Design, Synthesis, X-Ray Crystallography and Biological Activities of Covalent, Non-Peptidic Inhibitors of SARS-CoV-2 Main Protease

Abstract

Highly contagious SARS-CoV-2 coronavirus has infected billions of people worldwide with flu-like symptoms since its emergence in 2019. It has caused deaths of several million people. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and therefore a drug target. Several series of covalent inhibitors of Mpro were designed and synthesized. Structure-activity relationship studies show that 1) several chloroacetamide- and epoxide-based compounds targeting Cys145 are potent inhibitors with IC₅₀ values as low as 0.49 μM, and 2) Cys44 of Mpro is not nucleophilic for covalent inhibitor design. High resolution X-ray studies revealed the protein-inhibitor interactions and mechanisms of inhibition. It is of interest that Cys145 preferably attacks the more hindered C_α atom of several epoxide inhibitors. Chloroacetamide inhibitor 13 and epoxide inhibitor 30 were found to inhibit cellular SARS-CoV-2 replication with an EC₆₈ (half-log reduction of virus titer) of 3 and 5 μM. These compounds represent new pharmacological leads for anti-SARS-CoV-2 drug development.

Graphical Abstract

The COVID-19 pandemic, caused by a novel coronavirus SARS-CoV-2 identified in December 2019, has posed an unprecedented global health crisis in the modern history. With its extremely high infectivity, SARS-CoV-2 has rapidly spread to over 200 countries worldwide and infected billions of people^{1, 2}. While most patients experience various flu-like symptoms and can recover in 7–14 days, life-threatening severe illnesses, including severe pneumonia, sepsis, and lung and multiple organ failure, can occur at a significantly higher risk for people at age of ≥65 years or with certain medical conditions (e.g., chronic lung diseases and immunocompromised)^3–5. More than 6 million people have died of or with SARS-CoV-2, which demands effective countermeasures against this virus. With enormous, wholehearted efforts from the scientific community, pharmaceutical industry and governments, several vaccines and antiviral drugs have been available to prevent and treat SARS-CoV-2 infection. However, finding new antiviral therapeutics are still needed due to continued viral evolution and drug (nirmatrelvir)-resistant mutations⁶ as well as emergence of a new coronavirus in the future.

SARS-CoV-2 is a positive-sense, single-stranded RNA virus, with its ~30 kb genome encoding a handful of structural and non-structural proteins⁷. Because it is highly conserved during evolution and across the SARS-family coronaviruses, SARS-CoV-2 main protease (Mpro, also known as 3C-like protease or 3CLpro) has been considered a promising target in drug discovery and development⁸. In contrast to the frequent mutations of the spike protein, Mpro of SARS-CoV-2 has remained almost identical from the original to Omicron strains, particularly for the enzyme active site⁹. It also exhibits a high sequence identity of 96% to that of SARS-CoV found in 2003¹⁰. Functionally, Mpro is responsible for site-specific cleavage of the viral polyprotein to generate 11 non-structural proteins of SARS-CoV-2 and is essential for replication of the virus in host cells⁹.

The structure of Mpro consists of three domains: the first two with a chymotrypsin fold constitute the protein active site, while the third domain is for homodimerization critical to Mpro’s catalytic activity¹¹. Mpro has a similar overall structure to 3C-like proteases of the picornavirus family viruses, such as rhinovirus¹². The substrates of Mpro have a consensus sequence P2P1-P1’ with the amide bond between P1 and P1’ being hydrolyzed. The P1 residue is always Gln and the P1’ Ser or Ala, while a hydrophobic Leu, Phe or Val residue can be found at the P2 position. The protein active site is situated in the cleft between domains I and II, consisting of the S1’, S1 and S2 pockets that accommodate the corresponding P1’-P2 residues of the substrate. The -SH group of Cys145, which is deprotonated by His41, acts as a nucleophile to attack the P1-P1’ amide bond with the oxyanion transition state stabilized by hydrogen bond/electrostatic interactions with Gly143, Ser144, and Cys145 (oxyanion hole). The protonated His41 then acts as an acid to protonate the P1’ amine to facilitate its leaving. The resulting protein (Cys145)-bound thioester is rapidly hydrolyzed to release the P1 acid and completes a catalytic cycle^{13, 14}.

A number of inhibitors of SARS-CoV-2 Mpro have been developed, with most of the potent ones being peptidomimetic compounds bearing an electrophilic “warhead” group to covalently bind to Cys145 (Figure 1)^{8, 15–18}, including FDA-approved drug nirmatrelvir (PF-07321332)¹⁹. Nevertheless, studies towards finding more chemo-types of Mpro inhibitors are desirable, given Mpro’s essential roles in replication of SARS-CoV-2 and other coronaviruses. Here, we report design, synthesis, X-ray crystallography and biological activity studies of several novel series of covalent, non-peptidic Mpro inhibitors.

Figure 1.

Representative inhibitors of SARS-CoV-2 Mpro with their electrophilic “warhead” groups shown in red.

Results and Discussion

Inhibitor design targeting Cys44 in the S2 pocket of Mpro.

Peptidic or peptidomimetic inhibitors of Mpro, including nirmatrelvir, generally have poor metabolic stabilities caused by human proteases and cytochrome P450. In FDA-approved Paxlovid, ritonavir is used to inhibit cytochrome P450 and enhance nirmatrelvir’s metabolic stability. To this end, non-covalent Mpro inhibitor ML188 (Figure 1)^19–26 and its more potent derivative compound 23R²⁴ are of interest because of their potent activity and potential of this chemo-type of compounds to have an improved pharmacokinetics²⁷. Analysis of the X-ray structure of SARS-CoV-2 Mpro-23R complex²⁴ revealed that Cys44 in the S2 pocket could be exploited for covalent binding, with the distances between the S atom of Cys44 and the distal phenyl ring of the 23R biphenyl group being ~4 Å (Supporting Information Figure S1). A molecular dynamics simulation study also suggested that Cys44 is “hyper-reactive”²⁸. Derivatives of compound 23R with a variety of electrophilic groups were designed and synthesized to probe the possibility.

Synthesis.

The general methods for synthesis of compounds 1-42 are shown in Scheme 1. A four-component Ugi reaction using an acid 43, amine 44, aldehyde 45 and isocyanide 46 produced the target compounds 1–4, 11–14, 20–42 and 49 in 46–89% yield. To synthesize the epoxy acid 43a, which is not commercially available, ethyl crotonate 47 (or several substituted derivatives) was reacted with meta-chloroperoxybenzoic acid to give the epoxide compound 48, which was hydrolyzed to yield 43a. The tert-butoxycarbonyl (Boc)-protected compound 49 prepared using the Ugi reaction was deprotected to give the target compounds 11, 12, 20 and 21, whose primary -NH₂ group was subjected to an amide forming reaction to generate compounds 5–10, 15, 16, 18, and 19.

Scheme 1.

General methods for synthesis of compounds 1-42.^a

a Reagents and conditions: (a) MeOH, room temperature, overnight, (b) meta-chloroperoxybenzoic acid, 1,2-dichloroethane, reflux; (c) KOH, ethanol, room temperature, 1 h; (d) 4N HCl in dioxane, dichloromethane, room temperature; (e) 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro-phosphate, N,N-diisopropylethylamine, dichloromethane, room temperature.

Structure-activity relationship (SAR) study I.

An established biochemical assay²⁹ was used to determine the inhibitory activities of our synthesized compounds against SARS-CoV-2 Mpro and the results are shown in Table 1. Racemic compound ML188 exhibited 43% inhibition at 20 μM, while compound 1, the racemic analog of compound 23R without the methyl substituent in the R¹, was found to be a strong inhibitor with an IC₅₀ value of 0.74 μM. Activities of these known compounds are comparable to the reported values^{20, 24}. Compound 2 with an electrophilic epoxy group to replace the distal phenyl ring in compound 1 showed only 18% inhibition of Mpro at 20 μM, which is similar to its precursor compound 3 (26% inhibition) with a non-reactive vinyl group at this position. Analogous epoxy compound 4 with an additional -CH₂- was also inactive (10% inhibition at 20 μM). Changing to a chloroacetamide “warhead” group in compound 5 showed a modest inhibition of Mpro with an IC₅₀ of ~20 μM (47% inhibition at 20 μM). Compound 6 with a dichloroacetamide group exhibited a similar activity (41% inhibition at 20 μM). Compound 7 with an acrylamide group, whose electrophilic C_β atom is 1 more bond-length away from the phenyl ring, was found to be less inhibitory (22% inhibition). Next, moving these three “warhead” groups to the meta-position of the phenyl ring gave compounds 8-10, which were found to have similar or less activities (47%, 20% and 5% inhibition at 20 μM, respectively). In addition, the precursor compounds 11 and 12 with a para- and meta-NH₂ group were almost inactive (0% and 18% inhibition at 20 μM, respectively).

Table 1.

Structures and inhibitory activities of compounds 1-12 against SARS-CoV-2 Mpro.

Compound	R1	R2	R3	IC50 (μM) or % inhibition at 20 μM
ML188	t-Bu	t-Bu	H	43%
1	Bn	Ph	H	0.74 ± 0.07
2	Bn		H	18%
3	Bn		H	26%
4	t-Bu		H	10%
5	Bn		H	~20 (47%)
6	Bn		H	41%
7	Bn		H	22%
8	Bn	−H		~20 (47%)
9	Bn	−H		20%
10	Bn	−H		5%
11	Bn	−NH2	−H	0%
12	Bn	−H	−NH2	18%

Our X-ray crystallographic studies (described below, Figure 2c/d) revealed that chloroacetamide-containing, modest inhibitors 5 and 8 covalently bind to Cys145, but not Cys44 as designed. They exhibited a drastically distinct binding pose to compound 23R with less favorable interactions with Mpro. These could be responsible for the weak inhibitory activities of these two compounds.

Figure 2.

X-ray structures of SARS-CoV-2 Mpro in complex with compounds 5 (tube model with C atoms in purple), 8 (tube model in brown) and 13 (ball and stick model in green). (A) Three superimposed overall structures, with the Mpro backbones shown as curved lines; (B) The active site of the Mpro-13 structure, together superimposed with that of compound 23R (thin tube model in black); (C, D) The active site of the Mpro-5 (C) and −8 (D) structure, superimposed with that of compound 13 (thin tube model in green). Cys145 and Cys44 are shown in tube models, while hydrogen bonds are shown as dashed yellow lines.

To further probe whether Cys44 can be utilized for covalent binding, compounds in Table 2 with two electrophilic “warhead” groups were designed and investigated for their activity against SARS-CoV-2 Mpro. Based on the crystal structures of compounds 23R and 5, the chloroacetamide group in the backbone of these compounds was anticipated to covalently bind Cys145, while a variety of second electrophilic R² or R³ groups in the sidechain targeted Cys44. First, chloroacetamide compounds 13 and 14 (R² = t-Bu and Ph) without a second “warhead” served as controls. Both compounds were found to strongly inhibit the enzyme with IC₅₀ values of 0.6 and 0.95 μM, respectively. Our X-ray crystallography study (described below) indicated that the chloroacetamide group of compound 13 covalently binds to Cys145, with the -Cl atom being the leaving group. Other moieties of 13 exhibit a similar binding pose to that of ML188 (or 23R): the pyridin-3-yl and 4-tert-butylphenyl groups occupy the S1 and S2 pockets, respectively, with favorable hydrophobic and hydrogen bond interactions. However, compounds 15 and 16 (IC₅₀ = 3.2 and 3.6 μM) with an additional mono- or di-chloroacetamide (R²) group are ~5× less active than compound 13. Compound 17 with an electrophilic epoxy R² substituent showed a similarly reduced inhibition with an IC₅₀ of 3.6 μM. Moving the secondary mono- or di-chloroacetamide group to the meta-position gave compounds 18 or 19 (IC₅₀ = 3.6 and 2.2 μM), which exhibited comparable (or slightly improved) activities to compounds 15 and 16. In addition, compounds 20 and 21 with R² or R³ being an -NH₂ group were almost inactive (12% and 33% inhibition at 20 μM). These results indicate that Cys44 in the S2 pocket of Mpro is unreactive for covalent modification, despite in the presence of a nearby electrophilic chloroacetamide or epoxy group in compounds 15-19. Rather, Cys44 seems to merely constitute the hydrophobic S2 pocket, which is consistent with the decreasing activities of compounds 13-21 bearing a decreasing lipophilicity for t-Bu/Ph (for 13/14), chloroacetamide/epoxymethyl (for 15-19) and -NH₂ (for 20/21). Activity of compound 22 (IC₅₀ = 1.6 μM) having a -OMe R² substituent, with its lipophilicity falling between 13/14 and 15-19, also supports this SAR.

Table 2.

Structures and inhibitory activities of compounds 13–26 against SARS-CoV-2 Mpro.

Compound	R1	R2	R3	IC50 (μM) or % inhibition at 20 μM
13	t-Bu	t-Bu	H	0.60 ± 0.05
14	t-Bu	Ph	H	0.95 ± 0.04
15	t-Bu		H	3.2 ± 0.79
16	t-Bu		H	3.6 ± 0.19
17	t-Bu		H	3.6 ± 0.30
18	t-Bu	H		3.6 ± 0.60
19	t-Bu	H		2.2 ± 0.51
20	t-Bu	NH2	H	12%
21	t-Bu	H	NH2	33%
22	t-Bu		H	1.61 ± 0.41
23		t-Bu	H	41%
24		t-Bu	H	2.4 ± 0.30
25		t-Bu	H	6.7 ± 0.60
26		t-Bu	H	0.49 ± 0.22

Effects on varying the R¹ substituent in compound 13 were investigated. Compound 23 with a benzyl R¹ group was significantly less active (41% inhibition at 20 μM). Compounds 24 and 25 (IC₅₀ = 2.4 and 6.7 μM) with a linear or branched butyl R¹ group showed moderate activities. Compound 26 with a cyclohexyl R¹ exhibited the strongest potency with an IC₅₀ of 0.49 μM.

X-ray studies of chloroacetamide inhibitors of Mpro.

X-ray crystallography was performed to find out how these chloroacetamide inhibitors interact with SARS-CoV-2 Mpro. Crystal structures of the protein in complex with compounds 5, 8 and 13 were determined at a resolution of 1.73–1.90 Å, with the statistics for diffraction data and structure refinement shown in Supporting Information Table S1. The overall structures of the three complexes (Figures 2a) are very similar to each other with root mean square deviations (rmsd) of ~0.4 Å, which are also not deviated from that of the Mpro-23R complex (rmsd ~0.6 Å). Based on the F_o-F_c (omit) and 2F_o-F_c (electron density) maps (Figures S2 and S3), the three inhibitors were found to be located in the protein active site. Their protein-inhibitor interactions are illustrated in Figure S4. Close-up views of the binding sites of compounds 13, 5 and 8 (Figures 2b–d) clearly show that an SN2 substitution reaction occurred between their electrophilic chloroacetamide group and Cys145, resulting in covalent binding to the protein. The -Cl atom of these compounds, the leaving group, is absent in the structures. In addition, although obtained as a racemic mixture, only the R-enantiomers (at the asymmetric C2 atom, Table 2) of these three compounds bind to Mpro, presumably because of their higher affinity and activity. This is similar to compounds 23R and ML188^{20, 21, 24}, whose R-enantiomers were separated, determined to be more active, and crystallized with Mpro (while the S-enantiomers are inactive). For the potent inhibitor 13 (IC₅₀ = 0.6 μM), its 3-pyridinyl group occupies the S1 pocket with favorable hydrophobic interactions as well as a hydrogen bond with His163, while its 4-tert-butylphenyl group deeply inserts into the hydrophobic S2 pocket of Mpro, which includes Cys44. These two groups exhibit a similar binding pose to those of compounds 23R (Figure 2b) and ML188 and should contribute to the high-affinity binding for compound 13.

Unexpectedly, the two weak inhibitors 5 and 8 were found to covalently bind Cys145, despite that their chloroacetamide group was designed to target Cys44 in the S2 pocket. Consequently, these two compounds exhibit drastically different binding poses, as compared to compound 13 (Figure 2c/d). None of the furan and pyridine rings of compounds 5 and 8 have interactions with the S1 or S2 pocket of Mpro, which should account for their weak inhibitory activities. Lack of nucleophilicity of Cys44 is likely because there is no nearby basic residue (e.g., histidine) to help deprotonate and activate its -SH group.

SAR study II.

Next, the “warhead” group in compound 13 was optimized, in an effort to replace its chloroacetamide moiety, which is quite reactive and often associates with increased toxicities as well as non-specific, off-target activities. As shown in Table 3, compounds 27-29 with an acrylamide group were weakly active (23–35% inhibition at 20 μM). Compound 30, the epoxide derivative of compound 29, was found to be a potent inhibitor of SARS-CoV-2 Mpro with an IC₅₀ of 0.76 μM. Compound 31 with a benzyl ketone functionality was inactive. Compounds 32-34 with a hydroxy-, acetyloxy- and cyano-methyl group, respectively, were also almost inactive (0–28% inhibition at 20 μM). Thus, the potent epoxy inhibitor 30 was chosen for more SAR studies, since an epoxy group has been commonly used as an electrophile in drugs (e.g., carfilzomib).

Table 3.

Structures and inhibitory activities of compounds 27-34 against SARS-CoV-2 Mpro.

Compound	R1	R4	IC50 (μM) or % inhibition at 20 μM
27	t-Bu		35%
28	Bn		23%
29	c-hexyl		30%
30 a	c-hexyl		0.76 ± 0.18
31	t-Bu		0%
32	t-Bu		21%
33	t-Bu		0%
34	c-hexyl		28%

^aThe more active diastereomer of compound 30.

Compound 30 was synthesized as a pair of racemic diastereomers (~1:1, Table 4 and Figure S5), which can be separated with column chromatography. In addition, although we did not separate the two enantiomers of the more active diastereomer of 30 (IC₅₀ = 0.76 μM), its X-ray structure in complex with Mpro at a high resolution of 1.95 Å (described below) indicates that the absolute configurations of the more active enantiomer are R- at the C2 atom (same as compound 13) and S- at the epoxy C_α atom. The other diastereomer of compound 30, a racemic mixture of (R, R)- and (S, S)-30 (Table 4 and Figure S5), is 13× less active with an IC₅₀ of 9.9 μM. Racemic mixture of (R, S, R)-35 with a methyl group at the epoxy C_β atom inhibited Mpro with an IC₅₀ of 1.9 μM, while its diastereomer (R, R, S)-35 had only modest activity (42% inhibition at 20 μM). The diastereomeric mixtures of compound 36, which has two methyl groups at the C_β atom and are not separable by column chromatography, also exhibited a strong inhibitory activity with an IC₅₀ of 2.5 μM. However, the diastereomeric mixtures of compound 37, which has a methyl group at the C_α atom, exhibited a very weak inhibitory activity against Mpro (28% inhibition at 20 μM). These results suggest that the Cys145-mediated SN2 substitution reaction occurs preferably at the epoxy C_α atom, since steric hindrance at the C_α atom drastically reduces the activity (>25-fold activity reduction for compound 30 vs. 37), while that at the C_β atom is tolerable (≤3-fold activity reduction for 30 vs. 35/36). This SAR was confirmed by our X-ray crystallographic studies (described below). In addition, there are large activity differences (>10-fold) between the two separated diastereomers of 30 and 35, indicating the absolute configuration at the asymmetric epoxy-C_α atom is also an important factor for Mpro inhibition. Our subsequent X-ray crystallographic studies of these two compounds show that their (S)-C_α-enantiomers bind to Mpro and are likely more active than their corresponding (R)-enantiomers.

Table 4.

Structures and inhibitory activities of compounds 30, 35-42 against SARS-CoV-2 Mpro.^a (see Figure S5 for full structures)

Compound	R1	R4	IC50 (μM) or % inhibition at 20 μM
(R, S)-b and (S, R)-30	c-hexyl		0.76 ± 0.18
(R, R)- and (S, S)-30	c-hexyl		9.9 ± 1.6
(R, S, R)-b and (S, R, S)-35	c-hexyl		1.9 ± 0.21
(R, R, S)-b and (S, S, R)-35	c-hexyl		42%
diastereomic mixture of (R, S)- and (S, R)-36	c-hexyl		2.5 ± 0.28
diastereomic mixture of (R, R)-b and (S, S)-37	c-hexyl		28%
(R, S)- and (S, R)-38	t-Bu		1.24 ± 0.19
(R, R)- and (S, S)-38	t-Bu		21%
(R, S, R)- and (S, R, S)-39	t-Bu		4.33 ± 0.32
(R, R, S)- and (S, S, R)-39	t-Bu		5.32 ± 0.64
diastereomic mixtures of (R, S)- and (S, R)-40	t-Bu		31%
diastereomic mixtures of (R, R)- and (S, S)-41	t-Bu		22%
(R, S)-b and (S, R)-42	Bn		1.6 ± 0.32
(R, R)- and (S, S)-42	Bn		45%

^aThe absolute configurations refer to that of the C2 atom, followed by that of the epoxy C_α and C_β (if exists) atom.

^bThe absolute configurations of the more active enantiomer were determined by X-ray crystallography.

Similar SARs were observed for the epoxy compounds 38-41 having a tert-butyl R¹ group, with their (S)-C_α enantiomer being more active than its (R)-enantiomer. Also, compounds with less hindered epoxy C_α atom exhibited more activities (e.g., 38 vs. 41). In addition, for the R¹ substituent, (R, S)-/(S, R)-38 (IC₅₀ = 1.2 μM), as well as (R, S)-/(S, R)-42 (IC₅₀ = 1.6 μM) with a benzyl R¹ group, is less active than (R, S)-/(S, R)-30. These results show a cyclo-hexyl R¹ group is more favorable for this series of epoxy inhibitors of Mpro.

X-ray studies of epoxy inhibitors of Mpro.

Crystal structures of SARS-CoV-2 Mpro in complex with epoxy compounds 30 (more active diastereomer), 35 (both diastereomers), 37 (diastereomeric mixture) and 42 (more active diastereomer) were determined at a high resolution of 1.61–1.98 Å, respectively. The statistics for diffraction data and structure refinement of these five structures are shown in Table S2. Their omit and electron density maps are shown in Figures S2 and S3, which reveal the protein-inhibitor interactions (Figure S4). These high-resolution structures can be used to determine the mechanism of inhibition and the absolute configures at the C2 and epoxy C_α atoms of the inhibitors. The overall structures of the five Mpro-inhibitor complexes (Figures 3a) are very similar to each other with root mean square deviations (rmsd) of ≤0.3 Å, which are also not deviated from that of the non-covalent Mpro-23R complex (rmsd ~0.5 Å). Same as compound 13 (as well as 23R), the C2 atom of these five inhibitors invariably has the R-configuration. Moreover, both the 3-pyridinyl and 4-tert-butylphenyl group of these compounds adopt a similar binding pose and are almost superimposed with each other. The 3-pyridinyl group occupies the S1 pocket with favorable hydrophobic interactions as well as a hydrogen bond with His163 and 4-tert-butylphenyl is located in the hydrophobic S2 pocket of Mpro (Figures 3b–f).

Figure 3.

X-ray structures of SARS-CoV-2 Mpro in complex with compound 30 (tube model with C atoms in green), 42 (in pink), the more active diastereomer of 35 (in brown), the less active diastereomer of 35 (in purple) and 37 (in burgundy). (A) Five superimposed overall structures, with Mpro backbone shown as curved lines; (B-F) The active site of the Mpro-(R, S)-30 (B), -(R, S)-42 (C), -(R, S, R)-35 (D), -(R, R, S)-35 (E), and -(R, R)-37 (F) structure. Hydrogen bonds are shown as yellow dashed lines.

A close-up view of the inhibitor-binding site (Figures 3b–f) clearly indicates that an SN2 substitution reaction occurred between the electrophilic epoxy group of all these inhibitors and Cys145, resulting in covalent inhibition of SARS-CoV-2 Mpro. Interestingly, for the more active diastereomer of 30, 42 and 35 with IC₅₀ of 0.76–1.9 μM (Figures 3b,c,d), Cys145 attacks the more hindered epoxy C_α atom, despite the C_β atom with considerably less steric hindrance (particularly for 30 and 42) is more favorable for the SN2 reaction. The high-resolution X-ray structures show the C_α atom of the three bound products invariably has the R-configuration, indicating it in the precursor compounds 30, 42 and 35 has the S-configuration. X-ray crystallography (that cannot distinguish -OH from -CH₃) cannot determine the absolute configuration of the C_β atom of compound 35, which is retained for the SN2 reaction. Rather, it is deduced to be R-, based on the S-configuration of the C_α atom and the trans-C=C bond of the starting compound ethyl crotonate 47 (Scheme 1).

The crystal structure of Mpro in complex with the less active diastereomer of compound 35 (42% inhibition at 20 μM) shows Cys145 also forms a covalent bond with the more hindered epoxy C_α atom with the S-configuration (Figure 3e), which indicates the inhibitor was (R, R, S)-35. However, the superimposed structures of (R, R, S)-35 and the more active diastereomer (R, S, R)-35 (Figure S6) do not seem to provide an obvious explanation for the ~10× activity difference between these two diastereomers, presumably due to a relatively small energy difference (~1.5 kcal/mol).

For compound 37 with a tertiary epoxy C_α atom (at which an SN2 substitution reaction is prohibitive), X-ray crystallography shows Cys145 attacks and forms a covalent bond with the less hindered C_β atom (Figure 3f). Because the epoxy O atom, the leaving group, must be trans- to the S atom of Cys145, the bound product C_α atom is deduced to have the S-configuration, which indicates the inhibitor was (R, R)-37. It is noted that there was no configuration inversion at the C_α atom. The change from S- to R- is merely due to the nomenclature rule. With only a distance of 3.2 Å between the carbonyl C and the Cys145 S atom, the C(O)-C_α-C_β-S moiety in the Mpro-37 complex adopts a kinked conformation (Figure S7), as compared to an extended conformation for the corresponding C(O)-C_α-S moiety in the Mpro-30 complex. This could explain the low inhibitory activity of 37 as well as why the SN2 reaction preferably occurs at the more hindered epoxy C_α atom. Adjacent carbonyl group also activates the C_α atom for the reaction.

Anti-SARS-CoV-2 activity evaluation.

Anti-SARS-CoV-2 activity of selected Mpro inhibitors were evaluated. Replication of SARS-CoV-2 (Washington strain, 2020) in Vero cells causes significant cytopathic effects (CPE) and eventually, cell lysis, which can be clearly observed under the microscope. An MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay can be used to quantitate the number of viable cells. 0.01 Multiplicity of infection (MOI, the number of infectious viral particles per cell) of SARS-CoV-2 was added to infect a monolayer of the cells for 2h. Upon removal of the virus, cells were cultured in fresh media containing increasing concentrations of a compound for 48h. An end-point dilution assay was used to determine the viral titers in the supernatant. Half-log (0.32×) serial dilutions of the supernatant containing newly generated SARS-CoV-2 viruses were added to (uninfected) Vero cells in quadruplicate. Upon incubation for ~5 days, SARS-CoV-2 infection in each sample was determined with CPE followed by MTT assay. TCID₅₀ (tissue culture infective dose) was calculated based on the highest dilution in which ≥50% of the quadruplicate samples were infected with SARS-CoV-2. As compared to TCID₅₀ of the untreated samples, ability of a compound to reduce SARS-CoV-2 replication can be determined. As shown in Figures 4 and S8, chloroacetamide compound 13 (IC₅₀ = 0.6 μM) can inhibit replication of SARS-CoV-2 by 68% (0.5-log titer reduction) at 3 μM and 97% (1.5-log reduction) at 5 μM without significant cytotoxicity. However, it showed considerable cytotoxicity at 10 μM (~40% inhibition, CC₅₀ = 12.1 μM). The more active diastereomer of epoxy compound 30 (IC₅₀ = 0.76 μM) exhibited an EC₆₈ of 5 μM and EC₉₀ of 15 μM against SARS-CoV-2. The more active diastereomer of epoxy compound 35 (IC₅₀ = 1.9 μM) showed a weaker antiviral activity with an EC₆₈ of 15 μM, while compound 36 (mixture of diastereomers, IC₅₀ = 2.5 μM) was inactive at 15 μM. These three epoxy compounds at 15 μM had no or negligible cytotoxicity to Vero cells (Figure S8).

Figure 4.

Anti-SARS-CoV-2 activities of compounds 13, 30 (more active diastereomer), 35 (more active diastereomer) and 36 in Vero cells.

CONCLUSION

Evolution and drug-resistant mutations of SARS-CoV-2 require continued drug discovery targeting the viral protease Mpro. Based on non-covalent inhibitor 23R as well as its binding structure in Mpro, several series of covalent inhibitors were designed, synthesized, and tested for their activities against SARS-CoV-2 Mpro. The structure-activity relationships of these compounds are summarized and illustrated in Figure 5, underscoring that 1) several chloroacetamide- and epoxide-based inhibitors targeting Cys145 are potent inhibitors with IC₅₀ values as low as 490 nM, and 2) Cys44 in the S2 pocket is not nucleophilic for covalent inhibitor design. High resolution X-ray crystallographic studies show these inhibitors bind to the protein active site with their electrophilic “warhead” groups covalently modifying Cys145. For the epoxide inhibitors, it is interesting that Cys145 preferably attacks the more hindered epoxy C_α atom, rather than the sterically favored C_β atom. In addition, the absolute configurations of the bound inhibitors are determined. Chloroacetamide inhibitor 13 was found to have a strong anti-SARS-CoV-2 activity with an EC₆₈ of 3 μM, while compound 30 is the most potent epoxide inhibitor with an EC₆₈ of 5 μM. These compounds represent new pharmacological leads for further anti-SARS-CoV-2 drug development.

Figure 5.

Summary of structure-activity relationships.

Methods

All the chemicals used for synthesis were purchased from Aldrich (Milwaukee, WI) or Alfa Aesar (Ward Hill, MA). Unless otherwise stated, all solvents and reagents were used as received. All reactions were conducted with the use of a Teflon-coated magnetic stir bar at the indicated temperature and were performed under an inert atmosphere when stated. The identity of the synthesized compounds was characterized by ¹H and ¹³C NMR on a Varian (Palo Alto, CA) 400-MR spectrometer and mass spectrometer (Shimadzu LCMS-2020). Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). The identity of the potent inhibitors was confirmed with high resolution mass spectra (HRMS) using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer with electrospray ionization (ESI). The purities of the final compounds were determined to be >95% with a Shimadzu Prominence HPLC using a Zorbax C18 (or C8) column (4.6 × 250 mm) at 0.7 mL/min flow rate (water:acetonitrile with 0.1% formic acid, 90:10 – 5:95 in 3 min) monitored by UV at 254 nm.

Chemical synthesis.

General Procedure for the Ugi Reaction:

The Ugi reaction was performed using a reported method with modifications²⁰. An Amine 44 (1 mmol) and aldehyde 45 (1 mmol) were added to methanol (5 mL) and stirred at room temperature for 20 min, followed by adding a carboxylic acid 43 (1 mmol) and an isocyanide 46 (1 mmol) successively. The resulting mixture was stirred at room temperature overnight. Upon completion of the reaction monitored by TLC, the solvent was removed under reduced pressure and the oily residue was purified with flash column chromatography (silica gel, hexane/ethyl acetate, 10/1- 1/1) to give a product as a white to pale yellow powder in 46–89% yield.

General Procedure for synthesis of epoxy-carboxylic acid 43a³⁰.

A reaction mixture of a substituted ethyl crotonate (2 mmol) and meta-chloroperoxybenzoic acid (4 mmol) in dichloromethane (15 mL) was refluxed for 4 h. Upon cooling, it was filtered and washed with hexane (5 mL × 2). The filtrate was washed with saturated aqueous solution of NaHCO₃, Na₂CO₃ and Na₂S₂O₃, dried over MgSO₄, concentrated, and the residue purified with flash column chromatography (silica gel, hexane/ethyl acetate, 10/1- 1/1) to afford the ethyl ester of the product as a colorless oil, which was hydrolyzed by KOH (3 equiv.) in EtOH (5 mL) at room temperature for 1h and acidified with 3 N HCl to pH 2. The solvent was removed by reduced pressure and the crude product 43a was used for the Ugi reaction without purification.

General Procedure for synthesis of compounds 5–10, 15–16 and 18–19:

To a solution of Boc-protected compound 49 (1 mmol) in dichloromethane (10 mL), 4N HCl in dioxane (4 mmol) was added and stirred until deprotection was completed. The resulting precipitate was collected by filtration and used for the next step without purification. A reaction mixture of the amine thus obtained (1 mmol), a carboxylic acid (1 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium (1.2 equivalent), N,N-diisopropylethyl-amine (4 equivalent) in dichloromethane (5 mL) was stirred overnight at room temperature. Upon completion of the reaction, it was diluted in ethyl-acetate (30 mL) and washed with 1N HCl solution, saturated NaHCO₃ solution, brine, and dried over Na₂SO₄. Upon removal of the solvent, the residue was purified with column chromatography (silica gel, hexane/ethyl acetate, 10/1 – 1/1) to give a target compound as a white to pale yellow powder in 40–90% yield.

General Procedure for synthesis of compounds 2, 4 and 17:

A precursor Ugi compound with a vinyl group (obtained using the above method) was epoxidized by meta-chloroperoxybenzoic acid (4 mmol) using the method described above. The crude product thus obtained was purified with flash column chromatography (silica gel, hexane/ethyl acetate, 10/1- 1/1) to afford the target compound as a white to pale yellow powder in 40–90% yield.

N-([1,1’-Biphenyl]-4-yl)-N-(2-(benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide (1). ¹H NMR (CDCl₃, 400 MHz) δ 8.41 (s, 2H), 7.53 (t, J = 9.7 Hz, 3H), 7.42 (dd, J = 15.4, 8.0 Hz, 4H), 7.36 – 7.12 (m, 9H), 7.04 (d, J = 4.6 Hz, 1H), 6.32 (s, 1H), 6.13 (d, J = 1.9 Hz, 1H), 5.55 (d, J = 3.0 Hz, 1H), 4.50 (t, J = 5.9 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 168.9, 159.7, 151.5, 149.7, 146.2, 145.1, 141.5, 139.5, 138.3, 138.3, 138.1, 131.4, 128.9, 128.7, 128.7, 128.0, 127.7, 127.6, 127.4, 127.0, 123.2, 117.5, 111.3, 63.0, 43.8. MS (ESI) calculated for (C₃₁H₂₆N₃O₃)⁺ [M+H]⁺ 488.2, found 488.2.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(oxiran-2-yl)phenyl)furan-2-carboxamide (2). ¹H NMR (CDCl₃, 400 MHz) δ 8.19 (s, 1H), 8.01 – 7.77 (m, 1H), 7.65 (s, 1H), 7.24 (s, 11H), 6.14 (s, 2H), 5.52 (s, 1H), 4.47 (m, 2H), 3.81 (s, 1H), 3.14 (s, 1H), 2.74 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.9, 159.8, 146.7, 145.9, 145.5, 145.2, 139.1, 138.9, 138.0, 137.8, 136.6, 131.1, 128.8, 128.8, 127.9, 127.8, 127.6, 127.6, 127.4, 126.6, 125.3, 118.7, 118.0, 112.0, 111.5, 62.4, 51.9, 51.7, 44.0, 29.8. MS (ESI) calculated for (C₂₇H₂₄N₃O₄)⁺ [M+H]⁺ 454.2, found 454.2.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-vinylphenyl)furan-2-carboxamide (3). ¹H NMR (CDCl₃, 400 MHz) δ 8.36 (s, 2H), 7.67 (s, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.29 – 7.10 (m, 7H), 7.01 (dd, J = 13.8, 6.1 Hz, 3H), 6.60 (dd, J = 17.5, 10.9 Hz, 1H), 6.27 (s, 1H), 6.11 (s, 1H), 5.69 (d, J = 17.6 Hz, 1H), 5.48 (d, J = 3.1 Hz, 1H), 5.26 (d, J = 10.9 Hz, 1H), 4.44 (dd, J = 12.9, 7.4 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 168.9, 159.6, 151.4, 149.6, 146.1, 145.1, 138.4, 138.3, 138.1, 138.0, 135.7, 131.2, 128.6, 128.5, 127.6, 127.4, 127.4, 127.0, 126.8, 123.1, 117.5, 115.5, 111.3, 62.9, 43.8, 43.7. MS (ESI) calculated for (C₂₇H₂₄N₃O₃)⁺ [M+H]⁺ 438.2, found 438.2.

N-(2-(tert-Butylamino)-2-oxo-1-(pyridin-3-yl) ethyl)-N-(4-(oxiran-2-ylmethyl) phenyl)furan-2-carboxamide (4). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 8.07 (d, J = 6.0 Hz, 1H), 7.37 (s, 1H), 7.19 (d, J = 7.7 Hz, 3H), 7.10 – 7.07 (m, 3H), 6.33 (s, 1H), 6.18 (s, 1H), 5.98 (s, 1H), 5.56 (d, J = 3.5 Hz, 1H), 3.12 (s, 1H), 2.91 (d, J = 4.4 Hz, 1H), 2.80 (dd, J = 10.3, 6.0 Hz, 2H), 2.47 (s, 1H), 1.35 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 166.58, 159.65, 145.81, 145.30, 140.69, 138.71, 138.65, 138.62, 137.55, 134.37, 132.00, 130.45, 130.09, 127.84, 125.16, 117.75, 111.35, 62.98, 51.98, 51.83, 46.53, 38.04, 31.56, 28.57, 22.62, 14.10. MS (ESI) calculated for (C₂₅H₂₈N₃O₄)⁺ [M+H]⁺ 434.2, found 434.2.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(2-chloroacetamido)phenyl)furan-2-carboxamide (5). ¹H NMR (CDCl₃, 400 MHz) δ 8.61 (s, 1H), 8.38 (s, 2H), 7.46 (dd, J = 22.5, 8.0 Hz, 3H), 7.23 (ddd, J = 15.4, 13.0, 11.2 Hz, 5H), 7.11 – 6.88 (m, 3H), 6.27 (s, 1H), 6.18 – 6.04 (m, 1H), 5.55 (d, J = 3.0 Hz, 1H), 4.53 – 4.31 (m, 2H), 4.11 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 168.9, 164.2, 159.8, 151.5, 149.8, 146.1, 145.3, 138.5, 138.0, 137.6, 135.4, 131.9, 130.2, 128.8, 127.7, 127.6, 123.4, 120.1, 117.7, 111.5, 63.0, 43.9, 43.0. MS (ESI) calculated for (C₂₇H₂₄ClN₄O₄)⁺ [M+H]⁺ 503.1, found 503.2.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(2,2-dichloroacetamido)phenyl)furan-2-carboxamide (6). ¹H NMR (CDCl₃, 400 MHz) δ 9.35 (s, 1H), 8.36 (d, J = 19.6 Hz, 2H), 7.47 (dd, J = 19.7, 8.0 Hz, 3H), 7.28 – 7.09 (m, 9H), 6.27 (s, 1H), 6.12 (d, J = 16.0 Hz, 2H), 5.54 (s, 1H), 4.46 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 169.0, 162.3, 159.9, 151.3, 149.7, 146.0, 145.4, 138.5, 137.8, 137.6, 135.6, 131.8, 130.2, 128.8, 127.6, 127.6, 123.6, 120.4, 117.9, 111.6, 67.0, 63.1, 44.0. MS (ESI) calculated for (C₂₇H₂₃Cl₂N₄O₄)⁺ [M+H]⁺ 537.1, found 537.1.

N-(4-Acrylamidophenyl)-N-(2-(benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide (7). ¹H NMR (CDCl₃, 400 MHz) δ 9.27 (d, J = 38.8 Hz, 1H), 8.33 (s, 2H), 7.47 (s, 4H), 7.25 – 7.06 (m, 9H), 6.28 (dd, J = 25.8, 15.3 Hz, 2H), 6.09 (s, 1H), 5.58 (d, J = 9.9 Hz, 1H), 5.43 (s, 1H), 4.43 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 169.2, 168.6, 164.2, 159.9, 151.2, 149.4, 145.9, 145.2, 139.3, 139.1, 138.6, 137.9, 134.2, 131.5, 131.1, 130.4, 128.7, 128.0, 127.5, 123.5, 120.0, 119.9, 117.8, 111.5, 63.2, 43.9, 40.0, 39.9. MS (ESI) calculated for (C₂₈H₂₅N₄O₄)⁺ [M+H]⁺ 481.2, found 481.2.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(3-(2-chloroacetamido)phenyl)furan-2-carboxamide (8). ¹H NMR (CDCl₃, 400 MHz) δ 8.77 (s, 1H), 8.50 (s, 1H), 8.38 (s, 1H), 7.80 (d, J = 6.3 Hz, 1H), 7.49 (s, 1H), 7.33 – 6.96 (m, 9H), 6.24 (s, 1H), 6.11 (s, 1H), 5.58 (s, 1H), 4.50 – 4.28 (m, 2H), 4.12 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 169.1, 164.5, 159.7, 151.4, 149.6, 146.0, 145.4, 139.8, 138.5, 138.3, 137.9, 130.2, 129.9, 128.8, 127.6, 127.6, 127.3, 123.5, 122.5, 120.6, 117.8, 111.5, 63.6, 43.9, 43.1. MS (ESI) calculated for (C₂₇H₂₄ClN₄O₄)⁺ [M+H]⁺ 503.1, found 503.1.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(3-(2,2-dichloroacetamido)phenyl)furan-2-carboxamide (9). ¹H NMR (CDCl₃, 400 MHz) δ 9.71 (s, 1H), 8.60 (s, 1H), 8.20 (s, 1H), 7.96 (d, J = 5.5 Hz, 1H), 7.77 (s, 1H), 7.41 (s, 1H), 7.41 – 6.97 (m, 8H), 6.25 (d, J = 29.6 Hz, 2H), 6.05 (s, 1H), 5.45 (s, 1H), 4.30 – 4.18 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 169.2, 164.1, 159.9, 146.0, 145.3, 139.7, 138.0, 131.4, 130.0, 128.8, 128.1, 127.7, 127.5, 126.3, 122.2, 120.7, 117.9, 111.5, 63.6, 56.1, 43.9. MS (ESI) calculated for (C₂₇H₂₃Cl₂N₄O₄)⁺ [M+H]⁺ 537.1, found 537.1

N-(3-Acrylamidophenyl)-N-(2-(benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide (10). ¹H NMR (CDCl₃, 400 MHz) δ 8.68 (s, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 7.54 (s, 1H), 7.17 (s, 8H), 6.34 (d, J = 24.5 Hz, 3H), 6.06 (s, 1H), 5.68 (s, 1H), 5.51 (s, 1H), 5.10 (s, 1H), 4.35 (m, 2H), 3.33 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 169.2, 164.1, 159.9, 146.0, 145.3, 139.7, 138.0, 131.4, 130.0, 128.8, 128.1, 127.7, 127.5, 126.3, 122.2, 120.7, 117.9, 111.5, 56.1, 43.9, 32.1, 29.8, 29.5. MS (ESI) calculated for (C₂₈H₂₅N₄O₄)⁺ [M+H]⁺ 481.2, found 481.2.

N-(4-Aminophenyl)-N-(2-(benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide (11). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.95 – 8.83 (m, 1H), 8.75 (d, J = 7.3 Hz, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.84 – 7.75 (m, 1H), 7.69 (s, 1H), 7.29 – 7.13 (m, 9H), 6.37 (d, J = 1.8 Hz, 1H), 6.29 (s, 1H), 5.67 (d, J = 3.3 Hz, 1H), 4.31 (ddd, J = 27.5, 15.3, 5.9 Hz, 2H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 167.2, 158.5, 146.0, 145.8, 144.4, 138.9, 134.4, 131.7, 128.3, 127.2, 126.9, 126.1, 122.0, 117.0, 111.5, 66.4, 62.8, 42.6. MS (ESI) calculated for (C₂₅H₂₃N₄O₃)⁺ [M+H]⁺ 427.2, found 427.2.

N-(3-Aminophenyl)-N-(2-(benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide (12). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.67 (s, 1H), 8.37 (s, 2H), 7.73 (s, 1H), 7.47 (d, J = 7.1 Hz, 1H), 7.33 – 7.03 (m, 6H), 6.82 (s, 2H), 6.46 – 6.27 (m, 2H), 6.13 (s, 1H), 5.46 (s, 1H), 5.12 (s, 2H), 4.34 (s, 2H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 168.9, 158.1, 151.4, 149.3, 148.8, 146.2, 145.1, 139.7, 139.3, 137.7, 130.5, 128.8, 128.2, 127.1, 126.7, 122.7, 118.1, 116.3, 116.0, 113.8, 111.3, 62.6, 42.3. MS (ESI) calculated for (C₂₅H₂₃N₄O₃)⁺ [M+H]⁺ 427.2, found 427.2.

N-(tert-Butyl)-2-(N-(4-(tert-butyl)phenyl)-2-chloroacetamido)-2-(pyridin-3-yl)acetamide (13). ¹H NMR (CDCl₃, 400 MHz) δ 8.52 – 8.15 (m, 3H), 7.34 (d, J = 8.0 Hz, 2H), 7.19 (s, 2H), 6.98 (dd, J = 7.7, 4.8 Hz, 1H), 6.11 (s, 1H), 5.93 (s, 1H), 3.81 (s, 2H), 1.30 (s, 9H), 1.20 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.6, 167.2, 152.6, 151.4, 149.7, 137.9, 135.2, 130.3, 129.8, 126.4, 123.0, 63.3, 51.9, 42.7, 34.7, 31.2, 28.7, 28.7. HRMS (ESI) calculated for (C₂₃H₃₁ClN₃O₂)⁺ [M+H]⁺ 416.2105, found 416.2093.

N-([1,1’-Biphenyl]-4-yl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-chloroacetamide (14). ¹H NMR (CDCl₃, 400 MHz) δ 8.49 – 8.39 (m, 2H), 7.55 – 7.38 (m, 10H), 7.10 – 6.99 (m, 1H), 6.02 (s, 1H), 5.96 (s, 1H), 3.89 (s, 2H), 1.36 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.6, 167.2, 151.5, 150.0, 142.1, 139.5, 138.0, 137.0, 130.9, 130.2, 129.1, 129.0, 128.1, 127.3, 127.2, 123.3, 63.3, 52.1, 42.6, 28.8, 28.8. MS (ESI) calculated for (C₂₅H₂₇ClN₃O₂)⁺ [M+H]⁺ 436.2, found 436.2.

N-(tert-Butyl)-2-(2-chloro-N-(4-(2-chloroacetamido)phenyl)acetamido)-2-(pyridin-3-yl)acetamide (15). ¹H NMR (CDCl₃, 400 MHz) δ 8.46 (d, J = 3.6 Hz, 1H), 8.40 (s, 1H), 8.27 (s, 1H), 7.42 (d, J = 7.7 Hz, 3H), 7.26 (d, J = 1.2 Hz, 2H), 7.13 – 7.05 (m, 1H), 5.98 (s, 1H), 5.71 (s, 1H), 4.17 (s, 2H), 3.83 (s, 2H), 1.36 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.5, 167.2, 164.0, 151.5, 150.1, 137.9, 137.6, 134.3, 131.6, 130.0, 123.4, 120.5, 63.1, 52.2, 42.9, 42.4, 29.9, 28.8. MS (ESI) calculated for (C₂₁H₂₅Cl₂N₄O₃)⁺ [M+H]⁺ 451.1, found 451.2.

N-(tert-Butyl)-2-(2-chloro-N-(4-(2,2-dichloroacetamido)phenyl)acetamido)-2-(pyridin-3-yl)acetamide (16). ¹H NMR (CDCl₃, 400 MHz) δ 8.48 (d, J = 4.1 Hz, 1H), 8.40 (s, 1H), 8.25 (s, 1H), 7.52 – 7.35 (m, 3H), 7.11 (dd, J = 7.6, 4.9 Hz, 1H), 6.03 (s, 1H), 5.98 (s, 1H), 5.68 (s, 1H), 5.30 (s, 1H), 5.12 (s, 1H), 3.83 (s, 2H), 1.36 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.5, 167.2, 161.9, 151.5, 150.2, 137.9, 137.2, 134.7, 131.7, 130.0, 123.5, 120.7, 66.8, 63.1, 52.2, 42.4, 38.8, 29.9, 28.8. MS (ESI) calculated for (C₂₁H₂₄Cl₃N₄O₃)⁺ [M+H]⁺ 485.1, found 485.1.

N-(tert-Butyl)-2-(2-chloro-N-(4-(oxiran-2-ylmethyl)phenyl)acetamido)-2-(pyridin-3-yl)acetamide (17). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (s, 1H), 8.06 (s, 1H), 7.48 (s, 1H), 7.19 (s, 1H), 7.08 (s, 4H), 6.28 (s, 1H), 5.87 (s, 1H), 5.08 – 4.99 (m, 1H), 3.83 (s, 2H), 3.33 (s, 2H), 2.78 (s, 1H), 2.54 (s, 1H), 1.33 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.34, 167.27, 166.35, 144.76, 141.88, 140.79, 138.82, 136.20, 136.00, 135.38, 134.31, 134.08, 132.64, 132.11, 132.01, 130.35, 130.21, 130.04, 129.93, 129.80, 129.71, 129.57, 128.42, 127.95, 116.69, 77.21, 62.24, 52.06, 52.04, 51.79, 51.77, 46.63, 42.24, 39.53, 38.07, 35.09, 34.63, 33.17, 31.55, 28.51, 25.24, 22.62, 22.18, 20.67, 14.10, 13.86. MS (ESI) calculated for (C₂₂H₂₇ClN₃O₃)⁺ [M+H]⁺ 416.2, found 416.1.

N-(tert-Butyl)-2-(2-chloro-N-(3-(2-chloroacetamido)phenyl)acetamido)-2-(pyridin-3-yl)acetamide (18). ¹H NMR (CDCl₃, 400 MHz) δ 8.50 – 8.37 (m, 2H), 8.29 (s, 1H), 7.52 – 7.44 (m, 1H), 7.25 (s, 1H), 7.16 – 7.03 (m, 1H), 5.93 (s, 1H), 5.75 (s, 1H), 5.11 (s, 1H), 4.16 (s, 2H), 3.89 (s, 2H), 1.35 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.5, 167.0, 164.1, 151.5, 150.1, 138.0, 137.9, 133.8, 130.2, 129.9, 127.0, 123.4, 122.0, 120.8, 53.9, 52.2, 42.9, 42.5, 38.8, 29.9, 29.4, 28.8. MS (ESI) calculated for (C₂₁H₂₅Cl₂N₄O₃)⁺ [M+H]⁺ 451.1, found 451.1.

N-(tert-Butyl)-2-(2-chloro-N-(3-(2,2-dichloroacetamido)phenyl)acetamido)-2-(pyridin-3-yl)acetamide (19). ¹H NMR (CDCl₃, 400 MHz) δ 8.44 (d, J = 19.5 Hz, 2H), 7.71 (s, 1H), 7.52 (s, 1H), 7.42 (s, 1H), 7.11 (s, 2H), 6.21 (s, 1H), 5.90 (s, 1H), 5.65 (s, 1H), 5.11 (s, 1H), 4.19 (s, 2H), 3.94 (d, J = 13.8 Hz, 1H), 1.29 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 167.5, 162.1, 162.1, 151.4, 150.2, 137.8, 131.1, 130.3, 129.8, 129.0, 127.2, 123.6, 122.3, 120.9, 66.8, 63.8, 52.3, 42.9, 29.9, 28.7. MS (ESI) calculated for (C₂₁H₂₄Cl₃N₄O₃)⁺ [M+H]⁺ 485.1, found 485.1.

N-(4-Aminophenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-chloroacetamide (20). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.74 – 8.62 (m, 2H), 8.09 – 7.99 (m, 2H), 7.80 – 7.70 (m, 1H), 7.35 (s, 2H), 7.12 (d, J = 7.9 Hz, 2H), 6.16 (s, 1H), 5.75 (s, 1H), 4.01 (s, 2H), 1.19 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 166.2, 165.8, 151.6, 144.4, 144.2, 142.6, 134.7, 131.9, 125.9, 121.6, 117.8, 66.4, 61.7, 50.8, 43.1, 28.2. MS (ESI) calculated for (C₁₉H₂₄ClN₄O₂)⁺ [M+H]⁺ 375.2, found 375.1.

N-(3-Aminophenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-chloroacetamide (21). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.70 (d, J = 5.8 Hz, 2H), 8.12 – 8.00 (m, 2H), 7.82 – 7.71 (m, 1H), 7.36 – 7.25 (m, 2H), 7.20 (d, J = 7.7 Hz, 1H), 6.18 (s, 1H), 4.03 (d, J = 5.7 Hz, 2H), 1.19 (d, J = 9.4 Hz, 9H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 166.3, 165.4, 144.7, 144.1, 142.5, 138.7, 134.4, 130.2, 128.4, 126.0, 124.4, 122.4, 61.6, 50.8, 43.0, 28.2. MS (ESI) calculated for (C₁₉H₂₄ClN₄O₂)⁺ [M+H]⁺ 375.2, found 375.2.

N-(tert-Butyl)-2-(2-chloro-N-(4-methoxyphenyl)acetamido)-2-(pyridin-3-yl)acetamide (22). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (dd, J = 4.8, 1.6 Hz, 1H), 8.38 (d, J = 2.3 Hz, 1H), 7.59 (s, 1H), 7.39 (dt, J = 8.0, 2.0 Hz, 1H), 7.06 (dd, J = 8.0, 4.9 Hz, 1H), 6.83 (s, 1H), 6.59 (s, 1H), 6.40 (s, 1H), 5.97 (s, 1H), 5.80 (s, 1H), 3.83 (s, 2H), 3.74 (s, 3H), 1.34 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.54, 167.42, 159.75, 151.31, 149.66, 138.04, 131.54, 130.11, 123.10, 114.47, 62.84, 55.38, 51.90, 42.39, 28.60. MS (ESI) calculated for (C₂₀H₂₅ClN₃O₃)⁺ [M+H]⁺ 390.1, found 390.2.

N-Benzyl-2-(N-(4-(tert-butyl)phenyl)-2-chloroacetamido)-2-(pyridin-3-yl)acetamide (23). ¹H NMR (CDCl₃, 400 MHz) δ 8.36 (d, J = 28.7 Hz, 2H), 7.37–7.22 (m, 8H), 6.94 (d, J = 45.9 Hz, 3H), 6.06 (s, 1H), 4.46 (s, 2H), 3.82 (s, 2H), 1.22 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ 168.6, 167.3, 152.7, 151.4, 149.9, 138.1, 138.0, 135.1, 129.9, 128.8, 127.7, 127.6, 126.5, 123.1, 62.9, 43.9, 42.8, 34.8, 31.3. MS (ESI) calculated for (C₂₆H₂₉ClN₃O₂)⁺ [M+H]⁺ 450.2, found 450.2.

N-Butyl-2-(N-(4-(tert-butyl)phenyl)-2-chloroacetamido)-2-(pyridin-3-yl)acetamide (24). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (dd, J = 4.9, 1.6 Hz, 1H), 8.40 (d, J = 2.3 Hz, 1H), 7.42 (dt, J = 7.9, 1.9 Hz, 1H), 7.24 (s, 2H), 7.04 (dd, J = 8.1, 4.9 Hz, 1H), 6.08 (t, J = 5.8 Hz, 1H), 5.99 (s, 1H), 3.85 (s, 2H), 3.29 (td, J = 7.2, 5.7 Hz, 2H), 1.54 – 1.40 (m, 2H), 1.31 (dt, J = 14.9, 7.4 Hz, 2H), 1.24 (d, J = 1.6 Hz, 9H), 0.88 (td, J = 7.3, 1.6 Hz, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.22, 167.23, 152.66, 151.22, 149.78, 137.96, 135.18, 129.98, 129.58, 126.44, 122.96, 63.05, 42.51, 39.73, 34.67, 31.42, 31.16, 20.01, 13.67. MS (ESI) calculated for (C₂₃H₃₁ClN₃O₂)⁺ [M+H]⁺ 416.2, found 416.2.

N-(4-(tert-Butyl)phenyl)-2-chloro-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)acetamide (26). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (dd, J = 4.8, 1.7 Hz, 1H), 8.40 (d, J = 2.2 Hz, 1H), 7.41 (dt, J = 8.1, 2.0 Hz, 1H), 7.32 – 7.13 (m, 2H), 7.04 (dd, J = 8.0, 4.9 Hz, 1H), 5.98 (s, 1H), 5.91 (d, J = 8.1 Hz, 1H), 3.88 – 3.82 (m, 2H), 3.79 (dq, J = 11.5, 3.9 Hz, 1H), 2.03 – 1.89 (m, 1H), 1.89 – 1.78 (m, 1H), 1.73 – 1.51 (m, 3H), 1.44 – 1.28 (m, 2H), 1.24 (d, J = 1.6 Hz, 9H), 1.20 – 1.00 (m, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.30, 167.24, 152.62, 151.12, 149.56, 138.08, 135.18, 130.19, 129.62, 126.40, 122.99, 62.94, 48.94, 42.47, 34.66, 32.78, 32.72, 31.16, 25.41, 24.76, 24.69. MS (ESI) calculated for (C₂₅H₃₃ClN₃O₂)⁺ [M+H]⁺ 442.2, found 442.2.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)acrylamide (27). ¹H NMR (400 MHz, Chloroform-d) δ 8.43 (dd, J = 5.1, 1.7 Hz, 2H), 7.41 (dt, J = 8.0, 2.0 Hz, 1H), 7.33 – 7.12 (m, 2H), 7.07 – 7.00 (m, 1H), 6.90 (s, 2H), 6.39 (dd, J = 16.8, 1.9 Hz, 1H), 6.19 (s, 1H), 6.04 (s, 1H), 5.96 (ddd, J = 17.4, 10.2, 1.7 Hz, 1H), 5.54 (dd, J = 10.3, 1.9 Hz, 1H), 1.35 (d, J = 1.6 Hz, 9H), 1.25 (d, J = 1.6 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.95, 166.42, 151.84, 151.10, 149.27, 138.11, 136.02, 130.70, 129.74, 128.62, 128.43, 126.09, 122.75, 62.98, 51.68, 34.61, 31.21, 28.64. MS (ESI) calculated for (C₂₄H₃₂N₃O₂)⁺ [M+H]⁺ 394.2, found 394.3.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(tert-butyl)phenyl)acrylamide (28). ¹H NMR (400 MHz, Chloroform-d) δ 8.48 – 8.36 (m, 2H), 7.45 (dt, J = 8.0, 2.0 Hz, 1H), 7.26 (ddt, J = 23.5, 16.4, 8.3 Hz, 7H), 7.03 (dd, J = 8.1, 4.9 Hz, 1H), 6.86 (s, 2H), 6.75 (t, J = 5.8 Hz, 1H), 6.39 (dd, J = 16.8, 1.9 Hz, 1H), 6.14 (s, 1H), 5.96 (ddd, J = 16.8, 10.3, 1.6 Hz, 1H), 5.55 (dd, J = 10.4, 1.9 Hz, 1H), 4.50 (d, J = 5.8 Hz, 2H), 1.25 (d, J = 1.6 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.82, 166.56, 151.90, 151.25, 149.62, 138.02, 136.00, 130.31, 129.71, 128.85, 128.68, 128.29, 127.66, 127.47, 126.14, 122.82, 62.78, 43.81, 34.61, 31.21. MS (ESI) calculated for (C₂₇H₃₀N₃O₂)⁺ [M+H]⁺ 428.2, found 428.2.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)acrylamide (29). ¹H NMR (400 MHz, Chloroform-d) δ 8.44 (dd, J = 4.9, 1.7 Hz, 2H), 7.44 (dt, J = 8.0, 2.0 Hz, 1H), 7.30 – 7.17 (m, 2H), 7.04 (dd, J = 8.1, 5.0 Hz, 1H), 6.91 (s, 2H), 6.39 (dt, J = 16.8, 1.8 Hz, 1H), 6.25 – 6.16 (m, 1H), 6.08 (d, J = 1.6 Hz, 1H), 5.96 (ddd, J = 16.9, 10.3, 1.6 Hz, 1H), 5.54 (dt, J = 10.3, 1.8 Hz, 1H), 3.82 (ddt, J = 14.9, 10.5, 3.7 Hz, 1H), 2.02 – 1.90 (m, 1H), 1.90 – 1.80 (m, 1H), 1.79 – 1.50 (m, 4H), 1.42 – 1.30 (m, 2H), 1.25 (d, J = 1.6 Hz, 9H), 1.21 – 1.10 (m, 2H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.86, 166.50, 151.84, 151.11, 149.36, 138.01, 136.12, 130.69, 129.71, 128.69, 128.40, 126.11, 122.82, 62.78, 48.71, 34.60, 32.82, 32.76, 31.21, 25.45, 24.73, 24.68. MS (ESI) calculated for (C₂₆H₃₄N₃O₂)⁺ [M+H]⁺ 420.3, found 420.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)oxirane-2-carboxamide (more active diastereomer of 30). ¹H NMR (400 MHz, Chloroform-d) δ 8.47 (dd, J = 4.8, 1.6 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 7.52 (dt, J = 8.0, 2.0 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.08 (dd, J = 8.2, 4.9 Hz, 3H), 5.96 (s, 1H), 5.91 (d, J = 8.0 Hz, 1H), 3.87 – 3.73 (m, 1H), 3.14 (dt, J = 4.0, 1.9 Hz, 1H), 3.00 (dd, J = 6.7, 2.3 Hz, 1H), 2.71 (dd, J = 6.6, 4.2 Hz, 1H), 2.01 – 1.91 (m, 1H), 1.86 (d, J = 12.6 Hz, 1H), 1.63 (s, 3H), 1.43 – 1.28 (m, 3H), 1.26 (d, J = 1.5 Hz, 9H), 1.20 – 1.09 (m, 2H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.44, 167.18, 152.39, 151.24, 149.70, 137.96, 135.18, 130.21, 129.51, 126.40, 122.94, 62.99, 48.89, 47.28, 46.66, 34.65, 32.73, 32.68, 31.16, 25.40, 24.75, 24.71. HRMS (ESI) calculated for (C₂₆H₃₄N₃O₃)⁺ [M+H]⁺ 436.2600, found 436.2587.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)oxirane-2-carboxamide (less active diastereomer of 30). ¹H NMR (400 MHz, Chloroform-d) δ 8.54 – 8.33 (m, 2H), 7.39 (dd, J = 8.0, 2.1 Hz, 1H), 7.25 – 7.17 (m, 2H), 7.05 (dd, J = 8.0, 4.9 Hz, 3H), 6.09 (s, 1H), 5.95 (d, J = 8.1 Hz, 1H), 3.78 (tdd, J = 11.0, 7.5, 3.9 Hz, 1H), 3.12 (dt, J = 4.2, 2.0 Hz, 1H), 2.98 (dd, J = 6.7, 2.3 Hz, 1H), 2.70 (dd, J = 6.6, 4.2 Hz, 1H), 1.98 – 1.75 (m, 2H), 1.75 – 1.48 (m, 3H), 1.45 – 1.27 (m, 2H), 1.24 (d, J = 1.7 Hz, 9H), 1.19 – 1.02 (m, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.47, 167.46, 152.34, 151.27, 149.72, 137.97, 134.58, 130.01, 129.98, 126.25, 122.92, 62.54, 48.86, 47.28, 46.84, 34.63, 32.78, 32.71, 31.16, 25.39, 24.75, 24.66. MS (ESI) calculated for (C₂₆H₃₄N₃O₃)⁺ [M+H]⁺ 436.3, found 436.2.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-oxo-3-phenylpropanamide (31). ¹H NMR (400 MHz, Chloroform-d) δ 8.50 (dd, J = 4.9, 1.6 Hz, 1H), 8.43 (d, J = 2.2 Hz, 1H), 7.40 (dt, J = 8.0, 2.0 Hz, 2H), 7.23 – 7.10 (m, 4H), 7.07 (dd, J = 8.0, 4.9 Hz, 2H), 6.86 – 6.58 (m, 3H), 5.89 (s, 1H), 5.81 (s, 1H), 4.20 (s, 1H), 3.15 (s, 1H), 2.86 (dd, J = 13.6, 4.3 Hz, 1H), 2.58 (dd, J = 13.6, 7.6 Hz, 1H), 1.33 (d, J = 1.7 Hz, 9H), 1.27 (d, J = 1.7 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 175.08, 167.16, 152.50, 150.91, 149.42, 138.45, 136.92, 134.87, 130.44, 130.07, 129.21, 128.26, 126.65, 123.02, 70.41, 63.62, 51.83, 41.43, 34.70, 31.21, 28.59. MS (ESI) calculated for (C₃₀H₃₆N₃O₃)⁺ [M+H]⁺ 486.3, found 486.3.

N-(tert-Butyl)-2-(N-(4-(tert-butyl)phenyl)-2-hydroxyacetamido)-2-(pyridin-3-yl)acetamide (32). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (dd, J = 4.8, 1.6 Hz, 1H), 8.41 (d, J = 2.3 Hz, 1H), 7.36 (dt, J = 8.0, 2.0 Hz, 1H), 7.21 (s, 2H), 7.03 (dd, J = 8.0, 4.8 Hz, 2H), 5.94 (s, 1H), 5.75 (s, 1H), 3.79 (d, J = 2.5 Hz, 2H), 1.34 (d, J = 1.4 Hz, 9H), 1.23 (d, J = 1.4 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 173.06, 167.46, 152.52, 151.19, 149.70, 137.85, 133.65, 129.97, 129.68, 126.29, 122.92, 63.11, 61.04, 51.89, 34.63, 31.15, 28.60. MS (ESI) calculated for (C₂₃H₃₂N₃O₃)⁺ [M+H]⁺ 398.2, found 398.2.

2-((4-(tert-Butyl)phenyl)(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)amino)-2-oxoethyl acetate (33). ¹H NMR (400 MHz, Chloroform-d) δ 8.46 – 8.41 (m, 1H), 8.38 (d, J = 2.3 Hz, 1H), 7.37 (dt, J = 8.0, 2.0 Hz, 1H), 7.34 – 7.10 (m, 3H), 7.03 (dd, J = 8.0, 4.9 Hz, 1H), 6.08 (s, 1H), 5.96 (s, 1H), 4.39 – 4.21 (m, 2H), 2.12 (s, 3H), 1.37 (s, 9H), 1.24 (d, J = 1.7 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 170.64, 167.75, 167.51, 152.62, 151.25, 149.46, 138.14, 134.56, 130.07, 129.33, 126.54, 122.75, 62.88, 62.16, 51.83, 34.66, 31.16, 28.57, 20.49. MS (ESI) calculated for (C₂₅H₃₄N₃O₄)⁺ [M+H]⁺ 440.3, found 440.3.

N-(4-(tert-Butyl)phenyl)-2-cyano-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)acetamide (34). ¹H NMR (399 MHz, Chloroform-d) δ 8.44 (dt, J = 3.5, 1.7 Hz, 1H), 8.39 (d, J = 2.5 Hz, 1H), 7.45 – 7.27 (m, 2H), 7.20 – 6.93 (m, 2H), 6.04 (d, J = 7.9 Hz, 1H), 6.01 (d, J = 2.1 Hz, 1H), 3.75 (dtt, J = 11.6, 8.3, 4.0 Hz, 1H), 3.33 – 3.16 (m, 2H), 1.93 (d, J = 12.5 Hz, 1H), 1.81 (d, J = 12.8 Hz, 1H), 1.75 – 1.48 (m, 3H), 1.42 – 1.26 (m, 2H), 1.23 (d, J = 2.2 Hz, 9H), 1.18 – 1.00 (m, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.15, 163.09, 152.87, 151.21, 149.45, 137.94, 135.10, 130.07, 129.09, 123.01, 113.99, 62.83, 49.12, 34.67, 32.72, 32.69, 31.13, 26.40, 25.38, 24.80, 24.71. MS (ESI) calculated for (C₂₆H₃₃N₄O₂)⁺ [M+H]⁺ 433.3, found 433.2.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3-methyloxirane-2-carboxamide (more active diastereomer of 35). ¹H NMR (400 MHz, Chloroform-d) δ 8.54 – 8.36 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.17 – 7.09 (m, 1H), 7.05 (d, J = 25.6 Hz, 2H), 6.03 – 5.88 (m, 2H), 3.80 (d, J = 9.5 Hz, 1H), 3.28 – 3.10 (m, 1H), 2.84 (d, J = 2.0 Hz, 1H), 2.05 – 1.91 (m, 1H), 1.85 (d, J = 12.4 Hz, 1H), 1.48 – 1.30 (m, 2H), 1.26 (d, J = 1.6 Hz, 8H), 1.19 – 1.13 (m, 1H), 1.11 (dd, J = 5.5, 1.9 Hz, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.55, 167.19, 152.47, 151.02, 149.50, 138.22, 135.32, 130.33, 129.40, 126.38, 123.02, 62.87, 54.74, 54.55, 48.90, 34.68, 32.78, 32.72, 31.17, 25.41, 24.73, 24.70, 16.70. MS (ESI) calculated for (C₂₇H₃₆N₃O₃)⁺ [M+H]⁺ 450.3, found 450.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3-methyloxirane-2-carboxamide (less active diastereomer of 35). ¹H NMR (400 MHz, Chloroform-d) δ 8.47 (dt, J = 4.8, 1.4 Hz, 1H), 8.42 (d, J = 2.2 Hz, 1H), 7.38 (dt, J = 8.0, 2.1 Hz, 1H), 7.25 – 7.20 (m, 2H), 7.17 – 6.74 (m, 3H), 6.06 (s, 1H), 5.83 (d, J = 8.1 Hz, 1H), 3.86 – 3.69 (m, 1H), 3.18 (ddd, J = 7.2, 3.7, 2.1 Hz, 1H), 2.80 (s, 1H), 2.01 – 1.88 (m, 1H), 1.82 (d, J = 12.7 Hz, 1H), 1.71 (s, 2H), 1.43 – 1.28 (m, 2H), 1.24 (q, J = 1.4 Hz, 9H), 1.20 – 1.12 (m, 1H), 1.12 – 1.07 (m, 3H), 1.07 – 0.93 (m, 1H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.61, 167.51, 152.35, 151.37, 149.83, 137.95, 134.64, 130.04, 129.91, 126.13, 122.90, 62.35, 54.84, 54.69, 48.86, 34.64, 32.80, 32.72, 31.16, 25.40, 24.74, 24.66, 16.70. MS (ESI) calculated for (C₂₇H₃₆N₃O₃)⁺ [M+H]⁺ 450.3, found 450.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3,3-dimethyloxirane-2-carboxamide (36). ¹H NMR (400 MHz, Chloroform-d) δ 8.50 – 8.37 (m, 2H), 7.41 (dt, J = 7.9, 2.0 Hz, 1H), 7.32 – 7.19 (m, 2H), 7.13 – 6.78 (m, 3H), 6.14 – 5.88 (m, 2H), 3.82 – 3.67 (m, 1H), 2.98 – 2.90 (m, 1H), 1.93 (d, J = 11.7 Hz, 1H), 1.82 (d, J = 13.0 Hz, 1H), 1.75 – 1.49 (m, 3H), 1.43 – 1.27 (m, 4H), 1.23 (d, J = 2.2 Hz, 8H), 1.20 – 0.98 (m, 4H), 0.98 – 0.80 (m, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.34, 167.58, 152.50, 151.31, 149.60, 138.37, 134.24, 130.16, 129.99, 129.61, 126.05, 122.98, 122.90, 61.69, 60.21, 48.87, 34.65, 32.79, 31.15, 25.41, 24.80, 23.28, 18.59. HRMS (ESI) calculated for (C₂₈H₃₈N₃O₃)⁺ [M+H]⁺ 464.2913, found 464.2898.

N-(4-(tert-Butyl)phenyl)-N-(2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-methyloxirane-2-carboxamide (37). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (td, J = 5.1, 2.6 Hz, 1H), 8.41 (s, 1H), 7.47 – 7.36 (m, 1H), 7.25 – 7.20 (m, 2H), 7.06 (dd, J = 7.8, 4.8 Hz, 1H), 6.98 (s, 2H), 5.95 (d, J = 19.0 Hz, 1H), 5.90 – 5.82 (m, 1H), 3.89 – 3.70 (m, 1H), 2.90 (dd, J = 27.3, 5.0 Hz, 1H), 2.49 (t, J = 6.4 Hz, 1H), 1.90 (d, J = 26.6 Hz, 4H), 1.75 – 1.50 (m, 3H), 1.33 (d, J = 2.2 Hz, 3H), 1.30 (d, J = 2.2 Hz, 1H), 1.24 (d, J = 1.5 Hz, 9H), 1.19 – 1.03 (m, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 152.04, 150.87, 149.35, 149.28, 138.15, 130.08, 129.69, 125.81, 125.63, 122.98, 63.40, 57.06, 53.82, 53.64, 48.82, 34.62, 32.80, 32.72, 31.19, 25.41, 24.72, 24.65, 20.01. MS (ESI) calculated for (C₂₇H₃₆N₃O₃)⁺ [M+H]⁺ 450.3, found 450.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)oxirane-2-carboxamide (more active diastereomer of 38). ¹H NMR (399 MHz, Chloroform-d) δ 8.55 – 8.35 (m, 2H), 7.50 (dt, J = 8.1, 2.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 2H), 7.19 – 6.81 (m, 3H), 5.97 (s, 1H), 5.94 (d, J = 2.2 Hz, 1H), 3.15 (dd, J = 4.2, 2.3 Hz, 1H), 2.99 (dd, J = 6.6, 2.5 Hz, 1H), 2.71 (dt, J = 6.4, 3.0 Hz, 1H), 1.35 (d, J = 2.2 Hz, 9H), 1.25 (d, J = 2.2 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.41, 167.28, 152.44, 149.61, 138.08, 135.10, 130.22, 129.54, 126.43, 122.91, 63.31, 51.86, 47.28, 46.70, 34.67, 31.18, 28.59. MS (ESI) calculated for (C₂₄H₃₂N₃O₃)⁺ [M+H]⁺ 410.2, found 410.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)oxirane-2-carboxamide (less active diastereomer of 38). ¹H NMR (399 MHz, Chloroform-d) δ 8.45 (dt, J = 3.7, 1.7 Hz, 1H), 8.40 (d, J = 2.5 Hz, 1H), 7.38 (dt, J = 8.2, 2.1 Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.03 (dt, J = 7.3, 3.5 Hz, 3H), 6.00 (d, J = 2.2 Hz, 1H), 5.93 (s, 1H), 3.12 (dt, J = 4.8, 2.5 Hz, 1H), 2.98 (dd, J = 6.8, 2.5 Hz, 1H), 2.70 (dt, J = 6.4, 3.2 Hz, 1H), 1.32 (d, J = 2.1 Hz, 9H), 1.23 (d, J = 2.1 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.38, 167.55, 152.30, 151.21, 149.57, 138.01, 134.58, 130.07, 130.02, 126.24, 122.89, 62.93, 51.81, 47.32, 46.86, 34.63, 31.16, 28.60. MS (ESI) calculated for (C₂₄H₃₂N₃O₃)⁺ [M+H]⁺ 410.2, found 410.2.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3-methyloxirane-2-carboxamide (more active diastereomer of 39). ¹H NMR (400 MHz, Chloroform-d) δ 8.51 – 8.35 (m, 2H), 7.48 (dt, J = 8.1, 2.0 Hz, 1H), 7.27 (s, 1H), 7.05 (dt, J = 7.2, 3.6 Hz, 3H), 6.02 – 5.88 (m, 2H), 3.17 (tq, J = 5.6, 2.9, 2.4 Hz, 1H), 2.84 (d, J = 2.1 Hz, 1H), 1.39 – 1.32 (m, 9H), 1.27 – 1.24 (m, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.38, 152.40, 151.25, 149.67, 137.98, 135.21, 130.23, 129.51, 126.30, 122.89, 63.10, 54.72, 54.56, 51.85, 34.67, 31.17, 28.59, 16.71. MS (ESI) calculated for (C₂₅H₃₄N₃O₃)⁺ [M+H]⁺ 424.3, found 424.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3-methyloxirane-2-carboxamide (less active diastereomer of 39). ¹H NMR (400 MHz, Chloroform-d) δ 8.55 – 8.34 (m, 2H), 7.49 – 7.34 (m, 1H), 7.23 (d, J = 9.5 Hz, 3H), 7.14 – 6.82 (m, 3H), 5.98 (d, J = 2.0 Hz, 1H), 5.89 (s, 1H), 3.17 (dh, J = 5.4, 2.9, 2.4 Hz, 1H), 2.80 (d, J = 2.2 Hz, 1H), 1.32 (d, J = 2.0 Hz, 9H), 1.23 (d, J = 2.1 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.52, 167.60, 151.20, 149.59, 138.05, 134.60, 130.08, 126.11, 122.91, 62.70, 54.84, 54.75, 51.80, 34.63, 31.16, 28.60, 16.70. MS (ESI) calculated for (C₂₅H₃₄N₃O₃)⁺ [M+H]⁺ 424.3, found 424.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-3,3-dimethyloxirane-2-carboxamide (40). ¹H NMR (400 MHz, Chloroform-d) δ 8.52 – 8.43 (m, 2H), 7.65 – 7.50 (m, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.08 (ddd, J = 19.8, 7.9, 4.9 Hz, 1H), 6.98 (s, 2H), 6.12 (d, J = 33.8 Hz, 1H), 5.98 (dd, J = 25.5, 2.3 Hz, 1H), 2.94 (dd, J = 6.2, 2.3 Hz, 1H), 1.41 – 1.29 (m, 12H), 1.25 (d, J = 2.7 Hz, 9H), 0.94 (dd, J = 20.2, 2.4 Hz, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.47, 152.51, 151.08, 150.63, 149.28, 149.14, 138.74, 138.18, 135.29, 130.69, 130.11, 129.32, 126.30, 126.10, 123.08, 122.87, 63.15, 61.88, 61.63, 61.39, 60.18, 51.87, 51.72, 34.67, 31.15, 28.59, 28.52, 23.34, 23.27, 18.51, 18.41. MS (ESI) calculated for (C₂₆H₂₆N₃O₃)⁺ [M+H]⁺ 438.3, found 438.3.

N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-2-methyloxirane-2-carboxamide (41). ¹H NMR (400 MHz, Chloroform-d) δ 8.43 (ddd, J = 7.1, 5.0, 2.0 Hz, 1H), 8.38 (t, J = 2.4 Hz, 1H), 7.42 – 7.30 (m, 1H), 7.22 (dt, J = 9.0, 3.0 Hz, 2H), 7.06 – 6.84 (m, 3H), 5.96 – 5.73 (m, 2H), 2.94 (d, J = 2.3 Hz, 1H), 2.86 (d, J = 2.3 Hz, 1H), 1.34 (dd, J = 9.2, 1.9 Hz, 9H), 1.24 (d, J = 2.1 Hz, 12H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.52, 167.46, 151.30, 151.23, 149.68, 149.57, 137.99, 137.83, 130.19, 130.09, 129.77, 125.74, 125.55, 122.80, 63.66, 63.59, 53.84, 53.66, 51.78, 51.75, 34.60, 31.18, 28.60, 20.02. MS (ESI) calculated for (C₂₅H₃₄N₃O₃)⁺ [M+H]⁺ 424.3, found 424.3.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(tert-butyl)phenyl)oxirane-2-carboxamide (more active diastereomer of 42). ¹H NMR (399 MHz, Chloroform-d) δ 8.52 – 8.37 (m, 2H), 7.53 (dt, J = 8.0, 2.0 Hz, 1H), 7.37 – 7.20 (m, 7H), 7.06 (dt, J = 7.1, 3.3 Hz, 3H), 6.55 (t, J = 5.5 Hz, 1H), 6.03 (d, J = 2.0 Hz, 1H), 4.49 (d, J = 5.8 Hz, 2H), 3.15 (dt, J = 4.6, 2.3 Hz, 1H), 2.99 (dd, J = 6.7, 2.3 Hz, 1H), 2.70 (dt, J = 6.3, 3.0 Hz, 1H), 1.26 (d, J = 2.1 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.56, 168.08, 152.54, 151.30, 149.89, 138.10, 137.71, 135.18, 129.89, 129.43, 128.72, 127.73, 127.54, 126.51, 123.00, 63.13, 47.26, 46.70, 43.93, 34.68, 31.18. HRMS (ESI) calculated for (C₂₇H₃₀N₃O₃)⁺ [M+H]⁺ 444.2287, found 444.2276.

N-(2-(Benzylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(tert-butyl)phenyl)oxirane-2-carboxamide (less active diastereomer of 42). ¹H NMR (399 MHz, Chloroform-d) δ 8.50 – 8.29 (m, 2H), 7.36 (dd, J = 7.8, 2.1 Hz, 1H), 7.33 – 7.15 (m, 7H), 7.02 (dd, J = 7.9, 4.8 Hz, 3H), 6.72 (d, J = 6.0 Hz, 1H), 6.14 (d, J = 2.2 Hz, 1H), 4.52 (dd, J = 14.8, 5.8 Hz, 1H), 4.47 – 4.33 (m, 1H), 3.11 (dt, J = 4.8, 2.4 Hz, 1H), 2.95 (dd, J = 6.8, 2.5 Hz, 1H), 2.68 (dt, J = 6.5, 3.2 Hz, 1H), 1.24 (d, J = 2.2 Hz, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 168.55, 168.54, 152.38, 151.27, 149.70, 138.12, 137.85, 134.43, 130.10, 129.73, 128.66, 127.61, 127.48, 126.25, 122.92, 62.48, 47.24, 46.87, 43.78, 34.64, 31.17, 29.68. MS (ESI) calculated for (C₂₇H₃₀N₃O₃)⁺ [M+H]⁺ 444.2, found 444.2.

Expression and purification of SARS-CoV-2 Mpro.

The SARS-CoV-2 plasmid²⁹ was obtained as a gift from Professor Rolf Hilgenfeld at University of Luebeck, Germany. E. coli BL21 (Rosetta strain, Agilent) was transformed with the plasmids and cultured at 37 °C in LB medium in the presence of an appropriate antibiotics according to the vector. Upon reaching an optical density of ~1.3 at 600 nm, protein expression was induced by adding 0.5 mM isopropylthiogalactoside at 16 °C overnight. Cells were harvested, lysed, and centrifuged at 20,000 rpm for 20 min. The supernatant was collected and applied to an affinity column chromatography using immobilized metal affinity chromatography (IMAC) beads (GE Healthcare). HRV3C protease (Sigma-Aldrich) was used to remove the His-tag of the protein at 4°C overnight. The GST-tag of the protein, which was auto-cleaved by Mpro itself, was removed by incubation with immobilized glutathione (GSH) beads (0.5mL) for 1 hour. the protein was further purified to be >95% purity (SDS-PAGE) with a size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column.

Enzymatic Assays.

Activity and inhibition assay for Mpro²⁹ was performed using the enzyme (100 nM) and Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH₂ (20 μM) as the substrate in a HEPES buffer (20mM, pH 6.5) containing 0.05% Triton X-100. To determine IC₅₀, triplicate samples of a compound with concentrations ranging from 1 nM to 10 μM were incubated with the enzyme for 10 min before adding the substrate to initiate the reaction in 96-well plate (100 μL final volume). The fluorescence signal (Ex: 360 nm, Em: 460 nm) of each well was monitored every 30s, using a Tecan microplate reader. The initial velocity data were imported into Prism (version 5.0), and IC₅₀ values with standard deviation were obtained by using a standard dose-response curve fitting.

Crystallization and Structure Determination of Mpro-Inhibitor Complexes.

SARS-CoV Mpro (2–3mg/mL) was mixed with an inhibitor (with a final concentration of 450 μM) and incubated overnight at 4°C. The sample was centrifuged at 10,000 rpm for 20 min at 4°C. Crystallization screening was carried out using the TTP LabTech Mosquito crystallization robot instrument. The best co-crystallization conditions were chosen to be repeated and optimized. Optimized crystals were grown using the hanging-drop, vapor diffusion technique for 1–3 weeks at room temperature. Each Mpro-inhibitor complex required different crystallization buffers for optimal co-crystallization, with that consisting of 25% PEG 3350 and 0.1M Hepes at pH 7.5 for compound 13, that of 20% PEG3350 and 0.2M Sodium thiocyanate for compounds 5 and 8, that of 25% PEG1500 and 100mM PCB at pH 7.0 for compound 30, that of 20% PEG 6000, 200mM NaCl and 100mM Hepes at pH 7 for compound 42, that of 25% PEG 1500 and 100mM MMT buffer at pH 6.5 for compound 35, and that of 25% PEG 1500 and 100mM PCB buffer at pH 7.0 for compound 37.

X-ray diffraction data for the SARS-CoV-2 Mpro-inhibitor complex structures were collected remotely at SBC 19-ID and 19-BM beamlines of the Advanced Photon Source (APS), Argonne National Laboratory, or 19-ID NYX and 17-ID-2 FMX beamlines of the National Synchrotron Light Source II (NSLS II), Brookhaven National Laboratory. Diffraction data collected at both beamlines of APS was processed with the HKL3000 software³¹, while HKL2000³² and autoPRO³³ were used at the 19-ID NYX and 17-ID-2 FMX beamlines of NSLS II respectively. The Phaser-MR within the PHENIX software suite was used to solve the structures via molecular replacement using the PDB model 7ALH³⁴. Structural refinement was performed using phenix.refine and COOT³⁵. Figures were generated using Maestro or PyMOL in Schrödinger (Schrödinger Suite, version 2022, Schrödinger, LLC, New York, NY, 2022) The crystallographic statistics are shown in Tables S1 and S2. Coordinates of these eight Mpro-inhibitor complexes were deposited into Protein Data Bank with PDB ID codes 8TPB, 8TPC, 8TPD, 8TPE, 8TPF, 8TPG 8TPH and 8TPI.

Cellular antiviral activity testing.

Antiviral activity of a compound against SARS-CoV-2 (Washington strain) was evaluated in Vero cells. 1.5 × 10⁴ Vero cells/well were seeded in 96-well plates and cultured in DMEM media with 2% FBS overnight to form a monolayer of cells. Duplicate samples of the specified concentration of a compound and 0.01 MOI (multiplicity of infection) of the virus were added. Upon incubation for 2h, the supernatant was removed and cells were washed with the culture medium. Fresh medium (150 μL/well) containing the same concentration of a compound were added. Upon incubation at 37 °C for 48h, aliquots of the supernatant from each well were used to determine the virus titers using an end-point dilution assay. Half-log (0.32x) serial dilution of the viral supernatant (50 μL) was added to a monolayer of Vero cells in quadruplicate in 96-well plates and cultured for 7 days. CPE/cell lysis was determined with microscope followed by MTT assay. TCID50 was calculated based on the highest dilution in which ≥50% (i.e., ≥2 out of the 4 quadruplicate wells) of Vero cells were infected with the virus. Compared to controls, the ability for a compound to reduce TCID50 can be determined.

PDB ID Codes

8TPC: SARS-CoV-2 Mpro in complex with compound 5; 8TPD: SARS-CoV-2 Mpro in complex with compound 8; 8TPB: SARS-CoV-2 Mpro in complex with compound 13; 8TPF: SARS-CoV-2 Mpro in complex with compound (R, S)-30; 8TPG: SARS-CoV-2 Mpro in complex with compound (R, R, S)-35; 8TPH: SARS-CoV-2 Mpro in complex with compound (R, S, R)-35; 8TPI: SARS-CoV-2 Mpro in complex with compound (R, R)-37; and 8TPE: SARS-CoV-2 Mpro in complex with compound (R, S)-42. Authors will release the atomic coordinates and experimental data upon article publication.

Abbreviation:

Mpro

main protease

3CLpro

3C-like protease

Boc

tert-butoxycarbonyl

SAR

structure-activity relationship

rmsd

root mean square deviations

CPE

cytopathic effects

MOI

multiplicity of infection

TCID

tissue culture infective dose

HRMS

high resolution mass spectra

Q-TOF

quadrupole-time-of-flight

ESI

electrospray ionization

IMAC

immobilized metal affinity chromatography

APS

Advanced Photon Source

NSLS II

National Synchrotron Light Source II