l-2',3'-Didehydro-2',3'-dideoxy-3'-fluoronucleosides: synthesis, anti-HIV activity, chemical and enzymatic stability, and mechanism of resistance.

Article abstract of DOI:10.1021/jm0300274

As antiviral nucleosides containing a 2',3'-unsaturated sugar moiety with 2'-fluoro substitution are endowed with increased stabilization of the glycosyl bond, it was of interest to investigate the influence of the fluorine atom at the 3'-position. Variou

Full text of DOI:10.1021/jm0300274

J . Med. Chem. 2003, 46, 3245-3256
3245
L-2′,3′-Did eh yd r o-2′,3′-d id eoxy-3′-flu or on u cleosid es: Syn th esis, An ti-HIV Activity,
Ch em ica l a n d En zym a tic Sta bility, a n d Mech a n ism of Resista n ce
Youhoon Chong,† Giuseppe Gumina,† J udy S. Mathew,‡ Raymond F. Schinazi,‡ and Chung K. Chu*,†
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia,
Athens, Georgia 30602 and Emory University School of Medicine/ Veterans Affairs Medical Center,
Decatur, Georgia 30033
Received J anuary 16, 2003
As antiviral nucleosides containing a 2′,3′-unsaturated sugar moiety with 2′-fluoro substitution
are endowed with increased stabilization of the glycosyl bond, it was of interest to investigate
the influence of the fluorine atom at the 3′-position. Various pyrimidine and purine L-3′-fluoro-
2′,3′-unsaturated nucleosides were synthesized from their precursors, L-3′,3′-difluoro-2′,3′-
dideoxy nucleosides, by elimination of hydrogen fluoride. In the L-3′,3′-difluoro-2′,3′-dideoxy
nucleoside series, cytidine 16 and 5-fluorocytidine 18 analogues showed modest antiviral activity
(EC50 11.5 and 8.8 µM, respectively) when evaluated against HIV-1 in human peripheral blood
mononuclear (PBM) cells. In the 2′,3′-unsaturated series, L-3′-fluoro-2′,3′-didehydro-2′,3′-
dideoxycytidine 24 and 5-fluorocytidine 26 showed highly potent antiviral activity (EC50 0.089
and 0.018 µM, respectively) without significant cytotoxicity. The guanosine analogue 48 showed
only marginal anti-HIV activity with some cytotoxicity (EC50 38.5 µM, and IC50 17.4, 58.4,
36.5 µM in PBM, CEM, and Vero cells, respectively). The cytidine 24 and 5-fluorocytidine 26
analogues, however, showed significantly decreased antiviral activity against the clinically
important lamivudine-resistant variants (HIV-1M184V). Molecular modeling studies demonstrated
that the 3′-fluoro atom of the L-3′-fluoro-2′,3′-unsaturated nucleoside is within the hydrogen
bonding distance with the amide backbone of Asp185, which favors the binding of the nucleoside
triphosphate to the wild-type RT. This favorable binding mode, however, cannot be maintained
when the triphosphate of 3′-fluoro 2′,3′-unsaturated nucleoside binds to the active site of M184V
RT because the bulky side chain of Val184 occupies the space needed for the nucleotide. The
biological results suggest that, in addition to the sugar conformation, the base moiety may
also play a role in their interaction with the M184V RT.
In tr od u ction
electronegative fluorine atom (Scheme 4), it was of
interest to investigate the influence of the fluorine atom
at the 3′-position. In view of the fact that the L-isomers
tend to have potent antiviral activity with reduced
toxicity in comparison to their D-counterparts, in a
recent communication, we reported the stereoselective
synthesis of the â-L-3′-F-d4C which showed potent anti-
HIV activity (EC50 0.03 µM) in PBM cells.4 By using a
similar synthetic strategy, a series of L-nucleosides were
synthesized from the key intermediate, (4S)-1-O-acetyl-
5-O-benzoyl-2,3-dideoxy-2,2-difluoro-â-D-ribofurano-
side (7), and their antiviral activities were evaluated
against HIV-1 in human peripheral blood mononuclear
(PBM) cells. The cytidine 24 and 5-fluorocytidine 26
analogues were also evaluated against a lamivudine-
resistant HIV-1 RT (HIV-1M184V).
A number of fluorine-substituted nucleoside ana-
logues have demonstrated their potent antiviral activity
as well as favorable chemical and pharmacological
properties by virtue of the small but highly electro-
negative nature of the fluorine atom, which is also
capable of participating in hydrogen bonding. Thus,
fluorination at the 5-position of the pyrimidine base1
as well as at the 2′- and/or 3′-position of the sugar
moiety of the nucleoside analogue have been extensively
studied in the pursuit of safe, effective and chemically
stable antiviral agents.2,3 Among those, the most inter-
esting antiviral activities were found when a fluorine
was substituted in 2′,3′-dideoxy-2′,3′-didehydro nucleo-
sides. Both the 5-fluorocytidine analogue1c,e and the 2′-
fluoro analogue2b-d showed potent anti-HIV activity
without significant cytotoxicity. It is interesting to note
that the antiviral activities of nucleosides containing the
2′,3′-unsaturated sugar moiety with a 2′-fluoro substitu-
tion have increased stabilization of the glycosyl bond.
As this stabilization of the glycosyl bond might result
from the destabilization of the oxonium ion resulting
from the cleavage of the glycosyl bond by the highly
Resu lts a n d Discu ssion
Ch em istr y. Synthesis of the nucleoside analogues
with a fluorine atom at the 3′-position of a 2′,3′-
unsaturated sugar ring has been hampered by the lack
of an efficient method for introducing the fluorovinyl
group at the 3′-position. The only synthetic method
known to date employs oxidation followed by difluori-
nation of the 3′-OH group at the nucleoside level.5
Elimination of HF by sodium methoxide afforded the
desired 3′-fluoro-2′,3′-unsaturated thymidine analogue.
However, for the extensive structure-activity relation-
* To whom correspondence should be addressed. Tel: (706)-542-
5379. Fax: (706)-542-5381. E-mail: dchu@rx.uga.edu.
† University of Georgia.
‡ Emory University School of Medicine/Veterans Affairs Medical
Center.
10.1021/jm0300274 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/24/2003

3246 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Sch em e 1. Synthesis of the Key Intermediate 7a
Chong et al.
a
Keys: (a) BnOH, HCl (g); (b) 2,2-dimethoxypropane, p-TsOH, acetone; (c) CSCl2, PhOH, pyr, CH2Cl2; (d) nBu3SnH, AIBN, toluene,
reflux; (e) 4% TFA, 40 °C; (f) HCl, MeOH; (g) BzCl, pyr; (h) PDC, Ac2O, CH2Cl2; (i) DAST, CH2Cl2, reflux; (j) Ac2O, H2SO4, AcOH.
Sch em e 2. Synthesis of the Pyrimidine Nucleosidesa
a
Keys: (a) HMDS, CH3CN, pyrimidines, TMSOTf, (b) NH3, MeOH, rt, (c) NaOMe, DMF (or 2:1 DMF:dioxane), rt.
ship study, a more general synthetic method such as
the introduction of the 3,3-difluoro functionality before
condensation with the heterocyclic base needed to be
developed. In addition, the preparation of the key
intermediate 7 required improvement from our previous
report due to the high cost of L-xylose as well as lengthy
synthetic steps. Therefore, 2-deoxyribose, which can be
prepared from inexpensive L-arabinose in large quantity
and high yield,6 was methylated followed by benzoyla-
tion to give the 5-O-benzoyl methyl furanoside 4. Pyri-
dinium dichromate-mediated oxidation of compound 4
followed by difluorination with DAST provided the
intermediate 6, which was transglycosylated to the key
intermediate (4S)-1-O-acetyl-5-O-benzoyl-2,3-dideoxy-
3,3-difluoro-â-D-ribofuranoside (7) in high yield (55%
yield overall from 4, Scheme 1) by treatment with acetic
anhydride and acetic acid in the presence of sulfuric
acid. Condensation of 7 with various pyrimidine het-
erocyclic bases under Vorbru¨ggen conditions gave R/â
mixtures of the corresponding pyrimidine analogues
8-15 in 40-60% yield (Scheme 2). The stereochemistry
of condensation products has been established by NOE
experiments, where clear correlations between protons
H
1′ and H4′ were unequivocally observed for â-anomers.4

Anti-HIV Fluoronucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3247
Sch em e 3. Synthesis of the Purine Nucleosidesa
a
Keys: (a) HMDS, (NH4)2SO4, 6-chloropurine; TMSOTf, CH3CN, (b) HMDS, (NH4)2SO4, 2-fluoro-6-chloropurine; TMSOTf, CH3CN, (c)
NH4OH, 60 °C, (d) HSCH2CH2OH, NaOMe, 60 °C, (e) NaOMe, DMF, rt, (f) NH3 bubbling, DME, rt.
The 1H NMR data of the condensation products are
summarized in Table 5. The protecting groups on the
heterocyclic base and the 5′-position of the sugar moiety
were deblocked by methanolic ammonia to give the
corresponding 2′,3′-dideoxy-3′,3′-difluoro pyrimidine nu-
cleosides 16-23 (Scheme 2), which underwent smooth
elimination to the final 3′-fluoro-2′,3′-unsaturated
pyrimidine nucleosides 24-29 by treatment with NaOMe
in DMF. However, the instability of the uridine ana-
logues (30 and 31) under the reaction conditions re-
sulted in decomposition, which was circumvented by
using a 2:1 mixture of DMF and dioxane to give the
desired compounds (30 and 31) in moderate yields (54%
and 41%, respectively).
For the preparation of the purine nucleosides, the key
intermediate 7 was condensed with 6-chloropurine and
2-fluoro-6-chloropurine to give R/â mixtures of the
corresponding nucleosides 32/33 and 42/43 in 47% and
78% yield, respectively (Scheme 3). To achieve a clean
and high-yield conversion, the temperature of the reac-
tion mixture had to be carefully controlled in such a
manner that, after addition of TMSOTf at 0 °C, the
reaction mixture was stirred for 6 h at room tempera-
ture and then for another 2 h at 60 °C. Amination of
the mixture of 32 and 33 by treatment with ammonium
hydroxide at 60 °C in a steel bomb gave the adenosine
analogue (34 and 35), whereas hydrolysis in the pres-
ence of sodium methoxide and 2-mercaptoethanol in
refluxing methanol gave the inosine analogues (36 and
37). Dry ammonia gas was bubbled into a solution of
42 and 43 in ethylene glycol dimethyl ether (DME) at
room temperature for 16 h to give the 2-amino-6-
chloropurine derivatives (44 and 45) in 29% and 28%
yield, respectively, which were readily separated by
silica gel column chromatography. The 2-amino-6-
chloropurine derivatives (44 and 45), thus obtained,
were hydrolyzed by using NaOMe and 2-mercaptoetha-
nol in refluxing methanol to give 2′,3′-dideoxy-3′,3′-
difluoroguanosine derivatives (46 and 47) in 51% and
80% yield, respectively. The 2′,3′-dideoxy-3′,3′-difluoro-

3248 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Chong et al.
Ta ble 1. Anti-HIV-1 Activity of
L-2′-Deoxy-3′,3′-difluoronucleosides
Ta ble 3. Activity of Selected Nucleosides against
Lamivudine-Resistant Virus (HIV-1M184V) in Human PBM Cells
M184V
xxBRU
(EC50, µM) EC50, µM EC90, µM
compound
FIa
24 (â-cytidine)
26 (â-5F-cytidine)
L-d4C
0.023
0.009
0.17
2.4
10.9
2.9
15.7
67.4
28.2
104
1211
17
AZT
3TC
0.01
0.035
0.003
>100
0.025
>100
0.3
>2857
cytotoxicity (IC50, µM)
activity
(EC50, µM)
compd
base
PBM
CEM
Vero
a
FI is the fold increase (EC50 HIV-1M184V/EC50 HIV-1xxBRU).
Not available.
16
17
18
19
20
21
22
23
34
35
36
37
46
47
cytosine
cytosine
5-F-cytosine
5-F-cytosine
thymine
thymine
uracil
uracil
adenine
11.5
>100
8.8
>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
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
b
Ta ble 4. Comparison of the Calculated Energy Required for
the Dissociation of the Glycosidic Bond of the Protonated
Cytidine Nucleosides with Their Stabilities at pH 2 Buffer
Solution
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
compound
t1/2 (h)a
∆Ecalcd,b (kcal/mol)
D-2′-Fd4C2c
L-3′-Fd4C (24)
L-d4C
>72
48
1
57.55
50.71
40.56
adenine
hypoxanthine
hypoxanthine
guanine
a
b
Half-life at pH 2 buffer solution. Calculated energy required
for dissociation of the glycosidic bond, ∆Ecalcd ) E(R+) + E (BH) -
E(R-BH+) (Figure 1).
guanine
Ta ble 2. Anti-HIV-1 Activity of
L-2′,3′-Dideoxy-2′,3′-didehydro-3′-fluoronucleosides
selectivity index (SI, 976) is very high. No other syn-
thesized nucleoside showed significant cytotoxicity up
to 100 µM except the 2′,3′-unsaturated uridine 30 (IC50
30.1 µM for CEM) and guanosine 48 (IC50 17.4, 58.4,
and 36.5 µM in PBM, CEM, and Vero cells, respectively)
analogues.
An tivir a l Activity a ga in st La m ivu d in e-Resista n t
(HIV-1M184V) Mu ta n t Str a in . Single point mutation at
residue 184 (M184V) of the reverse transcriptase (RT)
in HIV causes high-level resistance to lamivudine (3TC)
and contributes to the failure of anti-AIDS therapy.7,8
Therefore, to discover novel drug candidates active
against the M184V mutant HIV-1 RT, the cytosine (24),
5-fluorocytosine analogues (26), and L-d4C along with
two positive controls, AZT and 3TC, were evaluated
against the lamivudine-resistant mutant strain (HIV-
cytotoxicity (IC50, µM)
activity
(EC50, µM)
compd
base
PBM
CEM
Vero
24
25
26
27
28
29
30
31
38
39
40
48
49
cytosine
cytosine
5-F-cytosine
5-F-cytosine
thymine
thymine
uracil
uracil
adenine
adenine
0.089
>100
86.9 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.018 >100
30.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
30.1 >100
>100
>100
>100
>100
>100
36.5
>100
>100
>100
>100
1
M184V) in human PBM cells in vitro (Table 3). Unfor-
hypoxanthine >100
tunately, the results of the study suggests that the 3′-
fluoro-2′,3′-unsaturated nucleosides are significantly
cross-resistant to the M184V mutant. Interestingly, the
5-fluorocytosine analogue (26) was 11.6-fold more cross-
resistant than the cytosine analogue (24), suggesting
that in addition to the sugar conformation, the base
moiety also plays a role. Compared with L-d4C, L-3′Fd4C
(24) showed more potent anti-HIV activity against wild-
type RT. However, L-d4C and L-3′Fd4C (24) showed
similar anti-HIV activity against M184V RT. Therefore,
it is apparent that the 3′-fluorine substituent of L-3′Fd4C
(24) can be specifically recognized by the wild-type RT,
but not by M184V RT (vide infra for molecular modeling
studies).
Ch em ica l Sta bility. It is conceivable that the 2′-
fluoro-2′,3′-unsaturated compound, whose fluorine atom
is closer to the glycosyl bond than the 3′-fluoro analogue,
is more effective in stabilizing the nucleoside in an acidic
environment. To prove this hypothesis, D-2′-Fd4C,2c L-3′-
Fd4C 24, and L-d4C1e were treated in a pH 2 buffer
solution, and their stabilities were measured by TLC
every 10 min (initial 3 h) to 6 h (Table 4). As expected,
L-d4C started decomposition to the corresponding sugar
moiety and aglycon (cytosine), marking its half-life at
1 h. Compared with D-2′-Fd4C, which showed indefinite
guanine
guanine
38.5
>100
17.4
>100
58.4
>100
purine nucleosides (34-37 and 46-47) were treated
with NaOMe in DMF to give the final 3′-fluoro-2′,3′-
unsaturated nucleosides (38-41 and 48-49). However,
these nucleosides slowly decomposed during purification
(column chromatography on silica gel followed by frac-
tional crystallization in EtOH) resulting in low yields
(33-46%).
Str u ctu r e-Activity Rela tion sh ip s. Using AZT as
a positive control, the EC50 values of the synthesized
nucleosides were evaluated against HIV-1 in human
PBM cells in vitro, and the results are summarized in
Tables 1 and 2. In the 3′,3′-difluoro series (Table 1), only
the cytidine 16 (EC50 11.5 µM) and 5-fluorocytidine 18
(EC50 8.8 µM) analogues showed moderate antiviral
activity. However, the 2′,3′-unsaturated nucleosides
(Table 2) showed more potent anti-HIV-1 activity in
comparison to the corresponding 3′,3′-difluoro ana-
logues, among which cytidine 24 (EC50 0.089 µM),
5-fluorocytidine 26 (EC50 0.018 µM), and guanosine 48
(EC50 38.5 µM) showed moderate to potent antiviral
activities. Even though the cytidine analogue 24 showed
minimal cytotoxicity in PBM cells (IC50 86.9 µM), the

Anti-HIV Fluoronucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3249
Sch em e 4. Dissociation of the Glycosyl Bond of 2′,3′-Unsaturated Nucleosides
stability under acidic buffer solution, L-3′-Fd4C 24
slowly decomposed, but its half-life (48 h) was much
longer than that for L-d4C.
commercial cytidine deaminase, only the adenosine
deaminase (ADA) binding efficiencies and deamination
kinetics studies of the adenosine analogue 38 were
conducted.12 As expected, the unnatural L-configured
adenine derivative 38 was found not to be deaminated
by the enantioselective adenosine deaminase ensuring
the metabolic stability of the unnatural L-nucleosides
against the enzyme. As other L-cytidine analogues,13 it
is unlikely that the L-cytidine derivatives with 3′-F
substitution are substrates for cytidine deaminase.
In summary, we have developed an efficient synthesis
of optically pure â-L-3′-Fd4 nucleosides in an attempt
to evaluate the effect of substitution of the fluorine atom
at the 3′-position of the 2′,3′-unsaturated nucleosides,
and it was found that 3′-F substitution also provides
stability to the glycosyl bond. The stability of the
glycosyl bond achieved by the 3′-fluorine substitution
was found to be strong enough to provide potent
antiviral activity to the cytidine 24 and 5-fluorocytidine
26 analogues even though it was not as strong as that
of 2′-fluorine. Additionally, the molecular modeling
study showed that the 3′-fluorine atom participates in
binding of the L-3′-Fd4CTP at the active site of the
HIV-1 RT by forming a hydrogen bond with the amide
backbone of Asp185. However, the 5-fluorocytidine
analogue 26 showed significant cross-resistance to the
lamivudine-resistant HIV-1 RT, which could be ex-
plained by the disrupted binding mode by the steric
hindrance with the bulky side chain of Val184.
In many cases, the stability of the nucleoside ana-
logue, particularly the stability of the glycosyl bond, is
an important factor governing the biological activity as
well as the therapeutic usefulness of the nucleoside drug
candidate. Therefore, for the purpose of designing novel
nucleoside antiviral agents, an indirect method of
measuring the stability of the glycosyl bond was of
interest. For this purpose, we tried to evaluate the
energy required for the dissociation of the glycosyl bond
as a result of a protonation under acidic conditions
(Scheme 4). The energy of each chemical species was
determined by quantum mechanical (RHF, 6-31G*)
method, and the energy required for the bond dissocia-
tion reaction (∆E) was calculated to find a potential
correlation with the chemical stability of the nucleoside
analogues. As shown in Table 4, the stability of the
resulting oxonium ion (Scheme 4) correlates with the
energy required for the bond dissociation. The fluorine
substitution at the 2′- or 3′-position destabilizes the
corresponding oxonium ion species, and the destabiliza-
tion was greater in the 2′-F substitution because of the
shorter distance between the oxonium ion center and
the fluorine atom.
Molecu la r Mod elin g. The triphosphate of the quan-
tum mechanically geometry-optimized L-3′-Fd4C was
docked into the active site of HIV-1 RT, and the
resulting complex was minimized9 to give a character-
istic binding mode of this nucleoside triphosphate, in
which the 3′-fluorine atom is strongly interacting with
the amide backbone of Asp185 by hydrogen bonding
(Figure 1a). The large binding pocket of the HIV-1 RT,
which accommodates the unnatural L-configured nucle-
osides such as 3TC,10 L-d4FC,1e and L-2′-Fd4C,2b was
found to provide enough space for L-3′-Fd4CTP to bind
at the active site without any steric hindrance (Figure
1b). This favorable binding mode, however, cannot be
maintained when the triphosphate of the synthesized
3′-fluoro 2′,3′-unsaturated nucleoside binds to the active
site of M184V RT because the bulky side chain of Val184
occupies the space needed for binding and pushes the
3′-fluorine atom of L-3′-Fd4CTP away from the amide
backbone of Asp185, out of hydrogen bonding distance,
resulting in poor binding energy and thereby cross-
resistance (Figure 1c).
Exp er im en ta l Section
Melting points were determined on a Mel-temp II apparatus
and are uncorrected. Nuclear magnetic resonance spectra were
recorded on a Bruker 400 AMX spectrometer at 400 MHz for
1H NMR and 100 MHz for 13C NMR with tetramethylsilane
as the internal standard. Chemical shifts (δ) are reported as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br s (broad singlet). UV spectra were recorded on a Beckman
DU-650 spectrophotometer. Optical rotations were measured
on a J asco DIP-370 digital polarimeter. High-resolution mass
spectra were recorded on a Micromass Autospec high-resolu-
tion mass spectrometer. TLC was performed on Uniplates
(silica gel) purchased from Analtech Co. Column chromatog-
raphy was performed using either silica gel-60 (220-440 mesh)
for flash chromatography or silica gel G (TLC grade, >440
mesh) for vacuum flash column chromatography. Elemental
analyses were performed by Atlantic Microlab Inc., Norcross,
GA.
Meth yl 2-Deoxy-L-er yth r op en tofu r a n osid e (3). Metha-
nolic HCl (1%, 50.4 mL made by adding 1.7 mL of AcCl to100
mL of MeOH) was added to a solution of 26 (25.2 g, 0.19 mol)
in MeOH (250 mL). The reaction mixture was stirred at room
temperature for 25 min and neutralized with Amberlite IRA
400 (OH-) resin. After filtration followed by evaporation of the
volatiles, the residual syrup was used for the next step without
further purifications.
Meta bolic Sta bility. The metabolic stability toward
deaminases such as cytidine deaminase and adenosine
deaminase is another measure for the successful thera-
peutic candidates because, from a therapeutic point of
view, along with nucleoside phosphorylases, these are
the two major deactivating enzymes of the nucleoside
catabolism pathway.11 Due to the unavailability of the
Meth yl 5-O-Ben zoyl-2-d eoxy-â-L-er yth r op en tofu r a n o-
sid e (4). Methyl 2-deoxy-L-ribofuranoside 3 (1.1 g, 7.46 mmol)

3250 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Chong et al.
F igu r e 1. (a) Binding mode of L-3′-Fd4CTP at the active site of HIV-1 RT. 3′-F is hydrogen bonded to the amide backbone of
Asp185. (b) Binding site of HIV-1 RT, deep and spacious enough to accommodate the unnatural L-nucleoside triphosphates. (c)
Steric hindrance of L-3′-Fd4CTP with the bulky side chain of Val184 in M184V RT (left). In wild-type RT, L-3′-Fd4CTP is in
favorable distance from Met184 which may enable a stabilizing hydrophobic interaction (right).
was dissolved in dry pyridine (10 mL), cooled to 0 °C, and
treated with BzCl. The mixture was stirred at room temper-
ature for 10 h and then diluted with cold water. The aqueous
phase was extracted with CH2Cl2, and the combined organic
layers were washed twice with aqueous NaHCO3 solution, 2
N HCl, and water. The organic layer was dried over MgSO4,
filtered, and evaporated to give a crude, which was purified
by column chromatography on silica gel (hexane:EtOAc ) 2:1)
to give 1.35 g (5.37 mmol, 72% yield) of 4 as a pale yellow oil
(1:1 epimeric mixture): 1H NMR (CDCl3, 400 MHz) δ 8.08-
8.07 (m, 2H), 8.03-8.01 (m, 2H), 7.60-7.56 (m, 2H), 7.47-
7.43 (m, 4H), 5.15 (d, J ) 4.5 Hz, 1H), 5.11 (dd, J ) 5.3, 1.5
Hz, 1H), 4.57 (td, J ) 6.8, 4.8 Hz, 1H), 4.43-4.36 (m, 4H),
4.26 (d, J ) 6.1 Hz, 1H), 4.20 (q, J ) 5.2 Hz, 1H), 3.41 (s, 3H),
3.32 (s, 3H), 2.32 (ddd, J ) 13.3, 6.8, 1.5 Hz, 1H), 2.22-2.10
(m, 3H); HRMS (FAB) obsd, m/z 253.1083, calcd for C13H17O5,
m/z 253.1076 (M + H+). Anal. (C13H16O5) C, H.
Met h yl 5-O-Ben zoyl-2-d eoxy-â-L-glycer o-p en t ofu r a -
n osid -3-u lose (5). Chromic anhydride (2.73 g, 27.30 mmol)
was added to a stirred mixture of anhydrous pyridine (4.5 mL,
55.64 mmol) and anhydrous dichloromethane (50 mL) at room
temperature, and stirring was continued at room temperature
for 15 min. A solution of 4 (1.72 g, 6.82 mmol) in anhydrous
dichloromethane (20 mL) was then added, followed immedi-
ately by acetic anhydride (2.6 mL, 27.51 mmol). After 15 min,
the dark brown solution was treated with ethyl acetate (400
mL), and the resulting mixture was filtered through a TLC-
grade silica gel pad, which was washed with ethyl acetate (one
portion of 500 mL). The filtrate was concentrated under
reduced pressure and coevaporated with toluene (3 × 100 mL),
and solvents were removed in vacuo to give 1.50 g (88%) of 5
as an orange oil: 1H NMR (CDCl3, 400 MHz) δ 8.12-8.10 (m,
4H), 7.63-7.58 (m, 2H), 7.49-7.45 (m, 4H), 5.41 (t, J ) 5.0
Hz, 2H), 4.80 (dd, J ) 12.1, 2.7 Hz, 1H), 4.69 (dd, J ) 11.7,
3.2 Hz, 1H), 4.51-4.47 (m, 2H), 4.44-4.42 (m, 1H), 4.31 (t, J
) 3.0 Hz, 1H), 3.50 (s, 3H), 3.43 (s, 3H), 2.85 (dd, J ) 18.3,
5.7 Hz, 1H), 2.71 (ddd, J ) 18.2, 5.4, 0.6 Hz, 1H), 2.56 (d, J )
12.6 Hz, 1H), 2.52 (d, J ) 12.4 Hz, 1H); HRMS (FAB) obsd,
m/z 251.0920, calcd for C13H15O5, m/z 251.0919 (M + H+). Anal.
(C13H14O5‚0.07C6H5CH3) C, H.
Meth yl 5-O-Ben zoyl-2-d id eoxy-3,3-d iflu or o-3-d eoxo-â-
L-glycer op en tofu r a n osid -3-u lose (6). (Diethylamino)sulfur
trifluoride (3.0 mL, 22.71 mmol) was added to a solution of 5
(1.40 g, 5.59 mmol) in anhydrous dichloromethane (25 mL),
and the reaction mixture was refluxed for 36 h. The resulting
solution was then carefully poured into an ice-cooled saturated
solution of sodium bicarbonate (50 mL) and extracted with
diethyl ether (4 × 100 mL). The combined organic extracts
were washed with brine (50 mL), dried over MgSO4, filtered,
and concentrated to a crude oil that was purified by silica gel
flash column chromatography (1:9 ethyl acetate/hexanes) to
give 1.01 g (66%) of 6 as a yellow oil: 1H NMR (CDCl3) δ 7.90-

Anti-HIV Fluoronucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3251
7.18(m, 4H), 5.10(m, 1H), 4.54-4.29(m, 3H), 3.31, 3.29(s, 3H),
3.28 (s, 3H), 2.67-2.21(m, 2H); HRMS (FAB) obsd, m/z
273.0950, calcd for C13H15F2O4, m/z 273.0938 (M + H+). Anal.
(C13H14F2O4) C, H.
procedure for condensation reaction of the acetate 7 with
pyrimidines. The title compounds 10 and 11 were obtained
on 1.67-mmol scale in 59% yield: For the compound 10; [R]28
D
-67.91° (c 0.87, CH2Cl2); UV (CHCl3) λmax 326.5 nm; 13C NMR
(CDCl3) δ 165.85, 152.25 (d, J ) 18.4 Hz), 146.90, 140.06 (d, J
) 240.6 Hz), 135.44, 133.60, 133.19, 129.90, 129.55, 129.01,
128.61, 128.34, 125.51 (t, J ) 253.9 Hz), 124.09, 123.75, 82.69,
79.50 (dd, J ) 31.4, 24.9 Hz), 60.58 (d, J ) 4.1 Hz), 40.80 (t,
J ) 23.8 Hz); Anal. (C23H18F3N3O5) C, H, N.; For the compound
1-O-Acet yl-5-O-b en zoyl-3,3-d iflu or o-3-d eoxo-â-L-glyc-
er op en top yr a n osid -3-u lose (7). Concentrated sulfuric acid
(80 µL, 1.50 mmol) was added to an ice-cold solution of 6 (200
mg, 0.73 mmol) and acetic anhydride (300 µL, 3.20 mmol) in
glacial acetic acid, and the reaction was stirred at 0 °C for 5
min, then at room temperature for 10 more min. The resulting
mixture was poured into an ice-cold saturated solution of
sodium bicarbonate (100 mL) and extracted with dichlo-
romethane (3 × 100 mL). The combined organic extracts were
washed with water (30 mL) and brine (30 mL), dried over
MgSO4, filtered, concentrated, and coevaporated with toluene
(3 × 10 mL) to give 210 mg (95%) of 5:3 epimeric mixture 7 as
a crude yellow oil which was used in the following reaction
without further purification: 1H NMR (CDCl3) δ 8.06-8.02
(m, 4H), 7.57 (m, 2H), 7.47-7.43 (m, 4H), 6.45 [d, 1H, H1
(major isomer), J ) 5.3 Hz], 6.41 [d, 1H, H1 (minor isomer), J
) 5.9 Hz], 4.65-4.49 (m, 6H, H4, H5), 2.11 (s, 3H), 2.04 (s,
3H); 13C NMR (CDCl3) (major isomer) δ 169.72, 165.91, 133.28,
129.68, 128.43, 127.50 (dd, J ) 254.9, 251.6 Hz), 95.34 (dd, J
) 7.7, 4.5 Hz), 79.10 (dd, J ) 33.0, 24.7 Hz), 61.01 (dd, J )
7.7, 2.4 Hz), 41.91 (t, J ) 25.1 Hz) 21.03; (minor isomer) δ
169.62, 165.91, 133.27, 129.73, 128.41, 127.35 (dd, J ) 255.1,
251.3 Hz), 95.83 (dd, J ) 7.6, 3.8 Hz), 80.31 (dd, J ) 32.0,
25.0 Hz), 62.12 (dd, J ) 7.8, 3.5 Hz), 41.27 (t, J ) 24.8 Hz)
21.00; MS (FAB) m/z 301 (M + H+).
11; [R]27 41.49° (c 0.15, CHCl3); ); UV (CHCl3) λmax 327.0 nm;
D
13C NMR (CDCl3) δ 165.80, 152.49 (d, J ) 16.5 Hz), 146.91,
139.98 (d, J ) 240.2 Hz), 135.53, 133.63, 133.23, 129.94,
129.62, 129.05, 128.68, 128.40, 126.35 (dd, J ) 257.7, 249.5
Hz), 124.53, 124.23, 85.60, 80.79 (dd, J ) 31.1, 25.1 Hz), 61.42
(d, J ) 5.6 Hz), 40.80 (t, J ) 24.3 Hz); Anal. (C23H18F3N3O5‚
0.2C6H14) C, H, N.
N4-Ben zoyl-1-[(1S,4S)-5-O-b en zoyl-2,3-d id eoxy-3,3-d i-
flu or o-â-L-r ibofu r a n osyl]th ym in e (12) a n d N4-Ben zoyl-
1-[(1R,4S)-5-O-ben zoyl-2,3-d id eoxy-3,3-d iflu or o-r-L-r ibo-
fu r a n osyl]th ym in e (13). See the general procedure for
condensation reaction of the acetate 7 with pyrimidines. The
title compounds 12 and 13 were obtained on 0.62-mmol scale
in 53% yield: UV (MeOH) λmax 263.5 nm; 13C NMR (CDCl3,
100 MHz) (major isomer) δ 165.87, 163.78, 150.25, 135.05,
133.47, 126.67 (dd, J C-F ) 255.7, 251.1 Hz), 111.71, 84.84 (t,
J C-F ) 5.9 Hz), 80.15 (dd, J C-F ) 31.3, 24.5 Hz), 61.51 (dd,
J C-F ) 6.7, 4.1 Hz), 40.34 (t, J C-F ) 24.3 Hz), 12.59; (minor
isomer) δ 165.84, 163.45, 150.25, 133.82, 133.59, 125.91 (dd,
J C-F ) 255.1, 252.3 Hz), 112.39, 81.18 (t, J C-F ) 5.9 Hz), 79.11
(dd, J C-F ) 31.6, 24.6 Hz), 60.88 (dd, J C-F ) 6.1, 3.1 Hz), 40.47
(t, J C-F ) 23.9 Hz), 12.32; HRMS (FAB) obsd, m/z 367.1122,
General procedure for condensation reaction of the acetate
7 with pyrimidines. The preparation of cytosine derivatives 8
and 9 is representative.
calcd for C17H17F2N2O5, m/z 367.1106 (MH+). Anal. (C17H16
F2N2O5‚0.03CHCl3) C, H, N.
-
(-)-N4-Ben zoyl-1-[(1S,4S)-5-O-ben zoyl-2,3-d id eoxy-3,3-
d iflu or o-â-L-r ibofu r a n osyl]cytosin e (8) a n d (+)-N4-Ben -
zoyl-1-[(1R,4S)-5-O-ben zoyl-2,3-d id eoxy-3,3-d iflu or o-r-L-
r ibofu r a n osyl]cytosin e (9). A mixture of N4-benzoylcytosine
(225 mg, 1.05 mmol) and ammonium sulfate (7 mg, 0.053
mmol) in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) was heated
under reflux for 4 h, and then the solvent was removed in
vacuo at 30-35 °C. To the residual oil, a solution of 7 (210
mg, 0.70 mmol) in anhydrous acetonitrile (10 mL) was added
followed, upon cooling to 0 °C, by trimethylsilyl trifluo-
romethanesulfonate (TMSOTf, 0.19 mL, 1.05 mmol). The
resulting mixture was stirred at room temperature overnight,
diluted to 50 mL with dichloromethane, and poured into an
ice-cold saturated solution of NaHCO3 (25 mL). The organic
phase was separated, washed with brine (5 mL), dried over
MgSO4, filtered, and concentrated to a crude that was purified
by flash column chromatography (3:97 MeOH/CHCl3) to give
80 mg of 9 (25%) as the first eluted product and 100 mg of 8
(31%) as the second eluted product. Both isomers are colorless
oils, which become white solids upon trituration with diethyl
ether and a drop of methanol. For the compound 8; white solid,
1-[(1S,4S)-5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-â-L-r i-
bofu r a n osyl]u r a cil (14) a n d 1-[(1R,4S)-5-O-Ben zoyl-2,3-
d id eoxy-3,3-d iflu or o-r-L-r ibofu r a n osyl]u r a cil (15). See
the general procedure for condensation reaction of the acetate
7 with pyrimidines. The title compounds 14 and 15 were
obtained on 1.67-mmol scale in 35% yield: UV (CHCl3) λmax
256.5 nm; Anal. (C16H14F2N2O5) C, H, N.
General procedure for the deprotection reaction of the
pyrimidine analogures 8-15 with methanolic ammonia. The
preparation of cytosine derivative 16 is representative.
(-)-1-[(1S,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ib ofu r a n -
osyl]cytosin e (16). A mixture of 8 (70 mg, 0.15 mmol) in
saturated ammonia/methanol solution (10 mL) was stirred at
room temperature for 4 h, and then concentrated to dryness
under reduced pressure. The residue was purified by prepara-
tive TLC (1:9 methanol/chloroform) to give 38 mg of 16 (100%)
as a white solid: mp 194-196 °C (dec); [R]24 -51.89° (c 1.15,
D
MeOH); UV (H2O) λmax 276.5 nm (ꢀ 18 160, pH 2), 268.0 nm (ꢀ
13 280, pH 7), 268.5 nm (ꢀ 13 580, pH 11); 13C NMR (DMSO-
d6, 100 MHz) δ 168.19, 158.46, 142.62, 128.71 (dd, J C-F
)
mp 166-167 °C (dec); [R]24 -89.75° (c 0.31, CHCl3); UV
255.1, 247.4 Hz), 97.04, 84.73 (dd, J C-F ) 6.7, 4.9 Hz), 83.74
(dd, J C-F ) 29.5, 25.0 Hz), 60.73 (t, J C-F ) 4.8 Hz), 42.14 (t,
J C-F ) 23.4 Hz), 12.29; HRMS (FAB) obsd, m/z 248.0850, calcd
for C9H12F2N3O3, m/z 248.0847 (MH+). Anal. (C9H11F2N3O3‚
0.1H2O) C, H, N.
D
(MeOH) λmax 259.5 nm, 301.0 nm; 13C NMR (CDCl3, 100 MHz)
δ 166.48, 165.91, 162.58, 154.55, 143.35, 133.59, 133.31,
129.63, 129.07, 128.60, 127.53, 125.71 (t, J C-F ) 253.6 Hz),
96.90, 84.39 (dd, J C-F ) 6.6, 3.6 Hz), 79.80 (dd, J C-F ) 31.5,
25.1 Hz), 60.77 (d, J C-F ) 4.8 Hz), 41.69 (t, J C-F ) 23.7 Hz);
HRMS (FAB) obsd, m/z 456.1377, calcd for C23H20F2N3O5, m/z
456.1371 (MH+). Anal. (C23H19F2N3O5) C, H, N. For the
(+)-1-[(1R,4S)-2,3-Did eoxy-3,3-d iflu or o-r-L-r ibofu r a n -
osyl]cytosin e (17). See the general procedure for the depro-
tection reaction of the cytidine analogue 8 with methanolic
ammonia. The title compound 17 was obtained on 0.09-mmol
compound 9: white solid, mp 176-178 °C (dec); [R]24 42.61°
D
(c 1.19, CHCl3); UV (MeOH) λmax 259.0 nm; 13C NMR (CDCl3,
100 MHz) δ 166.54, 165.80, 162.77, 154.62, 143.48, 133.51,
scale in 100% yield: White solid, mp 198-200 (dec); [R]25
D
28.23° (c 0.27, MeOH); UV (H2O) λmax 277.5 nm (ꢀ 15 010, pH
2), 269.0 nm (ꢀ 10 130, pH 7), 269.0 nm (ꢀ 10 150, pH 11); 13
C
133.19, 129.58, 128.94, 128.60, 127.58, 126.51 (dd, J C-F
)
255.7, 249.3 Hz), 96.71, 86.83 (dd, J C-F ) 8.1, 2.0 Hz), 80.78
(dd, J C-F ) 31.7, 25.2 Hz), 61.47 (t, J C-F ) 5.3 Hz), 41.14 (t,
J C-F ) 24.0 Hz); HRMS (FAB) obsd, m/z 456.1374, calcd for
NMR (CD3OD, 100 MHz) δ 168.37, 158.54, 142.57, 129.28 (dd,
J C-F ) 254.3, 247.2 Hz), 96.48, 87.75 (dd, J C-F ) 8.7, 3.6 Hz),
84.93 (dd, J C-F ) 29.3, 25.3 Hz), 61.24 (t, J C-F ) 5.4 Hz), 42.63
(t, J C-F ) 23.9 Hz), 12.29; HRMS (FAB) obsd, m/z 248.0829,
calcd for C9H12F2N3O3, m/z 248.0847 (MH+). Anal. (C9H11F2N3O3)
C, H, N.
C
23H20F2N3O5, m/z 456.1371 (MH+). Anal. (C23H19F2N3O5) C,
H, N.
(-)-N4-Ben zoyl-1-[(1S,4S)- 5-O-ben zoyl-2,3-d id eoxy-3,3-
d iflu or o-â-L-r ibofu r a n osyl]-5-flu or ocytosin e (10) a n d (+)-
N4-Ben zoyl-1-[(1R,4S)-5-O-ben zoyl-2,3-d id eoxy-3,3-d iflu -
or o-r-L-r ibofu r an osyl]-5-flu or ocytosin e (11). See the general
(-)-1-[(1S,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ibofu r a n o-
syl]-5-flu or ocytosin e (18) a n d (+)-1-[(1R,4S)-2,3-Did eoxy-
3,3-d iflu or o-r-L-r ibofu r a n osyl]-5-flu or ocytosin e (19). See

3252 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Chong et al.
the general procedure for the deprotection reaction of the
cytidine analogue 8 with methanolic ammonia. The title
compounds 18 and 19 were obtained on 1.29-mmol scale in
nm (ꢀ 8010, pH 7), 264.5 nm (ꢀ 8060, pH 11); 13C NMR (CD3-
OD, 100 MHz) δ 168.31, 163.40 (d, J C-F ) 284.8 Hz), 159.05,
144.05, 102.34 (d, J C-F ) 9.7 Hz), 95.68, 88.42 (d, J C-F ) 14.9
Hz), 81.96 (d, J C-F ) 24.7 Hz), 61.82 (d, J C-F ) 2.2 Hz); HRMS
(FAB) obsd, m/z 228.0778, calcd for C9H11FN3O3, m/z 228.0784
(M + H+). Anal. (C9H10FN3O3) C, H, N.
47% and 43% yield, respectively: For the compound 18; [R]25
D
-54.71° (c 0.99, CH3OH); UV (H2O) λmax 283.5 nm (ꢀ 10 050,
pH 2), 278.0 nm (ꢀ 8430, pH 7), 278.0 nm (ꢀ 8350, pH 11); 13
C
NMR (CD3OD) δ 150.30 (d, J ) 14.1 Hz), 146.82, 129.14 (d, J
) 241.0 Hz), 118.67 (dd, J ) 255.9, 247.1 Hz), 116.73 (d, J )
33.3 Hz), 74.93 (dd, J ) 7.8, 3.5 Hz), 73.82 (dd, J ) 29.6, 25.1
Hz), 50.69 (t, J ) 4.9 Hz), 32.02 (t, J ) 23.4 Hz); HRMS (FAB)
obsd, m/z 266.08, calcd for C9H11F3N3O3, m/z 266.07 (M + H)+;
Anal. (C9H10F3N3O3) C, H, N. For the compound 19; mp 159-
(+)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-r-L-
r ibofu r a n osyl]cytosin e (25). See the general procedure for
the elimination reaction of the cytidine analogue 16 with
sodium methoxide. The title compound 25 was obtained on
0.06-mmol scale in 51% yield: White solid: mp 135 °C (dec);
[R]24 169.67° (c 1.17, MeOH); UV (H2O) λmax 276.5 nm (ꢀ
D
160 °C; [R]25 19.68° (c 0.23, CH3OH); ); UV (H2O) λmax 284.0
D
14 140, pH 2), 268.0 nm (ꢀ 9510, pH 7), 268.5 nm (ꢀ 9660, pH
11); 13C NMR (CD3OD, 100 MHz) δ 167.67, 163.00 (d, J C-F
)
nm (ꢀ 7494, pH 2), 278.5 nm (ꢀ 5816, pH 7), 278.5 nm (ꢀ 6030,
pH 11); 13C NMR (CD3OD) δ 159.86 (d, J ) 14.0 Hz), 156.28,
138.51 (d, J ) 242.3 Hz), 128.65 (dd, J ) 255.1, 247.2 Hz),
126.20 (d, J ) 34.1 Hz), 87.27 (d, J ) 5.5 Hz), 84.44 (dd, J )
29.0, 25.5 Hz), 60.69 (t, J ) 5.3 Hz), 42.02 (t, J ) 24.1 Hz);
Anal. (C9H10F3N3O3) C, H, N.
285.1 Hz), 158.31, 142.36, 101.93 (d, J C-F )10.0 Hz), 96.82,
89.55 (d, J C-F ) 15.1 Hz), 81.80 (d, J C-F ) 24.9 Hz), 61.88 (d,
J C-F ) 2.2 Hz); HRMS (FAB) obsd, m/z 228.0773, calcd for
C9H11FN3O3, m/z 228.0784 (MH+). Anal. (C9H10FN3O3‚
0.82MeOH) C, H, N.
(-)-1-[(1S,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ib ofu r a n -
osyl]th ym in e (20) a n d (-)-1-[(1R,4S)-2,3-Did eoxy-3,3-d i-
flu or o-r-L-r ibofu r a n osyl]th ym in e (21). See the general
procedure for the deprotection reaction of the cytidine analogue
8 with methanolic ammonia. The title compounds 20 and 21
were obtained on 0.38 mmol scale in 55% and 45% yield,
respectively: For the compound 20; White solid: mp 140-148
(-)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]-5-flu or ocytosin e (26). See the general pro-
cedure for the elimination reaction of the cytidine analogue
16 with sodium methoxide. The title compound 26 was
obtained on 0.12-mmol scale in 95% yield: mp 124 °C; [R]25
D
-25.47° (c 0.10, CH3OH); UV (H2O) λmax 284.0 nm (ꢀ 10 057,
pH 2), 277.5 nm (ꢀ 8191, pH 7), 277.5 nm (ꢀ 8519, pH 11); 13
C
°C (dec); [R]22 -31.27° (c 0.21, CHCl3); UV (H2O) λmax 264.0
NMR (CD3OD) δ 162.99 (d, J ) 285.1 Hz), 159.91 (d, J ) 14.2
Hz), 156.91, 138.37 (d, J ) 242.0 Hz), 127.44 (d, J ) 33.0 Hz),
101.83 (d, J ) 10.0 Hz), 88.32 (d, J ) 15.0 Hz), 81.51 (d, J )
24.8 Hz), 61.08; Anal. (C9H9F2N3O3) C, H, N.
D
nm (ꢀ 11 700, pH 2), 263.5 nm (ꢀ 12 170, pH 7), 264.0 nm (ꢀ
8660, pH 11); 13C NMR (DMSO-d6, 100 MHz) δ 163.59, 150.36,
135.40, 127.26 (dd, J C-F ) 255.8, 246.7 Hz), 110.20, 80.97 (dd,
J C-F ) 28.5, 24.4 Hz), 80.32 (dd, J C-F ) 7.1, 4.1 Hz), 58.50,
38.54 (t, J C-F ) 23.6 Hz), 12.29; HRMS (FAB) obsd, m/z
263.0849, calcd for C10H13F2N2O4, m/z 263.0843 (MH+). Anal.
(+)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-R-L-
r ibofu r a n osyl]-5-flu or ocytosin e (27). See the general pro-
cedure for the elimination reaction of the cytidine analogue
16 with sodium methoxide. The title compound 27 was
obtained on 0.15-mmol scale in 94% yield: mp 146 °C (dec);
.
(C10H12F2N2O4 0.14 Et2O) C, H, N. For the compound 21;
White solid: mp 145-148 °C (dec); [R]24 -26.50° (c 0.15,
D
[R]26 143.04° (c 0.13, CH3OH ); UV (H2O) λmax 284.0 nm (ꢀ
CHCl3); UV (H2O) λmax 265.5 nm (ꢀ 12 820, pH 2), 263.5 nm (ꢀ
D
12 590, pH 7), 264.0 nm (ꢀ 10 030, pH 11); 13C NMR (DMSO-
7837, pH 2), 277.5 nm (ꢀ 6280, pH 7), 278.0 nm (ꢀ 6033, pH
11); 13C NMR (CD3OD) δ 163.23 (d, J ) 281.8 Hz), 159.89 (d,
J ) 14.0 Hz), 156.77, 138.77 (d, J ) 243.7 Hz), 126.25 (d, J )
31.8 Hz), 101.75 (d, J ) 10.1 Hz), 90.06 (d, J ) 15.2 Hz), 81.86
(d, J ) 25.2 Hz), 61.86; Anal. (C9H9F2N3O3) C, H, N.
d6, 100 MHz) δ 163.77, 150.38, 136.32, 127.77 (dd, J C-F
)
252.3, 249.2 Hz), 109.82, 83.47 (t, J C-F ) 6.0 Hz), 81.75 (dd,
J C-F ) 28.5, 24.3 Hz), 58.76, 38.57 (t, J C-F ) 23.6 Hz), 12.17;
HRMS (FAB) obsd, m/z 263.0835, calcd for C10H13F2N2O4, m/z
263.0843 (MH+). Anal. (C10H12F2N2O4‚0.5MeOH) C, H, N.
(+)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]th ym in e (28). See the general procedure for
the elimination reaction of the cytidine analogue 16 with
sodium methoxide. The title compound 28 was obtained on
0.11-mmol scale in 54% yield: White solid: mp 146-148 °C
(-)-1-[(1S,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ib ofu r a n -
osyl]u r a cil (22) a n d (-)-1-[(1R,4S)-2,3-Did eoxy-3,3-d i-
flu or o-r-L-r ibofu r a n osyl]u r a cil (23). See the general pro-
cedure for the deprotection reaction of the cytidine analogue
8 with methanolic ammonia. The title compounds 22 and 23
were obtained on 0.57-mmol scale in 42% and 56% yield,
(dec); [R]22 32.19° (c 0.20, CHCl3); UV (H2O) λmax 264.0 nm (ꢀ
D
10 080, pH 2), 264.5 nm (ꢀ 10 130, pH 7), 264.5 nm (ꢀ 7710,
pH 11); 13C NMR (CDCl3, 100 MHz) δ 164.01, 161.54 (d, J C-F
) 287.8 Hz), 150.50, 136.85, 110.83, 99.64 (d, J C-F ) 10.2 Hz),
85.65 (d, J C-F ) 14.8 Hz), 79.98 (d, J C-F ) 24.5 Hz), 60.56 (d,
J C-F ) 1.8 Hz), 12.28; HRMS (FAB) obsd, m/z 243.0789, calcd
for C10H12FN2O4, m/z 243.0781 (MH+). Anal. (C10H11FN2O4) C,
H, N.
respectively: For the compound 22; mp 138-140 °C; [R]26
D
-25.86° (c 0.21, MeOH); UV (H2O) λmax 259.0 nm (ꢀ 8725, pH
2), 259.0 nm (ꢀ 9145, pH 7), 259.0 nm (ꢀ 6638, pH 11); 13C
NMR (CD3OD) δ 165.92, 152.01, 141.79, 128.09 (dd, J ) 255.5,
247.4 Hz), 103.35, 83.20 (dd, J ) 29.6, 24.7 Hz), 83.08 (dd, J
) 8.0, 4.3 Hz), 60.21 (t, J ) 4.7 Hz), 40.87 (t, J ) 23.6 Hz);
Anal. (C9H10F2N2O4) C, H, N. For the compound 23; mp 194-
(-)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]th ym in e (29). See the general procedure for
the elimination reaction of the cytidine analogue 16 with
sodium methoxide. The title compound 29 was obtained on
0.10-mmol scale in 51% yield White solid: mp 146-148 °C
(dec); [R]22D -18.68° (c 0.22, CH3OH); UV (H2O) λmax 264.0 nm
(ꢀ 10 075, pH 2), 264.5 nm (ꢀ 10 130, pH 7), 264.5 nm (ꢀ 7708,
pH 11); 13C NMR (CDCl3, 100 MHz) δ 163.61, 161.53 (d, J C-F
) 289.0 Hz), 150.83, 134.66, 111.96, 99.99 (d, J C-F ) 10.1 Hz),
86.97 (d, J C-F ) 14.9 Hz), 80.25 (d, J C-F ) 24.6 Hz), 61.42,
12.56; HRMS (FAB) obsd, m/z 243.0773, calcd for C10H12FN2O4,
m/z 243.0781 (MH+). Anal. (C10H11FN2O4) C, H, N.
(+)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eoxy-3-flu or o-â-L-r i-
bofu r a n osyl]u r a cil (30). A solution of 22 (56 mg, 0.23 mmol)
in a 1:2 mixture of anhydrous dioxane (1 mL) and DMF (2
mL) was treated with sodium methoxide (49 mg, 0.90 mmol)
at room temperature, and the resulting mixture was stirred
at 50-55 °C for 6 h. After dilution with MeOH, the mixture
was filtered through a short silica gel pad washing with 10:1
CH2Cl2:MeOH. The filtrate was evaporated to dryness under
196 °C; [R]25 -5.3° (c 0.32, MeOH); UV (H2O) λmax 259.0 nm
D
(ꢀ 8833, pH 2), 259.0 nm (ꢀ 9150, pH 7), 259.0 nm (ꢀ 6629, pH
11); 13C NMR (CD3OD) δ 166.58, 152.42, 142.50, 129.14 (dd, J
) 254.4, 247.5 Hz), 103.23, 86.95 (dd, J ) 8.1, 4.0 Hz), 84.78
(dd, J ) 29.2, 25.2 Hz), 61.09 (t, J ) 5.4 Hz), 41.82 (t, J )
24.2 Hz); Anal. (C9H10F2N2O4) C, H, N.
General procedure for the elimination reaction of the
pyrimidine analogues 16-23 with sodium methoxide. The
preparation of cytosine derivative 24 is representative.
(+)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]cytosin e (24). A mixture of 16 (30 mg, 0.12
mmol) and sodium methoxide (50 mg, 0.37 mmol) in anhydrous
DMF (5 mL) was stirred at room temperature overnight and
then quenched with 1 mL of a saturated aqueous solution of
ammonium chloride. Evaporation of volatiles in vacuo gave a
crude, which was purified by a short silica gel flash column
chromatography (CHCl3 to 1:15 MeOH/CHCl3) to yield 15 mg
(54%) of 24 as a white solid.: mp 182-183 °C (dec); [R]22D 6.67°
(c 0.54, MeOH); UV (H2O) λmax 276.0 nm (ꢀ 11 990, pH 2), 267.5

Anti-HIV Fluoronucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3253
reduced pressure, and the residue was purified by a short silica
gel flash column chromatography (20:1 CH2Cl2/MeOH) to yield
20 mg (0.09 mmol, 54% yield) of 30 as a white solid: mp 92-
compound 36; mp 166-168 °C; [R]24 5.82° (c 0.21, CH3OH );
D
UV (H2O) λmax 252.5 nm (ꢀ 18 306, pH 2), 249.0 nm (ꢀ 19 825,
pH 7), 257.5 nm (ꢀ 20 988, pH 11); 13C NMR (CD3OD) δ 158.85,
149.75 (dd, J ) 276.7, 264.3 Hz), 147.10, 140.36, 125.74,
111.57, 83.80 (t, J ) 24.6 Hz), 83.00 (d, J ) 6.0 Hz), 60.68,
41.25 (t, J ) 24.5 Hz); Anal. (C10H10F2N4O3) C, H, N. For the
94 °C; [R]22 11.74° (c 0.61, MeOH); UV (H2O) λmax 259.0 nm
D
(ꢀ 26 681, pH 2), 258.5 nm (ꢀ 15 591, pH 7), 259.0 nm (ꢀ 11 073,
pH 11); 13C NMR (CD3OD) δ 166.27, 163.31 (d, J ) 285.4 Hz),
152.56, 143.16, 102.59, 101.19 (d, J ) 10.8 Hz), 87.13 (d, J )
15.2 Hz), 81.53 (d, J ) 24.5 Hz), 61.25 (d, J ) 2.6 Hz); Anal.
(C9H9FN2O4) C, H, N.
compound 37; mp 103-105 °C; [R]25 -39.39° (c 0.323, CH3-
D
OH ); UV (H2O) λmax 250.0 nm (ꢀ 19 258, pH 2), 250.0 nm (ꢀ
19 480, pH 7), 258.0 nm (ꢀ 22 384, pH 11); 13C NMR (CD3OD)
δ 159.28, 150.84 (t, J ) 227.0 Hz), 147.33, 140.76, 126.48,
109.46, 84.90 (d, J ) 5.8 Hz), 84.15 (dd, J ) 29.0, 24.6 Hz),
60.71 (t, J )4.0 Hz), 41.83 (t, J )24.9 Hz);Anal. (C10H10F2N4O3)
C, H, N.
(+)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-r-L-
r ibofu r a n osyl]u r a cil (31). See the procedure for the prepa-
ration of the uridine analogue 30 by elimination reaction of
22 with sodium methoxide. The title compound 31 was
obtained on 0.36-mmol scale in 41% yield: mp 88-90 °C; [R]23
(-)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]a d en in e (38). A solution of 34 (30 mg, 0.11
mmol) in a 2:1 mixture of anhydrous dioxane (1.5 mL) and
DMF (0.7 mL) was treated with NaOMe (18 mg, 0.33 mmol)
at 0 °C, and the resulting mixture was stirred for 15 h at room
temperature. The crude was filtered through a short silica gel
pad, and the filtrate was evaporated to dryness. The residue
was purified by column chromatography on silica gel (10:1 CH2-
Cl2:MeOH) to give the desired product 38 (20 mg, 0.08 mmol,
73% yield) as a white solid: mp 148 °C (dec); [R]25D -11.78° (c
0.18, CH3OH ); UV (H2O) λmax 258.0 nm (ꢀ 11 310, pH 2), 258.5
nm (ꢀ 12 814, pH 7), 258.5 nm (ꢀ 13 283, pH 11); 13C NMR
(CD3OD) δ 163.23 (d, J ) 285.1 Hz), 157.42, 153.84, 150.30,
141.56, 120.21, 101.33 (d, J ) 11.3 Hz), 86.39 (d, J ) 15.5 Hz),
82.16 (d, J ) 24.7 Hz), 61.64; Anal. (C10H10FN5O2) C, H, N.
D
184.6° (c 0.30, MeOH); UV (H2O) λmax 259.0 nm (ꢀ 28 099, pH
2), 258.5 nm (ꢀ 15 700, pH 7), 259.5 nm (ꢀ 12 669, pH 11); 13
C
NMR (CD3OD) δ 166.11, 163.41 (d, J ) 285.7 Hz), 152.31,
142.09, 103.34, 101.30 (d, J ) 10.8 Hz), 88.77 (d, J ) 15.4 Hz),
81.91 (d, J ) 25.2 Hz), 61.77; Anal. (C9H9FN2O4‚0.2C2H5-
OC2H5) C, H, N.
9-[(1S,4S)-5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-â-L-r i-
b ofu r a n osyl]-6-ch lor op u r in e (32) a n d 9-[(1R,4S)-5-O-
Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-r-L-r ib ofu r a n osyl]-6-
ch lor op u r in e (33). A mixture of 6-chloropurine (1.03 g, 6.66
mmol), ammonium sulfate (44 mg, 0.33 mmol), and HMDS (20
mL) was refluxed for 6 h. After being cooled to room temper-
ature, volatiles were evaporated under reduced pressure. To
the residue, a solution of 7 in anhydrous CH3CN was added,
and the mixture was cooled to 0 °C, whereupon TMSOTf was
added dropwise. The mixture was stirred for 8h at 0 °C, 10 h
at room temperature, and then 3 h at 60 °C. The reaction
mixture was poured into an aqueous NaHCO3 solution and
extracted three times with CH2Cl2. The combined organic
layers were dried over MgSO4, filtered, and evaporated to give
a yellow residue which was purified by column chromatogra-
phy on silica gel (1:2 hexane:EtOAc) to give 610 mg (1.55 mmol,
47% yield) of a inseparable mixture of 32 and 33 as a pale
yellow oil: UV (CH2Cl2) λmax 266.6 nm; HRMS (FAB) obsd, m/z
395.0723, calcd for C17H18ClF2N4O3, m/z 395.0719 (M + H+);
Anal. (C17H13ClF2N4O3‚0.3H2O) C, H, N.
(+)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-r-L-
r ibofu r a n osyl]a d en in e (39). See the procedure for the
preparation of the adenosine analogue 38 by elimination
reaction of 34 with sodium methoxide. The title compound 39
was obtained on 0.16-mmol scale in 62% yield: mp 146 °C
(dec); [R]24 86.06° (c 0.66, CH3OH ); UV (H2O) λmax 258.5 nm
D
(ꢀ 10 076, pH 2), 258.5 nm (ꢀ 11 042, pH 7), 258.5 nm (ꢀ 10 812,
pH 11); 13C NMR (CD3OD) δ 163.70 (d, J ) 285.5 Hz), 157.36,
154.08, 150.41, 140.64, 120.41, 101.10 (d, J ) 11.4 Hz), 87.19
(d, J ) 15.7 Hz), 81.88 (d, J ) 25.2 Hz), 61.66; Anal. (C10H10
FN5O2) C, H, N.
-
(+)-1-[(1R,4S)-2,3-d id eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]h yp oxa n th in e (40). A solution of 36 (52 mg,
0.19 mmol) in a 2:1 mixture of anhydrous dioxane (1 mL) and
DMF (0.5 mL) was treated with NaOMe (31 mg, 0.57 mmol)
at 0 °C, and the resulting mixture was stirred for 12 h at room
temperature. A mixture of NaOMe (31 mg, 0.57 mmol) in 0.5
mL of DMF was added to the mixture, and the mixture was
stirred for 12 h at room temperature. The crude was filtered
through a short silica gel pad, and the filtrate was evaporated
to dryness. The residue was purified by column chromatog-
raphy on silica gel (10:1 CH2Cl2:MeOH) to give the desired
product 40 (15 mg, 0.06 mmol, 32% yield) as a white solid and
the starting material 36 (17 mg, 0.06 mmol) recovered: mp
200 °C (dec); [R]24D 6.74° (c 0.17, CH3OH ); UV (H2O) λmax 247.5
nm (ꢀ 6346, pH 2), 248.5 nm (ꢀ 6505, pH 7), 256.5 nm (ꢀ 11 296,
pH 11); 13C NMR (CD3OD) δ 160.65 (d, J ) 271.7 Hz), 150.03,
147.00, 140.93, 125.25, 118.01, 101.19 (d, J ) 11.3 Hz), 86.26
(d, J ) 15.1 Hz), 82.20 (d, J ) 24.6 Hz), 61.45; Anal. (C10H9-
FN4O3) C, H, N.
9-[(1S,4S)-5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-â-L-r i-
bofu r a n osyl]-6-ch lor o-2-flu or op u r in e (42) a n d 9-[(1R,4S)-
5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-r-L-r ibofu r a n osyl]-
6-ch lor o-2-flu or op u r in e (43). A mixture of 6-chloro-2-
fluoropurine (1.26 g, 7.33 mmol), ammonium sulfate (220 mg,
1.67 mmol), and HMDS (40 mL) was heated under reflux for
4 h until a clear suspension was obtained. After being cooled
to room temperature, volatiles were evaporated under reduced
pressure. To the off-white solid, a solution of 7 (1 g, 3.33 mmol)
in anhydrous CH3CN was added, and the mixture was cooled
to 0 °C, whereupon TMSOTf (0.96 mL, 5.33 mmol) was added
dropwise. The mixture was stirred for 1 h at 0 °C, 24 h at room
temperature and then 3 h at 60 °C. To a separatory funnel
containing 100 mL of aqueous NaHCO3 solution was slowly
added the reaction mixture. After dilution with CH2Cl2 and
phase separation, the aqueous layer was extracted three times
(+)-1-[(1R,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ibofu r a n o-
syl]a d en in e (34) a n d (-)-1-[(1S,4S)-2,3-Did eoxy-3,3-d i-
flu or o-r-L-r ibofu r a n osyl]a d en in e (35). A mixture of com-
pound 32 and compound 33 (0.1 g, 0.25 mmol) was treated
with aqueous ammonia in a steel bomb at 60 °C for 8 h. Solvent
was evaporated, and the residue was purified by column
chromatography (20:1 CH2Cl2:MeOH) to give the desired
compounds 34 (95 mg, 0.35 mmol 42% yield) and 35 (100 mg,
0.37 mmol, 44% yield) as white solids. For the compound 34;
mp 198-200 °C; [R]25D 18.21° (c 0.45, CH3OH ); UV (H2O) λmax
256.0 nm (ꢀ 12 797, pH 2), 258.0 nm (ꢀ 13 143, pH 7), 258.5
nm (ꢀ 12 876, pH 11); 13C NMR (CD3OD) δ 160.70, 157.58,
153.86, 150.21, 141.17, 128.22 (t, J ) 257.6 Hz), 120.60, 83.81
(dd, J ) 29.2, 24.6 Hz), 83.35 (d, J ) 7.7 Hz), 60.96 (t, J ) 5.7
Hz), 40.90 (t, J ) 24.1 Hz); Anal. (C10H11F2N5O2) C, H, N.
For the compound 35; mp 152-154 °C; [R]24D -36.67° (c 0.47,
CH3OH ); UV (H2O) λmax 256.0 nm (ꢀ 14 121, pH 2), 258.0 nm
(ꢀ 14 226, pH 7), 258.5 nm (ꢀ 14 598, pH 11); 13C NMR (CD3-
OD) δ 157.40, 154.03, 150.38, 140.79, 128.88 (t, J ) 252.1 Hz),
120.61, 84.13 (t, J ) 6.0 Hz), 83.71 (dd, J ) 29.3, 24.7 Hz),
60.36 (dd, J ) 6.8, 3.1 Hz), 41.15 (t, J ) 25.0 Hz); Anal.
(C10H11F2N5O2) C, H, N.
(+)-1-[(1R,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ib ofu r a n -
osyl]h yp oxa n th in e (36) a n d (-)-1-[(1S,4S)-2,3-Did eoxy-
3,3-d iflu or o-r-L-r ibofu r a n osyl]h yp oxa n th in e (37). A mix-
ture of compounds 32 and 33 (0.222 g, 0.56 mmol) in anhy-
drous MeOH (20 mL) was treated with 2-mercaptoethanol
(0.16 mL, 2.25 mmol) and NaOMe (0.125 g, 2.31 mmol) at 60
°C for 6 h. The resulting mixture was quenched with 0.1 mL
of glacial AcOH, concentrated, and filtered through a short
pad of silica gel. The filtrate was concentrated and purified
by column chromatography (10:1 CH2Cl2:MeOH) to give the
desired compounds 36 (0.06 g, 0.22 mmol, 39% yield) and 37
(0.056 g, 0.21 mmol, 38% yield) as white solids: For the

3254 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Chong et al.
with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, evaporated, and purified by column chroma-
tography on silica gel (4:1 hexane:EtOAc) to give 1.07 g (2.6
mmol, 78% yield) of an inseparable mixture of 42 and 43 as a
0.13 mmol, 46% yield) as an off-white solid: mp > 250 °C;
[R]24 44.25° (c 0.677, CH3OH ); UV (H2O) λmax 248.5 nm (ꢀ
D
18 111, pH 2), 251.5 nm (ꢀ 17 481, pH 7), 260.0 nm (ꢀ 19 537,
pH 11); 13C NMR (CD3OD) δ 163.45 (d, J ) 285.0 Hz), 159.35,
149.12, 137.41, 117.96 (d, J ) 10.7 Hz), 109.70, 101.27 (d, J )
11.4 Hz), 86.87 (d, J ) 15.7 Hz), 81.73 (d, J ) 25.1 Hz), 61.67;
Anal. (C10H10FN5O3‚0.5H2O) C, H, N.
pale yellow foam: UV (CH2Cl2) λmax 269.5 nm; Anal. (C17H12
ClF3N4O3‚0.04C6H14) C, H, N.
-
(+)-1-[(1R,4S)-5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-â-
L-r ibofu r a n osyl]-2-a m in o-6-ch lor op u r in e (44) a n d (+)-1-
[(1S,4S)-5-O-Ben zoyl-2,3-d id eoxy-3,3-d iflu or o-r-L-r ib o-
(+)-1-[(1S,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-r-L-
r ibofu r a n osyl]gu a n in e (49). See the procedure for the
preparation of the guanosine analogue 48 from 46 by elimina-
tion reaction using sodium methoxide in DMF. The title
compound 49 was obtained on 0.10-mmol scale in 33% yield :
fu r a n osyl]-2-a m in o-6-ch lor op u r in e
(45). Anhydrous
ammonia was bubbled into a solution of a mixture of 42 and
43 (152 mg, 0.37 mmol) in dry dimethoxyethane (20 mL) at
room temperature for 6 h. After evaporation of volatiles, the
residue was filtered through a short silica gel pad to remove
inorganic salts, and the filtrate was concentrated and purified
by column chromatography on silica gel (3:1 hexane:EtOAc)
to give the desired products 44 (43 mg, 29% yield) and 45 (42
mp 200 °C (dec); [R]23 21.75° (c 0.15, DMF ); UV (H2O) λmax
D
253.0 nm (ꢀ 20 778, pH 2), 249.0 nm (ꢀ 11 259, pH 7), 266.5
nm (ꢀ 9759, pH 11); 13C NMR (CD3OD) δ 164.97, 162.14, 160.71
(d, J ) 215.84 Hz), 156.00, 138.86, 118.03, 101.48 (d, J ) 11.0
Hz), 86.17 (d, J ) 15.3 Hz), 82.42 (d, J ) 24.6 Hz), 62.15; Anal.
(C10H10FN5O3) C, H, N.
mg, 28% yield) as white foams: For the compound 44; [R]23
D
10.18° (c 0.17, CH2Cl2 ); UV (CH2Cl2) λmax 300.0 nm; 13C NMR
(CDCl3) δ 165.89, 159.11, 153.09, 151.73, 140.33, 133.35,
129.61, 129.15, 128.42, 126.83 (t, J ) 253.8 Hz), 125.59, 82.40
(t, J ) 5.3 Hz), 79.47 (dd, J ) 31.1, 24.1 Hz), 61.05 (d, J ) 7.3
Hz), 39.51 (t, J ) 25.2 Hz); Anal. (C17H14ClF2N5O3) C, H, N.
For the compound 45; [R]22D 6.27° (c 2.011, CH2Cl2 ); UV (CH2-
Cl2) λmax 300.0 nm; 13C NMR (CDCl3) δ 165.92, 159.16, 153.07,
151.64, 139.74, 133.38, 129.53, 129.10, 128.43, 126.19 (dd, J
) 256.4, 251.2 Hz), 125.33, 81.24 (t, J ) 5.8 Hz), 79.41 (dd, J
) 31.3, 24.3 Hz), 61.37 (t, J ) 4.3 Hz), 39.60 (t, J ) 24.4 Hz);
Anal. (C17H14ClF2N5O3) C, H, N.
Sta bility Stu d ies. Energy required for the cleavage of the
glycosyl bond: The initial conformations of L-d4C, L-â-2′-Fd4C,
and L-â-3′-Fd4C 24 were constructed by builder module in
Spartan 5.1.1 (Wave Functions, Inc., Irvine, CA), and all
calculations were performed on a Silicon Graphics O2 work-
station. The initial conformations were cleaned up and geom-
etry-optimized through quantum mechanical ab initio calcu-
lations using RHF/6-31G* basis in Spartan 5.1.1. Proton (H+)
was added to the O2 atom of the cytidine analogues to build
R-BH+(Scheme 4), which was again geometry-optimized by
using the same basis function (RHF/6-31G*). The aglycon (BH)
and oxonium ion of the sugar moiety (R+) were also indepen-
dently optimized to give the corresponding minimized energies.
The dissociation enthalpy of the glycosyl bond (Table 4) was
calculated by using the minimized energies: [∆Ecalcd ) E(R+)
+ E(BH) - E(R-BH+)].
Molecu la r Mod elin g Stu d y. Binding affinity study to
HIV-1 reverse transcriptase: All molecular modeling of the
enzyme-substrate complexes was carried out using Sybyl 6.7
(Tripos Associates, St. Louis, MO) on a Silicon Graphics
Octane2 workstation. The enzyme site of the enzyme-ligand
complex was constructed 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).14 A model of
the NRTI binding site was constructed which consisted of
residues between Lys1 and Pro243 in the p66 subunit, and a
7:4 (template-primer) duplex. The geometry-optimized struc-
tures of each inhibitor, obtained from the geometry optimiza-
tion study, were used as the initial Cartesian coordinates. The
heterocyclic moiety of n + 1th nucleotide in the template
strand overhang was modified to the base complementary to
the incoming NRTIs. Thus, the adenine moiety which was in
the original X-ray structure (1rtd)14 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.14 Ga¨steiger-Hu¨ckel charge
was given to the enzyme-ligand complex with formal charges
(+2) to two Mg atoms in the active site. Then, Kollman-All-
Atom charges were loaded to the enzyme site from the
biopolymer module in Sybyl. Fluorine parameters were ob-
tained from the literature15,16 and MM2 parameters and put
to the parameter files. To eliminate local strains resulting from
merging inhibitors and/or point mutations, residues inside 6
Å from the merged inhibitors and mutated residues were
annealed until energy change from one iteration to the next
was less than 0.05 kcal/mol. The annealed enzyme-inhibitor
complexes were minimized by using Kollman-All-Atom Force
Field until iteration number reached 5000.
(+)-1-[(1R,4S)-2,3-Did eoxy-3,3-d iflu or o-â-L-r ib ofu r a n -
osyl] gu a n in e (46). A solution of 44 (166 mg, 0.41 mmol) in
anhydrous MeOH (20 mL) was treated with 2-mercaptoethanol
(0.11 mL, 1.62 mmol) and NaOMe (90 mg, 1.66 mmol), and
the resulting mixture was stirred for 18 h at 60 °C. After the
mixture was cooled to room temperature, 0.2 mL of AcOH was
added. The volatiles were removed under reduced pressure,
and the remaining inorganic salts were removed by filtering
the residue through a short silica gel pad washing with 8:1
CH2Cl2:MeOH. The filtrate was concentrated to give a white
solid, which was dissolved in a small amount of DMF and
purified by column chromatography on silica gel (8:1 CH2Cl2:
MeOH) to give 60 mg (0.21 mmol, 51% yield) of 46 as a white
solid: mp > 300 °C; [R]25 25.49° (c 0.189, DMF ); UV (H2O)
D
λ
max 253.5 nm (ꢀ 12 628, pH 2), 251.5 nm (ꢀ 11 901, pH 7), 258.0
nm (ꢀ 10 140, pH 11); 13C NMR (DMSO-d6) δ 160.30, 157.48,
154.57, 138.49, 130.93 (dd, J ) 255.4, 246.4 Hz), 120.09, 84.83
(t, J ) 28.2 Hz), 82.80 (t, J ) 4.1 Hz), 62.36, 42.17 (t, J ) 23.7
Hz); Anal. (C10H11F2N5O3) C, H, N.
(+)-1-[(1S,4S)-2,3-Did eoxy-3,3-d iflu or o-r-L-r ib ofu r a n -
osyl] gu a n in e(47). See the procedure for the preparation of
the guanosine analogue 46 from 44 by hydrolysis reaction
using 2-mercaptoethanol and sodium methoxide. The title
compound 47 was obtained on 0.27-mmol scale in 80% yield:
mp > 250 °C; [R]24D 24.40° (c 0.157, DMF ); UV (H2O) λmax 254.0
nm (ꢀ 14 309, pH 2), 254.0 nm (ꢀ 14 464, pH 7), 258.0 nm (ꢀ
12 693, pH 11); 13C NMR (CD3OD) δ 165.97, 161.12 (dd, J )
272.3, 246.3 Hz), 152.70, 137.79, 128.81, 117.96, 83.56 (dd, J
) 29.5, 24.9 Hz), 82.55 (d, J ) 8.9 Hz), 60.84 (t, J ) 5.5 Hz),
40.83 (t, J ) 24.1 Hz); Anal. (C10H11F2N5O3‚0.9H2O) C, H, N.
(+)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-3-flu or o-â-L-
r ibofu r a n osyl]gu a n in e (48). A solution of 46 (110 mg, 0.38
mmol) in anhydrous DMF (1.5 mL) was treated with NaOMe
(83 mg, 1.53 mmol), and the resulting mixture was stirred for
24 h at room temperature. Two more equivalents (41 mg) of
sodium methoxide was added, and the mixture was stirred for
12 h at room temperature. The mixture was filtered through
a short silica gel pad washing with 7:1 CH2Cl2:MeOH. The
filtrate was evaporated to dryness, and the residue was
dissolved in a small amount of DMF. The undissolved solid
was filtered off, and the filtrate was evaporated to dryness
again. The residue was dissolved in a small amount of DMF
and purified by column chromatography on silica gel (CH2Cl2:
MeOH ) 8:1) to give a pale yellow gel, which was trituated
with CH2Cl2 and Et2O to give the desired product 48 (35 mg,
Ad en osin e Dea m in a se Stu d y. Assays were performed at
25 °C in phosphate buffer solution (pH 7.4) with substrate
concentrations in the range of 15-100 µM and with 0.15 unit
of adenosine deaminase (EC 3.5.4.4 from calf intestinal mu-
cosa). The assays were monitored with a UV spectrometer at
265 nm. Initially, the qualitative assays were performed with
D-â-2′-Fd4A 38 (200 µM) in the presence of 0.24 unit of
adenosine deaminase for 120 min to determine whether it is

Anti-HIV Fluoronucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3255
a substrate of this enzyme. The concentration (ct) of each
Ack n ow led gm en t. This research was supported by
the U.S. Public Health Service Grant (AI32351 and
AI41890) from the National Institutes of Health and the
Department of Veterans Affairs.
substrate at
a certain time (t) was calculated from the
absorbance (At) at that time (t), where it was assumed that
the total change of absorbance (A0 - A∞) was directly related
to the disappearance of the substrate.17
An tivir a l Assa y. Human peripheral blood mononuclear
(PBM) cells (obtained from Atlanta Red Cross) were isolated
by Ficoll-Hypaque discontinuous gradient centrifugation 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 standard
reference virus for the antiviral assays. The molecular infec-
tious clones HIV-1xxBru and HIV-1M184Vpitt were obtained from
Dr. J ohn 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-
terleukin-2 (Chiron Corp, Emeryville, CA). AZT and/or 3TC
were 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
Su p p or tin g In for m a tion Ava ila ble: 1H NMR data for
compounds 8-40 and 42-49. This material is available free
of charge via the Internet at http://pubs.acs.org.
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