Anti-herpetic and anti-dengue activity of abietane ferruginol analogues synthesized from (+)-dehydroabietylamine

Article abstract of DOI:10.1016/j.ejmech.2015.11.009

The abietane-type diterpenoid (+)-ferruginol (1), a bioactive compound isolated from several plants, has attracted much attention as consequence of its pharmacological properties, which includes antibacterial, antifungal, antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and antitumor actions. In this study, we report on the antiviral evaluation of ferruginol (1) and several analogues synthesized from commercial (+)-dehydroabietylamine. Thus, the activity against Human Herpesvirus type 1, Human Herpesvirus type 2 and Dengue Virus type 2, was studied. Two ferruginol analogues showed high antiviral selectivity index and reduced viral plaque-size in post-infection stages against both Herpes and Dengue viruses. A promising lead, compound 8, was ten-fold more potent (EC₅₀ = 1.4 μM) than the control ribavirin against Dengue Virus type 2. Our findings suggest that the 12-hydroxyabieta-8,11,13-triene skeleton, which is characteristic of the diterpenoid ferruginol (1), is an interesting molecular scaffold for development of novel antivirals. In addition, the cytotoxic and antifungal activities of the synthesized ferruginol analogues have also been investigated.

Full text of DOI:10.1016/j.ejmech.2015.11.009

Accepted Manuscript
Anti-Herpetic and Anti-Dengue Activity of Abietane Ferruginol Analogues Synthesized
from (+)-Dehydroabietylamine
Vicky C. Roa-Linares, Yaneth M. Brand, Lee S. Agudelo-Gomez, Verónica Tangarife-
Castaño, Liliana A. Betancur-Galvis, Juan C. Gallego-Gomez, Miguel A. González
PII:
S0223-5234(15)30346-9
10.1016/j.ejmech.2015.11.009
EJMECH 8199
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2015
Revised Date: 23 September 2015
Accepted Date: 5 November 2015
Please cite this article as: V.C. Roa-Linares, Y.M. Brand, L.S. Agudelo-Gomez, V. Tangarife-Castaño,
L.A. Betancur-Galvis, J.C. Gallego-Gomez, M.A. González, Anti-Herpetic and Anti-Dengue Activity
of Abietane Ferruginol Analogues Synthesized from (+)-Dehydroabietylamine, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.009.
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ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
1
Anti-Herpetic and Anti-Dengue Activity of Abietane Ferruginol
Analogues Synthesized from (+)-Dehydroabietylamine
a,b
a,b
a
Vicky C. Roa-Linares , Yaneth M. Brand , Lee S. Agudelo-Gomez ,Verónica Tangarife-
a
a,b
b
*,c
Castaño , Liliana A. Betancur-Galvis , Juan C. Gallego-Gomez and Miguel A. González,
a
Group of Investigative Dermatology, Institute of Medical Research, Medicine Faculty, University of Antioquia, Medellin, A.A1226,
Antioquia, Colombia
b
Translational and Molecular Medicine Group, Institute of Medical Research, Medicine Faculty, University of Antioquia, Medellin,
Colombia
c
Departamento de Química Orgánica, Universidad de Valencia, E-46100 Burjassot, Valencia, SPAIN
Abstract— The abietane-type diterpenoid (+)-ferruginol (1), a bioactive compound isolated from several plants, has attracted much
attention as consequence of its pharmacological properties, which includes antibacterial, antifungal, antimicrobial, cardioprotective, anti-
oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and antitumor actions. In this study, we report on the
antiviral evaluation of ferruginol (1) and several analogues synthesized from commercial (+)-dehydroabietylamine. Thus, the activity
against Human Herpesvirus type 1, Human Herpesvirus type 2 and Dengue Virus type 2, was studied. Two ferruginol analogues showed
high antiviral selectivity index and reduced viral plaque-size in post-infection stages against both Herpes and Dengue viruses. A promising
lead, compound 8, was ten-fold more potent (EC50 = 1.4 M) than the control ribavirin against Dengue Virus type 2. Our findings suggest
that the 12-hydroxyabieta-8,11,13-triene skeleton, which is characteristic of the diterpenoid ferruginol (1), is an interesting molecular
scaffold for development of novel antivirals. In addition, the cytotoxic and antifungal activities of the synthesized ferruginol analogues
have also been investigated. © 20155 Elsevier Science. All rights reserved
Keywords: Antiviral, Herpes, Dengue, abietane, diterpene, ferruginol, dehydroabietylamine
However, mutations in the viral DNA polymerase can
Introduction
also cause this resistance. In immunocompromised patients,
the presence of HHV species resistant to ACV, with a
prevalence of 4-10%, has complicated their clinical
management [3]. Moreover, Dengue virus (DENV) is an
Human Herpesvirus (HHV) types 1 and 2 are enveloped
DNA viruses characterized for producing latent infection in
sensory neurons and reactivation during periods of severe
immunosuppression of the host. Many factors such as
stress, fatigue, sexual relations with people who have active
lesions, excess exposure to heat or cold, fever, laser
treatments, local tissue trauma and nerve damage, can
predispose to reactivation, especially in neonates, transplant
and immunocompromised patients which tend to present
aggressive and recurrent lesions [1]. Also, it has been
reported that prolonged therapy with Acyclovir (ACV), and
its analogues, has induced the emergence of drug-resistant
virus strains. Human Herpesvirus predominantly develops
resistance, almost 95%, as a result of mutations in the genes
that encode the viral thymidine kinase (TK) [2].
enveloped ssRNA virus.
To date, there have been
described four serotypes established in humans (DENV-1
to DENV-4) and recently, a fifth Dengue subtype that
follows the sylvatic cycle has been identified [4]. Dengue is
the most important mosquito-borne disease worldwide,
since it is distributed over one hundred countries of the
tropical belt and the tropics, both the Old and New World
[
5]. Disease manifestations cover a wide spectrum ranging
from dengue with or without warning signs to severe
dengue in which plasma leakage, haemorrhage and organ
impairment can lead to death. In addition, unusual
manifestations such as cardiomyopathy, liver failure and
neurological disorders have been reported [6]. The major
problem of this pathology is that there are no drugs
—
——
*
To whom correspondence should be addressed. (M.A.G) Tel.: +34-96-3543880; fax: +34-96-3544328. E-mail: Miguel.A.Gonzalez@uv.es

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European Journal of Medicinal Chemistry
approved for human treatment or vaccine available for
prophylaxis. Also, antiviral drugs addressed to specific
genes or proteins of the virus are not a good alternative due
to the high mutation rate of RNA viruses, often because
viral mutants are selected for drug resistance [7]. During
the last years in Colombia, several reports had shown the
high rates of evolution of DENV, possibly related with the
new Asiatic-American genotypes found in Colombia and
Brazil [8].
The aromatic abietane-type diterpenoids comprise a
large number of natural metabolites of plant origin which
possess a wide variety of biological effects [9]. These
abietanes have been the target of several synthetic
campaigns towards both the natural products and synthetic
derivatives with interesting pharmacological properties
ferruginol (1) from commercially available (+)-
dehydroabietylamine (3), where a series of phthalimides
could have potential antiviral activity [13]. A fullerene
diterpenoid hybrid was also envisaged as potential inhibitor
[14]. Herein, we describe the antiviral activity of ferruginol
(1) and some analogues (5-14) (Scheme 1) synthesized
from dehydroabietylamine (3) against HHV-1 and HHV-2,
and DENV-2. Our aim is to identify new antiviral drug
candidates which act on cellular and/or molecular targets
instead on viral proteins. In this way, the concerns about
the viral mutants resistant to the treatments can be avoided.
This approach has been recently called host-targeted
antiviral in an evolutionary study of resistance of Dengue
viruses under selective pressure of several antiviral agents
[15].
[
10]. Among these known bioactive compounds, (+)-
ferruginol (1), some derivatives of (+)-dehydroabietic acid
2) and (+)-dehydroabietylamine (3) as well as (+)-
Results and Discussion
(
jiadifenoic acid C (4) (Fig. 1) have shown promising
Chemistry
The synthesis of (+)-ferruginol (1) and analogues 5-14
from commercial (+)-dehydroabietylamine (3) was
performed as outlined in the Scheme 1. Compounds 1 and
results, including antiviral properties [10b,11].
5
-9 were synthesized as reported in the literature using a
Friedel-Crafts acylation and a Baeyer-Villiger oxidation as
key steps [16]. Then, compound 6 was used as starting
material for the preparation of compounds 10 and 11. Later,
compound 11 was converted into compounds 12-14 as
described recently in the literature [17]. Thus, the synthesis
starts with the introduction of the phthalimide group on (+)-
dehydroabietylamine (3), followed by Friedel-Crafts
acylation and oxidation under Baeyer-Villiger conditions to
afford acetate 5 in 72% overall yield. Hydrolysis of the
acetate group in 5 gave phenol 8 in high yield, while
Figure 1. Examples of bioactive aromatic abietane diterpenoids.
The present study is a continuation of our research
programs to discover bioactive diterpenoids [12]. It
includes the synthesis of a focused library based on (+)-
Scheme 1. Synthesis of the tested compounds 1 and 5-14.

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European Journal of Medicinal Chemistry
3
3
overall deprotection of 5 afforded the amino-phenol 6 in
5% yield. Compound 6 was the intermediate of three
separate approaches. Firstly, tosylation under standard
conditions gave compound 7 in 90% yield. Secondly,
oxidative deamination of 6 gave 18-oxoferruginol (9) in
moderate yield (50%), which was converted into ferruginol
14.5 µM, high activity against HHV-2 (Rf=1x10 ) and
reduction of cytopathic effect during DENV-2 infection,
comparable to control of untreated cells, at concentration of
29.0 µM. Compound 9 (18-oxoferruginol) also showed
7
3
high activity against HHV-2 (Rf=1x10 ) at a concentration
of 41.7 µM, however, its ability to exert reduction of
cytopathic effect in the presence of DENV-2 infection was
lower than that of compound 8. Moreover, compound 9 did
not show activity against HHV-1.
(
1) by Wolff-Kishner reduction (90% yield). And finally,
the treatment of 6 with tetrachlorophthalic anhydride
TCPA) afforded phenol 10 in 75% yield, which was
acetylated with acetic anhydride in pyridine to give acetate
1 in quantitative yield. Acetate 11 was oxidized at C-7
(
1
In 2007, Wen and co-workers reported the antiviral
potential of certain diterpenoid derivatives, including the
abietane-type diterpenoid ferruginol (1), during in vitro
coronavirus infection [18]. However, at present, the
antiviral activity of the abietane class of compounds has not
been much investigated, especially the study of derivatives
and analogues; therefore, the activity against HHV-1,
HHV-2 and DENV-2 reported in this study is to our
knowledge, the first report of antiviral activity of ferruginol
analogues. Also, as the compounds 8 and 9 are active
against two different viruses, such as herpesvirus and
dengue virus, our findings suggest that these molecules
probably have a broad-spectrum of antiviral activity against
enveloped viruses with DNA and RNA genome.
with excess of t-BuOOH as oxidant and CrO /pyridine
3
mixture as a catalyst in DCM, the yield of ketone 12 was
6
6%. Subsequently, the reaction of 12 with p-
tosylhydrazide yielded the corresponding p-tosylhydrazone
3 (77% yield). The fullerene-terpenoid hybrid 14 was
1
obtained by the treatment with NaOMe of 13 in anhydrous
pyridine for 20 min at room temperature followed by
addition of a solution of C60 in chlorobenzene and heating
at 70 ºC for 24 h. This gave compound 14 in ca. 30% yield.
All the compounds showed spectroscopic data in agreement
with the assigned structures and purity >95%.
Biological Evaluation
The synthesized compounds 1 and 5-14 (Scheme 1) were
evaluated for antiviral activity against HHV-1, HHV-2 and
DENV-2 (see Table 1). Two ferruginol analogues,
compounds 8 and 9 (18-oxoferruginol), showed relevant
activity.
Later on, to check the specific antiviral activity of these
compounds, we carried out the evaluation of other
biological activities such as cytotoxic and antifungal,
because is well known that ferruginol (1) has promising
bioactivities, such as antifungal, antibacterial, miticidal
antiplasmodial, antileishmanial, nematicidal, and antitumor,
among others [9]. In Table 2, it is shown the cytotoxic
activity on Vero cells as well as on the tumor cells HeLa,
Jurkat and U937 of ferruginol 1 and analogues 5-14.
Table 1. Reduction of viral titer, inhibition of cytopathic effect
and antiviral activity against HHV-1, HHV-2 and DENV-2 of
ferruginol analogues 8 and 9.
a
Vero cell line
Compounds 1, 6, 7, 9, 10 and 12 produced a dose-
dependent inhibition on the growth of the three tumor cell
lines: Jurkat, U937 and HeLa, as well as the Vero cell line,
HHV-1
HHV-2
DENV-2
2
Antiviral
activity
Antiviral
activity
(µM)
Antiviral
activity
(µM)
with R (coefficient of linear regression) > 0.8. Most of
CPE
Rf
b
Compound Rf
b
these compounds showed cytotoxic activity against at least
one tumor cell line at concentrations below 30 µM.
However, only compound 10 against HeLa and Jurkat cell
lines, and compound 12 against the three tumor cell lines,
showed a great SI (SI ≥ 5), being compound 12 the most
selective agent against tumor cell lines respect the non-
tumor cell line (SI > 43.8, > 26.0 and > 38.5, for HeLa,
Jurkat and U937, respectively). The compounds 5, 8, 11, 13
and 14 were not active against the tumor cell lines at the
tested concentrations and hence they were not evaluated on
the Vero cell line.
d
c
c
Inhibition
c
(
µM)
2
3
3
3
8
10
14.5
10
10
10
29.0
41.7
6.7
+
29.0
41.7
NT
9
NA NA
+
4
ACV
10
6.7
NT
+
RIBA
NT NT
NT NT
123.0
a
Vero Cercopithecus aethiops African green monkey kidney cell line
ATCC CCL- 81.
b
Reduction factor of the viral titer
c
Maximal non-toxic concentration that showed viral reduction factor
d
Cytopathic effect inhibition (+)
In addition, the antifungal activity of all compounds
was studied against two dermatophytes (Trichophyton
rubrum and T. mentagrophytes), and the filamentous fungi
Fusarium oxysporum, Aspergillus fumigatus, A. flavus and
A. terreus. Only one ferruginol analogue, compound 9,
showed anti-dermatophyte activity against T. rubrum and
T. mentagrophytes with geometric mean of minimal
inhibitory concentration (GM-MIC) values of 41.7 µM and
HHV-1: Human Herpesvirus type 1 (CDC Atlanta strain). HHV-2:
Human Herpesvirus type 2 (VR-734-G strain). DENV-2: Dengue virus
(
New Guinea strain). NT: not tested. NA: not active.
These values represent the mean of two independent experiments.
In particular, the ferruginol analogue 8 showed moderate
activity against HHV-1 (Rf=1x10 ) at a concentration of
2
8
3.3 µM, respectively. None of the compounds showed

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Table 2. Cytotoxicity (IC -μM) of ferruginol (1) and analogues 5-14 on HeLa, Jurkat, U937 and Vero cell lines.
5
0
4
European Journal of Medicinal Chemistry
c
Cell lines
HeLa
Jurkat
U937
Vero
a
a
a
a
IC50
IC50
IC50
IC50
b
b
b
SI
SI
SI
(
μM)
(μM)
48.2
>50
(μM)
21.3
>50
>80
36.0
>50
33.2
>40
>40
8.3
(μM)
90.4
NE
1
5
6
7
8
9
1
1
1
1
1
64.9
>50
>80
>50
>50
60.0
29.0
>40
7.3
1.4
1.9
4.2
NA
NA
NA
NA
1.5
NA
0.8
NA
NA
2.3
49.4
30.9
>50
39.8
82.5
NE
2.7
NA
4.2
NA
2.7
21.6
21.9
>40
90.8
266.8
NE
0
1
2
3
4
9.2
12.2
NA
>26.0
NA
NA
NA
NA
NA
NA
>38.5
NA
NA
NA
NA
NA
>43.8
NA
NA
NA
NA
12.3
>30
>320
NE
>30
>30
0.099
0.834
>30
>30
0.006
0.077
>30
NE
PXT
DOX
0.0005
0.012
0.65
1.93
a
Inhibitory concentration 50 defined as concentration of compounds that induces 50% of growth inhibition at 48 h.
SI, selectivity index defined as VERO IC50 over either Jurkat, U937 or HeLa IC50
b
c
Jurkat, human acute T cell leukemia ATCC TIB-152; U937, human promonocytic cell line ATCC CRL-1593.2; HeLa, human
cervix epitheloid adenocarcinoma cells ATCC CRL-1958; Vero, Cercopithecus aethiops African green monkey kidney cells ATCC
CCL-81.
PXT: Paclitaxel; DOX: Doxorubicine. NA: Not apply; NE: Not evaluated. These values represent the geometric mean of two
independent experiments, each one made by quadruplicate.
activity against the filamentous fungus tested (data not
shown). Likewise, none of the compounds showed anti-
Candida activity against three yeast strains (Candida
albicans, C. parapsilosis and C. tropicalis) (data not
shown).
In order to define the stage of viral replication cycle in
which the ferruginol analogues 8 and 9 were active, we
performed two different conditions of treatment. Firstly, we
tested the effect of compounds on early stages of viral
replication cycle, such as cell-free viral particle, adhesion
and viral entry by treatment before infection (pre-infection
treatment). And secondly, we tested the antiviral activity on
several steps of viral cycle that includes traffic, replication,
assembly, egress of new viral particles and cell-to-cell
spread by treatment after infection (post-infection
treatment).
Although there are several studies showing that
ferruginol (1) and some related abietanes exhibit promising
antifungal and cytotoxic activity [19], our results reveals
that compound 8 lacks of antifungal activity in the fungal
models tested and cytotoxicity against HeLa, Jurkat and
U937 tumor cells at the evaluated concentrations.
Therefore, this compound could be a selective molecule
against enveloped viruses.
As can be seen in Table 3, compounds 8 and 9 were
effective during post-infection treatment and were not
active during pre-infection treatment.
Mechanism of antiviral activity of ferruginol analogues
8
and 9
With both pre- and post-infection treatments, a plaque
reduction assay was performed and the concentration of
compounds 8 and 9 that reduced the number of viral
plaques in 50% (EC ) was interpolated from the dose-
Previous studies suggest that the mechanism by which
ferruginol (1) exerts antitumor activity is related to the
enhanced expression of BAX proapoptotic protein,
inhibition of signaling pathways involved in cell survival
and proliferation (Ras / PI3K and Jak/STAT) and reduction
of expression of cyclin-dependent kinases [20]. However,
the cellular and molecular basis by which these compounds
exhibit antiviral activity has not been currently described.
50
response curves. Furthermore, the antiviral selectivity index
(SI) was calculated through the relation between the
inhibitory concentration 50 (IC ) in Vero cells and EC of
50
50
each virus (IC / EC ) (Table 3).
50
50

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European Journal of Medicinal Chemistry
5
Table 3. Antiviral activity (EC -μM) on pre and post-infection treatment of ferruginol analogues 8 and 9.
5
0
EC50 (μM) (SI)
EC50 (μM) (SI)
IC50
IC50
Pre-infection treatment
Post-infection treatment
Compound
(μM)
(μM)
8 days
7
2h
HHV-1
NI
HHV-2
NI
DENV-2
NI
HHV-1
NI *
HHV-2
19.2 (10.0)
16.6 (5.9)*
NT
DENV-2
1.4 (57.7)*
5.0 (10.4)
NT
8
190.7
97.8
>10
80.8
52.2
NT
9
NI
NI
NI
19.6 (4.9)
NT
DEX-S
ACV
RIBA
0.015
NT
0.012
NT
NT
>1700
NT
NT
NT
2.2
0.27
NT
NT
NT
NT
NT
NT
NT
13.5
EC50, 50% antiviral effective concentration; IC50, 50% inhibitory concentration of cell proliferation (µg/mL); SI, Antiviral selectivity index values
IC50/EC50) are presented between parenthesis; DEX-S, dextran sulfate; ACV, acyclovir; RIBA, ribavirin; *Reduction on viral plaque-size. NI, no
inhibitory activity; NT, not tested. These values represent the mean of three independent experiments.
(
Compound 8 reduced the 50% of plaque forming units at a
concentration of 19.2 μM against HHV-2 and showed a
high SI equal to 10, whereas compound 9 reduced the
number of plaques in 50% at concentrations of 19.6 μM
and 16.6 μM, for HHV-1 and HHV-2, respectively. As
expected the controls, DEX-S and ACV, did show antiviral
activity in pre-treatment and post-treatment, respectively.
The antiviral selectivity index is defined as the relative
effectiveness of a product to inhibit viral replication
compared to the capacity for inducing cell death. This
index results useful to make bioactivity comparisons
between the compounds and helps in designing more potent
compounds. In recent reports, SI values higher than 10 (SI
>10) are considered indicative of a potential therapeutic
agent that would merit further biopharmaceutical and pre-
clinical studies [22]. Therefore, compound 8 is promissory
to continue this type of study.
These results show that the compounds 8 and 9 have
important activity against HHV-2 strain at low
concentrations, in comparison to HHV-1. This is possibly
due to a smaller number of receptors available in the host
cell to carry out the infection with HHV-2, making this
virus more sensitive to the action of the compounds [21].
Additionally to the dose-dependent effect previously
described during post-infection treatment, we found that
compounds 8 and 9 induced a reduction on viral plaque
size. To determine the average of reduction of viral plaque
size during compound-treatment relative to mock-treated
control, we performed an image analysis using Image-Pro
Plus 6.0 software (Figure 2). The area of plaques was
measured in millimeters and statistically significant
differences (P value <0.001) were found in all cases.
Compound 8 showed a dramatic reduction on viral plaque
size (93.7%) at a concentration of 29.0 μM against HHV-1
(CDC-Atlanta acyclovir-sensitive strain) (Figure 2A).
Respect to HHV-2, compound 9 reduced the size of viral
plaques (68.2%) at a concentration of 20.8 μM (Figure 2B).
Differences in the activity of these compounds were found
against HHV-1 strain. In the end-point titration technique
(
(
EPTT) assay, compound 8 showed moderate activity
Table 1, Rf = 1x10 ) and in the plaque forming unit (PFU)
2
assay this compound did not show a dose-dependent effect.
However, it showed a significant reduction in viral plaque
size (Table 3, highlighted with an asterisk), related among
others, with a low rate of viral replication and reduction of
cytopathic effect, confirming the antiviral effect of this
compound.
On the other hand, compound 9 did not show reduction of
viral titer during screening (EPTT) against HHV-1, but
inhibits the number of plaque forming units during the PFU
assay. This difference can be explained due the amount of
virus used in each assay. In the EPTT assay a high amount
of virus (10 TCID ) was used and the concentration of
compound that showed antiviral activity could not be
detected. In the PFU assay, 100 PFU/well was used and the
EC50 value was determined, nevertheless, this compound
did not show a high SI value (4.9), demonstrating closeness
between the cytotoxic concentration and the antiviral
concentration.
Furthermore, during treatment with compound 8 at a
concentration of 29.0 μM (Figure 2C) on cell infection by
HHV-1 acyclovir-resistant strain 29R (thymidine kinase
mutant), we found a similar effect to that showed against
the acyclovir-sensitive strain, reducing the viral plaque size
in 87.1%. This finding may suggest that the
phosphorylation by viral thymidine kinase it is not required
for the effect exerted by this compound, thus, is possible
that anti-herpetic mechanism of action of ferruginol
analogue 8 differs to acyclovir and other nucleoside
analogues.
50
Likewise, the anti-dengue activity of these ferruginol
analogues was evaluated 8 days post-infection (d.p.i), being
both compounds 8 and 9 active at EC50 concentrations of
1
.4 μM (SI=57.7) and 5.0 μM (SI=10.4), respectively.

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European Journal of Medicinal Chemistry
(A) HHV-1
(E) HHV-1
Likewise, reduction on viral plaque-size was observed
when cells infected with DENV-2 were treated with
compound 8 at concentration of 3.6 µM (Figure 2D). The
reduction percentages reported for all viruses indicate the
reduction of viral plaque size during compound-treatment
relative to mock-treated control (100%). Representations of
plaque morphology of HHV-1(CDC Atlanta), HHV-1
(
29R) and DENV-2 after the treatment with compound 8
and mock-treated control, are shown in Figures 2E, 2F and
H, respectively. Figure 2G shows the effect after treatment
2
Control
8
with compound 9 against HHV-2 and mock-treated control.
(B) HHV-2
(F) HHV-2
The reduction on viral plaque size of HHV strains and
DENV-2 in the presence of compounds 8 and 9 suggests
that these ferruginol analogues may prevent the first-round
of viral replication, release and/or cell-to-cell spread, being
this last stage an important factor in infectivity, virulence,
establishment of latency and immune response evasion
during Herpesvirus infections [23]. Therefore, inhibition of
viral intercellular diffusion is an attractive target in the
search for new antiviral drugs, mainly to treat the latent
infections and drug-resistant strains.
Control
9
In recent years, several compounds have shown effects on
virus cell-to-cell spreading during Herpesvirus infection.
For example, Lin and co-workers reported that chebulagic
acid and punicalagin, two hydrolyzable tannins isolated
from of Terminalia chebula (Combretaceae), inhibit the
HHV-1 cell-to-cell spread. This fact was verified 24 h post-
treatment using a fluorescent plaque assay in the presence
of HHV-1 neutralizing antibodies to guarantee cell-to-cell
transmission via intercellular junctions between infected
and uninfected cells [24]. Meanwhile, Nyberg and Ekblad
and co-workers demonstrated that the low molecular
weight heparin sulfate-mimic, PI-88, which is a mixture of
highly sulfated mannose containing oligosaccharides,
reduced the area of viral plaques with IC50 values of 2
μg/mL and 0.7 μg/mL for HHV-1 and HHV-2, respectively
(
C) HHV-1 (29R)
(G) HHV-1 (29R)
Control
8
(D) DENV-2
(H) DENV-2
[
25]. Nevertheless, to date it has not been reported a similar
effect on viral plaque size of HHV and other viruses from
abietane diterpenoids, among these, ferruginol (1) and their
derivatives.
Several molecular and cellular components involved in
Herpesvirus cell-to-cell spread have been previously
reported. For example, viral glycoproteins such as E and M
play a relevant role in normal viral plaque phenotype of
HHV-1, because the plaque size of the double-deletion
mutant (∆gEgM) reduced over 5-fold less than wild type
strain [26]. In polarized epithelial cells, Johnson and co-
workers demonstrated by electron microscopy, that the
gE/gI glycoproteins complex is necessary to accumulate
virions at cell junctions, since mutants that not express gE
glycoprotein leads to particles accumulation more
frequently on apical surfaces. Thus, they concluded that the
gE/gI complex is required to sort selectively HHV-1
particles to lateral surfaces and specifically to cell junctions
Control
8
Figure 2. Effect of compounds 8 and 9 on HHV-1, HHV-2 and
DENV-2 plaque-size during Vero cell infection. Effect on viral
plaque-size of compounds 8 and 9 in presence of 100 PFU/mL of
HHV-1 (2A), HHV-2 (2B), HHV-1 29R strain (2C) and DENV-2
(
2D). Results were expressed as an average of plaque size area (mm)
of 80 viral plaques developed in compound-treated cells relative to
mock-treated controls. Statistically significant differences were
found in all cases with p values <0.001 (∗∗∗). Parallel, the image
analysis using Image-Pro Plus 6.0 software represents the reduction
of viral plaque size during treatment with compounds 8 and 9 of
HHV-1 (2E), HHV-2 (2F), HHV-1 29R strain (2G) and DENV-2
(
2H). Two separate experiments were carried out for each
[
27]. On the other hand, structures containing actin and
compound.
tubulin during Herpesviruses infection have the ability to
project virions towards adjacent cells. Additionally, both

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
7
the membrane composition and cytoskeletal structures of
egress sites are modified during infection in non-polarized
cells [28]. According to the findings of Dixit and co-
workers, HHV-1 also promotes cytoskeletal rearrangements
that facilitate viral spread in vitro, including dramatic
increases of filopodia in cultured neurons [29]. Multiple
and convergent ways by which several viruses such as
antiviral selectivity index against the viruses tested and a
ten-fold more potency (EC50 = 1.4 M) than the control
ribavirin against Dengue Virus type 2.
With regard to the cytotoxic properties of the tested
compounds, it should be noted that the chlorinated-
phthalimide analogue 12 exhibited moderate cytotoxicity of
broad spectrum, without toxicity towards the non-tumor
cell line (Vero). Additionally, the tested compounds did not
show relevant antifungal activity.
Vaccinia, Hepatitis
B
and
C
viruses, Human
Immunodeficiency virus, Human T-lymphotropic virus,
Murine leukemia virus and HHV cause subversion of
cytoskeleton for different objectives in viral cycle are
summarized by Taylor and co-workers [30]. The actin
cytoskeleton is reorganized by these viruses affecting every
stage of the viral life cycle, from entry through assembly to
egress. Using immunochemical assays, drug inhibition
assays and protein interaction profiling methods, Wang and
co-workers identified the ways in which DENV-2 interacts
with actin cytoskeleton. This study showed that dynamic
treadmilling of actin is necessary for Dengue virus entry,
production and release, indicating a direct effect of viral E
protein on the structural modifications of actin cytoskeleton
Based on the above, the results here reported are relevant
for further research of possible cellular and molecular
mechanisms involved in the effect of reduction on viral
plaque size. Also, these findings are important to promote
the realization of biopharmaceutical testing and finding
new highly selective antiviral drugs mainly directed
towards cellular targets.
Experimental
[
31].
Chemistry. General procedures
Considering that cytoskeletal components such as
microtubules and actin microfilaments are required for both
Herpesvirus and Flavivirus replication cycle (mainly
involved in the entry, traffic, release and cell-to-cell
spread), is possible that ferruginol analogues 8 and 9 cause
disruption of these cellular pathways which may be
involved in viral spread. Nonetheless, other experiments
are required to confirm this hypothesis.
Optical rotations were measured using a 5 cm cell in a
Schmidt-Haensch Polartronic-D polarimeter. NMR spectra
were recorded on a 300 MHz spectrometer. All spectra
were recorded in CDCl as solvent unless otherwise stated.
3
13
Complete assignments of C NMR multiplicities were
made on the basis of DEPT experiments. J values are given
in Hz. MS data were acquired on a QTOF spectrometer.
Reactions were monitored by TLC using Merck silica gel
60 F-254 in 0.25 mm-thick plates. Compounds on TLC
plates were detected under UV light at 254 nm and
visualized by immersion in a 10% sulfuric acid solution
and heating with a heat gun. Purifications were performed
by flash chromatography on Merck silica gel (230-400
mesh). Commercial reagent grade solvents and chemicals
were used as purchased unless otherwise noted. Combined
organic extracts were washed with brine, dried over
Although it is very risky assure that some viral or cellular
component are involved in our findings, the study of
ferruginol analogues will be an important tool from
medicinal chemistry for dissecting these complicated
cellular and molecular processes of the viral infections. In
the future, this research will be routed to understanding of
some cellular and molecular mechanisms where, two very
different viruses (Herpesvirus and Dengue), are converging
in a conserved evolutionary pathway during cell infection,
affecting cytoskeleton elements, endomembrane system
and signaling pathways involved in viral egress from the
host-cells and spreading infection toward neighbor cells.
anhydrous NaSO , filtered, and concentrated under reduced
4
pressure.
Materials. Compounds 1 and 5-9 have been prepared
according to known procedures [16]. Compound 6 was
used as starting material for the preparation of compounds
10 and 11. Later, compound 11 was converted into
compounds 12-14 as described recently in the literature
[17]. All compounds prepared in this work exhibit
spectroscopic data in agreement with the proposed
structures. Purity of all final compounds was 95% or
higher.
Conclusions
In summary, we have discovered important antiviral
properties in the molecular scaffold 12-hydroxyabieta-
8
,11,13-triene functionalized at C-18, which is reminiscent
of the abietane diterpenoid ferruginol (1). Two ferruginol
analogues, compounds 8 and 9, synthesized from readily
available (+)-dehydroabietylamine (3) were found to
consistently inhibit Human Herpesvirus (type 1 and 2) and
Dengue virus type 2 at low micromolar concentrations.
These compounds showed antiviral activity specifically in
post-infective stages and significantly reduced the viral
plaque-size during infection. The presence of a phthalimide
moiety in the molecule, compound 8, resulted in the highest
12-Hydroxy-N,N-
(tetrachlorophthaloyl)dehydroabietylamine (10)
A mixture of the tetrachlorophthalic anhydride (640 mg,
16a
2.14 mmol) and 12-hydroxydehydroabietylamine 6 (647
mg, 2.14 mmol) in acetic acid (7.5 mL) was heated at
reflux for 2 h, then cooled to rt and 50 mL of water was

ACCEPTED MANUSCRIPT
8
European Journal of Medicinal Chemistry
added. The resulting solid was filtered off, washed with
water, dried under vacuum and purified by column
chromatography eluting with toluene to give compound 10
Herpesvirus type 2 (HHV-2 VR-734-G acyclovir-sensitive
strain) was obtained from the Center for Disease Control.
All of them were amplified in Vero cells (African green
monkey kidney-Cercopithecus aethiops, ATCC: CCL 81
line). Virus stocks were titrated in Vero cells by plaque
assay and expressed as plaque forming units (PFU/mL).
Dengue virus type 2 (DENV-2 New Guinea strain) was
donated by Maria Elena Peñaranda and Eva Harris
(Sustainable Sciences Institute and the University of
California at Berkeley) was amplified in C6/36HT of Aedes
albopictus cells, from ATCC, and titrated in Vero cells
following our laboratory conditions [32].
2
0
(
913 mg, 75%) as a yellow solid: []
-10.0 (c 1.0,
D
1
CHCl ); H NMR (300 MHz)  6.87 (1H, s), 6.60 (1H, s),
3
2
3
.67 (1H, d, J = 15.0), 3.54 (1H, d, J = 15.0), 3.10 (1H, m),
.92 (2H, m), 2.15 (2H, m), 1.24 (3H, d, J = 6.0), 1.22 (3H,
1
3
d, J = 6.0), 1.22 (3H, s), 1.04 (3H, s); C NMR (75 MHz)
2 x 164.5 (s), 150.6 (s), 148.1 (s), 2 x 140.1 (s), 131.6

(
(
C
s), 2 x 129.5 (s), 2 x 127.4 (s), 127.1 (s), 126.8 (d), 110.5
d), 49.8 (t), 45.2 (d), 39.5 (s), 38.0 (t), 37.5 (s), 37.1 (t),
2
9.3 (t), 26.7 (d), 25.7 (q), 22.7 (q), 22.5 (q), 19.5 (t), 19.0
+
(
q), 18.4 (t). HRMS (ESI) m/z 568.0986 [M+1] , calcd for
C H Cl NO : 568.0980.
Cells were maintained in Dulbecco's Modified Eagle
Medium (DMEM) supplemented with 5% of inactivated
FBS to Vero cells and 10% of inactivated FBS to C6/36HT
cells, 100 units/mL of penicillin, 100 µg/mL of
streptomycin, 100 µg/mL of l-glutamine, 0.14% NaHCO3,
and 1% of each non-essential amino acids and minimum
essential medium vitamin solution (choline chloride, D-
calcium pantothenate, folic acid, nicotinamide, pyridoxal
hydrochloride, riboflavin, thiamine hydrochloride and i-
inositol). Vero cells were incubated at 37°C in humidified
2
8
30
4
3
1
2-Acetoxy-N,N-
(
tetrachlorophthaloyl)dehydroabietylamine (11)
A solution of phenol 10 (656 mg, 1.15 mmol) in a 1:1
mixture (14 mL) of acetic anhydride and pyridine was
stirred at rt for one day. Then, 150 mL of water were added
and the mixture was extracted with ethyl acetate (3 x 25
mL). The combined organic extracts were washed with 6%
NaHCO (2 x 50 mL), water and brine. The resulting
5% CO atmosphere and C6/36 HT at 34°C in humidified
3
2
organic extract was dried over sodium sulphate, filtered and
concentrated. The residue was chromatographed on silica
eluting with chloroform to afford acetate 11 (704 mg,
5% CO atmosphere.
2
To determine the cytotoxic activity, cell lines of human
cervix epitheloid adenocarcinoma cells (HeLa, ATCC
CCL-2), acute T cell leukemia (Jurkat, ATCC TIB-152)
and human promonocytic cell line (U937, ATCC CRL-
1593.2) were used, as well as, the non-tumor cell line Vero.
Vero and HeLa cells were grown in DMEM supplemented.
Jurkat and U937 cells were maintained in RPMI-1640
medium (supplemented with 10% FBS), 100 units/mL of
penicillin, 100 µg/mL of streptomycin, and 100 µg/mL of
neomycin and maintained at 37 ºC in humidified 5% CO2
atmosphere.
1
00%) as a white solid with identical spectroscopic data to
the reported in the literature [17].
Biology
Reagents and compounds
Dulbecco’s Modified Eagle’s Medium (DMEM) and 3-
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide (MTT) were obtained from Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA). Fetal bovine serum
(
FBS) and penicillin/streptomycin were purchased from
Antiviral activity
Invitrogen Life Technologies (Carlsbad, CA, USA).
Acyclovir was obtained from Biogen Laboratory. Ribavirin
was obtained from Calbiochem (La Jolla, CA, USA).
Screening against HHV-1 and HHV-2.
Paclitaxel,
Doxorubicine,
Amphotericine
B
and
The anti-herpetic activity of ferruginol analogues on Vero
cells against 10 TCID50 of HHV-1 and HHV-2, were
carried out using the end-point titration technique (EPTT).
Vero cells were harvested in 96-well plates at a density of
Itraconazole were purchased from Sigma-Aldrich Chemical
Co. Terbinafine was obtained from Recalcine Laboratories
(
Santiago de Chile, Chile). Dimethyl sulfoxide (DMSO)
4
was purchased from Merck KGaA (Darmstadt, Germany).
2.0x10 cells/well at 37°C in humidified 5% CO2
atmosphere until constituted ≥80% of the cell monolayer.
Viral suspension/compound mixture were performed in
DMEM with 2% FBS supplemented containing
carboxymethylcellulose (CMC) to 1% and 0.5% for HHV-1
and HHV-2, respectively and incubated for 15 minutes at
room temperature. Compounds were evaluated at
concentrations of 6.25-50 μg/mL. After 48h (HHV-1) and
Stock solutions of compounds were prepared in DMSO and
frozen at -70 °C. The concentration of DMSO in biological
assays was of 0.05%. Cell controls with DMSO at 0.05%
were used.
Cell culture and viruses
7
2h (HHV-2) of incubation at 37°C in a humidified 5%
Human Herpesvirus type
1
(HHV-1 CDC Atlanta
CO atmosphere, the cell monolayers were stained with a
2
acyclovir-sensitive strain) was obtained from the Center for
Disease Control (Atlanta, GA, USA); HHV-1 29R
solution of 3.5% formaldehyde with 0.2% crystal violet and
the plaques were counted. Two independent experiments by
quadruplicate were carried out for each viral serotype and
each compound. Controls were included untreated cells,
(
acyclovir-resistant strain) was donated by Prof. Claudia
Oliveira (University of Santacatarina-Brazil) and Human

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European Journal of Medicinal Chemistry
9
cells treated with compounds and cells infected with HHV-
or HHV-2. Positive control included in this assay was
defined as the concentration that reduces the 50% of plaque
forming units. EC50 for each compound were obtained from
dose-effect curves for linear regression methods using the
statistical GraphPad Prisma 5.0 and EC50 values are
expressed as the mean of at least four dilutions by
quadruplicate. To define which compounds were more
selective to infected cells than to non-infected cells, the
antiviral selectivity index (SI) was calculated and defined
as the ratio between the inhibitory concentration 50 (IC )
1
Acyclovir. According to the parameters established by
Vlietinck et al.[33] the relevant or moderate antiviral
activity of a purified natural product, is one whose3
reduction factor (Rf) of viral titer is respectively of ≥ 1x10
2
or 1x10 . This parameter was used in our study.
Screening against DENV-2.
50
in Vero cells and the EC for each virus on Vero cells.
50
To determine the anti-DENV activity we carried out the
protocol previously described by Tang et al.[34] with some
modifications. Briefly, Vero cells were harvested in 96-
Plaque size-reduction assay
4
well plates at a density of 2.0 x10 cells/well at 37°C in
For this, a PFU assay was carried out on Vero cells (2.5
4
humidified 5% CO atmosphere. After 24 h, medium was
x10 cells/well) and compounds previously prepared in
2
removed and two-fold serial dilutions of DENV-2 at
CMC, were added 2 h after infection with 100 PFU/well of
1
0TCID50 and non-cytotoxic concentrations of compounds
were incubated for 10 minutes at room temperature. Then,
0 μL of each dilution (compound/DENV-2) was added
virus [25b]. Compound 8 was added at concentrations from
1.56 μg/mL to 12.5 μg/mL and compound
9 at
5
concentrations from 0.78 μg/mL to 6.25 μg/mL, against
both HHV-1 (CDC-Atlanta and 29R) and HHV-2. To
determine the effect against DENV-2, compounds 8 and 9
were added in a concentration range of 0.05 μg/mL to 1.56
μg/mL. Finally, the plates were incubated for 72 h (HHV
strains) and 8 days (DENV-2), fixed and stained with a
solution of 3.5% formaldehyde/0.2% crystal violet. The
images of 20 plaques for each compound concentration and
each well were captured using a Nikon digital camera view
DS-L1 attached to a Nikon inverted microscope (Tokyo,
Japan) [25a]. The area of each plaque in millimeters was
determined using the Image-Pro Plus 6.0 software ®. Two
independent experiments were performed by duplicate.
and the plates were incubated at 37 °C for 3 days, time at
which the cytopathic effect (CPE) in control (infected cells)
was observed in 100% of cell monolayer. The cell
monolayers were stained with a solution of 3.5%
formaldehyde with 0.2% crystal violet and CPE was
observed under inverted microscope. Controls were
included untreated cells, cells treated with compounds and
cells infected with DENV-2. As positive control we used
Ribavirin. Compounds were considered active against
DENV-2 if has the ability to reduce or avoid the cytopathic
effect during viral infection. This assay was conducted by
quadruplicate and repeated twice for each compound.
Assays for pre- and post-infective stages of HHV-1,
HHV-2 and DENV-2.
Cytotoxic activity
Subsequently, the potential antiviral of active compounds
was evaluated by the plaque reduction assay as previously
described [35]. Vero cell monolayers grown in 24-well
plates were infected with 100 PFU per well of each virus.
Treatments were performed by adding compounds either
simultaneously with virus (pre-infection treatment) or after
viral infection (post-infection treatment). For pre-infection
treatment, a virus/compound mixture was added to cell
monolayers and was incubated for 1 h at 37 °C (5% CO2).
After incubation, washing was performed with PBS (pH =
Cell growth inhibition and/or cytotoxicity on Vero, HeLa,
Jurkat and U937 cells were measured using the technique
MTT.
The
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (Sigma, New Jersey, USA)
assay, according to the protocol previously described [12c]
with few modifications, was used. Briefly, Vero and HeLa
4
cells were plated at 2.0 x 10 cells per well in a 96-well
flat-bottomed plates and incubated for 24 h at 37 °C4.
Moreover, Jurkat and U937 cells were plated at 3.0 x 10
cells per well in a 96-well round-bottomed plate. Then each
diluted compound was added to the appropriate wells and
the plates were incubated for further 48 h at 37 °C in a
7
2
.0) and added CMC 1%, 0.75% and 2% for HHV-1, HHV-
and DENV-2, respectively. In post-infection treatment,
virus was added on cell monolayer and incubated for 1 h at
3
7 °C (5% CO ). After, washing was performed with PBS
humidified
incubator
with
5%
CO2.
Finally,
2
(
1
pH = 7.0) and compounds previously prepared in CMC
%, 0.75% and 2% for HHV-1, HHV-2 and DENV-2
spectrophotometric reading was carried out at 570 nm to
determine the inhibitory concentration 50 (IC ), defined as
50
respectively, were added. In both treatments, plates allowed
to incubate for 72 h (HHV-1 and HHV-2) and 8 days
the concentration for each compound that inhibits 50% of
cell growth. The IC50 values for each compound were
obtained by linear regression analysis of the dose-response
curves generated from the absorbance data with the
statistical GraphPad Prisma 5.0. IC50 values were expressed
as the geometric mean of two independent experiments
done in quadruplicate. To define which compounds were
more selective to cancerous cells than to non-cancerous
cells, it was calculated selectivity index (SI) defined as
(
DENV-2). Cells were then fixed and stained with a
solution of 3.5% formaldehyde with 0.2% crystal violet and
viral plaques were counted. Dextran sulfate was employed
as positive control in pre-infection assays. Ayclovir (ACV)
and Ribavirin (RIBA) were used as positive controls in
post-infection stages to HHV strains and DENV-2,
respectively. The fifty effective concentration (EC ) was
50

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0
European Journal of Medicinal Chemistry
Vero IC50 over HeLa, Jurkat or U937 IC50. A compound
References and Notes
with an IC50 < 30 M was considered active and with a SI
≥
5 was considered selective. Paclitaxel and Doxorubicine
were used as positive controls.
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dilutions of compounds were dispensed into 96-well flat-
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0% of inhibition of visible growth after incubation at 28
ºC to 48 h to F. oxysporum and 6 days to dermatophytes.
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Acknowledgments
Financial support from COLCIENCIAS-Grant RC-366-
2
011 and 111554531592 (Patrimonio Autónomo del Fondo
Nacional de Financiamiento para la Ciencia, la Tecnología
y la Innovación, Francisco José de Caldas) is gratefully
acknowledged. V. C. R.-L., L. S. A.-G. and V. T.-C. thank
COLCIENCIAS Programa Jóvenes Investigadores for their
fellowships.

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ACCEPTED MANUSCRIPT
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European Journal of Medicinal Chemistry
M. Antiherpetic activity of a sulfated polysaccharide from
Agaricus brasiliensis mycelia. Antiviral Res. 2011, 92, 108-
14.
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Chryssanthou, E.; Denning D. W.; Donnelly, J. P.; Dromer,
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Antifungal Susceptibility Testing Subcommittee of the
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(AFST-EUCAST). Clin. Microbiol. Infect. 2003, 9, 467-474.

ACCEPTED MANUSCRIPT
Highlights



Aromatic abietanes are a class of bioactive diterpenoids.
Article focuses on the antiviral activity of several ferruginol analogues.
A promising lead was ten-fold more potent (EC = 1.4 M) than the reference ribavirin against
5
0
Dengue Virus type 2 and exhibited a high antiviral selectivity index (SI= 58).