Synthesis and antiviral evaluation of novel 2′,2′-difluoro 5′-norcarbocyclic phosphonic acid nucleosides as antiviral agents

Article abstract of DOI:10.1080/15257770.2013.854380

A very efficient synthetic route to novel 2′,2′-difluoro 5′-norcarbocyclic phosphonic acid nucleosides from but-3-en-1-ol 5 is described. The discovery of 2′-fluorinated furanose nucleoside 1 as a potent anti-HIV-1 agent has led to the synthesis and biological evaluation of 2′-modified 5′-norversions of the carbocyclic phosphonate nucleosides. The synthesized nucleoside analogues 18, 19, 23a, 23b, and 24 were tested for anti-HIV activity as well as cytotoxicity. Adenine analogue 19 shows significant anti-HIV-1 activity (EC₅₀ = 13 μM).

Full text of DOI:10.1080/15257770.2013.854380

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Synthesis and Antiviral Evaluation of
Novel 2′,2′-Difluoro 5′-Norcarbocyclic
Phosphonic Acid Nucleosides as Antiviral
Agents
Guang Huan Shen a & Joon Hee Hong a
a BK-21 Project Team, College of Pharmacy , Chosun University ,
Kwangju , Republic of Korea
Published online: 03 Mar 2014.
To cite this article: Guang Huan Shen & Joon Hee Hong (2014) Synthesis and Antiviral Evaluation of
Novel 2′,2′-Difluoro 5′-Norcarbocyclic Phosphonic Acid Nucleosides as Antiviral Agents, Nucleosides,
Nucleotides and Nucleic Acids, 33:1, 1-17, DOI: 10.1080/15257770.2013.854380
To link to this article: http://dx.doi.org/10.1080/15257770.2013.854380
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Nucleosides, Nucleotides and Nucleic Acids, 33:1–17, 2014
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.854380
SYNTHESIS AND ANTIVIRAL EVALUATION OF NOVEL
2ꢀ,2ꢀ-DIFLUORO 5ꢀ-NORCARBOCYCLIC PHOSPHONIC ACID
NUCLEOSIDES AS ANTIVIRAL AGENTS
Guang Huan Shen and Joon Hee Hong
BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju, Republic of Korea
A very efficient synthetic route to novel 2ꢁ,2ꢁ-difluoro 5ꢁ-norcarbocyclic phosphonic acid nucle-
2
osides from but-3-en-1-ol 5 is described. The discovery of 2ꢁ-fluorinated furanose nucleoside 1 as
a potent anti-HIV-1 agent has led to the synthesis and biological evaluation of 2ꢁ-modified 5ꢁ-
norversions of the carbocyclic phosphonate nucleosides. The synthesized nucleoside analogues 18,
19, 23a, 23b, and 24 were tested for anti-HIV activity as well as cytotoxicity. Adenine analogue 19
shows significant anti-HIV-1 activity (EC50 = 13 μM).
Keyword Antiviral agent; 2ꢁ-modified nucleoside; 5ꢁ-norcarbocyclic nucleoside; phos-
phonic acid nucleoside
INTRODUCTION
Fluorinated nucleosides, containing fluorine atom or fluorine-
containing groups in the sugar moiety, have drawn increasing attention
due to the introduction of the fluorine into some nucleosides resulting
in a great improvement in the bioactivity and stability of the correspond-
ing compounds.[1] GS-9148 (1) has a promising antiviral resistance profile,
retaining potency toward HIV RT-resistant virus containing M184V, mul-
tiple thymidine analogue mutations (TAMs), and K65R resistance muta-
tions.[2] GS-9131 (2), an ethylalaninyl phosphonoamidate prodrug of GS-
9148, demonstrated excellent potency toward multiple subtype of HIV-1
clinical isolates (mean EC50 = 37 nM), several fold better than 3ꢁ-azido
2ꢁ,3ꢁ-dideoxythymidine (AZT)+.[3] Especially noteworthy is gemcitabine[4]
(2ꢁ-deoxy-2ꢁ,2ꢁ-difluorocytidine) (3), which has been approved by the FDA
for the treatment of inoperable pancreatic cancer and of 5-fluorouracil-
resistant pancreatic cancer Figure 1.[5] Recently, 6ꢁ-fluorocarbocyclic nucle-
oside (4) exhibited moderate activities against herpes simplex virus type
Received 22 April 2013; accepted 8 October 2013.
Address correspondence to Joon Hee Hong, BK-21 Project Team, College of Pharmacy, Chosun
University, Kwangju 501-759, Republic of Korea. E-mail: hongjh@chosun.ac.kr
1

2
G. H. Shen et al.
(HSV-1) and type (HSV-2) in vitro.[6] The 2ꢁ,3ꢁ-dideoxynucleosides (ddNs)
have been proved to the most effective therapeutic agents against human
immunodeficiency virus (HIV) and hepatitis B virus (HBV).[7]
The phosphonate has certain advantages over its phosphate counter-
part as it is metabolically stable because its phosphorus–carbon bond is
not susceptible to hydrolytic cleavage.[8] Moreover, the spacial location of
the oxygen atom, namely the β-position from the phosphorus atom in the
nucleoside analogue, has been demonstrated to play a critical role for an-
tiviral activity.[9] These atoms for antiviral activity may be attributed to the
increased binding capacity of the phosphonate analogues to target enzymes.
Phosphorylation by kinases and incorporation into nucleic acid (eventually
leading to chain termination) is considered as an important mechanism to
explain the antiviral activity of nucleosides.[10] The potent antiviral activity
of phosphonylated nucleobases is ascribed to their intracellular phosphory-
lation to their diphosphates and to refractory incorporation of the modified
nucleosides in nucleic acids.[11]
Stimulated by these findings that 2ꢁ-fluorinated furanose nucleoside
phosphonates has excellent biological activities, we sought to synthesize a
novel class of nucleosides comprising 2ꢁ,2ꢁ-difluorinated 5ꢁ-norcarbocyclic
nucleoside phosphonate analogues in order to search for more effective
therapeutics against HIV and to provide analogues for probing the confor-
mational preferences of enzymes associated with the nucleoside kinases of
nucleosides and nucleotides.
NH2
O
NH2
N
N
N
N
O
O
P
N
HN
N
N
O
HO P
HO
O
O
N
O
O
O
F
F
2 (GS-9131)
1 (GS-9148)
O
NH2
N
NH
N
F
N
NH2
N
HO
O
N
F
HO
O
HO
4
HO
3 (gemcitabine)
FIGURE 1 Rationale for the design of the target phosphonic acid necleosides
F

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
3
OH
OH
O
ii
i
82%
91%
5
6
7
79%
OH
iii
PMBO
PMBO
HO
PMBO
4
v
59%
iv
67%
3
and
1
2
8
HO
OH
OH
9
10b
10a
Reagents: i) DMSO, (COCl)2, TEA, CH2Cl2; ii) allylMgBr, THF; iii)
Grubbs cat.(II) CH2Cl2; iv) PMBCl, NaH, DMF; v) OsO4, NMO,
acetone/t-BuOH/H2O (6:1:1).
SCHEME 1 Synthesis of cyclopentandiol intermediate 10
RESULTS AND DISCUSSIONS
As shown in Scheme 1, the target compounds were prepared from but-
3-en-1-ol 5 through a cyclopentenol intermediate 8. The cyclopentandiol
10b was prepared via catalytic osmium tetroxide and N -methyl morpholine
N -oxide (NMO) of olefin 9. As shown in Figure 2, the stereochemistry was
readily determined by NOE experiment. On irradiation of C4-H, relatively
strong NOE was observed at C1-H and C2-H of 10a, which showed 1,2,4-cis re-
lationships. But relatively weak NOE was observed at C1-H and C2-H of 10b,
which means the 1,4- and 2,4-trans relationships. Selective benzoylation of
10b gave cyclopentanol derivative 11 as racemic mixture. PCC oxidation[12]
of alcohol 11 gave a ketone 12, which was subjected to DAST fluorination[13]
to give compound 13. Removal of the benzoyl protecting group of 13 in the
condition of methanolic ammonia gave the secondary alcohol 14
H
PMBO
H
PMBO
H
4
3
2
1
H
OH
HO
H
H
HO OH
10b
10a
FIGURE 2 NOE relationships between the proximal hydrogens of 10a and 10b

4
G. H. Shen et al.
(Scheme 2). To synthesize the 2ꢁ-difluorinated carbocyclic adenosine nu-
cleoside analogues, the protected cyclopentanol 14 was treated with 6-
chloropurine under Mitsunobu conditions[14] (DIAD and PPh3). Slow addi-
tion of diisopropylazodicarboxylate (DIAD) to a mixture of cyclopentanol
14, triphenylphosphine, and the 6-chloropurine in anhydrous solvent (THF)
gave a yellow solution, which was stirred for 30 minutes at 0◦C and fur-
ther stirred overnight at room temperature (rt) to give the protected 6-
chloropurine analogue 15 as an only N 9-regioisomer [UV (MeOH) λmax
263.0 nm].[15]
PMBO
PMBO
PMBO
ii
i
66%
63%
O
OBz
HO
OBz
11
HO
OH
10b
12
iii 46%
PMBO
F
PMBO
OH
iv
78%
F
OBz
F
13
F
14
Reagents: i) BzCl, DMAP, Pyridine; ii) PCC, CH2Cl2; iii) DAST,
CH2Cl2; iv) NH3, MeOH.
SCHEME 2 Synthesis of difluorinated cyclopentanol intermediate 14
The PMB protection group was removed with 2,3-dichloro-5,6-dicyano-
p-benzoquinone (DDQ)[16] to produce the 5ꢁ-nornucleoside analogue 16,
which was treated with diethylphosphonomethyl triflate[17] using lithium
t-butoxide to yield the nucleoside phosphonate analogue 17 (Scheme 3).
The chlorine group of purine analogue 17 was then converted to amine
with methanolic ammonia at 65◦C to give a corresponding adenosine phos-
phonate derivative 18. Hydrolysis of diethyl phosphonate functional groups
of 18 by treatment with bromotrimethylsilane in CH3CN in the presence of
2,6-lutidine gave a desired adenosine phosphonic acid derivative 19.[18]
For the synthesis of guanine analogues, 2-fluoro-6-chloropurine[19] was
condensed with glycosyl donor in the similar conditions used for the con-
densation of 6-chloropurine. Mitsunobu coupling of the alcohol 14 with
2-fluoro-6-chloropurine gives analogue 20, which was sequentially subjected
to DDQ hydrolysis and phosphonate alkylation reactions in the similar pro-
cedure described for 17 to provide 22.

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
5
Cl
N
N
PMBO
i
N
PMBO
OH
F
N
62%
F
14
F
15
F
ii
64%
Cl
N
Cl
N
O
EtO P
EtO
N
iii
54%
N
O
N
N
N
HO
N
F
F
17
63%
F
16
F
iv
NH2
N
N
NH2
N
O
HO P
HO
N
O
EtO P
EtO
N
O
v
51%
N
N
O
N
F
F
F
19
18
F
Reagents: i) 6-chloropurine, DEAD, PPh3, 1,4-dioxane/DMF; ii) DDQ,
CH2Cl2/H2O (10:1); iii) (EtO)2POCH2OTf, LiO-t-Bu, THF; iv) NH3/MeOH,
63 oC; v) TMSBr, 2,6-lutidine, CH3CN;
SCHEME 3 Synthesis of 2ꢁ,2ꢁ-difluoro cyclopentanyl adenine phosphonic acid 19
Bubbling ammonia into the compound 22 gave separable 2-fluoro-6-
aminopurine analogue[20] analogue 23a (13%) and 2-amino-6-chloropurine
analogue 23b (51%), respectively. Fluorine acts as a better leaving group
than chlorine in nucleophilic aromatic substitution. Phosphonate 23b was
treated with TMSBr to provide phosphonic acid and sequentially treated
sodium methoxide and 2-mercaptoethanol in methanol to give a desired
5ꢁ-norcarbocyclic guanine phosphonic acid 24, (Scheme 4).[21]
The antiviral activity of phosphonate nucleoside is mostly explained by
their intracellular metabolism to their diphosphates followed by incorpora-
tion into the viral genome and chain termination.[22] As shown in Table 1,
adenine nucleoside phosphonic ester 19 exhibits significant anti-HIV ac-
tivity. However, nucleoside analogues 18, 23a, 23b, and 24 showed weak
anti-HIV activity or cytotoxicity at concentrations up to 100 μM. Anti-HIV
activity was determined in human peripheral blood mononuclear (PBM)

6
G. H. Shen et al.
Cl
N
N
N
N
PMBO
OH
F
i
PMBO
F
65%
F
F
14
20
F
ii
61%
Cl
Cl
N
N
N
N
O
P
EtO
iii
59%
N
HO
N
F
N
F
EtO
O
N
F
F
21
F
22
F
iv
O
X
N
N
N
N
NH
N
O
P
HO
O
P
v
N
F
HO
O
N
EtO
O
NH2
41%
Y
EtO
F
F
24
F
23a: X = NH2, Y = F (13%)
23b: X = Cl, Y = NH2(51%)
Reagents: i) (EtO)2POCH2OTf, LiO-t-Bu, THF; ii) DDQ, CH2Cl2/H2O (10:1);
iii) 2-fluoro-6-chloropurine, DEAD, PPh3, 1,4-dioxane/DMF; iv) NH3/DME, rt;
v) (a) TMSBr, 2,6-lutidine, CH3CN; (b) NaOMe, HSCH2CH2OH, MeOH.
SCHEME 4 Synthesis of 2ꢁ,2ꢁ-difluoro cyclopentanyl guanine phosphonic acid 24
TABLE 1 The antiviral activities of the synthesized compounds
cytotoxicity
HIV-1
IC50(μM)
Compound
EC50(μM) EC90(μM)
PBM
CEM Vero
18
19
42
13
81
68
43
90
90
95
95
95
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
42.8
23a
23b
24
PMEA
AZT
4.4
0.112
ND
ND
35.8
>100 11.6
ND, Not Determined; PMEA, 9-[2-(Phosphonomethoxy)ethyl]adenine; AZT, Azidothymidine;
EC50 (μM): EC50 values are for 50% inhibition of virus production as indicated by supernatant RT
levels.
EC90 (μM): EC90 values are for 90% inhibition of virus production as indicated by supernatant RT
levels.
IC50 (μM): IC50 values indicate 50% inhibition of cell growth.

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
7
FIGURE 3 Superimpose of monophosphate of 1 and 19 (Color figure available online).
cells infected with HIV-1 strain LAI. PBM cells (1×105 cell/mL) were in-
fected with HIV-1 at a multiplicity of infection (MOI) of 0.02 and cultured
in the presence of various concentrations of the test compounds. After 4 days
of incubation at 37◦C, numbers of viable cells were determined using the
3-(4,5-di-methylthiazole-2-yl)-2,5-diphenyltetrazolium bromide method. The
cytotoxicities of the compounds were evaluated in parallel with their antiviral
activities, which were assessed on the basis of the viabilities of mock-infected
cells.[23]
CONCLUSIONS
Based on the potent anti-HIV activity of 2ꢁ-fluorinated nucleoside as well
as phosphonic acid nucleoside analogues, we have designed and success-
fully synthesized novel 2ꢁ,2ꢁ-difluorinated 5ꢁ-norcarbocyclic phosphonic acid
nucleosides starting from but-3-en-1-ol. Interestingly, adenine analogue 19
shows significant antiviral activity against HIV-1. As shown in Figure 3, su-
perimposed modeling of monophosphate of (1) and 19 shows significant
overlap between two parts such as phosphonic acid and the sugar (cyclopen-
tane) moiety. However, adenine base moiety is not positioned closer to that
of the adenine analogue (1). Energy minimization was optimized with the
framework of the density functional theory (DFT), with Spartan modeling
software. The B3LYP functional with 6–31G∗ basis set was employed. The in-
formation obtained in the present study will be useful for the development

8
G. H. Shen et al.
of a novel nucleoside analogue. To increase the cellular uptake of phospho-
nic acid analogues, developments of bis-SATE phosphonodiester prodrugs
are underway.
EXPERIMENTAL SECTION
Melting points were determined on a Mel-temp II laboratory device and
are uncorrected. NMR spectra were recorded on a JEOL 300 Fourier trans-
form 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 on a Beckman DU-7 spectrophotometer (Beckman,
South Pasadena, CA, USA). The mass spectra (HRMS) were taken on VAR-
IAN MAT 311 at an ionizing potential of 70 eV. The elemental analyses were
performed using a Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT,
USA). TLC was performed on Uniplates (silica gel) purchased from Anal-
tech Co. (7558, Newark, DE, USA). All reactions were carried out under
an atmosphere of nitrogen unless specified. Dry dichloromethane, benzene,
and pyridine were obtained by distillation from CaH2. Dry THF was obtained
by distillation from Na and benzophenone immediately prior to use.
But-3-enal (6)
To a stirred solution of oxalyl chloride (155 mg, 1.224 mmol) in CH2Cl2
(12 mL) was added a solution of DMSO (191 mg, 2.448 mmol) in CH2Cl2
(5.0 mL) dropwise at −78◦C. The resulting solution was stirred at −78◦C
for 30 minutes, and a solution of alcohol 7 (88 mg, 1.224 mmol) in CH2Cl2
(8 mL) was added dropwise. The mixture was stirred at −78◦C for 30 minutes
and TEA (0.684 mL, 4.88 mmol) was added. The resulting mixture was
warmed to 0◦C and stirred for 30 minutes. H2O (10 mL) was added, and the
solution was stirred for 30 minutes at rt. The mixture was diluted with water
(100 mL) and then extracted with EtOAc (2 × 100 mL). The combined
organic layer was washed with brine, dried over anhydrous MgSO4, and
filtered. The filtrate was concentrated in vacuo and the residue was subjected
1
to next reaction without further purification: Crude yield 91%; H NMR
(CDCl3, 300 MHz) δ 9.73 (m, 1H), 6.03–5.94 (m, 1H), 5.18–5.14 (m, 2H),
3.10 (t, J = 6.8 Hz, 2H); 13C NMR (CDCl3, 75 MHz) δ 202.0, 135.5, 118.3,
48.4.
Hepta-1,6-dien-4-ol (7)
To a solution of 6 (2.3 g, 32.81 mmol) in dry THF (10 mL), vinylmag-
nesium bromide (39.37 mL, 1.0 M solution in THF) was slowly added at
−10◦C and stirred 4 hours at 0◦C. Saturated NH4Cl solution (20 mL) was
added to the mixture, which was slowly warmed to rt. The mixture was di-
luted with water (150 mL) and extracted with EtOAc (150 mL) two times.

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
9
The combined organic layer was washed with brine, dried over anhydrous
MgSO4, filtered, and evaporated in vacuo. The residue was purified by silica
gel column chromatography (EtOAc/hexane, 1:10) to give 7 (3.02 g, 82%)
as a colorless oil: 1H NMR (CDCl3, 300 MHz) δ 5.73–5.69 (m, 2H), 5.05–4.98
(m, 4H), 3.31 (m, 1H), 2.13 (m, 4H); 13C NMR (CDCl3, 75 MHz) δ 140.4,
115.7, 74.7, 43.5.
Cyclopent-3-enol (8)
To a solution of 7 (270. mg, 2.406 mmol) in dry methylene chloride
(5 mL) was added 2nd generation Grubbs catalyst (15.0 mg, 0.0176 mmol).
The reaction mixture was refluxed overnight and cooled to rt. The mixture
was concentrated in vacuo, and the residue was purified by silica gel column
chromatography (EtOAc/hexane, 1:8) to give cyclopentenol 8 (159.8 mg,
1
79%): H NMR (CDCl3, 300 MHz) δ 5.63 (m, 2H), 3.65 (quint, 1H), 2.55
(m, 2H), 2.36 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 134.5, 72.4, 43.5.
1-[(Cyclopent-3-enyloxy)methyl]-4-methoxybenzene (9)
NaH (60% in mineral oil, 66.4 mg, 1.66 mmol) was added portion-wise
to a cooled (0◦C) solution of secondary alcohol 8 (116 mg, 1.385 mmol)
and p-methoxybenzyl chloride (0.206 mL, 1.52 mmol) in anhydrous DMF
(8 mL). The reaction mixture was stirred overnight at rt. The solvent was
removed in vacuo and the residue was diluted with H2O (50 mL) followed
by extraction with diethyl ether (60 mL) two times. The combined organic
layer was washed with brine, dried over anhydrous MgSO4, and concentrated
under vacuum. The residue was purified by silica gel column chromatogra-
1
phy (EtOAc/hexane, 1:20) to give 9 (189.6 mg, 67%) as a colorless oil: H
NMR (CDCl3, 300 MHz) δ 7.11 (d, J = 7.8 Hz, 2H), 6.72 (d, J = 7.9 Hz, 2H),
5.62 (m, 2H), 4.65 (s, 2H), 3.74 (s, 3H), 3.25 (quint, 1H), 2.57 (m, 2H),
2.38 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 159.7, 137.5, 134.5, 129.9, 115.7,
73.5, 72.6, 56.3, 42.5; Anal. Calc. for C13H16O2: C, 76.44; H, 7.90; Found: C,
76.52; H, 7.84.
4-(4-Methoxybenzyloxy)cyclopentane-1,2-diol (10a and 10b)
Compound 9 (118.5 mg, 0.58 mmol) was dissolved in a cosolvent system
(10 mL) (acetone: t-BuOH: H2O = 6:1:1) along with 4-methylmorpholine
N -oxide (135 mg, 1.16 mmol). Subsequently, OsO4 (0.29 mL, 4% wt.% in
H2O) was added. The mixture was stirred overnight at rt and quenched
with saturated Na2SO3 solution (5 mL). The resulting solid was removed by
filtration through a pad of Celite, and filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (MeOH/CH2Cl2,
1:5) to give 10a (40.08 mg, 29%) and 10b (41.46 mg, 30%) as a colorless
oils: spectroscopical data for 10a; 1H NMR (DMSO-d6, 300 MHz) δ 7.09 (d,
J = 7.7 Hz, 2H), 6.71 (d, J = 7.8 Hz, 2H), 4.89 (d, J = 5.0 Hz, 2H, D2O

10
G. H. Shen et al.
exchangeable), 4.64 (s, 2H), 3.72 (s, 3H), 3.38–3.32 (m, 2H), 2.87 (quint,
1H), 1.90 (m, 2H), 1.68 (m, 2H); 13C NMR (DMSO-d6, 75 MHz) δ 159.4,
131.8, 129.5, 114.8, 77.5, 73.4, 71.9, 55.7, 41.6; Anal. Calc. for C13H18O4: C,
65.53; H, 7.61; Found: C, 65.47; H, 7.55. data for 10b; 1H NMR (DMSO-d6,
300 MHz) δ 7.11 (d, J = 7.8 Hz, 2H), 6.71 (d, J = 7.8 Hz, 2H), 4.92 (d,
J = 5.2 Hz, 2H, D2O exchangeable), 4.67 (s, 2H), 3.56 (s, 3H), 3.30–3.24
(m, 2H), 2.81–2.78 (m, 1H), 1.94–1.90 (m, 2H), 1.70–1.67 (m, 2H); 13C
NMR (DMSO-d6, 75 MHz) δ 159.7, 131.3, 129.7, 115.1, 77.6, 73.6, 71.5, 54.8,
40.9; Anal. Calc. for C13H18O4: C, 65.53; H, 7.61; Found: C, 65.58; H, 7.59.
(rel)-(1S,2R,4R)-4-(4-Methoxybenzyloxy)-2-hydroxycyclopentyl benzoate(11)
To a solution of compound 10β (557 mg, 2.34 mmol) in anhydrous pyri-
dine (11 mL), benzoyl chloride (354 mg, 2.52 mmol) and DMAP (24.5 mg,
0.2 mmol) were added. The reaction mixture was stirred overnight at rt. The
reaction mixture was then quenched using a saturated NaHCO3 solution
(0.4 mL) and evaporated under reduced pressure. The residue was parti-
tioned between water and ethyl acetate and the organic layer was separated.
The aqueous layer was extracted with ethyl acetate, and the combined or-
ganic layer extracts were washed with brine, dried over MgSO4, and filtered.
The organic solvent was evaporated under reduced pressure and the residue
was purified by silica gel column chromatography (EtOAc-hexanes 1:12) to
1
yield compound 11 (504 mg, 63%) as a colorless oils. H NMR (CDCl3,
300 MHz) δ 7.98 (m, 2H), 7.46–7.38 (m, 3H), 7.09 (d, J = 6.8 Hz, 2H), 6.70
(d, J = 6.8 Hz, 2H), 4.64 (s, 2H), 4.09 (m, 1H), 3.84 (m, 1H), 3.74 (s, 3H),
2.87–2.84 (m, 1H), 2.21 (m, 1H), 2.12 (br s, 1H), 1.94–1.90 (m, 2H), 1.67
(m, 1H); 13C NMR (CDCl3, 75 MHz) δ 169. 7, 159.6, 134.5, 130.3, 129.5,
128.6, 115.2, 77.1, 73.5, 72.9, 71.3, 55.5, 41.4, 38.4; Anal. Calc. for C20H22O5:
C, 70.16; H, 6.48; Found: C, 70.08; H, 6.54.
(rel)-(1S,4S)-4-(4-Methoxybenzyloxy)-2-oxocyclopentyl benzoate (12)
To a solution of compound 11 (1.64 g, 4.8 mmol) in CH2Cl2 (50 mL), 4A
˚
molecular sieves (2.8 g) and PCC (2.58 g, 12.03 mmol) were added slowly at
0◦C, and stirred overnight at rt. To the mixture, excess diethyl ether (200 mL)
was then added. The mixture was stirred vigorously for 3 hours at the same
temperature, and the resulting solid was filtered through a short silica gel
column. The filtrate was concentrated under vacuum and the residue was
purified by silica gel column chromatography (EtOAc/hexane, 1:25) to give
compound 8 (1.07 g, 66%) as a colorless oil: 1H NMR (CDCl3, 300 MHz) δ
7.98 (d, J = 7.0 Hz, 2H), 7.45–7.37 (m, 3H), 7.08 (d, J = 6.8 Hz, 2H), 6.71
(d, J = 6.8 Hz, 2H), 4.65 (s, 2H), 4.55 (m, 1H), 3.74 (s, 3H), 3.38 (m, 1H),
2.71–2.64 (m, 3H), 2.18–2.14 (m, 1H); 13C NMR (CDCl3, 75 MHz) δ 211.2,
168. 9, 159.7, 134.4, 131.5, 129.7, 128.4, 114.7, 77.4, 72.8, 71.5, 54.8, 42.5,
28.1; Anal. Calc. for C20H20O5: C, 70.57; H, 5.92; Found: C, 70.65; H, 5.88.

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
11
(rel)-(1S,4S)-4-(4-Methoxybenzyloxy)-2,2-difluorocyclopentyl benzoate (13)
A solution of compound 12 (326 mg, 0.96 mmol) in anhydrous CH2Cl2
(40 mL) was treated with DAST (0.992 mL, 7.52 mmol) under argon at
room temperature. The reaction mixture was stirred overnight and was then
quenched by adding a saturated solution of NaHCO3 in water (20 mL).
The organic phase was separated, and the aqueous layer was extracted with
CH2Cl2 (3 × 100 mL). The organic layer was dried over anhydrous magne-
sium sulfate, filtered, and evaporated to dryness. The residue was purified
by silica gel column chromatography (EtOAc/hexane, 1:25) to give the di-
fluorinated product 13 (160 mg, 46%) as a colorless syrup. 1H NMR (CDCl3,
300 MHz) δ 8.01 (m, 2H), 7.48–7.37 (m, 3H), 7.08 (d, J = 7.0 Hz, 2H), 6.73
(d, J = 6.9 Hz, 2H), 4.64 (s, 2H), 4.51–4.45 (m, 1H), 3.74 (s, 3H), 2.89 (m,
1H), 2.19–1.94 (m, 4H); 19F NMR (CDCl3, 282 MHz) δ −101.4 (dm, J =
246.7 Hz, 1F), −119.6 (dm, J = 247.9 Hz, 1F); 13C NMR (CDCl3, 75 MHz)
δ 167.8, 159.5, 133.7, 130.4, 129.8, 129.3, 128.2, 114.6, 110.2 (dd, J = 234.6,
240.4 Hz), 78.1 (dd, J = 20.2, 22.4 Hz), 72.9, 71.5, 67.0, 55.4, 37.5 (dd, J =
19.5, 21.6 Hz), 31.5; Anal. Calc. for C20H20F2O4: C, 66.29; H, 5.56; Found:
C, 66.35; H, 5.61.
(rel)-(1S,4S)-4-(4-Methoxybenzyloxy)-2,2-difluorocyclopentanol (14)
A solution of 13 (205 mg, 0.566 mmol) in saturated methanolic ammonia
(10 mL) was stirred overnight at rt, and the volatiles were evaporated. The
residue was purified by silica gel column chromatography (EtOAc/hexane,
1
1:5) to give 14 (114 mg, 78%): H NMR (CDCl3, 300 MHz) δ 7.11 (d, J =
7.0 Hz, 2H), 6.72 (d, J = 6.9 Hz, 2H), 4.65 (s, 2H), 3.79–3.69 (m, 4H), 2.87
(m, 1H), 2.11–1.94 (m, 5H); 19F NMR (CDCl3, 282 MHz) δ −103.5 (dm, J =
251.1 Hz, 1F), −122.1 (dm, J = 251.9 Hz, 1F); 13C NMR (CDCl3, 75 MHz)
δ 159.8, 130.1, 128.4, 114.7, 114.7 (dd, J = 238.6, 242.6 Hz), 74.2 (dd, J =
19.8, 21.5 Hz), 72.3, 65.7, 56.1, 37.1 (dd, J = 20.2, 22.4 Hz), 33.8; Anal. Calc.
for C13H16F2O3: C, 60.46; H, 6.24; Found: C, 60.53; H, 6.48.
(rel)-(1ꢀS,4ꢀS)-9-[4-(4-Methoxybenzyloxy)-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl] 6-
chloropurine(15)
To a stirred solution of triphenylphosphine (281 mg, 1.07 mmol) in
dry THF (5 mL) at 0◦C was added dropwise the diisopropyl azodicarboxylate
(DIAD) (216 mg, 1.07 mmol) and the reaction mixture was stirred at this tem-
perature for 30 minutes. After that, a solution of the alcohol 14 (138.2 mg,
0.535 mmol) in THF (5 mL) was added and the reaction mixture was stirred
at 0◦C for 30 minutes. Then the cold bath was removed and the yellow
solution was stirred for 30 minutes at room temperature. 6-Chloropurine
(230 mg, 1.07 mmol) was then added and the reaction mixture was stirred
overnight at room temperature. The reaction mixture was concentrated
under reduced pressure and the residue was purified by silica gel column

12
G. H. Shen et al.
chromatography (EtOAc/hexane/MeOH, 1:3:0.02) to give compound 15
(131 mg, 62%); UV (MeOH) λmax 263.0 nm: 1H NMR (CDCl3, 300 MHz) δ
8.69 (s, 1H), 8.27 (s, 1H), 7.10 (d, J = 7.0 Hz, 2H), 6.69 (d, J = 7.0 Hz, 2H),
4.64 (s, 2H), 4.27 (m, 1H), 3.74 (s, 3H), 2.85 (m, 1H), 2.21–1.84 (m, 4H);
19F NMR (CDCl3, 282 MHz) δ −106.3 (dm, J = 236.4 Hz, 1F), −120.6 (dm,
J = 237.2 Hz, 1F); 13C NMR (CDCl3, 75 MHz) δ 159.6, 154.3, 148.8, 141.7,
132.6, 130.5, 128.4, 114.7, 110.7 (dd, J = 237.2, 244.4 Hz), 72.3, 67.8, 57.7
(dd, J = 20.4, 22.4 Hz), 55.3, 37.7 (dd, J = 20.3, 21.6 Hz), 30.2; Anal. Calc.
for C18H17ClF2N4O2 (+1.0 MeOH): C, 53.55; H, 4.97; N, 13.15. Found: C,
53.44; H, 4.91; N, 13.09.
(rel)-(1ꢀS,4ꢀS)-9-(4-Hydroxy-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) 6-chloropurine
(16)
To a solution of compound 15 (167 mg, 0.423 mmol) in CH2Cl2/H2O
(6 mL, 10:1 v/v) was added DDQ (143 mg, 0.632 mmol), and the mixture
was stirred overnight at room temperature. Saturated NaHCO3 (0.9 mL)
was added to quench the reaction, which was then stirred for 3 hours at rt.
The mixture was diluted with water (75 mL) and extracted with CH2Cl2 (3
× 80 mL). The combined organic layer was dried over anhydrous MgSO4
and filtered. The filtrate was concentrated in vacuo and the residue was puri-
fied by silica gel column chromatography (EtOAc/hexane/MeOH, 4:1:0.02)
to give compound 16 (74 mg, 64%) as a white solid: mp 167–169◦C; UV
(MeOH) λmax 263.0 nm; 1H NMR (DMSO-d6, 300 MHz) δ 8.69 (s, 1H), 8.26
(s, 1H), 4.89 (d, J = 5.0 Hz, 1H, D2O exchangeable), 4.31–4.24 (m, 1H),
3.26 (m, 1H), 2.21–1.85 (m, 4H); 19F NMR (DMSO-d6, 282 MHz) δ −104.7
(dm, J = 233.6 Hz, 1F), −119.6 (dm, J = 235.1 Hz, 1F); 13C NMR (DMSO-d6,
75 MHz) δ 151.9, 151.6, 150.9, 145.6, 132.5, 110.6 (dd, J = 228, 232.6 Hz),
60.5, 57.5 (dd, J = 20.2, 21.8 Hz), 41.6 (dd, J = 18.8, 20.6 Hz), 31.7; Anal.
Calc. for C10H9ClF2N4O (+0.5 MeOH): C, 43.48; H, 3.82; N, 19.32. Found:
C, 43.40; H, 3.77; N, 19.25.
(rel)-(1ꢀS,4ꢀS)- Diethyl [9-(4-hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) 6-
chloropurine] phosphonate (17)
Both LiOt-Bu (1.49 mL of 0.5 M solution in THF, 0.744 mmol) and a so-
lution of diethyl phosphonomethyltriflate (208 mg, 0.696 mmol) in 7.0 mL
of THF were slowly added to a solution of the 6-chloropurine analogue 16
(96 mg, 0.348 mmol) in 6.0 mL of THF at −10◦C and stirred overnight at rt
under nitrogen. The mixture was quenched by adding saturated NH4Cl solu-
tion (5 mL) and further diluted with additional H2O (100 mL). The aqueous
layer was extracted with EtOAc (3 × 100 mL). The combined organic layer
was dried over anhydrous MgSO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (MeOH/Hexane/EtOAc,

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
13
0.02:3:1) to give 17 (79 mg, 54%) as a foam: 1H NMR (DMSO-d6, 300 MHz)
δ 8.71 (s, 1H), 8.34 (s, 1H), 4.26 (m, 1H), 4.09–4.01 (m, 4H), 3.91 (d,
J = 8.2 Hz, 2H), 2.87 (m, 1H), 2.21–1.83 (m, 4H), 1.18 (m 6H); 19F NMR
(DMSO-d6, 282 MHz) δ −105.7 (dm, J = 231.6 Hz, 1F), −121.3 (dm, J =
232.9 Hz, 1F); 13C NMR (DMSO-d6, 75 MHz) δ 151.7, 151.2, 150.4, 146.5,
136.8, 111.4 (dd, J = 230.0, 238.4 Hz), 69.0, 63.2, 62.6, 62.1, 59.0 (dd,
J = 19.4, 21.2 Hz), 38.1 (dd, J = 20.2, 21.8 Hz), 29.4, 14.2 Anal. Calc. for
C15H20ClF2N4O4P (+1.0 MeOH): C, 42.13; H, 5.30; N, 12.28; Found: C,
42.06; H, 5.35; N, 12.21.
(rel)-(1ꢀS,4ꢀS)- Diethyl [9-(4-hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl)
adenine] phosphonate (18)
A solution of 17 (168 mg, 0.395 mmol) in saturated methanolic ammo-
nia (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 chromatog-
raphy (MeOH/CH2Cl2, 1:8) to give 18 (101 mg, 63%) as a white solid: mp
156–158◦C; UV (MeOH) λmax 261.5 nm; 1H NMR (DMSO-d6, 300 MHz) δ
8.30 (s, 1H), 8.12 (s, 1H), 6.12 (br s, 2H, D2O exchangeable), 4.23 (m, 1H),
4.08–4.02 (m, 4H), 3.89 (d, J = 8.0 Hz, 2H), 2.86 (m, 1H), 2.22–1.88 (m, 4H),
1.14 (m 6H); 19F NMR (DMSO-d6, 282 MHz) δ −101.4 (dm, J = 229.7 Hz,
1F), −118.9 (dm, J = 231.2 Hz, 1F); 13C NMR (DMSO-d6, 75 MHz) δ 155.3,
152.7, 150.5, 141.5, 120.5, 110.8 (dd, J = 228.4, 234.4 Hz), 68.9, 63.6, 62.7,
62.2, 57.9 (dd, J = 20.4, 21.8 Hz), 38.1 (dd, J = 21.7, 23.0 Hz), 29.6, 15.0;
Anal. Calc. for C15H22F2N5O4P (+1.0 MeOH): C, 43.96; H, 5.99; N, 16.02;
Found: C, 43.87; H, 6.05; N, 15.98.
(rel)-(1ꢀS,4ꢀS)- 9-[(4-Hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) adenine]
phosphonic acid (19)
To a solution of the phosphonate 18 (234 mg, 0.579 mmol) in anhy-
drous CH3CN (12 mL) and 2,6-lutidine (1.35 mL, 11.6 mmol) was added
trimethylsilyl bromide (0.888 mg, 5.79 mmol). The mixture was heated for
24 hours at 75◦C under nitrogen gas and then concentrated in vacuum to
give a brown residue, and co-evaporated from conc aqueous NH4OH (2 ×
30 mL). The resultant solid was triturated with acetone (2 × 10 mL) and
the residue was purified by reverse-phase chromatography. Lyophilization
of an appropriate fraction provided phosphonic acid salt 19 (108 mg, 51%)
as a white salt (ammonium salt): UV (H2O) λmax 262.0 nm; 1H NMR (D2O,
300 MHz) δ 8.22 (s, 1H), 8.13 (s, 1H), 4.29–4.24 (m, 1H), 3.78 (d, J = 8.0 Hz,
1H), 2.85 (m, 1H), 2.21–1.84 (m, 4H); 19F NMR (D2O, 282 MHz) δ −110.4
(dm, J = 234.2 Hz, 1F), −126.4 (dm, J = 235.4 Hz, 1F); 13C NMR (D2O,
75 MHz) δ 154.5, 152.5, 150.3, 141.6, 119.7, 110.3 (dd, J = 221.4, 234.8 Hz),

14
G. H. Shen et al.
69.1, 67.6, 58.2 (dd, J = 19.6, 20.4 Hz), 38.6 (dd, J = 18.7, 20.4 Hz), 30.2;
HPLC tR = 10.85 minutes HRMS [M-H]+ req. 348.0067, found 348.0065.
(rel)-(1ꢀS,4ꢀS)-9-[4-(4-Methoxybenzyloxy)-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl]
2-fluoro-6-chloropurine (20)
Coupling of 14 with 2-fluoro-6-chloropurine under the similar conden-
sation conditions as described for 15 to give 20 as a solid: yield 65%; UV
(MeOH) λmax 265.0 nm; 1H NMR (CDCl3, 300 MHz) δ 8.43 (s, 1H), 7.10 (d,
J = 6.8 Hz, 2H), 6.72 (d, J = 6.9 Hz, 2H), 4.65 (s, 2H), 4.27–4.23 (m, 1H),
3.74 (s, 3H), 2.87 (m, 1H), 2.19–1.83 (m, 4H); 19F NMR (CDCl3, 282 MHz)
δ −107.3 (dm, J = 241.5 Hz, 1F), −123.6 (dm, J = 242.7 Hz, 1F); 13C NMR
(CDCl3, 75 MHz) δ 159.6, 157.1 (d, J = 218.6 Hz), 153.4, 136.4, 129.6, 128.5,
120.7, 114.9, 110.5 (dd, J = 221.4, 234.8 Hz), 73.1, 67.5, 56.0 (dd, J = 19.2,
21.7 Hz), 37.8 (dd, J = 18.7, 19.3 Hz), 30.3; Anal. Calc. for C18H16ClF3N4O2
(+1.0 MeOH): C, 51.38; H, 4.54; N, 12.61; Found: C, 51.44; H, 4.49; N,
12.70.
(rel)-(1ꢀS,4ꢀS)-9-(4-Hydroxy-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) 2-fluoro-6-chloro
purine (21)
Deprotection of 20 was performed from 21 using the similar procedure
1
as described for 16 as a solid: yield 61%; H NMR (DMSO-d6, 300 MHz) δ
8.46 (s, 1H), 4.91 (d, J = 5.2 Hz, 1H, D2O exchangeable), 4.27–4.23 (m,
1H), 3.35 (m, 1H), 2.23–1.87 (m, 4H); 19F NMR (DMSO-d6, 282 MHz) δ
−105.7 (dm, J = 234.2 Hz, 1F), −123.8 (dm, J = 235.7 Hz, 1F); 13C NMR
(DMSO-d6, 75 MHz) δ 157.2 (d, J = 218.4 Hz), 153.2, 145.4, 136.1, 121.2,
110.2 (dd, J = 219.2, 220.4 Hz), 60.6, 59.3 (dd, J = 18.8, 21.6 Hz), 40.3 (dd,
J = 18.8, 20.2 Hz), 32.2; Anal. Calc. for C10H8ClF3N4O (+1.0 MeOH): C,
40.77; H, 3.73; N, 17.29; Found: C, 40.83; H, 3.68; N, 17.35.
(rel)-(1ꢀS,4ꢀS)- Diethyl [9-(4-hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl)
2-fluoro-6-chloropurine] phosphonate (22)
Phosphonation of 21 was performed by the similar procedure as de-
1
scribed for 17 as a form: yield 59%; H NMR (DMSO-d6, 300 MHz) δ 8.45
(s, 1H), 4.25–4.22 (m, 1H), 4.10–4.07 (m, 4H), 3.86 (d, J = 8.0 Hz, 2H),
2.85 (m, 1H), 2.21–1.85 (m, 4H), 1.38–1.30 (m, 6H); 19F NMR (DMSO-d6,
282 MHz) δ -111.2 (dm, J = 239.3 Hz, 1F), −127.8 (dm, J = 240.7 Hz,
1F); 13C NMR (DMSO-d6, 75 MHz) δ 157.5 (d, J = 219.2 Hz), 153.5, 144.9,
136.7, 122.0, 111.1 (dd, J = 218.4, 220.4 Hz), 69.2, 63.4, 63.2, 60.6, 58.8
(dd, J = 18.6, 21.4 Hz), 39.8 (dd, J = 18.9, 20.8 H), 30.2, 15.1; Anal. Calc.
for C15H19ClF3N4O4P (+0.5 MeOH): C, 40.64; H, 4.62; N, 12.23; Found: C,
40.55; H, 4.57; N, 12.18.

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
15
(rel)-(1ꢀS,4ꢀS)- Diethyl [9-(4-hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) 2-
fluoro-6-aminopurine] phosphonate (23a) and (rel)-(1ꢀS,4ꢀS)- Diethyl [9-(4-
hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) 2-amino-6-chloropurine]
phosphonate (23b)
Dry ammonia gas was bubbled into a stirred solution of 22 (250 mg,
0.564 mmol) in DME (10 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/CH2Cl2, 1:8) to give 23a (31 mg, 13%) and 23b (126 mg, 51%)
as solids: Data for 23a; UV (MeOH) λmax 261.0 nm; 1H NMR (DMSO-d6,
300 MHz) δ 8.20 (s, 1H), 6.67 (br d, 2H), 4.26–4.22 (m, 1H), 4.08 (m, 4H),
3.89 (d, J = 8.1 Hz, 2H), 2.88 (m, 1H), 2.19–1.85 (m, 4H), 1.35 (m, 6H); 19
F
NMR (DMSO-d6, 282 MHz) δ −108.2 (dm, J = 235.5 Hz, 1F), −129.6 (dm,
J = 237.0 Hz, 1F); 13C NMR (DMSO-d6, 75 MHz) δ 160.3 (d, J = 268.4 Hz),
155.3, 152.5, 141.5, 118.2, 110.7 (dd, J = 208.4, 210.6 Hz), 69.0, 63.4, 62.2,
58.2 (dd, J = 18.6, 20.4 Hz), 37.2 (dd, J = 16.7, 18.9 Hz), 29.6, 15.1; Anal.
Calc. for C15H21F3N5O4P (+1.0 MeOH): C, 42.22; H, 5.54; N, 15.39; Found:
C, 42.31; H, 5.47; N, 15.46; Data for 23b; UV (MeOH) λmax 308.0 nm; 1H NMR
(DMSO-d6, 300 MHz) δ 8.13 (s, 1H), 6.77 (br d, 2H), 4.27–4.23 (m, 1H),
4.08–4.05 (m, 4H), 3.86 (d, J = 8.0 Hz, 2H), 2.87–2.86 (m, 1H), 2.18–1.83
(m, 4H), 1.39–1.34 (m, 6H); 19F NMR (DMSO-d6, 282 MHz) δ −106.8 (dm,
J = 231.5 Hz, 1F), −126.8 (dm, J = 232.8 Hz, 1F); 13C NMR (DMSO-d6,
75 MHz) δ 158.3, 154.2, 151.1, 143.2, 124.7, 111.2 (dd, J = 210.4, 212.8 Hz),
69.1, 63.2, 62.4, 59.2 (dd, J = 19.6, 21.6 Hz), 38.4 (dd, J = 19.7, 10.8 Hz),
30.3, 14.8; Anal. Calc. for C15H21ClF2N5O4P (+1.0 MeOH): C, 40.79; H,
5.35; N, 14.86; Found: C, 40.68; H, 5.29; N, 14.78.
(rel)-(1ꢀS,4ꢀS)- 9-[(4-Hydroxymethyl-2ꢀ,2ꢀ-difluorocyclopentan-1ꢀ-yl) guanine]
phosphonic acid (24)
To a solution of 23b (278 mg, 0.632 mmol) dry CH3CN (20 mL) was
added trimethylsilyl bromide (0.146 mL, 11.04 mmol) at room tempera-
ture. After this mixture was stirred for 30 hours, the solvent was removed,
evaporating three times with methanol. The residue was dissolved in MeOH
(24.0 mL) and 2-mercaptoethanol (172.8 μL, 2.532 mmol) and NaOMe
(134.4 mg, 2.532 mmol) was added to the mixture. The mixture was re-
fluxed for 18 hours under N2, cooled, neutralized with glacial AcOH, and
evaporated to dryness under vacuum. The residue was co-evaporated from
conc NH4OH (2 × 32 mL) and the resultant solid was triturated with acetone
(2 × 12 mL). The residue was purified by chromatography on a prepara-
tive column of reversed-phase C18 silica gel eluting water. Lyophilization of
an appropriate fraction provided 24 (99 mg, 41%) as a yellowish salt (am-
monium salt). UV (H2O) λmax 253.5 nm; 1H NMR (D2O, 300 MHz) δ 7.81
(s, 1H), 4.25–4.21 (m, 1H), 3.67–3.62 (d, J = 8.0 Hz, 2H), 2.88 (m, 1H),

16
G. H. Shen et al.
2.18–1.81 (m, 4H); 19F NMR (D2O, 282 MHz) δ −109.5 (dm, J = 234.2 Hz,
1F), −122.4 (dm, J = 235.9 Hz, 1F); 13C NMR (D2O, 75 MHz) δ 157.6, 154.3,
152.0, 136.2, 117.7, 109.9 (dd, J = 210.4, 214.8 Hz), 70.1, 67.8, 48.2 (dd, J =
18.6, 20.3 Hz), 37.6 (dd, J = 18.6, 19.8 Hz), 30.2; HPLC tR = 9.29 minutes;
HRMS [M–H]+ req. 364.0247, found 364.0245.
REFERENCES
1. Qiu, X.-L.; Xu, X.-H.; Qing, F.-L. Recent advances in the synthesis of fluorinated nucleosides. Tetra-
hedron. 2010, 66, 789–843.
2. Boojamra, C.G.; Mackman, R.L.; Markevitch, D.Y.; Prasad, V.; Ray, A.S.; Douglas, J.; Grant, D.;
Kim, C.U.; Cihlar, T. Synthesis and anti-HIV activity of GS-9148 (2ꢁ-Fd4AP), a novel nucleoside
phosphonate HIV reverse transcriptase inhibitor. Bioorg. Med. Chem. Lett. 2008, 18, 1120–1123.
3. (a) Mackman, R.L.; Ray, A.S.; Hui, H.C.; Zhang, L.; Birkus, G.; Boojamra, C.G.; Desai, M. C.; Douglas,
J.L.; Gao, Y.; Grant, D.; Laflamme, G.; Lin, K.Y.; Markevitch, D.Y.; Mishra, R.; McDermott, M.;
Pakdaman, R.; Petrakovsky, O.V.; Vela, J.E.; Cihlar, T. Discovery of GS-9131: Design, synthesis and
optimization of amidate prodrugs of the novel nucleoside phosphonate HIV reverse transcriptase
(RT) inhibitor GS-9148. Bioorg. Med. Chem. 2010, 18, 3606–3617. b) Cihlar, T.; Ray, A.S.; Boojamra,
C.G.; Zhang, L.; Hui, H.; Laflamme, G.; Vela, J.E.; Grant, D.; Chen, J.; Myrick, F.; White, K.L.;
Gao, Y.; Lin, K.Y.; Douglas, J.L.; Parkin, N.T.; Carey, A.; Pakdaman, R.; Mackman, R.L. Design and
profiling of GS-9148, a novel nucleotide analog active against nucleoside-resistant variants of human
immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate prodrug, GS-9131.
Antimicrob Agents Chemother. 2008, 52, 655–665.
4. (a) Hertel, L.W.; Kroin, J.S.; Misner, J.W.; Tustin, J.M. Synthesis of 2-deoxy-2,2-difluoro-d-ribose
and 2-deoxy-2,2ꢁ-difluoro-d-ribofuranosyl nucleosides. J. Org. Chem. 1988, 53, 2406–2409. b) Hertel,
L.W.; Boder, G.B.; Kroin, J.S., Rinzel, S.M.; Poore, G.A.; Todd, G.C.; Grindey, G.B. Evaluation of the
antitumor activity of gemcitabine (2ꢁ,2ꢁ-difluoro-2ꢁ-deoxycytidine). Cancer Res. 1990, 50, 4417–4422.
5. Noble, S.; Goa, K.L. Gemcitabine. A review of its pharmacology and clinical potential in non-small
cell lung cancer and pancreatic cancer. Drugs. 1997, 54, 447–472.
6. Borthwick, A.D.; Kirk, B.E.; Biggadike, K.; Exall, A.M.; Butt, S.; Roberts, S.M.; Knight, D.J.;
Coates, J.A.; Ryan, D.M. Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2ꢁ- and
6ꢁ-fluorocarbocyclic 2ꢁ-deoxy guanosines. J. Med. Chem. 1991, 34, 907–914.
7. Mitsuya, H.; Broder, S. Inhibition of the in vitro infectivity and cytopathic effect of hu-
man T-lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2ꢁ,3ꢁ-
dideoxynucleosides. Proc. Natl. Acad. Sci. USA. 1986, 83, 1911–1915.
8. Wu, T.; Froeyen, M.; Kempeneers, V.; Pannecouque, C.; Wang, J.; Busson, R.; De Clercq, E.;
Herdewijn, P. Deoxythreosyl phosphonate nucleosides as selective anti-HIV agents. J. Am. Chem.
Soc. 2005, 127, 5056–5065.
9. Kim, C.U.; Luh, B.Y.; Misco, P.F.; Bronson, J.J.; Hitchcock, M.J.; Ghazzouli, I.; Martin, J.C. Acyclic
purine phosphonate analogues as antiviral agents. Synthesis and structure-activity relationships. J.
Med. Chem. 1990, 33, 1207–1213.
10. De Clercq, E.; Holy´, A.; Rosenberg, I. Efficacy of phosphonylmethoxyalkyl derivatives of adenine in
experimental herpes simplex virus and vaccinia virus infections in vivo. Antimicrob Agents Chemother.
1989, 33, 185–191.
11. Wang, W.; Jin, H.; Fuselli, N.; Mansour, T.S. Synthesis and anti HCMV activity of 3,4-disubstituted
tetrahydrofuran derived nucleosides and nucleotides: A tethered series of PME derivatives. Bioorg.
Med. Chem. Lett. 1997, 7, 2567–2572.
12. Mancuso, A.J.; Huang, S.L.; Swern, D. Oxidation of long-chain and related alcohols to carbonyls by
dimethyl sulfoxide “activated” by oxalyl chloride. J. Org. Chem. 1978, 43, 2480–2482.
13. Maryanoff, B.E.; Reitz, A.B. The Wittig olefination reaction and modifications involving phosphoryl-
stabilized carbanions. Stereochemistry, mechanism, and selected synthetic aspects. Chem. Rev., 1989,
89, 863–927.
14. (a) Tanaka, M.; Norimine, Y.; Fujita, T.; Suemune, H.; Sakai, K. Chemoenzymatic Synthesis of An-
tiviral Carbocyclic Nucleosides: Asymmetric Hydrolysis of meso-3,5-Bis(acetoxymethyl)cyclopentenes

2ꢁ,2ꢁ-Difluoro 5ꢁ-Norcarbocyclic Phosphonic Acid Analogues
17
Using Rhizopus delemar Lipase. J. Org. Chem. 1996, 61, 6952–6957. b) Liu, L.J.; Kim, S.W.; Lee, W.;
Hong, J.H. Selective ring-opening fluorination of epoxide: an efficient synthesis of 2ꢁ-C-fluoro-2ꢁ-C-
methyl carbocyclic nucleosides. Bull. Korean Chem. Soc. 2009, 30, 2989–2992.
15. Chong, Y.H.; Gumina, G.; Chu, C.K. A divergent synthesis of d- and l-carbocyclic 4ꢁ-fluoro-2ꢁ,3ꢁ-
dideoxynucleosides as potential antiviral agents. Tetrahedron: Asymmetry 2000, 11, 4853–4875.
16. Oh, H.S.; Kang, H.Y. Total synthesis of neomethymycin and novamethymycin. Tetrahedron 2010, 66,
4307–4317.
17. (a) Phillion, D.P.; Andrew, S.S. Synthesis and reactivity of diethyl phosphonomethyltriflate. Tetrahe-
dron Lett. 1986, 27, 1477–1480. b) Xu, Y.; Flavin, M.T.; Xu, Z.-Q. Preparation of new wittig reagents and
their application to the synthesis of α,β-unsaturated phosphonates. J. Org. Chem. 1996, 61, 7697–7701.
18. Hockova´, D.; Holy´, A.; Masoj´ıdkova´, M.; Keough, D.T.; De Jersey, J.; Guddat, L.W. Synthesis of
branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates
which inhibit Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.
Bioorg. Med. Chem. 2009, 17, 6218–6232.
19. Robins, M.J.; Uznanski, B. Non-aqueous diazotization with t-butyl nitrite. Introduction of fluorine,
chlorine, and bromine at C-2 of purine nucleoside. Can. J. Chem. 1981, 59, 2608–2611.
20. Montgomery, J.; Hewson, K. Nucleosides of 2-fluoroadenine. J. Med. Chem. 1969, 12, 498–504.
21. Tong, G.L.; Ryan, K.J.; Lee, W.W.; Acton, E.M.; Goodman, L. Nucleosides of thioguanine and other
2-amino-6-substituted purines from 2-acetamido-5-chloropurine. J. Org. Chem. 1967, 32, 859–862.
22. Holy, A.; Votruba, I.; Merta, A.; Cerny, J.; Vesely, J.; Vlach, J.; Sediva, K.; Rosenberg, I.; Otmar, M.;
Hrebabecky, H.; Travniekb, M.; Vonkac, V.; Snoeck, R.; De Clercq, E. Acyclic nucleotide analogues:
synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro.
Antiviral Res. 1990, 13, 295–311.
23. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.; De Clercq, E.
Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds.
J. Virol. Methods 1988, 20, 309–321.