China Paper Supporting Data

science.sciencemag.org/cgi/content/full/science.
abf1611/DC1
Supplementary Materials for

SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse
model
Jingxin Qiao, Yue-Shan Li, Rui Zeng, Feng-Liang Liu, Rong-Hua Luo, Chong Huang,
Yi-Fei Wang, Jie Zhang, Baoxue Quan, Chenjian Shen, Xin Mao, Xinlei Liu, Weining Sun,
Wei Yang, Xincheng Ni, Kai Wang, Ling Xu, Zi-Lei Duan, Qing-Cui Zou, Hai-Lin Zhang,
Wang Qu, Yang-Hao-Peng Long, Ming-Hua Li, Rui-Cheng Yang, Xiaolong Liu, Jing You,
Yangli Zhou, Rui Yao, Wen-Pei Li, Jing-Ming Liu, Pei Chen, Yang Liu, Gui-Feng Lin,
Xin Yang, Jun Zou, Linli Li, Yiguo Hu, Guang-Wen Lu, Wei-Min Li, Yu-Quan Wei,
Yong-Tang Zheng*, Jian Lei*, Shengyong Yang*
*Corresponding author. Email: yangsy@scu.edu.cn (S.Y.); leijian@scu.edu.cn (J.L.); zhengyt@mail.kiz.ac.cn

(Y.-T.Z.)
Published 18 February 2021 on Science First Release

DOI: 10.1126/science.abf1611
This PDF file includes:

Figs. S1 to S5
Tables S1 to S7
Materials and Methods
Supplementary Text
References
Other supplementary material for this manuscript includes:

MDAR Reproducibility Checklist (PDF)
Fig. S1. Boceprevir/Telaprevir binding pocket of SARS-CoV-2 Mpro. (A) Boceprevir and (B)
Telaprevir are displayed as sticks and colored by atom type, with carbon atoms in purple, oxygen
atoms in red, and nitrogen atoms in blue. S1', S1, S2 and S4 pockets of Mpro are marked. The
catalytic site residues Cys145 and His41 are also shown as sticks and colored by atom type, with
carbon atoms in green, sulfur atoms in orange, oxygen atoms in red, and nitrogen atoms in blue.
All figures were prepared using the program PyMOL (https://pymol.org).
2
Fig. S2. Chemical structures of designed SARS-CoV-2 Mpro inhibitors.
3
Fig. S3. Plasma concentration–time curves for MI-09 and MI-30. (A-D) The mean plasma
concentration-time curves of (A) MI-09 (i.p. 20 mg/kg), (B) MI-30 (i.p. 20 mg/kg), (C) MI-09
(p.o. 20 mg/kg) and (D) MI-30 (p.o. 20 mg/kg) in rats (n = 3). The EC50 and EC90 values of MI-
09 and MI-30 were calculated from the cell-based antiviral activity data obtained with HPAEpiC
(dashed lines) and Vero E6 cells (solid lines).
4
Fig. S4. Scanning images of whole lung tissue sections. (A-L) The whole lung slide scan images
from SARS-CoV-2 (5×106 TCID50) -infected hACE2 transgenic mice on 3 dpi (n = 3). [Note: (C),
(E), (G) and (J) correspond to the full images of the lung tissue sections of mice treated with
vehicle, MI-09 (p.o.), MI-09 (i.p.), and MI-30 (i.p.) shown in Fig. 4E, respectively].
5
Fig. S5. Antiviral activity of six compounds (MI-09, MI-12, MI-14, MI-28, MI-30 and MI-31)
and two positive controls (GC376 and 11b) against SARS-CoV-2 virus replication in Huh7
cells. Huh7 cells were infected with SARS-CoV-2 at an MOI of 0.01 and treated with different
concentrations of test compounds. At 48 h post infection, viral RNA copies (per mL) were
quantified from cell culture supernatants by the RT-qPCR method. Data are shown as mean ± SD,
n = 2 biological replicates.
6
Table S1. Biochemical and biophysical activities of Mpro inhibitors.a
Enzyme Mpro DSF Enzyme Mpro DSF

Compound Compound
(IC50, nM) Tm / ΔTm (°C) (IC50, nM) Tm / ΔTm (°C)
MI-01 453.0 ± 3.2 70.0 ± 0.2 / 18.7 MI-17 298.8 ± 2.3 66.6 ± 0.2 / 15.0
MI-02 52.1 ± 1.4 72.6 ± 0.2 / 21.2 MI-18 525.9 ± 8.3 69.3 ± 0.2 / 18.0
MI-03 16.5 ± 1.4 69.8 ± 0.1 / 18.5 MI-19 195.6 ± 0.8 67.1 ± 0.2 / 16.8
MI-04 18.5 ± 0.5 70.2 ± 0.0 / 18.8 MI-20 375.0 ± 1.0 64.9 ± 0.4 / 14.8
MI-05 13.2 ± 0.4 69.7 ± 0.0 / 19.7 MI-21 7.6 ± 0.2 69.2 ± 0.1 / 18.9
MI-06 14.5 ± 0.4 73.1 ± 0.2 / 21.7 MI-22 17.4 ± 0.4 67.3 ± 0.4 / 17.0
MI-07 43.3 ± 0.8 69.5 ± 0.4 / 18.1 MI-23 7.6 ± 0.1 70.5 ± 0.3 / 19.1
MI-08 37.2 ± 0.7 68.6 ± 0.5 / 18.3 MI-24 378.2 ± 2.3 63.9 ± 0.5 / 12.5
MI-09 15.2 ± 0.4 72.8 ± 0.1 / 21.5 MI-25 36.2 ± 0.7 72.5 ± 0.2 / 21.1
MI-10 50.8 ± 0.5 70.6 ± 0.1 / 19.2 MI-26 69.1 ± 1.6 67.2 ± 1.0 / 16.9
MI-11 13.3 ± 0.2 71.9 ± 0.1 / 20.5 MI-27 93.5 ± 1.2 72.3 ± 0.8 / 20.9
MI-12 19.0 ± 0.6 70.8 ± 0.3 / 19.5 MI-28 9.2 ± 0.2 72.5 ± 0.2 / 21.2
MI-13 12.4 ± 0.2 71.1 ± 0.2 / 20.1 MI-29 34.7 ± 0.4 73.0 ± 0.8 / 21.7
MI-14 13.0 ± 0.3 71.7 ± 0.1 / 20.7 MI-30 17.2 ± 0.6 68.2 ± 0.5 / 17.2
MI-15 748.5 ± 2.3 70.0 ± 0.5 / 17.1 MI-31 30.0 ± 0.4 71.4 ± 0.1 / 20.4
MI-16 153.1 ± 1.0 70.6 ± 0.1 / 19.2 MI-32 19.7 ± 0.7 69.2 ± 0.1 / 18.9
11b 27.4 ± 0.5 68.3 ± 0.0 / 17.0 GC376 37.4 ± 1.9 70.6 ± 0.1 / 20.5
a
Data are shown as mean ± SD, n = 3 biological replicates.
7
Table S2. Data collection and refinement statistics.
Mpro-MI-23 Mpro-Boceprevir Mpro-Telaprevir

Data collection statistics
Space group C2 P212121 P212121
Unit-cell dimensions
a, b, c (Å) 113.48, 52.46, 45.40 67.76, 99.43, 103.81 67.91, 98.73, 103.49
α, β, γ (°) 90, 103.3, 90 90, 90, 90 90, 90, 90
Wavelength (Å) 0.97620 0.97853 0.97853
Vm (Å3 /Da) 1.90 2.53 2.51
Solvent content (%) 35.30 51.33 50.95
Resolution range (Å) 47.38-2.00 (2.06-2.00) 19.85-2.25 (2.32-2.25) 19.80-1.74 (1.77-1.74)
No. of unique reflections 17,469 (1,217) 33,972 (3,097) 71,966 (3,883)
Rmerge 0.063 (0.520) 0.177 (1.423) 0.098 (1.925)
Rpim a 0.027 (0.213) 0.048 (0.405) 0.020 (0.390)
CC1/2 b 0.999 (0.888) 0.997 (0.830) 1.000 (0.794)
Mean I/σ(I) 16.6 (3.7) 11.7 (2.1) 22.8 (2.2)
Multiplicity 6.6 (6.7) 14.2 (13.3) 25.8 (24.5)
Completeness (%) 99.3 (94.4) 99.9 (100.0) 99.0 (100.0)
Wilson B-factor (Å2) 31.5 33.4 25.7
Refinement statistics
Rfactor (%) c 16.6 19.8 17.4
Rfree (%) c 20.9 24.6 21.6
No. of atoms
Protein 2,371 4,633 4,704
Inhibitor(s) 33 74 49/25*
Water 284 430 526
Clashscore d 2 2 4
r.m.s.d. in bond lengths (Å) 0.008 0.008 0.011
r.m.s.d. in bond angles (°) 1.010 1.080 1.748
B-factor for inhibitor(s) 33.5 46.8 40.5/69.4*
Average B-factor (Å2) 34.0 40.0 34.0
Ramachandran plot
Preferred regions (%) 98.0 98.0 97.0
Allowed regions (%) 2.0 2.0 3.0
Outlier regions (%) 0.0 0.0 0.0
PDB entry 7D3I 7COM 7C7P
8
a
R pim = ∑ℎ𝑘𝑘𝑘𝑘 �1/(𝑛𝑛 − 1) ∑𝑛𝑛𝑖𝑖=1|𝐼𝐼𝑖𝑖 (ℎ𝑘𝑘𝑘𝑘) − 𝐼𝐼 (̅ ℎ𝑘𝑘𝑘𝑘)|/ ∑ℎ𝑘𝑘𝑘𝑘 ∑𝑛𝑛𝑖𝑖=1 𝐼𝐼𝑖𝑖 (ℎ𝑘𝑘𝑘𝑘) (31).
b
CC1/2 is the correlation coefficient determined by two random half data sets (32).
c
R factor = ∑ℎ𝑘𝑘𝑘𝑘|𝐹𝐹𝑜𝑜 (ℎ𝑘𝑘𝑘𝑘) − 𝐹𝐹𝑐𝑐 (ℎ𝑘𝑘𝑘𝑘)|/ ∑ℎ𝑘𝑘𝑘𝑘 |𝐹𝐹𝑜𝑜 (ℎ𝑘𝑘𝑘𝑘)| . Rfree was calculated for a test set of
reflections (~ 5.0%) omitted from the refinement.
d
Clashscore is defined as the number of clashes calculated for the model per 1000 atoms (including
hydrogens) of the model. Hydrogens were added by MolProbity (33).
*In 7C7P, molecule A binds to the complete Telaprevir, while molecule B interacts with only
partial Telaprevir. Therefore, the statistical values for these two ligands are separated.
9
Table S3. Cytotoxicity of Mpro inhibitors against selected human cell lines.a
CC50 (μM)
Compound
HPAEpiC LO2 BEAS-2B A549 Huh7
MI-03 >500 >500 >500 >500 >500
MI-04 >500 >500 >500 >500 >500

MI-05 >500 >500 >500 >500 >500
MI-06 >500 >500 >500 >500 >500
MI-07 >500 >500 >500 >500 >500
MI-08 >500 >500 >500 >500 >500
MI-09 >500 >500 >500 >500 >500
MI-11 >500 >500 >500 >500 >500
MI-12 >500 >500 >500 >500 >500

MI-13 >500 >500 >500 >500 >500
MI-14 >500 >500 >500 >500 >500
MI-21 >500 >500 >500 >500 >500
MI-22 >500 >500 >500 >500 >500
MI-23 >500 >500 >500 >500 >500

MI-25 >500 >500 >500 >500 >500
MI-28 >500 >500 >500 >500 >500

MI-29 >500 >500 >500 >500 >500
MI-30 >500 >500 >500 >500 >500
MI-31 >500 >500 >500 >500 >500

MI-32 >500 >500 >500 >500 >500
11b 270 109 251 >500 >500
GC376 260 246 254 326 489
a
Data were obtained in three independent experiments.
10
Table S4. Cell-based antiviral activity and cytotoxicity of Mpro inhibitors on Vero E6 cells.a
Cell protection Toxicity Cell protection Toxicity

Compound SIb Compound SIb
EC50 (μM) CC50 (μM) EC50 (μM) CC50 (μM)
MI-03 2.23 ± 0.62 > 500 > 224 MI-14 0.66 ± 0.06 > 500 > 758
MI-04 1.64 ± 0.88 > 500 > 305 MI-21 2.97 ± 0.81 > 500 > 168
MI-05 5.57 ± 1.00 > 500 > 90 MI-22 30.49 ± 1.24 > 500 > 16
MI-06 1.73 ± 1.01 > 500 > 289 MI-23 5.63 ± 1.09 > 500 > 89
MI-07 16.95 ± 0.90 > 500 > 29 MI-25 24.72 ± 17.17 > 500 > 20
MI-08 4.68 ± 0.83 > 500 > 107 MI-28 0.67 ± 0.06 > 500 > 746
MI-09 0.86 ± 0.07 > 500 > 581 MI-29 1.65 ± 0.23 > 500 > 303
MI-11 1.18 ± 0.10 > 500 > 424 MI-30 0.54 ± 0.13 > 500 > 926
MI-12 0.53 ± 0.07 > 500 > 943 MI-31 0.83 ± 0.28 > 500 > 602
MI-13 2.08 ± 0.81 > 500 > 240 MI-32 1.50 ± 0.28 > 500 > 333
11b 0.89 ± 0.04 > 500 > 562 GC376 1.46 ± 0.14 > 100 > 68
a
Data are given as mean ± SD, n = 2 independent experiments.
b
SI values were calculated by CC50 / EC50.
11
Table S5. Primary pharmacokinetic (PK) evaluation of six selected compounds in SD rats.a
T1/2 Tmax Cmax AUClast AUCINF_obs MRTINF_obs CL Vssobs F

Compound Admin
(h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (mL/min/kg) (mL/kg) %
i.v. 16867 ± 7429 ± 7432 ±
1.58 ± 0.97 0.08 ± 0.00 0.41 ± 0.04 22.67 ± 2.97 559 ± 30 -
(10 mg/kg) 1453 928 926
i.p. 7597 ± 11581 ± 11590 ±
MI-09 4.53 ± 0.49 0.50 ± 0.00 1.33 ± 0.20 - - 78.0 ± 4.1
(20 mg/kg) 1665 618 613
p.o. 576 ± 1665 ± 1667 ±
0.77 ± 0.22 1.58 ± 2.10 2.72 ± 0.43 - - 11.2 ± 6.4
(20 mg/kg) 309 952 953
i.v. 5459 ± 1082 ± 1084 ±
0.47 ± 0.02 0.08 ± 0.00 - 31.17 ± 4.65 - -
(2 mg/kg) 892 152 152
MI-12
p.o. 127 ± 258 ± 262 ±
1.03 ± 0.06 0.33 ± 0.14 - - - 4.8 ± 0.8
(10 mg/kg) 45 45 47
i.v. 18959 ± 4681 ± 4685 ±
1.11 ± 0.14 0.08 ± 0.00 - 18.05 ± 2.57 - -
(5 mg/kg) 2564 2564 723
MI-14
p.o. 273 ± 381 ± 401 ±
0.91 ± 0.31 0.25 ± 0.00 - - - 4.1 ± 1.6
(10 mg/kg) 51 153 178
i.v. 16312 ± 5844 ± 5852 ±
1.59 ± 0.06 0.08 ± 0.00 0.34 ± 0.02 14.25 ± 0.54 293 ± 16 -
(5 mg/kg) 525 226 226
MI-28
p.o. 402 ± 842 ± 852 ±
0.93 ± 0.50 0.25 ± 0.00 2.22 ± 0.15 - - 7.2 ± 2.7
(10 mg/kg) 33 314 300
i.v. 23582 ± 9768 ± 9776 ±
2.35 ± 0.48 0.08 ± 0.00 0.38 ± 0.01 17.10 ± 1.12 387 ± 20 -
(10 mg/kg) 1755 649 652
i.p. 14573 ± 14878 ± 14894 ±
MI-30 3.88 ± 1.45 0.50 ± 0.00 0.92 ± 0.11 - - 76.2 ± 20.9
(20 mg/kg) 2853 4075 4072
p.o. 1017 ± 2843 ± 2846 ±
0.77 ± 0.22 1.50 ± 2.17 2.89 ± 0.44 - - 14.6 ± 1.0
(20 mg/kg) 94 191 191
i.v. 38529 ± 9539 ± 9545 ±
1.34 ± 1.13 0.08 ± 0.00 - 8.94 ± 1.63 - -
(5 mg/kg) 2771 1861 1863
MI-31
p.o. 715 ± 968 ± 976 ±
0.80 ± 0.03 0.33 ± 0.14 - - - 5.1 ± 0.5
(10 mg/kg) 280 94 92
a
Data are shown as mean ± SD; n = 3 animals.
12
Table S6. In vivo toxicity study of MI-09 and MI-30 in SD rats.
Acute toxicity study Repeated dose one week toxicity study
Compound MI-09 / MI-30 MI-09 / MI-30
Administration p.o. i.p. i.v. p.o. i.p. i.v.
250 mg/kg 24 mg/kg 100 mg/kg 100 mg/kg 6 mg/kg

Dosage 250 mg/kg
500 mg/kg 40 mg/kg 200 mg/kg 200 mg/kg 18 mg/kg
2 rats
Number of 4 rats per (24 mg/kg)
4 rats 6 rats per group
animals dosage group 10 rats
(40 mg/kg)
Frequency Single dose Single dose Single dose Twice daily Once daily Once daily
1. No abnormalities in body weight or general status were observed in each group during administration.
Results 2. At the end of administration, histological examination of heart, liver, spleen, lung and kidney was carried
out, and no abnormality was found in each group.
13
Table S7. Primer sequences for quantifying inflammatory gene expression.
Gene Genbank ID Sequence
AGAACATCATCCCTGCATCC
GAPDH NM_008084.3
CACATTGGGGGTAGGAACAC
AACCAAGAGATAAGCTGGAGTCAC
IL-6 NM_001314054.1
AACGCACTAGGTTTGCCGAG
TGCCACCTTTTGACAGTGATGA
IL-1β NM_008361.4
ATCAGGACAGCCCAGGTCAA
TGCAGGATGATGGTCAAGCC
CXCL-10 NM_021274.2
CCACTTGAGCGAGGACTCAG
CAGCAAGGCGAAAAAGGATGC
IFN-γ NM_008337.4
CTTCCTGAGGCTGGATTCCG
GTGGGAGATGTCCTCAACTGC
IFN-β NM_010510.1
TCTCTGCTCGGACCACCATC
14
Materials and Methods
Production of authentic SARS-CoV-2 main protease
The cDNA plasmid pET28b encoding SARS-CoV-2 (GenBank: MN908947) main protease (Mpro)
was synthesized for E. coli expression system (General Biosystems). To obtain the wild type
SARS-CoV-2 Mpro with authentic N- and C-termini, the designed Mpro construct contains residues
as below:
“MGTSAVLQSGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSED
MLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRI
QPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHH
MELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLN
RFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGR
TILGSALLEDEFTPFDVVRQCSGVTFQGPLEHHHHHH”.
At the N-terminus, this construct includes the Mpro cleavage site between Nsp4 and Nsp5 of
SARS-CoV-2 (TSAVLQ / SGFRK; “ / ” indicates the cleavage site). At the C-terminus, it
contains a modified PreScission cleavage site (SGVTFQ / GP). An authentic N-terminus of Mpro
was obtained by the auto-cleavage of Mpro during its gene expression, and the authentic C-terminus
was generated by digesting with PreScission protease.
The certified pET28b-Mpro plasmid was transformed into E. coli BL21(DE3) cells (Novagen)
and then cultured in Luria broth medium containing 50 μg/ml kanamycin at 37°C. When the cells
were grown to an optical density at 600 nm of ~ 0.8, 0.5 mM (final concentration) isopropyl-D-
thiogalactoside (IPTG) was added to the cell culture to induce the corresponding gene expression
at 18°C. In 16 ~ 18 h, the cells were harvested by centrifugation at 8,000 rpm. The cell pellets were
then resuspended in buffer A (20 mM Tris-HCl pH 8.0, 10 mM imidazole, 500 mM NaCl and 5%
15
Glycerol), lysed by sonication on ice and clarified by centrifuging at 20,000 rpm at 4°C to remove
debris. The supernatants were loaded onto HisTrap FF column (GE Healthcare) and eluted in
buffer B (20 mM Tris-HCl pH 8.0, 500 mM imidazole, 500 mM NaCl and 5% Glycerol) with a
step-gradient method (0-100% buffer B). Subsequently, Mpro was cleaved by PreScission protease
to remove the His-tag and dialyzed against buffer C (20 mM Tris-HCl pH 8.0, 150 mM NaCl and
5% Glycerol) overnight at 4°C. The target protein was loaded to GSTtrap FF and Nickel column
(GE Healthcare) again to remove GST-tagged PreScission protease (the GST-PreScission
plasmids were kindly provided by Prof. Yibei Xiao from China Pharmaceutical University,
Nanjing, China) and uncleaved His-tag protein. Then the authentic Mpro was further purified by gel
filtration (Superdex 75 Increase 10/300 GL, GE Healthcare) in buffer C. The quality of purified
Mpro was checked by SDS-PAGE.
Crystallization and data collection
The purified SARS-CoV-2 Mpro protein (concentrated to ~ 5 mg/mL) was incubated with
Boceprevir, Telaprevir, and MI-23 at a molar ratio of 1:20 at 4°C for 2 h, respectively. Each
mixture was crystallized at 291K by the sitting-drop vapor-diffusion method with the protein-to-
matrix ratio of 1:1 (v/v) using commercial screen kits: Index, PEGRx 1/2, PEG/Ion Screen 1/2,
SaltRx 1/2 (Hampton Research) together with PACT premier, Structure Screen 1/2, JCSG plus
(Molecular Dimensions). The crystals of both SARS-CoV-2 Mpro-Boceprevir and Mpro-Telaprevir
appeared under No. 32 (0.1 M MES pH6.0, 14% w/v PEG 4000) of PEGRx 1/2. The Mpro-
Telaprevir crystals were further produced under condition: 0.1 M MES pH 6.0 containing 13%
w/v PEG 4000. And the crystals of Mpro with Boceprevir were obtained with condition: 0.1 M
MES pH 6.5 containing 21% w/v PEG 4000 and 20% v/v PEG400. The crystals of Mpro-MI-23
were observed under condition No.73 of PEGRx1/2: 0.1% w/v n-octyl-β-D-glucoside, 0.1 M
16
sodium citrate tribasic dihydrate pH 5.5 and 22% w/v PEG 3,350. All crystals were fished and
flash-cooled in liquid nitrogen.
The diffraction data sets of both Mpro-Boceprevir and Mpro-Telaprevir were collected at an X-
ray wavelength 0.97853 Å at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL19U1
(Shanghai, China). The diffraction data sets of Mpro-MI-23 were collected at a wavelength 0.97620
Å at PETRA III beamline P11, DESY (Hamburg, Germany). Data sets were processed by XDS
(34) and scaled with Aimless (35) in CCP4. The statistics of diffraction data sets are listed in table
S2.
Phase determination and refinement
All three structures were determined by molecular replacement (MR). The initial phases of Mpro
in complex with Telaprevir were determined by MR with program MOLREP (36) using the SARS-
CoV Mpro (PDB entry: 3SNB (37)) as a search model. Telaprevir was subsequently built into the
model using Coot (38). Refinement of this structure was performed by program REFMAC5 (39).
The structures of Mpro-Boceprevir and Mpro-MI-23 were determined through the same method but
using the search model from Mpro-Telaprevir structure. In addition, the refinements of the latter
two structures were performed by program BUSTER (40). See table S2 for the detailed refinement
statistics.
Enzyme kinetics and correction of inner-filter effect
Enzyme kinetics experiments were performed at room temperature in a reaction buffer (20 mM
HEPES, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT and 20% Glycerol, pH 6.5). The Mpro (25 μL)
was pipetted into black 96-well plates at a final concentration of 100 nM. The reaction was initiated
by adding 25 μL of the fluorescent substrate MCA-AVLQSGFR-Lys (DNP)-Lys-NH2 (GL
Biochem) with different final concentrations (from 1.6 to 200 μM). The fluorescence signal of
17
released MCA was monitored at 405 nm with excitation at 320 nm using a CLARIOstar microplate
reader (BMG Labtech). The initial velocities were calculated from the first 1 min of the reaction
curve and then the variations of the fluorescence were converted to the amount of the cleaved
substrate via fitting to a calibration curve generated from the fluorescence measurements of free
MCA (0.01-20 μM).
To correct the inner-filter effect of the fluorescent substrate (41), the fluorescence values were
monitored for 1 min at background as well as different substrate concentrations in 50 μL of the
reaction buffer. These values are defined as f (0) and f (S), respectively. Afterwards, free MCA
(final concentration: 0.5 µM) was added to each well, and then the second measurements were
taken as f (0+MCA) and f (S+MCA). Therefore, the inner filter effect of each substrate
concentration was corrected according to the function:
f (S+MCA)-f (S)
Corr% = 100% ×
f (0+MCA)-f (0)
The corrected initial velocity of the reaction was calculated as
V= Vinitial velocity ⁄(Corr%)
The Km and Vmax values were derived by the initial velocity plotted against the FRET concentration
with the Michaelis-Menten equation in GraphPad Prism 8.0 software. kcat/Km was calculated using
the equation kcat ⁄Km = Vmax ⁄([E]×Km ) , where [E] is the total enzyme concentration. All
experiments were performed in triplicate.
IC50 measurement
The enzymatic inhibition assay was carried out with a final concentration of 100 nM recombinant
SARS-CoV-2 Mpro and 20 μM FRET substrate. For the determination of IC50 values, the enzyme
was pre-incubated with various concentrations of each compound for 10 min, and then the reaction
18
was initiated by adding the FRET substrate and monitored at 405 nm in the kinetic mode for 10
min on a CLARIOstar microplate reader (BMG Labtech). The initial velocities were calculated
from the first 1 min of each reaction curve. The IC50 values were calculated using a dose-response
model in GraphPad Prism 8.0 software. All experiments were performed in triplicate, and the
values are presented as mean ± standard deviation (SD).
Differential scanning fluorimetry assay
The differential scanning fluorimetry (DSF) assay was carried out on a CFX96 real-time PCR
Detection System (Bio-Rad). All the experiments were performed in the following buffer: 20 mM
HEPES pH 6.5 containing 120 mM NaCl. The Mpro protein (final concentration: 2 μM) was pre-
incubated with 40 μM of compounds for 30 min. 5×SYPRO Orange dye (Sigma) was added to
probe the thermal denaturation from 20°C to 95°C at a scan rate of 1.5 °C/min. The melt
temperature (Tm) was calculated by using a Boltzmann model in GraphPad Prism 8.0 software.
The thermal shift (ΔTm) was calculated using the equation ∆Tm=Tm(compound) -Tm(DMSO) . All
experiments were performed in triplicate, and the values are presented as mean ± SD.
Viruses and cells
The SARS-CoV-2 (strain 107) was provided by Guangdong Provincial Center for Disease Control
and Prevention (Guangzhou, China). This virus was propagated and titrated on African green
monkey kidney epithelial cells (Vero E6) (ATCC, no. 1586), which were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco).
A549 (human adenocarcinoma alveolar basal epithelial cell line) was purchased from ATCC (Lot#.
CCL-185). Huh7 (human hepatocarcinoma cell line), BEAS-2B (human bronchial epithelial cell
line) and LO2 (human normal hepatocyte cell line) were purchased from Cell Bank, Chinese
Academy of Sciences (SCSP-526, GNHu27 and GNHu6). HPAEpiC (human pulmonary alveolar
19
epithelial cell line) was purchased from the ScienCell Research Laboratory (San Diego, CA). All
cell lines except HPAEpiC were cultured in high glucose DMEM medium with 4.5 mM L-
glutamine (GE Life Sciences) supplemented with 10% FBS (Hyclone) and 1% penicillin–
streptavidin (Gibco). While HPAEpiC cells were cultured in the basal medium supplemented with
growth factors according to the instruction of manufacturer. HPAEpiC cells were used between
passages 5 and 10. All cell lines used in this study were cultured at 37°C in a humidified 5% CO2
atmosphere. Mycoplasma testing was performed at regular intervals and no mycoplasma
contamination was detected. All the infection experiments were performed at biosafety level-3
(BSL3) conditions at the Key Laboratory of Animal Models and Human Disease Mechanisms of
the Chinese Academy of Sciences, Kunming Institute of Zoology (Kunming, China).
Cytotoxicity assay
Vero E6, HPAEpiC, LO2, A549, BEAS-2B and Huh7 cells were respectively seeded in 96 well
plates and grown overnight. Various concentrations of compounds were then added to each well.
After incubation for 72 h, the cell viability was evaluated using CCK8 (Beyotime) according to
the manufacturer’s protocol. All experiments were performed in triplicate.
Cellular antiviral activity assay
Assays of cellular antiviral activity were performed using CCK8 and RT-qPCR methods. For the
CCK8 method, Vero E6 cells were seeded in 96-well plates and grown overnight. The cells were
then infected with SARS-CoV-2 at an MOI of 0.1. At the same time, the test compounds were
added to the wells with different concentrations. After incubation for 2 h, the medium was replaced
with fresh drug-containing medium. In 72 h, the cytopathic effect caused by SARS-CoV-2
infection was quantitatively analyzed using CCK8 (Beyotime) according to the manufacturer’s
protocol.
20
For the RT-qPCR method, HPAEpiC and Huh7 cells were respectively seeded in 48-well
plates (200 μL/well) at 8×105 cells/well and grown overnight. Cells were infected with SARS-
CoV-2 at an MOI of 0.01. Then the infected cells were treated with different concentrations of the
test compounds. After 1 h of incubation at 37°C, the virus-drug mixture was removed and replaced
with fresh medium containing compounds. In 48 h, the cell supernatants were collected to extract
viral RNA, which was subjected to RT-qPCR analysis. TaqMan primers for SARS-CoV-2 are 5'-
GGGGAACTTCTCCTGCTAGAAT-3' and 5'-CAGACATTTTGCTCTCAAGCTG-3' with
SARS-CoV-2 probe FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'. The EC50 values were
calculated by using a dose-response model in GraphPad Prism 8.0 software. All experiments were
performed for two times in triplicate, and the values are presented as mean ± SD.
Assessment of pharmacokinetic properties
All procedures related to animal handling, care and treatment in pharmacokinetic (PK) studies
were performed according to the guidelines approved by the Institute Animal Care and Use
Committee (IACUC) of Shanghai Medicilon Inc. and ZLA (Beijing) Pharmaceutical Technology
Co. Ltd. All animals used in this study were chosen randomly.
The PK properties of compounds were examined in male Sprague-Dawley (SD) rats (n = 3
per group, weight: 200-230 g). Compounds were dissolved in saline with 5% (v/v) DMSO (Sigma-
Aldrich) plus 3% (v/v) HS15 (GLPBIO). The animals were administered with a single dose of 2,
5 or 10 mg/kg (intravenous injection (i.v.)), 10 or 20 mg/kg (intraperitoneal injection (i.p.) or oral
gavage (p.o.)). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 24 h, and
centrifuged to isolate plasma. Subsequently, the plasma compound concentrations were
determined by LC-MS/MS-13 (TQ5500, SCIEX), and the PK parameters were calculated using
Phoenix WinNonlin 7.0.
21
In vivo toxicity study
The test compounds were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v) HS15
(GLPBIO) and 92% saline. Specific-pathogen free (SPF) SD rats (age: 7-11 weeks) consist of half
male (weight: 200-230 g) and half female (weight: 190-220 g). All animal experiment procedures
in this study were approved by the Institutional Committee for Animal Care and Biosafety at
Kunming Institute of Zoology, Chinese Academy of Sciences (SMKX-20200915-11). All animals
used in this study were chosen randomly.
For the acute toxicity study of MI-09 and MI-30, the SD rats were dosed at 24 mg/kg (n = 2)
and 40 mg/kg (n = 10) by i.v. via the tail vein or 250 mg/kg (n = 4) and 500 mg/kg (n = 4) via p.o.
or 250 mg/kg (n = 4) via i.p.
For the repeated dose toxicity study of MI-09 and MI-30, SPF SD rats were administered at 6
and 18 mg/kg i.v. via the tail vein once daily or 100 and 200 mg/kg p.o. twice daily or 100 and
200 mg/kg i.p. once daily for one week (n = 6). All animals were monitored twice a day for at least
7 days for toxic signs, including body weight loss, food intake reduction and abnormal behaviors.
At the end of the experiment, samples of heart, liver, spleen, lung and kidney were collected.
In vivo antiviral studies of MI-09 and MI-30
The in vivo antiviral studies were carried out in an animal biosafety level 3 (ABSL3) facility using
high-efficiency particulate air (HEPA) filtered isolators. All animal experimental procedures in
this study were approved by the Institutional Committee for Animal Care and Biosafety at
Kunming Institute of Zoology, Chinese Academy of Sciences (SMKX-20200915-11). All animals
used in this study were chosen randomly.
Mouse infection and sample collection.
22
The whole angiotensin-converting enzyme 2 (ACE2) humanized mice (hACE2 mice) weighing
18-31 g, aged 8-10 weeks, male, were purchased from Gempharmatech. Co., Ltd (#T037659). The
compounds (MI-09 and MI-30) were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v)
HS15 (GLPBIO) and 92% saline. The mice were treated with compounds 1 h prior to infection.
Then the mice were anesthetized by inhalation of isofluorane (RWD Life Science, Shenzhen,
China) and infected with 2×106 TCID50 (low dose) or 5×106 TCID50 (high dose) of SARS-CoV-2
(strain 107) by intranasal instillation. For the low challenge dose group, 50 mg/kg MI-09 was
given p.o. twice daily (n = 3) or i.p. once daily (n = 3), and 50 mg/kg MI-30 was given i.p. once
daily (n = 3). For the high challenge dose group, 100 mg/kg MI-09 was given p.o. twice daily (n
= 3) or i.p. once daily (n = 3), and 100 mg/kg MI-30 was given i.p. once daily (n = 3). The
conditions of mice including body weight were monitored daily until sacrifice. Lung tissues (n =
3, per dpi group) were collected on days 1, 3 and 5 post infection.
RNA extraction and measurement of viral loads in tissues
RNA was extracted from lung tissues using the TRIzol™ Reagent (Invitrogen) according to the
manufacturer’s instructions. Viral RNA was quantified by THUNDERBIRD® Probe One-step
qRT-PCR Kit (Toyobo) according to the manufacturer’s instructions and the TaqMan primers are
the same as those used in the previous section “Cellular antiviral activity assay”. The results were
expressed as copies per microgram tissue.
Histopathology
The tissues of hACE2 transgenic mice were collected, fixed in 4% paraformaldehyde, embedded
in paraffin and sectioned at 3 μm. Sections were stained with hematoxylin and eosin (H & E) and
analyzed by light microscopy. The lung injury was evaluated based on histological features
including alveolar septal thickening, hemorrhage, inflammatory cell infiltration and consolidation.
23
Assays for inflammatory cytokines and chemokines
To assess the expression of inflammatory cytokines and chemokines in the lung, we performed
real-time PCR assay. RNA extracted from the lung tissues was reversely transcribed into cDNA
using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara), and then gene expression was
quantified through TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara) and the ViiA™ 7
Real-Time PCR System. Primer sequences for quantification of inflammatory gene expression are
shown in table S7.
Immune cell fluorescence staining
Mouse lungs were fixed in 4% PFA for at least 7 days, and then were paraffin embedded and cut
into 4 μm sections following the standard procedure. After deparaffinization in xylene, antigen
retrieval and blocking, lung sections were incubated with rat monoclonal antibody F4/80 (Huabio,
1:100) or rabbit polyclonal antibody Ly6G (Servicebio, 1:300) overnight at 4°C and then with
horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody or HRP-conjugated
goat anti-rabbit secondary antibody at room temperature for 1 h to catalyze Cy3-tyramine and Cy5-
tyramine and to amplify the staining signal according to tyramide signal amplification (TSA).
After nucleus staining by DAPI, all sections were photographed using a LEICA DMI 4000B
microscope (Germany) and analyzed by ImageJ software (NIH, USA) and FlowJo software (BD,
USA). To measure the macrophage and neutrophil infiltration semi-quantitatively, 5 arbitrarily
chosen fields of the lung parenchyma in each lung section were examined by light microscopy for
the presence of neutrophils or macrophages. This assessment was performed in a blinded fashion.
The cumulative scores for each animal are expressed as the number of positive fields per 100 fields
(%).
24
Supplementary Text
Synthesis of Mpro Inhibitors
Scheme 1. Synthesis of compounds 11a-n.a
a
Reaction conditions: (i) BrCH2CN, LiHMDS, dry THF, -78°C, 5 h; (ii) PtO2, H2, MeOH, 0°C to
25°C, 24 h; (iii) NaOAc, MeOH, 65°C, 12 h; (iv) TFA, CH2Cl2, 25°C, 14 h; (v) HATU, DIEA,
dry DMF, 25°C, Ar, 12 h; (vi) 2 M NaOH, MeOH, 25°C, 2.5 h; (vii) HATU, DIEA, dry DMF,
0°C, Ar, 12 h; (viii) NaBH4, MeOH, 0°C to 25°C, 2 h; (ix) Dess-martin reagent, dry CH2Cl2, 25°C,
3.5 h.
25
Scheme 2. Synthesis of compounds 18a-r.a
O O O
O O O O OH
O i iii
R2
ii
HCl HN H N H R2 N H
R2 OH O
NH
H H H
12 13a - r 14a- r 15a - r

O
CF3COOH H 2N
O
4
O O O
NH NH NH
O O O
R2 R2 O v R2
O iv O
N O N OH N O
N N N
H H H
H O H H H
H H H
16a - r 17a - r 18a - r
R 2: O
O O O
O
N
N O N
N
H
O
13a 13b 13c 13d 13e
O O O
O O
F
F F
N N
F
13f 13g 13h F 13i 13j
O O O
O
O O
O O O
O
O O
O Cl Cl F
O
13k 13l 13m 13n 13o

O
O O
O
Cl O O
Cl Cl
Cl Cl
13p 13q 13r
a
Reaction conditions: (i) HATU, DIEA, dry DMF, 25°C, Ar, 12 h; (ii) 2 M NaOH, MeOH, 25°C,
2 h; (iii) HATU, DIEA, dry DMF, 0°C, Ar, 12 h; (iv) NaBH4, MeOH, 0°C to 25°C, 2 h; (v) Dess-
martin reagent, dry CH2Cl2, 25°C, 3.5 h.
26
General procedure:
Ethanol, methanol, petroleum ether, ethyl acetate, dichloromethane, 1,4-dioxane and DMF were
obtained from standard suppliers and used without further purification. Anhydrous solvents were
purchased from J&K Scientific. All chemicals were purchased from Bidepharm Technology Co.
Ltd, Energy Chemical or Alfa Aesar and used without further purification. All reactions were
monitored by thin layer chromatography (TLC) and visualization was achieved by using ultraviolet
light (254 nm) or displayed by iodine reagent and ninhydrin reagent. Column chromatography was
carried out using Biotage Isolera flash purification system under proper pressure. 1H NMR and 13C
NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz and 100 MHz,
respectively. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are
reported in parts per million (ppm) units. Low resolution ESI-MS readings were recorded on an
Agilent 1200-G6410A mass spectrometer. High-resolution mass spectra were recorded on Q-TOF
Premier mass spectrometer (Micromass, Manchester, UK). The purity of compounds was
determined to be over 95% by reversed-phase HPLC analysis.
Dimethyl (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)pentanedioate (2)
To a solution of N-Boc-L-glutamic acid dimethyl ester 1 (10.0 g, 36.32 mmol) in dry THF (100
mL) was added dropwise a solution of lithium bis(trimethylsilyl)amide (LHMDS) in THF (60 mL,
1 M) at -78°C under Argon, then the mixture was stirred at -78°C. Meanwhile, bromoacetonitrile
(2.66 mL, 38.2 mmol) was added dropwise to the mixture under -78°C. The reaction mixture kept
at -78°C was stirred for 5 h. After the reactant was consumed, the reaction was quenched by
saturated NH4Cl solution (40 mL). The reaction mixture was allowed to warm up to room
temperature and then poured into brine (40 mL). The organic layer was concentrated and purified
by Biotage flash column chromatography (5-25%, ethyl acetate/petroleum ether) to give product
27
2 (7.7 g, 67%) as a pale-yellow oil. 1H NMR (400MHz, CDCl3) δ 5.18 (d, J = 8.8 Hz, 1H), 4.42-
4.33 (m, 1H), 3.77(s, 3H), 3.75 (s, 3H), 2.97-2.91 (m, 1H), 2.87-2.76 (m, 2H), 2.20-2.14 (m, 2H),
1.47 (9H, s). ESI-MS (m/z): 315.09 (M + H)+.
Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (3)
Compound 2 (3.0 g, 9.5 mmol) was dissolved in MeOH (100 mL) in a round-bottomed flask before
the addition of PtO2 (1.40 g, 6.17 mmol) at 0°C, then the resulting mixture was stirred under
hydrogen at 25°C for 24 h. The mixture was filtered over bergmeal to remove the catalyst. NaOAc
(1.54 g, 18.8 mmol) was added to the filtrate before the mixture was stirred at 65°C for another 12
h. Next, the reaction was quenched with water and extracted with ethyl acetate. The organic layer
was washed by brine and dried over anhydrous Na2SO4. The organic layer was concentrated in
vacuo and purified via Biotage flash column chromatography (50-75%, ethyl acetate/petroleum
ether) to give the product 3 (1.5g, 55%) as a yellow solid. 1H NMR (400MHz, CDCl3) δ 6.12 (s,
1H), 5.49 (s, 1H), 4.35-4.27 (m, 1H), 3.70 (s, 3H), 3.37-3.29 (m, 2H), 2.47-2.40 (m, 2H), 2.15-
2.03 (m, 1H), 1.88-1.79 (m, 2H), 1.42 (s, 9H). ESI-MS (m/z): 287.03 (M + H)+.
Methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate (4)
To a solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-
yl)propanoate 3 (2.5 g) in DCM (30 mL) was added TFA (20 mL). The reaction mixture was stirred
at room temperature for 14 h, and then cautiously concentrated. The crude material was used in
the next step without further purification.
General procedure A (Compounds MI-01 – MI-14 were prepared with various acid derivatives
using General Procedure A):
Methyl (1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-
azabicyclo[3.1.0]hexane-2-carboxylate (7i)
28
To a stirred suspension of the 2-(4-(trifluoromethoxy)phenoxy)acetic acid 6i (0.24 g, 1.0 mmol),
HATU (0.49 g, 1.2 mmol) and DIEA (494 μL, 3 mmol) in dry DMF, (1R,2S,5S)-6,6-dimethyl-3-
aza-bicylo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride (0.21 g, 1.0 mmol) was
added. The reaction mixture was stirred under 25°C for 12 h under argon atmosphere. The solvent
was added into water (v/v = 1/4) and the mixture was extracted by DCM (50 mL×3). The organic
phases were combined and washed with saturated aqueous sodium thiosulfate solution (20 mL×3).
The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give
the yellow oil that was purified by Biotage flash column chromatography (15-20%, ethyl
acetate/petroleum ether) to afford 7i (0.34 g, 88%) as a white solid. 1H NMR (400 MHz, MeOD)
δ 7.19 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 4.80-4.71 (m, 2H), 4.78-4.72 (m, 1H) 3.89-
3.72 (m, 1H), 3.73 (s, 3H), 3.67-3.60 (m, 1H), 1.61-1.55(m, 1H), 1.49 (d, J = 7.4 Hz, 1H), 1.08 (s,
3H), 0.97 (s, 3H). ESI-MS (m/z): 388.18 (M + H)+.
(1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo
[3.1.0]hexane-2-carboxylic acid (8i)
A solution of methyl (1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy) acetyl)-3-
azabicyclo[3.1.0]hexane-2-carboxylate 7i (200 mg) in MeOH (30 mL) was treated with 2 M NaOH
aqueous solution (20 mL). The reaction mixture was stirred for 2.5 h at 25°C. After completion of
the reaction, MeOH was concentrated and then the solution was acidified to pH∼3 using
hydrochloric acid. The aqueous layer was extracted with DCM (30 mL×3) and the combined
organic layers were concentrated. The crude intermediate 8i was used in the next step without
further purification.
Methyl (S)-2-((1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-
azabicyclo[3.1.0]hexane-2-carboxamido)-3-((S)-2-oxopyrrolidin-3-yl) propanoate (9i)
29
To a solution of (1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-
azabicyclo[3.1.0]hexane-2-carboxylic acid 8i (0.45 g, 1.2 mmol) in dry DMF was added HATU
(0.61 g, 1.6 mmol) sequentially at 0°C. The solution was stirred for 30 mins, and then DIEA (0.59
mL, 3.6 mmol) was added. Next, the crude product 4 (0.27 g 1.45 mmol) was added, and the
reaction mixture kept at 0°C was stirred for 12 h under argon atmosphere. The reaction mixture
was quenched with water (v/v = 1/4) and extracted with ethyl acetate for 3 times. The organic
phases were combined and extracted with saturated NH4Cl solution (100 mL×3), saturated
NaHCO3 solution (100 mL×2) and brine (100 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated, and the residue was purified by reversed-phase column chromatography
(10-25%, acetonitrile/H2O) to afford 9i (0.48 g, 73%) as a white solid. 1H NMR (400 MHz, MeOD)
δ 7.17 (d, J = 8.0 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 4.80-4.67 (m, 2H), 4.55-4.31 (m, 1H), 3.94-
3.83 (m, 1H), 3.72 (s, 3H), 3.68-3.57 (m, 1H), 3.23-3.12 (m, 1H), 3.11-3.00 (m, 2H), 2.58-2.41 (m,
1H), 2.26-2.04 (m, 2H), 1.73-1.40 (m, 4H), 1.10 (s, 3H), 0.98 (s, 3H). ESI-MS (m/z): 542.13 (M
+ H)+.
(1R,2S,5S)-N-((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-(2-(4-
(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (10i)
To a solution of methyl (S)-2-((1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)
phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-((S)-2-oxopyrrolidin-3-yl)
propanoate 9i (0.56 mg, 1.1 mmol) in methanol (50 mL) was added NaBH4 (0.14 g, 8.8 mmol)
under 0°C. The reaction mixture was stirred at 25°C for 2 h. Then the reaction was quenched with
water (20 mL). The suspension was extracted with ethyl acetate (50 mL×3). The organic layers
were combined, dried over anhydrous Na2SO4 and filtered. The filtrate was evaporated and could
be used for the next step without further purification.
30
(1R,2S,5S)-6,6-dimethyl-N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-3-(2-(4-
(trifluoromethoxy) phenoxy) acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (11i, MI-09)
(1R,2S,5S)-N-((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-6,6-dimethyl-3-(2-(4-
(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide 10i (0.38 g, 0.75
mmol) was dissolved in dry DCM, and then Dess-martin reagent (0.95 mg, 0.79 mmol) was added
slowly. The resulting mixture became turbid and was stirred at 25°C for 3.5 h. The mixture was
filtered, concentrated and purified by reversed-phase preparative chromatography system (20-35%,
acetonitrile/H2O) to give the product 11i (MI-09, 0.28g, 45%) as a white solid. 1H NMR (400 MHz,
MeOD) δ 7.24-7.13 (m, 2H), 6.99 (d, J = 9.0 Hz, 2H), 4.86-4.64 (m, 2H), 4.57-4.31 (m, 2H), 4.04-
3.91 (m, 1H), 3.71-3.56 (m, 1H), 3.20-2.90 (m, 2H), 2.63-2.47 (m, 1H), 2.26-2.05 (m, 1H), 2.02-
1.91 (m, 1H), 1.71-1.43 (m, 4H), 1.11 (s, 3H), 1.00 (s, 3H). 13C NMR (101 MHz, MeOD) δ 181.60,
172.58, 167.18, 156.90, 142.99, 122.22, 121.99, 121.87, 119.34, 115.51, 115.43, 66.06, 61.05,
60.14, 51.26, 46.00, 39.94, 37.75, 30.87, 29.88, 27.75, 25.03, 19.04, 11.63. HRMS (m/z):
calculated for C24H28F3N3O6+ [M+H] + 512.1964; found, 512.2137.
General procedure B (MI-15 – MI-32 were prepared with various acid derivatives using General
procedure B):
Ethyl(1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenta[c]pyrrole-1-
carboxylate (14p)
A mixture of 2-(2,4-dichlorophenoxy)acetic acid 13p (0.58 g, 2.62 mmol), HATU (1.2 g, 3.14
mmol), DIEA (1.3 mL, 7.86 mmol), and (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1-
carboxylicacid ethyl ester hydrochloride (0.58 g, 2.62 mmol) in dry DMF (15 mL) was stirred at
25°C for 12 h under argon atmosphere, diluted with water and extracted with DCM (30 mL×3).
The organic layers were washed with brine, dried and filtered. The filter liquor was purified by
31
Biotage flash column chromatography (18-23%, ethyl acetate/petroleum ether) to furnish 14p
(0.60 g, 59%) as a yellow solid. 1H NMR (400 MHz, MeOD) δ 7.42 (d, J = 5.4 Hz, 1H), 7.26-7.19
(m, 1H), 6.97 (d, J = 8.9 Hz, 1H), 4.80-7.72 (m, 2H), 4.28 (d, J = 3.6 Hz, 1H), 4.22-4.10 (m, 2H),
3.87 (d, J = 10.6 Hz, 1H), 3.63-3.48 (m, 1H), 3.57 (d, J = 10.5 Hz, 2H), 2.71-2.61 (m, 1H), 2.08-
1.84 (m, 1H), 1.83-1.46 (m, 4H), 1.31-1.17 (m, 3H). ESI-MS (m/z): 386.02 (M + H)+.
(1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenta[c]pyrrole-1-carboxylic
acid (15p)
A mixture of ethyl (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclope-
nta[c]pyrrole-1-carboxylate 14p (200 mg, 0.52 mmol) and 2 M NaOH aqueous solution (10 mL)
in MeOH (20 mL) was stirred at 25°C for 2 h. The reaction mixture was made acidic with hydrogen
chloride aqueous solution and extracted with DCM (50 mL×3). The organic layers were washed
with brine, dried and filtered. The crude product 15p was used in the next step without further
purification.
Methyl (S)-2-((1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenta[c]pyrrole-
1-carboxamido)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (16p)
HATU (0.099 g, 0.26 mmol) was added to the solution of (1S,3aR,6aS)-2-(2-(2,4-
dichlorophenoxy)acetyl)octahydrocyclopenta[c]pyrrole-1-carboxylic acid 15p (0.071 g, 0.20
mmol) in DMF (10 mL). The solution was stirred for 30 mins, and then DIEA (100 μL, 0.60 mmol)
and the crude product methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate trifluoroacetate
4 (0.060 g, 0.32 mmol) were added. The reaction mixture was stirred at 0°C for 12 h under argon
atmosphere, and then quenched with water (30 mL) and extracted with ethyl acetate (30 mL) for
3 times. The organic phases were washed with saturated NH4Cl solution (100 mL×3), saturated
NaHCO3 solution (100 mL×2) and brine (100 mL), and the organic layer was washed with brine,
32
dried over anhydrous Na2SO4 and filtered. The solvent was evaporated under reduced pressure,
and the resultant residue was purified by reversed-phase column chromatography (10-15%,
acetonitrile/H2O) to afford the target compound 16p (0.063 g, 59%) as a white solid. 1H NMR
(400 MHz, MeOD) δ 7.40 (d, J = 2.5 Hz, 1H), 7.26-7.19 (m, 1H), 6.95 (d, J = 8.9 Hz, 1H), 4.79-
4.71 (m, 2H), 4.54-4.25 (m, 1H), 4.27-4.20 (m, 1H), 3.72 (s, 3H), 3.54-3.46 (m, 2H), 3.21-3.12 (m,
1H), 3.10-2.99 (m, 1H), 2.86-2.70 (m, 2H), 2.04-1.97 (m, 2H), 1.96-1.46 (m, 9H). ESI-MS (m/z):
526.03 (M + H)+.
(1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-
yl)propan-2-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide (17p)
To a solution of methyl (S)-2-((1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclop-
enta[c]pyrrole-1-carboxamido)-3-((S)-2-oxopyrrolidin-3-yl)propanoate 16p (1,00 g, 2.0 mmol) in
MeOH was added the NaBH4 (0.6 g, 16 mmol) under 0°C, then the reaction mixture was stirred at
25°C for 2 h. After TLC analysis completion, the reaction was quenched by water (20 mL). The
reaction mixture was extracted with ethyl acetate (50 mL×3) and the organic layers were washed
with H2O (50 mL×2) and brine (50 mL). The organic phase was dried over anhydrous Na2SO4 and
concentrated, and the residue was used without further purification.
(1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-
yl)propan-2-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide (18p, MI-30)
To a solution of the (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-hydroxy-3-((S)-2-
oxopyrrolidin-3-yl)propan-2-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide 17p (0.50 g, 1.0
mmol) in dry DCM, the Dess-martin reagent (0.55 g, 1.3 mmol) was added slowly and the reaction
mixture was stirred at 25°C for 3.5 h. The reaction was filtered and purified by reversed-phase
preparative chromatography system (30-35%, acetonitrile/H2O) to afford the target compound 18p
33
(0.21g, 42%) as a white solid. 1H NMR (400 MHz, MeOD) δ 7.48-7.38 (m, 1H), 7.27-7.20 (m,
1H), 7.02-6.94 (m, 1H), 4.97-4.90 (m, 1H), 4.85-4.80 (m, 1H), 4.55-4.44 (m, 1H), 4.32-4.22 (m,
1H), 4.03-3.89 (m, 2H), 3.55-3.47 (m, 1H), 3.21-3.11 (m, 1H), 3.04-2.94 (m, 1H), 2.93-2.85 (m,
1H), 2.73-2.63 (m, 1H), 2.61-2.50 (m, 1H), 2.18 (m, 1H), 1.96-1.51 (m, 8H). 13C NMR (101 MHz,
MeOD) δ 181.64, 173.39, 167.10, 152.81, 129.54, 127.47, 125.73, 123.12, 114.81, 66.81, 66.04,
60.14, 54.08, 52.20, 51.30, 43.34, 40.00, 37.73, 31.69, 31.14, 29.74, 27.67, 24.69. HRMS (m/z):
calculated for C23H27Cl2N3O5+ [M+H] + 496.1361; found, 496.0842.
34
Diastereomeric purity
O
NH
1
O S
F 3C O 2
HN H
O S
N 3 O
S O
5 4
H R
H
S
Stereocenters of compound MI-09
Here MI-09 is taken as an example to show the chiral control and diastereomeric purity of our
compounds. Intermediate 2 provided by the dianionic alkylation of N-Boc-L-glutamic acid
dimethyl ester (1) with bromoacetonitrile have the single S configuration (1S, 2S) at both chiral
centers (Scheme 1), as this reaction occurred in a highly stereoselective manner (9). Then,
subsequent hydrogenation and cyclization of the resulting intermediate 2 afforded the lactam 3,
which kept both stereocenters (1S, 2S) according to the literature (9). Intermediate 7i was prepared
through a condensation reaction (step v) between commercially available stereospecific starting
material 5 containing three stereocenters (3S, 4R, 5S) and 4-(trifluoromethoxy)phenoxyacetic acid.
In this condensation reaction (step v), we observed a slight racemization. But intermediate 7i is
the main product, and can be separated by Biotage flash column chromatography (15-20%, ethyl
acetate/petroleum ether). The following deprotection (step iv, yielding 4) and hydrolysis (step vi,
generating 8i) reactions are unlikely to lead to racemization under mild conditions. In step vii
(Scheme 1), which is the condensation reaction of intermediate 4 and 8i, we observed partial
racemization of 9i. However, our target, 9i (1S, 2S, 3S, 4R, 5S), is the main product, and can also
be separated by reversed-phase column chromatography (TLC rf, target product: 0.35, another
isomer: 0.38, DCM/MeOH = 25/1, ratio 5 to 1). The remaining two step reactions, viii and ix, were
35
monitored by TLC and are again unlikely to lead to racemization. Finally, compound MI-09 was
separated by reversed-phase preparative chromatography system (20-35%, acetonitrile/H2O).
MI-09 was determined by the Waters e2695 HPLC system with the use of a phenomenex-
C18 reversed-column (4.6 mm × 150 mm, 5 μm). MI-09 was analyzed using MeOH/H2O = 70:30
(v/v) (10 min, 1 mL/min) and integrated the peak areas at 252 nm. Diastereomeric purity (HPLC):
98.18%.
Other compounds derived from either Boceprevir (MI-01 – MI-08 and MI-10 – MI-14) or
Telaprevir (MI-15 – MI-32) were prepared and analyzed by very similar methods as those for MI-
09 (a main difference is that commercially available stereospecific starting material 12 (Scheme 2)
other than 5 (Scheme 1) was used in the syntheses of compounds derived from Telaprevir). As an
example, the structure of MI-23 was confirmed by the crystal structure of its complex with SARS-
CoV-2 Mpro (See Fig. 2). The purity of these compounds was determined to be over 95% by
reversed-phase HPLC analysis.
36
Characterization of Mpro Inhibitors
Synthetic
No. Structure NMR HRMS
procedure
1H NMR (400 MHz, DMSO) δ 10.86 (s,
O
NH
1H),8.75 (d, J = 7.8 Hz, 1H), 8.11 (s, 1H),
8.06 (t, J = 9.4 Hz, 2H), 7.97 (t, J = 7.3 Hz,
O H
2H), 7.34 (d, J = 8.7 Hz, 1H), 4.37-4.25 (m,
MI-01
HN
1H), 4.05-3.98 (m, 1H), 3.72-3.61 (m, 2H),
N O 448.2245 A
(11a) O
3.59-3.50 (m, 2H), 2.59-2.47 (m, 2H),
H
H 2.17-2.11 (m, 1H), 2.01-1.93 (m, 1H),
1.91-1.84 (m, 1H), 1.87-1.77 (m, 1H),
Exact Mass: 447.2158
1.54-1.49 (m, 1H),1.10 (s, 3H), 0.87 (s,
3H).
1H NMR (400 MHz, MeOD) δ 7.78 (td, J =
8.9, 8.5, 3.6 Hz, 3H), 7.47-7.40 (m, 1H),

7.34 (d, J = 7.5 Hz, 1H), 7.21 (dd, J = 8.9,
2.6 Hz, 1H), 7.14 (d, J = 2.7 Hz, 1H), 4.87-
O
NH 4.83 (m, 2H), 4.54-4.46 (m, 1H), 4.43-4.35
(m, 1H), 4.10-3.94 (m, 2H), 3.73-3.58 (m,
O
HN
H 1H), 3.42-3.34 (m, 1H), 2.68-2.41 (m, 2H),
MI-02
(11b)
O
N
O
O 2.02-1.90 (m, 1H), 1.75-1.36 (m, 2H), 478.2342 A
H
H 1.28-1.16 (m, 2H), 1.11 (s, 3H), 0.98 (s,
3H). 13C NMR (101 MHz, MeOD) δ 181.58,
172.64, 167.41, 155.92, 134.48, 129.36,
128.95, 127.12, 126.74, 125.94, 118.28,
106.97, 65.67, 61.07, 60.19, 51.27, 46.09,
39.64, 37.67, 30.92, 30.15, 29.91, 29.71,
27.84, 25.07, 19.15, 19.05.
1H NMR (400 MHz, MeOD) δ 7.15 (dd, J
= 9.5, 4.3 Hz, 1H), 6.86 (ddd, J = 33.8,

16.1, 7.0 Hz, 3H), 4.46–4.36 (m, 1H),
O
NH
4.20–4.11 (m, 1H), 3.93–3.73 (m, 1H),
F 3.64–3.44 (m, 1H), 3.19–3.11 (m, 1H),
O
HN
H 2.86–2.74 (m, 2H), 2.60–2.39 (m, 3H),
MI-03
(11c)
N
O
O
2.27–2.15 (m, 1H), 2.09–1.83 (m, 2H), 444.2303 A
H
H
1.65–1.30 (m, 4H), 0.95 (s, 3H), 0.75 (s,
3H).13C NMR (101 MHz, MeOD) δ 181.64,
173.02, 171.53, 164.15, 161.73, 143.78,
129.81, 123.96, 114.88, 112.55, 65.51,
60.70, 40.14, 37.86, 35.31, 31.33, 29.97,
27.40, 25.62, 25.00, 19.12, 18.88.
1H NMR (400 MHz, MeOD) δ 6.57-6.50
(m, 3H), 4.81-4.66 (m, 2H), 4.48-4.44 (m,

1H), 4.39-4.35 (m, 1H), 4.02-3.90 (m, 2H),
O
NH
3.66-3.57 (m, 1H), 3.18-2.98 (m, 2H),
F
O H
2.55-2.51 (m, 1H), 2.25-2.19 (m, 1H),
MI-04 HN
2.03-1.93 (m, 1H), 1.68-1.57 (m, 2H),
O N O 464.1999 A
(11d) F
H
O
1.52-1.47 (m, 1H), 1.09 (s, 3H), 0.99 (s,
H
3H).13C NMR (101 MHz, MeOD) δ 181.61,
Exact Mass: 463.1919 172.58, 166.66, 164.90, 162.46, 160.55,
98.59, 96.30, 95.78, 66.12, 61.05, 51.39,
48.46, 47.83, 45.91, 39.98, 37.79, 30.93,
30.00, 27.74, 25.03, 19.04, 11.63.
37
1H NMR (400 MHz, MeOD) δ 7.21–7.10
(m, 1H), 6.90 (ddd, J = 12.3, 6.7, 3.1 Hz,
1H), 6.73 (dp, J = 8.7, 3.0, 2.4 Hz, 1H),
4.81–4.63 (m, 2H), 4.56–4.34 (m, 2H),
O
NH
4.06–3.90 (m, 2H), 3.71–3.58 (m, 1H),
F
F O H
3.22–3.12 (m, 1H), 3.09–2.97 (m, 1H),
MI-05 HN
2.61–2.49 (m, 1H), 2.28–2.16 (m, 1H),
O N O 464.1996 A
(11e)
H
O
2.09–1.93 (m, 1H), 1.68–1.50 (m, 3H),
H
1.11 (s, 3H), 1.01 (s, 3H). 13C NMR (101
Exact Mass: 463.1919 MHz, MeOD) δ 181.58, 172.58, 167.01,
166.99, 116.93, 116.75, 110.32, 104.25,
104.04, 66.37, 61.04, 53.70, 51.27, 45.95,
39.96, 37.78, 30.85, 30.01, 27.74, 25.05,
19.03, 11.64.
1H NMR (400 MHz, MeOD) δ 6.82 (d, J =
8.8 Hz, 1H), 6.60 (s, 1H), 6.41 (dd, J = 8.8,

2.8 Hz, 1H), 4.66–4.63 (m, 1H), 4.63–4.52
(m, 1H), 4.52–4.44 (m, 1H), 4.01– 3.94 (m,
O
NH 1H), 3.81 (s, 3H), 3.75 (s, 3H), 3.66–3.43
O (m, 2H), 3.40–3.36 (m, 1H), 3.22–3.09 (m,
O O
HN
H 1H), 3.06–2.93 (m, 1H), 2.60–2.46 (m,
MI-06
(11f)
O N
O
O 1H), 2.23–2.15 (m, 1H), 2.04–1.96 (m, 488.2393 A
H
H 1H), 1.65–1.56 (m, 2H), 1.51-1.48 (m, 1H),
1.08 (s, 3H), 0.97 (s, 3H).13C NMR (101
MHz, MeOD) δ 181.64, 172.61, 167.77,
167.75, 152.99, 150.01, 143.88, 112.68,
104.57, 100.80, 66.39, 61.03, 55.85,
55.06, 53.42, 51.36, 46.01, 39.99, 37.77,
30.81, 29.78, 27.75, 25.05, 19.01, 11.62.
1H NMR (400 MHz, MeOD) δ 7.47 (t, J =
8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.23–

7.14 (m, 2H), 4.86–4.76 (m, 2H), 4.54–
O
4.47 (m, 1H), 4.11–3.88 (m, 2H), 3.72–
NH
F
F F 3.59 (m, 1H), 3.40–3.34 (m, 1H), 3.21–
O H
3.07 (m, 1H), 3.05–2.94 (m, 1H), 2.69–
MI-07
HN
2.48 (m, 1H), 2.29–2.15 (m, 1H), 2.07–
O N O 496.2054 A
(11g) O
1.94 (m, 1H), 1.73–1.59 (m, 2H), 1.55–
H
H 1.50 (m, 1H), 1.11 (s, 3H), 1.02 (s, 3H). 13C
NMR (101 MHz, MeOD) δ 181.61, 172.57
166.98, 158.46, 131.52, 131.20, 130.00,
125.37, 117.90, 117.45, 111.57, 65.85,
61.05, 45.93, 39.97, 37.76, 30.92, 29.76,
27.74, 27.53, 25.03, 24.84, 19.13, 19.02.
1H NMR (400 MHz, MeOD) δ 6.98 (t, J =
9.3 Hz, 1H), 6.76 (dd, J = 12.9, 2.9 Hz,

1H), 6.66 (dt, J = 8.9, 2.1 Hz, 1H), 4.74–
O
NH
4.55 (m, 2H), 4.51–4.44 (m, 1H), 4.40–
F 4.31 (m, 1H), 4.12–3.88 (m, 2H), 3.80 (s,
O O
HN
H 3H), 3.67–3.55 (m, 1H), 3.22–3.08 (m,
MI-08
(11h)
O N
O
O 1H), 3.05–2.95 (m, 1H), 2.63–2.46 (m, 476.2194 A
H
H 1H), 2.27–2.15 (m, 1H), 2.06–1.92 (m,
1H), 1.67–1.42 (m, 3H), 1.09 (s, 3H), 0.98
(s, 3H).13C NMR (101 MHz, MeOD) δ
181.61, 172.62, 167.43, 153.75, 152.38,
151.31, 114.42, 109.44, 103.91, 103.69,
98.49, 98.18, 66.47, 61.03, 56.03, 51.39,
38
45.98, 39.99, 37.78, 30.90, 29.81, 27.74,
25.03, 19.01, 11.61.
1H NMR (400 MHz, MeOD) δ 7.24-7.13
(m, 2H), 6.99 (dd, J = 9.0, 4.1 Hz, 2H),

4.86-4.64 (m, 2H), 4.57-4.31 (m, 2H),
O
NH
4.04-3.91 (m, 1H), 3.71-3.56 (m, 1H),
3.20-2.90 (m, 2H), 2.63-2.47 (m, 1H),
O O H 2.26-2.05 (m, 1H), 2.02-1.91 (m, 1H),
MI-09 F3C HN
(11i)
O
N
O
O 1.71-1.43 (m, 4H), 1.11 (s, 3H), 1.00 (s, 512.2137 A
H
H 3H). 13C NMR (101 MHz, MeOD) δ 181.60,
172.58, 167.18, 156.90, 142.99, 122.22,
121.99, 121.87, 119.34, 115.51, 115.43,
66.06, 61.05, 60.14, 51.26, 46.00, 39.94,
37.75, 30.87, 29.88, 27.75, 25.03, 19.04,
11.63.
1H NMR (400 MHz, MeOD) δ 7.28 (dd, J =
9.0, 6.4 Hz, 1H), 7.19-7.09 (m, 1H), 6.81

(dt, J = 9.1, 3.0 Hz, 1H), 4.72-4.53 (m, 2H),
O
NH
4.41-4.17 (m,1H), 3.94-3.63 (m, 2H), 3.58-
Br 3.45 (m, 1H), 3.30-3.26 (m, 1H), 3.22-3.18
Cl O
HN
H (m, 1H), 3.08-2.79 (m, 2H), 2.47-2.01 (m, [M + Na]+
MI-10
(11j)
O
N
O
O 1H), 1.59-1.32 (m, 4H), 0.99 (s, 3H), 0.89 562.0720 A
H
H (s, 3H).13C NMR (101 MHz, MeOD) δ
181.61, 172.54, 166.80, 166.79, 157.36,
130.22, 125.93, 121.79, 119.74, 115.39,
65.93, 61.08, 51.38, 51.21, 45.94, 39.99,
37.77, 30.92, 29.96, 27.77, 25.04, 19.04,
11.68.
1H NMR (400 MHz, MeOD) δ 7.52 (s, 1H),
7.35 (dd, J = 8.8, 2.3 Hz, 1H), 6.97–6.78

(m, 1H), 4.93–4.36 (m, 4H), 4.21–3.87 (m,
O
NH
2H), 3.74–3.62 (m, 1H), 3.33–3.25 (m,
Br O H
2H), 2.64–2.47 (m, 1H), 2.38–2.22 (m,
MI-11 HN
1H), 2.19–2.05 (m, 1H), 1.93–1.78 (m, [M + Na]+
O
N O
562.0717 A
(11k) Cl
H
O
1H), 1.70–1.53 (m, 2H), 1.07 (s, 3H), 0.96
H (s, 3H). 13C NMR (101 MHz, MeOD) δ
Exact Mass: 539.0823 181.60, 172.54, 166.45, 153.19, 132.10,
130.35, 123.42, 115.16, 112.52, 66.58,
61.11, 53.71, 51.38, , 46.99, 40.02, 37.73,
30.89, 29.90, 27.79, 25.06, 19.05, 11.70.
1H NMR (400 MHz, MeOD) δ 7.13 (d, J =
9.0 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H), 4.66–

O
NH
4.23 (m, 4H), 3.95–3.46 (m, 3H), 3.13–
2.98 (m, 1H), 2.97–2.76 (m, 1H), 2.51–
Cl O
HN
H 2.32 (m, 1H), 2.11–2.00 (m, 1H), 1.95–
MI-12
(11l)
O
N
O
O 1.82 (m, 1H), 1.62–1.40 (m, 3H), 0.99 (s, 462.1798 A
H
H 3H), 0.88 (s, 3H). 13C NMR (101 MHz,
MeOD) δ 181.60, 172.59, 167.22, 156.90,
128.86, 115.93, 61.04, 48.26, 48.05,
47.84, 47.63, 47.41, 47.20, 46.99, 46.00,
39.97, 37.75, 27.61, 25.05, 19.03, 11.64.
39
1H NMR (400 MHz, MeOD) δ 7.37 (d, J =
8.9 Hz, 1H), 7.09 (s, 1H), 6.86 (dd, J = 8.9,
3.0 Hz, 1H), 4.79–4.37 (m, 4H), 4.04–3.89
O
NH
(m, 2H), 3.61–3.55 (m, 1H), 3.17–3.07 (m,
Cl
O H
1H), 2.98–2.88 (m, 1H), 2.57–2.45 (m,
Cl
MI-13
HN
1H), 2.23–2.13 (m, 1H), 2.01–1.92 (m,
O
N O 496.1408 A
(11m)
H
O
1H), 1.69–1.52 (m, 3H), 1.08 (s, 3H), 0.99
H
(s, 3H). 13C NMR (101 MHz, MeOD) δ
Exact Mass: 495.1328 181.58, 157.50, 130.44, 116.56, 114.87,
65.94, 60.16, 48.30, 48.09, 47.88, 47.67,
47.45, 47.24, 47.03, 45.95, 39.97, 37.76,
29.97, 27.78, 25.09, 20.01, 11.71.
1H NMR (400 MHz, MeOD) δ 7.39 (s, 1H),
7.19 (dd, J = 8.9, 2.6 Hz, 1H), 6.91 (d, J =

8.9 Hz, 1H), 4.95–4.84 (m, 1H), 4.80–4.70
O
NH (m, 1H), 4.55–4.45 (m, 1H), 4.41–4.30 (m,
Cl
1H), 4.06–3.61 (m, 3H), 3.29–2.90 (m,
Cl O
HN
H 2H), 2.64–2.39 (m, 1H), 2.21–2.09 (m,
MI-14
(11n)
O
N
O
O 1H), 2.05–1.92 (m, 1H), 1.70–1.50 (m, 496.1406 A
H
H 3H), 1.08 (s, 3H), 0.98 (s, 3H). 13C NMR
(101 MHz, MeOD) δ 181.58, 152.73,
129.34, 127.37, 114.70, 66.72, 61.11,
53.74, 48.29, 48.08, 47.87, 47.66, 47.44,
47.23, 47.02, 46.08, 40.01, 37.75, 29.91,
27.79, 25.09, 19.05, 11.73.
O
NH
1H NMR (400 MHz, MeOD) δ 8.78-8.54
O
HN
H (m, 2H), 8.18-7.86 (m, 1H), 7.54-7.42 (m,
MI-15 N
(18a) N
O
O 1H), 4.57-4.27 (m, 2H), 4.00-3.60 (m, 2H), 399.2030 B
H 3.37-3.34 (m, 1H), 3.28-3.26 (m, 1H),
H
2.82-2.15 (m, 4H), 2.08-1.39 (m, 9H)
O
NH 1H NMR (400 MHz, MeOD) δ 7.59 (d, J =
O
39.2 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.21
H
MI-16 HN (t, J = 7.6 Hz, 1H), 7.10-6.83 (m, 2H), 4.56-
N
H N O 437.2186 B
(18b) O
4.22 (m, 2H), 4.11-3.62 (m, 2H), 3.30-3.14
H
H (m, 1H), 3.04-2.58 (m, 3H), 2.44-2.24 (m,
Exact Mass: 436.2111 1H), 2.16-1.23 (m, 10H).
1H NMR (400 MHz, MeOD) δ 8.60-8.45
(m, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.23 (dt,

O
NH
J = 14.3, 7.5 Hz, 1H), 6.94 (d, J = 26.3 Hz,
2H), 4.51-3.91 (m, 2H), 3.84-3.62 (m, 2H),
N
O
HN
H 3.15-3.02 (m, 1H), 2.94-2.62 (m, 3H),
MI-17
(18c)
N
N
O
O 2.42-1.99 (m, 2H), 1.98-1.39 (m, 8H). 13C 438.2143 B
H
H NMR (101 MHz, MeOD) δ 180.88, 173.90,
173.55, 148.38, 140.20, 134.62, 128.83,
124.28, 124.06, 99.24, 68.40, 54.33,
53.61, 40.16, 39.92, 39.74, 31.98, 31.65,
29.90, 27.57, 24.66.
O
NH 1H NMR (400 MHz, MeOD) δ 7.65-7.50
O H
(m, 3H), 7.41-7.27 (m, 3H), 6.84 (dd, J =
MI-18 HN 99.2, 15.5 Hz, 1H), 4.59-4.29 (m, 2H),
N O 424.2231 B
(18d) O 4.14-3.81 (m, 2H), 3.70-3.48 (m, 1H),
H
H
3.04-2.56 (m, 3H), 2.50-2.31 (m, 1H),
Exact Mass: 423.2158 2.17-1.77 (m, 4H), 1.76-1.43 (m, 5H).
40
1H NMR (400 MHz, MeOD) δ 7.86-7.72
(m, 1H), 7.48 (dd, J = 50.7, 8.7 Hz, 1H),
6.75 (dd, 1H), 6.58-6.47 (m, 2H), 4.51-
O
NH
4.29 (m, 2H), 4.09-3.90 (m, 2H), 3.87 (s,
O
3H), 3.82 (s, 3H), 3.62-3.47 (m, 1H), 3.05-
O O
HN
H 2.73 (m, 2H), 2.71-2.43 (m, 2H), 2.41-2.13
MI-19
(18e) N
O
O (m, 1H), 2.05-1.78 (m, 4H), 1.76-1.48 (m, 484.2445 B
H
H 5H). 13C NMR (101 MHz, MeOD) δ 181.68,
180.76, 173.87, 167.60, 162.95, 159.83,
138.03, 129.97, 116.55, 105.51, 67.10,
66.92, 54.65, 53.56, 42.97, 40.76, 40.16,
39.73, 37.91, 37.78, 32.37, 31.73, 31.27,
29.84, 27.60, 26.94, 24.67.
1H NMR (400 MHz, MeOD) δ 7.58-7.33
(m, 3H), 6.81-6.36 (m, 3H), 4.54-4.37 (m,

1H), 4.17-3.77 (m, 2H), 3.69-3.47 (m, 1H),
O
NH 3.41-3.32 (m, 2H), 2.97 (s, 6H), 2.91-2.73
(m, 2H), 2.70-2.46 (m, 1H), 2.41-2.14 (m,
O H
1H), 2.12-1.77 (m, 4H), 1.76-1.49 (m, 4H),
N
MI-20 HN
N O 467.2655 B
(18f) O
1.35-1.11 (m, 1H). 13C NMR (101 MHz,
H
H
MeOD) δ 181.64, 180.75, 173.92, 173.78,
Exact Mass: 466.2580 152.00, 143.30, 129.35, 129.11, 122.68,
122.15, 112.19, 111.86, 67.06, 53.51,
51.38, 50.74, 42.96, 40.17, 39.76, 38.94,
37.97, 32.45, 31.70, 27.58, 26.79, 24.67.
1H NMR (400 MHz, MeOD) δ 7.27 (q, J =
7.8 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 6.98

(d, J = 10.3 Hz, 1H), 6.90 (td, J = 8.5, 2.5
O
Hz, 1H), 4.50–4.40 (m, 1H), 4.22–4.12 (m,
NH
1H), 4.00–3.88 (m, 1H), 3.86–3.76 (m,
F
O H
1H), 3.28–3.19 (m, 2H), 2.97–2.85 (m,
MI-21 HN 2H), 2.79–2.57 (m, 4H), 2.40–2.25 (m,
N O 444.2292 B
(18g) O 1H), 2.11–1.43 (m, 9H), 1.40–1.31 (m,
H
H
1H). 13C NMR (101 MHz, MeOD) δ 181.68,
Exact Mass: 443.2220 173.79, 172.18, 164.15, 161.73, 143.89,
129.75, 124.01, 114.81, 112.45, 66.48,
53.63, 42.81, 40.14, 38.19, 37.85, 35.38,
31.64, 31.11, 30.34, 29.88, 29.58, 27.55,
24.54.
1H NMR (400 MHz, MeOD) δ 7.22–7.06
(m, 2H), 7.03 (s, 1H), 4.49–4.40 (m, 1H),

4.21–4.13 (m, 1H), 4.00–3.90 (m, 1H),
O
NH
3.87–3.79 (m, 1H), 3.29–3.23 (m, 2H),
F
2.94–2.83 (m, 2H), 2.80–2.57 (m, 4H),
F O H
MI-22 HN 2.37–2.26 (m, 1H), 2.13–1.48 (m, 9H),
N O 462.2202 B
(18h) O
1.45–1.33 (m, 1H). 13C NMR (101 MHz,
H
H MeOD) δ 181.59, 171.95, 124.60, 117.08,
Exact Mass: 461.2126 116.91, 116.63, 66.58, 53.64, 48.37,
48.15, 47.94, 47.51, 47.30, 47.08, 42.85,
40.18, 37.88, 35.36, 31.76, 31.23, 29.99,
27.60, 24.64.
41
1H NMR (400 MHz, MeOD) δ 6.75 (dt, J =
11.4, 5.7 Hz, 2H), 6.68–6.59 (m, 1H),
4.40–4.31 (m, 1H), 4.13–4.04 (m, 1H),
O
NH
3.91–3.80 (m, 1H), 3.79–3.69 (m, 1H),
F
O
3.20–3.15 (m, 2H), 2.88–2.77 (m, 2H),
H
MI-23 HN 2.71–2.49 (m, 4H), 2.28–2.15 (m, 1H),
N O 462.2202 B
(18i) F O
2.10–1.41 (m, 9H), 1.36–1.26 (m, 1H). 13C
H
H NMR (101 MHz, MeOD) δ 181.65, 145.70,
Exact Mass: 461.2126 111.14, 101.11, 100.86, 53.61, 48.27,
48.06, 47.85, 47.64, 47.42, 47.21, 47.00,
42.85, 40.16, 37.86, 34.89, 31.67, 31.17,
30.16, 29.61, 27.55, 24.59.
O
NH 1H NMR (400 MHz, MeOD) δ 7.22 (d, J =
8.5 Hz, 2H), 6.69 (d, J =7.9 Hz, 2H) 4.45-

O H
4.37 (m, 1H), 4.01-3.77 (m, 2H), 3.55-3.48
N
MI-24 HN
N O 469.2813 B
(18j)
H
O
(m, 2H), 3.23-2.99 (m, 3H), 3.08(s, 6H),
H
2.93-2.43 (m, 4H), 2.30-2.11 (m, 2H),
Exact Mass: 468.2737 1.70-1.32 (m, 9H).
1H NMR (400 MHz, MeOD) δ 6.93–6.77
(m, 4H), 4.76–4.55 (m, 2H), 4.49–4.41 (m,

1H), 4.30–4.17 (m, 1H), 4.00–3.92 (m,
O
NH
1H), 3.73 (s, 3H), 3.50–3.44 (m, 1H),
3.20–2.96 (m, 2H), 2.90–2.81 (m, 1H),
O O H
MI-25 HN 2.73–2.48 (m, 2H), 2.27–2.14 (m, 1H),
O
N O 458.2288 B
(18k) O 2.02–1.44 (m, 9H). 13C NMR (101 MHz,
H
H MeOD) δ 181.66, 173.49, 168.45, 154.41,
Exact Mass: 457.2213 152.32, 152.12, 115.48, 115.35, 114.33,
114.19, 66.90, 66.69, 54.71, 52.23, 51.21,
43.29, 40.03, 37.77, 31.71, 31.16, 29.84,
29.60, 27.61, 24.67.
1H NMR (400 MHz, MeOD) δ 6.90-6.69
(m, 5H), 4.68-4.38 (m, 2H), 4.29-4.17 (m,

1H), 4.09-3.84 (m, 2H), 3.72 (d, J = 4.6 Hz,
O
NH
3H), 3.66-3.52 (m, 1H), 3.36 (s, 3H), 3.02-
O
2.87 (m, 1H), 2.85-2.76 (m, 1H), 2.70-2.53
O H
MI-26 HN (m, 2H), 2.38-2.24 (m, 1H), 2.04-1.81 (m,
O
N O 472.2440 B
(18l) O
4H), 1.79-1.55 (m, 5H). 13C NMR (101
H
H MHz, MeOD) δ 181.62, 173.31, 173.19,
Exact Mass: 471.2369 171.84, 154.19, 151.51, 116.52, 115.45,
114.35, 114.31, 72.95, 72.36, 67.28,
54.79, 53.78, 53.23, 43.33, 40.17, 37.65,
32.27, 32.03, 31.66, 27.73, 24.80, 16.87.
1H NMR (400 MHz, MeOD) δ 6.70 (dd, J =
8.8, 3.9 Hz, 1H), 6.51-6.34 (m, 2H), 4.73-

O
NH
4.54 (m, 2H), 4.49-4.11 (m, 6H), 4.01-3.75
O (m, 2H), 3.54-3.33 (m, 2H), 3.25-2.99 (m,
O O
HN
H 3H), 2.90-2.76 (m, 1H), 2.68-2.14 (m, 4H),
MI-27
(18m)
O
N
O
O 2.10-1.46 (m, 7H). 13C NMR (101 MHz, 486.2242 B
H
H MeOD) δ 181.70, 173.49, 168.50, 168.37,
152.70, 143.83, 138.29, 116.85, 107.47,
103.55, 66.92, 66.68, 64.40, 63.96, 52.20,
51.09, 43.28, 40.06, 37.78, 31.73, 31.13,
29.83, 29.59, 27.59, 24.66.
42
1H NMR (400 MHz, MeOD) δ 7.26 (td, J =
6.8, 2.6 Hz, 2H), 6.97–6.87 (m, 2H), 4.84–
4.70 (m, 2H), 4.51–4.42 (m, 1H), 4.29–
O
NH 4.20 (m, 1H), 4.02–3.95 (m, 1H), 3.52–
3.42 (m, 1H), 3.21–3.10 (m, 1H), 3.05–
O H
2.83 (m, 2H), 2.73–2.47 (m, 2H), 2.24–
Cl
MI-28 HN
O
N O 462.1794 B
(18n) O
2.13 (m, 1H), 2.02–1.51 (m, 9H). 13C NMR
H
H
(101 MHz, MeOD) δ 181.64, 173.43,
Exact Mass: 461.1717 167.82, 156.98, 156.98, 129.04, 128.84,
125.69, 115.96, 66.90, 65.97, 52.16,
43.32, 39.97, 37.75, 31.69, 31.17, 29.60,
27.64, 24.69
1H NMR (400 MHz, MeOD) δ 7.18 (ddd, J
= 8.5, 6.2, 3.0 Hz, 1H), 7.02 (dt, J = 25.0,

8.4 Hz, 2H), 4.98–4.87 (m, 1H), 4.83–4.76
O
NH
(m, 1H), 4.52–4.40 (m, 1H), 4.30–4.20 (m,
O
1H), 4.10–3.76 (m, 2H), 3.52–3.41 (m,
Cl H
MI-29 HN 1H), 3.17–2.83 (m, 2H), 2.73–2.44 (m,
O
N O 480.1702 B
(18o) F
O
2H), 2.21–2.08 (m, 1H), 2.05–1.44 (m,
H
H 9H). 13C NMR (101 MHz, MeOD) δ 181.65,
Exact Mass: 479.1623 173.39, 167.30, 167.27, 153.33, 150.86,
145.28, 125.41, 124.07, 116.38, 116.16,
67.10, 66.94, 52.12, 51.30, 43.33, 39.98,
37.73, 31.65, 31.11, 29.78, 27.61, 24.71.
1H NMR (400 MHz, MeOD) δ 7.48–7.38
(m, 1H), 7.27–7.20 (m, 1H), 7.02–6.94 (m,

1H), 4.97–4.90 (m, 1H), 4.85–4.80 (m,
1H), 4.55–4.44 (m, 1H), 4.32–4.22 (m,
O
NH 1H), 4.03–3.89 (m, 2H), 3.55–3.47 (m,
Cl 1H), 3.21–3.11 (m, 1H), 3.04–2.94 (m,
O H
1H), 2.93–2.85 (m, 1H), 2.73–2.63 (m,
Cl
MI-30 HN
O
N O 496.0842 B
(18p) O
1H), 2.61–2.50 (m, 1H), 2.18 (m, 1H),
H
H
1.96–1.51 (m, 8H). 13C NMR (101 MHz,
Exact Mass: 495.1328 MeOD) δ 181.64, 173.39, 167.10, 152.81,
129.54, 127.47, 125.73, 123.12, 114.81,
66.81, 66.04, 60.14, 54.08, 52.20, 51.30,
43.34, 40.00, 37.73, 31.69, 31.14, 29.74,
27.67, 24.69.
1H NMR (400 MHz, MeOD) δ 7.37 (d, J =
8.9 Hz, 1H), 7.11 (d, J = 2.8 Hz, 1H), 6.88

(dd, J = 8.9, 2.9 Hz, 1H), 4.82–4.72 (m,
O
NH
2H), 4.50–4.19 (m, 2H), 4.02–3.86 (m,
Cl
O
2H), 3.46–3.39 (m, 1H), 3.17–2.83 (m,
Cl H
MI-31 HN 3H), 2.69–2.45 (m, 2H), 2.24–2.09 (m,
O
N O 496.1402 B
(18q) O
1H), 1.99–1.44 (m, 8H). 13C NMR (101
H
H MHz, MeOD) δ 181.64, 173.40, 167.36,
Exact Mass: 495.1328 157.59, 132.20, 130.41, 123.83, 116.61,
114.87, 66.91, 66.03, 60.16, 54.10, 52.09,
51.27, 43.33, 39.98, 37.76, 31.65, 31.14,
29.82, 27.64, 24.70.
43
1H NMR (400 MHz, MeOD) δ 7.14–7.03
(m, 2H), 6.82–6.73 (m, 1H), 4.82–4.47 (m,
3H), 4.33–4.21 (m, 1H), 4.14–3.97 (m,
O
NH
1H), 3.92–3.76 (m, 1H), 3.54–3.46 (m,
O
1H), 3.26–3.20 (m, 1H), 2.89–2.49 (m,
Cl H
MI-32 HN 3H), 2.32–2.26 (m, 1H), 2.23 (s, 3H),
O
N O 476.1949 B
(18r) O
2.15–1.77 (m, 5H), 1.70–1.49 (m, 4H). 13C
H
H NMR (101 MHz, MeOD) δ 181.06, 173.06,
Exact Mass: 475.1874 168.16, 155.08, 129.94, 128.71, 126.06,
125.39, 112.68, 67.10, 66.60, 52.38,
52.11, 50.31, 43.28, 40.18, 39.01, 31.86,
31.64, 31.11, 29.22, 24.80, 14.93.
44
1H and 13C NMR spectra
1H NMR spectrum of MI-03
13C NMR spectrum of MI-03
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
HRMS of MI-01 – MI-32
MI-01
14:22:46 08-Dec-2020
201208_M01 4 (0.068) Cm (2:23) TOF MS ES+
448.2245 1.14e4
100
%
449.2285
447.2410
429.2306
450.2337
430.2278 470.2066
462.2413 471.2039
413.2647 417.1658 427.2443 431.2394 437.2243 451.2336
402.2209 411.2370 441.3348 463.2522 475.6862 486.1868 492.1854 494.3103
0 m/z
400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500
MI-02
14:24:52 08-Dec-2020
201208_M02 24 (0.410) Cm (4:24) TOF MS ES+
478.2342 2.00e4
100
518.2278
%
479.2380
519.2303
500.2165
480.2408 501.2209
516.1954 520.2382
460.2245 492.2536
481.2475 502.2252
452.2513 461.2260 477.2482 493.2590 510.2578 521.2356
430.2382 437.1857 446.2328 448.2267 470.1980 486.1985
0 m/z
425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525
65
MI-03
17:39:24 03-Dec-2020
201203_M03 18 (0.308) Cm (1:23) TOF MS ES+
444.2303 474
100
466.2117
%
454.2083
468.2155
445.2334
480.2378
484.2237
455.2153
452.1896 488.1936
413.2659 469.2355
446.2242
414.2751 437.1830 471.2126
393.2178 397.0417 426.2233 429.3247
407.2030 447.2225 458.2525 466.1658
0 m/z
390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490
MI-04
17:41:25 03-Dec-2020
201203_M04 9 (0.154) Cm (1:24) TOF MS ES+
464.1999 82.3
100
%
465.1965
465.2336 478.2563
446.1966 464.1608 486.1757
402.2278 478.2105 488.1986
444.2575 447.2210 458.1361 464.0696 466.3193 485.5795
405.2487 428.4398 491.1491 496.2060
407.0248 412.1197 430.6216 498.1970
0 m/z
400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500
66
MI-05
17:43:41 03-Dec-2020
201203_M05 6 (0.103) Cm (1:24) TOF MS ES+
464.1996 1.07e3
100
%
465.1971
500.2174
488.1938
486.1791
446.2047 466.2155
478.2153 489.1996 500.1746
413.2588 417.1697 447.1778 452.2453 457.3493 467.2349 481.2656
402.1893 428.1997 437.2257 446.1685 472.1351
0 m/z
400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500
MI-06
14:26:35 08-Dec-2020
201208_M06 23 (0.393) Cm (3:24) TOF MS ES+
488.2393 3.84e3
100
510.2228
528.2316
489.2457
511.2282
%
529.2383
530.2532
490.2467 512.2316 526.2044
502.2506
503.2536 524.2351 531.2810
470.2293 491.2509 513.2370
448.2331 472.2492 480.2227 482.2046 504.2555 518.2262 540.2240
452.2452 458.2339 463.3008
0 m/z
440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540
67
MI-07
14:28:57 08-Dec-2020
201208_M07 3 (0.051) Cm (3:24) TOF MS ES+
496.2054 2.75e4
100
%
497.2094
520.2040
536.1970
518.1902
498.2171
521.2072 537.1998
478.1964 499.2199 510.2231

504.1724 523.1715 534.1649 538.2100
479.2004 492.3163 511.2268
442.2452 444.2393 452.2536 457.2560 464.1951 476.2740
0 m/z
440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540
MI-08
17:45:04 03-Dec-2020
201203_M08 8 (0.137) Cm (1:23) TOF MS ES+
476.2194 1.91e3
100
%
498.2072
477.2210
484.1845 499.2130
458.2043 478.2144 490.2309
475.2310 500.2264
417.1690 437.1993 450.3680 457.2380 514.1788
427.2503 429.3185 464.1967 478.2629 498.1710 503.1809 512.2401
446.2127
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
68
MI-09
09:49:23 28-Sep-2020
200928_MI_09 3 (0.051) Cm (3:24) TOF MS ES+
566.2231 1.22e4
100
%
567.2274
552.2068
512.2137
520.1794 553.2064 568.2272

578.2214
429.1367 534.1975
430.1380 441.1294 564.2082 582.1972
415.1134 482.2068 521.1832 535.2004
462.1216 494.1948 498.1896 596.2385
0 m/z
400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
MI-10
14:31:44 08-Dec-2020
201208_M10 7 (0.120) Cm (1:23) TOF MS ES+
564.0688 2.53e4
100
562.0720
%
565.0752
496.2232
567.0741
497.2283 519.2081
518.1891 521.2079 544.0660 546.0621 568.0812
498.2321 535.2588 550.2141 579.1084 581.1022 586.0591
502.2117 512.2323 524.0800 542.0917 552.0758
492.3200
0 m/z
490 495 500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590
69
MI-11
14:33:48 08-Dec-2020
201208_M11 13 (0.222) Cm (1:24) TOF MS ES+
564.0698 4.87e4
100
562.0717
%
566.0687
582.0809
580.0809
567.0713
542.0889 584.0779
540.0910 578.0748
516.1877 544.0767 568.0686 585.0796
498.1894 524.0781 526.0803 532.1546 550.0681 560.0604
502.1755 514.1807
0 m/z
490 495 500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590
MI-12
17:47:52 03-Dec-2020
201203_M12 23 (0.393) Cm (1:24) TOF MS ES+
462.1798 1.52e4
100
%
464.1779
502.1720
465.1815 484.1620
504.1695
476.1968 486.1606
413.2690 444.1702
446.1641 478.1948 505.1757
414.2714 456.1739 461.1885 472.1622 492.1928 494.2002
425.2271 427.2424 432.1758 439.0187
0 m/z
410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510
70
MI-13
17:49:46 03-Dec-2020
201203_M13 16 (0.274) Cm (1:24) TOF MS ES+
496.1408 1.07e3
100
498.1346
%
516.1927
504.1084
506.1128
518.1829
499.1456
520.1268
510.1584
462.1671 478.1373 480.1396 524.1082 526.1392 532.1339

470.1544 536.1339
450.3719 457.2437 492.3408 541.2010
0 m/z
445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540 545
MI-14
14:35:46 08-Dec-2020
201208_M14 18 (0.308) Cm (3:24) TOF MS ES+
496.1406 2.55e3
100
498.1399
%
499.1434
500.1388
536.1329
518.1211
516.2151 520.1229 538.1369
501.1377
478.1305 480.1252 510.1438 521.1296
462.2136 534.0969
447.3271 452.2467 457.2686 464.1861 476.2790 482.2296 492.3289 502.1668
0 m/z
440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540
71
MI-15
14:37:44 08-Dec-2020
201208_M15 8 (0.137) Cm (3:23) TOF MS ES+
399.2030 4.33e3
100
421.1848
400.2079
%
422.1892
360.3245
439.1974
423.1947
401.2118
361.3309
443.1664
403.1737
437.1715 444.1736
413.2548 430.2437
362.3276 404.1734 419.2741 431.2400
353.2650 379.1768 381.2975 391.1964 393.2044 445.1906
363.2001 374.1581
0 m/z
350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450
MI-16
14:39:50 08-Dec-2020
201208_M16 19 (0.325) Cm (3:24) TOF MS ES+
437.2186 1.53e4
100
%
459.2007
438.2220
453.2113
430.2437 454.2169 481.1886
460.2054
413.2669
427.2384 439.2204 463.2971 478.2341

391.2000 419.2732 431.2481 452.2438 482.1879
421.2003 464.1862 475.1743
399.2006 411.2410
436.2428 449.2198 469.1914 483.2319
0 m/z
385 390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485
72
MI-17
14:42:29 08-Dec-2020
201208_M17 24 (0.410) Cm (2:24) TOF MS ES+
460.1951 2.03e4
100
%
438.2143
461.1999
439.2174
442.1843 462.2071
404.1950 413.2662 430.2453
391.2021 419.2743 423.1960 437.2171 443.1844 453.2169 463.3013
379.1730 405.2009
393.2129 465.1749
385.1230 447.2880 474.2149
0 m/z
375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475
MI-18
14:45:06 08-Dec-2020
201208_M18 12 (0.205) Cm (4:24) TOF MS ES+
424.2231 1.76e3
100
%
425.2269
446.2091
426.2248 447.2058
427.2422 438.2328 464.2150

381.2918 384.2992 402.2187 413.2691 443.1898 451.1929 459.2130 468.1987
393.2231 406.2160 419.2862 473.3088
0 m/z
375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475
73
MI-19
14:47:32 08-Dec-2020
201208_M19 5 (0.086) Cm (3:23) TOF MS ES+
484.2445 2.98e3
100
%
485.2463
486.2555
506.2272
487.2655
508.2397
524.2337
478.2366 488.2625 505.2495 509.2453 525.2386
522.1929
437.2006
448.2148
452.2627 457.2469 463.3152 466.2432 478.1951 501.2838 529.2051
0 m/z
435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535
MI-20
19:51:49 09-Dec-2020
201209_MI20_1 10 (0.171) Cm (3:24) TOF MS ES+
467.2655 1.38e4
100
%
468.2709
507.2615
505.3022
469.2769
501.2308
470.2750 489.2505 508.2655
459.2410 494.2243
427.2327 430.2491 451.2687 453.2593 481.2787 509.2747
417.1559 438.2646 443.2645 461.2465
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
74
MI-21
17:51:36 03-Dec-2020
201203_M21 5 (0.086) Cm (1:23) TOF MS ES+
444.2292 4.18e3
100
%
445.2322
466.2121
484.2211
467.2167
413.2674 485.2224
446.2406 471.1864
458.2429 482.1945
391.2020 414.2950 426.2206 447.2402 472.1883 486.2310
406.3503 428.2423 438.2125 463.1851
395.1959
0 m/z
390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490
MI-22
15:10:21 08-Dec-2020
201208_MI22 23 (0.393) Cm (3:23) TOF MS ES+
462.2202 7.33e3
100
%
484.2007
463.2263
502.2127
485.2066
503.2193
464.2273 489.1806 498.2350
489.6813 504.2188 512.2490
444.2164 472.2432
427.2446 430.2488 445.2248 481.1903
419.2663 444.1903 457.2736
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
75
MI-23
14:54:55 08-Dec-2020
201208_M23 7 (0.120) Cm (2:24) TOF MS ES+
462.2202 1.45e4
100
%
502.2132
484.2016
463.2242
503.2148
485.2055
472.2423
464.2265
473.2392 486.2086 500.1813 504.2150
444.2118 466.1908
427.2382 461.2463 477.2446 513.2148
417.1596 430.2386 432.2125 444.1555 452.2586
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
MI-24
15:15:17 08-Dec-2020
201208_MI24 11 (0.188) Cm (3:24) TOF MS ES+
469.2813 5.94e4
100
%
509.2739
493.2791
470.2847
491.2641 494.2562
501.3086 510.2753
471.2903
472.2546 495.2497
455.2658 477.2495 483.2970 502.3111
513.2454
456.2696
419.2753 422.1693 430.2431 463.2950
437.2191 446.2635 452.2318
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
76
MI-25
17:53:06 03-Dec-2020
201203_M25 18 (0.308) Cm (2:23) TOF MS ES+
458.2288 748
100
%
459.2299
480.2123
460.2336 481.2049
440.2108 472.2324
413.2843 433.2975 480.1714 498.2420
402.1869 421.2089 423.1782 444.4967 450.6397 453.2153 461.3397 466.2292
485.1733
496.2078
0 m/z
400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500
MI-26
14:57:56 08-Dec-2020
201208_M26 10 (0.171) Cm (4:23) TOF MS ES+
472.2440 1.94e3
100
%
473.2487
496.2439
497.2444
474.2535
494.2401
498.2408
475.2687
451.2304 454.2356 456.2556 489.2848 494.2097 500.2033 512.2270
421.2125 423.2365 437.2379 444.2127 465.2313
0 m/z
415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515
77
MI-27
15:00:17 08-Dec-2020
201208_M27 2 (0.034) Cm (2:24) TOF MS ES+
486.2242 2.08e3
100
508.2034
%
487.2264 529.2736
530.2672
526.2498
509.2065
524.2243
488.2240 519.2410
531.2686
516.2282
496.2201
437.2273 445.2544 465.2369 472.2313 513.1957 532.2510
449.1988 456.2126 479.2325 494.2175 497.2137 505.2480
0 m/z
435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535
MI-28
17:54:31 03-Dec-2020
201203_M28 21 (0.359) Cm (1:23) TOF MS ES+
462.1794 614
100
%
464.1817
484.1632
500.2137
465.1775
486.1768
472.1631 476.1988 487.1681 500.1732

399.0469 406.3581 421.1957 423.8469 432.1695 437.2247 444.1385 446.1682 456.1556 482.1972 491.2663
413.2837
0 m/z
400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500
78
MI-29
15:02:39 08-Dec-2020
201208_M29 12 (0.205) Cm (2:24) TOF MS ES+
480.1702 2.64e3
100
%
520.1611
482.1690
502.1511
522.1589
483.1694
504.1503
500.2162 523.1589
462.1628 494.1997
450.3777 463.1709 472.2299 474.2798 486.2256 500.1777 507.1362 516.2001
430.2719 437.1814 446.2007 462.1258
0 m/z
425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525
MI-30
09:47:17 28-Sep-2020
200928_MI_30 3 (0.051) Cm (3:24) TOF MS ES+
550.0474 2.02e3
100
552.0455
%
496.0842
498.0806 553.0502
536.0419 562.0438
538.0410
518.0516 566.0940
520.0436
499.0770 540.0748 567.1037
482.1211 521.0457
408.2574 413.2660 429.1205 496.0402 568.9994 587.0771
441.1053 451.2645 462.0154 599.0911
0 m/z
400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
79
MI-31
15:05:42 08-Dec-2020
201208_M31 10 (0.171) Cm (3:24) TOF MS ES+
496.1402 2.60e3
100
498.1375
%
516.1863 519.2044
499.1434
536.1348
500.1369
521.2056
534.1711 538.1280
529.2709
478.1322 501.1525
462.1855 480.1247 507.1189
494.2416
445.1157 452.2426 457.2802 464.1654 472.2364 484.1640
0 m/z
440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540
MI-32
15:08:19 08-Dec-2020
201208_M32 20 (0.342) Cm (2:24) TOF MS ES+
476.1949 5.25e3
100
516.1887
498.1776
%
472.2441
478.1938
518.1887
494.2254
500.1750
484.1612
479.1983 519.1924
512.2304
486.1615 501.1763
458.1833 510.2062 520.1773

490.2173
430.2431 457.2417 462.1892 470.1813 521.2599
437.2131 442.2469 450.1974
0 m/z
425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525
80
References
1. P. Zhou, X.-L. Yang, X.-G. Wang, B. Hu, L. Zhang, W. Zhang, H.-R. Si, Y. Zhu, B. Li, C.-L.
Huang, H.-D. Chen, J. Chen, Y. Luo, H. Guo, R.-D. Jiang, M.-Q. Liu, Y. Chen, X.-R.
Shen, X. Wang, X.-S. Zheng, K. Zhao, Q.-J. Chen, F. Deng, L.-L. Liu, B. Yan, F.-X.
Zhan, Y.-Y. Wang, G.-F. Xiao, Z.-L. Shi, A pneumonia outbreak associated with a new
coronavirus of probable bat origin. Nature 579, 270–273 (2020). doi:10.1038/s41586-
020-2012-7 Medline
2. F. Wu, S. Zhao, B. Yu, Y.-M. Chen, W. Wang, Z.-G. Song, Y. Hu, Z.-W. Tao, J.-H. Tian, Y.-
Y. Pei, M.-L. Yuan, Y.-L. Zhang, F.-H. Dai, Y. Liu, Q.-M. Wang, J.-J. Zheng, L. Xu, E.
C. Holmes, Y.-Z. Zhang, A new coronavirus associated with human respiratory disease in
China. Nature 579, 265–269 (2020). doi:10.1038/s41586-020-2008-3 Medline
3. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, The
species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV
and naming it SARS-CoV-2. Nat. Microbiol. 5, 536–544 (2020). doi:10.1038/s41564-
020-0695-z Medline
4. P. Smith, Covid-19 in Australia: Most infected health workers in Victoria’s second wave
acquired virus at work. BMJ 370, m3350 (2020). doi:10.1136/bmj.m3350 Medline
5. M.-K. Looi, Covid-19: Is a second wave hitting Europe? BMJ 371, m4113 (2020).
doi:10.1136/bmj.m4113 Medline
6. Y. Chen, Q. Liu, D. Guo, Emerging coronaviruses: Genome structure, replication, and
pathogenesis. J. Med. Virol. 92, 418–423 (2020). doi:10.1002/jmv.25681 Medline
7. R. Hilgenfeld, From SARS to MERS: Crystallographic studies on coronaviral proteases enable
antiviral drug design. FEBS J. 281, 4085–4096 (2014). doi:10.1111/febs.12936 Medline
8. K. Anand, J. Ziebuhr, P. Wadhwani, J. R. Mesters, R. Hilgenfeld, Coronavirus main
proteinase (3CLpro) structure: Basis for design of anti-SARS drugs. Science 300, 1763–
1767 (2003). doi:10.1126/science.1085658 Medline
9. L. Zhang, D. Lin, Y. Kusov, Y. Nian, Q. Ma, J. Wang, A. von Brunn, P. Leyssen, K. Lanko, J.
Neyts, A. de Wilde, E. J. Snijder, H. Liu, R. Hilgenfeld, α-Ketoamides as broad-spectrum
inhibitors of coronavirus and enterovirus replication: Structure-based design, synthesis,
and activity assessment. J. Med. Chem. 63, 4562–4578 (2020).
doi:10.1021/acs.jmedchem.9b01828 Medline
10. T. Pillaiyar, M. Manickam, V. Namasivayam, Y. Hayashi, S.-H. Jung, An overview of severe
acute respiratory syndrome–coronavirus (SARS-CoV) 3CL protease inhibitors:
Peptidomimetics and small molecule chemotherapy. J. Med. Chem. 59, 6595–6628
(2016). doi:10.1021/acs.jmedchem.5b01461 Medline
11. Z. Jin, X. Du, Y. Xu, Y. Deng, M. Liu, Y. Zhao, B. Zhang, X. Li, L. Zhang, C. Peng, Y.
Duan, J. Yu, L. Wang, K. Yang, F. Liu, R. Jiang, X. Yang, T. You, X. Liu, X. Yang, F.
Bai, H. Liu, X. Liu, L. W. Guddat, W. Xu, G. Xiao, C. Qin, Z. Shi, H. Jiang, Z. Rao, H.
Yang, Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature 582,
289–293 (2020). doi:10.1038/s41586-020-2223-y Medline
12. L. Zhang, D. Lin, X. Sun, U. Curth, C. Drosten, L. Sauerhering, S. Becker, K. Rox, R.
Hilgenfeld, Crystal structure of SARS-CoV-2 main protease provides a basis for design
of improved α-ketoamide inhibitors. Science 368, 409–412 (2020).
doi:10.1126/science.abb3405 Medline
13. W. Dai, B. Zhang, X.-M. Jiang, H. Su, J. Li, Y. Zhao, X. Xie, Z. Jin, J. Peng, F. Liu, C. Li,
Y. Li, F. Bai, H. Wang, X. Cheng, X. Cen, S. Hu, X. Yang, J. Wang, X. Liu, G. Xiao, H.
Jiang, Z. Rao, L.-K. Zhang, Y. Xu, H. Yang, H. Liu, Structure-based design of antiviral
drug candidates targeting the SARS-CoV-2 main protease. Science 368, 1331–1335
(2020). doi:10.1126/science.abb4489 Medline
14. R. L. Hoffman, R. S. Kania, M. A. Brothers, J. F. Davies, R. A. Ferre, K. S. Gajiwala, M. He,
R. J. Hogan, K. Kozminski, L. Y. Li, J. W. Lockner, J. Lou, M. T. Marra, L. J. Mitchell
Jr., B. W. Murray, J. A. Nieman, S. Noell, S. P. Planken, T. Rowe, K. Ryan, G. J. Smith
3rd, J. E. Solowiej, C. M. Steppan, B. Taggart, Discovery of ketone-based covalent
inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of
COVID-19. J. Med. Chem. 63, 12725–12747 (2020). doi:10.1021/acs.jmedchem.0c01063
Medline
15. B. Boras et al., Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical
Candidate for the Potential Treatment of COVID-19. bioRxiv 293498 [preprint]. 13
September 2020.
16. M. Westberg et al., Rational design of a new class of protease inhibitors for the potential
treatment of coronavirus diseases. bioRxiv 275891 [preprint]. 16 September 2020.
17. C. Ma, M. D. Sacco, B. Hurst, J. A. Townsend, Y. Hu, T. Szeto, X. Zhang, B. Tarbet, M. T.
Marty, Y. Chen, J. Wang, Boceprevir, GC-376, and calpain inhibitors II, XII inhibit
SARS-CoV-2 viral replication by targeting the viral main protease. Cell Res. 30, 678–692
(2020). doi:10.1038/s41422-020-0356-z Medline
18. M. D. Sacco, C. Ma, P. Lagarias, A. Gao, J. A. Townsend, X. Meng, P. Dube, X. Zhang, Y.
Hu, N. Kitamura, B. Hurst, B. Tarbet, M. T. Marty, A. Kolocouris, Y. Xiang, Y. Chen, J.
Wang, Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for
developing dual inhibitors against Mpro and cathepsin L. Sci. Adv. 6, eabe0751 (2020).
doi:10.1126/sciadv.abe0751 Medline
19. W. Zhu, M. Xu, C. Z. Chen, H. Guo, M. Shen, X. Hu, P. Shinn, C. Klumpp-Thomas, S. G.
Michael, W. Zheng, Identification of SARS-CoV-2 3CL protease inhibitors by a
quantitative high-throughput screening. ACS Pharmacol. Transl. Sci. 3, 1008–1016
(2020). doi:10.1021/acsptsci.0c00108 Medline
20. K. S. Yang, X. R. Ma, Y. Ma, Y. R. Alugubelli, D. A. Scott, E. C. Vatansever, A. K. Drelich,
B. Sankaran, Z. Z. Geng, L. R. Blankenship, H. E. Ward, Y. J. Sheng, J. C. Hsu, K. C.
Kratch, B. Zhao, H. S. Hayatshahi, J. Liu, P. Li, C. A. Fierke, C. K. Tseng, S. Xu, W. R.
Liu, A quick route to multiple highly potent SARS-CoV-2 main protease inhibitors.
ChemMedChem 15, 1–8 (2020). Medline
21. A. D. Rathnayake, J. Zheng, Y. Kim, K. D. Perera, S. Mackin, D. K. Meyerholz, M. M.
Kashipathy, K. P. Battaile, S. Lovell, S. Perlman, W. C. Groutas, K.-O. Chang, 3C-like
protease inhibitors block coronavirus replication in vitro and improve survival in MERS-
CoV-infected mice. Sci. Transl. Med. 12, eabc5332 (2020).
doi:10.1126/scitranslmed.abc5332 Medline
22. R. Cannalire, C. Cerchia, A. R. Beccari, F. S. Di Leva, V. Summa, Targeting SARS-CoV-2
proteases and polymerase for COVID-19 treatment: State of the art and future
opportunities. J. Med. Chem. acs.jmedchem.0c01140 (2020).
doi:10.1021/acs.jmedchem.0c01140 Medline
23. C. Gampe, V. A. Verma, Curse or cure? A perspective on the developability of aldehydes as
active pharmaceutical ingredients. J. Med. Chem. 63, 14357–14381 (2020).
doi:10.1021/acs.jmedchem.0c01177 Medline
24. P. S. Dragovich, T. J. Prins, R. Zhou, S. E. Webber, J. T. Marakovits, S. A. Fuhrman, A. K.
Patick, D. A. Matthews, C. A. Lee, C. E. Ford, B. J. Burke, P. A. Rejto, T. F.
Hendrickson, T. Tuntland, E. L. Brown, J. W. Meador 3rd, R. A. Ferre, J. E. V. Harr, M.
B. Kosa, S. T. Worland, Structure-based design, synthesis, and biological evaluation of
irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam
moieties as L-glutamine replacements. J. Med. Chem. 42, 1213–1224 (1999).
doi:10.1021/jm9805384 Medline
25. R. P. Jain, H. I. Pettersson, J. Zhang, K. D. Aull, P. D. Fortin, C. Huitema, L. D. Eltis, J. C.
Parrish, M. N. G. James, D. S. Wishart, J. C. Vederas, Synthesis and evaluation of keto-
glutamine analogues as potent inhibitors of severe acute respiratory syndrome 3CLpro. J.
Med. Chem. 47, 6113–6116 (2004). doi:10.1021/jm0494873 Medline
26. Y. Yip, F. Victor, J. Lamar, R. Johnson, Q. M. Wang, D. Barket, J. Glass, L. Jin, L. Liu, D.
Venable, M. Wakulchik, C. Xie, B. Heinz, E. Villarreal, J. Colacino, N. Yumibe, M.
Tebbe, J. Munroe, S.-H. Chen, Discovery of a novel bicycloproline P2 bearing peptidyl
α-ketoamide LY514962 as HCV protease inhibitor. Bioorg. Med. Chem. Lett. 14, 251–
256 (2004). doi:10.1016/j.bmcl.2003.09.074 Medline
27. H. K. Ganguly, G. Basu, Conformational landscape of substituted prolines. Biophys. Rev. 12,
25–39 (2020). doi:10.1007/s12551-020-00621-8 Medline
28. P. Bazzini, C. G. Wermuth, in The Practice of Medicinal Chemistry, C. G. Wermuth, D.
Aldous, P. Raboisson, D. Rognan, Eds. (Academic Press, ed. 4, 2015), pp. 319–357.
29. Y. C. Martin, A bioavailability score. J. Med. Chem. 48, 3164–3170 (2005).
doi:10.1021/jm0492002 Medline
30. S.-H. Sun, Q. Chen, H.-J. Gu, G. Yang, Y.-X. Wang, X.-Y. Huang, S.-S. Liu, N.-N. Zhang,
X.-F. Li, R. Xiong, Y. Guo, Y.-Q. Deng, W.-J. Huang, Q. Liu, Q.-M. Liu, Y.-L. Shen, Y.
Zhou, X. Yang, T.-Y. Zhao, C.-F. Fan, Y.-S. Zhou, C.-F. Qin, Y.-C. Wang, A mouse
model of SARS-CoV-2 infection and pathogenesis. Cell Host Microbe 28, 124–133.e4
(2020). doi:10.1016/j.chom.2020.05.020 Medline
31. M. S. Weiss, R. Hilgenfeld, On the use of the merging R factor as a quality indicator for X-
ray data. J. Appl. Crystallogr. 30, 203–205 (1997). doi:10.1107/S0021889897003907
32. P. A. Karplus, K. Diederichs, Linking crystallographic model and data quality. Science 336,
1030–1033 (2012). doi:10.1126/science.1218231 Medline
33. V. B. Chen, W. B. Arendall 3rd, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral,
L. W. Murray, J. S. Richardson, D. C. Richardson, MolProbity: All-atom structure
validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
doi:10.1107/S0907444909042073 Medline
34. W. Kabsch, XDS. Acta Crystallogr. D 66, 125–132 (2010).
doi:10.1107/S0907444909047337 Medline
35. P. R. Evans, G. N. Murshudov, How good are my data and what is the resolution? Acta
Crystallogr. D 69, 1204–1214 (2013). doi:10.1107/S0907444913000061 Medline
36. A. Vagin, A. Teplyakov, Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–
25 (2010). doi:10.1107/S0907444909042589 Medline
37. L. Zhu, S. George, M. F. Schmidt, S. I. Al-Gharabli, J. Rademann, R. Hilgenfeld, Peptide
aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main
protease. Antiviral Res. 92, 204–212 (2011). doi:10.1016/j.antiviral.2011.08.001 Medline
38. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta
39. O. Kovalevskiy, R. A. Nicholls, F. Long, A. Carlon, G. N. Murshudov, Overview of
refinement procedures within REFMAC5: Utilizing data from different sources. Acta
40. G. Bricogne et al., BUSTER version 2.10.3 (Global Phasing Ltd., 2017).
41. Y. Liu, W. Kati, C.-M. Chen, R. Tripathi, A. Molla, W. Kohlbrenner, Use of a fluorescence
plate reader for measuring kinetic parameters with inner filter effect correction. Anal.
Biochem. 267, 331–335 (1999). doi:10.1006/abio.1998.3014 Medline

China Paper Supporting Data

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

China Paper Supporting Data

Uploaded by

Copyright:

Available Formats

science.sciencemag.org/cgi/content/full/science.

Supplementary Materials for

*Corresponding author. Email: yangsy@scu.edu.cn (S.Y.); leijian@scu.edu.cn (J.L.); zhengyt@mail.kiz.ac.cn

Published 18 February 2021 on Science First Release

This PDF file includes:

Other supplementary material for this manuscript includes:

Enzyme Mpro DSF Enzyme Mpro DSF

Mpro-MI-23 Mpro-Boceprevir Mpro-Telaprevir

MI-03 >500 >500 >500 >500 >500

MI-04 >500 >500 >500 >500 >500

MI-12 >500 >500 >500 >500 >500

MI-23 >500 >500 >500 >500 >500

MI-28 >500 >500 >500 >500 >500

MI-31 >500 >500 >500 >500 >500

Cell protection Toxicity Cell protection Toxicity

T1/2 Tmax Cmax AUClast AUCINF_obs MRTINF_obs CL Vssobs F

Acute toxicity study Repeated dose one week toxicity study

Compound MI-09 / MI-30 MI-09 / MI-30

Administration p.o. i.p. i.v. p.o. i.p. i.v.

250 mg/kg 24 mg/kg 100 mg/kg 100 mg/kg 6 mg/kg

Gene Genbank ID Sequence

Production of authentic SARS-CoV-2 main protease

SARS-CoV-2 (TSAVLQ / SGFRK; “ / ” indicates the cleavage site). At the C-terminus, it

was generated by digesting with PreScission protease.

(GE Healthcare) again to remove GST-tagged PreScission protease (the GST-PreScission

Mpro was checked by SDS-PAGE.

Crystallization and data collection

(Molecular Dimensions). The crystals of both SARS-CoV-2 Mpro-Boceprevir and Mpro-Telaprevir

flash-cooled in liquid nitrogen.

Phase determination and refinement

Enzyme kinetics and correction of inner-filter effect

by adding 25 μL of the fluorescent substrate MCA-AVLQSGFR-Lys (DNP)-Lys-NH2 (GL

MCA (0.01-20 μM).

monitored for 1 min at background as well as different substrate concentrations in 50 μL of the

concentration was corrected according to the function:

The corrected initial velocity of the reaction was calculated as

V= Vinitial velocity ⁄(Corr%)

experiments were performed in triplicate.

values are presented as mean ± standard deviation (SD).

Differential scanning fluorimetry assay

Viruses and cells

atmosphere. Mycoplasma testing was performed at regular intervals and no mycoplasma

the Chinese Academy of Sciences, Kunming Institute of Zoology (Kunming, China).

the manufacturer’s protocol. All experiments were performed in triplicate.

Cellular antiviral activity assay

with fresh drug-containing medium. In 72 h, the cytopathic effect caused by SARS-CoV-2

GGGGAACTTCTCCTGCTAGAAT-3' and 5'-CAGACATTTTGCTCTCAAGCTG-3' with

SARS-CoV-2 probe FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'. The EC50 values were

Assessment of pharmacokinetic properties

The PK properties of compounds were examined in male Sprague-Dawley (SD) rats (n = 3

5 or 10 mg/kg (intravenous injection (i.v.)), 10 or 20 mg/kg (intraperitoneal injection (i.p.) or oral

centrifuged to isolate plasma. Subsequently, the plasma compound concentrations were

Phoenix WinNonlin 7.0.

Kunming Institute of Zoology, Chinese Academy of Sciences (SMKX-20200915-11). All animals

used in this study were chosen randomly.

or 250 mg/kg (n = 4) via i.p.

In vivo antiviral studies of MI-09 and MI-30

Kunming Institute of Zoology, Chinese Academy of Sciences (SMKX-20200915-11). All animals

used in this study were chosen randomly.

Mouse infection and sample collection.

3, per dpi group) were collected on days 1, 3 and 5 post infection.

RNA extraction and measurement of viral loads in tissues

manufacturer’s instructions. Viral RNA was quantified by THUNDERBIRD® Probe One-step