WO2022265577A2 - Coronavirus enzyme modulators, methods of synthesis and uses thereof - Google Patents

Coronavirus enzyme modulators, methods of synthesis and uses thereof Download PDF

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WO2022265577A2
WO2022265577A2 PCT/SG2022/050408 SG2022050408W WO2022265577A2 WO 2022265577 A2 WO2022265577 A2 WO 2022265577A2 SG 2022050408 W SG2022050408 W SG 2022050408W WO 2022265577 A2 WO2022265577 A2 WO 2022265577A2
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optionally substituted
compound
mmol
equiv
alkyl
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WO2022265577A3 (en
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Cheng San Brian Chia
Qian Wen TAN
Subramanyam VANKADARA
Yi Yang SEE
Shuyi Pearly NG
Weijun Xu
Choon Heng LOW
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Agency For Science, Technology And Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates, in general terms, to coronavirus 3CL protease enzyme modulators, their methods of synthesis and uses thereof.
  • the enzyme modulators can be used for inhibiting coronavirus 3CL protease and thereby prevent replication of the virus.
  • Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses with the largest known viral RNA genomes (26 to 32 kilobases).
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • OC43, NL63, HKU1, 229E four human coronaviruses commonly cause mild-to moderate respiratory diseases in humans. It is therefore of strategic importance to increase the number of effective antivirals against coronaviruses by developing drugs against viral targets that are highly conserved within the family and crucial for virus replication.
  • the SARS-CoV-2 genome encodes for more than 20 proteins, with two proteases, the papain-like protease (PLpro) and 3C-like protease (3CLpro) that are vital for virus replication.
  • the first two open reading frames (ORFs) of the coronavirus genome encode for overlapping replicase polyproteins la (PP1A) and lab (PP1AB) that are approximately 450 kD and 750 kD respectively.
  • PP1A and PP1AB contain proteins that are required for virus replication and transcription.
  • Both PLpro and 3CLpro cleave PP1A and PP1AB into individual proteins.
  • Both virus polyproteins are cleaved at more than 10 junctions by 3CLpro to produce individual non-structural proteins critical for virus replication. It has been demonstrated that inhibiting 3CLpro and thus, proteolytic processing of virus polyproteins blocks viral replication effectively.
  • 3CLpro is a virally encoded protein that has no close human analogs, reducing the likelihood of drug
  • 3CLpro also known as the main protease
  • 3CLpro also known as the main protease
  • 3CLpro is considered a promising drug target since it is highly conserved across known coronavirus strains. Accordingly, designing inhibitors that are active against 3CLpro of various coronaviruses is highly desirable for future pandemic readiness.
  • Remdesivir is currently the only Food and Drug Administration-approved drug for the treatment of COVID-19. There is currently a lack of anti-viral drugs for use in treating COVID-19.
  • the present invention relates to modulators (and in particular inhibitors) of coronavirus 3-chymotrypsin-like protease (3CLpro).
  • the peptide-like modulators have an a-ketoamide moiety.
  • the peptide-like modulators also demonstrate inhibitory activity against various coronaviruses, including SARS-CoV-2, MERS, HCoV- 229E, HCoV-OC43 and MHVA59, by preventing replication.
  • the inhibitors also demonstrate inhibitory activity against the picornavirus 3Cpro, specifically coxsackievirus 3B 3Cpro which are cysteine proteases.
  • the present invention provides a compound of Formula (la) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl, or optionally
  • the compound is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof: ( l'a) wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalky
  • the compound is a compound of Formula (ll'a) pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; and R 7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally
  • R 7 is -N H 2 , -NH(methyl), -NH(ethyl), -NH(propyl), -NH(iso- propyl), -NH(cyclopropyl), -NH(cyclobutyl), -NH(cyclopentyl), -NH(cyclohexyl), - NH(benzyl), -N(methyl) 2 , -N(ethyl) 2 , -N(propyl) 2 , -N(iso-propyl) 2 , pyrrolidinyl, piperidinyl, azetidinyl, or aziridinyl.
  • R 2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyla Ikyl, optionally substituted aminosulfonyl and optionally substituted aminosulfonylalkyl.
  • the compound is a compound of Formula (Ill'b) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 3a , R 4 , R 5 , R 6 and R 7 are as disclosed herein; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • R 3 is selected from:
  • R 3a is selected from:
  • R 4 is selected from H, methyl, ethyl, propyl, isopropyl, and cyclopropyl.
  • R 5 is selected from:
  • R 6 is selected from:
  • the compound is a compound of Formula (IV'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R 4 is selected from H, optionally substituted alkyl; R 5 is selected from optionally substituted cycloalkyl; R 10 and R 11 are each independently selected from halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
  • the present invention also provides a method of synthesising a compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising : a) sequentially reacting a C-terminus an amino acid like compound comprising R 5 and an amino moiety of a compound comprising R 2 with a C-terminus and/or N-terminus of an amino acid like compound comprising R 3 ; and b) oxidising the intermediate of step a) with an oxidising agent in order to form R 1 .
  • a N-terminus of the amino acid like compound comprising R 5 is reacted with a carboxylate compound comprising R 6 .
  • the compound comprising R 2 comprises a lactamide moiety.
  • the method further comprises: a) oxidising a hydroxyl moiety of a compound comprising R 2 to an aldehyde; and b) reacting the aldehyde with an isocvano compound in order to form a lactamide compound comprising R 2 .
  • the present invention also provides a method of treating or preventing a virus infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), (l'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof.
  • the present invention also provides a use of a compound of Formula (I), (l'), (II), (ll'), (lll ), (lll' ), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a virus infection in a patient in need thereof.
  • the present invention also provides a compound of Formula (I), (l'), a (II), (ii), (III), (IIl ), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof for use in treating or preventing a virus infection in a patient in need thereof.
  • the virus infection is caused or associated with a virus selected from rhinovirus, Middle East Respiratory Syndrome coronavirus (MMRS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), common coronaviridae (including but not limited to OC43, HKU1, 229E and NL63), enterovirus, poliovirus, coxsackievirus, hepatitis A virus, foot-and-mouth disease virus (FMDV) belonging to the picornaviridae family and calicivirus from the caliciviridae family.
  • MMRS-CoV Middle East Respiratory Syndrome coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome coronavirus
  • common coronaviridae including but not limited to OC43, HKU1, 229E and NL63
  • enterovirus poliovirus
  • coxsackievirus hepatitis A virus
  • FMDV foot-and-mouth disease virus
  • Alkyl refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso- butyl, n-hexyl, and the like.
  • Halo or halogen refers to fluoro, chloro, bromo and iodo.
  • Aryl refers to an unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms.
  • aryl groups include phenyl, naphthyl and the like.
  • eroaryl refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (i.e. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within thS ring (and includes oxides of sulfur, selenium and nitrogen).
  • heteroaryl groups can have a single ring (e.g. pyridyl, pyrrolyl or N-oxides thereof or furyl) or multiple condensed rings (e.g. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).
  • a single ring e.g. pyridyl, pyrrolyl or N-oxides thereof or furyl
  • multiple condensed rings e.g. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl.
  • heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine,
  • Arylalkyl refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
  • Acyl refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Oxyacyl refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Amino refers to the group -NR"R" where each R" is independently hydrogen, sulfonyl, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of sulfonyl, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminoalkyl refers to the group -alkyl-NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminoacyl refers to the group -C(0)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Acylamino refers to the group -NR"C(0)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • “Amidoacyl” refers to the group -C(0)C(0)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein, or R"R" can be linked to form a N-heteroaryl or N-heterocyclyl.
  • Acyloxy refers to the groups -0C(0)-alkyl, -0C(0)-aryl, -C(0)0-heteroaryl, and -C(0)0-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Aminoacyloxy refers to the groups -OC(0)NR"-alkyl, -0C(0)NR"-aryl, -0C(0)NR"- heteroaryl, and -OC(0)NR"-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Oxyacylamino refers to the groups -NR"C(0)0-alkyl, -NR"C(0)0-aryl, -NR"C(0)0- heteroaryl, and NR"C(0)0-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Cycloalkyl refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 11 carbon atoms.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, indanyl, 1,2,3,4-tetrahydronapthalenyl and the like.
  • Heterocyclyl refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, R 2 or R 1 is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.
  • heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2, 3, 4-tetra hydroisoquinoline, 4,5,6,7-t
  • Sulfonyl refers to groups H-S(0) 2 -, amino-S(0) 2 -, alkyl-S(0) 2 -, cycloalkyl-S(0) 2 -, aryl-S(0) 2 -, heteroaryl-S(0) 2 -, and heterocyclyl-S(0) 2 -, where amino, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • “Sulfonylalkyl” refers to group sulfonyl-alkyl-, where sulfonyl and alkyl are as described herein.
  • a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from halo, hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalo
  • an "optionally substituted amino” group may include amino acid and peptide residues.
  • sequence analysis of SARS-CoV and SARS-CoV- 2 3CLpro shows about 96% identity in the protease amino acid sequences.
  • 3CLpro almost exclusively cleaves substrates after a Pl-Gln and its P2 substrate residue is typically a Leu, with exceptions such as Met, Val, lie and Phe.
  • the truncated protein fragments then fold into functional proteins/enzymes needed for viral replication.
  • a consensus cleavage site of P3-XLQ-(S/G/A/N)-P1' has been identified for 3CLpro across known coronaviruses.
  • the inventors have utilised this high sequence conservation and cleavage specificity amongst 3CLpro of known human coronaviruses to develop a pan- coronavirus 3CLpro inhibitor by i) mimicking the structure of Leu-Gin and ii) attaching an electrophilic moiety at its C-terminus which covalently binds to the protease's Cysl45, 3CLpro activity can be modulated, or at least inhibited.
  • the compounds are found to inhibit alpha and beta coronavirus including SARS Cov-2 virus.
  • the present invention provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally
  • the compound of Formula (I) is a compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally
  • R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
  • the compound of Formula (I) is a compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
  • the compound of Formula (I) is a compound of Formula (la) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted
  • the compound of Formula (I') is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
  • R 1 is selected from cyano, or optionally substituted amidoacyl
  • R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl
  • R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl
  • R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl
  • R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl
  • R 6 is selected from optionally substituted alky
  • the compound of Formula (I') is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof: wherein R 1 is selected from cyano, or optionally substituted amidoacyl; R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalky
  • R 1 is cyano. In other embodiment, R 1 is optionally substituted amidoacyl.
  • the optionally substituted amidoacyl can have a a-ketoamide structure or a structure selected from wherein R 7 , R 8 and R9 are as defined herein.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (II): wherein R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R 6 is selected from optionally substitute
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Il): wherein R 2 , R 3a , R 4 , R 5 , R 6 , and R 7 are as disclosed herein.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ila): wherein R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R 6 is selected from optionally substitute
  • R 7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'a)
  • R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl
  • R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl
  • R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl
  • R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl
  • R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'a): wherein R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R 6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; and R 7 is selected from optionally substituted amino, optionally substituted N-
  • R 7 is optionally substituted amino, or optionally substituted N- heterocyclyl.
  • R 7 is selected from -IMH 2 , optionally substituted alkylamino, optionally substituted cycloalkylamino, optionally substituted arylamino, optionally substituted arylalkylamino, optionally substituted heteroarylalkylamino, optionally substituted heterocycylalkylamino and optionally substituted N-heterocyclyl.
  • R 7 is selected from -NH 2 , optionally substituted C 1 -C 5 alkylamino, optionally substituted C 3 -C 6 cycloalkylamino, optionally substituted arylamino, optionally substituted aryl(C 1 -C 5 alkyl)amino, optionally substituted heteroaryl (C 1 -C 5 alkyl)amino, optionally substituted heterocycyl(C 1 -C 5 alkyl)amino and optionally substituted C 3 -C 6 N-heterocyclyl.
  • the amino is -NH 2 , or optionally substituted one or two times with methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, phenyl, tetrahydropyrany I methyl, oxazolylmethyl.
  • substituents can be further substituted by 1, 2 or 3 halogens such as fluoride, chloride and bromide.
  • the optionally substituted N-heterocyclyl is optionally substituted N- cyclobutyl or optionally substituted N-cyclohexanyl.
  • R 7 is -NH 2 , optionally substituted -NH(alkyl), optionally substituted -N(alkyl) 2 , optionally substituted -NH(cycloalkyl), or optionally substituted N-heterocyclyl.
  • R 7 is -NH 2 , optionally substituted -NH(C 1 -C 5 alkyl), optionally substituted -N(C 1 -C 5 alkyl>2, optionally substituted -NH(C3-Cs cycloalkyl), optionally substituted - NH(aryl-C 1 -C 5 alkyl) or optionally substituted C 3 -C 6 N-heterocyclyl.
  • R 7 is -NH 2 , -NH(methyl), -NH(ethyl), -NH(propyl), -NH(iso-propyl), - NH(cyclopropyl), -NH(cyclobutyl), -NH(cyclopentyl), -NH(cyclohexyl), -NH(benzyl), - NH(phenyl), -NH(tetrahydropyranylmethyl), -NH(oxazolylmethyl), -N(methyl) 2 , - N(ethyl) 2 , -N(propyl) 2 , -N(iso-propyl) 2 , or C3-Cs N-heterocyclyl.
  • the C 3 -C 6 N- heterocyclyl can be pyrrolidinyl, piperidinyl, azetidinyl, or aziridinyl.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (lib):
  • R 2 , R 3 , R , R 5 and R 6 are as disclosed herein;
  • R 8 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl;
  • R9 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl; or R 6 and R9 are linked to form an optionally substituted heterocyclyl, or optionally substituted heteroaryl.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'b): wherein R 2 , R 3a , R 4 , R 5 and R 6 are as disclosed herein;
  • R 8 is selected from H , optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl;
  • R9 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl; or R 8 and R9 are linked to form an optionally substituted heterocyclyl, or optionally substituted heteroaryl.
  • R 8 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl. In some embodiments, R 8 is selected from H , C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl. In other embodiments, R 8 is selected from H, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, or phenyl. In other embodiments, R 8 is H.
  • R9 is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted aryl, optionally substituted aryl(C 1 - C 5 alkyl). In other embodiments, R9 is selected from methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, or phenyl.
  • R 8 and R9 are linked to form an optionally substituted C 3 -C 6 heterocyclyl. In other embodiments, R 8 and R9 are linked to form optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted azetidinyl, or optionally substituted aziridinyl.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (lie): wherein R 2 , R 3 , R 4 , R 5 and R 6 are as disclosed herein.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (II'c): wherein R , R 3a , R , R 5 and R 6 are as disclosed herein.
  • the a-ketoamide moiety can act as a targeting ligand to reversibly bind with Cysl45 in the active site of 3CLpro to inhibit the protease activity, but having a higher binding affinity relative to the viral peptides.
  • Inhibition of 3CL proteases is expected to reduce the ability of the coronavirus to replicate inside a host cell. By disrupting the ability of the coronavirus to process its polyprotein precursors after the viral genome has been translated in a host cell, disease and symptoms resulting from rhinovirus and coronavirus infections can be treated and/or prevented.
  • the a-ketoamide moiety are also expected to similarly reversibly bind with 3Cpro, and thus can be also used for treating viruses in the picornaviridae family. This is based on the understanding that both 3CLpro and 3Cpro recognize the Gln- (Ser/Ala/Gly) sequence and also have a cysteine-histidine catalytic dyad at its active site that participate in peptide bond cleavage. In particular, the cyclopropylamino moiety of Formula (lie) and/or (II'ç) was found to be especially effective.
  • R 2 is selected from optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted C 4 -C 8 heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted amino(C 1 -C 6 alkyl), optionally substituted sulfonyl, and optionally substituted sulfonyl(C 1 -C 5 alkyl).
  • R 2 is selected from optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted C 4 -C 8 heteroaryl, optionally substituted sulfonylamino, optionally substituted sulfonylamino(C 1 -C 6 alkyl), optionally substituted aminosulfonyl and optionally substituted aminosulfonyl(C 1 -C 5 alkyl).
  • R 2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonylalkyl, optionally substituted sulfonylamino, optionally substituted sulfonylaminoalkyl, optionally substituted aminosulfonyl and optionally substituted aminosulfonylalkyl.
  • R 2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonylalkyl, and optionally substituted aminosulfonylalkyl.
  • R 2 is selected from optionally substituted C 3 -C 6 cycloalkyl having an amide moiety, optionally substituted (C 1 -C 5 alkyl)sulfonyl(C 1 -C 5 alkyl), optionally substituted sulfonylamino, optionally substituted sulfonylamino(C 1 -C 5 alkyl), optionally substituted aminosulfonyl and optionally substituted aminosulfonyl(C 1 -C 5 alkyl).
  • R 2 is selected from optionally substituted C 3 -C 6 cycloalkyl having an amide moiety, optionally substituted (C 1 -C 5 alkyl)sulfonyl(C 1 -C 5 alkyl), and optionally substituted aminosulfonyl(C 1 -C 5 alkyl).
  • R 2 is selected from optionally substituted phenyl, optionally substituted pyridinyl, optionally substituted sulfonylmethyl, optionally substituted cyclopentyl, and optionally substituted cycloalkyl having an amide moiety.
  • R 2 is optionally substituted cycloalkyl having an amide moiety.
  • R 2 is optionally substituted cycloalkyl having an amide moiety at the 2' position.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (III):
  • R 1 , R 3 , R 4 , R 5 and R 6 are as disclosed herein;
  • R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IIl): wherein R 1 , R 3a , R 4 , R 5 and R 6 are as disclosed herein; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ilia): wherein R 1 , R 3 , R 4 , R 5 and R 6 are as disclosed herein; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ill'a): wherein R 1 , R 3a , R 4 , R 5 and R 6 are as disclosed herein; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Illb): wherein R 3 , R 4 , R 5 , R 6 and R are as disclosed herein; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ill'b):
  • R 3a , R 4 , R 5 , R 6 and R 7 are as disclosed herein;
  • R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
  • R 10 and R 11 are each independently selected from F, Cl, Br, methyl, ethyl, propyl or iso-propyl. In other embodiments, R 10 and R 11 are each independently selected from F, methyl, ethyl, propyl or iso-propyl.
  • R 10 and R 11 are linked to form an optionally substituted cycloalkyl. In other embodiments, R 10 and R 11 are linked to form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • n 1 or 2.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IIIc): wherein R 3 , R 4 , R 5 , R 6 , R 10 and R 11 and n are as disclosed herein.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (III'c):
  • R 3a , R 4 , R 5 , R 6 , R 10 and R 11 and n are as disclosed herein.
  • R 3 is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted C 4 -C 8 heteroaryl. In other embodiments, R 3 is selected from optionally substituted C 1 -C 5 alkyl, or optionally substituted C 3 -C 6 cycloalkyl.
  • the cycloalkyl can be selected from cyclopentyl, cyclobutyl, cyclopropyl, and cyclohexyl.
  • the optional substituent can be halo, or C 1 -C 4 alkyl.
  • the alkyl can be optionally substituted with optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl.
  • the optional substituent can be halo, or C 1 -C 4 alkyl.
  • R 3 is selected from methyl, ethyl, trifluoroethyl, cyclohexanyl, cyclobutyl, cyclopropyl, cyclopentyl, and bicyclo[l.l.l]pentyl.
  • R 3 is selected from:
  • R 3 is selected from: In some embodiments, R 3a is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted C 4 -C 8 aryl, optionally substituted C 4 -C 8 heteroaryl. In other embodiments, R 3a is selected from optionally substituted C 1 -C 5 alkyl, or optionally substituted C 3 -C 6 cycloalkyl.
  • the cycloalkyl can be selected from cyclopentyl, cyclobutyl, cyclopropyl, and cyclohexyl.
  • the optional substituent can be halo, or C 1 -C 4 alkyl.
  • the alkyl can be optionally substituted with optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl.
  • the optional substituent can be halo, or C 1 -C 4 alkyl.
  • R 3a is selected from methyl, ethyl, trifluoroethyl, cyclohexanyl, cyclobutyl, cyclopropyl, cyclopentyl, and bicyclo[l.l.l]pentyl.
  • R 3a is selected from: In other embodiments, R 3a is selected from:
  • R 4 is selected from H, optionally substituted C 1 -C 5 alkyl, and optionally substituted C 3 -C 6 cycloalkyl. In other embodiments, R 4 is selected from H, and optionally substituted C 1 -C 5 alkyl. In other embodiments, R 4 is selected from H, optionally substituted methyl, optionally substituted ethyl, and optionally substituted cyclopropyl. In other embodiments, R 4 is selected from H, methyl, ethyl, propyl, isopropyl, and cyclopropyl.
  • N-alkylation for example, N-methylation
  • EC 50 values show that the EC 50 values are improved.
  • R 4 is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted aryl, optionally substituted C 4 -C 8 heteroaryl.
  • the aryl is phenyl.
  • R 4 is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 heterocyclyl, optionally substituted C 4 -C 8 heteroaryl.
  • R 4 is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, cyclopropylmethyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, indanyl, phenyl, benzyl, phenylethyl, diphenylmethyl, naphthalenyl, thiophenyl, pyridinyl, methylphenylmethyl, bisphenylmethyl, or phenylmethyl.
  • R 5 is selected from:
  • R 5 is selected from:
  • R 6 is selected from optionally substituted C 1 -C 5 alkyl, optionally substituted C 4 -C 8 aryl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 - C 8 heteroaryl, optionally substituted C 3 -C 6 heterocycyl.
  • R 6 is selected from optionally substituted methyl, optionally substituted ethyl, optionally substituted iso-propyl, optionally substituted iso-butyl, optionally substituted tert-butyl, optionally substituted cyclopropyl, optionally substituted phenyl, and optionally substituted phenylethyl.
  • R 6 is selected from:
  • R 6 is selected from:
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV):
  • R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl
  • R 4 is selected from H, optionally substituted alkyl
  • R 5 is selected from optionally substituted aryl, or optionally substituted cycloalkyl
  • R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl
  • n is an integer from 1 and 2.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IVa): wherein R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl; is selected from H, optionally substituted alkyl; R 5 is selected from optionally substituted aryl, or optionally substituted cycloalkyl; R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV'):
  • R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl
  • R 4 is selected from H, optionally substituted alkyl
  • R 5 is selected from optionally substituted cycloalkyl
  • R 10 and R 11 are each independently selected from H, halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl
  • n is an integer from 1 and 2.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV'a): wherein R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R 4 is selected from H, optionally substituted alkyl; R 5 is selected from optionally substituted cycloalkyl; R 10 and R 11 are each independently selected from H , halo or optionally substituted alkyl; or R 10 and R 11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V):
  • R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl
  • R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl
  • R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl
  • R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl
  • R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, optionally substituted cycloalkyl.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Va): wherein R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R 3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl,
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'): wherein R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl; and R
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'): wherein R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl.
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'a): wherein R 2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted hetero
  • the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'a): wherein R 2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyla Ikyl ; R 3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R 4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R 5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; and R 6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl.
  • the compound of Formula (I) is selected from:
  • the compound is selected from:
  • the compound is selected from:
  • the compound is selected from:
  • the present invention also provides a method of synthesising a compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising: a) sequentially reacting a C-terminus an amino acid like compound comprising R 5 and an amino moiety of a compound comprising R 2 with a C-terminus and/or N-terminus of an amino acid like compound comprising R 3 ; and b) oxidising the intermediate of step a) with an oxidising agent in order to form R 1 .
  • the method of synthesis can be via a general scheme as shown in general route A, B, D, E, F, H, and I.
  • the compound comprising R 2 comprises a lactamide moiety.
  • a N-terminus of the amino acid like compound comprising R 5 is reacted with a carboxylate compound comprising R 6 .
  • the amino acid like compounds can be coupled using amide coupling reagents.
  • amide coupling reagents For example,H ATU and DIPEA can be used.
  • the oxidation step is performed using Dess-Martin periodinane (DMP).
  • DMP Dess-Martin periodinane
  • primary alcohols are oxidised to aldehydes and secondary alcohols to ketones.
  • the method further comprises: a) oxidising a hydroxyl moiety of a compound comprising R 2 to an aldehyde; and b) reacting the aldehyde with an isocyano compound in order to form a lactamide compound comprising R 2 .
  • the isocyano compound has the functional group -N oC.
  • amino acid like compounds and intermediates can be protected using protecting groups.
  • the present invention also provides a method of treating and/or preventing a virus infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), (l'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof.
  • the present invention also provides a use of a compound of Formula (I), (l'), (II), (ll'), (III), (Ill'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating and/or preventing a virus infection in a patient in need thereof.
  • the present invention also provides a compound of Formula (I), (T), (II), (ll'), (III), (Ill'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof for use in treating and/or preventing a virus infection in a patient in need thereof.
  • the virus infection is caused at least in part by a virus in the picornaviridae family or coronaviridae family.
  • the virus infection is caused by a virus belonging to the following families: levi-, narna-, picorna-, dicistro- , marna-, sequi-, como-, poty-, calici-, astro-, noda-, tetra-, luteo-, tombus-, corona-, arteri-, roni-, flavi-, toga-, bromo-, tymo-, clostero-, flexi-, seco-, barna, ifla-, sadwa-, chera-, hepe-, sobemo- , umbra-, tobamo-, tobra-, hordei-, furo-, porno-, peclu-, beny- , ourmia-, and id
  • the virus infection is caused at least in part by the coxsackievirus from the family picornaviridae. Variations in virus family are also included within this scope.
  • the World Health Organisation recommends using a Greek naming system for SARS-CoV-2 variants.
  • the virus infection can be COVID-19, caused by SARS-CoV-2 and its variants thereof.
  • Viruses constantly change through mutation and these mutations can result in a new variant of the virus. Due to the mutations, some variants emerge and disappear, others persist, while some variants spread more easily and quickly.
  • Some variants of the SARS-CoV-2 coronavirus to date are alpha, beta, gamma, delta and omicron.
  • the variants can have sub-variants. For example, omicron has a few major offshoots (sublineages), including BA.l, BA.2 and BA.3.
  • the virus infection is caused or associated with a virus selected from rhinovirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), common coronaviridae (including but not limited to OC43, HKU1, 229E and NL63), enterovirus, poliovirus, coxsackievirus, hepatitis A virus, foot-and-mouth disease virus (FMDV) belonging to the picornaviridae family and calicivirus from the caliciviridae family.
  • the virus is alpha human coronaviruses.
  • the virus is beta human coronaviruses.
  • the virus is human enteroviruses A-D. In other embodiments, the virus is poliovirus. In other embodiments, the virus is coxackievirus. In other embodiments, the virus is echovirus. In other embodiments, the virus is hepatitis A virus. In other embodiments, the virus is calicivirus. In other embodiments, the virus is norovirus. In other embodiments, the virus is Norwalk virus.
  • the virus infection is caused or associated with coronavirus; i.e. coronavirus infection. In other embodiments, the virus infection is associated with rhinovirus. In other embodiments, the virus infection is from an RNA-based virus. In other embodiments, the virus infection is from a single-stranded RNA virus.
  • RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. This nucleic acid is usually single-stranded RNA (ssRNA). RNA viruses can be further classified according to the sense or polarity of their RNA into negative-sense and positive-sense. Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. As such, purified RNA of a positive-sense virus can directly cause infection though it may be less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not infectious by itself as it needs to be transcribed into positive- sense RNA; each virion can be transcribed to several positive-sense RNAs.
  • ssRNA single-stranded RNA
  • the virus infection is caused by a positive-sense, single-stranded RNA virus. In other embodiments, the virus infection is caused by a negative-sense, single-stranded RNA virus.
  • the method is a method of inhibiting progression of a virus infection in a patient in need thereof.
  • the use is a use in the manufacture of a medicament for inhibiting progression of a virus infection in a patient in need thereof.
  • the compound is for inhibiting progression of a virus infection in a patient in need thereof.
  • the compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') is an inhibitor of 3CL protease.
  • Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
  • the present invention includes within its scope cationic salts e.g. sodium or potassium salts, or alkyl esters (e.g. methyl, ethyl) of the phosphate group.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • prodrug any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention.
  • the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug.
  • pro-drug is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art.
  • Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G.
  • prodrugs of the compound can include a protecting group protecting the amino group, including but not limited to, Carbobenzyloxy (Cbz) group, p-Methoxybenzyl carbonyl (Moz or MeOZ) group, tert-Butyloxycarbonyl (BOC) group, 9-Fluorenylmethyloxycarbonyl (Fmoc) group, Acetyl (Ac) group, Benzoyl (Bz) group, Benzyl (Bn) group, Carbamate group, p-Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), p-Methoxyphenyl (PMP) group, Tosyl (Ts) group, Troc (trichlor
  • the compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention.
  • Methods of solvation are generally known within the art.
  • a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.
  • the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods.
  • Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
  • Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.
  • the compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition.
  • the formulation of such compositions is well known to those skilled in the art.
  • the composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
  • Carriers can include, for example, water, saline (e.g. normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added.
  • saline e.g. normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)
  • sodium lactate Ringer's solution sodium lactate Ringer's solution
  • dextrose dextrose
  • glycerol glycerol
  • ethanol ethanol
  • auxiliary substances such as wetting or emulsifying agents, buffers, and the like
  • Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining
  • the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1/2 or 3/8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).
  • a fine gauge hollow bore needle e.g., 30 gauge, 1/2 or 3/8 inch needle
  • a temporal approach e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens.
  • a person skilled in the art will appreciate that other means for injecting and/or administering the compound, composition or combinations to the vitreous of the eye can also be used.
  • These other means can include, for example, intravitreal medical delivery devices.
  • These devices and methods can include, for example, intravitreal medicine delivery devices, and biodegradable polymer delivery members that are inserted in the eye for long term delivery of medicaments.
  • These devices and methods can further include transscleral delivery devices.
  • solutions or suspensions of the compound, composition or combinations of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye.
  • Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.
  • the compound, composition or combinations of the invention may also be suitable for intravenous administration.
  • a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg/m 2 .
  • the compound, composition or combinations of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • the compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt, solvate or prodrug is orally administrable.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • a binder e.g. inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • the compound, composition or combinations of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum
  • pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum
  • mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • the compound, composition or combinations of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like.
  • suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Transdermal patches may also be used to administer the compounds of the invention.
  • the compound, composition or combination of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
  • composition or combination of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.
  • suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
  • Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
  • intermediate iii as a light yellow solid (1.85 g, 5.21 mmol).
  • intermediate iii (1.85 mg, 5.21 mmol) was added HCI (4M in 1,4-dioxane, 10 equiv.). The mixture was stirred for 1 h, before concentration under pressure to afford intermediate 1-C (1.52 g, 5.21 mmol) as a white solid.
  • intermediate v was purified by reversed-phase prep-HPLC to afford intermediate v as a colourless solid (123.20 mg, 49.5%).
  • acetone 3 mL
  • 5% aqueous NaHCCH 1.30 mL
  • TEMPO 69.92 mg, 0.447 mmol, 1.1 equiv.
  • a 5% aqueous solution of NaOCI 1.2 mL, 0.814 mmol, 2 equiv.
  • intermediate xv (175 mg, 0.47 mmol) was added HCI (4M in 1,4-dioxane, 10 equiv). The mixture was stirred for 1 h, before concentration under pressure to afford intermediate 1-H (119 mg, 0.47 mmol) as a white solid.
  • intermediate A-3 60 mg, 0.14 mmol, 1.0 equiv.
  • intermediate 1-A 40 mg, 0.14 mmol, 1.0 equiv.
  • /V,/V-diisopropylethylamine 126 ⁇ L, 0.72 mmol, 5.0 equiv.
  • HATU 55 mg, 0.14 mmol, 1.0 equiv.
  • reaction mixture was diluted with DMF (1 mL), filtered and the crude product was purified by Waters MDAP preparative H PLC reverse phase chromatography (20-70% MeCN/H 2 O, 0.1% formic acid) to afford compound 107 as white solid upon lyophilization (7.5 mg, 0.012 mmol).
  • intermediate 1-7 (39.9 mg, 0.117 mmol, 1.0 equiv.), HATU (53.5 mg, 0.141 mmol, 1.2 equiv.) and N,N V-diisopropylethylamine (0.14 ml, 0.821 mmol, 7.0 equiv.) at 0 °C and stirred at rt for 30 min.
  • the reaction mixture was diluted with EtOAc (5 mL) and washed with water (2 mL).
  • intermediate J-4 270 mg, 0.417 mmol, 1.0 equiv.
  • THF 2.1 mL
  • diethylamine 864 ⁇ L, 8.35 mmol, 20 equiv.
  • the reaction was stirred at room temperature for 0.5 h.
  • the reaction was concentrated under reduced pressure and purified by silica gel column chromatography (0-20% MeOH/CH 2 Cl 2 ) to afford intermediate J-5 as a yellow solid (195 mg, 0.459 mmol); MS (ESI) m/z 425.3 [C21H36N4O5 + H] + .
  • intermediate J-8 (126 mg, 0.236 mmol, 1.0 equiv.) in EtOAc (2.4 mL) was added acetic acid (16 ⁇ L, 0.284 mmol, 1.2 equiv.) followed by isocyanocyclopropane (19 ⁇ L, 0.284 mmol, 1.2 equiv.) at 0 °C.
  • acetic acid (16 ⁇ L, 0.284 mmol, 1.2 equiv.
  • isocyanocyclopropane (19 ⁇ L, 0.284 mmol, 1.2 equiv.) at 0 °C.
  • the reaction was warmed to room temperature and stirred for 17 h.
  • the reaction was concentrated under reduced pressure and the crude was purified by silica gel column chromatography (0- 10% MeOH/CH 2 Cl 2 ) to afford intermediate J-9 as a white solid (125 mg, 0.189 mmol);
  • intermediate L-1 130 mg, 0.456 mmol
  • 1,4-dioxane 0.4 mL
  • HCI 4 M in 1,4-dioxane, 1.5 mL
  • the reaction was reduced under pressure and dried under high vacuum overnight to afford intermediate L-2 as an off-white solid.
  • Some compounds herein may exist as mixtures of diastereomers with diastereomeric ratios of at least 9: 1.
  • the full-length gene encoding SARS-CoV-2 3CLpro from strain BetaCoV/Wuhan/WIV04/2019 was synthesized and cloned into Ndel and Xhol site of pET29a(+) vector by Genscript (Piscataway, NJ, USA), as described previously. 2
  • the full-length gene encoding a Human Coronavirus 229E (HCoV-229E, Accession X69721.1) was cloned in pET29a( + ) with a C-terminus His tag.
  • the codon optimized plasmid for E. coli expression was transformed into competent BL21(DE3) cells.
  • a single colony was picked to inoculate 10 mL of Terrific Broth (TB) supplemented with 50 mg/L Kanamycin and grown at 37 °C with shaking at 200 rpm.
  • the 10-mL inoculum was added to 1 L of TB with 50 mg /L Kanamycin and grown to an optical density at 600 nm of 2.5.
  • the culture was induced using 0.5mM Isopropyl b-D-l-thiogalactopyranoside (IPTG) and grown at 37 °C for an additional 2 hrs.
  • IPTG Isopropyl b-D-l-thiogalactopyranoside
  • the cell pellet was resuspended in lysis buffer (20mM Tris, pH 7.5, lOOmM NaCI, 2mM dithiothreitol (DTT) and 10 pg/mL DNase I), and lysed by sonication (25% amplitude, 2 seconds on/ 2 seconds off). Cell debris were removed by centrifugation at 39,191x g for 30 min at 4°C. The supernatant was loaded onto equilibrated HisTrap HP column (Cytiva). The column was washed with lysis buffer containing 5mM imidazole, followed by another wash with 30mM imidazole.
  • lysis buffer 20mM Tris, pH 7.5, lOOmM NaCI, 2mM dithiothreitol (DTT) and 10 pg/mL DNase I
  • Protein was eluted using buffer with 300mM imidazole and further purified by gel filtration chromatography on HiLoad 16/600 Superdex 200 prep grade column (Cytiva) with 20mM Tris, pH 7.5, lOOmM NaCI, 2mM DTT. Fractions from resulted peak were pooled and concentrated using centrifugal filter unit of 10,000-molecular-weight-cutoff. The purity and molecular weight of the protein was confirmed with SDS-PAGE and Mass Spectrometry (MS). A predominant peak at 34863 Da was observed by MS which matched with the calculated molecular weight of SARS-CoV-2 C-His-3CLpro without the N-terminal Methionine residue.
  • MS Mass Spectrometry
  • Recombinant MERS-CoV 3CLpro (accession K9N638) expressed in E.coli was purchased from R&D Systems (Cat No. E-719). The purity and molecular size of commercial MERS- CoV 3CL was confirmed in our lab by SDS-PAGE. Further validation by MS showed a predominant peak at 33361 Da that matched with theoretical molecular weight of 33360.3 Da.
  • the 3C protease domain from Non-Coronaviruses, Human Coxsackievirus B3, strain Beijing0811 (Accession GQ141875.1) and Norovirus Hu/GIi.6/GZ2010- L96/Guangzhou/CHN/2011 (Accession JX989075.1) were cloned into pET29a(+) and pET28a(+) vector, respectively.
  • the gene synthesis and protein purification were conducted by Genscript (Piscataway, NJ, USA). Briefly, the codon optimized plasmids for E. coli expression was transformed into competent BL21(DE3) cells.
  • the culture was induced at 0.5 mM IPTG and grown at 37°C for 4 hrs for CVB3 and overnight at 15°C for Norovirus. Both proteins were purified from soluble fraction using His affinity and size exclusion chromatography.
  • the CVB3 protease was stored in 50 mM Tris-HCI, 100 mM NaCI, 2 mM dithiothreitol, pH 7.5 and a similar storage buffer was used for Norovirus except the Tris-HCI concentration was 20mM with pH 8.0.
  • HRV-3C protease with a N-terminus His tag was purchased from Sigma-Aldrich (Cat no: SAE0045).
  • a highly sensitive FRET based protease assay was developed to identify inhibitors of 3CL proteases.
  • the substrate, Peptide 1 (Dabcyl)KTSAVLQSGFRKM(Glu)(EDANS) (2) was synthesized by Genscript.
  • the test compounds were 3-fold serially diluted in 100% DMSO to 15 concentrations, starting at 3.33 mM. 1.5 mI of the serially diluted compounds were transferred to a black 384 well assay plate (Cat. 781900, Greiner). 23.5 mI of 2.13X concentration of SARS-CoV-2 Chis-3CLpro, 229E-CoV Chis-3CLpro or MERS-CoV 3CLpro prepared in assay buffer was added to the compounds and incubated for 30 mins at 25°C.
  • the final assay contained 12.5 nM of SARS-CoV-2 Chis-3CLpro or 6 nM 229E-CoV Chis-3CLpro or 75 nM MERS-CoV 3CLpro with 6 mM peptide 1 substrate and 3% DMSO in assay buffer containing 50 mM HEPES at pH 7.5, 100 mM NaCI, and 0.01% Triton X-100 and ImM DTT.
  • the FRET signal was measured using an excitation wavelength of 340 nm (UV(TRF) 340/60 nm, Barcode 101), emission wavelength of 490 nm (DSPPsion 486/10 filter, Barcode 220) and Lance/DELFIA D400 single mirror (Barcode 412) on Envision plate reader (2104 Envision Multilabel Plate Readers, Perkin Elmer).
  • the dose-dependent inhibition curves were fitted with a variable slope using GraphPad Prism software (GraphPad, USA) to determine a compound's IC50.
  • a similar FRET assay was developed for the non-coronavirus 3C proteases.
  • the substrate peptides, Peptide 13: ⁇ Glu(Edans) ⁇ LEVLFQGP ⁇ Lys(Dabcyl) ⁇ , Peptide 12 ⁇ Glu(Edans) ⁇ DFHLQGP ⁇ Lys(Dabcyl) ⁇ 3 and Peptide 1 all synthesized by GenScript were used for HRV 3C, Norovirus 3C and CVB3 3C protease assays, respectively.
  • the compound dilution and assay buffer were similar to SARS-CoV-2 Chis-3CLpro assay.
  • the final assay condition for HRV 3C protease contained 100 nM of enzyme, 6 mM Peptide 13 with 1 hr incubation at 25°C.
  • the final assay condition for Norovirus 3C protease contained 1.25 mM of enzyme and 12.5 mM Peptide 12 and 1.5 hr incubation at 37°C.
  • the final assay condition for CVB3 protease contained 2.5 mM enzyme, 6 mM peptide 1 substrate 4 with 2 hrs incubation at 25°C.
  • MRC-5 ATCC® CCL-171TM cells. MRC-5 cells were seeded at a density of 10,000 cells per well into black, clear flat-bottom 96- well plates (Greiner) on the day before virus inoculation. Cells were infected with 229E or OC43 virus at multiplicity of infection (MOI) of 0.01 using 50 ⁇ l of virus inoculum.
  • MOI multiplicity of infection
  • virus inoculum was removed and 50mI of diluted test compounds in EMEM + 2% FBS, was added to each well.
  • the cells were then incubated at 35°C with 5% CO 2 for 4 days (229E) or 5 days (OC43).
  • Cell viability was measured with CellTiter Glo (Promega), post 4 or 5-day incubation according to manufacturer's protocol, using Tecan infinite M200 pro plate reader. Cytotoxicity of compounds was assessed in parallel on uninfected cells plated together with the cells for virus infection. The percent effect of test compounds at each concentration was calculated with the DMSO-treated uninfected control wells set as 100% cell viability and untreated virus-containing control wells as 0% virus inhibition.
  • Relative Cell-Titer Glo signals were plotted against log10 values of compound concentration.
  • concentration required for 50% inhibition of virus CPE (EC50 value) was determined using a 4-parameter logistic model in Prism 8.
  • Percent effect for cytotoxicity of test compounds on uninfected cells was calculated using values for DMSO-treated uninfected control wells set as 100% cell viability.
  • CC50 values were calculated using a 4-parameter logistic model in Prism 8 and selectivity index (SI) was calculated by dividing CC50 value over EC50 value for each compound.
  • A549-hACE2 cells that stably express the human angiotensin-converting enzyme 2 (hACE2) were plated at a density of 10,000 - 12,000 cells per well in white opaque 96- well plates (Corning). On the next day, cells were inoculated with SARS-CoV2-Nluc viruses (MOI 0.1) (Xie et a I . , 2020) together with diluted compounds in phenol red-free medium containing 2% FBS. 50pl of virus-compound mixture was added to each well and plates were incubated at 37°C with 5% C02for 48 hours. At 48 hours post-infection, 50mI of Nano-Glo luciferase substrates (Promega) were added to each well.
  • SARS-CoV2-Nluc viruses MOI 0.1
  • Xie et a I . , 2020 SARS-CoV2-Nluc viruses
  • 50pl of virus-compound mixture was added to each well and plates were incubated at 37°
  • Luciferase signals were measured using BioTek Synerge Neo2 plate reader (lid-on, Gain 100). The relative luciferase signals were calculated by normalizing the luciferase signals of compound-treated cells to that of the DMSO control wells (set as 100%). The relative luciferase signal was plotted against the loglO values of compound concentration. EC50 (compound concentration causing 50% reduction of luciferase signal) values were calculated using a 4-parameter logistic model in Prism 8.

Abstract

The present invention relates, in general terms, to coronavirus 3CL protease enzyme modulators, their methods of synthesis and uses thereof. In particular, the enzyme modulators can be used for inhibiting coronavirus 3CL protease and thereby prevent replication of the virus.

Description

Coronavirus Enzyme Modulators, Methods of Synthesis and Uses
Thereof
Technical Field
The present invention relates, in general terms, to coronavirus 3CL protease enzyme modulators, their methods of synthesis and uses thereof. In particular, the enzyme modulators can be used for inhibiting coronavirus 3CL protease and thereby prevent replication of the virus.
Background
Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses with the largest known viral RNA genomes (26 to 32 kilobases). The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in great morbidity and mortality in many countries worldwide. Until recently, there were no commercially available vaccines and there are few antiviral agents available to combat SARS-CoV-2 infections. The history of SARS-CoV-1, MERS and SARS-CoV-2 outbreaks in the past two decades suggests that future viral pandemics are inevitable. Additionally, four human coronaviruses (OC43, NL63, HKU1, 229E) commonly cause mild-to moderate respiratory diseases in humans. It is therefore of strategic importance to increase the number of effective antivirals against coronaviruses by developing drugs against viral targets that are highly conserved within the family and crucial for virus replication.
The SARS-CoV-2 genome encodes for more than 20 proteins, with two proteases, the papain-like protease (PLpro) and 3C-like protease (3CLpro) that are vital for virus replication. The first two open reading frames (ORFs) of the coronavirus genome encode for overlapping replicase polyproteins la (PP1A) and lab (PP1AB) that are approximately 450 kD and 750 kD respectively. PP1A and PP1AB contain proteins that are required for virus replication and transcription. Both PLpro and 3CLpro cleave PP1A and PP1AB into individual proteins. Both virus polyproteins are cleaved at more than 10 junctions by 3CLpro to produce individual non-structural proteins critical for virus replication. It has been demonstrated that inhibiting 3CLpro and thus, proteolytic processing of virus polyproteins blocks viral replication effectively. Additionally, 3CLpro is a virally encoded protein that has no close human analogs, reducing the likelihood of drug toxicity.
3CLpro, also known as the main protease, is considered a promising drug target since it is highly conserved across known coronavirus strains. Accordingly, designing inhibitors that are active against 3CLpro of various coronaviruses is highly desirable for future pandemic readiness.
Remdesivir is currently the only Food and Drug Administration-approved drug for the treatment of COVID-19. There is currently a lack of anti-viral drugs for use in treating COVID-19.
It would be desirable to overcome or ameliorate at least one of the above-described problems.
Summary
The present invention relates to modulators (and in particular inhibitors) of coronavirus 3-chymotrypsin-like protease (3CLpro). In certain embodiments, the peptide-like modulators have an a-ketoamide moiety. The peptide-like modulators also demonstrate inhibitory activity against various coronaviruses, including SARS-CoV-2, MERS, HCoV- 229E, HCoV-OC43 and MHVA59, by preventing replication. The inhibitors also demonstrate inhibitory activity against the picornavirus 3Cpro, specifically coxsackievirus 3B 3Cpro which are cysteine proteases.
The present invention provides a compound of Formula (la) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000003_0001
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000004_0001
( l'a) wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
In some embodiments, the compound is a compound of Formula (ll'a) pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000004_0002
wherein R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; and R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
In some embodiments, R7 is -N H2, -NH(methyl), -NH(ethyl), -NH(propyl), -NH(iso- propyl), -NH(cyclopropyl), -NH(cyclobutyl), -NH(cyclopentyl), -NH(cyclohexyl), - NH(benzyl), -N(methyl)2, -N(ethyl)2, -N(propyl)2, -N(iso-propyl)2, pyrrolidinyl, piperidinyl, azetidinyl, or aziridinyl.
In some embodiments, R2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyla Ikyl, optionally substituted aminosulfonyl and optionally substituted aminosulfonylalkyl.
In some embodiments, the compound is a compound of Formula (Ill'b) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000005_0001
wherein R3a, R4, R5, R6 and R7 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, R3 is selected from:
Figure imgf000006_0001
In some embodiments, R3a is selected from:
Figure imgf000006_0002
In some embodiments, R4 is selected from H, methyl, ethyl, propyl, isopropyl, and cyclopropyl.
In some embodiments, R5 is selected from:
In some embodiments, R6 is selected from:
Figure imgf000007_0001
In some embodiments, the compound is a compound of Formula (IV'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000007_0002
wherein R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R4 is selected from H, optionally substituted alkyl; R5 is selected from optionally substituted cycloalkyl; R10 and R11 are each independently selected from halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
The present invention also provides a method of synthesising a compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising : a) sequentially reacting a C-terminus an amino acid like compound comprising R5 and an amino moiety of a compound comprising R2 with a C-terminus and/or N-terminus of an amino acid like compound comprising R3; and b) oxidising the intermediate of step a) with an oxidising agent in order to form R1.
In some embodiments, a N-terminus of the amino acid like compound comprising R5 is reacted with a carboxylate compound comprising R6.
In some embodiments, the compound comprising R2 comprises a lactamide moiety.
In some embodiments, the method further comprises: a) oxidising a hydroxyl moiety of a compound comprising R2 to an aldehyde; and b) reacting the aldehyde with an isocvano compound in order to form a lactamide compound comprising R2.
The present invention also provides a method of treating or preventing a virus infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), (l'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof.
The present invention also provides a use of a compound of Formula (I), (l'), (II), (ll'), (lll ), (lll' ), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a virus infection in a patient in need thereof. The present invention also provides a compound of Formula (I), (l'), a (II), (ii), (III), (IIl ), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof for use in treating or preventing a virus infection in a patient in need thereof.
In some embodiments, the virus infection is caused or associated with a virus selected from rhinovirus, Middle East Respiratory Syndrome coronavirus (MMRS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), common coronaviridae (including but not limited to OC43, HKU1, 229E and NL63), enterovirus, poliovirus, coxsackievirus, hepatitis A virus, foot-and-mouth disease virus (FMDV) belonging to the picornaviridae family and calicivirus from the caliciviridae family.
Detailed description
"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso- butyl, n-hexyl, and the like.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
"Oxo/hydroxy" refers to groups =0, HO-.
"Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like. eroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (i.e. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within thS ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (e.g. pyridyl, pyrrolyl or N-oxides thereof or furyl) or multiple condensed rings (e.g. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl). Examples of heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine and the like.
"Arylalkyl" refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
"Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Amino" refers to the group -NR"R" where each R" is independently hydrogen, sulfonyl, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of sulfonyl, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminoalkyl" refers to the group -alkyl-NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminoacyl" refers to the group -C(0)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Acylamino" refers to the group -NR"C(0)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein. "Amidoacyl" refers to the group -C(0)C(0)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein, or R"R" can be linked to form a N-heteroaryl or N-heterocyclyl.
"Acyloxy" refers to the groups -0C(0)-alkyl, -0C(0)-aryl, -C(0)0-heteroaryl, and -C(0)0-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Aminoacyloxy" refers to the groups -OC(0)NR"-alkyl, -0C(0)NR"-aryl, -0C(0)NR"- heteroaryl, and -OC(0)NR"-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Oxyacylamino" refers to the groups -NR"C(0)0-alkyl, -NR"C(0)0-aryl, -NR"C(0)0- heteroaryl, and NR"C(0)0-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Cycloalkyl" refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 11 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, indanyl, 1,2,3,4-tetrahydronapthalenyl and the like.
"Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, R2 or R1 is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond. Examples of heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2, 3, 4-tetra hydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, and the like.
"Sulfonyl" refers to groups H-S(0)2-, amino-S(0)2-, alkyl-S(0)2-, cycloalkyl-S(0)2-, aryl-S(0)2-, heteroaryl-S(0)2-, and heterocyclyl-S(0)2-, where amino, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Sulfonylalkyl" refers to group sulfonyl-alkyl-, where sulfonyl and alkyl are as described herein.
In this specification "optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from halo, hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di- (substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues. Without wanting to be bound by theory, sequence analysis of SARS-CoV and SARS-CoV- 2 3CLpro shows about 96% identity in the protease amino acid sequences. 3CLpro almost exclusively cleaves substrates after a Pl-Gln and its P2 substrate residue is typically a Leu, with exceptions such as Met, Val, lie and Phe. The truncated protein fragments then fold into functional proteins/enzymes needed for viral replication. A consensus cleavage site of P3-XLQ-(S/G/A/N)-P1' has been identified for 3CLpro across known coronaviruses. The inventors have utilised this high sequence conservation and cleavage specificity amongst 3CLpro of known human coronaviruses to develop a pan- coronavirus 3CLpro inhibitor by i) mimicking the structure of Leu-Gin and ii) attaching an electrophilic moiety at its C-terminus which covalently binds to the protease's Cysl45, 3CLpro activity can be modulated, or at least inhibited. Advantageously, the compounds are found to inhibit alpha and beta coronavirus including SARS Cov-2 virus.
The present invention provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000013_0001
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl. In some embodiments, the compound of Formula (I) is a compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000014_0001
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I) is a compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000014_0002
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I) is a compound of Formula (la) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000015_0001
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000016_0001
herein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000016_0002
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
In some embodiments, R1 is cyano. In other embodiment, R1 is optionally substituted amidoacyl. The optionally substituted amidoacyl can have a a-ketoamide structure or a structure selected from
Figure imgf000017_0001
wherein R7, R8 and R9 are as defined herein.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (II):
Figure imgf000017_0002
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl; and R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl. In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Il):
Figure imgf000018_0001
wherein R2, R3a, R4, R5, R6, and R7 are as disclosed herein.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ila):
Figure imgf000018_0002
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl; and
R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'a)
Figure imgf000019_0001
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl; and R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'a):
Figure imgf000019_0002
wherein R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; and R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
In some embodiments, R7 is optionally substituted amino, or optionally substituted N- heterocyclyl. In other embodiments, R7 is selected from -IMH2, optionally substituted alkylamino, optionally substituted cycloalkylamino, optionally substituted arylamino, optionally substituted arylalkylamino, optionally substituted heteroarylalkylamino, optionally substituted heterocycylalkylamino and optionally substituted N-heterocyclyl. In other embodiments, R7 is selected from -NH2, optionally substituted C1-C5 alkylamino, optionally substituted C3-C6 cycloalkylamino, optionally substituted arylamino, optionally substituted aryl(C1-C5 alkyl)amino, optionally substituted heteroaryl (C1-C5 alkyl)amino, optionally substituted heterocycyl(C1-C5 alkyl)amino and optionally substituted C3-C6 N-heterocyclyl. In other embodiments, the amino is -NH2, or optionally substituted one or two times with methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, phenyl, tetrahydropyrany I methyl, oxazolylmethyl. These optional substituents can be further substituted by 1, 2 or 3 halogens such as fluoride, chloride and bromide. In other embodiments, the optionally substituted N-heterocyclyl is optionally substituted N- cyclobutyl or optionally substituted N-cyclohexanyl. In other embodiments, R7 is -NH2, optionally substituted -NH(alkyl), optionally substituted -N(alkyl)2, optionally substituted -NH(cycloalkyl), or optionally substituted N-heterocyclyl. In other embodiments, R7 is -NH2, optionally substituted -NH(C1-C5 alkyl), optionally substituted -N(C1-C5 alkyl>2, optionally substituted -NH(C3-Cs cycloalkyl), optionally substituted - NH(aryl-C1-C5 alkyl) or optionally substituted C3-C6 N-heterocyclyl. In other embodiments, R7 is -NH2, -NH(methyl), -NH(ethyl), -NH(propyl), -NH(iso-propyl), - NH(cyclopropyl), -NH(cyclobutyl), -NH(cyclopentyl), -NH(cyclohexyl), -NH(benzyl), - NH(phenyl), -NH(tetrahydropyranylmethyl), -NH(oxazolylmethyl), -N(methyl)2, - N(ethyl)2, -N(propyl)2, -N(iso-propyl)2, or C3-Cs N-heterocyclyl. The C3-C6 N- heterocyclyl can be pyrrolidinyl, piperidinyl, azetidinyl, or aziridinyl.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (lib):
Figure imgf000021_0001
wherein R2, R3, R , R5 and R6 are as disclosed herein;
R8 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl;
R9 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl; or R6 and R9 are linked to form an optionally substituted heterocyclyl, or optionally substituted heteroaryl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (ll'b):
Figure imgf000021_0002
wherein R2, R3a, R4, R5 and R6 are as disclosed herein;
R8 is selected from H , optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl;
R9 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl; or R8 and R9 are linked to form an optionally substituted heterocyclyl, or optionally substituted heteroaryl.
In some embodiments, R8 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl. In some embodiments, R8 is selected from H , C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl. In other embodiments, R8 is selected from H, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, or phenyl. In other embodiments, R8 is H.
In some embodiments, R9 is selected from optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted aryl, optionally substituted aryl(C1- C5 alkyl). In other embodiments, R9 is selected from methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexanyl, benzyl, or phenyl.
In some embodiments, R8 and R9 are linked to form an optionally substituted C3-C6 heterocyclyl. In other embodiments, R8 and R9 are linked to form optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted azetidinyl, or optionally substituted aziridinyl.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (lie):
Figure imgf000022_0001
wherein R2, R3, R4, R5 and R6 are as disclosed herein.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (II'c):
Figure imgf000022_0002
wherein R , R3a, R , R5 and R6 are as disclosed herein.
Without wanting to be bound by theory, it is thought that the a-ketoamide moiety can act as a targeting ligand to reversibly bind with Cysl45 in the active site of 3CLpro to inhibit the protease activity, but having a higher binding affinity relative to the viral peptides. Inhibition of 3CL proteases is expected to reduce the ability of the coronavirus to replicate inside a host cell. By disrupting the ability of the coronavirus to process its polyprotein precursors after the viral genome has been translated in a host cell, disease and symptoms resulting from rhinovirus and coronavirus infections can be treated and/or prevented. The a-ketoamide moiety are also expected to similarly reversibly bind with 3Cpro, and thus can be also used for treating viruses in the picornaviridae family. This is based on the understanding that both 3CLpro and 3Cpro recognize the Gln- (Ser/Ala/Gly) sequence and also have a cysteine-histidine catalytic dyad at its active site that participate in peptide bond cleavage. In particular, the cyclopropylamino moiety of Formula (lie) and/or (II'ç) was found to be especially effective.
In some embodiments, R2 is selected from optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted C4-C8 heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted amino(C1-C6 alkyl), optionally substituted sulfonyl, and optionally substituted sulfonyl(C1-C5 alkyl). In other embodiments, R2 is selected from optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted C4-C8 heteroaryl, optionally substituted sulfonylamino, optionally substituted sulfonylamino(C1-C6 alkyl), optionally substituted aminosulfonyl and optionally substituted aminosulfonyl(C1-C5 alkyl). In some embodiments, R2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonylalkyl, optionally substituted sulfonylamino, optionally substituted sulfonylaminoalkyl, optionally substituted aminosulfonyl and optionally substituted aminosulfonylalkyl. In some embodiments, R2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonylalkyl, and optionally substituted aminosulfonylalkyl. In some embodiments, R2 is selected from optionally substituted C3-C6 cycloalkyl having an amide moiety, optionally substituted (C1-C5 alkyl)sulfonyl(C1-C5 alkyl), optionally substituted sulfonylamino, optionally substituted sulfonylamino(C1-C5 alkyl), optionally substituted aminosulfonyl and optionally substituted aminosulfonyl(C1-C5 alkyl). In some embodiments, R2 is selected from optionally substituted C3-C6 cycloalkyl having an amide moiety, optionally substituted (C1-C5 alkyl)sulfonyl(C1-C5 alkyl), and optionally substituted aminosulfonyl(C1-C5 alkyl). In other embodiments, R2 is selected from optionally substituted phenyl, optionally substituted pyridinyl, optionally substituted sulfonylmethyl, optionally substituted cyclopentyl, and optionally substituted cycloalkyl having an amide moiety. In other embodiments, R2 is optionally substituted cycloalkyl having an amide moiety. In other embodiments, R2 is optionally substituted cycloalkyl having an amide moiety at the 2' position.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (III):
Figure imgf000024_0001
wherein R1, R3, R4, R5 and R6 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IIl):
Figure imgf000024_0002
wherein R1, R3a, R4, R5 and R6 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ilia):
Figure imgf000024_0003
wherein R1, R3, R4, R5 and R6 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ill'a):
Figure imgf000025_0001
wherein R1, R3a, R4, R5 and R6 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Illb):
Figure imgf000025_0002
wherein R3, R4, R5, R6 and R are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Ill'b):
wherein R3a, R4, R5, R6 and R7 are as disclosed herein; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
In some embodiments, R10 and R11 are each independently selected from F, Cl, Br, methyl, ethyl, propyl or iso-propyl. In other embodiments, R10 and R11 are each independently selected from F, methyl, ethyl, propyl or iso-propyl.
In some embodiments, R10 and R11 are linked to form an optionally substituted cycloalkyl. In other embodiments, R10 and R11 are linked to form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
In some embodiments, n is 1 or 2.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IIIc):
Figure imgf000026_0001
wherein R3, R4, R5, R6, R10 and R11 and n are as disclosed herein.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (III'c):
wherein R3a , R4, R5, R6, R10 and R11 and n are as disclosed herein.
In some embodiments, R3 is selected from optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted C4-C8 heteroaryl. In other embodiments, R3 is selected from optionally substituted C1-C5 alkyl, or optionally substituted C3-C6 cycloalkyl. The cycloalkyl can be selected from cyclopentyl, cyclobutyl, cyclopropyl, and cyclohexyl. The optional substituent can be halo, or C1-C4 alkyl. The alkyl can be optionally substituted with optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. The optional substituent can be halo, or C1-C4 alkyl. In other embodiments, R3 is selected from methyl, ethyl, trifluoroethyl, cyclohexanyl, cyclobutyl, cyclopropyl, cyclopentyl, and bicyclo[l.l.l]pentyl.
In other embodiments, R3 is selected from:
Figure imgf000027_0001
In other embodiments, R3 is selected from: In some embodiments, R3a is selected from optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted C4-C8 aryl, optionally substituted C4-C8 heteroaryl. In other embodiments, R3a is selected from optionally substituted C1-C5 alkyl, or optionally substituted C3-C6 cycloalkyl. The cycloalkyl can be selected from cyclopentyl, cyclobutyl, cyclopropyl, and cyclohexyl. The optional substituent can be halo, or C1-C4 alkyl. The alkyl can be optionally substituted with optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. The optional substituent can be halo, or C1-C4 alkyl. In other embodiments, R3a is selected from methyl, ethyl, trifluoroethyl, cyclohexanyl, cyclobutyl, cyclopropyl, cyclopentyl, and bicyclo[l.l.l]pentyl.
In other embodiments, R3a is selected from:
Figure imgf000028_0001
In other embodiments, R3a is selected from:
Figure imgf000029_0001
In some embodiments, R4 is selected from H, optionally substituted C1-C5 alkyl, and optionally substituted C3-C6 cycloalkyl. In other embodiments, R4 is selected from H, and optionally substituted C1-C5 alkyl. In other embodiments, R4 is selected from H, optionally substituted methyl, optionally substituted ethyl, and optionally substituted cyclopropyl. In other embodiments, R4 is selected from H, methyl, ethyl, propyl, isopropyl, and cyclopropyl.
The inventors have found N-alkylation (for example, N-methylation) can improve the pharmacokinetics of the compounds. For example, the cellular permeability of the compounds is improved, as shown by the EC50 values.
In some embodiments, R4 is selected from optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted aryl, optionally substituted C4-C8 heteroaryl. In some embodiments, the aryl is phenyl. In some embodiments, R4 is selected from optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 heterocyclyl, optionally substituted C4-C8 heteroaryl. In other embodiments, R4 is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, cyclopropylmethyl, cyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, indanyl, phenyl, benzyl, phenylethyl, diphenylmethyl, naphthalenyl, thiophenyl, pyridinyl, methylphenylmethyl, bisphenylmethyl, or phenylmethyl.
In other embodiments, R5 is selected from:
Figure imgf000030_0001
In other embodiments, R5 is selected from:
Figure imgf000030_0002
In some embodiments, R6 is selected from optionally substituted C1-C5 alkyl, optionally substituted C4-C8 aryl, optionally substituted C3-C6 cycloalkyl, optionally substituted C4- C8 heteroaryl, optionally substituted C3-C6 heterocycyl. In other embodiments, R6 is selected from optionally substituted methyl, optionally substituted ethyl, optionally substituted iso-propyl, optionally substituted iso-butyl, optionally substituted tert-butyl, optionally substituted cyclopropyl, optionally substituted phenyl, and optionally substituted phenylethyl.
In other embodiments, R6 is selected from:
Figure imgf000031_0001
In other embodiments, R6 is selected from:
Figure imgf000031_0002
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV):
wherein R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R4 is selected from H, optionally substituted alkyl; R5 is selected from optionally substituted aryl, or optionally substituted cycloalkyl; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IVa):
Figure imgf000032_0001
wherein R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl; is selected from H, optionally substituted alkyl; R5 is selected from optionally substituted aryl, or optionally substituted cycloalkyl; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV'):
wherein R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R4 is selected from H, optionally substituted alkyl; R5 is selected from optionally substituted cycloalkyl; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (IV'a):
Figure imgf000033_0001
wherein R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl; R4 is selected from H, optionally substituted alkyl; R5 is selected from optionally substituted cycloalkyl; R10 and R11 are each independently selected from H , halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 and 2.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V):
Figure imgf000034_0001
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (Va):
Figure imgf000034_0002
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'):
Figure imgf000035_0001
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'):
Figure imgf000035_0002
wherein R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'a):
Figure imgf000036_0001
wherein R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyl, optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I') or a pharmaceutically acceptable salt, solvate or prodrug thereof is a compound of Formula (V'a):
Figure imgf000036_0002
wherein R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted sulfonyla Ikyl ; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl.
In some embodiments, the compound of Formula (I) is selected from:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0002
In some embodiments, the compound is selected from:
Figure imgf000049_0001
In some embodiments, the compound is selected from:
In some embodiments, the compound is selected from:
Figure imgf000051_0001
The present invention also provides a method of synthesising a compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising: a) sequentially reacting a C-terminus an amino acid like compound comprising R5 and an amino moiety of a compound comprising R2 with a C-terminus and/or N-terminus of an amino acid like compound comprising R3; and b) oxidising the intermediate of step a) with an oxidising agent in order to form R1.
For example, the method of synthesis can be via a general scheme as shown in general route A, B, D, E, F, H, and I.
In some embodiments, the compound comprising R2 comprises a lactamide moiety.
In some embodiments, a N-terminus of the amino acid like compound comprising R5 is reacted with a carboxylate compound comprising R6.
The amino acid like compounds can be coupled using amide coupling reagents. For example,H ATU and DIPEA can be used.
In some embodiments, the oxidation step is performed using Dess-Martin periodinane (DMP). In Dess-Martin oxidation, primary alcohols are oxidised to aldehydes and secondary alcohols to ketones. In some embodiments, the method further comprises: a) oxidising a hydroxyl moiety of a compound comprising R2 to an aldehyde; and b) reacting the aldehyde with an isocyano compound in order to form a lactamide compound comprising R2.
The isocyano compound has the functional group -N ºC.
For example, this is shown in general route C, and G. DMP can be used.
The amino acid like compounds and intermediates can be protected using protecting groups.
The present invention also provides a method of treating and/or preventing a virus infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), (l'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof.
The present invention also provides a use of a compound of Formula (I), (l'), (II), (ll'), (III), (Ill'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating and/or preventing a virus infection in a patient in need thereof.
The present invention also provides a compound of Formula (I), (T), (II), (ll'), (III), (Ill'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof for use in treating and/or preventing a virus infection in a patient in need thereof.
In some embodiments, the virus infection is caused at least in part by a virus in the picornaviridae family or coronaviridae family. In other embodiments, the virus infection is caused by a virus belonging to the following families: levi-, narna-, picorna-, dicistro- , marna-, sequi-, como-, poty-, calici-, astro-, noda-, tetra-, luteo-, tombus-, corona-, arteri-, roni-, flavi-, toga-, bromo-, tymo-, clostero-, flexi-, seco-, barna, ifla-, sadwa-, chera-, hepe-, sobemo- , umbra-, tobamo-, tobra-, hordei-, furo-, porno-, peclu-, beny- , ourmia-, and idaeovirus. In other embodiments, the virus infection is caused at least in part by the coxsackievirus from the family picornaviridae. Variations in virus family are also included within this scope. For example, the World Health Organisation recommends using a Greek naming system for SARS-CoV-2 variants.
For example, the virus infection can be COVID-19, caused by SARS-CoV-2 and its variants thereof. Viruses constantly change through mutation and these mutations can result in a new variant of the virus. Due to the mutations, some variants emerge and disappear, others persist, while some variants spread more easily and quickly. Some variants of the SARS-CoV-2 coronavirus to date are alpha, beta, gamma, delta and omicron. The variants can have sub-variants. For example, omicron has a few major offshoots (sublineages), including BA.l, BA.2 and BA.3.
In some embodiments, the virus infection is caused or associated with a virus selected from rhinovirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), common coronaviridae (including but not limited to OC43, HKU1, 229E and NL63), enterovirus, poliovirus, coxsackievirus, hepatitis A virus, foot-and-mouth disease virus (FMDV) belonging to the picornaviridae family and calicivirus from the caliciviridae family. In other embodiments, the virus is alpha human coronaviruses. In other embodiments, the virus is beta human coronaviruses. In other embodiments, the virus is human enteroviruses A-D. In other embodiments, the virus is poliovirus. In other embodiments, the virus is coxackievirus. In other embodiments, the virus is echovirus. In other embodiments, the virus is hepatitis A virus. In other embodiments, the virus is calicivirus. In other embodiments, the virus is norovirus. In other embodiments, the virus is Norwalk virus.
In some embodiments, the virus infection is caused or associated with coronavirus; i.e. coronavirus infection. In other embodiments, the virus infection is associated with rhinovirus. In other embodiments, the virus infection is from an RNA-based virus. In other embodiments, the virus infection is from a single-stranded RNA virus.
An RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. This nucleic acid is usually single-stranded RNA (ssRNA). RNA viruses can be further classified according to the sense or polarity of their RNA into negative-sense and positive-sense. Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. As such, purified RNA of a positive-sense virus can directly cause infection though it may be less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not infectious by itself as it needs to be transcribed into positive- sense RNA; each virion can be transcribed to several positive-sense RNAs.
In some embodiments, the virus infection is caused by a positive-sense, single-stranded RNA virus. In other embodiments, the virus infection is caused by a negative-sense, single-stranded RNA virus.
In some embodiments, the method is a method of inhibiting progression of a virus infection in a patient in need thereof.
In some embodiments, the use is a use in the manufacture of a medicament for inhibiting progression of a virus infection in a patient in need thereof.
In some embodiments, the compound is for inhibiting progression of a virus infection in a patient in need thereof.
In some embodiments, the compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') is an inhibitor of 3CL protease.
The compound of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present invention includes within its scope cationic salts e.g. sodium or potassium salts, or alkyl esters (e.g. methyl, ethyl) of the phosphate group.
Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
It will be appreciated that any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention. Thus the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art. Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et ai, Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et ai., Chapter 5, (Harwood Academic Publishers). For example, when R1 comprises an amino group, prodrugs of the compound can include a protecting group protecting the amino group, including but not limited to, Carbobenzyloxy (Cbz) group, p-Methoxybenzyl carbonyl (Moz or MeOZ) group, tert-Butyloxycarbonyl (BOC) group, 9-Fluorenylmethyloxycarbonyl (Fmoc) group, Acetyl (Ac) group, Benzoyl (Bz) group, Benzyl (Bn) group, Carbamate group, p-Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), p-Methoxyphenyl (PMP) group, Tosyl (Ts) group, Troc (trichloroethyl chloroformate ) group, and Sulfonamides (Nosyl & Nps) groups.
The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.
The compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration. As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.
The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.
The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for example, water, saline (e.g. normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. By way of example, the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1/2 or 3/8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).
A person skilled in the art will appreciate that other means for injecting and/or administering the compound, composition or combinations to the vitreous of the eye can also be used. These other means can include, for example, intravitreal medical delivery devices. These devices and methods can include, for example, intravitreal medicine delivery devices, and biodegradable polymer delivery members that are inserted in the eye for long term delivery of medicaments. These devices and methods can further include transscleral delivery devices.
Other modes of administration including topical or intravenous administration may also be possible. For example, solutions or suspensions of the compound, composition or combinations of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.
The compound, composition or combinations of the invention may also be suitable for intravenous administration. For example, a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg/m2. The compound, composition or combinations of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt, solvate or prodrug is orally administrable.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
The compound, composition or combinations of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
The compound, composition or combinations of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention. The compound, composition or combination of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the active ingredients particularly mentioned above, the composition or combination of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
Examples Synthesis of intermediates 1-A to 1-1
Figure imgf000060_0001
1-A was synthesized following a procedure as disclosed in Michael Dominic Sacco et. al, Science Advances 2020, 6, 50, eabe0751, which is herein incorporated by reference. Following a similar procedure, intermediate 1-B was also synthesized.
Synthesis of intermediate 1-C
To a solution of i (4.4 g, 16.15 mmol) in CH2CI2 (65 mL) was added Dess-Martin periodinane (11 g, 25.85 mmol) at 0 °C and the reaction was left to stir at rt for 3 h. The crude reaction was filtered and reduced under pressure to afford intermediate ii as a light yellow solid (2.78 g, 10.28 mmol). To the crude solid in EtOAc (40 mL) was added cyclopropyl isocyanide (828 mg, 12.34 mmol, 1.2 equiv.) and acetic acid (710 μL, 12.34 mmol, 1.2 equiv.) at 0 °C and the reaction was left to stir at rt overnight. The crude mixture was azeotroped with MeOH (3x) and re-dissolved in MeOH (10 mL). To the solution was added K2CO3 (2.84 g, 20.56 mmol, 2.0 equiv.) at 0 °C and the reaction was left to stir at rt for 1 h. The crude mixture was filtered, reduced under pressure, and purified by silica gel column chromatography (0-10% MeOH/ CH2CI2) to afford intermediate iii as a light yellow solid (1.85 g, 5.21 mmol). To intermediate iii (1.85 mg, 5.21 mmol) was added HCI (4M in 1,4-dioxane, 10 equiv.). The mixture was stirred for 1 h, before concentration under pressure to afford intermediate 1-C (1.52 g, 5.21 mmol) as a white solid.
Following a similar procedure, intermediates 1-D, 1-E, 1-F, 1-J, 1-K, 1-L, 1-M and 1- N were also synthesized.
Synthesis of intermediate 1-G:
Scheme 2:
Figure imgf000062_0001
To a suspension of methyltriphenylphosphanium bromide (623.73 mg, 1.746 mmol, 1.6 equiv.), in THF (7 mL) at room temperature under N2 was added KHMDS (1.64 ml_, 1.637 mmol, 1.5 equiv., 1 M in THF) and allowed to stir for 1 h. The resulting solution was then cooled to -78 °C and ii (295 mg, 1.091 mmol, 1 equiv.) dissolved in THF (1 mL) was added dropwise. Upon complete addition, the reaction was allowed to warm to room temperature and stirred for 2 h. The reaction was quenched by the addition of saturated aqueous NH4CI (10 mL) and then extracted with Et20 (3 x 10 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by silica gel column chromatography (0-100% EtO Ac/ Hexanes) to afford intermediate iv as a colourless viscous oil (221.60 mg, 75.7%). To intermediate iv (221 mg, 0.824 mmol, 1 equiv.) in acetone (15 mL) and water (5 mL) cooled to 0 °C was added OSO4 (0.52 mL, 0.082 mmol, 0.1 equiv., 4 wt% in water). The reaction was allowed to warm to room temperature and stirred for an additional 4h. The reaction was quenched with Na2SO4 (20 mL, 1 M) and filtered through a pad of wet Celite. The filtrate was extracted with EtOAc (3 x 10 mL), dried over Na2SO4, and concentrated in vacuo. The crude was purified by reversed-phase prep-HPLC to afford intermediate v as a colourless solid (123.20 mg, 49.5%). To a solution of intermediate v (123.00 mg, 0.407 mmol, 1 equiv.) in acetone (3 mL) and 5% aqueous NaHCCH (1.30 mL) cooled to 0 °C was added KBr (4.84 mg, 0.041 mmol, 0.1 equiv.), followed by TEMPO (69.92 mg, 0.447 mmol, 1.1 equiv.) and then, dropwise, a 5% aqueous solution of NaOCI (1.2 mL, 0.814 mmol, 2 equiv.). The reaction was stirred at 0 °C for 2 h. The reaction was quenched by the addition of 5% NaHCO3 solution (2 mL) and the solvent volume was reduced in vacuo. The remaining aqueous layer was washed with ether (2 x 3 mL) and the extracts were discarded. The aqueous layer was then acidified to pH ~6 with 2 M HCI and purified by reversed-phase prep-HPLC to afford intermediate vi as a colourless glassy solid (63.50 mg, 49.3%). To a solution of intermediate vi (63.50 mg, 0.201 mmol, 1 equiv.) and N -methylcyclopropanamine (15.7 mg, 0.221 mmol, 1.1 equiv.) in DMF (2 mL) cooled to 0 °C was added N,N- diisopropylethylamine (104.89 μL, 0.602 mmol, 3 equiv.) followed by HATU (83.95 mg, 0.221 mmol, 1.1 equiv.). Upon complete addition, the reaction was stirred at 0 °C for 1 h. The reaction was quenched by the addition of saturated aqueous NH4CI (5 mL) and extracted with EtOAc (3 x 5 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by reversed-phase prep-HPLC to afford intermediate vii as a colourless glassy solid (50.70 mg, 68.4%). To intermediate vii (50 mg, 0.135 mmol) was added HCI (4M in 1,4-dioxane, 10 equiv). the mixture was stirred for 1 h, before concentration under pressure to afford intermediate 1-G (34.3 mg, 0.135 mmol) as a white solid.
Synthesis of intermediate 1-H: Scheme 3:
Figure imgf000064_0001
Intermediate viii (1.65 g, 6.0 mmol) dissolved in anhydrous THF (25 mL) under N2 was cooled to in -78 °C. LiHMDS (1M in THF, 14.4 mL, 14.4 mmol, 2.4 equiv.) was added dropwise at same temperature and stirred for 30 min. 3-bromo-2-methylprop-l-ene (1.82 mL, 18.0 mmol, 3 equiv.) was then added dropwise at -78 °C and stirring continued for a further 3 h at -78 °C. After 3h, the reaction was quenched with sat. NH4CI. The crude reaction was then extracted with EtOAc (3 x 15 mL) and the combined organic fractions washed with brine, dried over anhydrous MgSO4 and concentrated. The crude was purified by silica gel column chromatography (0-60% EtOAc/Hexanes) to afford intermediate ix as a light yellow liquid (1.68 g, 5.1 mmol). Iron(III) oxalate hexahydrate (2.42 g, 5 mmol, 2 equiv.) was dissolved in water (100 mL). The clear yellow solution was cooled to 0 °C and degassed for 10 min under N2. NalNb (490 mg, 7.5 mmol, 3 equiv.) and degassed EtOH (50 mL) were added under N2. An ethanolic solution (50 mL) of intermediate ix (824 mg; 2.5 mmol) was transferred by syringe to the reaction mixture. Solid NaBH4 (606 mg; 16.0 mmol, 6.4 equiv.) was added in 2 portions (5 min apart). The resulting mixture was stirred for 30 min before addition of 28-30% aqueous ammonium hydroxide (40 ml_). The reaction mixture was stirred for a further 10 min at 0 °C. The crude reaction was filtered over a Celite plug and washed with additional MeOH. The combined filtrate was concentrated to remove most of the alcoholic solvents and the resulting solution was diluted with water (lOmL) and extracted with EtOAc (3 x 25 ml_). The combined organic fractions were washed with brine, dried over anhydrous MgSO4 and concentrated. The crude was purified by silica gel column chromatography (0- 60% EtOAc/Hexanes) to yield intermediate x as a colourless gum (292 mg, 0.78 mmol). Intermediate x (292 mg, 0.78 mmol) was dissolved in MeOH (3 ml_). Pd/C (10 wt%, 60 mg) was added under N2 and the reaction was put under H2 (balloon) for 1 h. After 1 h, the reaction was put under N2 and DIPEA (69 μL, 0.39 mmol, 0.5 equiv.) was added. The reaction mixture was stirred at room temperature overnight. The crude reaction was then filtered over a Celite plug, concentrated, and dried under high vacuum to yield intermediate xi as a white solid (242 mg, 0.77 mmol) Intermediate xi (241 mg, 0.767 mmol), was dissolved in anhydrous THF (10 mL) and cooled to 0 °C under N2. L1BH4 (1M in THF, 2.3 ml_, 2.3 mmol, 3 equiv.) was added at 0 °C in dropwise, after which the reaction mixture was stirred at the same temperature for 5 h. After complete consumption of starting material, saturated NH4CI (10 mL) and EtOAc (20 mL) was added. The organic layer was collected, and the aqueous layer further extracted with EtOAc (3 x lOmL). The combined organic fractions were then washed with brine, dried over anhydrous MgS04 and concentrated to afford intermediate xii as a white solid (210 mg, 0.73 mmol). Intermediate xii (413 mg, 1.44 mmol) was dissolved in DCM/DMF (1:1, 6 mL) and cooled to 0 °C. Dess-Martin periodinane (979 mg, 2.31 mmol, 1.6 equiv.) was added in one portion and the reaction mixture was then stirred at the same temperature for 2 h. After complete consumption of the starting material, the reaction mixture was diluted with EtOAc (50 mL) and quenched with saturated NaHCO3 and NaS2O3 (1:1, 60 mL). The organic layer was collected and further washed with saturated NaHC03 and NaS2O3 (1:1, 20 mL), 5% LiCI (2 x 20 mL) and brine (2 x 25 mL). The organic layer was then dried over anhydrous Na2SO4 and to yield crude intermediate xiii as a white solid (300 mg, 1.06 mmol). Intermediate xiii (300 mg; 1.05 mmol) was dissolved in EtOAc (5 mL) and cooled to 0 °C. Glacial acetic acid (200 μL, 3.16 mmol, 3 equiv.) was added, followed by cyclopropyl isocyanide (250 μL, 3.16 mmol, 3 equiv.) at 0 °C and the reaction was stirred at room temperature for 18 hr. The crude reaction was concentrated to remove any volatile organics before redissolving in EtOAc (50 ml_). The material was washed sequentially with saturated NaHC03 (20 mL) and brine (20 ml_). The organic layer was dried over anhydrous Na2SO4 to yield crude intermediate xiv as a white solid (420 mg, 1.02 mmol). Intermediate xiv (420 mg, 1.02 mmol) was dissolved in MeOH (3 mL). A saturated methanolic K2CO3 solution (3 mL) was added in 1 portion and the reaction was stirred at room temperature for 1 h. The reaction was concentrated and purified by preparative HPLC (20-70% MeCN/H2O, 0.1% formic acid) to afford intermediate xv as a white solid (175 mg, 0.47 mmol). To intermediate xv (175 mg, 0.47 mmol) was added HCI (4M in 1,4-dioxane, 10 equiv). The mixture was stirred for 1 h, before concentration under pressure to afford intermediate 1-H (119 mg, 0.47 mmol) as a white solid.
Following a similar procedure, intermediate 1-1 was also synthesized.
GENERAL ROUTE A:
Figure imgf000066_0001
Specific exemplification of general route A
Preparation of tert-butyl (2S)-2-[(2R)-2-{[(9H-fluoren-9- ylmethoxy)carbonyl]amino>-3-phenylpropanamido]pentanoate (A-1)
Figure imgf000067_0001
To a mixture of tert-butyl (2S)-2-aminopentanoate hydrochloride (660 mg, 3.15 mmol, 1.05 equiv.), (2R)-2-{[(9H-/-fluoren-9-ylmethoxy)carbonyl]amino}-3-phenylpropanoic acid (1.16 g, 3.0 mmol, 1.0 equiv.), /V,/V-diisopropylethylamine (2.6 mL, 15.0 mmol, 5.0 equiv.) in anhydrous DMF (15 mL) was added HATU (1.14 g, 3.0 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. Water (30 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2 to afford intermediate A-l as a white solid (1.62 g, 3.0 mmol); MS (ESI) m/z 487.3 [C33H38N2O5 - C4H8 + H]+.
Preparation of tert-butyl (S)-2-((/?)-2-(l-naphthamido)-3- phenylpropanamido)pentanoate (A-2)
Figure imgf000067_0002
To a solution of intermediate A-l (200 mg, 0.37 mmol, 1.0 equiv.) in DMF (1 mL) was added DBU (57 μL, 0.37 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 10 min. The reaction mixture was concentrated under vacuo to its crude to afford crude tert-butyl (S)-2-((R)-2-amino-3-phenylpropanamido)pentanoate. To a mixture of crude tert-butyl (S)-2-((R)-2-amino-3-phenylpropanamido)pentanoate, 1-naphthoic acid (58 mg, 0.332 mmol, 0.9 equiv.) and /V,/V-diisopropylethylamine (193 μL, 1.1 mmol, 3.0 equiv.) in DMF (1 mL) was added HATU (140 mg, 0.37 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 20 min. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NaHCO 3, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (20-40% EtOAc/Flexanes) to afford intermediate A-2 as a white solid (103 mg, 0.22 mmol); MS (ESI) m/z 419.2 [C29H34N2O4 - C4H8 + H]+. Preparation of (S)-2-((R)-2-(l-naphthamido)-3- phenylpropanamido)pentanoic acid (A-3)
Figure imgf000068_0001
To a solution of intermediate A-2 (70 mg, 0.15 mmol, 1.0 equiv.) in CH2CI2 (0.2 mL) was added TFA in excess (1 mL, 13.1 mmol, 87 equiv.) at 0 °C and stirred at rt for 1 h. The reaction mixture was concentrated under vacuo to its crude to afford intermediate A-3 as an oil (61.7 mg, 0.15 mmol); MS (ESI) m/z 419.1 [C25H2SN2O4 + H]+.
Preparation of /V-((2/?)-l-(((2S)-l-(((2S)-4-(cyclopropylamino)-3-hydroxy-4- oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan-2- yl)amino)-l-oxo-3-phenylpropan-2-yl)-l-naphthamide (A-4)
Figure imgf000068_0002
To a mixture of intermediate A-3 (60 mg, 0.14 mmol, 1.0 equiv.), intermediate 1-A (40 mg, 0.14 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (126 μL, 0.72 mmol, 5.0 equiv.) in DMF (1 mL) was added HATU (55 mg, 0.14 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 1 h. Water (30 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NaHCO3 , brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by semi-preparative HPLC reverse phase chromatography (10-70% MeCN/H2O, 0.1% formic acid) to afford intermediate A-4 as a white solid upon lyophilisation (58 mg, 0.09 mmol); MS (ESI) m/z 642.3 [C36H43N5O6 + H]+.
Preparation of N -((/?)-l-(((S)-l-(((S)-4-(cyclopropylamino)-3,4-dioxo-l- ((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan-2-yl)amino)-l- oxo-3-phenylpropan-2-yl)-l-naphthamide (Compound 2) To a solution of intermediate A-4 (58 mg, 0.09 mmol, 1.0 equiv.) in anyhydrous CH2CI2 (3 mL) was added Dess-Martin periodinane (62 mg, 0.15 mmol, 1.6 equiv.) at 0 °C and stirred at rt for 1 h. The reaction was diluted with EtOAc, washed with Na2S2O3: NaHC03 (1 : 1). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2) to afford compound 2 as a white solid upon lyophilization (41 mg, 0.064 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.80-8.46 (m, 3H), 8.3-8.11 (m, 1H), 7.95 (dd, J = 17.5 Hz, 8.2 Hz, 2H), 7.86-7.73 (m, 1H), 7.64-7.55 (m, 1H), 7.50 (q, J = 8.0 Hz, 2H), 7.45-7.34 (m, 4H), 7.30 (t, J = 7.4 Hz, 2H), 7.27-7.21 (m, 1H), 5.17-4.95 (m, 1H), 4.95-4.83 (m, 1H), 4.47-4.25 (m, 1H), 3.15-2.85 (m, 4H), 2.79-2.70 (m, 1H), 2.45-2.35 (m, 1H), 2.22-2.09 (m, 1H), 1.99- 1.88 (m, 1H), 1.72-1.43 (m, 4H), 1.32-1.17 (m, 2H), 0.93-0.80 (m, 3H), 0.71-0.46 (m, 4H); MS (ESI) m/z 640.3 [C36H41N5O6 + H]+.
GENERAL ROUTE B:
Figure imgf000070_0001
Specific exemplification of general route B
Preparation of (9H-fluoren-9-yl)methyl ((2S)-l-(((2S)-4-(cyclopropylamino)- 3-hydroxy-4-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l- oxopentan-2-yl)carbamate (B-1)
Figure imgf000070_0002
A mixture of (2S)-2-{[(9/-/-fluoren-9-ylmethoxy)carbonyl]amino}pentanoic acid (597 mg, 1.759 mmol, 1.0 equiv.), (3S)-3-amino-N-cyclopropyl-2-hydroxy-4-((S)-2- oxopyrrolidin-3-yl)butanamide hydrochloride (424.4 mg, 1.76 mmol, 1.0 equiv.), HATU (668.9 mg, 1.76 mmol, 1.0 equiv.), /V,/V-diisopropylethylamine (0.5 mL, 8.80 mmol, 5.0 equiv.) in anhydrous DMF (1.5 mL) was stirred at rt for 2 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CFteCh) to afford intermediate B-l as a white solid (870 mg, 1.55 mmol); MS (ESI) m/z 563.2 [C31H38N4O6 + H]+.
Preparation of (2S)-2-amino-/V-((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo- l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)pentanamide (B-2)
Figure imgf000071_0001
A mixture of intermediate B-l (300 mg, 0.53 mmol, 1.0 equiv.) and DBU (80 μL, 0.53 mmol, 1.0 equiv.) in anhydrous DMF (1 mL) was stirred at rt for 10 min. The reaction mixture was concentrated under vacuo to its crude to afford intermediate B-2 as an oil (180 mg, 0.53 mmol); MS (ESI) m/z 341.2 [C1SH28N4O4 + H]+.
Preparation of (9H-fluoren-9-yl)methyl ((2R)-l-(((2S)-l-(((2S)-4- (cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2- yl)amino)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)carbamate (B-3)
Figure imgf000071_0002
A mixture of intermediate B-2 (180 mg, 0.53 mmol, 1 equiv.), (2R)-2-{[(9/-/-fluoren- 9-ylmethoxy)carbonyl]amino}-3-phenylpropanoic acid (205 mg, 0.53 mmol, 1.0 equiv.),H ATU (201 mg, 0.53 mmol, 1.0 equiv.), /V,/V-diisopropylethylamine (0.46 mL, 2.65 mmol, 5.0 equiv.) in anhydrous DMF (2 mL) was stirred at rt for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CFteCh) to afford intermediate B-3 as a clear oil (253 mg, 0.36 mmol); MS (ESI) m/z 710.3 [C H N O + H]+.
Preparation of (2S)-2-((/?)-2-amino-3-phenylpropanamido)-/V-((2S)-4- (cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2- yl)pentanamide (B-4)
Figure imgf000072_0001
A mixture of intermediate B-3 (50 mg, 0.07 mmol, 1.0 equiv.) and DBU (11 μL, 0.07 mmol, 1.0 equiv.) in DMF (0.15 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under vacuo to its crude to afford intermediate B-4 as an oil (34 mg, 0.07 mmol); MS (ESI) m/z 488.3 [C25H37N5O5 + H]+.
Preparation of (2S)-2-[(2/?)-2-{bicyclo[l.l.l]pentan-l-ylformamido}-3- phenylpropanamido]-/V-[l-(cyclopropylcarbamoyl)-l-hydroxy-3-(2- oxopyrrolidin-3-yl)propan-2-yl]pentanamide (B-5)
Figure imgf000072_0002
To a solution of intermediate B-4 , bicyclo[l. l.l]pentane-l-carboxylic acid (7.9 mg, 0.07 mmol, 1.0 equiv.), HATU (37.5 mg, 0.09 mmol, 1.4 equiv.), N,N- diisopropylethylamine (40 μL, 0.21 mmol, 3.0 equiv.) in anhydrous DMF (2 mL) was stirred at rt for 0.5 h. Water (5 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-12% MeOH/CH2Cl2) to afford intermediate B-5 as a cream solid (21.7 mg, 0.037 mmol); MS (ESI) m/z 582.3 [C H N O + H]+. Preparation of (2S)-2-[(2R)-2-{bicyclo[l.l.l]pentan-l-ylformamido}-3- phenylpropanamido]-/V-[l-(cyclopropylcarbamoyl)-l-oxo-3-(2-oxopyrrolidin- 3-yl)propan-2-yl]pentanamide (Compound 50)
Figure imgf000073_0001
A solution of intermediate B-5 (20.6 mg, 0.035 mmol, 1.0 equiv.) in DMF (1 mL) was cooled to 0 °C before addition of Dess-Martin periodinane (24 mg, 0.057 mmol, 1.6 equiv.). The reaction was stirred at rt for 1 h. The reaction was diluted with EtOAc, washed with Na2S203: NaHC03 (1 : 1). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2) to afford compound 50 as a white solid upon lyophilization (10 mg, 0.017 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.75-8.32 (m, 1H), 8.22-7.95 (m, 1H), 7.84-7.32 (m, 2H), 7.29-7.01 (m, 5H), 5.12-4.92 (m, 1H), 4.58-4.36 (m, 1H), 4.34-3.96 (m, 2H), 3.18- 3.00 (m, 2H), 2.97-2.65 (m, 4H), 2.24-1.92 (m, 2H), 1.95-1.78 (m, 6H), 1.74-1.47 (m, 3H), 1.47-1.33 (m, 1H), 1.32-0.97 (m, 3H), 0.89-0.72 (m, 3H), 0.69-0.45 (m, 4H); MS(ESI), m/z 580.3 [C31H41N5O6 + H]+
GENERAL ROUTE C:
Specific exemplification of general route C
Preparation of (9H-fluoren-9-yl)methyl ((S)-l-(((S)-l-hydroxy-3-((S)-2- oxopyrrolidin-3-yl)propan-2-yl)amino)-l-oxopentan-2-yl)carbamate (C-1)
Figure imgf000074_0001
To fe/t-butyl ((S)-l-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (lg, 3.87 mmol, 1.0 equiv.) in 1,4-dioxane (3 mL) was added 4N HCI in 1,4-dioxane (16 ml_, 77.4 mmol, 20 equiv.) at 0 °C and stirred at rt for 2 h. The reaction mixture was concentrated under vacuo to afford crude (S)-3-((S)-2-amino-3- hydroxypropyl)pyrrolidin-2-one hydrochloride. A mixture of crude (S)-3-((S)-2-amino- 3-hydroxypropyl)pyrrolidin-2-one hydrochloride (753 mg, 3.87 mmol, 1.0 equiv.), (S)- 2-((((9/-/-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid (1.31 g, 3.87 mmol, 1.0 equiv.), HATU (1.47 g, 3.87 mmol, 1.0 equiv.), A/,/V-diisopropylethylamine (3.37 mL, 19.3 mmol, 5.0 equiv.) in anhydrous DMF (15 mL) was stirred at rt for 0.5 h. Water (50 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NaHC03, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-12% MeOH/CH2Cl2) to afford intermediate C-l as a white solid (1.45 g, 3.02 mmol); MS (ESI) m/z 480.2 [C27H33N3O5 + H]+.
Preparation of (S)-2-amino-/V-((S)-l-hydroxy-3-((S)-2-oxopyrrolidin-3- yl)propan-2-yl)pentanamide
Figure imgf000075_0001
A mixture of intermediate C-l (858 mg, 1.79 mmol, 1.0 equiv.) and DBU (273 μL, 1.79 mmol, 1.0 equiv.) in DMF (4 mL) was stirred at rt for 0.5 h. The reaction mixture was concentrated under vacuo to its crude and purified by silica gel chromatography (40- 90% MeOH/CH2CI2) to afford intermediate C-2 as a white solid (395 mg, 1.54 mmol); MS (ESI) m/z 258.2 [C12H23N3O3 + H]+.
Preparation of benzyl ((/?)-l-(((S)-l-(((S)-l-hydroxy-3-((S)-2-oxopyrrolidin- 3-yl)propan-2-yl)amino)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropan-2- yl)carbamate (C-3)
Figure imgf000075_0002
To a mixture of (2R)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanoic acid (460 mg, 1.54 mmol, 1.0 equiv.), intermediate C-2 (395 mg, 1.54 mmol, 1.0 equiv.), N,N- diisopropylethylamine (0.8 mL, 4.61 mmol, 3.0 equiv.) in anhydrous DMF (6 mL) was addedH ATU (584 mg, 1.54 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. The crude mixture was purified by semi-preparative H PLC reverse phase chromatography (10-65% MeCN/hhO, 0.1% formic acid) to afford intermediate C-3 as a white solid upon lyophilisation (680 mg, 1.26 mmol); MS (ESI) m/z 539.2 [C29H38N4O6 + H]+.
Preparation of benzyl ((/?)-l-oxo-l-(((S)-l-oxo-l-(((S)-l-oxo-3-((S)-2- oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-3-phenylpropan- 2-yl)carbamate (C-4)
Figure imgf000076_0001
A solution of intermediate C-3 (680 mg, 1.26 mol, 1.0 equiv.) in DMF (7 mL) was cooled to 0 °C before addition of Dess-Martin periodinane (856.7 mg, 2.02 mmol, 1.6 equiv.). The reaction was stirred at rt for 1.5 h. The reaction was filtered and concentrated to afford its crude. The crude product was purified by semi-preparative HPLC reverse phase chromatography (20-60% MeCN/hhO, 0.1% formic acid) to afford intermediate C-4 as a white fluffy solid upon lyophilization (525 mg, 0.98 mmol); MS (ESI) m/z 537.2 [C29H36N4O6 + H]+.
Preparation of (benzyl ((2/?)-l-(((2S)-l-(((2S)-4-(cyclohexylamino)-3- hydroxy-4-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan- 2-yl)amino)-l-oxo-3-phenylpropan-2-yl)carbamate (C-5)
Figure imgf000076_0002
To a solution of intermediate C-4 (35 mg, 0.065 mmol, 1.0 equiv.) in DMF (lmL) was added AcOFI (16 μL, 0.26 mmol, 4.0 equiv.) and cyclohexylisocyanide (33 μL, 0.26 mmol, 4.0 equiv.) at 0 °C and stirred at rt for 18 h. The reaction was diluted with MeOH and concentrated to afford a crude yellow oil. To the crude yellow oil in MeOH (1.5mL) was added K2CO3 (27 mg, 0.20 mmol, 30 equiv.) at 0 °C and stirred at rt for 2 h. The reaction was filtered and concentrated to afford its crude. The crude product was purified by semi-preparative HPLC reverse phase chromatography (20-75% MeCN/H20, 0.1% formic acid) to afford intermediate C-5 as a white fluffy solid upon lyophilization (26 mg, 0.039 mmol); MS (ESI) m/z 664.3 [C36H49N5O7 + H]+. Preparation of benzyl ((R)-l-(((S)-l-(((S)-4-(cyclohexylamino)-3,4-dioxo-l- ((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan-2-yl)amino)-l- oxo-3-phenylpropan-2-yl)carbamate (Compound 39)
Figure imgf000077_0001
To a solution of intermediate C-5 (25 mg, 0.038 mmol, 1.0 equiv.) in DMF (0.8 mL) was added Dess-Martin periodinane (26 mg, 0.06 mmol, 1.6 equiv.) at 0 °C and stirred at rt for 1 h. The reaction mixture was quenched with MeOH (1 mL) and purified by semi-preparative HPLC reverse phase chromatography (20-85% MeCN/hhO, 0.1% formic acid) to afford compound 39 as a white fluffy solid upon lyophilization (17 mg, 0.026 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.6-8.29 (m, 2H), 8.25-8.04 (m, 1H), 7.64 (s, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.41-7.13 (m, 10H), 5.13-4.86 (m, 3H), 4.40-4.25 (m, 2H), 3.60-3.50 (m, 1H), 3.18-3.08 (m, 2H), 2.93 (dd, J = 13.5 Hz, 5.1 Hz, 1H), 2.81-2.71 (m, 1H), 2.45-2.31 (m, 1H), 2.26-2.11 (m, 1H), 2.04-1.85 (m, 1H), 1.79-1.51 (m, 8H), 1.47-1.37 (m, 1H), 1.34-1.05 (m, 7H), 0.81 (t, J = 7.3 Hz, 3H); MS (ESI) m/z 662.3 (ESI) [C H N O + H]+.
GENERAL ROUTE D:
Figure imgf000078_0001
D-7
Specific exemplification of general route D
Preparation of tert-butyl (2S)-2-({[(4a-methyl-9H-fluoren-9- yl)methoxy]carbonyl}amino)pentanoate (D-1)
Figure imgf000078_0002
To a solution of (S)-2-((((9/-/-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)pentanoic acid (4 g, 11.3 mmol, 1.0 equiv.) in CH2CI2 (64 mL) was added thionyl chloride (16 ml_, 22.0 mmol, 2.0 equiv.) DMF (10 μL) at 0 °C and the reaction was stirred at rt for 2 h. After 2 h, fBuOH (10.7 mL, 113 mmol, 10 equiv.) was added dropwise at 0 °C and the reaction was stirred at rt for 1.5 h. Water (60 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NaHCC>3, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-40% EtOAc/Hexanes) to afford intermediate D-l as a colourless oil (3.76 g, 9.18 mmol); MS (ESI) m/z 354.1 [C25H31NO4 - C4H8 + H]+.
Preparation of tert-butyl (S)-2-((/?)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-2-cyclohexyl-/V-methylacetamido)pentanoate
(D-2)
Figure imgf000079_0001
A solution of intermediate D-l (3 g, 7.33 mmol, 1.0 equiv.) and dimethylamine (29.58 mL, 440 mmol, 60 equiv.) in anhydrous THF (20 mL) was stirred at 40 °C for 2 h. The reaction mixture was concentrated under vacuo to its crude. To a solution of crude tert- butyl (S)-2-(methylamino)pentanoate (858 mg, 4.58 mmol, 1.0 equiv.), (R)-2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopentylacetic acid (3.48 g, 9.16 mmol, 2.0 equiv.) and /V,/V-diisopropylethylamine (7.8 mL, 4.58 mmol, 10.0 equiv.) in DMF (12 mL) was added HATU (3.48 g, 9.16 mmol, 2.0 equiv.) at 0 °C and stirred at rt for 1 h. Water (60 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NaFIC03, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-60% EtOAc/ eHxanes) to afford intermediate D-2 as a white solid upon lyophilization (1.5 g, 2.73 mmol); MS (APCI) m/z 548.4 (ESI) [C33FU4N2O5 + H]+.
Preparation of (S)-2-((/?)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- phenylpropanamido)pentanoic acid (D-3)
To a solution of intermediate D-2 (309 mg, 0.56 mmol, 1.0 equiv.) in CH2CI2 (0.1 mL) was added TFA in excess (0.9 mL, 11.8 mmol, 21 equiv.) at 0 °C and stirred at rt for 0.5 h. The reaction mixture was concentrated under vacuo to its crude to afford intermediate D-3 as an oil (277 mg, 0.56 mmol); MS (ESI) m/z 493.1 [C29FI36N2O5 + H]+.
Preparation of (9H-fluoren-9-yl)methyl ((l/?)-l-cyclohexyl-2-(((2S)-l- (((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopiperidin-3- yl)butan-2-yl)amino)-l-oxopentan-2-yl)(methyl)amino)-2- oxoethyl)carbamate
Figure imgf000080_0001
To a solution of intermediate D-3 (277 mg, 0.56 mmol, 1.0 equiv.), 3-amino -N- cyclopropyl-2-hydroxy-4-(2-oxopyrrolidin-3-yl)butanamide (143.7 mg, 0.56 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (490 μL, 2.82 mmol, 5.0 equiv.) in anhydrous DMF (2 mL) was added HATU (214 mg, 0.56 mmol, 1.0 equiv.) at 0 °C and stirred for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NFUCI, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2CI2) to afford intermediate D-4 as a white solid upon lyophilization (425 mg, 0.58 mmol); MS (ESI) m/z 730.3 [C41H55N5O7 + H]+.
Preparation of (2S)-2-((/?)-2-amino-2-cyclohexyl-/V-methylacetamido)-/V- ((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopiperidin-3- yl)butan-2-yl)pentanamide (D-5)
To a solution of intermediate D-4 (213 mg, 0.29 mmol, 1.0 equiv.) in anhydrous CH2CI2 (3 mL) was added DBU (22 μL, 0.29 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 30 min. The reaction mixture was concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-30% MeOH/CH2Cl2) to afford intermediate D-5 as a yellow oil (148 mg, 0.29 mmol); MS (ESI) m/z 508.3 [C26H45N5O5 + H]+.
Preparation of /V-((l/?)-l-cyclohexyl-2-(((2S)-l-(((2S)-4-
(cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopiperidin-3-yl)butan-2- yl)amino)-l-oxopentan-2-yl) (methyl )amino)-2- oxoethyl )bicyclo[ 1.1.1] pentane- 1-carboxamide (D-6)
Figure imgf000081_0001
To a solution of intermediate D-5 (70 mg, 0.14 mmol, 1.0 equiv.), bicyclo[l.l.l]pentane-l-carboxylic acid (15.5 mg 0.14 mmol, 1.0 equiv.) and N,N- diisopropylethylamine (120 μL, 0.69 mmol, 5.0 equiv.) in anhydrous DMF (0.5 mL) was added HATU (52.4 mg, 0.14 mmol, 1.0 equiv.) at 0 °C and stirred for 0.5 h. The reaction mixture was concentrated under vacuo to its crude. The crude product was purified by Waters MDAP preparative HPLC reverse phase chromatography (20-70% MeCN/hhO, 0.1% formic acid) to afford intermediate D-6 as a white solid upon lyophilisation (27 mg, 0.045 mmol); MS (ESI) m/z 602.3 [C32H51N5O6 + H]+.
Preparation of /V-((/?)-l-cyclohexyl-2-(((S)-l-(((S)-4-(cyclopropylamino)- 3,4-dioxo-l-((S)-2-oxopiperidin-3-yl)butan-2-yl)amino)-l-oxopentan-2- yl) (methyl )amino)-2-oxoethyl)bicyclo[ 1.1. l]pentane- 1-carboxamide (Compound 100)
To a solution of intermediate D-6 (27 mg, 0.045mmol, 1.0 equiv.) in DMF (0.5 mL) was added Dess-Martin periodinane (38 mg, 0.09 mmol, 2.0 equiv.) at 0 °C and stirred at rt for 4 h. The reaction mixture was quenched with MeOH (1 mL) and purified by Waters MDAP preparative HPLC reverse phase chromatography (20-70% MeCN/hhO, 0.1% formic acid) to afford compound 100 as a white solid upon lyophilization (26 mg, 0.043 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.58-8.08 (m, 2H), 7.34-7.21 (m, 2H), 5.02-4.49 (m, 3H), 3.18-3.16 (m, 2H), 2.95, 2.77 (2 x s, 3H), 2.75-2.70 (m, 1H), 2.38- 2.36 (m, 1H), 2.32-2.20 (m, 1H), 2.15-2.06 (m, 1H), 1.95 (s, 6H), 1.89-1.36 (m, 13H), 1.29-1.08 (m, 7H), 0.86 (t, J = 7.3 Hz, 3H), 0.68-0.63 (m, 2H), 0.61-0.58 (m, 2H); MS (ESI) m/z 600.3 [C32H49N5O6 + H]+.
General Route E:
Figure imgf000082_0001
Specific exemplification of general route E
Preparation of tert-butyl (2R)-3-phenyl-2-(3-phenylpropanamido)propanoate (E-l)
To a mixture of 3-phenylpropionic acid (58.3 mg, 0.39 mmol, 1.0 equiv.), tert- butyl (2R)-2-amino-3-phenylpropanoate hydrochloride (100 mg, 0.39 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (0.34 ml_, 1.94 mmol, 5.0 equiv.) in DMF (2 mL) was added HATU (147.5 mg, 0.39 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NFUCI, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2) to afford intermediate E-l as a yellow oil (96.9 mg, 0.27 mmol); MS (ESI) m/z 298.1 (ESI) [C22H27NO3 - C4H8 + H]+.
Preparation of (2R ))-3-phenyl-2-(3-phenylpropanamido)propanoic acid (E-2)
Figure imgf000083_0001
To a solution of intermediate E-l (49.9 mg, 0.14 mmol, 1.0 equiv.) in CH2CI2 (0.1 mL) was added TFA in excess (0.9 mL, 11.8 mmol, 84 equiv.) and stirred at rt for 0.5 h. The reaction mixture was concentrated under vacuo to its crude to afford intermediate E-2 as an oil (42 mg, 0.14 mmol); MS (ESI) m/z 298.1 [C18FI19NO3 + Fl]+.
Preparation of (2S)-/V-((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)-2-((/?)-N-methyl-3-phenyl-2-(3- phenylpropanamido)propanamido)pentanamide (E-3)
Figure imgf000083_0002
To a solution of (2S)-/V-((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)-2-(methylamino)pentanamide B-2 (30.8 mg, 0.087 mmol, 1.0 equiv.), (2R)-3-phenyl-2-(3-phenylpropanamido)propanoic acid (42 mg, 0.14 mmol, 1.6 equiv.) and L/,/V-diisopropylethylamine (80 μL, 0.43 mmol, 5.0 equiv.) in DMF (2 mL) was added HATU (33 mg, 0.087 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NhUCI, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2) to afford intermediate E-3 as clear oil (11.3 mg, 0.018 mmol); MS (ESI) m/z 634.3 [C35H47N5O6 + H]+.
Preparation of (S)-/V-((S)-4-(cyclopropylamino)-3,4-dioxo-l-((S)-2- oxopyrrolidin-3-yl)butan-2-yl)-2-((R)-/V-methyl-3-phenyl-2-(3- phenylpropanamido)propanamido)pentanamide (Compound 45)
Figure imgf000084_0001
To a solution of intermediate E-3 (11.3 mg, 0.018 mmol, 1.0 equiv.) in DMF (0.25 mL) was added Dess-Martin periodinane (12 mg, 0.029 mmol, 1.6 equiv.) at 0 °C and stirred at rt for 1 h. The reaction was diluted with EtOAc, washed with Na2S203:NaFIC03 (1 : 1). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOFI/CH2CI2) to afford compound 45 as a white solid upon lyophilization (4.1 mg, 0.006 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.82-8.16 (m, 4H), 8.00-7.61 (m, 2H), 7.31-7.05 (m, 8H), 5.12-4.72 (m, 3H), 3.20-3.03 (m, 2H), 2.97-2.64 (m, 6H), 2.43-2.26 (m, 3H), 2.24-2.07 (m, 1H), 2.03-1.83 (m, 1H), 1.80- 1.48 (m, 4H), 1.41-1.09 (m, 4H), 0.98-0.70 (m, 3H), 0.70-0.45 (m, 4H); MS (ESI) m/z 632.3 [C35H45N5O6 + H]+.
General Route F: Specific exemplification of gene
Preparation of tert-b -(cyclopentylformamido)-3- phenylpropanamido]pentan
Figure imgf000085_0001
A solution of (2R)-2-(cyclopentylformamido)-3-phenylpropanoic acid (147.6 mg, 0.57 mmol, 1.0 equiv.), tert-butyl (2S)-2-aminopentanoate hydrochloride (118.5 mg, 0.57 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (0.49 ml_, 2.83 mmol, 5.0 equiv.) in DMF (1 mL) was added HATU (214.8 mg, 0.57 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NH4CI, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-5% MeOH/CH2Cl2) to afford intermediate F-1 as a white solid (109.4 mg, 0.26 mmol); MS (ESI) m/z 361.2 [C24H36N2O4 - C4H8 + H]+.
Preparation of (2S)-2-[(2/?)-2-(cyclopentylformamido)-3- phenylpropanamido]pentanoic acid (F-2) To a solution of intermediate F-l (71 mg, 0.17 mmol, 1.0 equiv.) in CH2CI2 (0.1 mL) was added TFA in excess (0.9 mL, 11.8 mmol, 69 equiv.) at 0 °C and stirred at rt for 1 h. The reaction mixture was concentrated under vacuo to its crude to afford intermediate F-2 as an oil (61.8 mg, 0.17 mmol); MS (ESI) m/z 361.2 [C20H28N2O4 + H]+.
Preparation of /V-((2/?)-l-(((2S)-l-(((2S)-4-(cyclopropylamino)-3-hydroxy-4- oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan-2- yl)amino)-l-oxo-3-phenylpropan-2-yl)cyclopentanecarboxamide (F-3)
Figure imgf000086_0001
A solution of intermediate F-2 (61.6 mg, 0.17 mmol, 1.5 equiv.), 3-amino -N- cyclopropyl-2-hydroxy-4-(2-oxopyrrolidin-3-yl)butanamide hydrochloride (32.5 mg 0.12 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (0.1 mL, 0.59 mmol, 5.0 equiv.) in DMF (0.6 mL) was added AHTU (44.5 mg, 0.12 mmol, 1.0 equiv.) at 0 °C and stirred at rt for 0.5 h. Water (10 mL) was added to the reaction mixture and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with sat. NFUCI, brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOFI/CFhCb) to afford intermediate F-3 as a white solid (70.8 mg, 0.12 mmol); MS (ESI) m/z 584.3 [C31H45N5O6 + H]+.
Preparation of /V-((/?)-l-(((S)-l-(((S)-4-(cyclopropylamino)-3,4-dioxo-l- ((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-l-oxopentan-2-yl)amino)-l- oxo-3-phenylpropan-2-yl)cyclopentanecarboxamide (Compound 67)
To a solution of intermediate F-3 (70.8 mg, 0.12 mmol, 1.0 equiv.) in DMF (1.7 mL) was added Dess-Martin periodinane (82.3 mg, 0.19 mmol, 1.6 equiv.) at 0 °C and stirred at rt for 1 h. The reaction was diluted with EtOAc, washed with Na2S203:NaHC03 (1 : 1). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to its crude. The crude product was purified by silica gel chromatography (0-10% MeOH/CH2Cl2) to afford compound 67 as a white solid upon lyophilization (25.5 mg, 0.044 mmol); 1H NMR (400 MHz, DMSO-d6): d (ppm) 8.80-7.31 (m, 5H), 7.28-7.09 (m, 5H), 5.09-3.95 (m, 3H), 3.19-2.98 (m, 3H), 2.97-2.87 (m, 1H), 2.80-2.69 (m, 2H), 2.22-2.05 (m, 1H), 1.98-1.80 (m, 1H), 1.74-1.23 (m, 13H), 1.20- 1.03 (m, 2H), 0.91-0.73 (m, 3H), 0.70-0.42 (m, 4H); MS (ESI) m/z 582.2 [C31H43N5O6 + H]+.
General Route G:
Figure imgf000088_0001
Specific exemplification of general route G
Preparation of methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate hydrochloride (G-l)
Figure imgf000089_0001
A mixture of methyl (2S)-2-[(te/t-butoxycarbonyl)amino]-3-[(3S)-2-oxopyrrolidin-3- yl]propanoate (3.00 g, 10.48 mmol, 1.0 equiv.) in 4N HCI in 1,4-dioxane (26.2 ml_, 104.78 mmol, 10 equiv.) was stirred at rt for 40 min. The reaction mixture was concentrated under vacuo to its crude to afford intermediate G-l as a solid (3.32 g, 17.82 mmol); MS (APCI) m/z 187.1 [C8H14N2O3 + H]+.
Preparation of methyl (2S)-2-[(2S)-2-{[(9H-fluoren-9- ylmethoxy)carbonyl]amino}pentanamido]-3-[(3S)-2-oxopyrrolidin-3- yl]propanoate (G-2)
Figure imgf000089_0002
A mixture of (2S)-2-{[(9/-/-fluoren-9-ylmethoxy)carbonyl]amino}pentanoic acid (1.18 g, 3.49 mmol, 1.0 equiv.), methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3- yljpropanoate hydrochloride (1.10 g, 3.49 mmol, 1.0 equiv.), HATU (1.99 g, 5.24 mmol, 1.5 equiv.) in DMF (7.0 mL) and N,N V-diisopropylethylamine (3.04 ml_, 17.47 mmol, 5.0 equiv.) was stirred at rt for 2 h. Water (100 mL) was added to the reaction mixture and the aqueous layer was washed with EtOAc (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (50-100% EtOAc/Hexanes) to afford intermediate G- 2 as a solid (1.48 g, 2.91 mmol); MS (APCI) m/z 508.3 [C28H33N3O6 + H]+.
Preparation of 9H-fluoren-9-ylmethyl-/V-[(lS)-l-{[(2S)-l-hydroxy-3-[(3S)-2- oxopyrrolidin-3-yl]propan-2-yl]carbamoyl}butyl]carbamate (G-3)
A
Figure imgf000090_0002
ylmethoxy)carbonyl]amino}pentanamido]-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate (1.48 g, 2.93 mmol, 1.0 equiv.) in THF (5.9 mL) was stirred in 0 °C for 10 min, followed by the dropwise addition of 2M lithium borohydride in THF (4.4 mL, 8.80 mmol, 3.0 equiv.). The resultant mixture was stirred at rt for 2 h. The reaction was quenched with 2N HCI (5 mL) and diluted with water (20 mL). The aqueous layer was washed with EtOAc (50 mL) and the organic layer was dried over Na2SO4, filtered and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-10% MeOH/CH2Cl2) to afford intermediate G-3 as a solid (744 mg, 1.55 mmol); MS (APCI) m/z 480.3 [C27H33N3O5 + H]+.
Preparation of 9H-fluoren-9-yl methyl N -[(lS)-l-{[(2S)-l-oxo-3-[(3S)-2- oxopyrrolidin-3-yl]propan-2-yl]carbamoyl}butyl]carbamate (G-4)
Figure imgf000090_0001
To a solution of 9H -fluoren-9-ylmethyl N -[(lS)-l-{[(2S)-l-hydroxy-3-[((S)-2- oxopyrrolidin-3-yl]propan-2-yl]carbamoyl}butyl]carbamate (723 mg, 1.51 mmol, 1.0 equiv.) in DMF (15.5 mL) was added Dess-Martin periodinane (1.02 g, 2.41 mmol, 1.6 equiv.) at 0 °C. The suspension was stirred at rt for 2 h. NaS2C>3: NaHCCb (80 mL:80 mL) was added to the reaction mixture and the aqueous layer was washed with EtOAc (150 mL). The organic layer was dried over Na2SO4, filtered and concentrated under vacuo to its crude to afford intermediate G-4 as a solid (720 mg, 1.51 mmol); MS (APCI) m/z 478.2 [C27H31N3O5 + H]+.
Preparation of (2S)-l-(cyclopropylcarbamoyl)-2-[(2S)-2-{[(9H-fluoren-9- ylmethoxy)carbonyl]amino}pentanamido]-3-[(3S)-2-oxopyrrolidin-3- yl]propyl acetate (G-5)
Figure imgf000091_0002
To a solution of 9H-fluoren-9-yl methyl A/-[(lS)-l-{[(2S)-l-oxo-3-[(3S)-2- oxopyrrolidin-3-yl]propan-2-yl]carbamoyl}butyl]carbamate (739 mg, 1.55 mmol, 1.0 eq) in EtOAc (15.5 mL) was added acetic acid (106 μL, 1.86 mmol, 1.2 equiv.) followed by cyclopropyl isocyanide (125 μL, 1.86 mmol, 1.2 equiv.) at 0 °C The resultant mixture was stirred at rt overnight. The reaction was quenched with MeOH (4 x 20 mL) and concentrated under vacuo to its crude to afford intermediate G-5 as a solid (830 mg, 1.37 mmol); MS (APCI) m/z 605.3 [C33H40N4O7 + H]+.
Preparation of (2S)-2-[(2S)-2-aminopentanamido]-l-
(cyclopropylcarbamoyl)-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (G-6)
Figure imgf000091_0001
To a mixture of (2S)-l-(cyclopropylcarbamoyl)-2-[(2S)-2-{[(9/-/-fluoren-9- ylmethoxy)carbonyl]amino}pentanamido]-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (810 mg, 1.34 mmol, 1.0 equiv.) in THF (6.7 mL) was added diethylamine (2.77 mL, 26.79 mmol, 20.0 equiv.). The mixture was stirred at rt for 1 h and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-50% MeOH/CH2Cl2) to afford intermediate G-6 as a solid (451 mg, 1.18 mmol); MS (APCI) m/z 383.3 [C18H30N4O5 + H]+.
Preparation of (2S)-2-[(2S)-2-[(2/?)-2-cyclopentyl-2-{[(9H-fluoren-9- ylmethoxy)carbonyl]amino}acetamido]pentanamido]-l- (cyclopropylcarbamoyl)-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (G-7) A mixture of (2S)-2-[(2S)-2-aminopentanamido]-l-(cyclopropylcarbamoyl)-3-[(3S)-2- oxopyrrolidin-3-yl]propyl acetate (50 mg, 0.131 mmol, 1.0 equiv.), {R)- cyclopentyl({[(9/-/-fluoren-9-ylmethoxy)carbonyl]amino})acetic acid (47.8 mg, 0.131 mmol, 1.0 equiv.) and HATU (74.5 mg, 0.196 mmol, 1.5 equiv.) in DMF (1.30 mL) was added N,N V-diisopropylethylamine (114 μL, 0.654 mmol, 5.0 equiv.). The resultant mixture was stirred at rt overnight. Water (20 mL) was added to the reaction mixture and the aqueous layer was washed with EtOAc (30 mL). The organic layer was dried over Na2SO4, filtered and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-22% MeOH/CH2Cl2) to afford intermediate G-7 as a solid (60.4 mg, 0.083 mmol); MS (ESI) m/z 730.3 [C40H51N5O8 + H]+.
Preparation of (2S)-2-[(2S)-2-aminopentanamido]-l-
(cyclopropylcarbamoyl)-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (G-8)
Figure imgf000092_0001
(2S)-l-(cyclopropylcarbamoyl)-2-[(2S)-2-{[(9/-/-fluoren-9- ylmethoxy)carbonyl]amino}pentanamido]-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (60 mg, 0.099 mmol, 1.0 equiv.) in THF (0.82 mL) was added diethylamine (0.170 mL, 1.98 mmol, 20.0 equiv.). The mixture was stirred at rt for 1 h and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-50% MeOFI/CFhCb) to afford intermediate G-8 as a solid (30.6 mg, 0.080 mmol); MS (APCI) m/z 508.3 [C25H41N5O6 + H]+.
Preparation of (2S)-2-[(2S)-2-[(2/?)-2-[(4-chlorophenyl)formamido]-2- cyclopentylacetamido]pentanamido]-l-(cyclopropylcarbamoyl)-3-[(3S)-2- oxopyrrolidin-3-yl]propyl acetate (G-9) To a mixture of (2S)-2-[(2S)-2-[(2R)-2-amino-2-cyclopentylacetamido]pentanamido]- l-(cyclopropylcarbamoyl)-3-[(3S)-2-oxopyrrolidin-3-yl]propyl acetate (30 mg, 0.059 mmol, 1.0 equiv.), 4-chlorobenzoic acid (9.3 mg, 0.059 mmol, 1.0 equiv.) and HATU (33.7 mg, 0.089 mmol, 1.5 equiv.) in DMF (1.0 mL) was added N,N- diisopropylethylamine (31 μL, 0.177 mmol, 3.0 equiv.). The resultant mixture was stirred at rt for 1 h. Water (20 mL) was added to the reaction mixture and the aqueous layer was washed with EtOAc (30 mL). The organic layer was dried over Na2SO4, filtered and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-23% MeOH/CH2Cl2) to afford intermediate G-9 as a solid (26.2 mg, 0.040 mmol); MS (APCI) m/z 648.4 [C32H44CIN5O7 + H]+.
Preparation of (2S)-2-[(2/?)-2-[(4-chlorophenyl)formamido]-2- cyclopentylacetamido]-/V-[(2S)-l-(cyclopropylcarbamoyl)-l-hydroxy-3- [(3S)-2-oxopyrrolidin-3-yl]propan-2-yl]pentanamide (G-10)
Figure imgf000093_0001
(2S)-2-[(2S)-2-[(2R)-2-[(4-chlorophenyl)formamido]-2- cyclopentylacetamido]pentanamido]-l-(cyclopropylcarbamoyl)-3-[(3S)-2- oxopyrrolidin-3-yl]propyl acetate (26.0 mg, 0.0402 mmol, 1.0 equiv.) in MeOH (1.0 mL) was added K2CO3 (16.7 mg, 0.121 mmol, 3.0 equiv.). The suspension was stirred at rt for 30 min, filtered and concentrated under vacuo to its crude. Crude product was purified by silica gel flash chromatography (0-23% MeOH/CH2Cl2) to afford intermediate G-IO as a solid (19.2 mg, 0.032 mmol); MS (APCI) m/z 604.4 [C30H42CIN5O6 + H]+.
Preparation of (2S)-2-[(2/?)-2-[(4-chlorophenyl)formamido]-2- cyclopentylacetamido]-/V-[(2S)-l-(cyclopropylcarbamoyl)-l-oxo-3-[(3S)-2- oxopyrrolidin-3-yl]propan-2-yl]pentanamide (Compound 107)
To a solution of (2S)-2-[(2R)-2-[(4-chlorophenyl)formamido]-2-cyclopentylacetamido]- N-[(2S)-l-(cyclopropylcarbamoyl)-l-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2- yl]pentanamide (19.0 mg, 0.031 mmol, 1.0 equiv.) in DMF (0.50 mL) was added Dess- Martin periodinane (21.3 mg, 0.050 mmol, 1.6 equiv.) at 0 °C. The suspension was stirred at rt for 2 h. The reaction mixture was diluted with DMF (1 mL), filtered and the crude product was purified by Waters MDAP preparative H PLC reverse phase chromatography (20-70% MeCN/H2O, 0.1% formic acid) to afford compound 107 as white solid upon lyophilization (7.5 mg, 0.012 mmol). 1H NMR (400 MFIz, DMSO-ds): d ppm 8.70 (d, J = 5.1 Hz, 1H), 8.57 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 7.1 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 7.94-7.85 (m, 2H), 7.62 (s, 1H), 7.56-7.50 (m, 2H), 5.05-4.90 (m, 1H), 4.35-4.20 (m, 2H), 3.17 (s, 1H), 3.15-3.05 (m, 1H), 3.05-3.00 (m, 1H), 2.75-2.65 (m, 1H), 2.45-2.35 (m, 1H), 2.30-2.20 (m, 1H), 2.15-2.05 (m, 1H), 2.00-1.90 (m, 1H), 1.85-1.75 (m, 1H), 1.65-1.45 (m, 9H), 1.40-1.25 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H), 0.68-0.60 (m, 2H), 0.58-0.54 (m, 2H); MS (APCI) m/z 603.4 [C30H40CIN5O6 + H]+.
General Route H:
Figure imgf000095_0001
Specific exemplification of general route H
Preparation of methyl l-amino-3,3-difluorocyclobutane-l-carboxylate hydrochloride (H-l)
Figure imgf000095_0002
To a solution of l-[(fe/t-butoxycarbonyl)amino]-3,3-difluorocyclobutane-l-carboxylic acid (250 mg, 0.995 mmol, 1.0 equiv.) in MeOH (6.0 mL) was added thionyl chloride (433 μL, 5.97 mmol, 6.0 equiv.) at 0 °C under nitrogen. The reaction was warmed to rt and left to stir for 16 h before concentrating under reduced pressure to afford intermediate H-l as a white solid (201 mg, 0.997 mmol); MS (ESI) m/z 166.1 [C6H9F2NO2 + H]+.
Preparation of methyl 3,3-difluoro-l-pivalamidocyclobutane-l-carboxylate (H-2) To a solution of pivalic acid (122 mg, 1.19 mmol, 1.2 equiv.), methyl 1 -amino-3, 3- difluorocyclobutane-l-carboxylate hydrochloride (200 mg, 0.99 mmol, 1.0 equiv.) and N ,N -diisopropylethylamine (864 μL, 4.96 mmol, 5.0 equiv.) in DMF (1.0 mL) was added HATU (754 mg, 1.98 mmol, 2.0 equiv.). The reaction was stirred at 90 °C for 0.5 h. The reaction was diluted with sat. NaHCO3 (2.0 mL). The product was extracted with EtOAc (3 x 2.0 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (0- 40% EtOAc/Hexanes) to afford intermediate H-2 as a yellow solid (204 mg, 0.82 mmol); MS (APCI) m/z 250.1 [C11H17F2NO3 + H]+.
Preparation of 3,3-difluoro-l-pivalamidocyclobutane-l-carboxylic acid (H-3)
Figure imgf000096_0001
To a solution of methyl l-(2,2-dimethylpropanamido)-3,3-difluorocyclobutane-l- carboxylate (142 mg, 0.570 mmol, 1.0 equiv.) in TFIF (1.0 mL) and water (1.0 mL) was added lithium hydroxide monohydrate (47.8 mg, 1.14 mmol, 2.0 equiv.). The reaction was heated at 90 °C for 0.5 h. The reaction was concentrated under reduced pressure and acidified with 6 M HCI. The product was extracted with EtOAc (4 x 2.0 mL), dried over anhydrous Na2SO4and concentrated under reduced pressure to afford intermediate H-3 as a white solid (130 mg, 0.55 mmol); MS (APCI) m/z 236.1 [C10FI15F2NO3 + H ]+ .
Preparation of (2S)-l-(cyclopropylcarbamoyl)-2-[(2S)-2-{[l-(2,2- dimethylpropanamido)-3,3-difluorocyclobutyl]formamido}pentanamido]-3- [(3S)-2-oxopyrrolidin-3-yl]propyl acetate (H-4)
Figure imgf000096_0002
To a solution of (2S)-2-[(2S)-2-aminopentanamido]-l-(cyclopropylcarbamoyl)-3-[(3S)- 2-oxopyrrolidin-3-yl]propyl acetate (50.0 mg, 0.131 mmol, 1.0 equiv.), l-(2,2- dimethylpropanamido)-3,3-difluorocyclobutane-l-carboxylic acid (36.9 mg, 0.157 mmol, 1.2 equiv.) and A/,/V-diisopropylethylamine (114 μL, 0.654 mmol, 5.0 equiv.) in DMF (0.25 mL) was added HATU (99.4 mg, 0.261 mmol, 2.0 equiv.). The reaction was stirred at rt for 2 h. The reaction was diluted with water (0.25 mL) and sat. NaHCO3 (0.5 mL). The product was extracted with EtOAc (6 x 0.5 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (0-10% MeOH/CH2CI2) to afford intermediate H-4 as a white solid (25 mg, 0.042 mmol); MS (APCI) m/z 250.1 [C28H43F2N5O7 + H ]+ .
Preparation of (2S)-N -[(2S)-l-(cyclopropylcarbamoyl)-l-hydroxy-3-((3S)-2- oxopyrrolidin-3-yl]propan-2-yl]-2-{[l-(2,2-dimethylpropanamido)-3,3- difluorocyclobutyl]formamido}pentanamide (H-5)
Figure imgf000097_0001
To a solution of (2S)-l-(cyclopropylcarbamoyl)-2-[(2S)-2-{[l-(2,2- dimethylpropanamido)-3,3-difluorocyclobutyl]formamido}pentanamido]-3-[(3S)-2- oxopyrrolidin-3-yl]propyl acetate (25.0 mg, 0.042 mmol, 1.0 equiv.) in MeOFI (0.2 mL) was added K2CO3 (17.4 mg, 0.125 mmol, 3.0 equiv.). The reaction was stirred at rt for 0.5 h. The reaction was filtered, concentrated under reduced pressure and diluted with water (0.2 mL). The product was extracted with EtOAc (6 x 0.5 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford intermediate H- 5 as a white solid (21.0 mg, 0.038 mmol); MS (APCI) m/z 558.4 [C26FU1F2N5O6 + H]+ .
Preparation of (2S)-/V-[(2S)-l-(cyclopropylcarbamoyl)-l-oxo-3-[(3S)-2- oxopyrrolidin-3-yl ]propan-2-yl ]-2-{[l-( 2, 2-dimethyl propanamido)-3, 3- difluorocyclobutyl]formamido}pentanamide (Compound 109) To a solution of (2S)-/V-[(2S)-l-(cyclopropylcarbamoyl)-l-hydroxy-3-[(3S)-2- oxopyrrolidin-3-yl]propan-2-yl]-2-{[l-(2,2-dimethylpropanamido)-3,3- difluorocyclobutyl]formamido}pentanamide (20.0 mg, 0.036 mmol, 1.0 equiv.) in DMF (0.5 mL) was added Dess-Martin periodinane (30.4 mg, 0.072 mmol, 2.0 equiv.) at 0 °C. The reaction was warmed to rt and stirred for 4 h. The reaction was filtered and purified by preparative HPLC reverse phase chromatography (10-70% MeCN/hhO, 0.1% formic acid). The product was lyophilized and repurified by preparative HPLC reverse phase chromatography (5-95 % MeCN/H20, 0.1% formic acid) to afford compound 109 as a white solid upon lyophilization (3.1 mg, 0.006 mmol); ‘H NMR (400 MHz, DMSO- de): d ppm 8.77-8.76, 7.80-7.79 (d x 2, J = 4.8 Hz, 1H), 8.59-8.57, 7.48-7.46 (d x 2, J = 7.6 Hz, 1H), 8.27, 8.24 (s x 2, 1H), 7.67, 7.50 (s, 1H), 7.30-7.28, 7.28-7.26 (d x 2, J = 8.0 Hz, 1H), 5.04-4.99, 4.05-4.00 (ddd x 2, J = 3.6, 7.2, 10.8, 1H), 4.30-4.20 (m, 1H), 3.33-3.09 (m, 3H), 3.07-2.97 (m, 1H), 2.90-2.79 (m, 1H), 2.77-2.70 (m, 1H), 2.68-2.58 (m, 1H), 2.38-2.29, 2.06-1.98 (m x 2, 1H), 2.20-2.12 (m, 1H), 1.92-1.79 (m, 1H), 1.74-1.31 (m, 4H), 1.27-1.17 (m, 2H), 1.11 (s, 9H), 0.84-0.81 (t, J = 7.2 Hz, 3H), 0.67-0.61, 0.53-0.47 (m x 2, 2H), 0.59-0.56 (m, 2H); MS (APCI) m/z 558.4 [C26H39F2N5O6 + H]+.
General Route I:
Figure imgf000098_0001
Figure imgf000099_0001
THF (1 ml) was added diethylamine (0.39 ml, 3.741 mmol, 20.0 equiv.). The reaction was stirred at rt for 1 h before concentrating under vacuo to afford intermediate 1-1 (50.0 mg, 0.186 mmol); MS (ESI) m/z 313.3 [C17H32N2O3 + H]+.
Preparation of tert-butyl (S)-2-((R )-2-cyclopentyl-N -methyl-2- pivalamidoacetamido)pentanoate (1-2)
Figure imgf000100_0001
To a solution of intermediate 1-1 (120.0 mg, 0.384 mmol) in DMF was added pivalic acid (62.8 mg, 0.615 mmol, 1.6 equiv.),H ATU (233.6 mg, 0.615 mmol, 1.6 equiv.) and N,N -diisopropylethylamine (0.27 ml, 1.536 mmol, 4.0 equiv.) at 0 °C and stirred for 45 min. The reaction mixture was diluted with EtOAc (10 mL) and washed with water (5 mL). The aqueous phase was back-extracted with EtOAc (3 x 5 mL) and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by preparativeH PLC (20-70% MeCN/H2O , 0.1% formic acid) to afford intermediate 1-2 as an off-white solid upon lyophilization (100 mg, 0.252 mmol); MS (ESI) m/z 397.3 [C22H40N2O4 + H]+.
Preparation of (S)-2-((R )-2-cyclopentyl-N -methy l-2- pivalamidoacetamido)pentanoic acid (1-3)
Figure imgf000100_0002
To a solution of intermediate 1-2 (47.0 mg, 0.119 mmol) in CH2CI2 (0.5 ml_), was added TFA (0.60 ml, 2.370 mmol, 20.0 equiv.). The reaction was stirred at rt for 1 h before concentrating to its crude to afford intermediate 1-3 (40.0 mg, 0.118 mmol); MS (ESI) m/z 341.3 [C18H32N2O4 + H]+.
Preparation of (9H-fluoren-9-yl)methyl (S)-(l-oxo-3-(pyridin-3-yl)propan-2- yl)carbamate (1-4)
Figure imgf000101_0001
To a solution of (2S)-2-{[(9/-/-fluoren-9-ylmethoxy)carbonyl]amino}-3-(pyridin-3- yl)propanoic acid (500 mg, 1.29 mmol, 1.0 equiv.) in CH2CI2 (10 mL), was added CDI (271.4 mg, 1.673 mmol, 1.3 equiv.) at 0 °C and the reaction was stirred for 1 h. The reaction was then cooled down to -78 °C and DIBAL-H (1.0 M in THF, 2.96 mL, 2.961 mmol, 2.3 equiv) was added drop-wise and the reaction was stirred at -78 °C for 4 h. The reaction was quenched with 25% tartaric acid (20 mL) and the mixture was stirred at -78 °C for 10 min. The mixture was extracted with EtOAC (3 x 50mL) and the combined organic layer was washed with brine (20 ml), dried over anhydrous Na2SO4 and concentrated to afford intermediate 1-4 as white sticky residue (340mg, 0.91 mmol); MS (ESI) m/z 373.1 [C23H20N2O3 + H]+.
Preparation of (3S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-l- (cyclopropylamino)-l-oxo-4-(pyridin-3-yl)butan-2-yl acetate (1-5)
Figure imgf000101_0002
To a solution of intermediate 1-4 (170 mg, 0.456 mmol, 1.0 equiv.) in EtOAc (5 mL), was added acetic acid (30 μL, 0.547 mmol, 1.2 equiv.) and isocyanocyclopropane (40 μL, 0.547 mmol, 1.2 equiv.) at 0 °C. The reaction was stirred at rt for 16 h. The reaction was stopped and co-evaporate with methanol to its crude. Crude product was purified by silica gel flash chromatography (0-100% EtOAc/Hexanes) to afford intermediate I- 5 as a solid (70.0 mg, 0.140 mmol); MS (ESI) m/z 500.2 [C29H29N3O5 + H]+.
Preparation of (3S)-3-amino-/V-cyclopropyl-2-hydroxy-4-(pyridin-3- yl)butanamide (1-6)
Figure imgf000102_0001
To a solution of intermediate 1-5 (70.0 mg, 0.140 mmol) in MeOH (1.5 mL) was added K2CO3 (117.0 mg, 0.841 mmol, 6.0 equiv.) and the mixture was stirred at rt for 30 min. The reaction was filtered to remove remaining K2CO3 and filtrate was concentrated to its crude. The crude residue was re-dissolved in MeOH (1 mL) and was loaded onto ISOLUTE® SCX-2 cartridge (silica-based media with a chemically bonded propylsulfonic acid functional group). The impurities were eluted out through gravity and collected separately. 3N ammonia in MeOH (10 mL) was added to the cartridge and filtrate collected was concentrated to give intermediate 1-6 as white solid (27.6 mg, 0.117 mmol); MS (ESI) m/z 236.1 [C12H17N3O2 + H]+.
Preparation of (2S)-2-((/?)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)- /V-((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-l-(pyridin-3-yl)butan-2- yl)pentanamide (1-7)
Figure imgf000102_0002
To a solution of intermediate 1-6 (27.6 mg, 0.117 mmol) in DMF was added intermediate 1-7 (39.9 mg, 0.117 mmol, 1.0 equiv.), HATU (53.5 mg, 0.141 mmol, 1.2 equiv.) and N,N V-diisopropylethylamine (0.14 ml, 0.821 mmol, 7.0 equiv.) at 0 °C and stirred at rt for 30 min. The reaction mixture was diluted with EtOAc (5 mL) and washed with water (2 mL). The aqueous phase was back-extracted with EtOAc (3 x 5 mL) and the combined organic layers were dried over Na2S04, filtered and concentrated under reduced pressure. The crude product was purified by preparative HPLC (20-70% MeCN/H20, 0.1% formic acid) to afford compound 1-7 as an off-white solid upon lyophilization (32.1 mg, 0.057 mmol); MS (ESI) m/z 558.3 [C30H47N5O5 + H]+. Preparation of (S)-2-((/?)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)-/V- ((S)-4-(cyclopropylamino)-3,4-dioxo-l-(pyridin-3-yl)butan-2- yl)pentanamide (Compound 114)
Figure imgf000103_0001
To a solution of intermediate 1-7 (32 mg, 0.057 mmol) in anhydrous DMF (0.5 mL) was added Dess-Martin periodinane (43.63 mg, 0.115 mmol, 2.0 equiv.) was added at 0 °C and the reaction was stirred at rt for 2 h. The crude product was purified by preparative HPLC (20-70% MeCN/hhO, 0.1% formic acid) to afford compound 114 as an white solid upon lyophilization (21.1 mg, 0.038 mmol); 1H NMR (400 MHz, DMSO-i/6): d ppm 8.83-8.63 (m, 1H), 8.54-8.29 (m, 2H), 8.01, 7.86 (2 x dd, J = 8.0 Hz, 1H), 7.72-7.41 (m, 2H), 7.34-7.25 (m, 1H), 5.25-5.12 (m, 1H), 5.01-4.97 (m, 1H), 4.73-4.37 (m, 1H), 3.19-3.12 (m, 1H), 2.93, 2.76 (2 x s, 3H), 2.91-2.82 (m, 1H), 2.76-2.63 (m, 1H), 2.25 (sextet, J = 8.0 Hz, 1H), 1.84-1.64 (m, 2H), 1.57-1.32 (m, 6H), 1.27-1.13 (m, 3H), 1.12, 1.08 (2 x s, 9H), 1.10-1.07 (m, 1H), 0.84-0.79 (m, 3H), 0.68-0.54 (m, 4H); MS (ESI) m/z 556.3 [C30H45N5O5 + H]+.
General Route J:
Figure imgf000104_0001
J-11 Specific exemplification of general route J
Preparation of methyl (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanoate hydrochloride (J-l)
Figure imgf000105_0001
To a flask of methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-[(3S)-2-oxopyrrolidin-3- yl]propanoate (1 g, 3.493 mmol, 1.0 equiv.) was added HCI 20 equiv. (4 M in 1,4- dioxane, 17.5 mL, 69.9 mmol, 20 equiv.) The reaction was stirred at room temperature for 2 h. After complete conversion of starting material, the reaction was concentrated under reduced pressure to afford intermediate J-l as a brown foamy solid (1.13 g, 5.08 mmol); MS (ESI) m/z 187.1 [C8H14N2O3 + H]+.
Preparation of methyl (S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl) (methyl )ami no)pentanamido)-3-( (S)-2-oxopyrrolidin- 3-yl)propanoate (J-2)
Figure imgf000105_0002
To a solution of intermediate J-l (389 mg, 1.75 mmol, 1.0 equiv.), (2S)-2-{[(9H- fluoren-9-ylmethoxy)carbonyl](methyl)amino}pentanoic acid (741 mg, 2.10 mmol, 1.2 equiv.), and N,N-diisopropylethylamine (1.52 mL, 8.74 mmol, 5.0 equiv.) in DMF (3.5 mL) was added HATU (797 mg, 2.1 mmol, 1.2 equiv.) at 0 °C. The reaction was stirred at 0 °C for 0.5 h. The reaction was diluted with water (1 mL) and sat. NhUCI (7 mL). The crude was extracted with EtOAc (3 x 7 mL), washed with brine, dried over anhydrous NA2SO4 and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (0-100% EtOAc/Hexanes) to afford intermediate J-2 as a white foamy solid (1 g, 1.91 mmol); MS (ESI) m/z 522.3 [C29H35N3O6 + H]+.
Preparation of methyl (S)-2-((S)-2-(methylamino)pentanamido)-3-((S)-2- oxopyrrolidin-3-yl)propanoate (J-3) To a solution of intermediate J-2 (250 mg, 0.479 mmol, 1.0 equiv.) in THF (2.4 mL) was added diethylamine (992 μL, 9.59 mmol, 20 equiv.). The reaction was stirred at room temperature for 0.5 h. The reaction was concentrated under reduced pressure and purified by silica gel column chromatography (0-20% MeOH/CH2Cl2) to afford intermediate J-3 as a white solid (124 mg, 0.414 mmol); MS (ESI) m/z 300.2 [C14H25N3O4 + H]+.
Preparation of methyl (5/?,8S,llS)-5-cyclopentyl-l-(9H-fluoren-9-yl)-7- methyl-3,6,9-trioxo-ll-(((S)-2-oxopyrrolidin-3-yl)methyl)-8-propyl-2-oxa- 4,7,10-triazadodecan-12-oate (J-4)
Figure imgf000106_0001
To a solution of intermediate J-3 (125 mg, 0.418 mmol, 1.0 equiv.), (R)-2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopentylacetic acid (183 mg, 0.501 mmol, 1.2 equiv.) and /V,/V-diisopropylethylamine (364 μL, 2.09 mmol, 5.0 equiv.) in DMF (0.84 mL) was added HATU (1901 mg, 0.501 mmol, 1.2 equiv.) at 0 °C. The reaction was stirred at 0 °C for 0.5 h. The reaction was diluted with water (0.5 mL) and sat. NFUCI (2 mL). The crude was extracted with EtOAc (3 x 3 mL), dried over anhydrous NaS2C>4 and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (0-100% EtOAc/Flexanes) to afford intermediate J-4 as a white solid (318 mg, 0.492 mmol); MS (ESI) m/z 647.4 [C36FU6N4O7 + Fl]+.
Preparation of methyl (S)-2-((S)-2-((/?)-2-amino-2-cyclopentyl-N- methylacetamido)pentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (J- 5)
Figure imgf000107_0001
To a solution of intermediate J-4 (270 mg, 0.417 mmol, 1.0 equiv.) in THF (2.1 mL) was added diethylamine (864 μL, 8.35 mmol, 20 equiv.). The reaction was stirred at room temperature for 0.5 h. The reaction was concentrated under reduced pressure and purified by silica gel column chromatography (0-20% MeOH/CH2Cl2) to afford intermediate J-5 as a yellow solid (195 mg, 0.459 mmol); MS (ESI) m/z 425.3 [C21H36N4O5 + H]+.
Preparation of methyl (S)-2-((S)-2-((/?)-2-(4-chlorobenzamido)-2- cyclopentyl-/V-methylacetamido)pentanamido)-3-((S)-2-oxopyrrolidin-3- yl)propanoate (J-6)
Figure imgf000107_0002
To a solution of intermediate J-5 (177 mg, 0.417 mmol, 1.0 equiv.), 4-chlorobenzoic acid (78.3 mg, 0.5 mmol, 1.2 equiv.) and N,N -diisopropylethylamine (363 μL, 2.09 mmol, 5 equiv.) in DMF (0.83 mL) was added AHTU (190 mg, 0.5 mmol, 1.2 equiv.) at 0 °C. The reaction was stirred at 0 °C for 0.5 h. The reaction was diluted with water (0.5 mL) and sat. NFUCI (2 mL). The crude was extracted with EtOAc (3 x 3 mL), dried over anhydrous NaS2C>4 and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (0-100% EtOAc/Flexanes) to afford intermediate J-6 as a brown oil (234 mg, 0.417 mmol); MS (ESI) m/z 563.3 [C28H39N4O6CI + H]+.
Preparation of 4-chloro-/V-((/?)-l-cyclopentyl-2-(((S)-l-(((S)-l-hydroxy-3-
((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-l-oxopentan-2- yl) (methyl )amino)-2-oxoethyl)benzamide (J-7)
Figure imgf000108_0002
To a solution of intermediate J-6 (234 mg, 0.417 mmol, 1.0 equiv.) in THF (4.2 mL) was added LiBFU (2 M in TFIF, 1.04 mL, 2.08 mmol, 5.0 equiv.) at 0 °C. The reaction was warmed to room temperature and stirred for 1 h. The reaction was diluted with water (8 mL) and acidified with HCI (1 M). The product was extracted with EtOAc (3 xlO mL), washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (0- 15% MeOH/CH2CI2) to afford intermediate J-7 as a white solid (127 mg, 0.237 mmol); MS (ESI) m/z 535.3 [C27H39N4O5CI + H]+.
Preparation of 4-chloro-/V-((/?)-l-cyclopentyl-2-(methyl((S)-l-oxo-l-(((S)-l- oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-2- oxoethyl)benzamide
Figure imgf000108_0001
To a solution of intermediate J-7 (127 mg, 0.237 mmol, 1.0 equiv.) in DMF (2.4 mL) was added Dess-martin periodinane (201 mg, 0.475 mmol, 2.0 equiv.) portionwise at 0 °C. The reaction was warmed to room temperature and stirred for 5 h. The reaction was diluted with EtOAc (50 mL), followed by sat. NaS2C>3 (25 mL) and saturated NaHCCb (25 mL). The crude was further extracted with EtOAc (50 mL) and the combined organics were washed with brine, dried over anhydrous NaS204 and concentrated under reduced pressure to afford intermediate J-8 as a colourless oil (132 mg, 0.248 mmol); MS (ESI) m/z 533.3 [C27H37N4O5CI + H]+.
Preparation of (3S)-3-((S)-2-((/?)-2-(4-chlorobenzamido)-2-cyclopentyl-/V- methylacetamido)pentanamido)-l-(cyclopropylamino)-l-oxo-4-((S)-2- oxopyrrolidin-3-yl)butan-2-yl acetate (J-9)
Figure imgf000109_0002
To a solution of intermediate J-8 (126 mg, 0.236 mmol, 1.0 equiv.) in EtOAc (2.4 mL) was added acetic acid (16 μL, 0.284 mmol, 1.2 equiv.) followed by isocyanocyclopropane (19 μL, 0.284 mmol, 1.2 equiv.) at 0 °C. The reaction was warmed to room temperature and stirred for 17 h. The reaction was concentrated under reduced pressure and the crude was purified by silica gel column chromatography (0- 10% MeOH/CH2Cl2) to afford intermediate J-9 as a white solid (125 mg, 0.189 mmol);
Figure imgf000109_0003
Preparation of 4-chloro-N-((lR)-l-cyclopentyl-2-(((2S)-l-(((2S)-4- (cyclopropylamino)-3-hydroxy-4-oxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2- yl)amino)-l-oxopentan-2-yl) (methyl )amino)-2-oxoethyl)benzamide (J-10)
Figure imgf000109_0001
To a solution of intermediate J-9 (125 mg, 0.189 mmol) in MeOH (0.95 mL) was added K2CO3 (79 mg, 0.568 mmol, 3.0 equiv.). The reaction was stirred at room temperature for 1.5 h. The reaction was filtered, concentrated under reduced pressure, and diluted with water (1 mL). The product was extracted with EtOAc (3 x 1 mL), dried over anhydrous Na SC and concentrated under reduced pressure to afford intermediate J- 10 as a white solid (90 mg, 0.146 mmol); MS (ESI) m/z 618.4 [C31H44N5O6CI + H]+.
Preparation of 4-chloro-N-((R)-l-cyclopentyl-2-(((S)-l-(((S)-4-
(cyclopropylamino)-3,4-dioxo-l-((S)-2-oxopyrrolidin-3-yl)butan-2- yl)amino)-l-oxopentan-2-yl)( methyl )amino)-2-oxoethyl)benzamide (Compound 118)
Figure imgf000110_0001
To a solution of intermediate J-ll (90 mg, 0.146 mmol, 1.0 equiv.) in DMF (1.5 mL) was added Dess-martin periodinane (123.5 mg, 0.291 mmol, 2.0 equiv.) portion-wise at 0 °C. The reaction was warmed to room temperature and stirred for 2.5 h. The reaction was filtered, purified by preparative HPLC reverse phase chromatography (20- 70 % MeCN/hhO, 0.1% formic acid) to afford compound 118 as a white solid (33 mg, 0.054 mmol); ‘H NMR (400 MHz, DMSO-d6): d ppm 8.93-8.77 (m, 1H), 8.69-8.11 (m, 1H), 7.96-7.88 (m, 2H), 7.75-7.41 (m, 1H), 6.96-6.09 (m, 1H), 5.01-4.86 (m, 2H), 4.68-4.64, 4.11-4.00 (m, 1H), 3.29-2.94 (m, 2H), 3.11-2.81 (m, 3H), 2.78-2.69 (m, 1H), 2.56-2.35 (m, 2H), 2.18-1.75 (m, 4H), 1.72-1.43 (m, 8H), 1.38-1.10 (m, 5H), 0.91-0.78 (m, 3H), 0.69-0.45 (m, 4H); MS (ESI) m/z 616.3 [C31H42N5O6CI + H]+.
General Route K:
Figure imgf000111_0001
Specific exemplification of general route K
Preparation of (3S)-3-[(2S)-2-amino-3-hydroxypropyl]piperidin-2-one hydrochloride (K-l)
Figure imgf000111_0002
To tert-butyl ((S)-l-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)carbamate (210 mg, 0.771 mmol, 1.0 equiv.) in 1,4-dioxane (3.8 mL) was added HCI (4M in 1,4- dioxane, 10 equiv.) and the reaction was stirred at room temperature for 40 min. The - I l l - crude mixture was concentrated under reduced pressure to afford intermediate K-l (160.9 mg 0.771 mmol) as a white solid; MS (ESI) m/z 173.1 [C8H16N2O2 + H]+.
Preparation of (S)-2-((/?)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)-/V- ((S)-l-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)pentanamide (K-2)
Figure imgf000112_0001
To intermediate K-l (133 mg, 0.772 mmol, 1.02 equiv,) , ((S)-2-((/?)-2-cyclopentyl-A/- methyl-2-pivalamidoacetamido)pentanoic acid (1-3) (257 mg, 0.757 mmol, 1.0 equiv.) and A/,A/-diisopropylethylamine (1.32 mL, 3.79 mmol, 5.0 equiv.) in DMF (3.8 mL) was added HATU (288 mg, 0.757 mmol, 1.0 equiv.) and the reaction was stirred at 0 °C for 1 hr. The reaction was quenched with H2O (20 mL) and the aqueous layer was washed with EtOAc (3 x 20 mL). The organic layer was dried with Na2S04, filtered, and concentrated in vacuo. The crude was purified by silica gel column chromatography (0-20% EtOAC/Hexanes) to afford intermediate K-2 (302.2 mg, 0.611 mmol) as a white solid; MS (APCI) m/z 495.4 [C26H46N4O5 + H]+.
Preparation of (S)-2-((/?)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)-/V- ((S)-l-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)pentanamide (K-3)
Figure imgf000112_0002
To intermediate K-2 (302 mg, 0.611 mmol, 1.0 equiv.) in DMF (6.1 mL) at 0 °C was added Dess-martin periodinane (518 mg, 1.22 mmol, 2.0 equiv.) portion-wise and the reaction mixture was stirred at room temperature for 2 h. The reaction was diluted with EtOAc and washed with sat. Na2S203: NaHC03 (1 :1). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuo to afford intermediate K-3 as a crude brown solid (290 mg, 0.589 mmol); MS (ESI) m/z 493.2 [C26H44N4O5 + H]+.
Preparation of (3S)-3-((S)-2-((R)-2-cyclopentyl-/V-methyl-2- pivalamidoacetamido)pentanamido)-l-((3,3-difluorocyclobutyl)amino)-l- oxo-4-((S)-2-oxopiperidin-3-yl)butan-2-yl acetate (K-4)
Figure imgf000113_0001
To intermediate K-3 (100 mg, 0.204 mmol, 1.0 equiv.) in EtOAc (2 mL) at 0 °C was added acetic acid (14 μL, 0.244 mmol, 1.2 equiv.) and l,l-difluoro-3-isocyanocyclobutane (28.6 μL, 0.244 mmol, 1.2 equiv.) was added. The reaction was stirred at room temperature for 16 h. A second addition of l,l-difluoro-3-(28.6 μL, 0.244 mmol, 1.2 equiv.) was added and the reaction mixture was stirred for a further 3 h at room temperature. The reaction was quenched with MeOH (3 c 20 mL), concentrated in vacuo, and the crude was purified by silica gel column chromatography (0-10% MeOH/CH2Cl2) to afford intermediate K-4 as a white solid (65.7 mg, 0.098 mmol), MS (APCI) m/z 670.3 [C33H53F2N5O7 + H]+.
Preparation of (2S)-2-((/?)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)- N-((2S)-4-((3,3-difluorocyclobutyl)amino)-3-hydroxy-4-oxo-l-((S)-2- oxopiperidin-3-yl)butan-2-yl)pentanamide (K-5)
Figure imgf000113_0002
To intermediate K-4 (65 mg, 0.108 mmol, 1.0 equiv.) in MeOH (1 mL) was added K2CO3 (44.7 mg, 0.323 mmol, 3.0 equiv.). The reaction was stirred at room temperature for 1 hr. Upon reaction completion, the reaction mixture was filtered, diluted with H2O (5 mL) and the aqueous layer was washed with EtOAc (2 x 10 mL). The combined organics were dried with Na2SO4, filtered and concentrated in vacuo to afford intermediate K-5 as a crude brown solid (61 mg, 0.097 mmol); MS (ESI) m/z 628.3 [C31H51F2N5O6 + H]+.
Preparation of (S)-2-((R)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)-/V- ((S)-4-((3,3-difluorocyclobutyl)amino)-3,4-dioxo-l-((S)-2-oxopiperidin-3- yl)butan-2-yl)pentanamide (Compound 119)
Figure imgf000114_0001
To intermediate K-5 (61 mg, 0.097 mmol, 1.0 equiv.) in DMF (1 mL) was added Dess- martin periodinane (82.3 mg, 0.194 mmol, 2.0 equiv.) portion-wise at 0 °C before stirring at room temperature for 2 h. The crude mixture was filtered, purified by preparativeH PLC (20-70% MeCN/H2O, 0.1% formic acid) and lyophilized to afford compound 119 as a white solid (18 mg, 0.029 mmol); ‘H NMR (400 MHz, DMSO-cfa) d (ppm): 9.26-9.06 (m, 1H), 8.53-8.20 (m, 1H), 7.57-7.43 (m, 1H), 7.43-7.28 (m, 1H), 5.05-4.92 (m, 1H), 4.89-4.70 (m, 1H), 4.62-4.44 (m, 1H), 4.13-4.04 (m, 1H), 3.17 (d, J = 5.2 Hz, 1H), 3.13-3.08 (m, 1H), 2.95 (s, 2H), 2.90-2.81 (m, 2H), 2.77 (s, 1H), 2.32-2.22 (m, 2H), 2.16-2.03 (m, 1H), 1.90-1.65 (m, 5H), 1.61-1.38 (m, 9H), 1.29- 1.13 (m, 4H), 1.12-1.07 (m, 9H), 0.89-0.84 (m, 3H); MS (ESI) m/z 626.3 [C31H49F2N5O6 + H]+.
GENERAL ROUTE L:
Figure imgf000115_0003
Specific exemplification of general route L
Preparation of tert-butyl ((S)-l-amino-l-oxo-3-((S)-2-oxopiperidin-3- yl)propan-2-yl)carbamate (L-1)
Figure imgf000115_0001
A solution of methyl (S)-2-((te/t-butoxycarbonyl)amino)-3-((S)-2-oxopiperidin-3- yl)propanoate (100 mg, 0.333 mmol, 1.0 equiv.) in NH3 (7 M in MeOH, 2 mL) was sealed and stirred for 6.5 h at 50 °C. The reaction was reduced under pressure and dried under high vacuum overnight to afford intermediate L-1 as a white solid (93 mg, 0.326 mmol); MS (APCI) m/z 286.1 [C13H23N3O4 + H]+.
Preparation of (S)-2-amino-3-((S)-2-oxopiperidin-3-yl)propanamide hydrochloride (L-2)
Figure imgf000115_0002
To a solution of intermediate L-1 (130 mg, 0.456 mmol) in 1,4-dioxane (0.4 mL) was added HCI (4 M in 1,4-dioxane, 1.5 mL) at 0 °C and the reaction was stirred for 2 h. at room temperature. The reaction was reduced under pressure and dried under high vacuum overnight to afford intermediate L-2 as an off-white solid. ‘H NMR (400 MHz, DMSO -de) d (ppm): 8.33-8.28 (m, 2H), 7.97 (s, 1H), 7.85 (s, 1H), 7.58 (s, 1H), 3.87- 3.77 (m, 2H), 3.19-3.06 (m, 1H), 2.40 (dq, J = 12.5, 6.4 Hz, 1H), 2.08 (ddd, J = 14.5, 9.4, 7.5 Hz, 1H), 2.01-1.89 (m, 1H), 1.82-1.55 (m, 4H), 1.44 (dtd, J = 13.8, 10.8, 3.4 Hz, 1H); product could not be detected by MS.
Preparation of (S)-N-((S)-l-amino-l-oxo-3-((S)-2-oxopiperidin-3-yl)propan-
2-yl)-2-((R)-2-cyclopentyl-/V-methyl-2-pivalamidoacetamido)pentanamide
(L-3)
Figure imgf000116_0001
1. To a solution of ((S)-2-((/?)-2-cyclopentyl-A/-methyl-2-pivalamidoacetamido)pentanoic acid (1-3) (34 mg, 0.1 mmol, 1.0 equiv.), intermediate L-2 (23 mg, 0.1 mmol, 1.0 equiv.) and /V,/V-diisopropylethylamine (88 μL, 0.499 mmol, 5.0 equiv.) in DMF (1 mL) was added HATU (42 mg, 0.11 mmol, 1.1 equiv.) at 0 °C and the reaction was stirred for 0.5 h. The reaction mixture was quenched with H2O (1 mL) and MeOH (1 mL) at 0 °C and filtered. The crude was purified by preparative HPLC (20-70% MeCN/H20, 0.1% formic acid) and lyophilized to afford intermediate L-3 as a white solid (24 mg, 0.04 mmol); MS (APCI) m/z 508.3 [C26H45N5O5 + H]+.
Preparation of (S)-N-((S)-l-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-2-((/?)- 2-cyclopentyl-N-methyl-2-pivalamidoacetamido)pentanamide (Compound 180)
Figure imgf000116_0002
To a solution of intermediate L-3 (7 mg, 0.014 mmol, 1.0 equiv.) in CH2CI2 (1.5 mL) under N2 was added Burgess reagent (9mg, 0.034 mmol, 2.5 equiv.) and the mixture was stirred for 4 h at 45 °C. The reaction was reduced under pressure, purified by preparative HPLC (20-70% MeCN/H20, 0.1% formic acid) and lyophilized to afford compound 180 as a white solid (5.2 mg, 0.011 mmol); ‘H NMR (400 MHz, DMSO-d6) d (ppm): 8.22 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 6.5 Hz, 1H), 7.55-7.50 (m, 1H), 5.13 (q, J = 8.2 Hz, 1H), 4.96 (dd, J = 11.2, 4.7 Hz, 1H), 4.41 (dd, J = 9.5, 6.5 Hz, 1H), 3.14-3.03 (m, 2H), 2.94 (s, 2H), 2.77 (s, 1H), 2.31-2.13 (m, 3H), 1.94-1.36 (m, 14H), 1.30-1.14 (m, 3H), 1.12 (s, 7H), 1.09 (s, 2H), 0.87 (t, J = 7.3 Hz, 3H); MS (ESI) m/z 490.3 [C26H43N5O4 + H]+.
Some compounds herein may exist as mixtures of diastereomers with diastereomeric ratios of at least 9: 1.
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
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Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
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Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
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Figure imgf000166_0001
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Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
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Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Biochemical assays
Protein Expression and Purification
The full-length gene encoding SARS-CoV-2 3CLpro from strain BetaCoV/Wuhan/WIV04/2019 (Accession NC_045512) was synthesized and cloned into Ndel and Xhol site of pET29a(+) vector by Genscript (Piscataway, NJ, USA), as described previously.2 Similarly, the full-length gene encoding a Human Coronavirus 229E (HCoV-229E, Accession X69721.1) was cloned in pET29a( + ) with a C-terminus His tag. The codon optimized plasmid for E. coli expression was transformed into competent BL21(DE3) cells. A single colony was picked to inoculate 10 mL of Terrific Broth (TB) supplemented with 50 mg/L Kanamycin and grown at 37 °C with shaking at 200 rpm. The 10-mL inoculum was added to 1 L of TB with 50 mg /L Kanamycin and grown to an optical density at 600 nm of 2.5. The culture was induced using 0.5mM Isopropyl b-D-l-thiogalactopyranoside (IPTG) and grown at 37 °C for an additional 2 hrs. The cell pellet was resuspended in lysis buffer (20mM Tris, pH 7.5, lOOmM NaCI, 2mM dithiothreitol (DTT) and 10 pg/mL DNase I), and lysed by sonication (25% amplitude, 2 seconds on/ 2 seconds off). Cell debris were removed by centrifugation at 39,191x g for 30 min at 4°C. The supernatant was loaded onto equilibrated HisTrap HP column (Cytiva). The column was washed with lysis buffer containing 5mM imidazole, followed by another wash with 30mM imidazole. Protein was eluted using buffer with 300mM imidazole and further purified by gel filtration chromatography on HiLoad 16/600 Superdex 200 prep grade column (Cytiva) with 20mM Tris, pH 7.5, lOOmM NaCI, 2mM DTT. Fractions from resulted peak were pooled and concentrated using centrifugal filter unit of 10,000-molecular-weight-cutoff. The purity and molecular weight of the protein was confirmed with SDS-PAGE and Mass Spectrometry (MS). A predominant peak at 34863 Da was observed by MS which matched with the calculated molecular weight of SARS-CoV-2 C-His-3CLpro without the N-terminal Methionine residue.
A similar expression and purification condition were followed for HCoV-229E except that the cells were transformed in Rosetta (DE3), cultured in Luria Broth and after induction the cells were grown overnight at 18°C. The molecular weight of 34120 Da was observed using MS and matched with the calculated mass without the N-terminal Methionine residue.
Recombinant MERS-CoV 3CLpro (accession K9N638) expressed in E.coli was purchased from R&D Systems (Cat No. E-719). The purity and molecular size of commercial MERS- CoV 3CL was confirmed in our lab by SDS-PAGE. Further validation by MS showed a predominant peak at 33361 Da that matched with theoretical molecular weight of 33360.3 Da.
The 3C protease domain from Non-Coronaviruses, Human Coxsackievirus B3, strain Beijing0811 (Accession GQ141875.1) and Norovirus Hu/GIi.6/GZ2010- L96/Guangzhou/CHN/2011 (Accession JX989075.1) were cloned into pET29a(+) and pET28a(+) vector, respectively. The gene synthesis and protein purification were conducted by Genscript (Piscataway, NJ, USA). Briefly, the codon optimized plasmids for E. coli expression was transformed into competent BL21(DE3) cells. For protein expression, the culture was induced at 0.5 mM IPTG and grown at 37°C for 4 hrs for CVB3 and overnight at 15°C for Norovirus. Both proteins were purified from soluble fraction using His affinity and size exclusion chromatography. The CVB3 protease was stored in 50 mM Tris-HCI, 100 mM NaCI, 2 mM dithiothreitol, pH 7.5 and a similar storage buffer was used for Norovirus except the Tris-HCI concentration was 20mM with pH 8.0. HRV-3C protease with a N-terminus His tag was purchased from Sigma-Aldrich (Cat no: SAE0045).
Biochemical assay method for SARS-CoV2 3CL, HCoV-229E 3CL and MER5 3CL proteases
A highly sensitive FRET based protease assay was developed to identify inhibitors of 3CL proteases. The substrate, Peptide 1 (Dabcyl)KTSAVLQSGFRKM(Glu)(EDANS) (2) was synthesized by Genscript. The test compounds were 3-fold serially diluted in 100% DMSO to 15 concentrations, starting at 3.33 mM. 1.5 mI of the serially diluted compounds were transferred to a black 384 well assay plate (Cat. 781900, Greiner). 23.5 mI of 2.13X concentration of SARS-CoV-2 Chis-3CLpro, 229E-CoV Chis-3CLpro or MERS-CoV 3CLpro prepared in assay buffer was added to the compounds and incubated for 30 mins at 25°C. 25 mI of 2X concentration of Peptide 1 substrate was added to the assay plate. The SARS-CoV-2 Chis-3CLpro assay plate was incubated at 37°C, the 229E- CoV Chis-3CLpro assay plate and MERS-CoV 3CLpro assay plate were incubated at 25°C for 1.5 hrs. The final assay contained 12.5 nM of SARS-CoV-2 Chis-3CLpro or 6 nM 229E-CoV Chis-3CLpro or 75 nM MERS-CoV 3CLpro with 6 mM peptide 1 substrate and 3% DMSO in assay buffer containing 50 mM HEPES at pH 7.5, 100 mM NaCI, and 0.01% Triton X-100 and ImM DTT.
The FRET signal was measured using an excitation wavelength of 340 nm (UV(TRF) 340/60 nm, Barcode 101), emission wavelength of 490 nm (DSPPsion 486/10 filter, Barcode 220) and Lance/DELFIA D400 single mirror (Barcode 412) on Envision plate reader (2104 Envision Multilabel Plate Readers, Perkin Elmer). The dose-dependent inhibition curves were fitted with a variable slope using GraphPad Prism software (GraphPad, USA) to determine a compound's IC50.
Biochemical assay method for HRV 3C. Norovirus 3C and Coxsackievirus B3 3C proteases
A similar FRET assay was developed for the non-coronavirus 3C proteases. The substrate peptides, Peptide 13: {Glu(Edans)}LEVLFQGP{Lys(Dabcyl)}, Peptide 12 {Glu(Edans)} DFHLQGP{Lys(Dabcyl)}3 and Peptide 1 all synthesized by GenScript were used for HRV 3C, Norovirus 3C and CVB3 3C protease assays, respectively. The compound dilution and assay buffer were similar to SARS-CoV-2 Chis-3CLpro assay. The final assay condition for HRV 3C protease contained 100 nM of enzyme, 6 mM Peptide 13 with 1 hr incubation at 25°C. The final assay condition for Norovirus 3C protease contained 1.25 mM of enzyme and 12.5 mM Peptide 12 and 1.5 hr incubation at 37°C. The final assay condition for CVB3 protease contained 2.5 mM enzyme, 6 mM peptide 1 substrate4 with 2 hrs incubation at 25°C.
Legends:
+ : IC50 >20 mM + + : IC50 1 - 20 mM + + + : IC50 100 nM - 1 mM + + + + : IC50 <100 nM
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000192_0002
Comparator
Figure imgf000193_0001
Cellular assays
Drug titration in B5L2 human coronaviruses (229E and OC43)
The efficacy of compounds for inhibition of virus induced cytopathic effects (CPE) in human coronaviruses 229E (ATCC® VR-740™) and OC43 (ATCC® VR-1558™) was determined by monitoring host cell viability in MRC-5 (ATCC® CCL-171™) cells. MRC-5 cells were seeded at a density of 10,000 cells per well into black, clear flat-bottom 96- well plates (Greiner) on the day before virus inoculation. Cells were infected with 229E or OC43 virus at multiplicity of infection (MOI) of 0.01 using 50 μl of virus inoculum. After 1 hour of virus infection in EMEM containing 2% FBS, virus inoculum was removed and 50mI of diluted test compounds in EMEM + 2% FBS, was added to each well. The cells were then incubated at 35°C with 5% CO2 for 4 days (229E) or 5 days (OC43). Cell viability was measured with CellTiter Glo (Promega), post 4 or 5-day incubation according to manufacturer's protocol, using Tecan infinite M200 pro plate reader. Cytotoxicity of compounds was assessed in parallel on uninfected cells plated together with the cells for virus infection. The percent effect of test compounds at each concentration was calculated with the DMSO-treated uninfected control wells set as 100% cell viability and untreated virus-containing control wells as 0% virus inhibition. Relative Cell-Titer Glo signals were plotted against log10 values of compound concentration. The concentration required for 50% inhibition of virus CPE (EC50 value) was determined using a 4-parameter logistic model in Prism 8. Percent effect for cytotoxicity of test compounds on uninfected cells was calculated using values for DMSO-treated uninfected control wells set as 100% cell viability. CC50 values were calculated using a 4-parameter logistic model in Prism 8 and selectivity index (SI) was calculated by dividing CC50 value over EC50 value for each compound.
Drug titration in BSL3 SARS-CoV2-Nluc virus
A549-hACE2 cells that stably express the human angiotensin-converting enzyme 2 (hACE2) were plated at a density of 10,000 - 12,000 cells per well in white opaque 96- well plates (Corning). On the next day, cells were inoculated with SARS-CoV2-Nluc viruses (MOI 0.1) (Xie et a I . , 2020) together with diluted compounds in phenol red-free medium containing 2% FBS. 50pl of virus-compound mixture was added to each well and plates were incubated at 37°C with 5% C02for 48 hours. At 48 hours post-infection, 50mI of Nano-Glo luciferase substrates (Promega) were added to each well. Luciferase signals were measured using BioTek Synerge Neo2 plate reader (lid-on, Gain 100). The relative luciferase signals were calculated by normalizing the luciferase signals of compound-treated cells to that of the DMSO control wells (set as 100%). The relative luciferase signal was plotted against the loglO values of compound concentration. EC50 (compound concentration causing 50% reduction of luciferase signal) values were calculated using a 4-parameter logistic model in Prism 8.
Legends:
+ : EC50 >20 mM + + : EC50 1 - 20 mM + + + : EC50 <1 mM
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Comparator
Figure imgf000199_0002
It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims
1. A compound of Formula (la) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000201_0001
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted sulfonyl, or optionally substituted su Ifonyla I kyl ; R3 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycyl, or optionally substituted cycloalkyl.
2. The compound according to claim 1, wherein the compound is a compound of Formula (I'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000201_0002
wherein R1 is selected from cyano, or optionally substituted amidoacyl; R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl.
3. The compound according to claim 1 or 2, wherein the compound is a compound of Formula (ll'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000202_0001
wherein R2 is selected from optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted sulfonylalkyl; R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; and R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl.
4. The compound according to any one of claims 1 to 3, wherein R7 is -INH2, - NH(methyl), -NH(ethyl), -NH(propyl), -NH(iso-propyl), -NH(cyclopropyl), NH(cyclobutyl), -NH(cyclopentyl), -NH(cyclohexyl), -NH(benzyl), -N(methyl)2, - N(ethyl)2, -N(propyl)2, -N(iso-propyl)2, pyrrolidinyl, piperidinyl, azetidinyl, or aziridinyl.
5. The compound according to any one of claims 1 to 4, wherein R2 is selected from optionally substituted cycloalkyl having an amide moiety, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonylalkyl, optionally substituted aminosulfonyl, and optionally substituted aminosulfonylalkyl.
6 The compound according to any one of claims 1 to 5, wherein the compound is a compound of Formula (lll'b) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000203_0001
wherein R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R4 is selected from H, optionally substituted alkyl, or optionally substituted cycloalkyl; R5 is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; R6 is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted cycloalkyl; R7 is selected from optionally substituted amino, optionally substituted N-heterocyclyl or optionally substituted N-heteroaryl; R10 and R11 are each independently selected from H, halo or optionally substituted alkyl; or R10 and R11 are linked to form an optionally substituted cycloalkyl, optionally substituted halocycloalkyl, or optionally substituted heterocyclyl; and n is an integer from 1 to 3.
7. The compound according to claim 1, wherein R3 is selected from:
8. The compound according to any one of claims 2 to 6, wherein R3a is selected from:
Figure imgf000204_0001
9. The compound according to any one of claims 1 to 8, wherein R4 is selected from H, methyl, ethyl, propyl, isopropyl, and cyclopropyl.
10 The compound according to any one of claims 1 to 9, wherein R5 is selected from:
Figure imgf000204_0002
11. The compound according to any one of claims 1 to 10, wherein R6 is selected from:
Figure imgf000205_0001
12. The compound according to any one of claims 1 to 11, wherein the compound is a compound of Formula (IV'a) or a pharmaceutically acceptable salt, solvate or prodrug thereof:
Figure imgf000205_0002
wherein
R3a is selected from optionally substituted alkyl, optionally substituted cycloalkyl;
R4 is selected from H, optionally substituted alkyl;
R5 is selected from optionally substituted cycloalkyl;
R10 and R1 1 are eachh independently selected from halo or optionally substituted alkyl; and n is an integer from 1 and 2.
13. The compound according to any one of claims 1 to 12, wherein the compound is selected from:
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
14. A method of synthesising a compound of Formula (I), (I'), (II), (ll'), (III), (lll' ), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising : a) sequentially reacting a C-terminus an amino acid like compound comprising R5 and an amino moiety of a compound comprising R2 with a C-terminus and/or N-terminus of an amino acid like compound comprising R3; and b) oxidising the intermediate of step a) with an oxidising agent in order to form R1.
15. The method according to claim 14, wherein a N-terminus of the amino acid like compound comprising R5 is reacted with a carboxylate compound comprising R6.
16. The method according to claim 14 or 15, wherein the compound comprising R2 comprises a lactamide moiety.
17. The method according to claim 14 or 15, the method further comprises a) oxidising a hydroxyl moiety of a compound comprising R2 to an aldehyde; and b) reacting the aldehyde with an isocvano compound in order to form a lactamide compound comprising R2.
18. A method of treating or preventing a virus infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), (G), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof.
19. A use of a compound of Formula (I), (G), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for treating or preventing a virus infection in a patient in need thereof.
20. A compound of Formula (I), (I'), (II), (ll'), (III), (lll'), (IV), (IV'), (V) or (V') or a pharmaceutically acceptable salt, solvate or prodrug thereof for use in treating or preventing a virus infection in a patient in need thereof.
21. The method, use or compound according to any one of claims 18 to 20, wherein the virus infection is caused or associated with a virus selected from rhinovirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), common coronaviridae (including but not limited to OC43, HKU1, 229E and NL63), enterovirus, poliovirus, coxsackievirus, hepatitis A virus, foot-and-mouth disease virus (FMDV) belonging to the picornaviridae family and calicivirus from the caliciviridae family.
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