Stereoselective synthesis and antiviral activity of d-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-4 ′-thionucleosides

Article abstract of DOI:10.1021/jm020246+

As 2′,3′-didehydro-2′,3′-dideoxy-2′ -fluoronucleosides have exhibited interesting antiviral effects against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4′-thionucleosides in which 4′-oxygen is replaced by a sulfur a

Full text of DOI:10.1021/jm020246+

4888
J . Med. Chem. 2002, 45, 4888-4898
Ster eoselective Syn th esis a n d An tivir a l Activity of
D-2′,3′-Did eh yd r o-2′,3′-d id eoxy-2′-flu or o-4′-th ion u cleosid es
Youhoon Chong,† Hyunah Choo,† 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 J une 5, 2002
As 2′,3′-didehydro-2′,3′-dideoxy-2′-fluoronucleosides have exhibited interesting antiviral effects
against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4′-
thionucleosides in which 4′-oxygen is replaced by a sulfur atom. To study structure-activity
relationships, various pyrimidine and purine nucleosides were synthesized from the key
intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylse-
lenyl-4-thio-â-D-ribofuranoside 8, which was prepared from the 2,3-O-isopropylidene-D-glycer-
aldehyde 1 in 13 steps. The antiviral activity of the synthesized compounds were evaluated
against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which cytidine 17,
5-fluorocytidine 18, adenosine 24, and 2-fluoroadenosine 32 showed moderate to potent anti-
HIV activities (EC50 1.3, 11.6, 8.1, and 1.2 µM, respectively). It is noteworthy that 2-fluoro-
adenosine analogue 32 showed antiviral potency as well as high cytotoxicity (IC50 1.5, 1.1, and
7.6 µM for PBM, CEM, and Vero, respectively) whereas no other compound showed cytotoxicity
up to 100 µM. The cytidine 17 and 5-fluorocytidine 18 analogues showed significantly decreased
antiviral activity against the clinically important lamivudine-resistant variants (HIV-1M184V),
whereas the corresponding D-2′-Fd4 nucleosides showed limited cross-resistance. Molecular
modeling studies demonstrated that the larger van der Waals radius as well as the close
proximity to Met184 of the 4′-sulfur atom of D-2′-F-4′-Sd4C (17) may be the reasons for the
decreased antiviral potency of synthesized 4′-thio nucleosides against the lamivudine-resistant
variants (HIV-1M184V).
In tr od u ction
cally stable antiviral agents. Thus, it was of interest to
synthesize the nucleosides with isosteric replacement
of 4′-oxygen by sulfur atom. The first example of
4′-thionucleosides was reported in 1964 by Reist et al.,
who synthesized the 4′-thio counterpart of naturally
occurring adenosine.11 Since then, several classes of 4′-
thionucleosides have been reported,12 including 9-(4-
thio-D-xylofuranosyl)adenine,13 9-(4-thio-D-arabinofura-
nosyl)adenine,13 and 4′-thio-araC.14 However, the diffi-
culty of synthesizing optically pure 4′-thionucleosides
has impaired additional syntheses of these analogues,
and only a few examples have been known until
recently.15 Moreover, biologically interesting 2′-deoxy-
4′-thionucleosides had not been synthesized until
Walker16 and Secrist17 independently reported the
syntheses of pyrimidine 2′-deoxy-4′-thionucleosides in
1991, which were followed by an alternative synthesis
of 2′-deoxy-4′-thionucleosides using the Sharpless asym-
metric epoxidation,18 synthesis of 4′-thio-2′,3′-dideoxy-
nucleosides,19 and the syntheses of 4′-thioarabinonu-
cleosides20 as well as 2′-modified 2′-deoxy-4′-thio-
cytidines.21 The synthesized 4′-thio-2′-deoxy, 4′-thio-
2′,3′-dideoxy and 4′-thioarabino nucleosides have shown
to have potent anti-herpes, anti-HIV, and anti-cytome-
galovirus activities, respectively, and some analogues,
especially 4′-thiothymidine and 2′-deoxy-4′-thiocytidine,
have exhibited potent cytotoxicity.16-19 Interestingly, the
4′-thionucleosides are usually resistant to hydrolytic
cleavage of glycosyl linkage catalyzed by nucleoside
phosphorylase,22 which is one of the advantages of 4′-
Nucleoside analogue reverse transcriptase inhibitors
continue to be important drug regimens as part of highly
active antiretroviral therapy (HAART). However, be-
cause of the drug toxicity1,2 and the lack of a durable
response due to resistance,3 there is clearly a need for
new compounds to cope with these drawbacks. Particu-
larly, compounds with activity against HIV isolates that
are resistant to currently available therapies as well as
agents with beneficial pharmacokinetic profiles that
allow infrequent dosing are under active investigation.
The past decade has witnessed the emergence of a
class of 2′,3′-unsaturated nucleosides such as d4T,4,5
L-d4C,6,7 L-d4FC,6,7 and abacavir8,9 as interesting thera-
peutic candidates for anti-HIV therapy because of their
potent antiviral activity. The importance of this class
of compounds has been confirmed after a recent study
which showed that the instability of the purine ana-
logues can be remarkably improved by the isosteric
replacement of 2′-hydrogen by fluorine atom.10 There-
fore, agents containing the 2′,3′-unsaturated sugar
moiety with 2′-fluoro substitution has become one of the
rational targets in search for safe, effective, and chemi-
* Corresponding author: Dr. Chung K. Chu, Distinguished Research
Professor, Department of Pharmaceutical and Biomedical Sciences,
College of Pharmacy, The University of Georgia, Athens, GA 30602.
Tel: (706)-542-5379. Fax: (706)-542-5381. E-mail: dchu@rx.uga.edu.
† The University of Georgia.
‡ Emory University School of Medicine/Veterans Affairs Medical
Center.
10.1021/jm020246+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/24/2002

Synthesis and Antiviral Activity of 4′-Thionucleosides
Sch em e 1. Synthesis of the Key Intermediate 8a
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4889
a
Keys (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, touluene, -78 °C, (ii) Ac2O, TEA,
CH2Cl2.
thionucleosides compared with several metabolically
unstable “4′-oxy” antiviral agents which are substrates
for nucleoside phosphorylase.23 Even though 4′-thio-
nucleosides have recently received considerable atten-
tion as potential antiviral agents, their 2′,3′-unsaturated
analogues have not been well investigated probably
because of the synthetic difficulties. A report by Young
et al., however, reemphasized the importance of this
class of nucleosides, in which L-4′-thio-d4C analogues
showed marked anti-HBV as well and anti-HIV activ-
ity.24 In a recent communication, we reported the
stereoselective synthesis of the â-L-2′-F-4′-Sd4C which
showed potent anti-HIV activity (EC50 0.12 µM) in
human peripheral blood monomuclear (PBM) cells.25 By
using the same synthetic strategy, a series of D-coun-
terparts were synthesized for the structure-activity
relationship studies. Various pyrimidine and purine
nucleosides were synthesized from the key intermediate,
(2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-
dideoxy-2-fluoro-2-phenylselenyl-4-thio-â-D-ribofurano-
side 8, which was prepared from the 2,3-O-isopropyl-
idene-D-glyceraldehyde 1 in 13 steps. The antiviral
activities of the synthesized compounds were evaluated
against HIV-1 in human PBM cells. The antiviral
activity of cytidine 17 and 5-fluorocytidine 18 analogues
were also examined against the clinically important
lamivudine-resistant variants (HIV-1M184V). To under-
stand the reduced antiviral activity of the synthesized
â-D-2′-F-4′-Sd4N compared with â-D-2′-Fd4N10 and the
cross-resistance of the lamivudine-resistant variants
(HIV-1M184V) to â-D-2′-F-4′-Sd4C (17) and â-D-2′-F-4′-
Sd4FC (18), molecular modeling studies including a
quantum mechanical geometry optimization of â-D-2′-
F-4′-Sd4C (17) as well as energy-minimization of HIV-1
RT/â-D-2′-F-4′-Sd4C (17) complex were performed. Herein,
we report the full accounts of the synthesis, biological
evaluation, and molecular modeling studies of the titled
nucleosides.
known to date employ either nucleophilic attack of
dimesylate by disulfide anion,20,21 ring closure of dithio-
acetal,20 or reductive cyclization of thioacetic acid
ester.19 2-Fluoro-2-buten-4-olide 2, which was used as
the key intermediate for the synthesis of 2′-fluoro-2′,3′-
unsaturated nucleosides,10 can also be used for this
purpose because the butenolide ring can be converted
into the appropriate intermediates (dimesylate, dithio-
acetal, and thioacetic acid ester) for any of those three
reactions after some manipulations. The attempted
syntheses of these intermediates from the 2-fuoro-2-
buten-4-olide 2, however, gave very complicated mix-
tures presumably because of the instability of fluorovi-
nyl moiety. The transformation was possible only after
saturation of the butenolide 2 by catalytic hydrogena-
tion. Therefore, 2-fluoro-γ-butyrolactone 3 was used as
a platform for the generation of the key intermediate
(2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-
dideoxy-2-fluoro-2-phenylselenyl-4-thio-â-D-ribofurano-
side 8 (Scheme 1). (R)-2-Fluorobutenolide 2 was pre-
pared from 2,3-O-isopropylidene-D-glyceraldehyde 1 in
three steps by a known method (Scheme 1).26 (S)-2-
Fluorobutenolide 2 was hydrogenated to â-2-fluorolac-
tone 3 by treatment with 5% Pd/C under H2 in a
1
quantitative yield. The H NMR of the crude compound
3 showed no contamination by the epimer at C2. The
lactone 3 was converted to iodo ester 4 in three consecu-
tive steps. Hydrolysis using NaOH in aqueous EtOH
followed by methylation of the corresponding carboxylic
acid gave hydroxy methyl ester 3a (Scheme 2), which
was treated with iodine, triphenylphosphine, and imi-
dazole in toluene at 60 °C for 4 h to give a 6:1 epimeric
mixture of the iodo ester 4 in 83% overall yield (Scheme
1). To confirm which carbon center has epimerized, the
hydroxyl methyl ester 3a was subjected to reclosure of
the ring to give the starting 2-fluorobutenolide 3 under
basic conditions (Scheme 2). The spectroscopic data (1H
and 13C NMR) of this compound matched that of the
starting compound, which suggested that there was no
significant epimerization during saponification followed
by methylation. Iodination under several different reac-
tion conditions, however, confirmed that high temper-
ature and longer reaction time resulted in partial
Resu lts a n d Discu ssion
Ch em istr y. Introduction of a sulfur atom at the 4′-
position of a sugar ring has been hampered by the
laborious synthetic procedures. The synthetic methods

4890 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Sch em e 2. Epimerization at C4 during Iodinationa
Chong et al.
a
Keys: (a) (i) NaOH, EtOH, (ii) dimethyl sulfate, DMSO, (b) pyridine, rt, (c) I2, Ph3P, imidazole, toluene.
Sch em e 3. Synthesis of the Pyrimidine Nucleosides 17-20a
a
Keys: (a) HMDS, CH3CN, pyrimidines; TMSOTf, (b) mCPBA, -78 °C; pyridine, rt, (c) (i) TBAF, THF, (ii) NH3, MEOH, rt, (d) TBAF,
THF.
epimerization at C4. Treatment of the hydroxyl methyl
ammonia to give the desired 2′-fluoro-4′-thio-2′,3′-
unsaturated pyrimidine nucleosides 17-20 (Scheme 3).
The synthesis of purine analogues (24, 25, 30, and
32) needed more careful treatments (Scheme 4). The key
intermediate 8 was condensed with 6-chloropurine and
2-fluoro-6-chloropurine to give the corresponding nu-
cleosides 21 and 26 in 65% and 66% yield, respectively.
To achieve a clean and high-yield conversion, the
temperature of the reaction 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 temperature and then for 2 h at 60 °C. The
6-chloropurine derivative 21 was syn-eliminated by
successive treatment with mCPBA and pyridine to give
an inseparable mixture of 2′,3′-unsaturated nucleoside
22 and its ∆1,2-isomer 23 in 81% yield (3:1 determined
ester 3a under Mitsunobu conditions at 60 °C for 4 h
was found to be the optimum conditions to minimize
the epimerization. The epimeric mixture of iodo ester 4
was subjected to nucleophilic attack by potassium
thioacetate in DMF to give an epimeric mixture of
thioacetates 5. DIBAL-mediated reduction of the thio-
acetates 5 followed by Moffat-type oxidation provided
the corresponding thiolactone 6 and 6a , in 54% yield.
At this stage, the two epimers could be separated by
column chromatography on silica gel. During the ensu-
ing phenylselenylation, the C2 position was sp2-hybrid-
ized by trapping the enolate as silyl enol ether. The
phenylselenyl group approached the least hindered R
face of the silyl enol ether to give the corresponding
enantiomers 7 and 7a (Scheme 1). The optical rotation
values of these two compounds were matched with
1
by H NMR). Amination of the mixture of 22 and 23 by
treatment with methanolic ammonia at 100 °C in a steel
bomb gave the adenosine analogue, which was depro-
tected by TBAF in THF to give 2′-fluoro-4′-thio-2′,3′-
unsaturated adenosine 24 in 61% yield. The ∆1,2-isomer
23, however, did not survive under reaction conditions
and silica gel column chromatography due to its insta-
bility. On the other hand, the 2′,3′-unsaturated 6-chlo-
ropurine derivative 22 was hydrolyzed in the presence
of sodium methoxide and 2-mercaptoethanol in refluxing
methanol to give the hypoxanthine derivative, which
was converted to the desired 2′-fluoro-4′-thio-2′,3′-
unsaturated inosine 25 in 46% yield (yield is not
optimized). The 2-fluoro-6-chloropurine derivative 26
was also treated with mCPBA followed by pyridine to
give an inseparable mixture of the syn-eliminated
product 27 and its ∆1,2-isomer 28 in 71% yield. Dry
ammonia gas was bubbled into a solution of 27 and 28
in ethylene glycol dimethyl ether (DME) at room tem-
perature for 16 h to give the 2-amino-6-chloropurine
derivative 29 and 2-fluoro-6-aminopurine derivative 31
in 45% and 25% yield, respectively, which were readily
separated by silica gel column chromatography. Unfor-
tunately, the ∆1,2-isomer 28 was not stable enough to
survive the reaction and purification conditions. The
opposite signs {for 7; [R]24 54.0° (c 0.606, CHCl3) and
D
for 7a ; [R]24 -56.4° (c 0.542, CHCl3)}, which finally
D
confirmed the epimerization at C4 during iodination
under Mitsunobu conditions. The epimerized product
7a , therefore, can be recycled as the key intermediate
for the synthesis of L-antipodes. DIBAL reduction of the
2-fluoro-2-phenylselenothiolactone 7 followed by acetyl-
ation using Ac2O and triethylamine in CH2Cl2 gave the
key intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyl-
diphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylselenyl-4-
thio-â-D-ribofuranoside 8 in 90% yield (Scheme 1).
Condensation of 8 with various pyrimidine heterocyclic
bases under Vorbru¨ggen conditions gave the corre-
sponding pyrimidine analogues 9-12 in 40-60% yield
(Scheme 2). The condensation gave the â-anomer ex-
clusively by virtue of the bulky R-phenylselenyl group.27
Oxidation of the phenylselenide group by mCPBA at low
temperature followed by treatment with pyridine gave
the corresponding syn-eliminated 2′,3′-unsaturated com-
pounds 13-16 in high yields (70-80%) (Scheme 3). The
protecting groups on the heterocyclic base and the 5′
position of the sugar moiety were sequentially deblocked
by the treatment with TBAF in THF and methanolic

Synthesis and Antiviral Activity of 4′-Thionucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4891
Sch em e 4. Synthesis of the Purine Nucelosides 24-25, 30, and 32a
a
Keys: (a) HMDS, (NH4)2SO4, purines; TMSOTf, (b) mCPBA, -78 °C; pyridine, rt, (c) (i) NH3, MeOH, 80 °C, (ii) TBAF, THF, (d) (i)
HSCH2CH2OH, NaOH, 60 °C, (ii) TBAF, THF, (e) NH3 bubbling, DME,rt, (f) TBAF, THF.
Ta ble 1. Anti-HIV Activity and Cytotoxicity of
2-amino-6-chloropurine derivative 29, thus obtained,
D-2′,3′-Didehydro-2′,3′-dideoxy-2′-fluoro-4′-thionucleosides
was hydrolyzed by using sodium methoxide and 2-mer-
activity (EC50, µM)
cytotoxicity (IC50, µM)
captoethanol in refluxing methanol to give the final 2′-
fluoro-4′-thio-2′,3′-unsaturated guanosine 30 in 61%
yield. The 2-fluoro-6-aminopurine derivative 31, on the
other hand, was deprotected by TBAF in THF to afford
the 2′-fluoro-4′-thio-2′,3′-unsaturated 2-fluoroadenosine
32 in 70% yield.
Str u ctu r e-Activity Rela tion sh ip s. The synthe-
sized pyrimidine (17-20) and purine (24, 25, 30, and
32) nucleosides were evaluated against HIV-1 in human
PBM cells in vitro, and AZT was included as a positive
control. The results are summarized in Table 1. Among
the tested nucleosides, two pyrimidine nucleosides,
cytidine 17 (EC50 1.3 µM) and 5-fluorocytidine 18 (EC50
11.6 µM), and all purine nucleosides synthesized, ad-
enosine 24 (EC50 8.1 µM), inosine 25 (EC50 43.6 µM),
guanosine 30 (EC50 80.5 µM), and 2-fluoroadenosine 32
(EC50 1.2 µM), showed moderate to potent antiviral
activities. It is noteworthy that 2-fluoroadenosine ana-
logue 32 showed antiviral potency as well as high
cytotoxicity (IC50 1.5, 1.1, and 7.6 µM for PBM, CEM,
compound
HIV-1
PBM
CEM
Vero
17
18
19
20
24
25
30
32
AZT
1.3
11.6
92.3
>100
8.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
53.0
>100
>100
>100
>100
43.6
80.5
1.2
1.5
>100
1.1
14.3
7.6
28.0
0.004
and Vero, respectively) whereas no other compound
showed cytotoxicity up to 100 µM. Compared with the
â-D-2′-Fd4N nucleosides,10 there was a general trend of
antiviral activities throughout the series, where cyti-
dine, 5-fluorocytidine, and adenosine analogues were
most potent and almost all purine nucleosides showed
moderate to potent activities. However, the â-D-2′-Fd4N
nucleosides10 were consistently more potent than their
4′-thio congeners, which suggests that the two types of

4892 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Chong et al.
Ta ble 2. Activity of Selected Nucleosides against
Lamivudine-Resistant Virus (HIV-1M184V) in Human PBM Cells
shows that nucleoside inhibitors are located in a well-
defined binding pocket formed by Arg72, Met184, and
3′-OH pocket residues (Asp113, Tyr115, Phe116, and
Gln151) (Figure 1b). The role of Arg72 is notable
because, as it moves into the binding pocket, it stabilizes
the bound nucleotide through hydrogen bonding with
the triphosphate moiety as well as nonspecific hydro-
phobic interaction with the heterocyclic moiety of the
nucleoside triphosphate (Figure 1b). An additional
characteristic binding mode of these d4-nucleotides is
the possible π-π interaction32 between C2′-C3′ fluo-
rovinyl moiety of the nucleotide and the aromatic ring
of nearby Tyr115, which could be one reason for the high
antiviral activity of 2′,3′-unsaturated nucleosides4-9
(Figure 1b). Fluorine substitution at C2′ position would
make the C2′-C3′ double bond electron poor and, thus,
increase the π-π interaction with the electron-rich
aromatic ring of Tyr115. Despite almost the same
binding mode of â-D-2′-Fd4C and â-D-2′-F-4′-Sd4C 17,
the decreased antiviral activity of â-D-2′-F-4′-Sd4C can
be explained by the decrease in π-π interaction. The
replacement of electronegative oxygen by sulfur depo-
larizes charges inside the sugar ring in â-D-2′-F-4′-Sd4C
17, which may result in reduced π-π interaction
between the fluorovinyl moiety and aromatic ring of
Tyr115. Because of its out-of-plane location and large
van der Waals radius, the 4′-sulfur atom in the â-D-2′-
F-4′-Sd4N sugar moiety is in close contact to Met184
(Figure 1c). As a result, the mutation of Met184 to
Val184, which has a bulky side chain, results in a
significant steric hindrance between the 4′-sulfur atom
in the â-D-2′-F-4′-Sd4N sugar moiety and the side chain
of Val184, which might be one of the reasons for the
high cross-resistance of HIV-1M184V to â-D-2′-F-4′-Sd4C
(17) and â-D-2′-F-4′-Sd4FC (18) (Table 2, Figure 1c).
compound
xxBRU (EC90, µM) M184V (EC90, µM) FIa
17
5.0
9.9
0.01
0.08
≈125
25
3.5
0.3
6688
â-D-2′-F-d4FCb
34.7
AZT
3TC
0.003
535
a
FI is the fold increase (EC90 HIV-1M184V/EC90 HIV-1xxBRU).
b
Reference 11b: Lee et al. J . Med. Chem. 2002, 45, 1313-1320.
nucleosides may have similar structural features and
may be recognized in the same fashion at the kinase
level, but the way they interact with the viral poly-
merase (HIV-1 reverse transcriptase) must be somewhat
different (vide infra for molecular modeling studies).
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 . Lamivudine (3TC, (-)-
â-2′,3′-dideoxy-3′-thiacytidine) is an important compo-
nent of the highly active antiretroviral therapy (HAART).
However, single mutation at residue 184 (M184V) of the
reverse transcriptase (RT) in HIV causes high-level
resistance to 3TC and contributes to the failure of anti-
AIDS combination therapy.28,29 Therefore, there is an
urgent need to discover new drug candidates active
against the M184V mutant HIV-1 RT. The cytosine (17)
and 5-fluorocytosine analogues (18), along with two
positive controls, AZT and 3TC, were evaluated against
the lamivudine-resistant mutant strain (HIV-1M184V) in
human PBM cells in vitro (Table 2). Compared with the
4′-oxygen congeners such as â-D-2′-Fd4FC,10 the â-D-2′-
F-4′-Sd4C (17) showed significantly reduced anti-HIV
activity against HIV-1M184V (Table 2), which suggests
the isosteric substitution of 4′-oxygen with 4′-sulfur
made the â-D-2′-F-4′-Sd4N cross-resistant to the M184V
mutation in HIV-1 RT (vide infra for molecular modeling
studies).
Molecu la r Mod elin g. Since the conformational dif-
ference between the nucleoside analogues is one of the
critical factors in determining their antiviral activity,
it was of interest to compare the conformations of â-D-
2′-Fd4N and the corresponding 4′-thio congeners. For
this purpose, a quantum mechanical calculation (RHF/
3-21G*) was performed on cytidine analogues (â-D-2′-
Fd4C10 and â-D-2′-F-4′-Sd4C 17), and the geometry-
optimized structures were compared (Figure 1a). The
two structures were superimposable except for the 4′-
positions; the strain inside the 4′-thio sugar ring caused
by the longer C-S bond length resulted in movement
of the 4′-sulfur atom by 0.5 Å down to the plane formed
by C1′, C2′, C3′, and C4′. Another reason for this out-
of-plane movement of the 4′-sulfur atom is that, unlike
4′-oxygen, the 4′-sulfur atom cannot form a hydrogen
bond with 5′-OH due to the lack of the gauche effect.
However, the critically important configurations of the
N1 and C5′ atoms of a nucleoside are in good ac-
cordance, which may imply the similar tolerance to the
two types of nucleosides by the kinases. On the other
hand, when the triphosphates of â-D-2′-Fd4C10 and â-D-
2′-F-4′-Sd4C 17 bind to the active site of HIV-1 reverse
transcriptase,30 the isosteric replacement of 4′-oxygen
with the 4′-sulfur atom exerts effects on the interaction
between the nucleoside triphosphates and the active site
residues, particularly Tyr115 and Met184 (Figure 1,
parts b and c). The energy-minimized structures31 of â-D-
2′-Fd4C and â-D-2′-F-4′-Sd4C 17 bound to HIV-1 RT
Meta bolic Sta bility. In an attempt to understand
metabolic stability of the adenosine analogue 24, ad-
enosine deaminase (ADA) binding efficiencies and deam-
ination kinetics studies were performed.33 As shown in
Table 3, the adenine derivative 24 was found to be
tightly bound (KM ) 18.3 µM) to the mammalian
adenosine deaminase (from calf intestinal mucosa,
EC.3.5.4.4), but the turnover number (kcat ) 0.94 s-1
)
was very low, which indicates that even though the
adenine derivative 24 can strongly bind to ADA, it can
barely be catalyzed by ADA to the corresponding inosine
derivative. It is noteworthy that, compared with other
substrates of ADA, â-D-2′-F-4′-Sd4A 24 has very low KM
and kcat values, and the catalytic efficiency (kcat/KM) is
as low as that of â-FddA.34 From a therapeutic point of
view, this metabolic stability of the adenosine ana-
logue24 has important implications because adenosine
deaminase is one of the major deactivating enzymes of
the purine catabolism pathway.35
In summary, we have developed an efficient synthesis
of optically pure â-D-2′-F-4′-Sd4 nucleosides. The iso-
steric replacement of 4′-oxygen in â-D-2′-Fd4 nucleosides
with 4′-sulfur caused substantial changes in the binding
mode of â-D-2′-F-4′-Sd4 to the active site of HIV-1 RT,
resulting in reduced in vitro anti-HIV-1 activity. The
lamivudine-resistant mutant strain (HIV-1M184V) showed
significant cross-resistance to the â-D-2′-F-4′-Sd4 nu-
cleosides, which could be explained by the larger van

Synthesis and Antiviral Activity of 4′-Thionucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4893
F igu r e 1. (a) Superimposed, geometry-optimized structures of D-2′-F-4′-Sd4C (17) and D-2′-Fd4C. (b) Binding mode of D-2′-F-
4′-Sd4C (17) to the active site of HIV-1 RT. (c) Energy-minimized structure of D-2′-F-4′-Sd4C (17), complexed with the wild-type
HIV-1 RT (left) and M184V mutant RT (right).
Ta ble 3. Kinetic Constants for Adenosine Analogues as
mmol) in 200 mL of EtOAc was added 2.0 g of Pd/C (5% w/w)
under H2 atmosphere, and the mixture was stirred for 3 h.
After filtration of the reaction mixture through a Celite pad,
the filtrate was concentrated and purified by silica gel column
chromatography with 5% EtOAc in hexanes to give compound
3 (13.9 g, 37.5 mmol, 94% yield) as a white solid: mp 87-89
Substrates of ADAa
KM
(µM)
kcat/KM
t1/2
(min)
compound
kcat (s-1
)
(µM-1 s-1
)
D-2′-F-4′-Sd4A (24)
D-2′-F-d4A28
adenosine
18.3
23.0
24.5
0.94
NAb
76.4
0.05
NAb
3.1
33
42
0.5
°C; [R]24 15.5° (c 0.85, CHCl3); 1H NMR (CDCl3) δ 7.70-7.35
D
(m, 10H), 5.26 (dt, J ) 51.2, 8.8 Hz, 1H), 4.54-4.47 (m, 1H),
3.93 (dd, J ) 11.4, 2.3 Hz, 1H), 3.71 (dd, J ) 11.4, 3.6 Hz,
1H), 2.65 (ddt, J ) 13.1, 8.4, 6.6 Hz, 1H), 2.54 (ddt, J ) 24.2,
13.2, 9.0 Hz, 1H), 1.04 (s, 9H); 13C NMR (CDCl3) δ 171.30 (d,
J ) 21.3 Hz), 135.59, 135.30, 132.52, 132.20, 129.76, 129.35,
127.82, 127.63, 89.30 (d, J ) 191.0 Hz), 76.24 (d, J ) 6.1 Hz),
63.73, 30.14 (d, J ) 20.0 Hz), 26.58, 19.12; Anal. (C21H25FO3-
Si) C, H.
a
Kinetic data were obtained spectrophotometrically at 25 °C
by following the decrease in absorbance at 265 nm. b Not available.
der Waals radius as well as the close proximity to
Met184 of the 4′-sulfur atom.
Exp er im en ta l Section
(2S,4S/R)-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 (7.49 g, 20.1 mmol) in 5% aqueous EtOH was treated with
solid NaOH (0.885 g, 22.1 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 (2.29 mL, 24.2 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 (7.65 g, 30.1 mmol), imidazole (4.11 g, 60.4
mmol), and Ph3P (10.55 g, 40.2 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, to remove residual Ph3P. Aqueous
Na2S2O3 was added dropwise until the iodine color disap-
peared, to remove residual iodine. The resulting mixture was
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.
(2S,4S)-(+)-4-ter t-Bu tyldiph en ylsilyloxym eth yl-2-flu or o-
γ-bu tyr ola cton e (3). To a solution of lactone 2 (14.8 g, 39.9

4894 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Chong et al.
poured to a separatory funnel, diluted with 300 mL of toluene,
washed with brine, dried over MgSO4, filtered, concentrated,
and purified by silica gel column chromatography with 7%
EtOAc in hexanes to give compounds 4 (7.52 g, 14.6 mmol,
73% yield) as pale yellow oil: 1H NMR (CDCl3) for major δ
7.71-7.33 (m, 10H), 5.08 (ddd, J ) 47.9, 7.2, 5.0 Hz, 1H), 4.31-
4.17 (m, 1H), 3.96-3.76 (m, 2H), 3.79 (s, 3H) 2.75-2.08 (m,
2H), 1.10 (s, 9H), for minor δ 7.71-7.33 (m, 10H), 5.16 (ddd,
J ) 48.2, 10.3, 2.1 Hz, 1H), 4.31-4.17 (m, 1H), 3.96-3.76 (m
2H), 3.81 (s, 3H), 2.75-2.08 (m, 2H), 1.08 (s, 9H); HRMS (FAB)
obsd, m/z 515.0945, calcd for C22H29FIO3Si, m/z 515.0915 (M
+ H)+; Anal. (C22H28FIO3Si‚0.2C6H14) C, H.
(2S,4R/S)-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
componds 4 (11.72 g, 22.8 mmol) in 12 mL of DMF was treated
with solid KSAc (5.2 g, 45.7 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 (9.2 g, 19.9 mmol,
87% yield) as a red-brown oil: 1H NMR (CDCl3) for major δ
7.61-7.32 (m, 10H), 5.22-4.88 (m, 1H), 3.89-3.65 (m, 3H),
3.78 (s, 3H), 2.41-2.07 (m, 2H), 2.29 (s, 3H), 1.05 (s, 9H), for
major δ 7.61-7.32 (m, 10H), 5.22-4.88 (m, 1H), 3.89-3.65 (m,
3H), 3.78 (s, 3H), 2.41-2.07 (m, 2H), 2.27 (s, 3H), 1.06 (s, 9H);
HRMS (FAB) obsd, m/z 463.1770, calcd for C24H32FO4SSi, m/z
463.1775 (M + H)+; Anal. (C24H31FO4SSi) C, H, S.
2.49 (dd, J ) 13.4, 4.0 Hz, 1H), 2.22 (td, J ) 13.5, 10.5 Hz,
1H), 1.06 (s, 9H); 13C NMR (CDCl3) δ 196.32 (d, J ) 23.1 Hz),
137.01, 135.52, 132.63, 132.50, 130.02, 129.99, 129.38, 127.87,
124.78, 105.12 (d, J ) 260.6 Hz), 65.66, 44.58 (d, J ) 2.9 Hz),
39.13 (d, J ) 21.4 Hz), 26.69, 19.20; Anal. (C27H29FO2SSeSi)
C, H, S.
(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 (7a ). See the previous
procedure for reaction of the compound 7 with phenylselenyl
bromide. The title compound 7a was obtained on 9.69-mmol
scale in 74% yield as a pale yellow syrup: [R]24 -56.4° (c
D
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); 13C 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 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 C27H30FO2SSeSi, m/z 545.0885
(M + H)+; Anal. (C27H29FO2SSeSi‚0.15CHCl3) C, H, S.
(1R/S,2R,4S)-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-D-r ibofu r a -
n osid e (8). A solution of compounds 7 (4.77 g, 8.76 mmol) in
toluene (100 mL) was treated with 17.5 mL of 1 M DIBAL-H
in hexane at -78 °C for 1 h. The reaction was quenched with
4 mL of MeOH and warmed to room temperature for 1 h and
aq 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 (2.48 mL, 26.3
mmol), TEA (3.66 mL, 26.3 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 chroma-
tography with 3% Et2O in hexanes to give the acetate 8 (4.4
g, 7.48 mmol, 85% yield) as a pale yellow oil: 1H NMR (CDCl3)
δ 7.70-7.32 (m, 15H), 6.03, 5.99 (d and 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.
(2S,4S)-(+)-4-ter t-Bu tyldiph en ylsilyloxym eth yl-2-flu or o-
γ-t h iob u t yr ola ct on e (6) a n d (2S,4R)-(-)-4-ter t-Bu t yl-
diph en ylsilyloxym eth yl-2-flu or o-γ-th iobu tyr olacton e (6a).
A solution of compounds 5 (9.2 g, 19.9 mmol) in toluene (200
mL) was treated with 43.7 mL of 1 M DIBAL-H in hexane at
-78 °C for 1 h. The reaction was quenched with 9.6 mL of
MeOH and warmed to room temperature for 1 h, and aq
NaHCO3 (19 mL) and EtOAc (200 mL) were added to the
mixture. The resulting mixture was filtered, and the filtrate
was concentrated to driness. The crude thiolactol was treated
with Ac2O (19 mL) and DMSO (20 mL) at room temperature
for 24 h. The reaction mixture was poured to 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 chromatogra-
phy with 5% Et2O in hexanes to give the product 6 (4.2 g, 10.8
mmol, 54% yield) and 6a (0.7 g, 1.8 mmol, 9% yield) as yellow
Gen er a l P r oced u r e for Con d en sa tion Rea ction of th e
Aceta te 8 w ith P yr im id in es. The preparation of cytosine
derivative 9 is representative.
(+)-N4-Ben zoyl-1-[(1R,2R,4S)-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-D-r i-
bofu r a n osyl]cytosin e (9). A mixture of N4-benzoylcytosine
(0.505 g, 2.35 mmol) in HMDS (15 mL) and CH3CN (15 mL)
was heated under reflux for 5 h. After removal of solvent by
using a vacuum pump, a solution of the acetate 8 (0.460 g,
0.786 mmol) in 15 mL of CH3CN was added to the reaction
flask containing the silylated N4-benzoylcytosine, and then
TMSOTf (0.28 mL, 1.4 mmol) was added dropwise at room
temperature. After 16 h, the reaction was quenched with 1
mL of sat. NaHCO3 and the resulting mixture was concen-
trated. The crude mixture was diluted with 100 mL of CH2-
Cl2, washed with aq 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 (0.269 g, 0.362 mmol, 46% yield) as a
oil: For 6: [R]25 28.2° (c 1.08, CHCl3); 1H NMR (CDCl3) δ
D
7.68-7.37 (m, 10H), 5.07 (ddd, J ) 50.5, 10.2, 6.9 Hz, 1H),
3.96-3.78 (m, 3H), 2.70-2.62 (m, 1H), 2.18- 2.05 (m, 1H),
1.07 (s, 9H); 13C NMR (CDCl3) δ 200.69 (d, J ) 18.0 Hz),
135.53, 132.68, 132.60, 130.01, 127.86, 93.27 (d, J ) 197.0 Hz),
66.64, 43.98 (d, J ) 7.0 Hz), 32.61 (d, J ) 19.4 Hz), 26.69,
19.23; Anal. (C21H25FO2SSi) C, H, S, For 6a : [R]27 -46.7° (c
D
0.5, CHCl3); 1H NMR (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,4S)-(+)-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 (4.74 g, 12.2 mmol) in THF (60 mL) was added
14.7 mL of 1 M LiHMDS in THF slowly at -78 °C, and the
reaction mixture was stirred at the same temperature for 1 h.
TMSCl (2.01 mL, 15.9 mmol) was added dropwise to the
reaction mixture, and the mixture was allowed to warm to
room temperature. The resulting mixture was stirred at room
temperature for 30 min and cooled to -78 °C. A solution of
PhSeBr (4.37 g, 18.3 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 desired product 7 (4.77 g, 8.76 mmol, 72%
yield) as a pale yellow syrup: [R]24D 54.0° (c 0.606, CHCl3); 1H
NMR (CDCl3) δ 7.68-7.35 (m, 15H), 3.96-3.89 (m, 1H), 3.87
(dd. J ) 10.2, 5.1 Hz, 1H), 3.80 (dd, J ) 10.1, 7.1 Hz, 1H),
foam: [R]24 179.9° (c 0.50, CH2Cl2); UV(MeOH) λmax 308 nm;
D
13C NMR (CDCl3) δ 161.94, 155.42, 147.17, 136.96, 135.60,
135.54, 133.21, 132.83, 129.96, 129.92, 129.41, 129.20, 129.03,
127.78,0.127.60, 125.73, 106.39 (d, J ) 248.8 Hz), 96.87, 66.74
(d, J ) 17.6 Hz), 66.29, 47.21, 42.39 (d, J ) 22.2 Hz), 26.73,
19.15; Anal. (C38H38FN3O3SSeSi) C, H, N, S.
(+)-N4-Ben zoyl-1-[(1R,2R,4S)-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-D-r i-
bofu r a n osyl]-5-flu or ocytosin e (10). See the general proce-
dure for condensation reaction of the acetate 8 with pyrimidines.
The title compound 10 was obtained on 0.850-mmol scale in
52% yield: [R]24 202.6° (c 0.410, CH2Cl2); UV(CH2Cl2) λmax
D
332.5 nm; 13C NMR (CDCl3) δ 151.88 (d J ) 19.2 Hz), 147.15,
139.01 (d, J ) 237.9 Hz), 136.78, 135.90, 135.49, 135.47,
133.00, 132.71, 130.01, 129.96, 129.91, 129.51, 129.18, 128.24,
127.76, 126.85 (d, J ) 34.4 Hz), 124.74, 105.60 (d, J ) 247.5
Hz), 67.01 (d, J ) 18.6), 66.43, 47.19, 41.64 (d, J ) 21.0 Hz),

Synthesis and Antiviral Activity of 4′-Thionucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4895
26.68, 19.09;HRMS (FAB)obsd, m/z762, calcd for C38H38F2N3O3-
SSeSi, m/z 762.15 (M + H)+; Anal. (C38H37F2N3O3SSeSi) C, H,
N, S.
See the general procedure for syn-elimination reaction. The
title compound 16 was obtained on 0.391-mmol scale in 58%
yield: [R]25 -72.1° (c 0.35, CH2Cl2); UV(CH2Cl2) λmax 262.5
D
nm; 13C NMR (CDCl3) δ 162.78, 154.79 (d, J ) 280.9 Hz),
150.59, 139.55, 135.59, 135.49, 132.79, 132.37, 130.13, 130.10,
127.92, 127.90, 110.70 (d, J ) 16.9 Hz), 104.01, 67.33, 60.74
(d, J ) 23.7 Hz), 46.88 (d, J ) 7.2 Hz), 26.87, 19.31; FAB-MS
obsd, m/z 483, calcd for C25H28FN2O3SSi, m/z 483.1496 (M +
H)+; Anal. (C25H27FN2O3SSi) C, H, N, S.
(+)-1-[(1R ,2R ,4S )-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-D-r ibofu r a n o-
syl]th ym in e (11). See the general procedure for condensation
reaction of the acetate 8 with pyrimidines. The title compound
11 was obtained on 0.850-mmol scale in 45% yield: [R]25
D
137.8° (c 0.42, CH2Cl2); UV(CH2Cl2) λmax (CH2Cl2) 266.0 nm;
13C NMR (CDCl3) δ 163.25, 150.93, 137.22 (d, J ) 6.1 Hz),
136.88, 135.48, 132.94, 132.90, 129.87, 129.46, 129.15, 127.74,
125.16, 110.67, 106.15 (d, J ) 247.03 Hz), 66.76, 65.85 (d, J )
18.3 Hz), 46.79, 42.04 (d, J ) 21.0 Hz), 26.70, 19.17, 12.59;
HRMS (FAB) obsd, m/z 655, calcd for C32H36FN2O3SSeSi, m/z
655.1287 (M + H)+; Anal. (C32H35FN2O3SSeSi‚0.4H2O) C, H,
N, S.
Gen er a l P r oced u r e for Tw o Su ccessive Dep r otection s
of P r otected Un sa tu r a ted Nu cleosid es. This is representa-
tive of the preparation of cytidine analogues 17 and 18.
(-)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]cytosin e (17). A solution of the
protected cytidine 13 (0.135 g, 0.225 mmol) in 15 mL of THF
was treated with 0.31 mL of 1 M TBAF in THF for 2 h. The
mixture was concentrated and filtered through a short pad of
silica gel. After the filtrate was concentrated, 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 (0.048 g, 0.186 mmol, 84% yield) as a white solid: mp 89-
91 °C (dec); [R]24D -208.3° (c 0.44, MeOH); UV(H2O) λmax 279.5
nm (ꢀ 19900, pH 2). 272.0 nm (ꢀ 15900, pH 7), 272.5 nm (ꢀ
16200, pH 11); Anal. (C9H10FN3O2S 0.2H2O) C, H, N, S.
(-)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]-5-flu or ocytosin e (18). See the gen-
eral procedure for two successive deprotections. The title
compound 18 was obtained on 0.296-mmol scale in 90% yield:
(+)-1-[(1R ,2R ,4S )-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-D-r ibofu r a n o-
syl]u r a cil (12). See the general procedure for condensation
reaction of the acetate 8 with pyrimidines. The title compound
12 was obtained on 0.766-mmol scale in 53% yield: [R]23
D
136.5° (c 0.36, CH2Cl2); UV(CH2Cl2) λmax 262.5 nm; 13C NMR
(CDCl3) δ 162.77, 150.77, 141.80 (d, J ) 5.0 hz), 136.94, 135.58,
135.51, 132.80, 132.77, 129.93, 129.61, 129.26, 127.79, 125.12,
106.24 (d, J ) 248.7 Hz), 102.31, 66.06, 65.99 (d, J ) 14.6 Hz),
46.86, 41.58 (d, J ) 21.8 Hz), 26.75, 19.18; Anal. (C31H33FN2O3-
SSeSi) C, H, N, S.
Gen er a l P r oced u r e for Syn -Elim in a tion Rea ction Us-
in 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-[(1R,4S)-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-t h io-â-D-r ib ofu r a -
n osyl]cytosin e (13). To a solution of compound 9 (0.229 g,
0.310 mmol) in 5 mL of CH2Cl2 was added a solution of mCPBA
(57-86%, 69 mg) in 5 mL of CH2Cl2 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 aq NaHCO3, dried over MgSO4,
filtered, and concentrated. The crude product was purified by
silica gel column chromatography with 40% EtOAc in hexanes
to give eliminated product 13 (0.107 g, 0.225 mmol, 72% yield)
mp 187-189 °C; [R]25 -171.3° (c 0.42, MeOH); UV(H2O) λmax
D
286.0 nm (ꢀ 8600, pH 2). 282.0 nm (ꢀ 7500, pH 7), 282.0 nm (ꢀ
6900, pH 11); Anal. (C9H9F2N3O2S) 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
thymidine analogue 19 is representative.
(-)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]th ym in e (19). A solution of com-
pound 15 (0.119 g, 0.235 mmol) in THF (10 mL) was treated
with 0.25 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 CH2Cl2 to give the
desired product 19 (0.055 g, 0.206 mmol, 88% yield) as a white
solid: mp 174-176 °C; [R]25D -46.5° (c 0.29, MeOH); UV(H2O)
λmax 268.5 nm (ꢀ 5700, pH 2). 268.5 nm (ꢀ 5500, pH 7), 269.0
nm (ꢀ 4500, pH 11); Anal. (C10H11FN2O3S 0.1Et2O) C, H, N, S.
(-)-1-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]u r a cil (20). See the general proce-
dure for the desilylation reaction. The title compound 20 was
obtained on 0.193-mmol scale in 89% yield: mp 184-185 °C;
[R]25D -136.4° (c 0.47, MeOH); UV(H2O) λmax 263.0 nm (ꢀ 9100,
pH 2). 263.0 nm (ꢀ 9700, pH 7), 263.5 nm (ꢀ 7800, pH 11); Anal.
(C9H9FN2O3S‚0.3MeOH) C, H, N, S.
as a foam: [R]25 -140.2° (c 0.366, CH2Cl2); UV(MeOH) λmax
D
308 nm; 13C NMR (CDCl3) δ 171.09, 166.78, 162.14, 155.06,
155.04 (d, J ) 280.8 Hz), 144.49, 135.58, 135.50, 133.19,
132.96, 132.76, 132.43, 130.11, 128.97, 127.89, 127.58, 111.29
(d, J ) 16.6 Hz), 98.40, 67.75, 61.91 (d, J ) 23.6 Hz), 46.83 (d,
J ) 7.2 Hz), 26.82, 19.24; Anal. (C32H32FN3O3SSi) C, H, N, S.
(-)-N4-Ben zoyl-1-[(1R,4S)-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-t h io-â-D-r ib ofu r a -
n osyl]-5-flu or ocytosin e (14). See the general procedure for
syn-elimination reaction. The title compound 14 was obtained
on 0.465-mmol scale in 69% yield: [R]25D -150.5° (c 0.345, CH2-
Cl2); UV(CH2Cl2) λmax 331.0 nm; 13C NMR (CDCl3) δ 152.88
(d, J ) 281.3 Hz), 152.08 (d, J ) 19.1 Hz), 147.13, 140.42 (d,
J ) 241.0 Hz), 135.86, 135.59, 135.50, 133.17, 132.59, 132.39,
130.18, 130.15, 130.11, 128.33, 127.93, 124.05 (d, J ) 36.2 Hz),
111.24 (d, J ) 16.5 Hz), 67.77, 61.85 (d, J ) 23.54 hz), 47.05
(d, J ) 7.1 Hz), 26.79, 19.23; FAB-MS obsd, m/z 604, calcd for
Gen er a l P r oced u r e for Con d en sa tion Rea ction of th e
Aceta te 8 w ith P u r in es. The preparation of 6-chloropurine
derivative 21 is representative.
(+)-9-[(1R ,2R ,4S )-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-D-r ibofu r a n o-
syl]-6-ch lor op u r in e (21). A mixture of 6-chloropurine (0.947
g, 6.12 mmol) and ammonium sulfate (0.135 g, 1.02 mmol) in
60 mL of HMDS was refluxed for 5 h. HMDS was evaporated
to give a yellow solid. To this flask containing the silylated
6-chloropurine was added a solution of the acetate 8 (1.2 g,
2.04 mmol) in 1,2-dichloroethane (30 mL). The resulting slurry
was cooled to -25 °C, and TMSOTf (0.74 mL, 4.08 mmol) was
added dropwise at -25 °C. The reaction mixture was stirred
for 3 h at -25 f -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 aq 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 (1.01 g, 1.48 mmol, 73% yield) as a pale
C
32H32FN3O3SSi, m/z 604.1823 (M + H)+; Anal. (C32H31F2N3O3-
SSi) C, H, N, S.
(-)-1-[(1R,4S)-5-O-(ter t-Bu tyldiph en ylsilyl)-2,3-dideoxy-
2,3-d id eh yd r o-2-flu or o-4-t h io-â-D-r ib ofu r a n osyl]t h ym -
in e (15). See the general procedure for the syn-elimination
reaction. The title compound 15 was obtained on 0.375-mmol
scale in 69% yield: [R]26 -43.2° (c 0.34, CH2Cl2); UV(CH2-
D
Cl2) λmax 266.0 nm; 13C NMR (CDCl3) δ 163.47, 154.97 (d, J )
281.2 Hz), 150.78, 135.52, 135.43, 134.52, 132.79, 132.51,
130.03, 127.84, 112.65, 110.45 (d, J ) 16.8 Hz), 68.23, 60.49
(d, J ) 23.7 Hz), 46.74 (d, J ) 7.1 Hz), 26.74, 19.23, 12.54;
FAB-MS obsd, m/z 497, calcd for C26H30FN2O3SSi, m/z 497.1652
(M + H)+; Anal. (C26H29FN2O3SSi‚0.6H2O) C, H, N, S.
(-)-1-[(1R,4S)-5-O-(ter t-Bu tyldiph en ylsilyl)-2,3-dideoxy-
2,3-dideh ydr o-2-flu or o-4-th io-â-D-r ibofu r an osyl]u r acil (16).
yellow foam: [R]22 75.4° (c 1.08, CHCl3); UV(CH2Cl2) λmax
D
264.5 nm; 13C NMR (CDCl3) δ 151.84, 150.87, 144.98 (d, J )

4896 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Chong et al.
5.7 Hz), 136.32, 135.60, 135.53, 132.85, 132.76, 131.21, 130.02,
129.96, 129.44, 128.99, 128.79, 127.82, 124.34, 105.10 (d, J )
248.1 Hz), 66.64, 65.68 (d, J ) 20.3 Hz), 47.33, 41.68 (d, J )
20.7 Hz), 26.74, 19.17; Anal. (C32H32ClFN4OSSeSi) C, H, N,
S.
n osyl]-6-ch lor op u r in e (29) a n d 6-a m in o-9-[(1R,4S)-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-D-r ibofu r a n osyl]-2-flu or op u r in e (31). Dry
ammonia gas was bubbled into a stirred solution of a mixture
of compounds 27 and 28 (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 29 (0.190 g, 0.352
(+)-9-[(1R ,2R ,4S )-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-D-r ibofu r a n o-
syl]-6-ch lor o-2-flu or op u r in e (26). See the general procedure
for condensation reaction of the acetate 8 with purines. The
title compound 26 was obtained on 1.70-mmol scale in 67%
mmol, 45% yield) and a mixture of compound 31 and its ∆1,2
-
D
isomer (0.116 g, 0.22 mmol, 28% yield): for compound 29; [R]24
yield: [R]23 81.0° (c 1.37, CHCl3); UV(CHCl3) λmax 270.0 nm;
D
-70.4° (c 0.40, CH2Cl2); UV(CH2Cl2) λmax 303.5 nm; 13C NMR
(CDCl3) δ 159.11, 154.78 (d, J ) 281.2 Hz), 153.48, 151.49,
139.92, 135.57, 135.48, 132.76, 132.48, 130.12, 130.09, 127.91,
127.88, 125.30, 109.95 (d, J ) 16.5 Hz), 68.01, 58.33 (d, J )
13C NMR (CDCl3) δ 157.00 (d, J ) 220.8 Hz), 153.53 (d, J )
17.1 Hz), 152.4 (d, J ) 17.3 Hz), 145.53, 136.26, 135.59, 135.52,
132.80, 132.70, 130.03, 129.97, 129.79 (d, J ) 4.9 Hz), 129.49,
129.00, 127.83, 124.19, 104.99 (d, J ) 248.5 Hz), 66.53, 65.90
(d, J ) 20.7 Hz), 47.36, 41.48 (d, J ) 20.7 Hz), 26.75, 19.17;
Anal. (C32H31ClF2N4OSSeSi‚0.2hexane) C, H, N, S.
24.0 Hz), 47.46 (d, J ) 7.1 Hz), 26.76, 19.19; Anal. (C26H27
-
ClFN5OSSi) C, H, N, S: a mixture of compound 31 and its
∆
1,2-isomer; UV(CHCl3) λmax 261.0 nm.
9-[(1R,4S)-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-D-r ibofu r a n osyl]-6-ch lo-
r op u r in e (22) a n d 9-[(4S)-5-O-(ter t-Bu tyld ip h en ylsilyl)-
2,3-d id e o x y -1,2-d id e h y d r o -2-flu o r o -4-t h io -â-D -r ib o -
fu r a n osyl]-6-ch lor op u r in e (23). See the general procedure
for syn-elimination reaction. A mixture of title compounds 22
and 23 (3:1) were obtained on 1.48-mmol scale in 86% yield
as a pale yellow foam: UV(CH2Cl2) λmax 264.5 nm; Anal.
(C26H26ClFN4OSSi) C, H, N, S.
9-[(1R,4S)-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-D-r ibofu r an osyl]-6-ch lor o-
2-flu or op u r in e (27) a n d 9-[(4S)-5-O-(ter t-Bu tyld ip h en yl-
s ily l)-2,3-d id e o x y -1,2-d id e h y d r o -2-flu o r o -4-t h io -â-D -
r ibofu r an osyl]-6-ch lor o-2-flu or opu r in e (28). See the general
procedure for syn-elimination reaction. A mixture of title
compounds 27 and 28 were obtained on 1.14-mmol scale in
71% yield as a pale yellow foam: UV(CH2Cl2) λmax 266.0 nm;
Anal. (C26H25ClF2N4OSSi) C, H, N, S.
(-)-9-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]gu a n osin e (30). See the procedure
for the preparation of compound 25. The title compound 30
was obtained on 0.29-mmol scale in 61% yield: mp 210 °C
(dec); [R]25 -73.7° (c 0.152, DMSO); UV(H2O) λmax 254.0 nm
D
(ꢀ 11,600, pH 2). 255.0 nm (ꢀ 12,400, pH 7), 265.0 nm (ꢀ 12,-
500, pH 11); 13C NMR (DMSO-d6) δ 160.12, 157.49 (d, J )
278.1 Hz), 157.44, 154.47, 138.61, 119.83, 114.34 (d, J ) 17.0
Hz), 67.90, 60.99 (d, J ) 24.5 Hz), 51.73 (d, J ) 7.6 Hz), 44.06
(d, J ) 9.1 Hz); Anal. (C10H10FN5O2S) C, H, N, S.
(+)-9-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]-2-flu or oa d en osin e (32). See the
general procedure of desilylation. The title compound 32 was
obtained on 0.22-mmol scale in 70% yield: mp >300 °C; [R]26
D
17.1° (c 0.15, MeOH); UV(H2O) λmax 261.0 nm (ꢀ 12700, pH 2).
261.0 nm (ꢀ 12900, pH 7), 260.5 nm (ꢀ 13100, pH 11); 13C NMR
(MeOH-d4) δ 160.82 (d, J ) 269.8 Hz), 159.67, 159.02, 155.19,
152.65, 141.63, 118.69, 111.99 (d, J ) 17.1 Hz), 65.96 (d, J )
1.8 Hz), 60.77 (d, J ) 24.3 Hz), 50.04; Anal. (C10H9F2N5OS) C,
H, N, S.
(-)-9-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
th io-â-D-r ibofu r a n osyl]a d en in e (24). A mixture of com-
pound 22 and compound 23 (0.342 g, 0.65 mmol) was treated
with methanolic ammonia at 80 °C for 12 h using steel bomb.
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 0.3 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 24
(0.099 g, 0.37 mmol 57% yield) as a white solid: mp 190-191
Molecu la r Mod elin g Stu d y. (a) Conformational analy-
sis: The initial conformations of d-â-2′-F-d4C and D-â-2′-F-
4′-S-d4C 17 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 workstation. The
initial conformations were cleaned up and geometry-optimized
through quantum mechanical ab initio calculations using RHF/
3-21G* basis in Spartan 5.1.1. (b) 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).30 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 template overhang
was modified to the base complementary to the incoming
NRTIs. Thus, the adenine moiety which was in the original
X-ray structure (1rtd)30 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.30 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 enzyme site from the biopolymer module in Sybyl.
Fluorine parameters were obtained from the literatures36,37
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/
°C; [R]25 -33.7° (c 0.164, 7:1 CH2Cl2:MeOH); UV(H2O) λmax
D
257.5 nm (ꢀ 9,600, pH 2). 259.0 nm (ꢀ 9,500, pH 7), 259.0 nm
(ꢀ 9,800, pH 11); 13C NMR (MeOH-d4) δ 157.43, 156.31 (d, J )
277.9 Hz), 153.98, 150.51, 141.22, 120.10, 111.65 (d, J ) 17.1
Hz), 65.63, 60.38 (d, J ) 24.7 Hz), 49.73 (d, J ) 7.7 Hz); Anal.
(C10H10FN5OS) C, H, N, S.
(+)-9-[(1R,4S)-2,3-Did eoxy-2,3-d id eh yd r o-2-flu or o-4-
t h io-â-D-r ib ofu r a n osyl]h yp oxa n t h in e (25). A mixture of
compound 22 and compound 23 (0.323 g, 0.62 mmol) in
anhydrous MeOH (20 mL) was treated with 2-mercaptoethanol
(0.17 mL, 2.46 mmol) and NaOMe (0.136 g, 2.52 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 driness. The crude product was treated with
0.68 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 25 (0.135 g, 0.50 mmol, 81%
yield) as a white solid: mp 143-145 °C (dec); [R]27 21.5° (c
D
0.132, 7:1, CH2Cl2:MeOH); UV(H2O) λmax 247.5 nm (ꢀ 10200,
pH 2). 248.0 nm (ꢀ 10000, pH 7), 253.5 nm (ꢀ 10700, pH 11);
13C NMR (MeOH-d4) δ 158.85, 156.22 (d, J ) 248.4 Hz), 150.04,
147.13, 140.66, 125.29, 111.77 (d, J ) 17.0 Hz), 65.50, 60.51
(d, J ) 24.8 Hz), 49.83 (d, J ) 7.3 Hz); Anal. (C10H9FN4O2S)
C, H, N, S.
(-)-2-Am in o-9-[(1R,4S)-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-t h io-â-D-r ib ofu r a -

Synthesis and Antiviral Activity of 4′-Thionucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4897
mol. The annealed enzyme-inhibitor complexes were mini-
mized by using Kollman-All-Atom Force Field until iteration
number reached 5000.
(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 concen-
tration (IC50) was determined from the concentration-response
curve using the median effect method.38
Ad en osin e Dea m in a se Kin etics 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 units of adenosine deaminase (EC 3.5.4.4. from calf
intestinal mucosa). The assays were monitored with a UV
spectrometer at 265 nm. Initially, the qualitative assays were
performed with d-â-2′-F-4′-S-d4A 24 (200 µM) in the presence
of 0.24 units of adenosine deaminase for 120 min to determine
whether it is a substrate of this enzyme. The concentration
(ct) of each 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.35 Initial hydrolysis rates
for each substrate concentration were measured manually
through graphical curve fitting of the concentration vs time
data for reaction of each substrate. From Lineweaver-Burke
plot of these initial rates, Vmax (maximum velocity) and KM
(Michaelis constant) were obtained for each substrate and kcat
was also calculated with 0.15 units of the enzyme (MW 33000)
in 2 mL of buffer solution. The t1/2 of D-â-2′-F-4′-S-d4A 24 and
adenosine were also measured at 20 µM with 0.15 unit of the
enzyme.
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 infetious
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 TCID 50/6 × 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 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 supernatants were
collected for reverse transcriptase (RT) activity.
Ack n ow led gm en t. This research was supported by
the U.S. Public Health Service Grant (AI32351) from
the National Institutes of Health and the Department
of Veterans Affairs.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and el-
emental analysis data for compounds 9-22, 24-27, 29-30,
and 32. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Supernatants were centrifuged at 12000 rpm for 2 h to pellet
the virus. The pellet was solubilized with vortexing in 100 µL
of virus solubilization buffer (VSB) containing 0.5% Triton
X-100, 0.8 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride,
20% glycerol, and 0.05 M Tris, pH 7.8. Ten microliters of each
sample was added to 75 µL of RT reaction mixture (0.06 M
Tris, pH 7.8, 0.012 M MgCl2, 0.006 M dithiothreitol, 0.006 mg/
mL poly (rA)n oligo (dT)12-18, 96 µg/mL dATP, and 1 µM of 0.08
mCi/mL 3H-thymidine triphosphate (Moravek Biochemicals,
Brea, CA) and 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 pyrophosphate. The acid-insoluble
product was harvested onto filter paper using a Packard
Harvester (Meriden, CT), and the RT activity was read on a
Packard Direct Beta Counter (Meriden, CT). The RT results
were expressed in counts per minute (CPM) per milliliter. The
antiviral 50% effective concentration (EC50) and 90% effective
concentration (EC90) were determined from the concentration-
response curve using the median effect method.38
(5) Lin, T.-S.; Schinazi, R. F.; Prusoff, W. H. Potent and selective
in vitro activity of 3′-deoxyadenosine, deoxythymidine-2′-ene (3′-
deoxy-2′,3′-didehydrothymidine) against human immunodefi-
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Bergogne, M. C.; Aubertin, A.-M.; Kirn, A.; Imbach, J .-L.; Anti-
human immujnodeficiency virus activities of the â-L-enantiomer
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