Synthesis and anti-HSV-1 activity of quinolonic acyclovir analogues

Article abstract of DOI:10.1016/j.bmcl.2005.10.111

Several 1-[(2-hydroxy-ethoxy)methyl]-3-carbethoxy-4(1H)quinolones (2a-l) and l-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-carboxylic acids (3a-j and 3l) were synthesized and 2a-j, 2l and 3a-j, 3l were evaluated against herpes simplex virus type 1 (HSV-1), employing a one-pot reaction: silylation of the desired quinolone (BSTFA 1% TMCS) followed by equimolar amount addition of 1,3-dioxolane, chlorotrimethylsilane and KI, at room temperature. The acyclonucleosides 2a-l were obtained in 40-77% yields. The esters 2a-j and 2l were subsequently converted into the corresponding hydroxyacids 3 in 40-70% yields. Attempts of hydrolysis of 2k produced only a mixture of degradation products. Antiviral activity of 2 and 3 on HSV-1 virus infection was assessed by the virus yield assay. Except for compounds 2i and 3e, the acyclonucleosides were found to reduce the virus yield by 70-99% at the concentration of 50 μM, being the acids, in general, more effective inhibitors than their corresponding esters. Compounds 3j and 2d exhibited antiviral activity against HSV-1 virus with EC₅₀ of 0.7 ± 0.04 and 0.8 ± 0.09 μM, respectively. Both compounds were not toxic towards the Vero cell line.

Full text of DOI:10.1016/j.bmcl.2005.10.111

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1010–1013
Synthesis and anti-HSV-1 activity of quinolonic acyclovir analogues
Bianca dÕA. Lucero,a Claudia Regina B. Gomes,a Izabel Christina de P. P. Frugulhetti,b
a
a
a,
*
´
´
Letıcia V. Faro, Lise Alvarenga, Maria Cecılia B. V. de Souza,
Thiago M. L. de Souzab and Vitor F. Ferreiraa
a
ˆ
Universidade Federal Fluminense, Instituto de Quı´mica, Departamento de Quı´mica Organica, Outeiro de Sa˜o Joa˜o Batista
s/no, Centro, Nitero´i, CEP 24210-150, Rio de Janeiro, Brazil
bUniversidade Federal Fluminense, Instituto de Biologia, Programa de Neuroimunologia, Departamento de Biologia Celular
e Molecular, Outeiro de Sa˜o Joa˜o Batista s/no, Centro, Nitero´i, CEP 24210-150, Rio de Janeiro, Brazil
Received 8 July 2005; revised 25 October 2005; accepted 26 October 2005
Available online 29 November 2005
Abstract—Several 1-[(2-hydroxy-ethoxy)methyl]-3-carbethoxy-4(1H)quinolones (2a–l) and l-[(2-hydroxy-ethoxy)methyl]-4(1H)
quinolone-3-carboxylic acids (3a–j and 3l) were synthesized and 2a–j, 2l and 3a–j, 3l were evaluated against herpes simplex virus
type 1 (HSV-1), employing a one-pot reaction: silylation of the desired quinolone (BSTFA 1% TMCS) followed by equimolar
amount addition of 1,3-dioxolane, chlorotrimethylsilane and KI, at room temperature. The acyclonucleosides 2a–l were obtained
in 40–77% yields. The esters 2a–j and 2l were subsequently converted into the corresponding hydroxyacids 3 in 40–70% yields.
Attempts of hydrolysis of 2k produced only a mixture of degradation products. Antiviral activity of 2 and 3 on HSV-1 virus infec-
tion was assessed by the virus yield assay. Except for compounds 2i and 3e, the acyclonucleosides were found to reduce the virus
yield by 70–99% at the concentration of 50 lM, being the acids, in general, more effective inhibitors than their corresponding esters.
Compounds 3j and 2d exhibited antiviral activity against HSV-1 virus with EC50 of 0.7 0.04 and 0.8 0.09 lM, respectively. Both
compounds were not toxic towards the Vero cell line.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Herpes Simplex Virus type 1 (HSV-1) is a large envel-
oped virus containing double-stranded DNA genomes
of approximately 152 kb in size. HSV-1 is the primary
cause of oral and facial lesions in humans. Infection
by HSV-1 usually begins on the skin or mucosal epithe-
lium and subsequently spreads to the sensorial ganglia,
whose nerve process contacts the primary site of infec-
tion. Once inside these neurons of the sensorial ganglia,
the virus may continue to produce infection in certain
cells expressing three viral gene classes (IE, immediate
early; E, early; L, late) or the virus may enter a state
of latency characterized by the absence of lytic gene
transcription.1 Recently, there have been considerable
advances in the chemotherapy of virus infections.
Nevertheless, with the increase in the use of antiviral
drugs, a development of resistance to antivirals has been
observed. In the chemotherapy of herpes simplex virus
infections, acyclovir (1) is used clinically as an anti-
herpes drug. However, herpes viruses may sometimes
acquire resistance to acyclovir in immuno-compromised
patients.2 Many research approaches are currently
aimed at developing new types of antiviral agents that
have a wide range of efficacy without serious adverse
effects and are potent against viral strains resistant to
current antiviral agents. The search for more effective
antiviral agents has so far developed only a few
compounds that have reached prominence at clinical
level so far. Acyclic nucleosides are of interest because
of the antiviral activity of a large number of this class
of compounds.3–7 Up to now, there have only been a
few reports regarding the synthesis of quinolone
acyclonucleoside derivatives.8 We describe herein the
extension of Ubasawa9 one-pot synthesis method of
acyclonucleosides for the synthesis of 1-[(2-hydroxy-
ethoxy)methyl]-3-carbethoxy-4(1H)quinolones 2a–l and
1-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-car-
boxylic acids 3a–j and 3l, establishing now an efficient
and generally applicable methodology for the synthesis
of acyclovir quinolonic analogues. We have also studied
the effects of these acyconucleosides on herpes simplex
virus type 1 replication in Vero cells.
Keywords: Acyclonucleoside; Quinolone; HSV-1.
*
Corresponding author. Tel.: +55 21 26292361; fax: +55 21 22629
2135; e-mail: gqocica@vm.uff.br
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.111

B. d’A. Lucero et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1010–1013
1011
Attempts to introduce the acyclic chain into the hetero-
cycle by silylation of the quinolones in a refluxing mix-
ture of hexamethyldisilazane (HMDS) and ammonium
sulfate or in refluxing BSTFA/TMCS using acetonitrile
as solvent, and subsequent treatment with acetoxyethyl-
acetoxymethyl ether under trimethylsilyltrifluorome-
thanesulfonate (TMSOTf) catalysis were unsuccessful.
O
N
H
N
N
N
H2N
HO
O
1- Acyclovir
The synthesis of 1-[(2-hydroxy-ethoxy)methyl]-3-
The structures of the new compounds are supported by
spectroscopic analysis.
carbethoxy-4(1H)quinolones
ethoxy)methyl]-4(1H)quinolone-3-carboxylic acids
2
and 1-[(2-hydroxy-
3
was initiated from substituted 4-(1H) quinolones 4, pre-
pared in good yields (60–88%) employing the treatment
of appropriate meta- and para-substituted anilines 5
with diethyl ethoxymethylenemalonate to obtain the en-
amine-type derivatives 6 in 74–96% yields, which were
then cyclized in refluxing Dowtherm A (Scheme 1).10
NMR spectra were recorded on a Varian Unity Plus 300
spectrometer operating at 300.00 MHz (1H) and
75.0 MHz (13C). Chemical shifts are reported in ppm
(d) relative to tetramethylsilane (Supplementary data:
files a–d). Proton and carbon spectra were typically ob-
tained at room temperature. The two-dimensional
experiments were acquired using standard Varian Asso-
ciates automated programs for data acquisition and pro-
cessing. General procedures for the preparation of these
acyclonucleosides, along with analytical characteriza-
tion for 2d and 3d, are given.14
Acyclonucleosides 2a–l were prepared by previous
silylation of the quinolones by heating with bis(trimeth-
ylsilyl)trifluoroacetamide (BSTFA)11–13 containing 1%
of trimethylchlorosilane (TMCS), in acetonitrile, under
reflux, followed by addition of equimolar amount of
1,3-dioxolane, chlorotrimethylsilane, and potassium
iodide, at room temperature. Afterwards, the reaction
was quenched by addition of methanol followed by
neutralization with solid sodium bicarbonate (Scheme
1). Purification by crystallization from ethyl alcohol/
methylene chloride mixture (1:1) led to the desired pure
compounds 2a–l in 75%, 45%, 70%, 58%, 72%, 77%,
47%, 57%, 68%, 50%, 40% and 70% yields, respectively.
It is worth noting that the isolated nucleosides maintain
the carboxylic ethyl ester group at carbon C3 of the
quinolonic nucleus enabling further functionalization
of the free hydroxilic group of the acyclic unity, if
desired.
The antiviral effect of quinolone acyclonucleoside car-
boxylic acids 3 and their corresponding esters 2 on
HSV-1 virus replication was determined. Our results
show that except for compounds 2i and 3e, carboxylic
acids 3 and the esters 2 were found to reduce the virus
yield in 70–99% at the concentration of 50 lM, being
the acids, in general, more effective inhibitors than
their corresponding esters (Table 1). Compounds with
nearly the same antiviral effects were evaluated for
cytotoxicity in Vero cells. EC50 and the selectivity in-
dex (SI) were determined in parallel. Several of the
new compounds prevented the cytopathic effect of
HSV-1 in Vero cells, at micromolar concentrations,
and were minimally toxic to Vero cells resulting in a
good selectivity index. The MTT assay indicated that
compounds 3d, 3j and 2d exhibited a low cytotoxicity,
CC50 1400, 1200 and 1250 lM, respectively. Trypan
blue and MTT showed similar results (data not
showed). Acyclovir (1) results have been included for
comparison purposes. Compounds 3j and 2d were
the most effective anti-HSV-1 derivatives and present-
ed a 1.5- and 1.3-fold increase in their antiviral
activity in relation to acyclovir (1) (Table 2). The
The treatment of the compounds 2a–j and 2l with 0.1 N
ethanolic sodium hydroxide solution, at room tempera-
ture, followed by neutralization with Dowex 50 H+
produced the 3-carboxylic acyclonucleoside acids in
65%, 58%, 42%, 62%, 40%, 57%, 50%, 56%, 64%, 41%
(3a–j, respectively) and 70% (3l) yields (Scheme 1).
Hydrolysis reaction of 2k produced only a mixture of
degradation products.
O
EtO2C
CO2Et
ii
CO2Et
iii, iv, v
i
R
R
R
NH2
O
N
N
4
5
H
6
H
O
N
5
4a
CO2H
CO2Et
4
1
6
vi, vii
3
R
g) 7-Me
R
a) 6-Me
b) 6-OMe
c) 6-F
7
h) 7-OMe
i) 7-F
2
N
8
O
O
j) 7- Cl
k) 7-Br
d) 6-Cl
e) 6-Br
HO 3'
HO
2'
1'
3
2
l) 7-NO2
f) 6-NO2
Scheme 1. Synthesis of quinolonic acyclonucleosides 2 and 3. Reagents and conditions: (i) EtOH, diethyl ethoxymethyleremalonate, reflux; (ii)
Dowtherm A, reflux; (iii) BSTFA/TMSCl, CH3CN, reflux; (iv) 1,3-dioxolane/TMSCl/KI, rt; (v) NaHCO3; (vi) NaOEt//EtOH 0.6 M; (vii) Dowex-H+.

1012
B. d’A. Lucero et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1010–1013
Table 1. Antiviral activity of quinolonic acyclonucleosides 2a–j, 2l,
3a–j and 3l
African green monkey kidney cells (obtained from the
American Type Culture Collection), were grown in
DulbeccoÕs modified EagleÕs medium (DMEM-Gibco
Laboratories) supplemented with 2% heat-inactivated
foetal bovine serum (purchased from Fazenda Pig), 8%
calf serum (purchased from Centro Pan- Americano de
Febre Aftosa), 2.25% sodium bicarbonate, 500 U/mL
penicillin, 100 lg/mL streptomycin and 2.5 lg/mL
amphotericin B. Herpes simplex virus type 1 (HSV-1),
strain ACR-29,15 was kindly provided by Marcia Wigg
(Universidade Federal do Rio de Janeiro-Brasil) and
was routinely propagated in Vero cells. Virus stocks
were stored at ꢀ70 ꢁC until use. Acyclovir (1) was pur-
chased from Sigma (A 4669). It was dissolved in sterile
deionized water and further diluted in culture medium.
MTT (3-(4,5-dimethyltrazol-2yl)-2,5-diphenyl tetrazoli-
um bromide) was purchased from Sigma. Virus infectiv-
ity was measured by a dilution method using a 96-well
microtitre plate and expressed as 50% tissue culture
infections dose (TCID50) according to Reed and
Muench.16 Cells grown in 96-well microtitre were inocu-
lated with virus at input 1 PFU (plaque-forming unit)/
cell for 2 h at 37 ꢁC. After virus adsorption, virus inoc-
ulum was replaced by a culture medium containing
quinolone acyclonucleobases carboxylic acid and their
correspondent esters at the concentration of 50 lM.
Control cultures were incubated with media without
compounds. After 3 days of incubation at 37 ꢁC in 5%
CO2 atmosphere, the culture medium was harvested
and the virus titre of each sample was determined in
terms of 50% tissue culture dose (TCID 50/mL) by
endpoint dilution.
Compound
R-C6
R-C7
% of inhibition of
virus yield (HSV-1)
3a
3b
3c
3d
3e
3f
Me
O-Me
F
H
93
89
70
98
58
70
79
79
97
96
77
82
99
97
97
84
95
77
89
65
99
77
96
1
2
2
1
1
3
5
3
7
4
8
1
1
2
1
5
1
1
1
2
1
1
1
H
H
Cl
H
Br
NO2
H
H
H
3g
3h
3i
Me
O-Me
F
H
H
3j
H
Cl
3l
H
NO2
H
2a
2b
2c
2d
2e
2f
Me
O-Me
F
H
H
Cl
H
Br
NO2
H
H
H
2g
2h
2i
Me
O-Me
F
H
H
2j
H
Cl
2l
ACV
H
—
NO2
—
The experimental concentrations of quinolonic acyclonucleosides 2a–j,
2l, 3a–j and 3l were 50 lM and ACV concentration was 10 lM. Results
are presented as means of triplicate experiments. ACV-acyclovir has
been included for comparison purposes.
Table 2. Anti-HSV-1 activity, cytotoxicity and selectivity index in
Vero cells for some quinolonic acyclonucleosides 2 and 3
Compound R-C6
R-C7 EC50 (lM) CC50 (lM) S.I.c
a
b
The cytotoxicity of the compounds was tested in Vero
cells using two methods, MTT (3-(4,5-diethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide) and trypan blue
dye exclusion assay. Monolayers of uninfected cells were
incubated with culture medium containing different
concentrations of compounds for 72 h at 37 ꢁC. The
medium was then removed, the cells trypsinized and via-
ble cells counted by trypan blue dye exclusion test.17 The
50% cytotoxic concentration (CC50) was calculated by
regression analysis of the dose–response curves generat-
ed from these data. In the second method, monolayer of
Vero cells in 96-multiwell plates were incubated with
MTT (5 lg/mL) at 37 ꢁC for 4 h. After this period,
SDS 10% and 0.01 N HCl were added to each well
and incubated overnight. The plates were read using
an automatic plate reader with a 540 nm test wavelength
and a 690 nm reference wavelength.18 Plaque reduction
assay was performed utilizing Vero cells at a density of
3 · 105 infected with various dilutions of the superna-
tant from a yield reduction assay for 1 h at 37 ꢁC and
5% CO2. After adsorption, the plates were washed and
the medium was replaced with DMEM containing meth-
ylcellulose 1% and fetal bovine serum 5%. After incuba-
tion for 72 h, the monolayers were fixed with 1%
formaldehyde in PBS, methylcellulose removed, and cell
stained with a 0.1% solution of crystal violet in 70%
methanol. The virus yield assay was performed as fol-
lows. Confluent Vero cells were washed with PBS and
infected with HSV-1 at moi of 1 pfu/cell for 1 h at
37 ꢁC. The infected cells were washed with PBS and
3a
3d
3i
Me
Cl
H
H
F
2.2 0.02
1.l 0.03
1.4 0.8
>500
1400 238
>500
227
1273
357
H
3j
H
Cl
H
H
H
H
Cl
—
0.7 0.04
3.2 1.1
2.3 0.9
1200 205
>500
>500
1714
156
2b
2c
2d
2f
O-Me
F
Cl
217
0.8 0.09
1.5 0.6
1250 179
960 128
1196 30
960 156
1562
640
NO2
H
2j
ACV
0.87 0.1
1.09 0.25
1375
880
—
a 50% Effective concentration or concentration required to inhibit
HSV-1 virus yield.
b 50% Cytotoxic concentration or concentration required to reduce the
viability of host cells by 50%.
c Selective index (CC50/EC50).
quinolone nucleobases have not inhibited HSV-1 virus
replication (data not showed). Additionally, it seems
that the position and the nature of the substituent
on the quinolone ring, C6 or C7 carbons, are more
important than the type of the substituent at C3—a
carboxylic acid or an ethylcarboxylate group.
In conclusion, a new series of acyclovir analogues (2 and
3) was synthesized and some of them are potent anti-
HSV-1 agents.
The mechanism of antiviral activity of these compounds
is under investigation. For the biological assays, Vero,

B. d’A. Lucero et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1010–1013
1013
13. De Oliveira, M. R. P.; Alves, T. R. A.; Pinto, A. C.;
Pereira, H. deS.; Ferreira, L. R. L.; Nissin, M.; Frugulh-
etti, I. C. deP. P.; Ferreira, V. F.; De Souza, M. C. B. V.
Nucleosides Nucleotides Nucleic Acids 2004, 23, 735.
covered with a culture medium containing either no
compounds or a different concentration of compounds.
20 h after adsorption, cells were lysed by freezing and
thawing (three times), and the supernatant consisting
of culture medium and lysed cells was obtained by cen-
trifugation at 400g for 10 min at 4 ꢁC. Virus titre was
determined by the plaque assay in Vero cells as de-
scribed above. Data were statistically analyzed by Stu-
dentÕs t test for a significance level of p < 0.05.
14. General Procedures for the syntheses of 1-[(2-hydroxy-
ethoxy)methyl]-3-carbethoxy-4(1H)quinolones 2a–l and
1-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-carboxylic
acids 3a–j and 3l: The quinolone derivatives 4 were
prepared by treating the appropriate aniline with diethyl
ethoxymethylenemalonate to obtain the enamine deriva-
tives that were then cyclized in refluxing Dowtherm A.
These quinolones (3 mmol) were refluxed in bis(trimeth-
ylsilyl)trifluoroacetamide (BSTFA) (6.75 mmol) contain-
ing 1% TMSCl, in 10 mL acetonitrile, under nitrogen
atmosphere, for 4 h, followed by addition of potassium
iodide (3 mmol), 1,3-dioxolane (3 mmol) and TMSCl
(3 mmol). The resulting mixture was stirred for 24 h at
room temperature. Afterwards, it was poured into a
mixture of methyl alcohol/water (20 mL:10 mL), followed
by addition of solid sodium bicarbonate (3 mmol) and
subsequent stirring for 10 min, leading to the crude
products, which were purified by crystallization from a
ethyl alcohol/methylene chloride mixture (1:1), giving the
pure acyclonucleosides 2a–l. Reaction of these nucleo-
sides 2a–j and 2l (1 mmol) with 30 mL of 0.7 N ethanolic
sodium hydroxide solution, at room temperature, for 5 h,
followed by neutralization with Dowex 50-(acid form),
gave pure 1-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-
carboxylic acids 3a–j and 3l, respectively. Analytical data
for compounds 2d and 3j are showed. NMR spectra were
recorded on a Varian Unity Plus 300 spectrometer
operating at 300.00 MHz (1H) and 75.0 MHz (13C) in
CDC13, for 2d and DMSO-d6 for 3j, at room temper-
Acknowledgments
The fellowship granted to C.R.B.G. and B.A.L. from
CNPq (Brazil) is gratefully acknowledged. M.C.B.V.S.
and V.F.F. are grateful to CNPq for their individual re-
search fellowships. This work was partially supported by
CNPq (National Council of Research from Brazil) and
FAPERJ.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.10.111.
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2d—1-[(2-hydroxy-ethoxy)methyl]-3-car-
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bethoxy-6-chloro-4(1H)quinolone 58%, mp 146–
147 ꢁC; IR (KBr) cmꢀ1 3378, 1722, 1618; 1H NMR d
8.93 (s, H2), 8.27 (d, J = 2.1 Hz, H5), 8.00 (d, J = 8.7 Hz,
0
H8), 7.95 (dd, J = 9.0 and 2.4, H7), 5.89 (s, H1 ), 4.76 (t,
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0
0
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0
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6.07 (s, H1 ), 4.74 (t, J = 5.4 Hz, OH), 3.70–3.73 (m, H3 ),
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(COOH), 150.7 (C2), 140.2 (C7); 138.4 (C8a), 129.0 (C50),
127.1 (C4a), 126.4 (C6), 118.3 (C8), 110.6 (C3), 70.7 (C1 ),
0
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