Application of niclosamide and analogs as small molecule inhibitors of Zika virus and SARS-CoV-2 infection

Article abstract of DOI:10.1016/j.bmcl.2021.127906

Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2′-OMe and 2′-H substitutions were also advantageous. We found that the 4′-NO₂ substituent can be replaced with a 4′-CN or 4′-CF₃ substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.

Full text of DOI:10.1016/j.bmcl.2021.127906

Bioorg. Med. Chem. Lett. 40 (2021) 127906
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Application of niclosamide and analogs as small molecule inhibitors of Zika
virus and SARS-CoV-2 infection
,
,
Khalida Shamima *, Miao Xua, Xin Hua, Emily M Leea c, Xiao Lua, Ruili Huanga, Pranav Shaha,
Xin Xua, Catherine Z. Chena, Min Shena, Hui Guoa, Lu Chena, Zina Itkina, Richard T. Eastmana,
Paul Shinna, Carleen Klumpp-Thomasa, Sam Michaela, Anton Simeonova, Donald C. Loa,
Guo-li Mingb, Hongjun Songb, Hengli Tangc, Wei Zhenga, Wenwei Huanga
,
*
a National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20892-3370, USA
b Department of Neuroscience and Mahoney Institute for Neurosciences, University of Pennsylvania, Philadelphia, PA 19104, USA
c Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen
identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection.
However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very
limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medica-
tion. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve
physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that
the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties.
Other modifications in the anilide region with 2′-OMe and 2′-H substitutions were also advantageous. We found
that the 4′-NO2 substituent can be replaced with a 4′-CN or 4′-CF3 substituents. Together, these modifications
provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of
niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were
also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic
strategies targeting the COVID-19 pandemic.
Niclosamide
Flavivirus
Zika virus
Salicylanilide
Small molecule
NS-1 assay
The Zika virus (ZIKV) was first isolated in the forests of Uganda in
1947. It was circulating predominantly between primates and mosqui-
toes and had caused limited human infections in Asia and Africa.
However, the recent outbreak of ZIKV infection in humans in the
Americas and its potential link to fetal microcephaly poses an urgent
global concern.1–7 ZIKV is a vector-borne virus and is transmitted to
humans mainly by Aedes aegypti, a mosquito known also to transmit
yellow fever, dengue fever and chikungunya virus. ZIKV belongs to the
genus Flavivirus in the Flaviviridae family of RNA viruses and consists of a
10.7-kb single-stranded RNA genome that encodes a single polyprotein,
which is believed to be cleaved by host-cell proteases and viral NS2B/
NS3 protease into three structural (C, prM/M and E) and 7 non-
structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). ZIKV
infection has been linked with multiple adverse effects such as testis
damage,8,9 ocular damage,10,11 Guillain-Barre syndrome12–14 and other
neural complications in infected adults.15,16 Several screening efforts
had been directed towards employing synthetic small molecules, natural
products and FDA approved drugs for progression into clinic, but no
candidate has been progressed thus far.17–21
In a repurposing screen with existing drugs, we had identified the
antihelminthic drug niclosamide as a small molecule inhibitor of ZIKV.
Niclosamide inhibited ZIKV-induced caspase-3 activation and host cell
death at sub micromolar concentrations.2,22 The antiviral activity of
niclosamide was confirmed via a concentration-dependent reduction of
ZIKV NS1 protein. It inhibited replication of all three ZIKV strains
(MR766, FSS13025 and PRVABC59) as measured by ZIKV-NS1
Abbreviations: ZIKV, Zika virus; SAR, structure-activity relationship; RLM, rat liver microsomal stability; ADME, absorption; distribution, metabolism and
excretion; PAMPA, parallel artificial membrane permeability assay; MOI, multiplicity of infection.
* Corresponding authors at: 9800 Medical Center Drive, Rockville, MD 20892, USA.
E-mail addresses: khalida.shamim@nih.gov (K. Shamim), huangwe@mail.nih.gov (W. Huang).
https://doi.org/10.1016/j.bmcl.2021.127906
Received 30 November 2020; Received in revised form 15 February 2021; Accepted 21 February 2021
Available online 6 March 2021
0960-894X/Published by Elsevier Ltd.

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
In the present study, our goal was to systematically modify the
structure of niclosamide and examine the effect of these changes on in
vitro anti-ZIKV activity and ADME properties, such as aqueous solubility,
rat liver microsome (RLM) stability, and PAMPA permeability. Although
niclosamide displays good RLM stability (t1/2 > 30 min), it displays poor
kinetic solubility (1.1 µg/mL) and poor permeability (PAMPA < 1X
10ꢀ 6cm/s) (see Supporting information).46–48 Thus, it is desirable to
improve the aqueous solubility and passive permeability as a structure
optimization strategy.
We divided the structure of niclosamide into its salicylic acid and the
aniline motifs and systematically examined the effects of electron-
donating and electron-withdrawing substitutions in both regions
(Fig. 1). We also investigated the role of the hydroxyl group on the
salicylic acid side chain envisaging an internal hydrogen bonding
interaction between this hydroxyl and the amide carbonyl. In addition,
we also probed to find suitable replacement of the aniline 4′-NO2 group
as this group might be reduced and glucuronidated in vivo.24,49 A suit-
able substitution pattern and electronic properties might provide a
strategy to discover niclosamide based potent ZIKV inhibitors for
improving NS-1 potency and developing compounds with better ADME
properties. We used two complimentary assays to measure the expres-
sion of viral proteins as a surrogate marker for Zika replication. The non-
structural protein NS-1 assay was used to drive SAR and the E-protein
(envelope protein) read out was used as a secondary confirmatory assay.
While the ZIKV envelope (E) protein, covers the surface of the virus and
is responsible for membrane fusion and viral entry, the non-structural
NS-1 protein is needed for viral RNA replication and immune evasion.
Dose response inhibition of the viral replication was as was measured in
these phenotypic assays. The details of these assays are provided in the
Supplementary information.
Fig. 1. SAR regions of Niclosamide.
expression in a concentration-dependent manner.22
Niclosamide was approved by FDA for use in humans to treat tape-
worm infections in 198223,24 and is included on World Health Organi-
zation’s list of essential medicines.25 Niclosamide is also categorized as a
FDA Category B drug which implies that it did not demonstrate risk to
the fetus in animal reproduction studies, although there are no adequate
and well-controlled studies in pregnant women. Niclosamide has been
reported to have a number of biological activities such as anti-
tuberculosis activity,26 anti-bacterial activity,27 drug-resistant Staphy-
lococcus aureus activity,28 anti-cancer activity,29–32 and has also dis-
played broad anti-viral activity against coronaviruses (SARS-CoV,
MERS-CoV), mosquito-borne flaviviruses (ZIKV, dengue virus (DENV),
West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis
virus (JEV)), hepatitis C virus (HCV), ebola virus (EBOV), human
rhinovirus (HRV) and human adenovirus (HAdV) with micromolar to
nanomolar potencies.33–35 Niclosamide was shown to inhibit MERS-CoV
replication via SKP2 inhibition and subsequent autophagy induction.36
Similarly, niclosamide demonstrated SARS-CoV-2 inhibition while
rescuing autophagy signaling. SARS CoV-2 infection was shown to
hamper autophagosome/lysosome fusion efficiency and impede host
cell autophagy.37 The broad spectrum anti-viral response of niclosamide
appears to be related to its protonophoric activity which disrupts
membrane pH gradients and is also proposed to block endosomal acid-
ification, an important step for viral entry.33
To systematically evaluate the SAR associated with niclosamide
against Zika virus, we first focused our attention on the salicylic acid
region of the molecule (Table 1). Compounds were synthesized via PCl3-
mediated coupling of the suitably substituted salicylic acids with 2-
chloro-4-nitroaniline (Scheme 1).50,51 Experimental procedures and
analytical data are provided in the Supplementary Information.
As shown in Table 1, replacement of the hydroxyl group of salicylic
acid with a 2-methoxy substituent in 2 rendered the compound inactive.
Also 3 and 4 (O-ethylamine and O-piperidine substitutions) lost potency
in both NS-1 and E-protein assays thus validating that the hydroxyl
group is essential for activity. Halide substitutions were then evaluated
at the 5-chloro position of 5-chloro-2-hydroxybenzoicacid. It was
replaced by 5-F and 5-Br counterparts (5 and 6) and in both cases their
activities were similar to niclosamide. Compound 6 also showed
improvement in the ADME properties exhibiting better solubility (8 µg/
mL), rat liver microsomal stability (t1/2 > 30 min) and PAMPA perme-
ability (67 × 10ꢀ 6 cm/s). Moving the chloro substituent to the 4- posi-
tion (4-chloro-2-hydroxy benzamide) in compound 7 also seemed
acceptable. Though RLM stability (t1/2 = 21 min) was acceptable, this
change could not improve the solubility (<1 µg/mL) and permeability
issues associated with niclosamide. Electron-donating –OMe sub-
stituents either at the 4/5-position in 8 and 9 proved detrimental as the
IC50s increased to 6 µM in both cases. A smaller methyl substituent at the
5-position in 10 led to similar potency (IC50 = 3 µM) compared to
niclosamide but did not provide the required improvement in solubility
(<1 µg/mL). The 2, 6-dihydroxy benzamide substitution in 11, on the
other hand, resulted in good potencies in the NS-1 and E-protein assays
and showed better solubility (>7 µg/mL). Introduction of electron-
withdrawing substituents though, such as in the nitro substituent para
to the hydroxy in 12 and the acetyl substituent in 13 proved detrimental
At NCATS, niclosamide was also discovered in a screen for orthos-
teric inhibitors that directly target the interaction between the ZIKV
flavivirus proteases NS2B and NS3.38–40 Viral non-structural NS3 pro-
tein is the main driver for the viral protease activity and is conserved
among the flavivirus species. As NS2B co-factor binding is essential for
NS3 protease function, inhibitors preventing the NS2B-NS3 interaction
presented a novel approach. Niclosamide was shown to inhibit NS3-
NS2B interaction, inhibit ZIKV growth at early stages and 24 h post
infection and reduce ZIKV titer in human placental epithelial cells and
iPSC-derived human neural progenitor cells.38
Although the pharmacokinetic properties of niclosamide are appro-
priate for its use as an anthelmintic agent, its low solubility, low
bioavailability, and poor plasma exposure limit its use in studies that
require oral dosing and systemic exposure.29,34,41–44 Nevertheless,
niclosamide has been found to be potent and effective against a variety
of human diseases and at present is being investigated in multiple
clinical trials towards ulcerative colitis, diabetic nephropathy, colorectal
cancer, metastatic and recurrent prostrate carcinoma and now in
COVID-19.45 This emphasizes the need for potent and effective niclo-
samide analogs with better physicochemical properties for a swift
translation to clinic.
2

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 1
Structure-activity relationship in the salicylic acid ring motif.
Compound
ID
NS-1
IC50 (µM)
E-protein
IC50 (µM)
RLM, PAMPA, Solubility
(min, X10-6cm/s, µg/ml)
R =
1
0.57
0.88
>30, <1, 1.13
2
3
>46
9.1
>46
27, 1007, <1
8.51
>30, 1023, 1.9
4
11.9
20.1
>30, 517, 8.5
5
6
1.72
0.84
1.34
0.99
ND, ND, 3.3
>30, 67.1, 8.5
7
8
1.74
6.20
1.35
2.82
21.2, ND, 1.1
>30, <3, <1
9
6.80
3.0
8.53
2.74
1.53
>46
17.8, 67.1, 2.1
19.5, 134.3, <1
26.2, ND, 7.9
>30, 30.3, >50
>30, 33.1, >49
10
11
12
13
2.29
>46
13.37
11.14
14
15
>46
2.6
NT
NT
18, 499, <1
16.6, 90.2, 1.2
(continued on next page)
3

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 1 (continued)
IC50 values are average from three biological replicates, NT = Not tested, ND = Not Determined.
towards activity. These results were confirmed with IC50 values obtained
from the E-protein assay.
aniline region. As we were aware of potential liabilities of nitroani-
lines,24,49 we initiated our efforts with the removal of the NO2 group to
assess the physicochemical properties of the molecule in its absence.
This resulted in compound 24 which lost some potency (IC50 = 5 µM) as
compared to the previous bromide lead compound 6 (IC50 = 0.8 µM) but
gained in solubility (5 µg/mL) and PAMPA permeability (118 × 10ꢀ 6
cm/s). This result confirmed our hypothesis that a suitable replacement
of the nitro group might improve the physicochemical properties of
niclosamide. Encouraged by this result, we also tried the ‘H’ replace-
ment of 2′-chloro position of the aniline and synthesized 25. This analog
exhibited the desired ADME characteristics (RLM t1/2 = 22 min, solu-
bility = 6 µg/mL, PAMPA permeability = 127 × 10ꢀ 6 cm/s) while
maintaining potency in the NS-1 assay (IC50 = 2.5 µM), thus demon-
strating that electronic properties can play an important role in
providing the best combination of potency and ADME property
requirements.
Fused or extended aromatic groups in the salicylic acid region were
evaluated with 14, 15, 16 and 17. 4-hydroxyquinoline 14 was inactive
but the other two naphthalene derivatives 15 and 16 showed acceptable
potencies (2.6 and 1.6 µM, respectively). The biphenyl derivative 17
showed an IC50 value of 4.1 µM in NS-1 assay.
We introduced an amine in lieu of the phenolic hydroxyl anticipating
that it would be capable of forming an internal hydrogen bond. How-
ever, this rendered the compound inactive (18 and 19). However, while
the acetamide derivative 20 was also inactive, the derivatization of the
amine to the corresponding methyl and phenyl sulfonamides 21 and 22
respectively restored activity to the single digit micromolar range. This
suggested that the pKa of functional group in C-2 is important for activity
and for the analog’s ability to participate in an internal hydrogen bond.
Thus, our initial efforts in the salicylic acid region provided prom-
ising leads (5, 6, 7, 11, 16, 22) in terms of potency in the primary NS-1
assay and compounds 6 and 22 also exhibited better in vitro ADME
characteristics than niclosamide.
Encouraged by these results, we began investigating the effects of
electronic properties of substituents in the anilide ring system. Intro-
duction of an additional mildly electron-withdrawing chloro substituent
on niclosamide at the 6′ position in 26 dramatically increased solubility
(53 µg/mL) though showed a slight decrease in NS-1 inhibition (IC50 = 6
µM). Shifting the position of the chloro substituent from 2′ to the 3′
position (27) was tolerated. Changing the 2′-chloro substituents to the
electron-donating 2′-methoxy in the anilide region 28 provided a
promising compound in terms of potency and improved ADME charac-
teristics, with RLM t1/2 > 30 min and solubility at 9 µg/mL and a PAMPA
Next, we turned our attention to the anilide region (Table 2). Com-
pounds were synthesized either via PCl3-mediated coupling of 5-chloro-
2-hydroxybenzoic acid with corresponding amines (Scheme 1) or
reacting 5-chloro-2-hydroxy-benzoyl chloride with the corresponding
amines using pyridine as the solvent (Scheme 2) (see Supplementary
Information for experimental procedures).
First, we sought to identify bioisosteres of the 4′-NO2 group on the
4

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
structure–activity relationship of the modified derivatives, we generated
a binding model of niclosamide with viral protein target NS3 using an
ensemble docking approach.52 Our model as depicted in Fig. 2 predicts
that niclosamide binds to the NS3-NS2B interface in an inner, well-
defined hydrophobic pocket adjacent to a highly polar region. The sal-
icylic acid motif inserts into the pocket by forming extensive hydro-
phobic and aromatic stacking interactions with the surrounding residues
Tyr-23, Phe-46, Leu-58, Leu-65, and Tyr-79. The OH group on C-2 forms
a hydrogen bond with the backbone O atom of Arg-24 while the 5-Cl
points downward and forms halogen-binding interactions inside the
pocket. Both interactions are predicted to be key for the binding affinity
and activity of niclosamide. On the other side of the molecule, the ani-
lide motif points out of the pocket to the solvent exposed region with the
nitro group forming salt bridge with Arg-28 and Arg-59. These binding
interactions compares well to the NS2B-NS3 protein–protein interaction
(PPI) site53 with NS2B forming key interactions with residues Tyr-52,
Ile-53, and Glu-54 at the PPI interface (Also see Supplementary infor-
mation Suppl. S1).
Scheme 1. Synthesis of anilide derivatives.
permeability of 334 X 10ꢀ 6 cm/s. Thus, by addressing the electronic
properties in the anilide region we could identify 25 and 28 exhibiting
similar IC50s in NS-1 assay but much better physicochemical properties
than the parent niclosamide.
We next turned our attention to potential replacements of the nitro
group and other heteroaromatic/ aliphatic ring system replacements of
the anilide moiety. Introduction of the 5-isopropylthiazol-2-amine in 29
and of 5-nitrothiazol-2-amine in 30 as heterocyclic anilide replacements
proved ineffective, as did the 3-chloropyridin-4-amine in 31. A 4′-amino
substitution replacing 4′-NO2 on the aniline in 32 and the aliphatic 1-
methyl-piperidin-4-amine in 33 also produced inactive compounds.
Replacing the 4′-NO2 substituent with the electron-withdrawing 4′-
cyano substituent in 34, however, proved beneficial for potency and
showed an IC50 of 1.7 µM in the NS-1 inhibition assay.
These docking studies of the niclosamide derivatives with NS3 pro-
vided an understanding of the structural basis for the observed SAR. As
shown in Fig. 3, these analogs were predicted to adopt the same binding
conformation as niclosamide at the binding site. As the salicylic acid ring
was accommodated in a deep and restricted pocket, it was postulated
that substituents with bulky groups would pose steric hindrance and
thereby restrict potency. As expected, replacement of the OH group on
the salicylic acid ring significantly impaired potency (compounds 2, 3,
and 4). Similarly, replacement of the Cl with Me or OMe (9 and 10)
resulted in decreased potency, but substituents with 5-F and 5-Br (5 and
6) exhibited comparable potencies as niclosamide. Interestingly, other
substitutions on salicylic acid were tolerated (7, 11 and 16). For the
anilide motif the binding interaction with Arg-28 was predicted to be
key, and in fact replacement of the 4′-NO2 group with 4′-CN and 4′-CF3
(34, 41 and 42) showed comparable potencies as niclosamide, whereas
other substitutions disrupting the H-bond interaction dramatically
decreased activity (44, 45, 51, 55).
The 4′-fluoro replacement was then tried, and 5 analogs were syn-
thesized. Simple replacement of 4′-NO2 with 4′-F in 35 seemed to be
tolerated as was the 2′, 4′-difluoro analog 36, but the other fluorinated
analogs such as 37 and 38 were less potent in both NS-1 and E-protein
assays and compound 39 was inactive. Compound 40 with a 4′-sulfon-
amide replacement of the nitro moiety was also tried but failed to
improve potency compared to niclosamide as measured in the NS-1
assay.
A CF3 replacement of the 4′-NO2 group was then attempted and
compounds 41 and 42 were synthesized. This modification proved su-
perior and produced more potent compounds in both NS-1 and E-protein
assays (41, NS-1 = 0.8 µM and E-protein = 1.4 µM, and 42, NS-1 = 1.6
µM and E-protein = 1.7 µM) thus showing that the potential liability
associated with the nitro group in niclosamide can be removed without
compromising potency. The 2′-CF3 analog 43 had modest NS-1 and E-
protein inhibition values.
With these insights from our docking studies, advantageous modifi-
cations in the salicylamide and anilide region were then combined
(Table 3). To this end, the 5-bromo-2-hydroxybenzoic acid from 6 that
had shown good potency and ADME characteristics was combined with
the 4′-CF3 and 4′-CN replacements of 4′-NO2 on anilide (59–64). We
were encouraged to see that all of these compounds showed NS-1 IC50s
in the low micromolar range. However, these compounds did not show
improved solubility and PAMPA permeability over niclosamide. Bicyclic
naphthalene in the salicylic acid region and the biphenyl compounds 67
and 68 with extra aromatic appendage showed decreases in potency. An
indole replacement in 69 was also not effective in improving potency.
Homologation in the amine motif of niclosamide was also tried and
the results are shown in Table 1 in the Supplementary information.
Finally, we wanted to corroborate the SAR we had generated with a
Zika virus focus-forming assay to demonstrate actual reductions in viral
titer. To test this, we assayed a set of analogs that varied over a wide
range of potencies in the NS-1 and E-protein assays. As shown in Fig. 4
and Table 4, there was a good correlation between relative potencies
observed in the molecular assays compared to those observed in the viral
titer reduction assay. Viral titer IC50 comparison with cell viability in
SNB-19 cells is shown in Table 4 and plotted in Fig 5.
Introduction of a 4′-acetamido electron-withdrawing group to
replace the 4′-NO2 in 44 or a 3′-dimethyl carbamoyl group in 45 pro-
duced inactive compounds. A 4′-chloro substitution in the anilide was
then examined in 46, 47, and 48 but these compounds did not show
improvements in potencies over niclosamide. Compounds 49 and 50
with 2′-6′-dichloro and 2′-6′-difluoro substitution were inactive.
With electron-donating 4′-OMe and 3′-OMe substitutions in 51 and
52 the IC50s for inhibition were adversely affected. The same trend
continued in 53, 54, 55 and 56 where reduced potencies were observed.
Introduction of bicyclic nitro naphthalenes in the anilide region were
also investigated with compounds 57 and 58; these both showed
desirable inhibition values in the NS-1 assay.
Thus, by modifying the anilide region we found 4′-CN and 4′-CF3 as
suitable 4′-NO2 replacements in compounds 34, 41 and 42, all exhibit-
ing similar potencies as niclosamide in the NS-1 inhibition assay.
Modifying the electronic properties in the anilide region in 25 and 28
resulted in similar IC50s for inhibition in the NS-1 assay compared to
niclosamide but provided improved physicochemical characteristics in
terms of RLM stability, solubility, and PAMPA permeability.
To understand further the binding interaction of niclosamide and the
As we were preparing to report these SAR findings around niclosa-
mide against the Zika virus, our labs were also redirecting drug
screening and development resources against the SARS-CoV-2 virus,
5

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 2
Structure activity relationship in the aniline ring.
NS-1
IC50 (µM)
E-protein
IC50 (µM)
RLM, PAMPA, Solubility
Compound ID
24
R =
(min, X10-6cm/sec, µg/ml)
5.94
2.5
5.08
2.10
4.49
2.87
3.74
19, 118, 5
25
26
27
22, 127.1, 6.4
20, 53, 53
5.74
2.17
2.5
3.8, 68, <1
28
29
>30, 334.4, 9.2
41.71
40.41
4.49
>46
2, 117, 1
30
31
3, 37, >44
12.86
5.46
>30, 31, 6.5
32
33
34
44.15
>46
16.5
NT
30, 152, >44
1.8, 212, >40
>30, 5.1, <1
1.7
2.33
35
6.60
NT
19.3, 50.5, 1.2
36
37
38
5.56
8.37
5.29
9.19
8.32
18, ND, 1
11, ND, 2.4
11.36
18.4, 390.8, 1.6
39
>46
13.58
13, 25, >49
40
41
5.84
0.8
NT
>30, <1, >48
1.38
>30, <22.3, <1
42
1.6
1.72
>30, ND, <1
(continued on next page)
6

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 2 (continued)
43
44
13.05
>46
7.60
15, 26, >47
>30, 87, 5.1
16.42
45
46
47
>46
4.45
3.59
4.47
1.89
3.77
26, 5, >47
>30, <22.4, <1
14, 297, <1
48
49
50
3.53
>46
>46
4.52
>46
ND, ND, 8
24, 73, 21
>30, 32,16
>46
51
52
23.71
16.91
5.09
3.3, 81, 2.8
4, 43, 2.3
10.11
12.14
53
8.27
9.4, 48, 1.6
54
55
56
5.96
8.20
4.46
NT
5.4, 34, <1
1.6, 21, <1
15.59
5.85
ND, 153, 15.4
57
58
3.02
2.96
NT
NT
>30, 447.3, <1
10.7, 141, 1.7
IC50 values are average from three biological replicates, NT = Not tested, ND = Not Determined.
responsible for the COVID-19 pandemic. In high-throughput drug
repurposing screens we found that niclosamide displayed potent in vitro
activity against SARS-CoV-2.37,54 In a recent communication, an inde-
pendent group also confirmed niclosamide as a potent inhibitor of SARS-
CoV-2 (IC50 = 280 nM) with potency > 25X greater than chloroquine
and > 40X greater than remdesivir.55 We thus extended our studies to
explore the activity of key potent analogs against this strain of the novel
beta coronavirus.
Scheme 2. Synthesis of anilide derivatives.
Compounds 4, 6, 34, 41, 61, 28, 25 and niclosamide were examined
7

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Fig. 2. A) Predicted binding-model for niclosamide to NS3 at the NS3-NS2B PPI site. B) Key interactions of NS2B at NS2B-NS3 PPI site.
280 nM and a selectivity index of >176).55 Also, in our hands, the bromo
analog of niclosamide, compound 6, appeared slightly improved with an
IC50 of 101 nM and 27% efficacy. Compounds 34 and 41 displayed IC50
s
of 256 and 128 nM with 27% and 21% efficacy. Compounds 61, 28 and
25 showed activity that followed a similar trend to their ability to reduce
Zika virus titers with IC50s of 140 nM, 380 nM and 320 nM (with 24%,
44% and 27% corresponding efficacies) respectively. Compound 4, used
as a negative control, failed to show significant response in this assay
(Fig. 6).
General cytotoxicity of the selected compounds was measured in a
counter assay and is reported in the Supplemental information (Supp.
S2).
Taken together, our SAR analyses in the NS-1, E-protein, and Zika
viral titer assays identified compounds 6, 22, 25 and 28 that showed
comparable potencies to niclosamide but significantly improved in vitro
drug-like properties such as microsomal stability, solubility, and PAMPA
permeability for systemic exposure. Compound 6 had the best combi-
nation of activity in all three activity assays and developability assays,
positioning it as a promising candidate for in vivo evaluation. We also
discovered that the NO2 group, which is generally considered a liability
in drug-like compounds, on niclosamide could be replaced with a cyano
group (34) or a trifluoromethyl group (41 and 61) without losing po-
tency. We also found that 25 and 28 were equipotent in the viral titer
assay as the 4′-CN and 4′-CF3 replacements of the 4′-NO2 substituent (41
and 34). Advantageously, some of these novel leads have improved
developability properties as candidates for further in vivo pharmacoki-
netic profiling studies and advancing promising compounds to animal
models of Zika virus infection.
Fig. 3. Docking-based SAR modification of the salicylic acid motif at the inner
hydrophobic pocket and of the anilide motif at the open Arg-site.
for activity against the SARS-CoV-2 infection. As SARS-CoV-2 can
induce cell death after 48 to 72 h of infection, the activity of the com-
pounds to protect against virus-induced cell death was evaluated in a
CPE (cytopathic effect) assay.56 Vero-E6 cells were chosen as host cells
and infected with SARS-CoV-2 strain (USA_WA1/2020) at MOI of 0.002
which resulted in approximately 5% cell viability 72 h post infection.
The detailed assay protocol is described in the Supplementary infor-
mation, but it should be noted that cell culture conditions, MOI and the
incubation time can differ from reported literature protocols, and thus
can affect reported potencies. In our hands, niclosamide showed an IC50
of 112 nM with an efficacy of 30% (Fig. 6), whereas Jeon et al. have
We further tested potent analogs (compounds 6, 25, 28, 34, 41 and
61) along with a negative control (compound 4) against the novel beta
corona virus SARS-CoV-2. We observed that the broad anti-viral prop-
erties of niclosamide and its analogs were also carried against SARS-
CoV-2; the analogs with better drug-like properties will be suitable
candidates to be tested in in vivo models of COVID-19.
reported 100% efficacy in a similar CPE assay with niclosamide (IC50
=
8

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 3
Combining salicylic acid and aniline SAR.
NS-1
IC50
(µM)
RLM, PAMPA, Solubility
(min, X10-6cm/sec, µg/ml)
E-protein
IC50 (µM)
#
Structure
59
60
1.66
2.68
2.06
4.14
>30, ND, <1
>30, 23.2, <1
61
62
1.59
2.5
1.49
1.45
>30, ND, <1
>30, ND, <1
63
64
3.14
2.57
4.22
3.68
>30, ND, 1.6
20.3, ND, <1
65
66
67
6.04
6.83
10.5
NT
NT
NT
>30, ND, <1
27, 111.2, 6
>30, ND, <1
68
69
5.4
NT
NT
>30, ND, <1
15, 553, <1
12.5
IC50 values are average from three biological replicates. NT = Not tested, ND = Not Determined.
9

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Table 4
Correlation of virus NS-1 and virus titer assay.
Compound ID
NS-1
E-protein IC50 (µM)
Viral titer reduction
Cell Viability-Nuclei Counta
IC50 (µM)
IC50 (µM)
1
0.6
0.84
2.5
0.88
0.99
2.10
1.38
2.34
3.73
1.49
8.32
5.5
0.12
0.11
0.82
0.84
0.81
0.82
0.4
57% at 30 µM
59% at 30 µM
61% at 30 µM
59% at 30 µM
63% at 30 µM
59% at 30 µM
55% at 30 µM
68% at 30 µM
73% at 30 µM
6
25
41
34
28
61
38
31
0.8
1.7
2.5
1.6
11.4
12.9
1.8
8.6
a
Cytotoxicity was performed in SNB-19 cells, activity is normalized to DMSO control.
Fig. 4. Dose dependent reduction of virus production (as measured in focal forming units) in compound-treated SNB-19 cells.
Fig. 5. Dose dependent reduction of virus production (as measured in focal forming units) in compound treated SNB-19 host cells for niclosamide and compound 6
(purple), overlaid with cytotoxicity curves (blue) as measured by cell count in host SNB-19 cells.
10

K. Shamim et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127906
Fig. 6. Concentration response rescue curves for the key niclosamide analogs against SARS-CoV-2 CPE activity in Vero-E6 cells.
10 Miner JJ, Sene A, Richner JM, et al. Zika virus infection in mice causes Panuveitis
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13 Araujo LM, Ferreira ML, Nascimento OJ. Guillain-Barre syndrome associated with
the Zika virus outbreak in Brazil. Arq Neuropsiquiatr. 2016;74:253–255.
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Acknowledgements
This work was supported by the Intramural Research Program of the
National Center for Advancing Translational Sciences (NCATS) of the
NIH and NIH grant U19AI131130 (H.T. and G-I.M.). The authors thank
members of NCATS Analytical Chemistry, Drug Metabolism and Phar-
macokinetics and Compound Management (CoMa) for their support and
collaboration.
´
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Appendix A. Supplementary data
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broad-spectrum antivirals and NIH clinical collection compounds using a cell-based,
high-throughput screen assay. Antiviral Res. 2017;138:47–56.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127906.
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identified by a high-throughput screen for Zika protease inhibitors. Proc Natl Acad Sci
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