Synthesis, anti-HIV activity, and molecular mechanism of drug resistance of L-2′,3′-didehydro-2′,3′-dideoxy-2 ′-fluoro-4′-thionucleosides

Article abstract of DOI:10.1021/jm020376i

β-L-2′,3′-Didehydro-2′,3′-dideoxy-2 ′-fluoro-4′-thionucleosides β-L-2′-F-4′-S-d4Ns) have been synthesized and evaluated against HIV-1 in primary human lymphocytes. The key intermediate 8, which was prepared from 2,3-O-isopropylidene-L-glyceraldehyde 1 in

Full text of DOI:10.1021/jm020376i

J . Med. Chem. 2003, 46, 389-398
389
Syn th esis, An ti-HIV Activity, a n d Molecu la r Mech a n ism of Dr u g Resista n ce of
L-2′,3′-Did eh yd r o-2′,3′-d id eoxy-2′-flu or o-4′-th ion u cleosid es
Hyunah Choo,† Youhoon Chong,† Yongseok Choi,† J udy 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 Department of Pediatrics, Emory University School of Medicine/ Veterans Affairs Medical Center,
Decatur, Georgia 30033
Received September 4, 2002
â-L-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluoro-4′-thionucleosides (â-L-2′-F-4′-S-d4Ns) have been
synthesized and evaluated against HIV-1 in primary human lymphocytes. The key intermediate
8, which was prepared from 2,3-O-isopropylidene-L-glyceraldehyde 1 in 13 steps, was condensed
with various pyrimidine and purine bases followed by elimination and deprotection to give the
target compounds, â-L-2′-F-4′-S-d4Ns (17-20 and 27-30). The antiviral activity of the newly
synthesized compounds was evaluated against HIV-1 in human peripheral blood mononuclear
(PBM) cells, among which the cytosine 17, 5-fluorocytosine 18, and adenine 27 derivatives
showed potent anti-HIV activities (EC50 ) 0.12, 0.15, and 1.74 µM, respectively) without
significant cytotoxicity up to 100 µM in human PBM, CEM, and Vero cells. The cytosine
derivative 17 (â-L-2′-F-4′-S-d4C), however, showed cross-resistance to a 3TC-resistant variant
(HIV-1M184V). Molecular modeling studies suggest that the pattern of antiviral activity, similar
to that of â-L-2′-F-d4N, stemmed from their conformational and structural similarities. The
isosteric substitution of sulfur for 4′-oxygen was well tolerated in the catalytic site of HIV-1
reverse transcriptase in the wild-type virus. However, the steric hindrance between the sugar
moiety of the unnatural L-nucleoside and the side chains of Val184 of M184V RT in 3TC-
resistant mutant HIV strains destabilizes the RT-nucleoside triphosphate complex, which causes
the cross-resistance to 3TC (M184V mutant).
In tr od u ction
pyrimidine and purine nucleosides were synthesized for
the structure-activity relationship study. The cytosine
derivative 17 (â-L-2′-F-4′-S-d4C) was also evaluated
against a 3TC-resistant variant (HIV-1M184V). To un-
derstand the molecular basis of the antiviral activity
as well as the cross-resistance by the mutant RT strain
(HIV-1M184V), molecular modeling studies were con-
ducted using the published crystal structure of HIV-1
reverse transcriptase.11,12 Herein, we report the full
accounts of the synthesis and biological evaluation of
the titled nucleosides along with their molecular model-
ing studies.
A number of L-nucleosides such as 3TC,1,2 FTC,3
L-FMAU,4 L-d4FC,5 and L-dT6 have been discovered as
potent antiviral agents. Particularly, 2′,3′-unsaturated
nucleosides such as L-d4C,5 L-d4FC,5 and L-d4A7 have
drawn our attention because of their high antiviral
potency. A combination of the unnatural L-configuration
and the 2′,3′-unsaturated sugar moiety with 2′-fluoro
substitution was worth exploring, since the 2′-fluoro
moiety can stabilize the glycosidic bond. We have
recently discovered 2′,3′-unsaturated L-2′-fluoronucleo-
sides (â-L-2′-F-d4Ns) as potent antiviral agents against
HIV-1 and hepatitis B virus (HBV).8 Thus, to expand
our efforts to discover more potent and less toxic
antiviral agents, it was of interest to study the effect of
isosteric replacement of 4′-oxygen by a sulfur atom.
While there have been numerous efforts to modify the
sugar as well as the heterocyclic bases of natural
nucleosides for the purpose of developing new antiviral
agents, 2′,3′-unsaturated 4′-thionucleosides have not
been well investigated because of the synthetic difficul-
ties.9 In a recent communication,10 we reported a ster-
eoselective synthesis of L-2′,3′-didehydro-2′,3′-dideoxy-
2′-fluoro-4′-thiocytidine (â-L-2′-F-4′-S-d4C), which showed
potent anti-HIV activity (EC50 ) 0.12 µM) in human
peripheral blood monomuclear (PBM) cells without
cytotoxicity up to 100 µM. In this article, various
Resu lts a n d Discu ssion
Ch em istr y. Several synthetic strategies were ex-
plored for the synthesis of our target nucleosides: (1)
introduction of fluorine at the 2′-carbon by the modified
Horner-Emmons reaction using a fluorinated phospho-
nate reagent, which has been successfully used by our
group for syntheses of several fluorinated nucleo-
sides,13,14 (2) introduction of sulfur at the 4′-carbon by
double inversion,15 and (3) introduction of a phenylse-
lenyl group at the 2′-carbon, resulting in â-selectivity
during condensation with heterocyclic bases as well as
generation of a double bond to give d4 nucleosides.16 (R)-
2-Fluorobutenolide 2 was prepared from 2,3-O-isopro-
pylidene-L-glyceraldehyde 1 in three steps by a known
method (Scheme 1).13 Stereoselective hydrogenation of
(R)-2-fluorobutenolide 2 by using 5% Pd/C smooth-
ly occurred to give â-2-fluorolactone 3 in 95% yield.
Hydrolysis of â-2-fluorolactone 3 using NaOH in aque-
ous EtOH followed by methylation of the resulting
* To whom correspondence should be addressed. Phone: (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/jm020376i CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/20/2002

390 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Choo et al.
Sch em e 1a
a
(a) H2, Pd/C, EtOAc; (b) (i) NaOH, EtOH, (ii) dimethyl sulfate, DMSO, (iii) I2, Ph3P, imidazole, toluene; (c) KSAc, DMF; (d) (i) DIBAL-
H, toluene, -78 °C, (ii) DMSO, Ac2O; (e) LiHMDS, TMSCl, PhSeBr, -78 °C; (f) (i) DIBAL-H, toluene, -78 °C, (ii) Ac2O, TEA, CH2Cl2.
carboxylic acid gave a hydroxymethyl ester, which was
treated with iodine, triphenylphosphine, and imidazole
in toluene at 60 °C for 4 h to give a 6:1 epimeric mixture
of the iodo esters 4 in 82% overall yield. No significant
epimerization during saponification followed by methy-
lation was found when the hydroxymethyl ester was
treated with basic conditions to give the starting
compound 2-fluorobutenolide 3. The spectroscopic data
(1H and 13C NMR) of this compound matched the data
of the starting compound. Iodination at high tempera-
ture and long reaction time, however, resulted in partial
epimerization at C4. After optimization, the epimeriza-
tion could be minimized by treatment of the hydroxy-
methyl ester under Mitsunobu conditions at 60 °C for 4
h. The inseparable epimeric mixture of iodo esters 4 was
converted to an epimeric mixture of thiolacetates 5 in
91% yield by nucleophilic substitution with potassium
thiolacetate in DMF. Reductive cyclization of the thio-
lacetates 5 using DIBAL-H in toluene at -78 °C
followed by Moffatt-type oxidation provided the corre-
sponding thiolactone 6 in 54% yield as well as the
separable minor epimer 6a . The thiolatone 6 was
treated with LiHMDS and TMSCl followed by PhSeBr
to give the phenylselenylthiolatone 7 stereoselectively
(Scheme 1). Presumably, the phenylselenyl group ap-
proached the R-face of the TMS-enol ether plane pre-
dominantly because the â-face of the plane is sterically
hindered by the bulky 5-O-TBDPS group, resulting in
highly stereoselective addition. DIBAL reduction of the
2-fluoro-2-phenylselenothiolactone 7 followed by acety-
lation using Ac2O and triethylamine in CH2Cl2 gave the
key intermediate 8 in 83% yield. The acetate 8 was then
condensed with various pyrimidine heterocyclic bases
under Vorbru¨ggen conditions to give the corresponding
pyrimidine analogues 9-12 in 39-59% yield (Scheme
2). During the condensation, the â-anomer was obtained
exclusively by virtue of the bulky R-phenylselenyl
group.16 Oxidation of the phenylselenyl group by mCP-
BA at -78 °C followed by treatment with pyridine gave
the corresponding syn-eliminated 2′,3′-unsaturated com-
pounds 13-16 in moderate to good yields (68-80%). The
desired cytidine and 5-fluorocytidine analogues 17 and
18 were obtained by successive treatment with TBAF
in THF and methanolic ammonia. On the other hand,
the TBDPS protecting groups at 5′-positions of the sugar
moieties of compounds 15 and 16 were removed by
treatment with TBAF in THF to give the desired 2′-
fluoro-4′-thio-2′,3′-unsaturated uracil and thymine nu-
cleosides 19 and 20, respectively.
To synthesize the purine analogues 27-30, the key
intermediate 8 was condensed with 6-chloropurine and
2-fluoro-6-chloropurine to give the corresponding nu-
cleosides 21 and 22 in 73% and 67% yield, respectively
(Scheme 2). The 6-chloropurine derivative 21 was
treated with mCPBA followed by pyridine to give a syn-
eliminated inseparable mixture of 2′,3′-unsaturated
nucleoside 23 and its ∆1,2-isomer in 86% yield (3:1
determined by 1H NMR). Under the same reaction
conditions, the 2-fluoro-6-chloropurine derivative 22 also
gave an inseparable mixture of the syn-eliminated
product 24 and its ∆1,2-isomer in 71% yield (2:1 deter-
mined by 1H NMR). A mixture of compound 23, its ∆1,2
-
isomer, and methanolic ammonia was heated at 100 °C
in a steel bomb to give the corresponding adenosine
analogue, which was deprotected by TBAF in THF to
give the 2′-fluoro-4′-thio-2′,3′-unsaturated adenosine 27
in 57% yield. The ∆1,2-isomer, however, was unstable
under the reaction conditions and decomposed. The 2′,3′-
unsaturated 6-chloropurine derivative 23 was treated
with sodium methoxide and 2-mercaptoethanol in re-
fluxing methanol to give a hypoxanthine derivative,
which was converted to the final 2′-fluoro-4′-thio-2′,3′-
unsaturated inosine derivative 28 in 81% yield. On the
other hand, a solution of 24 and its ∆1,2-isomer in
ethylene glycol dimethyl ether (DME) was bubbled with
dry ammonia at room temperature for 16 h to give the
2-amino-6-chloropurine 25 and 2-fluoro-6-aminopurine
derivative 26 in 49% and 28% yield, respectively. The
∆
1,2-isomer of 24, however, decomposed under the
reaction as well as under purification conditions pre-
sumably because of its instability. The 2-amino-6-
chloropurine derivative 25 was hydrolyzed by using
sodium methoxide and 2-mercaptoethanol in refluxing
methanol to give the final 2′,3′-unsaturated 2′-fluoro-
4′-thioguanosine 29 in 61% yield. The 2′,3′-unsaturated
2′-fluoro-4′-thio-2-fluoroadenosine 30 was obtained by
deprotection of the TBDPS group of the 2-fluoro-6-
aminopurine derivative 26 by TBAF in THF in 70%
yield.

Drug Resistance of Thionucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 391
Sch em e 2a
a
(a) HMDS, CH3CN, pyrimidines or purines; TMSOTf, room temp, (b) mCPBA, -78 °C; pyridine, room temp; (c) (i) TBAF, THF, (ii)
NH3, MeOH, room temp; (d) TBAF, THF, room temp; (e) NH3 bubbling, DME, room temp; (f) (i) NH3, MeOH, 80 °C, (ii) TBAF, THF, room
temp; (g) (i) HSCH2CH2OH, NaOMe, 60 °C, (ii) TBAF, THF, room temp.
An ti-HIV Activity. The synthesized pyrimidine (17-
20) and purine (27-30) nucleosides were evaluated for
their anti-HIV activity and cytotoxicity in vitro, and
results are summarized in Table 1. The anti-HIV
activity was evaluated in PBM cells infected with HIV-
1, and AZT was included as a positive control.17 The
cytotoxicity of the synthesized nucleosides was also
assessed in human PBM, CEM, and Vero cells. Among
these nucleosides, two pyrimidine nucleosides, cytidine
30 (IC50 ) 13.0, 10.4, and 66.1 µM for PBM, CEM, and
Vero cells, respectively). However, all other synthesized
nucleosides showed no significant cytotoxicity. Despite
the replacement of an oxygen atom (â-L-2′-F-d4N) by a
sulfur atom (â-L-2′-F-4′-S-d4N), the anti-HIV activity
was generally maintained.
An tivir a l Activity a ga in st La m ivu d in e (3TC)-
Resista n t (HIV-1M184V) Mu ta n t Str a in . Although 3TC
is the most commonly used nucleoside in combination
therapy for HIV-1 infection,18 3TC monotherapy results
in the selection of a 3TC-resistant viral variant, result-
ing in prompt rebound in plasma viral load.19 High-level
resistance to 3TC is conferred by a single mutation at
codon 184 (M184V) in the catalytic domain of HIV-1 RT,
and the M184V substitution increases the 50% inhibi-
tory concentration of 3TC at least 1000-fold.20,21 This
17 (EC50 ) 0.12 µM) and 5-fluorocytidine 18 (EC50
)
0.15 µM), showed the most potent anti-HIV-1 activity,
and the purine nucleosides, adenosine 27 (EC50 ) 1.7
µM), inosine 28 (EC50 ) 15.5 µM), guanosine 29 (EC50
) 43.5 µM) and 2-fluoroadenosine 30 (EC50 ) 11.5 µM),
also showed moderate antiviral activity whereas sig-
nificant cytotoxicity was observed in 2-fluoroadenosine

392 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Choo et al.
Ta ble 1. Activity of L-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluoro-4′-thionucleosides
cytotoxicity
(IC50, µM)
configuration
and base
anti-HIV-1 activity
(EC50, µM) PBM
compd
PBM
CEM
Vero
17
18
19
20
27
28
29
30
AZT
L-cytosine
L-5-F-cytosine
L-uracil
L-thymine
L-adenine
L-hypoxanthine
L-guanine
L-2-F-adenine
0.12
0.15
>100
>100
1.7
>100
>100
>100
>100
>100
>100
>100
13.0
>100
>100
>100
>100
>100
>100
41.5
>100
>100
>100
>100
>100
>100
66.4
15.5
43.5
11.5
0.004
10.4
29.0
66.1
14.3
>100
Ta ble 2. Correlation of Relative Binding Energy Difference (∆ERel) with Fold Increase (FI)
WT (xxBRU) M184V
a
a
c
activity
(EC90, µM)
Erel
activity
(EC90, µM)
Erel
∆Erel
compd
(kcal/mol)
(kcal/mol)
FIb
(kcal/mol)
â-L-2′-F-4′-S-d4C 17
â-L-2′-F-d4C
â-D-2′-F-d4C
1.4
2.1
8.0
86.0
36.8
44.3
>100
>100
1.8
-21.5
-70.0
-6.5
>100
>100
107.5
106.8
50.8
0.2
a
b
Relative binding energy (Erel) is the binding energy of nucleoside triphosphate minus the binding energy of dCTP. EC90(HIV-1M184V)/
EC90(HIV-1xxBRU). c Relative binding energy difference: ∆Erel ) Erel(WT) - Erel(M184V).
viral resistance to 3TC prompted the discovery of
nucleosides with antiviral activity against HIV-1 iso-
lates containing common 3TC resistance mutation. It
is noteworthy that the recently U.S. FDA approved anti-
HIV drug, tenofovir, showed unique activity against
AZT- as well as 3TC-resistant mutant HIV strains.22
In view of this resistance issue, antiviral activity of the
cytosine analogue (17) was evaluated against the lami-
vudine-resistant mutant strain (HIV-1M184V) along with
other 2′,3′-unsaturated nucleosides such as â-L-2′-F-
d4C8 and â-D-2′-F-d4C23 (Table 2). Among these nucleo-
sides, only â-D-2′-F-d4C23 showed antiviral activity
against the M184V mutant, whereas its L-congener (â-
L-2′-F-d4C)8 and the 4′-sulfur-containing nucleoside (â-
L-2′-F-4′-S-d4C 17) were significantly cross-resistant.
From this study, it is apparent that the unnatural
L-sugar configuration of 2′-fluoro-2′,3′-unsaturated nu-
cleosides cannot be accommodated by the mutation at
the codon 184 of HIV-1 RT. Therefore, it was of interest
to investigate the molecular basis of drug resistance by
M184V mutant to various 2′-F-2′,3′-unsaturated nucleo-
sides (vide infra).
Molecu la r Mod elin g Stu d ies. To understand the
molecular basis of the antiviral activity and resistance
profiles of the two types of nucleosides (2′-F-4′-S-d4N
vs 2′-F-d4N) as well as the role of the isosterically
substituted sulfur atom, we conducted molecular model-
ing studies of the representative cytidine analogues (2′-
F-d4C and 2′-F-4′-S-d4C). Our molecular modeling
studies mainly focused on the interactions of the nucleo-
side triphosphates with the viral polymerase HIV-1 RT.
The crystal structure of HIV-1 RT, complexed with
thymidine triphosphate and DNA duplex reported by
Huang et al.,11 enabled us to perform molecular model-
ing studies of the triphosphates of several nucleoside
reverse transcriptase inhibitors (NRTIs). Previously, we
qualitatively demonstrated that the calculated binding
affinity of NRTIs correlates with anti-HIV activity.12,24
In our current studies, quantum mechanical ab initio
calculations at the level of RHF/3-21G* using Spartan
5.1.1 (Wavefunctions, Inc.) were performed on the
cytidine analogues (â-L-2′-F-4′-S-d4C 17 and â-L-2′-F-
d4C), and the geometry-optimized structures were
compared (Figure 1a). The two geometry-optimized
structures were nicely superimposed on each other
except for positions of the 4′-oxygen and 4′-sulfur.
Because the sulfur atom has a longer van der Waals
radius than the oxygen atom, the 4′-sulfur was posi-
tioned slightly outside of the 4′-oxygen when two cyti-
dine analogues were superimposed. However, the dis-
tance between the heterocyclic base and the 5′-hydroxy
group, which has been considered as the key factor25,26
in binding of the nucleosides to the nucleoside kinases,
is similar in the two classes, as shown in Figure 1a.
From this result, we may assume that both â-L-2′-F-4′-
S-d4C 17 and â-L-2′-F-d4C may be potentially the
substrates of nucleoside kinases, such as 2′-deoxycyti-
dine kinase. After the rate-limiting initial phosphory-
lation of the synthesized nucleosides, their triphos-
phates could be readily synthesized by cellular nucleotide
kinases.27 Recently it was also reported that L-nucleo-
side diphosphates are phosphorylated by 3-phospho-
glycerate kinase.28 The triphosphates of geometry-
optimized â-L-2′-F-4′-S-d4C 17 and â-L-2′-F-d4C, â-L-2′-
F-4′-S-d4CTP and â-L-2′-F-d4CTP were manually docked
into the truncated catalytic site of HIV-1 RT (Lys1-
Pro243 in p66 subunit), and the resulting enzyme-
inhibitor complexes were energy-minimized using the
Kollman all-atom force field.29 Both minimized struc-
tures showed that the triphosphates of â-L-2′-F-4′-S-d4C
17 and â-L-2′-F-d4C were bound tightly to the active site
of HIV-1 RT (parts 1c and 1d of Figure 1). In addition
to the structural similarity, which was shown in Figure
1a, their binding modes to the HIV-1 RT were also
similar in several aspects. Arg72 at the active site of
HIV-1 RT plays a key role in binding the triphosphates
of â-L-2′-F-4′-S-d4C 17 and â-L-2′-F-d4C. The guani-
dinium moiety of Arg72 approaches the active site in
order to establish multiple hydrogen bonds with the
triphosphates of â-L-2′-F-4′-S-d4CTP and â-L-2′-F-
d4CTP, resulting in stabilization of the nucleoside
triphosphates. The backbone amides, which are part of

Drug Resistance of Thionucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 393
F igu r e 1. (a) Superimposed structures of â-L-2′-F-d4C and â-L-2′-F-4′-S-d4C. (b) CPK structure showing interaction of â-L-2′-
F-4′-S-d4CTP with Arg72. (c) Minimized structure of â-L-2′-F-4′-S-d4CTP after docking to the active site of HIV-1 reverse
transcriptase. (d) Minimized structure of â-L-2′-F-d4CTP after docking to the active site of HIV-1 reverse transcriptase. (e) â-L-
2′-F-4′-S-d4CTP-RT complex after mutation from M184 to V184 showing steric hindrance between the sugar moiety of â-L-2′-
F-4′-S-d4CTP and the side chain of V184. (f) â-D-2′-F-d4CTP-RT complex after mutation showing no steric hindrance.
3′-OH pocket residues (Asp113 and Ala114), also sta-
bilize the triphosphates through the hydrogen bonding
interaction. Even though the 4′-sulfur atom in â-L-2′-
F-4′-S-d4C 17 is closer to Arg72 compared to the
4′-oxygen in â-L-2′-F-d4C, there was no destabilizing
steric hindrance between the 4′-sulfur atom and the
guanidinium moiety of Arg72, which allowed stable
orientation of â-L-2′-F-4′-S-d4C 17 at the active site
(Figure 1b). The conformational analysis and binding
affinity studies indicate that â-L-2′-F-4′-S-d4C 17 and
â-L-2′-F-d4C have similar structures, and the substitu-
tion of a sulfur atom is well tolerated at the polymerase
level, which could explain their similar antiviral potency
in the wild-type RT. However, the M184V mutation in
HIV-1 RT causes a serious problem in positioning the
L-configured nucleoside triphosphates at the active site
because the branched methyl groups of Val184 tend to
occupy the space where the sugar moiety of L-2′-F-2′,3′-
unsaturated nucleosides projects. The resulting steric
hindrance destabilized the L-2′-F-2′,3′-unsaturated nu-
cleoside triphosphate-RT complex (Figure 1e), which
can be confirmed by the highly decreased relative
binding energies of L-nucleosides (Table 2). On the other
hand, the structure of the â-D-2′-F-d4CTP-RT complex
does not show any significant steric crash between
Val184 and the sugar moiety of the nucleoside triphos-
phate because the natural D-configured sugar moiety
was located on the opposite side (Figure 1f). The potent
anti-HIV-1M184V activity of â-D-2′-F-d4C supports this
concept.
In summary, we have developed an efficient synthetic
methodology for â-L-2′,3′-didehydro-2′,3′-dideoxy-2′-

394 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Choo et al.
gel column chromatography with 7% EtOAc in hexanes to give
compounds 4 (13.7 g, 26.6 mmol, 82% yield) as a pale-yellow
oil: 1H NMR (CDCl3) for the major δ 7.75-7.35 (m, 10H), 5.10
(ddd, J ) 48.4, 7.5, 5.2 Hz, 1H), 4.33-4.18 (m, 1H), 3.95-3.78
(m 2H), 3.82 (s, 3H) 2.80-2.40 (m, 2H), 1.10 (s, 9H); 1H NMR
(CDCl3) for the minor δ 7.75-7.35 (m, 10H), 5.18 (ddd, J )
49.2, 10.8, 2.3 Hz, 1H), 4.33-4.18 (m, 1H), 3.95-3.78 (m 2H),
3.83 (s, 3H), 2.45-2.10 (m, 2H), 1.09 (s, 9H); HRMS (FAB)
obsd, m/z 515.0972, calcd for C22H29FIO3Si, m/z 515.0915 (M
+ H)+. Anal. (C22H28FIO3Si‚0.05C6H14) C, H.
fluoro-4′-thionucleosides (â-L-2′-F-4′-S-d4Ns) and dis-
covered that the pyrimidine analogues â-L-2′-F-4′-S-d4C
17 and â-L-2′-F-4′-S-d4-5FC 18 showed potent antiviral
activity against HIV-1. The cytidine analogue, â-L-2′-
F-4′-S-d4C 17, however, did not show any significant
antiviral activity against the 3TC-resistant mutant RT.
Our molecular modeling studies revealed that the same
pattern of the anti-HIV-1 activity of â-L-2′-F-4′-S-d4N
and â-L-2′-F-d4N can be explained by their conforma-
tional and structural similarities. The unnatural L-
configuration of the sugar moiety was found to provide
steric hindrance with the side chain of Val184 in 3TC-
resistant RT, which destabilized the RT-nucleoside
analogue complex.
(2R,4S/R)-4-Acetylsu lfan yl-5-ter t-bu tyldiph en ylsilyloxy-
2-flu or op en ta n oic Acid Meth yl Ester (5). A solution of
compounds 4 (13.7 g, 26.6 mmol) in 20 mL of DMF was treated
with solid KSAc (6.08 g, 53.2 mmol) at room temperature for
8 h. The resulting mixture was diluted with EtOAc (500 mL),
washed with water (2 × 200 mL), dried over MgSO4, filtered,
concentrated and purified by column chromatography with
10% EtOAc in hexanes to give products 5 (11.14 g, 24.1 mmol,
91% yield) as a red-brown oil: 1H NMR (CDCl3) for the major
δ 7.67-7.35 (m, 10H), 5.12-4.92 (m, 1H), 3.93-3.68 (m, 3H),
3.81 (s, 3H), 2.44-2.10 (m, 2H), 2.32 (s, 3H), 1.057 (s, 9H); 1H
NMR (CDCl3) for the minor δ 7.67-7.35 (m, 10H), 5.12-4.92
(m, 1H), 3.93-3.68 (m, 3H), 3.81 (s, 3H), 2.44-2.10 (m, 2H),
2.30 (s, 3H), 1.062 (s, 9H); HRMS (FAB) obsd, m/z 463.1789,
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 at 100 MHz for 13C NMR. 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. 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.
calcd for C24H32FO4SSi, m/z 463.1775 (M + H)+. Anal. (C24H31
FO4SSi) C, H, S.
-
(2R,4R)-(-)-4-ter t-Bu tyldiph en ylsilyloxym eth yl-2-flu or o-
γ-th iobu tyr ola cton e (6). A solution of compounds 5 (11.1 g,
24.1 mmol) in toluene (200 mL) was treated with 53 mL of 1
M DIBAL-H in hexane at -78 °C for 1 h. The reaction was
quenched with 12 mL of MeOH and warmed to room temper-
ature for 1 h, and aqueous NaHCO3 (23 mL) and EtOAc (200
mL) were added to the mixture. The resulting mixture was
filtered, and the filtrate was concentrated to dryness. The
crude thiolactol was treated with Ac2O (34 mL) and DMSO
(35 mL) at room temperature for 24 h. The reaction mixture
was poured into a separatory funnel containing ice-cooled
water (300 mL) and extracted with ethyl ether (3 × 300 mL).
The combined organic layer was washed with water (3 × 300
mL), dried over MgSO4, filtered, concentrated, and purified
by silica gel column chromatography with 5% Et2O in hexanes
to give the products 6 (5.08 g, 13.1 mmol, 54% yield) and 6a
(0.85 g, 2.2 mmol, 9% yield) as a yellow oil. For 6: [R]25D -29.4°
(2R,4R)-(-)-4-ter t-Bu tyldiph en ylsilyloxym eth yl-2-flu or o-
γ-bu tyr ola cton e (3). A solution of compound 2 (25 g, 67.5
mmol) in 500 mL of EtOAc was treated with 2.5 g of palladium
on carbon (5% w/w) under H2 atmosphere for 3 h. After
filtration through a Celite pad, the filtrate was concentrated
and purified by silica gel column chromatography with 7%
EtOAc in hexanes to give compound 3 (23.8 g, 64.1 mmol, 95%
yield) as a white solid: mp 88-89 °C; [R]24 -15.3° (c 0.835,
D
CHCl3); 1H NMR (CDCl3) δ 7.69-7.38 (m, 10H), 5.27 (dt, J )
51.4, 8.9 Hz, 1H), 4.54-4.47 (m, 1H), 3.92 (dd, J ) 11.7, 2.4
Hz, 1H), 3.74 (dd, J ) 11.7, 3.7 Hz, 1H), 2.67 (ddt, J ) 13.2,
8.6, 6.6 Hz, 1H), 2.54 (ddt, J ) 24.4,13.2, 9.0 Hz, 1H), 1.06 (s,
9H); 13C NMR (CDCl3) δ 171.19 (d, J ) 21.3 Hz), 135.62,
135.51, 132.73, 132.40, 129.97, 129.95, 127.97, 127.84, 89.82
(d, J ) 193.0 Hz), 76.31 (d, J ) 6.1 Hz), 63.94, 30.26 (d, J )
20.0 Hz), 26.64, 19.21; FABMS m/z 373 (M + H)+. Anal.
(C21H25FO3Si) C, H.
1
(c 1.18, CHCl3); H NMR (CDCl3) δ 7.68-7.37 (m, 10H), 5.07
(ddd, J ) 50.5, 10.2, 6.9 MHz, 1H), 3.96-3.78 (m, 3H), 2.72-
2.62 (m, 1H), 2.18- 2.07 (m, 1H), 1.07 (s,9H); 13C NMR (CDCl3)
δ 200.70 (d, J ) 17.4 MHz), 135.50, 132.66, 132.58, 129.99,
127.85, 93.26 (d, J ) 196.9 MHz), 66.61, 43.97 (d, J ) 7.1
MHz), 32.58 (d, J ) 19.6 MHz), 26.68, 19.21. Anal. (C21H25
-
1
FO2SSi) C, H, S. For 6a : [R]25 50.7° (c 1.3, CHCl3); H NMR
D
(CDCl3) δ 7.66-7.40 (m, 10H), 5.18 (dt, J ) 44.4, 7.0 Hz, 1H),
4.02 (quint, J ) 5.0 Hz), 3.86 (dd, J ) 10.8, 5.0 Hz, 1H), 3.84
(dd, J ) 10.8, 5.0 Hz, 1H), 2.88-2.78 (m, 2H), 1.07 (s, 9H).
Anal. (C21H25FO2SSi) C, H, S.
(2R,4R/S)-5-ter t-Bu tyld ip h en ylsilyloxy-2-flu or o-4-iod o-
p en ta n oic Acid Meth yl Ester (4). A mixture of compound
3 (12.2 g, 32.6 mmol) in 5% aqueous EtOH was treated with
solid NaOH (1.44 g, 36.0 mmol) at room temperature for 2 h.
The resulting mixture was concentrated and coevaporated two
times with 250 mL of toluene to dryness. The crude carboxylate
sodium salt was dissolved in 15 mL of DMSO and treated with
dimethyl sulfate (3.71 mL, 39.2 mmol) at 0 °C. After addition,
the ice bath was removed. The reaction mixture was stirred
for 1 h and then poured into ice-cooled water (500 mL) and
extracted with ethyl ether (3 × 200 mL). The combined organic
layer was washed with water (3 × 200 mL), dried over MgSO4,
and concentrated to dryness. The crude methyl ester was
treated with I2 (14.9 g, 58.7 mmol), imidazole (6.67 g, 98.0
mmol), and Ph3P (17.1 g, 65.3 mmol) in toluene (300 mL) at
60 °C for 4 h. Aqueous NaHCO3 (200 mL) was added to the
resulting mixture, and iodine was added portionwise until the
iodine color persisted, indicating the absence of remaining
Ph3P. Aqueous Na2S2O3 was added dropwise until the iodine
color disappeared, indicating the absence of the remaining
iodine. The resulting mixture was poured to a separatory
funnel, diluted with 300 mL of toluene, washed with brine,
dried over MgSO4, filtered, concentrated, and purified by silica
(2S,4R)-(-)-4-ter t-Bu tyldiph en ylsilyloxym eth yl-2-flu or o-
2-p h en ylselen yl-γ-th iobu tyr ola cton e (7). To a solution of
compound 6 (5.08 g, 13.1 mmol) in THF (60 mL), 15.7 mL of
1 M LiHMDS in THF was added slowly at -78 °C, and the
reaction mixture was stirred at the same temperature for 1 h.
TMSCl (2.16 mL, 17 mmol) was added dropwise to the reaction
mixture, and the mixture was warmed to room temperature.
The resulting mixture was stirred at room temperature for
30 min and cooled to -78 °C. A solution of PhSeBr (4.69 g,
19.6 mmol) in THF (20 mL) was rapidly added, and the
mixture was stirred at -78 °C for 1 h. The mixture was diluted
with ethyl ether (300 mL), washed with water (4 × 100 mL),
dried over MgSO4, filtered, concentrated, and purified by silica
gel column chromatography with 3% Et2O in hexanes to give
the desired product 7 (5.28 g, 9.69 mmol, 74% yield) as a pale-
yellow syrup: [R]24D -56.4° (c 0.542, CHCl3); 1H NMR (CDCl3)
δ 7.70-7.35 (m, 15H), 3.97-3.77 (m, 3H), 2.49 (dd, J ) 13.3,
4.5 Hz, 1H), 2.22 (td, J ) 14.4, 10.5 Hz, 1H), 1.05 (s, 9H); 13
C
NMR (CDCl3) δ 196.34 (d, J ) 22.7 Hz), 137.03, 135.53, 132.63,
132.50, 130.03, 129.39, 127.87, 124.79, 105.13 (d, J ) 260.6

Drug Resistance of Thionucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 395
Hz), 65.67, 44.59 (d, J ) 2.9 Hz), 39.13 (d, J ) 21.4 Hz), 26.70,
19.21; HRMS (FAB) obsd, m/z 545.0873, calcd for C27H30FO2-
SSeSi, m/z 545.0885 (M + H)+. Anal. (C27H29FO2SSeSi‚
0.01H2O) C, H, S.
mg) in 5 mL of CH2Cl2 was added at -78 °C, and the mixture
was stirred at -78 °C for 30 min. Pyridine (0.07 mL, 0.9 mmol)
was then added, and the resulting mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with
50 mL of CH2Cl2, washed with aqueous NaHCO3, dried over
MgSO4, filtered, and concentrated. The crude product was
purified by silica gel column chromatography with 30% EtOAc
in hexanes to give the eliminated product 13 (0.107 g, 0.183
(1S/R,2S,4R)-1-O-Acetyl-5-O-(ter t-bu tyld ip h en ylsilyl)-
2,3-d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r ibofu r a -
n osid e (8). A solution of compound 7 (4.98 g, 9.14 mmol) in
toluene (100 mL) was treated with 18.3 mL of 1 M DIBAL-H
in hexane at -78 °C for 1 h. The reaction was quenched with
4 mL of MeOH, the mixture was warmed to room temperature
for 1 h, and aqueous NaHCO3 (8 mL) and EtOAc (100 mL)
were added to the mixture. The resulting mixture was filtered,
and the filtrate was concentrated to dryness. A solution of the
crude thiolactol in CH2Cl2 (100 mL) was treated with Ac2O
(2.6 mL, 28 mmol), TEA (3.8 mL, 28 mmol), and a catalytic
amount of 4-DMAP at room temperature for 3 h. The resulting
mixture was concentrated and purified by silica gel column
chromatography with 3% Et2O in hexanes to give the acetate
8 (4.48 g, 7.61 mmol, 83% yield) as a pale-yellow oil: 1H NMR
(CDCl3) δ 7.70-7.32 (m, 15H), 6.03, 5.99 (d & s, J ) 7.6 Hz,
1H), 3.76-3.60 (m, 3H), 2.60-2.53 (m, 1H), 2.41-2.31 (m, 1H),
2.13, 2.01 (2s, 3H), 1.04, 0.99 (2s, 9H). Anal. (C29H28FO3SSeSi)
C, H, S.
mmol, 80% yield) as a foam: [R]24 128.81° (c 0.515, CH2Cl2);
D
UV(MeOH) λmax 308 nm. Anal. (C32H32FN3O3SSi‚0.1C6H14) C,
H, N, S.
(+)-N4-Ben zoyl-1-[(1S,4R)-5-O-(ter t-bu tyldiph en ylsilyl)-
2,3-d id eoxy-2,3-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n o-
syl]-5-flu or ocytosin e (14). See the general procedure for the
syn elimination reaction. The title compound 14 was obtained
in 73% yield (0.493 mmol): [R]26 152.58° (c 0.485, CH2Cl2);
D
UV(CH2Cl2) λmax 331.0 nm; FABMS m/z 604 (M + H)+. Anal.
(C32H31FN3O3SSi) C, H, N, S.
(+)-1-[(1S,4R)-5-O-(ter t-Bu tyldiph en ylsilyl)-2,3-dideoxy-
2,3-dideh ydr o-2-flu or o-4-th io-â-L-r ibofu r an osyl]u r acil (15).
See the general procedure for the syn elimination reaction.
The title compound 15 was obtained in 68% yield (0.411
mmol): [R]26 77.7° (c 0.492, CH2Cl2); UV(CH2Cl2) λmax 262.5
D
nm; HRMS (FAB) obsd, m/z 483.1572, calcd for C25H28FN2O3-
SSi, m/z 483.1574 (M + H)+. Anal. (C25H27FN2O3SSi) C, H, N,
S.
General procedure for condensation reaction of the acetate
8 with pyrimidines. The preparation of cytosine derivative 9
is representative.
(-)-N4-Ben zoyl-1-[(1S,2S,4R)-5-O-(ter t-bu tyld ip h en yl-
silyl)-2,3-d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r i-
bofu r a n osyl]cytosin e (9). A mixture of N4-benzoylcytosine
(0.548 g, 2.55 mmol) in HMDS (15 mL) and CH3CN (15 mL)
was refluxed for 5 h. After removal of the solvent using a
vacuum pump, a solution of the acetate 8 (0.500 g, 0.849 mmol)
in 15 mL of CH3CN was added to the reaction flask containing
the silylated N4-benzoylcytosine, and then TMSOTf (0.31 mL,
1.7 mmol) was added dropwise at room temperature. After 16
h, the reaction was quenched with 1 mL of saturated NaHCO3
and the resulting mixture was concentrated to one-fifth of the
volume. The crude mixture was diluted with 100 mL of CH2-
Cl2, washed with aqueous NaHCO3, dried over MgSO4, filtered,
and concentrated. The crude product was purified by silica gel
column chromatography with 30% EtOAc in hexanes to give
cytosine derivative 9 (0.300 g, 0.404 mmol, 48% yield) as a
(+)-1-[(1S,4R)-5-O-(ter t-Bu t yld ip h en ylsilyl)-2,3-d id e-
oxy-2,3-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n osyl]th y-
m in e (16). See the general procedure for the syn elimination
reaction. The title compound 16 was obtained in 76% yield
(0.326 mmol): [R]26 50.7° (c 0.530, CH2Cl2); UV(CH2Cl2) λmax
D
266.0 nm; HRMS (FAB) obsd, m/z 497.1725, calcd for C26H30
-
FN2O3SSi, m/z 497.1730 (M + H)+. Anal. (C26H29FN2O3SSi)
C, H, N, S.
Gen er a l P r oced u r e for Su ccessive Dep r otection s of
P r otected Un sa tu r a ted Nu cleosid es. The preparation of
cytidine analogue 17 is representative.
(+)-1-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]cytosin e (17). A solution of the
unsaturated cytidine 13 (0.180 g, 0.307 mmol) in 15 mL of THF
was treated with 0.37 mL of 1 M TBAF in THF for 2 h. The
mixture was concentrated and filtered through a short pad of
silica gel. The filtrate was concentrated, and without further
purification, the crude product was treated with methanolic
ammonia at room temperature for 30 h. After removal of
solvent, the residue was purified by silica gel column chroma-
tography with 5% MeOH in CH2Cl2 to give cytidine analogue
17 (0.066 g, 0.27 mmol, 88% yield) as a white solid: mp 89-
91 °C (dec); [R]24D 205.3° (c 0.140, MeOH); UV(H2O) λmax 279.5
nm (ꢀ 19 900, pH 2). 272.0 nm (ꢀ 15 900, pH 7), 272.5 nm (ꢀ
16 200, pH 11). Anal. (C9H10FN3O2S) C, H, N, S.
(+)-1-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]-5-flu or ocytosin e (18). See the gen-
eral procedure for successive deprotections. The title compound
18 was obtained in 88% yield (0.356 mmol): mp 189-190 °C;
[R]24D 169.2° (c 0.159, MeOH); UV(H2O) λmax 286.0 nm (ꢀ 8600,
pH 2). 282.0 nm (ꢀ 7500, pH 7), 282.0 nm (ꢀ 6900, pH 11). Anal.
(C9H9F2N3O2S) C, H, N, S.
foam: [R]24 -168.38° (c 0.486, CH2Cl2); UV(MeOH) λmax 308
D
nm. Anal. (C38H38FN3O3SSeSi) C, H, N, S.
(-)-N4-Ben zoyl-1-[(1S,2S,4R)-5-O-(ter t-bu tyld ip h en yl-
silyl)-2,3-d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r i-
bofu r a n osyl]-5-flu or ocytosin e (10). See the general proce-
dure for the condensation reaction of the acetate 8 with
pyrimidines. The title compound 10 was obtained in 59% yield
(0.849 mmol): [R]24 -209.1° (c 0.693, CH2Cl2); UV(CH2Cl2)
D
λ
max 332.5 nm; FABMS m/z 762 (M + H)+. Anal. (C38H37F2N3O3-
SSeSi) C, H, N, S.
(-)-1-[(1S ,2S ,4R )-5-O-(t er t -Bu t yld ip h e n ylsilyl)-2,3-
d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r ibofu r a n o-
syl]u r a cil (11). See the general procedure for the condensa-
tion reaction of the acetate 8 with pyrimidines. The title
compound 11 was obtained in 48% yield (0.849 mmol): [R]23
D
-129.2° (c 0.578, CH2Cl2); UV(CH2Cl2) λmax 262.5 nm. Anal.
(C31H33FN2O3SSeSi) C, H, N, S.
Gen er a l P r oced u r e for Desilyla tion Rea ction of ter t-
Bu tyld ip h en yl Eth er by Usin g TBAF . The preparation of
uridine analogue 19 from compound 15 is representative.
(+)-1-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]u r a cil (19). A solution of compound
15 (0.135 g, 0.280 mmol) in THF (10 mL) was treated with
0.33 mL of 1 M TBAF in THF at room temperature for 2 h.
The mixture was concentrated and purified by silica gel column
chromatography with 3% MeOH in CHCl3 to give the desired
product 19 (0.063 g, 0.258 mmol, 92% yield) as a white solid:
(-)-1-[(1S ,2S ,4R )-5-O-(t er t -Bu t yld ip h e n ylsilyl)-2,3-
d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r ibofu r a n o-
syl]th ym in e (12). See the general procedure forthe conden-
sation reaction of the acetate 8 with pyrimidines. The title
compound 12 was obtained in 38% yield (0.849 mmol): [R]26
D
-130.6° (c 0.464, CH2Cl2); UV(CH2Cl2) λmax (CH2Cl2) 266.0 nm;
FABMS m/z 655 (M + H)+. Anal. (C32H35FN2O3SSeSi) C, H,
N, S.
Gen er a l P r oced u r e for Syn Elim in a tion Rea ction
Usin g m CP BA to give 2′,3′-Un sa tu r a ted Nu cleosid es. The
preparation of cytosine derivative 13 is representative.
(+)-N4-Ben zoyl-1-[(1S,4R)-5-O-(ter t-bu tyldiph en ylsilyl)-
2,3-d id eoxy-2,3-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n o-
syl]cytosin e (13). To a solution of compound 9 (0.169 g, 0.228
mmol) in 5 mL of CH2Cl2, a solution of mCPBA (57-86%, 69
mp 184-185 °C; [R]24 132.9° (c 0.103, MeOH); UV(H2O) λmax
D
263.0 nm (ꢀ 9100, pH 2). 263.0 nm (ꢀ 9700, pH 7), 263.5 nm (ꢀ
7800, pH 11). Anal. (C9H9FN2O3S) C, H, N, S.
(+)-1-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]th ym in e (20). See the general pro-
cedure for desilylation reaction and trituation with Et2O. The

396 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
title compound 20 was obtained in 91% yield (0.248 mmol):
Choo et al.
Solvent was evaporated, and the residue was dried under
vacuum for 3 h. A solution of the dried crude product in 30
mL of THF was treated with 0.5 mL of 1 M TBAF in THF at
room temperature for 3 h. The resulting mixture was concen-
trated and purified by preparative TLC chromatography with
5-10% MeOH in CH2Cl2 to give the desired compound 27
(0.070 g, 0.26 mmol, 57% yield) as a white solid: mp 192-194
mp 174-176 °C; [R]24 50.4° (c 0.180, MeOH); UV(H2O) λmax
D
268.5 nm (ꢀ 5700, pH 2), 268.5 nm (ꢀ 5500, pH 7), 269.0 nm (ꢀ
4500, pH 11). Anal. (C10H11FN2O3S‚0.35Et2O) C, H, N, S.
General procedure for condensation reaction of the acetate
8 with purines. The preparation of 6-chloropurine derivative
21 is representative.
°C; [R]26 -38.1° (c 0.165, MeOH); UV(H2O) λmax 258.0 nm (ꢀ
D
(-)-9-[(1S ,2S ,4R )-5-O-(t er t -Bu t yld ip h e n ylsilyl)-2,3-
d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r ibofu r a n o-
syl]-6-ch lor op u r in e (21). A mixture of 6-chloropurine (0.789
g, 5.10 mmol) and ammonium sulfate (0.112 g, 0.85 mmol) in
30 mL of HMDS was refluxed for 5 h. HMDS was evaporated
to give a yellow solid. To this flask containing the silylated
6-chloropurine, a solution of the acetate 8 (1.0 g, 1.70 mmol)
in 1,2-dichloroethane (30 mL) was added. The resulting slurry
was cooled to -25 °C and TMSOTf (0.62 mL, 3.40 mmol) was
added dropwise at -25 °C. The reaction mixture was stirred
for 3 h at -25 to -10 °C, for 8 h at room temperature, and for
5 h at 40 °C. The resulting mixture was diluted with 300 mL
of CH2Cl2, washed with aqueous NaHCO3, dried over MgSO4,
filtered, and concentrated. The crude product was purified by
silica gel column chromatography with 11% EtOAc in hexanes
to give compound 21 (0.847 g, 1.24 mmol, 73% yield) as a pale-
9500, pH 2), 259.0 nm (ꢀ 8900, pH 7), 259.5 nm (ꢀ 8600, pH
11); HRMS (FAB) obsd, m/z 268.0669, calcd for C10H11FN5OS,
m/z 268.0668 (M + H)+. Anal. (C10H10FN5OS) C, H, N, S.
(-)-9-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]h yp oxa n th in e (28). A mixture of
compound 23 and its ∆1,2-isomer (0.216 g, 0.41 mmol) in
anhydrous MeOH (20 mL) was treated with 2-mercaptoethanol
(0.12 mL, 1.65 mmol) and NaOMe (0.091 g, 1.69 mmol) at 60
°C for 24 h. The resulting mixture was quenched with 0.1 mL
of glacial AcOH, concentrated, and filtered through a short
pad of silica gel with 4% MeOH in CH2Cl2. The filtrate was
concentrated to dryness. The crude product was treated with
0.84 mL of 1 M TBAF in THF at room temperature for 3 h.
The reaction mixture was concentrated and purified by
preparative TLC chromatography with 7% MeOH in CH2Cl2
to give the desired compound 28 (0.089 g, 0.33 mmol, 81%
yellow foam: [R]24 -90.0° (c 1.287, CHCl3); UV(CH2Cl2) λmax
D
yield) as a white solid: mp 143-145 °C (dec); [R]23 -23.7° (c
264.5 nm. Anal. (C32H32ClFN4OSSeSi) C, H, N, S.
D
0.324, 7:1 CH2Cl2/MeOH); UV(H2O) λmax 246.5 nm (ꢀ 12 000,
pH 2). 248.0 nm (ꢀ 10 600, pH 7), 253.5 nm (ꢀ 10 900, pH 11);
HRMS (FAB) obsd, m/z 269.0517, calcd for C10H9FN4O2S, m/z
269.0509 (M + H)+. Anal. (C10H9FN4O2S) C, H, N, S.
(-)-9-[(1S ,2S ,4R )-5-O-(t er t -Bu t yld ip h e n ylsilyl)-2,3-
d id eoxy-2-flu or o-2-p h en ylselen yl-4-th io-â-L-r ibofu r a n o-
syl]-6-ch lor o-2-flu or op u r in e (22). See the general procedure
for condensation reaction of the acetate 8 with purines. The
title compound 22 was obtained on in 67% yield (1.70 mmol):
[R]24D -86.5° (c 1.44, CHCl3); UV(CHCl3) λmax 266.0 nm. Anal.
(C32H31ClF2N4OSSeSi) C, H, N, S.
9-[(1S,4R)-5-O-(ter t-Bu t yld ip h en ylsilyl)-2,3-d id eoxy-
2,3-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n osyl]-6-ch lo-
r op u r in e (23) a n d 9-[(4R)-5-O-(ter t-bu tyld ip h en ylsilyl)-
2,3-d id eoxy-1,2-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n -
osyl]-6-ch lor op u r in e (∆1,2-Isom er ). See the general proce-
dure for syn elimination reaction. A mixture of compounds 23
and its ∆1,2-isomer (3:1) were obtained in 86% yield (1.10 mmol)
as a pale-yellow foam: UV(CH2Cl2) λmax 264.5 nm. Anal.
(C26H26ClFN4OSSi) C, H, N, S.
9-[(1S,4R)-5-O-(ter t-Bu t yld ip h en ylsilyl)-2,3-d id eoxy-
2,3-dideh ydr o-2-flu or o-4-th io-â-L-r ibofu r an osyl]-6-ch lor o-
2-flu or op u r in e (24) a n d 9-[(4R)-5-O-(ter t-Bu tyld ip h en yl-
silyl)-2,3-dideoxy-1,2-didehydro-2-fluoro-4-thio-â-L-ribofuran-
osyl]-6-ch lor o-2-flu or op u r in e (∆1,2-Isom er ). See the gen-
eral procedure for syn elimination reaction. A mixture of
compound 24 and its ∆1,2-isomer were obtained in 71% yield
(1.12 mmol) as a pale-yellow foam: UV(CH2Cl2) λmax 266.0 nm.
Anal. (C26H25ClF2N4OSSi) C, H, N, S.
(+)-9-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]gu a n osin e (29). See the procedure
for the preparation of compound 28. The title compound 29
was obtained in 61% yield (0.16 mmol): mp 210 °C (dec); [R]25
D
75.2° (c 0.15, DMSO); UV(H2O) λmax 256.0 nm (ꢀ 11 100, pH
2). 255.0 nm (ꢀ 12 500, pH 7), 266.0 nm (ꢀ 10 700, pH 11). Anal.
(C10H10FN5O2S‚0.2H2O) C, H, N, S.
(-)-9-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]-2-flu or oa d en osin e (30). See the
general procedure of desilylation and trituation with Et2O. The
title compound 30 was obtained in 70% yield (0.20 mmol): mp
>300 °C; [R]26 -17.8° (c 0.200, MeOH); UV(H2O) λmax 260.5
D
nm (ꢀ 12 700, pH 2). 260.5 nm (ꢀ 12 700, pH 7), 261.5 nm (ꢀ
12 200, pH 11). Anal. (C10H9F2N5OS) C, H, N, S.
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 TCID 50/1 × 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. The
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 recombi-
nant interleukin-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 a humidified 5% CO2/air at 37 °C for 5 days and superna-
tants were collected for reverse transcriptase (RT) activity.
(+)-2-Am in o-9-[(1S,4R)-5-O-(ter t-b u t yld ip h en ylsilyl)-
2,3-d id eoxy-2,3-d id eh yd r o-2-flu or o-4-th io-â-L-r ibofu r a n o-
syl]-6-ch lor op u r in e (25), 6-Am in o-9-[(1S,4R)-5-O-(ter t-
b u t yld ip h e n ylsilyl)-2,3-d id e oxy-2,3-d id e h yd r o-2-flu o-
r o-4-th io-â-L-r ibofu r an osyl]-2-flu or opu r in e (26) an d 6-Am i-
n o-9-[(4R)-5-O-(ter t-bu tyld ip h en ylsilyl)-2,3-d id eoxy-1,2-
d id eh yd r o-2-flu or o-4-t h io-â-L-r ib ofu r a n osyl]-2-flu or o-
p u r in e (∆1,2-Isom er ). Dry ammonia gas was bubbled into a
stirred solution of a mixture of compound 24 and its ∆1,2-isomer
(0.430 g, 0.79 mmol) in 1,2-dimethoxyethane (25 mL) at room
temperature for 5 h. The solvent was removed under reduced
pressure, and the residue was purified by preparative TLC
chromatography with 20% EtOAc in hexanes to give pure
compound 25 (0.209 g, 0.387 mmol, 49% yield) and a mixture
of compound 26 and its ∆1,2-isomer (0.116 g, 0.221 mmol, 28%
yield). For compound 25: [R]24 74.0° (c 0.236, CH2Cl2);
D
UV(CH2Cl2) λmax 300.0 nm. Anal. (C26H27ClFN5OSSi‚0.3EtOAc)
C, H, N, S. For a mixture of compounds 26 and ∆1,2-isomer:
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. An amount of
10 µL of each sample was added to 75 µL of RT reaction
mixture (0.06 M Tris, pH 7.8, 0.012 M MgCl2, 0.006 M
1
UV(CHCl3) λmax 261.0 nm. H NMR: see Supporting Informa-
tion.
(-)-9-[(1S,4R)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-L-r ibofu r a n osyl]a d en in e (27). A mixture of com-
pound 23 and its ∆1,2-isomer (0.241 g, 0.46 mmol) was treated
with methanolic ammonia at 80 °C for 12 h using a steel bomb.

Drug Resistance of Thionucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 397
dithiothreitol, 0.006 mg/mL poly(rA)noligo(dT)12-18, 96 µg/mL
dATP, and 1 µM of 0.08 mCi/ml 3H-thymidine triphosphate
(Moravek Biochemicals, Brea, CA)) and 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 â-counter (Meriden, CT).
The RT results were expressed in counts per minute (CPM)
inhibitor complexes were minimized by using the Kollman all-
atom force field until the iteration number reached 5000.
Ack n ow led gm en t. This research was supported by
the U.S. Public Health Service Research Grants AI32351
and AI25899 from the National Institute of Allergy and
Infectious Disease, NIH, and the Department of Veter-
ans Affairs.
1
per milliliter. The antiviral 50% effective concentration (EC50
)
Su p p or tin g In for m a tion Ava ila ble: Tables of H NMR
and 90% effective concentration (EC90) were determined from
the concentration-response curve using the median effect
method.30
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces
Cytotoxicity Assa ys. The compounds were evaluated for
their potential toxic effects on uninfected PHA-stimulated
human PBM cells, in CEM (T-lymphoblastoid cell line obtained
from American Type Culture Collection, Rockville, MD), and
in Vero (African green monkey kidney) cells. PBM cells were
obtained from the 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 a 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 incubated overnight. The reaction 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 concentra-
tion (IC50) was determined from the concentration-response
curve using the median effect method.30
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