Design and synthesis of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides as novel dual inhibitors of respiratory syncytial virus and influenza virus A

Article abstract of DOI:10.1016/j.ejmech.2019.111861

Respiratory syncytial virus (RSV) and influenza A virus (IAV) are two of the most common viruses that cause substantial morbidity and mortality in infants, young children, elderly persons, and immunocompromised individuals worldwide. Currently, there are

Full text of DOI:10.1016/j.ejmech.2019.111861

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Design and synthesis of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides as novel dual
inhibitors of respiratory syncytial virus and influenza virus A
Guo-Ning Zhang, Qiang Li, Jianyuan Zhao, Xuandi Zhang, Zhuxin Xu, Yujia Wang,
Yuanhui Fu, Qi Shan, Yanpeng Zheng, Juxian Wang, Mei Zhu, Ziqiang Li, Shan Cen,
Jinsheng He, Yucheng Wang
PII:
S0223-5234(19)31013-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111861
EJMECH 111861
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2019
Revised Date: 15 October 2019
Accepted Date: 5 November 2019
Please cite this article as: G.-N. Zhang, Q. Li, J. Zhao, X. Zhang, Z. Xu, Y. Wang, Y. Fu, Q. Shan,
Y. Zheng, J. Wang, M. Zhu, Z. Li, S. Cen, J. He, Y. Wang, Design and synthesis of 2-((1H-indol-3-
yl)thio)-N-phenyl-acetamides as novel dual inhibitors of respiratory syncytial virus and influenza virus A,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111861.
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Short communication
Design and synthesis of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides as novel
dual inhibitors of respiratory syncytial virus and influenza virus A
Guo-Ning Zhanga,#, Qiang Lia, #, Jianyuan Zhaoa, Xuandi Zhanga, b, Zhuxin Xuc, Yujia
Wanga, Yuanhui Fuc, Qi Shana, Yanpeng Zhengc, Juxian Wanga, Mei Zhua, Ziqiang Lib,
Shan Cena*, Jinsheng Hec*, Yucheng Wanga*
a.
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union
Medical College, Beijing 100050, China
；
b. School of Pharmacy, Jinzhou Medical University, Jinzhou, Liaoning 121001, China
c.
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing 100044,
China
* Corresponding author.
E-mail address: shancen@imb.pumc.edu.cn (S. Cen); jshhe@bjtu.edu.cn (J. He);
wangyucheng@imb.pumc.edu.cn (Y. Wang).
# Both authors contributed equally to this work.
Abbreviations
Respiratory syncytial virus, RSV; influenza A virus, IAV; human parainfluenza viruses, HPIV;
human metapneumoviruses, HMPV; acute respiratory infections, ARIs; plaque-forming units, pfu;
Gaussia luciferase, Gluc; Relative fluorescence units, RFU; Madin-Darby Canine Kidney, MDCK;
1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide, EDC; Hexafluorophosphate Azabenzotriazole
Tetramethyl Uronium, HATU.

ABSTRACT
Respiratory syncytial virus (RSV) and influenza A virus (IAV) are two of the most
common viruses that cause substantial morbidity and mortality in infants, young
children, elderly persons, and immunocompromised individuals worldwide. Currently,
there are no licensed vaccines or selective antiviral drugs against RSV infections and
most IAV strains become resistant to clinical anti-influenza drug. Here, we described
the discovery of a series of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamide as novel and
potent RSV and IAV dual inhibitors. Thirty-five derivatives were designed, prepared,
and evaluated for their anti-RSV and anti-IAV activities. Among the tested
compounds, 14’c, 14’e, 14’f, 14’h, and 14’i exhibited excellent activity against both
RSV and IAV, which showed low micromolar to sub-micromolar EC50 values. Further,
compounds 14’c and 14’e were identified as the most promising dual inhibitors with
lesser cytotoxicity than the clinical drug, ribavirin. These findings may contribute to
the development of a lead compound for the treatment of RSV and/or IAV infections.
Keywords: 2-((1H-indol-3-yl)thio)-N-phenyl-acetamide; respiratory syncytial virus;
influenza A virus; dual inhibitors

1. Introduction
Pneumonia is the leading cause of death for children under 5 years-old and accounts
for approximately 15% of total deaths in this age spectrum worldwide [1-3]. With the
availability of vaccines, the infection rate of Streptococcus pneumoniae
(pneumococcus) and Haemophilus influenzae type b has decreased, and viruses have
[4]
become the main pathogen of acute respiratory infections including pneumonia
.
Respiratory viruses are considered to be major contributors to acute respiratory
infections in children and are associated with almost 60% of acute respiratory
infections (ARIs) [4]. The most frequently reported viruses include respiratory
syncytial virus (RSV), Influenza A and B viruses (IAV, IBV), human rhinovirus
(HRV), parainfluenza viruses (PIVs), and adenovirus (ADV), which are responsible
for most episodes of ARIs in children.
RSV is a negative stranded non-segmented RNA virus of the pneumovirus family. To
add, it is the most common respiratory virus in infants, young children, elderly
persons, and immunocompromised persons worldwide [5]. RSV is the leading cause of
hospitalization for infants, especially those born premature or with chronic lung or
heart disease [6, 7]. Almost all infants are infected at least once by RSV by the age of
three, and severe lower respiratory tract infection by RSV is estimated to be the cause
of ∼3.4 million hospitalizations and ∼200,000 deaths worldwide [8]. RSV infection
has also been associated with a greater risk of long-term development of recurrent
wheezing and asthma in children [9]. Despite the substantial economic impact and the

medical needs associated with severe viral infection, neither vaccines nor a specific
antiviral therapy is currently available for RSV infection [10]. Instead, treatment
therapy is limited to the non-specific antiviral drug, ribavirin (Figure 1), which is
teratogenic and displays limited efficacy. The monoclonal antibody, palivizumab, has
demonstrated effectiveness as prophylaxis in infants who are premature or have
underlying comorbidities [11]. To add, some candidates, such as lumicitabine
(ALS-8176), presatovir (GS-5806), PC-786, JNJ-678, AK-0529, and TMC353121
(Figure 1) are in clinical development as anti RSVs [12-17]. In RSV-infected
hospitalized children under two years-old, higher viral loads on the third day after
hospitalization have been linked to an increased requirement for intensive care and
progression to respiratory failure [18]. This result suggests that early and efficacious
therapeutics given to hospitalized children may improve downstream morbidity and
reduce immunopathology therapeutics.

Figure 1 Structure of ribavirin, a non-specific anti-viral drug, and candidates in anti-RSV clinical
development.
Influenza A virus (IAV) is a negative-stranded segmented RNA virus. IAV infection is
one of the major causes of morbidity and mortality due to respiratory disease, and
outbreaks of infection sometimes occur in worldwide epidemics. Influenza is
estimated to be responsible for 291,000 to 646,000 deaths worldwide [19]. To add, IAV
is responsible for most cases of epidemic influenza [20]. Infrequently, novel or
reemerging strains of IAV have caused rapid and severe global pandemics, resulting in
millions of fatalities. Influenza B virus (IBV) almost exclusively infects humans and
is less common than IAV due to its lower genetic diversity. A degree of immunity to
IBV is usually acquired at an early age. IAV is often associated with more-severe
symptoms, morbidity, and mortality due to a greater genetic diversity typified by
chromosomal reassortment between human and avian viruses [21]. Currently, there are
only two main classes of antiviral drugs against influenza [22]: M2 ion channel
inhibitors, also called adamantanes (amantadine and rimantadine); and neuraminidase
inhibitors (oseltamivir and zanamivir) (Figure 2), to combat the spread of influenza A.
However, most of the influenza A strains have become resistant to adamantanes, and
strains resistant to oseltamivir and zanamivir have been reported [23, 24]. An urgent
need therefore exists to discover and develop new anti-influenza antivirals.

neuraminidase inhibitors:
O
M2 ion channel inhibitors:
HO
HO
OH
O
O
NH2
H2N
O
H
OH
O
HN
HN
HN
NH2
NH2
O
O
amantadine
rimantadine
NH
zanamivir
oseltamivir
Figure 2 Antiviral drugs against IAV in the clinic.
Patients infected with either influenza virus or RSV can present similar early
symptoms [25, 26]; however, the course of therapy and choice of antiviral agent differ
for both viruses [21]. The lack of a reliable and fast bedside test to rapidly confirm the
viral etiology makes it impossible to perform early administration of antiviral agent,
which is an important step toward reducing drug use, especially in hospitalized
children. Alternatively, discovering new active chemical entities against RSV and IAV
is a simple and efficient strategy for rapid treatment of respiratory infections.
In this work, we initially identified 29 compounds as potent RSV inhibitors using an
rRSV-mGFP high-throughput screening assay. After considering toxicity, druggability,
and feasibility of the synthesis of these compounds, compounds 1 and 2 were selected
as candidates for further optimization (Figure 3). These compounds showed moderate
to high inhibition (77.14% and 83.24% for 1 and 2, respectively) in a
fluorescence-based assay with little or no toxicity to HEp-2 cells at 50 µM.

Figure 3 RSV inhibitors 1 and 2 identified on rRSV-mGFP high-throughput screening platform.
As these two candidates share the common scaffold of 2-((1H-indol-3-yl)thio)
acetamide, we believe this fragment is crucial for RSV inhibition and thus, intend to
introduce different substituent groups to increase their anti-viral potency. Firstly, a
methyl group was introduced to the nitrogen-atoms of indole and a series of
derivatives from compound 1 was designed and synthesized. Thereafter, thioethers
were oxidized to sulfoxide and sulfone to yield 2 additional series of derivatives.
Finally, methoxy-, chlorine atoms, or nitrogen atom was successfully introduced to
the benzene ring in indole. These compounds were evaluated for their anti-RSV and
anti-IAV activity. Encouragingly, these compounds depicted excellent to moderate
activity toward these two viruses, with compounds 14’c, 14’e, 14’f, 14’h, and 14’i
exhibiting excellent activity and low micromolar to sub-micromolar EC50 values.
Among the compounds, 14’c and 14’e, and 14’f and 14’i were identified as the most
promising dual inhibitors with lower cytotoxicity than the clinical drug, ribavirin.
2. Materials and methods
2.1. Experimental chemistry
Reagents were purchased from commercial suppliers and used without further

purification, unless otherwise noted. NMR spectra were recorded on Bruker Avance
400 MHz (1H at 400 MHz) and 500 MHz (1H at 500 MHz, 13C at 125 MHz)
spectrometer. Chemical shift values are given in ppm with reference to the internal
standard, TMS (tetramethylsilane). The peak patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; q, quadruplet; m, multiple and dd, doublet of doublets.
Coupling constants (J) are reported in hertz (Hz). LC-MS data were recorded on
Agilent HPLC/MCD instrument. Silica gel column chromatography was performed
over silica gel 200-300 mesh, and the eluent was a mixture of ethyl acetate and
petroleum ether, or a mixture of methanol and dichloromethane.
2.2. Inhibitor design and synthesis
The structure of 2-((1H-indol-3-yl)thio) acetamide is considered to be a crucial moiety
for the RSV inhibition effect, thus methyl-, methoxy-, chlorine or nitrogen atom was
subsequently introduced to indole ring in this research.
The target products were synthesized mainly by two routes. In the first route (Scheme
1), the N-methyl substituted derivatives was obtained by the coupling of different
anilines and 2-((1-methyl-1H-indol-3-yl)thio)acetic acid, which was prepared by the
substitution of N-methyl indole in situ. The sulfur atom at position 2 of the indole ring
can be oxidized to sulfoxide and sulfone by meta-chloroperoxybenzoic acid.

I
NH2
S
H2N
O
a
b
c
S
OH
N
N
N
3
4
5
O
O
S
O
O
R1
d
d
R1
S
N
N
H
H
N
N
N
6a-h
7a-c
R1=
O
O
R1
a: 2-Cl
b: 3-Cl
c: 4-Cl
S
e: 2,4-di-Cl
f: 2,5-di-Cl
g: 2,6-di-Cl
h: 3,4-di-Cl
N
H
d: 2,3-di-Cl
8d-f
Scheme 1 Reagents and conditions: a). Thiourea, I2, KI, EtOH/H2O, 35-40oC; b).
o
Bromoacetic acid, NaOH, NH2NH2·H2O, 100 C, then 10% HCl; c). substituted
aniline, HATU, DIEA, dichloromethane; d). m-CPBA (1 equivalent for 7 series and 2
equivalents for 8 series), dichloromethane.
All other derivatives were obtained using a second route (Scheme 2).
Commercially-available anilines were acylated with bromoacetyl bromide to yield the
corresponding 2-bromo-N-(substituted phenyl) acetamides, which can be converted to
sodium S-(2-oxo-2-(phenylamino)ethyl) sulfurothioates using sodium thiosulfate.
Subsequently, the sodium sulfurothioates were reacted with substituted indole to yield
the target products.

o
Scheme 2 Reagents and conditions: a). Et3N, Dichloromethane, 0 C; b).
o
Na2S2O3·5H2O, MeOH: H2O=3:1(v/v), 60 C; c. Substituted indole or azaindole,
iodine, DMSO, 60 oC
Details regarding the syntheses and characterizations of the compounds can be found
in the Supplementary data.
2.3 Viruses and cells
Green Fluorescent Protein expressing recombinant RSV (RSV-mGFP; kindly
provided by Prof. Jean-Francois Eleouet, Unite de Virologie et Immunologie
Moleculaires (UR892), INRA, Jouy-en-Josas, France) and wild type subgroup A RSV
Long strain (wtRSV, kindly provided by Prof. Y. Qian, Capital Institute of Pediatrics,
Beijing, China) were propagated in HEp-2 cells (ATCC, Rockefeller, MD, USA) in
DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 2% fetal bovine
serum (FBS, Hyclone, Logan, UT, USA), L-glutamine (2 mmol/L), penicillin G (40
U/ml), streptomycin (100 µg/ml), and 0.2% sodium bicarbonate. RSV-mGFP and
wtRSV were purified by sucrose ultracentrifugation, titrated for infectivity by
immunoplaque assay, and expressed as plaque-forming units (pfu) per ml.
HEK293T-Gluc cells were generated by transfection of plasmid DNA pLenti6-Gluc

constitutively expressing the negative-strand RNA of Gaussia luciferase (Gluc) gene,
that is converted into the positive strand upon IAV infection, and expresses the Gluc
enzyme [27]
.
2.4 Cytotoxicity assay
Method 1 (for data in Table 1):
All compounds were individually subjected to cytotoxicity assay with MTS method.
HEp-2 cells were plated in 96-well plates, and infected with virus alone or together
with the test compound after 24 h. Following incubation for 48 h, 20 µl of 2 mg/ml
MTS (Promega, Madison, WI, USA) was added to 96-well plates, and cells were
further incubated for 3 h at 37 °C in a 5% CO2 incubator. After shaking the plate for
10 s, absorbance was measured with a microplate reader (Tecan, Mannedorf,
Switzerland) at a wavelength of 490 nm. Cells in the mock-treated control were left
uninfected by virus or infected but untreated with the test compound. Cell activity was
set as 100%.
Method 2 (for data in Table 2):
Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Briefly, 293T cells
were cultured in a 96-well plate and incubated with compounds. Six concentrations of
each compound, ranging from 12.5 µM to 200 µM, were used to treat cells for 48 h.
Cells cultured in DMSO only were used as the control. After a 48-h incubation, 10 µL
CCK-8 solution was added to each well and incubated for an additional 1 h at 37 °C.
Optical density (OD) of each well at 450 nm was recorded on a Microplate Reader

(Thermo, Varioskan Flash).
2.5 RSV replication assay
The compounds tested was dissolved in DMSO as a solution of 100mM and diluted to
the concentration indicated. HEp-2 cells in different concentrations were plated in
96-well flat bottom black plates. After 24 h, HEp-2 cells were infected with 3-fold
serially-diluted viral inocula starting from 3 × 104 pfu/well. Relative fluorescence
units (RFU) were measured using multi-mode microplate reader (Molecular Devices,
Silicon Valley, USA) with excitation wavelength of 479 nm and emission wavelengths
of 517 nm at 24 h, 48 h, and 72 h post-infection. Non-infected HEp-2 cells were
severed as background levels of the fluorescence.
2.6. IAV replication assay
HEK293T-Gluc and MDCK cells were incubated with IAV A/WSN/33 (H1N1) (or
H3N2) for 1 h at room temperature, and then cultured in fresh DMEM for 24 h at
[28]
37 °C. Gluc activity in culture medium was measured as described previously
.
Briefly, coelenterazine-h (16.7 µM in PBS) was equilibrated for 30 min in the dark at
room temperature. Thereafter, cell culture supernatants were added to the wells in
white and opaque 96-well plates, followed by automated injection of 60 µl of
coelenterazine-h per well. The 0.5-s photon counts were acquired using Centro XS3
LB 960 microplate luminometer (Berthold Technologies).

2.7. Statistical analysis
Data are presented as mean ± standard deviation (SD) from at least three independent
experiments. Statistical analyses were performed using two-tailed Student’s t-test.
3. Results
3.1. Chemistry
Using the commercially available substrate, 4 series of derivatives were designed and
synthesized. The obtained compounds were further evaluated for their anti-RSV and
anti-IAV activities.
The N-methyl-1H-indole analogues 6a-h, 7a-c, and 8d-f were synthesized according
to the route outlined in Scheme 1. This route started with the commercially-available
1-methyl-1H-indol. The key intermediate 5 was constructed in one pot using a method
[29]
reported
with good yield (85%). Subsequently, it was coupled with different
substitutive anilines to yield the N-methyl-1H-indole analogues 6a-h. Initially, 6d and
6f were obtained in a yield of no more than 10% when the coupling reaction was
conducted with 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC).
Considering the low reactivity of aniline, a more efficient coupling reagent,
Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU), was used
instead of EDC, and all target products 6a-h were yielded in moderate to high yields
(42.2% - 86.1%). At last, the analogues were oxidized to the corresponding sulfoxide
and sulfone using different amounts of meta-chloroperoxybenzoic acid quantitatively.

The analogues, 14a-i and 14’a-j, were synthesized according the route outlined in
Scheme 2. Compounds 3,4-di-chloroaniline 9 and 2-ethoxyaniline 10 were acylated
with bromoacetyl bromide 11 to yield the 2-bromo-N-phenylacetamides 12 and 12’
(yield:
88.7%
and
91.0%
respectively).
Subsequently,
the
2-bromo-N-phenylacetamides were converted to the corresponding sodium
sulfurothioates 13 and 13’, which reacted with the substituted indoles to yield the
target products 14a-h and 14’a-i by a nucleophilic attack in the presence of a catalytic
amount of iodine (yield: 68.3% - 81.4% for 2 steps from compound 12/12’). While
preparing compound 14’b, a byproduct 14’i with an additional 7-bromination
substitution was obtained (13.5%) with the target product. This byproduct was derived
by bromination of 6-methoxyl indole or compound 14’b by bromide ion generated in
the previous steps in the presence of iodine. Compound 14’i was also evaluated for its
anti-viral activity and as one of the most potent compounds with both anti-RSV and
anti-IAV activity. While preparing the azaindole analogues, the 7-azaindole analogues
14j and 14’j were obtained only in low yields of 8.4% and 6.7%, and the 4-, 5- and 6-
aza-indole analogues could not be formed under this reaction condition. This can be
explained by the oxidation of 1H-pyrrolo(2,3-c)pyridine, 1H-pyrrolo(3,2-c)pyridine,
and 1H-pyrrolo(3,2-b)pyridine in the presence of iodine (Scheme 3), and the
decreased nucleophilicity of the aromatic ring that caused a more difficult iodination.
Although 1H-pyrrolo(2,3-b)pyridine can be oxidized under this condition, the
intramolecular hydrogen bond between N and H-N obstructed the oxidation and hence

the same target product could be yielded.
Scheme 3. Oxidation of the azaindole in the present of iodine.
3.2. Biological results
3.2.1. Evaluation of anti-RSV activity and cytotoxicity
After the synthesis of the target products, they were evaluated for their anti-RSV
activity and cytotoxicity in an in vitro cell-based assay. Most of these derivatives
possessed a potent anti-RSV profile compared to that of the reference compound,
ribavirin (Table 1).
Compounds 6a-h were derived from lead 1. When an N-methyl was introduced to the
indole ring, compound 6h showed increased anti-RSV activity compared to that by
lead 1 (EC50=12.46±1.97 µM vs. EC50=32.70±2.09 µM). Compounds 6d-6h were
di-chlorine substituted derivatives, of which, 6g and 6h exhibited good anti-RSV

activities with an EC50 value of 17.27±0.73 and 12.46±1.97 µM. The activity of these
derivatives might be due to their symmetric substitution on the aromatic ring of
phenylamine. Besides, 6d, 6e, and 6f were not active with asymmetric substitution.
Derivatives 6a-c had one chlorine substituted on the phenylamine ring, of which, 6b
displayed similar activity to 6h while 6c was inactive. Such finding indicates that 3-Cl
is more important than 4-Cl for anti-viral activity. The 2-Cl substituted 6a was also
inactive. Compound 6a showed limited anti-RSV activity, but when oxidized to the
corresponding sulfoxide 7a, it possessed an anti-RSV activity with EC50 of
12.34±1.43 µM. For the comparison of the activity of thioether, sulfoxide 7a-c and
sulfone 8d-f indicated that when the oxygen atom was introduced into the molecules,
the orientation of phenylamine changed, ultimately altering the optimal position of
chlorine substitution on phenylamine for anti-RSV activity. The preliminary
structure-activity relationship revealed that the ability of a compound to inhibit RSV
is mainly dependent on the number and position of the chlorine substituent on the
phenylamine ring.
To explore the structure-activity relationship between the structure of indole ring and
its anti-RSV activity, compounds 14a-h, 14j, and 14’a-j were designed and
synthesized. Most of the compounds with a mono-chlorine or methoxy group (i.e.,
14b, 14g, 14’a, 14’c, 14’e, 14’f, and 14’h) on the indole ring depicted excellent to
moderate activity. Compound 14’a, with a 7-MeO on the indole ring, had the most
potent anti-RSV activity (EC50=0.43±0.10 µM), a value indicating 27-fold more

potent activity than the lead 2 and 76-fold greater activity than lead 1. With an
increase in anti-viral activity, toxicity of the mono-chlorine or methoxy substituted
derivatives decreased (i.e., 14b, 14’c, 14’d, 14’e, and 15’g). Compound 14’c was
identified as the most promising lead with a safety index (SI= CC50/ EC50) of 379.25.
When another nitrogen-atom was introduced onto the indole ring, the compounds
became inactive (14j vs. 1 and 14’j vs. 2).
Most of the compounds in series 14 and 14’ were more potent than those in series 6-8.
The EC50 values of the most potent compounds 14b, 14’a, 14’c, 14’e, 14’f, 14’h, and
14’i for RSV inhibition effect were 5.79±1.20, 0.43±0.10, 4.82±0.99, 4.46±0.28,
5.58±0.02, 2.45±0.40, and 2.92±0.77 µM, respectively. All compounds showed a
dose-dependent inhibition to RSV. Dose-response curve is shown in Figure 4.
Figure 4 Dose-response curve for compounds 8d, 14’d, 14’e, 14’g on HEp-2 cell
viability and RSV inhibition. The compounds tested were dissolved in DMSO as a
solution of 100mM and diluted to the concentration indicated. The concentration of
DMSO (v/v) used to determine the cells activity was 0.01‰, 0.1‰, 1‰, and 10‰,
respectively; and the concentration of DMSO used to determine the inhibition rate

was 0.001‰, 0.01‰, 0.1‰ and 1‰, respectively. Data are representative of three
independent experiments and values are expressed as mean ± SD.
To derive the stage of virus replication where these compounds exerted their antiviral
activity, the most promising compound 14’c was added before or after RSV infection.
As shown in Figure 5, pretreatment was unnecessary for full (99.94% and 99.89%)
antiviral activity. Treatment at times -1 h and 0 h with 20 µM of 14’c resulted in
similar and full virus inhibition effects. This result suggests that compound 14’c
inhibits RSV replication at post viral entry.
Figure 5 Anti-RSV effect of 14’c with and without pretreatment. Compound 14’c (20
µM) was added to the culture medium before infection (-1 h) or after infection (0 h).
DMSO and ribavirin were used as negative and positive controls, respectively. The
level of RSV-mGFP infection was determined by measuring fluorescence levels. Data
are normalized to control group, with the value of control arbitrarily set as 1. Data are
representative of three independent experiments and values are expressed as mean ±
SD.

3.2.1. Evaluation of anti-IAV activity
We proceeded to assess the anti-IAV effect of these compounds and found that the
most potent compounds 6g, 6h, 14’c, 14’f, and 14’i displayed inhibition greater than
85% against IAV A/WSN/33 (H1N1) at a concentration of 10 µM (data not shown).
This inspiring result encouraged us to further evaluate their anti-IAV effect at different
concentrations. Most of these compounds depicted moderate to excellent activity
(Table 2). Compounds 6g, 6h, 14’c, 14’e, 14’f, 14’h, and 14’i were identified as the
most potent IAV inhibitors with low micromolar to sub-micromolar EC50 values.
Compounds 6a-h, 7a-c, and 8d-f with a common N-Methyl substitution on the indole
ring showed relatively low activity and cytotoxicity unlike 14a-h and 14’a-i. This
result indicates that the substitution of the N-hydrogen on the indole ring may reduce
the toxicity of these IAV inhibitors. Compounds 14a-h and 14j have a common
3,4-di-Cl substitution on the aniline. When a methoxy group was introduced into these
molecules on the benzene ring in indole, compounds 14a-d displayed similar antiviral
effects with EC50 values in the 15-µM range. Such finding indicates that the position
of the methoxy group is not crucial. Higher activity was observed for derivatives
14e-h bearing the same chlorine atom on different positions of the benzene ring in
indole. When another nitrogen atom was introduced onto the indole ring, compound
14j showed decreased activity with an EC50 value of 32 µM. Compounds 14’a-j had a
common 2-ethoxyl substitution on the aniline and showed relatively higher inhibitory

effect than the corresponding 14a-h and 14j. This result demonstrates that a 2-ethoxyl
substitution on the aniline is superior to a 3,4-di-Cl substitution for the anti IAV effect.
The increased IAV inhibitory effect of compounds 14e-f compared to that of 14a-d
also demonstrate a chlorine atom substitution on different position of the benzene ring
in indole is crucial for their anti IAV effect.
Results obtained suggest that N-hydrogen on the indole ring may be related to its
cytotoxicity. All compounds with a 2-ethoxyl substitution on the aniline and a
chlorine/bromine substitution on the indole ring pose a high IAV inhibitory effect.
All compounds showed dose-dependent anti-IAV effect. The representative
dose-response curve for the most potent compounds 6g, 14’c, 14’f, 14’h and 14’i are
shown in Figure 6 and the EC50 values were 0.64, 1.26, 1.53, 0.60, and 1.90 µM,
respectively.
Figure 6 Dose-response curve for compounds 6g, 14’c, 14’f, 14’h, and 14’i. The
serially-diluted concentrations of test compounds were added to the culture medium.

Ribavirin was used as the positive control. The level of IAV A/WSN/33 (H1N1)
infection was determined by measuring Gluc activity. Data are normalized to control
group, with the value of control arbitrarily set as 100%. Data are representative of
three independent experiments and values are expressed as mean ± SD.
Compounds 6g, 14’e, 14’f, 14’h, and 14’i were selected to evaluate their antiviral
effect in MDCK cells against H1N1 and H3N2, which are important causes of human
influenza. All of these compounds showed a more potent inhibitory effect than
ribavirin against both of these two IAVs. (Figure 7). The EC50 values of compounds
6g, 14’e, 14’f, 14’h, and 14’i against H1N1 were 1.75, 4.33, 2.42, 2.67, and 3.25µM,
and that against H3N2 were 1.50, 1.83, 1.58, 2.33, and 1.50µM, while the EC50 values
of ribavirin against both virus were measured as 16.0 and 20.5µM, respectively.
Figure 7 Antiviral effect of compounds 6g, 14’e, 14’f, 14’h, and 14’i against H1N1
and H3N2 in MDCK cells. The serially-diluted concentrations of test compounds
were added to the culture medium. Ribavirin was used as the positive control. The
level of IAV A/WSN/33 (H1N1) and H3N2 infection was determined by measuring

Gluc activity. Data are normalized to control group, with the value of control
arbitrarily set as 100%. Data are representative of three independent experiments and
values are expressed as mean ± SD.
Notably, the most potent anti-IAV compounds 6g, 6h, 14’c, 14’e, 14’f , 14’h, and 14’i
showed potency in the anti-RSV assay above, and 14’c, 14’e, 14’f, 14’h, and 14’i
exhibited excellent activity against both RSV and IAV. Such finding suggests that
these compounds may employ a similar mechanism to control the replication of both
viruses. In support of this notion, we observed that compounds 14’f and 14’i inhibited
IAV replication at the post-entry stage, similar to that found for RSV. For instance,
treatment before or after IAV infection (at time -2 h and 0 h) with 20 µM of 14’i or
14’f resulting in similar and complete inhibitory effects on IAV replication
(99.72%-99.86%, see Figure 8).
Figure 8 Time of addition assay of 14’f and 14’i. Test compounds (20 µM) were
added to the culture medium before infection (-1 h) or during infection (0 h). DMSO
and ribavirin were used as negative and positive controls, respectively. The level of

IAV A/WSN/33 (H1N1) infection was determined by measuring Gluc activity. Data
are normalized to control group, with the value of control arbitrarily set as 100%.
Data are representative of three independent experiments and values are expressed as
mean ± SD.
To further explore the action of these active compounds, we investigated their effect
on IAV RNA transcription and replication using an IAV mini-genome replicon system.
IAV polymerase activity was reconstituted by expressing A/WSN/33 (H1N1) RdRp
subunits (PA, PB1 and PB2), NP, and a vRNA-like reporter gene Gluc (PolI-luc) in
293T cells as described previously [30]. The result showed that compounds 14’i and
14’f significantly inhibited the activity of luciferase in a dose-dependent manner, with
EC50 values of 1.98 µM and 3.89 µM, respectively (Figure 9). The similar potency of
compounds 14’i and 14’f for the inhibition of IAV infectivity and RdRp activity
suggests that these compounds may target viral RNA transcription and replication.
Figure 9 Effect of compounds 14’i and 14’f on IAV vRNP. HEK239T cells were
co-transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and
PolI-LUC in the absence or presence of compound 14’i or 14’f (1 µM, 2.5 µM, 5 µM,
10 µM, or 20 µM). At 24 h post-transfection, effects of vRNA transcription was
determined by measuring luciferase activity. Data are normalized to control group,

with the value of control arbitrarily set as 100%. Data are representative of three
independent experiments and values are expressed as mean ± SD.
4. Conclusions
RSV and IAV cause substantial morbidity and mortality, and mainly affect infants and
elderly persons worldwide. In this study, the anti-viral potential of a series of
2-((1H-indol-3-yl)thio)-N-phenyl-acetamide was revealed. The developed compounds
displayed moderate to high activity against both viruses, with compounds 14’c, 14’e,
14’f, 14’h, and 14’i exhibiting excellent activity at low micromolar to sub-micromolar
EC50 values. Among these compounds, 14’c and 14’e, and 14’f and 14’i were
identified as the most promising dual inhibitors, displaying lower cytotoxicity (with
highest safety index up to 379.25) than the clinical drug, ribavirin. The preliminary
results imply that these 2-((1H-indol-3-yl)thio)-N-phenyl-acetamide derivatives exert
their antiviral activity at the viral post-entry stage. Such findings can significantly
contribute to the discovery of novel and potent drugs against respiratory syncytial
virus and influenza A infection, thereby providing great protection for individuals at
both ends of the age spectrum.
Declarations of interest: None
Acknowledgments
The research was supported by the National Natural Science Foundation of China

(grant number 81703366 ZGN), The National Mega-Project for drug renovation
(grant numbers 2018ZX09711003-002-002 ZJM, 2019ZX09201001-003-007 WYC),
CAMS innovation fund for Medical Sciences (grant numbers 2016-I2M-1-011 WYC,
2016-I2M-3-014 WJX, 2017-I2M-3-019 BXG, and 2018-I2M-3-004 WJ), the
Fundamental Research Funds for the Central Universities (grant number 332017078
ZGN), and Fundamental Research Funds for Jinzhou Medical University (grant
number JYTQN201731 LZQ).
References
[1]. Liu, L., Oza, S., Hogan, D., et al., 2015. Global, regional, and national causes of child
mortality in 2000–13, with projections to inform post-2015 priorities: an updated systematic
analysis. Lancet 385, 430-440.
[2]. Jain, S., Williams, D.J., Arnold, S.R., et al., 2015. Community-acquired pneumonia requiring
hospitalization among U.S. children. N. Engl. J. Med. 372, 835-845.
[3]. Jain, S., Self, W.H., Wunderink, R.G., et al., 2015. Community-acquired pneumonia requiring
hospitalization among U.S. adults. N. Engl. J. Med. 373, 415-427.
[4]. Bhuyan, G. S., Hossain, M. A., Sarker, S. K., et al., 2017. Bacterial and viral pathogen
spectra of acute respiratory infections in under-5 children in hospital settings in Dhaka city.
PLoS ONE 12, e0174488.
[5]. Hardy, R. W., Wertz, G. W., 2000. The Cys3-His1 motif of the respiratory syncytial virus
M2-1 protein is essential for protein function. J. Virol. 13, 5880-5885.
[6]. Thompson, W. W., Shay, D. K., Weintraub, E., et al., 2003. Mortality associated with

influenza and respiratory syncytial virus in the United States. J. Am. Med. Assoc. 289,
179-186.
[7]. Elliot, A. J., Fleming, D. M., 2008. Influenza and respiratory syncytial virus in the elderly.
Expert Rev. Vaccines 7, 249-258.
[8]. Nair, H., Nokes, D. J., Gessner, B. D., et al., 2010. Global burden of acute lower respiratory
infections due to respiratory syncytial virus in young children: a systematic review and
meta-analysis. Lancet 375, 1545-1555.
[9]. Blanken, M. O., Rovers, M. M., Molenaar, J. M., et al., 2013. Respiratory syncytial virus and
recurrent wheeze in healthy preterm infants. N. Engl. J. Med. 368, 1791-1799.
[10].Palomo, C., Mas, V., Detalle, L., et al., 2016. Trivalency of a nanobody specific for the
human respiratory syncytial virus fusion glycoprotein drastically enhances virus
neutralization and impacts on escape mutant selection. Antimicrob. Agents Ch. AAC-00842.
[11].Cancellieri, M., Bassetto, M., Widjaja, I., et al., 2015. In silico structure-based design and
synthesis of novel anti-RSV compounds. Antivir. Res. 122, 46-50.
[12].Patel, K., Kirkpatrick, C. M., Nieforth, K. A., et al., 2019. Respiratory syncytial virus-A
dynamics and the effects of lumicitabine, a nucleoside viral replication inhibitor, in
experimentally infected humans. J. Antimicrob. Chemother. 2018, dky415.
[13].Hanfelt-Goade D., Maimon N., Nimer A., et al., 2018. A Phase 2b, randomized, double-blind,
placebo-controlled trial of presatovir (GS-5806), a Novel Oral RSV fusion inhibitor, for the
treatment of respiratory syncytial virus (RSV) in hospitalized adults//C17. New Insights in
Acute Pulmonary Infections. Am. Thorac. Soc. 2018: A4457
[14].Brookes, D. W., Coates M, Allen H, et al., 2018. Late therapeutic intervention with a

respiratory syncytial virus L-protein polymerase inhibitor, PC786, on respiratory syncytial
virus infection in human airway epithelium. Br. J. Pharmacol. 175, 2520-2534.
[15].Israel S., Rusch S., DeVincenzo J., et al., 2016. Effect of oral JNJ-53718678 (JNJ-678) on
disease severity in healthy adult volunteers experimentally inoculated with live respiratory
syncytial virus (RSV): a placebo-controlled challenge study//Oh, Those Pesky Viruses! 2016.
https://idsa.confex.com/idsa/2016/webprogram/Paper59339.html.
[16].Ark Biosciences, 2017. A study of AK0529 to evaluate pharmacokinetics and safety in
Chinese healthy volunteers. ClinTrials.gov, NCT03322800.
[17].Cockerill, G. S., Good, J. A., Mathews, N., 2019. State of the Art in Respiratory Syncytial
Virus Drug Discovery and Development. J. Med. Chem. 62, 3206-3227.
[18].El Saleeby, C. M., Bush, A. J., Harrison, L. M., et al., 2011. Respiratory syncytial virus load,
viral dynamics, and disease severity in previously healthy naturally infected children. J.
Infect. Dis. 204, 996-1002.
[19].Iuliano, A. D., Roguski, K. M., Chang, H. H., et al., 2018. Estimates of global seasonal
influenza-associated respiratory mortality: a modelling study. Lancet 391, 1285-1300.
[20].Zhou, H., Thompson, W. W., Belongia, E. A., et al., 2018. Estimated rates of influenza‐
associated outpatient visits during 2001‐2010 in 6 US integrated healthcare delivery
organizations. Influenza Other Resp. 12, 122-131.
[21].Jannetto, P. J., Buchan, B. W., Vaughan, K. A., et al., 2010. Real-time detection of influenza
A, influenza B, and respiratory syncytial virus A and B in respiratory specimens by use of
nanoparticle probes. J. Clin. Microbiol. 48, 3997-4002.
[22].Shen, Z., Lou, K., Wang, W., 2015. New small-molecule drug design strategies for fighting

resistant influenza A. Acta Pharm. Sin. B. 5, 419-430.
[23].Deyde, V. M., Xu, X., Bright, R. A., et al., 2007. Surveillance of resistance to adamantanes
among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J. Infect. Dis. 196,
249-257.
[24].Sheu T.G., Deyde V.M., Okomo-Adhiambo M., et al., 2008. Surveillance for neuraminidase
inhibitor resistance among human influenza A and B viruses circulating worldwide from
2004 to 2008. Antimicrob. Agents Chemother. 52, 3284–3292.
[25].Crowther, Z., 2012. Influenza and RSV: know the diagnostic options. MLO: Med. Lab. Obs.
44, 36.
[26].Gonzàlez-Parra, G., De Ridder, F., Huntjens, D., et al., 2018. A comparison of RSV and
influenza in vitro kinetic parameters reveals differences in infecting time. PloS ONE 13,
e0192645.
[27].Gao, Q., Wang, Z., Liu, Z., et al., 2014. A cell-based high-throughput approach to identify
inhibitors of influenza A virus. Acta. Pharm. Sin. B. 4, 301-306.
[28].Tannous, B. A., 2009. Gaussia luciferase reporter assay for monitoring biological processes in
culture and in vivo. Nat. Protoc. 4, 582-591.
[29].Mirskova, A. N., Levkovskaya, G. G., Kolesnikova, O. P., et al., 2010. Directed synthesis and
immunoactive properties of (2-hydroxyethyl) ammonium salts of 1-R-indol-3-ylsulfanyl
(sulfonyl) alkanecarboxylic acids. Russ. Chem. Bull. 59, 2236-2246.
[30].Wang, J., Wang, Y., Zhou, R., et al., 2018. Viruses 10, 330.