Potent inhibitors of SARS-CoV-2 3C-like protease derived from N-substituted isatin compounds

Article abstract of DOI:10.1016/j.ejmech.2020.112702

SARS-CoV-2 3C-like protease is the main protease of SARS-CoV-2 and has been considered as one of the key targets for drug discovery against COVID-19. We identified several N-substituted isatin compounds as potent SARS-CoV-2 3C-like protease inhibitors. Th

Full text of DOI:10.1016/j.ejmech.2020.112702

Journal Pre-proof
Potent inhibitors of SARS-CoV-2 3C-like protease derived from N-substituted isatin
compounds
Pei Liu, Hongbo Liu, Qi Sun, Hao Liang, Chunmei Li, Xiaobing Deng, Ying Liu, Luhua
Lai
PII:
S0223-5234(20)30674-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112702
EJMECH 112702
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2020
Revised Date: 26 July 2020
Accepted Date: 27 July 2020
Please cite this article as: P. Liu, H. Liu, Q. Sun, H. Liang, C. Li, X. Deng, Y. Liu, L. Lai, Potent inhibitors
of SARS-CoV-2 3C-like protease derived from N-substituted isatin compounds, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112702.
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Potent inhibitors of SARS-CoV-2 3C-like protease derived
from N-substituted isatin compounds
Pei Liu†#, Hongbo Liu†#, Qi Sun†#, Hao Liang†, Chunmei Li‡, Xiaobing Deng†, Ying Liu*,†,‡, Luhua
Lai*,†,‡,‖
†BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of
Chemistry and Molecular Engineering, ‡Center for Quantitative Biology, and ‖Peking-Tsinghua Center
for Life Sciences, Peking University, Beijing 100871, China
Abstract: SARS-CoV-2 3C-like protease is the main protease of SARS-CoV-2 and has been considered
as one of the key targets for drug discovery against COVID-19. We identified several N-substituted isa-
tin compounds as potent SARS-CoV-2 3C-like protease inhibitors. The three most potent compounds
inhibit SARS-CoV-2 3C-like protease with IC50’s of 45 nM, 47 nM and 53 nM, respectively. Our study
indicates that N-substituted isatin compounds have the potential to be developed as broad-spectrum anti-
coronavirus drugs.
Keywords: COVID-19, SARS-CoV-2 3C-like protease, N-substituted isatin compounds, in vitro assay,
Structure-activity relationship
1. Introduction
The coronavirus infectious disease 2019 (COVID-19) is a newly emerged infectious disease caused by
a novel coronavirus, SARS-CoV-2.[1, 2] COVID-19 has been recognized as a global threat as it rapidly
spreads and breaks out in many countries, causing significant health and economic impact. The SARS-
CoV-2 is a positive-strand RNA virus that uses a complex set of enzymes to replicate its RNA
genome.[3, 4] Among these, the 3C-like protease (3CLpro), also known as the main protease (Mpro), is
essential for processing the viral polyproteins that are translated from the viral RNA.[5] The active site
of 3CLpro contains Cys145 and His41 to constitute a catalytic dyad, in which cysteine functions as the
common nucleophile in the proteolytic process.[6, 7] The catalytic domain of 3CLpro in CoVs is highly
conserved.[8] Hence, 3CLpro has been considered as an attractive drug target for broad-spectrum anti-
coronavirus therapy.[4]
A variety of 3CLpro inhibitors have been reported in the literature over the past decade.[4, 9-12] To
date, several potential SARS-CoV-2 3CLpro inhibitors have been reported from compound library
screening,[8] rational design [8, 13], [14] and testing of ingredients from traditional Chinese
medicine.[15, 16] The chemical structures of the experimentally identified SARS-CoV-2 3CLpro inhibi-
1

tors are diverse, including α-ketoamide analogues[13], peptidomimetics compounds[8, 14], baicalein
and its derivatives[15, 16] and several repurposed approved drugs and drug candidates[8]. However,
only a few candidates have high inhibition activity against SARS-CoV-2 3CLpro and no effective thera-
py has been developed so far.
Previously, we reported a series of N-substituted 5-carboxamide-isatin compounds as inhibitors of
SARS CoV 3CLpro.[12] The best compound showed a sub-micromolar IC50 against SARS-CoV
3CLpro.[12] Apparently, the isatin scaffold with derivatization may also provide a good starting point for
SARS CoV-2 3CLpro inhibitor development, because the two proteases share high sequence identity and
the same active site. In order to verify whether isatin compounds can inhibit SARS-CoV-2 3CLpro, we
selected a series of isatin compounds from an in-house synthetic compound library, synthesized a few
new compounds, tested their inhibitory effects against SARS-CoV-2 3CLpro, and analyzed their struc-
ture-activity relationship (SAR).
2. Results and discussion
2.1. Chemistry.
The synthetic route used to prepare the test compounds 1-28 is shown in Scheme 1. Compound 26 was
resynthesized by a simple and effective synthetic route within three steps. First, 2-((4-
carbamoylphenyl)amino)-2-oxoacetimidic acid (I-26) was obtained by reaction of 4-aminobenzamide,
hydroxylamine hydrochloride, and chloral hydrate. Then, I-26 was converted into 2,3-dioxoindoline-5-
carboxamide (II-26) by treatment with concentrated sulfuric acid at 90 °C. Last, 26 was obtained by re-
action of 2,3-dioxoindoline-5-carboxamide (II-26) with 2-(bromomethyl)naphthalene.
The synthetic route used to prepare compound 29 is shown in Scheme 2, which is different from other
compounds. We started from 2-(4-aminophenyl)acetic acid using the general Sandmeyer synthetic
route.[12, 17] After got 2-(2,3-dioxoindolin-5-yl)acetic acid (II-29), additional carboxyl protection to
change to methyl 2-(2,3-dioxoindolin-5-yl)acetate (III-29) with methyl group, followed by the introduc-
tion of naphthyl group at N1 position was subjected to afford methyl 2-(1-(naphthalen-2-ylmethyl)-2,3-
dioxoindolin-5-yl)acetate (IV-29) and de-protection step to afford 2-(1-(naphthalen-2-ylmethyl)-2,3-
dioxoindolin-5-yl)acetic acid (V-29). Then through activating the carboxyl group using phosphorus ox-
ychloride, 29 was obtained by reaction of the activated V-29 and ammonium hydroxide. Compounds 1-
25 were collected from our synthesized in-house library.[12] Compounds 27, 28 and 29 are new com-
pounds that have not been reported before. The structures of the newly synthesized compounds 26, 27,
28 and 29 and the potent compound 23 were confirmed by 1H NMR, 13C NMR and HRMS.
Scheme 1a
2

aReagents and conditions: (a) Cl3CCH(OH)2, NH2OH•HCl, Na2SO4, H2O; (b) H2SO4; (c) K2CO3, R1X,
acetonitrile.
Scheme 2a
O
O
HO
HO
HO
H3CO
O
a
b
c
O
O
O
N
O
O
O
N
N
OH
N
H
NH2
H
H
I-29
II-29
III-29
O
O
O
H2N
H3CO
HO
O
O
O
f
e
O
O
d
O
N
N
N
IV-29
29
V-29
aReagents and conditions: (a) Cl3CCH(OH)2, NH2OH•HCl, Na2SO4, H2O; (b) H2SO4; (c) EDC, dime-
thylaminopyridine, methanol; (d) K2CO3, 2-(Bromomethyl)naphthalene, acetonitrile; (e) NaOH, metha-
nol; (f) i) POCl3; ii) ammonium hydroxide (30%), Et3N.
2.2. Biological Evaluation.
The inhibition activity was measured following the previously published procedure using a synthetic
peptide-pNA as substrate (Table 1).[12, 18] We used Tideglusib as a positive control for the enzyme as-
say. Our measured IC50 value (1.91 µM) is in consistent with the reported one (1.55 µM,Fig. S1). 8
Among all the 29 compounds that we tested, 12 showed inhibition activity over 50 % at 50 µM. Hydro-
phobic groups at the R1 position are required to insure the inhibitory effect. The carboxamide group at
R2 position (23-28) is essential for the high inhibition activity. Compounds 26 and 23 inhibit SARS-
CoV-2 3CLpro with IC50 values of 45 nM (Fig. 1) and 53 nM (Fig. S1), respectively. Further modifica-
tions at the 6-position of the naphthalene ring (27 and 28) were carried out to study the structure-activity
3

relationship (SAR) of these compounds. Small substituents like 6-bromo in the naphthalene ring can be
tolerated with a similar inhibition activity (IC50 of 47 nM, Fig. S1) of the parent compound 26. Howev-
er, due to space limitation in the enzyme active site, large substituents like 6-phenyl group significantly
reduced inhibitory effect (IC50 = 24.9 µM, Fig. S1). Substitution to other groups at R2 position like hal-
ogen (9), carboxylic acid N-hydroxysuccinimide ester (17) and ethanamide group (29) inactivate or de-
crease the activity of the compounds. The SARs of these compounds against SARS-CoV-2 3CLpro is
similar to that found for SARS-CoV 3CLpro,[12] further indicating that the substrate binding pocket in
3CLpro of CoVs is highly conserved and other classes of reported SARS-CoV or other CoVs 3CLpro in-
hibitors may also be effective against SARS-CoV-2 3CLpro.
Table 1. Inhibition activities of isatin derivatives against SARS-CoV-2 3CLpro
IC50 or percentage
Compds
R1
R2
(%) of inhibition at 50
µMb
Tideglusiba
1.91 ± 0.16
c
H
H
F
-
1
2
3
-CH2OH
-
-
F
-
-
-
4
5
6
Cl
NO2
CH3
CH3CH2CH2
n-C4H9
I
25% at 50 µM
51% at 50 µM
41.8 ± 8.0
7
8
I
I
9
CH3
CO2CH3
53% at 50 µM
10
4

CH3CH2CH2
CO2CH3
CO2CH3
53% at 50 µM
42% at 50 µM
11
12
n-C4H9
CO2CH3
CO2CH3
32% at 50 µM
45% at 50 µM
13
14
PhCH2
CH3
45% at 50 µM
47% at 50 µM
15.5 ± 1.2
15
16
17
n-C4H9
PhCH2
CH3
CH3CH2CH2
n-C4H9
CO2H
CO2H
CO2H
CO2H
CO2H
-
18
19
20
21
22
-
-
PhCH2
-
β-C10H7CH2
32% at 50 µM
CONH2
0.053 ± 0.010
23
CH3CH2CH2
CONH2
CONH2
10.2 ± 1.0
17.8 ± 0.7
24
25
26
27
28
29
n-C4H9
β
-C10H7CH2
-(6-Br)-C10H7CH2
-(6-Ph)-C10H7CH2
-C10H7CH2
CONH2
0.045 ± 0.007
0.047 ± 0.007
24.9 ± 4.6
β
CONH2
β
CONH2
β
CH2CONH2
39.2 ± 10.5
a Tideglusib was used as positive control. The reported IC50 was 1.55 µM,[8] which is in consistent with
the value measured in the current study. bDose-response curves of compounds Tideglusib, 9, 17, 23, 24, 25,
27, 28 and 29 are shown in Fig. S1. c Inhibition less than 25% at 50 µM.
5

IC50 = 0.045 ± 0.007 μM
100
80
60
40
20
0
1E-4
0.001
0.01
0.1
1
Compound 26
(µM
)
Fig. 1. Dose-response curve of 26 against SARS-CoV-2 3CLpro
2.3. Molecular docking study
To examine the potential binding mode of these compounds, we built the complex model structure of
26 and SARS-CoV-2 3CLpro using an induce-fit docking procedure. As shown in Fig. 2, 26 fits snugly
into the substrate-binding pocket. The carboxamide at C-5 makes H-bonds with the side-chain carboxyl
groups of Asn142 and Glu166, and the oxygens at C-2 and C-3 form H-bonds with the main-chain ami-
no group of Cys145. The naphthyl ring fits into the hydrophobic groove formed by Met49 and Met165
and forms π-π stacking interaction with His41.
6

Fig. 2. The complex structure of compound 26 and SARS-CoV-2 3CLpro from docking study. The pro-
tein was shown in ribbons, and the compound 26 (yellow) and interacting residues in stick.
Combing the information from structure-activity relationship and docking analysis, we conclude that:
(1) The carboxamide group at C-5 is essential for inhibition activity. Even adding one carbon between
the carboxamide group and the isatin ring (29) was not tolerated. Replacing the carboxamide with car-
boxylic acid or ester group would destroy the hydrogen bond with Asn142 and bring in charge repul-
sion, making them unsuitable for binding. (2) The N-1 position of the isatin ring favors hydrophobic ar-
omatic substituents. Aromatic group like naphthalene (26) or benzothiophene (23) made the molecule
more rigid, fit into the pocket well and lead to high inhibition activity. Large substituents in the naphtha-
lene ring significantly reduced activity (28).
3. Conclusion
In conclusion, we have tested the inhibition activity of 29 N-substituted isatin derivatives against
SARS-CoV-2 3CLpro. The most potent compound, 26, demonstrated an IC50 of 45 nM against SARS-
CoV-2 3CLpro, which is among the most potent SARS-CoV-2 3CLpro inhibitors known so far. We found
that isatin compounds with a carboxamide substitution at the C-5 position and aromatic substitution at
N-1 position are strong inhibitors of SARS-CoV-2 3CLpro. As these compounds also inhibit SARS-CoV
3CLpro, other known SARS-CoV 3CLpro inhibitors can be further tested for their inhibition activity
against SARS-CoV-2 3CLpro. The active compounds have relatively high cytotoxicity that hinder quan-
titatively measurement of their anti-SARS-CoV-2 activity (as shown in Fig. S2). Further optimization to
reduce the cytotoxicity and increase the cellular activity is necessary to develop this series of com-
pounds as broad-spectrum anti-coronavirus drugs.
4. Experimental section
4.1 Synthesis
Melting points were obtained using an X4 apparatus and are uncorrected. Yields refer to isolated
products. 1H NMR (400 MHz or 600 MHz) and 13C NMR (126 MHz or 151 MHz) spectra were meas-
ured on a Brucker APX 400 and Bruker-500 spectrometer using TMS as internal standard. The chemical
shift values (δ
) are reported in ppm relative to tetramethylsilane internal standard. 1H NMR spectra are
represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t=triplet, q = quartet, br =
broad, m = multiplet), coupling constant (J values) in Hz and integration. All reactions were monitored
by thin layer chromatography (TLC), carried out on silica gel 60 F-254 aluminum sheets using UV, light
(254 and 366 nm). The reagents and solvents were available commercially and purified according to
conventional methods.
4.1.1 General procedure of the synthesis of compounds 1-28
7

4.1.1.1. Synthesis of I. 2-((4-carbamoylphenyl)amino)-2-oxoacetimidic acid (I-26). Solution A: A 500
mL, three-necked, round-bottomed flask fitted with a condenser and a thermometer was charged with
12.4 g (0.178 mol) of hydroxylamine hydrochloride, 52 g (0.37 mol) of anhydrous sodium sulfate, 14 g
(0.085 mol) of chloral hydrate and 230 mL of water. To aid dissolution, the mixture was heated to ap-
proximately 70°C and stirred vigorously with the help of a mechanical stirrer. Solution B: 9.52 g (0.07
mol) of 4-aminobenzamide was added dropwise slowly into a 50-mL round-bottomed flask containing a
vigorously stirred mixture of 15 mL of water and 7.5 mL of concentrated sulfuric acid. Solution B was
added in one portion to solution A. The mixture was vigorously stirred and heated to reflux. After cool-
ing to room temperature the precipitate was collected by filtration and washed with ice-cold water.
12.31 g (85%) of product was obtained. The crude product is sufficiently pure for the next step.
4.1.1.2. Synthesis of II. 2,3-dioxoindoline-5-carboxamide (II-26). A 50-mL, two-necked, round-
bottomed flask fitted with a thermometer was charged with 10 mL of concentrated sulfuric acid. After
heating and stirring to 70°C, 10 g (0.048 mol) of I-26 was added slowly to keep the temperature at
70°C. The resulting deep red solution was heated to 90°C for 30 min and then was cooled to room tem-
perature (20°C) over an ice bath. The mixture was then added rapidly to a vigorously stirred mixture of
100m L of ice water and 20 mL of ethyl acetate. The organic phase was separated and the aqueous phase
was extracted twice with 250 mL of ethyl acetate. The combined red organic phases were dried with so-
dium sulfate. The solvent was removed under reduced pressure and the crude product was purified by
column chromatography. 0.092 g (1 %) of an orange powder was obtained.
4.1.1.3. Synthesis of 1-26. 1-(naphthalen-2-ylmethyl)-2,3-dioxoindoline-5-carboxamide (26). A mixture
of II-26 (0.092 g, 0.48 mmol), 2-(Bromomethyl)naphthalene (0.117 g, 0.53mmol) and K2CO3 (0.166 g,
1.20 mmol) in 40 mL of acetonitrile was heating to reflux for 10 h. After the reaction is completed
monitored by TLC. The solution was evaporated and purified by column chromatography. 0.079 g
(50 %) of an orange powder is obtained. m.p.: 290-292
℃ δ 8.10 (d, J
. 1H NMR (400 MHz, DMSO-d6)
= 1.8 Hz, 1H), 8.07 (dd, J = 8.2, 1.8 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.91 (dd, J = 9.0, 4.8 Hz, 2H),
7.88 – 7.81 (m, 1H), 7.61 – 7.54 (m, 1H), 7.54 – 7.46 (m, 2H), 7.42 (s, 1H), 7.06 (d, J = 8.3 Hz, 1H),
5.11 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
δ 183.08, 166.71, 152.83, 137.71, 133.35, 133.3, 132.82,
129.60, 128.79, 128.06, 126.82, 126.50, 126.18, 125.96, 123.73, 118.16, 111.17, 43.76. HRMS (ESI+)
calcd for C20H15N2O3 [M + H]+ : 331.1004, found: 331.1077.
4.1.1.4. 1-(benzo[b]thiophen-2-ylmethyl)-2,3-dioxoindoline-5-carboxamide
m.p.: 210-212 8.16 (dd, J = 8.3, 1.8 Hz, 1H), 8.11 (d, J = 1.7 Hz,
. 1H NMR (600 MHz, DMSO-d6)
1H), 8.05 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.80 – 7.74 (m, 1H), 7.59 (s, 1H), 7.43 (s, 1H), 7.34 (m, 3H),
5.25 (s, 2H). 13C NMR (151 MHz, DMSO-d6)
182.74, 166.68, 158.83, 152.34, 139.66, 139.53,
(23). An orange powder.
℃
δ
δ
8

139.03, 137.87, 129.83, 125.04, 125.00, 124.05, 123.99, 123.88, 122.98, 118.06, 111.19, 39.51. HRMS
(ESI+) calcd for C18H13N2O3S [M + H]+ : 337.0569, found: 337.0641.
4.1.1.5. 1-((6-bromonaphthalen-2-yl)methyl)-2,3-dioxoindoline-5-carboxamide (27). An orange pow-
der. m.p.: 269-271
℃ δ 8.19 (d, J = 1.9 Hz, 1H), 8.11 (d, J
. Yield: 35%. 1H NMR (400 MHz, DMSO-d6)
= 1.8 Hz, 1H), 8.07 (dd, J = 8.3, 1.9 Hz, 1H), 8.03 (s, 2H), 7.91 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.8 Hz,
1H), 7.62 (dd, J = 8.9, 1.9 Hz, 2H), 7.40 (s, 1H), 7.05 (d, J = 8.3 Hz, 1H), 5.10 (s, 2H). 13C NMR (101
MHz, DMSO-d6) δ 183.02, 166.71, 159.37, 152.75, 137.70, 134.12, 133.99, 131.86, 130.32, 129.95,
129.79, 129.60, 128.03, 127.18, 126.13, 123.74, 119.62, 118.20, 111.11, 43.65. HRMS (ESI+) calcd for
C20H14BrN2O3 [M + H]+ : 409.0110, found: 409.0182.
4.1.1.6. 2,3-dioxo-1-((6-phenylnaphthalen-2-yl)methyl)indoline-5-carboxamide (28). An orange powder.
m.p.: 263-265
℃ δ 8.21 (d, J = 1.8 Hz, 1H), 8.11 (d, J =
. Yield: 12%. 1H NMR (400 MHz, DMSO-d6)
1.9 Hz, 1H), 8.08 (dd, J = 8.2, 1.9 Hz, 1H), 8.05 – 7.92 (m, 4H), 7.83 (m, 3H), 7.60 (dd, J = 8.5, 1.8 Hz,
1H), 7.51 (t, J = 7.7 Hz, 2H), 7.44 – 7.35 (m, 2H), 7.07 (d, J = 8.3 Hz, 1H), 5.13 (s, 2H). 13C NMR (101
MHz, DMSO-d6)
129.49 , 129.24 , 128.78 , 128.06 , 127.40 , 126.43 , 125.95 , 125.54 , 123.74 , 118.18 , 111.19 , 43.78 .
13C NMR (101 MHz, DMSO-d6)
183.09, 166.72, 159.37, 152.83, 140.30, 138.03, 137.71, 133.49,
δ 166.72 , 159.37 , 152.83 , 140.30 , 138.03 , 137.71 , 133.49 , 133.17 , 132.58 ,
δ
133.17, 132.58, 129.59, 129.49, 129.24, 128.78, 128.06, 127.40, 126.43, 125.95, 125.54, 123.74,
118.18, 111.19, 43.78. HRMS (ESI+) calcd for C26H19N2O3 [M + H]+ : 407.1317, found: 407.1390.
4.1.2. Synthesis of compound 29.
4.1.2.1. Methyl 2-(2,3-dioxoindolin-5-yl)acetate (III-29). To a stirred solution of methanol (50 ml) were
added 2-(2,3-dioxoindolin-5-yl)acetic acid II-29 (2g, 9.7 mmol), 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (2.22 g, 11.6 mmol) and 4-dimethylaminopyridine
(0.061 g, 0.5 mmol) at 25
℃. After stirring for 3h, the reaction was quenched with 20ml water. The mix-
ture was extracted with EtOAc and the organic layer was washed with brine, dried over Na2SO4, fil-
tered, and concentrated. 1.46 g (70%) of product is obtained. The crude product is sufficiently pure for
the next step.
4.1.2.2. Methyl 2-(1-(naphthalen-2-ylmethyl)-2,3-dioxoindolin-5-yl)acetate (IV-29). A mixture of III-29
(1 g, 4.6 mmol), 2-(Bromomethyl)naphthalene (1.17 g, 5.3mmol) and K2CO3 (1.63 g, 11.8 mmol) in 100
mL of acetonitrile was heating to reflux for 10 h. After the reaction was completed monitored by TLC.
The solution was evaporated and purified by column chromatography. 1.062 g (63%) of a red powder is
obtained. 1H NMR (400 MHz, DMSO-d6)
δ 7.99 (s, 1H), 7.95 – 7.80 (m, 3H), 7.58 – 7.47 (m, 4H), 7.44
(dd, J = 8.0, 1.7 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 5.07 (s, 2H), 3.68 (s, 2H), 3.58 (s, 3H).
4.1.2.3. 2-(1-(naphthalen-2-ylmethyl)-2,3-dioxoindolin-5-yl)acetic acid (V-29). Methyl ester IV-29 (1g,
2.78 mmol) was dissolved in 100 ml methanol. NaOH (0.117 g, 2.92 mmol) was added to this solution.
9

The mixture was stirred at 55-60 °C for 1 h. After cooling to the room temperature, 20 ml 3 N HCl was
added. The precipitate was collected by filtration and washed with ice-cold water twice. 0.859 g (89%)
of an orange power is obtained.
4.1.2.4. 2-(1-(naphthalen-2-ylmethyl)-2,3-dioxoindolin-5-yl)acetamide (29). To a stirred solution of
EtOAc (5ml) and THF (5 ml) added compound V-29 (30 mg, 0.08mmol), POCl3 (110 mg, 0.71 mmol)
over an ice bath. The mixture was stirred for additional 30 min. Then the resulting solution was adder to
another mixture containing 5ml acetonitrile, 30% ammonium hydroxide (0.560 g, 10 mmol) and Et3N
(1.01 g, 10 mmol). After the reaction completed, the mixture was extracted with EtOAc and the organic
layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by
silica gel column chromatography to give the 29 (15 mg, 57%) as an orange powder. m.p.: 200-202
℃.
1H NMR (400 MHz, DMSO-d6)
δ
7.98 (s, 1H), 7.96 – 7.81 (m, 3H), 7.60 – 7.44 (m, 5H), 7.40 (dd, J =
8.2, 1.9 Hz, 1H), 6.92 (d, J = 8.1 Hz, 2H), 5.07 (s, 2H), 3.34 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
δ
172.94, 149.48, 139.36, 133.50, 133.35, 132.82, 130.85, 128.82, 128.08, 128.04, 126.81, 126.48,
126.22, 125.93, 118.16, 111.32, 43.65, 39.78. HRMS (ESI+) calcd for C21H17N2O3 [M + H]+ : 345.1161,
found: 345.1234.
4.2 Molecular Docking.
The crystal structure of SARS-CoV-2 3CLpro (PDB ID 6LU7) was used for molecular docking[8]. The
structures of the protein and 26 were prepared by Protein Preparation Wizard and LigPrep module, re-
spectively. The binding pocket was defined as a 20*20*20 Å3 cubic box centered to the centroid of
Cys145. Flexible docking was conducted using the Induced Fit Docking module in GLIDE. Standard
protocol and XP precision were used. Ten conformations were generated and scored by glidescore. The
best scored conformation was used for structural analysis. All the above mentioned modules were im-
plemented in Schrödinger version 2015-4 (Schrödinger software suite, L. L. C. New York, NY (2015).)
4.3 Biology
.
4.3.1 Cloning, expression and purification of SARS-CoV-2 3CLpro
.
The full-length gene encoding SARS-CoV-2 3CLpro was synthesized for Escherichia coli (E. coil) ex-
pression (Hienzyme Biotech). The expression and purification of SARS-CoV-2 3CLpro were carried out
using the reported protocol[15].
4.3.2 Enzyme assay
.
A colorimetric substrate Thr-Ser-Ala-Val-Leu-Gln-pNA (GL Biochemistry Ltd) and assay buffer (40
mM PBS, 100 mM NaCl, 1 mM EDTA, 0.1% Triton 100, pH 7.3) was used for the inhibition assay. To
evaluate the effects of compounds on SARS-CoV-2 3CL protease activity, compounds were first pre-
incubated with enzyme samples in the assay buffer for 30 min at room temperature. And then 10 µl of 2
10

mM substrate was added into the above system to the final concentration of 200 µM to initiate the reac-
tion. Increase in absorbance at 390 nm was recorded for 20 min at an interval of 30s with a kinetics
mode program using a plate reader (Synergy, Biotek). IC50 values were fitted with Hill1 function.
AUTHOR INFORMATION
Corresponding Author
#These authors contribute equally.
*Y.L.: e-mail, liuying@pku.edu.cn.
*L.L.: e-mail, lhlai@pku.edu.cn.
Acknowledgements
This work was supported in part by the National Natural Science Foundation of China
(21633001,21877003), and the Ministry of Science and Technology of China (2016YFA0502303).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
COVID-19, CoV infectious disease; 3CLpro, 3C-like protease; TLC, thin layer chromatography.
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• SARS-CoV-2 3C-like protease is a key target for drug discovery against COVID-19.
• N-substituted isatin compounds are potent SARS-CoV-2 3C-like protease inhibitors.
• Three compounds showed ten nanomolar level inhibitory potency.
• The most potent compound 26 demonstrates an IC50 of 45 nM.

Conflict of Interest Statement
The authors declare that they have no conflicts of interest to this work.