Design and evaluation of a potential mutagen for Hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2007.03.091

Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC₅₀ = 60 μM) and did not exhibit cytotoxicity (CC₅₀ > 100 μM). Investigation of phosphorylation events using nucleoside kinases and LC-MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.

Full text of DOI:10.1016/j.bmcl.2007.03.091

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5261–5264
Design and evaluation of a potential mutagen for Hepatitis C virus
,
Yung-Hyo Koh,* Jae Hoon Shim, Jean-Luc Girardet and Zhi Hong
Valeant Pharmaceutical Research & Development, 3300 Hyland Ave., Costa Mesa, CA 92626, USA
Received 6 February 2007; revised 23 March 2007; accepted 29 March 2007
Available online 2 April 2007
Abstract—Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B
enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug
of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC50 = 60 lM) and did not
exhibit cytotoxicity (CC50 > 100 lM). Investigation of phosphorylation events using nucleoside kinases and LC–MS analysis
revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.
Ó 2007 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection is a leading cause of
chronic liver diseases, such as cirrhosis and hepatocellular
carcinoma. It is estimated that more than 170 million peo-
ple are infected by HCV worldwide. HCV is a positive-
the viral genome in a dose-dependent manner, and a
small increase of mutation rate (2- to 6-fold) reduced
viral infectivity to as little as 0.00001% in cell culture.
In a primer-extension assay of poliovirus polymerase,
ribavirin triphosphate acted as a substrate and base-
paired equally well with either uridine or cytidine. As
a result, ribavirin monophosphate could be mis-incorpo-
rated into the nascent RNA product. Ribavirin in an
RNA template also served as a base for incorporation
of cytidine or uridine with equal efficiency, confirming
1
stranded RNA virus in the family of Flaviviridae. The
only FDA-approved HCV therapies are interferon or
pegylated interferon monotherapies or in combination
2
with ribavirin. However, their efficacy is less than ideal
as only 52–54% of patients achieve a sustained virological
3
response. Clearly, there is urgent medical need to develop
more antiviral agents to fight the HCV infection.
7
,8
that ribavirin mis-incorporation could be mutagenic.
The mutagenic effect of ribavirin on HCV infection
has been subsequently investigated using in vitro stud-
One idea to develop new antiviral agents is to exploit the
lack of fidelity or proof-reading mechanism during
RNA virus replication which is responsible for the
9
,10
ies.
In this paper, we report the design of a modified
cytidine base as a potential mutagen during HCV repli-
cation and evaluation of its biological activities.
4
extraordinary genetic variation among RNA viruses.
Such diverse RNA viral quasispecies are important to
promote escape from immune selection and to adapt
rapidly to new environments but also may put the
RNA viruses on the threshold of error catastrophe at
which a small increase of mutation rate may induce a ge-
We designed pyrrolo[2,3-d]pyrimidin-2-one nucleoside 1
as a potential mutagen by fusing a five-membered ring
on the pyrimidine of cytidine (Fig. 1). If this bicyclic ring
system tautomerizes between 1 and 1a in biological solu-
tions, structure 1 could act as a cytidine and structure 1a
5
,6
netic melt-down and eradicate a virus population.
Therefore, an RNA virus mutagen would be an effective
antiviral drug. In fact, recent studies have indicated that
ribavirin may act as such a mutagen. In the poliovirus
model system, ribavirin increased the mutation rate of
NH
N
N
NH
N
O
N
O
HO
HO
O
O
Keywords: Mutagen; Antiviral design; Nucleoside analog; HCV; RNA
virus.
HO OH
HO OH
1
1a
*
Corresponding author. Tel.: +1 714 729 5533; fax: +1 714 729
577; e-mail: ykoh@ardeabiosciences.com
5
Figure 1. Proposed mutagen, pyrrolopyrimidine nucleoside 1. The
pyrrolopyrimidine nucleoside could potentially mimic a cytidine and it
could also tautomerize to 1a to mimic a uridine.
Current address: Ardea Biosciences, 3300 Hyland Ave., Costa Mesa,
CA 92626, USA.
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.091

5262
Y.-H. Koh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5261–5264
could act as a uridine. Synthesis of 1 was achieved fol-
1
1
lowing a route depicted in Scheme 1. Hydrogenation
of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2) in
the presence of palladium on activated charcoal gave
3
. Protection of pyrrole nitrogen of 3 with benzyloxycar-
bonyl (Cbz) group was necessary for the next diazotiza-
tion. The diazotization of 4 with sodium nitrite in acetic
acid and water provided the pyrrolopyrimidine 5. Gly-
cosylation of 5 with ribofuranose 6 yielded nucleoside
7, and deprotection of benzoyl groups of 7 provided
the target nucleoside 1.
For biological evaluations, 1 was further converted to its
triphosphate form 8 and an S-acyl-2-thioethyl (SATE)
prodrug form 9 was synthesized following a well-estab-
1
2
lished literature procedure. The triphosphate 8 was
synthesized from the reaction of 1 with phosphorus oxy-
chloride in trimethylphosphate, followed by the reaction
with tributylammonium pyrophosphate. Synthesis of
prodrug 9 was accomplished by the treatment of 1 with
Scheme 2. Reagents: (a) POCl
3
, PO(OCH
3 3
) ; TBAPP, tributylamine,
20%; (b) 10, DMF, 1H-tetrazole; (CH )
3 3
COOH, 60%.
1
Lane 1
2
3
4
5
6
7
8
9
10
phosphoramidite 10 in the presence of H-tetrazole and
the subsequent oxidation with tert-butyl hydroperoxide
(
Scheme 2).
To test if 1 could mimic both cytidine and uridine, we
used triphosphate 8 in the HCV NS5B enzymatic nucle-
otide incorporation assay on different templates. Base-
pairing of 8 with A into a product was examined by
0
0
using an RNA template, 5 -AAAAAGGAGC-3 , and
a
3
3
P-labeled primer, pGpC. The reaction will give a tri-
nucleotide product or longer elongation products when
additional CTP is present. Similarly, incorporation
0
opposite to G was tested by using 5 -AAAAAAA
3
3
0
33
GAU-3 and pApU.
pGpC
3
3
pApU
The resulting products are shown in Figure 2 along with
products from control experiments using UTP or CTP.
It appears that 8 was not incorporated in significant
amount into a product through base-pairing with A
Figure 2. Incorporation of pyrrolopyrimidine triphosphate 8 by HCV
3
3
NS5B using AAAAAGGAGC and
pGpC (lanes 1–5), and
3
3
AAAAAAAGAU and pApU (lanes 6–10). Each reaction mixture
contained 5 lM of NS5B, 20 lM of the primer, and 20 lM of the RNA
template, and 1 mM of a nucleotide as the following: lane 1, none; lane
Cbz
NH
NH
N
a
b
2
, UTP; lane 3, UTP and CTP; lane 4, compound 8; lane 5, compound
and CTP; lane 6, none; lane 7, CTP; lane 8, CTP and UTP; lane 9,
N
N
N
8
compound 8; lane 10, compound 8 and UTP. The reactions were
Cl
N
NH2
N
NH2
N
NH2
2
3
4
performed in 50 mM HEPES, pH 7.3, 10 mM MgCl , and 10 mM
2
DTT at 30 °C for 1 h. The reaction products were resolved on a 25%
polyacrylamide-7 M urea-TBE gel and were scanned on
PhosphorImager.
Cbz
N
a
N
c
d
BzO
OAc
O
N
OH
(lane 4), nor were there any significant elongation prod-
5
BzO OBz
ucts (lane 5). However, the trinucleotide product was
clearly seen when it was incorporated through base-pair-
ing with G (lane 9). The intensity of the band is compa-
rable to that from CTP incorporation (lane 7).
Interestingly, elongation products from both CTP and
8 were not clearly visible (lanes 8 and 10). This result
indicates that the pyrrolopyrimidine base of 8 favors
the cytosine-like tautomer rather than the uracil-like
tautomer under our experimental conditions.
6
NH
NH
N
N
e
N
O
N
O
BzO
HO
O
O
BzO OBz
HO OH
7
1
Scheme 1. Reagents: (a) H
COCl, N(CH CH
CH COOH, 51%; (d) N,O-bis(trimethylsilyl)acetamide, CH
SnCl , 45%; (e) NH , CH OH, 98%.
2
, Pd–C, CH
3
OH, 90%; (b) C
6
H
5
CH
2
O-
O,
The SATE-prodrug 9 was tested by using a cell-based
HCV replication assay. It showed a weak inhibitory
activity in an HCV replicon system (EC50 = 60 lM)
2
3
)
3
,
DMAP, DMF, 52%; (c) NaNO
2
,
H
2
3
3
CN; 6,
4
3
3

Y.-H. Koh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5261–5264
5263
ATP
2
1.25
100
ADP
18.03
%
1-MP
AMP
1
9.90
16.48
14.28
-
0
18.18
1
1
00
1
10.01
-MP
21.50
%
1
16.66
3.33
-
1
16.74
00
1
%
-
1
Time
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
2.00
4.00
6.00
8.00
Figure 3. Chromatograms of UCK and NMPK reactions with pyrrolopyrimidine 1; lower panel, no enzyme, pyrrolopyrimidine 1 alone; middle
panel, UCK + 1; top panel, UCK + NMPK + 1. Reaction mixtures include 0.2 lM 1, 0.2 or 0.4 lM ATP, 2 lM UCK1 (human), and 0.014 U of
NMPK (bovine liver) in 50 mM HEPES, pH 7.3, and 2 mM MgCl . Reactions were performed at 37 °C for 30 min and quenched with 10 lM EDTA.
2
ADP
ATP
22.804
cytidine
2.85
1
9.40
1
00
AMP
CDP
%
15.45
6
.67 9.15
4.39
-
2
19.39
42.86
100
CMP
4
.37
%
22.81
-
3
Time
50.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
Figure 4. Chromatograms of UCK and NMPK reactions with cytidine; lower panel, UCK + cytidine; upper panel, UCK + NMPK + cytidine.
and did not exhibit any cytotoxicity (CC50 >100 lM).
The lack of both inhibitory and cytotoxic activity may
reflect poor phosphorylation events in the cells. Thus,
we decided to examine phosphorylation efficiency of 1
in vitro. The pyrrolopyrimidine nucleoside 1 was incu-
bated with uridine–cytidine kinase (UCK) and nucleo-
side monophosphate kinase (NMPK), and products
were analyzed on an LC–MS (Fig. 3). In parallel, a con-
trol reaction was performed with cytidine to confirm the
production of CMP and CDP (Fig. 4). The middle panel
in Figure 3 clearly shows that a reaction with UCK
alone produced the monophosphorylated product.
However, a reaction with both UCK and NMPK did
not produce any trace of a diphosphate product (top pa-
nel, Fig. 3). This is surprising because NMPK is known
to accommodate a wide range of analogs of nucleoside
monophosphates. Usually, the first phosphorylation of
nucleoside, producing NMP, is known to be the rate-
limiting step in cells, and to overcome this difficult step,
prodrug forms of NMP are frequently utilized.
acts as a CTP analog rather than as a UTP analog
under our experimental conditions. Testing the pyrrol-
opyrimidine nucleoside 1 as a mutagen in vivo may
not be feasible because, unlike other nucleoside ana-
logs, the second step of phosphorylation in cells, from
monophosphate ester to diphosphate ester, appears to
be unfavorable.
References and notes
1. World Health Organization: Hepatitis C-Global Preva-
lence (update). Weekly Epidemiological Record, 2000, 75,
1
8.
2
3
. Memon, M. I.; Memon, M. A. J. Viral Hepatitis 2002, 9, 84.
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V. K.; Shiffman, M.; Reindollar, R.; Goodman, Z. D.;
Koury, K.; Ling, M.; Albrecht, J. K. Lancet 2001, 358,
958.
. Hong, Z.; Cameron, C. E. Prog. Drug Res. 2002, 59, 41.
4
5. Biebricher, C. K.; Eigen, M. Virus Res. 2005, 107, 117.
6
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Rose, K. D.; Mullins, J. I. Proc. Natl. Acad. Sci. U.S.A.
In conclusion, an in vitro enzymatic assay indicated
that pyrrolopyrimidine triphosphate 8 preferentially
1
999, 96, 1492.

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Y.-H. Koh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5261–5264
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