Synthesis of N,O-homonucleosides with high conformational freedom

The 1,3-dipolar cycloaddition of vinyloxymethyl thymine with different nitrones has been exploited for the preparation of N,O -homonucleosides where the oxymethylene tether replaces the aminal linkage between the sugar moiety and the nucleobase


Introduction
During the past two decades, great strides have been made in the design of modified nucleoside drugs for the treatment of viral infections despite the stigma of toxicity and the development of drug resistance. 1,2In particular, isoxazolidine homonucleosides 3, synthesized by cycloaddition reaction between nitrones and allyl nucleobases in a diastereoselective or enantioselective way, have emerged as an important class of carbanucleoside analogues. 3,4The introduction of a carbon bridge between the nucleobase and the ribose unit or the isoxazolidine mimic leads to an increased resistance to hydrolytic or enzymatic cleavage and a more conformational flexibility and rotational freedom with respect to the natural nucleosides. 5Moreover, nucleoside mimics, characterized by higher flexibility, seem to be beneficial for the interaction with receptors; recently, it has been demonstrated that the binding sites of many enzymes are more flexible than previously thought and, as a direct consequence, more flexible inhibitors could show better features. 6ligonucleotides constructed with modified nucleosides containing a methylene or ethylene tether between the sugar moiety and the nucleobase, (compound 1 and 2 in Fig. 1), allow a lowering of the electrostatic repulsion while maintaining the ability to build Watson-Crick base pairs with unnatural DNA or RNA strain, due to a better alignment of complementary nucleobases. 7The creation of these artificial analogues is justified by their biological significance and by the pure scientific exploration eventually directed toward biomedical applications. 8ontinuing our interest in the synthesis of isoxazolidine nucleosides with high conformational freedom, we reports in this paper the first member of a new series of N,O nucleosides in which the oxymethylene tether replaces the aminal linkage between the isoxazolidine moiety and the thymine.Our target compounds were prepared by exploiting the reactivity of dipolarophile 9 in the 1,3-dipolar cycloaddition of three different nitrones: the reaction afforded to the direct construction of α-and β-oxymethylene N,O-nucleosides 4.

Results and Discussion
The key substrate 9 was synthesized by modifying the procedure described for the synthesis of purine derivatives (Scheme 1). 9 Thus, the commercially available diphenyl selenide was treated with sodium borohydride to give the corresponding not isolable selenol, which in situ was reacted with ethylencarbonate to give compound 6. 10 The treatment of 6 with paraformaldehyde and the subsequent bubbling with HCl furnishes in a quantitative yield the intermediate 7. 9 The coupling of 7 with silylated thymine, followed by treatment with sodium periodate and sodium bicarbonate, afforded the dipolarophile 9 with a yield of 70%.A study of the optimal conditions for the 1,3-dipolar cycloaddition of compound 9 was carried out by employing nitrones 10-12.(Scheme 2, Table  The reaction of nitrone 10 in THF at reflux, under traditional heating, proceeded slowly and with a low yield (entry 1).Switching to microwave irradiation at 100W for 1h at 80°C, an acceleration of the reaction time together with an increased yield was observed (entry 2).The same diastereomeric mixture of adducts 13a and 14a in a 1:3 ratio was obtained.The crude mixture was purified by MPLC on a Fluorosil column using as eluent a mixture of CHCl 3 /MeOH (99:1) and the structures of the cycloadducts have been assigned on the basis of 1 H NMR data and confirmed by NOE experiments.
Thus, the main product 14a shows the resonance of H 3' as a triplet at 3.48 ppm; the upfield proton H 4a' appears as a doublet of doublet at 2.62, while the downfield proton H 4b' appears as a doublet of doublets of doublets at 2.85 ppm.The H 5' proton resonates as a doublet at 5.55 ppm; the exocyclic methylene protons give rise to two doublets centered at 5.10 and 5.50 ppm.The irradiation of H 5' proton produced a significant enhancement for the resonances of H 4'b and the exocyclic methylene protons; conversely, when H 4'b was irradiated, a positive NOE effect was observed only for H 5' and H 4'a .Moreover, the irradiation of H 4'a induced a positive NOE effect for H 4'b and H 3' .These data support a cis relationship between H 3' and H 4'a protons and the substituent present at C-5 and allow to assign to compound 14a a trans configuration.According to the results reported for similar cycloaddition 11 reactions of nitrone 10, (E/Z ratio = 4:1), the stereochemical outcome of this cycloaddition reaction may be explained by considering that the major cycloadduct 14a could be formed by the E nitrone reacting in an exo mode.
As previously reported, 4 Z nitrone 11 reacts with allyl nucleobases in an exo mode leading to a cis adduct as main compound.Thus, in order to obtain the β-anomer 13b, the reaction between the nitrone 11 and the vinyloxymethyl thymine 9 was investigated (entry 3 and 4).Anyway, a lower reactivity together with a much longer reaction time, with respect to the reaction of nitrone 10, was observed.The 1 H NMR spectrum of the main product 13b shows the resonance of the H 3' proton centered at 2.85 ppm; the methylene protons at C-4 resonate at 2.01 (H 4'a ) and 2.45 ppm (H 4'b ), while H 5' proton resonates as a doublet centered at 5.30 ppm.The structural determination with the aid of NOE experiment was performed: a diagnostic NOE effect for proton H 5' was observed after irradiation of H 3' .Moreover, the irradiation of the upfield proton H 4'a induces a positive NOE effect for H 4'b , the methylene protons of the hydroxymethyl group and the vinyl proton of the thymine moiety.These data clearly indicated a cis configuration between the hydroxymethyl group at C-3 and the substituent at C-5 (Fig. 2) Recently, we have reported that isoxazolidine nucleosides bearing a phosphonate moiety at C-3' were able to completely inhibit the HTLV-1 infections. 12In this context we have tried to synthesize phosphonated N,O-homonucleosides 13c and 14c, starting from the phosphonated nitrone 12.However, in the cycloaddition reaction of 12 with compound 9 no significant amounts of cycloadducts were detected in the crude reaction mixture (entry 5) under traditional heating.Switching to microwave irradiation (entry 6), only a small amount of cycloadducts 13c and 14c is detectable in the 1 HNMR spectrum of the crude reaction mixture.Unfortunately, it was not possible to increase the yields by varying the reaction time, the microwave potency and the temperature, probably for the simultaneous acceleration of decomposition processes of nitrone 12. Thus, compound 4 could represent the alternative substrate for the preparation of compounds 13c and 14c by using the previously reported procedure. 13

Conclusions
We have explored the 1,3-dipolar cycloadditions of dipolarophile 9 with three different nitrones under conventional heating and microwave irradiation.The investigated routes allowed us to obtain α-and β-N,O-homonucleosides with an high conformational freedom.The procedure is general and may be extended to other nucleobases.Biological evaluations of the synthesized nucleosides are actually in progress.

Experimental Section
General Procedure.NMR spectra were measured on a 500 MHz Varian Unity Inova instrument in CDCl 3 as solvent.Chemicals shifts are in ppm (δ) from TMS as internal standard.NOE difference spectra were obtained subtracting alternatively right-off-resonance free induction decays (FIDS) from right-on-resonance induced FIDS.IR spectra were recorde using an FTIR-8300 (Shimadzu) spectrophotometer.MS Spectra were measured with a JEOL JMS-D 300 spectrometer.Elemental analyses were performed on a Perkin-Elmer 240B microanalyzer.The microwave reactions were carried out with a Discover Focused Microwave System (CEM Corporation).

Preparation of N,O-nucleosides 4α and 4β from 14a and 13a
To a solution of 13a or 14a (1 mmol) in dioxane/water mixture (10mL), NaBH 4 (10mmol) was added and the mixture was stirred for 6h at r.t.At the end of this time, the solvent was removed and the residue was subjected to purification by column chromatography on neutral alumina (CHCl 3 /MeOH 95:5).