Cyclic bis-benzyl tetrahydroisoquinolines, processes for their preparation and uses thereof

Abstract

The present application provides a cyclic dibenzyl tetrahydroisoquinoline compound represented by formula (I), a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemate, a crystalline hydrate and a solvate thereof, wherein R1 and R2 are as defined in the specification. The present application also provides a preparation method of the compound and the use of the compound for preparing an inhibitor for inhibiting viruses, inflammation, fibrosis, abnormal differentiation of T cells and / or preventing and / or treating diseases related to viral respiratory tract infection and pneumonia, inflammation-related diseases, fibrosis-related diseases and autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the fields of medicinal chemistry and chemical synthesis. Specifically, this invention relates to cyclic bisbenzyltetrahydroisoquinoline compounds, their preparation methods, and applications. Background Technology

[0002] The vast majority of acute infectious diseases are viral, which have high morbidity and mortality rates. Due to limited testing and diagnostic methods, outbreaks caused by new viruses are often characterized by suddenness, randomness, and unpredictability. Once an outbreak occurs, without effective prevention and control measures, it can easily lead to large-scale epidemics, seriously threatening people's health and lives.

[0003] The novel coronavirus (SARS-CoV-2) is the seventh coronavirus discovered in humans. Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by this virus. Currently, the virus has swept the globe, and the number of infections and deaths has far exceeded that of SARS in 2003, seriously threatening human health and socio-economic development.

[0004] Although vaccines and antiviral drugs have been approved for marketing to treat severe pneumonia caused by the SARS-CoV-2 coronavirus, their safety and efficacy remain unsatisfactory. As of November 2021, multiple variants of the novel coronavirus have emerged, such as the South African strain, the Delta strain, and the Omicron strain, becoming the major circulating variants globally. These variants are characterized by high viral load, strong infectivity, insidiousness, and immune evasion, necessitating effective prevention, treatment, and medication in clinical practice. Therefore, developing low-toxicity and highly effective antiviral drugs against the SARS-CoV-2 coronavirus to meet the clinical needs of SARS-CoV-2 infected patients both domestically and internationally is of great social significance.

[0005] Cyclic bisbenzyltetrahydroisoquinoline compounds, such as berberine, berberine, and tetrandrine, possess excellent anti-lung injury, anti-inflammatory, anti-fibrotic, and immunomodulatory effects, have attracted the attention of those skilled in the art. Since the outbreak of the COVID-19 pandemic, the inventors have screened a large number of natural products for antiviral activity and found that cyclic bisbenzyltetrahydroisoquinoline alkaloids such as berberine, berberine, and tetrandrine have anti-COVID-19 effects; however, their antiviral activity is weak and their cytotoxicity is relatively high, resulting in a very low selectivity index / therapeutic index (SI), posing potential safety risks.

[0006] In summary, there is an urgent need in this field to develop novel cyclic bisbenzyltetrahydroisoquinoline compounds with higher safety profiles to inhibit SARS-CoV-2 coronavirus and to treat pneumonia caused by novel coronavirus infection.

[0007] In addition, the inventors explored other potential uses for cyclic bisbenzyltetrahydroisoquinoline compounds, discovering that they inhibit CD4. + The role of T cell differentiation in vitro. CD4 + T cell (Th1 and Th17 cells)-mediated autoimmunity is considered a significant cause of multiple sclerosis. Th1CD4 cells produce interferon-γ (IFN-γ). + T cells and Th17CD4 cells that secrete interleukin-17 (IL-17A) + T cells play a crucial role in a mouse model of experimental allergic encephalomyelitis (EAE). T cell polarization, leukocyte migration, and infiltration into the central nervous system are essential steps in the pathogenesis of EAE. Increasing evidence suggests that Th17 cells (characterized by IL-17 production) are just as important as Th1 cells in the pathogenesis of multiple sclerosis (MS). For example, mice with low Th17 cell counts are less likely to develop EAE, and Th17 cells have been identified in brain lesions from MS patients. However, there are still no specific drugs for the treatment of MS. Most of the identified regulatory factors are transcription factors or cytokines. Small molecules that can regulate the differentiation of Th1 and Th17 cells, especially those that can simultaneously inhibit the differentiation of Th1 and Th17 cells, have not been reported. These molecules have potential applications in the treatment of multiple sclerosis.

[0008] Th17 cells are involved in maintaining immune homeostasis, particularly on the intestinal mucosa, and contribute to chronic inflammation associated with autoimmune diseases such as rheumatoid arthritis. Th17 cells play a crucial role in the pathogenesis of rheumatoid arthritis by mediating immune dysregulation, especially in the early stages of disease progression. Inhibiting excessive differentiation of Th17 cells can slow or prevent the onset and progression of rheumatoid arthritis.

[0009] Th17 cells play a crucial role in the host's defense against foreign pathogens. Besides their role in infectious diseases, Th17 cells are also involved in the pathogenesis of liver diseases such as viral hepatitis, autoimmune liver diseases, and metabolic liver diseases. Pathogenic Th17 cells also play a key role in liver diseases that develop in response to toxicity or metabolic damage, such as hepatitis B virus (HBV) / hepatitis C virus (HCV) infection, non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), hepatocellular carcinoma (HCC), and cholestatic liver disease. The number of Th17 cells is increased in the liver and serum of patients with various forms of acute and chronic liver injury. Th17 cells secrete two cytokines, IL-17 and IL-22, which are important for the development of inflammation. The IL-17 receptor is expressed in many different cell types, including monocytes, cholangiocellular cells, and hepatic stellate cells. Receptor activation induces the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, TNF, and TGFβ. IL-17 also directly induces the production of type I fibrillary collagen by hepatic stellate cells by activating the signal transducer and activator of transcription 3 (STAT3) signaling pathways. Additionally, IL-22 has a pro-inflammatory effect in hepatitis B virus infection. Therefore, compounds that inhibit excessive Th17 differentiation may have a role in alleviating liver fibrosis, hepatitis B virus (HBV) / hepatitis C virus (HCV) infectious liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), hepatocellular carcinoma (HCC), and cholestatic liver disease.

[0010] IL-17A, a glycoprotein secreted by Th17 cells, is also a pro-inflammatory cytokine involved in chronic inflammation and autoimmune diseases. IL-17A participates in the occurrence and progression of pulmonary fibrosis in a TGF-β1-dependent and independent manner, and the IL-17A signaling pathway is a potential therapeutic target for fibrotic lung disease. Inhibiting IL-17A secretion can also improve silica-induced lung dysfunction, lung inflammation, and fibrosis in mice. Therefore, inhibiting the excessive differentiation of Th17 cells that secrete IL-17A can reduce IL-17A levels, thereby alleviating the progression of various pulmonary fibrosis diseases, including silicosis.

[0011] Few studies have reported small molecules with good druggability that can simultaneously inhibit the differentiation of Th1 and Th17 cells. However, these molecules have potential applications in the treatment of autoimmune diseases such as rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, systemic sclerosis, neuromyelitis optica, autoimmune enteritis, and autoimmune hepatitis, as well as liver fibrosis, pulmonary fibrosis, renal fibrosis, scleroderma, and silicosis. They are also expected to alleviate liver diseases, including liver fibrosis, hepatitis B virus (HBV) / hepatitis C virus (HCV) infectious liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), hepatocellular carcinoma (HCC), and cholestatic liver disease. Summary of the Invention

[0012] The purpose of this invention is to provide a safer inhibitor for inhibiting the activity of viruses, especially SARS-CoV-2 coronavirus, as well as its preparation method and its use in treating, preventing and alleviating diseases caused by viral infections, especially pneumonia caused by novel coronavirus infection.

[0013] Another object of the present invention is to provide a compound with anti-inflammatory effects, a method for preparing the same, and its use in treating, preventing and alleviating inflammatory diseases such as pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, and inflammatory pain.

[0014] Another object of the present invention is to provide a compound with anti-fibrotic activity, a method for preparing the same, and its use in treating, preventing and alleviating fibrosis-related diseases such as pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis, and inflammatory pain.

[0015] Another object of the present invention is to provide an inhibitor of T cell differentiation, particularly an inhibitor of Th1 and Th17 cell differentiation, a method for preparing the inhibitor, and its use in treating, preventing and alleviating autoimmune diseases related to abnormal T cell differentiation, such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity reaction, and graft-versus-host disease.

[0016] In a first aspect of the invention, a cyclic bisbenzylisoquinoline compound of formula I is provided, or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, racemate thereof, or a hydrate, solvate thereof, or a mixture thereof.

[0017]

[0018] In the formula,

[0019] R1 and R2 are each independently selected from: hydrogen, halogen, nitro, hydroxyl, mercapto, C1-C6 alkoxy, halogenated C1-C6 alkoxy, hydroxylated C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkyl, halogenated C1-C6 alkyl, amino C1-C6 alkyl, hydroxylated C1-C6 alkyl, cyano C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, C2-C6 alkenyl, C2-C6 alkenylcarbonyl, C2-C6 alkenyloxy, C2-C6 alkynyl, C2-C6 alkynyloxy, amino, C1-C6 alkyl-substituted amino, benzyl-substituted amino, C1-C6 alkylacyl-substituted amino, C2-C6 alkenylacyl-substituted amino, cyano, C1-C6 carboxyl, C1-C6 aldehyde, C1-C6 alkylacyl, C3-C6 cycloalkylacyl The following are listed: carboxyl, halogenated C1-C6 alkyl acyl, sulfonylamino, C1-C6 alkyl-substituted sulfonylamino, carbamoyl, phenylcarbamoyl, N-methyl-N-methoxyamino, C1-C6 alkyl-substituted carbamoyl, C3-C6 cycloalkyl-substituted carbamoyl, adamantylcarbamoyl, pyridine or pyrimidinyl-substituted carbamoyl, carbamoyl substituted with hydroxy-C1-C6 alkoxy-C1-C6 alkyl, carbamoyl substituted with C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkoxycarbonyl, phenoxycarbonyl, hydroxymethyl substituted with C3-C6 cycloalkyl, carboxyl-C1-C6 alkyl, C1-C6 alkyl sulfonyl, halogenated C1-C6 alkyl sulfonyl, C1-C6 alkyl-substituted amino-C1-C6 alkyl, C1-C6... 20 Alkyl-substituted carbamoyloxy (or C1-C6 alkyl-substituted carbamoyloxy), C3-C 10 Cycloalkyl-substituted carbamoyloxy, C1-C6 alkanoyl-substituted aminoC1-C6 alkyl, C1-C6 alkoxycarbonyl, carbamoylC1-C6 alkyl, C1-C6 alkyl-substituted carbamoylC1-C6 alkyl, C2-C6 alkenyloxy (C2-C6 alkenyl-CO-O-), C2-C 10 ester group, Or -OZ, where Z is

[0020] Where n is 0, 1, 2, 3, or 4; m is 1, 2, 3, or 4;

[0021] The phenylcarbamoyl group and phenoxycarbonyl group thereon are not necessarily replaced by one or more substituents selected from F, Cl, Br, I, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy or halo-C1-C6 alkoxy.

[0022] Alternatively, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted, wherein the substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo groups, thio groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, and cyano groups; and the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0023] An additional condition is that the compound does not contain the following compounds:

[0024]

[0025] In another preferred embodiment, the compound does not contain the following compounds:

[0026]

[0027] In another preferred embodiment, the compound of formula I is a non-natural product.

[0028] In another preferred embodiment, the benzo[5-6 membered monocyclic heterocycle] is selected from:

[0029] In another preferred embodiment, in the compound, R1 and R2 are each independently selected from hydrogen, halogen, nitro, hydroxyl, mercapto, C1-C4 alkoxy, halogenated C1-C4 alkoxy, hydroxylated C1-C4 alkoxy, C1-C4 alkyl, C1-C4 alkyl, amino C1-C4 alkyl, hydroxylated C1-C4 alkyl, cyano C1-C4 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl, etc. Alkoxy, C2-C5 alkenyl, C2-C5 alkenylcarbonyl, C2-C5 alkenoxy, C2-C5 alkynyl, C2-C5 alkynoxy, amino, C1-C4 alkyl-substituted amino, benzyl-substituted amino, C1-C4 alkanoyl-substituted amino, C2-C5 alkenylyl-substituted amino, cyano, C1-C4 carboxyl, C1-C4 aldehyde, C1-C4 alkanoyl, C3-C5 cycloalkylyl, halogenated C 1–C4 alkyl acyl, sulfonamide (-SO2NH2), C1–C4 alkyl-substituted sulfonamide (-SO2NH2), carbamoyl (-CONH2), phenylcarbamoyl, N-methyl-N-methoxyamino, C1–C4 alkyl-substituted carbamoyl, C3–C5 cycloalkyl-substituted carbamoyl, adamantyl carbamoyl, pyridine or pyrimidinyl-substituted carbamoyl, carbamoyl substituted with hydroxy C1–C4 alkoxy C1–C4 alkyl, carbamoyl substituted with C1–C4 alkoxy C1–C4 alkyl, C1–C4 alkoxy carbonyl, phenoxy carbonyl, hydroxymethyl substituted with C3–C5 cycloalkyl, carboxyl C1–C4 alkyl, C1–C4 alkyl sulfonyl, halo-C1–C4 alkyl sulfonyl, C1–C4 alkyl-substituted amino C1–C4 alkyl, C1–C 10 Alkyl-substituted carbamoyloxy (or C1-C4 alkyl-substituted carbamoyloxy), C3-C8 cycloalkyl-substituted carbamoyloxy, C1-C4 alkanoyl-substituted aminoC1-C4 alkyl, C1-C4 alkoxycarbonyl, carbamoylC1-C4 alkyl, C1-C4 alkyl-substituted carbamoylC1-C4 alkyl, C2-C5 alkenyl acyloxy (C2-C5 alkenyl-CO-O-), C2-C6 ester group, Or -OZ, where Z is or n is 0, 1, 2, 3, or 4; m is 1, 2, 3, or 4;

[0030] Alternatively, R1 and R2 together with the adjacent benzene ring can form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 2 substituents, either substituted or unsubstituted; wherein the substituted substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo (=O), thio (=S), C1-C6 alkyl groups, C1-C6 alkoxy groups, and cyano groups; wherein the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0031] In another preferred embodiment, in the compound, R1 and R2 are each independently selected from: hydrogen, fluorine, chlorine, bromine, nitro, hydroxyl, mercapto, methoxy, ethoxy, trifluoromethoxy, -SCH3, -SCH2CH3, propyl, cyclopropyl, isopropyl, tert-butyl, trifluoromethyl, difluoromethoxy, bromomethyl, chloromethyl, vinyl, vinylmethyl, amino, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, cyano, carboxyl, aldehyde, -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CN, -CH2CH2CN, formyl, acetyl, propionyl, trifluoroacetyl, sulfonylamino, carbamoyl, N-methylamino N,N-dimethylcarbamoyl, N-ethylcarbamoyl, N,N-diethylcarbamoyl, -O(C=O)NH (C4 alkyl), cyclopentylcarbamoyloxy, cyclohexylcarbamoyloxy, -CH2CO2H, -CH2CH2CO2H, -SO2CH3, -SO2CF3, -CH2NHMe, -CH2NMe2, -CH2CONH2, -CONH2, -NHCOCH3, -CH2NHCOCH3, -(C=O)OCH2CH2OCH2CH2OH, -(C=O)OCH3, -CH2CONHMe, -CONHMe, -CONH(cyclopropyl), -CH2CONMe2, or -OZ, where Z is... or n is any integer from 0 to 4;

[0032] Alternatively, R1 and R2 together with the adjacent benzene ring can form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 2 substituents, either substituted or unsubstituted, wherein the substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo groups, thio groups, and C1-C6 alkyl groups; and the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0033] In another preferred embodiment, R1 is hydrogen, fluorine, chlorine, bromine, mercapto, methoxy, ethoxy, trifluoromethoxy, -SCH3, -SCH2CH3, propyl, cyclopropyl, isopropyl, tert-butyl, trifluoromethyl, difluoromethoxy, vinyl, vinylmethyl, amino, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, cyano, carboxyl, aldehyde, -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CN. -CH2CH2CN, formyl, acetyl, propionyl, trifluoroacetyl, sulfonylamino, carbamoyl, N-methylcarbamoyl, N,N-dimethylcarbamoyl, N-ethylcarbamoyl, N,N-diethylcarbamoyl, -CH2CO2H, -CH2CH2CO2H, -CH2NHMe, -CH2Nme2, -CH2CONH2, -NHCOCH3, -CH2NHCOCH3, -CH2CONHMe or -CH2CONMe2.

[0034] In another preferred embodiment, R1 is carbamoyl, N-methylcarbamoyl, N,N-dimethylcarbamoyl, N-ethylcarbamoyl, N-n-propylcarbamoyl, N-isopropylcarbamoyl, N-cyclopropylcarbamoyl, N,N-diethylcarbamoyl, or N-cyclopropylcarbamoyl.

[0035] In another preferred embodiment, R2 is hydrogen, fluorine, chlorine, bromine, nitro, amino, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, sulfonamide, or -NHCOCH3.

[0036] In another preferred embodiment, the compound of formula I is chiral or achiral.

[0037] In another preferred embodiment, the compound has the structure shown in formula Ia:

[0038]

[0039] In another preferred embodiment, the compound has the structure shown in formula Ib:

[0040]

[0041] In another preferred embodiment, the compound of formula (I) is selected from the following compounds:

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] In another preferred embodiment, the compound of formula (I) is selected from the following compounds:

[0048]

[0049] In a second aspect of the invention, a method for preparing the cyclic bisbenzylisoquinoline compounds described in the first aspect is provided, the method being selected from the group consisting of:

[0050] a) Using cyclic bisbenzylisoquinoline containing phenolic hydroxyl groups as raw materials, and alkylating agents, the cyclic bisbenzylisoquinoline compounds are obtained by alkylation reaction;

[0051] b) Using cyclic bisbenzylisoquinoline containing phenolic hydroxyl groups as raw materials, and acylation reagents are used to acylate the cyclic bisbenzylisoquinoline compounds to obtain the cyclic bisbenzylisoquinoline compounds;

[0052] c) Using spiky alkali as a raw material, the compound (I-1b) was obtained by reacting it with a sulfonating agent in the presence of a base.

[0053] (I-1b) and the coupling reagent undergo a coupling reaction to yield the cyclic bisbenzylisoquinoline compound I-1, as shown in reaction formula 1:

[0054]

[0055] In Formula I-1, R1 is selected from hydrogen, halogen, C1-C6 alkylthio, C1-C6 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyl-substituted amino, benzyl-substituted amino, cyano, carboxyl, aldehyde, and C1-C6 alkylacyl.

[0056] In formula 1-1b, L is selected from: leaving groups;

[0057] d) Using alkali as a raw material, compound (I-2) was prepared by nitration, reduction, and cyclization reactions, as shown in the following reaction formula 2:

[0058]

[0059] In Formula I-2, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted.

[0060] e) Using alkali as a raw material, compound of formula (I-3) is prepared through a multi-step reaction, as shown in reaction formula 3:

[0061]

[0062] In Formula I-3, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted.

[0063] f) The compounds obtained by methods a) to e) are subjected to functional group transformation to obtain the cyclic bisbenzylisoquinoline compounds.

[0064] In the above method, the raw material "sharpenone" exists in the form of sharpenone, its pharmaceutically acceptable salt, or its enantiomer, diastereomer, racemic mixture, or its hydrated crystals, solvates, or mixtures thereof.

[0065] In another preferred embodiment, the method is as follows: using spiky alkali as a raw material, a condensation acylation reaction is carried out with carboxylic acid to obtain the cyclic bisbenzyl isoquinoline compound.

[0066] In another preferred embodiment, the condensation acylation reaction is carried out in the presence of a condensing agent selected from the group consisting of N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), or combinations thereof.

[0067] In another preferred embodiment, the leaving group L is selected from the group consisting of C1-C6 alkylsulfonyloxy, halogenated C1-C6 alkylsulfonyloxy, benzenesulfonyloxy, naphthalenesulfonyloxy, or combinations thereof, preferably, L is methanesulfonyloxy or trifluoromethanesulfonyloxy.

[0068] In another preferred embodiment, the sulfonating agent is selected from: C1-C6 alkyl sulfonyl chloride, C1-C6 alkyl sulfonic anhydride, benzene sulfonyl chloride, benzene sulfonic anhydride, naphthalene sulfonyl chloride, naphthalene sulfonic anhydride, preferably methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride.

[0069] In another preferred embodiment, the solvent is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, acetonitrile, toluene, acetone, dioxane, and chloroform.

[0070] In another preferred embodiment, the base is selected from inorganic or organic bases.

[0071] In another preferred embodiment, the inorganic base is selected from: sodium hydroxide, potassium hydroxide, cesium hydroxide, barium hydroxide, potassium hydride, sodium hydride, sodium tert-butoxide, potassium tert-butoxide, potassium carbonate, sodium carbonate, or calcium carbonate; the organic base is selected from: pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline, and N,N-dimethylpyridine.

[0072] In another preferred embodiment, the coupling reaction is carried out in the presence of a palladium catalyst and a base.

[0073] In another preferred embodiment, the palladium catalyst is selected from: palladium acetate (Pd(OAc)2), bis(triphenylphosphine)palladium dichloride ((Ph3P)2PdCl2), bis(benzonitrile)palladium chloride ((PhCN)2PdCl2), tetra(triphenylphosphine)palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium acetate ((Ph3P)2Pd(Oac)2), 1,2-bis(diphenylphosphino)ethanepalladium dichloride ((PdCl2(d [1,3-bis(diphenylphosphine)ethane]palladium (Pd(dppe)2), [1,3-bis(diphenylphosphine)propane]palladium (PdCl2(dippp)) and [1,1'-bis(diphenylphosphine)ferrocene]palladium (Pd(dppf)Cl2), or combinations thereof.

[0074] In another preferred embodiment, reaction formula 1 may also include other reaction solvents that do not interfere with the reaction.

[0075] In another preferred embodiment, a suitable ligand may be added to reaction formula 1 as a reaction promoter.

[0076] In another preferred embodiment, the suitable ligand is selected from: 2,2'-diphenylphosphino-1,1'-binaphthyl (BINAP), tri-tert-butyl (P(t-Bu)3), 1,1'-di-(diphenylphosphino)ferrocene (dppf), 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (x-phos), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (Xantphos), tri-tert-butylphosphine tetrafluoroborate and tris(2-methylphenyl)phosphine (P(o-tolyl)3), or combinations thereof.

[0077] In another preferred embodiment, the coupling agent is selected from the group consisting of: C1-C6 alkylboronic acid, cyclopropylboronic acid, benzylamine, potassium cyanide, zinc cyanide, tributylvinyltin, CO, CO2, formic acid, sodium formate, lithium formate, C1-C6 alkylthiolate sodium, sodium methanesulfonate, or combinations thereof.

[0078] In another preferred embodiment, the benzo[5-6 membered monocyclic heterocycle] is selected from:

[0079] In another preferred embodiment, for benzo[5-6 membered monocyclic heterocycle], the substituent is selected from halogen, hydroxyl, mercapto, amino, oxo (=O), thio (=S), C1-C6 alkyl; the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0080] In another preferred embodiment, the nitrating agent in the nitration reaction is selected from: nitric acid, a mixture of concentrated sulfuric acid and nitric acid, a mixture of nitric acid, sodium nitrate and concentrated sulfuric acid, a mixture of potassium nitrate and concentrated sulfuric acid, a mixture of sodium nitrite and concentrated sulfuric acid, and a mixture of acetic acid and nitric acid.

[0081] In another preferred embodiment, the nitrating agent in the nitration reaction is a mixture of acetic acid and nitric acid.

[0082] In another preferred embodiment, the mixing ratio of the mixture is not limited.

[0083] In another preferred embodiment, the nitration reaction temperature is -20°C to room temperature.

[0084] In another preferred embodiment, the reduction reaction uses Pd / C as a catalyst.

[0085] In another preferred embodiment, the reduction reaction uses hydrogen, ammonium formate, or formic acid as the reducing agent.

[0086] In another preferred embodiment, the reduction reaction is carried out in a lower alcohol or a lower alcohol-water mixed solvent.

[0087] In another preferred embodiment, the lower alcohol is selected from methanol, ethanol, isopropanol, or combinations thereof.

[0088] In another preferred embodiment, the reduction reaction is carried out in the range of 0–100°C, preferably 0–40°C.

[0089] In another preferred embodiment, the reduction reaction is carried out at atmospheric pressure.

[0090] In another preferred embodiment, the reduction reaction is hydrogenated for 1 to 10 hours.

[0091] In another preferred embodiment, the cyclization reaction is carried out in the presence of a cyclization reagent.

[0092] In another preferred embodiment, the cyclization reagent includes: phosgene, triphosgene, 1,1'-carbonyldiimidazole (CDI), urea, carbon tetrabromide, formic acid, trimethyl orthoformate, trimethyl orthoacetate, triethyl orthoacetate, acetyl chloride, chloroacetyl chloride, bromoacetyl bromide, bromoacetyl chloride, or combinations thereof.

[0093] In another preferred embodiment, the ring-closing reaction is carried out in the presence or absence of a base.

[0094] In another preferred embodiment, the base is selected from: inorganic bases or organic bases; wherein the inorganic base is selected from sodium hydroxide, potassium hydroxide, potassium hydride, sodium hydride, sodium tert-butoxide, potassium tert-butoxide, potassium carbonate, sodium carbonate, cesium carbonate and sodium bicarbonate; wherein the organic base is selected from pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline and N,N-dimethylpyridine;

[0095] In another preferred embodiment, reaction formula 2 may also include other reaction solvents that do not interfere with the reaction.

[0096] In another preferred embodiment, for benzo[5-6 membered monocyclic heterocycle], the substituent is selected from halogen, hydroxyl, mercapto, amino, oxo (=O), thio (=S), C1-C6 alkyl; the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0097] In another preferred embodiment, the benzo[5-6 membered monocyclic heterocycle] is selected from:

[0098] In formula I-3b, L is a leaving group.

[0099] In another preferred embodiment, the leaving group L is selected from the group consisting of: C1-C6 alkylsulfonyloxy, halogenated C1-C6 alkylsulfonyloxy, benzenesulfonyloxy, and naphthalenesulfonyloxy; preferably methanesulfonyloxy and trifluoromethanesulfonyloxy.

[0100] In another preferred embodiment, method e) includes the following steps:

[0101] The compound of formula (I-1a) undergoes an ortho-nitration reaction with a nitrating agent to produce the compound of formula (I-3a);

[0102] The compound of formula (I-3a) and the sulfonating agent react in a suitable solvent in the presence of a base to give the compound of formula (I-3b);

[0103] Compound e3) and benzylamine undergo a Buchwald-Hartig reaction to give compound (I-3c);

[0104] The compound of formula (I-3c) undergoes a reduction reaction to give the compound of formula (I-3d);

[0105] Compound (e5) of formula (I-3d) is given by cyclization reaction to compound (I-3).

[0106] In another preferred embodiment, the nitrating agent in step e1) is a mixture of concentrated sulfuric acid and nitric acid, a mixture of nitric acid, sodium nitrate and concentrated sulfuric acid, a mixture of potassium nitrate and concentrated sulfuric acid, a mixture of sodium nitrite and concentrated sulfuric acid, a mixture of acetic acid and nitric acid, preferably a mixture of acetic acid and nitric acid.

[0107] In another preferred embodiment, the mixing ratio of the mixture is not limited.

[0108] In another preferred embodiment, the nitration reaction temperature is -20°C to room temperature.

[0109] In another preferred embodiment, the nitration reaction time is 10 minutes to 12 hours.

[0110] In another preferred embodiment, the sulfonating agent in step e2) is selected from: C1-C6 alkyl sulfonyl chloride, C1-C6 alkyl sulfonic anhydride, benzene sulfonyl chloride, benzene sulfonic anhydride, naphthalene sulfonyl chloride, naphthalene sulfonic anhydride, preferably methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride.

[0111] In another preferred embodiment, the solvent in step e2) is selected from: dichloromethane, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, acetonitrile, toluene, acetone, dioxane, and chloroform.

[0112] In another preferred embodiment, the base in step e2) is selected from inorganic or organic bases; wherein the inorganic base is selected from sodium hydroxide, potassium hydroxide, cesium hydroxide, barium hydroxide, potassium hydride, sodium hydride, sodium tert-butoxide, potassium tert-butoxide, potassium carbonate, sodium carbonate, or calcium carbonate; wherein the organic base is selected from pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline, and N,N-dimethylpyridine.

[0113] In another preferred embodiment, the coupling reaction in step e3) is carried out in the presence of a palladium catalyst and a base.

[0114] In another preferred embodiment, the palladium catalyst is selected from: palladium acetate (Pd(OAc)2), bis(triphenylphosphine)palladium dichloride ((Ph3P)2PdCl2), bis(benzonitrile)palladium chloride ((PhCN)2PdCl2), tetra(triphenylphosphine)palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium acetate ((Ph3P)2Pd(OAc)2), 1,2-bis(diphenylphosphino)ethanepalladium dichloride ((PdCl2(d [1,3-bis(diphenylphosphine)ethane]palladium (Pd(dppe)2), [1,3-bis(diphenylphosphine)propane]palladium (PdCl2(dippp)) and [1,1'-bis(diphenylphosphine)ferrocene]palladium (Pd(dppf)Cl2), or combinations thereof.

[0115] In another preferred embodiment, the base is selected from: sodium bis(trimethoxy)amino, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate, potassium phosphate, sodium phosphate, sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide, potassium fluoride, sodium fluoride, tetrabutylammonium fluoride (TBAF), sodium acetate, potassium acetate, cesium carbonate, potassium carbonate, and sodium carbonate, or combinations thereof.

[0116] In another preferred embodiment, the reaction may also include other reaction solvents that do not interfere with the reaction.

[0117] In another preferred embodiment, step e3) can also involve adding a suitable ligand as a reaction promoter to carry out the above reaction.

[0118] In another preferred embodiment, the suitable ligand is selected from the group consisting of: 2,2'-diphenylphosphino-1,1'-binaphthyl (BINAP), tri-tert-butyl (P(t-Bu)3), 1,1'-di-(diphenylphosphino)ferrocene (dppf), 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (x-phos), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (Xantphos), tri-tert-butylphosphine tetrafluoroborate, or tri(2-methylphenyl)phosphine (P(o-tolyl)3).

[0119] In another preferred embodiment, the reduction reaction in step e4) uses Pd / C, palladium hydroxide, or palladium hydroxide on carbon as a catalyst.

[0120] In another preferred embodiment, the reduction reaction in step e4) uses ammonium formate or hydrogen as a reducing agent.

[0121] In another preferred embodiment, the reduction reaction is carried out in a lower alcohol or a lower alcohol-water mixed solvent.

[0122] In another preferred embodiment, the lower alcohol is selected from methanol, ethanol, isopropanol, or combinations thereof.

[0123] In another preferred embodiment, the reduction reaction is carried out in the range of 20–100°C.

[0124] In another preferred embodiment, the reduction reaction is carried out at atmospheric pressure.

[0125] In another preferred embodiment, step e5) the cyclization reaction is carried out in the presence of a cyclization reagent.

[0126] In another preferred embodiment, the cyclization reagent is selected from the group consisting of phosgene, triphosgene, 1,1'-carbonyldiimidazole (CDI), urea, formic acid, sodium nitrite, carbon disulfide, thiophosgene, triethyl orthoacetate, chloroacetic acid, bromoacetic acid, ethyl bromoacetate, methyl bromoacetate, chloroacetamide, or combinations thereof.

[0127] In another preferred embodiment, the ring-closing reaction is carried out in the presence or absence of a base.

[0128] In another preferred embodiment, the base is selected from inorganic bases or organic bases.

[0129] In another preferred embodiment, the inorganic base is selected from sodium hydroxide, potassium hydroxide, potassium hydride, sodium hydride, sodium tert-butoxide, potassium tert-butoxide, potassium carbonate, sodium carbonate, cesium carbonate, or sodium bicarbonate; wherein the organic base is selected from pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline, and N,N-dimethylpyridine.

[0130] In another preferred embodiment, step e5) may also include other reaction solvents that do not interfere with the reaction.

[0131] In another preferred embodiment, the leaving group L is selected from the group consisting of: C1-C6 alkylsulfonyloxy, halogenated C1-C6 alkylsulfonyloxy, benzenesulfonyloxy, and naphthalenesulfonyloxy; preferably methanesulfonyloxy and trifluoromethanesulfonyloxy.

[0132] In another preferred embodiment, the sulfonating agent is selected from: C1-C6 alkylsulfonyl chlorides, C1-C6 alkylsulfonic anhydrides, benzenesulfonyl chlorides, benzenesulfonic anhydrides, naphthalenesulfonyl chlorides, and naphthalenesulfonic anhydrides, preferably methanesulfonyl chlorides, methanesulfonic anhydrides, trifluoromethanesulfonyl chlorides, and trifluoromethanesulfonic anhydrides. The solvent is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, acetonitrile, toluene, acetone, dioxane, and chloroform.

[0133] In another preferred embodiment, the base is selected from inorganic or organic bases.

[0134] In another preferred embodiment, the inorganic base is selected from sodium hydroxide, potassium hydroxide, cesium hydroxide, barium hydroxide, potassium hydride, sodium hydride, sodium tert-butoxide, potassium tert-butoxide, potassium carbonate, sodium carbonate, and calcium carbonate; the organic base is selected from pyridine, triethylamine, diisopropylethylamine, N,N-dimethylaniline, and N,N-dimethylpyridine.

[0135] In another preferred embodiment, the coupling reaction is carried out in the presence of a palladium catalyst and a base.

[0136] In another preferred embodiment, the palladium catalyst is selected from the group consisting of: palladium acetate (Pd(OAc)2), bis(triphenylphosphine)palladium dichloride ((Ph3P)2PdCl2), bis(benzonitrile)palladium chloride ((PhCN)2PdCl2), tetra(triphenylphosphine)palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium acetate ((Ph3P)2Pd(OAc)2), 1,2-bis(diphenylphosphino)ethanepalladium dichloride ((PdCl2) [1,3-bis(diphenylphosphine)ethane]palladium (Pd(dppe)2), [1,1'-bis(diphenylphosphine)ferrocene]palladium (Pd(dba)2), [1,1'-bis(diphenylphosphine)ferrocene]palladium (Pd(dppf)Cl2), or combinations thereof.

[0137] In another preferred embodiment, the base is selected from: sodium bis(trimethoxy)amino, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate, potassium phosphate, sodium phosphate, sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide, potassium fluoride, sodium fluoride, tetrabutylammonium fluoride (TBAF), sodium acetate, potassium acetate, cesium carbonate, potassium carbonate, sodium carbonate, or combinations thereof.

[0138] In another preferred embodiment, reaction formula 4 may also include other reaction solvents that do not interfere with the reaction.

[0139] In another preferred embodiment, a suitable ligand may be added to reaction formula 4 as a reaction promoter to carry out the above reaction.

[0140] In another preferred embodiment, the suitable ligand is selected from: 2,2'-diphenylphosphino-1,1'-binaphthyl (BINAP), tri-tert-butyl (P(t-Bu)3), 1,1'-di-(diphenylphosphino)ferrocene (dppf), 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (x-phos), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (Xantphos), tri-tert-butylphosphine tetrafluoroborate, and tris(2-methylphenyl)phosphine (P(o-tolyl)3). The coupling agent includes, but is not limited to, C1-C6 alkylboronic acid, cyclopropylboronic acid, benzylamine, potassium cyanide, zinc cyanide, tributylvinyltin, CO, CO2, formic acid, sodium formate, lithium formate, C1-C6 alkylthiolate sodium, and sodium methanesulfonate.

[0141] In another preferred embodiment, the functional group transformation reaction includes: oxidative hydrolysis reaction, borohydride-oxidation reaction, condensation acylation reaction, reduction reaction, acylation reaction, esterification reaction, Grignard reaction, chlorination reaction, or bromination reaction.

[0142] In another preferred embodiment, the oxidative hydrolysis reaction is carried out in the presence of an oxidant and a base.

[0143] In another preferred embodiment, the oxidative hydrolysis system includes: hydrogen peroxide / sodium hydroxide, hydrogen peroxide / potassium hydroxide, hydrogen peroxide / potassium carbonate, and hydrogen peroxide / sodium carbonate.

[0144] In another preferred embodiment, the hydroboration-oxidation reaction involves the addition of a boron reagent to an olefin group, followed by oxidative hydrolysis to an alcohol.

[0145] In another preferred embodiment, the boron reagent includes: borane, 9-BBN;

[0146] In another preferred embodiment, the condensation acylation reaction is carried out in the presence of a condensing agent.

[0147] In another preferred embodiment, the condensing agent includes: N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU).

[0148] In another preferred embodiment, the reduction reaction is carried out in the presence of a reducing agent.

[0149] In another preferred embodiment, the reducing agent includes: hydrogen, ammonium formate, sodium borohydride, potassium borohydride, diisobutylaluminum hydride (DIBAL), and borane.

[0150] In another preferred embodiment, the acylation reaction is carried out in the presence of an acylation reagent.

[0151] In another preferred embodiment, the acylation reagent includes acetyl chloride, acetic anhydride, propionyl chloride, propionic anhydride, methanesulfonyl chloride, etc.

[0152] In another preferred embodiment, the esterification reaction system includes, but is not limited to, thionyl chloride / methanol, thionyl chloride / ethanol, etc.

[0153] In another preferred embodiment, the chlorination reaction is carried out in the presence of a chlorination reagent.

[0154] In another preferred embodiment, the chlorination agent includes, but is not limited to, thionyl chloride, phosphorus pentachloride, and N-chlorosuccinimide (NCS).

[0155] In another preferred embodiment, the Grignard reaction is carried out in the presence of a Grignard reagent.

[0156] In another preferred embodiment, the Grignard reagent includes, but is not limited to, methyl magnesium bromide, methyl magnesium chloride, methyl magnesium iodide, etc.

[0157] In another preferred embodiment, the bromination reaction is carried out in the presence of a brominating reagent.

[0158] In another preferred embodiment, the brominating agent includes, but is not limited to, elemental bromine, N-bromosuccinimide (NBS), etc.

[0159] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0160] (A1) A first active ingredient, comprising a therapeutically effective amount of one or more of the following: a cyclic bisbenzylisoquinoline compound of formula I as described in any one of the first aspects, its enantiomers, diastereomers, racemates, and pharmaceutically acceptable salts, hydrates, and solvates; and

[0161] (B) Pharmaceutically acceptable carriers.

[0162] In another preferred embodiment, the pharmaceutical composition further contains (A2) a second active ingredient.

[0163] In another preferred embodiment, the second active ingredient is selected from: (Y1) RNA replication enzyme inhibitors (such as Remdesivir, Favipiravir, Molnupiravir); (Y2) 3CL enzyme inhibitors (such as nirmatrelvir); (Y3) antibodies; (Y4) any combination of Y1 to Y3 above.

[0164] In another preferred embodiment, the antibody includes an antibody against coronaviruses.

[0165] In a fourth aspect of the invention, the use of the cyclic bisbenzyltetrahydroisoquinoline compound described in the first aspect or the pharmaceutical composition described in the third aspect is provided, namely, for the preparation of inhibitors that inhibit viral replication, inflammation, fibrosis, and abnormal T-cell differentiation; and / or for the preparation of drugs for the prevention and / or treatment of diseases related to viral infection, inflammation-related diseases (pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, inflammatory pain), fibrosis-related diseases (pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis), autoimmune diseases related to abnormal differentiation of Th1 and / or Th17 (psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity reaction, graft-versus-host disease, etc.), osteoporosis, and neurodegenerative diseases.

[0166] In another preferred embodiment, the virus is selected from: filoviruses, flaviviruses, paramyxoviruses, arenaviruses, coronaviruses, or combinations thereof.

[0167] In another preferred embodiment, the filamentous virus is selected from Marburg virus and Ebola virus.

[0168] In another preferred embodiment, the coronavirus is selected from the group consisting of: coronaviruses that infect humans, Severe acute respiratory syndrome coronavirus (SARS-CoV), 2019 novel coronavirus and its variants (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), coronaviruses that cause the common cold, or combinations thereof.

[0169] In another preferred embodiment, the coronavirus that causes the common cold is selected from: human coronavirus OC. 43 (Humancoronavirus OC 43 Human coronavirus 229E, Human coronavirus NL63, and Human coronavirus HKUl.

[0170] In another preferred embodiment, the related diseases caused by the coronavirus infection are selected from the group consisting of: common cold, high-risk symptomatic infection, respiratory infection, pneumonia and its complications caused by human coronaviruses, novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2, novel coronavirus infectious disease caused by SARS-CoV-2, or combinations thereof.

[0171] In another preferred embodiment, the related diseases caused by infection with the 2019 novel coronavirus are selected from the group consisting of respiratory infections, pneumonia and its complications, or combinations thereof.

[0172] In another preferred embodiment, the use is for the preparation of (a) inhibitors that inhibit the replication of the 2019 novel coronavirus and its variants (SARS-CoV-2); and / or (b) medicaments for the treatment and / or prevention and relief of diseases related to infection with the 2019 novel coronavirus (SARS-CoV-2).

[0173] In another preferred embodiment, the disease is pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, inflammatory pain, pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity reaction, graft-versus-host disease, osteoporosis, neurodegenerative diseases, or combinations thereof.

[0174] In another preferred embodiment, the use is for preparing a medicament for treating and / or preventing or alleviating diseases related to inflammation, fibrosis, and abnormal T-cell differentiation.

[0175] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0176] Figure 1 The inhibitory effects of compounds 1, 2, 3, 4, 6, and 58 on Th1 / Th17 differentiation were demonstrated.

[0177] Figure 2 Compounds 1, 2, 3, 4, 6, and 58 were shown not to affect cell viability in LPS-induced inflammation models.

[0178] Figure 3Compounds 1, 2, 4, and 58 were shown to significantly inhibit inflammatory factors in an LPS-induced inflammation model.

[0179] Figure 4 Compounds 1, 2, 3, 4, 6, and 58 were shown not to affect cell viability in a TNF-α-induced inflammation model.

[0180] Figure 5 Compounds 1, 2, 3, 4, 6, and 58 were shown to significantly inhibit inflammatory factors in a TNF-α-induced inflammation model.

[0181] Figure 6 Compound 4 was shown to significantly inhibit LPS-induced acute inflammation in mice.

[0182] Figure 7 Compound 1 was shown to significantly improve cell survival in a TNF-α-induced A549 lung cell injury model.

[0183] Figure 8 Compounds 3 and 58 were shown to significantly inhibit the damage factor in a TNF-α-induced A549 lung cell injury model.

[0184] Figure 9 Compounds 1, 2, 3, 4, 6, and 58 were shown to significantly alleviate H2O2-induced ALM12 hepatocyte damage.

[0185] Figure 10 and Figure 11 Compounds 1, 2, and 4 were shown to alleviate TGF-β1-induced fibrosis in A549 lung epithelial cells.

[0186] Figure 12 Compounds 2 and 4 were shown to alleviate CCl4-induced acute liver fibrosis in mice. Detailed Implementation

[0187] Through extensive and in-depth research and screening, the inventors have unexpectedly developed, for the first time, a class of active ingredients that can effectively inhibit the replication of coronaviruses such as SARS-CoV-2 (2019 novel coronavirus). Experiments show that the active ingredients of this invention (cyclic bisbenzylisoquinoline compounds of Formula I or their pharmaceutically acceptable salts, or their enantiomers, diastereomers, racemates, or their hydrates, solvates, or mixtures thereof) can efficiently inhibit the replication and activity of coronaviruses such as SARS-CoV-2. This invention was completed based on this.

[0188] Surprisingly, compared with natural compounds such as berberine, berberine, and tetrandrine, the cyclic bisbenzylisoquinoline compounds described in Formula I have significantly improved anti-coronavirus activity (the antiviral activity can be increased by about 10 times or more) and / or have lower cytotoxicity. Therefore, they have a better therapeutic index (SI) and can be used for the prevention and treatment of coronaviruses such as SARS-CoV-2, showing good prospects for clinical application.

[0189] In addition, the inventors explored other potential effects of cyclic bisbenzylisoquinoline compounds, discovering that they inhibit CD4. + The role of T cell differentiation. Experiments have shown that the active ingredient of this invention (the cyclic bisbenzylisoquinoline compound of Formula I or a pharmaceutically acceptable salt thereof, or its enantiomers, diastereomers, racemates, or its hydrates, solvates, or mixtures thereof) can effectively inhibit diseases related to viral infection, inflammation-related diseases (pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, inflammatory pain), fibrosis-related diseases (pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis), autoimmune diseases related to abnormal differentiation of Th1 and / or Th17 (psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity, graft-versus-host disease, etc.), osteoporosis, and neurodegenerative diseases.

[0190] the term

[0191] As used herein, the terms "compounds of the present invention," "cyclic bisbenzylisoquinoline compounds," "compounds of the present invention for inhibiting coronavirus activity," and "compounds of the present invention for inhibiting coronavirus replication" are used interchangeably to refer to cyclic bisbenzylisoquinoline compounds with excellent inhibitory effects on coronavirus replication, including compounds represented by Formula I, or pharmaceutically acceptable salts thereof, or their enantiomers, diastereomers, racemates, or their crystalline hydrates, solvates, or mixtures thereof. The compounds of the present invention do not include natural compounds (such as tetrandrine, tetrandrine B, and scutellarin).

[0192] As used herein, "formulation of the present invention" refers to a formulation containing the compounds of the present invention.

[0193] As used herein, the term “comprising” or its variations such as “including” or “comprises” are to be understood as including the stated elements or components without excluding other elements or other components.

[0194] As used herein, the terms “novel coronavirus,” “2019-nCov,” or “SARS-CoV-2” are used interchangeably. The 2019 novel coronavirus is the seventh known coronavirus to infect humans and causes COVID-19, one of the most serious infectious diseases threatening human health worldwide.

[0195] As used herein, halogens typically refer to fluorine, chlorine, bromine, and iodine; preferably fluorine, chlorine, or bromine; more preferably fluorine or chlorine.

[0196] C1 to C6 alkyl refers to a straight-chain or branched saturated hydrocarbon group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, isohexyl, 3-methylpentyl or n-hexyl, etc., preferably methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl or tert-butyl.

[0197] Halogenated C1-C6 alkyl groups refer to straight-chain or branched saturated hydrocarbon groups containing 1-6 carbon atoms in which one or more identical or different halogen atoms are substituted for hydrogen atoms. Examples include trifluoromethyl, fluoromethyl, difluoromethyl, chloromethyl, bromomethyl, dichlorofluoromethyl, chloroethyl, bromopropyl, 2-chlorobutyl, or pentafluoroethyl.

[0198] C1 to C6 alkoxy groups refer to straight-chain or branched alkoxy groups containing 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentoxy, isohexoxy, 3-methylpentoxy, or n-hexoxy, etc., preferably methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy.

[0199] Halogenated C1-C6 alkoxy groups refer to straight-chain or branched alkoxy groups containing 1-6 carbon atoms in which the hydrogen atoms are replaced by one or more identical or different halogen atoms, such as -OCF3, -OCH2CH2Cl, -OCHBrCH2Cl or -OCF2CF3, etc.

[0200] C1-C6 alkylthio groups refer to straight-chain or branched alkylthio groups containing 1-6 carbon atoms, such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, sec-butylthio, n-pentylthio, isopentylthio, neopentylthio, or n-hexylthio, preferably methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, or tert-butylthio.

[0201] C2-C6 alkenyl groups refer to straight-chain or branched unsaturated hydrocarbon groups containing 1-3 double bonds and 2-6 carbon atoms, including both cis and trans configurations. Examples include vinyl, 1-propenyl, 2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-butadienyl, 1,3-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 3,3-dimethyl-1-propenyl, or 2-ethyl-1-propenyl, etc.

[0202] C2-C6 alkynyl refers to straight-chain or branched alkynyl groups containing 2-6 carbon atoms, such as ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 2-pentynyl, or 2-hexynyl.

[0203] C2-C6 alkenoxy groups refer to straight-chain or branched alkenoxy groups containing 1-3 double bonds and 2-6 carbon atoms, such as ethyleneoxy, 1-propenoxy, 1-methyl-1-propenoxy, 2-methyl-1-propenoxy, 1-pentenoxy, 1,3-pentadienoxy, or 2-pentenoxy.

[0204] C2-C6 alkynoxy groups refer to straight-chain or branched alkynoxy groups containing 2-6 carbon atoms, such as acetylenoxy, 2-propynoxy, 2-butynoxy, 3-butynoxy, 1-methyl-2-propynoxy, 2-pentynoxy, or 2-hexynoxy.

[0205] C1-C6 alkyl acyl groups refer to straight-chain or branched alkyl acyl groups containing 1-6 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, tert-butyryl, or hexanoyl.

[0206] Halogenated C1-C6 alkyl groups refer to straight or branched alkyl groups containing 1-6 carbon atoms in which the hydrogen atoms are replaced by one or more identical or different halogen atoms, such as trifluoroacetyl groups.

[0207] A carbamoyl group substituted with a C1-C6 alkyl group refers to a carbamoyl group in which one or two identical or different C1-C6 alkyl groups are substituted, such as -CONHMe, -CONHEt, -CON(Me)Et, -CONEt2 or -CONMe2, etc.

[0208] A carbamoyl group substituted with a hydroxyl C1-C6 alkoxy C1-C6 alkyl group refers to a carbamoyl group in which one or two identical or different hydroxyl C1-C6 alkoxy C1-C6 alkyl groups are substituted, such as -CONHCH2OCH2OH, -CONHCH2CH2OCH2CH2OH, etc.

[0209] A C1-C6 alkoxycarbonyl group substituted with a hydroxyl C1-C6 alkoxy group refers to a C1-C6 alkoxycarbonyl group in which an alkyl carbon atom is attached to an oxygen atom of a hydroxyl C1-C6 alkoxy group, such as -COOCH2OCH2OH, -COOCH2CH2OCH2CH2OH, etc.

[0210] Hydroxyl C1 to C6 alkyl refers to a straight-chain or branched alkyl group containing 1 to 6 carbon atoms, with one carbon atom connected to a hydroxyl group, such as -CH2OH, -CH2CH2OH, -CH(OH)CH3, -CH2CH2CH2OH, -CH2CH2CH2CH2OH or -CH2CH(CH3)CH2OH, etc.

[0211] Amino C1-C6 alkyl refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, with one carbon atom attached to an amino group, such as -CH2NH2, -CH2CH2NH2, -CH(NH2)CH3, -CH2CH2CH2NH2, or -CH2CH2CH2CH2NH2.

[0212] C1-C6 alkyl-substituted amino groups refer to amino groups in which one or two identical or different C1-C6 alkyl groups replace the hydrogen atom on the amino group, such as -CH2NHMe or -CH2CH2NEt2.

[0213] Carbamoyl C1-C6 alkyl refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, with one carbon atom attached to the carbonyl carbon of the carbamoyl group, such as -CH2CONH2, -CH2CH2CONH2, -CH(CONH2)CH3 or -CH2CH2CH2CONH2, etc.

[0214] C1-C6 alkyl-substituted carbamoyl C1-C6 alkyl refers to the amino hydrogen atom on the carbamoyl C1-C6 alkyl group being replaced by one or two identical or different C1-C6 alkyl groups, such as -CH2CONHMe, -CH2CH2CONHEt, -CH2CH2CONMe2 or -CH2CONEt2, etc.

[0215] Cyanoyl C1-C6 alkyl refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, with one carbon atom connected to a cyano group, such as cyanomethyl, 2-cyanoethyl, 1-cyanoethyl, 3-cyanopropyl, 4-cyanobutyl, or 5-cyanopentyl.

[0216] Carboxyl C1-C6 alkyl groups refer to straight-chain or branched alkyl groups containing 1-6 carbon atoms, with one carbon atom attached to a carboxyl group, such as carboxymethyl, 2-carboxyethyl, 1-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl, or 5-carboxypentyl, for example...

[0217] C1 to C6 alkyl sulfonyl groups refer to straight-chain or branched alkyl sulfonyl groups containing 1 to 6 carbon atoms, such as methanesulfonyl, ethanesulfonyl, or propanesulfonyl.

[0218] Halogenated C1-C6 alkylsulfonyl groups refer to straight-chain or branched alkylsulfonyl groups containing 1-6 carbon atoms, in which one or more identical or different halogen atoms are replaced by one or more identical or different halogen atoms, such as trifluoromethanesulfonyl groups.

[0219] C1-C6 alkyl-substituted amino groups refer to amino groups in which one or two identical or different C1-C6 alkyl or C1-C6 alkyl groups are substituted, such as -NHMe or -NEt2.

[0220] C3-C6 cycloalkyl refers to saturated cyclic hydrocarbon groups containing 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0221] C3-C6 cycloalkoxy groups refer to saturated cycloalkoxy groups containing 3-6 carbon atoms, such as cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0222] C2-C 10 Ester groups refer to saturated or unsaturated straight-chain or branched ester groups containing 2-10 carbon atoms, such as -COO-(C1-C9 alkyl), -COO-(C2-C8 alkenyl), or -COO-(C3-C9 cycloalkyl), or -O-CO-(C1-C9 alkyl), -O-CO-(C2-C8 alkenyl), or -O-CO-(C3-C9 cycloalkyl), etc., wherein the alkyl, alkenyl, or cycloalkyl groups can be substituted or unsubstituted (e.g., halogenated).

[0223] Cyclic bisbenzylisoquinoline compounds

[0224] Cyclic bisbenzylisoquinoline alkaloids are a very important class of natural products with a wide range of biological activities. Representative substances include oxyacanthine, tetrandrine, cepharanthine, and fangchinoline.

[0225]

[0226] Oxyacanthine is a white crystalline powder. It has a melting point of 216–217°C and an optical rotation of +131.5° (chloroform). It is practically insoluble in water but soluble in ethanol, chloroform, ether, and dilute acids. It is found in the roots of *Berberis thunbergii* (European barberry) and *Thalictrum angustifolium* (narrow-leaved pine) in the Ranunculaceae family. Oxyacanthine has antibacterial, antihypertensive, choleretic, and antileukemic effects.

[0227] The compounds of the present invention

[0228] The compounds of this invention are cyclic bisbenzylisoquinoline compounds of formula I, or pharmaceutically acceptable salts thereof, or their enantiomers, diastereomers, racemates, or their crystalline hydrates, solvates, or mixtures thereof, wherein the cyclic bisbenzylisoquinoline compounds are compounds of formula (I):

[0229]

[0230] In the formula, R1 and R2 are defined as above.

[0231] Preferably, the compound of formula I of the present invention is a non-natural compound.

[0232] In this invention, the inventors unexpectedly discovered that when the compound does not contain a phenolic hydroxyl group, its inhibitory activity against the coronavirus SARS-CoV-2 is significantly enhanced, and its EC50... 50 The value of EC of alkali hydrochloride 50 The value decreased by about 10 times.

[0233] Preferably, the compounds of the present invention are selected from Table A:

[0234] Table A

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] The term "pharmaceutically acceptable salt" refers to a conventional, non-toxic salt formed by reacting the active compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts can be prepared by reacting the active compound of the present invention with an inorganic or organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, and phosphoric acid, etc., and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid, etc.; or the active compound of the present invention... The active compound of the present invention forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid, or glutamic acid, and then forms a sodium, zinc, potassium, calcium, aluminum, or ammonium salt with an inorganic base; or the active compound of the present invention forms an ester with lysine, arginine, or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid, or forms a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid, or ethanesulfonic acid; or the active compound of the present invention forms a sodium, zinc, potassium, calcium, aluminum, or ammonium salt with an inorganic base by the carboxyl / phenolic hydroxyl group in the molecule.

[0242] Furthermore, the active ingredient of this invention is particularly suitable for use in combination with other anti-coronavirus drugs. Other representative anti-coronavirus drugs include (but are not limited to): interferon; RNA-dependent RNA polymerase inhibitors (such as Remdesivir (or GS-5734), favipiravir, Galidesivir, GS-441524, Molnupiavir (or EIDD-2801), NHC (EIDD-1931); 3CL protease inhibitors (nirmatrelvir, enstatrelvir, GC-376), lopinavir, ritonavir, nelfinavir; chloroquine, hydroxychloroquine, cyclosporine, carrimycin, baicalin, baicalein, naphthoquine, and ciclesonide. The interferon comprises ribavirin, penciclovir, leflunomide, teriflunomide, nafamostat, nitazoxanide, darunavir, arbidol, carmostat, niclosamide, ivermectin, baricitinib, ruxolitinib, dasatinib, saquinavir, beclabuvir, simeprevir, or pharmaceutically acceptable salts thereof, or combinations thereof. The interferon includes one or more of interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-1a, and interferon β-1b.

[0243] Furthermore, since SARS-CoV-2 infection can cause acute lung injury, inflammatory responses, and even cytokine storms, the active ingredients of this invention are particularly suitable for use in combination with drugs that improve acute lung injury, have anti-inflammatory effects, or modulate immunity. Representative drugs include, but are not limited to, zinc (Zinc), fingolimod, vitamin C, olmesartan medoxomil, valsartan, losartan, thalidomide, glycyrrhizic acid, artemisinin, dihydroartemisinin, artesunate, artemisone, azithromycin, escin, and naproxen.

[0244] Preferably, the active ingredient of the present invention is used in combination with artemisinin-based drugs (one or more of artemisinin, dihydroartemisinin, artesunate, and artemisinone). Numerous studies have shown that artemisinin-based drugs possess multiple anti-inflammatory and immunomodulatory mechanisms. They achieve their anti-inflammatory and immunomodulatory functions by inhibiting T cell proliferation and activation, inhibiting B cell activation and antibody production, increasing regulatory T cells, and reducing the release of pro-inflammatory cytokines. It is expected that these drugs can alleviate the symptoms of immune damage caused by SARS-CoV-2 infection.

[0245] Preferably, the active ingredient of the present invention is used in combination with artemisinin-based drugs (one or more of artemisinin, dihydroartemisinin, artesunate, and artemisinone) and azithromycin.

[0246] The active ingredient of this invention can inhibit the infectivity of novel coronaviruses such as SARS-CoV-2. Therefore, when the active ingredient of this invention is applied or administered for treatment, it can inhibit the infection of the 2019 novel coronavirus (SARS-CoV-2), thereby achieving an antiviral effect.

[0247] Preparation method

[0248] The method for preparing the cyclic bisbenzylisoquinoline compounds of the present invention is selected from one or more of the following methods:

[0249] a) Using cyclic bisbenzylisoquinoline containing phenolic hydroxyl groups as raw materials, and alkylating agents, the cyclic bisbenzylisoquinoline compounds are obtained by alkylation reaction;

[0250] b) Using cyclic bisbenzylisoquinoline containing phenolic hydroxyl groups as raw materials, and acylation reagents are used to acylate the cyclic bisbenzylisoquinoline compounds to obtain the cyclic bisbenzylisoquinoline compounds;

[0251] In another preferred embodiment, the method is as follows: using spiky alkali as a raw material, a condensation acylation reaction is carried out with carboxylic acid to obtain the cyclic bisbenzyl isoquinoline compound.

[0252] In another preferred embodiment, the condensation acylation reaction is carried out in the presence of a condensing agent, including but not limited to: N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), etc.

[0253] c) Using spiky alkali as a raw material, the compound (I-1b) was obtained by reacting it with a sulfonating agent in the presence of a base.

[0254] (I-1b) and the coupling reagent undergo a coupling reaction to yield the cyclic bisbenzylisoquinoline compound shown in Formula I-1, as shown in Formula 1:

[0255]

[0256] In Formula I-1, R1 is selected from hydrogen, halogen, C1-C6 alkylthio, C1-C6 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyl-substituted amino, cyano, carboxyl, aldehyde, and C1-C6 alkylacyl.

[0257] In Formula 1-1b, L is selected from: leaving groups, such as C1-C6 alkylsulfonyloxy, halogenated C1-C6 alkylsulfonyloxy, benzenesulfonyloxy, naphthalenesulfonyloxy, preferably methanesulfonyloxy and trifluoromethanesulfonyloxy;

[0258] d) Using alkali as a raw material, compound (I-2) was prepared by nitration, reduction, and cyclization reactions, as shown in the following reaction formula 2:

[0259]

[0260] In Formula I-2, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted.

[0261] In another preferred embodiment, the benzo[5-6 membered monocyclic heterocycle] is selected from:

[0262] In another preferred embodiment, the substituent is selected from halogen, hydroxyl, mercapto, amino, oxo (=O), thio (=S), and C1-C6 alkyl; the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S;

[0263] e) Using alkali as a raw material, compound of formula (I-3) is prepared through a multi-step reaction, as shown in reaction formula 3:

[0264]

[0265] In Formula I-3, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted.

[0266] The substituents are selected from halogens, hydroxyl groups, mercapto groups, amino groups, oxo (=O), thio (=S), and C1-C6 alkyl groups;

[0267] The heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

[0268] In another preferred embodiment, the benzo[5-6 membered monocyclic heterocycle] is selected from:

[0269] In formula I-3b, L is a leaving group, such as C1-C6 alkylsulfonyloxy, halogenated C1-C6 alkylsulfonyloxy, benzenesulfonyloxy, naphthalenesulfonyloxy, preferably methanesulfonyloxy and trifluoromethanesulfonyloxy.

[0270] In another preferred embodiment, method e) includes the following steps:

[0271] The compound of formula (I-1a) undergoes an ortho-nitration reaction with a nitrating agent to produce the compound of formula (I-3a);

[0272] The compound of formula (I-3a) and the sulfonating agent react in a suitable solvent in the presence of a base to give the compound of formula (I-3b);

[0273] Compound e3) and benzylamine undergo a Buchwald-Hartig reaction to give compound (I-3c);

[0274] The compound of formula (I-3c) undergoes a reduction reaction to give the compound of formula (I-3d);

[0275] Compound (e5) of formula (I-3d) is given by cyclization reaction to compound (I-3).

[0276] f) The compound obtained by any of methods a) to e) is subjected to functional group transformation to obtain the cyclic bisbenzylisoquinoline compound.

[0277] Pharmaceutical Compositions and Applications

[0278] The present invention also provides the use of one or more of the active compound of formula I of the present invention that inhibits coronavirus replication, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, as an active ingredient in the preparation of a medicament for treating and / or preventing and alleviating respiratory infections, pneumonia and other related diseases caused by coronavirus infections such as the 2019 novel coronavirus.

[0279] The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of an active ingredient, and the preferred proportion is that the active compound of the present invention accounts for 0.1 wt% to 90 wt% or 1 wt% to 50 wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent, solution or salt solution.

[0280] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of this invention. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., which are conventional in the pharmaceutical field.

[0281] The compounds and pharmaceutical compositions provided by this invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0282] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical preparation methods. The dosage form typically contains 0.05-400 mg of the active compound of the present invention per unit volume, preferably 1-500 mg per unit volume.

[0283] The compounds and pharmaceutical compositions of the present invention are suitable for clinical use in mammals, including humans and animals, and can be administered via oral, nasal, skin, lung, or gastrointestinal routes. Oral administration is preferred. The most preferred daily dose is 0.01-400 mg / kg body weight, administered as a single dose, or 0.01-200 mg / kg body weight, administered in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a low dose is started, and the dose is gradually increased until the most suitable dose is found.

[0284] The drugs or inhibitors of the present invention can be administered in various ways, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into the body such as muscles, intradermal, subcutaneous, veins, and mucous membranes; or by being mixed with or encapsulated by other substances and introduced into the body.

[0285] Typically, the active ingredient of the present invention or a pharmaceutical composition containing it can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0286] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0287] The active ingredients of this invention can be formulated into ordinary formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0288] In order to formulate the active ingredient of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0289] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0290] To formulate the drug delivery unit into capsules, the active ingredient of this invention can be mixed with a diluent and a flow aid, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and flow aids used to prepare the tablets of this invention can also be used to prepare the capsules of this invention.

[0291] To prepare the active ingredient of this invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0292] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0293] The active ingredients or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs.

[0294] When the active ingredient of this invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0295] The main advantages of this invention include:

[0296] 1) The compounds of this invention exhibit good inhibitory activity against coronavirus replication; some compounds inhibit the EC50 activity of SARS-CoV-2 RNA replication. 50 It can even reach levels of <1μM.

[0297] 2) The compounds of this invention do not contain phenolic hydroxyl groups, which not only significantly enhance the anti-SARS-CoV-2 activity, but also have good physicochemical properties, good metabolic properties, and high oral bioavailability.

[0298] 3) The compounds of this invention have good selectivity for hERG ion channels and Nav1.5 ion channels, and are expected to have low toxic side effects on the heart.

[0299] 4) The compound of this invention has strong activity, a high therapeutic index (SI), and is effective orally. It has the characteristics of low effective dose and few toxic side effects, and can be used to prevent and / or treat diseases caused by coronavirus infection, showing good clinical application prospects.

[0300] 5) The compounds of this invention also have therapeutic effects on diseases caused by viral infections, inflammation-related diseases (pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, inflammatory pain), fibrosis-related diseases (pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis), autoimmune diseases related to abnormal differentiation of Th1 and / or Th17 (psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity reaction, graft-versus-host disease, etc.), osteoporosis, and neurodegenerative diseases.

[0301] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0302] Example 1 Preparation of Compound 1

[0303]

[0304] The steps are as follows:

[0305]

[0306] Step 1:

[0307] Add 1.2 g (1.76 mmol) of alkali hydrochloride to a 50 mL round-bottom single-necked flask, followed by 12 mL of dichloromethane and 0.89 g (8.80 mmol, 5 eq). Stir for 10 min in an ice bath, then slowly add 0.58 mL (3.45 mmol, 1.96 eq) of trifluoromethanesulfonic anhydride. After the addition is complete, stir at room temperature for 2 h. The TLC reaction is complete. Add 5.3 mL of 1 N hydrochloric acid, stir, and separate the organic layer. Wash the organic layer once with saturated sodium bicarbonate solution, dry over anhydrous sodium sulfate, concentrate, and precipitate by silica gel column chromatography to obtain a pale yellow solid 1-a (1.08 g, 1.46 mmol), with a yield of 83%. 1H NMR (600MHz, Acetone-d6) δ7.50 (dd, J=8.5, 2.3Hz, 1H), 7.22 (d, J=8.4Hz, 1H), 7.06 (d d,J=8.2,2.3Hz,1H),7.00(dd,J=8.4,2.6Hz,1H),6.94(dd,J=8.3,2.0Hz,1H),6.71(s, 1H),6.49(s,1H),6.46(s,1H),6.33(dd,J=8.2,2.7Hz,1H),5.66(d,J=2.0Hz,1H),4.09(d,J=5.4Hz ,1H),3.76(s,3H),3.68(s,3H),3.62(d,J=3.5Hz,1H),3.31(d,J=14.5Hz,1H),3.24(dd,J=14.0,4.1 Hz,1H),3.16(s,3H),3.15–3.10(m,1H),3.01–2.88(m,2H),2.86–2.79(m,2H),2.77–2.71(m,1H),2 .63(dd,J=5.2,1.8Hz,1H),2.60(s,3H),2.56(s,3H),2.37–2.20(m,3H).ESI-MS(m / z):741.38[M+H] + .

[0308] Step 2:

[0309] Mix 1-a (850 mg, 1.15 mmol) with N,N-dimethylacetamide (10 mL), and then add zinc cyanide (200 mg, 1.70 mmol, 1.48 eq), tris(dibenzylacetone)palladium (55 mg, 0.06 mmol, 0.05 eq), 1,1'-bis(diphenylphosphine)ferrocene (65 mg, 0.12 mmol, 0.1 eq) and zinc powder (12 mg, 0.18 mmol, 0.16 eq) sequentially. Purge with nitrogen four times, heat to 140–150 °C and react for 2 h. After the starting material is completely converted by TLC, cool the reaction solution to 30–40 °C, dilute with ethyl acetate, filter to remove insoluble matter, wash the filtrate three times with 5% sodium bicarbonate aqueous solution, separate the ethyl acetate layer, dry with anhydrous sodium sulfate, filter, concentrate, and precipitate by silica gel column chromatography to obtain title compound 1 (583 mg, 0.94 mmol), a pale yellow solid, with a yield of 82%. 1H NMR (600MHz, Acetone-d6) δ7.51–7.45(m,2H),7.07(dd,J=8.2,2.3Hz,1H),7.01(dd,J=8.4,2.6Hz,1H),6.98(dd,J=8. 0,1.4Hz,1H),6.70(s,1H),6.48(s,1H),6.45(s,1H),6.34(dd,J=8.2,2.7Hz,1H),5.60(d,J=1.4Hz,1H),4.09(d,J=5. 4Hz,1H),3.76(s,3H),3.69(s,3H),3.67(m,1H),3.35–3.25(m,2H),3.15(s,3H),3.13–3.07(m,1H),3.02–2.88(m,2H) ,2.89–2.78(m,2H),2.73(m,1H),2.63(m,1H),2.60(s,3H),2.56(s,3H),2.38–2.19(m,3H).ESI-MS(m / z):618.37[M+H] + .

[0310] Example 2 Preparation of Compound 2

[0311]

[0312] 500 mg of alkali hydrochloride was suspended in acetonitrile (12 mL), and triethylamine (260 mg) was added at room temperature. Then, 114 mg of n-butyl isocyanate was slowly added dropwise to the reaction solution. After stirring at 25–28 °C for 2–3 h, ethyl acetate was added for dilution, and the mixture was washed three times with water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain 385 mg of the title compound 2 as a pale yellow solid, with a yield of 74%. 1H NMR (600MHz, Acetone-d6) δ7.43(dd,J=8.4,2.4Hz,1H),6.96(dd,J=8.2,2.3Hz,1H),6.92(dd,J=8.3,2.6Hz,1H),6.88(d,J=8.1Hz ,1H),6.79(dd,J=8.2,2.0Hz,1H),6.68(s,1H),6.47(s,1H),6.44(s,1H),6.26(dd,J=8.2,2.6Hz,1H),5.55(d,J=2.1Hz,1H),4.08( d,J=5.0Hz,1H),3.76(s,3H),3.67(s,3H),3.57(m,1H),3.30–3.10(m,5H),3.16(s,3H),2.96(m,1H),2.89–2.60(m,5H),2.59(s,3 H),2.55(s,3H),2.38–2.28(m,3H),1.51(p,J=7.1Hz,2H),1.36(h,J=7.4Hz,2H),0.87(t,J=7.3Hz,3H).ESI-MS(m / z):708.49[M+H] + .

[0313] Example 3 Preparation of compound 3

[0314]

[0315] The steps are as follows:

[0316]

[0317] Step 1:

[0318] Compound 1 (1.1 g, 1.78 mmol) was mixed with ethylene glycol (11 mL), potassium hydroxide (600 mg, 10.70 mmol, 6 eq) and water (0.5 mL) were added, the mixture was purged with nitrogen several times, and the temperature was raised to 150 °C for 7 h. After the reaction solution was cooled to room temperature, it was diluted with water (25 mL), the pH was adjusted to 7-8 with dilute hydrochloric acid, and the mixture was extracted with dichloromethane. The organic phase was separated, washed once with water and once with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 3-a (300 mg), a pale yellow solid, with a yield of 26%. 1H NMR(600MHz,Chloroform-d)δ7.67(d,1H),7.48(d,1H),6.92(m,1H),6.80(m,1H),6.58(s,1H ),6.55(m,1H),6.36(s,1H),6.20(s,1H),6.06(d,1H),5.51(s,1H),4.45(d,1H),3.98(m,1H), 3.78(s,3H),3.59–3.39(m,2H),3.50(s,3H),3.36–3.20(m,2H),3.17(s,3H),3.14–3.00(m,2H ),2.90–2.62(m,4H),2.75(s,3H),2.57(s,3H),2.43–2.25(m,2H).ESI-MS(m / z):637.36[M+H] + 635.37 [MH] - .

[0319] Step 2:

[0320] Compound 3-a (600 mg, 0.94 mmol) was mixed with methanol (6 mL), and concentrated sulfuric acid (352 mg, 3.59 mmol, 3.8 eq) was added at room temperature. After the addition was complete, the mixture was heated to reflux and reacted for another 7 h. The starting material was completely converted by TLC. Sodium bicarbonate (938 mg, 11.8 eq) was added and stirred for 10 min. Dichloromethane was added for dilution, and water was added and stirred to separate the layers. The organic phase was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain the title compound 3 (486 mg), an off-white solid, in 80% yield. 1 H NMR(600MHz,Chloroform-d)δ7.70(d,1H),7.45(brs,1H),6.95(m,2H),6.89(d,1H),6.60( s,1H),6.35(s,1H),6.30(dd,1H),6.30(s,1H),5.57(m,1H),4.22(brs,1H),3.85(s,3H),3 .78(s,3H),3.70(m,1H),3.61(s,3H),3.47–3.20(m,3H),3.17(s,3H),3.10–2.95(m,2H),2 .88–2.65(m,4H),2.68(s,3H),2.57(s,3H),2.46–2.22(m,3H).ESI-MS(m / z):651.47[M+H] + .

[0321] Example 4 Preparation of compound 4

[0322]

[0323] Compound 1 (1.1 g, 1.78 mmol) was suspended in tert-butanol (8 mL), and potassium tert-butoxide (1.4 g, 12.48 mmol, 7 eq) was added. The mixture was heated to 80 °C and reacted until the starting material was completely converted. After the reaction solution was cooled to room temperature, most of the tert-butanol was concentrated. Water and ethyl acetate were added to the residue, and the mixture was stirred to separate the layers. The ethyl acetate layer was separated, washed once with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain the title compound 4 (820 mg), a pale yellow solid, with a yield of 73%. 1 H NMR (600MHz, Acetone-d6) δ7.90(d,J=8.0Hz,1H),7.57(brs,1H),7.49(dd,J=8.4,2.3Hz,1H),7.03(td,J=8.0,2.4Hz,2H),6.96( dd,J=8.0,1.6Hz,1H),6.69(m,1H),6.68(s,1H),6.47(s,1H),6.45(s,1H),6.32(dd,J=8.2,2.7Hz,1H),5.63(d,J=1.5Hz,1H),4.0 9(d,J=5.3Hz,1H),3.76(s,3H),3.68(s,3H),3.63(m,1H),3.33–3.22(m,2H),3.16(s,3H),3.12(m,1H),2.97(m,1H),2.89(dd,J= 13.9,3.0Hz,1H),2.82(m,2H),2.73(m,1H),2.62(m,1H),2.60(s,3H),2.56(s,3H),2.37–2.24(m,3H).ESI-MS(m / z):636.38[M+H] + .

[0324] Example 5 Preparation of compound 5

[0325]

[0326] Compound 3-a (300 mg, 0.47 mmol) was mixed with tetrahydrofuran (2 mL), and CDI (230 mg, 1.42 mmol) was added at room temperature. The mixture was stirred at room temperature for 30 min, and then 28% aqueous methylamine (468 mg, 4.23 mmol) was added to the reaction mixture. After the addition was complete, the mixture was stirred for another 30 min. The reaction mixture was poured into water, extracted with ethyl acetate, and the organic layer was separated, concentrated, and purified by silica gel column chromatography to give the title compound 5 (230 mg), a pale yellow solid, in 75% yield. 1H NMR(600MHz,Chloroform-d)δ8.03(d,1H),7.79(q,1H),7.50(m,1H),7.00(m,3H),6.59(s,1 H),6.34(s,1H),6.31(m,2H),5.56(d,1H),4.15(d,1H),3.80(s,3H),3.68(t,1H),3.61(s,3 H),3.40–3.38(m,1H),3.35–3.22(m,2H),3.17(s,3H),3.03(m,1H),2.99(m,3H),2.98–2.82 (m,3H),2.70(m,2H),2.65(s,3H),2.55(s,3H),2.45–2.22(m,3H).ESI-MS(m / z):650.3[M+H] + .

[0327] Example 6 Preparation of Compound 6

[0328]

[0329] Compound 3-a (600 mg, 0.94 mmol) was dissolved in dichloromethane (5 mL), and N,N-dimethylformamide (8 μL) was added. Oxaloyl chloride (0.4 mL, 4.72 mmol) was then added dropwise at ambient temperature. After the addition was complete, nitrogen protection was applied, and stirring was continued for 40 min. The reaction solution was then concentrated to dryness and dissolved again in dichloromethane (5 mL). This solution was then added dropwise to a dichloromethane solution of cyclopropylamine (330 μL, 4.76 mmol) at ambient temperature. After the addition was complete, stirring was continued for 30 min. Saturated ammonium chloride aqueous solution (10 mL) was added, and the mixture was stirred to separate the layers. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 6 (504 mg, 0.75 mmol), a pale yellow solid, with a yield of 79%. 1H NMR(600MHz,Chloroform-d)δ8.07(d,1H),7.83(m,1H),7.64(m,1H),6.95(m,3H),6.60(s,1H),6.3 6(s,1H),6.30(s,1H),6.27(dd,1H),5.52(d,1H),4.30(m,1H),3.78(s,3H),3.71(m,1H),3.61(s,3 H),3.55(m,1H),3.42–3.20(m,2H),3.17(s,3H),3.15–3.00(m,2H),2.90–2.82(m,4H),2.71(s,3H) ,2.64(m,1H),2.57(s,3H),2.48–2.25(m,3H),0.77(m,2H),0.51(m,2H).ESI-MS(m / z):676.58[M+H] + .

[0330] Example 7 Preparation of Compound 7

[0331]

[0332] The preparation method of compound 7 (i.e. compound 3-a) has been described in Example 3.

[0333] Example 8 Preparation of Compound 8

[0334]

[0335] Compound 1 (500 mg, 0.81 mmol), formic acid (15 mL), and Raney nickel (1.03 g) were added sequentially to a 50 mL round-bottom single-necked flask. The mixture was purged with nitrogen, and the temperature was raised to 80–90 °C for 1–2 h. After the starting material was completely converted by TLC, the reaction mixture was cooled to ambient temperature. Insoluble matter was filtered off, the filtrate was concentrated to dryness, diluted with dichloromethane, washed with sodium bicarbonate solution, and the organic layer was separated. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain the title compound 8 (248 mg, 0.4 mmol), a yellow solid, in 49% yield. ESI-MS (m / z): 621.37 [M+H] + .

[0336] Example 9 Preparation of Compound 9

[0337]

[0338] Compound 8 (80 mg, 0.13 mmol) was dissolved in 1 mL of methanol, cooled to 5–10 °C in an ice bath, and sodium borohydride (9.3 mg, 0.245 mmol, 1.9 eq) was added. After the addition was complete, the temperature was slowly raised to ambient temperature. After 1 h, the reaction proceeded to TLC conversion. The reaction was quenched by carefully adding saturated ammonium chloride aqueous solution. Dichloromethane and water were added, and the mixture was stirred to separate the layers. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound 9 (52 mg, 0.084 mmol), a pale yellow solid, in 64% yield. ESI-MS (m / z): 623.38 [M+H] + .

[0339] Example 10: Preparation of compounds 10 and 33

[0340]

[0341] 1-a (250 mg, 0.34 mmol), palladium acetate (12 mg, 0.05 mmol, 0.15 eq), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (49 mg, 0.10 mmol, 0.3 eq), cesium carbonate (328 mg, 1.00 mmol, 3 eq), and benzylamine (75 μL, 0.69 mmol, 2 eq) were mixed in dioxane (4 mL), purged three times with nitrogen, heated to 100–105 °C, and reacted for 14 hours. TLC showed that the starting material reacted completely. After the reaction solution was cooled to room temperature, it was filtered with diatomaceous earth as an aid, the filter residue was washed with ethyl acetate, water and ethyl acetate were added to the filtrate, the mixture was stirred to separate the layers, the ethyl acetate layer was separated, washed once with brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give the title compound 10 (192 mg), a pale yellow solid, in 82% yield. ESI-MS (m / z): 698.47 [M+H] + .

[0342]

[0343] Similarly, during silica gel column chromatography, the main byproduct compound 33 (10 mg), a pale yellow solid, was also separated in 5% yield. ESI-MS (m / z): 593.48 [M+H] + .

[0344] Example 11 Preparation of compound 11

[0345]

[0346] Compound 10 (190 mg, 0.27 mmol), palladium on carbon (17 mg), ammonium formate (170 mg), and methanol (3 mL) were added sequentially to a 25 mL round-bottom single-necked flask. The mixture was purged twice with nitrogen, and the mixture was heated to reflux for 4–5 hours. After TLC conversion was complete, the mixture was filtered with diatomaceous earth as an aid. The filter residue was washed with ethyl acetate. Water and ethyl acetate were added to the filtrate, and the mixture was stirred to separate the layers. The ethyl acetate layer was separated, washed once with brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give compound 11 (114 mg), an off-white solid, in 69% yield. ESI-MS (m / z): 608.38 [M+H] + .

[0347] Example 12 Preparation of compounds 12 and 14

[0348]

[0349] The steps are as follows:

[0350]

[0351] Step 1:

[0352] Compound 11 dihydrochloride (300 mg, 0.44 mmol) was mixed with ethyl formate (10 mL), and triethylamine (400 mg, 3.95 mmol, 9 eq) was added at room temperature. The mixture was then heated to reflux and reacted overnight. After the starting material was converted by TLC, the reaction solution was cooled to room temperature, and 0.5 N dilute hydrochloric acid (7 mL) was added. The mixture was stirred and separated into layers. The organic layer was then washed with brine, concentrated, and purified by silica gel column chromatography to give compound 14 (216 mg), an off-white solid, in 77% yield. ESI-MS (m / z): 636.37 [M+H] + .

[0353] Step 2:

[0354] Compound 14 (100 mg, 0.157 mmol) was suspended in dry tetrahydrofuran (1.2 mL). Borane tetrahydrofuran (1 M in THF, 0.79 mL, 5 eq) was added dropwise under ice bath. After the addition was complete, the mixture was heated to reflux under nitrogen protection. After the TLC reaction was complete, the mixture was cooled to room temperature and then placed in an ice bath for 10 min. Methanol was carefully added dropwise with stirring. After no gas was generated, excess methanol and concentrated hydrochloric acid (95 μL) were added. The mixture was refluxed for 3 h. The reaction solution was concentrated, and the residue was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic layer was separated. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 12 (55 mg), an off-white solid, in 56% yield. ESI-MS (m / z): 622.48 [M+H] + .

[0355] Example 13 Preparation of compound 13

[0356]

[0357] Compound 11 dihydrochloride (150 mg, 0.220 mmol) was dissolved in pyridine (2.0 mL), and acetyl chloride (41 μL, 0.57 mmol, 2.6 eq) was added dropwise at ambient temperature. After the addition was complete, the mixture was heated to 60 °C and reacted for 2–3 h. The reaction solution was concentrated, and silica gel column chromatography was performed to give the title compound 13 (135 mg), an off-white solid, in 95% yield. ESI-MS (m / z): 650.48 [M+H] + .

[0358] Example 14 Preparation of Compound 15

[0359]

[0360] 1.0 g (1.47 mmol) of alkali hydrochloride was dissolved in acetic acid (8 mL). Concentrated nitric acid (65–68% aqueous nitric acid solution, 128 μL, 1.2 eq) was added dropwise at ambient temperature. After the addition was complete, the mixture was stirred at room temperature for 5 min. The reaction solution was then slowly poured into a saturated aqueous sodium bicarbonate solution and stirred until no gas was produced. The mixture was extracted with dichloromethane, and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 15 (486 mg) as a yellow solid, in 51% yield. ESI-MS (m / z): 654.36 [M+H] + 652.37 [MH] - .

[0361] Example 15 Preparation of Compound 16

[0362]

[0363] Compound 15 (525 mg, 0.80 mmol), 10% palladium on carbon (55 mg), ammonium formate (610 mg, 9.68 mmol, 12 eq), and methanol (12 mL) were added sequentially to a 25 mL round-bottom single-necked flask. The mixture was purged with nitrogen and heated to reflux for 1 h. The reaction proceeds were completely converted by TLC. Insoluble matter was filtered off, and the residue was washed with methanol. The filtrate was concentrated to a small volume, and the residue was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound 16 (360 mg), a pale yellow solid, in 72% yield. ESI-MS (m / z): 624.38 [M+H] + .

[0364] Example 16 Preparation of Compound 17

[0365]

[0366] The dihydrochloride salt of compound 16 (225 mg, 0.32 mmol) was dissolved in N,N-dimethylformamide (3 mL), and N,N'-carbonyldiimidazole (260 mg, 1.60 mmol, 5 eq) was added at room temperature. The mixture was stirred at ambient temperature for 2 h. After the starting material was converted by TLC, the reaction solution was poured into 12 mL of ice water and stirred for 10 min. The mixture was filtered, the filter cake was washed twice with water, and dried at 50 °C for 6 h to obtain the title compound 17 (127 mg), an off-white solid with a yield of 61%. 1 H NMR(500MHz,Chloroform-d)δ7.45(d,1H),6.90(dd,1H),6.84(dd,1H),6.69(s,1H),6.60(m, 1H),6.44(s,1H),6.40(m,1H),6.35(s,1H),5.31(d,1H),4.23(d,1H),3.77(s,3H),3.67(m,1 H),3.62(s,3H),3.34(m,1H),3.24–3.13(m,3H),3.22(s,3H),3.05–2.91(m,3H),2.90–2.81( m,2H),2.71(m,1H),2.66(s,3H),2.64(s,3H),2.56–2.45(m,2H).ESI-MS(m / z):650.30[M+H] + .

[0367] Example 17 Preparation of Compound 18

[0368]

[0369] The dihydrochloride salt of compound 16 (215 mg, 0.31 mmol) was dissolved in ethanol (3 mL). Trimethyl orthoformate (0.9 mL, 8.2 mmol, 27 eq) and p-toluenesulfonic acid monohydrate (60 mg, 0.32 mmol, 1 eq) were added at ambient temperature. The mixture was purged with nitrogen three times and reacted at 65–70 °C for 14 h. After complete conversion of the starting material by TLC, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic layer was separated. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound 18 (168 mg), a white solid, in 86% yield. ESI-MS (m / z): 634.48 [M+H] + .

[0370] Example 18 Preparation of Compound 19

[0371]

[0372] Compound 1 (605 mg, 0.98 mmol) was dissolved in dry tetrahydrofuran (5 mL) under nitrogen protection and reacted in an ice bath for 5 min. Methyllithium (1.53 mL, 1.6 M diethyl ether solution, 2.5 eq) was slowly added dropwise. After the addition was complete, the reaction was continued in an ice bath for 80 min. The reaction mixture was carefully poured into ice water, and the pH was adjusted to 2–3 with 2N sulfuric acid aqueous solution. The mixture was stirred at ambient temperature for 1 h, and the pH was adjusted to weakly alkaline with sodium bicarbonate. The mixture was extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound 19 (143 mg), a pale yellow solid, in 23% yield. ESI-MS (m / z): 635.38 [M+H] + .

[0373] Example 19 Preparation of Compound 21

[0374]

[0375] 500 mg of alkali hydrochloride was suspended in dichloromethane (5 mL), and 160 mg of lipoic acid, 220 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 15 mg of 4-dimethylaminopyridine were added sequentially. The mixture was stirred at 23–25 °C for 4–6 h. After the TLC reaction was complete, 4 mL of water was added and the mixture was stirred to separate the layers. The organic layer was separated, washed once with 2 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give 380 mg of the title compound 21 as a pale yellow solid, in 65% yield. ESI-MS (m / z): 797.48 [M+H] + .

[0376] Example 20 Preparation of compound 22

[0377]

[0378] Following the method of Example 6, title compound 22 was prepared by reacting compound 7 with cyclopentanamine; it was a pale yellow solid in 57% yield. ESI-MS (m / z): 704.48 [M+H] + .

[0379] Example 21 Preparation of compound 27

[0380]

[0381] The steps are as follows:

[0382]

[0383] Step 1:

[0384] Compound 15 (950 mg, 1.45 mmol) was dissolved in dichloromethane (7 mL), and pyridine (565 mg, 7.14 mmol, 4.9 eq) and 4-dimethylaminopyridine (14 mg, 0.115 mmol, 0.08 eq) were added. The mixture was stirred in an ice bath for 5–10 min, and trifluoromethanesulfonic anhydride (0.49 mL, 2 eq) was added dropwise. After the addition was complete, the mixture was kept at this temperature for 15 min. The reaction mixture was then converted by TLC. The mixture was washed with 0.5 N dilute hydrochloric acid, and the organic phase was separated. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give product 27-a (800 mg), a yellow solid, in 70% yield. ESI-MS (m / z): 786.28 [M+H] + .

[0385] Step 2:

[0386] In a 10 mL round-bottom single-necked flask, 27-a (205 mg, 0.26 mmol), palladium acetate (7 mg, 0.12 eq), 2-dicyclohexylphospho-2,4,6-triisopropylbiphenyl (27 mg, 0.21 eq), formic acid (33 μL, 3.3 eq), N,N-diisopropylethylamine (152 μL, 3.3 eq), and dioxane (2.0 mL) were added sequentially. The mixture was purged with nitrogen several times, stirred at 85–90 °C for 50 min, and the reaction mixture was allowed to complete TLC conversion. The reaction solution was cooled to room temperature, diluted with ethyl acetate, and the insoluble matter was filtered off. The filtrate was washed with water, and the ethyl acetate layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 27 (97 mg), a pale yellow solid, in 59% yield. ESI-MS (m / z): 638.24 [M+H] + .

[0387] Example 22 Preparation of compound 34

[0388]

[0389] Compound 1-a (120 mg, 0.162 mmol), palladium acetate (4 mg, 0.11 eq), 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (15 mg, 0.19 eq), isopropenylboronic acid benzoyl ester (60 μL, 2 eq), dioxane (1.5 mL), and water (0.4 mL) were added sequentially to a 5 mL round-bottom single-necked flask. Sodium carbonate (53 mg, 3 eq) was added last. The mixture was purged with nitrogen three times, heated to 70–75 °C, and reacted overnight. After cooling to room temperature, the reaction solution was diluted with dichloromethane, washed with water, and the organic phase was separated. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 34 (55 mg), a pale yellow solid, in 54% yield. ESI-MS (m / z): 633.57 [M+H] + .

[0390] Example 23 Preparation of compound 35

[0391]

[0392] Following the method of Example 22, compound 35, a pale yellow solid, was prepared by reacting compound 1-a with methylboric acid in a yield of 54%. ESI-MS (m / z): 607.38 [M+H] + .

[0393] Example 24 Preparation of compound 36

[0394]

[0395] Compound 11 (100 mg, 0.164 mmol) was dissolved in dichloromethane (2.0 mL), and pyridine (66 μL, 5 eq) and acryloyl chloride (27 μL, 2 eq) were added sequentially at ambient temperature. After the addition was complete, the reaction mixture was reacted at ambient temperature for 30 min. After TLC conversion of the starting material, the reaction solution was concentrated to dryness, and silica gel column chromatography was performed to give the title compound 36 (70 mg), an off-white solid, in 65% yield. ESI-MS (m / z): 662.48 [M+H] + .

[0396] Example 25 Preparation of compound 37

[0397]

[0398] Following the method of Example 6, title compound 37 was prepared by reacting compound 7 with n-pentylamine; the solid was pale yellow, in 67% yield. ESI-MS (m / z): 706.48 [M+H] + .

[0399] Example 26 Preparation of compound 38

[0400]

[0401] Following the method of Example 22, title compound 38 was prepared by reacting compound 1-a with cyclopropylboronic acid; the solid was pale yellow, with a yield of 59%. ESI-MS (m / z): 633.38 [M+H] + .

[0402] Example 27 Preparation of compound 39

[0403]

[0404] 600 mg (0.88 mmol) of alkali hydrochloride was suspended in 8 mL of dichloromethane. Triethylamine (0.62 mL, 5 eq) was added, and the mixture was stirred in an ice bath for 5–10 min. Acryloyl chloride (110 μL, 1.5 eq) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 50 min. TLC analysis showed complete conversion of the starting material. Saturated ammonium chloride solution was added, and the mixture was stirred to separate the layers. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 39 (240 mg), an off-white solid, in 41% yield. ESI-MS (m / z): 663.2 [M+H] + .

[0405] Example 28 Preparation of compound 40

[0406]

[0407] Under nitrogen protection, compound 8 (300 mg, 0.483 mmol) was mixed with methyl magnesium bromide (7.4 mL, 3.0 M tetrahydrofuran solution) in an ice bath. The mixture was slowly heated to room temperature and stirred for 2–3 h. The reaction solution was then poured into ice water, and dichloromethane and an appropriate amount of ammonium chloride were added. The mixture was stirred to separate the layers, and the organic layer was separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 40 (165 mg), a pale yellow solid, in 54% yield. ESI-MS (m / z): 637.2 [M+H] + .

[0408] Example 29 Preparation of Compound 41

[0409]

[0410] Following the method of Example 6, title compound 41 was prepared by reacting compound 7 with piperidine, as a pale yellow solid in 76% yield. ESI-MS (m / z): 704.68 [M+H] + .

[0411] Example 30 Preparation of compound 43

[0412]

[0413] Alkyl ether (480 mg, 0.79 mmol) and D-biotin (240 mg, 0.98 mmol, 1.2 eq) were suspended in dichloromethane (5 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230 mg, 1.20 mmol, 1.5 eq) and 4-dimethylaminopyridine (20 mg, 0.16 mmol, 0.2 eq) were added sequentially at room temperature. After the addition was complete, the reaction was allowed to proceed overnight at ambient temperature. After complete conversion of the starting material by TLC, a saturated ammonium chloride aqueous solution was added and the mixture was stirred to separate the layers. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give the title compound 43, a white solid, 435 mg, in 66% yield. ESI-MS (m / z): 835.48 [M+H] + .

[0414] Example 31 Preparation of compound 44

[0415]

[0416] Following the method of Example 6, title compound 44 was prepared by reacting compound 7 with dimethylhydroxylamine hydrochloride, as a pale yellow solid in 78% yield. ESI-MS (m / z): 680.3 [M+H] + .

[0417] Example 32 Preparation of compound 45

[0418]

[0419] Compound 7 (452 ​​mg, 0.709 mmol) was dissolved in dichloromethane (2 mL), and N,N-dimethylformamide (5.5 μL) was added. Oxaloyl chloride (0.305 mL, 3.61 mmol) was then added dropwise at ambient temperature. After the addition was complete, the reaction mixture was kept under nitrogen protection for 20 min. The reaction solution was then concentrated to dryness and dissolved again in dichloromethane (5 mL). This solution was then added dropwise to a dichloromethane solution of aniline (0.34 mL, 3.73 mmol) and pyridine (0.28 mL, 3.46 mmol) at room temperature. After the addition was complete, the mixture was stirred at ambient temperature for 10 min. TLC conversion of the starting material was completed. A saturated ammonium chloride aqueous solution was added, and the mixture was stirred to separate the layers. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give the title compound 45 (259 mg), a pale yellow solid, in 51% yield. ESI-MS (m / z): 712.2 [M+H] + .

[0420] Example 33 Preparation of compound 46

[0421]

[0422] Under nitrogen protection, compound 44 (393 mg, 0.573 mmol) was dissolved in dried tetrahydrofuran (5 mL), and the solution was cooled to 0–5 °C in an ice bath. Vinyl magnesium bromide (1.75 mL, 1.75 mmol, 3 eq, 1 M tetrahydrofuran solution) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed at ambient temperature for 1–2 h. TLC showed complete conversion of the starting material. The reaction mixture was carefully added to 1 N hydrochloric acid (5 mL) and stirred for 20 min. Sodium hydroxide aqueous solution was added to adjust the pH to approximately 8, followed by extraction with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain the title compound 46 (290 mg, 0.449 mmol), a pale yellow solid, in 78% yield. ESI-MS (m / z): 647.48 [M+H] + .

[0423] Example 34 Preparation of compound 47

[0424]

[0425] Following the method of Example 32, title compound 47 was prepared by reacting compound 7 with 4-trifluoromethylaniline as a pale yellow solid in 58% yield. ESI-MS (m / z): 780.48 [M+H] + .

[0426] Example 35 Preparation of compound 48

[0427]

[0428] Following the method of Example 6, title compound 48 was prepared by reacting compound 7 with isopropylamine as a pale yellow solid in 59% yield. ESI-MS (m / z): 678.48 [M+H] + .

[0429] Example 36 Preparation of compound 49

[0430]

[0431] Following the method of Example 6, title compound 49 was prepared by reacting compound 7 with 1-adamantaneamine as a pale yellow solid in 64% yield. ESI-MS (m / z): 770.58 [M+H] + .

[0432] Example 37 Preparation of compound 50

[0433]

[0434] Following the method of Example 6, title compound 50 was prepared by reacting compound 7 with cyclobutamine, as a pale yellow solid, in 58% yield. ESI-MS (m / z): 690.48 [M+H] + .

[0435] Example 38 Preparation of Compound 51

[0436]

[0437] Following the method of Example 32, title compound 51 was prepared by reacting compound 7 with 3-aminopyridine as a pale yellow solid in 71% yield. ESI-MS (m / z): 713.38 [M+H] + .

[0438] Example 39 Preparation of Compound 52

[0439]

[0440] Following the method of Example 32, compound 52, a pale yellow solid, was prepared by reacting compound 7 with p-methoxyaniline in a yield of 72%. ESI-MS (m / z): 742.52 [M+H] + .

[0441] Example 40 Preparation of compound 53

[0442]

[0443] Following the method of Example 28, title compound 53 was prepared by reacting compound 8 with cyclopropylmagnesium bromide. The compound was a pale yellow solid in 64% yield, and ESI-MS (m / z): 663.35 [M+H]. + .

[0444] Example 41 Preparation of compound 54

[0445]

[0446] Compound 53 (183 mg, 0.28 mmol) was dissolved in dichloromethane (2 mL), and Dess-Martin Periodinane (174 mg, 0.41 mmol, 1.5 eq) was added at ambient temperature. After the addition was complete, the reaction was continued for 1 h under nitrogen protection. The starting material was completely converted by TLC. The mixture was diluted with dichloromethane, washed once with saturated sodium bicarbonate solution, and the organic layer was separated. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound 54 (38 mg, 0.057 mmol), a pale yellow solid, in 20% yield. ESI-MS (m / z): 661.28 [M+H] + .

[0447] Example 42 Preparation of compound 55

[0448]

[0449] Following the method of Example 6, title compound 55 was prepared by reacting compound 7 with diethylene glycolamine, as a pale yellow solid in 61% yield. ESI-MS (m / z): 724.28 [M+H] + .

[0450] Example 43 Preparation of compound 56

[0451]

[0452] Following the method in step 2 of Example 3, title compound 56 was prepared by reacting compound 7 with n-pentanol. The compound was an off-white solid in 82% yield. ESI-MS (m / z): 707.46 [M+H] + .

[0453] Example 44 Preparation of compound 57

[0454]

[0455] Following the method of Example 32, title compound 57 was prepared by reacting compound 7 with p-fluorophenol, as a pale yellow solid in 55% yield. ESI-MS (m / z): 731.26 [M+H] + .

[0456] Example 45 Preparation of compound 58

[0457]

[0458] Following the method in step 2 of Example 3, title compound 58 was prepared by reacting compound 7 with diethylene glycol. It was an off-white solid with a yield of 72%. 1H NMR(500MHz,Chloroform-d)δ7.70(d,1H),7.34(m,1H),7.02(m,1H),6.94(dd,1H),6.89(dd,1H),6 .60(s,1H),6.38(s,1H),6.30–6.29(m,2H),5.49(d,1H),4.50(m,1H),4.42(m,2H),3.78(s,3H),3.7 7(m,2H),3.70(m,1H),3.63(m,2H),3.61(s,3H),3.58(m,2H),3.47(m,1H),3.29–3.05(m,3H),3.17 (s,3H),3.02–2.67(m,5H),2.77(s,3H),2.60(s,3H),2.55–2.26(m,3H).ESI-MS(m / z):725.26[M+H] + .

[0459] Example 46 Preparation of compound 59

[0460]

[0461] Following the method of Example 2, title compound 59 was prepared by reacting alkali hydrochloride with methyl isocyanate; the solid was pale yellow, with a yield of 73%. ESI-MS (m / z): 666.26 [M+H] + .

[0462] Example 47 Preparation of Compound 60

[0463]

[0464] Following the method of Example 2, title compound 60 was prepared by reacting alkali hydrochloride with ethyl isocyanate; the solid was pale yellow, with a yield of 69%. ESI-MS (m / z): 680.28 [M+H] + .

[0465] Example 48 Preparation of Compound 61

[0466]

[0467] Following the method of Example 2, title compound 61 was prepared by reacting alkali hydrochloride with n-propyl isocyanate. The solid was pale yellow, with a yield of 76%. ESI-MS (m / z): 694.24 [M+H] + .

[0468] Example 49 Preparation of Compound 62

[0469]

[0470] Following the method of Example 2, title compound 62 was prepared by reacting spiky alkali hydrochloride with isopropyl isocyanate. The solid was pale yellow, with a yield of 70%. ESI-MS (m / z): 694.28 [M+H] + .

[0471] Example 50 Preparation of compound 63

[0472]

[0473] Following the method of Example 5, title compound 63 was prepared by reacting compound 7 with an aqueous solution of ethylamine. The solid was pale yellow, with a yield of 80%. ESI-MS (m / z): 664.24 [M+H] + .

[0474] Example 51 Preparation of compound 64

[0475]

[0476] Following the method of Example 6, title compound 64 was prepared by reacting compound 7 with n-propylamine; it was a pale yellow solid in 73% yield. ESI-MS (m / z): 678.26 [M+H] + .

[0477] Example 52 Preparation of compound 65

[0478]

[0479] Following the method of Example 2, title compound 65 was prepared by reacting spicate hydrochloride with cyclopentyl isocyanate. It was an off-white solid in 76% yield. ESI-MS (m / z): 720.45 [M+H] + .

[0480] Example 53 Preparation of Compound 66

[0481]

[0482] Following the method of Example 2, title compound 66 was prepared by reacting spiky alkali hydrochloride with cyclohexyl isocyanate. It was an off-white solid in 71% yield. ESI-MS (m / z): 734.48 [M+H] + .

[0483] Pharmacological experiments

[0484] 1) Anti-COVID-19 activity test

[0485] Determination of the inhibitory activity of the compound against the replication of the 2019 novel coronavirus (SARS-CoV-2): Vero E6 cells were purchased from ATCC, and the SARS-CoV-2 virus was obtained from the National Virus Resource Center for Microbial Culture Collection. Vero E6 cells were cultured at a density of 5 × 10⁻⁶ cells / cells. 4 Cells were cultured overnight in 48-well cell culture dishes. Cells were pretreated with different concentrations of the test sample for 1 hour, then infected with virus (MOI of 0.01) for 1 hour. The virus-compound mixture was then removed, and cells were further cultured in fresh medium containing the test sample. At 24 h p, the cell supernatant was collected and lysed in lysis buffer. The viral copy number in the cell supernatant was quantitatively assessed by quantitative real-time RT-PCR (qRT-PCR).

[0486] The results (Table 1) showed that multiple compounds significantly inhibited SARS-CoV-2 virus replication and inhibited SARS-CoV-2 virus replication EC. 50 The values ​​were all significantly better than those of salicylic acid hydrochloride, being only about 1 / 10 or less. This suggests that these compounds of the present invention have significantly enhanced inhibitory activity against the coronavirus SARS-CoV-2. Furthermore, activity tests against SARS-CoV-2 variants showed that compounds 2 and 4 exhibited strong inhibitory effects against the delta strain, and compounds 5 and 6 also showed strong inhibitory effects against the South African strain.

[0487] 2) In vitro activity assay of SARS-CoV-2 envelope protein (2-E) ion channel

[0488] Determination of the inhibitory activity of compounds against the ion channel of the novel coronavirus envelope protein (2-E): The channel activity of the purified 2-E protein was detected at room temperature using a planar lipid bimolecular workstation (BLM). Phosphatidylcholine (PC) and phosphatidylserine (PS) stored in chloroform were mixed in a 3:2 ratio, dried under nitrogen, and then dissolved in decane to a final concentration of 50 mg / ml. The cup and tank of the BLM system were assembled, with the cup side defined as the cis side and the opposite side as the trans side. 1 ml of the inner and outer solutions were added to each side. The assembled device was placed on the operating stage of the planar lipid bilayer workstation. The small hole in the center of the cup was observed using a stereomicroscope. The prepared phospholipid was coated onto the cup hole, and then the purified 2-E protein was added to the cis side. Driven by an electrochemical gradient and voltage, the protein intercalated onto the artificial lipid bilayer membrane. The current was recorded using the voltage clamp mode of a patch-clamp amplifier, and the signal was acquired using Clampex 10.3 software. The conductance and turn-on frequency of a single channel were fitted using a Gaussian function, where current turn-on times less than 0.5-1.5 ms were considered noise. The current amplitude was recorded under different conditions by varying the voltage, and the reversal potential was observed.

[0489] When evaluating the channel inhibitory activity of compounds using the BLM system, after observing the appearance of current, the current was recorded for three minutes. Then, the test compound was added to the trans side, stirred for 3 seconds, and the current signal was recorded for ten minutes to observe the changes in current.

[0490] Recording drug inhibition at multiple concentrations and calculating the half-inhibitory concentration (IC50). 50 After the current was turned on and recorded for three minutes, compounds with final concentrations of 1, 10, 50, and 100 μM were added sequentially, and the recording was continued for three minutes at each concentration. The inhibition of this channel by specific compounds at different concentrations was observed, and the IC50 was calculated. 50 .

[0491] 3) In vitro activity assays of cardiotoxicity-related targets (hERG and Nav1.5)

[0492] hERG: CHO (Chinese Hamster Ovary) cells stably expressing the hERG potassium channel were used to record hERG potassium channel currents at room temperature using whole-cell patch-clamp technique. Glass microelectrodes were fabricated from glass electrode blanks (BF150-86-10, Sutter) using a stretching device. The tip resistance after perfusion with electrode fluid was approximately 2-5 MΩ. The glass microelectrodes were inserted into the amplifier probe to connect to the patch-clamp amplifier. Clamping voltage and data recording were controlled and recorded using pClamp 10 software via computer, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at -80 mV. The step voltage to induce hERG potassium current (IhERG) was applied from -80 mV with a 2-second depolarization voltage to +20 mV, then repolarized to -50 mV, held for 1 second, and then returned to -80 mV. This voltage stimulation was applied every 10 seconds. After confirming that the hERG potassium current was stable (at least 1 minute), the drug delivery process began. Each compound was administered for at least 1 minute at each test concentration, and at least 2 cells were tested for each concentration (n≥2).

[0493] Extracellular fluid formulation (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1.25 MgCl2, 10 HEPES and 10 Glucose, adjusted to pH 7.4 with NaOH.

[0494] Intracellular fluid formulation (mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA and 10 HEPES, pH adjusted to 7.2 with KOH.

[0495] Nav1.5: CHL cells stably expressing the hNav1.5 sodium channel were used to record hNav1.5 sodium channel currents at room temperature using whole-cell patch-clamp technique. Glass microelectrodes were fabricated from glass electrode blanks (BF150-86-10, Sutter) using a stretching device. The tip resistance after perfusion with electrode fluid was approximately 2-5 MΩ. The glass microelectrodes were inserted into the amplifier probe to connect to an Axopatch 200B (Molecular Devices) patch-clamp amplifier. Clamping voltage and data recording were controlled and recorded using pClamp 10 software via computer, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at -80 mV. A hyperpolarization voltage of 200 ms was applied to -120 mV, followed by depolarization to -20 mV for 20 ms to induce Nav1.5 current. This voltage stimulation was applied every 10 s. Once the hNav1.5 sodium current stabilized (1 minute), the drug delivery process began. The test sample concentration should be administered continuously starting from the lowest test concentration, with each test concentration administered for 1 minute to reach steady state or for a maximum of 3 minutes. At least 3 cells (n≥3) should be tested for each test sample concentration, and at least 2 cells (n≥2) should be tested for each positive control concentration.

[0496] Extracellular fluid formulation (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1.25 MgCl2, 10 HEPES and 10 Glucose, adjusted to pH 7.4 with NaOH.

[0497] Intracellular fluid formulation (mM): 130 CsF, 10 NaCl, 10 EGTA and 10 HEPES, pH adjusted to 7.2 with CsOH.

[0498] 4) Cytotoxicity test

[0499] In this embodiment, the half-maximal toxicity concentration (CMC) of each embodiment on Vero E6 cells was determined using the CCK8 assay kit. 50 ).

[0500] The results showed that compounds 2, 3, 4, 5, 6, and 58 exhibited weaker cytotoxicity than salicylate hydrochloride on Vero E6 cells, and their therapeutic index (SI) was significantly better than that of salicylate hydrochloride (Table 1). In particular, compounds 3, 4, 6, and 58 had SI values ​​greater than 50, which was much higher than those of the other compounds.

[0501] Table 1. Results of replication inhibitory activity of the original SARS-CoV-2 strain, the South African variant, the delta variant, and HCoV OC43, as well as the SARS-CoV-2 envelope protein (2-E) ion channel, cardiotoxicity-related targets, and cytotoxicity of the virus.

[0502]

[0503]

[0504] a SI = Cytotoxicity CC 50 Value / Inhibition of original strain virus replication EC 50 value.

[0505] As shown in Table 1, the IC50 of the compounds of the present invention inhibits the replication of the original SARS-CoV-2 virus. 50 The values ​​were significantly better than those of spiculine hydrochloride, with a large therapeutic index and good safety profile, especially compounds 3, 4, 6, and 58. The compounds of this invention also showed good inhibitory effects on the replication of SARS-CoV-2 variants; compounds 5 and 6 significantly inhibited the replication of the South African variant, while compounds 2 and 4 significantly inhibited the replication of the delta variant. The compounds of this invention also showed good inhibitory effects on the E protein ion channel of SARS-CoV-2, with an IC50 value of [missing value]. 50 The values ​​are significantly better than those of spiky alkali hydrochloride, especially compound 4 (IC). 50 (<1 μM). Furthermore, compounds 2 and 4 exhibit weak activity against hERG ion channels and Nav1.5 channels, which are highly associated with cardiotoxicity, suggesting minimal cardiotoxic side effects. Additionally, compounds 1, 2, 3, and 4 also show strong inhibitory activity against human coronavirus HCoV OC43.

[0506] 5) CD4 + T cell differentiation experiment

[0507] via CD4 + T cell in vitro differentiation system, detection of Th1CD4 + T-differentiation of IFN-γ and Th17CD4 + The study investigated the IL-17A content of T-differentiation and the effect of compounds on the inhibitory activity of Th1 / Th17.

[0508] Experimental methods:

[0509] CD4 + T cell isolation: using mouse CD4 + T-cell sorting kit ( CD4 cells were isolated from the spleen of 7–8 week old C57BL / 6 mice using the Untouched™ Mouse CD4 Cells Isolation Kit (Invitrogen, Catalog nos: 11415D). + T cells.

[0510] CD4 + In vitro differentiation of T cells: The isolated CD4 cells... +T cells were seeded into 96-well plates, and different concentrations of compounds, corresponding cytokines, and antibodies were added simultaneously: anti-CD28 (2 μg / ml), anti-CD3 (2 μg / ml), anti-IL-4 (10 μg / ml), IL-12 (10 ng / ml), anti-IFN-γ (10 μg / ml), anti-IL-4 (10 μg / ml), IL-6 (30 ng / ml), TGF-β1 (3 ng / ml), IL-1β (10 ng / ml), IL-23 (10 ng / ml), and TNF-α (10 ng / ml). Cells were cultured in a 5% CO2, 37°C cell culture incubator for 72 h. Cells were then collected, intracellular staining was performed, and flow cytometry analysis was conducted to determine IFN-γ / IL-17A levels and assess the effects of the compounds on CD4+. + The effect of T cells on Th1 / Th17 differentiation in vitro.

[0511] The results showed that compounds 1, 2, 3, 4, 6, and 58 all exhibited strong inhibitory effects on Th1 / Th17 differentiation at a concentration of 10 μM. Compound 2, even at a lower concentration of 1 μM, still showed a strong inhibitory effect on Th1 / Th17 differentiation. Figure 1 ).

[0512] 6) In vitro and in vivo anti-inflammatory test

[0513] 6.1) Anti-LPS-induced inflammation assay in Raw 264.7 cells

[0514] In this embodiment, the effects of the compounds in this invention on the inflammation level of an LPS-induced Raw 264.7 cell inflammation model were analyzed by cell viability and inflammatory factor assay; cell viability was analyzed by CCK-8 assay; and the mRNA expression levels of inflammation-related markers were detected by real-time quantitative PCR (Q-PCR). Raw 264.7 cells were purchased from ATCC; the CCK-8 kit was purchased from Meilunbio (catalog number MA0218-3); and LPS was purchased from Sigma (catalog number L2630).

[0515] 6.1.1) Effects of the compound on cell viability in an LPS-induced Raw 264.7 cell inflammation model

[0516] Experimental Methods: First, 8000 cells / well Raw 264.7 cells were cultured overnight in 96-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 1 μg / ml LPS and 0.1% DMSO, and the treatment group with 1 μg / ml LPS and 1 μM of the compounds (oxychloride, 1, 2, tetrandrine, tet, 3, 4, 6, 58). After sterilization, the 96-well plates were incubated in a constant temperature incubator for 16 hours. Finally, cell viability was assessed using a CCK-8 assay kit.

[0517] Experimental results are as follows Figure 2 As shown, no cell damage was observed in the LPS-induced inflammation model, and there was no difference in cell survival rate between the drug-treated group and the model group, indicating that the tested compound did not affect cell viability in the LPS-induced inflammation model.

[0518] 6.1.2) Detection of mRNA levels of relevant inflammatory markers in LPS-induced Raw264.7 cell inflammation model by the compound

[0519] Experimental Methods: First, 5*10⁴ cells / well Raw 264.7 cells were cultured overnight in 12-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 1 μg / ml LPS and 0.1% DMSO, and the drug treatment group with 1 μg / ml LPS and 1 μM of the compounds (oxychloride, 1, 2, tetrandrine, tet, 3, 4, 6, 58). The 12-well plates were sterilized and incubated in a constant temperature incubator for 16 hours. Cellular mRNA was extracted using the Trizol (Yisheng, 10606ES60) lysis method, reverse transcribed, and then detected by real-time quantitative PCR.

[0520] Table 2. Primer sequences for the target gene

[0521]

[0522] Experimental results are as follows Figure 3 As shown, under 1 μg / ml LPS induction, cytokines IL-6 and IL-1β increased significantly, and chemokine CXCL9 increased significantly, indicating that the inflammation model was effective. When different compounds (1 μM) were administered, the levels of inflammatory factors and chemokines decreased compared to the model group. Among them, compounds oxy, 1, 2, tet, 4, and 58 significantly inhibited different inflammatory factors, indicating that the above compounds have strong anti-inflammatory activity and that this is not achieved by affecting cell viability.

[0523] 6.2) Anti-TNF-α induced inflammation assay in Raw 264.7 cells

[0524] In this embodiment, the effect of the compound on the TNF-α-induced inflammation level of Raw 264.7 cells was analyzed by cell viability and inflammatory factor expression levels; cell viability was analyzed by CCK-8 assay; and the mRNA expression levels of inflammation-related markers were detected by real-time quantitative PCR (Q-PCR). Raw 264.7 cells were purchased from ATCC, the CCK-8 kit was purchased from Meilunbio (Catalog No. MA0218-3), and TNF-α was purchased from Merck (Catalog No. GF314).

[0525] 6.2.1) Effects of the compound on cell viability in a TNF-α-induced Raw 264.7 cell inflammation model

[0526] Experimental Methods: First, 8000 cells / well Raw 264.7 cells were cultured overnight in 96-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 10 ng / ml TNF-α and 0.1% DMSO, and the drug treatment group with 10 ng / ml TNF-α and 1 μM of the compounds (oxytocin hydrochloride, 1, 2, tetrandrine, tet, 3, 4, 6, 58). The 96-well plates were sterilized and incubated in a constant temperature incubator for 16 hours. Finally, cell viability was assessed using a CCK-8 assay kit.

[0527] Experimental results are as follows Figure 4 As shown, no cell damage was observed in the TNF-α-induced inflammation model, and there was no difference in cell survival rate between the drug-treated group and the model group, indicating that the tested compound did not affect cell viability in the TNF-α-induced inflammation model.

[0528] 6.2.2) Detection of mRNA levels of related inflammatory markers in a TNF-α-induced Raw264.7 cell inflammation model by the compound.

[0529] Experimental Methods: First, 5*10⁴ cells / well Raw 264.7 cells were cultured overnight in 12-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 10 ng / ml TNF-α and 0.1% DMSO, and the treatment group with 10 ng / ml TNF-α and 1 μM of the compounds (oxychloride, 1, 2, tetrandrine, tet, 3, 4, 6, 58). The 12-well plates were sterilized and incubated in a constant temperature incubator for 16 hours. Cellular mRNA was extracted using the Trizol (Yisheng, 10606ES60) lysis method, reverse transcribed, and then detected by real-time quantitative PCR. Primers were as shown in Table 2.

[0530] Experimental results are as follows Figure 5As shown, under 10 ng / ml TNF-α induction, cytokines IL-6 and IL-1β increased significantly, and chemokine CXCL9 increased significantly, indicating the effectiveness of the inflammation model. Administration of different compounds (1 μM) resulted in a decrease in inflammatory factors and chemokines compared to the model group. Compounds oxy, 1, 2, tet, 3, 4, 6, and 58 significantly inhibited different inflammatory factors. This indicates that the above compounds have strong anti-inflammatory activity, and this effect is not mediated by affecting cell viability.

[0531] 6.3) In vivo experiments on LPS-induced acute inflammation

[0532] In this embodiment, the effects of the compound on an LPS-induced in vivo acute inflammation model were analyzed by measuring the expression levels of inflammatory factors. The mRNA expression levels of tissue inflammation-related markers were detected by real-time quantitative PCR (Q-PCR), and serum inflammatory factor levels were detected by ELISA. C57BL / 6 mice were purchased from SLAC (Shanghai) Co., Ltd., and the ELISA kits were purchased from Novusbio (catalog numbers: IL-6, 890860; TNF-α, 266117).

[0533] Experimental methods:

[0534] Solution preparation: The LPS solution was prepared with PBS to a stock solution of 1 mg / ml and filtered through a 0.22 μm aqueous phase filter; the compound was diluted with 85% physiological saline + 5% DMSO + 5% HS-15 + 5% PEG400 as solvent to a concentration of 3 mg / ml.

[0535] Construction of an inflammation model mouse group: C57BL / 6 mice were randomly divided into three groups: one control group (n=5), one LPS modeling group (n=5), and two drug-treated groups (n=5). The control group received an intraperitoneal injection of 0.1 ml / 10 g PBS, while the model and drug-treated groups received an intraperitoneal injection of 3 mg / kg LPS, followed by drug administration two hours later. The control and model groups received an oral administration of 0.1 ml / 10 g solvent, while the drug-treated groups received an oral administration of 30 mg / kg compound 4. Blood was collected from the orbital sinus of each mouse 4 hours after drug administration. After euthanasia, spleen and lung tissues were harvested. mRNA was extracted from lung and spleen tissues, reverse transcribed, and then detected using real-time quantitative PCR to assess the mRNA expression levels of inflammation-related markers. Serum was extracted and analyzed using ELISA to assess the secretion levels of inflammatory factors.

[0536] Experimental results are as follows Figure 6As shown, stimulation with 3 mg / kg LPS significantly increased cytokines and chemokines, indicating the effectiveness of the in vivo inflammation model. Oral administration of compound 4 at 30 mg / kg significantly reduced the mRNA levels of inflammatory factors and chemokines, as well as the cytokine secretion levels, compared to the model group. This indicates that compound 4 possesses in vivo anti-inflammatory activity.

[0537] 7) Lung injury protective activity test

[0538] In this embodiment, the effects of cell viability combined with cell death-related molecular analysis compounds on TNF-α-induced damage levels in A549 lung epithelial cells were investigated. Cell damage was observed under a microscope; cell viability was analyzed using CCK-8 assay; and the mRNA expression levels of damage-related markers were detected using real-time quantitative PCR (Q-PCR). A549 cells were purchased from ATCC, the CCK-8 kit was purchased from Meilunbio (catalog number MA0218-3), and LPS was purchased from Sigma (catalog number L2630).

[0539] 7.1) Effects of the compound on TNF-α-induced A549 cell viability

[0540] Experimental Methods: First, 8000 A549 cells per well were cultured overnight in 96-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 10 ng / ml TNF-α and 0.1% DMSO, and the drug-treated groups with 10 ng / ml TNF-α and 1 μM of compounds (oxy, 1, 2, tet, 3, 4, 6, 58). The 96-well plates were sterilized and incubated in a constant temperature incubator for 24 hours. Finally, cell viability was assessed using a CCK-8 assay kit.

[0541] Experimental results are as follows Figure 7 As shown, in the TNF-α-induced lung injury model, the cell survival rate of the model group was affected, and the lung epithelial cells tended to die. Compared with the model group, some compounds could alleviate the lung epithelial cell damage mediated by TNF-α, among which compound 1 showed a significant difference.

[0542] 7.2) Detection of mRNA levels of TNF-α-induced A549 cell damage-related markers by the compound

[0543] Experimental Methods: First, 5*10⁴ cells / well of A549 cells were cultured overnight in 12-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 10 ng / ml TNF-α and 0.1% DMSO, and the drug-treated groups with 10 ng / ml TNF-α and 1 μM of compounds (oxy, 1, 2, tet, 3, 4, 6, 58). The 12-well plates were sterilized and incubated in a constant temperature incubator for 24 hours. Cellular mRNA was extracted using the Trizol (Yisheng, 10606ES60) lysis method, reverse transcribed, and then detected by real-time quantitative PCR.

[0544] Table 3. Primer sequences for the target gene

[0545]

[0546] Experimental results are as follows Figure 8 As shown, under 10 ng / ml TNF-α induction, lung epithelial cell damage and significant increases in TNF-α and IL-1β were observed, indicating the effectiveness of the inflammation model. After administration of different compounds (1 μM), the damage factors decreased compared to the model group, with compounds oxy, tet, 3, and 58 showing significant inhibition of different damage factors. This indicates that the compounds in this invention possess protective activity against lung epithelial cell injury, with oxy, tet, 3, and 58 exhibiting stronger protective activity.

[0547] 8) Experiment on H2O2-induced necrotizing injury of ALM12 hepatocytes

[0548] In this embodiment, the effects of the compounds in this invention on the level of H2O2-induced necrosis in ALM12 hepatocytes were analyzed by microscopic morphological observation, cell viability analysis, and analysis of cell death-related molecules. Cell viability was analyzed by CCK-8 assay. ALM12 cells were cultured under the following conditions: 1640 medium (gibco, c11995500BT) + 10% FBS (gibco, 10099141C), incubated at 37°C in a 5% CO2 incubator. The CCK-8 kit was purchased from Meilunbio, catalog number MA0218-3; H2O2 was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10011208.

[0549] Experimental Methods: First, 8000 ALM12 cells per well were cultured overnight in 96-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 2 mM H2O2 and 0.1% DMSO, and the drug-treated groups with 2 mM H2O2 and 1 μM of the compounds (oxy, 1, 2, tet, 3, 4, 6, 58). The 96-well plates were sterilized and incubated for 3 hours. Finally, cell damage and growth were observed under a microscope; cell viability was assessed using a CCK-8 assay kit.

[0550] Experimental results are as follows Figure 9 As shown, in the H2O2-induced liver injury model, the cell survival rate of the model group was affected, and the hepatocytes tended to die, showing a significant difference. Compared with the model group, the compounds of the present invention can be seen under the microscope to significantly alleviate H2O2-mediated hepatocyte injury. In the cell survival rate detection, all compounds can significantly protect against hepatocyte death.

[0551] 9) Experiment on TGF-β1-induced fibrosis in A549 lung epithelial cells

[0552] In this embodiment, the fibrosis-alleviating effect of the compounds in this invention on A549 cells was analyzed by microscopic observation and CCK-8 assay combined with Western Blot. Cell damage was observed under a microscope; cell viability was analyzed by CCK-8 assay; and the protein expression levels of fibrosis-related indicators were detected by Western Blot. A549 cells were purchased from ATCC; the CCK-8 assay kit was purchased from Meilunbio (MA0218-3); TGF-β1 was purchased from MCE (HY-P70543); primary antibodies: Collagen was purchased from ARG21965 (arigobio); α-smooth muscle actin was purchased from CST (mAb#19245); VIMENTN was purchased from CST (mAb#5741); and GAPDH was purchased from Yeasen (30201ES20).

[0553] Experimental Methods: First, 8000 cells / well A549 cells were cultured overnight in 96-well plates. The next day, the control group was treated with 0.1% DMSO, the model group with 10 ng / ml TGF-β1 and 0.1% DMSO, and the drug-treated groups with 10 ng / ml TGF-β1 and 1 μM of compounds (oxy, 1, 2, tet, 3, 4, 6, 58). The 96-well plates were sterilized and incubated in a constant temperature incubator for 72 hours. Finally, cell damage and growth were observed under a microscope; cell viability was assessed using a CCK-8 assay kit.

[0554] Experimental results are as follows Figure 10 and Figure 11 As shown, after stimulating A549 cells with 10 ng / ml TGF-β1, microscopic observation was performed at 24 h and 72 h. Deformation of A549 cells was observed at 24 h, with elliptical cells changing to elongated shapes, indicating fibrosis-like changes in lung epithelial cells. Microscopic observation in the drug-treated group showed no significant changes. Western blotting of fibrosis markers revealed upregulation of Collagen1 and α-SMA in the model group, indicating the model's effectiveness. Comparison between the drug-treated group and the model group showed that compounds oxy, 1, 2, tet, and 4 could partially alleviate fibrosis, indicating that these compounds have a certain protective effect.

[0555] 10) Animal experiments on resistance to carbon tetrachloride-induced acute liver fibrosis

[0556] In this embodiment, the effects of the compounds in this invention on a carbon tetrachloride-induced acute liver fibrosis model were analyzed by tissue observation, fibrosis indicators, HE staining, and Sirius red staining; mouse injury was assessed by body weight measurement and bright-field imaging; protein expression levels of fibrosis-related indicators were detected by Western blotting; and fibrosis progression was analyzed by HE and Sirius red staining. C57BL / 6 mice were purchased from Shanghai SLAC Corporation; CCl4 was purchased from Sinopharm Chemical Reagent Co., Ltd. (catalog number 10006464); olive oil was purchased from Sinopharm Group (catalog number 69018028); primary antibodies: Collagen was purchased from ARG21965 (catalog number arigobio); α-smooth muscle actin was purchased from CST (catalog number mAb#19245); VIMENTN was purchased from CST (catalog number mAb#5741); and GAPDH was purchased from Yeasen (catalog number 30201ES20).

[0557] Experimental methods:

[0558] Solution preparation: CCl4 was diluted with olive oil (1:9, vol:vol), and the compound was prepared with 85% physiological saline + 5% DMSO + 5% HS-15 + 5% PEG400 as solvent to a concentration of 3 mg / ml.

[0559] Establishment of a mouse model of fibrosis: Thirty-two C57 mice were randomly divided into three groups after one week of acclimatization: a control group, a model group, and two treatment groups. The control group consisted of 6 animals, while the other three groups each contained 8 animals. Except for the control group, the other three groups received intraperitoneal injections of CCl4 (0.6 ml / kg) on ​​days 1, 3, and 5, respectively. The control group also received an intraperitoneal injection of olive oil. Simultaneously, the treatment groups received continuous intragastric administration of compound 2 or compound 4 for 6 days, once daily, 2 hours after CCl4 injection. The treatment groups also received oral administration of 30 mg / kg of either compound 2 or compound 4. The control and model groups received an equal volume of physiological saline intraperitoneally. Twenty-four hours after the last administration, the mice were euthanized for sample analysis. A portion of liver tissue was cut to an appropriate size and placed in a 4% PFA container at -80°C for subsequent histological examination and Western blotting.

[0560] Detection methods: 1. After dissection, take in situ photographs of the liver; 2. Collect liver tissue, lyse it using RIPA, extract proteins for Western blotting; 3. After fixation, cut the liver tissue into sections and perform HE staining and Sirius red detection.

[0561] Experimental results:

[0562] a. No animals died until the end of the experiment; the animals' body weight decreased on the 6th day after the model was established. Figure 12 B);

[0563] b. After euthanasia of the experimental animals, the damage status of the mouse liver organs was observed in open field. Fibrotic lesions on the surface of the liver of the model group mice were clearly visible, while the roughness of the liver organ surface of the drug-treated group mice was alleviated. Figure 12 A);

[0564] c. HE staining showed that pseudolobules were clearly formed in the livers of mice in the model group, and the hepatocytes were dense, indicating that the model was successful. The drug-treated group showed some relief. Sirius red staining showed an increase in fibrous tissue in the model group and a decrease in the drug-treated group. Figure 12 C);

[0565] d. Western blotting (WB) revealed significant upregulation of liver fibrosis markers Collagen1 and α-SMA in the model group, indicating successful model construction. Administration of 30 mg / kg of compound 2 or compound 4 resulted in a downregulation of these liver fibrosis markers. Figure 12 D);

[0566] The above experimental results indicate that oral administration of compound 2 or compound 4 has a certain alleviating effect on acute liver fibrosis injury.

[0567] In summary, this invention discloses for the first time a novel class of cyclic bisbenzylisoquinoline alkaloids or their pharmaceutically acceptable salts, their preparation method, and discovers that they have a significant inhibitory effect on SARS-CoV-2 replication and inhibit ECV1. 50 The activity reaches single-digit micromolar levels or lower. Therefore, the cyclic bisbenzylisoquinoline alkaloids or their pharmaceutically acceptable salts described in this invention exhibit excellent anti-SARS-CoV-2 activity and have promising clinical application prospects. In addition, the cyclic bisbenzylisoquinoline alkaloids or their pharmaceutically acceptable salts described in this invention have anti-inflammatory, anti-fibrotic, and Th1 and Th17 cell differentiation-inhibiting effects, and are expected to be used to prevent and alleviate diseases caused by viral infections, inflammation-related diseases (pneumonia, non-alcoholic steatohepatitis, colitis, nephritis, pancreatitis, myocarditis, arthritis, inflammatory pain), fibrosis-related diseases (pulmonary fibrosis, silicosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis, retinal fibrosis, myelofibrosis), autoimmune diseases related to abnormal differentiation of Th1 and / or Th17 cells (psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neuromyelitis optica, myasthenia gravis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, delayed-type hypersensitivity, graft-versus-host disease, etc.), osteoporosis, and neurodegenerative diseases.

[0568] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A cyclic bisbenzyltetrahydroisoquinoline compound of Formula I or a pharmaceutically acceptable salt thereof, characterized in that, In the formula, R1 is selected from: hydrogen, C1-C6 alkyl, hydroxyC1-C6 alkyl, cyanoC1-C6 alkyl, C3-C8 cycloalkyl, cyano, C1-C6 alkylacyl, C3-C6 cycloalkylacyl, halogenated C1-C6 alkylacyl, carbamoyl, phenylcarbamoyl, C1-C6 alkyl-substituted carbamoyl, C3-C6 cycloalkyl-substituted carbamoyl, adamantylcarbamoyl, pyridine or pyrimidinyl-substituted carbamoyl, carbamoyl substituted with hydroxyC1-C6 alkoxyC1-C6 alkyl, carbamoyl substituted with C1-C6 alkoxyC1-C6 alkyl, C1-C6 alkoxycarbonyl substituted with hydroxyC1-C6 alkoxy, phenoxycarbonyl, hydroxymethyl substituted with C3-C6 cycloalkyl, carbamoyloxy substituted with C1-C6 alkyl, C1-C6 alkoxycarbonyl, carbamoylC1-C6 alkyl. ; R2 is selected from hydrogen or halogens; Where m is 1, 2, 3, or 4; The phenylcarbamoyl group and phenoxycarbonyl group thereon are not necessarily replaced by one or more substituents selected from F, Cl, Br, I, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy or halo-C1-C6 alkoxy. Alternatively, R1 and R2 together with the adjacent benzene ring form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 4 substituents, either substituted or unsubstituted, wherein the substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo groups, thio groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, and cyano groups; and the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

2. The compound according to claim 1, characterized in that: R1 is selected from the following group: hydrogen, C1-C4 alkyl, hydroxy C1-C4 alkyl, cyano C1-C4 alkyl, C3-C6 cycloalkyl, cyano, C1-C4 alkylacyl, C3-C5 cycloalkylacyl, halogenated C1-C4 alkylacyl, carbamoyl, phenylcarbamoyl, C1-C4 alkyl-substituted carbamoyl, C3-C5 cycloalkyl-substituted carbamoyl, adamantylcarbamoyl, pyridine or pyrimidinyl-substituted. Carbamoyl group, carbamoyl group substituted with hydroxyl C1-C4 alkoxy C1-C4 alkyl group, carbamoyl group substituted with C1-C4 alkoxy C1-C4 alkyl group, C1-C4 alkoxy carbonyl group substituted with hydroxyl C1-C4 alkoxy group, phenoxy carbonyl group, hydroxymethyl group substituted with C3-C5 cycloalkyl group, carbamoyloxy group substituted with C1-C4 alkyl group, C1-C4 alkoxy carbonyl group, carbamoyl C1-C4 alkyl group, ; R2 is selected from hydrogen or halogens; m is 1, 2, 3, or 4; Alternatively, R1 and R2 together with the adjacent benzene ring can form a benzo[5-6 membered monocyclic heterocycle] containing 1-2 substituents, either substituted or unsubstituted; wherein the substituted substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo (=O), thio (=S), C1-C6 alkyl groups, C1-C6 alkoxy groups, and cyano groups; wherein the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

3. The compound according to claim 1, characterized in that: R1 is selected from: hydrogen, propyl, cyclopropyl, isopropyl, tert-butyl, cyano, -CH2OH, -CH2CH2OH, formyl, acetyl, propionyl, trifluoroacetyl, carbamoyl, N-methylcarbamoyl, N,N-dimethylcarbamoyl, N-ethylcarbamoyl, N,N-diethylcarbamoyl, -O(C=O)NH(C4 alkyl), -CONH2, -(C=O)OCH3, -CONH(cyclopropyl); R2 is selected from: hydrogen, halogen; Alternatively, R1 and R2 together with the adjacent benzene ring can form a benzo[5-6 membered monocyclic heterocycle] containing 1 to 2 substituents, either substituted or unsubstituted, wherein the substituents are selected from halogens, hydroxyl groups, mercapto groups, oxo groups, thio groups, and C1-C6 alkyl groups; and the heterocycle contains 1 to 3 heteroatoms selected from N, O, and S.

4. The compound according to claim 1, characterized in that, The compound has the structure shown in formula Ia: R1 and R2 are as defined in claim 1.

5. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the group consisting of:

6. The compound according to claim 1, characterized in that, The compound of formula (I) is selected from the following compounds:

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (A1) A first active ingredient comprising a therapeutically effective amount of one or more mixtures of the compound of any one of claims 1 to 6 and a pharmaceutically acceptable salt thereof, and (B) Pharmaceutically acceptable carriers.

8. Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7, characterized in that, Inhibitors for the preparation of drugs that inhibit the replication of 2019 novel coronavirus, have anti-inflammatory, anti-fibrotic, and T-cell differentiation-inhibiting effects; and / or drugs for the prevention and / or treatment of diseases related to 2019 novel coronavirus infection, inflammation / fibrosis-related diseases, autoimmune diseases related to abnormal differentiation of Th1 and / or Th17 cells, osteoporosis, inflammatory pain, and neuroprotection.

9. The use as described in claim 8, characterized in that, The fibrosis mentioned refers to pulmonary fibrosis or liver fibrosis.

10. The use as described in claim 8, characterized in that, The autoimmune diseases associated with abnormal differentiation of Th1 and / or Th17 are selected from the following group: rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and neuromyelitis optica.

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