Cysteine ​​protease inhibitors and their uses

Abstract

Cysteine ​​protease inhibitors and their uses. A compound of formula C is disclosed, a pharmaceutically acceptable or physiologically acceptable salt thereof: wherein R a R b and R c As defined in this disclosure.

Description

Technical Field

[0001] This invention relates to cysteine ​​protease inhibitors and methods of using them, as well as the use of related compositions. Background Technology

[0002] Cysteine ​​proteases, also known as thiol proteases, are hydrolases that degrade proteins. These proteases share a common catalytic mechanism, involving a nucleophilic cysteine ​​thiol in one of the three or two catalytic elements. Inhibitors of cysteine ​​proteases have been used as antiviral drugs to treat HIV / AIDS and hepatitis C.

[0003] 3C-type proteases (3CL proteases) are cysteine ​​proteases and members of the PA family of proteases. 3CL proteases are the major proteases found in coronaviruses. They cleave coronavirus polyproteins at 11 conserved sites and are crucial in processing coronavirus replicase polyprotein (P0C6U8). While some 3CL protease inhibitors are already in use or undergoing clinical trials for the treatment of coronavirus infections such as COVID-19, there remains a need for novel cysteine ​​protease inhibitors that can effectively inhibit coronavirus replication. Summary of the Invention

[0004] Overview

[0005] On the one hand, this disclosure relates to compounds of formula C and their pharmaceutically acceptable salts or physiological salts:

[0006]

[0007] in

[0008] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0009] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、-S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0010] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, wherein R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0011] R a and R b They can fuse into rings; and

[0012] R c Selected from the following groups:

[0013] .

[0014] On the other hand, this disclosure relates to a compound of formula B, and its pharmaceutically acceptable salt or physiological salt:

[0015]

[0016] Each X is independently selected from O and S;

[0017] Y is independently selected from carbon-carbon single bonds or O, CH2, NR. b ;

[0018] A is selected from the following groups:

[0019] ;

[0020] R aSelected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0021] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0022] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)Rm -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0023] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0024] Where R a and R b They can be fused into a ring.

[0025] Detailed Explanation

[0026] The following description includes certain specific details to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that the embodiments can be implemented without employing one or more of these specific details, but using other methods, components, materials, etc.

[0027] Unless otherwise required in this disclosure, throughout the specification and the claims, the words “comprising” and “including” shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”.

[0028] When used in this disclosure and the appended claims, a singular designation without a quantity indication includes a plural designation unless the context clearly specifies otherwise.

[0029] Throughout this specification, the terms "an embodiment," "an embodiment," "in another embodiment," or "in some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature related to that embodiment. Therefore, the phrases "in an embodiment," "in another embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0030] It should be understood that the singular article “a” (corresponding to the English words “a,” “an,” and “the”) used in this disclosure and the appended claims includes plural objects unless otherwise expressly stated in the text. Thus, for example, a sustained-release tablet containing “pharmaceutically acceptable excipients” includes one, two, or more pharmaceutically acceptable excipients.

[0031] definition

[0032] Some chemical groups named herein are indicated by a simplified symbol preceding the total number of carbon atoms found in the indicated chemical group. For example, C7-C 12 Alkyl groups are described as having a total of 7 to 12 carbon atoms as defined below, and are C4-C6. 12 Cycloalkyl groups are described as having a total of 4 to 12 carbon atoms as defined below. The total number of carbon atoms in the simplified notation does not include carbons that may be present in substituents of the group.

[0033] Therefore, unless otherwise stated, the following terms used in the specification and appended claims shall have the following meanings:

[0034] As used in this disclosure, the term "oxo" refers to the =O group.

[0035] As used in this disclosure, the term "cyano" refers to the -CN group.

[0036] As used in this disclosure, the term "nitro" refers to the -NO2 group.

[0037] As used in this disclosure, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0038] As used in this disclosure, the term "alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturated bonds, having 1 to 12 carbon atoms, and being connected to the rest of the molecule by single bonds. In some embodiments, the alkyl group has 1 to 8 carbon atoms. In some embodiments, the alkyl group has 1 to 6 carbon atoms. In some embodiments, the alkyl group has 1 to 4 carbon atoms. In some embodiments, exemplary examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. In some embodiments, the alkyl group may be optionally substituted, i.e., substituted or unsubstituted.

[0039] Whenever a group is described as "optionally substituted," then the group may be unsubstituted or substituted with one or more of the shown substituents. Similarly, when a group is described as "unsubstituted or substituted," if substituted, then the substituent may be selected from one or more of the shown substituents. If no substituent is specified, it means that the shown "optionally substituted" or "substituted" group may be substituted with one or more groups selected individually or independently from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroarylalkyl, (heteroalicyclic)alkyl, hydroxyl, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamoyl, N-amino Carboxyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, protected C-carboxyl, O-carboxyl, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, thio, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, monosubstituted amino and disubstituted amino and their protected derivatives.

[0040] As used in this disclosure, the term "alkoxy" refers to the general formula -OR, where R is an alkyl group as defined above. In some embodiments, the alkoxy group has 1 to 8 carbon atoms. In some embodiments, the alkoxy group has 1 to 6 carbon atoms. In some embodiments, the alkoxy group has 1 to 4 carbon atoms. In some embodiments, exemplary examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, tert-pentoxy, etc. In some embodiments, the alkoxy group may be optionally substituted, i.e., substituted or unsubstituted.

[0041] As used in this disclosure, the term "cycloalkyl" refers to a stable, non-aromatic monocyclic or polycyclic alkyl group consisting only of carbon and hydrogen atoms. It may comprise a fused or bridged ring system having 3 to 18 carbon atoms, in some embodiments 3 to 15 carbon atoms, and in some embodiments 3 to 10 carbon atoms, and is saturated and connected to the remainder of the molecule by a single bond. Exemplary examples of monocyclic cycloalkyl groups in some embodiments include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Exemplary examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornel, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, etc. In some embodiments, the cycloalkyl group may be optionally substituted, i.e., substituted or unsubstituted.

[0042] As used in this disclosure, the term "heterocyclic alkyl" refers to monocyclic, bicyclic, and tricyclic systems of ternary, quaternary, pentaneary, hexanal, octaneary, nonanal, decacyclic, and up to 18 members, wherein a carbon atom forms the ring system with 1 to 5 heteroatoms. Heteroatoms are elements other than carbon, including but not limited to oxygen, sulfur, and nitrogen. The heterocycle may also contain one or more carbonyl or thiocarbonyl functional groups, thereby making the definition include oxocyclic and thiocyclic systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be linked together in a fused manner. Additionally, any nitrogen in the heterocyclic alkyl group may be quaternized. The heterocyclic alkyl group may be unsubstituted or substituted. Examples of such "heterocyclic groups" include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxopentane, 1,3-dioxopentane, 1,4-dioxopentane, 1,3-oxothiocyclohexane, 1,4-oxothiocyclohexadiene, 1,3-oxathiolane, 1,3-dithiocyclopentadiene, 1,3-dithiocyclopentane, 1,4-oxothiocyclohexane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, etc. Hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazoline, isoxazoline, isoxazoline, oxazoline, oxazoline, oxazolidinone, thiazoline, thiazoline, morpholine, ethylene oxide, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidone, 4-piperidinone, pyrzoline, pyrrolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiaran, thiomorpholine, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, and their benzo[a]-fused analogues (e.g., benzimidazolinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl).

[0043] As used in this disclosure, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system consisting only of hydrogen and carbon, containing 6 to 18 carbon atoms, wherein the ring system may be partially saturated. In some embodiments, the aryl group is C6-C. 14 Aryl group. In some embodiments, the aryl group is C6-C. 12 Aryl group. In some embodiments, the aryl group is C6-C. 10 Aryl. In some embodiments, exemplary examples of aryl groups include, but are not limited to, phenyl, naphthyl, and fluorenyl. In some embodiments, the aryl group may be optionally substituted, i.e., substituted or unsubstituted.

[0044] As used in this disclosure, the term "heteroaryl" refers to a 5- to 18-membered aromatic ring group comprising 1 to 17 carbon atoms and 1 to 10 heteroatoms selected from nitrogen, oxygen, and sulfur. For the purposes of this disclosure, the heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may comprise fused or bridged ring systems; and the nitrogen, carbon, or sulfur atom in the heteroaryl may optionally be oxidized; the nitrogen atom may optionally be quaternized. In some embodiments, the ring of the heteroaryl may contain 4 to 14 atoms. In some embodiments, the ring of the heteroaryl may contain 5 to 10 atoms. In some embodiments, the ring of the heteroaryl may contain 5 to 6 atoms. In some embodiments, exemplary examples of heteroaryl groups include, but are not limited to, azatril, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxolane, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxacycloheptyl, 1,4-benzodioxane, benzonaphthuronyl, benzodioxolane, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzothiophene, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazole, cenolinyl, dibenzofuranyl, dibenzothiophene, imidazopyridyl, imidazopiperazinyl, Imidazolyl, furanyl, furanoneyl, isothiazolyl, imidazolyl, indazole, indolyl, indazole, isoindolyl, dihydroindolyl, isodihydroindolyl, isoquinolinyl, indeneyl, isoxazolyl, naphthyl, diazanaphthyl, diazolinyl, 2-oxazapyryl, oxazolyl, ethylene oxide, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenothiazinyl, 2,3-diazanaphthyl, pteridinyl, purinyl, pyrroleyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrroleyl, quinazolinyl, quinoxolinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiopheneyl. In some embodiments, the heteroaryl group may be optionally substituted, i.e. substituted or unsubstituted.

[0045] As used in this disclosure, the term "physiologically acceptable" refers to a carrier, diluent, or excipient that does not eliminate the biological activity and properties of the compound.

[0046] As used in this disclosure, the term "carrier" refers to a substance that enables the inclusion of a compound into a cell or tissue.

[0047] As used in this disclosure, the term "excipient" refers to an inert substance added to a pharmaceutical composition to provide (not limited to) bulk, consistency, stability, binding capacity, lubricity, and disintegration capacity of the composition.

[0048] As used in this disclosure, the term "diluent" refers to an ingredient in a pharmaceutical composition that does not have pharmaceutical activity but may be pharmaceutically necessary or desired.

[0049] As used in this disclosure, the term "mammal" refers to animals including, for example, dogs, cats, cattle, sheep, horses, and humans. In some embodiments, mammals include humans.

[0050] As used in this disclosure, the term "patient" refers to animals (e.g., humans), companion animals (e.g., dogs, cats, or horses), and livestock (e.g., cattle, pigs, and sheep). In some embodiments, the patient is a mammal that includes both males and females. In some embodiments, the patient is a human.

[0051] As used in this disclosure, the terms "pharmaceutical-acceptable" or "medically acceptable" refer to carriers, loading agents, diluents, excipients, and / or salts that must be compatible with other components of the formulation and not be harmful to the recipient.

[0052] As used in this disclosure, the terms "optional" or "optionally" mean that the event or condition described below may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition.

[0053] As used in this disclosure, “pharmaceutical-acceptable carriers, diluents, or excipients” include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or animals and have no adverse effects on the composition of the pharmaceutical composition.

[0054] As used in this disclosure, the term "pharmaceutically acceptable salt" includes "acceptable acid adduct salt" and "acceptable base adduct salt".

[0055] "Acceptable acid adduct salts" refers to those salts that retain the biological validity and properties of a free base, wherein the acid adduct salt is biologically or otherwise suitable and is formed using inorganic or organic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such organic acids as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, phenylcarboxylic acid, 4-acetamidophenylcarboxylic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylaminosulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid. 2-Hydroxyethanesulfonic acid, formic acid, fumaric acid, viscous acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lacturonic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, viscous acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid, etc.

[0056] "Acceptable base addition salts" refers to those salts that retain the biological effectiveness and properties of the free acid, and which are suitable for biological or other purposes. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and salts of basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, heparin, choline, betaine, benzylamine, phenylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, aminobutanetriol, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. In some embodiments, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0057] Typically, crystallization produces solvates of the compounds of this invention. As used in this disclosure, the term "solvate" refers to an aggregate comprising one or more molecules of the compound of this disclosure and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Therefore, the compounds of this disclosure may exist in hydrated form, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and in corresponding solvated forms. The compounds of this disclosure may be true solvates, while in other cases, the compounds of this disclosure may contain only occasional water or be a mixture of water and a portion of occasional solvent.

[0058] As used in this disclosure, the term "pharmaceutical composition" refers to an formulation formed by the compound described in this disclosure with a medium generally accepted in the art for delivering a bioactive compound to a mammal such as a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients.

[0059] As used in this disclosure, "therapeuticly effective amount" refers to the amount of a compound or combination of compounds that improves, reduces, or eliminates a particular disease or condition and its symptoms, or avoids or delays the onset of a particular disease or condition or its symptoms. The amount of the compounds described in this disclosure constituting a "therapeuticly effective amount" will vary depending on the compound, the disease state and its severity, and the age and weight of the mammal to be treated, but those skilled in the art can determine the amount of the compounds described in this disclosure conventionally based on their knowledge and this disclosure.

[0060] As used in this disclosure, "to treat" or "to treat" encompasses the treatment of a related disease or condition in mammals, such as humans, suffering from a related disease or ailment, and includes:

[0061] (i) To prevent the occurrence of disease or disease state in mammals, especially when the mammal is susceptible to the disease state but has not yet been diagnosed with the disease state;

[0062] (ii) To suppress a disease or disease state, that is, to prevent its occurrence; or

[0063] (iii) Alleviate the disease or disease state, even if the disease or disease state subsides or does not progress.

[0064] As used in this disclosure, the terms “disease” and “disease state” may be used interchangeably or may be different, because a particular disease or disease state may not have a known causative agent (and therefore cannot be explained by etiology), and thus is not recognized as a disease, but rather as an undesirable disease state or symptom in which a clinician has identified a more or less specific set of symptoms.

[0065] The compounds described in this disclosure, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and thus may produce enantiomers, diastereomers, and other stereoisomers, which may be defined according to absolute stereochemistry as I- or (S)-, or (D)- or (L)- of amino acids. This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), I- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as HPLC using chiral columns. When the compounds described in this disclosure contain an alkene double bond or other geometrically asymmetric centers, unless otherwise stated, it means that the compounds include E and Z geometric isomers. Similarly, it also means that all tautomers are included.

[0066] "Stereoisomers" refer to compounds composed of identical atoms bonded by the same bonds, but having different three-dimensional structures that are not interchangeable. This disclosure covers a variety of stereoisomers and mixtures thereof.

[0067] "Ci-trans isomers" refer to molecules with the same molecular formula that have different spatial arrangements of relative distances between adjacent atoms or groups of atoms due to factors such as double bonds or rings that hinder the free rotation of bonds.

[0068] "Tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomers of any of the said compounds.

[0069] As used in this disclosure, the term "prodrug" is used to refer to a compound that can be converted into the bioactive compound of the present invention under physiological conditions or by solvent decomposition. Therefore, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of the compounds of the present disclosure. A prodrug may be inactive when administered to an individual in need, but is converted into the active compound of the present invention in vivo. Prodrugs are typically rapidly converted into the parent compound of the present disclosure in vivo, for example, by hydrolysis in the blood. Prodrug compounds often offer advantages in solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24, (Elsevier, Amsterdam)). Discussions of prodrugs are provided in Higuchi, T., et al, “Pro-drugs as Novel Delivery Systems,” ACSSymposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are cited in this paper.

[0070] As used herein, the term "prodrug" is also intended to include any covalently bound carrier that releases the active compound of this disclosure in vivo when such prodrugs are administered to a mammalian subject. Prodrugs of the compounds of this disclosure can be prepared by modifying functional groups present on the compounds of this disclosure in such a manner that, in conventional operation or in vivo, these modified substances are cleaved into the parent compound of this disclosure. Prodrugs include compounds of this disclosure wherein a hydroxyl, amino, or thiol group is attached to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free thiol group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol functional groups, or amide derivatives of amine functional groups, etc., of the compounds of this disclosure.

[0071] This disclosure also includes in vivo metabolites of the disclosed compounds. These products are obtained primarily through enzymatic processes, such as oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound. Therefore, this disclosure includes compounds produced by a method comprising exposing the compound of this disclosure to a mammal for a period sufficient to produce its metabolites. Identification of the metabolites typically involves preparing a radiolabeled isotope of the compound of the invention, administering it parenterally to an animal, such as a rat, mouse, guinea pig, monkey, or human, at a detectable dose (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours), and isolating the metabolites from urine, blood, or other biological samples. These products are readily isolated because they are labeled (others are isolated using antibodies capable of binding to antigenic epitopes present in the metabolites). The structure of the metabolites is determined in a conventional manner, for example, by MS, LC / MS, or NMR analysis. Typically, the analysis of the metabolites is performed using methods known to those skilled in the art for routine drug metabolism studies. Unless the metabolites are otherwise undetectable in the body, they can be used for the determination of therapeutic doses of the compounds disclosed herein.

[0072] On the one hand, this disclosure relates to a compound of formula C, and its pharmaceutically acceptable salt or physiological salt:

[0073]

[0074] in

[0075] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0076] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0077] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, wherein R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0078] R a and R b They can fuse into rings; and

[0079] R c Selected from the following groups:

[0080] .

[0081] In some implementations, R a or R a1 C6-C 10 Aryl and C5-C 10 The heteroaryl group is independently selected from group 2:

[0082]

[0083] Wherein, the group 2 is optionally surrounded by one or more R e replace;

[0084] Each R e Independently selected from H, halogen, OH, CF3, oxide, cyano, SF5, -NR m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl, C1-C8 alkoxy, C3-C10 cycloalkyl and C3-C 10 Heterocyclic groups;

[0085] Any two of the R e It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl or heterocyclic or C 6- C 10 Aryl or C 5- C 10 heteroaryl; and

[0086] The R e It may be optionally replaced by one or more halogens.

[0087] In another respect, this disclosure relates to a compound of formula B, and its pharmaceutically acceptable salt or physiological salt:

[0088]

[0089] X is independently selected from O and S;

[0090] Y is independently selected from carbon-carbon single bonds or O, CH2, NR. b ;

[0091] A is selected from the following groups:

[0092]

[0093] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0094] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0095] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0096] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0097] Where R a and R b They can be fused into a ring.

[0098] In some implementations, the compound of formula D, its pharmaceutically acceptable salt, or physiological salt:

[0099]

[0100] Ra Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0101] Each R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, and each R a1 It can be substituted by ≥1 substituent, which is independently selected from halogens, cyano groups and C1-C6 alkyl groups;

[0102] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m(C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0103] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0104] Where R a and R b They can be fused into a ring.

[0105] In some implementations, R c Selected from the following groups,

[0106] .

[0107] The group 3 is optionally surrounded by one or more R d Replaced

[0108] Each R d Independently selected from H, halogen, CF3, cyano, SF5, -NR m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0109] Any two R d It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl or heterocyclic or C6-C 10 Aryl or C5-C 10 Heteroaryl; and each R d It may optionally be replaced by one or more halogens.

[0110] In addition, this disclosure relates to the following compounds and their pharmaceutically acceptable salts or physiological salts:

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] In some embodiments, the alkyl group may be a C1-C6 alkyl group.

[0118] In some embodiments, the alkyl group may be a C1-C4 alkyl group.

[0119] In some implementations, the alkenyl group can be a C2-C6 alkenyl group.

[0120] In some embodiments, the alkenyl group can be a C2-C4 alkenyl group.

[0121] In some implementations, the alkynyl group can be a C2-C8 alkynyl group.

[0122] In some implementations, the alkynyl group can be a C2-C6 alkynyl group.

[0123] In some implementations, the alkynyl group can be a C2-C4 alkynyl group.

[0124] In some embodiments, the alkoxy group can be a C1-C8 alkoxy group.

[0125] In some embodiments, the alkoxy group can be a C1-C6 alkoxy group.

[0126] In some embodiments, the alkoxy group can be a C1-C4 alkoxy group.

[0127] In some embodiments, the general formulas B, C and D of this disclosure show that pharmaceutically acceptable salts, solvates, isomers, deuterated derivatives, prodrugs or metabolites have excellent therapeutic effects on viral infectious diseases.

[0128] In some embodiments, formulas B, C, and D of this disclosure indicate that their pharmaceutically acceptable salts, solvates, isomers, deuterated derivatives, prodrugs, or metabolites have excellent therapeutic effects against coronavirus infectious diseases.

[0129] In some embodiments, as shown in formulas B, C and D of this disclosure, the pharmaceutically acceptable salts, solvates, isomers, deuterated derivatives, prodrugs or metabolites are non-covalent small molecule inhibitors of 3Clpro with significant activity.

[0130] In some embodiments, the triazine derivatives of general formula I of this disclosure, their pharmaceutically acceptable salts, solvates, isomers, deuterated derivatives, prodrugs, or metabolites can effectively increase blood drug concentrations, prolong half-life, and significantly reduce single-dose dosage.

[0131] Pharmaceutical Composition

[0132] In another aspect, this disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula C, and a pharmaceutically acceptable salt or physiological salt thereof:

[0133]

[0134] in

[0135] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0136] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0137] The R bor R m Independently selected from H, C1-C3 alkyl, aryl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, wherein R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0138] R a and R b They can fuse into rings; and

[0139] R c Selected from the following groups:

[0140] ,

[0141] And pharmaceutically acceptable carriers, excipients or diluents.

[0142] In another aspect, this disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula B, and a pharmaceutically acceptable salt or physiological salt thereof:

[0143]

[0144] X is independently selected from O and S;

[0145] Y is independently selected from carbon-carbon single bonds or O, CH2, NR. b ;

[0146] A is selected from the following groups:

[0147] ;

[0148] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0149] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m(C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0150] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0151] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0152] Where R a and R b They can fuse into rings;

[0153] And pharmaceutically acceptable carriers, excipients or diluents.

[0154] In another aspect, this disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula D, and a pharmaceutically acceptable salt or physiological salt thereof:

[0155]

[0156] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0157] Each R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, and each R a1 It can be substituted by ≥1 substituent, which is independently selected from halogens, cyano groups and C1-C6 alkyl groups;

[0158] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0159] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0160] Where R a and R b They can fuse into rings;

[0161] And pharmaceutically acceptable carriers, excipients or diluents.

[0162] In another aspect, this disclosure relates to pharmaceutical compositions comprising therapeutically effective amounts of the following compounds, pharmaceutically acceptable salts or physiological salts thereof, and pharmaceutically acceptable carriers, excipients, or diluents:

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] In some embodiments, the pharmaceutical compositions of this disclosure include physiologically acceptable surfactants, carriers, diluents, excipients, lubricants, suspensions, film-forming substances, coating aids, or combinations thereof, as well as compounds of this disclosure, pharmaceutically acceptable salts, isomers, prodrugs, or metabolites thereof. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and, for example, at Remington's Pharmaceutical Sciences, 18 th The entire contents of Ed. Mack Publishing Co., Easton, PA (1990) are described here and are incorporated herein by reference.

[0171] Preservatives, stabilizers, dyes, sweeteners, flavorings, and fragrances can be provided in pharmaceutical compositions. For example, sodium benzoate, ascorbic acid, and esters of p-hydroxybenzoic acid can be added as preservatives. Additionally, antioxidants and suspensions can be used.

[0172] In different implementation schemes, alcohols, esters, sulfated aliphatic alcohols, etc., can be used as surfactants; sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicates, magnesium aluminate, magnesium aluminate methyl silicate, synthetic aluminum silicate, calcium carbonate, calcium bicarbonate, calcium hydrogen phosphate, calcium hydroxymethyl cellulose, etc., can be used as excipients; magnesium stearate, talc, hardened oil, etc., can be used as lubricants; coconut oil, olive oil, sesame oil, peanut oil, soybean oil, etc., can be used as suspensions or lubricants; cellulose acetate, as a derivative of sugars such as cellulose or sugar, or methyl acetate-isobutylene ester copolymer, as a derivative of polyethylene, can be used as suspensions; and plasticizers such as phthalates can be used as suspensions.

[0173] Suitable routes of administration may include, for example, oral, rectal, transmembrane, local, or enteral administration; parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary, intrathecal, direct intracardiac, intraperitoneal, intranasal, or intraocular injection. Compounds can also be administered at a predetermined rate and / or timed, pulsatilely in sustained-release or controlled-release dosage forms, including depot injections, osmotic pumps, pellets, and transdermal (including electromigration) patches.

[0174] The pharmaceutical compositions disclosed herein can be produced by known methods, such as conventional methods of mixing, dissolving, granulating, manufacturing tablets, grinding, emulsifying, encapsulating, retaining or compressing tablets.

[0175] Therefore, according to this disclosure, the pharmaceutical compositions used can be formulated using conventional methods with one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the treatment of the active compound into a pharmaceutically usable formulation. Suitable formulations depend on the chosen route of administration. Any known techniques, carriers, and excipients can be used as suitably understood and appreciated in the art.

[0176] Injectable formulations can be prepared in the following conventional forms: as solutions or suspensions, solid dosage forms suitable for preparation as solutions or suspensions prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, glucose, mannitol, lactose, lecithin, albumin, monosodium glutamate, cysteine ​​hydrochloride, etc. Additionally, if desired, the injectable pharmaceutical composition may contain small amounts of non-toxic excipients, such as wetting agents, pH buffers, etc. Physiologically suitable buffers include, but are not limited to, Hank's solution, Ringer's solution, or physiological saline buffer. If desired, absorption-enhancing agents (e.g., liposomes) may be used.

[0177] For transmembrane administration, a permeabilizing agent suitable for permeability barriers may be used in the formulation.

[0178] Parenteral drug formulations, for example, administered by bolus or continuous infusion, include aqueous solutions of active compounds in water-soluble form. Alternatively, suspensions of the active compounds can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or other organic oils such as soybean oil, grapefruit oil, or almond oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that enhance the solubility of the compound to prepare high-concentration formulations. The injectable formulation and additional preservatives may be present in a single dosage form, such as in ampoules or multi-dose containers. The composition may be in an oily or aqueous carrier in a dosage form such as a suspension, solution, or emulsion, and the composition may contain agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable carrier such as sterile pyrogen-free water before use.

[0179] For oral administration, the active compound can be readily formulated by combining it with a pharmaceutically acceptable carrier known in the art. Such a carrier allows the compounds of the invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, ointments, suspensions, etc., for oral ingestion by a method in which the active compound is mixed with a solid excipient, the resulting mixture is ground in any manner, and the granulated mixture is processed, if necessary, with the addition of a suitable excipient to obtain a tablet or lozenge core. Suitable excipients are particularly fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). Disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginate or alginate such as sodium alginate may be added if necessary. The lozenge core is then appropriately coated. For this purpose, a concentrated sugar solution may be used, optionally containing gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, shellac solution, and suitable organic solvents or solvent mixtures. To identify or characterize different combinations of active compound dosages, dyes or pigments may be added to the tablet or lozenge coating. For this purpose, a concentrated sugar solution may be used, optionally containing gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, shellac solution, and suitable organic solvents or solvent mixtures. To identify or characterize different combinations of active compound dosages, dyes or pigments may be added to the tablet or lozenge coating.

[0180] Oral pharmaceutical formulations include push-in capsules made of gelatin, and soft, sealed capsules made of gelatin and plasticizers such as glycerin or sorbitol. Push-in capsules may contain the active ingredient mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All oral formulations should be administered at a dose suitable for this administration.

[0181] For oral administration, the composition can be formulated into tablets or lozenges using conventional methods.

[0182] For inhalation administration, the compounds used in this disclosure are conveniently delivered in the form of a spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the metering amount. Capsules and cartridges, such as gelatin, used in inhalers or blowpipes can be formulated as powder mixtures containing the compound and a suitable powder matrix such as lactose or starch.

[0183] This disclosure also discloses a variety of pharmaceutical compositions known in the pharmaceutical industry for use in intraocular, intranasal, and intraauricular delivery. Suitable penetrants for these uses are generally known in the art. Pharmaceutical compositions for intraocular delivery include aqueous ophthalmic solutions of the active compound in a water-soluble form, such as eye drops, or in gellan gum or hydrogel form; ophthalmic ointments; ophthalmic suspensions, such as microparticles, small polymer particles suspended in a liquid carrier medium, lipid-soluble formulations, and microspheres; and ophthalmic inserts. For stability and comfort, these suitable pharmaceutical formulations are most often and preferably formulated as sterile, isotonic, and buffered pharmaceutical formulations. Pharmaceutical compositions for intranasal delivery may also include drops and sprays, typically formulated to mimic nasal secretions in many ways to ensure maintenance of normal ciliary function. As is known to those skilled in the art, suitable formulations are most often and preferably isotonic, mildly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and suitable pharmaceutical stabilizers. Drug formulations for intraocular delivery include suspensions and ointments for topical application within the ear. Common solvents used in these ocular formulations include glycerin and water.

[0184] The compounds can also be formulated into rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

[0185] In addition to the aforementioned formulations, the compounds can also be formulated as reservoir-type formulations. Such long-acting formulations can be administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, the compounds can be formulated, for example, using suitable polymers or hydrophobic materials (e.g., emulsions acceptable in oils) or ion exchange resins, or formulated as slightly soluble derivatives such as slightly soluble salts.

[0186] For hydrophobic compounds, a suitable drug carrier can be a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A common co-solvent system used is the VPD co-solvent system, which consists of 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80™, and 65% w / v polyethylene glycol 300, with the volume made up by anhydrous ethanol. Of course, the proportions of the co-solvent system can be significantly altered without compromising its solubility and toxicity characteristics. Furthermore, the co-solvent components can be modified: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80™; the fragment size of polyethylene glycol can be altered; other biocompatible polymers such as polyvinylpyrrolidone can replace polyethylene glycol; and other sugars or polysaccharides can replace glucose.

[0187] Alternatively, other delivery systems for hydrophobic drug compounds can be used. Known examples of delivery media or carriers for hydrophobic drugs are liposomes and emulsions. While often at the cost of higher toxicity, certain organic solvents, such as dimethyl sulfoxide, can also be used. Additionally, sustained-release systems can be used to deliver compounds, such as semi-permeable matrices containing solid hydrophobic polymers of therapeutic drugs. Many sustained-release materials are known and established by those skilled in the art. Depending on their chemical properties, sustained-release capsules can release the compound over weeks to 100 days.

[0188] Reagents for intracellular drug delivery are administered using techniques known to those skilled in the art. For example, such reagents can be encapsulated in liposomes. During liposome formation, all molecules present in the aqueous solution are incorporated into the aqueous interior. The contents of the liposomes are not only unaffected by the external microenvironment, but are also efficiently delivered to the cytoplasm due to the fusion of the liposomes with the cell membrane. The liposomes can be coated with tissue-specific antibodies. The liposomes will be targeted to the desired organ and selectively absorbed by the desired organ. Alternatively, small hydrophobic organic molecules can be administered directly intracellularly.

[0189] Treatment methods and uses

[0190] Furthermore, this disclosure relates to use in medicaments for the treatment and / or prevention of viral infectious diseases, comprising administering to an individual requiring the method a therapeutically effective amount of a compound of general formula C, a pharmaceutically acceptable salt thereof, or a physiological salt thereof, or administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of general formula C, a pharmaceutically acceptable salt thereof, or a physiological salt thereof:

[0191]

[0192] in

[0193] R aSelected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0194] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0195] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, wherein R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0196] R a and R b They can fuse into rings; and

[0197] R c Selected from the following groups:

[0198] .

[0199] In some implementations, exemplary examples of viruses that can be used in this disclosure include, but are not limited to, Middle East Syndrome-associated Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), Influenza A virus, Influenza B virus, Novel Coronavirus Infection (COVID-19), Spanish Flu virus, Arena virus, Bunyavirus, Rabies virus, Avian influenza virus, Poliovirus, Rhinovirus, Adenovirus, Ebola virus, Enterovirus, Hepatitis A virus, Hepatitis C virus, Hepatitis E virus, Enterovirus, HIV virus, Echovirus, Filovirus, Measles virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus, Newcastle disease virus, RS virus, Vesicular stomatitis virus, Mumps virus, Dengue virus, Coxsackie virus, Rotavirus, or Tobacco mosaic virus.

[0200] In some implementations, exemplary examples of individuals that can be used for the purposes of this disclosure include, but are not limited to, mammals.

[0201] In some implementations, the individuals that can be used in this disclosure are humans.

[0202] In some embodiments, the triazine derivatives of general formula I of this disclosure, their pharmaceutically acceptable salts, solvates, isomers, deuterated derivatives, prodrugs, or metabolites have excellent therapeutic effects against coronavirus infectious diseases.

[0203] On the other hand, this disclosure relates to use in medicaments for treating and / or preventing viral infectious diseases, comprising administering to an individual requiring the method a therapeutically effective amount of a compound of general formula B, a pharmaceutically acceptable salt thereof, or a physiological salt thereof, or administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of general formula B, a pharmaceutically acceptable salt thereof, or a physiological salt thereof:

[0204]

[0205] X is independently selected from O and S;

[0206] Y is independently selected from carbon-carbon single bonds or O, CH2, NR. b ;

[0207] A is selected from the following groups:

[0208] ;

[0209] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0210] The R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1 It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the R a1 Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, and C1-C6 alkyl groups;

[0211] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0212] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0213] Where R a and R b They can be fused into a ring.

[0214] On the other hand, this disclosure relates to use in medicaments for treating and / or preventing viral infectious diseases, comprising administering to an individual requiring the method a therapeutically effective amount of a compound of general formula D, a pharmaceutically acceptable salt thereof, or a physiological salt thereof, or administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of general formula D, a pharmaceutically acceptable salt thereof, or a physiological salt thereof:

[0215]

[0216] R a Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 A heteroaryl group comprising 1 to 4 heteroatoms, each heteroatom independently selected from N, O, and S; wherein, the R a Optionally by 1 or more R a1 replace;

[0217] Each R a1 Independently selected from halogen, oxo, cyano, SF5, -NR m R m -NR m (C=O)R m 、 -S(O)2-R m C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C8 alkyl, and C1-C8 alkoxy; any two of which R a1It can combine with the atoms they are attached to to form C3-C 10 cycloalkyl, heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, and each R a1 It can be substituted by ≥1 substituent, which is independently selected from halogens, cyano groups and C1-C6 alkyl groups;

[0218] Where R c Selected from H, C1-C6 alkyl, C1-C6 branched alkyl, C2-C6 alkenyl, C2-C6 branched alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, heterocyclic, C4-C 10 Bridged cycloalkyl, C5-C 12 Spirocycloalkyl, C6-C 10 Aryl and C5-C 10 Heteroaryl groups; the heteroaryl group comprises 1 to 4 heteroatoms, each heteroatom being independently selected from N, O, and S, wherein each R c Optionally substituted with one or more substituents, said substituents being independently selected from halogens, cyano groups, SF5, and -NR. m R m -NR m (C=O)R m -S(O)2-R m C1-C8 alkyl and C1-C8 alkoxy;

[0219] The R b or R m Independently selected from H, C1-C3 alkyl, aryl, carbonyl, -S(O)2-CH3, -(C=O)-CH3, -(C=O)-CF3, C1-C6 alkoxy, C3-C6 cycloalkyl, and C5-C6 heteroaryl, and R b or R m Optionally substituted with one or more substituents, said substituents being independently selected from halogens, C1-C6 alkyl groups, and C1-C6 alkoxy groups; and

[0220] Where R a and R b They can be fused into a ring.

[0221] Administration method

[0222] The compound or pharmaceutical composition may be administered to a patient by any suitable method. Non-limiting examples of administration methods include (a) oral administration, including administration in capsules, tablets, granules, sprays, syrups, or other such forms; (b) non-oral administration, such as rectal, vaginal, urethral, ​​intraocular, intranasal, or intraauricular administration, including administration in aqueous suspensions, oily preparations, or in drops, sprays, suppositories, ointments, creams, etc.; (c) administration by subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, intraorbital injection, intracapsular injection, intraspinal injection, intrasternal injection, etc., including delivery by infusion pump; (d) localized administration, such as injection directly into the renal or cardiac region, for example, via a reservoir implant; and topically administration; as would be considered appropriate by those skilled in the art, the compound of the invention is in contact with living tissue.

[0223] The most suitable route depends on the nature and severity of the disease state being treated. Those skilled in the art are also familiar with determining the method of administration (oral, intravenous, inhalation, subcutaneous, rectal, etc.), dosage form, appropriate pharmaceutical excipients, and other matters related to delivering the compound to the recipient.

[0224] Suitable pharmaceutical compositions for administration include compositions containing an effective amount of the active ingredient to achieve their intended effect. The therapeutically effective dose of the pharmaceutical compositions disclosed in this invention depends on the route of administration, the type of animal being treated (including humans), and the physical characteristics of the specific animal under consideration. The dose can be adjusted to achieve the desired effect, but this will depend on factors such as body weight, diet, concurrent drug treatment, and other factors recognized by those skilled in the art. More specifically, a therapeutically effective dose refers to the amount of compound that effectively prevents, alleviates, or improves disease symptoms, or prolongs the lifespan of the treated individual. The therapeutically effective dose can be well determined by the practical ability of those skilled in the art, particularly according to the detailed disclosure provided in this invention.

[0225] As will be apparent to those skilled in the art, the dosage and specific route of administration for in vivo administration will vary depending on age, weight, the species of mammal being treated, the specific compound used, and the specific purpose of those compounds. Those skilled in the art can determine the effective dose level—the dose level necessary to determine the desired effect—using conventional pharmacological methods. Typically, human clinical application of a compound is initiated at a lower dose level, increasing the dose level until the desired effect is achieved. Alternatively, established pharmacological methods can be used to establish the effective dose and route of administration of the compositions identified by this method using acceptable in vitro studies.

[0226] In non-human animal studies, the application of a potential compound begins at a higher dose level and is gradually reduced until the desired effect is no longer achieved or adverse side effects disappear. The dose range can be broad, depending on the expected effect and therapeutic indication. Typically, the dose can range from about 10 μg / kg body weight to 500 mg / kg body weight, preferably from about 100 μg / kg body weight to 200 mg / kg body weight. Alternatively, as those skilled in the art will understand, the dose can be based on and calculated according to the patient's surface area.

[0227] Physicians can select the exact formulation, route of administration, and dosage of the pharmaceutical composition of the present invention based on the patient's condition. Typically, the dosage range of the composition administered to the patient can be from about 0.5 mg / kg to 1000 mg / kg of patient body weight. Depending on the patient's needs, the dosage can be given once, twice, or more frequently over a day or several days. Where the human dosage of the compound has been established under at least certain conditions, the present invention will use those same dosages, or dosage ranges from about 0.1% to 500% of the established human dosage, more preferably from 25% to 250% of the established human dosage. In cases where no established human dosage exists, such as in the case of a newly discovered pharmaceutical compound, a suitable human dosage can be determined from the ED... 50 or ID 50 The value, or other appropriate value derived from in vitro or in vivo studies, is inferred, as is quantified in toxicity studies and efficacy studies in animals.

[0228] It should be noted that, due to toxicity and organ dysfunction, the attending physician will know and when to terminate, interrupt, or adjust medication. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The dosage administered in the treatment of the condition of interest will vary depending on the severity of the disease state and the route of administration. For example, the severity of the disease state can be assessed in part using standard prognostic methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response. Protocols equivalent to those discussed above can be used in veterinary medicine.

[0229] While an exact dosage can be determined based on drug-by-drug analysis, in most cases, some generalizations about the dosage can be made. Daily dosing regimens for adult patients include, for example, oral doses of 0.1 mg to 2000 mg of each active ingredient, preferably 1 mg to 1000 mg of each active ingredient, such as 5 to 500 mg of each active ingredient. In other embodiments, intravenous, subcutaneous, or intramuscular doses of each active ingredient used are 0.01 mg to 1000 mg, preferably 0.1 mg to 800 mg, such as 1 to 200 mg. In the case of administering pharmaceutically acceptable saline, the dosage can be calculated based on free base. In some embodiments, the composition is administered 1 to 4 times daily. Alternatively, the compositions of the invention can be administered by continuous intravenous infusion, preferably at doses up to 1000 mg of each active ingredient daily. As will be understood by those skilled in the art, in some situations, it is necessary to administer the compounds disclosed herein in amounts exceeding or far exceeding the preferred dosage ranges described above for effective and rapid treatment of rapidly progressing diseases or infections. In some embodiments, the compound is administered during continuous treatment, such as for one week or several weeks, or for several months or years.

[0230] Dosage and dosing intervals can be individually adjusted to provide plasma levels sufficient to maintain the modulated effect or minimum effective concentration (MEC) of the active moiety. The MEC varies for each compound, but it can be assessed from in vitro data. The required dose to achieve the MEC depends on individual characteristics and route of administration. However, plasma concentrations can be determined using HPLC or bioassays.

[0231] The MEC value can also be used to determine the dosing interval. The composition should be administered using a treatment regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably 30-90% of the time, and more preferably 50-90% of the time.

[0232] In cases of local administration or selective absorption, the effective local concentration of a drug is independent of its plasma concentration.

[0233] Of course, the amount of the administered composition depends on the individual being treated, the individual's weight, the severity of their pain, the route of administration, and the prescribing physician's judgment.

[0234] The efficacy and toxicity of the compounds disclosed in this application can be evaluated using known methods. For example, the toxicology of a specific compound or a subset of compounds sharing certain chemical motifs can be established by determining the toxicity of cell lines, such as mammalian cell lines, and preferably human cell lines, in vitro. The results of such studies can generally predict toxicity in animals such as mammals, or more specifically, toxicity in humans. Alternatively, the toxicity of a specific compound in animal models such as mice, rats, rabbits, or monkeys can be determined using known methods. The efficacy of a specific compound can be determined using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for almost every type of disease state, including but not limited to cancer, cardiovascular disease, and various immune disorders. Similarly, acceptable animal models can be used to determine the efficacy of chemical drugs for treating these disease states. When selecting a model to determine efficacy, a person skilled in the art can choose an appropriate model, dosage, route of administration, and treatment regimen under the guidance of prior art. Of course, human clinical trials can also be used to determine the efficacy of a compound in humans.

[0235] If desired, the composition may be placed in a packaging or dispensing device, which may contain one or more unit dosage forms containing the active ingredient. The packaging may include, for example, metal or plastic foil, such as blister packs. The packaging or dispensing device may include instructions for use. The packaging or dispensing device may also include precautions related to the container, provided by a government agency regulating the production, use, or sale of the drug, reflecting that the drug form has been approved by that agency for human or animal administration. Such precautions may, for example, be labels approved by the U.S. Food and Drug Administration for prescription drugs, or approved product instructions. Compositions containing the compounds of the present invention may also be prepared in suitable containers, formulated in compatible drug carriers, and labeled for the treatment of specified disease states. Detailed Implementation

[0236] The compounds of the present invention can be prepared using the methods disclosed herein and their conventional modifications. It is obvious that conventional and well-known synthetic methods can also be used in addition to the methods disclosed herein and those known in the art.

[0237] The synthesis of typical compounds of formulas B, C, and D, or pharmaceutically acceptable salts thereof, such as compounds having one or more of the structures described in formulas B, C, and D, or other compounds disclosed herein, can be accomplished using the methods described in the examples below.

[0238] Typical embodiments of the compounds disclosed herein can be synthesized using the general reaction schemes and / or examples described below. Given the description herein, it will be apparent that different products can be produced by substituting starting materials with materials having similar structures. The subsequent synthetic descriptions provide numerous examples illustrating how starting materials can be varied to yield corresponding products. Starting materials are typically obtained from commercial sources or synthesized using methods for synthesizing compounds already disclosed as examples of those disclosed herein. Group designations (e.g., R1, R2) used in the reaction schemes herein are for illustrative purposes only.

[0239] Synthesis reaction parameters

[0240] The compounds disclosed herein can be prepared from readily available starting materials using, for example, the following general methods and procedures. It should be understood that typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given therein; other process conditions may also be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through conventional process optimization.

[0241] Furthermore, conventional protecting groups may be necessary for those skilled in the art to prevent certain functional groups from undergoing side reactions. Suitable protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting specific functional groups, are well known in the art, and many protecting groups are described, for example, in TW Greene and GMWuts (1999), Protecting Groups in Organic Synthesis, 3rd Edition, Wiley, New York, and the references cited therein.

[0242] Furthermore, the compounds disclosed herein may contain one or more chiral centers.

[0243] Therefore, if desired, these compounds can be prepared or separated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as mixtures rich in stereoisomers. Unless otherwise stated, all such stereoisomers (and enriched mixtures) are included within the scope of this disclosure. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents known in the art, or racemic mixtures of these compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0244] The starting materials used in the following reactions are generally known compounds, or can be prepared by known methods or by obvious modifications thereof. For example, many starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA). Others can be prepared by processes or by obvious modifications thereof, as described in standard references, such as Fieser and Fieser's Organic Synthetic Reagents, Volumes 1–15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1–5 and Supplement (Elsevier Science Publishers, 1989) Organic Reactions, Volumes 1–40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry (John Wiley and Sons, 5th Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0245] The terms "solvent," "inert organic solvent," or "inert solvent" refer to solvents that are inert under the reaction conditions described herein (including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF"), N,N-dimethylformamide ("DMF"), chloroform, dichloromethane (or dichloromethane), diethyl ether, methanol, pyridine, etc.). Unless otherwise specified, the solvents used in the reactions of this invention are inert organic solvents, and the reactions are carried out under an inert gas, preferably nitrogen.

[0246] The term "qs" indicates the amount added sufficient to achieve the stated function, for example, to bring the solution to the desired volume (i.e., 100%). The compounds provided herein can be synthesized according to the general schemes provided below. In the schemes below, it should be understood that each compound shown can have the desired protecting group at any step. Standard protecting groups are entirely within the capabilities of those skilled in the art.

[0247] In another aspect, the present invention provides methods for preparing pharmaceutically acceptable salts, esters or stereoisomers and compounds of formulas B, C and D.

[0248] Preparation of intermediates

[0249] Intermediate A:

[0250]

[0251] (S)-2-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutyric acid (120 g, 0.520 mol, 1.06 equivalents) was added to DCM (1500 ml) and stirred for 5 minutes. Then, (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-carboxylic acid methyl ester hydrochloride (100 g, 0.488 mol, 1.0 equivalents), HOBt (81 g, 0.600 mol, 1.23 equivalents), and EDCI (100 g, 0.520 mol, 1.06 equivalents) were added. Then, TEA (172 g, 1.707 mol, 3.5 equivalents) was added dropwise to the reaction mixture. The reaction mixture was stirred at 20°C for 2 days, then diluted with water (1000 ml) and extracted with DCM (2 × 1000 ml). The organic layers were combined, and the organic phases were washed sequentially with brine, dried over anhydrous sodium sulfate, filtered, and the mother liquor was concentrated to obtain IntA-3 (140 g, yield 84%), which was a brown oily substance. LCMS [M+H]+: 383.2.

[0252] Int A-3 (140 g, 0.365 mol, 1.0 equivalent) was added to MeOH / H2O (700 / 700 ml), followed by NaOH (65.7 g, 1.643 mol, 4.5 equivalent). The reaction mixture was stirred at 20 °C for 2 hours. The pH of the reaction mixture was then adjusted to 4–5 with HCl (3N) under ice bath conditions. The reaction mixture was diluted with DCM (2500 ml) and extracted with DCM (2 × 1500 ml). The combined organic layers were washed sequentially with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The residue was recrystallized from EA / PE = 1 / 10 to give a white solid Int A-4 (85 g, 63% yield). LCMS [M+H]+: 369.2.

[0253] Int A-4 (85 g, 0.230 mol, 1.0 equivalent) was added to DCM (1500 ml) and stirred for 5 minutes. Then, Int A-5 (52.4 g, 0.253 mol, 1.1 equivalent), HOBt (38.9 g, 0.288 mol, 1.25 equivalent), and EDCI (48.6 g, 0.253 mol, 1.1 equivalent) were added, followed by dropwise addition of TEA (81.3 g, 0.805 mol, 3.5 equivalent). The reaction mixture was stirred at 20 °C for 1 day. The reaction mixture was diluted with water (1000 ml) and extracted with DCM (2 × 1000 ml). The combined organic layers were washed sequentially with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give Int A-6 (105 g, 87% yield) as a brown oil. LCMS [M+H]+: 522.3.

[0254] Int A-6 (105 g, 0.201 mol) was added to an ethyl hydrochloride solution (1100 mL, 1 N HCl, EA). The reaction mixture was stirred at 20 °C for 4 hours. The mixture was filtered, and the filter cake was washed with EA / PE (1:1, 500 mL) and dried to give a white solid Int A (86 g, 93% yield). LCMS [M+H]+: 458.2.

[0255] Intermediate B

[0256]

[0257] Int B-1 (2.72 g, 10 mmol, 1.0 equivalent) and N,O-dimethylhydroxylamine (0.92 g, 15 mmol, 1.5 equivalent) were added to DCM (25 ml) and stirred for 5 minutes. Then, HOBt (2.03 g, 15 mmol, 1.5 equivalent), EDCI (2.29 g, 12 mmol, 1.2 equivalent), and TEA (2.02 g, 20 mmol, 2 equivalent) were added. The reaction mixture was stirred at 20 °C for 6 hours. The reaction mixture was diluted with water (50 ml) and extracted with DCM (2 x 50 ml). The combined organic layers were washed successively with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give Int B-2 (2.7 g, 85.7% yield) as a brown oil. LCMS [M+H]+: 316.2.

[0258] Benzothiazole (2.57 g, 19 mmol, 3.0 eq) was added to THF (25 mL), cooled to -78 °C, and n-BuLi (36 mL, 1 M) was added dropwise under N2 protection. The reaction mixture was kept at this temperature for 1 hour. Then Int B-2 (2.0 g, 6.34 mmol, 1.0 equivalence) was added, and the mixture was stirred for 2 hours. The reaction mixture was brought to room temperature and quenched with ammonium chloride solution. DCM (50 mL) was then added for extraction. The organic phase was concentrated to give an oily compound Int B-3 (1.3 g), which was used directly in the next reaction without purification. LCMS [M+H]+: 390.1.

[0259] Add 1.3 g of Int B-3 to HCl / EA (20 ml, 2 M) and stir at room temperature for 4 hours. Filter out 0.5 g of white solid Int B. LCMS [M+H]+: 290.1.

[0260] Process 1

[0261] Preparation of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidone-3-yl)ethyl)-3-((S)-2-((4-fluorophenyl)sulfonamide)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound 53)

[0262]

[0263] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propyl-2-yl)-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100.0 mg, 0.23 mol, 1 equivalent) was added to DCM (5 ml), followed by the addition of DIEPA (91.7 mg, 0.70 mol, 3 equivalent) at 0 °C and stirring for 5 minutes. Then, 4-fluorobenzenesulfonyl chloride (55.2 mg, 0.28 mol, 1.2 equivalent) was added at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was extracted with EA (2 x 10 ml). The combined organic phases were washed sequentially with brine (10 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The crude product was purified by silica gel column chromatography (PE / EA = 3:1) to obtain a light yellow solid ((1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propane-2-yl)-3-((S)-2-((4-fluorophenyl)sulfonylamino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide) (125.0 mg, yield 91%). LCMS [M+H]+: 580.25.

[0264] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propane-2-yl)-3-((S)-2-((4-fluorophenyl)sulfonamido)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (125.0 mg, 0.22 mol, 1.0 equivalent) was added to DCM (10 mL), followed by the addition of Burgess reagent (154.1 mg, 0.65 mol, 3 equivalent). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was extracted with EA (2 x 10 mL). The combined organic phases were washed sequentially with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The product was separated by preparative liquid chromatography to obtain a yellow solid product (100.0 mg, 83% yield). LCMS[M+H]+:562.67.

[0265] Process 2

[0266] Preparation of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-2-(2,2-diphenylacetamido)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 18)

[0267]

[0268] 2,2-Diphenylacetic acid (278 mg, 1.31 mmol, 1.2 equivalents) was added to DMF (20 ml), followed by 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU: 500 mg, 1.31 mmol, 1.2 equivalents) and N,N-diisopropylethylamine (DIPEA: 430 mg, 3.4 mmol, 3 equivalents). The mixture was stirred at room temperature for 30 minutes. Then (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propyl-2-yl)-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (500 mg, 1.1 mmol, 1 equivalent) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was quenched with water (20 ml) and extracted with EA (3 x 30 ml). The combined organic phases were washed successively with brine (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was recrystallized from DCM / MeOH = 20 / 1 to give a pale yellow solid product (400 mg, 60% yield). LCMS [M+H]+: 616.3.

[0269] A solution of compound 18-1 (400 mg, 0.65 mmol, 1 equivalent) and Burgess reagent (320 mg, 1.3 mmol, 2 equivalents) in DCM (10 mL) was stirred at room temperature for 1 hour. After the reaction was complete, saturated sodium bicarbonate solution (20 mL) was added. The mixture was then extracted with DCM (20 mL). The combined organic layers were washed sequentially with saturated sodium chloride aqueous solution (3 × 10 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative liquid chromatography to give compound 18 (300 mg), in 77% yield. LCMS [M+H]+: 598.3.

[0270] Process 3

[0271] Preparation of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-2-(3,3-dimethylurea)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound 39)

[0272]

[0273] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propyl-2-yl)-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexyl-2-carboxamide (100.0 mg, 0.23 mol, 1 equivalent) was added to DMF (5 mL), followed by DBU (90.0 mg, 0.59 mol, 2.5 equivalent) and dimethylamine (12.7 mg, 0.28 mol, 1.2 equivalent). The mixture was stirred at 0 °C for 5 min, followed by CDI (46.0 mg, 0.28 mol, 1.2 equivalent). The reaction mixture was stirred at room temperature for 2 h, then extracted with EA (2 × 10 mL). The combined organic phases were washed sequentially with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a pale yellow solid (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propyl-2-yl)-3-((S)-2-(3,3-dimethylurea)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (85.0 mg, 70% yield). LCMS [M+H]+: 493.62.

[0274] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propyl-2-yl)-3-((S)-2-(3,3-dimethylurea)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (85.0 mg, 0.17 mol, 1.0 equivalent) was added to DCM (10 mL), followed by TFAA (59.0 mg, 0.52 mol, 3 equivalent). The reaction mixture was stirred at room temperature for 1 h and extracted with EA (2 x 10 mL). The combined organic phases were washed sequentially with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The residue was purified by preparative liquid chromatography to give a white solid (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-2-(3,3-dimethylurea)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (56.0 mg, 68% yield). LCMS [M+H]+: 475.61.

[0275] Process 4

[0276] Preparation of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidone-3-yl)ethyl)-3-((S)-2-(3,3-dimethylurea)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound 35)

[0277]

[0278] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propyl-2-yl)-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100.0 mg, 0.23 mol, 1 equivalent) was added to DCM (5 mL), followed by TEA (47.8 mg, 0.47 mol, 2 equivalents) and stirring at 0 °C for 5 min. Methyl isothiocyanate (34.6 mg, 0.47 mol, 2 equivalents) was then added. The reaction mixture was stirred at room temperature for 2 h and then extracted with EA (2 × 10 mL). The combined organic phases were washed sequentially with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain a pale yellow solid (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propane-2-yl)-3-((S)-3,3-dimethyl-2-(3-methylthiourea)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (95.0 mg, yield 81%). LCMS [M+H]+: 495.66.

[0279] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propyl-2-yl)-3-((S)-3,3-dimethyl-2-(3-methylthiourea)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (95.0 mg, 0.19 mol, 1.0 equivalent)) was added to DCM (10 mL), followed by TFAA (65.8 mg, 0.58 mol, 3 equivalent). The reaction mixture was stirred at room temperature for 1 h and extracted with EA (2 x 10 mL). The combined organic phases were washed sequentially with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the residue was purified by preparative liquid chromatography to give a white solid (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(3-methylthiourea)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (75.0 mg, 82% yield). LCMS [M+H]+: 477.64.

[0280] Process 5

[0281] Preparation of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-2-((N,N-dimethylamino)amino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (compound 47)

[0282]

[0283] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propyl-2-yl)-3-((S)-2-amino-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexyl-2-carboxamide (100.0 mg, 0.23 mol, 1 equivalence) was added to DCM (5 ml), followed by DIEA (61.2 mg, 0.47 mol, 2 equivalences), and the mixture was stirred at 0 °C for 5 min. Then, dimethylaminosulfonyl chloride (50.8 mg, 0.36 mol, 1.5 eq) was added. The reaction mixture was stirred at room temperature for 2 h, followed by extraction with EA (2 x 10 ml). The combined organic phases were washed sequentially with brine (10 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a pale yellow solid (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidine-3-yl)propane-2-yl)-3-((S)-2-((N,N-dimethylsulfonyl)amino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (96.0 mg, yield 76%). LCMS [M+H]+: 529.67.

[0284] (1R,2S,5S)-N-((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidone-3-yl)propyl-2-yl)-3-((S)-2-((N,N-dimethylaminosulfonyl)amino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (96.0 mg, 0.18 mol, 1.0 eq) was added to DCM (10 mL). Then TFAA (78.5 mg, 0.69 mol, 3 eq) was added. The reaction mixture was stirred at room temperature for 1 h, and then extracted with EA (2 x 10 mL). The combined organic phases were washed successively with brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the crude product was purified by preparative liquid chromatography to obtain a white solid (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidine-3-yl)ethyl)-3-((S)-2-((N,N-dimethylsulfonyl)amino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (75.0 mg, 80% yield). LCMS [M+H]+: 511.65.

[0285] All compounds in Table 1 can be synthesized using the steps described above. Data is summarized in Table 1.

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319] Example 2: Assessment of in vitro bioactivity

[0320] A. Evaluation of the in vitro inhibitory activity of the compounds against COVID-19 MPRO (WT) and MPRO (P132H)

[0321] 1. Purpose

[0322] The aim of this study was to evaluate the activity of the test compounds against SARS-CoV-2 WT and P132H mutant Mpro in an enzyme assay.

[0323] 2. Materials

[0324] 2.1. Test and control compounds

[0325] The control compounds were provided by Vucic Aptek. The compounds were determined at 10 different concentrations, in duplicate, and the IC50 was measured. 50 .

[0326] 2.2. Enzymes and substrates

[0327] C-His6-labeled COVID-19WT MPRO (NC_045512) and the P132H mutant MPRO were cloned, expressed in E. coli, and purified. The Dabcyl-KTSAVLQ‖SGFRKM-(Edans) substrate was synthesized. The assay buffer contained 20 mM Tris-HCl (pH 7.3), 100 mM NaCl, 1 mM EDTA, 5 mM TCEP, and 0.1% BSA. The final concentrations of MPRO protein and substrate in the MPRO enzyme activity were 25 nM and 25 μM, respectively.

[0328] 3. Methods

[0329] 3.1. 10 doses: The compound was diluted to 10 doses and added to a test plate (384W format), in duplicate.

[0330] 3.2. Add 25 μL of 30 nM Mpro protein to the assay plate containing the compound using a multiple drop method. Incubate the compound and Mpro protein at room temperature for 30 min. Add 5 μL of 150 μM substrate to the assay plate using a multiple drop method. The final concentrations of Mpro and substrate are 25 nM and 25 μM, respectively. For the 100% inhibition control (HPE, 100% efficacy), no enzyme or compound is added. For the no-inhibition control (ZPE, 0% efficacy), no compound is added. The final DMSO concentration is 1%. Each activity test site has a corresponding background control to normalize the fluorescence interference of the compound.

[0331] 3.3. After incubation at 30°C for 60 minutes, the fluorescence signal (RFU) was detected using a microplate reader M2e at Ex / Em = 340 nm / 490 nm.

[0332] 3.4. Inhibitory activity is calculated using the following formula, IC50. 50 The value is calculated using inhibition rate data.

[0333] 3.5. Calculate the IC50 of the compound using GraphPad Prism software. 50 The values ​​were obtained using a nonlinear regression model with logarithm (inhibitor) and response-variable slope (four parameters).

[0334] B. Evaluate the in vitro antiviral activity of the compound against SARS-CoV-2 in a cell-based replicon assay.

[0335] 1. Objective

[0336] In cell replicon assays, the in vitro antiviral activity of the compound against SARS-CoV-2 was evaluated.

[0337] 2. Materials

[0338] 2.1. Test compound and control compound

[0339] The test compounds were provided by the proposer. The control compound, remdesivir, was provided by WuXi AppTec. The compounds were determined in a debriefing at eight concentrations (as defined by the proposer). Remdesivir was measured starting at 1 μM.

[0340] 2.2. Cell lines

[0341] Huh7 cells were obtained from the JCRB cell bank and maintained in Dulbecco modified Eagle medium containing 10% FBS, 1% L-glutamine, 1% NEAA and 1% penicillin-streptomycin.

[0342] 2.3. Reagents and Instruments

[0343] The main reagent used in this method is the CellTiter Glo (Promega) luminescent cell viability assay kit.

[0344] The main instrument used in this method is TTP LabTech.

[0345] 3. Methods

[0346] SARS-CoV-2 replicon RNA was generated using the mMACHINE T7 Ultra Kit. The purified replicon RNA was transfected into Huh7 cells and seeded onto plates containing serially diluted compounds. Cells were incubated at 37°C and 5% CO2 for 1 day. The final cell culture volume was 60 μL / well, with a final concentration of 0.5%.

[0347] Fluorescence intensity was detected using TTP LabTech, and the antiviral activity of the compound was calculated based on the degree of inhibition of GFP expression. Cell viability was determined using CellTiter Glo according to the manufacturer's manual.

[0348] EC50 and CC50 values ​​were calculated using GraphPad Prism software, employing a nonlinear regression model of log (inhibitor) versus response with a variable slope (four parameters).

[0349] Table 2 summarizes the data on in vitro bioactivity assessment and the in vitro inhibitory activity against SARS-CoV-2:

[0350]

[0351]

[0352] Note: NA indicates no detection.

[0353] In vitro cell infection assay—testing the effectiveness of potential antiviral compounds against SARS-CoV-2.

[0354] 1. Objective:

[0355] The aim of this program was to test the in vitro efficacy of potential antiviral therapeutics against SARS-CoV-2 (the causative agent of COVID-19). The assay was conducted in Vero E6 cells using multiple concentrations of the test sample and wild-type strains of the virus, or delta or omicron variants. Following incubation, the cells were immunostained with coronavirus nucleoprotein-specific antibodies.

[0356] 2. Experimental materials:

[0357] Viral inoculation dosage: 48±4 h after infection, antiviral activity was observed using 0.005 TCID50 / cell as the target. For the Omicron variant, the MOI is pending and will be added through modification.

[0358] 3. Experimental Design:

[0359] The antiviral activity of each test item was determined using the treatment protocol. TA was mixed with the virus and incubated for 60-90 minutes to allow viral adsorption. After adsorption, the cells were washed with 1x PBS or culture medium, and then placed on 1x TA medium.

[0360] 4. Testing System:

[0361] Cell culture: African green monkey kidney (VeroE6) cells were preserved in Dulbecco's minimum essential medium containing 10% fetal bovine serum and antibiotics. For efficacy and cytotoxicity assays, FBS was reduced to 2%.

[0362] Viruses: The viruses listed in the table below used for research were stored at approximately -65°C before use.

[0363]

[0364] 5. Method:

[0365] African green monkey kidney (VeroE6) cells were preserved in Dulbecco's minimum essential medium containing 10% fetal bovine serum and antibiotics. For efficacy and cytotoxicity assays, the FBS was reduced to 2%.

[0366] Mix 50 μL of each 2-fold dilution of the test sample with 50 μL of 1-fold culture medium containing the virus, transfer to monolayer confluent Vero E6 cells, and allow for virus adsorption for 60–90 minutes. All groups were treated with CP-100356 to a final concentration of 2 µM. CP-100356 is a P-glycoprotein (P-gp) inhibitor that prevents P-gp from expelling Mpro inhibitors from the cells.

[0367] After adsorption, cells were washed with 1x PBS or culture medium and then placed on 1x TA medium. The concentration range was 10 μM to 0.003 μM, with a 3-fold dilution, for a total of 8 doses. All incubations were performed in a humidified chamber at 37 ± 2°C and 5 ± 2% CO2. Remdesivir was used as a positive control compound for parallel evaluation. Parallel evaluations were also performed using DMSO and CP-100356 cell control groups. 48 ± 4 hours post-infection, dose-dependent antiviral efficacy was investigated at MOI using 0.005 TCID50 / cell as the target.

[0368] After 48 ± 4 hours, cells were fixed with 80% cold acetone and stained with anti-coronavirus nucleoprotein monoclonal antibodies HM1056 and HM1057 (EastCoast Bio, or equivalent), followed by staining with peroxidase-conjugated goat anti-mouse IgG (Fitzgerald, or equivalent). Wells were then developed using the ABTS peroxidase substrate system (SeraCare, or equivalent). Development was stopped, and plate numbers were read at 405 nm using an ELISA plate reader (MolecularDevicesSpectraMax M2, or equivalent), with 490 nm as a reference.

[0369] This test was a non-GLP test performed according to IITRI standard operating procedures. Data are reported as the drug concentration (IC50) that resulted in a 50% reduction in staining intensity compared to the virus control group.

[0370] Control groups: Virus control (VC) wells contained only SARS-CoV-2 and Vero E6 cells and served as infection controls. Cell control (CC) wells contained only cells and no virus and served as background controls. VC and CC were loaded and repeated 12 times per 96-well plate.

[0371] Absorbance readings for each well were collected using Softmax Pro software (version 7.0.3; GXP; San Jose, CA) and imported into a Microsoft Excel spreadsheet for further calculations. Outliers were detected using the Grubbs test in the built-in analysis of Graphpad Prism9 and excluded from the study. For each well, the reduction in viral replication was measured as a percentage of the viral control group (n≥9 replicates), where viral replication was 100% and background was given by the cell control group (n≥9 replicates), where viral replication was 0%, using the following formula:

[0372] Virus reduction percentage = 100 - [((well A405 - average cell control A405) / (average virus control A405 - average cell control A405)) × 100]

[0373] Data was plotted using Graphpad Prism 9, and the concentration-response curve and effective inhibitory concentration (IC50) for each test sample were calculated by fitting a 4-parameter nonlinear regression curve. 50 IC 50 The dilution is defined as the inverse log10 dilution that causes a 50% decrease in the absorbance value of the virus control (a 50% decrease in A405).

[0374] Table 3 summarizes the dates of in vitro bioactivity assessments.

[0375] Table 3. In vitro inhibitory activity test of wild-type SARS-CoV-2

[0376]

[0377] In this disclosure, relational terms such as first and second are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0378] All patents, patent application publications, patent applications and non-patent publications cited in this specification are incorporated herein by reference in their entirety.

[0379] As can be understood from the foregoing, although specific embodiments of this disclosure have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of this disclosure. Therefore, the scope of this disclosure should be limited only by the appended claims.

Claims

1. A compound, or a pharmaceutically acceptable salt or physiological salt thereof, wherein the compound is selected from:

2. A pharmaceutical composition comprising: The compound of claim 1, or a pharmaceutically acceptable salt or physiological salt thereof; and Pharmaceutically acceptable carrier.

3. A compound, or a pharmaceutically acceptable salt thereof, said compound having the following structure:

4. A pharmaceutical composition comprising: The compound of claim 3, or a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable carrier.

Images