Synthesis and antiviral activity of 5-substituted cytidine analogues: Identification of a potent inhibitor of viral RNA-dependent RNA polymerases

Article abstract of DOI:10.1021/jm060872x

As part of our studies of lethal viral mutagens, a series of 5-substituted cytidine analogues were synthesized and evaluated for antiviral activity. Among the compounds examined, 5-nitrocytidine was effective against poliovirus (PV) and coxsackievirus B3

Full text of DOI:10.1021/jm060872x

6166
J. Med. Chem. 2006, 49, 6166-6169
causative factors in genomic mutagenesis.11 The addition of a
hydroxyl moiety to the 5-position of cytosine alters the relative
distribution of amino to imino nucleobase tautomers, thereby
increasing the abundance of imino 5-hydroxycytosine, which
can base-pair with adenine.12-14 Mispairing of this oxidative
lesion with A during DNA replication promotes transition
mutations and consequential scrambling of the encoded genetic
message. Oxidative DNA damage can also occur by the
introduction of oxidized deoxycytidine triphosphates (dCTPs)
into the genome during replication. The 5′-triphosphate of 2, a
product of dCTP oxidation, is incorporated into DNA by Klenow
DNA polymerase I.15,16 Fortunately, the integrity of DNA is
maintained by complex networks of repair machinery that target
such forms of DNA damage.17,18
On the basis of its mutagenic capacity toward genomic DNA,
5-hydroxy-2′-deoxycytidine (2) has been evaluated as an
antiviral lethal mutagen against the HIV retrovirus.9 Treatment
with 2 confers significant reductions in viral titer, including an
increase in G to A substitutions in the gene-encoding reverse
transcriptase (RT).9 In addition, the 5′-triphosphate of 2 functions
as a substrate for HIV RT and is incorporated opposite G and
A in the DNA template.9,19 To build upon these results, we
hypothesized that ribonucleoside 4 might function as an
analogous antiviral lethal mutagen against RNA viruses. To test
this hypothesis, we synthesized 5-hydroxycytidine (4) and
related analogues 5-7 and evaluated the antiviral activity of
these compounds against the RNA viruses poliovirus and
coxsackievirus B3 (CVB3).
An improved synthesis of 5-nitrocytidine (6) and 5-aminocy-
tidine (7) is shown in Scheme 1. Readily prepared 5-nitrocy-
tosine (10)20 was persilylated by reaction with HMDS and
catalytic TMSCl to provide 11. Vorbru¨ggen coupling condi-
tions21,22 afforded benzoyl-protected 5-nitrocytidine 13.23 Hy-
drogenation of 1323 provided the protected 5-aminocytidine 14.
Saponification of esters 13 and 14 as previously described23
delivered 6 and 7 in 40% and 26% overall yields. This approach
is more rapid than an earlier reported syntheses of 6 and 7 via
the common intermediate 13.23 Compound 7 has also been
synthesized by amination of 5-bromocytidine (5) with ammonia.
However, these approaches suffer from low yields or require
separation of the 5- and 6-amino regioisomers.24,25
Synthesis and Antiviral Activity of 5-Substituted
Cytidine Analogues: Identification of a Potent
Inhibitor of Viral RNA-Dependent RNA
Polymerases
Daniel A. Harki,‡,§ Jason D. Graci,†,§ Jessica E. Galarraga,†
William J. Chain,‡ Craig E. Cameron,† and
Blake R. Peterson*,‡
Department of Chemistry and Department of Biochemistry and
Molecular Biology, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802
ReceiVed July 24, 2006
Abstract: As part of our studies of lethal viral mutagens, a series of
5-substituted cytidine analogues were synthesized and evaluated for
antiviral activity. Among the compounds examined, 5-nitrocytidine was
effective against poliovirus (PV) and coxsackievirus B3 (CVB3) and
exhibited greater activity than the clinically employed drug ribavirin.
Instead of promoting viral mutagenesis, 5-nitrocytidine triphosphate
inhibited PV RNA-dependent RNA polymerase (Kd ) 1.1 ( 0.1 µM),
and this inhibition is sufficient to explain the observed antiviral activity.
Ribonucleoside analogues that enhance the basal mutation
frequency of RNA viruses constitute a promising new class of
antiviral therapeutics. Such compounds, termed lethal mutagens,
accelerate viral mutagenesis to intolerable levels, resulting in
“error catastrophe” and loss of viral viability.1-9 The mechanism
of antiviral activity for mutagenic ribonucleoside analogues
typically involves (i) in vivo conversion to ribonucleotides
facilitated by host cell enzymes, (ii) misincorporation into the
viral genome by error-prone viral RNA-dependent RNA poly-
merases (RdRPa), and (iii) indiscriminate nucleotide templating
during genomic replication. Over successive rounds of replica-
tion, the accrual of excessive mutations forces the virus into
“error catastrophe”, and viral viability is lost. Previously, we
demonstrated that ribavirin (1), a clinically employed antiviral
drug, functions as a lethal mutagen against poliovirus (PV)8 and
hepatitis C virus.10 Inspired by the known lethal mutagen for
HIV, 5-hydroxy-2′-deoxycytidine (2),6,9 we report here the
antiviral evaluation activity of a suite of 5-substituted cytidine
analogues (4-7).
The cytotoxicity of ribavirin (1), 5-hydroxy-2′-deoxycytidine
(2), 5-bromo-2′-deoxycytidine (3), and ribonucleoside analogues
4-7 was evaluated in HeLa S3 cells (Figure 1). 5-Hydroxy-
cytidine (4) was the most toxic ribonucleoside, with associated
host cell viability ranging from 31% to 40% across the four
concentrations tested. Interestingly, 2 was significantly less
cytotoxic, with >73% cell viability observed at all of the
concentrations examined. The least cytotoxic ribonucleoside
proved to be 5-nitrocytidine (6), a compound that yielded cell
viabilities greater than 76% for all concentrations tested.
The antiviral activity of 1-7 was evaluated against PV and
CVB3 in cell culture (Figure 1). In these experiments, HeLa
S3 cells were pretreated with 1-7 for 1 h, followed by
administration of a high multiplicity of infection (MOI) dose
Hydroxylated cytosines, such as 5-hydroxycytosine, are
hallmarks of oxygen radical induced DNA damage and early
a Abbreviations: RdRP, RNA-dependent RNA polymerase; PV, polio-
virus; CVB3, coxsackievirus B3; HIV, human immunodeficiency virus;
HCV, hepatitis C virus; RT, reverse transcriptase; S/S, symmetrical RNA
substrate; AcOH, acetic acid; dCTPs, deoxycytidine triphosphates; MeCN,
acetonitrile; MOI, multiplicity of infection; PFU, plaque-forming
unit; PAGE, polyacrylamide gel electrophoresis; TMSCl, trimethylsilyl
chloride.
* To whom correspondence should be addressed. Phone: (814) 865-
2969. Fax: (814) 863-5319. E-mail: brpeters@chem.psu.edu.
‡ Department of Chemistry.
§ These authors contributed equally to this work.
† Department of Biochemistry and Molecular Biology.
10.1021/jm060872x CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/15/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6167
Scheme 1a
a (a) TMSCl, HMDS; (b) SnCl4, MeCN; (c) 10% Pd/C, AcOH, THF;
(d) NaOH(aq), EtOH.
Figure 2. Incorporation of nucleotides derived from triphosphates 8
and 9 into symmetrical RNA substrates (S/S-N) in vitro. The 10-mer
substrates were end-labeled with 32P, complexed with PV RdRP, and
treated with the correct NTP, 8, or 9. An extended time period (15
min) was used to detect incorporation of 8 and 9. Products were
separated by denaturing PAGE.
infected HeLa S3 cells treated with 6 produced 33-fold and 12-
fold less viable PV and CVB3, respectively, at the highest
concentration tested.
To probe the antiviral mechanism of action of 6, we
synthesized its 5′-triphosphate 9 and evaluated the ability of 9
to function as a substrate for PV RdRP in a primer-extension
assay.27 Four distinct primer templates were utilized to probe
the in vitro incorporation of 9 opposite each RNA nucleobase
mediated by purified PV RdRP.28 For comparison, the incor-
poration of the structurally related 5-bromocytidine triphosphate
8 and natural nucleotides opposite each templating base was
examined. Both 8 and 9 were incorporated opposite guanine
and adenine (Figure 2). However, an unusually long, biologically
irrelevant time scale (15 min) was employed so that inefficient
incorporation by PV RdRP could be detected. Neither nucleotide
was incorporated opposite cytosine or uracil. Compared with
incorporation of CTP into S/S-G (kpol ) 157 ( 8 s-1, Kd )
19.2 ( 3.2 µM),8 5-nitrocytidine triphosphate 9 was added
approximately 1900-fold more slowly (kpol ) 0.082 ( 0.005
s-1) and bound the polymerase with an 18-fold higher affinity
(Kd ) 1.08 ( 0.08 µM). The slow rate of incorporation of 9
into viral RNA by PV RdRP, coupled with a negative result in
a previously described guanidine-resistance assay for poliovirus
mutagenesis (data not shown),8,29 suggests that the antiviral
activity observed for 5-nitrocytidine (6) does not result from
lethal mutagenesis. Alternatively, triphosphate 9, owing to its
18-fold higher affinity for the RdRP (compared with CTP), more
likely functions as an inhibitor of this enzyme.
For triphosphate 9 to confer an antiviral effect by inhibiting
PV RdRP, 6 must be metabolized to 9 in living HeLa cells. To
examine the presence of triphosphate 9 in cell extracts, HeLa
S3 cells were incubated with nucleoside 6, the intracellular
nucleotides were extracted, and phosphorylated metabolites were
analyzed by reverse-phase (RP) HPLC.30 As shown in Figure
3, analysis of the intracellular nucleotide pool revealed the
presence of 9 from the similarity in retention time and UV
absorbance compared with an authentic standard (details in the
Supporting Information). Co-injection of crude intracellular
material spiked with a known amount of 9 as a standard revealed
an enhancement in signal intensity, confirming that 9 is formed
from 6 in HeLa S3 cells.
Figure 1. (A) Cytotoxicity to HeLa S3 cells after treatment with 1-7
for 7 h, followed by recovery without compounds for 24 h. (B, C)
Antiviral effects of compounds against poliovirus (B) and coxsack-
ievirus B3 (C). HeLa S3 cells were incubated with 1-7 for 1 h at the
concentrations shown and subsequently infected with 106 PFU of PV
or CVB3. Fifteen minutes after the infection, fresh media containing
1-7 was added, and the infection progressed for 6 h. Cell-associated
virus was titered with plaque assays.
of either virus. After rapid association of virus with the host
cells (15 min), fresh media containing 1-7 was added at the
concentrations shown. The infection was allowed to progress
for an additional 6 h, and cell-associated virus was subsequently
titered by plaque assay as previously described.8,26 As expected,
the antiviral drug ribavirin (1) elicited a dose-dependent
reduction in viral titer in both PV and CVB3 infected cells
(Figure 1). Both 2′-deoxycytidines (2 and 3) failed to reduce
the titer of either virus at all concentrations tested. Surprisingly,
5-hydroxycytidine (4) also failed to significantly affect PV or
CVB3 titer at any concentration. Interestingly, 5-nitrocytidine
(6) and 5-aminocytidine (7) substantially decreased viral titer
in PV and CVB3-infected cells, with 6 surpassing the antiviral
activity of 1. Compared to treatment with ribavirin, virally

6168 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Letters
Figure 3. Analysis of HeLa cell extracts by RP HPLC. (A) Untreated
cells were lysed, nucleotides were extracted, and crude cellular material
was injected. (B) Cell extract after treatment with 6 (2 mM) for 3 h.
The bracketed region integrating for 226 mAU s-1 includes 9 (see
Supporting Information). (C) Analysis of 9 (0.56 nmol) as a standard
(120 mAU s-1). (D) Co-injection of the material shown in panel B
Figure 5. Incorporation of nucleotides derived from CTP, ATP, GTP,
and 9 opposite complementary bases of the symmetrical substrate S/S-
CGUA in vitro. The 10-mer substrates were end-labeled with 32P and
treated with PV RdRP and nucleotides. Products were separated by
denaturing PAGE.
spiked with 9 (0.56 nmol). The area under the bracket (342 mAU s-1
)
is the sum of materials from panels B and C analyzed separately.
In conclusion, we synthesized a 5-nitro derivative of the
ribonucleoside cytidine (6) and demonstrated that this compound
is a metabolic precursor to a potent inhibitor of PV RdRP.
Compound 6 is phosphorylated intracellularly to the 5′-
triphosphate (9), and this metabolite decreases the kinetics of
nucleotide incorporation during replication of PV in vitro. This
diminished viral replication yields a potent antiviral response
in human cells infected with polio- and coxsackieviruses.
Interestingly, a deoxy analogue of 6, 5-nitro-2′-deoxycytidine,
exhibits antiviral activity against herpes simplex viruses 1 and
233 and HIV-1,34 although its mechanism of antiviral activity
has not been fully elucidated. 5-Nitrocytidine (6) represents a
promising lead for the development of novel antiviral thera-
peutics.
Figure 4. Replication of PV quantified by a luciferase reporter assay.
HeLa S3 cells were transfected with pRLucRA. Cells were treated at
37 °C with DMSO (1%) vehicle control, 6 (1 mM), or 1 (2 mM) in the
presence or absence of 3 mM guanidine hydrochloride. Arrows illustrate
the lag in replication induced by 6.
Acknowledgment. We thank Dr. N. Chapman and Dr. S.
Tracy (University of Nebraska Medical Center) for CVB3 gene
constructs and Dr. Zhi Hong (Valeant Pharmaceuticals) for the
gift of ribavirin. We gratefully acknowledge the NIH (Grant
AI054776 to B.R.P. and C.E.C.) and the American Heart
Association (Grant 0340028N to C.E.C. and a predoctoral
fellowship to D.A.H.) for financial support.
To determine if 5-nitrocytidine (6) affects the kinetics of viral
replication, a luciferase-based reporter assay for PV replication
was performed. HeLa S3 cells were transfected with the
subgenomic replicon pRLucRA, which contains the wild-type
PV sequence with the capsid-coding region replaced by the
luciferase reporter gene.31,32 Cells were treated with 6, 1, or a
vehicle control (DMSO, 1%). Additionally, guanidine hydro-
chloride, a reversible inhibitor of PV replication,29 provided
another control. As shown in Figure 4, cells treated with 6
showed a lag in replication kinetics compared to the vehicle
and ribavirin.
Note Added after ASAP Publication. This manuscript was
released ASAP on September 15, 2006, with an error in the
caption (part D) of Figure 3. The correct version was posted on
September 19, 2006.
Given the high affinity of the 5-nitrocytidine triphosphate (9)
for PV RdRP, inhibition of this enzyme by metabolite 9 is a
potential mechanism of antiviral activity. To further measure
inhibition of PV RdRP in the presence of 9, we performed an
additional primer-extension assay to detect “stalling” of this
RdRP (Figure 5).27 The primer template utilized in this
experiment places G (a templating nucleotide for 9) in the
second position. As expected, extension of the nucleotide to
the +3 product (13-mer) is rapidly achieved by addition of three
nucleotides (GTP, CTP, and ATP) to corresponding templating
bases (C, G, and U). Conversely, template extension is not
observed in the presence of 9 alone. However, replacing CTP
with 9 in the presence of GTP and ATP results in significant
“stalling” at the +1 product, signifying rapid GTP incorporation
opposite C and slow incorporation of 9 opposite templating G.
In the context of the ∼1800 possible CTP incorporations that
occur during PV replication, this observed RdRP “stalling”
would greatly diminish the efficiency of viral replication, and
this inhibition is sufficient to explain the observed antiviral
activity of 6.
Supporting Information Available: Experimental procedures,
supporting spectra, and characterization data for synthetic com-
pounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
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