A novel imidazole nucleoside containing a diaminodihydro-S-triazine as a substituent: Inhibitory activity against the West Nile virus NTPase/helicase

Article abstract of DOI:10.1080/15257770500267063

□ The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2′-deoxy-β-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5- carbaldehyde (2) with guanidine resulted in the forma

Full text of DOI:10.1080/15257770500267063

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A Novel Imidazole Nucleoside Containing a
Diaminodihydro-S-triazine as a Substituent: Inhibitory
Activity Against the West Nile Virus NTPase/Helicase
Ravi K. Ujjinamatada a , Yankanagouda S. Agasimundin a , Peng Zhang a , Ramachandra S.
Hosmane a , Roman Schuessler b , Peter Borowski b , Kishna Kalicharran c & Ali Fattom c
a Department of Chemistry & Biochemistry , Laboratory for Drug Design and Synthesis,
University of Maryland, Baltimore County , Baltimore, Maryland, USA
b Abteilung für Virologie, Bernhard-Nocht-Institut für Tropenmedizin , Hamburg, Germany
c Nabi Biopharmaceuticals , Rockville, Maryland, USA
Published online: 16 Aug 2006.
To cite this article: Ravi K. Ujjinamatada , Yankanagouda S. Agasimundin , Peng Zhang , Ramachandra S. Hosmane ,
Roman Schuessler , Peter Borowski , Kishna Kalicharran & Ali Fattom (2005) A Novel Imidazole Nucleoside Containing a
Diaminodihydro-S-triazine as a Substituent: Inhibitory Activity Against the West Nile Virus NTPase/Helicase, Nucleosides,
Nucleotides and Nucleic Acids, 24:10-12, 1775-1788, DOI: 10.1080/15257770500267063
To link to this article: http://dx.doi.org/10.1080/15257770500267063
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1775–1788, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500267063
A NOVEL IMIDAZOLE NUCLEOSIDE CONTAINING A
DIAMINODIHYDRO-S-TRIAZINE AS A SUBSTITUENT:
Inhibitory Activity Against the West Nile Virus
NTPase/Helicase
Ravi K. Ujjinamatada, Yankanagouda S. Agasimundin, Peng Zhang, and
ᮀ
Ramachandra S. Hosmane
Laboratory for Drug Design and Synthesis, Depart-
ment of Chemistry & Biochemistry, University of Maryland, Baltimore County, Baltimore,
Maryland, USA
ᮀ
Roman Schuessler and Peter Borowski
Abteilung fu¨r Virologie, Bernhard-
Nocht-Institut fu¨r Tropenmedizin, Hamburg, Germany
ᮀ
Kishna Kalicharran and Ali Fattom
Nabi Biopharmaceuticals, Rockville, Mary-
land, USA
ᮀ
The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-
e][1,3]diazepine ring system by condensation of 1-(2ꢀ-deoxy-β-D-erythropentofuranosyl)-4-ethoxy-
carbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected
product, 1-(2ꢀ-deoxy-β-D-erythropentofuranosyl)-5-(2,4-diamino-3,6-dihydro-1,3,5-triazin-6-yl)im-
idazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated
by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed
promising in vitro anti-helicase activity against the West Nile virus NTPase/helicase with an
IC50 of 3-10 µg/mL.
Keywords Imidazole; s-Triazine; Nucleoside; West Nile Virus; Inhibition; Rearrange-
ment; Ring-expanded nucleoside
In honor and celebration of the life and career of John A. Montgomery.
Received 2 February 2005; accepted 28 April 2005.
The research was supported in part by a grant (#9RO1 AI55452) from the National Institute of
Allergy and Infectious Diseases (NIAID) of the National Institutes of Health, Bethesda, Maryland (to
R. S. H.), an unrestricted grant from Nabi Biopharmaceuticals, Rockville, Maryland (to R. S. H.), and
a grant (#BWB E/B31E/MO171/M5916-Rj. 2000) (to P. B.) from Bunesministerium der Vertedigung,
Germany.
Address correspondence to Ramachandra S. Hosmane, Laboratory for Drug Design and Synthesis,
Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop
Circle, Baltimore, MD 21250. E-mail: hosmane@umbc.edu
1775

1776
R. K. Ujjinamatada et al.
SCHEME 1
INTRODUCTION
We have outlined a broad program to synthesize and biologically screen
a variety of ring-expanded (“fat”) nucleosides and nucleotides as a potential
and proven source of antiviral and anticancer agents.[1] As part of this pro-
gram, we recently set out to synthesize a ring-expanded 2ꢁ-deoxyguanosine
analogue (1) (Scheme 1) by condensation of the imidazole nucleoside 2
with guanidine. To this end, compound 5 was prepared as a protected pre-
cursor to 2, using a literature procedure for the corresponding methyl
ester,[2,3] starting from ethyl 5-diethoxymethyl-1H-imidazole-4-carboxylate
(3), as outlined in Scheme 2. The condensation of diethoxyacetonitrile with
SCHEME 2

Inhibitory Activity Against West Nile Virus
1777
ethyl isocyanate, in the presence of sodium hydride in diglyme at 85◦C, pro-
vided 3 in 56% yield. The glycosylation of 3 with 2-deoxy-3,5-di-O-p-toluoyl-
α-D-erythropentofuranosyl chloride,[4,5] catalyzed by sodium hydride, pro-
duced the sugar-protected nucleoside 4 in 46% yield after purification by
flash chromatography on a silica gel column. The 1H and 13C NMR spectral
data as well as TLC analyses suggested that the product was a single isomer.
The sodium salt glycosylation procedure is known to be stereospecific and is
expected to produce mainly the β-anomer.[2,5,6] The β-anomeric structure
of 4 was corroborated by the observed splitting pattern, a doublet of dou-
blets with nearly equal coupling constants (J = 5.4 and 5.7 Hz), of the ano-
1
meric proton of 4 at δ 6.69 in its H NMR spectrum. The two-dimensional
1H-1H NOE spectroscopy (NOESY) of 4 provided the confirmatory evidence
for the β-configuration by revealing NOE between H-1ꢁ and H-4ꢁ. The ob-
served splitting mode and the coupling constants are generally considered
a diagnostic of β-anomeric configuration of nucleosides.[7] The regioiso-
meric structure of 4, as assigned, was confirmed by the nuclear Overhauser
effect (NOE) studies of the product aldehyde (5) formed by hydrolysis of
the acetal group of 4. The hydrolysis was accomplished by reaction of 4 with
80% aqueous acetic acid at room temperature for 22 h.
1
The two-dimensional H-1H NOE spectroscopy (NOESY) of 5 revealed
the presence of NOE between its aldehydic group and the sugar H-1ꢁ. The
observed NOE is not possible if the sugar moiety were to be attached to the
other N atom of the imidazole ring.
When compound 5 was reacted with excess guanidine and sodium
ethoxide in ethanol at room temperature for 15 hours, a new product was
obtained, which moved slower than the starting material on a silica gel
1
plate (Scheme 3). The H NMR, elemental microanalyses, and the mass
spectral data of the chromatographically purified product showed that the
compound had incorporated two molecules of guanidine in its structure
instead of one. Both structures 6 and 7 were consistent with the acquired
spectral and microanalytical data, and their formation by the reaction of 5
with guanidine could be rationalized by the two reaction pathways outlined
in Scheme 4.
The structure of the product was confirmed as 7 by the following
reaction sequences. Compound 4 was reacted with ammonia in ethanol
in a stainless steel vessel at 125◦C for 36 h to obtain the corresponding
carboxamide 8 with its free hydroxyl groups (Scheme 5). An attempted
conversion of the acetal groups of 8 into the corresponding carboxaldehyde,
however, resulted in concurrent deglycosylation to produce the heterocycle
9. Therefore, the two sugar hydroxy groups were reprotected by reaction
with toluoyl chloride in pyridine to obtain 10. The latter, upon treatment
with 80% acetic acid at room temperature for several hours, gave the desired
vinylogous aldehyde-amide 11. The reaction of 11 with excess guanidine in

1778
R. K. Ujjinamatada et al.
SCHEME 3
a solution of sodium ethoxide/ethanol afforded 7, which was identical in
all respects to the product formed by the reaction of ester 5 with excess
guanidine.
The above results raise another mechanistic possibility for the forma-
tion of 7 from 5, in which the latter’s carboxaldehyde group would react
with excess guanidine to form the triazine ring first, as in 10 (Scheme 6).
A molecule of ammonia released during the formation of 10 would subse-
quently react to produce 7. While this reaction pathway could not be com-
pletely ruled out, it appears less likely in view of the lack of formation of
equally plausible other products such as 11, especially when there is a large
molar excess of guanidine in the reaction mixture as compared to the tran-
siently formed ammonia. Finally, in an attempt to prepare the target 1, in-
termediate 2 was reacted with one equivalent of guanidine in anhydrous
ethanol. Unfortunately, the reaction produced an intractable mixture of
products that were difficult to separate and purify. The use of a base, change
of solvent, or increase or decrease in reaction temperature did not improve
the situation. Therefore, it appears that the targeted ring-expanded guano-
sine (1) has limited existence and stability.
Although our attempts to synthesize the desired 1 was unsuccessful,
the product 7, obtained easily from condensation of 5 or 2 with excess
guanidine, possessed novel structural characteristics worthy of further ex-
ploration, especially for its biological potential. In that context, nucleoside 7

Inhibitory Activity Against West Nile Virus
1779
SCHEME 4

1780
R. K. Ujjinamatada et al.
SCHEME 5
became an attractive choice for screening against the West Nile virus (WNV),
a topic of our current research focus.[5,8]
Nucleoside 7 was screened for antihelicase activity of WNV NTPase/
helicase harvested from the virus-infected Vero E6 cells, employing estab-
lished protocols.[5] The compound was tested against both RNA and DNA
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. The helicase activ-
ity 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. Compound 7 exhibited helicase inhibitory activity against WNV
NTPase/helicase with an IC50 value of 3–10 µg/mL when an RNA substrate
was employed. However, no inhibition could be detected when the same
experiment was repeated using a DNA substrate. As WNV is an RNA virus,
this result is not totally surprising. In that regard, a ribose analogue of 7
may carry a better prospect for WNV inhibition than 7 itself, and such an
endeavor is currently in progress.
EXPERIMENTAL
The 1H and 13C NMR spectra were recorded on a General Electric QE-
300 or a JEOL-400 NMR spectrometer, operating at 300 or 400 MHZ for
1H and 75 or 100 MHz for 13C, respectively. The data are reported in the

Inhibitory Activity Against West Nile Virus
1781
SCHEME 6

1782
R. K. Ujjinamatada et al.
following format: Chemical shift (all relative to Me4Si), multiplicity (s =
singlet, d = doublet, dt = double triplet, dd = double doublet, t = triplet,
q = quartet, m = multiplet, b = broad), coupling constants, integration and
assignment. Thin-layer chromatography was performed on Merck Kieselgel
60 F254 (0.2 mm thickness). Flash column chromatography was performed
using 32–63 mesh silica gel. Elemental microanalyses were performed by At-
lantic Microlab, Inc., Norcross, Georgia. Melting points were determined on
a Thomas Hoover capillary melting point apparatus and are uncorrected.
Mass spectra were recorded either at the Mass Spectral Facility, Department
of Biochemistry, Michigan State University, East Lansing, MI or the Uni-
versity of Maryland, College Park, Maryland. Anhydrous solvents were pur-
chased and used without further drying. All alcohols were dried over sodium
metal, distilled, and stored over molecular sieves.
Ethyl 5-Diethoxymethylimidazole-4-carboxylate (3). To a stirred sus-
pension of 60% sodium hydride (2.7 g) in freshly distilled diglyme (100
mL), a solution of diethoxyacetonitrile (6.45 g, 0.05 mol) and ethyl iso-
cyanoacetate (5.65 g, 0.05 mol) in diglyme (75 mL) was added slowly with
ice cooling under nitrogen atmosphere. The solution was heated at 80◦C
for 5 h and left overnight at room temperature. The reaction mixture was
quenched with saturated aqueous ammonium chloride solution and ex-
tracted with dichloromethane for three times. The combined organic ex-
tracts were dried over anhydrous sodium sulfate. The solid obtained after
removal of solvent under reduced pressure was purified by flash chromatog-
raphy over silica gel. The pure product obtained by elution with ethyl ac-
etate was finally crystallized from a mixture of dichloromethane-petroleum
ether (20:3) as colorless needles. Yield 6.82 g, 56%; mp 138◦C; Rf = 0.34
(chloroform:methanol, 10:1); IR, 1710, 1570, 1505, 1447, 1179, 1142 cm−1
;
1H NMR (CDCl3, 300 MHz) δ 12.50 (s, 1H, NH), 7.79 (s, 1H, imidazole
CH), 6.10 (s, 1H, acetal CH), 4.31 (q, J = 6.9 Hz, 2H, ester CH2), 3.72–3.50
(m, 4H, 2 CH2, acetal CH2), 1.29 (t, J = 6.9 Hz, 3H, ester CH3), 1.12 (t,
J = 6.9 Hz, 6H, 2 acetal CH3); 13C NMR (CDCl3, 75 MHz) 161.91, 137.00,
135.87, 126.25, 94.73, 62.20, 59.94, 14.49, 13.74; MS(FAB) m/z 243 (MH+).
Anal. Calcd for C11H18N2O4: C, 54.53; H, 7.49; N, 11.56. Found: C, 54.53,
H, 7.44, N, 11.50.
Ethyl 5-Diethoxymethyl-1-[(2 -deoxy-3 ,5 -di-O-p-toluoyl)-β-D-erythro-
pentofuranosyl]imidazole-4-carboxylate (4). To a solution of ethyl 5-dieth-
oxymethylimidazole-4-carboxylate (3) (2.42 g, 0.01 mol) in anhydrous
acetonitrile (75 mL) was added sodium hydride (60% in oil, 1.0 g, 25
mmol), and the mixture was stirred at room temperature under nitrogen
atmosphere for 30 min. To this mixture, 2-deoxy-3,5-di-O-p-toluoyl-α-D-
erythropentofuranosyl chloride (3.88 g, 0.01 mol) was added portion-wise
over a period of 1 h. After the addition was complete, the reaction mixture

Inhibitory Activity Against West Nile Virus
1783
was allowed to stir at room temperature for 2 h. It was then filtered and
evaporated under reduced pressure. The residue was purified by flash
chromatography on a silica gel column using a mixture of dichloromethane:
methanol (99:1) as an eluant. Appropriate fractions were pooled and
evaporated under reduced pressure to obtain a gummy residue. Yield: 2.71
g, 46%; Rf 0.40 (chloroform:methanol, 50:2); IR 1716, 1611, 1446, 1267,
1
1177, 1096 cm−1; H NMR (CDCl3, 300 MHz) δ 7.93–7.87 (m, 4H, Ar-H),
7.81 (s, 1H, imidazole CH), 7.26–7.20 (m, 4H, Ar-H), 6.69 (dd, J = 5.4 and
5.7 Hz ,1H, 1ꢁ-H,), 6.37 (s, 1H, CH(OEt)2), 5.59 (m, 1H, 3ꢁ-H), 4.73 (m, 2H,
4ꢁ, 5ꢁ-H), 4.68 (m, 1H, 5ꢁꢁ-H), 4.36 (q, J = 7.2 Hz, 2H, ester CH2), 3.86–3.48
(m, 4H, 2 CH2, acetal CH2), 2.90–2.87 (m, 1H, 2ꢁ-H), 2.50–2.42 (m, 2 H,
2ꢁꢁ-H), 2.41 (s, 3H, Ar-CH3), 2.38 (s, 3H, Ar-CH3), 1.38 (t, 3H, ester CH3),
1.22–1.14 (m, 6H, 2 acetal CH3; Mass (FAB), m/z 595.60 (MH+); Anal. Calcd
for C32H38N2O9: C, 64.63; H, 6.44; N, 4.71. Found: C, 64.64, H, 6.22, N, 4.70.
Ethyl 5-Formyl-1-[(2 -deoxy-3 ,5 -di-O-p-toluoyl)-β-D-erythropentofura-
nosyl]imidazole-4-carboxylate (5). A solution of ester-acetal 4 (2.37 g, 4
mmol) in 80% aqueous acetic acid (10 mL) was stirred at room tempera-
ture for 22 h. The reaction mixture was poured into ice water and the pre-
cipitated solid was extracted with dichloromethane. The organic extract was
dried over anhydrous sodium sulfate. The residue obtained after removal of
the solvent was purified by silica gel flash chromatography using a mixture
of dichloromethane-methanol (98:2) as the eluant. Appropriate fractions
were pooled and evaporated under reduced pressure. The residue was trit-
urated with methanol to get pure ester-aldhehyde 5. Yield 1.15 g, 55%; mp
125–127◦C; Rf = 0.44 (dichloromethane:methanol, 99:1); IR 1723, 1651,
1
1611, 1537, 1485, 1265, 1084 cm−1; H NMR (CDCl3, 400 MHz) δ 10.42
(s, 1H, CHO), 8.03 (s, 1H, imidazole CH), 7.89 (d, J = 6.8 Hz, 2H, Ar-H),
7.76 (d, J = 6.68 Hz, 2H, Ar-H), 7.20 (d, J = 8.72 Hz, 2H, Ar-H), 7.14 (d,
J = 7.36 Hz, 2H, Ar-H), 6.67 (t, J = 6.4 Hz, 1H, 1ꢁ-H), 5.51 (s, 1H, 3ꢁ-H),
4.69–4.59 (m, 3H, 4ꢁ, 5ꢁ, and 5ꢁꢁ-H), 4.40 (q, J = 6.88 Hz, 2H, ester CH2),
3.01–2.96 (m, 1H, 2ꢁ-H), 2.36 (s, 3H, Ar-CH3), 2.32 (s, 3H, Ar-CH3), 2.28–
2.23 (m, 1 H, 2ꢁꢁ-H), 1.36 (t, J = 6.88 Hz, 3H, ester CH3); 13C NMR (CDCl3,
100 MHz) 182.84, 166.21, 166.07, 161.89, 144.68, 144.45, 141.98, 138.27,
131.45, 129.94, 129.68, 129.50, 129.40, 126.43, 126.33, 88.53, 83.91, 74.53,
63.81, 61.89, 40.97, 21.85, 21.78, 14.37. Mass Spectrum (FAB) m/z 521.50
(MH+); Anal. Calcd for C28H28N2O8: C, 64.61; H, 5.42; N, 5.38. Found: C,
64.50, H, 5.43, N, 5.35.
5-(4,6-Diamino-2,5-dihydro-1,3,5-triazin-2-yl)-1-(2 -deoxy-β-D-erythro-
pentofuranosyl)imidazole-4-carboxamide (7). Method A: Guanidine hydro-
chloride (0.38 g, 0.006 mol) was neutralized with a solution of sodium (0.32
g, 0.02 mol) in anhydrous ethanol (10 mL) at ice cold temperature. This
guanidine solution was added to the aldehyde 5 or 2 (0.0015 mol) in anhy-

1784
R. K. Ujjinamatada et al.
drous ethanol (10 mL). This reaction mixture was stirred at room tempera-
ture under nitrogen atmosphere for 15 h. TLC showed the completion of re-
action (chloroform-methanol-ammonium hydroxide (1:1:0.5). Solvent was
evaporated and the residue was purified by silica gel flash chromatography,
using a mixture of chloroform-methanol-ammonium hydroxide (2:1:0.25);
Yield 0.32 g, 63%; mp 106◦C, Rf = 0.39 (chloroform:methanol:ammonium
hydroxide, 1:1:0.25); IR, 3302, m152, 3092, 1654, 1620, 1585, 1518, 1268
1
cm−1; H NMR (DMSO-d6, 300 MHz) δ 8.12 (s, 1H, imidazole CH), 7.90
(s, 1H, NH, exchangeable with D2O), 7.52 (s, 2H, NH2, exchangeable with
D2O), 7.10–7.40 (brs, 4H, NH2+NH2, exchangeable with D2O), 6.80 (s, 1H,
triazine CH), 6.24 (t, J = 6.0 Hz, 1H, 1ꢁ-H), 5.10–5.50 (brs, 2H, OH+OH,
exchangeable with D2O), 3.75 (m, 2H, 5ꢁH, 5ꢁꢁ-H), 2.35–2.18 (m, 2 H, 2ꢁ-
and 2ꢁꢁ-H); 13C NMR(DMSO-d6, 75 MHz); 164.44, 158.52, 158.32, 136.10,
134.29, 128.95, 87.59, 85.39, 69.48, 60.76, 55.54, 42.39; Mass (FAB) m/z
339.30 (MH+); Anal. Calcd for C12H18N8O4. 0.25 H2O: C, 42.02; H, 5.39;
N, 32.68. Found: C, 42.05, H, 5.74, N, 32.31.
Method B: Guanidine hydrochloride (20 mg, 0.2 mmol) was neutralized
with a solution of sodium (5 mg, 0.2 mmol) in ethanol (2 mL) at ice-cold
temperature. Separated sodium chloride was removed by filtration and the
guanidine solution was added to the aldehyde 11 (25 mg, 0.05 mmol) in
ethanol (3 mL). This reaction mixture was stirred at room temperature un-
der nitrogen atmosphere for 15 h. TLC (cholorform-methanol-ammonium
hydroxide, (1:1:0.5) showed the completion of reaction. Solvent was evapo-
rated and the residue was purified by silica gel flash chromatography, eluting
with a mixture of cholorform-methanol-ammonium hydroxide, (2:1:0.25).
The melting point and chromatographic mobility (mp 106◦C, Rf = 0.39)
as well as the spectral data of the compound were identical with those of 7
obtained by Method A above.
5-Diethoxymethyl-1-(2 -deoxy-β-D-erythropentofuranosyl)imidazole-4-
carboxamide (8). Compound 4 (0.59 g, 0.001 mol) was added to a satu-
rated ethanolic ammonia solution (5 mL) in a stainless steel pressure ves-
sel. The vessel was heated at 125◦C in an oil bath for 36 h. It was cooled to
room temperature, solvent was evaporated and the residue was purified by
silica gel flash chromatography, eluting first with a mixture of cholroform:
methanol (99:1) to remove p-toluamide, followed by elution with a mixture
of chloroform:methanol (10:1) to obtain 8 as a hygroscopic product. Yield
245 mg (73%); Rf 0.21 (9:1 chloroform:methanol); 1H NMR (DMSO-d6, 300
MHz) δ 8.03 (s, 1H, imidazole CH), 7.38 (s, 2H, NH2), 6.54 (t, J = 6.8 Hz,
1H, 1ꢁH,), 6.43 (s, 1H, acetal CH), 5.9 (s, 1H, OH), 5.18–5.08 (m, 1H, 4ꢁH),
4.55–4.43 (m, 1H, OH), 3.96–3.70 (m, 3H, 4ꢁ-, 5ꢁ-, and 5ꢁꢁ-H), 3.52–3.46 (m,
4H, acetal CH2), 2.65–2.54 (m, 1H, 2ꢁH), 2.35–2.24 (m, 1H, 2ꢁꢁH), 1.20–1.15
(m, 6H, 2 acetal CH3); Mass (FAB), m/z 330 (MH+).

Inhibitory Activity Against West Nile Virus
1785
5-Formylimidazole-4-carboxamide (9). Compound 8 (0.110 g, 0.33
mmol) was added to 1 mL of 80% aqueous acetic acid, and the mixture
was stirred at room temperature for 16 h. Then it was poured into ice-cold
water. Separated solid was filtered and washed with water. Yield 39 mg, 84%;
mp > 300◦C; Rf 0.23 (4:1 chloroform:methanol); 1H NMR (DMSO-d6, 300
MHz) δ 13.50 (s, 1H, NH), 10.17 (s, 1H, CHO), 7.97 (s, 1H, imidazole CH),
7.58 (s, 2H, NH2); 13C NMR (DMSO-d6, 75 MHz); 183.49, 163.37, 139.60,
134.25, 132.08; Mass (FAB), m/z 140 (MH+); Anal. Calcd for C5H5N3O2.
0.125 CH3OH: C, 42.97; H, 3.84; N, 29.34. Found: C, 42.67, H, 3.68, N,
29.38.
5-Diethoxymethyl-1-[(2 -deoxy-3 ,5 -di-O-p-toluoyl)-β-D-erythropentofu-
ranosyl]imidazole-4-carboxamide (10). To a mixture of compound 8 (0.083
g, 0.25 mmol) in anhydrous pyridine (5 mL), was added p-toluoyl chloride
(0.097 g, 0. 63 mmol) at ice cold temperature. The reaction mixture was
slowly brought to 50◦C and stirred for 2 h at this temperature. It was then
kept at room temperature for 12 h. The reaction mixture was poured onto
ice-cold water and extracted with ether. Ether layer was washed with water,
and dried over anhydrous sodium sulfate. The evaporation of ether gave
a gummy residue, which was purified by silica gel flash chromatography,
eluting with a mixture of 99:1 chloroform:methanol. Yield 0.078 g, 55%; Rf
1
0.25 (98:2 chloroform:methanol); H NMR (CDCl3, 400 MHz) δ 8.75 (s,
2H, NH2), 8.12–7.87 (m, 4H, ArCH), 7.81 (s, 1H, imidazole CH), 7.26–7.20
(m, 4H, ArCH), 6.47 (t, J = 6.8 Hz, 1H, 1ꢁH,), 6.09 (s, 1H, acetal CH),
5.60–5.54 (m, 1H, 3ꢁH), 4.68–4.52 (m, 3H, 4ꢁ-, 5ꢁ-, and 5ꢁꢁ-H), 3.79–3.50 (m,
4H, 2 CH2, 2.94–2.72 (m, 1H, 2ꢁ-, and 2ꢁꢁ-H), 2.44 (s, 3H, CH3), 2.42 (s,
3H, CH3), 1.21–1.16 (m, 6H, 2 CH3); 13C NMR (CDCl3, 100 MHz) 167.86,
163.34, 163.26, 143.51, 137.64, 136.92, 134.33, 129.84, 128.85, 95.81, 90.26,
86.68, 71.43, 71.32, 62.30, 61.68, 21.59, 21.41, 15.53, 15.39; Mass (FAB), m/z
566 (MH+).
5-Formyl-1-[(2 -deoxy-3 ,5 -di-O-p-toluoyl)-β-D-erythropentofuranosyl]
imidazole-4-carboxamide (11). A mixture of compound 10 (0.062 g, 0.11
mmol) and 0.5 mL of 80% aqueous acetic acid was stirred at room tem-
perature for 22 h. Then the reaction mixture was poured into ice cold wa-
ter. It was extracted with dichloromethane (3 × 10 mL), the extracts were
washed with water and dried over sodium sulfate. Evaporation of solvent
gave a semi solid, which was purified through flash chromatography over
silica gel, eluting with a mixture of 99:1 chlorform:methanol. Yield 0.032
1
g, 58%. Rf 0.42 (50:2 chloroform:methanol); H NMR (CDCl3, 400 MHz)
δ 10.01 (s, 1H, CHO) 8.2 (s, 2H, NH2), 7.98–7.90 (m, 4H, ArCH), 7.87 (s,
1H, imidazole CH), 7.29–7.19 (m, 4H, ArCH), 6.68 (t, J = 6.8 Hz, 1H, 1ꢁH,),
5.57–5.53 (m, 1H, 3ꢁ-H), 4.73–4.45 (m, 3H, 4ꢁ-, 5ꢁ-, and 5ꢁꢁ-H), 3.15–2.81 (m,

1786
R. K. Ujjinamatada et al.
1H, 2ꢁ- and 2ꢁꢁ-H), 2.46 (s, 3H, CH3), 2.43 (s, 3H, CH3); 13C NMR (CDCl3,
100 MHz) 184.41, 166.35, 165.76, 163.44, 144.08, 143.87, 140.75, 137.16,
131.57, 128.45, 128.20, 128.11, 128.00, 126.45, 126.34, 87.83, 83.98, 74.77,
64.06, 61.96, 41.34, 22.26, 22.03; Mass (FAB), m/z 492 (MH+).
Biochemical Methods
Materials: DNA oligonucleotides were prepared by Dr. M. Schreiber
(Bernhard Nocht Institute). RNA oligonucleotides were purchased from
HHMI Biopolymer/Keck Foundation, Biotechnology Resource Laboratory,
Yale University School of Medicine (New Haven, Connecticut). [γ- 32P]ATP
(220 Tbq/mmol) and [γ-33P]ATP (110 Tbq/mmol) were from Hartman
Analytic. All other chemicals were obtained from Sigma.
The WNV NTPase/helicase was purified from the cell culture medium
of virus-infected Vero E6 cells as described previously,[9] with some mod-
ifications. Briefly, the concentrated cell culture medium was mixed with
10 mL Reactive Red 120 agarose (Sigma) equilibrated with TGT buffer
for 4 h at 4◦C. The matrix was collected by sedimentation, transferred to
a column and washed with TGT buffer. Bound protein was eluted with 1 M
KCl in the same buffer, concentrated by ultrafiltration on a 30-kDa mem-
brane to a final volume of 2 mL, and subjected to gel exclusion chromatog-
raphy on a Superdex-200 column. Fractions expressing ATPase and heli-
case activities were chromatographed again on Reactive Red 120 agarose
(5 mL) as described above. The salt-eluted protein was precipitated with
polyethylene glycol (30% w/w), collected by centrifugation, solubilized with
TGT buffer, and applied to a hydroxyapatite (HA-Ultragel) column pre-
equilibrated with TGT buffer. The column was washed with 10 mL TGT
buffer, then with 2 mL TGT buffer containing 1 M KCl, and again with
5 mL TGT buffer. The NTPase/helicase was eluted with 1 mL TGT buffer
containing 50 mM KH2PO4, precipitated with PEG and dissolved in TGT
buffer. The final preparations of the enzymes were homogenous, as demon-
strated by Coomassie Blue staining of SDS/polyacrylamide gels.
Helicase activity was tested with 2 pmol WNV NTPase/helicase. Unwind-
ing of the partially hybridized DNA or RNA substrate (4.7 pM of nucleotide
base) was monitored in a reaction mixture (final volume 25 mL) contain-
ing 20 mM Tris/HCl, pH 7.5, 2 mM MgCl2, 1 mM β-mercaptoethanol, 10%
glycerol, 0.01% Triton X 100, 0.1 mg/mL BSA and 9.5 µM ATP. The reac-
tion was conducted for 30 min at 30◦C and stopped by addition of 5 mL
termination buffer (100 mM Tris/HCl, pH 7.5, 20 mM EDTA, 0.5% SDS,
0.1% Triton X-100, 25% glycerol and 0.1% bromophenol blue). Samples
were separated on a 15% Tris-borate-EDTA (TBE)-polyacrylamide gel con-
taining 0.1% SDS.[9] The gels were dried and exposed to Kodak X-ray films
at −70◦C. The areas of the gels corresponding to the released strand and
to the non-unwound substrate were cut out and 32P radioactivity counted.

Inhibitory Activity Against West Nile Virus
1787
Alternatively, the films were scanned and the radioactivity associated with
the released strand and with the non-unwound substrate quantified with
GelImage software (Amersham Pharmacia Biotech). The assays were car-
ried out with the same activity of the enzyme, determined with the DNA
substrate under conditions described above.
Substrates for Helicase Reaction
The RNA substrate for the helicase assays consisted of two partially hy-
bridized oligonucleotides with sequences as reported by Gallinari et al.[10]
The DNA substrate was obtained by annealing two DNA oligonucleotides
synthesized with sequences corresponding to the deoxynucleotide versions
of the aforementioned RNA strands. The release strands (26-mer) were 5ꢁ-
end labeled with [γ-32P]ATP, using T4 polynucleotide kinase (MBI, Fermen-
tas) as recommended by the manufacturer. For the annealing reaction, the
labeled oligonucleotide was combined at a molar ratio of 1:10 with the tem-
plate strand (40-mer), denatured for 5 min at 96◦C and slowly renatured as
elsewhere described.[9,11] The duplex DNA was electrophoresed on a 15%
native TBE-polyacrylamide gel, visualized by autoradiography and extracted
as described previously.[11] The amount of DNA or RNA duplex used as sub-
strates was determined by the ethidium bromide fluorescent quantitation
method.[12]
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