Synthesis, cytotoxicity and antiviral activity of podophyllotoxin analogues modified in the E-ring

Article abstract of DOI:10.1016/j.ejmech.2003.05.001

Several podophyllotoxin derivatives modified in the E-ring were prepared and evaluated for their cytotoxicity on four neoplastic cell lines (P-388, A-549, HT-29 and MEL-28) and for their antiherpetic activity against Herpes simplex virus type II. The trim

Full text of DOI:10.1016/j.ejmech.2003.05.001

European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis, cytotoxicity and antiviral activity of podophyllotoxin
analogues modified in the E-ring
M. Angeles Castro a,*, Jose´ M. Miguel del Corral a, Marina Gordaliza a,
M. Antonia Go´mez-Zurita a, M. Luz de la Puente a, Liliana A. Betancur-Galvis b,
Jelver Sierra b, Arturo San Feliciano a
a Departamento de Qu´ımica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
b Grupo Infeccio´n y Ca´ncer, Facultad de Medicina, Universidad de Antioquia, A.A1226 Medellin, Colombia
Received 3 March 2003; received in revised form 6 May 2003; accepted 26 May 2003
Abstract
Several podophyllotoxin derivatives modified in the E-ring were prepared and evaluated for their cytotoxicity on four neoplastic
cell lines (P-388, A-549, HT-29 and MEL-28) and for their antiherpetic activity against Herpes simplex virus type II. The
trimethoxyphenyl moiety was oxidized to ortho-quinone and further condensed with diamines and enamines to form different
heterocycles. Most of the compounds maintained their cytotoxicity at the mM level and some of them showed antiherpetic activity.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Podophyllotoxin; Cyclolignans; E-ring modifications; Cytotoxicity; Herpes simplex virus
1. Introduction
In the last few years, our research group has been
involved in the chemical modification of cyclolignans
and has prepared a large number of derivatives with
potent antiviral, cytotoxic and immunosuppressant
properties [2b]. It is worth stressing the selective
cytotoxicity of some derivatives [4] modified in the C-
and D-rings, with the general structure shown in Fig.
1B.
Cyclolignans constitute a family of natural products
with very interesting antiviral and cytotoxic properties.
Compounds in clinical use, such as the natural product
podophyllotoxin and the semisynthetic derivatives eto-
poside and teniposide (Fig. 1A), belong to this family
[1].
From podophyllotoxin to etoposide some chemical
modifications were made that also led to a change in the
mechanism of action, from the inhibition of microtubule
formation by the parent compound podophyllotoxin, to
In the majority of the studies related to cyclolignans,
the A- and E-rings were untouched and very little
research has tackled their influence on cytotoxic activity.
In a previous paper [5], we reported the result of
modifications affecting the A-ring. Here, we report
modifications performed on the E-ring that have been
related to active metabolites generated through an in
vivo oxidative pathway [6]. Indeed, the main modifica-
tions in the E-ring referred to in the literature imply
changes in the degree of oxidation.
It has been shown that the 3?,4?-catechol derivative of
etoposide can be formed in the presence of cytochrome
P-450 [7]a and that this catechol can be further oxidized
to the 3?,4?-ortho-quinone in the presence of oxygen [7b]
or under the influence of horseradish peroxidase or
prostaglandin E synthetase [7c]. Both catechol and
DNAꢀtopoisomerase II inhibition by etoposide and
/
congeners. This change is related to three main chemical
modifications [2]: demethylation at C-4? of the E ring, C-
7 epimerisation, and the presence of a glycosidic or
related moiety at the C-7 position on the C-ring. These
observations led to a great number of derivatives that
were synthesized and analysed by QSAR methods by
Lee and coworkers [3], while the cyclolignan skeleton
was virtually untouched in every case.
* Correspondence and reprints:.
E-mail address: macg@usal.es (M.A. Castro).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.05.001

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M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
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Fig. 1. Structures of podophylotoxin and related compounds. (A) Cyclolignans in clinical use. (B) Selective cytotoxic and immunosuppressant
cyclolignans lacking the lactone ring.
ortho-quinone bind strongly to purified calf thymus
DNA and this may contribute to the activity of these
compounds, through the formation of free radicals [8] or
even through the direct binding of the quinone to the
DNA [9].
Thus, a series of podophyllotoxin analogues with aza-
or oxa-heterocyclic systems, instead of the trimethox-
yphenyl ring, were prepared and evaluated for their
cytotoxicity. Some representative compounds were also
evaluated as antiviral agents.
Based on the possibility that the catechol/quinone
rings could be involved in the cytotoxicity mechanism, a
series of 3?,4?-O-didemethylepipodophyllotoxins and
3?,4?-didemethoxy-3?,4?-dioxopodophyllotoxins, with a
variety of substituents at C-7, were prepared and
evaluated as antitumour agents [9]. The ortho-quinones
were less cytotoxic than the catechols, and both were
less active than the 4?-O-demethyl series, although some
of them displayed activity comparable to the parent
compound and bound to both nucleic acids and
proteins.
2. Chemistry
The starting point for introducing different substitu-
ents on the cyclolignan E-ring skeleton was the trans-
formation of the trimethoxyphenyl subunit into the
quinonoid derivative. By treatment of cyclolignans 1
and 2 with nitric acid [14], the quinones 3 and 4 were
obtained (Fig. 2). It is well known that during chroma-
tography, the quinones can suffer further transforma-
tions that reduce the yields. As the reaction product was
sufficiently pure as shown by the NMR spectra, it was
used for the next steps without chromatographic pur-
ification. Numbering of compounds in the schemes and
in the NMR tables corresponds to the usual numbering
of lignans [15], for comparison purposes, although in
Section 4 the systematic name is given for those
derivatives with new heterocycles in ring E.
Other modifications performed in the E-ring imply
changes in the degree of oxygenation. Thus, cyclolignan
analogues in which one, two or all three methoxy groups
on the phenyl ring were replaced by hydrogen atoms or
an alkyl group, were prepared [10ꢀ12]. The activity
/
results showed that some of them were almost as potent
as the parent compound, suggesting that the presence of
the three oxygenated functions in E-ring of podophyllo-
toxin is not a strong determinant of cytotoxicity.
Since the hydroxyl group at C-7 of quinone 3 could
interfere with later transformations, we attempted to
acetylate it. It has been reported [14] that the quinone
system is unaffected by acetylation; however, when 3
was treated with acetic anhydride and pyridine at room
temperature, the only compound isolated was the
triacetate 5a. To obtain the quinone acetylated at C-7,
nitric acid demethylation was applied to podophyllo-
toxin acetate 1a yielding 3a. Reduction of 3a with
sodium dithionite yielded the catechol 5. Acetylation
On the other hand, the possibility of transforming the
ortho-quinone moiety into other rings has not been
explored (except for a study concerning the character-
ization of the ortho-quinone as its quinoxaline derivative
[13]) and nothing is reported about the effect of such
changes on cytotoxicity. We therefore decided to trans-
form the ortho-quinone into larger ring systems,
whether aromatic or not, and to analyse their influence
on cytotoxicity compared to podophyllotoxin.

M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ
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911
901
Fig. 2. Preparation of cyclolignans, nitrogenated in the E-ring, derived from podophyllotoxin and isoxazole analogues.
of this compound confirmed the structure of the
triacetate 5a.
isolated from the complex mixture of the crude reaction
product. Similar by-products were previously reported
by treatment of ortho-quinones with methanol under
weakly acidic conditions [14].
Another procedure reported for the preparation of
substituted benzodioxanes from ortho-quinones is the
reaction with enamines [18]. To apply this procedure, we
used commercially available heterocyclic enamines such
as pyrrolidine and morpholine enamines of cyclohex-
anone; these yielded a mixture of the two possible
regioisomers of the corresponding tricyclic analogues
14 and 15 (Fig. 3), as deduced from the presence of
several duplicated signals in their NMR spectra.
Quinones generally react with most nucleophiles [16]
and can undergo a wide range of reactions. Hence, this
intermediate offers many possibilities for the modifica-
tion of lignans. First, quinones served as substrates for
condensation with different diamines.
When differently substituted phenylenediamines were
used, the corresponding phenazines 6ꢀ
/
9 and 18ꢀ20 were
/
obtained (Fig. 2). When ethylenediamine was used as
reagent, the quinoxalines 10 and 16 were obtained in
which the new ring formed was aromatised. This result
can be explained through the oxidation of the expected
dihydroquinoxaline by the starting quinone, which was
reduced to the catechols 5 and 17; both were isolated as
by-products from the reaction.
3. Biological results and discussion
Catechol 5 can be transformed into different oxyge-
nated rings in a way similar to that used for the
transformation of the A-ring [5]. Thus, by reaction
with dihalogenated reagents such as 1,2-dibromoethane
and dibromomethane, dioxane 11 and dioxole 12 were
obtained (Fig. 3).
It has been reported that a-diketones, including some
ortho-quinones, can also form dioxane rings by reaction
with 1,2-diols [17]. We tried to obtain this kind of
derivative by treatment of quinone 3a with ethylene
glycol and 1,2-cyclohexanol in the presence of trimethy-
lorthoformate, but instead of the expected dioxanes,
only a small amount of the methylated derivative 13 was
3.1. Cytotoxicity
The compounds thus obtained were evaluated in vitro
[19] to establish their cytotoxicity for the following cell
cultures: murine leukaemia P-388, human lung carci-
noma A-549, human colon carcinoma HT-29 and
human melanoma MEL-28. Some general observations
can be made from the results shown in Table 1.
Transformation of the trimethoxyphenyl group into
the corresponding ortho-quinone leads to variable
cytotoxicity results, depending on the substituents pre-
sent in other parts of the molecule. If there is a free

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M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
Fig. 3. Preparation of cyclolignans, oxygenated in the E-ring, derived from podophyllotoxin.
hydroxyl group at C-7, a significant decrease in potency
is observed (1 vs. 3), while when this group is acetylated,
no change in the activity is observed (1a vs. 3a), 3a being
twice as potent as 3.
The catechol group in the E-ring is about four times
less potent than the ortho-quinone (5 vs. 3a) and this
potency is practically unchanged after acetylation (5a vs.
5) but, when one of the phenol groups is methylated, the
potency is partially recovered (13 vs. 5).
Transformation of the trimethoxyphenyl ring of the
acetyl podophyllotoxin series into polyheterocyclic sys-
tems decreases the potency several times, the effect
becoming more pronounced with increasing number of
substituents on the phenazine system (6 and 9 vs. 1a).
Only quinoxaline 10 retained an IC50 value below mM.
Those analogues in which new oxygenated rings are
fused to the E-ring (dioxole and dioxanes) were also less
cytotoxic than podophyllotoxin and slightly less potent
than the ortho-quinone precursor (11, 12, 14, 15 vs. 1
and 3a).
For the isoxazole series, the slight selectivity shown by
2 towards P-388 is lost in quinone 4, which had values of
IC50 in the same range as those for 2 on HT-29.
Quinoxaline 16 also retained the same range of cyto-
toxicity as the parent compound 2 and surprisingly,
phenazine 19, which has two methyl groups as sub-
stituents, maintained the cytotoxicity, while the other
derivatives (17, 18, 20) were the least potent derivatives
of all the series. This is difficult to explain on the basis
on the two proposed mechanisms of action of cyclo-
lignans; therefore, a third mechanism (such as that
postulated previously by Lee and coworkers [20]) could
Table 1
Cytotoxic activity of cyclolignans modified in the E-ring (IC50 mM)
Compound
P-388
A-549
HT-29
MEL-28
1
0.012
0.012
0.012
0.55
11
1a
2
0.55
2.3
0.55
5.7
0.55
5.7
3
1.3
0.59
1.3
0.59
1.3
0.59
1.3
0.59
12
3a
4
12
12
12
5
2.8
2.2
5.84
2.2
5.84
2.2
5.84
2.2
5a
6
1.0
2.0
1.0
2.0
1.0
2.0
1.0
2.0
7ꢁ
/8
9
4.8
4.8
4.8
4.8
10
11
12
13
14
15
16
17
18
19
20
0.56
2.2
0.56
2.2
0.56
2.2
0.56
2.2
2.27
0.11
4.33
2.02
2.8
2.27
0.23
8.66
2.02
2.8
2.27
0.23
8.66
2.02
2.8
2.27
0.23
8.66
2.02
2.8
20.0
20.0
20.0
20.0
10.0
1.0
10.0
1.0
18
10.0
1.0
18
10.0
1.0
18
ꢀ/
ꢀ/
ꢀ/
ꢀ18
/

M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ
/
911
903
be involved. More work will be necessary to clarify these
points.
which is practically inactive in the systems tested,
showed that the presence of catechol or quinone groups
induce some HSV antiviral activity in those molecules.
Compounds 1, 1a, 3a, 5, 6 and 10 were cytostatic when
evaluated on confluent monolayers of Vero cells that are
nonproliferative. Consequently the cytotoxic concentra-
tion needed to detach 100% (CC100) of the cell mono-
layer was not found below 150 mg mLꢃ1 (not shown).
The antiherpetic activity (HSV-I) of podophyllotoxin 1
and podophyllotoxin acetate 1a was previously reported
[6,22] using the plaque elimination assay. To analyse
their behaviour against HSV-2 both compounds were
submitted to the EPTT assay with a lower viral
3.2. Antiviral results
The evaluation of antiherpetic activity against Herpes
simplex virus type II (HSV-2) of some representative
compounds was carried out using the end-point titration
technique (EPTT) [21] in which the cytotoxic activity
and the antiviral effect were simultaneously evaluated
(Table 2). The authors stated that under defined
experimental conditions only those compounds showing
reduction factors (Rf) of the viral titre over 1ꢂ
103 could
/
challenge (1ꢂ
assay. By EPTT both compounds were shown to be
slightly active against HSV-2 with a Rf value of 1ꢂ
100.5
/
TCID50) and to the plaque elimination
be considered as having relevant antiviral activity.
Following this evaluation method, phenazines 18 and
19 exhibited the highest antiviral activity against HSV-2,
/
(not included). By the plaque elimination assay these
compounds were tested with a low viral concentration of
100 PFU (plaque forming units). The concentrations
needed for complete elimination of macroscopic plaque
formation (ED100 effective doses of 100%) without
toxicity to cell monolayers were 10 and 80 ng mLꢃ1
for podophyllotoxin 1 and podophyllotoxin acetate 1a,
respectively (not shown).
with a Rf of 1ꢂ
/
101.5 and 1ꢂ101.0, respectively, when
/
challenged with ten times the tissue culture infectious
dose 50 (TCID50) indicating a moderate activity against
HSV-2. For these compounds, the nontoxic concentra-
tion needed to obtain the largest reduction of the viral
titre was approximately half the cytotoxic concentration
needed to detach 100% (CC100) of the cell monolayer,
revealing that the antiviral activity, especially for
phenazine 19, is principally due to cytotoxicity. Com-
paring the antiviral activity of phenazines 18 and 20, we
found that in phenazine 20 the two chloro substituents
led to complete loss of activity. Furthermore, the
presence at C-7? of the 3?,4? ortho-quinone moiety
reduced the activity of the isoxazole derivatives (4 vs.
18).
4. Experimental
4.1. Chemistry
Melting points were determined by heating the
compounds in an external silicone bath and were
uncorrected. Optical rotations were recorded on a
Comparison of the antiviral activity of 3?,4?-catechol
podophyllotoxin (5) and 3?,4? ortho-quinone podophyl-
lotoxin (3a) with that of podophyllotoxin acetate (1a),
PerkinꢀElmer 241 polarimeter in chloroform solution
/
Table 2
Anti-HSV-2 activity of cyclolignan on Vero cells a determined by the end-point titration technique (EPTT) with 10TCDI50
CC100 (mg mLꢃ1
)
Viral reduction factor c
Antiviral activity (mg mLꢃ1
)
b
d
Cyclolignan
1
1a
ꢀ
/
20
20
23
20
25
25
NA
NA
101
NA
NA
23
ꢀ
/
3a
ꢀ
/
6
10
ꢀ
/
NA
NA
NA
100.5
101.5
101.0
NA
NA
NA
NA
101
NA
NA
NA
60
ꢀ
/
9
ꢀ
/
4
18
ꢀ/
120
120
60
30
30
19
20
30
NA
NA
NA
NA
25
11
15
ꢀ
/
25
25
25
25
ꢀ
/
14
5
acyclovir
ꢀ
/
ꢀ
/
ꢀ/600
104
6.0
a
VERO, Cercopithecus aethiops african green monkey kidney ATCC CCL 81.
Minimal toxic dose that detached 100% of the cell monolayer.
b
c
Ratio of the virus titre in the absence over virus titre in the presence of the tested compound.
Maximal nontoxic dose that showed the highest viral reduction factor. NA, no activity.
d

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M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
and UV spectra on a Hitachi 100-60 spectrophotometer
in ethanol. IR spectra were obtained on a Beckmann
(Acculab VIII) spectrophotometer. EIMS and HRMS
were run in a VG-TS-250 spectrometer working at 70
eV. NMR spectra were recorded at 200 MHz for 1H and
50.3 MHz for 13C in deuterochloroform using TMS as
internal reference, on a Bruker WP 200 SY. Chemical
shift values are expressed in ppm followed by multi-
plicity and coupling constants (J) in Hz. Column
chromatography was performed on silica gel (Merck
No 9385). TLC was carried out on silica gel 60 F245
(Merck, 0.25 mm thick). Solvents and reagents were
purified by standard procedures as required. Elementary
analyses were obtained with a LECO CHNS-932 and
NMR (Table 3). 13C-NMR (Table 4). HRMS (FAB-
POSI, Mꢁ1) Calc. for C22H19O9 427.1029. Found
427.1075. Anal. C22H18O9 (C, H).
/
4.1.2.4. Methyl 3?,4?-didemethoxy-3?,4?-
dioxoisoxazolopodophyllate 4. The isoxazole 4 (94%)
was obtained from compound 2. M.p.: 198ꢀ202 8C.
/
UV lmax(o): 217 (19600), 276 (10200), 314 (7100). IR
1
(cmꢃ1): 1734, 1665, 1625, 1484, 1257, 1037. H-NMR
(Table 3). 13C-NMR (Table 4).
4.1.3. 7-Acetyl-3?,4?-didemethylpodophyllotoxin 5.
Quinone 3a (214 mg, 0.5 mmol) was dissolved in
ethanol (60 mL) and water (10 mL). The solution was
stirred at room temperature (r.t.) and Na2S2O4 was
progressively added until the red solution became
yellow. The ethanol was partially evaporated and the
residual solution was extracted with EtOAc, washed
with water, dried over Na2SO4 and the solvent evapo-
rated. Column chromatography of the residue (eluent:
were within 90.4% of the theoretical values.
/
4.1.1. Sources of precursors
Podophyllotoxin 1 was obtained from the resin of
Podophyllum emodi by chromatographic means and was
converted to acetylpodophyllotoxin (1a) and methyl
isoxazolopodophyllate (2) by previously established
procedures [23].
Cl2CH2ꢀ
60%). M.p.: 248ꢀ
/
EtOAc 9:1) yielded the catechol 5 (126 mg,
102.48 (c,
/
252 8C (MeOH). [a]D22
0.5%). UV lmax(o): 209 (31900), 285 (3600), 325 (900).
ꢃ
/
4.1.2. Procedure for oxidative demethylation.
Compounds 3, 3a and 4
IR (cmꢃ1): 3450, 3352, 1775, 1726, 1610, 1518, 1237,
1
1034. H-NMR (Table 3). 13C-NMR (Table 4). Anal.
C22H20O9 (C, H).
4.1.2.1. 3?,4?-Didemethoxy-3?,4?-dioxopodophyllotoxin
(3). Nitric acid (60%, 1.2 mL) was rapidly added to a
solution of podophyllotoxin (1) (190 mg, 0.46 mmol) in
propionic acid (2 mL) at 0 8C. After exactly 4 min at
0 8C, the dark red solution was poured into water (40
mL) and extracted with EtOAc. The organic layer was
washed with aq. sat. NaHCO3 and brine, dried with
Na2SO4 and the solution concentrated to a reduced
volume. The residual solution was diluted with n-hexane
and the precipitate formed after 30 min of stirring, was
Acetylation of 5 with acetic anhydride and pyridine
gave the triacetate 5a.
4.1.4. General procedure for the condensation with
diamines. Compounds 6ꢀ
/
10 and 16ꢀ20
/
4.1.4.1. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(4-
methoxyphenazin-2-yl)-5,5a,6,8,8a,9-hexahydro-
furo[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-one
(6).
Quinone 3a (150 mg, 0.35 mmol) was dissolved in
ethanol (7 mL) and acetic acid (2 mL) and then 1,2-
phenylenediamine (76 mg, 0.70 mmol) was added. The
mixture was stirred overnight at r.t. and extracted with
EtOAc. The organic layer was washed with 2 N HCl, aq.
Sat. NaHCO3 and water. The reaction product obtained
after evaporation of the solvent, was chromatographed
filtered off to yield quinone 3 (150 mg, 85%). M.p.: 179ꢀ
/
181 8C. UV lmax(o): 251 (3200), 257 (3400). IR (cmꢃ1):
3450, 1770, 1698, 1662, 1627, 1484, 1237, 1038. 1H-
NMR (Table 3). 13C-NMR (Table 4).
4.1.2.2. Acetylation of 3. Quinone 3 (50 mg, 0.13 mmol)
was acetylated with acetic anhydride in pyridine. After
following the usual protocol, the reaction product was
on silica gel (Cl2CH2ꢀEtOAc 9:1) to give the phenazine
/
6 (60 mg, 35%). [a]D22
ꢃ35.58 (c, 0.11%). UV lmax(o):
/
chromatographed and eluted with Cl2CH2ꢀ
/
EtOAc 93:7
210 (35200), 259 (37700). IR (KBr, cmꢃ1): 1778, 1734,
1
to yield triacetate 5a (7,3?,4?-triacetyl-3?,4?-didemethyl-
1631, 1607, 1561, 1229, 1038. H-NMR (Table 3). 13C-
podophyllotoxin) (45 mg, 68%). IR (KBr, cmꢃ1): 1776,
NMR (Table 4). HRMS (FAB-POSI, Mꢁ1) Calc. for
/
1
1735, 1609, 1505, 1486, 1236. H-NMR (Table 3). 13C-
C28H23N2O7 499.1505. Found 499.1487. Anal.
C28H22O7N2 (C, H, N).
The same procedure outlined above was applied to
obtain the following compounds.
NMR (Table 4). Anal. C26H24O11 (C, H).
The same procedure was applied to obtain the
following compounds.
4.1.2.3. 7-Acetyl-3?,4?-didemethoxy-3?,4?-
dioxopodophyllotoxin (3a). The acetate 3a (78%) was
4.1.4.2. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(4-methoxy-
7(8)-methylphenazin-2-yl)-5,5a,6,8,8a,9-hexahydro-
obtained from 1a. M.p.: 144ꢀ
/
148 8C. IR (KBr, cmꢃ1):
furo[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-one (7ꢁ
/
8).
1775, 1734, 1702, 1665, 1628, 1486, 1235, 1038. 1H-
From quinone 3a (183 mg, 0.43 mmol) and 3,4-

Table 3
1H-NMR (CDCl3 ꢀ
/
TMS, d ppm (J Hz)) data of compounds 3ꢀ20
/
a
H
3
3a
5
5a
6
7ꢁ
/
8
9
10
2
5
7
8
9
7.16 s
6.64 s
4.76 m
2.90 m
6.71 s
6.51 s
6.75 s
6.50 s
6.77 s
6.54 s
6.78 s
6.52 s
6.76 s
6.50 s
6.81 s
6.56 s
6.79 s
6.51 s
5.81 d (9.4)
2.83 m
5.87 d (8.7)
2.87 m
5.87 d (9.1)
2.80 m
5.91 d (9.5)
2.90 m
5.87 d (9.2)
2.87 m
4.30
5.93 d (9.1)
2.97 m
4.35 m
5.10 d (9.4)
2.86 m
4.63 dd (8.3; 7.3); 4.24 4.50 dd (9.5; 7.3); 4.28 4.35 m; 4.20 m
4.40 dd (9.6; 6.9); 4.30 m
4.21 t (9.6)
4.33 dd; (9.4; 7.3); 4.23
dd (10.1; 9.4)
7.49 d (1.8)
7.03 d (1.8)
4.82 d (4.8)
dd (10.4; 8.6)
5.36 d (1.8)
6.47 d (1.8)
m
5.43 s
2?
6?
7?
8?
6.70 d (1.8)
6.05 d (1.8)
4.54 d (3.6)
2.87 m
7.02 d (1.8)
6.24 d (1.8)
4.64 d (4.4)
2.95 dd (14.0;
4.4)
7.14 d (1.6)
7.50 d (1.6)
7.09 bs
7.47 bs
7.12 d (1.8)
7.49 d (1.8)
4.86 d (4.8)
6.45 s
4.28 m
2.12 dd (14.8; 5.3)
4.41 d (5.5)
3.28 dd (14.2; 5.5)
4.84 d (4.2)
3.15 dd (14.2; 4.2)
4.81 d (4.7)
3.12 dd (14.5; 4.8)
3.15 dd (14.3; 4.8) 3.10 dd (14.5; 4.8)
CH3O-5?
OAc
3.75 s
6.01 s
3.84 s
2.17 s
3.86 s
2.20 s
3.81 s
4.21 s
2.20 s
4.20 s
2.17 s
4.21 s
2.21 s
4.13 s
2.19 s
2.28 s; 2.22 s;
2.19 s
5.98 s
Oꢀ/CH2 ꢀ
/
O
5.97 s; 5.99 s
5.97 s
5.96 s; 5.98 s
8.12 m; 8.36 m; 7.80
(2H) m
5.94 s; 5.96 s
8.11(7.97) d (8.9);
8.11(7.83) bs;
7.58(7.63) m
2.58 s
5.97 s; 6.00 s
8.13 s; 7.85 s
5.98 d (1.4); 5.96 d (1.4)
8.78 d (1.0); 8.76 d (1.0)
Others
CH3
H
2.53 s
11
12
13
14
15
2
5
7
8
9
6.76 s
6.56 s
6.75 s
6.54 s
6.76 s
6.54 s
6.74 s
6.50 s
6.76 s
6.54 s
5.70 d (4.9)
3.00 m
5.71 d (4.8)
3.00 m
5.87 d (8.0)
2.87 m
5.86 d (7.7)
2.89 m
5.85 d (7.7)
2.80 m
4.20ꢀ/4.50 m
4.43 dd (9.6; 6.9); 4.23 4.35 m; 4.20 m
dd (9.6; 3.3)
4.35 m; 4.20 m
4.36 m; 4.25 m
2?
6?
7?
8?
6.28 bs
6.40 bs
6.33 d (1.8)
6.40 d (1.8)
4.36 d (3.3)
3.25 dd (9.5; 3.7)
3.88 s
6.39 s
6.39 s
6.00 bs
6.75 bs
4.55 d (3.3)
2.90 m
3.87 s
6.05 m
6.74 bs
4.54 d (4.3)
2.80 m
3.83 s
4.20ꢀ/4.50 m
4.59 d (3.7)
2.87 m
3.79 s
3.27 dd (9.5; 3.2)
3.84 s
1.99 s
CH3O-5?/3?
OAc
2.02 s
2.18 s
2.21
2.20 s
Oꢀ
Others
/
CH2 ꢀ
/
O
5.94 bs
5.95 s
5.94 s
5.96 bs
5.97 bs
5.97 bs
4.20ꢀ
/
4.50 m
3.75 m; 2.89 m; 3.60 m; 2.80 m; 1.50ꢀ
/
1.40ꢀ/1.80 m
2.00 m
H
4
16
17
18
19
20
2
5
7
8
9
7.42 s
6.61 s
7.51 s
6.56 s
7.44 s
6.56 s
7.51 s
6.61 s
7.50 s
6.60 s
7.52 s
6.60 s
3.84 m
3.94 m
3.84 m
4.00 m
3.85 m; 4.83 dd 3.83 dd (8.3; 13.5); 4.81 3.84 m; 4.84 dd (8.0;
4.00 m
3.96 m
3.84 m; 489 dt (8.0;
2.1)
5.61 d (1.6)
3.82 dd (8.1; 13.5); 4.82 3.84 m; 4.83 m
dd (8.1; 9.1)
(8.0; 8.4)
7.33 d (1.5)
dd (8.3; 9.3)
7.25 d (1.5)
7.7)
7.30 bs
2?
7.24 d (1.5)
6.43 d (1.8)

906
M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
diaminotoluene (115 mg, 0.94 mmol). Column chroma-
tography of the reaction product with Cl2CH2ꢀEtOAc
85:15 yielded phenazines 7ꢁ8 (66 mg, 30%) and
/
/
catechol 5 (25 mg, 14%). UV lmax(o): 206 (22000), 269
(25600). IR (cmꢃ1): 1779, 1735, 1519, 1505 1229, 1038.
1H-NMR (Table 3). 13C-NMR (Table 4).
4.1.4.3. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(4-methoxy-
7,8-dimethylphenazin-2-yl)-5,5a,6,8,8a,9-hexahydro-
furo[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-one (9).
From 3a (300 mg, 0.54 mmol) and 4,5-dimethyl-1,2-
phenylenediamine (149 mg, 1.1 mmol). Column chro-
matography of the reaction product with Cl2CH2ꢀ
/
EtOAc 85:15 yielded phenazine 9 (60 mg, 21%). [a]D22
ꢁ18.58. UV lmax(o): 207 (28600), 270 (26000). IR
/
(cmꢃ1): 1779, 1735, 1614, 1559, 1505, 1234, 1127,
1038. 1H-NMR (Table 3). 13C-NMR (Table 4).
HRMS (FAB-POSI, Mꢁ1) Calc. for C30H27N2O7
/
527.1818. Found 527.1865. Anal. C30H26N2O7 (C, H,
N).
4.1.4.4. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(8-
methoxyquinoxalin-6-yl)-5,5a,6,8,8a,9-hexahydro-
furo[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-one (10).
From 3a (150 mg, 0.35 mmol) and ethylenediamine (46
mg, 0.76 mmol). Column chromatography of the
acetylated reaction product with Cl2CH2ꢀ
provided quinoxaline 10 (90 mg, 57%) and triacetate 5a
(27 mg, 18%). Analytical data of 10: [a]D22
75.68 (c,
/EtOAc 85:15
ꢃ
/
0.16%). UV lmax(o): 208 (29300), 255 (25300). IR (KBr,
cmꢃ1): 1779, 1734, 1682, 1616, 1504, 1235, 1126, 1035.
1H-NMR (Table 3). 13C-NMR (Table 4). Anal.
C24H20N2O7 (C, H, N).
4.1.4.5. (3aS, 4R, 5R) 5-(8-Methoxyquinoxalin-6-yl)-
3,3a,4,5-tetrahydro-[1,3] dioxolo [4?,5?:6,7] naphtho
[1,2-c]isoxazol-4-carboxylic acid methyl ester (16) and
(3aS, 4R, 5R) 5-(3,4-diacetyl-5-methoxyphenyl))-
3,3a,4,5-tetrahydro-[1,3]dioxolo[4?,5?:6,7]naphtho[1,2-
c]isoxazol-4-carboxylic acid methyl ester (17). From
quinone 4 (150 mg, 0.36 mmol) and ethylenediamine
(0.05 mL, 0.75 mmol). The reaction time was reduced to
2 h, the reaction product was acetylated with acetic
anhydride in pyridine, and the resulting acetylated
product was chromatographed on silica gel to give
quinoxaline 16 (42 mg, 27%) and diacetate 17 (48 mg,
31%).
Compound 16: m.p.: 120ꢀ
/
125 8C. [a]2D2
ꢃ121.78 (c,
/
0.23%). UV lmax(o): 214 (24700), 223 (25500), 256
(14100), 315 (12200). IR (cmꢃ1): 1735, 1615, 1573,
1
1500, 1236, 1127, 1037. H-NMR (Table 3). 13C-NMR
(Table 4). HRMS (FAB-POSI, Mꢁ1) Calc. for
/
C23H20N3O6 434.4352. Found 434.1323. Anal.
C23H19N3O6 (C, H, N).
Compound 17: m.p.: 224ꢀ
/
228 8C. [a]2D2
0.54%). UV lmax(o): 210 (18600), 217 (18500), 278
ꢃ117.08 (c,
/

Table 4
13C-NMR (CDCl3 ꢀ
/
TMS, d ppm) data of compounds 3ꢀ20
/
a
C
3
3a
4
5
5a
6
7ꢁ
/
8
9
10
1
129.5
107.4
148.6
148.1
109.8
136.0
72.2
128.4
107.1
148.4
148.0
109.6
128.4
72.7
119.3
104.5
148.5
150.9
108.6
130.6
155.0
44.0
128.3
106.7
147.6
146.5
109.8
131.4 b
73.7
128.3
107.0
148.2
147.8
109.8
131.5
73.6
128.7
106.9
148.3
148.0
109.7
131.1
73.4
128.6
128.6
106.9
148.2
147.9
109.7
131.3
73.4
129.5
106.9
148.2
147.9
109.6
131.2
73.3
2
106.8
3
148.2
4
147.8
5
109.6
6
131.1
7
73.3
8
41.6
39.1
38.6
38.4
38.6
38.5
38.6
38.5
9
72.2
71.6
74.6
71.4
71.5
71.4
71.3
71.4
71.5
1?
152.3
124.9
179.0
176.1
158.2
114.1
44.6
151.8
124.4
179.4
175.2
157.1
112.7
44.3
155.1
109.0
177.9
175.0
152.7
123.1
48.3
131.7 b
110.8
132.4
143.2
148.2
106.8
43.5
142.7
116.7
137.8
131.0
151.8
112.7
43.3
143.9
122.7
143.4
136.2
154.3
110.9
44.6
143.1(142.8)
122.6
142.9
122.6
142.7
135.6
154.3
110.4
44.5
143.1
122.5
143.0
134.4
154.4
112.1
44.2
2?
3?
143.3(142.3)
136.0
4?
5?
154.2
6?
110.8
7?
44.5
8?
45.3
44.9
48.5
45.5
45.5
45.3
45.2
45.3
45.2
9?
175.1
56.0
173.4
56.1
170.8
56.0
174.0
56.3
173.5
56.3
173.7
56.6
173.3
173.8
56.5
173.6
56.4
CH3O-5?
OAc
56.5
20.9, 171.5
21.2, 171.7
20.3, 20.6, 21.1, 167.72,11.06,8.107,1.5
171.4
21.0, 171.4
21.1, 171.5
21.0, 171.5
COOCH3
52.6
Oꢀ/CH2 ꢀ/O
102.3
101.8
102.0
101.6
101.6
101.6
101.5
101.6
101.6
Others
128.9, 130.1, 130.2, 143.5(143.6),
130.9, 142.3, 143.4 126.9(128.3),
140.8(128.3),
127.2, 128.6, 142.4, 145.3, 143.5
141.6, 142.9(2C),
20.7, 20.6
129.6(141.0),
133.1(133.9),
141.7(142.1)
C
11
12
13
14
15
16
17
18
19
20
1
126.1
104.3
148.4
147.2
110.0
131.5
72.3
126.1
102.4
148.4
147.5
109.8
131.0
72.1
128.2
106.9
148.0
147.5
109.6
132.4
73.6
128.2
106.7
148.0
147.5
109.9
132.2
73.5
128.2
106.7
148.0
147.5
109.8
131.5
73.7
119.3
104.3
147.9
150.6
109.1 b
133.8
156.0
43.4
118.9
103.9
147.8
150.5
109.3
134.0
156.1
43.2
119.2
104.2
147.9
150.6
107.9
133.7
156.0
43.4
119.3
104.3
147.9
150.6
109.1
134.0
156.1
43.5
119.3
104.3
148.0
150.7
109.0
133.5
155.9
43.5
2
3
4
5
6
7
8
39.5
39.5
38.6
38.5
38.5
9
70.7
70.6
71.3
71.3
71.3
74.4
74.4
74.3
74.3
74.4
1?
2?
3?
4?
5?
131.9
109.9
135.5
143.8
148.9
131.3
108.4
137.9
143.5
149.0
133.9
107.7
146.4
130.3
146.4
132.2
107.8
133.2
141.3
148.2
132.6
107.1
130.7
142.0
148.0
142.3
121.8
143.4
134.4
155.1
137.8
116.0
143.1
131.2
152.1
142.3 b
122.1
142.7 b
135.9
154.9
141.8 b
122.1
141.8 b
135.4
154.9
140.7
121.9
142.2
136.2
154.9

908
M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
(8100), 314 (5300). IR (cmꢃ1): 1774, 1735, 1610, 1504,
1269, 1131, 1037. 1H-NMR (Table 3). 13C-NMR (Table
4). HRMS (FAB-POSI, Mꢁ
/
1) Calc. for C25H24NO10
498.1400. Found 498.1421.
4.1.4.6. (3aS, 4R, 5R) 5-(4-Methoxyphenazin-2-yl)-
3,3a,4,5-tetrahydro-[1,3] dioxolo [4?,5?:6,7]
naphtho[1,2-c]isoxazol-4-carboxylic acid methyl ester
(18). From 4 (113 mg, 0.28 mmol) and phenylenedia-
mine (61 mg, 0.56 mmol) for 1 h. The reaction product
was dissolved in methanol and the precipitated product
was filtered yielding 18 (78 mg, 59%). M.p.: 124ꢀ128 8C.
/
[a]2D2
ꢃ167.68 (c, 0.38%). UV lmax(o): 212 (28700), 270
/
(28100), 314 (7100), 365 (3800). IR (cmꢃ1): 1735, 1610,
1520, 1504, 1257, 1129, 1038. H-NMR (Table 3). 13C-
1
NMR (Table 4). HRMS (FAB-POSI, Mꢁ1) Calc. for
/
C27H22N3O6 484.1508. Found 484.1492. Anal.
C27H21N3O6 (C, H, N).
4.1.4.7. (3aS,
4R,
5R)
dimethylphenazin-2-yl)-3,3a,4,5-tetrahydro-[1,3]
5-(4-Methoxy-7,8-
dioxolo [4?,5?:6,7] naphtho[1,2-c]isoxazol-4-carboxylic
acid methyl ester (19). From 4 (122 mg, 0.30 mmol) and
4,5-dimethyl-1,2-phenylenediamine (81 mg, 0.60 mmol)
for 1 h. Column chromatography of the reaction
product provided compound 19 (71 mg, 47%). M.p.:
165ꢀ170 8C. UV lmax(o): 265 (20300), 214 (11100), 376
/
(14000). IR (cmꢃ1): 1736, 1610, 1504, 1234, 1127, 1038.
1H-NMR (Table 3). 13C-NMR (Table 4). HRMS (FAB-
POSI, Mꢁ1) Calc. for C29H26N3O6 512.1821. Found
/
512.1852. Anal. C29H25N3O6 (C, H, N).
4.1.4.8. (3aS, 4R, 5R) 5-(7,8-Dichloro-4-methoxy-
dioxolo
phenazin-2-yl)-3,3a,4,5-tetrahydro-[1,3]
[4?,5?:6,7] naphtho[1,2-c]isoxazol-4-carboxylic acid
methyl ester (20). From 4 (121 mg, 0.29 mmol) and
4,5-dichloro-1,2-phenylenediamine (105 mg, 0.59 mmol)
during 4 h. The reaction product was crystallized in
methanol to yield 20 (132 mg, 81%). M.p.: 192ꢀ196 8C.
/
UV lmax(o): 223 (26800), 270 (28800), 314 (9200), 380
(1200). IR (cmꢃ1): 1737, 1625, 1504, 1236, 1127, 1038.
1H-NMR (Table 3). 13C-NMR (Table 4). HRMS (FAB-
POSI, Mꢁ
/
) Calc. for C27H19Cl2N3O6 552.0729. Found
552.0745.
4.1.5. Condensation of 5 with dihalogenated compounds.
Compounds 11 and 12
4.1.5.1. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(7-
methoxybenzo[1,3]dioxol-5-yl)-5,5a,6,8,8a,9-
hexahydro-furo[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-
one (12). A mixture of 5 (100 mg, 0.23 mmol),
dibromomethane (101 mg, 0.58 mmol), K2CO3 (81
mg) and sodium iodide (2 mg) in acetone (12 mL) was
refluxed for 24 h. After cooling, water was added and
the product extracted with EtOAc. The organic layer

M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ
/
911
909
was washed with brine, dried and evaporated. Column
chromatography on silica gel (eluent: CH2Cl2ꢀEtOAc
95:5) of the residue produced compound 12 (21 mg,
Et3N, eluent: C6H6ꢀEt2O 6:4) and compound 14 (124
/
/
mg, 54%) was obtained. UV lmax(o): 207 (39900), 290
(3700), 325 (800). IR (cmꢃ1): 1779, 1733, 1597, 1506,
1
21%). M.p.: 208ꢀ
/
210 8C (MeOH). UV lmax(o): 207
1237, 1125, 1038, 866, 735. H-NMR (Table 3). 13C-
NMR (Table 4).
(36100), 290 (5800), 325 (2000). IR (cmꢃ1): 1773,
1735, 1634, 1505, 1236, 1127, 1040. 1H-NMR (Table
3). 13C-NMR (Table 4).
4.1.7.2. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(4-methoxy-
5a(9a)-morpholin-4-yl-5a,6,7,8,9,9a-hexahydro-
dibenzo[1,4]dioxin-2-yl)-5,5a,6,8,8a,9-
4.1.5.2. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(8-methoxy-
2,3-dihydrobenzo[1,4]dioxin-6-yl)-5,5a,6,8,8a,9-
hexahydrofuro[3?,4?:6,7]naphtho[2,3-d][1,3] dioxol-6-
one (15). The same procedure was applied to 5 (127
mg, 0.30 mmol) together with 4-(1-cyclohexenyl)-mor-
pholine (101 mg, 0.60 mmol). The reaction mixture was
kept 3 h at 0 8C and 21 h at r.t. Chromatography of the
hexahydrofuro[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-
one (11). Following the same procedure, compound 11
(68%) was obtained from 5 (100 mg, 0.23 mmol) and
1,2-dibromoethane (109 mg, 0.58 mmol). M.p.: 240ꢀ
/
242 8C (MeOH). [a]D22
ꢁ
/
38.58 (c, 0.87%). UV lmax(o):
reaction product (eluent: hexeneꢀEtOAc 7:3) provided
/
210 (29200), 283 (1900), 326 (800). IR (cmꢃ1): 1771,
1735, 1596, 1506, 1236, 1127, 1037. 1H-NMR (Table 3).
compound 15 (64 mg, 36%). UV lmax(o): 210 (43900),
289 (4100), 326 (1000). IR (cmꢃ1): 1779, 1734, 1597,
13C-NMR (Table 4). HRMS (FAB-POSI, Mꢁ
/1) Calc.
1
1507, 1236, 1115, 1036, 867, 735. H-NMR (Table 3).
for C24H23O9 455.1342. Found 455.1363. Anal.
C24H22O9 (C, H).
13C-NMR (Table 4). HRMS (FAB-POSI, Mꢁ
1) Calc.
for C32H35NO10 593.2261. Found 593.2196. Anal.
C32H35O10N (C, H, N).
/
4.1.6. Reaction of quinone 3a with diols. 7-Acetyl-4?-
demethyl podophyllotoxin (13)
4.2. Bioactivity
Quinone 3a (150 mg, 0.35 mmol) was dissolved in
methanol (10 mL). Then ethylene glycol (26 mg, 0.42
mmol), trimethylorthoformate (107 mg, 1.0 mmol) and
4.2.1. Antineoplastic assay
A screening procedure [19] was used to assess the
cytotoxic activity against the following cell lines: P-388
(lymphoid neoplasms from DBA/2 mouse), A-549 (hu-
man lung carcinoma), HT-29 (human colon carcinoma)
and MEL-28 (human melanoma). Cells were seeded into
16 mm wells (multidishes NUNC 42001) at concentra-
(ꢃ) camphorsulfonic acid (10 mg, 0.05 mmol) were
/
successively added. The mixture was refluxed under
argon atmosphere for 21 h. The reaction mixture was
neutralized with triethylamine (5 mg, 0.05 mmol),
diluted with water and extracted with EtOAc. The
organic layer was washed with brine, dried and evapo-
rated providing a reaction product that was chromato-
tions of 1ꢂ
/
104 (P-388) or 2ꢂ104 (A-549, HT-29 and
/
MEL-28) cells/well, respectively, in 1-mL aliquots of
MEM supplement with of 10 FCS medium containing
the compound to be evaluated at the concentrations
tested. In each case, a set of control wells was incubated
in the absence of sample and counted daily to ensure the
exponential growth of cells. After 3 days at 37 8C, in
10% CO2, and 98% humidity, the P-388 cells were
observed through an inverted microscopy and the degree
of inhibition was determined by comparison with the
controls, whereas the A-549, HT-29 and MEL-28 cells
were stained with crystal violet before examination.
graphed on silica gel (eluent: CH2Cl2ꢀEtOAc 9:1) to
/
give derivative 13 (70 mg, 45%). IR (cmꢃ1): 3440, 1777,
1733, 1612, 1516, 1507, 1236, 1115, 1036. 1H-NMR
(Table 3). 13C-NMR (Table 4).
Column chromatography (eluent: CH2Cl2ꢀEtOAc
/
9:1) of the reaction product obtained in the same way
from 3a (151 mg, 0.35 mmol) and 1,2-cyclohexanediol
(53 mg, 0.45 mmol), provided 13 (64 mg, 42%).
4.1.7. Condensation of 5 with enamines. Compounds 14
and 15
4.2.2. Antiviral assays
4.1.7.1. (5R, 5aR, 8aS, 9R) 9-Acetoxy-5-(4-methoxy-
5a(9a)-pyrrolidin-1-yl-5a,6,7,8,9,9a-hexahydro-
dibenzo[1,4]dioxin-2-yl)-5,5a,6,8,8a,9-
4.2.2.1. Cell culture and virus. The cell line used was:
Cercopithecus aethiops African green monkey kidney
cells (VERO cell line ATCC CCL-81). Cells were grown
in MEM supplemented with 10% FBS, 100 units mLꢃ1
hexahydrofuro[3?,4?:6,7]naphtho[2,3-d][1,3]dioxol-6-
one (14). Quinone 5 (175 mg, 041 mmol) was dissolved
in chloroform (15 mL) under inert atmosphere at 0 8C.
Then, a solution of 1-(1-cyclohexenyl)pyrrolidine (93
mg, 0.62 mmol) in chloroform (8 mL) was added
dropwise and the mixture was stirred for 3 h at 0 8C.
The residue obtained after evaporation of the solvent
was chromatographed on silica gel (neutralized with 1%
of penicillin, 100 mg mLꢃ1 of streptomycin, 2 mM
L-
glutamine, 0.07% NaHCO3, 1% non-essential amino
acids and vitamin solution. The cultures were main-
tained at 37 8C in humidified 5% CO2.
HSV-2 was obtained from the Center for Disease
Control (Atlanta, GA). The virus stock was prepared

910
M.A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 899ꢀ911
/
from HSV-2-infected VERO cell cultures. The infected
cultures were subjected to three cycles of freezingꢀ
thawing, and centrifuged at 2000 rpm for 10 min. The
supernatant was collected, titrated, and stored at ꢃ
170 8C in 1-mL aliquots. To titrate the virus suspension,
confluent monolayer VERO cells were grown in 96-well
flat-bottomed plates, infected with 0.1 mL of serial
tenfold dilutions of the virus suspension in quadrupli-
cate and incubated for 48 h.
Acknowledgements
/
Financial support for this work came from Spanish
DGICYT (PPQ2000-1111) and Junta de Castilla y Leo´n
(SA-49/01). This work was carried out under the
auspices of the ?Programa CYTED (Programa Iberoa-
mericano de Ciencia y Tecnolog´ıa para el Desarrollo),
subprograma X?.
/
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