C. Chapron et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2699–2702
2701
M)
revealed and the 20-O,40-C-methylene bridge was closed in 55%
Table 1
Inhibition of HCV NS5B polymerase activity in vitro
over the two steps to give 26. Final, 50-O-mesylate displacement
with sodium benzoate and global deprotection provided the
20-C-methyl-modified guanine-LNA nucleoside 9 in 23% yield over
three steps.
Compound
1b WT IC50
(l
M)
1b S282T IC50 (l
LNA-A-TP, 4-TP
LNA-C-TP, 5-TP
39.83
1.39
86.27
1.44
Although 50-C-methyl-modified uridine-LNA nucleosides have
been synthesized, no reports exist of the analogous guanine-LNA
nucleosides 10a and 10b.20 Rather than beginning with the known
1,2,4-hydroxymethyl-tri-protected 30-O-naphthyl-allofuranose it
was anticipated in this case that the guanine-LNA nucleoside 20
would be a more convenient advanced intermediate on which to
attempt installation of the 50-C-methyl moiety (Scheme 4). Thus,
formation of the requisite N-benzoyl guanine-LNA nucleoside 28
bearing a free 50-hydroxyl was achieved via protecting group
manipulation in four steps from 20. Attempts to oxidize the
50-alcohol 28 to the corresponding aldehyde 29 resulted in almost
complete conversion to the undesired hydrate 30, which then
failed to react with methyl magnesium bromide under various
conditions.
LNA-U-TP, 6-TP
LNA-G-TP, 7-TP
ENA-G-TP, 8-TP
20-C-Me-LNA-G-TP, 9-TP
50-C-Me-LNA-G-TP, 10a-TP
50-C-Me-LNA-G-TP, 10b-TP
3.84
0.39
>100
0.20
15.86
>100
12.80
0.38
>100
0.48
25.11
>100
Table 2
Inhibition of HCV NS5B polymerase activity in vitro by LNA-G-TP, 7-TP
Enzyme
IC50
(l
M)
1a WT
2a WT
3a WT
4a WT
0.37
0.29
0.20
0.31
Conversely, a one-pot oxidation–Grignard process, analogous to
that utilized for 22, was found to be successful in providing the
desired 50-C-methyl guanine-LNA nucleosides 31a and 31b as a
1:1 mixture of 50-epimers. Neither flash column chromatography
on silica gel nor preparatory HPLC were effective in achieving sep-
aration of the epimers, however, they were isolable by repeated
counter current chromatography, providing the desired epimeric
alcohols 31a and 31b in 19% total yield.21 N-Benzoyl deprotection
with methanolic ammonia followed by transfer hydrogenation
using Pearlman’s catalyst and formic acid to remove the 30-O-ben-
zyl yielded the respective 50-C-methyl guanine-LNA nucleosides
10a and 10b.
As part of the initial screening evaluation, the eight correspond-
ing triphosphates 4-TP–9-TP, 10a-TP, and 10b-TP were prepared
to assess the inhibitory potential of these bicyclic ribonucleoside
systems against the purified HCV NS5B polymerase (Table 1).22
The two pyrimidines LNA-U-TP 6-TP and LNA-C-TP 5-TP were
discovered to inhibit the 1b wild type (WT) polymerase in vitro
Although the barrier to resistance for nucleosides in general is
considered to be high, the S282T mutation has been observed
in vitro for the 20-C-methyl class of nucleoside inhibitors.9 Accord-
ingly, LNA-G-TP 7-TP was assessed for its activity against the 1b
S282T mutant and found to be equipotent as against the WT.
Expansion of the alkyl bridge to incorporate a further CH2 unit
was found to be highly detrimental and no activity was observed
with the 6-membered ring ENA-G-TP 8-TP against the 1b WT
polymerase (Table 1). Modification of LNA-G by installation of a
50-C-methyl was also found to be detrimental, yielding IC50
15 lM for 10a-TP and no activity for the epimeric LNA-G-TP
10b-TP. In contrast, incorporation of the 20-C-methyl modification
into the LNA-G nucleoside template provided the 20-C-methyl-
LNA-G-TP 9-TP which displayed potent activity against the 1b
WT polymerase (IC50 0.20
l
M) with only two-fold decrease against
with IC50 values ranging from 1 to 4
lM, whereas the LNA-A-TP
the S282T mutant (IC50 0.48 l
M).
4-TP was found to be 10-fold less active. In contrast, potent inhibi-
tion of the 1b WT polymerase was observed for LNA-G-TP 7-TP
(IC50 0.39 lM). The pan-genotypic potential of the LNA-G-TP
The free nucleosides 4–9, 10a, 10b were also evaluated against
the whole cell-based replicon assay.23 Unsurprisingly, none of the
bicyclic ribonucleosides were found to possess inhibitory activity
7-TP was examined and found to be maintained across the 1a,
(EC50 >50
lM), although none were observed to be cytotoxic up
2a, 3a and 4a WT enzymes (Table 2).
to 100 M. It is well established that a common rate-limiting step
l
in the sequential processing of 20-C-methyl nucleosides to their
respective triphosphates is the initial phosphorylation by host cell
kinases to the corresponding nucleoside monophosphates.24
Although the second or third phosphorylations may potentially
be compromised in the case of these bicyclic ribonucleosides, it
is feasible that efficient nucleoside triphosphate formation may
require a monophosphate prodrug kinase by-pass strategy, which
will be investigated in due course.
R
R
R
O
O
O
a
b-d
HO
BzO
HO
70%
37%
BnO
O
BnO
O
BnO O
20
27
28, R = N-Bz-G
, R = G
, R = G
In summary, the synthesis of a variety of bicyclic ribonucleo-
sides containing a 20-O,40-C-alkylene-bridge has been performed
and their anti-HCV activity has been examined in vitro. Key to
the synthesis of the novel guanosine analog 9 was a practical
and high yielding one-pot oxidation–Grignard procedure to install
the 20-C-methyl functionality. This protocol was successfully
applied to the 50-C-methyl analog for which separation of the
resulting epimers was achieved via counter current chromatogra-
phy. Although none of the bicyclic ribonucleosides was found to
be active in the cell-based HCV replicon assay, both the LNA-G
7-TP and 20-C-methyl-LNA-G 9-TP were found to be highly potent
as the respective triphosphates against both NS5B 1b wild type
and S282T mutant polymerases. The potential utility of a mono-
phosphate prodrug strategy for these LNA nucleosides is under
investigation.
OH
R
R
O
O
O
O
e
HO
O
28
+
BnO
BnO
29
30
, R = N-Bz-G
, R = N-Bz-G
R
R
O
O
HO
HO
g, h
e, f
28
19%
51%, 31a
49%, 31b
BnO
31a, 31b
O
HO O
10a, 10b
, R = N-Bz-G
, R = G
Scheme 4. Reagents and conditions: (a) NH3, MeOH; (b) TBDMSCl, DMAP, pyridine,
DCM; (c) BzCl, pyridine; (d) HCl, 1,4-dioxane–THF; (e) trifluoroacetic anhydride,
DMSO, Et3N, THF, ꢀ78 °C to rt; (f) MeMgBr, THF, ꢀ78 °C; (g) NH3, MeOH; (h) 20%
Pd(OH)2/C, HCO2H, THF–MeOH, reflux.