Sterol analogues with diamide side chains interfere with the intracellular localization of viral glycoproteins

Article abstract of DOI:10.1016/j.bbrc.2012.09.019

The need to develop novel antiviral agents encouraged us to assess the antiviral activity of synthetic sterol analogues with a diamide side chains. Cytotoxicity and antiviral activity of a family of azasterol previously synthesized was evaluated against herpes simplex virus 1 (HSV-1) (KOS and B2006) and vesicular stomatitis virus (VSV). This family of compounds was extended by the synthesis of novel analogs using an Ugi multicomponent reaction and their ability to inhibit viral multiplication was also evaluated. The results show that some of the compounds tested exert an antiviral activity. Besides, the effect of the azasterols on the intracellular localization of viral glycoproteins was examined. Strikingly, alteration on the glycoprotein D (gD) of HSV-1 fluorescence pattern was observed with both the antiherpetic compounds and the inactive azasterols.

Full text of DOI:10.1016/j.bbrc.2012.09.019

Biochemical and Biophysical Research Communications 427 (2012) 107–112
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Sterol analogues with diamide side chains interfere with the intracellular
localization of viral glycoproteins
María Eugenia Dávola a, Fernando Alonso b, Gabriela M. Cabrera b, Javier A. Ramírez b, Andrea A. Barquero a,
⇑
a Laboratorio de Virología, Departamento de Química Biológica and IQUIBICEN (CONICET-Facultad de Ciencias Exactas y Naturales), Universidad de Buenos Aires, Pabellón 2, Piso
4, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina
b Departamento de Química Orgánica and UMYMFOR (CONICET-Facultad de Ciencias Exactas y Naturales), Universidad de Buenos Aires, Pabellón 2, Piso 3, Ciudad
Universitaria, C1428EGA Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2012
Available online 14 September 2012
The need to develop novel antiviral agents encouraged us to assess the antiviral activity of synthetic ste-
rol analogues with a diamide side chains. Cytotoxicity and antiviral activity of a family of azasterol pre-
viously synthesized was evaluated against herpes simplex virus 1 (HSV-1) (KOS and B2006) and vesicular
stomatitis virus (VSV). This family of compounds was extended by the synthesis of novel analogs using an
Ugi multicomponent reaction and their ability to inhibit viral multiplication was also evaluated. The
results show that some of the compounds tested exert an antiviral activity. Besides, the effect of the azas-
terols on the intracellular localization of viral glycoproteins was examined. Strikingly, alteration on the
glycoprotein D (gD) of HSV-1 fluorescence pattern was observed with both the antiherpetic compounds
and the inactive azasterols.
Keywords:
Azasterol
Ugi reaction
Antiviral
HSV-1
VSV
Ó 2012 Elsevier Inc. All rights reserved.
Glycoprotein transport
1. Introduction
of these plant and animal steroids reveals that cellular targets are
an important component of this activity, including modulation of
The development of antiviral agents with novel strategies of at-
tack continues to be a challenge since viral infections remain the
leading cause of worldwide death. New approaches are increas-
ingly focused to restrain specific host-cell functions that are re-
quired for viral replication instead of the traditional approach of
affecting virally-encoded molecules [1,2]. These strategies hold
further advantages particularly with respect to the prevention of
viral resistance and to obtaining antiviral drugs with a broad spec-
trum of action.
cell signaling pathways [10]. On the other hand, we have also re-
ported the isolation from Melia azedarach L. of the limonoid 1-cin-
namoyl-3,11-dihydroxymeliacarpin (CDM) which might be
affecting a cellular factor involved in the transport of viral glyco-
proteins [11]. The integrity of the viral glycoproteins is critical
for virion entry and/or assembly, therefore, cellular factors impli-
cated in vesicular transport, membrane trafficking and glycopro-
tein processing acquired much relevance as potential targets for
novel antiviral drugs [12].
In this sense, steroids are an attractive source for antiviral drug
discovery since they are known to possess several biological prop-
erties involving cellular targets. In particular, we have reported
that some synthetic polyfunctionalized stigmastane have in vitro
antiviral activity against several pathogenic viruses [3,4]. We have
also found that compounds 1 and 2 (Fig. 1) exhibit anti herpes sim-
plex virus 1 (HSV-1) activity in vitro and ameliorate the signs of
murine herpetic stromal keratitis [5,6]. Similar studies performed
in our laboratory have shown that dehydroepiandrosterone (3)
and some synthetic derivatives display a broad spectrum of antivi-
ral action against Junin virus, vesicular stomatitis virus (VSV) and
adenovirus [7–9]. The actual knowledge about the antiviral action
Otherwise, we have successfully applied a new strategy to
achieve sterol analogues with a high structural diversity by a sim-
ple and fast procedure [13,14]. The synthetic approach carried out
was the Ugi four-component reaction (U-4CR), in which an amino
component, an acid, a carbonyl compound and an isocyanide react
together to give an
a-aminoacylamide. Thus, we obtained a new
family of azasteroids with diamide side chains of general structure
4, some of which showed antifungal activity [15]. Several azaster-
oids are known to have interesting biological properties such as
anticancer, antiparasitic, antifungal and antibacterial activities
[16–19], however, there are no reports on their antiviral properties.
In this paper, previously synthesized [15] and novel sterol ana-
logues of general structure 4 (Fig. 1) were evaluated for their abil-
ity to inhibit viral multiplication and to affect the intracellular
localization of viral glycoproteins.
⇑
Corresponding author. Fax: +54 11 4576 3342.
E-mail address: alecab@qb.fcen.uba.ar (A.A. Barquero).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.09.019

108
M.E. Dávola et al. / Biochemical and Biophysical Research Communications 427 (2012) 107–112
2.1.4. N-((tert-butylcarbamoyl)methyl)-N-(2-chlorophenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4d)
Yield: 81%. M.p: 113–115 °C. 1H NMR (500 MHz): 0.60 (H-12
a,
1H, m); 0.81 (H-9a and H-14a, 2H, m); 0.83 (C-18, 3H, s); 0.92
(C-19, 3H, s); 1.37 (H-26, 9H, s); 2.20 (H-17, 1H, m); 3.48 (H-3,
1H, m); 3.56 (H-22a, 1H, d, J = 14.5); 4.62 (H-22b, 1H, d, J = 14.5);
5.28 (H-6, 1H, m); 6.45 (NH, 1H, b.s); 7.31 (H-30 and H-60, 2H, m);
7.42 (H-40 and H-50, 2H, m). 13C NMR (125 MHz): 13.9 (C-18);
19.3 (C-19); 20.8 (C-11); 24.6 (C-15); 26.3 (C-16); 28.7 (C-26);
31.5 (C-2); 31.7 (C-8); 31.8 (C-7); 36.5 (C-10); 37.2 (C-1); 38.7
(C-12); 42.2 (C-4); 45.5 (C-13); 49.8 (C-9); 51.1 (C-25); 52.6 (C-
17); 55.2 (C-14); 56.2 (C-22); 71.6 (C-3); 121.3 (C-6); 127.9 (C-
50); 129.4 (C-30); 130.2 (C-60); 131.1 (C-20); 132.8 (C-10); 140.7 (C-
5); 129.5 (C-40); 168.3 (C-23); 175.2 (C-20). Anal. C32H45ClN2O3
(C, H, N).
Fig. 1. Antiviral steroids.
2. Material and methods
2.1.5. N-((tert-butylcarbamoyl)methyl)-N-(4-methoxyphenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4e)
2.1. Synthesis
Yield: 78%. M.p: 116–118 °C. 1H NMR (500 MHz): 0.69 (H-12
a,
1H, m); 0.80 (H-9
a and H-14a, 2H, m); 0.83 (C-18, 3H, s); 0.92
2.1.1. General
(C-19, 3H, s); 1.38 (H-26, 9H, s); 2.48 (H-17, 1H, t, J = 9.9); 3.48
(H-3, 1H, m); 3.84 (-OMe, 3H, s); 3.88 (H-22a, 1H, d, J = 14.5);
4.39 (H-22b, 1H, d, J = 14.5); 5.29 (H-6, 1H, m); 6.44 (NH, 1H,
All solvents and reagents were purchased from Sigma–Aldrich
Chemical Co. and were of analytical grade. ESI-HRMS were mea-
sured on a Bruker micrOTOF-Q II. Melting points were determined
on a Fisher Johns apparatus and are uncorrected. All NMR spectra
b.s); 6.90 (H-3, 2H, d, J = 9.3 and 1.4); 7.12 (H-2 , 2H, b.s.). 13C
NMR (125 MHz): 13.8 (C-18); 19.3 (C-19); 20.8 (C-11); 24.7 (C-
15); 26.1 (C-16); 28.7 (C-26); 31.6 (C-2); 31.7 (C-8); 31.8 (C-7);
36.5 (C-10); 37.2 (C-1); 38.4 (C-12); 42.2 (C-4); 45.3 (C-13); 49.8
(C-9); 51.0 (C-25); 51.6 (C-17); 55.4 (-OMe); 56.1 (C-14); 56.2
(C-22); 71.6 (C-3); 114.6 (C-30); 121.3 (C-6); 129.0 (C-20); 136.3
(C-10); 140.7 (C-5); 158.9 (C-40); 168.8 (C-23); 175.2 (C-20). Anal.
0
´
were recorded on
a
Bruker AM-500 (500 MHz for 1H and
125.1 MHz for 13C). Chemical shifts (d) are given in ppm downfield
from TMS as the internal standard. Coupling constant (J) values are
in Hz. Assignments of the NMR signals of the side chain correspond
to the numbering shown in the Supplementary material. Combus-
tion analyses indicated by the symbols of the elements were deter-
mined with an Exeter CE 440 Elemental Analyzer and were within
0.4% of the theoretical values.
C33H48N2O4 (C, H, N).
2.1.6. N-((tert-butylcarbamoyl)methyl)-N-(4-methylphenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4f)
Yield: 81%. M.p: 111–112 °C. 1H NMR (500 MHz): 0.66 (H-12
a,
2.1.2. General synthetic procedure
The steroidal acid 5 (3b-hydroxyandrost-5-en-17b-carboxylic
acid, 50 mg, 0.16 mmol [20] was suspended in 1 mL of methanol
1H, m); 0.80 (H-9a and H-14a, 2H, m); 0.83 (C-18, 3H, s); 0.98
(C-19, 3H, s); 1.36 (H-26, 9H, s); 2.37 (H-50, 3H, s); 2.50 (H-17,
1H, t, J = 9.2); 3.48 (H-3, 1H, m); 3.88 (H-22a, 1H, d, J = 14.7);
4.38 (H-22b, 1H, d, J = 14.7); 5.29 (H-6, 1H, m); 6.44 (NH, 1H,
b.s); 7.06 (H-20, 2H, b.s.); 7.17 (H-30, 2H, d, J = 8.5). 13C NMR
(125 MHz): 13.8 (C-18); 19.3 (C-19); 20.8 (C-11); 21.1 (C-50);
24.7 (C-15); 26.0 (C-16); 28.7 (C-26); 31.6 (C-2); 31.7 (C-8); 31.7
(C-7); 36.5 (C-10); 37.2 (C-1); 38.4 (C-12); 42.2 (C-4); 45.4 (C-
13); 49.8 (C-9); 51.0 (C-25); 51.7 (C-17); 56.0 (C-14); 56.2 (C-
22); 71.7 (C-3); 121.3 (C-6); 127.6 (C-20); 130.2 (C-30); 137.8 (C-
40); 140.7 (C-5); 140.8 (C-10); 168.7 (C-23); 175.1 (C-20). Anal.
and 1.1 equivalent of the corresponding amine and 15 lL of form-
aldehyde (37% aq.) were added. The mixture was stirred for 15 min
at room temperature and then 1.1 equivalent of the isonitrile was
added. The reaction was kept under the same conditions until total
disappearance of the acid (usually 48 h). The solvent was evapo-
rated under reduced pressure and the residue was taken in EtOAc
and washed with NaOH (5% aq.). The compounds were purified by
silica gel column chromatography (hexane/EtOAc gradient).
C33H48N2O3 (C, H, N).
2.1.3. N-((tert-butylcarbamoyl)methyl)-N-(4-fluorophenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4c)
2.1.7. N-((tert-butylcarbamoyl)methyl)-N-(3,4-dichlorophenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4g)
Yield: 75%. M.p: 114–115 °C. 1H NMR (500 MHz): 0.62 (H-12
a,
Yield: 72%. M.p: 112–113 °C. 1H NMR (500 MHz): 0.65 (H-12
a,
1H, m); 0.81 (H-9
a
and H-14
a
, 2H, m); 0.82 (C-18, 3H, s); 0.98
1H, m); 0.80 (C-18, 3H, s); 0.85 (H-9a and H-14a, 2H, m); 0.98
(C-19, 3H, s); 1.38 (H-26, 9H, s); 2.45 (H-17, 1H, t, J = 9.9); 3.49
(H-3, 1H, m); 3.84 (H-22a, 1H, d, J = 14.5); 4.38 (H-22b, 1H, d,
J = 14.5); 5.29 (H-6, 1H, m); 6.32 (NH, 1H, b.s); 7.08 (H-30, 2H, m);
7.21 (H-20, 2H, b.s.). 13C NMR (125 MHz): 13.7 (C-18); 19.3 (C-
19); 20.8 (C-11); 24.7 (C-15); 26.0 (C-16); 28.7 (C-26); 31.5 (C-
2); 31.7 (C-8); 31.8 (C-7); 36.5 (C-10); 37.2 (C-1); 38.4 (C-12);
42.2 (C-4); 45.5 (C-13); 49.8 (C-9); 51.2 (C-25); 51.7 (C-17); 55.9
(C-14); 56.2 (C-22); 71.6 (C-3); 114.6 (C-30, d, JC–F = 25); 121.3 (C-
6); 129.7 (C-20, d, JC–F = 5); 139.6 (C-10); 140.7 (C-5); 161.8 (C-40,
d, JC–F = 250); 168.4 (C-23); 174.9 (C-20). Anal. C32H45FN2O3 (C, H,
N).
(C-19, 3H, s); 1.36 (H-26, 9H, s); 2.46 (H-17, 1H, t, J = 9.5); 3.49
(H-3, 1H, m); 3.83 (H-22a, 1H, d, J = 14.5); 4.34 (H-22b, 1H, d,
J = 14.5); 5.30 (H-6, 1H, m); 6.11 (NH, 1H, b.s); 7.15 (H-60, 1H,
b.s); 7.40 (H-20, 1H, b.s.); 7.47 (H-50, d, J = 8.5). 13C NMR
(125 MHz): 13.7 (C-18); 19.3 (C-19); 20.9 (C-11); 24.7 (C-15);
26.0 (C-16); 28.7 (C-26); 31.6 (C-2); 31.8 (C-8); 31.8 (C-7); 36.5
(C-10); 37.2 (C-1); 38.3 (C-12); 42.2 (C-4); 45.8 (C-13); 49.7 (C-
9); 52.0 (C-25); 52.0 (C-17); 55.5 (C-14); 56.2 (C-22); 71.6 (C-3);
121.3 (C-6); 127.6 (C-60); 129.0 (C-40); 131.1 (C-50); 130.0 (C-20);
133.2 (C-30); 140.7 (C-5); 143.0 (C-10); 168.3 (C-23); 175.2 (C-20).
Anal. C32H44Cl2N2O3 (C, H, N).

M.E. Dávola et al. / Biochemical and Biophysical Research Communications 427 (2012) 107–112
109
2.1.8. N-((tert-butylcarbamoyl)methyl)-N-(2-fluorophenyl)-3b-
2.4. Cytotoxicity assay
hydroxyandrost-5-ene-17b-carboxamide (4h)
Yield: 72%. M.p: 113–114 °C. 1H NMR (500 MHz): 0.56 (H-12
a,
The compounds were evaluated for cytotoxicity by a modifica-
tion of the crystal violet assay [21]. Vero cells were seeded at a con-
centration of 104 cells/well in 96-well plates and grown at 37 °C for
24 h. The culture medium was replaced by fresh medium contain-
ing compounds 4a–j at various concentrations and cells were fur-
ther incubated for 48 h. Then, monolayers were fixed in 10%
formaldehyde and stained with 0.05% crystal violet solution. After
that, the plates were washed with de-ionized water, and dried
1H, m); 0.80 (C-18, 3H, s); 0.97 (C-19, 3H, s); 1.35 (H-26, 9H, s);
2.36 (H-17, 1H, m); 3.47 (H-3, 1H, m); 3.99 (H-22a, 1H, d, J = 15);
4.32 (H-22b, 1H, d, J = 15); 5.28 (H-6, 1H, m); 6.39 (NH, 1H, b.s);
7.15 (H-40, 1H, m); 7.19 (H-50, 1H, m); 7.33 (H-60, 1H, m); 7.40
(H-30, 1H, m). 13C NMR (125 MHz): 13.9 (C-18); 19.3 (C-19); 20.8
(C-11); 24.6 (C-15); 26.3 (C-16); 28.7 (C-26); 31.5 (C-2); 31.7 (C-
8); 31.8 (C-7); 36.5 (C-10); 37.2 (C-1); 38.7 (C-12); 42.2 (C-4);
45.5 (C-13); 49.8 (C-9); 51.1 (C-25); 52.6 (C-17); 55.2 (C-14);
56.2 (C-22); 71.6 (C-3); 113.6 (C-30, d, JC–F = 30); 121.3 (C-6);
126.0 (C-50); 127.1 (C-60, d, JC–F = 7); 127.4 (C-40, d, JC–F = 7); 129,5
(C-10, d, JC–F = 28); 140.7 (C-5); 157.7 (C-20, d, JC–F = 255); 168.3
(C-23); 175.2 (C-20). Anal. C32H45FN2O3 (C, H, N).
prior to the solubilization of the bound dye with 100 lL of a 50%
ethanol 0.1% glacial acetic acid solution. The absorbance at
590 nm was measured on an Eurogenetics MPR-A 4i microplate
reader.
2.5. Antiviral activity
2.1.9. N-((tert-butylcarbamoyl)methyl)-N-(2-ethyl-6-methylphenyl)-
3b-hydroxyandrost-5-ene-17b-carboxamide (4i)
Vero cells grown in 96-well tissue culture plates were infected
with HSV-1 (strains KOS or B2006) or VSV at moi of 0.07 PFU/cell.
After 1 h adsorption at 37 °C the inoculum was removed and med-
ium containing different concentrations of compounds 4a–j was
added, by triplicate. The plates were incubated at 37 °C in a 5%
CO2 atmosphere until 100% cell death was observed microscopi-
cally in untreated infected control cells. Supernatants correspond-
ing to those triplicates were harvested after cell disruption by
three cycles of freezing and thawing and pooled. Virus yields were
titrated by plaque assay. For comparative purposes, acyclovir (ACV)
was tested as positive control against HSV-1.
Yield: 70%. M.p: 114–117 °C. 1H NMR (500 MHz): 0.67 (H-12
a,
1H, m); 0.72 and 0.78 (H-9
a and H-14a, 2H, m); 0.93 (C-18, 3H,
s); 0.98 (C-19, 3H, s); 1.21 (H-70, 2H, t, J = 7); 1.36 (H-26, 9H, s);
2.27 (H-90, 3H, s); 2.46 (H-80, 2H, q, J = 7); 2.60 (H-17, 1H, m);
3.48 (H-3, 1H, m); 3.85 (H-22a, 1H, d, J = 14.7); 4.23 (H-22b, 1H,
d, J = 14.7); 5.29 (H-6, 1H, m); 7.10 (H-30, 1H, m); 7.16 (H-50, 1H,
m); 7.22 (H-40, 1H, m); 7.46 (NH, 1H, b.s). 13C NMR (125 MHz):
14.1 (C-18); 14.4 (C-80); 19.3 (C-19); 19.4 (C-90); 20.8 (C-11);
24.1 (C-70); 24.7 (C-15); 26.0 (C-16); 28.7 (C-26); 31.6 (C-2); 31.7
(C-8); 31.7 (C-7); 36.5 (C-10); 37.2 (C-1); 38.4 (C-12); 42.2 (C-4);
45.1 (C-13); 49.7 (C-9); 50.9 (C-25); 52.1 (C-17); 56.1 (C-14);
56.8 (C-22); 71.7 (C-3); 121.4 (C-6); 126.5 (C-30); 128.4 (C-50);
128.9 (C-40); 135.6 (C-60); 135.7 (C-20); 140.7 (C-5); 141.4 (C-10);
168.9 (C-23); 177.5 (C-20). Anal. C35H52N2O3 (C, H, N).
2.6. Indirect immunofluorescence assay
For total glycoprotein staining, subconfluent cells grown on
glass coverslips in 24-well plates were fixed with methanol for
10 min at ꢀ20 °C. After washes with phosphate buffered saline
(PBS), the coverslips were incubated with primary antibody for
30 min at 37 °C, and then returned to culture dishes and subjected
to additional washes with PBS. Afterwards, cells were incubated
with secondary antibody for 30 min at 37 °C. Finally, coverslips
were rinsed, mounted and photographed with an Olympus BX51
microscope with epifluorescence optics. The mouse monoclonal
antibody anti-gD of HSV-1 was obtained from Santa Cruz Biotech-
nology, USA. The rabbit polyclonal anti-gG of VSV was kindly pro-
vided by Dr. Pablo Grigera (CEVAN Buenos Aires, Argentina).
Secondary goat anti-rabbit FluoroLinkTM CyTM2 and antimouse
FluoroLinkTM CyTM3 antibodies were purchased from GE Health-
care Bio-Sciences, Argentina.
2.1.10. N-((tert-butylcarbamoyl)methyl)-N-(3-trifluorophenyl)-3b-
hydroxyandrost-5-ene-17b-carboxamide (4j)
Yield: 71%. M.p: 111–113 °C. 1H NMR (500 MHz): 0.55 (H-12
a,
1H, m); 0.80 (H-9a and H-14a, 2H, m); 0.82 (C-18, 3H, s); 0.98
(C-19, 3H, s); 1.37 (H-26, 9H, s); 2.41 (H-17, 1H, t, J = 10); 3.48
(H-3, 1H, m); 3.90 (H-22a, 1H, d, J = 15); 4.39 (H-22b, 1H, d,
J = 15); 5.29 (H-6, 1H, m); 6.19 (NH, 1H, b.s); 7.50 (H-20 and H-60,
2H, b.s.); 7.54 (H-50, 1H, t, J = 8); 7.61 (H-40, 1H, d, J = 8). 13C NMR
(125 MHz): 13.7 (C-18); 19.3 (C-19); 20.8 (C-11); 24.7 (C-15);
26.0 (C-16); 28.7 (C-26); 31.6 (C-2); 31.8 (C-8); 31.8 (C-7); 36.5
(C-10); 37.2 (C-1); 38.4 (C-12); 42.2 (C-4); 45.7 (C-13); 49.7 (C-
9); 51.3 (C-25); 52.0 (C-17); 56.1 (C-14); 56.2 (C-22); 71.7 (C-3);
120.5 (C-20, q, JC–F = 20); 121.3 (C-6); 124.0 (-CF3, q, JC–F = 285);
124.3 (C-60); 124.4 (C-40, q, JC–F = 7); 131.2 (C-50, q, JC–F = 2); 128.9
(C-30, q, JC–F = 28); 140.7 (C-5); 145.4 (C-10, q, JC–F = 3); 168.0 (C-
23); 174.7 (C-20). Anal. C33H45F3N2O3 (C, H, N).
3. Results
2.2. Cells and viruses
Firstly, we performed a screening of the antiviral activity of five
known compounds of general structure 4 [15]. To cover a broader
spectrum of action, the assays were targeted against two mamma-
lian viruses, HSV-1 and VSV, which belong to DNA and RNA viruses,
respectively. The antiviral activity was expressed as the compound
concentration required to reduce the virus yield by 50% (EC50). The
cytotoxicity concentration required to reduce the Vero cell viability
by 50% (CC50) was also calculated. The results are summarized in
Table 1.
Vero cells were grown in Eagle’s minimal essential medium
(MEM) supplemented with 5% inactivated fetal bovine serum
(FBS) and 50 lg/ml gentamicin and maintained after monolayer
formation in MEM supplemented with 1.5% FBS (MEM 1.5%).
The Indiana strain of VSV, HSV-1 strain KOS and the TKꢀ mu-
tants B2006 strain were propagated at low multiplicity of infection
(moi) and plaque-assayed on Vero cells.
2.3. Antiviral compounds
This screening showed that only two of the compounds tested
(4a and 4b) have an interesting antiviral effect against HSV-1
(KOS strain) and VSV although at concentrations closely related
to their cytotoxicity (Table 1).
Compounds 4a–j were dissolved in dimethylsulfoxide (DMSO)
and diluted with MEM 1.5% for testing. The maximum concentra-
tion of DMSO tested (1% or 2%) exhibited no toxicity under
in vitro conditions.
These compounds share an androstane skeleton with an
a-ami-
noacylamide side chain involving a 17b-carboxyl group. Both are

110
M.E. Dávola et al. / Biochemical and Biophysical Research Communications 427 (2012) 107–112
Table 1
Cytotoxicity and antiviral activity of compounds in Vero cell cultures.
Compd.
H2N-R1
CC50
144
(l
M)a
EC50
VSV
12.4
(l
M)a
HSV-1 (KOS)
20
HSV-1(B2006)
20
4a
H2N
H2N
H2N
4b
4c
228
176
11.2
6
16
12
NA
NA
Cl
F
4d
136.8
10
12.8
12.8
H2N
Cl
4e
4f
154.4
140
NA
NA
NA
10.4
NA
NA
nd
nd
H2N
OMe
Cl
H2N
H2N
4g
244
NA
Cl
4h
4i
144
124
NA
NA
NA
NA
nd
nd
H2N
F
H2N
H2N
4j
196
NA
nd
NA
nd
CF3
ACV
>500
0.25
7.5
NA = not active at the highest concentration tested.
nd = Not determined.
a
Data represent mean values for three independent determinations.
substituted with a phenyl moiety at the nitrogen which is bound to
the C-17 and a t-butyl group at the second amidic nitrogen.
In order to expand these findings, we decided to synthesize no-
vel compounds structurally related to 4a and 4b, having a more di-
verse substitution pattern at the phenyl moiety. These derivatives
were prepared using the previously reported synthetic strategy
shown in Scheme 1 [15]. The steroidal acid 5 was treated with
formaldehyde, t-butylisocyanide and a set of anilines to lead to
the desired compounds 4c–j. As expected, the U-4CR generally
took place smoothly and in good yields (>75%). The structure of
all new compounds was assigned by 1D and 2D NMR experiments.
In some cases (for example compound 4i), the NMR spectra
showed that the compounds were present in the solution as a mix-
ture of two conformers. These conformers are expected to originate
from the cis–trans rotation around the N-substituted side chain
amide bond [22]. Because the interchange between the cis and
trans isomers of these bonds is generally slow in the NMR time
scale, the NMR spectrum is the composite of the NMR spectra of
the two configurational isomers. For clarity, only the chemical
shifts and coupling constants for the most populated conformer
in each case was described in the Experimental section. In order
to obtain an unambiguous characterization of the compounds, a di-
rect analysis via high resolution electrospray ionization mass spec-
trometry (ESI-HRMS) was performed, and is shown in the
Supplementary material.
The new azasterols were also evaluated for their cytotoxicity
and activity towards HSV-1 and VSV in Vero cells. The antiviral
assay showed that compounds 4c and 4d were active against both
HSV-1 (KOS strain) and VSV, whereas the derivative 4e displayed
activity only against HSV-1 (KOS strain) and compounds 4f–j were
found to be inactive (Table 1). In addition, the five anti-HSV-1 ac-
tive compounds (4a–e) were tested against the thymidine kinase-
deficient (TKꢀ) B2006 strain of HSV-1, which is resistant to acyclo-
vir. Unexpectedly, the results showed that only two of them (4a
and 4d) were able to inhibit the growth of the ACV-resistant strain.
Then, we examined the effect of all active compounds on viral
glycoprotein expression by immunofluorescence (IF) assay. Ini-
tially, Vero cells were infected with HSV-1 (KOS strain) and treated
Scheme 1. Synthesis of compounds 4c–j.

M.E. Dávola et al. / Biochemical and Biophysical Research Communications 427 (2012) 107–112
111
Fig. 2. Effect of compounds 4a–g on HSV-1 viral glycoprotein D localization, Vero cells infected with HSV-1 (KOS strain) at moi = 1 were treated or not (CV) with compounds
4a–g. IF staining was performed by adding anti-gD antibodies to cells fixed with methanol at 24 h pos-infection.
with compounds 4a–e and an IF staining using a mouse monoclo-
nal anti-glycoprotein D (gD) antibody was performed. By visual
inspection of the images, we observed that the gD protein was dis-
tributed throughout the cytoplasm and the plasma membrane of
untreated infected cells, whereas it appeared to be associated with
the perinuclear region in most of the treated cells (Fig. 2). Similarly,
when cells were infected with VSV and treated with compounds
4a–d, the glycoprotein G (gG) fluorescence was localized at the
juxtanuclear area of both the infected and treated cells (data not
shown). Strikingly, the alteration on the gD fluorescence pattern
was also observed for compounds 4f and 4g, which have no activity
against HSV-1 (Fig. 2).
Finally, we have also demonstrated that sterol analogues with
or without antiviral activity interfere with the intracellular trans-
port of viral glycoproteins. It is important to point out that there
are multiple potential targets on the glycoprotein traffic that might
be hampering by azasteroids. For example, alterations in Golgi and
ER cholesterol content would immediately impact on protein
transport and organelle structure. The sterol analogues could be
affecting the activity of the Oxysterol-binding protein (OSBP) and
OSBP-related proteins (ORPs) which constitute a large gene family
that participate in sterol signaling and/or transport. In particular,
ORP9L maintains cholesterol flux from the endosomal pathway
to the trans-Golgi/TGN by transferring cholesterol to the ER or
other organelles [23].
In conclusion, the azasteroids evaluated in this study show
interesting biological properties not previously reported, such as
antiviral drugs and compounds interfering with the glycoprotein
transport. Besides, our synthetic procedure allows a rapid synthe-
sis of derivatives with novel modifications to improve their activi-
ties. Further studies are under way to explore the potential cellular
targets of these sterol analogues that might explain both, the effect
on virus replication and the alteration on glycoprotein traffic.
4. Discussion
In the present paper we show that several synthetic azasteroids
are able to reduce the viral multiplication of HSV-1 and/or VSV, a
property that has not been previously described for this class of
molecules. Further, it is interesting to note that small changes in
the chemical structure of the compounds considerably modify
their biological properties. Results in Table 1 show that the intro-
duction of different groups at the para position in the phenyl moi-
ety alters both the cytotoxicity and the spectrum of the antiviral
action of the compounds. For example, addition of a chloro or a flu-
oro group (4b and 4c, respectively) resulted in cytotoxicity lower
than that of compound 4a, but did not affect their antiviral activity
against HSV-1 and VSV. Instead, compounds 4e and 4f in which the
substituent is a methoxy and a methyl group, respectively, dis-
played CC50 values similar to that of compound 4a although com-
pound 4e was active only against HSV-1 and compound 4f was
devoid of antiviral activity. Moreover, the influence of the substitu-
ent position was also evident with compound 4h, which has a flu-
oro group at ortho position instead of at para position and shows no
antiviral activity against any of the viruses tested, in contrast to
compound 4c. This feature allows us to design new compounds
with the aim of improving the antiviral properties and reduce
the cytotoxic effects of these analogues.
Acknowledgments
This work was supported by grants from the Universidad de
Buenos Aires (UBACyT X-029), the Agencia Nacional de Promoción
Científica y Técnica (ANPCyT PICT2008-1394 y PME 2007-354) and
the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP
0315, 100516, 1007). We are grateful to UMYMFOR (UBA-CONI-
CET) for the analytical and spectroscopic determinations.
Drs. A.A. Barquero, G.M. Cabrera and J.A. Ramírez are Research
Members of the CONICET.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bbrc.2012.09.019.
Another remarkable aspect of the sterol analogues is the differ-
ential susceptibility of the TKꢀ strain to the five anti-HSV-1 active
compounds. How some compounds can inhibit the wild type strain
of HSV but not TKꢀ strain is a question that still remains open. It is
hard to think that the thymidine kinase has a role in the mecha-
nism of action of these derivatives since the chemical structure
of azasterols is very different from that of nucleoside analogues.
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