WO2022066776A1

WO2022066776A1 - Compositions and methods for treatment of coronavirus infection

Info

Publication number: WO2022066776A1
Application number: PCT/US2021/051564
Authority: WO
Inventors: Florian ERMINI; Joseph Vacca; Leslie J. HOLSINGER; Casey C. LYNCH; Stephen S. Dominy
Original assignee: Cortexyme, Inc.
Priority date: 2020-09-23
Filing date: 2021-09-22
Publication date: 2022-03-31
Also published as: EP4216961A1; AR123583A1; TW202229309A

Abstract

Compounds and methods for the treatment of coronavirus infections are disclosed. Compounds according to Formula I as described herein, containing reactive functional groups for covalent binding to active site residues in target proteases, can be used for treating infection by SARS-CoV-2 and other coronaviruses.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF

CORONAVIRUS INFECTION

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Pat. Appl. No. 63/082,305, filed on September 23, 2020, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Initial symptoms of COVID-19 may include fever, fatigue, dry cough, aches and pains, nasal congestion, runny nose, sore throat, and diarrhea. While approximately 80% of infected patients are expected to recover from the disease without needing special treatment, around 1 in every 6 patients have become seriously ill and developed difficulty breathing and approximately 4.5 million people worldwide have died as of September 2021. Other long-term sequalae have been reported, including neurological and cardiovascular conditions. The risk of serious illness is elevated in senior citizens, as well as in persons having conditions such as high blood pressure, heart problems, and diabetes. Like other coronaviruses, SARS-CoV-2 expresses a viral protease termed 3 -chymotrypsin-like cysteine protease (3CLpro), also referred to as main protease (Mpro), that is understood to be vital for the viral life cycle and replication.

DETAILED DESCRIPTION OF THE INVENTION

[0003] Provided herein are compounds useful as inhibitors of coronavirus main proteases, exhibiting higher potency in enzyme inhibition and increased efficacy in viral replication assays than previously known compounds. The compounds can be used for treatment of infections by SARS-Co-V2 as well as previously known coronaviruses, such as MERS and SARS, or viruses that may emerge in the future. Covalent binding of the compounds to catalytic residues in the target protease provides long occupancy times on the active site and sustained protease inhibition. Because a high level of protease inhibition is often necessary to effectively block viral replication, the sustained protease inhibition is an advantage over the activity of protease inhibitors characterized by rapidly reversible binding. I. Definitions

[0004] As used herein, the term “alkyl,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C1-2, C1-3, Ci-4, C1-5, C1-6, C1-7, C1-8, C1-9, Ci-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, C1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted alkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0005] As used herein, the term “alkoxy,” by itself or as part of another substituent, refers to a group having the formula -OR, wherein R is alkyl.

[0006] As used herein, the term “cycloalkyl,” by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated.

Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbomadiene. When cycloalkyl is a saturated monocyclic C3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted cycloalkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy. The term “lower cycloalkyl” refers to a cycloalkyl radical having from three to seven carbons including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0007] As used herein, the terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0008] As used herein, the term “haloalkyl,” by itself or as part of another substituent, refers to an alkyl group where some or all of the hydrogen atoms are replaced with halogen atoms. As for alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, such as Ci-6. For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some instances, the term “perfluoro” can be used to define a compound or radical where all the hydrogens are replaced with fluorine. For example, perfluoromethyl refers to 1,1,1 -trifluoromethyl .

[0009] As used herein, the term “aryl,” by itself or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15 or Ci6, as well as Ce-io, C6-12, or Ce-14 . Aryl groups can be monocyclic, fused to form bicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted aryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0010] As used herein, the term “heteroaryl,” by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heteroaryl groups can include any number of ring atoms, such as C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4; or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. For example, heteroaryl groups can be Cs-s heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or Cs-s heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms; or C5-6 heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-6 heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms. The heteroaryl group can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3, 5 -isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted heteroaryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0011] The heteroaryl groups can be linked via any position on the ring. For example, pyrrole includes 1-, 2- and 3 -pyrrole, pyridine includes 2-, 3- and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazole includes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and 5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes 2-, 4-, 5- and 6- pyrimidine, pyridazine includes 3- and 4-pyridazine, 1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-, 5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiophene includes 2- and 3 -thiophene, furan includes 2- and 3 -furan, thiazole includes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and 5-isothiazole, oxazole includes 2-, 4- and 5- oxazole, isoxazole includes 3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindole includes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline, isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2- and 4-quinazoline, cinnoline includes 3- and 4-cinnoline, benzothiophene includes 2- and 3 -benzothiophene, and benzofuran includes 2- and 3 -benzofuran.

[0012] Some heteroaryl groups include those having from 5 to 10 ring members and from 1 to 3 ring atoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3, 5 -isomers), thiophene, furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include those having from 5 to 8 ring members and from 1 to 3 heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Some other heteroaryl groups include those having from 9 to 12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, benzofuran and bipyridine. Still other heteroaryl groups include those having from 5 to 6 ring members and from 1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.

[0013] Some heteroaryl groups include from 5 to 10 ring members and only nitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, and cinnoline. Other heteroaryl groups include from 5 to 10 ring members and only oxygen heteroatoms, such as furan and benzofuran. Some other heteroaryl groups include from 5 to 10 ring members and only sulfur heteroatoms, such as thiophene and benzothiophene. Still other heteroaryl groups include from 5 to 10 ring members and at least two heteroatoms, such as imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline, quinazoline, phthalazine, and cinnoline.

[0014] As used herein the term “heterocyclyl,” by itself or as part of another substituent, refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms of N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heterocyclyl groups can include any number of ring atoms, such as, C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms can be replaced with heteroatoms in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocyclyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. The heterocyclyl groups can also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocyclyl groups can be unsubstituted or substituted. Unless otherwise specified, “substituted heterocyclyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=0), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0015] The heterocyclyl groups can be linked via any position on the ring. For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2- azetidine, pyrrolidine can be 1-, 2- or 3 -pyrrolidine, piperidine can be 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or 4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine, piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1- or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine, isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be 2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or 5- isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

[0016] When heterocyclyl includes 3 to 8 ring members and 1 to 3 heteroatoms, representative members include, but are not limited to, pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane and dithiane. Heterocyclyl can also form a ring having 5 to 6 ring members and 1 to 2 heteroatoms, with representative members including, but not limited to, pyrrolidine, piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, and morpholine.

[0017] As used herein, the term “carbonyl,” by itself or as part of another substituent, refers to — C(O)-, i.e., a carbon atom double-bonded to oxygen and bound to two other groups in the moiety having the carbonyl.

[0018] As used herein, the term “amino” refers to a moiety -NR2, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation.

[0019] As used herein, the term “sulfonyl” refers to a moiety -SO2R, wherein the R group is alkyl, haloalkyl, or aryl. An amino moiety can be ionized to form the corresponding ammonium cation. “Alkyl sulfonyl” refers to an amino moiety wherein the R group is alkyl.

[0020] As used herein, the term “hydroxy” refers to the moiety -OH. [0021] As used herein, the term “cyano” refers to a carbon atom triple-bonded to a nitrogen atom (z.e., the moiety -ON).

[0022] As used herein, the term “carboxy” refers to the moiety -C(O)OH. A carboxy moiety can be ionized to form the corresponding carboxylate anion.

[0023] As used herein, the term “amido” refers to a moiety -NRC(O)R or -C(O)NR.2, wherein each R group is H or alkyl.

[0024] As used herein, the term “nitro” refers to the moiety -NO2.

[0025] As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (z.e., O=).

[0026] As used herein, the term “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to a subject. By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof. Pharmaceutical excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, glidants, coatings, sweeteners, flavors and colors.

[0027] As used herein, the term “salt” refers to an acid salt or base salt of an active agent such as a protease inhibitor. Acid salts of basic active agents include mineral acid salts (e.g., salts formed using hydrochloric acid, hydrobromic acid, phosphoric acid, and the like), and organic acid salts (e.g., salts formed using acetic acid, propionic acid, glutamic acid, citric acid, and the like). Quaternary ammonium salts may be formed using reagents such as methyl iodide, ethyl iodide, and the like. It is understood that the pharmaceutically acceptable salts are non-toxic.

[0028] Acidic active agents may be contacted with bases to provide base salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts.

[0029] The neutral forms of the active agents can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner if desired. In some embodiments, the parent form of the compound may differ from various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt forms may be equivalent to the parent form of the compound.

[0030] As used herein, the terms “treat,” “treatment,” and “treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., viral infection), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.

[0031] As used herein the terms “effective amount” and “therapeutically effective amount” refer to a dose of a compound such as a 3CL^pro inhibitor that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman ’s The Pharmacological Basis of Therapeutics, 11^th Edition, 2006, Brunton, Ed., McGraw-Hill; and Remington: The Science and Practice of Pharmacy, 21 ^st Edition, 2005, Hendrickson, Ed., Lippincott, Williams & Wilkins).

[0032] As used herein, the term “subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.

II. Protease Inhibitor Compounds

[0033] Provided herein are compounds according to Formula I:

and pharmaceutically acceptable salts thereof, wherein: R¹ is selected from the group consisting of Ce-io aryl, 5- to 12-membered heteroaryl, and Ci-6 haloalkyl, wherein Ce-io aryl is substituted with one or more R^la, and 5- to 12-membered heteroaryl is optionally substituted with one or more R^lb; each R^la is independently halogen; each R^lb is independently selected from the group consisting of halogen, Ci-3 alkyl, and C1-3 haloalkyl;

R² is selected from the group consisting of pyrrolidinyl, piperidinyl, azepanyl, and -CH2C(O)NH2, wherein pyrrolidinyl, piperidinyl, and azepanyl are optionally substituted with one or more oxo moieties;

R³ is selected from the group consisting of H and Ci-6 alkyl;

R⁴ is selected from the group consisting of Ci-6 alkyl and Ce-io aryl, each of which is optionally substituted with one or more R^4a; each R^4a is independently selected from the group consisting of Ci-6 alkyl, C3-8 cycloalkyl, and Ce-io aryl, each of which is optionally substituted with one or more R^4b; each R^4b is an independently-selected halogen;

R⁵ is selected from the group consisting of H and C1-6 alkyl; or R⁴ and R⁵ are taken together to form monocyclic or bicyclic

5- to 12-membered heterocyclyl, which is optionally substituted with one or more R^4a;

R⁶ is selected from the group consisting of Ce-io aryl, 5- to 12-membered heteroaryl, -OR⁷, -NHR⁷, -NHC(O)OR⁷, and -CHR⁷NHC(O)R⁷, wherein C₆-io aryl, 5- to 12-membered heteroaryl are optionally substituted with one or more R^6a; each R^6a is independently selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, hydroxy, C3-8 cycloalkyl, and Ce-io aryl; or R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with -NHR⁷ or -NHC(O)OR⁷;

R⁷ is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, and Ce-io aryl, each of which is optionally substituted with one or more R^7a; each R^7a is independently selected from the group consisting of halogen, C1-6 alkyl, Ce-io aryl, and C3-8 cycloalkyl, wherein C1-6 alkyl, Ce-io aryl, and C3-8 cycloalkyl are optionally substituted with one or more R^7b; and each R^7b is an independently-selected halogen. [0034] Some embodiments of the present disclosure provide compounds according to Formula I, and pharmaceutically acceptable salts thereof, wherein:

R¹ is selected from the group consisting of Ce-io aryl, 5- to 12-membered heteroaryl, and Ci-6 haloalkyl, wherein Ce-io aryl is substituted with one or more R^la, and 5- to 12-membered heteroaryl is optionally substituted with one or more R^lb; each R^la is independently halogen; each R^lb is independently selected from the group consisting of halogen, Ci-3 alkyl, and C1-3 haloalkyl;

R³ is selected from the group consisting of H and Ci-6 alkyl;

R⁵ is selected from the group consisting of H and C1-6 alkyl;

R⁶ is selected from the group consisting of Ce-io aryl, 5- to 12-membered heteroaryl, -OR⁷, -NHR⁷, and -NHC(O)OR⁷; or R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with -NHR⁷ or -NHC(O)OR⁷;

R⁷ is selected from the group consisting of C1-6 alkyl and Ce-io aryl, each of which is optionally substituted with one or more R^7a; each R^7a is independently selected from the group consisting of C1-6 alkyl, Ce-io aryl, and C3-8 cycloalkyl, each of which is optionally substituted with one or more R^7b; and each R^7b is an independently-selected halogen.

[0035] In some embodiments, R² -CH2C(O)NH2. In some embodiments, R² is pyrrolidinyl, piperidinyl, or azepanyl. In some embodiments, R² is substituted with one or more oxo moieties. Examples of R² groups include, but are not limited to:

wherein subscript m is 1, 2, or 3. In some embodiments, R² is selected from the group consisting of 2-oxopyrrolidin-3-yl, 5-oxopyrrolidin-3-yl , and 2,5-dioxopyrrolidin-3-yl.

[0036] In some embodiments, R¹ is Ci-6 haloalkyl. In such embodiments, R¹ can be, e.g., chloromethyl, dichloromethyl, tri chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, pentachloroethyl, pentafluoroethyl, 1,1, 1,3, 3, 3 -hexachloropropyl, 1,1, 1,3, 3, 3 -hexafluoropropyl, or the like.

[0037] In some embodiments, R¹ is 5- to 12-membered heteroaryl, which is optionally substituted with one or more R^lb. R¹ may be, for example, isoxazolyl, oxazolyl, imidazolyl, pyrazolyl, pyridinyl, oxazinyl, pyrimidinyl, pyrazinyl, or pyridazinyl. In some embodiments, R¹ is pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, or pyrimidin-6-yl. In some embodiments, R¹ is selected from isoxazol-3-yl, pyridin-3-yl, pyridin-4-yl, 2,6-dimethylpyridin-5-yl, and 2- methylpyrimidin-5-yl. In some embodiments, R¹ is selected from isoxazol-3-yl, pyridin-3-yl, pyridin-4-yl, 2,6-dimethylpyridin-5-yl, and 2-methylpyrimidin-5-yl.

[0038] In some embodiments, R¹ is phenyl substituted with 1-5 independently-selected halogen. The moiety -OR¹ may be, for example, 2-halophenoxy; 3-halophenoxy; 4- halophenoxy; 2,3-dihalophenoxy; 2,4-dihalophenoxy; 2,5-dihalophenoxy; 2,6- dihalophenoxy; 3,4-dihalophenoxy; 3,5-dihalophenoxy; 2,3,6-trihalophenoxy; 2,3,5- trihalophenoxy; or 2,3,5,6-tetrahalophenoxy. In some embodiments, each halogen in the halophenoxy moiety may be independently fluoro, chloro, or bromo.

[0039] In some embodiments, R¹ is selected from the group consisting of phenyl, hexafluoroisopropyl, 2,6-dimethylpipiderin-4-yl, 2-methylpyrimidin-5-yl, and isoxazol-3-yl, wherein phenyl is substituted with 1-5 independently-selected halogen.

[0040] In some embodiments, compounds having a structure according to Formula la:

and pharmaceutically acceptable salts thereof, are provided.

[0041] In some embodiments, compounds having a structure according to Formula lb:

and pharmaceutically acceptable salts thereof, are provided, wherein subscript n is an integer ranging from 1 to 5.

[0042] In some embodiments, subscript n is 3. In some embodiments, subscript n is 4. In some embodiments, each R^la is fluorine.

[0043] In some embodiments, R³ is H. In some embodiments, R³ is methyl (-CH3), ethyl (-CH2CH3), //-propyl, isopropyl, //-butyl, isobutyl, .scc-butyl, tert-butyl, //-pentyl, branched pentyl, //-hexyl, branched hexyl. In some embodiments, R⁵ is H. In some embodiments, R⁵ is methyl, ethyl, //-propyl, isopropyl, //-butyl, isobutyl, .scc-butyl, tert-butyl, //-pentyl, branched pentyl, //-hexyl, branched hexyl. In some embodiments, R⁵ is H or -CH3.

[0044] In some embodiments, R⁴ is C1-6 alkyl, which is optionally substituted with one or more R^4a. In some embodiments, R^4a is C3-8 cycloalkyl or halogen- substituted Ce-io aryl. In some embodiments, R⁴ and R⁵ are taken together to form monocyclic or bicyclic

5- to 12-membered heterocyclyl, which is optionally substituted with one or more R^4a. In some embodiments, R⁴ is methyl substituted with cycloalkyl. R⁴ may be, for example, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl. In some embodiments, R⁴ is unsubstituted C1-6 alkyl (e.g., //-propyl, isopropyl, //-butyl, isobutyl, secbutyl, or tert-butyl).

[0045] In some embodiments, R⁶ is selected from the group consisting of indol-2-yl, benzofuran-2-yl, and benzyloxy, each of which is unsubstituted or substituted with one or more R^6a. In some embodiments, R⁶ is unsubstituted indol-2-yl or indol-2-yl substituted with C1-6 alkoxy. In some embodiments, R⁶ is 4-alkoxyindol-2-yl, 5-alkoxyindol-2-yl, 6- alkoxyindol-2-yl, or 7-alkoxyindol-2-yl (e.g, 4-methoxyindol-2-yl, 5-methoxyindol-2-yl, 6- methoxyindol-2-yl, or 7-methoxyindol-2-yl).

[0046] In some embodiments, R⁶ is unsubstituted benzofuran-2-yl or benzofuran-2-yl substituted with C1-6 alkoxy. In some embodiments, R⁶ is 4-alkoxybenzofuran-2-yl, 5- alkoxybenzofuran-2-yl, 6-alkoxybenzofuran-2-yl, or 7-alkoxybenzofuran-2-yl (e.g., 4- methoxybenzofuran-2-yl, 5-methoxybenzofuran-2-yl, 6-methoxybenzofuran-2-yl, or 7- methoxybenzofuran-2-yl).

[0047] In some embodiments, R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with - NHR⁷ or

-NHC(O)OR⁷. For example, R⁵ and R⁶ may be taken together to form oxodihydropyrrolyl, oxodihydropyridinyl, oxodihydroimidazolyl, oxodihydropyrazolyl, oxodihydrotriazolyl, oxodihydropyrazinyl, or oxodihyrotriazinyl. In some embodiments, R⁵ and R⁶ are taken together with the atoms to which they are attached to form 3-oxo-3,4-dihydropyrazin-2-yl or 2-oxo- 1,2-dihydropyri din-3 -yl, each of which is substituted with -NHR⁷ or -NHC(0)0R⁷.

[0048] In some embodiments, R⁶ is -CHR⁷NHC(O)R⁷, wherein R⁷ is Ci-6 alkyl, each of which optionally substituted with halogen. In some embodiments, R⁶ is -OR⁷, wherein R⁷ is C3-8 cycloalkyl, which is optionally substituted with halogen.

[0049] In some embodiments, compounds of Formula I, Formula la, and/or Formula lb and pharmaceutically acceptable salts thereof are provided, wherein:

R³ is H;

R⁴ is unsubstituted C1-6 alkyl (e.g., w-propyl, isopropyl, //-butyl, isobutyl, ec-butyl, or Zc V-butyl), C3-8 cycloalkyl-methyl e.g., cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl), or optionally substituted benzyl e.g., halobenzyl);

R⁵ is H; and

R⁶ is indol-2-yl or benzofuran-2-yl, each of which is optionally substituted with one or more C1-6 alkoxy (e.g., methoxy).

[0050] In some embodiments, compounds of Formula I and/or Formula la and pharmaceutically acceptable salts thereof are provided, wherein:

R¹ is 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl, 2,4- difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 3,4-difluorophenyl, 3,5- difluorophenyl, 2,3,6-trifluorophenyl, 2,3,5-trifluorophenyl, 2,3,5,6-tetrafluorophenyl, 1,1, 1,3, 3, 3 -hexafluoroisopropyl, 2,6-dimethylpipiderin-4-yl, or isoxazol-3-yl;

R³ is H; R⁴ is unsubstituted Ci-6 alkyl (e.g., w-propyl, isopropyl, //-butyl, isobutyl, ec-butyl, or tert-butyl), C3-8 cycloalkyl-methyl (e.g., cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl), or optionally substituted benzyl (e.g., halobenzyl);

R⁵ is H; and

[0051] In some embodiments, compounds of Formula I and pharmaceutically acceptable salts thereof are provided, wherein:

R² is

subscript m is 1, 2, or 3;

R¹ is 2-fluorophenyl, 3 -fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl, 2,4- difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 3,4-difluorophenyl, 3,5- difluorophenyl, 2,3,6-trifluorophenyl, 2,3,5-trifluorophenyl, 2,3,5,6-tetrafluorophenyl, 1,1, 1,3, 3, 3 -hexafluoroisopropyl, 2,6-dimethylpipiderin-4-yl, or isoxazol-3-yl;

R³ is H;

R⁴ is unsubstituted C1-6 alkyl (e.g., w-propyl, isopropyl, //-butyl, isobutyl, sec-butyl, or tert-butyl), C3-8 cycloalkyl-methyl (e.g., cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl), or optionally substituted benzyl (e.g., halobenzyl);

R⁵ is H; and

[0052] In some embodiments, compounds of Formula I and pharmaceutically acceptable salts thereof are provided, wherein:

R² is 2-oxopyrrolidin-3-yl,

R³ is H;

R⁴ is isobutyl or cyclopropylmethyl; R⁵ is H; and

R⁶ is indol-2-yl or 4-methoxyindol-2-yl.

[0053] In some embodiments, the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

[0054] In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

[0056] In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

[0057] Compounds according to the present disclosure may be further substituted. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents are generally those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In general, “substituted,” as used herein, does not encompass replacement and/or alteration of a key functional group by which a molecule is identified, e.g., such that the “substituted” functional group becomes, through substitution, a different functional group. For example, a “substituted phenyl” group must still comprise the phenyl moiety and cannot be modified by substitution, in this definition, to become, e.g., a cyclohexyl group.

[0058] Examples of suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R^a; -(CH2)o-40R^a; -0(CH₂)o-4R^a, -0-(CH₂)o-4C(0)OR^a; -(CH₂)o-4CH(OR^a)₂; -(CH₂)o-4SR^a; -(CH₂)o-4Ph, wherein Ph is phenyl which may be substituted with R^a; -(CH2)o-40(CH2)o-iphenyl, which phenyl may be substituted with R^a; -CH=CHPh, wherein Ph is phenyl which may be substituted with R^a; -(CH2)o-40(CH2)o-i-Py, wherein Py is pyridyl which may be substituted with R^a; -NO₂; -CN; -N₃; -(CH₂)o-4N(R^a)₂; -(CH₂)o-4N(R^a)C(0)R^a; -N(R^a)C(S)R^a; -(CH₂)o- ₄N(R^a)C(O)NR^a ₂; -N(R^a)C(S)NR^a ₂; -(CH₂)o-4N(R^a)C(0)OR^a; -N(R^a)N(R^a)C(O)R^a; -N(R^a)N(R^a)C(O)NR^a ₂; -N(R^a)N(R^a)C(O)OR^a; -(CH₂)o-4C(0)R^a; -C(S)R^a;

-(CH₂)o-4C(0)OR^a; -(CH₂)o-4C(0)SR^a; -(CH₂)o-4C(0)OSiR^a ₃; -(CH₂)o-40C(0)R^a; -OC(0)(CH₂)o-4SR-SC(S)SR^a; -(CH₂)o-4SC(0)R^a; -(CH₂)o-4C(0)NR^a ₂; -C(S)NR^a ₂, -C(S)SR^a; -SC(S)SR^a, -(CH₂)o-40C(0)NR^a ₂; -C(O)N(OR^a)R^a; -C(O)C(O)R^a; -C(O)CH₂C(O)R^a; -C(NOR^a)R^a; -(CH₂)o-₄SSR^a; -(CH₂)o-₄S(0)₂R^a; -(CH₂)o-₄S(0)₂OR^a; -(CH₂)o-₄OS(0)₂R^a; -S(O)₂NR^a ₂; -(CH₂)o-₄S(0)R^a; -N(R^a)S(O)₂NR^a ₂; -N(R^a)S(O)₂R^a; -N(OR^a)R^a; -C(NH)NR^a ₂; -P(O)₂R^a; -P(O)R^a ₂; -OP(O)R^a ₂; -OP(O)(OR^a)₂; -SiR^a ₃;

-(Ci-4 straight or branched)alkylene)-O-N(R^a)₂; or -(Ci-₄ straight or branched)alkylene)- C(O)O-N(R^a)₂. Each R^a is independently hydrogen; Ci-s alkyl; -CH₂Ph, -0(CH₂)o-iPh; -CH₂-(5- to 6-membered heteroaryl); C₃-8 cycloalkyl; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl; and each R^a may be further substituted as described below.

[0059] Examples of suitable monovalent substituents on R^a are independently halogen, -₂C(0)R^P;

t or branched alkylene)C(O)OR^p; or -SSR^P; wherein each R¹’ is independently selected from Ci-4 alkyl; -CH₂Ph; -0(CH₂)o-iPh; C₃-s cycloalkyl; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl. Suitable divalent substituents on a saturated carbon atom of R^a include =0 and =S.

[0060] Examples of suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0; =S; =NNR⁷?; =NNHC(0)R^y; =NNHC(0)0R^y; =NNHS(O)₂R ; =NR⁷; =NOR⁷; -O(C(R ₂))_2-3O-; or -S(C(R ₂))_2-3S-; wherein each independent occurrence of R^y is selected from hydrogen; C1-8 alkyl, which may be substituted as defined below; C₃-s cycloalkyl; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR^p ₂)_2-3O-; wherein each independent occurrence of R¹’ is selected from hydrogen; C1-8 alkyl which may be substituted as defined below; C₃-s cycloalkyl; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

[0061] Examples of suitable substituents on the alkyl group of R⁷ include halogen; -R⁵; -OH; -OR⁵; -CN;-C(O)OH; -C(O)OR⁵; -NH₂; -NHR⁵; -NR⁵ ₂; or -NO₂; wherein each R⁵ is independently Ci-₄ alkyl; -CH?Ph; -0(CH₂)o-iPh; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl. [0062] Examples of suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R^E; -NR^E ₂; -C(O)R^E; -C(O)OR^E; -C(O)C(O)R^E;

-C(O)CH₂C(O)R^E; -S(O)₂R^E; -S(O)₂NR^E ₂; -C(S)NR^E ₂; -C(NH)NR^E ₂; or -N(R^E)S(O)₂R^E; wherein each R^E is independently hydrogen; Ci-s alkyl which may be substituted as defined below; C3-8 cycloalkyl; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

[0063] Examples of suitable substituents on the alkyl group of R^E are independently halogen; -R⁵; -OH; -OR⁵; -CN; -C(O)OH; -C(O)OR⁵; -NH₂; -NHR⁵; -NR⁵ ₂; or -NO₂; wherein each R⁵ is independently Ci-4 alkyl; -CH₂Ph; -0(CH₂)o-iPh; Ce-io aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

[0064] Compounds according to the present disclosure may be prepared as summarized in Scheme 1 and as described in the examples below. A protected aspartic acid (i) can be reacted with bromoacetonitrile and cyclized to form the corresponding oxopyrrolidone (iii), followed by installation of an a-halomethylene group which is displaced by an alcohol to afford oxymethylketone (v). Deprotection results in amine (vi) which can be further elaborated in subsequent acylation steps.

Scheme 1

[0065] The starting materials and reagents used in preparing the compounds of the present disclosure are either available from commercial suppliers or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Vol. 1-28 (Wiley, 2016); March’s Advanced Organic Chemistry, 7^th Ed. (Wiley, 2013); and Larock’s Comprehensive Organic Transformations, 2^nd Ed. (Wiley, 1999). The starting materials and the intermediates of the reaction can be isolated and purified if desired using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including measuring physical constants and obtaining spectral data.

[0066] The acylation steps may be conduction with one or more coupling agents include for example, carbodiimides (e.g., N,N '-dicyclohexylcarbodiimide (DCC), N,N- dicyclopentylcarbodiimide, A,A'-diisopropylcarbodiimide (DIC), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), etc.), phosphonium salts (HOBt, PyBOP, HO At, etc.), aminium/uronium salts (e.g., pyrimidinium uronium salts such HATU, tetramethyl aminium salts, bispyrrolidino aminium salts, bispiperidino aminium salts, imidazolium uronium salts, uronium salts derived from N, N, A'-trimethyl-A '-phenylurea, morpholino-based aminium/uronium coupling reagents, antimoniate uronium salts, etc.), organophosphorus reagents (e.g., phosphinic and phosphoric acid derivatives), organosulfur reagents (e.g., sulfonic acid derivatives), triazine coupling reagents (e.g., 2- chloro-4,6-dimethoxy-l,3,5- triazine, 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4 methylmorpholinium chloride, 4-(4,6- dimethoxy-l,3,5-triazin-2-yl)-4 methylmorpholinium tetrafluoroborate, etc.), pyridinium coupling reagents (e.g., Mukaiyama’s reagent, pyridinium tetrafluoroborate coupling reagents, etc.), polymer-supported reagents (e.g., polymer-bound carbodiimide, polymer- bound TBTU, polymer-bound 2,4,6-trichloro-l,3,5-triazine, polymer-bound HOBt, polymer- bound HOSu, polymer-bound IIDQ, polymer-bound EEDQ, etc.), and the like. Alternatively, acylation can be conducted using an activated carboxylic acid derivative such as an acid anhydride, a mixed anhydride an acid chloride, or an activated ester (e.g., a pentafluorophenyl ester or an A-hydroxysuccinimidyl ester).

[0067] Amines and other functional groups can be protected to prevent unwanted side reactions during various synthetic steps. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyl oxy carbonyl (Boc); allyloxycarbonyl (Alloc); -toluene sulfonyl (Tos); 2,2, 5,7, 8- pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5- sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamido; phthalimido; and the like. Examples of hydroxyl protecting groups include, but are not limited to, benzyl; tert-butyl; trityl; te/7-butyl di methyl silyl (TBDMS; TBS); 4,5- dimethoxy-2-nitrobenzyloxycarbonyl (Dmnb); propargyloxycarbonyl (Poc); and the like. Other alcohol protecting groups and amine protecting groups are known to those of skill in the art including, for example, those described by Green and Wuts (Protective Groups in Organic Synthesis, 4^th Ed. 2007, Wiley-Interscience, New York). The protecting groups can be removed using standard conditions so as to restore the original functional groups for further synthetic elaboration.

[0068] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure over a temperature range of from about -78 °C to about 250 °C. For example, reactions can be conducted at from about 0 °C to about 125 °C, or at about room (or ambient) temperature, e.g., about 20 °C. In some embodiments, reactions are conducted at about 0 °C, 20 °C, 25 °C, 90 °C, 100 °C, 110 °C, 125 °C, 150 °C, 175 °C, or 200 °C. In some embodiments, reactions are conducted starting at a first temperature (e.g., about -78 °C or about 0 °C), and allowed to warm to a higher second temperature (e.g., about 20 °C or about 25 °C). One of skill in the art will appreciate that various modifications to the procedures described herein can be made.

III. Pharmaceutical Compositions

[0069] In some embodiments, the 3CL^pro inhibitor is administered as a pharmaceutical composition containing at least one pharmaceutically acceptable excipient and the 3CL^pro inhibitor or a pharmaceutically acceptable salt thereof. A 3CL^pro inhibitor may be administered to the subject before administration of one or more additional actives, after administration of one or more additional actives, or concurrently with administration of one or more additional actives. A 3CL^pro inhibitor may be administered in a composition separate from one or more additional actives, or in a composition containing one or more additional actives. Also provided herein are compositions containing: (i) one or more 3CL^pro inhibitors; (ii) one or more pharmaceutically acceptable excipients; and optionally (iii) optionally one or more additional active agents, each of which is independently an anti-inflammatory agent, an analgesic agent, an antiviral agent, an antitussive agent, or a CYP3 A4 inhibitor. The compositions may be formulated, e.g., for oral administration, intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intrathecal administration, intraarterial administration, intranasal administration, or rectal administration.

[0070] The pharmaceutical compositions can be prepared by any of the methods well known in the art of pharmacy and drug delivery. In general, preparation of the compositions includes the step of bringing the active ingredients into association with a carrier containing one or more accessory ingredients. The pharmaceutical compositions are typically prepared by uniformly and intimately bringing the active ingredients into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. The compositions can be conveniently prepared and/or packaged in unit dosage form.

[0071] The pharmaceutical compositions may be in a form suitable for oral use. Suitable compositions for oral administration include, but are not limited to, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs, solutions, buccal patches, oral gels, chewing gums, chewable tablets, effervescent powders, and effervescent tablets. Such compositions can contain one or more agents selected from sweetening agents, flavoring agents, coloring agents, antioxidants, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

[0072] Tablets generally contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients, including: inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as corn starch and alginic acid; binding agents, such as polyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG), starch, gelatin, and acacia; and lubricating agents such as magnesium stearate, stearic acid, and talc. The tablets can be uncoated or coated, enterically or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Tablets can also be coated with a semi-permeable membrane and optional polymeric osmogents according to known techniques to form osmotic pump compositions for controlled release. Compositions for oral administration can be formulated as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules wherein the active ingredients are mixed with water or an oil medium (such as peanut oil, liquid paraffin, or olive oil).

[0073] The pharmaceutical compositions can also be in the form of an injectable aqueous or oleaginous solution or suspension. Sterile injectable preparations can be formulated using non-toxic parenterally-acceptable vehicles including water, Ringer’s solution, and isotonic sodium chloride solution, and acceptable solvents such as 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0074] Aqueous suspensions contain the active agents in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include, but are not limited to: suspending agents such as sodium carboxymethylcellulose, methylcellulose, oleagino- propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin, polyoxyethylene stearate, and polyethylene sorbitan monooleate; and preservatives such as ethyl, w-propyl, and p- hydroxybenzoate. Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin, or cetyl alcohol. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. Dispersible powders and granules (suitable for preparation of an aqueous suspension by the addition of water) can contain the active ingredients in admixture with a dispersing agent, wetting agent, suspending agent, or combinations thereof.

[0075] The pharmaceutical compositions of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, such as gum acacia or gum tragacanth; naturally-occurring phospholipids, such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. [0076] Transdermal delivery can be accomplished by means of iontophoretic patches and the like. The active ingredients can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the active agents with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

IV. Methods for Treatment of Coronavirus Infection

[0077] Also provided herein are methods for the treatment of coronavirus infections. The infection may be, but is not limited to, a SARS-CoV-2 infection, a SARS-CoV infection, a MERS-CoV infection, a HCoV-229E infection, a HCoV-OC43 infection, or a HCoV-NL63 infection. The methods include administering a therapeutically effective amount of a compound as described above to a subject in need thereof. In some embodiments, the subject has coronavirus disease 2019 (COVID-19). The compound may act as 3CL^pro inhibitor so as to slow or stop the replication of SARS-CoV-2 in the subject.

[0078] In some embodiments, the subject is a human, an agricultural animal (e.g., livestock such as cows, sheep, pigs, or the like), or a companion animal (e.g., a pet such as a cat or dog). In some embodiments, the subject is a human over the age of 50 years old.

[0079] 3CL^pro inhibitor compounds according to the present disclosure, as well as other active agents employed in combination therapy as described herein, can be administered to subject orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intrathecally, intraarterially, intranasally, rectally, or via other routes if indicated. In some embodiments, the 3CL^pro inhibitor is administered orally or via injection. Active agents can be administered at any suitable dose in the methods provided herein. In general, a 3CL^pro inhibitor or other active agent (e.g., a CYP3 A4 inhibitor) is administered at a dose ranging from about 0.1 milligrams to about 1000 milligrams per kilogram of a subject’s body weight (i.e., about 0.1-1000 mg/kg). The dose of the 3CL^pro inhibitor compound can be, for example, about 0.1-1000 mg/kg, or about 1-500 mg/kg, or about 25-250 mg/kg, or about 50- 100 mg/kg. The dose of the 3CL^pro inhibitor can be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg/kg. In some embodiments, the 3CL^pro inhibitor is administered in an amount ranging from about 0.1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the 3CL^pro inhibitor is administered in an amount ranging from about 0.1 mg/kg/day to about 1.0 mg/kg/day. In some embodiments, a subject is administered one or more (e.g., two or three) 3CL^pro inhibitor doses of 10-100 mg (e.g., 15-40 mg, or 45-85 mg) per day. In some embodiments, the total daily dose of the 3CL^pro inhibitor ranges from about 10 to about 100 mg (e.g., 15-40 mg, or 45-85 mg) per day.

[0080] The dosages can be varied depending upon the requirements of the patient, the severity of the infection, the route of administration, and the particular formulation being administered. The dose administered to a patient should be sufficient to result in a beneficial therapeutic response in the patient. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of the drug in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the typical practitioner. The total dosage can be divided and administered in portions over a period of time suitable to treat to the CO VID-19.

[0081] A 3CL^pro inhibitor or other active agent can be administered for periods of time which will also vary depending upon the severity of the CO VID-19, and the overall condition of the subject to whom the active agent is administered. Administration can be conducted, for example, hourly, every 2 hours, three hours, four hours, six hours, eight hours, or twice daily including every 12 hours, or any intervening interval thereof. Administration can be conducted once daily, or once every 36 hours or 48 hours, once per week, twice per week, or three times per week. Following treatment, a subject can be monitored for changes in their condition and for alleviation of the symptoms of COVID-19. The dosage of the active agent can either be increased in the event the subject does not respond significantly to a particular dosage level, or the dose can be decreased if an alleviation of symptoms is observed, or if the CO VID-19 has been remedied, or if unacceptable side effects are seen with a particular dosage. Treating COVID-19 according to the methods of present disclosure can include alleviating one or more symptoms including, but not limited to, fever, cough, and shortness of breath. In some embodiments, treating COVID-19 can prevent severe, life-threatening illnesses such as pneumonia.

[0082] The methods and compositions described herein also can be administered prophylactically in subjects at risk for infection with SARS-CoV-2, to reduce the risk of developing COVID-19.

[0083] In some embodiments, the levels of SARS-CoV-2 3CL^pro activity in a subject may be reduced by from about 25% to about 95% upon treatment of a subject according to the methods of the present disclosure. For example, 3CL^pro activity in the subject may be reduced by from about 35% to about 95%, or from about 40% to about 85%, or from about 40% to about 80% as compared to the corresponding levels of 3CL^pro activity prior to the first administration of the active agent (e.g., 24 hours prior to the first administration of the active agent).

[0084] In some embodiments, the methods further include administering an analgesic agent (including anti-inflammatory analgesic agents), an antiviral agent, and an antitussive agent, or a combination thereof to the subject. Examples of non-steroidal anti-inflammatory agents (NSAIDs) include, but are not limited to, aceclofenac, 5-amino salicylic acid, aspirin, celecoxib, dexibuprofen, diclofenac, diflunisal, etodolac, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, loxoprofen, mefenamic acid, nabumetone, naproxen, nimesulide, sulindac, and pharmaceutically acceptable salts thereof. NSAIDs can be effective for relieving symptoms such as fever and pain. Additional analgesic agents such as paracetamol (acetaminophen) may also be administered in conjunction with the 3CL^pro inhibitor. Examples of further antiviral agents include, but are not limited to, protease inhibitors (e.g., ritonavir, lopinavir, saquinavir, indinavir, or the like), nucleic acid polymerase inhibitors (e.g., acyclovir, foscarnet, ganciclovir, ribavirin or the like), interferons, antibodies or other biologies targeting coronavirus binding or entry, and other small molecules useful in the treatment of coronaviruses. Examples of antitussive agents include, but are not limited to, codeine, hydrocodone, benzonatate, dextromethorphan, and chi ophedi anol. In some embodiments, the methods include administering a CYP3A4 inhibitor to the subject. Administration of the CYP3A4 inhibitor can prevent premature metabolism of the 3CL^pro inhibitor and increase plasma concentration levels following administration orally or via another route. Examples of CYP3 A4 inhibitors include, but are not limited to, aprepitant, azamulin, boceprevir, chlorzoxazone, cilostazol, cimetidine, ciprofloxacin, clotrimazole, cobicistat, conivaptan, crizotinib, cyclosporine, diltiazem, dronedarone, erythromycin, fluconazole, fluvoxamine, fosaprepitant, grapefruit juice, imatinib, istradefylline, itraconazole, ivacaftor, ketoconazole, lomitapide, posaconazole, ranitidine, ranolazine, ritonavir (optionally administered with danoprevir, dasabuvir, elvitegravir, indinavir, lopinavir, ombitasvir, paritaprevir, saquinavir, tipranavir, and combinations thereof), telaprevir, telithromycin, ticagrelor, tofisopam, troleandomycin, verapamil, and voriconazole. It will be appreciated that an active agent may be classified in more than one category; ritonavir, for example, can be classified as a protease inhibitor and a CYP3A4 inhibitor.

[0085] Also provided herein are methods for inhibiting SARS-CoV-2 3CL^pro. The methods include contacting the 3CL^pro with an effective amount of a compound as described herein. Inhibiting the 3CL^pro generally includes contacting the 3CL^pro with an amount of the protease inhibitor sufficient to reduce the activity of the 3CL^pro as compared to the 3CL^pro activity in the absence of the protease inhibitor. For example, contacting the 3CL^pro with the protease inhibitor can result in from about 1% to about 99% 3CL^pro inhibition (i.e., the activity of the inhibited 3CL^pro ranges from 99% to 1% of the 3CL^pro activity in the absence of the compound). The level of 3CL^pro inhibition can range from about 1% to about 10%, or from about 10% to about 20%, or from about 20% to about 30%, or from about 30% to about 40%, or from about 40% to about 50%, or from about 50% to about 60%, or from about 60% to about 70%, or from about 70% to about 80%, or from about 80% to about 90%, or from about 90% to about 99%. The level of 3CL^pro inhibition can range from about 5% to about 95%, or from about 10% to about 90%, or from about 20% to about 80%, or from about 30% to about 70%, or from about 40% to about 60%. In some embodiments, contacting the 3CL^pro with a protease inhibitor as described herein will result in complete (i.e., 100%) 3CL^pro inhibition. Inhibiting 3CL^pro according to the methods of the present disclosure may occur in vitro or in vivo (e.g., following administration of a protease inhibitor to a subject in the course of treating CO VID-19).

V. Examples

Example 1. Preparation of \-((.S')-3-Cyclopropyl-l-oxo-l-(((.S')-3-oxo-l-((.S')-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2- yl)benzofuran-2-carboxamide (101)

[0086] Compound 2: To a mixture of Compound 1 (10 g, 36.32 mmol, 1 eq) in THF (100 mL) was added dropwise LiHMDS (1 M, 78.45 mL, 2.16 eq) at -78°C under N2. The mixture was stirred at -78°C for 1 h. Then Compound 1A (6.53 g, 54.48 mmol, 3.63 mL, 1.5 eq) was added dropwise to the mixture. The mixture was stirred at -78°C for 2 h. TLC (Petroleum ether: Ethyl acetate=l : 1 Rf = 0.43) showed that the reaction was completed. To the mixture was added MeOH (30 mL), then the mixture was poured into a mixed solution (5 mL HOAc/40 mL THF), then the resulting mixture was stirred at 30 min in -78°C. The mixture was poured into water (100 mL) and ethyl acetate (100 mL). The combined organic phase was washed with brine (100 mL><3), dried with anhydrous Na2SO₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=l/l to 0/1). Compound 2 (9 g, 28.63 mmol, 78.83% yield) was obtained as yellow oil.

[0087] Compound 3: To a mixture of Compound 2 (15.37 g, 48.90 mmol, 1 eq) in MeOH (160 mL) was added COCI2.6H2O (6.98 g, 29.34 mmol, 0.6 eq) at 0 °C. Then NaBH₄ (11.15 g, 294.85 mmol, 6.03 eq) was added to the mixture at 0°C. The mixture was stirred at 25 °C for 12 hrs. TLC (Petroleum ether/Ethyl acetate = 0: 1, Rf = 0.43) indicated one new spot formed. The reaction mixture was quenched by addition of NH4Q (200mL), and then the mixture was filtered and extracted with DCM 300 mL (100 mL *3). The combined organic layers were washed with saturated NaHCOs aqueous (200 mL), saturated NaiSCh aqueous (300 mL) and saturated brines (100 mL), dried over Na2SO₄, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si O2, Petroleum ether/Ethyl acetate = 1/1 to 0/1). Compound 3 (8 g, 27.94 mmol, 57.14% yield) was obtained as a yellow oil. ¹ H NMR (400 MHz, chloroform-t/) 3 4.30 - 4.48 (m, 1 H) 3.60 - 3.84 (m, 3 H) 3.24 - 3.42 (m, 2 H) 2.36 - 2.54 (m, 2 H) 2.06 - 2.24 (m, 1 H) 1.69 - 1.94 (m, 3 H) 1.44 (s, 9 H).

[0088] Compound 4: To a solution of DIPA (14.00 g, 138.31 mmol, 19.55 mL, 5.5 eq) in THF (50 mL) was added n-BuLi (2.5 M, 55.32 mL, 5.5 eq) at 0 °C. The mixture was stirred at 0 °C for 1 hr. And then the solution was added into a solution of Compound 3 (7.2 g, 25.15 mmol, 1 eq) and chloro(iodo)methane (24.39 g, 138.31 mmol, 10.04 mL, 5.5 eq) in

THF (10 mL) at -78 °C and the mixture was stirred at -78 °C for 3 hr. TLC (Petroleum ether: Ethyl acetate = 0: 1, Rf = 0.45) showed that the reaction was completed. The reaction mixture was quenched by addition of saturated aqueous NH4Q (30 mL), and then extracted with Ethyl acetate 300 mL (100 mL x 3). The combined organic layers were washed with saturated aqueous NaiSCh (30 mL), saturated aqueous NaHCCh (30 mL) and saturated brines (30 mL), dried over Na2SO4, filtered and the filtrate was concentrated under concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCb, Petroleum ether/Ethyl acetate=l/l to 0/1). Compound 4 (1.3 g, 4.27 mmol, 16.96% yield) was obtained as yellow oil.

4 5

[0089] Compound 5: To a solution of Compound 4 (1 g, 3.28 mmol, 1 eq) in DMF (10 mL) was added K2CO3 (906.96 mg, 6.56 mmol, 2 eq), KI (1.09 g, 6.56 mmol, 2 eq) and Compound 5A (817.37 mg, 4.92 mmol, 1.5 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hr. TLC (Petroleum ether: Ethyl acetate = 1 : 1, Rf = 0.43) showed the reaction was completed. The residue was diluted with H2O (20 mL), and extracted with EtOAc 90 mL (30 mL x 3). The combined organic layers were washed with brine 60 mL (20 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si O2, Petroleum ether/Ethyl acetate = 3/1 to 1/1). Compound 5 (1.3 g) was obtained as yellow oil. ’H NMR (400 MHz, methanol-d4) 3 7.03 - 7.20 (m, 1 H) 4.97 - 5.50 (m, 2 H) 4.23 - 4.39 (m, 1 H) 3.33 - 3.37 (m, 2 H) 2.29 - 2.52 (m, 2 H) 1.95 - 2.01 (m, 1 H) 1.72 - 1.92 (m, 2 H) 1.45 (s, 10 H)

5 6

[0090] Compound 6: A mixture of Compound 5 (800.00 mg, 1.84 mmol, 1 eq) in TFA (1 mL) and DCM (5 mL) was stirred at 25 °C. The mixture was stirred at 25°C for 1 hr. TLC (Petroleum ether: Ethyl acetate = 3 : 1, Rf = 0.43) showed a new spot was formed and the reaction was completed. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 6 ((5)-3-((5)-2-amino-3-oxo-4-(2, 3,5,6- tetrafluorophenoxy)butyl)pyrrolidin-2-one, 1.2 g, crude, TFA) was obtained as yellow oil.

[0091] Compound 8: To a stirred solution of Compound 7 (1.5 g, 11.61 mmol, 1 eq) in MeOH (15 mL) under N2 was added SOC12 (4.15 g, 34.84 mmol, 2.53 mL, 3 eq) at 0 °C dropwise. The resulting mixture was stirred at 70°C for 3 h. The reaction mixture was concentrated under reduced pressure to afford a yellow solid. And the solid was triturated with petroleum ether (10 mL><2) to afford an off-white solid. And the solid was used to next step without further purification. Compound 8 (1.95 g, 10.85 mmol, 93.46% yield, HC1) was obtained as an off-white solid. 1H NMR (400 MHz, methanol-d4) <5 = 4.16 - 4.07 (m, 1H), 3.85 (s, 3H), 1.97 - 1.85 (m, 1H), 1.83 - 1.70 (m, 1H), 0.88 - 0.72 (m, 1H), 0.63 - 0.54 (m, 2H), 0.22 - 0.12 (m, 2H).

9

8

[0092] Compound 9: To a solution of Compound 8 (0.2 g, 1.40 mmol, 1 eq) in DMF (2 mL) was added HATU (798.48 mg, 2.10 mmol, 1.5 eq) , Compound 8A (227.00 mg, 1.40 mmol, 1 eq) and DIPEA (361.87 mg, 2.80 mmol, 487.70 pL, 2 eq), the mixture was stirred at 25°C for 12 hr. TLC (Petroleum ether: Ethyl acetate = 3 : 1, Rt = 0.84) showed a new spot was formed and the reaction was completed. The residue was poured into water (3 mL) and ethyl acetate (3 mL). The aqueous phase was extracted with ethyl acetate (1 mL><3). The combined organic phase was washed with brine (1 mL><3), dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCb, Petroleum ether/Ethyl acetate=l/l to 3/1). Compound 9 (0.3 g, 1.04 mmol, 74.58% yield) was obtained as yellow oil. ^XH NMR (400 MHz, methanol-t/4) d 7.73 (d, J= 7.9 Hz, 1 H) 7.61 (d, J= 8.4 Hz, 1 H) 7.53 (s, 1 H) 7.43 - 7.49 (m, 1 H) 7.24 - 7.40 (m, 1 H) 4.73 (dd, J=8.4, 5.6 Hz, 1 H) 3.76 (s, 3 H) 1.69 - 2.00 (m, 2 H) 0.80 - 0.95 (m, 1 H) 0.41 - 0.55 (m, 2 H) -0.01 - 0.29 (m, 2 H)

9 10

[0093] Compound 10: To a solution of Compound 9 (0.28 g, 974.56 pmol, 1 eq) in MeOH (2.5 mL) and H2O (0.5 mL) was added LiOH.H2O (81.78 mg, 1.95 mmol, 2 eq) at 25°C. The mixture was stirred at 25°C for Ihr. TLC (Petroleum ether: Ethyl acetate = 1 : 1, Rf = 0.26) showed a new spot was formed and the reaction was completed. The mixture was adjust to pH = 8 with IN HC1. Then the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O 6 mL (2 mL x 3) and extracted with EtOAc 12 mL (4 mL x 3). The combined organic layers were dried over Na2SO₄, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 10 (0.2 g, 731.84 pmol, 75.09% yield) was obtained as yellow oil. ^XH NMR (400 MHz, methanol-t/4) 3 7.74 (d, J= 7.9 Hz, 1 H) 7.62 (d, J= 8.3 Hz, 1 H) 7.53 (s, 1 H) 7.44 - 7.49 (m, 1 H) 7.22 - 7.40 (m, 1 H) 4.72 (dd, J = 8.3, 5.5 Hz, 1 H) 1.75 - 1.97 (m, 2 H) 0.79 - 0.91 (m, 1 H) 0.46 - 0.59 (m, 2 H) 0.04 - 0.25 (m, 2 H)

[0094] A-((5)-3-Cyclopropyl-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-3-yl)-4- (2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)benzofuran-2-carboxamide (101): To a solution of Compound 10 (81.39 mg, 181.57 pmol, TFA) in DMF (2 mL) was added HATU (125.22 mg, 329.33 pmol, 1.5 eq) at 0°C. Compound 6 (0.06 g, 219.55 pmol, 1 eq) and DIPEA (85.12 mg, 658.66 pmol, 114.72 pL, 3 eq) was added to the mixture at 25°C. The mixture was stirred at 25°C for 12hr. The reaction mixture was partitioned between FEO (12 mL) and EtOAc (9 mL). The organic phase was separated, washed with brine 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75x30mmx3pm; mobile phase: [water (0.05% NH3H2O + 10mM NH₄HCO₃)-ACN]; B%: 40%-60%, 8min). Compound 101 (29 mg, 49.19 pmol, 22.41% yield, 100% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C29H28F4N3O6: 590.2; found 590.2; RT = 3.006 min. The observed isomeric ratio of isomer, as determined by supercritical fluid chromatography (SFC), was 2.45: 10.58: 1. ^TH NMR (400 MHz, chloroform-t/) 3 8.59 - 9.26 (m, 1 H) 7.62 - 7.70 (m, 1 H) 7.52 (d, J= 8.1 Hz, 1 H) 7.46 (s, 1 H) 7.35 - 7.45 (m, 2 H) 7.30 (d, J= 7.9 Hz, 1 H) 6.71 - 6.88 (m, 1 H) 5.79 - 6.18 (m, 1 H) 5.00 - 5.26 (m, 2 H) 4.66 - 4.93 (m, 2 H) 3.30 - 3.43 (m, 2 H) 2.38 - 2.56 (m, 2 H) 1.99 - 2.17 (m, 2 H) 1.82 - 1.95 (m, 3 H) 0.71 - 0.92 (m, 1 H) 0.55 (br d, J= 7.9 Hz, 2 H) 0.09 - 0.28 (m, 2 H).

Example 2. Preparation of Benzyl ((5)-4-methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2- yl)carbamate (102)

11 12

[0095] Compound 12: To a solution of Compound 5 (1.88 g, 7.09 mmol, 1 eq) in DMF (20 mL) was added EDCI (1.70 g, 8.87 mmol, 1.25 eq) and HOBt (1.20 g, 8.87 mmol, 1.25 eq) at 25°C, the solution was stirred at 25°C for 0.5 hr. This solution was named as A. To a solution of Compound 11 (2.13 g, 7.09 mmol, 1 eq, TFA) in DMF (5 mL) was added DIEA (3.67 g, 28.38 mmol, 4.94 mL, 4 eq) at 0°C in the separate flask, the solution was stirred at 0°C for 0.5 hr. This solution was named as B. Then solution B was added to the solution A. The mixture was stirred at 25°C for 16 hrs. The solution was poured into water (100 mL) and ethyl acetate (100 mL). The resulting mixture was extracted with ethyl acetate (100 mL><3). The combined organic phase was washed with brine (100 mL><3), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether: Ethyl acetate = 1/0 to 1/1) to afford Compound 12 (1.7 g, 3.92 mmol, 55.28% yield) as a white solid. ^XH NMR (400 MHz, chloroform-t/) 3 7.88 (br d, J=6.4 Hz, 1 H) 7.28 - 7.41 (m, 5 H) 6.06 (s, 1 H) 5.39 (br d, J=8.4 Hz, 1 H) 5.10 (s, 2 H) 4.43 - 4.53 (m, 1 H) 4.26 - 4.37 (m, 1 H) 3.64 - 3.81 (m, 3 H) 3.29 - 3.39 (m, 2 H) 2.34 - 2.50 (m, 1 H) 2.10 - 2.25 (m, 1 H) 1.81 - 1.97 (m, 2 H) 1.62 - 1.76 (m, 3 H) 1.44 - 1.59 (m, 1 H) 0.83 - 1.04 (m, 6 H).

[0096] Compound 13: To a mixture of DIPA (1.28 g, 12.69 mmol, 1.79 mL, 5.5 eq) in THF (3 mL) was added n-BuLi (2.5 M, 5.08 mL, 5.5 eq) at 0°C. The mixture was stirred at 0°C for 60 min. The mixture was added a solution of chloro(iodo)methane (2.24 g, 12.69 mmol, 920.93 pL, 5.5 eq) and Compound 12 (1 g, 2.31 mmol, 1 eq) in THF (2 mL) at -78°C. Then the resulting mixture was stirred at -78°C for 3 hrs. TLC (Petroleum ether: Ethyl acetate = 1 : 1, Rf = 0.51) showed that the reaction was completed. The reaction mixture was quenched by addition NH4Q (100 mL), and then extracted with ethyl acetate 300 mL (100 mL x 3). The combined organic layers were washed with NaHCCh (100 mL), Na2SCh (100 mL) and saturated brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiCh, Petroleum ether: Ethyl acetate = 1/0 to 1/1) to afford Compound 13 (0.89 g, crude) as a yellow oil.

13 102

[0097] Benzyl ((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2-oxopyrrolidin-3-yl)-4-(2, 3,5,6- tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)carbamate: To a mixture of Compound 13 (400 mg, 885.07 pmol, 1 eq) in DMF (8 mL) was added 2, 3,5,6- tetrafluorophenol (146.99 mg, 885.07 pmol, 1 eq), KI (146.92 mg, 885.07 pmol, 1 eq) and K2CO3 (244.64 mg, 1.77 mmol, 2 eq). The mixture was stirred at 25°C for 12 h. The mixture was poured into water (100 mL) and ethyl acetate (100 mL). The resulting mixture was extracted with ethyl acetate (100 mL><3). The combined organic phase was washed with brine (100 mL><3), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 80x40mmx3pm; mobile phase: [water (lOmM NH4HCO3)-ACN]; B%: 35%-65%, 8min) to afford Compound 102 (30 mg, 51.59 pmol, 5.83% yield, 100% purity) as a yellow solid. ’H NMR (400 MHz, methanol-^) d 7.22 - 7.47 (m, 5 H) 7.02 - 7.19 (m, 1 H) 4.95 - 5.25 (m, 3 H) 4.47 - 4.74 (m, 2 H) 4.07 - 4.21 (m, 1 H) 3.12 - 3.26 (m, 2 H) 2.20 - 2.58 (m, 2 H) 2.01 - 2.15 (m, 1 H) 1.65 - 1.92 (m, 3 H) 1.42 - 1.64 (m, 2 H) 0.76 - 1.04 (m, 6 H). LCMS (ESI): m/z: [M + H] calcd for C28H32F4N3O6: 581; found 581; RT = 3.082 min. The observed isomeric ratio, as determined by SFC, was 2.36: 1 : 1.89:2.02:12.07.

Example 3. Preparation of tert-Butyl (l-((5)-4-methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-2-oxo- l,2-dihydropyridin-3-yl)carbamate (103)

[0098] Compound 15: To a solution of Compound 14 (10 g, 90.82 mmol, 1 eq) in THF (150 mL) was added BOC2O (19.82 g, 90.81 mmol, 20.86 mL, 1 eq) at 25 °C. The mixture was stirred at 70 °C for 4 hrs. Then BOC2O (15.86 g, 72.65 mmol, 16.69 mL, 0.8 eq) was added and the mixture was refluxed at 70 °C for 12 hrs. TLC (Petroleum ether: Ethyl acetate = 1 : 1, Rf = 0.39) showed a new spot was formed and the reaction was completed. The reaction mixture was partitioned between H2O (300 mL) and EtOAc (180 mL). The organic phase was separated, washed with brine 120 mL (40 mLx 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCb, Petroleum ether/Ethyl acetate=l/O to 0/1). Compound 15 (19 g, 90.38 mmol, 99.52% yield) was obtained as a yellow solid. ’H NMR (400 MHz, DMSO-tA) d l 1.93 (br s, 1 H) 7.79 (br d, J= 6.4 Hz, 1 H) 7.69 (s, 1 H) 7.04 (dd, J = 6.5, 1.5 Hz, 1 H) 6.21 (t, J= 6.9 Hz, 1 H) 1.45 (s, 9 H).

16 17

[0099] Compound 17: Compound 16 (12.5 g, 95.29 mmol, 1 eq) was dissolved in H2SO4 (500 mL) at 0 °C. Then NaNCh (65.75 g, 952.94 mmol, 10 eq) in H2O (100 mL) was added dropwise to the solution. The resulting mixture was stirred at 0 °C for 3 hrs and then allowed to warm to 20 °C and stirred at 20 °C for 16 hrs. TLC (Petroleum ether: Ethyl acetate = 0: 1, Rt = 0.42) showed a new spot was formed and the reaction was completed. The reaction mixture was partitioned between H2O (600 mL) and EtOAc (600 mL). The organic phase was separated, washed with brines 300 mL (100 mL x 3), dried over Na2SO4 and concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 17 (17 g, 128.63 mmol, 67.49% yield) was obtained as a yellow oil. ’H NMR (400 MHz, DMSO-t/e) 3 3.94 (dd, J= 8.7, 4.9 Hz, 1 H) 1.68 - 1.80 (m, 1 H) 1.37 - 1.47 (m, 2 H) 0.83 - 0.91 (m, 6 H).

[0100] Compound 18: To a solution of Compound 17 (16 g, 121.07 mmol, 1 eq) in MeOH (40 mL) and 2,2-dimethoxypropane (160 mL) was added TMSC1 (1.32 g, 12.11 mmol, 1.54 mL, 0.1 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (300 mL) and extracted with EtOAc 150 mL (50 mL x 3). The combined organic layers were washed with brine 90 mL (30 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 18 (14 g, 95.77 mmol, 79.10% yield) was obtained as a yellow oil. ’H NMR (400 MHz, chloroform-r/) 3 4.22 (dd, J= 7.7, 5.7 Hz, 1 H) 3.71 - 3.83 (m, 2 H) 1.80 - 1.94 (m, 1 H) 1.52 - 1.61 (m, 2 H) 0.90 - 1.01 (m, 6 H).

[0101] Compound 19: To a solution of Compound 18 (20 g, 136.81 mmol, 1 eq) in DCM (200 mL) was added 2,6-lutidine (15.39 g, 143.65 mmol, 16.73 mL, 1.05 eq) and Tf2O (57.90 g, 205.22 mmol, 33.86 mL, 1.5 eq) at 0 °C under N2. The mixture was stirred at 0 °C for 30 min under N2. TLC (Petroleum ether: Ethyl acetate = 5: 1, Rt = 0.81) showed a new spot was formed and the reaction was completed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was extracted with EtOAc (300 mL) after washing with a mixture of brine and IN HC1 (3: 1 v/v), then dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 19 (40 g, crude) was obtained as a brown oil.

[0102] Compound 20: To a solution of Compound 15 (2.20 g, 10.46 mmol, 0.97 eq) in THF (40 mL) was added NaH (646.85 mg, 16.17 mmol, 60% purity, 1.5 eq) at 0 °C for 30 min. Then Compound 19 (3 g, 10.78 mmol, 1 eq) in THF (50 mL) was added. The mixture was stirred at 25 °C for 12 hrs. The reaction mixture was quenched by addition H2O (100 mL) at 0 °C, and extracted with EtOAc 90 mL (30 mL x 3). The combined organic layers were washed with brine 90 mL (30 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 20 (1 g, 2.96 mmol, 27.41% yield) was obtained as a brown oil. ^XH NMR (400 MHz, methanol-t/4) S 7.98 (br d, J= 7.7 Hz, 1 H) 7.26 (dd, J= 7.0, 1.8 Hz, 1 H) 7.18 - 7.29 (m, 1 H) 6.39 (t, J= 7.2 Hz, 1 H) 5.48 (dd, J= 10.5, 5.0 Hz, 1 H) 3.72 (s, 3 H) 1.97 - 2.13 (m, 2 H) 1.50 - 1.58 (m, 9 H) 1.33 - 1.44 (m, 1 H) 0.90 - 0.96 (m, 1 H) 0.93 (dd, J= 11.1, 6.7 Hz, 6 H)

[0103] Compound 21: To a solution of Compound 20 (0.8 g, 2.36 mmol, 1 eq) in MeOH (10 mL) and H2O (2 mL) was added LiOH.H2O (198.41 mg, 4.73 mmol, 2 eq) at 20 °C. The mixture was stirred at 20 °C for 1 hr. TLC (Petroleum ether: Ethyl acetate = 0: 1, Rt = 0.26) showed a new spot was formed and the reaction was completed. The mixture was adjust to pH = 8 with IN HC1. Then the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (15 mL) and extracted with EtOAc 12 mL (4 mL x 3). The combined organic layers were dried over Na2SO₄, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 21 (0.8 g, crude) was obtained as a yellow oil. 'H NMR (400 MHz, DMSO ) 3 7.79 (s, 1 H) 7.35 (dd, J= 7.0, 1.4 Hz, 1 H) 6.30 (t, J= 7.2 Hz, 1 H) 5.35 (dd, J= 11.2, 4.4 Hz, 1 H) 2.04 - 2.14 (m, 1 H) 1.79 - 1.96 (m, 1 H) 1.41 - 1.50 (m, 9 H) 1.21 - 1.30 (m, 1 H) 0.81 - 0.87 (m, 6 H).

21 103

[0104] terf-Butyl (l-((5)-4-methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-3-yl)-4- (2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-2-oxo-l,2-dihydropyridin-3- yl)carbamate (103): To a mixture of Compound 21 (0.1 g, 308.29 pmol, 1 eq) in DMF (3 mL) was added HATU (175.83 mg, 462.43 pmol, 1.5 eq) at 0 °C, the mixture was stirred at 0 °C for 1 h. Then Compound 6 (138.20 mg, 308.29 pmol, 1 eq, TFA) and DIPEA (79.69 mg, 616.58 pmol, 107.40 pL, 2 eq) was added to the mixture at 25 °C. The mixture was stirred at 25 °C for 12 h. The reaction mixture was partitioned between H2O (12 mL) and EtOAc (9 mL). The organic phase was separated, washed with brines 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (basic condition; column: Phenomenex Gemini-NX C18 75x30mmx3pm; mobile phase: [water (0.05% NH3H2O + lOmM NH₄HCO₃)-ACN]; B%: 50%-70%, 8min). Compound 103 (17 mg, 24.69 pmol, 8.01% yield, 93.03% purity) was obtained as a white solid. The observed ratio of isomers was 3.49: 1 : 8.71 : 5.25. LCMS (ESI): m/z: [M + H] calcd for C30H37F4N4O7: 641; found 641; RT= 3.321 min. 'H NMR (400 MHz, methanol-^) 3 7.95 (br s, 1 H) 7.33 (br d, J= 7.5 Hz, 1 H) 7.04 - 7.18 (m, 1 H) 6.32 - 6.42 (m, 1 H) 5.57 - 5.69 (m, 1 H) 4.98 - 5.22 (m, 1 H) 4.65 (br d, J= 11.2 Hz, 1 H) 3.35 (br s, 1 H) 3.19 - 3.30 (m, 2 H) 2.14 - 2.47 (m, 2 H) 1.94 - 2.12 (m, 2 H) 1.63 - 1.93 (m, 3 H) 1.47 - 1.56 (m, 9 H) 1.42 (br dd, J= 13.3, 6.7 Hz, 1 H) 0.84 - 0.99 (m, 6 H)

Example 4. Preparation of tert-Butyl (4-((5)-4-methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-3-oxo- 3,4-dihydropyrazin-2-yl)carbamate (104)

18 23

[0105] Compound 23: To a solution of Compound 18 (1 g, 9.00 mmol, 1 eq) in DMF (2 mL) was added NaH (359.97 mg, 9.00 mmol, 60% purity, 1 eq) at 0°C for 30 min. Then Compound 22 (2.50 g, 9.00 mmol, 1 eq) was added, the mixture was stirred at 25°C for 12hr. TLC (Petroleum ether: Ethyl acetate = 1 : 1, Rf= 0.43) showed the reaction was completed. The reaction mixture was quenched by addition H2O (10 mL) at 0°C, and extracted with EtOAc 30 mL (10 mL x 3). The combined organic layers were washed with brine 9 mL (3 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 1/1 to 1/1). Compound 23 (1 g, 4.18 mmol, 46.44% yield) was obtained as yellow oil. ’H NMR (400 MHz, methanol-^) d 6.64 - 6.85 (m, 2 H) 5.40 (dd, J= 10.8, 4.9 Hz, 1 H) 3.64 - 3.79 (m, 3 H) 2.03 - 2.10 (m, 1 H) 1.92 - 1.98 (m, 1 H) 1.34 - 1.46 (m, 1 H) 0.81 - 1.00 (m, 6 H).

[0106] Compound 24: To a solution of Compound 23 (0.5 g, 2.09 mmol, 1 eq) in THF (2 mL) was added TEA (422.91 mg, 4.18 mmol, 581.71 pL, 2 eq), DMAP (25.53 mg, 208.97 pmol, 0.1 eq) and BOC2O (912.13 mg, 4.18 mmol, 960.14 pL, 2 eq) at 0°C. The mixture was stirred at 25°C for 12hr. The reaction mixture was quenched by addition of H2O (10 mL) at 25°C, and then extracted with EtOAc 15 mL (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5/l to 3/1). Compound 24 (0.4 g, 1.18 mmol, 56.40% yield) was obtained as yellow oil.

[0107] Compound 25: To a solution of Compound 24 (250 mg, 736.62 pmol, 1 eq) in MeOH (5 mL) and H2O (1 mL) was added LiOH.H2O (61.82 mg, 1.47 mmol, 2 eq) at 25°C. The mixture was stirred at 25°C for Ihr. TLC (Petroleum ether/Ethyl acetate = 0: 1, Rf = 0.43) indicated one new spot formed. The mixture was adjust to pH = 8 with IN HC1. Then the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O 3 mL (1 mL x 3) and extracted with EtOAc 9 mL (3 mL x 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 25 (0.2 g, 614.71 pmol, 83.45% yield) was obtained as yellow oil. ’H NMR (400 MHz, methanol-^) d 7.67 (d, J= 4.5 Hz, 1 H) 7.31 (d, J= 4.5 Hz, 1 H) 5.36 - 5.63 (m, 1 H) 2.02 - 2.20 (m, 2 H) 1.99 (s, 1 H) 1.39 (s, 9 H) 0.85 - 0.99 (m, 6 H).

[0108] terf-Butyl (4-((5)-4-methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-3-yl)-4- (2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-3-oxo-3,4-dihydropyrazin-2- yl)carbamate (104): To a solution of Compound 25 (97.34 mg, 299.16 pmol, 1 eq) in DMF (1 mL) was added HATU (170.63 mg, 448.74 pmol, 1.5 eq) at 0°C. The mixture was stirred at 0°C for Ihr. Compound 6 (0.1 g, 223.07 pmol, 1 eq, TFA) and DIPEA (77.33 mg, 598.33 pmol, 104.22 pL, 2 eq) was added to the mixture at 25°C. The mixture was stirred at 25°C for 12hr. The residue was poured into water (3 mL) and ethyl acetate (3 mL). The aqueous phase was extracted with ethyl acetate (1 mL><3). The combined organic phase was washed with brine (1 mL><3), dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75x30mmx3pm; mobile phase: [water (0.05% NH3H2O + 10 mMNHiHCOi) - ACN]; B%: 40%-65%, 8min). Compound 104 (5 mg, 7.48 pmol, 2.50% yield, 95.74% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C29H36F4N5O7: 642.25; found 642.25; RT = 1.618 min. 95.74 % purity in QC_LCMS. ee value = 55.62% in SFC. ’H NMR (400 MHz, methanol-^) 3 7.20 (d, J= 4.9 Hz, 1 H) 6.96 - 7.11 (m, 2 H) 5.44 - 5.57 (m, 1 H) 5.04 - 5.18 (m, 2 H) 4.68 (dd, J = 10.9, 4.1 Hz, 1 H) 3.20 - 3.29 (m, 2 H) 2.38 - 2.48 (m, 1 H) 2.22 (br s, 1 H) 2.00 - 2.09 (m, 1 H) 1.85 - 1.98 (m, 3 H) 1.69 - 1.81 (m, 1 H) 1.54 (s, 9 H) 1.42 - 1.51 (m, 1 H) 1.42 - 1.51 (m, 1 H) 0.96 (br t, J= 5.8 Hz, 5 H) 0.92 - 0.99 (m, 1 H).

Example 5. Preparation of A-((5)-3-(3-Fluorophenyl)-l-oxo-l-(((5)-3-oxo-l-((5)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)-lH- indole-2-carboxamide (105)

[0109] Compound 27: To a solution of Compound 26 (1.5 g, 8.19 mmol, 1 eq) in MeOH (16 mL) was added SOCh (1.95 g, 16.38 mmol, 1.19 mL, 2 eq) dropwise at 0 °C and the resulting mixture was stirred at 70 °C for 3 h. The reaction mixture was concentrated under reduced pressure to afford a light yellow solid. And the solid was washed with petroleum ether (20 mLx3) and dried under reduced pressure to afford a white solid. And the solid was used to next step without further purification. Compound 27 (1.7 g, 7.28 mmol, 88.85% yield, HC1) was obtained as a white solid. 1H NMR (400 MHz, methanol-d4) 3 7.44 - 7.36 (m, 1H), 7.12 - 7.03 (m, 3H), 4.36 (t, J = 6.8 Hz, 1H), 3.82 (s, 3H), 3.27 (d, J = 6.2 Hz, 1H), 3.23 - 3.16 (m, 1H).

[0110] Compound 29: To a solution of Compound 28 (344.84 mg, 2.14 mmol, 1 eq) in DMF (1 mL) was added HATU (1.22 g, 3.21 mmol, 1.5 eq) at 0°C. Compound 27 (0.5 g, 2.14 mmol, 1 eq, HC1) and DIPEA (553.10 mg, 4.28 mmol, 745.41 pL, 2 eq) was added to the mixture at 25°C, The mixture was stirred at 25°C for 12hr. TLC (Petroleum ether: Ethyl acetate= 1 :1, Rf = 0.43) showed the reaction was completed. The reaction mixture was quenched by addition H2O 9 mL (3 mL x 3) at 0°C, and extracted with EtOAc 9 mL (3 mL x 3). The combined organic layers were washed with brine 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( Si O2, Petroleum ether/Ethyl acetate = 5/1 to 1/1). Compound 29 (0.7 g, 2.06 mmol, 96.12% yield) was obtained as a yellow oil. ^TH NMR (400 MHz, chloroform-;/) 3 7.46 - 7.55 (m, 1 H) 7.27 - 7.35 (m, 1 H), 7.22 - 7.26 (m, 1 H) 7.11 - 7.18 (m, 1 H) 6.90 - 6.99 (m, 2 H) 6.83 - 6.90 (m, 2 H), 6.73 (br d, J= 7.6 Hz, 1 H) 5.00 - 5.26 (m, 1 H) 3.79 (s, 3 H) 3.16 - 3.42 (m, 2 H).

29 30

[0111] Compound 30: To a solution of Compound 29 (0.4 g, 1.18 mmol, 1 eq) in MeOH (2.5 mL) and H2O (0.5 mL) was added LiOH.H2O (98.64 mg, 2.35 mmol, 2 eq) at 25°C. The mixture was stirred at 25°C for Ihr. The mixture was adjust to pH = 8 with IN HC1. Then the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O 15 mL (5 mL x 3) and extracted with EtOAc 12 mL (4 mL x 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 30 (0.3 g, 919.34 pmol, 78.22% yield) was obtained as yellow oil. ’H NMR (400 MHz, methanol-^) 3 7.60 (d, J= 8.1 Hz, 1 H) 7.41 (dd, J= 8.4, 0.7 Hz, 1 H) 7.17 - 7.31 (m, 2 H) 7.01 - 7.11 (m, 4 H) 6.92 (td, J= 8.6, 2.0 Hz, 1 H) 4.90 (br s, 1 H) 3.36 (dd, J= 13.9, 5.1 Hz, 1 H) 3.15 (dd, J= 13.9, 9.3 Hz, 1 H).

[0112] A-((5)-3-(3-Fluorophenyl)-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-3-yl)-4- (2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)-lH-indole-2-carboxamide

(105): To a solution of Compound 6 (122.92 mg, 274.20 pmol, TFA) in DMF (2 mL) was added HATU (174.78 mg, 459.67 pmol, 1.5 eq) at 0°C. Compound 30 (0.1 g, 306.45 pmol, 1 eq) and DIPEA (118.82 mg, 919.34 pmol, 160.13 pL, 3 eq) was added to the mixture at 25°C, The mixture was stirred at 25°C for 12hr. The reaction mixture was partitioned between FEO (12 Ml) and EtOAc (9 mL). The organic phase was separated, washed with brines 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 150x30mmx5pm; mobile phase: [water (0.1%TFA)-ACN]; B%: 45%-75%, 9min). Compound 105 (9.6 mg, 14.94 pmol, 4.88% yield, 100% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C32H28F5N4O5: 643.2; found 643.2; RT = 4.438 min. The observed ratio of isomers was 2.32: 1.73: 1 : 8.86: 10.40. ^XH NMR (400 MHz, methanol-^) 3 7.52 - 7.63 (m, 1 H) 7.35 - 7.44 (m, 1 H) 6.84 - 7.33 (m, 8 H) 4.90 - 5.27 (m, 2 H) 4.50 (dd, J = 11.5, 3.1 Hz, 1 H) 4.46 - 4.81 (m, 1 H) 3.00 - 3.29 (m, 4 H) 2.19 - 2.54 (m, 1 H) 2.02 - 2.17 (m, 1 H) 1.82 - 2.01 (m, 1 H) 1.61 - 1.82 (m, 2 H).

Example 6. Preparation of A-((5)-3-Cyclopropyl-l-oxo-l-(((5)-3-oxo-l-((5)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)-lH- indole-2-carboxamide (106)

[0113] Compound 8: To a stirred solution of Compound 7 (1.5 g, 11.61 mmol, 1 eq) in MeOH (15 mL) under N2 was added SOC12 (4.15 g, 34.84 mmol, 2.53 mL, 3 eq) at 0 °C dropwise. The resulting mixture was stirred at 70°C for 3 h. The reaction mixture was concentrated under reduced pressure to afford a yellow solid. And the solid was triturated with petroleum ether (10 mL><2) to afford an off-white solid. And the solid was used to next step without further purification. Compound 8 (1.95 g, 10.85 mmol, 93.46% yield, HC1) was obtained as an off-white solid. ’H NMR (400 MHz, methanol-d4) <5 = 4.16 - 4.07 (m, 1H), 3.85 (s, 3H), 1.97 - 1.85 (m, 1H), 1.83 - 1.70 (m, 1H), 0.88 - 0.72 (m, 1H), 0.63 - 0.54 (m, 2H), 0.22 - 0.12 (m, 2H).

[0114] Compound 31: To a solution of Compound 8 (0.5 g, 2.78 mmol, 1 eq, HC1) in DMF (5 mL) was added HATU (1.99 g, 5.24 mmol, 1.5 eq) at 0°C. Compound 28 (562.77 mg, 3.49 mmol, 1 eq) and DIPEA (902.62 mg, 6.98 mmol, 1.22 mL, 2 eq) was added to the mixture at 25°C. The mixture was stirred at 25°C for 12hr . TLC (Petroleum ether: Ethyl acetate= 1 : 1, Rf = 0.43) showed the reaction was completed. The reaction mixture was quenched by addition H2O (5 mL) at 0°C, and extracted with EtOAc 9 mL (3 mL x 3). The combined organic layers were washed with brine 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si O2, Petroleum ether/Ethyl acetate=5/l to 1/1). Compound 31 (0.7 g, 2.44 mmol, 70.01% yield) was obtained as yellow oil.

[0115] Compound 32: To a solution of Compound 31 (0.4 g, 1.40 mmol, 1 eq) in MeOH (2.5 mL) and H2O (0.5 mL) was added LiOH.FbO (117.24 mg, 2.79 mmol, 2 eq) at 25°C. The mixture was stirred at 25°C for Ihr. The mixture was adjust to pH=8 with IN HC1. Then the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (6 mL) and extracted with EtOAc 12 mL (4 mL x 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was used for the next step without purification. Compound 32 (0.2 g, 734.49 pmol, 52.58% yield) was obtained as yellow oil. ’H NMR (400 MHz, methanol-^) d 7.62 (d, J= 8.1 Hz, 1 H) 7.44 (d, J= 8.3 Hz,

1 H) 7.01 - 7.26 (m, 3 H) 4.71 (br dd, J= 8.3, 5.4 Hz, 1 H) 1.70 - 1.91 (m, 2 H) 0.81 - 0.98 (m, 1 H) 0.39 - 0.54 (m, 2 H) 0.02 - 0.27 (m, 2 H).

[0116] \-((.S)-3-Cy clopropy 1- 1-oxo- l-(((5)-3-oxo- l-((5)-2-oxopyrr olidin-3-yl)-4-

(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)-lH-indole-2-carboxamide

(106): To a solution of Compound 6 (147.31 mg, 440.69 pmol, 1.2 eq) in DMF (2 mL) was added HATU (209.46 mg, 550.87 pmol, 1.5 eq) at 0°C. Compound 32 (147.31 mg, 328.60 pmol, TFA) and DIPEA (142.39 mg, 1.10 mmol, 191.90 pL, 3 eq) was added to the mixture at 25°C, The mixture was stirred at 25°C for 12hr. The reaction mixture was partitioned between H2O (12 mL) and EtOAc (9 mL). The organic phase was separated, washed with brines 9 mL (3 mL x 3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 150x30mmx5pm; mobile phase: [water (0.1% TFA)-ACN]; B%: 40%-70%, 9min). Compound 106 (15 mg, 23.78 pmol, 6.48% yield, 93.32% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C29H29F4N4O5: 589; found 589; RT = 4.244 min. The observed ratio of isomers was 1.18: 1 : 2.50: 3.29. ^XH NMR (400 MHz, methanol-t/4) 3 7.50 - 7.71 (m, 1 H) 7.36 - 7.48 (m, 1 H) 7.12 - 7.26 (m, 2 H) 6.91 - 7.10 (m, 2 H) 5.07 - 5.35 (m, 1 H) 4.54 - 4.77 (m, 2 H) 3.18 - 3.31 (m, 2 H) 2.03 - 2.68 (m, 3 H) 1.61 - 1.93 (m, 4 H) 0.71 - 0.95 (m, 1 H) 0.37 - 0.59 (m, 2 H) 0.02 - 0.28 (m, 2 H).

Example 7. Preparation of A-((5)-4-Methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-

3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-lH-indole-2- carboxamide (107)

[0117] Compound 34: To a solution of Compound 28 (887.14 mg, 5.50 mmol, 1 eq) in DCM (5 mL) was added EDCI (1.16 g, 6.06 mmol, 1.1 eq), HOBt (818.21 mg, 6.06 mmol, 1.1 eq) slowly and followed by the addition of DIPEA (2.49 g, 19.27 mmol, 3.36 mL, 3.5 eq) dropwise at 0 °C and the resulting mixture was stirred at 0 °C for 20 mins. Then Compound 33 was added into the mixture at 0 °C and the resulting mixture was stirred at 25 °C for 1 h. TLC (Petroleum ether/Ethyl acetate = 3/1, product Rf = 0.3) showed that the starting material was consumed and one major spot was observed. Water (10 mL) was added into the mixture and the resulting mixture was layered. The inorganic phase was extracted with DCM (10 mLx3) and the combined organic phases were concentrated under reduced pressure to afford a yellow solid. And the solid was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 100/0 - 100/13) to give Compound 34 (1 g, 3.47 mmol, 63.00% yield) as a yellow solid. ’H NMR (400 MHz, chloroform-d) 3 9.78 (s, 1H), 7.97 (d, J= 8.0 Hz, 1H), 7.75 (d, J= 8.3 Hz, 1H), 7.67 - 7.57 (m, 1H), 7.51 - 7.42 (m, 1H), 7.04 (d, J= 8.4 Hz, 1H), 5.28 - 5.18 (m, 1H), 4.12 (s, 3H), 2.13 - 2.10 (m, 1H), 2.05 (s, 2H), 1.32 (t, J = 5.8 Hz, 6H).

[0118] Compound 35: To a solution of Compound 34 (500 mg, 1.73 mmol, 1 eq) in MeOH (2.5 mL) and H2O (2.5 mL) was added LiOH.EEO (109.15 mg, 2.60 mmol, 1.5 eq) at 25 °C and the resulting mixture was stirred at 25 °C for 12 h. The pH of reaction mixture was adjusted to 2-3 with HC1 (2 M) and then concentrated under reduced pressure to afford a white solid. And the solid was dissolved in ethyl acetate (5 mL) and water (3 mL) and the resulting mixture was layered. The aqueous phase was extracted with ethyl acetate (3 mL><2) and the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give Compound 35 (440 mg, 1.60 mmol, 92.50% yield) as a yellow solid. ^XH NMR (400 MHz, DMSO-de) 6 12.64 (s, 1H), 11.56 (s, 1H), 8.57 (d, J= 8.1 Hz, 1H), 7.73 - 7.55 (m, 1H), 7.51 - 7.36 (m, 1H), 7.31 - 7.15 (m, 2H), 7.12 - 6.96 (m, 1H), 4.55 - 4.42 (m, 1H), 1.85 - 1.66 (m, 2H), 1.65 - 1.54 (m, 1H), 1.05 - 0.76 (m, 6H).

[0119] N-((5)-4-Methyl-l-oxo-l-(((5)-3-oxo-l-((5)-2-oxopyrrolidin-3-yl)-4-(2, 3,5,6- tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-lH-indole-2-carboxamide (107): To a solution of Compound 35 (50 mg, 182.27 pmol, 1 eq) in DMF (1 mL) was added HATU (103.96 mg, 273.41 pmol, 1.5 eq), DIPEA (82.45 mg, 637.95 pmol, 111.12 pL, 3.5 eq) and Compound 6 (85 mg, 229.28 pmol, 1.26 eq, HC1, crude, calculated as 100% purity) at 0 °C and the resulting mixture was stirred at 25 °C for 2 h. TLC (Petroleum ether/Ethyl acetate = 1/2, product Rf= 0.5) showed that one major new spot was observed. The reaction mixture was diluted with water (2 mL) and the resulting mixture was extracted with ethyl acetate (2 mL><3) and the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford a yellow oil. And the oil was purified by column chromatography (SiCb, Petroleum ether/Ethyl acetate = 100/10 - 0/100) to afford a yellow oil. And the oil was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75x30mmx3pm; mobile phase: [water (lOmM NH4HCO3)-ACN]; B%: 30%-60%, 12 min) to give Compound 107 (12 mg, 20.32 pmol, 11.15% yield, 100% purity) as a white solid. LCMS (ESI): m/z: [M + H] calcd for C29H31F4N4O5: 591; found 591; RT = 2.570 min. ^XH NMR (400 MHz, methanol-d₄) 6 7.71 - 7.49 (m, 1H), 7.48 - 7.34 (m, 1H), 7.29 - 7.12 (m, 2H), 7.11 - 6.85 (m, 2H), 5.29 - 5.12 (m, 1H), 4.75 - 4.61 (m, 2H), 4.45 - 4.07 (m, 1H), 3.30 - 3.19 (m, 2H), 2.70 - 2.37 (m, 1H), 2.52 - 2.36 (m, 1H), 2.34 - 2.22 (m, 1H), 2.18 - 2.02 (m, 1H), 1.96 - 1.64 (m, 5H), 1.14 - 0.85 (m, 6H). ee value = 30.16%.

Example 8. Preparation of N-((S)-l-(((S)-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3- oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-lH- indole-2-carboxamide (108)

[0120] The title compound was prepared as described in Example 7, using (5)-3-((5)-2- amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in place of Compound 6 ((5)-3-((5)-2-amino-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)pyrrolidin-2- one) in the final step. The product was as a white solid (40 mg, 63.26 pmol, 28.48% yield, 93.703% purity). The observed isomeric ratio, as determined by SFC, was 2.25:1 : 14.05: 15.68:28.93:10.56. LCMS (ESI): m/z: [M + H] calcd for C26H31F6N4O5: 593; found 593. ^XH NMR (400 MHz, methanol-^) d 7.61 (d, J= 8.0 Hz, 1 H) 7.44 (d, J= 8.3 Hz, 1 H) 7.16 - 7.25 (m, 2 H) 7.03 - 7.11 (m, 1 H) 4.91 - 5.11 (m, 2 H) 4.53 - 4.82 (m, 3 H) 3.16 - 3.29 (m, 2 H) 2.22 - 2.62 (m, 2 H) 1.97 - 2.13 (m, 1 H) 1.63 - 1.91 (m, 5 H) 0.89 - 1.11 (m, 6 H).

Example 9. Preparation of N-((S)-3-cyclopropyl-l-(((S)-4-((l, 1,1, 3,3,3- hexafluoropropan-2-yl)oxy)-3-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l- oxopropan-2-yl)-lH-indole-2-carboxamide (109)

[0121] The title compound was prepared as described in Example 6, using (5)-3-((5)-2- amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in place of Compound 6 in the final step. The product (40 mg, 64.35 pmol, 19.47% yield, 95% purity) was obtained as white solid. LCMS (ESI): m/z: [M + H] calcd for C26H29F6N4O5: 591; found 591.3. The observed isomeric ratio, as determined by SFC, was 1 : 5.31 : 2.14: 6.05. ^XH NMR (400 MHz, methanol-t/4) 3 7.62 (d, J= 8.0 Hz, 1 H) 7.44 (d, J= 8.3 Hz, 1 H) 7.15 - 7.26 (m, 2 H) 7.02 - 7.10 (m, 1 H) 4.99 (dt, J= 12.0, 6.0 Hz, 1 H) 4.57 - 4.82 (m, 5 H) 3.21 - 3.29 (m, 2 H) 2.20 - 2.66 (m, 2 H) 1.96 - 2.09 (m, 1 H) 1.59 - 1.88 (m, 4 H) 0.87 (br s, 1 H) 0.52 (br s, 2 H) 0.21 (br s, 2 H).

Example 10. Preparation of N-((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2-oxopyrrolidin-

3-yl)-4-(2,3,6-trifluorophenoxy)butan-2-yl)amino)pentan-2-yl)-lH-indole-2- carboxamide (110)

[0122] The title compound was prepared as described in Example 7, using (5)-3-((5)-2- amino-3-oxo-4-(2,3,6-trifluorophenoxy)butyl)pyrrolidin-2-one in place of Compound 6 in the final step. The product (17 mg, 28.17 pmol, 9.66% yield, 94.88% purity) was obtained as white solid. LCMS (ESI): m/z: [M + H] calcd for C29H32F3N4O5:573.3; found 573.3. The observed isomeric ratio, as determined by SFC, was 2.5:3: 1 : 10.4: 14.2. ^XH NMR (400 MHz, methanol-t/i) 3 0.92 - 1.07 (m, 6 H) 1.19 - 2.69 (m, 9 H) 3.18 - 3.30 (m, 2 H) 4.02 - 4.80 (m, 2 H) 5.00 - 5.27 (m, 1 H) 6.57 - 6.99 (m, 2 H) 7.02 - 7.11 (m, 1 H) 7.12 - 7.27 (m, 2 H) 7.37 - 7.46 (m, 1 H) 7.50 - 7.66 (m, 1 H). Example 11. Preparation of N-((S)-3-cyclohexyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,6-trifluorophenoxy)butan-2-yl)amino)propan-2-yl)-lH- indole-2-carboxamide (111)

[0123] The title compound was prepared as described in Example 7, using benzyl (5)-2- amino-3 -cycloh exylpropanoate in place of methyl Z-leucinate (Compound 33) for coupling with lH-indole-2-carboxylic acid (Compound 28) in the first step and (5)-3-((S)-2-amino-3- oxo-4-(2,3,6-trifluorophenoxy)butyl)pyrrolidin-2-one in place of Compound 6 in the final step. The product (2 mg, 2.97 pmol, 1.87% yield, 90.88% purity) was obtained as white solid. LCMS (ESI): m/z: [M + H] calcd for C32H36F3N4O5: 613.3; found 613.3. ee value=84.98%. ^XH NMR (400 MHz, methanol-^) 3 0.88 - 1.13 (m, 2 H) 1.15 - 1.26 (m, 2 H) 1.27 - 1.35 (m, 1 H) 1.40 - 1.57 (m, 1 H) 1.64 - 1.88 (m, 7 H) 1.88 - 1.95 (m, 1 H) 2.06 - 2.16 (m, 1 H) 2.22 - 2.34 (m, 1 H) 2.35 - 2.53 (m, 1 H) 2.54 - 2.68 (m, 1 H) 3.18 - 3.29 (m, 2 H) 4.10 - 4.43 (m, 1 H) 4.63 - 4.68 (m, 1 H) 4.96 - 5.14 (m, 2 H) 6.81 - 7.03 (m, 2 H) 7.03 - 7.11 (m, 1 H) 7.13 - 7.28 (m, 2 H) 7.32 - 7.48 (m, 1 H) 7.53 - 7.72 (m, 1 H).

Example 12. Preparation of N-((S)-l-(((S)-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3- oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4- methoxy-lH-indole-2-carboxamide (112)

[0124] The title compound was prepared as described in Example 7, using 4-methoxy-lH- indole-2-carboxylic acid in place of lH-indole-2-carboxylic acid (Compound 28) for coupling with methyl Z-leucinate (Compound 33) in the first step, and using (5)-3-((5)-2- amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in place of Compound 6 in the final step. The product (15 mg, 22.89 pmol, 10.31% yield, 95.016% purity) was obtained as a white solid. The observed isomeric ratio, as determined by SFC, was 1:5.4:3.7:8.6. LCMS (ESI): m/z: [M + H] calcd for C27H33F6N4O6: 623; found 623. ^XH NMR (400 MHz, methanol-^) 3 7.28 (s, 1 H) 7.10 - 7.21 (m, 1 H) 7.03 (br d, J= 8.1 Hz, 1 H) 6.51 (br d, J= 7.5 Hz, 1 H) 4.93 - 5.09 (m, 1 H) 4.48 - 4.82 (m, 4 H) 3.93 (s, 3 H) 3.25 (br d, J = 6.3 Hz, 2 H) 2.20 - 2.64 (m, 2 H) 1.95 - 2.12 (m, 1 H) 1.60 - 1.92 (m, 5 H) 0.90 - 1.09 (m, 6 H).

Example 13. Preparation of 4-methoxy-N-((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-phenoxybutan-2-yl)amino)pentan-2-yl)-lH-indole-2-carboxamide (H3)

[0125] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-3-oxo-4-phenoxybutyl)pyrrolidin-2-one in place of (5)-3-((5)-2-amino-4-((l, 1,1, 3,3,3- hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (0.07 g, 116.75 pmol, 21.97% yield, 91.5% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C30H37N4O6: 549.3; found 549.3. The observed isomeric ratio, as determined by SFC, was 2.19: 1 :3.88. ^XH NMR (400 MHz, chloroform-t/) 3 10.78 (br s, 1 H) 9.58 - 9.81 (m, 1 H) 8.57 (br d, J= 6.0 Hz, 1 H) 7.28 - 7.33 (m, 1 H) 7.07 - 7.23 (m, 2 H) 6.66 - 7.04 (m, 5 H) 6.49 (d, J= 7.7 Hz, 1 H) 6.05 - 6.15 (m, 1 H) 4.51 - 5.01 (m, 4 H) 3.90 - 4.05 (m, 3 H) 2.92 - 3.36 (m, 2 H) 2.52 - 2.66 (m, 1 H) 2.28 - 2.49 (m, 1 H) 2.02 - 2.12 (m, 1 H) 1.94 (dt, J= 10.4, 5.2 Hz, 1 H) 1.73 - 1.84 (m, 3 H) 1.22 - 1.55 (m, 1 H) 0.92 - 1.00 (m, 6 H).

Example 14. Preparation of N-((S)-l-(((S)-4-(2-fluorophenoxy)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4-methoxy-lH- indole-2-carboxamide (114)

[0126] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-4-(2-fluorophenoxy)-3-oxobutyl)pyrrolidin-2-one in place of (5)-3-((5)-2-amino-4- ((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (10 mg, 16.64 pmol, 4.66% yield, 94.3 % purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C30H36FN4O6: 567.3; found 567.3. The observed isomeric ratio, as determined by SFC, was 2.82: 1 :4.13. ’H NMR. (400 MHz, chloroform-t/) 3 10.73 (br s, 1 H) 9.33 - 9.51 (m, 1 H) 8.58 (br d, J= 5.4 Hz, 1 H) 7.19 (q, J= 7.8 Hz, 1 H) 7.05 - 7.14 (m, 2 H) 6.91 - 7.04 (m, 3 H) 6.66 - 6.77 (m, 1 H) 6.39 - 6.53 (m, 1 H) 6.39 - 6.53 (m, 1 H) 5.80 - 5.98 (m, 1 H) 4.89 - 4.93 (m, 1 H) 4.86 (d, J= 3.6 Hz, 2 H) 4.74 - 4.82 (m, 1 H) 3.92 - 4.01 (m, 3 H) 2.90 - 3.39 (m, 2 H) 2.60 (br d, J= 11.7 Hz, 1 H) 2.32 - 2.50 (m, 1 H) 2.00 - 2.08 (m, 1 H) 1.68 - 1.93 (m, 4 H) 1.24 - 1.40 (m, 1 H) 0.99 (br d, J= 5.5 Hz, 6 H).

Example 15. Preparation of N-((S)-l-(((S)-4-(2,6-difluorophenoxy)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4-methoxy-lH- indole-2-carboxamide (115)

[0127] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-4-(2,6-difluorophenoxy)-3-oxobutyl)pyrrolidin-2-one in place of (5)-3-((5)-2-amino- 4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (55 mg, 85.33 pmol, 25.45% yield, 90.7% purity) was obtained as a yellow solid. LCMS (ESI): m/z: [M + H] calcd for C30H35F2N4O6: 585.3; found 585.3. The observed isomeric ratio, as determined by SFC, was 1 :4.7: 1 :6.8. ^XH NMR (400 MHz, chloroform-t/) 3 10.91 (br s, 1 H) 9.32 - 9.55 (m, 1 H) 8.44 (d, J= 6.1 Hz, 1 H) 7.15 - 7.25 (m, 1 H) 7.09 - 7.14 (m, 1 H) 7.00 - 7.04 (m, 1 H) 6.87 - 6.95 (m, 2 H) 6.79 (br d, J= 8.1 Hz, 1 H) 6.44 - 6.55 (m, 1 H) 5.94 (s, 1 H) 4.90 - 5.01 (m, 2 H) 4.63 - 4.89 (m, 1 H) 3.93 - 3.99 (m, 3 H) 2.81 - 3.36 (m, 2 H) 2.22 - 2.61 (m, 2 H) 2.04 - 2.11 (m, 1 H) 1.69 - 1.92 (m, 4 H) 1.25 - 1.50 (m, 1 H) 0.91 - 1.03 (m, 6 H).

Example 16. Preparation of 4-methoxy-N-((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,6-trifluorophenoxy)butan-2-yl)amino)pentan-2-yl)-lH-indole- 2-carboxamide (116)

[0128] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-3-oxo-4-(2,3,6-trifluorophenoxy)butyl)pyrrolidin-2-one in place of (5)-3-((5)-2- amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (55 mg, 87.06 pmol, 18.73% yield, 95.39% purity) was obtained as white solid. LCMS (ESI): m/z: [M + H] calcd for C3oH34F3N40e:603.3; found 603.3. The observed isomeric ratio, as determined by SFC, was 6.9: 1 :31.9:5.6:45.3. ^XH NMR (400 MHz, methanol-t/i) d 0.91 - 1.10 (m, 6 H) 1.61 - 1.92 (m, 5 H) 2.04 - 2.65 (m, 3 H) 3.19 - 3.30 (m, 2 H) 3.89 - 3.95 (m, 3 H) 3.99 - 4.44 (m, 1 H) 4.52 - 4.78 (m, 2 H) 4.98 - 5.25 (m, 1 H) 6.45 - 6.53 (m, 1 H) 6.55 - 7.07 (m, 3 H) 7.09 - 7.31 (m, 2 H).

Example 17. Preparation of 4-methoxy-N-((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2-yl)-lH- indole-2-carboxamide (117)

[0129] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)pyrrolidin-2-one in place of (5)-3-((5)-2- amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (21 mg, 32.13 pmol, 11.91% yield, 94.94% purity) was obtained as a white solid. LCMS (ESI): m/z: [M + H] calcd for C3oH33F4N40e:621.3; found 621.3. The observed isomeric ratio, as determined by SFC, was 2.6: 9.9: 1 : 7.9: 2.6. 'H NMR (400 MHz, methanol-t/4) 3 0.94 - 1.06 (m, 6 H) 1.64 - 1.95 (m, 5 H) 2.00 - 2.17 (m, 1 H) 2.23 - 2.65 (m, 2 H) 3.21 - 3.30 (m, 2 H) 3.86 - 3.95 (m, 3 H) 4.09 - 4.40 (m, 1 H) 4.54 - 4.79 (m, 2 H) 5.09 - 5.28 (m, 1 H) 6.45 - 6.57 (m, 1 H) 6.89 - 7.32 (m, 4 H).

Example 18. Preparation of N-((S)-l-(((S)-4-(isoxazol-3-yloxy)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4-methoxy-lH- indole-2-carboxamide (118)

[0130] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-4-(isoxazol-3-yloxy)-3-oxobutyl)pyrrolidin-2-one in place of (5)-3-((5)-2-amino-4- ((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (50 mg, 87.41 pmol, 10.64% yield, 94.33% purity) was obtained as a yellow solid. LCMS (ESI): m/z: [M + H] calcd for C27H34N5O?:540.3; found 540.3. The observed isomeric ratio, as determined by SFC, was 1.18:4.08:1 :7.25: 15.99. ^XH NMR (400 MHz, methanol-t/4) 3 0.87 - 1.09 (m, 6 H) 1.57 - 1.94 (m, 5 H) 2.01 - 2.22 (m, 1 H) 2.25 - 2.69 (m, 2 H) 3.09 - 3.26 (m, 2 H) 3.93 (s, 3 H) 4.52 - 4.73 (m, 2 H) 5.01 - 5.27 (m, 2 H) 5.98 - 6.23 (m, 1 H) 6.51 (d, J= 7.75 Hz, 1 H) 6.92 - 7.07 (m, 1 H) 7.12 - 7.19 (m, 1 H) 7.24 - 7.35 (m, 1 H) 8.35 (dd, J= 5.82, 1.81 Hz, 1 H).

Example 19. Preparation of N-((S)-l-(((S)-4-((2,6-dimethylpyridin-4-yl)oxy)-3-oxo-l- ((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4-methoxy-lH- indole-2-carboxamide (119)

[0131] The title compound was prepared as described in Example 12, using (S)-3-((S)-2- amino-4-((2,6-dimethylpyridin-4-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in place of (5)-3-((5)- 2-amino-4-((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (23 mg, 36.50 pmol, 7.40% yield, 91.68% purity) was obtained as a yellow solid, ee value = 32.32%. LCMS (ESI): m/z: [M + H] calcd for C3iH4oNs06:578.3; found 578.3. ’H NMR (400 MHz, methanol-^) 3 0.88 - 1.07 (m, 6 H) 1.59 - 1.94 (m, 5 H) 1.96 - 2.25 (m, 2 H) 2.26 - 2.43 (m, 6 H) 2.43 - 2.63 (m, 1 H) 3.17 - 3.30 (m, 2 H) 3.93 (s, 3 H) 4.27 - 4.70 (m, 2 H) 4.89 - 5.17 (m, 2 H) 6.51 (d, J= 1.15 Hz, 1 H) 6.54 - 6.72 (m, 2 H) 6.96 - 7.05 (m, 1 H) 7.09 - 7.19 (m, 1 H) 7.24 - 7.33 (m, 1 H).

Example 20. Preparation of N-((S)-l-(((S)-4-(isoxazol-4-yloxy)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-l-oxopentan-2-yl)-4-methoxy-lH- indole-2-carboxamide (120)

[0132] The title compound was prepared as described in Example 12, using (5)-3-((5)-2- amino-4-(isoxazol-4-yloxy)-3-oxobutyl)pyrrolidin-2-one in place of (5)-3-((5)-2-amino-4- ((l,l,l,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutyl)pyrrolidin-2-one in the final step. The product (80 mg, 146.69 pmol, 21.55% yield, 98.94% purity) as a white solid. The observed isomeric ratio, as determined by SFC, was 6.13:23.98:33.41 : 1. LCMS (ESI): m/z: [M + H] calcd for C27H₃4N₅O7:540.3; found 540.3. ’H NMR (400 MHz, methanol-^) 3 1.01 (dd, J= 16.44, 6.14 Hz, 6 H) 1.60 - 1.94 (m, 5 H) 2.03 - 2.15 (m, 1 H) 2.25 - 2.36 (m, 1 H) 2.37 - 2.62 (m, 1 H) 3.20 - 3.30 (m, 2 H) 3.93 (s, 3 H) 4.46 - 4.69 (m, 2 H) 4.80 - 4.88 (m, 2 H) 6.51 (d, J = 7.67 Hz, 1 H) 7.03 (d, J= 8.33 Hz, 1 H) 7.12 - 7.18 (m, 1 H) 7.26 - 7.34 (m, 1 H) 8.31 - 8.40 (m, 1 H) 8.44 - 8.56 (m, 1 H).

Example 21. Preparation of N-((S)-3-cyclopropyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2- yl)benzofuran-2-carboxamide (121)

[0133] The title compound was prepared as described in Example 6, using benzofuran-2- carboxylic acid in place of lH-indole-2-carboxylic acid (Compound 28) for coupling with methyl (5)-2-amino-3-cyclopropylpropanoate (Compound 8). The product (29 mg, 49.19 pmol, 22.41% yield, 100% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C29H28F4N3O6: 590.2; found 590.2. The observed isomeric ratio, as determined by SFC, was 2.45: 10.58: 1. ^XH NMR (400 MHz, chloroform^/) 3 8.59 - 9.26 (m, 1 H) 7.62 - 7.70 (m, 1 H) 7.52 (d, J= 8.1 Hz, 1 H) 7.46 (s, 1 H) 7.35 - 7.45 (m, 2 H) 7.30 (d, J= 7.9 Hz, 1 H) 6.71 - 6.88 (m, 1 H) 5.79 - 6.18 (m, 1 H) 5.00 - 5.26 (m, 2 H) 4.66 - 4.93 (m, 2 H) 3.30 - 3.43 (m, 2 H) 2.38 - 2.56 (m, 2 H) 1.99 - 2.17 (m, 2 H) 1.82 - 1.95 (m, 3 H) 0.71 - 0.92 (m, 1 H) 0.55 (br d, J= 7.9 Hz, 2 H) 0.09 - 0.28 (m, 2 H).

Example 22. Preparation of N-((S)-3-cyclopropyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)propan-2-yl)-N- methylbenzofuran-2-carboxamide (122)

[0134] The title compound was prepared as described in Example 21, using methyl (S)-3- cyclopropyl-2-(methylamino)propanoate in place of methyl (5)-2-amino-3- cyclopropylpropanoate for coupling with benzofuran-2-carboxylic acid. The product (0.06 g, 94.44 pmol, 12.33% yield, 95% purity) was obtained as yellow oil. LCMS (ESI): m/z: [M + H] calcd for C30H30F4N3O6: 604.3; found 604.3. ’H NMR (400 MHz, chloroforms/) 3 -0.20 - 0.23 (m, 2 H) 0.24 - 0.61 (m, 2 H) 0.63 - 0.91 (m, 1 H) 1.19 - 1.74 (m, 1 H) 1.79 - 2.20 (m, 4 H) 2.23 - 2.65 (m, 2 H) 2.92 - 3.29 (m, 2 H) 3.33 (br s, 3 H) 4.64 - 4.95 (m, 1 H) 5.00 - 5.34 (m, 3 H) 6.78 (br d, J= 8.50 Hz, 1 H) 7.28 - 7.34 (m, 1 H) 7.35 - 7.47 (m, 2 H) 7.54 (br d, J = 7.00 Hz, 1 H) 7.67 (d, J= 7.75 Hz, 1 H) 8.37 (br s, 1 H).

Example 23. Preparation of 4-methoxy-N-((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l-((S)-2- oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2- yl)benzofuran-2-carboxamide (123)

[0135] The title compound was prepared as described in Example 21, via coupling of 4- methoxybenzofuran-2-carboxylic acid and methyl Z-leucinate (Compound IB), followed by hydrolysis of the resulting methyl ester, to provide (4-methoxybenzofuran-2-carbonyl)-Z- leucine for reaction with Compound 6 ((5)-3-((5)-2-amino-3-oxo-4-(2, 3,5,6- tetrafluorophenoxy)butyl)-pyrrolidin-2-one) in the final step. The product (20 mg, 30.51 pmol, 5.65% yield, 94.81% purity) was obtained as a white solid. LCMS (ESI): m/z: [M + H] calcd for C3oH32F4N30?:622.2; found 622.2. The observed isomeric ratio, as determined by SFC, was 5.9: 23.7: 1. ^XH NMR (400 MHz, methanol-^) d 0.92 - 1.09 (m, 6 H) 1.62 - 1.97 (m, 5 H) 2.04 - 2.18 (m, 1 H) 2.26 - 2.63 (m, 2 H) 3.96 (s, 3 H) 4.07 - 4.45 (m, 1 H) 4.56 - 4.76 (m, 2 H) 5.11 - 5.26 (m, 1 H) 6.64 - 6.88 (m, 1 H) 6.91 - 7.24 (m, 2 H) 7.33 - 7.44 (m, 1 H) 7.46 - 7.57 (m, 1 H).

Example 24. Preparation of 4,4-difluorocyclohexyl ((S)-4-methyl-l-oxo-l-(((S)-3-oxo-l- ((S)-2-oxopyrrolidin-3-yl)-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)amino)pentan-2- yl)carbamate (124)

[0136] The title compound was prepared as described in Example 7, using 4,4- difluorocyclohexyl carbonochloridate in place of lH-indole-2-carboxylic acid (Compound 28) in the first step. The 4,4-difluorocyclohexyl carb onochlori date was obtained in 68.55% yield via reaction of 4,4-difluorocyclohexan-l-ol with triphosgene in di chloromethane solution containing pyridine (2.8 eq). The title compound (10 mg, 16.41 pmol, 4.01% yield, 100% purity) was obtained as a white solid. LCMS (ESI): m/z: [M + H] calcd for C27H34F6N3O6: 610.3; found 610.3. The observed enantiomeric excess, as determined by SF was 87.1%. ’H NMR (400 MHz, methanol-^) 3 0.83 - 1.10 (m, 6 H) 1.20 - 1.59 (m, 3 H) 1.65 - 2.19 (m, 12 H) 2.20 - 2.65 (m, 2 H) 2.97 - 3.29 (m, 1 H) 3.34 - 3.49 (m, 1 H) 3.69 - 4.45 (m, 2 H) 4.63 - 4.81 (m, 1 H) 5.16 (d, J= 8.58 Hz, 1 H) 7.12 (ddd, J= 10.46, 7.24, 3.28 Hz, 1 H).

Example 25. Preparation of (lR,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-N-((S)-3-oxo-l-((S)-2-oxopyrrolidin-3-yl)-4- (2,3,6-trifluorophenoxy)butan-2-yl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (125)

[0137] Compound 38: To a mixture of Compound 36 (2 g, 15.25 mmol, 1 eq) in MeOH (9 mL) was added TEA (2.18 g, 21.55 mmol, 3.00 mL, 1.41 eq) and Compound 37 (2.03 g, 15.86 mmol, 1.60 mL, 1.04 eq) at 25°C, and the mixture was stirred at 25°C for 12 hrs. TLC (Petroleum ether: Ethyl acetate = 0: 1, Rf = 0.43) showed that the reaction was completed. The residue was concentrated under reduced pressure to afford Compound 38 (3.2 g, 9.75 mmol, 63.91% yield, TEA) as a white solid. ^XH NMR (400 MHz, methanol-t/i) 3 1.01 (s, 9 H) 4.17 (s, 1 H).

38 40 [0138] Compound 40: To a mixture of Compound 38 (1 g, 3.05 mmol, 1 eq, TEA) in toluene (1 mL) and DMF (1 mL) was added Compound 39 (187.91 mg, 913.60 pmol, 0.3 eq, HC1), EDCI (758.94 mg, 3.96 mmol, 1.3 eq) and NMM (770.07 mg, 7.61 mmol, 837.03 uL, 2.5 eq) at 25°C. The mixture was stirred at 25°C for 5 hrs. LCMS showed that the reaction was completed. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75x30mmx3pm; mobile phase: [water (lOmM NH4HCO3)-ACN]; B%: 40%-60%, 12min) to afford Compound 40 (0.31 g, 819.27 pmol, 26.90% yield) as a white solid. ^TH NMR (400 MHz, chloroforms/) 3 0.98 - 1.04 (m, 7 H) 1.08 (s, 5 H) 1.49 - 1.52 (m, 1 H) 1.54 - 1.56 (m, 1 H) 3.72 (s, 1 H) 3.78 (s, 1 H) 3.94 (dd, J= 10.19, 4.06 Hz, 1 H) 4.09 (dd, J= 10.08, 5.48 Hz, 1 H) 4.30 - 4.49 (m, 1 H) 4.55 - 4.64 (m, 1 H) 6.79 - 6.92 (m, 1 H).

[0139] Compound 41: To a mixture of Compound 40 (0.3 g, 792.84 pmol, 1 eq) in H2O (1 mL) and MeOH (1 mL) was added LiOH.H2O (49.91 mg, 1.19 mmol, 1.5 eq) at 25°C, and the mixture was stirred at 25°C for 12 hrs. LCMS showed that the reaction was completed. The mixture was combined with IM HC1 (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford Compound 40 (0.21 g, 576.35 pmol, 72.69% yield) as a white solid.

[0140] (lR,2S,5S)-3-((S)-3,3-Dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6- dimethyl-N-((S)-3-oxo-l-((S)-2-oxopyrrolidin-3-yl)-4-(2,3,6-trifluorophenoxy)butan-2- yl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (125): Compound 42 was prepared as described for Compound 6 in Example 1, using 2,3,6-trifluorophenol in place of 2, 3,4,6- tetrafluorophenol. To a solution of Compound 41 (0.1 g, 274.45 pmol, 1 eq) and Compound 42 (118.10 mg, 274.45 pmol, 1 eq, TFA) in DMF (1 mL) was added HATU (208.71 mg, 548.91 pmol, 2 eq) and DIPEA (141.89 mg, 1.10 mmol, 191.22 uL, 4 eq) at 25°C. The mixture was stirred at 25°C for Ihr. LCMS showed that the reaction was completed. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge Prep OBD Cl 8 150x40mmx 10pm; mobile phase: [water (NH₃H2O+NH4HCO₃)-ACN]; B%: 25%-65%, 8min) to afford the product 125 (80 mg, 115.14 pmol, 41.95% yield, 95.37% purity) as yellow solid. LCMS (ESI): m/z: [M + H] calcd for C₃oH₃7F₆N406:663.3; found 663.3; RT = 3.152 min. The observed enantiomeric excess, as determined by SFC, was 11.6%. ^TH NMR (400 MHz, methanol-t/4) d 0.92 - 1.09 (m, 15 H) 1.42 - 1.48 (m, 1 H) 1.57 - 1.65 (m, 1 H) 1.71 - 1.86 (m, 1 H) 1.86 - 1.98 (m, 1 H) 2.01 - 2.16 (m, 1 H) 2.29 - 2.41 (m, 1 H) 2.60 - 2.70

(m, 1 H) 3.23 - 3.30 (m, 1 H) 3.32 - 3.37 (m, 1 H) 3.64 - 3.88 (m, 1 H) 3.93 - 4.13 (m, 1 H)

4.20 - 4.31 (m, 1 H) 4.32 - 4.43 (m, 1 H) 4.55 - 4.69 (m, 1 H) 4.69 - 4.84 (m, 1 H) 5.07 - 5.12

(m, 1 H) 6.95 - 7.04 (m, 2 H).

Example 26. Assay of SARS-CoV2 3CL^pro Inhibition

[0141] Compounds were prepared as 30 mM stock solutions in DMSO. Compounds were tested for their potency in inhibiting the cysteine protease activity of SARS-CoV-2 Mpro by monitoring the cleavage of a fluorogenic substrate (see, Reaction Biology SARS-CoV-2 MPro protease assay, Catalog No. Mpro) in 10-dose ICso curves with 3 -fold serial dilutions starting at a high test concentration of 10 pM. The substrate was included at a final concentration of 5 pM. Protease activity was measured as a time-course increase in fluorescence signal from the fluorogenic peptide substrate, and the initial linear portion of the slope (signal/min) was analyzed. Data were assess in terms of Hill slope, % enzyme activity with no inhibitor control as 100% activity, and ICso. Compound binding and inhibition was unaffected by the presence or absence of reducing agent (DTT), which was added to mimic the reducing environment of the cell and to prevent protease active site oxidation. Activity was compared to the activity of reference compound (2S)-2-((S)-2- (((benzyloxy)carbonyl)amino)-4-methylpentanamido)-l -hydroxy-3 -(fS')-2-oxopyrroli din-3- yl)propane-l -sulfonate (001). The compounds were potent inhibitors of SARS-CoV-2 3CL^pro as summarized in Table 1, with several compounds exhibiting IC50 values well below 15 nM.

Table 1

+++ : 1 nM < ICso < 15 nM

++ : 15 nM < IC50 < 150 nM

+: 150 nM < IC50 < 300 nM

Example 27. Main protease inhibition in various coronaviruses.

[0142] IC50 values were determined for compound 107 against the main proteases of SARS-CoV2, 229E, OC43, MERS, SARS, HKU1, and NL63 coronaviruses, using a FRET- based substrate cleavage assay to measure inhibition of the isolated Mpro/3CLpro proteases from each of these strains. Under the assay conditions employed, the measured IC50 values ranged from 7.9 nM to 96 nM. The potency of compound 107 was approximately 2- to 3.5- fold higher than the potency of comparator 001 for the main proteases of SARS-CoV2, 229E, MERS, HKU1, and NL63. The data indicate broad activity of these inhibitors against the Mpro/3CLpro proteases across most strains of coronavirus. Example 28. Effect of inhibitors on SARS-CoV-2 viral replication [0143] SARS-CoV-2, USA-WA1/2020, stocks were prepared by passaging the virus in Vero 76 cells using test media of MEM supplemented with 2% FBS and 50 pg/mL gentamicin. Test compounds were solubilized in DMSO to prepare 50 mM stock solutions. Compounds were serially diluted from the starting (high) test concentration of 10 pM. Each dilution was added to 5 wells of a 96-well plate with 80-100% confluent Vero 76 cells. Three wells of each dilution were infected with virus, and two wells remained uninfected to assess cytotoxicity of compounds. Six wells were infected and untreated as virus controls, and six wells were uninfected and untreated as cell controls. SARS-CoV-2 was prepared to achieve the lowest possible multiplicity of infection (MOI) that would yield >80% cytopathic effect (CPE) within 5 days. This assay assesses multiple rounds of viral infection, replication, and virus production. Plates were incubated at 37±2°C, 5% CO2. On day 5 post-infection, once untreated virus control wells reached maximum CPE, plates were stained with neutral red dye for approximately 2 hours (±15 minutes). Incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol for >30 minutes and the optical density was read on a spectrophotometer at 540 nm. This assay assessed virus-induced CPE and the ability of the compounds to inhibit this. Optical densities were converted to percent of cell controls and normalized to the virus control, then the concentration of test compound required to inhibit CPE by 50% (ECso) and 90% (EC90) was calculated by regression analysis.

[0144] For virus yield reduction assays, the supernatant fluid from wells treated with and without each compound at all tested concentrations was collected on day 3 post infection, before neutral red staining (3 wells pooled) and tested for virus titer using a standard endpoint dilution CCID50 assay and titer calculations using the Reed-Muench (1948) equation. The concentration of compound required to reduce virus yield by 1 log 10 was calculated by regression analysis (EC90).

[0145] Compounds provided herein exhibited high potency in viral replication assays, as assessed by EC90 values. Surprisingly, compound 102 exhibited a 3.7-fold improvement in the viral replication assay (EC90) as compared to reference compound 001, even though the IC50 of compound 102 for in vitro 3CL^pro inhibition was around an order of magnitude higher than the IC50 of reference compound 001. A similar increase in potency was observed for compound 102 in the CPE-based viral replication assay described above, as compared to control compound 001. [0146] Antiviral activity for compounds described herein is summarized in Table 2. Viral- induced CPE assay ECso values and virus yield reduction (VYR) EC90 values (concentration calculated to reduce virus yield by 1 log) were determined at three days. 50% cytotoxic concentration values (CC50) are also included. The observed antiviral potency for the compound was consistent with 3CLpro inhibitor activity. Antiviral activity was increased in the presence of CP- 100356, a Pgp inhibitor. Compounds according to the present disclosure exhibited significantly higher potency vs. known comparators remdesivir and 001. CC50 (50% cytotoxic concentration) values were determined to assess cytotoxicity. The CC50 values determined for compounds 105 and 107 were in the range of 17-19 pM. CC50 values determined for the other compounds in the table were greater than 30 pM.

Table 2

fft : IC50/EC90 < 500 nM ft : 500 nM < IC50/EC90 < 1 pM f: 1 pM < IC50/EC90

Example 29. Effect of inhibitors on SARS-CoV-2 viral replication in human lung cells. [0147] Compounds 107, 110, 116, and 117 were tested in a cell-based antiviral assay employing a SARS CoV-2 reporter virus with nanoluciferase inserted at ORF7 in the viral genome. The cell-based assay measures nanoluciferase enzyme activity as an index of viral replication in the human lung cell line A549 cells expressing ACE2 at 72 hours post infection. [0148] Test article preparation: Working stock solutions of each compound were prepared by diluting stock solutions (10 mM in DMSO) to 33.3 pM for compound 107 and to 100 pM for compounds 110, 116, and 117. An 80 pL aliquot of each working stock sample was transferred into wells of an empty ECHO plate (Labcyte cat # P-05525). These samples were diluted 2-fold by transferring 40 pL of each into an adjacent well containing 40 pL media and mixing. This process was repeated to create 8 more wells of serially diluted sample, each well containing a 2-fold diluted sample of the previous well. A 90-nL aliquot for each sample was then dispensed into corresponding wells of assay ready plates (ARPs) using an ECHO555 acoustic liquid handling system. The final assay concentration range in the assay was 0.2-100 nM for compound 107 and 0.6-300 nM for compounds 110, 116, and 117.

[0149] Measurement of antiviral activity: A549 cells expressing ACE-2 were grown in DMEM high glucose supplemented with 20% HI FBS, 1% NEAA, 100 pg/ml blasticidin and split 1 :6 twice per week. Blasticidin was removed from the media one passage before using the cells in the assay. On the day of assay, the cells are harvested in DMEM supplemented with 2% HI FBS, 1% HEPES, 1% Pen/Strep. Assay ready plates (ARPs) pre-drugged with test compounds were prepared in the BSL-2 lab by adding 5 pL assay media to each well. The plates and cells were then passed into the BSL-3 facility. A working stock of SARS CoV-2 nanoluciferase reporter virus (NLRV) passaged five times in A549 cells expressing ACE2 was diluted 6000-fold in media containing 160,000 cells per mL (MOI ~ 0.002) and stirred at 200 RPM for approximately 10 minutes. A 25 pL aliquot of virus inoculated cells (4000 cells) was added to each well in columns 3-24 of the assay plates. The wells in columns 23-24 did not contain test compounds, only virus infected cells for the 0% inhibition controls. Prior to virus inoculation, a 25 pL aliquot of cells was added to columns 1-2 (no test compounds) of each plate for the cell only 100% inhibition controls. After incubating plates at 37°C/5% CO2 and 90% humidity for 72 hours, 30 pL of NanoGio (Promega) was added to each well. Luminescence was read using a BMG CLARIOstar plate reader (bottom read) following incubation at room temperature for 10 minutes to measure luciferase activity as an index of virus titer. Plates were sealed with a clear cover and surface decontaminated prior to luminescence reading.

[0150] ECso values determined for compounds 107, 110, 116, and 117 in A549 human lung cells ranged from 22 nM to 35 nM. The EC50 value determined for remdesivir using the same assay conditions was 130 nM. Example 30. Inhibitor pharmacokinetics and selectivity.

[0151] Compounds 107 and 116 were administered to laboratory mice intravenously (1 mg/kg), orally (5 mg/kg), subcutaneously (5 mg/kg), and intranasally (5 mg/kg). The compounds were formulated in DMSO/PEG300/solutol/water (v:v:v:v, 5:20:5:70) for IV and oral administration, and in 0.5% HPMC (4000 cps), 0.5% polysorbate, 10 mM PBS (pH 7.4) for subcutaneous and intranasal administration. Cmax values of 51.4 ng/mL (po), 424 ng/mL (sc), and 737 ng/mL (in) were observed for compound 107; Cmax values of 538 ng/mL (sc) and 443 ng/mL (in) were observed for compound 116. The data indicate that this series of compounds provide efficacious plasma concentrations for treatment of coronavirus infection in vivo.

[0152] The selectivity of compounds 107, 110, 116, and 117 was assessed and is representative of inhibitors in this series of compounds. Selectivity was analyzed using an industry standard Adverse Enzyme Panel Screen of enzymes as well as a series of cellular proteases. The screen was performed at a single concentration (10 pM) for each compound. A high level of selectivity for the SARS-CoV-2 3Clpro/Mpro protease against off-targets was identified. The only off-target activity at any level was identified on a set of metabolic enzymes (cytochrome P450 1A2/3A4/2C19 and UGT1A1) as well a small subset of the cysteine cathepsin family of proteases and calpain-1. No other off-target activities were identified on other cellular proteases or pharmacologically important enzymes.

VI. Exemplary Embodiments

[0153] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. A compound according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R³ is selected from the group consisting of H and Ci-6 alkyl;

R⁶ is selected from the group consisting of Ce-io aryl, 5- to 12-membered heteroaryl, -OR⁷, -NHR⁷, -NHC(O)OR⁷, and -CHR⁷NHC(O)R⁷, wherein Ce-io aryl, 5- to 12-membered heteroaryl are optionally substituted with one or more R^6a; each R^6a is independently selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, hydroxy, C3-8 cycloalkyl, and Ce-io aryl; or R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with -NHR⁷ or -NHC(O)OR⁷;

R⁷ is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, and Ce-io aryl, each of which is optionally substituted with one or more R^7a; each R^7a is independently selected from the group consisting of halogen, C1-6 alkyl, Ce-io aryl, and C3-8 cycloalkyl, wherein C1-6 alkyl, Ce-io aryl, and C3-8 cycloalkyl are optionally substituted with one or more R^7b; and each R^7b is an independently-selected halogen.

2. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein R² is selected from the group consisting of 2-oxopyrrolidin-3-yl, 5- oxopyrrolidin-3-yl , and 2,5-dioxopyrrolidin-3-yl. 3. The compound of embodiment 1 or embodiment 2, wherein R¹ is selected from the group consisting of phenyl, hexafluoroisopropyl, 2,6-dimethylpipiderin-4- yl, 2-methylpyrimidin-5-yl, and isoxazol-3-yl, wherein phenyl is substituted with 1-5 independently-selected halogen.

4. The compound of any one of embodiments 1-3, having a structure according to Formula la:

or a pharmaceutically acceptable salt thereof.

5. The compound of any one of embodiments 1-4, having a structure according to Formula lb:

or a pharmaceutically acceptable salt thereof, wherein subscript n is an integer ranging from 1 to 5.

6. The compound of any one of embodiments 1-5, or a pharmaceutically acceptable salt thereof, wherein subscript n is 4 or 3.

7. The compound of any one of embodiments 1-6, or a pharmaceutically acceptable salt thereof, wherein each R^la is fluorine.

8. The compound of any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein R³ is H

9. The compound of any one of embodiments 1-8, or a pharmaceutically acceptable salt thereof, wherein R⁴ is Ci-6 alkyl, which is optionally substituted with one or more R^4a. 10. The compound of embodiment 9, or a pharmaceutically acceptable salt thereof, wherein R^4a is C3-8 cycloalkyl or halogen-substituted Ce-io aryl.

11. The compound of any one of embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein R⁵ is H or -CH3.

12. The compound of any one of embodiments 1-11, or a pharmaceutically acceptable salt thereof, wherein R⁶ is selected from the group consisting of indol-2-yl, benzofuran-2-yl, and benzyloxy, each of which is optionally substituted with one or more R^6a.

13. The compound of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein R⁶ is substituted with C1-6 alkoxy.

14. The compound of any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with - NHR⁷ or -NHC(O)OR⁷.

15. The compound of embodiment 14, or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁶ are taken together with the atoms to which they are attached to form 3-oxo-3,4-dihydropyrazin-2-yl or 2-oxo-l,2-dihydropyri din-3 -yl, each of which is substituted with -NHR⁷ or -NHC(O)OR⁷.

16. The compound of embodiment 1, which is selected from any of the compounds disclosed herein or a pharmaceutically acceptable salt thereof.

17. The compound of embodiment 1, which is selected from any single enantiomer or diastereomer of a compound disclosed herein or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition comprising a compound of any one of embodiments 1-17 and a pharmaceutically acceptable excipient.

19. A method for treating a coronavirus infection, the method comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1-17, or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of a pharmaceutical composition according to embodiment 18, to a subject in need thereof.

20. The method of embodiment 19, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV OC43, and HCoV NL63.

21. The method of embodiment 20, wherein the coronavirus is SARS- CoV-2.

22. The method of any one of embodiments 19-21, wherein the subject has coronavirus disease 2019 (COVID-19).

23. The method of any one of embodiments 19-22, wherein the compound is administered orally, intranasally, or via injection.

24. The method of any one of embodiments 19-23, further comprising administering one or more agents selected from the group consisting of an anti-inflammatory agent, an analgesic agent, an antiviral agent, and an antitussive agent to the subject.

25. The method of any one of embodiments 19-24, further comprising administering a CYP3A4 inhibitor to the subject.

26. The method of any one of embodiments 19-25, wherein the subject is a human, an agricultural animal, or a companion animal.

27. A method for inhibiting a coronavirus main protease, the method comprising contacting the protease with an effective amount of a compound according to any one of embodiments 1-17.

28. The method of embodiment 27, wherein the coronavirus main protease is SARS-CoV-2 3CL^pro.

[0154] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A compound according to Formula I:

R⁵ o r^R2 ^RY^NY^ I^

O R⁴ R³ O ^OR1 (I), or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from the group consisting of substituted phenyl, unsubstituted phenyl, other Ce-io aryl, 5- to 12-membered heteroaryl, and Ci-6 haloalkyl, wherein: phenyl is substituted with 4, 3, 2, or 1 R^la, Ce-io aryl is substituted with one or more R^la, and

5- to 12-membered heteroaryl is optionally substituted with one or more R^lb; each R^la is independently halogen; each R^lb is independently selected from the group consisting of halogen, Ci-3 alkyl, and C1-3 haloalkyl;

R² is selected from the group consisting of oxopyrrolidinyl, dioxopyrrolidinyl, pyrrolidinyl, piperidinyl, azepanyl, and -CH2C(O)NH2, wherein piperidinyl and azepanyl are optionally substituted with one or more oxo moieties;

R³ is selected from the group consisting of H and Ci-6 alkyl;

R⁴ is selected from the group consisting of propyl substituted with one or more R^4a, unsubstituted propyl, C1-2 alkyl, C4-6 alkyl, and Ce-io aryl, wherein C1-2 alkyl, C4-6 alkyl, and Ce-io aryl are optionally substituted with one or more R^4a; each R^4a is independently selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, and Ce-io aryl, each of which is optionally substituted with one or more R^4b; each R^4b is an independently-selected halogen;

R⁶ is selected from the group consisting of indolyl substituted with R^6a, other 5- to 12-membered heteroaryl, Ce-io aryl, -OR⁷, -NHR⁷, -NHC(O)OR⁷, and -CHR⁷NHC(O)R⁷, wherein Ce-io aryl and 5- to 12-membered heteroaryl are optionally substituted with one or more R^6a; each R^6a is independently selected from the group consisting of Ci-6 alkoxy, Ci-6 alkyl, hydroxy, C3-8 cycloalkyl, and Ce-io aryl; or R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with -NHR⁷ or - NHC(O)OR⁷;

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R² is selected from the group consisting of 2-oxopyrrolidin-3-yl, 5- oxopyrrolidin-3-yl , and 2,5-dioxopyrrolidin-3-yl.

3. The compound of claim 1, wherein R¹ is selected from the group consisting of phenyl, hexafluoroisopropyl, 2,6-dimethylpipiderin-4-yl, 2-methylpyrimidin-5- yl, and isoxazol-3-yl, wherein phenyl is substituted with 1-5 independently-selected halogen.

4. The compound of claim 1, having a structure according to Formula la:

or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, having a structure according to Formula lb:

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6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein subscript n is 4 or 3.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each R^la is fluorine.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is H

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁴ is Ci-6 alkyl, which is optionally substituted with one or more R^4a.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein R^4a is C3-8 cycloalkyl or halogen-substituted Ce-io aryl.

11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁵ is H or -CH3.

12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁶ is selected from the group consisting of indol-2-yl, benzofuran-2-yl, and benzyloxy, each of which is optionally substituted with one or more R^6a.

13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein R⁶ is substituted with C1-6 alkoxy.

14. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁶ are taken together with the atoms to which they are attached to form 5- to 12-membered heterocyclyl, which is optionally substituted with -NHR⁷ or -NHC(O)OR⁷.

15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁶ are taken together with the atoms to which they are attached to form 3-oxo-3,4-dihydropyrazin-2-yl or 2-oxo-l,2-dihydropyri din-3 -yl, each of which is substituted with -NHR⁷ or -NHC(O)OR⁷.

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16. The compound of claim 1, which is selected from the group consisting

2 of:

18. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient.

19. A method for treating a coronavirus infection, the method comprising administering a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

20. The method of claim 19, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV OC43, and HCoV NL63.

21. The method of claim 20, wherein the coronavirus is SARS-CoV-2.

22. The method of claim 19, wherein the subject has coronavirus disease 2019 (COVID-19).

23. The method of claim 19, wherein the compound is administered orally, intranasally, or via injection.

24. The method of claim 19, further comprising administering one or more agents selected from the group consisting of an anti-inflammatory agent, an analgesic agent, an antiviral agent, and an antitussive agent to the subject.

25. The method of claim 19, further comprising administering a CYP3A4 inhibitor to the subject.

26. The method of claim 19, wherein the subject is a human, an agricultural animal, or a companion animal.

27. A method for inhibiting a coronavirus main protease, the method comprising contacting the protease with an effective amount of a compound according to claim 1.

28. The method of claim 27, wherein the coronavirus main protease is SARS-CoV-2 3CL^pro.

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