Broad-spectrum antiviral drugs against coronaviruses

Abstract

Compounds that exhibit antiviral activity and / or inhibit viral replication against viruses, in particular viruses belonging to the picornavirus-like supergroup, including coronaviruses, having the following formula: (I) wherein X comprises a cyclic moiety, R2 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, amino acid side chain, bicyclic or tricyclic side chain, combinations and substituted forms thereof, and Z is selected from the group consisting of C1-C6 hydroxyalkyl, aldehyde, alpha-ketoamide, and bisulfite, and in particular -CH2OH, -CHO, -CH(OH)SO3 ‑ Na + , and -[0(C=0)R w ]SO3 ‑ Na + .

Description

[0001] Cross-references to related applications

[0002] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 001,781, entitled BROAD SPECTRUM ANTIVIRALS AGAINSTCORONAVIRUS, filed March 30, 2020, which is incorporated herein by reference in its entirety.

[0003] Statement on Federally Funded Research

[0004] This invention was completed with the support of the U.S. government under grant number R01 AI130092 granted by the National Institutes of Health. The government holds certain rights to this invention. Technical Field

[0005] This disclosure relates to broad-spectrum antiviral compounds that target 3C-like proteases of coronaviruses. Background Technology

[0006] Many viruses encode multiproteins using proteases, which catalyze the subsequent cleavage of these proteins into mature, functional proteins essential for viral replication. Previous attempts have attempted to inhibit viral activity by targeting these proteases. However, due to structural variations in viral proteases, most protease inhibitors have a narrow range of specificity—genus, species, or even strain specificity. Therefore, broad-spectrum antiviral drugs are rare and have proven elusive for researchers.

[0007] Highly pathogenic coronaviruses pose a significant threat to public health, such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the emerging SARS-CoV-2, the pathogen of coronavirus disease 2019 (COVID-19). Other members of the picornavirus-like supercluster, such as caliciviruses (including norovirus and Sapporovirus) and picornaviruses, share a common characteristic with coronaviruses: they also possess viral 3C or 3CL proteases, which are responsible for the majority of cleavage of the corresponding viral polyproteins. These 3C and 3CL proteases share several common features, including typical chymotrypsin-like folding and a catalytic triplet (or dummy) of Cys-His-Glu (or Asp) on the protease, as well as a preference for Glu or Gln residues at the P1 position on the substrate. Caliciviruses include norovirus (Norwalk virus [NV]), feline calicivirus, MD145, murine norovirus [MNV], swine vesicular herpesvirus, and rabbit hemorrhagic disease virus. Picornaviridae include enteroviruses (such as enterovirus 71), poliovirus, Coxsackievirus, foot-and-mouth disease virus (FMDV), hepatitis A virus (HAV), porcine swine flu virus, and rhinovirus (which causes the common cold).

[0008] Coronaviruses, in particular, are a large family of viruses that can cause a variety of diseases in humans and animals. Coronaviruses include human coronaviruses (which cause the common cold, such as strain 229E), transmissible gastroenteritis virus (TGEV), mouse hepatitis virus (MHV), bovine coronavirus (BCV), feline infectious peritonitis virus (FIPV), and SARS-CoV. Most coronaviruses typically cause the common cold, a mild upper respiratory illness. However, global outbreaks of novel human coronavirus infections with severe respiratory illness have periodically occurred in animals, including SARS-CoV, MERS-CoV, and most recently, SARS-CoV-2, which emerged in December 2019 and subsequently spread worldwide. Genetic analysis of SARS-CoV-2 indicates that it is closely related to bat-derived SARS-like beta coronaviruses bat-SL-CoVZC45 and bat-SL-CoVZXC21. Despite the periodic emergence of novel coronaviruses infecting humans, there are currently no FDA-approved, widely effective vaccines or antiviral drugs against these viruses, highlighting the urgent need to develop preventative and therapeutic measures against coronaviruses.

[0009] The SARS-CoV-2 genome is relatively large (~30 kb) and shares similarities with the genomes of SARS-CoV and MERS-CoV (~80% and ~50% sequence identity, respectively). It contains two open reading frames (ORF1a and ORF1b) that encode a variety of structural and non-structural proteins. Translation of the genomic mRNA of ORF1a produces a polyprotein (pp1a), while a second polyprotein (pp1b) is the product of a ribosomal frameshift that links ORF1a and ORF1b together. These two polyproteins are processed by a 3C-like protease (3CLpro, also known as the main protease, Mpro) (11 cleavage sites) and a papain-like cysteine ​​protease (PLpro) to produce 16 mature non-structural proteins, including an RNA-dependent RNA polymerase (RdRp) involved in the replication-transcriptional complex. Both 3CLpro and PLpro are essential for viral replication, making them attractive targets for drug development. Coronavirus 3CLpro is a chymotrypsin-like cysteine ​​protease with two N-terminal domains containing two β-barrel chymotrypsin-like folds. The active site of 3CLpro is located in the cleft between the two domains and is characterized by the catalytic Cys148-His41 dual.

[0010] Our efforts in this area have led to the discovery of a range of broad-spectrum inhibitors for viruses, including coronaviruses encoding 3CLpro and norovirus, and the first demonstration of a coronavirus 3CLpro inhibitor (…). GC376 Currently in clinical development, see U.S. Patent No. 9,474,759, issued October 25, 2016, which is incorporated herein by reference in its entirety for its clinical efficacy. Administration of the 3CLpro inhibitor to cats suffering from feline infectious peritonitis (FIP), a systemic disease caused by a 100% lethal coronavirus, reversed FIP progression and led to clinical remission. We also recently reported the following results: exploratory in vitro studies using a dipeptide-based family of MERS-CoV 3CLpro inhibitors containing a piperidine moiety as a novel design element, along with associated structural and biochemical studies. Summary of the Invention

[0011] The COVID-19 pandemic remains a major global public health issue, and there is an urgent need to develop effective treatments, including vaccines, biologics, and small molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Examination of the viral life cycle has revealed several virus- and host-based blockade points that could be leveraged to combat the virus. The SARS-CoV-2 3CL protease, an enzyme essential for viral replication, is an attractive viral blockade point, and the design of inhibitors to this protease could lead to the development of effective SARS-CoV-2-specific antiviral drugs.

[0012] Here, we report that the 3CLpro inhibitor is highly effective against a variety of coronaviruses, including MERS-CoV, SARS-CoV, and SARS-CoV-2, in enzyme and / or cell-based assays. In a mouse model of MERS-CoV infection, administration of the lead compound one day after viral infection significantly improved survival and reduced lung viral titers and histopathology, demonstrating proof-of-concept therapeutic efficacy. These results suggest that this line has the potential to be developed into broad-spectrum antiviral agents against these important human coronaviruses and other viruses belonging to the picornavirus-like supergroup, including caliciviruses and picornaviruses.

[0013] In one respect, an antiviral compound comprising Formula I or a pharmaceutically acceptable salt or prodrug thereof is provided:

[0014]

[0015] Each X contains at least one cyclic portion, and in particular, substituted or unsubstituted C3-C. 10 Cycloalkyl, substituted or unsubstituted 3-7 membered heterocycles having 1-3 cyclic heteroatoms selected from N, O and S, or substituted C 6-10 The aryl group can be directly attached to oxygen, or it can be linked via a branched or unbranched and substituted or unsubstituted C1-C6 alkyl bond, or a branched or unbranched C1-C6 alkylene bond; R1 is a glutamine or imidazole substitute; R2 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, natural (Leu) or non-natural (Cha) amino acid side chain, bicyclic or tricyclic side chain, or a combination thereof, and particularly a branched or unbranched C1-C6 alkyl, C3-C 10 cycloalkyl, C 6-10 Aryl, C1-C6 alkylene-C 6-10 aryl, C2-C6 alkenyl, C2-C6 alkynyl or combinations thereof, wherein the C1-C6 alkyl group is optionally surrounded by one or more halogens, –OH, –SH, –SCH3, –NH2, –COOH, –C(O)NH2, –NH(C=NH)NH2, C3-C 10 cycloalkyl groups, C groups optionally substituted with -OH 6-10 Aryl, or 5-9 membered heteroaryl substitution having 1-3 cyclic heteroatoms selected from N, O, and S; Z is selected from the group consisting of C1-C6 hydroxyalkyl, aldehyde, α-ketoamide, and bisulfite, and especially –CH2OH, -CHO, -SO3Na, -CH(OH)SO3 - Na + And -[O(C=O)R w SO3 - Na+ , where R w It is an alkyl or arylalkyl group, with -CH3 and -CH2CH3 being particularly preferred. Deuterated forms of the above compounds are also considered in this document.

[0016] A method for treating or preventing viral infection in a subject from one or more coronaviruses and other viruses belonging to the microRNA-like supergroup (including caliciviruses and microRNAs) is also provided. The method includes administering to the subject a therapeutically effective amount of a first antiviral compound according to various embodiments described herein.

[0017] Broad-spectrum antiviral compositions are also disclosed. These compositions comprise a first antiviral compound dispersed in a pharmaceutically acceptable carrier according to various embodiments described herein.

[0018] This document also provides a kit. The kit comprises: an antiviral compound according to the various embodiments described herein; and instructions for administering the compound to a subject in need.

[0019] A method for preventing or inhibiting viral replication in cells is also disclosed. The method involves contacting coronavirus, microRNA, or calicivirus cells with compounds according to various embodiments described herein.

[0020] The use of compounds according to various embodiments described herein in the preparation of therapeutic or preventive medicaments for treating or preventing viral infections of coronavirus, picornavirus, or calicivirus in subjects is also disclosed. Attached Figure Description

[0021] The patent or application documents contain at least one color drawing. Upon request and payment of the necessary fees, the patent office will provide a copy of the patent or patent application publication with color drawings.

[0022] Figure 1 Displayed with for 6a to 6k and 7a to 7k A stepwise compound synthesis reaction scheme 1 for intermediate compounds of a series of 3C-like protease (3CLpro) inhibitors. An alcohol input is reacted with (L) leucine isocyanate methyl ester or (L) cyclohexylalanine isocyanate methyl ester to generate a product, which is then hydrolyzed with lithium hydroxide in an aqueous tetrahydrofuran solution to the corresponding acid. The acid is subsequently reacted with a glutamine-substituted methyl ester… 8 "Couplet yields a compound." 4 "Lithium borohydride reduction produces alcohols." 5 Then it was oxidized to the corresponding aldehyde using the Des Martin periodane reagent.6 The bisulfite adduct is produced by treatment with sodium bisulfite and ethyl acetate in an aqueous ethanol solution. 7 Step a) Amino acid methyl ester isocyanate / TEA / CH3CN / reflux / 2 h; Step b) 1M LiOH / THF / RT / 3 h; Step c) EDCI / HOBT / glutamine substitute / DIPEA / DMF / RT / 24 h; Step d) 2M LiBH4 / THF / methanol / RT / 12 h; Step e) Des Martin periodoyl ether / DCM / 15°C to 18°C / 3 h; and Step f) NaHSO3 / ethyl acetate / ethanol / H2O / 44°C to 55°C.

[0023] Figures 2A-F The X-ray crystal structures of MERS-CoV 3CLpro with 6h (A, B, C) and 7j (D, E, F) are shown.

[0024] Figure 2G-L The X-ray crystal structures of SARS-CoV 3Clpro with compound 7j (G, H, I) and SARS-CoV-2 3Clpro with compound 7j (J, K, L) are shown. Figures 2A-F and 2G-L In the figures (A), (D), (G), and (J), the contouring at 3σ is shown. F o - F c Figures omitted (green grid); Figures (B), (E), (H), and (K) show the hydrogen-bonded interactions between the inhibitor and the 3CL protease (dashed lines); and Figures (C), (F), (I), and (L) show the electrostatic surface representation of the binding pocket occupied by the inhibitor. Adjacent residues are yellow (nonpolar), cyan (polar), and white (weakly polar).

[0025] Figure 3 A) was shown 7i B) 6b C) 6g and D) 6d Electrostatic surface representation of the 3CLpro pocket occupied by MERS-CoV. Adjacent residues are yellow (nonpolar), cyan (polar), and white (weakly polar).

[0026] Figure 4 It shows that it has A) 7i B) 6b C) 6g and D) 6d The inhibitor-binding structure of MERS-CoV 3CLpro is outlined at 3σ. F o- F c Image omitted (green grid).

[0027] Figure 5 It shows MERS-CoV 3CLpro and A) 7i B) 6b C) 6g and D) 6d Hydrogen bond interactions between them (dashed lines).

[0028] Figure 6 The compound was shown 6j or 6h In infection with MERS MA Data on therapeutic treatment in hDPP4-KI mice. (A) shows the compound. 6j Dose-dependent curves of the compound inhibiting MERS-CoV in cell culture. 6j Serial dilutions of the virus were added to confluent Huh-7 cells, which were immediately infected with MERS-CoV at a multiple of infection (MOI) of 0.01. After incubating the cells at 37°C for 48 hours, viral titers were determined using a plaque formation assay, and the 50% inhibitory concentration (EC50) was determined using GraphPadPrism software. 50 Values ​​(B and C) will be determined by mouse-adapted MERS-CoV (MERS). MA hDPP4-KI mice infected with -CoV n = 6) Start using the compound 1 day after viral infection (dpi). 6j or 6h Treatment lasted up to 10 days, with survival (B) and body weight (C) monitored for 15 days. Control mice received only the carrier. (D and E) MERS MA -CoV-infected hDPP4-KI mice were treated with the compound starting at 1, 2, or 3 dpi. 6j treat( n = 5), and monitor survival (D) and body weight (E) for 15 days. Includes untreated mice and mice treated with the drug ( n = 4) As a control. Data points represent the mean and SEM of an experiment. Survival curves in the groups were analyzed using the log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test.

[0029] Figure 7 Displayed MERS MA Lung virus titer and histopathology of infected hDPP4-KI mice. hDPP4-KI mice were treated with MERS at 0 dpi. MA-CoV infection, then starting 1 dpi with either a carrier as a control treatment or a compound. 6j Treatment until euthanasia (per group) n = 4 or 5). (A) Collect lung tissue and measure viral titers at 3 and 5 dpi. Examine lung edema and hyaline membrane formation (B), and perform histopathological staining of lung sections with hematoxylin and eosin at 6 dpi (C to F). (B) Rate tissue edema and hyaline membrane formation using the following scale: 0, none; 1, sparse (<5 alveoli); 2, <33% of lung fields; 3, 34% to 66% of lung fields; and 4, >66% of lung fields ( 30 (C) through (F) show the load control in (C) and (E) and the compounds in (D) and (F) at 40×[(C) and (D)] or 100×[(E) and (F)]. 6j Representative histopathological images of the treated tissue. An asterisk indicates multiple t-tests were performed. P <0.01.

[0030] Figure 8 Showing the use of S2- 1-7 Scheme 2, which involves the stepwise synthesis of a series of 3C-like protease (3CLpro) inhibitors using intermediate compounds, is similar to Scheme 1.

[0031] Figure 9 Compound S2- was shown 5g and S2- 6g In infection with MERS MA Therapeutic treatment in hDPP4-KI mice. MERS infection... MA hDPP4-KI mice (N=5) were given the compound starting at 1 dpi. S2-5g or S2-6g (From Protocol 2) Treatment was administered, and survival was monitored for 21 days. Each compound was administered 50 mg / kg once daily via intraperitoneal injection at 1–11 dpi. Control mice (ctrl) received the loading agent.

[0032] Figure 10A The reaction scheme for synthesizing precursor alcohols 12-16 is shown.

[0033] Figure 10B The reaction scheme for synthesizing inhibitor 1-24b-c is shown.

[0034] Figure 10C The reaction scheme for synthesizing amino alcohol A is shown.

[0035] Figure 11The binding modes of inhibitors containing nonpolar substituents are shown. A / E) AMJ-I-157 (5c), B / F) AMJ-I-158 (1c), C / G) AMJ-I-159 (3c), and D / H) NN-II-111 (8b) are shown bound to SARS-CoV-2 3CLpro. The binding modes are outlined at 3σ. F o - F c Polder diagram (AD) omitted. Hydrogen bonding interactions (EH) are plotted as dashed lines.

[0036] Figure 12 The surface representation shows the orientation of the nonpolar group near the S4 subsite of SARS-CoV-2 3CLpro, with adjacent residues in yellow (nonpolar), cyan (polar), and white (weakly polar). A) AMJ-I-157 (5c), B) AMJ-I-158 (1c), C) AMJ-I-159 (3c), D) NN-II-111 (8b). E) Superposition of gray AMJ-I-159 (3c) and coral AMJ-I-158 (1c). F) Superposition of gray AMJ-I-159 (3c) and magenta NN-II-111 (8b).

[0037] Figure 13 The binding modes of inhibitors containing a 4,4-difluorocyclohexyl group are shown. A / D) AMJ-I-108 (12b), B / E) AMJ-I-114 (13c), and C / F) AMJ-I-111 (14c) are shown with SARS-CoV-2 3CLpro. The binding modes are outlined at 3σ. F o - F c Polder diagram (AC) omitted. Hydrogen bonding interactions (DF) are plotted as dashed lines.

[0038] Figure 14 The surface representation shows the orientation of the 4,4-difluorocyclohexyl group near the S4 subsite of SARS-CoV-2 3CLpro, with adjacent residues appearing as yellow (nonpolar), cyan (polar), and white (weakly polar). A) AMJ-I-108 (12b), B) AMJ-I-114 (13c), and C) AMJ-I-111 (14c). D) Superposition of 12b (gold), 13c (coral), and 14c (gray). The ring between Gln 189 and Gly 195 is cyan in the structure with 13c and magenta in the structure with 12b / 14c.

[0039] Figure 15 The binding modes of inhibitors containing fluorinated aromatic groups are shown. A / C) CSD-III-028 (17c), B / D) CSD-III-029 (18c) with SARS-CoV-2 3CLpro. The binding modes are outlined at 3σ. F o - F c Polder diagram omitted (AB). Hydrogen bonding interactions (CD) are plotted as dashed lines. The 3.38 Å contact between the F atom in 17c and the O atom in the main chain of Glu 166 is plotted as a solid line in Figure C. Surface representation shows the orientation of the 4,4-difluorocyclohexyl group near the S4 subsite of SARS-CoV-2 3CLpro, with adjacent residues in yellow (nonpolar), cyan (polar), and white (weakly polar) colors. E) CSD-III-028 (17c) and F) CSD-III-029 (18c).

[0040] Figure 16 The binding mode of NN-II-123 (21c) containing a perfluorinated aromatic group is shown. A) Profiled at 3σ F o - F c Polder diagram omitted. B) Hydrogen bonding interactions are plotted as non-dashed lines. Close contacts with the perfluorinated ring that are longer than the typical polar contact distance are plotted as solid lines. C) Surface representation showing the orientation of NN-II-123 (21c) near the S4 subsite of SARS-CoV-2 3CLpro, with adjacent residues in yellow (nonpolar), cyan (polar), and white (weakly polar).

[0041] Figure 17 The binding modes of SARS-CoV-2 3CLpro associated with CSD-III-008 (19b) (A and B) and CSD-III-009 (20b) (C and D) are shown. Electron density is profiled at 3σ. F o - F c Polder diagram omitted (green grid). Hydrogen bonding interactions between SARS-CoV-2 3Clpro and CSD-III-008 (E and F) and CSD-III-009 (G and H) associated with subunits A (E / F) and B (G / H).

[0042] Figure 18The surface representation is shown, illustrating the orientation of CSD-III-008(19b) (A and B) and CSD-III-009 (C and D) associated with subunits A (A / C) and B (B / D) on the SARS-CoV-2 3CLpro surface. Adjacent residues are yellow (nonpolar), cyan (polar), and white (weakly polar). Detailed Implementation

[0043] A series of non-deuterated and deuterated 3CLpro protease inhibitors and prodrug forms have been synthesized and demonstrated broad-spectrum activity against multiple coronaviruses (including MERS-CoV, SARS-CoV, and SARS-CoV-2) and other viruses belonging to the picornavirus-like supergroup (including caliciviruses and picornaviruses) in enzyme and cell-based assays. The efficacy of these compounds in animal models of MERS-CoV infection has also been demonstrated. Members of this series of compounds are highly effective as antiviral therapies targeting specific viruses, or more importantly, they are broad-spectrum antiviral agents targeting multiple viruses. The latter's broad applicability constitutes a significant advance in antiviral research and public health.

[0044] The embodiments described herein include antiviral compounds with broad-spectrum (multivalent) activity against coronaviruses and other viruses belonging to the picornavirus-like supergroup (including caliciviruses and microRNAs). Not wishing to be bound by theory, these compounds are based on small-molecule antiviral drugs that effectively target and inhibit the activity of viral 3CL protease in a variety of viral species, strains, and subtypes, thereby preventing the formation of mature virus and inhibiting viral replication in host cells. In some embodiments, the compounds are prodrugs converted into active compounds that target and inhibit viral 3CL protease activity.

[0045] In some embodiments, an antiviral compound comprising (consisting substantially of or even consisting of) formula (I) or a pharmaceutically acceptable salt thereof is provided:

[0046] .

[0047] In the above structure, X comprises at least one ring-shaped portion, and in particular, substituted or unsubstituted C3-C. 10 Cycloalkyl, substituted or unsubstituted 3-7 membered heterocycles having 1-3 cyclic heteroatoms selected from N, O and S, or substituted C 6-10The aryl group can be directly attached to oxygen, or it can be linked via a branched or unbranched and / or substituted or unsubstituted C1-C6 alkyl bond, or a branched or unbranched C1-C6 alkylene group. Substituted or unsubstituted C3-C6 cycloalkanes or saturated heterocycles are particularly preferred cyclic moieties, with cyclohexane and 6-membered heterocycles being most preferred (most preferably oxoheterocyclic compounds). The cycloalkanes and heterocycles can be monosubstituted or disubstituted. In the case of a disubstituted ring, the two substituents are on the same ring atom. When X contains a substituted aryl group, para-substitution is particularly preferred. Preferred substituents include halogens (-F, -Cl, -Br, particularly -F) and C1-C6 branched or unbranched alkyl groups. The substituted aryl group is preferably phenyl, which is preferably monosubstituted by a halogen substituent (-F, -Cl, -Br, particularly -F). In such embodiments, meta-substitution is particularly preferred. As noted, the cyclic moiety may be directly or via a branched or unbranched C1-C6 alkyl bond to oxygen. In some embodiments, the bond may further comprise one or more side chain groups, including substituted or unsubstituted alkyl (C1-C6 alkyl), substituted or unsubstituted aryl (e.g., phenyl, substituted phenyl), arylalkyl (e.g., benzyl or a group wherein the aryl group is naphthalene), etc.

[0048] In the above structure, each R2 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, natural (e.g., Leu) or non-natural (e.g., Cha) amino acid side chain, bicyclic or tricyclic side chain, or combination thereof, and particularly branched or unbranched C1-C6 alkyl, C3-C 10 cycloalkyl, C 6-10 Aryl, C1-C6 alkylene-C 6-10 aryl, C2-C6 alkenyl, C2-C6 alkynyl or combinations thereof, wherein the C1-C6 alkyl group is optionally surrounded by one or more halogens, –OH, –SH, –SCH3, –NH2, –COOH, –C(O)NH2, –NH(C=NH)NH2, C3-C 10 cycloalkyl groups, C groups optionally substituted with -OH 6-10 The aryl group, or a 5-9 membered heteroaryl group having 1-3 cyclic heteroatoms selected from N, O and S, wherein, for example, R2 may be a bicyclic or tricyclic side chain, leucine (Leu), cyclohexylalanine (Cha) or a fluorinated side chain.

[0049] In the above structures, each Z is selected from the group consisting of C1-C6 hydroxyalkyl, aldehyde, α-ketoamide, and bisulfite, and particularly –CH2OH, -CHO, and -CH(OH)SO3. - Na + And -[O(C=O)R w SO3 - Na+ , where R w It is an alkyl or arylalkyl group, preferably -CH3 and -CH2CH3.

[0050] This disclosure includes the deuterated form of the above-described compounds.

[0051] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term C... n An alkyl group is defined as having "n" carbon atoms. For example, a C4 alkyl group is an alkyl group having 4 carbon atoms. C1-C6 alkyl groups are alkyl groups having a range of carbon atoms including the entire range (e.g., 1-6 carbon atoms) and all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and tert-butyl. Unless otherwise stated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group. The term "hydroxyalkyl" refers to an alkyl group substituted with 1-3 -OH moieties, such as 1, 2, or 3 -OH moieties. For example, C1-C6 hydroxyalkyl refers to a C1-C6 alkyl group substituted with 1, 2, or 3 -OH groups, such as a C1-C6 alkyl group substituted with 1 -OH group.

[0052] As used herein, the term "alkylene" refers to an alkyl group having a substituent. For example, an alkylene group can be -CH2CH2- or -CH2-. Term C n This refers to an alkylene group having "n" carbon atoms. For example, C1-C6 alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range and all subgroups, as previously described for "alkyl" groups. Unless otherwise stated, an alkylene group can be an unsubstituted alkylene group or a substituted alkylene group.

[0053] As used herein, the term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing 2 to 6 carbon atoms and having at least one carbon-carbon double bond.

[0054] As used herein, the term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and at least one triple bond. Examples of C2-C6 alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, and 3-hexyne.

[0055] As used herein, the terms "cycloalkane" and "cycloalkyl" refer to aliphatic cyclic hydrocarbon groups comprising three to ten carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms). The term C... nThis refers to a cycloalkyl group having "n" carbon atoms. For example, a C6 cycloalkyl group refers to a cycloalkyl group with 6 carbon atoms in the ring. (C3-C...) 10 A cycloalkyl group is a cycloalkyl group having a number of carbon atoms ranging from 3 to 10 carbon atoms in its entirety (e.g., 3 to 10 carbon atoms) as well as in all subgroups (e.g., 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 7-8, 6-9, 7-9, 8-9, 6-10, 7-10, 8-10, 9-10, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise stated, a cycloalkyl group may be an unsubstituted or substituted cycloalkyl group. The cycloalkyl groups described herein may be separate from or fused with another cycloalkyl group, heterocycloalkyl group, aryl group, and / or heteroaryl group. When one cycloalkyl group is fused with another cycloalkyl group, each cycloalkyl group may contain three to ten carbon atoms unless otherwise specified. Unless otherwise specified, the cycloalkyl group may be unsubstituted or substituted.

[0056] As used herein, the term "heterocyclic" or "heterocyclic alkyl" is similar to the definition of cycloalkyl, except that the ring contains one to three heteroatoms independently selected from oxygen, nitrogen, and sulfur. Specifically, the term "heterocyclic alkyl" refers to a ring containing a total of three to seven atoms (e.g., three to seven, or five to seven), wherein one, two, or three of these atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur, and the remaining atoms in the ring are carbon atoms. Non-limiting examples of heterocyclic alkyl groups include piperidine, pyrazolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, etc.

[0057] The cycloalkyl and heterocycloalkyl groups can be saturated or partially unsaturated cyclic systems, optionally composed of, for example, one to three groups independently selected from halogens, OH, C(O)-C. 1-6 Alkyl, C(O)NH2 and C 5-6 The heterocyclic alkyl group is substituted. Optionally, the heterocyclic alkyl group may be further N-substituted by an alkyl, alkylene-OH, alkylene aryl, or alkylene heteroaryl group. The heterocyclic alkyl group described herein may be separate from or fused with another heterocyclic alkyl, cycloalkyl, aryl, and / or heteroaryl group. When a heterocyclic alkyl group is fused with another heterocyclic alkyl group, each heterocyclic alkyl group may contain three to seven total ring atoms and one to three heteroatoms, for example, 6 to 14 total ring atoms, such as 12, 13, or 14 ring atoms and one to three heteroatoms. Unless otherwise stated, the heterocyclic alkyl group may be unsubstituted or substituted.

[0058] As used herein, the term "aryl" refers to a monocyclic or bicyclic aromatic ring having 5 to 10 total ring carbon atoms, such as a phenyl ring. Unless otherwise stated, the aryl group may be unsubstituted or composed of one or more, particularly one to four, independently selected, for example, halogens, OH, C(O)-C. 1-6 Alkyl, C(O)NH2 and C 5-6 Cycloalkyl groups are substituted. The aryl group may be separate from or fused with another aryl group (e.g., naphthyl, anthracene), a cycloalkyl group (e.g., tetrahydronaphthyl), a heterocyclic alkyl group, and / or a heteroaryl group (e.g., phenyl). Exemplary aryl groups include, but are not limited to, phenyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, etc.

[0059] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic aromatic ring having 5 to 10 total ring atoms and containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur atoms within the aromatic ring. Unless otherwise stated, the heteroaryl group may be unsubstituted or surrounded by one or more, particularly one to four, heteroatoms selected from, for example, halogens, OH, C(O)-C. 1-6 Alkyl, C(O)NH2 and C 5-6 Substituents of cycloalkyl groups. In embodiments, the heteroaryl group is substituted by one or more of alkyl and alkoxy groups. Examples of heteroaryl groups include, but are not limited to, thiophene, furanyl, pyridinyl, pyrroleyl, oxazolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazoleyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0060] As used herein, the term "substituted" when used to describe a modified chemical functional group means that at least one hydrogen radical on the functional group is substituted by a substituent. Substituents can include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, oxy, alkoxy, heteroalkoxy, ester, thioester, carboxyl, cyano, nitro, amino, amide, acetamide, and halogens (e.g., fluorine, chlorine, bromine, or iodine). When a chemical functional group includes more than one substituent, these substituents can be bonded to the same carbon atom or to two or more different carbon atoms.

[0061] As used herein, the phrase “substituted or unsubstituted” means unsubstituted (e.g., substituted by H) or substituted by a group, such as those defined herein. It should be understood that the substitution of a given atom is limited by its valence. Substituent (group) prefixes such as alkyl, without the modifiers “unsubstituted” or “substituted,” should be understood to indicate that the specific substituent is unsubstituted.

[0062] The term "pharmaceutically acceptable salt" refers to an acidic or basic salt of the compounds of the present invention that has the desired antiviral activity and is not undesirable biologically or otherwise. This disclosure also includes prodrugs (esters, amides, carbamates, carbonates, ethers, imines, phosphates, and other derivatives of the disclosed compounds) and deuterated derivatives of the compounds.

[0063] In some embodiments, the particularly preferred X portion includes the following structure in parentheses (wherein oxygen from formula (I) is described for clarity):

[0064]

[0065] Where m and n are 0, 1, 2, or 3, provided that at least one of n or m is 1; p is 0, 1-5; R, when present, is a halogen; Y1 is O or S (=O). o Where o is 0, 1, or 2; Y is (CR i R j ) o Where o can be 0 (meaning Y is absent and oxygen is directly bonded to the carbon in the ring) or 1 (meaning Y = CR). i R j , where R i and R j All can be H, or all can be methyl, or one can be H and the other can be methyl, or a deuterated derivative thereof; o can also be 3 or greater, but 0 and 1 are preferred; each Q is selected from the group consisting of branched or unbranched alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl), C(CH3)2, CHF, CF2 or CHCF3, but can also be O or S (=O). o Where o is 0, 1, or 2; Q can also be a carbon atom connected to different groups, for example, Q can be CR w R x , where each R w It is -H or alkyl, arylalkyl, phenyl or deuterated phenyl, substituted phenyl or F, and R x= H or alkyl or F; each R3 is selected from the group consisting of -OH, -H, branched or unbranched alkyl (e.g., methyl, ethyl, butyl, isobutyl), substituted or unsubstituted aryl (e.g., phenyl, substituted phenyl), arylalkyl (e.g., benzyl or a group wherein the aryl is naphthyl), and preferably at least one or both R3 moieties are alkyl (more preferably C1-C3 alkyl), or one R3 is alkyl and one R3 is a -OH group; each R4, R5 and R6 are independently selected from -H, branched or unbranched alkyl (e.g., methyl, ethyl, butyl, isobutyl), substituted or unsubstituted aryl ... For example, the group consisting of phenyl, substituted phenyl), arylalkyl (e.g., benzyl or a group wherein the aryl group is naphthyl), substituted or unsubstituted aryl (e.g., phenyl, substituted phenyl), arylalkyl and cyclopropane ring (cis and / or trans), wherein each R4 may also be a halogen (F, Cl, Br); each R5 is preferably -H, alkyl or halogen (F, Cl, Br); each R6 is preferably -H, branched or unbranched alkyl, haloalkyl, substituted or unsubstituted aryl (e.g. phenyl, substituted phenyl), arylalkyl, cyclopropane ring (cis and / or trans), or cyclohexane or substituted cyclohexane or bicyclic or tricyclic ring.

[0066] Certain cycloalkane compounds have structures including the following:

[0067]

[0068] The substituents are as defined above, and each R1 is a glutamine or imidazole substitute:

[0069]

[0070] Specific examples of the cycloalkanes series have the following structures:

[0071]

[0072] That is, where m and n are each equal to 2, and W is CF2, CHCF3, O, S, S(=O), SO2, or a C1-C6 alkyl group; and the following structures:

[0073]

[0074] Where m is 1 and n is 0.

[0075] Macrocyclic derivatives of cycloalkanes were also considered:

[0076]

[0077] Where m and n are 0, 1, 2 or 3 respectively, at least one of m or n is 1, and each q is 1-6, and the remaining substituents are as defined above.

[0078] In one or more embodiments, the particularly preferred [X] group comprises the following structure shown in parentheses, with the oxygen bond from formula (I) depicted for clarity:

[0079]

[0080]

[0081] Prophylactic and / or therapeutic compositions with specific or broad-spectrum antiviral activity are also disclosed. Combinations of one or more of the above-described compounds may also be used in this invention. The compositions comprise the antiviral compound described herein dispersed in a pharmaceutically acceptable carrier. As used herein, the term carrier refers to a diluent, excipient, loading agent, etc., in which an antiviral drug can be dispersed for administration. A suitable carrier will be pharmaceutically acceptable. As used herein, the term "pharmaceuticalally acceptable" means that it is not biologically or otherwise undesirable because it can be administered to a subject without excessive toxicity, irritation, or anaphylactic reactions, and does not cause unacceptable biological effects or interact harmfully with any other component of the composition containing it. Pharmaceutically acceptable carriers will be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the subject. Pharmaceutically acceptable ingredients include those acceptable for veterinary use as well as for human pharmaceutical use, and depend on the route of administration. For example, compositions suitable for administration by injection are typically solutions in sterile isotonic buffer solutions. Exemplary carriers include aqueous solutions, such as physiological (n.) saline (~0.9% NaCl), phosphate-buffered saline (PBS), sterile water / distilled hot-pressed water (DAW), various oil-in-water or water-in-oil emulsions, and dimethyl sulfoxide (DMSO) or other acceptable carriers.

[0082] The composition may comprise a therapeutically effective amount of the compound dispersed in a carrier. As used herein, a “therapeutically effective” amount means an amount that will elicit a biological or medical response in the tissue, system, or subject being sought by an investigator or clinician, and particularly by causing some desired therapeutic or preventative effect against a viral infection through slowing and / or inhibiting 3CL protease activity and / or viral replication. Those skilled in the art will recognize that an amount may also be considered therapeutically “effective” even if the condition is not completely eradicated or prevented, but its symptoms and / or effects are partially improved or alleviated in a subject. In some embodiments, based on the total weight of 100% of the composition, the composition will comprise about 5% to about 95% by weight of the antiviral compound described herein, preferably about 30% to about 90% by weight. In some embodiments, the composition may comprise a combination of more than one type of the aforementioned antiviral compound, in which case the total level of all such compounds will preferably fall within the range described above.

[0083] The composition may include other components such as adjuvants, other active agents, preservatives, buffers, salts, and other pharmaceutically acceptable ingredients. The term "adjuvant" is used herein to refer to a substance that has an immunomodulatory effect and is added to or co-formulated in a therapeutic composition to enhance, provoke, and / or modulate an innate, humoral, and / or cell-mediated immune response against the active ingredient. Other active agents that may be included in the composition include other antiviral compounds (e.g., cathepsins) or any immunogenic active component (e.g., antigens), such as those similar to pathogenic microorganisms or infectious agents, and / or those prepared from their attenuated or killed forms, their toxins, subunits, particles, and / or one of their surface proteins, thereby eliciting an immune response to that microorganism or infectious agent. In addition to live, modified, or attenuated vaccine components, active agents using synthetic peptides, carbohydrates, or antigens may also be used.

[0084] The compositions according to the embodiments disclosed herein can be used in subjects to treat and / or prevent viral infections from coronaviruses as well as other viruses belonging to the picornavirus-like supergroup, including caliciviruses and picornaviruses. Therefore, the embodiments described herein have broad-spectrum therapeutic and / or preventative uses. As used herein, the term “therapeutic” or “treatment” refers to a process designed to produce beneficial alterations to a subject’s existing condition (e.g., viral infection, disease, ailment), such as by reducing the severity of clinical symptoms and / or effects of infection, and / or reducing the duration of infection / symptoms / effects. As used herein, the term “prophylactic” or “prevention” refers to a process designed to suppress or improve the effects of future viral infections or diseases that a subject may be exposed to (but is not currently infected with). In some cases, the composition can prevent the development of observable morbidity caused by a viral infection (i.e., near 100% prevention). In other cases, the composition may only partially prevent and / or mitigate the severity of morbidity caused by a viral infection (i.e., reduce the severity of symptoms and / or effects of infection, and / or reduce the duration of infection / symptoms / effects). In any case, these compounds are still considered to be "preventing" the target infection or disease.

[0085] In use, a therapeutically effective amount of the antiviral compound is administered to the subject. In some embodiments, a composition comprising a therapeutically effective amount of the antiviral compound is administered to the subject. In any case, the compound or a pharmaceutically acceptable salt thereof is preferably administered to the subject in an amount sufficient to provide an antiviral compound level of about 0.1 mg to about 1,000 mg of the compound per kg of subject body weight, preferably about 1 mg / kg to about 100 mg / kg of subject body weight, more preferably about 10 mg / kg to about 50 mg / kg of subject body weight (independent of salt, if present). Therefore, it should be understood that, for example, in the case of a compound salt, the formulation may be administered in amounts greater than the above ranges to provide a sufficient level of the active compound.

[0086] In some embodiments, the subject has or suffers from a condition (e.g., infection, disease, or ailment) prior to administration of the compound, wherein the methods described herein can be used to treat the condition and / or mitigate its effects. Preferably, the antiviral compound is administered as soon as possible after infection, preferably within about 7 days of the onset of observable symptoms, more preferably within about 5 days of the onset of observable symptoms, and even more preferably within 3 days of the onset of observable symptoms. It should be understood that the earlier one or more compounds are administered, the greater the chance of successfully reducing the effects of viral infection. In other embodiments, the subject does not have a given condition prior to administration of the compound, wherein the methods described herein can be used to prevent the onset or occurrence of a condition and / or mitigate its effects, as described above. The disclosed embodiments are applicable to a variety of routes of administration, depending on the specific carrier and other components used. For example, preventive and / or therapeutic compounds or compositions can be administered intramuscularly, subcutaneously, intradermally, or intravenously. They can also be administered via mucous membranes such as intranasally or orally. Compounds or compositions can also be administered through the skin via transdermal patches.

[0087] In some embodiments, the compound or composition may be provided in a unit dosage form in a suitable container. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human or animal use. Each unit dosage form may contain a predetermined amount of the compound (and / or other active agent) of the invention calculated to produce the desired effect in a carrier. In other embodiments, the compound may be provided separately from the carrier (e.g., in its own vial, ampoule, sachet, or other suitable container) for on-site mixing prior to administration to a subject. Kits comprising one or more antiviral compounds are also disclosed herein. The kit also includes instructions for administering the compound to a subject. One or more antiviral compounds may be provided as part of a dosage unit already dispersed in a pharmaceutically acceptable carrier, or they may be provided separately from the carrier. The kit may further include instructions for preparing the antiviral compound for administration to a subject, including, for example, instructions for dispersing the compound in a suitable carrier.

[0088] It should be understood that the treatment and prevention methods described herein are applicable to humans and any suitable animals, including but not limited to dogs, cats and other pets, as well as rodents, primates, horses, cattle, pigs, etc. The methods can also be applied to clinical investigations and / or research. Further advantages of the various embodiments of this disclosure will be apparent to those skilled in the art after reading the disclosure herein and the working examples below. It should be understood that, unless otherwise stated herein, the various embodiments described herein are not necessarily mutually exclusive. For example, a feature described or depicted in one embodiment may be included in, but not necessarily in, other embodiments. Therefore, this invention covers various combinations and / or integrations of the specific embodiments described and claimed herein.

[0089] As used herein, when the phrase “and / or” is used in a list of two or more items, it means that any one of the listed items may be used alone, or any combination of two or more listed items may be used. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition may contain or exclude A alone; B alone; C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0090] This specification also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, these ranges are to be interpreted as providing textual support for claims that only state the lower limit of the range and claims that only state the upper limit of the range. For example, the disclosed numerical range of about 10 to about 100 provides textual support for claims stating "greater than about 10" (no upper limit) and claims stating "less than about 100" (no lower limit).

[0091] Abbreviations: ORF, Open Reading Frame; EDCI, 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide; HOBt, N-hydroxybenzotriazole; DIEA, Diisopropylethylamine; DTT, Dithiothreitol; DMSO, Dimethyl sulfoxide; DMF, N,N-Dimethylformamide; DMP, Dysmartin periodoylene; DSC, N,N'-Disuccinimidyl carbonate; TEA, Triethylamine; CDI, Carbonyldiimidazole; MNV, Norovirus; MOI, Multiple of Infection; CPE, Cytopathic Effect; TCID 50 50% tissue culture infection dose; IC 50 50% inhibition concentration in enzyme assay; EC 50 50% effective concentration in cell culture; CC 50 GESAMT, a universal and highly efficient assay for macromolecular targets, is based on 50% cytotoxicity concentrations in cell-based assays. structure Comparison; RMSD, root mean square deviation; XDS, X-ray detector software; MME, monomethyl ether; PK, pharmacokinetics.

[0092] Example

[0093] The following examples illustrate the method according to the present invention. However, it should be understood that these examples are provided for illustrative purposes only, and nothing contained herein should be considered as a limitation on the overall scope of the invention. Unless otherwise stated, the precursors, intermediates, and final compounds described in the following synthetic reactions are independently numbered in each example. For the avoidance of doubt, the structures are listed in the following table.

[0094] Example 1

[0095] SARS-CoV-2 3CLpro is a catalytically active Cys-His dual (Cys 145 -His 41 The protease exhibits a strong preference for the -haa-N-Leu-Gln-aa sequence, where aa is a small amino acid, haa is a hydrophobic amino acid, and N is solvent-exposed and quite diverse (V / T / K), corresponding to the subsite -S4-S3-S2-S1-S1'-. Cleavage occurs at P1-P... 1’ On easily broken bonds. The 3D structure of SARS-CoV-2 3CLpro is similar to that of SARS-CoV 3CLpro; however, the S2 subsite of SARS-CoV-2 3CLpro exhibits considerable plasticity and can accommodate native and non-native amino acids with smaller side chains. High-resolution crystal structures of the binding inhibitors have been determined, enabling the use of structure-directed approaches in inhibitor design. Continuing our research in this area, we report here preliminary results related to a series of inhibitors (I) that inhibit the SARS-CoV-2 protease, which introduce conformationally restricted cyclohexane moieties into their structures, aiming to develop novel chemical spaces and optimally engage in favorable binding interactions with the protease active site.

[0096] Materials and methods.

[0097] Synthesis of 3CLpro compound. Chemical compound. 6a-k and 7a-k As in Option 1 ( Figure 1 The syntheses shown are listed in Tables 1 and 2. In short, the alcohol input reacts with (L) leucine isocyanate methyl ester or (L) cyclohexylalanine isocyanate methyl ester to produce a dipeptide. 2 It is then hydrolyzed with lithium hydroxide in an aqueous solution of tetrahydrofuran to the corresponding acid. Subsequently, the acid is replaced with glutamine to replace the methyl ester. 8 Coupling produces compounds 4 Lithium borohydride reduction produces alcohols. 5 It is then oxidized to the corresponding aldehyde using the Des Martin periodoylane reagent. 6 Bisulfite adducts are generated by treatment with sodium bisulfite and ethyl acetate in an aqueous ethanol solution. 7 .

[0098] Synthesis of 3CLpro inhibitors

[0099] Overview. Reagents and drying solvents were purchased from various chemical suppliers (Sigma-Aldrich, Acros Organics, Chem-Impex, TCI America, Oakwood Chemical, APExBIO, SynQuest, Fisher, and Bachem) and used as is. Silica gel (230-450 mesh) for rapid chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin-layer chromatography was performed using Analtech silica gel plates. Visualization was performed using UV light and / or iodine. NMR spectra were recorded in CDCl3 or dimethyl sulfoxide (DMSO)-d6 using a Varian XL-400 spectrometer. Melting points were recorded on a Mel-Temp instrument and were uncalibrated. High-resolution mass spectrometry (HRMS) was performed at the University of Kansas Mass Spectrometry Laboratory using an LCT Premier mass spectrometer (Waters, Milford, MA) equipped with a time-of-flight mass analyzer and an electrospray ionization source. Compound purity was determined by high-performance liquid chromatography (HPLC) using a reversed-phase column (symmetry). ® The watersAlliance HPLC system (C 18 3.5 µm, 4.6 X 75 mm) was used for determination at 254 nm and >95%. The gradient started from 60% methanol: 40% water over a period of 15 minutes to 99% methanol: 1% water, with a mobile phase flow rate of 1.0 mL / min.

[0100] Synthesis of amino acid methyl ester isocyanate. General procedure. 100 mmol of amino acid methyl ester hydrochloride was placed in an oven-dried RB flask (500 mL) and dried overnight on a vacuum pump. The flask was purged with nitrogen, and 200 mL of dried dioxane was added, followed by 150 mmol of trichloromethyl chloroformate. The stirred reaction mixture was then refluxed for 10 h. The solvent was removed by rotary evaporation, and the residue was vacuum distilled to give pure isocyanate as a colorless oil.

[0101] (S)-2-isocyano-4-methylpentanoate methyl ester, (yield 66%) 1 H NMR (400 MHz, cdcl3) δ4.08 – 4.00 (m, 1H), 3.81 (s, 3H), 1.91 – 1.76 (m, 1H), 1.73 – 1.56 (m, 2H), 1.04 – 0.89 (m, 6H).

[0102] ( Smethyl 3-cyclohexyl-2-isocyanopropionate, (yield 68%) 1 H NMR (400 MHz, DMSO- d 6) δ4.38 (ddd, J = 9.5, 4.6, 1.2 Hz, 1H), 3.74 (s, 3H), 1.80 – 1.50 (m, 8H), 1.50 – 1.35 (m, 1H), 1.30 – 1.06 (m, 3H), 1.01 – 0.77 (m, 3H).

[0103] Synthesis of amino acid carbamate 2. General procedure. A solution of alcohol (20 mmol) in anhydrous acetonitrile (15 mL) was treated with triethylamine (40 mmol) and then with amino acid methyl ester isocyanate (20 mmol). The resulting reaction mixture was refluxed with stirring for 2 h and then cooled to room temperature. The solution was concentrated, and the residue was absorbed in ethyl acetate (100 mL). The organic layer was washed with 5% HCl (2 × 25 mL) and brine (25 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product as a yellow oil. The crude oil was purified by rapid chromatography to give the ester. 2 It is a colorless oily substance.

[0104] ((cyclohexyloxy)carbonyl)- L -Leucine methyl ester (2a) Yield (62.5%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.46 (d, J = 7.9 Hz, 1H), 4.46 (dd, J = 8.9, 4.9 Hz, 1H), 4.02 (ddd, J = 10.2, 7.9, 4.9 Hz, 1H), 3.61 (s, 3H), 1.84 – 1.75 (m, 2H), 1.68 (s, 2H), 1.62 – 1.48 (m, 1H), 1.42 (ddd, J = 13.6, 8.9, 4.8 Hz, 1H), 1.38 – 1.25 (m,4H), 1.29 (s, 1H), 1.19 (d, J = 10.4 Hz, 1H), 0.86 (dd, J = 12.5, 6.5 Hz, 7H).

[0105] (((4-ethylcyclohexyl)oxy)carbonyl)- L -Leucine methyl ester (2b) Yield (64.0%). 1 H NMR (400MHz, DMSO- d 6) δ 7.45 (dd, J = 16.7, 8.0 Hz, 1H), 4.39 (tt, J = 11.0, 4.3 Hz,1H), 4.02 (ddt, J = 10.2, 7.9, 5.3 Hz, 1H), 3.61 (s, 3H), 1.94 – 1.86 (m, 2H), 1.79 – 1.36 (m, 6H), 1.34 – 1.04 (m, 5H), 0.97 (ddd, J = 13.2, 3.5, 1.6 Hz,1H), 0.95 – 0.75 (m, 9H).

[0106] (((4-propylcyclohexyl)oxy)carbonyl)- L -Leucine methyl ester (2c) Yield (67.0%). 1 H NMR (400MHz, DMSO- d 6) δ 7.44 (dd, J = 18.5, 8.0 Hz, 1H), 4.71 (d, J = 14.5 Hz, 1H), 4.38 (tt, J = 11.0, 4.2 Hz, 1H), 4.02 (ddd, J = 10.2, 7.9, 5.0 Hz, 1H), 3.61(s, 3H), 3.31 (d, J = 0.5 Hz, 1H), 1.93 – 1.85 (m, 1H), 1.77 – 1.68 (m, 2H), 1.67 – 1.50 (m, 1H), 1.53 – 1.36 (m, 2H), 1.28 (ddt, J = 15.4, 11.5, 4.3 Hz,4H), 1.25 – 1.08 (m, 3H), 1.01 – 0.94 (m, 1H), 0.98 – 0.81 (m, 9H).

[0107] (((4-Isopropylcyclohexyl)oxy)carbonyl)- L -Leucine methyl ester (2d) Yield (62.0%). 1 H NMR (400MHz, DMSO- d 6) δ 7.47 (d, J = 7.9 Hz, 1H), 4.37 (tt, J = 11.1, 4.3 Hz, 1H), 4.02(ddd, J = 10.2, 7.9, 4.9 Hz, 1H), 3.61 (s, 3H), 1.93 (d, J = 11.7 Hz, 2H), 1.83– 1.66 (m, 2H), 1.65 – 1.49 (m, 1H), 1.48 – 1.34 (m, 2H), 1.26 (dq, J = 15.7,8.5, 6.2 Hz, 2H), 1.13 – 0.95 (m, 3H), 0.95 – 0.76 (m, 13H).

[0108] (((4-Butylcyclohexyl)oxy)carbonyl)- L -Leucine methyl ester (2e) Yield (69.0%). 1 H NMR (400MHz, DMSO- d 6) δ 7.44 (dd, J = 18.7, 8.0 Hz, 1H), 4.70 (q, J = 6.9, 3.8 Hz, 1H), 4.38 (tt, J = 11.0, 4.2 Hz, 1H), 4.10 – 3.91 (m, 1H), 3.61 (d, J = 1.6 Hz, 3H),1.97 – 1.82 (m, 1H), 1.82 – 1.66 (m, 2H), 1.66 – 1.56 (m, 1H), 1.56 – 1.36(m, 3H), 1.36 – 1.08 (m, 9H), 0.95 (td, J = 12.8, 11.8, 3.3 Hz, 1H), 0.90 –0.75 (m, 9H).

[0109] ((3-Cyclohexylpropoxy)carbonyl)- L -Leucine methyl ester(2f) Yield (63.5%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.51 (d, J = 8.0 Hz, 1H), 4.03 (ddd, J = 10.3, 8.0, 4.8 Hz, 1H), 3.62 (s, 3H), 1.76 – 1.57 (m, 6H), 1.53 (ddt, J = 8.6, 6.6, 3.2 Hz, 2H), 1.48– 1.35 (m, 1H), 1.28 – 1.04 (m, 9H), 0.86 (dd, J = 13.6, 6.5 Hz, 8H).

[0110] (((5-ethyl-1,3-dioxane-5-yl)oxy)carbonyl)- L -Leucine methyl ester (2g)。 Yield (51.0%). 1 HNMR (400 MHz, DMSO- d 6) δ 7.50 (d, J = 8.1 Hz, 1H), 4.77 (d, J = 5.9 Hz, 1H), 4.61 (d, J = 5.9 Hz, 1H), 4.13 – 4.05 (m, 1H), 4.06 – 3.98 (m, 1H), 3.69 (dd, J = 11.4, 0.8 Hz, 2H), 3.63 (s, 3H), 3.43 (d, J = 5.0 Hz, 2H), 1.74 – 1.63 (m,1H), 1.63 – 1.34 (m, 1H), 1.26 (q, J = 7.6 Hz, 2H), 0.86 (dd, J = 14.0, 6.4 Hz, 6H), 0.76 (t, J = 7.6 Hz, 3H).

[0111] ((4,4-difluorocyclohexyl)oxy)carbonyl)- L -Leucine methyl ester (2h) Yield (62.0%). 1 H NMR (400MHz, DMSO- d6) δ 7.59 (d, J = 8.0 Hz, 1H), 4.89 – 4.57 (m, 1H), 4.09 – 3.92 (m,1H), 3.62 (s, 3H), 2.14 – 1.87 (m, 4H), 1.82 (tq, J = 8.0, 4.2 Hz, 2H), 1.77 –1.59 (m, 3H), 1.59 – 1.49 (m, 1H), 1.48 – 1.34 (m, 1H), 0.86 (dd, J = 12.8, 6.5 Hz, 6H).

[0112] ( S methyl 3-cyclohexyl-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)propionate (2i) Yield (59.0%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.58 (d, J = 8.0 Hz, 1H), 4.81 – 4.64 (m,1H), 4.06 (td, J = 8.9, 5.4 Hz, 1H), 3.62 (s, 3H), 2.11 – 1.88 (m, 4H), 1.88 –1.78 (m, 1H), 1.78 – 1.55 (m, 7H), 1.49 (tq, J = 13.7, 6.9, 5.3 Hz, 2H), 1.39– 1.26 (m, 1H), 1.26 – 1.04 (m, 4H), 0.88 (dt, J = 33.1, 11.1 Hz, 2H).

[0113] ((4,4-difluorocyclohexyl)methoxy)carbonyl)- L -Leucine methyl ester (2j) Yield (61.0%). 1 H NMR (400 MHz, DMSO-) d 6) δ 7.59 (d, J = 8.0 Hz, 1H), 4.83 – 4.62 (m, 1H), 3.62 (s,3H), 3.26 (dd, J= 6.5, 2.7 Hz, 2H), 2.04 – 1.90 (m, 5H), 1.90 – 1.63 (m, 4H), 1.54 – 1.40 (m, 2H), 1.26 – 1.07 (m, 1H), 0.87 (dd, J = 13.0, 7.0 Hz, 6H).

[0114] ((3,3-difluorocyclobutyl)methoxy)carbonyl)- L -Leucine methyl ester (2k) Yield (63.0%). 1 H NMR (400 MHz, DMSO-) d 6) δ 7.56 (d, J = 8.2 Hz, 1H), 4.77 – 4.60 (m, 1H), 3.62 (s,3H), 3.48 – 3.40 (m, 2H), 2.60 – 2.45 (m, 4H), 2.45 – 2.28 (m, 3H), 2.28 –2.14 (m, 1H), 0.87 (dd, J = 13.0, 7.0 Hz, 6H).

[0115] acid 3 The synthesis of. General procedure. ester 2 A solution of 20 mmol in THF (30 mL) was treated with 1 M LiOH (80 mmol). The reaction mixture was stirred at room temperature for 3 h, and the disappearance of the ester was monitored by TLC. Most of the solvent was removed under vacuum, and the solution was acidified to pH 5% with 5% hydrochloric acid. 2. The aqueous layer was extracted with ethyl acetate (3 x 100 mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the compound. 3 .

[0116] ((cyclohexyloxy)carbonyl)- L -Leucine (3a) Yield (90.5%). 1 H NMR (400 MHz, DMSO- d 6)δ 12.44 (br s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 4.55 – 4.37 (m, 1H), 3.93 (ddd, J = 10.3, 8.2, 4.7 Hz, 1H), 1.80 (dd,J = 9.5, 4.9 Hz, 2H), 1.74 – 1.57 (m, 4H), 1.57 – 1.38 (m, 2H), 1.36 – 1.22 (m, 5H), 0.86 (dd, J = 12.6, 6.5 Hz, 6H).

[0117] (((4-ethylcyclohexyl)oxy)carbonyl)- L -Leucine (3b) Yield (87.0%). 1 H NMR (400 MHz, DMSO- d 6) δ 12.44 (br s, 1H), 7.47 (dd, J = 16.7, 8.0 Hz, 1H), 4.66 – 4.52 (m,1H), 4.08 (ddt, J = 10.2, 7.9, 5.3 Hz, 1H), 1.93 – 1.82 (m, 2H), 1. 82 – 1.30 (m, 6H), 1.30 – 1.01 (m, 5H), 0.92 – 0.77 (m, 10H).

[0118] (((4-propylcyclohexyl)oxy)carbonyl)- L -Leucine (3c) Yield (92.0%). 1 H NMR (400 MHz, DMSO- d 6) δ 12.45 (br s, 1H), 7.26 (dd, J = 16.2, 8.2 Hz, 1H), 4.77 – 4.61 (m,1H), 4.39 (td, J = 11.1, 5.4 Hz, 1H), 3.93 (tt, J = 9.5, 5.4 Hz, 1H), 1.89 (d, J = 12.0 Hz, 1H), 1.81 – 1.57 (m, 3H), 1.57 – 1.37 (m, 4H), 1.37 – 1.06 (m, 7H), 1.06 – 0.68 (m, 9H).

[0119] (((4-Isopropylcyclohexyl)oxy)carbonyl)- L -Leucine (3d) Yield (89.5%), mp 83-850 C. 1 HNMR (400 MHz, DMSO- d 6) δ 12.44 (br s, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.36 (tt, J = 11.1, 4.2 Hz, 1H), 3.89 (td, J = 9.0, 5.2 Hz, 1H), 1.92 (d, J = 11.8 Hz,2H), 1.82 – 1.55 (m, 4H), 1.55 – 1.33 (m, 4H), 1.33 – 1.11 (m, 2H), 1.11 –0.93 (m, 4H), 0.93 – 0.76 (m, 9H).

[0120] (((4-Butylcyclohexyl)oxy)carbonyl)- L -Leucine (3e) Yield (87.0%). 1 H NMR (400 MHz, DMSO- d 6) δ 12.44 (br s, 1H), 7.26 (dd, J = 16.4, 8.2 Hz, 1H), 4.71 (d, J = 16.3Hz, 1H), 4.38 (tt, J = 11.0, 4.2 Hz, 1H), 3.99 – 3.84 (m, 1H), 1.89 (dd, J =12.5, 3.4 Hz, 1H), 1.77 – 1.59 (m, 3H), 1.59 – 1.36 (m, 4H), 1.36 – 1.21 (m,7H), 1.22 – 1.06 (m, 4H), 0.94 (dd, J = 23.6, 11.5 Hz, 1H), 1.06 – 0.73 (m,6H).

[0121] ((3-Cyclohexylpropoxy)carbonyl)- L -Leucine (3f) Yield (93.0%), mp 48-50 0 C. 1 H NMR (400 MHz, DMSO-) d6) δ 12.46 (br s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 4.02 – 3.83(m, 1H), 1.80 – 1.58 (m, 6H), 1.58 – 1.49 (m, 2H), 1.49 – 1.31 (m, 1H), 1.27– 1.02 (m, 8H), 0.86 (dd, J = 13.8, 6.6 Hz, 9H).

[0122] (((5-ethyl-1,3-dioxane-5-yl)oxy)carbonyl)- L -Leucine (3g)。 Yield (51.5%). 1 H NMR (400 MHz, DMSO-) d 6) δ 12.52 (br s, 1H), 7.40 (d, J = 8.3 Hz, 1H), 4.82 (t, J =5.4 Hz, 1H), 4.63 (d, J = 5.9 Hz, 1H), 4.15 – 4.01 (m, 1H), 4.01 – 3.92 (m,1H), 3.80 – 3.64 (m, 2H), 3.54 – 3.40 (m, 2H), 1.76 – 1.60 (m, 1H), 1.60 –1.35 (m, 1H), 1.27 (q, J = 7.9 Hz, 2H), 0.87 (dd, J = 14.1, 6.6 Hz, 6H), 0.77(t, J = 7.6 Hz, 3H).

[0123] (((4,4-difluorocyclohexyl)oxy)carbonyl)- L -Leucine (3h) Yield (85.0%). 1 H NMR (400MHz, DMSO- d 6) δ 12.50 (br s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 4.87 – 4.59 (m,1H), 3.95 (ddd, J= 10.2, 8.2, 4.8 Hz, 1H), 2.13 – 1.87 (m, 4H), 1.87 – 1.77(m, 2H), 1.77 – 1.57 (m, 3H), 1.57 – 1.36 (m, 2H), 0.87 (dd, J = 12.9, 6.5 Hz, 6H).

[0124] ( S )-3-cyclohexyl-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)propionic acid (3i) Yield (83.0%). 1 H NMR (400 MHz, DMSO- d 6) δ 12.44 (br s, 1H), 7.41 (d, J = 8.2 Hz, 1H),4.79 – 4.64 (m, 1H), 4.10 – 4.00 (m, 1H), 2.09 – 1.88 (m, 4H), 1.90 – 1.72(m, 2H), 1.72 – 1.54 (m, 6H), 1.54 – 1.30 (m, 3H), 1.30 – 1.00 (m, 4H), 1.00 – 0.66 (m, 2H).

[0125] (((4,4-Difluorocyclohexyl)methoxy)carbonyl)- L -Leucine (3j) Yield (86.5%). 1 H NMR (400MHz, DMSO- d 6) δ 12.42 (br s, 1H), 7.41 (d, J = 8.2 Hz, 1H), 4.80 – 4.65 (m,1H), 3.87 – 3.67(m, 2H), 2.01 – 1.88 (m, 5H), 1.86 – 1.74 (m, 2H), 1.74 –1.56 (m, 3H), 1.56 – 1.29 (m, 2H), 0.86 (dd, J = 11.0, 7.0 Hz, 6H).

[0126] (((3,3-Difluorocyclobutyl)methoxy)carbonyl)- L -Leucine (3k) Yield (84.5%). 1 H NMR (400MHz, DMSO-d 6) δ 12.48 (br s, 1H), 7.39 (d, J 0.87 (dd, J = 12.0, 7.2 Hz, 6H).

[0127] Synthesis of dipeptide ester 4. General procedure. Add EDCI (12.5 mmol, 1.25 equivalence) and HOBt (12.5 mmol, 1.25 equivalence) to a solution of compound 3 (10 mmol) in anhydrous DMF (20 mL) in an oven-dried RB flask (250 mL), and stir the mixture at room temperature for 30 min. In a separate RB flask, treat a solution of deprotected glutamine substitute (10 mmol) in DMF (15 mL) cooled to 0–5 °C with diisopropylethylamine (DIEA) (40 mmol, 4 equivalence), stir for 30 min, and then add acid-containing... 3 The reaction mixture was stirred for 24 h while the reaction was monitored by TLC. The solvent was removed, and the residue was dissolved in ethyl acetate (200 mL). The organic layer was washed successively with 10% citric acid (2 × 40 mL), saturated NaHCO3 aqueous solution (40 mL), and then brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid product. The ester was purified by rapid chromatography. 4 It is a white solid.

[0128] ( S )-2-(( S )-2-(((cyclohexyloxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4a) Yield (50.5%), mp 92-94 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ8.37 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 6.6 Hz, 1H), 7.10 (d, J= 8.2 Hz, 1H),4.55 – 4.39 (m, 1H), 4.41 – 4.25 (m, 1H), 4.12 – 3.94 (m, 1H), 3.62 (s, 3H),3.23 – 3.02 (m, 2H), 2.32 (ddq, J = 13.6, 11.3, 3.4 Hz, 1H), 2.26 – 2.01 (m,2H), 1.78 (t, J = 4.7 Hz, 2H), 1.73 – 1.54 (m, 6H), 1.54 – 1.45 (m, 1H), 1.45– 1.37 (m, 1H), 1.37 – 1.25 (m, 4H), 1.23-1.11 (m, 1H), 0.87 (dd, J = 10.4, 6.6 Hz, 6H).

[0129] ( S )-2-(( S )-2-((((4-ethylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4b) Yield (58.0%), mp 62-64 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.36 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 4.53 –4.23 (m, 2H), 3.99 (q, J = 7.6 Hz, 1H), 3.62 (s, 3H), 3.23 – 3.01 (m, 2H), 2.37 – 2.24 (m, 1H), 2.24 – 1.96 (m, 2H), 1.96 – 1.78 (m, 2H), 1.78 – 1.66(m, 2H), 1.59 (q, J = 13.3, 12.0 Hz, 4H), 1.43 (ddd, J= 17.6, 10.3, 5.8 Hz,4H), 1.32 – 1.12 (m, 5H), 1.12 – 1.00 (m, 1H), 1.00 – 0.88 (m, 1H), 0.88 –0.78 (m, 6H).

[0130] ( S )-2-(( S )-4-methyl-2-(((((4-propylcyclohexyl)oxy)carbonyl)amino)pentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4c) Yield (59.0%), mp 58-60 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.37 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 18.6 Hz, 1H), 7.08 (d, J = 8.0Hz, 1H), 4.48 – 4.23 (m, 1H), 4.11 – 3.91 (m, 1H), 3.62 (s, 3H), 3.24 – 3.02(m, 2H), 2.40 – 2.23 (m, 1H), 2.23 – 2.00 (m, 2H), 1.88 (d, J = 11.3 Hz, 1H),1.78 – 1.66 (m, 3H), 1.65 – 1.51 (m, 3H), 1.51 – 1.33 (m, 4H), 1.28 (h, J =7.3 Hz, 4H), 1.21 – 1.08 (m, 4H), 1.03 – 0.88 (m, 1H), 0.90 – 0.78 (m, 8H).

[0131] ( S )-2-(( S )-2-((((4-isopropylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4d) Yield (56.0%), mp 70-72 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.37 (d,J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 8.1Hz, 1H), 4.35 (tq, J = 11.3, 4.2 Hz, 2H), 4.08 – 3.92 (m, 1H), 3.62 (s, 3H), 3.25 – 3.02 (m, 2H), 2.33 (qd, J = 10.5, 3.8 Hz, 1H), 2.25 – 2.02 (m, 2H), 1.96 – 1.85 (m, 2H), 1.82 – 1.64 (m, 3H), 1.64 – 1.51 (m, 3H), 1.41 (ttd, J =10.9, 8.2, 7.7, 3.4 Hz, 4H), 1.19 (q, J = 8.0, 7.1 Hz, 2H), 1.02 (dq, J = 9.0,5.7 Hz, 4H), 0.94 – 0.75 (m, 9H).

[0132] ( S )-2-(( S )-2-((((4-Butylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4e) Yield (55.5%), mp 63-65 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.36 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 4.35(dh, J = 11.8, 3.9 Hz, 1H), 4.09 – 3.90 (m, 1H), 3.61 (s, 3H), 3.24 – 2.99 (m,2H), 2.41 – 2.23 (m, 1H), 2.19 – 1.99 (m, 2H), 1.87 (d, J = 11.6 Hz, 2H), 1.72(d, J= 12.8 Hz, 2H), 1.66 – 1.52 (m, 3H), 1.52 – 1.31 (m, 3H), 1.31 – 1.21(m, 6H), 1.21 – 1.06 (m, 4H), 1.06 – 0.90 (m, 1H), 0.87 (dt, J = 10.4, 5.4 Hz, 9H).

[0133] ( S )-2-(( S )-2-((((3-cyclohexylpropoxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4f) Yield (59.0%), mp 59-61 0 C. 1 H NMR (400 MHz, DMSO- d 6)δ 8.38 (d, J = 7.9 Hz, 1H), 7.64 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H), 4.33 (ddd, J = 11.8, 7.8, 4.3 Hz, 1H), 4.11 – 3.95 (m, 1H), 3.90 (t, J = 6.6 Hz, 2H), 3.61(s, 3H), 3.12 (dtd, J = 18.5, 9.4, 7.2 Hz, 2H), 2.31 (ddd, J = 12.8, 9.1, 3.8Hz, 1H), 2.08 (tdd, J = 15.3, 10.1, 3.0 Hz, 2H), 1.75 – 1.57 (m, 8H), 1.57 –1.46 (m, 2H), 1.46 – 1.34 (m, 2H), 1.28 – 1.05 (m, 6H), 0.87 (dd, J = 10.7, 6.6 Hz, 8H).

[0134] ( S )-2-(( S )-2-((((5-ethyl-1,3-dioxane-5-yl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( Smethyl 2-oxopyrrolidone-3-yl)propionate (4g) Yield (30.5%), mp 60-62 0 C. 1 H NMR (400MHz, DMSO- d 6) δ 8.45 (d, J = 8.1 Hz, 1H), 7.65 (s, 1H), 7.26 (d, J = 8.2 Hz, 1H), 4.82 (d, J = 6.0 Hz, 1H), 4.62 (d, J = 6.0 Hz, 1H), 4.33 (ddd, J = 11.8,7.8, 4.3 Hz, 1H), 4.10 – 3.94 (m, 2H), 3.79 – 3.66 (m, 2H), 3.62 (s, 3H),3.53 – 3.40 (m, 1H), 3.13 (dtd, J = 18.4, 9.3, 7.3 Hz, 2H), 2.32 (ddd, J =13.8, 11.5, 7.2 Hz, 1H), 2.20 – 1.98 (m, 2H), 1.75 – 1.52 (m, 3H), 1.52 –1.31 (m, 2H), 1.26 (q, J = 7.6 Hz, 2H), 0.88 (dd, J = 11.5, 6.5 Hz, 6H), 0.75(t, J = 7.6 Hz, 3H).

[0135] ( S )-2-(( S )-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4h) Yield (57.0%), mp 77-79 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.42 (d, J = 7.9 Hz, 1H), 7.64 (s, 1H), 7.26 (d, J= 8.1 Hz, 1H),4.76 – 4.63 (m, 1H), 4.39 – 4.21 (m, 1H), 4.10 – 4.95 (m, 2H), 3.62 (s, 3H),3.23 – 3.02 (m, 2H), 2.39 – 2.24 (m, 2H), 2.17 – 1.85 (m, 5H), 1.86 – 1.73 (m, 2H), 1.73 – 1.54 (m, 4H), 1.53 – 1.32 (m, 2H), 0.98 – 0.79 (m, 6H).

[0136] ( S )-2-(( S )-3-cyclohexyl-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)propionamidyl)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate (4i) Yield (49.0%), mp 80-82 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.40 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 6.8 Hz, 1H), 7.24 (d, J = 8.1Hz, 1H), 4.75 – 4.64 (m, 1H), 4.34 (ddd, J = 11.8, 7.8, 4.2 Hz, 1H), 4.12 –3.96 (m, 2H), 3.62 (s, 3H), 3.21 – 3.01 (m, 2H), 2.37 – 2.25 (m, 1H), 2.25 –1.85 (m, 6H), 1.85 – 1.75 (m, 2H), 1.74 – 1.51 (m, 9H), 1.51 – 1.37 (m, 2H), 1.37 – 1.25 (m, 1H), 1.23 – 1.04 (m, 2H), 0.98 – 0.77 (m, 2H).

[0137] ( S )-2-(( S )-2-(((((4,4-difluorocyclohexyl)methoxy)carbonyl)amino)-4-methylpentanoylamino)-3-(( S methyl 2-oxopyrrolidone-3-yl)propionate(4j) Yield (52.5%), mp 59-61 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.41 (d, J = 8.9 Hz, 1H), 7.64 (s, 1H), 7.23 (d, J = 7.4 Hz, 1H), 4.33 (ddd, J = 11.7, 7.7, 4.3 Hz, 1H), 4.08 – 3.88 (m, 1H), 3.88 – 3.69 (m,2H), 3.61 (s, 3H), 3.23 – 3.04 (m, 2H), 2.36 – 2.25 (m, 1H), 2.25 – 1.92 (m,5H), 1.92 – 1.68 (m, 4H), 1.68 – 1.51 (m, 2H), 1.54 – 1.32 (m, 2H), 1.32 –1.10 (m, 3H), 0.87 (dd, J = 11.4, 6.5 Hz, 6H).

[0138] (2 S )-2-(( S methyl 2-((((3,3-difluorocyclobutyl)methoxy)carbonyl)amino)-4-methylpentanoylamino)-3-(2-oxopyrrolidone-3-yl)propionate (4k) Yield (53.0%), mp 55-57 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.42 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 4.33(ddd, J = 11.7, 7.8, 4.3 Hz, 1H), 4.10 – 3.89 (m, 4H), 3.62 (s, 3H), 3.22 –3.04 (m, 2H), 2.70 – 2.54 (m, 2H), 2.46 – 2.25 (m, 3H), 2.16 – 1.99 (m, 2H),1.61 (ddt, J= 15.0, 10.4, 7.0 Hz, 3H), 1.52 – 1.31 (m, 2H), 0.87 (dd, J =10.9, 6.6 Hz, 6H).

[0139] Synthesis of dipeptide alcohol 5. General procedure. To ester 4 Lithium borohydride (Aldrich) (2 M; 15 mmol in THF) was added dropwise to a solution of 5 mmol in anhydrous THF (30 mL), followed by the addition of anhydrous methanol (90 mL), and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then acidified by adding 5% HCl and the pH was adjusted to ~2. The solvent was removed, leaving a residue, which was absorbed in ethyl acetate (100 mL). The organic layer was washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude grayish-white solid, which was purified by rapid chromatography to give dipeptide alcohol. 5 It is a white solid.

[0140] (( S )-1-((( S )-1-hydroxy-3-(( S 2-Oxypyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate cyclohexyl (5a)。 Yield (91.0%), mp 132-134 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 7.55 (dd, J = 17.6, 9.7 Hz, 1H), 7.12 – 6.96 (m, 1H), 6.52 (S, 1H), 4.67 (q, J = 6.0, 5.5 Hz, 1H), 4.59 – 4.30 (m, 1H), 3.94 (td, J = 8.8, 5.6 Hz,1H), 3.85 – 3.70 (m, 1H), 3.42 – 3.28 (m, 1H), 3.24 (tq, J = 10.4, 5.8 Hz,1H), 3.18 – 3.00 (m, 1H), 2.31 – 2.03 (m, 2H), 1.87 – 1.72 (m, 3H), 1.72 –1.62 (m, 3H), 1.56 (dt, J= 12.2, 8.4 Hz, 1H), 1.52 – 1.34 (m, 4H), 1.34 –1.12 (m, 5H), 0.85 (dd, J = 9.4, 6.6 Hz, 6H).

[0141] (( S )-1-((( S )-1-hydroxy-3-(( S 4-ethylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5b)。 Yield (91.0%), mp 100-102 0 C. 1 H NMR (400MHz, DMSO- d 6) δ 7.66 – 7.46 (m, 1H), 7.12 – 6.96 (m, 1H), 6.52 (s, 1H), 4.66(t, J = 5.4 Hz, 1H), 4.36 (td, J = 10.8, 5.4 Hz, 1H), 3.99 – 3.82 (m, 1H), 3.76 (s, 1H), 3.43 – 3.29 (m, 1H), 3.22 (dt, J = 10.6, 6.2 Hz, 1H), 3.14 (t, J = 8.8Hz, 1H), 3.05 (p, J = 8.9, 8.4 Hz, 1H), 2.30 – 2.05 (m, 2H), 1.93 – 1.83 (m,2H), 1.83 – 1.65 (m, 4H), 1.65 – 1.52 (m, 2H), 1.52 – 1.30 (m, 4H), 1.30 –1.13 (m, 5H), 1.13 – 1.03 (m, 1H), 0.95 (t, J = 12.9 Hz, 1H), 0.85 (ddd, J =7.5, 5.7, 3.0 Hz, 6H).

[0142] (( S )-1-((( S )-1-hydroxy-3-(( S4-propylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5c)。 Yield (92.0%), mp 10⁵-10⁷ 0 C. 1 H NMR (400MHz, DMSO- d 6) δ 7.66 – 7.44 (m, 1H), 7.02 (dt, J = 27.5, 8.8 Hz, 1H), 6.52 (s,1H), 4.77 – 4.54 (m, 1H), 4.36 (tt, J = 10.2, 3.8 Hz, 1H), 3.94 (qd, J = 8.4,5.6 Hz, 1H), 3.76 (s, 1H), 3.47 – 3.30 (m, 1H), 3.22 (dd, J = 10.5, 6.6 Hz,1H), 3.18 – 3.09 (m, 1H), 3.04 (td, J = 9.5, 7.2 Hz, 1H), 2.34 – 2.04 (m, 2H), 1.89 (d, J = 18.0 Hz, 1H), 1.84 – 1.65 (m, 3H), 1.65 – 1.51 (m, 2H), 1.41(dddd, J = 27.3, 10.6, 8.4, 4.3 Hz, 5H), 1.28 (dt, J = 14.8, 7.5 Hz, 5H), 1.17(q, J = 7.4 Hz, 4H), 1.07 – 0.90 (m, 1H), 0.86 (ddd, J = 9.0, 6.5, 5.2 Hz, 6H).

[0143] (( S )-1-((( S )-1-hydroxy-3-(( S 4-Isopropylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5d)。 Yield (87.0%), mp 77-79 0 C. 1 H NMR (400MHz, DMSO- d6) δ 7.65 – 7.43 (m, 1H), 7.11 – 6.95 (m, 1H), 6.59 (d, J = 56.3Hz, 1H), 4.69 (s, 1H), 4.35 (tt, J = 11.1, 4.2 Hz, 1H), 3.93 (td, J = 9.0, 5.8Hz, 1H), 3.76 (s, 1H), 3.33 (dd, J = 10.6, 5.0 Hz, 1H), 3.22 (dd, J = 10.5, 6.5Hz, 1H), 3.18 – 3.09 (m, 1H), 3.05 (td, J = 9.3, 7.0 Hz, 1H), 2.33 – 2.03 (m,2H), 1.97 – 1.84 (m, 1H), 1.78 (ddd, J = 14.1, 11.6, 3.5 Hz, 1H), 1.73 – 1.62(m, 2H), 1.62 – 1.49 (m, 2H), 1.49 – 1.28 (m, 6H), 1.28 – 1.11 (m, 2H), 1.12– 0.93 (m, 4H), 0.93 – 0.76 (m, 9H).

[0144] (( S )-1-((( S )-1-hydroxy-3-(( S 4-Butylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5e)。 Yield (86.5%), mp 57-59 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 7.65 – 7.44 (m, 2H), 7.02 (dt, J = 26.8, 8.9 Hz, 1H), 4.66 (d, J =4.7 Hz, 2H), 4.36 (tt, J= 11.0, 4.2 Hz, 1H), 4.03 – 3.83 (m, 1H), 3.83 – 3.67(m, 1H), 3.33 (s, 1H), 3.28 – 3.19 (m, 1H), 3.19 – 3.10 (m, 1H), 3.10 (s,1H), 2.29 – 2.05 (m, 2H), 1.94 – 1.83 (m, 1H), 1.83 – 1.66 (m, 2H), 1.57 (tt, J = 12.3, 6.2 Hz, 2H), 1.50 – 1.30 (m, 6H), 1.30 – 1.05 (m, 8H), 1.05 – 0.74 (m, 9H).

[0145] (( S )-1-((( S )-1-hydroxy-3-(( S 3-Cyclohexylpropyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5f)。 Yield (89.0%), mp 45-47 0 C. 1 H NMR (400 MHz, DMSO- d 6) δ 7.60 (d, J = 8.9 Hz, 1H), 7.54 (s, 1H), 7.18 – 7.04 (m, 1H), 4.74 – 4.59 (m, 1H), 4.00 – 3.85 (m, 4H), 3.83 – 3.69 (m, 1H), 3.27 – 3.18 (m, 1H),3.18 – 3.01 (m, 2H), 2.27 – 2.06 (m, 2H), 1.78 (ddd, J = 14.6, 11.5, 3.6 Hz,1H), 1.71 – 1.57 (m, 5H), 1.57 – 1.48 (m, 3H), 1.48 – 1.31 (m, 3H), 1.27 –1.03 (m, 6H), 0.85 (dd, J = 9.8, 6.6 Hz, 8H).

[0146] (( S )-1-((( S )-1-hydroxy-3-(( S5-ethyl-1,3-dioxane-5-ester of 2-oxopyrrolidine-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5g)。 Yield (71.0%), mp 58-60 0 C. 1 H NMR (400 MHz, DMSO-) d 6) δ 7.65 (d, J = 8.9 Hz, 1H), 7.53 (s, 1H), 7.21 (d, J = 8.3Hz, 1H), 4.81 (d, J = 6.0 Hz, 1H), 4.71 – 4.58 (m, 2H), 4.12 – 3.91 (m, 3H), 3.85 – 3.65 (m, 2H), 3.47 (dd, J = 11.4, 2.8 Hz, 2H), 3.22 (dt, J = 10.4, 6.1Hz, 1H), 3.18 – 2.99 (m, 2H), 2.32 – 2.06 (m, 2H), 1.78 (ddd, J = 14.5, 11.5,3.5 Hz, 1H), 1.72 – 1.49 (m, 2H), 1.49 – 1.31 (m, 2H), 1.31 – 1.19 (m, 2H), 0.86 (dd, J = 10.8, 6.6 Hz, 6H), 0.75 (t, J = 7.6 Hz, 3H).

[0147] (( S )-1-((( S )-1-hydroxy-3-(( S 4,4-Difluorocyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5h)。 Yield (90.0%), mp 126-128 0 C. 1 H NMR (400MHz, DMSO- d 6) δ 7.62 (d, J = 9.0 Hz, 1H), 7.54 (s, 1H), 7.23 (d, J= 8.3 Hz,1H), 4.77 – 4.58 (m, 2H), 3.95 (td, J = 8.9, 5.6 Hz, 1H), 3.85 – 3.69 (m, 1H), 3.23 (t, J = 8.4 Hz, 1H), 3.09 (dq, J = 35.5, 9.0 Hz, 2H), 2.30 – 2.08 (m, 2H), 2.08 – 1.85 (m, 4H), 1.85 – 1.73 (m, 2H), 1.69 (p, J = 6.0 Hz, 2H), 1.63 –1.49 (m, 2H), 1.49 – 1.30 (m, 4H), 0.86 (dd, J = 10.0, 6.6 Hz, 6H).

[0148] (( S )-3-cyclohexyl-1-((( S )-1-hydroxy-3-(( S 4,4-Difluorocyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-1-oxopyrrolidone-2-yl)carbamate (5i)。 Yield (93.0%), mp 77-79 0 C. 1 H NMR (400MHz, DMSO- d 6) δ 7.61 (d, J = 8.2 Hz, 1H), 7.60 (s, 1H), 7.21 (d, J = 8.3 Hz,1H), 4.67 – 4.43 (m, 2H), 3.97 (td, J = 8.9, 5.6 Hz, 1H), 3.75 – 3.67 (m, 1H), 3.28 (t, J = 8.4 Hz, 1H), 3.10 (dq, J= 35.5, 9.0 Hz, 2H), 2.37 – 2.30 (m, 1H), 2.26 – 1.83 (m, 6H), 1.83 – 1.77 (m, 2H), 1.77 – 1.50 (m, 9H), 1.50 – 1.36(m, 2H), 1.36 – 1.23 (m, 1H), 1.23 – 1.04 (m, 2H), 0.98 – 0.77 (m, 3H).

[0149] (( S )-1-((( S )-1-hydroxy-3-(( S 4,4-Difluorocyclohexyl)methyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (5j)。 Yield (90.0%), mp 62-64 0 C. 1 H NMR (400 MHz, DMSO-) d 6) δ 7.60 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.20 (d, J = 8.3Hz, 1H), 4.66 (s, 1H), 3.95 (td, J = 8.9, 5.4 Hz, 1H), 3.90 – 3.69 (m, 3H), 3.23 (d, J = 5.7 Hz, 1H), 3.18 – 3.00 (m, 2H), 2.28 – 2.07 (m, 2H), 2.06 –1.93 (m, 2H), 1.90 – 1.64 (m, 6H), 1.56 (dq, J = 11.9, 8.8 Hz, 2H), 1.51 –1.30 (m, 3H), 1.30 – 1.12 (m, 2H), 0.86 (dd, J = 10.5, 6.6 Hz, 6H).

[0150] ((2 S )-1-(((2 S 1-Hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (3,3-difluorocyclobutyl)methyl ester (5k)。 Yield (91.0%), mp 47-49 0C. 1 H NMR (400MHz, DMSO- d 6) δ 7.62 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 4.66 (t, J = 5.5 Hz, 1H), 4.10 – 3.85 (m, 3H), 3.76 (d, J = 10.8 Hz, 1H),3.40 – 3.29 (m, 1H), 3.23 (dt, J = 10.4, 6.0 Hz, 1H), 3.18 – 3.00 (m, 2H), 2.70 – 2.52 (m, 1H), 2.45 – 2.27 (m, 3H), 2.28 – 2.05 (m, 2H), 1.78 (ddd, J =14.6, 11.6, 3.5 Hz, 1H), 1.72 – 1.50 (m, 2H), 1.47 – 1.30 (m, 3H), 0.86 (dd, J = 9.9, 6.6 Hz, 6H).

[0151] Synthesis of dipeptide aldehyde 6. General procedure. Under a nitrogen atmosphere, the compound 5 (5 mmol) was dissolved in excess anhydrous dichloromethane (300 mL) in a 500 mL RB flask dried in an oven and cooled to 0 °C. Dys-Martin periodane reagent (15 mmol, 3 equivalents) was added in portions over 30 minutes with stirring under nitrogen. The ice bath was removed, and the reaction mixture was stirred at approximately 15 °C for 3 h under nitrogen (as indicated by TLC monitoring of complete disappearance of the starting material). The reaction mixture was transferred to a separatory funnel, and the organic layer was increased to ~300 mL by adding fresh dichloromethane. The organic layer was thoroughly washed with 10% sodium thiosulfate aqueous solution (2 x 100 mL), followed by saturated sodium bicarbonate aqueous solution (100 mL) and brine (100 mL) over a period of less than 30 minutes. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give a grayish-white crude solid, which was purified by rapid chromatography over one hour to give pure aldehyde. 6 It is a white solid.

[0152] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S2-Oxypyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)cyclohexyl carbamate (6a)。 Yield (57.5%), mp 68-70 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.43 (d, 7.3 Hz, 1H), 7.64 (d, J = 7.0 Hz, 1H), 7.18 (d, J = 9.1 Hz, 1H), 4.66 (t, J = 5.4 Hz, 1H), 4.56 – 4.38 (m, 2H), 4.19 (ddd, J =11.4, 7.5, 4.1 Hz, 1H), 4.01 – 3.86 (m, 1H), 3.86 – 3.70 (m, 1H), 3.30 – 2.99(m, 2H), 2.36 – 2.05 (m, 2H), 1.96 – 1.84 (m, 1H), 1.84 – 1.72 (m, 2H), 1.72– 1.53 (m, 3H), 1.46 (dddd, J = 18.5, 17.1, 9.3, 4.6 Hz, 2H), 1.24 (dt, J =53.6, 6.7 Hz, 5H), 0.93 – 0.78 (m, 6H). HRMS (TOF MS ES+) calculation of M+H = C 20 H 34 N3O5 = 396.2498, measured mass = 396.2511, 3.2 ppm. Calculated M + Na = C. 20 H 33 N3O5Na = 418.2318, measured mass = 418.2337, 4.6 ppm.

[0153] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4-Ethylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6b)。 Yield (60.0%), mp 55-57 0 C, 1H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H),7.25 – 7.15 (d, J = 7.3 Hz, 1H), 4.77 – 4.61 (m, 1H), 4.38 (tt, J = 11.0, 4.2Hz, 1H), 4.23 – 4.09 (m, 1H), 4.09 – 3.94 (m, 1H), 3.27 – 3.00 (m, 2H), 2.36 – 2.07 (m, 2H), 1.96 – 1.81 (m, 3H), 1.73 (d, J = 12.8 Hz, 2H), 1.63 (ddd, J =13.1, 7.4, 2.6 Hz, 2H), 1.46 (ttd, J = 13.6, 9.1, 8.5, 4.4 Hz, 3H), 1.31 –1.12 (m, 3H), 0.91 – 0.80 (m, 12H). HRMS (TOF MS ES+) calculation of M+H = C 22 H 38 N3O5 = 424.2811, measured mass = 424.2833, 2.2 mmu. Calculated M + Na = C 22 H 37 N3O5Na = 446.2631, measured mass = 446.2649, 4.1 ppm.

[0154] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4-propylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6c)。 Yield (53.0%), mp 50-52 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.40 (d, J = 7.7 Hz, 1H), 7.64 (d, J= 7.6 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 4.76 – 4.60 (m, 1H), 4.37 (td, J = 10.9, 5.3 Hz, 1H), 4.25 – 4.07 (m, 1H), 4.09 – 3.91 (m, 1H), 3.12 (ddt, J = 25.3, 17.0, 8.6 Hz,2H), 2.35 – 2.04 (m, 2H), 1.95 – 1.78 (m, 2H), 1.78 – 1.56 (m, 4H), 1.46(ddt, J = 15.2, 8.7, 4.8 Hz, 4H), 1.28 (h, J = 7.1, 6.6 Hz, 5H), 1.21 – 1.08(m, 4H), 0.87 (dtd, J = 10.3, 7.1, 4.8 Hz, 8H). HRMS (TOF MS ES+) calculation of M+H = C 23 H 40 N3O5 = 438.2968, measured mass = 438.2990, 2.2 mmu. Calculated M + Na = C 23 H 39 N3O5Na = 460.2787, measured mass = 460.2811, 2.4 mmu.

[0155] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4-Isopropylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6d)。 Yield (57.5%), mp 47-49 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.41 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 4.36 (tt, J= 11.0, 4.2 Hz, 1H), 4.24 – 4.09 (m, 1H), 4.09 – 3.88 (m, 1H), 3.28 – 3.00 (m, 2H), 2.39 – 2.08 (m, 2H), 2.08 – 1.85 (m, 3H), 1.81 – 1.54 (m, 4H), 1.54 – 1.35 (m, 4H), 1.35 – 1.11 (m, 2H), 1.02 (s, 3H), 0.94 – 0.77 (m, 12H). M+H = C calculated by HRMS (TOF MS ES+). 23 H 40 N3O5 = 438.2968, measured mass = 438.2997, 2.9 mmu.

[0156] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4-Butylcyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6e)。 Yield (53.0%), mp 45-47 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.40 (d, J = 7.8 Hz, 1H), 7.63 (s, 1H), 7.24 – 7.12(d, J = 7.2 Hz, 1H), 4.78 – 4.63 (m, 1H), 4.37 (td, J = 10.9, 5.2 Hz, 1H), 4.19(ddt, J = 11.2, 7.7, 3.7 Hz, 1H), 4.11 – 3.91 (m, 1H), 3.11 (ddt, J = 27.6,19.2, 9.7 Hz, 2H), 2.35 – 2.05 (m, 2H), 1.97 – 1.77 (m, 2H), 1.77 – 1.55 (m,4H), 1.46 (ttd, J= 13.7, 9.0, 8.6, 4.6 Hz, 5H), 1.32 – 1.07 (m, 8H), 1.07 –0.79 (m, 10H). M+H = C calculated by HRMS (TOF MS ES+). 24 H 42 N3O5 = 452.3124, measured mass = 452.3146, 4.8 ppm.

[0157] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 3-Cyclohexylpropyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6f)。 Yield (61.0%), mp 48-50 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J =8.0 Hz, 1H), 4.19 (ddd, J = 11.4, 7.5, 4.1 Hz, 1H), 4.03 (ddt, J = 10.0, 7.0, 3.3 Hz, 1H), 3.96 – 3.81 (m, 2H), 3.23 – 3.02 (m, 2H), 2.36 – 2.06 (m, 2H), 1.97 – 1.75 (m, 1H), 1.75 – 1.56 (m, 7H), 1.56 – 1.30 (m, 4H), 1.30 – 1.02 (m, 7H), 0.97 – 0.74 (m, 8H). HRMS (TOF MS ES+) calculated M+H = C 23 H 40 N3O5 = 438.2968, measured mass = 438.2983, 3.4 ppm. Calculated M + Na = C. 23 H 39 N3O5Na = 460.2787, measured mass = 460.2803, 3.4 ppm.

[0158] (( S )-4-methyl-1-oxo-1-(((S )-1-oxo-3-(( S 5-ethyl-1,3-dioxane-5-ester of 2-oxopyrrolidine-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6g)。 Yield (51.0%), mp 41-43 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 9.40 (s, 1H), 8.47 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 4.82 (d, J = 6.0 Hz, 1H), 4.62 (d, J = 6.0 Hz, 1H), 4.19 (ddd, J = 11.3, 7.3, 4.0 Hz, 1H), 4.11 – 3.93 (m, 2H), 3.74 (dd, J = 12.0, 4.0 Hz, 2H), 3.47 (dd, J = 11.4, 2.1 Hz, 2H), 3.12 (dt, J = 16.3, 10.4 Hz, 2H),2.37 – 2.08 (m, 2H), 1.97 – 1.79 (m, 1H), 1.79 – 1.57 (m, 2H), 1.57 – 1.32(m, 2H), 1.26 (q, J = 7.5 Hz, 2H), 0.87 (ddd, J = 10.3, 9.2, 6.6 Hz, 6H), 0.76(t, J = 7.6 Hz, 3H). HRMS (TOF MS ES+) calculation of M+Na = C 20 H 33 N3O7Na = 450.2216, measured mass = 450.2233, 3.7 ppm.

[0159] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4,4-Difluorocyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6h)。Yield (50.5%), mp 44-46 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 7.34 (d, J =7.4 Hz, 1H), 4.76 – 4.63 (m, 1H), 4.20 (ddd, J = 11.4, 7.6, 4.1 Hz, 1H), 4.09– 3.95 (m, 2H), 3.25 – 2.99 (m, 2H), 2.41 – 2.09 (m, 2H), 2.09 – 1.86 (m,4H), 1.86 – 1.74 (m, 2H), 1.75 – 1.56 ( m , 5H), 1.55 – 1.29 (m, 2H), 0.98 –0.79 (m, 6H). HRMS (TOF MS ES+) calculation of M+H = C 20 H 32 F2N3O5 = 432.2310, measured mass = 432.2298, 2.8 ppm.

[0160] (( S )-3-cyclohexyl-1-oxo-1-((( S )-1-oxo-3-(( S 4,4-Difluorocyclohexyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)propyl-2-yl)carbamate (6i)。 Yield (51.0%), mp 52-54 0 C, 1 H NMR (400MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.33 (d, J = 7.9 Hz, 1H), 4.82 – 4.56 (m, 1H), 4.18 (ddt, J = 11.3, 7.7, 3.8 Hz, 1H),4.05 (dq, J= 12.3, 7.7, 7.1 Hz, 2H), 3.22 – 3.00 (m, 2H), 2.35 – 2.09 (m,2H), 2.09 – 1.85 (m, 4H), 1.81 (dt, J = 8.8, 3.6 Hz, 2H), 1.74 – 1.53 (m, 8H), 1.53 – 1.38 (m, 2H), 1.32 (q, J = 8.9, 7.9 Hz, 1H), 1.24 – 1.04 (m, 4H), 0.88(t, J = 13.3 Hz, 2H). M+H = C calculated by HRMS (TOF MS ES+). 23 H 36 F₂N₃O₅ = 472.2623, measured mass = 472.2640, 3.6 ppm. Calculated M + Na = C. 23 H 35 F2N3O5Na = 494.2442, measured mass = 494.2470, 2.8 mmu.

[0161] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4,4-Difluorocyclohexyl)methyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (6j)。 Yield (50.5%), mp 47-49 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 9.40 (s, 1H), 8.44 (d, J = 7.5 Hz, 1H), 7.95 (s, 1H),7.36 – 7.28 (m, 1H), 4.19 (ddd, J = 11.4, 7.6, 4.2 Hz, 1H), 4.03 (td, J = 8.8, 6.2 Hz, 1H), 3.84 (d, J= 6.1 Hz, 2H), 3.24 – 3.02 (m, 2H), 2.37 – 2.08 (m, 2H), 2.08 – 1.94 (m, 2H), 1.94 – 1.80 (m, 1H), 1.80 – 1.55 (m, 7H), 1.55 – 1.33 (m, 2H), 1.33 – 1.11 (m, 3H), 0.97 – 0.79 (m, 6H). M+H = C calculated by HRMS (TOF MS ES+). 21 H 34 F₂N₃O₅ = 446.2467 Measured mass = 446.2452, 3.4 ppm Calculated M + Na = C 21 H 33 F₂N₃O₅Na = 468.2286, Measured mass = 468.2281, 1.1 ppm

[0162] ((2 S )-4-methyl-1-oxo-1-(((2) S 1-O-3-(2-O-pyrrolidone-3-yl)prop-2-yl)amino)pent-2-yl)carbamate (3,3-difluorocyclobutyl)methyl ester (6k)。 Yield (50.5%), mp 40-42 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 9.39 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.39 (t, J = 7.9 Hz, 1H), 4.31 (td, J = 6.7, 4.2 Hz, 1H), 4.19 (ddd, J = 11.4,7.7, 4.1 Hz, 1H), 4.03 (ddt, J = 9.8, 7.1, 3.7 Hz, 4H), 3.26 – 3.00 (m, 2H), 2.60 (tq, J= 11.2, 8.8, 5.1, 2.8 Hz, 2H), 2.47 – 2.24 (m, 3H), 2.24 – 2.06(m, 1H), 1.98 – 1.79 (m, 1H), 1.72 – 1.55 (m, 2H), 1.55 – 1.28 (m, 2H), 0.87(ddd, J = 11.1, 7.8, 6.6 Hz, 6H). M+H = C calculated by HRMS (TOF MS ES+). 19 H 30 F₂N₃O₅ = 418.2154, measured mass = 418.2161, 1.8 ppm. Calculated M + Na = C. 19 H 29 F2N3O5Na = 440.1973, measured mass = 440.1991, 4.1 ppm.

[0163] Synthesis of dipeptidyl bisulfite adduct 7. General procedure. aldehyde under stirring 6 Anhydrous ethanol (12 mL) was added to a solution of sodium bisulfite (5 mmol) in 20 mL of anhydrous ethyl acetate, followed by a solution of sodium bisulfite (540 mg; 5 mmol) in 5 mL of water. The reaction mixture was stirred at 50 °C for 3 hours. The reaction mixture was then cooled to room temperature and filtered through an anhydrous sodium sulfate stopper. The remaining solid in the filter was thoroughly washed with anhydrous ethanol, and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to give a grayish-white solid, which was washed with anhydrous diethyl ether (3 x 50 mL), filtered under vacuum, and dried under vacuum to give the compound. 7 It is a white powder.

[0164] (2 S )-2-(( S )-2-(((cyclohexyloxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7a) Yield (77.0%), mp 95-97 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 7.70 – 7.32 (m, 2H), 7.30 – 6.93 (m, 1H), 4.66 (t, J= 5.6 Hz, 1H), 4.56 – 4.30 (m, 1H), 4.07 – 3.89 (m, 1H), 3.89 – 3.65 (m, 1H), 3.08 (dt, J =33.4, 7.9 Hz, 2H), 2.40 – 2.05 (m, 2H), 1.95 (dt, J = 31.2, 13.5 Hz, 1H), 1.84– 1.71 (m, 3H), 1.71 – 1.60 (m, 3H), 1.60 – 1.52 (m, 1H), 1.52 – 1.32 (m,2H), 1.32 – 1.13 (m, 6H), 0.84 (dt, J = 9.7, 6.5 Hz, 6H).

[0165] (2 S )-2-(( S )-2-((((4-ethylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7b) Yield (81.0%), mp 115-117 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 7.69 – 7.34 (m, 2H), 7.27 – 6.99 (m, 1H), 4.79 – 4.58(m, 1H), 4.37 (tt, J = 11.0, 4.2 Hz, 1H), 4.23 (tq, J = 9.4, 2.7 Hz, 1H), 4.12– 3.70 (m, 2H), 3.20 – 2.97 (m, 2H), 2.38 – 2.06 (m, 2H), 2.06 – 1.82 (m,2H), 1.73 (d, J = 12.8 Hz, 2H), 1.65 – 1.50 (m, 2H), 1.50 – 1.31 (m, 4H), 1.31 – 1.12 (m, 6H), 1.12 – 1.02 (m, 2H), 1.02 – 0.90 (m, 1H), 0.85 (td, J = 6.9, 4.0 Hz, 6H).

[0166] (2 S )-1-hydroxy-2-(( S )-4-methyl-2-((((4-propylcyclohexyl)oxy)carbonyl)amino)pentamido)-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7c) Yield (77.5%), mp 124-126 0 C, 1 HNMR (400 MHz, DMSO- d 6) δ 7.62 – 7.35 (m, 2H), 7.26 – 7.00 (m, 1H), 4.80 –4.59 (m, 1H), 4.35 (dd, J = 9.2, 4.1 Hz, 1H), 4.29 – 4.12 (m, 1H), 4.12 – 3.77(m, 2H), 3.07 (dq, J = 33.0, 8.7, 8.3 Hz, 2H), 2.28 – 2.03 (m, 2H), 2.04 –1.80 (m, 2H), 1.71 (d, J = 13.1 Hz, 2H), 1.65 – 1.50 (m, 2H), 1.51 – 1.35 (m,4H), 1.28 (h, J = 8.2, 7.8 Hz, 6H), 1.23 – 1.03 (m, 5H), 1.03 – 0.76 (m, 6H).

[0167] (2 S )-1-hydroxy-2-(( S )-2-((((4-isopropylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7d) Yield (78.0%), mp 10¹-10³ 0 C, 1 HNMR (400 MHz, DMSO- d 6) δ 7.67 – 7.26 (m, 2H), 7.16 (dt, J = 18.3, 9.0 Hz, 1H), 4.83 – 4.62 (m, 1H), 4.36 (dq, J= 11.2, 6.4, 5.3 Hz, 1H), 4.23 (td, J = 9.2,4.7 Hz, 1H), 4.07 – 3.70 (m, 2H), 3.21 – 2.96 (m, 2H), 2.43 – 2.04 (m, 2H),2.04 – 1.84 (m, 2H), 1.84 – 1.63 (m, 2H), 1.63 – 1.47 (m, 2H), 1.47 – 1.34 (m, 4H), 1.34 – 1.14 (m, 2H), 1.14 – 0.92 (m, 3H), 0.85 (dd, J = 10.5, 6.7 Hz, 12H).

[0168] (2 S )-2-(( S )-2-((((4-Butylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7e) Yield (50.5%), mp 128-130 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 7.65 – 7.33 (m, 2H), 7.14 (td, J = 19.7, 17.5, 8.3 Hz,1H), 4.80 – 4.63 (m, 1H), 4.37 (tt, J = 8.6, 4.0 Hz, 1H), 4.28 – 4.16 (m, 1H), 4.11 – 3.78 (m, 1H), 3.09 (dt, J = 34.3, 8.9 Hz, 2H), 2.24 – 2.05 (m, 2H), 2.05 – 1.82 (m, 2H), 1.72 (d, J = 12.7 Hz, 3H), 1.66 – 1.51 (m, 3H), 1.51 –1.34 (m, 4H), 1.32 – 1.03 (m, 8H), 1.01 – 0.75 (m, 10H).

[0169] (2 S )-2-(( S)-2-((((3-cyclohexylpropoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7f) Yield (76.0%), mp 10⁴-10⁶ 0 C, 1 H NMR (400MHz, DMSO- d 6) δ 7.73 – 7.34 (m, 2H), 7.32 – 7.08 (m, 1H), 4.23 (t, J = 10.6Hz, 1H), 4.13 – 3.68 (m, 3H), 3.10 (dt, J = 28.3, 9.6 Hz, 2H), 2.27 – 2.05 (m,2H), 1.96 (dt, J = 26.0, 12.3 Hz, 1H), 1.83 – 1.49 (m, 10H)1.49 – 1.30 (m,2H), 1.30 – 1.02 (m, 7H), 0.97 – 0.75 (m, 8H).

[0170] (2 S )-2-(( S )-2-((((5-ethyl-1,3-dioxane-5-yl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7g) Yield (72.0%), mp 92-94 0 C, 1 H NMR (400 MHz, DMSO- d 6) δ 7.60 (dd, J = 34.3, 9.2 Hz, 1H), 7.45 (d, J =3.6 Hz, 1H), 7.28 (dd, J = 27.2, 8.4 Hz, 1H), 4.81 (d, J = 6.0 Hz, 1H), 4.63(d, J = 6.0 Hz, 1H), 4.22 (t, J = 10.5 Hz, 1H), 4.10 – 3.88 (m, 2H), 3.79 –3.64 (m, 2H), 3.47 (dd,J = 11.5, 5.1 Hz, 2H), 3.18 – 2.98 (m, 2H), 2.25 –2.04 (m, 2H), 1.95 (dt, J = 22.7, 11.9 Hz, 1H), 1.69 – 1.50 (m, 2H), 1.45 (p, J = 7.3, 6.2 Hz, 2H), 1.34 – 1.16 (m, 2H), 1.16 – 0.99 (m, 1H), 0.85 (ddd, J =10.9, 6.5, 2.9 Hz, 6H), 0.76 (t, J = 7.6 Hz, 3H).

[0171] (2 S )-2-(( S )-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7h) Yield (69.0%), mp 74-76 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 7.75 – 7.38 (m, 2H), 7.30 (dt, J = 35.6, 7.5 Hz, 1H),4.80 – 4.60 (m, 1H), 4.56 – 4.27 (m, 1H), 4.07 – 3.88 (m, 1H), 3.73 (q, J =7.1 Hz, 1H), 3.63 (dt, J = 9.6, 6.7 Hz, 1H), 3.10 (dq, J = 25.2, 9.8, 8.5 Hz,2H), 2.37 – 2.08 (m, 2H), 2.08 – 1.86 (m, 4H), 1.80 (d, J = 10.7 Hz, 2H), 1.74– 1.55 (m, 4H), 1.55 – 1.32 (m, 3H), 0.99 – 0.75 (m, 6H).

[0172] (2 S )-2-(( S)-3-cyclohexyl-2-((((4,4-difluorocyclohexyl)oxy)carbonyl)amino)propionamidyl)-1-hydroxy-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7i) Yield (50.5%), mp 99-101 0 C, 1 HNMR (400 MHz, DMSO- d 6) δ 7.67 – 7.51 (m, 1H), 7.46 (d, J = 3.1 Hz, 1H), 7.43 –7.22 (m, 1H), 4.77 – 4.63 (m, 1H), 4.29 – 4.15 (m, 1H), 4.12 – 3.80 (m, 2H), 3.21 – 2.98 (m, 2H), 2.27 – 1.85 (m, 6H), 1.85 – 1.34 (m, 12H), 1.34 – 1.22 (m, 1H), 1.22 – 1.03 (m, 4H), 0.96 – 0.72 (m, 3H).

[0173] (2 S )-2-(( S )-2-((((4,4-difluorocyclohexyl)methoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(( S Sodium 2-oxopyrrolidine-3-yl)propane-1-sulfonate (7j) Yield (50.5%), mp 90-92 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 7.57 (t, J = 8.9 Hz, 1H), 7.45 (s, 1H), 7.38 – 7.17 (m,1H), 4.29 – 4.10 (m, 1H), 4.05 – 3.67 (m, 4H), 3.09 (dt, J = 29.8, 8.8 Hz,2H), 2.33 – 2.05 (m, 2H), 2.05 – 1.88 (m, 4H), 1.88 – 1.64 (m, 5H), 1.64 –1.48 (m, 2H), 1.43 (q, J= 7.3 Hz, 2H), 1.30 – 1.11 (m, 2H), 1.04 – 0.78 (m, 6H).

[0174] (2 S )-2-(( S )-2-((((3,3-difluorocyclobutyl)methoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (7k) Yield (50.5%), mp 77-79 0 C, 1 H NMR (400 MHz, DMSO-) d 6) δ 7.74 – 7.12 (m, 3H), 4.20 (d, J = 9.9 Hz, 1H), 4.12 –3.90 (m, 4H), 3.90 – 3.78 (m, 1H), 3.22 – 2.93 (m, 2H), 2.68 – 2.53 (m, 2H),2.47 – 2.24 (m, 4H), 2.24 – 2.05 (m, 1H), 2.05 – 1.70 (m, 1H), 1.70 – 1.52(m, 2H), 1.52 – 1.29 (m, 2H), 0.86 (ddd, J = 14.4, 11.0, 6.6 Hz, 6H).

[0175] Table 1.3 Structure of CLpro inhibitors and their application in FRET assay (ICP-C) 50 ) and cell culture system (EC 50 The effects of various coronaviruses on cell culture toxicity (CC) 50 ).

[0176]

[0177] # Not measured. Because the bisulfite adduct is converted to the aldehyde form, only one of the two forms, the bisulfite adduct and the aldehyde, was measured.

[0178] Table 2.3 Other compounds that inhibit CLpro and their effects on MERS-CoV in FRET assays (IC50) 50 ) and cell culture toxicity (CC) 50 ).

[0179]

[0180]

[0181] Fluorescence resonance energy transfer (FRET) enzyme assay. Expression and purification of 3CLpro from MERS-CoV, SARS-CoV, or FIPV were performed using standard methods previously described in our laboratory. We also cloned and expressed 3CLpro from SARS-CoV-2. A codon-optimized cDNA of the full-length 3CLpro of SARS-CoV-2, fused at the N-terminus with a sequence encoding 6 histidine residues, was synthesized via Integrated DNA (Coralville, IA) (GenBank ID MN908947.3, incorporated herein by reference). The synthesized gene was subcloned into the pET-28a(+) vector. Expression and purification of SARS-CoV-2 3CLpro were performed according to standard procedures described in our laboratory. In short, the compound was prepared in DMSO. 6a-k and 7a-k The stock solutions were prepared and diluted in assay buffer consisting of 20 mM HEPES buffer at pH 8 containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The protease (MERS-CoV, SARS-CoV, SARS-CoV-2, or FIPV 3CLpro) was mixed with serial dilutions of each compound or with DMSO in 25 µL of assay buffer and incubated at 37 °C for 30 min (MERS-CoV and FIPV) or at room temperature for 1 h (SARS-CoV and SARS-CoV-2). Then, 25 µL of assay buffer containing the substrate (FAM-SAVLQ / SG-QXL) was added. ® 520, AnaSpec, Fremont, CA). The substrate was derived from a cleavage site on a SARS-CoV viral polyprotein. Fluorescence readings were obtained 1 h after substrate addition using a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) with excitation at 360 nm and emission at 460 nm. Relative fluorescence units (RFU) were determined by subtracting background values ​​(from substrate-containing wells without the protease) from the original fluorescence values. The IC50 of the compound was determined by fitting a variable slope to a dose-dependent FRET inhibition curve using GraphPad Prism software (GraphPad, La Jolla, CA). 50 value.

[0182] Antiviral assays in cell-based systems. Compounds were also investigated. 6a-k and 7a-kAntiviral activity against MERS-CoV, FIPV, or MHV-1 replication in Huh-7, CRFK, or CCL1 cells, respectively. In simple terms, culture medium containing DMSO (<0.1%) or each compound (up to 100 µM) was added to confluent cells, and the cells were immediately infected with the virus at an MOI of 0.01. After incubating the cells at 37°C for 24 hours, they were then subjected to TCID using CRFK or CLL1 cells. 50 Viral titers were determined using either FIPV or MHV methods or a plaque assay using Vero81 cells (MERS-CoV). For SARS-CoV-2, confluent VeroE6 cells were seeded at ~50-100 plaque-forming units / well, and culture medium containing various concentrations of each compound and agar was applied to the cells. Plaques in each well were counted after 48-72 h. EC50 was determined using variable slope (GraphPad, La Jolla, CA) software. 50 value.

[0183] Non-specific cytotoxicity. Compounds were measured in Huh-7, CRFK, or CCL1 cells. 6a-k and 7a-k The cytotoxic dose (CC) that causes 50% cell death 50 Confluent cells grown in 96-well plates were incubated with each compound at various concentrations (1 to 100 µM) for 72 hours. Cell cytotoxicity was measured using the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI), and CC was calculated using variable slopes with GraphPad Prism software. 50 Value. By using CC 50 Divide by EC 50 To calculate the in vitro treatment index.

[0184] X-ray crystallography studies. Crystallization and data collection. MERS-CoV 3CLpro or SARS-CoV 3CLpro purified in 100 mM NaCl, 20 mM Tris pH 8.0, and SARS-CoV 3CLpro were concentrated to 10.6 mg / mL (0.3 mM) and 22 mg / mL (0.64 mM), respectively, for crystallization screening. All crystallization experiments were performed using an NT8 drop-setting robot (Formulatrix Inc.) and a UVXPO MRC (Molecular Dimensions) seated drop vapor diffusion plate at 18 °C. 100 nL of protein and 100 nL of crystallization solution were dispensed and equilibrated with 50 µL of the latter. Compounds were prepared in DMSO. 6b, 6d , 6g, 6h, 7iand 7j The stock solution (100 mM) of the MERS 3CLpro inhibitor was prepared, and the complex was generated by mixing 1 µL of the ligand (2 mM) with 49 µL (0.29 mM) of the protease and incubating on ice for 1 h. Crystals of the MERS 3CLpro inhibitor complex were obtained under the following conditions. 6b, 6d , 6g and 6h Complex screening (Molecular Dimensions) conditions: E2 (8% (w / v) PEG 8000, 100 mM sodium citrate, pH 5.0), compound 7i: Proplex screening (Molecular Dimensions) conditions B8 (15% (w / v) PEG 4000, 100 mM sodium citrate, pH 5.0, 100 mM magnesium chloride) and compounds 7j Index HT screening (Hampton Research) conditions F6 (25% (w / v) PEG 3350, 100 mM Bis-Tris, pH 5.5, 200 mM ammonium sulfate). [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 7j Crystals of the SARS-CoV 3CLpro complex were obtained from Index HT screening (Hampton Research) under condition H8 (15% (w / v) PEG 3350, 100 mM magnesium formate). Before storage in liquid nitrogen, the samples were transferred to fresh droplets containing 80% crystallizing agent and 20% (v / v) PEG 200. X-ray diffraction data were collected at Advanced Photon Source beamline 17-ID using a Decrispilatus 6M pixel array detector.

[0185] Structural analysis and refinement. Autoproc was used with XDS integration strength, and Laue-class analysis and data scaling were performed using Aimless. The previously determined MERS 3CLpro (PDB: 5WKK) was used. 21 The structures of SARS-CoV 3CLpro (PDB:1Q2W) and SARS-CoV 3CLpro were used as search models for structural resolution via molecular substitution using Phaser. Phenix and Coot were used for structural refinement and manual model building, respectively. Disordered side chains were truncated to a level where electron density was observable. Structure verification was performed using MolProbity, and the diagram was prepared using the CCP4MG package. Crystallographic data are provided in Table 3.

[0186] Table 3. Crystallographic data of MERS CoV 3CLPro and SARS CoV 3CLPro inhibitor complexes.

[0187]

[0188] 1) The value in parentheses is for the highest resolution shell.

[0189] 2) R 叠加 = Σ hkl Σ i | I i ( hkl ) -< I ( hkl )>| / Σ hkl Σ i I i ( hkl ),in I i ( hkl ) is the intensity of the i-th reflection measurement, and < I ( hkl )> is the average intensity of all reflections with index hkl.

[0190] 3) R 因子 = Σ hkl || F obs ( hkl ) | - | F calc ( hkl ) || / Σ hkl | F obs ( hkl Rfree uses 5% of randomly selected reflections not included in the refinement to compute in the same way.

[0191] 4) R meas = Multiweighting unrelated to redundancy R 叠加。 R pim = Precision indication (multi-weighted) R 叠加。

[0192] 5) CC 1 / 2 It is the correlation coefficient of the average strength between two random half-sets of data.

[0193] Animal care and ethics statements. In vivo studies were conducted at the University of Iowa's Biosafety Level 3 animal facility. All experiments were conducted in accordance with protocols approved by the University of Iowa's Institutional Animal Care and Use Committee, in accordance with guidelines developed by the Association for Assessment and Certification of Laboratory Animal Care and the U.S. Department of Agriculture.

[0194] Therapeutic treatment in a mouse model of MERS-CoV infection. Using MERS-infected mice. MA Two compounds in this series were examined in 10-week-old male hDPP4-KI mice. 6j and 6h The in vivo efficacy of the compound was investigated. In the first study, animals were divided into three groups (N=5-6), lightly anesthetized with ketamine / methathiazide, and infected with 50 µl of 750 plaque-forming units (pfu) of MERS-CoV via intranasal inoculation. 6j or 6h The drug was prepared in 10% ethanol and 90% PEG400 and administered to mice intraperitoneally at a dose of 50 mg / kg / day (once daily) from 1 to 10 dpi. Control mice received the drug. Animals were weighed daily and monitored for 15 days. Animals were euthanized when they lost 30% of their initial body weight or at 15 dpi.

[0195] In the next study, we will... 6j Treatment was delayed for up to 3 days post-viral challenge (dpi) to determine the effect of delayed treatment on mouse survival. Animals were divided into five groups (N=5), and the compound was administered to mice one, two, or three days post-viral challenge (1, 2, or 3 dpi, respectively). 6j (50 mg / kg / day, once daily) until 10 dpi. Following viral challenge, mice were monitored for weight loss and survival for 15 days as described above. As a control, the load (10% ethanol + 90% PEG400) was administered in equal amounts to the test compound, or the animals were left untreated (untreated).

[0196] A third study was conducted to evaluate the compounds. 6j Therapeutic effects in the lungs. For lung harvest and viral titration, animals were divided into three groups (N=4-5) of mice, and the compound was administered to mice at 1 dpi up to 10 dpi. 6j(50 mg / kg / day, once daily) or carrier. Animals were euthanized at 3 or 5 dpi, and lungs were aseptically removed, separated in 1X PBS using a manual homogenizer, briefly centrifuged, and the supernatant was removed. Samples were titrated on Vero-81 cells as reported elsewhere. For histopathology, mice were euthanized at 6 dpi, lungs were fixed in 10% formalin, and hematoxylin and eosin (HE) stained tissues by a veterinary pathologist using a post-examination masking method. Briefly, tissue edema and hyaline membrane parameters were scored sequentially using the following scale: 0 – none, 1 – sparse (<5 alveoli), 2 – <33% of lung field, 3 – 34–66% of lung field, and 4 ->66% of lung field.

[0197] Statistical analysis. Survival curves in the groups were analyzed using the Mantel-Cox test and the Gehan-Breslow-Wilcoxon test with GraphPad Prism Software (San Diego, California). Log-transformed viral titers in the lungs, edema, and hyaline membranes in the groups were analyzed using multiple t-tests with GraphPad Prism Software.

[0198] result

[0199] Crystallographic studies of 3CLpro. To establish the mechanism of action and elucidate the structural determinants related to the binding of the inhibitor (I) to the active site of MERS-CoV 3CLpro in general, particularly the interaction mode with the S4 subsite, several high-resolution co-crystal structures were determined. Examination of the crystal structures of the inhibitors binding to MERS-CoV 3CLpro revealed the potential to enhance the interaction with the S4 subsite using appropriately modified cyclohexane derivatives. The S4 pocket of MERS-CoV 3CLpro is surrounded by a series of predominantly hydrophobic residues, including Phe188, Val193, Ala171, and Leu170. Figures 2A-F , 2G-L and Figure 3 Hydrophobic and hydrogen-bonding functional groups were introduced into the inhibitor to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate homologues. The bisulfite adduct was restored to the corresponding aldehyde, which subsequently reacted with Cys 148 to form a nearly identical covalent complex with a tetrahedral arrangement at the newly formed stereocenter. Figures 2A-F and Figure 3-5 Compounds 6h The main chain (Table 1 and Figures 2A-FIt interacts with residues Gln192, Gln167, and Glu169 via H-bonds. Three additional side-chain H-bonds between the γ-lactam ring and His166, Phe143, and Glu169 are also evident. Figures 2A-F Furthermore, the P2Leu's side zipper is tightly fitted inside the hydrophobic S2 pocket. Figures 2A-F Interestingly, compounds 7j The extra methylene group in it (converts to an aldehyde, therefore with) 6j The same) leads to the reorientation of the difluorocyclohexyl group and the formation of three H bonds between Gln195 and Ala171 and the fluorine atom, while losing one hydrogen bond in Gln192 and a substitution in Phe143. Figures 2A-F In our enzyme and cell-based assays, P2Leu was replaced with P2Cha (Table 2, compounds). 7i This leads to a significant loss of inhibitory activity. Unwilling to be bound by theory, this appears to be a result of two additional hydrogen bond losses: the loss of hydrogen bonds with Gln192 and the substitution of Gln167 and Phe143. Figure 5 A). Figure 3-5 The text provides information about compounds. 7i, 6b, 6g and 6d The electron density, hydrogen bonding interactions, and electrostatic surface representation of MERS 3CLpro in the complex.

[0200] In SARS-CoV 3CLpro- compound 7j complex ( Figure 2G-L In ), compounds 7j The main chain forms direct hydrogen bonds with Cys 145, His 163, His 164, Glu 166, and Gln 189. The compound also forms additional hydrogen bonds with His 41 and a water-mediated contact with Gly 143. Figure 2G-L However, compared to MERS-CoV 3CLpro, there is a loss of three hydrogen bonds between Gln195 and Ala171 and the fluorine atom. Not wanting to be bound by theory, in FRET assays, these compounds showed a difference compared to MERS-CoV 3CLpro. 7j It showed moderate to low potency against SARS-CoV 3CLpro, suggesting that the H-bond forming portion adapted to the S4 subsite plays an important role in potency.

[0201] discuss

[0202] The 3CLpro compound exhibits potent activity in FRET enzyme assays and cell-based assays. The compound was evaluated in FRET assays. 6a-k and 7a-kThe activity of 3CLpro against various coronaviruses was investigated, and selected compounds were tested in a cell-based system. Table 1-2 shows the 50% inhibitory concentration (IC50) for various coronaviruses in the enzyme assay. 50 ) and 50% effective concentration in cell culture (EC 50 (The average of at least two measurements). The 50% cytotoxic concentration (CC) of the compound is also listed. 50 The results showed that inhibitors with a strong preference for the P2Leu residue of MERS-CoV 3CLpro exhibited submicromolar IC50. 50 Value, but replacing Leu with Cha (cyclohexylalanine) eliminates its activity ( 6h and 7h compared to 6i and 7i (Tables 1 and 2). Compounds tested against MERS-CoV in cell culture ( 7a, 6c, 7e, 7g, 7h and 6j It also shows submicromolar EC 50 It's worth it, but 7g Except for these compounds, 6j EC showed the most effective antiviral activity against MERS-CoV. 50 The value is 0.03µM. (Compared to...) 6j In comparison, GC376 with P2Leu residues and a non-fluorinated benzyl cap showed 1 / 3 potency against MERS-CoV in cell culture.

[0203] Selected amounts of the compound were also determined in cell culture. 6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j and 7j The activities of SARS-CoV-2, FIPV, and mouse hepatitis virus (MHV) against SARS-CoV, and the activities of 3CLpro against SARS-CoV and SARS-CoV-2 in enzyme assays (Table 1). They were effective against SARS-CoV-2, EC 50 Values ​​ranged from 100 to 200 nM (Table 1). These compounds were also found to be highly effective against FIPV and MHV, EC... 50 The values ​​ranged from 70 to 200 nM. In enzyme assays, these compounds showed activity against 3CLpro of SARS-CoV and SARS-CoV-2, although the effect was not very pronounced (i.e.,...). 6j IC50 of SARS-CoV-23CLpro 50 The activity against MERS-CoV was 4.5 times higher than that against MERS-CoV (Table 1). Notably, GC376 also showed a 5.6-fold decrease in activity against SARS-CoV-2 compared to FIPV 3CLpro. These findings suggest that these compounds possess broad-spectrum activity against a variety of human and animal coronaviruses. The low cytotoxicity of this series of compounds, coupled with the high potency of some compounds, provides impetus for in vivo studies.

[0204] Crystallographic studies of 3CLpro. Examination of the crystal structures of previously obtained inhibitors binding to MERS-CoV 3CLpro revealed the potential to enhance the interaction with the S4 subsite binding site using appropriately modified cyclohexane derivatives. The S4 pocket of MERS-CoV 3CLpro is surrounded by a series of major hydrophobic residues, including Phe188, Val193, Ala171, and Leu170 (Figure 2A-L-). Figure 4 Hydrophobic and hydrogen-bonding functional groups were introduced into the inhibitor to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate homologues. The supplementary material includes a variety of compounds including... 7j ( 6j The co-crystal structure of the bisulfite adduct of MERS-CoV or SARS-CoV 3CLpro.

[0205] Therapeutic treatment with 3CLpro inhibitors significantly improved survival and reduced lung viral load and pathology in mice. The compounds were identified using cell-based assays. 6j For further research, its EC50 against MERS-CoV 50 The concentration was 0.03 µM, and we identified the compound. 6j and another derivative 6h Efficacy of these two compounds in a mouse model of MERS-CoV infection. 6h and 6j It has high potency against MERS-CoV in enzyme or cell-based assays (Table 1 and ). Figure 6 A). The generation of transgenic mice expressing hDPP4 for MERS-CoV infection was previously reported. Infection with mouse-adapted virus (MERS-CoV) MA hDPP4 knock-in mice (hDPP4-KI) developed fatal lung disease, accompanied by severe inflammation, diffuse alveolar damage, and weight loss. In the first study, with MERS... MA Infect hDPP4-KI mice and administer medication from one day (1 dpi) to 10 dpi post-infection. 6h , 6j (50 mg / kg / day, once daily) or treatment with the loading agent (control). All mice treated with the loading agent (simulated) died at 8 dpi. Figure 6 B). In contrast, 40% of users 6h The treated mice survived, and all were treated with 6j Mice treated with (50 mg / kg / day, 1 to 10 dpi) all survived at the end of the study (15 dpi). Figure 6 B). Compared with the control, using 6j or 6hThe survival rate of treated mice was significantly improved (p<0.05), and compared with... 6h Compared to treated mice, 6j The survival rate of treated mice was also significantly improved (p<0.05). 6j All treated mice recovered rapidly from weight loss starting at 3 dpi. Figure 6 C). Use 6h The surviving mice treated continued to lose weight until 6 dpi, but began to gain weight from 9 dpi onwards. Figure 6 C). Interestingly, besides compounds 6j In addition to the presence of an extra methylene group, the compound 6j and 6h They have almost identical structures. We do not wish to be bound by theory, although the compounds... 6h and 6j All showed effective anti-3CLpro activity, but in these tests, the compounds... 6h The antiviral activity in cell culture was lower than that of the compound. 6j (Table 1), which may explain its lower therapeutic efficacy in mouse models. Figure 6 B).

[0206] In our observation 6j After the first therapeutic treatment resulted in the survival of infected mice, we conducted another study by delaying the start of treatment to a maximum of 3 dpi. Similar to the first study, neither the untreated mice nor the mice given the load survived, and there was no statistically significant difference between the two groups (0% survival). When treatment started at 1 dpi... 6j At the time of treatment, four out of five mice survived (80% survival). Mice treated starting at 1 dpi showed a significantly increased survival rate compared to untreated or drug-treated mice (P<0.05). 6j If treatment is delayed by another day (2 dpi), use 6j The survival rate of treated mice decreased to 40%, which is still higher than the 0% survival rate of untreated or drug-treated mice, but 6j There was no statistically significant difference between the treatment (2 dpi) and untreated or drug-treated groups. Figure 6 D)(p<0.05). Compared with the untreated or drug-treated group, at 3 dpi 6j Treatment also failed to statistically improve mouse survival. Figure 6 D)(p<0.05). All mice lost body weight after viral infection, but with 6j The surviving mice treated recovered their lost body weight by 15 dpi. Figure 6 E). Mice treated starting at 1 dpi recovered weight faster than those treated starting at 2 dpi. Figure 6E). These results indicate that when mice were administered [at 1 dpi] 6j At that time, the survival of mice increased significantly, and 6j The antiviral effect in mouse models is greater than 6h It has a better survival rate and faster weight recovery. Figure 6 (D and E)(p<0.05). These findings highlight the importance of early intervention for achieving positive clinical outcomes.

[0207] Using compounds 6j Treatment significantly reduced lung virus titers and edema. In hDPP4-KI mice, MERS-positive mice showed... MA The lung pathology caused by the infection is very similar to that of severe human MERS infection, with diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and lymphocytic infiltration of the alveolar septa. 24 A group of mice were infected with the virus and started treatment at 1 dpi. 6j Mice were treated with either a viral load or a carrier, and their lungs were collected for viral load determination (3 and 5 dpi) or histopathology (6 dpi). During these two days, the viral titer in the lungs of treated mice was significantly reduced (P<0.05). Figure 7 A). Compared with mice treated with the loading agent, the pulmonary edema in the treated mice was statistically significantly reduced (P<0.05). Figure 7 BF). 6j The treated mice showed a decreased clear membrane score, but this was not statistically different from that of mice treated with the load. As previously reported, MERS-CoV infection treated with the load resulted in patchy consolidation. Figure 7 C and E), can be variablely composed of cellular inflammation, vascular congestion, and atelectasis. The airways are usually intact, with only scattered, uncommon exfoliated cells (C and E). Figure 7 C and E). In some lungs, lymphatic vessels are filled with degenerated cells and cell debris (C and E). Figure 7 C and E). Alveolar edema was detected in some lung fields. Figure 7 (C and E). However, in 6j Almost no lesions caused by MERS-CoV infection were observed in the treated lungs. Figure 7 D and F), which supports 6j Survival analysis of treatment.

[0208] While there are currently no approved vaccines or small molecule therapies for treating MERS-CoV or SARS-CoV infection, many preventative and therapeutic options are under development. The most advanced clinically available antiviral compound with broad-spectrum efficacy is the nucleoside analog remdesivir. Originally developed as an antiviral agent for Ebola virus, it has also shown high efficacy against both MERS-CoV and SARS-CoV in cell culture and animal models. Using mouse models, prophylactic treatment (but not therapeutic) with remdesivir significantly reduced MERS-CoV-mediated weight loss, lung viral titer, and lung injury score compared to mice treated with the drug. In summary, we have developed a highly effective 3CLpro inhibitor against multiple coronaviruses, including SARS-CoV-2, and demonstrated proof-of-concept therapeutic efficacy of the inhibitor using an hDPP4-KI mouse model, significantly improving survival in mice infected with MERS-CoV. These studies lay a solid foundation for further advancement of this series along the development pipeline.

[0209] Data and material availability. The coordinates and structural factors of the MERS 3CLpro inhibitor complex were stored in the global protein database (wwPDB) with the accession code: MERS-CoV 3CLpro and inhibitor. 6b (6VGY) 6d (6VGZ) 6g (6VH0) 6h (6VH1) 7i (6VH2) and 7j (6VH3). SARS-CoV 3CLpro and its inhibitors 7j (6W2A).

[0210] Example 2

[0211] Other compounds

[0212] The compound was synthesized according to scheme 2 (hereinafter referred to as "S2"; see also...). Figure 8 Each compound includes a specified warhead (Z group) and a glutamine substitute:

[0213]

[0214] Using compounds S2-5g or S2-6g Treatment of MERS infection MA Results in hDPP4-KI mice (N=5) Figure 9 See also Table 4-6 and Scheme 2 (in Chinese). Figure 8Advantageously, these compounds also showed efficacy against norovirus in individual studies (see also Example 5). The inhibitory activity of the synthesized compounds against NV 3CLpro and their anti-norovirus activity in a cell-based replicon system were evaluated. IC50 was determined in an enzyme assay. 50 Values ​​of EC against NV in cells containing replicons (HG23 cells) 50 Values ​​and CC in HG23 cells 50 The values ​​are displayed in the corresponding table and are the average of at least two measurements.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221] Example 3

[0222] The synthesis of the compounds shown can be readily performed using previously disclosed procedures to prepare a suitable precursor, 2-methyl-2-aryloxypropionic acid, which is then treated with carbonyl diimidazole and sodium borohydride to provide the corresponding alcohol, which is then used in Scheme 2 to prepare the inhibitor. A precursor, thioaryloxy acid, is prepared using the disclosed procedure, followed by reduction to produce an alcohol, which is then used in Scheme 2 to prepare the inhibitor. Accordingly, X-position substituted phenyl compounds are synthesized:

[0223]

[0224] Where R is H, p-Cl, m-Cl, mF, pF, or m-OCH3, and Y1 is O or S (=O). o Where o is 0, 1, or 2; R1, R2, and Z are defined as above.

[0225] Specific examples include the following, which have been shown to be effective against SARS-CoV-2 3CLpro:

[0226]

[0227]

[0228] Other synthesized compounds include:

[0229]

[0230]

[0231] Other iterations include:

[0232]

[0233] R3 is a branched or unbranched alkyl group, preferably methyl, ethyl, or n-propyl.

[0234] Further cyclopropane compounds were synthesized according to the reaction scheme described herein, wherein X in the basic main chain design is further defined as follows.

[0235]

[0236] Each R1 is a glutamine or imidazole substitute:

[0237]

[0238] Each R2 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, natural or non-natural amino acid side chain, bicyclic or tricyclic side chain, or combination thereof, and in particular leucine (Leu), cyclohexylalanine (Cha), or fluorinated side chain.

[0239] Each R3 is selected from the group consisting of -H, branched or unbranched alkyl groups (e.g., methyl, ethyl, butyl, isobutyl), substituted or unsubstituted aryl groups (e.g., phenyl, substituted phenyl), arylalkyl groups (e.g., benzyl or groups in which the aryl group is naphthyl), etc.

[0240] Each R4, R5, and R6 is independently selected from the group consisting of -H, branched or unbranched alkyl groups (e.g., methyl, ethyl, butyl, isobutyl), halogens (F, Cl, Br), haloalkyl groups, substituted or unsubstituted aryl groups (e.g., phenyl, substituted phenyl), arylalkyl groups, and cyclopropane rings (cis and / or trans); and

[0241] Each Z is selected from the group consisting of C1-C6 hydroxyalkyl, aldehyde, α-ketoamide, and bisulfite, and especially –CH2OH, -CHO, and -CH(OH)SO3. - Na + And -[O(C=O)R w SO3 - Na + , where R w It is an alkyl or arylalkyl group, with -CH3 and -CH2CH3 being particularly preferred.

[0242] Other series of cycloalkane compounds were synthesized:

[0243]

[0244] Including its macrocyclic derivatives:

[0245]

[0246] Where m and n are each 1-3;

[0247] Each q is 1-6;

[0248] Each R1 is a Gln or imidazole substitute;

[0249] Each R2 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, natural or non-natural amino acid side chain, bicyclic or tricyclic side chain, or combination thereof, and in particular leucine (Leu), cyclohexylalanine (Cha), or fluorinated side chain.

[0250] Each R3 is selected from the group consisting of -H, branched or unbranched alkyl groups (e.g., methyl, ethyl, butyl, isobutyl), substituted or unsubstituted aryl groups (e.g., phenyl, substituted phenyl), arylalkyl groups (e.g., benzyl or groups in which the aryl group is naphthyl), etc.

[0251] Each Q is selected from branched or unbranched alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl), C(CH3)2, CHF, CF2, or CHCF3;

[0252] Y = (CR i R j )o, where o can be 0 (meaning Y is absent and oxygen is directly bonded to the carbon of the ring) or 1 (meaning Y = CRiRj, where Ri and Rj can both be H, or both are methyl, or one is H and the other is methyl, or their deuterated derivatives; o can also be 3 or greater, but 0 and 1 are preferred; and

[0253] Each Z is selected from the group consisting of C1-C6 hydroxyalkyl, aldehyde, α-ketoamide, and bisulfite, and especially –CH2OH, -CHO, and -CH(OH)SO3. - Na + And -[O(C=O)R w SO3 - Na + , where R w It is an alkyl or arylalkyl group, with -CH3 and -CH2CH3 being particularly preferred.

[0254] Example 4

[0255] Structure-guided design of potent inhibitors of SARS-CoV-2 3CL protease

[0256] Introduction

[0257] The SARS-CoV-2 life cycle encompasses multiple exploitable viral and host-based druggable targets, including inhibitors that block viral entry and fusion, as well as replication inhibitors targeting 3CL, papain-like proteases, and RNA-dependent RNA polymerases. Attractive host-based targets include transmembrane serine protease 2 (TMPRSS2), cathepsin L, and furin. Therefore, the development of small molecule therapies targeting host or viral targets essential for viral replication is a potentially fruitful research avenue. SARS-CoV-2 3CLpro is a catalytically linked Cys-His dual (Cys... 145 -His 41 The protease exhibits a strong preference for the -YZ-Leu-Gln-X sequence, corresponding to the subsite -S4-S3-S2-S1-S1'-, where X is a small amino acid (Ser, Ala, Gly), Y is a small hydrophobic amino acid, and Z is exposed to solvents and can tolerate polar or nonpolar amino acid chains. Therefore, SARS-CoV-2 3CLpro is an attractive target for drug development.

[0258] We recently described the structure-guided design of a dipeptide family of MERS-CoV and SARS-CoV-2 3CL protease inhibitors, incorporating piperidine or cyclohexyl moieties into their structures capable of favorable binding interactions with the S4 pocket (see examples above). We further demonstrated that members of the cyclohexyl family of compounds improved survival in a mouse model infected with MERS-CoV. Here we report the results of structure-guided studies aimed at exploring the effects of stereochemistry, conformation, and structure, including the systematic introduction of fluorine (F-walk) around the structure of the clinical candidate GC376, and the synthesis of deuterated inhibitors to modulate pharmacological activity, pharmacokinetic properties, and oral bioavailability.

[0259] Results and discussion

[0260] Chemistry. Inhibitors 1-24b / c The new synthetic scheme differs from Schemes 1 and 2, requiring the use of a diverse set of precursor alcohols (Table 10), some of which are commercially available. Schemes 1 and 2 rely on linear synthesis to prepare the inhibitor, involving more steps and more purification, and in some cases, lower final product yields. The new scheme involves pooling synthesis, which involves synthesizing two separate fragments and then linking them together, resulting in fewer steps, higher product yields, and a less cumbersome (less purification) process. In short, alcohols... 12-16 and 13The aldehyde is readily synthesized from 4,4-difluorocyclohexanecarboxylic acid by reduction to the corresponding alcohol via treatment with carbonyl diimidazole and sodium borohydride, followed by oxidation with a Dess-Martin periodoane (DMP) reagent. Subsequent treatment with a series of Grignard reagents yields the alcohol. 12 , 14-16 ( Figure 10A Alcohols are synthesized by reacting methyl 4,4-difluorocyclohexanecarboxylate with excess magnesium methyl iodide, followed by acid treatment. 13 The deuterated alcohol was obtained by treating the precursor carboxylic acid with carbonyl diimidazole and then adding sodium borodeuteride. 9 , 11 , 20 and 22 All trans-substituted alcohols were synthesized by reducing the precursor 4-substituted cyclohexanone with sodium borohydride / CeCl3.

[0261] By reacting each precursor alcohol with bis(succinimide) carbonate, and then with an amino alcohol A Conjugation to obtain inhibitors 1-24b / c The product was treated with Des Martin periodane to give an aldehyde. 1-24b After treatment with sodium bisulfite, it is converted into the corresponding bisulfite adduct. 1-24c ( Figure 10B Therefore, in Figure 10B In the end, the final compound was designated as an aldehyde. b ) and bisulfite ( c ), and was designated as a The compound refers to an intermediate. Therefore, for example, a compound 1b and 1c These refer to the aldehyde and bisulfite forms of compound 1, respectively.

[0262] In inhibitors 6-8、10-16、23、24 In this case, an alternative synthesis was used, involving the reaction of a precursor alcohol with (L)leucine methyl ester isocyanate, as described in detail above. Precursor amino alcohol A Synthesis such as Figure 10C As shown, this can be easily accomplished by coupling (L) Z-Leu with a glutamine substitute, followed by sequential reduction with LiBH4 and removal of the protecting group (H2 / Pd).

[0263] Biochemical research. As described in the experimental section, compounds were identified. 1-24b / c Inhibitory activity against SARS-CoV-2 3CL protease in biochemical assays and cell-based systems. IC50 in Huh-7, CRFK, or CCL1 cells. 50 Value and CC 50 The values ​​are summarized in Table 7; they are the average of at least two measurements.

[0264] Table 7. Inhibitors 1-24 IC50 of SARS-CoV-2 3CLpro 50 and CC 50 .

[0265]

[0266] For comparative purposes, the inhibitory activity of other compounds against the MERS-CoV 3CL protease was also determined as previously described, and the IC50 was... 50 The values ​​are listed in Table 8.

[0267] Table 8. IC50 of selected compounds against MERS-CoV 3CLpro 50 value.

[0268]

[0269] X-ray crystallography studies were conducted. A series of high-resolution cocrystal structures were determined to elucidate the interaction between the inhibitor and the active site of SARS-CoV-23CLpro. Specifically, we sought to confirm the mechanism of action, identify the structural determinants associated with the binding of the inhibitor to the protease active site, and ultimately utilize the accumulated structural information and insights gained to further optimize pharmacological activity and PK parameters. The functional groups interacting within the S4 subsite of three groups of inhibitor types were analyzed: 1) nonpolar substituents, 2) 4,4-difluorocyclohexyl groups attached to the stereocenter, and 3) fluorinated aryl compounds based on the GC376 structure.

[0270] For all structures described in this embodiment, the active sites contain significantly different electron densities consistent with those of the inhibitors covalently bound to Cys 145. Furthermore, the electron densities are consistent with the R and S enantiomers at the stereocenter formed by the covalent bonding of the Sg atoms of Cys 145, and are therefore modeled as each enantiomer having a 0.5 occupancy rate. The γ-lactam ring of the inhibitor forms direct hydrogen bonds with Glu166 and His163, while Glu166 and Gln189 form additional hydrogen bonds with the C=O and NH groups of the carbamate moiety in the inhibitor. The inhibitor undergoes hydrophobic interactions with the leucine side chain located within the S2 pocket. The eutectic structure confirms that the reaction of Cys 145 with the aldehyde tip results in the formation of a tetrahedral hemithioacetal, which is stabilized by hydrogen bonding with His164.

[0271] Table 9. Crystallographic data of the SARS-CoV-2 3CLpro inhibitor complex

[0272]

[0273] 1) The value in parentheses is for the highest resolution shell.

[0274] 2) R 叠加 = Σ hkl Σ i | I i ( hkl ) -< I ( hkl )>| / Σ hkl Σ i I i ( hkl ),in I i ( hkl ) is the intensity of the i-th reflection measurement, and < I ( hkl )> is the average intensity of all reflections with index hkl.

[0275] 3) R 因子 = Σ hkl || F obs ( hkl ) | - | F calc ( hkl ) || / Σ hkl | F obs ( hkl Rfree uses 5% of randomly selected reflections not included in the refinement to compute in the same way.

[0276] 4) R meas = Multiweighting unrelated to redundancy R 叠加。 R pim = Precision indication (multi-weighted) R 叠加

[0277] 5) CC 1 / 2 It is the correlation coefficient of the average strength between two random half-sets of data.

[0278] Nonpolar substituent. 5c (AMJ-I-157) 1c (AMJ-I-158) 3c (AMJ-I-159) and 8b The structure of (NN-II-111) exhibits a well-defined electron density and similar hydrogen bond interactions, such as Figure 11 As shown. For all structures, the nonpolar groups are mainly located in the S4 subsite, close to the hydrophobic ridge formed by residues Leu 167, Pro 168, Gly 170, and Ala 191. Figure 12 ).However, 1c The dimethylcyclohexyl ring in the S4 subsite is too short to fully bind to the hydrophobic ridge. Figure 12 B). In 3c The addition of a n-propyl group allows for further bonding with hydrophobic cracks, and 8b The extra carbon atom allows the propyl group to extend further. Figure 12 (C and 2D). 3c and 1c superposition ( Figure 12 E) indicates that the 4,4-dimethylcyclohexyl ring is relative to... 3c The n-propyl group in the sample slightly shifts out of the S4 subsite. Furthermore, 3c and 8b The superposition showed very similar binding patterns, although 8b The n-propyl group is located deeper in the S4 subsite (Figure 2F). Overall, the similar binding modes of the inhibitors and the accompanying high potency are reflected in their low IC50 values. 50 Values ​​and similar potency (Table 7, compounds) 1-5b / c Regarding inhibitors 8 The hypothesis is that a corresponding deuterated inhibitor, almost equivalent to the non-deuterated inhibitor 8, will be discovered. 9 (Table 7) may show improved PK characteristics.

[0279] 4,4-Difluorocyclohexyl compounds. In previous studies involving norovirus 3CL protease inhibitors, the strategic introduction of a gem-dimethyl group into the inhibitor structure enhanced potency by restricting rotation around nearby single bonds and reducing entropy loss associated with binding. Therefore, we sought to synthesize gem-dimethyl-substituted compounds. 13c To take advantage of this, in addition, by introducing a stereocenter (compound) 12c To achieve the same purpose. Those with SARS-CoV 3CLpro 12b , 13c and 14c The structure exhibits a well-defined electron density and the commonly observed hydrogen bond interactions. Figure 13 ).like Figure 14 As shown in AC, the 4,4-difluorocyclohexyl rings in all structures are located near the hydrophobic fracture at the S4 subsite. The superposition of these structures reveals... 12b and 13c Almost identical binding modes, with the 4,4-difluorocyclohexyl group located in the same region within the S4 subsite ( Figure 14 D). For14c The benzyl ring is oriented within a wide fissure formed by Asn 142 and Gln 189. However, 13c The 4,4-difluorocyclohexyl ring contacts residues Thr 190 and Ala 191 (3.0–3.2 Å) and forms new hydrogen bond interactions with the oxygen and nitrogen atoms of the main chain, respectively. Figure 13 E). This will 13c The 4,4-difluorocyclohexyl ring is more deeply positioned within the S4 pocket, leading to conformational changes across the ring from Gln 189 to Gly 195 to accommodate the new interactions and avoid spatial conflicts. However, why... 13c IC 50 Compare 12b and 14c The fact that it is about 4 times higher is not intuitive.

[0280] Fluorinated aryl compounds. Position analogue scanning is a widely used strategy for optimizing the binding affinity, selectivity, and physicochemical properties of lead compounds containing aromatic or heteroaromatic rings. For example, introducing fluorine (F-walk) or nitrogen (N-walk) by leveraging the beneficial effects of fluorine (or nitrogen) and minor structural changes is an effective means of multi-parameter optimization. To determine the effect of fluorine on the binding mode at the S4 subsite of GC376, the fluorinated benzyl compound was identified using SARS-CoV-2 3Clpro. 17c , 18c , 19b , 20b ( 19b (deuterated analogues) and 21c The structure of the inhibitor o-fluorobenzyl ( 17c ) and m-fluorobenzyl ( 18c The compound exhibits a defined electron density and similar hydrogen-bonded interactions, such as Figure 15 As shown. Interestingly, 17c The ortho-fluorobenzyl ring adopts a conformation in which the fluorine atom is far from Thr 190, but is located 3.38 Å away from the oxygen atom in the main chain at Glu 166. Figure 15 C). On the contrary 18c The fluorine atom in the S4 pocket is located between Thr 190 and Ala 191, and is 3.10 Å away from the nitrogen atom in the main chain of Ala 191. Figure 15 D). 17c and 18c The orientation of the fluorine atoms relative to the hydrophobic ridges in the S4 pocket is as follows: Figure 15 As shown in E-F15.

[0281] Inhibitors containing p-fluorobenzyl groups 19b and its deuterated analogues 20bAs expected, they employ very similar binding modes and hydrogen bond interactions, and as... Figure 17-18 As shown. Interestingly, the inhibitor adopts two conformations, in which the p-fluorobenzyl ring protrudes from the S4 subsite in subunit B and is located in the S4 pocket of subunit A. However, the electron density of the p-fluorobenzyl ring in subunit A is slightly weaker, suggesting that the orientation in subunit B is likely the dominant conformation. This is likely because the fluorine atom does not form any contact with the polar atom in the S4 subsite, resulting in a conformation where the aromatic ring is located outside the pocket, which is the same conformation observed for the parent compound GC376.

[0282] Perfluorinated compounds 21c It also shows a distinct difference in electron density consistent with the aromatic ring in one conformation ( Figure 16 A). Interestingly, an ortho-fluorine atom interacts with the main chain oxygen of Glu 166 (3.08 Å), which is greater than the interaction observed above for... 17c The observed (3.38 Å) is shorter. Another ortho-fluorine atom is located 2.92 Å from the main chain N atom at Thr 190 and 3.12 Å from the side chain N atom at Gln 189. Figure 16 B). Similarly, the inter-fluorine atom is located near the nitrogen atom in the main chain of Ala 191 (3.40 Å), which is comparable to... 18c The observed distance (3.10 Å) is longer. The pentafluorobenzyl ring is located at the top of the hydrophobic crack within the S4 subsite ( Figure 16 C), which is different from GC376, in which the benzene ring and γ-lactam ring undergo hydrophobic collapse and the inhibitor is in the shape of a "paperclip".

[0283] Finally, the GC376 variant 23b / c and 24b / c Synthesis and screening of mixtures of epimers. Inhibitors of aldehyde and bisulfite adducts were discovered. 23b / c Effectively suppresses 3CLpro (IC) 50 The concentrations were 0.15 and 0.18 µM, respectively, and their efficiencies were respectively... 24b / c The aldehyde and bisulfite adducts were 27 and 19 times stronger, respectively. These findings provide preliminary validation for designs that utilize chiral centers to achieve directional control and enhance binding interactions.

[0284] in conclusion

[0285] Effective management of SARS-CoV-2, the pathogen causing the COVID-19 pandemic, requires not only the availability of safe and effective vaccines but also the availability of small-molecule therapeutics and prophylactic agents targeting the virus and host-based druggable targets. The SARS-CoV-2 3CL protease is an attractive target for developing COVID-19 treatments due to its important role in viral replication. A range of approaches have been used to enhance potency and physicochemical parameters, including conformational and stereochemical control of inhibitors by introducing gem-dimethyl (Thorpe-Ingold effect) or stereocenters, deuteration, and fluorine. Almost all inhibitors were found to exhibit submicromolar potency without cytotoxicity. Furthermore, several deuterated inhibitors were found to be equivalent to their corresponding non-deuterated inhibitors, potentially exhibiting improved pharmacokinetics. The fluorine walk approach explored bioisosteric substitutions of the benzene ring in GC376 by replacing one or more hydrogen atoms. The effects of these modifications included unexpected binding modes of the F-substituted benzene ring and moderately enhanced potency. The introduction of multiple fluorine atoms resulted in orientations that allowed fluorine atoms to H-bond with residues in the S4 pocket, although the bond angles were not optimal. High-resolution cocrystal structures containing a range of inhibitors revealed the mechanism of action and provided valuable insights into the binding of inhibitors to the active site and the identity of the structural determinants involved in the binding. Overall, the findings described in this paper are important and timely, and provide an effective platform for further preclinical studies.

[0286] Experimental Section

[0287] Overview. Reagents and anhydrous solvents were purchased from various chemical suppliers (Sigma-Aldrich, Acros Organics, Chem-Impex, TCI America, Oakwood Chemical, APExBIO, Cambridge Isotopes, AlphaAesar, Fisher, and Advanced Chemblocks) and used as is. Silica gel (230-450 mesh) used for rapid chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin-layer chromatography was performed using Analtech silica gel plates. Visualization was performed using UV light and / or iodine. NMR spectra were recorded in CDCl3 or DMSO-d6 using a Varian XL-400 spectrometer. Melting points were recorded on a Mel-Temp instrument and were uncalibrated. High-resolution mass spectrometry (HRMS) was performed at the Wichita State University Mass Spectrometry Laboratory using an Orbitrap Velos Pro mass spectrometer equipped with an electrospray ionization source (ThermoFisher, Waltham, MA). As demonstrated by the NMR analysis, the purity of all final compounds was >95%.

[0288] Compound Synthesis

[0289] compound 1-5a, 9a, 17-22a Preparation. General procedure. Add N,N'-disuccinimidyl carbonate (DSC) (1.2 equivalents) and TEA (3.0 equivalents) to a solution of anhydrous acetonitrile (10 mL / g alcohol) (Table 10), and stir the reaction mixture at room temperature for 4 h. Remove the solvent under vacuum and dissolve the residue in ethyl acetate (40 mL / g alcohol). Wash the organic phase with a saturated aqueous solution of NaHCO3 (2 × 20 mL / g alcohol), followed by washing with brine (20 mL / g alcohol). Combine the organic layers and dry on anhydrous Na2SO4, filter, and concentrate under vacuum to obtain a mixed carbonate that can be used in the next step without further purification.

[0290] TEA (1.5 equivalents) was added to a solution of Leu-Gln substituted amino alcohol (1.0 equivalent) in anhydrous dichloromethane (10 mL / g amino alcohol), and the reaction mixture was stirred at room temperature for 20 min (solution 1). In a separate flask, a mixed carbonate was dissolved in anhydrous dichloromethane (10 mL / g carbonate) (solution 2). Solution 1 was added to solution 2, and the reaction mixture was stirred at room temperature for 3 h. Dichloromethane was added to the organic phase (40 mL / g carbonate), followed by washing with a saturated aqueous solution of NaHCO3 (2 × 20 mL / g alcohol), and then with brine (20 mL / g alcohol). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The resulting crude product was purified by rapid chromatography (hexane / ethyl acetate) to give dipeptide alcohol. a It is a white solid. The reaction scheme is described in... Figures 10A-10C middle.

[0291] Table 10 - Alcohol Inputs

[0292]

[0293] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 4,4-dimethylcyclohexyl ester (1a)。 Yield (36%) 1 H NMR (400 MHz, DMSO-d6)δ 7.57 (d, J = 8.9 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J= 8.3 Hz, 1H), 4.67 – 4.63(m, 1H), 4.47 – 4.40 (m, 1H), 3.98 – 3.88 (m, 1H), 3.78 – 3.74 (m, 1H), 3.36– 3.28 (m, 1H), 3.27 – 3.17 (m, 1H), 3.13 (t, J = 8.9 Hz, 1H), 3.09 – 2.98 (m,1H), 2.26 – 2.16 (m, 1H), 2.15 – 2.10 (m, 1H), 1.83 – 1.72 (m, 1H), 1.68 –1.63 (m, 2H), 1.62 – 1.50 (m, 2H), 1.48 – 1.30 (m, 6H), 1.25 – 1.13 (m, 3H), 0.92 – 0.81 (m, 12H).

[0294] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate(1r,4S)-4-isopropylcyclohexyl ester (2a)。 Yield (35%) 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 8.9 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 4.65 (t, J = 5.5 Hz, 1H), 4.34 (td, J = 11.0, 5.5 Hz, 1H), 3.97 – 3.88 (m, 1H), 3.79 – 3.74 (m, 1H), 3.38 – 3.28 (m, 1H), 3.27 – 3.18 (m, 1H), 3.14 (t, J =9.0 Hz, 1H), 3.05 (q, J = 8.5 Hz, 1H), 2.29 – 2.20 (m, 1H), 2.18 – 2.07 (m,1H), 1.91 (d, J= 7.9 Hz, 2H), 1.84 – 1.72 (m, 1H), 1.71 – 1.66 (m, 2H), 1.62 – 1.50 (m, 2H), 1.48 – 1.30 (m, 4H), 1.26 – 1.13 (m, 2H), 1.01 (s, 3H), 0.93– 0.80 (m, 12H).

[0295] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (1S,4S)-4-propylcyclohexyl ester (3a)。 Yield (38%) 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 9.0 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 4.65 (s, 1H), 4.36 (td, J = 11.0, 5.5 Hz, 1H), 3.98 – 3.88 (m, 1H), 3.78 –3.74 (m, 1H), 3.36 – 3.29 (m, 1H), 3.28 – 3.18 (m, 1H), 3.14 (t, J = 8.9 Hz,1H), 3.10 – 2.99 (m, 1H), 2.30 – 2.19 (m, 1H), 2.17 – 2.07 (m, 1H), 1.93 –1.84 (m, 2H), 1.77 – 1.67 (m, 3H), 1.62 – 1.48 (m, 2H), 1.48 – 1.30 (m, 3H), 1.33 – 1.21 (m, 3H), 1.21 – 1.08 (m, 4H), 0.97 – 0.90 (m, 2H), 0.90 – 0.81 (m, 9H).

[0296] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopyran-2-yl)carbamate (1S,4S)-4-butylcyclohexyl ester (4a)。 Yield (35%) 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J= 9.0 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 4.65 (t, J = 5.5 Hz, 1H), 4.36 (tt, J = 10.9, 4.2 Hz, 1H), 3.98 – 3.88 (m, 1H), 3.79 – 3.74 (m, 1H), 3.38 – 3.28 (m, 1H), 3.28 – 3.18 (m, 1H), 3.14 (t, J =8.9 Hz, 1H), 3.10 – 2.99 (m, 1H), 2.30 – 2.18 (m, 1H), 2.18 – 2.07 (m, 1H), 1.93 – 1.84 (m, 2H), 1.79 – 1.65 (m, 3H), 1.62 – 1.50 (m, 2H), 1.48 – 1.30 (m, 3H), 1.29 – 1.14 (m, 9H), 0.97 – 0.90 (m, 2H), 0.90 – 0.81 (m, 9H).

[0297] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (1r,4S)-4-phenylcyclohexyl ester (5a)。 Yield (51%) 1 H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J = 9.0 Hz, 1H), 7.51 (s, 1H), 7.32 – 7.10 (m, 6H), 4.66(t, J = 5.5 Hz, 1H), 4.52 (ddd, J = 15.2, 10.8, 4.3 Hz, 1H), 3.99 – 3.90 (m,1H), 3.80 – 3.75 (m, 1H), 3.41 – 3.28 (m, 1H), 3.28 – 3.18 (m, 1H), 3.14 (t, J= 8.9 Hz, 1H), 3.10 – 3.00 (m, 1H), 2.29 – 2.20 (m, 1H), 2.20 – 2.09 (m, 1H), 2.03 – 1.98 (m, 3H), 1.81 (d, J = 13.0 Hz, 3H), 1.69 – 1.50 (m, 4H), 1.49 –1.31 (m, 5H), 0.86 (dd, J = 8.8, 6.5 Hz, 6H).

[0298] ((2 S )-1-(((2 S 1-Hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (4-(trifluoromethyl)cyclohexyl)methyl ester (6a)。 Yield (83%). 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.16 (d, J = 8.3 Hz, 1H),4.66 (s, 1H), 3.99 – 3.89 (m, 1H), 3.83 – 3.71 (m, 2H), 3.27 – 3.19 (m, 2H),3.15 (t, 2H), 3.10 – 3.02 (m, 1H), 2.26 – 2.08 (m, 3H), 1.91 – 1.73 (m, 4H), 1.64 – 1.52 (m, 4H), 1.49 – 1.31 (m, 1H), 1.30 – 1.15 (m, 1H), 1.08 – 0.94(m, 4H), 0.90 – 0.81 (m, 6H).

[0299] ((2 S )-1-(((2 S )-1-hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamic acid((1 r 4 S 4-(trifluoromethyl)cyclohexyl)methyl ester (7a)。 Yield (80%). 1 H NMR (400MHz, DMSO-d6) δ 7.61 (d, J= 9.0 Hz, 1H), 7.53 (s, 1H), 7.19 (d, J = 8.2 Hz,1H), 4.67 (s, 2H), 4.07 – 3.86 (m, 2H), 3.76 (s, 1H), 3.26 – 3.18 (m, 1H), 3.17 – 3.11 (m, 2H), 3.09 – 3.01 (m, 2H), 2.33 – 2.19 (m, 3H), 2.18 – 2.09 (m, 1H), 1.89 – 1.85 (m, 2H), 1.84 – 1.72 (m, 2H), 1.64 – 1.32 (m, 9H), 0.90 – 0.81 (m, 6H).

[0300] ((2 S )-1-(((2 S )-1-hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamic acid((1 r 4 R 4-propylcyclohexyl)methyl ester (8a)。 Yield (86%). 1 H NMR (400 MHz, DMSO-d6) δ 7.62 – 7.57 (m, 1H), 7.52 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 4.65(s, 1H), 3.99 – 3.89 (m, 1H), 3.82 – 3.68 (m, 3H), 3.37 – 3.19 (m, 2H), 3.18– 3.02 (m, 2H), 2.26 – 2.07 (m, 4H), 1.76 – 1.65 (m, 4H), 1.62 – 1.51 (m,8H), 1.49 – 1.32 (m, 2H), 1.32 – 1.24 (m, 2H), 1.21 – 1.10 (m, 2H), 0.99 –0.78 (m, 9H).

[0301] ((2 S )-1-(((2 S )-1-hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamic acid((1 s 4 S 4-propylcyclohexyl)methyl ester d 2(9a)。 Yield (43%). 1 H NMR (400MHz, DMSO-d6) δ 7.59 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.12 (d, J = 8.3 Hz,1H), 4.65 (s, 1H), 3.98 – 3.89 (m, 1H), 3.80 – 3.70 (m, 1H), 3.27 – 3.10 (m,2H), 3.10 – 3.00 (m, 2H), 2.28 – 2.06 (m, 4H), 1.70 (s, 4H), 1.63 – 1.51 (m,2H), 1.49 – 1.34 (m, 8H), 1.32 – 1.23 (m, 2H), 1.20 – 1.10 (m, 2H), 0.90 –0.81 (m, 9H).

[0302] (( S )-1-((( S )-1-hydroxy-3-(( S 4,4-Difluorocyclohexyl)methyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (10a)。 Yield (90%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.60 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 4.66 (s, 1H), 3.95 (td, J = 8.9, 5.4 Hz, 1H), 3.90 – 3.69 (m, 3H), 3.23 (d, J =5.7 Hz, 1H), 3.18 – 3.00 (m, 2H), 2.28 – 2.07 (m, 2H), 2.06 – 1.93 (m, 2H), 1.90 – 1.64 (m, 6H), 1.56 (dq, J = 11.9, 8.8 Hz, 2H), 1.51 – 1.30 (m, 3H), 1.30 – 1.12 (m, 2H), 0.86 (dd, J = 10.5, 6.6 Hz, 6H).

[0303] ((2 S )-1-(((2 S 1-Hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (4,4-difluorocyclohexyl)methyl ester- d 2 (11a)。 Yield (81%). 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.20 (d, J = 8.2 Hz, 1H),3.99 – 3.90 (m, 1H), 3.77 (s, 1H), 3.37 – 3.19 (m, 2H), 3.18 – 3.00 (m, 2H),2.27 – 2.07 (m, 2H), 2.03 – 1.94 (m, 2H), 1.87 – 1.63 (m, 6H), 1.63 – 1.49 (m, 2H), 1.47 – 1.29 (m, 3H), 1.29 – 1.13 (m, 2H), 0.90 – 0.81 (m, 6H).

[0304] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 1-(4,4-difluorocyclohexyl)ethyl ester (12a)。 Yield (76%). 1 H NMR (400 MHz, DMSO-d6) δ 7.61 – 7.50 (m, 2H), 7.11 (d, J = 8.2 Hz, 1H), 4.66 (t, J= 5.3 Hz,1H), 4.59 – 4.50 (m, 1H), 3.98 – 3.90 (m, 1H), 3.79 – 3.75 (m, 1H), 3.39 –3.29 (m, 1H), 3.28 – 3.18 (m, 1H), 3.18 – 3.10 (m, 1H), 3.10 – 2.99 (m, 1H), 2.27 – 2.18 (m, 1H), 2.16 – 2.09 (m, 1H), 2.02 – 1.97 (m, 2H), 1.83 – 1.63(m, 5H), 1.63 – 1.51 (m, 3H), 1.47 – 1.31 (m, 3H), 1.27 – 1.22 (m, 2H), 1.12 (dd, 2H), 0.90 – 0.81 (m, 6H).

[0305] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 2-(4,4-difluorocyclohexyl)propyl-2-ester (13a)。 Yield (72%). 1 H NMR (400 MHz, DMSO6) δ 7.59 – 7.47 (m, 2H), 6.94 (d, J = 8.2 Hz, 1H), 4.66 (t, J = 5.5 Hz,1H), 3.93 – 3.83 (m, 1H), 3.79 – 3.75 (m, 1H), 3.42 – 3.29 (m, 1H), 3.27 –3.18 (m, 1H), 3.14 (t, J = 8.9 Hz, 1H), 3.10 – 2.99 (m, 1H), 2.27 – 2.09 (m,2H), 2.06 – 1.91 (m, 3H), 1.85 – 1.61 (m, 5H), 1.61 – 1.51 (m, 2H), 1.46 –1.34 (m, 3H), 1.33 (s, 6H), 1.31 – 1.18 (m, 2H), 0.90 – 0.81 (m, 6H).

[0306] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 1-(4,4-difluorocyclohexyl)-2-phenylethyl ester (14a)。 Yield (79%). 1 H NMR (400MHz, DMSO-d6) δ 7.61 – 7.54 (m, 1H), 7.52 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H), 4.66 (t, J = 5.3 Hz, 1H), 4.59 – 4.50 (m, 1H), 3.98 – 3.90 (m, 1H), 3.79 –3.75 (m, 1H), 3.39 – 3.29 (m, 1H), 3.28 – 3.18 (m, 1H), 3.18 – 3.10 (m, 1H),3.10 – 2.99 (m, 1H), 2.27 – 2.18 (m, 1H), 2.16 – 2.09 (m, 1H), 2.02 – 1.97(m, 2H), 1.83 – 1.63 (m, 5H), 1.63 – 1.51 (m, 3H), 1.47 – 1.31 (m, 3H), 1.27–1.22 (m, 2H), 1.12 (dd, 3H), 0.90–0.81 (m, 6H).

[0307] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (4,4-difluorocyclohexyl)(phenyl)methyl ester (15a)。 Yield (86%). 1H NMR (400MHz, DMSO-d6) δ 7.64 – 7.50 (m, 2H), 7.39 – 7.23 (m, 6H), 4.68 – 4.64 (m,1H), 4.00 – 3.89 (m, 1H), 3.87 – 3.73 (m, 2H), 3.39 – 3.29 (m, 1H), 3.27 –3.20 (m, 1H), 3.19 – 3.10 (m, 1H), 3.10 – 3.02 (m, 1H), 2.25 – 2.09 (m, 2H),2.06 – 1.92 (m, 2H), 1.87 – 1.66 (m, 5H), 1.65 – 1.51 (m, 2H), 1.49 – 1.32 (m, 4H), 1.32 – 1.18 (m, 2H), 0.93 – 0.71 (m, 6H).

[0308] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 1-(4,4-difluorocyclohexyl)pentyl ester (16a)。 Yield (91%). 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 9.0 Hz, 1H), 7.52 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 4.66 (t, J = 5.6 Hz, 1H), 4.00 – 3.90 (m, 1H), 3.90 – 3.77 (m, 1H), 3.77 –3.74 (m, 1H), 3.40 – 3.31 (m, 1H), 3.29 – 3.19 (m, 1H), 3.14 (t, J = 9.0 Hz,1H), 3.11 – 3.00 (m, 1H), 2.25 – 2.15 (m, 1H), 2.15 – 2.09 (m, 1H), 2.02 –1.97 (m, 3H), 1.88 – 1.80 (m, 1H), 1.79 – 1.65 (m, 8H), 1.63 – 1.51 (m, 2H), 1.49 – 1.31 (m, 4H), 1.26 – 1.18 (m, 3H), 0.93 – 0.81 (m, 9H).

[0309] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate-2-fluorobenzyl ester (17a)。 Yield (33%). 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.2 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.34 – 7.27 (m, 1H), 7.12 (td, J = 7.5,1.2 Hz, 1H), 7.09 – 7.00 (m, 1H), 6.23 (s, 1H), 5.56 (d, J = 8.3 Hz, 1H), 5.19– 5.14 (m, 2H), 4.27 – 4.22 (m, 1H), 4.01 – 3.96 (m, 1H), 3.77 – 3.46 (m,2H), 3.37 – 3.24 (m, 2H), 2.46 – 2.33 (m, 2H), 2.11 – 1.87 (m, 2H), 1.85 –1.74 (m, 1H), 1.74 – 1.58 (m, 2H), 1.57 – 1.46 (m, 1H), 0.94 (d, J = 5.8 Hz, 6H).

[0310] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate-3-fluorobenzyl ester (18a)。 Yield (41%). 1 H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 7.2 Hz, 1H), 7.35 – 7.28 (m, 1H), 7.12 – 7.02 (m, 2H), 7.02 – 6.94 (m,1H), 6.41 (s, 1H), 5.70 (d, J= 8.3 Hz, 1H), 5.16 – 5.00 (m, 2H), 4.29 – 4.22(m, 1H), 4.02 – 3.93 (m, 1H), 3.74 – 3.53 (m, 2H), 3.36 – 3.22 (m, 2H), 2.45– 2.30 (m, 2H), 2.07 – 1.95 (m, 1H), 1.79 (td, J = 9.4, 2.8 Hz, 1H), 1.73 –1.43 (m, 4H), 0.94 (d, J = 6.1 Hz, 6H).

[0311] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate-4-fluorobenzyl ester (19a)。 Yield (37%). 1 H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.4 Hz, 1H), 7.37 – 7.28 (m, 2H), 7.08 – 6.97 (m, 2H), 6.37 (s, 1H), 5.56(d, J = 8.1 Hz, 1H), 5.12 – 5.02 (m, 2H), 4.31 – 4.13 (m, 1H), 4.05 – 3.90 (m,1H), 3.66 – 3.53 (m, 2H), 3.39 – 3.25 (m, 2H), 2.46 – 2.31 (m, 2H), 2.05 –1.93 (m, 1H), 1.88 – 1.76 (m, 1H), 1.74 – 1.57 (m, 3H), 1.57 – 1.45 (m, 1H), 0.93 (d, J = 6.1 Hz, 6H).

[0312] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (4-fluorophenyl)methyl d2 (20a)。 Yield (37%). 1 H NMR (400 MHz, CDCl3) δ7.76 (d, J= 7.3 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.07 – 6.98 (m, 2H), 6.32 (s,1H), 5.55 (d, J = 8.3 Hz, 1H), 4.27 – 4.20 (m, 1H), 4.00 – 3.96 (m, 1H), 3.66 – 3.53 (m, 2H), 3.37 – 3.23 (m, 2H), 2.48 – 2.32 (m, 2H), 2.03 – 1.93 (m,1H), 1.87 – 1.76 (m, 1H), 1.73 – 1.57 (m, 3H), 1.55 – 1.45 (m, 1H), 0.93 (d, J = 6.1 Hz, 6H).

[0313] ((2 S )-1-(((2 S 1-Hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (perfluorophenyl) methyl ester (21a)。 Yield (21%). 1 H NMR (400 MHz, DMSO-d6) δ7.66 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 5.18 – 5.07(m, 2H), 4.64 (s, 1H), 3.99 – 3.90 (m, 1H), 3.75 (s, 1H), 3.25 – 3.02 (m,4H), 2.27 – 2.16 (m, 1H), 2.15 – 2.05 (m, 2H), 1.81 – 1.71 (m, 1H), 1.62 –1.50 (m, 2H), 1.47 – 1.31 (m, 2H), 0.90 – 0.78 (m, 6H).

[0314] ((2 S )-1-(((2 S 1-Hydroxy-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate (perfluorophenyl) methyl ester d 2 (22a)。 Yield (11%). 1H NMR (400 MHz, DMSO-d6) δ7.65 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 4.64 (s, 1H), 3.99 – 3.90 (m, 1H), 3.75 (s, 1H), 3.25 – 3.02 (m, 4H), 2.26 – 2.16 (m, 1H), 2.15 – 2.05 (m, 2H), 1.81 – 1.71 (m, 1H), 1.60 – 1.49 (m, 2H), 1.46 – 1.31 (m, 2H), 0.90 – 0.77 (m, 6H).

[0315] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate-1-phenylbutyl ester (23a)。 Yield (60%). 1 H NMR (400 MHz, CDCl3) δ 7.59(dd, J = 79.1, 7.3 Hz, 1H), 7.36 – 7.21 (m, 5H), 6.24 (d, J = 37.3 Hz, 1H), 5.60 (t, J = 7.1, 7.1 Hz, 1H), 5.41 (dd, J = 23.4, 8.0 Hz, 1H), 4.21 – 4.11 (m,1H), 4.04 – 3.89 (m, 1H), 3.69 – 3.47 (m, 2H), 3.36 – 3.19 (m, 2H), 2.53 –2.17 (m, 2H), 2.03 – 1.80 (m, 3H), 1.79 – 1.42 (m, 3H), 1.41 – 1.21 (m, 2H), 0.98 – 0.83 (m, 11H).

[0316] ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)-4-methyl-1-oxopent-2-yl)carbamate 1,2-diphenylethyl carbamate (24a)。 Yield (83%). 1 H NMR (400 MHz, CDCl3) δ7.58 (dd,J = 49.0, 7.4 Hz, 1H), 7.33 – 7.16 (m, 8H), 7.11 – 7.02 (m, 2H), 6.09 (d, J = 26.6 Hz, 1H), 5.88 – 5.75 (m, 1H), 5.42 – 5.32 (m, 1H), 4.17 –4.07 (m, 1H), 4.04 – 3.87 (m, 1H), 3.68 – 3.43 (m, 2H), 3.33 – 2.98 (m, 4H), 2.52 – 2.21 (m, 2H), 2.02 – 1.39 (m, 6H), 0.94 – 0.79 (m, 6H).

[0317] Preparation of compound 1-24b. General procedure. The dipeptide alcohol is maintained at 0-5°C under a nitrogen atmosphere. a Add (1 equivalent) Dys-Martin periodinane reagent (3.0 equivalent) to a solution of anhydrous dichloromethane (300 mL / g dipeptide alcohol) and stir the reaction mixture at 15–20 °C for 3 h. Wash the organic phase with 10% Na₂S₂O₃ aqueous solution (2 x 100 mL / g dipeptide alcohol), then with saturated NaHCO₃ aqueous solution (2 x 100 mL / g dipeptide alcohol), distilled water (2 x 100 mL / g dipeptide alcohol), and brine (100 mL / g dipeptide alcohol). Dry the organic phase on anhydrous Na₂SO₄, filter, and concentrate under vacuum. Purify the crude product by rapid chromatography (hexane / ethyl acetate) to obtain an aldehyde. b It is a white solid. The general reaction scheme is as follows: Figure 10B As shown.

[0318] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate 4,4-dimethylcyclohexyl ester (1b)。 Yield (80%). 1 H NMR (400 MHz, DMSO-d6) δ9.40 (s, 1H), 8.39 (d, J = 7.7 Hz, 1H), 7.62 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H),4.49 – 4.40 (m, 1H), 4.24 – 4.14 (m, 1H), 4.07 – 3.97 (m, 1H), 3.16 (t, J=9.2 Hz, 1H), 3.12 – 3.00 (m, 1H), 2.31 – 2.22 (m, 1H), 2.19 – 2.09 (m, 1H), 1.95 – 1.83 (m, 1H), 1.74 – 1.55 (m, 5H), 1.54 – 1.31 (m, 6H), 1.25 – 1.15 (m, 2H), 0.92 – 0.81 (m, 12H).

[0319] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (1r,4S)-4-isopropylcyclohexyl ester (2b)。 Yield (78%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.40 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.19 (d, J =8.0 Hz, 1H), 4.42 – 4.30 (m, 1H), 4.19 (ddd, J = 11.4, 7.6, 4.2 Hz, 1H), 4.07– 3.96 (m, 1H), 3.28 – 3.01 (m, 2H), 2.35 – 2.19 (m, 1H), 2.19 – 2.08 (m,1H), 1.95 – 1.84 (m, 3H), 1.73 – 1.57 (m, 5H), 1.53 – 1.36 (m, 3H), 1.27 –1.20 (m, 2H), 1.02 (s, 3H), 0.97 – 0.80 (m, 12H).

[0320] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (1S,4S)-4-propylcyclohexyl ester (3b)。 Yield (73%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.40 (d, J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.19 (d, J = 7.9Hz, 1H), 4.37 (td,J = 11.0, 5.5 Hz, 1H), 4.24 – 4.12 (m, 1H), 4.07 – 3.97 (m,1H), 3.21 – 3.01 (m, 2H), 2.35 – 2.22 (m, 1H), 2.19 – 2.08 (m, 1H), 1.95 – 1.85 (m, 3H), 1.76 – 1.55 (m, 5H), 1.54 – 1.37 (m, 2H), 1.35 – 1.19 (m, 4H), 1.19 – 1.09 (m, 3H), 1.01 – 0.91 (m, 2H), 0.91 – 0.81 (m, 9H).

[0321] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (1S,4S)-4-butylcyclohexyl ester (4b)。 Yield (65%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.40 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.19 (d, J = 7.9Hz, 1H), 4.37 (td, J = 11.0, 5.3 Hz, 1H), 4.19 (ddd, J = 11.5, 7.7, 4.1 Hz,1H), 4.02 (q, J = 8.5 Hz, 1H), 3.17 (t, J = 9.1 Hz, 1H), 3.13 – 3.01 (m, 1H), 2.32 – 2.23 (m, 1H), 2.20 – 2.08 (m, 1H), 1.96 – 1.84 (m, 3H), 1.76 – 1.72(m, 3H), 1.72 – 1.55 (m, 3H), 1.53 – 1.37 (m, 2H), 1.29 – 1.13 (m, 8H), 1.01 – 0.91 (m, 2H), 0.91 – 0.80 (m, 9H).

[0322] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (1r,4S)-4-phenylcyclohexyl ester (5b)。 Yield (63%). 1 H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.43 (d, J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.32 – 7.10 (m,6H), 4.53 (td, J = 10.9, 5.4 Hz, 1H), 4.20 (ddd, J = 11.4, 7.5, 4.0 Hz, 1H),4.07 – 3.92 (m, 1H), 3.29 – 3.02 (m, 2H), 2.36 – 2.23 (m, 1H), 2.21 – 2.10(m, 1H), 2.07 – 1.96 (m, 2H), 1.96 – 1.85 (m, 1H), 1.81 (d, J = 12.8 Hz, 2H),1.70 – 1.56 (m, 6H), 1.56 – 1.42 (m, 4H), 0.89 (dd, J = 9.5, 6.6 Hz, 6H).

[0323] ((2 S )-4-methyl-1-oxo-1-(((2) S 1-Oxo-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (4-(trifluoromethyl)cyclohexyl)methyl ester (6b)。 Yield (88%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.29 (d, J =8.0 Hz, 1H), 4.22 – 4.15 (m, 1H), 4.07 – 3.99 (m, 1H), 3.78 (d, J= 2.0 Hz,2H), 3.20 – 3.05 (m, 3H), 2.31 – 2.08 (m, 3H), 1.95 – 1.76 (m, 4H), 1.70 –1.40 (m, 5H), 1.29 – 1.16 (m, 1H), 1.02 (q, J = 13.0 Hz, 4H), 0.92 – 0.81 (m, 6H).

[0324] ((2 S )-4-methyl-1-oxo-1-(((2) S )-1-oxo-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamic acid((1 r 4 S 4-(trifluoromethyl)cyclohexyl)methyl ester 7b Yield (91%). 1 H NMR (400MHz, DMSO-d6) δ 9.40 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 4.24 – 4.14 (m, 1H), 4.08 – 3.99 (m, 1H), 3.98 – 3.92 (m,2H), 3.21 – 3.04 (m, 3H), 2.33 – 2.20 (m, 3H), 2.19 – 2.08 (m, 1H), 1.92 – 1.83 (m, 4H), 1.69 – 1.38 (m, 9H), 0.92 – 0.81 (m, 6H).

[0325] ((2 S )-4-methyl-1-oxo-1-(((2) S )-1-oxo-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamic acid((1 r 4 R 4-propylcyclohexyl)methyl ester 8b Yield (42%). 1 H NMR (400 MHz, DMSO-6) δ 9.40 (s, 1H), 8.42 (d, J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J=8.0 Hz, 1H), 4.22 – 4.15 (m, 1H), 4.05 – 4.01 (m, 1H), 3.78 – 3.74 (m, 2H), 3.20 – 3.05 (m, 2H), 2.19 – 2.10 (m, 3H), 1.94 – 1.85 (m, 3H), 1.75 – 1.60 (m, 10H), 1.54 – 1.40 (m, 2H), 1.33 – 1.23 (m, 2H), 1.17 – 1.10 (m, 2H), 0.97 – 0.81 (m, 9H).

[0326] ((2 S )-4-methyl-1-oxo-1-(((2) S )-1-oxo-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamic acid((1 s 4 R 4-propylcyclohexyl)methyl ester d 2 ( 9b Yield (96%). 1 H NMR (400MHz, DMSO-d6) δ 9.40 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 4.24 – 4.14 (m, 1H), 4.08 – 3.97 (m, 1H), 3.21 – 3.03 (m,4H), 2.31 – 2.10 (m, 4H), 1.75 – 1.67 (m, 4H), 1.55 – 1.35 (m, 8H), 1.35 –1.22 (m, 2H), 1.18 – 1.10 (m, 2H), 1.00 – 0.76 (m, 9H).

[0327] (( S )-4-methyl-1-oxo-1-((( S )-1-oxo-3-(( S 4,4-Difluorocyclohexyl)methyl 2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (10b)。 Yield (51%). 1 H NMR (400 MHz, DMSO- d 6) δ 9.40 (s, 1H), 8.44 (d,J = 7.5 Hz, 1H), 7.95 (s, 1H), 7.36 – 7.28 (m,1H), 4.19 (ddd, J = 11.4, 7.6, 4.2 Hz, 1H), 4.03 (td, J = 8.8, 6.2 Hz, 1H), 3.84 (d, J = 6.1 Hz, 2H), 3.24 – 3.02 (m, 2H), 2.37 – 2.08 (m, 2H), 2.08 –1.94 (m, 2H), 1.94 – 1.80 (m, 1H), 1.80 – 1.55 (m, 7H), 1.55 – 1.33 (m, 2H), 1.33 – 1.11 (m, 3H), 0.97 – 0.79 (m, 6H). HRMS m / z: [M+H] + C 21 H 34 Calculated value of F₂N₃O₅: 446.2467; Measured value: 446.2452, m / z: [M+Na] + C 21 H 33 Calculated value of F2N3O5Na: 468.2286; Measured value: 468.2281.

[0328] ((2 S )-4-methyl-1-oxo-1-(((2) S )-1-oxo-3-(2-oxopyrrolidone-3-yl)prop-2-yl)amino)pent-2-yl)carbamate (4,4-difluorocyclohexyl)methyl ester- d 2( 11b Yield (63%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.44 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.32 (d, J=8.1 Hz, 1H), 4.23 – 4.15 (m, 1H), 4.07 – 3.99 (m, 1H), 3.21 – 3.05 (m, 2H), 2.32 – 2.20 (m, 1H), 2.19 – 2.10 (m, 2H), 2.05 – 1.95 (m, 2H), 1.79 – 1.58 (m, 8H), 1.55 – 1.40 (m, 2H), 1.30 – 1.15 (m, 2H), 0.92 – 0.83 (m, 6H).

[0329] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate 1-(4,4-difluorocyclohexyl)ethyl ester (12b)。 Yield (70%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.46 – 8.36 (m, 1H), 7.63 (s, 1H), 7.23 (d, J = 8.2Hz, 1H), 4.59 – 4.52 (m, 1H), 4.23 – 4.18 (m, 1H), 4.06 – 3.98 (m, 1H), 3.21– 3.03 (m, 2H), 2.32 – 2.22 (m, 1H), 2.18 – 2.09 (m, 1H), 2.06 – 1.95 (m,3H), 1.95 – 1.76 (m, 2H), 1.72 – 1.59 (m, 5H), 1.55 – 1.40 (m, 2H), 1.31 –1.14 (m, 3H), 1.12 (d, J = 6.3 Hz, 3H), 0.92 – 0.81 (m, 6H).

[0330] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate 2-(4,4-difluorocyclohexyl)propyl-2-ester (13b)。 Yield (42%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.35 (d, J= 7.7 Hz, 1H), 7.70 – 7.60 (m, 1H), 7.06(d, J = 8.1 Hz, 1H), 4.02 – 3.89 (m, 2H), 3.21 – 3.00 (m, 2H), 2.31 – 2.11 (m,1H), 2.07 – 1.83 (m, 4H), 1.82 – 1.74 (m, 4H), 1.69 – 1.58 (m, 4H), 1.56 –1.36 (m, 3H), 1.34 (s, 6H), 1.30 – 1.23 (m, 1H), 0.92 – 0.81 (m, 6H).

[0331] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate 1-(4,4-difluorocyclohexyl)-2-phenylethyl ester (14b)。 Yield (80%). 1 H NMR (400MHz, DMSO-d6) δ 9.39 (s, 1H), 8.38 – 8.30 (m, 1H), 7.62 (s, 1H), 7.26 – 7.15(m, 5H), 4.77 – 4.73 (m, 1H), 4.22 – 4.18 (m, 1H), 3.93 – 3.87 (m, 1H), 3.18 – 2.99 (m, 2H), 2.92 – 2.83 (m, 1H), 2.78 – 2.68 (m, 1H), 2.32 – 2.18 (m,1H), 2.16 – 2.07 (m, 1H), 2.07 – 1.94 (m, 2H), 1.91 – 1.80 (m, 3H), 1.79 –1.68 (m, 1H), 1.67 – 1.41 (m, 7H), 1.41 – 1.27 (m, 3H), 0.92 – 0.80 (m, 4H), 0.75 (dd, J = 11.6, 6.5 Hz, 2H).

[0332] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (4,4-difluorocyclohexyl)(phenyl)methyl ester (15b)。 Yield (63%). 1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.48 – 8.35 (m, 1H), 7.65 (d, J = 5.4 Hz, 1H), 7.52 –7.43 (m, 1H), 7.39 – 7.20 (m, 5H), 4.22 – 4.17 (m, 1H), 4.00 – 3.96 (m, 1H), 3.85 (d, J = 6.0 Hz, 1H), 3.23 – 3.05 (m, 2H), 2.32 – 2.22 (m, 1H), 2.17 –2.13 (m, 1H), 2.05 – 1.87 (m, 3H), 1.84 – 1.72 (m, 2H), 1.70 – 1.59 (m, 4H), 1.55 – 1.38 (m, 3H), 1.33 – 1.16 (m, 3H), 0.97 – 0.74 (m, 6H).

[0333] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate 1-(4,4-difluorocyclohexyl)pentyl ester (16b)。 Yield (88%). 1 H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.32 (d, J =8.0 Hz, 1H), 4.24 – 4.14 (m, 1H), 4.05 – 4.00 (m, 1H), 3.87 – 3.81 (m, 1H), 3.21 – 3.04 (m, 2H), 2.30 – 2.22 (m, 1H), 2.19 – 2.08 (m, 1H), 2.06 – 1.94 (m, 3H), 1.94 – 1.80 (m, 3H), 1.79 – 1.71 (m, 5H), 1.69 – 1.58 (m, 4H), 1.55 – 1.41 (m, 3H), 1.26 – 1.18 (m, 3H), 0.92 – 0.81 (m, 9H).

[0334] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate-2-fluorobenzyl ester (17b)。 Yield (68%). 1 H NMR (400 MHz, CDCl3) δ 9.48 (s,1H), 8.35 (d, J = 5.9 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.35 – 7.27 (m, 1H), 7.18 – 7.00 (m, 2H), 6.02 (s, 1H), 5.38 (d, J = 8.5 Hz, 1H), 5.22 – 5.16 (m,2H), 4.44 – 4.26 (m, 2H), 3.39 – 3.27 (m, 2H), 2.49 – 2.30 (m, 2H), 2.01 –1.92 (m, 2H), 1.91 – 1.80 (m, 1H), 1.81 – 1.45 (m, 3H), 0.97 (d, J = 5.8 Hz, 6H). HRMS m / z: [M+Na] + C 21 H 28 Calculated value of FN3NaO5: 444.1911, measured value: 444.1907.

[0335] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate-3-fluorobenzyl ester (18b)。 Yield (68%). 1 H NMR (400 MHz, CDCl3) δ 9.48 (s,1H), 8.45 (d, J = 5.7 Hz, 1H), 7.35 – 7.24 (m, 1H), 7.13 – 7.05 (m, 2H), 7.05– 6.94 (m, 1H), 6.18 (s, 1H), 5.48 (d, J= 8.6 Hz, 1H), 5.16 – 5.03 (m, 2H), 4.41 – 4.26 (m, 2H), 3.41 – 3.27 (m, 2H), 2.56 – 2.29 (m, 2H), 2.00 – 1.79(m, 2H), 1.77 – 1.64 (m, 3H), 1.60 – 1.51 (m, 1H), 0.97 (d, J = 6.0 Hz, 6H). HRMS m / z: [M+Na] + C 21 H 28 Calculated value of FN3NaO5: 444.1911, Measured value: 444.1911.

[0336] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate-4-fluorobenzyl ester (19b)。 Yield (60%). 1 H NMR (400 MHz, CDCl3) δ 9.48 (s,1H), 8.36 (d, J = 5.7 Hz, 1H), 7.38 – 7.29 (m, 2H), 7.09 – 6.98 (m, 2H), 6.00(s, 1H), 5.36 (d, J = 8.6 Hz, 1H), 5.11 – 5.04 (m, 2H), 4.35 – 4.28 (m, 2H), 3.41 – 3.28 (m, 2H), 2.50 – 2.31 (m, 2H), 1.97 – 1.93 (m, 1H), 1.90 – 1.76(m, 1H), 1.74 – 1.65 (m, 3H), 1.59 – 1.51 (m, 1H), 0.96 (d, J = 6.0 Hz, 6H). HRMS m / z: [M+H] + C 21 H 29 Calculated FN3O5: 422.2091, Measured: 422.2085. HRMS m / z: [M+Na] + C 21 H 28 Calculated value of FN3NaO5: 444.1911, measured value: 444.1903.

[0337] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate (4-fluorophenyl)methyl d2 (20b)。 Yield (75%). 1 H NMR (400 MHz, CDCl3) δ9.48 (s, 1H), 8.36 (d, J = 5.7 Hz, 1H), 7.37 – 7.28 (m, 2H), 7.08 – 6.98 (m,2H), 6.06 (s, 1H), 5.38 (d, J = 8.6 Hz, 1H), 4.39 – 4.14 (m, 2H), 3.41 – 3.30(m, 2H), 2.48 – 2.29 (m, 2H), 2.01 – 1.92 (m, 1H), 1.92 – 1.80 (m, 1H), 1.80– 1.62 (m, 3H), 1.59 – 1.46 (m, 1H), 0.96 (d, J = 5.9 Hz, 6H). HRMS m / z: [M+H] + C 21 H 27 Calculated D₂FN₃O₅ value: 424.2217, measured value: 424.2210. HRMS m / z: [M+Na] + C 21 H 26 Calculated value of D2FN3NaO5: 446.2037, measured value: 446.2027.

[0338] ((2 S )-4-methyl-1-oxo-1-(((2) S 1-O-3-(2-O-pyrrolidone-3-yl)prop-2-yl)amino)pent-2-yl)carbamate (perfluorophenyl) methyl ester (21b)。 Yield (86%). 1 H NMR (400 MHz, DMSO-d6) δ9.39 (s, 1H), 8.47 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.59 (d, J= 7.8 Hz, 1H),5.14 (s, 2H), 4.23 – 4.14 (m, 1H), 4.07 – 3.98 (m, 1H), 3.22 – 3.03 (m, 2H),2.33 – 2.20 (m, 1H), 2.17 – 2.06 (m, 2H), 1.93 – 1.83 (m, 1H), 1.70 – 1.56 (m, 2H), 1.51 – 1.39 (m, 2H), 0.91 – 0.80 (m, 6H).

[0339] ((2S)-4-methyl-1-oxo-1-(((2S)-1-oxo-3-(2-oxopyrrolidone-3-yl)propyl-2-yl)amino)pent-2-yl)carbamate (perfluorophenyl)methyl ester- d 2 ( 22b Yield (83%). 1 H NMR (400 MHz, DMSO-d6)δ 9.38 (s, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.58 (d, J = 7.8 Hz,1H), 4.22 – 4.14 (m, 1H), 4.06 – 3.99 (m, 1H), 3.20 – 3.05 (m, 2H), 2.27 (d, J = 7.9 Hz, 1H), 2.16 – 2.07 (m, 2H), 1.92 – 1.83 (m, 1H), 1.68 – 1.58 (m, 2H), 1.50 – 1.41 (m, 2H), 0.90 – 0.80 (m, 6H).

[0340] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate-1-phenylbutyl ester (23b)。 Yield (71%). 1 H NMR (400 MHz, CDCl3) δ 9.44(d, J = 36.5 Hz, 1H), 8.21 (dd, J = 43.6, 6.1 Hz, 1H), 7.41 – 7.17 (m, 5H), 6.28 (d, J= 30.3 Hz, 1H), 5.68 – 5.54 (m, 1H), 5.36 (dd, J = 26.5, 8.5 Hz,1H), 4.40 – 4.19 (m, 2H), 3.41 – 3.15 (m, 2H), 2.56 – 2.14 (m, 2H), 2.01 –1.81 (m, 3H), 1.77 – 1.62 (m, 3H), 1.59 – 1.44 (m, 1H), 1.42 – 1.25 (m, 1H), 1.00 – 0.77 (m, 11H).

[0341] ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolid-3-yl)propyl-2-yl)amino)pentan-2-yl)carbamate 1,2-diphenylethyl carbamate (24b)。 Yield (82%). 1 H NMR (400 MHz, DMSO-d6) δ9.35 (dd, J = 24.5, 7.6 Hz, 1H), 8.48 – 8.32 (m, 1H), 7.61 (d, J = 21.6 Hz,1H), 7.53 – 7.42 (m, 1H), 7.36 – 7.09 (m, 10H), 5.83 – 5.71 (m, 1H), 4.24 –4.08 (m, 1H), 4.06 – 3.89 (m, 1H), 3.24 – 2.86 (m, 4H), 2.34 – 2.05 (m, 3H), 1.94 – 1.78 (m, 1H), 1.67 – 1.28 (m, 4H), 0.90 – 0.68 (m, 6H).

[0342] Preparation of compound 1-24c. General procedure. Stirring is used to add dipeptide aldehyde. b Anhydrous ethanol (5 mL / g dipeptide aldehyde) was added to a solution of sodium bisulfite (1 equivalent) in ethyl acetate (10 mL / g dipeptide aldehyde), followed by a solution of sodium bisulfite (1 equivalent) in water (1 mL / g dipeptide aldehyde). The reaction mixture was stirred at 50 °C for 3 h. The reaction mixture was cooled to room temperature and then filtered under vacuum. The solid was thoroughly washed with anhydrous ethanol, and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid. The white solid was stirred with anhydrous diethyl ether (3 x 10 mL / g dipeptide aldehyde), and the solvent was carefully removed using a pipette and dried under vacuum for 2 h to give the dipeptide bisulfite adduct.c It is a white solid. The general reaction scheme is as follows: Figure 10B As shown.

[0343] (2S)-2-((S)-2-((((4,4-dimethylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (1c) Yield (39%). 1 H NMR (400 MHz, DMSO-d6) δ 7.50 (dd, J = 13.4, 9.1 Hz, 1H), 7.44 (s, 1H), 7.21 – 7.11 (m, 1H), 5.45 – 5.27 (m, 1H), 4.53 – 4.39 (m, 1H), 4.01 – 3.92 (m, 1H), 3.95 – 3.78(m, 1H), 3.14 – 3.08 (m, 1H), 3.06 – 3.01 (m, 1H), 2.19 – 2.05 (m, 1H), 2.04– 1.86 (m, 1H), 1.70 – 1.65 (m, 3H), 1.62 – 1.52 (m, 3H), 1.52 – 1.31 (m, 6H), 1.28 – 1.09 (m, 2H), 0.92 – 0.80 (m, 12H).

[0344] (2S)-1-hydroxy-2-((S)-2-((((1r,4S)-4-isopropylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (2c) Yield (36%). 1H NMR (400MHz, DMSO-d6) δ 7.54 – 7.42 (m, 2H), 7.23 – 7.11 (m, 1H), 5.45 – 5.28 (m,1H), 4.41 – 4.30 (m, 1H), 4.00 – 3.89 (m, 1H), 3.89 – 3.78 (m, 1H), 3.17 –3.09 (m, 1H), 3.08 – 2.97 (m, 1H), 2.14 – 2.04 (m, 2H), 2.01 – 1.84 (m, 3H),1.73 – 1.65 (m, 2H), 1.62 – 1.51 (m, 3H), 1.48 – 1.34 (m, 3H), 1.30 – 1.14 (m, 2H), 1.01 (s, 3H), 0.89 – 0.80 (m, 12H).

[0345] (2S)-1-hydroxy-2-((S)-4-methyl-2-((((1S,4S)-4-propylcyclohexyl)oxy)carbonyl)amino)pentamido)-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (3c) Yield (40%). 1 H NMR (400 MHz, DMSO-d6) δ 7.54 – 7.39 (m, 2H), 7.23 – 7.10 (m, 1H), 5.45 – 5.28 (m, 1H), 4.42 – 4.32 (m, 1H), 4.03 – 3.88 (m, 1H), 3.89 – 3.78 (m, 1H), 3.18 – 3.08(m, 1H), 3.07 – 2.97 (m, 1H), 2.19 – 2.03 (m, 2H), 1.97 – 1.80 (m, 3H), 1.71(d, J = 13.2 Hz, 2H), 1.59 – 1.56 (m, 3H), 1.48 – 1.37 (m, 2H), 1.34 – 1.02(m, 7H), 0.97 – 0.89 (m, 2H), 0.92 – 0.80 (m, 9H).

[0346] (2S)-2-((S)-2-((((1s,4S)-4-butylcyclohexyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (4c)Yield (33%). 1 H NMR (400MHz, DMSO-d6) δ 7.55 – 7.42 (m, 2H), 7.22 – 7.12 (m, 1H), 5.48 – 5.30 (m,1H), 4.42 – 4.32 (m, 1H), 3.97 – 3.89 (m, 1H), 3.88 – 3.79 (m, 1H), 3.17 –3.08 (m, 1H), 3.07 – 2.98 (m, 1H), 2.19 – 2.02 (m, 2H), 1.99 – 1.83 (m, 3H),1.72 (d, J = 13.3 Hz, 3H), 1.60 – 1.55 (m, 3H), 1.48 – 1.37 (m, 2H), 1.27 –1.17 (m, 6H), 1.17 – 1.13 (m, 3H), 0.97 – 0.92 (m, 1H), 0.90 – 0.80 (m, 9H).

[0347] (2S)-1-hydroxy-2-((S)-4-methyl-2-((((1r,4S)-4-phenylcyclohexyl)oxy)carbonyl)amino)pentamido)-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (5c) Yield (35%). 1 H NMR (400 MHz, DMSO-d6) δ 7.57 – 7.47 (m, 1H), 7.44 (s, 1H), 7.32 – 7.10 (m, 6H), 5.52 –5.22 (m, 1H), 4.55 – 4.50 (m, 1H), 4.06 – 3.89 (m, 1H), 3.89 – 3.77 (m, 1H), 3.15 – 3.10 (m, 1H), 3.09 – 3.00 (m, 1H), 2.14 – 2.09 (m, 2H), 2.05 – 1.97(m, 2H), 1.81 (d, J = 13.1 Hz, 3H), 1.62 – 1.52 (m, 5H), 1.50 – 1.39 (m, 4H), 1.14 – 1.03 (m, 1H), 0.93 – 0.81 (m, 6H).

[0348] (2S)-1-hydroxy-2-((S)-4-methyl-2-((((4-(trifluoromethyl)cyclohexyl)methoxy)carbonyl)amino)pentamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium( 6c Yield (81%). 1 H NMR (400 MHz, DMSO-d6) δ 7.62 – 7.49 (m, 1H), 7.46 (s, 1H), 7.25 – 7.13 (m, 1H), 5.50 (d, J = 6.3 Hz, 1H), 5.34 (d, J = 6.0 Hz, 1H), 4.41 – 4.33 (m, 1H), 4.27 – 4.18 (m,1H), 3.81 – 3.70 (m, 2H), 3.13 (s, 2H), 3.08 – 2.99 (m, 1H), 2.20 – 2.06 (m,3H), 1.91 – 1.76 (m, 4H), 1.63 – 1.57 (m, 4H), 1.49 – 1.37 (m, 1H), 1.27 –1.16 (m, 1H), 1.13 – 0.92 (m, 4H), 0.93 – 0.80 (m, 6H).

[0349] (2S)-1-hydroxy-2-((S)-4-methyl-2-(((((1) r 4 S )-4-(trifluoromethyl)cyclohexyl)methoxy)carbonyl)amino)pentamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium( 7c Yield (76%). 1 H NMR(400 MHz, DMSO-d6) δ 7.58 – 7.50 (m, 1H), 7.46 (d, J = 9.6 Hz, 1H), 7.25 –7.16 (m, 1H), 5.41 (d, J = 6.3 Hz, 1H), 5.28 (d, J= 5.9 Hz, 1H), 4.45 – 4.32(m, 1H), 4.00 – 3.90 (m, 2H), 3.50 – 3.25 (m, 1H), 3.08 – 3.01 (m, 2H), 2.31– 2.26 (m, 3H), 2.15 – 2.08 (m, 1H), 1.90 – 1.85 (m, 4H), 1.67 – 1.33 (m, 10H), 0.91 – 0.81 (m, 6H).

[0350] (2S)-1-hydroxy-2-((S)-4-methyl-2-(((((1) r 4 R )-4-propylcyclohexyl)methoxy)carbonyl)amino)pentamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium( 8c Yield (82%). 1 H NMR (400MHz, DMSO-d6) δ 7.53 – 7.40 (m, 1H), 7.20 – 7.09 (m, 1H), 6.10 – 6.04 (m,1H), 5.41 (d, J = 6.2 Hz, 1H), 5.29 (d, J = 6.0 Hz, 1H), 4.43 – 4.32 (m, 1H), 4.00 – 3.89 (m, 1H), 3.74 (s, 2H), 3.16 – 3.01 (m, 2H), 2.15 – 2.05 (m, 6H), 1.71 (d, J = 11.4 Hz, 10H), 1.53 – 1.36 (m, 2H), 1.35 – 1.22 (m, 2H), 1.19 –1.03 (m, 2H), 0.98 – 0.77 (m, 9H).

[0351] (2S)-1-hydroxy-2-((S)-4-methyl-2-(((((1) s 4 S )-4-propylcyclohexyl)methoxy- d 2) Carbonyl) Amino) Pentamamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt ( 9c Yield (77%). 1H NMR (400MHz, DMSO-d6) δ 7.57 – 7.42 (m, 1H), 7.27 – 7.09 (m, 1H), 6.07 (d, J = 10.0Hz, 1H), 5.40 (d, J = 6.4 Hz, 1H), 5.29 (d, J = 6.0 Hz, 1H), 3.99 – 3.89 (m,1H), 3.78 – 3.69 (m, 1H), 3.17 – 2.98 (m, 4H), 2.20 – 2.06 (m, 4H), 1.71 (d, J = 11.6 Hz, 4H), 1.49 – 1.35 (m, 8H), 1.35 – 1.22 (m, 2H), 1.18 – 1.09 (m,2H), 0.90 – 0.80 (m, 9H).

[0352] (2S)-2-((S)-2-((((4,4-difluorocyclohexyl)methoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (10c) Yield (50.5%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.57 (t, J = 8.9 Hz, 1H), 7.45 (s, 1H), 7.38 – 7.17 (m, 1H), 4.29 –4.10 (m, 1H), 4.05 – 3.67 (m, 4H), 3.09 (dt, J = 29.8, 8.8 Hz, 2H), 2.33 –2.05 (m, 2H), 2.05 – 1.88 (m, 4H), 1.88 – 1.64 (m, 5H), 1.64 – 1.48 (m, 2H),1.43 (q, J = 7.3 Hz, 2H), 1.30 – 1.11 (m, 2H), 1.04 – 0.78 (m, 6H).

[0353] (2S)-2-((S)-2-((((4,4-difluorocyclohexyl)methoxy- d 2) Carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt(11c) Yield (81%). 1 H NMR (400MHz, DMSO-d6) δ 7.60 – 7.50 (m, 1H), 7.45 (s, 1H), 7.29 – 7.18 (m, 1H), 5.41(d, J = 6.3 Hz, 1H), 5.24 (d, J = 6.0 Hz, 1H), 4.37 – 4.32 (m, 1H), 3.98 – 3.89(m, 1H), 3.15 – 3.02 (m, 2H), 2.14 – 2.05 (m, 3H), 2.01 – 1.96 (m, 1H), 1.84– 1.70 (m, 8H), 1.62 – 1.53 (m, 4H), 1.45 – 1.38 (m, 1H), 0.90 – 0.80 (m, 6H).

[0354] (2S)-2-((2S)-2-(((1-(4,4-difluorocyclohexyl)ethoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (12c) Yield (48%). 1 H NMR (400MHz, DMSO-d6) δ 7.52 (d, J = 9.9 Hz, 1H), 7.43 (s, 1H), 7.28 – 7.13 (m, 1H), 5.36 – 5.17 (m, 1H), 4.58 – 4.51 (m, 1H), 3.96 – 3.91 (m, 1H), 3.83 – 3.78(m, 1H), 3.18 – 3.09 (m, 1H), 3.06 – 3.01 (m, 1H), 2.17 – 1.88 (m, 3H), 1.87 – 1.77 (m, 4H), 1.74 – 1.66 (m, 2H), 1.65 – 1.51 (m, 3H), 1.48 – 1.35 (m,2H), 1.30 – 1.16 (m, 3H), 1.16 – 1.04 (m, 3H), 0.89 – 0.80 (m, 6H).

[0355] (2S)-2-((S)-2-((((2-(4,4-difluorocyclohexyl)prop-2-yl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (13c) Yield (39%). 1 H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 10.6 Hz, 1H), 7.43 (s, 1H), 7.12 – 6.95 (m,1H), 5.47 – 5.26 (m, 1H), 4.06 – 3.71 (m, 2H), 3.17 – 3.08 (m, 1H), 3.08 –2.97 (m, 1H), 2.14 – 1.93 (m, 6H), 1.86 – 1.65 (m, 5H), 1.64 – 1.49 (m, 2H), 1.44 – 1.36 (m, 2H), 1.33 (s, 6H), 1.28 – 1.23 (m, 2H), 0.84 (ddd, J = 11.6, 6.5, 3.0 Hz, 6H).

[0356] (2S)-2-((2S)-2-(((1-(4,4-difluorocyclohexyl)-2-phenylethoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (14c) Yield (41%). 1 H NMR (400 MHz, DMSO-d6) δ 7.53 – 7.47 (m, 1H), 7.43 (s, 1H), 7.39 – 7.33 (m, 1H), 7.28 – 7.15 (m, 5H), 5.38 – 5.15 (m, 1H), 4.75 – 4.71 (m, 1H), 3.96 – 3.91(m, 1H), 3.87 – 3.68 (m, 1H), 3.13 – 3.09 (m, 2H), 3.07 – 2.96 (m, 1H), 2.90 – 2.82 (m, 1H), 2.00 (s, 4H), 1.92 – 1.80 (m, 3H), 1.76 (s, 3H), 1.59 – 1.51 (m, 4H), 1.49 – 1.33 (m, 3H), 0.89 – 0.79 (m, 4H), 0.79 – 0.67 (m, 2H).

[0357] (2S)-2-((2S)-2-((((4,4-difluorocyclohexyl)(phenyl)methoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (15c) Yield (66%). 1 H NMR(400 MHz, DMSO-d6) δ 7.64 – 7.50 (m, 1H), 7.49 – 7.44 (m, 1H), 7.42 – 7.19(m, 6H), 5.48 – 5.35 (m, 1H), 4.01 – 3.75 (m, 3H), 3.18 – 3.10 (m, 1H), 3.08 – 3.01 (m, 1H), 2.22 – 2.06 (m, 1H), 2.06 – 1.91 (m, 3H), 1.87 – 1.68 (m,4H), 1.61 – 1.54 (m, 3H), 1.49 – 1.40 (m, 3H), 1.35 – 1.14 (m, 3H), 0.93 –0.71 (m, 6H).

[0358] (2S)-2-((2S)-2-(((1-(4,4-difluorocyclohexyl)pentyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (16c) Yield (58%). 1 H NMR (400MHz, DMSO-d6) δ 7.60 – 7.51 (m, 1H), 7.44 (s, 1H), 7.38 – 7.23 (m, 1H), 5.46– 5.21 (m, 1H), 3.98 – 3.74 (m, 3H), 3.14 – 3.07 (m, 1H), 3.06 – 2.98 (m,1H), 2.16 – 2.04 (m, 2H), 2.04 – 1.92 (m, 4H), 1.86 – 1.66 (m, 8H), 1.64 –1.52 (m, 3H), 1.49 – 1.36 (m, 3H), 1.21 – 1.17 (m, 3H), 0.83 (ddd, J = 11.9, 6.5, 3.1 Hz, 9H).

[0359] (2S)-2-((S)-2-((((2-fluorobenzyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (17c) Yield (71%). 1 H NMR (400 MHz, DMSO-d6)δ 7.77 – 7.70 (m, 1H), 7.70 – 7.59 (m, 1H), 7.59 – 7.33 (m, 3H), 7.26 – 7.12(m, 2H), 5.65 (d, J = 78.6 Hz, 1H), 5.16 – 5.01 (m, 2H), 4.11 – 3.84 (m, 2H), 3.17 – 2.97 (m, 2H), 2.39 – 2.07 (m, 2H), 2.07 – 1.85 (m, 1H), 1.70 – 1.51 (m, 3H), 1.51 – 1.33 (m, 2H), 0.92 – 0.77 (m, 6H). HRMS m / z: [M] - C 21 H 29 Calculated value of FN3O8S: 502.1659, measured value: 502.1650. HRMS m / z: [M+Na] + C 21 H 29 Calculated value of FN3Na2O8S: 548.1455, Measured value: 548.1446.

[0360] (2S)-2-((S)-2-((((3-fluorobenzyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (18c) Yield (89%). 1 H NMR (400 MHz, DMSO-d6)δ 7.75 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 9.3 Hz, 1H), 7.57 (d, J= 7.9 Hz, 1H),7.54 – 7.35 (m, 2H), 7.25 – 7.08 (m, 2H), 5.75 – 5.46 (m, 1H), 5.15 – 4.98(m, 2H), 4.13 – 3.87 (m, 2H), 3.17 – 2.89 (m, 2H), 2.23 – 2.04 (m, 2H), 2.04 – 1.91 (m, 1H), 1.89 – 1.74 (m, 1H), 1.70 – 1.31 (m, 4H), 0.91 – 0.77 (m,6H). HRMS m / z: [M+Na] + C 21 H 29 Calculated value of FN3Na2O8S: 548.1455, Measured value: 548.1450. HRMS m / z: [M] - C 21 H 29 Calculated value of FN3O8S: 502.1659, measured value: 502.1655.

[0361] (2S)-2-((S)-2-((((4-fluorobenzyl)oxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (19c) Yield (68%). 1 H NMR (400 MHz, DMSO-d6)δ 7.70 – 7.57 (m, 1H), 7.57 – 7.49 (m, 1H), 7.48 – 7.37 (m, 3H), 7.23 – 7.14(m, 2H), 5.43 (d, J = 85.6 Hz, 1H), 5.09 – 4.92 (m, 2H), 4.12 – 3.81 (m, 2H), 3.14 – 2.94 (m, 2H), 2.23 – 1.91 (m, 2H), 1.62 – 1.50 (m, 4H), 1.50 – 1.38 (m, 2H), 0.90 – 0.80 (m, 6H). HRMS m / z: [M+Na] + C 21 H 29 Calculated value of FN3Na2O8S: 548.1455, measured value: 548.1448. HRMS m / z: [M] - C 21 H 29Calculated value of FN3O8S: 502.1659, measured value: 502.1645.

[0362] (2S)-2-((S)-2-(((((4-fluorophenyl)methoxy-d2)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (20c) Yield (90%). 1 H NMR (400 MHz, DMSO-d6) δ 7.68 (d, J = 9.2 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 7.8Hz, 1H), 7.48 – 7.35 (m, 2H), 7.23 – 7.13 (m, 2H), 5.52 (dd, J 0.92 – 0.78 (m, 6H). HRMS m / z: [M+Na] + C 21 H 27 Calculated value of D₂FN₃Na₂O₈S: 550.1581, Measured value: 550.1573. HRMS m / z: [M+Na] + C 21 H 27 Calculated value of D2FN3O8S: 504.1785, measured value: 504.1769.

[0363] (2S)-1-hydroxy-2-((S)-4-methyl-2-((((perfluorophenyl)methoxy)carbonyl)amino)pentamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium( 21c Yield (81%). 1 H NMR (400 MHz, DMSO-d6) δ7.68 – 7.42 (m, 2H), 6.06 (s, 1H), 5.41 (d, J = 6.3 Hz, 1H), 5.24 (d, J = 5.9Hz, 1H), 5.13 (s, 2H), 4.36 (d,J = 7.2 Hz, 1H), 4.01 – 3.89 (m, 1H), 3.48 –3.41 (m, 3H), 2.20 – 2.04 (m, 3H), 1.63 – 1.33 (m, 4H), 0.89 – 0.78 (m, 6H).

[0364] (2S)-1-hydroxy-2-((S)-4-methyl-2-((((perfluorophenyl)methoxy- d 2) Carbonyl) Amino) Pentamamido)-3-(2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt ( 22c Yield (89%). 1 H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J = 9.1 Hz, 1H), 7.58 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 5.12(d, J = 12.4 Hz, 1H), 4.42 – 4.36 (m, 1H), 3.99 – 3.90 (m, 2H), 3.16 – 3.00(m, 2H), 2.20 – 2.05 (m, 3H), 1.77 (s, 1H), 1.62 – 1.50 (m, 2H), 1.47 – 1.37 (m, 2H), 0.88 – 0.78 (m, 6H).

[0365] (2S)-1-hydroxy-2-((2S)-4-methyl-2-(((1-phenylbutoxy)carbonyl)amino)pentamido)-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (23c) Yield (63%). 1 H NMR (400 MHz, DMSO-d6)δ 7.60 (d, J = 9.8 Hz, 1H), 7.47 (d, J = 5.0 Hz, 1H), 7.41 (d, J = 7.4 Hz, 1H),7.38 – 7.22 (m, 5H), 5.54 (t, J= 6.6, 6.6 Hz, 1H), 5.37 – 5.25 (m, 1H), 4.12 – 3.78 (m, 2H), 3.17 – 2.95 (m, 2H), 2.31 – 1.88 (m, 3H), 1.85 – 1.38 (m,4H), 1.36 – 1.18 (m, 2H), 0.93 – 0.82 (m, 10H), 0.79 – 0.70 (m, 2H).

[0366] (2S)-2-((2S)-2-(((1,2-diphenylethoxy)carbonyl)amino)-4-methylpentamido)-1-hydroxy-3-((S)-2-oxopyrrolidone-3-yl)propane-1-sulfonate sodium salt (24c) Yield (69%). 1 H NMR (400 MHz, DMSO-d6) δ 7.69 – 7.36 (m, 2H), 7.36 – 7.07 (m, 11H), 5.82 – 5.71 (m, 1H), 5.59 –5.32 (m, 1H), 4.15 – 3.81 (m, 2H), 3.18 – 2.76 (m, 4H), 2.32 – 1.72 (m, 4H), 1.71 – 1.30 (m, 4H), 0.90 – 0.60 (m, 6H).

[0367] Enzyme assay and inhibition studies. Cloning and expression of SARS-CoV-2 3CL protease and assay of FRET enzyme. A codon-optimized cDNA of the full-length SARS-CoV-2 3CLpro, fused at the N-terminus with a sequence encoding 6 histidine residues, was synthesized via Integrated DNA (Coralville, IA) (GenBank ID MN908947.3). The synthesized gene was subcloned into the pET-28a(+) vector. SARS-CoV-2 3CLpro expression and purification were performed according to the previously described standard procedure. Briefly, an inhibitor stock solution was prepared in DMSO and diluted in assay buffer consisting of 20 mM HEPES buffer at pH 8 containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The SARS-CoV-2 protease was mixed with serially diluted inhibitors or with DMSO in 25 µL of assay buffer and incubated at 37 °C for 1 h, followed by the addition of 25 µL of assay buffer containing the substrate (FAM-SAVLQ / SG-QXL). ®520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage site on the SARS-CoV viral polyprotein. Fluorescence readings were obtained 1 h after substrate addition using a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) with excitation at 480 nm and emission at 520 nm. As previously described, relative fluorescence units (RFU) were determined by subtracting the background value (the substrate-containing wells without the protease) from the original fluorescence value. The IC50 of the compound was determined by fitting a variable slope to a dose-dependent FRET inhibition curve using GraphPad Prism software (GraphPad, La Jolla, CA). 50 Values. Expression and purification of MERS-CoV 3CLpro, and FRET enzyme assay, were performed as previously described.

[0368] Cell-based assays of antiviral activity were performed. Compounds were investigated. 2a and 3a Antiviral activity against SARS-CoV-2 replication. In short, confluent Vero E6 cells were seeded with SARS-CoV-2 at 50–100 plaque-forming units / well, and culture medium containing various concentrations of each compound and agar was applied to the cells. Plaques in each well were counted after 48–72 hours. The 50% effective concentration (EC50) was determined using variable slope (GraphPad, La Jolla, CA) with GraphPad Prism software. 50 )value.

[0369] Non-specific cytotoxicity / in vitro cytotoxicity. Confluent cells grown in 96-well plates were incubated with each compound at various concentrations (1 to 100 µM) for 72 hours. Cell cytotoxicity was measured using the CytoTox 96 Non-radioactive Cytotoxicity Assay Kit (Promega, Madison, WI), and CC was calculated using variable slopes via GraphPad Prism software. 50 Value. By using CC 50 Divide by EC 50 To calculate the in vitro safety index.

[0370] X-ray crystallography study. Crystallization and data collection. SARS-CoV-2 3CLpro purified in 100 mM NaCl, 20 mM Tris at pH 8.0 17Concentrate to 9.6 mg / mL (0.28 mM) for crystallization screening. All crystallization experiments were performed using an NT8 drop-setting robot (Formulatrix Inc.) and a UVXPO MRC (Molecular Dimensions) seated drop vapor diffusion plate at 18°C. Dispense 100 nL of protein and 100 nL of crystallization solution and equilibrate with 50 μL of the latter. Prepare a stock solution of 100 mM inhibitor in DMSO and prepare the SARS-CoV-2 3CLpro:inhibitor complex by mixing 1 µL of ligand (2 mM) with 49 µL (0.28 mM) SARS2 3CLpro and incubating on ice for 1 h. Crystals were obtained within 1–2 days for the following complexes under various conditions. 8b (NN-II-111) Complex HT screening (Molecular Dimensions) condition F7 (0.5 M ammonium sulfate, 100 mM MES, pH 6.5). 12b AMJ-I-106 13c (AMJ- I-114), 14c (AMJ-I-111) and 21c (NN-II-123) Index HT screening (Hampton Research) condition D10 (20% (w / v) PEG 5000 MME, 100 mM Bis-Tris pH 6.5). 19b (CSD-III-008) and 20b (CSD-III-009): Complex HT screening (Rigaku Reagents) condition C5 (20% (w / v) PEG 4000, 100 mM Tris pH 8.0). 1c (AMJ-I-158): Index HT screening (Hampton Research) condition F2 (20% (w / v) PEG 2000 MME, 100 mM Tris pH 8.5, 200 mM trimethylamine N-oxide dihydrate). 3c (AMJ-I-159): Index HT screening (Hampton Research) condition F5 (17% (w / v) PEG 10,000, 100 mM Bis-Tris pH 5.5, 100 mM ammonium acetate). 5c (AMJ-I-157): Index HT screening (Hampton Research) condition F1 (10% (w / v) PEG 3350, 100 mM Tris pH 7.5, 200 L-proline). 17c (CSD-III-028) and 18c (CSD- III-029) Index HT screening (Rigaku Reagents) condition H11 (30% (w / v) PEG 2000 MME, 100 mM potassium thiocyanate). Transfer the sample to a fresh droplet consisting of 80% crystallization solution and 20% (v / v) PEG 200 and store in liquid nitrogen. Before freezing, [the sample should have...] 8b Crystals of SARS-CoV-2 3CLpro were transferred to a cryoprotectant solution containing 80% crystallizing agent and 20% (v / v) glycerol. X-ray diffraction data were collected on the Advanced Photon Source beamline, but data for the SARS-CoV-2 3CLpro / 14c complex were collected on National Synchrotron LightSource II (NSLS-II) AMX beamline 17-ID-1. All diffraction data were collected using a Decris Eiger2 X 9M pixel array detector.

[0371] Structural resolution and refinement. XDS integration intensity was performed via Autoproc, and Laue-class analysis and data scaling were performed using Aimless. The previously determined structure of SARS2 3CLpro (PDB 6XMK) was used as the search model for structure resolution via molecular substitution by Phaser. Structural refinement and manual model building were performed using Phenix and Coot, respectively. Disordered side chains were truncated to a level where electron density was observable. Structure validation was performed using Molprobity, and graphs were prepared using the CCP4MG package.

[0372] The coordinates and structural factors of the SARS2 3CLpro-inhibitor complex have been saved to the global protein database (wwPDB), with the following access code: 8b (NN-II-111) , 12b (AMJ-I-106) 19b (CSD-III-008) 20b (CSD-III-009) 1c (AMJ-I-158) 3c (AMJ-I-159) 5c (AMJ-I-157) 13c (AMJ-I-114) 14c (AMJ-I-111) , 17c ( CSD-III-028) 18c (CSD-III-029) and 21c (NN-II-123) .

[0373] Example 5

[0374] Using the novel reaction scheme described in Example 4, additional phenyl derivatives were synthesized using the following alcohol inputs:

[0375]

[0376] Similar to MERS and SARS, the inhibitory activity of the synthesized compound against NV 3CLpro and its anti-norovirus activity in a cell-based replicon system were evaluated. The IC50 was determined in an enzyme assay. 50 Values ​​of EC against NV in cells containing replicons (HG23 cells) 50 Values ​​and CC in HG23 cells 50 The values ​​are shown in the table below (see also Tables 4B, 5B and 6B), and are the average of at least two measurements.

[0377] Table 11. Norovirus 3CL protease inhibitors 5、6 ( q , r In vitro and cell-based activity and cytotoxicity values.

[0378]

[0379] Table 12. Norovirus 3CL protease inhibitors 5 and 6 ( j , k , m , n , o )as well as 7 ( k , l , o In vitro and cell-based activity and cytotoxicity values

[0380]

[0381] Table 13. Norovirus 3CL protease inhibitors 5、6 ( s , t In vitro and cell-based activity and cytotoxicity values.

[0382]

Claims

1. A compound of formula I: Or its pharmaceutically acceptable salt, wherein: X is selected from the following structures shown in parentheses, depicting the oxygen bond from formula (I) for clarity: , , , , , , , and ; R2 is a branched or unbranched C1-C6 alkyl or cyclohexylalanine (Cha) side chain; and Z is selected from C1-C6 hydroxyalkyl groups, aldehydes, α-ketoamides, and -CH(OH)SO3. - Na + -[O(C=O)CH3]SO3 - Na + and -[O(C=O)CH2CH3]SO3 - Na + A group that is formed.

2. The compound or salt according to claim 1, wherein Z is a C1-C6 hydroxyalkyl or aldehyde.

3. The compound or salt according to claim 1, wherein Z is –CH2OH or -CHO.

4. The compound or salt according to claim 1, wherein Z is -CH(OH)SO3 - Na + -[O(C=O)CH3]SO3 - Na + or-[O(C=O)CH2CH3]SO3 - Na + .

5. The compound or salt according to any one of claims 1 to 4, wherein R2 is isobutyl.

6. The compound or salt according to claim 1, wherein X is: 。 7. The compound or salt according to claim 1, wherein X is: 。 8. Compounds selected from the following: 、 、 、 、 、 、 、 or ， in, Z is CH(OH)SO3Na or CHO, and R2 is a leucine (Leu) or cyclohexylalanine (Cha) side chain. Or its pharmaceutically acceptable salt.

9. Use of the compound or salt according to any one of claims 1 to 8 in the preparation of a medicament for inhibiting the replication of coronaviruses, caliciviruses, or microRNA viruses.

10. The use according to claim 9, wherein the virus is a coronavirus.

11. The use according to claim 9, wherein the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or SARS-CoV-2.

12. An antiviral composition comprising a first compound and a pharmaceutically acceptable carrier, wherein the first compound is the compound according to any one of claims 1 to 8.

13. The composition of claim 12, wherein the carrier is selected from the group consisting of: sterile isotonic buffer, physiological saline, phosphate-buffered saline, DMSO, sterile water, oil-in-water emulsion, water-in-oil emulsion, and mixtures thereof.

14. The composition of claim 12, wherein, based on the total weight of 100% of the composition by weight, the composition comprises about 5% to about 95% by weight of the first compound.

15. The composition according to any one of claims 12 to 14, further comprising a second compound.

16. The composition of claim 15, wherein the second compound is the compound of any one of claims 1 to 8, and the first compound is different from the second compound.

17. The composition according to any one of claims 12 to 14, further comprising one or more of an adjuvant, other active agents, preservatives, buffers, and salts.

18. Use of the compound or salt according to any one of claims 1 to 8 in the preparation of a medicament for inhibiting the activity of viral 3C-like proteases.

19. Use of the first compound in the preparation of a medicament for treating or preventing viral infection in a subject, wherein the first compound is the compound according to any one of claims 1 to 8, and wherein the viral infection is a coronavirus, calicivirus, or microRNA infection.

20. The use according to claim 19, wherein the viral infection is severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) infection, or SARS-CoV-2 infection.

21. The use according to claim 19, wherein the first compound is effective against MERS-CoV IC50. 50 Less than 1.

22. The use according to claim 19, wherein the first compound is effective against SARS-CoV-2 at IC50. 50 Less than 5.

23. The use according to claim 19, wherein the first compound is dispersed in a pharmaceutically acceptable carrier.

24. The use according to claim 23, wherein the drug is a unit dosage form of the first compound dispersed in the pharmaceutically acceptable carrier.

25. The use according to claim 19, wherein the medicament further comprises a second compound.

26. The use according to claim 25, wherein the second compound is the compound according to any one of claims 1 to 8, and the first compound is different from the second compound.

27. The use according to claim 25, wherein the pharmaceutical formulation comprises administering the first compound and the second compound together.

28. The use according to claim 25, wherein the first compound and the second compound are dispersed or dissolved in a pharmaceutically acceptable carrier.

29. The use according to any one of claims 19 to 28, wherein the medicament is formulated such that the first compound is administered via intramuscular, subcutaneous, intradermal, intranasal, intravenous, oral, or transdermal patch administration.

30. The use according to any one of claims 19 to 28, wherein the subject is a human.

31. The use according to any one of claims 19 to 28, wherein the subject is a non-human animal.

32. A kit comprising the compound according to any one of claims 1 to 8; and instructions for administering said compound to a subject in need of doing so.

33. The kit of claim 32, wherein the compound is provided in unit dosage form.

34. The kit according to claim 32 or 33, wherein the compound is provided in a first container, the kit further comprising a carrier in a second container; and instructions for preparing the compound for administration to the subject.

35. Use of the compound according to any one of claims 1 to 8 in the preparation of a medicament for preventing or inhibiting the replication of coronaviruses, caliciviruses or microRNA viruses in cells.

36. The use according to claim 35, wherein the virus is selected from the group consisting of MERS-CoV, SARS-CoV and SARS-CoV-2.

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