Discovery of a small molecule inhibitor of human adenovirus capable of preventing escape from the endosome

Article abstract of DOI:10.3390/ijms22041617

Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections in immunocompromised individuals. However, no antiviral drug has been approved specifically for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicy-lamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection. Compound 16 displays submicromolar IC₅₀ values, a higher selectivity index (SI > 100) and 2.5-fold virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide, our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating and the presentation of lytic protein VI.

Full text of DOI:10.3390/ijms22041617

International Journal of
Molecular Sciences
Article
Discovery of a Small Molecule Inhibitor of Human Adenovirus
Capable of Preventing Escape from the Endosome
Jimin Xu 1,† , Judith Berastegui-Cabrera 2,†, Marta Carretero-Ledesma 2, Haiying Chen 1, Yu Xue 1,
Eric A. Wold 1 , Jerónimo Pachón 2,3 , Jia Zhou 1,
*
and Javier Sánchez-Céspedes 2,
*
1
Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical
Branch (UTMB), Galveston, TX 77555, USA; jimxu@utmb.edu (J.X.); haichen@utmb.edu (H.C.);
yuxue1@utmb.edu (Y.X.); eawold@utmb.edu (E.A.W.)
Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville (IBiS),
University Hospital Virgen del Rocío, CSIC, University of Seville, E41013 Seville, Spain;
jbc9393@hotmail.com (J.B.-C.); marta.carreterole@gmail.com (M.C.-L.); pachon@us.es (J.P.)
Department of Medicine, University of Seville, E-41009 Seville, Spain
2
3
*
Correspondence: jizhou@utmb.edu (J.Z.); jsanchez-ibis@us.es (J.S.-C.); Tel.: +(1)-409-772-9748 (J.Z.);
+(34)-955-923-100 (J.S.-C.)
†
These authors contributed equally to this work.
Abstract: Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an
array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections
in immunocompromised individuals. However, no antiviral drug has been approved specifically
for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicy-
lamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection.
Compound 16 displays submicromolar IC50 values, a higher selectivity index (SI > 100) and 2.5-fold
virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide,
our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved
through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating
and the presentation of lytic protein VI.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Xu, J.; Berastegui-Cabrera,
J.; Carretero-Ledesma, M.; Chen, H.;
Xue, Y.; Wold, E.A.; Pachón, J.; Zhou,
J.; Sánchez-Céspedes, J. Discovery of
a Small Molecule Inhibitor of Human
Adenovirus Capable of Preventing
Escape from the Endosome. Int. J.
Mol. Sci. 2021, 22, 1617. https://
doi.org/10.3390/ijms22041617
Keywords: antiviral agent; adenovirus; salicylamide derivatives; entry inhibition
Academic Editors: Li Lin and
Kamalendra Singh
1. Introduction
Human adenoviruses (HAdVs) are common pathogens with broad tissue tropism
and are frequently responsible for infections displaying mild to moderate respiratory
and gastrointestinal symptoms as well as conjunctivitis, notably in pediatric populations
and among active duty military service members. HAdVs are characterized by a linear
double-stranded DNA genome, ranging from 34–36 kb in size, surrounded by a nonen-
Received: 2 January 2021
Accepted: 2 February 2021
Published: 5 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
veloped, icosahedral capsid [
serotypes distributed among seven species (HAdV A–G), which display a large variance
in infection and symptom severity [ ]. In healthy individuals with normal immune
1]. Presently, HAdVs compromise more than 100 classified
2,3
function, HAdV infections are often self-limiting and are rarely associated with severe
disease. However, in immunocompromised individuals and other at-risk groups, such as
solid-organ transplant (SOT) and allogenic hematopoietic stem cell transplant (allo-HSCT)
recipients, HAdV infections can result in life-threatening illness and infection-related medi-
Copyright:
© 2021 by the authors.
cal complications [
by a frequency of infections recorded as high as 42–47% with the mortality rate up to 80%
in cases with disseminated disease [ ]. Additionally, following increasingly accurate
molecular diagnostic technology development, HAdV infections have been found to be
involved in both isolated cases and outbreaks of community-acquired pneumonia (CAP)
in otherwise healthy individuals [8–13].
4,5]. Pediatric allo-HSCT recipients are especially high-risk, exemplified
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4,6,7
Int. J. Mol. Sci. 2021, 22, 1617. https://doi.org/10.3390/ijms22041617
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Despite the consequences of acquiring disseminated HAdV infection, there are no an-
tiviral drugs specifically approved for the treatment of HAdV infections [14–21]. Currently,
intravenous cidofovir (Figure 1) is used off-label to treat HAdV-associated diseases in
many transplant clinics. Cidofovir, an acyclic nucleotide phosphonate cytosine analogue, is
considered a broad-spectrum antiviral agent against DNA viruses and has shown efficacy
against all HAdV serotypes in vitro, acting as a chain terminator during DNA replica-
tion [22]. However, cidofovir lacks sufficient oral bioavailability and is highly subject to
renal elimination; thus, even its intravenous administration can result in notable nephrotox-
icity and myelotoxicity, necessitating the routine coadministration of probenecid to mitigate
nephrotoxicity [14]. In efforts to utilize the efficacy against HAdVs, a series of lipid ester
derivatives of cidofovir were synthesized that displayed meaningfully decreased nephro-
toxicity by reducing kidney exposure to the drug, and were also marked by increased oral
bioavailability and cellular uptake due to the lip moiety, which is cleaved inside the cell by
phospholipase to transform the prodrug into cidofovir [23–25]. Among these derivatives,
the alkoxyalkyl ester prodrug brincidofovir (BCV, CMX001, 3-hexadecyloxy-1-propanol-
cidofovir, 2) showed promising results in phase II and III clinical trials (NCT01231344
and NCT02087306), which heightened interest in its potential to be developed as the first
approved treatment for HAdV infections [26–29]. Unfortunately, long-term trials of BCV
treatment led to adverse effects such as diarrhea in some patients and also highlighted that
alternative administration strategies should be considered [29]. Therefore, there is still an
urgent need to develop highly effective antivirals against HAdV infections that are charac-
terized by a lack of toxicity, reduced adverse effects, and increased oral bioavailability.
Figure 1. Representative human adenovirus (HAdV) inhibitors.
We discovered that niclosamide (3), an FDA-approved anthelmintic drug that is a part
of our ongoing antiviral drug discovery and development program [30
inhibited HAdV infection with an IC50 value of 0.6 M, displayed moderate cytotoxicity
(CC50 = 22.9 M), and was characterized as having a narrow safety window, as shown by a
–36], significantly
µ
µ
selectivity index (SI) of 38.2 [37]. To improve potency and decrease toxicity, we focused
our previous strategies for chemical modification on the aniline moiety substituents and
changes to the amide linker, whereby a series of salicylamide derivatives were discovered
as potent anti-HAdV inhibitors with improved safety windows [38
derivatives, the 3’-fluoro-5’-trifluoromethyl substituted compound and the 2’-chloro-5’-
(1-methylcyclopentyl)amino substituted compound showed improvements in potency
(IC50 = 0.18 M and 0.27 M, respectively), decreased cytotoxicity (CC50 = 120.0 M and
156.8 M, respectively) and dramatically increased the selectivity index (SI = 666.7 and
580.7, respectively), whereas compound modified with a L-valine linker maintained the
,39]. Among these
4
5
µ
µ
µ
µ
6

Int. J. Mol. Sci. 2021, 22, 1617
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same level of potency (IC50 = 0.45 µM) with a decreased cytotoxicity (CC50 = 200.0 µM)
and increased selectivity index (SI = 444.4), compared to the hit compound niclosamide.
However, all of these lead compounds yielded from our previous optimization efforts
shared the similarity by containing an aniline group to form the amide scaffolds like
niclosamide, and whether this moiety can be altered with more conformationally flexible
alkyl groups remains unclear. Herein, as depicted in Figure 2, we further explored the
structure–activity relationship (SAR) studies by replacing the aniline moiety of niclosamide
with various alkylamine groups to identify new antiviral agents with potential action
mechanisms distinct from that of niclosamide. This work highlights the discovery of a
unique compound 16 (JMX0493), which displays an improved selectivity index while
acting as a potent inhibitor of HAdV infection by targeting the HAdV entry pathway that
prevents viral particle disassembly and subsequent release from the endosome.
Figure 2. The drug design for this work, which highlights the discovery of new compound 16 (JMX0493) with both improved
selectivity index and capability of inhibiting the escape from the endosome of the viral particles.
2. Results and Discussion
2.1. Chemistry
The general synthesis of salicylamide derivatives is shown in Scheme 1. Direct sub-
stitution of methyl 5-chloro-2-hydroxybenzoate with a variety of amines in methanol
afforded derivatives 11–15, 18–25, and 28. As shown in Scheme 2, condensation of 5-
chloro-2-hydroxybenzoic acid with cyclohexylmethanamine or (S)-1-cyclohexylethanamine
provided compounds 16 and 17, respectively. 5-Chloro-2-methoxybenzoic acid was cou-
pled with piperidine or 1-acetylpiperazine followed by demethylation with BBr3 to give
compounds 26 and 27, respectively. Efficient and accessible synthetic methodology is
imperative in antiviral discovery efforts and remains a consideration in our molecular
design along with robust SAR studies discussed in the following section.
2.2. In Vitro Evaluation of Human Adenovirus Inhibition
The newly synthesized compounds were first screened in the plaque assay, measuring
the inhibition of HAdV plaque formation. As shown in Table 1, we first substituted the
aniline moiety of niclosamide with simple cycloalkanamines (11–15). When we increased
the ring size of the cycloalkyl groups, the corresponding compounds 11
trend where ring size correlated with increased potency with inhibition values of 22.6%,
57.6%, and 99.6% at 10 M, respectively. Compound 13 with the N-cycloheptyl substitution
displayed micromolar potency (IC50 = 4.7 M) and similar cytotoxicity (CC50 = 30.9 M) to
niclosamide. Interestingly, the tetrahydropyran derivative 14 was completely inactive at
10 M, whereas the N-Boc-piperidine-substituted compound 15 inhibited HAdV plaque
formation with a percentage of 52.2%. Inserting one carbon atom as a linker between the
amide and cycloalkyl produced compounds 16 19. Excitingly, the N-cyclohexylmethyl
substituted derivative 16 exhibited submicromolar potency (IC50 = 0.78 M) and, in contrast
to niclosamide, displayed significantly decreased cytotoxicity (CC50 = 91.2 M) and an
–13 showed a
µ
µ
µ
µ
–
µ
µ

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increased selectivity index (SI = 116.9). The introduction of an (S)-methyl on the methylene
linker (17) retained the same level of potency (IC50 = 0.51
µM) but significantly increased
the cytotoxicity (CC50 = 23.0 M) as compared with derivative 16, steering our efforts away
µ
from similar such modifications. Compound 18 with a tetrahydropyran moiety showed no
anti-HAdV activity, whereas the N-Boc protected derivative 19 was active with an inhibition
percent of 88.5% against HAdV plaque formation at 10
atom in the linker to achieve the N-cyclohexylethyl derivative 20, we observed a slight
loss of potency (IC50 = 1.0 M) but also diminished cytotoxicity (CC50 = 66.3 µM) and
µM. By adding an additional carbon
µ
improved selectivity index (SI = 64.3) compared to niclosamide. However, building on
the two-carbon linker theme, we found that neither piperazine analogue (21 and 22) nor
the morpholine analogue 23 showed meaningful inhibitory activity against HAdV plaque
formation up to 10 µM. The lack of inhibitory activity by 23 and 14 may be associated
with limited tolerance for electronegative atoms within R2 position substituents and is
under subsequent investigation. Surprisingly, derivative 24 with a linear hexylamine
substitution maintained single digit micromolar potency with an IC50 value of 3.73
As expected, N-Boc protected derivative 25 was also active against HAdV with inhibition
of 74.0% at 10 M. To probe molecular space constraints, secondary amine intramolecular
ring moieties were applied but found not to be tolerated in any configuration tested, as
shown by the activity data for compounds 26 28. Based on our previous work and the SAR
µM.
µ
–
reported herein, we hypothesize that the target binding site accommodating the R2 moiety
is preferentially engaged by simple hydrophobic moieties that are either of moderate length
(e.g., one carbon linker) or shortened, bulky groups (e.g., N-cycloheptyl moiety), and are
devoid of terminal electronegative atoms as seen in tetrahydropyran and morpholine
derivatives. Larger, more complex moieties may be tolerated, but at a cost of incorporating
additional functional groups that are metabolically labile or contributors to cytotoxicity.
Thus, compound 16 was of keen interest due to the conservative N-cyclohexylmethyl
substitution and its noteworthy inhibition activity, low cytotoxicity, and high selectivity
index (SI > 100).
Scheme 1. Synthetic route to achieve compounds 11
–
15
, 18–25, and 28. Reagents and conditions: (i)
◦
R1NH2 or 1-methylpiperazine, CH3OH, r.t. to 80 C, 48–96 h.

Int. J. Mol. Sci. 2021, 22, 1617
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Scheme 2. Synthetic route to achieve compounds 16, 17,
26, and 27. Reagents and conditions: (ii) R1NH2, EDCI, DMAP,
◦
◦
DCM, r.t., 12 h. (iii) piperidine or 1-acetylpiperazine, EDCI, DMAP, DCM, r.t., 2 h; (iv) BBr3, DCM, −78 C to 0 C, 12 h.
Table 1. Inhibition of HAdV in plaque assay, cytotoxicity, and selectivity index for compounds 11–28.
Plaque Assay (10 µM) a
CC50 (µM) b
22.9 ± 9.8
R1
R2
Compound
3
Selectivity Index (SI) c
(%) Inhibition
IC50 (µM)
0.6 ± 0.05
NA
100 ± 0
38.2
NT d
11
12
H
H
22.6 ± 7.4
57.6 ± 0.2
NT
NT
NT
NT
NT
13
14
H
H
99.6 ± 0.2
0.0 ± 0.0
4.7 ± 0.1
30.9 ± 6.3
6.6
NT
NT
NT
15
16
17
H
H
H
52.2 ± 7.1
100 ± 0
100 ± 0
NT
NT
NT
116.9
45.1
0.78 ± 0.01
0.51 ± 0.14
91.2 ± 18.4
23.0 ± 1.3

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Table 1. Cont.
Plaque Assay (10 µM) a
CC50 (µM) b
R1
R2
Compound
Selectivity Index (SI) c
(%) Inhibition
IC50 (µM)
18
19
H
H
0.0 ± 0.0
NT
NT
NT
NT
NT
88.5 ± 1.9
97.8 ± 1.7
NT
20
21
22
23
H
H
H
H
1.0 ± 0.1
66.3 ± 7.9
NT
64.3
NT
NT
NT
22.2 ± 13.2
0.00 ± 0.00
0.00 ± 0.00
NT
NT
NT
NT
NT
24
25
26
H
H
98.5 ± 2.1
74.0 ± 5.7
0.00 ± 0.00
3.73 ± 0.64
NT
23.8 ± 3.1
NT
6.4
NT
NT
NT
NT
27
28
0.00 ± 0.00
NT
NT
NT
NT
NT
NT
0.00 ± 0.00
a
Percentage of control HAdV5-GFP inhibition at 10 µM and inhibitory concentration 50% (IC50) at low MOI in a plaque assay using
b
c
the 293β5 cell line. Cytotoxic concentration 50% (CC50) using A549 cell line. Selectivity index value was determined as the ratio of
CC50 to IC50 in a plaque assay for each compound. NT: not tested. The results represent means
independent experiments.
d
±
SD of triplicate samples from three
We next evaluated the anti-HAdV effect of compound 16 using a virus burst assay
that can measure the ability of a pharmacological agent to suppress the production of
new viral particles. As shown in Table 2, the addition of compound 16 resulted in a
208-fold overall reduction in virus yield, 2.5 times greater than the reduction observed by
niclosamide addition.
2.3. Antiviral Mechanism of Action Studies
The HAdV replicative cycle is a multistage process characterized by numerous, low-
redundancy steps that may be targeted to inhibit viral attachment, entry, replication,
and/or escape. Thus, we investigated several key points in its replicative cycle to identify
a potential target responsible for the antiviral activity of compound 16.

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Table 2. Virus yield reduction for derivative 16.
Compound
Virus Yield Reduction (fold) a
niclosamide
82 ± 35
16
208 ± 108
a
Fold reduction in virus yield as the ratio of particles produced in the presence of DMSO divided by the yield in
the presence of niclosamide (3) at 5 µM and derivative 16 at 10-fold IC50 concentration obtained in the plaque
assay. Virus yield reduction assay used A549 cell line and the MOI of HAdV was 100 vp/cell. The results represent
means ± SD of triplicate samples from three independent experiments.
2.3.1. Impact of Compound 16 on HAdV Replication
We first examined the effect that compound 16 had on the HAdV DNA replication pro-
cess. As viral replication is dependent upon efficient DNA replication, we performed quan-
titative real-time PCR to measure HAdV DNA replication in the presence of compound 16
.
After one round of infection, DNA was extracted at 24 h, avoiding the influence of newly
generated viral particles derived from subsequent rounds of infection occurring at 32–36 h
timepoints [19]. As shown in Figure 3, compound 16 displayed significant activity in the
qPCR assay, inhibiting or delaying HAdV-5 DNA replication by 50% at a concentration
of 7.8
µ
M (p
≤
0.001) when compared to the DMSO treated control, whereas niclosamide
prevented any detectable DNA replication. This result indicates that compound 16 likely
targets, at least partially, the HAdV DNA replication process. The decrease in HAdV
DNA copy number in the presence of compound 16 suggests that this molecule could be
interfering directly with proteins involved in HAdV DNA replication or with an earlier
step in its replicative cycle, through which interference would indirectly downregulate
DNA replication.
Figure 3. Effect of compound 16 on the HAdV DNA replication. Concentration of niclosamide
(3) was 5
µM and 10-fold IC50 concentration obtained in the plaque assay for compound 16. Bars
represent means ± SD of triplicate samples. *** p ≤ 0.001, **** p ≤ 0.0001.

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2.3.2. Time of Addition Assay
To obtain a more complete picture and pinpoint the potential step in the HAdV
replicative cycle targeted by compound 16, we measured the time dependence of compound
addition on its ability to inhibit HAdV. HAdV has been shown to be enter the cell within
5 min of viral attachment, to escape the endosome after 15 min, and to attach to the nuclear
pore complex after 35 45 min postinfection (p.i.). Our results showed that the antiviral
−
activity of compound 16 exhibited a time-dependent decrease in activity Figure 4, especially
apparent at early time points where we observed a decrease in its inhibitory activity from
95.3% inhibition at 0 min p.i. to 30.7% inhibition at 20 min p.i. Collectively, these results
indicate that compound 16 inhibits an early step in the virus cycle, after cell attachment
and before entry into the cell nucleus.
Figure 4. Effect of compound 16 on HAdV infection at different time points. The concentrations used
were 5 µM for niclosamide (3) and 10-fold IC50 concentration obtained in the plaque assay for analog
16. Line charts represent means ± SD of duplicate samples.
To confirm if the antiviral activity of compound 16 was dependent on a step between
HAdV entry and the arrival to the nucleus, we performed an assay to quantify the HAdV
genome accessibility to the nucleus. Once HAdV escapes from the endosome, the partially
uncoated capsid is transported to the cell nucleus, where further disassembly occurs
and the HAdV genome is released into the cell nucleus for early gene transcription and
subsequent replication. Thus, any inhibitory effect on that process will be reflected by
the number of HAdV genomes that reach the host nucleus after a synchronized infection.
Briefly, 45 min after infection, cells were recovered to separate the cytoplasmic and nuclear
fractions. Then, HAdV DNA was isolated from each fraction and quantify by the real-time
PCR. As shown in Figure 5, compound 16 inhibited more than 99% of the HAdV DNA
accessibility to the nucleus and showed a higher inhibitory effect compared to the DMSO
control experiment and niclosamide at 45 min postinfection (p
≤
0.0001). At this point, our
mechanistic approaches confirmed that the antiviral activity of compound 16 was targeting
a step before HAdV transcription of early genes and DNA replication. Going backwards
in the HAdV replicative cycle, the next process is to identify the precise step targeted by
compound 16 through evaluating the HAdV escape from the endosome.

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Figure 5. Percentage of nuclear-associated HAdV genome of compound 16. Concentration of niclosamide
(3) was 5 µM and 10-fold IC50 concentration obtained in the plaque assay for compound 16. Bars represent
means ± SD of triplicate samples. **** p ≤ 0.0001.
2.3.3. Impacts of Compound 16 on HAdV Escape from the Endosome
Upon attachment to its cellular receptors, HAdV particles are internalized by en-
docytosis into the cells and viral particles undergo partial disassembly inside the early
endosomes, resulting in the release of protein VI from the interior of the capsid, which
plays a key role in HAdV escape from the endosome [40]. To evaluate the potential influ-
ence of compound 16 in preventing the HAdV escape from the endosome, we used the
α
-sarcin co-delivery assay as a measurement of the ability of this compound to interfere
with virus-mediated endosome lysis. Since the -sarcin alone is unable to penetrate the
α
cell, in this assay, the successful virus-mediated lysis of the endosome would result in
ribotoxin-mediated inhibition of cellular protein synthesis. Increasing concentrations of
HAdV in the presence or absence of compound 16 were mixed with α-sarcin and incubated
with A549 cells. After metabolic labeling with methionine L-homopropargylglycine (HPG),
cell samples were assayed for HPG incorporation into cellular proteins. As shown in
Figure 6, cells treated with either the vehicle (DMSO) or niclosamide showed rates of
HPG incorporation under 50% compared to a control without α-sarcin, independent of the
virus concentration used. Interestingly, compound 16 showed a similar behavior as the
entry-defective ts1 mutant HAdV that contains a mutation in the protease gene and fails to
penetrate cell endosomes [41]. Compound 16 prevented HAdV-mediated endosome lysis
at concentrations of the virus below 12 ng, similar to the effects observed by the ts1 mutant.
This result, together with our time of addition assay, showing a decrease of the inhibitory
effect after 15–20 min p.i, suggests that the mechanism for inhibition of this compound is
likely related to the blockage of HAdV escape from the endosome.

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Figure 6. Activity of compound 16 on HAdV-mediated endosomolysis in a α-sarcin assay. The concentrations used were
5 µM for niclosamide (3) and 10-fold IC50 concentration obtained in the plaque assay for derivative 16. Results represent
means ± SD of triplicate assays.
The studies described above delineate the step of infection that is targeted by com-
pound 16; however, the precise molecular mechanism involved in its antiviral activity
remained to be established. One alternative mode of action was that compound 16 could
block HAdV-mediated lysis of the endosome by stabilizing the virus capsid, thereby
preventing uncoating. Indeed, human defensins and neutralizing antibodies have been
previously shown to act through this mechanism [42]. To determine whether compound 16
impacts HAdV uncoating, we used a thermostability assay that mimics virus disassembly
in the endosome, which was previously described by Wiethoff et al. [40], with a few modi-
fications. In this assay, temperatures above 48 ◦C promote selective removal of the virus
vertex region. HAdV-5 was incubated with or without 50 µM concentrations of compound
16 at temperatures from 37 ◦C to 52 ◦C and then added to cells to evaluate the viability
of the viruses. Viruses incubated with either compound 16 or DMSO at 37 ◦C, 40 ◦C, and
44.5 ◦C were largely intact, showing similar rates of infect◦ion measured by the number of
cells expressing GFP Figure 7. Upon heating HAdV-5 to 48 C or above, in those nontreated
with compound 16, there was no GFP expression 24 h postinfection. However, in wells
with the addition of 16, significant levels of GFP expression were observed for those viruses
heated to 48 ◦C, and some residual expression remained at 52 ◦C. These findings suggest
that compound 16 may prevent HAdV endosomal escape by stabilizing the viral capsid
and subsequently inhibiting endosomal membrane lysis [43].
2.4. Synergistic Activity of the Selected Compounds
In previous reports [38,39], we found three compounds—4 (JMX0312), 5 (JMX0510-2),
and
6 (JMX0281)—with high activity against HAdV, targeting different steps in the HAdV
replicative cycle. We hypothesize that the double and triple combination of these com-
pounds with compound 16 (JMX0493) should significantly increase their antiviral activity.
To evaluate our hypothesis, we conducted a combination study based on the Chou–Talalay
method for drug combination using the CalcuSyn software [44]. The constant ratio for
each combination was selected based on the IC50 values for each drug. The data for all the
combinations showed good conformity with the mass action law principle (r = 0.87–0.95
)

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Table 3. Two out of three double combinations showed a synergistic effect, and concretely,
the combination of compounds
and the combination of compounds
(ED50 = 0.40), while the combination of compounds
additive effect (ED50 = 1.09). For the triple combinations, one of them (the combination of
compounds , and 16; ratio 1:1.5:4.3) exhibited a synergistic effect with an ED50 value of
0.33, while the other triple combination (compounds , and 16; ratio 1:1.7:2.9) showed a
4
and 16 (ratio 1:4.3) showed strong synergism (ED50 = 0.26)
and 16 (ratio 1:1.73) was also classified as synergistic
and 16 (ratio 1:2.9) showed only an
6
5
4, 5
5, 6
clear antagonistic effect (ED50 = 2.01), probably due to compound interactions. It is worth
noting that in our previous report, the combinatory index also increased when compound
5 was added to the mix, reaching additive or antagonistic values.
Figure 7. Compound 16 stabilizes HAdV capsid. The concentration used was 50 µM. Data are representative of three experiments.
Table 3. IC50 values in a plaque assay for each derivative and its combination. Combination index
value was calculated with CalcuSyn software.
Combinatory Index (CI) Values in Plaque Assay
Combination (Ratio)
4 and 16 (1:4.3)
ED50
0.26
1.09
0.40
0.33
2.01
ED75
0.38
1.08
0.27
0.26
1.17
ED90
0.56
1.14
0.19
0.22
0.72
r
0.87
0.92
0.94
0.94
0.95
5 and 16 (1:2.9)
6 and 16 (1:1.73)
4, 5, and 16 (1:1.5:4.3)
5, 6, and 16 (1:1.7:2.9)
2.5. Impact of Compound 16 on HCMV Replication
The broad antiviral activity of compound 16 was tested using human cytomegalovirus
(HCMV) in a DNA replication assay. To avoid the influence of newly generated viral
particles from subsequent rounds of infection, HCMV DNA was extracted from HFF cells
at 72 h postinfection and quantified in a single round of infection as a measurement of the
DNA replication efficiency. The addition of compound 16 displayed significant reductions
(p
≤
0.001) in the quantification of total HCMV DNA, showing a 95% decrease compared
to the DMSO control, as depicted in Figure 8.

Int. J. Mol. Sci. 2021, 22, 1617
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Figure 8. Effect of compound 16 on HCMV replication. Concentration of niclosamide (3) was 5 µM
and compound 16 was tested at 10-fold IC50 concentration obtained in the HAdV plaque assay. Bars
represent means ± SD of triplicate samples. *** p ≤ 0.001.
3. Conclusions
In summary, salicylamide derivatives were designed, synthesized, and biologically
evaluated for their antiviral activity against HAdV. Among these new molecules, com-
pound 16 (JMX0493) maintained submicromolar potency against HAdV while displaying a
markedly higher selectivity index (SI > 100) compared to the hit compound niclosamide.
In addition, compound 16 was able to reduce virus yield to a 2.5-fold greater extent
than niclosamide, indicative of a safer and more effective profile in virus yield reduction.
Moreover, compound 16 demonstrated potential as a broad antiviral agent by showing a
significant inhibition of HCMV DNA replication. As for its mechanism of action, unlike
niclosamide, compound 16 showed a specific inhibition of HAdV infection via blocking the
escape of viral particles from the endosome by stabilizing the viral capsid and preventing
HAdV uncoating, thereby blocking subsequent viral migration to the nuclear membrane.
4. Materials and Methods
4.1. Chemistry
All commercially available starting materials and solvents were reagent grade and
used without further purification. Reactions were performed under a nitrogen atmosphere
in dry glassware with magnetic stirring. Preparative column chromatography was per-
formed using silica 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 on a Brucker-300 (1H, 600 and 300 MHz; 13C, 150 and 75 MHz)
1
spectrometer. H and 13C NMR spectra were recorded with TMS as an internal reference.
1H and 13C NMR spectra of representative compounds are shown in Supplemental Figures
(page S2–S19 in Supporting Information). Chemical shifts were expressed in ppm, and
J values were given in Hz. High-resolution mass spectra (HRMS) were obtained using a
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. Melting points were measured on a
Thermo Scientific Electrothermal Digital Melting Point Apparatus and were uncorrected.
Purities of final compounds were established using analytical HPLC, which was carried

Int. J. Mol. Sci. 2021, 22, 1617
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out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A UV/VIS). HPLC
analysis conditions: Waters
µ
Bondapak C18 (300
×
3.9 mm); flow rate 0.5 mL/min; UV
detection at 270 and 254 nm; linear gradient from 10% acetonitrile in water to 100% acetoni-
trile 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.
General procedure A. Methyl 5-chloro-2-hydroxybenzoate (1.0 eq) was dissolved in
methanol (10 mL/0.5 mmol) followed by addition of different amine (3.0 eq). The resulting
mixture was stirred at r.t. ~80 ◦C for 48~96 h, and then concentrated. The residue was
purified by preparative TLC to afford the final amide products.
5-Chloro-N-cyclopentyl-2-hydroxybenzamide (11). Compound ◦11 (116 mg, 90%)
was prepared as a beige solid according to general procedure A (60 C, 48 h), starting
from methyl 5-chloro-2-hydroxybenzoate and cyclopentylamine. HPLC purity 99.9%
1
(tR = 18.07 min). H NMR (300 MHz, CDCl3)
δ 12.34 (s, 1H), 7.37–7.26 (m, 2H), 6.90 (d,
J = 8.7 Hz, 1H), 6.39 (d, J = 4.2 Hz, 1H), 4.45–4.27 (m, 1H), 2.21–1.98 (m, 2H), 1.83–1.45 (m,
6H). 13C NMR (75 MHz, CDCl3)
δ 168.6, 160.0, 133.9, 125.2, 123.3, 120.1, 115.5, 51.8, 33.1 (2C),
23.9 (2C). HRMS (ESI) calculated for C12H15ClNO2, 240.0791 (M + H)+; found, 240.0785.
5-Chloro-N-cyclohexyl-2-hydroxybenzamide (12). Compound 12 (71 mg, 51%)
was prepared as a beige solid according to general procedure A (60 ◦C, 48 h), starting
from methyl 5-chloro-2-hydroxybenzoate and cyclohexylamine. HPLC purity 99.9%
(tR = 18.85 min)
J = 8.4 Hz, 1H), 6.21 (d, J = 5.4 Hz, 1H), 4.00–3.86 (m, 1H), 2.06–1.94 (m, 2H), 1.83–1.60
(m, 3H), 1.48–1.13 (m, 5H). 13C NMR (75 MHz, CDCl3)
168.1, 160.2, 134.0, 125.1, 123.3,
. δ 12.36 (s, 1H), 7.35–7.27 (m, 2H), 6.90 (d,
1H NMR (300 MHz, CDCl3)
δ
120.2, 115.6, 49.0, 33.0 (2C), 25.5, 25.0 (2C). HRMS (ESI) calculated for C13H17ClNO2,
254.0948 (M + H)+; found, 254.0941.
5-Chloro-N-cycloheptyl-2-hydroxybenzamide (13). Compound 13◦(75 mg, 52%) was
prepared as an off-white solid according to general procedure A (60 C, 48 h), starting
from methyl 5-chloro-2-hydroxybenzoate and cycloheptylamine. HPLC purity 99.5%
1
(
tR = 19.58 min). H NMR (300 MHz, CDCl3)
δ
12.36 (s, 1H), 7.34–7.27 (m, 2H), 6.90 (d,
J = 9.0 Hz, 1H), 6.27 (d, J = 5.7 Hz, 1H), 4.20–4.03 (m, 1H), 2.08–1.93 (m, 2H), 1.74–1.47 (m,
10H). 13C NMR (75 MHz, CDCl3)
δ 167.8, 160.2, 133.9, 125.1, 123.3, 120.2, 115.6, 51.2, 35.1
(2C), 28.0 (2C), 24.2 (2C). HRMS (ESI) calculated for C14H19ClNO2, 268.1104 (M + H)+;
found, 268.1097.
5-Chloro-2-hydroxy-N-(tetrahydro-2H-pyran-4-yl)benzamide (14). Compound 14
(
30 mg, 22%) was prepared as a light-yellow solid according to general procedure A (80 ◦C
,
96 h), starting from methyl 5-chloro-2-hydroxybenzoate and 4-aminotetrahydropyran.
1
HPLC purity 99.9% (tR = 15.48 min). H NMR (300 MHz, CDCl3)
δ 12.18 (s, 1H), 7.38–7.29
(m, 2H), 6.98–6.88 (m, 1H), 6.21 (d, J = 6.6 Hz, 1H), 4.26–4.12 (m, 1H), 4.07–3.96 (m, 2H),
3.59–3.46 (m, 2H), 2.05–1.91 (m, 2H), 1.69–1.52 (m, 2H). 13C NMR (75 MHz, CDCl3)
δ
168.4,
160.3, 134.3, 125.1, 123.4, 120.3, 115.3, 66.8 (2C), 46.5, 33.1 (2C). HRMS (ESI) calculated for
C12H15ClNO3, 256.0740 (M + H)+; found, 256.0734.
tert-Butyl 4-(5-chloro-2-hydroxybenzamido)piperidine-1-carboxylate (15). Compound
15 (24 mg, 13%) was prepared as an off-white solid according to general procedure A
(80 ◦C, 96 h), starting from methyl 5-chloro-2-hydroxybenzoate and 4-aminopiperidine-1-
1
carboxylic acid tert-butyl ester. HPLC purity 98.8% (tR = 18.51 min). H NMR (300 MHz
,
CDCl3)
δ 12.21 (s, 1H), 7.43–7.23 (m, 2H), 6.96–6.88 (m, 1H), 6.44 (d, J = 6.6 Hz, 1H),
4.20–4.02 (m, 3H), 2.97–2.80 (m, 2H), 2.05–1.94 (m, 2H), 1.52–1.40 (m, 11H). 13C NMR
(
75 MHz, CDCl3) δ 168.5, 160.3, 154.9, 134.2, 125.3, 123.4, 120.3, 115.4, 80.1, 47.5, 42.9
(2C), 32.0 (2C), 28.6 (3C). HRMS (ESI) calculated for C17H24ClN2O4, 355.1425 (M + H)+;
found, 355.1417.
5-Chloro-N-(cyclohexylmethyl)-2-hydroxybenzamide (16). To a solution of cyclohex-
anemethylamine (200 mg, 1.77 mmol), 5-chlorosalicylic acid (244 mg, 1.41 mmol) and
DMAP (18 mg, 0.14 mmol) in 20 mL of DCM were added EDCI (509 mg, 2.66 mmol) at 0 ◦C
.
The resulting mixture was stirred at r.t. for 24 h and then concentrated. The residue was
purified by column chromatography (Hex/EtOAc = 10/1) to afford compound 16 240 mg
(
,

Int. J. Mol. Sci. 2021, 22, 1617
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63%) as a light-yellow solid. HPLC purity 99.9% (tR = 19.68 min). 1H NMR (300 MHz
CDCl3) 12.22 (brs, 1H), 7.36–7.28 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 3.28 (t,
J = 6.3 Hz, 2H), 1.82–1.50 (m, 6H), 1.34–1.10 (m, 3H), 1.06–0.90 (m, 2H). 13C NMR (75 MHz,
,
δ
CDCl3) δ 169.0, 160.2, 134.0, 125.0, 123.4, 120.2, 115.5, 46.1, 38.0, 31.0 (2C), 26.4, 25.9 (2C).
HRMS (ESI) calculated for C14H19ClNO2, 268.1104 (M + H)+; found, 268.1101.
(S)-5-Chloro-N-(1-cyclohexylethyl)-2-hydroxybenzamide (17). Compound 17 was
prepared using a procedure similar to that used to prepare compound 16 starting from
5-chlorosalicylic acid and (S)-1-cyclohexylethanamine. The title compound was obtained
1
(
138 mg, 39%) as an off-white solid. HPLC purity 99.6% (tR = 20.25 min). H NMR
(300 MHz, CDCl3)
δ 12.36 (s, 1H), 7.33 (dd, J = 8.7, 2.4 Hz, 1H), 7.29 (d, J = 2.4 Hz, 1H), 6.93
(d, J = 8.7 Hz, 1H), 6.03 (d, J = 7.5 Hz, 1H), 4.12–3.98 (m, 1H), 1.85–1.61 (m, 5H), 1.50–1.38 (m,
1H), 1.32–0.95 (m, 8H). 13C NMR (75 MHz, CDCl3)
δ
168.3, 160.4, 134.0, 124.8, 123.3, 120.3,
115.6, 50.1, 43.2, 29.4, 29.2, 26.4, 26.2, 26.2, 18.0. HRMS (ESI) calculated for C15H21ClNO2,
282.1261 (M + H)+; found, 282.1257.
5-Chloro-2-hydroxy-N-((tetrahydro-2H-pyran-4-yl)methyl)benzamide (18). Compound 18
(117 mg, 80%) was prepared as a light-yellow solid according to general procedure A
(r.t., 48 h), starting from methyl 5-chloro-2-hydroxybenzoate and 4-(aminomethyl) tetrahy-
1
dropyran. HPLC purity 99.8% (tR = 16.12 min). H NMR (300 MHz, CDCl3)
δ 12.23 (s,
1H), 7.45 (d, J = 2.4 Hz, 1H), 7.31 (dd, J = 9.0, 2.4 Hz, 1H), 7.06 (s, 1H), 6.91 (d, J = 9.0 Hz,
1H), 4.05–3.94 (m, 2H), 3.49–3.27 (m, 4H), 1.99–1.81 (m, 1H), 1.73–1.61 (m, 2H), 1.47–1.29
(m, 2H). 13C NMR (75 MHz, CDCl3)
δ 169.1, 159.8, 134.0, 125.5, 123.5, 120.0, 115.6, 67.6
(2C), 45.4, 35.2, 30.6 (2C). HRMS (ESI) calculated for C13H17ClNO3, 270.0897 (M + H)+;
found, 270.0890.
tert-Butyl 4-((5-chloro-2-hydroxybenzamido)methyl)piperidine-1-carboxylate (19).
Compound 19 (74 mg, 37%) was prepared as an off-white solid according to general proce-
dure A (r.t., 48 h), starting from methyl 5-chloro-2-hydroxybenzoate and 4-(aminomethyl)-
1-N-Boc-piperidine. HPLC purity 99.8% (tR = 18.83 min). 1H NMR (300 MHz, CDCl3)
δ
12.23 (s, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.30 (dd, J = 8.7, 2.1 Hz, 1H), 7.08–6.86 (m, 2H),
4.20–4.02 (m, 2H), 3.32 (s, 2H), 2.79–2.59 (m, 2H), 1.87–1.67 (m, 3H), 1.45 (s, 9H), 1.25–1.09
(m, 2H). 13C NMR (75 MHz, CDCl3)
169.2, 160.1, 155.0, 134.1, 125.5, 123.5, 120.1, 115.6,
δ
79.8, 45.2, 43.7 (2C), 36.4, 30.0 (2C), 28.6 (3C). HRMS (ESI) calculated for C18H26ClN2O4,
369.1581 (M + H)+; found, 369.1572.
5-Chloro-N-(2-cyclohexylethyl)-2-hydroxybenzamide (20). Compound 20 (69 mg,
46%) was prepared as an off-white solid according to general procedure A (r.t., 48 h),
starting from methyl 5-chloro-2-hydroxybenzoate and 2-cyclohexylethylamine. HPLC
1
purity 99.9% (tR = 20.07 min). H NMR (300 MHz, CDCl3)
δ 12.33 (s, 1H), 7.38–7.27 (m,
2H), 6.91 (d, J = 8.7 Hz, 1H), 6.37 (s, 1H), 3.51–3.39 (m, 2H), 1.80–1.60 (m, 5H), 1.56–1.44 (m,
2H), 1.39–1.14 (m, 4H), 1.02–0.84 (m, 2H). 13C NMR (75 MHz, CDCl3)
δ
169.0, 160.1, 134.0,
125.1, 123.4, 120.2, 115.5, 37.9, 36.9, 35.5, 33.2 (2C), 26.5, 26.2 (2C). HRMS (ESI) calculated
for C15H21ClNO2, 282.1261 (M + H)+; found, 282.1253.
tert-Butyl 4-(2-(5-chloro-2-hydroxybenzamido)ethyl)piperazine-1-carboxylate (21).
Compound 21 (88 mg, 43%) was prepared as an off-white solid according to general
procedure A (r.t., 48 h), starting from methyl 5-chloro-2-hydroxybenzoate and 4-N-(2-
1
aminoethyl)-1-N-Boc-piperazine. HPLC purity 98.4% (tR = 15.08 min). H NMR (300 MHz,
CDCl3)
δ
7.35–7.25 (m, 2H), 7.15 (s, 1H), 6.89 (d, J = 8.7 Hz, 1H), 3.56–3.39 (m, 6H), 2.59 (t,
168.9, 160.1,
J = 6.0 Hz, 2H), 2.49–2.38 (m, 4H), 1.44 (s, 9H). 13C NMR (75 MHz, CDCl3)
δ
154.8, 134.0, 125.4, 123.3, 120.1, 115.5, 80.0, 56.3, 52.8 (2C), 43.8 (2C), 36.1, 28.5 (3C). HRMS
(ESI) calculated for C18H27ClN3O4, 384.1690 (M + H)+; found, 384.1680.
5-Chloro-2-hydroxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide (22). Compound 22
(62 mg, 37%) was prepared as a pale solid according to general procedure A (r.t., 48 h), start-
ing from methyl 5-chloro-2-hydroxybenzoate and 2-(4-methylpiperazin-1-yl)ethylamine.
1
HPLC purity 95.0% (tR = 11.25 min). H NMR (300 MHz, CDCl3)
δ
9.45 (br s, 1H, 7.34 (d,
J = 2.4 Hz, 1H), 7.29 (dd, J = 9.0, 2.7 Hz, 1H), 7.20 (s, 1H), 6.88 (d, J = 9.0 Hz, 1H), 3.53–3.44
(m, 2H), 2.70–2.33 (m, 10H), 2.29 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ
168.7, 160.0, 133.9,

Int. J. Mol. Sci. 2021, 22, 1617
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125.6, 123.3, 120.0, 115.7, 56.0, 55.2 (2C), 52.8 (2C), 46.1, 36.0. HRMS (ESI) calculated for
C14H21ClN3O2, 298.1322 (M + H)+; found, 298.1314.
5-Chloro-2-hydroxy-N-(2-morpholinoethyl)benzamide (23). Compound 23 (60 mg,
39%) was prepared as a grey solid according to general procedure A (r.t., 96 h), starting
from methyl 5-chloro-2-hydroxybenzoate and 4-(2-aminoethyl)morpholine. HPLC purity
99.6% (tR = 11.66 min). 1H NMR (300 MHz, CDCl3)
δ
7.35–7.28 (m, 2H), 7.03 (s, 1H),
6.94–6.88 (m, 1H), 3.74 (t, J = 4.5 Hz, 4H), 3.51 (t, J = 5.7 Hz, 2H), 2.61 (t, J = 6.0 Hz, 2H),
2.51 (t, J = 4.5 Hz, 4H). 13C NMR (75 MHz, CDCl3)
168.9, 160.2, 134.1, 125.3, 123.3, 120.2,
δ
115.4, 67.1 (2C), 56.5, 53.4 (2C), 35.8. HRMS (ESI) calculated for C13H18ClN2O3, 285.1006
(M + H)+; found, 285.0999.
5-Chloro-N-hexyl-2-hydroxybenzamide (24). Compound 24 (107 mg, 78%) was pre-
pared as a white solid according to general procedure A (r.t., 48 h), starting from methyl 5-
chloro-2-hydroxybenzoate and hexylamine. HPLC purity 98.0% (tR = 19.72 min). 1H NMR
(300 MHz, CDCl3)
6.90 (d, J = 8.7 Hz, 1H), 6.52 (s, 1H), 3.46–3.36 (s, 2H), 1.67–1.53 (m, 2H), 1.42–1.20 (m,
6H), 0.92–0.82 (m, 3H). 13C NMR (75 MHz, CDCl3)
169.0, 160.0, 134.0, 125.2, 123.4, 120.1,
δ 12.33 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 7.30 (dd, J = 8.7, 2.4 Hz, 1H),
δ
115.5, 40.1, 31.5, 29.4, 26.7, 22.6, 14.1. HRMS (ESI) calculated for C13H19ClNO2, 256.1104
(M + H)+; found, 256.1098.
tert-Butyl (4-(5-chloro-2-hydroxybenzamido)butyl)carbamate (25). Compound 25
(56 mg, 30%) was prepared as a white solid according to general procedure A (r.t., 48 h),
starting from methyl 5-chloro-2-hydroxybenzoate and N-Boc-1,4-butanediamine. HPLC
1
purity 98.5% (tR = 17.75 min). H NMR (300 MHz, CDCl3)
δ 12.53 (s, 1H), 7.63 (s, 1H), 7.53
(s, 1H), 7.29 (dd, J = 8.7, 2.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.77 (s, 1H), 3.48 (q, J = 6.0 Hz
,
2H), 3.16 (q, J = 6.3 Hz, 2H), 1.72–1.53 (m, 4H), 1.45 (s, 9H). 13C NMR (75 MHz, CDCl3)
δ
169.3, 160.2, 156.9, 133.9, 125.9, 123.4, 119.9, 115.6, 79.9, 39.9, 39.8, 28.6, 28.6 (3C), 25.2.
HRMS (ESI) calculated for C16H24ClN2O4, 343.1425 (M + H)+; found, 343.1417.
(5-Chloro-2-hydroxyphenyl)(piperidin-1-yl)methanone (26). To a solution of 5-chloro-
2-methoxybenzoic acid (210 mg, 1.13 mmol), piperidine (80 mg, 0.94 mmol) and DMAP
(
28 mg, 0.23 mmol) in DCM (20 mL) was added EDCI (433 mg, 2.26 mmol) at 0 ◦C. The
resulting mixture was stirred at r.t. for 12 h and concentrated. The residue was purified
by column chromatography to afford the amide intermediate (5-chloro-2-methoxyphenyl)
1
(piperidin-1-yl)methanone (230 mg, 96%) as colorless oil. H NMR (300 MHz, CDCl3)
δ
7.25–7.09 (m, 2H), 6.77 (d, J = 8.7 Hz, 1H), 3.74 (s, 3H), 3.69–3.52 (m, 2H), 3.19–3.03 (m, 2H),
1.69–1.31 (m, 6H). 13C NMR (75 MHz, CDCl3)
55.8, 47.9, 42.5, 26.3, 25.5, 24.5.
δ 165.9, 153.8, 129.7, 127.8, 127.5, 125.7, 112.2,
The amide intermediate (5-chloro-2-methoxyphenyl)(piperidin-1-yl)methanone (230 mg,
0.91 mmol) was dissolved in DCM (50 mL), and then BBr3 (4.53 mL, 4.53 mmol, 1 M in
DCM) was added at 0 ◦C. The mixture was stirred at r.t. for 2 h. The mixture was diluted
with DCM, washed with H2O and brine, dried (Na2SO4) and concentrated. The residue
was purified by preparative TLC to afford compound 26 (203 mg, 93%) as a white solid.
1
HPLC purity 95.1% (tR = 15.13 min). H NMR (300 MHz, CDCl3)
δ
9.57 (s, 1H), 7.26 (dd,
J = 8.7, 2.7 Hz, 1H), 7.19 (d, J = 2.7 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 3.67–3.60 (m, 4H),
1.77–1.61 (m, 6H). 13C NMR (75 MHz, CDCl3)
δ 169.4, 157.5, 132.3, 127.8, 123.4, 119.6, 118.9,
47.0 (2C), 26.2 (2C), 24.6. HRMS (ESI) calculated for C12H15ClNO2, 240.0791 (M + H)+;
found, 240.0786.
1-(4-(5-Chloro-2-hydroxybenzoyl)piperazin-1-yl)ethanone (27). Compound 27 was
prepared using a procedure similar to that used to prepare compound 26 starting from 5-
chlorosalicylic acid and 1-acetylpiperazine. The title compound was obtained (139 mg, 91%
1
in two steps) as a yellow solid. HPLC purity 97.2% (tR = 12.32 min). H NMR (300 MHz
,
CDCl3)
δ 9.51 (s, 1H), 7.23–7.14 (m, 2H), 6.86 (d, J = 8.7 Hz, 1H), 3.72–3.48 (m, 8H), 2.11
(s, 3H). 13C NMR (75 MHz, CDCl3)
δ
169.6, 168.9, 155.0, 132.0, 127.9, 124.2, 120.8, 118.8,
46.1, 45.3, 44.8, 41.5, 21.4. HRMS (ESI) calculated for C13H16ClN2O3, 283.0849 (M + H)+;
found, 283.0843.

Int. J. Mol. Sci. 2021, 22, 1617
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(5-Chloro-2-hydroxyphenyl)(4-methylpiperazin-1-yl)methanone (28). Compound 28
(39 mg, 28%) was prepared as a pale yellow solid solid according to general procedure A
(60 ◦C, 96 h), starting from methyl 5-chloro-2-hydroxybenzoate and 1-methylpiperazine.
1
HPLC purity 98.3% (tR = 10.32 min). H NMR (300 MHz, CDCl3)
δ
7.23 (dd, J = 8.7, 2.7 Hz
,
1H), 7.17 (d, J = 2.7 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 3.69 (t, J = 5.1 Hz, 4H), 2.45 (t, J = 5.1 Hz
,
4H), 2.32 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ
169.2, 156.7, 132.2, 127.8 (2C), 123.7, 119.4,
55.0 (2C), 46.0, 45.5 (2C). HRMS (ESI) calculated for C12H16ClN2O2, 255.0900 (M + H)+;
found, 255.0895.
4.2. Biology
4.2.1. Cell Lines and Virus Strain
Human A549 (ATCC® CCL-185
CRL-1573 ) cell lines were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). The 293 5 stable cell line overexpressing the human 5 integrin
™ ™
), HFF (ATCC® SCRC-1041 ) and HEK-293 (ATCC®
™
β
β
subunit and the HAdV-5 ts1 mutant were kindly provided by Dr. Glen Nemerow [45].
The cell lines were propagated in Dulbecco’s modified Eagle medium (DMEM, Life Tech-
nologies/Thermo Fisher, MA, USA) supplemented with 10% fetal bovine serum (FBS)
(Omega Scientific, Tarzana, CA, USA), 10 mM HEPES, 100 units/mL penicillin, 4 mM
L-glutamine, 100 µg/mL streptomycin, and 0.1 mM nonessential amino acids (complete
DMEM). Wild-type HAdV-5 and HCMV (AD169) were from the ATCC. The HAdV-GFP
used in this study is a replication-defective HAdV5 that contains a CMV promoter-driven
enhanced green fluorescent protein (eGFP) reporter gene cassette in place of the E1/E3
regions [46]. HAdVs were propagated in 293 β5 cells and separated from the cellular lysate
by cesium chloride density gradient centrifugation. We calculated virus concentration, in
mg/mL, with the Bio-Rad Protein Assay (Bio-Rad Laboratories, CA, USA) and converted
it to virus particles/mL (vp/mL) using 4 × 1012 vp/mg.
4.2.2. Plaque Assay
Compounds were measured at concentrations of 10
ranging from 10 to 0.3 M using low MOI infections (0.06 vp/cell) in a plaque assay. In brief,
293 5 cells were seeded in 6-well plates at a density of 4
105 cells per well in duplicate
µM and in a dose
−
response assay
µ
β
×
for each condition. Cells were infected with HAdV5-GFP (0.06 vp/cell) and rocked for 2 h
at 37 ◦C, when cells reached 80%–90% confluency. When the incubation was finished, the
inoculum was removed followed by washing the cells once with PBS. The cells were then
carefully overlaid with 4 mL/well of equal parts of 1.6% (water/vol) Difco Agar Noble
(Becton, Dickinson & Co., Sparks, MD, USA) and 2
Eagle, BioWhittaker, MD, USA) supplemented with 2
glutamine, and 10% FBS. The compounds were also added to the mixture in concentrations
ranging from 10 to 0.3
M. After incubation for 7 days at 37 ◦C, we scanned plates with a
×
EMEM (Minimum Essential Medium
×
penicillin/streptomycin, 2 L-
×
µ
Typhoon FLA 9000 imager (GE Healthcare Life Sciences, MA, USA) and quantified plaques
with ImageJ [47].
4.2.3. Cytotoxicity Assay
The cytotoxicity of these derivatives was tested by commercial kit AlamarBlue® (Ref.
DAL1025, Invitrogen, MA, USA). A549 cells at a density of 5
plates were seeded. Decreasing concentrations of each derivative (200
×
103 cells per well in 96-well
µM, 150
µM, 100
µM,
80
µ
M, 60
µ
M, 40
µ
M, 30
µ
M, 20
µ
M, 10
µ
M, 5
µ
M, 2.5
µ
M, 0
µ
M) were diluted in 100 µ
L of
Dulbecco’s Modified Eagle Medium (DMEM). Cells were then incubated at 37 ◦C for 48 h
according to the kit protocol. The cytotoxic concentration 50 (CC50) value was obtained by
using the statistical package GraphPad Prism. This assay was performed in duplicate.
4.2.4. Virus Yield Reduction
A549 cells (1.5
×
105 cells/well in a 24-well plate) were incubated for 24 h in 500
µL
of complete DMEM and then infected with wild-type HAdV5 (100 vp/cell) when more

Int. J. Mol. Sci. 2021, 22, 1617
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than 90% of confluency were observed. Infected cells were incubated for 48 h at 37 ◦C in
500 µL of complete DMEM containing niclosamide (3) at 5 µM and derivative 16 at 10-fold
IC50 concentration obtained in the plaque assay or the same volume of DMSO (positive
control). After 48 h, cells were harvested and subjected to three rounds of freeze/thaw.
Serial dilutions of clarified lysates were titrated on A549 cells (3
calculated TCID50 values using an endpoint dilution method [48].
×
104 cells/well), and we
4.2.5. DNA Quantification by Real-Time PCR
A549 cells (1.5
complete DMEM and then infected with wild-type HAdV5 (100 vp/cell) when more than
90% of confluency were observed. Infected cells were incubated 24 h at 37 ◦C in 500
L of
complete DMEM containing niclosamide (3) at 5 M concentration and compound 16 at
×
105 cells/well in a 24-well plate) were incubated for 24 h in 500
µL of
µ
µ
10-fold its IC50 concentration in the plaque assay or the same volume of DMSO ◦(positive
control). All samples were carried out in duplicate. After incubation for 24 h at 37 C, DNA
was purified from the cell lysate with the E.Z.N.A.® Tissue DNA Kit (Omega Bio-tek, Nor-
cross, GA, USA) following the manufacturer’s instructions. TaqMan primers and probes
for a common region of the HAdV5 hexon were designed with the GenScript Real-Time
PCR (TaqMan) Primer Design software (GenScript, Leiden, Netherlands). Oligonucleotides
sequences were: AQ1: 50-GCC ACG GTG GGG TTT CTA AAC TT-30; AQ2: 50-GCC CCA
0
0
GTG GTC TTA CAT GCA CAT-3 ; Probe: 6-FAM-5 -TGC ACC AGA CCC GGG CTC AGG
TAC TCC GA-30-TAMRA. Real-time PCR mixtures contained 9.5
µL of the purified DNA,
AQ1 and AQ2 at a concentration of 200 nM each and Probe at a concentration of 50 nM in a
◦
total volume of 25
µ
L. The PCR cycling protocol was 95 C for 3 min followed by 40 cycles
of 95 ◦C for 10 s and 60 ◦C for 30 s. Human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene was applied as internal control. Oligonucleotides sequences for GAPDH
and conditions were previously reported by Rivera et al. [49].
For quantification, gene fragments from hexon and GAPDH were cloned into the
pGEM-T Easy vector (Promega, MA, USA), and known concentrations of template were
used to produce a standard curve in parallel for each experiment. All assays were done in
thermal cycler LightCycler® 96 System (Roche, Basel, Switzerland).
4.2.6. Nuclear-Associated HAdV Genomes
The nuclear delivery of HAdV genomes was evaluated by real-time PCR following
nuclear isolation from infected cells. 1
wild-type HAdV5 at MOI 2000 vp/cell in the presence of 10-fold IC50 concentration ob-
tained in the plaque assay for compound 16, niclosamide (3) at 5 M or the same volume of
×
106 A549 cells in 6-well plates were infected with
µ
DMSO for positive control. Forty-five minutes after infection, A549 cells were trypsinized,
collected, and then washed twice with PBS. After that, we separated cytoplasmic and
nuclear fractions using a hypotonic buffer solution and NP-40 detergent and resuspended
the cell pellet in 500
µ
L of 1
×
hypotonic buffer ◦(20 mM Tris-HCl pH 7.4, 10 mM NaCl,
3 mM MgCl2). After incubation for 15 min at 4 C, 25 µL of NP-40 was added and the
samples were vortexed. The homogenates were centrifuged for 10 min at 835g and 4 ◦C.
After the removal of the cytoplasmic fraction (supernatant), HAdV DNA was separated
from the nuclear fraction (pellet) and from the cytoplasmic fraction using the E.Z.N.A.®
Tissue DNA Kit (Omega Bio-tek, Norcross, GA, USA).
4.2.7. Time of Addition Assay
The anti-HAdV effects of compounds
time-curve assay using 293 5 cells (3
105 cells/well in corning black wall, clear bottom
96-well plates) that were infected with HAdV5-GFP (2000 vp/cell) in the presence of 5
of compound and 10-fold IC50 concentration obtained in the plaque assay for derivative
16. Parallel samples of HAdV-5 were incubated with or without the selected compounds
on ice for 1 h. Virus was then added to 293
5 cells and incubated at 37 ◦C. Compounds
and 16 were added at the indicated time points before or during this incubation. After a
3 and 16 at different points were evaluated in a
β
×
µM
3
β
3

Int. J. Mol. Sci. 2021, 22, 1617
18 of 21
◦
◦
total of 2 h at 37 C, cells were incubated for an additional 48 h at 37 C and 5% CO2 before
analyzing GFP expression using the Typhoon 9410 imager (GE Healthcare Life Sciences,
MA, USA) as above.
4.2.8. HAdV-Mediated Endosome Disruption
A549 cells (~20,000 cells/well) were incubated in DMEM without cysteine or methion-
ine supplemented with 10% dialyzed FBS [DMEM (
infection. Threefold serial dilutions (0.45 ng to 1000 ng) of HAdV5, or HAdV-5 ts1 were
preincubated with cells in the presence of 5 M niclosamide (3), 10-fold the IC50 concentra-
tion obtained in the plaque assay for compound 16, or the same volume of DMSO (negative
control) for one hour. The medium was then removed and replaced with 50 L DMEM (
containing 0.1 mg/mL -sarcin (Santa Cruz Biotechnology, Dallas, TX, USA) and the virus
−
)] in black 96-well plates for 1 h before
µ
µ
−
)
α
and drug mixtures. After 2 h at 37 ◦C, the Click-iT HPG Alexa Fluor 488 Protein Synthesis
Assay Kits (Invitrogen, MA, USA) were used to analyze protein synthesis according to the
manufacturer’s instructions. The incorporation of the amino acid analog of methionine
L-homopropargylglycine (HPG) containing Alexa Fluor 488 azide was measured using a
Typhoon 9410 imager (GE Healthcare Life Sciences, MA, USA) and calculated subtracting
the background level of the control well containing L-homopropargylglycine (HPG) and
α-sarcin but not virus (100% incorporation).
4.2.9. Thermostability Assay
HAdV-GFP (37.5 ng) was incubated with compound 16 (50 µM) or the same volume
of DMSO for 1 h in complete DMEM. Parallel samples were incubated for 10 min at the
indicated temperatures, cooled to r.t., and added to A549 cells on ice to synchronize the
infection. After the incubation, cells were washed twice with PBS 1x to remove the nonat-
tached virus and the excess of compound 16 and DMSO. Cells were then incubated for
◦
24 h at 37 C and 5% CO2 before being visualized for GFP expression in an Olympus
inverted microscope Model IX71 (Hamburg, Germany) and analyzed by capturing rep-
resentative images of each condition using the CellSens Dimension platform (Olympus,
Hamburg, Germany).
4.2.10. Statistical Analyses
One-way ANOVA tests (Dunnet method) were performed using the GraphPad Prism 6.
A P value under 0.05 was considered a statistical significance. We pointed out this statistical
significance with asterisk in graphs, the numbers of which indicate the level of significance
(* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).
Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/1422
-0067/22/4/1617/s1, Copies of 1H and 13C NMR spectra.
Author Contributions: J.X., J.B.-C., M.C.-L. and H.C. performed research. J.X., J.B.-C., J.Z. and J.S.-C.
designed research. J.X., J.B.-C., M.C.-L., H.C., J.Z. and J.S.-C. analyzed data. J.X., J.B.-C., M.C.-L., H.C.,
Y.X., E.A.W., J.P., J.Z. and J.S.-C. wrote or contributed to the writing and revision of the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: This work was partially supported by the UTMB Technology Commercialization Program,
the John D. Stobo, M. D. Distinguished Chair Endowment Fund (to JZ) and John Sealy Memorial
Endowment Fund at UTMB, Plan Nacional de I + D + i 2013–2016 and Instituto de Salud Carlos III,
Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria
y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009),
cofinanced by the European Development Regional Fund “A way to achieve Europe”, Operative
program Intelligent Growth 20142020, the Instituto de Salud Carlos III, Proyectos de Investigación en
Salud (PI15/00489) and Proyectos de Desarrollo Tecnológico en Salud (DTS17/00130), the Spanish
Adenovirus Network (AdenoNet, BIO2015/68990-REDT), and the program “Nicolás Monardes”
(C-0059-2018) Servicio Andaluz de Salud, Junta de Andalucía. E.A.W. is supported by the National
Institutes of Health (NIH) National Research Service Award (NRSA) F31 DA04551. E.A.W. and J.Z.

Int. J. Mol. Sci. 2021, 22, 1617
19 of 21
have no financial connections with the above European funding sources, and thus there is no conflict
of interest to disclose.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
allo-HSCT allogenic hematopoietic stem cell transplant
BCV
brincidofovir
CAP
community-acquired pneumonia
half-maximal cytotoxic concentration
dichloromethane
4-(dimethylamino)pyridine
dimethyl sulfoxide
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
ethyl acetate
human adenoviruses
human cytomegalovirus
L-homopropargylglycine
high-performance liquid chromatography
high-resolution mass spectrometry
half-maximal inhibitory concentration
postinfection
CC50
DCM
DMAP
DMSO
EDCI
EtOAc
HAdVs
HCMV
HPG
HPLC
HRMS
IC50
p.i.
SAR
SOT
structure–activity relationship
solid-organ transplant
TLC
TMS
thin layer chromatography
tetramethylsilane
UV
ultraviolet
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