Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues

Article abstract of DOI:10.1016/j.bmc.2008.03.074

A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1-14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC₅₀ = 4.3 μM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC₅₀ = 18 μM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.

Full text of DOI:10.1016/j.bmc.2008.03.074

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5624–5634
Synthesis, cytostatic and anti-HIV evaluations of the
new unsaturated acyclic C-5 pyrimidine nucleoside analogues
a
a
a
b
´
´
Tatjana Gazivoda, Silvana Raic-Malic, Vedran Krisˇtafor, Damjan Makuc,
b
c
c
c
ˇ
´
´
´
ˇ
´
Janez Plavec, Sinisa Bratulic, Sandra Kraljevic-Pavelic, Kresimir Pavelic,
Lieve Naesens,d Graciela Andrei,d Robert Snoeck,d Jan Balzarinid and Mladen Mintasa,*
aDepartment of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
Marulic´ev trg 19, HR-10000 Zagreb, Croatia
bSlovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, POB 660, SL-1001 Ljubljana, Slovenia
c
-
Division of Molecular Medicine, Ruder Bosˇkovic´ Institute, Bijenicˇka 54, POB 1016, HR-10001 Zagreb, Croatia
dRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 8 January 2008; revised 20 March 2008; accepted 28 March 2008
Available online 1 April 2008
Abstract—A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1–14) in which the sugar moiety was replaced by the
conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using
Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in
general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all eval-
uated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC50 = 4.3 lM). However, this compound was
also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep
G2 (IC50 = 18 lM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity
against HIV-1 and HIV-2.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
thermore, thymine with a 2-butenyl spacer was the first
acyclic nucleoside analogue exhibiting potent inhibition
of thymidine kinase 2 (TK-2) which catalyzes phosphor-
ylation of certain antiviral drugs.7
Many nucleoside analogues with interesting biological
properties have arisen by substitution at the 5-position
of the uracil base in the 20-deoxyuridine series.1 Pyrimi-
dine nucleosides containing C-5 alkynyl groups have
been shown to possess significant antiviral and/or anti-
cancer properties.2 The 5-alkynyluracil nucleosides with
a longer alkynyl chain at the C-5 position expressed
appreciable antiviral activity in contrast to the corre-
sponding alkyl derivatives that showed decreasing anti-
viral activity with increasing C-5 side chain length.2–5
Introduction of very rigid allenic moiety as a linker be-
tween the heterocyclic base and hydroxymethyl group
led to compounds such as adenallene and cytallene
which effectively inhibited the replication of HIV.6 Fur-
We have reported that some pyrimidines and purines
containing
c-(Z)-ethylidene-2,3-dibenzylbutenolide
exhibited antiproliferative effects against malignant hu-
man tumour cell lines.8–10 The C-5 alkynyl substituted
pyrimidine derivatives of L-ascorbic acid had some,
although slight, inhibitory potency against cytomegalo-
virus (CMV) and varicella-zoster virus (VZV).11 The 5-
propynyl uracil derivative of L-ascorbic acid showed
pronounced cytostatic activity against cervical carci-
noma (HeLa).12 We have also reported that the (Z)-4-
amino-2-butenyladenine nucleoside analogue showed
selective activity against HIV-1.13 These results
prompted us to synthesize a series of the novel (Z)-
and (E)-2-butenyl-N-phthalimido pyrimidines contain-
ing an alkynyl longer side chain at C-5 (1–14) (Fig. 1)
and evaluate their cytostatic and anti-HIV potency in
cell culture.
Keywords: Unsaturated acyclic pyrimidine nucleoside analogues; Z-
and E-isomers; Cytostatic evaluations; Anti-HIV activity.
*
Corresponding author. Tel.: +385 1 4597 214; fax: +385 1 4597
224; e-mail: mladen.mintas@fkit.hr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.074

T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
5625
O
R
R
C
O
N
R
C
(CH2)3 CH3
(CH2)7 CH3
(CH2)2
C
HN
C
HN
O
N
O
O
O
Br
N
O
CH3
N
O
(CH2)3 CH3
(CH2)4 CH3
8-14
1-7
Figure 1. Unsaturated acyclic C-5 alkynyl substituted pyrimidine nucleoside analogues 1–14.
2. Results and discussion
2.1. Compounds preparation
due to small Dd/J ratios. As an example of our simula-
tions, multiplets of H20 and H30 in 14 are compared to
4
0
0
the experimental spectrum in Figure 3. Vinyl JH1 H3
4
0
0
and JH2 H4 couplings were below signal half width
The key precursors (Z)- (IIa) and (E)-1-[40-(N-phthalim-
ido)-20-butenyl]-5-iodouracil (IIb) for cross-coupling
with alkynes were synthesized by condensation of the
(Z)-4-chloro-2-butenyl-N-phthalimide (Ia) and (E)-4-
bromo-2-butenyl-N-phthalimide (Ib) with 5-iodoura-
cil.13 The (Z)-4-chloro-2-butenyl (Ia) and (E)-4-bromo-
2-butenyl (Ib) derivatives of N-phthalimide were synthe-
sized by using a classical Gabriel reaction.14 Different
substituents at C-5 position of the pyrimidine ring in
1–7 and 8–14 were introduced by the reaction of the 5-
iodouracil derivatives IIa and IIb under optimized Sono-
gashira Pd(0)-catalyzed reactions.15–20 It is important to
note that this reaction was performed at room tempera-
ture in order to avoid the formation of bicyclic furopyr-
imidine co-products produced by heating the reaction
mixture. Reaction of the (Z)- and (E)-1-[40-(N-phthalim-
ido)-20-butenyl]-5-iodouracil with various terminal al-
kynes in the presence of palladium(0) catalyst and
copper(I) iodide as co-catalyst in DMF at room temper-
ature gave 5-substituted Z- and E-uracil derivatives 1–7
and 8–14 (Scheme 1).
(<1 Hz). Smaller JH2 H3 coupling constants of 10.6,
10.6 and 11.1 Hz in 2, 3 and 7, respectively, are in accor-
dance with Z-isomers along C20@C30 double bonds. On
the other hand, larger JH2 H3 coupling constants of
15.7, 15.5 and 15.7 Hz in 8, 12 and 14, respectively,
are in conformity with E-isomers.
3
0
0
3
0
0
E-Isomers exhibit larger chemical shift difference be-
tween H20 and H30 (Dd 0.06–0.07 ppm) than Z-isomers
1
(Dd 0.02–0.04 ppm). The other significant H and 13C
chemical shift differences between E- and Z-isomers
were observed, and are presented in Figure 4. Perusal
of Figure 4 shows that there is a clear distinction in sev-
eral 1H and 13C chemical shifts with respect to Z- and E-
configurations along the C20@C30 double bond.
In our further analysis, we have focused on conforma-
tional properties of novel pyrimidine-2,4-dione nucleo-
3
3
0
0
0
0
side analogues. JH1 H2 and JH3 H4 coupling constants
showed unrestricted rotation along the involved single
bonds. In full agreement, protons of both methylene
groups are isochronous. Evaluation of NOE enhance-
ments between H6 and protons of N1 substituents did
not show any interaction that would indicate preference
for any of conformational states. In addition, NOE data
did not indicate close spatial proximity of moieties at-
tached to N1 and C5 of pyrimidine-2,4-dione.
1
2.2. H and 13C NMR studies
1H and 13C NMR data given in Table 1 and Section 5
are in full agreement with the proposed structures 1–
14 (Fig. 2).
1
Signals of H and 13C resonances were observed which
were characteristic for each of the moieties constituting
structures of N1, C5-substituted pyrimidine nucleoside
analogues (1–14). H10 and H40 protons exhibit distinct
chemical shifts between 4.09 and 4.60 ppm, whereas
H20 and H30 protons along C@C double bond were ob-
served between 5.50 and 5.80 ppm (Table 1). Chemical
shifts of phenyl protons are between 7.1 and 7.53 ppm,
while phthalimido protons are between 7.8 and
7.9 ppm. H6 chemical shifts were found in the range
from 7.82 to 8.47 ppm and H3 protons in the range from
3. Biological results
3.1. Cytostatic activity
Compounds 1–14 were evaluated for their cytostatic
activities against malignant human tumour cell lines:
murine leukemia (L1210), human T-lymphocyte
(Molt4/C8 and CEM), cervical carcinoma (HeLa), pan-
creatic carcinoma (MiaPaCa-2), colorectal adenocarci-
noma (SW 620), breast epithelial adenocarcinoma
(MCF-7) and hepatocellular carcinoma (Hep G2), as
well as normal diploid human fibroblasts (WI 38) (Table
2). Cytostatic activities of compounds 1–14 were com-
pared with those of 5-fluorouracil (5-FU). The novel
C-5 alkynyl substituted olefinic pyrimidine compounds
11.33 to 11.73 ppm. The configuration along C20@C30
3
0
0
double bond was evaluated through JH2 H3 coupling
constants (Table 1). The values of all coupling constants
involved in the six spin system of N1-substituent includ-
3
0
0
ing JH2 H3 had to be determined through simulation

5626
T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
O
O
N
O
O
N
Br
I
I
HN
HN
Cl
O
N
H
O
N
H
O
O
Ia
IU
Ib
IU
(i)
(i)
O
N
O
N
I
I
HN
HN
O
N
O
O
O
O
N
O
IIb
(ii)
IIa
(ii)
O
R
O
R
C
C
C
C
HN
HN
O
N
O
O
N
O
N
O
N
O
1-7
8-14
R
R
(CH ) CH
(CH ) CH
8
1
2 3
3
2 3
3
(CH ) CH
2 7
(CH ) CH
2 4
9
2
3
4
5
6
3
3
(CH )
2 2
(CH ) CH
2 5
10
11
3
Br
(CH ) CH
2 7
3
CH
3
(CH )
2 2
12
13
(CH ) CH
(CH ) CH
2 3
2 3
3
3
(CH ) CH
3
(CH ) CH
14
7
2 4
2 4
3
Scheme 1. Synthesis of the unsaturated acyclic C-5 substituted pyrimidine nucleoside analogues. Reagents and conditions: (i) NaH, DMF, argon
atmosphere, rt; (ii) R-C„CH, i-Pr2EtN, (PPh3)4Pd, CuI, DMF, rt.
(1–14) showed better antiproliferative activities on the
malignant tumour cells than their structurally related
C-5 unsubstituted uracil and C-5 fluoro-substituted ura-
cil derivatives containing 40-(N-phthalimido)-20-butenyl
side chain.13
than standard 5-fluorouracil (5-FU). Among E-isomers
of C-5 substituted alkynyl 1-[40-(N-phthalimido)-20-
butenyl]pyrimidine derivatives 8–14, compound 14 con-
taining the p-pentylphenylethynyl side chain at C-5
exhibited the best antiproliferative effect on all the eval-
uated cell lines (IC50 in the range of 4.3–39 lM) and nor-
mal fibroblasts (IC50 = 25 lM). Besides, compounds
with a C-5 hexynyl (8), heptynyl (9) and phenylbutynyl
(12) side chain showed cytostatic activity, particularly
against hepatocellular carcinoma (Hep G2; IC50 = 7.3–
9 lM) (Fig. 5). Thus, compounds 7 and 14 emerged as
the most interesting lead compounds with cytostatic
activities that could be used for further structural opti-
mization and biological studies on Hep G2 cells.21–23
In the series of Z-izomers of C-5 alkynyl 1-[40-(N-phtha-
limido)-20-butenyl] pyrimidine derivatives 1–7, com-
pounds containing a hexynyl (1), octynyl (2) and p-
butylphenylethynyl (6) side chain at C-5 exhibited mod-
erate cytostatic effects against all evaluated cell lines,
particularly against hepatocellular carcinoma (Hep G2;
IC50 = 16.6–40.2 lM) (Fig. 5). However, these com-
pounds were also cytotoxic against normal human fibro-
blasts (WI 38; IC50 = 16.7–49 lM) (Fig. 6).
3.2. Antiviral activities
On the contrary, compound 7 with the C-5 p-pentyl-
phenylethynyl side chain showed selective antiprolifera-
tive effect on the growth of hepatocellular carcinoma
(Hep G2, IC50 = 18 lM). However, this compound
had lesser cytostatic activity against Hep G2 cell line
Compounds 1–14 were evaluated for their inhibitory
activities against HIV-1 and HIV-2 in human T-lympho-
cyte (CEM) cells (Table 3). Compounds 3 and 4 showed
some specific albeit slight activity against HIV-1, while

Table 1. 1H NMR chemical shifts (d/ppm) and H–H coupling constants (J/Hz) of Z- and E-nucleoside analogues (1–7 and 8–14)
H40
H10
H20
H30
H300
/H2,H3,H4
Phth H4,H5 H6
7.8–7.9 (m) 8.07 (s) 11.67 (s)
H3
JH2 H3
0
0
Compound CH3
1
2
3
4
0.90 (t, J = 7.1) 4.38 (m)
4.6 (m)
5.5–5.7 (m)
2.40 (t, J = 6.7)
0.84 (t, J = 6.7) 4.36 (d, J = 6.6) 4.51 (d, J = 7.0) 5.60 (td, J = 7.0; 10.6) 5.63 (td, J = 6.6; 10.6) 2.36 (t, J = 6.7)
—
—
—
7.8–7.9 (m) 7.96 (s) 11.55 (s) 10.6
7.8–7.9 (m) 7.94 (s) 11.57 (s) 10.6
—
—
4.37 (d, J = 7.1) 4.52 (d, J = 6.9) 5.61 (td, J = 6.9; 10.6) 5.65 (td, J = 7.1; 10.6) 2.65 (m)
4.40 (d, J = 5.3) 4.56 (d, J = 4.7)
7.2–7.3 (m)
5.6–5.7 (m)
—
7.53 (d, J = 8.5) 7.8–7.9 (m) 8.24 (s) 11.73 (s)
7.42 (d, J = 8.6)
—
5
6
1.23 (s) 4.40 (m) 4.54 (m)
0.81 (t, J = 7.4) 4.31 (d, J = 5.3) 4.48 (d, J = 5.4)
5.7–5.8 (m)
5.6–5.7 (m)
—
—
7.3–7.4 (m)
7.30 (d, J = 8)
7.16 (d, J = 8)
7.23 (m)
7.38 (m)
—
7.8–7.9 (m) 8.27 (s) 11.43 (s)
7.8–7.9 (m) 8.11 (s) 11.60 (s)
—
—
7
0.84 (t, J = 6.7) 4.39 (d, J = 6.8) 4.56 (d, J = 6.8) 5.64 (td, J = 6.8; 11.1) 5.66 (td, J = 6.8; 11.1)
—
7.85 (m)
8.18 (s) 11.69 (s) 11.1
8
9
0.87 (t, J = 7.0) 4.17 (d, J = 5.0) 4.28 (d, J = 6.0) 5.66 (td, J = 6.0; 15.7) 5.73 (td, J = 5.0; 15.7) 2.35 (t, J = 6.7)
7.8–7.9 (m) 7.91 (s) 11.33 (s) 15.7
0.86 (t, J = 7.0) 4.15 (m)
4.30 (m)
4.52 (m)
4.30 (m)
5.6–5.7 (m)
5.6–5.7 (m)
5.6–5.7 (m)
2.34 (t, J = 6.9)
2.68 (m)
2.34 (t, J = 6.8)
—
—
7.8–7.9 (m) 7.91 (s) 11.41 (s)
7.8–7.9 (m) 8.47 (s) 11.50 (s)
7.8–7.9 (m) 7.91 (s) 11.33 (s)
—
—
—
10
11
12
13
0.84 (m)
0.85 (s)
—
4.14 (m)
4.15 (m)
—
7.1–7.3 (m)
4.18 (d, J = 5.1) 4.24 (d, J = 5.5) 5.66 (td, J = 5.5; 15.5) 5.72 (td, J = 5.1; 15.5) 2.63 (m)
7.8–7.9 (m) 7.82 (s) 11.52 (s) 15.5
—
0.78 (t, J = 7.3) 4.09 (d, J = 4.9) 4.18 (d, J = 5.1)
5.6–5.7 (m)
—
—
7.25 (d, J = 7.9) 7.7–7.8 (m) 7.97 (s) 11.55 (s)
7.12 (d, J = 7.9)
14
0.85 (t, J = 6.9) 4.19 (d, J = 5.5) 4.28 (d, J = 5.7) 5.69 (td, J = 5.7; 15.7) 5.75 (td, J = 5.5; 15.7)
7.35 (m)
7.22 (m)
7.8–7.9 (m) 8.07 (s) 11.64 (s) 15.7
Additional data: 1: H400 and H500 (1.4–1.5, m); 2: H400–H900 (1.2–1.5, m); 3: H400 (2.81, m); 6: H1000 (2.52, t, J = 7.7), H2000 (1.47, p, J = 7.6), H3000 (1.22, hex, J = 7.3); 7: H4000 (1.28, m), H3000 (1.27, m), H2000
(1.56, m), H1000 (2.58, t, J = 7.6); 8: H500 (1.42, m), H400 (1.45, m); 9: H400–H600 (1.1–1.5, m); 10: H400–H700 (1.2–1.5, m); 11: H400–H900 (1.2–1.4, m); 12: H400 (2.79, m); 13: H1000 (2.48, t, J = 7.6), H2000 (1.44, p,
J = 7.5), H3000 (1.18, hex, J = 7.3); 14: H3000 (1.26, m), H4000 (1.32, m), H2000 (1.56, m), H1000 (2.58, t, J = 7.6).

5628
T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
3'''
compound 14 exhibited moderate activity against both
1'''
4'''
HIV-1 and HIV-2. None of the other viruses were sensi-
tive to the inhibitory activity of the tested compounds
except for compounds 2 (EC50 = 2.5–4 lM) and 5
(EC50 = 20–21 lM) against influenza A (H3N2), for
φ
3
6
2'''
φ2
φ
4
5
φ1
O
4
φ
compound
14
against
Coxsackie
B4
virus
C
2''
φ
(EC50 = 12 lM) and vaccinia virus (EC50 = 12 lM) and
for compound 7 for varicella-zoster virus (EC50 = 9.4–
8.7 lM). However, the EC50 values of the compounds
were close to their toxicity threshold, and therefore, it
is unclear whether the observed activity is due to a spe-
cific antiviral effect, or indirectly, to a cytotoxic/cyto-
static activity of the tested compounds.
C
1''
HN
3
2
5
6
O
1
Phth4
Phth2
Phth9
O
N
Phth5
Phth6
Phth1
Phth3
Phth8
N
1'
4'
2'
3'
Phth7
O
4. Conclusions
Figure 2. Atom numbering given in Table
1 is exemplified on
The novel (Z)- (1–7) and (E)- (8–14) unsaturated acyclic
C-5-alkynyl uracil derivatives were prepared by using
Sonogashira coupling of (Z)- and (E)-1-[40-(N-phthalim-
ido)-20-butenyl]-5-iodouracil with various terminal
alkynes.
compound 6. Note that chemical shifts are equal for /2 and /6, /3
and /5, Phth2 and Phth9, Phth3 and Phth 8, Phth 4 and Phth7, Phth5
and Phth6 due to symmetry.
By comparison of the cytostatic activities of the Z- and
E-isomers of C-5 alkynyl olefinic pyrimidine deriva-
tives, we can infer that the E-isomers (8–14) showed
better cytostatic activities. Furthermore, introduction
of the phenyl ring in longer side chain caused an
increasing cytostatic effect. Thus, the C-5 pentylpheny-
lethynyl substituted olefinic pyrimidine derivative 14
showed the most pronounced inhibitory activity
against all the tested tumour cell lines (IC50 = 4.3–
39 lM), including human normal fibroblasts. On the
contrary, its Z-isomer (7) showed selective inhibitory
activity against the Hep G2 cell line (IC50 = 18 lM).
Thus, the results of the cytostatic examinations indi-
cated that compounds 7 and 14 are suitable candidates
for further biological studies on lead drug and struc-
tural optimization.
1
Figure 3. Part of H NMR spectra of 14 showing H30 and H20
multiplets. (a) Experimental spectrum acquired in DMSO-d6 at 298 K;
(b) simulated spectrum (A2BCD2 spin system).
Figure 4. Comparison of 1H and 13C chemical shift in Z- and E-isomers. 1H and 13C chemical shifts axes are indicated on the left and the right side of
13
the plot, respectively. 1H and C chemical shifts are labelled with filled (dH10, j H40) and blank symbols (s C10, h C40), respectively.

T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
5629
Table 2. Inhibitory effects of compounds 1–14 on the growth of malignant tumour cell lines
a
Compound
IC50 (lM)
L1210
Molt4/C8
CEM
HeLa
MCF-7
MiaPaCa-2
SW 620
Hep G2
WI 38
1
109
45
41
36
100
39
33
59
45
64
>100
68
82
67
17
39
28
49
2
3
143
448
245
31
146
89
168
80
>100
87
>100
>100
>100
43
>100
>100
>100
56
>100
>100
>100
>100
>100
>100
58
>100
89
68
>100
47
4
5
266
31
238
23
>100
85
>100
17
6
40
18
7
28
30
102
192
>500
28
>100
89
91
>100
8.8
44
>100
77
60
>100
35
40
8
236
>500
43
171
>500
35
7.9
9
9
10
11
12
13
14
34
24
66
30
32
87
14
43
40
53
>250
44
42
>250
39
29
>250
38
31
>100
29
94
>100
27
79
13
44
11
>100
30
7.3
9.1
4.3
31
40
39
17
23
27
5.6
26
25
5-FUb
0.69
20
9.23
16
4.5
6.5
8.7
9.2
10
a IC50, 50% inhibitory concentration, or compound concentration required to inhibit tumour cell proliferation by 50%.
b The cytostatic effects of 5-fluorouracil against all tumour cell lines except for Hep G2 were published.1
5. Experimental
5.1. General methods
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, H–H
coupling constants involved as well as with the use of a
series of 2D experiments (gHSQC and gHMBC). Chemi-
cal shifts and coupling constants for H10–H20–H30–H40
spin system were obtained by spin simulation using
VNMRJ 2.1B software. 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 chromatography (CLC) was per-
formed using silica gel (0.063–0.2 mm) Fluka; glass col-
umn was slurry-packed under gravity. Compounds
purity was analyzed by HPLC with DAD detector.
Melting points were determined on a Kofler micro hot-
stage apparatus (Reichert, Wien) and are uncorrected.
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 recorded on Var-
ian Gemini 300 MHz, Varian Unity Inova 300 MHz and
Varian NMR System 800 MHz NMR spectrometers. All
the data were recorded at 298 K. NMR samples of all
compounds were prepared in DMSO-d6. Chemical shifts
were referenced to the residual solvent signal of DMSO
Figure 5. Dose–response curves of the tested compounds 1–14 on hepatocellular carcinoma (Hep G2).

5630
T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
Figure 6. Dose–response curves of the tested compounds 1–14 on the normal diploid fibroblasts (WI 38).
Table 3. Anti-HIV-1 and HIV-2 activities of compounds 1–14 in
human T-lymphocyte (CEM) cells
ethyl acetate = 1:1) afforded IIa as white crystals
(735 mg, 48.1%, mp = 218–219 ꢁC) after recrystalliza-
tion from MeOH.
Compound
EC50 (lM)
HIV-1
HIV-2
1H NMR: d 11.59 (s, 1H, NH), 8.12 (s, 1H, H-6), 7.76–
7.80 (m, 4H, Phth), 5.52–5.57 (m, 2H, H-20, H-30), 4.43
(d, 2H, J = 6.0 Hz, H-10), 4.29 (d, 2H, J = 5.9 Hz, H-40)
ppm.
1
2
P50
>50
35
P50
>50
P50
>50
>250
>10
>2
3
4
10
5
>250
>10
>2
13C NMR: d 167.49 (C2-Phth), 160.93 (C4), 150.56 (C2),
149.42 (C6), 134.40 (C5-Phth), 131.67 (C3-Phth), 128.10
(C30), 127.26 (C20), 123.03 (C4-Phth), 68.71 (C5), 44.36
(C10), 34.45 (C40) ppm. MS m/z 438.1 (M+Å). Anal.
(C16H12IN3O4) C, H, N.
6
7
8
P50
>50
P10
>50
P10
>10
14
P50
>50
P10
>50
P10
>10
3.1
9
10
11
12
13
14
5.2.2. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-iodouracil
(IIb). To a stirred mixture of 5-iodouracil (1 g,
4.2 mmol) and 60% NaH (101 mg, 4.2 mmol) in DMF
(40 mL), (E)-4-bromo-2-butenyl-N-phthalimide (Ib,
980 mg, 3.5 mmol) was added. The reaction mixture
was stirred under argon atmosphere for 4 h at 50 ꢁC
and then at room temperature overnight. The solvent
was evaporated, saturated NH4Cl (100 mL) was added
and the solution was extracted with EtOAc (3·
70 mL). Combined organic extracts were dried over
MgSO4. The solvent was evaporated and the residue
purified by column chromatography (CH2Cl2/
MeOH = 60:1), which afforded IIb as white crystals
(810 mg, 52.9%, mp = 221–222 ꢁC) after recrystalliza-
tion from MeOH.
EC50, effective concentration or concentration required to protect
CEM cells against the cytopathogenicity of HIV by 50%.
5.2. Compounds preparation
(Z)-4-Chloro-2-butenyl-N-phthalimide (Ia) and (E)-4-
bromo-2-butenyl-N-phthalimide (Ib) were prepared as
described previously.13
5.2.1. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-iodouracil
(IIa). To a stirred mixture of 5-iodouracil (1 g,
4.2 mmol) and 60% NaH (101 mg, 4.2 mmol) in DMF
(40 mL) after 1 h (Z)-4-chloro-2-butenyl-N-phthalimide
(Ia, 824 mg, 3.5 mmol) was added at room temperature.
The reaction mixture was stirred under argon atmo-
sphere at 40 ꢁC for 3 h, then at room temperature over-
night and concentrated to dryness. Purification of the
residue by column chromatography (petroleum ether/
1H NMR: d 11.57 (s, 1H, NH), 8.02 (s, 1H, H-6), 7.77–
7.80 (m, 4H, Phth), 5.57–5.65 (m, 2H, H-20, H-30), 3.97–
4.21 (m, 4H, H-10, H-40) ppm.
13C NMR: d 167.42 (C2-Phth), 160.93 (C4), 150.38 (C2),
149.41 (C6), 134.55 (C5-Phth), 131.60 (C3-Phth), 128.06

T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
5631
(C30), 126.35 (C20), 123.09 (C4-Phth), 68.36 (C5), 48.24
(C10), 38.27 (C40) ppm. MS m/z 438.2 (M+Å). Anal.
(C16H12IN3O4) C, H, N.
13C NMR: d 168.06 (C2-Phth), 162.27 (C4), 150.35 (C2),
149.44 (C6), 134.93 (C5-Phth), 133.38 (C4-Ph), 132.32
(C2-Ph), 132.15 (C3-Phth), 128.92 (C30), 127.48 (C20),
123.54 (C4-Phth), 122.38 (C3-Ph), 122.15 (C1-Ph),
97.83 (C5), 91.26 (C200), 84.20 (C100), 45.09 (C10), 34.97
(C40) ppm.
5.2.3. General procedure for the preparation of (Z)- and
(E)-1-[40-(N-phthalimido)-20-butenyl]-5-alkynyluracil (1–
14). To a stirred solution of Z- or (E)-1-[40-(N-phthalim-
ido)-20-butenyl]-5-iodouracil (200 mg, 0.46 mmol) in
anhydrous dimethylformamide (15 mL) were added
diisopropylethylamine (0.16 mL, 0.92 mmol), the acety-
lene derivative (1.37 mmol), tetrakis(triphenylphos-
phine)palladium(0) (53 mg, 0.046 mmol) and copper(I)
iodide (17.4 mg, 0.09 mmol). The mixture was stirred
for 20 h at room temperature under nitrogen, then evap-
orated to dryness and purified by column chromatogra-
phy (initial eluent: CH2Cl2, followed by CH2Cl2/
MeOH = 60:1).
MS m/z 491.9 (M+Å). Anal. (C24H16BrN3O4) C, H, N.
5.2.8. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-methyl-
phenylethynyl)uracil (5). The procedure was carried out
using p-methylphenylacetylene (0.17 mL, 1.37 mmol)
which gave orange-brown powder of 5 (43 mg, 23.5%,
mp = 225–226 ꢁC).
13C NMR: d 167.54 (C2-Phth), 160.72 (C4), 149.90 (C2),
146.97 (C6), 138.41 (C4-Ph), 134.56 (C5-Phth), 131.67
(C3-Phth), 130.95 (C2-Ph), 129.38 (C3-Ph), 126.94 (C-
20), 127.13 (C-30), 123.03 (C4-Phth), 119.34 (C1-Ph),
97.22 (C5), 91.94 (C200), 73.11 (C100), 45.79 (C10), 34.48
(C40), 21.12 (CH3) ppm.
5.2.4. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-hexynyl-
uracil (1). The procedure was carried out using hexyne
(0.16 mL, 1.37 mmol) which gave brown powder of 1
(34 mg, 19.6%, mp = 175–176 ꢁC).
MS m/z 426.3 (M+Å). Anal. (C25H19N3O4) C, H, N.
13C NMR: d 168.03 (C2-Phth), 161.52 (C4), 151.33 (C2),
149.98 (C6), 134.92 (C5-Phth), 132.15 (C3-Phth), 131.88
(C30), 129.15 (C20), 123.53 (C4-Phth), 94.18 (C5), 88.17
(C200), 68.88 (C100), 44.85 (C10), 35.10 (C40), 34.93 (C400),
29.48 (C500), 21.11 (C300), 13.90 (CH3) ppm.
5.2.9. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-butyl-
phenylethynyl)uracil (6). The procedure was carried out
using p-butylphenylacetylene (0.24 mL, 1.37 mmol)
which gave white-yellow powder of 6 (107 mg, 51.5%,
mp = 163–165 ꢁC).
MS m/z 392.2 (M+Å). Anal. (C22H21N3O4) C, H, N.
13C NMR: d 167.53 (C2-Phth), 161.89 (C4), 149.87 (C2),
148.33 (C6), 143.11 (C4-Ph), 134.41 (C5-Phth), 131.66
(C3-Phth), 130.95 (C2-Ph), 128.66 (C3-Ph), 128.31 (C-
30), 127.07 (C20), 123.03 (C4-Phth), 119.66 (C1-Ph),
97.84 (C5), 92.00 (C200), 81.64 (C100), 44.56 (C10), 34.63
(C40), 34.47 (C1000), 32.76 (C3000), 21.67 (C2000), 13.70
(CH3) ppm.
5.2.5. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-decynyl-
uracil (2). The procedure was carried out using decyne
(0.25 mL, 1.37 mmol) which gave white-yellow powder
of 2 (83 mg, 41.8%, mp = 167–168 ꢁC).
13C NMR: d 167.54 (C2-Phth), 162.29 (C4), 149.93 (C2),
147.50 (C6), 134.44 (C5-Phth), 131.68 (C3-Phth), 128.16
(C30), 127.21 (C-20), 123.05 (C4-Phth), 98.50 (C5), 93.20
(C200), 72.71 (C100), 44.40 (C10), 34.45 (C40), 31.24 (C-800),
28.61, 28.52, 28.26, 28.19, (C400–C700), 22.07 (C900), 18.77
(C300) 13.93 (CH3) ppm.
MS m/z 468.1 (M+Å). Anal. (C28H25N3O4) C, H, N.
5.2.10. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-pentyl-
phenylethynyl)uracil (7). The procedure was carried out
using p-pentylphenylacetylene (0.27 mL, 1.37 mmol)
which gave yellow-brown crystals of 7 (101 mg, 47.2%,
mp = 175–176 ꢁC).
MS m/z 448.2 (M+Å). Anal. (C26H29N3O4) C, H, N.
5.2.6. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-(400-phenyl-
butynyl)uracil (3). The procedure was carried out using 4-
phenylbutyne (0.19 mL, 1.37 mmol) which gave white-
yellow powder of 3 (128 mg, 65.7%, mp = 174–175 ꢁC).
13C NMR: d 167.57 (C2-Phth), 161.93 (C4), 149.91 (C2),
148.38 (C6), 143.18 (C4-Ph), 134.44 (C5-Phth), 131.68
(C3-Phth), 130.98 (C2-Ph), 128.70 (C3-Ph), 128.34 (C-
30), 127.10 (C20), 123.06 (C4-Phth), 119.68 (C1-Ph),
97.85 (C5), 92.03 (C200), 81.66 (C100), 44.58 (C10), 34.49
(C40), 34.95 (C1000), 30.83 (C3000), 30.31 (C2000), 21.91
(C4000), 13.88 (CH3) ppm.
13C NMR: d 162.26 (C4), 149.94 (C2), 147.58 (C6),
140.44 (C1-Ph), 134.44 (C5-Phth), 131.67 (C3-Phth),
127.21 (C-20), 126.18 (C4-Ph), 123.06 (C4-Phth), 98.37
(C5), 92.70 (C200), 73.25 (C100), 44.47 (C10), 34.47 (C40),
34.37 (C400), 21.03 (C300) ppm.
MS m/z 482.2 (M+Å). Anal. (C29H27N3O4) C, H, N.
MS m/z 440.1 (M+Å). Anal. (C26H21N3O4) C, H, N.
5.2.11. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-hexynyl-
uracil (8). The procedure was carried out using hexyne
(0.16 mL, 1.37 mmol) which gave brown powder of 8
(52 mg, 30.1%, mp = 114–115 ꢁC).
5.2.7. (Z)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-bromo-
phenylethynyl)uracil (4). The procedure was carried out
using p-bromophenylacetylene (248.7 mg, 1.37 mmol)
which gave yellow powder of 4 (134 mg, 61.6%,
mp = 188–189ꢁC).
13C NMR: d 169.23 (C2-Phth), 160.91 (C4), 149.60 (C2),
146.12 (C6), 128.26 (C30), 97.67 (C5), 93.17 (C200), 72.72

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T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
(C100), 49.46 (C10), 38.34 (C40), 30.16 (C400), 21.30 (C500),
18.39 (C300), 13.44 (CH3) ppm.
13C NMR: d 167.95 (C2-Phth), 162.40 (C4), 150.17 (C2),
148.83 (C6), 143.61 (C4-Ph), 134.91 (C5-Phth), 132.08
(C3-Phth), 131.45 (C2-Ph), 129.15 (C3-Ph), 128.59
(C30), 126.59 (C20), 123.58 (C4-Phth), 120.08 (C1-Ph),
98.21 (C5), 92.50 (C200), 82.07 (C100), 48.98 (C10), 38.83
(C40), 35.12 (C300), 33.27 (C3000), 22.17 (C2000), 14.21
(CH3) ppm.
MS m/z 392.1 (M+Å). Anal. (C22H21N3O4) C, H, N.
5.2.12. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-heptynyl-
uracil (9). The procedure was carried out using heptyne
(0.18 mL, 1.37 mmol) which gave brown powder of 9
(39 mg, 21.8%, mp = 67–68 ꢁC).
MS m/z 468.1 (M+Å). Anal. (C28H25N3O4) C, H, N.
13C NMR: d 167.93 (C2-Phth), 161.39 (C4), 150.07 (C2),
146.60 (C6), 134.94 (C5-Phth), 132.08 (C3-Phth), 128.72
(C30), 127.19 (C20), 123.59 (C4-Phth), 98.15 (C5), 93.70
(C200), 73.18 (C100), 49.94 (C10), 42.28 (C40), 30.88 (C-400),
28.26 (C500), 22.08 (C600), 19.14 (C300), 14.30 (CH3) ppm.
5.2.17. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-pentyl-
phenylethynyl)uracil (14). The procedure was carried out
using p-pentylphenylacetylene (0.27 mL, 1.37 mmol)
which gave yellow powder of 14 (132 mg, 61.7%,
mp = 75–76 ꢁC).
MS m/z 406.4 (M+Å). Anal. (C23H23N3O4) C, H, N.
13C NMR: d 167.49 (C2-Phth), 161.93 (C4), 149.70 (C2),
148.37 (C6), 143.19 (C4-Ph), 131.62 (C3-Phth), 130.99
(C2-Ph), 128.69 (C3-Ph), 128.12 (C30), 126.09 (C-20),
119.60 (C1-Ph), 97.72 (C5), 92.03 (C200), 81.59 (C100),
48.51 (C10), 38.36 (C40), 34.93 (C1000), 30.83 (C3000),
30.30 (C2000), 21.90 (C4000), 13.89 (CH3) ppm.
5.2.13. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-octynyl-
uracil (10). The procedure was carried out using octyne
(0.2 mL, 1.37 mmol) which gave brown powder of 10
(34 mg, 18.3%, mp = 58–60 ꢁC).
13C NMR: d 167.77 (C2-Phth), 161.02 (C4), 154.69 (C2),
142.13 (C6), 134.92 (C5-Phth), 132.04 (C3-Phth), 128.79
(C30), 126.76 (C20), 123.56 (C4-Phth), 106.58 (C5), 97.65
(C200), 69.07 (C100), 51.85 (C10), 38.82 (C40), 31.16 (C400),
28.35 (C500), 25.30 (C600), 22.41 (C700), 19.31 (C300), 14.32
(CH3) ppm.
MS m/z 482.3 (M+Å). Anal. (C29H27N3O4) C, H, N.
5.3. Cytostatic activity assays
5.3.1. Antiproliferation assays. The experiments were
carried out on nine human cell lines, eight of which
are derived from eight cancer types and one normal,
fibroblast cell line. The following cell lines were used:
murine leukemia (L1210), human T-lymphocytes
(Molt4/C8 and CEM), cervical carcinoma (HeLa),
breast carcinoma (MCF-7), pancreatic carcinoma (Mia-
PaCa-2), hepatocellular carcinoma (Hep G2), colon car-
cinoma (SW 620) and diploid fibroblasts (WI 38).
MS m/z 420.2 (M+Å). Anal. (C24H25N3O4) C, H, N.
5.2.14. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-decynyl-
uracil (11). The procedure was carried out using decyne
(0.25 mL, 1.37 mmol) which gave yellow powder of 11
(61 mg, 30.1%, mp = 62–63 ꢁC).
13C NMR: d 167.92 (C2-Phth), 161.38 (C4), 150.07 (C2),
146.57 (C6), 134.93 (C5-Phth), 132.08 (C3-Phth), 128.72
(C30), 127.21 (C20), 123.40 (C4-Phth), 98.15 (C5), 93.70
(C200), 73.17 (C100), 49.93 (C10), 42.28 (C40), 31.70 (C400),
29.04 (C500), 28.94 (C600), 28.69 (C700), 28.58 (C800), 22.54
(C900), 19.18 (C300), 14.38 (CH3) ppm.
Murine L1210 and human Molt4/C8 and CEM cells
were seeded at 50–75 · 103cells/well in 96-well microtiter
plates in the presence of different concentrations of the
test compounds. After 2 (L1210) or 3 (Molt4/C8 and
CEM) days, the cell number was determined with a
Coulter counter (Coulter Electronics, England). The
cytostatic concentration was determined as the com-
pound concentration required to reduce L1210, Molt4/
C8 or CEM cell growth by 50% relative to the number
of cells in the untreated controls (IC50). IC50 values were
calculated from graphic plots of the number of cells
(percentage of control) as a function of the concentra-
tion of the test compounds.
MS m/z 448.4 (M+Å). Anal. (C26H29N3O4) C, H, N.
5.2.15. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-(400-phe-
nylbutynyl)uracil (12). The procedure was carried out
using 4-phenylbutyne (0.19 mL, 1.37 mmol) which gave
yellow powder of 12 (58 mg, 29.8%, mp = 84–86 ꢁC).
13C NMR: d 167.49 (C2-Phth), 162.22 (C4), 149.73 (C2),
147.60 (C6), 140.41 (C1-Ph), 134.47 (C5-Phth), 123.13
(C4-Phth), 98.25 (C5), 92.72 (C200), 73.20 (C100), 48.25
(C10), 38.35 (C40), 34.32 (C400), 21.00 (C300) ppm.
The HeLa, Hep G2, MCF-7, MiaPaCa-2, SW 620 and
WI 38 cells were cultured as monolayers and maintained
in Dulbecco’s modified Eagle’s medium (DMEM) sup-
plemented with 10% foetal bovine serum (FBS), 2 mM
L-glutamine, 100 U/mL penicillin and 100 lg/mL strep-
tomycin in a humidified atmosphere with 5% CO2 at
37 ꢁC. As reported previously,24 the panel cell lines were
inoculated onto a series of standard 96-well microtiter
plates on day 0 at 1 · 104 to 3 · 104 cells/mL, depending
on the doubling times of specific cell line. Test agents
were then added in five, 10-fold dilutions (10ꢀ8 to
10ꢀ4 M) and incubated for further 72 h. Working dilu-
MS m/z 440.2 (M+Å). Anal. (C26H21N3O4) C, H, N.
5.2.16. (E)-1-[40-(N-Phthalimido)-20-butenyl]-5-(p-butyl-
phenylethynyl)uracil (13). The procedure was carried
out using p-butylphenylacetylene (0.24 mL, 1.37 mmol)
which gave yellow powder of 13 (143 mg, 68.9%,
mp = 171–172 ꢁC).

T. Gazivoda et al. / Bioorg. Med. Chem. 16 (2008) 5624–5634
5633
tions were freshly prepared on the day of testing. The
compounds were prepared as 0.04 M solutions in
DMSO, and the solvent was also tested for eventual
inhibitory activity by adjusting its concentration to be
the same as in the working concentrations. After 72 h
of incubation the cell growth rate was evaluated by per-
forming the MTT assay (Sigma) which detects dehydro-
genase activity in viable cells. The MTT Cell
Proliferation Assay is a colorimetric assay system, which
measures the reduction of a tetrazolium component
(MTT) into an insoluble formazan product by the mito-
chondria of viable cells. For this purpose, the substance-
treated medium was discarded and MTT was added to
each well at a concentration of 20 lg/40 lL. After 4 h
of incubation the precipitates were dissolved in 160 lL
of DMSO. The absorbance (OD, optical density) was
measured on a microplate reader at 570 nm. The absorb-
ancy is directly proportional to the cell viability. The
percentage of growth (PG) of the cell lines was calcu-
lated according to one or the other of the following
two expressions:
herpesvirus in CRFK cell cultures was based on estimat-
ing the virus-induced cytopathicity in the cell cultures
under conditions and at a time point at which non-trea-
ted virus-exposed cell cultures reached a full virus-in-
duced cytopathogenicity effect.
Acknowledgements
Support for this study was provided by the Ministry of
Science of the Republic of Croatia (Projects #125-
0982464-2925, #125-0982464-2922 and #098-0982464-
2393), the ‘Fonds voor Wetenschappelijk Onderzoek’
(FWO) (#G.0188.07) and the ‘Geconcerteerde Ond-
erzoeksacties (GOA)’ of the K.U. Leuven (#05/15).
The authors thank Mrs. L. van Berckelaer, L. Ingels,
L. Persoons, V. Broeckx, F. Demeyer, A. Camps, S.
Carmans and L. Van den Heurck for excellent technical
assistance.
Supplementary data
If (mean ODtest ꢀ mean ODtzero) P 0 then
PG ¼ 100 ꢁ ðmean ODtest ꢀ mean ODtzeroÞ=
ðmean ODctrl ꢀ meanODtzeroÞ:
If (mean ODtest – mean ODtzero) < 0 then
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.03.074.
References and notes
PG ¼ 100 ꢁ ðmean ODtest ꢀ mean ODtzeroÞ=ODtzero
where mean ODtzero = the average of optical density
measurements before exposure of cells to the test com-
pound; mean ODtest = the average of optical density
measurements after the desired period of time; and mean
ODctrl = the average of optical density measurements
after the desired period of time with no exposure of cells
to the test compound.
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Antiviral activity against HIV-1 and HIV-2 was based
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