Synthesis of three triterpene series and their activity against respiratory syncytial virus

Article abstract of DOI:10.1002/ardp.201800108

The human respiratory syncytial virus (hRSV) is a leading cause of hospitalization due to acute lower respiratory infection especially in infants and young children, sometimes causing fatal cases. The monoclonal antibody palivizumab is one of the available options for preventing this virus, and at the moment there are several hRSV vaccine trials underway. Unfortunately, the only drug option to treat hRSV infection is ribavirin, which can be used in severe high-risk cases. For this reason, new medicines are needed and, in this context, the triterpenes and their derivatives are promising alternatives, since many of them have shown important antiviral activity, such as bevirimat. Therefore, we report three series of triterpene (betulin (BE), betulinic acid (BA), and ursolic acid (UA)) derivatives tested against hRSV. The derivatives were synthesized by using commercial anhydrides in an easy and inexpensive step reaction. For the antiviral assay, A549 cells were infected by hRSV and after 96 h of compound or ribavirin (positive control) treatment, the cell viability was tested by MTT assay. DMSO, non-infected cells and infected cells without treatment were used as negative control. The triterpene esterification at the hydroxyl group resulted in 17 derivatives. The 3,28-di-O-acetylbetulin derivative (1a) showed the best results for cell viability, and real-time PCR amplification was performed for 1a treatment. Remarkably, one new anti-hRSV prototype was obtained through an easy synthesis of BE, which shall represent an alternative for a new lead compound for anti-hRSV therapy.

Full text of DOI:10.1002/ardp.201800108

Received: 1 April 2018
Revised: 23 June 2018
Accepted: 29 June 2018
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DOI: 10.1002/ardp.201800108
FULL PAPER
Synthesis of three triterpene series and their activity against
respiratory syncytial virus
Gloria N. Santos da Silva1
Diana Monti Atik2
Jheini L. Antunes Fernandes2
Ana Paula Duarte de Souza2
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Deise de Freitas do Nascimento2
Simone C. Baggio Gnoatto1
Tiago Fazolo2
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1 Phytochemistry and Organic Synthesis
Abstract
Laboratory, School of Pharmacy, Federal
University of Rio Grande do Sul, Porto Alegre,
Brazil
The human respiratory syncytial virus (hRSV) is a leading cause of hospitalization due to
acute lower respiratory infection especially in infants and young children, sometimes
causing fatal cases. The monoclonal antibody palivizumab is one of the available
options for preventing this virus, and at the moment there are several hRSV vaccine
trials underway. Unfortunately, the only drug option to treat hRSV infection is ribavirin,
which can be used in severe high-risk cases. For this reason, new medicines are needed
and, in this context, the triterpenes and their derivatives are promising alternatives,
since many of them have shown important antiviral activity, such as bevirimat.
Therefore, we report three series of triterpene (betulin (BE), betulinic acid (BA), and
ursolic acid (UA)) derivatives tested against hRSV. The derivatives were synthesized by
using commercial anhydrides in an easy and inexpensive step reaction. For the antiviral
assay, A549 cells were infected by hRSV and after 96 h of compound or ribavirin
(positive control) treatment, the cell viability was tested by MTT assay. DMSO, non-
infected cells and infected cells without treatment were used as negative control. The
triterpene esterification at the hydroxyl group resulted in 17 derivatives. The 3,28-di-
O-acetylbetulin derivative (1a) showed the best results for cell viability, and
real-time PCR amplification was performed for 1a treatment. Remarkably, one new
anti-hRSV prototype was obtained through an easy synthesis of BE, which shall
represent an alternative for a new lead compound for anti-hRSV therapy.
2 Clinical and Immunology Laboratory,
Biomedical Research Institute, Pontifícia
Universidade Católica do Rio Grande do Sul,
Porto Alegre, Brazil
Correspondence
Dr. Gloria N. Santos da Silva, Phytochemistry
and Organic Synthesis Laboratory, School of
Pharmacy, Federal University of Rio Grande
do Sul, Porto Alegre 90610-000, Brazil.
Email: narjarags@gmail.com
Funding information
CNPq; CAPES
K E Y W O R D S
A549 cells, betulin, betulinic acid, respiratory syncytial virus, ursolic acid
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1
INTRODUCTION
(hRSV), which is a member of the Paramyxoviridae family and subfamily
Pneumovirinae.[5] Despite considerable efforts toward the develop-
Several viral diseases are a global public health burden, such as
dengue,[1] Zika,[2] Ebola,[3] viral hepatitis, and acquired immunodefi-
ciency syndrome (AIDS).[4] Another virus causing a global impact on
human health is the pathogen named human respiratory syncytial virus
ment of effective treatments for hRSV disease, this seasonal infection
is still a major cause of pneumonia and bronchiolitis in infants, in young
children, and even in adults.[6–8] A United States study estimated that
hRSV is the most common cause of viral mortality in children aged
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Arch Pharm Chem Life Sci. 2018;e1800108.
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© 2018 Deutsche Pharmazeutische Gesellschaft
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under 5 years.[9] hRSV is highly contagious and is transmitted by direct
contact with nasal secretions or by fomite spread on hands, cots, and
fluffy toys. Preventing the spread of the virus is possible by promoting
cough etiquette, hand washing, and cohorting hospitalized hRSV
patients.[10] The incubation period is typically 2–8 days, with virus
replication spreading from the upper respiratory tract epithelial cells to
the lower respiratory tract.[11]
active compound.[22] Consequently, BE and BA are of interest for
medicine as the basis for the development of new antiviral agents.
The therapeutic potential of UA, a pentacyclic triterpene found in
several traditional plants, has also attracted attention. The functional
properties have been described for UA, such as antibacterial,
antiprotozoal, anti-inflammatory, anti-HIV, and antitumor.[23,24]
However, the ability of these triterpenes for the inhibition of hRSV
was not evaluated. Since there is not a variety of medicine for hRSV
treatment, this current study shows efforts to find a new and effective
compound as an alternative. Thus, we assayed 20 compounds divided
in the three triterpenes series; BE, BA, and UA and their carbon 3 (C-3)
and carbon 28 (C-28 for BE) derivatives to verify their anti-hRSV
potential.
The available options for preventing and treating hRSV are limited
to select populations in high-resource settings.[12] One of this is the
monoclonal antibody palivizumab for prophylaxis.[13] The aerosolized
antiviral agent ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carbox-
amide) is the approved drug for the treatment of hRSV infections.
Ribavirin is a synthetic guanosine nucleoside analogue with in vitro
antiviral activity against a range of RNA and DNA viruses.[14] There is a
concern regarding the efficacy of ribavirin and based on the available
data it is not recommended in the current guidelines for management
of bronchiolitis.[15]
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2
RESULTS AND DISCUSSION
An example of therapeutic strategies that have been developed
for the treatment of hRSV infection is GS-5806, which is an hRSV
fusion and entry inhibitor that successfully completed a phase I safety
trial and a phase II hRSV challenge trial in adults.[16] Other studies have
been developing but the majority of the compounds are still in the
preliminary stages of research, such as emodin.[17]
Through an easy and inexpensive step reaction, it was possible to
obtain 17 triterpenes derivatives with satisfactory yield (range of
70–93%). The structures of all derivatives are depicted in Table 1. After
the synthesis of these compounds, their structural elucidation could be
deduced from 1H, 13C NMR, and HR-EI-MS data. The data of BA and
UA series were compared with those previously described in our prior
studies,[25,26] and 3,28-di-O-succinylbetulin (1d) was compared with
data published by Tian et al.[27] In addition, we have described for the
first time four new BE derivatives. The BE was modified at C-3 and C-
28 to determine if ester substituents influence anti-hRSV activity,
whereas the anti-HIV derivative from BA, bevirimat, is the successful
result of C-3 modification and in our previous studies against another
microorganism, malaria parasite, also displayed improvement of
activity when C-3 and C-28 were chemically modified.[25,26] The
1H NMR spectra of the BE derivatives have two singlets around δ
4.50 ppm suggesting the presence of an isoprenyl group at position H-
29. The loss of dd at δ 3.05 ppm suggests that in the presence of H-3
the carbon binding at hydroxyl was substituted for an ester group.
Also, after the change at C-3 the presence of ─CHOCO─ around δ
2.50 ppm suggested that esterification had occurred. The signals of
ester groups were observed in the 13C NMR spectrum at the range of δ
157–177 ppm. The isoprenyl was evidenced in signals around δ 109
(C-29) and `δ 150 ppm (C-20).
Although some agents have been reported to have anti-hRSV
activity,[16–19] new strategies for therapeutic intervention during
moderate to severe hRSV infection are evidently needed. It is clearly
seen that the progress made in hRSV vaccines and therapeutics since
the discovery of the virus in 1950 has been poor, particularly compared
to other viruses like influenza virus, hepatitis C virus, and HIV.[12] In this
context, natural compounds, as triterpenes, might be an alternative.
The triterpenes, betulin (BE), betulinic acid (BA), and ursolic acid (UA)
(Figure 1), have shown a variety of biological activity.
BE is a pentacyclic triterpene alcohol with a lupane skeleton. BE
can be used as such, or after chemical modification as a starting
compound for other useful materials and compounds, such as BA
which possess various interesting pharmacological properties.[20] For
instance, BA has described its anti-HIV, antibacterial, antimalarial, anti-
inflammatory, and anthelmintic activities.[21] Furthermore, a promising
BA derivative called bevirimat, the first-in-class HIV-1 maturation
inhibitor, has been modified at C-28 position and resulted in even more
FIGURE 1 Chemical structures of betulin (BE), betulinic acid (BA), and ursolic acid (UA)

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TABLE 1 Library of the three series of triterpene derivatives, cytotoxicity and anti-hRSV activity by MTT assay
Code
BE
R
CC50 (μM)
7.48
EC50 (μM)
69.72
TI
H
0.11
1a
2245.38
104.66
21.45
1b
1d
268.27
11.197
196.81
120.15
1.36
0.093
1e
1f
624.95
244.96
98.87
73.05
6.32
3.35
BA
H
30.31
13.74
***
***
2a
27.77
0.49
2b
33.14
38.51
0.86
2c
2d
120.48
30.79
33.81
93.77
3.56
0.33
2e
2f
16.02
22.19
84.57
0.72
1.66
140.08
UA
H
23.73
26.80
18.67
48.75
1.27
0.54
3a
(Continues)

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TABLE 1 (Continued)
Code
3b
R
CC50 (μM)
EC50 (μM)
TI
14.61
71.00
0.20
3c
3d
685.00
46.57
86.85
***
7.89
***
3e
12.77
12.22
7.02a
28.09
76.44
1.96a
0.45
0.16
3.57
3f
Ribavirin
–
CC50, 50% cytotoxic concentration; EC50, 50% inhibitory concentration of the viral effect; TI, therapeutic index defined as CC50 over EC50; ***, beyond the
highest tested concentration (100 μM).
aµg.
Regarding the biological assay, Table 1 presents the antiviral
activity expressed as the EC50 for all compounds against hRSV in A549
cells. EC50 is the compound concentration required to reduce virus-
induced cytopathogenicity or viral plaque formation by 50%. The 50%
cytotoxic concentration (CC50), causing a 50% decrease in cell viability
was determined using a colorimetric 3-[4,5-dimethylthiazol-2-y1]-2,5-
diphenyltetrazolium bromide (MTT) assay system. Ribavirin was the
reference compound included. Among the precursor triterpenes
tested, UA proved to be more active with EC50 = 18.67 μM, although
in the UA series the 3c derivative was more selective with TI = 7.89.
Furthermore, the BE derivative (1a) displayed the higher value for
TI = 21.45, showing the importance of lupane for the acetyl modifica-
tion. Based on the result of TI, we selected the BE derivative (1a) to
perform an experiment to evaluate antiviral activity associated with
virus replication. Real-time PCR amplification was performed to hRSV
protein F gene and β-actin from cells infected with hRSV or infected
and treated with 1a (12.5 and 50 μM) or ribavirin (0.3 and 1.2 μg)
during 96 h. We found that BE derivative (1a) was able to significantly
reduce the hRSV RNA gene expression suggesting a reduction in virus
replication (Figure 2). Thus, our results show that the derivative more
active is not selective. It is relevant research since few triterpene
derivatives have been evaluated against hRSV, such as described by Li
et al.[28] The planning of new derivatives from 1a may be a promising
alternative for obtaining a future drug, as alternative therapy against a
virus with high morbidly and mortality range.
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3
CONCLUSION
In summary, three series of triterpene derivatives (BE, BA, and UA)
were prepared in high yield and in a total of 17 derivatives. All
compounds were evaluated against hRSV and BE derivative 1a was the
most selective. Then real-time PCR amplification was performed for
hRSV protein F gene and β-actin of infected cells treated with 1a,
demonstrating an effect of this compound in reducing virus RNA gene
expression. Further, structure modifications of 1a might afford a novel
series of anti-hRSV compounds that hold promise for treating a burden
pathogen. These modifications will be planned in order to find a
compound activity and selective. However, here we display informa-
tion that may base further investigations, in the scenario where the

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processed without refluxing for 1 h (for acyclic anhydrides). The crude
residue was purified in column chromatography to give the expected
pure compounds. Column chromatography was carried out using silica
gel 60 (Merck). Analytical thin layer chromatography was performed
on silica gel 60 plates (Merck) and spots visualized by spraying with
anisaldehyde/sulfuric. All compounds obtained are illustrated in
Table 1.
The NMR spectra of compounds 1a, 1b, 1e, and 1f are provided as
Supporting Information. The InChI codes of the investigated
compounds together with some biological activity data are also
provided as Supporting Information.
3,28-Di-O-acetylbetulin (1a)
Compound 1a was prepared by using acetic anhydride. White powder,
yield = 90%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.03 (s, 15H, CH3-23;
CH3-24; CH3-25; CH3-26 and CH3-27); 1.06 (m, 1H CH-13); 1.09 (t,
1H, CH-18); 1.12 (t, 2H, CH2-1); 1.24 (t, 2H, CH2-16); 1.36 (t, 2H, CH2-
15); 1.39 (m, 4H, CH2-2 and CH2-21); 1.43 (t, 4H, CH2-11 and CH2-22);
1.50 (t, 2H, CH2-6); 1.51 (m, 2H, CH2-12); 1.56 (t, 2H, CH2-7); 1.62 (t,
1H, CH-5); 1.79 (br s, 3H, CH3-30); 1.84 (m, 2H, CH2-2); 1.86 (t, 1H,
CH-9); 2.07 (s, 6H, CH3-32 and CH3-34); 2.44 (ddd, 1H, CH-19); 4.24
(dd, 2H, CH2-28); 4.46 (dd, 1H, CH-3); 4.58 (s, 1H, CH-29b); 4.68 (s,
1H, CH-29a). 13C NMR (75 MHz, CDCl3), δ (ppm): 16.15 (C-25); 16.48
(C-26 and C-27); 18.15 (C-6); 19.09 (C-34); 20.78 (C-32); 21.06 (C-30);
21.32 (C-11); 25.12 (C-2); 26.90 (C-12); 23.68 (C-23 and C-24); 27.93
(C-15); 29.55 (C-21); 29.71 (C-16 and C-22); 35.53 (C-7); 37.03 (C-10);
37.53 (C-13); 37.77 (C-4); 38.36 (C-1); 40.86 (C-8); 42.66 (C-14); 48.74
(C-19); 46.28 (C-18); 47.69 (C-17); 50.25 (C-9); 55.35 (C-5); 62.78
(C-28); 80.89 (C-3); 109.88 (C-29); 150.09 (C-20); 170.99 (C-31);
171.60 (C-33). HRMS (ESI-MS, m/z); [M+Na]+ calcd. for C34H54O4Na:
549.3915. Found: 549.2620, mp: 210–214°C.
FIGURE 2 Antiviral activity of 1a. Real-time PCR amplification
was performed for hRSV protein F gene and β-actin gene from cells
infected with hRSV or infected and treated with 1a (12.5 and
50 μM) or ribavirin (0.3 and 1.2 μg) during 96 h. Relative expression
was obtained by subtracting the CT value of β-actin from CT value
of hRSV protein F gene. Statistical significance between the groups
and hRSV was determined with one-way ANOVA followed by
Bonferroni post-hoc test. **p < 0.01, ***p < 0.001
only drug available, ribavirin, is not selective and for this reason is not
safe to use.
3,28-Di-O-isobutylbetulin (1b)
The compound 1b was prepared by using isobutyric anhydride. White
powder, yield = 93%. 1H NMR (400 MHz, CDCl3), δ (ppm): 0.96 (t, 2H,
CH2-16); 1.02 (s, 15H, CH3-23, CH3-24, CH3-25, CH3-26 and CH3-27);
1.06 (t, 1H, CH-18); 1.13 (m, 3H, CH2-1 and CH-13); 1.17 (d, 12H, CH3-
33, CH3-34, CH3-37 and CH3-38); 1.38 (t, 4H, CH2-15 and CH2-21);
1.41 (m, 4H, CH2-2 and CH2-11); 1.49 (m, 4H, CH2-6 and CH2-12);
1.57 (t, 2H, CH2-7); 1.60 (t, 1H, CH-5); 1.67 (t, 2H, CH2-22); 1.79 (br s,
3H, CH3-30); 2.42 (m, 1H, CH-36); 2.53 (m, 1H, CH-32); 1.82 (t, 1H,
CH-9); 2.03 (ddd, 1H, CH-19); 2.26 (t, 2H, CH2-36); 2.38 (t, 2H, CH2-
32); 4.26 (dd, 2H, CH2-28); 4.44 (dd, 1H, CH-3); 4.57 (s, 1H, CH-29b);
4.67 (s, 1H, CH-29a). 13C NMR (75 MHz, CDCl3) δ (ppm): 16.05 (C-25);
16.07 (C-26); 16.59 (C-27); 19.12 (C-33, C-34, C-37 and C-38); 19.25
(C-6); 20.82 (C-11 and C-30); 23.68 (C-23 and C-24); 25.18 (C-2);
26.95 (C-12); 27.96 (C-15); 29.61 (C-21); 29.82 (C-16); 34.12 (C-22);
34.26 (C-36); 34.49 (C-32); 34.57 (C-7); 37.10 (C-10); 37.57 (C-13);
37.97 (C-1 and C-4); 38.36 (C-8); 40.91 (C-14); 42.73 (C-18); 46.57
(C-17); 48.83 (C-19); 50.29 (C-9); 55.39 (C-5); 62.42 (C-28); 80.35 (C-
3); 109.88 (C-29); 150.19 (C-20); 176.82 (C-35); 177.52 (C-31). HRMS
(ESI-MS, m/z); [M−H+K]+ calcd. for C38H61O4K: 619.4124. Found:
619.3745, mp: 110–112°C.
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 Obtaining of BE, BA, and UA
BA was isolated from Platanus acerifolia bark (voucher specimen: ICN
171329) and UA was isolated from waste apple (Malus domestica) peels
obtained from a local juice factory, as described by Silva et al.[26] Both
compounds were identified using full spectroscopy data, which are
consistent with those previously described.[25,26] BE was purchased
from Sigma.
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4.1.2 Semisynthesis of BE, BA, and UA derivatives
BE, BA, and UA derivatives were synthesized according to the general
procedure described in the literature.[25,26] In brief, for ester
derivatives the commercial anhydride (1.1 mmoL, 5 Eq) and DMAP
(0.22 mmoL, 1 Eq) were added to BA or UA (0.22 mmoL) in pyridine or
CH2Cl2 (2 mL) and refluxed for 24 h (for cyclic anhydrides) or

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3,28-Di-O-trifluoroacetylbetulin (1e)
an anti-hRSV antibody (Millipore, Billerica, MA, USA). Viral aliquots
The compound 1e was prepared by using trifluoroacetic anhydride.
White powder, yield = 80%. 1H NMR (400 MHz, CDCl3), δ (ppm): 0.83 (t,
2H, CH2-16); 0.99 (m, 1H, CH-13); 1.06 (s, 15H, CH3-23, CH3-24, CH3-
25, CH3-26 and CH3-27); 1.08 (t, 1H, CH-18); 1.15 (t, 2H, CH2-1); 1.30
(m, 2H, CH2-12); 1.33 (t, 2H, CH2-15); 1.42 (m, 2H, CH2-21); 1.43 (t, 4H,
CH2-11 and CH2-22); 1.45 (t, 2H, CH2-6); 1.62 (t, 1H, CH-5); 1.64 (t, 2H,
CH2-7); 1.78 (br s, 3H, CH3-30); 1.80 (m, 2H, CH2-2); 1.84 (t, 1H, CH-9);
1.99 (ddd, 1H, CH-19); 4.15 (dd, 2H, CH2-28); 4.58 (dd, 1H, CH-3); 4.62
(s, 1H, CH-29b); 4.71 (s, 1H, CH-29a). 13C NMR (75 MHz, CDCl3), δ
(ppm): 15.98 (C-26); 16.14 (C-27); 16.24 (C-25); 18.07 (C-6 and C-30);
19.08 (C-11); 20.77 (C-23 and C-24); 23.25 (C-2); 25.04
(C-12); 26.92 (C-15); 27.77 (C-21); 29.33 (C-16); 34.00 (C-22); 34.21
(C-7); 37.04 (C-10); 37.74 (C-13); 38.07 (C-4); 38.20 (C-1); 40.88 (C-8);
42.74 (C-14); 46.61 (C-18); 47.53 (C-17); 48.76 (C-19); 50.19 (C-9);
55.23 (C-5); 66.84 (C-28); 86.23 (C-3); 110.30 (C-29); 116.05 (C-32);
116.11 (C-34) 149.50 (C-20); 157.74 (C-31); 158.16 (C-33). HRMS (ESI-
MS, m/z); [M−H+3OH]+ calcd. for C34H50F6O7: 683.3383. Found:
683.3650, mp: 175–176°C.
were stored in −80°C.
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4.2.2 Cytotoxicity assay
A549 cells were seeded in a 96-well flat-bottom plate at 5 × 103 cells
per well. After 24 h incubation, each compound at 100, 50, 25, 12.5,
and 6.25 µM was added to the plates which were then incubated for a
further 96 h at 37°C in a humidified incubator with 5% CO2. Cell
viability was assessed by a colorimetric assay based on the reduction of
3-[4,5-dimethylthiazol-2-y1]-2,-diphenyltetrazolium bromide (MTT)
(Molecular Probes™, Thermo Fisher Scientific) by mitochondrial
enzymes. Briefly, 100 µL of medium was removed and 40 µL of
MTT reagent from a 5 mg/mL stock solution was added in each well.
Cells were incubated for 4 h and the precipitated formazan crystals
were dissolved in dimethyl sulfoxide (DMSO). Finally, the optic density
(OD) was analyzed at 570/620 nm using micro-plate reader (EZ Read
400, Biochrom). DMSO was added as a control in the same volume
used to dilute the compounds. The results were obtained from
triplicate assays. The percentage of cytotoxicity of each compound
was calculated considering 100% the OD of untreated cells and using
as reference the OD of cells treated with DMSO.
3,28-Di-O-dichloroacetylbetulin (1f)
The compound 1f was prepared by using chloroacetic anhydride.
White powder, yield = 85%. 1H NMR (400 MHz, CDCl3), δ (ppm): 0.99
(s, 15H, CH3-23, CH3-24, CH3-25, CH3-26 and CH3-27); 1.06 (m, 3H,
CH-13 and CH2-16); 1.06 (t, 1H, CH-18); 1.13 (t, 2H, CH2-1); 1.26 (m,
2H, CH2-21); 1.30 (t, 2H, CH2-15); 1.34 (m, 2H, CH2-11); 1.42 (t, 2H,
CH2-7); 1.45 (m, 4H, CH2-2 and CH2-12); 1.51 (t, 2H, CH2-6); 1.64 (t,
1H, CH-5); 1.74 (t, 2H, CH2-22); 1.79 (br s, 3H, CH3-30); 1.81 (t, 1H,
CH-9); 2.00 (ddd, 1H, CH-19); 4.07 (dd, 2H, CH2-28); 4.48 (dd, 1H, CH-
3); 4.61 (s, 1H, CH-29b); 4.70 (s, 1H, CH-29a); 5.92 (s, 1H, CH-34); 5.96
(s, 1H, CH-32). 13C NMR (75 MHz, CDCl3) δ (ppm): 15.96 (C-25); 16.11
(C-26); 16.32 (C-27); 18.01 (C-6); 19.08 (C-30); 20.74 (C-11); 23.19 (C-
23 and C-24); 25.06 (C-2); 26.95 (C-12); 27.80 (C-15); 29.41 (C-21);
29.48 (C-16); 33.98 (C-7 and C-22); 34.29 (C-10); 37.01 (C-13); 37.66
(C-4); 38.21 (C-1); 40.84 (C-8); 42.70 (C-14); 46.75 (C-18); 47.66
(C-17); 48.80 (C-19); 50.17 (C-9); 55.28 (C-5); 64.43 (C-34); 64.78
(C-32); 66.16 (C-28); 80.90 (C-3); 109.08 (C-29); 149.70 (C-20);
164.30 (C-31); 164.96 (C-33). HRMS (ESI-MS, m/z); [M−H+Na]+ calcd.
for C34H50Cl4O4Na: 683.2198. Found: 683.3828, mp: 166–170°C.
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4.2.3 Antiviral assay
To assess the effect of the compound in inhibiting the cell death caused
by the virus, A549 cells were seeded in 96-well flat-bottom plate
(5 × 103 per well) and after 24 h infected with 5 × 104 PFU of RSV.
After 2 h each compound at 100, 50, 25, 12.5, and 6.25 µM was added
to the plates which were then incubated for a further 96 h at 37°C in a
humidified incubator with 5% CO2. Cell viability was assessed by MTT
colorimetric assay as described above. Ribavirin was used as positive
control (TCI America) at 2.4, 1.2, 0.6, 0.3, 0.15 µg per well. The results
were obtained from triplicate assays. The percent protection of each
compound was calculated as [(A − B)/C − B)] × 100, where A, B, and C
are the OD values of treated infected, untreated infected, and
untreated uninfected cells, respectively.
|
4.2.4 Data analysis
The 50% cytotoxic concentration (CC50) and the 50% antiviral
effective concentration (EC50) for each compound were obtained
from dose-effect curves and determined by linear regression analysis
(not shown). The therapeutic index (TI) (i.e., selective index) is defined
as CC50/EC50.
|
4.2 Antiviral activity and cytotoxicity assay
|
4.2.1 Cell lines and virus
Human adenocarcinomic alveolar epithelial cells (A549) and Vero
cells were cultured in DMEM low glucose (Gibco™, Thermo Fisher
Scientific, Waltham, MA, USA) supplemented with 10% of heat-
inactivated fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil).
RSV A2 strain was kindly provided by Dr. Fernando Polack,
Fundación Infant, Argentina. The virus was grown on Vero cells in
Opti-MEM medium (Gibco™, Thermo Fisher Scientific) supple-
mented with 2% of FBS and was then purified from cell culture
supernatant. Viral plaque-forming units (PFU) were identified using
|
4.2.5 Viral load quantification by real-time PCR
Total RNA was extracted from cells infected with RSV or infected and
treated with 1a (12.5 and 50 μM) or ribavirin (0.3 and 1.2 μg).
Complementary DNA (cDNA) was synthesized using transcriptase kit
GoScrip™ (Promega™, Madison, WI, USA). Real-time PCR was
performed for the amplification of the RSV F protein gene using

SANTOS DA SILVA ET AL.
7 of 7
|
specific primers and probes: forward-5′-AACAGATGTAAGCAGCT
CCGTTATC-3′, reverse-5′-GATTTTTATTGGATGCTGTACATTT-3′,
and probe 5′-FAM/TGCCATAGCATGACACAATGGCTCCT-TAMR
A/-3′ and for amplification of human β-actin as endogenous control
gene using TaqMan assay (Hs00174103_m1 ACTB). The reaction was
performed in a StepOne® (Applied Biosystems®) under the following
conditions: 50°C for 2 min and 95°C for 10 min followed by for 40
cycles of 95°C for 15 s and 60°C for 1 min. The Delta cycle-threshold
(ΔCt) was obtained by subtracting the endogenous control gene Ct
value from the Ct value of the RSV protein F gene.[29]
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ACKNOWLEDGMENTS
This work was supported by grants and financial support from Brazilian
Agencies CNPq and CAPES. The authors are also grateful to the
Graduate Programme in Pharmaceutical Sciences (PPGCF/UFRGS).
GNSS received CAPES Fellowship.
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The authors have declared no conflict of interest.
ORCID
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Gloria N. Santos da Silva
http://orcid.org/0000-0002-6584-201X
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