Discovery and optimization of 2-((1H-indol-3-yl)thio)-N-benzyl-acetamides as novel SARS-CoV-2 RdRp inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113622

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective for controlling this pandemic to date. In this study,

Full text of DOI:10.1016/j.ejmech.2021.113622

European Journal of Medicinal Chemistry 223 (2021) 113622
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery and optimization of 2-((1H-indol-3-yl)thio)-N-benzyl-
acetamides as novel SARS-CoV-2 RdRp inhibitors
Guo-Ning Zhang 1, Jianyuan Zhao 1, Quanjie Li 1, Minghua Wang, Mei Zhu, Juxian Wang**,
Shan Cen***, Yucheng Wang*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the
global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective
for controlling this pandemic to date. In this study, several 2-((indol-3-yl)thio)-N-benzyl-acetamides
were identified as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors. After a two-round
optimization, a new series of 2-((indol-3-yl)thio)-N-benzyl-acetamides was designed, synthesized, and
evaluated for SARS-CoV-2 RdRp inhibitory effect. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 were iden-
Received 13 January 2021
Received in revised form
20 May 2021
Accepted 4 June 2021
Available online 10 June 2021
tified as potent inhibitors with IC50 values of 3.35
3.76 0.79 M, and 1.11 0.05 M, respectively; the IC50 of remdesivir (control) was measured as
1.19 0.36 M. All of the compounds inhibited RNA synthesis by SARS-CoV-2 RdRp. The most potent
0.21
m
M, 4.55
0.2
m
M, 1.65
0.05 mM,
Keywords:
SARS-CoV-2
COVID-19
m
m
m
compound 6d5, which showed a stronger inhibitory activity against the human coronavirus HCoV-OC43
than remdesivir, is a promising candidate for further investigation.
© 2021 Elsevier Masson SAS. All rights reserved.
RdRp inhibitor
2-((Indol-3-yl)thio)-N-Benzyl-acetamides
1. Introduction
time [10], regardless of the availability of effective vaccines.
Therefore, there is an urgent need to identify novel and efficient
Humans are currently affected by the coronavirus disease
(COVID-19) pandemic, caused by the emergence of severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in
~160 million infections and more than 3.3 million deaths world-
wide (until May 13, 2021) [1]. To date, no specific antiviral drug has
been approved to treat the infection caused by this coronavirus.
Furthermore, the interim results of the largest clinical trial of anti-
COVID-19 drugs to date show that the four most promising
candidate therapies, namely remdesivir [2e4], hydroxychloroquine
anti-COVID-19 drugs.
SARS-CoV-2 belongs to the Coronaviridae family and is a
positive-sense single-stranded RNA betacoronavirus [11e13]. In
addition to SARS-CoV-2, six other human coronavirusesdHCoV-
229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respira-
tory syndrome coronavirus (SARS-CoV), and Middle East respira-
tory syndrome coronavirus (MERS-CoV)dhave been identified
previously [14,15]. HCoV-229E, HCoV-OC43, HCoV-NL63, and
HCoV-HKU1 usually only evoke mild cold-like symptoms [16,17];
hence, they are commonly used as drug screening tools for viral
infectious diseases [18].
RNA-dependent RNA polymerase (RdRp), which mediates the
transcription and replication of the RNA genome during infection, is
a crucial enzyme in the life cycle of most RNA viruses, except ret-
roviruses [19,20]. The fact that this enzyme has no human coun-
terpart, together with its essentiality for the life cycle of the virus,
improves its potential as a drug target for antiviral development.
Thus, this is a very active field of research and a hot topic for
pharmaceutical companies and academic research groups [21e25].
A number of antiviral drugs, such as sofosbuvir, favipiravir,
ribavirin, beclabuvir, and dasabuvir (Fig. 1), are in clinical use for
different viruses (e.g., hepatitis C [26e28], influenza [29], and
[5], lopinavir/ritonavir [6], and interferon-b1a [7], have little effect
on the 28-day mortality or hospitalization course of hospitalized
patients. Although vaccines are officially approved for the preven-
tion of COVID-19 and massive vaccination has begun in many
countries, infections are still increasing worldwide [8,9]. It is
believed that SARS-CoV-2 may persist among humans for a long
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: wangjuxian@imb.pumc.edu.cn (J. Wang), shancen@imb.pumc.
edu.cn (S. Cen), wangyucheng@imb.pumc.edu.cn (Y. Wang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113622
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Fig. 1. Structure of FDA-approved antivirals targeting RdRp.
respiratory syncytial [30] viruses) on the basis of their anti-RdRp
activity [31,32]. Further, dozens of RdRp inhibitors are undergoing
clinical trials, which were comprehensively summarized by Tian
et al. in January 2021 [33]. In addition to the approved indications,
most RdRp inhibitors have also been studied for the treatment of
other viral infections [34].
In our previous work, we have reported a series of 2-((1H-indol-
3-yl)thio)-N-phenyl-acetamides (Fig. 2) as novel dual inhibitors of
respiratory syncytial virus (RSV) and influenza A virus (IAV) with
sub-micromolar EC50 values; the mechanism study further sug-
gested that these compounds act as RdRp inhibitors [35,36].
Considering that the RdRp structure is highly conserved in RNA
viruses, these compounds were evaluated for their SARS-CoV-2
RdRp inhibitory effect in a cell-based Gaussia luciferase (Gluc) re-
porter assay. Notably, most compounds showed moderate inhibi-
tory activity (15%e40%) at a concentration of 10 mM; however, an
increased inhibitory effect was observed when substituted aniline
was replaced with benzylamine (6a1) or 2-methoxybenzylamine
(6a5) (Fig. 2). Thus, a new series of 2-((indol-3-yl)thio)-N-benzyl-
acetamides was synthesized and evaluated for its SARS-CoV-2
RdRp inhibitory effect in the Gluc reporter system. After a two-
round optimization, compounds 6b2, 6b5, 6c9, 6d2, and 6d5
were identified as potent RdRp inhibitors with IC50 values of
3.35 0.21 mM, 4.55 0.2 mM, 1.65 0.05 mM, 3.76 0.79 mM, and
Fig. 2. Previously identified SARS-CoV-2 RdRp inhibitors and newly designed compounds.
2

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
1.11
0.05
m
M, respectively; the IC50 of the positive control
values [35,36]. After the outbreak of COVID-19, we screened these
compounds and found that several 2-((indol-3-yl)thio) acetamides
remdesivir was measured as 1.19 0.36
mM. All the selected com-
pounds 6b2, 6b5, 6c9, 6d2, and 6d5 showed dose-dependent in-
hibition of SARS-CoV-2 RdRp, and the activity was comparable to
that of remdesivir. They inhibit the efficiency of SARS-CoV-2 RdRp-
mediated RNA synthesis and diminish both plus- and minus-strand
Gluc RNA level more potently than remdesivir. All the tested
compounds showed good tolerance to nps14/nsp10 exoribonucle-
ase, which is necessary for their efficacy in vivo. Finally, compound
6d5 was selected to evaluate its antiviral effect against HCoV-OC43
and HCoV-NL63; it was identified as the most potent candidate
with a stronger inhibitory activity than remdesivir in vivo.
moderately inhibited SARS-CoV-2 RdRp at 10 mM. When
substituted aniline, at the right part of the molecule, was replaced
with a benzylanine, increased potency of the compounds was
observed (Fig. 2). Thus, a new series of 2-((indol-3-yl)thio)-N-
benzyl-acetamides was designed. Compounds 6a1-6, 6b1-7, and
6c1-8 were prepared via the substitution of ethyl acetate-2-sodium
thiosulfate with indole (or methyl-/bromo-substituted indole),
followed by hydrolysis and coupling with a series of benzylamines.
Subsequently, these compounds were tested for their SARS-
CoV-2 RdRp inhibitory effect in a Gluc reporter system specif-
ically initiated by the coronavirus RdRp, utilizing remdesivir as the
positive control. The IC50 values of compounds 6a1-6, 6b1-7, and
6c1-8 against SARS-COV-2 RdRp are listed in Table 1. The cytotox-
icity of these compounds was also tested under the same
conditions.
2. Results and discussion
The synthetic schemes for compounds 6a-d are illustrated in
Scheme 1. Briefly, ethyl bromoacetate 1 was substituted with so-
dium thiosulfate to form the Bunte salt ethyl acetate-2-sodium
thiosulfate 2, followed by substitution at the 3-position of indole
(or substituted indole) and hydrolysis to yield a key intermediate 5.
Finally, this intermediate was coupled with different substituted
benzylamines to afford the target products 6a-d. Compound 6c9
was also generated via Suzuki-Miyaura cross-coupling of 6c8 with
phenylboronic acid. All the target compounds were obtained in
high purity (>95%) via separation using a flash column. They were
fully characterized through NMR and high-resolution mass spec-
trometry. Details of the chemical synthesis and structural charac-
terization can be found in the Materials and Experimental Details
section. The 1H and 13C NMR spectra of 6a-d are presented in the
supplementary material.
As shown in Table 1, five out of the six compounds in the series
6a1-6 showed moderate SARS-CoV-2 RdRp inhibitory activity
(45%e76%) at 10
mM, and the IC50 value of the most potent com-
pound 6a6 was measured to be 7.50 1.68
mM. Compound 6a1 was
the simplest in the series and showed moderate inhibitory activity.
After the introduction of another benzyl group to the nitrogen atom
in the amide, the RdRp inhibitory activity was enhanced, as shown
by the IC50 values of 6a1 (26.92 mM) vs. 6a2 (12.30 mM). However,
after introducing an electron-withdrawing trifluoromethyl group at
the 4-position of the benzyl ring, the activity was completely lost
(6a3 vs. 6a1). The substitution with O-alkylated groups (e.g.,
methoxyl and dimethylaminoethoxy) or chlorine atom on the
benzyl ring is also beneficial for the SARS-CoV-2 RdRp inhibitory
effect (6a4-6 vs. 6a1).
When the starting material N-methyl indole was replaced by
indole, compounds in the series 6b1-7 were obtained. After the
removal of the N-methyl group, the RdRp inhibitory activity
significantly increased; compounds 6b1-2 were four times more
potent than 6a1-2, as indicated by their IC50 values. Similar to 6a3,
6b3 exhibited a significantly high inhibitory effect. These results
2.1. Design, synthesis, and structure-activity relationship research
of compounds 6a1-6, 6b1-7, and 6c1-8
In our previous work, we discovered that 2-((1H-indol-3-yl)
thio)-N-phenyl-acetamide specifically targets the RdRp of both RSV
and IAV, resulting in replication inhibition at sub-micromolar EC50
Scheme 1. General synthetic scheme for compounds 6a-d. Reagents and conditions: (a) Na2S2O3, MeOH/H2O; (b) Iodine, DMSO, 60 ꢀC; (c) NaOH, EtOH/H2O; (d) HATU, DIEA,
dichloromethane; (e) Phenylboronic acid, Pd2(dba)3, Sphos, cesium Carbonate, 1,4-dioxane.
3

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Table 1
Structure, log P value, and SARS-CoV-2 RdRp inhibitory activity of compounds 6a1-6, 6b1-6, and 6c1-8..
Compounds
6a1
R1
R2
Log Pa
3.09
Anti-SARS-CoV-2 RdRp IC50
(
m
M)
CC50 (m
M)b
b
-
26.92 3.54
95.50
b
6a2
-
4.74
12.3 1.40
91.20
b
6a3
6a4
6a5
-
3.99
3.18
3.10
NAc
>100
>100
>100
b
-
8.91 0.87
9.82 0.96
b
-
b
6a6
-
4.38
7.50 1.68
>100
6b1
6b2
H
H
3.03
4.67
6.81 1.03
3.35 0.21
>100
>100
6b3
6b4
H
H
3.92
3.11
7.94 1.02
9.08 1.38
>100
>100
6b5
6b6
H
H
3.27
3.28
4.55 0.23
7.64 0.54
>100
>100
6b7
6c1
6c2
6c3
H
3.58
3.80
5.07
3.82
6.68 0.86
8.71 0.33
7.21 0.59
10.72 1.11
>100
>100
>100
>100
4-Br
4-Br
5-Br
6c4
6c5
5-Br
6-Br
5.09
3.82
9.77 0.46
>100
>100
10.01 0.95
6c6
6-Br
5.09
8.71 0.63
67.61
4

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Table 1 (continued)
Compounds
6c7
R1
R2
Log Pa
3.80
Anti-SARS-CoV-2 RdRp IC50
(
m
M)
CC50 (m
M)b
7-Br
7.08 0.65
72.44
6c8
7-Br
5.07
2.82
4.55 0.25
1.56 0.12
>100
>100
Remdesivir
e
e
All data are expressed as mean SD of triplicate assays.
a
Log P ¼ Predicted octanol/water partition coefficient. The log P values were predicted using molinspiration (https://www.molinspiration.com/services/logp.html).
b
The half-maximal cytotoxic concentration (CC50) for each compound was calculated by comparing the viability of compounds-treated cells with that of DMSO-treated cells
in cell countingkit-8 (CCK-8) assay.
c
NA: not active.
indicate that the -NH- fragment is beneficial to their SARS-CoV-2
RdRp inhibitory activity, and this may be attributed to a hydrogen
bond interaction between the ligand and receptor. Furthermore,
the addition of another methyl or benzyl group at the nitrogen
atom of amide slightly enhanced SARS-CoV-2 RdRp inhibitory ac-
most promising candidate with a lower IC50 value than remdesivir
and an excellent safety index.
2.3. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 inhibit RNA synthesis
by SARS-CoV-2 RdRp
tivity, as indicated by the IC50 values of 6b1 (6.81
mM) vs. 6b2
(3.31 M) or 6b5 (4.55 M). Meanwhile, a methyl group introduced
m
m
to the methylene group of benzylamine caused negligible effects on
SARS-CoV-2 RdRp inhibitory activity, as evidenced by the IC50
values of 6b1 (6.81 mM) and 6b7 (6.68 mM).
In our previous study [35], we found that a bromine atom
introduced on the indole ring can significantly increase the RdRp
inhibitory effects of compounds in this series against IAV and RSV;
thus, derivatives with a bromine substitution (6c1-8) were also
designed and synthesized. All of these compounds exhibited potent
inhibitory effects, and their IC50 values were in the range of
After identifying the most potent SARS-CoV-2 RdRp inhibitors in
the CoV-RdRp-Gluc reporter assay, we decided to evaluate whether
they could also inhibit the efficiency of RNA synthesis by SARS-CoV-
2 RdRp. Treatment with 5
6b5, 6c9, 6d2, 6d5, or remdesivir decreased the levels of viral plus-
and minus-strand Gluc RNA in dose-dependent manner
mM and 10 mM of the compounds 6b2,
a
(Fig. 4AeF). Compounds 6b5, 6c9, and 6d5 diminished the levels of
both plus- and minus-strand Gluc RNA more potently than the
positive control (remdesivir). These data confirm that 6b2, 6b5,
6c9, 6d2, and 6d5 act as SARS-CoV-2 RdRp inhibitors.
4.55e10.72 mM. Among these brominated compounds, 6c1-2 and
6c7-8 displayed a more potent RdRp inhibitory activity than 6c3-6,
suggesting that bromine substitution at position 4- or 7- is highly
suitable for their interaction with RdRp.
2.4. 6b2, 6b5, 6c9, 6d2, and 6d5 are resistant to proofreading
activity of nsp14/nsp10
2.2. Design, synthesis, and SAR research of compounds 6c9 and
6d1-5
Coronavirus nonstructural protein 14 (nsp14) exoribonuclease
in complex with its activator nonstructural protein 10 (nsp10)
performs a proofreading function during coronavirus replication. It
can excise erroneous mutagenic nucleotides incorporated by nsp12
into viral RNA. Ribavirin was reported to be excised from the
nascent RNA strand of CoVs and showed limited efficacy in vivo
[37]. To confirm whether compounds 6b2, 6b5, 6c9, 6d2, and 6d5
are resistant to the proofreading activity of exoribonuclease, we co-
expressed nsp14 and nsp10 into the cell-based CoV-RdRp-Gluc
system. Gluc activity of these compounds slightly declined in the
presence of nsp14 and nsp10. All the compounds tested showed
good tolerance of nps14/nsp10 with slightly increased IC50 values
(Fig. 5 and Table 3), when compared with the data in Fig. 4. The IC50
values for 6b2, 6b5, 6c9, 6d2, and 6d5 increased to a lesser extent
than that of remdesivir, indicating that these compounds are more
tolerant to CoV exoribonuclease. Compounds 6c9 and 6d5 were
identified as the most promising inhibitors against SARS-CoV-2
To obtain highly efficacious SARS-CoV-2 RdRp inhibitors, com-
pounds 6d1-5 were designed and synthesized using 4-/7-
brominated indole and substituted benzylamine as the starting
materials in the second-round optimization. Meanwhile, 6c9 was
also synthesized to confirm whether another conjugation of ben-
zene ring with indole benefits RdRp inhibition.
After the synthesis of these derivatives, their SARS-CoV-2 RdRp
inhibition activities were assayed as described above. As listed in
Table 2 and depicted in Fig. 3, 6c9 shows significantly increased
SARS-CoV-2 RdRp inhibitory activity (IC50 ¼ 1.65
0.05 mM),
indicating that it could act as a lead compound for the development
of a novel SARS-CoV-2 RdRp inhibitor with high potency and
improved drug-likeness properties. Derivatives 6d1-4 were
designed based on the SAR obtained in the first-round optimiza-
tion, and most of them exhibited good activities except 6d3. These
results also proved that di-substitution on the amide nitrogen atom
is beneficial for RdRp inhibition. To improve the drug-likeness
properties of 6d1-2, another oxygen atom and ethyoxyl were
introduced into the molecule and thus compound 6d5 was ob-
tained. Notably, it was identified as the most potent SARS-CoV-2
RdRp; their IC50 values were 2.18
mM and 1.89 mM, respectively, in
the presence of nsp10/nsp14 exoribonuclease.
In our previous study [38], we suggested that nsp10/nsp14
exoribonuclease must be included in the cell-based CoV-RdRp-Gluc
assay to identify effective RdRp inhibitors, as the exoribonuclease
complex nsp14/nsp10 renders SARS-CoV-2 RdRp resistant to NA
inhibitors. Ribavirin can be excised by exoribonuclease from the
nascent RNA strand of CoVs to diminish its antiviral effect. Com-
pounds 6b2, 6b5, 6c9, 6d2, and 6d5 showed better tolerance to
nps14/nsp10 than remdesivir, indicating that these compounds
may be more potent inhibitors of SARS-CoV-2 than remdesivir.
RdRp inhibitor with an IC50 value of 1.11
acceptable log P value of 4.38.
As illustrated in Fig. 3, all the selected compounds 6b2, 6b5, 6c9,
6d2, and 6d5 showed dose-dependent inhibition of SARS-CoV-2
RdRp, and the activity was comparable to that of remdesivir, as
indicated by the IC50 values. Compound 6d5 was identified as the
0.05 mM and an
5

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Table 2
Structure, log P value, and SARS-CoV-2 RdRp inhibitory activity of compounds 6c9 and 6d1-5.
Compounds
6c9
R1
R2
Log Pa
4.78
Anti-SARS-COV-2 RdRp IC50
(m
M)
CC50 (m
M)b
7-Ph
1.65 0.05
95.52 3.11
6d1
6d2
4-Br
4-Br
4.03
5.43
4.73 0.67
3.76 0.79
25.12
25.7
6d3
6d4
7-Br
7-Br
4.03
5.43
20.89 2.68
7.08 0.32
52.48
>100
6d5
4-Br
4.38
2.82
1.11 0.05
1.19 0.36
>100
>100
Remdesivir
e
e
All data are expressed as mean SD of triplicate assays.
a
Log P ¼ Predicted octanol/water partition coefficient. The log P values were predicted using molinspiration (https://www.molinspiration.com/services/logp.html).
b
The half-maximal cytotoxic concentration (CC50) for each compound was calculated by comparing the viability of compounds-treated cells with that of DMSO-treated cells
in cell countingkit-8 (CCK-8, Beyotime) assay.
2.5. Evaluation of antiviral activity of 6d5 against HCoV-OC43 and
HCoV-NL63
palm and finger subdomains of SARS-CoV-2 RdRp. The hydrogen on
the indole nitrogen of 6d5 formed strong hydrogen bonds with
Asp760, a highly conserved catalytic residue that is required for
RNA synthesis. An H-bond interaction between the carbonyl oxy-
gen atoms of 6d5 and Cys622 was also observed. Moreover, the
Compound 6d5 was selected for evaluation of its antiviral effect
against the human coronavirus strains HCoV-OC43 and HCoV-
NL63, which are commonly used in antiviral drug screening, in a
cell-based assay in vitro. The data in Fig. 6 illustrate that replication
of HCoV-OC43 and HCoV-NL63 was effectively inhibited in a dose-
dependent manner after treatment with serially diluted 6d5. In
particular, 6d5 showed a more potent inhibitory effect than
remdesivir on HCoV-OC43 replication. Moreover, 6d5 exhibited a
higher antiviral activity against HCoV-OC43 (which belongs to the
benzene ring in 2-ethoxyaniline formed a
p-cation and an edge-to-
face interaction with residues Arg553 and Try445, respectively.
p-p
To gain further insight into the structure-activity relationships
of the 6d5 analogs, five 2-((1H-indol-3-yl)thio)-N-benzyl-acet-
amide derivatives (6a1, 6a2, 6b2, 6c7, and 6c9) that exhibited
diverse inhibitory activities were also docked to SARS-CoV-2 RdRp.
The calculated binding energies (DGADV) and predicted binding
poses of these derivatives are depicted in Fig. 8 and Fig. 9, respec-
tively. The values of DGADV were used to estimate the relative
binding affinity of the inhibitors. As seen in Fig. 8, there is a positive
linear relationship between DGADV of selected derivatives and their
corresponding experimental IC50 values against SARS-CoV-2 RdRp
(R2 ¼ 0.83), implying that the strong inhibitory effect of compounds
is associated with their high binding affinity to SARS-CoV-2 RdRp.
In addition, because of their common structural skeleton, it is
reasonable to assume that the binding sites of these derivatives are
similar to those of 6d5. As expected, all compounds presented a
similar arrangement within the central cavity of SARS-CoV-2 RdRp
(Fig. 9A).
same
coronavirus).
According to serology studies and genomic analysis, coronavirus
is divided into four genera: -coronavirus, -coronavirus, -coro-
navirus, and -coronavirus. HCoV-OC43 and SARS-CoV-2 are
members of the -coronavirus, whereas HCoV-NL63 is a member of
the -coronavirus. The higher antiviral activity of 6d5 against
b-coronavirus as SARS-CoV-2) than against HCoV-NL63 (an a-
a
b
g
d
b
a
HCoV-OC43 than against HCoV-NL63 implies that this compound
may act as a potent SARS-CoV-2 inhibitor.
2.6. Molecular modeling studies
Comparison of 6a1 and 6a2. As the simplest compound in the
series, 6a1 showed a moderate binding energy (ꢁ5.0 kcal/mol).
After the introduction of another benzyl group to the nitrogen atom
in the amide, a more favorable binding energy was obtained for 6a2
To explore the binding mode of identified inhibitors, molecular
docking study of the most potent compound 6d5 against SARS-
CoV-2 RdRp (PDB-ID: 6M7K) was performed using AutoDock Vina.
As shown in Fig. 7, the binding pocket of 6d5 is located in the
6

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Fig. 3. Dose-response inhibition curves of selected compounds 6b2, 6b5, 6c9, 6d2, and 6d5 against SARS-CoV-2 RdRp. HEK293T cells were transfected with CoV-Gluc, nsp12,
nsp7, and nsp8 plasmid DNA at the ratio of 1:10:30:30. At 12 h post-transfection, the cells were re-seeded in 96-well plates (104/well) and treated with serially diluted compounds
6b2 (A), 6b5 (B), 6c9 (C), 6d2 (D), 6d5 (E) and remdesivir (F). Gluc activity in the supernatants was measured. CC50 values in the cells were measured using CCK-8 kits. All data are
expressed as mean SD of triplicate assays.
(ꢁ6.1 kcal/mol). Although the H-bond interactions were lost, the
benzene ring of 6a2 contributed to additional hydrophobic in-
is essential for the life cycle of viruses, it a popular therapeutic drug
target pursued by pharmaceutical companies and academic
research groups. RdRp is considered a conserved protein within
RNA viruses; thus, scientists worldwide have repurposed preex-
isting drugs that target RdRp to identify candidates against SARS-
CoV-2. In this paper, we report the discovery of SARS-CoV-2 RdRp
inhibitors based on the structural optimization of the IAV and RSV
RdRp ligands. After a two-round optimization, compounds 6b2,
6b5, 6c9, 6d2, and 6d5 were identified as potent RdRp inhibitors
teractions with Phe793 and Met794. Additionally, strong
p-cation
interactions were observed between 6a2 and Arg553. These ob-
servations may explain why 6a2 had a stronger inhibitory effect
than 6a1.
Comparison of 6b2 and 6a2. The calculated binding energy of
6b2 was ꢁ7.1 kcal/mol, which is more favorable than that of 6a2.
The only structural difference between 6a2 and 6b2 is the sub-
stituent at the nitrogen atom of indole. As seen in Fig. 9C, there is an
additional H-bond between the -NH- fragment in the indole ring
and the carboxyl group of Asp623 and may account for the stronger
with IC50 values of 3.35 0.21
3.76 0.79 M, and 1.11 0.05
remdesivir (control) was measured as 1.19 0.36
m
m
M, 4.55 0.2
M, respectively, whereas the IC50 of
M. Compound
mM, 1.65 0.05 mM,
m
m
inhibitory activity of 6b2 (IC50 ¼ 3.35
m
M) than that of 6a2.
6d5 was identified as the most potent candidate, having a more
potent inhibitory activity than remdesivir against HCoV-OC43 in a
cell-based assay.
Comparison of 6c7 and 6c9. The only structural difference be-
tween 6c7 and 6c9 is the substituent on the benzene ring of indole.
When the bromine atom in 6c7 was replaced with a benzene ring,
additional hydrophobic interactions were formed between 6c9 and
palm subdomain residues Try800, Phe812, and Cys813. Conse-
quently, 6c9 exhibited more favorable binding energy (ꢁ7.2 kcal/
mol) than 6c7 (ꢁ6.1 kcal/mol). Thus, we speculated that the
introduction of a benzene ring at this position improves the
inhibitory effect of the compounds.
Molecular docking studies were carried out to explore the
binding mode and structure-activity relationships of 2-((1H-indol-
3-yl)thio)-N-benzyl-acetamide derivatives. As seen in Figs. 7A and
9A, the predicted binding sites of these inhibitors were located at
the palm and finger subdomains of SARS-CoV-2 RdRp. Specifically,
these active compounds interact with motif A residues D618-R624,
palm domain residues K794, S795, D760, and D761, and finger
domain residues D452, Y455, K551, and R553. Given that D618,
D760, and D761 are involved in binding two magnesium ions at the
catalytic center, the binding of these compounds may block the
conformational change required to coordinate the divalent cations
during the catalytic process. Notably, the strong correlation
3. Conclusion
RdRp is a crucial enzyme in the life cycle of most RNA viruses.
Considering that this enzyme has no human counterpart and that it
7

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Fig. 4. Inhibition of CoV-Gluc RNA expression by 6b2, 6b5, 6c9, 6d2, and 6d5. HEK293T cells were transfected with CoV-Gluc, nsp12, nsp7, and nsp8 plasmid DNA at the ratio of
1:10:30:30. At 6 h post-transfection, supernatants were replaced with fresh medium containing 6b2 (A), 6b5 (B), 6c9 (C), 6d2 (D), 6d5 (E), and remdesivir (F). The cells were
cultured for additional 24 h, total cellular RNA was extracted, and CoV-Gluc levels were determined via real-time qRT-PCR. Error bars indicate SD, **p < 0.01, ***p < 0.001.
Fig. 5. 6b2, 6b5, 6c9, 6d2, and 6d5 were resistant to the proofreading activity of nsp14/nsp10. HEK293T cells were co-transfected with CoV-Gluc, nsp12, nsp7, nsp8, nsp10, and
nsp14 plasmids at a ratio of 1:10:30:30:25:25. Gluc activity was determined using a cell-based assay. The IC50 values of 6b2 (A), 6b5 (B), 6c9 (C), 6d2 (D), 6d5 (E), and remdesivir (F)
were determined using a cell-based system with the nsp10/nsp14 proofreading function.
between the calculated binding energies and experimental IC50
values implies that the strong inhibitory effect of compounds is
associated with their high binding affinity to SARS-CoV-2 RdRp.
Structure-activity relationship analysis highlighted that the -NH-
fragment in the indole ring makes an important contribution to the
binding of RdRp through a hydrogen bond and that an additional
aromatic ring on the indole ring or the nitrogen in amide is
beneficial for the ligand-receptor interaction.
We often believe that drugs should be of nanomolar potency in
medicinal chemistry, and most approved drugs comply with this
“nanomolar rule”; however, there are a few exceptions such as
sofosbuvir, favipiravir, and ribavirin. Thus, further structural opti-
mization for the improvement of the activity is underway. The
current work is a successful attempt to identify new anti-SARS-
8

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
Table 3
IC50 values of 6b2, 6b5, 6c9, 6d2, and 6d5 for SARS-CoV-2 RdRp inhibitory activity with or without nsp14/nsp10.
Compounds
Nsp12 (IC50
/
m
M)a
Nsp12 þ 14/10 (IC50
/m
M)b
Ratio ((12 þ 14/10)/12)
6b2
6b5
6c9
6d2
6d5
Remdesivir
3.35
4.55
1.65
3.76
1.11
1.19
4.01
5.37
2.18
3.85
1.89
3.11
1.20
1.18
1.32
1.02
1.70
2.61
a
IC50 values of 6b2, 6b5, 6c9, 6d2, and 6d5 against SARS-CoV-2 RdRp without nsp14/nsp10 exoribonuclease.
IC50 values of 6b2, 6b5, 6c9, 6d2, and 6d5 against SARS-CoV-2 RdRp with nsp14/nsp104 exoribonuclease.
b
Fig. 6. Evaluation of antiviral activity of 6d5 against human coronavirus strains HCoV-OC43 and HCoV-NL63. HCT-8 and LLC-MK2 cells were infected by HCoV-OC43 (A) or
HCoV-NL63 (B) at a MOI (multiplicity of infection) of 0.1 and 0.01, respectively, and treated with serial dilutions of 6d5 and remdesivir at 1 h post-infection. The effect of the
treatments on cell viability was measured via MTS assay at 120 h post-infection.
Fig. 7. Binding pose analysis of compound 6d5. (A) Predicted structure of SARS-CoV-2 RdRp in complex with compound 6d5. (B) Key interactions between compound 6d5 and
SARS-CoV-2 RdRp. (C) Residues within 5 A of compound 6d5 are shown on the 2D ligand interaction diagram. For clarity, only polar hydrogen atoms are shown. Dark blue, red, and
green indicate positively charged, negatively charged, and hydrophobic residues, respectively. Hydrogen bonding is depicted as magenta arrows,
as red lines, and interactions are depicted as green lines.
p-cation interactions are depicted
p-p
CoV-2 candidates from other viral RdRp inhibitors.
RdRp inhibitors. They can inhibit the efficiency of RNA synthesis by
SARS-CoV-2 RdRp and diminish the level of both plus- and minus-
strand Gluc RNA more potently than the positive control remdesi-
vir. The most potent compound 6d5 showed stronger inhibitory
activity than remdesivir against HCoV-OC43 in cells and has been
In summary, a new series of 2-((indol-3-yl)thio)-N-benzyl-
acetamides was designed and synthesized based on the screening
results of our antiviral library and a two-round optimization, and
6b2, 6b5, 6c9, 6d2, and 6d5 were identified as potent SARS-CoV-2
9

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
4.2. Synthesis
4.2.1. Sodium S-(2-ethoxy-2-oxoethyl) sulfurothioate 2
To a solution of ethyl bromoacetate (5.5 mL, 50 mmol) in 75 mL
of methanol and 25 mL of H2O, sodium thiosulfate pentahydrate
(14.88g, 60 mmol) was added and the solution was stirred for 1h at
65 ꢀC. The solvent was evaporated under reduced pressure, and
50 mL of methanol and 50 mL of dichloromethane were added. The
mixture was washed with ultrasonic for 5 min and filtered with
suction. After concentration under reduced pressure, 10.86 g of
sodium S-(2-ethoxy-2-oxoethyl) sulfurothioate 2 was obtained as a
light-yellow solid which was used directly for the next step.
1H NMR (400 MHz, Methanol-d4)
d
4.19 (q, J ¼ 7.1 Hz, 2H), 3.84
(s, 2H), 1.28 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, Methanol-d4)
d
62.8, 37.5, 14.4. ESI-MS(m/z): 220.8 [M ꢁ Na]-.
Fig. 8. Correlation of calculated binding energies of selected compounds with their
corresponding experimental IC50 values against SARS-CoV-2 RdRp.
4.2.2. General procedure for the synthesis of 5a-f
To a solution of indole/substituted indole (49 mmol) and So-
dium S-(2-ethoxy-2-oxoethyl) sulfurothioate 2 (10.86g, 49 mmol)
in DMSO, iodine (1.42g, 5.6 mmol) was added. The solution was
stirred for 30min at 60 ꢀC and 200 mL of ethyl acetate was added
after the solution was cooled to 30 ꢀC. the mixture was washed with
100 mL of saturated sodium thiosulfate, 1000 mL of water, and
100 mL of saturated saline the organic phase was dried over
anhydrous sodium sulfate and concentrated under reduced pres-
sure. After purification on a silica column compounds 4a-f was
afforded.
To a solution of compounds 4a (5.31g, 25.62 mmol) in MeOH
and H2O (24 mL þ 6 mL), sodium hydroxide (1.23g, 30.75 mmol)
was added. The solution was stirred for 30min at 25 ꢀC and
extracted with ether. The pH of aqueous phase was adjusted to 3e4
and extracted with ethyl acetate (50 mL x 3). After removal of the
solvent under reduced pressure 5a was afforded (quantitative). The
synthesis of 5b-f was similar to that of 5a.
selected as a potential candidate for further investigation.
4. Materials and experimental details
4.1. Chemicals and instruments
All starting materials for chemical synthesis were purchased
from commercial suppliers (Shanghai Bide Pharmaceutical Tech-
nology Co., Ltd. & Beijing InnoChem Science & Technology Co., Ltd)
and used without further purification. NMR spectra were recorded
on Bruker Avance 400 MHz and 500 MHz spectrometers. LC-MS
and purity data were measured using an Agilent HPLC/MSD in-
strument. APCI high-resolution mass spectra (HRMS) were recor-
ded on an Autospec Ultima-TOF spectrometer.
Fig. 9. Predicted binding poses for five 2-((1H-indol-3-yl)thio)-N-benzyl-acetamides derivatives. (A) Superposition of binding sites of selected compounds. Color codes: 6a1
(yellow), 6a2 (blue), 6b2 (cyan), 6c7 (orange), 6c9 (green), and 6d5 (purple). (BeG) The 2D ligand interaction diagrams for selected compounds. For clarity, only polar hydrogen
atoms are shown. Dark blue, red, cyan and green indicate positively charged, negatively charged, polar, and hydrophobic residues, respectively. Hydrogen bonding is depicted as
magenta arrows,
p-cation interactions are depicted as red lines, and
p-
p
interactions are depicted as green lines.
10

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
4.2.3. 2-((1-Methyl-1H-indol-3-yl)thio)acetic acid 5a
4.2.11. N,N-dibenzyl-2-((1-methyl-1H-indol-3-yl)thio)acetamide
6a2
1H NMR (500 MHz, DMSO‑d6)
d
12.48 (s, 1H), 7.64 (d, J ¼ 7.9 Hz,
1H), 7.54 (s, 1H), 7.47 (d, J ¼ 8.2 Hz, 1H), 7.22 (t, J ¼ 7.6 Hz, 1H), 7.14
Purity: 96.6%.
(t, J ¼ 7.5 Hz, 1H), 3.78 (s, 3H), 3.38 (s, 2H). 13C NMR (126 MHz,
1H NMR (400 MHz, DMSO‑d6)
d
7.48 (dt, J ¼ 8.2, 0.9 Hz, 1H), 7.45
DMSO‑d6)
d
171.7, 137.3, 135.3, 129.6, 122.4, 120.3, 119.1, 110.8, 101.9,
(s, 1H), 7.37e7.25 (m, 6H), 7.25e7.21 (m, 1H), 7.19 (dt, J ¼ 8.0, 1.2 Hz,
2H), 7.14e7.05 (m, 3H), 4.45 (s, 2H), 4.42 (s, 2H), 3.76 (s, 3H), 3.67 (s,
39.4, 33.1.ESI-MS (m/z):220.2 [M ꢁ H]-.
2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.5, 137.8, 137.3, 135.3, 129.1,
128.9, 128.2, 127.8, 127.6, 127.0, 122.4, 120.3, 119.0, 110.8, 50.7, 48.6,
38.8, 33.1. HRMS (ESI) m/z calcd. for C25H24N2OSNa ([M þ Na]þ):
423.1501, found 423.1501.
4.2.4. 2-((1H-indol-3-yl)thio)acetic acid 5b
1H NMR (400 MHz, DMSO‑d6)
d
11.41 (s, 2H), 7.62 (dd, J ¼ 7.8,
1.1 Hz, 2H), 7.52 (d, J ¼ 2.6 Hz, 2H), 7.41 (dt, J ¼ 8.1, 0.9 Hz, 2H), 7.15
(ddd, J ¼ 8.1, 7.0, 1.4 Hz, 2H), 7.09 (ddd, J ¼ 8.0, 7.0, 1.2 Hz, 2H), 2.50
4.2.12. 2-((1-Methyl-1H-indol-3-yl)thio)-N-(4-(trifluoromethyl)
benzyl)acetamide 6a3
(p, J ¼ 1.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 171.23, 136.27,
130.92, 128.70, 121.79, 119.66, 118.40, 112.05, 102.37, 38.77. ESI-MS
Purity: 99.6%.
(m/z):206.2 [M ꢁ H]-.
1H NMR (400 MHz, DMSO‑d6)
d
8.49 (t, J ¼ 6.0 Hz, 1H), 7.66e7.58
(m, 3H), 7.48 (s, 2H), 7.36e7.28 (m, 2H), 7.22 (ddd, J ¼ 8.2, 7.0,1.2 Hz,
4.2.5. 2-((4-Bromo-1H-indol-3-yl)thio)acetic acid 5c
1H), 7.12 (ddd, J ¼ 8.0, 7.0,1.0 Hz,1H), 4.30 (d, J ¼ 5.9 Hz, 2H), 3.75 (s,
1H NMR (500 MHz, DMSO‑d6)
d 12.43 (s, 0H), 11.73 (s, 1H), 7.58
3H), 3.36 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.3, 144.6, 137.3,
(d, J ¼ 2.6 Hz,1H), 7.45 (d, J ¼ 8.1 Hz,1H), 7.27 (d, J ¼ 7.5 Hz,1H), 7.05
135.2, 129.7, 128.3, 125.5, 122.3, 120.2, 119.2, 110.7, 102.1, 42.4, 40.5,
33.0. HRMS (ESI) m/z calcd. for C19H17F3N2OSNa ([M þ Na]þ):
401.0906, found 401.0894.
(t, J ¼ 7.8 Hz, 1H), 3.47 (s, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 171.1,
138.3, 133.6, 125.8, 124.7, 123.5, 113.3, 112.5, 103.3, 40.9.ESI-MS (m/
z):284.1 [M ꢁ H]-.
4.2.13. N-(4-(2-(dimethylamino)ethoxy)benzyl)-2-((1-methyl-1H-
indol-3-yl)thio)acetamide 6a4
4.2.6. 2-((5-Bromo-1H-indol-3-yl)thio)acetic acid 5d
1H NMR (500 MHz, DMSO‑d6)
d 12.49 (s, 1H), 11.65 (s, 1H), 7.79
Purity: 96.0%.
1H NMR (500 MHz, DMSO‑d6)
d
8.27 (t, J ¼ 5.9 Hz, 1H), 7.62 (d,
(d, J ¼ 2.0 Hz, 1H), 7.63 (d, J ¼ 2.6 Hz, 1H), 7.42 (d, J ¼ 8.6 Hz, 1H),
7.29 (dd, J ¼ 8.6, 1.9 Hz, 1H), 3.41 (s, 2H). 13C NMR (126 MHz,
J ¼ 7.9 Hz, 1H), 7.50e7.41 (m, 2H), 7.25e7.18 (m, 1H), 7.16e7.08 (m,
1H), 7.04 (d, J ¼ 8.5 Hz, 2H), 6.84 (d, J ¼ 8.6 Hz, 2H), 4.14 (d,
J ¼ 5.8 Hz, 2H), 4.04 (t, J ¼ 5.7 Hz, 2H), 3.75 (s, 3H), 3.18 (s, 2H), 2.73
(t, J ¼ 5.7 Hz, 2H), 2.30 (s, 6H). 13C NMR (126 MHz, DMSO‑d6)
DMSO‑d6) d 171.7, 135.5, 133.2, 131.2, 124.9, 121.2, 114.6, 113.0, 102.7,
39.3. ESI-MS (m/z):284.1 [M ꢁ H]-.
d
169.0, 157.8, 137.3, 135.2, 131.8, 129.8, 129.2, 122.3, 120.3, 119.3,
4.2.7. 2-((6-Bromo-1H-indol-3-yl)thio)acetic acid 5e
114.7, 110.7, 102.3, 65.8, 57.9, 45.7, 40.7, 33.1. HRMS (ESI) m/z calcd.
for C22H27N3O2SNa ([M þ Na]þ): 420.1716, found 420.1748.
1H NMR (500 MHz, DMSO‑d6)
d 12.55 (s, 1H), 11.54 (s, 1H),
7.63e7.54 (m, 3H), 7.23 (dd, J ¼ 8.4,1.7 Hz,1H), 3.38 (s, 2H). 13C NMR
(126 MHz, DMSO‑d6)
d 171.1, 163.0, 137.1, 132.0, 127.8, 122.6, 120.3,
4.2.14. N-(2-methoxybenzyl)-2-((1-methyl-1H-indol-3-yl)thio)
acetamide 6a5
114.6, 114.5, 103.0, 38.7. ESI-MS (m/z):284.1 [M ꢁ H]-.
Purity: 97.9%.
1H NMR (400 MHz, DMSO‑d6)
d 8.21e8.13 (m, 1H), 7.63 (ddd,
4.2.8. 2-((7-Bromo-1H-indol-3-yl)thio)acetic acid 5f
1H NMR (500 MHz, DMSO‑d6)
d 12.48 (s, 1H), 11.69 (s, 1H), 7.65
J ¼ 8.0, 2.2, 1.2 Hz, 1H), 7.51e7.42 (m, 2H), 7.22 (td, J ¼ 7.8, 1.2 Hz,
2H), 7.12 (tt, J ¼ 7.1, 1.1 Hz, 1H), 6.99e6.89 (m, 2H), 6.86e6.77 (m,
1H), 4.22e4.15 (m, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 3.39e3.35 (m, 2H).
(d, J ¼ 7.9 Hz,1H), 7.59 (d, J ¼ 2.7 Hz,1H), 7.39 (d, J ¼ 7.5 Hz,1H), 7.06
(t, J ¼ 7.7 Hz,1H). 13C NMR (126 MHz, DMSO‑d6)
d 171.6,135.0,132.5,
131.0, 124.9, 121.6, 118.6, 105.1, 104.6, 39.1. ESI-MS (m/z):284.2
13C NMR (101 MHz, DMSO‑d6)
d 169.1, 157.0, 137.3, 135.1, 128.4,
[M ꢁ H]-.
128.0, 126.9, 122.3, 120.5, 120.2, 119.2, 110.8, 110.7, 102.2, 55.7, 40.5,
37.9, 33.1. HRMS (ESI) m/z calcd. for C19H20N2O2SNa ([M þ Na]þ):
363.1137, found 363.1130.
4.2.9. General procedure for the synthesis of 6a1-6, 6b1-7, 6c1-8,
6d1-5, and 6c9
4.2.15. N-(3,4-dichlorobenzyl)-2-((1-methyl-1H-indol-3-yl)thio)
To a solution of compound 5 in dichloromethane (0.2 mmol/mL)
was added DIEA (2.4 eq.), HATU (2.2 eq.) and substituted benzyl
amine (1.1eq.). The solution was stirred for 1h at 25 ꢀC and diluted
with dichloromethane and washed with 30 mL of saturated sodium
bicarbonate, sodium bicarbonate, 0.5 N hydrochloric acid and
saturated sodium chloride solution in sequence. After purification
on a silica column (PE/EA ¼ 10:1e5:1) the target product was
afforded.
acetamide 6a6
Purity: 99.5%.
1H NMR (400 MHz, DMSO‑d6)
d
8.46 (t, J ¼ 6.0 Hz, 1H), 7.64e7.57
(m, 1H), 7.53 (d, J ¼ 8.3 Hz, 1H), 7.48 (dt, J ¼ 8.1, 0.9 Hz, 1H), 7.46 (s,
1H), 7.41 (d, J ¼ 2.0 Hz, 1H), 7.22 (ddd, J ¼ 8.2, 7.0, 1.2 Hz, 1H), 7.11
(ddd, J ¼ 8.2, 5.2, 1.5 Hz, 2H), 4.21 (d, J ¼ 5.9 Hz, 2H), 3.76 (s, 3H),
3.36e3.34 (m, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.4, 141.0,
137.3, 135.1, 131.2, 130.8, 129.7, 129.7, 128.1, 122.3, 120.2, 119.2, 110.8,
102.1, 41.8, 40.5, 33.1. HRMS (ESI) m/z calcd. for C18H16Cl2N2OS
([M þ Na]þ): 401.0252, found 401.0253.
4.2.10. N-benzyl-2-((1-methyl-1H-indol-3-yl)thio)acetamide 6a1
Purity: 95.7%.
1H NMR (400 MHz, DMSO‑d6)
d
8.34 (t, J ¼ 5.9 Hz, 1H), 7.62 (dt,
4.2.16. N-benzyl-2-((1H-indol-3-yl)thio)acetamide 6b1
J ¼ 7.8, 1.0 Hz, 1H), 7.51e7.46 (m, 1H), 7.45 (s, 1H), 7.31e7.17 (m, 4H),
Purity: 95.2%.
7.16e7.06 (m, 3H), 4.21 (d, J ¼ 5.9 Hz, 2H), 3.75 (s, 3H), 2.53e2.47
1H NMR (400 MHz, DMSO‑d6)
d
11.39 (s, 1H), 8.36 (t, J ¼ 5.9 Hz,
(m, 2H). 13C NMR (101 MHz, DMSO‑d6)
d
169.1, 139.6, 137.3, 135.2,
1H), 7.62 (dt, J ¼ 7.7, 1.0 Hz, 1H), 7.47 (d, J ¼ 2.6 Hz, 1H), 7.45e7.40
(m, 1H), 7.30e7.19 (m, 3H), 7.15 (ddd, J ¼ 8.2, 7.0, 1.3 Hz, 1H),
7.13e7.06 (m, 3H), 4.22 (d, J ¼ 5.9 Hz, 2H), 3.36 (s, 2H). 13C NMR
128.6, 127.7, 127.2, 122.3, 120.2, 119.3, 110.7, 102.2, 42.8, 39.5, 33.1.
HRMS (ESI) m/z calcd. for C18H18N2OSNa ([M þ Na]þ): 333.1032,
found 333.1045.
(101 MHz, DMSO‑d6)
d 169.1, 139.6, 136.7, 131.2, 129.3, 128.6, 127.6,
11

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
127.1, 122.2, 120.0, 119.0, 112.4, 103.2, 42.8, 40.5. HRMS (ESI) m/z
calcd. for C17H16N2OSNa ([M þ Na]þ): 319.0875, found 319.0872.
amides in the structure which was similar to that reported in
literature [39,40]. HRMS (ESI) m/z calcd. for C19H20N2O2SNa ([M þ
Na]þ): 363.1137, found 363.1145.
4.2.17. N,N-dibenzyl-2-((1H-indol-3-yl)thio)acetamide 6b2
Purity: 92.7%.
4.2.22. (R)-2-((1H-indol-3-yl)thio)-N-(1-phenylethyl)acetamide
1H NMR (400 MHz, DMSO‑d6)
d 11.41 (s, 1H), 7.58e7.51 (m, 1H),
6b7
7.47 (d, J ¼ 2.7 Hz, 1H), 7.45e7.41 (m, 1H), 7.37e7.24 (m, 6H),
1H NMR (400 MHz, DMSO‑d6)
d
11.37 (s, 1H), 8.22 (d, J ¼ 8.0 Hz,
7.21e7.11 (m, 3H), 7.11e7.02 (m, 3H), 4.44 (d, J ¼ 6.9 Hz, 4H), 3.67 (s,
1H), 7.60 (ddd, J ¼ 7.8, 1.4, 0.7 Hz, 1H), 7.45e7.37 (m, 2H), 7.32e7.23
(m, 2H), 7.25e7.15 (m, 3H), 7.14 (ddd, J ¼ 8.2, 7.0, 1.3 Hz, 1H), 7.07
(ddd, J ¼ 7.9, 7.0, 1.1 Hz, 1H), 4.83 (p, J ¼ 7.2 Hz, 1H), 3.33 (s, 2H), 1.22
2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.6, 137.8, 137.3, 136.8, 131.4,
129.2,129.1,128.9,128.1, 127.8, 127.5, 127.0, 122.3, 120.2,118.8, 112.5,
102.5, 50.8, 48.5, 38.7. HRMS (ESI) m/z calcd. for C24H22N2OSNa
([M þ Na]þ): 409.1345, found 409.1336.
(d, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 168.2, 144.8,
136.7,131.2,129.4,128.6,127.0,126.4,122.2,120.0,119.1,112.4,103.2,
48.4, 38.7, 22.6. HRMS (ESI) m/z calcd. for C18H18N2OSNa ([M þ
Na]þ): 333.1032, found 333.1041.
4.2.18. 2-((1H-indol-3-yl)thio)-N-(4-(trifluoromethyl)benzyl)
acetamide 6b3
Purity: 95.5%.
1H NMR (400 MHz, DMSO‑d6)
d
11.43 (s, 1H), 8.49 (t, J ¼ 6.0 Hz,
4.2.23. 2-((4-Bromo-1H-indol-3-yl)thio)-N-(2-methoxybenzyl)
acetamide 6c1
1H), 7.67e7.57 (m, 3H), 7.52 (d, J ¼ 2.6 Hz, 1H), 7.44 (dt, J ¼ 8.1,
Purity: 95.2%.
1.0 Hz, 1H), 7.32e7.24 (m, 2H), 7.16 (ddd, J ¼ 8.2, 7.1, 1.3 Hz, 1H), 7.08
1H NMR (400 MHz, DMSO‑d6)
d 11.74e11.68 (bs, 1H), 8.22 (t,
(ddd, J ¼ 8.0, 7.0, 1.1 Hz, 1H), 4.31 (d, J ¼ 5.9 Hz, 2H), 3.39 (s, 2H). 13
C
J ¼ 5.9 Hz, 1H), 7.54e7.42 (m, 2H), 7.27 (dd, J ¼ 7.6, 0.8 Hz, 1H), 7.22
(ddd, J ¼ 8.2, 7.3,1.8 Hz,1H), 7.05 (t, J ¼ 7.8 Hz,1H), 6.95 (ddd, J ¼ 7.8,
4.9, 1.4 Hz, 2H), 6.84 (td,J ¼ 7.4, 1.1 Hz, 1H), 4.20 (d, J ¼ 5.9 Hz, 2H),
NMR (101 MHz, DMSO)
d 169.4, 144.6, 136.8, 131.3, 129.3, 128.1,
125.5, 125.5, 125.4, 125.4, 122.2, 120.1, 119.0, 112.5, 103.0, 42.4, 38.7.
HRMS (ESI) m/z calcd. for C18H15F3N2OSNa ([M þ Na]þ): 387.0749,
found 387.0743.
3.78 (s, 3H), 3.48 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.7,
156.9, 138.3, 133.2, 128.4, 127.8, 127.0, 125.8, 124.6, 123.4, 120.5,
113.4, 112.4, 110.8, 103.8, 55.7, 42.1, 37.8. HRMS (ESI) m/z calcd. for
C
4.2.19. N-(4-(2-(dimethylamino)ethoxy)benzyl)-2-((1H-indol-3-yl)
thio)acetamide 6b4
18H17BrN2O2SNa ([M þ Na]þ): 427.0086, found 427.0091.
Purity: 97.9%
1H NMR (400 MHz, DMSO‑d6)
d
8.28 (t, J ¼ 5.9 Hz, 1H), 7.61 (ddt,
4.2.24. 2-((4-Bromo-1H-indol-3-yl)thio)-N-(3,4-dichlorobenzyl)
acetamide 6c2
J ¼ 7.7,1.4, 0.7 Hz,1H), 7.47 (d, J ¼ 2.6 Hz,1H), 7.43 (dt, J ¼ 8.1, 0.9 Hz,
1H), 7.15 (ddd, J ¼ 8.2, 7.0, 1.3 Hz, 1H), 7.08 (ddd, J ¼ 8.0, 7.0, 1.1 Hz,
1H), 7.04e6.98 (m, 2H), 6.86e6.79 (m, 2H), 4.14 (d, J ¼ 5.8 Hz, 2H),
4.02 (t, J ¼ 5.8 Hz, 2H), 2.63 (t, J ¼ 5.8 Hz, 2H), 2.23 (s, 6H). 13C NMR
Purity: 95.7%.
1H NMR (400 MHz, DMSO‑d6)
d
11.75 (s, 1H), 8.51 (t, J ¼ 6.0 Hz,
1H), 7.57e7.50 (m, 2H), 7.46 (dd, J ¼ 8.2, 0.9 Hz, 1H), 7.43 (d,
(101 MHz, DMSO‑d6)
d 169.0, 157.8, 136.7, 131.6, 131.2, 129.3, 128.9,
J ¼ 2.0 Hz, 1H), 7.27 (dd, J ¼ 7.6, 0.8 Hz, 1H), 7.10 (dd, J ¼ 8.3, 2.1 Hz,
1H), 7.05 (t, J ¼ 7.8 Hz, 1H), 4.24 (d, J ¼ 6.0 Hz, 2H), 3.46 (s, 2H). 13
C
122.2, 120.0, 119.0, 114.6, 112.4, 103.2, 66.1, 58.1, 45.9, 42.2, 40.5.
HRMS (ESI) m/z calcd. for C21H26N3O2S ([M þ H]þ): 384.1740, found
384.1760.
NMR (101 MHz, DMSO‑d6)
d 169.0, 141.2, 138.3, 133.3, 131.3, 130.8,
129.6, 129.4, 127.8, 125.8, 124.6, 123.4, 113.4, 112.4, 103.7, 42.1, 41.6.
HRMS (ESI) m/z calcd. for C17H13BrCl2N2OSNa ([M þ Na]þ):
464.9201, found 464.9228.
4.2.20. 2-((1H-indol-3-yl)thio)-N-benzyl-N-methylacetamide 6b5
Purity: 99.7%.
1H NMR (500 MHz, DMSO‑d6)
d
11.41 (s, 1H), 7.59 (major) þ 7.55
4.2.25. 2-((5-Bromo-1H-indol-3-yl)thio)-N-(2-methoxybenzyl)
acetamide 6c3
(minor) (d, J ¼ 7.9 Hz, 1H), 7.51 (dt, J ¼ 15.7, 2.6 Hz, 1H), 7.43 (dd,
J ¼ 8.2, 2.7 Hz, 1H), 7.36e7.29 (m, 2H), 7.26 (dt, J ¼ 9.3, 4.7 Hz, 1H),
7.20e7.04 (m, 4H), 4.47 (minor) þ 4.45 (major) (bs, 2H), 3.66
(major) þ 3.62 (minor) (bs, 2H), 2.86 (major) þ 2.73 (minor) (s, 3H).
Purity: 94.9%.
1H NMR (400 MHz, DMSO‑d6)
d 11.64e11.58 (m, 1H), 8.19 (t,
J ¼ 5.8 Hz, 1H), 7.81e7.75 (m, 1H), 7.54 (d, J ¼ 2.6 Hz, 1H), 7.41 (dd,
J ¼ 8.6, 0.6 Hz, 1H), 7.27 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.21 (ddd, J ¼ 8.2,
7.3, 1.8 Hz, 1H), 6.95 (dd, J ¼ 8.2, 1.0 Hz, 1H), 6.90 (dd, J ¼ 7.5, 1.8 Hz,
1H), 6.83 (td, J ¼ 7.4, 1.1 Hz, 1H), 4.18 (d, J ¼ 5.8 Hz, 2H), 3.78 (s, 3H),
13C NMR (126 MHz, DMSO‑d6)
d 169.2, 138.0, 136.8, 131.5, 129.4,
129.1, 128.9, 128.0,127.8, 127.5, 127.1, 122.3, 120.2, 118.9, 112.6, 102.6,
53.3, 50.6, 38.9, 38.8, 35.7, 33.9. Rotamers formed because of ter-
tiary amides in the structure which was similar to that reported in
literature [39,40]. HRMS (ESI) m/z calcd. for C18H18N2OSNa ([M þ
Na]þ): 333.1032, found 333.1031.
3.38 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.1, 157.0, 135.5,
132.9,131.3,128.4,127.9,126.8,124.8,121.4,120.5,114.5,113.0,110.8,
103.0, 55.7, 38.7, 37.9. HRMS (ESI) m/z calcd. for C18H17BrN2O2SNa
([M þ Na]þ): 427.0086, found 427.0090.
4.2.21. 2-((1H-indol-3-yl)thio)-N-(2-methoxybenzyl)-N-
methylacetamide 6b6
Purity: 99.6%.
4.2.26. 2-((5-Bromo-1H-indol-3-yl)thio)-N-(3,4-dichlorobenzyl)
acetamide 6c4
1H NMR (500 MHz, DMSO‑d6)
d
11.40 (s, 1H), 7.64 (major) þ 7.54
(minor) (d, J ¼ 7.9 Hz, 1H), 7.50 (dd, J ¼ 7.7, 2.5 Hz, 1H), 7.42 (dd,
J ¼ 8.1, 4.8 Hz,1H), 7.25 (q, J ¼ 8.3, 7.7 Hz,1H), 7.19e7.12 (m,1H), 7.09
(dt, J ¼ 17.2, 7.5 Hz, 1H), 7.03e6.95 (m, 2H), 6.94e6.87 (m, 1H), 4.40
(major) þ 4.37 (minor) (s, 2H), 3.78 (major) þ3.72 (minor) (s, 3H),
3.67 (major) þ3.60 (minor) (s, 2H), 2.91 (major) þ 2.73 (minor) (s,
Purity: 98.5%.
1H NMR (400 MHz, DMSO‑d6)
d
11.62 (d, J ¼ 2.7 Hz, 1H), 8.47 (t,
J ¼ 5.9 Hz, 1H), 7.78e7.72 (m, 1H), 7.56 (d, J ¼ 2.6 Hz, 1H), 7.51 (d,
J ¼ 8.3 Hz, 1H), 7.40 (dd, J ¼ 8.6, 0.6 Hz, 1H), 7.37 (d, J ¼ 2.1 Hz, 1H),
7.26 (dd, J ¼ 8.6, 1.9 Hz, 1H), 7.04 (dd, J ¼ 8.3, 2.1 Hz, 1H), 4.21 (d,
3H). 13C NMR (126 MHz, DMSO‑d6)
d
169.2, 157.4, 157.3, 136.8, 131.4,
J ¼ 5.9 Hz, 2H), 3.36 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.4,
129.4, 129.2, 128.6, 127.9, 127.7, 125.4, 124.9, 122.3, 120.9, 120.7,
120.2, 120.1, 118.9, 118.8, 112.5, 111.3, 111.1, 102.8, 102.7, 55.8, 55.7,
46.0, 39.0, 38.9, 36.3, 33.8. Rotamers formed because of tertiary
140.9, 135.5, 133.0, 131.3, 131.2, 130.8, 129.7, 129.5, 127.8, 124.8,
121.3, 114.6, 113.0, 102.8, 41.7, 38.7. HRMS (ESI) m/z calcd. for
C
17H13BrCl2N2OSNa ([M þ Na]þ): 464.9201, found 464.9261.
12

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
4.2.27. 2-((6-Bromo-1H-indol-3-yl)thio)-N-(2-methoxybenzyl)
4.2.32. N,N-dibenzyl-2-((4-bromo-1H-indol-3-yl)thio)acetamide
acetamide 6c5
6d2
Purity: 95.5%.
Purity: 97.7%.
1H NMR (400 MHz, DMSO‑d6)
d
11.52 (s, 1H), 8.17 (t, J ¼ 5.9 Hz,
1H NMR (400 MHz, DMSO‑d6)
d
11.75 (d, J ¼ 2.8 Hz, 1H), 7.54 (d,
1H), 7.61 (dd, J ¼ 1.8, 0.5 Hz, 1H), 7.58e7.53 (m, 1H), 7.50 (d,
J ¼ 2.6 Hz, 1H), 7.27e7.16 (m, 2H), 6.95 (dd, J ¼ 8.2, 1.0 Hz, 1H), 6.89
(dd, J ¼ 7.5,1.9 Hz,1H), 6.82 (td, J ¼ 7.4,1.1 Hz,1H), 4.17 (d, J ¼ 5.8 Hz,
J ¼ 2.7 Hz, 1H), 7.45 (dd, J ¼ 8.1, 0.9 Hz, 1H), 7.38e7.28 (m, 5H),
7.28e7.22 (m, 2H), 7.15 (tt, J ¼ 6.3, 1.4 Hz, 4H), 7.05 (t, J ¼ 7.8 Hz,1H),
4.47 (d, J ¼ 12.3 Hz, 4H), 3.77 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
2H), 3.78 (s, 3H), 3.38 (s, 2H),. 13C NMR (101 MHz, DMSO‑d6)
d
169.1,
d 169.1, 138.3, 137.8, 137.3, 133.8, 129.2, 128.9, 128.0, 127.8, 127.5,
157.0, 137.6, 132.2, 128.4, 128.4, 128.0, 126.8, 123.0, 120.9, 120.5,
127.0, 124.7, 123.5, 112.5, 103.0, 50.7, 48.5, 41.0. HRMS (ESI) m/z
115.0, 114.9, 110.8, 103.8, 55.7, 38.7, 37.9. HRMS (ESI) m/z calcd. for
calcd. for C24H21BrN2OSNa ([M þ Na]þ): 487.0450, found 487.0474.
C
18H17BrN2O2SNa ([M þ Na]þ): 427.0086, found 427.0107.
4.2.33. N-benzyl-2-((7-bromo-1H-indol-3-yl)thio)-N-
methylacetamide 6d3
4.2.28. 2-((6-Bromo-1H-indol-3-yl)thio)-N-(3,4-dichlorobenzyl)
acetamide 6c6
Purity: 99.5%.
1H NMR (400 MHz, DMSO‑d6)
d 11.68 (s, 1H), 7.66e7.52 (m, 2H),
Purity: 97.8%.
7.38 (td, J ¼ 7.1, 6.6, 0.9 Hz, 1H), 7.35e7.29 (m, 2H), 7.29e7.22 (m,
1H), 7.19e7.09 (m, 2H), 7.06 (major) þ 7.03 (minor) (t, J ¼ 7.8 Hz,
1H), 4.51 (minor) þ 4.44 (major) (s, 2H), 3.70 (major) þ 3.66 (mi-
nor) (s, 2H), 2.88 major) þ 2.72 (minor) (s, 3H). 13C NMR (101 MHz,
1H NMR (400 MHz, DMSO‑d6)
d
11.53 (d, J ¼ 2.5 Hz, 1H), 8.45 (t,
J ¼ 6.0 Hz, 1H), 7.61 (dd, J ¼ 1.8, 0.5 Hz, 1H), 7.57e7.48 (m, 3H), 7.38
(d, J ¼ 2.0 Hz, 1H), 7.19 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.04 (dd, J ¼ 8.3,
2.0 Hz, 1H), 4.20 (d, J ¼ 5.9 Hz, 2H), 3.36 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) d 169.0, 168.9, 137.9, 137.6, 135.0, 132.5, 131.1, 129.1, 128.9,
DMSO‑d6) d 169.4,140.9,137.6,132.3,131.2,130.7,129.7,129.5,128.4,
127.9, 127.7, 127.5, 127.1, 124.9, 124.9, 121.6, 121.6, 118.6, 118.5, 105.1,
104.5, 104.3, 53.3, 50.5, 38.8, 38.7, 38.7, 35.7, 33.9. Rotamers formed
because of tertiary amides in the structure which was similar to
that reported in literature [39,40]. HRMS (ESI) m/z calcd. for
127.9, 123.0, 120.8, 115.1, 115.0, 103.6, 41.7, 38.7. HRMS (ESI) m/z
calcd. for
464.9234.
C
17H13BrCl2N2OSNa ([M
þ
Na]þ): 464.9201, found
C
18H17BrN2OSNa ([M þ Na]þ): 411.0137, found 411.0181.
4.2.29. 2-((7-Bromo-1H-indol-3-yl)thio)-N-(2-methoxybenzyl)
acetamide 6c7
4.2.34. N,N-dibenzyl-2-((7-bromo-1H-indol-3-yl)thio)acetamide
Purity: 99.3%.
6d4
1H NMR (400 MHz, DMSO‑d6)
d
11.69e11.63 (m, 1H), 8.17 (t,
Purity: 96.5%.
J ¼ 5.9 Hz, 1H), 7.66e7.59 (m, 1H), 7.53 (dd, J ¼ 2.8, 1.0 Hz, 1H), 7.38
(dd, J ¼ 7.6, 0.8 Hz, 1H), 7.21 (ddd, J ¼ 8.2, 7.1, 2.0 Hz, 1H), 7.04 (t,
J ¼ 7.7 Hz, 1H), 6.94 (dd, J ¼ 8.3, 1.0 Hz, 1H), 6.90e6.77 (m, 2H), 4.16
(d, J ¼ 5.7 Hz, 2H), 3.40 (d, J ¼ 1.1 Hz, 2H). 13C NMR (101 MHz,
1H NMR (400 MHz, DMSO‑d6)
d
11.68 (d, J ¼ 2.8 Hz, 1H),
7.59e7.51 (m, 2H), 7.38 (dd, J ¼ 7.6, 0.9 Hz, 1H), 7.34e7.22 (m, 6H),
7.15e7.11 (m, 2H), 7.10e7.06 (m, 2H), 7.01 (t, J ¼ 7.7 Hz, 1H), 4.44 (d,
J ¼ 8.3 Hz, 4H), 3.71 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.4,
DMSO‑d6)
d
169.0,157.0, 135.0,132.3, 131.1, 128.4,127.9, 126.8, 124.8,
137.7, 137.3, 135.0, 132.5, 131.0, 129.1 ꢂ 2, 128.9 ꢂ 2, 128.0 ꢂ 2, 127.8,
127.5, 127.0 ꢂ 2, 124.9, 121.6, 118.5, 105.1, 104.3, 50.8, 48.5, 38.6.
HRMS (ESI) m/z calcd. for C24H21BrN2OSNa ([M þ Na]þ): 487.0450,
found 487.0472.
121.5, 120.5, 118.7, 110.8, 105.0, 104.9, 55.7, 40.2, 37.8. HRMS (ESI) m/
z calcd. for C18H17BrN2O2SNa ([M þ Na]þ): 427.0086, found
427.0099.
4.2.35. N-(2-ethoxybenzyl)-2-((4-bromo-1H-indol-3-yl)thio)-N-
methoxyacetamide 6d5
4.2.30. 2-((7-Bromo-1H-indol-3-yl)thio)-N-(3,4-dichlorobenzyl)
acetamide 6c8
Purity: 96.2%.
Purity: 99.2%.
1H NMR (400 MHz, DMSO‑d6)
d 11.73 (s, 1H), 7.54 (s, 1H), 7.45
1H NMR (400 MHz, DMSO‑d6)
d 11.69e11.64 (m, 1H), 8.46 (t,
(dd, J ¼ 8.1, 0.8 Hz, 1H), 7.31e7.19 (m, 2H), 7.12e6.99 (m, 2H), 6.96
J ¼ 6.0 Hz, 1H), 7.61 (dt, J ¼ 7.9, 0.8 Hz, 1H), 7.54 (d, J ¼ 2.7 Hz, 1H),
(dd, J ¼ 8.3, 1.0 Hz, 1H), 6.87 (td, J ¼ 7.5, 1.1 Hz, 1H), 4.02 (q,
7.49 (d, J ¼ 8.3 Hz,1H), 7.41e7.34 (m, 2H), 7.07e6.96 (m, 2H), 4.19 (d,
J ¼ 6.9 Hz, 2H), 3.75 (s, 2H), 3.57 (s, 3H), 1.32 (t, J ¼ 6.8 Hz, 3H). 13
C
J ¼ 6.0 Hz, 2H), 3.38 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.3,
NMR (101 MHz, DMSO‑d6)
d 169.5, 156.7, 138.3, 133.8, 129.0, 128.8,
141.0, 135.0,132.4,131.2, 131.0, 130.7, 129.6,129.5,127.8, 124.9, 121.5,
125.9, 124.7, 124.5, 123.4, 120.5, 113.3, 112.5, 112.0, 103.4, 63.7, 62.0,
42.8, 38.7, 15.1. HRMS (ESI) m/z calcd. for C19H19BrN2O3SNa ([M þ
Na]þ): 471.0348, found 471.0437.
118.6, 105.1, 104.7, 41.7. HRMS (ESI) m/z calcd. for
C
17H13BrCl2N2OSNa ([M þ Na]þ): 464.9201, found 464.9249.
4.2.31. N-benzyl-2-((4-bromo-1H-indol-3-yl)thio)-N-
methylacetamide 6d1
4.2.36. Synthesis of compound N-(2-methoxybenzyl)-2-((7-phenyl-
1H-indol-3-yl)thio)acetamide 6c9
Purity: 96.8%.
Purity: 99.7%.
1H NMR (400 MHz, DMSO‑d6)
d
11.73 (s, 1H), 7.59 (minor) þ 7.53
6c8 (442 mg, 1 mmol), phenylboronic acid (146 mg, 1.2 mmol),
Pd2(dba)3(18 mg, 0.02 mmol), SPhps (16 mg, 0.04 mmol) and
Cs2CO3 (651 mg, 2 mmol) were dissolved in 1,4-dioxane (2 mL)
under Ar. The mixture was stirred for 3h at 125 ꢀC in a CEM mi-
crowave reactor. After purification on a silica column the target
product was afforded. (306 mg, 76.2%).
(major) (d, J ¼ 2.7 Hz, 1H), 7.46 (major) þ 7.44(minor) (dd, J ¼ 8.1,
0.9 Hz, 1H), 7.33 (m, 2H), 7.29e7.22 (m, 2H), 7.20e7.14 (m, 2H),
7.08e7.00 (m, 1H), 4.56 (minor) þ 4.46 (major) (s, 2H), 3.79
(major) þ 3.72 (minor) (s, 2H), 2.92 (major) þ 2.76 (minor) (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 168.7, 168.6, 138.3, 138.3, 138.0,
137.6, 133.6, 133.6, 129.1, 128.9, 127.9, 127.7, 127.5, 127.1, 126.1, 124.6,
124.6, 123.4, 113.3, 112.4, 112.4, 103.4, 103.2, 53.3, 50.5, 41.2, 40.8,
38.7, 35.7, 33.8. Rotamers formed because of tertiary amides in the
structure which was similar to that reported in literature [39,40].
HRMS (ESI) m/z calcd. for C18H17BrN2OSNa ([M þ Na]þ): 411.0137,
found 411.0181.
1H NMR (400 MHz, DMSO‑d6)
d
11.29 (d, J ¼ 2.8 Hz, 1H), 8.21 (t,
J ¼ 5.9 Hz, 1H), 7.68e7.59 (m, 3H), 7.57e7.49 (m, 2H), 7.48e7.41 (m,
2H), 7.25e7.15 (m, 3H), 6.96 (ddd, J ¼ 11.1, 7.8, 1.4 Hz, 2H), 6.82 (td,
J ¼ 7.4, 1.1 Hz, 1H), 4.19 (d, J ¼ 5.8 Hz, 2H), 3.42 (s, 2H), 2.50 (p,
J ¼ 1.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.2, 157.0, 138.7,
133.9, 131.9, 130.2, 129.4, 128.7, 128.4, 128.0, 127.8, 126.9, 126.4,
13

G.-N. Zhang, J. Zhao, Q. Li et al.
European Journal of Medicinal Chemistry 223 (2021) 113622
122.5, 120.7, 120.5, 118.5, 110.8, 103.9, 55.7, 38.7, 37.9. HRMS (ESI) m/
z calcd. for C24H22N2O2SNa ([M þ Na]þ): 425.1294, found 425.1294.
4.8. Statistical analysis
Data are presented as the means SD from at least three in-
dependent experiments and statistical analyses were performed
using a two-tailed Student's t-test. Differences between groups
4.3. Cells, viruses, and transfection
were considered statistically significant if
p < 0.001(***)
p
<
0.01(**),
Traditionally, HEK293T, HCT-8 and LLC-MK2 cells were grown in
Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher
Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal
bovine serum (FBS; Gibco). Cells were cultured at 37 ꢀC in the
incubator supplied with 5% of CO2. Plasmid were transfected using
the VigoFect transfection reagent (Beijing Vigorous, Beijing, China)
according to the manufacturer's instructions. HCT-8 cells were
infected with HCoV-OC43 (VR-1558) at a multiplicity of infection
(MOI) of 0.1and LLC-MK2 cells were infected with HCoV-NL63
strain Amsterdam I at an MOI of 0.01.
4.9. Molecular modeling studies
The cryo-electron microscopy structure of SARS-CoV-2 RdRp
(PBD ID: 6M71) was prepared for docking using AutoDockTools
(ADT, version: 1.5.6) by removing co-crystalized water molecules,
adding polar hydrogens, and merging Gasteiger charges. The active
site of SARS-CoV-2 RdRp was defined as a box of 26 A ꢂ 26 A ꢂ 26 A,
centered near the active sites (x ¼ 123.6 A, y ¼ 117.84 A, z ¼ 133.6
A). For each compound, we sampled the torsion angles of all
rotatable bonds to generate multiple conformations. Each confor-
mation was then assigned hydrogen appropriate for pH 7.4 and
converted to PDBQT format input file using Open Babel (version
2.3.2). Molecular docking study was performed by using Auto Dock
Vina software as previously described [41]. The final docked
4.4. Plasmids, compounds and reagents
The codon-optimized pCOVID19-nsp12, pCOVID19-nsp7, pCO-
VID19-nsp8, pCOVID19-nsp10 and pCOVID19-nsp14 was encoding
a flag-tagged nsp12, nsp7, nsp8, nsp10 and nsp14 at its C-terminus,
respectively. The plasmidpCoV-Gluc which is the transcription of
Gaussia-luciferase (Gluc) as the reporter gene flanked by 50 and 30
untranslated regions (UTRs) of SARS-CoV-2 is initiated by the CMV
(cytomegalovirus) promoter. Briefly, 50UTR-Gluc-30UTR was inser-
ted into the BamHl and Notl sites of pRetroX-tight-Pur vector
(kindly provided by Dr. Guo Fei). The primers are upper (50-GGC
GGA TCC ATT AAA GGT TTA TAC-30) and lower (50-TTA GCG GCC GCG
TCA TTC TCC TAA GAA-30). The compounds were synthesized in our
lab and Remdesivir (S8932) were obtained from Selleck chemicals
(Houston, TX, USA) and prepared in DMSO.
structure with the best binding energy (DGADV) was selected for
each compound. The protein-ligand interactions were visually
inspected by using PyMOL (version 2.3.4) and Free Maestro (version
11.8.012).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.5. Gluc activity assay and real-time RNA isolation and
quantitative RT-PCR
Acknowledgments
The research was supported by the National Natural Science
Foundation of China (grant number 81703366 ZGN), National Sci-
ence & Technology Major Project "Key New Drug Creation and
Manufacturing Program", China (Numbers: 2019ZX09201001-003
The experiments were performed in accordance with the stan-
dard procedure we previously reported [38].
̊
̊
̊
4.6. Cell toxicity assay
WYC, 2019ZX09721001-004-006) and the Fundamental Research
̊
̊
Funds for the Central Universities (33320200046).
̊
The cell viability was evaluated by cell countingkit-8 (CCK-8,
Beyotime) assay. HEK293T cells were cultured in a 96-well plate
and incubated with 1L of each tested compound ranging from
Appendix A. Supplementary data
0.78 mM to 100 mM and incubated for 24 h. Then 10 mL of CCK-8
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113622.
solution was added to each well and incubated for an additional
1 h at 37 ꢀC. The optical density (OD) at 450 nm of each well was
measured using the Enspire 2300 Multiable reader (PekinElmer).
Cells cultured in the medium with DMSO only were taken as the
control. The half-maximal cytotoxic concentration (CC50) for each
compound was calculated by comparing the viability of
compounds-treated cells with that of DMSO-treated cells.
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