The novel C-5 aryl, alkenyl, and alkynyl substituted uracil derivatives of l-ascorbic acid: Synthesis, cytostatic, and antiviral activity evaluations

Article abstract of DOI:10.1016/j.bmc.2006.10.046

The novel C-5 substituted uracil derivatives of l-ascorbic acid were synthesized by coupling of 5-iodouracil-4,5-didehydro-5,6-dideoxy-l-ascorbic acid with unsaturated stannanes under Stille reaction conditions. The new compounds were evaluated for their antitumoral and antiviral activities. Among all compounds evaluated the 5-propynyl substituted uracil derivative of l-ascorbic acid (7) exhibited the most pronounced cytostatic activities against all examined tumor cell lines (IC₅₀: 0.2-0.78 μM). However, this compound was also cytotoxic to human normal fibroblasts WI 38. The 5-(phenylethynyl)uracil-2,3-di-O-benzylated l-ascorbic acid derivative (4) exhibited an albeit slight (IC₅₀: 55-108 μM), but selective inhibitory effect toward all tumor cell lines except for cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), laryngeal carcinoma (Hep-2), and colon carcinoma (SW 620), and no cytotoxicity to normal human fibroblast (WI 38). Compound 7 showed some, not highly specific, inhibitory potential against vesicular stomatitis virus, Coxsackie B4 virus, and Sindbis viruses (EC₅₀: 1.6 μM).

Full text of DOI:10.1016/j.bmc.2006.10.046

Bioorganic & Medicinal Chemistry 15 (2007) 749–758
The novel C-5 aryl, alkenyl, and alkynyl substituted uracil
derivatives of L-ascorbic acid: Synthesis, cytostatic, and antiviral
activity evaluations
a
a
b
b
´
´
´
Tatjana Gazivoda, Silvana Raic-Malic, Marko Marjanovic, Marijeta Kralj,
b
c
c
a,
*
´
Kresˇimir Pavelic, Jan Balzarini, Erik De Clercq and Mladen Mintas
aDepartment of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulic´ev trg 19, HR-10000 Zagreb, Croatia
b
-
Division of Molecular Medicine, Ruder Bosˇkovic´ Institute, Bijenicˇka 54, HR-10001 Zagreb, Croatia
cRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 14 September 2006; revised 18 October 2006; accepted 23 October 2006
Available online 25 October 2006
Abstract—The novel C-5 substituted uracil derivatives of L-ascorbic acid were synthesized by coupling of 5-iodouracil-4,5-didehy-
dro-5,6-dideoxy-L-ascorbic acid with unsaturated stannanes under Stille reaction conditions. The new compounds were evaluated
for their antitumoral and antiviral activities. Among all compounds evaluated the 5-propynyl substituted uracil derivative of
L-ascorbic acid (7) exhibited the most pronounced cytostatic activities against all examined tumor cell lines (IC50: 0.2–0.78 lM).
However, this compound was also cytotoxic to human normal fibroblasts WI 38. The 5-(phenylethynyl)uracil-2,3-di-O-benzylated
L-ascorbic acid derivative (4) exhibited an albeit slight (IC50: 55–108 lM), but selective inhibitory effect toward all tumor cell lines
except for cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), laryngeal carcinoma (Hep-2), and colon carcinoma (SW
620), and no cytotoxicity to normal human fibroblast (WI 38). Compound 7 showed some, not highly specific, inhibitory potential
against vesicular stomatitis virus, Coxsackie B4 virus, and Sindbis viruses (EC50: 1.6 lM).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ure of C-5 substituents likely to confer activity are those
which are electron-withdrawing and conjugated to the
pyrimidine base.11,12 In addition, unsaturated analogues
of nucleosides have also been developed as antitumoral
and antiviral agents.13–16 We have found that some
pyrimidine and purine derivatives of 4,5-didehydro-
5,6-dideoxy-L-ascorbic acid possess pronounced cyto-
static activities against some malignant human tumor
cell lines.17–19 It has also been presumed that the biolog-
ical activity of such type of derivatives of L-ascorbic acid
has its origin in the reactivity of their double bond con-
jugated with the lactone ring toward biological nucleo-
philes.20 The intense search for clinically useful
nucleoside derivatives has resulted in a wealth of new
approaches for their synthesis. One of the most useful
synthetic approaches involves the use of palladium-cat-
alyzed addition and substitution reactions.21 Taking
into account the pharmacological potential of this class
of compounds, we have synthesized by Stille cross-cou-
pling the novel C-5 substituted pyrimidine derivatives of
2,3-di-O-benzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic
acid (Scheme 1) and evaluated their cytostatic and anti-
viral activities.
Vitamin C has been considered as a useful synthetic
precursor to many molecules of potential biological
utility on account of its inherent, varied chemical
functionality.1–3
On the other hand, many pyrimidine nucleoside ana-
logues substituted at the 5-position of the heterocycle
are known to possess potent biological properties and
have been investigated as anticancer and antiviral
drugs.4–6 The most active congeners among the 5-substi-
tuted 20-deoxyuridines are E-5-(2-halogenovinyl)-20-
deoxyuridines and 5-heteroaryl-20-deoxyuridines. These
compounds have emerged as potent and selective inhib-
itors of herpes viruses (HSV-1 and HSV-2) and varicel-
la-zoster virus (VZV).7–10 The structure–activity
relationship (SAR) studies seem to indicate that the nat-
Keywords: L-Ascorbic acid; C-5 substituted uracil derivatives; Cyto-
static activities; Antiviral activities.
*
Corresponding author. Tel.: +385 1 4597 214; fax: +385 1 4597
224; e-mail: mladen.mintas@fkit.hr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.046

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T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
O
O
I
I
HN
HN
O
N
H
O
N
(i)
IU
O
O
OAc
O
AcO
O
PhH CO
2
OCH Ph
2
IUAA
PhH CO
2
OCH Ph
2
(ii)
ABAA
O
N
R
HN
O
O
O
PhH CO
OCH Ph
2
2
1
2
3
4
5
6
7
8
9
10
R
C
S
O
(iii)
(v)
(iv)
O
O
N
O
N
Br
R
HN
HN
HN
R
O
O
O
N
Br
O
O
O
O
O
O
O
Br
PhH CO
2
OCH Ph
2
HO
OH
PhH CO
OCH Ph
2
2
11
12 13
14
15
16
17
18
19
Br Cl
R
R
S
O
Scheme 1. Synthesis of C-5 pyrimidine substituted 4,5-didehydro-5,6-dideoxy-L-ascorbic acid derivatives. Reagents and conditions: (i) HMDS,
(NH4)2SO4, reflux, then TMS-triflate, CH3CN, 60 ꢁC; (ii) R-SnBu3, (PPh3)2PdCl2, THF, reflux; (iii) Br2, CCl4, rt, 30 min; (iv) NBS/NClS, pyridine,
90 ꢁC, 2 h; (v) 1 M BCl3/CH2Cl2, ꢀ40 ꢁC to rt, 24 h.
2. Results and discussion
5-substituted uracil derivatives of 4,5-didehydro-5,6-
dideoxy-L-ascorbic acid 1–10 (Scheme 1).
2.1. Synthesis
Bromination of the 5-vinyl-L-ascorbic acid derivative (5)
with bromine gave 5-(2-bromovinyl)-6-bromouracil-2,3-
di-O-benzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid
(11) (Scheme 1). Halogenation of the 5-furyluracil deriv-
ative of L-ascorbic acid (1) with N-bromosuccinimide
(NBS) and N-chlorosuccinimide (NClS) gave 5-(bro-
Condensation of 5,6-di-O-acetyl-2,3-di-O-benzyl-L-
ascorbic acid (ABAA)22,23 and 5-iodouracil (IU) gave
5-iodouracil-2,3-di-O-benzyl-4,5-didehydro-5,6-dide-
oxy-L-ascorbic acid (IUAA)18 which, by the Stille reac-
tion24,25 with various unsaturated stannanes, afforded

T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
751
mofuryl)uracil (12) and 5-(chlorofuryl)uracil (13) deriv-
atives of L-ascorbic acid, respectively (Scheme 1).
pound exhibited also cytotoxic effect on normal human
fibroblasts (WI 38). Moreover, 5-furyluracil-(1), 5-vinyl-
uracil-(5), 5-ethynyluracil- (6), and 5-isopentenyluracil
(10) derivatives of 2,3-di-O-benzylated-L-ascorbic acid
and 5-(phenylethynyl)uracil-2,3-dihydroxy-L-ascorbic
acid derivative (17) exhibited rather marked inhibitory
activity against the growth of all tumor cell lines (IC50
within the concentration range of 1–8.3 lM), but also
cytotoxic activities toward normal human fibroblasts
(WI 38). However, the 5-(phenylethynyl)uracil-2,3-di-
O-benzylated L-ascorbic acid derivative (4) showed
slight (IC50: 55–108 lM) cytostatic activity toward all
tumor cell lines except for cervical carcinoma (HeLa),
pancreatic carcinoma (MiaPaCa-2), laryngeal carci-
noma (Hep-2), and colon carcinoma (SW 620), and no
cytotoxicity to normal human fibroblast (WI 38). Intro-
duction of a halogen in position 5 of the furan moiety of
5-bromofuryluracil- (12) and 5-chlorofuryluracil (13)
derivatives of L-ascorbic acid decreases their cytostatic
activities compared to the corresponding unsubstituted
5-furyluracil derivative (1). The 6-bromo-50-bromo-5-
(2-bromovinyl)uracil-2,3-di-O-benzylated L-ascorbic
acid derivative (11) showed better inhibitory activity
than its structural congener 20 without bromines at
positions C-6 and C-50. Comparison of cytostatic and
cytotoxic activities of compounds 1–20 (Table 1) indi-
cated low selectivity of most compounds in their cyto-
static potency.
Debenzylation of the condensation products (1–6) was
accomplished by treatment with solution of boron tri-
chloride in dichloromethane affording 14–19 (Scheme 1).
(E)-5-(2-Bromovinyl)uracil (BVU) needed for the synthe-
sis of L-ascorbic acid congener of (E)-5-(2-bromovinyl)-
20-deoxyuridine (BVDU) was prepared by a procedure
given in the literature.26 (E)-5-(2-Bromovinyl)uracil-2,3-
di-O-benzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid
(20) was obtained by Vorbruggen condensation of BVU
¨
and ABAA (Scheme 2). Silylation of 5-bromovinyluracil
with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and
trichloromethylsilane in the presence of ammonium sul-
fate, and subsequent condensation of the silylated prod-
uct thus obtained with ABAA in the presence of
trimethylsilyltrifluoromethanesulfonate (TMS-triflate)
as Friedel–Crafts catalyst, gave 20 (Scheme 2).
2.2. Cytostatic activity
Compounds 1–20 were evaluated for their cytostatic
activities against malignant human tumor cell lines:
murine leukemia (L1210), human T-lymphocytes
(Molt4/C8, CEM), cervical carcinoma (HeLa), breast
carcinoma (MCF-7), pancreatic carcinoma (MiaPaCa-
2), laryngeal carcinoma (Hep-2), and colon carcinoma
(SW 620), as well as human fibroblast cells (WI 38)
(Table 1).
2.3. Antiviral activity
The compounds 1–20 were also evaluated against herpes
simplex virus type 1 and 2, vaccinia virus, vesicular sto-
matitis virus, Coxsackie virus B4, Sindbis virus, Punta
Toro virus, respiratory syncytial virus, parainfluenza-3
virus, and reovirus-1, and their activities were compared
with those of 9-(3-hydroxyethoxymethyl)guanine (acyclo-
vir), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganci-
clovir), (E)-5-(2-bromovinyl)-20-deoxyuridine (brivudin),
9-(1,3-dihydroxypropyl)adenine [(S)-DHPA], and 1-(b-
D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide (riba-
virin). The results presented in Tables 2 and 3 show that
2,3-di-O-benzylated derivative of L-ascorbic acid con-
taining a propynyl side chain at C-5 of the uracil ring
(7) displayed borderline activity against vesicular stoma-
titis virus, Coxsackie B4 virus, and Sindbis viruses (IC50:
1.6 lM). Furthermore, the 5-isopentenyluracil-2,3-di-O-
benzylated L-ascorbic acid derivative (10) showed mod-
erate activity against HSV-1, HSV-2, vaccinia virus, and
Punta Toro virus (IC50: 4 lM) (Tables 2 and 3), while 5-
phenylethynyluracil-2,3-dihydroxy-L-ascorbic acid (17)
showed also a comparable activity against HSV-2, vac-
cinia virus, and vesicular stomatitis virus (IC50: 4 lM)
(Table 3). The 5-ethynyluracil derivative of 2,3-dihy-
droxy-L-ascorbic acid (19) showed possible activity only
against vaccinia virus (IC50: 4 lM) (Table 2). Determi-
nation of the cytotoxicity of 1–20 indicates that all
compounds showed relatively low selectivity in their
antiviral potencies. The antiviral activity levels were
usually close to the cytotoxic activity, and therefore it
is unclear whether the observed activity is due to a
selective antiviral activity, or to a toxic activity against
the cell cultures.
Of all evaluated compounds, 5-propynyluracil derivative
of 2,3-di-O-benzylated L-ascorbic acid (7) showed the
best cytostatic activity against all evaluated tumor cell
lines (IC50: 0.2–0.78 lM) (Fig. 1). However, this com-
OAc
O
Br
O
AcO
O
HN
+
O
N
PhH2CO
OCH2Ph
H
ABAA
BVU
(i)
O
N
Br
HN
O
O
O
PhH2CO
20
OCH2Ph
Scheme 2. The synthesis of (E)-5-(2-bromovinyl)uracil-2,3-di-O-ben-
zyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (20). Reagents and
conditions: (i) HMDS, (NH4)2SO4, Cl3MeSi, reflux, then TMS-triflate,
CH3CN, 60 ꢁC.

752
T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
Table 1. Inhibitory effects of compounds 1–20 on the growth of malignant tumor cell lines and diploid fibroblasts (WI 38)
a
Compound
IC50 (lM)
L1210
Molt4/C8
CEM
7.0
HeLa
MCF-7
MiaPaCa-2
Hep-2
SW 620
WI 38
1
2
6.8 0.4
142 27
142 37
6.6 0.2
149.8 13.6
159.4 7.9
0
2 0.03
18 0.5
2
0.4
4
1.6 0.05
16.5 2.5
2.2 1
18 0.1
2.4 0.4
21 0.08
1.8b
19b
28b
>100
117 9.7
122 11.8
101 11
7.2 0.6
4.6 0.2
0.77 0.18
17
18
3
25
>100
8
1
18
>100
1
22
>100
5
21
>100
2
4
108
7.2
1
108
7.6. 0.2
7.2
0.78 0.06
6
55 43
5
0
14.5 0.7
3.2 0.2
0.2 0.12
2.9 1.8
>100
3.8 0.6
2.9 1.3
0.2 0.03
4.7 1.3
77 21
22 0.4
8.7 2.7
0.5 0.1
14.2 1.9
4.6 1.2
0.7 0.09
4.7 1.3
>100
15.6 4.6
2
14.3 8.6
1.5 0.2
0.2 0.07
10.4 5.5
>100
6
6.8 0.6
0.47 0.27
0
5
7
0.3 0.2
2.7 1.6
>100
8
20
>250
1
20
>250
1
20
>250
1
5
>100
1
9
10
11
12
13
14
15
16
17
18
19
20
8.2 0.2
32 19
115 21
165 1.9
P500
8.3 0.3
110 8.7
125 6.9
157.9 26.3
P500
7.7 0.2
91 11.6
134 10
162 26
436 90
>500
2
0.4
7
4
2
5
0.2
2.6
22
1
4
0.8
2
0.3
22
24
15
29
4
6
20
27
2
9
4
P100
49 48
>100
49b
16b
42b
6
28
95
4
2
P100
>100
>100
>100
P100
>100
>100
>100
P100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>500
>500
354 205
8.1 0.2
309 111
322 84
8.2 0.1
309 11
282
2
>100
3
7.6 0.2
284 75
2
1
1
0.07
3
0.5
3 0.6
46 14
4
1
0.09
40
13
45
6
5
5
36
13
5
2
P100
13
>100
>100
17
>100
36b
11
38b
15 1
196 54.1
37
1
23
233 60
6
3
12
40
1
4
3
2
P200
40 15
a IC50—50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%.
b Only one experiment was carried out.
Compound 7
150
WI 38
100
HeLa
50
SW 620
H 460
0
MCF-7
-50
MiaPaCa-2
-100
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)
Figure 1. Dose–response profile for compound 7 tested in vitro.
3. Conclusion
most pronounced cytostatic activities against all exam-
ined tumor cell lines (IC50: 0.2–0.78 lM), but it was also
cytotoxic to human normal fibroblasts WI 38. The
5-propynyluracil derivative of L-ascorbic acid (7) had
low activity against Sindbis virus, Coxsackie virus B4,
and vesicular stomatitis virus (EC50: 1.6 lM). Moreover,
the 5-isopentenyluracil-2,3-di-O-benzylated- (10) and
5-(phenylethynyl)uracil- (17) and 5-ethynyluracil- (19)
2,3-dihydroxy-L-ascorbic acid derivatives showed bor-
derline activity (EC50: 4 lM against herpes simplex virus
type 1 and 2, vaccinia virus, and Punta Toro virus).
The new C-5 substituted uracil L-ascorbic acid deriva-
tives (1–10) were synthesized by palladium-catalyzed
cross-coupling Stille reaction of 5-iodouracil-2,3-di-O-
benzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid and
unsaturated aliphatic and aryl stannanes. Bromination
of the 5-vinyluracil derivative (5) with bromine gave
the tribrominated compound (11), while bromination
of 5-furyluracil derivative (1) with N-halosuccinimide
afforded corresponding monohalogenated compounds
12 and 13. Compounds with deprotected positions 2
and 3 of the lactone ring (14–19) were obtained by deb-
enzylation of 1–6 with borontrichloride. The (E)-5-(2-
bromovinyl)uracil derivative of L-ascorbic acid (20)
was prepared by condensation of silylated uracil base
and diacetylated derivative of L-ascorbic acid. Among
all evaluated compounds the 5-propynyl substituted
uracil derivative of L-ascorbic acid (7) exhibited the
4. Experimental
4.1. General methods
Melting points were determined on a Kofler micro hot-
stage apparatus (Reichert, Wien) and are uncorrected.

T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
753
Table 2. Antiviral activity and cytotoxicity of compounds 1–20 in E6SM and HELc cell cultures
Compound
Minimum inhibitory concentrationa (lM)
Minimum cytotoxic
concentrationb (lM)
Herpes simplex
virus-1 (KOS)
Herpes simplex
virus-2 (G)
Vaccinia
virus
Vesicular
stomatitis virus
Herpes simplex
virus-1 TKꢀ KOS ACV
1
>6.4
>156
>157
>40
>18
>18
>0.4
>8
>6.4
>156
>157
>40
>18
>18
>0.4
>8
>6.4
>156
95
>6.4
>156
>157
>40
> 6.4
>156
>157
>40
>18
>18
>0.4
>8
32
780
787
200
87
2
3
4
>40
>18
>18
>0.4
>8
5c
>18
>18
6c
P18
2
7
>0.4
>8
8
40
40
9
>8
4
>8
4
>8
4
>8
>4
>8
>4
10c
11
20
>116
>70
>150
>100
>100
>100
>4
>116
>70
>150
>100
>100
>100
4
>116
>70
>150
>100
>100
>100
4
>116
>70
>116
>70
>150
>100
>100
>100
>4
578
347
752
>100
>100
>100
20
12
13
>150
>100
>100
>100
4
14c
15c
16c
17c
18
>144
>4
>144
>4
>144
4
>144
>4
>144
>4
720
20
19c
20
>150
0.48
500
>150
300
300
>150
2.4
>150
>500
>500
>500
>100
>150
300
500
746
>500
>500
>500
>100
Brivudin
Ribavirin
Acyclovir
Ganciclovir
100
300
60
0.48
0.096
0.8
0.096
60
0.8
a Required to reduce virus-induced cytopathogenicity by 50%.
b Required to cause a microscopically detectable alteration of normal cell morphology.
c Cytotoxicity and antiviral activity in HEL cell cultures.
Table 3. Antiviral activity and cytotoxicity of compounds 1–20 in Vero and HeLac cell cultures
Compound
Minimum inhibitory concentrationa (lM)
Minimum cytotoxic
concentrationb (lM)
Sindbis virus
Coxsackie virus B4
>6.4
>156
Punta Toro virus
Vesicular stomatitis virusc
1
>6.4
>156
>157
120
>6.4
>156
>157
>200
>18
>6.4
>156
>157
>200
>87
>18
1.6
32
780
2
3
>157
>200
>18
787
>200
87
4
5
>18
>18
6
>18
>18
>1.6
>40
P18
8
7
1.6
1.6
8
24
>200
>4
24
>200
>4
24
200
9
10
>200
4
>40
>4
>200
20
578
11
12
>116
>70
>116
>70
>116
>70
>116
70
347
752
13
14
>150
>100
>100
>100
>4
>150
>100
>100
>100
>4
>150
>100
>100
>100
>4
>150
>100
>100
>100
>4
>100
>100
>100
20
15
16
17
18
>29
>4
>29
>4
>29
4
120
>4
720
20
19
20
>150
>500
>500
100
>150
>500
>500
>500
>150
>500
300
>150
>500
>500
100
746
Brivudin
(S)-DHPA
Ribavirin
>500
>500
>500
300
a Required to reduce virus-induced cytopathogenicity by 50%.
b Required to cause a microscopically detectable alteration of normal cell morphology.
c Antiviral activity in HeLa cell cultures.

754
T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
Precoated E. Merck Silica Gel 60F-254 plates were used
for thin-layer chromatography (TLC) and for prepara-
tive chromatography, and the spots were detected under
UV light (254 nm). Column chromatography (CLC) was
performed using silica gel (0.063–0.2 mm) Fluka; glass
column was slurry-packed under gravity. Compounds’
purity was analyzed by HPLC with DAD detector.
The electron impact mass spectra were recorded with
an EXTREL FT MS 2002 instrument with ionizing
energy of 70 eV. High field one- and two-dimensional
1H and 13C NMR spectra were recorded on a Varian
Gemini 300 spectrometer, operating at 75.46 MHz for
the 13C resonance. The samples were dissolved in
(C-10), 122.76 (C-20), 138.89 (C-30), 142.14 (C-40),
68.93 (C-50), 43.39 (C-60), 149.76 (C-2), 161.77 (C-4),
104.00 (C-5), 140.89 (C-6), 73.00 and 73.98 (C-70 and
C-700), 135.75 and 135.39 (C-80 and C-800 Ph), 128.77–
128.52 (C–Ph), 137.28 (C-200), 119.32 (C-300), 121.48
(C-400), 126.61 (C-500) ppm. MS m/z 514.6 (M+Å).
4.2.1.3. 1-(5-Phenyluracil-1-yl)-2-(2,3-di-O-benzyl-2-
butene-4-olidylidene)ethane (3). The procedure was car-
ried out using (tributylstannyl)benzene (734 mg, 2
mmol) for 2 days to yield colorless oil (41 mg, 31%).
1H NMR (DMSO-d6): d 11.48 (s, 1H, NH), 7.84 (s,
1H, H-6), 5.50 (t, J = 6.12 Hz, 1H, H-50), 4.06 (d,
J = 6.81 Hz, 2H, H-60), 5.15 and 4.92 (s, 2H, H-70 and
H-700), 7.56–7.21 (m, 10H, Ph) ppm. 13C NMR
(DMSO-d6): d 163.71 (C-10), 121.88 (C-20), 142.21 (C-
30), 147.98 (C-40), 101.22 (C-50), 43.24 (C-60), 149.12
(C-2), 161.66 (C-4), 106.26 (C-5), 142.91 (C-6), 73.90
and 72.86 (C-70 and C-700), 135.74 and 135.32 (C-80
and C-800 Ph), 128.76–128.54 (C–Ph), 126.51 (C-100)
ppm. MS m/z 508.5 (M+Å).
1
DMSO-d6 and measured in 5 mm NMR tubes. The H
and 13C NMR chemical shift values (d) are expressed
in ppm referred to TMS and coupling constants
(J) in Hz.
4.2. Compounds’ preparation
4.2.1. General procedure for the preparation of 5-substi-
tuted uracil derivatives of L-ascorbic acid (1–10). To a
solution of 5-iodouracil-2,3-di-O-benzyl-L-ascorbic acid
(0.27 mmol) in tetrahydrofuran (15 mL), organostann-
ane (2.7 mmol) and catalytic amounts of dichlorobis(tri-
phenylphosphine)palladium (0.027 mmol) were added.
The reaction mixture was refluxed for 2–20 h, then the
reduced palladium was filtered off and solvent was evap-
orated under reduced pressure. The oily residue was
twice purified by column chromatography (CH2Cl2/
MeOH = 40:1) to yield the oil which was then addition-
ally purified by thin-layer preparative chromatography
(CH2Cl2/MeOH = 60:1).
4.2.1.4. 1-[5-(Phenylethynyl)uracil-1-yl]-2-(2,3-di-O-
benzyl-2-butene-4-olidylidene)ethane (4). The procedure
was carried out using 2-phenyl(tributylstannyl)ethine
(938 mg, 2.4 mmol) for 24 h which gave yellow oil
1
(62 mg, 32.4%). H NMR (DMSO-d6): d 11.51 (s, 1H,
NH), 8.15 (s, 1H, H-6), 5.33 (d, J = 6.92 Hz, 1H, H-
50), 4.41 (d, J = 7.12 Hz, 2H, H-60), 5.20 and 5.08 (s,
2H, H-70 and H-700), 7.48–7.35 (m, 15H, Ph) ppm. 13C
NMR (DMSO-d6): d 164.12 (C-10), 121.41 (C-20),
137.53 (C-30), 146.59 (C-40), 99.17 (C-50), 42.11 (C-60),
152.43 (C-2), 161.31 (C-4), 100.73 (C-5), 148.13 (C-6),
73.61 and 73.18 (C-70 and C-700), 136.13 and 135.62
(C-80 and C-800 Ph), 127.16–128.56 and 131.42–132.00
(C–Ph), 88.21(C-100), 92.63 (C-200), 133.05 (C-300) ppm.
MS m/z 532.5 (M+Å).
4.2.1.1. 1-[5-(Furan-2-yl)uracil-1-yl]-2-(2,3-di-O-ben-
zyl-2-butene-4-olidylidene)ethane (1). The procedure
was carried out using 2-(tributylstannyl)furan (2.5 mL,
7.9 mmol) for 2 h which gave orange powder (216 mg,
1
33.2%, mp 71–73 ꢁC). H NMR (DMSO-d6): d 11.30
4.2.1.5. 1-(5-Vinyluracil-1-yl)-2-(2,3-di-O-benzyl-2-bu-
tene-4-olidylidene)ethane (5). The procedure was carried
out using (tributylstannyl)ethene (634 mg, 2 mmol) for
4 h under reflux and additionally stirred for 24 h at
room temperature. Compound 5 was obtained as white
powder (55 mg, 40%, mp 156–158 ꢁC). 1H NMR
(DMSO-d6): d 11.36 (s, 1H, NH), 7.80 (s, 1H, H-6),
5.45 (t, J = 6.67 Hz, 1H, H-50), 4.47 (d, J = 6.63 Hz,
2H, H-60), 5.24 and 5.08 (s, 2H, H-70 and H-700), 7.40–
7.31 (m, 10H, Ph), 6.26 (dd, J = 11.55, 6.1 Hz, 1H, H-
100), 5.45 (dd, J = 1.9, 11.5 Hz, 2H, H-200) ppm. 13C
NMR (DMSO-d6): d 163.71 (C-10), 122.96 (C-20),
142.25 (C-30), 148.08 (C-40), 103.80 (C-50), 43.04 (C-
60), 149.93 (C-2), 162.57 (C-4), 110.48 (C-5), 142.96
(C-6), 73.99 and 72.99 (C-70 and C-700), 135.76 and
135.38 (C-80 and C-800 Ph), 128.83–128.52 (C–Ph),
127.92 (C-100), 113.84 (C-200) ppm. MS m/z 458.5 (M+Å).
(s, 1H, NH), 7.91 (s, 1H, H-6), 5.48 (t, J = 6.54 Hz,
1H, H-50), 4.51 (d, J = 6.74 Hz, 2H, H-60), 5.31 and
5.14 (s, 2H, H-70 and H-700), 7.66–7.54 (m, 10H, Ph),
6.60 (d, J = 3.08 Hz, 1H, H-300), 6.46 (dd, J = 1.70,
3.10 Hz, 1H, H-400), 7.72 (dd, J = 1.80, 3.09 Hz, 1H,
13
H-500) ppm. C NMR (DMSO-d6): d 163.76 (C-10),
122.89 (C-20), 133.41 (C-30), 142.35 (C-40), 67.40
(C-50), 42.76 (C-60), 150.82 (C-2), 159.57 (C-4), 101.25
(C-5), 145.41 (C-6), 74.04 and 73.00 (C-70 and C-700),
135.76 and 135.42 (C-80 and C-800 Ph), 131.57–128.58
(C–Ph), 148.14 (C-200), 109.11 (C-300), 113.23 (C-400),
134.70 (C-500) ppm. MS m/z 498.5 (M+Å).
4.2.1.2. 1-[5-(Thiophen-2-yl)uracil-1-yl]-2-(2,3-di-O-
benzyl-2-butene-4-olidylidene)ethane (2). The procedure
was carried out using 2-(tributylstannyl)thiophene
(899 mg, 2.7 mmol) for 2 h yielding brown powder
1
(47 mg, 33.9%, mp 145–147 ꢁC). H NMR (DMSO-d6):
4.2.1.6. 1-(5-Ethynyluracil-1-yl)-2-(2,3-di-O-benzyl-2-
butene-4-olidylidene)ethane (6). The procedure was car-
ried out using (tributylstannyl)ethine (630 mg, 2 mmol)
for 4 h under reflux and additionally stirred for 24 h at
50 ꢁC. Compound 6 was isolated as yellow-brown oil
d 11.61 (s, 1H, NH), 8.23 (s, 1H, H-6), 5.51 (t,
J = 7.12 Hz, 1H, H-50), 4.43 (d, J = 7.24 Hz, 2H, H-
60), 5.24 and 5.07 (s, 2H, H-70 and H-700), 7.38–7.25
(m, 10H, Ph), 7,48 (d, J = 3.08 Hz, 1H, H-300), 6,98 (t,
J = 3.10 Hz, 1H, H-400), 7,55 (dd, J = 1.70, 3.10 Hz,
1H, H-500) ppm. 13C NMR (DMSO-d6): d 163.72
1
(52 mg, 43.8%). H NMR (DMSO-d6): d 11.56 (s, 1H,
NH), 8.02 (s, 1H, H-6), 5.47 (t, J = 6.60 Hz, 1H, H-50),

T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
755
4.47 (d, J = 6.63 Hz, 2H, H-60), 5.33 and 5.07 (s, 2H, H-
70 and H-700), 7.37–7.25 (m, 10H, Ph), 4.03 (s, 1H, H-200)
ppm. 13C NMR (DMSO-d6): d 169.03 (C-10), 122.98 (C-
20), 142.27 (C-30), 149.82 (C-40), 103.60 (C-50), 43.32 (C-
60), 157.85 (C-2), 163.68 (C-4), 97.03 (C-5), 149.47 (C-6),
73.98 and 72.88 (C-70 and C-700), 135.75 and 135.40 (C-80
and C-800 Ph), 128.83–127.70 (C–Ph), 83.49 (C-100), 75.08
(C-200) ppm. MS m/z 456.5 (M+Å).
d 11.28 (s, 1H, NH), 7.99 (s, 1H, H-6), 5.64 (t,
J = 6.88 Hz, 1H, H-50), 4.48 (d, J = 6,92 Hz, 2H, H-
60), 5.22 and 5.07 (s, 2H, H-70 and H-700), 7.47–7.32
(m, 10H, Ph), 2.68 (m, 1H, H-100), 5.99 (m, 1H, H-200),
2.21 (m, 6H, H-400, 400 0) ppm. 13C NMR (DMSO-d6): d
162.43 (C-10), 122.74 (C-20), 140.52 (C-30), 145.66 (C-
40), 101.23 (C-50), 43.14 (C-60), 151.83 (C-2), 159.65
(C-4), 104.12 (C-5), 142.16 (C-6), 72.38 and 72.23 (C-
70 and C-700), 135.96 and 135.11 (C-80 and C-800 Ph),
129.46–127.21 (C–Ph), 33.38 (C-100), 119.78 (C-200),
130.16 (C-300), 21.26 and 23.65 (C-400 and C-400 0) ppm.
MS m/z 500.5 (M+Å).
4.2.1.7. 1-(5-Propynyluracil-1-yl)-2-(2,3-di-O-benzyl-
2-butene-4-olidylidene)ethane (7). The procedure was
carried out using (tributylstannyl)-1-propine (789 mg,
2.4 mmol) for 4 h yielding brown oil (55 mg, 32.5%).
1H NMR (DMSO-d6): d 11.40 (s, 1H, NH), 8.13 (s,
1H, H-6), 5.43 (t, J = 6.64 Hz, 1H, H-50), 4.43 (d,
J = 7.01 Hz, 2H, H-60), 5.25 and 4.98 (s, 2H, H-70 and
H-700), 7.56–7.42 (m, 10H, Ph), 2.23 (s, 3H, H-300) ppm.
13C NMR (DMSO-d6): d 164.28 (C-10), 122.87 (C-20),
142.13 (C-30), 144.56 (C-40), 101.54 (C-50), 42.78 (C-
60), 149.77 (C-2), 162.32 (C-4), 100.11 (C-5), 143.73
(C-6), 72.55 and 72.41 (C-70 and C-700), 134.67 and
134.52 (C-80 and C-800 Ph), 128.60–127.44 (C–Ph),
82.33 (C-100), 97.09 (C-200), 24.65 (C-300) ppm. MS m/z
470.5 (M+Å).
4.2.1.11. 1-[5-(2-Bromovinyl)-6-bromouracil-1-yl]-2-
(2,3-di-O-benzyl-2-bromobutene-4-olidylidene)ethane
(11). To a solution of 5-vinyluracil-2,3-di-O-benzyl-L-
ascorbic acid 5 (210 mg, 0.46 mmol) in tetrachlorometh-
ane (15 mL), bromine (147.2 mg, 0.92 mmol) was added
dropwise. Reaction mixture was stirred for 30 min at
room temperature and solvent was evaporated in vacuo.
The oily residue was purified by column chromatogra-
phy (CH2Cl2/MeOH = 50:1) to yield yellow oil
(91.6 mg, 28.6%). 1H NMR (DMSO-d6): d 11.42 (s,
1H, NH), 5.11 (s, 1H, H-60), 5.14 and 5.09 (s, 2H, H-
70 and H-700), 7.33–7.19 (m, 10H, Ph), 7.45 (m, 2H, H-
100), 7.82 (d, J = 3.9 Hz, 1H, H-200) ppm. 13C NMR
(DMSO-d6): d 163.18 (C-10), 122.25 (C-20), 142.33 (C-
30), 148.62 (C-40), 100.87 (C-50), 42.54 (C-60), 148.16
(C-2), 160.11 (C-4), 109.72 (C-5), 132.24 (C-6), 73.27
and 73.16 (C-70 and C-700), 134.19 and 134.06 (C-80
and C-800 Ph), 128.52–127.26 (C–Ph), 132.11 (C-100),
104.88 (C-200) ppm. MS m/z 695.2 (M+Å).
4.2.1.8. 1-(5-Allyluracil-1-yl)-2-(2,3-di-O-benzyl-2-bu-
tene-4-olidylidene)ethane (8). The procedure was carried
out
using
(tributylstannyl)-2-propene
(794 mg,
2.4 mmol) for 2 days under reflux to yield yellow oil of
8 (48 mg, 28.2%). 1H NMR (DMSO-d6): d 11.31 (s,
1H, NH), 7.86 (s, 1H, H-6), 5.37 (t, J = 6.68 Hz, 1H,
H-50), 4.43 (d, J = 6,98 Hz, 2H, H-60), 5.22 and 5.06
(s, 2H, H-70 and H-700), 7.36–7.22 (m, 10H, Ph), 2.89
(m, 2H, H-100), 6.02 (m, 1H, H-200), 4.71 (m, 2H, H-300)
4.2.1.12. 1-[5-(5-Bromofuran-2-yl)uracil-1-yl]-2-(2,3-
di-O-benzyl-2-butene-4-olidylidene)ethane (12). To a
13
ppm. C NMR (DMSO-d6): d 165.14 (C-10), 123.06
(C-20), 140.12 (C-30), 147.22 (C-40), 100.76 (C-50),
43.21 (C-60), 155.34 (C-2), 162.88 (C-4), 98.66 (C-5),
142.75 (C-6), 73.21 and 73.07 (C-70 and C-700), 135.74
and 135.28 (C-80 and C-800 Ph), 128.40-127.25 (C-Ph),
33.12 (C-100), 126.23 (C-200), 109.84 (C-300) ppm. MS
m/z 472.2 (M+Å).
solution of 5-furyluracil-2,3-di-O-benzyl-L-ascorbic acid
1 (200 mg, 0.4 mmol) in pyridine (10 mL), N-bromosuc-
cinimide (71.2 mg, 0.4 mmol) was added. The reaction
mixture was stirred for 1 h at 90 ꢁC. Additional amount
of the N-bromosuccinimide (71.2 mg, 0.8 mmol) was
then added and reaction was continued for next 1 h at
90 ꢁC. The reaction mixture was stirred at room temper-
ature overnight and solvent was then evaporated under
reduced pressure. The oily residue was twice purified by
column chromatography (CH2Cl2/MeOH = 50:1) to
4.2.1.9. 1-(5-Alenyluracil-1-yl)-2-(2,3-di-O-benzyl-2-
butene-4-olidylidene)ethane (9). The procedure was car-
ried out using (tributylstannyl)alene (789 mg, 2.4 mmol)
for 2 days which gave orange oil of 9 (72 mg, 42.6%). 1H
NMR (DMSO-d6): d 11.43 (s, 1H, NH), 8.40 (s, 1H, H-
6), 5.52 (t, J = 7,02 Hz, 1H, H-50), 4.42 (d, J = 6,88 Hz,
2H, H-60), 5.18 and 4.96 (s, 2H, H-70 and H-700), 7.55–
7.31 (m, 10H, Ph), 6.06 (s, 1H, H-100), 5.12 (s, 2H, H-
1
yield yellow oil (41.9 mg, 18.3%). H NMR (DMSO-
d6): d 11.68 (s, 1H, NH), 8.10 (s, 1H, H-6), 5.57 (t,
J = 6.47 Hz, 1H, H-50), 4.65 (d, J = 6.44 Hz, 2H, H-
60), 5.31 and 5.16 (s, 2H, H-70 and H-700), 7.43–7.31
(m, 10H, Ph), 6.83 (d, J = 3.36 Hz, 1H, H-300), 6.62 (d,
J = 3.39 Hz, 1H, H-400) ppm. 13C NMR (DMSO-d6): d
163.76 (C-10), 122.89 (C-20), 133.41 (C-30), 142.35
(C-40), 67.40 (C-50), 42.76 (C-60), 150.82 (C-2), 159.57
(C-4), 101.25 (C-5), 145.41 (C-6), 74.04 and 73.00
(C-70 and C-700), 135.76 and 135.42 (C-80 and C-800 Ph),
131.57–128.58 (C–Ph), 148.14 (C-200), 109.11 (C-300),
113.23 (C-400), 134.70 (C-500) ppm. MS m/z 577.4 (M+Å).
13
300) ppm. C NMR (DMSO-d6): d 162.55 (C-10),
121.18 (C-20), 143.26 (C-30), 149.33 (C-40), 102.08
(C-50), 42.54 (C-60), 150.69 (C-2), 160.22 (C-4), 106.80
(C-5), 137.83 (C-6), 74.16 and 73.09 (C-70 and C-700),
134.18 and 133.65 (C-80 and C-800 Ph), 130.75–128.14
(C–Ph), 88.33 (C-100), 182.44 (C-200), 81.08 (C-300) ppm.
MS m/z 470.5 (M+Å).
4.2.1.10. 1-[5-(2-Isopentenyl)uracil-1-yl]-2-(2,3-di-O-
benzyl-2-butene-4-olidylidene)ethane (10). The procedure
was carried out using (tributylstannyl)-3-methyl-2-bu-
tene (862 mg, 2.4 mmol) for 48 h under reflux to yield
4.2.1.13. 1-[5-(5-Chlorofuran-2-yl)uracil-1-yl]-2-(2,3-
di-O-benzyl-2-butene-4-olidylidene)ethane (13). To
solution of 5-furyluracil-2,3-di-O-benzyl-L-ascorbic acid
1 (200 mg, 0.4 mmol) in pyridine (10 mL), N-chlorosuc-
cinimide (104.8 mg, 0.8 mmol) was added. The reaction
a
1
brown oil of 10 (41 mg, 22.8%). H NMR (DMSO-d6):

756
T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
mixture was stirred for 2 h at 90 ꢁC and then continued
at room temperature overnight. Solvent was evaporated
and the oily residue was twice purified by column chro-
matography (CH2Cl2/MeOH = 50:1) to yield brown oil
(99.4 mg, 46.7%). 1H NMR (DMSO-d6): d 11.31 (s,
1H, NH), 8.42 (s, 1H, H-6), 5.55 (t, J = 6.70 Hz, 1H,
H-50), 4.38 (d, J = 6.72 Hz, 2H, H-60), 5.19 and 5.06
(s, 2H, H-70 and H-700), 7.38–7.21 (m, 10H, Ph), 6.44
(d, J = 4.13 Hz, 1H, H-300), 5.99 (d, J = 3.87 Hz, 1H,
raphy (CH2Cl2/MeOH = 60:1) to yield oily compound
1
16 (64.3 mg, 49.4%). H NMR (DMSO-d6): d 11.51 (s,
1H, NH), 7.99 (s, 1H, H-6), 5.56 (t, J = 6,88 Hz, 1H,
H-50), 4.39 (d, J = 6,82 Hz, 2H, H-60), 7.38–7.22 (m,
13
5H, Ph) ppm. C NMR (DMSO-d6): d 164.15 (C-10),
121.82 (C-20), 141.87 (C-30), 147.33 (C-40), 102.46 (C-
50), 43.89 (C-60), 148.72 (C-2), 161.26 (C-4), 104.13 (C-
5), 143.31 (C-6), 129.53–128.88 (C–Ph), 127.43 (C-100)
ppm. MS m/z 328.2 (M+Å).
13
H-400) ppm. C NMR (DMSO-d6): d 164.23 (C-10),
123.18 (C-20), 135.37 (C-30), 141.18 (C-40), 66.92 (C-
50), 40.98 (C-60), 151.03 (C-2), 161.12 (C-4), 103.21 (C-
5), 145.22 (C-6), 74.27 and 73.66 (C-70 and C-700),
134.99 and 134.02 (C-80 and C-800 Ph), 130.43–128.27
(C–Ph), 146.07 (C-200), 114.23 (C-300), 108.11 (C-400),
139.65 (C-500) ppm. MS m/z 532.9 (M+Å).
4.2.1.17. 1-[5-(Phenylethynyl)uracil-1-yl]-2-(2,3-dihy-
droxy-2-buten-4-olidylidene)ethane (17). Compound 4
(435 mg, 0.818 mmol) was treated according to a proce-
dure that was analogous to that for the synthesis of
compound 14. The oily residue was purified by column
chromatography (CH2Cl2/MeOH = 60:1) to yield 17
(106.3 mg, 36.7%). 1H NMR (DMSO-d6): d 11.72 (s,
1H, NH), 7.97 (s, 1H, H-6), 5.28 (d, J = 6.90 Hz, 1H,
H-50), 4.46 (d, J = 7.08 Hz, 2H, H-60), 7.44–7.37 (m,
4.2.1.14.
1-[5-(Furan-2-yl)uracil-1-yl]-2-(2,3-dihy-
droxy-2-buten-4-olidylidene)ethane (14). To a stirred
solution of 1 (200 mg, 0.402 mmol) in anhydrous
CH2Cl2 (10 mL), 1 M solution of BCl3 in CH2Cl2
(0.5 ml) was added under argon at ꢀ78 ꢁC. The reaction
mixture was stirred at ꢀ40 ꢁC for 2 h, and then 1 M
solution of BCl3 in CH2Cl2 (0.5 ml) was added. It was
additionally stirred at 0 ꢁC for 2 h, then the temperature
was raised to room temperature and stirred overnight. A
solvent mixture of CH2Cl2/MeOH (1:1) was added to
deactivate unreacted BCl3 and the solvent was then re-
moved under reduced pressure. The oily residue was
purified by column chromatography (CH2Cl2/
MeOH = 60:1) to yield yellow oily compound 14
13
5H, Ph) ppm. C NMR (DMSO-d6): d 162.12 (C-10),
122.61 (C-20), 137.51 (C-30), 147.08 (C-40), 98.23 (C-
50), 42.78 (C-60), 152.43 (C-2), 160.01 (C-4), 102.44 (C-
5), 148.39 (C-6), 131.40–132.04 (C–Ph), 90.45 (C-100),
93.48 (C-200), 133.56 (C-300) ppm. MS m/z 352.3 (M+Å).
4.2.1.18.
1-(5-Vinyluracil-1-yl)-2-(2,3-dihydroxy-2-
buten-4-olidylidene)ethane (18). Compound 5 (90 mg,
0.197 mmol) was treated by a procedure analogous to
that for the preparation of compound 14. The crude oily
residue was purified by column chromatography
(CH2Cl2/MeOH = 60:1) to yield yellow oil 18 (18.2 mg,
33.3%). 1H NMR (DMSO-d6): d 11.81 (s, 1H, NH),
8.14 (s, 1H, H-6), 5.40 (t, J = 6,68 Hz, 1H, H-50), 4.42
(d, J = 6.75 Hz, 2H, H-60), 6.61 (dd, J = 11.40, 6.3 Hz,
1H, H-100), 5.77 (dd, J = 3.2, 11.54 Hz, 2H, H-200) ppm.
13C NMR (DMSO-d6): d 164.52 (C-10), 122.53 (C-20),
143.26 (C-30), 149.12 (C-40), 101.55 (C-50), 41.26 (C-
60), 151.27 (C-2), 162.34 (C-4), 108.77 (C-5), 142.97
(C-6), 126.54 (C-100), 115.22 (C-200) ppm. MS m/z 278.2
(M+Å).
1
(57 mg, 44.6%). H NMR (DMSO-d6): d 11.42 (s, 1H,
NH), 8.04 (s, 1H, H-6), 5.43 (t, J = 6.55 Hz, 1H, H-50),
4.42 (d, J = 6.67 Hz, 2H, H-60), 6.53 (m, 2H, H-300 and
H-400), 7.72 (d, J = 3.64 Hz, 1H, H-500) ppm. 13C NMR
(DMSO-d6): d 164.15 (C-10), 123.29 (C-20), 134.72
(C-30), 142.38 (C-40), 69.04 (C-50), 42.88 (C-60), 151.11
(C-2), 160.24 (C-4), 99.39 (C-5), 144.67 (C-6), 147.59
(C-200), 108.41 (C-300), 111.33 (C-400), 132.16 (C-500)
ppm. MS m/z 318.2 (M+Å).
4.2.1.15. 1-[5-(Thiophen-2-yl)uracil-1-yl]-2-(2,3-dihy-
droxy-2-buten-4-olidylidene)ethane (15). Compound 2
(260 mg, 0.498 mmol) was treated according to a proce-
dure that was analogous to that for the preparation of
compound 1 to give 14. The residue was purified by col-
umn chromatography (CH2Cl2/MeOH = 60:1) to yield
4.2.1.19. 1-(5-Ethynyluracil-1-yl)-2-(2,3-dihydroxy-2-
buten-4-olidylidene)ethane (19). Compound 6 (65 mg,
0.140 mmol) was treated according to a procedure that
was analogous to that for the preparation of compound
14. The crude oily residue was purified by column chro-
matography (CH2Cl2/MeOH = 60:1) to yield 19 as
1
1
brown oily compound 15 (96.6 mg, 55.7%). H NMR
brown oil (21.4 mg, 55.4%). H NMR (DMSO-d6): d
(DMSO-d6): d 11.39 (s, 1H, NH), 8.14 (s, 1H, H-6),
5.28 (t, J = 6.96 Hz, 1H, H-50), 4.46 (d, J = 6.68 Hz,
2H, H-60), 7.19 (d, J = 3.10 Hz, 1H, H-300), 6.71 (t,
J = 3.10 Hz, 1H, H-400), 7.58 (d, 3.10 Hz, 1H, H-500)
11.68 (s, 1H, NH), 7.89 (s, 1H, H-6), 5.45 (t,
J = 6.64 Hz, 1H, H-50), 4.45 (d, J = 6.72 Hz, 2H, H-
60), 3.87 (s, 1H, H-200) ppm. 13C NMR (DMSO-d6): d
164.15 (C-10), 122.34 (C-20), 141.17 (C-30), 150.02
(C-40), 98.15 (C-50), 43.38 (C-60), 155.58 (C-2), 161.32
(C-4), 103.56 (C-5), 147.69 (C-6), 85.76 (C-100), 74.38
(C-200) ppm. MS m/z 276.2 (M+Å).
13
ppm. C NMR (DMSO-d6): d 163.81 (C-10), 123.14
(C-20), 136.57 (C-30), 142.54 (C-40), 70.12 (C-50), 42.31
(C-60), 150.22 (C-2), 161.34 (C-4), 102.53 (C-5), 140.81
(C-6), 138.33 (C-200), 120.07 (C-300), 124.52 (C-400),
128.44 (C-500) ppm. MS m/z 334.3 (M+Å).
4.2.1.20. 1-[5-(2-Bromovinyl)uracil-1-yl]-2-(2,3-di-O-
benzyl-2-butene-4-olidylidene)ethane (20). The solution
of anhydrous 5-(2-bromovinyl)uracil (BVU) (500 mg,
2.3 mmol) and chlorotrimethylsilane (0.05 mL) in hex-
amethyldisilazane (10 mL) was refluxed for 3 h under ar-
gon atmosphere. Evaporation of unreacted HMDS
under reduced pressure gave oily product to which was
4.2.1.16. 1-[5-Phenyluracil-1-yl]-2-(2,3-dihydroxy-2-
buten-4-olidylidene)ethane (16). Compound 3 (200 mg,
0.394 mmol) was treated according to a procedure that
was analogous to that for the preparation of compound
14. The oily residue was purified by column chromatog-

T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
757
added 5,6-di-O-acetyl-2,3-di-O-benzyl-L-ascorbic acid
(ABAA) (880 mg, 2 mmol) dissolved in anhydrous ace-
tonitrile (10 mL). Trimethylsilyl triflate (1.7 mL) was
added dropwise to the reaction mixture and it was heat-
ed at 55–70 ꢁC for 16 h. The reaction was terminated by
diluting with CH2Cl2 (50 mL), solvent was evaporated
and oily residue purified by column chromatography
(CH2Cl2/MeOH = 40:1) affording 20 (93.1 mg, 8.7%).
1H NMR (DMSO-d6): d 11.06 (s, 1H, NH), 9.33 (s,
1H, H-6), 5.41 (t, J = 6.71 Hz, 1H, H-50), 4.31 (m, 2H,
H-60), 5.16 and 5.12 (s, 2H, H-70 and H-700), 7.39–7.47
(m, 10H, Ph), 7.68 (d, J = 3.6 Hz, 1H, H-100), 7.66 (d,
J = 3.4 Hz, 1H, H-200) ppm. 13C NMR (DMSO-d6): d
175.51 (C-10), 136.49 (C-20), 145.87 (C-30), 147.72 (C-
40), 75.08 (C-50), 38.83 (C-60), 169.45 (C-2), 172.88 (C-
4), 120.41 (C-5), 130.53 (C-6), 73.52 and 72.71 (C-70
and C-700), 136.46 and 136.40 (C-80 and C-800 Ph),
127.22–131.07 (C–Ph), 134.46 (C-100), 108.22 (C-200)
ppm. MS m/z 537.4 (M+Å).
and CEM) days, the cell number was determined
with a Coulter counter (Coulter Electronics, Eng-
land). The cytostatic concentration was determined
as the compound concentration required to reduce
L1210, Molt4/C8 or CEM cell growth by 50% rela-
tive 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 concentration of the test
compounds.
The HeLa, Hep-2, MCF-7, MiaPaCa-2, SW 620, and
WI 38 cells were cultured as monolayers and main-
tained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 U/mL penicillin, and
100 lg/mL streptomycin in a humidified atmosphere
with 5% CO2 at 37 ꢁC. As reported previously,17,23
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 (10ꢀ8–10ꢀ4 M) and incubated for a
further 72 h. Working dilutions 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 performing the MTT as-
say (Sigma) which detects dehydrogenase activity in
viable cells. The MTT Cell Proliferation Assay is a
colorimetric assay system, which measures the reduc-
tion of a tetrazolium component (MTT) into an insol-
uble formazan product by the mitochondria 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 four hours of incu-
bation, the precipitates were dissolved in 160 lL of
DMSO. The absorbance (OD, optical density) was
measured on a microplate reader at 570 nm. The
absorbancy is directly proportional to the cell viability.
The percentage of growth (PG) of the cell lines was
calculated according to one or the other of the follow-
ing two expressions:
4.3. Antiviral activity assays
Antiviral activity against HSV-1, HSV-2, vaccinia virus,
vesicular stomatitis virus, Coxsackie virus B4, Sindbis
virus, Punta Toro virus, respiratory syncytial virus,
parainfluenza-3 virus, and reovirus-1 was determined
essentially as described previously.26 After a 2-h incuba-
tion period, residual virus was removed and the infected
cells were further incubated with the medium containing
different concentrations of the tested compounds. After
incubation for 3 days at 37 ꢁC, virus-induced cytopath-
ogenicity was monitored microscopically. Antiviral
activity was expressed as the concentration required to
reduce virus-induced cytopathogenicity by 50% (EC50).
4.4. Cytotoxicity assays
Cytotoxicity measurements were based on the inhibition
of HEL, Vero or HeLa cell growth. Cells were seeded at
a rate of 5 · 103 cells/well into 96-well microtiter plates.
Then, medium containing different concentrations of the
test compounds was added. After 3 days of incubation
at 37 ꢁC, the alteration of morphology of the cell cul-
tures was recorded microscopically. Cytotoxicity was ex-
pressed as minimum cytotoxic concentration (MCC) or
the compound concentration that causes a microscopi-
cally detectable alteration of cell morphology.
If (mean ODtest ꢀ mean ODtzero) P 0 then
PG ¼ 100 ꢁ ðmean ODtest
4.5. Antiproliferative assays
ꢀ mean ODtzeroÞ=ðmean ODctrl ꢀ mean ODtzeroÞ;
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, CEM), cervical carcinoma
(HeLa), breast carcinoma (MCF-7), pancreatic carci-
noma (MiaPaCa-2), laryngeal carcinoma (Hep-2), colon
carcinoma (SW 620), and diploid fibroblasts (WI 38).
If (mean ODtest ꢀ mean ODtzero) < 0 then
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, mean
ODctrl = the average of optical density measurements
after the desired period of time with no exposure of cells
to the test compound.
Murine L1210 and human Molt4/C8 and CEM cells
were seeded at 50 · 103 cells/well in 96-well microtiter
plates in the presence of different concentrations of
the test compounds. After 2 (L1210) or 3 (Molt4/C8

758
T. Gazivoda et al. / Bioorg. Med. Chem. 15 (2007) 749–758
10. Ostrowski, T.; Wroblowski, B.; Busson, R.; Rozenski, J.;
De Clercq, E.; Bennett, M. S.; Champness, J. N.;
Summers, W. C.; Sanderson, M. R.; Herdewijn, P. J.
Med. Chem. 1998, 41, 4343.
11. Kumar, R.; Wiebe, L. I.; Knaus, E. E. Can. J. Chem. 1996,
74, 1609.
12. Wigerinck, P.; Pannecouque, C.; Snoeck, R.; Claes, P.; De
Clercq, E.; Herdewijn, P. J. Med. Chem. 1991, 34, 2383.
13. Megati, S.; Phadtare, S.; Zemlicka, J. J. Org. Chem. 1992,
57, 2320.
Each test point was performed in quadruplicate in two
individual experiments. The results were expressed as
IC50, a concentration necessary for 50% inhibition,
which were calculated from dose–response curves using
linear regression analysis by fitting the test concentra-
tions that give PG values above and below the respective
reference value.
Acknowledgment
14. Mitsuya, H.; Yarchoan, R.; Broder, S. Science 1990, 249,
1533.
15. Haines, D. R.; Tseng, C. K. H.; Marquez, V. E. J. Med.
Chem. 1987, 30, 943.
16. Larsson, A.; Stenberg, K.; Ericson, A. C.; Haglund, U.;
Support for this study was provided by the Ministry of
Science of the Republic of Croatia (Projects #0125003
and #0098093).
¨
Yisak, W. A.; Johansson, N. G.; Oberg, B.; Datema, R.
Antimicrob. Agents Chemother. 1986, 30, 598.
´
17. Gazivoda, T.; Plevnik, M.; Plavec, J.; Kraljevic, S.; Kralj,
M.; Pavelic, K.; Balzarini, J.; De Clercq, E.; Mintas, M.;
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