Application of Compound ATV014 in Anti-SARS-CoV-2 Infection

Abstract

Use of compound ATV014 or its pharmaceutically acceptable salt or its crystalline hydrate or its solvate in the preparation of a product for preventing, alleviating or treating infection with the Omicron strain of the novel coronavirus, or the replication or propagation of its homologous variant virus and the cytopathic effect produced thereby. It relates to the technical fields of pharmacy and viral infectious diseases. The structural formula of compound ATV014 is as follows:

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis and relates to the fields of pharmaceutical technology and viral infectious diseases technology. Specifically, it relates to a nucleoside derivative, its prodrug and / or its pharmaceutically acceptable salt, and their compositions and uses. Background Art

[0002] Currently, worldwide, in addition to vaccines, anti-SARS-CoV-2 drugs are effective means to reduce severe cases and mortality. Experts unanimously believe that treating with antiviral drugs within five days after infection to reduce the viral load is the best treatment window and the optimal treatment plan, which can avoid the aggravation of the condition caused by missing the treatment window.

[0003] Currently, there are three anti-SARS-CoV-2 drugs approved abroad: Remdesivir from Gilead, Paxlovid from Pfizer, and Molnupiravir from Merck. Remdesivir is currently an effective drug approved by the US FDA for use in children and for mild, moderate, and severe cases. In severe cases, only Remdesivir is recommended among the above three drugs. An important defect of Remdesivir is that its injection administration method must be carried out in the hospital.

[0004] Through the applicant's previous research on Remdesivir and its precursor compound GS-441524 (Li, et al, J. Med. Chem. 2020), it was found that GS-441524 showed better antiviral effects than Remdesivir in in vivo activity tests in mice. Although the compound GS-441524 has a similar mechanism of action to Remdesivir, it shows better safety. Therefore, the applicant has applied for a patent for the application of the compound GS-441524 in the prevention, alleviation, and / or treatment of SARS-CoV-2 (application number or patent number 202011000517.2). Later, through pharmacokinetic analysis of GS-441524 by the applicant, it was found that its oral bioavailability is very low and it can only be used in the form of an injection. Seeking research on orally available low-toxic nucleoside derivatives or prodrugs of GS-441524 will be of great significance.

[0005] Based on Remdesivir and GS-441524, the applicant carried out structural optimization and upgrading adjustments, developed a series of nucleoside compounds that can effectively reduce the liver and kidney toxicity caused by Remdesivir and can be taken orally, and applied for a related patent on a nucleoside compound for treating viral infections and its uses (application number 2021110837309). A series of compounds including the compound ATV014 have all shown activity against inhibiting SARS-CoV-2. Summary of the Invention General Overview of the Invention

[0007] The object of the present invention is to provide the use of a nucleoside derivative of the ATV014 structure or a pharmaceutically acceptable salt thereof.

[0008] Specifically, the use of compound ATV014 or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof in the preparation of a product for preventing, alleviating or treating coronavirus infection, or the replication or reproduction of its homologous variant virus and the cytopathic effect produced thereby.

[0009] To provide a method for preventing, alleviating or treating a disease related to the cytopathic effect caused by the infection of the novel coronavirus Omicron strain or the replication or reproduction of its homologous variant virus, comprising the step of administering a safe and effective amount of compound ATV014 or a pharmaceutically acceptable salt thereof to a subject in need.

[0010] To provide the use of compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing, alleviating or treating a disease related to the cytopathic effect caused by the infection of the novel coronavirus Omicron strain or the replication or reproduction of its homologous variant virus.

[0011] To provide the use of compound ATV014 or a pharmaceutically acceptable salt thereof for preventing, alleviating or treating a disease related to the cytopathic effect caused by the infection of the novel coronavirus Omicron strain or the replication or reproduction of its homologous variant virus.

[0012] To provide a pharmaceutical composition, the first active ingredient of which is compound ATV014 or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof.

[0013] To provide an active ingredient or a preparation containing the active ingredient, the active ingredient being compound ATV014 or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof.

[0014] The compound ATV014 has the following structural formula:

[0015] Detailed description of the invention

[0017] To achieve one of the above objects, the present invention adopts the following technical solutions:

[0018] The present invention provides the use of compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of a product for alleviating or treating coronavirus infection, or the replication or reproduction of its homologous variant virus and the cytopathic effect produced thereby.

[0019] Optionally, the novel coronavirus Omicron strain includes the Omicron original strain (B.1.1.529) and Omicron mutant strains.

[0020] Optionally, the Omicron mutant strains include BA.1, BA.2, BA.3, BA.4, BA.5 lineages, and future Omicron mutant strains that emerge.

[0021] Optionally, the infections caused by the Omicron strain include fever, headache, cough, sore throat, muscle soreness, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, and sepsis.

[0022] Optionally, the compound or its pharmaceutically acceptable salt can be applicable to humans or animals.

[0023] The animals may include bovines, equines, ovines, porcines, canines, felines, rodents, primates, avians, and fish.

[0024] Beneficial effects

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] (1) The ATV014 or its pharmaceutically acceptable salt of the present invention can effectively inhibit the replication and / or propagation of coronaviruses in cells, greatly enhancing the antiviral activity against SARS-CoV-2 and its viral variants. Among them, the inhibitory activities against the Delta strain and the Omicron strain are 17.8 and 103 times higher than those of remdesivir respectively, and both show better antiviral effects than other tested compounds.

[0027] (2) The compound ATV014 has a better inhibitory effect on the Omicron strain than other variants including the alpha, beta, and delta strains. The inhibitory activity of ATV014 against Omicron reached 13 nM on the BA.1 strain, 34 nM on the BA.5 strain, 139 nM on the XBB strain, and 93 nM on the EG.5.1 strain, all of which are superior to GS-441524 or remdesivir. This indicates that the compound ATV014 can effectively inhibit the replication and / or propagation of the Omicron strain of the novel coronavirus in cells, and the Omicron strain is more sensitive to ATV014 than other strains or compounds.

[0028] (3) The compound ATV014 has good pharmacokinetic properties, and its bioavailability in rats is as high as 49%.

[0029] (4) The structure of the ATV014 or its pharmaceutically acceptable salt of the present invention is simple, easy to synthesize, and conducive to production and distribution.

[0030] (5) The method for preparing ATV014 or a pharmaceutically acceptable salt thereof according to the present invention is simple in operation and is conducive to industrial production.

[0031] Term Definitions

[0032] Unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings:

[0033] "V / V" represents a volume ratio. EC 50 Represents the median effective concentration.

[0034] "Room temperature" in the present invention refers to the ambient temperature, with the temperature ranging from approximately 10°C to approximately 40°C. In some embodiments, "room temperature" refers to a temperature ranging from approximately 20°C to approximately 30°C; in other embodiments, "room temperature" refers to a temperature ranging from approximately 25°C to approximately 30°C; in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0035] The term "treatment" as used herein, unless otherwise indicated, means reversing, alleviating the disorder or disease to which the term applies or one or more symptoms of such a disorder or disease, inhibiting the progression of the disorder or disease or one or more of its symptoms, or preventing the disorder or disease or one or more of its symptoms. The term "treatment" as used herein refers to a treatment act as "treatment" was just defined above.

[0036] The compounds described in the present invention also include references to their physiologically acceptable salts, examples including salts derived from suitable bases, such bases as alkali metals or alkaline earth metals (e.g., Na + 、Li + 、K + 、Ca +2 and Mg +2 ), ammonium, and NR4 +(wherein R is as defined herein). Physiologically acceptable salts of the nitrogen atom or amino group include: (a) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; (b) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine, etc.; and (c) salts formed with elemental anions such as chlorine, bromine, and iodine. Physiologically acceptable salts of the hydroxy compound include combinations of the anion of the compound with suitable cations such as Na + and NR4 + .

[0037] For therapeutic use, salts of the active ingredient of the compounds of the present invention are physiologically acceptable, i.e., they are salts derived from physiologically acceptable acids or bases. However, salts that are not physiologically acceptable acids or bases can also be used, for example, in the preparation or purification of physiologically acceptable compounds. All salts, whether derived from physiologically acceptable acids or bases or not, are within the scope of the present invention.

[0038] Method for detecting the activity against the novel coronavirus

[0039] Another aspect of the present invention relates to a method for detecting the activity against the novel coronavirus, which includes the step of treating a sample suspected of containing the Coronaviridae family with the compounds described in the present invention.

[0040] The compounds of the present invention can be used as anti-SARS-CoV-2 compounds, as intermediates for such compounds or for other uses as described below. The anti-SARS-CoV-2 compounds will bind to positions on a surface or in a cavity having a geometry unique to SARS-CoV-2. The compounds that bind to anti-SARS-CoV-2 can bind with different degrees of reversibility. Those compounds that bind substantially irreversibly are ideal candidates for the method of the present invention. Once labeled, those compositions that bind substantially irreversibly can be used as probes for detecting SARS-CoV-2. Accordingly, the present invention relates to a method for detecting SARS-CoV-2 in a sample suspected of containing SARS-CoV-2, which comprises the steps of: treating the sample suspected of containing SARS-CoV-2 with a composition comprising a compound of the present invention bound to a label; and observing the effect of the sample on the activity of the label. Suitable labels are well known in the field of diagnostics and include stable free radicals, fluorophores, radioisotopes, enzymes, chemiluminescent groups and chromogens. The compounds herein are labeled in a conventional manner using functional groups such as hydroxyl, carboxyl, mercapto or amino groups.

[0041] In the context of the present invention, samples suspected of containing SARS-CoV-2 include natural or artificial materials such as living organisms; tissue or cell cultures; biological samples such as biomaterial samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.); laboratory samples; food, water or air samples; biological product samples such as cell extracts, especially recombinant cell extracts that synthesize the required glycoproteins, etc. Typically, the sample will be suspected of containing an organism that produces SARS-CoV-2, often a pathogenic organism such as the family Coronaviridae. The sample can be contained in any medium, including water and organic solvent / water mixtures. The sample includes living organisms such as humans and artificial materials such as cell cultures.

[0042] The treatment step of the present invention includes adding the composition of the present invention to the sample, or it includes adding a precursor of the composition to the sample. The addition step includes any of the administration methods described above.

[0043] If desired, the activity of SARS-CoV-2 after administration of the composition can be observed by any method, including direct and indirect methods for detecting anti-SARS-CoV-2 activity. Quantitative, qualitative and semi-quantitative methods for detecting SARS-CoV-2 activity are all contemplated. Typically, one of the above screening methods is applied, however, any other method can also be applied, such as observing the physiological performance of a living organism.

[0044] Screening of Compositions with Anti-SARS-CoV-2 Activity

[0045] The compounds of the present invention are suitable for treating or preventing infections by the family Coronaviridae in animals or humans. However, in the process of screening for compounds that can inhibit human coronaviruses, cell-based assays should be the primary screening tool.

[0046] Screening of the compositions of the present invention for compounds having anti-SARS-CoV-2 activity by any conventional technique for evaluating antiviral activity. In the context of the present invention, typically, compositions having anti-SARS-CoV-2 activity are first screened, and then the in vivo activity of the compositions showing antiviral activity is screened. Compositions having an in vitro Ki (inhibition constant) of less than about 5×10 -6 M and preferably less than about 1×10 -7 M are preferably used in vivo. Useful in vitro screening has been described in detail in the literature and will not be repeated here. However, the Examples describe suitable in vitro assays.

[0047] Pharmaceutical formulations

[0048] The compounds of the present invention are formulated with conventional carriers and excipients, which will be selected according to conventional practice. Although the active ingredients can be administered alone, they are preferably made into pharmaceutical formulations. The formulations of the present invention, whether for veterinary or human applications, comprise at least one active ingredient as defined above and one or more acceptable carriers therefor, and optionally comprise other therapeutic ingredients, especially those additional therapeutic ingredients as disclosed herein. The carrier must be "acceptable" in the sense that it is compatible with the other components in the formulation and is physiologically harmless to its recipient.

[0049] The formulations include those suitable for the abovementioned routes of administration. The formulations can be conveniently made into unit dosage forms and can be made by any method well known in the pharmaceutical art. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA.). Such methods include the step of mixing the active ingredient with a carrier which constitutes one or more accessory components. Generally, the formulations are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then shaping the product if necessary.

[0050] The present invention further provides veterinary compositions which comprise at least one active ingredient as defined above and a veterinary carrier therefor.

[0051] The veterinary carrier is a substance for veterinary composition purposes and can be a solid, liquid or gaseous substance, and further it is inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally or by any other desired route.

[0052] Route of administration

[0053] One or more compounds of the invention (referred to herein as the active ingredient) are administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), among others. It is to be understood that the preferred route may vary, for example, with the condition of the recipient. The advantage of the compounds of the invention is that they are orally bioavailable and can be administered orally.

[0054] Metabolites of the compounds of the invention:

[0055] In vivo metabolites of the compounds described herein also fall within the scope of the invention to the extent that such products are novel and non-obvious over the prior art. These products can result from, for example, oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, mainly due to enzymatic processes. Accordingly, the invention includes novel and non-obvious compounds produced by a method comprising contacting a compound of the invention with a mammal for a period of time sufficient to produce its metabolites. Such products are typically identified as follows: Prepare a radiolabeled (e.g., 14 C or 3 H) compound of the invention, administer it parenterally to an animal, such as a rat, mouse, guinea pig, monkey, or human, at a detectable dose (e.g., greater than about 0.5 mg / kg), allow sufficient time for metabolism to occur (typically, about 30 seconds to 30 hours), and isolate its transformation products from urine, blood, or other biological samples. Because they are labeled, these products are readily isolated (others are isolated using antibodies that can bind to epitopes remaining in the metabolite). The structure of the metabolite is determined in a conventional manner, e.g., by MS or NMR analysis. In general, the analysis of metabolites is carried out in the same manner as conventional drug metabolism studies known to those skilled in the art. Transformation products, provided they are not otherwise found in vivo, can be used in diagnostic assays for the therapeutic administration of the compounds of the invention even if they do not themselves have SARS-CoV-2 polymerase inhibitory activity.

[0056] Formulations and methods for determining the stability of a compound in surrogate gastrointestinal secretions are known. A compound is defined herein as being stable in the gastrointestinal tract where less than about 50 mole percent of the protected groups are deprotected in a surrogate of intestinal or gastric juice after incubation at 37 °C for 1 hour. Just because a compound is stable to the gastrointestinal tract does not mean that it will not hydrolyze in vivo. Prodrugs of the invention are typically stable in the digestive system, but they are generally hydrolyzed substantially to the parent drug in the digestive lumen, liver, or other metabolic organs or intracellularly.

[0057] It should be noted that the specific dosage and usage of the compound with the structure of Formula I, its prodrug and / or its pharmaceutically acceptable salts for different patients are determined by many factors, including the patient's age, weight, gender, natural health status, nutritional status, activity strength of the drug, taking time, metabolic rate, severity of the disease and the subjective judgment of the treating physician. The effective dose of the active ingredient depends at least on the nature of the disease to be treated, toxicity (whether the compound is used prophylactically or against active virus infection), delivery method and pharmaceutical formulation, and will be determined by clinicians using conventional dose escalation studies. It is expected that the dose will be about 0.0001 to about 100 mg / kg body weight per day; typically, about 0.01 to about 10 mg / kg body weight per day; more typically, about 0.01 to about 5 mg / kg body weight per day; most typically, about 0.05 to about 0.5 mg / kg body weight per day. For example, for an adult with a body weight of about 70 kg, the daily candidate dose will be in the range of 1 mg to 1000 mg, preferably 5 mg to 500 mg, and can be in the form of a single dose or multiple doses.

[0058] The drugs of the above various dosage forms can all be prepared according to the conventional methods in the pharmaceutical field.

[0059] When describing the experimental details, certain abbreviations and acronyms were used. Although most of them can be understood by those skilled in the art, the following table contains a list 1 of these abbreviations and acronyms.

[0060] Table 1

[0061] Abbreviation Meaning DCC Dicyclohexylcarbodiimide DCM Dichloromethane DMAP 4-Dimethylaminopyridine EA Ethyl acetate HCl Hydrochloric acid <![CDATA[H2SO4]]> Sulfuric acid rt Room temperature SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 THF Tetrahydrofuran TLC Thin layer chromatography Description of the Drawings

[0062] Figure 1 Shows the inhibitory effects of compounds ATV006 and ATV014 in Example 2 on the SARS-CoV-2 Omicron mutant strains BA.1 and BA.5 variants respectively in Vero-E6 cells. Among them, the horizontal axis is the drug concentration in μM; the vertical axis is the inhibition rate in %.

[0063] Figure 2 Shows the plasma concentration-time curve of ATV014 in rats in Example 3. Among them, the horizontal axis is time in hours, and the vertical axis is the drug concentration in plasma in μg / L.

[0064] Figure 3 Shows the inhibitory activities of ATV014, GS-441524 and Remdesivir against the Omicron XBB mutant strain on Vero cells in Example 4. Among them, the horizontal axis is the drug concentration in μM; the vertical axis is the inhibition rate in %.

[0065] Figure 4In Example 5, the inhibitory activities of ATV014 and GS-441524 against the Omicron EG.5.1 mutant strain on Vero cells are shown. Here, the horizontal axis represents the drug concentration in μM, and the vertical axis represents the inhibition rate in %. Detailed implementation manners

[0066] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting examples are further disclosed below for further detailed description of the present invention.

[0067] The reagents used in the present invention can all be purchased from the market or can be prepared by the methods described in the present invention.

[0068] In the present invention, μM represents micromoles per liter; mmol represents millimoles; and equiv represents equivalents.

[0069] Example 1. Synthesis of (((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl)cyclohexanecarboxylate (Compound ATV014)

[0070]

[0071] In a 500 mL reactor, install a stirrer, a thermometer, and a constant-pressure dropping funnel. Add GS-441524 (10 g, 0.03 mol), add magnesium sulfate-dried acetone (300 mL), then add 2,2-dimethoxypropane (17 g, 0.16 mol). At room temperature, slowly add concentrated sulfuric acid (2.4 mL, 0.04 mol) dropwise to the system. After 5 minutes, the addition is complete and the solid starts to dissolve. Heat the reaction mixture to 45 °C and continue the reaction for 4 h. Monitor the reaction by HPLC until it is complete (OD-3 column, mobile phase: n-hexane / isopropanol = 80:20, flow rate: 0.8 mL / min, injection volume: 1 μL). Stop the reaction. After cooling in an ice bath, add solid NaHCO3 (10 g) and water (30 mL) to the reaction solution, and continue to adjust the pH to 7 - 8 with sodium bicarbonate. Remove the solvent under reduced pressure. Dilute the residue with ethyl acetate (300 mL). Wash the ethyl acetate layer with water (80 mL) and saturated brine (80 mL) respectively, and dry over anhydrous sodium sulfate. Filter by suction. Distill the filtrate under reduced pressure until about 100 mL of solvent remains. Slowly pour the residue into ice-cooled petroleum ether and stir vigorously to precipitate a large amount of white solid. Filter by suction to obtain 10.5 g of white solid Compound 1, with a yield of 91%.

[0072] Dissolve 15.0 g of Compound 1 in 15 ml of dichloromethane, then add cyclohexanecarboxylic acid and 554.0 mg of 4-dimethylaminopyridine. After stirring for 10 min, add 10.2 g of dicyclohexylcarbodiimide and stir at room temperature for 24 h. After separation by column chromatography (eluent: petroleum ether / ethyl acetate (V / V) = 1 / 1), Compound 2 (white solid) is obtained. Dissolve Compound 2 in 30 mL of 37% hydrochloric acid aqueous solution and 150 mL of tetrahydrofuran. After stirring for 6 h, add sodium carbonate to adjust the pH to 8, and rotary evaporate to remove the organic solvent. After separation by column chromatography (eluent: petroleum ether / ethyl acetate (V / V) = 1 / 3), 2.8 g of ATV014 (free base crystal form I, yield 49%) is obtained, and the two-step yield is 45.8%. Take the obtained Compound ATV014 to detect the hydrogen spectrum and carbon spectrum, and the results are as follows:

[0073] Hydrogen spectrum: 1 H NMR(600MHz,DMSO-d6)δ(ppm):7.92(s,1H),7.86(br,1H),6.92(d,J=4.5Hz,1H),6.81(d,J=4.5Hz,1H),6.33(d,J=5.9Hz,1H),5.38(d,J=5.9Hz,1H),4.70(t,J=5.3Hz,1H),4.32-4.29(dd,J=12.2Hz,2.6Hz,1H),4.24-4.21(m,1H),4.16-4.13(dd,J=12.3Hz,4.8Hz,1H),3.98-3.95(q,J=5.9Hz,1H),2.26-2.22(m,1H),1.75-1.72(m,2H),1.64-1.56(m,3H),1.30-1.12(m,5H).

[0074] Carbon spectrum: 13 C NMR(150MHz,DMSO-d6)δ(ppm):175.34,156.06,148.4,124.0,117.4,117.0,110.7,101.2,81.7,79.4,74.5,70.6,63.0,42.6,29.0,28.9,25.7,25.2,25.1.

[0075] Example 2: Inhibitory effect of the compound on SARS-CoV-2 in Vero-E6 cells

[0076] The SARS-CoV-2 mutant strain B.1 is the hCoV-19 / CHN / SYSU-IHV / 2020 strain, and its Accession ID on GISAID is: EPI_ISL_444969; it was isolated from the sputum specimen of a woman. The SARS CoV-2 β (B.1.351, SARS_CoV-2_human_CHN_20SF18530_2020, Accession ID: GWH:WHBDSE01000000) and Delta (B.1.617.2, GDPCC 2.00096) variants were isolated from hospitalized COVID-19 patients. Two SARS-CoV-2 Omicron variants (BA.1 and BA.5) were isolated from hospitalized COVID-19 patients.

[0077] The compounds remdesivir, GS-441524, ATV006, and ATV014 were taken as test compounds and the operations were carried out according to the following steps:

[0078] Vero-E6 cells were inoculated into 48-well plates. When the cell density was about 70 - 80%, the supernatant was discarded and replaced with fresh DMEM medium, and then each compound was added to the medium respectively to make the final concentration of the compound 50 μM, 10 μM, 5 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, or 0.01 μM. The cells were infected with five SARS-CoV-2 mutant strains at a multiplicity of infection (MOI) of 0.05: three early mutant strains (B.1, B.1.351, and B.1.617.2), and two Omicron mutant strains BA.1 and BA.5. The antiviral activity was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) to quantify the viral copy number in the supernatant 48 hours after infection. We calculated the inhibitory effect of the test drugs at different concentrations on virus replication and calculated the EC 50 . This experiment consisted of three independent repeated experiments, and each experiment had 3 replicates. The EC of different compounds against different SARS-CoV-2 variants in Vero-E6 cells 50 See Table 2 and Figure 1 .

[0079] Table 2: EC of different compounds against different SARS-CoV-2 variants in Vero-E6 cells 50

[0080]

[0081] Compared with the control drug Remdesivir, ATV014 has greatly improved antiviral activity against SARS-CoV-2 and its viral variants. Among them, the inhibitory activities against the Delta strain and the Omicron strain are 17.8 and 103 times higher than those of Remdesivir respectively, showing better antiviral effects than other tested compounds. The inhibitory activity of ATV014 against Omicron reached 13 nM on the BA.1 strain and 34 nM on the BA.5 strain.

[0082] Example 3: Metabolism of Compound ATV014 and GS-441524 in Rats

[0083] 1. Dosage and administration method for each group:

[0084] Intravenous injection group of ATV014: Intravenous injection of 5 mg of ATV014 per kg of mouse body weight.

[0085] Oral administration group of ATV014: Gavage of 25 mg of ATV014 per kg of mouse body weight.

[0086] Intravenous injection group of GS-441524: Intravenous injection of 5 mg of GS-441524 per kg of mouse body weight.

[0087] Oral administration group of GS-441524: Gavage of 25 mg of GS-441524 per kg of mouse body weight.

[0088] 2. Operation:

[0089] Sixteen male SD rats weighing 220 g to 250 g were divided into 4 groups, namely the ATV014 intravenous injection group, the ATV014 oral administration group, the GS-441524 intravenous injection group, and the GS-441524 oral administration group, with 4 rats in each group (3 rats in each ATV014 group), and they were administered according to the description in "1. Dosage and administration method of each group". Blood was collected from the jugular vein. Approximately 0.3 mL of blood was collected into heparin tubes at 0.083 h (not collected for the oral group), 0.16 h (not collected for the oral group), 0.25 h, 0.5 h, 1 h (not collected for the intravenous injection group), 2 h, 4 h, 8 h, 24 h, and 48 h after administration, centrifuged at 4000 r / min for 10 min at 4 °C, and the upper plasma was transferred and stored frozen (about -20 °C) temporarily until determination. Take 50 μL of plasma sample, add 100 μL of 90% methanol aqueous solution, and vortex mix; then add 350 μL of methanol-acetonitrile mixed solution (1:1, V / V), and vortex mix; centrifuge at 10000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane before injection for detection; the blood samples within 0.5 hour after intravenous administration and 4 hours after oral administration were diluted 10-fold before injection for detection. The drug concentration in each sample was determined by high performance liquid chromatography (HPLC) / mass spectrometry (MS). The Waters UPLC / XEVO TQ-S chromatographic column and the InertSustain AQ-C18HP column (3.0 mm × 50 mm, 3.0 μm, GL) were used to separate the analytes. The DAS (Drug and Statistics) 3.0 software was used to calculate the pharmacokinetic parameters.

[0090] Results: See Table 3, Table 4 and Figure 2 .

[0091] Table 3: Pharmacokinetic parameters of SD rats after administration of ATV014 (detecting GS-441524, mean ± standard deviation, n = 3)

[0092]

[0093] Table 4: Pharmacokinetic parameters of SD rats after administration of GS-441524 (mean ± standard deviation, n = 4)

[0094]

[0095] Conclusion:

[0096] From Table 3, Table 4 and Figure 2 it can be seen that the oral bioavailability of ATV014 is 49.08%, and the oral bioavailability of GS-441524 is 22.63%. It shows that compared with GS-441524, the oral bioavailability of ATV014 is significantly improved, and it has better oral druggability.

[0097] Example 4: In vitro inhibitory effect of compound ATV014 on SARS-CoV-2 Omicron XBB mutant strain in Vero-E6 cells

[0098] African green monkey kidney cells (Vero) were purchased from ATCC, catalog number CCL-81. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin. DMEM medium supplemented with 2% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin was used as the test medium.

[0099] Virus strain: Isolated SARS-CoV-2 XBB.1.16 mutant strain, strain name: hCoV-19 / Guangdong / SZTH-070 / 2023.

[0100] Compound remdesivir, GS-441524, and ATV014 were taken as test compounds respectively, and the operations were carried out according to the following steps:

[0101] 1) Drugs used: Control drug remdesivir (RDV) and test drugs: GS-441524 (SHEN26-69-0), ATV014, and the stock solution concentration was 10 mM.

[0102] 2) Prepare Vero cells: 2 24-well plates, about 105 cells per well.

[0103] 3) Drug dilution: Dilution of samples was carried out in 2 mL EP tubes, and the diluent was 2% DMEM, with the following concentrations: 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064 μM.

[0104] 4) Add the corresponding volume of virus stock solution according to MOI equal to 0.05 to each EP tube containing different drug concentrations.

[0105] 5) Cell preparation: After culturing for about 24 hours, discard the medium and wash the cells once with 1× sterile PBS;

[0106] 6) Take 250 μL of the diluted virus and drug mixture and add it to the washed 24-well cell plate, with 3 replicates. The virus, drug, and cells were co-incubated at 37 °C for 1 hour. At the same time, set up blank control wells (without virus and drug) and virus control wells (only with virus).

[0107] 7) Maintenance solution preparation: Dilution of samples was carried out in 2 mL EP tubes, and the diluent was 2% DMEM, with the same concentration as before.

[0108] 8) After incubation for 1 hour, aspirate the supernatant, wash twice with PBS, then add the maintenance medium containing the corresponding concentration of the drug, and culture at 37 °C for 48 hours.

[0109] 9) Take the supernatant to extract RNA, detect the copy number of SARS-CoV-2 RNA, and calculate the inhibition rate. The antiviral activity calculation formula for the sample is as follows:

[0110] Inhibition rate (%) = (Average copy number of virus control - Average copy number after drug treatment) / (Average copy number of virus control) × 100

[0111] Use GraphPad Prism (version 8) to perform non-linear fitting analysis on the inhibition rate and cell viability of the sample, and calculate the half-maximal effective concentration (EC 50 ) value. The fitting formula is: log(inhibitor) vs. response--Variable slope.

[0112] Conclusion: The experimental results show that the EC 50 values of ATV014 and SHEN26-69-0 against the tested SARS-CoV-2 Omicron XBB.1.16 mutant strain are 139 nM and 778 nM respectively, and the EC 50 value of the control drug remdesivir (RDV) against the tested SARS-CoV-2 Omicron XBB.1.16 mutant strain is 2.336 μM ( Figure 3 ). Among the Omicron XBB variant strains, the antiviral activity of ATV014 is better than that of GS-441524 and remdesivir.

[0113] Example 5. In vitro inhibitory effect of compound ATV014 on SARS-CoV-2 Omicron EG.5.1 mutant strain in Vero-E6 cells

[0114] Vero-E6 cells:

[0115] Culture with DMEM high-glucose complete medium containing 10% fetal bovine serum. Passage the cells once 1 day before the experiment to make the cells in the logarithmic growth phase. The cells after adding the virus and the sample are maintained and cultured with DMEM high-glucose complete medium containing 3% fetal bovine serum.

[0116] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): EG.5.1, isolated, cultured and preserved by the Biosafety Level 3 Laboratory of Kunming Institute of Zoology, Chinese Academy of Sciences.

[0117] Respectively take compounds GS-441524 and ATV014 as the test compounds, and operate according to the following steps:

[0118] Vero E6 cells were inoculated into culture plates in advance and cultured overnight at 37°C with 5% CO2. When the monolayer cells grew to about 80%, they were ready for use. In a P3 laboratory, different concentrations of the drug to be tested were added to each well. The samples were set with 8 concentration gradients (200, 66.70, 22.20, 7.40, 2.50, 0.80, 0.30, 0.10 μM) and an equal volume of virus-diluted supernatant (MOI = 0.01). There were 3 replicate wells for each concentration gradient. At the same time, a solvent control, a negative control without drug and virus, and a positive control were set. After infection in an incubator at 37°C with 5% CO2 for 1 h, the virus-drug mixed medium was aspirated, and after washing with 1×PBS, it was replaced with a medium containing only the drug to be tested and continued to be cultured. 8 concentration gradients were set, with 3 replicate wells for each gradient. At the same time, a positive control, a solvent control, and a negative control were set. After culturing at 37°C with 5% CO2 for 48 h, the cell supernatant was collected, and the viral RNA was extracted for Real-time PCR virus quantification to calculate the inhibition rate of the drug on virus replication and the EC 50 value.

[0119] Conclusion: The experimental results showed that ATV014 showed good inhibitory effects on the SARS-CoV-2 variant EG.5.1, with an EC 50 = 0.093 μM ( Figure 4 ), and GS-441524 also had good inhibitory effects on the variant EG.5.1, with an EC 50 = 0.73 μM. Against the Omicron EG.5.1 variant, the antiviral activity of ATV014 was better than that of GS-441524.

[0120] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications described herein within the content, spirit, and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and alterations are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. Use of compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, alleviating or treating infection with the Omicron strain of the novel coronavirus; the Omicron strain of the novel coronavirus is an Omicron mutant strain; the Omicron mutant strain is of the BA.1 or BA.5 lineage; the compound ATV014 has the following structure: 。 2. The use according to claim 1, wherein The infection is caused by the Omicron strain and includes fever, headache, cough, sore throat, muscle soreness, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, and sepsis.

3. The use according to claim 1, characterized in that, The compound ATV014 or a pharmaceutically acceptable salt thereof is applicable to humans.

4. The use according to claim 1, characterized in that, The compound ATV014 or a pharmaceutically acceptable salt thereof is applicable to animals.

5. The use according to claim 4, characterized in that, The animals include bovines, equines, ovines, porcines, canines, felines, rodents, primates, avians or fish.

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