key: cord-0728373-qby1wy5f
authors: Dampalla, Chamandi S.; Rathnayake, Athri D.; Perera, Krishani Dinali; Jesri, Abdul-Rahman M.; Nguyen, Harry Nhat; Miller, Matthew J.; Thurman, Hayden A.; Zheng, Jian; Kashipathy, Maithri M.; Battaile, Kevin P.; Lovell, Scott; Perlman, Stanley; Kim, Yunjeong; Groutas, William C.; Chang, Kyeong-Ok
title: Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies
date: 2021-12-05
journal: J Med Chem
DOI: 10.1021/acs.jmedchem.1c01037
sha: 842b46d5233058b8590e884ff5526ffd8ea242d0
doc_id: 728373
cord_uid: qby1wy5f

[Image: see text] The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease . 1 The severity of the ongoing pandemic is having a major impact on public health worldwide and is further exacerbated by the emergence of more virulent strains. 2, 3 Intense worldwide efforts to combat the virus have led to the successful development of FDA-approved vaccines, and an array of potential therapeutics, such as monoclonal antibodies, repurposed drugs, and others, are currently being evaluated in clinical trials or are at various stages of clinical development. 4, 5 The SARS-CoV-2 life cycle encompasses multiple viral-and host-based druggable targets that can be exploited, including, for example, inhibitors that block virus entry and fusion, and replication inhibitors targeting the 3C-like protease (3CLpro), papain-like protease and the RNA-dependent RNA polymerase, among others. Attractive host-based targets include the proteases transmembrane serine protease 2 (TMPRSS2), cathepsin L, and furin. Thus, the development of small-molecule therapeutics that target host or viral targets essential for viral replication is a potentially fruitful avenue of investigation. 6−9 The SARS-CoV-2 genome contains two open reading frames (ORF1a and ORF1b). Translation of the genomic mRNA of ORF1a yields a polyprotein (pp1a), while a second polyprotein (pp1ab) is produced by a ribosomal frameshift that joins ORF1a together with ORF1b. The two polyproteins are processed by two cysteine proteases, a 3C-like protease (3CLpro) at 11 distinct cleavage sites and a papain-like protease (PLpro) at 3 distinct cleavage sites, resulting in 16 mature nonstructural proteins. The two proteases are essential for viral replication, making SARS-CoV-2 3CLpro an attractive target for therapeutic intervention. 9−16 SARS-CoV-2 3CLpro is a homodimer with a catalytic Cys− His dyad (Cys 145 −His 41 ) and an extended binding cleft. The protease displays a strong preference for a -Y−Z−Leu−Gln−X sequence, corresponding to the residues -P 4 −P 3 −P 2 −P 1 −P 1 ′-, 17 where X is a small amino acid (Ser, Ala, Gly), Y is a small hydrophobic amino acid, and Z is solvent-exposed and can tolerate polar or nonpolar amino acid chains. 18 Our foray in this area has focused on the structure-guided design of inhibitors of SARS-CoV-2 and MERS-CoV 3CLpro, 19−21 as well as feline infectious peritonitis virus (FIPV) protease inhibitors. 22, 23 We recently described the structure-guided design of a dipeptidyl series of MERS-CoV and SARS-CoV-2 3CLpro inhibitors incorporating in their structure a piperidine 20 or cyclohexyl 19 moiety capable of engaging in 19 In this report, we established a cellbased assay to screen inhibitors against SARS-CoV-2 3CLpro, which is safe (BSL2) and fast (takes less than 24 h). Furthermore, we report the results of structure-guided studies intended to interrogate the effects of stereochemistry, conformation, and structure, including the systematic introduction of fluorine (F-walk) 24, 25 around the structure of GC376 21−23 and the synthesis of deuterated inhibitors, 26−28 to modulate pharmacological activity, pharmacokinetic (PK) properties, and oral bioavailability. Chemistry. The synthesis of compounds 1−24b/c entailed the use of a structurally diverse set of precursor alcohols ( Table  1) , some of which were commercially available. Alcohols 12−16 were readily synthesized from 4,4-difluorocyclohexane carboxylic acid via reduction to the corresponding alcohol by treatment with carbonyl diimidazole and sodium borohydride, 29 followed by oxidation with Dess−Martin periodinane reagent to yield the aldehyde. Subsequent treatment with an array of Grignard reagents generated alcohols 12 and 14−16 (Scheme 1/panel A). Alcohol 13 was synthesized by reacting the methyl ester of 4,4difluorocyclohexane carboxylic acid with excess methyl magnesium iodide, followed by acidic workup (Scheme 1/ panel A). Deuterated alcohols 9, 11, 20, and 22 were obtained by treatment of the precursor carboxylic acid with carbonyl diimidazole followed by the addition of sodium borodeuteride. All trans-substituted alcohols were synthesized by reducing the Figure 1 . Generation of a cell-based assay for screening SARS-CoV-2 3CLpro inhibitors in HEK293T cells. Panel (A) Plasmid 1; pR-SA2−3CLpro encodes SARS-CoV-2 3CLpro from the PRRSV reverse genetics system. The gene of SARS-CoV-2 3CLpro is inserted between ORF1b and 2a of PRRSV genome. Plasmid 2; pGlo-VRLQS encodes firefly luciferase with coronavirus 3CLpro recognition sequences VRLQS. Active luciferase is generated by the cleavage with CoV 3CLpro. Panel (B) Semiconfluent HEK293T cells were transfected with two plasmids, and after overnight, various concentrations of each compound are applied to the cells. The inhibition of SARS-CoV-2 3CLpro is determined by measuring luciferase activity. Panel (C) Inhibition curves of selected compounds, 1c, 4c, 8c, 14c, 15c, 18c, 21c, and 23c, using the cell-based assay with pR-SA2−3CLpro and pGlo-VRLQS. precursor 4-substituted cyclohexanone with sodium borohydride/CeCl 3 . 30 Compounds 1−24b/c were readily obtained by reacting each precursor alcohol with disuccinimidyl carbonate, 31 followed by coupling with amino alcohol A. The resulting product was treated with Dess−Martin periodinane to yield aldehydes 1− 24b, which were converted to the corresponding bisulfite adducts 1−24c upon treatment with sodium bisulfite (Scheme 1/panel B). 32 An alternative synthesis was used in the case of compounds 6−8, 10−16, 23, and 24, which involved the reaction of the precursor alcohol with (L) leucine methyl ester isocyanate, as described in detail previously. 33 The synthesis of precursor amino alcohol A was readily accomplished by coupling (L) Z-Leu with a glutamine surrogate, followed by sequential reduction with LiBH 4 and removal of the protective group (H 2 /Pd) (Scheme 1/panel C).

Biochemical Studies. Enzyme Assays. The inhibitory activity of compounds 1−24b/c against SARS-CoV-2 3CLpro in biochemical assays was determined as described in the Experimental Section. The IC 50 values against SARS-CoV-2 and MERS-CoV-2 in the enzyme assays are summarized in Table 2 , and they are the average of at least two determinations. Most of the compounds potently inhibited SARS-CoV-2 3CLpro and displayed IC 50 values that ranged between 0.13 and 1.25 μM. Compounds 15b and 15c were the most effective against SARS-CoV-2 3CLpro, with IC 50 values 0.13 and 0.17 μM, respectively. The inhibitory activity of a select number of compounds against MERS-CoV 3CLpro was also investigated. The compounds were found to be 3−5-fold more potent against MERS-CoV-3CLpro, with IC 50 values in the 40−150 nM range (Table 2) . Interestingly, compounds 15b and 15c were the most effective against MERS-CoV-3CLpro as well, with IC 50 values 0.04 and 0.05 μM, respectively. The broad spectrum of inhibitory activity displayed by these compounds enhances their therapeutic potential.

Establishment of the Cell-Based Assay for SARS-CoV-2 3CLpro Inhibitors. We have previously reported EC 50 values determined by incubating SARS-CoV-2 3CLpro inhibitors and Vero E6 cells that were inoculated with SARS-CoV-2 at 50−100 plaque forming units/well. 19, 33 This cell-based assay requires a BSL3 facility and takes at least 2−3 days. As an alternative method, we report herein a relatively fast and safe cell-based assay system to screen SARS-CoV-2 3CLpro inhibitors using two plasmids. A similar cell-based assay has been reported; 34 however, in contrast, the present system utilizes the replication units of porcine respiratory and reproductive syndrome virus (PRRSV) 35 to express SARS-CoV-2 3CLpro. In this system, plasmid 1, pR-SARS-CoV-2 3CLpro, was used to express SARS-CoV-2 3CLpro, whereas plasmid 2, pGlo-VRLQS, was used to express luciferase-VRLQS in HEK293T cells (Figure 1 /panel A). The expressed inactive luciferase is activated by the catalytic mechanism of SARS-CoV-2 3CLpro in HEK293T cells. Hence, the inhibition of SARS-CoV-2 3CLpro was measured as a function of firefly luciferase activity ( Figure 1/panel B) . Plasmid 2 also contains intact Renilla luciferase gene as an expression control. As a negative control, the inactive form of the 3CLpro was introduced to pR-SARS-CoV-2 3CLpro by the mutagenesis, and the resulting plasmid was designated as pR-SARS-CoV-2 3CLpro C145A. The mock-transfection was also used as a negative control. The expression of coronavirus 3CLpro was reported to induce cytotoxicity in the transfected cells. 36, 37 The level of cytotoxicity in our assay system was about 7%, and the ratio of firefly to Renilla luminescence was adjusted to reduce variability due to cytotoxicity and transfection efficiency. The EC 50 values of a select number of compounds, including 1b/1c, 4b/4c, 8b/8c, 14b/14c, 15b/15c, 18b/18c, 21b/21c, and 23b/23c, were determined (Table 2) . Inhibition curves by each compound were consistent with a dose-dependent mode and R 2 > 0.8 ( Figure 1 /panel C). The IC 50 and EC 50 values of 15b and 15c were the lowest among tested compounds listed in Table 2 .

For comparative purposes, we determined EC 50 values using live SARS-CoV-2 in Vero E6 cells and the established twoplasmid system of two compounds (4c, 15c) from this work and a previously published 3CLpro inhibitor, GC376. Compounds 4c and 15c were selected on the basis of having the lowest IC 50 values in this series. The EC 50 of GC376 was found to be 0.23 ± 0.01 μM in live SARS-CoV-2 in Vero E6 cells. 33 In the twoplasmid system, the EC 50 of GC376 was determined to be 3.15 ± 0.67 μM (14-fold higher), and the R 2 values of the inhibition curves were >0.9. When the antiviral effects of compounds 4c and 15c were examined from live SARS-CoV-2 in Vero E6 cells, the EC 50 values were 0.85 ± 0.1 and 0.70 ± 0.08 μM, respectively ( Table 2 ). The results show that while compounds in this series are cell-permeable, the EC 50 values were higher from the two-plasmid system (2-fold for 15c and 5-fold for 4c) than those by live SARS-CoV-2 in Vero E6 cells. The higher EC 50 s may be due to various reasons including different cell types, presence of transfection reagent, incubation time, and overexpression of the 3CLpro and luciferase in HEK293T cells due to inhibition of cytotoxic 3CLpro in the two-plasmid system. 36,37 Most of the examined compounds showed minimal toxicity up to 100 μM; however, the CC 50 values for 4b/4c and 8b/8c were in the 40−60 μM range ( Table 2 ). Although the EC 50 values obtained from the two-plasmid system are higher than those obtained from live SARS-CoV-2 in Vero E6 cells, considering the feasibility of conducting the experiments under BSL2 laboratory conditions and the relatively short amount of time required (24 h), the cell-based two-plasmid method could be a useful initial screening tool for 3CLpro inhibitors against SARS-CoV-2.

X-ray Crystallographic Studies. A series of highresolution cocrystal structures were determined to elucidate the interaction of the inhibitors with the active site of SARS-CoV-2 3CLpro. Specifically, we sought to confirm the mechanism of action, identify the structural determinants associated with the binding of the inhibitors to the active site of the protease, and ultimately harness the accumulated structural information and insights gained to further optimize pharmacological activity and PK parameters. Three groups of inhibitor types were analyzed with respect to their structural elements that interact within the S 4 subsite environment, which are (1) nonpolar substituents ( Table 2 , entries 1−9), (2) 4,4difluorocyclohexyl groups that are connected to a stereocenter ( Table 2, For all structures described below, the active sites contained prominent difference electron density consistent with inhibitors covalently bound to Cys 145. Additionally, the electron density was consistent with both the R and S enantiomers at the stereocenter formed by covalent attachment of the Sγ atom of Cys 145 and were therefore modeled as each enantiomer with 0.5 occupancy. The γ-lactam ring of the inhibitor forms direct hydrogen bonds with Glu 166 and His163, and Glu 166 and Gln 189 form additional H-bonds with the CO and NH of the carbamate moiety in the inhibitor. The inhibitor engages in hydrophobic interactions with the leucine side chain, which is snugly accommodated in the S 2 pocket. The cocrystal structure confirms that the reaction of Cys145 with the aldehyde warhead results in the formation of a tetrahedral hemithioacetal that is stabilized by a H-bond to His164. Nonpolar Substituents. The structures of 5c, 1c, 3c, and 8b displayed well-defined electron densities and similar hydrogen bond interactions, as shown in Figure 2 . For all structures, the nonpolar groups are mainly positioned within the S 4 subsite near a hydrophobic ridge formed by residues Leu 167, Pro 168, Gly 170, and Ala 191 ( Figure 3 ). However, the dimethylcyclohexyl ring in 1c is too short to fully engage the hydrophobic ridge in the S 4 subsite ( Figure 3B ). The addition of an n-propyl group in 3c permits further engagement with the hydrophobic cleft, and the extra carbon atom in 8b allows the propyl group to extend even further ( Figure 3C ,D). Superposition of 3c and 1c ( Figure  3E ) shows that the 4,4-dimethylcyclohexyl ring is moved slightly out of the S 4 subsite relative to the n-propyl group in 3c. Additionally, the superposition of 3c and 8b revealed quite similar binding modes although the n-propyl group of 8b is positioned deeper within the S 4 subsite ( Figure 3F ). Overall, the similar binding modes and attendant high potency of the inhibitors are reflected in their low IC 50 values and similar potencies ( Table 2 , compounds 1−5b/c). With respect to compound 8, it was envisaged that the corresponding deuterated compound 9, found to be nearly equipotent to nondeuterated compound 8 (Table 2) , would likely display improved PK properties due to its enhanced in vivo stability arising from the greater strength of the C−D bond and the resulting deuterium kinetic isotope effect. 26−28 4,4-Difluorocyclohexyl Compounds. In previous studies related to norovirus 3CLpro inhibitors, the strategic introduction of a gem-dimethyl group into the inhibitor structure resulted in enhanced potency by restricting the rotation around the nearby single bonds and lowering the entropic penalty associated with binding. 38 Thus, we sought to capitalize on this by synthesizing gem-dimethyl-substituted compound 13c and, additionally, achieve the same end by introducing a Figures 4E and 5D ), which may explain why the IC 50 of 13c is ∼4-fold higher than those of 12b and 14c. Fluorinated Aryl Compounds. Positional analogue scanning is a widely used strategy for optimizing binding affinity, selectivity, and physicochemical properties of lead compounds containing aromatic or heteroaromatic rings. 24 For instance, the introduction of fluorine (F-walk) 25 or nitrogen (N-walk) 39 is an effective means for multiparameter optimization by leveraging the beneficial impact of fluorine (or nitrogen) and minor structural changes. In an effort to determine the effect of fluorine on the binding mode in the S 4 subsite of GC376, the structures of the fluorinated benzyl compounds 17c, 18c, 19b, 20b (deuterated analogue of 19b), and 21c were determined with SARS-CoV-2 3CLpro. The inhibitor o-fluorobenzyl (17c) and m-fluorobenzyl (18c) compounds displayed well-defined electron densities and similar hydrogen bond interactions, as shown in Figure 6 . Interestingly, the o-fluorobenzyl ring of 17c adopts a conformation in which the fluorine atom is directed Figure 6C) . Conversely, the fluorine atom in 18c is positioned between Thr 190/Ala 191 in the S 4 pocket and is 3.10 Å from the backbone nitrogen atom of Ala 191 ( Figure 6D ). The orientations of the fluorine atoms in 17c and 18c relative to the hydrophobic ridge in the S 4 pocket are shown in Figure 6E ,F.

The compounds that contain a p-fluorobenzyl group 19b and its deuterated analogue 20b not surprisingly adopt very similar binding modes and hydrogen bond interactions, as shown in Figures S1 and S2 . Interestingly, the inhibitor adopts two conformations in which the p-fluorobenzyl ring is projected away from the S 4 subsite in subunit B and is positioned in the S 4 pocket in subunit A. However, the electron density for the pfluorobenzyl ring is somewhat weaker in subunit A, which suggests that the pose in subunit B is likely the predominant conformation. This may be due to the fact that the fluorine atom does not form any contacts with polar atoms in the S 4 subsite and results in a conformation in which the aryl ring is positioned out of the pocket, which is the same conformation observed for the parent compound GC376.

The perfluorinated compound 21c also displayed a welldefined difference electron density consistent with the aryl ring in one conformation ( Figure 7A ). Interestingly, one of the ofluorine atoms interacts with the backbone oxygen of Glu 166 (3.08 Å), which is shorter than that observed for 17c described above (3.38 Å). The other o-fluorine atom is positioned 2.92 Å from the backbone N-atom of Thr 190 and 3.12 Å from the side chain N-atom of Gln 189 ( Figure 7B) . Similarly, the m-fluorine atom is positioned near the backbone nitrogen atom of Ala 191 (3.40 Å), which is longer than the distance observed for 18c (3.10 Å). The pentafluorobenzyl ring is positioned on top of the hydrophobic cleft within the S 4 -subsite ( Figure 7C ), unlike GC376 where the phenyl ring undergoes a hydrophobic collapse with the γ-lactam ring and the inhibitor assumes a "paper clip" shape.

Finally, GC376 variants 23b/c and 24b/c were synthesized and screened as mixtures of epimers. The aldehyde and bisulfite adduct compounds 23b/c were found to potently inhibit 3CLpro (IC 50 0.15 and 0.18 μM, respectively), and these were 27-and 19-fold more potent than the corresponding 24b/c aldehyde and bisulfite adducts, respectively. These findings provide tentative validation of the design regarding the use of a chiral center to attain directional control and augment binding interactions.

Effective management of SARS-CoV-2, the causative agent of the COVID-19 pandemic, would require the availability of safe and effective vaccines (already realized), as well as the availability of small-molecule therapeutics and prophylactics that target viral-and host-based druggable targets. SARS-CoV-2 3CLpro is an attractive target for the development of COVID-19 therapeutics because of its vital role in viral replication. An array of approaches was utilized to optimize potency and physicochemical parameters, including conformational and stereochemical control via the introduction of a gem-dimethyl group (Thorpe−Ingold effect) or stereocenter, deuteration, and fluorine, into the inhibitors. Virtually, all inhibitors were found to display a submicromolar potency against SARS-CoV-2 and MERS-CoV 3CLpro, and the inhibitory activities were confirmed by a newly established fast and safe cell-based assay. Furthermore, several deuterated inhibitors, which are likely to exhibit improved pharmacokinetics, were found to be equipotent with the corresponding nondeuterated inhibitors. The fluorine-walk approach was applied to explore bioisosteric replacements for the phenyl ring in GC376 by replacing one or more hydrogen atoms. The effects of these modifications included unanticipated binding modes of the F-substituted phenyl ring and modestly enhanced potency. The introduction of multiple fluorine atoms resulted in an orientation that allowed the fluorine atoms to engage in H-bonding with residues in the S 4 pocket, although with suboptimal bond angles. Highresolution cocrystal structures with an array of inhibitors unraveled the mechanism of action and provided valuable insights regarding the binding of the inhibitors to the active site and the identity of the structural determinants involved in binding. The 4,4-difluorocyclohexane methyl moiety connected to the benzylic carbon, coupled with the directional control imparted by the chiral center, resulted in a near-optimal fit in this series ( Table 2, entry 15 ). Collectively, the results of the studies described herein are significant and timely and provide an effective launching pad for conducting further preclinical studies.

General. Reagents and dry solvents were purchased from various chemical suppliers (Sigma-Aldrich, Acros Organics, Chem-Impex, TCI America, Oakwood Chemical, APExBIO, Cambridge Isotopes, Alpha Aesar, Fisher, and Advanced Chemblocks) and were used as obtained. Silica gel (230−450 mesh) used for flash chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin-layer chromatography was performed using Analtech silica gel plates. Visualization was accomplished using UV light and/or iodine. NMR spectra were recorded in CDCl 3 or DMSO-d 6 using a Varian XL-400 spectrometer. The purity of most of the aldehyde inhibitors was found to be ≥95%, determined by absolute qNMR analysis using a Bruker AV III 500 equipped with a CPDUL CRYOprobe and CASE autosampler at the KU NMR lab. 54 Dimethyl sulfone TraceCERT was used as the internal calibrant. High-resolution mass spectrometry (HRMS) was performed at the Wichita State University Mass Spectrometry lab using an Orbitrap Velos Pro mass spectrometer (ThermoFisher, Waltham, MA) equipped with an electrospray ion source.

Synthesis of Compounds. Preparation of Compounds 1−5a, 9a, and 17−22a. General Procedure. To a solution of alcohol (1 equiv) ( Table 1) in anhydrous acetonitrile (10 mL/g alcohol) were added N,N′-disuccinimidyl carbonate (1.2 equiv) and triethyl amine (TEA) (3.0 equiv), and the reaction mixture was stirred for 4 h at room temperature. The solvent was removed in vacuo, and the residue was dissolved in ethyl acetate (40 mL/g alcohol). The organic phase was washed with saturated aqueous NaHCO 3 (2 × 20 mL/g alcohol), followed by brine (20 mL/g alcohol). The organic layers were combined and dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to yield the mixed carbonate, which was used in the next step without further purification.

To a solution of Leu−Gln surrogate amino alcohol (1.0 equiv) in dry methylene chloride (10 mL/g of amino alcohol) was added TEA (1.5 equiv), and the reaction mixture was stirred for 20 min at room temperature (solution 1). In a separate flask, the mixed carbonate was dissolved in dry methylene chloride (10 mL/g of carbonate) (solution 2). Solution 1 was added to solution 2, and the reaction mixture was stirred for 3 h at room temperature. Methylene chloride was added to the organic phase (40 mL/g of carbonate) and then washed with saturated aqueous NaHCO 3 (2 × 20 mL/g alcohol), followed by brine 2-(4,4-Difluorocyclohexyl)propan-2-yl-((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (13a). Yield (72%). 1 Preparation of Compounds 1−24b. General Procedure. To a solution of dipeptidyl alcohol a (1 equiv) in anhydrous dichloromethane (300 mL/g dipeptidyl alcohol) kept at 0−5°C under a N 2 atmosphere was added Dess−Martin periodinane reagent (3.0 equiv), and the reaction mixture was stirred for 3 h at 15−20°C. The organic phase was washed with 10% aq Na 2 S 2 O 3 (2 ×100 mL/g dipeptidyl alcohol), followed by saturated aqueous NaHCO 3 (2 × 100 mL/g dipeptidyl alcohol), distilled water (2 × 100 mL/g dipeptidyl alcohol), and brine (100 mL/g dipeptidyl alcohol). The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The resulting crude product was purified by flash chromatography (hexane/ ethyl acetate) to yield aldehyde b as a white solid. 1-(4,4-Difluorocyclohexyl)ethyl-((S)-4-methyl-1-oxo-1-(((S)-1oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (12b). Yield (70%). 1 

The resultant crude product was purified by flash chromatography (hexane/ethyl acetate) to yield dipeptidyl alcohol a as a white solid

−3.28 (m, 1H), 3.27−3.17 (m, 1H), 3.13 (t, J = 8.9 Hz, 1H), 3.09− 2.98 (m, 1H)

S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-carbamate (2a

01 (s, 3H), 0.93−0.80 (m, 12H). (1s,4S)-4-Propylcyclohexyl-((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-carbamate (3a)

)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-carbamate (4a)

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

-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (6a)

-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (7a)

-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (9a)

)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (10a). Yield (90%). 1 H NMR (400 MHz

Hz, 2H), 1.51−1.30 (m, 3H), 1.30−1.12 (m, 2H), 0.86 (dd, J = 10.5, 6.6 Hz, 6H). (4,4-Difluorocyclohexyl)methyl-d 2 -((2S)-1-(((2S)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (11a)

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)

(S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (12a). Yield (76%). 1 H NMR (400 MHz

−1.63 (m, 5H), 1.63−1.51 (m, 3H), 1.47−1.31 (m, 3H), 1.27− 1.22 (m, 2H)

−3.28 (m, 2H), 2.50−2.31 (m, 2H), 1.97−1.93 (m, 1H), 1.90− 1.76 (m, 1H)

-Fluorophenyl)methyl-d 2 -((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)-carbamate (20b)

HRMS m/z: [M + H] + calculated for C 21 H 27 D 2 FN 3 O 5 : 424.2217, found: 424.2210. HRMS m/z: [M + Na] + calculated for C 21 H 26 D 2 FN 3 NaO 5 : 446.2037

Yield (86%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.39 (s, 1H), 8.47 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H)

HRMS m/z: [M + Na] + calculated for C 21 H 24 F 5 N 3 NaO 5 516.1534

Yield (83%). 1 H NMR (400 MHz, DMSO-d 6 ) 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)

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). HRMS m/z: [M + H] + calculated for C 21 H 23 D 2 F 5 N 3 O 5 496.1840; found 496.1837. HRMS m/ z: [M + Na

28 (d, J = 30.3 Hz, 1H), 5.68−5.54 (m, 1H), 5.36 (dd

Yield (82%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.35 (dd

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)

To a solution of dipeptidyl aldehyde b (1 equiv) in ethyl acetate (10 mL/g of dipeptidyl aldehyde) was added absolute ethanol (5 mL/g of dipeptidyl aldehyde) with stirring, followed by a solution of sodium bisulfite (1 equiv) in water (1 mL/g of dipeptidyl aldehyde). The reaction mixture was stirred for 3 h at 50°C. The reaction mixture was allowed to cool to room temperature and then vacuum-filtered. The solid was thoroughly washed with absolute ethanol, and the filtrate was dried over anhydrous sodium sulfate

2-oxopyrrolidin-3-yl)propane-1-sulfonate (2c). Yield (36%)

400 MHz, DMSO-d 6 ) δ 7.54−7.42 (m, 2H), 7.23−7.11 (m, 1H), 5.45−5

(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

DMSO-d 6 ) δ 7.54−7.39 (m, 2H), 7.23−7.10 (m, 1H), 5.45−5.28 (m, 1H

4S)-4-butylcyclohexyl)oxy)carbonyl)-amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (4c)

−3.08 (m, 1H), 3.07−2.98 (m, 1H), 2.19−2.02 (m, 2H), 1.99− 1.83 (m, 3H)

4S)-4-phenylcyclohexyl)oxy)carbonyl)amino)pentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (5c)

44 (s, 1H

−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)

-(trifluoromethyl)-cyclohexyl)methoxy)carbonyl)amino)pentanamido)-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate (6c)

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). HRMS m/z: [M + Na

Yield (76%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.58−7.50 (m, 1H), 7.46 (d, J = 9.6 Hz, 1H), 7.25−7.16 (m, 1H)

4R)-4-propylcyclohexyl)methoxy)carbonyl)amino)pentanamido)-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate (8c). Yield (82%). 1 H NMR (400 MHz

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

HRMS m/z: [M + Na] + calculated for C 21 H 32 D 2 F 2 N 3 Na2O 8 S 574

DMSO-d 6 ) 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)

Yield (39%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.49 (d, J = 10

HRMS m/z: [M + Na] + calculated for C 23 H 38 F 2 N 3 Na 2 O 8 S 600.2143

400 MHz, DMSO-d 6 ) 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)

−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). HRMS m/z

400 MHz, DMSO-d 6 ) 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

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)

HRMS m/z: [M] − calculated for C 21 H 29 FN 3 O 8 S: 502.1659, found: 502.1645. Sodium-(2S)-2-((S)-2-((((4-fluorophenyl)methoxy-d 2 )carbonyl)-amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (20c)

The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted following a standard procedure described previously. 19 Briefly, a stock solution of an inhibitor was prepared in dimethyl sulfoxide (DMSO) and diluted in assay buffer composed of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 8, containing NaCl (200 mM), ethylenediaminetetraacetic acid (EDTA) (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The SARS-CoV-2 3CLpro was mixed with serial dilutions of

Relative fluorescence units (RFUs) were determined by subtracting background values (substratecontaining well without protease) from the raw fluorescence values, as described previously. The dose-dependent FRET inhibition curves were fitted with a variable slope using GraphPad Prism software

Two plasmids, pR-SARS-CoV-2 3CLpro and pGlo-VRLQS, were used for the system (Figure 1/panel A). First, the open reading frame of SARS-CoV-2 3CLpro was cloned to the reverse genetics system of PRRSV with GFP 35 and designated as pR-SARS-CoV-2 3CLpro. The GFP gene was replaced with SARS-CoV-2 3CLpro gene with Af lII and MluI enzyme sites. As a negative control, the inactive form of the 3CLpro was introduced to pR-SARS-CoV-2 3CLpro by the mutagenesis, and resulting plasmid was designated as pR-SARS-CoV-2

One day old HEK293T cells in 48-well plates were cotransfected with two plasmids, pR-SARS-CoV-2 3CLpro and pGlo-VRLQS, or pR-SARS-CoV-2 3CLpro C145A (serves as a negative control) and pGlo-VRLQS. Following morning, the medium containing Mock-DMSO or serial concentrations of each compound were replaced to the transfected cells and incubated at 37°C for 6 h. Cell lysates were prepared for testing the levels of Firefly and Renilla luciferases (Dual Luciferase assay kit, Promega) in a luminometer (Promega). The expression levels of Firefly luciferase were normalized with Renilla luciferase levels. The transfection of pR-SARS-CoV-2 3CLpro C145A and pGlo-VRLQS resulted in minimal levels of Firefly luciferase, and this was applied to adjust all Firefly expression levels. The inhibition curve (Figure 1/panel C) for each compound was prepared, and the 50% effective concentration (EC 50 ) values were determined by GraphPad Prism software using a variable slope

Purified SARS-CoV-2 3CLpro 19 in 100 mM NaCl, 20 NT8 drop-setting robot (Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop vapor diffusion plates at 18°C. One hundred nanoliters of protein and 100 nL of crystallization solution were dispensed and equilibrated against 50 μL of the latter. A stock solution of 100 mM compound was prepared in DMSO, and the SARS-CoV-2 3CLpro:compound complex was prepared by mixing 1 the following complexes: 8b: Proplex HT screen (Molecular Dimensions) condition F7 (0.5 M ammonium sulfate, 100 mM MES pH 6.5); 12b, 13c, 14c, and 21c: Index HT screen (Hampton Research) condition D10 (20% (w/v) poly(ethylene glycol) (PEG) 5000 monomethyl ether (MME), 100 mM Bis−Tris pH 6.5); 19b and 20b: Proplex HT screen (Rigaku Reagents) condition C5 (20% (w/v) PEG 4000, 100 mM Tris pH 8.0); 1c: Index HT screen

Crystals of SARS-CoV-2 3CLpro with 8b were transferred to a cryoprotectant solution containing 80% crystallant and 20% (v/v) glycerol prior to freezing. X-ray diffraction data were collected at the Advanced Photon Source beamline except for the SARS-CoV-2 3CLpro complex with 14c, which were collected at the National Synchrotron Light Source II (NSLS-II) AMX beamline 17-ID-1. All diffraction data were collected using a Dectris Eiger2 X 9M pixel array detector. Structure Solution and Refinement. Intensities were integrated using XDS 40,41 via Autoproc, 42 and the Laue class analysis and data

Groutas − Department of Chemistry

Email: bill.groutas@wichita.edu Kyeong-Ok Chang − Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine

Rathnayake − Department of Chemistry

52242, United States Maithri M. Kashipathy − Protein Structure Laboratory

contributed equally to this work. Notes ■ ABBREVIATIONS USED CC 50 , 50% cytotoxic concentration in cell-based assays; CDI

DTT, dithiothreitol; EC 50 , the 50% effective concentration in cell culture

GESAMT, general efficient structural alignment of macromolecular targets; IC 50 , the 50% inhibitory concentration in the enzyme assay

MOI, multiplicity of infection; ORF, open reading frame

RMSD, root-mean-square deviation

A new coronavirus associated with human respiratory disease in China

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Vaccines and therapies in development for SARS-CoV-2 infections

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Drug development and medicinal chemistry efforts toward SARS-Coronavirus and Covid-19 therapeutics

COVID-19: Drug targets and potential treatments

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2): A global pandemic and treatments strategies

Targeting SARS-CoV-2 proteases and polymerase for COVID-19 treatment: State of the art and future opportunities

Advances in developing small molecule SARS 3CLpro inhibitors as potential remedy for Corona Virus infection

Potential of coronavirus 3C-like protease inhibitors for the development of new anti-SARS-CoV-2 drugs: Insights from structures of protease and inhibitors

Structure of M pro from SARS-CoV-2 and discovery of its inhibitors

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Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

where the residues on the N-terminus side of the peptide bond that is cleaved are designated as P 1 -P n and those on the C-terminus side are designated P 1 -P 1 ′. The corresponding active site subsites are designated S 1 -S n and S 1 -S n ′

Structure-Based Design of a Cyclic Peptide Inhibitor of the SARS-CoV-2 Main Protease. bioRixiv

Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element

Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses

Reversal of the progression of fatal coronavirus infection in cats by a broadspectrum coronavirus protease inhibitor

Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis

Positional analog scanning: an effective strategy for multiparameter optimization in drug design

Design and NMR-based screening of LEF, a library of chemical fragments with different local environments of fluorine

Applications of deuterium in medicinal chemistry

Deuterated drugs: updates and obviousness analysis

A decade of deuteration in medicinal chemistry

The synthesis of aminoazole analogs of lysine and arginine: the Mitsunobu reaction with lysinol and arginol

Cerium(III) chloride-mediated stereoselective reduction of a 4-substituted cyclohexanone using NaBH 4

N′-Disuccinimidyl carbonate: A useful reagent for alkoxycarbonylation of amines

A novel, nonaqueous method for regeneration of aldehydes from bisulfite adducts

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

Detecting SARS-CoV-2 3CLpro expression and activity using a polyclonal antiserum and a luciferase-based biosensor

Cellular Activity of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics. bioRxiv

A Simplified Cell-Based Assay to Identify Coronavirus 3CL Protease Inhibitors. bioRxiv

Natural products-inspired use of the gem-dimethyl group in medicinal chemistry

The necessary nitrogen atom: a versatile high-impact design element for multiparameter optimization

Automatic indexing of rotation diffraction patterns

Data processing and analysis with the autoPROC toolbox

An introduction to data reduction: space-group determination, scaling and intensity statistics

Phaser crystallographic software

PHENIX: a comprehensive Python-based system for macromolecular structure solution

Features and development of Coot

MolProbity: all-atom structure validation for macromolecular crystallography

Developments in the CCP4 molecular-graphics project

Scaling and assessment of data quality

Improved R-factors for diffraction data analysis in macromolecular crystallography

Global indicators of X-ray data quality

Linking crystallographic model and data quality

Resolving some old problems in protein crystallography

The purity of some of the aldehyde inhibitors ranged between 89−94% due to the facile epimerazation of the aldehyde α-carbon upon storage (see Supporting Information)

The authors declare no competing financial interest.