key: cord-1044071-4r6a3hf9
authors: Wang, Hao; Pei, Rongjuan; Li, Xin; Deng, Weilong; Xing, Shuai; Zhang, Yanan; Zhang, Chen; He, Shuai; Sun, Hao; Xiao, Shuqi; Xiong, Jin; Zhang, Yecheng; Chen, Xinwen; Wang, Yaxin; Guo, Yu; Zhang, Bo; Shang, Luqing
title: The structure-based design of peptidomimetic inhibitors against SARS-CoV-2 3C like protease as Potent anti-viral drug candidate
date: 2022-05-13
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2022.114458
sha: ac0cc13c4c5498d98dea4a52e5edc9c8b78dd760
doc_id: 1044071
cord_uid: 4r6a3hf9

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), as the pathogen of coronavirus disease 2019 (COVID-19), has infected millions of people and took hundreds of thousands of lives. Unfortunately, there is deficiency of effective medicines to prevent or treat COVID-19. 3C like protease (3CLPro) of SARS-CoV-2 is essential to the viral replication and transcription, and is an attractive target to develop anti-SARS-CoV-2 agents. Targeting on the 3CLPro, we screened our protease inhibitor library and obtained compound 10a as hit to weakly inhibit the SARS-CoV-2 3CLPro, and determined the co-crystal structure of 10a and the protease. Based on the deep understanding on the protein-ligand complexes between the hit and SARS-CoV-2 3CLPro, we designed a series of peptidomimetic inhibitors, with outstanding inhibitory activity against SARS-CoV-2 3CLPro and excellent anti-viral potency against SARS-CoV-2. The protein-ligand complexes of the other key inhibitors with SARS-CoV-2 3CLPro were explicitly described by the X-ray co-crystal study. All such results suggest these peptidomimetic inhibitors could be further applied as encouraging drug candidates.

Based on the insight on the above complex structures, the protein-ligand complexes of the typical inhibitors with the protease at P1 and P2 sites were summarized. For further check the validity of the model, a series of inhibitors with (S)-δ-lactam ring at the P1 site and various alkyl groups at P2 site were designed, synthesized and evaluated ( Figure 3 ). According to comparing the inhibitory activity of 10a with 10l, 10b with 10i, and 10c with 10d, it was firm to confirm (S)-δ-lactam ring with more contact area exhibited preferable inhibitory activity than (S)-γ-lactam ring. Following comparing the inhibitory activity of 10d-10l, long hydrophobic flexible chain alkyl group was suitable to occupy the S2 pocket of the protease (Table S1 ).

When further insight on the complex structure of 10d with the protease, a long narrow channel nearby the S1 and S2 pockets attracted our attention and thus the tripeptidomimetic inhibitors were designed to evolve into tetrapeptidomimetic inhibitors to occupy the channel as far as possible for improving the inhibitory activity of peptidomimetic inhibitors ( Figure 2E and 3) . In detail tetrapeptidomimetic inhibitors 14a-14d with branched alkyl groups at P3 position for preferably stucking the S3 pocket were synthesized and evaluated. As the results, the superior inhibitory activities were reflected by tetrapeptidomimetic inhibitors than tripeptidomimetic inhibitors ( Figure 4 and Table   S1 and S2), which illustrated the stereoscopic extension of the inhibitor is meaningful to improve the inhibitory potency of the inhibitor. Moreover, the superior inhibitory activity of 14c (IC50= 0.281 ± 0.033 μM) made it reasonable to fix the branched isopropyl group at P3 site of tetrapeptidomimetic inhibitors to further design and optimize P4 site of tetrapeptidomimetic inhibitors ( Figure 3 ).

For the discovery of the suitable group at P4 site (Table S2) , a series of aldehydes with various substituted cinnamic acids were obtained and evaluated. 17a (IC50= 0.196 ± 0.004 μM) and 17g (IC50= 0.173 ± 0.014 μM) exhibited excellent inhibitory activities against the 3CL Pro . Subsequently, the inhibitor 14c with cinnamic acid fragment was evolved into 17i (IC50=0.271 ± 0.001 μM) with naphthyl group and 17k (IC50= 0.221 ± 0.001 μM) with indole group via conformational restriction; 17l (IC50= 0.199 ± 0.001 μM) with phenylethyl group and 17m (IC50= 0.257 ± 0.024 μM) with phenyl group via scaffold hopping strategy; and 17q (IC50= 0.173±0.013 μM) with furanyl group and 17r (IC50= 0.172 ± 0.018 μM) with pyrrolyl group via bioisostere strategy, respectively. All of these inhibitors displayed preferable inhibitory activity than that of 14c. Considering of the ligand efficiency, 14c was evolved into 17m for further research. Previously, we proposed a strategy to introduce a hydrogen bond acceptor into the P4 site of inhibitor for the construction of tight interaction with Q195 and enhancement of the potency of For tetrapeptidomimetic inhibitors, we managed to obtain the complex structures of 3CL Pro -17i (PDB: 7DGH) and 3CL Pro -17x (PDB: 7DGI). In each complex structure, the inhibitor is covalently linked to the catalytic residue Cys145 of 3CL Pro via its aldehyde. The P4 group of each inhibitor fits in the S4 pocket of 3CL Pro consequently increasing the contact area between the enzyme and the inhibitor.

Therefore, the tetrapeptidomimetic inhibitors should exhibit better inhibitory activity than tripeptidomimetic inhibitors, such as 10d. However, although inhibitor 17x showed a lower IC50 than 10d, the tetrapeptidomimetic inhibitor 17i showed only a similar IC50 to 10d, indicating that there is selectivity between the S4 pocket and the P4 group ( Figure 6A ). To elucidate the selectivity, we compared the complex structures of 3CL Pro -17i and 3CL Pro -17x. As shown in Figure 6B -6E, the P4 naphthyl group of inhibitor 17i is sterically larger than the nitrophenyl group of 17x. The carbonyl group of 3CL Pro Gln192 is flipped in the 3CL Pro -17i complex structure due to steric effects, but not in the 3CL Pro -17x complex structure. As a result, the backbone of Gln192 adopts an unfavorable conformation in 3CL Pro -17i complex structure. Such energy consuming change in 3CL Pro may cancel out the benefits of the interaction between the P4 group and the S4 pocket. Therefore, the inhibitor 17i cannot significantly promote its inhibitory activity. From the IC50 data we noticed that a smaller aromatic group at the P4 site was prone to better improve the inhibitor's inhibitory activity. This phenomenon is consistent with our structural observations. J o u r n a l P r e -p r o o f The protein-ligand complexes of the naphthyl group of 17x with the S4 pocket of SARS-CoV-2 3CL Pro in detail. In the structure, the protease is presented as a white cartoon, and the significant residues and inhibitor are shown as sticks (the protease residues are in green and the inhibitor are in lime green and violet).

Encouraged by the excellent 3CL Pro inhibitory activities of the peptidomimetic inhibitors, we further explored the anti-SARS-CoV-2 activity of those inhibitors in vitro. First, we indicated that all of inhibitors did not manifest the obvious cytotoxicity at A549 cell lines, which was used for simulation of the lung environment (table S1 and table S2 ). These inhibitors were then screened at concentration of 10 μM in VeroE6 cells for their anti-SARS-CoV-2 activity. As the tripeptidomimetic aldehydes, 10b, 10c, 10g, and 10h reduced the viral RNA more than 2logs ( Figure 7) . Then, the EC50 and CC50 of 10b, 10c, 10g, and 10i were then tested ( Figure 7 ). While none of these compounds showed significant cytotoxity on VeroE6 cells, all compounds showed a dose-dependent inhibition effect on SARS-CoV-2 virus proliferation. Among the tested tripeptidomimetic inhibitors, 10b with (S)-γ-lactam ring at P1 site, isobutyl group at P2 site and cinnamic acid fragment at P3 site had the most potential anti-SARS-CoV-2 activity with EC50 of 1.06 μM, which makes it become an attractive potential clinic candidate medicine.

Though most of the tetrapeptidomimetic inhibitors have preferable inhibitory activity on 3CL pro than the tripeptidomimetic inhibitors, the tetrapeptidomimetic inhibitors did not show superior antiviral activity. Among the tetrapeptidomimetic aldehydes, only compounds 14d and 17i inhibited SARS-CoV-2 viral RNA more than 2logs at concentration of 10 μM (Figure 7) . The EC50 and CC50 of 14j and 17i were determined (figure 7). None of the tested compounds influencs the cell viability, while all compounds dose-dependently inhibit SARS-CoV-2 viral propagation. Though 14c with isopropyl group at P3 site and cinnamic acid fragment at P4 had no antiviral activity, 17i which changed cinnamic acid fragment to naphthyl group at P4 site could inhibit SARS-CoV-2 and the EC50 was calculated as 1.36 μM, indicated the possibility to adjust P4 site group and enhance the anti-viral activity of the tetrapeptidomimetic inhibitors.

J o u r n a l P r e -p r o o f diseases (SARS, . However, 3CL Pro remains the conserved key sites and catalytic mechanism during the virus evolution. This study comprehensively reveals the protein-ligand complexes of SARS-CoV-2 3CL Pro with peptidomimetic inhibitors, including the SAR of the peptidomimetic inhibitor and co-crystal structure of SARS-CoV-2 3CL Pro with inhibitors. The dimer cocrystal structure may afford a potential allosteric site for SARS-CoV-2 3CL Pro inhibitor design. We believe this work could provide bedrock to promote the development of antiviral agents against SARS-CoV-2 and even novel coronavirus in the future.

General methods: All reagents were purchased from commercial suppliers and used as received.

NMR spectra were recorded on a Bruker Ascend 400 in the indicated solvent. (400 MHz for 1 H and 101

MHz for 13 C) (Bruker, Karlsruhe, Germany) NMR spectrometer. Molecular mass was determined on a mass spectrometry (Shimadzu (China) Co., Ltd.). All tested compounds exhibited purities of > 95% as analyzed by HPLC (Dionex UltiMate 3000, Germany).

The synthesis procedure of (S)-2-cinnamamido-N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)pent-4-ynamide 10a was exhibited as an example to illustrate the general procedure to process the peptidomimetic inhibitors 10a-10l.

The SOCl2 (54.23 mL, 747.64 mmol) was added in drop-wise to a suspension of L-glutamic acid (50.00 g, 339.84 mmol) in anhydrous MeOH (500 mL) at 0 o C. After stirring for 30 min in the ice-bath, the reaction was heated to reflux status for 3 h. After cooling, the mixture was evaporated to remove the solvent and the redundant SOCl2. Then, the anhydrous THF was added to suspend the residue (1.0 L) and other reagents (ditertbutyl dicarbonate (111.25 g, 509.75 mmol) and triethylamine (51.58 g, 509.75 mmol)) were subsequently added into the reaction at ice-bath. Then, the reaction mixture was allowed to stir overnight at room temperature. Following removing the solvent of the mixture, the residue was dissolved in DCM (800 mL) and washed with H2O (400 mL×2), saturated citric acid solution (400 mL×2), saturated NaHCO3 solution (400 mL×2) and brine (400 mL×2). After the organic phase was concentrated, column chromatography (EtOAc: petroleum ether, 1: 5 v/v) was used to give the pure product as colorless oil dimethyl (tert-butoxycarbonyl)-L-glutamate compound 4 (92.06 g, 334.40 mmol, 98.40 %). 1 H NMR (400 MHz, CDCl3) δ: 5.44 (d, J = 8.1 Hz, 1H), 4.33 (dd, J = 12.6, 7.5 Hz, 1H) , 3.75 (s, 3H), 3.68 (s, 3H), 2.51 -2.34 (m, 2H), 2.24 -2.13 (m, 1H), 1.97 (td, J = 14.7, 8.2 Hz, 1H), 1.44 (s, 9H) . 13 C NMR (100 MHz, CDCl3) δ: 173.02, 172.58, 155.32, 79.64, 52.73, 52.18, 51.56, 29.92, 28.11, 27.42. pentanedioate 5a (13.50g, 42.86 mmol, 59.12 %) . 1 H NMR (400 MHz, CDCl3) δ: 5.34 (d, J = 8.8 Hz, 1H), 4.27 (dd, J = 14.2, 7.8 Hz, 1H), 6H), 1H), 2H), (m, 2H), 1.33 (s, 9H); 13 C NMR (100 MHz, CDCl3) δ: 172.49, 172.01, 155.55, 117.24, 80.19, 52.55, 52.52, 50.98, 38.16, 33.43, 28.11, 18.83 .

The similar procedure with compound 5a was executed to obtain compound yellow oil dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-(2-cyanoethyl)pentanedioate 5b via the replacement of 3bromopropionitrile into 3-Bromopropionitrile. 1 1.45 (s, 9H) . 13 C NMR (100 MHz, CDCl3) δ: 174.39, 172.34, 155.38, 118.71, 80.28, 52.54, 52.16, 51.56, 40.79, 34.37, 28.24, 27.30, 15.12. yield 57.50 %. To a solution of 5a (10 g, 31.81 mmol) dissolved into anhydrous MeOH (400 mL), CoCl2·6H2O (4.54 g, 19. 09 mmol) was added at -10 o C. Subsequently, NaBH4 (7.22 g, 31.81 mmol) was carefully added portion-wise at 0 °C. Following additional 48 h of stirring at 0 °C, saturated ammonium chloride solution (50 mL) was added to the reaction for quenching the reaction. Then, the mixture was filtered to remove insoluble substances. After removing the solvent, the residue was extracted with DCM (100 mL×3) and further purified by column chromatography (EtOAc: petroleum ether, 2.5:1 v/v) to give the pure yellow oil methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate 6a (4.22 g, 14.73 mmol, 46 04, 173.01, 155.79, 79.72, 52.32, 52.25, 40.46, 38.28, 33.91, 28.24, 27.96 .

The similar operation was applied to obtain methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2oxopiperidin-3-yl)propanoate 6b as yellow oil. 1 23, 155.91, 79.51, 52.12, 51.68, 42.05, 37.85, 34.01, 28.21, 26.42, 21.43 .

To a solution of 6a (1.0 g, 3.49 mmol) dissolved in anhydrous DCM (50 mL), CF3COOH (2.5 mL) was added slowly at 0 °C. Subsequently, the reaction mixture was allowed to continuously stir at room temperature for 3 h and concentrated to remove the redundant trifluoroacetic acid. Then, the triethylamine was added to the solution of the residue dissolved in DCM (60 mL) to adjust the pH value Compound 7b (methyl (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-3-((S)-2oxopyrrolidin-3-yl)propanoate) : 1 H NMR (400 MHz, CDCl3) δ: 7.94 (d, J = 6.7 Hz, 1H), 7.30 (s, 1H), 5.41 (d, J = 8.3 Hz, 1H), 1H), 1H) 13 C NMR (100 MHz, CDCl3) δ: 179.91, 173.40, 172.25, 155.61, 79.54, 52.82, 52.24, 50.89, 42.06, 40.44, 38.26, 33.05, 28.23, 27.90, 24.55, 22.81, 22.07 . 178.31, 172.85, 172.41, 155.68, 79.69, 54.31, 52.26, 50.44, 42.53, 38.90, 33.61, 33.46, 28.38, 27.21, 26.58, 22.16, 13.98 . 62, 172.90, 172.42, 155.55, 79.57, 54.26, 52.24, 50.40, 42.18, 37.90, 33.26, 33.09, 28.32, 27.37, 26.53, 22.46, 21.59, 13.96 . 155.91, 79.29, 61.46, 52.03, 50.38, 41.86, 37.77, 34.82, 32.57, 28.27, 26.41, 26.19, 21.75 .

Compound 7i (methyl (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-cyclopropylpropanamido) -3-((S)- 172.75, 172.46, 155.41, 79.32, 54.64, 52.13, 50.08, 42.02, 37.99, 37.62, 33.34, 28.28, 26.22, 21.43, 7.09, 4.30, 4.17 . 172.40, 172.07, 155.41, 133.10, 118.59, 79.64, 53.56, 52.25, 50.25, 42.16, 37.77, 37.50, 33.43, 28.29, 26.35, 21.45 . 54, 172.25, 170.68, 155.23, 80.15, 79.49, 71.42, 52.72, 52.34, 50.24, 42.26, 37.75, 33.74, 28.29, 26.37, 23.11, 21.50 .

The synthesis of 8a-8l is referred to the procedure of preparation of 7a-7l. 165. 84, 141.26, 134.81, 129.69, 128.78, 127.85, 120.71, 52.31, 51.36, 51.34, 42.61, 40.55, 38.42, 32.74, 27.94, 24.72, 22.90, 22.20 . 59, 173.43, 172.15, 165.98, 140.95, 134.91, 129.57, 128.73, 127.84, 120.97, 52.92, 52.24, 51.29, 40.55, 38.40, 33.26, 32.75, 27.85, 27.59, 22.46, 13.98 . 141.08, 134.84, 129.64, 128.78, 127.83, 120.78, 52.92, 52.25, 50.65, 42.19, 37.88, 33.25, 33.12, 27.38, 26.47, 22.49, 21.53, 13.98 . 173.68, 172.46, 165.59, 140.87, 134.89, 129.55, 128.73, 127.80, 120.88, 52.23, 50.50, 48.74, 42.10, 37.77, 33.18, 26.24, 21.40, 19.10 . 56, 171.43, 165.73, 141.18, 134.80, 129.66, 128.79, 127.80, 120.90, 59.96, 52.10, 50.81, 42.12, 37.87, 35.47, 32.95, 26.62, 26.47, 21.65 .

Compound 8i (methyl (S)-2-((S)-2-cinnamamido-3-cyclopropylpropanamido)-3-((S)-2-oxopiperidin- 3-yl) 172.43, 165.58, 141.03, 134.86, 129.62, 128.77, 127.83, 120.82, 53.47, 52.26, 50.68, 42.21, 38.23, 37.90, 33.28, 26.52, 21.49, 7.26, 4.48, 4.30 . 174.47, 172. 14, 171.09, 165.40, 141.35, 138.04, 134.86, 129.64, 128.77, 128.60, 127.89, 127.29, 127.08, 120.64, 56.40, 52.14, 50.78, 42.13, 37.79, 33.27, 26.27, 21.35 . 23, 170.57, 165.75, 141.68, 134.67, 129.80, 128.80, 127.90, 120.30, 79.61, 71.35, 52.38, 51.35, 50.82, 42.29, 38.02, 33.47, 26.58, 23.14, 21.45 .

To a solution of 8a (370 mg, 0.90 mmol) dissolved into anhydrous MeOH (30.0 mL), NaBH4 (0.51 g, 13.53 mmol) was added at 0 °C. Then, the reaction mixture was stirred at ambient temperature for 2 h.

Saturated NH4Cl solution (20 mL) was added to the mixture for quench the reaction. Following evaporating the solvent, EA (60 mL×2) was added to extract the aqueous components. The organic phase was washed with H2O (30 mL×2), saturated brine (30 mL×2), and concentrated. Finally, the residue was purified by column chromatography (DCM: MeOH, 25:1 v/v) to afford the pure product as 13, 141.11, 134.80, 129.61, 128.60, 127.55, 120.03, 79.09, 71.15, 64.09, 52.83, 49.52, 40.15, 38.19, 32.03, 27.57, 21.45 .

The procedure to obtain 9b-9l is similar to the procedure of preparation of 9a. 62, 166.17, 141.22, 134.74, 129.72, 128.80, 127.83, 120.67, 65.47, 52.26, 50.41, 42.16, 40.64, 38.31, 32.22, 28.32, 24.92, 23.06, 22.07 . 10, 140.67, 134.86, 129.53, 128.60, 127.55, 120.27, 64.19, 54.12, 49.31, 40.17, 38.18, 32.13, 31.75, 27.90, 27.60, 22.14, 13.03 . 140.68, 134.88, 129.51, 128.57, 127.48, 120.23, 64.22, 49.52, 48.40, 41.62, 37.26, 32.50, 25.71, 20.66, 16.88 . 134.93, 129.48, 128.57, 127.48, 120.29, 64.27, 59.56, 48.40, 41.58, 37.30, 32.32, 30.29, 25.66, 20.70, 18.42, 17.45 . 167.12, 140.72, 134.88, 129.51, 128.58, 127.49, 120.20, 64.23, 52.40, 48.45, 41.60, 40.75, 37.25, 32.43, 25.68, 24.64, 22.07, 20.67, 20.65 . 56, 171.47, 166.97, 140.80, 134.99, 129.46, 128.58, 127.52, 120.44, 64.29, 61.71, 61.66, 41.59, 37.30, 33.62, 32.33, 25.98, 25.70, 20.71 . 170.78, 164.59, 138.27, 132.47, 127.08, 126.15, 125.07, 117.87, 61.75, 52.14, 45.96, 39.19, 34.82, 34.40, 30.05, 23.28, 18.25, 4.87, 1.42, 1.08 . 172.43, 167.05, 140.81, 134.86, 133.33, 129.53, 128.58, 127.49, 120.17, 117.29, 64.23, 53.60, 48.46, 41.61, 37.25, 36.16, 32.45, 25.68, 20.66 . For obtaining the purity aldehyde as far as possible, abundant CCl4 and hexane were added into the eluent to form azeotropes. Then, the eluent was concentrated at 44 o C and give the residue which mainly contain aldehyde. Following the abundant hexane was added into the solution of residue dissolved into chloroform, the precipitation was filtered and gave the purity aldehyde. 199.92, 180.88, 171.18, 165.94, 141.85, 134.60, 130.97, 128.84, 127.92, 120.19, 79.38, 71.73, 57.89, 54.97, 41.27, 38.06, 29.68, 28.42, 23.85 . HRMS (m/z): 382.1690 (M+H) + .

The similar procedure with the preparation of 10a were applied to obtain compound 10b-10l. 199.73, 179.85, 174.02, 165.94, 141.46, 134.70, 129.79, 128.82, 127.85, 120.46, 57.65, 51.55, 42.57, 40.63, 38.14, 29.70, 28.30, 24.92, 22.94, 22.12 . HRMS (m/z): 199.65, 179.90, 173.53, 165.88, 141.37, 134.71, 129.78, 128.82, 127.85, 120.53, 57.68, 53.09, 40.62, 38.14, 33.21, 29.70, 28.35, 27.67, 22.45, 13.94 . HRMS (m/z): 400.2159 199.86, 174.92, 173.35, 165.83, 141.29, 134.74, 129.74, 128.81, 127.84, 120.55, 57.13, 53.20, 42.23, 37.29, 33.09, 30.70, 27.65, 27.11, 22.47, 21.31, 13.95 . HRMS (m/z): 414.2317 (M+ H) + . 199.89, 174.99, 173.75, 165.54, 141.34, 134.72, 129.78, 128.83, 127.85, 120.45, 57.32, 48.92, 42.32, 37.60, 30.80, 27.42, 21.33, 19.40 . HRMS (m/z): 372.1847 (M+H) + .

J o u r n a l P r e -p r o o f 199.70, 174.94, 172.35, 165.90, 141.43, 134.75, 129.76, 128.83, 127.84, 120.56, 58.19, 57.55, 42.33, 37.64, 31.81, 30.74, 27.49, 21.35, 19.26, 18.11 . HRMS (m/z): 400.2158 (M+H) + .

Compound 10g 174.70, 173.88, 165.85, 141.29, 134.77, 129.71, 128.79, 127.84, 120.57, 57.01, 51.64, 42.48, 42.21, 37.17, 30.59, 27.01, 24.93, 22.94, 22.18, 21.43 199.64, 174.73, 171.62, 165.77, 141.50, 134.72, 129.79, 128.84, 127.84, 120.63, 60.31, 57.50, 42.27, 37.46, 35.01, 30.57, 27.24, 26.74, 21.45 199.82, 174.92, 173.11, 165.68, 141.28, 134.75, 129.74, 128.81, 127.85, 120.57, 57.22, 53.69, 42.26, 38.07, 37.31, 30.80, 27.21, 21.34, 7.38, 4.63, 4.36 199.88, 174.91, 171.35, 165.18, 141.67, 138.13, 134.69, 129.78, 129.01, 128.80, 128.37, 127.86, 127.26, 120.21, 57.54, 56.96, 42.34, 37.74, 30.89, 27.49, 21.26 172.35, 165.74, 141.43, 134.69, 132.90, 129.79, 128.82, 127.86, 120.42, 118.91, 57.21, 52.51, 42.25, 37.64, 37.36, 30.77, 27.15, 21.33 18, 175.09, 170.88, 165.70, 141.91, 134.60, 129.92, 128.86, 127.94, 120.07, 79.33, 71.72, 57.70, 51.50, 42.39, 37.80, 30.88, 27.64, 23.17, 21.37 . HRMS (m/z): 396.1846 (M+ H) + .

The compound 14a-14d were prepared by following scheme 2, and the detailed synthetic procedure was similar with the synthesis of compound 10a-10l. the relevant NMR data were exhibited below:

Compound 11a (methyl (6S,9S,12S)-9-isobutyl-2,2-dimethyl-4,7,10-trioxo-12-(((S)-2-oxopiperidin-3yl)methyl)- 6-propyl-3-oxa-5,8,11-triazatridecan-13- 172.71, 172.40, 172.05, 155.68, 79.86, 54.45, 52.19, 51.58, 49.79, 42.25, 42.12, 37.58, 34.45, 33.40, 28.25, 26.12, 24.55, 22.95, 21.95, 21.53, 18.79, 13.75 . 40, 172.66, 172.31, 171.53, 155.94, 79.86, 59.27, 52.19, 51.62, 49.47, 42.41, 42.11, 37.49, 36.71, 33.47, 28.24, 25.92, 24.84, 24.62, 22.94, 21.95, 21.50, 15.41, 10.95 .

Compound 11c (methyl (6S,9S,12S)-9-isobutyl-6-isopropyl-2,2-dimethyl-4,7,10-trioxo-12-(((S)-2oxopiperidin-3-yl)methyl)-3-oxa- 5,8,11-triazatridecan-13- 172.63, 172.29, 171.54, 79.88, 60.18, 52.24, 51.64, 50.09, 42.18, 40.59, 37.74, 33.50, 30.80, 28.26, 26.31, 24.61, 22.89, 22.00, 21.44, 19.20, 18.03 .

Compound 11d (methyl (6S,9S,12S)-6-(cyclohexylmethyl)-9-isobutyl-2,2-dimethyl-4,7,10-trioxo-12-(((S)-2-oxopiperidin-3-yl)methyl)-3-oxa- 5,8,11-triazatridecan-13- 172.86, 172.52, 167.13, 141.21, 134.77, 129.66, 128.67, 127.69, 120.15, 53.32, 51.97, 51.93, 50.02, 41.81, 40.74, 37.37, 34.19, 33.18, 25.77, 24.50, 22.40, 21.36, 20.86, 18.82, 13.28 . 80, 129.68, 128.78, 127.89, 120.48, 52.18, 52.04, 51.33, 50.06, 42.34, 41.80, 39.85, 37.79, 34.01, 33.82, 33.58, 32.80, 26.44, 26.35, 26.12, 26.01, 24.73, 22.88, 22.10, 21.40 . 173.15,167.41, 140.93, 134.89, 129.51, 128.55, 127.54, 120.05, 64.22, 53.74, 52.21, 48.45, 41.61, 40.37, 37.26, 33.87, 32.76, 25.70, 24.53, 22.05, 20.70, 20.60, 18.80, 12.76 . 92, 173.50, 172.42, 167.46, 140.96, 134.93, 129.50, 128.56, 127.55, 120.18, 64.27, 58.37, 52.35, 48.41, 41.61, 40.52, 37.23, 36.85, 32.74, 25.73, 24.73, 24.49, 22.02, 20.73, 20.64, 14.68, 10 .08. 172.28, 167.39, 140.95, 134.93, 129.50, 128.56, 127.56, 120.16, 64.28, 59.22, 52.23, 48.36, 41.61, 40.50, 37.22, 32.71, 30.67, 27.34, 25.71, 24.48, 22.02, 20.69, 18.47, 17.49 . 199.80, 174.93, 173.62, 171.99, 166.02, 141.67, 134.70, 129.84, 128.86, 127.89, 121.01, 58.74, 57.27, J o u r n a l P r e -p r o o f 52. 00, 42.39, 41.56, 37.08, 31.60, 31.36, 30.98, 24.85, 22.86, 22.06, 21.32, 19.31, 18.34 . HRMS (m/z):

513.3001 (M+H) + . 199.83, 174.83, 173.14, 172.47, 166.09, 141.55, 134.73, 129.78, 128.82, 127.89, 120.36, 56.96, 52.17, 51.51, 42.32, 41.52, 39.73, 37.39, 34.11, 33.62, 32.79, 31.00, 27.14, 26.38, 26.18, 26.04, 24.87, 22.91, 22.04, 21.29 . HRMS (m/z): 567.3469

(M+H) + .

The compound 17a-17e were obtained by via scheme 3, and the detailed synthetic procedure was similar with the synthesis of compound 10a-10m. the relevant NMR data were exhibited below: 172.48, 172.08, 167.18, 141.37, 140.06, 132.00, 129.43, 127.74, 119.18, 58.79, 52.07, 52.00, 50.07, 41.92, 40.89, 37.40, 33.30, 31.14, 25.87, 24.50, 22.44, 21.62, 20.97, 20.88, 18.98, 17.98 . 10, 173.39, 172.50, 172.14, 167.32, 151.01, 141.36, 132.38, 127.86, 126.80, 119.19, 58.84, 52.02, 52.00, 50.02, 41.88, 40.80, 37.36, 33.97, 33.25, 31.05, 25.81, 24.48, 23.40 (double peak height), 22.39, 21.51, 20.86, 18.93, 17.89 . 173.41, 172.52, 172.14, 167.09, 142.45, 140.80, 140.11, 133.82, 128.71, 128.26, 127.57, 127.24, 126.74, 120.23, 58.90, 52.00, 51.95, 50.02, 41.84, 40.86, 37.38, 33.21, 31.10, 25.80, 24.50, 22.36, 21.55, 20.91, 18.93, 17. 96.

Compound 172.50, 172.17, 167.43, 161.06, 141.02, 129.32, 127.49, 117.69, 114.12, 58.77, 54.94, 52.00, 51.95, 50.02, 41.84, 40.80, 37.37, 33.21, 30.99, 25.81, 24.47, 22.34, 21.46, 20.87, 18.90, 17.86 . 173.38, 172.52, 172.30, 167.99, 151.60, 141.88, 129.30, 122.58, 114.73, 111.88, 58.74, 53.42, 51.99, 49.99, 41.86, 40.87, 39.73, 37.36, 33.20, 31.14, 25.80, 24.48, 22.39, 21.60, 20.92, 18.96, 17.98 . 81, 51.82, 49.95, 49.84, 41.77, 40.70, 37.33, 33.14, 30.94, 25.73, 24.45, 22.25, 21.36, 20.89, 18.81, 17.79 . 87, 40.87, 37.38, 33.25, 31.08, 25.83, 24.49, 22.38, 21.57, 20.89, 18.94, 17.95 . 86, 52.12, 52.02, 50.10, 41.94, 40.89, 37.41, 33.36, 31.13, 25.89, 24.51, 22.46, 21.62, 20.84, 18.97, 17.96 . 21, 173.55, 172.57, 172.24, 168.80, 134.90, 132.57, 131.23, 128.69, 127.93, 127.71, 127.50, 127.39, 126.46, 123.74, 59.62, 52.07, 51.50, 50.41, 41.59, 40.54, 38.10, 32.48, 30.90, 26.28, 24.38, 21.88, 21.14, 18.65, 18. 14. 12, 173.27, 172.47, 171.95, 162.16, 136.90, 130.09, 127.42, 124.33, 121.84, 120.28, 112.07, 104.01, 58.65, 53.39, 52.24, 50.06, 41.98, 40.81, 37.43, 33.25, 31.44, 25.86, 24.57, 22.46, 21.67, 20.79, 19.04, 18.32 . 173.41, 172.53, 172.47, 169.91, 138.15, 127.56, 126.14, 124.73, 120.37, 120.26, 111.08, 102.50, 59.03, 52.11, 52.01, 50.04, 41.85, 40.73, 37.36, 33.21, 31.41, 25.81, 24.49, 22.36, 21.50, 20.85, 19.02, 18.13 . 94, 173.71, 173.45, 172.44, 172.19, 140.77, 128.09, 128.03, 125.80, 58.62, 51.78, 51.40, 49.57, 41.56, 40.55, 37.23, 37.20, 32.82, 31.45, 30.58, 25.49, 24.35, 21.92, 21.04, 20.93, 18.41, 17.47 . 172.53, 172.26, 171.19, 167.54, 134.20, 131.53, 128.54, 127.26, 59.01, 52.11, 51.99, 50.04, 42.24, 41.97, 37.85, 33.64, 31.49, 26.22, 24.75, 22.63, 22.02, 21.67, 19.38, 18.59 . 11, 173.30, 172.48, 172.11, 168.08, 144.60, 139.84, 132.48, 128.82, 127.95, 127.71, 127.05, 127.03, 59.05, 52.10 (double peak height), 50.07, 41.93, 40.83, 37.40, 33.32, 31.40, 25.90, 24.52, 22.44, 21.61, 20.87, 19.05, 18.19 . 48, 175.09, 173.34, 172.46, 172.18, 58.19, 52.00, 51.92, 50.02, 45.20, 41.88, 40.89, 37.38, 33.28, 30.92, 29.77, 29.14, 25.86 (double peak height), 25.57, 25.45, 24.47, 22.40, 21.46, 20.87, 18.90, 17.79 . δ: 174.43, 172.73, 172.30, 170.84, 158.18, 147.52, 144.27, 114.69, 112.14, 58.04, 52.21, 51.82, 50.04, 42.23, 41.91, 37.74, 33.44, 31.67, 26.22, 24.68, 22.74, 22.11, 21.53, 19.24, 18.27 . 172.70, 172.50, 171.65, 162.63, 145.44, 143.43, 122.26, 108.75, 58.74, 52.17, 51.86, 49.83, 42.17, 41.78, 37.65, 33.54, 31.14, 26.10, 24.62, 22.64, 22.23, 21.47, 19.26, 18.82 . 173.52, 172.55, 172.51, 161.81, 125.05, 122.21, 111.04, 109.33, 58.52, 51.99, 51.96, 49.98, 41.84, 40.71, 37.35, 33.13, 31.31, 25.73, 24.50, 22.30, 21.50, 20.86, 18.91, 18.22 . 175. 45, 173.03, 172.92, 171.55, 166.25, 147.38, 141.04, 122.15, 57.47, 53.39, 52.17, 51.44, 42.12, 40.28, 37.14, 32.47, 30.39, 25.76, 24.37, 22.07, 20.49, 20.75, 18.74, 17.95. J o u r n a l P r e -p r o o f 6 Hz, 1H), 8.70 (dd, J = 4.9, 1.6 Hz, 1H), 1H), 7.51 (ddd, J = 8.0, 5.0, 0.6 Hz, 1H), 4.58 (dd, J = 11.7, 4.0 Hz, 1H), 4.50 (dd, J = 8.7, 4.3 Hz, 2H) 174.94, 173.26, 172.51, 171.86, 166.13, 151.51, 148.14, 135.94, 130.25, 123.75, 59.31, 51.99, 51.90, 49.79, 41.83, 40.78, 37.32, 33.16, 30.98, 25.75, 24.49, 22.35, 21.58, 20.94, 18.97, 18.35 . 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.86 (td, J = 7.7, 1.8 Hz, 1H), 7.44 (ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 4.76 (td, J = 8.4, 5.2 Hz, 1H), 4.57 (td, J = 10.2, 9.5, 5.2 Hz, 2H) , 3.71 (s, 3H), 3.33 (d, J = 5.6 Hz, 2H), 2.51 (ddd, J = 14.0, 12.1, 4.2 Hz, 1H), 2.29 (ddt, J = 20.4, 13.6, 5.8 Hz, 2H) , 2.08 -1.98 (m, 1H), 1.87 (ddd, J = 13.6, 9.9, 3. 37, 172.86, 172.40, 170.85, 164.32, 149.34, 148.27, 137.33, 126.35, 122.37, 58.60, 52.21, 51.61, 49.85, 42.12, 41.97, 37.55, 33.10, 31.52, 25.94, 24.62, 22.69, 22.21, 21.58, 19.30, 18.25 . 174.63, 172.60, 172.30, 171.28, 166.42, 164.82 (d, J = 252.2 Hz), 130.26 (d, J = 2.6 Hz), 129.62 (d, J = 8.9 Hz), 115.49 (d, J = 21.8 Hz), 59.06, 52.24, 51.97, 50.23, 42.28, 41.74, 37.82, 33.67, 31.46, 26.38, 24.69, 22.71, 22.10, 21.36, 19.25, 18.52 . 174.54, 172.51, 172.29, 171.02, 166.43, 137.90, 132.46, 128.76, 128.70, 59.06, 52.30, 51.92, 50.30, 42.34, 41.89, 37.93, 33.64, 31.49, 26.52, 24.70, 22.75, 22.13, 21.48, 19.26, 18.53 . 14, 173.31, 172.47, 171.80, 166.31, 149.62, 139.66, 128.60, 123.54, 59.37, 52.14, 52.11, 50.06, 41.94, 40.80, 37.39, 33.35, 31.16, 25.89, 24.52, 22.44, 21.57, 20.83, 19.01, 18.25 . 173.31, 172.47, 171.93, 167.13, 137.39, 133.34, 133.01, 127.82, 125.34 (dd, J = 7.3, 3.6 Hz, 1H), 125.04, 122.34, 59.26, 52.10, 52.01, 50.07, 41.88, 40.81, 37.40, 33.30, 31.15, 25.88, 24.51, 22.36, 21.51, 20.86, 18.96, 18.17 . 2, 8.8, 5.5 Hz, 12H) ; 13 C NMR (101 MHz, MeOD) δ: 175.61, 175.09, 173.28, 172.45, 172.03, 67.09, 58.34, 52.00, 51.78, 49.86, 41.86, 41.62, 40.80, 37.35, 33.22, 30.86, 29.29, 28.68, 25.80, 24.46, 22.38, 21.43, 20.85, 18.88, 17.79 . 16, 172.53, 167.68, 140.87, 140.16, 132.30, 129.63, 127.69, 120.01, 64.29, 58.99, 52.37, 49.07, 42.16, 41.25, 37.49, 33.38, 31.22, 25.91, 24.63, 22.51, 21.66, 20.99, 20.85, 19.39, 18.27 . 173.41, 172.28, 167.70, 150.98, 141.38, 132.44, 127.86, 126.75, 119.08, 64.48, 59.36, 52.26, 48.86, 41.86, 40.65, 37.34, 33.96, 32.97, 30.69, 25.94, 24.61, 23 .30 (double peak height), 22.47, 21.10, 20.68, 18.84, 17.85 . 11, 173.25, 172.14, 167.31, 142.55, 141.06, 140.14, 133.74, 128.77, 128.31, 127.65, 127.34, 126.83, 119.96, 64.64, 59.29, 52.29, 52.25, 42.02, 40.74, 37.46, 33.01, 30.82, 26.12, 24.67, 22.65, 21.30, 20.69, 18.99, 17.98 . 173.31, 171.95, 166.62, 149.54, 142.23, 121.77, 64.28, 59.80, 52.27, 49.23, 48.27, 41.62, 40.55, 37.21, 32.64, 30.57, 25.73, 24.48, 21.97, 20.80, 20.78, 18.51, 17.98 . 88, 173.39, 172.09, 166.63, 151.37, 147.99, 135.92, 130.42, 123.71, 64.31, 59.76, 52.29, 48.31, 41.63, 40.61, 37.24, 32.67, 30.56, 25.75, 24.49, 22.00, 20.82, 18.56, 18. 12, 170.97, 164.70, 149.26, 148.30, 137.42, 126.45, 122.38, 65.68, 58.68, 52.00, 49.52, 42.28, 41.90, 37.83, 32.54, 31.58, 26.54, 24.87, 22.83, 22.08, 21.51, 19.30, 18.33 . 173.33, 172.25, 167.78, 137.49, 132.62, 128.89, 128.33, 64.27, 59.73, 52.26, 48.25, 41.62, 40.56, 37.24, 32.65, 30.60, 25.75, 24.48, 21.96, 20.79, 20.75, 13 C NMR (101 MHz, CDCl3) δ: 199.72, 174.77, 173.24, 171.84, 162.70, 145.45, 143.55, 122.23, 108.70, 58.60, 56.78, 52.00, 42.20, 41.48, 37.08, 31.26, 30.80, 26.80, 24.80, 22.67, 22.18, 21.29, 19.26, 18.74 173.47, 171.23, 161.67, 125.00, 123.00, 110.05, 109.63, 58.85, 56.81, 51.76, 42.37, 41.20, 37.50, 31.34, 30.50, 26.64, 24.98, 22.78, 21.90, 21.12, 19.29, 18.41 199.49, 174.86, 173.03, 171.08, 165.66, 150.52, 141.17, 121.18, 59.08, 57.08, 52.04, 42.30, 41.64, 37.31, 31.52, 30.87, 27.12, 24.85, 22.74, 22.15, 21.28, 19.25, 18.54 199.56, 174.83, 173.10, 171.33, 165.80, 152.14, 148.50, 135.69, 129.93, 123.53, 59.24, 57.01, 52.03, 42.24, 41.57, 37.11, 31.33, 29.70, 27.03, 24.82, 22.76, 22.11, 21.31, 19.34, 18.58 88, 174.87, 173.31, 171.09, 164.52, 149.33, 148.28, 137.39, 126.41, 122.39, 58.57, 56.68, 51.87, 42.15, 41.61, 36.90, 31.43, 30.57, 26.74, 24.83, 22.75, 22.12, 21.41, 19.35, 18.17 column (GE Healthcare). Following washing the resin with the washing buffer contain 20 mM imidazole (pH 8), PPase was added to generate the mature SARS-CoV-2 3CL Pro . Crude protein was purified by Superdex 200 gel filtration chromatography (GE Healthcare), and verified by SDS-PAGE analysis. Finally, the target protein was concentrated into 50 mg/mL and stored at -80 °C.

Compound 16b ((S)-N-((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)-2-((S)-2-((E)-3-(4- isopropylphenyl)acrylamido)-3-methylbutanamido)-4-

Purified SARS-CoV-2 3CL Pro was pre-incubated with the inhibitor in 5:1 stoichiometric ratio at 4 °C overnight and incubated with the diverse crystallization conditions. Following iterative rounds of optimization of the crystallization conditions, the crystals of SARS-CoV-2 3CL Pro was suitable to grow in 0.1 M MES, pH 6.0, 10% PEG 6000 buffer condition and in hanging-drop, vapor diffusion method at 16 °C.

For collection of X-Ray diffraction data, the crystals were flash-cooled in liquid nitrogen followed by dragging the crystals through the crystallization solution supplemented with 20% glycerol. The X-ray diffraction data were collected at the SSRF Beamline BL19U1. The data were processed by the HKL3000 package. The crystal structure of SARS-CoV-2 3CL Pro (PDB code: 6LZE) was used as the initial searching model to determine the complex structure. Subsequently, the manual model was refined by performing COOT and PHENIX software through rigid-body refinement, energy minimization and individual bfactor refinement. Finally, the quality of the final refined model was verified by the program PHENIX validation module and the statistics information were summarized in Table S3 -S8.

The FRET-based peptide MCA-TSAVLQSGFRK(DNP)M was synthesized as a substrate via a solidphase method. When the peptide was cleaved by 3CL Pro at the Gln-Ser bond, the fluorescence was released. In detailed, a 2.0 μM solution of SARS-CoV-2 3CL Pro dissolved in 25 μL assay buffer (pH = 8.0, 20 mM Tris-HCl, 150 mM NaCl) was incubated with 25 μL seven different concentrations of the inhibitor (2-fold dilution), respectively, and DMSO only as blank control at 37 °C for 30 min. Subsequently, a 50 μL solution contain 30 μM substrate was added to the mixture and the reaction was initiated. After 2h incubation, the change of relative fluorescence units was recorded by a microplate reader (Thermo Varioskan Flash, USA) at λex of 340 nm and λem of 440 nm. Finally, following inhibitory curve fitting by GraphPad Prism 7.0, the IC50 value of the inhibitor was obtained.

For matching the similar environment with the SARS-CoV-2 infection, the lung tumor cell line A549 was chosen to test the toxicity of peptidomimetic inhibitor. In brief, the cell (5000 per well) was incubated with the 25 μL seven different concentrations (2-fold dilution) of the peptidomimetic inhibitor, respectively, at 37 °C for 24h. Subsequently, the solution contained MTT was added to the plate and incubated with the cell for 4 h. Then, the super supernatant was discarded and the cell was washed by PBS buffer at 3 times. 200 μL DMSO was added and the absorbance at 490nm was recorded J o u r n a l P r e -p r o o f by a microplate reader (Thermo Varioskan Flash, USA). Finally, the cellular viability rate was calculated and the CC50 value of the peptidomimetic was obtained by the survival curve fitting.

SARS-CoV-2 (WIV04) was passaged in Vero E6 cells and titered by a plaque assay. Vero E6 cells 

Detailed synthetic procedures, compound characterization, and biology assays. This material is available free of charge via the Internet at http://pubs.acs.org.

* For L.S.: shanglq@nankai.edu.cn

The authors declare no competing financial interests.

(S)-2-oxopiperidin-3-yl)propan-2-yl)-4-methylpentanamide

m, 1H), 2.20 -1.95 (m, 3H), 1.90 -1.76 (m, 1H), 1.73 -1.57 (m, 5H), 1.51 (dd, J = 9.8, 3.4 Hz, 1H), 1.05 -0.83 (m, 12H) ; 13 C NMR (101 MHz, MeOD) δ: 176

)-2-oxopiperidin-3-yl)propan-2-yl) amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-2-naphthamide) : 1 H NMR (400MHz, MeOD) δ: 8.42 (s, 1H)

(m, 1H), 2.31 -2.19 (m, 1H), 2.18 -2.08 (m, 1H), 2.08 -1.99 (m, 1H), 1.83 -1.71 (m, 2H), 1.71 -1.58 (m, 4H), 1.51 -1.38 (m, 1H), 1.08 (dd

(m, 1H), 1.72 -1.57 (m, 5H), 1.55 -1.44 (m, 1H), 1.06 (dd, J = 6.7, 2.2 Hz, 6H), 0.91 (dd, J = 12.6, 6.0 Hz, 6H); 13 C NMR (101 MHz, CDCl3: MeOD = 1:2) δ: 176.07

m, 2H), 2.93 (t, J = 7.7 Hz, 2H), 2.60 (t, J = 7.7 Hz, 2H), 2.38 -2.23 (m, 1H)

)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl

)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)cyclohexanecarboxamide) : 1 H NMR (400MHz

)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)furan-2-carboxamide) : 1 H NMR (400MHz, MeOD) δ: 7.69 (dd, J = 1.6, 0.6 Hz, 1H), 7.19 (dd, J = 3.5, 0.7 Hz, 1H), 6.60 (dd

2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-4-(trifluoromethyl)benzamide) : 1 H NMR (400MHz, MeOD) δ: 8.00 (d

)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino

(S)-2-oxopiperidin-3-yl)propan-2-yl)pentanamide) : 1 H NMR (400MHz, CDCl3) δ: 9

m, 3H), 2.07 -1.94 (m, 1H), 1.94 -1.79 (m, 2H), 1.73 -1.56 (m, 4H), 1.54 -1.42 (m, 1H

3-methylbutanamido)-4-methyl-N-((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)pentanamide) : 1 H NMR (400MHz, CDCl3) δ: 9

(4-methoxyphenyl)acrylamido)-3-methylbutanamido)-4-methyl-N-((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)pentanamide) : 1 H NMR (400MHz, CDCl3) δ: 9

73 (s, 1H), 6.54 (s, 1H)

27 (s, 2H), 2.99 (s, 6H), 2.44 -2.27 (m, 1H), 2.25 -2.10 (m, 2H), 2.08 -1.95 (m, 1H), 1.93 -1.79 (m, 2H), 1.74 -1.47 (m, 5H), 1.01 -0.83 (m, 12H); 13 C NMR (101 MHz, CDCl3) δ: 199

49 (s, 1H), 8.32 (d, J = 6.8 Hz, 1H), 7.71 (d, J = 15.8 Hz, 1H), 7.49 (t, J = 7.3 Hz, 1H), 7.30 (dd, J = 13

Hz, 1C), 140.31 (d, J = 2.6 Hz, 1C), 137.73 (d, J = 2.2 Hz, 1C) 130.35 (d, J = 8.4 Hz, 1C), 123.88 (d, J = 2.6 Hz, 1C), 122.21 (d, J = 2.0 Hz, 1C), 116.60 (d, J = 21.9 Hz, 1C)

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)-2-naphthamide) : 1 H NMR (400MHz, CDCl3) δ: 9.51 (s, 1H), 8.38 (d, J = 7.0 Hz, 1H), 8.31 (s, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.91 -7.77 (m, 4H), 7.50 (dt, J = 14.7, 6.9 Hz, 2H)

82 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)-1H-indole-2-carboxamide) : 1 H NMR (400MHz, CDCl3) δ: 9

61 (s, 1H), 6.35 (s, 1H), 4.65 -4.50 (m, 2H), 4.44 -4.33 (m, 1H), 3.30 -3.16 (m, 2H), 2.35 -2.23 (m, 2H), 2.23 -2.10 (m, 1H

Hz, 1H), 7.41 -7.31 (m, 1H)

m, 2H), 3.01 -2.83 (m, 2H), 2.61 -2.48 (m, 2H), 2.38 -2.27 (m, 1H

)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)benzamide) : 1 H NMR

Hz, 3H), 7.47 (t, J = 6.8 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.22 (d, J = 8.3 Hz, 1H)

1 H NMR (400MHz, CDCl3) δ: 9.50 (s, 1H), 8.36 (d, J = 7.0 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.60 (dd, J = 16.9, 7.8 Hz, 3H)

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)cyclohexanecarboxamide) : 1 H NMR (400MHz, CDCl3) δ: 9

Hz, 1H), 4.35 -4.25 (m, 1H), 3.40 -3.23 (m, 2H), 2.42 -2.27 (m, 1H), 2.25 -2.10 (m, 2H), 2.10 -1.98 (m, 2H), 1.95 -1.75 (m, 6H), 1.73 -1.60 (m, 4H), 1.60 -1.49 (m, 2H), 1.49 -1.37 (m, 2H

99 (s, 1H), 6.50 (d, J = 1.5 Hz, 1H)

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)furan-3-carboxamide) : 1 H NMR (400MHz, CDCl3) δ: 9.50 (s, 1H), 8.35 (d, J = 7.1 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.40 (d, J = 1.5 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H)

HRMS (m/z): 505.2751 (M+H) + Compound 17w (4-chloro-N-((S)-3-methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)benzamide) : 1 H NMR (400MHz, CDCl3) δ: 9

J = 20.0, 8.1 Hz, 1H), 1.91 -1.84 (m, 1H)

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)-4-nitrobenzamide) : 1 H NMR (400MHz, CDCl3) δ: 9

m, 2H), 2.42 -2.28 (m, 1H), 2.28 -2.10 (m, 2H), 2.08 -1.95 (m, 1H), 1.95 -1.82 (m, 2H), 1.79 -1.47 (m, 5H), 0.98 (t, J = 6.4 Hz, 6H), 0.90 (dd, J = 11.6, 5.9 Hz, 6H

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)-4-(trifluoromethyl)benzamide) : 1 H NMR (400MHz, CDCl3) δ: 9.49 (s, 1H), 8.39 (d, J = 6.8 Hz, 1H), 7.92 (d, J = 8.1 Hz, 2H), 7.66 (d, J = 8.2 Hz, 3H), 7.36 (d, J = 8.7 Hz, 1H)

yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)tetrahydro-2H-pyran-4-carboxamide

NMR (400MHz, CDCl3) δ: 9.47 (s, 1H), 8.29 (d, J = 6.4 Hz, 1H)

A Novel Coronavirus from Patients with Pneumonia in China

Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro

Antiviral activities of type I interferons to SARS-CoV-2 infection

Pfizer's novel COVID-19 antiviral heads to clinical trials

Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19

An Oral SARS-CoV-2 Mpro Inhibitor Clinical Candidate for the Treatment of COVID-19

The Architecture of SARS-CoV-2 Transcriptome

Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach

The SARS-CoV-2 main protease as drug target

Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Study Group of the International Committee on Taxonomy of, The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors

Mechanism of the Maturation Process of SARS-CoV 3CL Protease

An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

Comprehensive Insights into the Catalytic Mechanism of Middle East Respiratory Syndrome 3C-Like Protease and Severe Acute Respiratory Syndrome 3C-Like Protease

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

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

Picornaviral 3C protease inhibitors and the dual 3C protease/coronaviral 3C-like protease inhibitors

Cyanohydrin as an Anchoring Group for Potent and Selective Inhibitors of Enterovirus 71 3C Protease

4-Iminooxazolidin-2-one as a Bioisostere of the Cyanohydrin Moiety: Inhibitors of Enterovirus 71 3C Protease

Application of Dually Activated Michael Acceptor to the Rational Design of Reversible Covalent Inhibitor for Enterovirus 71 3C Protease

A large number of compounds were synthesized and most of the inhibitors exhibited excellent inhibitory activity against SARS-CoV-2 3CL Pro

The protein-ligand complexes of SARS-CoV-2 3CL Pro with peptidomimetic inhibitors were revealed, including the SAR of the peptidomimetic inhibitor and cocrystal structure of SARS-CoV-2 3CL Pro with inhibitors

We thank the staff of SSRF BL19U1 for their support in collecting the diffraction data. 

J o u r n a l P r e -p r o o f

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:J o u r n a l P r e -p r o o f