Synthesis and Antiviral Activity Evaluation of 2′,5′,5′-Trifluoro-Apiosyl Nucleoside Phosphonic Acid Analogs

Article abstract of DOI:10.1080/15257770.2015.1115522

Racemic synthesis of novel 2′,5′,5′-trifluoro-apiose nucleoside phosphonic acid analogs were performed as potent antiviral agents. Phosphonation was performed by direct displacement of triflate intermediate with diethyl (lithiodifluoromethyl) phosphonate

Full text of DOI:10.1080/15257770.2015.1115522

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Antiviral Activity Evaluation of
2′,5′,5′-Trifluoro-Apiosyl Nucleoside Phosphonic
Acid Analogs
Eunae Kim & Joon Hee Hong
To cite this article: Eunae Kim & Joon Hee Hong (2016) Synthesis and Antiviral Activity
Evaluation of 2′,5′,5′-Trifluoro-Apiosyl Nucleoside Phosphonic Acid Analogs, Nucleosides,
Nucleotides and Nucleic Acids, 35:3, 130-146, DOI: 10.1080/15257770.2015.1115522
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1115522
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Date: 09 March 2016, At: 08:38

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Synthesis and Antiviral Activity Evaluation of
ꢀ,ꢀ,ꢀ-Trifluoro-Apiosyl Nucleoside Phosphonic Acid
Analogs
Eunae Kim and Joon Hee Hong
BK- Project Team, College of Pharmacy, Chosun University, Kwangju ,Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received  September 
Accepted  October 
Racemic synthesis of novel 2ꢀ,5ꢀ,5ꢀ-trifluoro-apiose nucleoside
phosphonic acid analogs were performed as potent antiviral
agents. Phosphonation was performed by direct displacement of
triflate intermediate with diethyl (lithiodifluoromethyl) phospho-
nate to give the corresponding (α,α-difluoroalkyl) phosphonate.
Condensation successfully proceeded from a glycosyl donor with
persilylated bases to yield the nucleoside phosphonate analogs.
Deprotection of diethyl phosphonates provided the target nucle-
oside analogs. An antiviral evaluation of the synthesized com-
pounds against various viruses such as HIV, HSV-1, HSV-2, and
HCMV revealed that the pyrimidine analogues have significant
anti-HCMV activity.
KEYWORDS
antiviral agents;
ꢀ,ꢀ,ꢀ-trifluoro-apiose
nucleoside phosphonic acid
analogues; Vorbrüggen
reaction
Introduction
The modification of the nucleosides and/or sugar moiety of a natural nucleoside is
an obvious choice for developing new antiviral compounds, and apiose-based nucle-
oside could serve this purpose.
Recently, apiose 5ꢀ-nor nucleoside phosphonate,[1] such as, PMDTA (1), has been
synthesized and has shown promising anti-HIV properties. Herdewijn et al reported
the synthetic procedure of 3ꢀ-C-ethynyl analogue of PMTA (2).[2]This absence of a
4ꢀ-hydroxymethyl group avoids problems of steric hindrance during phosphoryla-
tion reactions with kinases.
Phosphonates and structurally modified phosphonatesisosters can mimic phos-
phates in biological system.[3] The resistance of the phosphorus-carbon phospho-
nate linkage to hydrolysis by chemical agents or esterases is one of the features
responsible for their increasing popularity. Fluoro-substitution at the α-carbon of
phosphonates may increase the effectiveness of these phosphate mimetics as a result
both geometric and electronic factors.[4]The replacement of phosphonates by fluo-
rophosphonates has provided a number of analogues showing significant biological
activity.[5]
CONTACT Joon Hee Hong
Republic of Korea.
hongjh@chosun.ac.kr
College of Pharmacy, Chosun University, Kwangju –,
©  Taylor & Francis Group, LLC

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
131
Scheme . Synthesis of fluorinated apioseglycosyl donor intermediate 10.
9-(5,5-Difluoro-5-phosphonopentyl)guanine (3) has been utilized as a substrate
analogue inhibitor of purine nucleoside phosphorylase.[6] 2-Chloro-2ꢀ,5ꢀ-dideoxy-
5ꢀ-difluoromethylphosphinyl adenosine (2CDPA, 4), the nonhydrolyzable analogue
of 2-chlorodeoxyadenosine monophosphate was prepared for the treatment of
refractory chronic leukemia and hairy cell leukemia to overcome the undesired
metabolic pathway of 2CDA.[7] However, biological testing performed on various
T cells showed that 2CDPA does not exhibit expected cytotoxic effect. The lack of
cytotoxicity is probably caused by an insufficient level of phosphorylation inside T
cells.
On the basis of the above encouraging results, we undertook the synthesis
of isosteric and isopolar 5ꢀ-difluoromethylphosphonatederivatives of apiosyl 5ꢀ-
nornucleosidephosphonateto find more effective antiviral agents.
Results and Discussion
Target compounds were synthesized from lactone derivative 6, which was readily
obtained from 1,3-dihydroxyacetone, as previously described (Scheme 1).[8] Hydro-
genation of 2-fluoro-butenolide 6 to 2-fluorolactone 7 was achieved with 5% Pd/C
under H2 treatment with a yield of 90%. Protection[9] of 7 with TBDMSCl in methy-
lene chloride at 25°C furnished the desired O-silyl ether 8, which was converted
to lactol9 by DIBALH reduction in toluene at −78°C for 1.0 h in 74% two step
yield. Protection of anomeric position was needed prior to phosphonation. Hence,
methoxylation of anomeric position furnished glycoside 10 as anomeric mixture in
a 89% yield using the conditions [CH(OMe)3, BF3/Et2O] even in the presence of
acid labile silyl protection group.[10] Removal of the TBDMS group of glycoside 10
by TBAF furnished alcohol 11 as anomeric mixture with a 90% yield which was
converted to anomeric mixture of difluorophosphonate13 using triflation followed
by a triflate displacement according to the procedure of Berkowitz et al.[11] The
preparation of a suitable glycosylating agent 14 was attempted by direct acetolysis
of 13 under acidic conditions (Ac2O, AcOH, H2SO4, EtOAc, 0°C)[12] to afford an
anomeric mixture of 1-O-acetyl-furanoside 14 in a 83% yield (Scheme 2). The syn-
thesis of adenine nucleoside was performed using a Vorbrüggencondensation[13] of

132
E. KIM AND J. H. HONG
Scheme . Synthesis of fluorinated apioseglycosyl donor intermediate 14.
compound 14 with silylated 6-chloropurine and trimethylsilyltriflate(TMSOTf) as
a catalyst in dichloroethane (DCE) to yield the protected 6-chloropurine deriva-
tives, 15α and 15β, respectively. A complete nuclear overhauser effect (NOE) study
between proximal hydrogens verified their relative stereochemistry (Figure 2). NOE
experiments of both products showed that glycosylation in α-direction is isomer
15α(NOE: H1β/H3α, 0.9%), and glycosylation of β-direction is isomer 15β(NOE:
H1α/H3α, 1.7%). The chlorine group from purine analog 15β was then converted
to an amine with methanolic ammonia at 68°C to produce the adenosine phos-
phonate derivative 16 in 67%yield. Hydrolysis of the diethyl phosphonate func-
tional groups of 16 with bromotrimethylsilane treatments in CH3CN in the pres-
ence of 2,6-lutidine yielded adenosine phosphonic acid derivative 17 (Scheme 3).[14]
The structure of 17 was unambiguously determined by spectroscopical analysis: 1H
NMR, 13NMR, 31P NMR, UV, HRMS.
Figure . Synthesisrationaleofꢀ,ꢀ-difluoroandapiosenucleosidephosphonicacidsshowingpotent
biological activity.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
133
Scheme . Synthesis of ꢀ,ꢀ,ꢀ-trifluoro-apiosyl adenosine phosphonic acid analogues.
Condensation of 2-fluoro-6-chloropurine[15] with glycosyl donor 14 proceeded
under conditions similar to those used for synthesis of analogues 15α and 15β to
yield 18α (35%) and 18β (36%), respectively. The relative stereo chemistries of
purine analogs18α and 18β were determined by the study of NOE experiments
between proximal diastereotropichydrogens.
Mild bubbling ammonia into compound 18β in DME yielded 2-fluoro-
6-aminopurine[16] analogue 19 (11%) and 2-amino-6-chloropurine analogue
20(41%), respectively. In nucleophilic aromatic substitution, fluorine atom is bet-
ter leaving group than chlorine atom. The 2-amino-6-chloropurine derivative 20
was treated with TMSBr and 2,6-lutidine to yield phosphonic acid and was then
treated with sodium methoxide and 2-mercaptoethanol in methanol to produce
guanosinephosphonic acid 21 (Scheme 4).[17]
Figure . NOE differences between the proximal hydrogens of 15α and 15β.

134
E. KIM AND J. H. HONG
Scheme . Synthesis of ꢀ,ꢀ,ꢀ-trifluoro-apiosyl guanosinephosphonic acid analogues.
Condensation of N4-benzoyl cytosine with glycosyl donor 14 proceeded under
conditions similar to those used for the synthesis of adenine analogues to yield
22α (33%) and 22β (34%), respectively. Ammonolysis of 22β followed by depro-
tection of diethyl phosphonate furnished the target cytosine phosphonic acid 24
(Scheme 5). Also, uracil and thymine nucleoside analogues 27 and 28 were also pre-
pared from 14 via condensation and deprotection procedures (Scheme 6).
Biological activity evaluation
The antiviral assay against several viruses such as the human immunodeficiency
virus 1 (HIV-1), herpes simplex virus-1,2 (HSV-1,2) and human cytomegalovirus
(HCMV) was performed. As shown in Table 1, compound pyrimidine analog 28
exhibited moderate or weak antiviral activity against HCMV in the Davis cell with-
out any cytotoxicity up to 100 μmol.[18]The reason for the lower antiviral activity
of final phosphonic acids than their ester analogies might be the result of poor cell
membrane diffusion of phosphonate ions in vitro conditions. This suggests that this

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
135
Scheme . Synthesis of ꢀ,ꢀ,ꢀ-trifluoro-apiosyl cytosine phosphonic acid analogues.
class of apiosyl nucleoside, which has fluorine group in the 2ꢀ-position, can be a
novel structural template for the development of new anti-HCMV agents.
In summary, based on the potent biological activities of the fluorinated phos-
phonate nucleosides and apiosylnucleoside phosphonic acid analogues, we designed
and successfully synthesized novel 5ꢀ,5ꢀ-difluoro-2ꢀ-fluoroapiosyl nucleosidephos-
phonic acid analogues from 1,3-diacetylacetone. Among them, pyrimidine analog
28 showed significant anti-HCMV activity.
Experimental Section
Uncorrected melting points were determined using a Mel-temp II laboratory device.
Nuclear magnetic resonance (NMR) spectra were recorded using 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), or dd (doublet of doublets). Ultraviolet (UV) spectra were
obtained using a Beckman DU-7 spectrophotometer (Beckman, South Pasadena,
CA, USA). Mass spectra (MS) were collected in electrospray ionization (ESI) mode.
Elemental analyses were performed using a Perkin–Elmer 2400 analyzer (Perkin–
Elmer, Norwalk, CT, USA). Thin layer chromatography (TLC) was performed on

136
E. KIM AND J. H. HONG
Scheme . Synthesis of ꢀ,ꢀ,ꢀ-trifluoro-apiosyl uracil and thymine phosphonic acid analogues.
Table . The antiviral activity of the synthesized compounds
HIV-EC(μM)
HSV-EC(μM)
HSV-EC(μM)
HCMVEC(μM)
CytotoxicityCC(μM)







β
β

>
>
.
.
.
>
>
>
>
>
>
.
ND
>
>
>
>
>
>
>
>
>
>
>
ND
>
>
>
>
>
>
>
>
>
>
>
ND
>
>
>
>
>
>
>
.
.
.
.
ND
>
>
>
>
>
>
>
>

>

.
>

AZT
GCV
ACV
ND
.
ND
ND
.
ND
ND
>
AZT: Azidothymidine; GCV: Ganciclovir; ACV: Acyclovir
ND: Not Determined
EC(μM): Concentration required to inhibit % of the virus induced cytopathicity
CC(μM): Concentration required to reduce the cell viability by %

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
137
Uniplates (silica gel) purchased from Analtech Co. (7558, Newark, DE, USA). All
reactions were performed in a nitrogen atmosphere unless otherwise specified. Dry
dichloromethane, benzene, and pyridine were obtained by distillation from CaH2.
Dry tetrahydrofuran (THF) was obtained by distillation from Na and benzophenone
immediately prior to use.
(rel)-(2S,3S)-2-Fluoro-dihydro-3-(hydroxymethyl)furan-1(3H)-one (7)
To a solution of lactone 6 (3.168 g, 24mmol) in 120 mL of EtOAc, 1.2 g of Pd/C
(5% w/w) was added under H2 atmosphere; the mixture was stirred for 6 h. After
filtration of the reaction mixture through a celite pad, the filtrate was concentrated
and purified using silica gel column chromatography (EtOAc/hexane, 1:3) to yield
compound 7 (2.89 g, 90%). 1H NMR (CDCl3, 300 MHz) δ 4.41 (dd, J= 8.8, 7.2 Hz,
1H), 4.25–4.08 (m, 2H), 3.64 (dd, J=9.2, 6.8 Hz, 1H), 3.35 (dd, J=9.2, 8.2 Hz, 1H),
2.47–2.39 (m, 1H); 13C NMR (CDCl3, 75 MHz) δ 172.6 (d, J= 22.4 Hz), 104.9 (d,
J= 208.6 Hz), 64.7, 63.2, 34.6 (d, J= 21.8 Hz); Anal. Calcd. for C5H7FO3: C, 44.78;
H, 5.26; found: C, 44.65; H, 5.34; MS m/z135 (M + H)+.
(rel)-(2S,3R)-2-Fluoro-dihydro-3-(t-butyldimethylsilyloxymethyl)furan-1(3H)-one
(8)
t-Butyldimethylsilyl chloride (TBDMSCl) (1.54 g, 10.24 mmol) was added slowly
at 0°C to a solution of 7 (1.15 g, 8.62 mmol) and imidazole (1.17 g, 17.24 mmol)
in CH2Cl2 (40 mL), and stirred for 6 h at room temperature. The solvent was
evaporated under reduced pressure. The residue was diluted with H2O (100 mL)
and extracted twice with ethyl acetate (EtOAc) (100 mL ×2). The combined
organic layer was dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified using silica gel column chromatogra-
1
phy (EtOAc/hexane, 1:3) to yield compound 8 (1.94 g, 91%): H NMR (CDCl3,
300 MHz) δ 4.40–4.35 (dd, J= 11.4, 8.8 Hz, 1H), 4.26–4.12 (m, 2H), 3.86 (dd,
J= 10.8, 6.6 Hz, 1H), 3.65 (dd, J= 10.8, 8.2 Hz, 1H), 2.48–2.35 (m, 1H), 0.87
(m, 9H), 0.03 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 171.4 (d, J= 23.6 Hz),
103.7 (d, J= 202.6 Hz), 65.4, 63.9, 35.5 (d, J= 20.4 Hz), 25.6, 18.4, −4.6; Anal.
Calcd. for C11H21FO3Si: C, 53.19; H, 8.52; found: C, 53.24; H, 8.59; MS m/z 249
(M + H)+.
(rel)-(1R/1S,2S,3R)-[(2-Fluoro-tetrahydro-1-hydroxyfuran-3-yl)methoxy](t-butyl)
dimethylsilane (9)
A solution of compound 8 (1.05 g, 4.26mmol) in toluene (50 mL) was treated with
8.52 mL of 1 M DIBAL-H in hexane at −78°C for 1 h. The reaction was quenched
with 2 mL of methanol (MeOH) and warmed to room temperature for 1 h before
aqueous (aq) NaHCO3 (4 mL) and EtOAc (50 mL) were added to the mixture.

138
E. KIM AND J. H. HONG
The resulting mixture was filtered and the filtrate was concentrated to dryness. The
residue was purified using silica gel column chromatography (EtOAc/hexane, 1:6)
to yield compound 9 (863mg, 81%). 1H NMR (CDCl3, 300 MHz) δ 5.78 (d, J = 16.4
Hz, 0.5H),5.68 (d, J =14.8 Hz, 0.5H), 3.89–3.84 (m, 2H), 3.67–3.51 (m, 3H), 2.31–
2.24 (m, 1H), 0.89 (m, 9H), 0.02 (m, 6H); Anal. Calcd. for C11H23FO3Si: C, 52.77;
H, 9.26; found: C, 52.90; H, 9.34.
(rel)-(1R/1S,2S,3R)-[(2-Fluoro-tetrahydro-1-methoxyfuran-3-yl)methoxy](t-butyl)
dimethylsilane (10)
Lactol9 (1.84 g, 7.38 mmol) was dissolved in anhydrous diethyl ether (25 mL),
and powdered anhydrous molecular sieves (4 A˚, 0.18 g) were added. With stirring,
methyl orthoformate (1.62 mL, 14.4 mmol) and BF3ꢀOEt2 (224 μL) were added,
and stirred for 40 min. The reaction mixture was quenched with Et3N and brine
until neutral. The mixture was extracted with diethyl ether, dried over anhydrous
MgSO4, and concentrated to give a residue. The residue was purified by using silica
gel column chromatography (EtOAc/hexane, 1:20) to yield compound 10 (1.73 g,
89%) as diastereomeric mixture. 1H NMR (CDCl3, 300 MHz) δ 5.46 (d, J=17.7Hz,
0.5H), 5.37 (d, J=15.6 Hz, 0.5H), 3.97–3.82 (m, 3H), 3.73–3.56 (m, 2H), 2.25–2.18
(m, 1H), 0.89 (m, 9H), 0.01 (m, 6H); Anal. Calcd. for C12H25FO3Si: C, 54.51; H,
9.53. Found: C, 54.39; H, 9.47; MS m/z265 (M + H)+.
(rel)-(1R/1S,2S,3S)-(2-Fluoro-tetrahydro-1-methoxyfuran-3-yl)methanol (11)
To a solution of compound10 (620 mg, 2.34 mmol) in THF (15 mL), TBAF (3.52
mL, 1.0 M solution in THF) at 0°C was added. The mixture was stirred at room
temperature for 5 h, and concentrated. The residue was purified by silica gel col-
umn chromatography (EtOAc/hexane, 1:5) to give compound11 (316 mg, 90%) as
1
diastereomeric mixture. H NMR (CDCl3, 300 MHz) δ 5.42–5.31 (m, 1H), 4.02–
3.98 (m, 1H), 3.85–3.76 (m, 1H), 3.64–3.47 (m, 2H), 3.38–3.29 (m, 1H), 3.25, 3. 24
(s, s, 3H), 2.21–2.12 (m, 1H); Anal. Calcd. for C6H11FO3: C, 48.00; H, 7.38. Found:
C, 48.12; H, 7.45; MS m/z151 (M + H)+.
(rel)-(1R/1S,2S,3R)-(2-Fluoro-tetrahydro-1-methoxyfuran-3-yl)methyl
trifluoromethanesulfonate (12)
To a cooled solution (−78°C) of glycoside 11 (324 mg, 2.16 mmol) in pyridine
(0.854 mL, 10.8 mmol) and CH2Cl2 (25 mL), triflic anhydride (730 mg, 2.59
mmol) was slowly added. After 3.5 h, the reaction mixture was poured onto a
mixture of ice and sodium hydrogen carbonate. The aqueous layer was extracted
with CH2Cl2 (3× 50 mL), and the combined CH2Cl2 solution were dried, and
rapidly and repeatedly concentrated with toluene to remove any residual pyridine.
The residue was extracted with light petroleum (3× 50 mL), and the combined
extracted were filtered and cooled. After careful evaporation of additional solvent,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
139
the crude residue 12 (597 mg, ∼98%) was subjected to next reaction without further
purification.
(rel)-Diethyl 5,5-difluoro-4-[(1R/1S,2S,3S)-2-fluoro-tetrahydro-1-methoxyfuran-3-
yl] ethylphosphonate (13)
To a solution of diisopropylamine (471 μL, 3.36 mmol) and HMPA (584 μL, 3.36
mmol) at −78°C in THF (7 mL) under Ar was added n-butyllithium (2.1 mL of a
1.6 M solution in hexane, 3.36 mmol). The resulting solution was allowed to stir for
30 min at 0°C and then cooled to −78°C. To this solution of LDA at −78°C were
added via cannula, a (−78°C) solution of diethyl (α,α-difluoromethyl) phospho-
nate (631 mg, 3.36 mmol) in THF (2.8 mL), and, 3 min later, a (−78°C) solution
of triflate12 (270 mg, 0.96 mmol) in THF (2.8 mL), dropwise, via cannula. After
10 min at −78°C, the reaction was quenched by adding aqueous NH4Cl (16.0 mL)
and Et2O (16.0 mL). The aqueous layer was further extracted with EtOAc (2× 65
mL), and the combined organic extracts were dried, filtered, and evaporated. Silica
gel flash chromatography (EtOAc/hexane, 1:2) gave 13 (202 mg, 66%) as a form. 1H
NMR (CDCl3, 300 MHz) δ 5.44–4.32 (m, 1H), 4.25–4.18 (m, 4H), 3.99–3.78 (m,
2H), 3.61–3.56 (m, 1H), 3.25, 3.24 (s, s, 3H), 2.35–2.16 (m, 3H), 1.24 (m, 6H); Anal.
Calcd. for C11H20F3O5P: C, 41.26; H, 6.29. Found: C, 41.14; H, 6.32; MS m/z321 (M
+ H)+.
(rel)-Diethyl 4-[(1R/1S,2S,3S)-1-acetoxy-2-fluoro-tetrahydrofuran-3-yl]-5,5-difluo
roethylphosphonate (14)
Glycoside 13 (505 mg, 1.58 mmol)was dissolved in EtOAc (13 mL), mixed with
a solution of EtOAc (25 mL), acetic anhydride (14.5 mL), acetic acid (11.0 mL)
and conc H2SO4 (0.066 mL) at −10°C, and stirred for 24 h at room temperature.
The reaction was diluted with CHCl3 (100 mL) and poured into cold 5% aque-
ous NaHCO3 (165 mL). The organic layer was separated and the aqueous layer
extracted with CHCl3 (3× 33 mL). The combined organic layers were washed with
brine, dried, and evaporated to dryness. The residue was purified using silica gel col-
umn chromatography (EtOAc/hexane, 1:2) to yield compound 14 (456mg, 83%) as
a form. 1H NMR (CDCl3, 300 MHz) δ 6.55–6.42 (m, 1H), 4.27–4.19 (m, 5H), 3.86–
3.70 (m, 1H), 2.63–2.53 (m, 1H), 2.29–2.18 (m, 3H), 2.03, 2.01 (s, s, 3H), 1.26–1.21
(m, 6H); Anal. Calcd. for C12H20F3O6P: C, 41.39; H, 5.79. Found: C, 41.44; H, 5.86;
MS m/z349 (M + H)+.
(rel)-Diethyl 4-[(1S,2S,3S)-1-(6-chloro-9H-purin-9-yl)-2-fluoro-tetrahydrofuran-3-
yl]-5,5-difluoroethylphosphonate (15α) and (rel)-diethyl 4-[(1R,2S,3S)-1-(6-chloro-
9H-purin-9-yl)-2-fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate
(15β)
6-Chloropurine (266 mg, 1.72 mmol), anhydrous HMDS (12 mL), and a catalytic
amount of ammonium sulfate (12 mg) were refluxed to a clear solution (18 h);
the solvent was then distilled under anhydrous conditions. The residue obtained

140
E. KIM AND J. H. HONG
was dissolved in anhydrous 1,2-dichloroethane (10 mL), and to this mixture, a
solution of 14 (299 mg, 0.86 mmol) in dry DCE (10 mL) and TMSOTf (382 mg,
1.72mmol) was added, and stirred for 6 h at rt. The reaction mixture was quenched
with 10.0 mL of saturated NaHCO3, stirred for 2 h, filtered through a Celite pad,
and the filtrate obtained was then extracted twice with CH2Cl2 (2×100 mL). Com-
bined organic layers were dried over anhydrous MgSO4, filtered, and concentrated
under vacuum. The residue was purified using silica gel column chromatography
(EtOAc/hexane/MeOH, 3:1:0.03) to yield compounds 15α (121 mg, 32%) and 15β
1
(117 mg, 31%), respectively. Data for 15α: H NMR (CDCl3, 300 MHz) δ 8.78
(s, 1H), 8.26 (s, 1H), 6.23 (dd, J=16.4, 6.0 Hz, 1H), 4.27–4.23 (m, 4H), 3.87 (dd,
J= 10.8, 6.2 Hz, 1H), 3.68–3.51 (m, 2H), 2.22–2.13 (m, 1H), 1.74–1.61 (m, 2H),
1.27 (m, 6H); 31P (121.5 MHz, CDCl3) δ7.64 (t, JP,F = 101.6 Hz); Anal. Calc. for
C15H19ClF3N4O4P: C, 40.69; H, 4.33; N, 12.65. Found: C, 40.82; H, 4.40; N, 12.78;
MS m/z443 (M + H)+; Data for 15β: 1H NMR (CDCl3, 300 MHz) δ 8.73 (s, 1H), 8.24
(s, 1H), 6.19 (dd, J=18.2, 6.8 Hz, 1H), 4.30–4.25 (m, 4H), 3.92 (dd, J= 11.0, 6.4 Hz,
1H), 3.71–3.53 (m, 2H), 2.25–2.14 (m, 1H), 1.80–1.67 (m, 2H), 1.29–1.28 (m, 6H);
31P (121.5 MHz, CDCl3) δ7.70 (t, JP,F = 104.2 Hz); Anal. Calc. for C15H19ClF3N4O4P
(+0.5 MeOH): C, 40.64; H, 4.62; N, 12.23. Found: C, 40.55; H, 4.57; N, 12.32; MS
m/z443 (M + H)+.
(rel)-Diethyl 4-[(1R,2S,3S)-1-(6-amino-9H-purin-9-yl)-2-fluoro-tetrahydrofuran-3-
yl]-5,5-difluoroethylphosphonate (16)
A solution of 15β (362 mg, 0.82mmol) in saturated methanolic ammonia (15 mL)
was stirred overnight at 65°C in a steel bomb and the volatiles were evaporated.
The residue was purified using silica gel column chromatography (MeOH/CH2Cl2,
1:12) to yield 16 (232 mg, 67%) as a white solid: UV (MeOH) λ
262.0 nm;
max
1H NMR (DMSO-d6, 300 MHz) δ8.41 (s, 1H), 8.17 (s, 1H), 7.40 (br s, 2H, D2O
exchangeable), 6.25 (dd, J=17.8, 6.2 Hz, 1H), 4.26–4.22 (m, 4H), 3.87–3.70 (m,
2H), 3.58 (dd, J= 10.6, 6.4 Hz, 1H), 2.26–2.18 (m, 1H),2.03–1.84 (m, 2H), 1.31–
1.28 (m, 6H);31P (121.5 MHz, DMSO-d6) δ7.54 (app t, JP,F = 103.2 Hz); Anal. Calc.
for C15H21F3N5O4P (+1.0 MeOH): C, 42.22; H, 5.53; N, 15.39; Found: C, 42.43; H,
5.65; N, 15.49; MS m/z424 (M + H)+.
(rel)-4-[(1R,2S,3S)-1-(6-Amino-9H-purin-9-yl)-tetrahydrofuran-3-yl]-2-fluoro-5,5-
difluoroethyl-phosphonic acid sodium salt (17)
To a solution of compound 16 (139 mg, 0.33 mmol) and 2,6-lutidine (2.3 mL,
19.8 mmol) in 24 mL of dry CH3CN was added bromotrimethylsilane (1.01 g, 6.6
mmol) at room temperature under nitrogen. The reaction mixture was continu-
ously refluxed for 24 h. The reaction mixture was concentrated under high vac-
uum at room temperature, and the residue was coevaporated with MeOH and 0.5

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
141
M TEAB solution. Purification by HPLC using reverse phase C18 and ion exchange
with Dowex-Na+ resin offered 17 (63 mg, 49%) as a colorless solid (sodium salt)
after lyophilization. 1H NMR (D2O, 300 MHz) δ8.38 (s, 1H), 8.18 (s, 1H), 6.19 (dd,
J=18.0, 6.2 Hz, 1H), 3.82–3.63 (m, 2H), 3.58 (dd, J= 10.2, 6.6 Hz, 1H), 2.61 (dd, J=
9.8, 6.4 Hz, 1H), 2.26–2.18 (m, 2H), 1.84–1.65 (m, 1H); 13C NMR (D2O, 75 MHz)
δ 155.8, 153.1, 149.4, 140.2, 125.4 (dt, J=211.2,270.6 Hz), 119.7,96.2 (d, J= 202.6
Hz), 89.6, (d, J= 24.7 Hz), 68.4, 24.4 (d, J= 22.8 Hz), 19.2 (dd, J= 25.4, 19.7 Hz);
31P (121.5 MHz, D2O) δ5.82 (dd, JP,F = 105.3, 90.2 Hz); HPLC tR= 10.24; HRMS
[M-H]+ req. 366.0745, found 366.0743.
(rel)-Diethyl 4-[(1S,2S,3S)-1-(2-fluoro-6-chloro-9H-purin-9-yl)-2-fluoro-
tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (18α) and (rel)-diethyl
4-[(1R,2S,3S)-1-(2-fluoro-6-chloro-9H-purin-9-yl)-2-fluoro-tetrahydrofuran-3-yl]-
5,5-difluoroethylphosphonate (18β)
Condensation of 14 with 2-fluoro-6-chloropurine under Vorbrüggen condensation
conditions similar to those described for 15α and 15β yielded 18α and 18β, respec-
tively. Data for 18α: yield 35%; UV (MeOH) λmax 268.0 nm; 1H NMR (CDCl3, 300
MHz) δ 8.26 (s, 1H), 6.25 (dd, J=18.8, 6.0 Hz, 1H), 4.29–4.27 (m, 4H), 3.87–3.66
(m, 2H), 3.57 (dd, J= 10.4, 7.2 Hz, 1H), 2.21–2.09 (m, 1H), 1.84–1.78 (m, 2H),
1.31 (m, 6H); 31P (121.5 MHz, CDCl3) δ7.32 (t, JP,F = 106.8 Hz); Anal. Calc. for
C15H18ClF4N4O4P: C, 39.10; H, 3.94; N, 12.16; Found: C, 39.17; H, 3.89; N, 12.18;
1
MS m/z461 (M + H)+.data for 18β: yield 36%; UV (MeOH) λ
268.5 nm; H
max
NMR (CDCl3, 300 MHz) δ 8.30 (s, 1H), 6.22 (dd, J=19.0, 6.2 Hz, 1H), 4.31–4.29
(m, 4H), 3.85 (dd, J= 10.4, 7.0 Hz, 1H), 3.60–3.43 (m, 2H), 2.28–2.14 (m, 1H), 1.78–
1.62 (m, 2H), 1.29 (m, 6H); 31P (121.5 MHz, CDCl3) δ7.25 (t, JP,F = 105.6 Hz); Anal.
Calc. for C15H18ClF4N4O4P: C, 39.10; H, 3.94; N, 12.16; Found: C, 39.03; H, 3.98;
N, 12.11; MS m/z461 (M + H)+.
(rel)-Diethyl 4-[(1R,2S,3S)-1-(2-fluoro-6-amino-9H-purin-9-yl)-2-fluoro-
tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (19) and (rel)-diethyl
4-[(1R,2S,3S)-1-(2-amino-6-chloro-9H-purin-9-yl)-2-fluoro-tetrahydrofuran-3-yl]-
5,5-difluoroethylphosphonate (20)
Dry ammonia gas was bubbled into a stirred solution of 18β (450 mg, 0.98mmol)
in DME (12.0 mL) at room temperature overnight. Salts were removed by filtration
and the filtrate was concentrated under reduced pressure. The residue obtained was
purified using silica gel column chromatography (MeOH/CH2Cl2, 1:10) to produce
19 (47 mg, 11%) and 20 (183 mg, 41%). Data for 19; UV (MeOH) λmax 261.0 nm; 1H
NMR (DMSO-d6, 300 MHz) δ 8.32 (s, 1H), 7.70 (br s, NH2, 2H, D2O exchangeable),
6.21 (dd, J=18.5, 6.2 Hz, 1H), 4.30–4.28 (m, 4H), 3.88–3.69 (m, 2H), 3.61–3.55 (dd,
J= 9.8, 6.6 Hz, 1H), 2.26–2.12 (m, 1H),1.70–1.58 (m, 2H), 1.31 (m, 6H); 31P (121.5

142
E. KIM AND J. H. HONG
MHz, DMSO-d6) δ 7.13 (t, JP,F = 109.5 Hz); Anal. Calc. for C15H20F4N5O4P (+1.0
MeOH): C, 40.61; H, 5.11; N, 14.80; Found: C, 40.83; H, 5.19; N, 14.76; MS m/z
442 (M + H)+. Data for 20; UV (MeOH) λmax 307.0 nm; 1H NMR (DMSO-d6, 300
MHz) δ 8.14 (s, 1H), 7.67 (br s, NH2, 2H, D2O exchangeable), 6.21 (dd, J=19.1, 6.4
Hz, 1H),4.30 (m, 4H), 3.86 (dd, J= 9.7, 7.4 Hz, 1H), 3.65–3.51 (m, 2H), 2.32–2.25
(m, 1H),1.82–1.76 (m, 2H), 1.30–1.28 (m, 6H); 31P (121.5 MHz, DMSO-d6) δ7.11
(t, JP,F = 107.2 Hz); Anal. Calc. for C15H20ClF3N5O4P (+1.0 MeOH): C, 39.29; H,
4.94; N, 14.32; Found: C, 39.44; H, 4.87; N, 15.22; MS m/z 458 (M + H)+.
(rel)-4-[(1R,2S,3S)-1-(2-Amino-6-oxo-9H-purin-9-yl)-tetrahydrofuran-3-yl]-2-
fluoro-5,5-difluoroethyl-phosphonic acid sodium salt(21)
To a solution of 20 (278 mg, 0.61 mmol) and 2,6-lutidine (3.92 g, 36.6 mmol) in
dry CH3CN (24.4 mL), trimethylsilyl bromide (1.87 g, 12.2 mmol) was added at
room temperature. The mixture was stirred for 24 h and the solvent was removed
using evaporation with MeOH three times. The residue was dissolved in MeOH
(24.4 mL) and 2-mercaptoethanol (190 mg, 2.44 mmol), and then NaOMe (131 mg,
2.44 mmol) was added. The mixture was refluxed for 18 h under N2, cooled, neu-
tralized with glacial AcOH, and evaporated to dryness under vacuum. The residue
was purified by a C18 reverse-phase column chromatography with a gradient from
100% triethylammonium acetate buffer (TEAA buffer, 50 mM) to a mixture of 80%
TEAA buffer and 20% CH3CN (tR 9.90 min), and ion exchange with Dowex-Na+
resin yielded 21 (133 mg, 54%) as a colorless solid (sodium salt) after lyophilization.
1H NMR (D2O, 300 MHz) δ 7.95 (s, 1H), 6.18 (dd, J=17.8, 6.2 Hz, 1H), 3.90–3.75
(m, 2H), 3.60–3.56 (dd, J= 9.8, 7.2 Hz, 1H), 2.26–2.17 (m, 1H), 1.78–1.65 (m, 2H);
13C NMR (D2O, 75 MHz) δ 157.8, 154.7, 152.5, 135.6, 128.4 (ddd, J = 208.2, 260.8,
Hz), 118.3, 97.4 (d, J = 210.5 Hz),79.3 (d, J = 26.0 Hz), 65.9, 24.6 (d, J = 24.8 Hz),
19.6 (dd, J = 23,8, 18.6 Hz); 31P (121.5 MHz, D2O) δ5.89 (dd, JP,F = 110.2, 88.4 Hz);
HPLC tR= 9.90 min; HRMS [M-H]+ req. 382.0756, found 382.0754.
(rel)-Diethyl 4-[(1S,2S,3S)-1-(N4-benzoylamino-2-oxo-3,4-dihydropyrimidin-
1(2H)-yl)-2-fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (22α) and
(rel)-diethyl 4-[(1R,2S,3S)-1-(N4-benzoylamino-2-oxo-3,4-dihydropyrimidin-
1(2H)-yl)-2-fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (22β)
Condensation of 14 with N4-benzoyl cytosine under Vorbrüggen condensation con-
ditions similar to those described for 15α and15β yielded 22α and 22β as solids.
Data for 22α: yield 33%; 1H NMR (CDCl3, 300 MHz) δ 8.18 (d, J = 7.0 Hz, 1H),
8.03–7.98 (m, 2H), 7.65–7.54 (m, 4H), 6.21 (dd, J =18.6, 6.2 Hz, 1H), 4.29–4.26 (m,
4H), 4.18–4.10 (m, 1H), 3.86 (dd, J = 10.2, 7.6 Hz, 1H), 3.59 (dd, J = 10.2, 6.8 Hz,
1H), 2.34–2.25 (m, 1H), 1.78–1.65 (m, 2H), 1.32 (m, 6H); 31P (121.5 MHz, CDCl3)
δ7.32 (t, JP,F = 106.2 Hz); Anal. Calc. for C21H25F3N3O6P: C, 50.10; H, 5.01; N, 8.35;

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
143
Found: C, 50.21; H, 4.95; N, 8.28; MS m/z504 (M + H)+.data for 22β: yield 34%;
1H NMR (CDCl3, 300 MHz) δ 8.20 (d, J = 7.2 Hz, 1H), 8.05–7.99 (m, 2H), 7.63–
7.55 (m, 4H), 6.19 (dd, J = 18.4, 6.4 Hz, 1H), 4.24–4.19 (m, 4.5H), 4.16–4.11 (m,
0.5H), 3.88 (dd, J= 10.4, 7.0 Hz, 1H), 3.65 (dd, J = 10.4, 6.2 Hz, 1H), 2.37–2.28 (m,
1H), 1.72–1.63 (m, 2H), 1.32–1.29 (m, 6H); 31P (121.5 MHz, CDCl3) δ7.27 (t, JP,F
= 107.4 Hz); Anal. Calc. for C21H25F3N3O6P (+0.5 MeOH): C, 49.74; H, 5.24; N,
8.09; Found: C, 49.87; H, 5.18; N, 8.12; MS m/z504 (M + H)+.
(rel)-Diethyl 4-[(1R,2S,3S)-1-(4-amino-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)-2-
fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (23)
Compound 22β (442 mg, 0.88 mmol) was treated with saturated methanolic ammo-
nia (12 mL) overnight at rt. The solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography (MeOH/CH2Cl2/1:10) to
give compound 23 (305 mg, 87%): UV (MeOH) λmax 270.5 nm; 1H NMR (DMSO-
d6, 300 MHz) δ 7.75 (d, J = 7.0 Hz, 1H), 7.27 (br d, 2H, D2O exchangeable), 6.15
(dd, J = 19.2, 7.0 Hz, 1H), 5.75 (d, J = 7.0 Hz, 1H), 4.24–4.20 (m, 4.5H), 4.13–4.09
(m, 0.5H), 3.86 (dd, J= 10.8, 6.8 Hz, 1H), 3.70 (dd, J = 10.8, 7.2 Hz, 1H), 2.36–2.23
(m, 1H), 2.01–1.92 (m, 2H), 1.27–1.24 (m, 6H); 31P (121.5 MHz, DMSO-d6) δ7.08
(t, JP,F = 108.2 Hz); Anal. Calc. for C14H21F3N3O5P (+1.0 MeOH): C, 41.78; H, 5.84;
N, 9.74; Found: C, 41.85; H, 5.77; N, 9.68; MS m/z400 (M + H)+.
(rel)-4-[(1R,2S,3S)-1-(4-Amino-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)-tetrahy
drofuran-3-yl]-2-fluoro-5,5-difluoroethyl-phosphonic acid sodium salt(24)
Final cytosine analogue 24 was synthesized from 23 by the similar deprotection pro-
cedure as described for 17: Yield 50%; UV (H2O) λ
271.0 nm; 1H NMR (D2O,
max
300 MHz) δ 7.47 (d, J= 7.0 Hz, 1H), 6.11 (dd, J=18.4, 6.8 Hz, 1H), 5.56 (d, J= 7.0
Hz, 1H), 4.27–4.18 (m, 1H), 3.89 (dd, J= 10.4, 6.0 Hz, 1H), 3.67 (dd, J= 10.4, 8.0
Hz, 1H), 2.36–2.25 (m, 1H), 2.02–1.93 (m, 2H); 13C NMR (D2O, 75 MHz) δ 165.7,
155.6, 141.5, 123.7 (dt, J = 211.7, 265.2 Hz), 96.2 (d, J= 211.6 Hz, 1H), 87.2 (d, J =
24.7 Hz), 66.2, 23.4 (d, J = 20.2 Hz), 19.2 (dd, J = 25.8, 20.8 Hz); 31P (121.5 MHz,
D2O) δ5.76 (dd, JP,F = 113.4, 88.4 Hz); HPLC tR = 9.42 min; HRMS [M-H]+ req.
342.0637, found 342.0635.
(rel)-Diethyl 4-[(1S,2S,3S)-1-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-fluoro-
tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (25α) and (rel)-diethyl 4-[
(1R,2S,3S)-1-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-fluoro-tetrahydrofuran-
3-yl]-5,5-difluoroethylphosphonate (25β)
Uracil analogues were synthesized using the similar Vorbrüggen condensation con-
ditions as described for the synthesis of 6-chloropurine analogues 15α and 15β.
Data for 25α: yield 36%; 1H NMR (DMSO-d6, 300 MHz) δ 11.19 (br s, 1H, D2O
exchangeable), 7.52 (d, J = 7.4 Hz, 1H), 6.17 (dd, J =18.2, 6.6 Hz, 1H), 5.58 (d, J

144
E. KIM AND J. H. HONG
= 7.4 Hz, 1H), 4.28–4.25 (m, 4.5H), 4.18–4.14 (m, 0.5H), 3.88 (dd, J = 10.8, 6.8
Hz, 1H), 3.75 (dd, J = 10.8, 7.8 Hz, 1H), 2.23–2.15 (m, 1H), 1.98–1.85 (m, 1H),
1.30 (m, 6H); 31P (121.5 MHz, DMSO-d6) δ 7.54 (t, JP,F = 112.0 Hz); Anal. Calc.
for C14H20F3N2O6P: C, 42.01; H, 5.04; N, 7.00; Found: C, 42.13; H, 5.08; N, 7.09;
MS m/z401 (M + H)+. Data for 25β: yield 37%;1H NMR (DMSO-d6, 300 MHz)
δ 11.21 (br s, 1H, D2O exchangeable), 7.56 (d, J = 7.4 Hz, 1H), 6.19 (dd, J = 18.8,
6.8 Hz, 1H), 5.55 (d, J = 7.4 Hz, 1H), 4.29–4.18 (m, 5H), 3.91 (dd, J = 10.2, 7.0 Hz,
1H), 3.72 (dd, J = 10.2, 6.2 Hz, 1H), 2.21–2.13 (m, 1H), 1.95–1.81 (m, 1H), 1.32–
1.29 (m, 6H); 31P (121.5 MHz, DMSO-d6) δ7.59 (t, JP,F = 109.8 Hz); Anal. Calc. for
C14H20F3N2O6P (+0.5 MeOH): C, 41.85; H, 5.33; N, 6.73; Found: C, 41.95; H, 5.28;
N, 6.83; MS m/z401 (M + H)+.
(rel)-Diethyl 4-[(1S,2S,3S)-1-(2,4-dioxo-5-methyl-3,4-dihydropyrimidin-1(2H)-yl)-
2-fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (26α) and
(rel)-diethyl 4-[(1R,2S,3S)-1-(2,4-dioxo-5-methyl-3,4-dihydropyrimidin-1(2H)-yl)-
2-fluoro-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate (26β)
Thymine analogues were synthesized using the similar Vorbrüggen condensation
conditions as described for the synthesis of 6-chloropurine analogues 15α and 15β.
1
Data for 26α: yield 35%; H NMR (DMSO-d6, 300 MHz) δ 11.18 (br s, 1H, D2O
exchangeable), 7.69 (s, 1H), 6.19 (dd, J = 19.2, 7.0 Hz, 1H), 4.31–4.25 (m, 4.5H),
4.17–4.12 (m, 0.5H), 3.87 (dd, J = 10.4, 7.0 Hz, 1H), 3.60 (dd, J = 10.4, 7.6 Hz, 1H),
2.35–2.28 (m, 1H), 2.02–1.93 (m, 2H), 1.79 (s, 3H), 1.32–1.29 (m, 6H); 31P (121.5
MHz, DMSO-d6) δ 7.44 (t, JP,F = 109.6 Hz); Anal. Calc. for C15H22F3N2O6P: C,
43.48; H, 5.35; N, 6.76; Found: C, 43.56; H, 5.43; N, 6.87; MS m/z415 (M + H)+.
Data for 27β: yield 36%; 1H NMR (DMSO-d6, 300 MHz) δ 11.14 (br s, 1H, D2O
exchangeable), 7.71 (s, 1H), 6.21 (dd, J = 18.6, 7.2 Hz, 1H), 4.29–4.20 (m, 4.5H),
4.15–4.11 (m, 0.5H), 3.83 (dd, J = 10.2, 7.2 Hz, 1H), 3.62 (dd, J = 10.2, 7.2 Hz, 1H),
2.23–2.16 (m, 1H), 1.94–1.83 (m, 2H), 1.72 (s, 3H), 1.30–1.27 (m, 6H); 31P (121.5
MHz, DMSO-d6) δ 7.56 (t, JP,F = 111.3 Hz); Anal. Calc. for C15H22F3N2O6P (+1.0
MeOH): C, 43.07; H, 5.87; N, 6.28; Found: C, 43.16; H, 5.78; N, 6.36; MS m/z 415
(M + H)+.
(rel)-4-[(1R,2S,3S)-1-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-
3-yl]-2-fluoro-5,5-difluoroethyl-phosphonic acid sodium salt (27)
Uracil phosphonic acid analogue 27 was synthesized from 25β using the similar
hydrolysis conditions as described for 18: Yield 49%; UV (H2O) λ
260.5 nm;
max
1H NMR (D2O, 300 MHz) δ 7.81 (d, J = 7.0 Hz, 1H), 6.13 (dd, J =17.6, 6.8 Hz,
1H), 5.85 (d, J = 7.0 Hz, 1H), 4.28 (dd, J = 9.8, 6.8 Hz, 1H), 4.19 (dd, J = 9.8, 8.2
Hz, 1H), 3.89 (dd, J = 10.8, 7.2 Hz, 1H), 3.71 (dd, J = 10.8, 6.4 Hz, 1H), 2.25–2.16
(m, 1H), 1.96–1.82 (m, 2H); 13C NMR (D2O, 75 MHz) δ 166.5, 152.5, 142.3, 126.4

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
145
(dt, J = 206.2, 268.4 Hz), 103.5, 94.6 (d, J = 205.8 Hz), 87.0 (d, J = 23.8 Hz), 67.3,
24.7 (d, J = 22.6 Hz), 18.5 (dd, J = 24.8, 19.4 Hz); 31P (121.5 MHz, D2O) δ 5.75
(dd, JP,F = 108.5, 86.7 Hz); HPLC tR=10.38 min; HRMS [M-H]+ req. 343.0637,
found 343.0635.
(rel)-4-[(1R,2S,3S)-1-(2,4-Dioxo-5-methyl-3,4-dihydropyrimidin-1(2H)-yl)-
tetrahydrofuran-3-yl]-2-fluoro-5,5-difluoroethyl-phosphonic acid sodium salt
(28)
Thymine analogue 28 was synthesized from 26β using the similar hydrolysis con-
ditions as described for 18: Yield 54%; UV (H2O) λ
267.0 nm; 1H NMR (D2O,
max
300 MHz) δ 7.73 (s, 1H), 6.18 (dd, J=17.4, 7.6 Hz, 1H), 4.26 (dd, J = 8.8, 7.6 Hz,
1H), 4.18 (dd, J = 7.8, 6.8 Hz), 3.83 (dd, J = 10.4, 7.2 Hz, 1H), 3.75 (dd, J = 10.4, 6.2
Hz), 2.24–2.17 (m, 1H), 2.03–1.92 (m, 2H), 1.77 (s, 3H); 13C NMR (D2O, 75 MHz)
δ 163.5, 150.1, 135.9, 125.6 (dt, J = 204.4, 263.6 Hz), 109.3, 86.5 (d, J = 210.4 Hz),
66.1, 23.7 (d, J = 24.4 Hz), 18.2 (dd, J = 23.4, 19.6 Hz), 12.7; 31P (121.5 MHz, D2O)
δ5.72 (t, JP,F = 109.4 Hz); HPLC tR=10.78 min; HRMS [M-H]+ req. 357.0635, found
357.0633.
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