A pyrimidine-pyrazolone nucleoside chimera with potent in vitro anti-orthopoxvirus activity

Article abstract of DOI:10.1016/j.bmcl.2006.03.043

Synthetic hybridization of two privileged drug scaffolds, pyrazolone on the one hand and pyrimidine nucleoside on the other, resulted in the generation of two novel 5-substituted pyrimidine nucleosides with potent in vitro antiviral activity against two representative orthopoxviruses, vaccinia virus and cowpox virus.

Full text of DOI:10.1016/j.bmcl.2006.03.043

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3224–3228
A pyrimidine–pyrazolone nucleoside chimera with potent
in vitro anti-orthopoxvirus activity
Xuesen Fan,a Xinying Zhang,a Longhu Zhou,a Kathy A. Keith,b
Earl R. Kernb and Paul F. Torrencea,*
aDepartment of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011, USA
bDepartment of Pediatrics, University of Alabama School of Medicine, Birmingham, AL 35233, USA
Received 12 February 2006; revised 13 March 2006; accepted 14 March 2006
Available online 5 April 2006
Abstract—Synthetic hybridization of two privileged drug scaffolds, pyrazolone on the one hand and pyrimidine nucleoside on the
other, resulted in the generation of two novel 5-substituted pyrimidine nucleosides with potent in vitro antiviral activity against two
representative orthopoxviruses, vaccinia virus and cowpox virus.
ꢀ 2006 Elsevier Ltd. All rights reserved.
In 1983, the World Health Organization (WHO) de-
clared smallpox, a disease responsible for more deaths
than all the wars of all time, eradicated.1,2 By 1983, all
known stocks of variola virus were in two World Health
Organization (WHO) collaborating centers: the US Cen-
ters for Disease Control and Prevention (CDC) in
Atlanta and (after a transfer in 1994) the Russian State
Research Center of Virology and Biotechnology (the
Vektor Institute) in Novosibirsk. The WHO Committee
on Orthopoxvirus Infections voted on several occasions
to recommend destruction of the stocks, but each time
the decision was deferred to permit more research on
live variola virus.
more in the development pipeline. Only one drug,
cidofovir, is available for treatment of smallpox vaccina-
tion complications.12–14
To discover novel antivirals for orthopoxvirus infec-
tions, we have employed two different ‘‘privileged’’ drug
scaffolds;15,16 that is, those molecular frameworks that
have spawned a significant number of drugs and other
biologically active agents and can be used to discover
molecular ‘masterkeys.’17 The first is the nucleoside scaf-
fold that has yielded many valuable therapeutics for
HIV and herpes virus infections. The second scaffold is
the pyrazolone ring that is the basis of agents with var-
ious biological activities including antihyperglycemic
properties,18 anti-tumor necrosis factor activity,19,20
non-steroidal anti-inflammatory drugs (NSAIDs),21
inhibition of human telomerase,22 and antibacterial
activity.23
Meanwhile, intelligence estimates have suggested the
possible existence of clandestine stocks of variola and
the possibility of bioengineered strains resistant to the
classical vaccination protection. When these concerns
are combined with the established fact that the present
form of smallpox vaccination is the most dangerous vac-
cination extant in terms of morbidity and mortality, it is
patently obvious that novel antiviral agents for this
threat be discovered.3–14 The goal of the US Govern-
ment is to provide two FDA approved drugs with two
Entry to such chimeric structures centered on the 5-po-
sition of pyrimidine nucleosides can be gained most
readily through the intermediacy of the previously de-
scribed 5-formyl-20-deoxyuridine.24–27 The aldehyde
precursor,
30,50-di-O-acetyl-5-formyl-20-deoxyuridine
(3, Scheme 1), was prepared through the oxidation of
30,50-di-O-acetylthymidine (2, Scheme 1) with potassium
peroxysulfate (K2S2O8) in the presence of CuSO4Æ5H2O
and 2,6-lutidine in aqueous acetonitrile. The corre-
sponding deacetylated counterpart, 5-formyl-20-deoxy-
uridine (8, Scheme 2), was prepared by first protecting
Keywords: Smallpox; Vaccinia virus; Cowpox virus; 5-Formyl-20-de-
oxyuridine; Privileged drug scaffold.
*
Corresponding author. Tel.: +1 928 523 0298; e-mail: Paul.Torrence@
nau.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.043

X. Fan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3224–3228
3225
Scheme 1. Reagents and conditions: (a) (CH3CO)2O, DMAP, rt, overnight, 92%; (b) K2S2O8, CuSO4Æ5H2O, 2,6-lutidine, CH3CN/H2O (1:1, v/v),
65 ꢁC, 2 h, 35%; (c) ethanol, rt, overnight, molar ratio of 3 and 4: 1:1; (d) ethanol, rt, overnight, molar ratio of 3 and 4: 1:2.5.
the aldehyde group of 3 as the dimethyl acetal (7), fol-
lowed by sequential treatment with NaOMe/MeOH
and AcOH/H2O to give 8. The pyrazolone derivatives
were prepared through the condensation of an appropri-
ate aldehyde (3, Scheme 1 or 8, Scheme 2) with 1-phenyl-
3-methyl-2-pyrazolin-5-one (4). Condensation product
528 was obtained by stirring a solution of 3 with one
equivalent of 4 in ethanol at room temperature over-
night (Scheme 1). Compound 628 was obtained from
the condensation of 3 with 2.5 equiv of pyrazolone 4
(Scheme 1). The other two pyrazolone derivatives 928
and 1028 (Scheme 2) were prepared in a similar manner.
Condensation of the pyrimidine aldehyde and the pyraz-
olone led to a new double bond exocyclic to the pyraz-
olone ring C2 and in conjugation with the pyrimidine
ring. This alkene substructure can exist as two isomers,
In vitro activities (Table 1) were determined according
to previously described methodologies.29 None of the
compounds described here had any significant cytopath-
ic effect on uninfected cells under these conditions. The
CC50’s all were in excess of 250 lM. These novel agents
possessed in vitro antiviral activities that exceeded the
activity of cidofovir against VV and CV under these
in vitro test conditions. For the pyrimidinylidene
monopyrazolone (9), the EC50, as defined by the CPE
assay, was 0.3 lM. These compounds (9 and 10) were
somewhat more active against CV than against VV.
Since CV is often considered to be a closer model to
the smallpox variola virus than is VV, these results have
to be considered to be encouraging. The potent antiviral
activities of compounds 9 and 10 provide strong leads to
novel orthopoxvirus antivirals; moreover, the novelty of
these structures implies a new paradigm in the structure–
activity driven search for nucleosides with antiviral
activity. Such potent activity in the context of pyrimi-
dine nucleoside 5-substituent hypermodification has
not been observed previously. Studies are in progress
to ascertain the spectrum of activity of 9 and 10, and
to elucidate the mechanism of inhibition of virus replica-
tion and the in vivo potential of these novel agents.
1
namely E and Z. From the H NMR and 13C NMR re-
sults, it was clear that only one isomer was formed since
there was one signal each for the newly formed vinylic
proton and for the pyrimidine H6. The identification
of the Z-configuration was based on NOE experiments
which showed that the vinylic proton exhibited a strong
NOE effect with the protons of the pyrazolone 3-methyl
group.

3226
X. Fan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3224–3228
Scheme 2. Reagents and conditions: (e) MeOH, Amberlite IR-120 (H+), reflux, 2 h; (f) NaOMe/MeOH, rt, 3 h; (g) AcOH/H2O, 50 ꢁC, overnight;
(h) ethanol, 60 ꢁC, 2 h; rt, overnight, molar ratio of 8 and 4: 1:1; (i) ethanol, 60 ꢁC, overnight, molar ratio of 8 and 4: 1:2.5.
Table 1. Inhibition of orthopoxvirus replication by pyrazolo-pyrimidine nucleosidesa
b
c
Compound
Efficacy EC50 (lM)
Vacciniad PRe Cowpoxd CPE
Toxicity CC50 (lM)
Neutral red uptake
Vacciniad CPE
Cowpoxd PRe
Cidofovir
3.2
32
20 11
56 5.1
119 81
6.9 0.9
11 1.0
7.1
33.7
37.1
0.3
32 10
48 20
>317
0
5
6
>252 37
>288 13
>286 25
>292 14
43
25 1.1
5.6 5.2
9.0 7.0
9
10
1.7
20
1.8
a Assays were performed according to the procedures described previously.29
b EC50, effective concentration to reduce viral cytopathogenicity (CPE) or plaque formation (PR) by 50%. Values without a standard deviation refer
to experiments carried out once in triplicate.
c CC50, concentration which causes a cytotoxic effect (as ascertained by neutral red uptake) on 50% of uninfected cells.
d Virus used for challenge: vaccinia virus (Copenhagen) or cowpox virus (Brighton).
e Values are the means standard deviation of 2 or more assays.
Acknowledgments
References and notes
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This research was supported by funds from the US
Army Medical Research Institute of Infectious Diseases
(USARMRIID DAMD 17-03-C-0081 US Army Medi-
cal Research Materiel Command) and Public Health
Service Contract NO1-AI-30049 from NIH, NIAID,
Bethesda, MD (ERK). The technical assistance of
Robert Smith and Shalisa Sanders is gratefully
acknowledged.
3. Alibek, K. Int. J. Infect. Dis. 2004, 8, S3.

X. Fan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3224–3228
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28. Selected data for 1-(2-deoxy-3,5-di-O-acetylpentofurano-
syl)-5-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-
4-ylidene)pyrimidine-2,4(1H,3H)-dione (5): mp 228–230 ꢁC;
1H NMR (CDCl3) d: 1.97 (s, 3H, CH3,on the pyrazolone
ring), 2.14 (s, 3H, CH3, –OCOCH3), 2.34 (s, 3H, CH3, –
OCOCH3), 2.54–2.59 (m, 2H, H20), 4.33–4.57 (m, 3H,
H40, H50), 5.38–5.41 (m, 1H, H30), 6.36–6.40 (m, 1H,
H10), 7.20 (t, 1H, J = 7.6 Hz, PhH), 7.42 (t, 2H,
J = 7.6 Hz, PhH), 7.69 (s, 1H, @CH), 7.92 (d, 2H, J =
7.6 Hz, PhH), 8.70 (br s, 1H, NH), 10.93 (s, 1H, H6).
13C NMR (CDCl3) d: 13.34, 20.92, 21.15, 37.72, 63.99,
74.35, 83.14, 86.60, 109.01, 119.48, 125.40, 126.62,
129.08, 136.23, 138.31, 148.43, 149.18, 151.06, 161.58,
162.71, 170.41, 170.61. ESI LRMS m/e: 497 (MH+), 519
(MNa+). HRMS (ESI) Calcd for C24H25N4O8: 497.1673
(MH)+, found 497.1687. Selected data for 5-[bis(3-methyl-
5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-yl)methyl-1-
(2-deoxy-3,5-di-O-acetylpentofuranosyl)pyrimidine-2,4(1H,
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1
3H)-dione (6): mp 158–160 ꢁC; H NMR (CDCl3) d: 1.83
(s, 3H, CH3 of pyrazolone ring), 2.04 (s, 3H, CH3, of
pyrazolone ring), 2.19 (s, 3H, CH3, –OCOCH3), 2.28–2.39
(m, 5H, CH3,–OCOCH3, H20), 4.06–4.18 (m, 3H, H40,
H50), 4.49 (s, 1H, –CH), 5.15–5.17 (m, 1H, H30), 6.10–6.14
(m, 1H, H10), 7.10–7.16 (m, 2H, PhH), 7.25–7.32 (m, 4H,
PhH), 7.54–7.58 (m, 4H, PhH), 7.84 (s, 1H, H6), 9.82 (br s,
1H, NH). 13C NMR (CDCl3) d: 11.96, 12.21, 20.76, 21.12,
26.32, 37.00, 63.82, 74.71, 82.60, 86.94, 114.50, 121.37,
121.57, 126.35, 126.52, 129.11, 129.20, 136.89, 137.46,
138.04, 147.09, 147.41, 150.13, 163.89, 170.42, 170.93. ESI
LRMS m/e: 671 (MH+), 693 (MNa+). HRMS (ESI) Calcd
for C34H35N6O9: 671.2484 (MH)+, found 671.2454.
Selected data for 1-(2-deoxypentofuranosyl)-5-[(3-methyl-
5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-ylidene)pyrimi-
dine-2,4(1H,3H)-dione (9): mp >275 ꢁC; 1H NMR (DMF-
d7) d: 2.32 (s, 3H, CH3, on the pyrazolone ring), 2.38–2.48
(m, 2H, H20), 3.86–3.89 (m, 2H, H50), 4.05–4.08 (m, 1H,
H40), 4.52–4.53 (m, 1H, H30), 4.84 (t, 1H, J = 6.4 Hz, 50-
OH), 5.46 (d, 1H, J = 4.0 Hz, 30-OH), 6.32–6.36 (m, 1H,
H10), 7.22 (t, 1H, J = 7.6 Hz, PhH), 7.46 (t, 2H,
J = 8.0 Hz, PhH), 7.76 (s, 1H, @CH), 8.02 (d, 2H,
J = 8.0 Hz, PhH), 10.79 (s, 1H, H6), 12.01 (br s, 1H,
NH). 13C NMR (DMF-d7) d: 12.58, 40.67, 62.54, 71.51,
86.86, 89.36 108.04, 118.71, 123.80, 124.91, 129.09, 138.82,
138.98, 149.56, 150.05, 151.76, 162.98. ESI LRMS m/e:
413 (MH+), 435 (MNa+). HRMS (FAB) Calcd for
C20H21N4O6: 413.1462 (MH)+, found 413.1452. Com-
pound 9 was easily soluble in DMSO. In addition, a
0.1 mM solution of 9 in 1% DMSO–phosphate-buffered
saline (pH 7.5) or in 1% DMSO/H2O could be prepared
readily. Selected data for 5-[bis(3-methyl-5-oxo-1-phenyl-
4,5-dihydro-4H-pyrazol-4-yl)methyl-1-(2-deoxypentofur-
anosyl)pyrimidine-2,4(1H,3H)-dione (10): mp 182–184 ꢁC;
1H NMR (CD3OD) d: 2.13–2.20 (m, 1H, H20-1), 2.25–2.32
(m, 4H, H20-2, CH3 of pyrazolone ring), 2.38 (s, 3H, CH3
of pyrazolone ring), 3.48–3.50 (m, 2H, H50), 3.81–3.83 (m,
1H, H40), 4.26–4.29 (m, 1H, H30), 4.95 (s, 1H, –CH), 6.26–
6.29 (m, 1H, H10), 7.27–7.31 (m, 2H, PhH), 7.42–7.47 (m,
4H, PhH), 7.61–7.65 (m, 4H, PhH), 7.85 (s, 1H, H6). 13C
NMR (DMSO-d6) d: 12.39, 25.99, 62.65, 71.16, 79.86,
84.44, 87.96, 115.12, 121.4,4 121.69, 126.38, 129.57,
136.37, 146.51, 146.85, 150.70, 163.32. ESI LRMS m/e:
587 (MH+), 609 (MNa+). HRMS (ESI) Calcd for
C30H31N6O7: 587.2255 (MH)+, found 587.2228.
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X. Fan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3224–3228
trations. Infection with vaccinia virus (VV) Copenhagen
neutral red in PBS and incubated for 5–6 h. After
aspiration of the stain, viral plaques were counted using
a stereomicroscope at 10· magnification. The concentra-
tion of agent that inhibited viral CPE or plaque formation
by 50% was defined as the EC50 and was calculated using
standard methods. The effect of the potential antiviral
agent on uninfected host cell viability was evaluated using
HFF cells seeded in 96-well plated incubated with various
concentrations of drug for 7 days. After incubation, cell
monolayers were stained with a solution of neutral red and
the concentration of agent that reduced neutral red uptake
by 50% was defined as the CC50.
or cowpox virus (CV) Brighton at 1000 PFU per well was
followed by incubation at 37 ꢁC for 7 days. After
incubation, the plates were stained with a crystal violet
solution for 4 h, rinsed, allowed to dry for 24 h, and then
read on a BioTek Plate Reader at 620 nm. Confirmatory
assays using a plaque reduction (PR) assay were per-
formed using HFF cells seeded in 6-well plates 2 days
prior to use and infected with VV or CV at 20–30 PFU per
well. After a 1 h incubation period, various concentrations
of drug were added in triplicate and the plates were
incubated at 37 ꢁC for 3 days. The cells were stained with a