The unsaturated acyclic nucleoside analogues bearing a sterically constrained (Z)-4′-benzamido-2′-butenyl moiety: Synthesis, X-ray crystal structure study, cytostatic and antiviral activity evaluations

Article abstract of DOI:10.1016/j.bmc.2010.07.035

A series of the novel acyclic unsaturated pyrimidine (1-12) and adenine (13) nucleoside analogues bearing conformationally restricted (Z)-2′-butenyl moiety were synthesized and evaluated for their antiviral and cytostatic activity potency against malignant tumor cell lines and normal human fibroblast (WI38). The N-1 and/or N-3 acyclic side chain substitution in pyrimidine ring in N-3 substituted 5-trifluoromethyluracil derivative (11), N-1, N-3 disubstituted 5-fluorouracil derivative (12) and adenine derivative (13) was deduced from their ¹H and ¹³C NMR spectra and confirmed by single crystal X-ray structure analysis. The X-ray crystal structure analysis 11-13 revealed also supramolecular self-assemblies, in which infinite chains or dimers built two- and three-dimensional networks. The results of the in vitro cytostatic activity evaluations of 1-13 indicate that the majority of the compounds tested exhibited a non-specific and moderate antiproliferative effect at the highest concentration (100 μM). Of all evaluated compounds on the cell lines tested only the N-1 4″-fluoro- substituted-benzamide uracil derivative (7) showed rather marked and selective inhibitory activity against the growth of MCF-7 cells at a concentration of 2.7 μM and no cytotoxic effect on normal fibroblasts WI38. This compound can be therefore considered as a potential antitumor lead compound for further synthetic structure modification.

Full text of DOI:10.1016/j.bmc.2010.07.035

Bioorganic & Medicinal Chemistry 18 (2010) 6249–6257
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The unsaturated acyclic nucleoside analogues bearing a sterically
constrained (Z)-40-benzamido-20-butenyl moiety: Synthesis, X-ray crystal
structure study, cytostatic and antiviral activity evaluations
a
a
a
b
c
d
´
´
´
Krešimir Benci , Karlo Wittine , Malajka Radan , Mario Cetina , Mirela Sedic , Sandra Kraljevic Pavelic ,
d
e
a,
*
´
Krešimir Pavelic , Erik De Clercq , Mladen Mintas
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 19, 10000 Zagreb, Croatia
b Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, Prilaz baruna Filipovi´ca 28a, 10000 Zagreb, Croatia
c
-
ˇ
´
Division of Molecular Medicine, Laboratory for Systems Biomedicine, Ruder Boškovic Institute, Bijenicka cesta 54, PO Box 1016, 10001 Zagreb, Croatia
d
ˇ
´
´
Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, 51000 Rijeka, Croatia
e Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2010
Revised 7 July 2010
Accepted 15 July 2010
Available online 7 August 2010
A series of the novel acyclic unsaturated pyrimidine (1–12) and adenine (13) nucleoside analogues
bearing conformationally restricted (Z)-20-butenyl moiety were synthesized and evaluated for their
antiviral and cytostatic activity potency against malignant tumor cell lines and normal human fibro-
blast (WI38). The N-1 and/or N-3 acyclic side chain substitution in pyrimidine ring in N-3 substituted
5-trifluoromethyluracil derivative (11), N-1, N-3 disubstituted 5-fluorouracil derivative (12) and ade-
nine derivative (13) was deduced from their 1H and 13C NMR spectra and confirmed by single crystal
X-ray structure analysis. The X-ray crystal structure analysis 11–13 revealed also supramolecular self-
assemblies, in which infinite chains or dimers built two- and three-dimensional networks. The results
of the in vitro cytostatic activity evaluations of 1–13 indicate that the majority of the compounds tested
Keywords:
Unsaturated acyclic nucleoside analogues
X-ray diffraction
Supramolecular self-assembling
Cytostatic activity
exhibited a non-specific and moderate antiproliferative effect at the highest concentration (100 lM). Of
all evaluated compounds on the cell lines tested only the N-1 400-fluoro-substituted-benzamide uracil
Antiviral activity
derivative (7) showed rather marked and selective inhibitory activity against the growth of MCF-7
cells at a concentration of 2.7 lM and no cytotoxic effect on normal fibroblasts WI38. This compound
can be therefore considered as a potential antitumor lead compound for further synthetic structure
modification.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
acyclic nucleoside analogues is a structural feature important for
strong antiherpetic activity of the guanine analogue.7 Thymidine
Nucleoside analogues have been the cornerstone of antiviral
chemotherapy over the past decades.1 Although structure–activity
relationship studies have not led to a uniform pharmacophore
model for the antiviral activities of nucleosides, particular struc-
tural features such as the presence of unsaturated acyclic side
chain as a substitute for nucleosides sugar have proved to be effec-
tive for specific antiviral activities.2 The diverse range of com-
pounds containing this structural motif includes the anti-HIV
agent carbovir,3 the antibiotic neplanocin A4 and many other
examples of such type of compounds with significant antiviral or
antibacterial activity. Furthermore, the introduction of a rigid
structural element into nucleoside or carbocyclic nucleoside struc-
ture have been shown to lead to synthesis of effective antiviral
nucleoside analogues.5,6 Thus, the presence of a double bond in
with a 20-butenyl spacer was the first acyclic nucleoside analogue
exhibiting potent inhibition of thymidine kinase 2 (TK-2) which
catalyzed phosphorylation of antiviral drugs.8 Among them, (Z)-
configuration of the cyclopropyl guanine nucleoside showed anti-
herpetic potency (HSV-1 and HSV-2) comparable to that of
acyclovir.9
In this connection and related to our previous studies on acyclic
nucleoside analogues2,10 we prepared a series of the novel cis-ole-
finic pyrimidine (1–12) and adenine (13) nucleoside analogues
(Fig. 1). The initial aim of this study was to evaluate the antiviral
activity potency of this new type of acyclic nucleoside analogues
as well as their inhibitory activities against human tumor cell lines.
Besides, we also wanted to examine by X-ray crystal structure
analysis how the fluorinated pyrimidine (11 and 12) nucleoside
bases and its adenine (13) analogue influence the formation of
supramolecular self-assembling which play an important role in
drug receptor interactions.
* Corresponding author. Tel.: +385 1 4597 214; fax: +385 1 4597 224.
E-mail addresses: mladen.mintas@fkit.hr, mmintas@fkit.hr (M. Mintas).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.035

6250
K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
O
4
NH2
R5
R5
4
5
5
3N
HN3
2
2
3''
R1
3''
6''
R1
6
6
4''
4''
O
1N
2''
1''
O
1N
2''
1''
H
H
4'
4'
N
N
1'
1'
5''
5''
6''
2'
3'
2'
3'
O
O
5,6 and 9,10
1-4 and 7,8
5
6
2
7
10
8
9
3
4
1
R5
R1
F
CH3
H
CF3
H
H
H
F
H
F
CH3
F
H
F
F
F
H
H
H
H
4''
4''
6''
3''
2''
3''
2''
5''
5''
1''
1''
6''
O
O
NH
NH
4'
4'
3'
3'
2'
NH2
6
5
O
O
9
4
4
3
1N
2'
F
3N
CF3
N
5
5
N
2
8
1'
1'
3''
6''
2
3''
2
6
4''
4''
4
6
N 7
2''
1''
2''
1''
3 N
1
N
O
1 N
O
H
4'
H
H
4'
N
1'
5''
N
1'
5''
2'
3'
2'
3'
6''
O
O
12
11
13
Figure 1. The benzamide derivatives of unsaturated acyclic pyrimidine (1–12) and adenine (13) nucleoside analogues.
(12) and adenine derivative (13) was undertaken primarily to deter-
mine the position of nitrogen substitution in the pyrimidine ring of
(11) and (12) and to examine the ability of those compounds to form
supramolecular self-assembling. The molecular structures of 11 and
12 are shown in Figures 2 and 3. Equivalent bond lengths in N-3
substituted carboxamide derivative 11 and N-1, N-3 disubstituted
derivative 12 are within the range in closely related 2-butenyl uracil
derivatives,11,12 and thymine derivative of 1-aminocyclopropane-1-
carboxylic acid we published previously.12 Butenyl moiety adopts
cis conformation in these two structures; the C7–C8–C9–C10 torsion
angle in 11 amounts ꢀ1.9(3)°, while the C7–C8–C9–C10 and C18–
C19–C20–C21 torsionanglesin 12 are 1.4(2) and 5.1(2)°, respectively.
The biggest conformational difference between these two struc-
tures is in the orientation of the carboxamide moiety. The conforma-
tion of this moiety in 11, defined by the C9–C10–N2–C11 torsion
angle of 127.08(17)°, is anticlinal. In 12, two carboxamide moieties
have two different conformations, synclinal and anticlinal [C9–
C10–N2–C11 = 78.47(17)°; C20–C21–N4–C22 = ꢀ143.90(14)°]. The
2-butenyl-benzamido moiety attached to the N3 atom of the pyrim-
idine ring in 11 has different orientation compared to 12, which is
accompanied by formation of intramolecular hydrogen bonds
between moiety and ring (Figs. 2 and 3).
2. Results and discussion
2.1. Chemistry
The new unsaturated acyclic uracil (1–4, 7, and 8), cytosine (5,
6, 9, and 10) and adenine (13) nucleoside analogues bearing a con-
formationally restricted (Z)-20-butenyl moiety were synthesized by
condensation of nucleoside bases with (Z)-4-chloro-2-butenylben-
zamide (15) or (Z)-4-chloro-2-butenyl-4-fluorobenzamide (16),
respectively (Scheme 1). These precursors were readily prepared
by reaction of (Z)-40-chloro-20-butenylamine hydrochloride (14)
with benzamide or 4-fluorobenzamide. Besides N-1 regioselectivi-
ty of the coupling of heterocycle with acyclic residue which is con-
sidered essential as natural pyrimidine nucleosides are substituted
at N-1, the N-3 substitution has also been found in the N-3 alkyl-
ated 5-trifluoromethyluracil derivative 11 as well as the N-1 and
N-3 dialkylated 5-fluorouracil derivative 12.
2.2. 1H and 13C NMR spectra
Structures of the newly synthesized compounds were deduced by
analysis of their 1H and 13C NMR as well as mass spectra. The assign-
ment of 1H NMR spectra was performed on the basis of the chemical
shifts, substituent-induced chemical shifts, and signal intensities,
magnitude and multiplicity of H–H coupling constants. The 1H and
13C NMR data given in Table 1 and the experimental part are in full
agreement with the proposed structures. DMSO-d6 was used as a
solvent for all compounds; chemical shifts are referred to TMS.
The molecules of N-3 trifluoromethyluracyl derivative 11 are
assembled by N1ꢁ ꢁ ꢁO3 hydrogen bond [Dꢁ ꢁ ꢁA = 2.737(3) Å] into
infinite chains which can be described by graph-set notation as
C(6)13 (Fig. 4). This hydrogen bond is reinforced by C6ꢁ ꢁ ꢁO1 hydro-
gen bond [Dꢁ ꢁ ꢁA = 3.460(2) Å], so forming new chain motif and
hydrogen-bonded sheets.
The sheets are further linked by two weak aromatic
ing interactions14 between nearly parallel pyrimidine and phenyl
rings of the neighboring molecules. An interplanar angle ( ) be-
tween the rings is 9.90(10)°. These two ꢁ ꢁ ꢁ interactions form di-
mers and a complete two-dimensional network.
p
ꢁ ꢁ ꢁ
p stack-
2.3. X-ray crystal structure analysis
a
p
p
The single X-ray crystal structure analysis of 5-trifluoromethyl-
uracil derivative (11), N-1, N-3-dialkylated 5-fluorouracil derivative

K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
6251
R1
4'
Cl
Cl
H
N
i
NH3+Cl-
O
14
15 (R1 = H)
16 (R1 = F)
ii
R1
4''
Base
1'
H
4'
N
O
R1
R1
Base
Base
F
U
T
C
5-FC
7
8
9
H
H
H
H
H
H
H
U
5-FU
T
5-CF3
C
5-FC
A
1
2
3
4
5
F
F
F
10
6
13
Scheme 1. Synthesis of carboxamide derivatives of unsaturated acyclic uracil (1–4, 7, and 8), cytosine (5, 6, 9, and 10), and adenine (13) nucleoside analogues. Reagents and
conditions: (i) THF, H2O, benzoylchloride or 4-fluorobenzoylchloride, rt; (ii) uracil (U), 5-fluorouracil (5-FU), 5-trifluoromethyluracil (5-CF3U), thymine (T), cytosine (C), 5-
fluorocytosine (5-FC) or adenine (A), DMF, NaH, rt.
Table 1
1H NMR chemical shifts (d/ppm) and H–H coupling constants (J/Hz) in 1H NMR spectra for compounds 1–13 (c.f. Fig. 1)
H-3
H-5
5.58 (d, 1H, 7.69 (d, 1H, 4.45 (d, 2H, 5.65
J3 = 7.86) J3 = 6.72)
8.15 (d, 1H, 4.43 (d, 2H, 5.68
J3 = 6.72) J3 = 6.84) (m, 1H) (m, 1H) J3 = 6.00)
7.59 (s, 1H) 4.41 (d, 2H, 5.67 5.50 4.03 (t, 2H, 8.72
J3 = 6.78) (m, 1H) (m, 1H) J3 = 5.92) (br, 1H)
8.46 (s, 1H) 4.55 (d, 2H, 5.66 5.58 4.20 (t, 2H, 8.75
J3 = 6.73) (m, 1H) (m, 1H) J3 = 5.97) (br, 1H)
5.67 (d, 1H, 7.63 (d, 1H, 4.43 (d, 2H, 5.63 5.52 4.04 (t, 2H, 8.71
(m, 1H) (m, 1H) J3 = 5.97) (br, 1H)
7.99 (d, 1H, 4.41 (d, 2H, 5.64 5.54 4.04 (t, 2H, 8.72
J3 = 6.72) J3 = 6.84) (m, 1H) (m, 1H) J3 = 5.97) (br, 1H)
H-6
H-10
H-20
H-30
H-40
4.03 (t, 2H, 8.70
(br, 1H)
4.02 (t, 2H, 8.81
(br, 1H)
NH
H-300
7.85 (d, 2H, J3 = 6.87) 7.46 (t, 2H, 7.52 (d, 1H,
J3 = 7.08) J3 = 6.99)
7.87 (d, 2H, J3 = 7.38) 7.46 (t, 2H, 7.53 (d, 1H,
J3 = 7.00) J3 = 7.26)
7.85 (d, 2H, J3 = 7.08) 7.46 (t, 2H, 7.52 (d, 1H,
J3 = 7.13) J3 = 6.93)
7.85 (d, 2H, J3 = 6.84) 7.47 (t, 2H, 7.53 (d, 1H,
J3 = 7.11) J3 = 6.96)
7.86 (d, 2H, J3 = 6.87) 7.46 (t, 2H, 7.52 (d, 1H, 7.03 (br, 2H)
J3 = 7.14) J3 = 6.96)
7.86 (d, 2H, J3 = 6.81) 7.47 (t, 2H, 7.52 (d, 1H, 7.53 (br,
H-200
H-400
NH2
1
2
11.27
(br, 1H) J3 = 7.83)
11.71
5.53
/
(m, 1H) (m, 1H) J3 = 5.87)
5.53
/
/
/
/
/
/
(br, 1H)
3ª 11.26
(br, 1H)
11.86
(br, 1H)
/
4
5
6
J3 = 7.14)
/
J3 = 7.14)
J3 = 6.75)
/
J3 = 7.14)
J3 = 6.96)
1H) + 7.40 (br,
1H)
/
7
11.27
5.57 (d, 1H, 7.67 (d, 1H, 4.44 (d, 2H, 5.65
J3 = 7.86) J3 = 6.79) (m, 1H) (m, 1H) J3 = 5.98)
7.59 (s, 1H) 4.42 (d, 2H, 5.65 5.53 4.03 (t, 2H, 8.75
J3 = 6.78) (m, 1H) (m, 1H) J3 = 5.84)
5.66 (d, 1H, 7.61 (d, 1H, 4.41 (d, 2H, 5.62 5.49
(m, 1H) (m, 1H) J3 = 6.00)
5.55
4.02 (t, 2H, 8.72
7.91 (dd, 2H, J1–
7.29 (t, 2H,
J3 = 8.79)
7.31 (t, 2H,
J3 = 8.84)
7.29 (t, 2H,
J3 = 8.79)
/
/
/
(br, 1H) J3 = 7.86)
8b 11.27
(br, 1H)
(br, 1H) 3 = 8.70; J2–4 = 8.70)
/
7.93 (dd, 2H, J1–
/
(br, 1H) 3 = 8.67; J2–4 = 8.67)
4.02 (t, 2H, 8.74
9
/
7.92 (dd, 2H, J1–
7.05 (br,
1H) + 6.97 (br,
1H)
J3 = 7.14)
J3 = 7.14)
J3 = 6.84)
(br, 1H) 3 = 8.70; J2–4 = 8.70)
10
/
/
7.98 (d, 1H, 4.40 (d, 2H, 5.64
J3 = 6.69) J3 = 6.75) (m, 1H) (m, 1H) J3 = 5.93)
5.53
4.03 (t, 2H, 8.75
7.93 (dd, 2H, J1–
7.30 (t, 2H,
J3 = 8.81)
/
7.62 (br,
1H) + 7.40 (br,
1H)
(br, 1H) 3 = 8.61; J2–4 = 8.61)
11c
12
/
/
/
/
/
/
8.15 (d, 1H, 4.43 (m, 2H) 5.68
J3 = 6.72) (m, 1H) (m, 1H)
8.15 (s, 2H) 4.43 (m, 4H) 5.68 5.53
(m, 2H) (m, 2H)
4.94 (d, 2H, 5.71 5.68
J3 = 6.30) (m, 1H) (m, 1H) J3 = 5.28)
5.53
4.02 (m, 2H) 8.81
(br, 1H)
4.02 (m, 4H) 8.81
(br, 2H)
4.14 (t, 2H, 8.78
(br, 1H)
7.87 (d, 2H, J3 = 7.38) 7.46 (tm,
7.53 (m, 1H)
7.53 (m, 2H)
/
2H)
7.87 (d, 4H, J3 = 7.38) 7.46 (tm,
4H)
7.86 (d, 2H, J3 = 6.78) 7.48 (t, 2H, 7.50 (d, 1H, 7.22 (br, 2H)
J3 = 8.67) J3 = 6.70)
/
13d
/
a
b
c
Compound 3: signal for –CH3: 1.75 ppm (s, 3H).
Compound 8: signal for –CH3: 1.76 ppm (s, 3H).
Compound 12: signal for H-1: 8.37 ppm (br, 1H).
d
Compound 13: signal for H-2: 8.18 ppm (s, 1H); signal for H-8: 8.15 ppm (s, 1H).

6252
K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
lecular aggregation as hydrogen-bonded acceptor via N2ꢁ ꢁ ꢁO2
hydrogen bond [Dꢁ ꢁ ꢁA = 2.965(2) Å], and as double donor via
O2ꢁ ꢁ ꢁN3 [Dꢁ ꢁ ꢁA = 3.039(2) Å] and O2ꢁ ꢁ ꢁO1 hydrogen bond
[Dꢁ ꢁ ꢁA = 2.828(2) Å], thus generating a two-dimensional network
(Fig. 7). The sixmembered and fivemembered rings of neighboring
purine moieties are nearly parallel, so enabling three ꢁ ꢁ ꢁ
p p interac-
tions0 formation (
a
= 0°; = 2.16(7)°). A fourth p p interaction
a
ꢁ ꢁ ꢁ
between phenyl rings with an interplanar angle of 0° expands
the two-dimensional network into a three-dimensional network
(Fig. 7).
3. Biological results
3.1. Cytostatic activity
The compounds were evaluated for their antiproliferative effect
against several malignant tumor cell lines: cervical carcinoma
(HeLa), colorectal adenocarcinoma, metastatic (SW 620), pancre-
atic carcinoma (MiaPaCa-2), breast epithelial adenocarcinoma,
metastatic (MCF-7), breast adenocarcinoma metastatic (SK-BR-3)
and larynx carcinoma (HEp-2), and compared with their effects
on the growth of normal human fibroblasts (WI38) (Table 2, Sup-
plementary data).
The results of the in vitro cytostatic activity evaluations re-
vealed that the majority of the acyclic pyrimidine (1–10) and ade-
nine nucleoside analogues (13) exhibited a non-specific and
moderate antiproliferative effect at the highest concentration
Figure 2. A molecular structure of 11, with the atom-numbering scheme.
Displacement ellipsoids for nonhydrogen atoms are drawn at the 30% probability
level. The intramolecular hydrogen bonds are shown dashed.
(100 lM) on the tested cell line panel. Strikingly, some compounds
Two N–Hꢁ ꢁ ꢁO hydrogen bonds, N2ꢁ ꢁ ꢁO4 [Dꢁ ꢁ ꢁA = 2.9244(17) Å]
and N4ꢁ ꢁ ꢁO2 [Dꢁ ꢁ ꢁA = 2.9762(16) Å] link the molecules of the N-1,
N-3 disubstituted 5-fluorouracil derivative 12 into C(4) and C(11)
chains, respectively (Fig. 5). Supramolecular structure of 12 also
contains two C–Hꢁ ꢁ ꢁO hydrogen bonds, C18ꢁ ꢁ ꢁO3 [Dꢁ ꢁ ꢁA =
3.4615(17) Å] and C28ꢁ ꢁ ꢁO2 [Dꢁ ꢁ ꢁA = 3.482(2) Å], which link infinite
sharing structural similarity differed in the selectivity of their cyto-
static activities (Supplementary data). For example, among uracil
derivatives compound 7 showed selective and rather marked
inhibitory activity against the growth of the MCF-7 cells
(IC50 = 2.7 0.05 lM), whereas compound 1 exerted pronounced
selective cytostatic activity on HeLa cells (IC50 = 4.9 0.04). These
compounds resulted to have diverse effects on the growth of nor-
mal human fibroblasts as well. Compound 7 showed no cytotoxic-
ity on WI38 while compound 1 showed strong cytotoxic effect on
this cell line (IC50 = 6.6 0.05) It is however interesting that intro-
duction of Fluor abolished cytotoxic effects of compound 7 on
WI38 (Fig. 1, Supplementary data). Contrary, comparing compound
3 (bearing a CH3 moiety) that showed high cytotoxicity on WI38
(4.3 0.07) with a structurally related compound 8 where Fluor
has been introduced, strong cytotoxic effect on WI38 (0.1 0.08)
has not been abolished.
chains into two-dimensional network. Four
p
ꢁ ꢁ ꢁ
p
stacking interac-
= 3.49(7)°) and
= 9.00(8)°) complete
tions between pyrimidine and phenyl rings (
a
between neighboring phenyl rings
three-dimensional network.
(
a
a
Adenine derivative 13 (Fig. 6), in which 20-butenyl-benzamido
moiety is bound to the N9 atom of the adenine moiety crystallizes
as monohydrate. Bond lengths in 13 are close to equivalent ones in
structurally related compounds.12,15,16 The exception is C4–C5
bond length in (Z)-methyl 1-{N-[(tert-butoxy)carbonyl]amino}-2-
(6-aminopurine-9-yl)cyclopropanecarboxylate16 which is ca.
0.03 Å shorter.
And again, cytosine derivatives 5, 6 and 9 all exerted a strong
cytotoxic effect on WI38 while introduction of Fluor at two posi-
tions in compound 10 seems to abolish cytotoxicity in this cell line
(Fig. 1, Supplementary material).
The molecules of 13 are linked into dimers by two N–Hꢁ ꢁ ꢁN
hydrogen bonds, N6ꢁ ꢁ ꢁN7 [Dꢁ ꢁ ꢁA = 3.090(2) Å] and N6ꢁ ꢁ ꢁN1
[Dꢁ ꢁ ꢁA = 2.949(2) Å]. Water molecule participates also in supramo-
Figure 3. A molecular structure of 12, with the atom-numbering scheme. Displacement ellipsoids for nonhydrogen atoms are drawn at the 30% probability level. The
intramolecular hydrogen bonds are shown dashed.

K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
6253
Figure 4. A part of the crystal structure of 11, showing hydrogen bonds and pyrimidine and phenyl rings’ stacking which form two-dimensional network. Hydrogen bonds are
indicated by dashed lines.
Figure 5. A crystal packing diagram of 12, viewed along the a axis, and showing hydrogen bonds and parallel arrangements of pyrimidine and phenyl rings which enable
three-dimensional network formation. Hydrogen bonds are indicated by dashed lines.
3.2. Antiviral activity
viruses. Unfortunately, none of the compounds showed pro-
nounced antiviral activity at subtoxic concentrations. Cytotoxicity
of all evaluated compounds on HEL, Vero, HeLa, and MDCK cell cul-
tures were greater then 100% (data not shown).
Compounds 1–10 and 13 were evaluated for their antiviral
activity against a wide variety of DNA and RNA viruses, including
HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vac-
cinia virus, and vesicular stomatitis virus], Vero (parainfluenza-3,
reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), HeLa
(vesicular stomatitis virus, Coxsackie virus B4, and respiratory syn-
cytial virus) or MDCK [influenza A (H1N1; H3N2) and influenza B]
4. Conclusions
We have successfully prepared a series of the novel unsaturated
acyclic pyrimidine (1–12) and adenine (13) nucleoside analogues

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K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
WI38. This compound would be therefore regarded as a potential
antitumor lead compound for further synthetic structure
modification.
5. Experimental
5.1. General methods
Melting points (uncorrected) were determined with Kofler mi-
cro hot-stage (Reichert, Wien). Precoated Merck Silica Gel 60F-
254 plates were used for thin layer chromatography (TLC) and
the spots were detected under UV light (254 nm). Column chroma-
tography (CLC) was performed using silica gel (0.063–0.2 mm) Flu-
ka; glass column was slurry-packed under gravity. The electron
impact mass spectra were recorded with an EXTREL FT MS 2002
instrument with ionizing energy of 70 eV. 1H and 13C NMR spectra
were obtained on a Bruker 300 MHz NMR spectrometer. All data
were recorded in DMSO-d6 at 298 K. Chemical shifts were refer-
enced to the residual solvent signal of DMSO at d 2.50 ppm for
1H and d 39.50 ppm for 13C. Individual resonances were assigned
on the basis of their chemical shifts, signal intensities, multiplicity
of resonances, and H–H coupling constants.
Figure 6. A molecular structure of monohydrate of compound 13, with the atom-
numbering scheme. Displacement ellipsoids for nonhydrogen atoms are drawn at
the 30% probability level.
5.2. Procedures for the preparation of compounds
bearing a conformationally restricted (Z)-20-butenyl moiety. X-ray
structure analysis revealed supramolecular self-assemblies of 5-
trifluoromethyluracyl derivative (11), N-1, N-3-dialkylated 5-fluo-
rouracil derivative (12) and adenine derivative (13). In all of these
structures, the main hydrogen-bonded motifs, chains or dimers,
are extended into two-dimensional network (for 11) or three-
5.2.1. General procedure for the preparation of (Z)-4-chloro-2-
butenyl-benzamide (15) and (Z)-4-chloro-2-butenyl-4-fluoro-
benzamide (16)
To a stirred suspension of (Z)-4-chloro-2-butenylaminehydro-
chloride (14) (1.0 g, 7.0 mmol) in THF (35 ml) and water (0.2 ml)
N,N-diisopropylethylamine (2.7 ml, 15.4 mmol) was added fol-
lowed by addition of benzoylchloride (0.76 ml, 8.4 mmol) or 4-
fluorobenzoylchloride (0.99 ml, 8.4 mmol). The resultant solution
was stirred overnight at room temperature. Reaction mixture
was evaporated, dissolved in CH2Cl2 (100 ml) and extracted with
dimensional network (for 12 and 13) by
pꢁ ꢁ ꢁp interactions. The
400-fluorosubstituted benzamide uracil derivative (7) showed selec-
tive cytostatic inhibitory activity in MCF-7 cell lines at micromolar
concentrations and no cytotoxicity to human normal fibroblasts
Figure 7. A crystal packing diagram of 13, viewed along the b axis, and showing two-dimensional network formed by hydrogen bonds, which are extended by
interactions into three-dimensional network. Hydrogen bonds are indicated by dashed lines.
pꢁ ꢁ ꢁp

K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
6255
H2O (4 ꢂ 20 ml), dried (MgSO4), concentrated in vacuo. Purification
by column chromatography (silica gel, PE/EtOAc = 3/1) afforded 15
(900 mg, 61%) and 16 (1100 mg, 69%) both as white crystals.
Compound 4: 13C NMR (DMSO) d: 166.66 (C@O), 152.17 and
150.83 (C-4), 150.11 (C-2), 140.90 (C-5), 134.71 (C-100), 131.71 (C-
400), 131.60 (C-200), 130.37 and 129.00 (C-6), 128.88 (C-20), 127.80
(C-30), 125.82 (C-300), 44.70 (C-10), 36.81 (C-40); MS m/z 353.12
[MH]+. Anal. (C16H14F3N3O3) C, H, N.
1
Compound 15: H NMR (DMSO) d: 4.43 (d, 2H, J3 = 6.84, H-10);
5.68 (m, 1H, H-20); 5.53 (m, 1H, H-30); 4.02 (t, 2H, J3 = 6.00, H-40);
8.81 (br, 1H, NH); 7.87 (d, 2H, J3 = 7.38, H-300); 7.46 (d, 2H,
J3 = 7.00, H-200); 7.53 (d, 1H, J3 = 7.26, H-400); 13C NMR (DMSO) d:
134.71 (C-100), 131.71 (C-400), 131.60 (C-200), 128.88 (C-20), 127.80
(C-30), 125.82 (C-300), 42.46 (C-10), 36.81 (C-40); mp = 51–53 °C.
Compound 11: 13C NMR (DMSO) d: 166.40 (C@O), 152.20 and
150.74 (C-4), 150.15 (C-2), 140.70 (C-5), 134.75 (C-100), 131.39 (C-
400), 131.20 (C-200), 130.14 and 129.50 (C-6), 127.82 (C-20), 127.30
(C-30), 125.49 (C-300), 44.30 (C-10), 36.51 (C-40); MS m/z 353.10
[MH]+. Anal. (C16H14F3N3O3) C, H, N.
1
Compound 16: H NMR (DMSO) d: 4.41 (d, 2H, J3 = 6.84, H-10);
5.62 (m, 1H, H-20); 5.49 (m, 1H, H-30); 4.02 (t, 2H, J3 = 6.00, H-40);
8.74 (br, 1H, NH); 7.92 (dd, 2H, J1–3 = 8.70; J2–4 = 8.70, H-300); 7.29
5.2.3.5. (Z)-1-(40-Benzamide-20-butenyl)cytosine (5). The proce-
dure was carried out by using cytosine (166 mg, 1.5 mmol) which
gave 5 (123 mg, 43%, mp = 207–212 °C). 13C NMR (DMSO) d: 165.98
(C@O), 165.89 (C-4), 155.74 (C-2), 145.60 (C-6), 134.30 (C-100),
131.15 (C-400), 129.98 (C-200), 128.26 (C-20), 127.11 (C-30), 126.90
(C-300), 93.55 (C-5), 45.22 (C-10), 36.20 (C-40); MS m/z 284.13
[MH]+. Anal. (C15H16N4O2) C, H, N.
13
(d, 2H, J3 = 8.79, H-200); C NMR (DMSO) d: 166.13 (C-400), 130.45
(C-20), 131.27 and 131.22 (C-100), 130.34 and 130.22 (C-300),
127.43 (C-30), 115.84 and 115.55 (C-200), 94.12 (C-5), 42.75 (C-10),
36.77 (C-40); mp = 47–49 °C.
5.2.2. General procedure for the preparation of (Z)-1-(40-benz-
amide-20-butenyl)pyrimidine (1–6) and adenine (13) or (Z)-1-[40-
(400-fluoro)benzamide-20-butenyl]pyrimidine (7–10) derivatives
Compound 15 (210 mg, 1.0 mmol) or 16 (228 mg, 1.0 mmol) were
added after 2 h to a stirred mixture containing pyrimidine or purine
bases and NaH (1.2 equiv) in DMF (20 ml) the. Reaction mixture
was then stirred overnight at room temperature. The resultant solu-
tion was concentrated to dryness in vacuo and purified by column
chromatography (silica gel, CH2Cl2/MeOH = 20:1) which gave pure
white crystals of pyrimidine (1–12) and adenine (13) derivatives.
5.2.3.6. (Z)-1-(40-Benzamide-20-butenyl)-5-fluorocytosine (6).
The procedure was carried out by using cytosine (195 mg,
1.5 mmol) which gave 6 (152 mg, 50%, mp = 186–193 °C). 13C
NMR (DMSO) d: 166.04 (C@O), 157.45 and 157.37 (C-4), 154.00 s
(C-2), 134.28 (C-100), 131.17 (C-400), 130.37 (C-200), 130.22 and
130.02 (C-6), 128.27 (C-20), 127.10 (C-30), 126.41 (C-300), 136.46
(C-5), 45.25 (C-10), 36.21 (C-40); MS m/z 302.12 [MH]+. Anal.
(C15H15FN4O2) C, H, N.
5.2.3. Compounds preparation
5.2.3.7. (Z)-1-[40-(400-Fluoro)benzamide-20-butenyl]uracil (7).
The procedure was carried out by using uracil (448 mg, 4.0 mmol)
which gave 7 (156 mg, 51%, mp = 151–154 °C).
5.2.3.1. (Z)-1-(40-Benzamide-20-butenyl)uracil (1). The proce-
dure was carried out using uracil (448 mg, 4.0 mmol) which gave
1 (153 mg, 54%, mp = 143–157 °C). 13C NMR (DMSO) d: 166.67
(C@O), 164.17 (C-4), 151.39 (C-2), 145.72 (C-6), 134.77 (C-100),
131.69 (C-400), 131.50 (C-200), 128.78 (C-20), 127.63 (C-30), 126.16
(C-300), 101.67 (C-5), 44.54 (C-10), 36.86 (C-40); MS m/z 285.11
[MH]+. Anal. (C15H15N3O3) C, H, N.
13C NMR (DMSO) d: 165.97 (C@O), 165.59 (C-400), 164.15 (C-4),
151.37 (C-2), 145.70 (C-6), 131.40 (C-20), 131.25 and 131.21 (C-
100), 130.37 and 130.22 (C-300), 126.21 (C-30), 115.84 and 115.50
(C-200), 101.66 (C-5), 44.53 (C-10), 36.91 (C-40); MS m/z 303.10
[MH]+. Anal. (C15H14FN3O3) C, H, N.
5.2.3.2. (Z )-1-(40-Benzamide-20-butenyl)-5-fluorouracil (2) and
(Z)-1,3-di-(40-benzamide-20-butenyl)-5-fluorouracil (12). The
procedure was carried out by using 5-fluorouracil (650 mg,
5.0 mmol) which gave 2 (145 mg, 48%, mp = 139–141 °C) and 12
(90 mg, 39%, mp = 152–154 °C).
5.2.3.8. (Z)-1-[40-(400-Fluoro)benzamide-20-butenyl]thymine (8).
The procedure was carried out by using thymine (504 mg,
4.0 mmol) which gave 8 (139 mg, 44%, mp = 199–204 °C). 13C
NMR (DMSO) d: 166.00 (C@O), 165.54 (C-400), 164.70 (C-4),
151.32 (C-2), 141.48 (C-6), 131.37 (C-30), 131.32 and 131.25 (C-
100), 130.32 and 130.20 (C-300), 126.37 (C-20), 115.86 and 115.57
(C-200), 109.25 (C-5), 44.14 (C-10), 36.89 (C-40); MS m/z 317.12
[MH]+. Anal. (C16H16FN3O3) C, H, N.
Compound 2: 13C NMR (DMSO) d: 166.66 (C@O), 158.17 and
157.83 (C-4), 150.11 (C-2), 138.68 (C-5), 134.71 (C-100), 131.71
(C-400), 131.60 (C-200), 130.37 and 129.92 (C-6), 128.88 (C-20),
127.80 (C-30), 125.82 (C-300), 44.70 (C-10), 36.81 (C-40); MS m/z
303.10 [MH]+. Anal. (C15H14FN3O3) C, H, N.
5.2.3.9. (Z)-1-(40-400-Fluorobenzamide-20-butenyl)cytosine (9).
The procedure was carried out by using cytosine (166 mg,
1.5 mmol) which gave 9 (126 mg, 42%, mp = 187–197 °C). 13C
NMR (DMSO) d: 166.39 (C@O), 166.00 (C-400), 165.52 (C-4),
156.31 (C-2), 146.11 (C-6), 130.37 (C-20), 131.25 and 131.21 (C-
100), 130.32 and 130.20 (C-300), 127.43 (C-30), 115.84 and 115.55
(C-200), 94.12 (C-5), 44.75 (C-10), 36.77 (C-40); MS m/z 302.12
[MH]+. Anal. (C15H15FN4O2) C, H, N.
Compound 12: 13C NMR (DMSO) d: 166.21 (C@O), 157.87 and
156.54 (C-4), 151.34 (C-2), 139.08 (C-5), 134.21 (C-100), 132.21
(C-400), 131.83 (C-200), 131.07 and 129.63 (C-6), 127.88 (C-20),
126.92 (C-30), 124.88 (C-300), 44.74 (C-10), 36.87 (C-40); MS m/z
476.20 [MH]+. Anal. (C26H25FN4O4) C, H, N.
5.2.3.3. (Z)-1-(40-Benzamide-20-butenyl)thymine (3). The proce-
dure was carried out using thymine (504 mg, 4.0 mmol) which
gave 3 (138 mg, 49%, mp = 130–138 °C). 13C NMR (DMSO) d:
166.59 (C@O), 164.70 (C-4), 151.33 (C-2), 141.49 (C-100), 134.72
(C-6), 131.71 (C-400), 131.46 (C-300), 128.79 (C-20), 127.61 (C-30),
126.31 (C-200), 109.25 (C-5), 44.14 (C-10), 36.83 (C-40); MS m/z
299.13 [MH]+. Anal. (C16H17N3O3) C, H, N.
5.2.3.10. (Z)-1-[40-(400-Fluoro)benzamide-20-butenyl]-5-fluorocy-
tosine (10). The procedure was carried out by using 5-fluorocyto-
sine (195 mg, 1.5 mmol) which gave 10 (157 mg, 49%, mp = 170–
13
174 °C). C NMR (DMSO) d: 165.97 (C@O), 165.51 (C-400), 158.00
and 157.82 (C-4), 154.51 (C-2), 130.41 (C-6), 130.78 (C-20),
131.25 and 131.22 (C-100), 130.30 and 130.18 (C-300), 126.96 (C-
30), 115.84 and 115.55 (C-200), 137.76 (C-5), 44.77 (C-10), 36.78 (C-
40); MS m/z 320.11 [MH]+. Anal. (C15H14F2N4O2) C, H, N.
5.2.3.4. (Z)-1-(40-Benzamide-20-butenyl)-5-trifluoromethyluracil
(4) and (Z)-3-(40-benzamide-20-butenyl)-5-trifluoromethyluracil
(11). The procedure was carried out by using 5-trifluoromethyl-
uracil (716 mg, 4.0 mmol) which gave 4 (151 mg, 43%, mp = 135–
138 °C) and 11 (24 mg, 3.4%, mp = 131–133 °C).
5.2.3.11. (Z)-9-(40-Benzamide-20-butenyl)adenine (13). The pro-
cedure was carried out by using adenine (203 mg, 1.5 mmol) which

6256
K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
gave 13 (139 mg, 45%, mp = 150–152 °C). 13C NMR (DMSO) d:
166.69 (C@O), 156.43 (C-4), 152.94 (C-8), 149.7 (C-6), 141.69 (C-
2), 134.83 (C-100), 131.69 (C-400), 131.34 (C-300), 128.78 (C-30),
127.64 (C-20), 131.34 (C-300), 119.14 (C-5), 40.82 (C-10), 36.85 (C-
40); MS m/z 308.14 [MH]+. Anal. (C16H16N6O) C, H, N.
noma), HEp-2 (larynx carcinoma), MCF-7 (breast carcinoma), SK-
BR-3 (adenocarcinoma metastatic), and WI38 (normal diploid hu-
man fibroblasts), were cultured as monolayers and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 2 mM
L-glutamine, 100 U/ml penicil-
lin and 100 g/ml streptomycin in a humidified atmosphere with
l
5.3. X-ray determination of compounds 11, 12, and 13
5% CO2 at 37 °C.
Colorless single crystals of 11 and 12 suitable for X-ray single
crystal analysis were obtained at room temperature by partial
evaporation from a methanol solution. Colorless single crystal of
13 was obtained from an ethanol solution applying the same crys-
tallization method. The intensities for 12 and 13 were collected at
295 K on a Oxford Diffraction Xcalibur2 diffractometer using
5.4.2. Proliferation assays
The cytostatic activity against L1210, Molt4/C8, and CEM cells
were measured in 200 ll-wells of a 96-well microtiter plate (initial
cell number: 5–7.5 ꢂ 104 cells/well) essentially as originally de-
scribed.9. After 48 (L1210) or 72 h (CEM, Molt4/C8), the tumor cell
number was determined by a Coulter counter. For the antiprolifer-
ative assays, a panel of monolayer tumor cell lines (larynx carci-
noma HEp-2, cervical carcinoma HeLa, breast carcinoma MCF-7,
metastatic breast adenocarcinoma SK-BR-3, pancreatic carcinoma
MiaPaCa-2, colon carcinoma SW 620, and human normal fibro-
blasts (WI38) were inoculated in standard 96-well microtiter
plates on day 0, at 3000–6000 cells per well according to the dou-
bling times of the specific cell line. Test agents were then added in
five, 10-fold dilutions (1 ꢂ 10ꢀ8–1 ꢂ 10ꢀ4 M) and incubated for an-
other 72 h. The solvent (DMSO) was also tested for eventual inhib-
itory activity by adjusting its concentration to be the same as in the
working concentrations (DMSO concentration never exceeded
0.1%). After 72 h of incubation, the cell growth rate was evaluated
by performing the MTT assay: experimentally determined absor-
bance values were transformed into a cell percentage growth
(PG) using the formulas proposed by NIH and described previ-
ously.6 This method directly relies on control cells behaving nor-
mally at the day of assay because it compares the growth of
treated cells with the growth of untreated cells in control wells
on the same plate—the results are therefore a percentile difference
from the calculated expected value.
graphite-monochromated MoKa radiation (k = 0.71073 Å). The
intensities for 11 were collected on the same instrument at
130 K. CrysAlis17 programs were used for data collection and reduc-
tion. The crystal structures were solved by direct methods.18 All
nonhydrogen atoms were refined anisotropically by full-matrix
least-squares calculations based on F2.18 The hydrogen atoms of
the N1 and N2 atoms in 11, N2 and N4 atoms in 12, and N2, N6
and O2 atoms in 13 were found in a difference Fourier map and
their coordinates and isotropic thermal parameters have been re-
fined freely. All other hydrogen atoms were included in calculated
positions as riding atoms, with SHELXL9718 defaults. Examination of
the refined structure of 12 revealed that this structure contains sol-
vent accessible void of 72.00 Å3. This compound crystallizes with a
solvent molecule which is severely disordered (the maximum elec-
tron density at the center of the void is 0.57 eÅꢀ3), and could not be
refined satisfactorily. The data are therefore corrected using the
19
19
SQUEEZE routine in PLATON
.
PLATON program was used for structure
analysis and drawings preparation. CCDC 773593–773595 contain
the supplementary crystallographic data for this Letter. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Crystal data for 11: crystal dimension 0.17 ꢂ 0.26 ꢂ 0.70 mm3;
The IC50 values for each compound were calculated from dose–
response curves using linear regression analysis by fitting the test
concentrations that give PG values above and below the reference
value. If, however, all of the tested concentrations produce PGs
exceeding the respective reference level of effect (e.g., PG value
of 50) for a given cell line, the highest tested concentration is as-
signed as the default value (in the screening data report that de-
fault value is preceded by a ‘>’ sign). Each test point was
performed in quadruplicate in three individual experiments. Final-
ly, the effects of the tested substances were evaluated by plotting
the mean percentage growth for each cell type in comparison to
control on dose–response graphs.
C16H14F3N3O3, Mr = 353.30, monoclinic space group P21/c; a =
8.4391(6), b = 21.2803(18), c = 11.9798(10) Å, b = 133.542(5)°, V =
1559.5(2) Å3; Z = 4; dx = 1.505 g cmꢀ3 ) = 0.129 mmꢀ1
(MoK
S = 0.976; R/wR = 0.0476/0.1164 for 234 parameters and 2690
reflections with I P 2 (I), R/wR = 0.0751/0.1262 for all 3727 inde-
;
l
a
;
r
pendent reflections measured in the range 8.80°–2h–56.00°.
Crystal data for 12: crystal dimension 0.19 ꢂ 0.36 ꢂ 0.60 mm3;
ꢀ
C
26H25FN4O4, Mr = 476.50, triclinic space group P; a = 9.4313(3),
b = 10.9594(4), c = 12.4687(4) Å,
c
a
= 89.210(3), b = 87.665(2),
= 70.720(3)°, V = 1215.49(7) Å3; Z = 2; dx = 1.302 g cmꢀ3
(Mo-
) = 0.095 mmꢀ1; S = 0.803; R/wR = 0.0387/0.0846 for 324 param-
(I), R/wR = 0.0953/0.1048 for
; l
Ka
eters and 3035 reflections with I P 2
r
5.4.3. Antiviral activity assays
all 5856 independent reflections measured in the range 7.62°–2h–
56.00°.
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity in HEL [herpes simplex
virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular
stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis, Cox-
sackie B4, and Punta Toro virus), HeLa (vesicular stomatitis virus,
Coxsackie virus B4, and respiratory syncytial virus) or MDCK [influ-
enza A (H1N1; H3N2) and influenza B] cell cultures. Confluent cell
cultures (or nearly confluent for MDCK cells) in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1 CCID50 being the
virus dose to infect 50% of the cell cultures). After a 1 h virus
adsorption period, residual virus was removed, and the cell cul-
tures were incubated in the presence of varying concentrations
Crystal data for 13: crystal dimension 0.27 ꢂ 0.34 ꢂ 0.47 mm3;
C
16H16N6OꢁH2O, Mr = 326.36, monoclinic space group P21/c; a =
15.5183(5), b = 8.2078(2), c = 13.2945(4) Å, b = 107.517(3)°, V =
1614.81(8) Å3; Z = 4; dx = 1.342 g cmꢀ3 ) = 0.094 mmꢀ1
(MoK
S = 0.954; R/wR = 0.0369/0.0627for 237 parameters and 2535
reflections with I P 2 (I), R/wR = 0.0984/0.1041 for all 3873 inde-
;
l
a
;
r
pendent reflections measured in the range 7.40°–2h–56.00°.
5.4. Cytostatic and antiviral activity assays
5.4.1. Cell culturing
(200, 40, 8. . . lM) of the test compounds. Viral cytopathicity was
The suspension cell lines L1210, Molt4/C8, and CEM were cul-
tured in RPMI-1640 medium supplemented with 10% fetal bovine
serum, 2 mM L-glutamine and 0.075% NaHCO3 in a humidified
atmosphere with 5% CO2 at 37 °C. The cell lines HeLa (cervical car-
cinoma), SW 620 (colon carcinoma), MiaPaCa-2 (pancreatic carci-
recorded as soon as it reached completion in the control virus-in-
fected cell cultures that were not treated with the test compounds.
The methodology of the anti-HIV assays was as follows: human
CEM cells (ꢃ3 ꢂ 105 cells/ml) were infected with 100 CCID50 of HI-
V(IIIB) or HIV-2(ROD)/ml and seeded in 200-ll wells of a microtiter

K. Benci et al. / Bioorg. Med. Chem. 18 (2010) 6249–6257
6257
plate containing appropriate dilutions of the test compounds. After
Supplementary data
4 days of incubation at 37 °C, HIV-induced giant cell formation was
examined microscopically.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.035.
5.4.4. Cytotoxicity assays
Cytotoxicity measurements were based on the inhibition of HEL
cell growth. HEL cells were seeded at a rate of 5 ꢂ 103 cells/well
into 96-well microtiter plates and allowed to proliferate for 24 h.
Then, medium containing different concentrations of the test com-
pounds was added. After 3 days of incubation at 37 °C, the cell
number was determined by a Coulter counter. The cytostatic con-
centration was calculated as the CC50, the compound concentration
required to reduce cell growth by 50% relative to the number of
cells in the untreated controls. CC50 values were estimated from
graphic plots of the number of cells (percentage of control) as a
function of the concentration of the test compounds. Cytotoxicity
was expressed as minimum cytotoxic concentration (MCC) or the
compound concentration that causes a microscopically detectable
alteration of cell morphology.
References and notes
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´
´
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Support for this study was provided by the Ministry of Science,
Education and Sports of the Republic of Croatia (Projects # 125-
0982464-2922, # 098-0982464-2393, # 335-098-703532 and #
119-1193079-3069) and by the ‘Geconcerteerde Onderzoeksacties’
of the Katholieke Universiteit Leuven (project # 05/19). We thank
Lizette van Berckelaer for excellent technical assistance in per-
forming part of the antitumor cell activity assays, as well as Leen
Ingels, Leentje Persoons, Frieda De Meyer, Vicky Broeckx, Anita
Camps and Lies Vandenheurck for excellent technical assistance
in performing the antiviral activity assays.
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