Azabicyclo[3.1.0]hexane derivatives as 3c-like protease inhibitors

Abstract

This invention relates to an azabicyclo[3.1.0]hexane derivative that can be used as a 3C-like protease inhibitor. This azabicyclo[3.1.0]hexane derivative can be used to treat fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, olfactory and gustatory disturbances and their complications, or combinations thereof, caused by novel coronavirus infection.

Description

[0001] Related applications

[0002] This application claims priority to patent application No. 202210217412.5 filed with the State Intellectual Property Office of China on March 7, 2022, entitled "A azabicyclo[3.1.0]hexane derivative used as a 3C-like protease inhibitor", which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to a azirbicyclo[3.1.0]hexane derivative that can be used as an inhibitor of 3C-like proteases. Background Technology

[0004] Among all known RNA viruses, coronaviruses have a maximum genome length between approximately 26 and 32 kb. Besides encoding structural proteins, a large portion of the coronavirus genome is also transcribed and translated into polypeptides that encode proteins essential for viral replication and gene expression. The major protease (Mpro), approximately 306 aa long, is a key enzyme in coronavirus replication and is also encoded by this polypeptide, responsible for processing it into functional proteins. Mpro possesses cleavage site specificity similar to that of the parvovirus 3C protease (3Cpro), and is therefore also called a 3C-like protease (3CLpro). Studies have shown that 3CLpro from different coronaviruses is highly conserved in both sequence and 3D structure. These characteristics and their functional importance make 3CLpro a target for anti-coronavirus drug design.

[0005] The role of 3CLpro protease is to hydrolyze and cleave the expressed peptide chain at appropriate sites, preparing it for the formation of a three-dimensional or four-dimensional structure, thus forming the enzyme required for viral replication. The enzyme itself does not change during the catalytic process, but the activation energy of the hydrolysis reaction is lowered, thereby accelerating the rate of hydrolysis. The sulfhydryl group on cysteine ​​residues plays a crucial role in the entire catalytic hydrolysis process. (See Thanigaimalai et al., An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy, Journal of Medicinal Chemistry, 59(14):6595-6628.)

[0006] Public literature on 3CLpro inhibitors exists in the prior art. For example, WO2021 / 250648A1 discloses a compound currently known as Nirmatrelvir (PF-07321332). As one of the active ingredients of Paxlovid, it, when used in combination with ritonavir, has been shown to reduce the risk of death and hospitalization due to the novel coronavirus. Nirmatrelvir has the following structure:

[0007]

[0008] Furthermore, WO2021 / 205290A1 also discloses a method for treating diseases caused by the novel coronavirus via a 3C-like protease inhibitor-mediated pathway with a similar structure, specifically disclosing a compound having the following structure, which is claimed to be administered via injection for severe novel coronavirus infection:

[0009]

[0010] However, these existing compounds all have drawbacks. For example, parviride also inhibits the CYP3A4 enzyme, which may interfere with the metabolism of other drugs by this enzyme, altering the half-life and clearance rate, reducing efficacy, or causing adverse reactions. For instance, when patients take parviride and terfenadine simultaneously, parviride inhibits the oxidative metabolism of terfenadine by CYP3A4, leading to an abnormally high concentration of the latter in the patient's body, causing QT wave prolongation and arrhythmia. Furthermore, the compounds disclosed in WO2021 / 205290A1 also face the problem of ineffectiveness when administered orally. Therefore, the need to develop novel 3C-like protease inhibitors is becoming increasingly urgent. Summary of the Invention

[0011] In view of the shortcomings of the prior art, the object of the present invention is to provide a novel 3C-like protease inhibitor that overcomes the disadvantages of prior art compounds and can be used to treat novel coronavirus infection. This object is achieved by the subject matter described in the following aspects and embodiments of this application.

[0012] In a first aspect, the present invention provides a compound of formula (I) or a physiologically / pharmaceutically acceptable salt or ester thereof, including its stereoisomers or tautomers, racemates, nitrides, solvates, isotope-labeled substances, prodrugs, or metabolites:

[0013]

[0014] Where Cy represents C without substitution or optionally substituted by one, two or more Rs. 3-12Carbocyclic or 3-12 membered heterocyclic groups, where each R is independently selected from oxo (=O), C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, halogen, hydroxyl, amino, cyano, nitro or formyl.

[0015] In some embodiments, Cy in the compound of formula (I) represents an unsubstituted or optionally substituted C with one, two or more Rs. 3-12 Carbocyclic or 3-12 membered heterocyclic groups, where each R is independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, halogen, hydroxyl, amino, cyano, nitro or formyl.

[0016] In some embodiments, the compound of formula (I) has the structure of formula (IA):

[0017]

[0018] Cy is defined as in equation (I).

[0019] In some embodiments, the compound of formula (I) has the structure of formula (IB):

[0020]

[0021] Cy is defined as in equation (I).

[0022] In some embodiments, the compound of formula (I) has the structure of formula (IC):

[0023]

[0024] Cy is defined as in equation (I).

[0025] In some embodiments, the compound of formula (I) has the structure of formula (ID):

[0026]

[0027] Cy is defined as in equation (I).

[0028] In some embodiments, the compound of formula (I) has the structure of formula (IE):

[0029]

[0030] Cy is defined as in equation (I).

[0031] In some implementations, Cy represents C that is unsubstituted or optionally substituted with one, two or more Rs. 3-12 Carbocyclic or 3-12 membered heterocyclic group. Preferably, Cy represents an unsubstituted or optionally substituted C with one, two or more R groups. 4-8 A carbocyclic group or a 5-8 membered heterocyclic group. More preferably, Cy represents an unsubstituted or optionally substituted C with one, two or more R groups. 5-7 Carbocyclic or 5-7 membered heterocyclic group. More preferably, Cy represents an unsubstituted C5 or C6 cycloalkyl or a 5, 6 or 7 membered heterocyclic group, optionally substituted with one, two or more Rs; or Cy represents an unsubstituted C8 cycloalkyl, such as a C8 bridged cycloalkyl, like adamantyl; or Cy represents an unsubstituted 8, 9 or 10 membered heterocyclic group, optionally substituted with one, two or more Rs. More preferably, Cy represents an unsubstituted or optionally R-substituted C6 carbocyclic group, morpholino, piperidinyl, homopiperidinyl, or tetrahydropyranyl; or Cy represents an unsubstituted or optionally R-substituted thiazocycloheptanyl, bicyclo[2.2.2]octyl, azabicyclo[3.1.0]hexyl, azaspiro[4.5]decyl, oxo-2,8-diazaspiro[4.5]decyl, aminobicyclo[2.2.2]octyl, azabicyclo[2.2.1]heptyl, azaspiro[2.5]octyl, diazaspiro[4.5]decyl, or dioxothiazospiro[3.4]octyl. Most preferably, Cy represents methylcyclohexyl, tetrahydropyranyl, cyclohexyl, piperidinyl, morpholinyl, or homopiperidinyl; or Cy represents 1,1-dioxo-1,2-thiazaspiro[2.2.2]octyl, 3-azabicyclo[3.1.0]hexyl, 8-azaspiro[4.5]decyl, 1-oxo-2,8-diazaspiro[4.5]decyl, 4-aminobicyclo[2.2.2]octyl, 2-azabicyclo[2.2.1]heptyl, 6-azaspiro[2.5]octyl, 2,8-diazaspiro[4.5]decyl, or 5,5-dioxo-5-thia-2-azaspiro[3.4]octyl.

[0032] In some implementations, Cy represents C that is unsubstituted or optionally substituted by one, two or more Rs. 3-12 Carbocyclic or 3-12 membered heterocyclic group. Preferably, Cy represents an unsubstituted or optionally substituted C with one, two or more R groups. 4-8 Carbocyclic or 5-8 membered heterocyclic group. More preferably, Cy represents an unsubstituted or optionally substituted C with one, two or more R groups. 5-7The group is a carbocyclic or 5-7 membered heterocyclic group. More preferably, Cy represents an unsubstituted C5 or C6 cycloalkyl or 5, 6 or 7 membered heterocyclic group, optionally substituted with one, two or more R groups. More preferably, Cy represents an unsubstituted C6 carbocyclic group, morpholino, piperidinyl, homopiperidinyl or tetrahydropyranyl, optionally substituted with one R group. Most preferably, Cy represents methylcyclohexyl, tetrahydropyranyl, cyclohexyl, piperidinyl, morpholino, or homopiperidinyl.

[0033] In some implementations, R is independently selected from oxo (=O), C 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 The R group can be a halogenated alkoxy group, a halogenated group, a hydroxyl group, an amino group, a cyano group, a nitro group, or a formyl group. Preferably, each R group is independently selected from an oxo (=O), C, or C group. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 Halogenated alkoxy, halogen, hydroxyl, or amino. More preferably, each R is independently selected from oxo (=O), C, ... 1-3 Alkyl, C 1-3 Halogenated alkyl, halogen, hydroxyl, or amino. Most preferably, each R is independently selected from oxo (=O), methyl, ethyl, or propyl.

[0034] In some implementations, R are each independently selected from C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 The R group consists of a haloalkoxy group, a halogen group, a hydroxyl group, an amino group, a cyano group, a nitro group, or a formyl group. Preferably, each R group is independently selected from C10. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Haloalkyl, C 1-3 Halogenated alkoxy, halogen, hydroxyl, or amino groups. More preferably, each R is independently selected from C. 1-3 Alkyl, C 1-3 Halogenated alkyl, halogen, hydroxyl, or amino. Most preferably, each of R is independently selected from methyl, ethyl, or propyl.

[0035] In some implementations, Cy has the following structure:

[0036]

[0037] Preferably, Cy has the following structure:

[0038]

[0039] In some embodiments, the compound of formula (I) has the following structure:

[0040]

[0041]

[0042] In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as defined herein, or a salt or ester thereof, a stereoisomer or tautomer, a racemic mixture, a nitrogen oxide, a solvate, an isotope label, a prodrug, or a metabolite thereof.

[0043] In some preferred embodiments of the invention, the pharmaceutical composition according to the invention may optionally also contain at least one physiologically / pharmaceutically acceptable excipient.

[0044] In some preferred embodiments of the invention, the pharmaceutical composition according to the invention may optionally contain additional active ingredients. These additional active ingredients are, for example, remdesivir (or GS-5734), lopinavir, molnupiravir, ritonavir, chloroquine (or Sigma-C6628), hydroxychloroquine, and / or alpha-interferon.

[0045] In some preferred embodiments of the invention, the pharmaceutical composition according to the invention comprises a therapeutically effective amount of a compound of formula (I) or a salt or ester thereof, their stereoisomers or tautomers, racemates, nitrides, solvates, isotope labels, prodrugs or metabolites.

[0046] In some preferred embodiments of the invention, the pharmaceutical composition according to the invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor, or a host-targeting antiviral drug.

[0047] In some preferred embodiments of the invention, the pharmaceutical compositions according to the invention are used for the prevention or treatment of diseases, symptoms, syndromes and / or disorders selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory infection, olfactory and gustatory disturbances and their complications, or combinations thereof, caused by novel coronavirus infection.

[0048] According to the present invention, the pharmaceutical composition according to the present invention can be formulated into a dosage form suitable for administration by methods known in the art.

[0049] In a third aspect, the present invention provides the use of compounds of formula (I) according to the present invention, or salts or esters thereof, their stereoisomers or tautomers, racemates, nitrides, solvates, isotope labels, prodrugs or metabolites in the preparation of pharmaceuticals.

[0050] In some preferred embodiments of the invention, the medicament prepared according to the invention may optionally contain additional active ingredients. These additional active ingredients are, for example, remdesivir (or GS-5734), lopinavir, molnupiravir, ritonavir, chloroquine (Sigma-C6628), hydroxychloroquine, and / or alpha-interferon.

[0051] In some preferred embodiments of the present invention, the drug prepared according to the present invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor, or a host-targeting antiviral drug.

[0052] In some preferred embodiments of the invention, the medicament prepared according to the invention is used to prevent or treat diseases, symptoms, syndromes and / or disorders selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory infection, olfactory and gustatory disorders and their complications, or combinations thereof, caused by novel coronavirus infection.

[0053] According to the present invention, the drug prepared according to the present invention can be further formulated into a dosage form suitable for administration by methods known in the art.

[0054] In a fourth aspect, the present invention provides a method for treating or preventing diseases, symptoms, syndromes and / or disorders caused by novel coronavirus infection, the method comprising administering to an individual in need a compound of formula (I) according to the invention, or a salt or ester thereof, a stereoisomer or tautomer thereof, a racemic mixture, a nitrogen oxide, a solvate, an isotope label, a prodrug or metabolite, or a pharmaceutical composition as described in the second aspect of the invention.

[0055] In some preferred embodiments of the invention, diseases, symptoms, syndromes and / or disorders caused by novel coronavirus infection include: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory infection, olfactory and gustatory disturbances and their complications, or combinations thereof.

[0056] Those skilled in the art will understand that the features listed in various aspects and embodiments of the present invention can be freely combined, as long as they do not conflict with or are incompatible with each other.

[0057] Beneficial effects of the present invention

[0058] This invention provides a novel 3C-like protease inhibitor that can be used to treat novel coronavirus infection. The compound of formula (I) of this invention showed superior antiviral efficacy compared to PF-07321332 in in vitro anti-novel coronavirus assays. Detailed Implementation

[0059] The invention will be described in further detail below.

[0060] Unless otherwise specified, the following terms as used herein shall have the meanings as explained below, and their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., may be combined and combined with each other in any way; terms not explained in detail shall have the same meaning as commonly understood by one of ordinary skill in the art; all patent and non-patent literature or other materials disclosed herein, whether cited in whole or in part, are incorporated herein by reference.

[0061] For the purposes of this invention, the chemical elements and the periodic table (CAS edition) are consistent with the *Handbook of Chemistry and Physics*, 75th edition, 1994. Furthermore, general principles of organic chemistry can be found in *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito: 1999, and *March's Advanced Organic Chemistry* by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0062] the term

[0063] In this document, the terms “comprising,” “including,” and / or “containing” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0064] In this article, when describing one, two, or more species, "more species" should refer to the case of more than 2, such as the case of integers greater than or equal to 3, such as 3, 4, 5, 6, 7, 8, 9, or 10 species.

[0065] In this document, the term "optional" means either the presence or absence of the stated feature, implying that the event subsequently described may but is not necessarily to occur, and thus includes both cases where the event occurs or does not occur. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but is not necessarily present, and thus includes cases where the heterocyclic group is alkyl-substituted and cases where the heterocyclic group is not alkyl-substituted.

[0066] In this paper, expressions such as "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B and / or C", and "X is A, B and / or C" all express the same meaning, that is, X can be any one, two or more of A, B and C.

[0067] In this document, the term "unsubstituted" means that one or more hydrogen atoms on an atom, residue, group, or part thereof are not substituted by atoms or groups of atoms other than hydrogen atoms (i.e., substituents), and thus the atom, residue, group, or part retains its original structure. The term "substituted" means that one, two, or more hydrogen atoms in a group, preferably up to five hydrogen atoms, more preferably one to three hydrogen atoms, are each independently substituted by a corresponding number of substituents. When substituted by more than one substituent, these substituents are independent of each other; that is, the one or more substituents may be identical, but this is not excluded. Unless specifically stated otherwise, a substituent group may be substituted at any substituted position of the substituted group. When more than one position in a given structural formula can be substituted by one, two, or more substituents, then these substituents may be substituted independently at those positions. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions experimentally or theoretically without excessive effort.

[0068] In this article, the phrase "each independently of the other" should be understood as meaning that the individual items described are independent of each other and can be independently selected from the same or different options. For example, "each independently of the other" can mean that in different groups, the specific options expressed by the same symbol do not affect each other; or it can mean that in the same group, the specific options expressed by the same symbol do not affect each other.

[0069] In this document, two or more groups with distinct meanings are sometimes used together to describe a larger part, which includes the structural combinations formed by the two or more groups chosen independently of each other. For example, "alkylaryl" means "alkyl" and "aryl" linked together, and each "alkyl" and "aryl" has an independent meaning described for them, thus together forming the combined group "alkylaryl".

[0070] In this article, the mark "C" x-y "When used with a group, it indicates the upper and lower limits of the number of carbon atoms contained in that group. For example, "C 1-12 "Alkyl refers to an alkyl group containing a minimum of one carbon atom and a maximum of twelve carbon atoms. Those skilled in the art will understand that such a number does not include the number of carbon atoms contained in the substituents to which these groups are attached when they are further substituted."

[0071] In this document, the term "XY-membered" when used in conjunction with a cyclic group indicates the upper and lower limits of the number of ring atoms contained in that cyclic group. For example, a "3-20-membered" heterocyclic group refers to a heterocyclic group containing a minimum of three ring atoms and a maximum of twenty ring atoms. Those skilled in the art will understand that such numbers do not include the number of carbon atoms contained in the substituents attached to these heterocyclic groups when they are further substituted.

[0072] In this document, the term "halogen" refers to fluorine, chlorine, bromine, and / or iodine. Accordingly, the term "halogenated" refers to fluorination, chlorination, bromination, and / or iodination. Within the scope of this document, when an atom, residue, group, or part is halogenated, the atom at the halogenated position can be monosubstituted, disubstituted, or polysubstituted up to fully substituted by the halogen atom.

[0073] In this document, the term "alkyl" refers to a straight-chain or branched monovalent saturated aliphatic hydrocarbon group. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl. 2,3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc.

[0074] The term "carbocyclic (group)" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group. The carbocyclic group may contain 3 to 20 carbon atoms, preferably 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, and more preferably 3 to 6 carbon atoms. The carbocyclic group may be monocyclic or polycyclic, and may be a saturated cycloalkyl group or may optionally contain one, two, or more double and / or triple bonds, thereby forming so-called cycloalkenyl or cycloynyl groups. In the case of multiple rings, these rings may form spirocyclic, fused, and bridged ring structures. Preferably, the carbocyclic group is a cycloalkyl group, i.e., a saturated monocyclic or polycyclic cyclic hydrocarbon group, more preferably a saturated monocyclic hydrocarbon group. For example, non-limiting examples of monocyclic carbocyclic rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptanetrienyl, cyclooctyl, cyclooctatetraenyl, etc.; non-limiting examples of polycyclic carbocyclic rings include decahydronaphthyl or isobornyl.

[0075] The term "alkoxy" refers to -O-alkyl, wherein an alkyl group is defined as described herein and includes cycloalkyl groups. Non-limiting examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, etc. Alkoxy groups may be unsubstituted or optionally substituted.

[0076] The term "heterocyclic (group)" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms or groups selected from N, O, NH, S, S(O) or S(O)2, but excluding ring portions of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms (e.g., 1, 2, 3, and 4); more preferably, it comprises 3 to 6 ring atoms (e.g., 3, 4, 5, and 6). The heterocyclic group can be connected to the rest of the molecule via any one of the carbon atoms, or a nitrogen atom (if present), or an oxygen or sulfur atom (especially in the case of forming ononium salts). The heterocyclic group can include fused or bridged rings and / or spirocyclic rings. Non-limiting examples of monocyclic heterocyclic groups include azirrobutyl, oxacyclobutyl, pyrrolyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, dioxacyclopentenyl, tetrahydropyranyl, pyrrolinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, trithiaalkyl, homopiperazinyl, diazacycloheptyl, etc., preferably piperidinyl, morpholinyl, homopiperridinyl, tetrahydropyranyl, and pyrrolyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups, and may also be benzofused heterocyclic groups such as dihydroisoquinolinyl. The heterocyclic group may be bicyclic, and non-limiting examples include hexahydrocyclopenta[c]pyrrolo-2(1H)-yl and hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl. Heterocyclic groups can also be partially unsaturated, meaning they can contain one or more double bonds. Non-limiting examples include dihydrofuranyl, dihydropyranyl, 2,5-dihydro-1H-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl.

[0077] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0078] Unless otherwise stated, heterocyclic groups, heteroaryl groups, or heteroaromatic rings include all possible isomers, such as their positional isomers. Thus, for some illustrative, non-limiting examples, forms may include those in which one, two, or more of the following positions (if present) are substituted or bonded to other groups, including pyridin-2-yl, pyridinoid-2-yl, pyridinoid-3-yl, pyridinoid-3-yl, pyridinoid-4-yl, and pyridinoid-4-yl; thiophene groups or thiophene groups include thiophene-2-yl, thiophene-2-yl, thiophene-3-yl, and thiophene-3-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

[0079] Unless otherwise stated, the definitions of terms in this document also apply to expressions containing those terms, such as C. 1-6 The definition of alkyl also applies to C 1-6 Alkyloxy group, -N(C) 1-6 Alkyl)2、-NHC 1-6 Alkyl, -SO-C 1-6 Alkyl or -S(O)2-C 1-6 The alkyl part in alkyl groups, etc.

[0080] In this document, "physiologically / pharmaceutically acceptable salt" refers to the salts of the compounds of the present invention, which are safe and effective when used in mammals and have the intended biological activity.

[0081] Physiologically / pharmaceutically acceptable salts include acid addition salts of compounds of the present invention that have sufficient basicity and contain a nitrogen atom in the chain or ring. Additionally, the basic nitrogen-containing group can be quaternized with reagents such as: lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; long-chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides such as benzyl and phenethyl bromides, etc. As examples, physiologically / pharmaceutical acceptable salts include, but are not limited to, hydrochlorides, sulfates, nitrates, hydrogen sulfates, hydrobromic acids, acetates, oxalates, citrates, methanesulfonates, formates, or meglumine salts, etc.

[0082] Since the compounds of the present invention can have multiple salt-forming sites, the physiologically / pharmaceutically acceptable salts include not only salts formed at one salt-forming site of the compounds of the present invention, but also salts formed at two, three, or all of the salt-forming sites. Therefore, in the physiologically / pharmaceutically acceptable salts, the molar ratio of the compound of formula (I) to the anion of the acid or the cation of the base required for salt formation can vary within a wide range, for example, it can be 4:1 to 1:4, such as 3:1, 2:1, 1:1, 1:2, 1:3, etc.

[0083] In this document, the term "nitrogen oxide" refers to a compound containing several nitrogen-containing functional groups, to which one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. N-oxides can be formed by treating the corresponding nitrogen-containing compounds with oxidizing agents such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) (see Advanced Organic Chemistry, Wiley Interscience, 4th ed., Jerry March, pages). In particular, N-oxides can be prepared using the LWDeady method (Syn. Comm. 1977, 7, 509-514), in which the nitrogen-containing compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

[0084] In this document, the term "ester" refers to an ester that is hydrolyzable in vivo, formed from compounds containing a hydroxyl or carboxyl group. Such esters are, for example, physiologically / pharmaceutically acceptable esters that, upon hydrolysis in humans or animals, produce a parent alcohol or acid. Compounds of formula (I) of this invention contain a carboxyl group and can form hydrolyzable esters in vivo with suitable groups, including, but not limited to, alkyl, arylalkyl, etc.

[0085] Depending on the position and nature of the substituents, the compounds of the present invention may also contain one or more asymmetric centers. The asymmetric carbon atom may exist in (R) or (S) configuration; a racemic mixture is produced when there is only one asymmetric center, and a mixture of diastereomers is obtained when multiple asymmetric centers are present. In some cases, asymmetry may also exist due to hindered rotation around a specific bond, for example, where the central bond connects two substituted aromatic rings of a particular compound. Furthermore, the substituents may also exist in cis or trans isomeric forms.

[0086] The compounds of the present invention also include all possible stereoisomers thereof, which are either single stereoisomers or mixtures thereof in any proportion of said stereoisomers (e.g., R-isomers or S-isomers, or E-isomers or Z-isomers). The separation of single stereoisomers (e.g., single enantiomers or single diastereomers) of the compounds of the present invention can be achieved by any suitable prior art method (e.g., chromatography, particularly chiral chromatography).

[0087] The term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds of this invention can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Proton-transfer tautomers arise from the migration of covalently bonded hydrogen atoms between two atoms. Tautomers generally exist in equilibrium form; attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This invention encompasses all tautomeric forms of the compounds.

[0088] In this invention, the compounds according to the invention also include isotopically labeled compounds, which are the same as those shown in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of H, C, N, O, S, F and Cl, respectively such as 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 32 P, 35 S, 18 F and 36 Cl. Compounds of the present invention, their prodrugs, or physiologically / pharmaceutically acceptable salts of said compounds or prodrugs containing the aforementioned isotopes and / or other isotopes are within the scope of the present invention. Isotope-labeled compounds according to the present invention can generally be prepared according to the methods described herein by replacing non-isotope-labeled reagents with isotope-labeled reagents. Certain isotope-labeled compounds of the present invention, for example, incorporate radioactive isotopes (such as… 3 H and 14 Compounds in (C) can be used for drug and / or substrate tissue distribution assays. Tritium (i.e., 3 H) and carbon-14 (i.e.14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Furthermore, heavier isotopes (such as deuterium, i.e., 2 H) substitution can provide certain therapeutic advantages derived from greater metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and is therefore preferred in some cases. The compounds of the invention claimed in the claims are particularly defined as being substituted with deuterium or tritium. Furthermore, the presence of hydrogen in the substituents, without separately listing the terms deuterium or tritium, does not imply the exclusion of deuterium or tritium, but may also include deuterium or tritium.

[0089] In this document, the term "prodrug" or "prodrug precursor" refers to the conversion of a compound into a compound of the aforementioned formula (I) or the specific compound represented in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug of this invention can be an ester, and in this invention, esters that can serve as prodrugs include phenyl esters, aliphatic esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound in this invention contains a hydroxyl / carboxyl group, which can be acylated to yield the prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a hydroxyl group on the parent compound.

[0090] In this document, the term "metabolite" refers to the product obtained in vivo by the metabolism of a specific compound or its salt. A metabolite of a compound can be identified using techniques known in the art, and its activity can be characterized by experimental methods as described in this invention. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this invention includes metabolites of compounds, including metabolites produced by sufficiently exposing the compounds of this invention to mammals for a period of time.

[0091] Unless otherwise stated, any abbreviations for protecting groups, amino acids and other compounds used in this invention shall be those that are commonly used and recognized, or refer to the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 1972, 11: 942-944).

[0092] In this document, the term "solvate" refers to an association formed by one or more solvent molecules with the compounds of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. Therefore, the term "hydrate" refers to an association formed by solvent molecules that are water.

[0093] In this document, the term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein, or their physiologically / pharmaceuticalally acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmaceuticalally acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity. The term "physiologically / pharmaceuticalally acceptable" refers to a molecular entity and composition that, when administered to a human, is physiologically tolerable and generally does not produce allergic or similar adverse reactions, such as gastrointestinal upset, dizziness, etc. The term "carrier" refers to a diluent, excipient, or matrix administered with the compound. These drug carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water and aqueous solutions, saline solutions, and aqueous glucose and glycerol solutions are preferred as carriers, particularly injectable solutions. Suitable drug carriers are described in EW Martin's "Remington's Pharmaceutical Sciences".

[0094] In this document, the term "treatment" refers to any disease or condition, and in some embodiments, it means improving the disease or condition (i.e., slowing down or stopping or alleviating the development of the disease or at least one of its clinical symptoms). In other embodiments, "treatment" means alleviating or improving at least one bodily parameter, including bodily parameters that may not be perceptible to the patient. In still other embodiments, "treatment" means regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. In still other embodiments, "treatment" means preventing or delaying the onset, occurrence, or nausea of ​​the disease or condition.

[0095] In this document, the term "effective amount" or "therapeutic effective amount" refers to the amount of the compound described in this invention sufficient to achieve the intended application (including, but not limited to, the treatment of diseases as defined below). Therapeutic effective amounts may vary depending on factors such as the intended application (in vitro or in vivo), the subject being treated, and the condition of the disease, such as the subject's weight and age, the severity of the disease, and the route of administration, which can be readily determined by those skilled in the art. Specific dosages will vary depending on factors such as the specific compound selected, the administration regimen, whether it is administered in combination with other compounds, the timing of administration, the tissue to which the drug is administered, and the physical delivery system used.

[0096] In some preferred embodiments of the invention, the pharmaceutical excipients may be those widely used in the pharmaceutical manufacturing field. The excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers, or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0097] Substances that can be used as physiologically / pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicates, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-blocking polymers, lanolin, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose. Sodium cellulose, ethyl cellulose, and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol, phosphate buffer solutions, and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants, release agents, coatings, sweeteners, flavorings and spices, preservatives, and antioxidants.

[0098] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0099] The dosage form of the drug of this invention can be selected according to specific circumstances. A drug dosage form typically consists of a drug, excipients, and a container / sealing system. One or more excipients (also known as inactive ingredients) can be added to the compounds of this invention to improve or enhance the manufacture, stability, administration, and safety of the drug, and to provide a method for obtaining the desired drug release profile. Therefore, the type of excipient added to the drug can be determined by various factors, such as the physical and chemical properties of the drug, the route of administration, and the preparation steps. Pharmaceutical excipients exist in this field and include those listed in various pharmacopoeias. (See the United States Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP); publications of the Center for Drug Evaluation and Research (CEDR) of the US Food and Drug Administration (www.fda.gov), such as the Inactive Ingredient Guide (1996); Handbook of Pharmaceutical Additives (2002) by Ash; Synapse Information Resources, Inc., Endicott NY; etc.)

[0100] The pharmaceutical compositions of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate the processing of active molecules into formulations for pharmaceutical use.

[0101] The appropriate formulation depends on the desired route of administration. Routes of administration include intravenous injection, administration via mucosa or nose, and oral administration. For oral administration, compounds can be formulated into liquid or solid dosage forms and presented as immediate-release or controlled-release / sustained-release formulations. Suitable dosage forms for individual oral intake include tablets, pills, sugar-coated pills, hard-shell and soft-shell capsules, liquids, gels, syrups, ointments, suspensions, and emulsions.

[0102] Solid oral dosage forms can be obtained using excipients, including fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, flow aids, anti-adhesion agents, cation exchange resins, humectants, antioxidants, preservatives, colorants, and flavoring agents. These excipients can be synthetic or of natural origin. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium lauryl sulfate / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silica, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugars (i.e., dextrose, sucrose, lactose, etc.), talc, tragacanth gum, hydrogenated vegetable oils, and waxes. Ethanol and water can be used as granulation aids. In some cases, tablets need to be coated with, for example, a taste-masking film, an acid-resistant film, or a delayed-release film. Natural and synthetic polymers are often combined with colorants, sugars, and organic solvents or water to coat tablets, resulting in sugar-coated pills. When capsules are preferred over tablets, their drug powders, suspensions, or solutions can be delivered in compatible hard-shell or soft-shell capsule forms.

[0103] The effective therapeutic dose can first be estimated using various methods well known in the art. The initial dose for animal studies can be based on the effective concentration established in cell culture assays. A suitable dose range for humans can be determined, for example, using data obtained from animal studies and cell culture assays. In some embodiments, the compounds of the present invention can be prepared as oral formulations.

[0104] The appropriate formulation, route of administration, dosage, and dosing interval can be selected based on methods known in the art and taking into account the specific circumstances of the individual.

[0105] Example

[0106] The preparation method of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technical solutions implemented based on the content of the present invention are covered within the scope of protection intended by the present invention.

[0107] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art; the reagents, raw materials, instruments, equipment, etc. used in the following examples are all commercially available.

[0108] Reagents used

[0109] Main protease: NCBI Reference Sequence: NC_045512, protein sequence: SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ; prepared using E. coli expression system.

[0110] Fluorescent substrate MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2: purchased from Beyotime, product number: P9731

[0111] African green monkey kidney cells Vero E6 (ATCC, CRL-1586): purchased from the American Tissue Culture Collection (ATCC), catalog number: CRL-1586.

[0112] Human colorectal adenocarcinoma cells Caco-2 (ATCC, HTB-37): purchased from the American Tissue Culture Collection (ATCC), catalog number: HTB-37

[0113] Human lung adenocarcinoma cells Calu-3 (ATCC, HTB-55): purchased from the American Tissue Culture Collection (ATCC), catalog number: HTB-55

[0114] 2019-nCoV-WIV04 (IVCAS 6.7512) Virus Strain: The original strain of the novel coronavirus (2019-nCoV-WIV04, accession number: IVCAS 6.7512) was preserved by the Virus and Microbial Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0115] Instruments, equipment and measurement methods

[0116] (1) Centrifuges: Eppendorf 5810R benchtop refrigerated centrifuge and 5424 benchtop ambient temperature centrifuge (sample processing)

[0117] (2) Olympus IX51 inverted microscope (cell observation)

[0118] (3) Dual-Luciferase assay instrument: Turner BioSystems Modulus (USA) TM Single-tube multi-functional analyzer (cell viability and toxicity testing)

[0119] (4) ABI 6th Generation Real-Time Quantitative PCR Instrument (Absolute Quantitative Virus Copy Number)

[0120] (5) Clean bench, biosafety cabinet, incubator and -80℃ / -20℃ / 4℃ refrigerator, etc. (for experimental operation and sample preservation)

[0121] (6) Microplate reader: BioTek Synergy Neo2

[0122] (7) 96-well plate: Costa flat-bottom all-black microplate, catalog number 3916

[0123] Abbreviations and abbreviations

[0124] Boc: tert-butyloxycarbonyl;

[0125] MeOH: Methanol;

[0126] DCM: Dichloromethane;

[0127] EtOAc: Ethyl acetate;

[0128] DIPEA / DIEA: N,N-diisopropylethylamine;

[0129] HATU: 2-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0130] TEA: Triethylamine.

[0131] Preparation Examples

[0132] Intermediate Example 1

[0133]

[0134]

[0135] 1.1 Synthesis of compound M2:

[0136] The reaction mixture of compound M1 (10 g) and a methanol solution of methylamine (7 M, 100 mL) was stirred at 20 °C until complete. Thin-layer chromatography (ethyl acetate:methanol = 5:1) revealed compound M1 and detected a new spot with relatively high polarity (compound M1:R). f =0.71; Compound M2: R f =0.41). The reaction solution was concentrated under reduced pressure to obtain a yellow solid compound M2 (10 g). The crude product was used directly in the next step.

[0137] 1 H NMR (400MHz, CDCl3) δppm 7.17(br s,1H),6.71(br s,1H),6.07(br s,2H),4.36(br d,J=6.0Hz,1H),3.32-3.39(m,2H),2.51-2.55(m,1H),2.35-2.38(m,1H),1.99-2.17(m,1H),1.77-1.95(m,2H),1.43(s,9H).

[0138] 1.2 Synthesis of Compound M3

[0139] Burgess reagent (13.2 g) was added to a DCM (100 mL) solution of compound M2 (10 g), and the mixture was stirred at 20 °C for 12 hours. Thin-layer chromatography (ethyl acetate:methanol = 5:1) showed that compound M2 had completely reacted and detected a new spot with lower polarity (compound M2: Rf = 0.41; compound M3: Rf = 0.68). The reaction mixture was quenched with water (500 mL), diluted with DCM (500 mL), and extracted with DCM solvent (100 mL × 3). The combined organic phases were washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound M3 (8.3 g, yield: 88.9%) as a white solid.

[0140] 1.3 Synthesis of Compound M4

[0141] HCl / EtOAc (4M, 83mL) was added to a solution of compound M3 (8.3g) in ethyl acetate (30mL). The mixture was stirred at 20°C for 5 hours. Thin-layer chromatography (ethyl acetate:methanol = 5:1) showed that compound M3 had completely reacted and a new spot with greater polarity was detected (compound M3:R). f =0.68; Compound M4: R f=0.00). The reaction mixture was filtered, and the filter cake was washed with ethyl acetate (100 mL × 3) to give a white solid compound M4 (5 g, yield: 80.5%, hydrochloride form), which was used directly for the next step without further purification.

[0142] 1 H NMR(400MHz,DMSO-d6)δppm 9.32(br s,3H),4.79(br s,1H),3.16-3.22(m,2H),2.27-2.37(m,1H),2.18-2.25(m,1H),1.96-2.01(m,1H),1.67-1.83(m,2H).

[0143] 1.4 Synthesis of Compound M6

[0144] Compound M5 (10 g) was dissolved in a mixed solvent of 1,4-dioxane (80 mL) and water (20 mL). Under conditions controlled at 0–5 °C in an ice-water bath, an aqueous solution of sodium hydroxide (2 M, 20 mL) and Boc anhydride (15.5 g) were added. The reaction mixture was then stirred at 15–20 °C for 2 hours. The reaction mixture was slowly quenched in ice water (200 mL), and the aqueous phase was extracted with DCM (100 mL, 80 mL). The combined organic phases were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a pale yellow oily compound M6 (15 g).

[0145] 1 H NMR(400MHz,DMSO-d6)δppm:4.00(d,J=9.8Hz,1H),3.68(d,J=1.2Hz,3H),3.48-3.53(m,1H), 3.25-3.28(m,1H),1.39-1.43(m,2H),1.30-1.38(m,9H),1.00(s,3H),0.90(d,J=5.6Hz,3H).

[0146] 1.5 Synthesis of Compound M7

[0147] Compound M6 (10 g) was dissolved in 1,4-dioxane (200 mL). Under conditions of controlled temperature of 0–5 °C in an ice-water bath, an aqueous solution of lithium hydroxide (1 M, 100 mL) was added. The reaction mixture was then heated to 80–85 °C and stirred for 4 hours. The reaction mixture was slowly poured into ice water (300 mL) to quench the reaction. The pH was adjusted to 1–2 with dilute hydrochloric acid (2 M), and the mixture was extracted with DCM (200 mL, 100 mL). The combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily compound M7 (8 g, yield: 84.4%).

[0148] 1 H NMR(400MHz,DMSO-d6)δppm:12.7(s,1H),3.88-3.92(m,1H),3.44-3.54(m,1H),3.24- 3.28(m,1H),1.38-1.43(m,2H),1.30-1.38(m,9H),1.00(s,3H),0.90(d,J=6.0Hz,3H).

[0149] 1.6 Synthesis of Compound M8

[0150] Compound M7 (4 g) was dissolved in anhydrous DCM (40 mL). Under conditions of controlled temperature (0–5 °C) in an ice-water bath, N,N-diisopropylethylamine (20.3 g) and 2-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (8.94 g) were added, followed by compound M4 (3.57 g, HCl salt). The reaction mixture was then stirred at 15–20 °C for 12 hours. The reaction mixture was slowly quenched in ice water (100 mL) and extracted with DCM (50 mL, 40 mL). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by column chromatography (silica, petroleum ether:ethyl acetate = 100:1–0:1) to obtain a yellow oily compound M8 (4 g, yield: 65.4%).

[0151] 1H NMR (400MHz, DMSO-d6) δppm:8.85-8.91(m,1H),7.67-7.75(m,1H),4.50-4.51(m,1H),3.87-3.94(m,1H),3.62-3.67(m,1H),3.28(d,J=10.8H z,1H),3.14-3.19(m,2H),2.13-2.36(m,3H),1.67-1.87(m,2H),1.40( m,1H),1.30-1.37(m,9H),1.22(s,1H),1.01(s,3H),0.87-0.90(m,3H).

[0152] 1.7 Synthesis of Compound M9

[0153] Compound M8 (1.00 g) was added to an ethyl acetate solution (4 M, 10 mL) of hydrochloric acid at 0–5 °C. The reaction mixture was then stirred at 15–20 °C for 12 hours. The reaction solution was directly evaporated to dryness using a rotary evaporator to obtain a yellow oily compound M9 (700 mg).

[0154] 1 H NMR (400MHz, DMSO-d6)ppmδ=9.53-9.72(m,1H),7.74-7.84(m,1H),4.97-5.08(m,1H),3.59-3.64(m, 3H),3.15-3.22(m,2H),2.30-2.45(m,2H),2.12-2.25(m,2H),1.65-1.93(m,4H),1.03-1.08(m,6H).

[0155] Preparation Example 1: Synthesis of Compound SWD001

[0156]

[0157] 3-Methylcyclohexanecarboxylic acid (7 mg) was dissolved in DCM (2 mL), and DIEA (89 mg), HATU (20 mg), and compound M9 (10 mg) were added. The reaction was carried out under nitrogen protection at 20 °C with stirring for 12 hours. Thin-layer chromatography (ethyl acetate:methanol = 5:1) showed that compound M9 reacted completely, forming a principal spot with low polarity. The reaction solution was directly purified by preparative thin-layer chromatography (silica, ethyl acetate:methanol = 5:1). Further purification was performed by high-performance liquid chromatography (HPLC) (chromatographic instrument: ACSTJ-GX-AG, column: C18-1150 x 30 mm x 5 μm, mobile phase: water-acetonitrile, acetonitrile %: 30%-60%, 10 min) to obtain a white solid SWD001 (2.85 mg, yield: 20.0%). LCMS (ESI) m / z: 415 [M+H] + .

[0158] 1 H NMR(400MHz,CD3OD)δppm 4.94-5.07(m,1H),4.14-4.26(m,1H),3.89-4.10(m,1H),3.54-3.66(m,1H),3.33-3.41(m,2H),2.58-2.77(m,1H), 2.08-2.55(m,3H),1.15-2.03(m,12H),1.05-1.13(m,3H),1.00-1.03(m,1H),0.95-1.00(m,3H),0.83-0.94(m,3H).

[0159] Preparation Example 2: Synthesis of SWD002

[0160]

[0161] (S)-Tetrahydro-2H-pyran-2-carboxylic acid (10.7 mg) was dissolved in anhydrous DCM (1 mL). DIEA (89 mg), HATU (39.3 mg), and compound M9 (20 mg) were added sequentially at 15–20 °C, and the reaction mixture was stirred at 15–20 °C for 12 hours. The reaction mixture was quenched by slowly pouring it into ice water (10 mL), and then extracted with DCM (5 mL, 4 mL). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by high performance liquid chromatography (HPLC) (column: Phenomenex C18-1150*30 mm*5 μm; mobile phase: water (10 mM ammonium bicarbonate solution); B%: 20%–50%, 10 min) to obtain a white solid compound SWD002 (4.26 mg, yield: 14.4%).

[0162] LCMS(ESI) m / z: 403 [M+H] + ;

[0163] 1 H NMR(400MHz,CD3OD)δppm 4.96-5.06(m,1H),4.17-4.28(m,1H),4.03-4.14(m,1H),3.79-4.01(m,3H),3.47-3.58(m,1H),3.32-3.44(m,2H) ,2.58-2.74(m,1H),2.21-2.51(m,2H),1.76-2.03(m,3H),1.31-1.74(m,7H),1.05-1.10(m,3H),0.89-1.00(m,3H)

[0164] Preparation Example 3: Synthesis of SWD003

[0165]

[0166] Compound M9 (10.6 mg) was dissolved in anhydrous DCM (1 mL), and DIEA (89 mg), HATU (39.3 mg), and cyclohexanecarboxylic acid (20 mg) were added sequentially at 15–20 °C. The reaction mixture was then stirred at 15–20 °C for 12 hours. The reaction mixture was quenched by slowly pouring it into ice water (10 mL), and then extracted with DCM (5 mL, 4 mL). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by high performance liquid chromatography (HPLC) (column: Phenomenex C18-1150*30 mm*5 μm; mobile phase: water (10 mM ammonium bicarbonate solution); B%: 30%–60%, 10 min) to obtain a white solid compound SWD003 (2.85 mg, yield: 8.85%).

[0167] LCMS(ESI)m / z:401[M+H] + ;

[0168] 1H NMR(400MHz,CD3OD)δppm 4.94-5.10(m,1H),4.15-4.25(m,1H),3.91-4.09(m,1H),3.58-3.76(m,1H),3.32-3.43(m,2H),2.58-2.75(m,1H), 2.08-2.50(m,3H),1.66-2.04(m,7H),1.53-1.63(m,1H),1.23-1.46(m,6H),1.06-1.11(m,3H),0.94-1.00(m,3H).

[0169] Preparation Example 4: Synthesis of SWD004

[0170]

[0171] To a solution of compound M9 (0.1 g) and TEA (70 mg) in DCM (2 mL), p-nitrophenyl chloroformate (76 mg) was added, and the mixture was stirred at 20 °C for 12 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated saline solution (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain crude product M10 (105 mg). LCMS (ESI) m / z: 456 [M + H] + .

[0172] Compound M10 (0.12 g) was placed in a reaction flask, piperidine (561 mg) was added, and the mixture was stirred at 20 °C for 12 hours. The reaction solution was diluted with water (60 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated saline solution (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by high performance liquid chromatography (HPLC) (column: C18-1150*30 mm*5 μm; mobile phase: [water (ammonium bicarbonate solution)-acetonitrile]; B%: 20%-50%, 8 min) to obtain a white solid compound SWD004 (7.17 mg, yield: 6.78%).

[0173] LCMS(ESI)m / z:402[M+H] + ;

[0174] 1H NMR(400MHz,CD3OD)δppm 0.97-1.06(m,3H),1.07-1.11(m,3H),1.26(br d,J=7.6Hz,1H),1.50-1.66(m,4H),1.67-1.75(m,2H),1.76-1.98(m,3H),2.16-2.47(m,2H),2.55-2 .73(m,1H),3.33-3.44(m,3H),3.46-3.62(m,4H),3.62-3.76(m,2H),4.67(s,1H),4.79-4.83(m,1H)

[0175] Preparation Example 5: Synthesis of SWD005

[0176]

[0177] Compound M10 (90 mg) was placed in a reaction flask, and morpholine (430 mg) was added. The mixture was stirred at 20 °C for 12 hours. The reaction solution was diluted with water (60 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated sodium chloride aqueous solution (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by high performance liquid chromatography (column: C18-1150 x 30 mm x 5 μm; mobile phase: [water-acetonitrile]; acetonitrile %: 20%-50%, 10 min) to obtain a white solid compound SWD005 (1.71 mg, yield: 2.14%).

[0178] LCMS(ESI) m / z: 404 [M+H] + ;

[0179] 1 H NMR(400MHz,CD3OD)δppm 0.98(d,J=7.6Hz,3H),1.08(s,3H),1.43(dd,J=18.0,7.6Hz,1H),1.54-1.62(m,1H),1.88-1.93(m,1H),2.05(d,J=11.2Hz,2H),2. 21-2.46(m,3H),2.56-2.72(m,1H),3.33-3.45(m,3H),3.49-3.83(m,8H),3.86-4.01(m,1H),4.18-4.30(m,1H),4.95-5.09(m,1H)

[0180] Preparation Example 6: Synthesis of SWD009

[0181]

[0182] Step 1: (S)-1-(tert-Butoxycarbonyl)perpiperidine-2-carboxylic acid (0.2 g) was dissolved in DCM (3 mL), and DIEA (531 mg) and HATU (469 mg) were added. N2 gas was introduced, and the mixture was purged three times. The temperature was then lowered to 0 °C, and compound M11 (203 mg) was added at this temperature. After warming to room temperature, the mixture was stirred at 20 °C for 12 hours. LC-MS analysis showed that the reactant (S)-1-(tert-Butoxycarbonyl)perpiperidine-2-carboxylic acid was completely consumed, and a mass spectrum peak matching that of product M12 was detected. The reaction solution was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain crude compound M12 (400 mg). LCMS (ESI) m / z: 395 [M+H] + .

[0183] Step 2: Compound M12 (0.3 g) was dissolved in a mixed solution of methanol (3 mL) and water (3 mL), and lithium hydroxide monohydrate (64 mg) was added. The mixture was stirred at 20 °C for 2 hours. LCMS analysis showed that reactant M12 was completely consumed, and a mass spectrometry peak matching that of product M13 was detected. The reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL, 10 mL × 3), the pH of the aqueous phase was adjusted to 6-7 with 1N HCl, and then extracted with ethyl acetate (30 mL, 10 mL × 3). The combined ethyl acetate phases were washed with 30 mL, 10 mL × 3 of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound M13 (230 mg). LCMS (ESI) m / z: 381 [M + H] + .

[0184] Step 3: Compound M13 (0.23 g) was dissolved in dichloromethane (5 mL), and DIEA (469 mg) and HATU (345 mg) were added. N2 gas was bubbled through the solution, and the mixture was purged three times. The temperature was then lowered to 0 °C. At this temperature, (2S)-2-amino-3-(2-oxopyrrolidone-3-yl)propionitrile hydrochloride (138 mg) was added. The mixture was then heated to room temperature and stirred at 20 °C for 12 hours. LC-MS analysis showed that reactant M13 was completely consumed, and a mass spectrum peak matching that of product M14 was detected. The reaction solution was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated sodium chloride aqueous solution (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain crude compound M14 (300 mg). The residue was purified by preparative thin-layer chromatography (silica, petroleum ether:ethyl acetate = 1:1) to obtain M14 (150 mg). LCMS(ESI) m / z: 516 [M+H] + .

[0185] Step 4: Dissolve compound M14 (0.08 g) in DCM (2 mL), add trifluoroacetic acid (106 mg), and stir the mixture at 20 °C for 6 hours. Extract the reaction solution with ethyl acetate (10 mL). Wash the combined organic layers with saturated sodium chloride aqueous solution (10 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain the residue. Purify the residue by high performance liquid chromatography (HPLC) (column: C18-1150*30 mm*5 μm; mobile phase: [water (ammonium bicarbonate solution)-acetonitrile]; B%: 20%-50%, 10 min) to obtain a white solid SWD009 (3.8 mg, yield: 5.89%). LCMS (ESI) m / z: 416 [M+H] + ;

[0186] 1 H NMR(400MHz,CD3OD)δppm 4.94-5.07(m,1H),4.23-4.31(m,1H),3.83-3.97(m,1H),3.66-3.76(m, 2H),2.99-3.11(m,1H),2.74-2.86(m,1H),2.66(s,2H),2.25-2.46(m,2 H),1.95-2.24(m,2H),1.84-1.94(m,1H),1.56-1.82(m,8H),1.38-1.46 (m,1H),1.27-1.36(m,1H),1.09(d,J=5.2Hz,3H),1.00(d,J=6.0Hz,3H).

[0187] Preparation Example 7: Synthesis of SWD013

[0188]

[0189] Step 1: Dissolve (R)-2-aminopent-4-enoic acid (4.5 g) in methanol (31.5 mL), and slowly add thionyl chloride (36.9 g) dropwise while maintaining the reaction system temperature at 0–5 °C in an ice-water bath. Then, stir the reaction system at 15–20 °C for 12 hours. Concentrate the reaction mixture directly to obtain a yellow solid M15 (3 g, yield: 78.3%). 1 H NMR (400MHz, DMSO-d6) δppm: 8.59 (s, 2H), 5.64-5.91 (m, 1H), 5.07-5.28 (m, 2H), 4.13 (t, J = 6.0Hz, 1H), 3.73 (s, 3H), 2.59 (t, J = 6.4Hz, 2H).

[0190] Step 2: Dissolve M15 (1.2 g) in dichloromethane (8.4 mL) and add triethylamine (2.2 g) while maintaining the temperature at 0–5 °C with dry ice. Cool the reaction system to -5–5 °C and slowly add allyl sulfonyl chloride (1.22 g). Stir the reaction system at 15–20 °C for 2 hours. Quench the reaction mixture slowly in 30 mL of ice water, then extract with dichloromethane (20 mL, 10 mL). Wash the combined organic phases with saturated sodium chloride aqueous solution (30 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. Column chromatography (silica, petroleum ether:ethyl acetate = 8:1–6:1) yields a yellow oil M16 (1 g, yield: 59.2%).

[0191] 1 H NMR(400MHz,MeOD)δppm:5.71-5.99(m,2H),5.30-5.46(m,2H),5.06-5.21(m,2 H),4.04-4.15(m,1H),3.76-3.82(m,2H),3.71-3.75(m,3H),2.39-2.60(m,2H).

[0192] Step 3: Dissolve compound M16 (300 mg) in dichloromethane (3000 mL) and add Hoveyda-Grubbs second-generation catalyst (24.2 mg) at 15–20 °C. Then stir the reaction mixture at 15–20 °C for 12 hours. Use the reaction mixture directly for the next step.

[0193] Step 4: Add wet palladium on carbon (30 mg) to the reaction system of compound M17, and replace with hydrogen three times. Under a hydrogen (15 PSi) atmosphere, stir the reaction system at 15–20 °C for 12 hours. Filter the reaction system with diatomaceous earth, wash the filter cake with methanol (200 mL, 100 mL, 80 mL), and concentrate the filtrate to obtain a white solid M18 (120 mg, yield: 45.7%).

[0194] 1 H NMR (400MHz, MeOD) δppm: 3.90-4.00(m,1H),3.74(s,3H),3.19-3.28(m,2H),2.21-2.35(m,1H),1.99-2.14(m,1H),1.70-1.96(m,4H).

[0195] Step 5: Dissolve M18 (120 mg) in a mixed solvent of methanol (1 mL), tetrahydrofuran (1 mL), and water (1 mL). Add lithium hydroxide monohydrate (49 mg) at 0–5 °C and stir at 15–20 °C for 2 hours. Slowly pour the reaction mixture into ice water (20 mL) to quench the reaction. Adjust the pH to 1–2 with dilute hydrochloric acid (2 M) and extract with dichloromethane (10 mL, 8 mL, 6 mL). Wash the combined organic phases with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a white solid M19 (70 mg, yield: 62.6%).

[0196] 1 H NMR (400MHz, DMSO-d6) δppm: 3.91-3.95(m,1H), 3.23-3.26(m,2H), 2.09-2.34(m,2H), 1.70-1.97(m,4H).

[0197] Step 6: Dissolve M19 (13.3 mg) in dichloromethane (2 mL), add N,N-diisopropylethylamine (26.7 mg) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (39.3 mg) at 0–5 °C, and finally add compound 5001_6a (20 mg). Stir the reaction system at 15–20 °C for 2 hours. Quench the reaction mixture slowly in ice water (20 mL) and extract with dichloromethane (10 mL, 8 mL). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. This crude product was then purified by high-performance liquid chromatography (HPLC) (instrument: ACSTJ-GX-AG, column: C18-1150 x 30 mm x 5 μm, mobile phase: water (10 mM ammonium bicarbonate solution)-acetonitrile, B%: 20%-50%, 10 min) to obtain SWD0013 (20 mg, yield: 56.7%). LCMS (ESI) m / z: 466 [M+H] + ;

[0198] 1 H NMR(400MHz,DMSO-d6)δppm 8.80-9.41(m,1H),7.62-7.88(m,1H),7.11-7.42(m,1H),4.85-5.01(m,1H),3.73-4.17(m,2H),3.45-3.62(m,2H),3.10-3.2 7(m,4H),2.09-2.43(m,3H),1.65-1.88(m,7H),1.27-1.50(m,1H),1.24-1.59(m,2H),0.97-1.08(m,3H),0.83-0.94(m,3H).

[0199] Preparation Example 8: Synthesis of SWD021

[0200]

[0201] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 443 [M+H] + ;

[0202] 1H NMR(400MHz,CD3OD)δppm 4.94-5.02(m,1H),4.26(s,1H),3.96-4.00(m,1H),3.86(d,J=10.8Hz,1H),3.35-3.36(m,1H),3.28-3.31(m,1H),2.64-2.67(m,1 H),2.31-2.33(m,1H),1.92-1.97(m,8H),1.64-1.65(m,8H),1.28(t,J=10.0Hz,1H),1.06(d,J=5.2Hz,3H),0.94(d,J=6.4Hz,3H)

[0203] Preparation Example 9: Synthesis of SWD023

[0204]

[0205] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 400 [M+H] + ;

[0206] 1 H NMR(400MHz,CD3OD)δppm 4.97-5.04(m,1H),4.26-4.31(m,1H),3.96-4.10(m,1H),3.70-3.79(m,1H),3.34 -3.37(m,2H),3.17-3.21(m,1H),2.86-3.03(m,3H),2.56-2.74(m,1H),2.37-2.43 (m,1H),2.26-2.33(m,1H),2.11-2.18(m,1H),1.79-2.06(m,3H),1.57-1.63(m,1 H),1.39-1.49(m,1H),1.09(d,J=8.0Hz,3H),0.93-0.99(m,3H),0.82-0.87(m,1H)

[0207] Preparation Example 10: Synthesis of SWD024

[0208]

[0209] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 456 [M+H] + ;

[0210] 1H NMR(400MHz,CD3OD)δppm 4.98-5.05(m,1H),4.22-4.37(m,1H),3.92-4.00(m,1H),3.60-3.74(m,2H),3.35-3.37(m,1H),3.01-3.08(m,4H),2.63-2.69( m,1H),2.23-2.39(m,3H),1.79-2.00(m,6H),1.56-1.71(m,7H),1.39-1.44(m,1H),1.09(d,J=4.4Hz,3H),0.97(d,J=6.0Hz,3H)

[0211] Preparation Example 11: Synthesis of SWD030

[0212]

[0213] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 471 [M+H] + ;

[0214] 1 H NMR(400MHz,CD3OD)δppm 4.94-5.04(m,2H),4.22-4.42(m,1H),3.99-4.03(m,1H),3.68-3.82(m,1H),3.36-3.51(m,3H),2.92-3.24(m,4H) ,2.62-2.65(m,1H),2.25-2.41(m,2H),1.79-2.02(m,5H),1.57-1.68(m,2H),1.29-1.46(m,2H),0.96-1.11(m,6H)

[0215] Preparation Example 12: Synthesis of SWD031

[0216]

[0217] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 442 [M+H] + ;

[0218] 1H NMR(400MHz,CD3OD)δppm 4.92-5.03(m,1H),4.27-4.61(m,1H),3.79-4.06(m,2H),3.34-3.37(m,1H),2.17-2.6 8(m,3H),1.87-2.05(m,8H),1.58-1.72(m,7H),1.27-1.36(m,2H),0.94-1.12(m,6H).

[0219] Preparation Example 13: Synthesis of SWD036

[0220]

[0221] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 414 [M+H] + ;

[0222] 1 H NMR(400MHz,CD3OD)δppm 5.11-4.94(m,1H),4.28(d,J=7.2Hz,1H),4.02-3.89(m,1H),3.73-3.51(m,2H),3.41-3.33(m,2H),2.85-2 .55(m,2H),2.51-2.11(m,2H),2.06-1.77(m,2H),1.75-1.21(m,8H),1.17-1.05(m,3H),1.03-0.88(m,3H).

[0223] Preparation Example 14: Synthesis of SWD037

[0224]

[0225] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 428 [M+H] + ;

[0226] 1 H NMR(400MHz,CD3OD)δppm 5.08-4.94(m,1H),4.30-4.14(m,1H),4.14-3.98(m,1H),3.90-3.65(m,1H),3.42-3.33(m,1H),3.30-3.19(m,1H), 3.16-2.54(m,5H),2.50-2.11(m,2H),2.01-1.33(m,9H),1.22-1.07(m,4H),1.06-0.93(m,3H),0.91-0.79(m,1H).

[0227] Preparation Example 15: Synthesis of SWD038

[0228]

[0229] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 457 [M+H] + ;

[0230] 1 H NMR(400MHz,D2O)δppm 5.09-4.81(m,1H),4.48(q,J=8.2Hz,1H),4.31-4.14(m,1H),4.14-3.86(m,1H),3.84-3.64(m,1H),3.63-3.48(m,1H ),3.43-3.29(m,2H),3.27-3.06(m,4H),3.00-2.50(m,2H),2.48-2.15(m,3H),2.12-1.37(m,9H),1.07-0.83(m,6H).

[0231] Preparation Example 16: Synthesis of SWD039

[0232]

[0233] The experimental procedures were the same as those for the synthesis of compound SWD001 in Example 1. LCMS (ESI) m / z: 478 [M+H] + ;

[0234] 1 H NMR(400MHz,CD3OD)δppm 4.93-5.14(m,1H),4.34(d,J=14.6Hz,1H),4.10-4.23(m,2H),3.86-3.99(m,2H),3.61-3.85(m,3H),3.07-3.22(m,2H),2.63-2. 75(m,1H),2.10-2.46(m,5H),1.82-1.99(m,2H),1.61-1.70(m,1H),1.49-1.64(m,1H),1.07-1.16(m,4H),1.01(d,J=6.0Hz,3H).

[0235] Biological Example 1 The inhibition of the representative compound of this invention against the major protease (Mpro) of the novel coronavirus was determined using FRET.

[0236] This experiment used a continuous kinetic method to measure the activity of the novel coronavirus Mpro. The substrate used was MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2, the excitation wavelength was 320 nm, and the emission wavelength was 405 nm.

[0237] This experiment determined the inhibition rate of a representative compound on the main protease by comparing the initial reaction rate of the enzyme with that of the control group at different drug concentrations. The IC50 value was then calculated using a GraphPad Prism nonlinear fitting curve. In the experiment, the measurement system contained 0.15 μM main protease, 20 μM fluorescent substrate, and substrate concentrations (0-1 μM) of different inhibitors.

[0238] Inhibition rate (%) = (RFU) 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%

[0239] This experiment used a 120 μl assay system with the following composition: 108 μl protease, 10 μl 240 μM substrate, and 2 μl inhibitor compound to be tested. The buffer solution consisted of (50 mM Tris, pH 7.4, 1 mM EDTA, and 0.01% Triton X-100).

[0240] This experiment tested the representative compound at 40 μM to preliminarily evaluate the inhibitory effect of the compound on SARS-CoV-2 Mpro. Subsequently, the inhibitory rate of the representative compound at different concentrations of the present invention on SARS-CoV-2 was tested to calculate the IC50 value.

[0241] The inhibition rates and IC50 values ​​of the representative compounds of this invention against the novel coronavirus Mpro at a concentration of 40 μM, as determined therein, are shown in Table 1 below:

[0242] Table 1

[0243] compound Inhibition rate % IC50 (μM) PF-07321332 118.05 0.0245 SWD001 101.8 2.230 SWD002 76.2 15.08 SWD003 88.1 28.46 SWD004 14.2 - SWD005 82.5 16.93 SWD009 98.7 6.631 SWD013 112.9 0.2575 SWD021 87.4 5.788 SWD023 42.5 20.25 SWD024 71.57 13.35 SWD030 6.4 - SWD031 63.4 - SWD036 86.6 2.874 SWD037 75.6 10.06 SWD038 88.1 3.849 SWD039 30.4 -

[0244] "-" indicates that the measurement was not performed.

[0245] Biological Example 2: Cytotoxicity Test

[0246] The cytotoxicity of the representative compounds of this invention was tested in African green monkey kidney cells Vero E6 (ATCC, CRL-1586), human colorectal adenocarcinoma cells Caco-2 (ATCC, HTB-37), and human lung adenocarcinoma cells Calu-3 (ATCC, HTB-55). The specific steps included adding 100 μL of cells to each well of a 96-well plate to achieve a cell count of 2 × 10⁶ cells per well. 4Cells were serially diluted 3-fold at the highest concentration of 1000 μM, with two replicates for each dilution (n=10). Cell viability was assessed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) after 48 h. The half-cytotoxic concentration (CC50) at which the representative compound of the present invention elicited a cytotoxic response in 50% of the cells was calculated based on the luminescence values. The results are shown in Table 2 below.

[0247] Table 2

[0248]

[0249] "-" indicates that the measurement was not performed.

[0250] The above results demonstrate that the representative compounds of this invention exhibit CC50 values ​​greater than 100 μM in different cell lines, indicating very low cytotoxicity and significant development potential. Except for compound SWD001, the CC50 values ​​of the other compounds were higher in Caco-2 and Calu-3 cells than in Vero E6 cells, suggesting that these two cell lines are less toxic and more tolerant to these compounds. In Vero E6 cells, the CC50 values ​​of compounds SWD001 and SWD003 were comparable to those of PF-07321332; in Caco-2 and Calu-3 cells, the CC50 values ​​of compounds SWD002 and SWD003 were comparable to those of PF-07321332.

[0251] Biological Example 3: In vitro anti-novel coronavirus test

[0252] In this study, African green monkey kidney cell Vero E6 (ATCC, CRL-1586), human colorectal adenocarcinoma cell line Caco-2 (ATCC, HTB-37), and human lung adenocarcinoma cell line Calu-3 (ATCC, HTB-55), as well as the 2019-nCoV-WIV04 (IVCAS 6.7512) virus strain, were used to test the infection dose.

[0253] Add 250 μL of cells to each well of a 48-well plate to make the cell count 5 × 10⁻⁶ cells per well. 4Cells were serially diluted 3-fold at the highest concentration of 100 μM, with each dilution (n=8) set up in triplicate. 2019-nCoV-WIV04 (MOI=0.01) was added to each well to infect cells for 48 h. After 48 h, RNA was extracted from the supernatant using the QIAamp Viral RNA Mini Kit (Qiagen), and viral copy number was detected by qRT-PCR. The half-maximal effective concentration (EC50) and selectivity index (SI) were calculated based on the absolute copy number. SI = AVA / TOX, where AVA (antiviral activity) refers to EC50 and TOX (cytotoxicity) refers to CC50. The results are shown in Table 3 below.

[0254] Table 3

[0255]

[0256] "-" indicates that the measurement was not performed.

[0257] The above results demonstrate that the representative compounds SWD001, SWD002, SWD005, and SWD009 of this invention exhibit varying degrees of inhibitory activity against the novel coronavirus at the cellular level, with compounds SWD001 and SWD009 showing better antiviral effects than the other two compounds. Based on CC50 and EC50, the selectivity index (SI) values ​​for SARS-CoV-2 against the representative compounds SWD001, SWD002, SWD005, and SWD009 of this invention were calculated. The SI values ​​of SWD001 and SWD009 against the novel coronavirus at the cellular level were higher than those of SWD002 and SWD005. It is well known that Vero E6 cells possess a strong drug pump pathway, resulting in lower SI values ​​for the representative compounds of this invention and PF-07321332 compared to the other two cell lines. In conclusion, SWD001 and SWD009 effectively inhibit the novel coronavirus at the cellular level and possess the potential to be used as anti-coronavirus drugs.

[0258] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can freely combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0259] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound of formula (I) or a physiologically / pharmaceutically acceptable salt thereof, their tautomers: wherein, The compound of formula (I) has the following structure: or .

2. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to claim 1 or a salt thereof, their tautomers, and optionally at least one physiologically / pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, optionally comprising additional active ingredients.

4. The pharmaceutical composition according to claim 3, wherein the additional active ingredient is selected from remdesivir, lopinavir, monopivir, ritonavir, chloroquine, hydroxychloroquine and / or alpha-interferon.

5. The use of a compound of formula (I) of claim 1 or a salt thereof and their tautomers or the pharmaceutical composition of any one of claims 2 to 4 in the preparation of a medicament for the treatment or prevention of diseases, symptoms, syndromes and / or disorders caused by novel coronavirus infection.

6. In accordance with the use of claim 5, the drug is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor, or a host-targeting antiviral drug.

7. The use according to claim 5, wherein the medicament optionally comprises an additional active ingredient, wherein the additional active ingredient is selected from remdesivir, lopinavir, monoupivir, ritonavir, chloroquine, hydroxychloroquine and / or alpha-interferon.

8. The use according to claim 5, wherein the medicament is used to prevent or treat diseases, symptoms, syndromes and / or disorders selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory infection, olfactory or gustatory disturbances and their complications, or combinations thereof, caused by novel coronavirus infection.

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