Synthesis, Structural Studies and Antitumoral Evaluation of C-6 Alkyl and Alkenyl Side Chain Pyrimidine Derivatives

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.


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 hydrogenbond 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.

Cell cycle perturbations
Compounds 12 and 15 were tested at various concentrations close or slightly higher than their IC 50 (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 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.  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 as shown in Figure 3a. In fact, strongly deshielded 1 H-NMR signal at δ 15.08 ppm corresponding to hydroxyl group supports its involvement in hydrogen bond (Table 2).  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/φ6protons 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 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.

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. 1 H-, 13 C-and 19 F-NMR spectra were acquired on a Varian Unity Inova 300 MHz NMR spectrometer.
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 IC 50 , which is the concentration necessary for 50% of inhibition. The IC 50 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 × 10 5 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 LT TM 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 (IC 50 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: IC 50 ≈ 69 µM, 10: IC 50 ≈ 47 µM) than their saturated pyrimidine derivatives 7 and 9. Furthermore, among C-6 fluorophenylalkyl 5-phenylpyrimidines, compounds 13 and 15 with a p-fluorophenyl moiety in the side chain exhibited the most pronounced antiproliferative activity (13: IC 50 ≈ 2.5 µM, 15: IC 50 ≈ 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 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.