Substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues potently inhibit respiratory syncytial virus (RSV) replication and RSV infection-associated inflammatory responses

Article abstract of DOI:10.1016/j.bmc.2021.116157

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children, and specific treatment for RSV infections remains unavailable. We herein reported a series of substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent RSV inhibitors. Among them, six low cytotoxic compounds (11, 12, 15, 22, 26, and 28) have been identified and selected to study associated inhibitory mechanisms. All these compounds suppressed not only the viral replication but also RSV-induced IRF3 and NF-κB activation and associated production of cytokines/chemokines. The two most potent compounds (15 and 22) were selected for further molecular mechanism studies associated with their suppression effect on RSV-activated IRF3 and NF-κB. These two compounds decreased RSV-induced IRF3 phosphorylation at serine 396 and p65 phosphorylation at serine 536 at both early and late infection phases. In addition, compound 22 also inhibited RSV-induced p65 phosphorylation at serine 276 at the late phase of RSV infection.

Full text of DOI:10.1016/j.bmc.2021.116157

Bioorg. Med. Chem. 39 (2021) 116157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide
analogues potently inhibit respiratory syncytial virus (RSV) replication and
RSV infection-associated inflammatory responses
,
,
,
c
,
d
,
,c,*
Jimin Xua 1, Wenzhe Wub 1, Haiying Chena, Yu Xuea, Xiaoyong Baob
*, Jia Zhoua
a Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States
b Department of Pediatrics, University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States
c Sealy Center for Molecular Medicine, and University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States
d Institute for Human Infections and Immunity, University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children, and
specific treatment for RSV infections remains unavailable. We herein reported a series of substituted N-(4-amino-
2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent RSV inhibitors. Among them, six low cyto-
toxic compounds (11, 12, 15, 22, 26, and 28) have been identified and selected to study associated inhibitory
mechanisms. All these compounds suppressed not only the viral replication but also RSV-induced IRF3 and NF-κB
activation and associated production of cytokines/chemokines. The two most potent compounds (15 and 22)
were selected for further molecular mechanism studies associated with their suppression effect on RSV-activated
IRF3 and NF-κB. These two compounds decreased RSV-induced IRF3 phosphorylation at serine 396 and p65
phosphorylation at serine 536 at both early and late infection phases. In addition, compound 22 also inhibited
RSV-induced p65 phosphorylation at serine 276 at the late phase of RSV infection.
Salicylamide derivatives
RSV
Inflammation
RSV replication
1. Introduction
admissions and 59,600 in-hospital deaths in children younger than 5
years old globally. In addition, in-hospital deaths, due to RSV-caused
Respiratory syncytial virus (RSV) is an enveloped non-segmented
negative-sense RNA virus with two major subgroups A and B,
belonging to the Orthopneumovirus genus, the Pneumoviridae family.1 It
is the most significant cause of lower respiratory tract infection (LRTI) in
the pediatric population.2 Almost all children are infected by RSV by the
age of two and re-infection may occur through life, demonstrating
incomplete immunity.3,4 It is currently estimated that 33.1 million ep-
isodes of RSV-associated LRTI lead to about 3.2 million hospital
LRTI, contribute to about 45% of hospital admitted patients younger
than 6 months old, demonstrating a considerable burden of RSV infec-
tion on health-care services.5,6 RSV infection is also a common cause of
upper respiratory tract infection (URTI) associated with significant
outpatient visits. In high-risk populations, such as premature infants, the
elderly, immunocompromised individuals, and patients with chronic
medical conditions, RSV infection is also associated with an increased
chance of morbidity and mortality.7–10 Moreover, RSV infection has
Abbreviations: RSV, respiratory syncytial virus; LRTI, lower respiratory tract infection; URTI, upper respiratory tract infection; mAb, monoclonal antibody; F
protein, fusion protein; N protein, nucleoprotein; G protein, glycoprotein; P protein, phosphoprotein; M protein, matrix protein; RdRp, RNA-dependent RNA po-
lymerase; Boc, t-butyloxy carbonyl; THP, tetrahydropyran; CC50, cytotoxicity concentration 50%; MOI, multiplicity of infection; RLRs, RIG-I-like receptors; ds,
double-stranded; IRF, interferon regulatory factor; TLRs, Toll-like receptors; SAECs, small airway epithelial cells; MIP, macrophage inflammatory proteins; MCP-1,
monocyte chemoattractant protein 1; IP-10, interferon gamma-induced protein 10; Ser, serine; RANTES, regulated on activation, normal T cell expressed and
secreted; TNF, tumor necrosis factor; p.i., post-infection, TLC, thin layer chromatography; UV, ultraviolet; TMS, tetramethylsilane; HRMS, high-resolution mass
spectrometry; HPLC, high-performance liquid chromatography; DCE, 1,2-dichloroethane; DCM, dichloromethane; DMAP, 4-(dimethylamino)pyridine; DMF, dime-
thylformamide; DMSO, dimethyl sulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc, ethyl acetate.
* Corresponding authors at: Department of Pediatrics, University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States (X. Bao). Chemical Biology
Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, TX 77555, United States (J. Zhou).
E-mail addresses: xibao@utmb.edu (X. Bao), jizhou@utmb.edus (J. Zhou).
1
These two authors contributed equally and should be considered as co-first authors.
https://doi.org/10.1016/j.bmc.2021.116157
Received 6 November 2020; Received in revised form 25 March 2021; Accepted 30 March 2021
Available online 18 April 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
42
been also associated with long-term sequelae such as wheezing and
asthma in children.11–13
exfoliated cells arising from immune-inflammatory response.38,41,
Lung autopsy studies for infants who died of RSV infection revealed that
in addition to the detection of RSV antigen in exfoliated alveolar cells,
proinflammatory neutrophils and macrophages are abundantly present
throughout the airway lumen, supporting the acute inflammatory
sequelae fatal resulting from uncontrolled inflammatory disease.43,44
Other human pathology studies, from the findings from the airways of
mechanically ventilated infants and various infection models, also
support significant inflammatory pathology after the infection.45,46 In
some cases, which demonstrated an effective suppression on RSV
replication by antiviral treatment, the clinical impact of antiviral
treatment was minimal.47,48 Therefore, it is possible that one or more
components of the inflammatory response, which is independent of
ongoing virus replication, can be initiated by the viral infection and
amplified, leading to an uncontrollable condition by antiviral treatment
alone. Thus, developing a therapeutic strategy that can suppress both
virus replication and RSV-induced inappropriate inflammatory response
may serve as a unique and promising avenue for RSV infection treat-
ment. Niclosamide, an FDA-approved anthelminthic drug, displayed
broad-spectrum antiviral activities against various viruses such as
coronavirus, flavivirus and Ebola virus.49,50 Our group has been
devoting to niclosamide drug repurposing and new analog optimization
studies with a long-term interest51–55 and previously identified a series
Despite the significant clinical impact, there currently remains no
effective and specific treatment option for RSV infection.2 Palivizumab,
a monoclonal antibody (mAb) targeting fusion protein (F protein) of
RSV, was licensed in 1998 for prophylactic use in high-risk infants.
However, it is not very cost-effective, with a limited application to high-
risk infants for their first RSV season.14,15 Ribavirin is the only approved
antiviral therapy as an inhaled formulation for RSV infection, but its use
in the clinic is restricted due to the very limited efficacy and significant
safety concerns.16 Currently, anti-RSV drug discovery projects have
mainly targeted the F protein for viral entry, the nucleoprotein (N), and
RSV RNA-dependent RNA polymerase (RdRp) complex.2 In the last two
decades, numerous highly potent and structurally different RSV fusion
inhibitors have been reported, and some of them have successfully
progressed to clinical development exemplifying compounds 1–4
(Fig. 1).2,17–25 Presatovir (2) is one of the most advanced candidates
which is orally available and reduced viral load and disease severity in
phase II clinical trial, demonstrating proof of concept for fusion in-
hibitors in RSV infection treatment.19,26 Replication inhibitors occupy a
key position in anti-RSV drug discovery and recent efforts in developing
new assays for screening have enabled the identification of a series of
RdRp inhibitors with new chemotypes.27–35 Among them, nucleoside
polymerase inhibitor 5 (Lumicitabine) and non-nucleoside polymerase
inhibitor 6 (PC786) have progressed into clinical development.2,28 In
addition, benzodiazepine 7 was reported as potent RSV inhibitors by
targeting the N protein.36,37 While significant efforts and progress have
been made in anti-RSV drug discovery, it seems that there is more, in
addition to the control of viral replication and entry, to be considered for
the anti-RSV therapeutic development, as accumulating data support
that both direct damages from viral replication and the host immune-
inflammatory response contribute to RSV-induced respiratory disease
although their relative weight remains controversial.38
of
N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide
de-
rivatives as potent inhibitors of human adenovirus infection.56 Herein,
we reported these novel chemotype analogues as promising candidates
against not only RSV replication but also RSV infection-associated in-
flammatory responses.
2. Results and discussion
2.1. Chemistry
While initial wave of innate cytokines/chemokines in response to
RSV infection is essential for controlling RSV replication, accumulating
evidence also supports that the host inflammatory responses to RSV
infection can be too strong and long-lasting after the initial wave,
leading to profound negative consequences to the host.39,40 Especially
for the infected infants, the small diameter of bronchioles makes them
particularly susceptible to obstruction by edema, secretions, and
The analogues were developed as outlined in Scheme 1. Briefly,
reduction of the nitro group of niclosamide 8 with zinc dust provided the
amine analog 9, which was then readily converted to compounds 10–13,
16–23, 26, and 29 via acetylation followed by hydrolysis or reductive
amination with responding aldehydes or ketones. Boc-deprotection of
23 under acidic conditions afforded compound 24. Alcohol analogue 25
was accessed from THP ether 29 via acidic depyranylation. Direct
Fig. 1. The representative RSV inhibitors under development.
2

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
Scheme 1. Synthesis of Compounds 9–28. Reagents
and conditions: (a) Zn, NH4Cl, H2O, MeOH, 0 ◦C to r.t,
16 h, 87–99%; (b) i. AcCl, Et3N, acetone, 50 ◦C, 2 h,
98%; ii. LiOH, H2O, MeOH, r.t, 1 h, 97%; (c) aldehyde
or ketone, NaBH3CN, AcOH, MeOH, 0 ◦C to r.t, 12 h,
7–84%; (d) 1 M HCl/Et2O, MeOH, r.t, 12 h, 96%; (e)
TsOH, MeOH, r.t, 12 h, 22% in two steps; (f) i. 2-
methyl-1-pentene or 1-methyl-1-cyclohexene, Fe
(acac)3, PhSiH3, EtOH, 60 ◦C, 1 h; ii. Zn, 2 M HCl (aq.),
60 ◦C, 1 h, 35–44%; (g) pyrrolidine or morpholine,
K2CO3, DMF, 100 ◦C, 1 h, 88–98%; (h) i. 5-chlorosali-
cylic acid, EDCI, DMAP, DCM, 0 ◦C to r.t, overnight;
ii. NaOH, H2O, MeOH, r.t, 1 h, 23% in two steps; (j) i. 5-
chloro-2-methoxybenzoic acid, EDCI, DMAP, DCM, r.t,
2 h; ii. BBr3, DCM, ꢀ 78 ◦C to r.t, 12 h, 83% in two
steps.
hydroamination of niclosamide with 2-methyl-1-pentene or 1-methyl-1-
cyclohexene afforded compounds 14 and 15, respectively.57 Substitu-
tion of 2-chloro-4-fluoro-1-nitrobenzene with pyrrolidine or morpholine
gave the intermediates 31 and 32, respectively, which were then
reduced to provide amino derivatives 33 and 34. Condensation of
amines 33, 34 with 5-chlorosalicylic acid followed by hydrolysis or 2-
methoxy-5-methylbenzoic acid followed by demethylation afforded
the final products 27 and 28, respectively.
displayed the most potent anti-RSV activity, reducing viral titer by 2.59
log, compared with vehicle treatment. However, it also showed mod-
erate cytotoxicity (CC50 = 22.9 µM). Unexpectedly, 3-pyridyl substitu-
tion (19) led to a significant loss of potency. Notably, 2-furanyl
substitution (22) reduced viral titer by 2.13 log. This compound also
showed low cytotoxicity (CC50 = 76.3 µM). We also found that polar
substitutions were not tolerated, as N-Boc protected derivative 23 was
effective in viral replication suppression, while its corresponding amine
24 and hydroxyalkyl analogue 25 were completely incapable of inhib-
iting replication at 10 µM. Intriguingly, N,N-dialkylated derivatives
26–28 all showed potent inhibitory activities against RSV with reduc-
tion in virus titer ranging from 1.51 to 1.91 log, while compounds 26
and 28 also displayed very low cytotoxicity (CC50 = 322.6 µM and 89.9
µM, respectively).
2.2. In vitro evaluation of RSV replication inhibition
Twenty diversified substituted N-(4-amino-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide analogues were selected and screened for
their potential inhibitory activities against RSV. Briefly, human A549
epithelial cells were infected with RSV at a multiplicity of infection
(MOI) of 1 for 2 h, and then the medium containing virus was removed
and the cells were treated with the tested compound at 10 µM for 15 h.
The production of progeny virus was detected by titration assays, as we
previously described.58,59 As shown in Table 1, cells treated with most
substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide
derivatives at 10 µM produced more than a log less infectious particles
than vehicle-treated cells. Meanwhile, these compounds did not affect
cell viability at 10 µM (Fig. 2), excluding the possibility of induced cell
toxicity that led to a less favored environment for RSV replication.
Although niclosamide displayed 1.47 log reduction in virus titer, it also
showed high cytotoxicity with about 24% cell death at 10 µM. Consistent
with our previous results observed for HAdV infection,56 amino substi-
tution (9) and acetamide substitution (10) led to a complete loss of
potency. However, N-alkylated derivatives 11–17 exhibited their
capability in suppressing RSV replication by more than a log. Among
these derivatives, Compounds 11 with n-propyl, 12 with isopropyl, and
15 with 1-methylcyclohexyl possessed potent anti-RSV activities with
about 1.8 log reduction in virus titer meanwhile displaying low cyto-
toxicity (CC50 = 69.7 µM, 82.7 µM, and 86.5 µM, respectively). Long
length alkyl groups seemed not to be favorable as shown by the activity
of compound 13. Derivatives 18–22 were selected to investigate the
effect of various aryl groups. Compounds 18 with benzyl and 20 with 4-
flurobenzyl exhibited similar potency with about 1.5 log reduction in
virus titer, while 3,5-bis(trifluoromethyl) substituted derivative 21
2.3. Modulation of RSV-activated IRF and NF-κB by derivatives
Cell intrinsic RIG-I-like receptors (RLRs) can detect RSV double-
stranded (ds) RNA and stimulate a signaling cascade, leading to the
activation of interferon regulatory factor (IRF) and NF-κB. The activated
IRFs and NF-κB would translocate to nuclear and initiate the transcrip-
tion of numerous genes involved in the immune/inflammatory re-
sponses to viral infections. Moreover, Toll-like receptors (TLRs) on the
cell membrane can also sense RSV ssRNA, dsRNA, and viral envelope
protein, resulting in the activation of IRFs and NF-κB.60 To study the
effect of compounds on RSV-induced IRF and NF-κB, six potent com-
pounds (11, 12, 15, 22, 26 and 28) with low cytotoxicity and one
negative compound (19) were selected. A549 cells were transfected with
luciferase reporter plasmids harboring the binding sites of transcrip-
tional factors IRF3 or NF-κB. After 20 h, cells were infected with RSV at a
MOI of 1, and the viruses were removed 2 h later, followed by treatment
with 10 µM compounds for 15 h. As shown in Fig. 3A and 3B, five
compounds (11, 12, 22, 26 and 28) dramatically suppressed both RSV-
induced IRF3 and NF-κB activation. However, compound 15 only sup-
pressed RSV-activated IRF3 with no effect on NF-κB. Compound 19 as a
negative control, which did not affect the production of progeny RSV,
showed no effect on RSV-induced IRF3 and NF-κB activities.
As discussed, besides the replication-dependent inflammation, there
are also inflammatory components which are independent of the
3

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
replication. To investigate whether there is replication-independent
inflammation and whether the compounds suppress such inflamma-
tion, we assessed the reporter activity at 4 h posttreatment when the
significant replication has not started yet. As shown in Fig. 3C, RSV
genome copies were comparable between each group at 4 h posttreat-
ment. Interestingly, all six active compounds (11, 12, 15, 22, 26 and 28)
significantly inhibited IRF3 and NF-κB activation, indicating that they
also could suppress RSV replication independent signaling (Figs. 3D and
3E). The negative control (19), as expected, displayed no suppression on
either RSV-induced IRF3 or NF-κB activity. Intriguingly, compound 15,
which failed to inhibit NF-κB activation at 15 h post-infection (p.i.,
Fig. 3B.), could suppress RSV-induced NF-κB activation at the early
phase of RSV infection (Fig. 3E). Overall, these data showed the com-
pounds’ suppression on viral replication-independent inflammatory re-
sponses. In addition, the cells were treated with the compounds after
RSV entry, suggesting that these compounds inhibit RSV-induced IRF3
and NF-κB activity independent from viral entry.
Table 1
Antiviral
Activity
and
Cytotoxicity
of
Compounds
8–28.
Compd
R1
R2
H
Virus titer
log
CC50
(µM)b
reductiona
Niclosamide
1.47 ±
0.04
NTc
NT
NT
9
H
H
ꢀ 0.01 ±
0.20
10
0.09 ±
0.08
11
12
H
H
1.82 ±
0.10
69.7
2.4. The impact of derivatives on RSV-induced inflammatory response
± 0.9
82.7
1.88 ±
0.43
The impact of compounds on the early inflammation was also
investigated in small airway epithelial cells (SAECs), the primary human
airway cells, by investigating their effect on RSV-induced cytokines/
chemokines in SAECs. The SAECs were infected by RSV at MOI = 5. At 2
h post infection, viruses were removed, followed by the treatment with
10 µM compounds individually. At 4 h posttreatment, the cytokines/
chemokines in the supernatant were detected. As shown in Fig. 4A, RSV-
induced MCP-1, IP-10, and RANTES were significantly decreased by
these six active compounds. Regarding the effect of compounds on RSV-
± 1.6
13
14
15
H
H
H
1.16 ±
18.6
0.08
± 0.5
1.81 ±
23.7
0.22
± 0.8
1.82 ±
86.5
0.14
± 3.7
induced MIP-1β, IL-6 and TNF-
inhibit MIP-1β induction, only compound 15 could inhibit IL-6 induc-
tion, and compound 26 and 15 were able to inhibit TNF- . Our negative
α, compounds 11 and 15 were capable to
16
17
H
H
1.42 ±
141.4
0.21
± 3.9
α
1.44 ±
NT
control 19 did not show any inhibitory effect on RSV-induced cytokines/
chemokines. Given the result showing that RSV genome copies were
comparable among groups (Fig. 4B), the study supported that the active
compounds could be a regulator of induced early inflammation which
was not RSV replication dependent.
0.23
18
19
H
H
1.53 ±
81.3
0.32
± 5.6
0.54 ±
NT
NT
The nature of inflammation seen in severe bronchiolitis indicates
that enhanced disease in infants is associated with an imbalanced or
dysregulated immune response to viral infection.43,61 MCP-1, MIP-1β,
IP-10, and RANTES are important proinflammatory chemokines and
involved in the up-regulation of inflammatory response. The increase of
nasal MCP-1 and MIP-1β in RSV patients is positively associated with
severity.62 The administration of IP-10 in RSV infected mice enhanced
the severity of pneumonia.63 RSV-induced RANTES contributes to the
exacerbation of allergic airway inflammation. The selected active
compounds significantly suppressed the induction of these four proin-
flammatory chemokines, implying their potential roles in easing RSV-
0.09
20
21
H
H
1.54 ±
0.31
2.59 ±
22.9
0.15
± 0.3
22
23
H
H
2.13 ±
76.3
0.23
± 2.3
induced inflammation. TNF-
α and IL-6 are well known proin-
1.53 ±
NT
NT
NT
flammatory cytokines. TNF-a levels are highest during the acute phase of
infection and then decline during recovery. The association of IL-6 with
RSV severity is elusive and controversial. Some groups reported high IL-
6 nasopharyngeal concentrations are associated with the severity of RSV
infection.64 In contrast, in a cohort of children with RSV bronchiolitis,
higher nasal IL-6 was associated with a shorter requirement for sup-
plemental oxygen.65 It was also reported that IL-6 in BAL fluid of pre-
term infants with RSV bronchiolitis was lower than infants born at term
and the low IL-6 may reflect the prolonged clinical course.66 Although
all six active compounds can suppress MCP-1, IP-10 and RANTES, they
0.37
24
H
H
ꢀ 0.07 ±
0.37
25
26
27
ꢀ 0.16 ±
0.88
1.91 ±
0.33
322.6
± 45.8
NT
1.51 ±
0.08
28
1.74 ±
89.9
0.33
± 4.7
exhibit different impacts on MIP-1β, IL-6 and TNF-α induction. Together
a
with the results showing different impacts of compounds on the acti-
vation of IRF-3 and NF-κB, these data suggest that the compounds likely
employ substantially different antiviral and anti-inflammatory
strategies.
Virus titer log reduction was calculated by subtracting the log10 means of the
production of progeny virus in the presence of 10 µM compounds from the log10
means of the production of progeny virus in the vehicle-treated cells.
b
Cytotoxic concentration 50% (CC50).
c
NT: not tested.
4

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
30
20
****
****
10
*
0.0
e
9
0
1
2
3
4
5
6
7
8
9
0
1
2
2
3
4
5
6
7
8
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
d
i
m
S
M
a
D
s
o
l
c
i
N
Fig. 2. Cytotoxicity assay of compounds. A549 cells were treated with the tested compounds at 10 µM for 15 h. Cell damage was determined by the LDH release
assay. DMSO was used as a vehicle control. P < 0.05 (*) and P < 0.0001 (****) relative to the DMSO group.
Fig. 3. The effect of compounds on the activation of IRF and NF-κB by RSV. (A-B) A549 cells were transiently transfected with IRF or NF-κB luciferase reporter
plasmids. At 20 h post-transfection, the cells were infected with RSV (MOI = 1), and then treated with 10 µM compounds. At 15 h posttreatment, the cells were
harvested to measure luciferase expression to assess the activation of IRF-3 (A) or NF-κB (B). (C) A549 cells were infected with RSV (MOI = 5), and then treated with
10 µM compounds. At 4 h posttreatment, RSV genome copies in the cells were detected by RT-qPCR. (D-E) The experiments were carried out similarly to what is
described for A and B, except that the cells were infected with RSV (MOI = 5), and the activation of IRF-3 (C) and NF-κB (D) were investigated at 4 h posttreatment. P
< 0.05 (*), P < 0.01 (**), P < 0.001(***) and P < 0.0001 (****) relative to the DMSO group.
2.5. The impact of compounds 22 and 15 on RSV replication and RSV-
activated IRF and NF-κB pathway
chemokines in SAECs, we further explored their effect on RSV replica-
tion as well as IRF3 and NF-κB pathways. Various doses of compounds
15 or 22 were used to treat RSV-infected cells for 15 h, and then the
production of infectious progeny virus was detected. As shown in Fig. 5A
As shown in Fig. 4, compound 15 inhibited the induction of all tested
immune mediators, while the other five active compounds showed
inhibitory effects on several tested cytokines/chemokines. Compounds
and B, compounds 15 at 2.5 μM and 22 at 7.5 μM still led to a 1.57 and
1.22 log reduction in virus titer, respectively. To explore the impact of
compounds on viral RNA replication at different times, the RSV genome
copies in the infected cells were detected at 6 h, 15 h and 24 h post-
12, 22, and 28 displayed no suppression on MIP-1α, Il-6, and TNF-α,
while compound 22 indeed enhanced RSV-induced IL-6. As compounds
15 and 22 displayed intriguing effects on RSV-induced cytokines/
treatment. JNJ-53718678,
a
fusion inhibitor under clinical
5

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
Fig. 4. The impact of compounds on the cytokine/chemokine induction in SAECs by RSV. (A) SAECs were infected with RSV (MOI = 5), and then treated with 10 μM
compounds. At 4 h posttreatment, the supernatant was collected, and the level of cytokines/chemokines was measured by Bio-Plex. (B) RSV genomic copies were also
measured by RT-qPCR for samples from panels A. P < 0.05 (*), P < 0.01 (**), P < 0.001(***), or P < 0.0001 (****) shown on the bars is relative to the DMSO group.
A P-value of < 0.05 was considered significant. Mean ± SE is shown.
development, was employed as a control.22 The viral genome copies in
each group were comparable at 6 h posttreatment, and compounds 15
and 22 significantly decreased viral genome copies at 15 h and 24 h
posttreatment (Fig. 5C). JNJ-53718678 did not affect viral genome
copies at 15 h posttreatment and slightly decreased viral genome copies
at 24 h posttreatment, possibly due to the entry inhibition of progeny
virus. Moreover, compounds 15 and 22 decreased the expressions of
viral phosphoprotein (P), matrix (M) and N proteins, and fusion protein
(F) (Fig. 5D). These results indicated that compounds 15 and 22
significantly suppressed the generation of progeny viruses, viral RNA
replication and viral protein expression.
suppression of Ser396 phosphorylation observed for compounds 15 and
22 was consistent with their inhibitory effects on RSV-induced IRF3
activity at the early and late viral infection (Figs. 3D and A). Inducible
phosphorylation on Ser276 and Ser536 can regulate p65 transcriptional
activity without modification of nuclear translocation or DNA-binding
activity.69 Thus, we investigated the phosphorylation levels of Ser276
and Ser536 of p65 in treated cells. At 4 h posttreatment, RSV-induced
Ser536 phosphorylation and nuclear translocation of p65 were sup-
pressed by both compounds, while RSV-induced Ser276 phosphoryla-
tion was not affected by either compound (Figs. 6C and E). The
suppression of Ser536 phosphorylation observed for compounds 15 and
22 was consistent with their inhibitory effects on RSV-induced NF-κB
activity at the early viral infection (Fig. 3E), suggesting both compounds
suppressed the early activation of NF-κB by inhibiting Ser536 phos-
phorylation. At 15 h posttreatment, compound 22 robustly suppressed
RSV-induced Ser276 and Ser536 phosphorylation and nuclear trans-
location of p65 (Fig. 6E). In contrast, compound 15 only impaired RSV-
induced Ser536 phosphorylation of p65, and decreased nuclear trans-
location of p65, but with no effect on Ser276 phosphorylation of p65
(Fig. 6E). Together with the findings on the distinct impact of com-
pounds 15 and 22 on the later activation of NF-κB (Fig. 3B), the result of
Fig. 6E suggested that, at the late infection phase, compound 22 sup-
pressed NF-κB activation by impairing the phosphorylation of Ser276
and Ser536, while compound 15 targeted only Ser536 phosphorylation.
We also found that compound 22 or 15 had undetectable or minimal
impact on p65 or IRF-3 total protein levels in the cell (Fig. 6F).
NF-κB is a dimeric transcription factor composed of various combi-
nations of the Rel family of proteins, including RelA/p65, RelB, c-Rel,
p50/p105, and p52/p100. Owing to its abundance in most cell types and
the presence of a strong transactivation domain, p65 is thought to be
responsible for most transcriptional activity of NF-κB.67 Phosphoryla-
tion and nuclear translocation are critical to IRF3 and p65 transcrip-
tional activity.68 To investigate whether compounds 15 and/or 22
affected RSV-induced IRF3 or/and NF-κB activity via influencing the
phosphorylation and nuclear translocation of IRF3 and p65, we pre-
pared the nucleus fractions for cells after the treatment of compound 15
or 22. Consistent with comparable RSV replication, determined by the
genome copies shown Fig. 4B, we found RSV protein expression was also
comparable in control cells and cells treated with compound 15 or 22, at
early time point of infection (4 h p.i., Fig. 6A). We also found that
compounds 15 and 22 significantly decreased RSV-induced IRF3 phos-
phorylation at serine 396 (Ser396) and nuclear translocation of IRF3 at 4
and 15 h posttreatment, shown in Figs. 6B and D, respectively. The
In this study, we also investigated the effects of compounds 22 and
15 on Poly I:C and TNF-α induced NF-κB and IRF-3 activation to confirm
6

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
Fig. 5. Dose- and time-dependent suppression of compounds 15 and 22 in RSV replication. (A) and (B) A549 cells were infected with RSV (MOI = 1) and treated with
compound 15 (A) or compound 22 (B) at various doses as indicated for 15 h. Total viruses were harvested, and titers determined. (C) A549 cells were infected with
RSV (MOI = 1), and treated with 10 μM compound 15, compound 22 or JNJ-53718678 for 6, 15 or 24 h. RSV genomic copies were measured by RT-qPCR. (D) Viral
proteins were detected by western blot using anti-RSV (upper panel) or anti-F (middle panel) for 15 h post-treatment samples from panels C. The loading control
β-actin was also investigated. P < 0.05 (*) and P < 0.01 (**) relative to the DMSO group.
the suppression role of both compounds in viral replication-independent
cellular signaling. We found that Poly I:C-activated IRF-3 was sup-
pressed by both compounds 22 and 15 (Fig. 7A). However, Poly I:C-
induced NF-κB activation was only impacted by compound 22, not 15
replication-independent and -dependent cytokine/chemokine induction
by RSV, highlighting their potential acting as anti-inflammatory agents
to alleviate RSV-associated symptoms. The potential controller, at the
transcriptional factor level, was also investigated and discussed. Overall,
this is the first proof-of-concept description on using N-(4-amino-2-
chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as a therapeu-
tic agent to potentially treat RSV infection and associated inflammation.
In the future, we will 1) investigate whether substituted N-(4-amino-2-
chlorophenyl)-5-chloro-2-hydroxybenzamide analogues also play a role
in RSV-induced host response of immune cells; 2) identify the molecular
mechanisms used by compounds for their suppressive role in viral
replication and associated inflammation; and 3) test these potent com-
pound candidates in inhibiting RSV replication and pulmonary inflam-
mation in the animal models of RSV.
(Fig. 7B). We also used TNF-α to activate NF-κB, which again was not
impacted by compound 15 (Fig. 7C). These results were consistent with
the effects of compounds on RSV- induced NF-κB and IRF-3 activation at
late infection phases and meanwhile demonstrated the suppression role
of both compounds on viral replication-independent inflammatory
response.
The overall suppressive role of compound 22 in p65 and IRF-3 could
not explain why the IL-6 induction was enhanced by compound 22 at the
early time point post-infection. Since many inflammatory/immune
mediators are regulated by other transcription factors, such as C/EBP
and AP-1, in addition to p65 and IRF-3, compound 22 was possibly
unable to suppress the activation of other transcription factors control-
ling the IL-6 induction. Nevertheless, our results identified several
candidates that can suppress inflammatory responses to RSV at various
levels.
4. Experimental section
4.1. Chemistry
4.1.1. General chemistry methods
3. Conclusion
All commercially available starting materials and solvents were re-
agent grade and used without further purification. Reactions were per-
formed under a nitrogen atmosphere in dry glassware with magnetic
stirring. Preparative column chromatography was performed using sil-
ica gel 60, particle size 0.063–0.200 mm (70–230 mesh, flash).
Analytical TLC was carried out employing silica gel 60 F254 plates
(Merck, Darmstadt). Visualization of the developed chromatograms was
performed with detection by UV (254 nm). NMR spectra were recorded
In summary, a series of substituted N-(4-amino-2-chlorophenyl)-5-
chloro-2-hydroxybenzamide analogues was discovered as potent RSV
inhibitors. Six selected potent compounds 11 (JMX0439-2), 12
(JMX0447), 15 (JMX0509), 22 (JMX0457), 26 (JMX0439-1) and 28
(JMX0490) with low cytotoxicity were found to inhibit RSV replication
substantially and effectively, supported by their suppression on the
generation of progeny viruses, viral genome copies, and/or viral pro-
teins expression. We also found that these compounds can decrease
on a Bruker-300 (1H, 300 MHz; 13C, 75 MHz) spectrometer. 1H and 13
C
NMR spectra were recorded with TMS as an internal reference. Chemical
7

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
Fig. 6. The impact of compounds 15 and 22 on RSV-induced IRF and NF-κB modifications. (A)-(C) A549 cells were infected with or without RSV (MOI = 5), and then
treated with 10 M compound 15 or 22 for 4 h. (A) Viral proteins were detected by western blot. (B) and (C) Nuclear extracts of cells were prepared and subjected to
μ
Western blotting. (D and E) A549 cells were infected with or without RSV (MOI = 1), and then treated with 10 μM compound 15 or 22 for 15 h. Nuclear extracts of
cells were prepared and subjected to Western blotting. (F) The total IRF-3 and p65 from the cell lysates at 4 h (upper panel) or 15 h (lower panel) of infection were
also investigated. In all blots, β-actin was used as loading controls.
Fig. 7. The impact of compounds 15 and 22 on Poly I:C and TNF-α induced IRF-3 and/or NF-κB activation. HEK-293 cells were transiently transfected with IRF-3 (A)
or NF-κB (B and C) luciferase reporter plasmids. At 15 h post-transfection, the cells were transfected with Poly I:C (A and B) or treated with 10 ng/ml TNF-
h post-treatment, the luciferase activity was investigated. P < 0.01 (**) and P < 0.001(***) relative to the DMSO group.
α (C). At 4
shifts were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra (HRMS) were obtained from Thermo Fisher
LTQ Orbitrap Elite mass spectrometer. Parameters include the
following: Nano ESI spray voltage was 1.8 kV; Capillary temperature
was 275 ◦C and the resolution was 60,000; Ionization was achieved by
positive mode. The most intense molecular ion peaks of these derivatives
shown in ESI-MS matched well with the calculated mass values by using
the 35Cl isotope. Purities of final compounds were established by
8

J. Xu et al.
Bioorganic & Medicinal Chemistry 39 (2021) 116157
analytical HPLC, which was carried out on a Shimadzu HPLC system
(model: CBM-20A LC-20AD SPD-20A UV/VIS). HPLC analysis condi-
4.1.5. 5-Chloro-N-(2-chloro-4-((furan-2-ylmethyl)amino)phenyl)-2-
hydroxybenzamide (22)
tions: Waters
μBondapak C18 (300 × 3.9 mm); flow rate 0.5 mL/min; UV
To a solution of compound 9 (100 mg, 0.34 mmol) and 2-furaldehyde
(48 mg, 0.51 mmol) in 10 mL of MeOH was added AcOH (81 mg, 1.35
mmol) at 0 ◦C. The mixture was stirred at 0 ◦C for 30 min, then NaBH3CN
(85 mg, 1.35 mmol) was added. The resulting mixture was stirred at r.t.
overnight. The pH of the mixture was adjusted to 9 ~ 10 with NaHCO3
(aq.) at 0 ◦C. The mixture was extracted with DCM, dried (Na2SO4) and
concentrated. The residue was purified by column chromatography
(Hex/EtOAc) to give compound 22 (105 mg, 82%) as a white solid.
HPLC purity 99.2% (tR = 18.88 min). 1H NMR (300 MHz, CDCl3) δ 11.90
(s, 1H), 8.10 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H),
7.43 – 7.32 (m, 2H), 6.97 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H),
6.61 (dd, J = 8.7, 2.4 Hz, 1H), 6.33 (s, 1H), 6.25 (d, J = 2.7 Hz, 1H), 4.30
(s, 2H), 4.17 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 166.9, 160.4, 151.9,
146.2, 142.3, 134.6, 126.6, 125.2, 124.8, 123.8, 123.6, 120.5, 115.8,
113.0, 112.5, 110.6, 107.5, 41.4. HRMS (ESI) calcd for C18H15Cl2N2O3,
377.0460 (M+H)+; found, 377.0456.
detection at 270 and 254 nm; linear gradient from 10% acetonitrile in
water to 100% acetonitrile in water in 20 min followed by 30 min of the
last-named solvent (0.1% TFA was added into both acetonitrile and
water). All biologically evaluated compounds are > 95% pure. The
detailed syntheses of compounds 9–13, 16–18, 20, 21 and 23–26 were
reported in our previous publication.56
4.1.2. 5-Chloro-N-(2-chloro-4-((2-methylpentan-2-yl)amino)phenyl)-2-
hydroxybenzamide (14)
To a solution of niclosamide (150 mg, 0.46 mmol) and Fe(acac)3 (49
mg, 0.14 mmol) in EtOH (4 mL) was added donor olefin 2-methyl-1-pen-
tene (113 mg, 1.38 mmol), and PhSiH3 (99 mg, 0.92 mmol). The
resulting mixture was heated in an oil bath preheated to 60 ◦C with
stirring for 1 h. The reaction mixture was then cooled to room temper-
ature and Zn (598 mg, 9.20 mmol) and 2 N HCl (2 mL) was added to the
◦
reaction mixture. After stirring at 60 C for another 1 h, the reaction
mixture was cooled to room temperature and filtered through Celite®.
After the filter cake was washed with EtOAc, the filtrate was neutralized
with sat. NaHCO3 (aq.) and extracted with EtOAc for three times. The
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The resulting crude
product was then purified on SiO2 to furnish compound 14 (62 mg, 35%)
as a yellow solid. HPLC purity 98.5% (tR = 16.70 min). 1H NMR (300
MHz, CDCl3) δ 8.14 (s, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 2.4 Hz,
1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.76 (d, J =
2.4 Hz, 1H), 6.62 (dd, J = 9.0, 2.4 Hz, 1H), 1.66 – 1.57 (m, 2H), 1.41 –
1.24 (m, 8H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
166.8, 160.3, 145.6, 134.5, 126.1, 125.2, 124.4, 123.8, 123.1, 120.4,
115.8, 115.8, 115.3, 54.2, 44.0, 28.2 (2C), 17.4, 14.6. HRMS (ESI) calcd
for C19H23Cl2N2O2, 381.1137 (M+H)+; found, 381.1130.
4.1.6. 5-Chloro-N-(2-chloro-4-(pyrrolidin-1-yl)phenyl)-2-
hydroxybenzamide (27)
To a solution of 2-chloro-4-fluoronitrobenzene (520 mg, 2.96 mmol)
and pyrrolidine (256 mg, 3.55 mmol) in 10 mL of DMF was added K2CO3
(818 mg, 5.92 mmol). The resulting mixture was stirred at 100 ◦C for 1
h. Then the mixture was cooled to r.t and poured into 50 mL of H2O.
Yellow precipitate was isolated by filtration and dried to afford 1-(3-
chloro-4-nitrophenyl)pyrrolidine (31) (660 mg, 98%). 1H NMR (300
MHz, CDCl3) δ 8.06 (d, J = 9.3 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 6.39
(dd, J = 9.3, 2.4 Hz, 1H), 3.41 – 3.33 (m, 4H), 2.10 – 2.04 (m, 4H).
To a solution of compound 31 (660 mg, 2.91 mmol) in 20 mL of
MeOH was added 4 mL of saturated NH4Cl (aq.). Zinc dust (946 mg,
14.56 mmol) was added to the solution at 0 ◦C. The reaction was stirred
at r.t for 16 h. TLC indicated that the starting material was gone. 200 mL
of EtOAc was added to the solution. The Zinc solid was filtered, and the
filtrate was washed with 30 mL of brine, dried (Na2SO4) and concen-
trated under vacuum. The residue was purified by column chromatog-
raphy to afford 2-chloro-4-(pyrrolidin-1-yl) (33) (500 mg, 87%) as a
brown solid. 1H NMR (300 MHz, CDCl3) δ 6.72 (d, J = 8.7 Hz, 1H), 6.53
(d, J = 2.7 Hz, 1H), 6.39 (dd, J = 8.7, 2.7 Hz, 1H), 3.56 (s, 2H), 3.23 –
3.15 (m, 4H), 2.01 – 1.94 (m, 4H).
4.1.3. 5-Chloro-N-(2-chloro-4-((1-methylcyclohexyl)amino)phenyl)-2-
hydroxybenzamide (15)
Compound 15 was prepared by a procedure similar to that used to
prepare compound 14, starting from niclosamide and 1-methyl-1-cyclo-
hexene. The title compound (80 mg, 44%) was obtained as a light-yellow
solid. HPLC purity 98.9% (tR = 24.32 min). 1H NMR (300 MHz, CDCl3) δ
11.92 (br s, 1H), 8.15 (s, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 2.4
Hz, 1H), 7.37 (dd, J = 9.0, 2.4 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.78 (d,
J = 2.4 Hz, 1H), 6.64 (dd, J = 9.0, 2.7 Hz, 1H), 3.61 (br s, 1H), 1.90 –
1.77 (m, 2H), 1.59 – 1.42 (m, 8H), 1.33 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 166.8, 160.3, 145.4, 134.5, 126.1, 125.2, 124.4, 123.8, 123.0,
120.4, 115.9, 115.9, 115.5, 53.5, 38.1 (2C), 26.8, 25.7, 22.1 (2C). HRMS
(ESI) calcd for C20H23Cl2N2O2, 393.1137 (M+H)+; found, 393.1130.
To a solution of compound 33 (129 mg, 0.66 mmol), 5-chlorosali-
cylic acid (119 mg, 0.69 mmol) and DMAP (10 mg, 0.082 mmol) in
30 mL of DCM was added EDCI (251 mg, 1.31 mmol) at 0 ◦C. The
resulting mixture was stirred at r.t overnight. The mixture was
concentrated and then to the residue was added H2O (10 mL) and 2 N
NaOH (aq., 3 mL). The mixture was stirred at r.t for 1 h. The pH of the
mixture was adjusted to 6 ~ 7 with 2 N HCl (aq.). Then the mixture was
extracted with EtOAc (2 × 80 mL) and dried (Na2SO4) and concentrated
under vacuum. The residue was purified by column chromatography
(Hex/EtOAc = 5/1 to 3/1) followed by crystallization in MeOH to give
4.1.4. 5-Chloro-N-(2-chloro-4-((pyridin-3-ylmethyl)amino)phenyl)-2-
hydroxybenzamide (19)
Compound 9 (100 mg, 0.34 mmol) and 3-pyridinecarboxaldehyde
(54 mg, 0.51 mmol) were suspended in DCE (20 mL) and treated with
AcOH (61 mg, 1.01 mmol). NaBH(OAc)3 (179 mg, 0.84 mmol) was
added in portions at 0 ◦C, and the mixture was stirred at r.t overnight.
The pH of the mixture was adjusted to 9 ~ 10 with NaHCO3 (aq.) at 0 ◦C.
The yellow solid was isolated by filtration to afford compound 19 (52
compound 27 (50 mg, 23%) as a yellow solid. HPLC purity 97.4% (tR
=
1
20.09 min). H NMR (300 MHz, CDCl3) δ 12.01 (s, 1H), 8.08 (s, 1H),
7.91 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.37 (dd, J = 8.7, 2.1
Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.48 (d, J = 8.7 Hz, 1H),
3.33 – 3.19 (m, 4H), 2.08 – 1.94 (m, 4H). 13C NMR (75 MHz, CDCl3) δ
166.9, 160.4, 146.6, 134.4, 126.8, 125.2, 124.9, 123.7, 121.2, 120.4,
115.9, 111.5, 110.7, 47.8 (2C), 25.6 (2C). HRMS (ESI) calcd for
1
mg, 40%). HPLC purity 99.2% (tR = 15.66 min). H NMR (300 MHz,
DMSO‑d6) δ 12.20 (s, 1H), 10.43 (s, 1H), 8.59 (d, J = 1.2 Hz, 1H), 8.46
(dd, J = 4.5, 1.2 Hz, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.79 – 7.69 (m, 2H),
7.46 (dd, J = 8.7, 2.7 Hz, 1H), 7.36 (dd, J = 7.8, 4.8 Hz, 1H), 7.01 (d, J =
8.7 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.66 – 6.53 (m, 2H), 4.33 (d, J =
6.0 Hz, 2H). 13C NMR (75 MHz, DMSO‑d6) δ 163.7, 156.6, 148.9, 148.1,
146.9, 135.0 (2C), 133.1, 128.9, 126.8, 126.0, 123.5, 123.2, 122.9,
119.1, 118.8, 112.0, 111.5, 43.9. HRMS (ESI) calcd for C19H16Cl2N3O2,
388.0620 (M+H)+; found, 388.0612.
C
17H17Cl2N2O2 351.0667 (M+H)+, found 351.0662.
4.1.7. 5-Chloro-N-(2-chloro-4-morpholinophenyl)-2-hydroxybenzamide
(28)
To a solution of 2-chloro-4-fluoronitrobenzene (640 mg, 3.65 mmol)
and morpholine (381 mg, 4.38 mmol) in 10 mL of DMF was added
K2CO3 (1007 mg, 7.29 mmol). The resulting mixture was stirred at
100 ◦C for 1 h. Then the mixture was cooled to r.t and poured into 50 mL
of H2O. Yellow precipitate was isolated by filtration and dried to afford
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Bioorganic & Medicinal Chemistry 39 (2021) 116157
4-(3-chloro-4-nitrophenyl)morpholine (32) (780 mg, 88%). 1H NMR
(300 MHz, CDCl3) δ 8.03 (d, J = 9.3 Hz, 1H), 6.86 (d, J = 2.7 Hz, 1H),
6.73 (dd, J = 9.3, 2.7 Hz, 1H), 3.89 – 3.82 (m, 4H), 3.37 – 3.31 (m, 4H).
To a solution of compound 32 (780 mg, 3.21 mmol) in 20 mL of
MeOH was added 4 mL of saturated NH4Cl (aq.). Zinc dust (1040 mg,
16.07 mmol) was added to the solution at 0 ◦C. The reaction was stirred
at r.t for 16 h. TLC indicated that the starting material was gone. 200 mL
of EtOAc was added to the solution. The Zinc solid was filtered, and the
filtrate was washed with 30 mL of brine, dried (Na2SO4) and concen-
trated under vacuum. The residue was purified by column chromatog-
raphy to afford 2-chloro-4-morpholinoaniline (34) (650 mg, 95%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 6.86 (t, J = 1.5 Hz, 1H), 6.74 –
6.71 (m, 2H), 3.86 – 3.81 (m, 4H), 3.77 (s, 2H), 3.03 – 2.98 (m, 4H).
To a solution of compound 34 (166 mg, 0.78 mmol), 5-chloro-2-
methoxybenzoic acid (218 mg, 1.17 mmol) and DMAP (10 mg, 0.082
mmol) in 20 mL of DCM was added EDCI (449 mg, 2.34 mmol) at 0 ◦C.
The resulting mixture was stirred at r.t overnight. The mixture was
diluted with 100 mL of DCM, washed with H2O (2 × 30 mL), dried
(Na2SO4) and concentrated to afford the crude product, which was used
for the next step without further purification. The crude product was
dissolved in 20 mL of DCM, and then BBr3 (3.91 mL, 3.91 mmol, 1 M in
DCM) was added dropwise at 0 ◦C. The resulting mixture was stirred at r.
t for 1 h, and then the mixture was poured into 50 mL of ice water. The
pH of mixture was adjusted to 6 ~ 7 with Na2CO3 (aq.). The mixture was
extracted with DCM (2 × 80 mL), dried (Na2SO4) and concentrated. The
residue was purified by column chromatography to afford compound 28
4.2.3. Cell growth assay
A549 cells were seeded into 96-well plates at a density of 1.2 × 104
cells/well. After 24 h, the whole medium was replaced with fresh me-
dium containing compounds (10 ~ 200
μM) and 2% FBS. At 15 h
posttreatment, the cells were washed with PBS 3 times, followed by
staining with trypan blue. For each sample, four non-overlapping fields
at 100× magnification were randomly captured using a video camera,
and the number of the unstained (viable) cells was counted in a blinded
fashion. Two biological replicates were evaluated.
4.2.4. Cytokine and chemokine quantification
RSV-induced chemokines and cytokines were quantified by using a
multi-analytic human (M50-0KCAF0Y) cytokine/chemokine profiling
kit from Bio-Rad (Hercules, CA) according to the manufacturer’s in-
structions. Data were analyzed using the multiplex analysis software
from Bio-Rad.
4.2.5. qRT-PCR
Total cellular RNA was extracted using TRIzol reagents (Thermo
Fisher Scientific, Waltham, MA). qRT-PCR, used to examine viral
replication, was performed using SYBR as we previously described for
RSV or its family member human metapeumovirus.70,71 In brief, to
quantify viral antigenomic copies in the context of RSV infection, syn-
thetic transcripts of the genome were generated from Topo plasmid
– –
containing N P M genes, using the T7 MegaScript kit, following the
digestion with PmeI. The reaction mixture was then treated with Turbo
DNase and purified using the MegaScript kit. Primers were designed to
span the N and P regions of the viral genome and incorporated a Cmr tag.
First-strand cDNA was transcribed with a P-specific primer, 5′-
CTGCGATGAGTGGCAGGCACTACAGTGTATTAGACTTRACAGCAGAAG
ꢀ 3′. The underlined letters indicate the Cmr tag sequence. QPCRs were
performed using the following primers: forward, 5′- CTGCGAT-
GAGTGGCAGGC ꢀ 3′, and reverse, 5′- GCATCTTCTCCATGRAATTCAGG
ꢀ 3′. RT-PCRs and QPCRs were performed as described above.
(240 mg, 83% in two steps) as a brown solid. HPLC purity 98.1% (tR
=
18.12 min). 1H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 8.10 (d, J = 9.3
Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.7, 2.4 Hz, 1H), 6.98 (d,
J = 9.0 Hz, 1H), 6.95 (d, J = 2.7 Hz, 1H), 6.86 (dd, J = 9.0, 2.7 Hz, 1H),
3.91 – 3.81 (m, 4H), 3.20 – 3.11 (m, 4H). 13C NMR (75 MHz, CDCl3) δ
167.0, 160.5, 149.5, 134.7, 126.0, 125.4, 125.2, 124.1, 123.9, 120.6,
115.9, 115.8, 114.8, 66.8 (2C), 49.1 (2C). HRMS (ESI) calcd for
C
17H17Cl2N2O3 367.0616 (M+H)+, found 367.0614.
4.2.6. Western blot analysis
Total cellular lysates or cytosol and nuclear extracts were prepared
for uninfected or infected cells as previously described.70,72 Proteins
were then quantified with a protein quantification kit from Bio-Rad,
followed by fractionation using SDS-PAGE denaturing gels and protein
transfer to polyvinylidene difluoride membranes as previously
described. Membranes were blocked with 5% milk in TBS-Tween 20 and
incubated with the proper primary antibodies according to the manu-
facturer’s instructions.
4.2. Biology
4.2.1. Cell lines, virus, and antibodies
HEp-2 and A549 cells were purchased from the ATCC (Manassas,
VA), and were maintained in MEM and F12K medium respectively,
containing 10% (v/v) FBS. SAECs, isolated from the normal human lung
distal portion, were purchased from Lonza (Pittsburgh, PA), and were
maintained according to the manufacturer’s protocol. RSV long strain
was propagated in HEp-2 cells at 37 ◦C and purified by sucrose gradient
as previously described.58,70 Viral titer was determined by immuno-
staining in HEp-2 cells using polyclonal biotin-conjugated goat anti-RSV
antibody (7950–0104; Bio-Rad, Hercules, CA) and streptavidin peroxi-
dase polymer (S2438; Sigma-Aldrich, St. Louis, MO) sequentially, as
previously described.58,70 The polyclonal biotin-conjugated goat anti-
RSV antibody was also used for Western blot to detect viral protein
expression. The monoclonal antibody against β-actin was from Sigma
(A1978). Primary antibodies against IRF-3(CST#4302), phospho-IRF3
4.2.7. Reporter gene assays
The cells were transfected in triplicate with luciferase reporter gene
plasmids containing multiple copies of NF-κB binding sites (NF-κB-Luc)
or the IRF-3 binding site (PRDIII-I-Luc and IRF-3-Luc) using FuGene 6
(Roche, Indianapolis, IN), as previously described.70,72, 73 At 20 h post-
transfection, cells were infected with RSV for various times and lysed to
measure luciferase reporter activity.
4.2.8. Statistical analysis
(Ser396)(#29047),
p65(CST#4764),
phospho-p65(Ser276)
All experiments were carried out at least twice. The test results were
analyzed by Graphpad Prism 5 software. Two groups comparison was
evaluated using an unpaired two-tailed t-test. A one-way ANOVA test
was performed to analyze differences among multiple groups. A p-value
of<0.05 was considered significant. Means ± standard errors (SE) are
shown.
(CST#3037), and phospho-p65(Ser536)(CST# 3033) were purchased
from Cell Signaling Technology (Denvers, MA), and goat anti-rabbit IgG-
HRP (4050–05) was purchased from SouthernBiotech (Birmingham,
AL).
4.2.2. Lactate dehydrogenase (LDH) assay
A549 cells were seeded into 96-well plates at a density of 1.5 × 104
cells/well. After 24 h, the whole medium was replaced with fresh me-
Declaration of Competing Interest
dium containing 10 μM compounds and 2% FBS. At 15 h posttreatment,
The authors have no conflict of interest to disclose.
the cells were harvested and subjected to LDH assay using LDH-Glo™
Cytotoxicity Assay Kit (Promega, Madison, WI) according to the man-
ufacturer’s protocol. Three biological replicates were evaluated.
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Bioorganic & Medicinal Chemistry 39 (2021) 116157
25 Zheng X, Gao L, Wang L, et al. Discovery of ziresovir as a potent, selective, and orally
bioavailable respiratory syncytial virus fusion protein inhibitor. J Med Chem. 2019;
62:6003–6014.
Acknowledgements
This work was supported by the grants R01 AI116812 and R21
AG069226 from the National Institutes of Health, and FAMRI Clinical
Innovator Award (#160020) to X.B. J.Z. is also partly supported by the
John D. Stobo, M. D. Distinguished Chair Endowment Fund, and the
John Sealy Memorial Endowment Fund at UTMB.
26 DeVincenzo JP, Whitley RJ, Mackman RL, et al. Oral GS-5806 activity in a
respiratory syncytial virus challenge study. N Engl J Med. 2014;371:711–722.
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27 Jimenez-Somarribas A, Mao S, Yoon J-J, et al. Identification of non-nucleoside
inhibitors of the respiratory syncytial virus polymerase complex. J Med Chem. 2017;
60:2305–2325.
28 Wang G, Deval J, Hong J, et al. Discovery of 4’-chloromethyl-2’-deoxy-3’,5’-di-O-
isobutyryl-2’-fluorocytidine (ALS-8176), a first-in-class RSV polymerase inhibitor for
treatment of human respiratory syncytial virus infection. J Med Chem. 2015;58:
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Appendix A. Supplementary data
29 Matharu DS, Flaherty DP, Simpson DS, et al. Optimization of potent and selective
quinazolinediones: Inhibitors of respiratory syncytial virus that block RNA-
dependent RNA-polymerase complex activity. J Med Chem. 2014;57:10314–10328.
30 Deval J, Hong J, Wang G, et al. Molecular basis for the selective inhibition of
respiratory syncytial virus RNA polymerase by 2’-fluoro-4’-chloromethyl-cytidine
triphosphate. PLoS Pathog. 2015;11:e1004995.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116157.
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