Syntheses and antitumor evaluation of C(6)-isobutyl- and C(6)-isobutenyl-substituted pyrimidines, and dihydropyrrolo[1,2-c]pyrimidine-1, 3-diones

Article abstract of DOI:10.1002/cbdv.201000202

A growing body of evidence supports that pyrimidine derivatives, in which the sugar residues have been replaced by acyclic side chains, might be developed as promising anticancer agents that interfere with tumor cell proliferation, survival, and metastati

Full text of DOI:10.1002/cbdv.201000202

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Syntheses and Antitumor Evaluation of C(6)-Isobutyl- and C(6)-Isobutenyl-
Substituted Pyrimidines, and Dihydropyrrolo[1,2-c]pyrimidine-1,3-diones
by Svjetlana Krisˇtafora), Tatjana Gazivoda Kraljevic´ a), Simon M. Ametameyb), Mario Cetinac),
Ivana Ratkajd), Romana Tandara Hacˇeke), Sandra Kraljevic´ Pavelic´ d), and Silvana Raic´-Malic´*a)
a) Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
´
University of Zagreb, Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb
(phone: þ385-1-4597213; fax: þ385-1-4597224; e-mail: silvana.raic@fkit.hr)
b) Center for Radiopharmaceutical Sciences, ETH Zurich (Swiss Federal Institute of Technology),
Wolfgang-Pauli-Strasse 10, CH-8093 Zurich
c) Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb,
´
Prilaz baruna Filipovica 28a, HR-10000 Zagreb
d
´
ˇ
´
) Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, HR-51000 Rijeka
e) Institute of Immunology, Rockefellerova 2, HR-10000 Zagreb
A growing body of evidence supports that pyrimidine derivatives, in which the sugar residues have
been replaced by acyclic side chains, might be developed as promising anticancer agents that interfere
with tumor cell proliferation, survival, and metastatic formation. In this work, we prepared novel
pyrimidines bearing i-Bu (i.e., 3, 4, and 7–9) and isobutenyl (i.e., 5 and 10) side chains at C(6) and
examined their in vitro effects on tumor cell lines. The dihydropyrrolo[1,2-c]pyrimidine-1,3-diones 6 and
11 were obtained as products of intramolecular cyclization, which occurred during the removal of Bn in 5
or MeO protecting groups in 10. Fluorination of 3 with diethylaminosulfur trifluoride (DAST) and then
dehydrohalogenation of the resulting fluorinated derivative 4 afforded 6-isobut-2’-enyl pyrimidine
derivative 5 with a C(2’)¼C(3’) bond. For the preparation of 6-isobut-1’-en-1-yl pyrimidine 10, a synthetic
strategy involving acetylation of the 1,3-diols was applied. Antitumor evaluation of compounds 3–11
showed that 2,4-dimethoxypyrimidine containing 6-[(1,3-dibenzyloxy)-2-hydroxy]methyl side chain, 3,
exerted a strong antiproliferative effect on the studied tumor cell lines. Additionally, it was shown that
the mechanism of antiproliferative effect of 3 in HeLa cells include early G2/M arrest and apoptosis, as
well as a p53-independent S-phase arrest upon prolonged treatment.
Introduction. – Pyrimidines are biologically important molecules and valuable
heterocyclic nuclei for the design of pharmaceutical agents [1][2]. Pyrimidine
nucleoside analogs are widely used as chemotherapeutic agents in the treatment of
cancer [3–6]. In particular, the pyrimidine analog 5-fluorouracil (5-FU) is one of the
most widely used drugs for the treatment of breast, gastrointestinal, and liver
carcinoma [7]. Furthermore, carmofur is a 5-FU derivative that bears a potentiated
antitumor activity, while at the same time being clinically less toxic [8][9]. In anticancer
treatment regimens, pyrimidine nucleoside analogs are widely used in combinational
chemotherapy. These compounds, mainly due to their DNA-damaging properties that
cause arrest of cells in the S phase, are suitable for combinational treatments with other
cell-cycle inhibitors targeting different signalling pathways. Such approach might
enhance cytotoxicity of drugs and improve clinical response of patients [10]. Moreover,
pyrrolo-pyrimidines have been attracting considerable attention in a view of the high
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
biological activity of these bicyclic compounds [11]. The excellent biological activities
exhibited by C(5)- and/or C(6)-substituted uracil derivatives provide a good rationale
for further exploration of chemistry and antitumoral activities of these pyrimidines
[12–19].
In addition, we have reported that fluorinated pyrimidine derivatives containing the
3,3,3-trifluoro-2-hydroxyprop-1-enyl side chain at C(6) exhibited a pronounced effect
against breast carcinoma (MCF-7), while the compound with the 2-(fluoromethyl)-2-
acetoxypropyl chain exhibited moderate effect against cervical carcinoma (HeLa) [20].
C(6)-Substituted pyrimidines containing the 4-fluorophenyl moiety in the side chain
showed pronounced antiproliferative effect on colon (SW620 and HCT116) and lung
carcinoma (H460) [21]. The 6-(bromomethyl)-substituted pyrimidine derivatives
showed marked cytotoxic activity against all evaluated tumor cell lines, particularly
against colon carcinoma (SW620) [22].
Besides, findings from the molecular docking of some C(6)-substituted pyrimidines
into the active site of Herpes simplex virus type 1 thymidine kinase (HSV-1 TK)
indicated that these compounds showed the same interactions known for natural
substrate thymidine [23][24]. Biological phosphorylation-pattern assays revealed that
6-(2,3-dihydroxypropyl)pyrimidine, 6-[3-hydroxy-2-(hydroxymethyl)propyl]pyrimi-
dine, and its N-Me analog represent promising precursors of nontoxic reporter probes
for the monitoring of HSV-1 TK gene expression by means of positron-emission
tomography (PET) [23][25].
Taking into account the pharmacological potential of the cited class of compounds,
we performed the presented study with the primary aim to evaluate the cytostatic
potencies of pyrimidine derivatives bearing 6-isobutyl (in 3, 4, and 7–9), and
isobutenyl (in 5 and 10) side chain, in addition to structurally related bicyclic
compounds 6 and 11. Moreover, among the tested compounds, 3 exerted a strong
antiproliferative effect, and the mechanisms underlying this property were studied in
further details on HeLa cells.
Results and Discussion. – 1. Chemistry. The syntheses of the C(6)-substituted
pyrimidine derivatives were performed as indicated in Schemes 1 and 2. Pyrimidine
derivative 1 was synthesized in analogy to [23], and ketone 2 was prepared using a
modified CoreyꢀKim oxidation [26] of 1,3-bis(benzyloxy)propan-2-ol [27] with N-
chlorosuccinimide (NCS) and Me2S. The treatment of 2,4-dimethoxy-5,6-dimethylpyr-
imidine (1) with lithium diisopropylamide (LDA) in THF at ꢀ558 afforded the
lithiated pyrimidine precursor of 1, which reacted in situ with ketone 2 to give
pyrimidine derivative 3 [28] (Scheme 1).
To obtain the unsaturated side chain at C(6) of the pyrimidine derivative I,
dehydration of tertiary alcohol 3 was attempted. The reaction of 3 with H2SO4 (20%
(v/v)) did not yield the expected product with the isobutenyl side chain at C(6).
Therefore, compound 3 was converted into its fluorinated analog 4 using diethylami-
nosulfur trifluoride (DAST) albeit in 20% yield (Scheme 1). In addition, compound 5,
derived from dehydrohalogenation of 4, was obtained in 10% yield. Subsequently, 4
was treated with an excess of MeONa to afford the unsaturated pyrimidine derivative 5
in 75% yield. The presence of the C(2’)¼C(3’) bond in 5 was deduced from the pattern

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1457
Scheme 1
i) 1. POCl3, reflux; 2. MeONa/MeOH, reflux. ii) N-Chlorosuccinimide (NCS), CH2Cl2, Me2S, r.t. iii)
Lithium diisopropylamide (LDA), THF, reagent 2, ꢀ558. iv) Diethylaminosulfur trifluoride (DAST),
CH2Cl2, ꢀ408. v) MeONa/MeOH. vi) BCl3, CH2Cl2, ꢀ408.
of chemical shifts, and on the basis of the magnitude and multiplicity of H,H spin-spin
coupling constants (cf. Exper. Part).
1
The methylene groups CH2(1’) and CH2(3’’) in 5 display singlets in the H-NMR
spectrum, while the corresponding CH2 groups in 4 show doublets caused by H,F
coupling. Besides, the vinyl H-atom (HꢀC(3’)) in 5 is deshielded and appears as a
singlet. Moreover, the CH2 H-atoms (PhCH2O) of the two Bn moieties in 5 display two
signals due to chemical non-equivalency. On the contrary, corresponding H-atoms
(PhCH2O) in benzylated pyrimidine derivatives 3 and 4 are equivalent, and each
exhibits one singlet. The removal of the Bn protecting groups in 5 was accomplished
using BCl3 at ꢀ408 but was accompanied by an intramolecular cyclization and allylic
rearrangement, which resulted in the formation of the bicyclic chloromethyl compound
6 (Scheme 1). The formation of 6 may be explained by the nucleophilic attack of N(1)
at C(3’’) of the chlorinated side chain at C(6). The structure of 6 was confirmed by X-
ray crystallographic analysis.
The strategy for the preparation of 6-isobut-1’-enyl, instead of 6-isobut-2’-enyl
pyrimidine derivatives, involved isopropylidene ketal formation as the most commonly
used protection for 1,3-diols. However, in the attempted synthesis of the acetonide of 7
using acetone and AcCl, a mixture of 1,2- and 1,3-acetonides was obtained, in which the
1,2-acetonide was favored over the 1,3-derivative. An alternative route to obtain 6-
isobut-1’-enyl pyrimidine derivative with 1,3-diols protected as acetates was then used
as depicted in Scheme 2.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 2
i) BCl3, CH2Cl2, ꢀ408. ii) Ac2O; 4-(Dimethylamino)pyridine (DMAP), pyridine, MeCN, r.t. iii)
Diethylaminosulfur trifluoride (DAST), CH2Cl2, ꢀ408. iv) MeONa/MeOH. v) Me3SiCl (TMSCl), NaI,
MeCN, r.t.
Removal of the Bn groups was accomplished with BCl3 to afford triol 7 in 70%
yield. Conversion of the primary OH groups in 7 to ester functionalities was
accomplished using Ac2O and 4-(dimethylamino)pyridine (DMAP) to yield compound
8 with a free tertiary OH group. Treatment of 8 with DAST gave the fluorinated
derivative 9, which was then submitted to dehydrohalogenation with MeONa. The
desired unsaturated pyrimidine derivative 10 with a C(1’)¼C(2’) bond was obtained in
1
42% yield. The C(6)-side-chain H,H coupling patterns in H-NMR spectra for 10 are
doublets corresponding to CH2(3’) and CH2(3’’), triplets for OH groups, and a singlet
for HꢀC(1’).
Demethoxylation [29] of 10 using trimethylsilyl iodide (TMSI) provided bicyclic
compound 11 in 67% yield. Intramolecular cyclization may be explained by
nucleophilic attack of N(1) at C(3’) of the iodinated isobutenyl side chain.
2. X-Ray Crystal Structure Study. The structures of C(6)-substituted pyrimidine 3
and bicyclic compound 6 were unequivocally confirmed by single-crystal X-ray
diffraction method. Compound 3 (Fig. 1) crystallizes with two independent molecules
in the asymmetric unit in the orthorhombic space group Pca21. These two independent
molecules, denoted as A and B, differ in the conformation of one of two BnOCH2
groups, and thus, C(20) in molecule A is synclinal, whereas in molecule B is
antiperiplanar with respect to C(22) of the Ph ring (C(20A)ꢀO(5A)ꢀC(21A)ꢀC(22A),
62.7(5)8; C(20B)ꢀO(5B)ꢀC(21B)ꢀC(22B), 172.1(4)8). Furthermore, C(10) is anti-
periplanar in A and synclinal in B with respect to O(5) (C(10A)ꢀC(11A)ꢀ
C(20A)ꢀO(5A), 179.2(4)8; C(10B)ꢀC(11B)ꢀC(20B)ꢀO(5B), ꢀ54.7(5)8). The ori-
entation of two Ph rings towards the pyrimidine ring is almost equal in molecule A. The

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1459
Fig. 1. Molecular structure of 3, with the atom-numbering scheme. For clarity, only one independent
molecule is shown. Displacement ellipsoids for non-H-atoms are drawn at the 30% probability level.
dihedral angles between mean planes of the pyrimidine ring and Ph rings, C(14)ꢀC(19)
and C(22)ꢀC(27), are 79.5(2) and 73.7(2)8, respectively. On the other hand, the
orientation of the Ph rings in molecule B is slightly different, with the corresponding
dihedral angles being 82.7(2) and 65.0(2)8, respectively. The bond lengths in 2,4-
dimethoxypyrimidine are within the values of the equivalent ones in similar structures
we published recently [30][31]. It should be pointed out that the C(4)ꢀC(5) bond in
2,4-dimethoxy-6-(3,3,4,4,4-pentafluoro-2-hydroxybut-1-en-1-yl)pyrimidine [22] is ca.
0.04 ꢂ shorter compared to all other structures.
Two intermolecular H-bonds, C(16B)· · ·O(2A) and C(7B)· · ·O(5A) (Table 1),
join two independent molecules of 3, so forming finite D(2) motifs [32]. The H-donor
atoms are exclusively atoms of molecule B, whereas the O-atoms of molecule A are
exclusively H-acceptors. The combination of these two CꢀH· · ·O H-bonds generates
infinite chains (Fig. 2).
The supramolecular aggregation is augmented by aromatic p· · ·p stacking
interactions. The pyrimidine rings of the two independent molecules are mutually
parallel (Fig. 3), with an interplanar angle of 0.758, an interplanar spacing of 3.35 ꢂ and
3.36 ꢂ, and a corresponding centroidꢀcentroid offset of 1.18 ꢂ and 1.20 ꢂ. Five

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 1. Intermolecular H-Bonding Geometries for 3 and 6
DꢀH· · ·A
DꢀH [ꢂ] H· · ·A [ꢂ] D· · ·A [ꢂ] DꢀH· · ·A [8] Symmetry codes
3
6
C(16B)ꢀH(16B)· · ·O(2A) 0.95
2.58
2.47
2.01(2)
2.54
2.54
3.481(7)
3.384(6)
2.807(3)
3.332(3)
3.356(3)
158
155
171(2)
139
141
x, y, 1þz
C(7B)ꢀH(72B)· · ·O(5A) 0.98
x, ꢀ1þy, z
3ꢀx, 1ꢀy,1ꢀz
N(3)ꢀH(3)· · ·O(2)
0.80(2)
0.97
0.97
1
C(10)ꢀH(10B)· · ·O(1)
C(11)ꢀH(11B)· · ·O(1)
2ꢀx, ꢃ2 þy,1/2ꢀz
1
2ꢀx, ꢃ2 þy,1/2ꢀz
Fig. 2. Crystal packing diagram of 3, viewed along the a axis, showing an infinite chain formed by two
CꢀH· · ·O H-bonds. H-Bonds are indicated with dashed lines.
CꢀH· · ·p interactions participate also in supramolecular aggregation of 3. The
H· · ·Centroid (Cg) distances range from 2.63 to 3.00 ꢂ, and CꢀH· · ·Cg angles from
1538 to 1758. The p· · ·p and CꢀH· · ·p interactions complete the three-dimensional
framework.
In bicyclic compound 6 (Fig. 4), in which the six-membered and five-membered
rings are linked via C(6) and N(1), crystallizes in the monoclinic space group P21/c.
These two rings are nearly parallel, as the dihedral angle between their mean planes is
0.30(10)8. The ClCH2 group is in the plane of the five-membered ring, defined by the
C(8)ꢀC(9)ꢀC(11)ꢀCl(1) torsion angle of 3.7(3)8. A survey of Cambridge Structural
Database [33] revealed that this is the first structure in which at least one of three non-
common ring C-atoms of the five-membered ring (C(8), C(9), or C(10)) is not sp3-
hybridized. Thus, the C(8) and C(9) are sp2-hybridized, and the C(8)ꢀC(9) bond of
1.343(3) ꢂ has a character of a Csp2¼Csp2 bond.
The molecules of 6 are linked by N(3)· · ·O(2) Hꢀbonds (Table 1 and Fig. 5) into
centrosymmetric dimers via eight-membered rings. This characteristic motif for

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1461
Fig. 3. Crystal packing diagram of 3, viewed along the c axis, showing the packing of the molecules. H-
Bonds are indicated with dashed lines.
Fig. 4. Molecular structure of 6, with the atom-numbering scheme. Displacement ellipsoids for non-H-
atoms are drawn at the 30% probability level.
pyrimidine derivatives can be defined by a graph-set notation as R22(8). In addition, two
CꢀH· · ·O H-bonds form R12(6) ring, so forming a three-dimensional network.
3. Antitumor Activities. Compounds 3–11 were evaluated for their cytostatic
activities against human malignant tumor cell lines: cervical carcinoma (HeLa), breast
epithelial adenocarcinoma, metastatic (MCF-7), hepatocellular carcinoma (HepG2),
colorectal adenocarcinoma (SW620), pancreatic carcinoma (MiaPaCa-2), and normal

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 5. Crystal packing diagram of 6, viewed along the b axis, showing NꢀH· · ·O and CꢀH· · ·O H-bonds.
H-Bonds are indicated with dashed lines.
human fibroblasts (WI38) (Table 2). Their effects were compared to antiproliferative
activity of a compound regularly used for chemotherapy of cancer patients, 5-
fluorouracil (5-FU). Among all synthesized compounds, only 2,4-dimethoxypyrimidine
with 6-[(1,3-dibenzyloxy)-2-hydroxy]methyl side chain, 3, showed a prominent
concentration-dependent inhibitory activity similar to those obtained by 5-FU against
all tested cell lines. Similarly to 5-FU, compound 3 exhibited also a cytostatic activity
Table 2. Inhibitory Effects of C(6)-Substituted Pyrimidine Derivatives 3–11 and 5-Fluorouracil (5-FU)
as Control Compound on the Growth of Malignant Tumor Cell Lines and Normal Fibroblasts
IC50 [mm]a)
HeLa
MCF-7
4.22ꢁ0.43
>100
HepG2
2.42ꢁ0.69
>100
48.02ꢁ0.23
SW620
3.48ꢁ0.9
>100
64.61ꢁ0.36
MiaPaCa-2
6.08ꢁ0.37
>100
WI38
3.84ꢁ0.39
>100
3
4
5
6
7
3.67ꢁ0.45
>100
>100
>100
>100
>100
85.15ꢁ0.13
67.81ꢁ0.35
78.78ꢁ0.16
>100
>100
>100
94.6ꢁ0.16 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8
9
>100
>100
>100
>100
95.89ꢁ0.13 >100
10
11
5-FU
>100
>100
4.4ꢁ1.5
>100
>100
42.14ꢁ11.43 8ꢁ1.7
3.38ꢁ2.49
11.45ꢁ6.5
18.5ꢁ17.1
a) IC50; 50% Inhibitory concentration, or compound concentration required to inhibit tumor cell
proliferation by 50%.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1463
(IC50 3.84 mm) against normal human fibroblasts (WI38). The pyrimidine derivative 5
bearing C(6)-isobutenyl side chain with the C(2’)¼C(3’) bond showed moderate
cytostatic activity against HepG2, MCF-7, SW620, MiaPaCa-2, and WI38 cell lines at
the highest tested concentrations (IC50 ranging from 48.02 to 85.15 mm) (Table 2). In
contrast, compounds 4, 7, 8, 10, and 11 did not exhibit any inhibitory effects. Due to the
obtained results, compound 3, bearing two benzylated and one free OH group in the
C(6)-side chain, was chosen for further biological evaluation on HeLa cells that have
the inactivated p53 protein [34]. It has been widely documented that many tumors
bearing inactivated p53 protein and/or mutated p53 gene do not react well on therapy
with common chemotherapeutic agents such as 5-FU [35][36]. Thus, we were
interested in elucidation of the mechanism of antiproliferative effect on HeLa cells
exerted by compound 3 in comparison to 5-FU. It is well-known that the main tumor
suppressor activity of the functional gene p53 involves activation of the G1 cell-cycle
checkpoint and/or apoptosis [37][38]. The results obtained by flow cytometric analysis
of HeLa cells treated with compound 3 revealed a strong influence on the cell cycle,
expectedly not involving a rise in G1 cell population (Table 3). After the 24-h
treatment with compound 3, an increase of cells in G2/M phase (12.8%) has been
observed at a concentration of 1ꢂ10ꢀ5 m, as well as an increase of cells in subG1
(11.7%) at a concentration of 5ꢂ10ꢀ5 m, indicative for apoptosis. Interestingly, the
main antiproliferative mechanism for compound 3 might be linked with the cell-cycle
arrest in the S phase (ranging from 10.2–33.7%) observed upon the 48-h treatment at
all tested concentrations accompanied with a concomitant decrease in G1 and subG1
cell populations. A similar effect was observed for HeLa cells treated with 5-FU
(Table 3).
Table 3. Flow Cytometric Analysis of HeLa Cells upon the 24 h and 48 h Treatments with Compound 3
and 5-FU
Cell percentage ([%]ꢁstandard deviation)
subG1
G1
S
G2/M
3
Control 24 h
45.1ꢁ2.3
59.9ꢁ3.4
47.3ꢁ5.4
52.7ꢁ1.4
61.2ꢁ1.1
61.9 ꢁ4.2
52.9ꢁ1.3a)
32.6ꢁ4.8a)
23.9ꢁ4.1a)
25.7ꢁ4.9
30.5ꢁ4.1
20.1ꢁ2.8
19.4ꢁ1.5
21.9ꢁ2.3
32.1ꢁ2.9a)
49.7ꢁ4.1a)
55.6ꢁ3.2a)
14.4ꢁ3.7
22.2ꢁ4.8
27.2ꢁ1.4a)
19.5ꢁ2.5
16.2ꢁ2.6
15.1ꢁ2.3
17.8ꢁ0.7
20.5ꢁ4.3
5ꢂ10ꢀ6 m 24 h
1ꢂ10ꢀ5 m 24 h
5ꢂ10ꢀ5 m 24 h
Control 48 h
43.2ꢁ2.1
50.4ꢁ2.7
56.8ꢁ0.7a)
53.6ꢁ1.5
5ꢂ10ꢀ6 m 48 h
1ꢂ10ꢀ5 m 48 h
5ꢂ10ꢀ5 m 48 h
49.8ꢁ0.7a)
39.9ꢁ1.1a)
21.6ꢁ0.8a)
5-FU
Control 24 h
6.96ꢁ1.28
9.35ꢁ0.51a)
7.96ꢁ0.59
4.42ꢁ0.38
4.97ꢁ0.64
5.20ꢁ0.81
56.67ꢁ1
30.7ꢁ0.89
6.69ꢁ1.28
6.00ꢁ0.35a)
12.23ꢁ1.17
4.42ꢁ0.38
5.73ꢁ0.57a)
11.1ꢁ1.78
5ꢂ10ꢀ5 m 24 h
1ꢂ10ꢀ6 m 24 h
Control 48 h
40.7ꢁ0.68a)
55.1ꢁ1.31
53.27ꢁ1.42a)
32.73ꢁ0.89a)
33.1ꢁ1.1
54.87ꢁ0.4
38.77ꢁ2.62a)
49.33ꢁ1.02a)
5ꢂ10ꢀ5 m 48 h
55.1ꢁ3.1a)
1ꢂ10ꢀ6 m 48 h
39.57ꢁ2.17a)
a) Statistically significant at p<0.05. The results are given in percentages of cells (%ꢁstandard
deviation) in a particular cell cycle phase.

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Fig. 6. HeLa Cells treated with compound 3 after 24 h and 48 h treatment
Observed morphological changes of HeLa cells treated with compound 3 are in line
with cell-cycle analysis results (Fig. 6). Untreated HeLa cells formed an integrated
confluent monolayer of large, flat cells. However, a change of cell morphology upon 24-
h treatment that points to apoptosis (shrinkage of cells, chromatin condensation) has
been induced with compound 3 at the highest tested concentration (5ꢂ10ꢀ5 m) which is
in concordance with the cell-cycle analysis showing a rise in subG1 population under
the same treatment condition. On the contrary, cells treated with lower concentrations
(5ꢂ10ꢀ6 and 1ꢂ10ꢀ5 m) revealed a more spread appearance with widen cellꢀcell
interspaces probably due to the cell-cycle arrest monitored by flow cytometry.
HeLa Cells on the other hand exerted strong apoptotic features upon the 48-h
treatment at concentrations of 5ꢂ10ꢀ6 and 1ꢂ10ꢀ5 m confirmed by rise of subG1
population detected by flow cytometry as well. However, the highest tested concen-
tration (5ꢂ10ꢀ5 m) induced formation of enlarged cells probably due to a strong S
phase arrest. The damaged cells thus block the cell cycle, and if the damage is too high
they ultimately die.
Conclusions. – Novel pyrimidine derivatives bearing isobutyl (in 3, 4, and 7–9), and
isobutenyl (in 5 and 10) side chains at C(6), and dihydropyrrolo[1,2-c]pyrimidine-1,3-
diones 6 and 11 were successfully synthesized and characterized. The position of the
C¼C bond in the side chains of compounds 3–5 and 7–10 depended on the nature or
type of functionality used to protect the OH groups in the side chain. Thus, the Bn
protecting groups gave rise to a C(2’)¼C(3’) bond (compound 5), whereas the Ac
protecting groups led to a C(1’)¼C(2’) bond (compound 10). The dihydropyrrolo[1,2-
c]pyrimidine-1,3-diones 6 and 11 were obtained as products of intramolecular
cyclization, which occurred during the removal of the Bn (in 5) or MeO protecting
groups (in 10).
Compounds 3–11 were shown to exert none or moderate antiproliferative effect on
the tested tumor cell lines. However, 6-(1,3-dibenzyloxy)isobutyl-substituted 2,4-
dimethoxypyrimidine derivative 3 showed the strongest concentration-dependent
inhibitory activity against all tested cell lines similar to the effect of the control
compound 5-FU. Furthermore, the results of the flow cytometric analysis revealed a
strong influence of compound 3 on the cell cycle of HeLa cells. The mechanism of its

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1465
antiproliferative effect includes early increase of cells in G2/M phase, and apoptosis as
well as the cell cycle arrest in the S phase upon prolonged treatment, accompanied with
a concomitant decrease in G1 and subG1 cell populations. The increase of cells in the S
phase after a prolonged treatment with compound 3 observed in our experiments might
be a result of hampered DNA replication process due to a structural similarity of
compound 3 with natural nucleosides. Besides, the pyrimidine derivative 5 with a
C(2’)¼C(3’) bond exhibited moderate non-specific cytostatic activity against HepG2,
MCF-7, SW620, MiaPaCa-2, and WI38 cell lines at the highest tested concentration.
The biological results for 3 indicated that this compound is a lead compound for
further synthetic structural optimization.
Support for this study was provided by the Ministry of Science of the Republic of Croatia (Projects
No. 125-0982464-2925, No. 119-1193079-3069, No. 335-0982464-2393, and No 335-0000000-3532).
Experimental Part
1. General. M.p.: Kofler micro hot-stage (Reichert, Wien); uncorrected. TLC: Precoated Merck
silica-gel 60 F-254 plates; detection of the spots under UV light (254 nm). Column chromatography
(CC): silica gel (0.063–0.2 mm) Fluka; glass column slurry-packed under gravity. High-performance
liquid chromatography (HPLC): Agilent 1100 series system with UV detection (photodiode array
detector) using Zorbax C18 reversed-phase (RP) anal. column (2.1ꢂ30 mm, 3.5 mm); all compounds used
for biological evaluation showed >95% purity in this HPLC system. 1H- and 13C-NMR spectra: Bruker
300 MHz NMR spectrometer; in (D6)DMSO at 298 K, chemical shifts referenced to the residual solvent
signal of DMSO at d 2.50 ppm for 1H and d 39.50 ppm for 13C; individual resonances were assigned on the
basis of their chemical shifts, signal intensities, multiplicity of resonances and H,H coupling constants.
MS: Agilent 6410 instrument equipped with electrospray interface and triple quadrupole analyzer (LC/
MS/MS).
2. Synthesis. 2.1. 1,3-Bis(benzyloxy)propan-2-one (2) [26]. N-Chlorosuccinimide (NCS; 3.69 g,
0.27 mol) was suspended in dry CH2Cl2 (190 ml), and the mixture was cooled at ꢀ258 in a dry-ice bath.
Me2S (20.4 ml, 0.27 mol) was added under Ar. 1,3-Bis(benzyloxy)propan-2-ol (23 ml, 0.10 mol) in
CH2Cl2 (100 ml) was added to the mixture, which was stirred at ꢀ258 for 2 h. Then, Et3N (65.1 ml,
0.47 mol) was added, and the cooling bath was removed. The mixture was stirred overnight at r.t. and
neutralized with 1m HCl. The org. layer was separated and washed with sat. aq. NaCl soln. (70 ml). The
aq. layer was extracted with AcOEt (2ꢂ100 ml), and the org. layers were combined and dried (MgSO4).
After the removal of the solvents, the residue was purified using CC (hexane/AcOEt 4 :1) to give 2
(18.0 g, 72.6%). Crude colorless oil. 1H-NMR: 4.28 (s, CH2(1), CH2(3)); 4.52 (s, 2 PhCH2O); 7.26–7.38
(m, 2 Ph). MS: 271.1 ([MþH]þ ).
2.2. 1,3-Bis(benzyloxy)-2-[(2,6-dimethoxy-5-methylpyrimidin-4-yl)methyl]propan-2-ol (3) [28].
LDA (28.64 ml, 2m in THF/heptane/ethylbenzene) was added dropwise to the soln. of 1 (4.81 g,
45 mmol) in THF (57 ml) at ꢀ708. The temp. was raised to ꢀ558, and the mixture was stirred for 30 min.
To the mixture, the soln. of 2 (7.54 g, 28 mmol) in THF (41 ml) was added, and stirring was continued for
2.5 h at ꢀ558. The soln. was neutralized with AcOH and evaporated. The oily residue was extracted with
AcOEt and H2O. The org. layer was dried (MgSO4) and purified by CC (hexane/AcOEt 3 :1 to afford 3
(4.4 g, 58.4%). M.p. 46–488. 1H-NMR: 2.03 (s, Me); 2.85 (s, CH2(1’)); 3.33 (s, CH2(3’), CH2(3’’)); 3.78 (s,
MeO); 3.88 (s, MeO); 4.49 (s, 2 PhCH2O); 5.10 (s, OH); 7.26–7.34 (m, 2 Ph). 13C-NMR: 10.52 (Me);
37.84 (C(1’)); 54.37 (MeO); 54.42 (MeO); 72.98 (C(3’,3’’)); 73.72 (PhCH2O); 74.81 (C(2’)); 109.96
(C(5)); 127.76 (CH of Ph); 127.78 (CH of Ph); 128.60 (CH of Ph); 138.98 (Cq of Ph); 161.92 (C(6)); 165.94
(C(2)); 169.60 (C(4)). MS: 439.1 ([MþH]þ ).
2.3. 4-[2-Fluoro-3-(phenylmethoxy)-2-[(phenylmethoxy)methyl]propyl]-2,6-dimethoxy-5-methyl-
pyrimidine (4) and 2,4-Dimethoxy-5-methyl-6-{(2Z)-3-(phenylmethoxy)-2-[(phenylmethoxy)methyl]-
prop-2-en-1-yl}pyrimidine (5). A soln. of 3 (3.23 g, 7.4 mmol) in dry CH2Cl2 (20 ml) was cooled to

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
ꢀ788 and stirred for 15 min under Ar. DAST (7.5 mmol) was added dropwise, and the mixture was kept
at ꢀ788 for additional 15 min after which the cooling bath was removed. After 4 h of stirring at r.t., sat.
aq. NaHCO3 soln. (10 ml) was added, and the mixture was partitioned. The org. layer was separated,
dried (MgSO4), and evaporated to dryness. The raw product was purified by CC (hexane/AcOEt 3 :1) to
afford 4 (642 mg, 19.8%) as colorless and 5 (348 mg, 11.2%) as yellow oil.
Data of 4. 1H-NMR: 2.00 (s, Me); 3.11 (d, 3J(H,F)¼18.1, CH2(1’)); 3.65 (dd, 2J(H,H)¼10.7,
3J(H,F)¼22.8, CH2(3’)); 3.72 (s, MeO); 3.81 (d, 2J(H,H)¼10.8, 3J(H,F)¼22.2, CH2(3’’)); 3.88 (s, MeO);
4.52 (s, 2 PhCH2O); 7.26–7.38 (m, 2 Ph).13C-NMR: 10.45 (Me); 37.75 (d, J(C,F)¼23.1, C(1’)); 54.42
(MeO); 54.40 (MeO); 71.14 (d, J(C,F)¼24.3, C(3’,3’’)); 73.08 (PhCH2O); 98.11 (d, J(C,F)¼177.4,
C(2’)); 110.07 (C(5)); 127.92–128.88 (CH of Ph); 138.50 (Cq of Ph); 162.25 (C(2)); 163.48 (d, J(C,F)¼7.0,
C(6)); 169.69 (C(4)). MS: 441.1 ([MþH]þ ).
Data of 5. 1H-NMR: 1.94 (s, Me); 3.42 (s, CH2(1’)); 3.75 (s, MeO); 3.81 (s, CH2(3’’)); 3.85 (s, MeO);
4.27 (s, PhCH2O); 4.87 (s, PhCH2O); 6.42 (s, HꢀC(3’)); 7.15–7.22 (m, Ph); 7.27–7.33 (m, Ph).13C-NMR:
10.05 (Me); 31.23 (C(1’)); 54.21 (MeO); 54.30 (MeO); 70.20 (C(3’’)); 70.74 (PhCH2O); 73.65
(PhCH2O); 108.13 (C(5)); 111.12 (C(2’)) 127.67–129.29 (CH of Ph); 138.01 (Cq of Ph); 139.04 (Cq of Ph);
146.28 (C(3’)); 162.53 (C(6)); 167.39 (C(2)); 169.38 (C(4)). MS: 421.1 ([MþH]þ ).
2.4. 2,4-Dimethoxy-5-methyl-6-{(2Z)-3-(phenylmethoxy)-2-[(phenylmethoxy)methyl]prop-2-en-1-
yl}pyrimidine (5). The mixture of 4 (642 mg, 1.46 mmol) and MeONa (395 mg, 7.3 mmol) in dry MeOH
(20 ml) was heated to reflux for 3 d. The solvent was removed under reduced pressure, and the oily
residue was extracted with CH2Cl2 and H2O. The org. layer was dried (MgSO4), and evaporation to
dryness afforded 5 (464 mg, 75.6%) as yellow oil.
2.5. 6-(Chloromethyl)-4-methylpyrrolo[1,2-c]pyrimidine-1,3(2H,7H)-dione (6). A mixture of 5
(205 mg, 0.5 mmol) in dry CH2Cl2 (10 ml) was cooled to ꢀ788, and 1m BCl3 soln. in CH2Cl2 (2 ml)
was added under Ar. The mixture was stirred at ꢀ788 for 2 h, then the temp. was raised to ꢀ408, and the
mixture was stirred for additional 2 h. The reaction was quenched by addition of CH2Cl2/MeOH 1:1
(5 ml), and the cooling bath was removed. The mixture was evaporated to dryness, and the crude product
was purified by CC (CH2Cl2/MeOH 50 :1) to give 6 (27 mg, 25.4%). Yellow crystals. M.p. >3008.
1H-NMR: 1.86 (s, Me); 4.59 (s, CH2Cl); 4.64 (s, CH2(7)); 6.91 (s, HꢀC(5)); 11.11 (s, NH).13C-NMR: 10.33
(Me); 29.47 (CH2Cl); 54.62 (C(7)); 103.61 (C(4)); 112.94 (C(6)); 121.30 (C(5)); 148.79 (C(6)); 150.32
(C(1)); 165.97 (C(3)). MS: 213.1 ([MþH]þ ), 215.1 ([MþHþ2]þ ).
2.6. 2-[(2,6-Dimethoxy-5-methyl-4-pyrimidinyl)methyl]propane-1,2,3-triol (7). A soln. of 3 (1.54 g,
3.52 mmol) in dry CH2Cl2 (40 ml) was cooled to ꢀ788, and BCl3 (1m in CH2Cl2, 14.1 ml) was added under
Ar. The mixture was stirred at ꢀ408 for 2 h, then CH2Cl2/MeOH 1:1 (70 ml) was added, and the cooling
bath was removed. The solvents were evaporated under reduced pressure, and the residue was purified by
CC (CH2Cl2/MeOH 10 :1) to give 7 (639 mg, 70.4%). Yellow oil. 1H-NMR: 2.07 (s, Me); 2.84 (s, CH2(1’));
2
2
3.28 (d, J¼11.0, CH2(3’)); 3.35 (d, J¼11.0, CH2(3’’)); 3.95 (s, MeO); 3.98 (s, MeO); 4.48 (br. s, OH).
13C-NMR: 10.52 (Me); 34.22 (C(1’)); 54.28 (MeO); 55.17 (MeO); 64.99 (C(3’,3’’)); 76.00 (C(2’)); 107.02
(C(5)); 148.22 (C(6)); 150.73 (C(2)); 165.30 (C(4)). MS: 259.1 ([MþH]þ ).
2.7. 2-[(2,6-Dimethoxy-5-methylpyrimidin-4-yl)methyl]-2-hydroxypropane-1,3-diyl Diacetate (8). To
the mixture of 7 (207.6 mg, 0.81 mmol), DMAP (15.5 mg, 0.12 mmol), and Et3N (0.3 ml, 2.01 mmol) in
dry MeCN (20 ml), Ac2O (0.16 ml, 1.69 mmol) was added, and the mixture was stirred at r.t. for 40 min.
The reaction was quenched with MeOH (1.3 ml), and the mixture was stirred for additional 5 min. After
evaporation of volatile materials and CC (CH2Cl2/MeOH 10 :1), 8 (163.4 mg, 54.3%) was isolated.
Yellow oil. 1H-NMR: 2.01 (s, 2 MeCO); 2.04 (s, Me); 2.87 (s, CH2(1’)); 3.81 (s, MeO); 3.89 (s, MeO); 4.05
(s, CH2(3’), CH2(3’’)); 5.38 (s, OH).13C-NMR: 10.60 (Me); 21.09 (MeCO); 37.98 (C(1’)); 54.42 (MeO);
66.65 (C(3’)); 72.67 (C(2’)); 110.24 (C(5)); 162.11 (C(6)); 164.68 (C(2)); 169.80 (C(4)); 170.57 (MeCO).
MS: 343.1 ([MþH]þ ).
2.8. 2-[(2,6-Dimethoxy-5-methylpyrimidin-4-yl)methyl]-2-fluoropropane-1,3-diyl Diacetate (9). A
soln. of 8 (348.5 mg, 0.93 mmol) in dry CH2Cl2 (40 ml) was cooled to ꢀ788 and stirred for 15 min under
Ar. DAST (1 mmol) was added dropwise, and the mixture was kept at ꢀ408 for additional 2 h after
which cooling bath was removed. To the mixture, MeOH (2 ml) was added, and stirring was continued for
15 min. The volatile materials were removed, and the residue was purified by CC (CH2Cl2/MeOH 40 :1)
1
3
to give 9 (90.8 mg, 26.0%). Colorless oil. H-NMR: 2.04 (s, Me); 2.05 (s, 2 MeCO); 3.14 (d, J(H,F)¼

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1467
16.9, CH2(1’)); 3.83 (s, MeO); 3.90 (s, MeO); 4.31 (dd, 2J(H,H)¼12.4, 3J(H,F)¼22.7, CH2(3’)); 4.42 (dd,
2J(H,H)¼12.2, 3J(H,F)¼22.1, CH2(3’’)). 13C-NMR: 9.89 (Me); 20.43 (MeCO); 35.77 (d, 2J(C,F)¼23.4,
C(1’)); 54.01 (MeO); 54.03 (MeO); 64.92 (d, 3J(C,F)¼23.5, C(3’)); 95.14 (d, 1J(C,F)¼179.9, C(2’));
3
109.72 (C(5)); 161.85 (d, J(C,F)¼7.8, C(6)); 169.40 (C(2)); 169.83 (C(4)); 170.20 (MeCO). MS: 345.1
([MþH]þ ).
2.9. 2-[(2,6-Dimethoxy-5-methylpyrimidin-4-yl)methylidene]propane-1,3-diol (10). To a cold dry
MeOH (10 ml), Na (28.2 mg, 1.21 mmol) was added, followed by compound 9 (91 mg, 0.24 mmol)
dissolved in MeOH (2 ml). The mixture was refluxed for 1 h and became pale yellow. After removal of
the solvent, the residue was purified using CC (CH2Cl2/MeOH 15 :1) to give 10 (24.2 mg, 41.9%). White
solid. M.p. 90–928. 1H-NMR: 2.03 (s, Me); 3.86 (s, MeO); 3.90 (s, MeO); 4.22 (d, 2J¼4.3, CH2(3’)); 4.43
2
3
(d, J¼5.8, CH2(3’’)); 4.87 (t, 3J¼5.9, OH); 5.06 (t, J¼5.6, OH); 6.58 (s, HꢀC(1’)).13C-NMR: 10.27
(Me); 54.40 (MeO); 54.54 (MeO); 59.45 (C(3’)); 62.97 (C(3’’)); 107.94 (C(5)); 117.52 (C(1’)); 152.81
(C(2’)); 161.97 (C(6)); 162.36 (C(2)); 169.94 (C(4)). MS: 241.1 ([MþH]þ ).
2.10. 6-(Hydroxymethyl)-4-methylpyrrolo[1,2-c]pyrimidine-1,3(2H,7H)-dione (11). TMSCl (0.2 ml,
1.58 mmol) and NaI (82 mg, 0.54 mmol) were added under Ar to a stirred mixture of 10 (19 mg,
0.08 mmol) in dry MeCN (2 ml) at r.t. The mixture was stirred at 658 for 72 h, and the solvent was
removed under reduced pressure. The residue was purified by CC (CH2Cl2/MeOH 10 :1) to give 11
(10.4 mg, 67.1%). Oil. 1H-NMR: 1.56 (s, Me); 3.93 (s, CH2OH); 4.12 (s, CH2(7)); 5.14 (t, 3J¼5.4, OH);
6.18 (s, HꢀC(5)); 10.99 (NH). 13C-NMR: 10.91 (Me); 58.51 (CH2OH); 62.55 (C(7)); 105.80 (C(4));
115.16 (C(6)); 145.70 (C(5)); 150.08 (C(4a)); 151.09 (C(1)); 165.27 (C(3)). MS: 195.1 ([MþH]þ ).
3. X-Ray Determination of Compounds 3 and 61). Crystals suitable for X-ray single crystal study were
grown from very dil. MeOH soln. for both compounds. The intensities were collected on an Oxford
Diffraction Xcalibur2 diffractometer with a Sapphire 3 CCD detector using graphite-monochromated
MoKa radiation (l¼0.71073 ꢂ) at different temps., 100 K for 3 and 295 K for 6. The data collection and
reduction were carried out with the CrysAlis programs [39]. The crystal structures were solved by direct
methods [40]. All non-H-atoms were refined anisotropically by full-matrix least-squares calculations
based on F2 [40]. The H-atom attached to N(3) in 6 was found in a difference Fourier map, and its
coordinates and isotropic thermal parameter have been refined freely. All other H-atoms in 3 and 6 were
treated using appropriate riding models, with SHELXL97 defaults [40]. The choice of H-position of the
OH group in both independent molecules of 3 was determined by best H-bond that could be created.
Examination of the refined structure of 3 using ADDSYM option in PLATON [41] revealed possible
additional symmetry, but the structure could not be refined satisfactorily in space group Pbca. In the
absence of significant anomalous scattering, the Flack parameter was inconclusive, and the Friedel
equivalents were, therefore, merged prior to the final refinement of this structure. PLATON program was
used for analysis, and molecular and crystal structure drawings preparation [41].
Crystal Data of 3. C25H30N2O5, Mr 438.51, orthorhombic space group Pca21; a¼24.9959(10), b¼
17.1603(7), c¼10.7305(5) ꢂ, V¼4602.7(3) ꢂ3; Z¼8; Dx ¼1.266 g/cm3; m(MoKa)¼0.088 mmꢀ1; S¼
1.013; R/wR¼0.0692/0.1750 for 585 parameters and 3571 reflections with Iꢃ2s(I), R/wR¼0.0844/0.1877
for all 4747 independent reflections measured in the range 7.928–2q–52.008.
Crystal Data of 6. C9H9Cl1N2O2, Mr 212.63, monoclinic space group P21/c; a¼7.1834(2), b¼
8.3351(3), c¼15.9522(5) ꢂ, b¼98.681(3)8, V¼944.19(5) ꢂ3; Z¼4; Dx ¼1.496 g/cm3; m(MoKa)¼
0.378 mmꢀ1; S¼1.043; R/wR¼0.0510/0.1583 for 132 parameters and 1576 reflections with Iꢃ2s(I); R/
wR¼0.0697/0.1674 for all 2271 independent reflections measured in the range 7.688–2q–56.008.
4. Antitumor Activity Assays. 4.1. Cell Culturing. The cell lines HeLa (cervical carcinoma), SW620
(colorectal adenocarcinoma, metastatic), MiaPaCa-2 (pancreatic carcinoma), MCF-7 (breast epithelial
adenocarcinoma, metastatic), HepG2 (hepatocellular carcinoma), and WI38 (normal human fibro-
blasts) were cultured as monolayers and maintained in Dulbeccoꢄs modified Eagle medium (DMEM),
supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, 100 U/ml penicillin, and 100 mg/ml
streptomycin in a humidified atmosphere with 5% CO2 at 378.
1
)
CCDC-784295 and 784296 contain the supplementary crystallographic data for this article. These
data can be obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
4.2. Proliferation Assays. The panel cell lines were inoculated onto a series of standard 96-well
microtiter plates on day 0, at 3000 to 6000 cells per well according to the doubling times of specific cell
line. Test agents and the control compound 5-FU were then added in five- to tenfold dilutions (1ꢂ10ꢀ8 to
1ꢂ10ꢀ4 m) 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 concentration never exceeded
0.1%). After 72 h of incubation, the cell growth rate was evaluated by performing the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay: experimentally determined
absorbance values were transformed to a cell percentage growth (PG) using the formulas proposed by
NIH and described in [42]. 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 – the results are, therefore, a percentile difference from the calculated expected value.
The IC50 and LC50 values for each compound were calculated from doseꢀresponse 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 ꢅ>ꢄ sign).
Each test point was 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 evaluated by plotting the mean percentage growth for each cell type in comparison to
control on doseꢀresponse graphs.
4.3. Cell-Cycle Analyses. A total of 3ꢂ104 cells/well were seeded in a 6-well plate (Falcon, USA).
After 24 h, HeLa cells were treated with the compound 3 at concentrations of 5ꢂ10ꢀ6, 1ꢂ10ꢀ5, and 5ꢂ
10ꢀ5 m, and 5-FU at concentrations of 5ꢂ10ꢀ5 and 1ꢂ10ꢀ6 m. After 24 and 48 h, the attached cells were
trypsinized, combined with floating cells, washed with PBS, and fixed with 70% EtOH. Immediately
before the analysis, the cells were washed again with PBS and stained with 1 mg/ml of propidium iodide
(PI) with the addition of 0.2 mg/ml of RNAse A. The stained cells were then analyzed with Becton
Dickinson FACScalibur flow cytometer (10,000 counts were measured). Each test point was performed
in triplicate. The percentage of the cells in each cell cycle phase was based on the obtained DNA
histograms and determined using the WinMDI 2.9 and Cylchred software. Statistical analysis was
performed in Microsoft Excel by using the ANOVA at p<0.05.
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Received July 19, 2010