Novel C-6 fluorinated acyclic side chain pyrimidine derivatives: Synthesis, 1H and 13C NMR conformational studies, and antiviral and cytostatic evaluations

Article abstract of DOI:10.1021/jm0614329

The synthetic route for introduction of a fluoroalkyl (7-12,14), fluoroalkenyl (15 and 16), fluorophenylalkyl (17, 19, 20, and 22), and fluorophenylalkenyl (18, 21) side chain at C-6 of the pyrimidine involved the lithiation of the pyrimidine derivatives

Full text of DOI:10.1021/jm0614329

J. Med. Chem. 2007, 50, 3037-3045
3037
1
13
Novel C-6 Fluorinated Acyclic Side Chain Pyrimidine Derivatives: Synthesis, H and C NMR
Conformational Studies, and Antiviral and Cytostatic Evaluations
†
‡
‡
§
§
§
|
Svjetlana Prekupec, Damjan Makuc, Janez Plavec, Lidija Sˇ uman, Marijeta Kralj, Kre sˇ imir Paveli c´ , Jan Balzarini,
|
†
,†
Erik De Clercq, Mladen Mintas, and Silvana Rai c´ -Mali c´ *
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, UniVersity of Zagreb, Maruli c´ eV trg 20, P.O. Box 177,
HR-10000 Zagreb, Croatia, SloVenian NMR Centre, National Institute of Chemistry, HajdrihoVa 19, P.O. Box 660, SL-1001 Ljubljana,
SloVenia, DiVision of Molecular Medicine, Rudjer Bo sˇ koVi c´ Institute, Bijeni cˇ ka 54, P.O. Box 1016, HR-10001 Zagreb, Croatia, and Rega
Institute for Medical Research, Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, B-3000 LeuVen, Belgium
ReceiVed December 15, 2006
The synthetic route for introduction of a fluoroalkyl (7-12, 14), fluoroalkenyl (15 and 16), fluorophenylalkyl
(17, 19, 20, and 22), and fluorophenylalkenyl (18, 21) side chain at C-6 of the pyrimidine involved the
lithiation of the pyrimidine derivatives 3 and 3a and subsequent nucleophilic addition or substitution reactions
of the organolithium intermediate thus obtained with various electrophiles. Conformational properties of
the novel fluorinated pyrimidine derivatives were assessed by the use of 1D difference NOE enhancements
and C-F coupling constants. Compounds 4-22 were evaluated for their antiviral and cytostatic activities.
Of all compounds evaluated, the 5-bromopyrimidine derivatives 5 and 6 showed the highest inhibitory
activities. Among the series of fluoroalkylated pyrimidines, which is generally more active than the series
of fluorophenylalkylated pyrimidines, compounds 8 and 14 displayed moderate cytostatic activities against
the tested tumor cell lines. Moreover, compound 8 containing a 2-fluoromethylpropyl side chain expressed
some but not highly specific activity against varicella-zoster virus (VZV). From C-6 fluorophenylalkylated
pyrimidine derivatives, 17a and 21 showed a slight activity against cytomegalovirus (CMV), VZV, and
Coxsackie B4 virus, respectively. Besides, compounds 17a and 21 showed no cytotoxic effect.
Introduction
Pyrimidines are biologically important molecules and valuable
primary tumors, and monitoring the efficacy of chemo- or
radiotherapy response.
1
9-21
Recently, we have reported that, among fluorinated propyl
or propenyl acyclic pyrimidine derivatives, compounds contain-
ing the 2-hydroxy-3,3,3-trifluoro-1-propenyl side chain exhibited
a pronounced effect against breast carcinoma (MCF-7), while
the compound with a 2-fluoromethyl-2-acetoxypropyl chain
1
,2
heterocyclic nuclei for the design of pharmaceutical agents.
A great number of C-5 and C-6 substituted pyrimidine nucleo-
sides have been prepared in view of their various biological
3
activities. Uracil derivatives substituted either at the C-5 or
the C-6 position and their nucleosides have considerable
22
exhibited a moderate effect against cervical carcinoma (HeLa).
4
importance in the field of antiviral chemotherapy. Furthermore,
18
We have also reported that F-radiolabeled pyrimidine-based
6
-substituted pyrimidines, as for example 1-[(2-hydroxyethyl)-
acyclic nucleosides in which the acyclic sugar moiety is attached
in the 6-position rather that at N-1 of the pyrimidine ring can
be suitable candidates for the development of nontoxic PET-
tracer molecules that are specifically and efficiently phospho-
5-8
methyl]-6-(phenylthio)thymine (HEPT)
and 3,4-dihydro-2-
9
alkoxy-6-benzyl-4-oxopyrimidines (DABOs), show a potent
and selective activity against human immunodeficiency virus
type-1 (HIV-1). The excellent biological activities exhibited by
these 6-substituted uracil derivatives provide a new emphasis
to explore the chemistry and biological activities of these
pyrimidine derivatives.10 A large number of nucleosides such
as thymidine analogues and acyclic guanosine derivatives show
antiviral activities against herpes viruses. The antiviral activity
of these compounds is due to their selective and efficient
activation through monophosphorylation by the viral enzyme
in the intact cells.1 Therefore, radiolabeling of these antiviral
agents, such as ganciclovir and penciclovir, with the positron-
rylated by the herpes simplex virus type 1 thymidine kinase
14,15
(HSV-1 TK).
The pronounced biological activities exhibited
by this class of compounds led us to synthesize new types of
C-6 fluoroalkylated (7-12 and 14-16) and fluorophenylalky-
lated (17-22) pyrimidine derivatives (Figure 1) as model
compounds for development of tracer molecules in positron-
emission tomography (PET).
11
2,13
Chemistry
Synthesis. 2,4-Dimethoxy-6-methylpyrimidine (3) as a key
1
8
emitting isotope F allows noninvasive imaging of the viral
precursor was prepared according to the procedure described
thymidine kinase enzyme activity by means of positron-emission
21,23
in the literature.
Radical reaction of 3 with N-bromosuc-
tomography (PET).1
4-17
Besides, the PET technique has proven
cinimide in acetic acid gave mono-, di-, and tribrominated
pyrimidine derivatives 4-6. The new 6-acyclic chain (7-12
and 14-16, Scheme 1) and C-6 fluorophenylalkyl chain (17-
to be vital in early detection of cancer. This is critical to
successful treatment and long-term cure of cancer, staging of
2
2, Scheme 2) substituted pyrimidine derivatives were synthe-
sized by lithiation, which has been studied extensively as an
*
Corresponding author. Tel: +38 51 4597 213. Fax: +38 51 4597
14,24
2
24. E-mail: Silvana.Raic@fkit.hr.
important carbon-carbon bond-forming reaction.
Lithiation
†
University of Zagreb.
National Institute of Chemistry.
Rudjer Bo sˇ kovi c´ Institute.
Katholieke Universiteit Leuven.
of the 6-methyl group of the pyrimidine (3) and subsequent
addition of the lithiated derivative to fluoroacetone and 1,1,1-
trifluoroacetone afforded 2-fluoromethyl-2-hydroxypropyl (7)
‡
§
|
1
0.1021/jm0614329 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/01/2007

3
038 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Prekupec et al.
shift ranges for uracil (δ 5.26-5.61) and its dimethoxy
1
9
derivatives (δ 6.21-6.61, Table 1). F NMR resonances were
1
13
well resolved (Supporting Information). H decoupled C NMR
spectra showed C-F coupling constants that enabled straight-
forward identification of fluorinated carbon atoms and their
neighbors.
Conformational studies of 8 and 19 were carried out as a
1
function of temperature. H NMR spectra were collected at -90,
-
50, and 20 °C and showed no significant temperature changes.
Conformational properties of 11, 12, 18, and 19 were assessed
with the use of 1D difference NOE enhancements. The
saturation of H-1′a (δ 2.82) and H-1′b (δ 3.00) protons in 11
resulted in different NOE enhancements at H-5 (Figure 2).
Furthermore, the saturation of H-1′a also showed higher NOE
enhancement at the C2′-Me group than saturation of H-1′b.
These NOE enhancements suggest a predominant conformation
in which H-1′a is spatially closer to the C2′-Me group and H-1′b
is closer to the H-5 proton. Restricted rotation across the C6-
C1′ bond is also confirmed by the fact that H-1′ protons are
not isochronous. This conformer is probably stabilized by the
Figure 1. The new C-6 fluoroalkylated (7-12 and 14-16) and
fluorophenylalkylated (17-22) pyrimidine derivatives.
22
N1···HO hydrogen bond (Figure 2). The saturation of H-1′a
(δ 2.64) and H-1′b (δ 2.83) in 12 resulted in NOE enhancements
of 5.4% and 4.1% at the H-5 proton, respectively. H-1′a also
showed stronger NOE of 1.4% at the C2′-Me group, while there
was no NOE between H-1′b and C2′-Me. In 12, H-1′a is
therefore spatially closer to both H-5 and C2′-Me protons in
comparison to H-1′b. NOE enhancement of 8.8% at H-1′ was
observed upon saturation of H-5 in 18. Furthermore, the
saturation of the H-1′ proton showed 8.2% NOE enhancement
at H-5 protons. These strong NOE enhancements clearly indicate
the trans geometry along the C1′dC2′ double bond. 18 adopts
a conformation where H-5 and H-1′ protons are spatially close.
The hydroxyl proton of 15 was found at δ 13.01 ppm, which
offers a clear indication of a relatively strong hydrogen bond.
As a result, C2′ is strongly deshielded (δ 171.40). Immediately
after 15 was dissolved in DMSO-d6, the predominant set of
signals (>96%) was observed (Table 1 and Supporting Informa-
tion). After ca. 1 day the second form (∼30%) with sharp NMR
signals appeared, which has been assigned to dihydroxy form
and 2-trifluoromethyl-2-hydroxypropyl (11) pyrimidine deriva-
tives. Nucleophilic substitution reaction of the organolithium
intermediate with methyl trifluoroacetate and ethyl pentafluoro-
propionate gave 2,4-dimethoxypyrimidine derivatives containing
a fluoro-substituted 2-hydroxy-1-propenyl side chain at C-6 (15
and 16, Scheme 1). Treatment of 7 and 11 with acetyl chloride
effected a conversion to the 2,4-dimethoxypyrimidine with an
acetylated side chain (8), pyrimidine derivatives containing free
2
,4-diketo and hydroxyl functionalities (9 and 12), and a 2,4-
dihydroxypyrimidine derivative with an acetylated hydroxyl
group (10). The Stille reaction,2
5-27
which is another efficient
carbon-carbon bond-forming reaction, was applied for the
introduction of the phenyl ring at C-5 of the pyrimidine moiety
in 13 using tributylphenylstannane and dichlorobis(triphenyl-
phosphine)palladium(II) as a catalyst. Subsequent nucleophilic
addition of lithiated 13 to 1,1,1-trifluoroacetone afforded the
pyrimidine derivative 14 with a trifluoroalkylated C-6 side chain
1
5H (Figure 3, Supporting Information).
(Scheme 1).
A fluorophenylalkyl side chain at C-6 of the pyrimidine
Biological Results
moiety was introduced by lithiation and subsequent nucleophilic
addition of pyrimidine derivative 3 and 3a to 4-fluoroacetophe-
none, 2-fluorophenylacetone, and 4,4′-difluorbenzophenone to
give 17, 17a, 19, and 22, respectively (Scheme 2). Reaction of
lithiatied intermediate 3 with ethyl 4-fluorobenzoate afforded
the C-6 substituted pyrimidine derivative containing unsaturated
side chain, which exists as both the keto (21K) and enol (21E)
tautomer (Scheme 2, Supporting Information). In the reaction
with acetyl chloride, compound 17 yielded unsaturated 2,4-
dimethoxypyrimidine 18 as a product of the elimination reaction,
while compound 19 gave acetylated pyrimidine-2,4-dione
derivative 20 (Scheme 2).
Antiviral Activities. The compounds 4-22 were evaluated
for their inhibitory activities against cytomegalovirus (CMVa
AD-169 and Davis strain) in human embryonic lung (HEL)
+
cells; varicella-zoster virus (TK VZV, thymidine kinase positive
-
and TK VZV, thymidine kinase deficient strains), parainfluenza
virus-3, reovirus-1, Sindbis, Punta Toro virus in Vero cell
cultures; and Coxsackie B4 virus in HeLa cell cultures (Tables
2
and 3). Their activities were compared with those of acyclovir,
ganciclovir, cidofovir, brivudin, (S)-DHPA, and ribavirin (Tables
2
and 3).
5
-Bromo-6-dibromomethyl-substituted pyrimidine derivative
+
6
)
showed moderate inhibitory potential against TK VZV (EC50
8.8 µM) and TK VZV (EC50 ) 10.5 µM). Besides,
compound 8 containing a 2-fluoromethylpropyl side chain
showed a slight but not highly specific activity against TK -
VZV (EC50 ) 8.1 µM) and TK VZV (EC50 ) 7.4 µM).
Deprotection of the 2,4-diketo and hydroxyl functionalities of
NMR Assignment and Conformational Studies. The chemi-
-
1
19
13
cal identity of 4-22 has been confirmed by H, F, and
C
NMR measurements. Respective chemical shifts are reported
in Table 1 and the Supporting Information. The analysis of the
+
-
1
H NMR spectra of all compounds was relatively simple because
they contain a small number of signals. H-1′ chemical shifts in
8
caused a loss of activity for 7, 9, and 10. The compound
7, 8, 10, 11, 12, 14, 17, 17a, 19, 20, 21K, and 22 were observed
containing the C-6 fluorophenylalkyl chain in C-5 methyl-
from 2.59 to 4.40 ppm, which correspond to C1′-C2′ single-
bond derivatives. In contrast, analogs with a C1′dC2′ double
bond (15, 16, 18, and 21E) exhibit H-1′ chemical shifts between
a
+
Abbreviations: TK VZV, varicella-zoster virus (thymidine kinase
-
positive); TK VZV, varicella-zoster virus (thymidine kinase deficient
strains); CMV, cytomegalovirus.
6
.09 and 6.59 ppm. H-5 resonances exhibit distinct chemical

C-6 Fluorinated Acyclic Side Chain Pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3039
Scheme 1a
a
Reagents and conditions: (i) LDA, THF, fluoroacetone; (ii) acetyl chloride, H2O; (iii) NBS, acetic acid; (iv) LDA, THF, 1,1,1-trifluoroacetone; (v)
Bu3SnPh, (PPh3)2PdCl2, THF; (vi) LDA, THF, methyl trifluoroacetate; (vii) LDA, THF, ethyl pentafluoropropionate.
Scheme 2a
a
Reagents and conditions: (i) LDA, THF, 4-fluoroacetophenone; (ii) acetyl chloride, H2O; (iii) LDA, THF, 2-fluorophenylacetone; (iv) LDA, THF, ethyl
4
-fluorobenzoate; (v) LDA, THF, 4,4′-difluorobenzophenone.
substituted pyrimidine 17a had marginal activity against the
CMV AD-169 strain (EC50 ) 45 µM), CMV Davis strain (EC50
congener 17 was virtually devoid of antiviral activity. Moreover,
C-6 fluorophenylalkenyl pyrimidine 21 displayed moderate
activity against Coxsackie B4 virus (EC50 ) 24 µM) and CMV
Davis strain (EC50 ) 38 µM).
+
-
)
20 µM), TK VZV (EC50 ) 80 µM), and TK VZV (EC50 )
4
5 µM). On the other hand, its C-5 unsubstituted pyrimidine

3
040 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Prekupec et al.
Table 1. 1H NMR Chemical Shifts (δ/ppm) and H-H/H-F Coupling Constants (J/Hz)
compd
-CH3
H-1
-OCH3
H-5
Ph
OH
4
-
-
-
2.51 (s)
4.55 (s)
7.27 (s)
2.72 (s)
3.97 (s)
-
-
-
-
-
-
-
-
-
-
-
-
-
3
.99 (s)
3.89 (s)
.99 (s)
3.97 (s)
.00 (s)
5
-
3
6
-
4
7a
8b
9c
1.08 (d)
3.86 (s)
3.87 (s)
3.96 (s)
3.97 (s)
6.45 (s)
6.35 (s)
5.28 (s)
5.31 (s)
6.47 (s)
5.61 (s)
-
5.05 (s)
4
JHF ) 2.0
4
2
1.50 (d)
3.15 (dd) JHF ) 1.6, J ) 13.4
-
4
4
2
JHF ) 2.3
3.18 (dd) JHF ) 1.4, J ) 13.4
1.04 (d)
(covered with H2O)
-
-
4
JHF ) 1.3
0d
1.43 (d)
2.88 (d) JHF ) 13.8
2
-
-
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
4
2
JHF ) 2.2
2.74 (d) J HF) 13.9
2
1
1.29 (s)
1.37 (s)
-
2.82 (d) J ) 13.6
3.97 (s)
3.98 (s)
-
-
2
3
.00 (d) J ) 13.6
2
2
2.64 (d) J ) 14.0
2
2.14 (s)
-
2
.83 (d) J ) 14.0
3
3.80 (s)
.91 (s)
7.55-7.63 (m)
-
3
2
4
1.28 (s)
-
2.70 (d) J ) 15.5
3.94 (s)
4.03 (s)
3.95 (s)
-
7.17-7.44 (m)
-
2
2.93 (d) J ) 15.5
5
6.09 (s)
2.99 (s)
6.47 (s)
6.52 (s)
6.61 (s)
6.32 (s)
6.21 (s)
-
-
-
-
13.01 (s)
4
.00 (s)
3.88 (s)
.89 (s)
4.01 (s)
.07 (s)
5He
6
-
7.25 (s)
3
-
-
4
2
7
1.53 (s)
1.57 (s)
2.58 (d)
3.06 (d) J ) 14.0
3.89 (s)
3.90 (s)
3.91 (s)
3.94 (s)
3.96 (s)
4.01 (s)
3.95 (s)
3.96 (s)
H3: 6.98 (m)
H2: 7.44 (m)
H3: 6.89 (m)
H2: 7.34 (m)
H3: 7.11 (m)
H2: 7.57 (m)
7.06-7.36 (m)
-
2
3
.12 (d) J ) 14.0
7af
8
3.29 (d) J ) 15.3
3
6.59 (m)
2
6.80 (s)
2
.02 (d) J ) 15.3
6.44 (s)
6.42 (s)
5.26 (s)
6.35 (s)
6.56 (s)
6.34 (s)
-
7
JHF ) 1.4
9g
0h
1E
1K
2
1.10 (s)
2.76 (d) J ) 13.7
2
-
2
2
.82 (d) J ) 13.7
2
1.02 (s)
2.95 (d) J ) 14.0
2
-
7.14-7.35 (m)
-
2
.76 (d) J ) 13.9
-
-
-
6.33 (s)
4.40 (s)
3.60 (s)
3.92 (s)
.97 (s)
3.81 (s)
.89 (s)
3.85 (s)
H3: 7.31 (m)
H2: 7.88 (m)
H3: 7.37 (m)
H2: 8.10 (m)
H3: 6.98 (m)
H2: 7.46 (m)
14.31 (s)
3
-
-
3
3.88 (s)
a
2
2
b
2
Additional signals: 4.21 (CH2F, d, JHF ) 47.8 Hz), 4.22 (CH2F, d, JHF ) 47.8 Hz). Additional signals: 1.98 (COCH3, s), 4.63 (CH2F, d, JHF ) 47.3
Hz). c Additional signals: 4.09 (CH2F, d, JHF )47.6 Hz), 4.10 (CH2F, d, JHF ) 47.7 Hz). Additional signals: 2.00 (COCH3, s), 4.56 (CH2F, dd, JHF )
2
2
d
2
2
2
2
e
3
4.8 Hz, J ) 9.9 Hz), 4.72 (CH2F, dd, JHF ) 35.3 Hz, J ) 9.9 Hz), 10.75 and 11.04 (NH). Initial ratio between 15/15H was 25:1. It dropped to 7:3 after
f g 4 2
1
day and to 1:1 after 4 days. Additional signal: 1.97 (CH3-5, s) Additional signals: 2.85 (H-3′, dd, JHF ) 1.3 Hz, J ) 13.7 Hz), 2.91 (H-3′, dd,
2 h 2 2
4
JHF ) 1.3 Hz, J ) 13.7 Hz). Additional signals: 1.91 (COCH3, s), 3.17 (H-3′, d, J ) 13.9 Hz), 3.24 (H-3′, d, J ) 14.1 Hz); 10.73 and 11.02 (NH).
Figure 3. Hydration equilibrium of 15. The predominant set of signals
that corresponded to 15 (structure on the left) was observed immediately
after it was dissolved in DMSO-d
the second form appeared, which has been assigned to dihydroxy form
5H. After 4 days the ratio of 15:15H was 1:1.
6
. After ca. 1 day at room temperature,
1
compounds 4-6 (CC50 ) 1.7-38.7 µM) increased their
cytotoxicity. Replacement of 5-bromine in 4 (CC50 ) 18 µM)
with 5-phenyl moiety in 13 and 14 resulted in somewhat reduced
cytotoxicity (CC50 ∼ 35 µM). Comparison of cytotoxicity and
antiviral potency of targeted fluoroalkylated and fluoropheny-
lalkylated derivatives showed that fluorophenylalkylated deriva-
tives 17a and 21 possessed moderate antiviral activity and no
cytotoxic effect (CC50 > 100 µM).
Figure 2. The predominant conformation of 11 in solution as
established by 1D NOE experiments. The key NOE enhancements are
shown. The proposed conformation is probably stabilized through the
formation of the N1···HO hydrogen bond.
Results of cytotoxicities showed that introduction of bromine
at the C-5 and/or C-6 position of pyrimidine in parent

C-6 Fluorinated Acyclic Side Chain Pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3041
Table 2. Antiviral Activity of Compounds 4-22 against CMV and
laryngeal carcinoma (Hep-2), cervical carcinoma (HeLa), pan-
creatic carcinoma (MiaPaCa-2), colon carcinoma (SW 620),
breast carcinoma (MCF-7), murine leukemia (L1210), and
human T-lymphocytes (Molt4/C8 and CEM) (Table 4). The
VZV in Human Embryonic Lung (HEL) Cells
antiviral activity EC50 (µM)a
CMV
cytotoxicity (µM)
6
-dibromomethyl-substituted pyrimidine derivative 6 showed
cell
cell
AD-169 Davis TK+ VZV TK- VZV morphology growth
pronounced cytotoxic activity, particularly against colon car-
cinoma (SW 620; IC50 ) 0.4 µM). Its 6-bromomethyl pyrimi-
dine derivative 5 displayed somewhat lower inhibitory activity
than 6, while 6-methyl-2,4-dimethoxy-5-bromopyrimidine (4)
showed no activity whatsoever. In the series of fluoroalkyl
pyrimidines, compounds 8 and 14 containing 2-fluoromethyl-
propyl and trifluoromethylpropyl chain, respectively, displayed
reasonable cytostatic activities. On the contrary, compounds
structurally related to 8, with a deprotected hydroxyl group (7),
2,4-diketo groups (10), and both hydroxyl and 2,4-diketo groups
c
compd
strain strain OKA strain 07/1 strain
(MCC)b
(CC50)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
2
2
2
>100 >100
>100 >100
>100
>100
8.8
>100
8.1
>100
>100
>100
>100
>20
>20
>100
>100
>100
80
88
g100
>100
100
>100
73.5
>100
>100
>100
>100
100
18
38.7
1.7
>100
50
>100
>100
>100
>100
34
>100
10.5
>100
7.4
>20
>20
>100 >100
>14.7 >14.7
>100 >100
>100 >100
>100 >100
>100 >100
>100
>100
>100
>100
>20
>20
>100
>100
>100
45
0
1
2
3
4
5
6
7
7a
8
9
0
1
2
>20
>0.8
>80
>20
>0.8
>100
4
35
(9) showed a lack of cytostatic activity. Interestingly, comparison
>100
>100
>100
>100
100
g100
>100
>100
>100
>1778
>1575
g1270
g1201
>100
g60
>100
>100
88
>100
>100
>100
>100
649
of the cytostatic effect of the 6-(2-trifluoromethyl-2-hydrox-
ypropyl)-2,4-dimethoxypyrimidine (11) with that of its phenyl-
substituted analogue 14 indicated that introduction of the phenyl
substitutent in the pyrimidine ring strongly enhanced the
cytostatic activity of 14. Besides, compound 16 with a multiple
fluoro-substituted butenyl chain displayed some inhibitory
activity, particularly against T-lymphocytes (CEM; IC50 ) 9.7
µM).
>100 >100
>100 59
45
20
>20
>20
>20
>100
>100
>100
>100
1.0
-
-
0.014
>20
>100
>100
>20
>100
32
-
-
168
>100 >100
>100 >100
>100 38
>100 >100
-
acyclovir
-
ganciclovir 4.8
1.3
0.41
-
172
51
408
cidofovir
brivudin
0.41
-
Among the C-6 fluorophenylalkyl pyrimidine derivatives, the
2-fluorophenylpropenyl pyrimidine 18 showed cytostatic activity
a
Effective concentration required to reduce virus plaque formation by
0%. Virus input was 100 plaque-forming units (PFU). Minimum cytotoxic
concentration that causes a microscopically detectable alteration of normal
cell morphology at day 7 after addition of the compounds. c Cytostatic
concentration required to inhibit cell proliferation by 50% at day 3 after
addition of the compounds.
against Hep-2 (IC50 ) 20 µM), HeLa (IC50 ) 17 µM), and
MCF-7 (IC50 ) 16 µM) cells. Replacement of methyl with
hydroxyl group in 21 abolished activity toward all tumor cells.
Finally, the activity of the most active pyrimidine derivatives
was also evaluated on the normal human fibroblast cell line
b
5
(
WI 38) to determine potential selectivity with regard to
Table 3. Antiviral Activity of Compounds 4-22 against
nontumor cells. These results clearly demonstrated that the
compounds 5, 14, 17a, and 18 are less toxic to normal than
tumor cells (Figure 4). Compound 5 has no influence on WI
Parainfluenza-3 Virus, Reovirus-1, Sindbis Virus, and Punta Toro Virus
in Vero Cell Cultures and Coxsackie B4 Virus in HeLa Cells
antiviral activity EC50 (µM)a
-5
3
8 fibroblasts up to 10 M concentration, while being strongly
Punta
Toro
virus
cytotoxic to all other tumor cell types. The influence of
compounds 14 and 17a is still more pronounced on tumor than
on normal cells, although it is evident mostly at the highest
tested concentration (10 M). Comparable growth-inhibitory
differences are also seen with compound 18, except on Mia-
Parainfluenza-3
virus
Sindbis
Reovirus-1 virus
Coxsackie
B4 virus
compd
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
2
2
2
>200
>8
>200
>8
>200 >200
>200
>40
>40
>8
>8
>40
-4
40
>40
40
>200
>29
>200
>29
>200 >200
>29 >29
>196 >196 (588)
>200 >200
>200 >200
>200 >200
>200
>147
>200
>200
>200
>200
>40
PaCa-2 cell line.
>196
>200
>200
>200
>40
>196
>200
>200
>200
>40
0
Conclusions
1
2
3
4
5
6
7
7a
8
9
0
1
2
The novel type of nonconventional C-6 fluoroalkylated (7-
2, 14-16) and fluorophenylalkylated (17-22) pyrimidine
>40
>40
40
>40
1
>40
>40
>40
>100
>100
120
>100
>100
200
>100 >100
>100 >100
>200 120
>200
>100
40
nucleoside mimetics as model compounds for development of
tracer molecules in positron-emission tomography (PET) were
synthesized. Compounds 4-22 were evaluated for their antiviral
and cytostatic activities. Among the all tested compounds, the
5-bromo-6-dibromomethyl-substituted pyrimidine derivative 6
exhibited the most prominent cytotoxic activity against the
malignant tumor cells tested. In general, fluoroalkyl pyrimidine
derivatives showed more pronounced cytostatic effects than the
fluorophenyl pyrimidine series. Thus, fluoroalkylated com-
pounds 8 and 14 showed appreciable inhibitory activity against
all tested tumor cell lines. The lack of activities of analogues
>200
>40
>130
>124
>200
>40
>200
>40
>130
>124
>200
>40
>200 >200
>40
>40
>200
>200
24
>40
40
>130 >130
>124 >124
200
>40
>200
>40
>40
brivudin
S)-DHPA
ribavirin
>250
>250
150
>250
>250
250
>250 >250
>250 >250
>250
>250
150
(
50
150
a
Effective concentration required to reduce virus plaque formation by
0%. Virus input was 100 PFU.
5
7
, 9, and 10 with deprotected functional groups might be
In addition, none of the compounds described in this study
explained by their lower lipophilicity and cell penetration.
were active against herpes simplex virus type 1 (HSV-1), herpes
simplex virus type 2 (HSV-2), vaccinia virus, vesicular stomatitis
virus, or respiratory syncytial virus (data not shown).
Cytostatic Activities. The compounds 4-22 were evaluated
for their activities against human malignant tumor cell lines:
5-Bromo-6-dibromomethyl-substituted pyrimidine 6 was 3-fold
more potent against TK VZV than acyclovir and 16-fold more
-
potent than brivudin. Besides, compound 8 was 16-fold more
-
active against TK VZV than acyclovir and 84-fold more active
+
than brivudin and 2-fold less active than acyclovir against TK -

3
042 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Prekupec et al.
Table 4. Inhibitory Effects of Compounds 4-22 on the Growth of Malignant Tumor Cell Lines
IC50 (µM)a
compd
Hep-2
HeLa
MiaPaCa-2
SW 620
MCF-7
L1210
>200
38 ( 36
8.8 ( 0.2
>200
Molt4/C8
CEM
4
5
6
7
8
9
>100
7 ( 4
2 ( 0.3
>200
>100
>100
2 ( 1
2 ( 0.2
>200
39 ( 9
>200
>200
>200
>100
>100
20 ( 10
>200
g100
>100
65 ( 36
>100
>200
>200
>100
>100
>100
1 ( 1
>100
>200
97 ( 81
8.5 ( 0.4
>200
>200
18 ( 6
1 ( 0.1
>200
8 ( 5
28 ( 28
7.1 ( 1.4
>200
0.4 ( 0.09
>200
6 ( 2
>200
17.7 ( 1.6
>200
11.7 ( 0.8
>200
30 ( 19
>200
18 ( 2
>200
21.7 ( 4.0
>200
47.8 ( 0
>200
20.6 ( 15
>200
10
11
12
13
14
15
16
17
17a
18
19
20
21
22
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>500
>500
>500
>100
90 ( 4
17 ( 2
>200
>100
>100
77 ( 23
82 ( 53
>200
70 ( 20
118 ( 26
80 ( 4
43 ( 7
>200
43 ( 11
70 ( 2
>200
19 ( 4
>200
20 ( 1
>200
17 ( 10
>200
55.4 ( 8
>100
26.3 ( 10.8
>100
45 ( 17
>100
76 ( 20
>100
80 ( 3
>200
9.7 ( 7
>200
43 ( 17
20 ( 13
>200
35 ( 10
17 ( 1
>200
g100
38 ( 30
16 ( 0.1
>200
195 ( 18
>200
144 ( 11
>200
79 ( 9
>200
>200
>500
136 ( 19
74 ( 15
156 ( 34
>200
56 ( 27
>200
>200
>200
>200
>200
>200
g100
>100
>200
>100
>100
g100
>200
192 ( 6
>500
>100
>100
>100
g500
a
50% inhibitory concentration, or compound concentration required to inhibit cell proliferation by 50%.
Figure 4. Dose-response profiles for compounds 5, 14, 17a, and 18 tested on human tumor cell lines and normal fibroblasts (WI 38) in vitro.
VZV. Unlike C-5-unsubstituted alkylated pyrimidine derivatives
4 and 17, their C-5 methyl-substituted congener 17a showed
(TLC) and the spots were detected under UV light (254 nm).
Column chromatography (CLC) was performed using silica gel
1
(0.063-0.2 mm, Kemika); the glass column was slurry-packed
some activity against CMV and VZV. C-6 fluorophenylalkenyl
pyrimidine 21 was 6-fold more potent against Coxsackie B4
virus than ribavirin. Furthermore, fluorophenylalkylated pyri-
midine derivatives 17a and 21 showed no cytotoxic effect. In
conclusion, from the fluoroalkylated pyrimidine series, com-
pound 8 emerged as the most interesting leading compound with
cytostatic and antiviral activities that could be used for further
structural optimization.
under gravity. Solvent systems used for the TLC and CLC were as
follows: pethroleum ether:ethyl acetate ) 5:1, pethroleum ether:
ethyl acetate ) 3:1, dichloromethane:methanol ) 20:1, and
dichloromethane:methanol ) 5:1. The electron impact mass spectra
were recorded with an EXTREL FT MS 2001 instrument with
ionizing energy of 70 eV. Elemental analyses were performed in
the Central Analytic Service, Rudjer B o_s kovi c´ Institute, Zagreb,
1
13
19
Croatia. H, C, and F NMR spectra were collected on a Varian
Unity Inova 300 MHz NMR spectrometer equipped with 5 mm
Experimental Section
1
15
31
H{ N- P} indirect-detection probe with gradients. All data were
recorded at 25 °C unless specified otherwise. NMR samples of all
compounds were prepared in deuterated DMSO-d , except for 8,
11, 12, 16, 17, 18, 19, and 22, which were dissolved in CD OD.
General Methods. Melting points (uncorrected) were determined
with a Kofler micro hot-stage (Reichert, Wien). Precoated Merck
silica gel 60F-254 plates were used for thin-layer chromatography
6
3

C-6 Fluorinated Acyclic Side Chain Pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3043
Fluorine chemical shifts were externally referenced relative to CCl
3
F
6-Methyl-2,4-dimethoxy-5-phenylpyrimidine (13). To the solu-
tion of compound 4 (1.03 g, 4.41 mmol) in dry THF (35 mL) were
added Bu SnPh (11.06 mL, 33.90 mmol) and PdCl (PPh ) (0.33
3 2 3 2
g, 0.44 mmol). The reaction mixture was refluxed overnight and
then the solvent was evaporated. The residue was chromatographed
(δ 0.0 ppm). Individual resonances were assigned on the basis of
their chemical shifts; signal intensities; multiplicity of resonances;
and H-H, C-H, and H-F coupling constants involved, as well
as with the use of a series of 2D experiments (gHSQC and
gHMBC).
on silica gel column using CH
2 2
Cl as eluent and compound 13 was
isolated as a solid (290 mg, 28.2%). 13: mp ) 70-75 °C; MS
6
-Methyl-2,4-dimethoxy-5-bromopyrimidine (4), 6-Bromo-
+
2
31 m/z [MH] . Anal. (C13
14 2 2
H N O ) C, H, N.
methyl-2,4-dimethoxy-5-bromopyrimidine (5), and 6-Dibromo-
methyl-2,4-dimethoxy-5-bromopyrimidine (6). A mixture of
compound 3 (5.0 g, 21.45 mmol) and acetic acid (115.5 mL) was
refluxed for 20 min and N-bromosuccinimide was then added (11.75
g, 66.01 mmol). The reaction mixture was refluxed for an additional
h and then stirred overnight at room temperature. Acetic acid
was evaporated at reduced pressure and the residue was dissolved
in CH Cl and then washed with NaHCO and Na solution.
The organic layer was dried over MgSO and the solvent was
evaporated. After column chromatography with CH Cl as eluent,
compounds 4 (450 mg, 29.9%), 5 (100 mg, 4.8%), and 6 (470 mg,
6-(2-Trifluoromethyl-2-hydroxypropyl)-2,4-dimethoxy-5-phen-
ylpyrimidine (14). The synthesis of 14 was the same as described
for 7. Reagents used were 13 (0.19 g, 0.82 mmol), THF (8 mL),
LDA (0.63 mL, 2 M in THF/heptane/ethylbenzene), and trifluo-
roacetone (0.09 mL, 0.99 mmol). After column chromatography
with dichloromethane:methanol ) 5:1 as eluent, oily compound
3
+
2
2
3
S
2 2
O
3
14 was isolated (40 mg, 12.5%). 14: MS 343 m/z [MH] . Anal.
17 2 3 3
(C16H N O F ) C, H, N.
4
2
2
6-(2-Hydroxy-3,3,3-trifluoro-1-propenyl)-2,4-dimethoxypy-
rimidine (15). The synthesis of 15 was performed by a procedure
analogous to that of 7 using compound 3 (1.090 g, 7.08 mmol)
dissolved in THF (15 mL), LDA (5.24 mL, 2 M in THF/heptane/
ethylbenzene), and methyl trifluoroacetate (0.85 mL, 8.49 mmol).
After column chromatography with pethroleum ether:ethyl acetate
) 5:1 as eluent, compound 15 was isolated as a colorless oil (751
+
1
8.3%) were isolated. 4: mp ) 69-70 °C; MS m/z 232, 234 [M ,
M + 2]. Anal. (C Br) C, H, N. 5: mp ) 77-82 °C; MS
m/z 299, 301, 303 [M - 2, M , M + 2]. Anal. (C
H, N. 6: mp ) 79-83 °C; MS m/z 386, 388, 390, 392 [M - 2,
7 9 2 2
H N O
+
7 8 2 2 2
H N O Br ) C,
+
M , M + 2, M + 4]. Anal. (C
-(2-Fluoromethyl-2-hydroxypropyl)-2,4-dimethoxypyrimi-
dine (7). The solution of 2,4-dimethoxy-6-methylpyrimidine (3)
502 mg, 3.26 mmol) in THF (10 mL) was cooled at -70 °C
7 7 2 2 3
H N O Br ) C, H, N.
+
6
mg, 42.4%). 15 and 15H: mp ) 70-72 °C; MS m/z 251 [MH] .
9 9 2 3 3
Anal. (C H N O F ) C, H, N.
(
6-(2-Hydroxy-4,4,4-trifluoro-3,3-difluoro-1-butenyl)-2,4-
dimethoxypyrimidine (16). In the same manner as described for
7, the synthesis of 16 was performed with 3 (737 mg, 4.78 mmol),
LDA (3.54 mL, 2 M in THF/heptane/ethylbenzene), THF (12 mL),
and ethyl pentafluoropropionate (0.85 mL, 5.74 mmol). After
column chromatography using pethroleum ether:ethyl acetate ) 3:1
as eluent, compound 16 (960 mg, 67.0%) was isolated as a yellow
and lithium diisopropylamide (2.4 mL, 2 M in THF/heptane/
ethylbenzene) was added dropwise to the reaction mixture. The
temperature was then raised to -55 °C and the reaction mixture
was stirred for 30 min. Fluoroacetone (0.22 mL, 3.91 mmol) was
added and the mixture was additionally stirred for 3 h and then
neutralized with glacial acetic acid. The temperature was raised to
room temperature and the reaction mixture was stirred further for
5 min. The solvent was evaporated and the residual yellow oily
product was extracted with CH Cl and water. The organic layer
was dried over Na SO and purified by silica gel column
+
solid oil. 16: mp ) 57-60 °C; MS m/z 301 [MH] . Anal.
1
9 2 3 5
(C10H N O F ) C, H, N.
2
2
6
-[2-(4′-Fluorophenyl)-2-hydroxypropyl]-2,4-dimethoxypy-
2
4
rimidine (17). The procedure was the same as for compound 7.
Reagents used were compound 3 (606 mg, 3.9 mmol), LDA (2.91
mL, 2 M in THF/heptane/ethylbenzene), THF (10 mL), and
chromatography using pethroleum ether:ethyl acetate ) 5:1 as
eluent. After column chromatography, compound 7 (357 mg,
7.6%) was isolated as yellow oil. 7: MS m/z 231 [MH] . Anal.
10
+
4
4-fluoroacetophenone (0.57 mL, 4.7 mmol). After column chro-
(C
H
15
2 3
N O F) C, H, N.
matography with pethroleum ether:ethyl acetate ) 5:1 as eluent,
compound 17 (659 mg, 57.8%) was isolated as a yellow oil. 17:
6
-(2-Fluoromethyl-2-acetoxypropyl)-2,4-dimethoxypyrimi-
dine (8), 6-(2-Fluoromethyl-2-hydroxypropyl)-2,4-dihydroxy-
pyrimidine (9), and 6-(2-Fluoromethyl-2-acetoxypropyl)-2,4-
dihydroxypyrimidine (10). The compound 7 (197 mg, 0.85 mmol)
was dissolved in acetyl chloride (5 mL). The reaction mixture was
refluxed for 5 h. Water (1.5 mL) was then added and the reaction
mixture was stirred overnight at room temperature. The solvent
was evaporated at the reduced pressure and the remaining yellow
oil was chromatographed on silica gel column using dichlo-
romethane:methanol ) 20:1 as eluent. Compounds 8 (93 mg,
17 2 3
MS m/z 291 [M - H]. Anal. (C15H N O F) C, H, N.
6-[2-(4′-Fluorophenyl)-2-hydroxypropyl]-2,4-dimethoxy-5-
methylpyrimidine (17a). The procedure was the same as for
compound 7. Reagents used were compound 3a (610 mg,
3.70 mmol), LDA (2.71 mL, 2 M in THF/heptane/ethylbenzene),
THF (10 mL), and 4-fluoroacetophenone (0.54 mL, 4.44 mmol).
After column chromatography with pethroleum ether:ethyl acetate
) 5:1 as eluent, compound 17a (240 mg, 19.4%) was isolated as
a yellow oil. 17a: MS m/z 305 [M - H]. Anal. (C H N O F) C,
16
19
2
3
4
8
1
1
0.3%), 9 (35 mg, 20.4%), and 10 (50 mg, 24.1%) were isolated.
H, N.
+
: MS m/z 273 [MH] . Anal. (C12
H
17
N
2
O
4
F) C, H, N. 9: mp )
F) C, H, N.
0: mp ) 187-190 °C; MS m/z 245 [MH] . Anal. (C10 F)
6
-[2-(4′-Fluorophenyl)-1-propenyl]-2,4-dimethoxypyrimi-
98-201 °C; MS m/z 201 [M - H]. Anal. (C
8 11 2 3
H N O
dine (18). Compound 17 (320 mg, 1.09 mmol) was dissolved in
acetyl chloride (5 mL). The reaction mixture was refluxed for 5 h.
Water (1.5 mL) was added and the reaction mixture was stirred
overnight at room temperature. The solvent was evaporated at
reduced pressure and the remaining yellow oil was chromatographed
on silica gel column using dichloromethane:methanol ) 20:1 as
eluent and 18 (63 mg, 21.1%) was isolated as a white solid. 18:
+
13 2 4
H N O
C, H, N.
6-(2-Trifluoromethyl-2-hydroxypropyl)-2,4-dimethoxypyrimi-
dine (11). The synthesis of 11 was performed analogously to that
of 7, using compound 3 (790 mg, 5.12 mmol) dissolved in THF
(11 mL), LDA (3.8 mL, 2 M in THF/heptane/ethylbenzene), and
+
trifluoroacetone (0.58 mL, 6.14 mmol) as reagents. After column
mp ) 55-60 °C; MS m/z 275 [MH] . Anal. (C H N O F)
1
5
15
2
2
chromatography with pethroleum ether:ethyl acetate ) 5:1 as eluent,
C, H, N.
compound 11 (432 mg, 31.7%) was isolated as a yellow oil. 11:
6
-[2-(2′-Fluorobenzyl)]-2-hydroxypropyl-2,4-dimethoxypy-
+
MS m/z 267 [MH] . Anal. (C10
H N
13 2
O
3
3
F ) C, H, N.
rimidine (19). A procedure analogous to that of 7 was used for
the synthesis of 19. Reagents used were 3 (968 mg, 6.28 mmol),
LDA (4.65 mL, 2 M in THF/heptane/ethylbenzene), 2-fluorophe-
nylacetone (1.05 mL, 7.54 mmol), and THF (15 mL). After
column chromatography using pethroleum ether:ethyl acetate
) 3:1 as eluent, compound 19 (743 mg, 37.3%) was isolated as a
6-(2-Trifluoromethyl-2-hydroxypropyl)-2,4-dihydroxypyrimi-
dine (12). A procedure analogous to the deprotection of 7 was used
for the synthesis of 12. Reagents used were 11 (307 mg, 1.15
mmol), acetyl chloride (5.5 mL), and water (2.5 mL). After column
chromatography using dichloromethane:methanol ) 20:1 as eluent,
1
2 (15.3 mg, 5.6%) was obtained. 12: mp ) 206-209 °C; MS
19 2 3
colorless oil. 19: MS m/z 305 [M - H]. Anal. (C16H N O F) C,
H, N.
m/z 237 [M - H]. Anal. (C ) C, H, N.
8
H
9
N
2
O
3
F
3

3
044 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
-[2-(2′-Fluorobenzyl)-2-acetoxypropyl]-2,4-dihydroxypyrimi-
Prekupec et al.
6
and incubated for a further 72 h. Working dilutions were freshly
prepared on the day of testing. The solvent was also tested for
eventual inhibitory activity by adjusting its concentration to be the
same as working concentrations. After 72 h of incubation, the cell
growth rate was evaluated by MTT assay, as described previ-
ously.2
dine (20). A procedure analogous to deprotection of 7 was used
for the synthesis of 20. Reagents used were 19 (353 mg, 1.15
mmol), acetyl chloride (5.5 mL), and water (2.5 mL). After column
chromatography using dichloromethane:methanol ) 20:1 as eluent,
2,31
2
0 (237 mg, 64.3%) was obtained. 20: mp ) 149-152 °C; MS
+
m/z 321 [MH] . Anal. (C16
H
17
2 4
N O F) C, H, N.
Each test point was performed in quadruplicate in three in-
dividual experiments. The results are expressed as IC , a
6-[2-(4′-Fluorophenyl)-2-hydroxy-1-etenyl]-2,4-dimethoxypy-
5
0
rimidine (21). In the same manner as described for 7, the synthesis
of 21 was performed with 3 (613 mg, 4.0 mmol), LDA (2.9 mL, 2
M in THF/heptane/ethylbenzene), THF (10 mL), and ethyl 4-fluo-
robenzoate (0.7 mL, 4.8 mmol). After column chromatography
using pethroleum ether:ethyl acetate ) 5:1 as eluent, 21 (576 mg,
concentration necessary for 50% of inhibition. Each result is a mean
value from three separate experiments. The IC50 values for each
compound were calculated from dose-response curves using linear
regression analysis by fitting the test concentrations that gave PG
(percentage of growth) values above and below the reference value
(i.e., 50%).
5
2
2.1%) was isolated. 21E and 21K: mp ) 98-102 °C; MS m/z
+
77 [MH] . Anal. (C14
13 2 3
H N O F) C, H, N.
6
-[2,2-(Di-4-fluorophenyl)-2-hydroxyethyl]-2,4-dimethoxypy-
Acknowledgment. Support for this study was provided by
the Ministry of Science of the Republic of Croatia (Projects
#125-0982464-2925, #125-0982464-2922, #098-0982464-2514
and #098-0982464-2393). We thank Lizette van Berckelaer for
excellent technical assistance in performing (part of) the
cytostatic cell activity assays, as well as Ann Absillis, Anita
Van Lierde, Frieda De Meyer, Leentje Persoons, Anita Camps,
and Lies Vandenheurck for excellent technical assistance in
performing the antiviral activity assays.
rimidine (22). The synthesis of 22 was performed by a procedure
analogous to that of 7 using compound 3 (607 mg, 3.9 mmol)
dissolved in THF (10 mL), LDA (2.91 mL, 2 M in THF/heptane/
ethylbenzene), and 4,4′-difluorobenzophenone (1.026 g, 4.7 mol).
After column chromatography with pethroleum ether:ethyl acetate
5:1 as eluent, compound 22 (564 mg, 38.8%) was isolated. 22:
mp ) 150-154 °C; MS m/z 371 [M - H]. Anal. (C20
C, H, N.
)
18 2 3 2
H N O F )
Antiviral Activity Assays. Antiviral activity against HCMV,
VZV, parainfluenza-3 virus, reovirus-1, Sindbis virus, Punta Toro
virus, Coxsackie B4 virus, HSV-1, HSV-2, vaccinia virus, vesicular
Supporting Information Available: 19F and 13C NMR chemical
shift values and elemental analysis results. This material is available
free of charge via the Internet at http://pubs.acs.org.
stomatitis virus, and respiratory syncytial virus was determined
essentially as described previously.2
8,29
Confluent human embryonic
lung (HEL) fibroblasts were grown in 96-well microtiter plates and
infected with the human cytomegalovirus (HCMV) strains Davis
and AD-169 at 100 PFU per well. After a 2-h incubation period,
residual virus was removed, and the infected cells were further
incubated with the medium containing different concentrations of
the compounds tested (in duplicate). After incubation for 7 days at
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