Synthesis and antiviral activity of 2′-deoxy-2′-fluoro- 2′-C-methyl-7-deazapurine nucleosides, their phosphoramidate prodrugs and 5′-triphosphates

Article abstract of DOI:10.1016/j.bmcl.2011.09.089

Thirty novel α- and β-d-2′-deoxy-2′-fluoro-2′- C-methyl-7-deazapurine nucleoside analogs were synthesized and evaluated for in vitro antiviral activity. Several α- and β-7-deazapurine nucleoside analogs exhibited modest anti-HCV activity and cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2011.09.089

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7094–7098
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiviral activity of 20-deoxy-20-fluoro-20-C-methyl-7-
deazapurine nucleosides, their phosphoramidate prodrugs and 50-triphosphates
Junxing Shi a, Longhu Zhou b,c, Hongwang Zhang b,c, Tamara R. McBrayer a, Mervi A. Detorio b,c
,
Melissa Johns b,c, Leda Bassit b,c, Megan H. Powdrill d, Tony Whitaker a, Steven J. Coats a,
Matthias Götte d, Raymond F. Schinazi b,c,
⇑
a RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
b Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
c Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA
d Department of Microbiology and Immunology, McGill University, 3775 University St., Montreal, QC, Canada H3A 2B4
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty novel
a
- and b-
D
-20-deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleoside analogs were synthe-
Received 9 August 2011
Revised 19 September 2011
Accepted 21 September 2011
Available online 29 September 2011
sized and evaluated for in vitro antiviral activity. Several
a- and b-7-deazapurine nucleoside analogs
exhibited modest anti-HCV activity and cytotoxicity. Four synthesized 7-deazapurine nucleoside phos-
phoramidate prodrugs (18–21) showed no anti-HCV activity, whereas the nucleoside triphosphates
(22–24) demonstrated potent inhibitory effects against both wild-type and S282T mutant HCV polymer-
ases. Cellular pharmacology studies in Huh-7 cells revealed that the 50-triphosphates were not formed at
significant levels from either the nucleoside or the phosphoramidate prodrugs, indicating that insuffi-
cient phosphorylation was responsible for the lack of anti-HCV activity. Evaluation of anti-HIV-1 activity
revealed that an unusual
1 activity (EC50 = 0.71 0.25
different cell lines.
Keywords:
HCV
Antiviral
7-Deazapurine
Nucleoside
Nucleotide
Prodrug
a-form of 7-carbomethoxyvinyl substituted nucleoside (10) had good anti-HIV-
l
M; EC90 = 9.5 3.3 M) with no observed cytotoxicity up to 100 M in four
l
l
Published by Elsevier Ltd.
Mitsunobu
Hepatitis C virus (HCV) is an important pathogen affecting
nearly 170 million people worldwide.1 HCV infections become
chronic in about 50% of cases,2 and about 20% of these chronic pa-
tients develop liver cirrhosis that can lead to hepatocellular carci-
anti-HCV agents.7 Although the base-modifications have not
produced any clinically significant nucleosides, 7-deazapurine
base-modified nucleosides have exhibited some usefulness with
20-C-methyl-7-deazapurine nucleosides demonstrating the most
potent anti-HCV activity in an HCV replicon.8 Based on the potent
activity of some 20-deoxy-20-fluoro-20-C-methyl nucleosides,9 we
hypothesized that the combination of 20-deoxy-20-fluoro-20-C-
methyl ribosyl sugars and 7-deazapurine bases could produce
potent anti-HCV nucleosides. Therefore, a series of novel 20-
deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleoside analogs
were designed and synthesized. To better understand the anti-
HCV assay data, some phosphoramidate prodrugs and triphos-
phates of selected nucleosides were also prepared. These nucleo-
sides and their phosphoramidate prodrugs were evaluated in a
Huh-7 cell-based HCV replicon for anti-HCV activity and cytotoxic-
ity. The compounds were also tested for anti-HIV-1 activity and
cytotoxicity in three additional cell lines. In addition, some se-
lected nucleosides were evaluated for anti-HBV activity. Herein
we report the synthesis and biological evaluation of a series of
20-deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleoside analogs
along with the phosphoramidate prodrugs and triphosphates of
selected compounds.
noma.3 Because current therapies such as interferon-alpha (IFN-
a)
and ribavirin carry limited efficacy and are associated with signif-
icant side-effects even when used with the newly approved HCV
protease inhibitors Incivek and Victrelis, there is a need for more
effective anti-HCV agents that can be used in IFN-
a/ribavirin spar-
ing regimens.4
Nucleosides that target HCV NS5B have demonstrated advanta-
ges of broader activity against various HCV genotypes and a higher
barrier to development of resistant viruses when compared to
known HCV protease inhibitors.5 A number of modified nucleoside
analogs have been reported to have anti-HCV activity.6 Among the
sugar-modifications, 20-C-methyl featured nucleosides have shown
the most promise, and several of these nucleosides and nucleoside
monophosphate prodrugs are at various stages of clinical trials as
⇑
Corresponding author. Tel.: +1 404 728 7711; fax: +1 404 417 1535.
E-mail address: rschina@emory.edu (R.F. Schinazi).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.09.089

J. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7094–7098
7095
R2
N
R2
N
Cl
R1
R1
R1
N
BzO
N
N
BzO
OH
b
c
O
O
a
O
+
R3
N
R3
N
R3
N
N
BzO
HO
HO
O
O
O
OBz
F
OBz
F
OBz
F
OH
F
OH
F
1
2
3
4a
R1 = R3 = H, R2 =NH2
5a
4b
4c
4d
4e
4f
4g
4h
4i
R1 =F, R2 =NH2, R3 = H
R1 = Cl, R2= NH2, R3 = H
5b
5c
5d
5e
5f
R1 = Br, R2 = NH2, R3 = H
R1 = I, R2 = NH2, R3 = H
R1 = H, R2 = R3 = NH2
R1 = I, R2 = R3 = NH2
5g
R1 = F, R2 = NHMe, R3 = H 5h
R1 = R3 = H, R2 = OMe
5i
Scheme 1. Synthesis of
a- and b-D
-20-deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleosides. Reagents and conditions: (a) LiAl(t-BuO)3H, THF, 0 °C, 3 h, 99%; (b) 7-
deazapurine (R3 = H or NHPiv), Ph3P, DEAD, THF, rt, 2 d, 40–60%; (c) (i) NH4OH, 1,4-dioxane, 120 °C, 20 h, 60–80%; or (ii) MeNH2, 1,4-dioxane, 120 °C, 20 h, 63–67%; or (iii)
MeOH, NaOMe, reflux, 3 h, 55–91%.
The synthesis of 20-deoxy-20-fluoro-20-C-methyl-7-deazapurine
nucleosides was challenging due to either a lengthy synthesis or
poor yields followed by a difficult separation based on using two dif-
ferent synthetic approaches. One approach is more linear in which
the 20-C-methyl and 20-fluoro substituent are added to a 7-deazapu-
rine ribonucleoside, and the other approach is more convergent
where the completed moieties are brought together by a sugar-base
condensation. Since the 20-C-methyl-20-fluoro sugar lactone 1 can be
readily prepared using a recently published method,10 it appeared
advantageous to choose the sugar-base condensation approach.
We first attempted to utilize Vörbrügen sugar-base condensations
under various conditions. Two sugars (1-O-acetyl-3,5-di-O-ben-
zoyl-2-deoxy-2-fluoro-2-C-methylriboside and its1-bromo analog)
and two 7-deazapurine bases (6-chloro-7-deazapurine and 6-
chloro-7-flouro-7-deazapurine) were used for the condensation,
and the silylating agent was either N,O-bis(trimethylsilyl)acetamide
(BSA) or hexamethyldisilazane (HMDS). The solvent was chosen
from CH2Cl2, CHCl3, ClCH2CH2Cl, or CH3CN, in the presence or ab-
sence of a Lewis acid catalyst (trimethylsilyl trifluoromethanesulfo-
nate (TMSOTf), SnCl4, or trimethylsilyl iodide). The reaction
temperature ranged from room temperature to 70 °C, and the reac-
tion time varied from a few hours to several days. However, all these
condensation conditions failed to generate detectable amounts of
the desired product. Another reported method11 for condensation
of 7-deazapurines was tested using 1-chloro-3,5-di-O-benzoyl-2-
deoxy-2-fluoro-2-C-methylriboside with the catalysts of tris(2-(2-
methoxyethoxy)ethyl)amine (TDA-1) and KOH, without success.
The direct SN2 substitution using 1-bromo-3,5-di-O-benzoyl-2-
deoxy-2-fluoro-2-C-methylriboside and 7-deazapurine under basic
conditions (NaH) also did not work. Ultimately, Mitsunobu reaction
conditions were explored, which resulted in successful couplings
(Scheme 1). The required lactol 2 was prepared from lactone 1 by
careful LiAl(t-BuO)3H reduction. Under relatively standard Mitsun-
obu conditions,12 the condensation of several different 7-substi-
tuted 6-chloro-7-deazapurines11b,13 with lactol 2 produced 6-
chloro-7-deazapurine nucleosides 3. In the case that the 7-deazapu-
rine contained an amino moiety, the amino group was protected
with a pivaloyl group. The coupling products were obtained in about
NH2
N
NH2
N
40–60% yield, as an approximate 1:1 mixture of
a and b isomers.
a
b
N
N
4e or 5e
Heating of 3 with NH4OH and 1,4-dioxane in a sealed steel vessel
resulted in amination and deprotection, giving rise to the 7-deazap-
urine nucleosides 4a–g and 5a–g.14 When MeNH2 was used instead
N
N
HO
HO
O
O
OH
F
OH
F
6( ) or 7( )
8( ) or 9( )
Scheme 2. Synthesis of 7-deaza-7-vinyl and -7-ethyl-purine nucleosides. Reagents
and conditions: (a) Tributyl(vinyl)tin, Pd(PPh3)4, DMF, 100 °C, 5 h, 53–75%; (b) Pd-C,
H2, MeOH, rt, 20 h, 60–70%.
R4
NH2
a or b
N
4e, 4g, 5e, or 5g
R3
N
N
HO
O
MeO2C
NH2
OH
F
12( ): R3 = R4 = H
13( ): R3 = R4 = H
N
a
4e or 5e
N
N
HO
14( ): R3 = NH2, R4 = H
15( ): R3 = NH2, R4 = H
16( ): R3 = H, R4 = Ph
17( ): R3 = H, R4 = Ph
O
OH
F
10( ) or 11( )
Scheme 4. Synthesis of 7-ethynyl-substituted nucleosides. Reagents and condi-
tions: (a) (i) Pd(PPh3)Cl2, THF, Cul, Et3N, triethylsilylacetelene, 45 °C, 20 h, 78–86%;
(ii) K2CO3, MeOH, rt, 20 h, 30–71%; (b) Pd(PPh3)2Cl2 ,THF, CuI, ET3N, phenylacete-
lene, 45 °C, 20 h, 89–95%.
Scheme 3. Synthesis of 7-carbomethoxyvinyl-7-deazapurine nucleosides. Reagents
and conditions: (a) Pd(OAc)2, PPh3, 1,4-dioxane, Et3N, methyl acrylate, reflux, 16 h,
83–100%.

7096
J. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7094–7098
NH2
N
of NH4OH, 6-methylamino-7-deazapurine nucleosides 4h and 5h
were obtained. Deprotection of 3 with NaOMe/MeOH produced
NH2
N
R1
R1
O
P
O
P
O
P
a
6-methoxy products 4i and 5i. The
a and b isomers were isolated
R3
N
R3
N
N
N
MO
O
O
O
HO
by flash chromatography, and the configuration was assigned based
O
O
OM OM OM
on NOE experiments. The 1H NMR signals for 20-C-methyl group are
also very distinctive between the
a and b isomers: all the a-isomers
OH
F
OH
F
had a doublet at around 1.29 ppm with a coupling constant JH,F of
21.95 Hz, whereas the b-isomers doublets were around 0.94 ppm
with JH,F of 22.33 Hz.
5a
5b
5f
R1 = R3 = H
22
23
24
R1 = F, R3 = H
R1 = H, R3 = NH2
7-Iodo-7-deazapurine nucleosides (4e, 4g, 5e, and 5g) were
used as building blocks for the preparation of 7-carbon substituted
nucleosides under the catalytic effects of various palladium re-
agents. For example, using Pd(PPh3)4 as a catalyst,15 4e and 5e
were reacted with tributyl(vinyl)tin, giving 7-vinyl substituted
nucleosides 6 and 7, respectively, in moderate to good yield
(Scheme 2). Hydrogenation of the vinyl compound (6 or 7) resulted
in the 7-ethyl nucleoside (8 or 9). Using Pd(OAc)2 and methyl acry-
late, 4e and 5e were converted to 7-carbomethoxyvinyl-7-deazap-
urine nucleosides 10 and 11 in excellent yield (Scheme 3).
Coupling of 7-iodo compounds (4e, 4g, 5e, or 5g) with triethylsily-
lacetelene in the presence of Pd(PPh3)2Cl2 and CuI produced, upon
desilylation with K2CO3 in methanol, moderate yield of 7-ethynyl-
substituted nucleosides 12–15. Similarly, 7-phenylethynyl-substi-
tuted nucleosides 16 and 17 were also prepared in very good yield
by replacing triethylsilylacetelene with phenylacetelene (Scheme
4).
Some nucleosides were selected for preparation of phenyl phos-
phoramidate prodrugs using a published procedure.16 Thus, two
6-amino-7-deazapurine nucleosides (5b and 5e) and two 2,6-dia-
mino-7-deazapurine nucleosides (5f and 5g) were converted to
their corresponding phosphoramidates 18–21 (Scheme 5). For an
HCV polymerase enzyme kinetics study, three 50-triphosphates
(22–24) were also prepared (Scheme 6) and purified by HPLC [DIO-
NEX NucleoPac PA-100 (9 ꢀ 250 mm) column; buffer A, 0.05 M
TEAB; buffer B, 0.5 M TEAB; flow rate, 7.5 mL/min; gradient,
increasing buffer B from 0% at 0 min to 50% at 10 min, and then
100% at 12 min and maintained to 17 min].
Scheme 6. Synthesis of nucleoside triphosphates. Reagents and conditions:
M@NH(n-Bu)3; (a) (i) POCl3, PO(OCH3)3, 2,4,6-collidine, 0 °C, 2 h; (ii)
(Bu3N)2H2P2O7, Bu3N, rt, 30 min; (iii) TEAB, rt, 45 min.
modest anti-HCV activity with EC50 values less than 10
ever, their EC90 values were all greater than 10 M, except one
compound, 7-vinyl-7-deaza-adenine nucleoside (7, b-form), which
demonstrated anti-HCV activity with an EC90 of 7.6 M, compara-
lM. How-
l
l
ble to the control compound, 20-C-methyl-cytidine (NM-107).
However, compound 7 also exhibited toxicity in the ribosomal
RNA assay and in three independent cell lines, so it appears that
the antiviral activity is secondary to cytotoxic effects. Interestingly,
its
a-isomer 6, also showed moderate anti-HCV activity and strong
toxicity across all cell lines. Similarly, both
a
and b forms of 7-iodo
substituted nucleosides (4e and 5e) also showed modest anti-HCV
activity and toxicity. It seems that the activity and toxicity are
associated mainly with the 7-position substituent, and not related
to the
a or b configuration. Moreover, these antiviral activities are
almost certainly due to the secondary toxicity of the compounds,
which could cause cell death or cytostatic effects, thus slowing
down or stopping viral replication.
Some 7-substituted nucleosides demonstrated anti-HIV-1 activ-
ity albeit again with cytotoxic effects in PBM cells, and in most
cases all cell lines tested. These include 7-vinyl (6 and 7,
b-isomers, respectively), 7-ethynyl (14, -isomer), and 7-phenyl-
ethynyl (16 and 17, and b-isomers, respectively) substituted
nucleoside analogs. Only one compound, 7-carbomethoxyvinyl
substituted nucleoside (10, -isomer), exhibited strong anti-HIV-
1 activity without any apparent cytotoxicity up to 100 M. Again,
it showed that the anti-HIV-1 activity and cytotoxicity are not re-
lated to the - or b-configuration of these nucleoside analogs.
a and
a
a
All synthesized nucleosides and phosphoramidate prodrugs
were evaluated for in vitro anti-HIV and anti-HCV activities, and
toxicity. For determination of anti-HCV activity and toxicity (de-
creased rRNA), the compounds were tested in an HCV replicon sys-
tem (96 h) using a standard published protocol.17 The anti-HIV
activity and cytotoxicity were assayed as previous described.18
The three synthesized triphosphates (22–24) were tested against
both wild-type and S282T mutant HCV NS5B polymerases.19 In
addition, some selected compounds were also evaluated against
HBV in hepatocytes.20
a
l
a
The enzymatic assay results of the nucleoside triphosphates
(22–24) with HCV RNA-dependent-RNA-polymerases are pre-
sented in Table 2. These triphosphates were potent inhibitors of
HCV NS5B polymerase. It is worth noting that these compounds
had almost equal potency against wild-type and S282T mutant
polymerases. This is in contrast to 20-C-methylcytidine-TP
(NM-107-TP), where a 17-fold increase in the S282T mutant com-
pared to wild-type virus was observed.
The results of antiviral activities and toxicities of these nucleo-
side analogs and prodrugs are summarized in Table 1. Among the
34 compounds tested in HCV replicon cells, 12 compounds showed
Five selected nucleosides (4e, 10, 14, 16, and 17) were evaluated
for their in vitro antiviral activity against wild-type HBV. Com-
pounds 4e and 17 exhibited weak anti-HBV activity and toxicity
to HepG2 cells (data not shown). Compounds 10, 14 and 16
showed no anti-HBV activity.
NH2
N
NH2
N
R1
R1
a
O
P
OPh
Since the selected nucleoside 50-triphosphates demonstrated
potent inhibitory activity against HCV polymerases, it is surprising
that the nucleoside phosphoramidate prodrugs 18–21 did not
show any anti-HCV activity. In many cases, the phosphorylation
step of nucleoside analogs to the monophosphate is the rate-limit-
ing step. Therefore, a monophosphate prodrug approach would by-
pass initial phosphorylation and potentially increase the amount of
nucleoside 50-triphosphate produced intracellularly. In order to
understand the mechanism of these nucleoside analogs, a cellular
pharmacology study was conducted. The nucleoside 5f and its
phosphoramidate prodrug 20 were separately incubated at
EtO
R3
N
R3
N
H
N
N
N
O
HO
O
O
O
OH
F
OH
F
5b
5e
5f
R1 = F, R3 = H
R1 = I, R3 = H
18
19
20
21
R1 = H, R3 = NH2
R1 = I, R3 = NH2
5g
Scheme 5. Synthesis of nucleoside phosphoramidate prodrugs. Reagents and
conditions: (a) Phenyl
THF, rt, 14 h, 11–47%.
L-ethoxyalaninyl phosphorochloridate, 1-methylimidazole,

J. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7094–7098
7097
Table 1
In vitro antiviral activity and cytotoxicity of 20-deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleosides (4a–17) and phosphoramidate prodrugs (18–21)a
b
Compd #
Anti-HCV (
l
M)
EC90
rRNA (lM) CC50
Anti-HIV-1 (
l
M)
Cytotoxicity, IC50
CEM
(lM) in:
EC50
EC50
EC90
PBM
VERO
4a
4b
4c
4d
>10
>10
<10
<10
>10
>10
>10
>10
>10
>10
>10
>10
>100
>100
>100
>100
>100
>100
>100
>100
>100
89.1
>100
8.1
96.0
76.0
>100
>100
>100
71.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4e
4f
4g
4h
4i
7.0
24.9
>10
>10
>10
>10
>33
>10
>10
>10
>10
>100
>100
>100
>100
>100
40.9
65.9
>10
>10
>10
>10
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
82.4
>100
94.0
>100
99.5
>100
24.8
35.5
14.7
95.5
19.0
0.71
>100
95.2
59.1
7.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
85.6
5a
5b
5c
5d
5e
5f
5g
5h
5i
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
NM-107
AZT
>10
>10
<10
<10
1.1
>10
>10
>10
>10
5.6
>10
>10
>10
>10
>33
>10
>10
>10
>10
18.4
7.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
9.6
>10
>10
>10
<10
10.4
>10
>10
>10
>10
8.3
>100
>100
18.5
74.2
53.2
>100
72.9
>100
>100
21.0
4.8
>100
20.7
>100
>100
>100
94.1
54.0
>100
40.8
41.1
>100
17.4
>100
67.4
47.4
>100
>100
>100
>100
50.5
24.0
>100
85.4
>100
>100
16.0
3.5
>100
>100
>100
>100
>100
>100
26.5
>100
15.7
11.6
>100
13.5
>100
>100
54.4
14.3
>100
>100
>100
53.1
21.4
>100
62.8
>100
>100
14.6
10.1
>100
>100
>100
>100
>100
>100
61.7
>100
51.3
45.7
>100
17.8
>100
65.1
>100
56.0
65.2
44.8
1.8
4.5
>10
<10
>10
>10
<10
<10
>10
>10
>10
<10
>10
>10
>10
>10
2.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
<10
>10
>10
>10
>10
>10
>10
>100
>100
9.5
>100
>100
>100
59.1
>100
38.0
31.1
84.4
7.7
10.7
39.4
58.1
88.0
26.7
47.5
0.0033
98.8
>100
>100
>100
100.0
0.028
>10
>10
a
All values are based on mean of replicate assays (N P2).
CC50: cytotoxic concentration that reduced the rRNA levels by 50% in 96 h.
b
50
l
M with Huh-7 cells at 37 °C for 4 h, and then the cells were
Although it is possible that these compounds are acting as non-
nucleoside inhibitors, it is more likely that they are unstable and
degrade to the 7-deazapurine base. Moreover, the fact that both
washed with phosphate-buffered saline. The intracellular metabo-
lites were extracted with 60% methanol in water and identified by
LC–MS/MS. In these experiments, only a trace amount of nucleo-
side triphosphate was found following incubation of the nucleo-
side 5f or its phosphoramidate prodrug 20. In the case of
compound 5f, neither the 50-monophosphate nor the diphosphate
was detected. In contrast, with the prodrug 20 a large amount of
the monophosphate and the parent nucleoside 5f were detected.
In addition, no corresponding 7-deazaguanosine nucleoside or
nucleoside phosphate was found after incubation of the two com-
pounds, suggesting that no deamination had occurred. These re-
sults provide an explanation for the lack of antiviral activity of
these nucleoside analogs and phosphoramidate prodrugs. This
work confirmed that not only the first phosphorylation step from
the nucleoside analog to monophosphate, but also the second
phosphorylation step from the monophosphate to the diphosphate
was problematic in Huh-7 cells at least for nucleoside 5f, and prob-
ably for all other nucleoside analogs synthesized. It is not clear if
the third step of the phosphorylation, from the diphosphate to
the triphosphate was also a problem. Nonetheless, a monophos-
phate prodrug approach is not sufficient, and a diphosphate pro-
drug, or most likely a triphosphate prodrug would be needed to
exert the anti-HCV activity of these nucleoside analogs.
a
and b nucleosides with the same 7-substitutent had similar anti-
viral activity and toxicity profiles suggests that the biological activ-
ity may be associated with the 7-substituent moieties of the
nucleosides. The only compound with good anti-HIV-1 activity
and no cytotoxicity,
worth further investigation to determine its mechanism of action.
In conclusion, 30 novel and b-
-20-deoxy-20-fluoro-20-C-
a-7-carbomethoxyvinyl nucleoside 10, is
a
D
methyl-7-deazapurine nucleoside analogs were synthesized and
evaluated for in vitro anti-HCV activity in a replicon assay. The
key step in the synthesis, the sugar-base condensation, was
achieved via a Mitsunobu reaction. Several a- and b-7-deazapurine
nucleoside analogs exhibited modest anti-HCV activity however
this activity was most likely due to their intrinsic toxicity. The four
Table 2
Inhibitory activity of 20-deoxy-20-fluoro-20-C-methyl-7-deazapurine nucleoside tri-
phosphates against wild-type and S282T mutant HCV NS5B polymerases
Nucleotide #
HCV NS5B pol IC50
(
l
M)
Fold resistance relative to
wild-type HCV pol
Wild-type
S282T
22
23
24
15
4
3
15
6
6
1
1.5
2
It is unusual that some a-nucleoside analogs exhibited antiviral
activity and/or toxicity, as these nucleoside analogs are not
NM-107-TP
3
51
17
typically efficiently phosphorylated to the triphosphate.21

7098
J. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7094–7098
Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K.;
LaFemina, R. L.; Leone, J.; MacCoss, M.; McMasters, D. R.; Tomassini, J. E.; Von
Langen, D.; Wolanski, B.; Olsen, D. B. J. Med. Chem. 2004, 47, 5284.
9. Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish, P. M.; Lostia, S.;
McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto, M. J.; Furman, P. A.; Stec,
W. J.; Patterson, S. E.; Pankiewicz, K. W. J. Med. Chem. 2005, 48, 5504.
10. Wang, P.; Chun, B. K.; Rachakonda, S.; Du, J.; Khan, N.; Shi, J.; Stec, W.; Cleary,
D.; Ross, B. S.; Sofia, M. J. J. Org. Chem. 2009, 74, 6819.
11. (a) Ugarkar, B. G.; Castellino, A. J.; DaRe, J. S.; Ramirez-Weinhouse, M.; Kopcho,
J. J.; Rosengren, S.; Erion, M. D. J. Med. Chem. 2003, 46, 4750; (b) Seela, F.; Peng,
X. J. Org. Chem. 2006, 71, 81.
12. But, T. Y. S.; Toy, P. H. Chem. Asian J. 2007, 2, 1340.
13. Wang, X.; Seth, P. P.; Ranken, R.; Swayze, E. E.; Migawa, M. T. Nucleosides
Nucleotides Nucleic Acids 2004, 23, 161.
synthesized 7-deazapurine nucleoside phosphoramidate prodrugs
18–21 showed no anti-HCV activity, whereas the nucleoside tri-
phosphates 22–24 demonstrated good inhibitory activity against
both wild-type and S282T mutant HCV polymerases (IC50
3–15 M). These 7-deazapurine nucleosides and prodrugs were
also evaluated for in vitro anti-HIV-1 activity and cytotoxicity. Only
one compound, an -form of 7-carbomethoxyvinyl substituted
nucleoside (10), showed good anti-HIV-1 activity (EC50 and EC90
of 0.71 0.25 M and 9.5 3.3 M, respectively) with no cytotoxic-
ity up to 100 M. Some selected compounds were evaluated against
:
l
a
l
l
l
HBV, and none showed significant in vitro anti-HBV activity. A cel-
lular pharmacology study of the nucleosides and prodrugs in Huh-7
cells utilizing LC–MS/MS revealed that the triphosphates were not
formed at therapeutically significant levels, indicating problems
in the first and second phosphorylation steps for these nucleoside
analogs by the cellular phosphorylation enzymes.
14. Selected synthetic procedures and spectroscopic data: 4-amino-7-iodo-1-(2-
deoxy-2-fluoro-2-C-methyl-
(4e) and 4-amino-7-iodo-1-(2-deoxy-2-fluoro-2-C-methyl-b-
1H-pyrrolo[2,3-d]pyrimidine (5e). To solution of 4-chloro-7-iodo-1H-
pyrrolo[2,3-d]pyrimidine (339 mg, 1.21 mmol), 2-deoxy-2-fluoro-2-C-methyl-
/b- -ribofuranose (500 mg, 1.34 mmol) and triphenylphosphine (698 mg,
a-
D
-ribofuranosyl)-1H-pyrrolo[2,3-d]pyrimidine
D-ribofuranosyl)-
a
a
D
2.66 mmol) in anhydrous THF (5 mL) was added diisopropyl azodicarboxylate
(DIAD) (0.53 mL, 2.66 mmol), and the reaction mixture was stirred at rt for 2 d.
The solvent was evaporated, and the residue was purified by flash
chromatography on silica gel eluting with hexane–EtOAc (9:1–2:1) to give
390 mg (51%) of the nucleoside 3e as a white solid, which contained a 1:1 ratio
Acknowledgments
of
a and b anomers. The compound 3e (390 mg, 0.61 mmol) was placed in a
We thank Emilie Fromentin, Aleksandr Obikhod, Sarah Solo-
mon, and Jason Grier for excellent technical assistance. This work
was supported in part by 2P30-AI-050409 (RFS), and the Depart-
ment of Veterans Affairs (RFS), and by the Cancer Research Society
(MG). Dr. Schinazi is a founder and major shareholder of RFS Phar-
ma, LLC and his laboratory received no funding from RFS Pharma or
vice versa. Dr. Götte received research grants from Tibotec, Merck,
Gilead Sciences, AstraZeneca, Pfizer, and GlaxoSmithKline. All
other authors have no competing interests.
steel vessel, and 1,4-dioxane (10 mL) was added followed by NH4OH (28%,
20 mL). The steel vessel was sealed and heated at 120 °C for 14 h. After cooling
to rt, the solvent was evaporated and the residue was purified by flash
chromatography on silica gel eluting with CH2Cl2–MeOH (95:5–9:1) to give
108 mg (43%) of 4e and 82 mg (33%) of 5e both as a white solid. 4e: Rf 0.30
(CH2Cl2–MeOH 9:1); 1H NMR (DMSO-d6) d 8.12 (s, 1H, H-2), 7.38 (d, J = 3.47 Hz,
1H, H-6), 6.70 (br s, 2H, NH2), 6.38 (d, J = 20.79 Hz, 1H, H-10), 5.71 (d,
J = 6.93 Hz, 1H, OH-30), 4.88 (t, J = 5.78 Hz, 1H, OH-50), 4.12–4.10 (m, 1H, H-30),
4.07–4.05 (m, 1H, H-40), 3.70, 3.50 (2 m, 2H, H-50), 1.29 (d, J = 21.95 Hz, 3H,
CH3). LC–MS calcd for C12H15FIN4O3 (M+1): 409.0, found: 409.0. 5e: Rf 0.42
(CH2Cl2–MeOH 9:1); 1H NMR (DMSO-d6) d 8.13 (s, 1H, H-2), 7.77 (s, 1H, H-6),
6.75 (br s, 2H, NH2), 6.32 (d, J = 18.10 Hz, 1H, H-10), 5.65 (d, J = 6.93 Hz, 1H, OH-
30), 5.31 (t, J = 4.62 Hz, 1H, OH-50), 4.14–4.12 (m, 1H, H-30), 3.87, 3.67 (2 m, 3H,
H-40, H-50), 0.94 (d, J = 22.72 Hz, 3H, CH3). LC–MS calcd for C12H15FIN4O3
(M+1): 409.0, found: 409.0.
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