Synthesis and biological properties of 2-oxabicyclo[4.1.0]heptane nucleosides containing uracil, and thymine

α - and β -2-Oxabicyclo[4.1.0]heptane nucleosides 8 - 13 containing uracil and thymine have been synthesized starting from L - threo -hex-4-enopyranosides 5 . The nucleosidation reaction performed with the O -acetyl derivative 7 affords only the β -anomer 8 while the nucleosidation of the O -methyl derivative 7 leads to the formation of α - and β -anomers. Antiviral, cytotoxicity and apoptotic activity have been investigated: no significant activity has been observed.


Introduction
There has recently been a renewed interest in the synthesis of nucleosides with a six-membered carbohydrate moiety (Figure 1), due to their potential antiviral [1][2][3][4] and antibiotic 5 activities and as building blocks in nucleic acid synthesis. 6Antiviral properties have been found in 1,5anhydrohexitols 1 which exhibit a marked and selective activity against HSV-1, HSV-2, VZV, and CMV at relatively low concentrations.This activity is, probably, linked to a conformational flexibility comparable to that of natural nucleosides.The biological activity of nucleoside analogues is often modulated by the nature of the spacer (the furanose ring in natural compounds) which exerts a profound effect on the conformation and puckering of the molecule in natural nucleosides and which is able to control the outcome of the interactions between these compounds and the cellular kinases.
The correlation between the conformational preference required by the enzyme and the particular sugar conformation proves troublesome due to the flexibility of the furanose ring.Such flexibility is responsible for significant differences between the conformation in solid state and in solution. 7For this reason, conformationally restricted nucleoside analogues were synthesized with the aim to reduce their flexibility, so improving the interaction between substrates and enzymes.For instance, conformationally locked oxa-or carbocyclic nucleosides, built on a bicyclo[3.1.0]hexanestemplate, have demonstrated conformational preferences for a number of enzymes. 8Furthermore, the biological effects of modified nucleosides depend importantly from the relative distance and arrangement of the hydroxymethyl group and the base moiety.
In this context, we have devised the synthesis of a series of new nucleosides featuring the presence of a pyranose ring fused with a cyclopropane system, in order to construct a spacer unit which could control the conformational mobility while maintaining the base unit and the hydroxymethyl group in a disposition suitable to interact with the enzymes.In fact AM1 calculations performed on the synthesized 2-oxabicyclo[4.1.0]heptanenucleoside 13b and on the 1,5-β-pyranose nucleoside 2 (Figure 2) chosen as references, indicate that the introduction of the cyclopropane ring at C4 and C5, into 13b with respect to 2, induces a puckering of the system which leads to a reduced conformational freedom and a lower distance between the base moiety and the hydroxymethyl group.

13b
Figure 2. AM1 optimized structures for compounds 2 and 13b According to these considerations, and following our interest in the chemical valorization of lactose, 9 a natural disaccharide easily and cheaply available in large amounts as a waste product of the cheese industry, we report here the synthesis and the biological properties of the 2oxabicyclo[4.1.0]heptanenucleosides 8-13, not yet reported in the literature.

Results and Discussion
Recently we have described the cyclopropanation reactions 10 of L-threo-hex-4-enopyranosides 5, according to a Furukawa-modified 11a Simmons-Smith procedure, 11b as a valuable tool to obtain 2-oxabicyclo[4.1.0]heptanederivatives 6, with a nearly quantitative yield (Scheme 1).These derivatives have then been utilized as the starting material for the preparation of nucleosides 8-13.

Scheme 1
The cyclopropane derivative 6 was converted in the more reactive peracetylated compound 7 (Scheme 1) and used for the subsequent nucleosidation reaction with silylated uracil, under Vorbrüggen conditions, 12 with TMSOTf in dry acetonitrile (Scheme 2).The reaction afforded exclusively the nucleoside 8 with good yield (80% isolated) and high stereoselectivity.The stereochemistry of 8 was assessed on the basis of mono-and two-dimensional 1 H-NMR experiments, and NOE effects.In particular, the diagnostic NOE enhancement observed for H-1 (4.4%) when irradiating H-3 proton and vice versa, clearly indicates a trans relationship between the nucleobase and the substituent at C-6.Moreover, the H-1 proton resonates as a doublet (5.88 ppm) with a coupling constant J 1,2 = 10.0 Hz, typical of a trans ax/ax disposition between H-1 and H-2 protons.
The deprotection of 8 with Et 3 N in methanol afforded the modified nucleoside 9 with a good yield (85%) (Scheme 2).

Scheme 2
The exclusive production of 8 is explainable in terms of the reversible formation of the σcomplex A which undergoes the attack of the persilylated base on the same side of the cyclopropane ring (Figure 3).With the aim to obtain the modified nucleoside featuring a cis-disposition between the base and the hydroxymethyl group at C-6, and in order to compare the biological activities of cis-and trans-derivatives, we have performed the nucleosidation reaction of the more hindered cyclopropanated dibenzyl-methyl ether 6 10 with silylated uracil and thymine (Scheme 3).A mixture of the two α− and β− anomers 10a,b and 11a,b in a 3:1 ratio (40% total yield) was obtained: the pure compounds were isolated by flash and HPLC chromatography.

Scheme 3
The stereochemistry was confirmed by mono-and two-dimensional 1 H-NMR experiments, and NOE measurements.In the case of the α-nucleosides (10a,b), the anomeric proton appears as a doublet (5.66 ppm) with a coupling constant J 1,2 = 9.5 Hz, typical of a trans ax / ax disposition between H-1 and H-2 protons; on the contrary in β-nucleosides (11a,b), H-1 appears as a doublet (5.93 ppm) with a small coupling constant J ax / eq (2.5 Hz).Moreover, for 10a,b diagnostic NOE enhancements were observed for H-1 when irradiating H-3 (2.5%).No NOE effect was observed in β-anomers 11a,b between H-1 and H-3 protons (Figure 4).

Biological assays ISSN 1424-6376
The synthesized compounds were tested for their antiviral activity against HSV-1, HSV-2 in Vero (African Green Monkey) cells, HTLV-1 in human cells (lymphomonocytes of peripheral blood) and for their toxicity in vitro.None of these derivatives reached the inhibitory concentration 50 at the highest concentrations tested (e.g.320 lM), indicating a lack of significant antiviral activity.
The apoptotic activity was also tested in Molt-3 cells.Only 13b showed a moderate activity (32%) at the concentration 500 µM after 48 h.No significant toxicity, when evaluated using the classical trypan blue test, was detected until 3 days of treatment in lymphoid Molt-3 cell line assayed.
Only 13b exhibits a moderate apoptotic activity (28%) at the concentration 500 µM after 48 h.

Antiviral and cytotoxicity assay for HSV
The newly synthesized nucleosides were evaluated for their activity against HSV-1 and HSV-2 by plaque reduction assay in VERO cells using a methodology reported in the literature. 13ytotoxicity assays were conducted in rapidly dividing Vero cells, as reported. 13

Evaluation of toxicity and apoptosis
Toxicity was evaluated by a standard viability assay, using the trypan blue exclusion test.Normally, apoptosis was evaluated by morphological analysis of the cells, performed following staining with acridine orange as previously described. 14Briefly, over 600 cells, including those showing typical apoptotic characteristics, were counted using a fluorescence microscope.The identification of apoptotic cells was based on the presence of uniformly stained nuclei showing chromatin condensation and nuclear fragmentation.In some experiments, apoptosis was detected by flow cytometric analysis of isolated nuclei, following staining with propidium iodide, on a Becton Dickinson FAC Scan Analytic Flow Cytometer, as previously described. 15