An analogue of AICAR with dual inhibitory activity against WNV and HCV NTPase/helicase: Synthesis and in vitro screening of 4-carbamoyl-5-(4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl)imidazole-1- β-d-ribofuranoside

Article abstract of DOI:10.1016/j.bmcl.2007.01.074

The title compound (4) was synthesized by the reaction of ethyl 1-(2,3,5-tri-O-benzoyl-β-d-ribofuranosyl)-5-formylimidazole-4-carbo xylate with excess guanidine in ethanol at reflux. Compound 4 was evaluated in vitro against NTPases/helicases of four diff

Full text of DOI:10.1016/j.bmcl.2007.01.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2285–2288
An analogue of AICAR with dual inhibitory activity against WNV
and HCV NTPase/helicase: Synthesis and in vitro screening
of 4-carbamoyl-5-(4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl)
imidazole-1-b-D-ribofuranoside
Ravi K. Ujjinamatada,a Andrea Baier,b Peter Borowskib and Ramachandra S. Hosmanea,*
aLaboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland, Baltimore County,
1000 Hilltop Circle, Baltimore, MD 21250, USA
bThe Faculty of Mathematics and Natural Sciences, The John Paul II Catholic University of Lublin, 20-718 Lublin, Poland
Received 15 December 2006; revised 13 January 2007; accepted 18 January 2007
Available online 27 January 2007
Abstract—The title compound (4) was synthesized by the reaction of ethyl 1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-5-formylimi-
dazole-4-carboxylate with excess guanidine in ethanol at reflux. Compound 4 was evaluated in vitro against NTPases/helicases
of four different viruses of the Flaviviridae family, including the West Nile virus (WNV), hepatititis C virus (HCV), dengue virus
(DENV), and the Japanese encephalitis virus (JEV), employing both an RNA and a DNA substrate. The compound showed activity
against NTPase/helicase of WNV and HCV with an IC50 of 23 and 37 lM, respectively, when a DNA substrate was employed, while
no activity was observed when an RNA substrate was used. There was no activity against the NTPase/helicase of either DENV or
JEV irrespective of whether an RNA or a DNA substrate was employed. Considering that Flaviviridae are RNA viruses, the
observed absence of activity against an RNA substrate, but the presence of activity against a DNA substrate is intriguing and
somewhat surprising. The preliminary studies show that compound 4 does not form a tight complex with either an RNA or a
DNA substrate, suggesting that its mechanism of action may involve direct interaction with the enzyme.
ꢀ 2007 Elsevier Ltd. All rights reserved.
We have recently reported1 the synthesis of novel imid-
azole analogues (III) from the corresponding imidazoles
O
NH
N
NH
N
(I), employing an interesting functional group transfor-
mation2 mediated by guanidine (Scheme 1). We also
reported that compound III (R = deoxyribosyl) exhibit-
ed a potent in vitro inhibitory activity against the West
Nile Virus (WNV) NTPase/helicase when an RNA
substrate was employed, but no activity with a DNA
substrate.1 Since WNV is an RNA virus, the observed
inactivity with a DNA substrate was not too surprising,
but instead gave further impetus to synthesize and
screen the ribose analogue of III (R = ribosyl) against
not only WNV3–5 but also a few other similarly dreadful
viruses belonging to the same Flaviviridae family, which
are of current global health threat, including the hepati-
O
O
NH
N
H2N NH2
(excess)
N
R
OEt
H
N
R
II
EtOH
H2N
H2N
I
NH
O
N
NH2
N
NH2
NH
NH2
N
R
N
Keywords: AICAR analogue; Synthesis; In vitro screening; Inhibition
of Flaviviridae NTPase/helicase; West Nile virus (WNV) and hepatitis
C virus (HCV).
III
R = CH2C6H5, CH2OCH2C6H5, Deoxyribosyl
*
Corresponding author. Tel.: +1 410 455 2520; fax: +1 410 455
1148; e-mail: hosmane@umbc.edu
Scheme 1.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.074

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R. K. Ujjinamatada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2285–2288
Table 1. Inhibitory effect of nucleoside 4 against the helicase activity of
O
O
O
WNV, HCV, JEV, and DENV NTPases/helicases, using a DNA
substratea
RO
RO
OCOCH3
N
N
OEt
OEt
N
N
OEt
OEt
OR
WNV
IC50 (lM)b
HCV
IC50 (lM)b
JEV
IC50 (lM)b
DENV
IC50 (lM)b
O
OEt
OEt
(CH3)3SiI /Benzene
RO
OR
23
37
>500
>430
87%
(R = COC6H5)
RO
2
a The helicase activity was determined as a function of increasing
concentrations of the compound in the presence of ATP adjusted to
the respective KM values equal to 9.5 lM, 105 lM, 4.2 lM, and
165 lM WNV, HCV, JEV, and DENV NTPase/helicase, and 4.7 pM
DNA substrate.
b The inhibitory effect of the compound was expressed as the concen-
tration at which 50% of the unwinding activity was observed. The
helicase activity of the enzyme measured in the absence of the
compound was referred to as 100%. The term IC50 is defined as the
concentration of the compound required for 50% inhibition of
enzyme activity.
O
NaH
CH3CN
N
80%AcOH
78%
OEt
OEt
N
H
O
N
NH
O
O
OEt
N
1
N
NH2
N
H2N NH2
(excess)
OEt
H
NH2
N
O
N
O
EtOH
NH
NH2
61%
HO
OH
RO
OR
HO
RO
3
4
(R = COC6H5)
Scheme 2.
substrates consisting of two annealed RNA or DNA
oligonucleotides. The unwinding activity of the enzyme
was assessed by monitoring the release of the shorter
labeled strand of the RNA or DNA duplex, employing
the protocol as described.27,28 The helicase activity
was calibrated with an RNA or a DNA substrate that
was unwound at an ATP concentration equal to the
KM value determined for the NTPase reaction.27,28 The
anti-helicase activity of Nucleoside 4 against the
mentioned four different NTPases/helicases is listed in
Table 1 employing a DNA substrate.
tis C virus (HCV),6–11 dengue virus (DENV),12–16 and
the Japanese encephalitis virus (JEV).17–20
Synthesis of the target nucleoside 4 (Scheme 2) com-
menced from ethyl 5-diethoxymethylimidazole-4-car-
boxylate 1.1,21,22 The latter was converted into its
sodium salt using sodium hydride in acetonitrile and
was further reacted with 2,3,5-tri-O-benzoyl-b-D-ribo-
furanosyl-1-iodide under standard conditions of glyco-
sylation (acetonitrile/50 ꢁC/2 h).21,23 The iodide itself
was prepared by reaction of 1-acetyl-2,3,5-tri-O-benzo-
yl-b-D-ribofuranose and iodotrimethylsilane at room
temperature in benzene.24 The glycosylation procedure
is known to be stereospecific giving predominantly the
b-anomeric product.24,25 The product ethyl 1-(2,3,5-tri-
O-benzoyl-b-D-ribofuranosyl)-5-(diethoxymethyl)imid-
azole-4-carboxylate (2) was isolated in 87% yield. The
acetal 2 was reacted with 80% aqueous acetic acid to
obtain the corresponding carboxaldehyde 3 in 78%
yield. The reaction of the latter with excess guanidine
in ethanol at reflux for 12 h provided the target
nucleoside 4. All intermediates as well as the final com-
pound were fully characterized by spectroscopic and
microanalytical data.26 Compound 4 can be considered
as an analogue of 5-aminoimidazole-4-carboxamide-1-
b-D-ribofuranoside (AICAR), an important biosynthetic
precursor of purine nucleosides, with a diaminodihydro-
triazine substituent replacing the 5-amino group of
AICAR.
However, no inhibition could be detected when the same
experiments were repeated using an RNA substrate. As
Flaviviridae are RNA viruses, the observed results are
intriguing, especially considering that the 20-deoxyribose
analogue of 4 (i.e., III; R = deoxyribosyl in Scheme 1)
has shown activity against the NTPase/helicase of
WNV with an RNA substrate, but no activity against
a DNA substrate as we reported recently.1 The signifi-
cance and implications of the observed contrasting re-
sults between the ribose analogue 4 and its 20-deoxy
counterpart with respect to an RNA or DNA substrate
of viral helicase are not clear at the moment.
Since the observed antiviral activity of 4 against
NTPase/helicase is very specific to the type of nucleic
acid (DNA vs RNA) substrate employed, we wondered
if the mechanism of action of 4 is dependent upon its
ability to form a tight complex with a DNA substrate
but not with RNA. There are many documented reports
demonstrating non-covalent, tight-binding interactions
of analogues of nucleobases, nucleosides, and nucleo-
tides, which simply bind to major or minor grooves of
DNA or RNA double helices.35,36 We ourselves have
recently reported such interactions with some ring-ex-
panded nucleoside (REN) analogues that were found
to form tight complexes with DNA or RNA substrates
of viral helicases, as evidenced by the observed complete
stability of the complexes in the presence of 0.5%
sodium dodecyl sulfate (SDS) as well as by the observed
severe hindrance to migration of the DNA or RNA sub-
strates in TBE–polyacrylamide gel electrophoresis in the
presence of REN analogues with or without the enzyme
The NTPases/helicases from four closely related
Flaviviridae, including the West Nile virus (WNV), hep-
atitis C virus, Japanese encephalitis virus (JEV), and
dengue virus (DENV), were employed for the helicase
inhibition studies. The WNV NTPase/helicase was iso-
lated and purified from the cell culture medium harvest-
ed from virus-infected Vero E6 cells as described by us
previously.27–30 The NTPase/helicase domains of
HCV,31,32 JEV,33 and DENV34 NS3 were expressed in
Escherichia coli and purified according to the protocol
for the HCV enzyme, which we reported earlier.27 The
compound was tested against both RNA and DNA

R. K. Ujjinamatada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2285–2288
2287
being present.27,28 By contrast, and to our surprise, no
such tight complex formation was observed with either
a DNA or an RNA substrate when the same experi-
ments were conducted using the nucleoside analogue 4.
This observation is indeed exciting as it implies that
the compound must be interacting directly with the en-
zyme. The observed difference in activity resulting from
the different types of substrates employed may well be
due either to the differential conformational effects
exerted by the enzyme–substrate complex upon subse-
quent binding of the inhibitor to the protein or due to
those caused by the enzyme–inhibitor complex upon
eventual binding of a DNA or an RNA substrate. These
speculations, however, are only tentative at this point.
20. Abe, M.; Shiosaki, K.; Hammar, L.; Sonoda, K.; Xing, L.;
Kuzuhara, S.; Kino, Y.; Cheng, R. H. Virus Res. 2006,
121, 152.
21. Ramesh, K.; Panzica, R. P. J. Chem. Soc., Perkin Trans. 1
1989, 1769.
22. Murakami, T.; Otsuka, M.; Ohno, M. Tetrahedron Lett.
1982, 23, 4729.
23. Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.;
Robins, R. K. J. Am. Chem. Soc. 1984, 106, 6379.
24. Tocik, Z.; Earl, R. A.; Beranek, J. Nucleic Acids Res. 1980,
8, 4755.
25. Marquez, V. E.; Liu, P. S.; Linevsky, J. K. J. Org. Chem.
1982, 47, 1712.
26. Experimental. Preparation and physicochemical proper-
ties of the compounds are as follows: Ethyl 5-diethoxym-
ethyl-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl) imidazole-
4-carboxylate (2).
Step 1: Preparation of sodium salt of ethyl 5-diethoxym-
ethyl-imidazole-4-carboxylate: To a solution of ethyl 5-
diethoxymethylimidazole-4-carboxylate (0.267 g, 1 mmol)
in dry acetonitrile (10 mL), sodium hydride (80 mg,
2 mmol) was added and the mixture was stirred at room
temperature under nitrogen atmosphere for 30 min.
Step 2: Reaction of sodium salt of ethyl 5-diethoxymethyl-
imidazole-4-carboxylate with 1-O-acetyl-2,3,5-O-benzoyl-
b-D-ribofuranosyl iodide: To a solution of 1-O-acetyl-
2,3,5-tri-O-benzoyl-b-D-ribofuranose (0.567 g, 1 mmol) in
dry benzene (5 mL), iodotrimethylsilane (0.225 mL,
1.6 mmol) was added dropwise. The mixture was swirled
for 10 min at room temperature and then it was added
dropwise to a stirred suspension of the above sodium salt
of ethyl 5-diethoxymethylimidazole-4-carboxylate. The
temperature of the reaction mixture was gradually raised
to 50 ꢁC and was stirred for 2 h. The reaction mixture was
then brought to room temperature, acetonitrile was
removed under vacuum, and the residue was purified by
column chromatography using hexanes/ethyl acetate (3:1)
as an eluting solvent. Appropriate fractions were pooled
and evaporated under vacuum. Gummy residue was
crystallized from benzene-pet ether to obtain colorless,
flaky solid. Yield 0.595 g, 87%; mp 43–45 ꢁC, Rf = 0.21
(hexanes/ethyl acetate 3:1); IR, 1718, 1703, 1577,
Acknowledgments
The research was supported in part by Grants (# 5 RO1
AI55452 and #1 R21 AI071802) from the National
Institute of Allergy and Infectious Diseases (NIAID)
of the National Institutes of Health, Bethesda,
Maryland, and by an unrestricted grant from Nabi
Biopharmaceuticals, Rockville, Maryland.
References and notes
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1501 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.1 (m, 7H,
;
Ar–H and imidazole CH), 7.59–7.29 (m, 9H, Ar–H), 6.82
(d, J = 3.9 Hz, 1H, 10-H), 6.37 (s, 1H, CH), 5.60 (m, 2H,
20-H, and 30-H), 4.8–4.4 (m, 3H, 40-, 50-, and 500-H), 4.40
(q, J = 1.8 Hz, 2H), 3.85–3.45 (m, 4H, 2CH2, 1.39 (t,
J = 6.9 Hz, 3H, CH3), 1.19 (t, J = 7.2 Hz, 3H, CH3), 1.02
(t, J = 6.9 Hz, 3H, CH3); HRMS (FAB) Calcd for
C37H38N2O11, 687.2554 (MH+); observed m/z 687.2562
(MH+).
Ethyl 5-formyl-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)
imidazole-4-carboxylate (3). A solution of ethyl 5-dieth-
oxymethyl-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)imid-
azole-4-carboxylate (0.515 g 0.75 mmol) in 80% aqueous
acetic acid (5 mL) was stirred at room temperature for
15 h. The solution was then poured into ice-water (25 mL)
and the precipitated solid was extracted with chloroform
(3 · 50 mL). The chloroform layer was washed with water
(2 · 25 mL) and dried over anhyd sodium sulfate. The
solvent was removed under vacuum and the residue was
purified by flash chromatography on silica gel, using
hexanes/ethyl acetate (2:1) as an eluting solvent. Appro-
priate fractions were pooled and evaporated under vacu-
um. The residue was triturated with methanol to obtain a
white solid. Yield 0.360 g, 78%; mp 167–169 ꢁC, Rf = 0.34
(hexanes/ethyl acetate 2:1); IR 1729, 1706, 1532,
14. Green, S.; Rothman, A. Curr. Opin. Infect. Dis. 2006, 19,
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;
1480 cmꢀ1 1H NMR (300 MHz, CDCl3) d 10.5 (s, 1H,
CHO), 8.17 (s, 1H, imidazole CH), 8.08–7.90 (m, 6H,

2288
R. K. Ujjinamatada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2285–2288
Ar–H), 7.62–7.32 (m, 9H, Ar–H), 6.92 (d, J = 4.5 Hz, 1H,
(DMSO-d6, 75 MHz) d 164.9, 161.0, 159.6, 134.4, 131.5,
126.2, 87.9, 83.5, 77.9, 71.1, 60.8, 57.9; HRMS (FAB)
Calcd for C12H18N8O5, 355.1401 (MH+); observed m/z
355.1418 (MH+).
10-H), 5.88-5.76 (m, 2H, 20-H, and 30-H), 4.89–4.41 (m,
3H, 40-, 50- and 500-H), 4.40 (q, J = 7.2 Hz, 2H), 1.42
(t, J = 6.9 Hz, 3H, CH3); HRMS (FAB) Calcd for
C33H28N2O10, 613.1822 (MH+); observed m/z 613.1828
(MH+).
27. Zhang, N.; Chen, H.-M.; Koch, V.; Schmitz, H.; Liao, C.-
L.; Bretner, M.; Bhadti, V. S.; Fattom, A. I.; Naso, R. B.;
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5-(4,6-Diamino-2,5-dihydro-1,3,5-triazin-2-yl)-1-(b-D-ribo-
furanosyl-)imidazole-4-carboxamide (4). Guanidine hydro-
chloride (0.192 g, 2 mmol) was neutralized with a solution
of sodium (46 mg, 2 mmol) in anhyd ethanol (5 mL) by
stirring at 5 ꢁC for 30 min. The precipitated sodium
chloride was removed through filtration and the fil-
trate was added to a solution of ethyl 5-formyl-1-(2,3,5-
tri-O-benzoyl-b-D-ribofuranosyl)imidazole-4-carboxylate
(0.307 g, 0.5 mmol) in dry ethanol (10 mL). The reaction
mixture was heated at reflux for 12 h. The reaction
mixture was cooled, mixed with flash silica gel (2 g), and
the solvent was evaporated. The residue was purified by
flash chromatography, using a mixture of chloroform/
methanol/ammonium hydroxide (1:1:0.25) as an eluting
solvent. The appropriate fractions were pooled and the
solvents were removed under vacuum to obtain a colorless
solid. Yield 0.108 g, 61%; mp 144–146 ꢁC, Rf = 0.24
(chloroform/methanol/ammonium hydroxide, 1:1:0.25);
IR 3341, 3305, 3296, 1647, 1621 cmꢀ1
;
1H NMR
(DMSO-d6, 300 MHz) d 8.03 (s, 1H, imidazole CH),
7.44 (s, 1H, CONH), 7.21 (s, 1H, CONH), 6.5 (s, 1H, CH),
6.92 (d, J = 5.1 Hz, 1H, 10-H), 6.10–5.90 (br s, 2H, 2OH),
5.55–4.90 (br s, 5H, OH+ 2 NH2), 4.10–3.81 (m, 2H, 20-H,
and 30-H), 3.62–3.38 (m, 3H, 40-, 50- and 500-H); 13C NMR