TWI899785B - Application of compound atv014 in anti-coronavirus infection - Google Patents

Abstract

Compound ATV014 or its pharmaceutically acceptable salt or its crystalline hydrate or its solvate is prepared for use in preventing, alleviating or treating infection by the novel coronavirus Omicron strain, or the replication or propagation of its homologous variant virus and its cytopathic effects, involving the fields of pharmaceutical technology and viral infection disease technology. The structural formula of compound ATV014 is as follows:

Description

Application of compound ATV014 in anti-new coronavirus infection

The present invention belongs to the field of drug synthesis, and relates to pharmaceutical technology and viral infection disease technology. Specifically, it relates to a nucleoside derivative, its prodrug and/or its pharmaceutically acceptable salt, and its composition and use.

Globally, Omicron has caused over 1.1 million deaths in 2022. Currently, in addition to vaccines, anti-COVID-19 medications are an effective means of reducing severe illness and mortality worldwide. Experts agree that treatment with antiviral drugs within five days of infection to reduce viral load represents the optimal treatment window and the most effective approach, avoiding missed treatment windows and the resulting worsening of the disease.

Currently, three COVID-19 drugs have been approved internationally: Gilead's remdesivir, Pfizer's pasirovix, and Merck's monapivir. Remdesivir is currently FDA-approved for use in children and for both mild, moderate, and severe cases. Of the three, only remdesivir is recommended for use in severe cases. A significant drawback of remdesivir is that it must be administered by injection in a hospital, which can easily strain hospital medical resources.

The applicant's previous research on remdesivir and its precursor compound, GS-441524 (Li et al ., J. Med. Chem. 2020), revealed that GS-441524 exhibited superior antiviral activity compared to remdesivir in mouse in vivo activity tests. While its mechanism of action is similar to that of remdesivir, GS-441524 exhibits a better safety profile. Consequently, the applicant has applied for a patent (Application No. 202011000517.2) describing the use of GS-441524 as a drug for the prevention, alleviation, and/or treatment of SARS-CoV-2. Subsequent pharmacokinetic analysis of GS-441524 revealed that its oral bioavailability is very low, requiring its use only as an injectable solution. It would be of great significance to seek for orally available, low-toxicity nucleoside derivatives or prodrugs of GS-441524.

The applicant has optimized and upgraded the structure of remdesivir and GS-441524, developing a series of orally administered nucleoside compounds that effectively reduce the hepatotoxicity caused by remdesivir. The applicant has also applied for a related patent, "A nucleoside compound for the treatment of viral infections and its use" (Application No. CN 2021110837309). These compounds, including compound ATV014, have demonstrated activity against the novel coronavirus.

This application claims priority to Chinese patent application No. 202310011667.0 filed on January 5, 2023. The full text of the above-mentioned Chinese patent application is hereby incorporated by reference as a part of this application.

Invention Overview

The purpose of the present invention is to provide the use of nucleoside derivatives of the ATV014 structure or pharmaceutically acceptable salts thereof.

Specifically, the compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof is used to prepare a product for preventing, alleviating or treating coronavirus infection, or the replication or reproduction of its homologous variants and the resulting cytopathic effects.

Provided is a method for preventing, alleviating, or treating diseases caused by infection with the novel coronavirus Omicron strain, or the cytopathic effects resulting from the replication or propagation of its homologous variants, comprising the steps of administering a safe and effective amount of the compound ATV014 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

Provided is the use of the compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing, alleviating, or treating diseases caused by infection with the novel coronavirus Omicron strain, or the cytopathic effects resulting from the replication or propagation of its homologous variants.

Provided are uses of the compound ATV014 or a pharmaceutically acceptable salt thereof for preventing, alleviating, or treating diseases caused by infection with the novel coronavirus Omicron strain, or cytopathic effects resulting from the replication or propagation of its homologous variants.

Provided is a pharmaceutical composition, the first active ingredient of which is compound ATV014 or a pharmaceutically acceptable salt thereof, or a crystalline hydrate thereof, or a solvate thereof.

Provided is an active ingredient or a preparation containing the active ingredient, wherein the active ingredient is compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof.

The compound ATV014 has the following structural formula: .

Description of the invention

To achieve one of the above objectives, the present invention adopts the following technical solutions:

The present invention provides the use of the compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of a product for alleviating or treating coronavirus infection, or the replication or propagation of its homologous variants and the resulting cytopathic effects.

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

Optionally, the Omicron mutants include BA.1, BA.2, BA.3, BA.4, BA.5 lineages and Omicron mutants produced in the future.

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

Alternatively, the compound or a pharmaceutically acceptable salt thereof may be suitable for use in humans or animals.

The animals may include bovines, equines, ovines, porcines, canines, felines, rodents, primates, birds, and fish.

Beneficial effects

Compared with the existing technology, the present invention has the following technical effects:

(1) ATV014 or a pharmaceutically acceptable salt thereof described in the present invention can effectively inhibit the replication and/or reproduction of coronaviruses in cells, and greatly enhances the antiviral activity against SARS-CoV-2 and its viral variants. The inhibitory activity against the Delta strain and the Omicron strain is 17.8 and 103 times higher than that of remdesivir, respectively, showing a better antiviral effect than other tested compounds.

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

(3) The compound ATV014 has good pharmacokinetic properties, and the bioavailability of ATV014 in rats is as high as 49%.

(4) The ATV014 or its pharmaceutically acceptable salt described in the present invention has a simple structure, is easy to synthesize, and is conducive to production and distribution.

(5) The method for preparing ATV014 or a pharmaceutically acceptable salt thereof described in the present invention is simple to operate and is conducive to industrial production.

Definition of terms

Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

“V/V” indicates volume ratio. _EC50 indicates the concentration at which the drug has a 50% effective effect.

As used herein, "room temperature" refers to ambient temperature, which is from about 10°C to about 40°C. In some embodiments, "room temperature" refers to a temperature from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature from about 25°C to about 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.

As used herein, the term "treating," unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder, or one or more symptoms of, the condition or disorder to which such term applies, or one or more symptoms of such condition or disorder. The term "treating," as used herein, refers to the act of treating, as "treating" is defined immediately above.

Reference to the compounds of the present invention also includes reference to their physiologically acceptable salts, examples of which include salts derived from suitable bases such as alkaline 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 nitrogen atoms or amino groups 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, hydroxyethylsulfonic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalene Physiologically acceptable salts of hydroxy compounds include hydroxyl groups ^{, ...}

For therapeutic use, salts of the active ingredients of the compounds of the present invention are physiologically acceptable, i.e., they are salts derived from physiologically acceptable acids or bases. However, salts of acids or bases that are not physiologically acceptable may also be used, for example, to prepare or purify physiologically acceptable compounds. All salts, whether or not derived from physiologically acceptable acids or bases, are within the scope of the present invention.

Anti-COVID-19 activity detection method

Another aspect of the present invention relates to a method for detecting activity against the new coronavirus, comprising the step of treating a sample suspected of containing the novel coronavirus family with the compound described in the present invention.

The compounds of the present invention can be used as anti-COVID-19 compounds, as intermediates for such compounds, or have other uses as described below. The anti-COVID-19 compounds will bind to a surface or location in a cavity having a geometry unique to the COVID-19. Compounds that bind to the COVID-19 can bind to varying degrees of reversibility. Compounds that bind substantially irreversibly are ideal candidates for use in this method of the present invention. Once labeled, compositions that bind substantially irreversibly can be used as probes for detecting COVID-19. Therefore, the present invention relates to a method for detecting COVID-19 in a sample suspected of containing COVID-19, comprising the steps of treating a sample suspected of containing COVID-19 with a composition comprising a compound of the present invention bound to a marker; and observing the effect of the sample on the activity of the marker. Suitable markers 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, hydroxyl, or amino groups.

In the context of the present invention, samples suspected of containing the new coronavirus include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples, such as biological material 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 desired glycoprotein, etc. Typically, the sample will be suspected of containing an organism that produces the new coronavirus, often a pathogenic organism, such as the Coronavirus family. The sample can be contained in any medium, including water and organic solvent/water mixtures. Samples include living organisms, such as humans and artificial materials, such as cell cultures.

The treatment step of the present invention comprises adding the composition of the present invention to the sample, or it comprises adding a precursor of the composition to the sample. The adding step comprises any of the administration methods described above.

If desired, the activity of the composition against SARS-CoV-2 can be observed after administration 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-described screening methods is employed, however, any other method, such as observing physiological properties of living organisms, may also be employed.

Screening of active compositions against new coronavirus

The compounds of the present invention are useful for treating or preventing SARS-CoV-2 infection in animals or humans. However, cell-based assays should be the primary screening tool for screening compounds that inhibit SARS-CoV-2 in humans.

The compositions of the present invention are screened for compounds having anti-COVID-19 activity by any conventional technique for evaluating antiviral activity. In the context of the present invention, typically, compositions having anti-COVID-19 activity are first screened, and then compositions exhibiting antiviral activity are screened for in vivo activity. 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.

pharmaceutical preparations

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, it is preferred to formulate them as pharmaceutical formulations. The formulations of the present invention, whether for veterinary or human use, contain at least one active ingredient as defined above together with one or more acceptable carriers therefor, and optionally other therapeutic ingredients, especially those disclosed herein. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically not harmful to the recipient thereof.

Formulations include those suitable for the above-mentioned route of administration. Formulations can be conveniently prepared in unit dosage form and can be prepared 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 that constitutes one or more auxiliary components. In general, the formulation is prepared as follows: by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely dispersed solid carrier or both, and then, if necessary, shaping the product.

The present invention further provides veterinary compositions comprising at least one active ingredient as defined above and a veterinary carrier for use therewith.

Veterinary carriers are substances used in veterinary compositions and can be solid, liquid, or gaseous substances that are inert or veterinary acceptable and compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or by any other desired route.

Application Path

One or more compounds of the present invention (referred to herein as active ingredients) are administered by any route appropriate to 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 will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient. Advantages of the compounds of the present invention are that they are orally bioavailable and can be administered orally.

Metabolites of the compounds of the present invention:

Also within the scope of the present invention are the in vivo metabolites of the compounds described herein, to the extent such products are novel and unobvious relative to the prior art. These products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily due to enzymatic processes. Thus, the present invention includes novel and unobvious compounds produced by a method comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce its metabolites. Such products are typically identified as follows: a radiolabeled (e.g., ¹⁴ C or ³ H) compound of the invention is prepared and administered 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), sufficient time for metabolism to occur (typically, about 30 seconds to 30 hours), and its conversion products are isolated from urine, blood, or other biological samples. Because they are labeled, these products are easily isolated (others are isolated using antibodies that bind to epitopes remaining in the metabolites). The structure of the metabolites is determined in a conventional manner, such as by MS or NMR analysis. In general, analysis of metabolites is performed using the same methods as conventional drug metabolism studies known to those skilled in the art. Conversion products, provided they are not otherwise found in vivo, can be used in diagnostic assays for therapeutic dosing of the compounds of the invention even if they do not themselves possess SARS-CoV-2 polymerase inhibitory activity.

Formulas and methods for determining the stability of compounds in surrogate gastrointestinal secretions are known. A compound is defined herein as gastrointestinal stable if, after incubation at 37°C for 1 hour, less than about 50 mole percent of the protected group is unprotected in a surrogate for intestinal or gastric fluid. Simply because a compound is gastrointestinal stable does not mean it will not hydrolyze in vivo. Prodrugs of the present invention are typically stable in the digestive system, but they are generally substantially hydrolyzed to the parent drug in the digestive lumen, liver, or other metabolic organs, or within cells.

It should also be noted that the specific dosage and method of use of the compound of Formula I, its prodrug, and/or pharmaceutically acceptable salt thereof for each patient will depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, potency of the drug, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The effective dose of the active ingredient will depend at least on the nature of the condition being treated, toxicity (whether the compound is used prophylactically or against active viral infection), the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using routine dose escalation studies. It is contemplated that the dosage will be about 0.0001 to about 100 mg/kg of body weight per day; typically, about 0.01 to about 10 mg/kg of body weight per day; more typically, about 0.01 to about 5 mg/kg of body weight per day; and most typically, about 0.05 to about 0.5 mg/kg of body weight per day. For example, for an adult weighing about 70 kg, the daily dosage will be in the range of 1 mg to 1000 mg, preferably 5 mg to 500 mg, and can be taken in a single dose or in multiple doses.

The above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

In describing the experimental details, certain abbreviations and acronyms are used. Although most of them are understood by those skilled in the art, the following table contains a list of these abbreviations and acronyms1. Table 1 Abbreviation Meaning DCC Dicyclohexylcarbodiimide DCM dichloromethane DMAP 4-Dimethylaminopyridine EA Ethyl acetate HCl hydrochloric acid H ₂ SO ₄ sulfuric acid rt Room temperature SARS-CoV-2 Novel coronavirus THF Tetrahydrofuran TLC Thin-layer chromatography

In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

In the present invention, μM means micromole per liter; mmol means millimole; and equiv means equivalent.

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)methylcyclohexanecarboxylate (Compound ATV014)

In a 500 mL reactor, a stirrer, a thermometer, and a constant pressure dropping funnel were installed. GS-441524 (10 g, 0.03 mol) was added, and acetone (300 mL) dried over magnesium sulfate was added. Then, 2,2-dimethoxypropane (17 g, 0.16 mol) was added. Concentrated sulfuric acid (2.4 mL, 0.04 mol) was added dropwise to the system at room temperature. After 5 minutes, the solid began to dissolve. The temperature was raised to 45 ^° C and the reaction was continued for 4 h. The reaction was complete as monitored by HPLC (OD-3 column, mobile phase: n-hexane/isopropanol = 80:20, flow rate: 0.8 mL/min, injection volume 1 μL). The reaction was stopped. After cooling in an ice bath, solid NaHCO ₃ (10 g) and water (30 mL). Sodium bicarbonate was then added to adjust the pH to 7-8. The solvent was removed by distillation under reduced pressure. The residue was diluted with ethyl acetate (300 mL). The ethyl acetate layer was washed with water (80 mL) and saturated brine (80 mL), respectively, and dried over anhydrous sodium sulfate. The filtrate was filtered and distilled under reduced pressure until approximately 100 mL of solvent remained. The residue was slowly poured into ice-cooled petroleum ether and stirred vigorously. A large amount of white solid was washed out. 10.5 g of compound 1 was obtained as a white solid by filtration, with a yield of 91%.

Dissolve 15.0 g of compound 1 in 15 ml of dichloromethane, then add cyclohexanecarboxylic acid and 554.0 mg of 4-dimethylaminopyridine. Stir for 10 minutes, then add 10.2 g of dicyclohexylcarbodiimide. Stir at room temperature for 24 hours. Separate by column chromatography (eluent: petroleum ether/ethyl acetate (v/v) = 1/1) to obtain compound 2 (white solid). Compound 2 was dissolved in 30 mL of 37% (by weight) hydrochloric acid and 150 mL of tetrahydrofuran. After stirring for 6 hours, sodium carbonate was added to adjust the pH to 8. The organic solvent was removed by rotary evaporation. Column chromatography (eluent: petroleum ether/ethyl acetate (v/v) = 1/3) yielded 2.8 g of compound ATV014 (free base Form I, 49% yield), with a two-step yield of 45.8%. Hydrogen spectroscopy and carbon chromatographic analysis of the obtained compound ATV014 revealed the following results:

Hydrogen spectrum: ¹ H NMR (600 MHz, DMSO- d ₆ ) δ (ppm): 7.92 (s, 1H), 7.86 (br, 1H), 6.92 (d, J =4.5 Hz, 1H), 6.81 (d, J =4.5 Hz, 1H), 6.33 (d, J =5.9 Hz, 1H), 5.38 (d, J =5.9 Hz, 1H), 4.70 (t, J =5.3 Hz, 1H), 4.32-4.29 (dd, J =12.2 Hz, 2.6 Hz, 1H), 4.24-4.21 (m, 1H), 4.16-4.13 (dd, J =12.3 Hz, 4.8 Hz, 1H), 3.98-3.95 (q, J =5.9 Hz, 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).

Carbon spectrum: ¹³ C NMR (150 MHz, DMSO- d ₆ ) 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.

Example 2: Inhibitory effect of compounds on SARS-CoV-2 in Vero-E6 cells

SARS-CoV-2 variant B.1, identified as hCoV-19/CHN/SYSU-IHV/2020 (GISAID Accession ID: EPI_ISL_444969), was isolated from a sputum specimen obtained from a woman admitted to Guangzhou Eighth People's Hospital. The SARS-CoV-2 β (B.1.351, SARS_CoV-2_human_CHN_20SF18530_2020, Accession ID: GWH: WHBDSE01000000) and Δ (B.1.617.2, GDPCC 2.00096) variants were isolated by the Guangdong Provincial Center for Disease Control and Prevention from a COVID-19 patient admitted to Guangzhou Eighth People's Hospital. Two SARS-CoV-2 Omicron variants (BA.1 and BA.5) were isolated from COVID-19 patients hospitalized at Shenzhen Third People's Hospital.

Remdesivir, GS-441524, ATV006, and ATV014 were used as test compounds and the following steps were performed:

Vero-E6 cells were seeded in 48-well plates. When the cell density reached approximately 70-80%, the supernatant was discarded and the culture medium was replaced with fresh DMEM. Each compound was then added to the culture medium at a final concentration of 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 at a multiplicity of infection (MOI) of 0.05 with five SARS-CoV-2 mutants: three earlier mutants (B.1, B.1.351, and B.1.617.2) and two omikonium mutants, BA.1 and BA.5. Antiviral activity was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) to quantify viral copy numbers in the supernatant 48 hours after infection. The inhibitory effect of the test drugs at different concentrations on viral replication was calculated, and the _EC50 was calculated. This experiment consisted of three independent replicates, each with three replicate wells. The _EC50 values of different compounds against different SARS-CoV-2 variants in Vero-E6 cells are shown in Table 2 and Figure 1.

Table 2: _EC50 of different compounds against different SARS-CoV-2 variants in Vero-E6 cells Compound Alpha mutant strain B.1 Beta mutant strain B.1.351 Delta mutant strain B.1.617.2 Omicron mutant strain BA.1 Omicron mutant strain BA.5 Remdesivir 5.902 2.344 2.725 1.342 GS-441524 3.573 0.697 0.924 ATV006 1.360 1.127 0.349 0.106 0.071 ATV014 0.790 0.075 0.153 0.013 0.034

Compared to the control drug remdesivir, ATV014 exhibits significantly enhanced antiviral activity against SARS-CoV-2 and its variants. Its inhibitory activity against the Delta and Omicron strains was 17.8-fold and 103-fold higher than remdesivir, respectively, demonstrating superior antiviral efficacy compared to other tested compounds. ATV014's inhibitory activity against Omicron reached 13 nM against the BA.1 strain and 34 nM against the BA.5 strain.

Example 3: Metabolism of Compounds ATV014 and GS-441524 in Rats

1. Dosage and Administration for Each Group: ATV014 Intravenous Injection Group: 5 mg of ATV014 per kg of mouse body weight was administered intravenously. ATV014 Oral Group: 25 mg of ATV014 per kg of mouse body weight was administered orally. GS-441524 Intravenous Injection Group: 5 mg of GS-441524 per kg of mouse body weight was administered orally. GS-441524 Oral Group: 25 mg of GS-441524 per kg of mouse body weight was administered orally.

2. Operation:

Sixteen male Sprague-Dawley rats weighing 220 to 250 g were divided into four groups: ATV014 intravenous injection group, ATV014 oral group, GS-441524 intravenous injection group, and GS-441524 oral group, with four rats in each group (three rats in each ATV014 group). Drugs were administered as described in "1. Dosage and Administration Method for Each Group." Blood was collected from the cervical vein. Approximately 0.3 mL of blood was collected into heparin tubes at 0.083 h (not collected in the oral group), 0.16 h (not collected in the oral group), 0.25 h, 0.5 h, 1 h (not collected in the intravenous group), 2 h, 4 h, 8 h, 24 h, and 48 h after drug administration. The blood was centrifuged at 4000 r/min for 10 min at 4°C. The supernatant plasma was transferred to a refrigerator (approximately -20°C) and stored temporarily until assayed. A 50 μL plasma sample was added to 100 μL of a 90% aqueous methanol solution and vortexed. Then, 350 μL of a methanol/acetonitrile mixture (1:1, v/v) was added and vortexed. The sample was centrifuged at 10,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and injected for analysis. Blood samples collected 0.5 hours after intravenous administration and within 4 hours after oral administration were diluted 10-fold and injected for analysis. Drug concentrations in each sample were determined by high-performance liquid chromatography (HPLC)/mass spectrometry (MS). Analytes were separated using a Waters UPLC/XEVO TQ-S column and an InertSustain AQ-C18HP column (3.0 mm × 50 mm, 3.0 μm, GL). Pharmacokinetic parameters were calculated using DAS (Drug and Statistics) 3.0 software.

Results: See Tables 3, 4 and Figure 2.

Table 3: Pharmacokinetic parameters of SD rats after administration of ATV014 (testing GS-441524, mean ± SD, n = 3) Parameters ATV014 intravenous injection set ATV014 oral group Name Unit AUC(0-t) μg/L*h 3015.06±156.96 7398.72±78.07 AUC(0-∞) μg/L*h 3036.30±158.66 7470.18±189.98 T1/2z (terminal elimination half-life) h 1.65±0.92 1.94±0.65 Tmax (time of maximum blood drug concentration) h 0.08±0.0 2.67±1.15 Cmax (maximum blood concentration) ng/mL 2466.44±73.54 1427.20±438.46 F (bioavailability) % / 49.08±.52

Table 4: Pharmacokinetic parameters of SD rats after administration of GS-441524 (mean ± SD, n = 4) Parameters GS-441524 intravenous injection set GS-441524 oral group Name Unit AUC(0-t) μg/L*h 3443.5±460.6 3896±1795.7 AUC(0-∞) μg/L*h 3478.8±455.5 3913.5±1778.2 T1/2z (terminal elimination half-life) h 12.69±7.34 6.85±6.76 Tmax (time of maximum blood drug concentration) h 0.08±0.0 0.75±0.29 Cmax (maximum blood concentration) ng/mL 2384.6±282.4 1071.7±147.2 F (bioavailability) % / 22.63±10.43

Conclusion:

As shown in Tables 3 and 4 and Figure 2, the oral bioavailability of ATV014 is 49.08%, while that of GS-441524 is 22.63%. This indicates that ATV014 has significantly improved oral bioavailability and better oral drugability compared to GS-441524.

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

African green monkey kidney cells (Vero) were purchased from ATCC, catalog number CCL-81. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. DMEM supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin was used as the experimental medium.

Virus strain: SARS-CoV-2 XBB.1.16 mutant isolated from Shenzhen Third People's Hospital, strain name: hCoV-19/Guangdong/SZTH-070/2023.

Remdesivir, GS-441524, and ATV014 were used as test compounds, and the following procedures were performed: 1) Drugs used: Control drug remdesivir (RDV) and test drugs: GS-441524 (SHEN26-69-0) and ATV014, with a stock concentration of 10 mM. 2) Vero cells were prepared: Two 24-well plates, with approximately 105 cells per well. 3) Drug dilution: Samples were diluted in 2 mL EP tubes using 2% DMEM at the following concentrations: 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 μM. 4) Add the corresponding volume of virus stock solution to each EP tube containing different drug concentrations at an MOI of 0.05. 5) Cell Preparation: After approximately 24 hours of incubation, discard the culture medium and wash the cells once with 1x sterile PBS. 6) Add 250 μL of the diluted virus and drug mixture to a washed 24-well plate in triplicate. Incubate the virus, drug, and cells at 37°C for 1 hour. Also set up blank control wells (without virus or drug) and virus control wells (with virus only). 7) Maintenance Solution Preparation: Samples were diluted in 2 mL EP tubes using 2% DMEM at the same concentration as above. 8) After 1 hour of incubation, aspirate the supernatant, wash twice with PBS, then add maintenance solution containing the corresponding drug concentration and incubate at 37°C for 48 hours. 9) Extract RNA from the supernatant, measure the SARS-CoV-2 RNA copy number, and calculate the inhibition rate. The antiviral activity of the sample is calculated as follows:

Inhibition rate (%) = (average copy number of virus control - average copy number after drug treatment) / (average copy number of virus control) × 100

GraphPad Prism (version 8) was used to perform a nonlinear fitting analysis of the inhibition rate and cell viability of the samples, and the median effective concentration (EC ₅₀ ) of the samples was calculated. The fitting formula was: log(inhibitor) vs. response -- variable slope.

Conclusion: Experimental results showed that the _EC50 values of ATV014 and SHEN26-69-0 against the tested SARS-CoV-2 omicorex XBB.1.16 variant were 139 nM and 778 nM, respectively. The _EC50 value of the control drug remdesivir (RDV) against the tested SARS-CoV-2 omicorex XBB.1.16 variant was 2.336 μM (Figure 3). ATV014 exhibited superior antiviral activity against the omicorex XBB variant compared to GS-441524 and remdesivir.

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

Vero-E6 cells:

Culture the cells in DMEM (high-glucose complete medium) supplemented with 10% fetal bovine serum. Passage the cells once one day before the experiment to maintain them in the logarithmic growth phase. After addition of virus and sample, maintain the cells in DMEM (high-glucose complete medium) supplemented with 3% fetal bovine serum.

Novel coronavirus (SARS-CoV-2): EG.5.1, isolated, cultured and preserved in the Biosafety Level 3 laboratory of the Kunming Institute of Zoology, Chinese Academy of Sciences.

Compounds GS-441524 and ATV014 were used as test compounds, and the following steps were followed:

Vero E6 cells were seeded into culture plates in advance and incubated overnight at 37°C, 5% _CO₂. The cells were used when the monolayer reached approximately 80% confluence. In the P3 laboratory, the test drug was added to each well at varying concentrations. Samples were prepared at eight concentration gradients (200, 66.70, 22.20, 7.40, 2.50, 0.80, 0.30, and 0.10 μM) along with an equal volume of diluted virus supernatant (MOI = 0.01). Each concentration gradient was replicated in triplicate. A solvent control, a negative control lacking drug or virus, and a positive control were also included. After 1 hour of infection at 37°C, 5% _CO₂, the virus-drug mixed medium was aspirated, the cells were washed with 1× PBS, and then replaced with medium containing the drug alone for continued incubation. An 8-well concentration gradient was established, with three replicates per gradient. Positive, solvent, and negative controls were also included. After 48 hours of incubation at 37°C, 5% _CO₂ , cell supernatants were collected, and viral RNA was extracted for real-time PCR quantification. The inhibition rate of drug on viral replication and the _EC₅₀ value were calculated.

Conclusion: Experimental results show that ATV014 exhibits potent inhibitory activity against the SARS-CoV-2 variant EG.5.1, with an _EC50 of 0.093 μM (Figure 4). GS-441524 also exhibits significant inhibitory activity against the EG.5.1 variant, with an _EC50 of 0.73 μM. ATV014 exhibits superior antiviral activity against the EG.5.1 variant of Omicron.

While the methods of the present invention have been described through preferred embodiments, it will be apparent to those skilled in the art that modifications, variations, and combinations of the methods and applications described herein can be made within the scope, spirit, and spirit of the present invention to implement and apply the present technology. Those skilled in the art can, based on the present disclosure, modify the process parameters as appropriate. It is specifically noted that all similar substitutions and modifications that would be obvious to those skilled in the art are considered encompassed by the present invention.

Figure 1 shows the inhibitory effects of compounds ATV006 and ATV014 in Example 2 against SARS-CoV-2 omega-3 mutants BA.1 and BA.5, respectively, in Vero-E6 cells. The horizontal axis represents drug concentration in μM, and the vertical axis represents inhibition rate in %. Figure 2 shows the drug-time curve of ATV014 in rats in Example 3. The horizontal axis represents time in hours, and the vertical axis represents plasma drug concentration in μg/L. Figure 3 shows the inhibitory activity of ATV014, GS-441524, and remdesivir against the omikonin XBB mutant in Vero cells from Example 4. The horizontal axis represents drug concentration in μM, and the vertical axis represents inhibition rate in %. Figure 4 shows the inhibitory activity of ATV014 and GS-441524 against the omikonin EG.5.1 mutant in Vero cells from Example 5. The horizontal axis represents drug concentration in μM, and the vertical axis represents inhibition rate in %.

Claims (6)

A compound ATV014, a pharmaceutically acceptable salt thereof, or a crystalline hydrate thereof is used in the preparation of a product for preventing, alleviating, or treating diseases caused by infection with the novel coronavirus Omicron strain, or the cytopathic effects resulting from the replication or propagation of its homologous variants. The compound ATV014 is (((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexanecarboxylate, having the following structure: . The use as described in claim 1, wherein the novel coronavirus Omicron strain includes the original Omicron strain (B.1.1.529) and the Omicron mutant strain. The use as described in claim 2, wherein the Omicron mutant strains include BA.1, BA.2, BA.3, BA.4, and BA.5 genealogies. The use as described in claim 1, wherein the infection is caused by the Omicron strain, including fever, headache, cough, sore throat, muscle aches, pneumonia, acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome (ARDS), and sepsis. The use as described in claim 1, wherein the compound ATV014 or a pharmaceutically acceptable salt thereof is suitable for use in humans or animals. The use according to claim 5, wherein the animal comprises bovines, equines, ovines, porcines, canines, felines, rodents, primates, birds or fish.