From sugars to modified nucleosides

Two strategies are reported for the diastereoselective synthesis of isoxazolidinyl nucleosides, as potential antiviral agents — a one-step approach based on 1,3-dipolar cycloaddition of sugar-derived nitrones with vinyl nucleobases derived from uracil and adenine, as well as a two-step methodology based on the Vorbrüggen nucleosidation of the 5-acetoxyisoxazolidines. The 1,3-dipolar cycloadditions of sugar-derived nitrones with vinyl acetate proceed with very good diastereoselectivity to give the diastereoisomeric isoxazolidines. Condensation of the major diastereomerically pure acetoxyisoxazolidines with silylated uracil, thymine, cytosine, N - acetylguanine and purines occurs with moderate to excellent stereoselectivity with the formation of the expected isoxazolidinyl nucleosides. The stereoselectivity of the addition of the silylated nucleobase is dependent on the structure of the substituent at C-3 originating from the starting chiral nitrone and on the attacking nucleobase.


Introduction
Nitrogen-containing heterocycles and their derivatives have broad application in synthetic materials, and biological chemistry and, as a result, their synthesis and reactivity is subject of considerable interest.][3][4][5][6][7][8][9][10] Nucleosides are generally defined as DNA or RNA subunits and consist of both a base moiety such as adenine, thymine, guanine, cytosine and uracil, and a sugar moiety such as D-ribose or 2-deoxy-D-ribose. 11Many nucleoside analogues have been synthesized with modification of the base, sugar, and phosphate moieties.In particular, nucleoside analogues in which the furanose ring has been replaced by different carbon or heterocyclic systems, have attracted special interest by virtue of their biological action as antiviral and/or anti-cancer agents. 12Among these, the uracil-, thymine-, cytosine-, and adenine-nucleosides 1 possessing an isoxazolidinyl moiety (carbocyclic-2´-oxo-3´-azanucleosides) are emerging as an interesting class of dideoxynucleoside analogues with potential pharmacological activity. 12For the synthesis of modified isoxazolidinyl nucleosides 1 two strategies can be used -in particular, a one-step approach based on the 1,3-dipolar cycloaddition of nitrones with vinyl nucleobases, and a twostep methodology based on the Vorbrüggen nucleosidation of 5-acetoxyisoxazolidines. [13][14][15][16][17][18][19][20] Figure 1

Sugar-derived Nitrones
Glycosides are important as enzyme inhibitors, and as chiral synthons suitable for the synthesis of many natural products.Since the 1,3-dipolar cycloaddition has a nearly singular capability of establishing large numbers of stereogenic centers in one synthetic step in recent years attention has been focused on the preparation of chiral sugar-derived nitrones.The configuration of the newly generated stereogenic centers would be determined by the nitrone.Asymmetric induction in 1,3-dipolar cycloaddition has been achieved efficiently by using nitrones with chiral groups at either the nitrogen atom or the carbon atom. 4Among nitrones, the sugar-derived nitrones represent versatile substrates as they provide a polyhydroxylated carbon framework with multiple avenues of chirality. 92][23][24][25][26][27] In connection with our synthetic studies on the utility of chiral nitrones for the synthesis of biologically interesting compounds, we have prepared the sugarderived nitrones 2-8 possessing structures suitable for building nitrogen-containing heterocycles (Scheme 1).
The aim of the present account is to describe the diastereoselective synthesis of isoxazolidinyl nucleosides by means of a 1,3-dipolar cycloaddition of chiral sugar-derived nitrones as the key step.The use of C-chiral nitrones allows diastereoselective synthetic access to homochiral isoxazolidinyl nucleosides.The enantioselective synthesis of isoxazolidinyl nucleosides by using chiral N-glycosyl nitrones is not included in this account. 15,20,28

From D-glycero nitrone and vinylthymine
0][31] This approach should be a highly promising reaction for the straightforward synthesis of a variety of isoxazolidinyl nucleosides.The first chiral version of that reaction has been investigated by Merino. 16The cycloaddition of the nitrone 9, which is readily available from 2,3-O-isopropylidene-D-glyceraldehyde, with vinylthymine (10) in refluxing toluene proceeded smoothly to give a 73:18:9 mixture of three adducts 11-13 in 88% yield (Scheme 3).Deprotection of the separated anomers was achieved by using catalytic p-TsOH in MeOH.The resulting 1,2-diols were treated sequentially with sodium periodate and sodium borohydride to give the hydroxymethyl-substituted isoxazolidinyl nucleosides 14-16 in good yields.

From D-xylo nitrones and vinyluracil and vinyladenine
We have prepared from D-xylose the new sugar-derived nitrones 2a and 2b possessing structures suitable for the building of pyrrolidines. 32Their cycloadditions to vinylated nucleobases 17, derived from uracil and adenine, proceeded regioselectively and led to the isoxazolidines 18-20 as a mixture of four diastereoisomers in all cases (Scheme 4).In the case of cycloadditions of nitrones 2a and 2b with 9-vinyladenine 17b an inseparable mixture of the adenosine diastereoisomers has been obtained.To improve the separation the free amino group of the adenine moiety was protected.The cycloaddition of nitrone 2a with N,N-dimesylated 9vinyladenine 17b proceeded with better selectivity, in favor of the major anti-cis isomer 20a. 32RKAT USA, Inc.

Scheme 3 Scheme 4
X-ray analysis of the major diastereoisomer 18a prepared from uracil reveals a C-3/C-1 ' antiand C-3/C-5 cis-configuration, and therefore indicates that the cycloaddition arises from the more sterically accessible si face of the Z-nitrone 2a, via an exo-transition state for the anti-cis diastereoisomer 18a and via an endo transition state for the anti-trans 19a isomer (Fig. 2).

From D-lyxo nitrone and vinyladenine
The 1,3-dipolar cycloaddition of the readily available chiral sugar-derived D-lyxosyl nitrone 3, with N,N-dibenzyl-9-vinyladenine has been also investigated in our group. 33Nitrone 3 reacted with N,N-dibenzyl-9-vinyladenine to give a mixture of all possible 5-substituted isoxazolidines 22-25 in the ratio of 39:27:19:15 in 98% yield, the anti-cis isomer 22 being the major product (Scheme 5).Two of them, with a C-3/C-5 trans configuration of the isoxazolidine moiety, are αanomers and the other two possess a C-3/C-5 cis configuration of the isoxazolidine moiety are βanomers.Whereas the regioselectivity of the reaction was very high, the corresponding 4substituted regioisomers were not detected, and both the cis/trans selectivity (cis/trans ratio 58:42) and the diastereofacial selectivity (anti/syn ratio 66:34) were rather low.

Acetoxyisoxazolidines prepared from Sugar-derived Nitrones
Since we found that the cycloadditions of chiral sugar-derived nitrones to vinylated nucleobases derived from uracil and adenine led to the isoxazolidines as a mixture of four diastereoisomers in all cases, for the synthesis of modified isoxazolidinyl nucleosides we have chosen a two-step methodology based on the Vorbrüggen nucleosidation of the 5-acetoxyisoxazolidines.Therefore, the cycloaddition of the chiral nitrones 2-8 with vinyl acetate was investigated.The reaction of the nitrone 9 with vinyl acetate has been described by Merino et al. 16 It is known that 1,3-dipolar cycloadditions of electron-rich alkenes to chiral α-alkoxy nitrones give preferentially antiadducts as a result of dipolarophile attack from the less sterically hindered si diastereotopic face of the nitrone.). 34In contrast to the cycloadditions of these nitrones with vinylated nucleobases, the anti-trans isoxazolidines 27b and 28b, are respectively the major products, (entries 1,2, Table 1).The attack of the dipolarophile on the Z configuration of the nitrones 2a-c proceeded preferentially through an endo transition state (Fig. 2, R 2 = OAc).The activation energy of the possible Z/E-isomerization for the sugar-derived nitrones is too high and therefore the nitrones reacted without any isomerization. 21,224-O-Benzyl-substituted D-xylo nitrone 2c reacted smoothly in vinyl acetate at reflux over 24 h to give an 83:10:7 mixture of the diastereoisomeric isoxazolidines 29a, 29b and 29d in 85% yield (entry 3, Table 1). 35The cycloaddition proceeded with very good diastereoselectivity for the anti-trans isoxazolidine 29b and is completely regioselective, with only the sterically favored 5-substituted isoxazolidines being detected.ARKAT USA, Inc.
On the other hand, cycloaddition of nitrone 3 with vinyl acetate afforded a mixture of four diastereoisomers 30a-d in 86% yield in favor of the major isomer 30a possessing the anti-cis configuration (Scheme 8, entry 4, Table 1). 33

Scheme 8
The D-erythro nitrone 4, prepared starting from D-glucose, reacted smoothly in refluxing vinyl acetate over 20 h to give a 56:28:16 mixture of diastereoisomeric isoxazolidines 31a, 31b and 31d in 78% yield (Scheme 9). 36The cycloaddition proceeded with very good anti-facial (84:16) and endo-facial (72:28) diastereoselectivity (entry 5, Table 1).It is noteworthy that in this case the reaction -probably for steric reasons -proceeded with reversed diastereoselectivity as expected for an inverse-demand cycloaddition reaction where the corresponding 3,5-cis-adducts are the major products. 9,16The structure of the major diastereomer as anti-trans-31b was established unambiguously by X-ray diffraction studies. 36n the other hand, the reaction of the D-threo nitrone 5, prepared starting from D-galactose, with vinyl acetate proceeded diastereoselectively and gave only two diastereoisomers 32a and 32b in a 84:16 ratio, with anti-cis 32a being predominant -although four diastereoisomers are possible -showing now a preference for exo-attack, as expected for an inverse-demand cycloaddition reaction (Scheme 3). 9,16Moreover, in this case an excellent anti-diastereofacial induction was observed; the corresponding syn-adducts have not been detected in the reaction mixture (entry 5, Table 1).The structure of the minor trans-anti isoxazolidine 32b was established unambiguously by X-ray diffraction studies. 36Thus, the observed excellent facial diastereoselectivity for the D-galactose-derived nitrone 5 is in contrast with the afore-mentioned results obtained by the nitrone cycloadditions to vinyl acetate.

Isoxazolidinyl nucleosides derived from D-glyceraldehyde
Merino and his colleagues have described the transformation of a mixture of anti-adducts 26a/26b into isoxazolidinyl-nucleoside analogues as depicted in Scheme 11. 16 The condensation of a mixture of 26a and 26b with silylated thymine, using the glycosylation methodology developed by Vorbrüggen, 38,39 resulted in a nucleoside product consisting of cis-36b and trans-37b isomers (75:25) which were isolated in 80% combined yield.

Scheme 11
Zhao and his coworkers have obtained cis-36b and trans-37b isomers in a 94:6 ratio in 83% combined yield. 40Condensation of acetate 26a with silylated uracil and N-benzoylcytosine in acetonitrile at room temperature, using TMSOTf as Lewis acid afforded the β-anomers 36a,c with excellent stereoselectivity (95:5).The acetoxy-isoxazolidine 26a was prepared in this case by Michael addition of N-methyl hydroxylamine to the α,β-unsaturated ester derived from 2,3-O-isopropylidene-D-glyceraldehyde, followed by reduction with DIBAL and subsequent acetylation.

Scheme 12
A surprising and unexpected fact is that, in the case of the anti-trans-5-acetoxy-substituted isoxazolidine 28b, prepared by the 1,3-dipolar cycloaddition of nitrone 2b with vinyl acetate, the Vorbrüggen nucleosidations with silylated nucleobases proceeded at 65 °C with excellent stereoselectivity.The corresponding β-anomers 40a,b,d are now the exclusive nucleosides for all of the used nucleobases (Scheme 13). 352][43] The attack on the oxonium intermediate from either the α-or β-side is possible, and hence the product distribution is sensitive to structural changes of the reactants.In our case, the stereoselectivity of the addition of the silylated nucleobase is evidently more dependent on the structure of the substituent at C-3 originating from the starting chiral nitrone.Moreover, in this case the product of elimination, the corresponding isoxazoline derivative 42, was also isolated (Scheme 13).It is noteworthy that in the case of silylated 5-fluorouracil, the isoxazoline 42 is formed as the major product (40a/42 ratio 58:42, 40b/42 ratio 81:19, 40d/42 ratio 90:10, 40f/42 ratio 9:91).To the best of our knowledge, such a formation of 2,3-dihydro-isoxazoles by the Vorbrüggen nucleosidation has not been observed.2,3-Dihydro-isoxazoles represent a class of heterocycles that may be employed as useful building blocks for synthesis. 44,45
As has already been mentioned, the best stereoselectivity in the nucleosidation was achieved in the case of benzoyl substituted isoxazolidine 28b.To exclude or to support the possibility of the participation of the neighboring benzoyl group, we have prepared the corresponding Dxylose-derived benzyl-substituted nitrone 2c.The Vorbrüggen nucleosidation of anti-trans benzyloxy-substituted isoxazolidine 29b with silylated uracil, thymine, N-acetylcytosine and guanine at RT in methylene chloride in the presence of trimethylsilyl triflate as catalyst, afforded the β-anomeric nucleosides 43 with the best diastereoselectivities in this series.Uracil and acetylated cytosine reacted with 90% diastereomeric excess (43a/44a ratio 95:5, 43b/44b ratio 80:20, 43d/44d ratio 94:6, 43e/44e ratio 63:37, Scheme 14).

Isoxazolidinyl nucleosides derived from L-threose
Gratifyingly, the nucleosidation of the anti-cis isoxazolidine 34a, derived from L-threose with silylated uracil, -thymine or N-acetylcytosine in the presence of trimethylsilyl triflate as catalyst, afforded only the β-anomeric nucleosides 54a-c as single products in good yields (62-65%).The corresponding α-anomeric nucleosides 55a-c have not been detected in the reaction mixture (Scheme 19). 37This is the best observed diastereoselectivity for the nucleosidation of 5-acetoxysubstituted isoxazolidines achieved from sugar-derived nitrones.

Conclusions
From two strategies for the diastereoselective synthesis of isoxazolidinyl nucleosides a two-step methodology based on the Vorbrüggen nucleosidation of the 5-acetoxyisoxazolidines proceeded more selectively than a one-step approach based on the 1,3-dipolar cycloaddition of C-glycosyl nitrones with vinyl nucleobases.We have made the potentially useful observation that the anomeric distribution of the formed nucleosides may be dependent not just on the nature of the attacking nucleobase but also on the 3-substituent of the isoxazolidine.This observation might be exploited in future to allow the generation of anomerically pure nucleosides via the Vorbrüggen method.