Synthesis and antiviral activities of new pyrazolo[4,3-c]quinolin-3-ones and their ribonucleoside derivatives

Article abstract of DOI:10.1081/NCN-120038009

Several new pyrazolo[4,3-c]quinolin-3-one ribonucleosides (5a-g) and their corresponding heterocycle moieties (3a-g) were synthesized and evaluated against vaccinia virus (VV) and herpes simplex virus type 1 (HSV-1). The derivatives 3c and 3d showed modes

Full text of DOI:10.1081/NCN-120038009

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Synthesis and Antiviral Activities of New
Pyrazolo[4,3‐c]quinolin‐3‐ones and Their
Ribonucleoside Derivatives
Mara R. P. de Oliveira a , Thatyana R. Alves c , Angelo C. Pinto a , Helena de S. Pereira
b , Luiz R. Leão‐Ferreira d , Nissin Moussatché b , Izabel Christina de P. P. Frugulhetti d
Vitor F. Ferreira c & Maria Cecília B. V. de Souza c
,
a Departamento de Química Orgânica, Instituto de Química , Universidade Federal do Rio
de Janeiro , Ilha do Fundão, Rio de Janeiro, CEP 21945‐970, Brazil
b Instituto de Biofísica Carlos Chagas Filho , Universidade Federal do Rio de Janeiro , Ilha
do Fundão, Rio de Janeiro, CEP 21941‐590, Brazil
c Departamento de Química Orgânica, Instituto de Química , Universidade Federal
Fluminense , Outeiro de São João Batista s/n°, Centro, Niterói, Rio de Janeiro, CEP
24210‐150, Brazil
d Programa de Neuroimunologia, Departamento de Biologia Celular e Molecular, Instituto
de Biologia , Universidade Federal Fluminense , Outeiro de São João Batista s/n°,
Centro, Niterói, Rio de Janeiro, CEP 24210‐150, Brazil
Published online: 17 Aug 2006.
To cite this article: Mara R. P. de Oliveira , Thatyana R. Alves , Angelo C. Pinto , Helena de S. Pereira , Luiz R.
Leão‐Ferreira , Nissin Moussatché , Izabel Christina de P. P. Frugulhetti , Vitor F. Ferreira & Maria Cecília B. V. de Souza
(2004) Synthesis and Antiviral Activities of New Pyrazolo[4,3‐c]quinolin‐3‐ones and Their Ribonucleoside Derivatives,
Nucleosides, Nucleotides and Nucleic Acids, 23:5, 735-748, DOI: 10.1081/NCN-120038009
To link to this article: http://dx.doi.org/10.1081/NCN-120038009
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 5, pp. 735–748, 2004
Synthesis and Antiviral Activities of New
Pyrazolo[4,3-c]quinolin-3-ones and Their
Ribonucleoside Derivatives
Mara R. P. de Oliveira,1 Thatyana R. Alves,3 Angelo C. Pinto,1
Helena de S. Pereira,2 Luiz R. Lea˜o-Ferreira,4 Nissin Moussatche´,2
Izabel Christina de P. P. Frugulhetti,4 Vitor F. Ferreira,3
and Maria Cec´ılia B. V. de Souza3,
*
1Departamento de Qu´ımica Orgaˆnica, Instituto de Qu´ımica and
2Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal
do Rio de Janeiro, Rio de Janeiro, Brazil
4
3Departamento de Qu´ımica Orgaˆnica, Instituto de Qu´ımica and Programa de
Neuroimunologia, Departamento de Biologia Celular e Molecular, Instituto de
Biologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil
ABSTRACT
Several new pyrazolo[4,3-c]quinolin-3-one ribonucleosides (5a–g) and their cor-
responding heterocycle moieties (3a–g) were synthesized and evaluated against
vaccinia virus (VV) and herpes simplex virus type 1 (HSV-1). The derivatives 3c and
3d showed modest inhibitory activity against vaccinia virus reaching 70% at a
concentration of 100 mM. All heterocyclic compounds (3a–f) showed a modest
inhibition against HSV-1, reaching the maximal inhibitory effect around 20–30%.
*
Correspondence: Maria Cec´ılia B. V. de Souza, Departamento de Qu´ımica Orgaˆnica, Instituto
de Qu´ımica, Universidade Federal Fluminense, Outeiro de Sa˜o Joa˜o Batista s/n°, Centro, Nitero´i,
CEP 24210-150, Rio de Janeiro, Brazil; E-mail: gqocica@vm.uff.br.
735
DOI: 10.1081/NCN-120038009
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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The antiviral effects of most of the pyrazolo[4,3-c]quinolin-3-one ribonucleosides
(5a–f) on VV and HSV were not impressive.
Key Words: Antiviral activities; Pyrazolo[4,3-c]quinolinone; Ribonucleoside
derivatives; Vaccinia virus; HSV-1.
INTRODUCTION
Pyrazole derivatives have been found to exhibit biological activities such as
antibacterial, anti-inflammatory, hypotensive and antitumor properties (Paul, S.; Gupta,
M.; Gupta, R.; Loupy, A. Microwave assisted solvent-free synthesis of pyrazolo[3,4-
b]quinolines and pyrazolo[3,4-c]pyrazoles using p-TsOH. Tetrahedron Lett. 2001, 42,
3827–3829). Condensed pyrazoles with other heterocyclic systems are known to
possess various important biological activities e.g. some 2-phenylpyrazolo[4,3-
c]quinolin-3-ones are ligands for benzodiazepine receptors (Fryer, R.I.; Zhang, P.;
Rios, R.; Gu, Z.Q.; Basile, A.S.; Skolnick, P. Structure-activity relationship studies at
the benzodiazepine receptor (BZR). A comparison of the substituent effects of
pyrazoloquinolinone analogs. J. Med. Chem. 1993, 36 (11), 1669–1673). Pyrazolo[3,4-
b]quinoline derivatives also possess antiviral (Smirnoff, P.; Crenshaw, R.R. Stimulation
of interferon-production in mice and in mouse spleen leukocytes by analogs by BL-
20803. Antimicrob. Agents Chemother. 1977, 11 (3), 571–573) and antimalarial (Stein,
R.G.; Beil, J.H.; Singh, T. Antimalarials 4-substituted 1H-pyrazolo-3,4-betaquinolines.
J. Med. Chem. 1970, 13 (1), 153) activities and pyrazolo[3,4-c]pyrazole derivatives are
useful antitumor agents. (Taylor, E.C.; Patel, H.; Kumar, H. Synthesis of pyrazolo[3,4-
d]pyrimidine analogs of the potent antitumor agent N-(4[2-(2-amino-4(3H)-oxo-7H-
pyrrolo[2,3-d]pyrimidin-5-yl-ethyl]benzoyl)-L-glutamic acid-(LY231514). Tetrahedron
1992, 48 (37), 8089–8100). As part of an ongoing program for the synthesis of
potential antiviral agents based on the fused pyrazoloquinoline system, we prepared and
investigated the antiviral and cytotoxic activities of several new pyrazolo[4,3-c]quinolin-3-
one derivatives (3a–g), as well as their respective ribonucleosides (5a–g).
RESULTS AND DISCUSSION
The quinolone derivatives 1a–g were prepared according to a procedure described
in the literature,[1] which includes treatment of the appropriate aniline with diethyl
ethoxymethylenemalonate to obtain the enamine derivatives that were then thermally
cyclized in Dowtherm A.[2] Refluxing these quinolones in thionyl chloride afforded the
corresponding chloro-derivatives 2a–g. By using a slightly modified procedure[3,4]
described in the literature, which involves the reaction of 2a–g with the desired
phenylhydrazine in toluene followed by refluxing in acetic acid, the corresponding 2-
phenylpyrazolo[4,3-c]quinolin-3-ones 3a–g were obtained in 78%, 85%, 70%, 80%,
74%, 72%, 84% yields, respectively (Scheme 1).
None of our attempts to silylate 3a–g in a refluxing mixture of hexamethyldi-
silazane (HMDS) and trimethylchlorosilane (TMSCl)[5,6] were successful. Heating 3a–
g with bis(trimethylsilyl)trifluoroacetamide (BSTFA)[7–9] containing 1% of TMSCl at

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Scheme 1. Synthetic route used for preparing the pyrazolo[4,3-c]quinolin-3-one derivatives 3a–g
and their ribonucleosides 5a–g.
110–120°C, under nitrogen atmosphere, followed by addition of a solution of 1-O-
acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose in acetonitrile, in the presence of tri-
methylsilyltrifluoromethanesulfonate (TMSOTf)[10] as a catalyst. This mixture was
stirred for 2–4 h and then poured into ice-cold water. Afterwards, it was neutralized
with saturated aqueous solution of sodium bicarbonate and the resulting solid was
collected by filtration, washed with water (4 ꢀ 30 mL) and hexane (3 ꢀ 20 mL).
Purification by column chromatography on silica gel, eluting with 20% of dichloro-
methane in ethyl acetate followed by crystallization from ethanol/acetone (9:1) afforded
the pure protected ribonucleosides 4a–g in 88%, 85%, 80%, 80%, 85%, 82% and 87%
yields, respectively. Deprotection reaction of these nucleosides 4a–g was carried out by

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de Oliveira et al.
treatment with 0.1 N sodium methoxide solution (40 mL), at room temperature, for 16–
24 h, followed by neutralization with saturated ethanolic ammonium chloride solution.
The resulting solids were washed with cold water and ethyl ether, giving pure 1-b-D-
ribofuranosylpyrazolo[4,3-c]quinolin-3-ones 5a–g in 90%, 88%, 89%, 92%, 82%, 87%
and 86% yields, respectively.
The structures of the new compounds are supported by 1D and 2D NMR
techniques [1H (Tables 1 and 2), 13C (Tables 3 and 4), 1Hx1H-COSY, 1Hx13C-
1
n
HETCOR J and JCH, n = 2,3, nOedif], IV, UV and FABHRMS analysis. The position
of glycosylation was established on the basis of cross peak between C4 and the
anomeric hydrogen H1’ (3JCH) in the HETCOR spectrum of 5a, clearly indicating that
the ribosylation occurred at N5. Compounds 5a–g afforded practically identical UV
curves, confirming the glycosylation at N5 for all heterocycles. Saturation of H1’
(riboside) resulted in nuclear Overhauser enhancements of H2’, H4’, H6 and H4
signals for 4a–g, also confirming N5 as the ribosylated nitrogen and establishing b-
configuration (H4’) for all nucleosides. Furthermore, all 13C NMR assignments of
the ribosyl moiety of compounds 4 and 5 are very similar in their respective series.
13C chemical shifts of their corresponding heterocyclic bases also show the same
pattern, of which the signal in the 161 ppm region corresponds to carbon C3 of the
pyrazole moiety.
BIOLOGICAL STUDIES
Antiviral Activity
The antiviral effects of pyrazolo[4,3-c]quinolin-3-one nucleobases and ribonucleo-
sides on HSV-1 and vaccinia virus replication were determined as described in the
Experimental section. Our results suggest that most of the nucleobases were more
effective inhibitors of vaccinia virus replication than the ribonucleoside counterparts.
Heterocycles 3c and 3d were able to inhibit VV infection near to 70%. It is important
to emphasize that compounds 3c and 3d were not cytotoxic to host cells at the same
concentration they inhibited VV infections, suggesting they may be selectively targeted
to virus replication. However, these compounds showed less significant inhibitory
effects on HSV-1 replication, reaching the maximal inhibitory effect around 20–30%
(Table 5). The antiviral activities of the pyrazolo[3,4-b]pyridine derivatives against
vaccinia virus have been recently described.[11] Comparatively, the antiviral agents
described here are more efficient against vaccinia virus. Altogether, these results
provide important information for future design of more potent antiviral pyrazolo
derivatives. Furthermore, all compounds in question, synthesized by our group,
probably present a common mechanism of inhibition against vaccinia virus replication,
which is currently under investigation in our laboratory.
EXPERIMENTAL
General Procedures. Melting points were determined on a Fisher-Johns
apparatus and are uncorrected. The IR spectra were recorded on a Perkin-Elmer

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Pyrazolo[4,3-cc]quinolin-3-ones Nucleosides
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Table 4. Carbon (75.0 MHz) chemical shift assignments for 5a–g [(DMSO-d6/TMS), J (Hz)].
C
5a
5b
5c
5d
5e
5f
5g
3
161.6 161.1
106.3 106.3
137.2 137.1
133.4 133.6
161.5
106.1
137.5
133.4
161.8 161.8
106.0 106.0
137.5 137.5
133.4 133.4
161.8
107.5
138.4
137.0
161.5
3a
4
106.8
138.4
136.5
5a
or 133.3
6
7
8
117.5 117.5
131.5 131.5
136.4 136.5
122.2 122.2
117.6
131.7
136.7
117.6 117.6
131.7 131.6
105.3
120.4
(J = 27.6)
163.0
(J = 300.0)
115.4
(J = 22.4)
125.0
or 120.3
123.7
129.7
or 131.7
136.7 136.7
9
122.3
119.5
142.7
93.0
122.3 122.3
119.4 119.3
143.0 142.9
124.5
118.5
142.5
92.9
9a
9b
1’
2’
3’
4’
5’
1@
119. 119.5
142.5 142.1
116.8
142.9
93.1
92.7
75.0
68.6
84.7
59.5
92.9
75.1
68.5
84.6
59.4
93.0
75.2
68.5
84.6
59.4
93.0
75.2
68.4
84.6
59.3
75.1
68.5
75.0
68.6
75.1
68.7
84.6
59.4
84.9
59.4
84.9
59.5
133.4 133.6
or 133.3
136.6
139.0 139.4.
120.0 120.3
128.9 131.6
139.0
139.1
(J = 2.5)
120.5
(J = 7.3)
115.5
(J = 22.2)
158.8
(J = 239.4)
2@
3@
4@
118.6 120.5
128.6 113.9
124.0 156.1
120.5
120.4
or 120.3
116.0
131.7
116.1
or 131.7
127.9 116.0
(J = 22.5)
159.7
(J = 293.9)
OCH3
CH3
–
55.4
20.7
–
–
–
–
–
–
–
20.5
20.7
20.7
20.7
1420 spectrometer as potassium bromide pellets and frequencies are expressed in cmꢁ1
.
Ultraviolet (UV) spectra were obtained on a Shimadzu spectrophotometer; l are in nm
and e in molꢁ1cmꢁ1. High resolution mass spectra (FABHRMS) were recorded in a 3-
nitrobenzylalcohol matrix in the positive ion mode on a VG ZAB-E mass spectrometer,
by the Department of Chemistry and Biochemistry, University of Oklahoma. NMR
spectra were recorded on a Varian Unity Plus 300 spectrometer operating at 300.00 MHz
(1H) and 75.0 MHz (13C), in specified solvents. Chemical shifts are reported in ppm (d)
relative to tetramethylsilane. Proton and carbon spectra were typically obtained at room
temperature. The two dimensional experiments were acquired using standard Varian
Associates automated programs for data acquisition and processing.
General Procedures for the Syntheses of: 2-Phenyl-2H-pyrazolo[4,3-c]quinolin-
3(5H)-ones (3a–g); 2-Phenyl-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[4,3-c]quinolin-3(5H)-ones (4a–g) and 2-Phenyl-5-b-D-ribofuranosyl-2H-
pyrazolo[4,3-c]quinolin-3(5H)-ones (5a–g). The quinolone derivatives 1a–g were

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Table 5. Antiviral activity of pyrazolo[4,3-c]quinolin-3-ones
(3a–3f) and ribonucleoside derivatives (5a–5f).
% of inhibition of
virus yield
Compounds 100 mM
HSV-1
VV
3a
3b
3c
3d
3e
3f
20
22
31
27
27
26
20
30
15
35
25
35
47
33
68
70
42
55
30
48
18
8
5a
5b
5c
5d
5e
5f
10
9
prepared by treating the appropriate aniline with diethyl ethoxymethylenemalonate to
obtain the enamine derivatives that were then cyclized in refluxing Dowtherm A.[1]
These quinolones (13 mmol) were refluxed in thionyl chloride (20 mL), for 17 h,
affording the corresponding chloro-derivatives 2a–g.[3,4] Reaction of 2a–g (4 mmol)
with the desired phenylhydrazine (8 mmol) in toluene (30 mL), for 3 h, followed by a
2 h-reflux in acetic acid gave the corresponding 2-phenylpyrazolo[4,3-c]quinolin-3-
ones 3a–g. Heating 3a–g (1 mmol) with bis(trimethylsilyl)trifluoroacetamide (BSTFA)
(3.0 mL)[7–9] containing 1% of TMSCl at 110–120°C, under nitrogen atmosphere, for
5 h, followed by addition of a solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribo-
furanose (1.1 mmol) in 15 mL of acetonitrile, in the presence of trimethylsilyltri-
fluoromethanesulfonate (TMSOTf) (1.2 mmol in 5 mL of acetonitrile)[10] as a catalyst,
at room temperature, and subsequent stirring for 2–4 h led to the crude product, which
was poured into ice-cold water (20 mL). After neutralization with saturated aqueous
solution of sodium bicarbonate, the resulting solid was collected by filtration, washed
with water (4 ꢀ 30 mL) and hexane (3 ꢀ 20 mL). Purification by column chroma-
tography on silica gel, eluting with 20% of dichloromethane in ethyl acetate followed
by crystallization from ethanol/acetone (9:1) afforded the pure protected ribonucleo-
sides 4a–g. Deprotection reactions of these nucleosides 4a–g (0.5 mmol) were carried
out by treatment with 0.1 N sodium methoxide solution (40 mL), at room temperature,
for 16–24 h, followed by neutralization with saturated ethanolic ammonium chloride
solution, affording solid products which were washed with cold water and ethyl ether,
giving pure 1-b-D-ribofuranosylpyrazolo[4,3-c]quinolin-3-ones 5a–g.
4a: 8-Methyl-2-phenyl-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-pyra-
zolo[4,3-c]quinolin-3(5H)-one- 88%, m.p. 201°C; FABHRMS m/z calcd. for
C43H34N3O8 (M + H)+ 720.2343, found 720.2346; IR 3060, 1720, 1630, 1260; UV
1
lmax (CHCl3) 244 (e 33,018), 411 (e 5,312); H NMR (Table 1); 13C NMR (Table 2).

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4b: 8-Methyl-2-(4-methoxyphenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
2H-pyrazolo[4,3-c]quinolin-3(5H)-one- 85%, m.p. 248–249°C; FABHRMS m/z calcd.
for C44H36N3O9 (M + H)+ 750.2449, found 750.2451; IR 3060, 1715, 1630, 1260; UV
1
lmax (CHCl3) 243 (e 18,311), 411 (e 2,711); H NMR (Table 1); 13C NMR (Table 2).
4c: 8-Methyl-2-(4-fluorophenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
2H-pyrazolo[4,3-c]quinolin-3(5H)-one- 80%, m.p. 225–226°C; FABHRMS m/z calcd.
for C43H33N3O8F (M + H)+ 738.2250, found 738.2251; IR 3060, 1720, 1630, 1260;
UV lmax (CHCl3) 243 (e 21,731), 418 (e 2,934); 1H NMR (Table 1); 13C NMR
(Table 2).
4d: 8-Methyl-2-(4-chlorophenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[4,3-c]quinolin-3(5H)-one- 80%, m.p. 237–238°C; FABHRMS m/z calcd. for
C43H33N3O837Cl (M + H)+ 756.1898, found 756.1926; IR 3050, 1720, 1635, 1260;
UV lmax (CHCl3) 243 (e 13,233), 411 (e 1,718); 1H NMR (Table 1); 13C NMR
(Table 2).
4e: 8-Methyl-2-(4-bromophenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[4,3-c]quinolin-3(5H)-one- 85%, m.p. 238°C; FABHRMS m/z calcd. for
C43H33N3O881Br (M + H)+ 798.1550, found 798.1551; IR 2970, 1720, 1635, 1260; UV
1
lmax (CHCl3) 243 (e 61,700), 411 (e 8,660); H NMR (Table 1); 13C NMR (Table 2).
4f: 7-Fluoro-2-(4-fluorophenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[4,3-c]quinolin-3(5H)-one- 82%, m.p. 212°C; FABHRMS calcd. for 798.1550,
found 798.1551; IR 2970, 1720, 1635, 1260; UV lmax (CHCl3) 285 (e 55,384), 421
1
(e 7,024); H NMR (Table 1); 13C NMR (Table 2).
4g: 7-Bromo-2-(4-bromophenyl)-5-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[4,3-c]quinolin-3(5H)-one- 87%, m.p. 227°C; FABHRMS m/z calcd. for
C42H30N3O881Br2 (M + H)+ 866.0357, found 866.0358; IR 3060, 1725, 1615, 1260;
UV lmax (CHCl3) 296 (e 23,726), 421 (e 2,975); 1H NMR (Table 1); 13C NMR
(Table 2).
5a: 8-Methyl-2-phenyl-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-
one- 90%, m.p. 282°C; FABHRMS m/z calcd. for C22H22N3O5 (M + H)+ 408.1560,
found 408.1559; IR 3660–2700, 1630, 1260; UV lmax (CH3OH) 393 (e 5,392), 223
1
(e 30,400); H NMR (Table 3); 13C NMR (Table 4).
5b: 8-Methyl-2-(4-methoxyphenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]qui-
nolin-3(5H)-one- 88%, m.p. 258–259°C; FABHRMS m/z calcd. for C23H24N3O5
(M + H)+ 438. 1663, found 438.1664; IR 3620–2800, 1625; UV lmax (CH3OH) 391 (e
1
5,500) 225 (e 43,800); H NMR (Table 3); 13C NMR (Table 4).
5c. 8-Methyl-2-(4-fluorophenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quinolin-
3(5H)-one- 89%, m.p. 274°C; FABHRMS m/z calcd. for C22H21N3O5F
(M + H)+426.1463 found 426.1465; IR 3620–2600, 1630; UV lmax (CH3 OH) 391
1
(e 4,262), 223 (e 24,900); H NMR (Table 3); 13C NMR (Table 4).

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5d: 8-Methyl-2-(4-chlorophenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quino-
lin-3(5H)-one- 92%, m.p. 250°C; FABHRMS m/z calcd. for C22H21N3O535Cl
(M + H)+ 444.1168, found 444.1169, C22H21N3O537Cl (M + H)+ 444.1141, found
1
444.1140; IR 3650–2800, 1610; UV lmax (CH3OH) 393 (e 3,243), 225 (e 31,500); H
NMR (Table 3); 13C NMR (Table 4).
5e: 8-Methyl-2-(4-bromophenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quino-
lin-3(5H)-one- 82%, m.p. 256°C; FABHRMS m/z calcd. for C22H21N3O579Br (M + H)+
486.0763, found 486.0664, C22H21N3O581Br (M + H)+ 488.0644, found 488.0643; IR
3630–2800, 1625; UV lmax (CH3OH) 389 (e 3,724), 225 (e 23,797); 1H NMR
(Table 3); 13C NMR (Table 4).
5f: 7-Fluoro-2-(4-fluorophenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quinolin-
3(5H)-one- 87%, m.p. 241–242°C; MS m/z 419 (5); IR 3660–2800, 1620; UV lmax
1
(CH3 OH) 391 (e 8,603), 223 (e 48,168); H NMR (Table 3); 13C NMR (Table 4).
5g: 7-Bromo-2-(4-bromophenyl)-5-b-D-ribofuranosyl-2H-pyrazolo[4,3-c]quino-
lin-3(5H)-one- 86%, mp. 258°C; FABHRMS m/z calcd. for C21H18N3O579Br2 (M + H)+
549.9612, found 549.9613, C21H18N3O579Br281Br2 (M + H)+ 551.9691, found 551.9592;
UV lmax (CH3OH) 388 (e 6,450), 226 (e 34,919); 1H NMR (Table 3); 13C NMR
(Table 4).
Cells and Viruses: BSC-40 and Vero, epithelial-derived cell lines from the
African green monkey kidney, were obtained from the American Type Culture
Collection and were grown in monolayer cultures in Dulbecco’s modified Eagle’s
(Gibco laboratories) containing 2% heated-inactivated fetal bovine serum (purchased
from Fazenda Pig), 8% calf serum (purchased from Centro Pan-Americano de Febre
Aftosa), 2.25% sodium bicarbonate, 500 U/mL penicilin, 100 mg/mL streptomycin,
50 mg/mL gentamycin, 2.5 mg/mL amphotericin B. Cell cultures were incubated at
37°C in humidified air atmosphere containing 5% CO2.
The vaccinia virus (WR strain) was obtained from the American Type Culture
Collection and was routinely propagated and titrated in confluent BSC-40 cells, as
described by Daˆmaso and Moussatche´.[12] Herpes simplex virus type 1 (HSV-1) 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.
Tissue Culture Inhibitory Dose (TCID50) for HSV-1 Titration: Virus
infectivity was measured by a dilution method using 96-well microtitre plate and
expressed as 50% tissue culture infections dose (TCID50) according to Reed and
Muench.[13] Sub-confluent VERO cells, grown in 96 well plates (106 cells/well), were
infected with HSV-1 at 1 PFU (plaque forming unit)/cell for 120 minutes at 37°C in
100 mL of DMEM). After this period, virus inoculum was replaced by a culture
medium, containing pyrazolo[4,3-c]quinolin-3-one derivatives or their respective
ribonucleosides at the concentration of 100 mM. Control cultures were incubated with
media without compounds. After 72 hours post-infection, the culture medium was
harvested and the virus titer of each sample was determined in terms of the 50 % tissue
culture dose (TCID 50/mL) by endpoint dilution.

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Pyrazolo[4,3-cc]quinolin-3-ones Nucleosides
747
Yield Reduction Assay for Vaccinia Virus Titration: Sub-confluent BSC-40
cells, grown in 6-well plates (106 cells/mL), were infected with 2.5 PFU of VV per
cell. After 20 minutes of virus adsorption at 37°C in 200 ml of DMEM, virus inoculum
was replaced by culture medium, containing or not either pyrazolo[4,3-c]quinolin-3-one
heterocycle moieties or their respective ribonucleosides at the concentration of 100 mM.
Infection proceeded for 12 hours at 37°C in humidified air containing 5% CO2, when
the culture medium was aspirated and cell monolayers were harvested in 500 mL of
1 mM Tris-HCL pH 9.0 and submitted to two cycles of freezing thawing for
homogenization. Virus titer of each sample was determined by fixing and staining
infected cell monolayers with PBS containing 0.1% crystal violet, 3.7% formaldehyde,
for 30 minutes. Afterwards, cell monolayers were thoroughly washed with water and
virus plaque number was determined. The yield reduction was determined by using the
virus yield obtained in the absence of drugs as a reference.
ACKNOWLEDGMENTS
The fellowship granted to M. R. P. O. from CNPq (Brazil) is gratefully
acknowledged. A. C. P., M. C. B. V. S., and V. F. F. are grateful to CNPq for their
individual research fellowships. This work was partially supported by CNPq (National
Council of Research from Brazil) and FAPERJ.
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Received June 30, 2003
Accepted June 1, 2004

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