N 4 Ester derivatives of hydroxyl cytidine and uses thereof

Abstract

This disclosure relates to N 4 Ester derivatives of hydroxycytidine (NHC), pharmaceutical compositions comprising thereof, methods of their preparation, and matters relating to N 4 Uses and methods of treating viral infections with β-hydroxycytidine ester derivatives.

Description

Technical Field

[0001] This disclosure relates to N 4 Ester derivatives of 1-hydroxycytidine (NHC), relating to pharmaceutical compositions comprising thereto, and N 4 Ester derivatives of β-hydroxycytidine are used to treat viral infections. These compounds can be administered orally to provide N... 4 -Hydroxycytidine. Background Technology

[0002] Currently, the SARS-CoV-2 virus that causes COVID-19 has infected more than 240 million people globally and caused approximately 5 million deaths, with no signs of slowing down. The global economy and human activity have been negatively impacted. Despite the recent introduction of vaccines, oral medications for treating infected patients remain in high demand and can supplement vaccine use. 4 1,3-hydroxycytidine (NHC) is a ribonucleoside analog with broad-spectrum antiviral activity against a variety of unrelated RNA viruses, including influenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV), and most importantly, human SARS-CoV-2. Although the exact molecular mechanism of action of NHC remains undetermined, it has been proposed that viral malfeasance is the basis of its antiviral activity [“Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia”, SciTransl Med. 2019 Oct, 23; 11(515):eaax5866], stemming from the tautomerization properties of NHC.

[0003]

[0004] The oxime form of the NHC mimics uridine and matches adenosine (structure on the left below), while another tautomer mimics cytidine and matches guanosine (structure on the right below). This mismatch could lead to viral catastrophic errors.

[0005]

[0006] N 4Monopravir / EIDD2801 / MK4486, a prodrug of hydroxycytidine (NHC), has just completed a clinical trial for the treatment of SARS-CoV-2 (the virus that causes COVID-19). A Phase III clinical trial reportedly treating early-stage SARS-CoV-2 infection at a dose of 800 mg twice daily for 5 days showed a 50% reduction in the number of patients progressing to hospitalization. High doses and BID administration require sustained effective concentrations of NHC in the body to induce viral erroneous catastrophe. Therefore, more and potentially better prodrugs (i.e., smaller doses, less frequent dosing, and higher efficacy) are still needed to treat viral infections, especially in the urgent treatment of the current global human catastrophe. Summary of the Invention

[0007] The inventors discovered a series of N 4 An ester derivative of 1,4-hydroxycytidine (NHC) can deliver NHC in the bloodstream of animals with improved bioavailability and prolonged exposure time compared to the parent molecule NHC.

[0008] This disclosure relates to certain ester prodrugs of NHC, combinations thereof, pharmaceutical compositions, uses and methods thereof.

[0009] This disclosure provides compounds of formula (I):

[0010]

[0011] Or its tautomers, stereoisomers, or racemates or pharmaceutically acceptable salts thereof, wherein

[0012] R is Ra-(C=O)-,

[0013] Where Ra is selected from C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 Alkyl, wherein each of the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups is optionally substituted by one or more substituents selected from the following groups: halogen, acyl, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7 Alkyl)2, -CO-NH2, -CO-NH(C 1-7Alkyl), -CO-N(C 1-7 Alkyl)2, -NH (acyl), -N (acyl)2, NH2-acyl, NHRy-acyl, N(Ry)2-acyl, C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-7 Alkyloxy, aryloxy, heteroaryloxy, halogenated-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy, halogenated C 2-6 alkenyl, halogenated -C 2-6 alkynyl, hydroxyl-C 1-7 Alkyl, C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 3-8 cycloalkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 Cycloalkoxy or 3 to 12-membered heterocyclic hydroxyl groups,

[0014] Ry is independently selected from C 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 alkyl.

[0015] In a preferred embodiment, Ra is selected from C. 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 12-membered heterocyclic groups, each optionally substituted by one or more substituents selected from the following groups: halogen, acyl, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7 Alkyl)2, -CO-NH2, -CO-NH(C 1-7 Alkyl), -CO-N(C 1-7 Alkyl)2, -NH (acyl), -N (acyl)2, NH2-acyl, NHRy-acyl, N(Ry)2-acyl, C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C1-7 Alkoxy, halogenated -C 1-7 Alkyl, Halogenated -C 1-7 Alkyloxy, aryloxy, heteroaryloxy, halogenated-C 2-6 alkenyl, halogenated -C 2-6 alkynyl, hydroxyl-C 1-7 Alkyl, C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 3-8 cycloalkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 Cycloalkoxy or 3 to 12-membered heterocyclic hydroxyl groups,

[0016] Ry is independently selected from C 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 alkyl.

[0017] In a further preferred embodiment, Ra is selected from C. 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, halogenated-C 1-7 Alkyl, C 1-7 Alkyl-OC 1-7 Alkyl, C 1-7 Alkyl-O-aryl, C 1-7 Alkyl-O-heteroaryl, halogenated-C 3-8 cycloalkyl, C 1-6 Alkyl-O-(CH2) n -、C 1-6 Alkyl-OC 1-6 Alkyl-O-(CH2) n -, Halogenated-C 1-6 Alkyl-O-(CH2) n -、C 3-6 Cycloalkyl-O-(CH2) n -, Halogenated-C 3-6 Cycloalkyl-O-(CH2) n -, 3 to 12-membered heterocyclic groups -O-(CH2)n - and 3 to 12-membered halogenated heterocyclic groups -O-(CH2) n -

[0018] The compounds described above, as well as the compounds disclosed below (including compounds of formula (I) and specific compounds, particularly the compounds of the examples), or their tautomers, stereoisomers, enantiomers, diastereomers, racemates, geometric isomers, hydrates or solvates or pharmaceutically acceptable salts thereof, are collectively referred to as "compounds of the present invention" or "compounds of this disclosure".

[0019] This disclosure also provides compounds of the invention for use as pharmaceuticals.

[0020] This disclosure also provides compounds of the present invention for the treatment or prevention of RNA virus infection.

[0021] This disclosure also provides pharmaceutical compositions comprising the compounds of the present invention, and optionally comprising pharmaceutically acceptable excipients.

[0022] This disclosure also provides a kit for treating or preventing RNA virus infection, which contains the pharmaceutical composition of this disclosure and instructions for use.

[0023] This disclosure also provides the use of the compounds of the present invention in the preparation of medicaments for treating or preventing RNA virus infections.

[0024] This disclosure also provides the use of the compounds of the present invention for the treatment or prevention of RNA virus infection.

[0025] This disclosure also provides a method for treating or preventing RNA virus infection in an individual, comprising administering an effective amount of the compound of the present invention to the individual in need.

[0026] This disclosure also provides for adding N. 4 Methods for the bioavailability of hydroxycytidine to treat or prevent RNA virus infection, comprising administering an effective amount of the compound of the present invention to an individual in need.

[0027] This disclosure also provides pharmaceutical combinations comprising the compounds of the present invention and at least one additional therapeutic agent.

[0028] This disclosure also provides a method for preparing the compounds of the present invention, as well as intermediates for preparing the compounds of the present invention.

[0029] Further advantages will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and achieved by the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and not restrictive. Attached Figure Description

[0030] The accompanying drawings described herein are for illustrative purposes only. The drawings are not intended to limit the scope of this disclosure.

[0031] Figure 1 The mean ± SD plasma concentration-time data of EX-2, monoravir, and NHC after oral administration of EX-2 and monoravir to beagle dogs are shown (EX-2 = CH2101, monoravir = CH2017, NHC = CH2018).

[0032] Figure 2 The inhibitory activity curves of the test compounds against the SARS-CoV-2 omeprón B.1.1.529 variant are shown.

[0033] Figure 3 The changes in animal body weight in study p26262-15 are shown.

[0034] Figure 4 The clinical scores in study P26262-15 are shown.

[0035] Figure 5 The survival rate in study P26262-15 is shown.

[0036] Figure 6 The results show the pulmonary virus titer in study p26262-15.

[0037] Figure 7 The single plasma concentration-time data of CH2101 in beagle dogs after oral administration of 10 mg / kg CH2101 are shown.

[0038] Figure 8 Individual plasma concentration-time data for EX-1 / NHC / CH2018 in beagle dogs after oral administration of 10 mg / kg CH2101 are shown.

[0039] Figure 9 The single plasma concentration-time data of CH2101 in beagle dogs after oral administration of 20 mg / kg CH2101 are shown.

[0040] Figure 10 Individual plasma concentration-time data for EX-1 / NHC / CH2018 in beagle dogs after oral administration of 20 mg / kg CH2101 are shown.

[0041] Figure 11 This study presents single plasma concentration-time data for CH2107 (monoravir) in beagle dogs following oral administration of 22 mg / kg of CH2107 (monoravir).

[0042] Figure 12 Individual plasma concentration-time data for EX-1 / NHC / CH2018 in beagle dogs after oral administration of 22 mg / kg CH2107 (monoravir). Invention Details

[0044] definition

[0045] As used herein, words, phrases and symbols are generally intended to have the meanings described below, unless otherwise stated in the context in which they are used.

[0046] As used in this article, the singular forms “a,” “an,” and “the” are also intended to include the plural forms unless the context explicitly indicates otherwise.

[0047] The compounds of this invention can be identified by their chemical structure and / or chemical name. When the chemical structure and chemical name conflict, the chemical structure plays a decisive role in identifying the compound.

[0048] Symbols in this article or This refers to the related structures being tautomers, existing in equilibrium, and readily converting from one isomer to another. The compounds of this invention can exist in oxime and other forms. Therefore, the chemical structures described herein include all possible tautomer forms of the illustrated compounds, particularly oxime tautomers and other forms. Regardless of the tautomer shown or the nature of the equilibrium between the tautomers, those skilled in the art will understand that the compounds of this invention include oxime and other forms.

[0049] "Bioavailability" refers to the rate and amount of drug that reaches an individual's systemic circulation after administration of a drug or its prodrug. It can be determined by evaluating, for example, the plasma or blood concentration-time curve of the drug. Parameters that can be used to characterize the plasma or blood concentration-time curve include the area under the curve (AUC), the time to reach maximum concentration (Tmax), and the maximum drug concentration (Cmax). Cmax is the maximum concentration of the drug in an individual's plasma or blood after administration of a certain dose of the drug or its prodrug form, and Tmax is the time to reach the maximum concentration (Cmax) of the drug in an individual's plasma or blood after administration of a certain dose of the drug or its prodrug form.

[0050] A prodrug is a derivative form of a drug that, after administration, is converted or metabolized in the body to the active form of the parent drug. Prodrugs are used to modify one or more aspects of a drug's pharmacokinetics to improve the therapeutic effect of the parent drug. For example, prodrugs are often used to improve the oral bioavailability of a drug. To achieve a therapeutic effect, drugs with poor oral bioavailability may require frequent dosing, high doses, or may need to be administered via routes other than oral, such as intravenous administration. Examples of prodrugs that can be used to improve bioavailability include esters, optionally substituted esters, branched esters, and optionally substituted branched esters.

[0051] "Metabolic intermediates" refer to compounds formed in vivo through the metabolism of a parent compound, and which further react in vivo to release an active agent. Compounds of formula (I) are protected ester prodrugs that, upon metabolism in vivo, provide the corresponding metabolic intermediate, such as N4-hydroxycytidine (NHC). It is desirable that the reaction product or its metabolites are non-toxic.

[0052] "Individual" refers to mammals, such as humans.

[0053] "Pharmaceutical acceptable" means approved or permitted by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0054] A "pharmacologically acceptable salt" is a salt of a compound that possesses the pharmacological activity required by the parent compound. Such salts include acid addition salts, formed from an inorganic acid and one or more protonable functional groups from the parent compound, such as hydroxylamine. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. It can form salts with organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheponic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthalene acid, salicylic acid, stearic acid, mucoconic acid, etc. "Pharmaceutically acceptable salts" also include base addition salts formed by the compound of the present invention carrying the acidic moiety with pharmaceutically acceptable cations, such as sodium, potassium, calcium, aluminum, lithium, and ammonium.

[0055] As used herein, "drug combination" refers to a product consisting of a mixture or combination of one or more therapeutic agents, including fixed and non-fixed combinations of therapeutic agents. The term "fixed combination" means that the therapeutic agent (e.g., the compound of the present invention) and at least one other therapeutic agent are administered simultaneously to an individual in a single entity or dose. The term "non-fixed combination" means that the therapeutic agent (e.g., the compound of the present invention) and at least one other therapeutic agent are administered simultaneously, jointly, or sequentially to an individual as separate entities without a specific time limit, wherein such administration provides a therapeutically effective level of active agent within the individual.

[0056] "Prevention" refers to reducing the risk of acquiring a disease or disorder, such as a viral infection (even if an individual who may be exposed to the disease or is predisposed to the disease but has not yet experienced or shown symptoms of the disease does not exhibit at least one clinical symptom of the disease). In some embodiments, "prevention" refers to reducing the symptoms of the disease by taking the compound in a preventative manner. Therapeutic applications for the prevention of disease or disorder are referred to as prevention. The compounds provided in this disclosure can provide superior preventative effects due to their antiviral activity.

[0057] "Treatment" of a disease or disorder, such as a viral infection, means preventing or improving the disease or at least one clinical symptom of the disease or disorder, reducing the risk of acquiring the disease or at least one clinical symptom of the disease, reducing the development of the disease or at least one clinical symptom of the disease, or reducing the risk of developing the disease or at least one clinical symptom of the disease. "Treatment" also means suppressing a disease, which can be physical (e.g., stabilizing identifiable symptoms), physiological (e.g., stabilizing bodily parameters), or both, and suppressing at least one bodily parameter or manifestation, which may or may not be identifiable to the individual. "Treatment" also means delaying the onset of a disease (e.g., a viral infection), or at least one or more of its symptoms, in an individual who may be exposed to the disease or disorder or is predisposed to the disease or disorder, even if the subject has not yet experienced or displayed symptoms of the disease.

[0058] As used herein, the term "effective amount" refers to the amount of the compound of the present invention that is effective against viral infection in an individual as defined above for "treatment" or "prevention." An effective amount may cause any observable or measurable change in the individual as defined above for "treatment" or "prevention." An "effective amount" can vary depending on, for example, the compound, the disease and / or disease symptoms, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health status of the individual to be treated, and the judgment of the prescribing physician. In any given case, the appropriate amount can be determined by those skilled in the art or can be determined by routine testing.

[0059] As used herein, "alkyl" refers to a straight-chain or branched saturated hydrocarbon moiety, such as those containing 1-7 carbon atoms (C1-C2). 1-7Preferably, it has 1-6 carbon atoms (C). 1-6 ), 1-4 carbon atoms (C 1-4 ) or 1-3 carbon atoms (C 1-3 Those of the kind. For example, "C" 1-7 "Alkyl" refers to an alkyl group having 1 to 7 (including 1, 2, 3, 4, 5, 6, or 7) carbon atoms. A representative C... 1-7 Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, etc.

[0060] As used herein, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon moiety containing at least one double bond, for example, containing 2-7 carbon atoms (C2-C5). 2-7 ), 2-6 carbon atoms (C 2-6 ), 2-4 carbon atoms (C 2-4 ) or 2-3 carbon atoms (C 2-3 Those of the kind. For example, "C" 2-6 "Alkenyl" refers to an alkenyl group having 2-6 (including 2, 3, 4, 5, or 6) carbon atoms. A representative C... 2-6 Alkenyl groups include vinyl, propenyl, allyl, butenyl, pentenyl, etc.

[0061] As used herein, "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon moiety containing at least one triple bond, for example, containing 2-7 carbon atoms (C60-C75). 2-7 ), 2-6 carbon atoms (C 2-6 ), 2-4 carbon atoms (C 2-4 ) or 2-3 carbon atoms (C 2-3 Those of the kind. For example, "C" 2-6 "Alynyl" refers to an alkynyl group having 2-6 (including 2, 3, 4, 5, or 6) carbon atoms. A representative C... 2-6 Alkyne groups include ethynyl, propynyl, propynyl, butynyl, etc.

[0062] As used herein, "alkoxy" refers to -O-alkyl, wherein the alkyl group has the meaning defined above, for example, containing 1 to 7 carbon atoms (C... 1-7 ), 1 to 6 carbon atoms (C 1-6 ), 1-4 carbon atoms (C 1-4 ) or 1-3 carbon atoms (C 1-3 ) alkoxy groups. For example, "C 1-7 "Alkoxy group" refers to an alkoxy group having 1-7 (including 1, 2, 3, 4, 5, 6, or 7) carbon atoms. A representative C... 1-7Alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexyloxy, etc.

[0063] As used in this article, the term "cycloalkyl" refers to a ring containing 3 to 8 carbon atoms (C1 to C2). 3-8 For example, 3-6 ring carbon atoms (C 3-6 ) or 5-6 ring carbon atoms (C 5-6 The saturated cyclic hydrocarbon moiety of cycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, or bicyclic systems, including spirocyclic and bridged rings, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, spiro[3.4]octyl, bicyclic [3.1.1]hexyl, bicyclic [3.1.1]heptyl, or bicyclic [3.2.1]octyl. The term “halo-cycloalkyl” herein refers to a cycloalkyl group as defined above, wherein one or more, for example, 1, 2, or 3 hydrogen atoms are replaced by halogen atoms.

[0064] As used herein, the term "heterocyclic group" refers to a saturated ring having 3-12 ring atoms (3-12-membered), 3-10 ring atoms (3-10-membered), 3-6 ring atoms (3-6-membered), 4-6 ring atoms (4-6-membered), or 5-6 ring atoms (5-6-membered), wherein one or more, for example, 1, 2, 3, or 4, preferably 1 or 2 ring atoms are heteroatoms independently selected from N, O, and S, preferably O, and the remaining ring atoms are carbon. Examples of heterocyclic groups include, but are not limited to, morpholino, pyrrolidone, pyrrolyl, piperidinyl, ethylene oxide, propylene oxide, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidinyl, tetrahydrothiophene, tetrahydrothioranyl, tetrahydropyrimidinyl, 1,3-dioxolane ring moiety, etc. Preferably, the heterocyclic group is tetrahydrofuranyl or tetrahydropyranyl. For example, the heterocyclic group may be selected from the following groups:

[0065]

[0066] It should be understood that structures having one or more asymmetric centers encompass their racemic mixtures and / or single enantiomers or mixtures thereof. For example, structures Covering

[0067] As used herein, the term "heterocyclic group" also includes "heterocyclic alkenyl group," which refers to a "heterocyclic group" as defined herein that contains at least one (e.g., 1, 2, or 3) double bonds. Examples of heterocyclic alkenyl groups include, but are not limited to:

[0068]

[0069] Each W is selected from CH2, NH, O, and S; each Y is selected from NH, O, C (=O), SO2, and S; and each Z is selected from N and CH, provided that each ring contains at least one heteroatom selected from N, O, or S. For example, the heterocyclic alkenyl group is pyrrolinyl (e.g., 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, 4-pyrrolinyl, or 5-pyrrolinyl), dihydrofuranyl (e.g., 1-, 2-, 3-, or 4-dihydrofuranyl), dihydrothiophenyl (e.g., 1-, 2-, 3-, or 4-dihydrothiophenyl), tetrahydropyridyl (e.g., 1-, 2-, 3-, 4-, 5-, or 6-tetrahydropyridyl), tetrahydropyranyl (e.g., 4-tetrahydropyranyl), or tetrahydrothiophenyl (e.g., 4-tetrahydrothiophenyl).

[0070] As used herein, the term "aryl" refers to a monovalent aromatic hydrocarbon group obtained by removing a hydrogen atom from a single carbon atom in an aromatic ring system. An aryl group refers to a monocyclic or fused polycyclic aromatic ring structure having a specific number of ring atoms. Specifically, the term includes groups containing 6 to 14, for example 6 to 10, preferably 6 ring members. Representative aryl groups include phenyl and naphthyl, preferably phenyl. The term "aryl" also includes biaryl groups, such as biphenyl and naphthyl.

[0071] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic ring structure, or its N-oxide, or its S-oxide or S-dioxide, comprising one or more (e.g., 1, 2, 3, or 4) heteroatoms independently selected from O, N, and S and a specific number of ring atoms. Specifically, the aromatic ring structure may have 5 to 10 ring members. Typically, a heteroaryl ring contains up to 4 heteroatoms, up to 3 heteroatoms, up to 2 heteroatoms, or, for example, one heteroatom, independently selected from O, N, and S, wherein N and S may be in an oxidized state, such as S=O or S(O)2. For example, the heteroaryl group can be a fused ring containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, or S, such as benzofuran, benzothiophene, indole, benzimidazole, indole, benzotriazole, pyrrolo[2,3-b]pyridine, pyrrolo[2,3-c]pyridine, pyrazolo[4,3-c]pyridine, pyrazolo[3,4-c]pyridine, pyrazolo[3,4-b]pyridine, isoindole, purine, indazine, imidazo[1,2-a]pyridine. Imidazolo[1,5-a]pyridine, 1H-pyrazolo[3,4-d]pyrimidine, 7H-pyrrolo[2,3-d]pyrimidine, quinoline, isoquinoline, cinnarine, quinazoline, quinoxaline, phthalazine, 1,6-naphthidine, 1,7-naphthidine, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine, pyrimido[5,4-d]pyrimidine, pyrazido[2,3-b]pyrazine, and pyrimido[4,5-d]pyrimidine. For example, the heteroaryl group can be a 5- or 6-membered heteroaryl group containing one or two heteroatoms independently selected from N, O, or S. Examples of 5-6 membered monocyclic heteroaryl groups include, but are not limited to, pyrrole, furanyl, thiophene, imidazolyl, furazonyl, oxazolyl, oxadiazolyl, oxtriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and triazinyl.

[0072] The term "acyl" refers to the group Rx-(C=O)-, where Rx is C. 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 Alkyl, wherein each of the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups is optionally substituted by one or more substituents selected from the following groups: halogen, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7Alkyl group 2, -NH (acyl), -N (acyl) 2, amino-acyl, C 1-7 Alkyl, C 1-6 Alkoxy, halogenated -C 1-7 Alkyl or halogenated -C 1-7 Alkyl group.

[0073] The terms “halogen” and “halogenated” refer to fluorine, chlorine, bromine, or iodine.

[0074] The term “halogenated-alkyl” in this document means an alkyl group as defined herein, wherein one or more, such as 1, 2, 3, 4, 5, or all of the hydrogen atoms are replaced by halogen atoms.

[0075] The term "substitution" refers to the replacement of at least one hydrogen atom in a molecule by a substituent. When substituted, one or more groups are "substituents". Molecules can be multiple-substituted.

[0076] As used herein, the term “optionally” means that the event or situation subsequently described may or may not occur, and the description includes both the possibility that the event or situation will occur and the possibility that it will not occur.

[0077] The term "lower aliphatic alcohols" refers to C1-C4 alcohols, which represent aliphatic alcohols with 1-4 carbon atoms, such as methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, etc.

[0078] All numerical ranges in this document should be understood as disclosing every and all numerical values ​​within that range, as well as every and all subsets of numerical values ​​within that range, whether or not they are specifically disclosed. For example, when referring to any numerical range, it should be considered as referring to every and all numerical values ​​within that range, such as every and all integers within that range. This disclosure includes all numerical values ​​falling within these ranges, all smaller ranges, and the upper or lower bounds of those ranges.

[0079] The technical and scientific terms used herein, unless otherwise defined, have the meanings commonly understood by those skilled in the art as per the scope of this disclosure.

[0080] The implementation scheme disclosed herein

[0081] Implementation Scheme 1. Compound of Formula (I):

[0082]

[0083]

[0084] Or its tautomers, stereoisomers, or racemates or pharmaceutically acceptable salts thereof, wherein

[0085] R is Ra-(C=O)-;

[0086] Where Ra is selected from C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 Alkyl, wherein each of the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups is optionally substituted by one or more substituents selected from the following groups: halogen, acyl, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7 Alkyl)2, -CO-NH2, -CO-NH(C 1-7 Alkyl), -CO-N(C 1-7 Alkyl)2, -NH (acyl), -N (acyl)2, NH2-acyl, NHRy-acyl, N(Ry)2-acyl, C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-7 Alkyloxy, aryloxy, heteroaryloxy, halogenated-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy, halogenated -C 2-6 alkenyl, halogenated -C 2-6 alkynyl, hydroxyl-C 1-7 Alkyl, C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 3-8 cycloalkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 Cycloalkoxy or 3 to 12-membered heterocyclic hydroxyl groups,

[0087] Ry is independently selected from C 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 alkyl.

[0088] Preferably, Ra is selected from C 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 12-membered heterocyclic, wherein each is optionally substituted by one or more substituents selected from the following groups: halogen, acyl, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7 Alkyl)2, -CO-NH2, -CO-NH(C 1-7 Alkyl), -CO-N(C 1-7 Alkyl)2, -NH (acyl), -N (acyl)2, NH2-acyl, NHRy-acyl, N(Ry)2-acyl, C 1-7 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-7 Alkoxy, halogenated -C 1-7 Alkyl, Halogenated -C 1-7 Alkyloxy, aryloxy, heteroaryloxy, halogenated-C 2-6 alkenyl, halogenated -C 2-6 alkynyl, hydroxyl-C 1-7 Alkyl, C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 1-7 Alkoxy-C 1-7 Alkyl, Halogenated -C 3-8 cycloalkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 Cycloalkoxy or 3 to 12-membered heterocyclic hydroxyl groups,

[0089] Ry is independently selected from C 1-7 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, C 3-8 cycloalkyl-C 1-7 Alkyl, C 6-10 Aryl-C 1-7 Alkyl, 5 to 10-membered heteroaryl-C 1-7 Alkyl groups and 3 to 12-membered heterocyclic groups -C 1-7 alkyl.

[0090] Implementation Scheme 2. A compound of formula (I) according to Implementation Scheme 1, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein R is selected from the following groups:

[0091]

[0092]

[0093] Implementation Scheme 3. The compound according to Implementation Scheme 1, or its tautomer, stereoisomer, or racemic mixture, or a pharmaceutically acceptable salt thereof, wherein

[0094] R is Ra-(C=O)-,

[0095] Ra is a methyl group substituted with Ra1, Ra2 and Ra3;

[0096] Each of Ra1, Ra2, and Ra3 is independently selected from H and C. 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 1-6 Alkyl-O-(CH2) n -、C 1-7 Alkyl-O-aryl, C 1-7 Alkyl-O-heteroaryl, C 1-6 Alkyl-OC 1-6 Alkyl-O-(CH2) n -、C 1-6 Haloalkyl-O-(CH2) n -、C 3-6 Cycloalkyl-O-(CH2) n - and 3-6 membered heterocyclic groups -O-(CH2) n - wherein each of the alkyl, cycloalkyl, and heterocyclic groups is optionally substituted by one or more substituents selected from the following groups: halogen, acyl, hydroxyl, cyano, nitro, amino, -NH(C 1-7 alkyl), -N(C) 1-7 Alkyl)2, C 1-7 Alkyl, C 1-6 Alkoxy, halogenated -C 1-7 Alkyl or halogenated -C 1-7 alkoxy groups; and

[0097] n is 0 or 1.

[0098] Implementation Scheme 4. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0099] R is Ra-(C=O)-,

[0100] Ra-(C=O)- is selected from:

[0101]

[0102] Raa is selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl-OC 1-6 Alkyl-, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups; preferably C 1-6 alkyl.

[0103] Implementation Scheme 5. The compound according to Implementation Scheme 4, or its tautomer, stereoisomer, or racemate, or its pharmaceutically acceptable salt, wherein Ra-(C=O)- is selected from:

[0104]

[0105]

[0106] Raa is defined as above.

[0107] Implementation Scheme 6. The compound according to Implementation Scheme 4, or its tautomer, stereoisomer, or racemate, or its pharmaceutically acceptable salt, wherein Ra-(C=O)- is selected from:

[0108]

[0109] Raa is defined as above.

[0110] Implementation Scheme 7. The compound according to Implementation Scheme 4, or its tautomer, stereoisomer, or racemate, or its pharmaceutically acceptable salt, wherein Ra-(C=O)- is selected from:

[0111]

[0112] Raa is defined as above.

[0113] Implementation Scheme 8. The compound according to Implementation Scheme 4, or its tautomer, stereoisomer, or racemate, or its pharmaceutically acceptable salt, wherein Ra-(C=O)- is selected from:

[0114]

[0115] Raa is defined as above.

[0116] Implementation Scheme 9. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkyl-OC 1-4 Alkyl-, C 3-5 Cycloalkyl and 4-6 membered heterocyclic groups.

[0117] Implementation Scheme 10. A compound according to any one of the foregoing embodiments, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, 2-methoxyethyl, fluorinated ethyl, fluorinated propyl, cyclopropyl, cyclobutyl, cyclopentyl, glycidyl, tetrahydro-2-furanyl, tetrahydro-3-furanyl, or tetrahydro-2H-pyran-4-yl; preferably methyl, ethyl, propyl, isopropyl, glycidyl, and tetrahydro-2H-pyran-4-yl.

[0118] Implementation Scheme 11. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and sec-butyl.

[0119] Implementation Scheme 12. A compound according to any one of Implementation Schemes 1-3, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0120] Ra1 and Ra3 are independently selected from H and C. 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-;

[0121] Ra2 is selected from C 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-;

[0122] Alternatively, Ra2 and Ra3 together with the carbon atoms they are attached to form C. 3-6 Cycloalkyl, or a 5-6 membered halocyclic heterocyclic group containing a cyclic heteroatom selected from O.

[0123] Implementation Scheme 13. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0124] Ra1 is selected from C 1-6 Alkyl, C 1-6 Alkyl-O- and C1-6 Alkyl-O-CH2-.

[0125] Implementation Scheme 14. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0126] Ra3 is selected from C 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-.

[0127] Implementation Scheme 15. A compound according to any one of Implementation Schemes 1-3, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0128] Ra1 is C 1-6 Alkyl-O- or C 1-6 Alkyl-O-CH2-,

[0129] Ra2 is selected from C 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-, and

[0130] Ra3 is selected from H and C. 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-.

[0131] Implementation Scheme 16: A compound according to any one of Implementation Schemes 1-3, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0132] Ra1 is C 1-6 Alkyl-O-,

[0133] Ra2 is C 1-6 Alkyl, and

[0134] Ra3 is H or C 1-6 alkyl.

[0135] Implementation Scheme 17. A compound according to any one of Implementation Schemes 1-3, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0136] Ra1 is C 1-6 Alkyl-O-CH2-, and

[0137] Each of Ra2 and Ra3 is C 1-6 alkyl.

[0138] Implementation Scheme 18. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0139] Ra1 is C 1-6 Alkyl-O- or C 1-6 Alkyl-O-CH2-, and

[0140] Each of Ra2 and Ra3 is C 1-6 Alkyl-O-CH2-.

[0141] Implementation Scheme 19. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0142] Ra1 is C 1-6 Alkyl-O-CH2, and

[0143] One of Ra2 and Ra3 is C. 1-6 Alkyl group, and another one is C. 1-6 Alkyl-O-CH2.

[0144] Implementation Scheme 20. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0145] Ra1 is C 1-6 Alkyl-O-, and

[0146] Ra2 and Ra3 are independently C 1-3 Alkyl groups, preferably Ra2 and Ra3 are the same.

[0147] Implementation Scheme 21. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0148] Ra1 is C 1-6 Alkyl-O- or C 1-6 Alkyl-O-CH2-, and

[0149] Ra2 and Ra3, together with the carbon atoms they are attached to, form C. 3-6 Cycloalkyl.

[0150] Implementation Scheme 22. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein

[0151] Ra1 is selected from H and C. 1-6 Alkyl, C 1-6 Alkyl-O- and C1-6 Alkyl-O-CH2-; and

[0152] Ra2 and Ra3, together with the carbon atoms to which they are attached, form 5-6 membered halocyclic heterocycles containing one cyclic heteroatom selected from O;

[0153] Preferably, Ra1 is selected from C 1-6 Alkyl, C 1-6 Alkyl-O- and C 1-6 Alkyl-O-CH2-.

[0154] Implementation Scheme 23. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 1 It is RaC=O, and R 2 and R 3 Each of them is H.

[0155] Implementation Scheme 24. A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 1 R 2 and R 3 Each of them is RaC=O.

[0156] Implementation Scheme 25: A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein Ra1 is C 1-6 Alkyl-O-.

[0157] Implementation Scheme 26: A compound according to any one of the foregoing implementation schemes, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, wherein Ra-(C=O)- is selected from:

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Implementation Scheme 27. The compound according to Implementation Scheme 1, or its tautomer, stereoisomer, or racemic mixture, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Implementation Scheme 28. A method for preparing a compound of formula I according to any one of Implementation Schemes 1-27, comprising the following steps:

[0193] Reaction of NHC with the anhydride of formula II yields the compound of formula I.

[0194]

[0195] Ra is defined as in any of the implementation schemes 1-27.

[0196] Implementation Scheme 29. The method according to Implementation Scheme 28, wherein the reaction is carried out in water or a mixture of water and an organic solvent, preferably the reaction solvent is selected from pure water, methanol, ethanol, propanol, isopropanol, other lower aliphatic alcohols or mixtures of aliphatic alcohols, DMF, DMSO, NMP, water-methanol mixture, water-ethanol mixture, water-propanol mixture, water-isopropanol mixture, water-n-butanol mixture, water-sec-butanol mixture, water-isobutanol mixture, water-THF mixture, water-ACN mixture, water-DMF mixture, water-DMSO mixture, water / 2-methylTHF mixture, or any mixture of water and an organic solvent capable of completely or partially dissolving NHC; more preferably water, lower aliphatic alcohols, water-lower aliphatic alcohol mixtures, water-THF mixtures, water / 2-methylTHF mixtures, water / ACN mixtures.

[0197] Implementation Scheme 30. The method according to Implementation Scheme 28 or 29, wherein the reaction is carried out without the addition of any inorganic or organic base (or catalyst), such as alkali metal hydroxide, carbonate, bicarbonate, alkoxide, or hydride, such as sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, sodium ethoxide, or sodium hydride, or organic tertiary amine, such as tri-C 1-4 Alkylamines, such as TEA, diisopropylethylamine, tripropylamine, tributylamine, or heterocyclic bases, such as pyridine, methylpyridine, dimethylpyridine, DMAP, DBU, etc.

[0198] Implementation Scheme 31. The method according to Implementation Scheme 28 or 29, wherein the reaction is carried out in the presence of an inorganic or organic base (or catalyst), such as an alkali metal hydroxide, carbonate, bicarbonate, alkoxide, or hydride, such as sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, sodium ethoxide, or sodium hydride, or an organic tertiary amine, such as tri-C 1-4 Alkylamines, such as TEA, diisopropylethylamine, tripropylamine, tributylamine, or heterocyclic bases, such as pyridine, methylpyridine, dimethylpyridine, DMAP, DBU, etc.

[0199] Implementation Scheme 32. The method according to any one of Implementation Schemes 28-31, wherein the product is obtained in solid crystalline form by cooling the reaction mixture without adding an antisolvent.

[0200] Implementation Scheme 33. The method according to any one of Implementation Schemes 28-32, wherein the product is obtained in solid crystalline form without any chromatographic purification.

[0201] Implementation Scheme 34. The method according to any one of Implementation Schemes 28-33, wherein the purity of the product generated in the reaction solution is approximately 90%-98%.

[0202] Implementation Scheme 35. The method according to any one of Implementation Schemes 28-33, wherein the purity of the product generated in the reaction solution exceeds 98%.

[0203] Implementation Scheme 36. A pharmaceutical composition comprising any one of the compounds of embodiments 1-27 or their tautomers, stereoisomers or racemates or their pharmaceutically acceptable salts, and optionally comprising a pharmaceutically acceptable excipient.

[0204] Implementation Scheme 37. Use of any compound of any one of Implementation Schemes 1-27, or its tautomer, stereoisomer, or racemate, or its pharmaceutically acceptable salt, in the preparation of a medicament for the treatment or prevention of RNA virus infection.

[0205] Implementation Scheme 38. As per the use of Implementation Scheme 37, wherein the RNA virus is a coronavirus, such as human coronavirus, SARS coronavirus, or MERS coronavirus; an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, Ross River virus, or Balma Forest virus; a filoviridae virus, such as Ebola virus; an orthomyxoviridae virus, such as influenza virus, influenza A virus, or influenza B virus; a paramyxoviridae virus, such as respiratory syncytial virus (RSV); a flavivirus, such as Zika virus or Poissan virus; preferably SARS-CoV-2 / COVID-19 virus, alpha variant SARS-CoV-2 / COVID-19 virus, beta variant SARS-CoV-2 / COVID-19 virus, gamma variant SARS-CoV-2 / COVID-19 virus, delta variant SARS-CoV-2 / COVID-19 virus, or any other variant SARS-CoV-2 / COVID-19 virus.

[0206] Implementation Scheme 39. A method for treating or preventing an individual from being infected with an RNA virus, comprising administering to the individual in need an effective amount of any one of Implementation Schemes 1-27, or a tautomer, stereoisomer, or racemic mixture thereof, or a pharmaceutically acceptable salt thereof.

[0207] Implementation Scheme 40. The method according to Implementation Scheme 39, wherein the RNA virus is a coronavirus, such as human coronavirus, SARS coronavirus or MERS coronavirus; an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus or Ross River virus; a fibrinoviridae virus, such as Ebola virus; an orthomyxoviridae virus, such as influenza virus, influenza A virus or influenza B virus; a paramyxoviridae virus, such as respiratory syncytial virus (RSV); a flavivirus, such as Zika virus; preferably SARS-CoV-2 / COVID-19 virus, alpha variant SARS-CoV-2 / COVID-19 virus, beta variant SARS-CoV-2 / COVID-19 virus, gamma variant SARS-CoV-2 / COVID-19 virus, delta variant SARS-CoV-2 / COVID-19 virus or any other variant SARS-CoV-2 / COVID-19 virus.

[0208] Implementation Scheme 41. A compound of any one of Implementation Schemes 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, used as a medicine.

[0209] Implementation Scheme 42. A compound of any one of Implementation Schemes 1-27 or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, used for the treatment or prevention of RNA virus infection.

[0210] Implementation Scheme 43. The compound for the stated use according to Implementation Scheme 42, wherein the RNA virus is a coronavirus, such as human coronavirus, SARS coronavirus, or MERS coronavirus; an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, or Ross River virus; a filoviridae virus, such as Ebola virus; an orthomyxoviridae virus, such as influenza virus, influenza A virus, or influenza B virus; a paramyxoviridae virus, such as respiratory syncytial virus (RSV); a flavivirus, such as Zika virus; preferably SARS-CoV-2 / COVID-19 virus, alpha variant SARS-CoV-2 / COVID-19 virus, beta variant SARS-CoV-2 / COVID-19 virus, gamma variant SARS-CoV-2 / COVID-19 virus, delta variant SARS-CoV-2 / COVID-19 virus, or any other variant SARS-CoV-2 / COVID-19 virus.

[0211] Implementation Plan 44. For increasing N for the treatment or prevention of RNA virus infection. 4 A method for the bioavailability of -hydroxycytidine, comprising administering to an individual in need an effective amount of any one of embodiments 1-27 of the compound or its tautomer, stereoisomer or racemate or its pharmaceutically acceptable salt.

[0212] Implementation Scheme 45. A pharmaceutical combination comprising a compound of any one of Implementation Schemes 1-27 or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent.

[0213] Implementation Plan 46. The drug combination according to Implementation Plan 45, wherein the additional therapeutic agents are selected from:

[0214]

[0215] How to use

[0216] According to this disclosure, RNA viruses are coronaviruses, such as human coronaviruses, SARS coronaviruses, or MERS coronaviruses; alpha viruses, such as eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, or Ross River virus; fibrinoviridae viruses, such as Ebola virus; orthomyxoviridae viruses, such as influenza viruses, influenza A viruses (including H1N1, H3N2, H7N9, or H5N1 subtypes), influenza B viruses, or influenza C viruses; paramyxoviridae viruses, such as respiratory syncytial virus (RSV); and flaviviruses, for example... Such as Zika virus, rotavirus, such as rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E; preferably SARS-CoV-2 / COVID-19 virus, α variant SARS-CoV-2 / COVID-19 virus, β variant SARS-CoV-2 / COVID-19 virus, γ variant SARS-CoV-2 / COVID-19 virus, δ variant SARS-CoV-2 / COVID-19 virus or any other variant SARS-CoV-2 / COVID-19 virus.

[0217] Preferably, according to this disclosure, the RNA virus is a human coronavirus, SARS coronavirus, MERS coronavirus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, Ross River virus, orthomyxoviridae virus, paramyxoviridae virus, RSV virus, influenza A virus, influenza B virus, filoviridae virus, or Ebola virus.

[0218] More preferably, according to the present invention, the RNA virus is a human coronavirus, SARS-CoV-2 / COVID-19 virus, α variant SARS-CoV-2 / COVID-19 virus, β variant SARS-CoV-2 / COVID-19 virus, γ variant SARS-CoV-2 / COVID-19 virus, δ variant SARS-CoV-2 / COVID-19 virus, or any other variant SARS-CoV-2 / COVID-19 virus.

[0219] According to this disclosure, an individual is at risk of infection with the following viruses, exhibits symptoms of the following viral infections, or is diagnosed with the following viral infections: SARS-CoV-2 / COVID-19 virus, influenza A virus including subtypes H1N1, H3N2, H7N9, or H5N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, human coronavirus, SARS coronavirus, MERS coronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, Coxsackie A virus, norovirus, rubella virus, lymphocytic choriomeningitis virus (LCMV), dengue virus, Zika virus, and so on. Kunkenya virus, Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Ross River Virus, Balma Forest Virus, Yellow Fever Virus, Measles Virus, Mumps Virus, Respiratory Syncytial Virus, Rinderpest Virus, California Encephalitis Virus, Hantavirus, Rabies Virus, Ebola Virus, Marburg Virus, Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2), Varicella-Zoster Virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Herpes Lymphotropic Virus, Roseola Virus or Kaposi's Sarcoma-Associated Herpesvirus, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus or Human Immunodeficiency Virus (HIV), Human T-Lymphotropic Virus Type I (HTLV-1), Flanders Splenic Focal Lesion Virus (SFFV) or Heterophilic MuLV-Associated Virus (XMRV). In some implementation schemes, an individual is at risk of Zika virus infection, exhibits symptoms of Zika virus infection, or is diagnosed with Zika virus infection.

[0220] According to the present invention, an individual is diagnosed with SARS-CoV-2 / COVID-19 virus infection, including α variant SARS-CoV-2 / COVID-19 virus, β variant SARS-CoV-2 / COVID-19 virus, γ variant SARS-CoV-2 / COVID-19 virus, δ variant SARS-CoV-2 / COVID-19 virus or any variant SARS-CoV-2 / COVID-19 virus infection, which can be treated by a compound of formula (I) or a drug containing a compound of formula (I).

[0221] According to the present invention, an individual is diagnosed with influenza A virus, including subtypes H1N1, H3N2, H7N9, H5N1 (low path) and H5N1 (high path), influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, MERS-CoV, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, Coxsackie A virus, norovirus, rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, and respiratory syncytial virus. The following viruses are contraindicated: parainfluenza virus 1 and 3, rinderpest virus, chikungunya virus, eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), California encephalitis virus, Japanese encephalitis virus, Rift Valley fever virus (RVFV), Hantavirus, dengue virus serotypes 1, 2, 3, and 4, Zika virus, West Nile virus, tacarribé virus, Junin virus, rabies virus, Ebola virus, Marburg virus, adenovirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes-lymphotropic virus, rose virus or Kaposi's sarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, or human immunodeficiency virus (HIV) infection. In some implementations, an individual is diagnosed with Zika virus infection.

[0222] According to the present invention, an individual is diagnosed with gastroenteritis, acute respiratory disease, severe acute respiratory syndrome, post-viral fatigue syndrome, viral hemorrhagic fever, acquired immunodeficiency syndrome, or hepatitis.

[0223] Pharmaceutical composition and administration

[0224] The compounds of the present invention (such as any of the compounds in the examples herein) can be formulated into pharmaceutical compositions, alone or in combination with one or more other therapeutic agents. A pharmaceutical composition comprises: (a) an effective amount of the compound of the present invention; (b) a pharmaceutically acceptable excipient (such as one or more pharmaceutically acceptable carriers); and optionally (c) at least one other therapeutic agent.

[0225] Pharmaceutically acceptable excipients are those that are compatible with (and in some embodiments, can stabilize) the active ingredient in a composition and are harmless to the individual to be treated. Suitable pharmaceutically acceptable excipients are disclosed in standard references in the art (e.g., Remington's Pharmaceutical Sciences, Remington: The Science and Practice of Pharmacy.) and include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavoring agents, tasters, diluents, and other known additives to provide a perfect presentation of the medicament (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to aid in the preparation of a pharmaceutical product (i.e., a drug).

[0226] The compounds of this invention can be administered in a variety of known ways, such as orally, enterically, by inhalation or through the lungs (i.e., pulmonary administration), nasally, sublingually, through the tongue, oral cavity, rectum, skin, transdermal, conjunctival, ear, or as implants or scaffolds. The term "entrantly" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion.

[0227] Oral or parenteral administration is preferred, especially oral administration.

[0228] The compounds of this invention can be administered in any convenient formulation, such as tablets, powders, capsules, pills, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, aqueous buffers, such as saline or phosphate buffers. Such compositions may contain conventional components of pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, fillers, and additional active agents.

[0229] Typically, it has been found that, in the case of parenteral administration, the dosage is about 0.001 to 20 mg / kg body weight, preferably about 0.01 to 10 mg / kg body weight, to achieve an effective effect. In the case of oral administration, the dosage is about 0.01 to 100 mg / kg body weight, preferably about 0.01 to 20 mg / kg body weight, and most preferably 0.1 to 15 mg / kg body weight.

[0230] Combination therapy

[0231] The compounds described herein can be administered adjunctly with at least one other therapeutic agent.

[0232] Other therapeutic agents include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraines, antimuscarinic agents, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, antiasthmatics, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastrointestinal drugs, muscle relaxants, nutritional supplements, vitamins, parasympathomimetic agents, stimulants, anorexia nervosa, antisleep aids, and antiviral agents. In particular embodiments, antiviral agents are non-CNS-targeting antiviral compounds. As used herein, "adjunctive administration" means that the compound can be administered with one or more other active agents in the same or different dosage forms. Other therapeutic agents may be formulated for immediate release, controlled release, or a combination thereof.

[0233] The compounds and pharmaceutical compositions of the present invention can be administered in combination with at least one other therapeutic agent, such as an antiviral agent, such as abacavir, acyclovir, adefovir, amantadine, amprenavir, amprolium, arbidol, atazanavir, atripla, balapiravir, BCX4430, berberine, cidofovir, dapoxetine, daclatasvir, darunavir, dasabuvir, deraviridine, didanoxin, docosanol, eduridine, efavirenz, emtricitabine, enfuviride, entecavir, famciclovir, favipiravir, fomivir, fosanavir, phosphonoformic acid, sodium phosphonoacetate, ganciclovir, GS-5734, ibatabin, isopromine, idoxuridine, imiquimod, indinavir, inosine, type III interferon, type II interferon, and type I interferon. Lamivudine, Leadipasvir, Lopinavir, Loviramide, Malawiro, Moroxydine, Mefenoxate, Nafinavir, Nevirapine, Nexavir, NITD008, Orbitavir, Oseltamivir, Palipvir, Pegylated Interferon Alpha-2a, Penciclovir, Peramivir, Procanalil, Podophyllotoxin, Retegvir, Ribavirin, Amantadine, Ritonavir, Pyramid Including ine, saquinavir, cimetvir, sofosbuvir, stavudine, telbivudine, tenofovir, tenofovir disoproxil fumarate, tenofovir Exalidex, telanavir, trifluuridine, triamcinolone, triamcinolone, tremafenamide, truvada, valacyclovir, valganciclovir, velivirol, vidarabine, viramidin, zalcitabine, zanamivir, monorapirvir, or zidovudine and combinations thereof.

[0234] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with any of the compounds disclosed in WO2012119559 for the treatment of SARS-CoV-2 / COVID-19 infection.

[0235] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with any of the compounds disclosed in WO2012119559 for the prevention of SARS-CoV-2 / COVID-19 infection.

[0236] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with proxalutamide for the treatment of SARS-CoV-2 / COVID-19 infection.

[0237]

[0238] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with proxalutamide for the prevention of SARS-CoV-2 / COVID-19 infection.

[0239] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with compound-X for the treatment of SARS-CoV-2 / COVID-19 infection.

[0240]

[0241] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with compound-X for the prevention of SARS-CoV-2 / COVID-19 infection.

[0242] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with PF-07321332 for the treatment of SARS-CoV-2 / COVID-19 infection.

[0243]

[0244] The compounds and pharmaceutical compositions disclosed herein can be administered in combination with PF-07321332 for the prevention of SARS-CoV-2 / COVID-19 infection.

[0245] Therefore, this disclosure also provides pharmaceutical combinations comprising the compounds of the present invention and at least one additional therapeutic agent. Examples of additional therapeutic agents include, but are not limited to, the active agents mentioned above, preferably proxalutamide, compound-X, and PF-07321332.

[0246] Unless otherwise stated, percentages in the tests and examples below are weight percentages; portions are weight portions. Solvent ratios, dilution ratios, and concentration data for liquid / liquid solutions are on a volume basis in each case.

[0247] Each embodiment and technical solution described in this disclosure, as well as the features in each embodiment and technical solution, should be understood as being able to be combined with each other in any way, and those technical solutions obtained by such combinations are included within the scope of this disclosure, just as each technical solution obtained by such combinations is specifically and separately listed, unless the context clearly shows otherwise.

[0248] To the extent permitted by law, all patents, patent applications, publications, and other references cited or mentioned herein are incorporated herein by reference in their entirety. The discussion of these references is solely for the purpose of summarizing the arguments presented therein. No such patent, patent application, publication, or reference, or any part thereof, is acknowledged as relevant material or prior art. The right to challenge the accuracy and relevance of any claims made regarding these patents, patent applications, publications, and other references as relevant material or prior art is specifically reserved. Example

[0249] The examples listed below are intended to illustrate compositions, methods, and results based on the disclosed subject matter. These examples are not intended to encompass all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the invention, as will be apparent to those skilled in the art.

[0250] Efforts have been made to ensure the accuracy of numerical values ​​(e.g., quantities, temperatures, etc.), but some errors and deviations should be taken into account. Unless otherwise stated, parts are weight portions. Many variations and combinations of reaction conditions, such as component concentrations, temperatures, pressures, and other reaction ranges and conditions, can be used to optimize the purity and yield of the product obtained from the process. Such process conditions can be optimized with only reasonable routine experiments.

[0251] Unless otherwise stated, all reagents and starting materials used in this invention are commercially available or prepared according to prior art.

[0252] 1 ¹H NMR spectra were measured on a Bruker 400MHz instrument. Chemical shifts were measured relative to the corresponding solvent peaks: CDCl₃ (δ 7.27), DMSO-d₆ (δ 2.50), CD₃OD (δ 3.31), D₂O (δ 4.79). The following abbreviations are used to describe coupling: s = singlet, d = doublet, t = triplet, q = quartet, quintet, m = multiplet, br = broad peak. 13 C NMR spectra were measured at 100 MHz on a Bruker instrument, with chemical shifts measured relative to the corresponding solvent peaks: CDCl3 (δ77.0), DMSOd6 (δ39.5), CD3OD (δ49.0).

[0253] Abbreviations and acronyms:

[0254] aq. aqueous solution

[0255] calc. calculated value

[0256] br s broad singlet (in NMR)

[0257] Direct chemical ionization of DCI (in MS)

[0258] decomposition point

[0259] DMF (dimethylformamide)

[0260] DMSO (dimethyl sulfoxide)

[0261] DSC differential scanning calorimetry

[0262] eq. equivalent

[0263] ESI electrospray ionization (in MS).

[0264] Et ethyl

[0265] fnd. Measured value

[0266] h hours

[0267] HPLC (High-Performance Liquid Chromatography)

[0268] HRMS high-resolution mass spectrometry

[0269] Conc. concentrated

[0270] LC-MS (Liquid Chromatography-Coupling Mass Spectrometry)

[0271] LiHMDS hexamethyldisilamide lithium

[0272] Memethyl

[0273] Min minutes

[0274] MS mass spectrometry

[0275] NMR nuclear magnetic resonance spectroscopy

[0276] Pd2 dba3 tris(dibenzylacetone)dipalladium

[0277] Phphenyl

[0278] PLM polarization microscope

[0279] RT room temperature

[0280] Rt retention time (in HPLC)

[0281] TGA thermogravimetric analysis

[0282] THF Tetrahydrofuran

[0283] UV spectroscopy

[0284] v / v volume ratio (for solutions)

[0285] Preparation of starting materials and intermediates

[0286] Preparation 1: Synthesis of alkoxy-substituted propionic acid and anhydride

[0287]

[0288] Synthesis methods of (R)-2-methoxypropionic acid (I-3) and acid anhydride (I-4):

[0289] Under nitrogen atmosphere, (S)-2-chloropropionic acid (80.0 g, 738 mmol, 1 equivalent, 98%) was added to a two-necked round-bottom flask. 25 wt% sodium methoxide (506 mL, 2.212 mol, 3 equivalents) was slowly added. The reaction was heated to 60 °C for 16 h, and the conversion was monitored until the remaining starting material was <2%. When sufficient conversion was achieved, the reaction vessel was cooled to room temperature, and the pH was adjusted with 4 M hydrochloric acid (200 mL, 99%) in dioxane to just change from >12 to 7, indicating that excess sodium methoxide was neutralized without protonating the sodium carboxylate. The reaction mixture was filtered to remove the salt, and the salt cake was washed twice with 5 mL of methanol. The filtrate was concentrated, redissolved in water, acidified with 6 M HCl to pH ~2, and extracted with EtOAc. The organic layer was dried over sodium sulfate and concentrated to give compound (I-3) (73 g, 95%), a liquid of sufficient purity for use without further purification. 1 H NMR (CD3OD) δ 3.67 (q, 1H), 3.33 (s, 3H), and 1.33 ppm (d, 3H).

[0290] In a 2-liter four-necked glass reactor equipped with a thermometer and a stirrer, 500 g of dichloromethane, 104.1 g (1.0 mol) (R)-2-methoxypropionic acid (3) and 57.3 g (0.5 mol) methanesulfonyl chloride were charged under a nitrogen atmosphere. The mixture was cooled to 5 °C.

[0291] Then, 101.3 g (1.0 mol, equivalent to 1 equivalence of the acid generated from methanesulfonyl chloride) of triethylamine was added dropwise over 2 hours, with the temperature of the reaction mixture controlled at 30 °C or lower. After the addition was complete, the mixture was stirred for 1 hour at the same temperature. Gas chromatography (GC) analysis of the reaction mixture showed that the conversion of (R)-2-methoxypropionic acid (3) was >95%.

[0292] After the reaction was complete, 200 g of water was added to the reaction mixture to wash it. The reaction mixture was washed twice more, each time with 200 g of water, followed by distillation to remove dichloromethane. 85.6 g of (R)-2-methoxypropionic anhydride (I-4), a yellow liquid, was obtained and used in the acylation step without further purification.

[0293] The following 2-alkoxy-substituted propionic anhydrides were prepared in the same manner as described in Preparation 1.

[0294]

[0295] Preparation 2: Synthesis of alkoxy-substituted isobutyric acid and acid anhydrides

[0296] Synthetic methods for 2-ethoxyisobutyric acid / 2-ethoxy-2-methylpropionic acid (I-21) and acid anhydride (I-22):

[0297]

[0298] 2-Ethoxyisobutyric acid was prepared according to the references (Ragan, John A.; Ide, Nathan D.; Cai, Weiling; Cawley, James J.; Colon-Cruz, Roberto; Kumar, Rajesh; Peng, Zhihui; Vanderplas, Brian C. [Organic process research and development, 2010, Vol. 14, #6, pp. 1402-1406]): In a 500 mL three-necked round-bottom flask, 2-bromo-2-methylpropionic acid (I-20) (40 g, 239.5 mmol) was dissolved in ethanol (320 mL) and cooled to 0–5 °C. Then, DIPEA (87.4 mL, 502.9 mmol) was added dropwise at 0–5 °C, and the reaction mixture was stirred at 0 °C for 30 minutes. The reaction mixture was then warmed to room temperature for 16 hours. After 16 hours, the reaction mixture was cooled to room temperature, and the ethanol was removed under vacuum, leaving a thick white slurry. Add diethyl ether and water to the slurry and cool to 0°C. Acidify the mixture with 10% HCl (50 mL), separate the organic layer, and wash with brine. Add 10% NaHSO3 aqueous solution to the organic phase and stir the mixture at room temperature for 6 hours. Acidify the biphase mixture with 10% HCl (50 mL) to pH 1.0 ± 0.5. Wash the organic phase with brine (100 mL), dry over sodium sulfate, filter, and concentrate to give 30 g of 2-ethoxy-2-methylpropionic acid (I-21). The product 2-ethoxy-2-methylpropionic acid (I-21) is used in the next step without further purification.

[0299] In a 2-liter four-necked glass reactor equipped with a thermometer and a stirrer, 300 g of dichloromethane, 66.1 g (0.5 mol) of 2-ethoxy-2-methylpropionic acid (I-21) and 28.65 g (0.25 mol) of methanesulfonyl chloride were charged under a nitrogen atmosphere. The mixture was cooled to 5°C.

[0300] Then, 50.65 g (0.5 mol, equivalent to 1 equivalence of the acid generated from methanesulfonyl chloride) of triethylamine was added dropwise over 2 hours, with the temperature of the reaction mixture controlled at 30 °C or lower. After the addition was complete, the mixture was stirred for 1 hour at the same temperature. Gas chromatography (GC) analysis of the reaction mixture showed that the conversion of 2-ethoxy-2-methylpropionic acid (I-21) was >95%.

[0301] After the reaction was complete, 100 g of water was added to the reaction mixture to wash it. The reaction mixture was washed twice more with 100 g of water each time, followed by distillation to remove dichloromethane. 51 g of 2-ethoxy-2-methylpropionic anhydride (I-22), a yellow liquid, was obtained and used in the acylation step without further purification.

[0302] The following 2-alkoxy-substituted 2-methylpropionic acid / 2-alkoxy-substituted isobutyric anhydride were prepared in the same manner as in Preparation 1:

[0303]

[0304] Preparation of 3:4-alkoxytetrahydro-2H-pyran-4-carboxylic acid and its anhydride

[0305] Synthetic methods for 4-methoxytetrahydro-2H-pyran-4-carboxylic acid (I-36) and its anhydride (I-37):

[0306] Commercially available methyl tetrahydro-2H-pyran-4-carboxylate was brominated according to the method described in Organic Letters, 2020, Vol. 22, #10, pp. 3922-3925. The ester was then hydrolyzed to the corresponding α-bromic acid (I-35). The α-bromic acid (I-35) was then converted to the corresponding acid (I-36) and anhydride (I-37) according to the method described in Preparation 2.

[0307]

[0308] The following 4-alkoxytetrahydro-2H-pyran-4-carboxylic acid and anhydride were prepared by a similar method.

[0309]

[0310] Preparation of 4:4-alkyltetrahydro-2H-pyran-4-carboxylic acid and anhydride: Synthetic methods for 4-methyltetrahydro-2H-pyran-4-carboxylic acid (I-46) and anhydride (I-47):

[0311]

[0312] Commercially available methyl tetrahydro-2H-pyran-4-carboxylate (I-33) was methylated in the same manner as described in Example 64.1A of US9434690. The methyl ester was then hydrolyzed with aqueous NaOH and acidified with HCl to give 4-methyltetrahydro-2H-pyran-4-carboxylic acid (I-46), an off-white solid.

[0313] 4-Methyltetrahydro-2H-pyran-4-carboxylic anhydride (I-47) was prepared according to method 2 and was a pale yellow oil.

[0314] The following 4-alkyltetrahydro-2H-pyran-4-carboxylic anhydrides were prepared by a similar method.

[0315]

[0316]

[0317] Preparation of 5: Synthesis of 2-ethyl-2-alkoxy-butyric acid and acid anhydride

[0318] Synthesis of 2-ethyl-2-methoxy-butyric acid (I-62) and acid anhydride (I-63):

[0319]

[0320] 2-Ethyl-2-bromo-butyric acid (I-61) is commercially available, or can be prepared according to the method described by Doran and Shonle in Journal of Organic Chemistry, 1938, Vol. 3, p. 195.

[0321] 2-Ethyl-2-bromo-butyric acid (I-61) is first converted to ethyl-2-methoxy-butyric acid (I-62), and then to ethyl-2-methoxy-butyric anhydride (I-63), which is a pale yellow oil, as described in preparation 2.

[0322] The following acids and anhydrides are prepared in a similar manner.

[0323]

[0324] Preparation of 6: Synthesis of 2-methyl-2-alkoxy-butyric acid and acid anhydride

[0325] A method for synthesizing 2-methyl-2-methoxybutyric acid (I-72) and anhydride (I-73). Commercially available (R,S)-2-hydroxy-2-methylbutyric acid (I-70) was resolved into enantiomeric pure R and S isomers (I-71), which were then esterified to a methyl ester (I-72) according to the method described in Preparation 74 of US2008114005.

[0326]

[0327] Alternatively, according to the method disclosed in Preparation 2, commercially available 2-bromo-2-methylbutyric acid is converted to (R,S)-2-methoxy-2-methylbutyric acid (I-78), and (I-78) is resolved into enantiomers (I-80) and (I-77) according to the method described in Preparation 74 of US2008114005. Then, the chiral acid (I-75) is converted to an anhydride (I-76) as described in Preparation 2, resulting in an oily substance.

[0328]

[0329] The following acids and anhydrides are prepared in a similar manner.

[0330]

[0331]

[0332] Preparation of 7: Synthesis of 2-alkyltetrahydrofuran-2-carboxylic acid and acid anhydride.

[0333] Synthesis of 2-methyltetrahydrofuran-2-carboxylic acid (I-112), (I-114) and its anhydrides (I-113), (I-115): Enantiomerically pure 2-methyltetrahydrofuran-2-carboxylic acid (I-112) and (I-114) were prepared according to the method described by Pohl; Wollweber in the European Journal of Medicinal Chemistry, 1976, Vol. 11, pp. 163, 168, 169. The acid was then converted to the corresponding anhydrides (I-113) and (I-115) in a manner similar to that described in Preparation 2.

[0334]

[0335] The following 2-alkyltetrahydrofuran-2-carboxylic acid and anhydride are prepared by a similar method.

[0336]

[0337] Preparation of 8: Synthesis of 2-methyl-2-alkoxymethylpropionic acid and acid anhydride.

[0338] Synthetic methods for 2-methyl-2-methoxymethylpropionic acid (I-130) and its anhydride (I-131):

[0339]

[0340] First, commercially available methyl 2-methyl-2-hydroxymethylpropionate (I-128) is methylated, and then the ester is hydrolyzed using the methods described in Examples 55 and 56 of WO2009 / 77608, 2009 to give 2-methyl-2-methoxymethylpropionate (I-130).

[0341] Then, according to the method of preparation 2, 2-methyl-2-methoxymethylpropionic acid (I-130) was converted into anhydride (I-131) to obtain an oily substance.

[0342] The following 2-methyl-2-alkoxymethylpropionic anhydride was prepared by a similar method.

[0343]

[0344]

[0345] Preparation 9: Synthesis of 1-alkyl-2,2-dialkoxy-isobutyric acid and acid anhydride

[0346] Synthetic methods for 1-methyl-2,2-dimethoxy-isobutyric acid (I-142) and its anhydride (I-143):

[0347]

[0348] 1-Methyl-2,2-dimethoxy-isobutyric acid (I-142) was prepared according to the method described in Reference Example 14 of US2004248941.

[0349]

[0350] Alternatively, 1-methyl-2,2-dimethoxy-isobutyric acid (I-142) can be prepared from commercially available 2,2-di(hydroxymethyl)propionic acid according to Reference Example 14 of EP1437352.

[0351] Then, following the method of preparation 2, 1-methyl-2,2-dimethoxy-isobutyric acid (I-142) was converted into an anhydride (I-143) to obtain an oily substance.

[0352] The following 1-alkyl-2,2-dialkoxy-isobutyric acid and anhydride were prepared by a similar method.

[0353]

[0354]

[0355]

[0356]

[0357] Synthesis of 10:1-(alkoxymethyl)cyclopropane-1-carboxylic acid and its anhydride.

[0358] Synthesis of 1-(methoxymethyl)cyclopropane-1-carboxylic acid (I-225) and its anhydride (I-226):

[0359]

[0360] Methyl 1-(hydroxymethyl)cyclopropane-1-carboxylic acid (I-223) was prepared according to the method described in Reference Example 22-1 of US9546155. The hydroxyl groups were then alkylated with iodomethane using a similar method described by Shen, Peng-Xiang et al. in Journal of the American Chemical Society, 2018, Vol. 140, #21, pp. 6545-6549. The ester was then hydrolyzed to give 1-(methoxymethyl)cyclopropane-1-carboxylic acid (I-225).

[0361] Then, following the method in preparation 2, 1-(methoxymethyl)cyclopropane-1-carboxylic acid (I-225) was converted into an anhydride (I-226) to obtain (I-226), which is an oily substance.

[0362] The following 1-(alkoxymethyl)cyclopropane-1-carboxylic acid and anhydride were prepared by a similar method.

[0363]

[0364] Synthesis of 11:1-(alkoxymethyl)cyclobutane-1-carboxylic acid and acid anhydride.

[0365] Synthetic methods for 1-(methoxymethyl)cyclobutane-1-carboxylic acid (I-238) and its anhydride (I-239):

[0366]

[0367] Methyl 1-(hydroxymethyl)cyclobutane-1-carboxylate (I-236) was prepared according to the method described in Reference Examples 22-4 of US9546155. Then, using a similar method to that described in Reference Example K-19 of US10040791, the hydroxyl groups were alkylated with iodomethane, followed by ester hydrolysis to give 1-(methoxymethyl)cyclobutane-1-carboxylic acid (I-238).

[0368] Then, following the method in Preparation 2, 1-(methoxymethyl)cyclobutane-1-carboxylic acid (I-238) was converted into an anhydride (I-239) to obtain an oily substance.

[0369] The following 1-(alkoxymethyl)cyclobutane-1-carboxylic acid and anhydride were prepared by a similar method.

[0370]

[0371] Synthesis of 12:1,2,2-trialkoxy-isobutyric acid and acid anhydride

[0372] Synthetic methods for 1-methoxy-2,2-diethoxy-isobutyric acid (223) and acid anhydride (224):

[0373]

[0374] Ethyl 1-hydroxy-2,2-diethoxy-isobutyrate (I-249) was prepared according to the method described by Bernardon, C. et al. in Comptes Rendus des Seances de l'Academie des Sciences, Serie C: Sciences Chimiques, 1968, Vol. 266, pp. 1502-1505. The hydroxyl groups were then alkylated with methyl iodoform using a method similar to that described in Example K-19 of US10040791. The ester was then hydrolyzed to give 1-methoxy-2,2-diethoxy-isobutyric acid (I-251).

[0375] Then, according to the method of preparation 2, 1-methoxy-2,2-diethoxy-isobutyric acid (I-251) was converted into an anhydride (I-252) to obtain an oily substance.

[0376] The following 1-alkoxy-2,2-dialkoxy-isobutyric acid and acid anhydride were prepared by a similar method.

[0377]

[0378]

[0379] Synthesis of 13:1-alkoxycyclobutanecarboxylic acid and acid anhydride

[0380] Synthesis of 1-methoxycyclopropaneformic acid (I-291) and its anhydride (I-292):

[0381] In the same manner as described in Example 26 3A of US10464914, commercially available methyl 2-methoxyacetate (I-289) was alkylated with dibromoethane to give methyl 1-methoxycyclopropanecarboxylate, which was then hydrolyzed under alkaline conditions to give the corresponding acid (I-291). The acid (I-291) was then converted to the corresponding acid anhydride (I-292) according to the method of Preparation 2, resulting in an oily substance.

[0382]

[0383] The following 1-methoxycyclopropanecarboxylic acid and anhydride, as well as 1-alkoxycyclobutanecarboxylic acid and anhydride, can be prepared in the same manner described above:

[0384]

[0385] Example 1:

[0386] Synthesis of N4-hydroxycytidine (NHC) or 1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)pyrimidin-2-one.

[0387]

[0388] A mixture of cytidine (20.0 g, 82.24 mmol, 1.0 equivalent) and NH₂OH·AcOH (23 g, 246.7 mmol, 3.0 equivalent) in H₂O (350 mL) was stirred at 40 °C for 48 h. The reaction was monitored by HPLC. After the reaction was complete, the water was evaporated under vacuum in a rotary evaporator to obtain a thick slurry, which was then suspended in 100 mL of water and crystallized in a refrigerator for 24 h. The crystallized solid was filtered, washed with cold H₂O (approximately 15.0 mL), and dried under vacuum to give the desired N₄-hydroxycytidine (NHC / EX-1) as a white solid (8.48 g, 40% yield). ¹H NMR (400 MHz, DMSO) δ 9.97 (br.s, ¹H), 9.45 (bs, ¹H), 7.04 (d, ¹H), 5.74 (d, ¹H), 5.57 (d, ¹H), 5.52 (m, 2H), 4.98–5.03 (m, 2H), 3.91–4.00 (m, 2H), 3.78 (dd, ¹H), 3.56 (m, 2H); Purity: 98% (evaluated by HPLC).

[0389] Example 2: Preparation of compound (EX-2)

[0390]

[0391] In a 250 mL round-bottom flask equipped with a magnetic stirrer, N4-hydroxycytidine (20 g, 72.2 mmol) was added to 30 mL of water with stirring, and the mixture was heated to 40 °C. Then, acetic anhydride (8.1 g, 0.794 mmol) was added dropwise. The reaction was stirred at this temperature for 2 hours until HPLC indicated that the reaction was complete. The reaction mixture was slowly cooled to 0 °C, and the resulting solid was filtered off and washed with methanol to give EX-2-1, a white solid. ¹H NMR (400 MHz, DMSO) showed two sets of peaks due to tautomerization of the NH=CNHO bond: δ 10.9 (br.s, ¹H), 7.4 (d, ¹H), 5.7 (m, 2H), 5.3 (br.s, ¹H), 5.0 (s, 2H), 4.0 (m, 2H), 3.8 (s, ¹H), 3.6 (m, 2H), 2.10 (s, 3H); purity: 99% (evaluated by HPLC).

[0392] The following examples illustrate a novel method for selective acylation reactions using acetic anhydride. Single solvents or solvent mixtures (binary, ternary, or quaternary mixtures, etc.) can be used for acylation reactions in varying proportions. By HPLC, the formation rate of the acylation product in the reaction solution is typically greater than 95%.

[0393]

[0394]

[0395] The following are examples illustrating the industrial feasibility of this new method:

[0396] In a 2-liter four-necked round-bottom flask equipped with a stirrer and thermometer, 160 g of NHC / N4-hydroxycytidine was added, followed by 1.52 kg of methanol. The reaction mixture was heated to 45-50 °C, and acetic anhydride (65 g) was added dropwise over 10-15 minutes at this temperature. Approximately 50% of the methanol was distilled off under vacuum at 40-45 °C. The reaction mixture was cooled to 30 °C, and another 5 g of acetic anhydride was added dropwise. The reaction mixture was further cooled to below 10 °C and stirred at this temperature for 2 hours. The resulting solid was filtered and washed with 100-150 g of methanol. The solid was dried to give 160 g of compound EX-2 as a white solid with a purity of 99.5%.

[0397] Example 3: Preparation of compound (EX-3)

[0398]

[0399] In a 100 mL round-bottom flask equipped with a magnetic stirrer, N4-hydroxycytidine (20 g, 72 mmol) was added to 36 mL of water with stirring, and the mixture was heated to 45 °C. Isobutyric anhydride (12.5 g, 0.79 mmol) was then added dropwise over 5–10 minutes. The reaction was stirred at this temperature for 1–2 hours until HPLC indicated completion. The water in the reaction flask was evaporated to dryness, and then methanol (20 mL) was added. The reaction mixture was heated to 50–60 °C to completely dissolve the solid, then slowly cooled to 0 °C. The resulting solid was filtered and washed with methanol to give EX-3-1 as a white solid (20.5 g) with a purity of 99.3% and a yield of 86%. ¹H NMR (400 MHz, DMSO) showed two sets of peaks due to tautomerization of the NH=CNHO bond: δ 10.9 (br.s, ¹H), 7.4 (d, ¹H), 5.7 (m, 2H), 5.3 (br.s, ¹H), 5.0 (s, 2H), 4.0 (m, 2H), 3.8 (s, ¹H), 3.6 (m, 2H), 2.8 (m, ¹H), 1.1 (s, 6H).

[0400] The following examples illustrate a novel method for selective acylation reactions using isobutyric anhydride. Single solvents or solvent mixtures (binary, ternary, or quaternary mixtures, etc.) can be used for acylation reactions in varying proportions. By HPLC, the formation rate of the acylation product in the reaction solution is typically greater than 95%.

[0401]

[0402]

[0403] Example 4: Preparation of compound (EX-4)

[0404]

[0405] In a 100 mL round-bottom flask with magnetic stirring, N4-hydroxycytidine (20 g, 72 mmol) was added to 40 mL of pyridine at room temperature with stirring, followed by dropwise addition of benzoyl chloride (11.1 g, 0.79 mmol) over 5–10 minutes. The reaction was stirred overnight at 40–50 °C until HPLC indicated completion. Excess pyridine was removed under vacuum, and the reaction residue was dissolved in EtOAc. The organic layer was washed with saturated sodium chloride solution. The organic layer was dried, concentrated, and purified on a silica gel column (DCM and MeOH, gradient) to give EX-4-1 as a white solid. ¹H NMR (400 MHz, DMSO) showed two sets of peaks due to tautomerization of the NH=CNHO bond: δ 11.14 (s, ¹H), 8.22 (d, ¹H), 7.96 (dd, ¹H), 7.4–7.6 (m, 5H), 5.80 (m, ¹H), 5.3 (br.s, ¹H), 5.0 (br.s, 2H), 3.83–4.03 (m, 2H), 3.8 (s, ¹H), and 3.5 (m, 2H).

[0406] The following example compounds are prepared similarly to those described in Examples 2, 3 or 4, using commercially available acid anhydrides or acyl chlorides.

[0407] For those carboxylic anhydrides and acyl chlorides that are not commercially available, they can be readily prepared by well-known standard methods.

[0408]

[0409]

[0410]

[0411]

[0412]

[0413] The following exemplary compounds can be prepared using similar methods to those described in the examples above, using commercially available acid anhydrides or acyl chlorides. For those carboxylic anhydrides and acyl chlorides that are not commercially available, they can be readily prepared using well-known standard methods.

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429] Example 170: Plasma stability

[0430]

[0431] Solution preparation: A stock solution (10 mM) of each test compound was prepared in DMSO. The stock solution of each compound was then diluted to 100 μM with acetonitrile.

[0432] Plasma warming: Plasma incubation was performed in duplicate at 37°C in 96-well plates. Plasma was preheated to 198 μL at 37°C for 5 minutes. Then, 2 μL of the 100 μM test compound was added to each well containing plasma, and the mixture was pipetted to obtain a homogeneous suspension. Immediately afterward, 20 μL of the incubation solution was transferred as the 0-minute sample to the well of the quenching plate, followed by the addition of 200 μL of acetonitrile and metoprolol as an internal standard (IS), and pipetted. At 2, 5, 60, and 90 minutes, the incubation solution was pipetted, and a 20 μL sample of the incubation solution at each time point was serially transferred to the well of the other quenching plate, followed by the addition of 200 μL of acetonitrile and metoprolol as an internal standard, and pipetted.

[0433] Sample analysis: Centrifuge the 96-well plate at 6000g for 10 minutes. Inject the supernatant into an LC-MS / MS system for analysis.

[0434] Example 171: Microparticle Stability

[0435]

[0436] Solution preparation: A stock solution (10 mM) of each test compound was prepared in DMSO. The stock solution of each compound was then diluted to 100 μM with acetonitrile.

[0437] Microparticle thermotherapy: The incubation mixture was prepared to a total volume of 200 μL, with the following final component concentrations: 0.1 M PBS (pH 7.4), NADPH (2 mM), and liver microsomes (0.2 mg / mL), and either the test compound (1 μM) or monoravir (1 μM) as a positive control. NADPH was added after all other components had been pre-incubated at 37°C for 5 minutes. The mixture was pipetted to obtain a homogeneous suspension, and immediately 20 μL of the incubation solution was transferred as the 0-minute sample to the well of a "quench" plate. Then, 200 μL of acetonitrile with trimethoprim was added as an IS, and the mixture was pipetted. At 2, 5, 10, and 45 minutes, the incubation solution was mixed with a pipette, and 20 μL of the incubation solution sample at each time point was continuously transferred to a separate well of a "quench" plate. Then, 200 μL of acetonitrile with metoprazor was added as an IS, and the mixture was pipetted.

[0438] Sample analysis: Centrifuge the 96-well plate at 6000g for 10 minutes. Inject the supernatant into an LC-MS / MS system for analysis.

[0439] Example 172: In vitro activity of compound EX-2 (CH2101) and monopravir against SARS-CoV-2 omeprazole B.1.1.529 variant.

[0440] This study aims to investigate the antiviral activity of the compounds of this invention against the SARS-CoV-2 Omeprone B.1.1.529 variant in VERO E6 cells.

[0441] Table 1. Test Methods

[0442] Virus cell Duration of drug treatment (days) / Method of termination Positive control SARS-CoV-2 Omeprón B.1.1.529 Vero E6 4 / CPE Monoravir

[0443] Vero E6 cells were transferred and seeded into 96-well plates at a density of 10,000 cells per well. The plates were then incubated overnight at 37°C with 5% CO2. Diluted test compound EX-2 (CH2101) and monoravir (3-fold serial dilution, 8 concentrations, triplicate) and virus (MOI = 0.1) were added, along with parallel blank controls (Vero E6 cells, without virus or test compound) and virus controls (Vero E6 cells, with virus but without test compound). The 96-well plates were incubated at 37°C with 5% CO2 for 4 days. Cell viability was measured for each cell using Celltiter Glo. Cell viability was used to calculate the antiviral activity of the test compound. If the cell viability in wells treated with the test compound was higher than that in wells treated with virus but not with the test compound (CPE at one week), this indicated that the test compound had inhibitory activity against the virus.

[0444] EC of the test compound 50 and CC 50 The value is calculated using GraphPad Prism (version 8) by applying log(inhibitor) to the response-variable slope. EC 90 The formula for calculating the value is: EC 90 =EC 50 ×9^(1 / slope).

[0445] The test results are shown in Table 2 and Figure 2 middle.

[0446] Table 2. Activity of test compounds against SARS-CoV-2 Omeprone B.1.1.529 variant

[0447] test compounds <![CDATA[EC 50 (μM)]]> <![CDATA[EC 90 (μM)]]> <![CDATA[CC 50 (μM)]]> <![CDATA[SI(CC 50 / EC 50 )]]> EX-2 / CH2101 1.60 2.483 18.20 11.38 Monoravir 15.05 23.355 >100 >6.65

[0448] Example 173: In vivo efficacy evaluation of test compound CH2101 (compound EX-2) against SARS-CoV-2 in a K18-hACE2 transgenic mouse infection model.

[0449] This study aimed to evaluate the in vivo efficacy of the test compound CH2101 (EX-2) against SARS-CoV-2 in a K18-hACE2 transgenic mouse infection model. The primary endpoint readings were changes in body weight, clinical symptom scores, death, and viral titer in the lungs.

[0450] 1.1 Male, specific pathogen-free K18 hACE2 transgenic mice (B6.Cg-Tg(K18-ACE2)2Primn / J mice) were purchased from Jackson Laboratories. Mice were bred and cared for according to IACUC-approved protocol #20003. Eligible mice were used for research after an acclimatization period of at least 3 days.

[0451] 1.2 Virus: SARS-CoV-2, the Hong Kong strain was derived from BEI Resources (NR-52282) and was internally amplified.

[0452] 1.3 Cells: Vero E6 were purchased from ATCC and internally propagated.

[0453] The carrier for EX-2 / CH2101 is 10% (v / v) PEG400 + 0.5% (w / v) CMC in pure water. The desired concentration of compound EX-2 / CH2101 in this carrier is 45 mg / mL, considering purity.

[0454] The carrier for Remdesivir is 5% DMSO + 10% Solutol + 85% saline (0.9% sodium chloride), and the required concentration is 2.5 mg / mL, taking purity into consideration.

[0455] Animal grouping: According to the study design, eligible mice were randomly and evenly divided into 6 groups.

[0456] Virus inoculation: Mice were anesthetized and inoculated with SARS-CoV-2 virus via intranasal route on day 0 at a dose of 5,000 pfu / mice / 50 μL.

[0457] Compound / carrier administration: Mice were treated with the carrier, remdesivir, or the test compound EX-2 / CH2101 from day 0 to day 6 or day 4 (groups 4-6) twice daily, 8-16 hours apart, via oral or subcutaneous route. The first dose was administered 2 hours after infection. See Table 3 for details.

[0458] Animal monitoring: Body weight and mortality were monitored daily throughout the study. Clinical signs were observed, and 1 point was awarded for any positive sign: fur wrinkles, kyphosis, lethargy / incontinence, and respiratory distress.

[0459] Sample Harvest: Endpoint Sample Harvest: Mice in groups 4-6 were sacrificed on day 5, and lung samples were collected in containers containing 1 ml of LEMEM. The vials were weighed before and after lung sample collection to calculate the net lung weight. The lung samples were frozen for virus titration via plaque detection.

[0460] The endpoint of the in vivo study: Day 14 is the predetermined endpoint day, during which all surviving animals will be euthanized.

[0461] Humane endpoint: Any mouse that suffers ≥20% or / and 3 points is euthanized and counted as dead.

[0462] Design and timeline: Detailed study designs for the in vivo portion are listed in Table 3.

[0463] Table 3. Study Design and Compound Administration Protocol

[0464]

[0465] Viral titer: Lung virus titer was determined by plaque assay. The plaque assay was performed using VERO E6 cells. Lung samples were homogenized using a tissue lyser, and the supernatant of the lung homogenate was serially diluted 10-fold. 0.2 mL of each homogenate was pipetted into pre-inoculated 6-well plates. After incubation for 3 days, cells were fixed with 4% paraformaldehyde and stained with crystal violet. Plaques were visually counted, and the viral titer was calculated using the following formula:

[0466] Virus titer / g lung tissue = Log 10(plaque / pore / 0.2*dilution factor / lung weight*1000).

[0467] Results: The in vivo efficacy of the test compounds was determined by changes in body weight, clinical symptom scores, survival rate, and pulmonary virus titer.

[0468] Protection of mouse body weight: Throughout the in vivo study, mouse body weight and weight changes were monitored daily, normalized to day 0, as described below, and... Figure 3 and Figure 4 Draw in the middle.

[0469] Infection control group (medium): Weight loss was observed from day 5 and continued thereafter without any signs of recovery, accompanied by death or euthanasia.

[0470] Remdesivir-25mpk group: Weight loss was observed from day 2 and continued thereafter, with a decrease of -13.6% by day 6, accompanied by death or euthanasia.

[0471] In the test compound (EX-2 / CH2101-450 mpk) group: the weight of all mice remained stable, and no significant weight loss was observed.

[0472] Clinical symptom observation: Clinical symptoms of mice were monitored daily throughout the in vivo study. Any positive signs of fur wrinkling, kyphosis, lethargy, and respiratory distress were scored as 1 point. The total score is described below and plotted on [the graph / chart]. Figure 4 middle.

[0473] Control group (vector): Mice began to show visible clinical symptoms from day 5, which progressed rapidly and peaked on day 6, with a maximum clinical symptom score of 2 (mean, similar below).

[0474] Remdesivir group (25mpk): Mice began to show visible clinical symptoms from day 6, with the highest clinical symptom score being 2 points.

[0475] In the test compound (CH2101-450 mpk) group, the mice were in good health and no clinical symptoms related to infection were observed.

[0476] Survival rate: Mice mortality was monitored daily throughout the in vivo study. Mice survival status is described below. Figure 5 The data are plotted in the diagram and summarized in Table 4.

[0477] Infected control group (vector): Death was observed between day 5 and day 7. The survival rate was 0%, and the median survival time was 5 days.

[0478] In the remdesivir group (25 mpk): death was observed between day 6 and day 7. The survival rate was 0%, and the median survival time was 6 days.

[0479] The test compound (CH2101-450 mpk) group: No deaths were observed throughout the study, and all mice survived to the end of the study, i.e., 100% survival rate.

[0480] Table 4. Survival Status

[0481]

[0482] Lung virus titer: For groups 4–6, lung samples were harvested on day 5, and lung virus titers were determined by plaque assay. Results are described below and plotted on… Figure 6 The results are summarized in Table 5.

[0483] The infection control group (vector) had a mean viral titer of 5.94 Log 10 (plaques / g lung, similar below), which met the study design and inclusion criteria and was consistent with historical data.

[0484] The remdesivir group (25 mpk) had an average viral titer of 5.11 Log, which was 0.83 Log lower than the vector group, showing a significant difference (P>0.05), indicating a good antiviral effect in vivo.

[0485] The test compound group (CH2101-450 mpk) had an average viral titer of 2.64 Log, which was 3.31 Log lower than that of the vector group, and the difference was statistically significant (P<0.01). Among them, two samples reached the detection limit, indicating that the antiviral effect was the best.

[0486] Figure 6The pneumovirus titer of p26262-15 is shown. Mice were treated as instructed and sacrificed on day 5 for pneumovirus titer determination by plaque assay. Data are presented as mean ± SEM and analyzed by t-test: *, p < 0.05; ***, p < 0.001, compared with the vector group; and ###, p < 0.001, compared with the remdesivir group. Data marked with an asterisk are less than or equal to LLOD.

[0487] Table 5. Summary of Pneumovirus Titer

[0488]

[0489] in conclusion:

[0490] Data showed that mice in the vector group exhibited the designed infection symptoms, weight loss, and ultimately died from the infection. Furthermore, the viral titer in lung samples from the vector group was 5.94 Log, while remdesivir significantly reduced the lung viral titer by 0.83 Log. All these results indicate the successful establishment of a SARS-CoV-2 mouse infection model and provide a platform for evaluating the efficacy of the experimental compounds in vivo.

[0491] The test compound EX-2 (CH2101) significantly inhibited viral replication in the lungs, protected against weight loss and clinical symptoms in infected mice, and improved survival rates. In the current model, it showed the best antiviral efficacy under the set conditions.

[0492] Example 174: Pharmacokinetic studies of EX-2 (CH2101), monopravir (CH2017), and their metabolite NHC (CH2018) after a single oral administration of EX-2 (CH2101) or monopravir (CH2017) in male and female beagle dogs.

[0493] Both test compounds were prepared in a solution of 1% methylcellulose (400 cps, SIGMA, SLCF9694) in pure water.

[0494] A total of 6 male and 6 female beagles, weighing approximately 7-13 kg, were initially purchased from Marshall Biotechnology Co., Ltd. The animals were fasted overnight before administration and were reintroduced 4 hours after administration.

[0495] Animals assigned to research

[0496]

[0497] Animals were artificially restrained, and approximately 1 mL of blood was collected at each time point via the cephalic or saphenous vein into pre-cooled EDTA-K2 tubes. Blood samples were centrifuged at 4°C (4000 rpm, 5 min), and plasma was obtained within 30 minutes of sample collection. All samples were stored at approximately -80°C until analysis. Backup samples were discarded two months after completion of the in vivo assay unless otherwise requested.

[0498] The test results are shown in Table 6-11 and Figure 7-12 .

[0499] Table 6. Plasma concentration-time data and p-values ​​of EX-2 / CH2101 after oral administration of 10 mg / kg CH2101 to beagles.

[0500]

[0501] Table 7. Plasma concentration-time data and pharmacokinetic parameters of EX-1 / NHC / CH2018 after oral administration of 10 mg / kg CH2101 to beagles.

[0502]

[0503] Table 8. Plasma concentration-time data and p-values ​​of EX-2 / CH2101 after oral administration of 20 mg / kg CH2101 to beagles.

[0504]

[0505] Table 9. Plasma concentration-time data and pharmacokinetic parameters of EX-1 / NHC / CH2018 after oral administration of 20 mg / kg CH2101 to beagles.

[0506]

[0507] Table 10. Plasma concentration-time data and p-values ​​of CH2107 (monoravir) after oral administration of 22 mg / kg to beagle dogs.

[0508]

[0509] Table 11. Plasma concentration-time data and p-values ​​of EX-1 / NHC / CH2018 after oral administration of 22 mg / kg CH2107 (monoravir) to beagles

[0510]

[0511] From Table 6-11 and Figure 7-12It can be seen that the AUC values ​​of EX-1 / NHC / CH2018 after oral administration of 20 mg / kg CH2101 in beagles are very similar to those after oral administration of 22 mg / kg CH2107 (monoravir) in beagles. Therefore, a single administration of CH2101 (EX-2) to beagles has comparable pharmacokinetic characteristics to a single administration of CH2107 (monoravir).

Claims

1. Compound of formula (I) Or its tautomers, stereoisomers, or racemates or pharmaceutically acceptable salts thereof, wherein R is Ra-(C=O)-. Ra is a methyl group.

2. A pharmaceutical composition comprising the compound of claim 1 or a tautomer, stereoisomer, or racemate thereof or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable excipient.

3. Use of the compound of claim 1 or its tautomer, stereoisomer or racemate or its pharmaceutically acceptable salt in the preparation of a medicament for the treatment or prevention of SARS-CoV-2 virus infection in an individual.

4. The use according to claim 3, wherein the individual is a mammal.

5. The use according to claim 3, wherein the individual is a person.

6. The use according to any one of claims 3-5, wherein the virus is selected from SARS-CoV-2 virus α variant, SARS-CoV-2 virus β variant, SARS-CoV-2 virus γ variant and SARS-CoV-2 virus δ variant.

7. A pharmaceutical combination comprising the compound of claim 1 or a tautomer, stereoisomer, or racemate thereof or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent.

8. The pharmaceutical combination of claim 7, wherein the additional therapeutic agent is selected from: 、 and 。 9. A method for preparing the compound of formula (I) according to claim 1, comprising the following steps: Reaction of NHC with the anhydride of formula (II) yields the compound of formula (I). Ra is as defined in claim 1.

10. The method of claim 9, wherein the reaction is carried out in water or a mixture of water and an organic solvent.

11. The method of claim 9, wherein the reaction is carried out in a solvent selected from pure water, methanol, ethanol, propanol, isopropanol, DMF, DMSO, NMP, water-methanol mixtures, water-ethanol mixtures, water-propanol mixtures, water-isopropanol mixtures, water-n-butanol mixtures, water-sec-butanol mixtures, water-isobutanol mixtures, water-THF mixtures, water-ACN mixtures, water-DMF mixtures, water-DMSO mixtures, and water / 2-methylTHF mixtures.

12. The method of claim 9, wherein the reaction is carried out in a solvent selected from water, lower aliphatic alcohols, water-THF mixtures, water / 2-methylTHF mixtures, water / ACN mixtures, or water-lower aliphatic alcohol mixtures.

13. The method according to any one of claims 9-12, wherein the product is obtained in solid crystalline form by cooling the reaction mixture without adding an antisolvent.

Images