Synthesis, structural studies and antitumoral evaluation of C-6 alkyl and alkenyl side chain pyrimidine derivatives

Article abstract of DOI:10.3390/molecules14124866

The synthetic route for introduction of fluorophenylalkyl (compounds 5, 7, 14 and 15) and fluorophenylalkenyl (compounds 4E and 13) side chains at C-6 of the pyrimidine nucleus involved the lithiation of the pyrimidine derivatives 1, 2 and 11 and subseque

Full text of DOI:10.3390/molecules14124866

Molecules 2009, 14, 4866-4879; doi:10.3390/molecules14124866
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Structural Studies and Antitumoral Evaluation of C-6
Alkyl and Alkenyl Side Chain Pyrimidine Derivatives
Svjetlana Krištafor 1, Tatjana Gazivoda Kraljević 1, Damjan Makuc 2, Janez Plavec 2,
Lidija Šuman 3, Marijeta Kralj 3 and Silvana Raić-Malić 1,*
1
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of
Zagreb, Marulićev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia;
E-Mails: prekupec@fkit.hr (S.K.); tgazivod@fkit.hr (T.G.K.)
2
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, P.O. Box 660, SI-1001
Ljubljana, Slovenia; E-Mails: damjan.makuc@ki.si (D.M.); janez.plavec@ki.si (J.P.)
3
Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička 54, P.O. Box 1016, HR-
10001, Zagreb, Croatia; E-Mails: lidija.suman@irb.hr (L.S.); marijeta.kralj@irb.hr (M.K.)
* Author to whom correspondence should be addressed; E-Mail: silvana.raic@fkit.hr;
Tel.: +38 51 4597 213; Fax: +38 51 4597 224.
Received: 30 September 2009; in revised form: 19 November 2009 / Accepted: 25 November 2009 /
Published: 27 November 2009
Abstract: The synthetic route for introduction of fluorophenylalkyl (compounds 5, 7, 14
and 15) and fluorophenylalkenyl (compounds 4E and 13) side chains at C-6 of the
pyrimidine nucleus involved the lithiation of the pyrimidine derivatives 1, 2 and 11 and
subsequent nucleophilic addition or substitution reactions of the organolithium
intermediate thus obtained with 2-fluorophenylacetone, 4-fluoroacetophenone or ethyl
4-fluorobenzoate as electrophiles. The structures of novel compounds were confirmed by
1H-, 19F- and 13C-NMR and MS. Compounds 8 and 10 containing unsaturated
fluorophenylalkyl side chains showed better inhibitory effect than their saturated
fluorophenylalkylated pyrimidine counterparts 7 and 9. A conformational study based on
NOE enhancements showed the importance of the double bond and substitution in the side
chain for the conformational preferences in relation to inhibitory activity. Among all tested
compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives
showed significant cytostatic activities against all tested tumor cell lines.

Molecules 2009, 14
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Keywords: C-6 alkyl and alkenyl pyrimidine derivatives; NMR conformational analysis;
cytostatic activity evaluations
Introduction
The application of fluorine-containing compounds in the pharmaceutical and agrochemical fields
has a very short history [1,2]. The small size of the fluorine substituent, combined with its high
electronegativity and its impact upon bond strengths give rise to the observed distinctive effect of
fluorine substituents on the biological activity of compounds [3]. In the area of medicinal chemistry,
incorporation of fluorine has played a significant role in the development of new anti-cancer and
anti-viral agents, anti-inflammatory and anti-hyperintensive agents, anti-fertility drugs and central
nervous system drugs. Fluorine affects the biological activity of compounds in a number of important
ways. It has been reported that covalently bonded fluorine is a very weak intermolecular hydrogen-
bond acceptor [4]. The presence of fluorine at a particular position in a molecule can enhance its
metabolic stability or modulate its physicochemical properties, such as its lipophilicity, acidity or
basicity. Fluorination can increase molecules’ binding affinity to a target protein, and by a
combination of factors interfere with specific enzyme action [2,5]. As a result, the introduction of
fluorine atoms, trifluoromethyl, difluoromethyl or other fluorinated and fluoroalkyl groups into
heterocyclic compounds may have significant influence on their biological and physical properties.
Therefore, fluorinated compounds in general and fluorinated heterocycles in particular are the focus of
much research [2]. Pyrimidines are biologically important molecules and valuable heterocyclic nuclei
for the design of pharmaceutical agents [6,7]. A great number of C-5 and C-6 substituted pyrimidine
nucleosides have been prepared in view of their various biological activities [8]. For this reason, the
development of synthetic methods for fluorine-containing heterocyclic compounds has been an
important field in both organofluorine chemistry and organic synthesis.
Recently, we have reported that fluorinated propyl or propenyl C-6 acyclic pyrimidine derivatives
containing the 2-hydroxy-3,3,3-trifluoro-1-propenyl side chain exhibited a pronounced effect against
breast carcinoma (MCF-7), while the compound with a 2-fluoromethyl-2-acetoxypropyl chain
exhibited moderate effect against cervical carcinoma (HeLa) [9]. We have also reported the synthesis
and biological results of a new type of C-6 fluoroalkylated and fluorophenylalkylated pyrimidine
derivatives as model compounds for development of tracer molecules in positron-emission
tomography (PET) [10]. In this study we present the syntheses and antitumoral evaluations of C-6
fluorophenylalkyl (6, 9, 14 and 15), fluorophenylalkenyl (4, 8, 10 and 13) and 5-furyl-6-methyl (12)
pyrimidine derivatives.
Results and Discussion
Chemistry
Key precursors 2,4-dimethoxy-6-methyl pyrimidine (1), 2,4-dimethoxy-5,6-dimethylpyrimidine (2)
and 5-bromo-2,4-dimethoxy-6-methylpyrimidine (3) were prepared according to the procedure

Molecules 2009, 14
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described in the literature [11,12]. The C-6 fluorophenylalkyl substituted pyrimidine derivatives 4, 5, 7
and 13-15 (Schemes 1 and 2) were synthesized by lithiation, which has been studied extensively as an
important carbon-carbon bond forming reaction [13,14]. Fluorophenyl-propyl and –ethyl side chaind
were introduced by lithiation and subsequent nucleophilic addition of pyrimidine derivatives 1 and 2 to
2-fluorophenylacetone and 4-fluoroacetophenone to give 5 and 7, respectively (Scheme 1).
Scheme 1. Synthesis of C-6 alkyl and alkenyl side chain pyrimidine derivatives (4-10).
OCH3
OCH3
CH3
OH
CH3
N
N
O
H3CO
N
H3CO
N
F
F
4E
4K
ii
OCH3
OCH3
N
OH
iii
R5
N
H3CO
N
H3C
F
H3CO
N
CH3
5
1
2
3
iv
H
CH3 Br
i
R5
O
HN
OH
v
O
N
H
H3C
F
OCH3
6
CH3
OH
N
H3CO
N
H3C
F
7
vi
iv
O
O
OCH3
CH3
CH3
CH3
CH3
CH3
HN
HN
N
OH
+
+
O
N
H
H3CO
N
H
O
N
H3C
F
F
F
8
9
10
Reagents and conditions: (i) NBS, acetic acid; (ii) LDA, THF, ethyl 4-fluorobenzoate; (iii) LDA,
THF, 2-fluorophenylacetone; (iv) NaI, (CH3)3SiCl, CH3CN; (v) LDA, THF, 4-fluoroacetophenone;
(vi) acetyl chloride, H2O.
Reaction of lithiatied intermediate 2 with ethyl 4-fluorobenzoate afforded the C-6 substituted
pyrimidine derivative containing unsaturated side chain, which exists as both the keto (4K) and enol
(4E) tautomers (Scheme 1). The deprotection of 2,4-dimethoxypyrimidine derivative bearing C-6
fluorophenylalkyl side chain (5) with sodium iodide and trimethylchlorosilane gave pyrimidin-2,4-
dione derivative 6. Using the same synthetic procedure compound 7 yielded its unprotected pyrimidin-
2,4-dione derivatives with unsaturated (8) and saturated (9) fluorophenylalkyl side chain. Reaction of
7 with acetyl chloride and water gave deprotected pyrimidin-2,4-dione 9 and the 2,4-
dimethoxypyrimidine derivative 10 with unsaturated side chain as a product of dehydration reaction.
The reason for production of unsaturated derivatives 8 and 10 is probably in enhanced stability of

Molecules 2009, 14
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those compounds due to conjugated π-system of a pyrimidine and phenyl ring via side chain double
bond, which is not present in compound 6 with the longer C-6 side chain.
The Stille reaction [15–17] was applied for the introduction of the phenyl and furyl rings at C-5 of
pyrimidine moiety in 11 and 12, respectively, using tributylphenylstannane and tributylfurylstannane
with dichlorobis(triphenylphosphine)palladium(II) as a catalyst. C-6 alkylation of 5-furylpyrimidine
derivative 12 did not give desired fluorophenylalkylated 5-furylpyrimidine product. Nucleophilic
substitution and addition reactions of lithiated 5-phenylpyrimidine derivative 11 with corresponding
electrophile as reagent afforded pyrimidine derivatives containing p-fluorophenyl-2-hydroxyethenyl
(13), o-fluorobenzyl-2-hydroxypropyl (14) and p-fluorophenyl-2-hydroxypropyl (15) side chain
(Scheme 2).
Scheme 2. Synthesis of 5-phenylpyrimidine derivatives with C-6 fluorophenylalkyl side chain (13-15).
OCH3
N
ii
OH
H3CO
N
13
F
OCH3
OCH3
N
Br
R5
OCH3
N
i
N
N
iii
N
OH
CH3
H3CO
N
CH3
H3CO
CH3
H3CO
3
11
12
F
14
R5
O
OCH3
N
iv
N
OH
H3C
H3CO
F
15
Reagents and conditions: (i) tributylphenylstannane/tributylfurylstannane, (PPh3)2PdCl2, THF; (ii)
LDA, THF, ethyl 4-fluorobenzoate; (iii) LDA, THF, 2-fluorophenylacetone; (iv) LDA, THF,
4-fluoroacetophenone.
Antitumoral activities
Compounds 4, 6, 8-10 and 12-15 were evaluated for their activities against human malignant tumor
cell lines: Molt-4 (acute lymphoblastic leukemia), colon carcinoma (HCT 116 and SW 620), breast
carcinoma (MCF-7) and lung carcinoma (H 460) (Table 1, Figure 1).
The results point to a strong impact of methyl, phenyl, or furyl C-5 substituents. For example,
5-unsubstituted pyrimidine derivatives bearing the same C-6 fluoroalkyl side chain as their structurally
related 5-substituted pyrimidine derivatives did not exhibit any cytostatic effect, as shown in our
previous paper [10]. In addition, pyrimidine derivative with fluorophenylalkylated side chain 4
exhibited moderate cytotoxic activity against all malignant tumor cells tested, particularly against
human breast carcinoma (MCF-7, IC50 = 2 µM). Furthermore, replacing of 5-methyl group (in 4 and 7)
with 5-phenyl moiety (in 13 and 15) improves the antiproliferative activity by an order of magnitude.
Moreover, comparing the activities of 2,4-dimethoxy-6-methyl-5-phenylpyrimidine (11) [10] and

Molecules 2009, 14
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5-furyl-2,4-dimethoxy-6-methylpyrimidine (12) presented here (IC50 = 2 – 10 μM), it is obvious that
replacing a phenyl with a furyl moiety strongly potentiates the antiproliferative activity. However,
introduction of C-6 side chain in 5-phenylpyrimidine derivative 11 significantly increased the
cytostatic effects of compounds 13-15. Conformationally restricted compounds 8 (IC50 ≈ 69 µM) and
10 (IC50 ≈ 47 µM) containing C-6 unsaturated side chain showed higher antiproliferative effect than
their analogues with saturated side chain 7 (IC50 > 100 µM) [10] and 9 (IC50 > 100 µM). Deprotection
of 2,4-diketo functionalities of 5 and 7 caused a loss of activity for 6 and 9.
In conclusion, from C-6 fluorophenylalkyl substituted 2,4-dimethoxy-5-phenylpyrimidines (13-15),
compounds 13 (IC50 ≈ 2.5 µM) and 15 (IC50 ≈ 1.6 µM) containing a p-fluorophenyl moiety in the side
chain showed the most pronounced activity. The effect of the 5-furylpyrimidine 12 and 15, as the best
representatives of the 5-phenylpyrimidine derivatives, were additionally tested on the cell cycle phases
perturbation.
Table 1. Inhibitory effects of compounds 4, 6, 8-10 and 12-15 on the growth of human
tumor cell lines.
IC50a (μM)
Compd
Molt-4
43 ± 41
> 100
HCT 116
41 ± 10
> 100
SW 620
32 ± 19
> 100
MCF-7
2 ± 1
H 460
19 ± 5
> 100
≥ 100
> 100
75 ± 3
4 ± 3
3 ± 0.1
17 ± 2
1 ± 0.4
4
6
> 100
51 ± 18
> 100
42 ± 1
10 ± 6
3 ± 1
8
9
78 ± 21
> 100
79 ± 21
≥ 100
35 ± 6
> 100
10
12
13
14
15
50 ± 45
6 ± 4
37 ± 0.03
3 ± 0.3
2 ± 0.2
14 ± 0.1
1 ± 0.5
32 ± 10
2 ± 1.6
2 ± 0.2
17 ± 0.1
2 ± 0.4
N.T.b
N.T. b
2 ± 0.03
14 ± 3
2 ± 1
a
IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumor cell
proliferation by 50%; b not tested.
Figure 1. Dose-response profiles for compounds 12 and 15. PG = percentage of growth.
Cell cycle perturbations
Compounds 12 and 15 were tested at various concentrations close or slightly higher than their IC50
(1, 5 and 10 μM) on HCT 116 cells (Figure 2). Both compounds showed similar influence on the cell
cycle, whereby strong accumulation of cells in G1 cell cycle phase accompanied with drastic reduction

Molecules 2009, 14
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of cells in S phase was detected. These effects were strongly dose-dependent. Moreover, compound 15
dose-dependently increased the percentage of cells in sub G1 after 48 hours of treatment, pointing to
the induction of apoptosis. In addition, both compounds at 10 μM concentration induced cell death of
more than 30% of cells after 24 hours, while all cells were dead after 48 hours (not shown).
These results unambiguously point to the effects of here presented compounds on the cell cycle
phenomena that occur probably during G1 phase and inhibit DNA synthesis, i.e. inhibit the
progression of cell to next cell cycle phase. Being unable to progress through S phase to mitosis
(M phase), the cells die, most probably by apoptosis.
Figure 2. Cell cycle analysis of HCT 116 cells treated with 1, 5 and 10 μM compounds 12
and 15, 24 h (A and C, respectively), or 48 h (B and D, respectively). The histograms show
percentages of live cells in G0/G1, S or G2/M phase, along with the number of dead
(subG1) cells, where subG1 population is expressed as a percentage of total number of
measured cells/counts.
A
B
C
D
Structural and conformational studies by NMR
1H-, 19F- and 13C-NMR data given in Table 2 and in the Experimental section are in full agreement
1
with the structures of 4E, 4K, 6, 8-10, 13 and 14. The observed chemical shifts of H- and
13C- resonances are characteristic of a C-6 substituted pyrimidine moiety.

Molecules 2009, 14
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Table 2. 1H- and 19F-NMR chemical shifts (δ/ppm), signal multiplicities and H-H coupling
constants (J/Hz).
C2'-
OH
Compd
4Ea
N1-H
N3-H
H5
–
H1'
C2'-Me
C5-Me
-OCH3
phenyl
19F
15.08
(s)
7.97 (m, Hφ2/Hφ6)
7.29 (m, Hφ3/Hφ5)
–
–
–
–
6.29 (s)
4.48 (s)
–
–
2.09 (s) 3.95 (s)
–110.31 (m)
–105.21 (m)
3.75 (s), 8.10 (m, Hφ2/Hφ6)
3.91 (s) 7.37 (m, Hφ3/Hφ5)
7.14 (m, Hφ3)
4Ka
–
–
1.97 (s)
10.92
(s)
10.33
(s)
5.37
(d)
4.73
(s)
7.27 (m, Hφ4)
6b
2.46 (d)
6.39 (m)
1.02 (s)
–
–
–115.83 (m)
7.14 (m, Hφ5)
7.33 (m, Hφ6)
11.06
(s)
10.59
(s)
7.64 (m, Hφ2/Hφ6)
8c
9d
–
–
–
–
2.02 (d)
1.52 (s)
2.43 (d)
1.70 (d)
1.50 (s)
2.06 (s)
–
–113.36 (m)
–
7.24 (m, Hφ3/Hφ5)
10.93
(s)
10.33
(s)
2.73 (d)
2.82 (d)
5.52
(b)
7.51 (m, Hφ2/Hφ6)
–
7.13 (m, Hφ3/Hφ5)
3.89 (s), 7.66 (m, Hφ2/Hφ6)
3.92 (s) 7.24 (m, Hφ3/Hφ5)
10e
–
–
6.78 (q)
–
–113.79 (m)
7.40 (m, Hφ2/Hφ6)
3.79 (s)
11.67
(s)
13
–
–
–
–
6.93 (s)
–
–
–
7.33 (m, Hφ3/Hφ5)
3.90 (s)
–
–
7.22 (m, 5Hφ)
7.17 (m, Hφ3)
7.31 (m, Hφ4)
3.80 (s)
5.19
(s)
14f
–
–
2.61 (s)
0.95 (s)
7.19 (m, Hφ5)
3.92 (s)
7.38 (m, Hφ6)
7.02 (m, 5Hφ)
a The 4K : 4E ratio is ca. 55 : 45 as observed in 1H and 19F NMR spectra; b 4JH1’-H5 = 1.5, multiplet
for H3': 2.74 (d), 2.79 (d) with 2JH gem = 13.4 Hz; c 4JH1'-C2'Me = 1.2 and 5JH1'-C5Me = 0.6 Hz; d 2JH1' gem
= 13.9 Hz; e 4JH1'-C2'Me = 1.2 Hz; f Multiplet for H3': 2.79 (d), 2.68 (d) with 2JH gem = 13.7 Hz.
The conformational properties of 4E, 8 and 10 and the configuration along C1'=C2' double bond
were assessed with the use of 1D difference NOE experiments. The saturation of H1' (δ 6.29) in 4E
resulted in strong NOE enhancements at C5-Me and φ2/φ6-protons which confirmed the
Z-configuration along C1'=C2' double bond (Figure 3a). In addition, the saturation of C5-Me protons
(δ 2.09) resulted in NOE at H1'. The observed NOE enhancements are in agreement with a
conformation in which C2'-OH group is predisposed for the formation of the N1⋅⋅⋅HO hydrogen bond
1
as shown in Figure 3a. In fact, strongly deshielded H-NMR signal at δ 15.08 ppm corresponding to
hydroxyl group supports its involvement in hydrogen bond (Table 2).

Molecules 2009, 14
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Figure 3. The key NOE enhancements and configuration along C1'=C2' double bond for a)
4E, b) 8 and c) 10 in DMSO-d6 solution.
a)
OCH3
15.6%
CH3
N
HN
N
4.3%
H
28.1%
H3CO
N
H
O
4E
F
b)
O
3.2%
CH3
1.5%
H
13.2%
O
N
H
1.0%
H3C
F
8
c)
OCH3
10.9%
CH3
4.9%
H
15.4%
H3CO
N
H3C
F
10
The saturation of H1' (δ 6.39) in 8 showed strong NOE enhancements at φ2/φ6-protons of
fluorinated phenyl ring and none at C2'-Me which is in agreement with E-configuration along C1'=C2'
double bond. On the other hand, the saturation of H1' resulted in a weak NOE enhancement at C5-Me
group (Figure 3b). The saturation of C5-Me protons (δ 1.70) showed weak NOE enhancements at both
H1' and C2'-Me group which suggested conformational freedom along C6-C1' single bond (Figure 3b).
The saturation of H1' (δ 6.78) in 10 resulted in strong NOE enhancements at C5-Me and φ2/φ6-
protons of fluorinated phenyl ring (Figure 3c). The absence of NOEs with C2'-Me protons confirmed
E-configuration along C1'=C2' double bond as shown in Figure 1c. Furthermore, the saturation of

Molecules 2009, 14
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C5-Me protons (δ 2.06) showed NOE enhancement at H1'. These results are in agreement with the
predominant conformation in which H1’ is spatially closer to the C5-Me group.
Experimental
General
Melting points (uncorrected) were determined on a Kofler micro hot-stage (Reichert, Wien).
Precoated Merck silica gel 60F-254 plates were used for thin layer chromatography (TLC) and the
spots were detected under UV light (254 nm). Column chromatography (CLC) was performed using
silica gel (0.063-0.2 mm) Fluka; glass column was slurry-packed under gravity. The electron impact
mass spectra were recorded with an EXTREL FT MS 2001 instrument with ionizing energy 70 eV.
Elemental analyses were performed in the Central Analytic Service, Ruđer Bošković Institute, Zagreb.
13
19
1H-, C- and F-NMR spectra were acquired on a Varian Unity Inova 300 MHz NMR spectrometer.
All data were recorded in DMSO-d6 at 298 K. Chemical shifts were referenced to the residual solvent
signal of DMSO at δ 2.50 ppm for 1H and δ 39.50 ppm for 13C, whereas fluorine chemical shifts were
referenced relative to external CCl3F (δ 0.0 ppm). Individual resonances were assigned on the basis of
their chemical shifts, signal intensities, multiplicity of resonances, H-H coupling constants involved as
well as with the use of a series of 2D NMR experiments (gHSQC and gHMBC).
Procedures for the preparation of compounds
2,4-Dimethoxy-6-methylpyrimidine (1), 2,4-dimethoxy-5,6-dimethylpyrimidine (2), 2,4-di-
methoxy-6-methyl-5-bromopyrimidine (3), 6-[2-(2’-fluorobenzyl)-2-hydroxypropyl]-2,4-dimethoxy-
pyrimidine (5), 6-[2-(4’-fluorophenyl)-2-hydroxypropyl]-2,4-dimethoxy-5-methyl-pyrimidine (7), 2,4-
dimethoxy-6-methyl-5-phenylpyrimidine (11), 5-furyl-2,4-dimethoxy-6-methylpyrimidine (12) and 6-
[2-(4’-fluorophenyl)-2-hydroxypropyl]-5-phenyl-2,4-dimethoxypyrimidine (15) were synthesized in
accord with original procedures given in the literature [10-12,18].
6-[2-(4’-Fluorophenyl)-2-hydroxyethenyl]-2,4-dimethoxy-5-methylpyrimidine (4)
The solution of 2,4-dimethoxy-5,6-dimethylpyrimidine (2) (0.66 g, 3.94 mmol) in anhydrous THF
(10 mL) was cooled at −70 °C and lithium diisopropylamide (LDA, 2.91 mL, 2 M in
THF/heptane/ethylbenzene) was added dropwise to the reaction mixture. The temperature was then
raised to −55 °C and the reaction mixture was stirred for 30 min. Ethyl 4-fluorobenzoate (0.69 mL,
4.73 mmol) was added and the mixture was additionally stirred for 3 h and then neutralized with
glacial acetic acid. The temperature was raised to room temperature and the reaction mixture was
stirred for an additional 15 min. The solvent was evaporated and the residual yellow oily product was
extracted with CH2Cl2 and water. The organic layer was dried over Na2SO4 and purified by column
chromatography using petroleum ether-ethyl acetate = 7:1 as eluent. After column chromatography,
compound 4 was isolated as a mixture of 4E and 4K. Yield = 0.28 g (24.7%); mp = 85-88 °C.
13
1
5
4E: C-NMR (DMSO-d6) δ: 168.84 (C4), 163.24 (Cφ4, d, JCF = 247.8), 163.13 (C2', d, JCF = 0.6),
4
3
162.39 (C6), 160.28 (C2), 131.49 (Cφ1, d, JCF = 3.0), 128.84 (Cφ2 and Cφ6, d, JCF = 8.5), 115.46

Molecules 2009, 14
4875
2
6
(Cφ3 and Cφ5, d, JCF = 21.7), 102.74 (C5), 90.66 (C1', d, JCF = 1.4), 54.38 (C2-OCH3), 54.08 (C4-
OCH3), 8.98 (C5-CH3).
13
1
4K: C-NMR (DMSO-d6) δ: 194.57 (C2'), 169.09 (C4), 165.11 (Cφ4, d, JCF = 251.6), 163.19 (C6),
162.14 (C2), 133.12 (Cφ1, d, 4JCF = 3.3), 131.39 (Cφ2 and Cφ6, d, 3JCF = 9.6), 115.73 (Cφ3 and Cφ5, d,
2JCF = 22.0), 109.08 (C5), 54.01 (C2-OCH3), 53.95 (C4-OCH3), 45.00 (C1'), 9.75 (C5-CH3); MS m/z
291 (MH)+; Anal. calcd. for C15H15FN2O3: C, 62.06; H, 5.21; N, 9.65. Found: C, 61.94; H, 5.22; N,
9.69.
6-[2-(2’-Fluorobenzyl)-2-hydroxypropyl]pyrimidin-2,4-dione (6)
A mixture of compound 5 (0.08 g, 0.27 mmol), chlorotrimethylsilane (0.12 mL, 0.95 mmol) and
NaI (0.14 g, 0.95 mmol) in anhydrous acetonitrile (6 mL) was stirred at room temperature for 18 h and
then at 60 °C for 3 h under argon atmosphere. The solvent was evaporated under reduced pressure, and
the residue was chromatographed using dichloromethane-methanol = 25:1 as eluent to give 6.
Yield = 50 mg (66.4%); mp = 157-159 °C. 13C-NMR (DMSO-d6) δ: 163.97 (C4), 160.91 (Cφ2, d, 1JCF
3
3
= 243.4), 152.95 (C6), 151.32 (C2), 133.38 (Cφ6, d, JCF = 5.0), 128.32 (Cφ4, d, JCF = 8.2), 124.25
(Cφ1, d, 2JCF = 15.9), 123.78 (Cφ5, d, 4JCF = 3.3), 114.88 (Cφ3, d, 2JCF = 23.1), 100.81 (C5), 71.52 (C2',
4
5
3
5
d, JCF = 1.1), 43.66 (C1', d, JCF = 0.6), 40.86 (C3', d, JCF = 5.5), 25.13 (C2'-CH3, d, JCF = 1.4); MS
m/z 277 (M-H); Anal. calcd. for C14H15FN2O3: C, 60.42; H, 5.43; N, 10.07. Found: C, 60.61; H, 5.41;
N, 10.11.
6-[2-(4’-Fluorophenyl)-1-propenyl]-5-methylpyrimidin-2,4-dione (8) and 6-[2-(4’-fluorophenyl)-2-
hydroxypropyl]-5-methylpyrimidin-2,4-dione (9)
The deprotection of 7 was performed in the same manner as described for 6. Reagents used were:
compound 7 (0.26 g, 0.84 mmol), chlorotrimethylsilane (0.37 mL, 0.84 mmol) and NaI (0.44 g,
2.94 mmol) in anhydrous acetonitrile (7 mL). After column chromatography using dichloromethane-
methanol = 40:1 as eluent crystalline compounds 8 and 9 were isolated. 8: Yield = 50 mg (24.4%);
mp = 140-142 °C; 9: Yield = 60 mg (27.3%); mp = 192–194 °C.
13
1
8: C-NMR (DMSO-d6) δ: 164.89 (C4), 162.14 (Cφ4, d, JCF = 245.0), 150.79 (C2), 145.92 (C6),
4
3
141.77 (C2'), 137.08 (Cφ1, d, JCF = 3.3), 128.09 (Cφ2 and Cφ6, d, JCF = 8.2), 118.05 (C1', d,
6JCF = 1.1), 115.28 (Cφ3 and Cφ5, d, 2JCF = 21.4), 105.55 (C5), 17.79 (C2'-CH3), 10.85 (C5-CH3); MS
m/z 261 [MH]+; Anal. calcd. for C14H13F2N2O2: C, 64.61; H, 5.03; N, 10.76. Found: C, 64.48; H, 5.04;
N, 10.78.
13
1
9: C-NMR (DMSO-d6) δ: 165.34 (C4), 161.42 (Cφ4, d, JCF = 242.4), 150.84 (C2), 147.54 (C6),
4
3
144.63 (Cφ1, d, JCF = 2.6), 127.42 (Cφ2 and Cφ6, d, JCF = 8.0), 114.84 (Cφ3 and Cφ5, d,
2JCF = 21.0), 107.22 (C5), 74.14 (C2'), 43.97 (C1'), 29.46 (C2’-CH3), 10.64 (C5-CH3); MS m/z 277
[M-H]; Anal. calcd. for C14H15FN2O3: C, 60.42; H, 5.43; N, 10.07. Found: C, 60.30; H, 5.45; N, 10.09.

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6-[2-(4’-Fluorophenyl)-2-hydroxypropyl]-5-methylpyrimidin-2,4-dione (9) and 6-[2-(4’-fluorophenyl)-
1-propenyl]-2,4-dimethoxy-5-methylpyrimidine (10)
Compound 7 (0.16 g, 0.52 mmol) was dissolved in acetyl chloride (7 mL) and the reaction mixture
was refluxed for 5 h. Water (2 mL) was then added and the reaction mixture was stirred overnight at
room temperature. The solvent was evaporated at the reduced pressure and the remaining yellow oil
was chromatographed using dichloromethane as eluent, which yielded compounds 9 and 10.
9: Yield = 60 mg (44.3%); 10: Yield = 20 mg (9.8%); mp = 66–68 °C.
13
1
10: C-NMR (DMSO-d6) δ: 169.52 (C4), 162.01 (Cφ4, d, JCF = 245.0), 162.0 (C6), 162.0 (C2),
4
3
143.03 (C2'), 139.22 (Cφ1, d, JCF = 3.0), 128.20 (Cφ2 and Cφ6, d, JCF = 8.2), 122.11 (C1', d,
6JCF = 1.7), 115.24 (Cφ3 and Cφ5, d, JCF = 21.4), 108.42 (C5), 54.10 (C2-OCH3), 53.92 (C4-OCH3),
2
17.85 (C2'-CH3), 9.89 (C5-CH3); MS m/z 289 [MH]+; Anal. calcd. for C16H17FN2O2: C, 66.65; H, 5.94;
N, 9.72. Found: C, 66.92; H, 5.93; N, 9.74.
6-[2-(4’-Fluorophenyl)-2-hydroxy-1-ethenyl]-2,4-dimethoxy-5-phenylpyrimidine (13)
The synthesis of 13 was performed in the same manner as described for 4. Reagents used were:
compound 11 (138 mg, 0.59 mmol), LDA (0.58 mL, 2 M in THF/heptane/ethylbenzene) and ethyl
4-fluorobenzoate (0.09 mL, 0.58 mmol) in anhydrous THF (6 mL). After column chromatography
using cyclohexane-ethyl acetate = 6:1 as eluent compound 13 was isolated as crude oil. Yield = 44.3
mg (16.6%). 13C-NMR (DMSO-d6) δ: 167.99 (C4), 163.55 (Cφ4, d, 1JCF = 233.5), 163.03 (C2'), 162.35
4
3
(C6), 160.52 (C2), 134.00 (Cφ1, d, JCF = 2.7), 132.88 (Cφ1'), 131.08 (Cφ2 and Cφ6, d, JCF = 8.8),
2
130.79 (Cφ2'), 129.12 (Cφ3'), 128.00 (Cφ4'), 127.87 (Cφ3 and Cφ5, d, JCF = 18.3), 122.33 (C1'),
114.08 (C5), 54.79 (C2-OCH3), 54.55 (C4-OCH3); MS m/z 353 [MH]+; Anal. calcd. for C20H17FN2O3:
C, 68.17; H, 4.86; N, 7.95. Found: C, 68.37; H, 4.84; N, 7.97.
6-[2-(2’-Fluorobenzyl)-2-hydroxypropyl]-2,4-dimethoxy-5-phenylpyrimidine (14)
According to the procedure described for 4, the synthesis of 14 was performed using compound 11
(95 mg, 0.41 mmol), LDA (0.40 mL, 2 M in THF/heptane/ethylbenzene) and 2-fluorophenylacetone
(0.09 mL, 0.41 mmol) in anhydrous THF (6 mL) as reagents. After column chromatography using
petroleum ether-ethyl acetate = 6:1 as eluent compound 14 was isolated. Yield = 40.3 mg (25.7%).
13C-NMR (DMSO-d6) δ: 169.19 (C4), 161.95 (Cφ2, d, 1JCF = 245.7), 166.56 (C6), 163.02 (C2), 133.91
(Cφ6, d, 3JCF = 4.7), 133.54 (Cφ1’), 131.25 (Cφ2’), 130.90 (Cφ4, d, 3JCF = 7.2), 129.58 (Cφ3’), 125.16
2
4
2
(Cφ4’), 116.24 (Cφ1, d, JCF = 14.0), 123.77 (Cφ5, d, JCF = 3.3), 115.00 (Cφ3, d, JCF = 22.7), 116.25
3
(C5), 72.91 (C2'), 44.28 (C1'), 54.89 (C2-OCH3), 54.51 (C4-OCH3). 40.85 (C3', d, JCF = 5.6), 27.14
(C2'-CH3); MS m/z 383 [MH]+; Anal. calcd. for C22H23FN2O3: C, 69.09; H, 6.06; N, 7.33. Found: C,
69.29; H, 6.04; N, 7.31.
Antitumor activity assays
The colon carcinoma (HCT 116 and SW 620), breast carcinoma (MCF-7) and lung carcinoma (H
460) cells were cultured as monolayers and maintained in Dulbecco's modified Eagle's medium

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(DMEM), while MOLT-4 cells (acute lymphoblastic leukemia) were cultured in suspension in RPMI
medium, both supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL
penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C.
The growth inhibition activity was assessed as described previously, according to the slightly
modified procedure of the National Cancer Institute, Developmental Therapeutics Program [9,10].
Briefly, the cells were inoculated onto standard 96-well microtiter plates on day 0. Test agents were
then added in five consecutive 10-fold dilutions (10-8 to 10-4 mol/L) and incubated for further 72 hours.
Working dilutions were freshly prepared on the day of testing. The solvent (DMSO) was also tested
for eventual inhibitory activity by adjusting its concentration to be the same as in working
concentrations (maximal concentration of DMSO was 0.25%). After 72 hours of incubation, the cell
growth rate was evaluated by performing the MTT assay [10] which detects dehydrogenase activity in
viable cells. The absorbency (OD, optical density) was measured on a microplate reader at 570 nm.
Each test point was performed in quadruplicate in three individual experiments. The results are
expressed as IC50, which is the concentration necessary for 50% of inhibition. The IC50 values for each
compound are calculated from dose-response curves using linear regression analysis by fitting the test
concentrations that give PG (percentage of growth) values above and below the reference value (i.e.
50%). Each result is a mean value from three separate experiments.
Cell cycle analysis
2 × 105 cells were seeded per well in a 6-well plate. After overnight incubation, tested compounds
were added. After desired length of time, the attached cells were trypsinized, combined with floating
cells, washed with phosphate buffered saline (PBS) and fixed with 70% ethanol. Immediately before
the analysis, cells were washed with PBS and incubated with 0.1 μg/μL RNAse A at 37 °C for
15 minutes. Subsequently, cells were stained with 50 µg/ml of propidium iodide (PI) and analyzed by
Becton Dickinson FACScalibur flow cytometer. For each analysis, 20,000 events were measured.
Measurements were performed in duplicate for two independent experiments. The percentage of the
cells in each cell cycle phase was based on the obtained DNA histograms and determined using the
ModFit LTTM software (Varity House). Statistical analysis was performed in Microsoft Excel by using
the ANOVA at p < 0.05.
Conclusions
C-6 fluorophenylalkyl (6, 9, 14 and 15), fluoropenylakenyl (4, 8, 10 and 13) and 5-furyl-6-methyl
(12) pyrimidine derivatives were synthesized and evaluated for their cytostatic activities. Among all
tested compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives showed
the most significant cytostatic activities against all tumor cell lines (IC50 are in low micromolar range).
In the series of C-6 fluorophenylalkyl 5-methylpyrimidines, compounds 8 and 10 containing
unsaturated side chains showed better inhibitory effects (8: IC50 ≈ 69 µM, 10: IC50 ≈ 47 µM) than their
saturated pyrimidine derivatives 7 and 9. Furthermore, among C-6 fluorophenylalkyl 5-phenyl-
pyrimidines, compounds 13 and 15 with a p-fluorophenyl moiety in the side chain exhibited the most
pronounced antiproliferative activity (13: IC50 ≈ 2.5 µM, 15: IC50 ≈ 1.6 µM). Besides, 12 and 15
induced strong changes in the cell cycle of tumor cells (accumulation of cells in G1 phase and drastic

Molecules 2009, 14
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decrease in the number of cells in S phase), which eventually caused death of tumor cells. Thus, these
compounds emerged as the most interesting leading compounds that could be used for further
structural optimization.
Acknowledgments
Support for this study was provided by the Ministry of Science of the Republic of Croatia (Projects
#125-0982464-2925 and # 098-0982464-2514).
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Sample Availability: Samples of compounds 1-15 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).