D- and L-2′,3′-didehydro-2′,3′-dideoxy-3′- fluoro-carbocyclic nucleosides: Synthesis, anti-HIV activity and mechanism of resistance

Article abstract of DOI:10.1021/jm061304k

Introducing 2′-fluoro substitution on the 2′,3′-double bond in carbocyclic nucleosides has provided biologically interesting compounds with potent anti-HIV activity. As an extension of our previous works in the discovery of anti-HIV agents, D- and L-2′,3′

Full text of DOI:10.1021/jm061304k

1828
J. Med. Chem. 2007, 50, 1828-1839
D- and L-2′,3′-Didehydro-2′,3′-dideoxy-3′-fluoro-carbocyclic Nucleosides: Synthesis, Anti-HIV
Activity and Mechanism of Resistance
Jianing Wang,† Yunho Jin,† Kimberly L. Rapp,‡ Raymond F. Schinazi,‡ and Chung K. Chu*,†
College of Pharmacy, The UniVersity of Georgia, Athens, Georgia 30602, and Emory UniVersity School of Medicine/Veterans Affairs Medical
Center, Atlanta, Georgia 30033
ReceiVed NoVember 9, 2006
Introducing 2′-fluoro substitution on the 2′,3′-double bond in carbocyclic nucleosides has provided biologically
interesting compounds with potent anti-HIV activity. As an extension of our previous works in the discovery
of anti-HIV agents, D- and L-2′,3′-unsaturated 3′-fluoro carbocyclic nucleosides were synthesized and evaluated
against HIV-1 in human peripheral blood mononuclear (PBM) cells. Among the synthesized L-series
nucleosides, compounds 18, 19, 26 and 28 exhibited moderate antiviral activity (EC50 7.1 µM, 6.4 µM, 10.3
µM, and 20.7 µM, respectively), while among the D-series, the guanosine analogue (35, D-3′-F-C-d4G)
exhibited the most potent anti-HIV activity (EC50 0.4 µM, EC90 2.8 µM). However, the guanosine analogue
35 was cross-resistant to the lamivudine-resistant variants (HIV-1M184V). Molecular modeling studies suggest
that hydrophobic interaction as well as hydrogen-bonding stabilize the binding of compound 35 in the active
site of wild type HIV reverse transcriptase (HIV-RT). In the case of L-nucleosides, these two effects are
opposite which results in a loss of binding affinity. According to the molecular modeling studies, cross-
resistance of D-3′-F-C-d4G (35) to M184V mutant may be caused by the realignment of the primer and
template in the HIV-RTM184V interaction, which destabilizes the RT-inhibitor triphosphate complex, resulting
in a significant reduction in anti-HIV activity of the D-guanine derivative 35.
Introduction
constructed followed by coupling with various heterocyclic
bases. However, this method was found to be unsuccessful in
the synthesis for the 3′-fluoro congeners due to the instability
of the final products under the same conditions used. Therefore,
condensation of a gem-3′,3′-difluoro sugar 8 or 29 with base
moieties followed by an elimination reaction in the last step
was successful to obtain the target purine nucleosides. Pyrimi-
dine nucleosides were, however, synthesized via the linear
method using the intermediate 11 or 30. Herein, synthesis, anti-
HIV activity, and molecular modeling studies of D- and L-2′,3′-
didehyhydro-2′,3′-dideoxy-3′-fluoro-carbocyclic nucleosides are
reported.
Nucleoside reverse transcriptase inhibitors (NRTI) have
played an important role in the treatment of HIV infections.1
However, major drawbacks of NRTI include the emergence of
drug resistant variants and toxicity.2-5 Therefore, conservative
efforts have been made to improve the antiviral efficacy as well
as to reduce the toxicity by modifying the structure.
The structures of several potent NRTIs, such as stavudine
(d4T), abacavir, reverset (D-d4FC), and elvucitabine (L-d4FC)
highlight the important role of a 2′,3′-double bond to enhance
the antiviral activity (Figure 1).1 Additionally, carbocyclic
nucleosides such as abacavir have attracted considerable atten-
tion due to their potent antiviral activity as well as the stability
toward metabolic degradation.6 In view of these facts, it was
of interest to incorporate these structural features into nucleoside
analogues. Several interesting compounds with potent anti-HIV
activity have been discovered as the result of this concept.7-13
In connection to these efforts, our laboratory recently reported
the stereoselective synthesis and anti-HIV activity of D- and
L-2′,3′-didehyhydro-2′,3′-dideoxy-2′-fluoro-carbocyclic nucleo-
sides.14 Among the series, the adenosine analogue with L-
configuration showed the most potent anti-HIV activity (EC50
0.77 µM). On the basis of molecular modeling studies, it was
found that both the double bond on the carbocyclic ring as well
as the 2′-fluoro substitution contribute to the favorable binding
affinity between the inhibitor and the HIV-RT. Hence, further
exploration of the antiviral activity of 3′-fluorine congeners was
of interest to expand our knowledge on the structure-activity
relationships of the same class of nucleosides.
Results and Discussion
Chemistry. Both D- and L-nucleosides were synthesized;
however, the following descriptions are mainly based on the
L-series according to Schemes 1-3, unless otherwise indicated.
The epoxide 1 was synthesized from D-ribose by the known
method in our laboratory.14 The desired regioisomer was
obtained by reductive ring opening of the epoxide with judicious
selection of reducing reagents (Scheme 1). The preliminary study
suggested that compound 4 was the major product when epoxide
2 was treated with LAH. The selective opening of the epoxide
by the hydride may be due to the steric hindrance of the bulky
trityl group adjacent to the 3′-position. On the basis of this result,
several other reducing reagents were investigated (Table 1).
Among them, Super-Hydride gave predominantly compound 4
(4:3 ) 15.3:1) in excellent yield (98%). Oxidation of the alcohol
4 with PDC gave the ketone 5 as an unstable compound. It was
interesting to note that introducing a difluorine group at the 3′-
position to compound 5 was quite difficult in comparison to
the 2′-fluoro isomer.14 A harsh condition of neat diethylami-
nosulfur trifluoride (DAST) with reaction temperature at 40 °C
for 36 h was needed to convert the ketone 5 to difluoro
compound 6 in 68% yield. The following elimination reaction
also gave significant problems: Treating compound 6 with
For the synthesis of 2′-fluoro carbocyclic nucleosides, the
fluorine substituted unsaturated carbocyclic ring was first
* Corresponding author. Tel.: (706) 542-5379. Fax: (706) 542-5381.
E-mail: DCHU@RX.UGA.EDU.
† The University of Georgia.
‡ Emory University School of Medicine/Veterans Affairs Medical Center.
10.1021/jm061304k CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/21/2007

2′,3′-Unsaturated 3′-Fluoro Carbocyclic Nucleosides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1829
Figure 1. Several potent NRTIs with 2′,3′-double bond.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) (i) ref 14, (ii) R-AIBBr, CH3CN, (iii)
K2CO3, MeOH; (b) TrCl, DMAP, Et3N, CH2Cl2; (c) Super-Hydride (1.0
M in THF), 0 °C to rt; (d) PDC, AcOH, 4 Å molecular sieve, CH2Cl2; (e)
neat DAST, 40 °C; (f) TMSI, CH2Cl2; (g) TBDPSCl, imidazole, CH2Cl2;
(h) MsCl, Et3N, CH2Cl2, rt; (i) NaN3, DMF, 130 °C; (j) H2/Pd/C, 30 psi, rt.
Table 1. Ring-Opening Reaction of Epoxide 2
yield
entry
reducingagent
4:3
(%)
1
2
3
4
5
Super-Hydride
LAH
DIBAL-H
Red-Al
15.3:1
5.1:1
-
98
88
0
0
0
a Reagents and conditions: (a) (i) â-methoxyacryloyl isocyanate, THF,
-30 °C to rt (for 12) or â-methoxy-R-methacryloyl isocyanate, THF, -30
°C to rt (for 16); (b) NH4OH, 1,4-dioxane/EtOH, steel bomb, 90-100 °C;
(c) (i) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN,
rt, (ii) NH4OH or NH3/MeOH, rt; (d) HCl/MeOH, rt; (e) 3 N HCl/1,4-
-
LiAl(t-butoxy)3
-
t
dioxane, reflux 3 h; (f) BuOK, THF/1,4-dioxane, 90 °C.
potassium tert-butoxide (tBuOK) in THF at 50 °C did not
produce the desired 3′-fluorovinyl moiety, and only the starting
material was recovered. Hence, a modified synthetic sequence
was adopted as illustrated in Scheme 1 (6 to 11). Both trityl
and benzyl groups were removed using iodotrimethylsilane
(TMSI) in 70% yield. The resulting diol 7 was selectively
protected by tert-butyldiphenylsilyl chloride (TBDPSCl) to give
the key intermediate 8 which was converted to amine 11 in
three steps. For the pyrimidine nucleosides, the linear synthetic
methodology reported by Shealy et al.15,16 was used as a direct
coupling reaction using the alcohol 8 under the Mitsunobu
conditions resulting in the decomposition of the starting material.
The amine 11 was coupled with substituted isocynate to give
the corresponding urea 12 or 16 (Scheme 2). Reaction of 12
with concentrated ammonium hydroxide/ethanol/1,4-dioxane in
a steel bomb gave the uridine analogue 13 in 41% yield.
Amination of 13 followed by deprotection afforded the cytidine
analogue 15. For the thymidine analogue 17, ring closure and
deprotection were accomplished in one step under acidic
condition from compound 16. Last, the 2′,3′-double bond was
obtained under basic elimination conditions to afford the target
Table 2. Elimination Reactions Using Traditional or Microwave
(MW)-Assisted Methods
isolated
temp
(°C)
reaction
time
yield
(%)
entry
1
substrate
methods
L-cytidine
analogue
D-cytidine
analogue
L-guanosine
analogue
D-guanosine
analogue
90
70
70
70
9 h
35
84
45
72
traditional oil
bath heating
MW-assisted
2
3
4
6 min
24 h
traditional oil
bath heating
MW-assisted
10 min
cytidine 18 and thymidine 19 analogues in 35% and 46% yield,
respectively (Table 2). To synthesize adenosine analogues,
triphenylphosphine (TPP) and diisopropyl azodicarboxylate
(DIAD) were first mixed in the THF:1,4-dioxane cosolvent at

1830 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Wang et al.
Scheme 3a
column, which makes it extremely difficult to identify and
separate. Second, the difluoro compounds are inert to the
conventional methods of elimination using BuOK in aprotic
t
solvent, and therefore, the reaction mixture has to be heated
for long periods of time to convert all the starting material to
the product (Table 3, entries 1 and 3). Unfortunately, under these
conditions, the newly formed target nucleosides decomposed,
which resulted in low yields. Fortunately, in the search for better
conditions for the elimination step during the synthesis of the
D-compounds, it was found that the microwave-assisted method
had several advantages over the traditional method. Upon
irradiation of the difluoro-nucleosides in the microwave syn-
thesizer with maximum output power of 300 W, the elimination
reaction was completed within 5-10 min at 70 °C and gave
70-80% yield, while a lower yield was obtained after a longer
time in the traditional thermally assisted conditions (Table 2).
This methodology may provide an efficient way for preparing
molecules which have a fluorovinyl moiety. Assignment of the
structures of newly synthesized nucleosides was accomplished
by NMR, elemental analysis, mass, UV, and IR spectroscopy.
Anti-HIV Activity. All the synthesized pyrimidine (18, 19,
and 37-40) and purine (26-28, 31-36) nucleosides were
evaluated against HIV-1 in human PBM cells. The EC50, EC90,
and toxicity data are listed in Table 3. In the D-3′,3′-difluoro
series, none of the compounds showed any antiviral activity
nor cytotoxicity when tested up to 100 µM. Among the D- and
L-3′-fluoro-2′,3′-unsaturated nucleosides, some of them exhibited
moderate to potent anti-HIV activity. The cytidine 18, thymidine
19, adenosine 26 and guanosine 28 analogues in the L-series
inhibited HIV-1 in PBM cells with EC50 that ranged from 6.4
to 20.7 µM. The guanosine analogue 35, bearing D-configura-
tion, is the most active compound among all the synthesized
nucleosides (EC50 0.41 µM, EC90 2.8 µM), although it exhibited
moderate cytotoxicity in PBM cells (IC50 21.1 µM). The antiviral
activities of 3′-fluoro-2′,3′-unsaturated carbocyclic nucleosides
in the current studies were generally maintained or enhanced,
in comparison to that of 2′-fluoro congeners.14 Thus, the role
of the fluorine substitution on the 3′-position of the double bond
may have significant effects (vide infra for molecular modeling
studies).
a Reagents and conditions: (a) DIAD, Ph3P, purines, THF or THF/1,4-
dioxane; (b) NH3/MeOH, steel bomb, 110 °C; (c) 3 N HCl, MeOH; (d) (i)
t
formic acid, (ii) NH4OH or NH3/MeOH, rt; (e) BuOK, THF/1,4-dioxane,
t
90 °C or BuOK, DMF, 70 °C.
0 °C and then further cooled to -78 °C. The key intermediate
8 and the 6-chloropurine were added sequentially, and the
reaction was allowed to gradually warm up to room temperature
until all the starting material was consumed (Scheme 3). The
crude product 20, which was contaminated with reduced DIAD
species, was directly treated with methanolic ammonia in a steel
bomb at 100 °C to give the adenosine analogue 21 in 37% yield
in two steps. After the silyl group was removed under acidic
condition, compound 22 was treated with tBuOK in THF at 90
°C to furnish the adenine derivative 26 in 50% yield. The
compound 21 could also be converted to inosine analogue 23
by treating with formic acid followed by ammonium hydroxide
in 39% yield in two steps. After the elimination reaction similar
to the method described for the adenosine analogue, the final
inosine analogue 27 was obtained in 49% yield. Condensation
of alcohol 8 with 2-amino-6-chloropurine or 6-chloro-N2-
isobutyrylpurine, under the Mitsunobu condition as described
above, failed to give the corresponding nucleoside. However,
when a mixture of TPP, 6-chloro-N2-isobutyrylpurine and
alcohol 8 in dry THF was treated with DIAD at 0 °C, the desired
product 24 was able to be isolated. The compound 24 was
converted to the guanosine analogue 25 using formic acid
followed by ammonium hydroxide in 18% yield from 8 in two
steps. The nucleoside 25 was further subjected to an elimination
Antiviral Activity against Lamivudine-Resistant (HIV-
1M184V) Mutant Strain. One of the drawbacks of the NRTI is
the emergence of drug-resistant mutant strains during the
extended treatment period, which may significantly compromise
the clinical efficacy. Lamivudine, an important component of
the highly active antiretroviral therapy (HAART), confers a
single mutation at residue 184 (M184V), which caused at least
a 1000-fold decrease in its antiviral activity.18,19 Discovery of
novel NRTI agents against lamivudine-resistant mutant strain
is of great interest. Unfortunately, all the potent NRTI with
L-configuration against wild type HIV-1 are always cross-
resistant to the lamivudine resistant mutant (M184V), which
may be due to the steric hindrance between the bulky side chain
of Val184 and the adjacent NRTI’s sugar ring.20 The situation
is generally better in D-nucleosides, as their sugar rings project
far away from the residue 184. However, the M184V mutant
has also been isolated after using the abacavir, a prodrug of
carbovir, which is a D-nucleoside.21-26 In view of the structural
similarity between the compound 35 and carbovir, it was of
interest to understand its resistance profile. Hence, we further
evaluated compound 35 against HIVM184V using carbovir (Table
4) as well as 3TC/AZT (data not shown) as control. On the
basis of this study, compound 35 appears to confer resistance
t
reaction using BuOK in DMF at 70 °C to give the guanosine
analogue 28 in 45% yield.
Similar procedures were conducted to synthesize the D-series
(Scheme 4). As the difluoro-substituted nucleosides on the
carbohydrate moiety exhibit interesting biological activity,17 the
difluoro-nucleosides 31-33, 37 and 38 in the D-series were also
evaluated against HIV-1 (Table 3). It is noteworthy that the
elimination reaction in the last step proceeded with significant
difficulties. First of all, the starting material and product have
almost identical Rf values on a silica gel plate as well as on a

2′,3′-Unsaturated 3′-Fluoro Carbocyclic Nucleosides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1831
Scheme 4a
t
t
a Reagents and conditions: (a) BuOK, THF/1,4-dioxane, 90 °C, conventional oil bath heating for 34, 36; BuOK, DMF, 70 °C, microwave-assisted for
t
t
35; (b) BuOK, THF/1,4-dioxane, 60 °C, traditional oil bath heating for 40; BuOK, DMF, 70 °C, microwave-assisted for 39.
Table 3. In Vitro Anti-HIV-1 Activity and Toxicity of D-3′,3′-Difluoro-2′,3′-dideoxy-carbocyclic Nucleosides and D- and
L-3′-Fluoro-2′,3′-didehydro-carbocyclic Nucleosides
anti-HIV-1 activity
cytotoxicity
(µM)a
(µM)
B
configuration
EC50
>100
>100
>100
>100
>100
EC90
>100
>100
>100
>100
>100
72.0
>100
33.5
PBM
CEM
Vero
adenine 31
D
D
D
D
D
L
L
L
L
L
D
D
D
D
D
D
95.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
N/A
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
N/A
guanosine 32
hypoxanthine 33
cytosine 37
thymine 38
cytosine 18
thymine 19
adenine 26
hypoxanthine 27
guanine 28
adenine 34
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
21.1
7.1
6.4
10.3
>100
>100
>100
20.7
14.8
40.6
2.8
guanine 35
0.41
>100
>100
68.8
0.087b
hypoxanthine 36
cytosine 39
thymine 40
carbovir
>100
>100
>100
>100
N/A
>100
>100
0.27b
a Anti-HIV activity evaluated in PBM cells against HIV-1LAI unless otherwise indicated. b Anti-HIV activity evaluated in PBM cells against HIV-1xxBRU
.
Table 4. Activity of D-3′-F-C-d4G against Lamivudine-Resistant Virus
(HIV-1M184V) in Human PBM Cells Using Carbovir as Control
Table 5. In Vitro Anti-HIV Activity of Selected 3′-FC-d4Ns and
Carbovir against HIV Wild Type Virus and Correlation with Calculated
Energy of Complex (Inhibitor-TP)/HIV-RT
xxBRU
M184V
EC50
Erel
EC50
(µM)
EC90
(µM)
EC50
(µM)
EC90
(µM)
compd
(µM)a
(kcal/mol)b
compd
FIa
D-3′-F-C-d4G 35
L-3′-F-C-d4G 28
carbovir
0.41
20.7
-24.4
+18.8
-10.8
D-3′-F-C-d4G 35
0.098
0.087
0.58
0.27
3.8
0.20
14.9
1.1
38.8
2.3
carbovirb
0.087c
a FI is the fold increase (EC50 HIV-1M184V/EC50 HIV-1xxBRU). b We also
performed the experiment using AZT and 3TC as control, in which AZT is
not but 3TC is highly cross-resistant to M184V mutant, and compound 35
is cross-resistant to M184V mutant (data not shown).
a EC50 in PBM cells against HIV-1LAI unless otherwise indicated. Erel
b
) (binding energy of inhibitor-TP) - (binding energy of natural 2′-dNTP).
c EC50 in PBM cells against HIV-1xxBRU
.
ship between the binding affinity and the antiviral activity.10-14
From present studies, the most active compound D-3′-F-C-d4G
35 has the most favorable relative binding energy (-24.4 kcal/
mol, Table 5), which is significantly higher than that of its lower
activity L-counterpart 28 (+18.8 kcal/mol, Table 5).
The minimized structure showed that D-3′-F-C-d4G was
bound tightly in the well-defined binding pocket inside the wild
type HIV-RT (Figure 2a). The triphosphate moiety is stabilized
by the extensive hydrogen bonds with amino acids Arg65,
Lys70, Lys72, Asp113 and Ala114. The carbocyclic ring stacks
right over the phenyl ring of Tyr115 forming a favorable
hydrophobic π-π interaction, which has been observed in our
previous reports.12-14 Also, the 3′-fluorine is strongly interacting
to HIV-1M184V (Table 4). Molecular modeling was thus per-
formed to understand the potential mechanism of the cross-
resistance (vide infra).
Molecular Modeling Studies. Among all the synthesized
nucleosides, D-3′-F-C-d4G is the most active compound, while
its L-form exhibited only marginal activity against HIV-1. From
the studies of drug resistant mutant, it was found that the anti-
HIV activity of D-3′-F-C-d4G to HIV-RTM184V significantly
decreased in comparison to the wild type virus. To understand
the molecular basis of antiviral activity as well as the drug-
resistance, molecular modeling studies were conducted on the
interactions between the NRTI and HIV-RT. Our previous
modeling studies have qualitatively demonstrated the relation-

1832 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Wang et al.
Figure 2. (a) Binding mode of D-3′-F-C-d4G-TP/HIV-RTWT complex. The triphosphate moiety is stabilized by hydrogen bonding with residues
Lys65, Arg72, Lys70, Asp113, and Ala114. The other strong hydrogen bond is detected between the 3′-fluoro and the backbone amide of Tyr115.
Also, the sugar ring is located right over the phenyl ring of Tyr115, forming a favorable hydrophobic interaction. (b) Comparing the binding mode
of D- and L-3′-F-C-d4G-TP, a decreased hydrophobic interaction is observed for the latter due to the hydrogen bond of its 3′-fluoro with backbone
amide of Asp185 pulling the sugar ring away from the top of Tyr115 (indicated by the blue arrow).
Table 6. In Vitro Anti-HIV-1 Activity of D-3′-F-C-d4G against Wild
Type (WT) and M184V Virus in Human PBM Cells Using Carbovir as
Positive Control and Correlation with Calculated Energy of Complex
(Inhibitor-TP)/HIV-RT after Molecular Dynamics Simulations
with the backbone amide of Tyr115 (Figure 2a). Stabilized by
the combined effects of hydrophobic interaction and hydrogen
bonding with Tyr115, D-3′-F-C-d4G is thus bound tightly with
HIV-RT, reflecting a higher level of anti-HIV activity, although
the initial kinase might have also played a significant role in
determining the observed anti-HIV potency. In the case of L-3′-
F-C-d4G, the 3′-fluorine is at a reasonable distance (2.0 Å) to
interact in a hydrogen bond with backbone amide of Asp185
in lieu of Tyr115. However, we noticed that this interaction
pulls the carbocyclic ring away from the Tyr115, which
decreases the hydrophobic π-π interaction (Figure 2b) and leads
to a lower binding affinity (Table 5).
xxBRU (WT)
M184V
a
a
EC50
Erel
EC50
Erel
c
compd
D-3-F-C-d4G 35 0.098 -270.2d
carbovir
0.087 -310.0d
(µM) (kcal/ mol) (µM) (kcal/ mol) FIb
∆Erel
3.8
0.20
84.9d
38.8 -355.1d
-63.4d
2.3 -246.6d
a Erel ) (binding energy of inhibitor-TP) - (binding energy of natural
2′-dNTP). b FI is the fold increase (EC50 HIV-1M184V/EC50 HIV-1xxBRU).
c ∆Erel ) Erel(WT) - Erel(M184V). d These values were calculated based
on the molecular dynamics results (refer to Experimental Section).
It has been well understood that the M184V mutation causes
serious problems in positioning the L-nucleoside triphosphate
at the active site by interfering the sugar ring with the bulky
side chain of Val184. However, reports for the D-nucleosides,
which confer significant cross-resistant to M184V, are rare. The
antiviral activity of D-3′-F-C-d4G in the current report showed
a marked decrease in the HIV-1M184V in comparison to the HIV-
the steric hindrance as we observed for the L-nucleosides.
However, a steric clash was noticed between the Val184 side
chain and the sugar ring of the final residue of the primer. In
the D-3′-F-C-d4G/HIV-RTM184V complex, Val184 pushes the last
residue of primer away and changes the conformation of
adjacent Asp185. The conformational change of Asp185
propagates to magnesium atoms, Asp110, and triphosphate
moiety of D-3′-F-C-d4G, through the strong electrostatic interac-
tions between two magnesium atoms and nearby negatively
charged residues such as Arg72 (Figure 3a). Although the D-3′-
1WT (Tables 4 and 6). To understand the underlying mechanism,
we further conducted the molecular dynamics studies of D-3′-
F-C-d4G-TP, carbovir-TP and dGTP binding with the wild type
HIV-RT as well as M184V mutant. According to our model, it
is unlikely that the resistance of M184V mutant is caused by

2′,3′-Unsaturated 3′-Fluoro Carbocyclic Nucleosides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1833
Figure 3. (a) Mutation of methionine to valine at position 184 induces conformational changes of the key residues inside the active site, such as
Asp185, Asp110, Val111, and Arg72. (b) Comparison of the bind modes of D-3′-F-d4G-TP with HIV-RTWT (yellow color, left) and HIV-RTM184V
(atom type, right). The propagated effect from the mutation on the codon 184 lifted the D-3′-F-d4G-TP from the surface of Tyr115 and causes a
decrease of the hydrophobic interaction, resulting in a loss of binding affinity. Furthermore, the catalytic distance lengthened significantly (3.9 to
5.1 Å). (c) The comparison of the bind modes of carbovir-TP with HIV-RTWT (yellow color, left) and HIV-RTM184V (atom type, right). The binding
mode is almost maintained in the mutant enzyme compared with the wild type enzyme.
F-C-d4G still maintains the hydrogen bond between the 3′-
fluorine and backbone amide of Tyr115, inhibitor’s sugar ring
was lifted away from the surface of the Ty115 aromatic ring,
resulting in the loss of hydrophobic stacking interaction which
may decrease the relative binding energy (Figure 3b right, Table
6). Furthermore, the primer/template reposition results in the

1834 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Wang et al.
zyloxy-3-hydroxyl-4-(O-triphenylmethyloxymethyl)-cyclopen-
tane (4). Epoxide 2 (26.0 g, 56.2 mmol) was dissolved in a 1.0 M
THF solution of Super-Hydride (180 mL, 180 mmol) at 0 °C. The
suspension was allowed to warm up to room temperature and stirred
for 30 min, and EtOAc/H2O was added to quench the reaction.
The organic layer was collected, and the aqueous layer was extracted
with EtOAc. The combined organic layer was dried over MgSO4
and concentrated in vacuo. The residue was purified by column
chromatography on a silica gel (EtOAc:hexanes ) 1:8) to give 3
(1.5 g, 6%) as a colorless oil and 4 (24.0 g, 92%) as a colorless
oil. Compound 3: 1H NMR data is identical to the literature.14
Compound 4: [R]25D +18.98° (c 0.93, CHCl3); 1H NMR (500 MHz,
CDCl3) δ 7.43-7.22 (m, 20H), 4.48 (s, 2H), 4.01-3.96 (m, 2H),
3.25-3.22 (m, 1H), 2.96 (t, J ) 8.5 Hz, 1H), 2.82 (d, J ) 6.0 Hz,
1H), 2.49-2.44 (m, 1H), 2.17-2.04 (m, 2H), 1.94-1.89 (m, 1H),
1.44-1.25 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 144.1, 138.4,
128.7, 128.5, 127.9, 127.7, 127.6, 127.1, 86.8, 79.1, 76.6, 70.7,
66.2, 46.9, 40.7, 34.0. Anal. calcd for (C32H32O3) C, H.
(-)-(2R,4R)-4-O-Benzyloxy-2-(O-triphenylmethyloxymethyl)-
cyclopentan-1-one (5). To a solution of alcohol 4 (23.5 g, 50.6
mmol) in anhydrous CH2Cl2 (300 mL) were added 4 Å molecular
sieve (40.5 g), pyridinium dichromate (37.6 g, 101.2 mmol), and
acetic acid (4.4 mL, 76.0 mmol). After the mixture was stirred at
room temperature for 3 h, Celite was added and stirred for another
30 min. The resulting brown slurry mixture was filtered over a
Celite pad. The filtrate was concentrated in vacuo, and the residue
was purified by column chromatography on a silica gel (EtOAc:
hexanes ) 1:50 to 1:20) to give ketone 5 (19.0 g, 81%) as a white
solid: mp 106-108 °C; [R]25D -59.32° (c 0.62, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 7.38-7.20 (m, 20H), 4.52 (s, 2H), 4.35 (t, J
) 5.0 Hz, 1H), 3.48-3.45 (m, 1H), 3.21-3.19 (m, 1H), 2.67-
2.62 (m, 1H), 2.57-2.38 (m, 3H), 2.17-2.10 (m, 1H); 13C NMR
(125 MHz, CDCl3) δ 184.7, 143.9, 138.1, 128.7, 128.5, 127.8,
127.7, 127.6, 127.0, 86.6, 74.8, 70.6, 62.1, 46.6, 45.6, 33.0. Anal.
calcd for (C32H30O3) C, H.
significant increase of the catalytic distance (3.9 Å in the wild
type vs 5.1 Å in the M184V mutant) between the 3′-OH (last
residue of the primer) and R-phosphate (D-3′-F-C-d4G-TP)
(Figure 3b). Consequently, the incorporation of D-3′-F-C-d4G-
MP into viral DNA chain in HIV-1M184V would be expected to
be more difficult than in the HIVWT, reflecting its decreased
antiviral activity against the mutant. In the case of carbovir-
TP, the relative binding energy to the M184V mutation also
decreased, but to a less extent than the D-3′-F-C-d4G-TP, which
is in accordance with the biological data (Table 6). Binding
mode analysis of carbovir-TP/HIV-RTM184V complex revealed
that the hydrophobic interaction was almost maintained and
catalytic distance experienced only a small increase (3.2 Å in
wild type vs 3.8 Å in M184V mutant, Figure 3c). These changes
may not significantly affect the incorporation of carbovir-MP
into viral DNA.
In summary, molecular modeling studies illustrated the
important roles of π-π interaction and the additional hydrogen
bond in the binding affinity of D- and L-nucleosides in the HIV-
RT activity site. The cross-resistance of compound 35 to HIV-
RTM184V may be partially due to the primer/template reposi-
tioning and resultant increased catalytic distance and the loss
of hydrophobic interaction.
Experimental Section
General Methods. Melting points were determined on a Mel-
temp II apparatus and were uncorrected. Nuclear magnetic reso-
nance spectra were recorded on a Varian Mercury 400 spectrometer
1
at 400 MHz for H NMR and 100 MHz for 13C NMR or Varian
1
Inova 500 spectrometer at 500 MHz for H NMR and 125 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 bs (broad singlet). UV spectra were
recorded on a Beckman DU-650 spectrophotometer. Optical rota-
tions were measured on a Jasco DIP-370 digital polarimeter. High-
resolution mass spectra were recorded on a Micromass Autospec
high-resolution mass spectrometer. TLC was performed on Uni-
plates (silica gel) purchased from Analtech Co. Column chroma-
tography 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.
(+)-(2R,4R)-4-O-Benzyloxy-1,1-difluoro-2-(O-triphenylmethy-
loxymethyl)-cyclopentane (6). Ketone 5 (19.0 g, 41.1 mmol) was
dissolved in neat diethyl aminosulfur trifluoride (DAST, 86.0 mL,
656.2 mmol) at room temperature. After being stirred at 40 °C for
36 h, the reaction mixture was diluted with 300 mL of CH2Cl2 and
then slowly added into saturated NaHCO3 (600 mL) solution. The
organic layer was collected, and the aqueous layer was extracted
with CH2Cl2. The combined organic layer was dried over MgSO4
and concentrated in vacuo. The residue was purified by column
chromatography on a silica gel (EtOAc:hexanes ) 1:100) to give
Microwave-Assisted Synthesis. Reactions were run in the
Discover reactor module (CEM Corporation) of focused microwaves
with a magnetron operating at a frequency at 2.45 GHz and a
maximum power output of 300 W. The thick-wall tube was heated
in a closed cavity located inside the instrument with continuous
stirring. The temperature was measured by an IR pyrometer inside
the reactor.
6 (13.5 g, 68%) as a pale yellow syrup: [R]27 +3.00° (c 0.90,
D
CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.48-7.21 (m, 20H), 4.47
(s, 2H), 4.05 (s, 1H), 3.37-3.34 (m, 1H), 3.11-3.08 (m, 1H), 2.83-
2.78 (m, 1H), 2.44-2.32 (m, 2H), 2.22-2.18 (m, 1H), 1.75-1.69
(m, 1H); 13C NMR (125 MHz, CDCl3) δ 144.0, 138.1, 128.9, 130.7
(t, J ) 252.5 Hz), 128.8, 128.5, 127.9, 127.8, 127.7, 127.6, 127.0,
86.8, 75.0, 70.8, 61.0 (d, J ) 7.6 Hz), 44.7 (t, J ) 21.5 Hz), 43.0
(t, J ) 11.2 Hz), 34.3 (d, J ) 6.1 Hz). Anal. calcd for (C32H30
F2O2) C, H.
(+)-(1R,2S,3S,4R)-2,3-Anhydro-1-O-benzyloxy-4-(O-triphe-
nylmethyloxymethyl)-cyclopentane (2). To a suspension of ep-
oxide 114 (16.0 g, 72.6 mmol) in anhydrous CH2Cl2 were added
DMAP (4.4 g, 36.3 mmol), triethylamine (15.1 mL, 109 mmol),
and trityl chloride (30.4 g, 109 mmol) at room temperature. The
reaction mixture was stirred for 24 h at room temperature and
concentrated in vacuo. EtOAc/H2O was added to the residue, and
the organic layer was collected, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
a silica gel (EtOAc:hexanes ) 1:40 to 1:20) to give 2 as a white
(-)-(2R,4R)-1,1-Difluoro-4-hydroxy-2-hydroxymethyl-cyclo-
pentane (7). To a solution of 6 (12.7 g, 26.2 mmol) in anhydrous
CH2Cl2 (300 mL) was added iodotrimethylsilane (11.2 mL, 78.6
mmol) at -20 °C. The reaction mixture was allowed to warm up
to room temperature and stirred for 6 h. An additional portion of
iodotrimethylsilane (5.6 mL, 39.3 mmol) was added, and the
mixture was stirred for another 8 h. The reaction was quenched
with MeOH at -20 °C, and the mixture was carefully neutralized
with solid NaHCO3. The resulting brown mixture was filtered, and
the filtrate was concentrated in vacuo. The residue was purified by
column chromatography on a silica gel (MeOH:CH2Cl2 ) 1:100
solid (30.6 g, 91%): mp 102-103 °C; [R]24 +46.71° (c 1.47,
D
CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.39-7.24 (m, 20H), 4.59
(d, J ) 2.5 Hz, 2H), 4.17 (t, J ) 7.5 Hz, 1H), 4.19-4.16 (m, 1H),
3.54 (s, 1H), 3.45 (s, 1H), 3.14-3.15 (m, 1H), 2.96-2.99 (m, 1H),
2.57 (d, J ) 7.0 Hz, 1H), 1.66-1.55 (m, 2H); 13C NMR (CDCl3,
125 MHz) δ 143.9, 138.4, 128.7, 128.4, 127.9, 127.8, 127.7, 127.1,
86.8, 79.1, 71.7, 64.2, 57.9, 56.7, 39.2, 28.7. Anal. calcd for
(C32H30O3) C, H.
to 1:10) to give diol 7 (2.8 g, 70%) as a pale brown oil: [R]25
D
1
-21.53° (c 0.90, MeOH); H NMR (500 MHz, CDCl3) δ 4.46-
4.43 (m, 1H), 3.83-3.76 (m, 2H), 2.82-2.70 (m, 1H), 2.48-2.38
(m, 1H), 2.28-2.19 (m, 1H), 2.02-1.90 (m, 2H), 1.77 (bs, 2H);
13C NMR (125 MHz, CDCl3) δ 131.8 (dd, J ) 252.4 and 248.0
Hz) 68.6 (t, J ) 5.2 Hz), 60.5 (d, J ) 8.6 Hz), 46.2 (t, J ) 21.5
(+)-(1R,2S,4R)-1-O-Benzyloxy-2-hydroxyl-4-(O-triphenylm-
ethyloxymethyl)-cyclopentane (3) and (+)-(1R,3S,4R)-1-O-Ben-

2′,3′-Unsaturated 3′-Fluoro Carbocyclic Nucleosides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1835
Hz), 45.7 (t, J ) 23.9 Hz), 36.3 (d, J ) 5.8 Hz). Anal. calcd for
(C6H10 F2O2) C, H.
(d, J ) 2.4 Hz), 133.4 (d, J ) 1.2 Hz), 130.0 (dd, J ) 252.8 and
3.2 Hz), 129.7, 127.8, 61.6 (dd, J ) 7.6 and 1.9 Hz), 48.0 (dd, J
) 23.4 and 20.2 Hz), 47.9 (dd, J ) 6.1 and 4.2 Hz), 45.8 (t, J )
22.4 Hz), 37.3 (d, J ) 4.8 Hz) 26.8, 19.3. HR-MS calcd for
(C22H29F2NOSi + H)+ 390.2065, found 390.2030. Anal. calcd for
(C22H29F2NOSi) C, H, N.
(-)-(2R,4R)-2-(O-tert-Butyldiphenylsilyloxymethyl)-1,1-difluoro-
4-hydroxy-cyclopentane (8). To a solution of diol 7 (2.6 g, 17.1
mmol) and imidazole (1.9 g, 27.4 mmol) in anhydrous CH2Cl2 (100
mL) was slowly added tert-butyldiphenylsilane chloride (4.8 mL,
18.8 mmol) at 0 °C during 1 h. The resulting mixture was stirred
at 0 °C for another 45 min and quenched with MeOH. The mixture
was concentrated in vacuo, and the residue was dissolved in EtOAc/
H2O. The organic layer was collected, and the aqueous layer was
extracted with EtOAc. The combined organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo, and
the residue was purified by column chromatography on a silica gel
(EtOAc:hexanes ) 1:20 to 1:4) to give 8 (4.9 g, 74%) as a colorless
oil: [R]27d -14.62° (c 0.90, CHCl3); 1H NMR (500 MHz, CDCl3)
δ 7.67-7.37 (m, 10H), 4.44 (s, 1H), 3.76 (ddd, J ) 450.0, 10.5
and 5.5 Hz, 2H), 2.81-2.69 (m, 1H), 2.50-2.40 (m, 1H), 2.24-
2.16 (m, 1H), 2.04-1.95 (m, 2H), 1.05 (s, 9H); 13C NMR (125
MHz, CDCl3) δ 135.6, 133.4, 133.4, 131.1, 129.7, 127.7, 68.7 (d,
J ) 6.7 Hz), 61.5 (d, J ) 8.1 Hz), 46.4 (t, J ) 22.4 Hz), 45.8 (t,
J ) 23.9 Hz), 37.1 (d, J ) 5.2 Hz), 26.8, 19.2. Anal. calcd for
(C22H28F2O2Si) C, H.
(-)-(2R,4R)-2-(O-tert-Butyldiphenylsilyloxymethyl)-1,1-difluoro-
4-[(methylsulfonyl)oxy]cyclopentane (9). Methanesulfonyl chlo-
ride (0.4 mL, 5.12 mmol) in 10 mL of anhydrous CH2Cl2 (10 mL)
was slowly added to a solution of alcohol 8 (1.00 g, 2.56 mmol)
and triethylamine (1.4 mL, 10.2 mmol) in anhydrous CH2Cl2 (10
mL) at 0 °C. The reaction mixture was allowed to warm up to
room temperature and kept for 4 h. After the solvent was removed
in vacuo, the residue was dissolved in EtOAc and washed with
water. The organic layer was dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromatography
on a silica gel (EtOAc:hexanes ) 1:20 to 1:10) to give 9 (1.17 g,
98%) as a colorless oil: [R]25D -19.66° (c 0.32, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 7.67-7.38 (m, 10H), 5.26 (m, 1H), 3.76 (d,
J ) 5.5 Hz, 2H), 3.03 (s, 3H), 2.74-2.50 (m, 3H), 2.36-2.32 (m,
1H), 2.18-2.12 (m, 1H), 1.05 (s, 9H); 13C NMR (125 MHz, CDCl3)
δ 135.6, 133.0 (d, J ) 2.0 Hz), 129.9, 129.8 (t, J ) 250.9 Hz),
127.8, 77.8 (dd, J ) 3.75 and 7.6 Hz), 61.0 (dd, J ) 2.9 and 4.8
Hz), 46.4 (dd, J ) 21.5 and 23.9 Hz), 43.7 (t, J ) 26.8 Hz), 34.8,
34.7 (t, J ) 4.8 Hz), 26.8, 19.2. HR-MS calcd for (C23H30NF2O4-
SSi + H)+ 469.1680, found 469.1662.
(+)-(1S,4R)-9-[6-(O-tert-Butyldiphenylsilyloxymethyl)-2,3-
dideoxy-3,3-difluoro-cyclopentanyl]uracil (13). To a suspension
of silver cyanate (810 mg, 5.4 mmol) in anhydrous benzene (20
mL) was added â-methoxyacryloyl chloride (650 mg, 5.4 mmol).
The mixture was heated under reflux for 30 min and cooled to
room temperature. The supernatant solution was added into the
solution of amine 11 (700 mg, 1.8 mmol) in anhydrous THF (30
mL) at -30 °C during 15 min. The mixture was allowed to
gradually warm up to room temperature and kept overnight. After
the solvent was removed in vacuo, the residue was purified by
column chromatography on a silica gel (EtOAc:hexanes ) 1:3 to
1:1) to give crude 12 (600 mg) as a yellow syrup which was directly
used for the next step. Crude compound 12 (600 mg) was dissolved
in 1,4-dioxane/ethanol (20 mL/20 mL) and treated with 28%
solution of ammonium hydroxide (20 mL) in a steel bomb at 90-
100 °C for 17 h. After the solvent was removed in vacuo, the residue
was purified by column chromatography on a silica gel (MeOH:
CH2Cl2 ) 1:200 to 1:100) to give 13 (360 mg, 41%) as a pale
yellow syrup: [R]26d +13.12° (c 0.31, CHCl3); UV (H2O) λmax 266.0
1
(MeOH); H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 7.67-7.38
(m, 10H), 7.22 (d, J ) 8.5 Hz, HzH1H), 5.68 (d, J ) 8.0 Hz, 1H),
5.26-5.19 (m, 1H), 3.88 (dtd, J ) 11.0, 10.0 and 5.0 Hz, 2H),
2.70-2.60 (m, 1H), 2.2.52-2.44 (m, 2H), 2.27-2.16 (m, 1H),
1.79-1.70 (m, 1H), 1.07 (s, 9H); 13C NMR (125 MHz, CD3OD) δ
162.8, 150.9, 140.0 (d, J ) 2.0 Hz), 135.6, 133.1, 133.0, 129.9,
128.3 (t, J ) 250.9 Hz), 127.9, 103.6, 61.0 (d, J ) 5.2 Hz), 49.6,
47.0 (t, J ) 21.9 Hz), 40.7 (t, J ) 24.9 Hz), 32.3 (d, J ) 4.2 Hz),
26.8, 19.3. HR-MS calcd for (C26H30F2N2O3Si + H)+ 485.2072,
found 485.2169 Anal. calcd for (C26H30F2N2O3Si) C, H, N.
(+)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-(O-tert-butyldiphen-
ylsilyloxymethyl)-cyclopentanyl]cytosine (14). To a solution of
uracil derivative 13 (360 mg, 0.74 mmol) in anhydrous acetonitrile
(25 mL) were added 2,4,6-triisopropyl benzenesulfonyl chloride
(450 mg, 1.48 mmol), 4-(dimethylamino)pyridine (90.4 mg, 0.74
mmol), and triethylamine (0.42 mL, 3.0 mmol) at 0 °C. After the
mixture was stirred at room temperature for 12 h, a 28% solution
of ammonium hydroxide (15 mL) was added to the brown mixture
and stirred at room temperature for another 12 h. The reaction
mixture was concentrated in vacuo, and the residue was purified
by column chromatography on a silica gel (MeOH:CH2Cl2 ) 1:30)
(+)-(2R,4S)-4-Azido-2-(O-tert-butyldiphenylsilyloxymethyl)-
1,1-difluoro-cyclopentane (10). Compound 9 (1.17 g, 2.5 mmol)
was dissolved in anhydrous DMF (45 mL) and heated at 130 °C
for 1.5 h. After the solvent was removed in vacuo, the residue was
dissolved in EtOAc and washed with water. The organic layer was
dried over MgSO4 and concentrated. The residue was purified by
column chromatography on a silica gel (EtOAc:hexanes ) 1:200
to 1:100) to give 10 (0.97 g, 93%) as a colorless oil: [R]26D +4.2°
to give 14 (300 mg, 84%) as a white solid: mp 250-252 °C; [R]24
D
1
+15.86° (c 0.31, MeOH); UV (MeOH) λmax 272.0 nm; H NMR
(500 MHz, CD3OD) δ 7.59-7.30 (m, 10H), 7.44 (d, J ) 7.0 Hz,
1H), 5.75 (d, J ) 7.5 Hz, 1H), 5.00-4.92 (m, 1H), 3.88 (ddd, J )
56.0, 10.5 and 6.0 Hz, 2H), 2.59-2.45 (m, 2H), 2.30-2.19 (m,
2H), 1.78-1.71 (m, 1H), 0.96 (s, 9H); 13C NMR (125 MHz, CD3-
OD) δ 165.8, 157.4, 142.0, 135.4, 133.1, 133.0, 129.7, 128.8 (t, J
) 250.9 Hz), 127.5, 95.2, 61.1 (d, J ) 8.1 Hz), 52.0 (d, J ) 5.8
Hz), 40.2 (t, J ) 24.8 Hz), 32.0 (d, J ) 4.2 Hz), 25.9, 18.6. HR-
MS calcd for (C26H31F2N3O2Si + H)+ 484.2232, found 484.2212.
Anal. calcd for (C26H31F2N3O2Si) C, H, N.
1
(c 0.51, CHCl3); H NMR (500 MHz, CDCl3) δ 7.67-7.38 (m,
10H), 3.96 (m, 1H), 3.78 (ddd, J ) 38.0, 11.0 and 5.5 Hz, 2H),
2.56-2.34 (m, 1H), 2.21-2.10 (m, 1H), 1.82-1.75 (m, 1H), 2.04-
1.95 (m, 2H), 1.06 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 135.6,
133.2 (d, J ) 3.4 Hz), 129.8, 128.9 (dd, J ) 248.5 and 254.2 Hz),
127.8, 61.2 (dd, J ) 7.1 and 2.9 Hz), 56.4 (dd, J ) 6.1 and 3.8
Hz), 47.2 (dd, J ) 23.4 and 21.0 Hz), 41.9 (t, J ) 24.8 Hz), 32.9
(t, J ) 1.5 Hz), 26.8, 19.2. HR-MS calcd for (C22H27F2N3OSi +
H)+ 416.1970, found 416.2022.
(+)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]cytosine (15). To a solution of 14 (300 mg, 0.62 mmol)
in MeOH (2 mL) was added 3 N HCl (2 mL). After being stirred
at room temperature for 17 h, the resulting mixture was coevapo-
rated with EtOH, and the residue was purified by column chro-
matography on a silica gel (MeOH:CH2Cl2 ) 1:15) to give 15 (110
mg, 72%) as a white solid: mp 134-136 °C; [R]23d +4.07° (c 0.22,
(-)-(2R,4S)-2-(O-tert-Butyldiphenylsilyloxymethyl)-1,1-difluoro-
cyclopentanamine (11). A suspension of azido compound 10 (0.97
g, 2.33 mmol) and 10% Pd/C (330 mg) in absolute EtOH was
shaken under 30 psi of H2 at room temperature for 2.0 h. Celite
was added into the solution, and the slurry was filtered through a
Celite pad. The volatile was removed in vacuo, and the residue
was purified by column chromatography on a silica gel (MeOH:
1
MeOH); UV (H2O) λmax 275.0 nm (MeOH); H NMR (400 MHz,
CD3OD) δ 7.72 (d, J ) 8.0 Hz, 1H), 5.95 (d, J ) 7.0 Hz, 1H),
5.14-5.07 (m, 1H), 3.88 (ddd, J ) 47.0, 11.5 and 5.5 Hz, 2H),
2.70-2.35 (m, 4H), 1.97-1.90 (m, 1H); 13C NMR (100 MHz, CD3-
OD) δ 165.8, 157.4, 142.0, 128.8 (t, J ) 249.2 Hz), 95.2, 59.0 (d,
J ) 7.6 Hz), 52.0 (t, J ) 7.6 Hz), 40.1 (t, J ) 25.1 Hz), 32.0 (d,
CH2Cl2 ) 1:50 to 1:10) to give 11 (0.86 g, 95%) as a colorless oil:
1
[R]26 -9.98° (c 0.79, MeOH); H NMR (500 MHz, CDCl3) δ
D
7.68-7.36 (m, 10H), 3.78 (ddd, J ) 55.0, 10.0 and 5.0 Hz, 2H),
3.46-3.39 (m, 1H), 2.51-2.28 (m, 3H), 1.93-1.82 (m, 1H), 1.45-
1.39 (m, 3H), 1.05 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 135.6

1836 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Wang et al.
J ) 3.8 Hz). HR-MS calcd for (C10H13F2N3O2 + H)+ 246.1054,
found 246.0975 Anal. calcd for (C10H13F2N3O2·0.65H2O) C, H, N.
(+)-(1S,4R)-9-[2,3-Dideoxy-2,3-didehydro-3,3-fluoro-6-hy-
droxymethylcyclopent-2-enyl]cytosine (18). To a suspension of
15 (110 mg, 0.45 mmol) in anhydrous THF:1,4-dioxane (10 mL:
10 mL) cosolvent was added potassium tert-butoxide (121 mg, 1.0
mmol). The reaction mixture was stirred at 90 °C for 9 h. The
yellow suspension was filtered through a short silica gel pad, and
the filtrate was concentrated in vacuo. The residue was purified by
column chromatography on a silica gel (MeOH:CH2Cl2 ) 1:30 to
1:15) to give 18 (35 mg, 35%) as a white solid: mp 244-248 °C;
residue was purified by column chromatography on a silica gel
(MeOH:CH2Cl2 ) 1:50 to 1:20) to give 21 as a colorless syrup
(305 mg, 37% from 8): [R]25 -10.89° (c 0.45, CHCl3); UV
d
1
(MeOH) λmax 260.0; H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H),
7.84 (s, 1H), 7.68-7.38 (m, 10H), 5.07 (m, 1H), 3.92 (ddd, J )
31.0, 10.5 and 5.0 Hz, 2H), 2.91-2.81 (m, 1H), 2.73-2.56 (m,
3H), 2.28-2.22 (m, 1H), 2.06 (bs, 1H), 1.08 (s, 9H); 13C NMR
(125 MHz, CDCl3) δ 155.6, 153.0, 150.2, 138.2, 135.6, 135.6,
133.2, 133.1, 129.9, 128.4 (dd, J ) 252.4 and 250.0 Hz), 127.8,
120.0, 61.1 (d, J ) 5.8 Hz), 49.8 (t, J ) 4.8 Hz), 47.5 (t, J ) 21.5
Hz), 33.7 (d, J ) 17.0 Hz), 26.8, 19.3. Anal. calcd for (C27H31F2N5-
OSi) C, H, N.
[R]25 +127.49° (c 0.36, MeOH); UV (H2O) λmax 284.0 nm (ꢀ
D
17115, pH 2), 274.0 nm (ꢀ 11975, pH 7), 274.0 nm (ꢀ 11680, pH
11); 1H NMR (400 MHz, CD3OD) δ 7.83 (d, J ) 7.5 Hz, HzH1H),
5.90 (d, J ) 7.5 Hz, 1H), 5.62-5.61 (m, 1H), 5.19 (s, 1H), 3.70
(ddd, J ) 132.5, 11.5 and 3.5 Hz, 2H), 2.95-2.93 (m, 1H), 2.86-
2.79 (m, 1H), 1.72-1.69 (m, 1H); 13C NMR (100 MHz, CD3OD)
δ 166.1, 166.0 (d, J ) 282.8 Hz), 157.6, 142.5, 103.3 (d, J ) 13.0
Hz), 94.3, 59.8, 55.9 (d, J ) 12.2 Hz), 42.9 (d, J ) 19.0 Hz), 31.7
(d, J ) 6.1 Hz). Anal. calcd for (C10H12F1N3O2) C, H, N.
(-)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]adenine (22). Compound 21 (300 mg, 0.59 mmol) was
dissolved in MeOH (5 mL) and treated with 3 N HCl (10 mL) at
room temperature for 16 h. After the mixture was neutralized with
solid NaHCO3, the resulting suspension was filtered and the filtrate
was concentrated in vacuo. The residue was purified by column
chromatography on a silica gel (MeOH:CH2Cl2 ) 1:50 to 1:20) to
give 22 (140 mg, 88%) as a white solid: mp 156-158 °C; [R]25
d
-9.86° (c 0.56, MeOH); UV (H2O) λmax 260.0 (MeOH); 1H NMR
(500 MHz, CD3OD) δ 8.28 (s, 1H), 8.24 (s, 1H), 5.13 (m, 1H),
3.86 (ddd, J ) 50.0, 11.5 and 6.0 Hz, 2H), 2.89-2.77 (m, 2H),
2.71-2.57 (m, 2H), 2.36-2.30 (m, 1H); 13C NMR (125 MHz, CD3-
OD) δ 156.0, 152.3, 149.4, 139.5, 128.9 (dd, J ) 251.8 and 248.0
Hz), 119.0, 59.1 (dd, J ) 8.1 and 1.9 Hz), 50.4 (dd, J ) 7.6 and
4.4 Hz), 41.1 (t, J ) 25.8 Hz), 33.0 (d, J ) 3.9 Hz). Anal. calcd
for (C11H13F2N5O) C, H, N.
(+)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]thymine (17). To a suspension of silver cyanate (400
mg, 2.7 mmol) in anhydrous benzene (8 mL) was added â-methoxy-
R-methacryloyl chloride (360 mg, 2.7 mmol). The mixture was
heated under reflux for 30 min and cooled to room temperature.
The supernatant solution was added into the solution of amine 11
(350 mg, 0.89 mmol) in anhydrous THF (8 mL) at -30 °C during
15 min. The mixture was allowed to gradually warm up to room
temperature and kept overnight. After removing the solvent in
vacuo, the residue was purified by column chromatography on a
silica gel (EtOAc:hexanes ) 1:3 to 1:1) to give crude 16 (360 mg)
as a yellow syrup which was directly used for the next step. Crude
compound 16 (360 mg) was dissolved in 1,4-dioxane (60 mL) and
treated with 3 N HCl (15 mL) at the refluxed temperature for 2.5
h. After the solvent was removed in vacuo, the residue was purified
by column chromatography on a silica gel (MeOH:CH2Cl2 ) 1:50
(-)-(1S,4R)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]adenine (26). Compound 22 (60 mg, 0.22
mmol) was converted to adenine derivative 26 (27 mg, 49%) as a
white solid using the same procedure as for 18: mp 225 °C (dec);
[R]29 -43.80° (c 0.18, DMSO); UV (MeOH) λmax 261.0 nm (ꢀ
D
17931, pH 2), 261.0 nm (ꢀ 17780, pH 7.4), 261.0 nm (ꢀ 18556,
pH 11); 1H NMR (500 MHz, CD3OD) δ 8.31 (s, 1H), 8.24 (s, 1H),
5.69-5.64 (m, 1H), 5.44 (s, 1H), 3.75 (ddd, J ) 126.5, 11.0 and
4.0 Hz, 2H), 3.09-3.04 (m, 1H), 3.02-3.00 (m, 1H), 2.05-2.00
(m, 1H); 13C NMR (125 MHz, CD3OD) δ 166.3 (d, J ) 283.2
Hz), 156.0, 152.2, 148.8, 139.8, 118.9, 103.2 (d, J ) 13.8 Hz),
60.1, 54.0 (d, J ) 12.4 Hz), 43.2 (d, J ) 18.6 Hz), 32.2 (d, J ) 6.2
Hz). Anal. calcd for (C11H12FN5O) C, H, N.
to 1:20) to give 17 (145 mg, 63%) as a white solid: mp 140-142
1
°C; [R]23 +3.9° (c 0.15, MeOH); UV (H2O) λmax 270.0 nm; H
D
NMR (400 MHz, CD3OD) δ 7.52 (s, 1H), 5.07-4.98 (m, 1H), 3.76
(ddd, J ) 33.2, 10.8 and 5.2 Hz, 2H), 2.63-2.32 (m, 4H), 1.98-
1.89 (m, 4H); 13C NMR (100 MHz, CD3OD) δ 164.8, 151.4, 137.6,
128.7 (dd, J ) 250.8 and 248.5 Hz), 110.6, 58.9 (dd, J ) 7.6 and
2.3 Hz), 50.8 (t, J ) 6.8 Hz), 39.5 (t, J ) 25.1 Hz), 31.3 (d, J )
3.8 Hz), 11.0. Anal. calcd for (C11H14F2N2O3) C, H, N.
(-)-(1S,4R)-9-[2,3-Dideoxy-2,2-difluoro-6-hydroxymethylcy-
clopentanyl]hypoxanthine (23). A crude 6-chloropurine analogue
20 (850 mg) was treated with 85% formic acid (40 mL) at 90 °C
for 3 h. After the volatile was completely removed in vacuo, the
residue was dissolved in concentrated ammonium hydroxide (35%)
and stirred at room temperature overnight. The solution was
evaporated in vacuo, and the residue was purified by column
chromatography on a silica gel (MeOH:CH2Cl2 ) 1:30 to 1:15) to
(+)-(1S,4R)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]thymine (19). Compound 17 (140 mg,
0.54 mmol) was converted to thymine derivative 19 (60 mg, 46%)
as a white solid using the same procedure as for 18: mp 182-184
°C (dec); [R]26 +23.99° (c 0.26, MeOH); UV (H2O) λmax 272.0
d
nm (ꢀ 14258, pH 2), 272.0 nm (ꢀ 14240, pH 7), 271.0 nm (ꢀ 11651,
give 23 (93 mg, 39% from 8) as a white solid: mp 244-246 °C;
1
1
pH 11); H NMR (400 MHz, CD3OD) δ 7.69 (s, 1H), 5.58-5.53
[R]24 -10.08° (c 0.23, MeOH); UV (MeOH) λmax 248.0 nm; H
D
(m, 1H), 5.15 (s, 1H), 3.69 (ddd, J ) 121.6, 11.6 and 3.2 Hz, 2H),
2.91-2.90 (m, 1H), 2.78-2.70 (m, 1H), 1.76-1.70 (m, 1H); 13C
NMR (100 MHz, CD3OD) δ 165.2, 166.0 (d, J ) 282.7), 151.6,
138.2, 109.7, 103.4 (d, J ) 13.7 Hz), 59.6 (d, J ) 1.5 Hz), 54.7
(d, J ) 12.2 Hz), 42.7 (d, J ) 18.3 Hz), 30.9 (d, J ) 6.1 Hz), 11.0.
Anal. calcd for (C11H13FN2O3‚0.1H2O) C, H, N.
(-)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-(O-tert-butyldiphen-
ylsilyloxymethyl)-cyclopentanyl]adenine (21). Triphenylphosphine
(2.12 g, 8.1 mmol) and diisopropyl azodicarboxylate (1.59 mL, 8.1
mmol)) were dissolved in anhydrous THF:1,4-dioxane (14 mL:7
mL) cosolvent and cooled to 0 °C. The resulting yellowish
suspension was further cooled to -78 °C. 6-Chloropurine (1.25 g,
8.1 mmol) and a solution of alcohol 8 (630 mg, 1.61 mmol) in
THF (14 mL) were added successively. The resulting mixture was
kept at -78 °C for 0.5 h and then stirred at room temperature for
24 h. MeOH was added to quench the reaction, and the mixture
was evaporated in vacuo. The residue was purified by column
chromatography on a silica gel (EtOAc:hexanes ) 1:10) to give
20 as a crude product, which was treated with methanolic ammonia
in a steel bomb at 100 °C for 24 h. After evaporation in vacuo, the
NMR (500 MHz, CD3OD) δ 8.22 (s, 1H), 8.09 (s, 1H), 5.18-5.10
(m, 1H), 3.84 (ddd, J ) 53.5, 11.5 and 6.0 Hz, 2H), 2.93-2.76
(m, 2H), 2.70-2.58 (m, 2H), 2.36-2.29 (m, 1H); 13C NMR (125
MHz, CD3OD) δ 157.6, 148.8, 145.0, 139.0, 128.8 (dd, J ) 251.9
and 248.0 Hz), 124.3, 59.1 (d, J ) 6.2 Hz), 50.7 (dd, J ) 7.1 and
4.2 Hz), 41.3 (t, J ) 25.8 Hz), 33.1 (d, J ) 4.2 Hz). Anal. calcd
for (C11H12F2N4O2) C, H, N.
(-)-(1S,4R)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]hypoxanthine (27). Compound 23 (22 mg,
0.081 mmol) was converted to inosine derivative 27 (10 mg, 49%)
as a white solid using the same procedure as for 18: mp 226-230
°C; [R]27 -33.05° (c 0.20 MeOH); UV (H2O) λmax 249.0 nm (ꢀ
D
12220, pH 2), 249.0 nm (ꢀ 12820, pH 7), 255.0 nm (ꢀ 13552, pH
1
11); H NMR (500 MHz, CD3OD) δ 8.26 (s, 1H), 8.08 (s, 1H),
5.66-5.72 (m, 1H), 5.43 (s, 1H), 3.75 (ddd, J ) 124.5, 11.5 and
4.0 Hz, 2H), 3.03-3.09 (m, 1H), 2.97 (dt, J ) 14.0 and 9.5 Hz,
1H), 2.05-2.00 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 166.4
(d, J ) 283.9 Hz), 157.6, 148.4, 145.1, 139.2, 123.9, 103.1 (d, J )
14.8 Hz), 60.0, 54.2 (d, J ) 12.9 Hz), 43.3 (d, J ) 18.6 Hz), 32.4
(d, J ) 5.8 Hz). Anal. calcd for (C11H11FN4O2‚0.1H2O) C, H, N.

2′,3′-Unsaturated 3′-Fluoro Carbocyclic Nucleosides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1837
(-)-(1S,4R)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]guanine (25). A solution of alcohol 8 (600 mg, 1.54
mmol), tripheylphosphine (1.61 g, 6.1 mmol), and 6-chloro-N2-
isobutyrylpurine (1.47 g, 6.1 mmol) in anhydrous THF (20 mL)
was cooled to 0 °C, and then diisopropyl azodicarboxylate (1.2
mL, 6.1 mmol) was slowly added during 2 h. The reaction mixture
was allowed to warm up to room temperature and stirred for 4 h.
The clear yellowish solution was concentrated in vacuo, and the
residue was purified by column chromatography on a silica gel
(EtOAc:hexanes ) 1:10 to 1:1) to give the corresponding nucleoside
24 as a crude product, which was treated with 85% formic acid
(30 mL) at 90 °C for 4 h. After the volatile was completely
removed, the residue was further treated with methanolic ammonia
at room temperature for 24 h. After the concentration in vacuo,
the residue was purified by column chromatography on a silica gel
(MeOH:CH2Cl2 ) 1:20 to 1:10) to give 25 (80 mg, 18% from 8)
as a white solid: mp: 253 °C (dec); [R]27d -9.30° (c 0.25, MeOH);
UV (MeOH) λmax 253.0 nm; 1H NMR (500 MHz, CD3OD) δ 7.86
(s, 1H), 3.83 (ddd, J ) 50.5, 11.5 and 5.5 Hz, 2H), 2.81-2.72 (m,
2H), 2.61-2.51 (m, 2H), 2.30-2.22 (m, 1H); 13C NMR (125 MHz,
CD3OD) δ 158.0, 153.7, 151.8, 136.4, 128.9 (dd, J ) 252.2 and
248.0 Hz), 116.5, 59.1 (dd, J ) 8.1 and 2.4 Hz), 50.0 (dd, J ) 7.1
and 3.4 Hz), 41.1 (t, J ) 25.8 Hz), 32.8 (d, J ) 3.4 Hz). HR-MS
calcd for (C11H13N5F2O2 + H)+ 286.1116, found 286.1137.
(+)-(1S,4R)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]guanine (28). To a suspension of 25 (70
mg, 0.25 mmol) in anhydrous DMF (8 mL) was added potassium
tert-butoxide (120 mg, 1.0 mmol). The reaction mixture was stirred
at 70 °C for 24 h. The yellow suspension was filtered through a
short silica gel pad and the filtrate was concentrated in vacuo. The
residue was purified by column chromatography on a silica gel
(MeOH:CH2Cl2 ) 1:20 to 1:10) to give 28 (30 mg, 45%) as a white
solid: mp 224-227 °C (dec); [R]27d +40.13° (c 0.11, MeOH); UV
(H2O) λmax 253.0 nm (ꢀ 13343, pH 2), 251.0 nm (ꢀ 13635, pH 7),
260.0 and 268.0 nm (ꢀ 11470 and 11893, respectively, pH 11); 1H
NMR (500 MHz, CD3OD) δ 7.92 (s, 1H), 5.52-5.46 (m, 1H), 5.37
(s, 1H), 3.74 (ddd, J ) 114.0, 11.0 and 4.0 Hz, 2H), 3.05-3.00
(m, 1H), 2.90 (dt, J ) 14.0 and 9.0 Hz, 1H), 2.01-1.96 (m, 1H);
13C NMR (125 MHz, CD3OD) δ 165.9 (d, J ) 282.9 Hz), 158.0,
153.8, 151.2, 136.6, 116.2, 103.1 (d, J ) 13.9 Hz), 60.1, 53.2 (d,
J ) 12.9 Hz), 43.2 (d, J ) 19.1 Hz), 32.2 (d, J ) 5.6 Hz). Anal.
calcd for (C11H12FN5O2‚1.1H2O) C, H, N.
MeOH); UV (H2O) λmax 254.0 nm (ꢀ 10631, pH 2), 252.0 nm (ꢀ
1
11008, pH 7), 256.0 nm (ꢀ 9234, pH 11); mp >250 °C; H NMR
(500 MHz, CD3OD) δ 7.83 (s, 1H), 3.79 (ddd, J ) 51.0, 14.5 and
6.5 Hz, 2H), 2.77-2.70 (m, 2H), 2.57-2.54 (m, 2H), 2.32-2.21
(m, 1H); 13C NMR (100 MHz, CD3OD) δ 158.0, 153.7, 151.8,
136.4, 128.9 (dd, J ) 251.5 and 246.9 Hz), 116.5, 59.1 (d, J ) 8.4
Hz), 49.9 (d, J ) 3.8 Hz), 41.0 (t, J ) 25.9 Hz), 32.8 (d, J ) 3.8
Hz). Anal. calcd for (C11H13F2N5O2‚0.3H2O) C, H, N.
(+)-(1R,4S)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]hypoxanthine (33): mp 227 °C; [R]23 +9.97° (c
D
1
0.45, MeOH); UV (MeOH) λmax 247.0 nm; H NMR (500 MHz,
CD3OD) δ 8.25 (s, 1H), 8.09 (s, 1H), 5.14 (m, 1H), 3.84 (ddd, J )
55.0, 11.5 and 6.0 Hz, 2H), 2.82 (m, 2H), 2.65 (m, 2H), 2.32 (m,
1H); 13C NMR (125 MHz, CD3OD) δ 157.6, 148.8, 145.0, 139.0,
128.7 (dd, J ) 251.5 and 247.5 Hz), 124.3, 59.0 (dd, J ) 8.0 and
2.4 Hz), 50.7, 41.3 (t, J ) 25.9 Hz), 33.1 (d, J ) 4.8 Hz). Anal.
calcd for (C11H12F2N4O2) C, H, N.
(+)-(1R,4S)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]adenine (34): mp 218-220 °C (dec);
[R]22 +41.00° (c 0.13, DMSO); UV (MeOH) λmax 260.0 nm (ꢀ
d
10925, pH 2), 261.0 nm (ꢀ 10807, pH 7), 261.0 nm (ꢀ 11780, pH
1
11); H NMR (500 MHz, CD3OD) δ 8.31 (s, 1H), 8.24 (s, 1H),
5.66 (br, 1H), 5.44 (s, 1H), 3.75 (ddd, J ) 130.0, 11.0 and 3.5 Hz,
2H), 3.05 (br, 1H), 2.98 (m, 1H), 2.00 (m, 1H); 13C NMR (125
MHz, CD3OD) δ 166.2 (d, J ) 287.5 Hz), 155.9, 152.1, 148.8,
139.8, 118.8, 103.2 (d, J ) 14.3 Hz), 60.0, 53.9 (t J ) 12.9 Hz),
43.2 (d, J ) 18.6 Hz), 32.2 (d, J ) 5.8 Hz); MS: m/z 250 (M +
1). Anal. calcd for (C11H12FN5O) C, H, N.
(-)-(1R,4S)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]guanine (35). To a suspension of 33 (60
mg, 0.21 mmol) in anhydrous DMF (5 mL) was added potassium
tert-butoxide (82 mg, 0.69 mmol). The reaction mixture in the thick-
walled tube was placed in a microwave synthesizer and irradiated
at maximum output power of 300 W with air-cooling at 70 °C for
10 min. The brown suspension was filtered through a short silica
gel pad, and the filtrate was concentrated in vacuo. The residue
was purified by column chromatography on a silica gel (MeOH:
CH2Cl2 ) 1:20 to 1:10) to give 35 (40 mg, 72%) as a white solid:
mp 220 °C (dec); [R]25d -44.12° (c 0.11, MeOH); UV (H2O) λmax
253.0 nm (ꢀ 13553, pH 2), 252.0 nm (ꢀ 14393, pH 7), 256.0 nm
and 268.0 (ꢀ 11186 and 11829, respectively, pH 11); 1H NMR (500
MHz, CD3OD) δ 7.92 (s, 1H), 5.49 (m, 1H), 5.37 (s, 1H), 3.74
(ddd, J ) 113.5, 11.5 and 4.0 Hz, 2H), 3.02 (m, 1H), 2.90 (dt, J )
14.0 and 9.0 Hz, 1H), 2.00-1.97 (m, 1H); 13C NMR (125 MHz,
CD3OD) δ 165.9 (d, J ) 282.0 Hz), 158.0, 153.8, 151.2, 136.6,
116.2, 103.1 (d, J ) 13.7 Hz), 60.0, 53.2 (d, J ) 12.9 Hz), 43.2
(d, J ) 18.3 Hz), 32.2 (d, J ) 5.4 Hz). Anal. calcd for (C11H12-
FN5O2‚0.9H2O) C, H, N.
(+)-(1R,4S)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]hypoxanthine (36): mp 254-256 °C;
[R]27D +34.01° (c 1.51, MeOH); UV (H2O) λmax 249.0 nm (ꢀ 10354,
pH 2), 248.5 nm (ꢀ 13925, pH 7), 254.0 nm (ꢀ 10142, pH 11); 1H
NMR (500 MHz, CD3OD) δ 8.15 (s, 1H), 8.08 (s, 1H), 5.68 (m,
1H), 5.44 (s, 1H), 3.86 (dd, J ) 10.5 and 4.5 Hz, 1H), 3.60 (m,
1H), 2.96-2.85 (m, 2H), 1.97 (m, 1H); 13C NMR (125 MHz, CD3-
OD) δ 166.4 (d, J ) 283.9 Hz), 157.6, 148.4, 145.1, 139.2, 123.9,
103.1 (d, J ) 14.8 Hz), 60.0, 54.2 (d, J ) 12.9 Hz), 43.3 (d, J )
18.6 Hz), 32.4 (d, J ) 5.8 Hz). Anal. calcd for (C11H11FN4O2) C,
H, N.
(+)-(2S,4S)-2-(O-tert-Butyldiphenylsilyloxymethyl)-1,1-difluoro-
4-hydroxy-cyclopentane (29): [R]24 +14.97° (c 0.83, CHCl3);
D
1H NMR (500 MHz, CD3OD) δ 7.67-7.66 (m, 4H), 74.5-7.37
(m, 11H), 4.44 (bs, 1H), 3.80 (dd, J ) 5.0 and 10.5 Hz, 1H), 3.72
(dd, J ) 6.0 and 10.5 Hz, 1H), 2.75 (m, 1H), 2.45 (m, 1H), 2.20
(q, J ) 14.0 Hz, 1H), 2.02-1.98 (m, 2H), 1.05 (s, 9H); 13C NMR
(125 MHz, CD3OD) δ 135.6, 133.3 (d, J ) 3.9 Hz), 133.2, 131.2,
129.8 (d, J ) 1.4 Hz), 129.1, 127.8 (d, J ) 1.4 Hz), 68.8, 61.5,
46.4 (t, J ) 21.4 Hz), 46.0 (t, J ) 28.1 Hz), 37.1 (d, J ) 5.3 Hz),
26.8, 19.3. Anal. calcd for (C22H28F2O2Si) C, H, N.
(+)-(2S,4R)-2-(O-tert-Butyldiphenylsilyloxymethyl)-1,1-difluoro-
cyclopentanamine (30): [R]26D +10.02° (c 0.68, MeOH); 1H NMR
(500 MHz, CDCl3) δ 7.68-7.37 (m, 10H), 3.86-3.70 (m, 2H),
3.49-3.39 (m, 1H), 2.52-2.28 (m, 3H), 1.94-1.79 (m, 1H), 1.45-
1.39 (m, 3H), 1.05 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 135.6
(d, J ) 2.4 Hz), 133.4, 130.0 (dd, J ) 253.1 and 249.2 Hz), 129.7,
127.7, 61.6 (dd, J ) 7.7 and 2.3 Hz), 48.1 (m), 48.0 (dd, J ) 22.9
and 21.3 Hz), 46.1 (t, J ) 22.9 Hz), 37.3 (d, J ) 4.6 Hz) 26.8,
19.2. Anal. calcd for (C22H29F2NOSi‚0.2H2O) C, H, N.
(-)-(1R,4S)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]cytosine (37): mp 132-134 °C; [R]26D -4.62° (c 0.3,
MeOH); UV (H2O) λmax 282.0 nm (ꢀ 16381, pH 2), 274.0 nm (ꢀ
(+)-(1R,4S)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
clopentanyl]adenine (31): mp 158-160 °C; [R]25D +9.50° (c 0.33,
MeOH); UV (H2O) λmax 260.0 (MeOH); 1H NMR (500 MHz, CD3-
OD) δ 8.24 (s, 1H), 5.12 (m, 1H), 3.92 (ddd, J ) 50.0, 11.5 and
5.5 Hz, 2H), 2.85 (m, 2H), 2.65 (m, 2H), 2.32 (m, 1H); 13C NMR
(125 MHz, CD3OD) δ 155.9, 152.3, 149.3, 139.5, 128.7 (dd, J )
251.6 and 248.0 Hz), 119.0, 59.2 (t, J ) 5.8 Hz), 50.3, 41.1 (t, J
) 25.8 Hz), 32.9. Anal. calcd for (C11H13F2N5O) C, H, N.
(+)-(1R,4S)-9-[2,3-Dideoxy-3-difluoro-6-hydroxymethylcyclo-
1
10363, pH 7), 273.0 nm (ꢀ 11099, pH 11); H NMR (500 MHz,
CD3OD) δ 7.72 (d, J ) 7.5 Hz, 1H), 5.95 (d, J ) 7.0 Hz, HzH1H),
5.14-5.07 (m, 1H), 3.81 (ddd, J ) 47.0, 11.5 and 5.5 Hz, 2H),
2.70-2.35 (m, 4H), 1.97-1.90 (m, 1H); 13C NMR (125 MHz, CD3-
OD) δ 165.8, 157.5, 142.2, 128.9 (dd, J ) 250.9 and 249.0 Hz),
95.2, 59.0 (t, J ) 5.6 Hz), 52.0 (t, J ) 6.2 Hz), 40.1 (t, J ) 25.2
Hz), 32.0 (d, J ) 4.4 Hz). Anal. calcd for (C10H13F2N3O2‚0.6H2O)
C, H, N.
pentanyl]guanine (32): mp 258 °C (dec); [R]28 +9.29° (c 0.15,
d

1838 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
(-)-(1R,4S)-9-[2,3-Dideoxy-3,3-difluoro-6-hydroxymethylcy-
Wang et al.
energy change from one iteration to the next was less than 0.05
kcal/mol. The annealed enzyme-inhibitor complexes were mini-
mized by using Kollman-All-Atom force field until iteration number
reached 5000.
clopentanyl]thymine (38): [R]26 -3.2° (c 0.25, MeOH); mp
D
142-144 °C; UV (H2O) λmax 271.0 nm (ꢀ 8478, pH 2), 271.0 nm
1
(ꢀ 8509, pH 7), 270.0 nm (ꢀ 7587, pH 11); H NMR (400 MHz,
CD3OD) δ 7.56 (s, 1H), 5.08-5.02 (m, 1H), 3.80 (ddd, J ) 33.2,
The structures (D-3′-F-C-d4G-TP/HIV-RTWT, D-3′-F-C-d4G-TP/
9.2 and 4.8 Hz, 2H), 2.65-2.37 (m, 4H), 2.01-1.92 (m, 4H); 13
C
HIV-RTM184V, carbovir-TP/HIV-RTWT, carbovir-TP/HIV-RTM184V
,
NMR (125 MHz, CD3OD) δ 164.9, 151.4, 137.7, 128.8 (dd, J )
250.9 and 248.0 Hz), 110.7, 58.9 (dd, J ) 8.0 and 2.4 Hz), 50.8 (t,
J ) 4.2 Hz), 39.5 (t, J ) 23.6 Hz), 31.4 (d, J ) 3.8 Hz), 11.0.
Anal. calcd for (C11H14F2N2O3) C, H, N.
GTP/ HIV-RTWT, and GTP/HIV-RTM184V) were further confirmed
by the molecular dynamics studies using MACROMODEL, version
9.1 (Schrodinger, Inc.). The complex was minimized until there
was no significant movement in atomic coordinates using MMFF94s
force field in the presence of GB/SA continuum water model before
performing molecular dynamics simulations. A conjugate gradient
Polak-Ribiere first derivative method was used for energy
minimization. Molecular dynamics simulations on nucleoside-TP/
RT complex was performed with MMFF94s in the presence of GB/
SA continuum water model on a SGI Origin 300 workstation
running the IRIX 6.5 operating system by heating from 0 to 300 K
over 5 ps and equilibrating at 300 K for an additional 10 ps.
Production dynamics simulations were carried out for 500 ps with
a step size of 1.5 fs at 300 K. A shake algorithm was used to
constrain covalent bonds to hydrogen atoms. A distance constrain
was used to constrained the two magnesium atoms with Asp110,
Val111, Asp185, and R- and â-phosphate of nucleotide. For
simulation of the nucleoside-TP/RT complex, the residues further
away than 15 Å from the active site were not considered and the
residues from 6 to 15 Å were constrained by harmonic constraints.
Only residues inside the 6 Å sphere from the bound nucleoside-TP
were allowed to move freely.
(-)-(1R,4S)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]cytosine (39). Compound 37 (60 mg, 0.24
mmol) was converted to cytosine derivative 39 (46 mg, 84%) as a
white solid using the same procedure as for 35: mp 220-230 °C;
[R]25 -124.63° (c 0.33, MeOH); UV (H2O) λmax 283.0 nm (ꢀ
D
15638, pH 2), 274.0 nm (ꢀ 10805, pH 7), 274.0 nm (ꢀ 10512, pH
11); 1H NMR (500 MHz, CD3OD) δ 7.83 (d, J ) 7.5 Hz, HzH1H),
5.90 (d, J ) 7.5 Hz, 1H), 5.63-5.59 (m, 1H), 5.19 (s, 1H), 3.70
(ddd, J ) 132.5, 10.5 and 3.5 Hz, 2H), 2.95-2.93 (m, 1H), 2.82
(td, J ) 14.0 and 9.0 Hz, 1H), 1.73-1.68 (m, 1H); 13C NMR (125
MHz, CD3OD) δ 166.1, 166.0 (d, J ) 283.2 Hz), 157.5, 142.6,
103.3 (d, J ) 13.4 Hz), 94.3, 59.8 (d, J ) 1.9 Hz), 55.9 (d, J ) 8.1
Hz), 42.9 (d, J ) 18.6 Hz), 31.7 (d, J ) 6.1 Hz). Anal. calcd for
(C10H12FN3O2) C, H, N.
(-)-(1R,4S)-9-[2,3-Dideoxy-2,3-didehydro-3-fluoro-6-hydroxy-
methylcyclopent-2-enyl]thymine (40): mp 184-186 °C (dec);
[R]25D -24.17° (c 0.15, MeOH); UV (H2O) λmax 272.0 nm (ꢀ 15633,
pH 2), 273.0 nm (ꢀ 15750, pH 7), 271.0 nm (ꢀ 12548, pH 11); 1H
NMR (400 MHz, CD3OD) δ 7.69 (d, J ) 1.2 Hz, 1H), 5.57-5.54
(m, 1H), 5.15 (s, 1H), 3.69 (ddd, J ) 121.6, 11.6 and 3.2 Hz, 2H),
2.93-2.90 (m, 1H), 2.78-2.70 (td, J ) 14.4 and 9.2 Hz, 1H), 1.76-
1.70 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 165.2, 166.0 (d, J
) 282.7), 151.6, 138.2, 109.7, 103.4 (d, J ) 13.7 Hz), 59.6 (d, J
) 2.3 Hz), 54.7 (d, J ) 12.2 Hz), 42.7 (d, J ) 18.3 Hz), 30.9 (d,
J ) 6.1 Hz), 11.0. Anal. calcd for (C11H13FN2O3) C, H, N.
Antiviral and Cytotoxicity Assay. HIV drug susceptibility
assays were performed as previously described.28 Cytotoxicity
assays in PBM, CEM, and Vero cells were conducted as previously
described.29
Acknowledgment. This research was supported by the U.S.
Public Health Service grants (AI25899, AI32351, and AI41980)
from the National Institute of Allergy and Infectious Diseases,
Emory’s CFAR Grant 5-P30-AI-50409, and the Department of
Veterans Affairs.
Supporting Information Available: Elemental analysis data
for compounds 2-40. This material is available free of charge via
the Internet at http://pubs.acs.org.
Molecular Modeling Study. (a) Conformational Analysis: The
initial conformations of inhibitors were constructed by builder
module in MACROMODEL, version 8.5 (Schrodinger, Inc.), based
on the crystal structure of carbovir. The Monte Carlo conformational
search was performed in 5000-step, in the presence of GB/SA water
model using MMFFs force field in MACROMODEL.
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