WO2024146588A1 - Use of compound atv014 for resisting sars-cov-2 infection - Google Patents

Abstract

The use of compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof in the preparation of a product for the prevention, remission or treatment of SARS-CoV-2 omicron strain infection, or the replication or reproduction of a homologous variant virus thereof and a cytopathic effect caused thereby. The present invention relates to the technical field of pharmaceutical technology and viral infectious diseases. The structural formula of the compound ATV014 is as follows:

Description

Application of compound ATV014 in anti-new coronavirus infection

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310011667.0 filed on January 5, 2023. The entire text of the above Chinese patent application is hereby cited as part of this application.

Technical Field

The present invention belongs to the field of drug synthesis, relates to pharmaceutical technology and the technical field of viral infection diseases, and specifically relates to a nucleoside derivative, a prodrug thereof and/or a pharmaceutically acceptable salt thereof, and a composition and use thereof.

Background technique

Globally, Omicron has caused more than 1.1 million deaths in 2022. Currently, in addition to vaccines, anti-COVID-19 drugs are an effective means to reduce severe illness and mortality worldwide. Experts agree that using antiviral drugs to reduce viral load within five days of infection is the best treatment window and the best treatment plan, which can avoid missing the treatment window and causing worsening of the disease.

Currently, three new crown drugs have been approved abroad: Gilead's remdesivir, Pfizer's pasirovir and Merck's monaprevir. 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 for use among the above three drugs. The important drawback of remdesivir is that its injection method must be carried out in the hospital, which is easy to cause a run on the hospital's medical resources.

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 produced an antiviral effect superior to remdesivir in the activity test 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 describing the application of compound GS-441524 in the prevention, relief and/or treatment of SARS-CoV-2 (application number 202011000517.2). The applicant later conducted a pharmacokinetic analysis of GS-441524 and found that its biological oral availability was very low and it could only be used in the form of an injection. It would be of great significance to seek oral, low-toxicity nucleoside derivatives or prodrugs of GS-441524.

The applicant has upgraded and adjusted the structure optimization based on remdesivir and GS-441524, and 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, a nucleoside compound for the treatment of viral infection and its use (application number 2021110837309). This includes compounds A series of compounds, including ATV014, have shown activity in inhibiting the new coronavirus.

Summary of the invention

SUMMARY OF THE INVENTION

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

Specifically, the compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof is used in the preparation of a product for preventing, alleviating or treating coronavirus infection, or the replication or reproduction of its homologous variant viruses and the cytopathic effects produced therefrom.

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

Provided is the use of 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 cytopathic effects caused by the replication or reproduction of its homologous variant viruses.

Provided is the use of 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 caused by the replication or reproduction of its homologous variant viruses.

Provided is a pharmaceutical composition, wherein the first active ingredient 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:

DETAILED DESCRIPTION OF THE INVENTION

In order to achieve one of the above purposes, the present invention adopts the following technical solution:

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

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 genealogies and Omicron mutants produced in the future.

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

Optionally, the compound or a pharmaceutically acceptable salt thereof can 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 prior art, 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 better antiviral effects 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 13nM on the BA.1 strain and 34nM on the BA.5 strain. The antiviral activity on the XBB strain was 139nM and the antiviral activity on the EG.5.1 strain was 93nM, which were better than GS-441524 or remdesivir. This shows that the compound ATV014 can effectively inhibit the replication and/or reproduction of the Omicron strain of the new coronavirus 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 convenient for production and distribution.

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

Definition of Terms

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

“V/V” means volume ratio. EC ₅₀ means half effective concentration.

In the present invention, "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.

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

The compounds described herein also include reference to their physiologically acceptable salts, examples of which include salts derived from appropriate bases, such 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 nitrogen atoms or amino groups include: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, and the like; (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, naphthalene, and the like; (c) salts formed with elemental anions, such as chlorine, bromine and iodine. Physiologically acceptable salts of hydroxy compounds include combinations of anions of the compounds with suitable cations such as Na ⁺ and _NR4 ⁺ .

For therapeutic use, the salts of the active ingredients of the compounds of the 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 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 using the compounds described in the present invention to treat a sample suspected of containing the new coronavirus family.

The compounds of the present invention can be used as anti-new coronavirus compounds, as intermediates for such compounds, or have other uses as described below. The anti-new coronavirus compounds will bind to a position on a surface or in a cavity having a geometry unique to the new coronavirus. The compounds that bind to the anti-new coronavirus can bind to different degrees of reversibility. Those compounds that are essentially irreversibly bound are ideal candidates for this method of the present invention. Once labeled, those compositions that are essentially irreversibly bound can be used as probes for detecting the new coronavirus. Therefore, the present invention relates to a method for detecting the new coronavirus in a sample suspected of containing the new coronavirus, comprising the following steps: treating a sample suspected of containing the new coronavirus 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 (e.g., hydroxyl, carboxyl, sulfhydryl, or amino).

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 for synthesizing 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 may 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.

The treating 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 novel coronavirus after administration of the composition can be observed by any method, including direct and indirect methods of detecting anti-novel coronavirus activity. Quantitative, qualitative and semi-quantitative methods for detecting novel coronavirus activity are all contemplated. Typically, one of the above-mentioned screening methods is applied, however, any other method may also be applied, such as observing the physiological properties of living organisms.

Screening of active compositions against new coronavirus

The compounds described herein are suitable for treating or preventing COVID-19 infections in animals or humans. However, in the screening of compounds that can inhibit COVID-19 viruses in humans, cell-based assays should be the primary screening tool.

The present invention compositions are screened for compounds having anti-new coronavirus activity by any conventional technique for evaluating antiviral activity. In the context of the present invention, typically, the composition having anti-new coronavirus activity is first screened, and then Compositions that exhibit antiviral activity are then screened for in vivo activity. Compositions with 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 screens have 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 in accordance with conventional practice. Although the active ingredients can be administered alone, it is preferred that they are formulated into pharmaceutical preparations. The preparations of the present invention, whether for veterinary or human use, contain at least one active ingredient as defined above and one or more acceptable carriers therefor, and optionally contain 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 of the formulation and is physiologically harmless to its recipient.

Formulations include those suitable for the above-mentioned routes of administration. The formulations can be conveniently prepared in unit dosage form and can be formulated 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 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, if necessary, shaping the product.

The invention further provides veterinary compositions comprising at least one active ingredient as defined above together with a veterinary carrier therefor.

Veterinary carriers are substances useful for the purpose of the veterinary composition and may be solid, liquid or gaseous substances which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

Route of administration

One or more compounds of the present invention (referred to herein as active ingredients) are administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including oral and sublingual) and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), etc. It should be understood that the preferred route may vary with, for example, the condition of the recipient. The benefits 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:

The in vivo metabolic products of the compounds described herein also fall within the scope of the present invention to the extent that such products are novel and non-obvious relative to the prior art. These products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily due to enzymatic processes. Thus, the present invention includes novel and non-obvious compounds produced by the following method, which comprises contacting a compound of the present invention with a mammal for a period of time sufficient to produce its metabolic products. Such products are typically identified as follows: a radiolabeled (e.g., ¹⁴ C or ³ H) compound of the present invention is prepared, 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 is allowed 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 in the same manner as conventional drug metabolism studies known to those skilled in the art. Conversion products, provided that they are not otherwise found in vivo, can also 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. Compounds are 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 fluid after incubation at 37°C for 1 hour. It cannot be assumed that compounds will not hydrolyze in vivo simply because they are stable to the gastrointestinal tract. The 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 having the structure of Formula I, its prodrug and/or its pharmaceutically acceptable salt for different patients are determined by many factors, including the patient's age, weight, sex, natural health, nutritional status, drug activity, time of administration, metabolic rate, severity of the disease, and 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 for prevention or against active viral infection), the method of delivery and the drug formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected that the dose is 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 weighing 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.

The above-mentioned drugs in various dosage forms 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 acronyms 1.

Table 1

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibitory effects of compounds ATV006 and ATV014 in Example 2 on SARS-CoV-2 Omicron mutants BA.1 and BA.5 in Vero-E6 cells, respectively. The horizontal axis is the drug concentration in μM, and the vertical axis is the inhibition rate in %.

FIG2 shows a drug-time curve of ATV014 in rats in Example 3, wherein the horizontal axis is time in hours, and the vertical axis is drug concentration in plasma in μg/L.

Figure 3 shows the inhibitory activity of ATV014, GS-441524 and remdesivir against the Omicron XBB mutant in Vero cells in Example 4, wherein the horizontal axis is the drug concentration in μM; the vertical axis is the inhibition rate in %.

Figure 4 shows the inhibitory activity of ATV014 and GS-441524 against the Omicron EG.5.1 mutant in Vero cells in Example 5, wherein the horizontal axis is the drug concentration in μM; the vertical axis is the inhibition rate in %.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution 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]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexanecarboxylate (Compound ATV014)

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 acetone (300 mL) dried over magnesium sulfate, and then add 2,2-dimethoxypropane (17 g, 0.16 mol). Add concentrated sulfuric acid (2.4 mL, 0.04 mol) dropwise to the system at room temperature. After 5 min, the addition is complete and the solid begins to dissolve. The temperature is raised to 45 °C and the reaction is continued for 4 h. The reaction is complete after HPLC monitoring (OD-3 column, mobile phase: n-hexane/isopropanol = 80:20, flow rate: 0.8 mL/min, injection volume 1 μL). Stop the reaction, cool in an ice bath, and add NaHCO ₃ solid (10 g), water (30 mL), continue to adjust pH to 7-8 with sodium bicarbonate, remove solvent by reduced pressure distillation, dilute the residue with ethyl acetate (300 mL), wash the ethyl acetate layer with water (80 mL) and saturated brine (80 mL), 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 petroleum ether cooled in an ice bath, and stir vigorously to wash out a large amount of white solid, and filter by suction to obtain 10.5 g of compound 1 as a white solid, with a yield of 91%.

15.0g of compound 1 was dissolved in 15ml of dichloromethane, and then cyclohexanecarboxylic acid and 554.0mg of 4-dimethylaminopyridine were added. After stirring for 10min, 10.2g of dicyclohexylcarbodiimide was added and stirred at room temperature for 24h. After column chromatography separation (eluent: petroleum ether/ethyl acetate (V/V) = 1/1), compound 2 (white solid) was obtained. Compound 2 was dissolved in 30mL of 37% hydrochloric acid aqueous solution and 150mL of tetrahydrofuran, stirred for 6 hours, sodium carbonate was added to adjust the pH to 8, and the organic solvent was removed by rotary evaporation. After column chromatography separation (eluent: petroleum ether/ethyl acetate (V/V) = 1/3), compound 2.8g ATV014 (free base crystal form I, yield 49%) was obtained, and the two-step yield was 45.8%. The obtained compound ATV014 was tested for hydrogen spectrum and carbon spectrum, and the results were as follows:

Hydrogen spectrum: ¹ H NMR (600 MHz, DMSO-d ₆ )δ(ppm):7.92(s,1H),7.86(br,1H),6.92(d,J＝4.5Hz,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).

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 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 a sputum specimen of a woman admitted to the Eighth People's Hospital of Guangzhou. SARS CoV-2 β (B.1.351, SARS_CoV-2_human_CHN_20SF18530_2020, Accession ID: GWH:WHBDSE01000000) and Delta (B.1.617.2, GDPCC2.00096) variants were isolated by the Guangdong Provincial Center for Disease Control and Prevention from COVID-19 patients hospitalized in the Eighth People's Hospital of Guangzhou. Two SARS-CoV-2 Omicron variants (BA.1 and BA.5) were isolated from COVID-19 patients hospitalized in the Third People's Hospital of Shenzhen.

Respectively take compounds Remdesivir, GS-441524, ATV006, and ATV014 as test compounds and perform the following steps:

Vero-E6 cells were seeded in 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 to make the final concentration of the compound 50μM, 10μM, 5μM, 2μM, 1μM, 0.5μM, 0.25μM, 0.1μM or 0.01μM. The cells were infected with five SARS-CoV-2 mutants at a multiplicity of infection (MOI) of 0.05: three early mutants (B.1, B.1.351 and B.1.617.2), two Omicron mutants BA.1 and BA.5. The antiviral activity was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) to quantify the number of viral copies in the supernatant 48 hours after infection. We calculated the inhibitory effect of different concentrations of the tested drugs on viral replication and calculated the EC ₅₀ . This experiment consisted of three independent replicates, each with 3 replicates. The _EC50 values of different compounds against different variants of SARS-CoV-2 in Vero-E6 cells are shown in Table 2 and Figure 1.

Table 2: _EC50 of different compounds against different variants of SARS-CoV-2 in Vero-E6 cells

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

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

1. Dosage and method of administration for each group:

ATV014 intravenous injection group: 5 mg of ATV014 per kg of mouse body weight was intravenously injected.

ATV014 oral group: 25 mg ATV014 per kg mouse body weight was administered intragastrically.

GS-441524 intravenous injection group: 5 mg of GS-441524 per kg of mouse body weight was injected intravenously.

GS-441524 oral group: 25 mg of GS-441524 per kg of mouse body weight was administered intragastrically.

2. Operation:

16 SD rats (male) weighing 220g-250g were divided into 4 groups, namely ATV014 intravenous injection group, ATV014 oral group, GS-441524 intravenous injection group, and GS-441524 oral group, with 4 rats in each group (3 rats in each ATV014 group), and the drugs were administered according to "1. Dosage and administration method of each group". Blood was collected from the jugular vein. About 0.3mL of blood was collected into heparin tubes at 0.083h (no blood was collected in the oral group), 0.16h (no blood was collected in the oral group), 0.25h, 0.5h, 1h (no blood was collected in the intravenous injection group), 2h, 4h, 8h, 24h, and 48h after administration, and centrifuged at 4000r/min for 10min at 4℃. The upper plasma was transferred to a refrigerator (about -20℃) and temporarily stored until the measurement. 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), vortex mix; centrifuge at 10000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm filter membrane before sampling and testing; blood samples taken 0.5 hours after intravenous administration and 4 hours after oral administration were diluted 10 times and then sampled for testing. The drug concentration in each sample was determined by high performance liquid chromatography (HPLC)/mass spectrometry (MS). A Waters UPLC/XEVO TQ-S column, InertSustain AQ-C18HP column (3.0 mm×50 mm, 3.0 μm, GL) was used to separate the analytes, and DAS (Drug and Statistics) 3.0 software was used to calculate the pharmacokinetic parameters.

Results: See Table 3, Table 4 and Figure 2.

Table 3: Pharmacokinetic parameters of SD rats after administration of ATV014 (detection of GS-441524, mean ± SD, n = 3)

Table 4: Pharmacokinetic parameters of GS-441524 administered to SD rats (mean ± standard deviation, n = 4)

in conclusion:

It can be seen from Table 3, Table 4 and Figure 2 that the oral bioavailability of ATV014 is 49.08%, and the oral bioavailability of GS-441524 is 22.63%, indicating that compared with GS-441524, the oral bioavailability of ATV014 is significantly improved and has better oral drugability.

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. The cells were cultured in 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 culture medium.

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

Take the compounds remdesivir, GS-441524, and ATV014 as test compounds, and perform the operation according to the following steps:

1) Drugs used: control drug remdesivir (RDV) and test drugs: GS-441524 (SHEN26-69-0), ATV014, the storage solution concentration is 10mM.

2) Prepare Vero cells: 2 24-well plates, approximately 105 cells per well.

3) Drug dilution: Sample dilution was carried out in a 2 mL EP tube. The diluent was 2% DMEM with the following concentrations: 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064 μM.

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

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

6) Take 250uL of the diluted virus and drug mixture and add it to the cleaned 24-well cell plate, in triplicate, and incubate the virus, drug and cells at 37°C for 1 hour. At the same time, set up blank control wells (without virus and drug) and virus control wells (only virus).

7) Preparation of maintenance solution: Sample dilution is carried out in a 2mL EP tube, the diluent is 2% DMEM, and the concentration is the same as before.

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

9) Take the supernatant to extract RNA, detect the number of SARS-CoV-2 RNA copies, 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 nonlinear fitting analysis on the inhibition rate and cell viability of the samples, and the half effective concentration (EC ₅₀ ) value of the samples was calculated. The fitting formula was: log (inhibitor) vs. response--Variable slope.

Conclusion: The experimental results showed that the EC50 values of ATV014 and SHEN26-69-0 against the tested SARS-CoV-2 Omicron XBB.1.16 mutant were 139nM and 778nM, respectively. The _EC50 value of the control drug Remdesivir (RDV) against the tested SARS _- CoV-2 Omicron XBB.1.16 mutant was 2.336μM (Figure 3). In the Omicron XBB mutant, the antiviral activity of ATV014 was better than that of 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:

The cells were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum, and the cells were passaged once 1 day before the experiment to keep the cells in the logarithmic growth phase. After the addition of virus and sample, the cells were maintained in DMEM high-glucose complete medium containing 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.

Take compounds GS-441524 and ATV014 as test compounds respectively, and perform the operation according to the following steps:

Vero E6 cells were inoculated into the culture plate in advance, cultured overnight at 37°C, 5% CO ₂ , and used when the monolayer cells grew to about 80%. In the P3 laboratory, different concentrations of the drug to be tested were added to each well. The samples were set up 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 dilution supernatant (MOI = 0.01). Each concentration gradient had 3 replicate wells, and a solvent control, a negative control without drugs and viruses, and a positive control were set up. After infection in a 37°C, 5% CO ₂ incubator for 1 hour, the virus-drug to be tested mixed culture medium was aspirated and discarded, and the culture medium containing only the drug to be tested was replaced with 1×PBS after washing and continued to be cultured. 8 concentration gradients were set up, each with 3 replicate wells, and positive controls, solvent controls, and negative controls were set up. After culturing for 48 hours at 37°C, 5% CO ₂ , the cell supernatant was collected, Viral RNA was extracted and used for real-time PCR virus quantification to calculate the inhibition rate of drug on viral replication and EC ₅₀ value.

Conclusion: The experimental results show that ATV014 has a good inhibitory effect on the SARS-CoV-2 variant EG.5.1, EC ₅₀ = 0.093μM (Figure 4), and GS-441524 also has a good inhibitory effect on the variant EG.5.1, EC ₅₀ = 0.73μM. In the case of the Omicron EG.5.1 variant, the antiviral activity of ATV014 is better than that of GS-441524.

The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine 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 refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims (10)

1. A method for preventing, alleviating or treating diseases related to cytopathic effects caused by infection with the novel coronavirus Omicron strain, or the replication or reproduction of its homologous variant viruses, characterized in that it comprises the steps of administering a safe and effective amount of compound ATV014 or a pharmaceutically acceptable salt thereof to a subject in need.
2. The use of compound ATV014 or a pharmaceutically acceptable salt thereof in the preparation of products for preventing, alleviating or treating diseases caused by infection with the novel coronavirus Omicron strain, or cytopathic effects caused by the replication or reproduction of its homologous variant viruses.
3. The compound ATV014 or a pharmaceutically acceptable salt thereof is used to prevent, alleviate or treat diseases caused by infection with the novel coronavirus Omicron strain, or cytopathic effects caused by the replication or reproduction of its homologous variant viruses.
4. The method according to claim 1, or the use according to claim 2 or 3, characterized in that the compound ATV014 is (((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl cyclohexanecarboxylate, or a pharmaceutically acceptable salt thereof, or a crystalline hydrate thereof, or a solvate thereof, having the following structure:
   
5. The method according to claim 1 or the use according to claim 2 or 3 is characterized in that the novel coronavirus Omicron strain includes the original Omicron strain (B.1.1.529) and the Omicron mutant strain.
6. The method according to claim 3 or the use according to claim 2 or 3, characterized in that the Omicron mutant strains include BA.1, BA.2, BA.3, BA.4, BA.5 genealogies and Omicron mutant strains produced in the future.
7. The method according to claim 1 or the use according to claim 2 or 3, characterized in that the infection is caused by the Omicron strain, including fever, headache, cough, pharyngitis, muscle aches, pneumonia, acute respiratory tract infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis.
8. The method according to claim 1, or the use according to claim 2 or 3, characterized in that the compound ATV014 or a pharmaceutically acceptable salt thereof is suitable for humans or animals; and/or the animals include bovines, equines, ovines, porcines, canines, felines, rodents, primates, birds or fish.
9. A pharmaceutical composition, characterized in that the pharmaceutical composition contains: a first active ingredient, wherein the first active ingredient is compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof.
10. An active ingredient or a preparation containing the active ingredient, characterized in that the active ingredient is compound ATV014 or a pharmaceutically acceptable salt thereof or a crystalline hydrate thereof or a solvate thereof.