Synthesis, antiviral activity, and mechanism of drug resistance of D- and L-2′,3′-didehydro-2′,3′-dideoxy-2′- fluorocarbocyclic nucleosides

Article abstract of DOI:10.1021/jm050096d

Carbocyclic nucleosides have received much attention due to their interesting biological activity and metabolic stability. Among nucleoside analogues, a fluorine substitution on the carbohydrate moiety or introduction of a 2′,3′-unsaturated structure moti

Full text of DOI:10.1021/jm050096d

3736
J. Med. Chem. 2005, 48, 3736-3748
Synthesis, Antiviral Activity, and Mechanism of Drug Resistance of D- and
L-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluorocarbocyclic Nucleosides
Jianing Wang,† Yunho Jin,† Kimberly L. Rapp,‡ Matthew Bennett,‡ Raymond F. Schinazi,‡ and Chung K. Chu*,†
College of Pharmacy, The University of Georgia, Athens, Georgia 30602, and Emory University School of Medicine/Veterans
Affairs Medical Center, Atlanta, Georgia 30033
Received February 1, 2005
Carbocyclic nucleosides have received much attention due to their interesting biological activity
and metabolic stability. Among nucleoside analogues, a fluorine substitution on the carbohydrate
moiety or introduction of a 2′,3′-unsaturated structure motif has been proven to be successful
in producing effective antiviral agents. By combining these structural features, both D- and
L-2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-carbocyclic nucleosides (D- and L-2′F-C-d4Ns) were
synthesized as potential anti-HIV agents. The target D- and L-carbocyclic nucleosides were
both stereospecifically synthesized from D-ribose. The structure-activity relationships of
synthesized compounds against HIV-1 in activated human peripheral blood mononuclear (PBM)
cells were studied, from which we found that the L-2′,3′-dideoxy-2′3′-didehydro-2′-fluoro-
adenosine analogue (L-2′F-C-d4A) 46 showed potent anti-HIV activity (EC50 ) 0.77 µM),
although it is cross-resistant to the lamivudine-resistant variant (HIV-1M184V). Modeling studies
demonstrated a good correlation between calculated relative binding energies and activity/
resistance data. The modeling study also indicated that an additional hydrogen bond and a
favorable van der Waals interaction contribute to the higher antiviral activity of L-2′F-C-d4A
in comparison to its D-counterpart. Also, like other L-nucleosides, the unfavorable steric
hindrance of the sugar moiety of L-2′F-C-d4A and the side chain of Val184 could explain the
cross-resistance of L-2′F-C-d4A with the M184V mutant. The significant difference of antiviral
activity between carbovir and its analogue L-2′F-C-d4G 25 may be due to distortion of the
phenyl ring of Tyr115 in the L-2′F-C-d4G-TP/HIV-RT complex, which resulted in a poor π-π
interaction.
Introduction
During the past 2 decades, extensive work has been
conducted in the field of nucleosides in search of novel
anti-HIV agents. As a result, eight clinically useful
nucleosides/nucleotide have been approved by the US
FDA for the treatment of HIV infection that are being
Figure 1. D- And L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro(-4′-
thio)nucleosides.
activity (EC50 ) 0.51, 0.17, and 1.5 µM, respectively) as
well as anti-HBV activity (EC50 ) 0.8, 0.22, and 1.7 µM,
respectively).6a,b D-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluoro-
5-fluorocytosine and -adenine were also active against
HIV in PBM cells without significant cytotoxicity (EC50
) 0.82 and 4.4 µM, respectively).6c In the case of the
4′-thio series, D-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-
4′-thiocytosine, L-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-
4′-thiocytosine, and -5-fluorocytosine demonstrated sig-
nificant anti-HIV activity (EC50 ) 1.3, 0.12, and 0.15
µM, respectively).6d,6e
Carbocyclic nucleoside is an analogue of natural
nucleoside in which the methylene group replaces the
oxygen atom of a furanose ring. As a consequence, the
glycosidic bond is resistant to nucleoside phosphorylases
and hydrolases, which makes the carbocyclic nucleosides
more stable toward metabolic degradation.7 Interest-
ingly, in some cases the substitution of the sugar ring
to a carbocyclic ring does not affect the enzyme recogni-
tion (especially kinases and target enzymes).7 Due to
these features, carbocyclic nucleosides have received
much attention as potential chemotherapeutic agents,7,8
such as abacavir and entecavir (Figure 2).9 Recently,
carbocyclic nucleosides have also been reported to
used as part of the highly active antiretroviral treat-
ment (HAART).1 However, the efficacy of nucleoside
reverse transcriptase inhibitors (NRTIs), as well as
other classes of anti-HIV agents, has been compromised
by the emergence of drug-resistant variants2 and toxic-
ity.3 Consequently, new classes of NRTIs with less
toxicity and less cross-resistance with existing regimes
are highly desirable.
A fluorine substitution on the carbohydrate moiety
has been proven to be successful in producing effective
antiviral agents4 and the 2′,3′-unsaturated analogues
have also demonstrated promising antiviral activity.5
On the basis of this information, D- and L-2′,3′-di-
dehydro-2′,3′-dideoxy-2′-fluoronucleosides (D- and L-2′F-
d4Ns) and their 4′-thio counterparts (D- and L-2′F-4′-
Sd4Ns) have been synthesized for the structure-activity
relationships (Figure 1).6 Among them, L-2′,3′-dide-
hydro-2′,3′-dideoxy-2′-fluorocytosine, -5-fluorocytosine,
and -adenine derivatives exhibited potent anti-HIV
* Corresponding author. Tel: (706) 542-5379. Fax: (706) 542-5381.
E-mail: dchu@rx.uga.edu.
† The University of Georgia.
‡ Emory University School of Medicine/Veterans Affairs Medical
Center.
10.1021/jm050096d CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/03/2005

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3737
hydride (LAH) was selected as the most efficient reduc-
ing reagent, which gave the 2- and 3-hydroxyl compound
in a ratio of 4.3:1. These conditions were also confirmed
in the L-series, which provided a ratio of 6:1. Further-
more, the 3-hydroxyl compound 5 could also be used as
the intermediate for the synthesis of 3-fluorine-substi-
tuted isomers for which synthesis is in progress and will
be reported in the future. The primary hydroxyl group
of 4 was selectively protected with trityl group, followed
by the oxidation of the 2-hydroxy group to give ketone
7, which was further treated with diethylaminosulfur
trifluoride (DAST) to give difluorinated compound 8 in
69% yield in three steps. Treatment of 8 with potassium
tert-butoxide in THF at 50 °C gave allylic alcohol 9 in
97% yield. After removing the trityl group under acidic
conditions, a tert-butyl diphenyl silyl group (TBDPS)
was used to protect the primary alcohol, which was
further subjected to a debenzylation reaction using
sodium metal in liquid ammonia with a small amount
of 1,4-dioxane as the solvent at -78 °C to afford the key
intermediate alcohol 12 in 64% yield in three steps.
However, the phenyl groups of TBDPS sometimes were
also reduced by using sodium/liquid ammonia. Ad-
ditionally, 1,4-dioxane was solidified under -78 °C,
which made the reaction take place under heteroge-
neous conditions. Hence, very careful control of the
debenzylation reaction conditions was needed to obtain
good yield. Due to these difficulities as described in
Scheme 3, a tert-butyldimethylsilyl (TBDMS) group was
used instead of the TBDPS group as the protecting
group, and THF was used instead of 1,4-dioxane in the
L-series. These modifications made the reaction easier
to control and the reaction was completed within 5 min
with improved yield from 70% to 90% in comparison to
the D-series.
Figure 2. Important carbocyclic nucleosides.
exhibit antiviral activity against smallpox, monkeypox,
as well as West Nile virus.10
Previously, optically active neplanocin A and D- and
L-aristeromycin (Figure 2) have been synthesized in our
laboratory.10a,11 More recently, the efficient and practical
synthetic methodology of the key intermediates D- and
L-cyclopent-2-enone has been also accomplished by our
group.10b,10c Therefore, efficient synthesis of carbocyclic
nucleosides with the 2′-fluoro-2′,3′-unsaturated struc-
ture motif becomes feasible. Herein, we report the full
accounts of the synthesis, antiviral activity, and molec-
ular modeling studies of the both D- and L-form of the
title nucleosides.
Results and Discussion
Chemistry. Several different synthetic approaches
have been explored for the synthesis of carbocyclic
nucleosides. Basically, there are two synthetic methods
to construct carbocyclic nucleosides: (1) a convergent
approach to attach a heterocyclic base to an appropriate
carbocyclic ring by a substitution reaction7,8 and (2)
linear construction of a base moiety from an amino-
subsistent on a carbocyclic moiety.7,8 Fully protected D-
and L-enantiomeically pure cyclopentane 1 and 26 were
synthesized from D-ribose via 12 and 13 steps, respec-
tively, by the reported method.10c Further modifications
of 1 and 26 gave the key intermediates 12 and 37 using
the procedure shown in Schemes 1 and 3, respectively.
Due to the similarity between the syntheses of D- and
L-isomers, the following discussion is based on Scheme
1 unless otherwise described. Compound 1 was de-
protected under the acidic condition to give triol 2 in
74% yield which was treated with 1-bromocarbonyl-1-
methylethyl acetate at -30 °C followed by the addition
of potassium carbonate at room temperature to give
epoxide 3, which in turn was ready for the ring-opening
reaction. In the case of D-isomers, lithium aluminum
Corresponding D- and L-carbocyclic nucleosides were
synthesized as shown in Schemes 2 and 4, respectively.
The following description is based on Scheme 2 unless
otherwise indicated. To synthesize the uridine and
thymidine analogues, Mitsunobu reaction was used to
condense the key intermediate 12 with protected uracil
and thymine to give the corresponding nucleosides as
crude products, which were contaminated with reduced
diisopropyl azodicarboxylate. These crude compounds
Scheme 1a
a Reagents and conditions: (a) 6 N HCl, H2O; (b) R-AIBBr, CH3CN, and then K2CO3; (C) LAH, anhydrous THF; (d) TrCl, pyridine; (e)
PDC, AcOH, CH2Cl2; (f) DAST, anhydrous CH2Cl2; (g) tBuOK, THF; (h) 3 N HCl, MeOH; (i) TBDPSCl, imidazole, CH2Cl2; (j) Na/liq NH3;
(k) synthesis of 3′-fluoro isomers.

3738 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Wang et al.
Scheme 2a
a Reagents and conditions: (a) DIAD, Ph3P, pyrimidines, THF or THF/1,4-dioxane; (b) NH3/MeOH, rt; (c) TBAF, THF, rt; (d) (i) 2,4,6-
triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, (ii) NH4OH; MeOH, room temp; (e) NH3, MeOH, steel bomb, 110 °C; (f)
HOCH2CH2SH, NaOMe, 70 °C; (g) 3 N HCl, MeOH; (h) (1) HCOOH, 80 °C, (2) NH4OH, MeOH, rt.
Scheme 3a
a Reagents and conditions: (a) 6 N HCl, H2O; (b) R-AIBBr, CH3CN, and then K2CO3; (C) LAH, anhydrous THF; (d) TrCl, pyridine; (e)
PDC, AcOH, CH2Cl2; (f) DAST, anhydrous CH2Cl2; (g) tBuOK, THF; (h) 3 N HCl, MeOH; (i) TBDMSCl, imidazole, CH2Cl2; (j) Na/liq NH3;
(k) synthesis of 3′-fluoro isomers.
were directly treated with methanolic ammonia to give
debenzoylated compounds 13 and 17 in 41% and 54%
yield, respectively, in two steps. To synthesize the
cytidine analogue, the uridine analogue 13 was further
subjected to the ammonolysis using 2,4,6-triisopropyl-
benzenesulfonyl chloride, 4-(dimethylamino)pyridine,
and triethylamine in acetonitrile to give 15 in 65% yield.
The uridine, cytidine, and thymidine analogues 14, 16,
and 18 were obtained by the deprotection of the silyl
group using 3 N HCl in 88-92% yield. The synthesis of
the purine analogues followed a similar procedure. The
key intermediate 12 was condensed with 6-chloropurine
to give the corresponding nucleoside 19, which was
further treated with methanolic ammonia in a steel
bomb at 110 °C to obtain adenine derivative 20 in 36%
yield in two steps, and the deprotection of silyl group
gave the target compound 21 in 93% yield. Compound
19 was also treated with 2-mercaptoethanol and sodium
methoxide in refluxing methanol, and the silyl group
was then removed under acidic conditions to give an
inosine analogue 23 in 64% yield in two steps. Conden-
sation of 2-amino-6-chloropurine with 12 needed careful
control of the solvent and reaction temperature. Al-
though treatment of 12 with 2-amino-6-chloropurine in

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3739
Scheme 4a
a Reagents and conditions: (a) DIAD, Ph3P, pyrimidines, THF; (b) NH3/MeOH, room temp; (c) TBAF, THF, rt; (d) (i) 2,4,6-
triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, (ii) NH4OH; 3 N HCl, MeOH, rt; (e) NH3, MeOH, steel bomb, 110 °C; (f)
HOCH2CH2SH, NaOMe, 70 °C; (g) 3 N HCl, MeOH; (h) (1) HCOOH, 80 °C, (2) NH4OH, MeOH, rt.
Table 1. In Vitro Anti-HIV-1 Activity and Toxicity of D- and
L-2′-Fluoro-2′,3′-didehydrocarbocyclic Nucleosides
Table 2. Activity of Selected Nucleosides against
Lamivudine-Resistant Virus (HIV-1M184V) in Human PBM Cells
xxBRU
M184V
EC50
(µM)
75.3
>100
EC50
(µM)
EC90
(µM)
EC90
compd
(µM)
FIa
98
>100
L-2′F-C-d4A
3TC
0.77
0.027
8.34
0.25
>100
>100
anti-HIV-1
activity,
a FI is the fold increase (EC50 HIV-1M184V/EC50 HIV-1xxBRU).
cytotoxicity (µM)
EC50
B
configuration
(µM)
PBM CEM Vero
shown in Scheme 4), syntheses were quite similar to
the D-isomers except the condensation conditions ap-
plied in the Mitsunobu reaction: triphenylphosphine
and diisopropyl azodicarboxylate were first mixed in
THF at 0 °C to form the yellowish suspension and then
cooled to -78 °C. The key intermediate 37 and the
corresponding protected base moiety were then added,
and the reaction was allowed to gradually warm to room
temperature. The conditions could be applied for both
purines and pyrimidines to provide the desired products.
The synthetic variations applied between the D- and L-
series may be due to the different 5′-protecting groups.
Assignment of the structures of newly synthesized
nucleosides was based on NMR, mass spectroscopy,
elemental analysis, and UV spectroscopy.
Anti-HIV Activity. The newly synthesized carbo-
cyclic nucleosides were tested for anti-HIV activity as
well as cytotoxicity using AZT as the positive control,
and the results are summarized in Table 1. Anti-HIV
activity was performed in human peripheral blood
mononuclear (PBM) cells infected with HIV-1. Cytotox-
icity was tested in three cell lines (PBM, CEM, and
Vero). Among the target nucleosides, L-adenosine ana-
uracil (14)
D
D
D
D
D
D
L
L
L
L
L
L
D
>100
>100
>100
72.8
>100
37.8
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 91.3 >100
>100 >100 >100
>100 14.3 28.0
cytosine (16)
thymine (18)
adenine (21)
hypoxanthine (23)
guanine (25)
thymine (39)
uracil (41)
>100
>100
37.7
cytosine (43)
adenine (46)
hypoxanthine (48)
guanine (50)
AZT
0.77
>100
>100
0.0018
the presence of triphenylphosphine and diisopropyl
azodicarboxylate in THF or DMF at 0 °C failed to
provide the corresponding 2-amino-6-chloropurine nu-
cleoside, the desired compound was obtained in a
mixture of THF and 1,4-dioxane (7:1) at -78 °C. The
removal of the silyl group using tetrabutylammonium
fluoride (TBAF) gave 24 in 40% yield in two steps. The
guanosine analogue 25 was obtained by treatment of
24 with formic acid at 80 °C followed by 28% ammonium
hydroxide solution in 84% yield. For the L-series (as

3740 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Wang et al.
Table 4. In Vitro Anti-HIV Activity of Selected 2′F-C-d4Ns
against Wild Type (WT) and M184V Virus in Human PBM
Cells and Correlation with Calculated Energy of the Complex
(2′F-C-d4N-TPs)/HIV-RT
Table 3. In Vitro Anti-HIV Activity of Selected 2′F-C-d4Ns
against HIV Wild Type (WT) Virus and Correlation with
Calculated Energy of the Complex (2′F-C-d4N-TPs)/HIV-RT
EC50
Erel
compd
(µM)a
(kcal/mol)b
xxBRU (WT)
M184V
EC50 Erel
b
b
L-2′F-C-d4A
D-2′F-C-d4A
D-2′F-C-d4G
0.77
-39.5
-22.7
-5.2
EC50
Erel
(µM) (kcal/mol) (µM) (kcal/mol) FIa ∆Erel
c
72.8
compd
L-2′F-C-d4A 0.77
3TC 0.027
37.8
-39.5
-51.4
75.3
>100
-26.3
-18.3
98 -13.2
>3700 -33.1
4.6c
-65.2
carbovir
0.55d
0.027
0.0018
a FI is the fold increase (EC50 HIV-1M184V/EC50 HIV-1xxBRU).
b Erel ) (binding energy of inhibitor-TP) - (binding energy of
natural 2′-dNTP). c ∆Erel ) Erel(WT) - Erel(M184V).
3TC
AZT
-51.4
-101.8
b
a EC50 in PBM cells unless otherwise indicated. Erel ) (binding
energy of inhibitor-TP) - (binding energy of natural 2′-dNTP).
c IC50 in MT4 cells.15 d EC50 in PBM cells (communicated biological
data).
1996, lamivudine (3TC) has been widely used in HIV
treatment. However, the rapidly emerged lamivudine-
resistant mutant strain compromised its efficacy. The
single point mutation at codon 184 (M184V) in the
YMDD motif increases the 50% inhibitory concentration
at least 1000-fold.12,13 Consequently, discovering effec-
tive novel NRTIs against these resistant variants is of
great interest. Thus, the most potent compound 46 was
studied with the M184V mutant using 3TC as control
(Table 2). From this study, it was found that there was
significantly reduced antiviral activity against HIV-
logue 46 showed the most potent activity against wild-
type HIV-1 (EC50 ) 0.77 µM), although its D-counterpart
was inactive. In the D-series, only cytidine 21 (EC50
)
72.8 µM) and guanosine 25 (EC50 ) 37.8 µM) showed
weak anti-HIV activity. It is interesting to note that,
although compound 25 has a structure similar to
carbovir, it was not as potent as carbovir. The lower
antiviral potency may be due to the decreased level of
phosphorylation by the initial nucleoside kinase and/or
by reduced binding of the triphosphate to the HIV
reverse transcriptase at the catalytic site (vide infra for
molecular modeling studies).
1
M184V for compound 46. Thus, its mechanism was also
investigated by molecular modeling as below.
Molecular Modeling. Considering the anti-HIV
activities of D- and L-2′,3′-dideoxy-2′,3′-didehydro-2′-
fluorocarbocyclic nucleosides (D- and L-2′F-C-d4Ns),
there are several interesting points that need to be
Antiviral Activity against Lamivudine-Resistant
(HIV-1M184V) Mutant Strain. Since its introduction in
Figure 3. (a) Binding mode of L-2′F-C-d4A-TP in the catalytic site of HIV-RT. Blue dotted lines indicate the π-π interaction.
The 2′-fluorine involves in the hydrogen bonding with OH on the phenyl ring of Tyr115. (b) Binding mode of L-2′F-C-d4A-TP in
the catalytic site of HIV-RT. The 2′-fluorine of D-2′F-C-d4A-TP does not have the hydrogen bond. (c) A favorable van der Waals
interaction between the sugar ring of L-2′F-C-d4A-TP and the side chain of Met184 which increases its binding affinity to HIV-
RT. (d) The side chain of Met184 is far away from the D-2′F-C-d4A-TP.

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3741
Figure 4. (a) Carbovir and D-2′F-C-d4G superimposed very nicely on each other, indicating the similar affinity to nucleoside
kinases. (b) Green one is the binding mode of carbovir-TP and the corresponding Tyr115 which has the good π-π interaction
between the phenyl ring and 2′,3′-double bond. D-2′F-C-d4G-TP and the corresponding Tyr115 are indicated in CPK mode in
which Tyr115 moves away from the bottom of D-2′F-C-d4G-TP and decrease the π-π interaction. (c) Side view of carbovir-TP and
Tyr115. As can be seen in the figure, carbovir-TP has a favorable π-π interaction with Tyr115. (d) Side view of D-2′F-C-d4G-TP
and Tyr115. Trp115 is distorted and moves away from the bottom of the sugar ring, which indicated an unfavorable π-π interaction.
understood: (a) L-2′F-C-d4A is significantly more active
than its D-counterpart, (b) it is cross-resistant to HIV-
RT(M184V), (c) D-2′F-C-d4G is significantly less potent
compared to carbovir, although these two compounds
share similar structural features.
bonding to OH in the phenyl ring of Tyr115 which is
not observed in the D-counterpart (Figure 3b). Addition-
ally, the carbocyclic ring of L-2′F-C-d4A-TP shows an
additional favorable van der Waals interaction between
the side chain of Met184 (Figure 3c) that is absent in
the D-counterpart (Figure 3d). The increased favorable
van der Waals interaction as well as the additional
hydrogen bond may result in the L-form binding more
tightly to HIV-RT, reflecting the higher relative binding
energy and consequently higher level of anti-HIV activ-
ity, although the initial kinase might have also played
a significant role in determining the observed anti-HIV
potency.
In view of the fact that carbovir is a potent anti-HIV
agent with an EC50 value of 4.6 µM, it is interesting to
find out the significantly lower binding energy of D-2′F-
C-d4G on HIV-RT.15 The two optimized-structures of
D-2′F-C-d4G and carbovir superimposed nicely on each
other (Figure 4a). As indicated by the modeling studies,
D-2′F-C-d4G might be also a good substrate for nucleo-
side kinase, like carbovir.16 Studies showed that carbo-
vir can be stereoselectively phosphorylated by 5′-
nucleotidase and GMP kinase to its triphosphate.17
Hence, D-2′F-C-d4G might also be converted to its
triphosphate by these kinases and then interacts with
HIV-RT. Upon analysis of the binding mode of D-2′F-
C-d4G-TP with HIV-RT, we found that Tyr115, which
is involved in the π-π interaction with the 2′,3′-double
bond of D-2′F-C-d4G, is distorted and moves away from
the bottom of the sugar ring (Figure 4b), in contrast to
the favorable π-π interaction in carbovir-TP/HIV-RT
complex (Figure 4c). Decrease of the favorable π-π
To understand the molecular basis of different anti-
HIV potency as described above, we conducted molec-
ular modeling studies to analyze the binding energy as
well as the interaction between HIV-RT and NRTI
triphosphates (Tables 3 and 4). In our previous studies,
the relative binding energy is proved to be qualitatively
correlated with anti-HIV activity.6,14 In this study, we
found that L-2′F-C-d4A had the most favorable relative
binding energy (-39.5 kcal/mol) among the synthesized
carbocyclic nucleosides in this report, which is in agree-
ment with its antiviral activity. The relative binding
energy of carbovir is significantly higher than that of
D-2′F-C-d4G by nearly 60 kcal/mol, which is also in
accordance with the difference of their antiviral activity.
Analogously, in L-2′F-C-d4A-TP/HIV-RT(M184V) complex,
a less favorable relative binding energy indicated a
nearly 100-fold decrease of anti-HIV activity. As ex-
pected, our model provided a qualitative, but not a
quantitative, correlation with the experimental data
(Table 3).
The energy minimized structures of L- and D-2′F-C-
d4A-TP were bound to the HIV-RT catalytic site, in
which Arg72, Lys65, Ala 114, and Asp 113 stabilize the
triphosphate moiety by multiple hydrogen bonds (Fig-
ures 3-5). A π-π interaction between the 2′,3′-double
bond and the aromatic ring of Tyr115 may also contrib-
ute to a positive binding (Figure 3a). In the case of L-2′F-
C-d4A-TP, the 2′-fluorine is stabilized by a hydrogen

3742 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Wang et al.
Figure 5. (a) In L-2′F-C-d4A-TP/HIV-RT(WT) complex, threre is no unfavorable steric hindrance between Met184 and the sugar
ring. (b) In L-2′F-C-d4A-TP/HIV-RT(M184V) complex, the side chain of Val184 and the sugar ring of the inhibitor experience steric
hindrance. (c) Compared with L-2′F-C-d4A-TP/HIV-RT(WT) complex, the minimized structure of L-2′F-C-d4A-TP/HIV-RT(M184V)
complex shows the movement of 2′-fluorine which cause loss of hydrogen bonding with Tyr115 and a disrupted base-pairing with
the complementary base in the template strand.
column chromatography. Elemental analyses were performed
by Atlantic Microlab Inc., Norcross, GA.
interaction (Figure 4d) implies the loss of binding
affinity, resulting in lower anti-HIV activity.
Synthesis of the D-Series, Compounds 1-25.
(+)-(1S,2R,3R,4S)-1-O-Benzyloxy-2,3-dihydroxyl-4-
hydroxymethylcyclopentane (2). A solution of fully pro-
tected cyclopentane 1 (29.9 g, 89.4 mmol)10c in MeOH (150 mL)
was treated with 3 N HCl (150 mL). After refluxing for 4 h,
the resulting brown mixture was coevaporated in vacuo with
EtOH (100 mL × 2) and the residue was purified by column
chromatography on silica gel (MeOH:CH2Cl2 ) 1:20) to afford
2 (15.7 g, 73.7%) as an off-white solid: mp 115.7-116.9 °C;
[R]22D +11.37° (c 0.71, MeOH); 1H NMR (400 MHz, MeOH-d4)
δ 7.38-7.24 (m, 5H), 4.55 (dd, J ) 11.9 and 35.1 Hz, 1H), 4.00
(t, J ) 3.9 Hz, 1H), 3.85 (dt, J ) 3.7 and 7.4 Hz, 1H), 3.72 (dd,
J ) 4.2 and 7.6 Hz, 1H), 3.60 (dd, J ) 4.6 and 10.7 Hz, 2H),
3.50 (dd, J ) 6.1 and 10.7 Hz, 2H), 2.23 (m, 1H), 2.00 (m, 1H),
1.72 (m, 1H); 13C NMR (100 MHz, MeOH-d4) δ 137.8, 128.8,
128.3, 128.0, 78.7, 75.8, 72.9 (d, J ) 11.8 Hz), 72.1, 65.1 (d, J
) 11.4 Hz), 45.9, 30.1, 0.2. Anal. Calcd for (C12H18O4) C, H.
Similar to other L-nucleosides,18 in the L-2′F-C-d4A-
TP/HIV-RT(M184V) complex, the binding pocket of sugar
ring points toward the side chain of Val184 and tends
to provide the steric hindrance when L-2′F-C-d4A-TP
binds to M184V RT (Figure 5b). To circumvent this
unfavorable binding, the L-2′F-C-d4A-TP/HIV-RT(M184V)
must undergo significant conformational change, result-
ing in a loss of the hydrogen bond between 2′-fluorine
and OH of Tyr115 and a disrupted base-pairing with
the complementary base in the template strand, which,
in turn, results in a decrease in the relative binding
energy (Figure 5c).19
Experimental Section
(-)-(1S,2R,3R,4S)-1-O-Benzyloxy-2,3-anhydro-4-hy-
droxymethylcyclopentane (3). A solution of triol 2 (15.7 g,
65.9 mmol) in anhydrous acetonitrile (200 mL) was cooled to
-30 °C and then treated with 1-bromocarbonylmethylethyl
acetate (24.2 mL, 164.7 mmol). After stirring at room temper-
ature for 1 h, H2O (150 mL) and EtOAc (150 mL) were added
to the resulting mixture and the organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo
and the residue was dissolved in MeOH (200 mL). Potassium
carbonate (36.4 g, 263.6 mmol) was added and reacted for 12
h at room temperature. The reaction mixture was filtered over
a Celite pad (∼5 cm) and the filtrate was concentrated under
reduced pressure. The residue was dissolved in EtOAc (200
mL), washed with H2O (100 mL), dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo and the residue
was purified by column chromatography on silica gel (EtOAc:
Melting points were determined on a Mel-temp II apparatus
and are uncorrected. Nuclear magnetic resonance spectra were
recorded on a Bruker AMX 400 spectrometer at 400 MHz for
1H NMR and 100 MHz for 13C NMR or a Varian Inova 500
1
spectrometer at 500 MHz for H NMR and 125 MHz for 13C
NMR with tetramethylsilane as the internal standard. Chemi-
cal shifts (δ) are reported as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), or bs (broad singlet). UV spectra
were recorded on a Beckman DU-650 spectrophotometer.
Optical rotations were measured on a Jasco DIP-370 digital
polarimeter. High-resolution mass spectra were recorded on
a Micromass Autospec high-resolution mass spectrometer. TLC
was performed on Uniplates (silica gel) purchased from
Analtech Co. Column chromatography was performed using
either silica gel 60 (220-440 mesh) for flash chromatography
or silica gel G (TLC grade, >440 mesh) for vacuum flash

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3743
hexanes ) 1:3 to 1:1) to give 3 (11.5 g, 79.3%) as a colorless
oil: [R]22D -54.06° (c 0.11, CHCl3); 1H NMR (400 MHz, MeOH-
d4) δ 7.39-7.27 (m, 5H), 4.63 (s, 2H), 4.14 (t, J ) 8.0 Hz, 1H),
3.61 (dd, J ) 5.3 and 10.6 Hz, 1H), 3.52 (m, 2H), 3.45 (d, J )
2.6 Hz, 1H), 2.52 (q, J ) 6.7 Hz, 1H), 1.76 (dd, J ) 8.1 and
13.2 Hz, 1H), 2.00 (m, 1H), 1.60 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 137.5, 128.7, 128.0, 127.9, 79.2, 71.9, 64.0, 57.7, 56.7,
41.1, 28.5. Anal. Calcd for (C12H16O3) C, H.
the reaction mixture was poured into a saturated NaHCO3
(150 mL) solution and extracted with CH2Cl2 (100 mL × 2).
The combined organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo
and the residue was purified by column chromatography on
silica gel (EtOAc:hexanes ) 1:100) to give difluorinated
compound 8 (10.0 g, 85.5%) as a colorless oil: [R]22 -12.95°
D
1
(c 0.16, MeOH); H NMR (400 MHz, CDCl3) δ 7.46-7.27 (m,
20H), 4.84 (d, J ) 11.9 Hz, 1H), 4.62 (d, J ) 11.9 Hz, 1H),
3.94 (m, 1H), 3.05 (m, 2H), 2.64 (m, 1H), 2.40 (m, 1H), 2.04-
1.94 (m, 2H), 1.78 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 217.5,
144.1, 138.3, 128.9, 128.7, 128.0, 128.0, 127.8, 127.3, 86.8, 74.9,
70.8, 62.3, 46.7, 45.8, 33.1. Anal. Calcd for (C32H30F2O2) C, H.
(-)-(1S,2R,4S)-1-O-Benzyloxy-2-hydroxyl-4-hydroxy-
methylcyclopentane (4) and (+)-(1S,3S,4S)-1-O-Benzyl-
oxy-3-hydroxyl-4-hydroxymethylcyclopentane (5). To a
solution of epoxide 3 (10.0 g, 45.4 mmol) in anhydrous THF
(150 mL) at -78 °C was slowly added lithium aluminum
hydride (LAH, 5.4 g, 136.2 mmol). After addition of LAH, the
reaction mixture was warmed to room temperature and stirred
for 4 h. Celite (20 g) was added to the reaction mixture and
iced H2O (150 mL) was added slowly to quench the reaction.
The slurry was filtered over a Celite pad (∼10 cm) and the
filtrate was extracted with EtOAc (100 mL × 4). The combined
organic layer was dried over MgSO4 and filtered. The filtrate
was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (EtOAc:hexanes ) 1:2)
to give 4 (6.9 g, 68.6%) and 5 (1.5 g, 14.8%) as colorless oils.
Compound 4: [R]23D -6.08° (c 0.38, MeOH); 1H NMR (400 MHz,
CDCl3) δ 7.38-7.29 (m, 5H), 4.56 (dd, J ) 11.7 and 29.3 Hz,
2H), 4.17 (bs, 1H), 3.90 (m, 1H), 3.48 (m, 2H), 2.60 (bs, 1H),
2.49 (m, 1H), 1.97-1.87 (m, 2H), 1.66 (m, 1H), 1.48 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 138.0, 128.5, 127.8, 127.7, 81.0,
(-)-(3S,5S)-1-Fluoro-3-(O-trityloxymethyl)-5-O-benzyl-
oxycyclopent-1-ene (9). To a solution of compound 8 (10.9
g, 22.5 mmol) in anhydrous THF (150 mL) was added potas-
sium tert-butoxide (13.3 g, 112.5 mmol) at room temperature.
After being stirred at 50 °C for 28 h (the completion of reaction
1
was monitored by H NMR), H2O (150 mL) was added to the
resulting dark brown mixture and it was extracted with EtOAc
(150 mL × 2). The combined organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo
and the residue was purified by column chromatography on
silica gel (EtOAc:hexanes ) 1:30) to give vinyl compound 9
(9.1 g, 87.5%) as a colorless oil: [R]23D -74.99° (c 0.95, CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.44-7.24 (m, 20H), 5.40 (s, 1H),
4.62 (dd, J ) 11.7 and 20.7 Hz, 2H), 4.54 (d, J ) 5.67 Hz, 1H),
3.09 (m, 2H), 2.97 (t, J ) 7.33 Hz, 1H), 2.08 (dd, J ) 8.1 and
13.9 Hz, 1H), 1.97 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 144.5,
129.1, 128.8, 128.2, 128.0, 127.4, 110.2 (d, J ) 8.9 Hz), 78.6
(d, J ) 20.9 Hz), 71.5, 67.7, 38.9 (d, J ) 7.7 Hz), 33.1 (d, J )
7.8 Hz). Anal. Calcd for (C32H30FO2) C, H.
72.3, 71.6, 66.9, 36.8, 34.3, 31.1. Anal. Calcd for (C12H18O3) C,
1
H. Compound 5: [R]22 +6.00° (c 0.15, MeOH); H NMR (400
D
MHz, CDCl3) δ 7.36-7.27 (m, 5H), 4.48 (s, 2H), 4.03 (m, 2H),
3.71 (dd, J ) 5.5 and 10.4 Hz, 1H), 3.53 (dd, J ) 8.0 and 10.4
Hz, 1H), 2.34 (m, 1H), 2.09 (m, 2H), 1.92 (m, 1H), 1.40 (m,
1H); 13C NMR (100 MHz, CDCl3) δ 138.2, 128.4, 127.6, 78.9,
76.1, 70.6, 65.4, 48.7, 40.8, 33.4. Anal. Calcd for (C12H18O3) C,
H.
(-)-(3S,5S)-1-Fluoro-3-hydroxymethyl-5-O-benzyloxy-
cyclopent-1-ene (10). To a solution of vinyl 9 (1.0 g, 2.15
mmol) in THF (15 mL) were added 6 N HCl (4 mL) solution
and MeOH (4 mL) at room temperature. After being heated
at 50 °C for 5 h, the resulting mixture was cooled to 0 °C and
neutralized by 1 N NaOH solution. After evaporation of half
the volume, the residue was extracted with EtOAc (50 mL ×
2). The combined organic layer was dried over MgSO4 and
filtered. The filtrate was concentrated in vacuo and the residue
was purified by column chromatography on silica gel (EtOAc:
hexanes ) 1:10 to 1:5) to give alcohol 10 (429 mg, 89.7%) as a
(-)-(1S,2R,4S)-1-O-Benzyloxy-2-hydroxyl-4-(O-trityl-
oxymethyl)cyclopentane (6). A mixture of diol 4 (7.27 g,
32.7 mmol) and trityl chloride (10.0 g, 32.7 mmol) in pyridine
(100 mL) was refluxed for 4 h. The reaction mixture was
concentrated in vacuo and the residue was dissolved in EtOAc
(200 mL), washed with brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo and the residue was
purified by column chromatography on silica gel (EtOAc:
colorless oil: [R]23 -154.82° (c 0.76, MeOH); 1H NMR (400
D
hexanes ) 1:30) to give 6 (15.1 g, 99.4%) as a colorless oil:
MHz, CDCl3) δ 7.35-7.28 (m, 5H), 5.28 (s, 1H), 4.61 (dd, J )
13.0 and 24.7 Hz, 2H), 4.57 (bs, 1H), 3.58 (dd, J ) 5.5 and
10.5 Hz, 1H), 3.51 (dd, J ) 5.8 and 10.5 Hz, 1H), 2.97 (m, 1H),
2.04 (m, 2H), 1.37 (t, J ) 5.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 138.3, 128.7, 128.0, 128.0, 108.8 (d, J ) 7.3 Hz), 78.4
(d, J ) 16.8 Hz), 71.5, 66.6 (d, J ) 2.7 Hz), 40.6 (d, J ) 5.7
Hz), 32.4 (d, J ) 5.7 Hz). Anal. Calcd for (C13H15 FO2‚0.3CH2Cl2)
C, H.
1
[R]24 -11.18° (c 0.30, MeOH); H NMR (400 MHz, CDCl3) δ
D
7.32-7.12 (m, 20H), 4.47 (dd, J ) 11.8 and 29.0 Hz, 2H), 4.07
(bs, 1H), 3.81 (dd, J ) 6.6 and 10.7 Hz, 1H), 2.86 (m, 2H),
2.54 (m, 1H), 2.45 (d, J ) 3.6 Hz, 1H), 1.86 (m, 2H), 1.55 (m,
1H), 1.43 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 144.3, 128.7,
128.5, 127.8, 127.7, 126.9, 81.0, 72.4, 71.6, 67.2, 35.1 (d, J )
19.8 Hz), 31.8. Anal. Calcd for (C32H32O3) C, H.
(-)-(2S,4S)-2-O-Benzyloxy-4-(O-trityloxymethyl)cyclo-
pentan-1-one (7). To a solution of alcohol 6 (15.1 g, 32.5
mmol) in anhydrous CH2Cl2 (250 mL) were respectively added
4 Å molecular sieve (15 g), pyridinium dichromate (24.5 g, 65.0
mmol), and acetic acid (0.7 mL, 0.05 mol %). After being stirred
at room temperature for 12 h, the resulting brown slurry was
filtered over a silica gel pad (∼15 cm) with EtOAc. The filtrate
was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (EtOAc:hexanes ) 1:50
to 1:20) to give ketone 7 (12.2 g, 81.3%) as a white solid: mp
(-)-(3S,5S)-1-Fluoro-3-(O-tert-butyldiphenylsilyloxy-
methyl)-5-O-benzyloxcyclopent-1-ene (11). To a solution
of vinyl alcohol 10 (3.0 g, 13.5 mmol) in CH2Cl2 (50 mL) were
added tert-butyldiphenylsilane chloride (3.9 mL, 14.8 mmol),
and imidazole (1.38 g, 20.2 mmol) at room temperature. After
being stirred at room temperature for 1 h, H2O (150 mL) was
added to the resulting white suspension mixture and the
organic layer was dried over MgSO4 and filtered. The filtrate
was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (EtOAc:hexanes ) 1:100)
to give 11 (6.2 g, 99%) as a colorless oil: [R]24D -74.99° (C 0.79,
MeOH); 1H NMR (400 MHz, CDCl3) δ 7.64-7.61 (m, 4H),
7.40-7.34 (m, 11H), 5.29 (s, 1H), 4.61 (dd, J ) 131.7 and 22.1
Hz, 2H), 4.55 (bs, 1H), 3.52 (m, 2H), 2.97 (bs, 1H), 1.99 (m,
2H), 1.03 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 163.2, 160.9,
138.2, 135.6 (d, J ) 8.4 Hz), 129.7 (d, J ) 2.2 Hz), 128.4, 127.8,
127.8, 127.7, 127.7, 109.5 (d, J ) 6.8 Hz), 78.3 (d, J ) 16.4
Hz), 71.1, 67.5 (d, J ) 2.7 Hz), 40.4 (d, J ) 5.7 Hz), 32.2 (d, J
) 5.7 Hz), 26.8, 19.2. Anal. Calcd for (C29H33FO2Si) C, H.
100-102 °C; [R]23 -27.05° (c 0.14, MeOH); 1H NMR (400
D
MHz, CDCl3) δ 7.41-7.24 (m, 20H), 4.82 (d, J ) 11.9 Hz, 1H),
4.35 (d, J ) 11.9 Hz, 1H), 3.91 (t, J ) 6.6, 1H), 3.13 (m, 2H),
2.70 (m, 1H), 2.53 (dd, J ) 8.8 and 19.0 Hz, 1H), 2.17 (dd, J )
6.0 and 18.6 Hz, 1H), 2.09-1.99 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 216.0, 143.9, 86.7, 78.3, 71.9, 66.4, 39.6, 33.6, 32.6.
Anal. Calcd for (C32H30O3) C, H.
(-)-(2S,4S)-1-Difluoro-2-O-benzyloxy-4-(O-trityloxy-
methyl)cyclopentane (8). To a solution of ketone 7 (11.2 g,
24.2 mmol) in anhydrous CH2Cl2 (100 mL) was added diethyl
aminosulfur trifluoride (DAST, 14.8 mL, 121.5 mmol) at room
temperature. After being stirred at room temperature for 4 h,
(-)-(3S,5S)-1-Fluoro-3-(O-tert-butyldiphenylsilyloxy-
methyl)-5-hydroxylcyclopent-1-ene (12). Liquid ammonia
(20 mL) was trapped in a three-neck round-bottom flask (50

3744 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Wang et al.
mL) at -78 °C and then sodium (825 mg, 35.8 mmol) was
added slowly. To the resulting dark blue solution was added
slowly a solution of 10 (660 mg, 1.43 mmol) in 1,4-dioxane (3
mL) at -78 °C over 5 min. After being stirred at the same
temperature for 10 min, CH2Cl2 (50 mL) was added slowly to
the reaction mixture. The resulting brown mixture was care-
fully poured into iced H2O (50 mL). (Caution: The remaining
sodium causes flames!) The mixture was extracted with CH2Cl2
(50 mL × 2). The combined organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo
and the residue was purified by column chromatography on
silica gel (EtOAc:hexanes ) 1:5) to give vinyl alcohol 12 (380
pH 7), 272.0 nm (ꢀ 16 461, pH 11); MS m/z 227 (M + 2). Anal.
Calcd for (C10H12FN3O2) C, H, N.
(-)-(1′R,4′S)-1-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6-(O-tert-butyldiphenylsilyloxymethyl)cyclopent-2-enyl]-
thymine (17). Compound 12 (400 mg, 1.08 mmol) was
converted to 17 (243 mg, 46% from 12) as a white foam using
the same procedure as that described for 13: [R]22 -24.72°
D
(c 0.61, CHCl3); UV (MeOH) λmax 267.5 nm. Anal. Calcd for
(C27H31FN2O4Si) C, H, N.
(+)-(1′R,4′S)-1-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
cyclopent-2-enyl]thymine (18). Compound 17 (225 mg, 0.46
mmol) was converted to the thymine derivative 18 (104 mg,
88%) as a white foam using the same procedure as that
described for 14: [R]23D +28.19° (c 0.76, CHCl3); UV (H2O) λmax
269.0 nm (ꢀ 5678, pH 2), 271.0 nm (ꢀ 6971, pH 7), 279.0 nm (ꢀ
6911, pH 11); MS m/z 241 (M + 1). Anal. Calcd for (C11H13-
FN2O3) C, H, N.
mg, 71.7%) as a colorless oil: [R]25 -90.52° (c 0.47, MeOH);
D
1H NMR (400 MHz, CDCl3) δ 7.66 (m, 4H), 7.42-7.36 (m, 6H),
5.23 (s, 1H), 4.79 (bs, 1H), 3.55 (m, 2H), 2.96 (bs, 1H), 2.12
(m, 2H), 1.91 (m, 1H), 1.04 (s, 9H); 13C NMR (100 MHz, CDCl3)
δ 164.2, 161.4, 155.2, 135.8 (d, J ) 4.5 Hz), 133.8 (d, J ) 9.1
Hz), 130.0 (d, J ) 2.3 Hz), 127.9, 108.5 (d, J ) 8.4 Hz), 72.1
(d, J ) 22.9 Hz), 71.9, 67.6 (d, J ) 3.0 Hz), 40.4 (d, J ) 7.6
Hz), 34.9 (d, J ) 6.8 Hz), 27.0, 19.5. Anal. Calcd for (C22H27-
FO2Si) C, H.
(+)-(1′R,4′S)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6-(O-tert-butyldiphenylsilyloxymethyl)cyclopent-2-enyl]-
adenine (20). A solution of vinyl alcohol 12 (700 mg, 1.89
mmol), tripheylphosphine (990 mg, 3.78 mmol), and 6-chloro-
purine (613 mg, 3.96 mmol) in anhydrous THF (10 mL) was
cooled to 0 °C and then diisopropyl azodicarboxylate (760 mg,
3.78 mmol) was added slowly. The reaction mixture was slowly
allowed to warm to room temperature and stirred for 12 h.
The yellowish resulting mixture was concentrated in vacuo and
the residue was purified by column chromatography on silica
gel (EtOAc:hexanes ) 1:10) to give the corresponding nucleo-
side 19 as crude product (570 mg), which was used for the next
reaction without further purification. The crude product (520
mg) was treated with methanolic ammonia and heated in a
steel bomb at 100 °C for 24 h. After the reaction mixture was
concentrated in vacuo, the residue was purified by column
chromatography on silica gel (MeOH:CH2Cl2 ) 1:20) to give
(-)-(1′R,4′S)-1-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6-(O-tert-butyldiphenylsilyloxymethyl)cyclopent-2-enyl]-
uracil (13). A solution of vinyl alcohol 12 (1.0 g, 2.70 mmol),
triphenyl phosphine (2.83 g, 10.80 mmol), and N3-benzoyluracil
(1.17 g, 5.40 mmol) in anhydrous THF (10 mL) was cooled to
0 °C and then diisopropyl azodicarboxylate (2.17 g, 10.80
mmol) was added slowly. The reaction mixture was slowly
allowed to warm to room temperature and stirred for 12 h.
The yellowish resulting mixture was concentrated in vacuo and
the residue was purified by column chromatography on silica
gel (EtOAc:hexanes ) 1:10) to the corresponding nucleoside
as crude product, which was used for the next reaction without
further purification. The crude product (700 mg) was treated
with methanolic ammonia and stirred at room temperature
for 12 h. After the reaction mixture was concentrated in vacuo,
the residue was purified by column chromatography on silica
gel (EtOAc:hexanes ) 1:5) to give 13 (530 mg, 41% from 12)
20 (335 mg, 36% from 12) as a colorless oil: [R]24 +2.18° (c
D
0.40, CHCl3); UV (MeOH) λmax 260.5 nm. Anal. Calcd for
(C27H30N5FOSi‚0.7H2O) C, H, N.
(+)-(1′R,4′S)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6′-hydroxymethylcyclopent-2-enyl]adenine (21). Com-
pound 20 (180 mg, 0.37 mmol) was converted to adenine
derivative 21 (85 mg, 93%) as a white solid using the same
as a white foam: [R]24 -14.76° (c 0.41, CHCl3); UV (MeOH)
D
λmax 263.5 nm. Anal. Calcd for (C26H29FN2O3Si‚0.7H2O) C, H,
N.
procedure as that described for 14: mp 218-220 °C; [R]23
D
(-)-(1′R,4′S)-1-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
cyclopent-2-enyl]uracil (14). To a solution of 13 (130 mg,
0.27 mmol) in MeOH (5 mL), 3 N HCl (5 mL) was added at
room temperature. After being stirred at room temperature
for 1 h, the resulting mixture was coevaporated with EtOH
and the residue was purified by column chromatography on
silica gel (MeOH:CH2Cl2 ) 1:15) to give 14 (45 mg, 68%) as a
+79.60° (c 0.35, MeOH); UV (H2O) λmax 258.0 nm (ꢀ 11 106,
pH 2), 260.5 nm (ꢀ 8708, pH 7), 260.5 nm (ꢀ 10 234, pH 11);
MS m/z 250 (M + 1). Anal. Calcd for (C11H12FN5O‚HCl) C, H,
N.
(+)-(1′R,4′S)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6-(O-tert-butyldiphenylsilyloxymethyl)cyclopent-2-enyl]-
inosine (22). To a solution of 6-chloropurine analogue 19 (240
mg, 0.47 mmol) in MeOH (10 mL) were added 2-mercapto-
ethanol (130 mg, 1.65 mmol) and sodium methoxide (94 mg,
1.65 mmol) at room temperature. After being refluxed for 12
h, the reaction mixture was neutralized with acetic acid and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (MeOH:CH2Cl2 ) 1:50) to give
white foam: [R]23 -20.97° (c 0.56, MeOH); UV (H2O) λmax
D
264.5 nm (ꢀ 11 145, pH 2), 264.5 nm (ꢀ 8932, pH 7), 264.5 nm
(ꢀ 13 131, pH 11); MS m/z 227(M + 1). Anal. Calcd for (C10H11-
FN2O3‚0.2H2O) C, H, N.
(-)-(1′R,4′S)-1-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6-(O-tert-butyldiphenylsilyloxymethyl)cyclopent-2-enyl]-
cytosine (15). To a solution of uracil derivative 13 (120 mg,
0.26 mmol) in anhydrous acetonitrile (5 mL) were added 2,4,6-
triisopropyl benzenesulfonyl chloride (156 mg, 0.52 mmol),
4-(dimethylamino)pyridine (32 mg, 0.26 mmol), and triethyl-
amine (0.15 mL, 1.04 mmol), respectively, at 0 °C. After the
reaction stirred at room temperature for 12 h, a 28% solution
of ammonium hydroxide (5 mL) was added to the resulting
brown mixture and it stirred at room temperature for another
12 h. The reaction mixture was concentrated in vacuo and the
residue was purified by column chromatography on silica gel
(MeOH:CH2Cl2 ) 1:30) to give 15 (100 mg, 83%) as a colorless
oil: [R]24D -45.68° (c 0.76, CHCl3); UV (MeOH) λmax 272.5 nm.
Anal. Calcd for (C26H30FN2O2Si) C, H, N.
22 (180 mg, 78%) as a colorless oil: [R]22 +22.28° (c 1.65,
D
CHCl3); UV (MeOH) λmax 220.5 and 249.5 nm. Anal. Calcd for
(C27H39FN4O2Si‚0.6H2O) C, H, N.
(+)-(1′R,4′S)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6′-hydroxymethylcyclopent-2-enyl]inosine (23). Com-
pound 22 (180 mg, 0.37 mmol) was converted to inosine
derivative 23 (85 mg, 93%) as a white solid using the same
procedure as for 14: [R]23D +81.45° (c 0.22, MeOH); mp 195-
197 °C; UV (H2O) λmax 249.0 nm (ꢀ 11 354, pH 2), 248.5 nm (ꢀ
15 927, pH 7), 254.0 nm (ꢀ 12 152, pH 11); MS m/z 251 (M+).
Anal. Calcd for (C11H12FN4O2‚0.6H2O) C, H, N.
(+)-(1′R,4′S)-2-Amino-6-chloro-9-[2′,3′-dideoxy-2′,3′-
didehydro-2′-fluoro-6-hydroxymethylcyclopent-2-enyl]-
purine (24). A solution of tripheylphosphine (1.25 g, 4.75
mmol) in anhydrous THF (10 mL) was cooled to 0 °C and then
treated with a solution of diisopropyl azodicarboxylate (956
mg, 4.75 mmol) in anhydrous 1,4-dioxane (2 mL) slowly. The
resulting suspension was stirred at 0 °C for 30 min and then
cooled to -78 °C. A solution of vinyl alcohol 12 (440 mg, 1.19
(-)-(1′R,4′S)-2-O-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
cyclopent-2-enyl]cytosine (16). Compound 15 (100 mg, 0.22
mmol) was converted to the cytosine derivative 16 (45 mg,
92%) as a white solid using the same procedure as described
for 14. mp 185-187 °C (dec); [R]23 -36.92° (c 0.64, MeOH);
D
UV (H2O) λmax 281.5 nm (ꢀ 9661, pH 2), 272.5 nm (ꢀ 13 881,

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3745
mmol) in anhydrous THF (5 mL) was added slowly, followed
by the addition of 2-amino-6-chloropurine (806 mg, 4.75 mmol).
The reaction mixture was slowly allowed to warm to room
temperature and stirred for 6 h. The precipitate was filtered
off and the filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (EtOAc:
hexanes ) 1:4) to the corresponding nucleoside (970 mg) as a
crude product, which was used for the next reaction without
further purification. The crude product (970 mg) was dissolved
in THF (10 mL) and then treated with a 1.0 M solution of
tetrabutylammonium fluoride in THF. After being stirred at
room temperature for 3 h, the reaction mixture was concen-
trated in vacuo and the residue was purified by column
chromatography on silica gel (MeOH:CH2Cl2 ) 1:40) to give
(CDCl3, 125 MHz) δ 138.24, 128.48, 127.69, 79.05, 76.34, 70.70,
65.58, 48.76, 40.89, 33.41. Anal. Calcd for (C13H18O3‚0.5H2O)
C, H.
(+)-(1R,2S,4R)-O-Benzyloxy-2-hydroxyl-4-(O-trityloxy-
methyl)cyclopentane (31) was prepared from 29 on a 54
mmol scale in 83% yield by a procedure similar to that
described for 6: [R]24 +12.00° (c 0.65, MeOH); 1H NMR
D
(MeOH-d4, 500 MHz) δ 7.42-7.20 (m, 20H), 4.78 (d, J ) 11.5
Hz, 1H), 4.51 (d, J ) 12 Hz, 1H), 4.15 (s, 1H), 3.88-3.86 (m,
1H), 2.98-2.92 (m, 2H), 2.61 (m, 1H), 2.56 (d, J ) 3.5 Hz, 1H),
1.96-1.63 (m, 2H), 1.67-1.63 (m, 1H), 1.51 (m, 1H); 13C NMR
(MeOH-d4, 125 MHz) δ 144.36, 138.16, 128.78, 128.57, 127.90,
127.80, 127.78, 126.96, 86.24, 81.13, 72.44, 71.63, 67.31, 35.18,
34.98, 31.85. Anal. Calcd for (C32H32O3) C, H.
24 (135 mg, 40% from 12) as a white foam: [R]23 +9.76° (c
D
(+)-(2R,4R)-2-O-Benzyloxy-4-(O-trityloxymethyl)cyclo-
pentan-1-one (32) was prepared from 31 on a 43 mmol scale
in 72% yield (14.3 g) by a procedure similar to that described
for 7: mp 102-103 °C; [R]26D +37.29° (c 0.32, MeOH); 1H NMR
(MeOH-d4, 500 MHz) δ 7.37-7.22 (m, 20H) 4.79 (d, J ) 12
Hz, 1H), 4.60 (d, J ) 11.5 Hz, 1H), 3.88 (t, J ) 7 Hz, 1H),
3.13-3.06 (m, 2H), 2.68-2.65 (m, 1H), 2.52-2.47 (dd, J ) 8.5
and 19 Hz, 1H), 2.14-2.08 (dd, J ) 6 and 19 Hz, 1H), 2.08-
2.04 (m, 1H), 1.98-1.93 (m, 1H); 13C NMR (MeOH-d4, 125
MHz) δ 216.11, 143.90, 137.71, 128.65, 128.45, 128.08, 127.87,
127.84, 127.08, 86.68, 78.30, 71.90, 66.42, 39.66, 33.63, 32.64.
Anal. Calcd for (C32H30O3) C, H.
1.09, MeOH); UV (MeOH) λmax 247.0 and 309.0 nm. Anal. Calcd
for (C11H11ClFN5O‚0.3MeOH) C, H, N.
(+)-(1′R,4′S)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6′-hydroxymethylcyclopent-2-enyl]guanine (25). A mix-
ture of 24 (95 mg, 0.33 mmol) and formic acid (4 mL) was
heated at 90 °C for 2 h and then concentrated in vacuo. The
residue was dissolved in methanol (4 mL) and treated with a
28% solution of ammonium hydroxide (1 mL). After being
stirred at room temperature for 2 h, the reaction mixture was
concentrated in vacuo and the residue was purified by column
chromatography on silica gel (MeOH:CH2Cl2 ) 1:30) to give
25 (15.5 g, 85%) as a white solid: mp 252 °C (dec); [R]24
(+)-(2R,4R)-1-Difluoro-2-O-benzyloxy-4-(O-trityloxy-
methyl)cyclopentane (33) was prepared from 32 on a 30
mmol scale in 85% yield by a procedure similar to that
D
+23.13° (c 0.45, MeOH); UV (H2O) λmax 253.5 nm (ꢀ 1259.2,
pH 2), 252.5 nm (ꢀ 12 600, pH 7), 252.0 nm (ꢀ 12 779, pH 11);
MS m/z 266 (M+). Anal. Calcd for (C11H12FN5O2‚1.6H2O) C,
H, N.
described for 8: [R]28 +12.29° (c 0.24, MeOH); 1H NMR
D
(CDCl3, 500 MHz) δ 7.44-7.22 (m, 20H), 4.77 (d, J ) 11.5 Hz,
1H), 4.57 (d, J ) 11.5 Hz, 1H), 3.89 (t, J ) 5.5 Hz, 1H), 3.03-
2.97 (m, 2H), 2.62-2.59 (m, 1H), 2.39-2.34 (m, 1H), 1.99-
1.88 (m, 2H), 1.75-1.71 (m, 1H); 13C NMR (CDCl3, 125 MHz)
δ 144.07, 137.91, 131.98, 129.98, 128.69, 128.48, 127.85,
127.05, 86.41, 79.58 (dd, J ) 15.2 and 23.6 Hz), 72.39, 66.41,
35.88 (t, J ) 18.3 Hz), 33.14, 32.59. Anal. Calcd for (C32H30F2O2)
C, H.
Synthesis of the L-Series, Compounds 26-50.
(-)-(1R,2S,3S,4R)-1-O-Benzyloxy-2,3-dihydroxyl-4-
hydroxymethylcyclopentane (27) was prepared from the
fully protected cyclopentane 2610c on a 18.9 mmol scale in 83%
yield by a procedure similar to that described for 2: mp 118.5-
1
119.5 °C; [R]26 -13.62° (c 1.03, MeOH); H NMR (MeOH-d4,
D
500 MHz) δ 7.38-7.26 (m, 5H), 4.60 (d, J ) 11.5 Hz, 1H). 4.52
(d, J ) 12.0 Hz, 1H), 4.01 (t, J ) 5.0 Hz, 1H), 3.86 (td, J ) 4.0
and 7.5 Hz, 1H), 3.72 (dd, J ) 4.0 and 7.5 Hz, 1H), 3.61 (dd,
J ) 5.0 and 11 Hz, 1H), 3.51 (dd, J ) 6.0 and 10.5 Hz, 1H),
3.31 (t, J ) 1.5 Hz, 1H), 2.25-22 (m, 1H), 2.04-1.98 (m, 1H),
1.76-1.71 (m, 1H); 13C NMR (MeOH-d4, 125 MHz) δ 138.63,
128.15, 127.78, 127.46, 78.50, 73.52, 72.83, 71.18, 63.24, 45.01,
30.12. Anal. Calcd for (C13H18O4) C, H.
(+)-(3R,5R)-1-Fluoro-3-(O-trityloxymethyl)-5-O-benzyl-
oxycyclopent-1-ene (34) was prepared from 33 on a 24.8
mmol scale in 95% yield by a procedure similar to that
described for 9: [R]28 +93.52° (c 0.61, CHCl3); 1H NMR
D
(CDCl3, 500 MHz) δ 7.42-7.21 (m, 20H), 5.37 (s, 1H), 4.62-
4.56 (dd, J ) 11.5 and 24.5 Hz, 2H), 4.52-4.50 (m, 1H), 3.05-
2.92 (m, 2H), 1.88-1.85 (m, 1H); 13C NMR (CDCl3, 125 MHz)
δ 162.05 (d, J ) 225.1 Hz) 144.14, 138.29, 128.94, 128.85,
128.72, 128.60, 128.53, 128.51, 128.47, 128.00, 127.97, 127.85,
127.83, 127.77, 127.71, 127.67, 127.05, 109.81 (d, J ) 7.3 Hz),
86.42, 78.11 (d, J ) 16.8 Hz), 71.17, 67.38 (d, J ) 2.7 Hz),
38.48 (d, J ) 6.1 Hz), 32.72 (d, J ) 5.4 Hz). Anal. Calcd for
(C32H29FO2) C, H.
(+)-(1R,2S,3S,4R)-1-O-Benzyloxy-2,3-anhydro-4-hy-
droxymethylcyclopentane (28) was prepared from 27 on a
110 mmol scale in 80% yield by a procedure similar to that
described for 3: [R]26 +76.91° (c 0.71, CHCl3); 1H NMR
D
(MeOH-d4, 500 MHz) δ 7.39-7.26 (m, 5H), 4.62 (s, 2H), 4.19-
4.16 (m, 1H), 3.63-3.60 (m, 1H), 3.55-3.52 (m, 2H), 3.45 (d,
J ) 3 Hz, 1H), 2.52 (dd, J ) 6.5 and 13 Hz, 1H), 1.76 (dd, J )
8 and 13 Hz, 1H), 1.67-1.54 (m, 2H); 13C NMR (MeOH-d4, 125
MHz) δ 138.46, 128.68, 127.99,127.97, 79.16, 71.93, 63.98,
57.69, 56.69, 41.15, 28.53. Anal. Calcd for (C13H16O3‚0.24H2O)
C, H.
(+)-(3R,5R)-1-Fluoro-3-hydroxymethyl-5-O-benzyloxy-
cyclopent-1-ene (35) was prepared from 34 on a 24 mmol
scale in 89% yield by a procedure similar to that described for
10: [R]27D +166.85° (c 1.0, MeOH); 1H NMR (CDCl3, 500 MHz)
δ 7.39-7.26 (m, 5H), 5.27 (d, J ) 1.5 Hz), 4.65-4.57 (m, 2H),
3.59-3.48 (m, 2H), 2.98-2.95 (m, 2H), 2.09-1.97 (m, 2H), 1.43
(t, J ) 5.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 162.58 (dd,
J ) 8 and 225.9 Hz) 138.15 (d, J ) 8 Hz), 128.57, 128.49,
128.46, 128.08, 128.00, 127.95, 127.87, 127.83, 127.82, 127.79,
108.79 (t, J ) 7.6 Hz), 78.20 (dd, J ) 8 and 16.8 Hz), 71.35 (d,
J ) 6.9 Hz), 66.34 (dd, J ) 2.3 and 8 Hz), 40.44 (dd, J ) 6.1
and 8.1 Hz), 32.21 (dd, J ) 5.3 and 8 Hz). Anal. Calcd for
(C13H15FO2) C, H.
(+)-(1R,2S,4R)-O-Benzyloxy-2-hydroxyl-4-hydroxy-
methylcyclopentane (29) and (-)-(1R,3R,4R)-O-Benzyl-
ox-3-hydroxyl-4-hydroxymethylcyclopentane (30) were
prepared from 28 on a 78 mmol scale in 72% and 14.5% yield,
respectively, by procedures similar to those described for 4 and
5. 29: [R]26 +12.69° (c 0.72, MeOH); 1H NMR (CDCl3, 500
D
MHz) δ 7.38-7.27 (m, 5H), 4.60 (d, J ) 11.5 Hz, 2H), 4.53 (d,
J ) 11.5 Hz, 1H), 4.19-4.16 (m, 1H), 3.90 (td, J ) 4 and 6.5
Hz, 1H), 3.53-3.45 (m, 2H), 2.58 (d, J ) 3.5 Hz, 1H), 2.53-
2.46 (m, 1H), 1.99-1.89 (m, 2H), 1.69-1.64 (m, 2H), 1.52-
1.46 (m, 1H); 13C NMR (CDCl3, 125 MHz) δ 138.03, 128.56,
127.92, 127.75, 81.06, 72.38, 71.64, 67.01, 36.90, 34.36, 31.18.
(+)-(3R,5R)-1-Fluoro-3-(O-tert-butyldimethylsilyloxy-
methyl)-5-O-benzyloxycyclopent-1-ene (36) was prepared
from 35 on a 5 mmol scale in 83% yield by a procedure similar
1
to that described for 11: [R]25 +120.19° (c 0.73, CHCl3); H
D
NMR (CDCl3, 500 MHz) δ 7.37-7.26 (m, 5H), 5.27 (s, 1H), 4.61
(dd, J ) 12 and 24.5 Hz, 2H), 4.57-4.54 (m, 1H), 3.52-3.42
(m, 2H), 2.93-2.89 (m, 1H), 2.04-1.98 (m, 1H), 1.94-1.89 (m,
1H), 0.87 (t, J ) 3 Hz, 9H), 0.025 (d, J ) 1 Hz, 6H); 13C NMR
(CDCl3, 125 MHz) δ 168.44 (d, J ) 225.1 Hz), 143.66, 133.79,
133.17, 133.02, 114.83 (d, J ) 7.3 Hz), 83.57 (d, J ) 16.8 Hz),
Anal. Calcd for (C13H18O3‚0.18H2O) C, H. 30: [R]25 -36.43°
D
1
(c 0.85, MeOH); H NMR (CDCl3, 500 MHz) δ 7.36-7.26 (m,
5H), 4.48 (s, 1H), 4.06-4.00 (m, 2H), 3.72-3.68 (m, 1H), 3.54-
3.49 (m, 1H), 2.86 (d, J ) 7 Hz, 1H), 2.35-2.30 (m, 1H), 2.13-
2.05 (m, 2H), 1.95-1.90 (m, 1H), 1.43-1.37 (m, 1H); 13C NMR

3746 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Wang et al.
76.48, 72.32 (d, J ) 2.6 Hz), 45.90 (d, J ) 5.7 Hz), 37.52 (d, J
) 5.4 Hz), 31.28 (d, J ) 5.3 Hz), 23.66, 0.01 (d, J ) 2.3 Hz).
Anal. Calcd for (C19H29FO2Si) C, H.
pared from 42 on a 0.05 mmol scale in 85% yield by a procedure
similar to that described for 39: mp 182 °C (dec); [R]24
D
+34.01° (c 0.32, MeOH); UV (H2O) λmax 282.0 nm (ꢀ 22 250,
pH 2), 273.0 nm (ꢀ 15 123, pH 7), 273.0 nm (ꢀ 14 938, pH 11).
MS m/z 226 (M + 1). Anal. Calcd for (C10H12FN3O2‚0.38H2O)
C, H, N.
(+)-(3R,5R)-1-Fluoro-3-(O-tert-butyldimethylsilyloxy-
methyl)-5-O-hydroxylcyclopent-1-ene (37). Liquid am-
monia (30 mL) was trapped in a three-neck round-bottom flask
at -78 °C and then sodium (220 mg, 9.6 mmol) was added. To
the resulting dark blue solution was quickly added a solution
of 36 (500 mg, 1.5 mmol) in anhydrous THF (10 mL). After
keeping the reaction mixture shaken very well for 4.5 min at
same temperature, a saturated NH4Cl solution was added to
quench the reaction, and the mixture was extracted with
EtOAc. The organic layer was combined, dried over MgSO4,
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc:hexanes ) 1:30) to give
(-)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
(O-tert-butyldimethylsilyloxymethyl)cyclopent-2-enyl]-
adenine (45) was prepared from 37 on a 0.93 mmol scale in
55% yield by a procedure similar to that described for 20: mp
167-168 °C; [R]23 -47.35° (c 0.49, MeOH); UV (H2O) λmax
D
258.5 nm (ꢀ 18 037, pH 2), 260.0 nm (ꢀ 19 612, pH 7), 260.0
nm (ꢀ 17 301, pH 11). Anal. Calcd for (C17H26FN2OSi) C, H,
N.
(-)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
hydroxymethylcyclopent-2-enyl]adenine (46) was pre-
pared from 45 on a 0.3 mmol scale in 82% yield by a procedure
similar to that described for 39: mp 224 °C (dec); [R]24D -83.80°
(c 0.32, MeOH); UV (H2O) λmax 259.0 nm (ꢀ 13 344, pH 2), 260.0
nm (ꢀ 13 894, pH 7), 260.0 nm (ꢀ 13 144, pH 11); MS m/z 250
(M + 1). Anal. Calcd for (C11H12FN5O) C, H, N.
37 as colorless oil (330 mg, 90%): [R]25 +128.07° (c 0.92,
D
1
CHCl3); H NMR (CDCl3, 500 MHz) δ 5.22 (d, J ) 2 Hz, 1H),
4.78-4.76 (m, 1H), 3.53-3.43 (m, 2H), 2.91-2.88 (m, 1H),
2.08-2.03 (m, 1H), 1.93-1.85(m, 1H), 0.88 (t, J ) 3 Hz, 9H),
0.034 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 162.52 (d, J )
223.5 Hz), 108.14 (d, J ) 7.3 Hz), 71.51 (d, J ) 18.3 Hz), 66.82
(d, J ) 2.6 Hz), 40.12 (d, J ) 5.7 Hz), 34.47 (d, J ) 5 Hz),
25.80, 18.20, -5.46 (d, J ) 1.9 Hz). Anal. Calcd for (C12H23-
FO2Si) C, H.
(-)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
hydroxymethylcyclopent-2-enyl]inosine (48) was pre-
pared from 37 on a 1.0 mmol scale in 33% yield by a procedure
similar to that described for 23: mp 226-228 °C (dec) [R]24
(+)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
(O-tert-butyldimethylsilyloxymethyl)cyclopent-2-enyl]-
thymine (38). Triphenyl phosphine (320 mg, 1.2 mmol) and
diisopropyl azodicarboxylate (790 mg, 3.0 mmol) were dissolved
in anhydrous THF and cooled to 0 °C. After forming a yellowish
suspension, the mixture was further cooled to -78 °C. N3-
Benzoyluracil (700 mg, 3.0 mmol) and a solution of alcohol 12
(250 mg, 1.0 mmol) in THF were added successively. The
resulting mixture was kept at -78 °C for 0.5 h and then stirred
at room temperature for 1 h. MeOH was added to quench the
reaction and the mixture was evaporated in vacuo. The residue
was purified by column chromatography on silica gel (EtOAc:
hexanes ) 1:10) to give the corresponding nucleoside as a
crude product, which was treated directly with methanolic
ammonia at room temperature for 24 h. After evaporation in
vacuo, the residue was purified by column chromatography
on silica gel (EtOAc:hexanes ) 1:6) to give 38 (192 mg, 54%
from 37) as a white solid: mp 86.5-88.5 °C; [R]23D +39.56° (c
0.30, CHCl3); UV (H2O) λmax 271.0 nm (ꢀ 12 158, pH 2), 271.0
nm (ꢀ 11 977, pH 7), 270.0 nm (ꢀ 9799, pH 11). Anal. Calcd for
(C17H27FN2O3Si) C, H, N.
D
-83.20° (c 0.34, MeOH); UV (H2O) λmax 249.0 nm (ꢀ 15 157,
pH 2), 252.0 nm (ꢀ 24 290, pH 7), 255.0 nm (ꢀ 16 164, pH 11);
MS m/z 251 (M + 1). Anal. Calcd for (C11H11FN4O2) C, H, N.
(-)-(1′S,4′R)-2-Amino-6-chloro-9-[2′,3′-Dideoxy-2′3′-
didehydro-2′-fluoro-6′-hydroxymethylcyclopent-2-enyl]-
purine (49). Triphenyl phosphine (630 mg, 2.4 mmol) and
diisopropyl azodicarboxylate (479 mg, 2.4 mmol) were dissolved
in anhydrous THF and cooled to 0 °C. After forming a yellowish
suspension, the mixture was further cooled to -78 °C. 2-Amino-
6-chloropurine (407 mg, 2.4 mmol) and a solution of alcohol
12 (150 mg, 0.61 mmol) in THF were added successively. The
resulting mixture was kept at -78 °C for 0.5 h and then stirred
at room temperature for 4 h. MeOH was added to quench the
reaction and the mixture was evaporated in vacuo. The residue
was purified by column chromatography on silica gel (EtOAc:
hexanes ) 1:10) to give 18 as crude product, which was treated
with tetrabutylammonium fluoride (1.0 M in THF, 0.6 mL, 0.6
mmol) for 1 h. After evaporation in vacuo, the residue was
purified by column chromatography on silica gel (MeOH:
CH2Cl2 ) 1:20) to give 49 as white foam (105 mg, 61%): [R]23
D
(-)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′,3′-didehydro-2′-fluoro-
6′-hydroxymethylcyclopent-2-enyl]thymine (39) Com-
pound 38 was treated with 3 N HCl in MeOH at room
temperature for 45 min. NaHCO3 was added to neutralize the
solution. The mixture was evaporated under reduced pressure
and the resulting mixture was dissolved in H2O and extracted
with i-PrOH:CHCl3 ) 1:4. The organic layer was dried over
MgSO4 and evaporated in vacuo. The residue was purified by
column chromatography on silica gel (MeOH:CH2Cl2 ) 1:20)
to give 39 as white foam (65 mg, 82%): [R]24D -25.76° (c 0.42,
CHCl3); UV (H2O) λmax 271.0 nm (ꢀ 14 280, pH 2), 271.0 nm (ꢀ
14 676, pH 7), 269.0 nm (ꢀ 12 382, pH 11); MS m/z 241 (M +
1). Anal. Calcd for (C11H13FN2O3‚0.1H2O) C, H, N.
(+)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
hydroxymethylcyclopent-2-enyl]uracil (41) was prepared
from 37 on a 0.41 mmol scale in 34% yield by a procedure
similar to that described for 39: [R]23D +19.08° (c 0.48, MeOH);
UV (H2O) λmax 265.0 nm (ꢀ 11 713, pH 2), 265.0 nm (ꢀ 11 887,
pH 7), 264.0 nm (ꢀ 9153, pH 11); MS m/z 227 (M + 1). Anal.
Calcd for (C10H11FN2O3‚0.2H2O) C, H, N.
-12.65° (c 0.42, MeOH); UV (H2O) λmax 308.0 nm (ꢀ 9044, pH
2), 308.0 nm (ꢀ 9349, pH 7), 308.0 nm (ꢀ 9201, pH 11). Anal.
Calcd for (C11H11ClFN5O‚0.33H2O) C, H, N.
(-)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
hydroxymethylcyclopent-2-enyl]guanosine (50) was pre-
pared from 49 on a 0.3 mmol scale in 81% yield by a procedure
similar to that described for 25: mp 256 °C (dec); [R]23
D
-23.80° (c 0.35, MeOH); UV (H2O) λmax 254.0 nm (ꢀ 14 452,
pH 2), 252.0 nm (ꢀ 15 511, pH 7), 257.0 nm (ꢀ 12 473, pH 11);
MS m/z 266 (M + 1). Anal. Calcd for (C11H12FN5O2‚1.7H2O)
C, H, N.
Antiviral Assay. Human peripheral blood mononuclear
(PBM) cells (obtained from the Atlanta Red Cross) were
isolated by Ficoll-Hypaque discontinuous gradient centrifuga-
tion from healthy seronegative donors. Cells were stimulated
with phytohemagglutinin A (Difco, Sparks, MD) for 2-3 days
prior to use. HIV-1LAI obtained from the Centers for Disease
Control and Prevention (Atlanta, GA) was used as the stan-
dard reference virus for the antiviral assays. The molecular
infectious clones HIV-1xxBru and HIV-1M184Vpitt were obtained
from Dr. John Mellors (University of Pittsburgh). Infections
were done in bulk for 1 h, either with 100 TCID50/1 × 107 cells
for a flask (T25) assay or with 200 TCID50/6 × 105 cells/well
for a 24-well plate assay. Cells were added to a plate or flask
containing a 10-fold serial dilution of the test compound. Assay
medium was RPMI-1640 supplemented with heat-inactivated
16% fetal bovine serum, 1.6 mM L-glutamine, 80 IU/mL
penicillin, 80 µg/mL streptomycin, 0.0008% DEAE-dextran,
0.045% sodium bicarbonate, and 26 IU/mL recombinant in-
(+)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
(O-tert-butyldimethylsilyloxymethyl)cyclopent-2-enyl]-
cytosine (42) was prepared from 37 on a 0.6 mmol scale in
34% yield by a procedure similar to that described for 15: mp
99-102 °C; [R]22D +42.65° (c 0.30, MeOH); UV (H2O) λmax 282.0
nm (ꢀ 20 683, pH 2), 273.0 nm (ꢀ 14 192, pH 7), 274.0 nm (ꢀ
13 886, pH 11). Anal. Calcd for (C16H26FN3O2Si) C, H, N.
(+)-(1′S,4′R)-9-[2′,3′-Dideoxy-2′3′-didehydro-2′-fluoro-6′-
hydroxymethylcyclopent-2-enyl]cytosine (43) was pre-

Antiviral Fluorocarbocyclic Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3747
terleukin-2 (Chiron Corp., Emeryville, CA). AZT was used as
a positive control for the assay. Untreated and uninfected PBM
cells were grown in parallel at equivalent cell concentrations
as controls. The cell cultures were maintained in humidified
5% CO2-air at 37 °C for 5 days and supernatants were
collected for reverse transcriptase (RT) activity.
in the original X-ray structure (1rtd)21 was modified to
guanine. The inhibitor triphosphates were manually docked
to the active site of the enzyme by adjusting the torsional
angles to those found in the X-ray structure.21 Ga¨steiger-
Hu¨ckel charges were given to the enzyme-ligand complex with
formal charges (+2) to the two Mg atoms in the active site.
Then, Kollman-all-atom charges were loaded to the enzyme
site using the biopolymer module in Sybyl. Fluorine param-
eters were obtained from the literature22 and MM2 parameters
were entered into the parameter files. To eliminate local
strains resulting from merging inhibitors and/or point muta-
tions, residues inside 6 Å from the merged inhibitors and
mutated residues were annealed until the energy change from
one iteration to the next was less than 0.05 kcal/mol. The
annealed enzyme-inhibitor complexes were minimized by
using the Kollman-all-atom force field until the iteration
number reached 5000.
Supernatants were centrifuged at 12 000 rpm for 2 h to
pellet the virus. The pellet was solubilized with vortexing in
100 µL of virus solubilization buffer (VSB) containing 0.5%
Triton X-100, 0.8 M NaCl, 0.5 mM phenylmethylsulfonyl
fluoride, 20% glycerol, and 0.05 M Tris, pH 7.8. Ten microliters
of each sample was added to the 75-µL RT reaction mixture
(0.06 M Tris, pH 7.8, 0.012 M MgCl2, 0.006 M dithiothreitol,
0.006 mg/mL poly (rA)n oligo (dT)12-18, 96 µg/mL dATP, and 1
µM of 0.08 mCi/mL [3H]thymidine triphosphate (Moravek
Biochemicals, Brea, CA) and the mixture was incubated at 37
°C for 2 h. The reaction was stopped by the addition of 100 µL
of 10% trichloroacetic acid containing 0.05% sodium pyrophos-
phate. The acid insoluble product was harvested onto filter
paper using a Packard Harvester (Meriden, CT), and the RT
activity was read on a Packard Direct Beta Counter (Meriden,
CT). The RT results were expressed in counts per minute (cpm)
per milliliter. The antiviral 50% effective concentration (EC50)
and 90% effective concentration (EC90) were determined from
the concentration-response curve using the median effect
method.20
Cytotoxicity Assays. The compounds were evaluated for
their potential toxic effects on uninfected PHA-stimulated
human PBM cells, CEM cells (T-lymphoblastoid cell line
obtained from American Type Culture Collection, Rockville,
MD) and Vero cells (African green monkey kidney cells). PBM
cells were obtained from whole blood of healthy seronegative
donors (HIV-1 and hepatitis B virus) by single-step Ficoll-
Hypaque discontinuous gradient centrifugation. Log phase
Vero, CEM, and PHA-stimulated human PBM cells were
seeded at a density of 5 × 103, 2.5 × 103, and 5 × 104 cells/
well, respectively. All of the cells were plated in 96-well cell
culture plates containing 10-fold serial dilutions of the test
drug. The cultures were incubated for 3, 4, and 5 days for Vero,
CEM, and PBM cells, respectively in humidified 5% CO2-air
at 37 °C. At the end of incubation, MTT tetrazolium dye
solution (Cell titer 96, Promega, Madison, WI) was added to
each well, and the plates were incubated overnight. The reac-
tion was stopped with stop solubilization solution (Promega,
Madison, WI). The plates were incubated for 5 h to ensure that
the formazan crystals were dissolved. The plates were read
at a wavelength of 570 nm using an ELISA plate reader (Bio-
tek instruments, Inc., Winooski, VT, Model # EL 312e). The
50% inhibition concentration (IC50) was determined from the
concentration-response curve using the median effect method.20
Molecular Modeling Study. (a) Conformational Analy-
sis. The initial conformation of D-2′F-C-d4A 21, D-2′F-C-d4G
25, L-2′F-C-d4A 46 was constructed by the builder module in
MACROMODEL version 8.5 (Schrodinger, Inc.) based on the
crystal structure of carbovir. The Monte Carlo conforma-
tional search was performed in 5000 steps, in the presence
of GB/SA water model using the MMFFs force field in
MACROMODEL.
(b) Study of Binding Affinity to HIV-1 Reverse Tran-
scriptase. All molecular modeling studies of the enzyme-
substrate complexes were performed using Sybyl 6.91 (Tripos
Associates, St. Louis, MO) on a Silicon Graphics Tezro
workstation. The enzyme site of the enzyme-ligand complex
was built based on the X-ray structure of the covalently
trapped catalytic complex of HIV-1 RT with TTP and primer-
template duplex (PDB entry 1rtd).21 A model of the NRTI
binding site was built, which consisted of residues between
Lys1 and Pro243 in the p66 subunit and a 7:4 (template-
primer) duplex. The conformationally optimized structure of
2′-fluoro-2′,3′-dideoxy-2′,3′-didehydrocarbocyclic nucleosides
was used to define the initial Cartesian coordinates. The
heterocyclic moiety of the (n + 1)th nucleotide in the template
overhang was modified to the base complementary to the
incoming NRTIs if needed; i.e., the adenine moiety which was
Acknowledgment. This research was supported by
the U.S. Public Health Service Grants (AI32351 &
AI25899) from the National Institute of Allergy and
Infectious Diseases & the Department of Veterans
Affairs.
Supporting Information Available: 1H and 13C NMR
data for compounds 13-25 and 38-50 and elemental analysis
data for compounds 2-50. This material is available free of
charge via the Internet at http://pubs.acs.org.
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