Stereoselective synthesis and biological evaluations of novel 3′-deoxy-4′-azaribonucleosides as inhibitors of hepatitis C virus RNA replication

Article abstract of DOI:10.1021/jm900197j

3′-Deoxy-4′-azaribonucleosides (15a-d) were synthesized starting from the commercially available (4R)-trans-4-hydroxy-L-proline 7. From biological evaluations, 15b and 15d emerged as potent inhibitors of HCV replication on a replicon assay. These findings demonstrate that synthesized pyrrolidine nucleosides represent a new template for antiviral or other biological studies and could be considered for novel combination therapy against HCV infection using nucleoside inhibitors and non-nucleoside inhibitors of HCV NS5B.

Full text of DOI:10.1021/jm900197j

4
054
J. Med. Chem. 2009, 52, 4054–4057
Brief Articles
Stereoselective Synthesis and Biological Evaluations of Novel 3′-Deoxy-4′-azaribonucleosides as
Inhibitors of Hepatitis C Virus RNA Replication
,
†
†
†
†
§
#
#
Ugo Chiacchio,* Luisa Borrello, Lia Crispino, Antonio Rescifina, Pedro Merino, Beatrice Macchi, Emanuela Balestrieri,
|
‡
,‡
Antonio Mastino, Anna Piperno, and Giovanni Romeo*
Dipartimento di Scienze Chimiche, UniVersit a` di Catania, Viale A. Doria 6, Catania 95125, Italy, Departamento de Quimica Organica,
UniVersidad de Zaragoza, Zaragoza E-50009, Spain, Dipartimento di Neuroscienze, UniVersit a` di Roma “Tor Vergata”, Via Montpellier 1,
Roma 00133, Italy, Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, UniVersit a` di Messina, Salita Sperone 31,
Messina 98168, Italy, and Dipartimento Farmaco-Chimico, UniVersit a` di Messina, Via SS. Annunziata, Messina 98168, Italy
ReceiVed February 17, 2009
3
′-Deoxy-4′-azaribonucleosides (15a-d) were synthesized starting from the commercially available (4R)-
trans-4-hydroxy-L-proline 7. From biological evaluations, 15b and 15d emerged as potent inhibitors of
HCV replication on a replicon assay. These findings demonstrate that synthesized pyrrolidine nucleosides
represent a new template for antiviral or other biological studies and could be considered for novel combination
therapy against HCV infection using nucleoside inhibitors and non-nucleoside inhibitors of HCV NS5B.
Introduction
Different RNA polymerase inhibitors have been reported in
literature; these compounds can be classified as nucleoside
inhibitors and non-nucleoside inhibitors. Nucleoside and nucle-
Hepatitis C virus infection constitutes one of the major
problems for public health. Current estimates suggest that HCV
9
a
otide analogues of DNA or RNA substrates can inhibit HCV
replication by acting as chain terminators, viral mutagens, or
simple competitive inhibitors. Although the biological activity
of NIs is strictly dependent on their conversion to the corre-
sponding triphosphates, they possess advantages over the NNIs
in the mode of action and, acting by chain termination, they
bind in the highly conserved active-site region of HCV NS5B,
and thus, they have a high likelihood of affecting RNA
infects 3.5% of the world population, and fatalities associated
with HCV infection result from chronic liver disease and
1
,2
hepatocellular carcinoma. HCV infection is also responsible
3
for the majority of liver transplants currently performed.
Current therapies, based on the combination of ribavirin and
pegylated interferon-R, have improved the sustained response
rates for patients infected with HCV genotypes 2 and 3 but suffer
from high cost and relapse rates, and the success rate for current
1
0
4,5
replication for all HCV genotypes.
therapy is <50% in genotype 1 patients. Thus, the development
Different nucleoside analogues, structurally modified both on
the heterocyclic base and on the sugar moiety, have been
reported as new anti-HCV agents. The best results have been
achieved by small modifications at 2′ and 4′ positions of the
ribose nucleus such as the introduction of a methyl group at
of new therapies for the treatment of HCV infection is an
6
intensive area of research.
A rational approach for the treatment of this pathology is
the design of new agents that can specifically act against viral
enzymes. HCV encodes a series of viral proteins such as the
NS2/3 autoprotease, the NS3 serine protease/NTPase/helicase,
1
1
C2′ or an azido group at C4′ (Figure 1).
7
In addition, new templates of 4′-oxonucleosides, such as 4′-
and the NS5B RNA-dependent RNA polymerase. The NS5B
12
13
14
thio, 4′-carbo, and 4′-seleno analogues, were also synthe-
sized, but they did not exhibit significant anti-HCV activity.
Thus, as part of our efforts toward novel templates for the
development of new therapeutic agents, we turned our attention
to 4′-aza-nucleosides. Literature data report that nucleosides
containing pyrrolidine and 4-hydroxyproline rings possess
substantial similarity to products that contain the tetrahydrofuran
ring. Thus, small geometric perturbations could significantly
influence the antiviral activity of these compounds.
On the basis of these considerations, we report in this paper
the first results on the synthesis and the biological evaluation
of the novel template, the 3′-deoxy-4′-azaribonucleosides. The
synthesized compounds have been shown to be HCV inhibitors
in a cell-based replicon assay at nanomolar order with no or
low toxicity.
RNA-dependent RNA polymerase is essential for viral genome
replication and therefore represents a good target in the search
of new potential drugs.
8
*
To whom correspondence should be addressed. For U.C.: phone: +39-
0
95-7385014; fax: +39-095-580138; e-mail: uchiacchio@unict.it. For G.R.:
phone: +39-090-356230; fax: +39-090-6766562; e-mail: gromeo@unime.it.
†
Universit a` di Catania.
Universidad de Zaragoza.
Universit a` di Roma “Tor Vergata”.
Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari,
1
5
§
#
|
Universit a` di Messina.
‡
Dipartimento Farmaco-Chimico, Universit a` di Messina.
Abbreviations: AM1, Austin model 1; Boc, tert-butoxycarbonyl; CC50
a
,
cytotoxic concentration required to cause 50% toxicity detected by MTS
assay; HB 1, heparin-binding protein 1; HCV, hepatitis C virus; IC50
inhibitory concentration that reduces by 50% the viral replication in replicon
assay; IFN, interferon; LiBEt H, lithium triethylborohydride; MTS, [3-(5,5-
,
3
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium]; NIs, nucleoside inhibitors; NNIs, non-nucleoside inhibitors;
NS2, nonstructural protein 2; NS3, nonstructural protein 3; NS5B, non-
structural protein 5B; NTPase, nucleoside 5′-triphosphatase; TBAF tet-
rabutylammonium fluoride; TBDMSCl, tert-butyldimethylsilyl chloride.
Results and Discussion
Chemistry. The synthetic approach toward 3′-deoxy-4′-
azaribonucleosides 15a-d, reported in Scheme 1, starts from
1
0.1021/jm900197j CCC: $40.75  2009 American Chemical Society
Published on Web 05/06/2009

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4055
Figure 1. Representative examples of nucleoside inhibitors.
Scheme 1. Synthesis of 3′-Deoxy-4′-azaribonucleosides 15
Scheme 2. Synthesis of 3′-Deoxy-4′-azaribonucleosides 23 and 24
1
6
(
4R)-trans-4-hydroxy-L-proline 7, which was converted into 9
nucleosides 15a-d in enantiomerically pure form were obtained
and reduced with LiBEt
Further reaction with TBAF and subsequently with Ac
3
H to a 4:1 mixture of hemiaminals 10.
4
by reduction with NaBH of 14a-d.
2
O
3′-Deoxy-4′-azaribonucleosides 23 and 24, containing the
ethoxycarbonyl group at the nitrogen atom of pyrrolidine ring,
were obtained by the procedure described in Scheme 2. Thus,
afforded the di-O-acetyl derivatives 12. Nucleosidation with
silylated pyrimidine nucleobases 13a-d gave compounds
1
7
1
4a-d as single diastereomers.
7 was reacted with ethyl chloroformate in methanol and
subsequently protected with TBDMSCl, giving 17. Oxidation
The relative configurational assignments for 14a-d were
determined on the basis of H NMR and NOE experiments (see
1
of 17 with RuO
2
/NaIO
4
, followed by reduction with LiBEt
3
H
Supporting Information). Finally, the target 3′-deoxy-4′-aza-
and reaction with TBAF and Ac
2
O, afforded 20. Reaction of

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056 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Brief Articles
Table 1. Inhibitory Concentration (IC50) of Azaribonucleosides on HCV
RNA Replication in a Subgenomic Replicon Harbored in HB-1 Cells
and Cytotoxycity (CC50) As Measured by MTS Assay
ribonucleosides, we hypothesize that these compounds act as
nucleoside analogues at the active site of the NS5B polymerase.
compd
HCV replicon IC50 (µM)
MTS CC50 (µM)
Conclusions
1
1
1
1
2
2
5a
5b
5c
5d
3
40.6
0.018
96
0.00035
4.8
>1000
0.01-0.008 U
>1000
>1000
>1000
623
>1000
200
In summary, we have accomplished the stereoselective
synthesis of novel 3′-deoxy-4′-azaribonucleosides, starting from
the chiral template (4R)-trans-4-hydroxy-L-proline, together with
their biological evaluations. From these investigations, thymine
and 5-fluouracil derivatives emerged as potent inhibitors of HCV
replication on a replicon assay. Considering that few different
classes of nucleoside analogues have been shown to inhibit HCV
replication, the reported results seem to be relevant to discover
new potent and safe antiviral agents possessing a nucleoside
structure. The findings reported here demonstrate that the
synthesized pyrrolidine nucleosides represent a new tem-
plate for antiviral or other biological studies that at the moment
are largely lacking. Particularly, similar to what occurs in HIV
infection, this new class of compounds could be considered for
novel combination therapy against HCV infection using NIs and
NNIs of HCV NS5B.
4
IFN
>100
2
0 with silylated fluorouracil yielded a nearly equimolar mixture
of pyrrolidine derivatives ꢀ-21 and R-22, which, reduced with
NaBH , gave the corresponding pure azaribonucleosides 23 and
4 (Scheme 2).
4
2
Interestingly, the difference between the alkyl groups of the
carbamate moiety (ethyl vs tert-butyl) exerted a complete
diastereofacial differentiation in the N-glycosylation reaction.
Whereas the tert-butyl carbamate only led to one isomer, the
ethyl group led to a mixture of isomers. The explanation, as
supported by preliminary AM1 semiempirical calculations, could
be found in a different conformational disposition of both
groups, which conditions the attack of the nucleophile on the
iminium ion intermediate (see Supporting Information).
Experimental Section
The purity of all the compounds was tested by combustion
analysis, and it is g95%.
General Procedure for Reduction of Nucleosides 15a-d. To
a stirred solution of 14a-d (0.11 mmol) in a 1:1 methanol/dioxane
Biological Evaluation. The synthesized 3′-deoxy-4′-azari-
bonucleosides 15a-d, 23, and 24 have been tested in a cell-
based subgenomic HCV replicon assay to evaluate their ability
to inhibit HCV RNA replication (Table 1). In addition,
cytotoxicity, expressed as CC50, was also evaluated with a MTS
based assay. The screening of all the compounds was
performed up to the fixed concentration of 10 µM and compared
mixture (10 mL), NaBH (40 mg, 1.05 mmol) was added at 0 °C,
4
and the obtained suspension was stirred overnight at room tem-
perature. At the end of this time the solvent was removed and the
residue was extracted with ethyl acetate (2 × 5 mL). The collected
organic phases, dried over sodium sulfate, gave after evaporation
of the solvent at reduced pressure a sticky oil, which was purified
by flash chromatography on silica gel.
18
1
9
3
tert-Butyl-(2R,3R,5S)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)3-hydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate (15a).
with IFN.
As shown in Table 1, all the modified nucleosides with a cis
relationship between the hydroxymethyl group and the nucleo-
base, such as compounds 15a-d and 23, are endowed with a
relevant antiviral activity while showing low or no toxicity.
Compound 24, which possesses a trans configuration not present
in natural nucleosides, shows no antiviral activity. The biological
activity of the evaluated compounds is strictly correlated to their
bonded nucleobase, in the order 5-fluorouracil > thymine > uracil
Eluent mixture: methanol/chloroform 10:90, R
mp ) 177-179 °C (27.5 mg, 0.084 mmol, 76% yield); [R] -87.8
3
(c 0.71, CH OH).
f
) 0.2. White solid:
25
D
tert-Butyl-(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)-2-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)pyrrolidine-1-carboxy-
late (15b). Eluent mixture: methanol/chloroform 7:93, R ) 0.13.
f
White solid: mp ) 119-122 °C (28.7 mg, 0.084 mmol, 76% yield);
2
5
[
R]
D
3
-80.0 (c 0.2, CH OH).
tert-Butyl-(2R,3R,5S)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-
-hydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate (15c).
>
cytosine. The potency of the 5-substituted pyrimidines
3
increases when the hydrogen atom is replaced by a methyl or
fluoride group. Thus, the more active compound 15d, which
possesses a 5-fluorouracil moiety as nucleobase, shows an IC50
in the nanomolar range (0.35 nM), even if it possesses a
moderate level of toxicity. The toxicity seems to be also related
to the substituent bonded at the nitrogen atom: when the Boc
group in 15d is substituted by an ethoxycarbonyl group, as in
Eluent mixture: methanol/chloroform 15:85, R ) 0.14. White solid:
f
25
mp ) 140-141 °C (29 mg, 0.084 mmol, 76% yield); [R]
D
-35,34
(c 0.57, CH OH).
3
tert-Butyl-(2R,3R,5S)-2-(5-fluoro-2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-3-hydroxy-5-(hydroxymethyl)pyrrolidine-1-car-
boxylate (15d). Eluent mixture: methanol/chloroform 10:90, R
f
)
0
.21. White solid: mp ) 60-61 °C (27.4 mg, 0.084 mmol, 76%
25
yield); [R]
D
3
-52.63 (c 0.38, CH OH).
23, a decreased toxicity is observed. Despite a drastic reduction
(
2R,3R,5S)-Ethyl-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
in the antiviral activity, the selective index of compound 23 is
still good (CC50/IC50 g 208). To gain a better understanding of
the mechanism of action of these nucleosides, triphosphates of
compounds 15d and 23 are necessary to evaluate the inhibition
of HCV NS5B-mediated RNA replication. However, the
synthesis of triphosphates by literature methods requires the use
of acidic conditions that are not compatible with the acidic labile
substituents present at the nitrogen atom. Thus, the mechanism
of action of these compounds cannot be elucidated in detail,
but by considering that the R-anomer is completely inactive and
the close relation between the active ꢀ-anomers and the natural
1
(2H)-yl)-3-hydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxy-
late (23) and (2S,3R,5S)-Ethyl-2-(5-fluoro-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)-3-hydroxy-5-(hydroxymethyl)pyrrolidine-
1-carboxylate (24). To a stirred solution of 21 and 22 (1.0 g; 2.58
4
mmol) in a 1:1 methanol/dioxane (50 mL), NaBH (0.98 g, 25.8
mmol) was added at 0 °C. The obtained mixture was further stirred
for 5 h. At the end of this time, the solvent was removed and the
residue was subjected to flash chromatography on RP-18 reverse
phase using water as eluent. The first eluted product was identified
as 23 (0.33 g, 1.05 mmol, 40% yield); white solid, mp ) 56-58
°
C. The second eluted product was identified as 24 (0.32 g, 1.05
mmol, 40% yield); white solid, mp ) 55-57 °C.

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4057
Acknowledgment. We gratefully acknowledge IRBM “P.
Angeletti”, Pomezia-Roma, Italy, M.I.U.R., and C.I.N.M.P.I.S.
for their generous support for this work.
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Supporting Information Available: Experimental conditions
and spectral data of intermediates 10-12, 14a-d, 17-22; spectral
data of target compounds 15a-d, 23, and 24; NOEDS for 14d;
explanation for diastereoselective N-glycosylation reaction; details
of cell culture, antihepatitis C virus, and cytotoxicity assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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