key: cord-0861551-8p0rp175
authors: Piotrowska, Dorota G.; Balzarini, Jan; Andrei, Graciela; Schols, Dominique; Snoeck, Robert; Wróblewski, Andrzej E.; Gotkowska, Joanna
title: Novel isoxazolidine analogues of homonucleosides and homonucleotides
date: 2016-12-15
journal: Tetrahedron
DOI: 10.1016/j.tet.2016.10.073
sha: 5d5a8cbd97e16906a58f62f9ab5a01c044ab2af5
doc_id: 861551
cord_uid: 8p0rp175

Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived nitrones 20a-20e (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing 1,3-dipolar cycloadditions with allyl alcohol as well as with alkenylphosphonates (allyl-, allyloxymethyl- and vinyloxymethyl- and vinylphosphonate). Besides reactions with vinylphosphonate the additions proceeded regioselectively to produce mixtures of major cis and minor trans 3,5-disubstituted isoxazolidines (d.e. 28–82%). From vinylphosphonate up to 10% of 3,4-disubstituted isoxazolidines was additionally produced. Vicinal couplings, shielding effects and 2D NOE correlations were employed in configurational assignments as well as in conformational analysis to find out preferred conformations for several isoxazolidines and to observe anomeric effects (pseudoaxial orientation of phosphonylmethoxy groups) for those obtained from vinyloxymethylphosphonate. None of the tested compounds were endowed in vitro with antiviral activity against a variety of DNA and RNA viruses at subtoxic concentrations (up to 250 μM) nor exhibited antiproliferative activity towards L1210, CEM, and HeLa cells (IC(50) = ≥100 μM).

A significant number of antiviral and anticancer drugs can be classified as close structural analogues of nucleosides or nucleotides. A search for new compounds has resulted in obtaining many active molecules which showed different levels of similarities to natural nucleosides. 1e10 Modifications of a nucleoside scaffold are practically unlimited since not only the sugar and nucleobase units could be altered but also additional linkers within the structure of the nucleoside can be incorporated. A list of commonly used ribofuranoside replacers includes 2 0 ,3 0 -dideoxyfuranose, cyclopentane, cyclopentene, 1,3-dioxolane, 1,3-oxathiolane, isoxazolidine rings and also acyclic entities.

The idea of incorporating the isoxazolidine ring into a nucleoside framework as a sugar replacer, first proposed by Tronchet, 11 has been explored to provide several biologically active compounds (Fig. 1) . A fluorouracil-containing isoxazolidine 1 was found to induce apoptosis on lymphoid and monocytoid cells and at the same time showed low cytotoxicity. 12 Antiviral nucleotides were also discovered among phosphonylated isoxazolidines 2 13 and 3 14 as well as among their analogues having the 1,2,3-triazole linker 4. 15 While nucleotides 2 have been found to be potent inhibitors of the reverse transcriptase of different retroviruses, 13 its truncated analogues 3 appeared even more potent exhibiting the inhibitory activity at concentrations in the nanomolar range. 14 High cytotoxicity toward several cancer cell lines was observed for isoxazolidine nucleosides of the general formula 5. 16 On the other hand, it is worth mentioning that the biological activity of compounds containing the isoxazolidine ring is not restricted to anticancer and antiviral properties, since it was found that they also posses antimicrobial, 17, 18 antifungal, 18e21 anti-inflammatory, 22, 23 antioxidant 24, 25 and insecticide activity, 26 among others.

Structural modifications of nucleosides may also influence stereoelectronic effects and contribute to the anomeric effect and thus control a conformational behavior of the sugar ring and affect the biological properties of nucleosides. This is exemplified by a replacement of the ring oxygen atom by a carbon atom leading to the formation of carbanucleosides. 6, 27, 28 This modification results in a greater metabolic stability of nucleoside analogues lacking the natural N-glycoside bond. A similar increase in stability can be achieved in 1 0 -homonucleosides which are formed by insertion of the methylene group between the nucleobase and the sugar or sugar mimetics as illustrated by 1 0 -homoadenosine 6 29, 30 Moreover, the biological activity of 1 0 -homonucleosides is also influenced by greater conformational flexibility and slightly improved lipophilicity. Among 1 0 -homonucleosides 31, 32 containing fivemembered rings as ribofuranoside mimics several compounds showing pronounced biological activities were identified 7e13 (Fig. 2) . 33e38 In most cases these compounds retain the hydroxymethyl group to allow for their sequential phosphorylation to active triphosphate metabolites.

Furthermore, 1 0 -homonucleotides containing a nonhydrolyzable PeC bond have been also synthesized to obtain analogues which could be phosphorylated to the active form, thereby omitting the first and less effective monophosphorylation step as exemplified by compounds 14e19 (Fig. 3) . 39e45 Recently, we have reported the synthesis of isoxazolidinecontaining analogues of homonucleosides cis-21/trans-22 having a nucleobase (B) at C3 of the isoxazolidine ring. 46 The synthetic approach relied on the application of the 1,3-dipolar cycloaddition of allyl alcohol to the nucleobase-derived nitrones 20. In this paper, a full account of an already communicated preliminary study 46 is given and the reactivity of nitrones 20 with selected alkenylphosphonates 23e26 leading to a new series of nucleotide analogues cis-27/trans-28 to cis-33/trans-34 is described together with the results of their antiviral and cytostatic activities (Scheme 1).

The synthesis of nucleobase-derived nitrones 20 has been recently described. 46 The 1,3-dipolar cycloadditions of the nitrones 20 to allyl alcohol were carried out at 60 C or under MW irradiation (Scheme 2, Table 1 ). The reactions were regiospecific and produced cis/trans mixtures of diastereoisomeric cycloadducts 21 and 22 in moderate to good diastereoselectivities (d.e. 82e28%). The cis/trans ratios of the isoxazolidines were calculated from the 1 H NMR spectra of the reaction mixtures by comparison of integrations of diagnostic resonances of the H 2 C-4 protons in the isoxazolidine ring as well as the signals of the respective protons of nucleobase moieties. The relative configurations in homonucleosides cis-21a and trans-22a have already been established based on 2D NOE experiments. 46 These assignments have been extended on cis-21b and trans-22b, cis-21c and trans-22b, cis-21d and trans-22d as well as cis-21e and trans-22e pairs of diastereoisomers due to almost identical spectral patterns for HC3, H 2 C4 and H5 protons but also for diastereotopic protons in H 2 CeB and H 2 CeOH moieties in the respective 1 H NMR spectra.

In continuation of our studies on the reactivity of the nitrones 20, allylphosphonate 23, allyloxymethylphosphonate 24, vinyloxymethylphosphonate 25 and vinylphosphonate 26 were selected as dipolarophiles to synthesize 1 0 -homonucleotide analogues having non-hydrolyzable PeC bonds separated by none, one, two or three bonds from C5 in the isoxazolidine ring in compounds 27/28, 29/30, 31/32 and 33/34, respectively. The installation of CeOeCeP(O)(OR) 2 fragments in the designed compounds is additionally substantiated by their presence in nucleoside phosphonate drugs like adefovir, tenofovir and cidofovir and several other drug candidates. 47, 48 Heating the nitrone 20a with an excess (3 equiv.) of allylphosphonate 23 at 60e80 C for 24 h did not result in the formation of even traces of the expected products. However, cycloadditions of nitrones 20 with alkenes 23e26 were successfully carried out under microwave irradiation (Scheme 3).

The progress of the reactions was monitored by the 1 H NMR spectroscopy until the disappearance of the starting nitrone. The ratios of diastereoisomeric cycloadducts were calculated from the respective 31 P NMR spectra of the crude reaction mixtures. The 1,3dipolar cycloadditions of the nitrones 20 with alkenylphosphonates 23, 24 and 25 (Scheme 3, Table 2 ) were regiospecific and gave cis/ trans mixtures of diastereoisomeric cycloadducts cis-27/trans-28, cis-29/trans-30 and cis-31/trans-32 with diastereoselectivities (d.e. 78e40%, Table 2 ) comparable to that found for analogous reactions with allyl alcohol (d.e. 82e28%, Table 1 ). In most cases chromatographic removal of the unreacted alkenylphosphonates was difficult and less effective than distilling-off an excess of allyl alcohol, and thus led to lower overall yields. In general, longer reaction times were required to achieve a full conversion of the nitrones 20a with less reactive dipolarophiles such as 23e25 when compared to Table 1 ; b) allyl alcohol MW, 60e85 C, see Table 1. an analogous reaction with allyl alcohol. Moreover, during the reaction of the adenine-derived nitrone 20e with allylphosphonate 23 decomposition of the starting nitrone was observed and the unreacted dipolarophile 23 was recovered almost quantitatively. When the same nitrone 20e was treated with vinyloxymethylphosphonate formation of a complex reaction mixture was noticed from which expected pure isoxazolidine cycloadducts could not be isolated.

On the other hand, traces of 5-fluorouracil were found in crude reaction mixtures when the nitrone 20b was treated with alkenylphosphonates 23e26 under MW irradiation. To verify the stability of this nitrone under conditions of the cycloaddition reaction a solution of 20b in acetonitrile was heated under MW irradiation and the progress of the reaction was monitored by the 1 H NMR spectroscopy. Indeed, the formation of 5-fluorouracil was observed after 7 h (1%) and increased to 6% after an additional 14 h. The amount of 5-fluorouracil reached 15% after 18 h but the solution was contaminated with other unidentified decomposition products (up to 46%). Similarly, slow decomposition of the adenine-derived nitrone 20e during MW irradiation of the solution in acetonitrile was observed. 1 H NMR spectra taken after 12 h revealed decomposition of the nitrone 20e (c.a. 15%), since additional signals appeared in a region characteristic of adenine protons.

The relative configurations in cis-27 and trans-28 as well as in cis-29 and trans-30 can again be deduced taking into account almost identical 1 H NMR spectral patterns when compared to those of cis-21 and trans-22. This could be predicted because the spatial and stereoelectronic influence of the substituents at C3 (CH 2 eBase) and at C5 (CH 2 eOH in 21/22, CH 2 eP in 27/28 and CH 2 eOCH 2 P in 29/30) have an indistinguishable impact on the preferred conformations of the isoxazolidine rings in the cis and trans isomers. Although we were unable to unequivocally establish these conformations in addition to 2D NOE spectral data 22 further support for our configurational assignments comes from the comparison of the chemical shifts of H-C5 protons in the cis and trans diastereoisomers (Fig. 4) . Thus, in the 1 H NMR spectra of all transconfigured isoxazolidines (22, 28, 30) resonances of H-C5 are significantly shifted upfield in comparison to the cis isomers (21, 27, 29) , e.g. 4.12 ppm in 22a vs. 4.40 ppm in 21a, because the H-C5 protons in the trans isomers are positioned in the shielding cone of the heteroaromatic ring. The same phenomenon can be observed for the Hb-C4 protons in both the cis and trans isoxazolidines but the shielding effects are much better pronounced for the cis isomers, e.g. 1.79 ppm for Hb-C4 vs. 2.59 ppm for Ha-C4 in 21a and Tables 2 and 3. 2.10 ppm for Hb-C4 vs. 2.31 ppm for Ha-C4 in 22a. Although 1 H and 13 C NMR spectra of isoxazolidines cis-31 and trans-32 prepared from vinyloxymethylphosphonate resembled each other regardless of a nucleobase present they significantly differed from those of the already discussed cis-21/27/29 and trans- On the other hand, when diethyl vinylphosphonate 26 was applied, in addition to major 3,5-disubstituted isoxazolidines cis-33a-e and trans-34a-e (Scheme 3, Table 3 ), the formation of minute amounts (less than 10%) of regioisomeric 3,4-disubstituted products 35a-e and 36a-e was also noticed. Their presence in the crude products as well as in the fractions obtained after column chromatography was detected by the 31 P NMR spectroscopy (Table 4) and additionally proved by careful analyses of the 1 H NMR spectra where diagnostic signals of nucleobase protons could be assigned to four different cycloadducts, namely cis-33a-e, trans-34a-e, and 35a-e/36a-e.

As observed previously, 1 H NMR spectra of the major (cis-33) and minor (trans-34) derivatives were also similar within the series (a-e). In 2D NOE spectrum of cis-33c interactions between H 2 C-B and HbC4, HaC4 and HC3 as well as HaC4 and HC5 protons were noticed thus proving their locations on the same sides of the iso- 13 C NMR spectra of cis-33c one can conclude that the isoxazolidine ring adopts the E 2 conformation 39 (Fig. 6) . To establish the trans configuration in 34c it is again worth noting the meaningful upfield shift (0.3 ppm) of HC5 proton in this isomer (4.18 ppm) in comparison to cis-33c (4.48 ppm) and NOE correlation peaks between H 2 C-B and HC5 (not detected in cis-33c) as well as between H 2 C-B and HbC4 (medium intensity) and H 2 C-B and HaC4 (weak). Furthermore, the preferred conformation 40 Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virusinduced cytopathicity by 50% (other viruses). None of the tested compounds showed appreciable antiviral activity toward any of the tested DNA and RNA viruses at the concentration up to 250 mM.

The 50% cytostatic concentration (IC 50 ) causing a 50% inhibition of cell proliferation was determined against murine leukemia L1210, human CD 4

þ T-lymphocyte CEM, human cervix carcinoma HeLa and human dermal microvascular endothelial cells (HMEC-1). Among all compounds evaluated, marginal, if any cytostatic activity was observed. Not only compounds containing adenine, uracil, 5bromouracil and thymine substituents as nucleobases were found inactive, but also the analogues bearing a 5-fluorouracil (5-FU) moiety showed no significant antiproliferative activity. These findings indicate that the 5-FU-containing compounds are not efficiently taken-up by the intact tumor cells and/or do not enzymatically release free 5-FU and/or do not inhibit thymidylate synthase, one of the most important target enzymes for 5-fluorodeoxyuridine-5ʹ-monophosphate.

New nucleobase-derived nitrones 20a-e were efficiently applied in the synthesis of isoxazolidine analogues of homonucleosides and homonucleotides which relied on the 1,3-dipolar cycloadditions of 20a-e first to allyl alcohol and next to allyl-, allyloxymethyl-, vinyloxymethyl-and vinylphosphonates. In general cycloadditions were regioselective and led to the formation of cis and trans mixtures of 3,5-disubstituted isoxazolidines with moderate to good diastereoselectivities. However, in cycloadditions to vinylphosphonate in addition to major 3,5-disubstituted isoxazolidines also 3,4-disubstituted isomers were formed (up to 10%).

Relative (cis and trans) configurations of 3,5-disubstituted isoxazolidines were established based on the detailed analysis of 1 H and 13 C NMR spectral data (vicinal couplings, shielding effects and 2D NOE correlations). Several isoxazolidines exist in preferred conformations including those obtained from vinyloxymethylphosphonate in which the phosphonylmethoxy groups are oriented pseudoaxially due to the anomeric effect.

All synthesized compounds were evaluated against a broadspectrum of DNA and RNA viruses but they were found to be inactive at concentrations up to 250 mM. Also, the compounds did not show significant cytostatic activity against murine leukemia L1210, human CD 4

þ T-lymphocyte CEM, human cervix carcinoma HeLa and human dermal microvascular endothelial cells.

Although the tested compounds contained biologically relevant fragments (nucleobases, the isoxazolidine ring and a phosphonate) they surprisingly did not show appreciable antiviral and anticancer activities. Since the isoxazolidine subunit can be also found in several structures endowed with antibacterial and antifungal activities we would progress along this line soon to hopefully discover new therapeutic applications for this class of compounds.

1 H NMR spectra were taken in CDCl 3 , CD 3 OD and D 2 O on the following spectrometers: Varian Gemini 2000BB (200 MHz), Varian Mercury-300 and Bruker Avance III (600 MHz) with TMS as internal standard. 13 The following adsorbents were used: column chromatography, Merck silica gel 60 (70e230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F 254 .

Preparative HPLC experiment was performed on a Waters apparatus equipped with Waters 2545 binary gradient module and Waters 2998 photodiode array detector (190e600 nm).

A mixture of nitrone 20 (1.0 mmol) and allyl alcohol (1.0 mL) was stirred at 60 C or irradiated in a Plazmatronika RM800 microwave reactor at 60e85 C for the time shown in Table 1 . All volatiles were removed in vacuo and the crude product was purified on silica gel column using chloroformeMeOH (10:1, 5:1, v/v) as the eluent to afford pure isoxazolidines 21 and 22. For details see Table 1 .

Yield: 68% (0. 

Inhibition of HIV-1 (NL4.3)-and HIV-2 (ROD)-induced cytopathicity in CD4 þ T-lymphocyte MT-4 cell cultures was determined in microtiter 96-well (200-ml) plates containing~10 6 MT-4 cells/ml and a variety of test compound concentrations. Thirty min after exposure of the MT-4 cells to the test compounds, the cell cultures were infected with HIV-1 (NL4.3) at 3 pg p24/well (or 60 pg/ml). The virus dose affords full cytopathicity after 4e5 days of incubation in the absence of the test compounds (control). Therefore, after 4e5 days incubation at 37 C in a CO 2 -controlled atmosphere, cytopathicity was microscopically recorded. Concomitantly, 100 ml of the supernatants of each of the cell cultures was removed from the wells and 50 ml of a MTS solution was added to the remaining cell suspension. After 2e3 h incubation at 37 C, 50 ml Triton X-100 (0.5%) was added and absorbancy measured using a Soft Max Pro programme.

Cytostatic measurements were based on the inhibition of murine leukemia L1210, human CD 4

þ T-lymphocyte CEM, human cervix carcinoma HeLa and human dermal microvascular endothelial cell proliferation. Cells were seeded at~5 Â 10 3 cells/well into 96-well (200 ml) microtiter plates. Then, medium containing different concentrations of the test compounds was added. After 2e4 days of further incubation at 37 C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC 50 , or the compound concentration required to inhibit cell proliferation by 50% relative to the number of cells in the untreated controls. Alternatively, cytotoxicity of the test compounds in confluent (HEL, Vero, HeLa and CRFK) cell cultures (used for the antiviral assays) was expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that caused a microscopically detectable alteration of cell morphology.

-methyl-3-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)isoxazolidin-5-yl)methoxy)

Yellowish oil; IR (film, cm À1 ) n max : 3488

53 (dd, 1H, J ¼ 12.6 Hz, J ¼ 8.3 Hz, HCHN), 3.33e3.28 (m, 1H, H-C3), 2.68 (s, 3H, CH 3 N), 2.37 (ddd, 1H, J ¼ 11.6 Hz, J ¼ 8.2 Hz, J ¼ 8.2 Hz, H a -C4), 2.10 (ddd, 1H, J ¼ 11.6 Hz

-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)isoxazolidin-5-yl)oxy)methyl) phosphonate (31d) Yield: 27% (0.110 g from 0.98 mmol of the nitrone 20d)

CH(CH 3 ) 2 ), 4.02 (dd, 1H, J ¼ 13.6 Hz, J ¼ 9.9 Hz, HCHP), 3.91 (dAB, 1H, J ¼ 13.8 Hz, J ¼ 8.3 Hz, HCHN), 3.89 (dAB, 1H, J ¼ 13.8 Hz, J ¼ 5.3 Hz, HCHN), 3.68 (dd, 1H, J ¼ 13.6 Hz, J ¼ 8.9 Hz, HCHP), 3.44 (dddd

102.7 (d, J ¼ 12.0 Hz, C5), 71.1 (d, J ¼ 5.5 Hz, 2 Â CH(CH 3 ) 2 ), 63.4 (C3), 61.2 (d, J ¼ 170.7 Hz, CP), 51.6 (CH 2 N)

CDCl 3 ) d: 19.57. Anal. Calcd. for C 17 H 30 N 3 O 7 P: C

-methyl-3-((5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)isoxazolidin-5-yl)oxy)methyl) phosphonate (32d) Yield: 2% (0.008 g from 0.98 mmol of the nitrone 20d)

4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl)-2-methylisoxazolidin-5-yl)phosphonate (33a) Yield: 14% (0.073 g from 1.69 mmol of the nitrone 20a); white amorphous solid; mp 114e115 C; IR (KBr, cm À1 ) n max : 3445

C3), 63.4 (d, J ¼ 6.5 Hz, CH 2 OP), 62.5 (d, J ¼ 7.1 Hz, CH 2 OP)

4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate (34a) Yellowish oil; IR (film, cm À1 ) n max : 3422

Hz, HCHN), 2.83 (dddd, 1H, J ¼ 20.9 Hz, J ¼ 13.0 Hz, J ¼ 10.1 Hz, J ¼ 7.3 Hz, H a -C4), 2.71 (s, 3H, CH 3 N), 2.30 (dddd, 1H, J ¼ 13.0 Hz

CH 3 CH 2 OP); 31 P NMR (80 MHz

-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate (33b) Yield: 33% (0.106 g from 0.89 mmol of the nitrone 20b); white amorphous solid, mp 126e127 C; IR (KBr, cm À1 ) n max : 3403

CDCl 3 ) d: 9.78 (brs, 1H, NH), 7.64 (d, 1H, J ¼ 5.9 Hz), 4.45 (dd, 1H, J ¼ 10.4 Hz, J ¼ 6.4 Hz, H-C5), 4.27e4.17 (m, 4H, CH 2 OP), 3.94e3.88 (M part of ABM system, 1H, HCHN) and 3.65e3.57 (AB part of ABM system, 2H, HCHN and H-C3), 2.79 (dddd, 1H, J ¼ 15.4 Hz, J ¼ 13.4 Hz, J ¼ 10.5 Hz, J ¼ 7.6 Hz, H a -C4)

d, J ¼ 172.4 Hz, C5), 65.2 (d, J ¼ 3.7 Hz, C3), 63.5 (d, J ¼ 6.5 Hz, CH 2 OP), 62.6 (d, J ¼ 6.7 Hz, CH 2 OP

-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-2-methylisoxazolidin-5-yl)

CH 2 OP), 4.01 (dd, 1H, J ¼ 13.8 Hz, J ¼ 3.5 Hz, HCHN), 3.52e3.45 (m, 1H, H-C3), 3.28 (dd, 1H, J ¼ 13.8 Hz

mmol of the nitrone 20c); colorless crystalline solid (crystallized from ethyl acetate/hexane) mp 209e213 C with decomposition; IR (KBr, cm À1 ) n max : 3369

-bromo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-2-methylisoxazolidin-5-yl) phosphonate (34c)

yellowish amorphous solid

72 (s, 3H, CH 3 N), 2.30 (dddd, 1H

CH 2 OP), 62.6 (d, J ¼ 7.0 Hz, CH 2 OP)

CDCl 3 ) d: 22.05. Anal. Calcd. for C 13 H

-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)isoxazolidin-5-yl)phosphonate (33d) Yield: 2% (0.009 g from 1.11 mmol of the nitrone 20d)

27 (q, 1H, J ¼ 1.2 Hz), 4.44 (dd, 1H, J ¼ 10.3 Hz, J ¼ 6.6 Hz, H-C5), 4.29e4.15 (m, 4H, 2 Â CH 2 OP

3 (d, J ¼ 6.4 Hz, CH 2 OP), 62.5 (d, J ¼ 6.7 Hz, CH 2 OP), 51.0 (CH 2 N)

CDCl 3 ) d: 22.73. Anal. Calcd

-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)isoxazolidin-5-yl)phosphonate (34d) Yield: 1% (0.005 g from 1.11 mmol of the nitrone 20d)

H-C3), 3.30 (dd, 1H, J ¼ 13.7 Hz, J ¼ 9.1 Hz, HCHN), 2.79 (dddd, 1H, J ¼ 20.7 Hz

CDCl 3 ) d: 22.11. Anal. Calcd

-amino-9H-purin-9-yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate (33e) Yellow oil; IR (film, cm À1 ) n max : 3323

(m, 6H, CH 2 N, 2 Â CH 2 OP), 3.74e3.68 (m, 1H, H-C3), 2.84e2.72 (m, 1H, H a -C4), 2.56 (s, 3H, CH 3 N), 2.29 (dddd, 1H, J ¼ 19.9 Hz, J ¼ 13.2 Hz, J ¼ 6.7 Hz

CDCl 3 ) d: 22.08. Anal. Calcd. for C 14 H 23 N 6 O 4 P: C, 45

-amino-9H-purin-9-yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate (34e) Yellow oil; IR (film, cm À1 ) n max : 3323

70 (s, 3H, CH 3 -N), 2.35 (dddd, 1H, J ¼ 11.9 Hz, J ¼ 9.1 Hz, J ¼ 9.1 Hz, J ¼ 2.9 Hz, H b -C4), 1.37 (t, 3H, J ¼ 6.7 Hz, CH 3 CH 2 OP), 1.35 (t, 3H, J ¼ 6.7 Hz, CH 3 CH 2 OP); 13 C NMR (150 MHz

-bromo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate (35c) A 22:2:49:27 mixture of isoxazolidines 33c, 34c, 35c and 36c (0.030 g) was subjected to the separation on a X Bridge Prep

2 Hz, H 2 C5), 4.08 (dd, 1H, J ¼ 13.3 Hz, J ¼ 2.5 Hz, HCHN), 3.49 (dddd, J ¼ 16.1 Hz, J ¼ 9

1 (C5), 65.1 (C3), 62.8 (d, J ¼ 6.7 Hz, CH 2 OP)

herpes simplex virus type 2 (HSV-2) strain G, varicellazoster virus (VZV) strains Oka and YS, TK-VZV strains 07-1 and YS-R, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as feline herpes virus (FHV), the poxvirus vaccinia virus (Lederle strain), para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, respiratory syncytial virus (RSV), feline coronavirus (FIPV) and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and influenza B virus (B/HK/5/ 72) and human immunodeficiency virus (HIV-1/III B and HIV-2/ ROD)

Antiviral Nucleosides: Chiral Synthesis and Chemotherapy

Their Chemistry and Biological Properties

Chemical Synthesis of Nucleoside Analogues

Antimicrob Agents Chemother

The authors wish to express their gratitude to Mrs. Leentje