Serinol-Based Benzoic Acid Esters as New Scaffolds for the Development of Adenovirus Infection Inhibitors: Design, Synthesis, and in Vitro Biological Evaluation

Article abstract of DOI:10.1021/acsinfecdis.0c00515

Over the years, human adenovirus (HAdV) has progressively been recognized as a significant viral pathogen. Traditionally associated with self-limited respiratory, gastrointestinal, and conjunctival infections, mainly in immunocompromised patients, HAdV is currently considered to be a pathogen presenting significant morbidity and mortality in both immunosuppressed and otherwise healthy individuals. Currently available therapeutic options are limited because of their lack of effectivity and related side effects. In this context, there is an urgent need to develop effective anti-HAdV drugs with suitable therapeutic indexes. In this work, we identified new serinol-derived benzoic acid esters as novel scaffolds for the inhibition of HAdV infections. A set of 38 compounds were designed and synthesized, and their antiviral activity and cytotoxicity were evaluated. Four compounds (13, 14, 27, and 32) inhibited HAdV infection at low micromolar concentrations (2.82-5.35 μM). Their half maximal inhibitory concentration (IC50) values were lower compared to that of cidofovir, the current drug of choice. All compounds significantly reduced the HAdV DNA replication process, while they did not block any step of the viral entry. Our results showed that compounds 13, 14, and 32 seem to be targeting the expression of the E1A early gene. Moreover, all four derivatives demonstrated a significant inhibition of human cytomegalovirus (HCMV) DNA replication. This new scaffold may represent a potential tool useful for the development of effective anti-HAdV drugs.

Full text of DOI:10.1021/acsinfecdis.0c00515

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Article
Serinol-Based Benzoic Acid Esters as New Scaffolds for the
Development of Adenovirus Infection Inhibitors: Design, Synthesis,
and In Vitro Biological Evaluation
∇
∇
∇
Sarah Mazzotta, Judith Berastegui-Cabrera, Gabriele Carullo, Margarita Vega-Holm,*
Marta Carretero-Ledesma, Lara Mendolia, Francesca Aiello, Fernando Iglesias-Guerra,*
Jeronimo Pachon, Jose Manuel Vega-Perez, and Javier Sanchez-Cespedes*
́ ́ ́ ́ ́ ́
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ABSTRACT: Over the years, human adenovirus (HAdV) has
progressively been recognized as a significant viral pathogen.
Traditionally associated with self-limited respiratory, gastrointesti-
nal, and conjunctival infections, mainly in immunocompromised
patients, HAdV is currently considered to be a pathogen presenting
significant morbidity and mortality in both immunosuppressed and
otherwise healthy individuals. Currently available therapeutic
options are limited because of their lack of effectivity and related
side effects. In this context, there is an urgent need to develop
effective anti-HAdV drugs with suitable therapeutic indexes. In this
work, we identified new serinol-derived benzoic acid esters as novel
scaffolds for the inhibition of HAdV infections. A set of 38
compounds were designed and synthesized, and their antiviral
activity and cytotoxicity were evaluated. Four compounds (13, 14,
2
7, and 32) inhibited HAdV infection at low micromolar concentrations (2.82−5.35 μM). Their half maximal inhibitory
concentration (IC ) values were lower compared to that of cidofovir, the current drug of choice. All compounds significantly
50
reduced the HAdV DNA replication process, while they did not block any step of the viral entry. Our results showed that
compounds 13, 14, and 32 seem to be targeting the expression of the E1A early gene. Moreover, all four derivatives demonstrated a
significant inhibition of human cytomegalovirus (HCMV) DNA replication. This new scaffold may represent a potential tool useful
for the development of effective anti-HAdV drugs.
KEYWORDS: serinol derivatives, benzoic acid esters, adenovirus, antivirals agents, urea derivatives, chemical synthesis
uman adenoviruses (HAdVs) belong to the family
Adenoviridae that includes more than 100 types
associated with human infections and are classified into 7
species (HAdV A−G). They are nonenveloped and icosahedral
viruses with a linear double-stranded DNA (dsDNA) genome
commonly associated with ocular diseases, while 40 and 41 are
16
H
frequently involved in enteric infections. Immunosuppressive
therapies used during transplantations can cause an increase in
the frequency of HAdV infections that can be acquired from
the transplanted organ or by the reactivation of a latent
17,18
1
−3
infection in the recipient.
In pediatric hematopoietic stem
of 34−36 kb.
HAdVs are responsible for a wide range of
cell transplantation (HSCT) patients, the incidence of HAdV
global clinical diseases, mostly in immunocompromised
patients, although a number of cases in healthy individuals
with severe community-acquired pneumonia have been
described.
Young children and immunosuppressed adult patients are
4,5
infections ranges from 15% to 44% with high mortality rates
8
,19,20
for those with disseminated disease.
6
,7
At this time, there is no formally approved antiviral therapy
for the treatment of HAdV infections. Nowadays, antiviral
4
,8
more susceptible to serious HAdV infections.9 HAdVs usually
,10
infect the epithelium of the respiratory tract
but also cause
Special Issue: Antiviral Therapeutics
Received: July 18, 2020
1
1
12
14
conjunctivitis, urinary tract infections, gastrointestinal
1
3
diseases, and multiple organ failures. Among them,
pneumonia and hemorrhagic cystitis are the most common
complications. Respiratory infections are traditionally
1
5
associated with types 1−3, 5, and 7. Types 8, 19, and 37 are
©
XXXX American Chemical Society
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A
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drugs such as nucleoside analogues cidofovir (CDV; 1, Figure
quinazoline-2,4(1H,3H)-diones against Vaccinia virus and
HAdV 2 (4, Figure 1). Regarding the HAdV type, compounds
with fluorine or methoxy groups on the aromatic ring were
1
), ganciclovir (2, Figure 1), and ribavirin provide no
34
considered the most active.
A range of benzoyl esters and amides have been identified as
interesting scaffolds due to their ability to block HAdV
infection. Salicylamides with electron withdrawing substituents
on the phenyl ring represented excellent inhibitors of HAdV 5
replication; some of them showed IC values in the
5
0
35,36
nanomolar range (5, Figure 1).
Benzoic acid esters were
also developed as potential antiviral agents against HAdV 7 (6,
37
Figure 1). Our previous research was focused on the
preparation of new piperazine-derived ureas/thioureas with
inhibitory activity against HAdV type 5 (7, Figure 1).
Optimized compounds blocked HAdV infection at nanomolar
or low micromolar ranges, showing low cytotoxicity and acting
38−40
in several phases of its life cycle.
On the basis of our
previous work and experience in the synthesis of new small
libraries of compounds with other potential biological
41,42
activity
and focused on searching novel scaffolds for the
preparation of potential anti-HAdV agents, the major aim of
this report was to present the design, synthesis, and in vitro
biological evaluation of a small library (38 compounds) of
different serinol derivatives. Biological screening and SAR
Figure 1. Some reported antiviral and anti-HAdV agents (1−8) and
the design of the general backbone of serinol derivatives.
(
structure activity relationships) studies were developed in
satisfactory results in terms of efficacy or safety. 1−23 CDV is
a cytosine analogue (acyclic nucleoside phosphonate) that
represents the most common antiviral agent used for HAdV-
related diseases. It showed broad spectrum antiviral properties,
acting as a false substrate for the viral DNA polymerase
2
order to identify novel lead compounds.
RESULTS AND DISCUSSION
■
As the source for our central backbone, the cheap and
commercially available reactive serinol was employed. Three
structural considerations were behind this choice (Figure 1).
(i) The aminoalcohol skeleton: Small molecules based on an
isopropanolamine core have been prepared and evaluated as
HIV inhibitors. They possessed simple nonpeptidic heteroaryl
structures, where the benzenesulfonamide group presented a
suitable moiety to be linked to the isopropanolic core to obtain
24
blocking DNA replication.
Unfortunately, the clinical use of CDV is limited because of
its low oral bioavailability, nephrotoxicity, and myelotox-
2
5,26
icity.
Over the years, several lipid conjugates of CDV have
been developed, like brincidofovir (CMX001, BCV), which
displayed greater in vitro antiviral activity and a good inhibition
of viral replication in animal models. Furthermore, it
demonstrated an increase in oral bioavailability and reduced
43,44
biological activity (8, Figure 1).
In this work, we have
employed an isomeric core, 2-amino-propane-1,3-diol. (ii) The
question of the symmetry: In the field of the development of
different scaffolds with antiviral activities, different types of
symmetric compounds have been described, such as novel
symmetrical phenylenediamines targeting the viral NS3
helicase being potential anti-HCV agents, symmetrically
disposed stilbenes as potent inhibitors of NS5A, and complex
homodimeric structures with large molecular weights (daclas-
27,28
nephrotoxicity compared to CDV.
Phases II and III of
clinical trials (NCT01231344, NCT02087306) confirmed the
anti-HAdV activity of BCV with reductions in HAdV-related
symptoms; nevertheless, it induced some gastrointestinal
29,30
45
disorders, limiting its use in therapy.
46
These features support the need for the development of new
antiviral agents with activity against HAdV and a good
therapeutic index. Different new nucleoside derivatives or
nitrogen base-based analogues have been described. A series of
octadecyloxyethyl esters of (S)-[(3-hydroxy-2-
phosphonomethoxy)propyl] nucleoside (ODE nucleoside)
showed antiviral activity against HAdV 14, an emerging type
associated with a high rate of serious complications. 5-
Aminouracil derivatives were tested against HAdV 5, and one
of them, the morpholino-substituted compound (3, Figure 1),
displayed a significant in vitro inhibition, achieving a half
47,48
tavir and other related symmetric compounds).
To the
best of our knowledge, no symmetric compounds have been
described as potential anti-HAdV agents. (iii) The function-
alization: The serinol scaffold allowed us to develop new
molecules keeping many important features present in the
reported anti-HAdV agents mentioned above, mainly
31
32,34,38
37
urea
and ester functions, that have been identified as
important for the antiviral activity (Figure 1). In our general
backbone, the urea function was introduced at the nitrogen
(this moiety could be considered an acyclic modification of the
nitrogen base present in some anti-HAdV agents) (1, 3), while
aromatic esters at the hydroxyls as an isosteric modification
with respect to the aromatic amide function, which is present
in niclosamide (5) and its derivatives, and also because of their
presence in effective anti-HAdV agents (6).
32
maximal inhibitory concentration (IC ) value of 0.5 μM.
50
In the past few years, also, non-nucleoside and structural
diversified compounds have been reported as new potential
antiviral candidates; many of them presented amide/urea
functions and aromatic moieties as common features in their
structures. Pyrimidine−nitrile derivatives with amide moieties
were prepared as new potential adenain inhibitors, an
As it is shown in Figure 1, our original framework has three
points for different structural modifications to generate
chemical diversity. Two of them are related to the substituents
33
important viral protease involved in the virus propagation.
Kang et al. reported the antiviral activity of 3-hydroxy-
B
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a
Table 1. Diester (13−37), Monoester (38−40), Dicarbamate (41−49), and Monocarbamate (50) Serinol Derivatives
b
entry
comp.
13
R1
R2
R3
R4
R5
CH3
H
OCH3
CN
R6
percentage of plaque-formation inhibition
structure
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
66.33 ± 2.49
89.69 ± 7.13
97.17 ± 0.77
7.66 ± 10.83
0.00 ± 0.00
55.83 ± 4.31
47.33 ± 38.86
9.40 ± 13.29
0.00 ± 0.00
6.78 ± 13.90
0.00 ± 0.00
41.48 ± 1.23
34.58 ± 6.23
25.68 ± 36.31
98.25 ± 2.48
91.51 ± 11.9
6.25 ± 8.84
5.81 ± 8.22
0.00 ± 0.00
97.58 ± 4.73
44.76 ± 2.69
43.29 ± 11.86
58.79 ± 3.43
0.00 ± 0.00
0.00 ± 0.00
30.0 ± 27.85
34.93 ± 16.67
27.51 ± 59.28
0.00 ± 0.00
99.74 ± 0.36
0.00 ± 0.00
98.59 ± 2.00
99.54 ± 0.87
0.00 ± 0.00
100 ± 0.00
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
D
D
D
C
C
C
C
C
C
C
C
C
E
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
cidofovir
CH3
H
H
H
NO2
OCH3
H
OCH3
OCH3
CH3
H
OCH3
NO2
OCH3
OCH3
CH3
H
OCH3
CN
OCH3
H
OCH3
H
H
CH3
H
H
H
H
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
37
37
38
41
H
OCH3
H
H
OCH3
H
OCH3
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
CF3
H
H
CF3
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
NO2
OCH3
H
H
OCH3
OCH3
N(CH3)2
OCH3
H
OCH3
H
OCH3
H
H
H
H
H
Cl
CF3
CH3
Cl
H
H
H
OCH3
OCH3
OCH3
CH3
H
OCH3
CN
NO2
CH3
H
H
H
CH3
CH3
CF3
CF3
CF3
CF3
CF3
Cl
Cl
Cl
CH3
CF3
OCH3
H
OCH3
H
OCH3
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
H
CH3
H
NO2
CH3
NO2
37.43 ± 3.12
0.00 ± 0.00
99.45 ± 0.77
3.51 ± 4.97
H
H
H
H
H
c
a
b
Inhibition of HAdV infection in the plaque assay. Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 μM using the 293β5 cell
c
line. See the Methods section for details. The data of cidofovir, as a positive clinical drug candidate, have been listed.
of the aromatic rings of both urea and ester functions. The
third consists of the replacement of the ester function by a
carbamate one.
Chemistry. General synthetic routes for the preparation of
new derivatives (Table 1) are summarized in Schemes 1 and 2
and included two reactions starting from commercial 2-amino-
1,3-propanediol. As it is shown in Scheme 1, in the first step,
the urea function was introduced by the reaction with
appropriate substituted phenyl isocyanate in dichloromethane
at room temperature (9−12). Three different substituted
C
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Scheme 1. Synthetic Pathways for the Preparation of Urea
chloride by strictly controlling the time (1 h), the
stoichiometry (1 equiv), and the temperature (−15 °C).
For the third structural diversification, dicarbamate deriva-
tives (41−50) were prepared by the reaction of the urea
derivative with the isocyanate. When commercial isocyanates
were employed, the reaction was performed at 110 °C in
Derivative Precursors (9−12) and Serinol Derivative Esters
(
13−40)
5
4
toluene. In the case of compounds 42 and 47, the
corresponding isocyanate was previously prepared from o-
toluidine by the reaction with triphosgene in basic conditions
55
(
Na CO ). In a second step, dicarbamate was generated in
2 3
milder conditions, using DMAP in dichloromethane at rt
Table 1). These compounds were prepared with the aim to
(
increase their potential ability to form hydrogen interactions
through both urethane functions (Scheme 2).
Biological Evaluation. Compounds 13−50 were screened
for their potential anti-HAdV activity through the plaque assay,
quantifying HAdV plaque formation in the presence of
candidate molecules (Table 1). As CDV remains the drug of
choice for the treatment of these infections, it was also
evaluated in parallel to our compounds, following our own
38,39,56
methodologies.
used the HAdV5-GFP vector in combination with the cell line
93β5 to generate the infection plaques to be quantified. This
In our protocol for the plaque assay, we
2
Scheme 2. Synthetic Pathway for the Preparation of Serinol
Derivative Dicarbamates (41−49)
may be a limitation in our initial screening since this vector
itself does not express the E1A gene, which is expressed by the
cells. Thus, there is a possibility that some potential molecules
were missed in our screening.
Among the serinol-derived diesters, the subfamily with a
methyl group at the phenylurea function (20−25) showed very
little plaque-formation inhibition. Only two of them, 24 and 25
(
dimethoxy and trimethoxy benzoyl derivatives) displayed an
appreciable inhibitory activity (41.48% and 34.58%, respec-
tively). On the other hand, trifluoromethyl and disubstituted
(
3-CF -4-Cl) urea analogues (13−19 and 26−33) presented
3
significantly higher levels of inhibition. Depending on the
substituent at the benzoyl moiety, different behaviors were
observed. In particular, the presence of electron-withdrawing
groups (p-CN, p-NO ) resulted in very low or no active
2
compounds (16, 17, 23, 29, and 30) with percentages of
plaque formation inhibition ranging from 0% to 7.66%. Higher
inhibition was detected among those compounds with electron
phenyl isocyanates (p-CF and p-CH as models of electron-
withdrawing and electron-releasing groups and disubstituted 3-
3
3
CF -4-Cl) were employed. In the second reaction, ester or
3
donating groups (13−15, 18, 19, 24−28, and 33). p-CH , o-
3
carbamate derivatives were generated from compounds 9−12.
Diester derivatives of serinol (13−34) were synthesized by an
acylation reaction of both primary hydroxyl groups, using acyl
chloride and DMAP in dichloromethane at rt. At this step, the
pursued chemical diversity was introduced by the different
types of substituent on the aromatic ester. It was decorated
with electron-withdrawing or -donating groups to assess the
effect of electronic properties, with a special focus on the
methoxy group (mono-, di-, and trimethoxy substituted), since
these benzoyl derivatives (amides and esters) are privileged
structural scaffolds, which are widely distributed in different
CH , p-OCH , 2,4-dimethoxy, and 3,4,5-trimethoxy derivatives
3
3
displayed plaque formation inhibition values ranging from 66%
to 97%. o-CH and p-OCH analogues with p-CF and 3-CF -
3
3
3
3
4-Cl ureas (14, 15, 27, and 28) were the most active
compounds with percentages of inhibition >90%, together with
compound 32, the trimethoxy derivative with a disubstituted
urea that showed an almost complete inhibition (97.58% of
inhibition; Table 1).
As compounds 15 and 28, both p-methoxy diesters with p-
CF3 and 3-CF -4-Cl substituted urea, showed 90% of
3
34,44,49−52
inhibition, their analogue p-Cl substituted on the urea function
anticancer and antiviral compounds.
Other relevant
(
34) was prepared to diversify the structure. It reached a
scaffolds broadly distributed in natural products with diverse
and important biological functions (among them antiviral
activities) are trimethoxycynnamic derivatives (esters and
moderate level of inhibition (43.29%).
Similarly, the presence of a p-dimethylamino group, a
different electron donor moiety (33), did not improve the
activity (44.76% of inhibition).
The introduction of the trimethoxycinnamic esters in our
skeleton did not offer active compounds, 0% of inhibition for
compounds 36 and 37 and 58.79% of inhibition for 35. This
group failed to provide any improvement over their
5
3
amides). For the preparation of these serinol derivative
esters (35−37), the condensation took place using carboxylic
acid as acylating agent under Steglich conditions (EDCI,
DMAP).
Monoester derivatives (38−40) were obtained through a
selective O-acylation reaction of serinol ureas with acyl
D
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Table 2. IC , CC , SI, and Virus Yield Reduction Values for Selected Compounds and for Cidofovir as a Positive Clinical
5
0
50
a
Drug Candidate
b
c
d
e
entry
comp.
13
14
15
27
28
32
42
44
45
47
50
cidofovir
IC50 (μM)
CC50 (μM)
selectivity index (SI)
yield reduction (fold-reduction)
1
2
3
4
5
6
7
8
9
1
1
1
5.35 ± 0.66
4.53 ± 0.64
2.63 ± 0.26
2.82 ± 0.31
8.96 ± 1.12
3.67 ± 1.46
3.76 ± 0.23
7.05 ± 2.65
2.05 ± 0.02
7.78 ± 2.02
9.74 ± 0.90
24.06 ± 5.9
93.24 ± 7.7
17.42
44.15
4.46
8.90
1.25
17.37
5.27
2.04
5.64
2.37
4.23
7.5
7.23 ± 5.38
92.28 ± 33.77
200.00 ± 33.87
11.73 ± 0.26
25.10 ± 0.16
11.24 ± 4.48
63.73 ± 0.50
19.8 ± 0.16
14.4 ± 0.63
11.56 ± 4.30
18.4 ± 0.60
42.83 ± 15.68
1.22 ± 0.63
0
1
2
41.16 ± 3.17
179.09 ± 35.10
82.5 ± 21.4
a
b
The results represent means ± SD of triplicate samples from three independent experiments. See the Methods section for details. Inhibitory
concentration 50 at a low MOI in a plaque assay. Cytotoxic concentration 50. The selectivity index value was determined as the ratio of 50%
cytotoxic concentration (CC ) to half maximal inhibitory concentration (IC ) in a plaque assay for each compound. Fold-reduction in virus yield
c
d
e
5
0
50
as the ratio of particles produced in the presence of DMSO divided by the yield in the presence of each compound (50 μM).
trimethoxybenzoyl esters analogues 19 (47.33%), 25 (34.58%),
and 32 (97.58%), respectively.
the current drug of choice for the treatment of HAdV
57
infections. Only compounds 13, 14, 27, and 32 showed a
CC50 value at least 10 times over their IC50 values, presenting
selectivity indexes ranging from 8.9 to 44.15, in all cases higher
than that obtained for cidofovir (Table 2). Thus, they were
selected for further biological analysis to gain some knowledge
regarding their potential mechanism of action.
By strict control of the acylation reaction, monoester
derivatives were prepared (38−40). The rationale behind
this structural modification was the evaluation of the presence
of a free primary hydroxyl group, as in CDV, to provide active
compounds (high% of inhibition) that could act through a
different mechanism. As depicted in Table 1, the plaque
formation inhibitions observed for monoacylated compounds
First, we evaluated their anti-HAdV potency using a virus
burst assay to measure their efficacy in blocking the production
of new HAdV viral particles. The treatment with these
compounds was associated with overall reductions in virus
yield between 1.22-fold and 92.3-fold (Table 2). These values
lie in the same range as those for cidofovir (82.5-fold).
Next, we evaluated the capacity of these compounds to
block the HAdV DNA replication process. For this purpose,
we carried out a real-time PCR assay to evaluate the HAdV
DNA replication efficiency in the presence of these compounds
in a single round of infection for 24 h. The presence of
compound 14 blocked almost completely (99.2%) the
synthesis of new copies of the HAdV DNA, while compounds
13 and 27 showed lightly lower inhibitions, 95.1% and 92.1%,
respectively. Compound 32, although to a lesser extent, also
showed a significant inhibition of HAdV DNA replication
(73.5%) (Figure 2)
3
8−40 were 30.0%, 34.93%, and 27.51%, respectively.
For the preparation of dicarbamate derivatives (41−49),
both p-CF and 3-CF -4-Cl urea derivatives were employed as
3
3
they led to most active diesters. The dicarbamate derivatives
provided different results in terms of the structure−activity
relationship. Compounds with the p-CF substituted urea and
3
electron-withdrawing substituents on the aromatic carbamate
function (44 p-CN and 45 p-NO ) showed a high percentage
2
of inhibition (98.59% and 99.45%, respectively). Moreover,
both ortho methyl derivatives from p-CF and disubstituted
3
phenyl urea (42 and 47) inhibited 99.54% and 100% of plaque
formation, respectively. Finally, the monocarbamate derivative
50, isolated during the synthetic preparation of compound 45,
was also evaluated for its antiviral activity and demonstrated to
be the only active monoacyl compound (99.45% of inhibition;
Table 1).
Interestingly, these results suggest that the presence of a
carbamate function seems to be crucial for the inhibition of
HAdV infection when electron-withdrawing groups are
present.
It is important to note that precursors 9−11 were also
assessed in the plaque assay, resulting in plaque formation
inhibitions of 6%, 0%, and 58%, respectively, which suggests
that the presence of acyl functions on primary hydroxyl groups
could be important for the antiviral activity.
Those compounds showing a HAdV inhibition >60% in the
plaque assay (a total of 11 out of 38, 28%) were selected to
evaluate their specificity, measured as the quotient between the
To elucidate if any of these compounds was interfering with
some steps between HAdV entry and HAdV DNA import into
the nucleus, we carried out a nuclear association assay to
quantitatively measure the HAdV genome accessibility to the
host nucleus. After binding to the cellular receptors, internal-
ization, and endosomal escape mediated by protein VI, the
partially uncoated HAdV capsids are transported to the nuclear
membrane via the microtubule network and their genome is
58,59
imported into the nucleus via the nuclear pore complex.
So, if the mechanism of action of these molecules would
involve interference with any of these steps of the HAdV entry,
this would be reflected in the amount of HAdV genomes that
reach the nucleus after a synchronized infection. As depicted in
Figure 3, the treatment with any of these four molecules did
not show a significant difference in the number of HAdV
genomes that reached the nucleus compared with those treated
with DMSO. The DNA copy number of the cellular
housekeeping gene GAPDH in both the nucleus and the
5
0% cytotoxic concentration (CC ) and their IC values. All
50 50
1
1 compounds inhibited HAdV5 infection in a dose-dependent
manner showing IC values from 2.05 μM (compound 45) to
50
9
.74 μM (compound 50). These values were significantly
lower than those observed for the drug cidofovir (24.06 μM),
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Figure 4. Effect of the selected compounds on HAdV E1A gene
expression. Compounds 13, 14, and 32 significantly blocked the
expression of the E1A gene. Compounds 13 and 14 were evaluated at
50 μM concentration; compound 27 was evaluated at a 15 μM
concentration and compound 32, at a 37 μM concentration. Results
are expressed as the relative copy number of HAdV E1A mRNA
normalized to GAPDH copy number, and they are presented as the
mean ± SD of duplicate assays. *p ≤ 0.05; **p ≤ 0.01.
Figure 2. Effect of the selected compounds on HAdV DNA
replication. All four compounds significantly reduced the HAdV
DNA replication process. Compounds 13 and 14 were evaluated at a
50 μM concentration; compound 27 was evaluated at a 15 μM
concentration and compound 32, at a 37 μM concentration. CDV was
used as a positive control for the inhibition of HAdV DNA replication
(
48 μM). Results are expressed as the relative copy number of HAdV
DNA normalized to GAPDH copy number, and they are presented as
the mean ± SD of duplicate assays. **p ≤ 0.01; ***p ≤ 0.001.
may be the cause of their DNA replication inhibition. On the
other hand, since compound 27 did not display inhibition of
the early gene transcription, it may suggest that this compound
could interact with viral proteins essential for HAdV DNA
replication, including the HAdV DNA polymerase, the
precursor of the terminal protein (pTP), or the DNA-binding
protein (DBP).
As other compounds have been demonstrated to inhibit
both HAdV and human cytomegalovirus (HCMV) DNA
38,39
replication,
we decided to perform an inhibitory assay
against HCMV with these four anti-HAdV compounds. As
shown in Figure 5, all of them were able to significantly block
the HCMV DNA replication in a 72 h assay. Compounds 13
and 27 showed a total inhibition of HCMV replication, while
compounds 14 and 32 gave percentages of inhibition of 91.8%
and 90%, respectively. This result suggests a broad antiviral
mechanism of action for these molecules and, since they seem
to act at different stages of the HAdV replicative cycle, it
supports high similarities in HAdV and HCMV routes of
infection. However, further studies will be needed to clarify
their specific mechanism for antiviral activity.
Figure 3. Effect of the selected compounds on HAdV genome nuclear
association. Compounds 13, 14, 27, and 32 did not block any of the
steps during the HAdV entry phase. Compounds 13 and 14 were
evaluated at a 50 μM concentration; compound 27 was evaluated at a
15 μM concentration and compound 32, at a 37 μM concentration.
Results are expressed as the mean ± SD of duplicate assays.
CONCLUSIONS
cytoplasm was also measured as a control for the purity of
nuclear isolation, indicating that we specifically quantified the
HAdV genomes that reached the nuclear membrane. On the
basis of this assay, we could confirm that any of the studied
compounds was interfering with the early steps that span the
entry phase of the HAdV viral particles.
Since the mechanism of action of these four compounds
seems to be related to the early steps after HAdV genome
entry into the nucleus, we decided to go a step forward in our
mechanistic studies. Once HAdV genomes enter the nucleus of
the host cell, the transcription of the immediate, early gene
E1A takes place first, and this product will be essential for the
transcription of other early proteins and the subsequent
replication of the HAdV DNA. We quantified the mRNA copy
number of the E1A gene using quantitative reverse tran-
scription PCR (RT-PCR). As shown in Figure 4, only
compounds 13, 14, and 32 significantly blocked the expression
of the E1A gene in a 6 h assay. Compound 27 did not show
any decrease in the expression of the E1A gene compared with
a control treated with DMSO. The ability of compounds 13,
■
In summary, a set of 38 novel serinol derivatives were designed
and synthesized as new potential anti-HAdV agents. According
to the biological evaluation data, serinol-based aromatic
diesters emerged as promising derivatives. Those compounds
having electron-withdrawing groups at the phenyl urea
function and electron-donor groups at the benzoic moiety
were the most active. In particular, we have identified four
compounds (13, 14, 27, and 32) that significantly inhibited
HAdV infection in a dose-dependent manner. These
compounds showed IC50 values at low micromolar concen-
tration (from 2.82 to 5.35 μM), being more active than
cidofovir in our in vitro assays (24.06 μM). Our mechanistic
studies suggest that all four compounds interfered with HAdV
DNA replication. Specifically, 13, 14, and 32 seemed to be
targeting the E1A early gene expression. The antiviral activity
of these derivatives was also evaluated against HCMV,
demonstrating a significant inhibition of HCMV DNA
replication, which suggests an antiviral broad-spectrum activity
for these compounds. Our work did not include additional
approaches such as genomics or proteomics methods that
14, and 32 to interfere with HAdV early gene transcription
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Figure 5. Effect of the selected compounds on HCMV DNA replication. All four compounds significantly inhibited HCMV DNA replication.
Compounds 13 and 14 were evaluated at 50 μM concentration; compound 27 was evaluated at a 15 μM concentration and compound 32, at a 37
μM concentration. Results are expressed as the relative copy number of HCMV DNA and normalized to GAPDH copy number, and they are
presented as the mean ± SD of duplicate assays. **p ≤ 0.01.
could give us more solid information regarding the specific
mechanism of action of these derivatives. Thus, alternative
mechanisms involving interactions with specific proteins
involved in the HAdV DNA replication and transcription
processes should be considered. Further investigations will be
necessary in order to fully clarify the specific mode of action
for their antiviral activity and to confirm their potential utility
as antiviral drugs after the evaluation of their stability in serum
and efficacy and safety in an animal model of infection. It will
also be necessary to prepare additional monoacylated
derivatives, following both structural aspects (analogues of
interest to be compared to the diacylated derivatives) and
stereochemical considerations (the racemic form and both
pure enantiomers) in order to thoroughly study structure−
activity relationships.
measurements with resolutions of 10 000. FAB mass spectra
were recorded using a thioglycerol matrix. NMR spectra were
recorded at 25 °C on a Bruker AV500 spectrometer at 500
1
13
MHz for H and 125 MHz for C. COSY, DEPT, HSQC, and
NOESY experiments were performed to assign the signals in
the NMR spectra. The chemical shifts (d) reported are given in
parts per million (ppm) on the δ scale relative to TMS, and the
1
coupling constants (J) are in hertz (Hz). H chemical shift
values (δ) are referenced to the residual nondeuterated
components of the NMR solvents (δ 2.54 ppm for DMSO).
The spin multiplicities are reported as s (singlet), d (doublet),
t (triplet), q (quadruplet), m (multiplet), or br s (broad
singlet). The purity of final compounds was evaluated by
elemental analysis (C, H, and N). The purity of all the final
compounds was confirmed to be ≥95% by combustion.
General Procedure 1: Synthesis of Urea Derivatives of
Serinol (9−12). To a solution of the corresponding isocyanate
When one takes into consideration all the results collected
so far and the novelty of the scaffold proposed, it is possible to
consider these four serinol-derived benzoic acid esters as
promising hits for the development of a new class of anti-
HAdV agents.
(
3.6 mmol) in dry dichloromethane (20 mL) was added
dropwise a solution of the aminoalcohol (3 mmol) in methanol
1 mL). A white precipitate appeared, and it was isolated by
(
filtration in vacuum.
METHODS
General Procedure 2: Synthesis of the Diester Derivatives
■
(
(
(
13−37). Method A. To a solution of the urea derivative 9−12
Chemistry. General Methods. All reagents, solvents, and
starting materials were obtained from commercial suppliers
and used without further purification. The crude reaction
mixtures were concentrated under reduced pressure by
removing the organic solvents in a rotary evaporator. Reactions
were monitored by thin layer chromatography (TLC) using
Kieselgel 60 F254 (E. Merck) plates and a UV detector for
visualization. Flash column chromatography was performed on
Silica Gel 60 (E. Merck). All reported yields are of purified
products. Melting points were obtained on a Stuart Melting
Point Apparatus SMP 10 and are uncorrected. Mass spectra
were recorded on a Micromass AUTOSPECQ mass
spectrometer: EI at 70 eV and CI at 150 eV and HR mass
0.54 mmol) in dry dichloromethane (20 mL) and DMAP
1.35 mmol), the appropriate acylating agent (1.1 mmol) in
dry dichloromethane (5 mL) was added. The reaction mixture
was stirred at rt until TLC showed that all the starting material
had reacted; then, it was evaporated to dryness. The resulting
residue was treated with a 1 N HCl aqueous solution and
extracted with ethyl acetate (3 × 50 mL). The combined
organic phases were washed with saturated NaHCO
solution and brine. The organic layer was dried (Na
filtered, and evaporated in vacuum. The compound was further
purified through flash column chromatography on silica gel
using the appropriate eluent.
aqueous
SO ),
3
2
4
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6
0
Method B. Carboxylic acid (2.87 mmol) was dissolved in
dichloromethane, and EDCI (3.55 mmol) was added. The
mixture was stirred for 1 h at rt (mixture 1). At the same time,
to a suspension of urea derivative (9−11) (0.78 mmol) in
dichloromethane was added DMAP (2.13 mmol), and the
mixture was stirred for 1 h at rt (mixture 2); then, mixture 2
was added dropwise into mixture 1. The reaction was stirred
for 24 h at rt. The organic layer was washed with saturated
units/mL penicillin, 100 μg/mL streptomycin, and 0.1 mM
nonessential amino acids (complete DMEM).
Wild-type HAdV-5 and HCMV (AD169) were obtained
from the ATCC. The HAdV-5-GFP used in this study is a
replication-defective virus in the noncomplementing A549 cell
line containing a CMV promoter-driven enhanced green
fluorescent protein (eGFP) reporter gene cassette in place of
62
the E1/E3 regions. HAdV was propagated in 293β5 cells and
isolated from the cellular lysate by cesium chloride density
centrifugation. Virus concentration, in mg/mL, was calculated
with the Bio-Rad Protein Assay (Bio-Rad Laboratories) and
NaHCO aqueous solution and brine; then, it was dried over
Na SO , filtered, and evaporated in vacuum. The compound
was further purified through flash column chromatography
using the appropriate mixture of hexane−ethyl acetate as
eluent.
3
2
4
1
2
converted to virus particles/mL (vp/mL) using 4 × 10 vp/
mg.
General Procedure 3: Selective Mono-O-acylation Re-
action of Serinol Ureas (38−40). To a cooled solution (−15
HAdV Plaque Assay. Derivatives were tested in a dose−
response assay using an MOI of 0.06 vp/cell and drug
concentrations ranging from 10 to 0.3125 μM in a plaque
assay. Briefly, 293β5 cells were seeded in 6-well plates at a
°C) of urea derivative (10 or 11, 1 mmol) and DMAP (0.9
mmol) in dry dichloromethane (20 mL) and dimethylfor-
mammide (1 mL) was added dropwise a solution of acyl
chloride (0.9 mmol) in dry dichloromethane (15 mL). The
reaction mixture was stirred at −15 °C for 1 h; then, it was
washed with a 1 N HCl aqueous solution (2 × 15 mL),
5
density of 4 × 10 cells per well in duplicates for each
condition. At 80−90% confluency, they were infected with
HAdV5-GFP and rocked for 2 h at 37 °C. After this
incubation, wells were washed once with PBS. Then, cells
were carefully overlaid with 4 mL/well of equal parts of 1.6%
(water/vol) bacteriological agar (EULABOR S.L.) and 2×
EMEM (Minimum Essential Medium Eagle, BioWhittaker)
supplemented with 2× penicillin/streptomycin, 2× L-gluta-
mine, and 10% FBS. The mixture also contained the drugs in
concentrations ranging from 10 to 0.3125 μM. After
incubation for 7 days at 37 °C, virus plaques were scanned
with a Typhoon 9410 imager (GE Healthcare Life Sciences)
saturated NaHCO (2 × 15 mL), and brine. The organic layer
3
was dried (Na SO ), filtered, and evaporated to dryness. The
2
4
compound was purified through flash column chromatography
using the appropriate mixture of hexane−ethyl acetate as
eluent.
General Procedure 4: Synthesis of Dicarbamate Deriva-
5
4
tives (41−49). Method A. Urea derivatives (9−11) (0.64
mmol) and isocyanate (1.47 mmol) were mixed in toluene (10
mL). The resulting suspension was refluxed (110 °C) for 24 h;
then, the reaction mixture was filtered and washed with fresh
toluene. The obtained solid was dried at rt and analyzed as
pure compound or further purified through flash column
chromatography using hexane−ethyl acetate as eluent. In the
case of 4-nitrophenyl isocyanate, after column chromatography
of the mixture, the monoderivative was also isolated as a side
compound.
63
and quantified with ImageJ.
Cytotoxicity Assay. The cytotoxicity of the cidofovir
derivatives was measured using the AlamarBlue (Invitrogen)
according to the manufacturer’s instructions. A549 cells at a
3
density of 5 × 10 cells per well in 96-well plates were seeded.
Decreasing concentrations of each compound (200, 150, 100,
80, 60, 40, 30, 20, 10, 5, 2.5, and 0 μM) were diluted in 100 μL
of DMEM, and cells were then incubated at 37 °C for the
following 48 h. After the incubation, the AlamarBlue reagent
was added to the cells (1/10th Alamar Blue reagent in culture
55
Method B. First, 2-methylphenyl isocyanate was prepared.
To a solution of o-toluidine (1.87 mmol) in dichloromethane
(
2
20 mL) was added an aqueous solution of Na CO (3 mmol,
0 mL), and the heterogeneous mixture was vigorously stirred
medium) for an extra 4 h. The CC value was obtained using
2
3
50
the statistical package GraphPad Prism. This assay was
performed in duplicate. The selectivity index (SI) was
evaluated as the ratio of CC50 to IC , where the IC is
for 5 min at rt. Triphosgene (0.62 mmol) was added to the
flask and stirred for 30 min. The phases were manually
separated, and the organic layer was dried (Na SO ), filtered,
50
50
defined as the concentration of compound that inhibits HAdV
infection by 50%.
2
4
and evaporated to dryness. The isocyanate was used without
further purification in the next reaction.
HAdV DNA and mRNA E1A Quantification by Real-Time
PCR. For DNA quantification, A549 cells (1.5 × 10 cells/well
5
2
-Methylphenyl isocyanate (1 mmol) was dissolved in
dichloromethane (20 mL); then, DMAP (1 mmol) and the
appropriate urea derivative (9 or 11) (0.42 mmol) were added.
The reaction mixture was stirred for 48 h and then
concentrated under reduced pressure. The crude product was
purified through flash column chromatography using hexane−
ethyl acetate as eluent.
in a 24-well plate) were incubated for 24 h in 500 μL of
complete DMEM, and they were infected with wild-type
HAdV5 (100 vp/cell) when more than 90% of confluency was
observed. Infected cells were incubated for 24 h at 37 °C in
500 μL of complete DMEM containing a 50 μM concentration
of either compounds 13 or 14 and 15 and 37 μM for
compounds 27 and 32, respectively, or the same volume of
DMSO (positive control). All samples were done in duplicate.
Biological Methods. Cell Lines and Virus Strain. Human
A549 (ATCC CCL-185), HEK-293 (ATCC CRL-1573), and
HFF (ATCC SCRC-1041) cell lines were from the American
Type Culture Collection (ATCC, Manassas, VA). The 293β5
stable cell line overexpressing the human β5 integrin subunit
After 24 h of incubation at 37 °C and 5% CO , DNA was
2
purified from the cell lysate with the E.Z.N.A. Tissue DNA Kit
(Omega Biotek, Norcross, GA) following the manufacturer’s
instructions. TaqMan primers and probes for a common region
of the HAdV5 were designed with the GenScript Real-Time
PCR (TaqMan) Primer Design software (GenScript).
Oligonucleotide sequences were as follows: AQ1: 5′-GCC
ACG GTG GGG TTT CTA AAC TT-3′; AQ2: 5′-GCC CCA
61
was kindly provided by Dr. Glen Nemerow. The cell lines
were propagated in Dulbecco’s modified Eagle medium
(
DMEM, Life Technologies/Thermo Fisher) supplemented
with 10% fetal bovine serum (FBS) (Omega Scientific,
Tarzana, CA), 10 mM HEPES, 4 mM L-glutamine, 100
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GTG GTC TTA CAT GCA CAT-3′; Probe: 6-FAM-5′-TGC
ACC AGA CCC GGG CTC AGG TAC TCC GA-3′-
TAMRA′. Real-time PCR mixtures consisted of 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 12.5 μL mixed with 12.5 μL of FastGene 2× PROBE
Universal (NIPPON Genetics). The PCR cycling protocol was
incubated in complete DMEM at a 50 μM concentration of
either compounds 13 or 14 and 15 and 37 μM for compounds
27 and 32, respectively, or the same volume of DMSO
(positive control) in duplicate. Then, cells were incubated for
72 h at 37 °C and 5% CO , and HCMV DNA was purified
2
from the cell lysate using the E.Z.N.A. Tissue DNA Kit
(Omega Biotek, Norcross, GA) following the manufacturer’s
instructions. Real-time PCR primers, mixtures, and protocols
9
5 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 60
38
°C for 30 s.
were the same as previously reported, and the assay was
performed in a thermal cycler LightCycler 96 System (Roche).
Statistical Analyses. One-way ANOVA tests (Dunnet
method) were carried out using the GraphPad Prism 6. We
considered statistical significance with a p value under 0.05.
This statistical significance was pointed out with asterisks in
the graphs, and the numbers of them indicate the level of
significance (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).
For the evaluation of RNA expression, the same conditions
of infection applied for the DNA quantification were used. Six
hours after infection, RNA was purified with the RNeasy Mini
Kit (QIAGEN, Valencia, CA) following the manufacturer’s
instructions. Quantification of RNA copy numbers was
performed using primers and conditions previously reported
6
4
for E1A.
The internal control used was the human glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) gene. Oligonucleotide
sequences for GAPDH and the conditions were those
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00515.
■
*
sı
64
previously reported.
For quantification, gene fragments from hexon and GAPDH
were cloned into the pGEM-T Easy vector (Promega), and
known concentrations of template were used to generate a
standard curve in parallel for each experiment. All assays were
performed in a thermal cycler LightCycler 96 System (Roche).
Virus Yield Reduction. A burst assay was used to assay the
Full description of the chemical characterization of
compounds 9−50; copies of NMR spectra of com-
pounds 9−50, Figures S1−S39 (PDF)
effect of selected compounds on virus production. A549 cells
AUTHOR INFORMATION
Corresponding Authors
■
5
(1.5 × 10 cells/well in a 24-well plate) were infected with
wild-type HAdV5 (100 vp/cell) when more than 90% of
confluency was observed. Infected cells were incubated for 48
h at 37 °C in 500 μL of complete DMEM at a 50 μM
concentration of either compounds 13 or 14 and 15 and 37
μM for compounds 27 and 32, respectively, or the same
volume of DMSO (positive control). After 48 h, cells were
harvested and subjected to three rounds of freezing/thawing.
Margarita Vega-Holm − Department of Organic and Medicinal
Chemistry, Faculty of Pharmacy, University of Seville, E-41071
Seville, Spain; orcid.org/0000-0003-2197-2004;
Email: mvegaholm@us.es
Fernando Iglesias-Guerra − Department of Organic and
Medicinal Chemistry, Faculty of Pharmacy, University of Seville,
E-41071 Seville, Spain; Email: iglesias@us.es
́ ́
Javier Sanchez-Cespedes − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
University of Seville, E41013 Seville, Spain; Email: jsanchez-
Serial dilutions of clarified lysates were titrated on A549 cells
4
(
3 × 10 cells/well), and TCID50 values were calculated using
65
an end-point dilution method.
́
Nuclear-Associated HAdV Genomes. The nuclear delivery
of the HAdV genomes was assessed with real-time PCR
ibis@us.es
following nuclear isolation from infected cells. A549 cells (1 ×
6
1
0 cells/well) in 6-well plates were infected with HAdV5 wild-
Authors
Sarah Mazzotta − Department of Organic and Medicinal
Chemistry, Faculty of Pharmacy, University of Seville, E-41071
Seville, Spain; Department of Pharmacy, Health and
Nutritional Sciences, University of Calabria, 87036 Arcavacata
di Rende, Cosenza, Italy
Judith Berastegui-Cabrera − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
́
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
University of Seville, E41013 Seville, Spain
Gabriele Carullo − Department of Pharmacy, Health and
Nutritional Sciences, University of Calabria, 87036 Arcavacata
di Rende, Cosenza, Italy; Department of Biotechnology,
Chemistry and Pharmacy, DoE 2018-2022, University of Siena,
53100 Siena, Italy; orcid.org/0000-0002-1619-3295
Marta Carretero-Ledesma − Unit of Infectious Diseases,
Microbiology and Preventive Medicine, Institute of Biomedicine
́
of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/
CMV DNA Quantification by Real-Time PCR. To test the
anti-HCMV activity of these compounds, HFF cells were
seeded in a 24-well plate (1 × 10 cells/plate) and infected
with HCMV (MOI of 0.05 vp/cell). Infected cells were
University of Seville, E41013 Seville, Spain
Lara Mendolia − Department of Organic and Medicinal
Chemistry, Faculty of Pharmacy, University of Seville, E-41071
Seville, Spain
6
I
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Francesca Aiello − Department of Pharmacy, Health and
Nutritional Sciences, University of Calabria, 87036 Arcavacata
di Rende, Cosenza, Italy; orcid.org/0000-0001-6846-5582
spectroscopy; DEPT, distortionless enhancement by polar-
ization transfer; HSQC, heteronuclear single quantum
correlation; NOESY, nuclear Overhauser effect spectroscopy;
TMS, TMS, tetramethylsilane; ATCC, American Type Culture
Collection; DMEM, Dulbecco’s modified Eagle medium; FBS,
fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid; EMEM, minimum essential medium Eagle;
TCID50, 50% tissue culture infective dose
Jero
and Preventive Medicine, Institute of Biomedicine of Seville
nimo Pachon − Unit of Infectious Diseases, Microbiology
́ ́
́
IBiS), University Hospital Virgen del Rocio/CSIC/University
(
of Seville, E41013 Seville, Spain; Department of Medicine,
University of Seville, E-41009 Seville, Spain
Jose Manuel Vega-Perez − Department of Organic and
́ ́
Medicinal Chemistry, Faculty of Pharmacy, University of Seville,
E-41071 Seville, Spain
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HAdVs, human adenoviruses; dsDNA, double-stranded DNA;
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