Synthesis, antiviral activity, cytotoxicity and cellular pharmacology of l-3′-azido-2′,3′-dideoxypurine nucleosides

Article abstract of DOI:10.1016/j.ejmech.2011.05.051

Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified l-3′-azido-2′,3′-dideoxypurine nucleosides. These l-nucleoside analogs were evaluated against HIV and hepatitis B virus. The l-3′-azido-2′,3′-dideoxypurines nuc

Full text of DOI:10.1016/j.ejmech.2011.05.051

European Journal of Medicinal Chemistry 46 (2011) 3832e3844
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiviral activity, cytotoxicity and cellular pharmacology
of
-30-azido-20,30-dideoxypurine nucleosides
L
Hong-wang Zhang a, Mervi Detorio a, Brian D. Herman b, Sarah Solomon a, Leda Bassit a, James H. Nettles a,
Aleksandr Obikhod a, Si-jia Tao a, John W. Mellors b, Nicolas Sluis-Cremer b, Steven J. Coats c,
,
Raymond F. Schinazi a
*
a Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, and the Veterans Affairs Medical Center, Decatur, GA 30033, USA
b Department of Medicine, Division of Infectious Diseases, University of Pittsburg School of Medicine, Pittsburgh, PA 15261, USA
c RFS Pharma, LLC, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified L
-30-
Article history:
azido-20,30-dideoxypurine nucleosides. These
L-nucleoside analogs were evaluated against HIV and
Received 20 April 2011
Received in revised form
19 May 2011
Accepted 22 May 2011
Available online 30 May 2011
hepatitis
B virus. The L
-30-azido-20,30-dideoxypurines nucleosides were metabolized to nucleoside
50-triphosphates in primary human lymphocytes, but exhibited weak or no antiviral activity against HIV-1.
The nucleosides were also inactive against HBV in HepG2 cells. Pre-steady state kinetic experiments
demonstrated that the L
-30-azido-20,30-dideoxypurine triphosphates could be incorporated by purified
HIV-1 reverse transcriptase, although their catalytic efficiency (kpol/Kd) of incorporation was low. Inter-
Keywords:
estingly, a phosphoramidate prodrug of
L
-30-azido-20,30-dideoxyadenosine exhibited anti-HIV-1 activity
L-Nucleoside analogs
without significant toxicity.
Antiviral agents
HIV
Published by Elsevier Masson SAS.
HBV
30-Azido purine nucleosides
1. Introduction
infection with mild or no toxicity (Fig. 1). Further, L
-20-deoxy-
thymidine (LdT, Telbivudine, TyzekaÒ) is a specific and selective
anti-HBV agent [18]. Lamivudine, emtricitabine, and telbivudine
are FDA-approved for clinical use. This experience has revealed
Nucleoside analogs continue to be the cornerstone of anti-
HIV-1 therapy [1], but the emergence of drug-resistant mutants
that render current therapies ineffective [2], drives interest in the
development of new and novel nucleoside analogs with high
potency, low toxicity, and favorable resistance profiles. The first
that
to, and in some cases greater than their
-nucleoside analogs may provide a more favorable toxicological
L-nucleoside analogs may display antiviral activity comparable
D-counterparts. Moreover,
L
synthesis of an
for decades it was assumed that only natural
L
-nucleoside was reported in 1964 [3]; however,
-configuration
profile along with greater stability against cellular enzymes such as
deaminases [19].
D
nucleosides could exhibit biological activity due to the stereo-
specificity of enzymes in living systems [4,5]. As a consequence,
little attention was paid to L-nucleoside analogs until the 1990s
with the discovery of lamivudine (3TC, Fig. 1) that exhibited
potent antiviral activity against HIV-1 and HBV [6,7]. Following
We recently identified 30-azido-20,30-dideoxyguanosine (30-
azido-ddG) as a lead compound [20] that exhibits potent activity
against HIV-1 variants containing either discrimination mutations
such as K65R, L74V or M184V or multiple thymidine analog muta-
tions (TAMs) that enhance nucleotide analog excision. Furthermore,
30-azido-ddG does not exhibit cytotoxicity in primary lymphocytes
or epithelial and T-cell lines, and does not decrease the mitochon-
drial DNA content of HepG2 cells. We have refined and improved
this discovery, a large number of L-nucleoside analogs were
synthesized and their antiviral activities evaluated [8e15]. In
addition to lamivudine (3TC, EpivirÒ) [7,16], emtricitabine (FTC,
EmtrivaÒ) [17] exhibited antiviral activity toward HIV-1 and HBV
the synthetic methodology to prepare purine modified D
-30-azido
nucleosides [21] and subsequently prepared a number of analogs
which were studied extensively at both the cellular and HIV-1 RT
level [22]. The low toxicity and broad antiviral activity against
* Corresponding author. Tel.: þ1 404 728 7711; fax: þ1 404 728 7726.
E-mail address: rschina@emory.edu (R.F. Schinazi).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.05.051

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3833
O
N
O
N
Cl
N
NH2
N
NH2
N
N
N
F
HN
HN
N
N
N
TBSO
O
OH
NH2
TBSO
O
1) BSA
O
OH
O
OH
O
O
O
O
O
2) Transglycosylation
Conditions
S
S
N3
OH
Telbivudine
(LdT, Tyzeka)
N3
Lamivudine
(3TC, Epivir)
Emtricitabine
(FTC, Emtriva)
2
1
Scheme 1. Model reaction for the optimization of the transglycosylation reaction with
Fig. 1. Clinically approved antiviral L-nucleoside analogs.
a 30-azido nucleoside.
With knowledge of the reported microwave-assisted Vor-
brüggen glycosylation reaction which was optimized to a 5 min
130 ꢀC ribosylation reaction [24], we began our study with variation
of the reaction temperature. The stability of the azido group to
TMSOTf at elevated temperature drove us to study temperatures up
to but not higher than 130 ꢀC. The best yields of 2 were obtained at
105 ꢀC and 120 ꢀC with 10 min of heating and 2.7 equiv of TMSOTf.
At 90 ꢀC the incomplete reaction gave a 32% yield while at 130 ꢀC
some decomposition was noted. All temperatures studied had no
effect on the 1:1 anomer ratio. We next studied the effect of reaction
time on the yield of 2. We chose to reduce the amount of TMSOTf to
1 equiv and use 130 ꢀC in an effort to reduce the reaction time to
5 min or less. Starting with a 10-min reaction time the yield was
increased to 41% of 2 versus 25% obtained with 2.7 equiv of TMSOTf.
Reducing the reaction time to 5 min had little effect on the reaction
outcome while a reaction time of 3 min increased the yield to 61% of
2. Further reductions in reaction time resulted in incomplete reac-
tions. Sub-stoichiometric amounts of TMSOTf gave reduced yields of
2; however, it is worth noting that even 0.1 equiv of TMSOTf gave
a 47% yield of 2, again with no effect on anomer ratio.
clinically relevant mutation profiles discovered with the
purine series led us to investigate the synthesis and antiviral
activity of the corresponding L-nucleosides.
D
-30-azido
2. Results and discussion
2.1. Chemistry
Our previous work [21] identified transglycosylation as the most
efficient method by which the desired 30-azido purine analogs
could be prepared. Problems of trace AZT leading to false positives
was identified as an issue in the
-30-azido-20,30-dideoxyuridine (AZU, CS-87) as the source of the
30-azido sugar in the transglycosylation reaction. Our best condi-
tions led to yields of 30e45% as a 1.1:1 mixture of anomers
D-series and ultimately led us to use
b-D
a/b
when 1.7 equiv of silylated base was reacted with 1 equiv of 50-
protected AZU and 5.2 equiv of TMSOTf at 85 ꢀC under positive
nitrogen pressure for 18 h. Our observations of extreme water
sensitivity and negative effects of extended reaction time were
most likely related to the stability of the azide group to these
conditions and ultimately led us to examine the use of microwave
irradiation as a method to reduce the overall reaction time and
With partially optimized conditions in hand we evaluated
several solvents for their effect on the outcome of the microwave-
assisted transglycosylation reaction. Surprisingly the choice of
solvent had a large effect on anomer ratio. We evaluated dichloro-
methane, chloroform, dichloroethane, THF, and acetonitrile for yield
and anomer ratio under a standard set of transglycosylation condi-
improve the yield of the desired
b anomer. Microwave heating has
the potential to significantly shorten reaction time, increase
product yields, and enhance product purities by reducing
unwanted side reactions compared to conventional heating
methods. The short reaction times provided by microwave
synthesis make it an ideal tool for rapid reaction scouting and
optimization of reaction conditions [23]. Herein, we report our
optimization of the microwave-assisted transglycosylation meth-
tions. The yields and a/b anomer ratios were determined to be as
follows: dichloromethane (80%, 1.6:1); chloroform (85%, 1.9:1);
dichloroethane (70%, 1.5:1); THF (50%, 1.9:1); and acetonitrile (61%,
1:1). Cleaner and higher yielding reactions were noted in the chlo-
rinated solvents; however, all solvents except acetonitrile favored or
odology, its application to the synthesis of
L
-30-azido purine
strongly favored the undesired
a anomer.
analogs, and evaluation of their antiviral activity, cytotoxicity and
incorporation by HIV-1 RT.
To test the generality of the optimized transglycosylation
conditions we evaluated several different bases and obtained the
following yields and
a/b anomer ratios: 2-amino-6-chloropurine
(40%, 1:1), 6-chloropurine (82%, 1:2), 2,6-dichloropurine (34%,
1:1.4), 2-fluoro-6-aminopurine (56%, 1:1). The consistently
acceptable yields obtained from these optimized microwave-
assisted transglycosylation conditions confirmed their generality
and broad synthetic usefulness. It is quite interesting that the ratio
2.2. Microwave transglycosylation optimization
To rapidly optimize the transglycosylation conditions, an HPLC
analysis of the crude reaction was used to determine the amount
of co-eluting
with 20-methyl-20,30,50-tribenzoyl-6-chloropurine-furanosyribose
as the internal standard. The ratio of and anomers was estimated
a/b
anomers based on a calibration curve (r2 of 0.9998)
of
a and b anomers shows a clear dependence on the purine base
employed and warrants further investigation.
a
b
by analysis of the 1H NMR of the crude reaction mixture. Because
AZU is readily available in three steps from dUrd, we chose its
transglycosylation to compound 2 as the model reaction to optimize
the microwave-assisted transglycosylation reaction (Scheme 1).
We examined SnCl4, TMSI, TMSOTf, and Ti(Oi-Pr)4 as potential
Lewis acids to affect the transglycosylation of 1 to 2. SnCl4 was found
to be incompatible with 1 and led to complex mixtures. On the other
end of the reactivity spectrum, the use of Ti(Oi-Pr)4 resulted in no
reaction. When TMSI was utilized a low yield of 2 was formed that
2.3. L-Nucleoside synthesis
We next turned our attention to the synthesis of
purine nucleoside analogs. Although -AZA [25,26] and
L
-30-azido
L-AZG [27]
L
have both been studied previously and found to have little anti-
viral activity against HIV-1 and HBV, we questioned if that was due
to lack of binding or other factors. Our plan included cellular phar-
macology in human PBM cells, preparation of the corresponding
50-triphosphate (TP) forms, incorporation studies with purified
HIV-1 RT, preparation and evaluation of purine analogs and mono-
phosphate prodrugs. To take advantage of the transglycosylation
favored the a-isomer as a 1.2:1 mixture. TMSOTf provided a 1:1
anomer mixture and the yield of 2 was higher than any other Lewis
acid tested.

3834
H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
4 with lithium azide in DMF at 130 ꢀC for 2 days furnished
compound 5 in 82% yield, which is the key transglycosylation syn-
O
N
O
N
HN
HN
O
thon for the synthesis of various L
-30-azido purine nucleosides.
a
b
O
With 30-azido 5 in hand, the 30-azido 6-chloropurine analog, 6
was prepared by the above microwave transglycosylation reaction
OTBS
OH
O
O
conditions in a very satisfying 81% yield as a 1:2 mixture of a/b
OH
OH
3
LdT
anomers. Deprotection by TBAF/acetic acid and separation of the
two anomers delivered 30-azido-6-chloropurine 7, which was
further converted to its prodrug 8 with t-BuMgCl and phenyl
ethoxyalaninyl phosphorochloridate in 27% isolated yield as a 1:1
mixture of phosphorous diastereomers [28].
O
N
O
HN
N
d
c
O
O
N
OTBS
OTBS
O
O
From the 6-Cl purine nucleoside 7, 6-substituted-2-H-purine
compounds 9, 10 [29], and 12 were prepared by reaction with
sodium methoxide, ammonia, and methylamine, respectively, with
yields ranging from 40 to 80% (Scheme 3). The phosphoramidate 11
was prepared in 35% yield, as described above.
N3
4
5
Cl
Cl
N
N
N
From 50-TBS-
L
-AZT, 5, protected -AZG 13 was obtained using the
L
N
N
f
e
developed transglycosylation conditions in 37% yield (Scheme 4).
Sequential deprotection of the 2-amino group with ammonia in
methanol followed by 50-hydroxyl group unmasking with TBAF
N
N
OH
OTBS
N
O
O
N3
N3
afforded
amidate 16 in 46% yield.
For the synthesis of -AZG analogs the 2-amino-6-chlororpurine
nucleoside 17 as the pure -anomer was prepared in 15% yield in
L-AZG, 15, which was further converted to the phosphor-
6
7
Cl
L
N
N
O
b
N
O
P
PhO
two steps from 2-amino-6-chloropurine as described above
(Scheme 5). Reaction with different nucleophilic reagents afforded
the 6-methoxypurine, 19, 2,6-diaminopurine, 20 and 6-N-methyl
aminopurine, 21 in 71%, 87%, 71% yields, respectively. The
2,6-diaminopurine analog, 20, was transformed to its phosphor-
amidate prodrug in 60% yield.
The synthesis of the 2,6-dichloropurine nucleoside 24 [30], and
the 2-fluoro-6-aminopurine nucleoside, 26, was efficiently carried
out in two steps as shown inScheme 6. In the case of 26, it proved to
N
N
H
COOC2H5
O
N3
8
Scheme 2. Transglycosylation to L-3-azido purines starting from LdT. Reagents and
conditions: (a) 1 equiv LdT, 1.02 equiv TBSCl, pyridine, CH2Cl2, rt, 18 h, 92%; (b) DIAD,
TPP, tol, 80 ꢀC, 30 min, 79%; (c) LiN3, DMF, 130 ꢀC, 2 days, 67%; (d) BSA, TMSOTf,
acetonitrile, 85 ꢀC, 18 h, 81%; (e) TBAF, rt, 16 h, 35%; (f) t-BuMgCl, phenyl ethox-
yalaninyl phosphorochloridate, THF, rt, 18 h, 27%.
be quite difficult to separate the mixture of
a and b anomers.
conditions developed above, we identified LdT as an inexpensive
readily available source to prepare the 30-azido sugar for the trans-
glycosylation. While trace LdT could lead to false positives when
screening for HBV activity, at least five chemical steps and as many as
Ultimately, repeated development of a preparative TLC plate with
dichloromethane/methanol/ammonium hydroxide in a ratio of
10:1:0.1 provides sufficient resolution to obtain pure
b anomer 26.
five purification steps separated the desired
L
-30-azido purine
2.4. Pharmacological profiling
nucleoside analog from the starting LdT.
As illustrated in Scheme 2, 50-TBS protected
L-AZT, 5 was
2.4.1. Anti-HIV activity
In total, eleven L
-30-azido purine nucleosides were prepared
synthesized with 40e50% overall yield in three steps through
a straightforward procedure. In brief, compound 3, which was
synthesized from LdT by reaction with TBSCl, was treated with
a mixture of triphenylphosphine and diisopropyl azodicarboxylate
at 80 ꢀC for 30 min to form the anhydride 4. Treatment of compound
and five were also further converted to their corresponding phos-
phoramidate prodrug forms. All were evaluated against HIV-1 in
human PBM cells and cytotoxicity was determined in PBM, CEM
and Vero cells [31]. The EC50, EC90, and cytotoxicity are listed in
OCH3
N
N
a
N
N
OH
O
N3
N3
N3
9
NH2
N
NH2
N
Cl
N
N
N
N
N
N
N
N
O
P
PhO
b
d
c
N
N
N
O
OH
OH
OH
N
H
COOC2H5
O
O
O
N3
N3
10
11
7
NHCH3
N
N
N
O
12
Scheme 3. Synthesis of some L-30-azido adenosine analogs from 6-chloropurine 7. Reagents and conditions: (a) NaOCH3, CH3OH, rt, 18 h, 51%; (b) NH3/CH3OH, CH3OH, 110 ꢀC, 12 h,
72%; (c) t-BuMgCl, phenyl ethoxyalaninyl phosphorochloridate, THF, rt, 18 h, 35%; (d) methylamine, THF, 80 ꢀC, 61%.

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3835
O
N
Cl
N
O
N
Cl
N
N
N
N
N
N
HN
NH
HN
H2N
N
N
a
c
b
d
a
b
N
N
N
OTBS
OTBS
OTBS
OTBS
N
OH
5
Cl
F
Cl
O
O
O
O
O
N3
N3
N3
5
N3
23
24
14
13
NH2
N
NH2
N
N
O
N
N
O
N
N
N
N
N
HN
N
HN
OH
HN
CO2Et
F
O
O
d
N
c
N
O
P
OPh
OH
H2N
H2N
O
O
O
N3
N3
25
26
N3
N3
16
15
Scheme 6. Synthesis of some L-2-halo 6-substituted 30-azido purines. Reagents and
conditions: (a) 2,6-dichloropurine, BSA, TMSOTf, acetonitrile, 85 ꢀC, 18 h, 26%; (b) TBAF,
rt, 16 h, 41%; (c) 2-fluoro-6-aminopurine, BSA, TMSOTf, acetonitrile, 85 ꢀC, 18 h, 46%;
(d) TBAF, rt, 16 h, 34%.
Scheme 4. Synthesis of L-AZG and its phosphoramidate 16. Reagents and conditions:
(a) 2-isopropylguanine, BSA, TMSOTf, acetonitrile, 85 ꢀC, 18 h, 37%; (b) NH3/CH3OH,
CH3OH, 110 ꢀC, overnight, 85%; (c) TBAF, rt, 16 h, 30%; (d) t-BuMgCl, phenyl ethox-
yalaninyl phosphorochloridate, THF, rt, 18 h, 46%.
in this series was realized by conversion of
L-AZA, 10, to its phos-
phoramidate, 11, resulting in an EC50 value of 1.4
observed toxicity up to 100 mM in human PBM cells.
m
M with no
Table 1. The 6-Cl, 7, and 2,6-dichloro, 24, purine analogs showed
weak anti-HIV activity that may result from the cytotoxicity dis-
played toward PBM cells. The phosphoramidate prodrug of 7
(compound 8) had little impact on antiviral potency or cytotoxicity.
The same antiviral activity and cytotoxicity relationship was
observed for the 6-Cl, 2-amino purine analog 17 and its prodrug 18.
The 6-OMe, 9, 6-N-Me-amino, 12, 2-amino, 6-OMe, 19, 2-amino,
2.4.2. Anti-HBV activity
All compounds were evaluated against wild-type HBV using the
HepG2 AD38 system [32,33]. Unfortunately, none demonstrated
significant anti-HBV activity (EC50 > 50
toward HepG2 cells (data not shown). Lamivudine, used as a posi-
tive control, had an EC50 of 0.067 M.
mM, Table 1) or cytotoxicity
6-N-Me amino, 21, and 2,6-diamino, 20 analogs as well as
and -AZT, 27, and their phosphoramidates, 16 and 28, respectively,
were all inactive against HIV-1 and also did not display cytotoxicity
up to 100 M in PBM, CEM, or Vero cells. However, conversion of
the 2,6-diamino analog, 20 to the phosphoramidate, 22 resulted in
an EC50 value of 16 M with no observed toxicity up to 100 M.
-AZA, 10, displayed very weak anti-HIV-1 activity with an EC50
L-AZG, 15,
m
L
2.4.3. Cellular pharmacology in human PBM cells
m
As a first step toward understanding the antiviral data obtained
with these compounds we undertook a study of their cellular phar-
m
m
macology in PBM cells. Briefly, the
analogs were incubated for 4 h at 50
L
-30-azido purine nucleoside
mM in PHA activated human
L
value of 62 mM with no observed toxicity up to 100 mM; fluorine
PBM cells at 37 ꢀC. After incubation, cells were washed with PBS,
intracellular nucleoside and nucleotide metabolites were then
extracted with 60% ice-cold MeOH, samples were dried,
re-suspended in HPLC mobile phase, and analyzed by LCeMS/MS.
Quantification of intracellular nucleoside and nucleotide metabolites
substitution at the 2-position gave nucleoside analog 26 with no
effect on HIV-1 activity or cytotoxicity. The most potent compound
Cl
N
Cl
N
was based on calibration curves generated from the
L-nucleoside or
N
N
N
N
HN
CO2Et
OPh
a
b
from the mono-, di-, or tri-phosphate nucleotides prepared from the
5
N
N
OH
O
P
H2N
H2N
O
corresponding
We studied
D
-nucleoside series.
O
O
L-AZA, 10, -AZG, 15, along with three compounds: 9,
L
N3
N3
12, and 20, which showed no antiviral activity in our assays. In the
-series, compounds 9, 12, and 20 were found to possess anti-HIV
18
17
D
activity, and were efficiently converted to their corresponding 6-
hydroxy purines in PBM cells [21]. We also studied three addi-
tional compounds: 7, 17, and 18, which displayed weak anti-HIV
activity. For each nucleoside analog we looked for intracellular
levels of the nucleoside, nucleotides, and possible metabolism to
hypoxanthine or guanine nucleoside analogs.
In contrast to the broad 6-position metabolism observed in
the D-series, the seven analogs that could potentially undergo 6-
position metabolism remained intact under these conditions.
Five of the eight nucleosides/prodrugs tested were successfully
phosphorylated to the NTP (Table 2). However, intracellular NTP
levels were much lower (fmol) than was previously observed
d
c
f
NH2
N
N
OCH3
NHCH3
N
N
N
OH
H2N
H2N
OH
N
N
N
O
N
N
OH
H2N
N
N
O
O
N3
20
N3
N3
21
e
19
NH2
N
N
HN
CO2Et
OPh
N
O
P
O
H2N
N
with -nucleosides (pmol) (except for 10 and 11). The detection
D
O
of intracellular NTPs correlated fairly well with antiviral activity.
Specifically, compounds 10, 11, and 18 had detectable NTP levels
and also displayed anti-HIV-1 activity while compounds 9 and 12
had nucleoside TP levels below the level of detection and were
N3
22
Scheme 5. Synthesis of some L-2-amino 6-substituted 30-azido purines. Reagents and
conditions: (a) (i) 2-amino-6-chloropurine, BSA, TMSOTf, acetonitrile, 85 ꢀC, 18 h, 54%;
(ii) TBAF, THF, 5 h, rt, 27%; (b) t-BuMgCl, phenyl ethoxyalaninyl phosphorochloridate,
THF, rt, 18 h, 40%; (c) NaOCH3, CH3OH, 80 ꢀC, 16 h, 71%; (d) NH3/CH3OH, CH3OH, 110 ꢀC,
18 h, 87%; (e) t-BuMgCl, phenyl ethoxyalaninyl phosphorochloridate, THF, rt, 18 h, 60%;
(f) methylamine, THF, 80 ꢀC, 18 h, 71%.
devoid of anti-HIV-1 activity.
L
-AZG, 15, and the 2,6-
diaminopurine nucleoside 20 had detectable TP levels, but
perhaps are not ideal substrates for HIV-1 RT as they did not show
any anti-HIV-1 activity in our assay. Compounds 7 and 17 displayed

3836
H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
Table 1
Antiviral activity and cytotoxicity of L
-30-azido purine nucleosides.
Cmpd
R1
H
R2
Cl
R3
H
Anti-HIV activity (
EC50 EC90
18.1 46.3
m
M)
Anti-HBV activity (
mM)
Cytotoxicity (IC50
PBM CEM
23.8 5.3
, mM)
EC50
98
Vero
36.5
7
8
H
Cl
16.5
83.1
>100
69.2
5.8
>100
9
H
H
OCH3
NH2
H
H
>100
>100
>100
>100
>100
>100
>100
>100
10
62.3
1.4
>100
>100
>100
>100
>100
11
12
H
NH2
15.6
>100
>100
H
NHCH3
OH
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15 (
16 (
17
L
-AZG)
NH2
>100
>100
L-AZG-PD)
NH2
NH2
NH2
OH
Cl
>100
>100
>100
>100
>100
>100
>100
>100
>100
H
13.2
78.3
59.1
46.2
18
Cl
16.5
55.9
71
23.1
19
20
21
NH2
NH2
NH2
OCH3
NH2
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NHCH3
22
NH2
NH2
15.8
>100
>100
>100
>100
>100
24
Cl
Cl
F
H
H
e
11.3
55.4
26.7
51
>100
>100
10.7
1.4
19
>100
>100
26
NH2
e
>100
>100
>100
>100
>100
>100
27 (
L-AZT)
e
>100
28 (
L-AZT-PD)
e
e
>100
>100
>100
>100
>100
>100
AZT
e
e
e
e
e
e
0.005
0.02
>10
100
14.3
50.6
Lamivudine
0.067
0.32
0.06
>100
>100
>100

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3837
Table 2
calculated for the natural dNTPs. The low catalytic efficiencies of
30-azido-ddNTP analogs suggest selective incorporation of natural
dNTPs, which is consistent with a lack of -nucleotide triphos-
L-
Cellular pharmacology of L
-30-azido purine nucleosides.
L
phate incorporation observed under steady state kinetic condi-
tions in the presence of natural dNTP (data not shown). The
decreased kpol/Kd values for the
primarily by exceptionally slow rates of incorporation (kpol). The
nucleotide incorporation rates of -AZA-TP, -AZT-TP, and -AZG-TP
were also 2307-, 915-, and 2740-fold lower than values previously
reported for their
-enantiomer 30-azido-20,30-ddNTP counter-
parts [36]. By contrast 3TC-TP was incorporated 12- to 500-fold
better than the
-30-azido-ddNTPs. This suggests that the 30-
azido group in the -configuration may be the dominant structural
L-nucleoside analogs were driven
L
L
L
D
Cmpd
R1
R2
Cl
R3
Cellular pharmacology in
human PBM cells
L
Metabolites
detected
Amount
(fmol/106 cells)
7.8
L
characteristic decreasing incorporation. Interestingly, the M184V
7
H
H
H
H
H
H
7
mutation that confers high levels of resistance to 3TC and (ꢁ)-FTC
9
OCH3
NH2
None
BLDa
also conferred resistance to the L
-30-azido-20,30-dideoxypurine-50-
10
10
4400
1400
76
triphosphates, albeit at lower levels of resistance. Similar to 3TC-
TP, the M184V mutation in HIV-1 RT discriminates against the
10-MP
10-DP
10-TP
L
-30-azido nucleotides by decreasing their affinity for the
670
enzyme’s active site without significantly impacting their rates of
incorporation.
11
H
NH2
10
7500
4200
1200
7400
10-MP
10-DP
10-TP
2.5. Modeling studies
12
15
H
NHCH3
OH
H
H
12-MP
17
Model structures were generated as described in Experimental
section. Surprisingly, examination of the nucleotide binding site
NH2
15
15-MP
15-TP
3200
46
54
revealed pockets that allow for binding of either D- or L
-30-azido
substituted analogs in different modes (Fig. 2). While binding to
the active site is necessary, full activity of chain terminators
17
18
NH2
NH2
Cl
Cl
H
17
17-DP
17-TP
7.6
38.3
BLD
depends upon bond formation between nucleotide
a-phosphate
and 30-oxygen of the primer. The -sugars guide the 30-azido
D
18
17
17-MP
17-DP
17-TP
12
49
430
190
10
moiety into a complimentary region of the receptor pocket
formed by the phenylalanine side chain of F116 and backbone
interaction of Y115 similar to that shown in crystallographic
ꢀ
studies [37]. This positions the
a
-phosphate about 3.4 A from the
primer 30-oxygen. Tight association between the positive residues
R72 and K65, the Mgþþ, and the
-phosphate are thought to
mediate the phosphotransfer reaction. However, the 30-azido of
-nucleotides, such as -AZG-TP (Fig. 2b), best fill a receptor cavity
20
NH2
NH2
H
20-MP
20-TP
3.5
7.6
a
a
BLD ¼ below the limit of detection.
L
L
flanked by methylene substituents of M184 and D185 above the
F160 pi system. While providing relatively high affinity binding
M), the 30-azido group
anti-HIV-1 activity with no detectable TPs indicating that the
activity may be more related to cytotoxicity toward PBM cells. The
most potent compound in the series, phosphoramidate 11, dis-
played significantly higher levels of nucleoside 10 and its phos-
phates. At the triphosphate level, prodrug 11 gave 11 times higher
levels of triphosphate relative to its parent nucleoside 10. This
correlates relatively well with the observed 45-fold increase in
anti-HIV activity seen with 11 versus 10 (Table 1).
for certain base pairs (
impairs the 30-OH/
-phosphate bond formation (Kpol) for all
analogs tested (Table 2). The models suggest the
-30-azido
substitution sterically inhibits movement to the expected transi-
L
-AZG-TP Kd ¼ 0.23
m
a
L
-30
L
ꢀ
tion state geometry where the reacting
between the primer 30-oxygen and
-phosphate. The difference in
binding mode sheds light on the observed effect of the M184V
mutation in reducing -AZG-TP binding. A comparison between
the mutant binding site for - and
-30-azido nucleotides suggests
that the
analogs have room to bind and react with the primer 30-
oxygen, while the
-30-azido version would have a van der Waals
a-phosphate is 2.9 A
b
L
D
L
2.4.4. HIV-1 RT incorporation studies
D
Pre-steady state kinetic analyses were performed to elucidate,
L
in detail, the interactions between the
purine-5-triphosphates and the polymerase active site of HIV-1
RT. The TP forms of -AZA, -AZG, -AZT, and the 2,6-diamino
L
-30-azido-20,30-dideoxy-
(VDW) steric collision with the most favored rotamer of V184
(Fig. 2c and d). A less favored rotomer (<11%) of V184 may allow
incorporation to occur at low rates similar to Fig. 2b, and consis-
tent with experimental results (Table 2).
L
L
L
analog, 20, were synthesized and purified using the methods of
Ludwig and Eckstein [34]. The results (Table 3) demonstrated that
Base pair hydrogen bonding also appears to be critical for
binding of the L
-30-azido nucleotides to HIV-1 RT. The three
each of the
into the nascent DNA by purified wild-type HIV-1 RT. Interestingly
the -2,6-diamino analog (20-TP) was incorporated opposite
thymine indicating that the molecule acts as an adenosine
mimetic. We previously reported that the -analog of this nucle-
L-nucleoside triphosphate analogs was incorporated
hydrogen bonds forming between G and C pairs stabilize a planar
relationship between substrate/template bases and the 30-azido.
This stable relationship precisely in the binding pocket (Fig. 2b)
must be associated with the observed strong binding affinity (Kd).
Conversely, the out of plane wobble between A and T due to loss of
L
D
otide also acted as an adenosine mimetic [35]. In general, the
catalytic efficiencies of incorporation (kpol/Kd) of each of the
the 2 position H-bond is transferred through the
azido group causing a collision with either M184, D184, or the pi
L
-sugar to the 30-
L
-nucleotide analogs were markedly lower than the kpol/Kd values

3838
H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
Table 3
Pre-steady state kinetic constants of
L-AZA-TP,
L-AZG-TP, L-AZT-TP, and 20-TP.
dNTP
L
-30-Azido-ddNTP
kpol (minꢁ1
L-AZA-TP
Selectivityb
Resistancec
kpol (minꢁ1
dATPd
)
Kd
(
m
M)a
kpol/Kd
(m
Mꢁ1 minꢁ1
)
)
Kd
(m
M)a
kpol/Kd (m )
Mꢁ1 minꢁ1
a
a
WT
M184V
1464 ꢂ 234
3.2 ꢂ 1.2
2.5 ꢂ 0.8
455
355
0.34 ꢂ 0.05
15 ꢂ 1.8
31 ꢂ 3.7
0.023
0.006
19,783
59,167
e
888 ꢂ 384
0.19 ꢂ 0.04
3
dATPd
20-TP
WT
M184V
1464 ꢂ 234
3.2 ꢂ 1.2
2.5 ꢂ 0.8
455
355
0.14 ꢂ 0.01
2.3 ꢂ 1.9
20 ꢂ 15
0.062
0.009
7339
39,444
e
888 ꢂ 384
0.18 ꢂ 0.02
5.4
TTPd
L-AZT-TP
WT
M184V
485 ꢂ 59
2.1 ꢂ 0.7
2.9 ꢂ 0.8
231
138
0.58 ꢂ 0.11
55 ꢂ 6.6
>>60
0.011
e
21,000
e
e
e
394 ꢂ 53
nde
dGTPd
452 ꢂ 65
nd
L-AZG-TP
WT
M184V
4.2 ꢂ 1.4
108
e
0.10 ꢂ 0.02
0.23 ꢂ 0.17
>>60
0.458
e
235.8
e
e
nd
nd
e
dCTPd
3TC-TP
WT
72 ꢂ 42
1.1 ꢂ 0.4
3.0 ꢂ 1.5
65
26
0.71 ꢂ 0.06
0.13 ꢂ 0.05
18 ꢂ 4.7
5.46
0.017
11.9
1529
e
M184Vf
78 ꢂ 24
0.3 ꢂ 0.06
128
a
Values are the mean ꢂ S.D. of three independent experiments.
b
c
d
e
f
Selectivity is defined as (kpol/Kd)dNTP/(kpol/Kd)analog
.
Resistance (n-fold) is defined as selectivityM184V/selectivityWT
.
Data are adapted from Ref. [20]. The experimental conditions and T/P sequences were identical to those used in this study.
nd ¼ not determined.
Data are adapted from Ref. [47]. The experimental conditions and T/P sequences were identical to those used in this study.
system of F160. The negative effects of this collision can be over-
come by restoring the 2-position hydrogen bonding interaction as
3. Conclusion
demonstrated by the
L
-2,6-diamino analog (20-TP) binding with
M for -AZA-TP. The -analog
counterpart (not
We have described a rapid, simple, and general microwave-
assisted transglycosylation reaction for the synthesis of 30-azido
a Kd of 2.26 M compared to 14.8
m
m
L
L
20-TP, incorporated across from T like its
D
purine nucleosides of both
D- and L-configuration. The synthesized
shown).
compounds were evaluated for antiviral activity against both HIV
Fig. 2. Proposed models of D- and L-AZG-TP interacting with HIV-RT WT and M184V active sites. a) D-AZG-TP (green carbons) bound0to HIV-1 WT RT. The 30-azido Van der Waals
ꢀ
(VDW) surface (blue) is interacting with the receptor at Y115 and F116 as described in the text. A space of 3.4 A between the primer 3 -oxygen and
a
-phosphate (dashed orange) is
unencumbered for bond formation; b) L-AZG-TP (green carbons) bound to HIV-1 WT RT. The 30-azido binds to the receptor above F160 in the space between the primer 30-oxygen
and the -phosphate in a way that enhances affinity, but inhibits incorporation. The formation of three hydrogen bonds (light blue) between bases (GeC) of the NTP and the
a
template act through the L-sugar to stabilize a planar alignment of the 30-azido within the pocket; c) D-AZG-TP is bound to the M184V mutant and has no conflict with the VDW
steric surface of the dominant V rotomer; d) L-AZG-TP binding to the M184V mutant does show a severe clash between the 30-azido group and the steric surface (beige) of valine
consistent with the relatively high calculated Kd value (Table 2). Mgþþ atom is hidden for clarity (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3839
and HBV and in addition cytotoxicity was studied in five different
cell lines. The cellular pharmacology in PBM cells was studied for
a representative subset of the synthesized compounds. We also
(3 ꢃ 5 mL), dried, and purified by silica gel chromatography to
obtain the desired product as an oil.
examined incorporation of
triphosphates by WT and M184V RT. Our data and modeling
suggest that
-30-azido substituted nucleotides having C, G, or
diamino bases can bind to WT HIV-RT with high affinity, however,
the bulky
-30 group interferes with incorporation due to steric
conflict at residue 184. Increased affinity does not correlate with
efficacy for this class of inhibitors. The -AZA prodrug, 11, demon-
strated significant anti-HIV activity with an EC50 value of 1.4 M,
but incorporation studies with HIV-RT show a lack of significant
incorporation of -AZA. Cellular pharmacology studies with 11 in
L
-30-azido purine nucleoside-
4.1.2. General procedure B for the preparation of phosphoramidate
prodrugs
L
To a solution of the appropriate nucleoside analog (50 mg,
0.17 mmol) in THF (10 mL) was added t-BuMgCl (1 M, 0.6 mL)
dropwise. The reaction mixture was stirred for 30 min at rt, then
phenyl ethoxyalaninyl phosphorochloridate (0.6 mL, 0.6 mmol)
was added. The reaction mixture was stirred for 3 h, then ethanol
(1 mL) and ammonium chloride (0.2 mL) were added to quench the
reaction. The reaction mixture was purified by column chroma-
tography on silica gel to afford pure phosphoramidate as an
approximate 1:1 mixture of phosphorous diastereomers.
L
L
m
L
PBM cells resulted in a significant enhancement in the delivery of
nucleoside 10 and of its phosphates that may explain the observed
anti-HIV activity. However, these
L-nucleosides may be acting by
4.1.2.1. 50-O-tert-Butyldimethylsilyl-20-deoxy-
b-L-thymidine (3). To
a mechanism other than chain termination of HIV-RT. Further
exploration of 30-azido nucleosides including modification of
30-azido sugar continues in our laboratories.
a solution of LdT (1 g, 4.12 mmol) in DMF (1 mL) at 0 ꢀC was
added imidazole (0.56 g, 8.24 mmol) followed by dropwise
addition of TBSCl (0.62 g, 4.12 mmol) over a period of 10 min. The
cooling bath was removed and the reaction mixture was stirred
toward room temperature overnight. The solvent was evapo-
rated; the residue was diluted with ethyl acetate (20 mL), washed
with water, then saturated sodium bicarbonate, and dried over
sodium sulfate. The solvent was removed and the resulting solid
was purified by column silica gel chromatography. Elution with
ethyl acetate/hexane (1:1) gave 3 as a white solid; yield 1.25 g,
4. Experimental
4.1. Chemistry
Unless otherwise stated, all reactions were carried out under an
atmosphere of dry argon or nitrogen in oven-dried glassware.
Anhydrous solvents were purchased from EMD Chemicals Inc
(E. Merck, Darmstadt, Germany). Unless noted otherwise, the
reagents and materials used were obtained from commercial
suppliers. Thin layer chromatography (TLC) was carried out on
85%. 1H NMR (CDCl3):
d
8.49 (br s, 1H, NH), 7.47 (d, J ¼ 1.2 Hz, 1H,
H-6), 6.32 (dd, J ¼ 5.6 Hz, J ¼ 8.0 Hz, 1H, H-10), 4.44e4.45 (m, 1H,
H-30), 4.00 (q, J ¼ 2.4 Hz, 1H, H-40), 3.85 (dd, J ¼ 2.8 Hz, J ¼ 11.2 Hz,
1H, H-50), 3.80 (dd, J ¼ 2.4 Hz, J ¼ 11.2 Hz, 1H, H-50), 2.34 (dd,
J ¼ 2.8 Hz, J ¼ 5.6 Hz, 0.5H, H-50), 2.31 (dd, J ¼ 2.8 Hz, J ¼ 6.0 Hz,
0.5H, H-50), 2.20 (br s, 1H, OH), 2.05 (m, 1H, H-20), 1.89 (d,
J ¼ 1.2 Hz, 3H, CH3), 0.90 (s, 9H, 3 ꢃ CH3), 0.099 (s, 3H, CH3), 0.094
(s, 3H, CH3). LRMS (ESI): m/z calcd for C16H29N2O5Si [M þ H]þ:
357.2, observed 357.1.
GHLF 250 mm silica gel plates, item 21521 from Analtech, Inc.
(Newark, DE, USA). Plate layer chromatography (PLC) was employed
for purification of some products, item 02013 from Analtech, Inc. 1H
NMR spectra were taken on a Varian Unity Plus 400 spectrometer
(Varian, Inc., Polo Alto, CA, USA) at room temperature and reported
in parts per million downfield from internal tetramethylsilane.
Signal multiplicities are represented by s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quadruplet), br (broad), br s
(broad singlet), m (multiplet). All J values are in hertz. Mass spectral
analyses were performed on a Micromass TOF instrument (Hew-
lett-Packard HPLC driven electrospray MS instrument). Purity of
final compounds was determined to be >95%, using an analytical
HPLC analyses performed on a Hewlett-Packard 1100 HPLC with
4.1.2.2. 50-O-tert-Butyldimethylsilyl-20,30-dideoxy-2,30-anhydro-
b-L-
thymidine (4). To a solution of 3 (0.8 g, 2.24 mmol) and triphenyl-
phosphine (0.73 g, 2.78 mmol) at 80 ꢀC in toluene (20 mL) was
added diisopropyl azodicarboxylate (0.83 mL, 4.21 mmol). After
30 min, the reaction mixture was concentrated, dissolved in ethyl
acetate (10 mL), filtered, and again concentrated to a white solid,
which was purified by column silica gel chromatography. Elution
with ethyl acetate/hexane (1:1 to 1:0) gave 4 as a white solid; yield
a Phenomenex Gemini-NX column (2 mm ꢃ 50 mm, 3
mm, C18,
110 A) and further supported by clean NMR spectra. Mobile phase
flow was 0.5 mL/min with a 3.5 min initial hold, a 6.5 min gradient
from 96% aqueous media (0.05% formic acid) to 96% CH3CN (0.05%
formic acid), and a 15 min total acquisition time. Photo diode array
detection was from 190 to 360 nm. Microwave reactions were
carried out in a Biotage Initiator EXPUS.
200 mg, 79%. 1H NMR (CDCl3):
d
6.92 (d, J ¼ 1.2 Hz, 1H, H-6), 5.46 (d,
ꢀ
J ¼ 4.0 Hz, 1H, H-10), 5.15 (s, 1H, H-30), 4.21 (dt, J ¼ 2.4 Hz, J ¼ 7.6 Hz,
1H, H-40), 3.69 (m, 2H, H-50), 2.66 (d, J ¼ 13.2 Hz, 1H, H-20), 2.38 (dt,
J ¼ 3.6 Hz, J ¼ 12.8 Hz, 1H, H-20), 1.89 (s, 3H, CH3), 0.84 (s, 9H,
3 ꢃ CH3), 0.03 (s, 3H, CH3), 0.02 (s, 3H, CH3). LRMS (ESI): m/z calcd
for C16H27N2O4Si [M þ H]þ: 339.2, observed 339.1.
4.1.1. General procedure A for the microwave-assisted
4.1.2.3. 50-O-tert-Butyldiemthylsilyl-30-azido-20,30-dideoxy-
b-L-thy-
transglycosylation reaction
midine (5). A solution of 4 (2 g, 5.90 mmol) and lithium azide (0.5 g,
10.21 mmol) in DMF (10 mL) at 120 ꢀC was stirred for 2 days, then
the solvent was removed and the resulting residue was dissolved in
ethyl acetate (30 mL). The organic layer was washed with water,
dried over sodium sulfate and purified by column silica gel chro-
matography. Elution with ethyl acetate/hexane (1:1) gave 5 as
To a solution of 5 (0.47 g, 1.24 mmol) and an appropriate purine
base (1.48 mmol) in acetonitrile (3 mL) in an open air undried
microwave reaction vessel was added bis(trimethylsilyl)acetamide
(BSA) (1.4 mL, 5.7 mmol) and the resulting heterogeneous mixture
was sealed with a crimp cap and heated near reflux with a heat gun
until a clear solution resulted (w2e5 min). The microwave reaction
vessel was placed in an ice bath for 5 min before TMSOTf (ranged
from 0.14 mL, 0.77 mmol to 0.24 mL, 1.24 mmol) was added drop-
wise with gentle agitation while remaining in the ice bath. The
resulting reaction mixture was heated to 130 ꢀC under microwave
irradiation for 3 min, cooled to room temperature, poured into
saturated sodium bicarbonate (5 mL), extracted with ethyl acetate
a white solid; yield 1.5 g, 66.6%. 1H NMR (CDCl3):
d 8.68 (s, 1H, NH),
7.40 (d, J ¼ 1.2 Hz, 1H, H-6), 6.18 (t, J ¼ 6.0 Hz, 1H, H-10), 4.19e4.23
(m, 1H, H-30), 3.90e3.95 (m, 2H, H-40 and H-50), 3.76 (dd, J ¼ 2.0 Hz,
J ¼ 11.2 Hz, 1H, H-50), 2.38e2.44 (m, 1H, H-20), 2.17e2.23 (m, 1H, H-
20), 1.90 (d, J ¼ 1.2 Hz, 3H, CH3), 0.91 (s, 9H, 3 ꢃ CH3), 0.11 (s, 6H,
2 ꢃ CH3). LRMS (ESI): m/z calcd for C16H28N5O4Si [M þ H]þ: 382.2,
observed 382.1.

3840
H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
4.1.2.4. 9-(3-Azido-50-O-tert-butyldimethylsilyl-2,3-dideoxy-
ribofuranosyl)-6-chloro-9H-purine (6). General procedure A was
employed, purified by silica gel chromatography. Elution with ethyl
a
,
b
-
L
-
H-20), 1.22e1.26 (m, 3H, CH3), 1.12e1.19 (m, 3H, CH3). HRMS (EI): m/
z calcd for C21H26N9O6P [M þ H]þ: 532.1821, found 532.1814.
acetate/hexane (1:3); yield 410 mg, 81%.
b
-isomer/
a
-isomer ¼ 2:1.
4.1.2.9. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-6-methoxyl-9H-
b
-isomer: 1H NMR (CDCl3):
d
8.72 (s, 1H, H-8), 8.41 (s, 1H, H-2), 6.47
purine (9). A solution of 7 (30 mg, 0.11 mmol) in methanol (2 mL)
was added sodium methoxide (25%, 1 mL). The reaction mixture
was stirred overnight, carefully neutralized with Dowex 25 resin,
filtered. The solvent was concentrated and the resulting residue
was purified by column silica gel chromatography. Elution with
dichloromethane/methanol (10:1) gave 9 as a clear oil; yield 15 mg,
(dd, J ¼ 2.4 Hz, J ¼ 6.8 Hz, 1H, H-10), 4.39e4.41 (m, 1H, H-30), 4.37 (q,
J ¼ 2.8 Hz, 1H, H-40), 3.72e3.80 (m, 2H, H-50), 2.90 (m, 1H, H-20),
2.63e2.66 (m, 1H, H-20), 0.87e0.90 (m, 9H, 3 ꢃ CH3), 0.084 (s, 6H,
2 ꢃ CH3).
a d
-isomer: 1H NMR (CDCl3): 8.71 (s, 1H, H-80), 8.46 (s, 1H,
H-20), 6.41 (t, J ¼ 6.0 Hz, 1H, H-10), 4.45 (q, J ¼ 6.8 Hz, 1H, H-40),
4.06e4.08 (m, 1H, H-30), 3.93 (dd, J ¼ 3.2 Hz, J ¼ 11.2 Hz, 1H, H-50),
3.80 (dd, J ¼ 2.4 Hz, J ¼ 10.8 Hz, 1H, H-50), 2.80e2.83 (m, 1H, H-20),
2.57e2.64 (m, 1H, H-20), 0.87 (s, 9H, 3 ꢃ CH3), 0.08 (s, 6H, 2 ꢃ CH3).
LCMS (ESI): m/z calcd for C16H25ClN7O2Si [M þ H]þ: 410.1 observed
410.1.
51%. 1H NMR (CD3OD):
d 8.50 (s, 1H, H-8), 8.49 (s, 1H, H-2), 6.41 (t,
J ¼ 6.4 Hz, 1H, H-10), 4.58e4.62 (m, 1H, H-30), 4.15 (s, 3H, OCH3),
4.03 (q, J ¼ 3.2 Hz, 1H, H-40), 3.81 (dd, J ¼ 3.6 Hz, J ¼ 12.4 Hz, 1H, H-
50), 3.71 (dd, J ¼ 4 Hz, J ¼ 12.4 Hz, 1H, H-50), 2.94e3.01 (m, 1H, H-20),
2.54e2.61 (m, 1H, H-20). HRMS (EI): m/z calcd for C11H13N7O3
[M þ H]þ: 292.1158, found 292.1152.
4.1.2.5. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-6-chloro-9H-purine
(7). A mixture of TBAF (1 mL, 1 M in THF) and acetic acid (0.2 mL)
was added to a solution of 6 (150 mg, 0.36 mmol) in THF (3 mL) and
the resulting solution was stirred for 5 h. After removal of the
solvent the resulting solid was purified by column silica gel chro-
matography. Elution first with ethyl acetate/hexane (1:1) then with
4.1.2.10. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-6-methylamino-
9H-purine (12). A solution of 7 (50 mg, 0.16 mmol) in methylamine
(1 M in THF, 3 mL) was heated at 80 ꢀC in a steel bomb with stirring
for 24 h. The reaction mixture was concentrated to a residue and
purified by column chromatography on silica gel. Elution with
dichloromethane/methanol (10:1) gave 12 as a oily product; yield
methanol/dichloromethane (1:50) gave the
b
-isomer 7 as a white
solid; yield 18.8 mg, 34.8%. 1H NMR (CDCl3):
d
8.79 (s, 1H, H-8), 8.72
30 mg, 61.2%. 1H NMR (CD3OD):
d 8.22 (s, 1H, H-8), 8.19 (s, 1H, H-2),
(s, 1H, H-2), 6.47 (dd, J ¼ 5.2 Hz, J ¼ 6.4 Hz, 1H, H-10), 4.60 (q,
J ¼ 6.0 Hz, 1H, H-40), 4.02e4.05 (m, 1H, H-30), 3.81 (dd, J ¼ 3.6 Hz,
J ¼ 12.4 Hz, 1H, H-50), 3.72 (dd, J ¼ 3.2 Hz, J ¼ 12.0 Hz, 1H, H-50), 2.98
(m, 1H, H-20), 2.59 (m, 1H, H-20). 13C-NMR (CDCl3): 151.7, 151.2,
150.2, 145.7, 131.7, 85.7, 85.1, 61.3, 60.6, 37.2. HRMS (EI): m/z calcd
for C10H10ClN7O2 [M þ H]þ: 296.0622, found 296.0657.
6.30 (t, J ¼ 6.8 Hz, 1H, H-10), 4.54e4.57 (m, 1H, H-30), 4.03 (q,
J ¼ 2.8 Hz, 1H, H-40), 3.81 (dd, J ¼ 3.2 Hz, J ¼ 12.8 Hz, 1H, H-50), 3.70
(dd, J ¼ 3.2 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.05 (br s, 3H, CH3), 2.88e2.95
(m, 1H, H-20), 2.47e2.53 (m, 1H, H-20). HRMS (EI): m/z calcd for
C11H12N8O2 [M þ H]þ: 291.1318, found 291.1312.
4.1.2.11. N2-Isobutyryl-50-O-tert-butyldimethylsilyl-30-azido-20,30-
4.1.2.6. 9-(3-Azido-2,3-dideoxy-
b
-
L
-ribofuranosyl)-6-chloro-9H-purine-
dideoxy-a,b-L-guanosine (13). General procedure B was employed
50-O-[phenyl-(ethoxy-
L
-alaninyl)]-phosphate (8). General procedure
and purified by silica gel chromatography, Elution with dichloro-
B was employed and purified by silica gel chromatography. Elution
methane/methanol (30:1); yield 700 mg, 37%. 1H NMR (CDCl3):
with ethyl acetate/hexane (1:1); yield 20 mg, 27.0%. 1H NMR
d 12.14 (s, 1H, NH-3), 9.02 (s, 1H, NH), 7.94 (s, 1H, H-8), 6.09 (t,
(CD3OD):
d
8.72, 8.69 (ds, 1H, H-8), 8.71, 8.65 (ds, 1H, H-2), 7.25 (t,
J ¼ 6.4 Hz, 1H, H-10), 4.37 (q, J ¼ 4.8 Hz, 1H, H-30), 3.99 (q, J ¼ 4 Hz,
1H, H-40), 3.82 (dd, J ¼ 3.6 Hz, J ¼ 11.6 Hz, 1H, H-50), 3.77 (dd,
J ¼ 2.8 Hz, J ¼ 11.2 Hz, 1H, H-50), 2.60e2.75 (m, 2H, CH and H-20),
2.42e2.48 (m, 1H, H-20), 1.25 (d, J ¼ 3.6 Hz, 3H, CH3), 1.23 (d,
J ¼ 3.6 Hz, 3H, CH3), 0.87 (s, 9H, 3 ꢃ CH3), 0.067 (s, 3H, CH3), 0.060
J ¼ 8.0 Hz, 2H, phenyleH), 7.08e7.16 (m, 3H, phenyleH), 6.45e6.51
(m, 1H, H-10), 4.74 (q, J ¼ 6.0 Hz, J ¼ 0.4 Hz, H-40), 4.63 (q, J ¼ 0.6 Hz,
J ¼ 0.6 Hz, H-40), 4.26e4.39 (m, 2H, H-30 and H-50), 4.16e4.23 (m,
1H, H-50), 3.99e4.11 (m, 2H, CH2), 3.71e3.88 (m, 1H, H-20),
3.01e3.12 (m, 1H, H-20), 2.60e2.72 (m, 1H, CH), 1.13e1.34 (m, 6H,
2 ꢃ CH3). HRMS (EI): m/z calcd for C21H24ClN8O6P [M þ H]þ:
551.1323, found 551.1316.
(s, 3H, CH3).
a
/b
¼ 1:4. LCMS (ESI): m/z calcd for C20H33N8O4Si
[M þ H]þ: 477.2, observed 477.0.
4.1.2.12. 50-O-tert-Butyldimenthylsilyl-30-azido-20,30-dideoxyguanosine
(14). A solution of 13 (140 mg, 0.29 mmol) in saturated NH3/CH3OH
(5 mL) was heated at 110 ꢀC overnight with stirring in steel bomb.
The reaction mixture was concentrated to a residue and purified by
column chromatography on silica gel. Elution with dichloro-
methane/methanol (10:1) gave 14 as a white solid; yield 0.10 g, 85%.
4.1.2.7. 30-Azido-20,30-dideoxy-
b-L-adensoine (10). A solution of 7
(100 mg, 0.33 mmol) in saturated ammonia in methanol (10 mL)
was heated to 110 ꢀC in steel bomb. The reaction mixture was
heated overnight then concentrated and purified by column chro-
matography on silica gel. Gradient elution with dichloromethane/
methanol (20:1 to 10:1) gave 10 as a white solid; yield 67 mg, 72%.
a/b
¼ 1:4. 1H NMR (CD3OD):
d
7.90 (s, 1H, H-8), 6.14 (dd, J ¼ 5.2 Hz,
1H NMR (DMSO-d6):
d
8.34 (s, 1H, H-8), 8.13 (s, 1H, H-2), 7.34 (br s,
J ¼ 6.8 Hz, 1H, H-10), 4.50 (q, J ¼ 6.4 Hz, 1H, H-30), 3.94 (q, J ¼ 4.4 Hz,
1H, H-40), 3.83e3.85 (m, 2H, H-50), 2.77e2.84 (m, 1H, H-20),
2.46e2.53 (m, 1H, H-20), 0.88 (s, 9H, 3 ꢃ CH3), 0.05 (s, 3H, CH3), 0.06
(s, 3H, CH3). LCMS (ESI) calcd for C16H27N8O3Si [M þ H]þ: 407.2,
observed 407.1.
2H, NH2), 6.29 (t, J ¼ 6.4 Hz, 1H, H-10), 5.34 (t, J ¼ 6.0 Hz, 1H, OH),
4.61e4.65 (m, 1H, H-30), 3.92 (q, J ¼ 4.0 Hz, 1H, H-40), 3.54e3.64 (m,
2H, H-50), 2.92e2.99 (m, 1H, H-20), 2.45e2.51 (m, 1H, H-20). HRMS
(EI): m/z calcd for C10H12N8O2 [M þ H]þ: 277.1161, found 277.1155.
4.1.2.8. 30-Azido-20,30-dideoxy-
b
-L
-adenosine-50-O-[phenyl-(ethoxy-
4.1.2.13. 30-Azido-20,30-dideoxy-
b-L-guanosine (15). To a solution of
L
-alaninyl)]-phosphate (11). General procedure B was employed
14 (200 mg, 0.49 mmol) in THF (5 mL) at 0 ꢀC was added TBAF/
AcOH (2 mL, 1 M in THF/0.4 mL). The reaction mixture was stirred
for 6 h at 0 ꢀC. The reaction mixture was concentrated to a residue
and purified by gradient column chromatography on silica gel.
Elution with dichloromethane/methanol (50:1 to 50:5) gave
and purified by silica gel chromatography. Elution with dichloro-
methane/methanol (10:1); yield 20 mg, 35%. 1H NMR (CD3OD):
d
8.28, 8.22 (ds, 1H, H-8), 8.19, 8.18 (ds, 1H, H-2), 7.27 (t, J ¼ 8.4 Hz,
2H, phenyl-H), 7.12e7.20 (m, 3H, phenyleH), 6.33e6.39 (m, 1H, H-
10), 4.57e4.71 (m, 1H, H-30), 4.32e4.37 (m, 1H, H-50), 4.24e4.29 (m,
1H, H-50), 4.11e4.18 (m, 1H, H-40), 3.99e4.10 (m, 2H, CH2),
3.77e3.90 (m, 1H, CH), 2.89e3.01 (m, 1H, H-20), 2.53e2.64 (m, 1H,
b
isomer 15 as a white solid; yield 43 mg, 30%. 1H NMR (CD3OD):
7.93 (s, 1H, H-8), 6.16 (t, J ¼ 6.4 Hz, 1H, H-10), 4.52e4.56 (m, 1H, H-
d
30), 3.96 (q, J ¼ 4.4 Hz, 1H, H-40), 3.77 (dd, J ¼ 3.2 Hz, J ¼ 11.6 Hz, 1H,

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3841
H-50), 3.70 (dd, J ¼ 4.0 Hz, J ¼ 12.0 Hz, 1H, H-50), 2.80e2.87 (m, 1H,
4.1.2.17. 9-(3-Azido-2,3-dideoxy-
b
-
L
-ribofuranosyl)-2-amino-6-met-
H-20), 2.44e2.50 (m, 1H, H-50). HRMS (EI): m/z calcd for C10H12N8O3
[M þ H]þ: 293.1110, found 293.1105.
hoxypurine (19). To a solution of 17 (50 mg, 0.16 mmol) in meth-
anol (5 mL) was added sodium methoxide in methanol (4.6 M,
0.2 mL) at rt. The reaction mixture was stirred overnight, concen-
trated to a residue and purified by column chromatography on
silica gel. Elution with ethyl acetate/hexane (1:1) gave 19 as a oily
4.1.2.14. 30-Azido-20,30-dideoxy-
b-L
-guanosine-50-O-[phenyl-(ethoxy-
L
-alaninyl)]-phosphate (16). General procedure B was employed
and purified by column chromatography on silica gel. Elution with
product; yield 35 mg, 71%. 1H NMR (CD3OD):
d 8.01 (s,1H, H-8), 6.20
dichloromethane/methanol (6:1), yield 26 mg, 46%. The faster
(t, J ¼ 6.8 Hz, 1H, H-10), 4.55e4.58 (m, 1H, H-30), 4.02 (s, 4H, CH3 and
H-40), 3.80 (dd, J ¼ 3.6 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.70 (dd, J ¼ 3.6 Hz,
J ¼ 12.4 Hz, 1H, H-50), 2.87e2.94 (m, 1H, H-20), 2.43e2.49 (m, 1H, H-
20). HRMS (EI): m/z calcd for C11H14N8O3 [M þ H]þ: 307.1267, found
307.1261.
moving compound: 1H NMR (CD3OD):
d 7.82 (s, 1H, H-8), 7.14e7.34
(m, 5H, phenyleH), 6.15 (t, J ¼ 6.4 Hz, 1H, H-10), 4.55e4.59 (m, 1H,
H-30), 4.33e4.38 (m, 1H, H-40), 4.25e4.30 (m, 1H, H-50), 4.03e4.21
(m, 3H, CH2 and H-50), 3.82e3.88 (m, 1H, CH), 2.77e2.84 (m, 1H, H-
20), 2.44e2.51 (m, 1H, H-20), 1.15e1.26 (m, 6H, CH3 and CH3). HRMS
(EI): m/z calcd for C21H26N9O7P [M þ H]þ: 548.1771, found 548.1765.
4.1.2.18. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl) -2,6-diaminopuri
The slower moving compound: 1H NMR (CD3OD):
d
7.86 (s, 1H,
-ne (20). A solution of 17 (25 mg, 0.08 mmol) in saturated ammonia
in methanol (3 mL) was heated with stirring in a pressure tube at
120 ꢀC for 14 h. The reaction mixture was cooled to room temper-
ature, concentrated to a residue and purified by column chroma-
tography on silica gel. Elution with dichloromethane/methanol
(10:1) gave 20 as a white solid; yield 20 mg, 87%. 1H NMR (CD3OD):
H-8), 7.29e7.34 (m, 2H, phenyleH), 7.14e7.20 (m, 3H, phenyleH),
6.17e6.21 (t, J ¼ 4.8 Hz, 1H, H-10), 4.65e4.70 (m, 1H, H-30),
4.28e4.41 (m, 2H, H-40 and H-50), 4.05e4.14 (m, 3H, CH2 and H-50),
3.88e3.92 (m, 1H, CH), 2.90e2.96 (m, 1H, H-20), 2.48e2.54 (m, 1H,
H-20), 1.24 (dd, J ¼ 1.2 Hz, J ¼ 7.2 Hz, 3H, CH3), 1.15 (t, J ¼ 7.2 Hz, 3H,
CH3). LRMS (ESI): m/z calcd for C21H27N9O7P [M þ H]þ: 548.2,
observed 548.0.
d
7.91 (s, 1H, H-8), 6.17 (t, J ¼ 6.8 Hz, 1H, H-10), 4.53e4.57 (m, 1H, H-
30), 4.03 (q, J ¼ 2.8 Hz, 1H, H-50), 3.82 (dd, J ¼ 3.2 Hz, J ¼ 12.4 Hz, 1H,
H-50), 3.70 (dd, J ¼ 3.2 Hz, J ¼ 12.4 Hz,1H, H-50), 2.87e2.94 (m,1H, H-
20), 2.41 (dq, J ¼ 3.6 Hz, J ¼ 14 Hz, 1H, H-20). HRMS (EI): m/z calcd for
C10H13N9O2 [M þ H]þ: 292.1270, found 292.1263.
4.1.2.15. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-2-amino-6-chlo-
ropurine (17). General procedure A was employed and purified by
column chromatography on silica gel. Elution with ethyl acetate/
hexane (1:1); yield 300 mg, 54% as a 1/1
a
/
b
mixture.
b
-isomer: 1H
4.1.2.19. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-2,6-diaminopuri
NMR (CDCl3):
d
8.05 (s, 1H, H-8), 6.21 (t, J ¼ 6.0 Hz, 1H, H-10), 5.12 (s,
-ne-5-O-(phenyl-[ethoxyl- -alaninyl])-phosphate (22). General pro-
L
2H, NH2), 4.38e4.42 (m, 1H, H-30), 4.02 (q, J ¼ 4.0 Hz, 1H, H-40), 3.86
(dd, J ¼ 4.0 Hz, J ¼ 11.2 Hz, 1H, H-50), 3.79 (dd, J ¼ 3.2 Hz, J ¼ 8.4 Hz,
1H, H-50), 2.70e2.77 (m, 1H, H-20), 2.47e2.54 (m, 1H, H-20), 0.89 (s,
9H, 3 ꢃ CH3), 0.08 (s, 6H, 2 ꢃ CH3).
cedure B was employed and purified by silica gel chromatography.
Elution with dichloromethane/methanol (10:1) to afford 22 as
a white solid (20 mg, 60%). 1H NMR (CDCl3):
d 7.55 and 7.57 (s, 1H,
H-8), 7.10e7.33 (m, 5H, phenyl-H), 6.06 (q, J ¼ 6.8 Hz, 1H, H-10), 5.51
(br s, 2H, NH2), 5.12 (br s, 1H, H-30), 5.01 (br s, 1H, H-40), 4.52e4.74
(m, 3H, NH and H-50), 3.93e4.27 (m, 5H, CH2, NH2, and CH),
2.89e3.17 (m, 1H, H-20), 2.34e2.41 (m, 1H, H-20), 1.29e1.33 (m, 3H),
1.18e1.22 (m, 3H). HRMS (EI): m/z calcd for C21H27N10O6P [M þ H]þ:
547.1930, found 547.1923.
a
-isomer: 1H NMR (CDCl3):
d 8.04 (s, 1H, H-8), 6.24 (dd,
J ¼ 3.2 Hz, J ¼ 7.2 Hz, 1H, H-10), 5.12 (s, 2H, NH2), 4.29e4.32 (m, 2H,
H-30 and H-40), 3.76 (dd, J ¼ 3.2 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.71 (dd,
J ¼ 4.4 Hz, J ¼ 11.2 Hz, 1H, H-50), 2.81e2.88 (m, 1H, H-20), 2.61 (dt,
J ¼ 3.6 Hz, J ¼ 14.0 Hz, 1H, H-20), 0.90 (s, 9H, 3 ꢃ CH3), 0.08 (s, 6H,
2 ꢃ CH3). LRMS (ESI): m/z calcd for C16H26ClN8O2Si [M þ H]þ: 425.2,
observed 425.0.
4.1.2.20. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-2-amino-6-met-
To
dideoxy-
0.23 mmol) from above (as the
added a mixture of TBAF (1 mL,1 M in THF) and acetic acid (0.2 mL).
The reaction mixture was stirred for 4 h, concentrated to a residue,
and purified by column chromatography on silica gel. Elution with
a
solution of 9-(5-O-tert-butyldimethylsilyl-3-azido-2,3-
-ribofuranosyl)-2-amino-6-chloropurine (100 mg,
mixture) in THF (2 mL) was
hylaminopurine (21). A solution of 17 (50 mg, 0.16 mmol) in
methylamine (1 M in THF, 3 mL) was heated with stirring in a steel
bomb at 110 ꢀC overnight. The reaction mixture was concentrated
to a residue and purified by column chromatography on silica gel.
Elution with dichloromethane/methanol (10:1) gave 21 as a white
a,b-L
a/b
solid; yield 35 mg, 71%. 1H NMR (CD3OD):
d 7.85 (s, 1H, H-8), 6.15 (t,
ethyl acetate/hexane (1:1 to 5:1) gave the
solid; yield 20 mg, 27%.
b
isomer 17 as a white
J ¼ 7.2 Hz, 1H, H-10), 4.52e4.56 (m, 1H, H-30), 4.03 (q, J ¼ 2.8 Hz, 1H,
H-40), 3.82 (dd, J ¼ 3.2 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.70 (dd, J ¼ 2.8 Hz,
J ¼ 12.0 Hz, 1H, H-50), 3.00 (br s, 3H, CH3), 2.87e2.94 (m, 1H, H-20),
2.39 (dq, J ¼ 3.6 Hz, J ¼ 14 Hz, 1H, H-20). HRMS (EI): m/z calcd for
C11H15N9O2 [M þ H]þ: 306.1427, found 306.1420.
1H NMR (CD3OD):
d
8.27 (s, 1H, H-8), 6.24 (t, J ¼ 6.4 Hz,1H, H-10),
4.57e4.61 (m, 1H, H-30), 3.98 (q, J ¼ 3.6 Hz, 1H, H-40), 3.79 (dd,
J ¼ 4.0 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.71 (dd, J ¼ 4.0 Hz, J ¼ 12.0 Hz, 1H,
H-50), 2.89e2.95 (m, 1H, H-20), 2.48 (m, 1H, H-20). HRMS (EI): m/z
calcd for C10H11ClN8O2 [M þ H]þ: 311.0771, found 311.0766.
4.1.2.21. 9-(5-tert-Butyldimethylsilyl-3-azido-2,3-dideoxy-
a,
b
-L
-rib
was
-ofuranosyl)-2,6-dichloropurine (23). General procedure
A
4.1.2.16. 9-(3-Azido-2,3-dideoxy-
b
-L
-ribofuranosyl)-2-amino-6-chlo-
employed and purified by column chromatography on silica gel.
ropurine-5-O-[phenyl-(ethoxy- -alaninyl)]-phosphate (18). General
procedure B was employed and purified by column chromatog-
L
Elution with ethyl acetate/hexane (1:2); yield 400 mg, 26%.
a
isomer/
b
isomer ¼ 1/1.4.
b d 8.40 (s, 1H,
-isomer: 1H NMR (CDCl3):
raphy on silica gel. Elution with ethyl acetate/hexane (1:1 to 5:1);
H-8), 6.42 (dd, J ¼ 2.0 Hz, J ¼ 7.2 Hz, 1H, H-10), 4.38e4.43 (m, 2H,
H-30 and H-40), 3.76 (dd, J ¼ 3.2 Hz, J ¼ 11.6 Hz, 1H, H-50), 3.70 (dd,
J ¼ 4.4 Hz, J ¼ 11.6 Hz, 1H, H-50), 2.88e2.95 (m, 1H, H-20), 2.54e2.59
yield 10 mg, 40%. 1H NMR (CD3OD):
d 8.18 and 8.14 (ds, 1H),
7.25e7.32 (m, 2H), 7.12e7.18 (m, 3H), 6.23e6.28 (m, 1H), 4.61e4.75
(m, 1H), 4.25e4.43 (m, 2H), 4.02e4.17 (m, 3H), 3.79e3.90 (m, 1H),
2.91e3.06 (m, 1H), 2.49e2.58 (m, 1H), 1.22e1.26 (m, 3H), 1.14e1.18
(m, 3H). HRMS (ESI) calcd for C21H25ClN9O6P: 565.14, observed
(M þ 1): 566.33. HRMS (EI): m/z calcd for C21H25ClN9O6P [M þ H]þ:
566.1432, found 566.1425.
(m, 1H, H-20), 0.88 (s, 9H, 3 ꢃ CH3), 0.07 (s, 6H, 2 ꢃ CH3).
a-isomer:
1H NMR (CDCl3):
d
8.48 (s, 1H, H-8), 6.37 (t, J ¼ 5.6 Hz, 1H, H-10),
4.37 (q, J ¼ 2.4 Hz, 1H, H-40), 4.06e4.09 (m, 1H, H-30), 3.95 (dd,
J ¼ 3.2 Hz, J ¼ 11.2 Hz, 1H, H-50), 3.80 (dd, J ¼ 2.8 Hz, J ¼ 11.6 Hz, 1H,
H-50), 2.72 (m, 1H, H-20), 2.61e2.64 (m, 1H, H-20), 0.86 (s, 9H,

3842
H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3 ꢃ CH3), 0.084 (s, 3H, CH3), 0.080 (s, 3H, CH3). LRMS (ESI): m/z
calcd for C16H24Cl2N7O2Si [M þ H]þ: 444.1, observed 444.1.
stirred at rt overnight, diluted with ethyl acetate (10 mL), washed
with sat. sodium bicarbonate, dried over sodium sulfate, concen-
trated, purified by column chromatography on silica gel, eluting with
ethyl acetate/hexane (1:1), to get 28 as oily product; yield 34 mg,
4.1.2.22. 9-(30-Azido-20,30-dideoxy-
b-L-ribofuranosyl) -2,6-dichlorop
-urine (24). To a solution of 23 (200 mg, 0.45 mmol) in THF
(10 mL) was added TBAF/AcOH (2 mL, 1 M in THF/0.4 mL) at rt. The
resulting solution was stirred for 10 h, then concentrated to
a residue and purified by column chromatography on silica gel.
43%. 1H NMR (CD3OD):
d
7.55(d, J ¼ 1.2 Hz, 0.5H, H-6), 7.47 (d,
J ¼ 1.2 Hz, 0.5H, H-6), 7.32e7.36 (m, 2H, phenyleH), 7.16e7.24 (m,
3H, phenyleH), 6.13 (q, J ¼ 6.8 Hz, 1H, H-10), 4.25e4.44 (m, 3H, H-30,
H-50), 4.04e4.16 (m, 3H, H-40, CH2, H-40), 3.89e3.95 (m, 1H, CH),
2.22e2.39 (m, 2H, H-20), 1.85 and 1.81 (d, J ¼ 1.2 Hz, 3H, CH3),
1.18e1.35 (m, 6H, 2 ꢃ CH3). LRMS (ESI): m/z calcd for C21H28N6O8P
[M þ H]þ: 523.1, observed 523.06.
Elution with ethyl acetate/hexane (1:1) gave the
b
d
-isomer 24 as
8.79 (s, 1H, H-
a white solid; yield 60 mg, 40.5%. 1H NMR (CD3OD):
8), 6.40 (dd, J ¼ 5.2 Hz, J ¼ 6.8 Hz, 1H, H-10), 4.55 (q, J ¼ 6.4 Hz, 1H,
H-40), 4.01e4.04 (m, 1H, H-30), 3.81 (dd, J ¼ 3.6 Hz, J ¼ 12.4 Hz, 1H,
H-50), 3.72 (dd, J ¼ 3.2 Hz, J ¼ 12 Hz, 1H, H-50), 2.92e2.99 (m, 1 H, H-
20), 2.59e2.66 (m, 1H, H-20). HRMS (EI): m/z calcd for C10H9Cl2N7O2
[M þ H]þ: 330.0273, found 330.0267.
4.2. Pharmacological profiling
4.2.1. HIV-1 RT inhibition assays
The WT HIV-1 RT (EC 2.7.7.49) and M184V RT enzymes were
purified as described previously [38,39]. The protein concentration
of these enzymes was determined spectrophotometrically at
4.1.2.23. 9-(5-O-tert-Butyldimethylsilyl-3-azido-2,3-dideoxy-a,b-L-
ribofuranosyl)-2-fluoro-6-aminopurine (25). General procedure A
was employed purified by column chromatography on silica gel.
Elution with dichloromethane/methanol (10:1); yield 500 mg, 46%.
280 nm using an extinction coefficient (3280) of 260,450 Mꢁ1 cmꢁ1
,
and by Bradford protein assays (SigmaeAldrich, St. Louis, MO). The
RNA- and DNA-dependent DNA polymerase activities of the
purified WT and mutant enzymes were essentially identical (data
not shown). Deoxynucleotide triphosphates were purchased from
a
/
b
isomer ¼ 1/1.
b d 8.07 (s, 1H, H-8), 6.52
isomer: 1H NMR (CDCl3):
(br s, 2H, NH2), 6.25 (t, J ¼ 6.0 Hz, 1H, H-10), 4.34 (m, 1H, H-30), 4.00
(q, J ¼ 4.0 Hz, 1H, H-40), 3.79 (dd, J ¼ 3.2 Hz, J ¼ 11.2 Hz, 1H, H-50),
3.72 (dd, J ¼ 4.4 Hz, J ¼ 13.2 Hz, 1H, H-50), 2.74e2.79 (m, 1H, H-20),
2.50e2.55 (m, 1H, H-20), 0.89 (s, 9H, 3 ꢃ CH3), 0.07 (s, 6H, 2 ꢃ CH3).
GE Healthcare (Piscataway, NJ), and [
PerkinElmer Life Sciences (Boston, MA). DNA heteropolymeric
oligonucleotides were synthesized by IDT (Coralville, IA). A
g-
32P]ATP was acquired from
a
-isomer: 1H NMR (CDCl3):
d
8.06 (s, 1H, H-8), 6.55 (s, 2H, NH2),
6.31 (dd, J ¼ 2.8 Hz, J ¼ 7.2 Hz, H-10), 4.40 (q, J ¼ 5.6 Hz, 1H, H-40),
4.34 (m, 1H, H-30), 3.92 (dd, J ¼ 4.0 Hz, J ¼ 11.2 Hz, 1H, H-50), 3.69
(dd, J ¼ 4.4 Hz, J ¼ 11.2 Hz, 1H, H-50), 2.83 (m, 1H, H-20), 2.56 (m, 1H,
H-20), 0.89 (s. 9H, 3 ꢃ CH3), 0.08 (s, 6H, 2 ꢃ CH3). LRMS (ESI): m/z
calcd for C16H26FN8O2Si [M þ H]þ: 409.2, observed 409.0.
20-nucleotide (nt) DNA primer (50-TCGGGCGCCACTGCTAGAGA-30)
and a 57-nt DNA template derived from the HIV-1 PBS region
(50-CTCAGACCCTTTTAGTCAGAATGGAAANTCTCTAGCAGTGGCGCCC
GAACAGGGACA-30) were used in steady state and pre-steady state
DNA synthesis experiments. Four DNA templates were synthe-
sized, each of which contained a different nucleotide at position
30 (indicated by N in the sequence). This strategy allowed us to
4.1.2.24. 9-(3-Azido-2,3-dideoxy-b-L-ribofuranosyl)-2-fluoro-6-ami-
nopurine (26). To a stirred solution of 25 (120 mg, 0.29 mmol) in
THF (4 mL) was added a mixture of TBAF/AcOH (1 mL, 1 M in THF/
0.1 mL). The resulting solution was stirred at 0 ꢀC for 30 min then
the cooling bath was removed and stirred toward room
temperature for 4 h. The reaction mixture was concentrated to
a residue and purified by column chromatography on silica gel.
Elution with ethyl acetate/hexane/methanol (60:20:3.5) with
trace ammonium hydroxide gave 26 as a white solid; yield
evaluate the kinetics of single nucleotide incorporation for each
L-
30-azido purine nucleotide triphosphate -30-azido-NTP) in
(L
essentially the same sequence context and using the same 20-nt
primer.
4.2.2. Pre-steady state kinetic analyses
The typical experiment was performed at 37 ꢀC in 50 mM
TriseHCl (pH 7.5) containing 50 mM KCl, 10 mM MgCl2, and
varying concentrations of nucleotide. All concentrations reported
refer to the final concentrations after mixing. HIV-1 RT (200 nM)
was pre-incubated with 20 nM T/P substrate prior to mixing with
nucleotide and divalent metal ions to initiate the reaction. At
30 mg, 34%. 1H NMR (CD3OD):
d 8.24 (s, 1H, H-8), 6.24 (t,
J ¼ 6.4 Hz, 1H, H-10), 4.52e4.55 (m, 1H, H-30), 4.00 (q, J ¼ 3.6 Hz,
1H, H-40), 3.80 (dd, J ¼ 3.2 Hz, J ¼ 12 Hz, 1H, H-50), 3.70 (dd,
J ¼ 4.0 Hz, J ¼ 12.4 Hz, 1H, H-50), 2.87e2.93 (m, 1H, H-20),
2.49e2.55 (m, 1H, H-20). HRMS (EI): m/z calcd for C10H11FN8O2
[M þ H]þ: 295.1067, found 295.1060.
various timepoints (1e60 min) 5
mL aliquots were removed and
quenched with 10 L of gel loading buffer. Products were sepa-
m
rated from substrates as described above. The disappearance of
substrate (20mer) and the formation of product (21mer) were
quantified using Quantity One Software. Data were fitted by
nonlinear regression with Sigma Plot software (Systat Software,
Inc., San Jose, CA) using the appropriate equations [40]. The
apparent burst rate constant (kobs) for each particular concentra-
tion of nucleotide was determined by fitting the time courses for
the formation of product (21mer) using the following equation:
4.1.2.25. 30-Azido-20-30-dideoxy-
b-L-thymidine (27). To a mixture of
TBAF (1 mL, 1 M in THF) and acetic acid (0.2 mL) was added to
a solution of 5 (200 mg, 0.52 mmol) in THF (10 mL). The reaction
mixture was stirred at rt for 5 h, evaporated the solvent, purified by
column chromatography on silica gel, eluting with ethyl acetate/
hexane (1:1 to 2:1) gave 27 as a white solid; yield 100 mg, 71%. 1H
NMR (CD3OD):
d
7.78 (d, J ¼ 1.2 Hz, 1H, H-6), 6.13 (t, J ¼ 6.4 Hz, 1H,
H-10), 4.31e4.36 (m, 1H, H-30), 3.87e3.90 (m, 1H, H-40), 3.79 (dd,
J ¼ 2.8 Hz, J ¼ 12.4 Hz, 1H, H-50), 3.70 (dd, J ¼ 2.8 Hz, J ¼ 11.6 Hz, 1H,
H-50), 2.31e2.43 (m, 2H, H-20), 1.85 (d, J ¼ 1.2 Hz, 3H, CH3). LRMS
(ESI): m/z calcd for C10H14N5O4 [M þ H]þ: 268.1, observed 268.0.
[21mer] ¼ A[1 ꢁ exp(ꢁkobst)], where
A represents the burst
amplitude. The turnover number (kpol) and apparent dissociation
constant for nucleotide (Kd) were then obtained by plotting the
apparent catalytic rates (kobs) against nucleotide concentration
and fitting the data with the following hyperbolic equation:
kobs ¼ (kpol[dNTP])/([dNTP] þ Kd). Catalytic efficiency was calcu-
lated as the ratio of turnover number over dissociation constant
4.1.2.26. 1-(30-Azido-20,30-dideoxy-
xyl- -alaninyl])-phosphate (28). To
b
-
L
-thymidine)-O-(phenyl-[etho-
solution of 27 (40 mg,
L
a
0.15 mmol) in THF (2 mL) at 0 ꢀC was added N-methylimidazole
(0.1 mL, 1.2 mmol), then slowly added phenyl ethoxyalaninyl phos-
phorochloridate (0.45 mL, 0.45 mmol). The reaction mixture was
([kpol/Kd]). Selectivity for natural dNTP versus L
-30-azido-NTP was
calculated as the ratio of catalytic efficiency of dNTP over that of L-
30-azido-NTP.

H.-w. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3832e3844
3843
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4.3.1. Preparation of reference systems
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Starting crystal structure coordinates of HIV-1 RT/DNA duplex
complexed with different ligands, (1RTD, 1TO5, 1N6Q) [37,41,42]
were downloaded from the RCSB Protein Data Bank at www.pdb.
org. Structure 1RTD, with T-TP bound was chosen as the reference
receptor system for fitting all others using the Matchmaker func-
tion in Chimera [43]. An oxygen atom was added to the 30-position
of the primer and hydrogen atoms were added using the “Protonate
3D” function in MOE 2009.1 with default settings [44]. Residues
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ꢀ
within 8 A of the active site were minimized within the MMFF94x
force field to a shallow 1.0 kcal gradient using stepwise decreasing
tether strengths. A 30-azido group was added to T-TP, typed for
MMFF [45] and similarly optimized to produce a “pre-incorpora-
tion” model of D
-AZT-TP liganded in relation to the primer 30-OH
and the 2 catalytic Mg atoms. Catalytic site interactions were
compared to the experimental structure of “post-incorporation”/
pre-translocation AZT-MP-terminated DNA (1N6Q) [37]. The bases
of the bound TP and its’ complementary base of the template were
pair-wise modified from TeA to form, CeG, AeT, and GeC models
using “Nucleic Acid Explorer “ in MOE.
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4.3.2. Analysis of
Sugar geometries of all
described were modified to the
D
/
L
substrate interactions
-30-azido ligand interaction models
-stereochemistry in Sybyl 8.1 while
D
L
keeping base and triphosphate positions fixed [46]. 30-Azido
substitution was fit into the receptor pocket and the entire ligand
geometry was minimized to a 1 kcal gradient with MMFF95 atom
types and charges. Steric surfaces, atomic distances, H-bond inter-
actions, and 184 mutant rotamers were analyzed for all models
using Chimera 1.4.1 and Discovery Studio 3.0. [43,47].
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Acknowledgements
This work was supported primarily by NIH grant 5R01-AI-
071846, and in part by 5R37-AI-025899, 2P30-AI-050409, 5R37-AI-
041980, and the Department of Veterans Affairs (to RFS). RFS is the
founder and a major shareholder in RFS Pharma, LLC. JWM is
a consultant to RFS Pharma, LLC, and holds share options in the
company. Computational modeling was performed with assistance
of the Emory School of Medicine Biomolecular Computing Resource
(BIMCORE). Hardware and software was supported in part by
Academic Excellence Grants from Sun Microsystems and Accelrys
Corporation.
[31] (a) R.F. Schinazi, J.P. Sommadossi, V. Saalmann, D.L. Cannon, M.-W. Xie,
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HepAD38 wild type cells as previously described (reference). Compounds
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