The new 5- or 6-azapyrimidine and cyanuric acid derivatives of l-ascorbic acid bearing the free C-5 hydroxy or C-4 amino group at the ethylenic spacer: CD-spectral absolute configuration determination and biological activity evaluations

Article abstract of DOI:10.1016/j.ejmech.2011.03.066

We report on the synthesis of the novel types of cytosine and 5-azacytosine (1-9), uracil and 6-azauracil (13-18) and cyanuric acid (19-22) derivatives of l-ascorbic acid, and on their cytostatic activity evaluation in human malignant tumour cell lines vs. their cytotoxic effects on human normal fibroblasts (WI38). The CD spectra analysis revealed that cytosine (5 and 6), uracil (14-16), 6-azauracil (17) and cyanuric acid (21) derivatives of l-ascorbic acid bearing free amino group at ethylenic spacer existed as a racemic mixture of enantiomers, whereas L-ascorbic derivatives containing the C-5 substituted hydroxy group at the ethylenic spacer were obtained in (4R, 5S) enantiomeric form. The stereochemistry of 6-azauracil derivative of l-ascorbic acid (13) was confirmed by X-ray crystal structure analysis. The molecules are self-assembled by one N-H?O hydrogen bond, two C-H?O hydrogen bonds and two C-H?π interactions into three-dimensional framework. Cytostatic activity evaluation indicated that compounds did not show distinctive antiproliferative effects on tested cell line panel. However, the cytosine derivative of l-ascorbic acid (1) containing the C4-C5 double bond conjugated with the lactone moiety produced rather marked growth inhibitory effect on hepatocellular carcinoma (HepG2), metastatic breast epithelial carcinoma (MCF-7) and cervical carcinoma (HeLa) cell lines at micromolar concentrations, but also exerted strong cytostatic effect on WI38. 5-Azacytosine derivative of l-ascorbic acid (2) with a double bond at the C4-C5 conjugated with the lactone moiety displayed potent antitumour activity against tested tumour cell lines with meanIC₅₀ values ranging from 0.92 to 5.91 μM. However, this compound also exhibited pronounced cytotoxicity towards WI38. Flow cytometric analysis of the cell cycle revealed that compound 2 triggers S phase arrest, which clearly demonstrates its interference with DNA replication, a key event of cell proliferation. Marked anticancer efficacy of compound 2 supports further in vivo investigation into its possible clinical utility.

Full text of DOI:10.1016/j.ejmech.2011.03.066

European Journal of Medicinal Chemistry 46 (2011) 2770e2785
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The new 5- or 6-azapyrimidine and cyanuric acid derivatives of -ascorbic acid
L
bearing the free C-5 hydroxy or C-4 amino group at the ethylenic spacer:
CD-spectral absolute configuration determination and biological activity
evaluations
a
b
,
c
c
ꢀ
ꢀa
ꢁ
ꢀ a
ꢀ d
ꢀ
ꢀd
K. Wittine , M. Stipkovic Babic , M. Kosutic , M. Cetina , K. Rissanen , S. Kraljevic Pavelic ,
a,
ꢀd
ꢀ e
*
ꢀ
A. Tomljenovic Paravic , M. Sedic , K. Pavelic , M. Mintas
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20, HR-10000 Zagreb, Croatia
a
ꢀ
b
ꢀ
Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, Prilaz baruna Filipovica 28a, HR-10000 Zagreb, Croatia
c Department of Chemistry, NanoScience Center, University of Jyväskylä, P.O. Box 35, FI-40014 Jyväskylä, Finland
d
ꢀ
ꢁ
ꢀ
Department of Biotechnology, University of Rijeka, trg brace Mazuranica 10, 51000 Rijeka, Croatia
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, HR-10000 Zagreb, Croatia
e
ꢁ
ꢀ
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on the synthesis of the novel types of cytosine and 5-azacytosine (1e9), uracil and 6-azauracil
(13e18) and cyanuric acid (19e22) derivatives of L-ascorbic acid, and on their cytostatic activity evaluation
in human malignant tumour cell lines vs. their cytotoxic effects on human normal fibroblasts (WI38). The
CD spectra analysis revealed that cytosine (5 and 6), uracil (14e16), 6-azauracil (17) and cyanuric acid (21)
derivatives of L-ascorbic acid bearing free amino group at ethylenic spacer existed as a racemic mixture of
Received 17 December 2010
Received in revised form
17 March 2011
Accepted 31 March 2011
Available online 6 April 2011
enantiomers, whereas L-ascorbic derivatives containing the C-5 substituted hydroxy group at the ethylenic
spacer were obtained in (4R, 5S) enantiomeric form. The stereochemistry of 6-azauracil derivative of
Keywords:
L
-ascorbic acid (13) was confirmed by X-ray crystal structure analysis. The molecules are self-assembled by
one NeH/O hydrogen bond, two CeH/O hydrogen bonds and two CeH/ interactions into three-
dimensional framework. Cytostatic activity evaluation indicated that compounds did not show distinc-
tive antiproliferative effects on tested cell line panel. However, the cytosine derivative of -ascorbic acid (1)
Pyrimidine and cyanuric acid derivatives
p
L-ascorbic acid
Circular dichroism
Cytostatic activity evaluation
X-ray diffraction
L
containing the C4-C5 double bond conjugated with the lactone moiety produced rather marked growth
inhibitory effect on hepatocellular carcinoma (HepG2), metastatic breast epithelial carcinoma (MCF-7) and
cervical carcinoma (HeLa) cell lines at micromolar concentrations, but also exerted strong cytostatic effect
Cell cycle analysis
on WI38. 5-Azacytosine derivative of
the lactone moiety displayed potent antitumour activity against tested tumour cell lines with meanIC50
values ranging from 0.92 to 5.91 M. However, this compound also exhibited pronounced cytotoxicity
L-ascorbic acid (2) with a double bond at the C4eC5 conjugated with
m
towards WI38. Flow cytometric analysis of the cell cycle revealed that compound 2 triggers S phase arrest,
which clearly demonstrates its interference with DNA replication, a key event of cell proliferation. Marked
anticancer efficacy of compound 2 supports further in vivo investigation into its possible clinical utility.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
b-D-arabino-pentofuranosyl) cytosine (CNDAC) [3], have been
developed as potent antitumour agents effective not only in
leukemias and lymphomas, but also in a wide variety of solid
tumours in vitro and in vivo. L-ascorbic acid (vitamin C), a water-
soluble vitamin essential for hundreds of vital metabolic reac-
tions, and its isomers have garnered great attention as anticancer
agents, although the mechanisms underlying their antitumour
activity have not been fully elucidated. In spite of the fact that it
cannot be produced by the human body, vitamin C is integral to the
formation of collagen, connective tissue, and immune system
factors, and also serves important anti-inflammatory and
Nucleoside analogues represent a valuable source that contrib-
utes significantly to the arsenal of chemotherapeutic agents against
viruses and cancer. In recent years, a number of five-membered
cytosine nucleoside analogues, such as 1-(2-deoxy-2-methylene-
b-D
-erythro-pentofuranosyl) cytosine (DMDC) [1], 20-deoxy-20,
20-difluorocytidine (gemcitabine) [2], and 1-(2-C-cyano-2-deoxy-
* Corresponding author. Tel.: þ385 1 4597 214; fax: þ385 1 4597 250.
E-mail address: mladen.mintas@fkit.hr (M. Mintas).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.066

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2771
antioxidant functions. Cameron and Pauling believed that
acid combats cancer by promoting collagen synthesis that prevents
tumours from invading other tissues [4]. However, researchers now
believe that L-ascorbic acid fights cancer by neutralizing free radicals
before they can damage DNA and initiate tumour growth, and/or
may act as a pro-oxidant helping body’s own free radicals to destroy
tumours in their early stages [5e7]. For example, a mixture of L-
ascorbic acid and cupric sulfate significantly inhibited human
mammary tumour growth in mice when administered orally [8]. A
number of ascorbic acid isomers/derivatives have been synthesized
L
-ascorbic
then subjected to either debenzylationwith boron-trichloride to give
3, or to ammonolysis with ammonium in methanol to yield
compound 5. Besides removing the N4-benzoyl protecting group,
ammonolysis created in this reaction a new chiral stereocenter with
the amino group at the C-4 position of the lactone moiety (5).
5-Azacytosine (5-AzaC) was silylated in an analogous way by using
1,1,1,3,3,3-hexamethyldisilazane (HMDS) and catalytic amount of
ammonium-sulphate. The resulted silylated base was reacted in situ
withdAdBAto give 2withtheassistanceof theFriedel-Crafts catalyst,
trimethylsilyltrifluoromethane sulphonate (TMSOTf). Debenzylation
with 1 M boron-trichloride in dichloromethane yielded 4 with free
and tested on tumour cell lines so far. It was shown that L-ascorbic
acid modulates the in vitro growth of colonies of human and mouse
myeloma progenitor/stem cells and leukemic colony-forming cells
from bone marrow of patients with acute myelocytic leukemia [9].
enolic hydroxyl group at C-2 of
with ammonia in methanol/dioxane mixture gave compound 6
bearing the amino group at the C-4 of -ascorbic acid lactone moiety.
Reaction of silylated N4-benzoylcytosine with the 4-(5,6)-
epoxy-2,3-di-O-benzyl- -ascorbic acid (EdBA) gave cytosine
-ascorbic acid derivative (7) bearing the free hydroxy group at the
C-5 position of the ethylenic spacer. Analogous reaction of 5-aza-
cytosine with EdBA gave -ascorbic acid derivative with the free
L
-ascorbic acid moiety. Reaction of 2
L
Furthermore,
suppressed the growth of malignant leukemia cell line P388D1 in
vivo in the following order of potency: 6-deoxy ¼ dihydroxy-
crotonolactone (eCl, eBr, eNH2) > -ascorbic acid and its stereo-
isomer -ascorbic acid, -isoascorbic acid and dehydroascorbic
L-Ascorbic acid and its derivatives were cytotoxic and
L
g-
L
L
D
D
L
acid > 6-substituted (ePO4, eSO4, -palmitate, -stearate) > 2,
6-disubstituted (dipalmitate) > 2-substituted (ePO4, eSO4, O-Me,
O-octadecyl > dihydroxy-g-butyrolactone [10e12]).
hydroxyl group at the C-5 of the ethylenic spacer (8). Subsequent
removal of N4-benzoyl protecting group in 7 with methanolic
ammonia gave 9 containing free hydroxy group at the C-5 of the
ethylenic spacer (Scheme 1).
Several studies have provided evidence for cytotoxicity of
-ascorbic acid in some human tumour cells, neuroblastoma [13],
L
Pyrimidine derivatives of 4,5-didehydro-5,6-dideoxy-L-ascorbic
osteosarcoma and retinoblastoma [14]. Substitution at 2-, 6- and 2,6-
positions in -ascorbic acid modulates its cytotoxic potential against
acid (10e12) were prepared by condensation of uracil, 5-fluoro-
uracil, thymine and 6-azauracil with 5,6-di-O-acetyl-2,3-di-O-
L
malignantcells. Furthermore, ascorbate-6-palmitateandascorbate-6-
stearate are more potent growth inhibitors of murine leukemia cells
thanascorbate 2-phosphate, ascorbate-6-phosphate and ascorbate-6-
sulphate [15]. In addition, ascorbyl palmitate and ascorbyl stearate
have attracted considerable interest in view of their potential use as
anticancer compounds, whose lipophilic nature allows them to easily
benzyl-
of -ascorbic acid (13) was synthesized in an analogous manner.
Ammonolysis of compounds 10e13 gave uracil (14), 5-fluorouracil
(15), thymine (16) and 6-azauracil (17) derivatives of -ascorbic acid
with the amino group at the C-4 position of the lactone moiety.
Reaction of silylated 6-azauracil with EdBA and TMSOTf furnished
L
-ascorbic acid (dAdBA) [23]. Related 6-azauracil derivative
L
L
traverse cell membranes and bloodebrain barrier [16].
L
-Ascorbic acid
6-azauracil derivative of
hydroxy group at the C-5 of the ethylenic spacer (Scheme 2).
In a similar manner, reaction of cyanuric acid (CA) and -ascorbic
acid derivatives (dAdBA and EdBA) afforded 19 and 22. Reaction of
19 with methanolic ammonia yielded 21 with the free amino group
at C-4, and with BCl3 gave 20 with the free hydroxyl groups at the
C2 and C3 positions of the lactone moiety (Scheme 3).
L
-ascorbic acid (18) bearing the free
and ascorbyl esters were shown to inhibit the proliferation of mouse
glioma and human brain tumour cells, glioma (U-373) and glioblas-
toma (T98G) cells, and renal carcinoma cells [17e19]. The role of
L
sodium-L-ascorbate in angiogenesis was ascertained by the study
demonstrating its inhibitory effect on the formation of vessel-like
tubular structures of endothelial cells cultured on Matrigel [20].
Related to our previous findings that some pyrimidine and purine
The structures of the novel 5- or 6-azapyrimidine and cyanuric
derivatives of 4,5-didehydro-5,6-dideoxy-L-ascorbic acid posses
acid derivatives of L
-ascorbic acid were deduced from their 1H NMR
pronounced cytostatic activity against some malignant tumour cell
lines [21e23], we have synthesized a series of novel pyrimidine
derivatives containing C4-C5 double bond conjugated with the
lactone moiety (1e4, 13, 19, 20), and their congeners bearing free
hydroxy group at C-5 of the ethylenic spacer (7e9, 18, 22), as well as
pyrimidine derivatives (5, 6, 14e17, 21) with the amino group at the
C-4 of thelactonemoiety(Fig.1). Besidessynthesis, thepresentstudy
providesinformation on theircytostatic activitiesin different human
tumour cell lines and cytotoxic effects on human normal fibroblasts.
spectra (Table 1), as well as their 13C NMR and mass spectra.
The Z-configuration was deduced on the basis of comparison of
coupling constants and chemical shifts in their 1H and 13C NMR
spectra with those of related L-ascorbic acid derivatives in which
the Z- and E-configuration were assessed by the use of decoupling
experiments and 2D NOESY and HMBC correlation experiments
[21,22]. An unambiguous proof of the (Z)-configuration of 6-
azauracil derivative of -ascorbic acid (13) was obtained by its X-ray
L
crystal structure analysis (c.f. Section 2.3).
2. Results and discussion
2.2. UV and CD spectra
2.1. Chemistry
We analysed the circular dichroism (CD) spectra of L-ascorbic
acid derivatives containing C-5 hydroxy group (8, Fig. 2A and 7,
Fig. 2B) or C-4 amino group (5, 6, 14e17 and 21) at the ethylenic
spacer. This investigation was undertaken to deduce their absolute
configurations and stereochemical course of condensation reac-
tions (Schemes 1e3).
Cytosine and 5-azacytosine derivatives of -ascorbic acid (1e9)
(Scheme 1) were prepared by the reaction of the silylated cytosine
bases (BzCt: benzoylated cytosine and 5-AzaC: 5-azacytosine) with
L
5,6-di-O-acetyl-2,3-di-O-benzyl-
L-ascorbic acid (dAdBA) or epoxy-
2,3-di-O-benzyl-L-ascorbic acid (EdBA) under Vorbrüggen reaction
L-ascorbic acid moiety consists a g-lactone ring in which ene-
conditions.
diol portion and the oxygen of the lactone moiety are conjugated
to the carbonyl group. The UV spectra of enones are characterized
by an intense absorption band (K-band) in the 215e250 nm region
[24]. 5-Azacytosine derivative (8, Fig. 2A) exhibits UV-band at
Silylated N4-benzoylcytosine was reacted with 5,6-di-O-acetyl-
2,3-di-O-benzyl- -ascorbic acid (dAdBA) to give cytosine derivative 1
as a mixture of (Z)- and (E)-isomers. Crystallization of this diaste-
reomeric mixture from ethanol gave pure (Z)-isomer of 1, which was
L
205 nm (band II,
e
¼ 32859 mmolꢀ1 cm2) resulting from n /
p
*

2772
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
A Cytosine and 5-azacytosine derivatives of L-ascorbic acid (1-9)
4
NHR
H N
2
5'
X
N
X
X
HO
4
R HN
6'
N
O
N
O
N
N
O
O
O
N
6
O
O
O
5
4
H N
2
O
BnO
OBn
3
R O
2
OR
BnO
OBn
2
3
4
4
Comp.
Comp.
R X
Comp.
R
R
R
X
X
1
2
3
4
5
6
7
8
9
Bn Bn Bz CH
CH
N
Bz CH
H N
H CH
Bn Bn H
N
H
H
H
H
CH
N
Bn H
B Pyrimidine and 6-azauracil derivatives of L-ascorbic acid (13-18)
O
5
R
5
R
O
HN
X
X
HN
O
N
N
HO
N
N
O
O
O
O
O
O
O
O
HN
H N
2
BnO
OBn
O
BnO
18
OBn
BnO
OBn
5
5
Comp.
14
15
Comp.
R
X
R
X
H
F
CH
CH
13
H
N
CH CH
3
16
H
N
17
C Cyanuric acid derivatives of L-ascorbic acid (19-22)
O
O
H
N
HN
NH
O
HN
H
N
O
N
O
O
N
HO
O
O
O
O
O
O
O
N
O
HN
H N
2
BnO
3
R O
2
OBn
O
BnO
OBn
OR
2
3
21
22
Comp.
19
R
R
Bn Bn
H
H
20
Fig. 1. The novel pyrimidine derivatives of L-ascorbic acid (1e9 and 13e22).
transition and 239 nm (band I,
p
e
¼ 14902 mmolꢀ1 cm2) arising from
In the CD spectrum of 8 (Fig. 2A) the negative Cotton effect at
248 nm (band I, De ¼ ꢀ71.43 mmolꢀ1 cm2) may be assigned to the
/ p
* transition. The UV spectrum of N4-benzoylcytosine deriv-
ative of
L-ascorbic acid (7, Fig. 2B) has 3 electronic transitions. The
p / p
* transition (K-band), while the one located at 209 nm (band II,
weak band at 307 nm (band I,
e
¼ 7121 mmolꢀ1 cm2) belongs to the
De ¼ 59.60 mmolꢀ1 cm2) can be attributed to the n /
p
* transition
lowest energy transition. Empirically this band would be described
(R-band). This assignment is in accord with that of structurally related
cyclopentenone systems [24]. The lowest energy transition in the CD
spectrum of 7 (Fig. 2B) gives rise to a negative Cotton effect around
304nm (bandI, De ¼ ꢀ10.96 mmolꢀ1 cm2) whichcanbe assigned tothe
as a carbonyl n /
character [25]. The other two absorption maxima at 257 nm (band
p / pCO
* transition but it actually has pPh *
II,
e
e
¼
18858 mmolꢀ1cm2) and 205 nm (band III,
¼ 31749 mmolꢀ1 cm2) can be assigned to the
p
/
p
* and n /
p
*
n / p
* of the carbonyl group. This rather broad band appears usually
of the enone chromophore. They probably cover more then one
electronic transition but can be assigned as designated above.
between 320 and 350 nm but conjugation leads to a red shift of the
n /
* carbonyl band [25]. The pPh CO* excitation of the benzoyl
p
/ p

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2773
moiety is located at 264 nm (band II, De ¼ ꢀ8.81 mmolꢀ1 cm2). A
positive Cotton effect around 241 nm (band III, De ¼ 6.75 mmolꢀ1 cm2)
torsion angle is ꢀ3.0(7)ꢁ (Fig. 3) indicating the Z-configuration of
C7eC8 double bond.
may be assigned to the
p
/
p
* transition of the enone chromophore
The bond lengths match very well with the corresponding ones
in 5-(trifluoromethyl)uracil [22] and 6-chloropurine [23] analogues
of 13. The oxygen atoms O5 and O6 are oriented in a synperiplanar
fashion; the O5eC11eC10eO6 torsion angle is 1.0(9)ꢁ. The confor-
mation of the benzyloxy groups is antiperiplanar, defined by the
C11eO5eC12eC13 and C10eO6eC19eC20 torsion angles
of ꢀ171.5(4) and 174.3(4)ꢁ, respectively. Two phenyl rings have
different orientation with respect to the lactone ring. While the
C20eC25 ring is nearly parallel with the lactone ring [dihedral
angle is 7.6(2)ꢁ], the C13eC18 ring is almost perpendicular to it
[dihedral angle is 73.5(2)ꢁ]. Such perpendicular orientation with
respect to the lactone ring is also observed for 6-azauracil ring, as
dihedral angle between their mean planes is 88.6(2)ꢁ. Three
intramolecular hydrogen bonds lock the conformation of the
molecule (Fig. 3; Table 2). The C6/O3 and C6/O1 hydrogen bonds
form five-membered rings, while the third intramolecular
hydrogen bond, C12/O6, forms six-membered ring.
which considerably overlaps with the n /
p
* of the benzamide
chromophore [26] at 223 nm (band IV, De ¼ 5.86 mmolꢀ1 cm2). This
band is generally assigned to a 1La aromatic transition [27] although the
intramolecular charge transfer transition has also been proposed as the
origin of this band [28]. The band at 207 nm (band V,
De ¼ 2.75 mmolꢀ1 cm2) can be attributed to the n /
p
* transition (R-
band) but may also result from a transition within the aromatic ring.
We have previously assigned the 4R, 5S configuration to the 4-
(5,6-epoxy) derivative of L-ascorbic acid on the basis of combined
use of CD spectroscopy and X-ray crystal structure analysis [29].
Comparison of that spectrum with the CD spectra of C-5 hydroxy
derivatives of -ascorbic acid (7 and 8) shows that all three spectra
L
are quite similar with negative Cotton effect trend but at different
wavelengths due to difference in molecular structure or solvent
effects. Thus, their configuration consequently has to be the same
4R, 5S. Ring opening of epoxy-
a stereospecific manner giving rise to 5-azacytosine-(8) and N4-
benzoylcytosine derivative of -ascorbic acid (7) with the same
L-ascorbic acid derivative occurs in
The N3/O1 hydrogen bond self-assemble the molecules of 13
into dimers (Fig. 4; Table 2), so forming characteristic synthon for
structures of pyrimidine derivatives, eight-membered ring.
The same hydrogen-bonding motif, centrosymmetric dimer,
forms C6/O3 hydrogen bond, but this time via ten-membered
ring. The C16/O4 hydrogen bond generates infinite chains and
L
stereostructure at C-4 and C-5 of the ethylenic spacer. This is sup-
ported also by mechanistic consideration of the nucleophile attack
of the epoxide derivative from the less hindered side.
Cytosine (5 and 6), uracil (14e16), 6-azauracil (17) and cyanuric
acid (21) derivatives of
L
-ascorbic acid with the free amino group at
links hydrogen-bonded dimers (Fig. 4). Two CeH/p interactions
the C-4 of the lactone moiety did not show Cotton effect which is
supported by a generally adopted view that the nucleophilic attack
takes place with equal probability from both sides of the C4eC5
double bond resulting in racemic mixtures of enantiomers (CD
spectra not shown).
participate also in supramolecular aggregation, so forming three-
dimensional framework (Fig. 5).
2.4. Biological results
2.4.1. Cytostatic activity
2.3. Molecular and crystal structure of 6-azauracil derivative of
Pyrimidine (1e9, 13e17) and cyanuric acid derivatives of
L
-ascorbic acid (13)
L-ascorbic acid (19e22) were evaluated in vitro for their cytostatic
effects against malignant tumour cell lines including cervical
carcinoma (HeLa), colorectal adenocarcinoma (SW620), pancreatic
carcinoma (MiaPaCa-2), metastatic breast epithelial adeno
In order to establish the exact stereostructure of 13 its single
crystal X-ray structure analysis was carried out. The C6eC7eC8eO3
Scheme 1. Synthesis of cytosine and 5-azacytosine derivatives of L-ascorbic acid (1e9). Reagentsand conditions: (i) HMDS, (NH4)2SO4/argon/reflux/3 h; TMSOTf/CH3CN/55e70ꢁC/12 h;
(ii) BCl3/CH2Cl2/ꢀ78 ꢁC/2 h; (iii) NH3/MeOH-dioxane/r.t./16 h.

2774
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
Scheme 2. Synthesis of pyrimidine derivatives of L-ascorbic acid (10e18). Reagents and conditions: (i) HMDS, (NH4)2SO4/argon/reflux/3 h; TMSOTf/CH3CN/55e70 ꢁC/12 h; (ii) NH3/
MeOH-dioxane/r.t./16 h.
carcinoma (MCF-7) and hepatocellular carcinoma (HepG2), and
compared with their effects on normal diploid human fibroblasts
(WI38) (Table 3).
and 3.49 mM, respectively). However, this compound was highly
cytotoxic to normal human fibroblasts as well. Marked cytostatic
effects were also observed in HepG2, MCF-7 and HeLa cell lines
Although tested compounds did not have, in general, distinct
and specific antiproliferative activity, some of them produced
rather marked effects on HepG2, MCF-7, MiaPaCa-2 and HeLa cells
when treated with cytosine derivative of L-ascorbic acid (1) con-
taining the C4-C5 double bond conjugated with the lactone moiety,
with the IC50 values of 1.98, 2.03 and 7.20, respectively. This
compound also inhibited the growth of human fibroblasts. Finally,
cytosine derivatives containing either enolic hydroxy groups of the
lactone moiety (3) or free C-5 hydroxy group at the ethylenic spacer
(7 and 9) showed stronger antiproliferative activity towards the
(Fig. 6). Among these, 5-azacytosine derivative of -ascorbic acid (2)
L
with a double bond at the C4eC5 conjugated with the lactone
moiety showed the most pronounced cytostatic activity, in partic-
ular towards MiaPaCa-2, HepG2 and MCF-7 cells (IC50 0.92, 2.72
Scheme 3. Synthesis of cyanuric acid derivatives of L-ascorbic acid (19e22). Reagents and conditions: (i) HMDS, (NH4)2SO4/argon/reflux/3 h; TMSOTf/CH3CN/55-70 ꢁC/12 h;
(ii) BCl3/CH2Cl2/-78 ꢁC/2 h; (iii) NH3/MeOH-dioxane/r.t./16 h.

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2775
Table 1
1H NMR chemical shifts (
d
/ppm) and HeH coupling constants (J/Hz) of compounds 1e9 and 13e22 (for enumeration of atoms in molecular skeleton c.f. Fig. 1A).
Comp. H5
H6
OCH2-2
OCH2-3
H-4
H50
H60
Ph
NH2-40
NH
NH2-4
OH-5
1
5.55 (t, 1H) 4.67
5.15
(s, 2H)
5.32
(s, 2H)
e
7.33
(d, 1H)
J ¼ 6.60
8.16
(d, 1H)
J ¼ 6.30
7.35e7.45
(m, 10H)
7.50e8.01 Phcyt
(m, 5H)
e
11.20
(s, 1H)
e
e
J ¼ 6.84)
(d, 2H)
J ¼ 6.72
2
3
4
5
5.50
4.49
5.14
(s, 2H)
5.31
(s, 2H)
e
e
e
e
e
7.39
(s, 1H)
7.29e7.44
(m, 10H)
7,33/8,26
(s, 2 ꢂ 1H)
e
e
e
e
e
e
e
e
e
e
e
(t, 1H)
J ¼ 6.70
5.40
(t, 1H)
J ¼ 6.95
5.34
(t, 1H)
J ¼ 6.87
1.85e2.03
(m,2H)
(d, 2H)
J ¼ 6.74
4.59
(d, 2H)
J ¼ 6.96
4.46
(d, 2H)
J ¼ 6.84
3.60
(t, 2H)
J ¼ 7.35
e
e
e
6.08
7.99
e
8.50/9.44
(s, 2 ꢂ 1H)
(d, 1H)
J ¼ 7.62
e
(d, 1H)
J ¼ 7.62)
7.38
5.44
(s, 2H)
7.35e7.44
(m, 10H)
7.48/8.08
(s, 2 ꢂ 1H)
(s, 1H)
5,01
(s, 2H)
5.13
5.61
(d, 1H)
J ¼ 7,14
7.37
(d,1H)
J ¼ 7,15
7.32e7.40
(m, 10H)
6.93/6.99
(s, 2 ꢂ 1H)
6,11/8,17
(s, 2x1H)
(dd, 2H)
J1 ¼ 11,83
J2 ¼ 22,93
5.13
(dd, 2H)
J1 ¼ 11.83
J2 ¼ 41.37
5.23
6
7
8
9
1.92e2.06
(m, 2H)
3.62e3.72
(m, 2H)
5.01
(s, 2H)
e
e
7,31
(s, 1H)
7.30e7.41
(m, 10H)
7.35/8.17
(s, 2 ꢂ 1H)
e
6.12/8.09
(s, 2x1H)
e
4.11e4.16
(m, 1H)
3.70/4.23
4.96
(dd, 2H)
J1 ¼ 11,13 J1 ¼ 11.64
4.99
(s, 1H)
5.58
(d, 1H)
J ¼ 6.90
8.07
(d, 1H)
J ¼ 7.20) 7.49e8.00 Phcyt
7.29e7.41
(m, 10H)
e
11.16
(s, 1H)
e
e
e
5.42
(d, 1H)
J ¼ 6.77
(2 ꢂ dd, 2H)
(dd, 2H)
J1 ¼ 9.78/2.46
J2 ¼ 13.08/13.08 J2¼17,49
J2 ¼ 51.13
(m, 5H)
7.26e7.40
(m, 10H)
3.98e4.08
(m, 1H)
3.49e4.08
4.95
(s, 2H)
5.20
4.98
(s, 1H)
e
7.35
(s, 1H)
7.38/8.12
(s, 2 ꢂ 1H)
e
e
5.47
(d, 1H)
J ¼ 6.75
(m, 2H)
(dd, 2H)
J1 ¼ 11.69
J2 ¼ 26.58
5.22
4.00e4.07
3.45e4.07
4.90
4.96
5.62
7.48
7.28e7.38
6.95/7.02
5.54
(m, 1H)
(m, 2H)
(s, 2H)
(dd, 2H)
J1 ¼ 11.71
J2 ¼ 24.73
5.33
(d, 2H)
J ¼ 1.68 J ¼ 7.14
(d, 1H)
(d, 1H)
J ¼ 7.16
(m, 10H)
(s, 2 ꢂ 1H)
(d, 1H)
J ¼ 6.84
13
14
5.51
4.69
5.16
(s, 2H)
e
e
7.47
(s, 1H)
e
7.35e7.45
(m, 10H)
e
e
12.16
(s, 1H)
e
e
(t, 1H)
J ¼ 6.74
1.96
(d, 2H)
J ¼ 6.71
3.64
(s, 2H)
5.01
5.14
7.45
5.51
7.33e7.42
11.21
6.16/8.17
e
(m, 2H)
(dd, 2H)
J1 ¼ 6.20
J2 ¼ 13.71
3.58e3.72
(m, 2H)
(s, 2H)
(dd, 2H)
J1 ¼ 11.91
J2 ¼ 22.41
5.14
(dd, 2H)
J1 ¼ 11.87
J2 ¼ 39.36
5.11
(dd, 2H)
J1 ¼ 11.89
J2 ¼ 24.01
5.15
(dd, 2H)
J1 ¼ 11.94
J2 ¼ 41.12
5.20
(d, 1H)
J ¼ 7.86) J1 ¼ 1.68
(dd, 1H) (m, 10H)
(s, 1H) (s, 2 ꢂ 1H)
J2 ¼ 7.77
15
16
17
18
1.93e2.08
(m, 2H)
5.01
(s, 2H)
e
e
e
e
e
7.91
7.31e7.41
e
e
e
e
11.73
6.17/8.16
e
e
e
(d, 1H)
J ¼ 3.39
(m, 10H)
(s, 1H) (s, 2 ꢂ 1H)
1.86e2.03
(m, 2H)
3.52e3.66
(m, 2H)
4.99
(s, 2H)
7.33
7.27e7.40 (m, 10H)
11.19
6.13/8.13
(s, 1H)
(s, 1H) (s, 2 ꢂ 1H)
1.94e2.08
(m, 2H)
3.82e3.92
(m, 2H)
5.03
(s, 2H)
7.46
(s, 1H)
e
e
7.20e7.44
(m, 10H)
1208
6.14/8.12
(s, 1H) (s, 2 ꢂ 1H)
3.93e4.04
(m, 1H)
3.93e4.04
(m, 1H)
4.97
(s, 2H)
4.96
(s, 1H)
7.44
(s, 1H)
7.29e7.41
12, 05
(s, 1H)
e
5.49
(dd, 2H)
J1 ¼ 11,79
J2 ¼ 35, 05
5.31
(m, 10H)
(d, 1H)
J ¼ 6.16
19
20
21
5.42
4.48
5.14
(s, 2H)
e
e
e
e
e
e
e
e
e
7.30e7.45
(m, 10H)
e
e
e
11.43
e
e
e
e
(t, 1H)
J ¼ 6.18
5.31
(t, 1H)
J ¼ 6.37
1.83e2.01
(m, 2H)
(d, 2H)
J ¼ 6.10
4.47
(d, 2H)
J ¼ 6.31
3.58e3.76
(m, 2H)
(s, 2H)
(s, 2H)
e
e
e
11.47
e
(s, 2H)
5.00
(s, 2H)
5.11
7.34e7.39
11.34
6.08/8.17
(dd 2H)
J1 ¼ 11.85
J2 ¼ 37.33
5.20
(m, 10H)
(s, 2H) (s, 2 ꢂ 1H)
22
3.96e4.05
3.46e4.05
4.94
4.98
e
e
7.27e7.42
e
11.45
e
e
(m, 1H)
(m, 2H)
(s, 2H)
(dd, 2H)
J1 ¼ 11.67
J2 ¼ 26.54
(s, 1H)
(m, 10H)
(s, 2H)
Compounds were dissolved in DMSO-d6. 3: 9.67 ppm (OH-3, s, 1H), 11.32 ppm (OH-2, s, 1H); 4: 9.75 ppm (OH-2; s, 1H); 16: 1.72 ppm (CH3, s, 3H); 20: 9.53 ppm (OH-3, s, 1H),
11.24 ppm (OH-2, s, 1H).

2776
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
interfere with DNA replication mechanisms in cancer cells at low
pharmacological concentrations (1
M and 5 M) even if these
m
m
micromolar concentrations failed to induce apoptosis in all cell
lines (data not shown). Knowing that drug potency (IC50 values
obtained in the in vitro studies) was found to be predictive of Phase
II performance/toxicity of the tested drugs [34,35], additional in
vivo studies should be preformed to investigate the possible use of
compound
2 as a novel anticancer pro-drug. On contrary,
compound 1 showed neither specific nor significant effect on HeLa
and MCF-7 cell cycle (Table 4) and apoptosis induction at micro-
molar concentrations (5
mM and 10
mM), except for inducing a rise
in S- phase HeLa cells after 24-h treatment at 10
mM. This finding
raises a question whether higher pharmacological concentrations
of this substance would induce non-specific cytotoxic effects, and
might not be suitable to achieve desired biological effect in vivo.
3. Experimental section
3.1. Materials and general methods
Melting points were determined on a Kofler micro hot-stage
instrument (Reichter, Wien) and were uncorrected. 1H and 13C
NMR spectra were recorded on a Varian Gemini 300 spectrometer,
operating at 300 and 75.5 MHz for the 1H and 13C nuclei, respec-
tively. Samples were measured in DMSO-d6 solutions at 20 ꢁC in
5 mm NMR tubes. Chemical shifts (d) in ppm were referred to TMS.
The electron impact mass spectra were recorded with an EXTERNAL
FT MS 2002 instrument with ionizing energy of 70 eV. Precoated
Merck silica gel 60 F254 plates were used for thin-layer chroma-
tography. Spots were visualized by shortwave (254 nm) UV light.
Column chromatography was performed on Fluka silica gel
(0.063e0.200 mm), with dichloromethane/methanol and petro-
lether/ethylacetate as eluent. Additional purification of some
compounds by recrystallization from ethanol afforded the analyt-
ical samples.
Fig. 2. A. CD (d,
c
¼
3000
ꢂ
10ꢀ5 mol dmꢀ3
cm) and UV (——,
cm) spectra of 8 in EtOH. B. CD (d,
,
l
¼
1
c
¼
2442
ꢂ
10ꢀ3 mol dmꢀ3
,
l
¼
1
c ¼ 2993 ꢂ 10ꢀ5 mol dmꢀ3, l ¼ 1 cm) and UV (——, c ¼ 2993 ꢂ 10ꢀ5 mol dmꢀ3, l ¼ 1 cm)
spectra of 7 in EtOH.
All chemicals were purchased from Sigma Aldrich (Germany).
All solvents were of analytical grade purity and dryed. Dichloro-
methane (CH2Cl2) was distilled from phosphorus pentoxide (P2O5)
and stored over 4Å molecular sieves. Acetonitrile (CH3CN) was
distilled from calcium hydride (CaH2) and stored over 3 Å molecular
sieves. Methanol (CH3OH) was not distilled but stored over 3 Å
molecular sieves.
MCF-7 cells in comparison with other tested tumour cell lines, but
it also exerted rather marked cytotoxic effect on normal fibroblasts.
2.4.2. Cell cycle progression
As compounds 1 and 2 exerted the most pronounced cytostatic
activities in tested cell lines, we monitored their effects on the cell
cycle (Tables 4 and 5). Compound 2 had specific impact on the
progression of MiaPaCa-2, SW620 and HeLa cell cycle causing
significant decline in G1 phase cells accompanied by marked
accumulation of cells in S phase (Table 5). This cell cycle pattern was
uniformly observed across all three tested cell lines, except for
MiaPaCa-2 cells treated with lower concentration of compound 2
for 24 h. Based on obtained data, it seems reasonable to believe that
observed cell growth inhibition upon treatment with compound 2
is attributable to its interference with cell replication. Observed
effect indeed, might be partially attributed to the 5-azacytidine’s
general toxicity reported previously [30]. 5-azacytidine could be
activated to the nucleoside triphosphate and incorporated into
both DNA and RNA resulting in the inhibition of DNA, RNA and
protein synthesis [31]. When applied at doses low enough to avoid
induction of cell death, incorporation of 5-azacytidine into DNA of
cultured cells promotes a rapid loss of activity of DNA (cytosine-C5)
methyltransferase, as this enzyme binds irreversibly to ZCyt resi-
dues in DNA [32]. If not repaired, this complex is toxic and muta-
genic in bacteria, cultured cells and rodents [33]. It still remains to
be elucidated whether 5-azacytidine inhibits growth primarily
through cytotoxicity or through the effects on DNA methylation.
Anticancer potential of compound 2 is corroborated by its ability to
3.2. Synthesis
3.2.1. Method A. General procedure for the preparation of pyrimidine
derivatives of 2,3-di-O-benzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic
acid (1, 2, 13 and 19)
Suspension of anhydrous base (1.5 g) and (NH4)2SO4 (110 mg) in
HMDS (30 mL) was refluxed overnight under the inert atmosphere
of argon. The excess HMDS was removed under the reduced pres-
sure (0.1 mmHg). To the oily product thus obtained 5,6-di-O-acetyl-
2,3-di-O-benzyl-L-ascorbic acid (nbase/1.507 mmol) dissolved in dry
CH3CN (15 mL) was added. Then TMSOTf (4 ꢂ 0.2 mL) was added
dropwise at room temperature and the mixture stirred for 16 h at
55e70 ꢁC. The reaction mixture was evaporated and the crude oily
product washed with CH2Cl2 and several times with aq. NaHCO3.
Organic layer was dried over MgSO4, evaporated, and the crude
product purified by silica gel column chromatography.
3.2.2. Method B. General procedure for the preparation of pyrimidine
derivatives with the amino group at the C-4 of -ascorbic acid (5, 6,
L
14e17 and 21)
To a solution of pyrimidine derivatives of
L-ascorbic acid
(0.13 mmol) in dioxane (10 mL) and methanol (10 mL) ammonia (g)

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2777
Table 2
Hydrogen-bonding and CeH/p interactions geometry for 13.
DeH/A
DeH (Å) H/A (Å) D/A (Å) DeH/A (ꢁ) Symmetry
codes
C6eH6A/O3
C6eH6B/O1
C12eH12B/O6 0.99
0.99
0.99
2.50
2.37
2.50
2.912(6) 105
2.777(5) 104
2.989(6) 110
N3eH3/O1
C6eH6A/O3
C16eH16/O4 0.95
0.89(6) 1.97(6) 2.849(5) 168(4)
1 ꢀ x, 1 ꢀ y, ꢀz
0.99
2.60
2.49
3.422(5) 141
3.359(5) 152
2 ꢀ x, 1 ꢀ y, 1 ꢀ z
ꢀ1 þ x, 3/2 ꢀ y,
ꢀ1/2 þ z
C15eH15/Cg1a 0.95
C21eH21/Cg2a 0.95
2.60
2.78
3.489(5) 157
3.708(5) 168
ꢀ1 þ x, y, z
1 þ x, 3/2 ꢀ y, 1/2 þ z
a
Cg1 and Cg2 are the centroids of C20eC25 and C13eC18 rings, respectively.
3.2.3. Method C. General procedure for the preparation of
pyrimidine derivatives of 2,3-di-O-benzyl-6-deoxy-L-ascorbic acid
(7, 8, 18 and 22)
The suspension of anhydrous pyrimidine base (1.5 g) and
(NH4)2SO4 (100 mg) in HMDS (30 mL) was refluxed for 3 h in the
inert atmosphere of argon. Excess HMDS was evaporated under the
reduced pressure. To the oily product thus obtained, 4-(5,6-epoxy)-
2,3-di-O-benzyl-L-ascorbic acid (0.30 mmol) in CH3CN (5 mL) was
added. Then, TMSOTf was added inportions (0.5 mL; 2.76 mmol) and
the reaction mixture heated for 12 h at 55e70 ꢁC. The solvent was
evaporated and the residue portioned between CH2Cl2 and satu-
rated aq. NaHCO3. Organic layer was dried over MgSO4, evaporated
and the residue submitted to silica gel column chromatography.
Fig. 3. A view of 13, with the atom-numbering scheme. Displacement ellipsoids for
non-hydrogen atoms are drawn at the 20% probability level. Intramolecular hydrogen
bonds are indicated by dashed lines.
3.2.4. Method D. General procedure for the preparation of
debenzylated pyrimidine derivatives of 4,5-didehydro-5,6-dideoxy-
was introduced at 0 ꢁC. The reaction mixture was stirred at room
temperature for 24 h, then concentrated under reduced
pressure and the crude product purified by silica gel column
chromatography.
L
-ascorbic acid (3, 4 and 20)
To a solution of 2,3-O-dibenzylated derivatives of L-ascorbic acid
(1, 2 and 19) (0.42 mmol) in anhydrous CH2Cl2 (7 mL) 1M solution of
BCl3 in CH2Cl2 (0.5 mL) was added under argon at ꢀ78 ꢁC. The
reaction mixture was stirred at ꢀ40 ꢁC for 4 h, and additional
amount of 1M BCl3 in CH2Cl2 (0.5 mL) was added. The reaction
mixture was then stirred at 0 ꢁC for 2 h. Thereafter, the temperature
was raised to room temperature and the reaction mixture stirred
Fig. 5. A crystal packing diagram of 13, viewed along the a axis, showing three-
dimensional framework formed by one NeH/O and two CeH/O hydrogen bonds,
Fig. 4. A part of the crystal structure of 13, showing NeH/O and CeH/O hydrogen
as well as two CeH/p interactions. Hydrogen bonds and CeH/p interactions are
bonds. Hydrogen bonds are indicated by dashed lines.
indicated by dashed lines.

2778
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
Table 3
Inhibitory effects of pyrimidine (1e9, 13e17) and cyanuric acid derivatives of
effects on the growth of normal diploid human fibroblasts (WI38).
L-ascorbic acid (19e22) on the growth of malignant tumor cell lines in comparison with their
a
Compd.
Tumor cell growth IC50 (mM)
HeLa
MCF-7
HepG2
1.98
SW620
MiaPaCa-2
24.24
WI38
8.49
NHBz
N
N
O
7.20
2.03
>100
O
O
1
BnO
OBn
NH
N
N
O
N
5.91
3.49
2.72
5.62
0.92
4.67
O
O
BnO
OBn
2
NH
N
N
O
>100
>100
>100
>100
35.88
8.92
23.50
>100
>100
>100
62.19
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.27
>100
88.89
>100
8.82
O
O
HO
OH
3
NH2
N
N
O
N
>100
31.99
>100
4.79
O
O
BnO
OH
4
H N
N
N
O
O
O
H N
BnO
OBn
5
H N
N
N
N
O
O
O
H N
BnO
OBn
6
HO
BzHN
O
O
N
N
7
O
BnO
OBn

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2779
Table 3 (continued )
a
Compd.
Tumor cell growth IC50
(
mM)
HeLa
MCF-7
HepG2
SW620
MiaPaCa-2
71.68
WI38
N
HO
H N
O
O
N
>100
73.76
>100
>100
>100
N
O
BnO
OBn
8
HO
H N
O
O
N
>100
7.34
3.77
44.31
41.28
>100
>100
53.94
N
O
BnO
OBn
9
O
HN
N
O
N
44.96
36.51
20.37
24.62
>100
36.24
>100
>100
O
O
BnO
N
OBn
13
H2N
O
O
>100
>100
>100
>100
>100
20.87
80.35
>100
>100
42.80
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
BnO
O
OBn
N
H
14
O
H2N
O
O
N
F
BnO
O
OBn
N
H
15
O
H2N
O
O
N
H3C
BnO
O
OBn
N
H
O
16
O
HN
N
N
O
O
O
H2N
BnO
OBn
17
O
HN
NH
O
N
O
>100
17.84
44.75
>100
37.94
0.01
O
(continued on next page)
O
OBn
BnO
19

2780
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
Table 3 (continued )
a
Compd.
Tumor cell growth IC50
(
mM)
HeLa
MCF-7
HepG2
SW620
MiaPaCa-2
WI38
35.26
O
HN
NH
O
N
O
>100
>100
>100
>100
>100
O
O
20
21
HO
OH
O
H
N
O
HN
N
77.12
84.91
>100
>100
>100
>100
>100
>100
>100
>100
O
O
O
H2N
BnO
OBn
H
N
O
HO
O
O
>100
>100
O
N
HN
O
BnO
OBn
22
a
IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%.
overnight. A solvent mixture of CH2Cl2/CH3OH (1:1) was added to
deactivate the unreacted BCl3 and the solvent was subsequently
removed under reduced pressure. The oily residue was purified by
silica gel column chromatography.
3.2.8. 4-Amino-1-[2-(3-benzyloxy-4-hydroxy-5-oxo-5H-furan-2-
ylidene)-ethyl]-1H-[1,3,5]triazin-2-one (4)
To a solution of 2 (197 mg; 0.46 mmol) in dry dichloromethane
according to method D the crude product was obtained and sepa-
rated by silica gel column chromatography (CH2Cl2/CH3OH ¼ 10:1)
to afford 4 as colorless oil (25 mg; 15.88%).
3.2.5. N-{1-[2-(3,4-bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-benzamide (1)
13C NMR (DMSO-d6):
d 41.74 (C-6), 72.18 (OCH2-3), 101.18 (C-5),
Using a procedure described in method A a mixture of N4-ben-
zoylcytosine (1.5 g; 6.97 mmol) and dAdBA (2.01 g; 4.57 mmol) gave
the crude product that was purified by silica gel column cromatog-
raphy (CH2Cl2/CH3OH ¼ 90:1). Recrystallization from ethanol affor-
dedZ-isomerof 1aswhitecrystals(349mg;13.89%;m.p.175e177 ꢁC).
123.24 (C-2), 127.90e128.54 (C6H5), 136.14 (C-3a), 141.22 (C-3),
143.45 (C-4), 153.61 (C-40), 158.99 (C-60), 164.61 (C-1), 166.40 (C-20).
MS m/z 343.2 [M þ 1].
3.2.9. 4-Amino-1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-
dihydro-furan-2-yl)-ethyl]-1H-pyrimidin-2-one (5)
13CNMR(DMSO-d6):
d45.37(C-6), 73.53(OCH2-2), 74.57(OCH2-3),
96.68 (C-50), 104.02 (C-5), 123.43 (C-2), 128.43e133.33 (C6H5), 135.87
(C-2a), 135.90 (C-3a), 143.10 (C-3), 148.58 (C-4), 150.71 (C-60), 163.76
(C-1), 164.23 (C-40), 165.45 (C-20), 166.12 (C-40). MS m/z 536.5 [M þ 1].
Compound 1 was treated with saturated methanolic ammonia
in accordance with method B. The reaction mixture was then
concentrated under reduced pressure and the residue was purified
by silica gel column chromatography (CH2Cl2/CH3OH ¼ 5:1) to give
5 as white crystals (110 mg; 43.79%; m.p. 168e170 ꢁC).
3.2.6. 4-Amino-1-[2-(3,4-bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-[1,3,5]triazin-2-one (2)
13C NMR (DMSO-d6):
d 36.47 (C-5), 45.08 (C-6), 72.52 (OCH2-2),
Reaction of silylated 5-azacytosine (1.5 g; 13.4 mmol) and
dAdbA (2 g; 4.54 mmol) according to method A gave a crude
product which was submitted to silica gel column chromatography
(CH2Cl2/CH3OH ¼ 60:1) and subsequent recrystallization from
ethanol to afford compound 2 (Z-isomer) as white crystals (60 mg;
3.06%; m.p. 177e178 ꢁC).
73.53 (OCH2-3), 82.33 (C-4), 93.75 (C-50), 123.53 (C-2), 128.19e129.23
(C6H5),136.90 (C-2a),137.29 (C-3a),146.38 (C-60), 154.02 (C-3), 156.18
(C-20), 166.38 (C-40), 168.40 (C-1). MS m/z 449.3 [M þ 1].
3.2.10. 4-Amino-1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-
dihydro-furan-2-yl)-ethyl]-1H-[1,3,5]triazin-2-one (6)
13C NMR (DMSO-d6):
d
42.40 (C-6), 73.44 (OCH2-2), 74.49 (OCH2-
Solution of 3 (149 mg; 0.28 mmol) was treated as described in
method B. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/CH3OH ¼ 5:1). The pale-yellow crystals obtained were
additionally recrystallized from ethanol and washed with distilled
water to yield 6 as white crystals (52 mg; 18.71%, m.p. 194e196 ꢁC).
3), 104.52 (C-5), 123.35 (C-2), 128.38e135.89 (C6H5), 135.89 (C-2a),
136.24 (C-3a), 142.61 (C-3), 148.61 (C-4), 154.10 (C-40), 159.54 (C-60),
164.20 (C-1), 166.92 (C-20). MS m/z 433.1; 434.1 [M þ 1; M þ 2].
3.2.7. 4-Amino-1-[2-(3,4-dihydroxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-pyrimidin-2-one (3)
13C NMR (DMSO-d6):
d 35.39 (C-5), 42.59 (C-6), 72.08 (OCH2-2),
73.27 (OCH2-3), 81.97 (C-4), 123.14 (C-2), 127.66e128.49 (C6H5),
136.34 (C-2a), 136.78 (C-3a), 153.46 (C-3), 153.82 (C-20), 159.11 (C-
60), 166.39 (C-40), 167.86 (C-1). MS m/z 450.1; 451.1 [M þ 1; M þ 2].
To a solution of 1 (250 mg; 0.47 mmol) in dry CH2Cl2 in accor-
dance with method D the crude product obtained was purified by
column chromatography (CH2Cl2/CH3OH ¼ 5:1) yielded 3 as white
crystals (20 mg; 17.05%; m.p. > 300 ꢁC).
3.2.11. N-{1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-
2-hydroxy-ethyl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-benzamide (7)
A mixture of N4-benzoylcytosine (220 mg; 1.01 mmol) and 4-(5,6-
13C NMR (DMSO-d6):
d
44.39 (C-6), 94.01 (C-50), 99.50 (C-5),
122.03 (C-2), 143.09 (C-3), 145.36 (C-4), 147.97 (C-20), 150.02 (C-60),
160.12 (C-40), 164.94 (C-1). MS m/z 252.2 [M þ 1].
epoxy)-2,3-O,O-dibenzyl-L-ascorbic acid (100 mg; 0.30 mmol)

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2781
Fig. 6. Concentration-response curves for compounds 1, 2, 3, 7 and 9 on tested tumour cell lines (HeLa, HepG2, MCF-7, MiaPaCa-2 and SW620) and normal human fibroblasts
(WI38). The percentages of growth (PG) were calculated.
according to method C gave yellow oil. The crude product was sepa-
rated by silica gel column chromatography (CH2Cl2/CH3OH ¼ 50:1) to
afford 7 as white crystals (53 mg; 32.40%; m.p. 89e91 ꢁC).
3.2.12. 4-Amino-1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-2-hydroxy-ethyl]-1H-pyrimidin-2-one (8)
The removal of the benzoyl protecting group of the compound 7
(220 mg; 0.4 mmol) was achieved using methanolic ammonia
(method C). The residue was submitted to silica gel column chro-
matography (CH2Cl2/CH3OH ¼ 10:1) to afford 8 as white crystals
(130 mg; 72.30%; m.p. 118e120 ꢁC).
13C NMR (DMSO-d6):
d 53.29 (C-6), 64.82 (C-5), 72.79 (OCH2-2),
73.62 (OCH2-3), 75.88 (C-4), 95.43 (C-50),120.79 (C-2),127.83e133.13
(C6H5),135.56 (C-2a),136.12 (C-3a),151.55 (C-60),155.36 (C-3),157.41
(C-20),163.19 (C-40),167.16 (C-1),169.08 (C-400). MS m/z 554.5 [M þ 1].
Table 4
Effect of compound 1 on the cell cycle progression. The results are presented as percentage of particular cell population (%) ꢃ standard deviation. Significant results (p > 0.05)
are denoted in bold and marked with *.
Cell line
HeLa
Treatment
Duration (h)
Treatment regimen
no treatment
Cell cycle phase (%)
G1
G2/M
S
SubG1
24
48
24
48
44.8 ꢃ 11.2
47.1 ꢃ 0.1
37.9 ꢃ 1.8
56.1 ꢃ 0.1
60.8 ꢃ 0.1
52.3 ꢃ 0.1
50.7 ꢃ 3.1
57.9 ꢃ 0.1
54.3 ꢃ 5.9
52.3 ꢃ 6.1
50. 6 ꢃ 7
10.9 ꢃ 2.6
13.1 ꢃ 0.1
8.45 ꢃ 6.3
3.25 ꢃ 0.1
2.8 ꢃ 0.1
44.4 ꢃ 8.7
39.9 ꢃ 0.1
53.7 ꢃ 4.5*
40.6 ꢃ 0.1
36.5 ꢃ 0.1
39.5 ꢃ 0.7
33.4 ꢃ 0.4
28.3 ꢃ 0.1
39.2 ꢃ 1
0.49 ꢃ 0.2
0.04 ꢃ 0.01
0.20 ꢃ 0.01
0.16 ꢃ 0.01
0.27 ꢃ 0.01
0.56 ꢃ 0.01
0.68 ꢃ 0.4
0.42 ꢃ 0.01
0.20 ꢃ 0.1
0.51 ꢃ 0.01
0.37 ꢃ 0.2
0.32 ꢃ 0.3
5 mM
10
mM
No treatment
5 mM
10
m
M
8.3 ꢃ 0.1
MCF-7
No treatment
5 mM
15.9 ꢃ 2.7
13.8 ꢃ 0.1
6.54 ꢃ 5.1
6.82 ꢃ 0.3
13.9 ꢃ 6.4
5.52 ꢃ 0.7
10
mM
No treatment
5 mM
40.9 ꢃ 5.7
35.6 ꢃ 0.6
38 ꢃ 1.5
10
m
M
56.6 ꢃ 0.9

2782
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
Table 5
Effect of compound 2 on the cell cycle progression. The results are presented as percentage of particular cell population (%) ꢃ standard deviation. Significant results (p > 0.05)
are denoted in bold and marked with *.
Cell line
Treatment Duration (h)
24
Treatment regimen
no treatment
Cell cycle phase (%)
G1
G2/M
S
SubG1
MiaPaCa-2
55.4 ꢃ 0.01
55.6 ꢃ 0.14
35.6 ꢃ 2.76*
44.7 ꢃ 7.21
22.9 ꢃ 0.07*
29 ꢃ 2.05*
10.3 ꢃ 0.07
14.9 ꢃ 0.07
7.05 ꢃ 1.34
9.35 ꢃ 0.92
12.9 ꢃ 0.01
10.2 ꢃ 0.85
10.7 ꢃ 0.07
8.15 ꢃ 2.9
34.3 ꢃ 0.07
29.5 ꢃ 0.07
57.4 ꢃ 1.41*
46 ꢃ 8.13
0.32 ꢃ 0.009
0.20 ꢃ 0.008
0.21 ꢃ 0.05
0.20 ꢃ 0.06
0.18 ꢃ 0.009
0.31 ꢃ 0.15
0.37 ꢃ 0.36
0.56 ꢃ 0.31
0.20 ꢃ 0.004
0.18 ꢃ 0.036
0.05 ꢃ 0.0024
0.21 ꢃ 0.05
0.02 ꢃ 0.008
0
1
5
m
m
M
M
48
24
48
24
48
no treatment
1
5
m
m
M
M
64.2 ꢃ 0.01*
60.9 ꢃ 1.2*
37.7 ꢃ 0.01
50.5 ꢃ 1.98*
58.3 ꢃ 0.07*
41.7 ꢃ 2.47
58.9 ꢃ 0.07*
51.8 ꢃ 4.88*
34.8 ꢃ 3.82
58.8 ꢃ 1.06*
72.1 ꢃ 1.41*
24.5 ꢃ 3.18
65.6 ꢃ 0.01*
62.3 ꢃ 0.01*
SW620
HeLa
no treatment
51.6 ꢃ 0.07
41.3 ꢃ 4.81*
24.1 ꢃ 0.07*
46.6 ꢃ 4.88
23.2 ꢃ 0.07*
39.1 ꢃ 1.06*
56 5 ꢃ 8.27
29.4 ꢃ 5.59*
16.5 ꢃ 0.21*
64.1 ꢃ 2.62
20.1 ꢃ 0.07*
23.9 ꢃ 0.01*
1
5
m
m
M
M
17.7 ꢃ 0.07*
11.9 ꢃ 2.33
17.9 ꢃ 0.07*
9.2 ꢃ 5.94
No treatment
1
5
m
m
M
M
no treatment
9.2 ꢃ 4.38
1
5
m
m
M
M
11.9 ꢃ 6.65
11.5 ꢃ 1.63
11.5 ꢃ 0.57
14.4 ꢃ 0.01
13.9 ꢃ 0.01
0
No treatment
0.02 ꢃ 0.016
0.02 ꢃ 0.005
0.23 ꢃ 0.005
1
5
m
m
M
M
13C NMR (DMSO-d6):
d
52.27 (C-6), 66.52 (C-5), 72.96 (OCH2-2),
(42.30%; m.p. 75e77 ꢁC) as white crystals, and 17 (4.2 mg; 4.05%) as
transparent yellow oil.
73.93 (OCH2-3), 75.24 (C-4), 94.63 (C-50), 120.65 (C-2), 129.47e133.64
(C6H5),135.29 (C-2a),136.31 (C-3a),152.83 (C-60),154.27 (C-3),157.41
(C-20), 162.59 (C-40), 166.92 (C-1). MS m/z 450.4 [M þ 1].
3.4.1. 1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-ethyl]-1H-pyrimidine-2,4-dione (14)
3.2.13. 4-Amino-1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-2-hydroxy-ethyl]-1H-[1,3,5]triazin-2-one (9)
Reaction of 5-azacytosine (678 mg; 6.05 mmol) and epoxy
derivative of L-ascorbic acid (600 mg; 1.77 mmol) as described in
1H NMR (DMSO-d6):
d 1.96 (H-5, 2H, m), 3.64 (H-6, 2H, dd,
J1 ¼ 6.20, J2 ¼ 13.71), 5.01 (OCH2-2, 2H, s), 5.14 (OCH2-3, 2H, dd,
J1 ¼11.91, J2 ¼ 22.41), 5.51 (H-60,1H, dd, J1 ¼1.68, J2 ¼ 7.77), 6.16/8.17
(NH2-4, 2 ꢂ 1H, s), 7.33e7.42 (Ph,10H, m), 7.45 (H-50,1H, d, J ¼ 7.86),
11.21 (NH, 1H, s).
method C afforded yellow oil. The crude product was separated by
silica gel column chromatography (CH2Cl2/CH3OH ¼ 20:1) to obtain
9 as white crystals (217 mg; 27.20%; m.p. 148e150 ꢁC).
13C NMR (DMSO-d6):
d 35.93 (C-5), 44.01 (C-6), 72.57 (OCH2-2),
73.75 (OCH2-3), 82.22(C-4),101.31(C-50),123.59(C-2),128.12e129.03
(C6H5),136.85 (C-2a),137.25 (C-3a),146.13 (C-60),151.28 (C-20),153.84
(C-3), 164.21 (C-40), 168.37 (C-1). MS m/z 432.2 [M e NH3]þ.
13C NMR (DMSO-d6):
d 50.47 (C-6), 65.33 (C-5), 73.29 (OCH2-2),
74.12 (OCH2-3), 76.29 (C-4), 121.29 (C-2), 128.25e129.17 (C6H5),
136.09 (C-2a), 136.62 (C-3a), 154.55 (C-3), 157.90 (C-20), 160.16
(C-60), 167.00 (C-1), 169.54 (C-40). MS m/z 451.1 [M þ 1].
3.4.2. 1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-ethyl]-5-fluoro-1H-pyrimidine-2,4-dione (15)
3.3. General procedure for the synthesis of pyrimidine derivatives of
1H NMR (DMSO-d6):
d 1.93e2.08 (H-5, 2H, m), 3.58e3.72 (H-6,
4,5-didehydro-5,6-dideoxy-
L
-ascorbic acid
2H, m), 5.01 (OCH2-2, 2H, s), 5.14 (OCH2-3, 2H, dd, J1 ¼ 11.87,
J2 ¼ 39.36), 6.17/8.16 (NH2-4, 2 ꢂ 1H, s), 7.31e7.41 (Ph, 10H, m), 7.91
(H-60, 1H, d, J ¼ 3.39), 11.73 (NH, 1H, s).
The synthesis of pyrimidine derivatives of
(10e12) with a double bond in the C4-C5 position was previously
L
-ascorbic acid
13C NMR (DMSO-d6):
d 35.17 (C-5), 43.72 (C-6), 72.08 (OCH2-2),
described [23].
73.29 (OCH2-3), 81.75 (C-4), 123.15 (C-2), 127.58e128.48 (C6H5),
130.21 (C-60, JCF ¼ 33.37), 136.34 (C-2a), 136.76 (C-3a), 139.39 (C-50,
JCF ¼ 228.94), 149.41 (C-3), 153.39 (C-20), 157.41 (C-40, JCF ¼ 25.43),
167.86 (C-1). MS m/z 466.4 [M ꢀ 1].
3.3.1. 2-[2-(3,4-bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-ethyl]-
2H-[1,2,4]triazine-3,5-dione (13)
Condensation of 6-azauracil (1.5 g; 13.27 mmol) and dAdBA in
accordance with method A gave yellow oil as a crude product which
was purified by silica gel column chromatography (petrolether/
EtOAc ¼ 3:1). Compound 13 was isolated as white crystals that
were recrystallized from ethanol (180 mg; 9.15%; m.p. 80e84 ꢁC).
3.4.3. 1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-ethyl]-5-methyl-1H-pyrimidine-2,4-dione (16)
1H NMR (DMSO-d6):
d 1.72 (CH3, 3H, s), 1.86e2.03 (H-5, 2H, m),
3.52e3.66 (H-6, 2H, m), 4.99 (OCH2-2, 2H, s), 5.11 (OCH2-3, 2H, dd,
J1 ¼ 11.89, J2 ¼ 24.01), 6.13/8.13 (NH2-4, 2 ꢂ 1H, 2xs), 7.27e7.40
(Ph þ H-60, 11H, m), 11.19 (NH, 1H, s).
13C NMR (DMSO-d6):
d 45.20 (C-6), 72.99 (OCH2-2), 73.97
(OCH2-3), 103.33 (C-5), 122.89 (C-2), 127.94e128.78 (C6H5), 135.38
(C-2a),135.74 (C-3a),135.74 (C-50), 142.29 (C-3),148.04 (C-4),148.06
(C-20),150.71 (C-60),157.08 (C-40),163.68 (C-1). MS m/z 434.1 [M þ 1].
13C NMR (DMSO-d6):
d 12.38 (CH3), 35.96 (C-5), 43.66 (C-6),
72.54 (OCH2-2), 73.77 (OCH2-3), 82.21 (C-4), 108.88 (C-50), 123.66
(C-2), 128.09e128.99 (C6H5), 136.85 (C-2a’), 137.28 (C-3a’), 141.87
(C-60), 151.21 (C-20), 153.79 (C-3), 164.75 (C-40), 168.37 (C-1). MS m/z
446.1 [M ꢀ NH3]þ.
3.4. General procedure for ammonolysis of pyrimidine derivatives
10e13
Pyrimidine derivatives (10e13) (0.13 mmol) were treated with
methanolic ammonia as described in method B. The residue was
subjected to column chromatography (CH2Cl2/CH3OH ¼ 10:1) to
give 14 (36.82%; m.p. 168e170 ꢁC), 15 (36.17%; m.p. 165e167 ꢁC), 16
3.4.4. 2-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-ethyl]-2H-[1,2,4]triazine-3,5-dione (17)
1H NMR (DMSO-d6):
d
1.94e2.08 (H-5, 2H, m), 3.82e3.92 (H-6,
2H, m), 5.03 (OCH2-2, 2H, s), 5.15 (OCH2-3, 2H, dd, J1 ¼ 11.94,

K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
2783
J2 ¼ 41.12), 6.14/8.12 (NH2-4, 2 ꢂ 1H, s), 7.20e7.46 (Ph, 10H, m), 7.46
temperature. Data were collected on a Bruker-Nonius Kappa Apex II
diffractometer using graphite-monochromated CuK radiation
¼ 1.54184 Å) at 123 K. COLLECT [36] software was used for the
(H-50, 1H, s), 12.08 (NH, 1H, s).
a
13C NMR (DMSO-d6):
d
34.32 (C-5), 45.40 (C-6), 71.54 (OCH2-2),
(l
72.75 (OCH2-3), 81.65 (C-4), 122.74 (C-2), 127.22e133.18 (C6H5),
134.56 (C-50), 134.96 (C-20), 135.84 (C-2a), 136.29 (C-3a), 153.08
(C-3), 169.12 (C-1), 169.37 (C-40). MS m/z 451.2 [M þ 1].
data collection and DENZO-SMN [37] for data processing. The
intensities were corrected for absorption using the SADABS2008
[38]. The crystal structure was solved by direct methods [39] and all
non-hydrogen atoms were refined anisotropically by full-matrix
least squares calculations based on F2 [40] using the programs
integrated in WinGX program package [41]. The hydrogen atom
attached to the N3 atom was found in a difference Fourier map and
was refined isotropically. All other hydrogen atoms were treated
using appropriate riding models, with SHELXL97 [40] defaults.
PLATON [42] program was used for structure analysis and drawings
preparation. Crystallographic data for the structure reported in this
paper have been deposited with the Cambridge Crystallographic
Data Centre. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
3.4.5. 2-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-
hydroxy-ethyl]-2H-[1,2,4]triazine-3,5-dione (18)
Reaction of 6-azauracil (230 mg; 2.02 mmol) and EdBA (200 mg;
0.59 mmol) as described in method C yielded yellow oil. Crude
product was purified by silica gel column chromatography (CH2Cl2/
CH3OH ¼ 20:1) to give 18 (12.6 mg; 4.73%) as yellow oil.
13C NMR (DMSO-d6):
d 52.48 (C-6), 64.98 (C-5), 72.70 (OCH2-2),
73.52 (OCH2-3), 75.16 (C-4), 107.57 (C-50), 120.57 (C-2),
127.74e128.75 (C6H5), 135.61 (C-2a), 136.10 (C-3a), 148.46 (C-3),
157.18 (C-20), 157.57 (C-40), 169.14 (C-1). MS m/z 452.1 [M þ 1].
3.4.6. 1-[2-(3,4-bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-ethyl]-
[1,3,5]triazinane-2,4,6-trione (19)
Crystal data for 13: crystal dimension 0.15 ꢂ 0.20 ꢂ 0.60 mm3
(colourless prism); C23H19N3O6, Mr ¼ 433.41, monoclinic space
group P 21/c (No. 14); a ¼ 9.8162(9), b ¼ 23.191(2), c ¼ 9.1177(7) Å,
Reaction of cyanuric acid (1.5 g; 11.57 mmol) and dAdBA as
described by method A afforded yellow oil as a crude product. Silica
gel column chromatography (petrol ether/EtOAc ¼ 2:1) yielded 19
as white crystals (172 mg; 96.26%; m.p. 128e130 ꢁC).
b
¼ 95.206(5)o; V ¼ 2067.1(3) Å3; Z ¼ 4; dx ¼ 1.393 g cmꢀ3
; m(Cu
K
a
) ¼ 0.856 mmꢀ1; 14087 reflections measured, R/wR ¼ 0.0740/
0.1788 for 294 parameters and 2146 reflections with I ꢄ 2
s(I),
13C NMR (DMSO-d6):
d
36.50 (C-6), 73.45 (OCH2-2), 74.48
R/wR ¼ 0.1147/0.2092 for all 3338 unique reflections measured in
(OCH2-3), 104.96 (C-5), 123.07 (C-2), 128.47e129.35 (C6H5), 135.89
(C-2a), 136.25 (C-3a), 144.67 (C-3), 148.72 (C-4), 149.07 (C-40),
150.10 (C-20), 150.10 (C-60), 164.27 (C-1). MS m/z 450.1 [M þ 1].
the range 7.62ꢁꢀ 2
q
ꢀ126.54ꢁ; S ¼ 0.961.
3.6. Cell culturing
3.4.7. 1-[2-(3,4-dihydroxy-5-oxo-5H-furan-2-ylidene)-ethyl]-
[1,3,5]triazinane-2,4,6-trione (20)
The cell lines HeLa (cervical carcinoma), SW620 (colorectal
adenocarcinoma, metastatic), MiaPaCa-2 (pancreatic carcinoma),
MCF-7 (breast epithelial adenocarcinoma, metastatic), HepG2
(hepatocellular carcinoma) and WI 38 (normal diploid human fibro-
blasts), were cultured as monolayers and maintained in Dulbecco’s
modifiedEaglemedium(DMEM)supplementedwith10%fetalbovine
Compound 19 (170 mg; 0.38 mmol) was dissolved in dry CH2Cl2
as per described in method D the crude product was submitted to
silica gel column chromatography (CH2Cl2/CH3OH ¼ 10:1) to afford
20 as white crystals (11.4 mg; 11.15%; m.p. 184e187 ꢁC).
13C NMR (DMSO-d6):
d
35.94 (C-6), 100.79 (C-5), 121.13 (C-2),
serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/mL
143.27 (C-3), 143.45 (C-40), 148.55 (C-4), 149.58 (C-20), 149.58 (C-60),
streptomycin in a humidified atmosphere with 5% CO2 at 37 ꢁC.
164.80 (C-1). MS m/z 270.1 [M þ 1].
3.7. Proliferation assays
3.4.8. 1-[2-(2-amino-3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-
2-yl)-ethyl]-[1,3,5]triazinane-2,4,6-trione (21)
The panel cell lines were inoculated onto a series of standard 96-
well microtiter plates on day 0, at 3000 cells to 6000 cells per well
according to the doubling times of specific cell line. Test agents were
To a solution of 19 (150 mg; 0.32 mmol) in methanol (15 mL) and
dioxane (10 mL) ammoniawas introduced as described in method B.
The crude product was purified bysilica gelcolumn chromatography
(CH2Cl2/CH3OH ¼ 10:1) to give 21 as colorless oil (21.3 mg; 13.84%).
then added in five, 10-fold dilutions (0.01e100 mM) and incubated
for further 72 h. Working dilutions were freshly prepared on the day
of testing in the growth medium. The solvent (DMSO) was also
tested for eventual inhibitory activity by adjusting its concentration
to be the same as in the working concentrations (DMSO concen-
tration never exceeded 0.1%). After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT assay: experi-
mentally determined absorbance values were transformed into
a cell percentage growth (PG) using the formulas proposed by NIH
and described previously [43]. This method directly relies on control
cells behaving normally 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 e the results are therefore a percentile
difference from the calculated expected value.
13C NMR (DMSO-d6):
d 36.54 (C-6), 72.55 (OCH2-2), 73.78
(OCH2-3), 82.29 (C-5), 123.63 (C-2), 128.12e129.13 (C6H5), 136.89
(C-2a),137.25 (C-3a),149.14 (C-3),150.19 (C-20),150.19 (C-60),150.39
(C-40), 153.77 (C-4), 168.26 (C-1). MS m/z 467.2 [M þ 1].
3.4.9. 1-[2-(3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-
hydroxy-ethyl]-[1,3,5]triazinane-2,4,6-trione (22)
Reactionofcyanuricacid (260mg;2.01mmol)andEdBA(200mg;
0.59 mmol) as described in method C gave crude product in the form
of yellow oil that was submitted to silica gel column chromatography
(CH2Cl2/CH3OH ¼ 50:1) to afford 22 as yellow oil (36.7 mg; 13.31%).
13C NMR (DMSO-d6):
d 44.60 (C-6), 66.27 (C-5), 72.71 (OCH2-2),
73.53 (OCH2-3), 120.43 (C-2), 127.87e129.15 (C6H5), 135.58 (C-2a),
136.47 (C-3a), 142.06 (C-20), 146.14 (C-40), 150.94 (C-60), 156.93
(C-3), 167.67 (C-1). MS m/z 468.1 [M þ 1].
The IC50 values for each compound were calculated from dose-
response curves using linear regression analysis by fitting the mean
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 assigned as the default value (in the screening data report that
default value is preceded by a “>” sign). Each test point was
3.5. X-ray crystal structure determination of 13
The crystal suitable for X-ray single crystal structure study was
grown by slow evaporation from ethanol solution at room

2784
K. Wittine et al. / European Journal of Medicinal Chemistry 46 (2011) 2770e2785
performed in quadruplicate in three individual experiments. The
results were statistically analyzed (ANOVA, Tukey post-hoc test at
p < 0.05). Finally, the effects of the tested substances were evalu-
ated by plotting the mean percentage growth for each cell type in
comparison to control on dose-response graphs.
Comparison of the CD spectra of 7 and 8 with their synthetic
precursor 4-(5,6)-epoxy derivative of -ascorbic acid (EdBA,
L
Scheme 1) permitted to deduce their absolute configuration. Thus,
the derivatives with the hydroxy group at the C-5 of ethylenic
spacer (7, Fig. 2B and 8, Fig. 2A) exist in 4R, 5S enantiomeric form.
This means that the ring-opening reaction step (Schemes 2 and 3)
proceeds in enantiospecific manner with the retention of
configuration.
3.8. DNA cell cycle analysis
Flow cytometric analysis was used to measure the DNA content
On the other hand, derivatives of L-ascorbic acid with the free
of the cells. Briefly, 3 ꢂ 105 cells were seeded per well in 6-well
amino group at the C-4 of the lactone moiety (5, 6,14e17 and 21), in
general, do not show Cotton effect supporting the assumption that
the attack goes equally from both sides of the C4eC5 double bond
resulting in racemic mixtures of enantiomers.
plates. After an overnight incubation, compounds 1 (5
mM and
10 mM) and 2 (1 mM and 5 mM) were added, and the cells were
cultured for 24 and 48 h. The cells were then harvested by trypsi-
nization, washed three times with phosphate buffered saline (PBS),
fixed with cold ethanol and stored at ꢀ20 ꢁC. Immediately before
analysis, cell suspension was centrifuged, the pellet was washed
X-ray crystal structure study of 6-azauracil derivative of L-
ascorbic acid 13 confirms the Z-configuration of the double bond.
Both benzyloxy groups adopt the same, antiperiplanar conforma-
tion. One NeH/O hydrogen bond, two CeH/O hydrogen bonds
twice with PBS and incubated with 0.2
15 min, followed by staining with propidium iodide (PI) at 10
m
g/
m
L RNase A at 37 ꢁC for
g/mL
m
and two CeH/p interactions self-assemble the molecules into
final concentration for 30 min in the dark. Analysis was carried out
using FACScalibur instrument (BD Biosciences, San Jose, CA),
whereby approximately 20,000 events were recorded for each
sample. WinMDI 2.8 (The Scripps Institute, La Jolla, CA) and
Cylchred (Cardiff University, Cardiff, UK) software were used to
determine the percentage of the cells in different phases of the
cell cycle.
three-dimensional framework.
Acknowledgments
Support for this study was provided by the Ministry of Science of
the Republic of Croatia (Projects #125-0982464-2922, # 335-
0982464-2393 and 335-0000000-3532).
3.9. Annexin V test
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