Enantioselective Synthesis of Homo-N-Nucleosides Containing a 1,4-Dioxane Sugar Analog

A dioxane homo-sugar analog, (2S,5S)-and (2R,5S)-5-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-iodomethyl-1,4-dioxane was prepared from (2R,3R)-dimethyl tartrate, and further elaborated into the corresponding homo-N-nucleoside analogs by its reactions with uracil and adenine, respectively.


Introduction
There has been an increasing interest in the synthesis of nucleoside analogs with modifications of the sugar moiety for the purpose of obtaining new antiviral and antitumor agents [1][2][3]. A well known class of modified nucleosides are the homo-N-and C-glycosidic nucleosides [4][5][6][7][8] The insertion of a methylene group between the heterocyclic base and the sugar moiety results in a more flexible structure, and due to the lack of an anomeric acetal position, these nucleosides are in general resistant to enzymatic degradation [8]. For these reasons, we decided to pursue the synthesis of nucleoside analogs, however, based on the conformationally more flexible, optically active homo-1,4-dioxane sugar analogs. The objective was thus to construct novel homo-N-nucleoside analogs containing a 1,4-

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dioxane sugar moiety. A representative structure is shown in Figure 1, where the 1,4-dioxane homosugar analog is substituted with uracil or adenine, respectively.

Results and Discussion
The formation of the 1,4-dioxane homo-sugar analog 4 was readily achieved starting from (2R,3R)dimethyl tartrate, an inexpensive and commercial available chiral starting material. Thus, (2R,3R)dimethyl tartrate was converted into the corresponding enantiomerically pure allyl ether 1 [9] either by the reaction with allyl bromide in the presence of silver oxide [10] or in a tin assisted reaction with dibutyltin oxide [11][12]. The dimethyl (2R,3R)-2-O-allyltartrate 1 was then reduced by LiAlH 4 [13][14][15] or NaBH 4 [16][17] to give triol 2. The two vicinal hydroxyl groups in 2 were next protected through formation of acetal 3 by the reaction with 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid (Scheme 1). The use of the tartrates as chiral starting materials conveniently allows for the synthesis of all the possible, optically active stereoisomers of 3 and subsequently the corresponding homo-sugar analogs. Scheme 1. Synthesis of the partially protected (2S,3S)-2(allyloxy)butane-1,3,4-triol, 3. Structures 4a and 4b were elucidated and verified by 1 H-, 13 C-, and DEPT-NMR experiments in combination with 2D NMR spectroscopy techniques (COSY, HSQC, HMBC, NOESY). The assigned structures were in full agreement with the NMR data. Product 4a was assigned the trans-configuration as the coupling constants J AC and J AB were measured to 10.2Hz and 2.4Hz, respectively. This was in agreement with the -CH 2 I group being in an equatorial position. The corresponding coupling values for the other isomer were 3.6Hz and 3.3Hz, respectively, being in agreement with the structure of the cis-isomer 4b.
The uracil homo-N-nucleoside analog 5a was obtained by reacting uracil with sodium hydride in DMF [22], followed by the reaction with trans-iodide 4a. A byproduct containing two 1,4-dioxane rings was also obtained and assigned the structure 6a. This byproduct was not easily separated from product 5a. The acetal functions in compounds 5a and 6a were then removed using Amberlyst 15 in methanol providing a mixture of homo-N-nucleoside analog 7a and dimer 8a, Scheme 3. Compounds 7a and 8a were now readily separated by flash chromatography. The cis-nucleoside analogs 7b and 8b were obtained from 4b by the same sequence of reactions. NMR spectra of the crude reaction mixtures gave indication of an additional byproduct, which as a working hypothesis was assumed to be the corresponding 3-regioisomer 10. To confirm the identity of the two regioisomers 5 and 10, uracil was first selectively protected as the 3-N position by benzoylation with benzoyl chloride in pyridine to provide pure N-3-benzoyluracil 9, [23]. Compound 9 was next reacted with iodide 4b in DMF to give product 5b (Scheme 4). The NMR spectral data of 5b prepared by the two different routes were in good agreement. Interestingly, the N-3 alkylation compound 10 was also observed in the product from the protected uracil 9. A 6:5 ratio of products 5b and 10 was observed. The results imply that a benzoyl-walk reaction probably took place under the reaction conditions. The pure 10 was isolated from the mixture of 5b and 10 by preparative TLC and its structure was confirmed by NMR spectroscopy. The detailed nature of these transformations was not further investigated. Using anhydrous potassium carbonate [24][25] as the base, the transiodide 4a and cisiodide 4b respectively were reacted with adenine to give compound 11a and 11b in 42% and 30% isolated yields, respectively, after flash chromatography. Deprotection of acetals 11a and 11b in the presence of Amberlyst-15 in methanol gave compound 12a and 12b in 85% and 70% yields (Scheme 5). Different from natural occurring purine nucleosides, 12a and 12b were stable under acidic conditions. The depurination reaction was avoided due to the presence of the methylene group between adenine and 1,4-dioxane homo-sugar analog moiety.
The structures of the four adenine nucleoside analogs 11a, 11b, 12a, 12b were verified to be the N-9 adenine regioisomers by HMBC-NMR spectroscopy technique. In the case of 11a, three bond correlations, between C4 and H A and between C8 and H A , were observed, while three bond correlation between C5 and H A was not found ( Figure 2). These findings were in agreement with e.g. product 11a be the N-9 adenine regioisomer.

Conclusions
In conclusion, optically active homo-N-sugar nucleoside analogs containing a 1,4-dioxane moiety as the sugar analog and substituted with uracil or adenine as the base were synthesized from dimethyl tartrate. These nucleoside analogs were stable under acidic conditions. Plans for the biological screening of the produced nucleoside analogs are currently in progress.

Experimental
General NMR spectra were recorded on Bruker Avance DPX 300 or DPX 400 instruments. Chemical shifts are reported in ppm using TMS as the internal standard in CDCl 3 or relative to 2.50 ppm for 1 H and 39.99 ppm for 13 C in DMSO-d 6 or 3.31 ppm for 1 H and 49.15 ppm for 13 C in CD 3 OD. Structural assignments were based on 1 H, 13 C, DEPT135 and 2D spetra, COSY, HSQC, HMBC, NOESY. EI-Mass and ESI spectra were recorded on a Finnigan MAT 95XL spectrometer. IR spectra were obtained on a Thermo Nicolet FT-IR Nexus spectrometer using a Smart Endurance reflection cell. Silica gel Kieselgel 60G (Merck) was used for Flash Chromatography. The solvents were purified by standard methods. The preparations of compounds 1 were described elsewhere [9][10][11][12].
(2S,3S)-3-(allyloxy)butane-1,2,4-triol, (2) This product was obtained by reduction of 1 with either LiAlH 4 or NaBH 4 . LiAlH 4 reduction: To a suspension of LiAlH 4 (3.72 g, 95 %, 93 mmol) in dry diethyl ether (50 mL) was drop wise added a solution of 1 (4.36 g, 20 mmol) in 4 mL of diethyl ether at 0-5 °C. The reaction mixture was refluxed for 18 hours and then cooled in an ice bath. Then 5 mL of water was added and the mixture stirred for 20 minutes, followed by addition of a 15 % NaOH solution (12 mL) and then 10 mL of water. The resulting mixture was stirred and the granular salt formed, was separated by filtration, washed with hot THF (200 mL), and the filtrate concentrated under reduced pressure. The residue was purified by flash chromatography (CHCl 3 /CH 3 OH, 9:1 mixture) to give 0.83 g, 26 % of the pure product 2. NaBH 4 reduction. Sodium borohydride (3.45 g, 93 mmol) in ethanol (50mL) was stirred for half an hour and then dropwise added a solution of 1 (4.35 g, 20 mmol) in ethanol (15 mL). The resulting solution was refluxed gently for 5 hours. The solution was cooled in an ice bath and added 10 mL of acetic acid. The mixture was stirred for 20 minutes and filtered. The solid was washed with 2x50 ml ethanol. The combined organic phase was concentrated under reduced pressure. The crude product was purified by flash chromatography using a 19:1 mixture of CH 2 Cl 2 / MeOH as the eluent yielding 2.88 g, 88 % of the pure product, which exhibited the following spectroscopic properties: 1 A solution of 2 (6.20 g, 38 mmol), 2,2-dimethoxylpropane (4.00 g, 38.5 mmol) and p-TsOH (223 mg, 1.2 mmol) in 100 mL acetone was stirred overnight at room temperature. The solvent was then removed and the residue was purified by flash chromatography using a 3:2 mixture of Et 2 O / n-hexane as the eluent to provide product 3 as acolorless oil (5.11 g, 85 %). Unreacted starting material 2 (1.05 g crude product) was recovered by continued elusion with a 19:1 mixture of CH 2 Cl 2 / MeOH. Product 3 exhibited the following spectroscopic properties: 1  To a solution of 3 (3.20 g, 15.8 mmol) in dry acetonitrile (50 mL) was added NaHCO 3 (4.19 g, 49.9 mmol) at -15°C. The mixture was stirred for 10 minutes and iodine (12.10 g, 47.7 mmol) was added. The reaction mixture was stirred for 68 hours with exclusion of light at -15 to -0°C. Ethyl acetate (80 mL) was added to the mixture and the solution was neutralized by saturated sodium thiosulfate solution until a colorless solution was obtained. The aqueous phase was extracted with ethyl acetate and the combined organic phase was dried over anhydrous sodium sulfate. The solution was filtered and evaporated. The residue was purified by gradient column chromatography using Et 2 O/n-hexane (1:4, 1:1) as eluent. The two diastereomers were separated in yields of 26.4% (4a) and 25.4% (4b).
The pure compounds were white solid. R f was 0.43 and 0.36 respectively (n-hexane/Et 2 O 1:1). The product 4a exhibited the following spectroscopic properties: 1

5a (2R,5S)-5-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-(uracil-1-yl-methyl)-1,4-dioxane (5b)
Method 1: The preparation of 5b was the same as used for the synthesis of 5a. Method 2: Sodium hydride (18 mg, 0.75 mmol) in 10 mL of dry DMF was stirred for half an hour at room temperature. N-3-benzoyluracil 9 (130 mg, 0.60 mmol) was added and stirred for an hour. To this suspension, iodide 4b (98 mg, 0.30 mmol) was added. The resulting stirred mixture was heated at 90°C overnight. The mixture was concentrated under reduced pressure to remove DMF. To the residue was added 20 mL methanol and the resulting mixture was stirred for 5 minutes. The solution was concentrated and purified by flash chromatography using CH 2 Cl 2 /CH 3 OH as the eluent. The obtained product (30 mg, 32 %) containing products 5b and 10 in a 5:6 ratio and was further purified by preparative TLC. The isolated product 5b exhibited the following spectroscopic properties: 1   The mixture of 5a and 6a (60mg, 5/6 ratio) was dissolved in methanol (10 mL), Amberlyst 15 (50 mg) was added and the mixture refluxed. The reaction was monitored by TLC until no more 7a was observed. The solution was filtered and the solvent was evaporated. The obtained product was further purified by flash chromatography to give pure 5a (12 mg, 34 %) and 8a (25 mg, 38 %) using CH 2 Cl 2 /MeOH (13:1) as eluent. The product 7a exhibited the following spectroscopic properties: 1   The product 8a exhibited the following spectroscopic properties: 1 The method for preparation of 7b was as same as applied for the synthesis of 7a. The product 7b exhibited the following spectroscopic properties: 1