Anti-SARS-cov-2 arylnaphthalene lignan compound, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are an anti-SARS-CoV-2 arylnaphthalene lignan compound, and a preparation method therefor and the use thereof. Results show that ANL-1, ANL-2, and ANL-4 to ANL-7 have no toxicity to Vero E6 cells, and ANL-3 has low toxicity to Vero E6 cells. In a Vero E6 cell model, the in vitro anti-SARS-CoV-2 activities of ANL-7 and ANL-2, in TI value, are respectively 19 times and 16 times that of remdesivir, and are higher than the in-vitro anti-SARS-CoV-2 activity of a market-available drug. ANL-2 has a significant inhibitory effect on RNA-dependent RdRp activity of SARS-CoV-2, and ANL-2 also effectively limits the replication of various pathogenic coronaviruses in Caco2 cells or hamsters. It can be seen that the arylnaphthalene lignan compound has a good anti-coronavirus activity and can be used for treating and preventing SARS-CoV-2 infection or delaying the progress of SARS-CoV-2 infection in a patient.

Description

A class of aryl naphthyl lignan compounds for resisting novel coronavirus, and preparation method and application thereof Technical Field

[0001] The present invention relates to a class of inhibitors of the new coronavirus, and specifically to a class of arylnaphthyl lignan compounds with anti-new coronavirus activity, as well as a preparation method and application thereof. Background Art

[0002] Coronaviruses (CoV) are enveloped, single-stranded, positive-sense RNA viruses belonging to the families Coronaviridae, Arteriviridae, and Roniviridae. SARS-CoV-2, the virus that causes the current COVID-19 pandemic, is a beta-coronavirus. Currently, much effort is focused on screening existing and emerging compounds for inhibitors of key enzymes in the viral life cycle. However, from a scientific perspective, the possibility of mutating SARS-CoV-2 into a more pathogenic variant cannot be ruled out. Therefore, highly effective viral inhibitors are urgently needed to combat coronaviruses.

[0003] Compared to vaccines and antibodies, oral small molecule drugs offer advantages such as low cost, ease of production, convenient transportation, and ease of use. They are a key focus in the development of therapeutics against the novel coronavirus, which is likely to persist in humans for a long time. In the field of small molecule drug research, most pharmacists are searching for targeted, specific drugs against the novel coronavirus. The main targets include the pathogen's spike protein (S protein), E protein (envelope protein), M membrane protein, N nucleocapsid protein, and various enzymes involved in viral replication; there are also host targets such as ACE2. Remdesivir is the first small molecule drug to treat COVID-19, approved by the US FDA in October 2020. Remdesivir is a nucleoside analog prodrug that is metabolized to its triphosphate form in the body and specifically inhibits the RNA-dependent RNA polymerase (RdRp) to exert its antiviral activity. However, its limited intravenous administration limits its early and widespread use. Merck's Monovaccin and Pfizer's Paxlovid received emergency authorization from the FDA for the treatment of COVID-19 in December 2021. Molnupiravir is a small molecule RdRp inhibitor with a chemical structure similar to that of Remdesivir. It is effective against gamma and delta variants of the novel coronavirus and can effectively reduce the mortality rate. Because it can be taken orally, it can be used to treat mild and moderate patients in the early stages of infection to prevent them from developing severe symptoms. Paxlovid is composed of two small molecules, one of which is M proThe first is the inhibitor Nirmatrelvir (PF-07321332), and the second is the oral bioavailability enhancer of Nirmatrelvir. The drug contains 20 tablets of 150mg Nirmatrelvir and 10 tablets of 100mg ritonavir. It has quickly become a first-line prescription drug worldwide, replacing the use of remdesivir, but the price is higher. Azvudine (2'-deoxy-2'-β-fluoro-4'-azidocytidine nucleoside; FNC; C9H) was created by Professor Chang Junbiao and others from Zhengzhou University in my country. 11 FN6O4) is a small molecule RdRp inhibitor. Azvudine was approved by the National Medical Products Administration (NMPA) on July 25, 2022, becoming China's first oral medication approved for the treatment of COVID-19 under "emergency conditional" approval. Azvudine, a nucleoside analog drug, was approved earlier (in July 2021) by the NMPA as a new anti-HIV-1 drug. It has a specific dual inhibitory effect (dual target) on HIV-1 reverse transcriptase (RT) and accessory protein (Vif).

[0004] In modern drug discovery, natural products have become a rich source for discovering lead compounds. By screening more than 3,500 plant extracts, we determined that Justicia cf. patentiflora has effective anti-HIV activity. The fractionation-guided separation of the methanol extracts of the stems and bark of this plant under the guidance of biological activity produced three ANL (aryl naphthalene) glycoside compounds. The compounds showed effective inhibitory activity against a variety of viruses. They also showed a significant inhibitory effect on drug-resistant HIV strains. Some aryl naphthalene lignans have been reported in the literature to have antiviral activity. Although some of these compounds showed significant antiviral activity against various viral strains, they were not considered to be potential antiviral drug candidates due to their low selectivity index (SI).

[0005] Therefore, there is a need to develop improved antiviral agents that can meet at least some of the aforementioned needs. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a class of aromatic naphthyl lignan compounds that are resistant to the new coronavirus, as well as a preparation method and application thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention discloses a novel class of arylnaphthalene analogs, the preparation of related compounds and novel intermediates, and their use in treating novel coronavirus infections such as SARS-CoV-2.

[0009] Therefore, the first aspect of the present invention is an aryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof for treating, preventing, or delaying the progression of infection with a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV), wherein the compound has formula (I):

[0010] or a pharmaceutically acceptable salt or prodrug thereof, wherein

[0011] X is oxygen or sulfur;

[0012] R 1 R 15 、-OR 15 、-C(O)R 15 , or -C(O)OR 15 ;

[0013] R 2 、R 5 、R 6 、R 10 、R 13 and R 14 each is hydrogen or halogen;

[0014] R 3 and R 4 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 3 and R 4 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group;

[0015] R 7 、R 8 and R 9 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 7 and R 8 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted by a group; or R 8 and R 9 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group;

[0016] R 11and R 12 Together form an oxo group; or in R 11 and R 12 When one of them is hydrogen or halogen, R 11 and R 12 The other one is selected from R 15 、-OR 15 、-C(O)R 15 and -C(O)OR 15 ;

[0017] R 15 is independently selected at each occurrence from hydrogen, alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, heteroaryl, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N=C(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18 , 1,3,2-dioxaborolane optionally substituted by 1, 2, 3 or 4 groups independently selected from alkyl, a glycoside group, an alkynyl group optionally substituted by a trialkylsilane, a 1,2,3,4 or 5 group independently selected from R 16 A hydrocarbon group substituted with a group, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from R 16 and optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 -(CH2) substituted by k -heterocyclyl, wherein k is an integer from 1 to 6;

[0018] R 16 is independently selected at each occurrence from alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, oxo, ═NR 17 、-OR 17 、-C(O)R 18 、-C(O)N(R 17 )R18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 and -N(R 17 )S(O)2R 18 ;and

[0019] R 17 and R 18 is independently hydrogen, alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl at each occurrence, or is selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4, or 5 groups independently selected from halogen, cyano, amino, hydroxy, C 1-6 Alkyl and C 1-6 Alkoxy group substitution;

[0020] The glycosidic groups are typically carbohydrates, particularly monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or polysaccharides, and may exist in various isomeric forms, such as α-D, α-L, β-D, or β-L. The carbohydrate groups may optionally be substituted with other types of substituents or even additional glycosidic groups. However, the total number of monosaccharide groups and substituted monosaccharide groups contained in the chemical structure of the compound may not exceed 10.

[0021] The glycosidic group may be a group of formula (i) or (ii):

[0022] in

[0023] R 19 and R 20 can form an oxo group together; or when R 19 and R 20 When one of the is hydrogen or halogen, R 19 and R 20 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0024] R 21and R 22 can form an oxo group together; or when R 21 and R 22 When one of the is hydrogen or halogen, R 21 and R 22 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0025] R 23 and R 24 can form an oxo group together; or when R 23 and R 24 When one of the is hydrogen or halogen, R 23 and R 24 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0026] R 25 and R 26 can form an oxo group together; or when R 25 and R 26 When one of the is hydrogen or halogen, R 25 and R 26 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 、-CH2R 27 and -C(O)R 27 ;

[0027] R 27 independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 The hydrocarbon group substituted by a group, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 -(CH2) substituted by k -heterocyclic group, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides.

[0028] In particular, the compound of formula (I) is:

[0029] In particular, the compound described by formula (I) is:

[0030] The second aspect of the present invention is a method for preparing ANL-2, and the preparation reaction formula is as follows:

[0031] The preparation method of ANL-2 comprises the following steps:

[0032] Using 2-bromo-4,5-dimethoxybenzaldehyde (1) as a raw material, acetal (2) is prepared by ethylene glycol protection, (2) reacts with piperonal to obtain compound (3), compound (3) is rapidly converted into isobenzofuran by heating in the presence of acetic acid, and then a diester (4) is obtained by Diels-Alder reaction, which is reduced to obtain salvinol (5); salvinol (5) is then reacted with trifluoromethanesulfonic anhydride to obtain product (6); product (6) reacts with trimethylethynylsilane to obtain product (7); product (7) is reacted in the presence of potassium carbonate to obtain the target product ANL-2.

[0033] The specific steps include:

[0034] (1) Ethylene glycol and p-toluenesulfonic acid are added to a toluene solution of 2-bromo-4,5-dimethoxybenzaldehyde (1), and the mixture is heated to 140°C overnight; the mixture is cooled to room temperature, and the toluene is removed by evaporation under reduced pressure; the reaction mixture is dissolved in ethyl acetate, and extracted with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution in sequence, and the organic phase is dried over anhydrous sodium sulfate; the crude product is evaporated to dryness under reduced pressure, and then subjected to silica gel column chromatography to obtain the acetal intermediate (2);

[0035] Preferably, in step (1), the molar ratio of bromo-4,5-dimethoxybenzaldehyde, ethylene glycol, and p-toluenesulfonic acid is 1±0.5:2±1:0.2±0.1; further 1:2:0.2;

[0036] (2) The acetal intermediate (2) is dissolved in anhydrous tetrahydrofuran, cooled, and n-butyl lithium solution is added dropwise, stirred, and a tetrahydrofuran solution of piperonal is added dropwise and slowly warmed to room temperature; the reaction solution is quenched with water and extracted with ethyl acetate; the combined organic phase is extracted with water and saturated sodium chloride solution, respectively, and dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a light yellow oily liquid intermediate (3) which is quickly used in the next Diels-Alde reaction;

[0037] Preferably, in step (2), the molar ratio of the acetal intermediate (2), n-butyl lithium, and piperonal is 1±0.5:1.2±0.5:1±0.5; further 1:1.2:1;

[0038] Preferably, in step (2), the cooling is cooling to -70°C to -80°C; further cooling to -78°C;

[0039] Preferably, in step (2), the stirring time is 10-60 minutes; further 30 minutes;

[0040] (3) dissolving the intermediate (3) in dichloromethane, adding glacial acetic acid and dimethyl butynedioate; reacting; cooling the reaction solution to room temperature and diluting with water, extracting the reaction solution with dichloromethane; combining the organic phases, drying with anhydrous sodium sulfate, and separating by silica gel column chromatography to obtain the diester compound intermediate (4);

[0041] Preferably, in step (3), the molar ratio of the intermediate (3) to dimethyl butynedioate is 1±0.5:1±0.5; further 1:1;

[0042] Preferably, in step (3), the reaction conditions are reflux at 100°C-160°C for 6-20 hours; further reflux at 140°C for 12 hours.

[0043] (4) The diester compound intermediate (4) is dissolved in anhydrous tetrahydrofuran, sodium borohydride is slowly added and heated to reflux; the reaction solution is cooled to room temperature and acidified dropwise with hydrochloric acid to a pH value close to 2; after acidification, the reaction solution is extracted with ethyl acetate, the organic phases are combined, washed with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography to obtain a light yellow solid (5) (hederin);

[0044] Preferably, in step (4), the molar ratio of the diester compound intermediate (4) to sodium borohydride is 1±0.5:5±2.5; further 1:5;

[0045] Preferably, in step (4), the heating reflux condition is heating reflux at 60°C-100°C for 6-20 hours; further heating reflux at 80°C for 12 hours;

[0046] Preferably, in step (4), the hydrochloric acid is 1-5M hydrochloric acid; further 2M hydrochloric acid;

[0047] (5) Under nitrogen protection, schizofiarin (5) and 4-dimethylaminopyridine were dissolved in ultra-dry dichloromethane, cooled, and trifluoromethanesulfonic anhydride was added dropwise, and stirred at room temperature; thin layer chromatography showed that the reaction was complete and a new spot was generated; the reaction solution was spin-dried to obtain a light yellow solid, which was washed with ethanol and purified to obtain the intermediate (6);

[0048] Preferably, in step (5), the molar ratio of the schizofiarin (5), 4-dimethylaminopyridine and trifluoromethanesulfonic anhydride is 1±0.5:2±1:1.5±0.5; further 1:2:1.2;

[0049] Preferably, in step (5), the cooling is cooling to -5°C to 5°C; further to 0°C;

[0050] Preferably, in step (5), the stirring time is 2-8 hours; further 4 hours.

[0051] (6) Add dichlorobis(triphenylphosphine)palladium, copper iodide, and intermediate (6) to the reaction vessel; add trimethylethynylsilane and triethylamine in dimethylformamide solution dropwise to the above reaction solution under nitrogen protection; heat the reaction solution and stir; after cooling to room temperature, quench the reaction solution with water and extract with dichloromethane; dry the organic phase over anhydrous sodium sulfate, and separate and purify it by silica gel column chromatography to obtain intermediate (7);

[0052] Preferably, in step (6), the molar ratio of the intermediate (6), dichlorobis(triphenylphosphine)palladium, copper iodide, trimethylethynylsilane and triethylamine is 1±0.5:0.05±0.01:0.15±0.06:2±1:3±1; further 1:0.05:0.15:2:3;

[0053] Preferably, in step (6), the heating and stirring conditions are stirring at 50°C-100°C for 5-16 hours; further stirring at 80°C for 10 hours.

[0054] (7) The intermediate (7) was dissolved in methanol, and potassium carbonate was added and stirred at room temperature; the reaction mixture was separated and purified by silica gel column chromatography to obtain the target product ANL-2.

[0055] Preferably, in step (7), the molar ratio of the intermediate (7) to potassium carbonate is 1±0.5:2±1; further 1:2.

[0056] Preferably, in step (7), the stirring time is 4-20 hours; further 12 hours.

[0057] The third aspect of the present invention is a method for preparing ANL-3, comprising the following steps:

[0058] The product ANL-3 was obtained by reacting galangin (5) with bromoacetonitrile;

[0059] The specific steps include:

[0060] (8) The schizofiarin (5) and bromoacetonitrile prepared in step (4) above were dissolved in acetone, Cs2CO3 was slowly added, and the mixture was stirred. TLC showed the formation of a spot with lower polarity. The mixture was purified to obtain the desired product ANL-3.

[0061] Preferably, in step (8), the molar ratio of the schizofiarin (5), bromoacetonitrile and Cs2CO3 is 1±0.5:2±1:2±1; further 1:2:2;

[0062] Preferably, in step (8), the stirring time is 5 to 20 hours, further 12 hours.

[0063] The fourth aspect of the present invention is a method for preparing ANL-4, comprising the following steps:

[0064] D-xylose is used as raw material and reacted with acetic anhydride and hydrogen bromide to obtain glycosyl bromide; anisin (ANL-1) is reacted with glycosyl bromide in the presence of TBAB to obtain the target product ANL-4.

[0065] The specific steps include:

[0066] (9) D-xylose was dissolved in pyridine, acetic anhydride was added and stirred at room temperature overnight; after the reaction was completed, CH2Cl2 was added for dilution and washed with HCl solution; the organic phases were combined and extracted with a saturated solution of NaHCO3, the organic phases were taken, dried over anhydrous Na2SO4, and concentrated to obtain a crude product of fully acetylated xylose, which was directly used in the next step without further purification; that is, dissolved in CH2Cl2, cooled, and then hydrogen bromide was added dropwise; the reaction was heated to room temperature and stirred; the reaction was then quenched with water, and the aqueous layer was extracted with CH2Cl2; the organic layers were combined, and washed with water, NaHCO3 and saturated The reaction mixture was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to obtain glycosyl bromide; the crude glycosyl bromide was concentrated and used directly in the next step without further purification; the galangin (5) was dissolved in CHCl3 and an aqueous sodium hydroxide solution, tetrabutylammonium bromide (TBAB) was added, the reaction was heated, glycosyl bromide was added, and the mixture was stirred; after cooling to room temperature, the aqueous layer was extracted with CHCl3, the combined organic layer was washed with saturated brine, dried over Na2SO4, and concentrated under reduced pressure; the reaction mixture was separated using a silica gel column (n-hexane / ethyl acetate = 1 / 1) to obtain the target product ANL-4;

[0067] Preferably, the molar ratio of D-xylose to acetic anhydride is 1±0.5:8±2; further 1:7;

[0068] Preferably, washing with 1% to 15% HCl solution; further washing with 10% HCl solution;

[0069] Preferably, the cooling is cooling to -5°C to 5°C; further to 0°C;

[0070] Preferably, the stirring time is 1 to 10 hours; further 4 hours;

[0071] Preferably, the molar ratio of schizofiarin to glycosyl bromide is 1±0.5:1±0.5; further 1:1;

[0072] Preferably, the concentration of the sodium hydroxide aqueous solution is (0.1±0.05)M; further 0.1M;

[0073] Preferably, the reaction is heated to 30° C. to 60° C., further to 40° C.; maintained for 10–60 min; further maintained for 10 min.

[0074] The fifth aspect of the present invention is a method for preparing ANL-5, ANL-6 and ANL-7, which specifically comprises the following steps:

[0075] (10) The -O-acetyl group of the glycosyl portion of ANL-4 was removed to obtain 7-O-xyloside of schizoflavin (8);

[0076] (11) Dioscorea 7-O-xyloside (8) was reacted with acetic anhydride Ac2O and tetrabutylammonium acetate TBAOAc to give xylose 3″-acetylated glycoside derivative ANL-5.

[0077] Specifically, 7-O-xyloside (8) of schizofiarin was dissolved in dry acetonitrile, and acetic anhydride and tetrabutylammonium acetate were added and stirred. After the reaction was completed, the solvent was concentrated by rotary evaporator and then separated and purified by silica gel column chromatography (eluted with petroleum ether / ethyl acetate in a volume ratio of 1.5:1) to obtain the main product ANL-5.

[0078] (12) ANL-5 was treated with allyl chloroformate, and the product was separated by silica gel column chromatography (eluted with petroleum ether / ethyl acetate in a volume ratio of 2:1) to obtain the glycosylated products of schizoferrin xyloside, ANL-6 (2″,4″-bis-O-formylpropenyl-3″-O-acetyl schizoferrin xyloside) and ANL-7 (4″-O-formylpropenyl-3″-O-acetyl schizoferrin xyloside).

[0079] Preferably, in step (10), an alkaline solution is used to remove the -O-acetyl group of the glycosyl portion of ANL-4, and the alkaline solution is at least one of an aqueous sodium hydroxide solution and an aqueous potassium hydroxide solution;

[0080] The molar ratio of ANL-4 to sodium hydroxide or potassium hydroxide is 1±0.1:1.5±0.4; further 1:1.2;

[0081] Preferably, in step (11), the molar ratio of the schizofiarin 7-O-xyloside (8), acetic anhydride, and tetrabutylammonium acetate is 1±0.1:1±0.2:0.5±0.2; further 1:1.11:0.3.

[0082] Preferably, in step (11), the reaction time is 2 to 24 hours; further 12 hours.

[0083] Preferably, in step (11), the reaction temperature is room temperature to 60°C; further 40°C.

[0084] Preferably, in step (12), the molar ratio of ANL-4 to allyl chloroformate is 1±0.1:2.5±0.5; further 1:2.

[0085] Preferably, in step (12), the treatment conditions are to use triethylamine Et3N and dimethylaminopyridine DMAP as catalysts.

[0086] Preferably, in step (12), the treatment time is 2 to 24 hours; further 15 hours.

[0087] Preferably, in step (12), the reaction temperature is -5°C to 40°C; further, 0°C to room temperature.

[0088] In the present invention, the room temperature refers to 20 to 30°C.

[0089] The sixth aspect of the present invention is a pharmaceutical preparation comprising an aryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.

[0090] The seventh aspect of the present invention is a pharmaceutical preparation comprising an alkynylaryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.

[0091] The eighth aspect of the present invention is a pharmaceutical preparation comprising a schizofiarin analogue or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.

[0092] The ninth aspect of the present invention is a pharmaceutical preparation comprising a schizofiarin analogue or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.

[0093] The tenth aspect of the present invention is a pharmaceutical formulation comprising an ANL-2 analogue or a pharmaceutically acceptable salt or prodrug thereof for use in treating, preventing or delaying the progression of a viral infection in a patient.

[0094] The novel coronavirus described in the present invention refers, in a broad sense, to a coronavirus that can cause severe symptoms, is lethal, and is contagious, including but not limited to at least one of the viruses that cause severe acute respiratory syndrome, Middle East respiratory syndrome, and the novel coronavirus disease COVID-19.

[0095] Further preferably, the novel coronavirus includes but is not limited to at least one of SARS-CoV-1 or SARS-CoV-2 or MERS-CoV or SARS-CoV-2 Omicron strain.

[0096] Other aspects of the present invention relate to providing methods for synthesizing ANL-2, ANL-3, ANL-4, ANL-5, ANL-6 or ANL-7 compounds and intermediate compounds during the synthesis. In addition, the present invention relates to intermediate compounds that can be used to prepare the compounds of the present invention.

[0097] The compounds of the present invention can exist in different forms, such as free acids, free bases, esters and other prodrugs, salts, and tautomers, and the present disclosure includes all variant forms of these compounds.

[0098] The degree of protection includes counterfeit or fraudulent products that contain or purport to contain compounds of the invention, regardless of whether they actually contain such compounds and regardless of whether any such compounds are contained in a therapeutically effective amount.

[0099] Included within the scope of protection are packages that include descriptions or instructions indicating that the package contains a species or pharmaceutical formulation of the present invention, as well as products that are or contain, or purport to be or contain, such formulations or species. Such packages may, but are not necessarily, counterfeit or false.

[0100] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0101] The present invention has the following advantages and effects compared to the prior art:

[0102] In order to verify the anti-coronavirus activity of the aryl naphthalene lignan compounds (Formula I), we present the synthesized series of aryl naphthalene lignan compounds ANL-1, ANL-2, ANL-3, ANL-4, ANL-5, ANL-6 or ANL-7 in the present invention. Experimental results showed that ANL-1, ANL-2, ANL-4, ANL-5, ANL-6 or ANL-7 were non-toxic to Vero E6 cells, and ANL-3 had low toxicity to Vero E6 cells; in the Vero E6 cell model, the in vitro anti-novel coronavirus (SARS-CoV-2) activity of ANL-7 and ANL-2 was 19 times and 16 times that of remdesivir, respectively, which was much higher than the in vitro anti-novel coronavirus activity of marketed drugs; the in vitro anti-novel coronavirus activity of ANL-4, ANL-5, and ANL-6 was much higher than that of remdesivir, and the in vitro anti-novel coronavirus activity of ANL-3 was close to that of remdesivir; ANL-2 had a significant inhibitory effect on the activity of the RNA-dependent RNA polymerase (RdRp) of the new coronavirus; ANL-2 also effectively restricted the replication of various pathogenic coronaviruses in Caco2 cells or hamsters. It can be seen that aryl naphthyl lignan compounds represented by ANL-2 and ANL-7 have good anti-coronavirus activity and can be used to treat, prevent or delay the progression of novel coronavirus (such as SARS-CoV-2) infection in patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG1 is a high performance liquid chromatography (HPLC) chart of ANL-2 provided in Example 2 of the present invention;

[0104] FIG2 is a high-resolution mass spectrum (HR-ESIMS) of ANL-2 provided in Example 2 of the present invention;

[0105] FIG3 is a diagram of ANL-2 provided in Example 2 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0106] FIG4 is a diagram of ANL-2 provided in Example 2 of the present invention. 13 C NMR ( 13 C NMR) spectrum;

[0107] FIG5 is a diagram of ANL-3 provided in Example 3 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0108] FIG6 is a diagram of ANL-3 provided in Example 3 of the present invention. 13 C NMR (13 C NMR) spectrum;

[0109] FIG7 is a diagram of ANL-4 provided in Example 4 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0110] FIG8 is a diagram of ANL-4 provided in Example 4 of the present invention. 13 C NMR ( 13 C NMR) spectrum;

[0111] FIG9 is a diagram of ANL-5 provided in Example 5 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0112] FIG10 is a diagram of ANL-5 provided in Example 5 of the present invention. 13 C NMR ( 13 C NMR) spectrum;

[0113] FIG11 is a diagram of ANL-6 provided in Example 5 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0114] FIG12 is a diagram of ANL-6 provided in Example 5 of the present invention. 13 C NMR ( 13 C NMR) spectrum;

[0115] FIG13 is a diagram of ANL-7 provided in Example 5 of the present invention. 1 H NMR ( 1 H NMR) spectrum;

[0116] FIG14 is a diagram of ANL-7 provided in Example 5 of the present invention. 13 C NMR ( 13 C NMR) spectrum;

[0117] FIG15 shows the effect of ANL-2 on Caco2 cell viability provided in Example 6 of the present invention;

[0118] Figure 16 shows the effect of ANL-2 provided in Example 6 of the present invention on viral gene copies of Caco2 cells infected with SARS-CoV-1, MERS-CoV, SARS-CoV-2 wild type or BA.5.2 variant;

[0119] Figure 17 shows the RdRp gene copy number in the lung and nasal turbinate tissues of hamsters infected with SARS-CoV-2 WT virus as provided in Example 6 of the present invention;

[0120] FIG18 is a particle size distribution diagram of the ANL-2 fat emulsion provided in Example 7 of the present invention measured by a particle size analyzer;

[0121] FIG19 shows the particle size of the ANL-2 fat emulsion provided in Example 7 of the present invention as measured by transmission electron microscopy;

[0122] FIG20 is a curve showing changes in body weight of mice after oral administration of ANL-2 suspension and fat emulsion provided in Example 8 of the present invention;

[0123] FIG21 is a blood drug concentration-time curve of ANL-2 fat emulsion administered by tail vein to three groups of rats provided in Example 9 of the present invention;

[0124] FIG22 is a blood drug concentration-time curve of three groups of rats orally administered with ANL-2 fat emulsion provided in Example 9 of the present invention. DETAILED DESCRIPTION

[0125] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0126] Throughout the description and claims of this specification, the word "comprising" means including but not limited to, and is not intended to exclude, for example, other additives, components, integers or steps.

[0127] As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "the compound" includes mixtures of two or more such compounds, reference to "an agent" includes mixtures of two or more such agents, etc.

[0128] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0129] As used herein, "subject" refers to an individual. Thus, "subjects" may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, hamsters, etc.), and birds. "Subjects" may also include mammals, such as primates or humans.

[0130] By "reduce" is meant a decrease in an event or characteristic (e.g., tumor growth). It will be understood that this is usually relative to some standard or expected value, in other words, it is relative, but does not always need to refer to that standard or relative value. For example, "reduce tumor growth" means a decrease in the rate of tumor growth relative to a standard or control.

[0131] So-called "prevention" or other forms of the term, such as "prevention" means stopping a particular event or characteristic, stabilizing or slowing the development or progression of a particular event or characteristic, or minimizing the chance of a particular event or characteristic occurring. Prevention does not need to be compared to a control because it is usually more absolute than, for example, reduction. As used herein, something can be reduced but not prevented, but something that is reduced can also be prevented. Similarly, something can be prevented but not reduced, but something that is prevented can also be reduced. It should be understood that where reduction or prevention is used, unless otherwise specifically noted, the use of the other terms is also explicitly disclosed.

[0132] By "treating" is meant administering a composition or performing a method to reduce, prevent, inhibit, or eliminate a particular characteristic or event (eg, tumor growth or survival). The term "controlling" is used synonymously with the term "treating."

[0133] The term "antiviral" refers to the ability to inhibit the replication of a specific virus, inhibit viral transmission, or prevent a virus from establishing itself in its host, as well as to ameliorate or alleviate the symptoms of disease caused by viral infection. Treatment is considered therapeutic if viral load is reduced, mortality and / or morbidity is reduced.

[0134] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only requires reduction or modification, not elimination.

[0135] As used herein, the term "pharmaceutically acceptable salt" refers to any salt of a compound of the invention which retains its biological properties and is not toxic or otherwise undesirable for pharmaceutical use. Such salts can be derived from and include a variety of organic and inorganic counterions known in the art. Such salts include: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxyphenyl)-1-ol benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, or (2) salts formed when an acidic proton present in the parent compound is (a) replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), or an alkali metal or alkaline earth metal hydroxide (e.g., sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide), ammonia, or (b) coordinated with an organic base, such as an aliphatic, alicyclic, or aromatic organic amine, for example, ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.In addition, examples of salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids such as hydrohalides (e.g., hydrochloride and hydrobromide), sulfates, phosphates, sulfamates, nitrates, acetates, trifluoroacetates, trichloroacetates, propionates, hexanoates, cyclopentylpropionates, glycolates, glutarates, pyruvates, lactates, malonates, succinates, sorbates, ascorbates, malate, maleates; fumarates, tartrates, citrates, benzoates, 3-(4-hydroxybenzoyl)benzoate, picrates, cinnamates, mandelates, phthalates. , laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethylsulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, pivalate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate, and the like.

[0136] As used herein, the terms "glycoside" or "glycosidic" compound are interchangeable and include any of the classes of compounds mentioned that upon hydrolysis produce a sugar and an aglycone.

[0137] As used herein, the terms "ANL" or "aryl naphthalene lignan" or "arylnaphthalene lignan" compounds are interchangeable.

[0138] As used herein, the term "aryl naphthalene lignan" or "arylnaphthalene lignan" or "ANL" includes reference to compounds containing the base structure of 2,3-dimethyl-1-phenyl-naphthalene as shown below:

[0139] As used herein, carbon numbering of arylnaphthyl lignan molecules includes reference to compounds comprising the numbering system shown below:

[0140] In the core structure of one type of arylnaphthalene compound, two methyl groups form a γ-lactone ring to become an arylnaphthofuran-2-one lignan or an arylnaphthofuran-3-one lignan as shown below:

[0141] As used herein, the carbon numbering of the arylnaphthyl lignan side chains includes reference to compounds comprising the numbering system shown below:

[0142] As used herein, the term "hydrocarbyl" includes reference to moieties consisting solely of hydrogen and carbon atoms; such moieties may contain aliphatic and / or aromatic moieties. The moieties may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Examples of hydrocarbyl groups include C 1-6 Alkyl (e.g., C1, C2, C3 or C4 alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl); C1, C2, C3 or C4 alkyl substituted by aryl (e.g., benzyl) or by cycloalkyl (e.g., cyclopropylmethyl); 1-6 Alkyl; cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); aryl (e.g., phenyl, naphthyl or fluorenyl); C 1-6 Alkenyl (e.g., ethenyl, 2-propenyl, or 3-butenyl); C 1-6 alkynyl (e.g., ethynyl, 2-propynyl or 3-butynyl) and the like.

[0143] As used herein, the terms "alkyl" and "C 1-6 "Alkyl" includes references to straight or branched chain alkyl moieties having 1, 2, 3, 4, 5 or 6 carbon atoms. The term includes references to groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl, etc. In particular, the alkyl moiety may have 1, 2, 3 or 4 carbon atoms.

[0144] As used herein, the term "alkenyl" includes reference to straight or branched chain alkyl moieties having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and additionally having at least one double bond, and, where applicable, E or Z stereochemistry. The term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl, and 3-hexenyl.

[0145] As used herein, the term "alkynyl" includes references to straight or branched chain alkyl moieties having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and additionally having at least one triple bond. The term includes references to groups such as ethynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, and 3-hexynyl.

[0146] As used herein, the terms "alkoxy" and "C 1-6 "Alkoxy" includes references to -O-alkyl groups, wherein the alkyl group is straight or branched and contains 1, 2, 3, 4, 5, or 6 carbon atoms. In one class of embodiments, the alkoxy group has 1, 2, 3, or 4 carbon atoms. The term includes references to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0147] As used herein, the term "cycloalkyl" includes references to alicyclic moieties having 3, 4, 5, 6, 7, or 8 carbon atoms. Such groups may be bridged or polycyclic ring systems. More common cycloalkyl groups are monocyclic. The term includes references to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and bicyclo[2.2.2]octyl.

[0148] As used herein, the term "aryl" includes reference to aromatic ring systems containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring carbon atoms. Aryl is typically phenyl, but can be a polycyclic ring system having two or more rings, at least one of which is aromatic. The term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthracenyl, and the like.

[0149] "Cyclic group" means a ring or ring system which may be unsaturated or partially unsaturated, but is typically saturated, and typically contains 5 to 13 ring atoms, such as a 5- or 6-membered ring. It includes carbocyclyl and heterocyclyl moieties.

[0150] As used herein, the term "carbocyclyl" includes reference to saturated (e.g., cycloalkyl) or unsaturated (e.g., aryl) ring moieties having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon ring atoms. In particular, carbocyclyl includes 3- to 10-membered rings or ring systems, particularly 5- or 6-membered rings, which may be saturated or unsaturated. The carbocyclyl moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthracenyl, and the like.

[0151] As used herein, the term "heterocyclyl" includes reference to saturated (e.g., heterocycloalkyl) or unsaturated (e.g., heteroaryl) heterocyclic moieties having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from boron, nitrogen, oxygen, phosphorus, silicon, and sulfur. In particular, heterocyclyl includes 3- to 10-membered rings or ring systems, more particularly 5- or 6-membered rings, which may be saturated or unsaturated.

[0152] The heterocyclic moiety is selected, for example, from oxiranyl, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furanyl, tetrahydrofuranyl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, in particular thiomorpholinyl, indolizinyl, isoindolyl, 3H-indolyl quinolinyl, benzothiophene, dibenzothiophene, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, B-carbolinyl, phenanthridinyl, acridinyl, furidinyl, phenanthrolinyl, furazolyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl, 1,3,2-dioxaborolane, and the like.

[0153] As used herein, the term "heterocycloalkyl" includes reference to saturated heterocyclic moieties having 3, 4, 5, 6, or 7 ring carbon atoms and 1, 2, 3, 4, or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. The group can be a polycyclic ring system, but is more typically a monocyclic ring. The term includes groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, tetrahydrothiazolyl, morpholinyl, thiomorpholinyl, quinolizinyl, and the like.

[0154] As used herein, the term "heteroaryl" includes references to aromatic heterocyclic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, and sulfur. The group may be a polycyclic ring system having two or more rings, at least one of which is aromatic, but is more typically a monocyclic ring. The term includes references to groups such as pyrimidinyl, furanyl, benzothienyl, thienyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridyl, benzofuranyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinazolinyl, pteridinyl, and the like.

[0155] As used herein, the term "halogen" includes reference to F, Cl, Br, or I.

[0156] As used herein, the expression "halogen-containing moiety" includes reference to a moiety containing from 1 to 30 polyvalent atoms selected from carbon, nitrogen, oxygen and sulfur, which moiety includes at least one halogen. The moiety may be a hydrocarbon group, for example, C 1-6 Alkyl or C 1-6 an alkoxy group, or a carbocyclic group such as an aryl group.

[0157] As used herein, the term "substituted" with reference to a moiety means that one or more, in particular up to 5, more particularly 1, 2 or 3 hydrogen atoms in the moiety are replaced independently of one another by the corresponding number of substituents described. As used herein, the term "optionally substituted" means substituted or unsubstituted. Of course, it should be understood that the substituents are only in chemically possible positions, and a person skilled in the art will be able to determine (experimentally or theoretically) whether a particular substitution is possible without undue effort.

[0158] When two or more moieties are described as being "each independently" selected from a list of atoms or groups, this means that the moieties may be the same or different. Thus, the identity of each moiety is independent of the identity of one or more other moieties.

[0159] As used herein, the term "enantiomer" means one of two stereoisomers that are mirror images of one another.

[0160] As used herein, the term "stereoisomer" refers to a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms but different three-dimensional orientations of their atoms in space.

[0161] The term "tautomer" refers to isomeric molecules that are readily interconvertible via chemical reactions. The reaction typically results in the migration of a hydrogen atom, which results in the switching of a single bond and an adjacent double bond.

[0162] A prodrug is a drug that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted in vivo to the active agent, typically by normal metabolic processes.

[0163] CC 50 It is a measure of cytotoxicity at the concentration of test drug that inhibits cell growth by 50%.

[0164] EC 50 or IC 50 It is a measure of antiviral activity at the effective concentration of the test drug that inhibits 50% of viral growth.

[0165] The term "selection index" or "SI" means the selection of 50 The value was divided by the EC of the test drug 50 or IC 50 Value (CC 50 / EC 50 or CC 50 / IC 50 ) to measure the ratio of the window between cytotoxicity and antiviral activity. A higher SI ratio means that the test drug will be more effective and safer against a given viral infection in in vitro experiments.

[0166] Symbols in chemical structures Indicates where a given chemical structure is bonded to another chemical structure.

[0167] The symbol "β" in a chemical structure indicates that the bond is attached above (or in front of) the plane of the paper or screen. The symbol "α" in a chemical structure indicates that the bond is attached below (or behind) the plane of the paper or screen.

[0168] A solid wedge in a chemical structure indicates that the bond is above (or in front of) the plane of the paper or screen toward the viewer. A dashed (or broken) wedge in a chemical structure indicates that the bond is below (or behind) the plane of the paper or screen away from the viewer.

[0169] Provided herein are a compound and its use in the manufacture of a medicament for treating a viral infection, as well as a method of using the compound to treat a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound, wherein the compound has formula (I):

[0170] or a pharmaceutically acceptable salt or prodrug thereof, wherein

[0171] X is oxygen or sulfur;

[0172] R 1 R 15 、-OR 15 、-C(O)R 15, or -C(O)OR 15 ;

[0173] R 2 、R 5 、R 6 、R 10 、R 13 and R 14 are each independently hydrogen or halogen;

[0174] R 3 and R 4 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 3 and R 4 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group;

[0175] R 7 、R 8 and R 9 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 7 and R 8 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted by a group; or R 8 and R 9 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group;

[0176] R 11 and R 12 Together form an oxo group; or in R 11 and R 12 When one of them is hydrogen or halogen, R 11 and R 12 The other one is selected from R 15 、-OR 15 、-C(O)R 15 and -C(O)OR 15 ;

[0177] R 15 is independently selected at each occurrence from hydrogen, alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, heteroaryl, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N=C(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18 , 1,3,2-dioxaborolane optionally substituted by 1, 2, 3, or 4 groups independently selected from alkyl, a glycoside group, an alkynyl group optionally substituted by a trialkylsilane, a 1,2,3,4 or 5 group independently selected from R 16 A hydrocarbon group substituted with a group, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from R 16 and optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 -(CH2) substituted by k - heterocyclyl, wherein k is an integer from 1 to 6 (e.g., 1, 2, 3, 4 or 5);

[0178] R 16 is independently selected at each occurrence from alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, oxo, ═NR 17 、-OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 and -N(R 17 )S(O)2R 18 ;and

[0179] R17 and R 18 is independently hydrogen, alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl at each occurrence, or is selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4, or 5 groups independently selected from halogen, trichloromethyl, trifluoromethyl, cyano, amino, hydroxy, C 1-6 Alkyl and C 1-6 Alkoxy groups are substituted.

[0180] The glycosidic groups are typically carbohydrates, particularly monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or polysaccharides, and may exist in various isomeric forms, such as α-D, α-L, β-D, or β-L. The carbohydrate groups may optionally be substituted with other types of substituents or even additional glycosidic groups. However, the total number of monosaccharide groups and substituted monosaccharide groups contained in the chemical structure of the compound may not exceed 10.

[0181] The glycosidic group may be a group of formula (i) or (ii):

[0182] in

[0183] R 19 and R 20 can form an oxo group together; or when R 19 and R 20 When one of the is hydrogen or halogen, R 19 and R 20 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0184] R 21 and R 22 can form an oxo group together; or when R 21 and R 22 When one of the is hydrogen or halogen, R 21 and R 22 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0185] R 23 and R 24can form an oxo group together; or when R 23 and R 24 When one of the is hydrogen or halogen, R 23 and R 24 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides;

[0186] R 25 and R 26 can form an oxo group together; or when R 25 and R 26 When one of the is hydrogen or halogen, R 25 and R 26 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 、-CH2R 27 and -C(O)R 27 ;

[0187] R 27 independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 The hydrocarbon group substituted by a group, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 -(CH2) substituted by k -heterocyclic group, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18, monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides.

[0188] In certain embodiments, the compound of formula (I) is a compound selected from ANL-2:

[0189] In certain embodiments, the compound of formula (I) is a compound selected from ANL-3, ANL-4, ANL-5, ANL-6, or ANL-7:

[0190] In certain embodiments, R 1 is hydrogen, alkyl, aryl, heteroaryl (such as furan, thiophene, pyridine, etc.), -N(R 17 )R 18 、-N=C(R 17 )(R 18 ), an alkynyl group optionally substituted with a trialkylsilane, a glycoside group, 1,3,2-dioxaborolane optionally substituted with 1, 2, 3, or 4 groups independently selected from alkyl, or -O-(CR2) m -R 16 , wherein m is an integer selected from 1-4, 1-3, or 1-2; R is independently hydrogen, alkyl, cycloalkyl, or aryl at each occurrence; and R 16 Alkynyl, cyano, -OR 17 、-N(R 17 )R 18 、-C(O)N(R 17 )R 18 , or -C(O)OR 17 .

[0191] In certain embodiments, R 1 -O-CH2-R 16 , where R 16 Alkynyl, cyano, -OR 17 、-N(R 17 )R 18 、-C(O)N(R 17 )R 18 , or -C(O)OR 17 .

[0192] In certain embodiments, R 3 and R 4 Each of which is independently selected from -OR 15 and -OC(O)R 15 , where R 15 is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or R 3and R 4 Together with the carbon atoms to which they are attached, they form a 5-6 membered heterocyclic group. 3 and R 4 For-OR 15 , where R 15 It is a C1-C6 alkyl group, a C1-C4 alkyl group, or a C1-C2 alkyl group.

[0193] In certain embodiments, R 8 and R 9 Each of which is independently selected from -OR 15 and -OC(O)R 15 , where R 15 is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or R 8 and R 9 Together with the carbon atoms to which they are attached, they form a 5-6 membered heterocyclic group. 8 and R 9 Together with the carbon atoms to which they are attached they form a methylenedioxy ring.

[0194] In certain embodiments, R 11 and R 12 Together they form an oxo group.

[0195] In certain embodiments, R 3 and R 4 Each of R is -O-alkyl; 8 and R 9 Together with the carbon atoms to which they are attached, they form a methylenedioxy ring; R 1 is heteroaryl, -OR 15 、-C(O)R 15 、-N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N=C(R 17 )R 18 , pinacol boron group, -OS(O)2CF3, glycoside group, optionally 1 or 2 independently selected from R 16 A heterocyclic group substituted with a group, or an alkynyl group optionally substituted with a trialkylsilane; or R 1 -OCH2-cyano, -OCH2-C≡CH, -OCH2-C(O)N(R 17 )R 18 , or -C(O)OR 17 ; and R 11 and R 12 Together they form an oxo group.

[0196] In certain embodiments, when R 19 and R 20 When one of the R 19 and R 20 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 .

[0197] In certain embodiments, when R 21 and R 22 When one of the R 21 and R 22 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 .

[0198] In certain embodiments, when R 23 and R 24 When one of the R 23 and R 24 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 .

[0199] In certain embodiments, when R 25 and R 26 When one of the R 25 and R 26 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 、-CH2R 27 and -C(O)R 27 .

[0200] Examples of the compounds of the present invention include those shown below. Of course, it will be understood that each compound may be in the form of a free compound, an acid or base addition salt, or a prodrug, where appropriate.

[0201] The present invention also provides a pharmaceutical composition comprising at least one compound described herein and at least one pharmaceutically acceptable excipient (such as an excipient); the pharmaceutical composition can be prepared according to a pharmaceutically described preparation method.

[0202] The compounds described herein and their pharmaceutically acceptable salts can be administered to a subject alone or in combination with a pharmaceutically acceptable excipient, carrier, and / or diluent as a pharmaceutical composition according to standard pharmaceutical practice. The compounds can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous, and topical administration, with intravenous and topical administration being preferred.

[0203] Thus, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more compounds described herein formulated together with one or more pharmaceutically acceptable excipients, carriers (additives) and / or diluents. The pharmaceutical compositions of the present invention can be formulated in particular for administration in solid or liquid form, including those suitable for: (1) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, such as, for example, sterile solutions or suspensions, or sustained release formulations; and (2) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes for application to the tongue.

[0204] As stated herein, some embodiments of the compound described herein may contain a basic functional group, such as an amino group, and therefore can form a pharmaceutically acceptable salt with a pharmaceutically acceptable acid. In this respect, the term "pharmaceutically acceptable salt" refers to the relatively nontoxic inorganic and organic acid addition salts of the compound of the present invention. These salts can be prepared in situ during administration of a vehicle or dosage form manufacturing process, or by reacting the purified compound of the present invention in its free base form with a suitable organic or inorganic acid individually, and separating the salt thus formed during subsequent purification to prepare. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate and laurylsulfonate etc.

[0205] Pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothioic acid, and the like.

[0206] In other cases, the compounds described herein may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these cases, the term "pharmaceutically acceptable salt" refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ during the preparation of a dosing vehicle or dosage form, or by reacting a purified compound in its free acid form with a suitable base (such as a pharmaceutically acceptable hydroxide, carbonate or bicarbonate of a metal cation), with ammonia, or with a pharmaceutically acceptable organic primary amine, secondary amine or tertiary amine alone. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, etc. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc.

[0207] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, solubilizers, buffers and antioxidants can also be present in the compositions.

[0208] The method for preparing these preparations or compounds includes the step of combining the compounds described herein with a carrier or excipient and optionally one or more auxiliary ingredients. Typically, the compounds of the present invention are uniformly and closely combined with a liquid carrier (liquid preparation), a liquid carrier followed by lyophilization (for powder preparations reconstituted with sterile water, etc.), or a fine-grained solid carrier, or both, and then the product is shaped or packaged as needed to prepare the preparation.

[0209] Pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more compounds described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, chelating agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0210] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant.

[0211] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can ensure that the effect of microorganisms on the compound of the present invention is prevented by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid etc. It is also desirable to include isotonic agents, such as sugar, sodium chloride etc. in the composition. In addition, the prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0212] The present invention provides aryl naphthalene analogs with anti-SARS-CoV-2 activity and their synthesis. The compounds of the present invention were synthesized and evaluated for their anti-SARS-CoV-2 activity, toxicity in mice, and pharmacokinetic studies.

[0213] Example

[0214] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative work are all within the scope of protection of the present invention. Unless otherwise stated, parts are parts by weight, temperature is ° C or ambient temperature, and pressure is equal to or close to atmospheric pressure. There are many variations and combinations of reaction conditions, such as component concentrations, temperature, pressure, and other reaction ranges and conditions that can be used to optimize the purity and yield of the product obtained by the described process. Only reasonable and routine experiments are required to optimize such process conditions. Examples of compounds of the present invention include those shown below. Of course, it should be understood that, where appropriate, each compound can be in the form of a free compound, an acid or base addition salt, or a prodrug.

[0215] Example 1 Total Synthesis of Dioscorea Indole (Codename ANL-1 or 2.15)

[0216] Using 2-bromo-4,5-dimethoxybenzaldehyde (1) as the raw material, acetal (2) is prepared by ethylene glycol protection, (2) reacts with piperonal to obtain compound (3), (3) is rapidly converted into isobenzofuran by heating in the presence of acetic acid, and then the diester (4) is obtained by Diels-Alder reaction, which is reduced to obtain salviae quinoxaline (5), namely ANL-1 or 2.15.

[0217] To a toluene solution (1 L) of 2-bromo-4,5-dimethoxybenzaldehyde (1) (30 g, 125 mmol), ethylene glycol (14 mL, 250 mmol) and p-toluenesulfonic acid (5.2 g, 25 mmol) were added. A water separator was installed and the mixture was heated to 140°C overnight. After cooling to room temperature, the toluene was removed by vacuum evaporation using a rotary evaporator. The reaction mixture was dissolved in ethyl acetate and extracted with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution in sequence. The organic phase was dried over anhydrous sodium sulfate. The crude product was evaporated to dryness under reduced pressure and then purified by silica gel column chromatography to obtain the acetal 2-(2-bromo-4,5-dimethoxy)-1,3-dioxolane (2) (33.5 g, 95%). The above acetal 2-(2-bromo-4,5-dimethoxy)-1,3-dioxolane (2) (30 g, 104 mmol) was dissolved in 1 L of anhydrous tetrahydrofuran, cooled to -78 ° C, and n-butyl lithium solution (1.8 M, 69.3 mL, 124.8 mmol) was added dropwise. After stirring for 30 minutes, a tetrahydrofuran solution of piperonal (15.6 g, 104 mmol) was added dropwise and slowly warmed to room temperature. After 2 hours, the reaction solution was quenched with water and extracted with ethyl acetate. The combined organic phase was extracted with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a light yellow oily liquid (3) which was quickly used in the next Diels-Alde reaction. The above intermediate 3 was dissolved in dichloromethane (100 mL), and glacial acetic acid (35 mL) and dimethyl butynedioate (16.7 mL, 104 mmol) were added. The reaction was refluxed at 140 ° C for 12 hours. The reaction solution was cooled to room temperature and diluted with water, and the reaction solution was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain a yellow solid (4) (30 g, 62%). The above diester compound intermediate (4) (5 mmol) was dissolved in anhydrous tetrahydrofuran (70 mL), sodium borohydride (0.945 g, 25 mmol) was slowly added and heated under reflux at 80°C for 12 hours. The reaction solution was cooled to room temperature and acidified dropwise with 2M hydrochloric acid to a pH value close to 2. After acidification, the reaction solution was extracted with ethyl acetate, the organic phases were combined, washed with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain a light yellow solid (5), namely ANL-1 or 2.15 (saturnin, 1.8 g, 95%), which is a known organic compound. HR-EIMS: m / z [M+H] + 381.0914(calcd 381.0912;M=C 21 H 16 O7). 1H NMR(400MHz,acetone-d6)δ7.69(1H,s,H-6),7.09(1H,s,H-3),6.96(1H,d,J=7.9Hz ,H-5'),6.85(1H,d,J=1.6Hz,H-2'),6.91(1H,dd,J=7.9,1.7Hz,H-6'),6.09(1H,br s,OCH2O),6.07(1H,br s,OCH2O),5.37(2H,s,H-9),3.99(3H,s,5-OMe),3.73(3H,s,4-OMe).

[0218] Example 2 Total Synthesis of 7-Deoxy-7-ethynyl-annuin (ANL-2)

[0219] Under nitrogen protection, schizoferrin (5) (3.8 g, 10 mmol), 4-dimethylaminopyridine (2.44 g, 20 mmol) were dissolved in 100 mL of ultra-dry dichloromethane, cooled to 0°C, and trifluoromethanesulfonic anhydride (2.5 mL, 12 mmol) was added dropwise. The mixture was stirred at room temperature for 4 hours. Thin layer chromatography showed that the reaction was complete and a new spot was generated. The reaction solution was dried to obtain a light yellow solid, which was washed with ethanol and purified to obtain product (6) (4.61 g, 90%). Dichlorobis(triphenylphosphine)palladium (70 mg, 0.1 mmol), copper iodide (57.2 mg, 0.3 mmol), and compound (6) (1 g, 2 mmol) were added to a 100 mL round-bottom flask. Under nitrogen protection, a dimethylformamide solution of trimethylethynylsilane (0.4 g, 4 mmol) and triethylamine (0.6 g, 6 mmol) was added dropwise to the above reaction solution. The reaction solution was heated at 80°C and stirred for 10 hours. After cooling to room temperature, the reaction solution was quenched with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and separated and purified by silica gel column chromatography to obtain product (7) (0.8 g, 90%). Compound (7) (240 mg, 0.52 mmol) was dissolved in 25 mL of methanol, and potassium carbonate (138 mg, 1.04 mmol) was added and stirred at room temperature for 2 hours. The reaction mixture was separated and purified by silica gel column chromatography to obtain the target product ANL-2 (122 mg, 70%). Purity 99.12% (HPLC) (Figure 1). HR-EIMS: m / z [M+H] + 389.1015(calcd.389.1020;M=C 23 H 16 O6); [M+Na] + 411.0833 (calcd.411.0839) (Figure 2). 1H NMR (400MHz, CDCl3) δ7.68(1H,s,H-6),7.14(1H,s,H-3),6.97(1H,d,J=8.0H z,H-5′),6.85(1H,d,J=1.2Hz,H-2′),6.84(1H,dd,J=8.0,4.4Hz,H-6′),6.1 0(1H,d,J=1.2Hz,-OCH2O-),6.06(1H,d,J=1.2Hz,-OCH2O-),5.43(2H,d,J=5 .2Hz,H-9),4.11(3H,s,5-OMe),3.82(3H,s,4-OMe),3.802(1H,s,H-2″)(Figure 3). 13 C NMR(100MHz, CDCl3)δ169.6(C-9'),152.7(C-5),150.3(C-4),147.8(C-3'),147.6(C- 4'),143.8(C-7),140.4(C-7'),133.3(C-2),128.8(C-1'),127.9(C-1),123.5(C-6') ,121.4(C-8'),118.4(C-8),110.5(C-2'),108.3(C-5'),106.4(C-3),104.1(C-6),101.4(-OCH2O-),87.7(C-2″),77.7(C-1″),68.2(C-9),56.2(5-OMe),55.9(4-OMe) (Figure 4).

[0220] Example 3 Total Synthesis of 7-O-acetonitrile-Sandrocin (ANL-3)

[0221] Dissolve schizoferrin 5 (38 mg, 0.1 mmol) and bromoacetonitrile (24 mg, 0.2 mmol) in 1 mL of acetone, and slowly add Cs2CO3 (65 mg, 0.2 mmol). Stir for 12 hours. TLC shows the formation of a less polar spot. The mixture is purified on preparative silica gel to yield the desired product ANL-3 (40 mg, 95%). HR-EIMS m / z [M+H] + 420.1061(calcd.420.1079;M=C 23 H 17 NO7). 1H NMR(400MHz,DMSO-d6)δ7.52(1H,s,H-6),7.05(1H,d,J=7.9Hz,H-5'),7.01(1H,s,H-3),6.91(1H,d,J=1.6Hz,H-2'),6.79(1H,dd . 13 C NMR(100MHz,DMSO-d6)δ168.8(C-9'),151.8(C-5),150.2(C-4),147.1(C-3'),147.0(C-4 '),144.7(C-7),135.5(C-7'),129.8(C-2),128.4(C-1'),127.9(C-8'),126.0(C-1),123. 6(C-6'),118.8(C-8),117.1(C-2″,C≡N),110.8(C-2'),108.1(C-5'),105.8(C-3),101.2 (C-6), 100.3 (-OCH2O-), 66.1 (C-9), 58.0 (C-1″, CH2), 55.9 (5-OCH3), 55.3 (4-OCH3) (Figure 6).

[0222] Example 4 Total Synthesis of Dioscorea Indole Glycoside Derivative ANL-4

[0223] D-xylose (91.6 mg, 0.61 mmol) was dissolved in pyridine (2 mL) and acetic anhydride (Ac2O, 0.4 mL, 4.21 mmol) was added and stirred at room temperature overnight. After the reaction was completed, CH2Cl2 was added for dilution, washed with 10% HCl solution, the organic phases were combined, extracted with a saturated solution of NaHCO3, the organic phases were taken, dried over anhydrous Na2SO4, and concentrated to obtain a crude product of fully acetylated xylose. Without further purification, it was directly used in the next step, i.e., dissolved in CH2Cl2 (5 mL), cooled to 0°C, and then hydrogen bromide (HBr, 33% AcOH, 1.5 mL) was added dropwise. The reaction was heated to room temperature and stirred for another 4 hours. The reaction was then quenched with water and the aqueous layer was extracted with CH2Cl2. The organic layers were combined, washed with water, 10% NaHCO3 and saturated brine, dried over Na2SO4, and concentrated under reduced pressure to obtain glycosyl bromide. The crude glycosyl bromide was concentrated and used directly in the next step without further purification. Aneurole (ANL-1, 215 mg, 0.61 mmol) was dissolved in CHCl₃ (15 mL) and aqueous sodium hydroxide (NaOH, 0.1 M, 20 mL). Tetrabutylammonium bromide (TBAB) (306 mg, 0.95 mmol) was added. The reaction was heated to 40°C for 10 min. Glycosyl bromide (0.61 mmol) was added and stirred at 40°C for 12 h. After cooling to room temperature, the aqueous layer was extracted with CHCl₃. The combined organic layers were washed with saturated brine, dried over Na₂SO₄, and concentrated under reduced pressure. The reaction mixture was separated by silica gel column chromatography (n-hexane / ethyl acetate = 1 / 1) to obtain the desired product, Aneurole-2″,3″,4″-triacetoxy-7-O-β-D-xylopyranoside ANL-4 (233.7 mg, 60% yield over three steps). HR-EIMS m / z [M+H] + 639.1684(calcd.639.1708;M=C 32 H 30 O 14 ). 1H NMR (400MHz, CDCl3) δ7.51(1H,d,J=1.0Hz,H-6),7.07(1H,br s,H-3),6.95(1H,d,J=7.6Hz,H-5'),6.82(1H,m,H-6'),6.80(H,br s, H-2'), 6.09 and 6.05 (each 1H, m, OCH2O), 5.48–5.38 (5H, m, H-2″, H-3″, H-4″, OCH2O), 5.32 (1H, d, J = 8.8Hz, H-1″), 5.12 (2H, m, H-9), 4.13 (1H, dd, J=7.2, 4.8Hz, Hb-5″), 4.07(3H,s,5-OCH3), 3.80(3H,s,4-OCH3), 3.41(1H,m,Ha-5″), 2.12(3H,s,Ac), 2.10(3H,s,Ac), 2.07(3H,s,Ac) (Figure 7). 13 C NMR(100MHz, CDCl3)δ170.2(Ac),170.0(Ac),169.6(Ac),169.5(C-9'),152.1(C-5),150.5(C-4),149.6(C-3') ,147.7(C-4'),144.1(C-7),136.9(C-7'),136.5(C-1),130.9(C-2),128.3(C-1'),127.9(C-6'),126.5(C-8), 123.7(C-6),119.4(C-3),110.8(C-2'),108.4(C-8'),106.4(C-5'),101.6(C-1″),101.4(-OCH2O-),80.8(C-2 ″), 79.9(C-4″), 71.8(C-3″), 71.5(C-5″), 68.8(C-9), 56.4(5-OMe), 56.0(4-OMe), 21.0(Ac), 20.9(Ac×2) (Figure 8).

[0224] Example 5 Total Synthesis of Dioscorea Indole Glycoside Derivatives ANL-5, ANL-6 and ANL-7

[0225] ANL-4 was first treated with a NaOH aqueous solution (the molar ratio of ANL-4 to NaOH was 1:1.2) at room temperature to remove the three O-acetyl groups on its glycosyl moiety. The product was then transferred to dichloromethane to obtain 7-O-xyloside (8) of schizoflavin. Compound 8 (150 mg, 0.3 mmol) was then dissolved in dry acetonitrile (1.5 mL), and acetic anhydride (32 μL, 0.32 mmol) and tetrabutylammonium acetate (27 mg, 0.09 mmol) were added. The reaction solution was stirred at 40°C for 12 h. After the reaction was completed, the solvent was concentrated by rotary evaporation and then separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1.5 / 1, isocratic elution) to obtain the product, 3″-acetoxy-β-D-xylopyranoside ANL-5 (99.8 mg, yield 60%). In the presence of triethylamine / 4-dimethylaminopyridine (Et3N / DMAP), ANL-5 was treated with allyl chloroformate (the molar ratio of ANL-5 to allyl chloroformate was 1:2) from 0°C to room temperature for 15h. The product was purified by Silica gel column chromatography separation gave allyloxycarbonyloxy (O-alloc) and oxyacetyl (O-Ac) substituted products of schizoferrin xyloside, including schizoferrin-3″-acetoxy-2″,4″-diallyloxycarbonyloxy-β-D-xylopyranoside ANL-6, and 3″-acetoxy-4″-allyloxycarbonyloxy-β-D-xylopyranoside 4-β-D-xylopyranoside ANL-7 (ANL-6, 5 mg, yield 20%; ANL-7, 9.5 mg, yield 41%).

[0226] ANL-5: HR-EIMS m / z[M+H] + 555.1471(calcd.555.1497;M=C 28 H 24 O 12 ). 1 H NMR (400 MHz, CDCl3) δ 7.86 (1H, s, H-6), 6.98 (1H, m, H-3), 6.87 (1H, m, H-5'), 6.75 (1H, m, H-6'), 6.68 (H, m, H-2'), 6.03 and 5.99 (each 1H, br s, OCH2O), 5.44 and 5.34 (each 1H, br d, J = 15.2 Hz, H-9), 4.88 (1H, t, J = 8.8 Hz, H-3"), 4.82 (1H, m, H-1"), 4.04 and 4.01 (each 1H, br d, J = 4.8 Hz, H-5), 3.97 (3H, s, 5-OCH3), 3.74 (3H, s, 4-OCH3), 2.04 (3H, s, Ac) (Figure 9). 1313C NMR (100 MHz, CDCl3) δ 173.3 (Ac), 170.2 (C-9'), 152.1 (C-5), 150.3 (C-4), 147.6 (C-3'), 147.6 (C-4'), 144.4 (C-7), 136.7 (C-7'), 131.0 (C-1), 130.8 (C-2), 128.3 (C-1'), 128.3 (C-6'), 119.1 (C-8), 110.8 (C-6), 110.8 (C-3), 108.38 (C-2'), 106.3 (C-8'), 105.4 (C-5'), 101.4 (-OCH2O-), 101.0 (C-1″), 79.2 (C-4″), 72.3 (C-2″), 68.6 (C-3″), 67.6 (C-9), 65.7 (C-5″), 56.4 (5-OMe), 55.9 (4-OMe), 21.2 (Ac) (Figure 10).

[0227] ANL-6: HR-EIMS: m / z [M+H] + 723.1897 (calcd. 723.1920; M = C 36 H 34 O 16 ). 1 1H NMR (400 MHz, CDCl3) δ 7.55 (1H, s, H-6), 7.10 (1H, s, H-3), 6.97 (1H, d, J = 8.0 Hz, H-5'), 6.83 - 6.79 (2H, m, H-2', 6'), 6.10 - 6.05 (2H, m, -OCH2O-), 5.93 and 5.88 (each 1H, m, H-9), 5.50 - 5.22 (8H, m, O-Alloc, H-2″, H-3″), 5.06 (1H, d, J = 8.0 Hz, H-1″), 5.00 (1H, br td, J = 9.2, 5.6 Hz, H-4″), 4.74 - 4.62 (4H, m, O-Alloc), 4.24 (1H, dd, J = 11.2, 5.6 Hz, Ha-5″), 4.06 (3H, s, 5-OMe), 3.81 (3H, s, 4-OMe), 3.36 (1H, br t, J = 11.2 Hz, Hb-5″), 2.11 (3H, s, OAc) (Figure 11). 13C NMR(100MHz, CDCl3)δ170.1(Ac),169.7(C-9'),154.3(Alloc),154.0(Alloc),152.4(C-5),150.4(C-4),147.7(C-3') ,147.7(C-4'),137.1(C-7),131.2(Alloc),131.1(Alloc),131.0(C-7'),130.9(C-1),128.2(C-2),126.7(C-1'),123 .7(C-6'),120.1(C-8),119.7(Alloc),119.4(Alloc),110.8(C-2'),110.7(C-8'),108.4(C-5',C-6),106.5(C-3),10 2.3(C-1″),101.4(-OCH2O-),75.6(C-3″),72.2(C-2″),71.9(C-4″),69.6(Alloc),69.3(Alloc),67.2(C-9),63.0(C-5 ″ ), 56.7(5-OMe), 56.0(4-OMe), 20.9(Ac) (Figure 12).

[0228] ANL-7: HR-EIMS: m / z[M+H] + 639.1680(calcd.639.1708;M=C 32 H 30 O 14 ). 1 H NMR (400 MHz, CDCl3) δ7.90 (1H, s, H-6), δ7.07 (1H, d, s, H-3), δ6.94 (1H, br d, J=8.0 Hz, H-5′), 6.82-6.76 (2H, m, H-2′, 6′), 6.09 and 6.04 (each 1H, br s, -OCH2O-), 5.96–5.87 (1H, m, Alloc olefinic hydrogen), 5.50-5.28 (5H, m, Ha-9, Alloc olefinic hydrogen, H-3″, H-4″), 5.13 (1H, t, J=8.0 Hz, H-1″), 4.95 (1H, m, Hb-9), 4.88 (1H, d, J=8 Hz, H-2″), 4.65-4.63 (2H, m, Alloc alkyl hydrogen), 4.24 (1H, m, Ha-5″), 4.02 (3H, s, 5-OMe), 3.80 (3H, s, 4-OMe), 3.35 (1H, m, Hb-5″), 2.18 (3H, s, OAc) (Figure 13). 13C NMR(100MHz, CDCl3)δ172.1(Ac),169.9(C-9'),154.1(Alloc),152.2(C-5),150.4(C-4),147.7(C-3'),147.6(C-4') ,144.2(C-7),137.0(C-7'),131.2(C-6),131.1(Alloc),130.9(C-8),128.3(C-1),127.2(C-2),123.7(C-1'),119.7( Alloc),119.3(C-3),110.8(C-2'),108.3(C-5'),106.4(C-6'),105.5(C-1″),101.4(-OCH2O-),100.9(C-8'),75.4( C-3″), 73.1(C-4″), 72.1(C-2″), 69.3(Alloc), 67.4(C-9), 62.8(C-5″), 56.5(5-OMe), 56.0(4-OMe), 21.1(Ac) (Figure 14).

[0229] Example 6

[0230] Determination of anti-SARS-CoV-2 activity in vitro (Method 1)

[0231] (I) Evaluation of the cytotoxicity of seven aryl naphthyl lignans, including ANL-1, ANL-2, ANL-3, ANL-4, ANL-5, ANL-6, and ANL-7, against Vero E6 cells (Method 1)

[0232] In 96-well plate, 2×10 4 Vero E6 cells were inoculated per well and cultured overnight at 37°C and 5% CO2. When the monolayer cells grew to about 70%, different concentrations of the drug to be tested (7 types of feroxin or its glycoside derivatives ANL-1-ANL-7, or the positive control drug remdesivir) were added at 100 μL / well, with 3 replicate wells. At the same time, a negative control well without drug was set up. Cultured at 37°C and 5% CO2 for 72 hours, the cell viability was detected using the CCK8 kit. The OD value was measured using a Bio-Tek Synergy 2 multi-function microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. The CC50 value (50% Cytotoxic Concentration) was calculated, which is the drug concentration that produces toxicity to 50% of Vero E6 cells.

[0233] (II) Determination of the in vitro anti-SARS-CoV-2 activity of seven aryl naphthyl lignans, including ANL-1, ANL-2, ANL-3, ANL-4, ANL-5, ANL-6, or ANL-7 (Method 1)

[0234] Vero E6 cells (common commercial products) were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum. The cells were passaged once 1 day before the experiment to keep the cells in the logarithmic growth phase. SARS-CoV-2 was amplified in Vero E6 cells, and the culture medium was collected, filtered through a 0.22 μm filter, and aliquoted into 0.5 mL / tubes and stored at -80°C. Vero E6 cells were seeded in 96-well plates at 2 × 10 4 / well, culture overnight at 37°C and 5% CO2. When the monolayer cells grow to about 70%, transfer them to the P3 laboratory for use. Drug dilution: set up 6 concentration gradients, with 3 replicate wells for each gradient. In the P3 laboratory, 50 μL of pre-prepared drugs (7 types of schizoflavin or its glycoside derivatives ANL-1 to ANL-7, or, the positive control drug remdesivir) and 50 μL of virus dilution supernatant (MOI = 0.1) were added to each well of the cell culture plate. A negative control without drugs and viruses, and a positive control without schizoflavin or its glycoside derivatives but containing remdesivir were set up. Cultured at 37°C and 5% CO2 for 72 hours, the cell viability was detected by CCK8 kit, and the OD value was measured by Bio-Tek EON microplate reader. The measurement wavelength was 450 nm and the reference wavelength was 630 nm. Calculate the inhibition rate of drug on viral replication and IC 50 IC 50 The value (50% Inhibitory Concentration) refers to the drug concentration that can inhibit the growth of Vero E6 by 50%.

[0235] Method 1 experimental results:

[0236] The in vitro anti-SARS-CoV-2 activity of schizoflavin or its glycoside derivatives ANL-7, ANL-2 and ANL-6 was the strongest, with a half inhibitory concentration IC 50 The in vitro anti-new coronavirus activity of the positive control drug Remdesivir was lower, and the IC 50 The in vitro anti-COVID-19 activity of compounds ANL-7, ANL-2, and ANL-6, measured by TI values, was 19-, 16-, and 10-fold that of remdesivir, respectively. See Table 1.

[0237] Table 1. In vitro anti-COVID-19 activity of seven arylnaphthyl lignan compounds and remdesivir (Method 1) Note: CC 50 : half inhibitory concentration of the sample on Vero E6 cells; IC50 : The concentration of the sample that causes half of the Vero E6 cells to die of SARS-CoV-2; therapeutic index TI = CC 50 / IC 50 .

[0238] According to literature reports, the in vitro anti-new coronavirus activity of the phosphate active structure of Monoclavir and Namatevir in the Vero E6 cell model is 1.2 times and 0.4 times that of Remdesivir, respectively (Fiaschi L, et al. Viruses 2022; 14: 1374.); the IC of Azivudine (phosphate active structure) is 50 The in vitro antiviral activity of these marketed drugs against the novel coronavirus is much lower than that of ANL-7, ANL-2, and ANL-6 in terms of therapeutic index.

[0239] (III) Determination of anti-coronavirus activity in vivo and in vitro (Method 2)

[0240] Viruses and Biosafety: SARS-CoV-1 GZ50 (GenBank: AY304495), MERS-CoV (EMC / 2012, GenBank: JX869059), SARS-CoV-2 wild-type (WT, HKU-001a, GenBank: MT230904), and Omicron BA 5.2 (GISAID: EPI_ISL_13777658) viruses were obtained from the Department of Microbiology, University of Hong Kong (HKU). SARS-CoV-1 and MERS-CoV were propagated in Vero E6 cells. All variants of SARS-CoV-2 were cultured in Vero E6-TMPRSS2 cells and titrated by plaque assay. All live virus culture, in vivo, and in vitro experiments were performed in a Biosafety Level 3 laboratory at the University of Hong Kong, following strict protocols.

[0241] Cell culture: Caco2 cells were obtained from ATCC (ATCC HTB-37) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to the supplier's guidelines. VeroE6-TMPRSS2 cells were obtained from the Japan Center for Bioresource Research (JCRB) Cell Bank (JCRB1819) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to the manufacturer's instructions. All cell lines used in this study were routinely tested for mycoplasma and cultured in a mycoplasma-free environment.

[0242] Determination of the half-inhibitory concentration of ANL-2 against coronavirus in vitro:

[0243] SARS-CoV-1, MERS-CoV, SARS-CoV-2WT, or SARS-CoV-2BA.5.2 (MOI = 0.1) were added to Caco2 cells. 2 hours after infection, the viral inoculum was removed, the cells were washed three times with phosphate-buffered saline (PBS), and then different concentrations of ANL-2 (0-10 μM) were added. 24 hours after infection, RNA extraction and qRT-PCR were performed using the QIAsymphony RNA kit (Qiagen, Germany) to quantify RdRp, NP, or sgRNA gene copies, and then IC was calculated using GraphPad Prism 6. 50 .

[0244] Cell viability was quantified using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, USA). Caco2 cells were incubated for 24 hours with different concentrations of ANL-2 (0–10 μM) and then processed according to the manufacturer's instructions. Luminescent signals were detected using a Victor X3 2030 Multi-Purpose Microplate Analyzer (Perkin Elmer, USA).

[0245] Determination of in vivo activity against SARS-CoV-2:

[0246] Animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong. Male and female Syrian hamsters, 6–8 weeks old, were obtained from the Laboratory Animal Services Center of the Chinese University of Hong Kong through the Centre for Comparative Medicine Research (CCMR) of the University of Hong Kong. Hamsters were maintained at 65% humidity and an ambient temperature of 21–23°C with free access to food and water, and a 12-h light–dark cycle.

[0247] Hamsters were anesthetized by intraperitoneal injection of ketamine (200 mg / kg) and xylazine (10 mg / kg), and 50 μL of SARS-CoV-2 WT was inoculated intranasally. The SARS-CoV-2 WT stock solution was diluted with PBS to a concentration of 3 × 10 per hamster. 3 PFU. Six hours after infection, each hamster was intraperitoneally injected with ANL-2 (50 mg / kg or 25 mg / kg) or 35% PEG400 aqueous solution in a final volume of 1000 μL. The infected hamsters were then intraperitoneally injected with ANL-2 (50 mg / kg or 25 mg / kg) or 35% PEG400 aqueous solution on days 1, 2, and 3 after infection, for a total of 4 doses. All hamsters were sacrificed on day 4 after infection for virological evaluation. After collecting hamster lung and nasal turbinate tissues, RNA was extracted from hamster lung tissue using the RNeasy Mini kit (Qiagen, Germany), and the viral gene copies of SARS-CoV-2 were quantified by RNA-dependent RNA polymerase (RdRp) using the QuantiNova Probe RT-PCR kit (Qiagen, Germany).

[0248] 50 mg / kg or 25 mg / kg of ANL-2 was administered by dissolving ANL-2 in a 35% PEG400 aqueous solution.

[0249] We also studied and evaluated the effect of ANL-2 on the inhibition of novel coronavirus replication in host Caco2 cells (Method 2). First, the effect of ANL-2 on the viability of host Caco2 cells was determined using a luminescent cell viability assay kit. It was found that ANL-2 had no effect on Caco2 cell viability within its detection concentration range (0-10 μM). 50 The value is greater than 10 μM (Figure 15). In the determination of the half-inhibitory concentration of ANL-2 against coronavirus in vitro, Caco2 cells were infected with various coronaviruses (including SARS-CoV-1, MERS-CoV, SARS-CoV-2WT and SARS-CoV-2BA.5.2) and added with different concentrations of ANL-2 (0-10 μM). After incubation for 24 hours, RNA extraction and qRT-PCR were performed to quantify RdRp, NP or sgRNA gene copies. According to the results of Figure 16, ANL-2 effectively reduced SARS-CoV-1 (IC 50 =2.69nM), MERS-CoV (IC 50 =1.49 nM), SARS-CoV-2 wild type (IC 50 <0.64 nM) and variant BA.5.2 (IC 50=9.06~16.3nM) replication in Caco2 cells.

[0250] The Syrian hamster animal model was used to evaluate the in vivo anti-SARS-CoV-2 activity of ANL-2. As shown in Figure 17, ANL-2 (25 mg / kg) significantly reduced the viral load in the lung tissue of hamsters infected with SARS-CoV-2 WT, but had no effect on the viral load in the nasal concha. In addition, the high-dose group (50 mg / kg) of ANL-2 had no significant effect on the viral load in the lung / nasal concha of hamsters infected with SARS-CoV-2 WT. This may be due to the lower solubility of ANL-2 in the injection solution of the high-dose group (50 mg / kg) than that of the low-dose group (25 mg / kg).

[0251] Example 7 Preparation of ANL-2 Test Solution for Animal In Vivo Safety and Pharmacokinetic Evaluation

[0252] A 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution was used to prepare an ANL-2 suspension as test solution 1, with doses of 500 mg / kg, 2000 mg / kg, and 5000 mg / kg. A 0.3% (w / v) ANL-2 fat emulsion was prepared as test solution 2. The preparation methods of the two test solutions are as follows:

[0253] ANL-2 suspension: Weigh 3.00 g of ANL-2 and add a certain volume of 0.5% CMC-Na aqueous solution. Vortex and ultrasonically disperse the mixture evenly. Dilute with 0.5% CMC-Na aqueous solution to prepare ANL-2 suspensions with concentrations of 50.00 mg / mL, 66.67 mg / mL, and 166.67 mg / mL, respectively, which are test solution 1.

[0254] ANL-2 fat emulsion:

[0255] (1) Weigh 4.50 g of ANL-2, add 300.00 g of soybean oil and 1.50 g of oleic acid, stir and ultrasonicate to disperse the drug evenly to obtain the oil phase.

[0256] (2) Weigh 30.00 g of soybean lecithin, 15.00 g of poloxamer P-68, 33.75 g of glycerol, and 0.75 g of vitamin E, add 1100 mL of ultrapure water, and disperse evenly at 75°C to obtain an aqueous phase.

[0257] (3) The oil phase and the water phase were preheated to 75°C, and the oil phase was slowly added dropwise to the water phase while stirring. The volume was then made up to 1500 mL with ultrapure water. The mixture was stirred for 10 min and ultrasonicated in an ultrasonic cell disruptor at an intensity of 600 W for 5 min to obtain colostrum.

[0258] (4) The colostrum was homogenized in a high-pressure homogenizer for 15 min with the parameter set to 1000 Bar to obtain a 3.00 mg / mL C180A-P1 fat emulsion, i.e., a 3.00 mg / mL ANL-2 fat emulsion, which was test solution 2.

[0259] (5) 20 μL of ANL-2 fat emulsion was diluted to 1 mL with normal saline injection. The particle size was measured using a particle size analyzer. The particle size was (145.0 ± 82.4) nm. The results are shown in Figure 18. The emulsion particle size meets the particle size requirements for fat emulsion for injection.

[0260] (6) The ANL-2 fat emulsion was observed and photographed under a transmission electron microscope, as shown in Figure 19. The droplets were uniform in size, ranging from 10 nm to 160 nm in diameter, and spherical in shape.

[0261] Example 8 Acute toxicity evaluation of ANL-2 in mice

[0262] The present invention uses the median lethal dose (LD50) Bliss method specified by the State Food and Drug Administration to evaluate the acute toxicity of the samples. ICR mice that have passed the adaptability observation were selected and randomly divided into 5 groups based on weight and gender, with 10 animals in each group, half male and half female.

[0263] An acute toxicity test was conducted in mice using ANL-2 test solution 1. ICR mice were orally administered 1 to 3 times within 8 hours, with a volume of 10 mL / kg each time and a cumulative dose of 500 mg / kg, 2000 mg / kg, and 5000 mg / kg.

[0264] An acute toxicity test of ANL-2 test solution 2 was conducted on mice by intravenous administration, intraperitoneal injection or oral administration. ICR mice were administered intravenously, intraperitoneally or orally three times within 8 hours, with a dosage volume of 16.7 mL / kg each time. ANL-2 test solution 2 at a concentration of 3.00 mg / mL was used, and the cumulative dose for three times was 150 mg / kg.

[0265] Animals in each group were observed continuously for 4 hours after administration, and then observed 1 to 2 times a day for 14 days. Observation contents: physical signs, behavioral activities, glandular secretions, respiration, excrement characteristics, diet, poisoning reactions and deaths, etc. After administration, the deaths of all animals, poisoning symptoms and the onset time, severity, duration and reversibility of poisoning reactions were recorded in detail. The observation contents of poisoning symptoms refer to the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs". At the same time, body weight and food intake were checked as planned. If an animal was found dead or in a dying state during the experiment, a gross anatomical examination was performed in time, and abnormal organs were fixed for histopathological examination. The surviving animals underwent gross anatomical examination as planned, and abnormal organs were fixed for histopathological examination.

[0266] The results of the in vivo acute toxicity study of ANL-2 (Figure 20) show that ANL-2 fat emulsion had no significant adverse reactions when administered orally to mice at a dose of 150 mg / kg; nor did ANL-2 CMC-NA suspension have significant adverse reactions when administered orally to mice at a dose of 5000 mg / kg. Therefore, it can be concluded that the no-observed-adverse-effect level (NOAEL) for intravenous, intraperitoneal, or oral administration of ANL-2 fat emulsion to mice is greater than 150 mg / kg; and the NOAEL for oral administration of ANL-2 CMC-NA suspension is greater than 5000 mg / kg, indicating that ANL-2 CMC-NA suspension does not exhibit acute oral toxicity in mice.

[0267] Example 9 Pharmacokinetic evaluation of ANL-2 in rats

[0268] The present invention uses SPF-grade SD rats, male, 220-260g, animal certificate: No. 44005800013136. The above animals were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Experimental Animal Production License No.: SCXK (Guangdong) 2018-0034), and the use of experimental animals was approved by the Ethics Committee (Approval No.: 20211012004). The animals were raised in the Experimental Animal Center of Guangzhou University of Chinese Medicine (Experimental Animal Use License No.: SYXK (Guangdong) 2018-0001), with a temperature of 22±3°C, a relative humidity of 30-70%, and a light-dark cycle of 12 hours. Free food and water, and feed was provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. 36 healthy SD rats, male, weighing (220-260g), were adapted to the SPF environment for one week. The subjects were randomly divided into six groups and administered three doses: 2 mg / kg, 4 mg / kg, and 8 mg / kg via tail vein injection, and three doses: 15 mg / kg, 25 mg / kg, and 50 mg / kg orally. Each group consisted of six animals. All subjects were fasted for 12 hours before blood collection, but were not allowed to drink water. Both tail vein injection and oral administration consisted of a 0.3% ANL-2 lipid emulsion.

[0269] (1) Intravenous injection of drug into rats and blood sampling

[0270] A 3.00 mg / mL ANL-2 fat emulsion was diluted with normal saline injection to 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL, and directly used for intravenous injection; a single tail vein injection of 2 mg / kg, 4 mg / kg, and 8 mg / kg was given with a dosing volume of 10 mL / 1 kg. 200 μL of blood was collected before administration (0 h) and 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, and 12 h after administration; blood was collected in prefabricated EP tubes containing EDTA·2K, centrifuged at 3000 rpm for 15 min, plasma was separated, and frozen at -80°C.

[0271] (2) Oral administration to rats and blood sampling

[0272] A 3.00 mg / mL ANL-2 fat emulsion was diluted with normal saline injection to 1.5 mg / mL, 2.5 mg / mL, and 3 mg / mL for oral administration. A single oral gavage of 15 mg / kg, 25 mg / kg, and 50 mg / kg was administered at a volume of 10 mL / 1 kg (the 50 mg / kg dose group had a volume of 16.67 mL / 1 kg). 200 μL of blood was collected before administration (0 h) and 10 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 24 h, and 36 h after administration. Blood was collected in prefabricated EP tubes containing EDTA·2K, centrifuged at 3000 rpm for 15 min, and plasma was separated and stored frozen at -80°C.

[0273] (3) Collection of blank plasma from rats: Six healthy male SD rats, 6 to 8 weeks old, weighing 220 to 260 g, were anesthetized with 2% sodium pentobarbital at a dose of 40 to 50 mg / kg (0.2 mL / 100 g). Blood was collected from the abdominal aorta in a vacuum tube containing EDTA·2K and centrifuged at 3000 rpm for 15 min to separate the plasma. The blood was then divided into centrifuge tubes, sealed, and stored frozen at -80°C.

[0274] The established UPLC-MS / MS method was used to determine the concentration of ANL-2 in the drug-containing plasma of rats after administration. The concentration and corresponding time points were input into EXCEL for statistical analysis. Mean ± SD was calculated for each group of quantitative data, and a blood drug concentration-time curve was drawn. All data were formatted into a data table and imported into DAS 3.0 statistical software. The non-compartmental model was used to process the data for each dose group and calculate AUC, C max , t max , t 1 / 2、The main pharmacokinetic parameters such as MRT, Vz / F, Clz / F, C0, etc. were statistically analyzed using Excel statistical software to calculate the oral bioavailability and evaluate the test results.

[0275] The plasma concentration-time curves (Figures 21 and 22) and pharmacokinetic parameters (Tables 2-4) indicate that oral absorption of ANL-2 fat emulsion is poor, with low peak concentrations, prolonged time to peak concentration, slow elimination, and a low area under the concentration-time curve. This suggests that the compound has low oral bioavailability but a prolonged in vivo retention time.

[0276] Table 2. Pharmacokinetic parameters of ANL-2 fat emulsion administered via tail vein in rats (n=6)

[0277] Table 3. Pharmacokinetic parameters of ANL-2 fat emulsion administered orally to rats (n=6)

[0278] Table 4. Oral bioavailability of ANL-2 fat emulsion

[0279] The compounds described herein can exhibit activity against SARS-CoV-2. According to the methods of the present invention, a compound of formula (I) is administered to a patient to inhibit the replication of SARS-CoV-2 or reduce the cytopathic effect of the virus.

[0280] The compounds, pharmaceutical compositions, and treatment methods described herein can be used to prevent, treat, or ameliorate infections caused by coronaviruses, including but not limited to SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

[0281] The present invention has now been fully described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that the same may be carried out by modifying or varying the present invention within a wider and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or variations are intended to be encompassed within the scope of the appended claims. The terms and expressions employed are used as terms of description and not of limitation, and it is not intended that the use of such terms and expressions exclude any equivalents of the features shown and described, or portions thereof, but it will be recognized that various modifications are possible within the scope of the invention as claimed. It will be understood, therefore, that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art and that such modifications and variations are deemed to be within the scope of the invention.

[0282] As used herein, "comprising" is synonymous with "including." "Containing" or "characterized by" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms.

[0283] When a group of materials, compositions, components or compounds is disclosed herein, it should be understood that all individual members of those groups and all subgroups thereof are disclosed individually. When Markush groups or other groupings are used herein, all individual members of the group and all possible combinations and subcombinations of the group are intended to be included individually in the present invention. Unless otherwise indicated, each formulation or combination of components described or illustrated herein can be used to practice the present invention. Whenever a range is given in the specification, such as a temperature range, time range, or composition range, all intermediate ranges and subranges, and all individual values ​​included in the given range are intended to be included in the present invention. In the present invention and claims, "and / or" means in addition to or alternatively. In addition, any singular term used also encompasses plural forms.

[0284] All references cited herein are hereby incorporated by reference in their entirety, to the extent that there is no inconsistency with the disclosure of this specification. Some references provided herein are incorporated by reference to provide details about the source of starting materials, additional starting materials, additional reagents, additional synthesis methods, additional analytical methods, additional biological materials, additional cells, and additional uses of the present invention. All titles used herein are only for convenience. All patents and publications mentioned in the specification indicate the technical level of those skilled in the art to which the present invention belongs, and are incorporated herein by reference to the extent that each publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. The references cited herein are incorporated herein by reference in their entirety, to indicate the state of the art as disclosed or filed on the date of application, and it is intended that this information may be adopted herein as needed to exclude specific embodiments in the prior art. For example, when claiming a composition of matter, it should be understood that compounds known and available in the art before the applicant's invention (including compounds permitted to be disclosed provided in the references cited herein) are not intended to be included in the composition of matter claim herein.

[0285] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of an aryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof, characterized in that: At least one of the following applications: (1) Use in the preparation of drugs for treating, preventing or delaying the progression of novel coronavirus infection; (2) Application in the preparation of novel coronavirus inhibitors; The novel coronavirus includes at least one of the viruses that cause severe acute respiratory syndrome, Middle East respiratory syndrome, and novel coronavirus disease COVID-19; The aryl naphthyl lignan compound has formula (I): in X is oxygen or sulfur; R 1 is R 15 、 -OR 15 、 -C(O)R 15 、 or -C(O)OR 15 ; R 2 、R 5 、R 6 、R 10 、R 13 and R 14 each is hydrogen or halogen; R 3 and R 4 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 3 and R 4 Together with the carbon atom to which they are attached, they form a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group; R 7 、R 8 and R 9 Each independently selected from -OR 15 and -OC(O)R 15 ; or R 7 and R 8 Together with the carbon atom to which it is attached, it forms a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted by a group; or R 8 and R 9 Together with the carbon atom to which it is attached, it forms a group optionally substituted by 1, 2, 3, 4 or 5 independently selected R 16 A 5-6 membered heterocyclic group substituted with a group; R 11 and R 12 Together form an oxo group; or in R 11 and R 12 When one of them is hydrogen or halogen, R 11 and R 12 The other one is selected from R 15 、-OR 15 、-C(O)R 15 and -C(O)OR 15 ; R 15 is independently selected at each occurrence from hydrogen, alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, heteroaryl, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N=C(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18 , 1,3,2-dioxaborolane optionally substituted by 1, 2, 3 or 4 groups independently selected from alkyl, a glycoside group, an alkynyl group optionally substituted by a trialkylsilane, a 1,2,3,4 or 5 group independently selected from R 16 A hydrocarbon group substituted with a group, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from R 16 or optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 -(CH2) substituted by k -heterocyclyl, wherein k is an integer from 1 to 6; R 16 is independently selected at each occurrence from alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, oxo, ═NR 17 、-OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 and -N(R 17 )S(O)2R 18 ;and R 17 and R 18 is independently hydrogen, alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl at each occurrence, or is selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4, or 5 groups independently selected from halogen, cyano, amino, hydroxy, C 1-6 Alkyl and C 1-6 Alkoxy groups are substituted.

2. The use according to claim 1, characterized in that: The glycosidic group is a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, or a substituted tetrasaccharide group.

3. The use according to claim 1, characterized in that: The glycoside group is selected from the group of structural formula (i) or (ii): in R 19 and R 20 can form an oxo group together; or when R 19 and R 20 When one of the is hydrogen or halogen, R 19 and R 20 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides; R 21 and R 22 can form an oxo group together; or when R 21 and R 22 When one of the is hydrogen or halogen, R 21 and R 22 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides; R 23 and R 24 can form an oxo group together; or when R 23 and R 24 When one of the is hydrogen or halogen, R 23 and R 24 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides; R 25 and R 26 can form an oxo group together; or when R 25 and R 26 When one of the is hydrogen or halogen, R 25 and R 26 The other one is selected from R 15 、-OR 15 、-C(O)R 15 、-C(O)OR 15 、-CH2R 27 and -C(O)R 27 ; R 27 independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 The hydrocarbon group substituted by a group, optionally substituted by 1, 2, 3, 4 or 5 independently selected from R 16 -(CH2) substituted by k -heterocyclic group, -OR 17 、-C(O)R 18 、-C(O)N(R 17 )R 18 、-C(O)OR 17 、-OC(O)R 17 、-S(O)2R 17 、-S(O)2N(R 17 )R 18 、-N(R 17 )R 18 、-N(R 17 )N(R 17 )R 18 、-N(R 17 )C(O)R 18 、-N(R 17 )S(O)2R 18 , monosaccharides, substituted monosaccharides, disaccharides, substituted disaccharides, trisaccharides, substituted trisaccharides, tetrasaccharides, and substituted tetrasaccharides.

4. The use according to claim 1, characterized in that: The aromatic naphthyl lignan compound is selected from at least one of ANL-2 to ANL-7:

5. The use according to any one of claims 1 to 4, characterized in that: The novel coronavirus includes but is not limited to at least one of SARS-CoV-1, SARS-CoV-2, MERS-CoV and SARS-CoV-2 Omicron strain.

6. The arylnaphthyl lignan compound according to any one of claims 1 to 5, characterized in that The aryl naphthyl lignan compound is not dioscorea oleiferin.

7. A pharmaceutical composition, characterized in that Contains at least one aryl naphthyl lignan compound according to claim 6 or a pharmaceutically acceptable salt or prodrug thereof.

8. The pharmaceutical composition according to claim 7, characterized in that It also contains at least one pharmaceutically acceptable excipient.

9. Use of the pharmaceutical composition according to claim 7 or 8, characterized in that: At least one of the following applications: 1) Use in the preparation of drugs for treating, preventing or delaying the progression of novel coronavirus infection; 2) Application in the preparation of novel coronavirus inhibitors.

10. The method for preparing the aryl naphthalene lignan compound according to claim 6, characterized in that: The preparation method of ANL-2 comprises the following steps: Using 2-bromo-4,5-dimethoxybenzaldehyde (1) as a raw material, acetal (2) is prepared by ethylene glycol protection, (2) reacts with piperonal to obtain compound (3), compound (3) is rapidly converted into isobenzofuran by heating in the presence of acetic acid, and then a diester (4) is obtained by Diels-Alder reaction, which is reduced to obtain salvinol (5); salvinol (5) is then reacted with trifluoromethanesulfonic anhydride to obtain product (6); product (6) reacts with trimethylethynylsilane to obtain product (7); product (7) is reacted in the presence of potassium carbonate to obtain the target product ANL-2; The preparation reaction formula of ANL-2 is as follows: The preparation method of ANL-3 comprises the following steps: The product ANL-3 was obtained by reacting galangin (5) with bromoacetonitrile; The preparation method of ANL-4 comprises the following steps: Using D-xylose as raw material, glycosyl bromide is obtained by reacting with acetic anhydride and hydrogen bromide; in the presence of TBAB, galangin (5) reacts with glycosyl bromide to obtain the target product ANL-4; The preparation method of ANL-5, ANL-6 and ANL-7 comprises the following steps: The -O-acetyl group of the glycosyl part of ANL-4 was removed to obtain schizoferrin 7-O-xyloside; schizoferrin 7-O-xyloside was reacted with acetic anhydride Ac2O and tetrabutylammonium acetate TBAOAc to obtain xylose 3″-acetylated glycoside derivative ANL-5; ANL-5 was treated with allyl chloroformate to obtain different acylated schizoferrin xylosides ANL-6 and ANL-7.

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