science.sciencemag.org/cgi/content/full/science.abb4489/DC1

Supplementary Materials for

Structure-based design of antiviral drug candidates

targeting the SARS-CoV-2 main protease

Wenhao Dai, Bing Zhang, Xia-Ming Jiang, Haixia Su, Jian Li, Yao Zhao, Xiong Xie, Zhenming Jin, Jingjing Peng, Fengjiang Liu, Chunpu Li, You Li, Fang Bai, Haofeng Wang,

Xi Cheng, Xiaobo Cen, Shulei Hu, Xiuna Yang, Jiang Wang, Xiang Liu, Gengfu Xiao, Hualiang Jiang, Zihe Rao, Lei-Ke Zhang*, Yechun Xu*, Haitao Yang*, Hong Liu*

*Corresponding author. Email: hliu@simm.ac.cn (H.L.); yanght@shanghaitech.edu.cn (H.Y.); zhangleike@wh.iov.cn (L.-K.Z.); ycxu@simm.ac.cn (Y.X.)

Published 22 April 2020 on Science First Release DOI: 10.1126/science.abb4489

This PDF file includes: Materials and Methods Scheme S1 Figs. S1 to S5 Tables S1 to S4 References

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Materials and Methods:

Chemistry

General procedures for compounds synthesis: The materials and solvents were purchased from

commercial sources and used without further purification. All products were characterized by their

NMR and MS spectra. 1H and 13C NMR spectra were recorded on a 400 MHz, 500 MHz or 600

MHz instrument. Compounds were purified by chromatography with silica gel (300-400 mesh).

Analytical thin layer chromatography (TLC) was HSGF 254 (0.15-0.2 mm thickness). High-

resolution mass spectra (HRMS) were measured on Micromass Ultra Q-TOF spectrometer. All

target compounds possessed a purity of ≥95% as determined by HPLC.

2

Scheme S1. Reagents and Conditions: (a) LiHMDS, THF, -78 °C; (b) NaBH4, CoCl2·6H2O, 0 °C;

(c) 4 M HCl, 12 h; (d) HATU, DIPEA, CH2Cl2, -20 °C, 12 h; (e) 4 M HCl, 12 h ; (f) HATU,

DIPEA, CH2Cl2, -20 °C, 12 h; (g) NaBH4, THF; (h) Dess-Martin Periodinane, CH2Cl2.

(2S,4R)-dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanomethyl) pentanedioate (2)

The solution of lithium bis(trimethylsilyl)amide (LiHMDS) (94 mL, 1 M in THF) was added

dropwise to a solution of N-Boc-L-glutamic acid dimethyl ester 1 (12.0 g, 43.6 mmol) in THF (100

mL) at -78 °C, then the mixture was stirred at -78 °C for 1 h. Subsequently, bromoacetonitrile

(3.24 mL, 46.6 mmol) was added dropwise to the mixture under the temperature of -78 °C, and

the reaction was kept at -78 °C for additional 4 h. After the reactant was consumed, the reaction

was quenched by saturated NH4Cl solution (40 mL). The reaction mixture was warmed up to room

temperature and extracted with ethyl acetate (50 mL×3). The organic layers were concentrated and

purified by flash column chromatography (petroleum ether/ethyl acetate = 4/1) to give product 2

(7.58 g, 55%) as colorless oil.

ESI-MS m/z 215.1 [M–Boc+H]+.

1H NMR (600 MHz, CDCl3) δ 5.11 (d, J = 7.5 Hz, 1H), 4.38 (s, 1H), 3.77 (s, 3H), 3.75 (s, 3H),

2.92-2.82 (m, 1H), 2.81-2.71 (m, 2H), 2.24-2.08 (m, 2H), 1.44 (s, 9H).

(S)-methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (3)

In a round-bottomed flask, the compound 2 (6.0 g, 19.09 mmol) was dissolved in anhydrous MeOH

(100 mL) before CoCl2·6H2O (2.72 g, 11.45 mmol) was added at 0 °C. Subsequently, NaBH4 (4.35

g, 114.78 mmol) was added protion-wise, and the reaction mixture was warmed up to room

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temperature and stirred for 12 h. After the reactant was consumed, the reaction was quenched by

saturated NH4Cl solution (30 mL). MeOH in the mixture was evaporated and the residual mixture

was extracted with ethyl acetate (50 mL×3). The organic layers were washed by saturated NH4Cl

solution (100 mL×3) and brine (100 mL×3), then the organic phase was dried (MgSO4) and

concentrated. The residue was purified by flash column chromatography (petroleum ether/ethyl

acetate = 2/1) to give the product 3 (2.18g, 40%) as white solid.

ESI-MS m/z 187.7 [M–Boc+H]+.

1H NMR (600 MHz, CDCl3) δ 6.64 (s, 1H), 5.56 (s, 1H), 4.29 (d, J = 9.1 Hz, 1H), 3.71 (s, 3H),

3.37- 3.26 (m, 2H), 2.47-2.42 (m, 2H), 2.13-2.08 (m, 1H), 1.84-1.81 (m, 2H), 1.41 (s, 9H).

Methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate hydrochloride (4)

Compound 3 (1.0 g, 3.5 mmol) was dissolved in 10 mL DCM, then the HCl (9 mL, 4M in dioxane)

was added. The reaction mixture was stirred at ambient temperature for 12 h, and the mixture was

concentrated in vacuo to get a white solid 4, which could be used for the following step without

purification.

Methyl (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-cyclohexylpropanamido)-3-((S)-2-

oxopyrrolidin-3-yl)propanoate (6a)

4

To the solution of Boc-L-Cyc-OH 5a (0.95 g, 3.5 mmol) in DCM (40 mL) was added the HATU

sequentially (1.9 g, 4.9 mmol) at -20 °C. The solution was kept at -20 °C for 20 mins, and then the

crude product 4 (0.77 g 3.5 mmol) was added. After 30 min later, DIPEA (1.7 mL, 10.5 mmol)

was added dropwise. And the reaction mixture was stirred at -20 °C for 12 h. The reaction mixture

was washed by saturated NH4Cl solution (100 mL×3), saturated NaHCO3 solution (100 mL×3)

and brine (100 mL×3). The organic phase layer was dried over Na2SO4 and concentrated in vacuo.

The resulting residue was purified by flash column chromatography (DCM: CH3OH, 40: 1 v/v) to

afford the pure product 6a (1.23 g, 80%) as white solid.

ESI-MS m/z 440.2 [M+H]+.

1H NMR (600 MHz, DMSO-d6) δ 8.29 (d, J = 8.1 Hz, 1H), 7.60 (s, 1H), 6.83 (d, J = 8.1 Hz, 1H),

4.39-4.28 (m, 1H), 3.97-3.93 (m, 1H), 3.60 (s, 3H), 3.13 (t, J = 9.0 Hz, 1H), 3.06-3.04 (m, 1H),

2.36-2.24 (m, 1H), 2.11-2.02 (m, 2H), 1.70-1.55 (m, 7H), 1.47-1.38 (m, 1H), 1.35 (s, 9H), 1.32-

1.19 (m, 2H), 1.17-1.04 (m, 3H), 0.85-0.81 (m, 2H).

Methyl (S)-2-((S)-2-amino-3-cyclohexylpropanamido)-3-((S)-2-oxopyrrolidin-3-yl) propano-

ate hydrochloride (7a)

To a solution of 6a (1.05 g, 2.4 mmol) in dry DCM was added the HCl (6 mL, 4M in dioxane) and

the reaction mixture was stirred at ambient temperature for 12 h. Solvent was removed in vacuo

and the crude product 7a was directly used in next step without further purification.

Methyl (S)-2-((S)-3-cyclohexyl-2-(1H-indole-2-carboxamido)propanamido)-3-((S)-2-

oxopyrrolidin-3-yl)propanoate (9a)

5

To a solution of the indole-2-carboxylic acid 8 (0.78 g, 2.4 mmol) in DCM was added the HATU

(1.09 g, 2.88 mmol) sequentially at -20 °C. The solution was kept at -20 °C for 20 mins, and then

the crude product 7a (0.90 g 2.4 mmol) was added. DIPEA (1.17 mL, 7.2 mmol) was added drop-

wise after 30 mins later. Then, the reaction mixture was stirred at -20 °C for 12 h, followed by

washing with saturated NH4Cl solution (100 mL×3), saturated NaHCO3 solution (100 mL×3) and

brine (100 mL×3). The organic phase was dried over Na2SO4 and concentrated, and the residue

was purified by column chromatography (CH2Cl2: CH3OH, 30: 1 v/v) to afford the pure product

9a (0.98 g, 85%) as white solid.

ESI-MS m/z 483.1 [M+H]+.

1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.57 (d, J = 7.9 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H),

7.64 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.26 (d, J = 1.5 Hz, 1H), 7.18 (t, J

= 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.59-4.57 (m, 1H), 4.40-4.31 (m, 1H), 3.62 (s, 3H), 3.16-

3.08 (m, 2H), 2.37-2.35 (m, 1H), 2.14-2.04 (m, 2H), 1.76-1.72 (m, 2H), 1.70-1.55 (m, 8H), 1.45-

1.35 (m, 1H), 1.20-1.12 (m, 2H), 0.97-0.88 (m, 2H).

N-((S)-3-cyclohexyl-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amin-o)-1-

oxopropan-2-yl)-1H-indole-2-carboxamide (10a)

6

To a solution of 9a (0.96 g, 2.0 mmol) in dry THF was added the NaBH4 (0.45 g, 12 mmol) protion-

wise at 0 °C and the CH3OH (2mL) was added dropwise, then the reaction mixture was stirred at

room temperature for 3 h. The completion of the reaction was confirmed by TLC then the reaction

was quenched by saturated NH4Cl solution (20 mL). The reaction mixture was extracted with ethyl

acetate (50 mL×3) and the organic layers were washed with saturated NH4Cl solution (50 mL×3)

and brine (50 mL×3). The organic phase was dried over Na2SO4 and concentrated, and the residue

was purified by column chromatography (DCM: CH3OH, 20: 1 v/v) to afford the pure product 10a

(0.82 g, 90%) as light solid.

ESI-MS m/z 455.3 [M+H]+.

1H NMR (400 MHz, Methanol-d4) δ 11.58 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 9.0 Hz,

1H), 7.62 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.26 (d, J = 1.2 Hz, 1H), 7.17

(t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.68 (t, J = 5.6 Hz, 1H), 4.53-4.51 (m, 1H), 3.83-3.76

(m, 1H), 3.38-3.33 (m, 1H), 3.28-3.21 (m, 1H), 3.11 (t, J = 8.7 Hz, 1H), 3.05-3.01 (m, 1H), 2.30-

2.34 (m, 1H), 2.17-2.09 (m, 1H), 1.85-1.78 (m, 1H), 1.76-1.72 (m, 2H), 1.69-1.61 (m, 3H), 1.61-

1.53 (m, 3H), 1.41-1.34 (m, 2H), 1.26 -1.12 (m, 2H), 1.04-0.78 (m, 3H).

N-((S)-3-cyclohexyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)

propan-2-yl)-1H-indole-2-carboxamide (11a).

To a solution of the 10a (0.45 g, 1.0 mmol) in DCM, the DMP (0.55 g, 1.2 mmol) was added

slowly and the reaction mixture was stirred at room temperature. When the reactant was consumed,

the reaction was filtered and washed with saturated Na2S2O3 solution (50 mL×3), saturated

NaHCO3 solution (50 mL×3) and brine (50 mL×3). The organic phase was dried over Na2SO4 and

7

concentrated, then the residue was purified by flash column chromatography (DCM: CH3OH, 20:

1 v/v) to afford the pure product 11a (0.34 g, 76%) as white solid.

HRMS (m/z): calculated for C25H31N4O4–[M-H]

– 451.2351; found, 451.2345.

HPLC purity ： 99.88%. 11a was determined by Agilent-1100 HPLC with binary pump,

photodiode array detector (DAD), using Agilent Extend-C18 column (150 x 4.6 mm, 5 μm). 11a

was analyzed using MeOH/H2O = 65:35 (v/v) (30 min, 1 mL/min) and calculated the peak areas

at 230 nm.

1H NMR (500 MHz, DMSO) δ 11.58 (s, 1H), 9.44 (s, 1H), 8.59 (d, J = 7.6 Hz, 1H), 8.49 (d, J =

7.9 Hz, 1H), 7.68-7.57 (m, 2H), 7.44 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 1.2 Hz, 1H), 7.19 (t, J = 7.5

Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 4.64-4.59 (m, 1H), 4.25-4.20 (m, 1H), 3.20-3.06 (m, 2H), 2.37-

2.30 (m, 1H), 2.18-2.13 (m, 1H), 1.96-1.92 (m, 1H), 1.81-1.58 (m, 9H), 1.42 (d, J = 2.9 Hz, 1H),

1.24-1.10 (m, 3H), 1.02-0.88 (m, 2H).

13C NMR (126 MHz, DMSO) δ 201.30, 178.76, 173.52, 161.54, 136.93, 131.76, 127.51, 123.85,

122.01, 120.18, 112.73, 103.97, 56.85, 51.18, 39.89, 39.56, 37.79, 34.21, 33.65, 32.31, 29.78,

27.76, 26.56, 26.27, 26.11

N-((S)-3-(3-fluorophenyl)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-

yl)amino)propan-2-yl)-1H-indole-2-carboxamide. (11b).

The synthesis procedure of 11b is similar as 11a.

8

HRMS (ESI) m/z: calculated for C25H26FN4O4＋[M＋H]

＋: 465.1933, found: 465.1936.

HPLC purity：99.20%. 11b was determined by Agilent-1100 HPLC with binary pump,

photodiode array detector (DAD), using Agilent Extend-C18 column (150 x 4.6 mm, 5 μm). 11b

was analyzed using MeOH/H2O = 65:35 (v/v) (20 min, 1 mL/min) and calculated the peak areas

at 254 nm.

1H NMR (500 MHz, Acetone) δ 10.92 (s, 1H), 9.44 (s, 1H), 8.57 (d, J = 6.8 Hz, 1H), 8.09 (d, J =

8.4 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.50 (dd, J = 8.3, 0.6 Hz, 1H), 7.28 (td, J = 7.9, 6.2 Hz, 1H),

7.22-7.11 (m, 5H), 7.07-7.03 (m, 1H), 6.93 (td, J = 8.3, 1.9 Hz, 1H), 5.08 (td, J = 8.4, 5.6 Hz, 1H),

4.38-4.34 (m, 1H), 3.36 (dd, J = 13.8, 5.6 Hz, 1H), 3.28-3.17 (m, 3H), 2.49-2.40 (m, 1H), 2.31-

2.25 (m, 1H), 2.02-1.96 (m, 1H), 1.85-1.79 (m, 1H), 1.78-1.71 (m, 1H).

13C NMR (126 MHz, Acetone) δ 200.80, 180.25, 172.63, 163.60 (d, J = 243.6 Hz), 162.40, 141.55

(d, J = 7.5 Hz), 137.87, 131.92, 130.89 (d, J = 8.3 Hz), 128.61, 126.37 (d, J = 2.7 Hz), 124.77,

122.63, 120.94, 117.03 (d, J = 21.3 Hz), 114.13 (d, J = 21.1 Hz) 113.17, 104.24, 58.57, 55.42,

40.89, 38.88, 38.37, 30.51, 29.10.

19F NMR (376 MHz, Acetone) δ -113.99- -114.92 (m).

Fig. S1. Spectral data for compounds 11a and 11b.

1H and 13C NMR, HRMS and HPLC spectra of 11a

9

10

11

1H, 13C and 19F NMR, HRMS and HPLC spectra of 11b.

12

13

Cloning, expression and purification of SARS-CoV-2 Mpro

The full-length gene encoding SARS-CoV-2 Mpro was optimized and synthesized for

Escherichia coli (E. coil) expression (GENEWIZ). The method of cloning and producing authentic

SARS-CoV-2 Mpro was followed by the protocol published for SARS-CoV Mpro previously (18).

Enzymatic activity and inhibition assays

The enzyme activity and inhibition assays have been described previously (22, 25). The

recombinant SARS-CoV-2 Mpro (30 nM at a final concentration) was mixed with serial dilutions

of each compound in 80 µL assay buffer (50 mM Tris–HCl, pH 7.3, 1 mM EDTA) and incubated

for 10 min. The reaction was initiated by adding 40 µL fluorogenic substrate with a final

concentration of 20 µM. After that, the fluorescence signal at 320 nm (excitation)/405 nm

(emission) was immediately measured every 30 s for 10 min with a Bio-Tek Synergy4 plate reader.

The Vmax of reactions added with compounds at various concentrations compared to the reaction

added with DMSO were calculated and used to generate IC50 curves. For each compound, IC50

values against SARS-CoV-2 Mpro were measured at 9 concentrations and three independent

experiments were performed. All experimental data was analyzed using GraphPad Prism software.

Crystallization

SARS-CoV-2 Mpro was incubated with 10 mM 11a or 11b for 30 min and the complex (5

mg/ml) was crystallized by hanging drop vapor diffusion method at 20 °C. The best crystals were

14

grown with well buffer containing 2% polyethylene glycol (PEG) 6000, 3% DMSO, 1 mM DTT,

0.1 M MES (pH 6.0). The cryo-protectant solution contained 30% PEG 400, 0.1 M MES (pH 6.0).

Data collection and structure determination

All data were collected on beamline BL19U1 at Shanghai Synchrotron Radiation Facility

(SSRF) at 100 K and at a wavelength of 0.9785 Å using a Pilatus3 6M image plate detector. Data

integration and scaling were performed using the program XDS (26). The structures were

determined by molecular replacement (MR) with the SARS-CoV Mpro (PDB ID: 2H2Z) as a search

model using the program PHASER (27). The output model from MR was subsequently subjected

to iterative cycles of manual model adjustment with Coot (28) and refinement was finished with

Phenix (29). The inhibitors 11a and 11b was built according to the omit map. Data collection and

structure refinement statistics are summarized in Table S1.

Coordinates and structure factors for SARS-CoV-2 Mpro in complex with the inhibitors 11a

and 11b have been deposited in Protein Data Bank with accession number 6LZE and 6M0K,

respectively.

Table S1. Data collection and refinement statistics

Mpro-11a Mpro-11b

PDB code 6LZE 6M0K

Data collection

Space group C2 C2

Cell dimensions

a, b, c (Å) 97.70, 80.94, 51.74 98.15, 81.70, 51.67

α, β, γ (°) 90, 114.27, 90 90, 114.69, 90

Wavelength (Å) 0.97852 0.97852

Resolution (Å) 50.00-1.51 (1.54-1.51) a 50.00-1.50 (1.54-1.50) a

Completeness (%) 98.0 (91.6) 98.8 (90.3)

Rmerge (%) 4.2 (55.5) 3.0 (77.1)

I/σ(I) 13.99 (1.80) 18.69 (1.30)

Refinement

Resolution (Å) 47.16-1.50 43.45-1.50

15

No. of reflections 57,378 58,412

Rwork/Rfree (%) 17.80/20.10 18.34/19.66

No. of atoms

Protein 2,340 2,347

Ligand 49 50

Water 209 163

B factor (Å2)

Protein 28.75 31.92

Ligand 37.60 52.56

Water 37.95 40.62

R.m.s deviations

Bond lengths (Å) 0.014 0.017

Bond angles (°) 1.280 1.440

Ramachandran plot (%)

Favored 98.0 98.0

Allowed 2.0 2.0

Outliers 0.0 0.0

a Values in parentheses are for highest-resolution shell.

Fig. S2. Overall structure of SARS-CoV-2 Mpro.

Cartoon representation of SARS-CoV-2 Mpro in complex with 11a in two different views. Protomer

A is shown in gold; protomer B in pale cyan. The catalytic dyad (His41 and Cys145) is indicated

16

as green and red spheres, respectively. The compound 11a is shown as magenta sticks.

Fig. S3. Electron density maps for Mpro-inhibitor interactions.

(A, C) The Fo -Fc omit maps of 11a (panel A), 11b (panel C) and water molecules are shown as

pale green mesh and contoured at 3 σ.

(B, D) The 2Fo -Fc electron density maps for residues involved in inhibitor binding are shown as

light blue mesh and contoured at 1σ.

17

18

Fig. S4. Comparison of the inhibitor binding modes in SARS-CoV and SARS-CoV-2 Mpros.

(A) Comparison of 11a and 11b binding pockets. Residues in Mpro-11a structure and Mpro-N3

structure are colored wheat and pale cyan, respectively. 11a and 11b are shown as sticks colored

magenta and yellow, respectively.

(B) Comparison of binding modes of 11a and 11b in SARS-CoV-2 Mpro with those of N1, N3 and

N9 in SARS-CoV Mpro. SARS-CoV-2 Mpro-11a (wheat, PDB code: 6LZE), SARS-CoV-2 Mpro-

11b (pale cyan, PDB code: 6M0K), SARS-CoV Mpro-N1(sky blue, PDB code:1WOF), SARS-CoV

Mpro-N3 (gray, PDB code: 2AMQ) and SARS-CoV Mpro-N9 (olive, PDB code: 2AMD).11a, 11b,

N1, N3 and N9 are shown in magenta, yellow, cyan, dirty violet and salt, respectively.

(C) Comparison of the 11a and N1 binding pockets. Residues in Mpro-11a structure and Mpro-

N1structure are colored wheat and sky blue, respectively. 11a and N1 are shown as sticks colored

in magenta and cyan, respectively.

(D) Comparison of the 11a and N9 binding pockets. Residues in Mpro-11a structure and Mpro-N9

structure are colored wheat and olive, respectively. 11a and N9 are shown as sticks colored in

magenta and salt, respectively.

(E) Comparison of the 11b and N1 binding pockets. Residues in Mpro-11b structure and Mpro-N1

structure are colored pale cyan and sky blue, respectively. 11b and N1 are shown as sticks colored

in yellow and cyan, respectively.

(F) Comparison of the 11b and N9 binding pockets. Residues in Mpro-11b structure and Mpro-N3

structure are colored pale cyan and gray, respectively. 11a and N3 are shown as sticks colored in

yellow and dirty violet, respectively.

(G) The chemical structures of N1, N3 and N9

Antiviral assay for 11a and 11b

African green monkey kidney Vero E6 cell line was obtained from American Type Culture

Collection (ATCC, no. 1586) and maintained in Dulbecco's Modified Eagle Medium (DMEM;

Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen), 1%

antibiotic/antimycotic (Gibco Invitrogen), at 37 °C in a humidified 5% CO2 incubator. A clinical

isolate of SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019) was propagated in Vero E6

cells, and viral titer was determined as described previously (11). All the infection experiments

were performed at biosafety level-3 (BLS-3).

19

To assess the antiviral activity of compounds, pre-seeded Vero E6 cells (5×104 cells/well)

were treated with the different concentration of the indicated compounds for 1 hour, and then were

infected with SARS-CoV-2 at a MOI of 0.05. Two hours later, the virus-drug mixture was removed

and cells were further cultured with drug containing medium. At 24 h p.i., cell supernatant was

collected and viral titer in supernatant was also detected by plaque assay. Vero E6 cells (1×105

cells/well) were incubated with supernatant. At 1 hour post infection, the supernatant was removed,

and the cells were incubated under an overlay consisting of DMEM supplemented with 2% FBS,

0.9% CMC (Calbiochem) and appropriate concentration of compounds. At 4 days post infection,

the overlay was discarded and cells were fixed for 30 min in 4% polyoxymethylene and stained

with crystal violet working solution. At 24 h p.i., we also monitored intracellular NP using

immunofluorescenc assay and measured viral RNA copy number in cell supernatant using real

time PCR, as described previously(11).

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Fig. S5. Intracellular levels of NP in SARS-CoV-2 infected Vero E6 cells.

Vero E6 cells were treated with a series concentration of indicated compounds 11a and 11b and

infected with SARS-CoV-2. Chloroquine (CQ, 10 μM) was used as positive control. At 24 hours

post infection, cells were fixed and NP expression in infected cells was analyzed by

immunofluorescence with anti-NP sera. Bars: 400 μm.

Animal studies

All procedures relating to animal handling, care, and treatment were performed according to

the guidelines approved by the Institute Animal Care and Use Committee at Shanghai Institute of

21

Materia Medica (2019-02-YY-08) and the Institute Animal Care and Use Committee (IACUC) of

Westchina Frontier PharmaTech. Co., Ltd (IACUC-A2020036-E005-01, IACUC-A2020036-

E011-01, IACUC-A2020036-E012-01). All animals used in this study were chosen randomly.

Pharmacokinetics of 11a and 11b

Male CD-1 mice (n = 3 per group, weight 25-30g and age: 6-8 weeks) were treated with a

solution of compounds 11a and 11b (DMSO/EtOH/PEG300/NaCl (5/5/40/50, v/v/v/v)) at doses

of 5 mg/kg or 20 mg/kg, 5 mg/kg and 5 mg/kg via intraperitoneal (ip), subcutaneous (sc) and

intravenous (iv), respectively. Blood samples were collected at 0.05, 0.25, 0.75, 2, 4, 8, and 24 h

after administration. Serum samples were obtained through common procedures and the

concentrations of compound in the supernatant were analyzed by LC-MS/MS.

The solution of compound 11a (HS15/NaCl (7.5/100, v/v)) was injected to SD rats and beagle

dogs (n = 2 per group) at doses of 10 mg/kg and 5 mg/kg via intravenous (iv). Blood samples were

collected at 0.05, 0.5, 1, 2, 4, 6, 8, 10. 12 and 24 h after administration. Serum samples were

obtained through common procedures and the concentrations of compound in the supernatant were

analyzed by LC-MS/MS.

Table S2. Preliminary pharmacokinetic (PK) evaluation of compounds 11a and 11b in mice.a

F

(%)

87.8

85.9

81.8

aThe value of mice represented the results (mean ± SD) from three independent experiments.

Table S3. Preliminary pharmacokinetic (PK) evaluation of compound 11a in SD rat and Beagle dog. a

Vss_obs

(mL/kg)

203.5

489.5

aThe value of SD rats and Beagle dogs represented the results (mean) from two independent experiments.

Vss_obs

(mL/kg)

-

1845 ±

211

-

-

768 ± 96

MRTINF_obs

(h)

1.85 ± 0.13

1.78 ± 0.19

2.23 ± 0.34

1.41 ± 0.81

0.63 ±

0.128

MRTINF_obs

(h)

0.8

1.4

CL

(mL/min/kg)

-

17.4 ± 2.76

-

-

20.6 ± 2.0

CL

(mL/min/kg)

4.01

5.80

AUCINF_obs

(h*ng/mL)

4272 ± 366

4871 ± 766

14090 ± 2677

3360 ± 973

4076 ± 381

AUCINF_obs

(h*ng/mL)

41500

14900

AUClast

(h*ng/mL)

4252 ± 367

4844 ± 757

13877 ± 2756

3307 ± 939

4041 ± 370

AUClast

(h*ng/mL)

41500

14900

Cmax

(ng/mL)

2394 ± 288

-

12783 ± 421

3019 ± 665

-

C3min

(ng/mL)

81500

21900

Tmax

(h)

0.25 ± 0.00

-

0.25 ± 0.00

0.25 ± 0.00

-

T1/2

(h)

7.6

5.5

T1/2

(h)

4.27 ± 1.23

4.41 ± 0.30

5.21 ± 1.35

2.22 ± 1.96

1.65 ± 0.45

Admin

i.v (10mg/kg)

i.v (5mg/kg)

Admin

i.p (5mg/kg)

i.v (5mg/kg)

i.p (20mg/kg)

s.c (5mg/kg)

i.v (5mg/kg)

Animal

SD Rat

Beagle Dog

Comp

d.

11a

11b

Comp

d.

11a

23

In vivo toxicity study of 11a

Acute toxicity study of 11a

SPF SD rats (age: 7-11 weeks) were half male and half female, which were weighted of 180-

220 g for females and 210-250 g for males. 11a was dissolved in HS15/NaCl (4 mg/mL, 7.5/100,

v/v), and the 11a was administrated once daily via intravenous drip. The SD rats were dosed 24

mg/kg (one rat), 40 mg/kg (ten rats) and 60 mg/kg (four rats) via intravenous drip administrated

for acute toxicity study.

Dose range toxicity study for one week of 11a

SPF SD rats were half male and half female, which were weighted of 180-220 g for females

and 200-240 g for males. Beagle dogs (age: 9-12 months) were half male and half female, which

were weighted of 9-11 kg. Dose range toxicity studies for one week were performed. SD rats were

assigned to four groups which contained one vehicle group (four SD rats) and three intravenous

drip administrated (0.2 mL/min) groups (six SD rats per group), the dosage of 11a were 2, 6, 18

mg/kg, respectively. The Beagle dogs (four dogs) were dosed via intravenous drip administration

(1.5 mL/min) (10 mg/kg (the first day, four dogs), 15 mg/kg (the second day, four dogs), 20 mg/kg

(the third day, four dogs), 25 mg/kg (the fourth day, four dogs), 25 mg/kg (the fifth to seventh

days, randomly two dogs), 40 mg/kg (the fifth to seventh days, other two dogs)). All animals were

clinically observed once a day at least during 7 days for toxic signs which including bodyweight,

food intake, and hematology. At the end of the experiment, samples of heart, liver, spleen, lung,

kidney and administration site were collected.

Table S4. In vivo toxicity study of 11a

Dose range studies for one week

intravenous drip (0.2 mL/min)

SD rats

2 mg/kg

6 mg/kg

18 mg/kg

Six rats for per group

Repeat dose

No obvious toxicity

1. During the administration

period, no anomalies of weight

and general state were observed

in each group.

2. At the end of administration, the

rats in each group underwent

hematological and biochemical

examination, and no anomalies

were observed.

3. At the end of the administration,

histological examination of the

heart, kidney and lung were

conducted, and no anomalies

were observed in each group

intravenous drip (1.5 mL/min)

Beagle dogs

10 mg/kg (the first day, four dogs)

15 mg/kg (the second day, four dogs)

20 mg/kg (the third day, four dogs)

25 mg/kg (the fourth day, four dogs)

25 mg/kg (the fifth to seventh days, randomly

two dogs)

40 mg/kg (the fifth to seventh days, other two

dogs)

Four dogs

Dose escalation

No obvious toxicity

1. When administered at a dosage of 25

mg/kg or more, the skin of the extremities

(two of four dogs) developed allergic

symptoms (lumpy, transient and recovery

on the same day) during the

administration period.

2. At the end of administration, there were

no anomalies in hematology, blood

biochemistry.

3. At the end of the administration,

histological examination of the heart,

lung, kidney, spleen and liver were

conducted, and no anomalies were

observed in each group.

Acute toxicity

intravenous drip

(0.2 mL/min)

SD rats

24 mg/kg

40 mg/kg

60 mg/kg

One rat for 24 mg/kg

Ten rats for 40 mg/kg

Four rats for 60 mg/kg

Single dose

No rats died after receiving

24, 40 mg/kg, and one rat

died after receiving 60

mg/kg

Study

Administration

Species

Dosage (mg/kg)

Number of animals

Frequency

Results

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