A phenazin derivative, its preparation method and use

Abstract

The present application relates to phenazepine derivatives with specific structures, preparation methods and uses thereof, and pharmaceutical compositions comprising the phenazepine derivatives. The phenazepine derivatives of the present application have multi-targeted activities of preventing viral infections and / or antiviral activities, and have good application prospects in the preparation of drugs for preventing viral infections and in the preparation of antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical industry, specifically to a phenylphenazine derivative, its preparation method and uses, and a pharmaceutical composition comprising the phenylphenazine derivative. Background Technology

[0002] The COVID-19 pandemic has spurred intensive research and development of vaccines and neutralizing antibodies against SARS-CoV-2, particularly those based on viral vectors, nucleic acids, and whole viral particles, which have achieved significant success and received emergency authorization. However, RNA viruses mutate rapidly, and off-target effects will become an increasingly serious problem as the virus evolves. Small molecule inhibitors designed to target viral targets may be ineffective against novel variants. Therefore, developing antiviral drugs that can address viral mutations is more meaningful (see Non-Patent Literature 1). Currently, drugs based on multi-target designs are all in the early stages of development.

[0003] Nitrogen-containing heterocyclic compounds possess a wide range of pharmacological activities and exhibit good drug-like properties. For example, clofazimine (hereinafter sometimes abbreviated as CFZ) (CAS No.: 2030-63-9) is clinically used for anti-tuberculosis treatment, and in recent years, this drug has been reported to have anti-SARS-CoV-2 activity (see Patent Document 1). However, CFZ has shortcomings such as relatively weak activity and does not yet meet the efficacy requirements of drugs for combating the novel coronavirus.

[0004] (Chroma CFZ)

[0005] Existing technical documents

[0006] Non-patent document 1: Narayanan A, Narwal M, Majowicz SA, et al. Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-proteaseassay [J]. Commun Biol. 2022, 5(1): 169.

[0007] Patent Document 1: CN112336724A Summary of the Invention

[0008] The problem the invention aims to solve

[0009] The technical problem of this invention is to provide a novel phenylphenazine derivative with multi-targeted and excellent anti-SARS-CoV-2 activity, its uses and preparation method; furthermore, to provide a novel phenylphenazine derivative with multi-targeted and excellent anti-SARS-CoV-2 activity and capable of preventing SARS-CoV-2 infection, its uses and preparation method.

[0010] The inventors have developed a series of novel phenylphenazine derivatives. Based on structure-activity relationship studies, they optimized the compound structures, resulting in enhanced antiviral activity and reduced toxicity of the compounds claimed in this invention. Furthermore, their in-depth research revealed that phenylphenazine compounds with specific structures not only exhibit antiviral activity by interfering with viral invasion and replication phases but also demonstrate significant preventative effects, suggesting a multi-target mechanism that simultaneously targets both the host and viral components.

[0011] means of solving technical problems

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0013] Firstly, a compound of general formula (I) or a pharmaceutically acceptable salt thereof is provided:

[0014]

[0015] In formula (I), X1 represents N or CH; R1 represents H or C. 1-5 alkoxy; R2 represents either benzyl or furan-2-ylmethyl optionally having a substituent, wherein the substituent is selected from halogen atoms, C 1-3 Alkyl or C 1-3 alkoxy group; R3 represents H, C 3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino; when R2 represents benzyl, R3 represents a C group with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino; when R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0016] Furthermore, the compound is represented by the following general formula (I-1):

[0017]

[0018] In formula (I-1), R1 represents H, R2 represents either benzyl or furan-2-ylmethyl with a substituent, wherein the substituent is selected from halogen atoms, methyl or methoxy; R3 represents H, C3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino;

[0019] When R2 represents benzyl, R3 represents C with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino; when R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0020] Furthermore, the compound is represented by the following general formula (I-2):

[0021]

[0022] In equation (I-2), R1 represents H or C. 1-5 alkoxy, R2 represents a benzyl group with a substituent selected from halogen atoms, methyl or methoxy; R3 represents H, C 3-5 cycloalkyl or C 1-5 alkyl.

[0023] Furthermore, the compound is selected from the group consisting of:

[0024]

[0025]

[0026] In a second aspect, a pharmaceutical composition is provided, characterized in that it contains an effective dose of the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0027] Thirdly, a method for preparing a compound of general formula (I) is provided, characterized by comprising the following synthetic process:

[0028]

[0029] In the above synthesis process:

[0030] The conditions indicated in step a are: alkali, organic solvent, 60 ~ 200 ℃;

[0031] The conditions indicated in step b are: 10% Pd / C, H2, organic solvent, 10 ~ 60 ℃;

[0032] The conditions represented by step c are: 1,5-difluoro-2,4-dinitrobenzene, base, organic solvent, 10 ~ 60 °C;

[0033] The conditions represented by step d are: R2NH2, organic solvent, 30 ~ 100 ℃;

[0034] The conditions indicated in step e are: 10% Pd / C, H2, organic solvent, 10 ~ 60 °C;

[0035] The conditions represented by step f are: atmospheric conditions, organic solvents, and 10 ~ 60 °C.

[0036] The conditions for step g are: R3NH2, organic acid, organic solvent, 50 ~ 150 °C.

[0037] In the above formulas, X1 represents N or CH; R1 represents H or C. 1-5 alkoxy; R2 represents either benzyl or furan-2-ylmethyl optionally having a substituent, wherein the substituent is selected from halogen atoms, C 1-3 Alkyl or C 1-3 alkoxy group; R3 represents H, C 3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino;

[0038] When R2 represents benzyl, R3 represents C with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino;

[0039] When R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0040] Fourthly, the use of compounds of general formula (I) or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention of SARS-CoV-2 infection and / or for the treatment of SARS-CoV-2.

[0041]

[0042] In formula (I), X1 represents N or CH; R1 represents H or C. 1-5 alkoxy; R2 represents either benzyl or furan-2-ylmethyl optionally having a substituent, wherein the substituent is selected from halogen atoms, C 1-3 Alkyl or C 1-3 alkoxy group; R3 represents H, C 3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino;

[0043] When R2 represents benzyl, R3 represents C with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino;

[0044] When R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0045] Furthermore, the use of compounds from the following group of compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for preventing and / or treating SARS-CoV-2 infection is provided.

[0046]

[0047]

[0048] Fifthly, the use of the pharmaceutical composition in the preparation of medicaments for preventing SARS-CoV-2 infection and / or for treating SARS-CoV-2 is provided.

[0049] Technical effects achieved by the present invention

[0050] Multi-target antiviral drugs can simultaneously intervene at multiple key targets in the viral replication process. Compared with single-target antiviral drugs, multi-target antiviral drugs have the following advantages: 1. High efficacy: Multi-target antiviral drugs can simultaneously intervene at multiple key targets, effectively inhibiting viral replication and transmission. 2. Low drug resistance: Because they intervene at multiple targets, multi-target antiviral drugs are more difficult to induce drug resistance, prolonging the drug's effectiveness. 3. High safety: Multi-target antiviral drugs have diversified effects, and the dosage of single drugs is lower, thus resulting in higher safety. 4. Strong adaptability: Because they can target multiple targets simultaneously, they have broad applicability in treating different types of viral infections.

[0051] The specific phenylphenazine derivatives of this invention exert their anti-SARS-CoV-2 effects by targeting the S protein and Nsp13 protein during membrane fusion and intracellular biosynthesis, respectively, thus affecting the membrane fusion and biosynthesis stages of SARS-CoV-2 and achieving excellent antiviral activity. Furthermore, the compounds with specific structures involved in this invention act not only on viral target proteins but also on host targets, exhibiting excellent anti-SARS-CoV-2 activity and preventative effects through a multi-target mechanism. In particular, the preventative effect can be used in a wider range of populations, and the safety is improved due to the reduced dosage. Attached Figure Description

[0052] Figure 1 This is a schematic diagram illustrating the experimental results of time-of-addition for a representative compound of the present invention.

[0053] Figure 2 This is a schematic diagram illustrating the safety test results of representative compounds of the present invention. Detailed Implementation

[0054] First, some of the statements in this invention will be explained.

[0055] "Optionally having substituents" means that the substituents are replaced at one or more positions by any one or any combination of the groups listed below.

[0056] "Halogen atom" can be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom;

[0057] “C 1～X (e.g., C) 1～5 "alkyl" refers to a straight-chain or branched alkyl group having 1 to X (e.g., 1 to 5) carbon atoms; examples include methyl, ethyl, propyl, isopropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-ethylpropyl, n-butyl, isobutyl, tert-butyl, pentyl, etc., which can be interpreted as including C1, C2, C3, ... C X Alkyl groups of various forms and structures, including (e.g., C5), are not limited to the examples listed above;

[0058] The alkyl groups mentioned above refer to various possible groups, including straight-chain or branched groups, such as butyl, including n-butyl, isobutyl, and tert-butyl.

[0059] “C 3～5 "Cycloalkyl" refers to a cycloalkyl group having 3 to 5 cyclic carbon atoms, and examples include cyclopropyl, cyclobutyl, and cyclopentyl cycloalkyl groups with various morphologies and structures.

[0060] “C 1～5 "Alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 5 carbon atoms; examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, etc. It can be interpreted as alkoxy groups with various morphologies including C1, C2, C3, C4, and C5, and is not limited to the examples listed above.

[0061] When optical isomers and geometric isomers are present in the compounds of this invention, they include all isomer categories.

[0062] In one aspect of the invention, a compound of the following general formula (I) or a pharmaceutically acceptable salt thereof is provided:

[0063]

[0064] In formula (I), X1 represents N or CH; R1 represents H or C. 1-5 alkoxy; R2 represents either benzyl or furan-2-ylmethyl optionally having a substituent, wherein the substituent is selected from halogen atoms, C 1-3 Alkyl or C 1-3 alkoxy group; R3 represents H, C 3-5cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino;

[0065] When R2 represents benzyl, R3 represents C with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino;

[0066] When R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0067] In formula (I):

[0068] R1 preferably represents H or C. 1-3 Alkoxy, particularly preferably H or methoxy.

[0069] R2 preferably represents benzyl or furan-2-ylmethyl with a substituent selected from halogen atoms, methyl or methoxy; more preferably it represents o-methylbenzyl, m-methylbenzyl, p-methylbenzyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, or furan-2-ylmethyl.

[0070] R3 preferably represents H, C 3-5 cycloalkyl, optional C-shaped with substituents 1-4 Alkyl group, wherein the optional substituent is N,N-dimethylamino; more preferably H, cyclopropyl, isopropyl, N,N-dimethylaminoethyl, and particularly preferably H, isopropyl.

[0071] In one aspect of the invention, a compound of the following general formula (I-1) or a pharmaceutically acceptable salt thereof is provided:

[0072]

[0073] In formula (I-1), R1 represents H, R2 represents either benzyl or furan-2-ylmethyl with a substituent, wherein the substituent is selected from halogen atoms, methyl or methoxy; R3 represents H, C 3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino.

[0074] In formula (I-1):

[0075] R2 preferably represents benzyl or furan-2-ylmethyl with a substituent selected from halogen atoms, methyl or methoxy; more preferably it represents o-methylbenzyl, m-methylbenzyl, p-methylbenzyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, or furan-2-ylmethyl.

[0076] R3 preferably represents H, C 3-5 cycloalkyl, optional C-shaped with substituents 1-4 Alkyl group, wherein the optional substituent is N,N-dimethylamino; more preferably H, cyclopropyl, isopropyl, N,N-dimethylaminoethyl, and particularly preferably isopropyl.

[0077] In one aspect of the invention, compounds of the following general formula (I-2) or pharmaceutically acceptable salts thereof are provided:

[0078]

[0079] In equation (I-2), R1 represents H or C. 1-5 alkoxy, R2 represents a benzyl group with a substituent selected from halogen atoms, methyl or methoxy; R3 represents H, C 3-5 cycloalkyl or C 1-5 alkyl.

[0080] In equation (I-2):

[0081] R1 preferably represents H or methoxy group.

[0082] R2 preferably represents o-methylbenzyl, m-methylbenzyl, p-methylbenzyl, 2,4-dimethoxybenzyl, p-fluorobenzyl, p-chlorobenzyl, or p-bromobenzyl.

[0083] R3 preferably represents H, C 1-5 Alkyl; H and isopropyl are further preferred.

[0084] In one aspect of the invention, compounds selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof are provided:

[0085]

[0086]

[0087] The compounds of the present invention are not limited to the examples listed above.

[0088] The term "pharmaceutically acceptable salt" in this invention refers to a salt that ensures the biological efficacy of the compounds of this invention and typically does not exhibit adverse properties in biological or other respects. In many cases, the compounds of this invention can form acid and / or base salts through the presence of amino and / or carboxyl groups or similar groups.

[0089] Pharmaceutically acceptable salts include both inorganic and organic salts.

[0090] Inorganic bases that can form inorganic salts with the compounds of this invention include, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonia.

[0091] Inorganic acids that can form inorganic salts with the compounds of this invention include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.

[0092] Organic bases that can form organic salts with the compounds of the present invention include, for example, primary amines, secondary amines, tertiary amines, ethylenediamine, propylenediamine, butanediamine, phenylmethylamine, phenylethylamine, etc.

[0093] Organic acids that can form organic salts with the compounds of this invention include, for example, formic acid, acetic acid, propionic acid, oxalic acid, citric acid, benzoic acid, etc.

[0094] In one aspect of the invention, a pharmaceutical composition is provided, characterized in that it contains an effective dose of the novel compound described herein or a pharmaceutically acceptable salt thereof.

[0095] In one aspect of the present invention, a method for preparing a compound of general formula (I) is provided, characterized by comprising the following synthetic process:

[0096]

[0097] In the above synthesis process:

[0098] The conditions indicated in step a are: alkali, organic solvent, 60 ~ 200 ℃;

[0099] The conditions indicated in step b are: 10% Pd / C, H2, organic solvent, 10 ~ 60 ℃;

[0100] The conditions represented by step c are: 1,5-difluoro-2,4-dinitrophenyltriethylamine, base, organic solvent, 10 ~ 60 °C;

[0101] The conditions represented by step d are: R2NH2, organic solvent, 30 ~ 100 ℃;

[0102] The conditions indicated in step e are: 10% Pd / C, H2, organic solvent, 10 ~ 60 °C;

[0103] The conditions represented by step f are: atmospheric conditions, organic solvents, and 10 ~ 60 °C.

[0104] The conditions represented by step g are: R3NH2, organic acid, organic solvent, 50 ~ 150 ℃.

[0105] The base in the conditions indicated in step a can be any organic or inorganic base. Examples of organic bases include primary amines, secondary amines, tertiary amines, ethylenediamine, propylenediamine, butanediamine, benzylamine, phenethylamine, etc., with triethylamine being preferred. Examples of inorganic bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, sodium carbonate, potassium fluoride, sodium bicarbonate, etc., with potassium fluoride and sodium bicarbonate being preferred. Examples of organic solvents in the conditions indicated in step a include methanol, ethanol, propanol, THF (tetrahydrofuran), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), dichloromethane, dioxane, acetone, etc., with DMSO being preferred. The temperature range in the conditions indicated in step a is preferably 70 ~ 150 ℃, more preferably 80 ~ 130 ℃.

[0106] The organic solvent in the conditions indicated in step b can be, for example, methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., with ethanol being preferred; the temperature range in the conditions indicated in step b is preferably 20 to 50°C, more preferably 20 to 40°C.

[0107] The base in the conditions indicated in step c is preferably an organic base, such as primary amines, secondary amines, tertiary amines, ethylenediamine, propylenediamine, butanediamine, benzylamine, phenethylamine, etc., and more preferably triethylamine; the organic solvent in the conditions indicated in step c can be such as methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., and is preferably ethanol; the temperature range in the conditions indicated in step c is preferably 20 ~ 50 ℃, and more preferably 20 ~ 40 ℃.

[0108] The organic solvent in the conditions indicated in step d can be, for example, methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., and is preferably THF; the temperature range in the conditions indicated in step d is preferably 40 ~ 90 ℃, more preferably 50 ~ 80 ℃.

[0109] The organic solvent in the conditions indicated in step e can be, for example, methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., and is preferably methanol / THF; the temperature range in the conditions indicated in step e is preferably 20 ~ 50 °C, more preferably 20 ~ 40 °C.

[0110] The organic solvent in the conditions indicated in step f can be, for example, methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., and is preferably ethanol / dichloromethane; the temperature range in the conditions indicated in step f is preferably 20 ~ 50 ℃, more preferably 20 ~ 40 ℃.

[0111] The organic acid in the conditions represented by step g can be, for example, formic acid, acetic acid, tartaric acid, oxalic acid, benzoic acid, salicylic acid, etc., and is preferably acetic acid; the organic solvent in the conditions represented by step g can be, for example, methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, acetone, etc., and is preferably dioxane; the temperature range in the conditions represented by step g is preferably 60 ~ 140 ℃, more preferably 80 ~ 100 ℃.

[0112] In the above formulas, X1 represents N or CH; R1 represents H or C. 1-5 alkoxy; R2 represents either benzyl or furan-2-ylmethyl optionally having a substituent, wherein the substituent is selected from halogen atoms, C 1-3 Alkyl or C 1-3 alkoxy group; R3 represents H, C 3-5 cycloalkyl or optionally C-shaped with substituents 1-5 Alkyl group, wherein the optional substituent is selected from halogen atoms or N,N-dimethylamino;

[0113] When R2 represents benzyl, R3 represents C with a substituent. 1-5 Alkyl group, wherein the substituent is N,N-dimethylamino;

[0114] When R3 represents isopropyl, R2 is not p-fluorobenzyl.

[0115] The preferred forms of R1, R2, and R3 are as described above.

[0116] In one aspect of the invention, the use of a compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection is provided. Example

[0117] The present invention will now be described in more detail using manufacturing examples and test examples, but the present invention is not limited to these examples.

[0118] <Manufacturing Example>

[0119] (1) Example of synthesis in step a

[0120] A mixture of 2-fluoronitrobenzene (150 mmol, Leyan), substituted aniline (180 mmol, Leyan), and anhydrous potassium fluoride (8.7 g, 150 mmol, Inokai) was dissolved in DMSO and stirred at 120 °C for 8 h. The mixture was then cooled, and water and ethyl acetate were added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with 2 N HCl and dried over anhydrous Na₂SO₄. The filtrate was concentrated under vacuum, and the crude product was recrystallized in 100 mL of 95% ethanol to give a product that was mostly a red solid, with a yield of 75–80%.

[0121] (2) Example of the synthesis of steps b and c

[0122] The product obtained in step a (115 mmol) and a mixture of 10% Pd / C (2.4 g) in ethanol were shaken for 1.5 h at room temperature under a hydrogen atmosphere (40 psi). After filtration, DDFNB (1,5-difluoro-2,4-dinitrobenzene, Leyen) (115 mmol) and triethylamine (115 mmol, Titan) were added to the filtrate; the mixture was stirred at room temperature for 3 h, filtered, and washed with ethanol to obtain product 19a as a red solid in yield of 85–90%.

[0123] The product obtained in step c is, for example, 19a below:

[0124] (19a)

[0125] (3) Example of synthesis in step d (synthesis of 3-(phenyl)-4,6-dinitro-3',4'-dimethoxybenzylaminodiphenylamine (20c))

[0126] The product obtained in step c, Example 19a (14.73 g, 40 mmol), 3',4'-dimethoxybenzylamine (6.69 g, 40 mmol), triethylamine (4.04 g, 40 mmol), and THF were refluxed for 18 h. After cooling to room temperature, the mixture was concentrated under vacuum, CH2Cl2 was added to the residue, and the resulting solid was filtered to give 17.5 g of a red solid, yield 85%, mp: 132–133 °C. 1 H NMR (600 MHz, DMSO- d 6 ) d 9.43 (s, 1H), 8.96 (s, 1H), 8.76 (t, J = 5.8 Hz, 1H), 7.62 (s, 1H), 7.30–7.21 (m, 2H), 7.16 (t, J= 7.7 Hz, 2H),7.07–6.99 (m, 1H), 6.89 (dd, J = 7.4, 4.4 Hz, 3H), 6.83 (t, J = 7.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.36 (dd, J = 8.2, 2.4Hz, 1H), 5.77 (s, 1H), 4.21 (d, J = 5.9 Hz, 2H), 3.72 (s, 3H), 3.68 (s, 3H).

[0127]

[0128] Example 1: 2-(2,4-dimethoxybenzyl)amino-3-imino-5-(phenyl)-3,5-dihydrophenazine (30a)

[0129] At -10 °C, zinc powder (7.1 g, 109 mmol, saturated) was added to a mixture of 20c (2.86 g, 6 mmol) in proportion. 15 mL of glacial acetic acid was slowly added until the mixture was stirred until the color turned light green (if the color did not change, add 5-10 drops of concentrated hydrochloric acid). The reaction was complete upon color change, and the mixture was transferred to room temperature. The reaction solution was filtered, the filtrate was concentrated, the residue was treated with water, and the crude product was alkalized with ammonia. The solid was filtered, washed with water, and then dissolved in anhydrous methanol. The solution was stirred in air overnight, the reaction solution was concentrated and mixed, and column chromatography was used to separate product 30a, a brown solid of 394 mg, yield 15%, mp: 174–176 °C. 1 H NMR (600 MHz, DMSO- d 6 ) d 7.90 (s, 1H), 7.80 (dd, J = 6.0, 4.6 Hz,2H), 7.77–7.69 (m, 1H), 7.58–7.51 (m, 2H), 7.39–7.32 (m, 2H), 7.14 (d, J =8.3 Hz, 1H), 6.63 (d, J = 2.4 Hz, 1H), 6.60–6.54 (m, 1H), 6.49 (dd, J= 8.3,2.4 Hz, 1H), 6.25 (s, 1H), 5.57 (s, 1H), 4.41 (s, 2H), 3.88 (s, 3H), 3.74 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) d 160.4, 158.4, 158.2, 148.9, 145.7, 137.3,136.8, 134.3, 131.8(2), 130.8, 130.7, 129.1, 128.9(2), 128.8, 128.5, 124.6,117.7, 115.6, 105.1, 99.0, 98.2, 95.3, 56.1, 55.7, 49.0. HRMS: calculated forC 27 H 25 N4O2 [M+H] + : 437.1972, found: 437.1977.

[0130] Example 2: 2-(2,4-dimethoxybenzyl)amino-3-isopropylimino-5-(phenyl)-3,5-dihydrophenazine (30b)

[0131] Compound 30a (0.5 mmol) was added to a tetrahydrofuran (2 mL) solution of isopropylamine (11.7 mmol, calcd.) in a sealed tube. The mixture was heated at 100 °C for 10 h, and then purified by vacuum concentration column chromatography (CH2Cl2 / CH3OH = 50:1) to give 100 mg of the product as a red solid, with a yield of 21%. mp: 220 – 222 °C. 1 H NMR (600 MHz, CDCl3) d 9.39 (br, 1H), 7.93 (dd, J = 8.3, 1.4 Hz, 1H), 7.84–7.67 (m, 3H), 7.49 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.39 (ddd, J = 8.5, 7.0, 1.4 Hz, 1H), 7.34–7.30(m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 6.86 (dd, J= 8.6, 1.2 Hz, 1H), 6.63 (s,1H), 6.37 (d, J = 2.3 Hz, 1H), 6.29 (dd, J = 8.4, 2.3 Hz, 1H), 5.58 (s, 1H), 4.53 (d, J = 3.1 Hz, 2H), 3.84 (s, 3H), 3.64 (s, 3H), 3.43 (p, J = 6.5 Hz,1H), 1.29 (s, 3H), 1.28 (s, 3H). 13 C NMR (151 MHz, CDCl3) d 159.0, 157.0,150.5, 145.1, 142.7, 137.6, 135.2, 133.8, 130.6, 130.4, 128.9, 128.4, 128.0,127.9, 127.9, 126.4, 125.8, 116.2, 116.2, 115.5, 103.4, 103.4, 99.3, 97.2,88.3, ​​54.5, 54.3, 46.5, 40.0, 20.1. HRMS (ESI) m / z: calculated for C 30 H 31 N4O2[M+H] + : 479.2441, found: 479.2445.

[0132] Example 3: 2-(o-methylbenzyl)amino-3-imino-5-(phenyl)-3,5-dihydrophenazine (23a)

[0133] Following the preparation procedure described in 30a, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 445 mg, with a yield of 19%. mp: 208–210 ℃. 1 H NMR (600 MHz, CDCl3) d 7.73(dd, J = 8.0, 1.5 Hz, 1H), 7.68 (t, J = 7.7 Hz, 2H), 7.61 (t, J = 7.5 Hz,1H), 7.32–7.27 (m, 3H), 7.22–7.09 (m, 5H), 6.51 (dd, J= 8.3, 1.3 Hz, 1H), 6.27 (s, 1H), 5.23 (s, 1H), 4.46 (s, 2H), 2.36 (s, 3H). 13 C NMR (151 MHz, CDCl3) d 159.8, 149.5, 146.1, 136.6, 135.3, 134.8, 134.2, 133.2, 130.2(2),130.0, 129.5, 128.8, 127.7(2), 127.2, 126.8, 126.5, 126.5, 125.2, 122.1,113.4, 96.6, 95.5, 44.1, 17.9. HRMS (ESI) m / z: calculated for C 26 H 23 N4 [M+H] + 391.1917, found: 391.1923.

[0134] Example 4: 2-(o-methylbenzyl)amino-3-cyclopropylimino-5-(phenyl)-3,5-dihydrophenazine (23c)

[0135] Following the preparation procedure described in 30b, cyclopropylamine was added to the reaction solution, and the reaction was carried out under the aforementioned conditions with appropriate post-treatment to obtain the target compound as a red solid, 56.0 mg, with a yield of 26%. mp: 199 – 202 ℃. 1 H NMR (600 MHz, CDCl3) d 8.01 (s, 1H), 7.82 (t, J = 7.5 Hz, 2H), 7.78 (d, J = 7.2Hz, 1H), 7.53 (m, 1H), 7.46–7.37 (m, 3H), 7.29 (d, J = 7.5 Hz, 1H), 7.19–7.17(m, 1H), 7.14 (td, J = 7.4, 1.4 Hz, 1H), 7.12–7.08 (m, 1H), 6.92 (m, 1H), 6.53 (m, 1H), 6.18–5.98 (m, 1H), 4.62 (s, 2H), 2.42 (s, 3H), 1.26 (m, 1H),1.01–0.93 (m, 2H), 0.71 (m, 2H). 13C NMR (151 MHz, CDCl3) d 173.0, 152.3,136.8, 135.2, 134.5, 133.7, 130.2(2), 129.4, 128.8, 127.8, 126.9, 126.7,126.4, 126.1(2), 125.1(2), 123.5, 123.0, 122.2, 118.2, 113.4, 95.7, 88.7,44.2, 28.7, 18.0, 5.5. HRMS (ESI) m / z: calculated for C 29 H 26 N4 [M+H] + :431.2230, found: 431.2221.

[0136] Example 5: 2-(p-chlorobenzyl)amino-3-isopropylimino-5-phenyl-3,5-dihydrophenazine (29b)

[0137] Following the preparation procedure of 30b, isopropylamine was added to the reaction solution, and the reaction was carried out under the above reaction conditions with appropriate post-treatment to obtain the target compound as a red solid, 48 mg, with a yield of 17%. mp: 128–130 ℃; 1 H NMR (500MHz, CDCl3) d 7.73–7.68 (m, 2H), 7.66–7.62 (m, 2H), 7.33–7.27 (m, 6H), 7.13(t, J = 7.3 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H), 6.07(s, 1H), 5.22 (s, 1H), 4.47 (s, 2H), 3.41–3.34 (m, 1H), 1.02 (d, J = 6.2 Hz, 6H); 13 C NMR (126 MHz, CDCl3) d151.0, 150.7, 147.9, 137.8, 136.6, 135.5,134.8, 132.8, 131.4, 131.2 (2), 129.6, 128.8 (2), 128.7 (2), 128.5 (2),127.6, 126.8, 122.5, 114.0, 96.4, 89.1, 49.2, 46.0, 23.5; HRMS (ESI) m / z:calculated for C 28 H 26 ClN4 [M+H] + : 453.1841, found 453.1840.

[0138] Example 6: 2-(p-methylbenzyl)amino-3-imino-5-(phenyl)-3,5-dihydrophenazine (33a)

[0139] Following the preparation procedure described in 30a, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 561 mg, with a yield of 23%. mp: 190–192 ℃. 1 H NMR (600 MHz, DMSO- d 6) d 9.00 (br, 1H), 8.10–7.98 (m, 1H), 7.85 (td, J = 7.3, 6.2, 1.6 Hz, 2H), 7.83–7.80 (m, 1H), 7.66–7.61 (m, 2H), 7.59 (td, J = 7.7, 7.0, 1.6 Hz, 1H), 7.36(d, J = 7.8 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 6.88–6.81 (m, 1H), 6.63 (s, 1H), 6.03 (s, 1H), 4.57 (s, 2H), 2.27 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6) d155.7, 146.6, 143.1, 138.3, 136.8, 136.7, 135.1, 135.1, 131.7(2), 131.4,130.8, 130.0, 129.6(2), 129.3, 128.3(2), 127.7(2), 126.8, 117.0, 100.6, 93.6,49.0, 21.2. HRMS (ESI) m / z: calculated for C 26 H 23 N4 [M+H] + : 391.1917, found:391.1923.

[0140] Example 7: 2-(m-methylbenzyl)amino-3-isopropylimino-5-(phenyl)-3,5-dihydrophenazine (34b)

[0141] Following the preparation procedure of 30b, isopropylamine was added to the reaction solution, and the reaction was carried out under the above reaction conditions with appropriate post-treatment to obtain the target compound as a red solid, 45.46 mg, with a yield of 21%. mp: 172–174 ℃. 1 H NMR (600 MHz, CDCl3) d 7.88 (d, J = 8.1 Hz, 1H), 7.73 (p, J = 7.0 Hz, 3H), 7.41(s, 1H), 7.34–7.26 (m, 3H), 7.18 (d, J = 7.8 Hz, 2H), 7.11 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.76 (s, 1H), 6.49 (s, 1H), 5.51 (s, 1H), 4.52(s, 2H), 3.41 (p, J = 6.5 Hz, 1H), 2.24 (s, 3H), 1.24 (d, J = 6.3 Hz, 6H). 13 CNMR (151 MHz, CDCl3) d150.4, 146.1, 143.5, 137.2, 137.0, 136.3, 135.4,133.8, 130.5(2), 130.1, 128.3, 128.1, 127.5, 127.1, 127.0 (2), 126.6, 125.2,123.5(2), 115.1, 98.6, 88.3, ​​49.5, 46.8, 45.9, 20.4 (2). HRMS (ESI) m / z:calculated for C 29 H 29 N4 [M+H] + : 433.2387, found: 433.2387.

[0142] Example 8: 2-(p-fluorobenzyl)amino-3-imino-5-(phenyl)-3,5-dihydrophenazine (31a)

[0143] Following the preparation procedure described in 30a, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 402.9 mg, with a yield of 17%. mp: 134–136 ℃. 1 H NMR (500 MHz, DMSO- d 6 ) d 9.36 (s, 2H), 8.95 (t, J = 5.0 Hz, 1H), 8.16 (s, 1H), 7.86 (t, J = 5.0 Hz, 3H), 7.68 (dd, J = 5.0 Hz, 4H), 7.55 (t, J = 5.0 Hz, 2H), 7.24 (t, J = 5.0Hz, 2H), 6.91 (d, J = 5.0Hz, 1H), 6.77 (s, 1H), 6.1 (s, 1H), 4.63 (d, J = 5.0Hz, 2H); 13 C NMR (126 MHz, DMSO- d 6 ) d162.4, 160.5, 154.6, 145.7, 142.0,138.2, 136.3, 134.8, 131.3(2), 129.5(2), 129.4(2), 129.4, 129.1, 127.3(2),126.1, 116.9, 115.4, 115.3, 100.6, 92.9, 45.0; HRMS (ESI) m / z calculated forC 25 H 20 FN4 [M+H] + 395.1665, found 395.1666.

[0144] Example 9: 2-(methylenefuranyl)amino-3-isopropylimino-5-phenyl-3,5-dihydrophenazine (36b)

[0145] Following the preparation procedure of 30b, isopropylamine was added to the reaction solution, and the reaction was carried out under the above reaction conditions with appropriate post-treatment to obtain the target compound as a red solid, 49 mg, with a yield of 18%. mp: 161–163 ℃; 1 H NMR (400 MHz, CDCl3) d 7.75–7.66 (m, 3H), 7.65–7.57 (m, 1H), 7.38 (s, 1H), 7.35–7.27 (m, 2H), 7.14 (t, J = 7.0 Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 6.45 (d, J = 7.9 Hz, 1H), 6.34–6.29 (m, 2H), 6.24 (s, 1H), 5.21 (s, 1H), 4.46 (s, 2H), 3.41–3.31 (m, 1H), 1.01 (d, J = 5.8 Hz, 6H); 13 C NMR (101 MHz, CDCl3) d151.4,151.2, 150.8, 147.8, 142.1 (2), 137.9, 135.6, 134.8, 131.5, 131.2 (2), 129.6,128.9, 127.7, 126.8, 122.5, 114.0, 110.4, 107.4, 96.3, 89.1, 49.2, 40.0, 23.5(2); HRMS (ESI) m / z calculated for C 26 H 25 N4O [M+H] + 409.2023 was found as 409.2022.

[0146] Example 10: 2-(p-Bromobenzyl)amino-3-isopropylimino-5-(phenyl)-3,5-dihydrophenazine (28b)

[0147] Following the preparation procedure of 30b, isopropylamine was added to the reaction solution, and the reaction was carried out under the above reaction conditions with appropriate post-treatment to obtain the target compound as a red solid, 62.1 mg, with a yield of 25%. mp: 159 – 161 ℃. 1 H NMR (600 MHz, CDCl3) d 10.25 (d, J = 6.8 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.79– 7.72 (m, 3H), 7.54 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.43 (ddd, J = 8.5,7.0, 1.4 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.35–7.29 (m, 4H), 6.89 (d, J =8.6 Hz, 1H), 6.56 (s, 1H), 5.62 (s, 1H), 4.57 (d, J = 5.7 Hz, 2H), 3.44 (h, J= 6.6 Hz, 1H), 1.32 (s, 3H), 1.31 (s, 3H). 13 C NMR (151 MHz, CDCl3) d150.6,145.0, 142.5, 137.6, 135.3, 135.2, 133.9, 130.7(2), 130.5(2), 130.4, 129.2,128.7, 128.3(2), 128.0, 126.4(2), 126.0, 120.1, 115.5, 99.6, 88.4, 46.6,45.4, 28.7, 20.1. HRMS (ESI) m / z: calculated for C 28 H 26 BrN4 [M+H] + : 497.1335, found: 497.1337.

[0148] Example 11: 2-(p-chlorobenzyl)amino-3-imino-5-(p-methoxyphenyl)-3,5-dihydropyridinephenazine (27a)

[0149] Following the preparation procedure described in 30a, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 505.0 mg, with a yield of 19%. mp: 228 ℃ (dec.). 1 H NMR (600 MHz, CDCl3) d 8.21–8.01 (m, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.25–7.21 (m, 3H), 7.19 (s, 1H), 7.12–7.05 (m, 5H), 6.98–6.74 (m, 1H), 6.08 (s, 1H), 5.25 (s, 1H), 4.42 (s, 2H), 3.84 (s, 3H). 13 C NMR (151 MHz, CDCl3) d 159.9, 158.9, 150.4, 146.3,146.1, 141.6, 134.9, 134.7, 133.8, 132.2, 129.9(2), 128.6, 128.5, 127.9(2),127.6(2), 118.1, 114.8(2), 97.5, 96.7, 54.5, 45.2. HRMS (ESI) m / z: calculatedfor C 25 H 21 ClN5O [M+H] + : 442.1434, found: 442.1429.

[0150] Example 12: 2-(p-chlorobenzyl)amino-3-cyclopropylimino-5-(p-methoxyphenyl)-3,5-dihydropyridinephenazine (27c)

[0151] Following the preparation procedure described in 30b, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 96 mg, in 40% yield. mp: 141–143 °C. 1 H NMR (600 MHz, CDCl3) δ 8.19(d, J = 1.8 Hz, 1H), 7.95–7.92 (m, 1H), 7.35–7.11 (m, 10H), 6.19–6.03 (m,1H), 5.80 (s, 1H), 4.47 (s, 2H), 3.92 (s, 3H), 2.70 (m, 1H), 0.85 (m, 2H), 0.82–0.78 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 158.8, 151.8, 146.2, 145.8,135.3, 135.2, 135.1, 133.6, 133.5, 132.1, 128.6(2), 127.8(2), 127.6(2),124.4, 122.5, 118.3, 114.8(2), 114.0, 96.2, 90.4, 54.5, 45.2, 28.7, 8.5.HRMS(ESI) m / z: calcd for C 28 H 25 ClN5O [M+H] + : 482.1742, found: 482.1729.

[0152] Example 13: 2-(2,4-dimethoxybenzyl)amino-3-N,N-dimethylaminoethylimino-5-(phenyl)-3,5-dihydrophenazine (30d)

[0153] Following the preparation procedure described in 30b, N,N-dimethylethylenediamine was added to the reaction solution, and the reaction was carried out under the aforementioned conditions with appropriate post-treatment to obtain the target compound as a red solid, 55.9 mg, with a yield of 22%. mp: 190 – 192℃. 1 H NMR (600 MHz, CDCl3) d 7.89 (d, J = 8.3 Hz, 1H), 7.72 (t, J= 7.6 Hz,2H), 7.69–7.64 (m, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.29 (d, J = 7.5 Hz, 3H), 7.18 (d, J = 8.3 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 6.39 (d, J = 2.3 Hz, 1H), 6.32 (d, J = 8.4 Hz, 1H), 5.56 (s, 1H), 4.52 (s, 2H), 3.83 (d, J = 1.7 Hz, 3H), 3.68 (d, J = 1.7 Hz, 3H), 3.28 (t, J = 7.2 Hz, 2H), 2.67 (t, J = 7.1 Hz, 2H), 2.16 (s, 6H). 13 C NMR (151 MHz, CDCl3) d 165.7, 162.8, 162.0,159.1, 157.1, 152.2, 143.9, 137.0, 135.7, 133.9, 130.5, 129.9, 129.1, 128.5,128.1, 127.9, 127.4, 126.7, 116.5, 114.9, 103.1, 98.3, 97.3, 88.1, 56.2,54.4, 54.3, 50.0, 47.7, 44.2, 40.4. HRMS (ESI) m / z: calculated for C 31 H 33 N5O2[M+H] + : 508.2707, found: 508.2704.

[0154] Example 14: 2-(benzyl)amino-3-N,N-dimethylaminoethylimino-5-(phenyl)-3,5-dihydrophenazine (35d)

[0155] Following the preparation procedure described in 30b, N,N-dimethylethylenediamine was added to the reaction solution, and the reaction was carried out under the aforementioned conditions with appropriate post-treatment to obtain the target compound as a red solid, 51 mg, with a yield of 23%. mp: 169 – 171 ℃. 1 H NMR (600 MHz, CDCl3) d 8.81 (br, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.71 (t, J = 7.2 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 7.5 Hz, 3H), 7.28 (t, J = 7.8 Hz, 3H), 7.24 (t, J = 7.6 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H), 6.69 (d, J = 8.5 Hz, 1H), 6.45 (s, 1H), 5.51 (s, 1H), 4.57 (s, 2H), 3.33–3.23 (m, 2H), 2.73 (t, J = 6.8 Hz, 2H), 2.23 (s, 6H). 13 C NMR (151 MHz, CDCl3) d 152.4,146.6, 144.2, 136.7, 136.5, 135.7, 133.9, 130.7, 130.5, 129.8, 128.7, 128.0,127.9, 127.7(2), 127.6, 126.8, 126.6, 126.5, 126.4, 126.2, 124.7, 114.8,98.1, 88.1, 56.0, 46.0, 43.7, 42.4. HRMS (ESI) m / z: calculated for C 29 H 30 N5 [M+H] + : 448.2495, found: 448.2509.

[0156] Comparative Example 1: 2-Benzylamino-3-imino-5-phenyl-3,5-dihydrophenazine (Comparative Compound 1)

[0157] Following the preparation procedure described in 30a, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid, 48.6 mg, with a yield of 19%. mp: 185–187 ℃; 1 H NMR (500 MHz, DMSO- d 6) d 9.27 (s, 1H), 7.78–7.72 (m, 2H), 7.68–7.64 (m, 1H), 7.55–7.52 (m, 1H), 7.45–7.42 (m, 2H), 7.38–7.32 (m, 5H), 7.27–7.22 (m, 1H), 7.14–7.10 (m, 2H), 6.34–6.30 (m, 1H), 5.94 (s, 1H), 5.16 (s, 1H), 4.51 (d, J = 5.4 Hz, 2H); 13 C NMR (126 MHz, DMSO-) d 6) d 159.6, 150.2, 147.3, 138.6, 137.1, 134.8, 132.8, 131.2(2), 130.8, 129.5, 128.7(2), 128.4(2), 127.1, 126.9(3), 126.7, 122.1, 113.7,96.4, 96.3, 45.3; HRMS (ESI) m / z calculated for C 25 H 21 N4 [M+H] + 377.1761, found 377.1760.

[0158] Comparative Example 2: 2-(p-Fluorobenzylamino)-3-isopropylimino-5-phenyl-3,5-dihydrophenazine (Comparative Compound 2)

[0159] Following the preparation procedure described in 30b, the reaction was carried out under the above reaction conditions, and appropriate post-treatment was performed to obtain the target compound as a red solid; mp: 166–168 °C; 1 H NMR (500 MHz, CDCl3) d 7.73–7.68 (m, 2H),7.67–7.60 (m, 2H), 7.34–7.29 (m, 4H), 7.13 (t, J= 7.3 Hz, 1H), 7.09–7.00 (m,3H), 6.45 (d, J = 8.1 Hz, 1H), 6.10 (s, 1H), 5.22 (s, 1H), 4.47 (s, 2H), 3.41–3.33 (m, 1H), 1.02 (d, J = 6.3 Hz, 6H); 13 C NMR (126 MHz, CDCl3) d 162.0,151.1, 150.7, 147.9, 137.8, 135.6, 134.8, 133.8, 131.5, 131.2(2), 129.6,128.9(2), 128.8, 128.7, 127.6, 126.7, 122.5, 115.5(2), 114.0, 96.3, 89.1,49.2, 46.0, 23.5(2); HRMS (ESI) m / z calculated for C 28 H 26 N4F [M+H] + 437.2136, found 437.2136.

[0160] <Experimental Example>

[0161] HCoV-OC43, which is very similar to SARS-CoV-2 and is also a β-coronavirus, was selected as a screening model for activity evaluation to examine the activity of this type of compound.

[0162] 1. Determination of the antiviral activity of the compound

[0163] H460 cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. During the experiment, H460 cells were seeded at an appropriate multiplicity of infection (MOI = 0.35) in 96-well plates and cultured in an incubator at 37 ℃, 5% CO2, and saturated humidity. 100 μL of DMEM complete medium was added to each well, consisting of 89% DMEM basal medium, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution. All cell culture reagents were purchased from Thermo Fisher Scientific, USA. After overnight culture and reaching approximately 80% confluence, subsequent infection and drug treatments were performed.

[0164] HCoV-OC43 (VR1558) was kindly provided by Beijing Ditan Hospital, Capital Medical University (Beijing, China). H460 cells were infected with HCoV-OC43 for 24 h, and maintenance medium containing different concentrations (starting from 100 μM and diluted 3-fold) of sample compounds or the positive control drug CFZ was added. Cell control wells and virus control wells were also established, and the cells were cultured at 5% CO2 and 37 °C. When the cytopathic effect (CPE) in the virus control group reached 4+, the CPE performance of each group was observed and recorded. Total cytotoxicity (TC) was calculated using the Reed-Muench method. 50 (medium toxic concentration, half-maximal toxic concentration) and IC50 50 (medium inhibition concentration, half-maximal inhibitory concentration), and based on TC 50 / IC 50 The ratio is used to calculate the SI (selectivity index) of a compound.

[0165]

[0166]

[0167] a TC 50 The half-maximal toxicity concentration (MCC) refers to the concentration at which, under the experimental conditions, the sample compound causes a toxic effect in 50% of H460 cells. b IC 50 The half-maximal inhibitory concentration (MCIC) refers to the concentration at which the sample compound can inhibit 50% of HCoV-OC43-induced CPE under the experimental conditions. c SI, the ratio of half-maximal cytotoxic concentration to half-maximal inhibitory concentration. d NA, no activity.

[0168] Therefore, compared with comparative compounds 1-2 and clofazimine, most of the compounds of this invention exhibit significantly enhanced antiviral activity due to their specific structural features. Among them, compounds 29b and 36b show the best activity.

[0169] 2. Time-of-addition experiments of representative compounds

[0170] We selected compounds 29b and 36b and conducted further studies using a time-of-addition approach. Clofazimine was used as a positive control, and DMSO as a negative control. Compounds 29b and 36b were added to H460 cells (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) or Huh7.5 cells (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) before (-2 h), during (0 h), and after (+2 h, +4 h, +8 h) HCoV-OC43 infection and incubated. Results are as follows: Figure 1 As shown. By Figure 1 The results show that compounds 29b and 36b of the present invention can simultaneously block the invasion and biosynthesis stages of the virus, and the blocking effect is superior to that of CFZ.

[0171] Notably, their blocking effect on viral invasion was more significant. When compound 29b was administered during the -2h to 0h and 0 to 2h time periods, its inhibition rate against viral proliferation reached 90%, suggesting that the compound also has antiviral activity. To further evaluate the preventive activity of compound 29b, we adjusted the dosing regimen of the time-of-addition experiment, administering the drug 12h and 24h earlier, and found that the antiviral activity was even stronger, with an inhibition rate close to 100%, suggesting that compound 29b can effectively prevent viral infection.

[0172] Figure 1 A) represents the time-of-addition experimental results of each compound in the H460-OC43 cell model (-2h ~ 8h); Figure 1 B) represents the results of time-of-addition experiments of each compound in the Huh7.5-OC43 cell model (-2 h ~ 8 h); Figure 1 C) represents the results of time-of-addition experiments of compound 29b in the H460-OC43 cell model (-24 h ~ -2 h).

[0173] 3. Safety experiments of representative compounds

[0174] We conducted acute toxicity studies of compound 29b on a Kunming mouse model (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The compound was administered via single gavage at doses of 0, 100 mg / kg, 200 mg / kg, 400 mg / kg, and 800 mg / kg. Figure 2 As shown, after 7 days of close observation, there were no significant changes in the survival status and body weight of the mice, suggesting that the median lethal dose (LD50) of compound 29b was low. 50The dosage was greater than 800 mg / kg. Furthermore, serum biochemical parameters were measured after treatment with compound 29b at all dosages. The results showed that the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (Cre) in the fasting serum of mice did not change significantly, indicating that compound 29b has good safety.

[0175] Industrial availability

[0176] This application develops a series of novel phenylphenazine derivatives, and through structural optimization, yields compounds with multi-targeted activities for preventing viral infection and / or antiviral activity. As multi-target anti-SARS-CoV-2 drugs, they can simultaneously intervene at multiple key targets in the viral replication process, achieving excellent antiviral activity. Furthermore, since the compounds with specific structures involved in this invention also act on host targets, they play a crucial role in applications for preventing SARS-CoV-2 virus infection.

Claims

1. The compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: ， in, The compound is selected from the following group of compounds: 。 2. A pharmaceutical composition, characterized in that, Contains an effective dose of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The method for preparing the compound according to claim 1, characterized in that, The synthesis process includes the following: ， In the above synthesis process: The conditions indicated in step a are: alkali, organic solvent, 60 ~ 200 ℃; The conditions indicated in step b are: 10% Pd / C, H2, organic solvent, 10 ~ 60 ℃; The conditions represented by step c are: 1,5-difluoro-2,4-dinitrobenzene, base, organic solvent, 10 ~ 60 °C; The conditions represented by step d are: R2NH2, organic solvent, 30 ~ 100 ℃; The conditions indicated in step e are: 10% Pd / C, H2, organic solvent, 10 ~ 60 °C; The conditions represented by step f are: atmospheric conditions, organic solvents, and 10 ~ 60 °C. The conditions for step g are: R3NH2, organic acid, organic solvent, 50 ~ 150 °C. In the above formulas, X1, R1, R2, and R3 are as described in claim 1.

4. The preparation method according to claim 3, wherein, The bases used in steps a and c are selected from tertiary amines. The organic acid in step g is selected from formic acid, acetic acid, tartaric acid, oxalic acid, benzoic acid, and salicylic acid. The organic solvents used in steps a to g are selected from methanol, ethanol, propanol, THF, DMF, DMSO, dichloromethane, dioxane, and acetone.

5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for preventing and / or treating SARS-CoV-2 infection.

6. Use of the pharmaceutical composition of claim 2 in the preparation of medicaments for preventing SARS-CoV-2 infection and / or for treating SARS-CoV-2.

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