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Synthesis and Antiviral Evaluation of Novel 4′-Trifluoromethylated 5′Deoxyapiosyl Nucleoside Phosphonic Acids a

a

a

a

Seyeon Kim , Eunae Kim , Wonjae Lee & Joon Hee Hong a

BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju, Republic of Korea Published online: 05 Nov 2014.

To cite this article: Seyeon Kim, Eunae Kim, Wonjae Lee & Joon Hee Hong (2014) Synthesis and Antiviral Evaluation of Novel 4′-Trifluoromethylated 5′-Deoxyapiosyl Nucleoside Phosphonic Acids, Nucleosides, Nucleotides and Nucleic Acids, 33:12, 747-766, DOI: 10.1080/15257770.2014.938753 To link to this article: http://dx.doi.org/10.1080/15257770.2014.938753

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Nucleosides, Nucleotides and Nucleic Acids, 33:747–766, 2014 C Taylor and Francis Group, LLC Copyright  ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2014.938753

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SYNTHESIS AND ANTIVIRAL EVALUATION OF NOVEL 4 -TRIFLUOROMETHYLATED 5 -DEOXYAPIOSYL NUCLEOSIDE PHOSPHONIC ACIDS

Seyeon Kim, Eunae Kim, Wonjae Lee, and Joon Hee Hong BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju, Republic of Korea 2

On the basis of the discovery that the threosyl nucleoside phosphonate PMDTA is a potent anti-HIV compound, we synthesized several 4 -trifluoromethyl-5 -deoxyapiosyl nucleoside phosphonic acids and evaluated their anti-HIV activity. An efficient synthetic route was optimized, starting from an α-trifluoromethyl-α,β-unsaturated ester. Glycosylation of the purine nucleosidic bases with a glycosyl donor yielded modified nucleoside intermediates, which were then phosphonated and hydrolyzed to provide the targeted nucleoside analogs. Once synthesized, the anti-HIV and cytotoxic activities of each analog were evaluated. None of the analogs showed significant anti-HIV activity at concentrations up to 100 μM. Keywords Antiviral agent; 4 -trifluoromethylated nucleoside; 5 -deoxyapiosyl nucleoside; nucleoside phosphonic acid

INTRODUCTION Nucleoside reverse transcriptase inhibitors (NRTIs) continue to be the cornerstone of anti-HIV therapy. Unfortunately, many of the existing treatments have significant drawbacks,[1] necessitating the continued optimization of new NRTIs. Introduction of a lipophilic moiety at the 4 -position of nucleosides, particularly purine nucleosides, results in interesting biological activity, as exemplified by 4 -ethynyl-cpAP.[2] Although monofluorinated[3] and gem-difluorinated[4] nucleosides have been widely studied, only a few trifluoromethylated[5] nucleosides have been reported, perhaps owing to a lack of efficient synthetic methods. Johnson and Kozak reported an efficient strategy for introducing a trifluoromethyl (-CF3 ) group at the C-4 position of ribose derivatives, and the strategy was used to synthesize a collection of 4 -trifluoromethylated nucleoside analogs (1).[6]

Received 21 March 2014; accepted 22 June 2014. Address correspondence to Dr. Joon Hee Hong, College of Pharmacy, Chosun University, Kwangju 501-759, Republic of Korea. E-mail: [email protected]

747

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S. Kim et al. NH2 N

N

N

N HO

NH2

N

N

O HO P

O

F 3C

N

O

O

OH

HO 1

N

2 (PMDTA) O

O

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H3C O HO P

NH N

O

O

OH 3 (PMDTT)

O

NH O HO P

N

O

O

HO 4

FIGURE 1 Synthesis rationale of 4 -trifluoromethylated 5 -deoxythreosyl phosphonic acid nucleoside analogs.

Since antiviral activity is most often associated with phosphonomethoxylated nucleosides, comparatively little attention has been focused on exploring the structure-activity relationships of phosphonate derivatives. In one study, nucleoside phosphonate analogs were demonstrated to have potent antiviral activity.[7] In another study, phosphonated threose nucleosides[8] such as PMDTA (2) and PMDTT (3) were synthesized (Figure 1) by assembling them from natural precursors.[9] It has also been demonstrated that the oligomerized form of threose-phosphate nucleosides, threose nucleic acid (TNA), can form duplexes with DNA or RNA with thermal stability similar to that of naturally occurring nucleic acid duplexes. Superimposition of a model of a phophonomethoxylated threose nucleoside onto the threedimensional structure of TNA mixed helices can be used to predict that the phosphonoalkoxy group would be bound to the 3 -position, bringing the phosphorus atom and the nucleobase closer to one another than is observed in the structures of ribose-phosphonates, wherein the phosphonate group is bound to the primary hydroxyl group of the nucleoside. In the literature, several 5 -phosphate isosteres have been utilized to prepare various nucleoside phosphonate derivatives. As shown in Figure 1, compound (4)[10] is a 5 -deoxynucleoside phosphonic acid in which the 5 oxygen of the nucleoside phosphate is replaced by a methylene group. The inhibitory mechanism of most nucleoside antivirals is thought to depend on intracellular phosphorylation, incorporation into DNA, and subsequent chain termination.[11] It is therefore likely that the antiviral potency of phosphonylated nucleobases may also relate to intracellular phosphorylation to the diphosphate form and to refractory incorporation into nucleic acid oligomers.[12]

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Phosphonate analogs have certain advantages over the corresponding phosphate, one of which is higher metabolic stability due to the phosphorous-carbon bond being inert to hydrolytic cleavage.[13] In addition, the carbon atom in phosphonate analogs is placed at the β-position relative to the phosphorous atom, which is known to play a critical role in antiviral activity.[14] A carbon atom at this position is thought to enhance the binding of phosphonate analogs to their target enzymes.[15] Based on the observations that 4 -branched and threose-modified nucleosides and 5 -deoxynucleoside phosphonic acids are all potent antivirals, we sought to synthesize a novel class of 4 -trifluoromethyl- 5 -deoxythreosyl nucleoside phosphonic acid analogs and to evaluate their antiviral activity against HIV. We envisioned that these analogs would also be useful for probing the conformational preferences of nucleoside and nucleotide kinases.

RESULTS AND DISCUSSIONS The α-trifluoromethyl-α,β-unsaturated ester 6[16] has been previously used as a building block for preparing trifluoromethylated compounds,[17] which prompted us to utilize it as the starting material for our synthetic route (Scheme 1). Ester 6 was reacted with diisobutylaluminum hydride

ref.16

O O

O

CF3

O

O 5

CO2Et

6

i 88%

CF3

O O

OH 7 90% ii

CF3 O

iv OP

OH

CF3

HO

OP 9

10

iii O 68%

CF3

O

OP 8

P = TBDMS

70% v 5

CF3 OP

HO 11

vi 81%

F3C

CO2Et OP 12

vii 62%

3

2

4

F3C

O

O1 13

Reagents: i) DIBALH, CH2Cl2; ii) TBDMSCl, imidazole, CH2Cl2; iii) 1,4dioxane, 2 N HCl solution; iv) NaIO4, MeOH, H2O; v) NaBH4, MeOH; vi) (EtO)3CCH3, CH3CH2CO2H; vii) TBAF, THF. SCHEME 1 Synthesis of 4-trifluoromethylated lactone intermediate 13.

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FIGURE 2 NOE differences between the proximal hydrogens of 16a and 16b.

(DIBALH) to form trifluoromethyl fluoroallylic alcohol 7. The alcohol was silylated with tert-butylchlorodiphenylsilane (TBDPSCl), and the isopropylidene group was hydrolyzed in 2 N HCl to give diol derivative 9. Oxidative cleavage of the diol with sodium periodate (NaIO4 ) followed by reduction of aldehyde 10 with sodium borohydride (NaBH4 ) yielded allylic alcohol derivative 11.[18] This substrate was subjected to Claisen rearrangement conditions in the presence of excess triethyl orthoacetate and a catalytic amount of propionic acid to give γ ,δ-unsaturated tertiary trifluoromethylated ethylester 12 with an 81% yield.[19] Lactone derivative 13 was prepared via desilylative cyclization from 12 with a 62% yield. Lactone 13 was reduced using DIBALH in toluene at –78◦ C to give lactol 14, which was acetylated in pyridine to give a key intermediate, 15, as a glycosyl donor. Synthesis of the adenine nucleoside was carried out by condensation of 15 with persilylated 6-chloropurine by using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a catalyst in dichloroethane (DCE) to give protected 6-chloropurine derivatives 16a and 16b. A complete nuclear Overhauser effect (NOE) NMR study enabled clear determination of the relative stereochemistry of 16a and 16b (Figure 2). For compound 16a, the spectrum showed a strong NOE (1.2%) corresponding to H-2 ↔ vinyl H-5 coupling, thus confirming that the compound was the 2 ,5 -cis isomer. On the basis of this finding, we concluded that the 4 -CF3 and the 2 -purine base of 16b were located on the β face. In contrast, the spectra of 16b only showed a weak H-2 ↔ vinyl H-5 NOE (0.7%), thus resulting in it being defined as the 2 ,4 -trans isomer. Cross-metathesis[20] of 16b with vinyl diethylphosphonate by using a second-generation Grubbs catalyst[21] gave vinylidene nucleoside phosphonate analog 17 in a 67% yield. The chlorine group of the purine analog 17 was then converted to an amine in methanolic ammonia heated at 65◦ C to give the corresponding adenosine phosphonate derivative 18, which was then hydrolyzed by treatment with bromotrimethylsilane (TMSBr) in acetonitrile in the presence of 2,6-lutidine to give adenosine phosphonate derivative 19.[22] Vinylidene phosphonate 17 was saturated under catalytic transfer hydrogenation conditions to yield ethyl phosphonate nucleoside analog 20 (73% yield). Adenine phosphonate analog 22 was prepared using reaction

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conditions similar to those used for the preparation of 19, with respect to complete ammonolysis and hydrolysis (Scheme 2). To synthesize the guanine analogs, 2-fluoro-6-chloropurine[23] was condensed with the glycosyl donor 15 in conditions similar to those used for the ¨ condensation of 6-chloropurine. Vorbruggen coupling[24] of the acetate 15

Cl N

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13

i 89%

iii

OR

F3C

F3C ii

O 14: R = H 80% 15: R = Ac

ON

N

and

N

N

N

N

N

F3C

Cl

O 16b (34%)

16a (33%)

67% iv Cl Cl N O EtO P EtO

N

N N

N

vii 61%

O EtO P EtO

N F3C

F3C

N

O

N

17

O 20 v 57% v 59% NH2 N

NH2 N O EtO P EtO

N

N

N

O EtO P EtO

N F3C

F3C

O

O

N N

18

21 vi 70%

vi 67% NH2

NH2 N O HO P HO

N F3C

N N

N O HO P HO

N F3C

O 22

O

N N

19

Reagents: i) DIBALH, toluene; ii) Ac2O, DMAP, pyridine; iii) silylated 6-chloropurine, TMSOTf, DCE; iv) vinyl diethylphosphonate, Grubbs cat.(II) CH2Cl2; v) NH3/MeOH; vi) TMSBr, 2,6-lutidine, CH3CN; vii) Pd/C, cyclohexene, MeOH. SCHEME 2 Synthesis of 4 -trifluoromethylated-5 -deoxythreosyl adenine nucleoside phosphonic acids.

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F3C

OAc F3C

O

O 15

N

N

F

N

F

and N

N 23a (30%)

N

N F3C

O

23b (31%)

Cl ii 64% Cl

Cl

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N O EtO P EtO

N N

N

F

N

v 61%

O EtO P EtO F3C

F3C

24

O iii

X

X N N

N

N Y

N

O EtO P EtO

O 28a: X = NH2, Y = F (9%) 28b: X = Cl, Y = NH2 (40%)

O

iv 49% O

O N

F3C

O 29

Y

N

25a: X = NH2, Y = F (8%) 25b: X = Cl, Y = NH2 (39%)

iv 43%

N

N

N F3C

F3C

O HO P HO

F

N

O 27 iii

O EtO P EtO

N

N

NH N

NH2

N O HO P HO

N F3C

NH N

NH2

O 26

Reagents: i) silylated 2-fluoro-6-chloropurine, TMSOTf, DCE; ii) vinyl diethylphosphonate, Grubbs cat.(II) CH2Cl2; iii) NH3, DME, rt; iv) (a) TMSBr, 2,6-lutidine, CH3CN; (b) NaOMe, HSCH2CH2OH, MeOH; v) Pd/C, cyclohexene, MeOH. SCHEME 3 Synthesis of 4 -trifluoromethylated-5 -deoxythreosyl guanine nucleoside phosphonic acids.

with 2-fluoro-6-chloropurine gave analogs 23a (30%) and 23b (31%). Crossmetathesis of 23b and reaction with vinyl diethylphosphonate gave 24 in a 64% yield. A complete nuclear Overhauser effect spectroscopy (NOESY) NMR study enabled clear determination of their relative stereochemistry, as in the case of 16a and 16b.

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TABLE 1 Antiviral activity of the synthesized compounds HIV-1 Compound

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19 22 26 29 PMEA AZT

Cytotoxicity IC50 (μM)

EC50 (μM)

EC90 (μM)

PBM

CEM

Vero

48 36 42 53 4.25 0.026

90 90 95 95 ND ND

>100 >100 >100 >100 >100 >100

>100 >100 >100 >100 40.0 12.9

>100 >100 >100 >100 >100 50.0

ND: Not determined. PMEA: 9-[2-(Phosphonomethoxy)ethyl]adenine. AZT: Azidothymidine. EC50 (μM): concentration that caused 50% maximal inhibition of virus production as indicated by supernatant RT levels. EC90 (μM): concentration that caused 90% maximal inhibition of virus production as indicated by supernatant RT levels. IC50 (μM): concentration that caused 50% maximal inhibition of cell growth.

Bubbling ammonia into compound 24 yielded 2-fluoro-6-aminopurine analog 25a[25] (8%) and 2-amino-6-chloropurine analog 25b (39%), which were readily separable. For the transesterification of phosphonate 25b, the mixture was treated with TMSBr and 2,6-lutidine to give the phosphonic acid. Without purification, the hydrolysis of 2-amino-6-chloropurine into guanine base was also done by using sodium methoxide and 2-mercaptoethanol in methanol (MeOH) to provide the desired guanine nucleoside phosphonic acid 26 in a 49% yield (Scheme 3).[26] The hydrolysis is initiated by displacement of chlorine by thiolate anion of 2-mercaptoethanol via nucleophilic

FIGURE 3 Superimpose of PMDTA and 22. (Color figure available online).

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aromatic substitution route. Guanine phosphonate 29 was synthesized from 24 via transfer catalytic hydrogenation, ammonolysis, and hydrolysis by using conditions similar to those described for the synthesis of 26. The antiviral activity of nucleoside phosphonic acid is mostly attributable to their intracellular conversion to the diphosphate form, which is incorporated into the viral genome, causing chain termination.[27] Herein, we evaluated the anti-HIV-1 activity of 19, 22, 26, and 29 in human peripheral blood mononuclear (PBM) cells infected with the HIV-1 strain LAI . Concurrently, we evaluated the cytotoxicity of the compounds by measuring their effect on the viability of mock-infected cells.[28] We found that none of the analogs had significant antiviral or cytotoxic activity at concentrations up to 100 μM (Table 1). It is possible that the sugar moiety of the analogs either inhibited diphosphorylation or binding to viral polymerases. A model of PMDTA (2) superimposed onto the corresponding adenine analog 22 (Figure 3) helped predict that the phosphonic acid and base moieties would be in quite different conformations for the two ligands. It is also worth noting that the furanose pucker of PMDTA (2) was similar to that of adenine analog 22.[29]

CONCLUSIONS Based on the potent anti-HIV activity of 4 -branched nucleosides and of threosyl phosphonic acid nucleosides, we designed and synthesized novel 4 -trifluoromethyl-5 -deoxythreosyl-phosphonic acid nucleoside analogs, starting from an α-trifluoromethyl-α,β-unsaturated ester. Biological evaluation indicated that these phosphonic acid nucleosides did not have any significant anti-HIV activity at concentrations up to 100 μM.

EXPERIMENTAL SECTION The melting points for all compounds were determined using a Mel-temp II laboratory device and are uncorrected. NMR spectra were recorded on a JEOL 300 Fourier transform spectrometer (JEOL, Tokyo, Japan); chemical shifts are reported in parts per million (δ), and signals are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd (doublet of doublets). UV spectra were obtained using a Beckman DU-7 spectrophotometer (Beckman, South Pasadena, CA, USA). Mass spectra were obtained in electrospray ionization (ESI) mode. Elemental analyses were performed using a Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA). Thin layer chromatography was performed on Uniplates (silica gel) purchased from Analtech Co. (7558, Newark, DE, USA). All reactions were carried out under an atmosphere of nitrogen unless specified. Dry dichloromethane

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(DCM), benzene, and pyridine were obtained by distillation of CaH2 . Dry tetrahydrofuran was obtained by distillation of Na and benzophenone immediately prior to use. (E) and (Z)-2-(Trifluoromethyl)-3-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl] prop-2-en-1-ol (7): DIBALH (19.5 mL, 1.0 M solution in hexane) was slowly added at −20◦ C to a solution of 6 (2.5 g, 9.32 mmol) in DCM (100 mL), and the mixture was stirred for 1 hour at the same temperature. MeOH (10 mL) was added to the resulting mixture, and the mixture was stirred for 3 hours at room temperature. The resulting solid was filtered through a Celite pad; the filtrate was concentrated under vacuum, and the residue was purified by silica gel column chromatography (EtOAc/hexane, 1:4) to give alcohol 7 (1.85 g, 88%) as a colorless oil: 1H NMR (CDCl3 , 300 MHz) as mixture δ 6.54–6.49 (m, 1H), 4.73 (m, 1H), 4.25 (m, 2H), 4.07–3.92 (m, 2H), 1.42 (s, 3H), 1.37 (s, 3H); Anal. calcd for C9 H13 F3 O3 : C, 47.79; H, 5.79; Found: C, 47.63; H, 5.86. (E) and (Z)-{2-(Trifluoromethyl)-3-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]all yloxy}(t-butyl)dimethylsilane (8): TBDMSCl (1.59 g, 10.56 mmol) was slowly added at 0◦ C to a solution of alcohol 7 (2.17 g, 9.6 mmol) and imidazole (0.98 g, 14.4 mol) in dry DCM (90 mL), and the mixture was stirred for 3 hours at room temperature. The reaction mixture was quenched using a saturated aqueous sodium bicarbonate (NaHCO3 ) solution (10 mL) and further diluted with water (100 mL). The mixture was extracted with DCM (100 mL) twice, and the combined organic layers were washed with brine, dried over anhydrous magnesium sulfate (MgSO4 ), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate, EtOAc/hexane, 1:18) to give compound 8 (2.94 g, 90%) as a colorless oil: 1H NMR (CDCl3 , 300 MHz) δ 6.47–6.42 (m, 1H), 4.59 (m, 1H), 4.47 (s, 2H), 4.05 (m, 1H), 3.83 (m, 1H), 1.44 (s, 3H), 1.39 (s, 3H), 0.86 (s, 9H), 0.02 (s, 6H); Anal. calcd for C15 H27 F3 O3 Si: C, 52.92; H, 7.99. Found: C, 52.84; H, 8.05. (E) and (Z)-(S)-5-(t-Butyldimethylsilanyloxy)-4-(trifluoromethyl)-pent-3en-1,2-diol (9): Aqueous HCl solution (25 mL, 2 N) was added to a solution of compound 8 (1.73 g, 5.1 mmol) in 1,4-dioxane (10 mL) and stirred for 3 hours at 0◦ C. The mixture was neutralized by slow addition of a saturated NaHCO3 solution and diluted with 50 mL of brine. The mixture was extracted with EtOAc (60 mL × 3), and the combined organic layer was dried over anhydrous MgSO4 , and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc/hexane, 4:1) to give diol 9 (1.04 g, 68%) as a colorless oil: 1H NMR (CDCl3 , 300 MHz) δ 6.51–6.48 (m, 1H), 4.47 (s, 2H), 4.01–3.98

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(m, 1H), 3.99 (m, 1H), 3.86–3.83 (m, 1H), 3.62–3.59 (m, 1H), 0.90 (s, 9H), 0.01 (s, 6H); Anal. calcd for C12 H23 F3 O3 Si: C, 47.98; H, 7.72. Found: C, 48.09; H, 7.65. (E) and (Z)-4-(t-Butyldimethylsilanyloxy) 3-trifluoromethyl-but-2-en-1-ol (11): A solution of NaIO4 (560 mg, 2.5 mmol) in H2 O (8.0 mL) was added dropwise to a solution of 9 (525 mg, 1.75 mmol) in MeOH (8.0 mL) over 30 minutes at 0◦ C and stirred for further 20 minutes at the same temperature. NaBH4 (198 mg, 5.25 mmol) was added, and the reaction mixture was stirred for 20 minutes at 0◦ C. The white solid was filtered out and washed with MeOH (15 mL). The combined filtrate was carefully neutralized using 0.5 N HCl and concentrated to dryness, and the residue was purified by silica gel column chromatography (EtOAc/hexane, 1:2) to give allylic alcohol 11 (472 mg, 70%) as a colorless oil: 1H NMR (CDCl3 , 300 MHz) δ 6.50–6.47 (m, 1H), 4.48 (s, s, 2H), 4.21 (s, s, 2H), 0.95 (s, 9H), 0.02 (s, 6H); Anal. calcd for C11 H21 F3 O2 Si: C, 48.87; H, 7.83; Found: C, 48.76; H, 7.90. (±)-3-(t-Butyldimethylsilanyloxymethyl) 3-trifluoromethyl-pent-4-enoic acid ethyl ester (12): A solution of allylic alcohol 11 (1.23 g, 4.55 mmol) in triethylorthoacetate (20 mL) and 0.2 mL of propionic acid was heated at 135–140◦ C overnight with constant stirring to allow for the removal of ethanol. An excess of triethyl orthoacetate was removed by distillation, and the residue was purified by silica gel column chromatography (EtOAc/hexane, 1:30) to give compound 12 (1.25 g, 81%) as a colorless oil: 1 H NMR (CDCl3 , 300 MHz) δ 5.74 (dd, J = 12.7, 5.6 Hz, 1H), 5.05-4.98 (m, 2H), 4.13 (q, J = 7.3 Hz, 2H), 3.90 (d, J = 9.2 Hz, 1H), 3.65 (d, J = 9.3 Hz, 1H), 2.36 (d, J = 10.4 Hz, 1H), 1.18 (t, J = 7.2 Hz, 3H), 0.91 (s, 9H), 0.07 (s, 6H); 13C NMR (CDCl3 , 75 MHz) δ 171.3, 154.2, 123.6 (q, J = 281 Hz), 110.2, 61.3, 52.7, 43.9 (q, J = 28.6 Hz), 27.5, 25.4, 18.6, 14.2, −5.3; Anal. calcd for C15 H27 F3 O3 Si: C, 52.92; H, 7.99; Found: C, 52.81; H, 8.06; MS m/z 341 (M + H)+. (±)-4-Trifluoromethyl-4-vinyl-dihydrofuran-2-one (13): To a solution of 12 (650 mg, 1.91 mmol) in THF (10 mL), TBAF (2.1 mL, 1.0 M solution in THF) was added at 0◦ C. The mixture was stirred overnight at room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/EtOAc, 15:1) to give 13 (213 mg, 62%): 1H NMR (CDCl3 , 300 MHz) δ 5.73 (dd, J = 10.4, 7.8 Hz, 1H), 5.15-5.07 (m, 2H), 4.46 (dd, J = 12.6 Hz, 1H), 4.21 (dd, J = 12.7 Hz, 1H), 2.35 (d, J = 11.8 Hz, 1H), 2.14 (dd, J = 11.8 Hz, 1H); 13C NMR (CDCl3 , 75 MHz) δ 173.7, 155.4, 131.3, 111.6, 59.8 (q, J = 278.4 Hz), 44.3 (q, J = 29.2 Hz), 37.5; MS m/z 181 (M+H)+.

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(±)-4-Trifluoromethyl-4-vinyl-tetrahydrofuran-2-ol (14): To a cooled (−78◦ C) and stirred solution of lactone 13 (350 mg, 1.94 mmol) in dry toluene (8 mL), a 1.0 M solution of DIBALH (2.2 mL, 2.2 mmol) was added dropwise. The mixture was stirred for 20 minutes at −78◦ C followed by dropwise addition of MeOH (2.5 mL) and dilution with EtOAc (40 mL). The reaction mixture was warmed to room temperature and stirred for 1 hour, and the precipitate was removed by filtration through a pad of Celite and washed with ethyl acetate. The filtrate and washings were concentrated in vacuo, and the residue was purified by silica gel column chromatography (EtOAc/hexane, 1:8) to give 14 (314 mg, 89%) as a diastereomeric mixture: 1H NMR (CDCl3 , 300 MHz) δ 5.78–5.69 (m, 1H), 5.49–5.46 (m, 1H), 5.11–5.02 (m, 2H), 3.87–3.80 (m, 1H), 3.60 (dd, J = 10.8, 6.2 Hz, 1H), 2.14–2.09 (m, 1H), 1.86–1.80 (m, 1H). (±)-Acetic acid 4-trifluoromethyl-4-vinyl-tetrahydrofuran-2-yl ester (15): To a solution of compound 14 (382 mg, 2.1 mmol) in anhydrous pyridine (12 mL) with DMAP (13 mg), acetic anhydride (321 mg, 3.15 mmol) was slowly added, and the mixture was stirred overnight under nitrogen. The pyridine was evaporated under reduced pressure and co-evaporated with toluene. The residue was diluted with H2 O (120 mL) and extracted with EtOAc (2 × 120 mL). The organic layers were combined, dried over MgSO4 , and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc/hexane, 1:20) to give compound 15 (376 mg, 80%) as a diastereomeric mixture: 1H NMR (CDCl3 , 300 MHz) δ 6.25–6.19 (m, 1H), 5.80–5.68 (m, 1H), 5.14–5.05 (m, 2H), 3.90–3.80 (m, 1H), 3.61–3.57 (m, 1H), 2.21–2.16 (m, 1H), 2.04 (s, s, 3H), 1.90–1.87 (m, 1H). (±)-6-Chloro-9-(4-(trifluoromethyl)-tetrahydro-4-vinylfuran-2-yl)-9Hpurine (16a) and its isomer (16b): 6-Chloropurine (219 mg, 1.42 mmol), anhydrous hexamethyldisilizane (HMDS, 12 mL), and a catalytic amount of ammonium sulfate (10 mg) were refluxed overnight, and the solvent was distilled under anhydrous conditions. The residue was dissolved in dry DCE (12 mL). To this mixture, a solution of 15 (159 mg, 0.71 mmol) in dry DCE (12 mL) and TMSOTf (315 mg, 1.42 mmol) was added, and the resulting mixture was stirred for 4 hours at room temperature. The reaction mixture was quenched with 2.5 mL of saturated NaHCO3 and stirred for 1 hour. The resulting solid was filtered through a Celite pad, and the filtrate was diluted with water (90 mL) and extracted with DCM (2 × 90 mL). The combined organic layers were dried over anhydrous MgSO4 , filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc/hexane/MeOH, 4:1:0.01) to give compound 16a (74 mg, 33%) and 16b (76 mg, 34%): data for 16a: 1H NMR

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(DMSO-d 6 , 300 MHz) δ 8.69 (s, 1H), 8.20 (s, 1H), 5.99 (dd, J = 5.4, 2.0 Hz, 1H), 5.81–5.78 (dd, J = 6.0, 4.2 Hz, 1H), 5.15–5.08 (m, 2H), 3.85 (d, J = 10.6 Hz, 1H), 3.61 (d, J = 10.5 Hz, 1H), 2.38 (dd, J = 11.4, 6.2 Hz, 1H), 2.19 (dd, J = 11.5, 8.2 Hz, 1H); 13C NMR (DMSO-d 6 , 75 MHz) δ 154.4, 151.6, 151.1, 150.3, 143.9, 132.8, 125 (q, J = 281.6 Hz), 109.6, 88.2, 60.7, 49.2 (q, J = 27.3 Hz), 24.7; Anal. calcd for C12 H10 ClF3 N4 O (+0.5 MeOH): C, 44.94; H, 3.62; N, 16.77; Found: C, 44.90; H, 3.57; N, 16.68; MS m/z 319 (M+H)+. Data for 16b: 1H NMR (DMSO-d 6 , 300 MHz) δ 8.69 (s, 1H), 8.20 (s, 1H), 5.99 (dd, J = 5.4, 2.0 Hz, 1H), 5.81–5.78 (dd, J = 6.0, 4.2 Hz, 1H), 5.15–5.08 (m, 2H), 3.85 (d, J = 10.6 Hz, 1H), 3.61 (d, J = 10.5 Hz, 1H), 2.38 (dd, J = 11.4, 6.2 Hz, 1H), 2.19 (dd, J = 11.5, 8.2 Hz, 1H); 13C NMR (DMSO-d 6 , 75 MHz) δ 155.8, 151.9, 151.5, 151.1, 144.8, 132.7, 124 (q, J = 278.2 Hz), 110.5, 86.9, 62.0, 48.8 (q, J = 29.1 Hz), 23.9; Anal. calcd for C12 H10 ClF3 N4 O (+0.5 MeOH): C, 44.94; H, 3.62; N, 16.77; Found: C, 44.85; H, 3.57; N, 16.67; MS m/z 319 (M+H)+. (±)-Diethyl 2-[2-(6-chloropurin-9-yl)-4-trifluoromethyltetrahydrofuran-4yl]-(trans)-vinylphosphonate (17): A second-generation Grubbs catalyst (20.4 mg, 0.0241 mmol) was added to a solution of 6-chloropurine derivative 16b (153 mg, 0.482 mmol) and diethyl vinylphosphonate (395 mg, 2.41 mmol) in DCM (12 mL). The reaction mixture was refluxed for 48 hours under dry argon gas and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc/nhexane/MeOH, 4:1:0.03) to give 17 (98 mg, 67%) in pure form: 1H NMR (DMSO-d 6 , 300 MHz) δ 8.74 (s, 1H), 8.32 (s, 1H), 6.71–6.67 (dd, J = 22.4, 17.2 Hz, 1H), 6.16 (dd, J = 20.8, 17.2 Hz), 6.03 (t, J = 5.8 Hz, 1H), 4.10-4.04 (m, 4H), 3.87 (d, J = 10.8 Hz, 1H), 3.59 (d, J = 10.7 Hz, 1H), 2.36 (dd, J = 11.2, 8.4 Hz, 1H), 2.15 (dd, J = 11.3, 9.2 Hz, 1H), 1.23–1.18 (m, 6H); 13 C NMR (DMSO-d 6 , 75 MHz) δ 151.8, 151.4, 151.1, 149.2 (d, J = 19.8 Hz), 144.5, 132.8, 124.6 (q, J = 286.2 Hz), 114.6, 88.7, 62.3, 61.8, 60.9, 50.2 (d, J = 27.9 Hz), 23.6, 14.2; Anal. calcd for C16 H19 ClF3 N4 O4 P: C, 42.15; H, 4.50; N, 11.92; Found: C, 42.25; H, 4.40; N, 11.85; MS m/z 455 (M+H)+. (±)-Diethyl 2-[2-(adenin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl](trans)-vinylphosphonate (18): A solution of 17 (205 mg, 0.451 mmol) in saturated methanolic ammonia (10 mL) was stirred overnight at 65◦ C in a steel bomb, and the volatiles were evaporated. The residue was purified by silica gel column chromatography (MeOH/DCM, 1:10) to give 18 (111 mg, 57%) as a white solid: mp 180–182◦ C; UV (MeOH) λmax 262.0 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.31 (s, 1H), 8.13 (s, 1H), 6.69 (dd, J = 17.2, 21.6 Hz, 1H), 6.14 (dd, J = 17.2, 19.7 Hz, 1H), 5.97 (dd, J = 5.6, 2.0 Hz, 1H), 4.11-4.07 (m, 4H), 3.86 (d, J = 10.8 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 2.38 (dd, J = 11.2, 9.2 Hz, 1H), 2.12 (dd, J = 11.2, 7.2 Hz, 1H), 1.25-1.20 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 155.5, 152.6, 151.2, 150.5 (d,

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J = 20.7 Hz), 141.6, 125.4 (q, J = 280.2 Hz), 119.0, 113.6, 88.5, 63.2, 62.7, 60.9, 51.1 (q, J = 28.4 Hz), 24.3, 14.1; Anal. calcd. for C16 H21 F3 N5 O4 P (+1.0 MeOH): C, 43.70; H, 5.39; N, 14.99; Found: C, 43.61; H, 5.45; N, 15.09; MS m/z 436 (M+H)+. (±)-2-[2-(Adenin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl]-(trans)vinylphosphonic acid (19): TMSBr (0.61 mL, 4.62 mmol) was added to a solution of phosphonate 18 (201 mg, 0.462 mmol) in anhydrous acetonitrile (12 mL) with 2,6-lutidine (1.076 mL, 9.24 mmol). The mixture was heated overnight at 75◦ C under nitrogen gas and then concentrated in vacuo. The residue was co-evaporated from concentrated aqueous ammonium hydroxide (NH4 OH, 2 × 28 mL). The resultant material was purified by triturating the residue in acetone (10 mL) twice and removing the acetone by evaporation. The residue was then purified by preparative reverse-phase chromatography. Lyophilization of the appropriate fraction provided phosphonic acid salt 19 (128 mg, 70%) in the form of a white salt (ammonium salt): 1H NMR (D2 O, 300 MHz) δ 8.31 (s, 1H), 8.11 (s, 1H), 6.69 (dd, J = 17.6, 22.0 Hz, 1H), 6.14 (dd, J = 17.6, 18.8. Hz, 1H), 6.01 (dd, J = 5.9, 2.4 Hz, 1H), 3.81 (d, J = 10.2 Hz, 1H), 3.60 (d, J = 10.2 Hz, 1H), 2.36 (dd, J = 9.8, 8.2 Hz, 1H), 2.15 (dd, J = 9.9, 6.4 Hz, 1H): 13C NMR (D2 O, 75 MHz) δ 154.5, 151.7, 151.3, 148.9 (d, J = 19.6 Hz), 142.5, 125. 2 (q, J = 279.8 Hz), 120.5, 112.8, 89.0, 62.5, 50.5 (q, J = 28.4 Hz), 23.7; HPLC t R = 10.31; HRMS [M−H]+ req. 378.0762, found 378.0763. (±)-Diethyl 2-[2-(6-chloropurin-9-yl)-4-trifluoromethyltetrahydrofuran-4yl]-ethylphosphonate (20): To a solution of vinyl phosphonate nucleoside analog 17 (272 mg, 0.6 mmol) in MeOH (12 mL) was added 10% Pd/C (12 mg) and cyclohexene (6 mL) under argon gas. The reaction mixture was refluxed for 48 hours, after which the mixture was filtered through a pad of Celite, evaporated, and purified by silica gel column chromatography (EtOAc/n-hexane/MeOH, 2:1:0.1) to give ethyl phosphonate analog 20 (167 mg, 61%) as a white solid: mp 179–181◦ C; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.66 (s, 1H), 8.21 (s, 1H), 5.94 (dd, J = 5.6, 2.0 Hz, 1H), 4.11–4.08 (m, 4H), 3.84 (d, J = 10.4 Hz, 1H), 3.58 (d, J = 10.5 Hz, 1H), 2.36 (dd, J = 11.0, 7.2 Hz, 1H), 2.12 (dd, J = 11.1, 8.6 Hz, 1H), 2.18–2.07 (m, 4H), 1.23–1.19 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 151.7, 151.3, 150.9, 144.8, 132.5, 126.2 (q, J = 268.8 Hz), 89.0, 63.0, 62.6, 48.3 (q, J = 27.8 Hz), 28.5, 19.1, 14.3; Anal. calcd for C16 H21 ClF3 N4 O4 P (+1.0 MeOH): C, 41.83; H, 5.16; N, 11.47; Found: C, 41.89; H, 5.20; N, 11.39; MS m/z 457 (M+H)+. (±)-Diethyl 2-[2-(adenin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl]ethylphosphonate (21): Transformation of the 6-chloropurine to the adenine derivative was accomplished from 20 by using the same ammonolysis

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procedure as that described for 18: yield 59%; mp 172–174◦ C; UV (MeOH) λmax 260.5 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.38 (s, 1H), 8.23 (s, 1H), 6.00 (t, J = 5.6 Hz, 1H), 4.15–4.10 (m, 4H), 3.86 (d, J = 10.6 Hz, 1H), 3.53 (d, J = 10.5 Hz, 1H), 2.35 (dd, J = 10.6, 6.8 Hz, 1H), 2.15 (dd, J = 10.7, 8.2 Hz, 1H), 2.12-1.87 (m, 4H), 1.21–1.17 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 155.2, 152.3, 150.5, 140.9, 126.2 (q, J = 278.8 Hz), 119.2, 64.4, 63.1, 62.7, 48.4 (q, J = 26.8 Hz), 28.7, 23.5, 18.9, 14.7; Anal. calcd for C16 H23 F3 N5 O4 P (+0.5 MeOH): C, 4.73; H, 5.56; N, 15.45; Found: C, 43.78; H, 5.50; N, 15.33; MS m/z 438 (M+H)+. (±)-2-[2-(Adenin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl]ethylphosphonic acid (22): Adenine phosphonic acid 22 was synthesized from 21 by using the same hydrolysis procedure as that described for 19: yield 67%, 1H NMR (D2 O, 300 MHz) δ 8.35 (s, 1H), 8.16 (s, 1H), 5.98 (t, J = 5.7 Hz, 1H), 3.79 (d, J = 10.6 Hz, 1H), 3.57 (d, J = 10.5 Hz, 1H), 2.38 (dd, J = 11.2, 8.0 Hz, 1H), 2.16 (dd, J = 11.2, 6.8 Hz, 1H), 2.11-1.93 (m, 4H): 13C NMR (D2 O, 75 MHz) δ 154.2, 151.4, 150.9, 148.4, 141.8, 124. 7 (q, J = 268.2 Hz), 118.7, 89.4, 65.2, 47.8 (q, J = 27.7 Hz), 24.1; HPLC t R = 10.34; HRMS [M−H]+ req. 380.0693, found 380.0694. (±)-6-Chloro-2-fluoro-[9-(4-(trifluoromethyl)-tetrahydro-4-vinylfuran-2yl)]-9H-purine (23a) and its isomer (23b): Coupling of 15 with 2-fluoro6-chloropurine was accomplished using the same conditions as those described for the synthesis of 16a and 16b to give 23a and 23b, respectively. Data for 23a: yield 30%; UV (MeOH) λmax 270.0 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.44 (s, 1H), 6.00 (t, J = 5.4 Hz, 1H), 5.71 (dd, J = 9.8, 8.0 Hz, 1H), 5.04–4.93 (m, 2H), 3.85 (d, J = 10.6 Hz, 1H), 3.52 (d, J = 10.5 Hz, 1H), 2.52 (dd, J = 11.2, 8.8 Hz, 1H), 2.37 (dd, J = 11.1, 7.6 Hz, 1H); 13C NMR (DMSO-d 6 , 75 MHz) δ 157.6 (d, J = 218.2 Hz), 154.4, 153.2, 145.3, 135.9, 125.3 (q, J = 278.0 Hz), 120.2, 109.7, 89.2, 62.3, 49.6 (q, J = 26.6 Hz), 24.2; Anal. calcd for C12 H9 ClF4 N4 O (+1.0 MeOH): C, 42.42; H, 3.56; N, 15.22; Found: C, 42.51; H, 3.64; N, 15.35; MS m/z 337 (M+H)+. Data for 23b: yield 31%; UV (MeOH) λmax 269.0 nm; 1H NMR (DMSOd 6 , 300 MHz) δ 8.42 (s, 1H), 6.02 (dd, J = 5.6, 1.8 Hz, 1H), 5.69 (dd, J = 9.8, 8.2 Hz, 1H), 5.03–4.95 (m, 2H), 3.82 (d, J = 10.2 Hz, 1H), 3.55 (d, J = 10.3 Hz, 1H), 2.56 (dd, J = 10.6, 8.2 Hz, 1H), 2.34 (dd, J = 10.7, 7.8 Hz, 1H); 13 C NMR (DMSO-d 6 , 75 MHz) δ 158.2 (d, J = 219.4 Hz), 155.0, 153.4, 145.4, 136.1, 126.0 (q, J = 282.0 Hz), 120.5, 108.9, 89.1, 48.6 (q, J = 27.8 Hz), 24.7; Anal. calcd for C12 H9 ClF4 N4 O (+1.0 MeOH): C, 42.42; H, 3.56; N, 15.22; Found: C, 42.34; H, 3.59; N, 15.13; MS m/z 337 (M+H)+.

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(±)-Diethyl 2-[2-(6-chloro-2-fluoropurin-9-yl)-4trifluoromethyltetrahydrofuran-4-yl]-(trans)-vinylphosphonate (24): The phosphonate nucleoside analog 24 was prepared from 23b by using the same cross-metathesis procedure as that described for the preparation of 17: yield 64%; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.46 (s, 1H), 6.68 (dd, J = 17.8, 19.4 Hz, 1H), 6.21 (dd, J = 17.9, 21.4 Hz, 1H), 5.99 (dd, J = 5.2, 1.8 Hz, 1H), 4.12-4.08 (m, 4H), 3.87 (d, J = 8.8 Hz, 1H), 3.54 (d, J = 8.8 Hz, 1H), 2.34 (dd, J = 11.0, 8.4 Hz, 1H), 2.19 (dd, J = 11.1, 9.4 Hz, 1H), 1.23–1.18 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 157.2 (d, J = 225.0 Hz), 153.6, 149.5, 146.8, 145.2, 137.2, 125.6 (q, J = 278.7 Hz), 121.3, 116.7, 88.4, 63.1, 62.7, 60.9, 50.4 (q, J = 28.4 Hz), 24.2, 14.1; Anal. calcd for C16 H18 ClF4 N4 O4 P (+1.0 MeOH): C, 40.50; H, 4.39; N, 11.11; Found: C, 40.59; H, 4.4391; N, 11.20; MS m/z 473 (M+H)+. (±)-Diethyl 2-[2-(2-fluoro-6-aminopurin-9-yl)-4trifluoromethyltetrahydrofuran-4-yl]-(trans)-vinylphosphonate (25a) and (±)-diethyl 2-[2-(6-chloro-2-aminopurin-9-yl)-4-trifluoromethyltetrahydro furan-4-yl]-(trans)-vinylphosphonate (25b): Dry ammonia gas was bubbled into a stirred solution of 24 (280 mg, 0.59 mmol) in dimethoxyethane (DME, 13 mL) at room temperature overnight. The salts were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH/DCM, 1:10) to give 25a (21 mg, 8%) and 25b (99 mg, 39%), respectively: data for 25a; UV (MeOH) λmax 260.0 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.45 (s, 1H), 6.70 (dd, J = 17.8, 21.4 Hz, 1H), 6.23 (dd, J = 17.6, 20.2 Hz, 1H), 6.04 (t, J = 5.8 Hz, 1H), 4.11–4.07 (m, 4H), 3.82 (d, J = 10.2 Hz, 1H), 3.55 (d, J = 10.3 Hz, 1H), 2.39 (dd, J = 10.4, 8.2 Hz, 1H), 2.18 (dd, J = 10.2, 7.2 Hz, 1H), 1.23-1.18 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 161.1 (d, J = 256.6 Hz), 155.3, 152.7, 148.9, 141.7, 125.3 (q, J = 277.8 Hz), 119.0, 114.8, 89.0, 63.7, 62.9, 60.7, 51.1 (q, J = 27.6 Hz), 23.5, 14.6; Anal. calcd for C16 H20 F4 N5 O4 P (+1.0 MeOH): C, 42.08; H, 4.98; N, 14.43; Found: C, 42.16; H, 4.95; N, 14.48; MS m/z 454 (M+H)+. Data for 25b; UV (MeOH) λmax 309.5 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.15 (s, 1H), 6.68 (dd, J = 17.2, 21.8 Hz, 1H), 6.19 (dd, J = 17.3, 19.8 Hz, 1H), 5.99 (dd, J = 5.7, 2.0 Hz, 1H), 4.10–4.06 (m, 4H), 3.79 (d, J = 10.4 Hz, 1H), 3.52 (d, J = 10.3 Hz, 1H), 2.35 (dd, J = 11.0, 7.2 Hz, 1H), 2.14 (dd, J = 11.1, 6.0 Hz, 1H), 1.21–1.16 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 158.5, 154.6, 151.4, 142.8, 141.7, 124.7 (q, J = 280.3 Hz), 122.3, 113.5, 87.3, 62.8, 62.3, 61.4, 49.7 (q, J = 28.2 Hz), 24.0, 14.2; Anal. calcd for C16 H20 ClF3 N5 O4 P (+0.5 MeOH): C, 40.85; H, 4.57; N, 14.43; Found: C, 40.95; H, 4.42; N, 14.54; MS m/z 470 (M+H)+.

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(±)-2-[2-(Guanin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl]-(trans)vinylphosphonic acid (26): To a solution of 25b (198 mg, 0.424 mmol), dry acetonitrile (15 mL), 2,6-lutidine (0.987 mL, 8.48 mmol), and TMSBr (0.56 mL, 4.24 mmol) were added at room temperature. After stirring this mixture for 36 hours, the solvent was removed, coevaporating three times with MeOH. The residue was dissolved in MeOH (15.0 mL), and 2-mercaptoethanol (148.6 μL, 2.12 mmol), and sodium methoxide (114.5 mg, 2.12 mmol) were added to the mixture. The mixture was refluxed for 24 hours under N2 , cooled, neutralized with glacial acetic acid, and evaporated to dryness under vacuum. The residue obtained was co-evaporated from concentrated NH4 OH (2 × 15 mL) and the resultant solid was triturated with acetone (2 × 15 mL). After evaporating the acetone, the residue was purified by preparative column chromatography using reverse-phase C18 silica gel, eluting with water. Lyophilization of the appropriate fraction provided compound 26 (85 mg, 49%) in the form of a yellowish salt (ammonium salt): mp 184–186◦ C; UV (H2 O) λmax 254.0 nm; 1H NMR (D2 O, 300 MHz) δ 7.81 (s, 1H), 6.70 (dd, J = 21.5, 17.4 Hz, 1H), 6.21 (dd, J = 18.4, 17.5 Hz, 1H), 6.01 (dd, J = 5.6, 2.2 Hz, 1H), 3.81 (d, J = 10.6 Hz, 1H), 3.56 (d, J = 10.5 Hz, 1H), 2.39 (dd, J = 10.8, 8.2 Hz, 1H), 2.17 (dd, J = 10.8, 6.8 Hz, 1H); 13C NMR (D2 O, 75 MHz) δ 158.0, 154.8, 152.5, 149.3 (d, J = 19.9 Hz), 136.9, 125.4 (q, J = 278.8 Hz), 118.2, 116.7, 78.8, 62.1, 50.3 (q, J = 28.6 Hz), 24.6, 13.9; HPLC t R = 9.31 min; HRMS [M−H]+ req. 394.0685, found 394.0686. (±)-Diethyl 2-[2-(6-chloro-2-fluoropurin-9-yl)-4trifluoromethyltetrahydrofuran-4-yl]-ethylphosphonate (27): Compound 27 was synthesized from 24 by the same catalytic hydrogenation procedure that was described for the preparation of 20: yield 61%; UV (MeOH) λmax 268.5 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.43 (s, 1H), 5.97 (dd, J = 5.6, 1.8 Hz, 1H), 4.13–4.09 (m, 4H), 3.84 (d, J = 10.6 Hz, 1H), 3.54 (d, J = 10.7 Hz, 1H), 2.53 (dd, J = 11.0, 8.0 Hz, 1H), 2.19 (dd, J = 11.2, 9.2 Hz, 1H), 2.13–1.78 (m, 4H), 1.20–1.16 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 158.5 (d, J = 256.7 Hz), 153.4, 145.8, 136.5, 126.2 (q, J = 268.7 Hz), 120.7, 88.4, 65.2, 62.3, 61.7, 47.5 (q, J = 27.6 Hz), 28.6, 25.0, 19.1, 14.3; Anal. calcd for C16 H20 ClF4 N4 O4 P (+0.5 MeOH): C, 40.43; H, 4.52; N, 11.43; Found: C, 40.56; H, 4.48; N, 11.52; MS m/z 475 (M+H)+. (±)-Diethyl 2-[2-(2-fluoro-6-aminopurin-9-yl)-4trifluoromethyltetrahydrofuran-4-yl]-ethylphosphonate (28a) and (±)diethyl 2-[2-(6-chloro-2-aminopurin-9-yl)-4-trifluoromethyltetrahydrofuran4-yl]-ethylphosphonate (28b): Ammonolysis of 27 was performed using the same procedure as that described for the preparation of 28a and 28b. Data for 28a; yield 9%; UV (MeOH) λmax 262.0 nm; 1H NMR (DMSO-d 6 , 300 MHz) δ 8.20 (s, 1H), 5.94 (dd, J = 5.8, 2.0 Hz, 1H), 4.13–4.05 (m, 4H), 3.85 (d, J =

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10.2 Hz, 1H), 3.56 (d, J = 10.3 Hz, 1H), 2.55 (dd, J = 11.2, 8.0 Hz, 1H), 2.25 (dd, J = 11.3, 6.8 Hz, 1H), 2.12–1.83 (m, 4H), 1.23–1.17 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 159.3 (d, J = 252.5 Hz), 154.8, 152.7, 141.8, 124.5 (q, J = 278.4 Hz), 121.0, 89.2, 64.2, 63.3, 62.7, 47.7 (q, J = 28.2 Hz), 28.6, 23.6, 18.6, 14.3; Anal. calcd for C16 H22 F4 N5 O4 P (+1.0 MeOH): C, 41.91; H, 5.38; N, 14.37; Found: C, 41.85; H, 5.45; N, 14.29; MS m/z 456 (M+H)+. Data for 28b; yield 40%; UV (MeOH) λmax 309.5 nm; 1H NMR (DMSOd 6 , 300 MHz) δ 8.16 (s, 1H), 5.97 (t, J = 5.7 Hz, 1H), 4.19–4.14 (m, 4H), 3.90 (d, J = 10.3 Hz, 1H), 3.56 (d, J = 10.4 Hz, 1H), 2.36 (dd, J = 11.2, 8.0 Hz, 1H), 2.23–1.91 (m, 5H), 1.22–1.17 (m, 6H); 13C NMR (DMSO-d 6 , 75 MHz) δ 158.7, 154.3, 151.1, 143.2, 125 (q, J = 287.3 Hz), 124.1, 89.1, 64.6, 63.2, 62.5, 47.7 (q, J = 28.7 Hz), 29.2, 23.6, 19.0, 15.1; Anal. calcd for C16 H22 ClF3 N5 O4 P (+1.0 MeOH): C, 40.59; H, 5.21; N, 13.92; Found: C, 40.71; H, 5.06; N, 13.88; MS m/z 472 (M+H)+. (±)-2-[2-(Guanin-9-yl)-4-trifluoromethyltetrahydrofuran-4-yl]ethylphosphonic acid (29): Guanine nucleoside phosphonic acid 29 was prepared from 28b using the same hydrolysis conditions as those described for the preparation of 26: yield 43%; UV (H2 O) λmax 254.5 nm; 1 H NMR (D2 O, 300 MHz) δ 7.84 (s, 1H), 5.98 (t, J = 5.8 Hz, 1H), 3.79 (d, J = 10.5 Hz, 1H), 3.53 (d, J = 10.4 Hz, 1H), 2.36 (dd, J = 10.0, 6.8 Hz, 1H), 2.18 (dd, J = 10.1, 8.8 Hz, 1H), 2.05–1.97 (m, 4H); 13C NMR (D2 O, 75 MHz) δ 158.1, 154.7, 152.6, 137.0, 124.9 (q, J = 272.6 Hz), 117.6, 77.3, 65.2, 48.4 (q, J = 27.8 Hz), 28.6, 24.2, 18.7, 14.3; HPLC t R = 9.33 min; HRMS [M−H]+ req. 396.0765, found 396.0766.

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