Structure-activity relationships of 2′-fluoro-2′,3′-unsaturated d-nucleosides as anti-hiv-1 agents

Article abstract of DOI:10.1021/jm010418n

We studied the structure-activity relationships of a series of 2′-fluoro-2′,3′-unsaturated D-nucleosides against HIV-1 in human peripheral blood mononuclear (PBM) cells. The target compounds 10-21 and 28-33 were prepared by N-glycosylation of the acetate

Full text of DOI:10.1021/jm010418n

J . Med. Chem. 2002, 45, 1313-1320
1313
Str u ctu r e-Activity Rela tion sh ip s of 2′-F lu or o-2′,3′-u n sa tu r a ted D-Nu cleosid es a s
An ti-HIV-1 Agen ts
Kyeong Lee,† Yongseok Choi,† Giuseppe Gumina,† Wen Zhou,† Raymond F. Schinazi,‡ and Chung K. Chu*,†
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, rdag, Athens, Georgia 30602, and
Emory University School of Medicine/ Veterans Affairs Medical Center, Decatur, Georgia 30033
Received September 7, 2001
We studied the structure-activity relationships of a series of 2′-fluoro-2′,3′-unsaturated
D-nucleosides against HIV-1 in human peripheral blood mononuclear (PBM) cells. The target
compounds 10-21 and 28-33 were prepared by N-glycosylation of the acetate 4, which was
readily prepared from 2,3-O-isopropylidene-D-glyceraldehyde in five steps. Among the newly
synthesized nucleosides, 2-amino-6-chloropurine (11), adenine (14), inosine (16), guanine (18),
2,6-diaminopurine (20), and 5-fluorocytosine (30) derivatives were found to exhibit interesting
anti-HIV activities with EC50 values of 4.3, 0.44, 1.0, 2.6, 3.0, and 0.82 µM, respectively. The
implications for drug resistance of the titled nucleosides with respect to lamivudine-resistant
variants (M184V) were also examined, and no significant cross-resistance with the variants
was observed with the D-series.
In tr od u ction
type moiety (uracil, cytosine, and thymine deriva-
tives).25-27 However, relatively little effort has been
devoted to the synthesis of purine analogues with 2′-
fluorinated ene-type modification, presumably due to
the difficulty to access the corresponding purine nucleo-
sides. Recently, we have reported the synthesis and
anti-HIV and anti-HBV (hepatitis B virus) activities of
2′-fluoro-2′,3′-unsaturated L-nucleosides. Of significance
was that â-L-2′-F-d4C, â-L-2′-F-d4FC, and â-L-2′-F-d4A
exhibited potent activities against HIV-1 and HBV.28
Encouraged by these promising biological results of
L-enantiomers, it was of interest to synthesize hitherto
unknown purine D-nucleosides for the study of the
structure-activity relationship. Herein, we report the
synthesis and anti-HIV activities as well as the activity
against a lamivudine-resistant mutant. It is critical to
discover antiviral agents that can demonstrate effective
antiviral activity against these resistant viruses (M184V
mutant) for combination chemotherapy since lamivu-
dine is currently widely used in the treatment of HIV
infection.
Nucleoside reverse transcriptase inhibitors (NRTIs)
continue to be the cornerstone of anti-HIV (human
immunodeficiency virus) therapy. Side effects of the
existing regimens, however, have been one of the
drawbacks.1-4 Additionally, the efficacy of the NRTIs
has been compromised by the development of resistant
variants.5-7 Approaches to address these problems
include using drugs with different resistance profiles
and mechanisms, such as a combination therapy of 3TC,
AZT, and indinavir.8 Indeed, it has become increasingly
clear that in order to achieve the greatest benefit in
terms of reduction of viral load and delay of resistance,
a combination of multiple-drug regimens is required.9
Therefore, there is a need for new RT inhibitors with
high potency and different resistance profiles, thereby
providing a greater choice of drug combinations.
Since the discovery of 2′,3′-dideoxy (ddN)- and 2′,3′-
didehydro-2′,3′-dideoxynucleosides (d4N), including 2′,3′-
dideoxycytidine (ddC),10,11 2′,3′-dideoxyinosine (ddI),11-16
2′,3′-didehydro-3′ deoxythymidine (d4T),17,18 and aba-
cavir19,20 as antiviral agents, a number of modified
dideoxynucleoside analogues have been synthesized and
identified as anti-HIV agents, such as L-2′,3′-didehydro-
2′,3′-dideoxycytidine (â-L-d4C), and its 5-fluoro congener
(â-L-Fd4C).21 It has also been found that the introduc-
tion of a fluorine atom into the 2′-position of nucleosides
in general, and of purine nucleosides in particular,
results in the stabilization of the glycosyl bond and
confers interesting biological activity as exemplified by
2′-â-fluoro-2′,3′-dideoxyadenosine (F-ddA).22-24 So far,
there have been limited reports on the synthesis of
pyrimidine nucleosides possessing a 2′-fluorinated ene-
Resu lts a n d Discu ssion
Ch em istr y. Following previously described method-
ologies,28-30 we have synthesized 2′-vinyl fluoride con-
taining D-nucleoside analogues (Scheme 1). 2,3-O-Iso-
propylidene-D-glyceraldehyde, prepared by oxidative
cleavage of 1,2:5,6-di-O-isopropylidene-D-mannitol, was
reacted with triethyl R-fluorophosphonoacetate to give
alkene 1, mainly as the E-isomer,32 along with a small
amount of Z-isomer, which was subsequently trans-
formed to the 2-fluorobutenolide intermediate 2.31-33
Selective reduction of 2 to lactol 3 by DIBAL-H in CH2-
Cl2 followed by treatment with acetic anhydride and
triethylamine gave the key intermediate 4. Condensa-
tion of acetate 4 with various trimethylsilylated purines
and pyrimidines was carried out at room temperature
in 1,2-dichloroethane (DCE). Completion of the reaction
took ca. 1 h in the presence of trimethylsilyl trifluo-
romethane sulfonate (TMSOTf) as a catalyst. The
* Corresponding author: Dr. C. K. Chu, Distinguished Research
Professor, The University of Georgia, College of Pharmacy, 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/jm010418n CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/09/2002

1314 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Lee et al.
Sch em e 1a
a
Reagents: (a) (EtO)2P(O)CHFCO2Et, NaHMDS, THF, -78 °C; (b) c-HCl, EtOH; (c) TBDMSCl, imidazole, CH2Cl2; (d) DIBAL-H, CH2Cl2,
-78 °C; (e) Ac2O, TEA, CH2Cl2; (f) silylated 6-Cl-purine, TMSOTf, DCE; (g) silylated 6-Cl-2-F-purine, TMSOTf, DCE; (h) TBAF, CH3CN;
(i) NH3/DME; (j) NH3/MeOH, 90 °C; (k) HSCH2CH2OH, NaOMe, reflux; (l) silylated N4-Bz-cytosine derivatives, TMSOTf, DCE; (m) NH3/
MeOH.
coupling reactions provided a mixture of R- and â-ano-
mers in 50-58% yield. For instance, acetate 4 was
reacted with silylated 6-chloropurine to give 5′-tert-
butyldimethylsiyl protected nucleosides 5 as an ano-
meric mixture. Deprotection of 5 with tetrabutylammo-
nium fluoride (TBAF) in acetonitrile facilitated the
separation of isomers on silica gel (3% MeOH/CH2Cl2)
to provide the precursors 8 (â-isomer) and 9 (R-isomer)
for adenine and hypoxanthine derivatives. Acetonitrile
gave smoother conversion and better yields than tet-
rahydrofuran (THF) during deprotections of the silyl
groups. Conversion of the â-6-chloropurine derivative
8 to the â-adenine derivative by ammonolysis in a steel
bomb at 90-100 °C produced 14 in 90% yield. Com-
pound 8 was also converted to the â-inosine analogue
16 by refluxing with mercaptoethanol and sodium
methoxide in methanol. For the preparation of 2,6-
disubstituted purine nucleosides, the acetate 4 was
condensed with silylated 2-fluoro-6 chloropurine in DCE
to give silyl protected nucleosides 6 and 7, which were
readily separated by column chromatography (12%
EtOAc/hexanes). Compound 6 was then converted to the
2-fluoroadenine derivative 10 and the 6-amino-2-chlo-
ropurine derivative 11 by treatment with ammonia in
ethylene glycol dimethyl ether (DME) followed by TBAF
in acetonitrile. The guanine derivative 18 was readily
prepared from compound 11 analogously to the inosine
analogue 16. Compound 11 was also further converted
to the 2,6-diaminopurine derivative 20 by ammonolysis.
The R-purine nucleosides 12, 13, 15, 17, 19, and 21 were
prepared analogously to their corresponding â-isomers.
Similarly, protected cytosines and their 5-substituted
congeners, 22-27, were synthesized by N-glycosylation
of the key intermediate 4 with appropriate N4-benzo-
ylated bases. Removal of the tert-butyldimethylsiyl
group of compounds 22-27, followed by ammonolysis
in the presence of methanolic ammonia, afforded the
desired free nucleosides 28-33, as described in Scheme
1. Assignment of the structures of the newly synthesized
nucleosides was based on the comparison of the chemi-
cal, physiological, and optical data to those of the
corresponding L-series.28
All the spectral properties of the newly synthesized
compounds (Table 1) were in good agreement with those
published for their corresponding L-enantiomers.
An tivir a l Activity. Anti-HIV activities of the syn-
thesized nucleoside analogues were determined in hu-
man peripheral blood mononuclear (PBM) cells acutely
infected with HIV-1 and compared to those of AZT and
FTC. The cytosine derivative 28, which has been
published by several laboratories,25-27 was also included
to compare the antiviral activities in different cell lines.
Results in Table 2 indicate the median antiviral potency
and growth inhibition of the newly synthesized com-
pounds expressed as EC50 and IC50, respectively. It was
found that D-2′-fluorinated unsaturated nucleosides (D-

SAR of 2′-Fluoro-2′,3′-unsaturated D-Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1315
Ta ble 1. Physical Properties of the Synthesized Nucleosides
no.
mp °C (solv)a
[R]D, deg
formula
anal.
8
9
128-130 (B)
157-159 (A)
188-190 (C)
157-160 (dec, B)
183-185 (dec, C)
148-150 (C)
>191 (dec, C)
173-174 (B)
129-131 (dec, B)
>202 (dec, B)
198-201 (B)
>212 (dec, B)
146-148 (B)
184-186 (B)
172-174(D)
+40.79 (c 0.18, MeOH)
-162.32 (c 0.13, MeOH)
+56.62 (c 0.13, CHCl3)
-10.99 (c 0.17, MeOH)
-154.01 (c 0.19, MeOH)
-134.78 (c 0.12, MeOH)
+53.91 (c 0.12, MeOH)
-163.83 (c 0.34, MeOH)
+52.77 (c 0.27, MeOH)
-159.85 (c 0.19, MeOH)
-24.64 (c 0.02, DMF)
-57.16 (c 0.09, DMF)
+7.18 (c 0.23, MeOH)
-102.18 (c 0.11, MeOH)
+21.94 (c 0.68, MeOH)
-157.86 (c 0.30, MeOH)
+34.35 (c 0.15, MeOH)
-153.51 (c 0.11, MeOH)
+39.65 (c 0.33, MeOH)
-169.31 (c 0.37, MeOH)
C
C
10H8FClN4O2‚0.1EtOH
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
10H8FClN4O2
10
11
12
13
14
15
16
17
18
19
20
21
28
29
30
31
32
33
C10H9F2N5O2
C
C
C
C
C
C
C
C
C
C
C
10H9FClN5O2
10H9F2N5O2
10H9FClN5O2‚0.3H2O
10H10FN5O2
10H10FN5O2
10H10FN4O3
10H10FN4O3
10H9FN5O3
10H9FN5O3‚0.3H2O
10H11FN6O2‚0.1CH2Cl2
10H11FN6O2‚0.4H2O
C9H10FN3O3
C9H10FN3O3
C9H9F2N3O3
C9H9F2N3O3
C
C
182-183 (B)
201-203 (D)
158-161 (B)
179-182 (dec, B)
180-182 (B)
10H12FN3O3
10H12FN3O3
a
Solvents: A, EtOAc-hexanes; B, CH2Cl2-MeOH; C, MeOH; D, hexanes-MeOH-CH2Cl2.
Ta ble 2. Median Effective (EC50) and Inhibitory (IC50
Concentration of D-2′-F-d4Ns in Human PBM Cells and
Cytotoxicity in Vero and CEM Cells
)
â-isomer 10 was inactive. These data, not fully explain-
able at this stage, were confirmed by repeated biological
essays and stereochemical assignments of the two
isomers. Furthermore, other examples where R-nucleo-
sides have shown anti-HIV activity have been re-
ported.28,35 Interestingly, it was found that in the
D-series several purine derivatives, such as the adenine
14 and hypoxanthine 16, show significant antiviral
activity whereas, in the corresponding L-series,29 the
pyrimidine derivatives such as the cytosine and 5-fluo-
rocytosine are the most potent. The anti-HIV activity
and cytotoxicity of cytosine derivative 28 were in ac-
cordance with the reported data.25-27 In the case of the
5-fluorocytosine derivative 30, however, the L-enanti-
omer exhibited higher antiviral potency than the newly
synthesized D-counterpart (0.17 vs 0.82 µM). It is
interesting to note that 5-fluoro substitution on the
cytosine base moiety retained potent antiviral activity
compared to the parent derivative. In contrast, the
5-methylcytosine derivatives showed no antiviral activ-
ity.
EC50 (µM)
PBM Vero CEM
HIV-1 cells cells cells
>100 >100 >100 >100
IC50 (µM)
compd
B
10
11
12
13
14
15
16
17
18
19
20
21
28
29
30
31
32
33
AZT
2-F-adenine (â)
6-Cl-2-NH2-purine (â) 4.3
98.8
41.2
77.4
68.3
>100 >100
2-F-adenine (R)
3.97
16.1 28.3
>100 30.2
>100 57.6
6-Cl-2-NH2-purine (R) g75
adenine (â)
adenine (R)
0.44
>100 >100 >100 >100
hypoxanthine (â)
hypoxanthine (R)
guanine (â)
1.0
92.7
87.7
>100
>100 >100 >100 >100
2.6
18.3
3.0
>100 >100 >100
>100 >100 >100
>100 >100 >100
guanine (R)
2,6-di-NH2-purine (â)
2,6-di-NH2-purine (R)
cytosine (â)25-27
cytosine (R)
>100 >100 >100 >100
2.6 7.3 >100 35.3
>100 >100 >100 >100
0.82 >100 >100 >100
5-F-cytosine (â)
5-F-cytosine (R)
5-Me-cytosine (â)
5-Me-cytosine (R)
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
0.004 >100 29.0
14.3
Although both 3TC and FTC showed potent antiviral
activities against HIV and HBV, several investigators
have reported rapid emergence of resistant viruses, in
vitro as well as in clinic, characterized by a mutation
at the 184 M codon (ATG) of the reverse transcriptase
(RT).36-39 However, this mutation had no effects on the
susceptibility toward AZT or nevirapine and minimal
effects on susceptibility to ddI and ddC. Huang et al.
have described the structure of HIV-1 RT complexed
with thymidine triphosphate and its implications in
drug resistance, and they suggested a correlation be-
tween mutations and the chemical structure of the
inhibitor.40 The M184V mutant can influence both the
triphosphate of the inhibitor as well as the primer
terminus in the active site. It is reported that interfer-
ence with valine at 184 is enhanced by the oxathiolane
ring with respect to the 2′-deoxynucleoside triphosphate,
which may account for the decreased antiviral activity
of 3TC. In view of these findings, it was anticipated that
â-D-2′-F-d4Ns may not be significantly affected by point
mutation at M184 by the sugar moiety. Preliminary
biological evaluations suggest this is indeed the case.
2′-F-d4N) had significant anti-HIV-1 activities. The
toxicity of these nucleosides has been assessed in human
PBM, Vero, and CEM cells (Table 2). No significant
toxicity was detected for most of the evaluated com-
pounds. Within the class of â-D-2′-F-d4N, purine deriva-
tives (adenine, hypoxanthine, 2-fluoroadenine, 6-chloro-
2-aminopurine, 2,6-diaminopurine, and guanine) showed
moderate to potent anti-HIV-1 activities, including two
R-anomers, and generally they were more potent than
their L-counterparts.28 The adenine derivative 14 ex-
hibited 10-fold greater potency than its corresponding
L-enantiomer (EC50 0.44 µM vs 4.7 µM). One probable
explanation is that these D-purine derivatives may be
better substrates for nucleoside kinases than their
corresponding L-nucleosides.34 Particularly, in the case
of â-guanine and its 6-chloro-congener, the D-series (18
and 11) was found to exhibit moderate anti-HIV activity
(EC50 2.6 and 4.3 µM, respectively), while the L-
nucleosides showed no antiviral activity against HIV-1
(>100 µM).28 Surprisingly, R-2′-F-adenine 12 displayed
potent anti-HIV activity (EC50 3.97 µM), whereas its

1316 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Lee et al.
Ta ble 3. Activity of Selected â-D-2′-F-d4Ns against M184V Pitt
Virus in Human PBM Cells
Exp er im en ta l Section
Melting points were determined on a Mel-temp II laboratory
device and are uncorrected. Nuclear magnetic resonance
spectra were recorded on a Bruker AMX400 400 MHz spec-
trometer with tetramethylsilane as the internal reference. UV
spectra were obtained on a Beckman DU 650 spectrophotom-
eter. Optical rotations were measured on a J asco DIP-370
digital polarimeter. Elemental analyses were performed by
Atlantic Microlab, Inc., Norcross, GA. All reactions were
monitored using thin-layer chromatography on Analtech 200
mm silica gel GF plates.
EC50
XxBRU Pitta M184V Pittb
compd
14
16
18
30
AZT
(-)-FTC
B
(µM)
(µM)
FIc
adenine
hypoxanthine
guanine
0.46
2.8
4.4
3.1
0.005
0.0046
0.8
2.5
11.2
9.61
0.004
21.5
1
1
3
3
1
5-F-cytosine
4674
(S)-(-)-4-[(ter t-Bu tyldim eth ylsilyloxy)m eth yl]-2-flu or o-
2-bu ten -4-olid e (2). A solution of triethyl 2-fluorophospho-
noacetate (40.5 g, 167.5 mmol) in THF (70 mL) was cooled to
-78 °C, and sodium bis(trimethylsilyl)amide (1.0 M solution
in THF, 167.5 mL, 167.5 mmol) was added dropwise. The
mixture was kept for 30 min at -78 °C, and then a solution of
D-(R)-glyceraldehyde acetonide (19.79 g, 152 mmol) in THF (70
mL) was added. After being stirred for 1 h at -78 °C, the
reaction mixture was treated with aqueous NH4Cl and ex-
tracted with ether. The organic phase was washed with brine,
dried over MgSO4, and concentrated. The residue was chro-
matographed on silica gel to give a mixture of E-1 and Z-1
a
b
(-)-FTC-sensitive isolates. (-)-FTC resistant isolates. c FI
(fold increase) EC50 ) EC50 data resistant virus/EC50 data from
xxBRU Pitt virus.
Ta ble 4. Kinetic Parameters of the Adenosine Analogues with
Mammalian Adenosine Deaminase Relative to Adenosine
compd
KM (M)
t1/2
14
2.3 × 10-5
41.6 min
NDb
25 s
â-L-2′-F-d4A28
NSa
adenosine
5 × 10-6
a
b
â-L-2′-F-d4A is not a substrate of adenosine deaminase. Not
1
(9:1 by H NMR) as a yellowish oil (34.4 g, 97.1%).
determined.
A solution of E/Z-1 (10.2 g, 46.7 mmol) in 60 mL of
anhydrous EtOH was treated with 15 mL of concentrated HCl
and stirred at room temperature for 2.5 h. The solvent was
removed in vacuo and the residue coevaporated with toluene
(3 × 200 mL) to give a lactone intermediate, along with
uncyclized ester intermediate. The resulting yellowish syrup
was used as such for the next reaction without further
purification. tert-Butyldimethylsilyl chloride (17.5 g, 116.5
mmol) and imidazole (8.0 g, 117 mmol) were added to a
solution of hydrolyzed products in CH2Cl2 (150 mL), and the
mixture was reacted overnight at room temperature. The
resulting mixture was treated with ice and diluted with CH2-
Cl2. The organic layers were combined, dried over MgSO4, and
concentrated to dryness. Purification on silica gel (4% EtOAc/
hexanes) provided 18.89 g (70.5% from D-(R)-glyceraldehyde
acetonide) of a crystalline solid 2: mp 48-50 °C; [R]27D -105.9
(c 0.5, CHCl3). Anal. Calcd for C10H19FO3Si: C, 53.63; H, 7.77.
Found: C, 53.68; H, 7.82.
1-Acetyl-4-[(ter t-bu tyldim eth ylsilyloxy)m eth yl]-2-flu or o-
2-bu ten -4-olid e (4). 2-Fluorobutenolide 2 (10.47 g, 42.5 mmol)
was dissolved in 150 mL of CH2Cl2 under a nitrogen atmo-
sphere, and then the mixture was cooled to -78 °C and treated
with 1.0 M solution of DIBAL-H in CH2Cl2 (63.6 mL, 63.6
mmol). The resulting mixture was reacted for 2 h at -78 °C.
The cold mixture was treated with dilute nitric acid, washed
with water, and dried (Na2SO4). Evaporation of the solvent
gave lactol intermediate 3 (anomeric mixture) as a pale
yellowish oil (8.6 g, crude yield 82%), which was used as such
for the next step.
To a solution of the lactol 3 (5.18 g, 20.85 mmol) and
triethylamine (11.6 mL, 83.4 mmol) in CH2Cl2 (40 mL) was
added acetic anhydride (7.9 mL, 83.4 mmol) at 0 °C, and the
resulting mixture was kept for 2 h at room temperature. The
reaction mixture was concentrated under vacuo and purified
by column chromatography (6.5% EtOAc/hexanes) to give 4
(4.79 g, 79.1%) as a yellowish oil.
Gen er a l P r oced u r e for Con d en sa tion of Aceta te 4
w ith Heter ocycles. A mixture of 6-chloropurine (1.04 g, 6.72
mmol), hexamethyldisilazane (25 mL), and ammonium sulfate
(catalytic amount) was refluxed for 4 h under a nitrogen
atmosphere. The clear solution obtained was concentrated in
vacuo, and the residue was dissolved in DCE (5 mL) and
reacted with a solution of 4 (1.50 g, 5.17 mmol) in DCE (40
mL), followed by trimethylsilyl triflate (1.3 mL, 6.72 mmol)
at 0 °C. After the mixture was stirred for 1 h at room
temperature under a nitrogen atmosphere, the reaction solu-
tion was poured into an ice-cold saturated NaHCO3 solution
(20 mL) and stirred for 15 min. The organic layer was washed
with water and brine and dried over MgSO4. The solvent was
removed under reduced pressure, and the resulting residue
The anti-HIV-1 activity of selected â-D-2′-F-d4Ns was
confirmed in human PBM cell lines with different virus
strains including an HIV-1 strain resistant to 3TC/FTC
(M184V Pitt.) as shown in Table 3. AZT, lamivudine,
and (-)-FTC (emtricitabine) were included as positive
controls. The decreasing order of potency of the selected
nucleosides in xxBRU Pitt.-infected human PBM cells
was â-D-adenine 14 (0.46 µM) > â-D-inosine 16 (2.8 µM)
> â-D-5-F-cytosine 30 (3.1 µM) > â-D-guanine 18 (4.4
µM). As expected, high levels of cross-resistance with
M184V isolates were evident with lamivudine and (-)-
FTC, not with AZT. However, no significant cross-
resistance was found for the newly synthesized com-
pounds.
In an attempt to understand metabolic properties, the
adenine derivative 14, which showed a favorable resis-
tance profile, was studied with respect to adenosine
deaminase, along with the previously reported enanti-
omer â-L-2′-F-d4A28 and adenosine. As shown in Table
4, the adenine derivative 14 was found to be a substrate
for mammalian adenosine deaminase (from calf intes-
tinal mucosa, EC. 3.5.4.4.), while its L-enantiomer was
not converted to the corresponding inosine derivative.
Compound 14 is deaminated more effectively than the
nonfluorinated analogue D-d4A (KM 30 µM, t1/2 3.1
days).34 Compared to adenosine, however, the D-isomer
14 is deaminated about 100 times slower. This property
of 14 may be favorable as a therapeutic agent, since
adenosine deaminase is one of the major deactivating
enzymes in vivo. As for chemical stability, as expected,
compound 14 has a half-life of ca. 3 days at pH 2.0 and
is stable at pH 7.4 and 11.0, whereas D-d4A has been
reported to have a half-life of less than 1 day at pH 7.0.41
In summary, from the structure-activity relation-
ships of 2′-fluoro-2′,3′-unsaturated D-nucleosides, it was
found that the â-D-2′-F-d4N series exhibited moderate
to potent anti-HIV-1 activities with high selectivity in
human PBM cells. Preliminary cross-resistance studies
indicated that no significant cross-resistance with 3TC/
FTC resistant virus (M184V) was detected. In view of
these interesting preliminary antiviral data, additional
biological studies are warranted.

SAR of 2′-Fluoro-2′,3′-unsaturated D-Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1317
was separated by silica gel column chromatography (17%
EtOAc/hexanes) to give anomeric mixture 5 (â:R ) 1:1.3, 1.15
g, 58.1%) as a syrup.
filtration, and the filtrate was evaporated under reduced
pressure. The residue was purified by column chromatography
(25-50% EtOAc/hexanes) to give two compounds, 5′-protected
2-fluoro-6-aminopurine derivative [130 mg, 27.5%: 1H NMR
(CDCl3) δ 0.08, 0.093 (2s, 2 × CH3), 0.91, (s, t-Bu), 3.65-3.76
(m, H-5′), 5.13 (ps t, J ) 4.4, 4.8 Hz, H-4′), 5.80 (s, H-3′), 6.76
(s, H-1′), 7.84 (s, H-8); UV (MeOH) λmax 268.5 nm. Anal. Calcd
for C16H23F2N5O2Si: C, 50.11; H, 6.05; N, 18.26. Found: C,
50.23; H, 5.99; N, 18.18] as a white solid and 5′-protected
2-amino-6-chloropurine derivative [199.3 mg, 44.3%: 1H NMR
(CDCl3) δ 0.082, 0.093 (2s, 2 × CH3), 0.91, (s, t-Bu), 3.73-3.84
(m, H-5′), 5.09 (m, H-4′), 5.12 (s, NH2), 5.80 (s, H-3′), 6.74 (s,
H-1′), 7.84 (s, H-8); UV (MeOH) λmax 307.5 nm. Anal. Calcd
for C16H23FClN5O2Si: C, 48.05; H, 5.80; N, 17.51. Found: C,
48.30; H, 5.72; N, 17.23] as a white solid.
6-Ch lor o-9-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-d id eoxy-
2-flu or o-D-glycer o-pen t-2-en ofu r an osyl]pu r in e (5): 1H NMR
(CDCl3) δ 0.09-0.11 (m, 4 × CH3), 0.91, 0.92 (2s, t-Bu), 3.85
(m, H-5′), 5.19 (ps t, J ) 4.4 Hz, H-4′), 5.02 (m, H-4′), 5.78,
5.85 (2s, H-3′), 6.93 (t, J ) 4.8 Hz, H-1′), 7.01 (m, H-1′), 8.21,
8.60 (2s, H-2), 8.78, 8.79 (2s, H-8); UV (CHCl3) λmax 263.5 nm.
Anal. Calcd for C16H22FClN4O2Si: C, 49.93; H, 5.76; Cl, 9.21;
N, 14.56. Found: C, 50.04; H, 5.73; Cl, 9.33; N, 14.45.
6-Ch lor o-2-flu or o-9-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-
d id e oxy-2-flu or o-â-D-glycer o-p e n t -2-e n ofu r a n osyl]p u -
r in e (6) a n d Its r-Isom er (7). A mixture of silylated
2-fluoro-6 chloropurine [prepared from 1.3 g (7.6 mmol) of
2-fluoro-6-chloropurine and 20 mL of HMDS], 4 (1.7 g, 5.85
mmol), and TMSOTf (1.5 mL, 5.85 mmol) in DCE (40 mL) was
stirred at room temperature overnight. After extractive work-
up, purification by silica gel column chromatography (12%
EtOAc/hexanes) gave â-anomer 6 (718 mg, 30.4%) as a white
foam and R-anomer 7 (473.7 mg, 20.1%) as a yellowish syrup.
6: UV (MeOH) λmax 268.5 nm. Anal. Calcd for C16H21ClF2N4O2-
Si‚0.1acetone: C, 47.90; H, 5.33; N, 13.71. Found: C, 48.02;
H, 5.30; N, 13.41. 7: UV (MeOH) λmax 268.0 nm. Anal. Calcd
for C16H21F2ClN4O2Si‚0.6 acetone: C, 48.84; H, 5.66; N, 12.80.
Found: C, 48.89; H, 5.60; N, 12.53.
These two isolated isomers were desilylated to give free
nucleosides 12 and 13 by the same methodology described for
compounds 8 and 9. After evaporation of the solvent, the
residues were purified by column chromatography to give 12
(36.3 mg, 89.9%, 9% MeOH/CH2Cl2) as a white solid and 13
(110.4 mg, 92%, 5% MeOH/CH2Cl2) as a white solid. 12: UV
(H2O) λmax 260.5 nm (ꢀ 11200) (pH 7), 261.0 nm (ꢀ 9800) (pH
2), 261.0 nm (ꢀ 10000) (pH 11). Anal. (C10H9F2N5O) C, H, N.
13: UV (H2O) λmax 305.5 nm (ꢀ 9800) (pH 7), 308.0 nm (ꢀ 9800)
(pH 2), 305.0 nm (ꢀ 5400) (pH 11). Anal. (C10H9ClFN5O2‚0.3H2O)
C, H, N.
6-Ch lor o-9-(2,3-dideoxy-2-flu or o-â-D-glycer o-pen t-2-en o-
fu r a n osyl)p u r in e (8) a n d Its r-Isom er (9). A solution of 5
(800 mg, 2.96 mmol) in CH3CN (20 mL) was treated with
TBAF (1 M solution in THF) (3.8 mL, 3.8 mmol) and stirred
for 30 min at room temperature. After evaporation of the
solvent, the residue was purified by column chromatography
(3% MeOH/CH2Cl2) to obtain â-anomer 8 (283.6 mg, 35.4%)
as a white solid and R-anomer 9 (341.3 mg, 42.6%) as a white
solid. 8: UV (MeOH) λmax 262.5 nm. Anal. (C16H8ClFN4O2‚
0.1EtOH). 9: UV (MeOH) λmax 262.5 nm. Anal. (C16H8ClFN4O2)
C, H, N.
2-F lu or o-6-a m in o-9-(2,3-d id eoxy-2-flu or o-â-D-glycer o-
p en t-2-en ofu r a n osyl)p u r in e (10) a n d 2-Am in o-6-ch lor o-
9-(2,3-d id eoxy-2-flu or o-â-D-glycer o-p en t-2-en ofu r a n osyl)-
p u r in e (11). Dry ammonia was bubbled into a stirred solution
of 6 (330 mg, 0.82 mmol) in dry DME (35 mL) at room
temperature overnight. The salts formed were removed by
filtration, and the filtrate was evaporated under reduced
pressure. The residue was purified by preparative TLC
(20∼30% EtOAc/hexanes) to give two compounds, 5′-protected
2-fluoro-6-aminopurine derivative [86 mg, 27.4%: 1H NMR
(CDCl3) δ 0.084, 0.09 (2s, 2 × CH3), 0.91, (s, t-Bu), 3.80-3.90
(m, H-5′), 4.96 (s, H-4′), 5.73 (s, H-3′), 5.86 (m, OH), 6.81 (s,
H-1′), 8.19 (s, H-8); UV (MeOH) λmax 268.5 nm. Anal. Calcd
for C16H23F2N5O2Si: C, 50.11; H, 6.05; N, 18.26. Found: C,
50.18; H, 6.05; N, 18.24] as a white solid and 5′-protected
2-amino-6-chloropurine derivative [141.7 mg, 43.2%: 1H NMR
(CDCl3) δ 0.072, 0.078 (2s, 2 × CH3), 0.92, (s, t-Bu), 3.81 (m,
H-5′), 4.95 (m, H-4′), 5.12 (s, NH2), 5.75 (s, H-3′), 6.78 (s, H-1′),
9-(2,3-Did eoxy-2-flu or o-â-D-gycer o-p en t -2-en ofu r a n o-
syl)a d en in e (14). A solution of 8 (75.5 mg, 0.28 mmol) and
saturated NH3/MeOH (50 mL) was heated at 90-100 °C in a
steel bomb for 36 h. After cooling to room temperature, the
solvent was removed under vacuum, and the residue was
purified by column chromatography using 6% MeOH/CH2Cl2
as eluent to give 14 (48.1 mg, 68.4%) as a white solid: UV
(H2O) λmax 259.0 nm (ꢀ 22500) (pH 7), 258.0 nm (ꢀ 23900) (pH
2), 259.0 nm (ꢀ 21400) (pH 11). Anal. (C10H10FN5O2) C, H, N.
9-(2,3-Did eoxy-2-flu or o-r-D-glycer o-p en t-2-en ofu r a n o-
syl)a d en in e (15). Compound 15 was prepared from 7 on a
0.29 mmol scale by the same procedure for its â-isomer 14.
Purification by column chromatography using 6% MeOH/CH2-
Cl2 as eluent afforded 15 (72 mg, 78%) as a white solid: UV
(H2O) λmax 258.5.0 nm (ꢀ 24200) (pH 7), 256.5 nm (ꢀ 25300)
(pH 2), 258.0 nm (ꢀ 27100) (pH 11). Anal. (C10H10FN5O2)
C, H, N.
9-(2,3-Did eoxy-2-flu or o-â-D-glycer o-p en t-2-en ofu r a n o-
syl)h yp oxa n th in e (16). A mixture of 8 (84.4 mg, 0.31 mmol),
NaOMe (1 M solution in MeOH) (2.18 mL, 1.25 mmol), and
2-mercaptoethanol (0.09 mL, 1.25 mmol) in MeOH (20 mL)
was refluxed for 7 h under a nitrogen atmosphere. The reaction
mixture was cooled, neutralized with glacial AcOH, and
evaporated to dryness under vacuum. The residue was purified
by silica gel column chromatography (11% MeOH/CHCl3) to
afford 16 (61 mg, 78%) as a white solid: UV (H2O) λmax 247.5
nm (ꢀ 20000) (pH 7), 247.5 nm (ꢀ 19600) (pH 2), 252.5 nm
(ꢀ 22900) (pH 11). Anal. (C10H9FN4O3) C, H, N.
9-(2,3-Did eoxy-2-flu or o-r-D-glycer o-p en t-2-en ofu r a n o-
syl)h yp oxa n th in e (17). Compound 17 was prepared from 9
on a 0.30 mmol scale by the same procedure used for its
â-isomer 16. Purification by silica gel column chromatography
(10% MeOH/CH2Cl2) afforded 17 (53.8 mg, 71.1%) as a white
solid: UV (H2O) λmax 248.5 nm (ꢀ 24200) (pH 7), 247.5 nm (ꢀ
25200) (pH 2), 253.0 nm (ꢀ 27100) (pH 11). Anal. (C10H9FN4O3)
C, H, N.
8.14 (s, H-8); UV (MeOH) λmax 307.5 nm. Anal. Calcd for C16H23
FClN5O2Si: C, 48.05; H, 5.80; N, 17.51. Found: C, 48.26; H,
5.88; N, 17.51] as a white solid.
These two isolated isomers were desilylated to give free
nucleosides 10 and 11 by similar methodology described for
compounds 8 and 9. After evaporation of the solvent, the
residues were purified by column chromatography to give 10
(36.3 mg, 89.9%, 9% MeOH/CH2Cl2) as crystals and 11 (110.4
mg, 92%, 5% MeOH/CH2Cl2) as a white solid. 10: UV (H2O)
-
9-(2,3-Did eoxy-2-flu or o-â-D-glycer o-p en t-2-en ofu r a n o-
syl)gu a n in e (18). A mixture of 11 (75.6 mg, 0.27 mmol),
2-mercaptoethanol (0.07 mL, 1.06 mmol), and 1 N NaOMe
(1.06 mL, 1.06 mmol) in MeOH (10 mL) was refluxed for 9 h
under a nitrogen atmosphere. The mixture was cooled, neu-
tralized with glacial AcOH, and evaporated to dryness under
vacuum. Purification on silica gel (12% MeOH/CH2Cl2) and
recrystallization from hot EtOH afforded 18 (43.4 mg, 60.2%)
as a white solid: UV (H2O) λmax 252.0 nm (ꢀ 9600) (pH 7), 249.5
λmax 260.5 nm (ꢀ 11200) (pH 7), 261.0 nm (ꢀ 9800) (pH 2), 261.0
nm (ꢀ 10000) (pH 11). Anal. (C10H9F2N5O2) C, H, N. 11: UV
(H2O) λmax 305.5 nm (ꢀ 9800) (pH 7), 308.0 nm (ꢀ 9800) (pH 2),
305.0 nm (ꢀ 5400) (pH 11). Anal. (C10H9ClFN5O2) C, H, N.
2-F lu or o-6-a m in o-9-(2,3-d id eoxy-2-flu or o-r-D-glycer o-
p en t-2-en ofu r a n osyl)p u r in e (12) a n d 2-Am in o-6-ch lor o-
9-(2,3-d id eoxy-2-flu or o-r-D-glycer o-p en t-2-en ofu r a n osyl)-
p u r in e (13). Dry ammonia was bubbled into a stirred solution
of 7 (454.6 mg, 1.23 mmol) in dry DME (40 mL) at room
temperature for 6 h. The salts formed were removed by
nm (ꢀ 7400) (pH 2), 263.5 nm (ꢀ 6600) (pH 11). Anal. (C10H10
-
FN5O3) C, H, N.

1318 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Lee et al.
9-(2,3-Did eoxy-2-flu or o-r-D-gycer o-p en t-2-en ofu r a n o-
syl)gu a n in e (19). Compound 19 was prepared from 10 on a
0.26 mmol scale following the same procedure used for its
â-isomer 18. Purification on silica gel using 12% MeOH/CH2-
Cl2 as eluent gave 52.1 mg (75%) of 19 as white solid: UV
(H2O) λmax 252.0 nm (ꢀ 9600) (pH 7), 249.5 nm (ꢀ 7400) (pH 2),
263.5 nm (ꢀ 6600) (pH 11). Anal. (C10H10FN5O3‚0.3 H2O)
C, H, N.
2,6-Dia m in o-9-(2,3-d id eoxy-2-flu or o-â-D-glycer o-p en t -
2-en ofu r a n osyl)p u r in e (20). A mixture of 11 (42 mg, 0.147
mmol) and saturated NH3/MeOH (20 mL) was heated at 90-
100 °C in a steel bomb for 36 h. After cooling to room
temperature, the solvent was removed under vacuum, and the
residue was purified by column chromatography using 10%
MeOH/CH2Cl2 as eluent to give 20 (30.5 mg, 78.2%) as a white
solid: UV (H2O) λmax 279.5 nm (ꢀ 9300) (pH 7), 288.5 nm
(ꢀ 9700) (pH 2), 279 nm (ꢀ 9700) (pH 11). Anal. (C10H11FN6-
O2‚0.1CH2Cl2) C, H, N.
2,6-Dia m in o-9-(2,3-d id eoxy-2-flu or o-r-D-glycer o-p en t-
2-en ofu r a n osyl)p u r in e (21). Compound 21 was prepared
from 13 on a 0.158 mmol scale following the same procedure
used for its â-isomer 20. Purification on silica gel using 10%
MeOH/CH2Cl2 as eluent gave 33 mg (78.5%) of 21 as white
solid: UV (H2O) λmax 279 nm (ꢀ 8000) (pH 7), 290 nm (ꢀ 11600)
(pH 2), 279 nm (ꢀ 11100) (pH 11). Anal. (C10H11FN6O2‚0.4H2O)
C, H, N.
1-[5-O-(ter t-Bu tyld im eth ylsilyl)-2,3-d id eoxy-2-flu or o-â-
D-glycer o-p en t-2 en ofu r a n osyl]-N4-ben zoylcytosin e (22)
a n d Its r-Isom er (23). Silylated N4-benzoylcytosine [prepared
from 1.56 g (7.24 mmol) of N4-benzoylcytosine and 30 mL of
HMDS], 2 (1.5 g, 5.17 mmol), and TMSOTf (1.4 mL, 1.4 mmol)
in dry DCE (20 mL) were reacted for 1 h at room temperature
under a nitrogen atmosphere. After extractive workup, isola-
tion by silica gel column chromatography (30% EtOAc/hexanes)
afforded â-anomer 22 (1.10 g, 47.8%) and R-anomer 23 (0.87
g, 38.1%) as white solids. 22: 1H NMR (CDCl3) δ 0.12, 0.13
(2s, 2 × CH3), 0.94, (s, t-Bu), 3.80-3.97 (m, H-5′), 4.94 (m,
H-4′), 5.61 (s, H-3′), 7.12 (m, H-1′), 7.50-7.92 (m, H-Bz), 7.91
(d, J ) 6.4 Hz, H-5), 8.41 (d, J ) 5.6 Hz, H-6), 8.72 (s, NH);
UV (MeOH) λmax 302.0 nm. Anal. Calcd for C22H28FN3O4Si: C,
59.30; H, 6.33; N, 9.43. Found: C, 59.39; H, 6.37; N, 9.42. 23:
1H NMR (CDCl3) δ 0.08, 0.09 (2s, 2 × CH3), 0.91, (s, t-Bu),
3.70-3.82 (m, H-5′), 5.05 (m, H-4′), 5.75 (s, H-3′), 7.08 (m, H-1′),
7.51-7.90 (m, 7H, H-5, H 6, H-Bz); UV (MeOH) λmax 302.0
nm. Anal. Calcd for C22H28FN3O4Si: C, 59.30; H, 6.33; N, 9.43.
Found: C, 59.14; H, 6.19; N, 9.31.
nitrogen atmosphere. After extractive workup, isolation by
silica gel column chromatography (12.5% EtOAc/cyclohexane)
afforded â-anomer 26 (0.54 g, 43.2%) and R-anomer 27 (0.48
g, 38.6%) as white solids. 26: 1H NMR (CDCl3) δ 0.11, 0.12
(2s, 2 × CH3), 0.93, (s, t-Bu), 3.80-3.89 (m, H-5′), 4.87 (m,
H-4′), 5.68 (s, H-3′), 6.94 (m, H-1′), 7.43-7.55 (m, 4H, H-Bz),
8.31 8.33 (m, 2H, H-6, H-Bz); UV (CHCl3) λmax 326.0 nm. Anal.
Calcd for C23H30FN3O4Si: C, 60.11; H, 6.58; N, 9.14. Found:
C, 59.92; H, 6.69; N, 8.94. 27: 1H NMR (CDCl3) δ 0.08, 0.09
(2s, 2 × CH3), 0.91, (s, t-Bu), 3.70-3.80 (m, H-5′), 5.03 (m,
H-4′), 5.76 (m, H-3′), 7.11 (m, H-1′), 7.43-7.56 (m, 4H, H-Bz),
8.31-8.33 (m, 2H, H-6, H-Bz), 13.15 (s, NH); UV (CHCl3) λmax
326.5 nm. Anal. Calcd for C23H30FN3O4Si: C, 60.11; H, 6.58;
N, 9.14. Found: C, 60.13; H, 6.53; N, 9.11.
1-(2,3-Did eoxy-2-flu or o-â-D-glycer o-p en t-2-en ofu r a n o-
syl)cytosin e (28). A solution of 22 (418,8 mg, 0.94 mmol) in
dry CH3CN (20 mL) was treated with TBAF (1 M solution in
THF, 1.4 mL, 1.4 mmol) and stirred for 1 h at room temper-
ature. After evaporation of the solvent, the resulting residue
was purified by preparative TLC (2.5% MeOH/CH2Cl2) to give
the N4-benzoylcytosine intermediate (256.0 mg, 82.2%, Anal.
Calcd for C16H14FN3O4: C, 58.01; H, 4.26; N, 12.68. Found:
C, 57.88; H, 4.27; N, 12.99) as a white solid, which was treated
with saturated methanolic ammonia solution (20 mL). The
reaction mixture was allowed to stir at room temperature until
TLC showed the disappearance of the starting material (8 h).
The reaction mixture was concentrated under reduced pres-
sure, and the residue was purified by silica gel column
chromatography (10% MeOH/CH2Cl2) to give 28 (144.6 mg,
83%) as a solid, which was crystalized from hexanes-MeOH-
CH2Cl2: UV (H2O) λmax 266.0 nm (ꢀ 7500) (pH 7), 276.0 nm (ꢀ
11100) (pH 2), 266.5 nm (ꢀ 9500) (pH 11). Anal. (C9H10FN3O3)
C, H, N.
1-(2,3-Did eoxy-2-flu or o-r-D-glycer o-p en t-2-en ofu r a n o-
syl)cytosin e (29). Compound 29 was prepared from 23 on a
0.87 mmol scale in two steps by the method described for
compound 28. Flash column chromatography (silica gel, 12%
MeOH/CH2Cl2) gave 119.8 mg (60.2% from compound 23) of
29 as a white solid: UV (H2O) λmax 266.0 nm (ꢀ 7500) (pH 7),
276.0 nm (ꢀ 12600) (pH 2), 267.0 nm (ꢀ 8900) (pH 11). Anal.
(C9H10FN3O3) C, H, N.
5-Flu or o-1-(2,3-dideoxy-2-flu or o-â-D-glycer o-pen t-2-en o-
fu r a n osyl)cytosin e (30). Compound 30 was prepared from
24 on a 0.50 mmol scale in two steps by the method described
for compound 28. Flash column chromatography (silica gel,
10% MeOH/CH2Cl2) and subsequent recrystallization (hex-
anes-MeOH-CH2Cl2) gave 71.1 mg (58.6% from compound
24) of 30 as a white solid: UV (H2O) λmax 277.5 nm (ꢀ 9800)
(pH 7), 282.0 nm (ꢀ 12600) (pH 2), 277.0 nm (ꢀ 8900) (pH 11).
Anal. (C9H9F2N3O3) C, H, N.
5-F lu or o-1-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-d id eoxy-
2-flu or o-â-D-glycer o-p en t-2-en ofu r a n osyl]-N4-ben zoylcy-
t osin e (24) a n d It s r-Isom er (25). Silylated N4-benzoyl-5
fluorocytosine [prepared from 822.1 mg (3.52 mmol) of N4-
benzoyl-5-fluorocytosine and 20 mL of HMDS], 2 (787 mg, 2.71
mmol), and TMSOTf (0.5 mL, 2.73 mmol) in dry DCE (20 mL)
were reacted for 1.5 h at room temperature under a nitrogen
atmosphere. After extractive workup, isolation by silica gel
column chromatography (12-25% EtOAc/hexanes) afforded
â-anomer 24 (0.55 g, 44.4%) and R-anomer 25 (0.39 g, 31.5%)
as white solids. 24: 1H NMR (CDCl3) δ 0.145, 0.151 (2s, 2 ×
CH3), 0.94, (s, t-Bu), 3.81-4.01 (m, H-5′), 4.92 (m, H-4′), 5.62
(s, H-3′), 7.93 (m, H-1′), 7.44-7.58 (m, 4H, H-Bz), 8.29 8.32
(m, 2H, H-6, H-Bz); UV (CHCl3) λmax 325.0 nm. Anal. Calcd
for C22H27F2N3O4Si: C, 57.00; H, 5.87; N, 9.06. Found: C,
56.95; H, 5.83; N, 8.96. 25: 1H NMR (CDCl3) δ 0.08, 0.09 (2s,
2 × CH3), 0.91, (s, t-Bu), 3.70-3.80 (m, H-5′), 5.30 (m, H-4′),
5.77 (s, H-3′), 6.92 (m, H-1′), 7.31 (d J ) 7.8 Hz, H-5), 7.44-
8.31 (m, H-Ph); UV (CHCl3) λmax 325.5 nm. Anal. Calcd for
5-Flu or o-1-(2,3-dideoxy-2-flu or o-r-D-glycer o-pen t-2-en o-
fu r a n osyl)cytosin e (31). Compound 31 was prepared from
25 on a 0.64 mmol scale in two steps by the method described
for compound 28. Flash chromatography (silica gel, 11%
MeOH/CH2Cl2) and subsequent recrystallization (MeOH-CH2-
Cl2-hexanes) gave 93.7 mg (59.7% from compound 25) of 31
as a white solid: UV (H2O) λmax 277.0 nm (ꢀ 8800) (pH 7), 282.5
nm (ꢀ 11400) (pH 2), 276.0 nm (ꢀ 7800) (pH 11). Anal.
(C9H9F2N3O3) C, H, N.
5-Meth yl-1-(2,3-dideoxy-2-flu or o-â-D-glycer o-pen t-2-en o-
fu r a n osyl)cytosin e (32). Compound 32 was prepared from
26 on a 0.47 mmol scale in two steps by the method described
for compound 28. Flash chromatography (silica gel, 10%
MeOH/CH2Cl2) and subsequent trituration with EtOAc gave
69.5 mg (61.3% from compound 26) of 32 as a white solid: UV
(H2O) λmax 274.5 nm (ꢀ 7000) (pH 7), 282.5 nm (ꢀ 9600) (pH 2),
274.5 nm (ꢀ 6000) (pH 11). Anal. (C10H12FN3O3) C, H, N.
C
22H27F2N3O4Si: C, 57.00; H, 5.87; N, 9.06. Found C, 56.87;
H, 5.86; N, 9.09.
5-Meth yl-1-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-d id eoxy-
2-flu or o-â-D-glycer o-p en t-2-en ofu r a n osyl]-N4-ben zoylcy-
t osin e (26) a n d It s r-Isom er (27). Silylated N4-benzoyl-5
methylcytosine [prepared from 806 mg (3.50 mmol) of N4-
benzoyl-5-methylcytosine and 25 mL of HMDS], 2 (780 mg,
2.69 mmol), and TMSOTf (0.68 mL, 3.50 mmol) in dry DCE
(25 mL) were reacted for 5 h at room temperature under a
5-Met h yl-1-(2,3-d id eoxy-2-flu or o-r-D-glycer o-p en t -2-
en ofu r a n osyl)cytosin e (33). Compound 33 was prepared
from 27 on a 0.40 mmol scale in two steps by the method
described for compound 28. Flash chromatography (silica gel,
10% MeOH/CH2Cl2) and subsequent trituration with EtOAc
gave 61.3 mg (63.5% from compound 27) of 33 as a white

SAR of 2′-Fluoro-2′,3′-unsaturated D-Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1319
(12) Dahlberg, J . E.; Mitsuya, H.; Blam, S. B.; Broder, S.; Aaronson,
S. A. Broad spectrum antiviral activity of 2′,3′-dideoxynucleo-
sides. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2469-2473.
(13) Mitsuya, H.; Broder, S. Inhibition of the in vitro infectivity and
cytopathic effect of human T lymphotrophic virus type III/
lymphadenopathy associated virus in vitro. Proc. Natl. Acad. Sci.
U.S.A. 1986, 82, 1911-1915.
(14) Yarchoan, R.; Mitsuya, H.; Thomas, R. V.; Pluda, J . M.; Hart-
man, N. R.; Perno, C. F.; Marczyk, K. S.; Allain, J . P.; J ohns, D.
G.; Broder, S. In vivo activity against HIV and favorable toxicity
profile of 2′,3′-dideoxyinosine. Science 1989, 245, 412-415.
(15) Yarchoan, R.; Mitsuya, H.; Broder, S. Clinical and basic advances
in the retroviral therapy of human immunodeficiency virus
infection. Am. J . Med. 1989, 87, 191-200.
(16) Faulds, D.; Brogden, R. N. Didanosine: A review of its antiviral
activity, pharmacokinetic properties and therapeutic potential
in human immunodeficiency virus infection. Drugs 1992, 44 (1),
94-116.
(17) Hamamoto, Y.; Yamamoto, N.; Matsui, T.; Matsuda, A.; Ueda,
T.; Yamamoto, N. Inhibitory effect of 2′,3′-didehydro-2′,3′-
dideoxynucleosides on infectivity, cytopathic effects and replica-
tion of human immunodeficiency virus. Antimicrob. Agents
Chemother. 1987, 31, 907-910.
(18) 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-
ciency virus in vitro. Biochem. Pharmacol. 1987, 36, 2713-2718.
(19) Daluge, S. M.; Good, S. S.; Faletto, M. B.; Miller, W. H.; St. Clair,
M. H.; Boone, L. R.; Tisdale, M.; Parry, N. R.; Reardon, J . E.;
Dornsife, R. E.; Averet, D. R.; Krenitsky, T. A. 1592U89, A novel
carbocyclic nucleoside analogue with potent, selective anti-
human immunodeficiency virus activity. Antimicrob. Agents
Chemother. 1997, 41, 1082-1093.
solid: UV (H2O) λmax 274.0 nm (ꢀ 6900) (pH 7), 283.0 nm (ꢀ
11300) (pH 2), 274.5 nm (ꢀ 6800) (pH 11). Anal. (C10H12FN3O3)
C, H, N.
Ad en osin e Dea m in a se Assa y.24a Assays were performed
at 25 °C in phosphate buffer solution (pH 7.4) with substrate
concentrations in the range of 15-200 µM and with 0.15-0.24
unit of adenosine deaminase (EC 3.5.4.4. from calf intestinal
mucosa) depending on the substrate. The assays were moni-
tored with a UV spectrometer at 265 nm, and every reaction
medium was checked by TLC or UV spectrometry to ensure
that the sole reaction product was the deaminated substrate.
Initially, the qualitative assays were performed with D-â-2′F-
d4A 14 and its l-enantiomer29 (200 µM) in the presence of 0.24
unit of adenosine deaminase for 120 min to determine whether
they were substrates of this enzyme. L-â-2′F-d4A was further
observed for 3 days by monitoring on TLC in the same
conditions. From the Michaelis-Menten equation, Vmax (maxi-
mal velocity) and KM (the Michaelis constant) were obtained
for compound 14 and adenosine (15 and 159 µM) with 0.15
unit of the enzyme. The t1/2 values of D-â-2′F-d4A 14 and
adenosine were also measured at 0.15 µM with 0.15 unit of
the enzyme.
Ack n ow led gm en t. This research was supported by
the U.S. Public Health Service Grant (AI 32351 and AI
25899) from the National Institutes of Health and the
Department of Veterans Affairs.
(20) Tisdale, M.; Alnadaf, T.; Cousens, D. Combination of mutations
in human immunodeficiency virus type 1 reverse transcriptase
required for resistance to the carbocyclic nucleoside 1592U89.
Antimicrob. Agents Chemother. 1997, 41, 1094-1098.
(21) (a) Gosselin, G.; Schinazi, R. F.; Sommadossi, J .-P.; Mathe, C.;
Bergogone, M. C.; Aubertin, A.-M.; Kirn, A.; Imbach, J .-L. Anti-
human immunodeficiency viruses activities of the â-L-enanti-
omer of 2′,3′-dideoxycytidine and its 5-fluoro derivative in vitro.
Antimicrob. Agents Chemother. 1994, 38, 1292-1297. (b) Lin,
T. S.; Luo, M. Z.; Liu, M. C.; Zhu, Y. L.; Gullen, E.; Dutchman,
G. E.; Cheng, Y.-C. Design and synthesis of 2′,3′-dideoxy-2′,3′-
didehydro-â-L-cytidine (â-L-d4C) and of 2′,3′-dideoxy-2′,3′-dide-
hydro-â-L-5-fluorocytidine (â-L-Fd4C), two exceptionally potent
inhibitors of hepatitis B virus (HBV) and potent inhibitors of
human immunodeficiency virus (HIV) in vitro. J . Med. Chem.
1996, 39, 1757-1759.
(22) Watanabe, K. A.; Su, T.-L.; Lein, R. S.; Chu, C. K.; Matsuda, A.;
Chun, M. W.; Lopez, C.; Fox, J . J . Nucleosides. 123. Synthesis
of antiviral nucleosides: 5-Substituted-1-(2-deoxy-2 halogeno-
â-D-arabinofuranosyl)cytosines and -uracils. Some structure-
activity relationships. J . Med. Chem. 1983, 26, 152-156.
(23) (a) Chu, C. K.; Ma, T. W.; Shanmuganathan, K.; Wang, C. G.;
Xiang, Y. J .; Pai, S. B.; Yao, G. Q.; Sommadossi, J .-P.; Cheng,
Y.-C. Use of 2′-fluoro-5-methyl-â-L-arabinofuranosyluracil as a
novel antiviral agents for hepatitis B virus and epstein barr
virus. Antimicrob. Agents Chemother. 1995, 39, 979-981. (b) Ma,
T. W.; Pai, S. B.; Zhu, Y. L.; Lin, J . S.; Shamuganathan, K.; Du,
J . F.; Wang, C.; Kim, H. B.; Newton, M. G.; Cheng, Y.-C.; Chu,
C. K. Structure-activity relationships of 1-(2-deoxy-2-fluoro-â-
L-arabinofuranosyl)pyrimidine nucleosides as anti-Hepatitis B
virus agents. J . Med. Chem. 1996, 39, 2835-2843.
(24) (a) Marquez, V. E.; Tseng, C. K.-H.; Mitsuya, H.; Aoki, S.; Kelley,
J . A.; Ford, H., J r.; Roth, J . S.; Broder, S.; J ohns, D. G.; Driscoll,
J . S. Acid-stable 2′-fluoro purine dideoxynucleosides as active
agents against HIV. J . Med. Chem. 1990, 33, 978-985. (b)
Ruxrungtham, K.; Boone, E.; Ford, H., J r.; Driscoll, J . S.; Davey,
R. T.; Lane, H. C. Potent activity of 2′-â-fluoro-2′,3′ dideoxy-
adenosine against human immunodeficiency virus type 1 infec-
tion in hu PBM-SCID mice. Antimicrob. Agents Chemother.
1996, 40, 2369-2374.
(25) Sterzycki, R. Z.; Ghazzouli, I.; Brankovan, V.; Martin, J . C.;
Mansuri, M. M. Synthesis and anti HIV activity of several 2′-
fluoro-containing pyrimidine nucleosides. J . Med. Chem. 1990,
33, 2150-2157.
Refer en ces
(1) Richman, D. D.; Fischl, M. A.; Grieco, M. H.; Gottlieb, M. S.;
Volberding, P. A.; Laskin, O. L.; Leedom, J . M.; Groopman, J .
E.; Mildvan, D.; Hirsch, M. S.; J ackson, G. G.; Durack, D. T.;
Phil, D.; Lusinoff-Lehrman, S. The AZT collaborative working
group. The toxicity of azidothymidine (AZT) in the treatment of
patients with AIDS and AIDS-related complex. N. Engl. J . Med.
1987, 317, 192-197.
(2) Strarnes, M. C.; Cheng, Y.-C. Cellular metabolism of 2′,3′-
dideoxycytidine, a compound active against human immunode-
ficiency virus in vitro. J . Biol. Chem. 1987, 262, 988-991.
(3) Lambert, J . S.; Seidlin, M.; Reichman, R. C.; Plank, C. S.;
Laverty, M.; Morse, G. D.; Knupp, C.; McLaren, C.; Pettineli,
C.; Valentine, F. T.; Dolin, R. 2′,3′-Dideoxyinosine (ddI) in
patients with the acquired immunodeficiency syndrome or AIDS-
related complex. N. Engl. J . Med. 1990, 322, 1333-1340.
(4) Mansuri, M. M.; Hitchcock, M. J . M.; Starrett, J . E.; Sterzycki,
R. Z.; Martin, J . C. Comparison of in vitro biological properties
and mouse toxicities of three thymidine analogues active against
human immunodeficiency virus. Antimicrob. Agents Chemother.
1990, 34, 637-641.
(5) Larder, B. A.; Darby, G.; Richman, D. D. HIV with reduced
sensitivity to zidovudine (AZT) isolated during prolonged therapy.
Science 1989, 243, 452-454.
(6) St Clair, M. H.; Martin, J . L.; Tudor-Williams, G.; Bach, M. C.;
Vavro, C. L.; King, D. M.; Kelham, P.; Kemp, S. D.; Larder, B.
A. Resistant to ddI and selectivity to AZT-induced by a mutation
in HIV-1 reverse transctiptase. Science 1991, 253, 1557-1559.
(7) Richman, D.; Shih, C. K.; Lowy, I.; Rose, J .; Prodanovich, P.;
Goff, S.; Griffin, J . Human immunodeficiency virus type 1
mutants resistant to nonnucleoside inhibitors of reverse tran-
scriptase arise in tissue culture. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 11241-11245.
(8) Gulick, R. M.; Mellers, J . W.; Havlir, D.; Eron, J . J .; Gonzalez,
C.; McMahon, D.; Richman, D. D.; Valentine, F. T.; J onas, L.;
Emini, E. A.; Chodakewitz, J . A. Treatment, with indinavir,
zidovudine, and lamivudine in adults with human immunode-
ficiency virus infection and prior antiviral therapy. N. Engl. J .
Med. 1997, 337, 734-739.
(9) De Clercq, E. In Search of a selective antiviral chemotherapy.
Clin. Microbiol. Rev. 1997, 10, 674-693.
(10) Yarchoan, R.; Perno, C. F.; Thomas, R. V.; Klecker, R. W.; Allain,
R. P.; Wills, R. J .; McAtee, N.; Fishl, M. A.; Dubinsky, R.;
McNeely, M. C.; Mitsuya, H.; Pluda, J . M.; Lawley, T. J .;
Leuther, M.; Safai, B.; Collins, J . M.; Myers, C. E.; Broder, S.
Phase I studies of 2′,3′ dideoxycytidine in human immunodefi-
ciency virus infection as a single agent and alternating with
zidovudine (AZT). Lancet 1988, 1, 76-81.
(26) Martin, J . A.; Bushnell, D. J .; Duncan, I. B.; Dunsdon, S. J .;
Hall, M. J .; Machin, P. J .; Merret, J . H.; Parkes, K. E. B.;
Roberts, N. A.; Thomas, G. J .; Galpin, S. A.; Kinchington, D.
Synthesis and antiviral activity of monofluoro and difluoro
analogs of pyrimidine deoxyribonucleosides against human
immunodeficiency virus (HIV-1). J . Med. Chem. 1990, 33, 2137-
2145.
(11) Balzarini, J .; Cooney, D. A.; Dalad, M.; Kang, G.-J .; Cupp, J .
E.; De Clerq, E.; Broder, S.; J ohns, D. G. 2′,3′-Dideoxycytidine;
regulation of its metabolism and antiretroviral potency by
natural pyrimidine nucleosides inhibitors of pyrimidine nucleo-
side synthesis. Pharmacology 1987, 32, 798-806.

1320 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Lee et al.
(27) Koshida, R.; Cox, S.; Harmenberg, J .; Gilljam, G.; Wahren, B.
Structure-activity relationships of fluorinated nucleoside ana-
logs and their synergestic effect in combination with phospho-
noformate against human immunodeficiency virus type 1.
Antimicrob. Agents Chemother. 1989, 33, 2083-2088.
(28) Lee, K.; Choi, Y.; Gullen, E.; Schlueter-Wurtz, S.; Schinazi, R.;
Cheng, Y.-C.; Chu, C. K. Synthesis and anti-HIV and anti-HBV
activities of 2′-fluoro-2′,3′-unsaturated L-nucleosides. J . Med.
Chem. 1999, 42, 1320-1328.
(29) Wilson, L. J .; Hager, M. W.; El-Kattan, Y. A.; Liotta, D. C.
Nitrogen glycosylation reactions involving pyrimidine and purine
nucleoside bases with furanoside sugars. Synthesis 1995, 1465-
1479.
(36) Schinazi, R. F.; Lloyd, R. M., J r.; Nguyen, M. H.; Cannon, D. L.;
McMillan, A.; Ilksoy, N.; Chu, C. K.; Liotta, D. C.; Bazmi, H. Z.;
Mellors, J . W. Characterization of human immunodeficiency
viruses resistant to oxathiolane-cytosine Nucleosides. Antimi-
crob. Agents Chemother. 1993, 37, 875-881.
(37) Boucher, C. A. B.; Gu, Z.; Cammack, N.; Schipper, P., Gao, Q.;
Rouse, P.; Schurrman, R.; Cameronm J . M.; Wainberg, M. A.
High level resistance in vitro to the nucleoside analogue 2′ deoxy-
3′-thiacytidine (3TC) is due to one amino acid change substitu-
tion at codon 184 located within the catalytic site of HIV-1
reverse transcriptase. IXth International Conference on AIDS,
Berlin, 1993, I, 242, Abstract PO-A26-0645.
(38) Tisdale, M.; Kemp, S. D.; Parry, N. R.; Larder, B. A. Rapid in
vitro selection of human immunodeficiency virus type 1 resistant
to 3′-thiacytidine inhibitors due to a mutation in the YMDD
region of reverse transcriptase. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 5653-5656.
(39) Wainberg, M. A.; Hsu, M.; Gu, Z.; Borkow, G.; Parniak, M. A.
Effectiveness of 3TC in HIV clinical trials may be due in part to
the M184 substitution in 3TC-resistant HIV-1 reverse tran-
scriptase. AIDS 1996, 10, S3-S10 (Suppl 5).
(40) Huang, H.; Chopra, R.; Verdine, G.; Harrison, S. Structure of a
covalently trapped catalytic complex of HIV-1 reverse tran-
scriptase; implications for drug resistance. Science 1998, 282,
1669-1675.
(41) Balzarini, J .; Kang, G.-J .; Dalal, M.; Herdewijn, P.; De Clerq,
E.; Broder, S.; J ohns, D. G. The anti-HTLV-III (anti-HIV) and
cytotoxic activity of 2′,3′-didehydro-2′,3′-dideoxyribonucleo-
sides: a comparison with their parental 2′,3′-dideoxyribonucleo-
sides. Mol. Pharm. 1987, 32, 162-167.
(30) Sujino, K.; Yoshida, T.; Sugimura, H. Facile synthesis of 2′,3′-
unsaturated nucleosides from 2-deoxyribose. Tetrahedron Lett.
1996, 37, 6133-6136.
(31) Morikawa, T.; Sasaki, H.; Mori, K.; Shiro, M.; Taguchi, T.
Simmons-Smith reactions of fluoroallyl alcohol derivatives.
Chem. Pharm. Bull. 1992, 40, 3189-3193.
(32) Ha¨fele, B.; J a¨ger, V. A short synthesis of (S)-5-hydroxy-2-penten-
4-olide from D-mannitol via 2,3-O-isopropylidene-D-glyceralde-
hyde. Liebigs Ann. Chem. 1987, 85-87.
(33) Choi, Y.; Lee, K.; Hong, J . H.; Schinazi, R. F.; Chu, C. K.
Synthesis and anti-HIV activity of L-2′-fluoro-2′,3′-unsaturated
purine nucleosides. Tetrahedron Lett. 1998, 39, 4437-4440.
(34) Pe´licano, H.; Pierra, C.; Eriksson, S.; Gosselin, G.; Imbach, J .-
L.; Maury, G. Enzymatic properties of the unnatural â-L-
enantiomers of 2′,3′-dideoxyadenosine and 2′,3′-didehydro-2′,3′-
dideoxyadenosine. J . Med. Chem. 1997, 40, 3969-3973.
(35) (a) Chu, C. K.; Ma, L.; Olgen, S.; Pierra, C.; Du, J .; Gumina, G.;
Gullen, E.; Cheng, Y.-C.; Schinazi, R. F. Synthesis and antiviral
activity of oxaselenolane nucleosides. J . Med. Chem. 2000, 43,
3906-3912.
J M010418N