1H), 2.85 (t, J = 6.7 Hz, 1H), 2.57–2.49 (m, 4H), 2.03 (p, J = 6.6 Hz, 1H).

[0549] Example 90

[0550]

[0551] 3-(piperidin-4-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (90)

[0552] Using 3-(piperidin-4-yl)prop-1-ol as the starting material, a white solid 90 was obtained according to the preparation method of compound 31, in 60% yield, mp: 100.1-102.1℃. ¹H NMR (500MHz, Chloroform-d) δ 8.22 (d, J = 2.0Hz, 1H), 7.74 (dd, J = 7.9, 2.1Hz, 1H), 7.54 (d, J = 2.0Hz, 1H), 7.01 (dd, J = 7.7, 2.1Hz, 1H), 4.33 (t, J = 6.3Hz, 2H), 3.80 (s, 2H), 3.13 (dddd, J = 1 3.4, 5.3, 3.9, 2.6Hz, 2H), 2.77 (dddd, J=13.4, 5.3, 4.0, 2.6Hz, 2H), 2.00 (p, J=3.8Hz, 1H), 1 .82 (tt, J=8.9, 6.3Hz, 2H), 1.77-1.68 (m, 2H), 1.40 (hept, J=6.1Hz, 1H), 1.25-1.15 (m, 4H).

[0553] Example 91

[0554]

[0555] 4-(dimethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (91)

[0556] Using 4-(dimethylamino)but-1-ol as a starting material, a colorless oil 91 was obtained according to the preparation method of compound 31, with a yield of 83%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, 1H), 7.74 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.01 (dd, J = 7.7, 2.1 Hz, 1H), 4.33 (t, J = 6.1 Hz, 2H), 3.80 (s, 2H), 2.54–2.48 (m, 2H), 1.82 (tt, J = 7.3, 5.9 Hz, 2H), 1.58 (tt, J = 7.3, 5.9 Hz, 2H).

[0557] Example 92

[0558]

[0559] 4-(diethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (92)

[0560] Using 4-(diethylamino)but-1-ol as a starting material, a pale yellow oil 92 was obtained according to the preparation method of compound 31, with a yield of 65%. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=2.0Hz, 0H), 7.74 (dd, J=7.9, 2.1Hz, 0H), 7.54 (d, J=1.9Hz, 0H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4.33 (t, J=6.1Hz, 1H), 3.80 (s, 1H), 3.01 (q, J=6.9Hz, 2H), 2.38 (t, J=6.0Hz, 1H), 1.87-1.77 (m, 1H), 1.63-1.53 ​​(m, 1H), 1.02 (t, J=6.9Hz, 3H).

[0561] Example 93

[0562]

[0563] 4-(pyrrolidone-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (93)

[0564] Using 4-(pyrrolidone-1-yl)but-1-ol as a starting material, a pale yellow oil 93 was obtained according to the preparation method of compound 31, with a yield of 89%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.8, 2.1 Hz, OH), 4.33 (t, J = 6.1 Hz, 1H), 3.80 (s, 1H), 2.60 (td, J = 3.6, 1.9 Hz, 2H), 2.38 (t, J = 6.0 Hz, 1H), 1.88–1.77 (m, 2H), 1.63–1.53 (m, 1H).

[0565] Example 94

[0566]

[0567] 4-(piperidin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate (94)

[0568] Using 4-(piperidin-1-yl)but-1-ol as a starting material, a white solid 94 was obtained according to the preparation method of compound 31, in 70% yield, mp: 121.5-124.5℃. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.8, 2.1 Hz, OH), 4.33 (t, J = 6.1 Hz, 1H), 3.80 (s, 1H), 2.41-2.35 (m, 2H), 1.87-1.77 (m, 1H), 1.65-1.53 ​​(m, 3H), 1.46-1.38 (m, 1H).

[0569] Example 95

[0570]

[0571] 4-Morpholinyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester (95)

[0572] Using 4-morpholinobutane-1-ol as a starting material, a colorless oil 95 was obtained according to the preparation method of compound 31, with a yield of 57%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.0 Hz, OH), 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.33 (t, J = 6.1 Hz, 1H), 3.80 (s, 1H), 3.77–3.71 (m, 2H), 2.51–2.45 (m, 2H), 2.38 (t, J = 6.0 Hz, 1H), 1.87–1.77 (m, 1H), 1.63–1.53 (m, 1H).

[0573] Example 96

[0574]

[0575] 4-(piperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate (96)

[0576] Using 4-(piperazin-1-yl)but-1-ol as a starting material, a colorless oil 96 was obtained according to the preparation method of compound 31, with a yield of 35%. 1H NMR (500MHz, Chloroform-d) δ8.22 (d, J=1.9Hz, 1H), 7.74 (dd, J=7.9, 2.1Hz, 1H), 7.54 (d, J=1.9Hz, 1H), 7.01 (dd, J=7.7, 2.1Hz, 1H), 4.33 (t, J=6 .1Hz, 2H), 3.80 (s, 2H), 2.92-2.86 (m, 4H), 2.57-2.51 (m, 4H), 2.40 (t, J= 6.0Hz, 2H), 1.87-1.77(m, 2H), 1.63-1.53(m, 2H), 1.50(p, J=3.4Hz, 1H).

[0577] Example 97

[0578]

[0579] 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate (97)

[0580] Using 4-(4-methylpiperazin-1-yl)but-1-ol as a starting material, a colorless oil 97 was obtained according to the preparation method of compound 31, with a yield of 75%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 1.9 Hz, OH), 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.33 (t, J = 6.1 Hz, 1H), 3.80 (s, 1H), 2.57–2.49 (m, 4H), 2.40 (t, J = 6.0 Hz, 1H), 1.87–1.77 (m, 1H), 1.63–1.53 (m, 1H).

[0581] Example 98

[0582]

[0583] 2-(diethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (98)

[0584] Using 2-(diethylamino)ethane-1-ol as a starting material, a colorless oil 98 was obtained according to the preparation method of compound 31, with a yield of 56%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.60 (t, J = 5.6 Hz, 1H), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.35 (t, J = 5.4 Hz, 1H), 4.13 (d, J = 5.5 Hz, 1H), 3.80 (s, 1H), 2.97 (t, J = 5.4 Hz, 1H), 2.89 (q, J = 6.9 Hz, 2H), 1.05 (t, J = 6.9 Hz, 3H).

[0585] Example 99

[0586]

[0587] 2-Morpholinoethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (99)

[0588] Using 2-morpholinoethanol-1-ol as the starting material, a colorless oil 99 was obtained according to the preparation method of compound 31, with a yield of 63%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.8, 2.1 Hz, OH), 7.60 (t, J = 5.6 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.8, 2.1 Hz, OH), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.5 Hz, 1H), 3.80 (s, 1H), 3.72 (t, J = 4.7 Hz, 2H), 2.97 (t, J = 6.1 Hz, 1H), 2.54–2.42 (m, 2H).

[0589] Example 100

[0590]

[0591] 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (100)

[0592] Using 2-(pyrrolidine-1-yl)ethane-1-ol as the starting material, a colorless oil 100 was obtained according to the preparation method of compound 31, with a yield of 47%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.60 (t, J = 5.6 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.5 Hz, 1H), 3.80 (s, 1H), 3.25–3.19 (m, 2H), 2.97 (t, J = 6.1 Hz, 1H), 1.96 (p, J = 2.1 Hz, 2H).

[0593] Example 101

[0594]

[0595] 2-(piperidin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (101)

[0596] Using 2-(piperidin-1-yl)ethane-1-ol as a starting material, a colorless oil 101 was obtained according to the preparation method of compound 31, with a yield of 48%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.60 (t, J = 5.6 Hz, 1H), 7.54 (d, J = 1.9 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.5 Hz, 1H), 3.80 (s, 1H), 2.97 (t, J = 6.1 Hz, 1H), 2.45–2.39 (m, 2H), 1.53–1.38 (m, 3H).

[0597] Example 102

[0598]

[0599] 2-(piperazin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine (102)

[0600] Using 2-(piperazin-1-yl)ethane-1-ol as a starting material, a colorless oil 102 was obtained according to the preparation method of compound 31, with a yield of 51%. ¹H NMR (500 MHz, Chloroform-d) δ 7.74 (dd, J = 7.9, 2.1 Hz, OH), 7.60 (t, J = 5.6 Hz, OH), 7.54 (d, J = 2.0 Hz, OH), 7.01 (dd, J = 7.7, 2.1 Hz, OH), 4.35 (t, J = 6.0 Hz, 1H), 4.13 (d, J = 5.5 Hz, 1H), 3.80 (s, 1H), 2.97 (t, J = 6.1 Hz, 1H), 2.92–2.86 (m, 2H), 2.57–2.51 (m, 2H).

[0601] Example 103

[0602]

[0603] 2-(diethylamino)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxylate (103)

[0604] Thiophene[2′,3′:4,5]benzo[1,2-d][1,3]dioxane ethyl 6-carboxylate (1.2 g, 5 mmol) was dissolved in 20 mL of dioxane, and 8.0 mL of 1N NaOH was added. The solution initially became turbid but became clear after 30 minutes when the reaction was complete. The dioxane was removed by distillation under reduced pressure, and the remaining product was dissolved in water. The solution was mixed with dichloromethane, and after separation, the organic phase was discarded, and the aqueous phase was adjusted to pH 3 with 10% HCl. The precipitate was collected and recrystallized in dry methanol. 0.51 g of white solid was finally obtained, and SOCl2 (2.8 mL, 0.04 mol) was dissolved in 50 mL of toluene and 2 drops of DMF. The solution was connected to the apparatus with stirring at 80 °C using a drying tube. The solution was then evaporated to dryness under reduced pressure, dissolved in pure toluene, and evaporated to dryness again. The product, thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxane-6-carboxyl chloride, was immediately used in the next reaction. 10 mL of pure toluene was added to the compound. Triethylamine (2.0 mL, 0.013 mol) and N,N-dimethylacetamide (1.3 mL, 0.013 mol) were added to the solution under cooling; the reaction was completed with stirring at room temperature for 1 hour. Ethyl acetate was added to the reaction system, and after shaking and separation, the aqueous phase was removed. The mixture was washed twice with water. The organic phase was collected, washed with saturated NaCl solution, and evaporated to dryness. The resulting solid was dissolved in diethyl ether and recrystallized to give 1.90 g of yellow crystals 103, yield 73%, mp: 38.1–39.5 °C. 1HNMR (500MHz, Chloroform-d) δ8.22 (d, J=2.3Hz, 0H), 7.08 (s, 0H), 5.93 (s, 1H), 4.43 (t, J=5.2Hz, 1H), 3.15 (t, J=5.3Hz, 1H), 2.89 (q, J=6.9Hz, 2H), 1.05 (t, J=6.9Hz, 3H).

[0605] Example 104

[0606]

[0607] 2-(pyrrolidone-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxen-6-carboxylate (104)

[0608] Using 2-(pyrrolidone-1-yl)ethane-1-ol as a starting material, a colorless oil 104 was obtained according to the preparation method of compound 103, with a yield of 47%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.4 Hz, OH), 7.08 (s, OH), 5.93 (s, 1H), 4.43 (t, J = 6.0 Hz, 1H), 3.25–3.19 (m, 2H), 3.15 (t, J = 6.0 Hz, 1H), 1.96 (p, J = 2.1 Hz, 2H).

[0609] Example 105

[0610]

[0611] 2-(piperidin-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxy-6-carboxylate (105)

[0612] Using 2-(piperidin-1-yl)ethane-1-ol as a starting material, colorless oil 105 was obtained according to the preparation method of compound 103, with a yield of 73%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.4 Hz, 1H), 7.22 (s, OH), 7.08 (s, 1H), 5.93 (s, 1H), 4.43 (t, J = 6.0 Hz, 2H), 3.15 (t, J = 6.0 Hz, 2H), 2.45–2.39 (m, 4H), 1.53–1.46 (m, 3H), 1.46–1.38 (m, 2H).

[0613] Example 106

[0614]

[0615] 2-Morpholinoethylthiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxocyclo-6-carboxylate (106)

[0616] Using 2-morpholinoethanol-1-ol as a starting material, a pale yellow oil, 106, was obtained according to the preparation method of compound 103, with a yield of 72%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.3 Hz, OH), 7.08 (s, OH), 5.93 (s, 1H), 4.43 (t, J = 5.9 Hz, 1H), 3.72 (t, J = 4.7 Hz, 2H), 3.15 (t, J = 5.9 Hz, 1H), 2.54–2.42 (m, 2H).

[0617] Example 107

[0618]

[0619] 2-(4-methylpiperazin-1-yl)ethylthiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxen-6-carboxylate (107)

[0620] Using 2-(4-methylpiperazin-1-yl)ethane-1-ol as a starting material, a pale yellow oil, 107, was obtained according to the preparation method of compound 103, with a yield of 25%. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d, J = 2.3 Hz, OH), 7.08 (s, OH), 5.93 (s, 1H), 4.43 (t, J = 5.9 Hz, 1H), 3.15 (t, J = 5.9 Hz, 1H), 2.57–2.48 (m, 4H).

[0621] Example 108

[0622]

[0623] (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxy-6-carbonyl)glycine (108)

[0624] Using glycine as a raw material, a pale yellow oil 108 was obtained according to the preparation method of compound 103, with a yield of 19%.

[0625] 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.3Hz, 1H), 7.63-7.57 (m, 1H), 7.22 (s, 0H), 7.08 (s, 1H), 5.93 (s, 2H), 3.84 (d, J=6.0Hz, 2H).

[0626] Example 109

[0627]

[0628] (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-alanine (109)

[0629] Using D-alanine as a starting material, compound 109 was prepared according to the method described for compound 103, yielding a pale yellow oil, 109, in 26% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.3 Hz, 1H), 7.22 (s, 1H), 7.08 (s, 1H), 6.95 (d, J = 8.2 Hz, 1H), 5.93 (s, 2H), 4.46 (dq, J = 8.1, 6.3 Hz, 1H), 1.41 (s, 2H).

[0630] Example 110

[0631]

[0632] (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-valine (110)

[0633] Using D-valine as a starting material, compound 103 was prepared according to the method described for compound 103 to obtain a pale yellow oil, 110, in 16% yield. ¹H NMR (500 MHz, Chloroform-d) δ 9.72 (s, 1H), 8.51 (d, J = 2.3 Hz, 1H), 7.22 (s, OH), 7.08 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 5.93 (s, 1H), 4.30–4.22 (m, 1H), 2.16–2.04 (m, J = 6.6 Hz, 1H), 0.93 (ddd, J = 6.5, 5.0, 1.5 Hz, 6H).

[0634] Example 111

[0635]

[0636] (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-leucine (111)

[0637] Using D-leucine as a raw material, colorless oil 111 was obtained according to the preparation method of compound 103, with a yield of 38%.

[0638] 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.4Hz, 1H), 7.22 (s, 0H), 7.08 (s, 1H), 6.95 (d, J=8.0Hz , 1H), 5.93 (s, 2H), 4.40 (dt, J=8.0, 7.0Hz, 1H), 1.76-1.55 (m, 3H), 0.93 (dd, J=6.4, 5.0Hz, 6H).

[0639] Example 112

[0640]

[0641] (thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-phenylalanine (112)

[0642] Using D-phenylalanine as a raw material, colorless oil 112 was obtained according to the preparation method of compound 103, with a yield of 41%.

[0643] 1H NMR (500MHz, Chloroform-d) δ8.51 (d, J=2.4Hz, 1H), 7.31-7.17 (m, 6H), 7.08 (s, 1H), 6.95 (d, J=8.6Hz, 1H), 5. 93 (s, 2H), 4.78 (dt, J=8.6, 6.8Hz, 1H), 3.17 (ddt, J=14.1, 6.8, 0.8Hz, 1H), 2.92 (ddt, J=14.1, 6.8, 0.8Hz, 1H).

[0644] Example 113

[0645]

[0646] 2-(dimethylamino)ethyl(thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)glycine ester (113)

[0647] Using 2-(dimethylamino)ethane-1-ol as a starting material, a colorless oil, 113, was obtained according to the preparation method of compound 103, with a yield of 58%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.4 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.22 (s, OH), 7.08 (s, 1H), 5.93 (s, 2H), 4.35 (t, J = 6.8 Hz, 2H), 4.13 (d, J = 5.5 Hz, 2H), 3.33 (t, J = 6.7 Hz, 2H).

[0648] Example 114

[0649]

[0650] N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide (114)

[0651] Using N1,N1-dimethylethane-1,2-diamine as a starting material, compound 103 was prepared according to the method described for compound 103 to obtain a brownish-yellow oil, 114, in 76% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.3 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.22 (s, OH), 7.08 (s, 1H), 6.26 (t, J = 4.3 Hz, 1H), 5.93 (s, 2H), 3.77 (d, J = 5.5 Hz, 2H), 3.35 (td, J = 6.0, 4.2 Hz, 2H), 2.46 (t, J = 6.0 Hz, 2H).

[0652] Example 115

[0653]

[0654] N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxen-6-carboxamide (115)

[0655] Using 2-(pyrrolidine-1-yl)ethane-1-amine as a starting material, colorless oil 115 was obtained according to the preparation method of compound 103, with a yield of 64%. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.4 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.08 (s, 1H), 6.26 (t, J = 4.2 Hz, 1H), 5.93 (s, 1H), 3.77 (d, J = 5.5 Hz, 2H), 3.14 (td, J = 5.4, 4.2 Hz, 2H), 2.67 (t, J = 5.4 Hz, 2H), 2.63–2.57 (m, 4H), 1.85–1.79 (m, 2H).

[0656] Example 116

[0657]

[0658] N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)thiopheno[2′,3′:4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide (116)

[0659] Using 2-(piperidin-1-yl)ethane-1-amine as a starting material, compound 103 was prepared according to the method described for compound 103, yielding a pale yellow oil, 116, in 69% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.4 Hz, OH), 7.60 (t, J = 5.6 Hz, 1H), 7.08 (s, OH), 6.26 (t, J = 4.2 Hz, 1H), 5.93 (s, 1H), 3.77 (d, J = 5.5 Hz, 1H), 3.14 (td, J = 5.4, 4.3 Hz, 1H), 2.67 (t, J = 5.4 Hz, 1H), 2.43–2.37 (m, 2H), 1.61–1.53 (m, 2H), 1.46–1.38 (m, 1H).

[0660] Example 117

[0661]

[0662] N-(2-(pyrrolidone-1-yl)ethyl)thieno[2′,3′:4,5]benzo[1,2-d][1,3]dioxen-6-carboxamide (117)

[0663] Using 2-(pyrrolidone-1-yl)ethane-1-amine as a starting material, compound 103 was prepared according to the method described for compound 103, yielding a pale yellow oil, 117, in 42% yield. ¹H NMR (500 MHz, Chloroform-d) δ 8.51 (d, J = 2.4 Hz, 1H), 7.08 (s, 1H), 6.58 (t, J = 4.0 Hz, 1H), 5.93 (s, 1H), 3.54 (td, J = 5.3, 4.0 Hz, 2H), 2.67 (t, J = 5.3 Hz, 2H), 2.63–2.57 (m, 4H), 1.85–1.79 (m, 2H).

[0664] Example 118: Western blot detection of the expression of aging-related proteins P16, P21, and P27

[0665] Experimental materials: MC3T3-E1 cells (adherent cells), culture medium: α-MEM. Culture conditions: 37℃, 5% CO2 cell culture incubator, compound 1-117 in DMSO solvent.

[0666] Cellular protein extraction and quantification (the entire process should ideally be performed on ice):

[0667] 1. After drug treatment, use a suction pump to remove the supernatant from each well of the cell plate.

[0668] 2. Wash with PBS to remove protein interference from the serum.

[0669] 3. Add RIPA lysis buffer (RIPA:PMSF = 1:100) to the cell plate for adherent cells (100 μL per well for a six-well plate and 80 μL per well for a twelve-well plate).

[0670] 4. Shake well to ensure the lysis buffer is in full contact with the cells in the well, and place on ice for lysis for 30 min.

[0671] 5. Use a clean cell scraper to scrape the lysed cells onto one side of the culture dish, and then use a pipette to transfer the mixture of cells and RIPA lysis buffer into a pre-cooled 1.5 mL centrifuge tube.

[0672] 6. Place the centrifuge tubes in a pre-cooled 4°C centrifuge, centrifuge at 12,000 rpm for 25 minutes, and then collect the supernatant for protein quantification.

[0673] 7. Protein Concentration Determination: The BCA method is used to determine protein concentration (the basic principle of the reaction is that under alkaline conditions, proteins complex with copper ions in the chromogenic reagent, reducing the copper ions to a +1 valence. The BCA complex forms a stable purple-blue compound with the +1 valence copper ions, exhibiting the highest absorbance at a wavelength of 562 nm, which is directly proportional to the protein concentration. Finally, a colorimetric method is used for quantitative analysis).

[0674] a) Establish a standard curve by adding reagents to the 96-well plate according to Table 1 below:

[0675] Table 1. Preparation of reagents for the standard curve

[0676] BSA (μL) 0 2 4 6 8 12 16 20 Purified water (μL) 20 18 16 14 12 8 4 0 BCA reagent (μL) 180 180 180 180 180 180 180 180 Concentration (μg / μL) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5

[0677] The standard curve was prepared using BSA protein standard solutions at 8 concentration points (initial concentration of 5 μg / μL).

[0678] (BCA reagent: Solution A: Solution B = 50:1)

[0679] b) Take 2 μL of the supernatant from the centrifuged sample and add it to the well. Add 18 μL of purified water (diluted 10 times) and then add 180 μL of BCA reagent.

[0680] c) Cover the plate and gently tap the side wall a few times to ensure the sample is thoroughly mixed.

[0681] d) Place in a 37℃ constant temperature incubator for 30 minutes.

[0682] e) Use an ELISA reader to measure the absorbance of each well at a wavelength of 560 nm.

[0683] f) After measuring the OD value, plot a standard curve with protein content as the x-axis and OD value as the y-axis. Substitute the measured sample OD value as the y-value into the equation of the standard curve to calculate the sample content (x-value), and then multiply it by the dilution factor (10 in this experiment) to obtain the total protein content in the original sample solution.

[0684] g) Calculate the minimum protein sample volume based on the minimum concentration and the required sample volume. Calculate the required sample volume for other samples based on the minimum sample volume and sample concentration, and add RJPA lysis buffer. Then add 5x loading buffer (sample to buffer volume ratio is 4:1).

[0685] 8. Heat in a 98℃ metal bath for 10 minutes, then centrifuge at low speed for 10 seconds.

[0686] 9. After cooling, load the sample onto the electrophoresis apparatus, or store it in a -20℃ refrigerator and wait for electrophoresis the next day.

[0687] 10. SDS-PAGE electrophoresis, membrane transfer and development:

[0688] 1) Preparation of SDS-polyacrylamide gel, Table 2:

[0689] Table 2. Preparation of two 10-well SDS polyacrylamide gel separating and stacking solutions

[0690]

[0691] 2) Load 10 μL of sample into the sample well, and use the concentration markers on the left and right sides to distinguish between the positive and negative sides.

[0692] 3) Insert the electrodes and start electrophoresis: upper layer 80V, 30min; lower layer 120V, 90min.

[0693] 4) Stop electrophoresis and remove the gel plate according to the positions of the protein marker, target band, and internal control band.

[0694] 5) Activate the PVDF membrane: Cut the PVDF membrane into rectangular pieces of 8.5cm*5.5cm with a utility knife, soak them in methanol or anhydrous ethanol for 15 seconds, activate them, and then soak them in water for later use.

[0695] 6) Place the transfer clamp in the ceramic bowl pre-filled with transfer buffer, black side down, and immerse it in the buffer. Place a sponge pad on top, and then place filter paper on top. Then pry open the gel plate and cut the gel according to the protein marker instructions. Place the cut gel, distinguishing the front and back, on the filter paper. Place the prepared membrane face upside down on the gel (make sure to mark the front and back of the membrane). Cover with filter paper and sponge pad. Use a roller to remove air bubbles each time you cover. After aligning, fix the clamp and place it in the transfer tank pre-filled with transfer buffer (black to black, red to red). Place ice cubes and an ice box around the transfer tank to maintain a low temperature and prevent the heat generated during the transfer process from affecting the transfer effect.

[0696] 7) Insert the electrode, 400mA, 100min to start the membrane transfer.

[0697] 8) Remove the PVDF membrane from the transfer clip and place it in a dish containing 5% skim milk powder (prepared by TBST). Shake slowly at room temperature for 30 minutes.

[0698] 9) Place it in a sealing plastic film and cut it to the corresponding band of the target protein with a utility knife. Seal it with a heat sealer, add the primary antibody diluted with 5% BSA (the primary antibody dilution ratio is generally 1:1000), and incubate overnight at 4°C.

[0699] 10) Take it out the next day, put the PVDF membrane in TBST washing solution, and shake and wash it 3 times on a shaker for 10 minutes each time.

[0700] 11) Incubate the secondary antibody at room temperature for 2 hours (the dilution ratio of the secondary antibody is generally 1:2000).

[0701] 12) After incubation, place the PVDF membrane in TBST solution and wash it three times on a shaker for 10 minutes each time;

[0702] 13) Turn on the exposure device and prepare the ECL chemiluminescence solution (solution A:solution B = 1:1) for use.

[0703] 14) During development, the PVDF membrane is placed with the protein side facing up in the dark chamber of the developing apparatus. ECL luminescent solution is dropped onto the target protein area of ​​the membrane, and the membrane is exposed for photographing. The experimental results are shown below. Figure 1 Tables 3, 4 and 5.

[0704] Table 3. Effects of 50 compounds on p16 protein expression as detected by Western blot.

[0705]

[0706]

[0707]

[0708] Note: The default value for the Con group is 1, and the values ​​for other groups are the ratios of the grayscale values ​​calculated with the Con group.

[0709] Table 4. Effects of 50 compounds on p21 protein expression as detected by Western blot.

[0710]

[0711]

[0712]

[0713] Note: The default value for the Con group is 1, and the values ​​for other groups are the ratios of the grayscale values ​​calculated with the Con group.

[0714] Table 5. Effects of 50 compounds on p27 protein expression as detected by Western blot.

[0715]

[0716]

[0717]

[0718] Note: The default value for the Con group is 1, and the values ​​for other groups are the ratios of the grayscale values ​​calculated with the Con group.

[0719] Experiments have shown that the compounds of this invention can reduce the protein expression of aging-promoting proteins P21, P16, and P27.

[0720] Example 119 β-galactosidase detection

[0721] Mouse bone marrow mesenchymal stem cells (Pronoseu, CP-M131) and complete culture medium for mouse bone marrow mesenchymal stem cells (Pronoseu, CM-M131). Culture conditions: 37℃, 5% CO2 cell culture incubator, compound 1-117 in DMSO solvent. Cell senescence β-galactosidase staining kit (Beyotime, C0602) was used for detection.

[0722] 1) Mouse bone marrow mesenchymal stem cells were cultured in 96-well plates and treated with the drug for 48 hours;

[0723] 2) Remove the cell culture medium, wash once with PBS, add 100 μL of β-galactosidase staining and fixation solution, and fix at room temperature for 15 minutes.

[0724] 3) Remove the cell fixative and wash the cells three times with PBS for three minutes each time.

[0725] 4) Remove the PBS and add 100 μL of staining working solution to each well according to Table 6 below.

[0726] Table 6. Preparation method of staining working solution

[0727] β-galactosidase staining solution A 10μl β-galactosidase staining solution B 10μl β-galactosidase staining solution C 930μl X-Gal solution 50μl

[0728] 5) Incubate at 37℃ for 48 hours, then seal the perforated plate with plastic wrap to prevent evaporation.

[0729] 6) Observe under a regular optical microscope, and randomly select 3 fields of view from each well to take pictures. The results are shown in […]. Figure 2 .

[0730] 7) The ImageJ software was used for counting analysis, and the results are shown in Table 7.

[0731] Table 7 shows the effects (%) of 50 compounds on β-galactosidase expression in mouse bone marrow mesenchymal stem cells.

[0732]

[0733]

[0734]

[0735] Example 120 Safety Test

[0736] This study used 45 C57BL / 6J mice (30 females and 15 males), purchased from Jiangsu Airingfei Company at approximately 16 months of age. After approximately one week of acclimatization, the mice were used to collect baseline data for the measured variables and then randomly assigned to three treatment groups, each consisting of 10 females and 5 males. Group 1 served as the solvent control group, receiving 0.1 ml of distilled water by gavage; Group 2 received compound 4 (30 mg / kg / day) by gavage; and Group 3 received compound 8 (30 mg / kg / day) by gavage. The mice were housed in a specific pathogen-free room, with a maximum of 5 mice per cage, and were examined daily. They were identified by ear tags and weighed.

[0737] Throughout the 10-month study period, the overall health of the mice was monitored daily. Mice exhibiting poor health, such as severe lethargy, inability to eat or drink, significant respiratory distress at rest, and / or a weight loss exceeding 20%, were euthanized with carbon dioxide. Mice found dead during routine checks or euthanized with carbon dioxide underwent necropsy, and the time of death was recorded. Survival curves are shown in [Figure number missing]. Figure 3 .

[0738] Example 121 Serum IL-10 Levels

[0739] 1. Sample preparation

[0740] This study used 45 C57BL / 6J mice (30 females and 15 males), purchased from Jiangsu Airingfei Company at approximately 16 months of age. After acclimatization to the housing for about one week, the mice were used to collect baseline data for the measured variables and then randomly assigned to three treatment groups, each consisting of 10 females and 5 males. Group 1 served as the solvent control group, receiving 0.1 ml of distilled water by gavage; Group 2 received compound 4 (30 mg / kg / day) by gavage; and Group 3 received compound 8 (30 mg / kg / day) by gavage. Whole blood was collected from the tails of all three groups (n=6, females) and allowed to stand at room temperature for 30 minutes to 2 hours until natural coagulation and serum separation. The blood was then centrifuged at approximately 1000-2000g for 10 minutes at 4°C, and the yellow supernatant was collected as serum. The prepared serum was kept on ice until use.

[0741] 2. Preparations before testing

[0742] a. After removing the reagent kit from the refrigerator, allow it to equilibrate at room temperature (25-28℃) for 20 minutes; after each test, store any remaining reagent at 4℃ promptly.

[0743] b. Prepare an appropriate amount of washing solution: Dilute the washing solution (20X) with deionized water to 1X. For example, 10ml of washing solution (20X) can be mixed with 190ml of water to obtain a 1X washing solution.

[0744] c. Add the standard diluent to one vial of standard according to the volume indicated on the standard label, and incubate at room temperature for 15 minutes. Then gently mix and pipette several times to completely dissolve the standard, achieving a final concentration of 200 pg / ml.

[0745] d. Take five clean 1.5 mL centrifuge tubes, add 250 μl of standard diluent to each tube in advance, and perform serial dilution of the standard to obtain six standard concentrations of 200, 100, 50, 25, 12.5, and 6.25 pg / mL. Finally, add the diluted standard to the wells of the pre-coated plate in sequence, and add the standard diluent directly as the 0 pg / mL concentration, for a total of seven standard concentrations.

[0746] 3. Washing

[0747] The automatic plate washer uses 300 μl of washing solution per well, with an interval of 15-30 seconds between injection and withdrawal. The plate is washed 5 times. After the final wash, the plate is placed upside down on thick absorbent paper and gently patted dry.

[0748] 4. Operating Procedures

[0749] a. Calculate and determine the number of pre-coated strips required for one experiment. Take out the required strips and place them in a 96-well frame. If the strips are not needed for the time being, put them back in the aluminum foil bag and seal them. Store at 4°C.

[0750] b. Standards should be prepared and standard curves plotted for each experiment. It is also recommended to set up background correction wells, i.e. blank wells, by adding only TMB solution and stop solution to these wells.

[0751] c. Add 100 μl of the sample or standard of different concentrations to the corresponding well, seal the reaction well with a sealing film (transparent), and incubate at room temperature for 120 minutes.

[0752] d. Wash the plate 5 times, and pat it dry on thick absorbent paper on the last wash.

[0753] e. Add 100 μl of biotinylated antibody per well (Note: This biotinylated antibody has been pre-prepared and can be used directly without further dilution). Seal the reaction wells with a clear sealing film and incubate at room temperature for 60 minutes.

[0754] f. Wash the plate 5 times, and pat it dry on thick absorbent paper on the last wash.

[0755] g. Add 100 μl of horseradish peroxidase-labeled streptavidin per well. Seal the wells with white sealing film and incubate at room temperature in the dark for 20 minutes. If the room temperature is low (below 25°C), the incubation time needs to be extended appropriately.

[0756] h. Wash the plate 5 times, and on the last wash, place it on thick absorbent paper and pat it dry.

[0757] i. Add 100 μl of TMB solution as a colorimetric reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 15-20 minutes. If the room temperature is too low, the incubation time needs to be extended appropriately.

[0758] j. Add 50 μl of stop solution per well, mix well, and immediately measure the A450 value.

[0759] See results Figure 4 As shown, compounds 4 and 8 provided by this invention can effectively increase the level of anti-inflammatory factor IL-10 in mouse serum.

[0760] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions 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. A substituted benzo[a]penta-unsaturated heterocyclic compound or its pharmaceutical salt, characterized in that, Selected from the following compounds: Compound 31: 2-(diethylamino)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate; Compound 32: 2-(pyrrolidone-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate; Compound 33: 2-(piperidin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate; Compound 35: 2-(4-methylpiperazin-1-yl)ethyl 6-methoxybenzo[b]thiophene-2-carboxylate; Compound 38: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-valine; Compound 39: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-leucine; Compound 40: (6-methoxybenzo[b]thiophene-2-carbonyl)-D-phenylalanine; Compound 41: 2-(dimethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 42: N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-6-methoxybenzo[b]thiophene-2-carboxamide; Compound 43: 6-methoxy-N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide; Compound 44: 6-methoxy-N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)benzo[b]thiophene-2-carboxamide; Compound 46: 6-methoxy-N-(2-(pyrrolidone-1-yl)ethyl)benzo[b]thiophene-2-carboxamide; Compound 47: 6-methoxy-N-(2-(piperidin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide; Compound 49: 6-methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide; Compound 53: 6-methoxy-N-(2-(piperazin-1-yl)ethyl)benzo[b]thiophene-2-carboxamide; Compound 56: (6-methoxybenzo[b]thiophen-2-yl)(4-(trifluoromethyl)piperidin-1-yl)methyl ketone; Compound 58: (4-(4-fluorophenyl)piperidin-1-yl)(6-methoxybenzothiophene-2-yl)methyl ketone; Compound 59: (6-methoxybenzothiophene-2-yl)(4-(pyridin-4-yl)piperidin-1-yl)methyl ketone; Compound 62: 2-(piperazin-1-yl)ethyl 6-methoxybenzofuran-2-carboxylate; Compound 68: 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzofuran-2-carboxylate; Compound 69: 3-(piperidin-4-yl)propyl-6-methoxybenzofuran-2-carboxylate; Compound 75: 4-(piperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate; Compound 76: 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzofuran-2-carboxylate; Compound 77: 2-(diethylamino)ethyl(6-methoxybenzofuran-2-carbonyl)glycine; Compound 78: 2-morpholinoethyl(6-methoxybenzofuran-2-carbonyl)glycine; Compound 79: 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine; Compound 80: 2-(piperidin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine; Compound 81: 2-(piperazin-1-yl)ethyl(6-methoxybenzofuran-2-carbonyl)glycine; Compound 83: Ethyl 2-(piperazin-1-yl)6-methoxybenzo[b]thiophene-2-carboxylate; Compound 86: 3-(pyrrolidone-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 87: 3-(piperidin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 89: 3-(4-methylpiperazin-1-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 90: 3-(piperidin-4-yl)propyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 91: 4-(dimethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 92: 4-(diethylamino)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 93: 4-(pyrrolidone-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 94: 4-(piperidin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylic acid ester; Compound 96: 4-(piperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate; Compound 97: 4-(4-methylpiperazin-1-yl)butyl-6-methoxybenzo[b]thiophene-2-carboxylate; Compound 98: 2-(diethylamino)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 99: 2-morpholinoethyl (6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 100: 2-(pyrrolidone-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 101: 2-(piperidin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 102: 2-(piperazin-1-yl)ethyl(6-methoxybenzo[b]thiophene-2-carbonyl)glycine; Compound 103: 2-(diethylamino)ethylthiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carboxylate; Compound 104: 2-(pyrrolidone-1-yl)ethylthiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxene-6-carboxylate; Compound 105: 2-(piperidin-1-yl)ethylthiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxy-6-carboxylate; Compound 107: 2-(4-methylpiperazin-1-yl)ethylthiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxane-6-carboxylate; Compound 108: (thieno[2',3':4,5]benzo[1,2-d][1,3]dioxy-6-carbonyl)glycine; Compound 109: (thieno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-alanine; Compound 110: (thieno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-valine; Compound 111: (thieno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)-d-leucine; Compound 112: (thieno[2',3':4,5]benzo[1,2-d][1,3]dioxy-6-carbonyl)-d-phenylalanine; Compound 113: 2-(dimethylamino)ethyl(thieno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carbonyl)glycine ester; Compound 114: N-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)thiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide; Compound 115: N-(2-oxo-2-((2-(pyrrolidone-1-yl)ethyl)amino)ethyl)thiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxen-6-carboxamide; Compound 116: N-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)thiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxoen-6-carboxamide; Compound 117: N-(2-(pyrrolidone-1-yl)ethyl)thiopheno[2',3':4,5]benzo[1,2-d][1,3]dioxene-6-carboxamide.

2. A pharmaceutical composition, characterized in that, It comprises the substituted benzo[a] pentagonal unsaturated heterocyclic compound of claim 1 or its pharmaceutical salt, and one or more pharmaceutically acceptable carriers or excipients.

3. The use of the substituted benzo[a]penta-unsaturated heterocyclic compound of claim 1 or its pharmaceutical salt in the preparation of products for treating or preventing aging.

4. The application according to claim 3, characterized in that, The products include pharmaceuticals and cosmetics.

5. The use of the substituted benzo[a]penta-unsaturated heterocyclic compound of claim 1 or its pharmaceutical salt in the preparation of a medicament for clearing or reducing senescent cells.

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