Omikexin F as well as preparation method and application thereof

Abstract

The invention relates to Omikexin F and a preparation method and application thereof, the structure of the Omikexin F is shown as a formula (1), and the invention further relates to a synthesis method of the Omikexin F and application of the Omikexin F in virus infection resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to an Omicin F and a preparation method and application thereof. Background Art

[0002] Acute respiratory infections are a major cause of morbidity and mortality from infectious diseases worldwide. Human coronaviruses (CoV) and influenza viruses (IAV) are common pathogens that cause acute respiratory infections. Since the beginning of the new century, acute respiratory syndromes caused by SARS-CoV, MERS-CoV, and SARS-CoV-2 have seriously endangered global health due to their strong infectivity and high lethality. The global influenza epidemic caused by influenza virus infection each year, as well as the occasional highly pathogenic avian influenza, has become a major problem plaguing public health security. Both coronaviruses and influenza viruses are RNA viruses that are highly susceptible to mutation, and the continued emergence of new viral variants may pose a long-term threat to human health. Currently, there are no broad-spectrum preventive and therapeutic drugs for acute respiratory viral infections, and there is an urgent need to develop new, safe, effective, and broad-spectrum antiviral drugs.

[0003] When respiratory viruses such as coronavirus, influenza virus and parainfluenza virus enter respiratory epithelial cells, they need to use the host cell protease to cut and activate the viral protein before entering the cell and starting to replicate. [1] Coronavirus is a type of positive-sense single-stranded RNA enveloped virus characterized by spikes on its surface that bind to receptors on the surface of host cells, thereby initiating membrane fusion. Coronavirus uses the host's protease to activate the spike protein (S protein) and then invade the host cell. Blocking this process can effectively inhibit viral infection. [2] Influenza virus surface hemagglutinin (HA) exists in the form of precursor protein HA0. After being hydrolyzed into HA1 and HA2 by proteases in host cells, the virus particles acquire infectivity. [3] Host cell proteases are crucial for viral infection, so inhibitors of host proteases may have the potential to broadly resist respiratory-related viruses and are expected to become an important means of intervening in respiratory viral infections. More importantly, compared with traditional drugs targeting viruses, Host-targeted antiviral drugs can, from the perspective of mechanism of action, Effectively avoid the problem of viruses developing drug resistance through genetic mutations and escaping drug inhibition .

[0004] Natural products derived from microorganisms are a major source of new anti-infective antibiotics. Streptomyces sp. CPCC200451 is a strain of Streptomyces isolated from soil samples in southern my country in the 1960s by scientists at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Its fermentation broth and extracts have shown excellent anti-influenza virus activity, but the antiviral active components have remained elusive. With the advent of the "post-genomic era," high-throughput sequencing-based transcriptomics, metabolomics, and other omics technologies have emerged and are widely used in genomic research. Starting with the whole genome of Streptomyces CPCC 200451, the research team, based on comparative transcriptomics and metabolomics results directed at anti-influenza activity, located the biosynthetic gene cluster of its active secondary metabolites and ultimately identified its active component as a pseudotetrapeptide compound, named omicsynin. A total of 18 compounds across three categories (A, B, and C) were identified from the producing bacteria. In vitro studies have shown that omicsyn B compounds have excellent anti-influenza virus (H3N2) and coronavirus HCoV-229E activity (IC 50 :1~3μM) [4] Some of the antiviral active compounds in the class of omixins are known protease inhibitors. For example, omixin B4 is an antipain protease inhibitor, suggesting that it may target proteases encoded by host cells and / or viruses, which may be the reason why it has a broad-spectrum antiviral effect against respiratory viruses such as influenza virus and coronavirus.

[0005] Omicron B4 showed good anti-influenza virus activity in vitro, and also had significant broad-spectrum protective effects on mice infected with influenza A H1N1 (pR8), H3N2, and highly pathogenic avian influenza viruses H5N1 and H7N9 in vivo. In addition, Omicron B4 showed significant inhibitory activity against human coronaviruses HCoV-229E and HCoV-OC43 on a variety of cells, and could significantly reduce the replication of the original strain of SARS-CoV-2 virus and a variety of new coronavirus variants including Omicron, showing a broad-spectrum anti-coronavirus effect. The study further clarified that Omicron B4 can significantly inhibit the replication of coronavirus HCoV-OC43 in mice. In vitro biochemical enzymology experiments confirmed that Omicron B4 has inhibitory activity against multiple host proteases, among which it has a very strong inhibitory effect on Cathepsin L, and also has good inhibitory activity on TMPRSS2, suggesting that the compound may be an inhibitor that simultaneously targets two key host proteases. [5] .

[0006] During in vitro and in vivo studies of microbial-derived omixin-like compounds, it was difficult to isolate and purify sufficient amounts of each active compound through microbial fermentation for in-depth drugability evaluation. Therefore, in further research, the inventors attempted to synthesize omixin-like compounds using chemical synthesis methods, resulting in the generation of novel, structurally similar compounds.

[0007] Based on this, the present invention is proposed.

[0008] References

[0009] [1].Laporte M,Naesens L.Airway proteases: an emerging drug target forinfluenza and other respiratory viruses

[0010] infections.Current Opinion in Virology,2017,24:16-24.

[0011] [2].Li G, Hilgenfeld R, Whitley R, De Clercq E. Therapeutic strategies for COVID-19: progress and lessons

[0012] learned.Nature Reviews Drug Discovery.2023,22(6):449-475.

[0013] [3].Garten W,Braden C,Arendt A,et al.Influenza virus activating hostproteases:Identification,localization and

[0014] inhibitors as potential therapeutics.European journal of cellbiology,2015,94(7-9):375-383.

[0015] [4].Sun H, Li X, Chen M, et al. Multi-omics-guided discovery ofomicsynins produced by Streptomyces sp.1647:

[0016] pseudo-tetrapeptides active against influenza a viruses and coronavirus HCoV-229E. Engineering, 2022, 16(9):176-186.

[0017] [5].Li Y, Wang K, Sun H, et al. Omicsynin B4 potently blocks coronavirusinfection by inhibiting host proteases

[0018] cathepsin L and TMPRSS2. Antiviral Research,2023,214:105606. Summary of the Invention

[0019] The present invention first relates to a compound Omixin F as shown in formula (1),

[0020]

[0021] The present invention also relates to a medicine or pharmaceutical composition comprising the Omicron F, wherein the medicine or pharmaceutical composition comprises a therapeutically effective amount of Omicron F and necessary pharmaceutical excipients.

[0022] The drug or pharmaceutical composition is a broad-spectrum antiviral drug. Preferably, the virus is a respiratory virus; most preferably, the virus is a coronavirus or influenza virus.

[0023] Furthermore, the present invention also relates to the use of the aforementioned Omicin F in the preparation of a drug, wherein the drug is a broad-spectrum antiviral drug. Preferably, the virus is a respiratory virus; most preferably, the virus is a coronavirus or influenza virus.

[0024] Furthermore, the present invention also relates to a method for preparing the Omicin F compound, which comprises the following steps:

[0025] (1) Synthesis of (3-chlorophenyl)diphenylmethyl (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate from 2-chlorotrityl chloride resin in a solid phase reactor;

[0026] (2) (3-chlorophenyl)benzhydryl (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate is reacted with piperidine to obtain (3-chlorophenyl)benzhydryl (2S)-2-amino-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate;

[0027] (3) (3-chlorophenyl)benzhydryl (2S)-2-amino-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate is reacted with bis(4-nitrophenyl) carbonate and 2,6-lutidine to obtain (3-chlorophenyl)benzhydryl (2S)-2-{[(4-nitrophenoxy)carbonyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate;

[0028] (4) (3-chlorophenyl)benzhydryl (2S)-2-{[(4-nitrophenoxy)carbonyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate, L-phenylalanine tert-butyl ester, 4-dimethylaminopyridine and N,N-diisopropylethylamine to give (3-chlorophenyl)benzhydryl (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate;

[0029] (5) (3-chlorophenyl) benzhydryl (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoate was deprotected to give (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoic acid ( Compound 11b );

[0030] (6) Under nitrogen protection, Fmoc-Arg(Pbf)-OH was dissolved in ethylene glycol monomethyl ether, and then 2-iodobenzoic acid and N-methylmorpholine were added to react to obtain (4S)-4-{[((9H-fluoren-9-yl)methoxy)carbonyl]amino}-5-{[(2-methylpropyloxy)carbonyl]oxy}-5-oxo-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]pentane-1-guanidine;

[0031] (7) (4S)-4-{[((9H-fluoren-9-yl)methoxy)carbonyl]amino}-5-{[(2-methylpropyloxy)carbonyl]oxy}-5-oxo-N′-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]pentane-1-guanidine (33.94 g, 45.32 mmol, 1 equivalent) was dissolved in ethylene glycol monomethyl ether, and sodium borohydride was added to react to obtain (9H-fluoren-9-yl)methyl N-[(2S)-1-hydroxy-5-{N′-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamate;

[0032] (8) (9H-fluoren-9-yl)methyl N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamate and 1,8-diazabicyclo[5.4.0]undec-7-ene are reacted to obtain N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine;

[0033] (9) reacting N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine, N-(tert-butoxycarbonyl)-L-phenylalanine, 1-hydroxybenzotriazole, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine to obtain tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate;

[0034] (10) Tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate and hydrochloric acid were dissolved in 1,4-dioxane to react and obtain (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropanamide ( Compound 11a );

[0035] (11) (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropionamide ( Compound 11a ), (2S)-2-{[((2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl)carbamoyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentanoic acid ( Compound 11b ), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine are reacted to obtain tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}butyl]carbamoyl}amino}-3-phenylpropanoate;

[0036] (12) Tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl [(1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid is dissolved in trifluoroacetic acid, triisopropylsilane, water and 2-[2-(2-mercaptoethoxy)ethoxy]ethanethiol to give (2S)-2-{[(1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid;

[0037] (13) (2S)-2-{[((1S)-4-imidocarboxamido-1-{[(1S)-1-{[(2S)-5-imidocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid and DESS-MARTIN reagent are dissolved in dimethyl sulfoxide and reacted to obtain (2S)-2-{[(1S)-4-imidocarboxamido-1-{[(1S)-1-{[(2S)-5-imidocarboxamido-1-oxopentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid trifluoroacetate (Omixin F).

[0038]

[0039] The beneficial effects of the present invention are:

[0040] (1) The amino acid residue at the position of valine in the pseudo-tetrapeptide structure of omexin B4 (phenylalanine-carbonyl-arginine-valine-arginine aldehyde) was replaced to synthesize the structural analogue omexin F (phenylalanine-carbonyl-arginine-phenylalanine-arginine aldehyde);

[0041] (2) The antiviral pharmacodynamics of Omicin F in vitro and in vivo were studied, and the results showed that it has significant anti-coronavirus and influenza virus activity and is a new drug candidate with broad-spectrum anti-respiratory virus activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 , The results of the effect of nasal administration of Omicin F on the survival rate of mice infected with HCoV-OC43.

[0043] Figure 2 , The results of the effect of nasal administration of Omicin F on the viral RNA level in the brain tissue of mice infected with HCoV-OC43.

[0044] Figure 3 , The results of the effect of nasal administration of Omicin F on the level of viral N protein in the brain tissue of mice infected with HCoV-OC43.

[0045] Figure 4 , the results of the effect of Omicin F on the body weight of mice infected with influenza virus.

[0046] Figure 5 , the results of the effect of Omicin F on the survival rate of mice infected with influenza virus.

[0047] Figure 6 , nuclear magnetic resonance spectrum (NMR) of Omicin F. DETAILED DESCRIPTION

[0048] The synthetic route of Aomixin F is as follows:

[0049]

[0050] Example 1. Synthesis of (2S)-2-{[((2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl)carbamoyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentanoic acid

[0051]

[0052] 2-Chlorotrityl chloride resin (3.76 g, 100%, 12 mmol, 0.5 equiv) was added to a solid phase reactor. Dimethylformamide (DMF, 100 mL) was added. The suspended resin was stirred under a nitrogen stream for 10 minutes. The solvent was then filtered off. Dichloromethane (DCM, 100 mL) was added. The suspended resin was stirred under a nitrogen stream for 3 minutes. The solvent was then filtered off. The resin was washed three times with dichloromethane. Fmoc-Arg(Pbf)-OH (15.89 g, 98%, 24 mmol, 1 equiv) and N,N-diisopropylethylamine (DIPEA, 15.67 g, 99%, 19.83 mL, 120 mmol, 5 equiv) were added to a solid phase reactor. The suspended resin was stirred under a nitrogen stream for 3 hours. The solvent was then filtered off. Dichloromethane (100 mL) and methanol (30 mL) were added to the solid phase reactor. The suspended resin was stirred under nitrogen flow for 30 min, washed with dimethylformamide (DMF, 100 mL) three times, and the product was used directly in the next reaction;

[0053]

[0054] Under nitrogen, (3-chlorophenyl)benzhydryl (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoate (11.11 g, 12 mmol, 1 equivalent) and piperidine (5.16 g, 99%, 5.93 mL, 60 mmol, 5 equivalents) were dissolved in dimethylformamide (DMF, 100 mL). The reaction was stirred at 25 ° C for 1 hour. The resin was washed with dimethylformamide (DMF, 100 mL) three times and the product was used directly in the next reaction.

[0055]

[0056] (3-Chlorophenyl)benzhydryl (2S)-2-amino-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoate (16.88 g, 24 mmol, 1 equivalent) was dissolved in dimethylformamide (DMF, 200 mL), followed by the addition of bis(4-nitrophenyl) carbonate (26.34 g, 97%, 17.56 mL, 84 mmol, 3.5 equivalents) and 2,6-lutidine (18.37 g, 98%, 19.97 mL, 168 mmol, 7 equivalents). The mixture was stirred at 25°C for 18 hours under nitrogen. Liquid chromatography-mass spectrometry (LC-MS) showed that the starting material was almost completely consumed, with the target product, Ms, being the primary product. The resin was washed three times with dichloromethane (DCM, 100 mL) and the product was used directly in the next reaction. MS [M+H]+: 592.3;

[0057]

[0058] Under nitrogen, (3-chlorophenyl)benzhydryl (2S)-2-{[(4-nitrophenoxy)carbonyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carboximido}valerate (20.84 g, 24 mmol, 1 eq), L-phenylalanine tert-butyl ester (13.98 g, 95%, 60 mmol, 2.5 eq), 4-dimethylaminopyridine (DMAP, 2.96 g, 99%, 2.96 mL, 24 mmol, 1 eq), and N,N-diisopropylethylamine (DIPEA, 15.67 g, 99%, 19.83 mL, 120 mmol, 5 eq) were dissolved in dimethylformamide (DMF, 250 mL). The reaction was stirred at 25°C for 16 hours. Liquid chromatography-mass spectrometry (LC-MS) showed that the starting material was almost completely consumed, and the target product Ms was mainly produced. The resin was washed three times with dichloromethane (DCM, 45 mL), and the product was directly used in the next reaction. MS [M+H] +: 674.4

[0059]

[0060] Under nitrogen, (3-chlorophenyl)benzhydryl (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoate (22.81 g, 24 mmol, 1 equivalent) was dissolved in hexafluoroisopropanol (HFIP, 200 mL, 98%) and dichloromethane (DCM, 200 mL). The reaction was stirred at 25°C for 4 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by flash silica gel column chromatography (C18) (ISCO, 80 g SepaFlash silica flash column, eluent: 0-90% water / acetonitrile, flow rate 50 mL / min). Finally, (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentanoic acid (5 g, yield: 30.92%) was obtained as a white solid. MS [M+H]+: 674.4

[0061] Example 2: Synthesis of (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropanamide

[0062]

[0063] Under nitrogen, Fmoc-Arg(Pbf)-OH (30 g, 98%, 45.32 mmol, 1 equiv) was dissolved in ethylene glycol monomethyl ether (DME, 300 mL, 99.5%). 2-Iodobenzoic acid (IBCF, 8.21 g, 98%, 7.68 mL, 58.91 mmol, 1.3 equiv) and N-methylmorpholine (NMM, 7.02 g, 98%, 7.47 mL, 67.97 mmol, 1.5 equiv) were then added at 0°C. The reaction was stirred at 25°C for 4 hours. Thin-layer chromatography (TLC, developing solvent: ethyl acetate:petroleum ether = 1:0) showed almost complete consumption of the starting material, with the formation of a new spot of high purity. The reaction mixture was filtered, and the filtrate was used directly in the next reaction.

[0064]

[0065] Under nitrogen, (4S)-4-{[((9H-fluoren-9-yl)methoxy)carbonyl]amino}-5-{[(2-methylpropyloxy)carbonyl]oxy}-5-oxo-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]pentane-1-guanidine (33.94 g, 45.32 mmol, 1 equivalent) was dissolved in ethylene glycol monomethyl ether (DME, 300 mL). Sodium borohydride (NaBH4, 5.25 g, 98%, 5.44 mL, 135.96 mmol, 3 equivalents) was added at 0°C. The reaction was stirred at 25°C for 16 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed, and the target product Ms was mainly produced. The reduction reaction was then quenched with a saturated sodium bicarbonate solution (NaHCO3), and the mixture was extracted three times with ethyl acetate (500 mL). The residue was purified by flash column chromatography (FCC, eluent: dichloromethane / methanol = 5:1, Rf = 0.3) to give a yellow oil. (9H-fluorene-9-yl)methyl N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamylamino}pentan-2-yl]carbamate (21.59 g, yield: 75.05%) was a yellow solid. MS [M+H] +: 635.6

[0066]

[0067] Under nitrogen, (9H-fluoren-9-yl)methyl N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamate (21.59 g, 34.01 mmol, 1 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 8.01 g, 97%, 7.62 mL, 51.02 mmol, 1.5 equiv) were dissolved in dichloromethane (DCM, 250 mL). The reaction was stirred at 25°C for 2 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed, and the target product, Ms, was mainly produced. The reaction solution was concentrated under vacuum, and the residue was purified by flash column chromatography (FCC, eluent: dichloromethane / methanol = 1:1, Rf = 0.4) to give a white solid. Finally, N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine (9.60 g, yield: 68.42%) was obtained as a white solid. MS [M+H]+: 413.5

[0068]

[0069] Under nitrogen protection, N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine (3 g, 7.27 mmol, 1 equivalent), N-(tert-butoxycarbonyl)-L-phenylalanine (2.56 g, 98%, 9.45 mmol, 1.3 equivalents), 1-hydroxybenzotriazole (HOBt, 1.28 g, 99.96%, 851.90 μL, 9 equivalents) were added. The reaction mixture was stirred at 25°C for 16 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed, with the target product Ms being the primary product. The reaction solution was concentrated under vacuum, and the residue was purified by flash column chromatography (FCC, eluent: dichloromethane / methanol = 6:1, Rf = 0.4) to yield a yellow oil. Finally, tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate (4.70 g, yield: 97.95%) was obtained as a white solid. MS [M+H]+: 660.3

[0070]

[0071] Under nitrogen, tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate (4.70 g, 7.12 mmol, 1 equivalent) and hydrochloric acid (4.38 g, 30 mL, 120 mmol, 16.847 equivalents, 4 M solution in 1,4-dioxane) were dissolved in 1,4-dioxane (30 mL). The reaction was stirred at 25°C for 4 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed, with the target product, Ms, being the primary product. The reaction solution was concentrated under vacuum, and the residue was used in the next reaction without further treatment. (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carboimido}pentan-2-yl]-3-phenylpropanamide (5 g, crude product, yield: 125.41%) was obtained as a white solid. MS [M+H]+: 560.3

[0072] Example 3, tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-

[0073] Synthesis of [(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}butyl]carbamoyl}amino}-3-phenylpropionate

[0074]

[0075] Under nitrogen protection, (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropionamide ( Synthesized from Example 2 get , 4.50 g, 8.04 mmol, 1 equivalent), (2S)-2-{[((2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl)carbamoyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentanoic acid ( Obtained by synthesis in Example 1, 5.69 g, 8.44 mmol, 1.05 equiv), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 3.43 g, 98%, 8.84 mmol, 1.1 equiv), 1-hydroxybenzotriazole (HOBT, 1.20 g, 99.96%, 796.95 μL, 8.84 mmol, 1.1 equiv), and N,N-diisopropylethylamine (DIPEA, 5.25 g, 99%, 6.71 mL, 40.20 mmol, 5 equiv) were dissolved in dimethylformamide (DMF, 15 mL) and dichloromethane (DCM, 30 mL). The reaction was stirred at 25°C for 2 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed, and the target product Ms was mainly produced. The reaction solution was concentrated under vacuum, and the residue was purified by flash column chromatography (FCC, eluent: dichloromethane / methanol = 6:1, Rf = 0.4) to give a yellow oil. Finally, tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}butyl]carbamoyl}amino}-3-phenylpropanoate (9.50 g, yield: 97.21%) was obtained as a yellow oil. MS [M+H]+: 608.4

[0076]

[0077] Under nitrogen protection, tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}butyl]carbamoyl}amino}-3-phenylpropanoate (9.50 g, 7.82 mmol, 1 Equivalent) was dissolved in trifluoroacetic acid (TFA, 92.50 mL), triisopropylsilane (TIPS, 2.50 mL), water (2.50 mL) and 2-[2-(2-mercaptoethoxy)ethoxy]ethanethiol (2.50 mL). The reaction was stirred at 25 ° C for 4 hours. Liquid chromatography-mass spectrometry (LCMS) showed that the starting material was almost completely consumed and the target product Ms was mainly generated. The reaction solution was concentrated under vacuum, and then the pH value of the mixture was adjusted to 7 with N, N-diisopropylethylamine (DIEA, 3 mL). The residue was purified by flash silica gel column chromatography (C18) (ISCO, 80 g The reaction mixture was purified by SepaFlash silica gel flash column with 0-90% water / acetonitrile as eluent at a flow rate of 50 mL / min. (2S)-2-{[((1S)-4-imidocarboxamido-1-{[(1S)-1-{[(2S)-5-imidocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropanoic acid (2 g, yield: 39.08%) was obtained as a yellow oil. MS [M+H]+: 655.4

[0078]

[0079] Under nitrogen protection, (2S)-2-{[(1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropanoic acid (1.27 g, 1.94 mmol, 1 equiv) and DESS-MARTIN reagent (1.27 g, 97%, 906.02 μL, 2.91 mmol, 1.5 equiv) were dissolved in dimethyl sulfoxide (DMSO, 15 mL). The reaction was stirred at 25° C. for 18 hours. Liquid chromatography-mass spectrometry (LCMS) showed The starting material was almost completely consumed, with the target product, Ms, being primarily produced. The residue was purified by preparative high-performance liquid chromatography (prep-HPLC, eluent: water (containing 0.1% trifluoroacetic acid)-acetonitrile) to afford (2S)-2-{[((1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-oxopentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropanoic acid trifluoroacetate (218 mg, yield: 13.92%, purity: 95%) as a white solid. MS [M+H]+: 653.4.

[0080] The synthetic product is named Aomixin F and its NMR spectrum is shown in Figure 6 .

[0081]

[0082] Experimental Example 4: Determination of the Antiviral Activity of Aumikexin F against HCoV-229E by CPE Method

[0083] Virology-related experimental operations were performed in a BSL-2 biosafety laboratory. Huh7 or Huh7.5 cells 2×10 4 Each well was inoculated into a 96-well plate and cultured overnight with 100 TCID 50The cells were infected with HCoV-229E virus solution, and maintenance solution containing samples of different dilutions and positive control drugs (Nimategravir) was added (3-fold serial dilution). Cell control wells and virus control wells were set up at the same time. The cells were cultured at 35°C with 5% CO2 for about 2 days. When the degree of cytopathic effect of the virus control group reached 75% to 100%, the cytopathic effect of each group was observed. The cell death ratio was marked as 4+ (cell death ratio 75% to 100%), 3+ (cell death ratio 50% to 75%), 2+ (cell death ratio 25% to 50%), 1+ (cell death ratio 0 to 25%), and 0+ (all cells survived). The Reed-Muench method was used to calculate the 50% inhibitory concentration (IC50) of the drug against the virus. 50 , the formula is shown below).

[0084]

[0085] Where: A = log drug concentration at which cumulative inhibition rate is less than 50%, B = inhibition rate at which cumulative inhibition rate is greater than 50%, C = inhibition rate at which cumulative inhibition rate is less than 50%, D = log dilution factor

[0086] Cytotoxicity assay (CPE) method: cells were seeded in 96-well plates at the above concentrations. After overnight culture, maintenance medium containing the drug to be tested was added. The drug to be tested was diluted 3-fold to 8 doses for the experiment. After 2 days of culture, the drug toxicity to the cells was observed under an inverted microscope. The 50% cytotoxic concentration (CC) was calculated using the Reed-Muench method. 50 ) Calculate the selection index (SI = CC 50 / IC 50 ).

[0087] The results are shown in Table 1. Under the experimental conditions,

[0088] (1) The anti-coronavirus HCoV-229E activity of namatevir is comparable to the results in the literature and previous experiments, indicating that the experimental system is established;

[0089] (2) Omicin F has an inhibitory effect on HCoV-229E strain. In Huh7.5 cells, the IC of Omicin F against HCoV-229E strain was determined by CPE method. 50 The IC of Omicin F against HCoV-229E was 2.69 μM, and the selectivity index SI was greater than 74.3. 50 The selectivity index SI was greater than 19.4.

[0090] Table 1. Anti-HCoV-229E virus activity of each compound in Huh7 and Huh7.5 cells (CPE method)

[0091]

[0092] Experimental Example 5: Determination of the Anti-Influenza Virus Activity of Omicin F by CPE Method

[0093] Virology-related experiments were performed in a BSL-2 biosafety laboratory. MDCK cells were cultured at 2.5×10 4 The cells were seeded at a density of 100 TCID / well in a 96-well culture plate and cultured in a 37°C, 5% CO2 incubator for 24 h. 50 MDCK cells were infected with a virus solution (influenza virus A / Hanfang / 359 / 95, H3N2). Two hours after infection, the virus solution was discarded and virus maintenance solution (MEM medium supplemented with 2 μg / mL TPCK-treated trypsin and 0.08% BSA) containing varying concentrations of Omicin F and the positive control compound baloxavir (starting with a 3-fold serial dilution starting from the maximum non-toxic concentration) was added. Culture was continued at 37°C and 5% CO2 for approximately 48 hours. When the cytopathic effect of the virus control group reached 4+ (75% to 100%), the cytopathic effect of each group was observed and the IC value of the drug was calculated using the Reed-Muench method. 50 and selection index SI.

[0094] As shown in Table 2, the anti-influenza virus activity IC of the positive control drug baloxavir 50 The selectivity index SI is greater than 333.3; Omicin F has an inhibitory effect on influenza A virus (H3N2 / Hanfang / 359 / 95) strains, IC 50 The selectivity index SI was greater than 128.2.

[0095] Table 2. Anti-influenza virus (IAV, H3N2) activity of each compound in MDCK cells (CPE method)

[0096]

[0097] Example 6 Effect of Nasal Administration of Aomixin F on the Survival of Mice Infected with HCoV-OC43 Virus

[0098] Preparation of Omicron F solution: Dissolve Omicron F in sterile ultrapure water to prepare working solutions with concentrations of 28.8 mg / mL and 72 mg / mL, corresponding to dosing concentrations of 12 mg / kg and 30 mg / kg, respectively.

[0099] The experimental animals were specific pathogen-free (SPF) BALB / c suckling mice, 4-6 days old, with an average weight of 3.8 g. They were randomly divided into 4 groups, with 8 mice in each group. All animals were raised in an animal biosafety level 2 laboratory (ABSL-2).

[0100] Mice were anesthetized with isoflurane and the experimental animals were divided into groups and administered as follows (the average weight of mice in each administration group was: virus control group, 3.9 g; normal control group, 3.4 g; Omicron F 12 mg / kg group, 4.0 g; Omicron F 30 mg / kg group, 3.9 g):

[0101] (1) Virus control group: 2.5 μL HCoV-OC43 + 2.5 μL sterile ultrapure water were administered intranasally;

[0102] (2) Normal control group: 5 μL of sterile ultrapure water was administered intranasally;

[0103] (3) Omicron F 12 mg / kg group: 2.5 μL HCoV-OC43 + 2.5 μL 28.8 mg / mL Omicron F were administered intranasally;

[0104] (4) Omicron F 30 mg / kg group: 2.5 μL HCoV-OC43 + 2.5 μL of 72 mg / mL Omicron F were administered intranasally.

[0105] The infection dose of HCoV-OC43 (ATCC VR1558, kindly provided by Beijing Ditan Hospital) in all groups of mice was 7-8 LD 50 , and the survival of the mice was recorded for 14 consecutive days.

[0106] The survival curve was drawn according to the survival days of mice in each group. Figure 1 As shown,

[0107] (1) All mice in the normal control group survived the 14-day experimental period, while all mice in the virus control group died on day 8. Compared with the virus control group, both the 12mg / kg and 30mg / kg groups of Aumikexin F were able to improve the survival rate of suckling mice after HCoV-OC43 virus infection.

[0108] (2) The average survival days of the Omicron F 30 mg / kg group was 12.0±1.9 days, which was significantly increased by 64.4% compared with the virus control group (7.3±0.5 days).

[0109] The above results show Omicin F has a protective effect against HCoV-OC43 virus infection in suckling mice .

[0110] Example 7 Effect of Nasal Administration of Aomixin F on the Brain Virus Load in HCoV-OC43-Infected Suckling Mice

[0111] Preparation of Omicron F solution: Dissolve Omicron F in 10% Solutol HS-15 (polyethylene glycol-15 hydroxystearate) to prepare working solutions with concentrations of 60 mg / mL and 100 mg / mL, corresponding to dosing concentrations of 30 mg / kg and 50 mg / kg, respectively.

[0112] Twenty-two specific pathogen-free (SPF) BALB / c suckling mice, 4-6 days old, with an average weight of 4.6 g, were randomly divided into four groups: a normal control group of two mice and six to seven mice in each of the remaining groups. All animals were housed in an ABSL-2 laboratory. Mice were anesthetized with isoflurane and administered the following doses (average weights of mice in each treatment group: virus control group, 5.0 g; normal control group, 4.0 g; 30 mg / kg omeprazole F group, 4.7 g; 50 mg / kg omeprazole F group, 4.8 g):

[0113] (1) Normal control group: mice were given 6 μL of ultrapure water intranasally;

[0114] (2) Virus control group: 3 μL HCoV-OC43 and 3 μL ultrapure water containing 10% Solutol were mixed and administered intranasally;

[0115] (3) Omicron F 30 mg / kg group: 3 μL HCoV-OC43 and 3 μL 60 mg / mL Omicron F were mixed and administered intranasally;

[0116] (4) Omicron F 50 mg / kg group: 3 μL HCoV-OC43 and 3 μL 100 mg / mL Omicron F were mixed and administered intranasally.

[0117] The final HCoV-OC43 viral infection dose for all mice was 7-8 LD 50 On the 6th day after virus infection, the mice were euthanized, and the brain tissues were collected, placed in cryopreservation tubes, and stored at -80°C.

[0118] 1. Effect of Omicin F on viral RNA levels in brain tissue of mice infected with HCoV-OC43 virus

[0119] Brain tissue was collected and placed in a tissue grinding tube, and total RNA was extracted using the Animal Tissue Magnetic Universal Total RNA Kit (ROA3302, Novazom). The HCoV-OC43 nucleocapsid protein (NP) RNA content and the internal reference gene GAPDH RNA content were detected using the TransScript II Probe One-Step qRT-PCR SuperMix Kit (Quanshijin Company) on an ABI7500Fast high-throughput real-time fluorescence quantitative PCR (qPCR) instrument.

[0120] Primers used in the one-step detection of HCoV-OC43 NP RNA content:

[0121] SEQ ID NO. 1: 5′-CGATGAGGCTATTCCGACTAGGT-3′ (upstream);

[0122] SEQ ID NO. 2: 5′-CCTTCCTGAGCCTTCAATATAGTAACC-3′ (downstream);

[0123] SEQ ID NO. 3: 5'-TAMRA-TCCCGCTTGGCACGGTACTCCCT-BHQ2-3' (Probe).

[0124] Primers used to determine GAPDH RNA content:

[0125] SEQ ID NO. 4: 5′-GAAGGTGAAGGTCGGAGTC-3′ (upstream);

[0126] SEQ ID NO. 5: 5'-GAAGATGGTGATGGGATTTC-3' (downstream).

[0127] The reaction system is as follows

[0128]

[0129] The reaction conditions are as follows

[0130]

[0131] Based on the Ct values ​​of viral RNA in each brain tissue sample obtained experimentally, relative quantitative analysis of the target gene was performed using the housekeeping gene GAPDH as an internal reference. The Ct values ​​were normalized and the 2-ΔΔCt method was used to calculate the expression difference of the target gene. The calculation method is as follows:

[0132] ΔCt 实验组 =Ct 目的基因，实验组 -Ct内参基因，实验组

[0133] ΔCt 对照组 =Ct 目的基因，对照组 -Ct 内参基因，对照组

[0134] ΔΔCt=ΔCt 实验组 -ΔCt 对照组

[0135] The expression level of the target gene in the experimental group relative to the control group is fold change = 2 -ΔΔCt

[0136] The results are as follows Figure 2 , it can be seen that

[0137] (1) Compared with the virus control group, Omicin F can significantly reduce the level of HCoV-OC43 RNA in mouse brain tissue Flat and dose-dependent .

[0138] (2) Compared with the viral control group, the viral RNA level in the brain tissue of the 30 mg / kg group of Atomixin F was reduced by an average of 3 Log (P < 0.05), and the viral RNA level in the brain tissue of the 50 mg / kg group of Atomixin F was reduced by an average of 4 Log (P < 0.01).

[0139] 2. Effect of Omicin F on viral protein levels in brain tissue of mice infected with HCoV-OC43 virus

[0140] The total protein of the brain tissue was extracted by mechanical grinding, and then SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the protein was subsequently transferred to a PVDF membrane. After electrotransfer, the PVDF membrane was immersed in a 5% skim milk powder blocking solution and slowly shaken at room temperature for 1 hour. According to the position indicated by the protein molecular weight standard, the membrane was cut according to the molecular weight of the target protein, and the corresponding primary antibody (internal reference GAPDH: 1:1000 dilution (TA-08, Zhongshan Jinqiao Company), coronavirus N protein: 1:5000 dilution (40643-T62, Yiqiao Shenzhou Company) was added and shaken overnight at 4°C. Rinse with TBST. Add 1:5000 secondary antibody and incubate at room temperature for 1 hour. After rinsing, expose and image.

[0141] The test results show that ( Figure 3 ):

[0142] (1) Compared with the virus control group, Omicin F can significantly inhibit the content of HCoV-OC43 virus N protein in brain tissue in a dose-dependent manner (30 mg / kg, P < 0.05; 50 mg / kg, P < 0.01), which is consistent with the experimental results at the RNA level.

[0143] (2) The viral N protein content in the brain tissue of the Aumikexin F 50 mg / kg group was reduced by an average of 90%.

[0144] Example 8 Effects of Nasal Administration of Aomixin F on Body Weight and Survival Rate of Mice Infected with Influenza A Virus

[0145] Preparation of Omicron F solution: Dissolve Omicron F in sterile ultrapure water to prepare working solutions with concentrations of 9.6 mg / mL and 48 mg / mL, corresponding to dosing concentrations of 12 mg / kg and 60 mg / kg, respectively.

[0146] The experimental animals were 2-week-old specific pathogen-free (SPF) KM mice with an average weight of 15.3 g. They were randomly divided into 5 groups (n=10). All animals were housed in an animal biosafety level 2 laboratory (ABSL-2). Except for the normal group, the mice were anesthetized with isoflurane and intranasally administered with 20 μL of influenza A virus H1N1 (PR8) (3LD 50 ) infection, and the body weight and survival of the mice were recorded for 14 consecutive days. The experimental animals were divided into groups and administered as follows (average body weight of mice in each treatment group: virus control group, 15.3g; normal control group, 15.7g; omixin F 12mg / kg group, 15.5g; omixin F 60mg / kg group, 15.5g; oseltamivir phosphate control group, 14.5g):

[0147] (1) Virus control group: 20 μL virus + 20 μL sterile ultrapure water were administered intranasally;

[0148] (2) Normal control group: 40 μL of sterile ultrapure water was administered intranasally;

[0149] (3) Omicron F 12 mg / kg group: 20 μL virus + 20 μL of 9.6 mg / mL Omicron F were administered intranasally;

[0150] (4) Omicron F 60 mg / kg group: 20 μL of virus + 20 μL of 48 mg / mL Omicron F were administered intranasally.

[0151] (5) Oseltamivir phosphate (Tamiflu) control group: 20 μL of virus was administered intranasally and 27.5 mg / kg of Tamiflu was administered orally, once a day.

[0152] 1. Effect of Omicin F on the body weight of mice infected with influenza A virus

[0153] The results show that ( Figure 4 ),

[0154] (1) The weight of mice in the virus control group did not increase after infection, but gradually decreased from the 5th day and then began to slowly recover from the 10th day.

[0155] (2) Omicron F can significantly alleviate the weight loss of mice caused by influenza A virus H1N1 infection. The performance of the Omicron F 12 mg / kg group and the oseltamivir phosphate (Tamiflu) control group in improving the weight of mice is basically the same. The weight of mice began to recover from the 8th day. By the 14th day, there was no difference in weight between the two groups, and both were significantly higher than the virus control group.

[0156] (3) In the Omicron F-60 mg / kg group, the weight gain was almost the same as that of the normal group, indicating that at this dose, Omicron F could basically eliminate the effects of the virus and the mice maintained normal growth.

[0157] 2. Protective effect of Omicin F on mice infected with influenza A virus

[0158] The results are as follows Figure 5 As shown,

[0159] (1) The survival rate of the virus control group was 60%;

[0160] (2) Compared with the virus control group, both the 12mg / kg and 60mg / kg groups of Omicin F significantly increased the survival rate of mice infected with influenza A virus H1N1. The survival rate of the 12mg / kg Omicin F group was 90%, the same as that of the control drug oseltamivir phosphate, and the mortality protection rate reached 75%; There was no death in the Aomixin F 60mg / kg group during the entire experimental period, and the survival rate 100％ .

[0161] Death protection rate = (mortality rate of virus control group - mortality rate of drug administration group) / mortality rate of virus control group × 100%.

[0162] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

Claims

1. A compound represented by formula (1): Omixin F, 2. A medicament or pharmaceutical composition comprising the Omicron F according to claim 1, characterized in that: The medicine or pharmaceutical composition comprises a therapeutically effective amount of Omicin F and necessary pharmaceutical excipients.

3. The drug or pharmaceutical composition according to claim 2, characterized in that The drug or pharmaceutical composition is a broad-spectrum antiviral drug. Preferably, the virus is a respiratory virus; most preferably, the virus is a coronavirus or influenza virus.

4. The use of the Omimaxin F according to claim 1 in the preparation of medicines, characterized in that: The drug is a broad-spectrum antiviral drug. Preferably, the virus is a respiratory virus; most preferably, the virus is a coronavirus or influenza virus.

5. The method for preparing the Omicron F according to claim 1, characterized in that: The method comprises the following steps: (1) Synthesis of (3-chlorophenyl)diphenylmethyl (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate from 2-chlorotrityl chloride resin in a solid phase reactor; (2) (3-chlorophenyl)benzhydryl (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate is reacted with piperidine to obtain (3-chlorophenyl)benzhydryl (2S)-2-amino-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate; (3) (3-chlorophenyl)benzhydryl (2S)-2-amino-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate is reacted with bis(4-nitrophenyl) carbonate and 2,6-lutidine to obtain (3-chlorophenyl)benzhydryl (2S)-2-{[(4-nitrophenoxy)carbonyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate; (4) (3-chlorophenyl)benzhydryl (2S)-2-{[(4-nitrophenoxy)carbonyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate, L-phenylalanine tert-butyl ester, 4-dimethylaminopyridine and N,N-diisopropylethylamine to give (3-chlorophenyl)benzhydryl (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}valerate; (5) (3-chlorophenyl) benzhydryl (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoate was deprotected to give (2S)-2-({[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]carbamoyl}amino)-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentanoic acid ( Compound 11b ); (6) Under nitrogen protection, Fmoc-Arg(Pbf)-OH was dissolved in ethylene glycol monomethyl ether, and then 2-iodobenzoic acid and N-methylmorpholine were added to react to obtain (4S)-4-{[((9H-fluoren-9-yl)methoxy)carbonyl]amino}-5-{[(2-methylpropyloxy)carbonyl]oxy}-5-oxo-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]pentane-1-guanidine; (7) (4S)-4-{[((9H-fluoren-9-yl)methoxy)carbonyl]amino}-5-{[(2-methylpropyloxy)carbonyl]oxy}-5-oxo-N′-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]pentane-1-guanidine (33.94 g, 45.32 mmol, 1 equivalent) was dissolved in ethylene glycol monomethyl ether, and sodium borohydride was added to react to obtain (9H-fluoren-9-yl)methyl N-[(2S)-1-hydroxy-5-{N′-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamate; (8) (9H-fluoren-9-yl)methyl N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamate and 1,8-diazabicyclo[5.4.0]undec-7-ene are reacted to obtain N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine; (9) reacting N-[(4S)-4-amino-5-hydroxypentyl]-N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]guanidine, N-(tert-butoxycarbonyl)-L-phenylalanine, 1-hydroxybenzotriazole, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine to obtain tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate; (10) Tert-butyl N-[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamate and hydrochloric acid were dissolved in 1,4-dioxane to react and obtain (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropanamide ( Combination Object 11a ); (11) (2S)-2-amino-N-[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]-3-phenylpropionamide ( Compound 11a ), (2S)-2-{[((2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl)carbamoyl]amino}-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarbamoylamino}pentanoic acid ( Compound 11b ), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine are reacted to obtain tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]iminocarboxamido}butyl]carbamoyl}amino}-3-phenylpropanoate; (12) Tert-butyl (2S)-2-{[((1S)-1-{[(1S)-1-{[(2S)-1-hydroxy-5-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}-4-{N'-[(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl]carbamoylamino}pentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl [(1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid is dissolved in trifluoroacetic acid, triisopropylsilane, water and 2-[2-(2-mercaptoethoxy)ethoxy]ethanethiol to give (2S)-2-{[(1S)-4-iminocarboxamido-1-{[(1S)-1-{[(2S)-5-iminocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid; (13) (2S)-2-{[((1S)-4-imidocarboxamido-1-{[(1S)-1-{[(2S)-5-imidocarboxamido-1-hydroxypentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid and DESS-MARTIN reagent were dissolved in dimethyl sulfoxide and reacted to obtain (2S)-2-{[((1S)-4-imidocarboxamido-1-{[(1S)-1-{[(2S)-5-imidocarboxamido-1-oxopentan-2-yl]carbamoyl}-2-phenylethyl]carbamoyl}butyl]carbamoyl}amino}-3-phenylpropionic acid trifluoroacetate ( Omichin F ).

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