3cl protease inhibitors and methods thereof

Abstract

The present disclosure relates to a compound of formula (I) or a salt, solvate, stereoisomer or prodrug thereof as a 3CL protease inhibitor. The compound can be used for treating coronavirus-related diseases or disorders. (I).

Description

Technical Field

[0001] This disclosure generally relates to 3CL protease inhibitors and their preparation methods. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a pandemic due to its high transmissibility and pathogenicity. Despite the availability of SARS-CoV-2 vaccines, the 2022 COVID-19 pandemic continues to ravage the world because emerging SARS-CoV-2 variants are more likely to evade the immune protection provided by existing vaccines. To date, the number of clinically approved antiviral drugs against SARS-CoV-2 and other coronavirus infections remains very limited. The history of outbreaks of SARS-CoV-1, MERS, and SARS-CoV-2 over the past two decades suggests that future pandemics are inevitable, making the need for novel antiviral drugs urgent. Furthermore, four existing human coronaviruses (OC43, NL63, HKU1, and 229E) cause common cold-like respiratory illnesses in humans. Developing novel compounds against coronaviruses is a strategic priority; these compounds should target highly conserved viral proteins that are crucial for viral replication.

[0003] The SARS-CoV-2 genome encodes more than 20 proteins, including two proteases—papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro)—that function to cleave the viral multiprotein into individual proteins essential for viral replication. 3CL protease, as the primary protease, is considered an important drug target. Viral replication can be blocked by inhibiting the proteolytic process of the viral multiprotein. Functionally, 3CL protease cleaves the viral multiprotein at more than 10 junctions, producing non-structural proteins crucial for viral replication. Given the high conservation of 3CL protease among known coronavirus strains, designing inhibitors targeting this protease would enhance our ability to prepare drugs for the next coronavirus outbreak. 3CL protease is also a virally encoded protein and does not share high similarity with human proteins, thus reducing the possibility of off-target activity.

[0004] It is hoped that at least one of the above problems can be overcome or improved. Summary of the Invention

[0005] This disclosure relates to compounds that can inhibit the activity of coronavirus 3CL protease, as shown herein, which are active against SARS-CoV-2, MERS, HCoV-229E, and HCoV-OC43.

[0006] This disclosure provides a compound of formula (I) or a salt, solvate, stereoisomer, or prodrug thereof:

[0007] (I)

[0008] R1 is selected from substituted acyl and cyano groups;

[0009] R2 is selected from optionally substituted alkyl and optionally substituted alkenyl groups;

[0010] X is selected from N and C-R4;

[0011] R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0012] R4 is selected from H and optionally substituted alkyl groups;

[0013] R5 is independently selected from H and optionally substituted alkyl groups;

[0014] R6 is selected from H and optionally substituted alkyl groups;

[0015] Alternatively, R2 and R6 can be linked to form an optionally substituted heterocyclic group;

[0016] m is an integer selected from 1 to 4; and

[0017] n is an integer selected from 1 to 4.

[0018] In some implementations, R1 is selected from HC(O)- and cyano.

[0019] In some implementations, R2 is a C1-C5 alkyl group.

[0020] In some embodiments, R2 is selected from butyl, isobutyl, pentyl, isopentyl, neopentyl, halopentyl, haloisopentyl, haloneopentyl, cyclopentylmethyl, and cyclopentyl.

[0021] In some embodiments, R3 is selected from H, optionally substituted benzyl, optionally substituted halobenzyl, optionally substituted trihalomethylbenzyl, optionally substituted cyclopentyl, optionally substituted cyclopentylmethyl, optionally substituted tert-butoxy, optionally substituted cyclohexyloxy, optionally substituted tetrahydropyranylamide, optionally substituted tert-butylamide, optionally substituted trihalotert-butylamide, optionally substituted trihalomethylamide, optionally substituted neopentylamide, optionally substituted dihalocyclobutylmethylamide, optionally substituted cyclopentylmethylamide, optionally substituted pyrimidinylamide, optionally substituted phenylamide, optionally... Substituted halophenylamides, optionally substituted dihalophenylamides, optionally substituted isoxazolylamides, optionally substituted methylisooxazolylamides, optionally substituted trihalomethylisooxazolylamides, optionally substituted thiazolylamides, optionally substituted methylthiazolylamides, optionally substituted oxazolylamides, optionally substituted methyloxazolylamides, optionally substituted phenylamides, optionally substituted halophenylamides, optionally substituted dihalophenylamides, optionally substituted trihalomethylphenylamides, optionally substituted halotrihalomethylphenylamides, optionally substituted halophenylpropionamides, etc. Optionally substituted cyclopropionylamino, optionally substituted trihalomethylcyclopropionylamino, optionally substituted cyclobutyrylamino, optionally substituted methylcyclobutyrylamino, optionally substituted dihalocyclobutyrylamino, optionally substituted bicyclic [1.1.1]pentanoylamino, optionally substituted trihalomethylbicyclic [1.1.1]pentanoylamino, optionally substituted oxacyclobutyrylamino, optionally substituted methyloxacyclobutyrylamino, optionally substituted bicyclic [2.1.1]hexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted dihalocyclohexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted spiro[3.3]heptanoylamino, optionally Substituted tert-butoxyacylamino (or Boc-NH or Boc-N(methyl)), optionally substituted cyclopentoxyacylamino, optionally substituted neopentoxyacylamino, optionally substituted cyclobutoxyacylamino, optionally substituted trihalomethylcyclobutoxyacylamino, optionally substituted benzenesulfonylamino, optionally substituted dihalobenzenesulfonylamino, optionally substituted cyclopropanesulfonylamino, optionally substituted methanesulfonylamino, optionally substituted trihalomethylsulfonylamino, optionally substituted cyclobutanesulfonylamino, optionally substituted amino, optionally substituted pyrimidinylamino, optionally substituted trihalomethylpyrimidinylamino, and optionally substituted tert-butaminoacyl.

[0022] In some implementations, R4 is H or methyl.

[0023] In some implementations, R5 is H or methyl.

[0024] In some embodiments, the compound is a compound of formula (Ia):

[0025] (Ia)

[0026] R1 is selected from substituted acyl and cyano groups;

[0027] Ht is an optionally substituted heterocyclic group;

[0028] X is selected from N and C-R4;

[0029] R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0030] R4 is selected from H and optionally substituted alkyl groups;

[0031] R5 is independently selected from H and optionally substituted alkyl groups;

[0032] m is an integer selected from 1 to 4; and

[0033] n is an integer selected from 1 to 4.

[0034] In some implementations, Ht is an optionally substituted 5-7 membered heterocyclic group.

[0035] In some embodiments, the optional substituents on the heterocyclic group are selected from halogen, methyl, methoxy, ethoxy, cyclopropyl, fused dimethylcyclopropyl, fused cyclopentyl, bridged methylene, and bridged ethylene.

[0036] In some implementations, m is an integer selected from 1 to 3.

[0037] In some implementations, n is an integer selected from 1 to 2.

[0038] This disclosure also provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, optionally combined with a pharmaceutically acceptable carrier, excipient or diluent.

[0039] This disclosure provides a method for treating a coronavirus-related disease or condition in a patient in need, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

[0040] This disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in the preparation of a medicament for treating coronavirus-related diseases or conditions.

[0041] This disclosure provides compounds of formula (I) or pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof for the treatment of coronavirus-related diseases or conditions.

[0042] In some implementations, the compound of formula (I) is a 3CL protease inhibitor.

[0043] In some implementations, the compound of formula (I) is characterized by IC 50 The value is less than 20 µM.

[0044] In some implementations, the coronavirus-related disease or condition is caused by a virus selected from SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-229E, and HCoV-OC43.

[0045] In some implementations, the compound of formula (I) is characterized by its ability to inhibit protease activity at IC50 assay. 50 The value is less than 20 µM.

[0046] In some implementation schemes, the compound of formula (I) is characterized by its viral cytopathic effect assay EC. 50 The value is less than 20 µM.

[0047] This disclosure provides a method for preparing a compound of formula (I), comprising:

[0048] i) Reacting compound (II) with compound (III) under dehydration conditions to form an amide bond:

[0049] (II)

[0050] (III);

[0051] ii) To undergo a closed-ring olefin metathesis reaction to form a compound of formula (IV):

[0052] (IV); and

[0053] iii) The alkoxyacyl moiety in formula (IV) is converted into the R1 moiety to form the compound of formula (I).

[0054] In some embodiments, step ii) further includes a hydrogenation reaction following the olefin metathesis reaction. Detailed Implementation

[0055] The currently disclosed compounds can be used as inhibitors of the 3CL protease, acting as competitive inhibitors of the protease's active site and exhibiting pancoronavirus activity. The 3CL protease cleaves its substrate almost specifically after P1-glutamine, and its P2 substrate residue is typically leucine, with methionine, valine, and phenylalanine in rare cases. A conserved cleavage site, P3-XLQ-(S / G / A / / N)-P1', is known to exist in coronaviruses. Due to its high sequence conservation and specificity in the recognition of cleavage sites in known human coronavirus 3CL proteases, the stated compounds are believed to be potential inhibitors of the pancoronavirus 3CL protease.

[0056] "Alkyl" refers to a monovalent alkyl group, which can be straight-chain or branched, preferably having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-hexyl, etc.

[0057] "alkylene" refers to a divalent alkyl group, preferably having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of such alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), and propylene isomers (such as -CH2CH2CH2- and -CH(CH3)CH2-).

[0058] "Alkenyl" refers to a monovalent alkenyl group, which can be straight-chain or branched, preferably having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and having at least one (preferably 1-2) carbon-carbon double bond. Examples include vinyl (-CH=CH2), n-propenyl (-CH2CH=CH2), isopropenyl (-C(CH3)=CH2), 2-butenyl (-CH2CH=CHCH3), etc.

[0059] "Alkoxy" refers to an alkyl-O- group, wherein the alkyl group is as described above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, etc.

[0060] "Cycloalkoxy" refers to a cycloalkyl-O- group, wherein the cycloalkyl group is as described herein.

[0061] "Halogens" refers to fluorine, chlorine, bromine, and iodine.

[0062] "Oxygenated / hydroxyl group" refers to the =O or HO- group.

[0063] "Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (such as phenyl) or multiple fused rings (such as naphthyl or anthracene), preferably having 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl, etc.

[0064] "Heteroaryl" refers to a monovalent aromatic heterocyclic group that conforms to Hückel's aromaticity rule (i.e., contains 4n+2 π electrons), preferably having 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur (including oxides of sulfur, selenium, and nitrogen). Such heteroaryl groups can be monocyclic (such as pyridyl, pyrroleyl, or their N-oxides or furanyl) or polycyclic (such as indoleazinyl, benzimidazolyl, coumarinyl, quinolinyl, isoquinolinyl, or benzothiopheneyl).

[0065] Examples of heteroaryl compounds include, but are not limited to: oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthridine, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenthiazine, thiazole, thiadiazole, oxadiazole, oxtriazole, tetrazolium, thiophene, benzo[b]thiophene, triazole, imidazopyridine, etc.

[0066] "Aryloxy group" refers to the aryl-O- group, where the aryl group is as described above.

[0067] "Aryl group" refers to an alkylene-aryl group, wherein the alkylene moiety preferably has 1 to 10 carbon atoms and the aryl moiety preferably has 6 to 10 carbon atoms. Examples of such aryl groups include benzyl, phenethyl, etc.

[0068] "Acyl" refers to HC(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)-, and heterocyclic-C(O)- groups, wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0069] "Acylalkyl" refers to an acyl-alkyl group, wherein the acyl and alkyl groups are as described above.

[0070] "Oxyacyl" refers to HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclic-OC(O)- groups, of which alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0071] "Amino" refers to the -NR"R" group, wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0072] "Aminoacyl" refers to the -C(O)NR"R" group, wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic is as described herein.

[0073] "Acylamino" refers to the -NR"C(O)R" group, wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0074] "Acyloxy group" refers to -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl and -C(O)O-heterocyclic groups, wherein alkyl, aryl, heteroaryl and heterocyclic groups are as described herein.

[0075] "Aminoacyloxy" refers to -OC(O)NR"-alkyl, -OC(O)NR"-aryl, -OC(O)NR"-heteroaryl and -OC(O)NR"-heterocyclic groups, wherein R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0076] "Oxylammonium" refers to -NR"C(O)O-alkyl, -NR"C(O)O-aryl, -NR"C(O)O-heteroaryl and NR"C(O)O-heterocyclic groups, wherein R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0077] "Oxykoxy" refers to -OC(O)O-alkyl, -OC(O)O-aryl, -OC(O)O-heteroaryl, and -OC(O)O-heterocyclic groups, wherein the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups are as described herein.

[0078] "Cycloalkyl" refers to a cyclic alkyl group having a single ring or multiple fused rings, preferably containing 3 to 11 carbon atoms. Such cycloalkyl groups include, but are not limited to: monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, etc.; or polycyclic structures such as adamantyl, indanyl, 1,2,3,4-tetrahydronaphthyl, etc.

[0079] "Cycloalkenyl" refers to a cyclic alkenyl group having a single ring or multiple fused rings and containing at least one internal unsaturation point, preferably containing 4 to 11 carbon atoms. Suitable examples of cycloalkenyl groups include, but are not limited to: cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4-enyl, cyclooct-3-enyl, indenyl, etc.

[0080] "Heterocyclic group" refers to a monovalent saturated or unsaturated group having a single ring or multiple fused rings, preferably containing 1 to 8 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium, or phosphorus. The most preferred heteroatom is nitrogen. It should be understood that, for example, when R2 or R' is an optionally substituted heterocyclic group containing one or more cyclic heteroatoms, the heterocyclic group can be linked to the core molecule of the compound of the present invention via a C-C bond or a C-heteroatom bond (especially a CN bond).

[0081] Examples of heterocyclic and heteroaryl groups include, but are not limited to: oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazole, isothiazine, phenoxazine, phenthiazine, imidazoline, imidazoline, piperidine, piperazine, dihydroindole, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazole, oxadiazole, oxtriazole, tetrazolium, thiazoline, thiophene, benzo[b]thiophene, morpholine, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, etc.

[0082] "Sulfinyl group" refers to HS(O)-, alkyl-S(O)-, cycloalkyl-S(O)-, aryl-S(O)-, heteroaryl-S(O)-, and heterocyclic-S(O)- groups, wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups are as described herein.

[0083] "Sulfoyl" refers to HS(O)2-, alkyl-S(O)2-, cycloalkyl-S(O)2-, aryl-S(O)2-, heteroaryl-S(O)2-, and heterocyclic-S(O)2- groups, wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups are as described herein.

[0084] "Sulfinamide" refers to HS(O)-NR"-, alkyl-S(O)-NR"-, cycloalkyl-S(O)-NR"-, aryl-S(O)-NR"-, heteroaryl-S(O)-NR"- and heterocyclic-S(O)-NR"- groups, wherein R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0085] "Sulfoamide" refers to HS(O)2-NR"-, alkyl-S(O)2-NR"-, cycloalkyl-S(O)2-NR"-, aryl-S(O)2-NR"-, heteroaryl-S(O)2-NR"- and heterocyclic-S(O)2-NR"- groups, wherein R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0086] "Aminosulfonyl" refers to the R"R"NS(O)2- group, wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic group is as described herein.

[0087] In this specification, "optional substitution" means that a group may, but must, be further substituted or fused (thus forming a fused polycyclic group) by one or more groups selected from the following: hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, amino, aminoacyl, thio, aralkyl, aralkoxy, aryl, aroxy, carboxyl, amide, cyano, halogen, nitro, phosphonyl, sulfonyl, phosphoramide, phosphonyl oxo, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclic, heterocyclic alkyl, heterocyclic oxy, oxoacyl, Oximes, oxime ethers, hydrazones, oxyacylaminos, oxysulfonylaminos, aminoacyloxys, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethylthio, trifluorovinyl, mono- and dialkylaminos, mono- and di(substituted alkyl)aminos, mono- and diarylaminos, mono- and diheteroarylaminos, mono- and diheterocyclic aminos, and asymmetric disubstituted amines having various substituents selected from alkyl, aryl, heteroaryl, and heterocyclic groups, and may also include bonds with a solid support material (e.g., substituted onto a polymer resin). For example, "optionally substituted amino" groups may include amino acid and peptide residues.

[0088] "Isomers" specifically include optical isomers (such as essentially pure enantiomers, essentially pure diastereomers and mixtures thereof) as well as conformational isomers (i.e. isomers that differ only at certain chemical bond angles), positional isomers (especially tautomers) and geometric isomers (such as cis-trans isomers).

[0089] The compounds described herein may contain one or more asymmetric centers and therefore may exist in a variety of isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may exist as a single enantiomer, diastereomer, or geometric isomer, or as a mixture of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferably, isomers can be prepared by asymmetric synthesis. The invention also includes compounds described herein as a single isomer substantially free of other isomers, or compounds as mixtures of various isomers. The term "optical enrichment" as used herein means that the compound consists of a significantly larger proportion of one enantiomer. In some embodiments, the compounds of the invention consist of at least about 90% by weight of the preferred enantiomer. In other embodiments, the compound consists of at least about 95%, 98%, or 99% by weight of the preferred enantiomer. Preferred enantiomers can be separated from racemic mixtures by any method known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0090] This disclosure provides a compound of formula (I) or a salt, solvate, stereoisomer, or prodrug thereof:

[0091] (I)

[0092] R1 is selected from substituted acyl and cyano groups;

[0093] R2 is selected from optionally substituted alkyl and optionally substituted alkenyl groups;

[0094] X is selected from N and C-R4;

[0095] R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0096] R4 is selected from H and optionally substituted alkyl groups;

[0097] R5 is independently selected from H and optionally substituted alkyl groups;

[0098] R6 is selected from H and optionally substituted alkyl groups;

[0099] Alternatively, R2 and R6 can be linked to form an optionally substituted heterocyclic group; m is an integer selected from 1 to 4; and

[0100] n is an integer from 1 to 4.

[0101] In some embodiments, R1 is selected from acyl and cyano groups. In some embodiments, R1 is selected from oxymethyl and cyano groups. In some embodiments, R1 is selected from carbonyl and cyano groups. In some embodiments, R1 is HC(O)-. In some embodiments, R1 is a cyano group.

[0102] In some embodiments, R2 is selected from optionally substituted alkyl groups. In some embodiments, R2 is selected from alkyl groups. In some embodiments, R2 is a C1-C5 alkyl group. Optional substituents may be halogens. In some embodiments, R2 is selected from butyl, isobutyl, pentyl, isopentyl, neopentyl, halopentyl, haloisopentyl, haloneopentyl, cyclopentylmethyl, and cyclopentyl.

[0103] In some embodiments, X is N. Therefore, the compound can be represented as shown in formula (I'):

[0104] (I').

[0105] In some embodiments, X is C-R4. Therefore, the compound can be represented as shown in formula (I''):

[0106] (I'').

[0107] In some embodiments, R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoacyloxy, optionally substituted amide, optionally substituted aminoacyl, and optionally substituted sulfonamide. In some embodiments, R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted oxyamide, optionally substituted aminoacyloxy, optionally substituted amide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0108] In some embodiments, the optional substituents on R3 are selected from haloalkyl, alkyl, cycloalkyl, alkylcycloalkyl, halocycloalkyl, haloalkylcycloalkyl, cycloalkylalkyl, heterocyclic, haloheterocyclic, alkylheterocyclic, haloalkylheterocyclic, heterocyclic alkyl, haloheterocyclic alkyl, heteroaryl, haloheteroaryl, alkylheteroaryl, haloalkylheteroaryl, heteroarylalkyl, haloheteroarylalkyl, aryl, haloaryl, alkylaryl, haloalkylaryl, arylalkyl, haloarylalkyl, bridged cycloalkyl optionally substituted with halogen and / or alkyl, spirocycloalkyl optionally substituted with halogen and / or alkyl, and protecting groups. In some embodiments, the optional substituents on R3 are selected from trihalomethyl, butyl, isobutyl, trihalomethylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, trihalomethylcyclopropyl, methylcyclobutyl, dihalocyclobutyl, trihalomethylcyclobutyl, dihalocyclobutylmethyl, cyclopentyl, dihalocyclohexyl, tetrahydrofuranyl, pyrimidinyl, trihalomethylpyrimidinyl, phenyl, halophenylpropyl, halophenyl, dihalophenyl, trihalomethylhalophenyl, trihalomethylisoxazole, methylisoxazole, bicyclo[2.1.1]hexyl, trihalomethylbicyclo[1.1.1]pentyl, methyloxacyclobutyl, spiro[3.3]heptyl, and tert-butoxyamino.

[0109] In some embodiments, R3 is selected from H, optionally substituted benzyl, optionally substituted halobenzyl, optionally substituted trihalomethylbenzyl, optionally substituted cyclopentyl, optionally substituted cyclopentylmethyl, optionally substituted tert-butoxy, optionally substituted cyclohexyloxy, optionally substituted tetrahydropyranylamide, optionally substituted tert-butylamide, optionally substituted trihalotert-butylamide, optionally substituted trihalomethylamide, optionally substituted neopentylamide, optionally substituted dihalocyclobutylmethylamide, optionally substituted cyclopentylmethylamide, optionally substituted pyrimidinylamide, optionally substituted phenylamide, optionally... Substituted halophenylamides, optionally substituted dihalophenylamides, optionally substituted isoxazolylamides, optionally substituted methylisooxazolylamides, optionally substituted trihalomethylisooxazolylamides, optionally substituted thiazolylamides, optionally substituted methylthiazolylamides, optionally substituted oxazolylamides, optionally substituted methyloxazolylamides, optionally substituted phenylamides, optionally substituted halophenylamides, optionally substituted dihalophenylamides, optionally substituted trihalomethylphenylamides, optionally substituted halotrihalomethylphenylamides, optionally substituted halophenylpropionamides, etc. Optionally substituted cyclopropionylamino, optionally substituted trihalomethylcyclopropionylamino, optionally substituted cyclobutyrylamino, optionally substituted methylcyclobutyrylamino, optionally substituted dihalocyclobutyrylamino, optionally substituted bicyclic [1.1.1]pentanoylamino, optionally substituted trihalomethylbicyclic [1.1.1]pentanoylamino, optionally substituted oxacyclobutyrylamino, optionally substituted methyloxacyclobutyrylamino, optionally substituted bicyclic [2.1.1]hexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted dihalocyclohexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted spiro[3.3]heptanoylamino, optionally Substituted tert-butoxyacylamino (or Boc-NH or Boc-N(methyl)), optionally substituted cyclopentoxyacylamino, optionally substituted neopentoxyacylamino, optionally substituted cyclobutoxyacylamino, optionally substituted trihalomethylcyclobutoxyacylamino, optionally substituted benzenesulfonylamino, optionally substituted dihalobenzenesulfonylamino, optionally substituted cyclopropanesulfonylamino, optionally substituted methanesulfonylamino, optionally substituted trihalomethylsulfonylamino, optionally substituted cyclobutanesulfonylamino, optionally substituted amino, optionally substituted pyrimidinylamino, optionally substituted trihalomethylpyrimidinylamino, and optionally substituted tert-butaminoacyl.

[0110] In some embodiments, when X is N, R3 is an optionally substituted oxoamide.

[0111] In some embodiments, when X is CR4, R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0112] In some embodiments, R4 is selected from H and optionally substituted alkyl groups. In some embodiments, R4 is selected from H and optionally substituted C1-C5 alkyl groups. In some embodiments, R4 is selected from H and C1-C5 alkyl groups. In some embodiments, R4 is H.

[0113] In some embodiments, R5 is independently selected from H and optionally substituted C1-C5 alkyl groups. In some embodiments, R5 is independently selected from H and C1-C5 alkyl groups. In some embodiments, R5 is independently H or methyl. In some embodiments, R5 is independently H.

[0114] In some embodiments, R6 is selected from H and optionally substituted C1-C5 alkyl groups. In some embodiments, R6 is H.

[0115] R2 and R6 can be linked to form an optionally substituted heterocyclic group. In this respect, R2 can form an optionally substituted heterocyclic group with the N atom at the β-position relative to R2. Therefore, in some embodiments, the compound is a compound of formula (Ia):

[0116] (Ia)

[0117] R1 is selected from substituted acyl and cyano groups;

[0118] Ht is an optionally substituted heterocyclic group;

[0119] X is selected from N and C-R4;

[0120] R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide.

[0121] R4 is selected from H and optionally substituted alkyl groups;

[0122] R5 is independently selected from H and optionally substituted alkyl groups;

[0123] m is an integer selected from 1 to 4; and

[0124] n is an integer selected from 1 to 4.

[0125] In some embodiments, the optionally substituted heterocyclic group (Ht) is an optionally substituted 5-7 membered heterocyclic group. In some embodiments, the heterocyclic group (Ht) is a 5-6 membered heterocyclic group. In some embodiments, the heterocyclic group (Ht) is a 5-membered heterocyclic group. In some embodiments, the heterocyclic group (Ht) is a 6-membered heterocyclic group.

[0126] In some embodiments, the optional substituents on the heterocyclic group are selected from halogens, alkyl groups, alkoxy groups, cycloalkyl groups, alkylcycloalkyl groups, and bridged alkylene groups. In some embodiments, the optional substituents on the heterocyclic group are selected from halogens, methyl groups, methoxy groups, ethoxy groups, cyclopropyl groups, fused dimethylcyclopropyl groups, fused cyclopentyl groups, bridged methylene groups, and bridged ethylene groups.

[0127] In some implementations, m is an integer selected from 1 to 3.

[0128] In some implementations, n is an integer selected from 1 to 2.

[0129] In some embodiments, the compound is a compound of formula (I'''):

[0130] (I''').

[0131] In some embodiments, the compound is of formula (I) iv Compounds:

[0132] (I iv ).

[0133] In some embodiments, the compound is of formula (I) iv a) or (I iv b) Compounds:

[0134] (I iv a) or (I iv b).

[0135] Formula (I iv a) is a cis isomer of formula (I), formula (I) iv b) is the trans isomer of formula (I). Studies have found that both isomers have potent activity, but the trans isomer is generally considered to be more active than the cis isomer.

[0136] Based on structure-activity relationships, it is believed that at least one chiral center on the lactam ring may play a key role in the activity of these compounds. This structural feature also differs from previously reported structures. In some embodiments, compounds of formula (I) or their salts, solvates, stereoisomers, or prodrugs as described in formula (I) v a) or (I v As shown in b):

[0137] (I v a) or (I v b).

[0138] In some embodiments, a compound of formula (I) or a salt, solvate, stereoisomer, or prodrug of formula (I) vi a) or (I vi As shown in b):

[0139] (I vi a) or (I vi b).

[0140] In some embodiments, a compound of formula (I) or a salt, solvate, stereoisomer, or prodrug of formula (I) vii a) or (I vii As shown in b):

[0141] (I vii a) or (I vii b).

[0142] In some embodiments, a compound of formula (I) or a salt, solvate, stereoisomer, or prodrug of formula (I) iix a) or (I iix As shown in b):

[0143] (I iix a) or (I iix b).

[0144] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) i As shown in the image:

[0145] (Ia i ).

[0146] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) iiAs shown in the image:

[0147] (Ia ii ).

[0148] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) iii As shown in the image:

[0149] (Ia iii ).

[0150] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) iv As shown in the image:

[0151] (Ia iv ).

[0152] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) v a) or (Ia v As shown in b):

[0153] (Ia v a) or (Ia v b).

[0154] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) vi a) or (Ia vi As shown in b):

[0155] (Ia vi a) or (Ia vi b).

[0156] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) vii a) or (Ia vii As shown in b):

[0157] (Ia vii a) or (Ia vii b).

[0158] In some embodiments, a compound of formula (Ia) or a salt, solvate, stereoisomer, or prodrug of formula (Ia) iixa) or (Ia iix As shown in b):

[0159] (Ia iix a) or (Ia iix b).

[0160] In some embodiments, the compound of formula (I) is selected from:

[0161] .

[0162] In some embodiments, the compound of formula (I) or its salts, solvates, stereoisomers or prodrugs are selected from:

[0163] .

[0164] This disclosure also provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, optionally combined with a pharmaceutically acceptable carrier, excipient or diluent.

[0165] The compounds of the present invention can be administered to subjects in the form of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include, but are not limited to: salts formed with pharmaceutically acceptable inorganic acids, such as hydrochlorides, sulfates, phosphates, nitrates, carbonates, borates, aminosulfonates, and hydrobroms; or salts formed with pharmaceutically acceptable organic acids, such as acetates, propionates, butyrates, tartrates, maleates, hydroxymaleates, fumarates, maleates, citrates, lactates, mucilages, gluconates, benzoates, succinates, oxalates, phenylacetates, methanesulfonates, toluenesulfonates, benzenesulfonates, salicylates, sulfanamides, aspartates, glutamates, edetates, stearates, palmitates, oleates, laurates, pantothenates, tannates, ascorbic acid salts, and valerates.

[0166] Basic salts include, but are not limited to, salts that form with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium salts. In particular, the invention includes cationic salts, such as sodium or potassium salts of phosphate groups, or alkyl esters (such as methyl esters, ethyl esters).

[0167] Nitrogen-containing basic groups can be quaternized by the following reagents: lower alkyl halides, such as chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl; dialkyl sulfates such as dimethyl sulfate and diethyl sulfate; and other reagents.

[0168] It should be understood that any compound that serves as a prodrug of formula (I) is also within the scope and spirit of this invention. Therefore, the compounds of this invention may be administered to a subject in a pharmaceutically acceptable prodrug form. The term "prodrug" is used in its broadest sense to include those derivatives that are converted in vivo into the compounds of this invention. These derivatives will be apparent to those skilled in the art. Other literature commonly describing prodrugs (and their preparation) includes: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al., Chapter 5 (Harwood Academic Publishers). For example, the amino moiety may be protected. The oxygen group may also be esterified.

[0169] The compounds of this invention can exist in crystalline form, either as free compounds or as solvates (e.g., hydrates), both of which are within the scope of protection of this invention. Methods of solvation are generally known in the art.

[0170] The compound of the present invention or its pharmaceutically acceptable salt, solvate or prodrug is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to achieve at least a partial effect of the intended effect, or delay the onset of the disease, or inhibit the progression of the disease, or completely prevent / reverse the onset or progression of macular degeneration.

[0171] As used herein, the term "effective amount" refers to the amount of compound that, when administered according to the intended dosing regimen, provides the desired therapeutic activity. Dosing may be administered at intervals of minutes, hours, days, weeks, months, or years, or continuously over any of these time periods. A suitable dose may range from about 0.1 ng to 1 g per kilogram of body weight per dose, for example, from 1 mg to 1 g per kilogram of body weight per dose. In one embodiment, the dose may range from 1 mg to 500 mg per kilogram of body weight per dose. In another embodiment, the dose may range from 1 mg to 250 mg per kilogram of body weight per dose. In yet another embodiment, the dose may range from 1 mg to 100 mg per kilogram of body weight per dose, for example, up to 50 mg per kilogram of body weight per dose.

[0172] The appropriate dosage and administration regimen can be determined by the attending physician and may depend on the severity of the condition as well as the patient’s overall age, health status and weight.

[0173] The compounds of the present invention can be administered in a single dose or in multiple doses. While the active ingredient can be administered alone, it is preferred to administer it as a composition, more preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carrier, diluent, or excipient. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antimicrobial agents, skin penetrants, surfactants, isotonic agents, and absorbents, etc. It should be understood that the compositions of the present invention may also contain other auxiliary physiologically active agents.

[0174] The carrier must be pharmaceutically "acceptable," meaning it is compatible with other components in the composition and harmless to the patient. The composition can be readily provided in unit dose form and can be prepared by any method known in the pharmaceutical field. These methods involve the step of combining the active ingredient with a carrier that constitutes one or more excipients. Typically, the composition is prepared by uniformly and thoroughly mixing the active ingredient with a liquid carrier or a finely chopped solid carrier, or both, and then shaping the product as needed.

[0175] The routes of administration include local administration or intravenous administration. For example, solutions or suspensions of the compounds, compositions, or combinations thereof of the present invention can be formulated. Local administration typically involves administering the compounds of the present invention at a dose of 0.1 ng to 10 mg.

[0176] The compounds or compositions of the present invention are also suitable for intravenous administration. For example, compounds of formula (I) or their pharmaceutically acceptable salts, solvates, or prodrugs can be administered at a maximum dose of 16 mg / m². 2 Administer intravenously at the prescribed dose.

[0177] Injectable formulations for such applications can be prepared in conventional forms, including liquid solutions or suspensions, solid forms suitable for reconstitution into liquid solutions or suspensions prior to injection, or emulsions. Carriers may include, but are not limited to: water, physiological saline (such as standard physiological saline (NS), phosphate-buffered saline (PBS), balanced salt solution (BSS)), lactated Ringer's solution, glucose, glycerol, ethanol, etc.; small amounts of auxiliary substances, such as wetting agents, emulsifiers, and buffers, may be added if necessary. Appropriate flowability can be maintained by using coatings such as lecithin, maintaining the desired particle size in the dispersed state, and using surfactants.

[0178] The compounds or compositions of the present invention are also suitable for oral administration and can be formulated into discrete unit dosage forms such as capsules, pouches, or tablets containing a predetermined dose of the active ingredient; or into powders or granules; or into solutions or suspensions of aqueous or non-aqueous liquids; or into oil-in-water or water-in-oil liquid emulsions. The active ingredient may also be present in the form of pills, saccharides, or pastes. In another embodiment, the compound of formula (I) or its pharmaceutically acceptable salt, solvate, or prodrug may be administered orally.

[0179] Tablets can be prepared by compression or molding, optionally with the addition of one or more excipients. Compressed tablets can be prepared by compressing a free-flowing active ingredient (such as powder or granules) in suitable equipment, optionally mixed with a binder (such as an inert diluent, preservative), a disintegrant (such as sodium carboxymethyl starch, crospovidone, or crospovidone carboxymethyl cellulose), a surfactant, or a dispersant. Molded tablets can be formed by molding a mixture of powdered compounds moistened with an inert liquid diluent in suitable equipment. Tablets can optionally be coated or scored, and can be formulated using, for example, different proportions of hydroxypropyl methylcellulose to provide a slow or controlled release of the active ingredient to provide a desired release profile. Tablets can be selectively enteric-coated to allow release of the active ingredient at intestinal sites outside the stomach.

[0180] The compounds or compositions of the present invention are suitable for topical oral administration, including: lozenges (containing the active ingredient in a flavoring matrix such as sucrose and gum arabic or tragacanth gum), tablets (containing the active ingredient in an inert matrix such as gelatin and glycerin or sucrose and gum arabic), and mouthwashes (containing the active ingredient in a suitable liquid carrier).

[0181] The compounds or compositions of this invention are suitable for topical skin administration, and may comprise compounds dissolved or suspended in any suitable carrier or matrix, and may be in the form of lotions, gels, creams, pastes, ointments, etc. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsified waxes, sorbitan monostearate, polysorbate 60, cetyl wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water. Transdermal patches can also be used to administer the compounds of this invention.

[0182] The compounds or compositions of the present invention are suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, bactericides, and solutes that make the compound, composition, or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners. The compounds, compositions, or combinations may be present in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Injectable solutions and suspensions can be prepared ad hoc from the aforementioned sterile powders, granules, and tablets.

[0183] Preferred unit dose compositions or combinations are formulations containing daily doses, unit doses, daily sub-dose (as described above), or suitable amounts of the active ingredient.

[0184] It should be understood that, in addition to the active ingredients specifically mentioned above, the compositions of the present invention may also contain other agents conventional to the type of composition or combination, such as binders, sweeteners, thickeners, flavoring agents, disintegrants, coating agents, preservatives, lubricants, and / or delay agents for oral administration. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginate, or agar. Suitable flavoring agents include peppermint oil, wintergreen oil, cherry flavor, orange flavor, or raspberry flavor. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and their esters, waxes, fatty alcohols, corn gluten, shellac, or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable slow-release agents include glyceryl monostearate or glyceryl distearate.

[0185] Those skilled in the art will understand that other methods of injection and / or administration of the compound, composition, or combination may also be used. These other methods may include, for example, medical delivery devices. These devices and methods may include, for example, biodegradable polymer delivery components for long-term drug administration.

[0186] This disclosure provides a method for preparing a compound of formula (I), comprising:

[0187] i) Reacting compound (II) with compound (III) under dehydration conditions to form an amide bond:

[0188] (II)

[0189] (III);

[0190] ii) To undergo a closed-ring olefin metathesis reaction to form a compound of formula (IV):

[0191] (IV); and

[0192] iii) The alkoxyacyl moiety in formula (IV) is converted into the R1 moiety to form the compound of formula (I).

[0193] In some embodiments, the amide bond is formed via an activated ester intermediate. In some embodiments, the amide bond is formed in the presence of HATU and a base. The base may be N,N-diisopropylethylamine.

[0194] In some embodiments, the olefin metathesis reaction is carried out in the presence of a Grubbs(II) catalyst.

[0195] In some embodiments, step ii) further includes a hydrogenation reaction following the olefin metathesis reaction. The hydrogenation reaction may be carried out using a palladium-on-carbon catalyst in the presence of hydrogen.

[0196] In some embodiments, the alkoxyacyl moiety is converted to a primary amide before being reacted with the Burgess reagent.

[0197] In some embodiments, the alkoxyacyl moiety is converted to an acylalkyl moiety in the presence of a reducing agent and a Dess-Martin oxidant.

[0198] In some embodiments, the method further includes the step of synthesizing a compound of formula (II), comprising:

[0199] a) Reacting compound (V) with a base to form compound (VI)

[0200] (V)

[0201] (VI)

[0202] c) Reacting compound (VI) with phthalimide to form compound (VII)

[0203] (VII)

[0204] d) React the compound of formula (VI) with methylamine hydrochloride to form the compound of formula (II).

[0205] This disclosure provides a method for treating a coronavirus-related disease or condition in a patient in need, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

[0206] This disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for the preparation of a medicament for treating coronavirus-related diseases or conditions in patients in need.

[0207] This disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for the treatment of coronavirus-related diseases or conditions in patients in need.

[0208] In some embodiments, the compound of formula (I) is a 3CL protease inhibitor. In contrast, the PL protease recognizes glycine-glycine at the P2-P1 position.

[0209] In some implementations, the coronavirus-related disease or condition is caused by a virus selected from SARS-CoV-1, SARS-CoV-2, MERS-CoV, CoV-229E, and HCoV-OC43. Coronaviruses may include four genera of coronaviruses: α, β, γ, or δ coronaviruses. They may be coronaviruses that infect humans, including but not limited to coronavirus 229E, coronavirus NL63, coronavirus OC43, or coronavirus HKU1. SARS-CoV-2 includes the initially discovered viral strains and subsequent variants, including but not limited to B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.427 / B.1.429 (Epsilon), P.2 (Zeta), B.1.525 (Eta), P.3 (Theta), B.1.526 (Iota), B.1.617.1 (Kappa), C.37 (Lambda), B.1.621 (Mu), B.1.1.529 (Omicron), and Omicron variants: BA.1, BA.2, BA.2 subvariants such as XBB (including XBB.1.16 (Arcturus) and XE), BA.3, BA.4, BA.5, and BA.5 subvariants such as BQ.1 and BQ.1.1.

[0210] In some implementations, the compound of formula (I) is characterized by its ability to inhibit protease activity at IC50 assay. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its IC50 resistance to SARS CoV-2. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its IC50 response to 229E. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its IC50 resistance to MERS. 50 The value is less than 20 µM.

[0211] In some implementation schemes, the compound of formula (I) is characterized by its viral cytopathic effect assay EC. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its EC50 response to 229E. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its EC value for OC43. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its resistance to the EC50 of SARS-CoV-2. 50 The value is less than 20 µM. In some embodiments, the compound of formula (I) is characterized by its resistance to EC50 of MERS.50 The value is less than 20 µM.

[0212] Example

[0213] Experimental steps

[0214] Unless otherwise specified, all reactions were carried out with continuous stirring under a nitrogen atmosphere. Where appropriate, the pre-reaction apparatus was dried in an oven at 100°C for 16 hours using anhydrous solvents (Sure-Seal™ product, Aldrich Chemical Company, Milwaukee, Wisconsin). Unless otherwise specified, solvents and reagents were purchased commercially and used directly without further purification. Acetonitrile was purchased from VWR (Germany). Deuterated solvents were purchased from Cambridge Isotope Laboratories (USA).

[0215] The reaction progress was monitored by thin-layer chromatography (TLC) and / or liquid chromatography-mass spectrometry (LCMS). TLC was performed on pre-coated silica gel plates with fluorescent indicators (excitation wavelength 254 nm), observed under UV light and / or stained with KMnO4, phosphomolybdic acid and / or cerium ammonium molybdate. LCMS data were recorded on an Agilent 1290 Infinity series single quadrupole mass spectrometer (ESI ion source) using acetonitrile / water or methanol / water gradients and trifluoroacetic acid or formic acid modifiers. Rapid column chromatography was performed using an automated system (Teledyne Isco Combiflash RF200) with silica gel columns (RediSepRF, silica gel 230-400 mesh, average pore size 60 Å, catalog number #69-2203-312). The crude target inhibitor was purified using a reversed-phase C18 column (Phenomenex Luna 5 µm C18(2), 100 × 4.6 mm, 100 Å) on a high-performance liquid chromatography (HPLC) system equipped with a UV detector. Unless otherwise specified, the mobile phase consisted of water and acetonitrile containing 0.1% formic acid.

[0216] NMR spectra were acquired using a 400 MHz spectrometer (Bruker Ascend 400, Germany). Chemical shifts are expressed as parts per million (ppm) relative to the solvent peak. Peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quintet; m, multiplet; br s, broad singlet; 30 app, apparent peak. The purity of the compound was ≥95% as determined by HPLC (Shimadzu) using acetonitrile / water (containing 0.1% formic acid) as eluent.

[0217] General Route 1

[0218]

[0219] Example 1

[0220] Example of General Route 1

[0221] Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate

[0222] and

[0223] Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3R,4S)-2-oxo-4-vinylpyrrolidine-3-yl)propionate

[0224]

[0225] Step 1-1. (E)-4-((tert-butyldimethylsilyl)oxy)but-2-en-1-ol.

[0226]

[0227] A solution of 2-buten-1,4-diol (trans) (100 g, 11.34 mol, 1 equivalent) in THF (2200 mL) was added in portions to NaH (54 g, 60 wt%) at 0°C under a nitrogen atmosphere and stirred at room temperature for 4 hours. Subsequently, TBDMSCl (171 g, 11.34 mol, 1.00 equivalent) was added dropwise at 0°C. The resulting mixture was stirred at room temperature for 12 hours. After confirming the completeness of the reaction by TLC (20% ethyl acetate / hexane, KMnO4 colorimetric assay), the reaction was quenched by slow addition of a saturated sodium bicarbonate aqueous solution (2000 mL). The resulting mixture was extracted with EtOAc (3 × 2000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with ethyl acetate:hexane (1:10) to give (2E)-4-[(tert-butyldimethylsilyl)oxy]but-2-en-1-ol (155 g, 64%) as a colorless oil.

[0228] Steps 1-2. (E)-5-(4-((tert-butyldimethylsilyl)oxy)but-2-en-1-yl)1-methyl(tert-butyloxycarbonyl)-L-glutamate.

[0229]

[0230] Under a nitrogen atmosphere, a solution of (2E)-4-[(tert-butyldimethylsilyl)oxy]but-2-en-1-ol (155 g, 7.65 mol, 1 equivalent) in CH2Cl2 (3000 mL) was added at 0°C to Boc-L-glutamic acid α-methyl ester (199.91 g, 7.65 mol, 1 equivalent), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (220.24 g, 11.48 mol, 1.5 equivalent), and DMAP (46.79 g, 3.82 mmol, 0.5 equivalent), and stirred at room temperature for 12 hours. The complete consumption of the starting materials was confirmed by LC / MS. The reaction was quenched by adding saturated ammonium chloride solution (2000 mL). The resulting mixture was extracted with CH2Cl2 (3 × 3000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate:hexane (1:5) to give 5-(2E)-4-[(tert-butyldimethylsilyl)oxy]but-2-en-1-yl-1-methyl(2S)-2-[(tert-butyloxycarbonyl)amino]glutarate (310 g, 82%) as a colorless oil. MS (ESI) m / z 446 [C 12 H 39 NO7Si + H] + .

[0231] Steps 1-3. Synthesis of (2S,3R)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-3-(((tert-Butyldimethylsilyl)oxy)methyl)pent-4-enoic acid and (2R,3S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-3-(((tert-Butyldimethylsilyl)oxy)methyl)pent-4-enoic acid.

[0232]

[0233] Under a nitrogen atmosphere, an anhydrous THF solution (3800 mL) of 5-(2E)-4-[(tert-butyldimethylsilyl)oxy]but-2-en-1-yl-1-methyl(2S)-2-[(tert-butyloxycarbonyl)amino]glutarate (310 g, 6.97 mol, 1 equivalent) was reacted with LiHMDS (2089 mL, 1 M THF solution, 2.1 mol) at -78°C for 60 min. Freshly distilled TMSCl (264.5 g, 24.5 mol, 3.5 equivalent) was then added dropwise at -78°C. The resulting mixture was stirred at -78°C for 1 h. Then, anhydrous triethylamine (246.4 g, 24.3 mol, 3.5 equivalent) was added dropwise at -78°C. The resulting mixture was stirred at room temperature for 12 h. The reaction was quenched by adding a saturated ammonium chloride solution (2000 mL). The pH of the mixture was adjusted to 3 with 2 M HCl. The resulting mixture was extracted with EtOAc (3 × 3000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate:hexane (1:1) to give 2-[(2S)-2-[(tert-butyloxycarbonyl)amino]-3-methoxy-3-oxopropyl]-3-{[(tert-butyldimethylsilyl)oxy]methyl}pent-4-enoic acid (290 g, 93%), as a pale yellow oil. MS (ESI) m / z 446 [C 12 H 39 NO7Si + H] + .

[0234] Steps 1-4. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-((tert-butyldimethylsilyl)oxy)but-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-((tert-butyldimethylsilyl)oxy)but-3-en-2-yl)glutarate.

[0235]

[0236] A solution of 2-[(2S)-2-[(tert-butyloxycarbonyl)amino]-3-methoxy-3-oxopropyl]-3-{[(tert-butyldimethylsilyl)oxy]methyl}pent-4-enoic acid (290 g, 6.5 mol, 1 equivalent) in anhydrous DMF (1500 mL) was reacted with potassium carbonate (179.9 g, 13.01 mol, 2 equivalent) at 0°C under a nitrogen atmosphere for 30 min, followed by dropwise addition of iodomethane (184.7 g, 13.01 mol, 2 equivalent) at 0°C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The reaction was quenched by adding water (1500 mL). The resulting mixture was extracted with EtOAc (3 × 2000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate:hexane (1:10) to give 1,5-dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-{1-[(tert-butyldimethylsilyl)oxy]but-3-en-2-yl}glutarate (265 g, 88%), as a colorless oil. MS (ESI) m / z 460 [C 22 H 41 NO7Si M + H] + .

[0237] Steps 1-5. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-hydroxybut-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-hydroxybut-3-en-2-yl)glutarate.

[0238]

[0239] Under a nitrogen atmosphere, anhydrous TBAF solution (3460 mL, 1 M THF solution) was mixed with glacial acetic acid (436 mL) at 0°C. At -5°C, 1,5-dimethyl(2S)-2-[(tert-butyloxycarbonyl)amino]-4-{1-[(tert-butyldimethylsilyl)oxy]but-3-en-2-yl}glutarate (265 g, 5.76 mol, 1 equivalent) was added dropwise to the mixture over 60 minutes. The resulting mixture was stirred at 0°C for 12 hours. The mixture was then alkalized to pH 7 with saturated sodium bicarbonate solution. The resulting mixture was extracted with EtOAc (3 × 3000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate:hexane (2:1) to give 1,5-dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-(1-hydroxybut-3-en-2-yl)glutarate (168 g, 84%) as a colorless oil. MS (ESI) m / z 346 [C 16 H 27 NO7 + H] + .

[0240] Steps 1-6. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-(1,3-dioxoisoindoline-2-yl)but-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-(1,3-dioxoisoindoline-2-yl)but-3-en-2-yl)glutarate.

[0241]

[0242] A THF solution (2600 mL) of 1,5-dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-(1-hydroxybut-3-en-2-yl)glutarate (265 g, 7.67 mol, 1 equivalent) was reacted with 2,3-dihydro-1H-isoindole-1,3-dione (226 g, 1.53 mmol, 2 equivalents) and triphenylphosphine (302 g, 1.15 mmol, 1.5 equivalents) at 0°C under a nitrogen atmosphere for 30 min. DIAD (240 g, 11.9 mol, 1.55 equivalents) was then added dropwise at 0°C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The reaction was quenched by adding a saturated ammonium chloride solution (3000 mL) at room temperature. The resulting mixture was extracted with EtOAc (3 × 3000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate:hexane (1:1) to give 1,5-dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-[1-(1,3-dioxoisoindol-2-yl)but-3-en-2-yl]glutarate (405 g, crude) as a yellow solid. MS (ESI) m / z 475 [C 24 H 30 N2O8 + H] + .

[0243] Steps 1-7. Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3R,4S)-2-oxo-4-vinylpyrrolidine-3-yl)propionate and methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate.

[0244]

[0245] A MeOH solution (4000 mL) of 1,5-dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-[1-(1,3-dioxoisoindol-2-yl)but-3-en-2-yl]glutarate (405 g, crude) was reacted with methylamine hydrochloride (170 g, 25.2 mol) and N,N-diisopropylethylamine (320 g, 24.8 mol). The resulting mixture was stirred at 80°C for 3 hours under a nitrogen atmosphere. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The reaction was quenched with a saturated ammonium chloride solution (3000 mL). The resulting mixture was extracted with EtOAc (3 × 3000 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate / hexane (2:1) to give methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate (115 g) as a yellow oil. LCMS (ESI) m / z 313 [C 15 H 24 N2O5 + H] + .

[0246] The substance was treated with MTBE (3 times) to remove triphenylphosphine oxide (about 35 g), and then purified twice by silica gel column chromatography, eluted with dichloromethane / ethyl acetate (1:1) to give 65 g of crude product.

[0247] The crude product (300 mg batch) was purified by preparative chiral supercritical fluid chromatography (Prep-Chiral-SFC) under the following conditions:

[0248] Column: CHIRALPAK IH, 3×25 cm, 5 μm; Mobile phase A: CO2; Mobile phase B: Methanol (0.1% 2 M ammonia-methanol solution); Flow rate: 100 mL / min; Gradient: 15% B isocratic; Column temperature (°C): 35; Back pressure (bar): 100; Wavelength: 210 nm; RT1: 3.8 min; RT: 8.652 min; Sample solvent: Methanol-preparative grade; Injection volume: 1 mL.

[0249] Isomer 1 (73 mg, off-white solid) was isolated. 1H NMR (CDCl3, 400 MHz) δ (ppm):6.12 (s, 1H), 5.87-5.77 (m, 2H), 5.23-5.19 (m, 2H), 4.46 (s, 1H), 3.76-3.74(d, J = 7.2 Hz, 3H), 3.59 LCMS (ES, m / z): 313 [M+H] + .

[0250] Isomer 2 (47.5 mg, off-white solid) was isolated. 1 H NMR (CDCl3, 400 MHz) δ (ppm):6.32 (s, 1H), 5.85-5.76 (m, 1H), 5.69-5.67 (m, 1H), 5.30-5.20 (m, 2H), 4.32-4.31 (m, 1H), 3.75-3.73 LCMS (ES, m / z): 313[M+H] + .

[0251] General Route 2

[0252]

[0253] Example 2

[0254] Specific examples of general route 2

[0255] Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4S)-2-oxo-4-vinylpyrrolidine-3-yl)propionate

[0256] and

[0257] Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3R,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate

[0258]

[0259] Step 2-1. Synthesis of (Z)-4-((tert-butyldimethylsilyl)oxy)but-2-en-1-ol.

[0260]

[0261] NaH (60 wt%, 3.0 g, 75 mmol) was suspended in anhydrous THF (125 mL) under a nitrogen atmosphere and cooled to 0°C. Cis-but-2-en-1,4-diol (6.67 g, 75 mmol, 1.0 equivalent) was added in a single addition, maintaining the temperature <5°C. After stirring at 0°C for 15 minutes, the reaction was brought to room temperature and stirred for 4 hours, forming a thick white slurry. The reaction was then cooled back to 0°C, followed by the addition of TBSCl (11.41 g, 75 mmol, 1.0 equivalent) in a single addition. The mixture was stirred at this temperature for 15 minutes, then slowly brought to room temperature over 30 minutes and stirred for another 4 hours. After confirming the completeness of the reaction by TLC (20% EtOAc / hexane, KMnO4 colorimetric analysis), the reaction was quenched by the slow addition of a saturated sodium bicarbonate aqueous solution (150 mL). The crude reactant was diluted with Et2O (100 mL). After removing the organic phase, the resulting aqueous phase was extracted with Et2O (3 × 50 mL). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a thick, pale yellow oil. The product was purified by rapid silica gel column chromatography, eluting with ethyl acetate:hexane (4:1), and concentrated to obtain the target product as a transparent, colorless oil (11.54 g, 81.8%).

[0262] Step 2-2. Synthesis of (Z)-5-(4-((tert-butyldimethylsilyl)oxy)but-2-en-1-yl)-1-methyl(tert-butyloxycarbonyl)-L-glutamate ester.

[0263]

[0264] A mixture of (S)-4-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (13.22 g, 50.6 mmol, 1.0 equivalent), cis-2-buten-1,4-diol mono-TBS ester (10.24 g, 50.6 mmol, 1.0 equivalent), and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (19.4 g, 101.2 mmol, 1.5 equivalent) was dissolved in anhydrous DCM (300 mL). 4-Dimethylaminopyridine (3 g, 25.3 mmol, 0.5 equivalent) was added in a single batch, and the mixture was stirred at room temperature for 12 hours. Complete consumption of the starting materials was confirmed by LC / MS. The reaction mixture was diluted with saturated ammonium chloride aqueous solution (200 mL) and extracted with CH2Cl2 (5 × 60 mL). The organic extract was dried over anhydrous sodium sulfate, filtered, and the volatiles were removed under vacuum to obtain a colorless, oily crude product. Purification was achieved by silica gel column chromatography, with the product eluted in ethyl acetate:hexane (3:7). The concentrate was then used to obtain the desired product, a colorless oil (20.3 g, 90%). MS (ESI) m / z 346.2 [M-Boc+H] + .

[0265] Steps 2-3. Synthesis of (2S,3S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-3-(((tert-Butyldimethylsilyl)oxy)methyl)pent-4-enoic acid and (2R,3R)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-3-(((tert-Butyldimethylsilyl)oxy)methyl)pent-4-enoic acid.

[0266]

[0267] 5-(2Z)-4-[(tert-butyldimethylsilyl)oxy]but-2-en-1-yl-1-methyl(2S)-2-[(tert-butyloxycarbonyl)amino]glutarate (20.0 g, 44.9 mmol, 1.0 equivalent) was dissolved in anhydrous THF (250 mL) and cooled to -78°C under a nitrogen atmosphere. LiHMDS (100 mL, 1 M THF solution, 100 mmol, 2.1 equivalent) was added dropwise, and the pale orange reaction mixture was stirred at -78°C for 1 hour. Anhydrous TMSCl (17.1 mL, 134.6 mmol, 3.0 equivalent, freshly distilled with CaH2) was then added, and the reaction was continued to be stirred at -78°C for 30 minutes. Anhydrous triethylamine (18.8 mL, 134.6 mmol, 3.0 equivalent) was then added. The reaction mixture was heated to room temperature and stirred overnight. After stirring overnight, the reaction was heated to 50°C and maintained for 4 hours. The reaction was cooled to room temperature, quenched with 200 mL of saturated ammonium chloride solution, acidified to pH 3 with 2 M HCl, and stirred at room temperature for 30 minutes. The mixture was then extracted with EtOAc (3 × 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification was performed by silica gel column chromatography, with the starting material eluted with ethyl acetate:hexane (3:7) and the target product eluted with ethyl acetate:hexane (1:1), yielding a viscous, pale yellow oil (13.8 g, 69.0%). MS (ESI) m / z 346.2 [M-Boc+H] + .

[0268] Steps 2-4. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-((tert-butyldimethylsilyl)oxy)but-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-((tert-butyldimethylsilyl)oxy)but-3-en-2-yl)glutarate.

[0269]

[0270] The starting acid (13.8 g, 31.0 mmol, 1.0 equivalent, a mixture of (S), (S), (S), and (S), (R), (R) diastereomers) was suspended in anhydrous DMF (60 mL) with potassium carbonate (8.62 g, 61.9 mmol, 2.0 equivalent) and cooled to 0°C. Iodomethane (3.86 mL, 61.9 mmol, 2.0 equivalent) was added in a single addition, and the mixture was stirred at room temperature for 3 hours in the dark. TLC showed complete consumption of the starting material. fThe target product was formed at 0.7°C (ethyl acetate:hexane = 1:1, KMnO4 colorimetric assay). The reaction was quenched by adding water (100 mL) and extracted with Et2O (3 × 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give the desired product as a yellow oil (17.8 g), which was assumed to be quantitative and used in the next step without further purification.

[0271] Steps 2-5. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-hydroxybut-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-hydroxybut-3-en-2-yl)glutarate.

[0272]

[0273] Anhydrous TBAF solution (310 mL, 1 M THF solution, 310 mmol, 10.0 equivalent) was cooled to 0°C under a N2 atmosphere. Glacial acetic acid (18.6 mL, 325 mmol, 10.5 equivalent) was added and stirred until the internal temperature was <5°C. This solution was then poured directly into anhydrous THF (20 mL) solution containing the TBS ether obtained in step 4 (17.8 g of crude product from the previous step), and the entire process was carried out under a N2 atmosphere. The reaction was stirred at 0°C for 3 to 6 hours. The reaction was monitored by TLC (RL in ethyl acetate:hexane (1:1)). f The reaction mixture was quenched immediately after the starting material was observed to be almost completely consumed (0.4; KMnO4 colorimetric assay). Water (200 mL) was added to dilute the reaction mixture, followed by extraction with EtOAc (3 × 200 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification was achieved by rapid silica gel column chromatography, eluting with ethyl acetate:hexane (7:3), to give a colorless oily target product (8.75 g, overall yield of 81.7%).

[0274] Steps 2-6. Synthesis of dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-((S)-1-(1,3-dioxoisoindoline-2-yl)but-3-en-2-yl)glutarate and dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-((R)-1-(1,3-dioxoisoindoline-2-yl)but-3-en-2-yl)glutarate.

[0275]

[0276] 1,5-Dimethyl(2S)-2-[(tert-butoxycarbonyl)amino]-4-(1-hydroxybut-3-en-2-yl)glutarate (6.90 g, 20.0 mmol, 1.0 equivalent), phthalimide (5.88 g, 40.0 mmol, 2.0 equivalent), and triphenylphosphine (7.86 g, 30.0 mmol, 1.5 equivalent) were dissolved in anhydrous THF (150 mL) and cooled to 0°C. DIAD (6.10 mL, 31.0 mmol, 1.55 equivalent) was added dropwise, forming a yellow solution. The reaction was continued at 0°C with stirring for 15 min, then heated to room temperature and stirred for 2 h. Complete consumption of the starting material (product R in ethyl acetate:hexane (1:1)) was observed by TLC. f 0.6 (UV active and colorimetrically determined by KMnO4). The reaction solution was diluted with saturated ammonium chloride aqueous solution (200 mL) and extracted with EtOAc (3 × 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification was achieved by rapid silica gel column chromatography, eluting with ethyl acetate:hexane (1:1), yielding the target product as a viscous, pale yellow solid (15 g). NMR showed the presence of trace amounts of phthalimide and DIAD byproducts. This material was directly fed into the next reaction step based on quantitative yield.

[0277] Steps 2-7. Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4S)-2-oxo-4-vinylpyrrolidine-3-yl)propionate and methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((3R,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate.

[0278]

[0279] A MeOH solution (120 mL) of phthalimide obtained in step 1 (15 g of the product from the previous step) was placed in a 150 mL pressure vessel, and methylamine hydrochloride (6.74 g, 100 mmol, 5.0 equivalent) and N,N-diisopropylethylamine (17.4 mL, 100 mmol) were added sequentially. The vessel was sealed and heated at 80°C for 3 hours. LC / MS analysis showed that the reaction was complete. The reaction volume was reduced to approximately 40 mL under vacuum. A saturated aqueous solution of ammonium chloride (100 mL) was added, and the mixture was extracted with EtOAc (3 × 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification was achieved by rapid silica gel column chromatography, eluting with ethyl acetate:hexane (3:1) to give two separate fractions of the target product. Methyl (S)-2-((tert-Butoxycarbonyl)amino)-3-((3S,4S)-2-oxo-4-vinylpyrrolidine-3-yl)propionate was separated as a less polar fraction: 2.02 g, 32.4%, a transparent viscous gel; methyl (S)-2-((tert-Butoxycarbonyl)amino)-3-((3R,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate was separated as a more polar fraction: 1.56 g, 25.0%, a pale yellow solid, with an overall yield of 57% for both steps.

[0280] Isomer 1: 1 H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 7.4 Hz, 1H), 5.81 -5.53 (m, 2H), 5.24 - 5.08 (m, 2H), 4.49 - 4.40 (m, 1H), 3.72 (s, 3H), 3.44(ddd, J = 9.5, 8.0, 1.4 Hz, 1H), 3.15 (t, J = 9.5 Hz, 1H), 2.77 (p, J = 8.9Hz, 1H), 2.30 (ddd, J = 11.2, 7.4, 4.1 Hz, 1H), 2.12 - 1.97 (m, 2H), 1.44 (s,9H). MS (APCI) m / z 213.1 [M-Boc+H] + .

[0281] Isomer 2: 1H NMR (400 MHz, CDCl3) δ 6.18 (d, J = 6.9 Hz, 1H), 5.78 (dddd, J = 14.9, 13.0, 9.0, 4.1 Hz, 1H), 5.61 (s, 1H), 5.28 - 5.13 (m, 2H),4.27 (dt, J = 10.8, 5.9 Hz, 1H), 3.81 - 3.67 (m, 3H), 3.62 - 3.40 (m, 1H), 3.15 (t, J = 9.3 Hz, 1H), 2.78 (p, J = 8.6 Hz, 1H), 2.30 (dt, J = 11.1, 5.9Hz, 1H), 2.13 - 1.89 (m, 2H), 1.44 (d, J = 3.7 Hz, 9H). MS (APCI) m / z 213.1[M-Boc+H] + .

[0282] General Route 3

[0283]

[0284] Example 3

[0285] Specific examples of general route 3

[0286] ((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)tert-butyl carbamate

[0287]

[0288] Step 3-1. Synthesis of (S)-2-((tert-butoxycarbonyl)amino)hex-5-enoic acid.

[0289]

[0290] Methyl (S)-2-((tert-butoxycarbonyl)amino)hex-5-enoate (1 g, 4.11 mmol, 1.0 equivalent) was dissolved in a tetrahydrofuran:water (4:1, 20 mL) mixture and cooled to 0°C. A 3 mL solution of lithium hydroxide monohydrate (519 mg, 12.33 mmol, 3 equivalent) in water was added to this solution. The reaction was stirred at 0°C for 4 hours, then slowly heated to room temperature. After the reaction was complete, the solution was neutralized to pH 2 with 0.2 M HCl, and the resulting suspension was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over sodium sulfate and concentrated under vacuum to obtain the crude target compound as a viscous yellow oil. 1 H NMR (400 MHz, CDCl3)δ 5.80 (ddt, J = 16.8, 10.2, 6.6 Hz, 1H), 5.07 (dq, J = 17.1, 1.6 Hz, 1H), 5.02 (dq, J = 10.2, 1.4 Hz, 1H), 4.38 - 4.29 (m, 1H), 2.17 (td, J = 7.8, 4.0Hz, 2H), 2.03 - 1.91 (m, 1H), 1.84 - 1.71 (m, 1H), 1.45 (s, 9H); MS (ESI) m / z130.1 [C6H 11 NO2 + H] + .

[0291] Step 3-2. Synthesis of methyl (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate.

[0292]

[0293] Crude (S)-2-((tert-Butoxycarbonyl)amino)hex-5-enoic acid (from step 1; 1 g, 4.36 mmol, 1 equivalent) was dissolved in anhydrous N,N-dimethylformamide (10 mL). Methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride (986 mg, 4.8 mmol, 1.1 equivalent) and HATU (1825 mg, 1.1 mmol, 1.1 equivalent) were added, and the reaction was cooled to 0°C. N,N-diisopropylethylamine (3.80 mL, 5.0 mmol, 5 equivalent) was added, and the resulting yellow solution was stirred at 0°C for 30 min, followed by stirring at room temperature for another 15 min. After the reaction was complete, the solution was quenched with saturated ammonium chloride solution (50 mL), and the resulting mixture was extracted with ethyl acetate (4 × 10 mL). The organic layers were combined and washed successively with saturated sodium bicarbonate, water, and brine, then dried over anhydrous magnesium sulfate. Purification was achieved by rapid silica gel column chromatography, eluting with ethyl acetate:hexane (3:2), yielding the target compound as a transparent gel (1.35 g, 3.55 mmol, 78%). 1 H NMR (400 MHz, CDCl3)δ 5.82 (ddt, J = 16.9, 10.1, 6.6 Hz, 1H), 5.16 - 4.96 (m, 3H), 4.45 (s, 1H), 4.39 (dt, J = 9.0, 6.7 Hz, 1H), 3.92 - 3.72 (m, 5H), 2.22 - 2.13 (m, 1H), 1.86 (ddt, J = 15.0, 8.5, 6.6 Hz, 1H), 1.72 - 1.60 (m, 1H), 1.52 - 1.40 (m,11H), 1.05 (s, 3H), 0.94 (s, 3H); MS (ESI) m / z 381.1 [C 20 H 32 N2O5+H] + .

[0294] Step 3-3. Synthesis of (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid.

[0295]

[0296] Methyl (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (250 mg, 0.657 mmol, 1 equivalent)) was dissolved in tetrahydrofuran (3.3 mL) and cooled to 0°C. A solution of lithium hydroxide monohydrate (82.7 mg, 1.97 mmol, 3 equivalents) in water (0.8 mL) was added to this solution. The reaction was brought to room temperature and stirred for 20.5 hours. After the reaction was complete, it was cooled to 0°C and quenched with saturated ammonium chloride. The resulting solution was acidified to pH 2 with 2 M HCl and extracted with ethyl acetate (4 × 25 mL). The organic layers were combined and dried over anhydrous sodium sulfate. The solution was concentrated under vacuum and purified by silica gel column chromatography. It was eluted with ethyl acetate:hexane (9:1) to give the target compound as a transparent oil (294.5 mg, 0.803 mmol, quantitative yield, containing trace amounts of solvent). 1 H NMR (400 MHz, MeOD)δ 5.85 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.10 (dq, J = 17.2, 1.6 Hz, 1H), 5.02 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 4.34 (s, 1H), 4.32 - 4.26 (m, 1H), 3.98 (d, J = 10.4 Hz, 1H), 3.86 (dd, J = 10.3, 5.2 Hz, 1H), 2.22 - 2.13 (m,2H), 1.82 (dtd, J = 13.7, 7.6, 5.9 Hz, 1H), 1.70 - 1.61 (m, 1H), 1.58 (dd, J= 7.4, 5.0 Hz, 1H), 1.51 (d, J = 7.5 Hz, 1H), 1.43 (s, 9H), 1.10 (s, 3H), 1.01 (s, 3H); MS (ESI) m / z 365.0 [C 19 H 30 N2O5 - H] - .

[0297] Steps 3-4. Synthesis of methyl (S)-2-amino-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate hydrochloride.

[0298]

[0299] Methyl (S)-2-((tert-Butoxycarbonyl)amino)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate (150 mg, 0.480 mmol, 1 equivalent) was dissolved in a 4 M HCl solution (2.4 mL, 9.60 mmol, 20 equivalents) of dioxane and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under vacuum to give the crude target compound. MS (ESI) m / z 213.0 [C 10 H 16 N2O3+ H] + .

[0300] Steps 3-5. Synthesis of methyl propionate (S)-2-((1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate.

[0301]

[0302] Methyl (S)-2-amino-3-((3S,4R)-2-oxo-4-vinylpyrrolidone-3-yl)propionate hydrochloride (from the crude product of steps 3-4, 0.480 mmol, 1 equivalent) was dissolved in N,N-dimethylformamide (4.80 mL) with (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (211.1 mg, 0.576 mmol, 1.2 equivalent) and cooled to 0°C. N,N-diisopropylethylamine (0.418 mL, 2.40 mmol, 5 equivalent) and HATU (200.8 mg, 0.528 mmol, 1.1 equivalent) were added, and the resulting yellow solution was stirred for 30 minutes. After the reaction was complete, the solution was diluted with ethyl acetate and washed successively with saturated ammonium chloride and brine. The aqueous layers were combined and back-extracted with ethyl acetate (2 × 25 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solution was concentrated under vacuum and purified by semi-preparative reversed-phase HPLC (30-60% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a transparent oil (199.4 mg, 0.356 mmol, 74%). 1H NMR (400 MHz, MeOD) δ 8.87 (d, J = 7.4 Hz, 1H), 5.92 - 5.78 (m,2H), 5.23 (dd, J = 17.1, 1.7 Hz, 1H), 5.18 (dd, J = 10.2, 1.8 Hz, 1H), 5.09(dq, J = 17.1, 1.7 Hz, 1H), 5.01 (dd, J = 10.2, 2.0 Hz, 1H), 4.69 (ddd, J =9.6, 6.6, 4.5 Hz, 1H), 4.35 (s, 1H), 4.29 (dt, J = 8.7, 5.4 Hz, 1H), 3.94 (d,J = 3.4 Hz, 2H), 3.74 (s, 3H), 3.57 (dd, J = 10.0, 6.0 Hz, 1H), 3.21 - 3.09(m, 2H), 2.65 (td, J = 8.0, 5.6 Hz, 1H), 2.20 - 2.10 (m, 2H), 2.06 (ddd, J =14.0, 8.1, 5.7 Hz, 1H), 1.88 - 1.75 (m, 2H), 1.70 - 1.61 (m, 1H), 1.58 (ddd,J = 6.2, 4.3, 2.3 Hz, 1H), 1.50 (d, J = 7.7 Hz, 1H), 1.43 (s, 9H), 1.10 (s,3H), 1.01 (s,3H); MS (ESI) m / z 561.2 [C 29 H 44 N4O7 + H] + .

[0303] Steps 3-6. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-((tert-butoxycarbonyl)amino)-12,12-dimethyl-1,9,13-trioxo-1,2,3,3a,6,7,8,9,11,11a,12,12a,12b,13,14,15,16,16a-octadecylcyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester.

[0304]

[0305] Methyl (S)-2-((1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-((3S,4R)-2-oxo-4-vinylpyrrolidine-3-yl)propionate (199.4 mg, 0.355 mmol, 1 equivalent) was dissolved in anhydrous dichloromethane (71.1 mL) under a nitrogen atmosphere. A second-generation Grubbs catalyst (30.2 mg, 0.0355 mmol, 10 mol%) and 2,6-dichlorobenzoquinone (6.3 mg, 0.0355 mmol, 10 mol%) were added, and the reaction was stirred at 45°C for 16.5 h. After cooling to room temperature, Si-TMT (1.5 g) was added to the reaction mixture, and the mixture was stirred for 1 h. The resulting suspension was filtered through a diatomaceous earth pad and washed with methanol. The filtrate was concentrated under vacuum to give the crude target compound as a brown solid. MS (APCI) m / z 533.3 [C 27 H 40 N4O7+ H] + .

[0306] Steps 3-7. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-((tert-butoxycarbonyl)amino)-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester.

[0307]

[0308] (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-((tert-butoxycarbonyl)amino)-12,12-dimethyl-1,9,13-trioxo-1,2,3,3a,6,7,8,9,11,11a,12,12a,12b,13,14,15,16,16a-octadecylcyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylate (from the crude product of steps 3-6, 0.355 mmol, 1 equivalent) was dissolved in methanol (20 mL) under a nitrogen atmosphere. Palladium on carbon (75.6 mg, 0.0711 mmol, 10 w / w%, 0.2 equivalents) and hydrogen gas (introduced via a balloon) were added, and the reaction was stirred at room temperature for 16.5 hours. After the reaction was complete, the mixture was filtered through a diatomaceous earth mat and washed with methanol. The filtrate was concentrated under vacuum and purified by semi-preparative reversed-phase HPLC (30-60% MeCN / H2O, 0.1% formic acid) to give the target compound as a white solid (65.4 mg, 0.122 mmol, 35%). 1 H NMR (400MHz, MeOD) δ 8.33 (d, J = 8.9 Hz, 1H), 4.74 (td, J = 8.5, 3.4 Hz, 1H), 4.58(s, 1H), 4.28 (dd, J = 9.2, 3.5 Hz, 1H), 4.12 (d, J = 10.7 Hz, 1H), 3.84 (dd,J = 10.6, 5.3 Hz, 1H), 3.77 (s, 3H), 3.39 - 3.29 (m, 1H), 3.01 (dd,J = 9.9,5.8 Hz, 1H), 2.55 (q, J = 7.2 Hz, 1H), 2.41 (h, J = 6.8 Hz, 1H), 1.43 (s, 9H), 1.40 - 1.18 (m, 5H), 1.10 (s, 3H), 0.96 (s, 3H); MS (APCI) m / z 535.3 [C 27 H 42 N4O7 + H] + .

[0309] Steps 3-8. Synthesis of tert-butyl carbamate ((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-carbamoyl-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)carbamate.

[0310]

[0311] (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-((tert-butoxycarbonyl)amino)-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylate (32.7 mg, 0.0611 mmol, 1 equivalent) was dissolved in 7 N ammonia-methanol solution (3 mL), and the reaction was stirred at 60°C. 7 N ammonia-methanol solution (2 × 2 mL) was added periodically during the reaction (4.5 h and 7.5 h), and the reaction was completed after a total of 23.5 hours. After the reaction was completed, the mixture was concentrated under vacuum and purified by semi-preparative reversed-phase HPLC (30-60% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (20.8 mg, 0.040 mmol, 65%). 1 H NMR (400 MHz, MeOD) δ 8.23 ​​(d, J = 8.7 Hz, 1H), 4.70 - 4.62 (m, 1H), 4.59 (s, 1H), 4.30 (dd, J = 8.4, 3.2 Hz, 1H), 4.05 (d, J = 10.7 Hz, 1H), 3.83 (dd, J = 10.6, 5.3Hz, 1H), 3.38 - 3.29 (m, 1H), 3.00 (dd, J = 9.9, 6.0 Hz, 1H), 2.68 (q, J =7.1 Hz, 1H), 2.41 (h, J = 6.9 Hz, 1H), 2.09 (ddd, J = 14.7, 7.4, 3.6 Hz, 1H),1.99 - 1.88 (m, 1H), 1.78 - 1.57 (m, 4H), 1.52 - 1.47 (m, 1H), 1.43 (s, 9H),1.37 - 1.19 (m, 5H), 1.10 (s, 3H), 0.97 (s, 3H); MS (APCI) m / z 520.3 [C 26 H41 N5O6+ H] + .

[0312] Steps 3-9. Synthesis of tert-butyl carbamate ((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)carbamate.

[0313]

[0314] ((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-carbamoyl-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl) tert-butyl carbamate (20.8 mg, 0.040 mmol, 1 equivalent) was dissolved in anhydrous N,N-dimethylformamide (0.8 mL). Burgess reagent (28.6 mg, 0.120 mmol, 3 equivalents) was added, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was quenched with water (0.1 mL) and purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid). The target compound was obtained by lyophilization as a white solid (10.6 mg, 0.021 mmol, 53%). 1H NMR (400 MHz, MeOD) δ 5.14(dd, J = 7.5, 4.2 Hz, 1H), 4.54 (s, 1H), 4.28 (dd, J = 8.9, 3.6 Hz, 1H), 4.08(d, J = 10.7 Hz, 1H), 3.84 (dd, J = 10.7, 5.1 Hz, 1H), 3.38 - 3.34 (m, 1H), 3.00 (dd, J = 9.9, 6.1 Hz, 1H), 2.52 - 2.36 (m, 2H), 2.17 (ddd, J = 14.9,7.4, 4.3 Hz, 1H), 2.10 - 1.98 (m, 1H), 1.95 - MS (ESI) m / z 502.2[C 26 H 39 N5O5 + H] + .

[0315] Example 4

[0316] Specific examples of general route 3

[0317]

[0318]

[0319]

[0320]

[0321] Step 4-1. Synthesis of methyl (S)-2-amino-3-((3S,4R)-2-oxo-4-vinylpiperidin-3-yl)propionate hydrochloride.

[0322]

[0323] A solution of 1,4-dioxane (0.1 mL) in 4.0 M HCl was added to a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-((3S,4R)-2-oxo-4-vinylpiperidin-3-yl)propionate (200 mg, 0.61 mmol) at 0°C. The reaction was stirred at room temperature for 1 hour. The reaction mixture was then concentrated under vacuum and used directly without further purification; MS (ESI) m / z 227.1 [C 11 H 18 N2O3 + H] + .

[0324] Step 4-2. Synthesis of (1S,3aR,6aS)-2-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)octahydrocyclopentano[c]pyrrole-1-carboxylic acid.

[0325]

[0326] In 0 o At C, a solution of LiOH (73 mg, 3.04 mmol) in water (1 mL) was added dropwise to a THF (4 mL) solution of (1S,3aR,6aS)-2-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)octahydrocyclopentano[c]pyrrole-1-carboxylate (400 mg, 1.01 mmol). The reaction was stirred at 0°C and slowly heated to room temperature over 5 hours. The reaction mixture was recooled to 0°C and quenched by adding saturated ammonium chloride (15 mL). The crude mixture was acidified to pH 3 with 0.5 M HCl solution and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a pale yellow paste. Purified by rapid silica gel column chromatography, eluted with ethyl acetate:hexane (1:1), the target compound was obtained as a colorless gel (355 mg, 0.97 mmol). 1H NMR (400 MHz, MeOD) δ 5.85(ddt, J = 16.9, 10.1, 6.7 Hz, 1H), 5.10 (dq, J = 17.1, 1.7 Hz, 1H), 5.02 (dq,J = 10.3, 1.2 Hz, 1H), 4.43 - 4.28 (m, 2H), 3.80 (qd, J = 10.6, 5.8 Hz, 2H), 2.89 - 2.68 (m, 2H), 2.18 (q, J = 7.5 Hz, 2H), 2.08 - 1.98 (m, 1H), 1.97 -1.89 (m, 1H), 1.88-1.75 (m, 2H), 1.73-1.54 (m, 4H), 1.46 (s, 9H); MS (ESI) m / z365.2 [C 19 H 30 N2O5 + H] + .

[0327] Step 4-3. Synthesis of methyl propionate (S)-2-((1S,3aR,6aS)-2-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)octahydrocyclopentano[c]pyrrole-1-carboxamido)-3-((3S,4R)-2-oxo-4-vinylpiperidin-3-yl)propionate.

[0328]

[0329] HATU (236 mg, 0.62 mmol) was added to a mixture of (2S)-2-aminovalerate tert-butyl hydrochloride (161 mg, 0.61 mmol), (2R)-2-{[(9H-fluorene-9-ylmethoxy)carbonyl]amino}-3-phenylpropionic acid (227 mg, 0.62 mmol), and N,N-diisopropylethylamine (0.61 mL, 3.05 mmol) in anhydrous DMF (2 mL) at 0°C. The reaction mixture was stirred at 0°C for 0.5 h. Water (5 mL) was added to the reaction mixture, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (155 mg, 0.27 mmol); MS (ESI) m / z 575.3 [C 30 H 46 N4O7 + H] + .

[0330] Step 4-4. Synthesis of (9S,12aR,15aS,15bS,18S,19aS)-9-((tert-butoxycarbonyl)amino)-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-18-carboxylic acid methyl ester.

[0331]

[0332] A mixture of degassed (S)-2-((1S,3aR,6aS)-2-((S)-2-((tert-butoxycarbonyl)amino)hex-5-enoyl)octahydrocyclopentano[c]pyrrolo-1-carboxamido)-3-((3S,4R)-2-oxo-4-vinylpiperidin-3-yl)propionate (155 mg, 0.27 mmol), second-generation Grubbs catalyst (23 mg, 0.027 mmol), and 2,6-dichlorocyclohexane-2,5-diene-1,4-dione (5 mg, 0.027 mmol) was dissolved in anhydrous dichloromethane (100 mL). The reaction mixture was degassed with nitrogen and then heated to 45°C. o The reaction was maintained at C for 18 hours. The reaction was then cooled to room temperature. Sl-TMT (571 mg) was added, and the resulting suspension was stirred at room temperature for 2 hours before being filtered through diatomaceous earth. The filter cake was washed with MeOH (50 mL), and the filtrates were combined and concentrated under vacuum. The residue was purified by semi-preparative reversed-phase HPLC (25-65% MeCN / H2O, 0.1% formic acid) to give the target compound as a white solid (23 mg, 0.042 mmol); MS (ESI) m / z 547.2 [C 28 H 42 N4O7+ H] + Simultaneously, a mixture of other 14-membered cyclic olefins (12 mg, 0.021 mmol) was collected, MS (ESI) m / z 547.2 [C 28 H 42 N4O7+ H] + , and a mixture of 13-membered cyclic olefins (37 mg, 0.069 mmol), MS (ESI) m / z 533.2 [C 27 H 40 N4O7+ H] + .

[0333] Steps 4-5. ((4aR,9S,12aR,15aS,15bS,18S,19aS,Z)-18-carbamoyl-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicosicohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl) tert-butyl carbamate and ( Synthesis of tert-butyl carbamate (4aR,9S,12aR,15aS,15bS,18S,19aS,E)-18-carbamoyl-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl)carbamate.

[0334]

[0335] (9S,12aR,15aS,15bS,18S,19aS)-9-((tert-butoxycarbonyl)amino)-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclic tetradec-18-carboxylic acid methyl ester (23 mg, 0.042 mmol) was dissolved in 7 N ammonia-methanol solution (3 mL). The reaction medium was at 55 °C. o The mixture was stirred at C for 16 hours. After concentration, the reaction solution was purified by semi-preparative reversed-phase HPLC (25-65% MeCN / H2O, 0.1% formic acid) to obtain ((4aR,9S,12aR,15aS,15bS,18S,19aS,Z)-18-carbamoyl-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicosicohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl) tert-butyl carbamate (15 mg, 0.028 mmol), a white solid. 1H NMR (400 MHz, MeOD) δ5.50 - 5.35 (m, 2H), 4.53 (d, J = 2.5 Hz, 1H), 4.44 - 4.34 (m, 2H), 3.90 (dd,J = 10.9, 4.3 Hz, 1H), 3.77 (dd, J = 10.8, 8.1 Hz, 1H), 3.78 - 3.23 (m, 2H), 3.09-2.99 (m, 1H), 2.97 - 2.85 (m, 1H), 2.85-2.76 (m, 1H), 2.66 - 2.52 (m, 2H), 2.44 - 2.32 (m, 1H), 2.26 - 2.04 (m, 2H), 2.03-1.87 (m, 3H), 1.87 - 1.71(m, 3H), 1.70 - 1.39 (m, 13H); MS (ESI) m / z 532.3 [C 27 H 41 N5O6 + H] + .

[0336] ((4aR,9S,12aR,15aS,15bS,18S,19aS,E)-18-carbamoyl-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicosicohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl)tert-butyl carbamate was also obtained as a white solid (5 mg, 0.0094 mmol). 1H NMR (400 MHz, MeOD) δ 5.46 (dd, J = 15.2, 8.2 Hz, 1H), 5.29 (ddd, J = 14.6, 8.3, 4.9 Hz, 1H), 4.72 (dp, J = 11.9, 4.0 Hz, 1H), 4.61 (s, 1H), 4.48 (d, J = 2.5 Hz, 1H), 4.37 (d, J = 6.9 Hz, 1H), 3.95 (dd, J= 10.5, 8.1 Hz, 1H), 3.70 (dd, J = 10.6, 3.5 Hz, 1H), 3.22 (ddd, J = 13.5,6.2, 3.0 Hz, 2H), 2.98 (tq, J = 8.0, 4.2 Hz, 1H), 2.77 (tdd, J = 8.2, 5.2,2.4 Hz, 1H), 2.59 - 2.52 (m, 2H), 2.29 (dd, J = 11.6, 8.2 Hz, 1H), 2.21 -1.68 (m, 9H), 1.67-1.39 (m, 12H); MS (ESI) m / z 532.3 [C 27 H 41 N5O6 + H] + .

[0337] Steps 4-6. Synthesis of (4aR,9S,12aR,15aS,15bS,18S,19aS,Z)-18-cyano-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-ylcarbamate tert-butyl ester.

[0338]

[0339] Add Burgess reagent (20 mg, 0.084 mmol) to an anhydrous DMF (3 mL) solution of ((4aR,9S,12aR,15aS,15bS,18S,19aS,Z)-18-carbamoyl-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl)carbamate (15 mg, 0.028 mmol). The reaction system was stirred at room temperature for 1 hour, quenched with water (0.05 mL), and purified by semi-preparative reversed-phase HPLC (25-65% MeCN / H2O, 0.1% formic acid). After lyophilization, the target compound was obtained as a white solid (10 mg, 0.019 mmol). 1 H NMR (400 MHz, MeOD) δ5.45 - 5.28 (m, 2H), 5.01 (dd, J = 7.2, 4.2 Hz, 1H), 4.39 (dd, J = 8.2, 2.4Hz, 1H), 4.32 (d, J = 2.3 Hz, 1H), 3.82 (d, J = 4.4 Hz, 2H), 3.39 - 3.34 (m,1H), 3.28 (ddt, J = 12.6, 6.7, 3.0 Hz, 1H), 3.05 - 2.69 (m, 4H), 2.47 (dt, J= 10.7, 4.0 Hz, 1H), 2.36 (dt, J = 15.8, 8.1 Hz, 1H), 2.27 - 1.51 (m, 12H), 1.45 (s, 9H); MS (ESI): m / z = 514.3 [M+H + C 27 H 39 N5O5+ H + ].

[0340] Steps 4-7. Synthesis of (4aR,9S,12aR,15aS,15bS,18S,19aS,E)-18-cyano-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicoserohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-ylcarbamate tert-butyl ester.

[0341]

[0342] The procedure is similar to steps 4-6. The target compound (3 mg) is prepared from the corresponding starting material (5 mg).

[0343] 1 H NMR (400 MHz, MeOD) δ 5.43 (dd, J = 15.2, 8.5 Hz, 1H), 5.27 (ddd,J = 17.6, 10.4, 4.9 Hz, 2H), 4.61 (s, 1H), 4.43 - 4.35 (m, 1H), 4.33 (d, J =3.1 Hz, 1H), 3.97 (dd, J = 10.5, 7.8 Hz, 1H), 3.69 (dd, J = 10.5, 3.3 Hz,1H), 3.32 - 3.26 (m, 1H), 3.20 (ddd, J = 12.6, 5.9, 2.3 Hz, 1H), 2.97 (tq, J= 11.3, 4.3 Hz, 1H), 2.73 (tdd, J = 8.1, 5.0, 3.0 Hz, 1H), 2.51 (ddt, J =22.9, 8.3, 3.9 Hz, 2H), 2.35 - 1.86 (m, 8H), 1.85 - 1.58 (m, 4H), 1.46 (s,10H). MS (ESI): m / z = 514.3 [M+H + C 27 H 39 N5O5+ H + ].

[0344] Steps 4-8. Synthesis of (4aS,8S,11aR,14aS,14bS,17S,18aS)-8-((tert-butoxycarbonyl)amino)-1,9,15-trioxodocosano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotridecane-17-carboxylate.

[0345]

[0346] Under a nitrogen atmosphere, Pd / C (10 wt%, 7 mg, 0.0069 mmol) was added to a methanol (4 mL) solution of the 13-membered cyclic olefin mixture (37 mg, 0.069 mmol) obtained in step 4-4. The reaction system was purged with hydrogen for 15 minutes and then stirred at room temperature under a hydrogen atmosphere for 5 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with methanol (5 mL). The combined filtrates were concentrated under vacuum and purified by semi-preparative reversed-phase HPLC (25-65% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (29 mg, 0.054 mmol). 1 H NMR (400 MHz, MeOD) δ 5.25 - 5.15 (m, 1H), 4.85 -4.82 (m, 1H), 4.35 (dd, J = 11.7, 4.3 Hz, 1H), 4.01 (dd, J = 11.0, 3.9 Hz,1H), 3.77-3.58 (m, 4H), 3.30 - 3.23 (m, 2H), 3.19 (tdd, J = 8.2, 6.4, 1.5 Hz,1H), 2.89 (tt, J = 8.3, 4.2 Hz, 1H), 2.32 - 2.23 (m, 1H), 2.08 - 1.37 (m,23H), 1.36 - 1.16 (m, 2H), 1.15 - 1.01 (m, 1H); MS (ESI): m / z = 535.3 [M+H + ;C 27 H 42 N4O7 + H + ].

[0347] Steps 4-9. Synthesis of (4aS,9S,12aR,15aS,15bS,18S,19aS)-18-carbamoyl-1,10,16-trioxododocohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-ylcarbamate tert-butyl ester.

[0348]

[0349] The operation steps are similar to those in steps 4-5.

[0350] 1H NMR (400 MHz, MeOD) δ 7.33 (d, J = 9.8 Hz, 1H), 5.11 - 5.01 (m,1H), 4.35 (dd, J = 11.6, 4.3 Hz, 1H), 4.00 (dd, J = 11.0, 3.8 Hz, 1H), 3.63(dd, J = 10.9, 8.5 Hz, 1H), 3.30 - 3.23 (m, 2H), 3.19 (tdd, J = 8.2, 6.5, 1.6Hz, 1H), 2.90 (tq, J = 8.3, 4.1 Hz, 1H), 2.34 - 2.26 (m, 1H), 2.00 - 1.37 (m, 24H), 1.27 (tdd, J = 12.9, 10.7, 4.7 Hz, 2H), 1.09 (q, J = 11.1 Hz, 1H); MS(ESI) m / z 520.2 [C 26 H 41 N5O6+ H] + .

[0351] Steps 4-10. Synthesis of tert-butyl carbamate ((4aS,8S,11aR,14aS,14bS,17S,18aS)-17-cyano-1,9,15-trioxodocosico[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotridecane-8-yl)carbamate.

[0352]

[0353] The operation steps are similar to those in steps 4-6.

[0354] 1H NMR (400 MHz, MeOD) δ 5.62 (dd, J = 9.3, 2.6 Hz, 1H), 4.85 - 4.81 (m, 1H), 4.33 (dd, J = 11.5, 4.2 Hz, 1H), 3.99 (dd, J = 10.9, 3.8 Hz, 1H), 3.61 (dd, J = 11.0, 8.5 Hz, 1H), 3.37 (m, 4H), 3.30 - 3.23 (m, 2H), 3.22 -3.14 (m, 1H), 2.90 (dq, J = 8.3, 4.2 Hz, 1H), 2.17 (dt, J = 8.5, 3.9 Hz, 1H),2.12 - 1.35 (m, 19H), 1.35 - 1.17 (m, 2H), 1.01 (q, J = 11.3 Hz, 1H); MS(ESI): m / z = 502.3 [M+H + C 26 H 39 N5O5+ H + ]

[0355] Steps 4-11. Synthesis of (4aS,9S,12aR,15aS,15bS,18S,19aS)-9-((tert-butoxycarbonyl)amino)-1,10,16-trioxododocohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-18-carboxylic acid methyl ester.

[0356]

[0357] The operation steps are similar to those in steps 4-8.

[0358] 1H NMR (400 MHz, MeOD) δ 6.83 (d, J = 7.4 Hz, 1H), 5.00 (dd, J = 9.0,2.9 Hz, 1H), 4.62 (s, 1H), 4.47 - 4.38 (m, 1H), 3.74 (m, 5H), 3.31 - 3.24 (m,2H), 3.10 (tdd, J = 8.5, 6.4, 2.2 Hz, 1H), 2.90 (ddt, J = 12.0, 8.2, 4.3 Hz,1H), 2.46 (dt, J = 8.7, 4.1 Hz, 1H), 2.18 (ddd, J = 14.7, 9.0, 2.8 Hz, 1H),2.06 - 1.84 (m, 4H), 1.82 - 1.54 (m, 7H), 1.54-1.25 (m, 14H), 1.19 (p, J =7.6 Hz, 2H); MS (ESI): m / z = 549.3 [M+H + C 28 H 44 N4O7 + H + ]

[0359] Steps 4-12. Synthesis of (4aS,9S,12aR,15aS,15bS,18S,19aS)-18-carbamoyl-1,10,16-trioxododocohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-ylcarbamate tert-butyl ester.

[0360]

[0361] (9S,12aR,15aS,15bS,18S,19aS)-9-((tert-butoxycarbonyl)amino)-1,10,16-trioxo-2,3,4,4a,7,8,9,10,12,12a,13,14,15,15a,15b,16,17,18,19,19a-eicosano-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclic tetradec-18-carboxylic acid methyl ester (6 mg, 0.011 mmol) was dissolved in 7N ammonia-methanol solution (1 mL). The reaction medium was at 55 °C. o Stir at C for 16 hours. Concentrate the reaction solution; it can be used without further purification. 1H NMR (400 MHz, MeOD) δ 4.72 (d, J = 3.9 Hz, 1H), 4.52 (d, J = 2.1 Hz,1H), 4.30 (dd, J = 7.6, 3.3 Hz, 1H), 3.71 - 3.53 (m, 2H), 3.17 (td, J = 6.5,2.6 Hz, 2H), 3.01 - 2.89 (m, 1H), 2.79 (tt, J = 8.2, 4.0 Hz, 1H), 2.43 (dt, J= 8.9, 4.2 Hz, 1H), 2.01 - 1.71 (m, 5H), 1.69 - 1.14 (m, 21H), 1.12 - 0.99(m, 2H); MS (ESI) m / z 534.2 [C 27 H 43 N5O6 + H] + .

[0362] Steps 4-12. Synthesis of tert-butyl carbamate ((4aS,9S,12aR,15aS,15bS,18S,19aS)-18-cyano-1,10,16-trioxododocohydro-1H-cyclopentano[3,4]pyrrolo[1,2-a]pyrido[3,4-g][1,4]diazacyclotetradecane-9-yl)carbamate.

[0363]

[0364] The operation steps are similar to those in steps 4-6.

[0365] 1H NMR (400 MHz, MeOD) δ 6.86 (d, J = 7.4 Hz, 1H), 5.47 (dd, J = 8.1,3.2 Hz, 1H), 4.56 (d, J = 2.2 Hz, 1H), 4.41 (tt, J = 7.4, 3.2 Hz, 1H), 3.74(qd, J = 10.9, 5.9 Hz, 2H), 3.31 - 3.22 (m, 2H), 3.07 (tdd, J = 8.4, 6.2, 2.2Hz, 1H), 2.92 (tq, J = 8.2, 4.3 Hz, 1H), 2.39 (dt, J = 8.8, 4.0 Hz, 1H), 2.14(ddd, J = 14.8, 9.6, 3.3 Hz, 1H), 2.08 - 1.84 (m, 5H), 1.82 - 1.24 (m, 19H),1.13 (p, J = 7.8 Hz, 2H); MS (ESI): m / z = 516.3 [M+H + C 27 H 41 N5O5+ H + MS(ESI): m / z = 502.3 [M+H] + C 26 H 39 N5O5+ H + ]

[0366] General Route 4

[0367]

[0368] Example 5

[0369] Specific examples of general route 4

[0370] N-((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)-3,3-dimethylbutyramide

[0371]

[0372] Step 5-1. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-amino-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester hydrochloride.

[0373]

[0374] (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-((tert-butoxycarbonyl)amino)-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylate (30 mg, 0.0561 mmol, 1 equivalent) was dissolved in a 4 M HCl solution of dioxane (0.280 mL, 1.12 mmol, 20 equivalents) and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under vacuum to give the crude target compound. MS (ESI) m / z 435.2 [C 22 H 34 N4O5+ H] + .

[0375] Step 5-2. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-(3,3-dimethylbutamido)-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester.

[0376]

[0377] (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-amino-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester hydrochloride (from the crude product of step 5-1, 0.0561 mmol, 1 equivalent) and 3,3-dimethylbutyric acid (8.6 µL, 0.0673 mmol, 1.2 equivalent) were dissolved in anhydrous DMF (0.561 mL) and cooled to 0°C. N,N-diisopropylethylamine (48.8 µL, 0.280 mmol, 5 equivalents) and HATU (23.4 mg, 0.0617 mmol, 1.1 equivalents) were added, and the resulting yellow solution was stirred for 30 minutes. After the reaction was complete, the solution was diluted with ethyl acetate and washed successively with saturated ammonium chloride and brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (20.8 mg, 0.039 mmol, 70%). 1 H NMR (400 MHz, MeOD) δ 8.37 (d, J = 8.9 Hz, 1H), 8.11 (d, J = 7.3 Hz, 1H), 4.77 - 4.67 (m, 1H), 4.57 (s, 1H), 4.53 (dd, J =9.2, 3.6 Hz, 1H), 4.13 (d, J = 10.7 Hz, 1H), 3.87 (dd, J = 10.6, 5.2 Hz, 1H), 3.77 (s, 3H), 3.37 - 3.34 (m, 1H), 3.02 (dd, J = 10.0, 5.6 Hz, 1H), 2.58 (q,J = 7.2 Hz, 1H), 2.42 (h, J = 6.7 Hz, 1H), 2.23 (ddd, J = 14.7, 7.2, 3.7 Hz,1H), 2.14 - 2.04 (m, 2H), 1.99 - 1.85 (m, 1H), 1.80 - 1.59 (m, 4H), 1.57 -1.45 (m, 1H), 1.42 - 1.19 (m, 5H), 1.10 (s, 3H), 1.01 (s, 9H), 0.96 (s, 3H); MS (ESI) m / z 533.2 [C 28 H 44 N4O6 + H] + .

[0378] Step 5-3. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-(3,3-dimethylbutyramamido)-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxamide

[0379]

[0380] The target compound was prepared using a method similar to steps 3-8. The compound was purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (16.6 mg, 0.032 mmol, 82%). 1 HNMR (400 MHz, MeOD) δ 8.28 (d, J = 8.6 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 4.70 - 4.60 (m, 1H), 4.58 (s, 1H), 4.57 - 4.52 (m, 1H), 4.07 (d, J = 10.7 Hz, 1H), 3.85 (dd, J = 10.6, 5.3 Hz, 1H), 3.35 (d, J = 6.7 Hz, 1H), 3.01 (dd, J =10.0, 5.7 Hz, 1H), 2.71 (q, J = 7.1 Hz, 1H), 2.42 (h, J = 6.7 Hz, 1H), 2.16 -2.05 (m, 3H), 1.97 (ddd, J = 13.8, 8.2, 6.2 Hz, 1H), 1.79 - 1.64 (m, 3H),1.61 (dd, J = 7.7, 5.1 Hz, 1H), 1.59 - 1.48 (m, 1H), 1.41 - 1.20 (m, 5H), 1.10 (s, 3H), 1.01 (s, 9H), 0.96 (s, 3H); MS (APCI) m / z 518.4 [C 27 H 43 N5O5 + H] + .

[0381] Step 5-4. Synthesis of N-((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)-3,3-dimethylbutyramide 1

[0382]

[0383] The target compound was prepared using a method similar to steps 3-9. The compound was purified by semi-preparative reversed-phase HPLC (20-70% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (12.4 mg, 0.025 mmol, 46%). 1 H NMR (400 MHz, MeOD) δ (ppm) 8.70 (d, J = 8.6 Hz, 0.3H), 8.09 (d, J = 7.2 Hz, 1H), 5.18 - 5.08 (m, 1H), 4.59 - 4.47 (m, 2H), 4.09 (d, J = 10.7 Hz, 1H), 3.86(dd, J = 10.6, 5.0 Hz, 1H), 3.42 - 3.35 (m, 1H), 3.01 (dd, J = 10.0, 6.0 Hz,1H), 2.47 (dq, J = 23.4, 7.1 Hz, 2H), 2.19 (ddd, J = 14.8, 7.4, 4.4 Hz, 1H),2.12 - 1.99 (m, 3H), 1.98 - 1.86 (m, 1H), 1.72 - 1.59 (m, 3H), 1.56 - 1.40(m, 1H), 1.40 - 1.18 (m, 5H), 1.10 (s, 3H), 1.01 (s, 9H), 0.97 (s, 3H); MS(ESI) m / z 500.2 [C 27 H 41 N5O4 + H] + .

[0384] General Route 5

[0385]

[0386] Example 6

[0387] Specific examples of general route 5

[0388] N-((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)benzenesulfonamide

[0389]

[0390] Step 6-1. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-12,12-dimethyl-1,9,13-trioxo-8-(benzenesulfonamide)eicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylic acid methyl ester.

[0391]

[0392] (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-amino-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylate hydrochloride (prepared as in step 5-1, 0.0841 mmol, 1 equivalent) was dissolved in dichloromethane (0.841 mL) and cooled to 0°C. Benzenesulfonyl chloride (12.9 µL, 0.101 mmol, 1.2 equivalent) and N,N-diisopropylethylamine (44 µL, 0.252 mmol, 3 equivalent) were added, and the reaction was stirred for 3.5 hours while being heated to room temperature. After the reaction was complete, it was quenched with saturated ammonium chloride solution and extracted with dichloromethane (4 × 5 mL). The organic layers were combined, dried with anhydrous sodium sulfate, concentrated under vacuum, and purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to obtain the target compound as a white solid (38.7 mg, 0.067 mmol, 80%). 1H NMR (400 MHz, MeOD) δ 8.28 (d, J = 8.9 Hz, 1H), 7.92 - 7.85 (m, 2H), 7.68 - 7.60 (m, 1H), 7.60 - 7.47 (m, 2H), 4.75 (tt, J = 7.6, 4.6 Hz, 1H), 4.46 (s, 1H), 4.04 (dd, J = 7.5, 3.3 Hz, 1H), 3.82 - 3.72 (m, 1H), 3.75 (s,3H), 3.68 (d, J = 10.8 Hz, 1H), 2.97 (dd, J = 9.9, 5.9 Hz, 1H), 2.56 (q, J =7.1 Hz, 1H), 2.38 (h, J = 6.7 Hz, 1H), 2.20 (ddd, J = 14.6, 7.4, 4.5 Hz, 1H), 1.88 (dq, J = 13.2, 6.3 Hz, 1H), 1.73 - 1.61 (m, 2H), 1.58 - 1.46 (m, 2H), 1.40 - 1.16 (m, 5H), 1.10 (d, J = 8.1 Hz, 1H), 1.04 (s, 3H), 0.66 (s, 3H); MS(ESI) m / z 575.1 [C 28 H 38 N4O7S + H] + .

[0393] Step 6-2. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-12,12-dimethyl-1,9,13-trioxo-8-(benzenesulfonamido)eicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxamide.

[0394]

[0395] The target compound was prepared according to the same method as steps 5-3 in general route 5. The compound was purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to give the target compound as a white solid (28.4 mg, 0.051 mmol, 75%). 1H NMR (400 MHz, MeOD) δ 8.20 (d, J = 8.6 Hz, 1H), 7.92 - 7.85 (m,2H), 7.65 - 7.52 (m, 4H), 4.69 - 4.60 (m, 1H), 4.45 (s, 1H), 4.04 (dd, J =6.9, 3.1 Hz, 1H), 3.75 (dd, J = 10.7, 5.1 Hz, 1H), 3.64 (d, J = 10.7 Hz, 1H), 2.96 (dd, J = 9.9, 6.7 Hz, 1H), 2.69 (td, J = 7.8, 5.8 Hz, 1H), 2.39 (h, J =6.9 Hz, 1H), 2.09 - 1.96 (m, 1H), 1.91 (dt, J = 13.0, 6.7 Hz, 1H), 1.69 (dt,J = 14.7, 6.3 Hz, 1H), 1.63 (d, J = 7.6 Hz, 1H), 1.58 - 1.49 (m, 2H), 1.45 -1.33 (m, 1H), 1.31 - 1.20 (m, 4H), 1.18 - 1.08 (m, 1H), 1.04 (s, 3H), 0.64(s, 3H); MS (APCI) m / z 560.3 [C 27 H 37 N5O6S + H] + .

[0396] Step 6-3. Synthesis of N-((3aR,8S,11aS,12aR,12bS,15S,16aS)-15-cyano-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-8-yl)benzenesulfonamide.

[0397]

[0398] The target compound was prepared according to the same method as steps 5-4 in general route 5. The compound was purified by semi-preparative reversed-phase HPLC (25-55% MeCN / H2O, 0.1% formic acid) to give the target compound as a white solid (23.1 mg, 0.043 mmol, 84%). 1H NMR (400 MHz, MeOD) δ (ppm) 7.87 (dd, J = 7.2, 1.7 Hz, 2H), 7.67- 7.59 (m, 1H), 7.59 - 7.50 (m, 2H), 5.12 (dd, J = 6.5, 4.9 Hz, 1H), 4.39 (s,1H), 4.04 (dd, J = 7.5, 3.2 Hz, 1H), 3.76 (dd, J = 10.6, 4.8 Hz, 1H), 3.68(d, J = 10.6 Hz, 1H), 3.37 - 3.34 (m, 1H), 2.97 (dd, J = 9.9, 6.2 Hz, 1H),2.41 (dp, J = 27.2, 7.0 Hz, 2H), 2.26 - 2.11 (m, 1H), 1.96 (dt, J = 14.9, 6.4Hz, 1H), 1.85 (dq, J = 13.4, 6.6 Hz, 1H), 1.66 - 1.54 (m, 2H), 1.54 - 1.39 (m, 1H), 1.39 - 1.14 (m, 5H), 1.12 - 0.96 (m, 4H), 0.70 (s, 3H); MS (ESI) m / z542.1 [C 27 H 35 N5O5S + H] + .

[0399] General Route 6

[0400]

[0401] Example 7

[0402] Specific examples of general route 6

[0403] (3aR,8S,11aS,12aR,12bS,15S,16aS)-12,12-dimethyl-1,9,13-trioxo-8-((4-(trifluoromethyl)pyrimidin-2-yl)amino)eicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxynitrile

[0404]

[0405] Step 7-1. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-8-amino-12,12-dimethyl-1,9,13-trioxoeicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylonitrile.

[0406]

[0407] Under a nitrogen atmosphere, sodium iodide (85.2 mg, 0.568 mmol, 5.0 equivalent) was added to a solution of trimethylchlorosilane (62 mg, 0.568 mmol, 5.0 equivalent) in anhydrous acetonitrile (0.5 mL) to prepare a trimethyliodosilane solution. The reaction was stirred at room temperature for 30 minutes, during which a white solid precipitate was observed. To the above turbid solution, N-[(1S,4S,6S,10R,15S,19S,21R)-4-cyano-20,20-dimethyl-2,7,16-trioxo-3,8,17-triazatetracyclo[15.4.0.0^{6,10}.0^{19,21}]cuicosan-15-yl]tert-butyl carbamate (57 mg, 0.114 mmol, 1.0 equivalent) dissolved in anhydrous acetonitrile (1.0 mL) was added. The resulting suspension was stirred at room temperature for 15 minutes. The crude reaction mixture was then filtered and concentrated under vacuum for 3 hours to obtain the target compound as a transparent membrane. MS (ESI) m / z 402.3 [C 21 H 31 N5O3 + H] + .

[0408] The substance can be further purified by semi-preparative reversed-phase HPLC (5-35% MeCN / H2O, 0.1% formic acid), and the target compound is obtained by lyophilization as a white solid. 1H NMR (400 MHz, MeOD) δ 5.21 (dd, J = 6.5, 4.9 Hz, 1H), 4.57 (s, 1H), 3.96 (t, J = 4.4 Hz, 1H), 3.83 (dd, J = 10.7, 5.1 Hz, 1H), 3.75 (d, J = 10.7 Hz, 1H), 3.42 - 3.35 (m, 1H), 2.99 (dd, J = 9.9, 6.8 Hz,1H), 2.57 - 2.33 (m, 2H), 2.25 - 2.11 (m, 1H), 2.00 - 1.91 (m, 1H), 1.91 -1.76 (m, 2H), 1.72 (d, J = 7.6 Hz, 1H), 1.65 (dd, J = 7.7, 4.9 Hz, 1H), 1.41- 1.28 (m, 5H), 1.19 - 1.13 (m, 1H), 1.12 (s, 3H), 0.98 (s, 3H).

[0409] Step 7-2. Synthesis of (3aR,8S,11aS,12aR,12bS,15S,16aS)-12,12-dimethyl-1,9,13-trioxo-8-((4-(trifluoromethyl)pyrimidin-2-yl)amino)eicocyclopropano[3,4]pyrrolo[1,2-a]pyrrolo[3,4-g][1,4]diazacyclotetradecane-15-carboxylonitrile.

[0410]

[0411] (1S,4S,6S,10R,15S,19S,21R)-15-amino-20,20-dimethyl-2,7,16-trioxo-3,8,17-triazatetracyclo[15.4.0.0^{6,10}.0^{19,21}]cosicosane-4-carboxynitrile (from the crude product of step 1; 45 mg, 0.112 mmol, 1.0 equivalent), 2-chloro-4-(trifluoromethyl)-pyrimidine (24.6 mg, 0.134 mmol, 1.2 equivalent), and N,N-diisopropylethylamine (99 μL, 0.56 mmol, 5.0 equivalent) were dissolved in anhydrous dimethylacetamide (1.1 mL). Sodium iodide (1 crystal, catalytic amount) was added, and the reaction was stirred at room temperature for 24 hours under a nitrogen atmosphere. Water (0.2 mL) was added, and the crude product was purified by semi-preparative reversed-phase HPLC (20-70% MeCN / H2O, 0.1% formic acid). After lyophilization, the target compound was obtained as a white solid (22 mg, 0.040 mmol, 36%).1 H NMR (400 MHz, MeOD) δ 8.67 - 8.31 (m, 1H), 6.96 (d,J = 4.9 Hz, 1H), 5.27 - 5.05 (m, 1H), 4.83 - 4.59 (m, 1H), 4.52 (s, 1H), 4.15(d, J = 10.6 Hz, 1H), 3.92 (dt, J = 10.6, 2.6 Hz, 1H), 3.42 - 3.35 (m, 1H), 3.01 (dd, J = 9.9, 6.3 Hz, 1H), 2.63 - 2.40 (m, 2H), 2.31 - 2.12 (m, 1H),2.12 - 1.94 (m, 2H), 1.87 - 1.72 (m, 1H), 1.67 (s, 2H), 1.60 - 1.45 (m, 1H), 1.45 - 1.19 (m, 6H), 1.15 - 1.01 (m, 4H), 0.97 - 0.75 (m, 2H) (This compound exists as a mixture of tautomers); MS (ESI) m / z 548.1 [C 21 H 31 N5O3 + H] + .

[0412] General Route 7

[0413]

[0414] Example 8

[0415] Specific examples of general route 7

[0416] (3aR,5S,8S,11S,14aS)-11-benzyl-8-isobutyl-3,7,10-trioxohexadecylpyrrolo[3,4-g][1,4]diazacyclotetrazane-5-carboxaldehyde

[0417]

[0418] Step 8-1. Synthesis of (3aR,5S,8S,11S,14aS)-11-benzyl-5-(hydroxymethyl)-8-isobutyldodecylpyrrolo[3,4-g][1,4]diazacyclotridecane-3,7,10(2H)-trione.

[0419]

[0420] (3aR,5S,8S,11S,14aS)-11-benzyl-8-isobutyl-3,7,10-trioxahexadecylpyrrolo[3,4-g][1,4]diazacyclotridecane-5-carboxylate (13.9 mg, 0.0295 mmol, 1 equivalent) was dissolved in 0.147 mL of 2 M LiBH4 THF solution, and the reaction was stirred at room temperature for 30 min. After the reaction was complete, the solution was diluted with ethyl acetate and washed four times with saturated ammonium chloride solution. The aqueous layers were combined and back-extracted with fresh ethyl acetate solution. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to give the crude target compound as a white solid (12.3 mg, 0.028 mmol, 94%). MS (APCI) m / z 444.3 [C 25 H 37 N3O4 + H] + .

[0421] Step 8-2. Synthesis of (3aR,5S,8S,11S,14aS)-11-benzyl-8-isobutyl-3,7,10-trioxohexadecylpyrrolo[3,4-g][1,4]diazacyclotridecane-5-carboxaldehyde.

[0422]

[0423] (3aR,5S,8S,11S,14aS)-11-benzyl-5-(hydroxymethyl)-8-isobutyldodecylpyrrolo[3,4-g][1,4]diazacyclotridecane-3,7,10(2H)-trione (3.7 mg, 0.00834 mmol, 1 equivalent) was dissolved in DMF (0.2 mL) and cooled to 0°C. Dess-Martin oxidant (7.07 mg, 0.0166 mmol, 2 equivalents) was added, and the reaction was stirred at room temperature for 2 hours. After the first hour, a second portion of Dess-Martin oxidant was added. The completed reaction was filtered through a syringe filter and washed with methanol. The filtrate was purified by semi-preparative reversed-phase HPLC (20-70% MeCN / H2O, 0.1% formic acid), lyophilized, and the target compound was obtained as a white solid (2.4 mg, 0.005 mmol, 65%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 9.59-9.54 (m, 0.01H), 7.87 (d, J = 7.7 Hz, 0.3H), 7.51 (d,J = 7.1 Hz, 0.2H), 7.37 (t, J = 7.3 Hz, 0.2H), 7.31-7.12 (m, 5H), 7.04 (t, J= 7.6 Hz, 0.2H), 4.67-4.54 (m, 1H), 4.17-4.04 (m, 1H), 3.28-3.22 (m, 1H), 3.07-2.99 (m, 1H), 2.93-2.50 (m, 4H), 2.47-2.38 (m, 1H), 2.05-1.89 (m, 1H), 1.89-1.74 (m, 1H), 1.66-1.12 (m, 9H), 0.92-0.64 (m, 6H); MS (APCI) m / z 442.3[C 25 H 35 N3O4 + H] + .

[0424] Some of the compounds in this article can exist as mixtures of hydrates and parent compounds.

[0425] .

[0426] Biochemical data

[0427] Protein expression and purification

[0428] The full-length gene encoding the SARS-CoV-2 3CL protease (derived from the BetaCoV / WIV04 / 2019 strain, accession number NC_045512) was synthesized in Genscript (Piscataway, NJ, USA) and cloned into the NdeI and XhoI sites of the pET29a(+) vector as previously described (Huang Y et al.; Acta Pharmacol Sin. 2020 Sep;41(9):1141-1149), which is incorporated herein by reference. Similarly, the full-length gene encoding human coronavirus 229E (HCoV-229E, accession number X69721.1) was cloned into pET29a(+) with a C-terminal His tag. Codon-optimized plasmids for E. coli expression were transformed into competent BL21(DE3) cells. Single colonies were picked and inoculated into 10 mL of Terrific Broth (TB) containing 50 mg / L kanamycin and cultured at 37°C with shaking at 200 rpm. 10 mL of the inoculum was added to 1 L of TB containing 50 mg / L kanamycin and cultured until the optical density at 600 nm reached 2.5. The culture was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and cultured for another 2 hours at 37°C. The cell pellet was resuspended in lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 2 mM dithiothreitol (DTT), and 10 μg / mL LDnase I) and lysed by sonication (25% amplitude, 2 sec on / 2 sec off). Cell debris was removed by centrifugation at 39,191 × g for 30 min at 4°C. The supernatant was loaded onto a pre-equilibrated HisTrap HP column (Cytiva). The column was first washed with lysis buffer containing 5 mM imidazole, followed by a second wash with 30 mM imidazole. The protein was eluted with buffer containing 300 mM imidazole and further purified by gel filtration chromatography on a HiLoad 16 / 600 Superdex 200 preparative-grade column (Cytiva) using an elution buffer of 20 mM Tris (pH 7.5), 100 mM NaCl, and 2 mM DTT. The fractions corresponding to the obtained peaks were collected and concentrated using a 10,000 molecular weight cutoff centrifugation filter. The purity and molecular weight of the protein were confirmed by SDS-PAGE and mass spectrometry (MS). MS showed a main peak at 34863 Da, which corresponds to the calculated molecular weight of the SARS-CoV-2 C-His-3CL protease after removing the N-terminal methionine residue.

[0429] HcoV-229E was expressed and purified under similar conditions, the difference being that Rosetta (DE3) was used for cell transformation, and cells were cultured in Luria Broth followed by overnight induction at 18°C. MS analysis showed its molecular weight to be 34,120 Da, consistent with the calculated mass after removing the N-terminal methionine residue.

[0430] The recombinant MERS-CoV 3CL protease (accession number K9N638) expressed in *E. coli* was purchased from R&D Systems (catalog number E-719). The purity and molecular size of the commercially available MERS-CoV 3CL were confirmed in our laboratory by SDS-PAGE. Further MS validation showed a main peak at 33361 Da, matching the theoretical molecular weight of 33360.3 Da.

[0431] Biochemical detection methods for SARS-CoV-2 3CL, HCoV-229E 3CL and MERS 3CL proteases

[0432] A highly sensitive FRET-based protease assay was developed to identify inhibitors of the 3CL protease. Substrate peptide 1 (Dabcyl)KTSAVLQSGFRKM(Glu)(EDANS) (2) was synthesized by Genscript. The test compound was serially diluted 3-fold in 100% DMSO, starting at 3.33 mM, for a total of 15 concentration gradients. 1.5 µL of the serially diluted compound was transferred to a black 384-well assay plate (catalog number: 781900, Greiner). 23.5 µL of SARS-CoV-2 Chis-3CL protease, H229E-CoV Chis-3CL protease, or MERS-CoV 3CL protease at a 2.13-fold concentration prepared according to the assay buffer was added to the compound, and the plate was incubated at 25°C for 30 min. 25 µL of peptide 1 substrate at a 2-fold concentration was added to the assay plate. The SARS-CoV-2 Chis-3CL protease detection plates were incubated at 37°C, while the H229E-CoV Chis-3CL protease detection plates and MERS-CoV 3CL protease detection plates were incubated at 25°C for 1.5 hours. The final detection system contained: 12.5 nM SARS-CoV-2 Chis-3CL protease, or 6 nM H229E-CoV Chis-3CL protease, or 75 nM MERS-CoV 3CL protease, as well as 6 µM peptide 1 substrate and 3% DMSO. The detection buffer consisted of 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.01% Triton X-100, and 1 mM DTT.

[0433] FRET signals were measured using an Envision ELISA reader (2104 EnVision Multilabel Plate Readers, PerkinElmer) with an excitation wavelength of 340 nm (UV(TRF) 340 / 60 nm, barcode 101) and an emission wavelength of 490 nm (DSPPsion 486 / 10 filter, barcode 220), using a Lance / DELFIA D400 single-lens microscope (barcode 412). The IC50 of the compounds was determined using GraphPad Prism software (GraphPad, USA) by fitting dose-dependent inhibition curves with variable slopes. 50 .

[0434] +: >20 µM

[0435] ++: 1 – 20 µM

[0436] +++: 100 nM – 1 µM

[0437] ++++: <100 nM

[0438]

[0439] Cell experiments

[0440] Drug titration of BSL2 human coronaviruses (229E and OC43)

[0441] MRC-5 cells (ATCC® CCL-171™) were seeded at a density of 10,000 cells per well in black clear 96-well plates (Greiner) and incubated overnight. The following day, the culture medium was removed from each well, and 229E (ATCC® VR-740™) or OC43 (ATCC® VR-1558™) virus was infected with 50 µL of virus inoculation medium per well at a multiple of infection (MOI) of 0.01. After 1 hour of virus infection in EMEM containing 2% FBS, the virus inoculation medium was removed, and 50 µL of the test compound diluted in EMEM + 2% FBS + 0.5% DMSO was added to each well. Compound assays were performed at a starting concentration of 50 µM, with 8 concentration points and 5-fold serial dilutions. Cells were then incubated for 4 days (229E) or 5 days (OC43), and cell viability was then assessed using a Tecaninfinite M200 pro microplate reader via CellTiter Glo (Promega) according to the manufacturer's instructions. Simultaneously, the cytotoxicity of the compound was evaluated on uninfected cells (co-platened with virus-infected cells). The concentration of cytotoxicity (CC) was determined by nonlinearly fitting the luminescent readings from uninfected, compound-treated wells to the compound concentration. 50 Value. Similarly, EC 50 The values ​​were determined by nonlinear fitting of the luminescence readings in the viral infection and compound treatment wells. Data analysis was performed using GraphPad Prism8, and SI was calculated as CC. 50 With EC 50 The ratio of .

[0442] Drug titration of BSL3 human coronaviruses (SARS-CoV-2 and MERS)

[0443] Vero E6 cells were seeded at a density of 20,000 cells per well in 96-well plates and cultured overnight in MEM + 10% FBS. The following day, the culture medium was removed from each well before adding the virus containing the compound. SARS-CoV-2 or MERS virus was immediately mixed with the test compound at a concentration of 100 TCID50 and then added to cells containing MEM + 2% FBS + 0.5% DMSO. Compound assays were performed using an 8-point, 5-fold serial dilution at a starting concentration of 50 µM in the presence of the efflux inhibitor CP100356. Virus ToxGlo assays were performed after an incubation period of 96 hours (SARS-CoV-2) or 120 hours (MERS), according to the manufacturer's protocol. Readings were detected using a Tecan Spark microplate reader with luminescence measurement.

[0444] CC was determined by nonlinearly fitting the luminescence readings in uninfected, compound-treated wells to the compound concentration. 50 Value. Similarly, EC 50The values ​​were determined by nonlinear fitting of the luminescence readings in the viral infection and compound treatment wells. Data analysis was performed using GraphPad Prism 8, and SI was calculated as CC. 50 With EC 50 The ratio of .

[0445] +: >20 µM

[0446] ++: 1 - 20 µM

[0447] +++: 100 nM - 1 µM

[0448]

[0449]

[0450]

[0451] It should be understood that numerous further modifications and arrangements of the various aspects of the described embodiments are possible. Therefore, the described aspects are intended to cover all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

[0452] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” shall be understood to imply inclusion of the said integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0453] Throughout this specification and the following claims, unless the context otherwise requires, the phrase “consistently composed of…” and its variations such as “consistently made of…” shall be understood to indicate that the listed elements are essential elements of the invention, i.e., indispensable elements. This phrase allows for the presence of other unlisted elements that do not substantially affect the features of the invention, but excludes other unspecified elements that would affect the fundamental and novel features of the defined method.

[0454] References to any prior disclosure (or information derived therefrom) or any known matter in this specification shall not and should not be construed as an admission or implication that such prior disclosure (or information derived therefrom) or known matter constitutes part of the general knowledge in the art to which this specification pertains.

Claims

1. A compound of formula (I) or a salt, solvate, stereoisomer, or prodrug thereof: (I)， R1 is selected from substituted acyl and cyano groups; R2 is selected from optionally substituted alkyl and optionally substituted alkenyl groups; X is selected from N and C-R4; R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide. R4 is selected from H and optionally substituted alkyl groups; R5 is independently selected from H and optionally substituted alkyl groups; R6 is selected from H and optionally substituted alkyl groups; Alternatively, R2 and R6 can be linked to form an optionally substituted heterocyclic group; m is an integer selected from 1 to 4; and, n is an integer selected from 1 to 4.

2. The compound according to claim 1, wherein R1 is selected from HC(O)- and cyano.

3. The compound according to claim 1 or 2, wherein R2 is a C1-C5 alkyl group.

4. The compound according to any one of claims 1 to 3, wherein R2 is selected from butyl, isobutyl, pentyl, isopentyl, neopentyl, halopentyl, haloisopentyl, haloneopentyl, cyclopentylmethyl and cyclopentyl.

5. The compound according to any one of claims 1 to 4, wherein R3 is selected from H, optionally substituted benzyl, optionally substituted halobenzyl, optionally substituted trihalomethylbenzyl, optionally substituted cyclopentyl, optionally substituted cyclopentylmethyl, optionally substituted tert-butoxy, optionally substituted cyclohexyloxy, optionally substituted tetrahydropyranylamide, optionally substituted tert-butylamide, optionally substituted trihalotert-butylamide, optionally substituted trihalomethylamide, optionally substituted neopentylamide, optionally substituted dihalocyclobutylmethylamide, optionally substituted cyclopentylmethylamide, optionally substituted pyrimidinylamide, etc. Optionally substituted phenylamide, optionally substituted halophenylamide, optionally substituted dihalophenylamide, optionally substituted isoxazolylamide, optionally substituted methylisooxazolylamide, optionally substituted trihalomethylisooxazolylamide, optionally substituted thiazolylamide, optionally substituted methylthiazolylamide, optionally substituted oxazolylamide, optionally substituted methyloxazolylamide, optionally substituted phenylamide, optionally substituted halophenylamide, optionally substituted dihalophenylamide, optionally substituted trihalomethylphenylamide, optionally substituted halotrihalomethylphenylamide, optionally substituted halogen Benzylpropionylamino, optionally substituted cyclopropionylamino, optionally substituted trihalomethylcyclopropionylamino, optionally substituted cyclobutyrylamino, optionally substituted methylcyclobutyrylamino, optionally substituted dihalocyclobutyrylamino, optionally substituted bicyclic [1.1.1]pentanoylamino, optionally substituted trihalomethylbicyclic [1.1.1]pentanoylamino, optionally substituted oxacyclobutyrylamino, optionally substituted methyloxacyclobutyrylamino, optionally substituted bicyclic [2.1.1]hexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted dihalocyclohexanoylamino, optionally substituted cyclohexanoylamino, optionally substituted spiro[3.3]heptanoylamino The group includes, optionally substituted tert-butoxyacylamino (or Boc-NH or Boc-N(methyl)), optionally substituted cyclopentoxyacylamino, optionally substituted neopentoxyacylamino, optionally substituted cyclobutoxyacylamino, optionally substituted trihalomethylcyclobutoxyacylamino, optionally substituted benzenesulfonylamino, optionally substituted dihalobenzenesulfonylamino, optionally substituted cyclopropanesulfonylamino, optionally substituted methanesulfonylamino, optionally substituted trihalomethylsulfonylamino, optionally substituted cyclobutanesulfonylamino, optionally substituted amino, optionally substituted pyrimidinylamino, optionally substituted trihalomethylpyrimidinylamino, and optionally substituted tert-butaminoacyl.

6. The compound according to any one of claims 1 to 5, wherein R4 is H or methyl.

7. The compound according to any one of claims 1 to 6, wherein R5 is H or methyl.

8. The compound according to any one of claims 1 to 7, wherein the compound is a compound of formula (Ia): (I), R1 is selected from substituted acyl and cyano groups; Ht is an optionally substituted heterocyclic group; X is selected from N and C-R4; R3 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted amino, optionally substituted aryl, optionally substituted alkoxy, optionally substituted cycloalkoxy, optionally substituted oxyamide, optionally substituted aminoamide, optionally substituted aminoacyl, and optionally substituted sulfonamide. R4 is selected from H and optionally substituted alkyl groups; R5 is independently selected from H and optionally substituted alkyl groups; m is an integer selected from 1 to 4; and, n is an integer selected from 1 to 4.

9. The compound according to any one of claims 1 to 8, wherein when R2 and R6 are connected to form an optionally substituted heterocyclic group or Ht, the heterocyclic group is an optionally substituted 5-7 membered heterocyclic group.

10. The compound according to any one of claims 1 to 9, wherein the optional substituent on the heterocyclic group is selected from halogen, methyl, methoxy, ethoxy, cyclopropyl, fused dimethylcyclopropyl, fused cyclopentyl, bridged methylene and bridged ethylene.

11. The compound according to any one of claims 1 to 10, wherein m is an integer selected from 1 to 3.

12. The compound according to any one of claims 1 to 11, wherein n is an integer selected from 1 to 2.

13. The compound according to any one of claims 1 to 12, wherein the compound of formula (I) or its salt, solvate, stereoisomer or prodrug of formula (I) iix As shown in b): (I iix b)。 14. The compound according to any one of claims 1 to 12, wherein the compound of formula (Ia) or its salt, solvate, stereoisomer or prodrug of formula (Ia) iix As shown in b): (It iix b).

15. The compound according to any one of claims 1 to 14, wherein the compound is selected from any one of the following compounds: ； ； ； ； ； ； ； ； ； ； ； ； ； 。 16. A pharmaceutical composition comprising an effective amount of a compound of formula (I) according to any one of claims 1 to 15 or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, and optionally in combination with a pharmaceutically acceptable carrier, excipient or diluent.

17. A method of treating a patient with coronavirus-related disease or condition, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

18. Use of a compound of formula (I) according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof, in the preparation of a medicament for treating coronavirus-related diseases or conditions.

19. The compound of formula (I) according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, for the treatment of coronavirus-related diseases or conditions.

20. The method, use, or compound according to any one of claims 17 to 19, wherein the compound of formula (I) is a 3CL protease inhibitor.

21. The method, use, or compound according to any one of claims 17 to 20, wherein the coronavirus-related disease or condition is caused by a virus selected from SARS-CoV-1, SARS-CoV-2, MERS-CoV, CoV-229E, and HCoV-OC43.

22. The method, use, or compound according to any one of claims 17 to 21, wherein the compound of formula (I) is characterized in that the protease inhibition assay IC 50 The value is less than 20 µM.

23. The method, use, or compound according to any one of claims 17 to 22, wherein the compound of formula (I) is characterized in the viral cytopathic effect assay EC. 50 The value is less than 20 µM.

24. A method for preparing a compound of formula (I), comprising: i) Reacting compound (II) with compound (III) under dehydration conditions to form an amide bond: (II), (III); ii) To undergo a closed-ring olefin metathesis reaction to form a compound of formula (IV): (IV); and iii) The alkoxyacyl moiety in formula (IV) is converted into the R1 moiety to form the compound of formula (I).

25. The method of claim 24, wherein step ii) further comprises a hydrogenation reaction following the olefin metathesis reaction.

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