Potent Inhibition of NTPase/Helicase of the West Nile Virus by Ring-Expanded ("Fat") Nucleoside Analogues

Article abstract of DOI:10.1021/jm030277k

A series of ring-expanded ("fat") nucleoside analogues (RENs)(RENS) containing the 6-aminoimidazo-[4,5-e][1,3] diazepine-4,8-dione ring system have been synthesized and screened for inhibition of NTPase/ helicase of the West Nile Virus (WNV). To assess th

Full text of DOI:10.1021/jm030277k

4776
J . Med. Chem. 2003, 46, 4776-4789
P oten t In h ibition of NTP a se/Helica se of th e West Nile Vir u s by Rin g-Exp a n d ed
(“F a t”) Nu cleosid e An a logu es
Ning Zhang,† Huan-Ming Chen,† Verena Koch,‡ Herbert Schmitz,‡ Michal Minczuk,§ Piotr Stepien,§
Ali I. Fattom,| Robert B. Naso,| Kishna Kalicharran,| Peter Borowski,*,‡ and Ramachandra S. Hosmane*,†
Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland,
Baltimore County, Baltimore, Maryland 21250; Abteilung fu¨r Virologie, Bernhard-Nocht-Institut fu¨r Tropenmedizin,
Bernhard-Nocht-Strasse 74, Hamburg, D-20359 Germany; Department of Genetics, University of Warsaw Pawinskiego 5A,
02-106, Poland; and W. W. Karakawa Microbial Pathogenesis Laboratory, Nabi Biopharmaceuticals,
Rockville, Maryland 20852
Received J une 9, 2003
A series of ring-expanded (“fat”) nucleoside analogues (RENs) containing the 6-aminoimidazo-
[4,5-e][1,3]diazepine-4,8-dione ring system have been synthesized and screened for inhibition
of NTPase/helicase of the West Nile Virus (WNV). To assess the selectivity of RENs against
the viral enzymes, a truncated form of human enzyme Suv3(∆1-159) was also included in the
study. Ring-expanded nucleosides 16, 17, and 19, which possess the long C12, C14, and C18
side-chains, respectively, at position 6, as well as the ring-expanded heterocycle 39, which
contains aralkyl substitution at position 1, were all found to have excellent profiles of activity
and selectivity toward the viral versus human enzymes against the West Nile Virus (IC50
ranging 1-10 µM). Compound 30, while being an equally potent inhibitor of WNV, was found
to be somewhat less selective, whereas compound 36, which is an R-anomeric counterpart of
30, exhibited potent and selective inhibition of WNV (IC50 1-3 µM). The same compounds
that showed potent inhibition of viral helicase activity completely failed to show any activity
against the viral NTPase reaction even up to 500 µM. However, at concentrations >500 µM of
RENs and the ATP concentrations >10 times the Km value of the enzyme, a significant
activation of NTPase activity was observed. This activating effect underwent further dramatic
enhancement (>1000%) by further increases in ATP concentration in the reaction mixture,
suggesting that the viral helicase and NTPase reactions are not coupled. A tentative mechanistic
model has been proposed to explain the observed results.
In tr od u ction
survivors still reported muscle weakness, 75% suffered
from memory loss, 60% from confusion, and more than
half could no longer live at home, although most were
healthy, active, and lived normal lives before the WNV
attack. Many of the patients end up with lingering
neurological damage as often occurs with encephalitic
infections. There are currently no drugs nor vaccines
available to treat or prevent the disease. Although the
drug ribavirin, a broad spectrum antiviral nucleoside
possessing a triazole ring, was initially reported to halt
the viral replication, the need to use very high doses of
the drug proved too toxic to be clinically useful.
With a number of Science Focus and News Focus
articles that have appeared in recent issues of Science
magazine,1-4 the West Nile Virus (WNV) is gaining wide
attention in North America.5-7 Nearly four years after
the 1999 outbreak of the West Nile virus in New York
City, which sickened 62 people, most of them elderly,
and killed 7, the virus has been detected in more than
60 bird species and about a dozen mammals and has
spread to almost every state and the District of Colum-
bia, with more than 1300 human cases of WNV infection
resulting in several dozen mortalities. West Nile virus
is mainly a bird virus that is spread by mosquitoes.
Humans and horses, as well as a dozen other mammals
are its dead-end hosts. Crows are the virus’s most
conspicuous hosts as they have been dying en masse
with WNV infection. Most humans infected with WNV
don’t even know it, or they experience only mild, flu-
like symptoms. Those over 65 are especially vulnerable
to WNV. Three months after the outbreak, 70% of the
The West Nile virus belongs to the family of viruses
called Flaviviridae that are small, enveloped, spherical
particles of 40 to 50 nm in diameter with single-
stranded, positive sense RNA genomes.8-11 They are
known to be the cause of severe encephalitic, hemor-
rhagic, hepatic, and febrile illnesses in humans. The
viral genome encodes a polyprotein of 3000 to 4000
amino acids that is processed by proteases into three
structural (C, prM, and E) and seven nonstructural (NS)
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
NS5).10-12 Among these proteins, the NS3 appears to
be a promising target for antiviral agents because of the
multiple enzymatic activities associated with this pro-
tein. NS3 exhibits serine protease, RNA-stimulated
nucleoside triphosphatase (NTPase), and RNA helicase
activities.13-15 The catalytic domain of the chymot-
* The correspondence concerning organic synthesis should be ad-
dressed to the University of Maryland. Tel: 410-455-2520. Fax: 410-
455-1148. E-mail: hosmane@umbc.edu. The correspondence concerning
virology should be addressed to the Bernhard-Nocht-Institut. Tel: 040/
42 818-458. Fax: 040/42 818-378. E-mail: borowski@bni.uni-ham-
burg.de.
† University of Maryland.
‡ Bernhard-Nocht-Institut.
§ University of Warsaw Pawinskiego 5A.
| W. W. Karakawa Microbial Pathogenesis Laboratory.
10.1021/jm030277k CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/23/2003

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4777
Sch em e 1
rypsin-like NS3 protease has been mapped to the NH2-
terminus region of the NS3, whereas the NTPase and
the helicase activities are associated with the COOH-
terminus of NS3.12,13 Helicases are capable of unwinding
duplex RNA and DNA structures by disrupting the
hydrogen bonds that keep the two strands together.16,17
This unwinding activity is essential for the virus
replication. Recently reported “knock out” experiments
demonstrated unambiguously that the switch-off of the
helicase activity abolishes the virus propagation of
bovine diarrhea virus (BVDV) and of dengue fever virus
(DENV).18,19 According to the data, the inhibition of the
helicase activity associated with NS3 protein may be
an effective tool for reduction of virus replication. In a
recent study, we have demonstrated that some imidazo-
[4,5-d]pyridazine nucleosides act as inhibitors of WNV
NTPase/helicase and reduce the unwinding activity of
the enzyme with an IC50 value in the micromolar
range.20 A comparable inhibitory potency was also
observed in tissue cultures of the virus.20 Furthermore,
closely related compounds that did not influence the
helicase activity, did not also exert any effect on the
virus replication.20
site,27 and (e) inhibition of unwinding by steric interfer-
ence in translocation of the enzyme along the poly-
nucleotide chain.28 There are even more mechanistic
possibilities by which the helicase activity could be
inhibited.29,30
We present here a class of nucleoside analogues,
called ring-expanded (REN or “fat”) nucleosides, which
are found to modulate, i.e., activate or inhibit, the
unwinding reaction of WNV NTPase/helicase. Although
many of the compounds tested also seem to affect the
respective ATPase activity, it nevertheless appears to
bear no significant consequence on the overall antihe-
licase activity.
Ch em istr y
We have recently reported the in vitro antiviral
activity of the parent ring-expanded nucleoside, 6-ami-
noimidazo[4,5-e][1,3]diazepine-4,8-dione (1), against NT-
Two alternative mechanisms of RNA/DNA unwinding
by NTPase/helicase have been postulated.21-24 Both
models predict that the enzymes bind and hydrolyze
NTP by a well characterized NTP binding pocket. The
energy released is used for the “march” of the enzyme
along the DNA or RNA structures and the unwinding
reaction results from capturing single strand (ss) regions
which arise due to thermal fluctuations at the fork.16,21
Alternatively, the energy could be transferred to the fork
and used for disruption of the hydrogen bonds that keep
the strands together.16,21 Consistent with the proposed
models, the following mechanisms of inhibition of the
helicase activity could be considered: (a) inhibition of
the NTPase activity by interference with NTP bind-
ing,25,26 (b) inhibition of NTPase activity by an allosteric
mechanism,25 (c) inhibition of the coupling of NTP
hydrolysis to unwinding reaction,26 (d) interference in
the interaction of helicase with its RNA or DNA
substrate via competitive blockade of substrate binding
Pases/helicases of a family of Flaviviridae, including the
hepatitis C virus (HCV), the West Nile Virus (WNV),
and the J apanese encephalitis virus (J EV).31 The ob-
served promising activities of 1 prompted us to further
explore the structure-activity relationship (SAR) of 1
against each of the above-mentioned viruses. In this
particular study, we have focused on the West Nile
virus. Two conspicuous loci of 1 that are viable for
further structural modifications, but remained largely
unexplored thus far, are position 6 and 1, where the
amino and the sugar group are attached, respectively.
Therefore, in exploring the potential anti-Flaviviridae
activity of the ring system contained in 1, we decided

4778 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Zhang et al.
Sch em e 2
Sch em e 3
to focus on variation of substituents at position 6, along
with variations of sugar moieties at position 1, including
the type of sugar (ribo-, 2′-deoxyribo-, or acyclic) as well
as its mode of attachment to the base (R or â configu-
ration).
in Scheme 3, or by (b) base-catalyzed glycosylation of
an imidazole diester with a pure, crystalline 1-R-chloro-
2-deoxy-D-ribofuranosyl-3,5-bis(p-toluate), as outlined in
Scheme 4. The deoxygenation procedure listed in
Schemes 3 consisted of selectively protecting the 3′,5′-
positions of the concerned nucleoside using the Mark-
iewicz reagent,39 1,3-dichloro-1,1,3,3-tetraisopropyldis-
iloxane, to form the corresponding 3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl) (TIPDS) derivative 27.
Further functionalization at O-2′ with phenyl chlo-
rothionocarbonate (phenoxythiocarbonyl chloride), em-
ploying 4-(dimethylamino)pyridine (DMAP) as a cata-
lyst, afforded the respective 3′,5′-O-TIPDS-protected 2′-
O-(phenoxythiocarbonyl) ester 28. Free radical-mediated
Barton deoxygenation37 with tributylstannane (tribu-
tyltin hydride), employing R,R′-azobis(isobutyronitrile)
(AIBN) as a radical initiator in toluene at reflux, gave
satisfactory conversion into the respective 3′,5′-TIPDS-
protected-2′-deoxynucleoside 29. The silyl deprotection
of the latter compound was achieved by treatment with
tetra-n-butylammonium fluoride, which gave the de-
sired 2′-deoxynucleoside 30.
The general method for the synthesis of nucleosides
(6-24) containing the parent structure 1 with different
alkyl, aralkyl, and aromatic substituents at position 6
(see Scheme 1) involved Vorbru¨ggen ribosylation32,33 of
dimethyl imidazole-4,5-dicarboxylate (2),34,35 followed by
condensation of the resulting imidazole nucleoside (3)
with the appropriately substituted guanidine deriva-
tives (4). The products were fully characterized by
elemental microanalyses, NMR, and mass spectral data.
The necessary guanidine derivatives, when not com-
mercially available, were synthesized using the general
synthetic procedure36 outlined in Scheme 2. The proce-
dure consisted of reacting 3,5-dimethylpyrazole-1-car-
boxamidine nitrate (5) with the appropriately substi-
tuted amine in methanol at reflux for a brief period. We
discovered that the use of methanol, instead of water
as reported in the literature, greatly improves both yield
and purity, in addition to significantly reducing the
reaction time because of the increased solubility of most
amines in methanol compared with water. Aromatic
amines required more reaction time than aliphatic
amines.
The glycosylation procedure outlined in Scheme 4
warranted the use of a highly pure and crystalline
sugar, 1-R-chloro-2-deoxy-D-ribofuranosyl-3,5-bis(p-tolu-
ate) (32),40 strongly basic medium, as well as the
appropriate imidazole diester in order to achieve both
high yield and high anomeric purity. For example, the
standard Vorbru¨ggen method of glycosylation of the
dimethyl ester 2 with the above chloro sugar (32),
The 2′-deoxy analogue (30) of nucleoside 16 was
synthesized by either (a) direct deoxygenation, employ-
ing the Barton deoxygenation procedure,37,38 as outlined

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4779
Sch em e 4
employing 1,1,1,3,3,3-hexamethyldisilazane (HMDS),
chlorotrimethylsilane (CTMS) and trimethylsilyl triflate
(TMSOTf) catalysis, only yielded a mixture of R and â
nucleosides with a 2:1 (R:â) ratio. Direct base-catalyzed
glycosylation without presilylation using a polar solvent
such as DMF, acetonitrile, or pyridine only made the
matter worse by formation of increasing amounts of the
R anomer in the mixture (R:â g 3:1). It was discovered
that the use of even the purest form of the R-chloro
sugar 32 for glycosylation under these conditions yielded
a mixture of both R and â anomers of the nucleoside
product, with predominance of the R-anomer in the
mixture. Apparently, the R anomer of the chloro sugar
(32) undergoes facile anomerization in a polar solvent
such as DMF or acetonitrile and is mostly transformed
into the thermodynamically more favored â-chloro ano-
mer if allowed to stand for a long enough time. Since
the displacement of the halide by the nucleobase is
believed to proceed by an SN2 mechanism, the resultant
nucleoside product would have the undesired R ano-
meric configuration. It was, therefore, concluded that
in order to form the desired â-nucleoside as the major
or the exclusive product, the rate of glycosylation has
to be significantly faster than the rate of anomerization.
Thus, the problem was finally solved by (a) using a
strong base such as sodium hydride, instead of bases
such as proton sponge or triethylamine, for condensa-
tion, (b) portionwise addition of the R-chloro sugar to
the suspension of the heterocyclic base and sodium
hydride over a prolonged period of a couple of hours
instead of just a few minutes, and (c) substitution of the
dimethyl ester 2 with its dibutyl analogue 31 for better
solubility. Indeed, this experimental modification yielded
the desired â-anomer 33 in greater than 95% anomeric
purity. The final ring-closure of 33 with N-dodecylguani-
dine yielded the target 30, which was identical in all
respects to that obtained by direct deoxygenation of the
ribonucleoside 16 as outlined in Scheme 3. The overall
yields of 30 by the two methods shown in Schemes 3
and 4 are 53% and 71%, respectively, suggesting that
the glycosylation procedure is preferred over the direct
deoxygenation of ribonucleoside both in terms of the
small number of synthetic steps and the product yield.
The R anomer 36 of nucleoside 30 was synthesized
(Scheme 5) by sequential reactions involving condensa-
tion of the imidazole diester 31, in the presence of proton
sponge, with the â-chloro sugar anomer 34, generated
in situ from the corresponding R-chlorosugar 32, to
obtain the desired R-anomer 35, followed by ring-closure
with N-dodecylguanidine. The distinction between R-
1
and â-anomers was achieved by H NMR and NOESY
studies. The major difference between â- and R-anomers
of the imidazole precursors 33 and 35, respectively, lay
in the absorption of the H-4′ signal which was 0.26 ppm
(78 Hz) more downfield in the R-anomer relative to that
of the â-anomer. Such an observation has been docu-
mented with other nucleoside analogues.41 The major
distinction between â- and R-anomers of the target
RENs 30 and 36, respectively, lay in the absorption of
the imidazole proton H-2, which was 0.27 ppm (81 Hz)
upfield in the R-anomer relative to that of the â-anomer.
However, neither the difference in chemical shifts nor
the pattern of coupling of H-1′ or H-2′ that are normally
employed to distinguish the two anomers41,42 were
discriminatory enough in case of RENs to allow defini-
tive assignments of their configurations. For example,
while the â-anomer 30 revealed its H-1′ signal at δ 6.62
as a pseudo triplet with J 1′,2′a. J 1′,2′b ) 5.9 Hz, the
corresponding R-anomer 36 revealed its H-1′ also at δ
6.62 but only as a doublet with J ) 6.2 Hz. The
R-anomers of 2′-deoxynucleosides normally exhibit their
H-1′ as a doublet of doublets with unequal coupling
constants, one being considerably larger than the other
(e.g., 5-7 Hz vs 1-3 Hz). The fact that only a doublet
with a coupling constant of 6.2 Hz was observed in 36
suggests that the second coupling constant must be near
or equal to zero. This further implies the presence of a
possible “north” (C2′-exo-C3′-endo) conformation for the
sugar pucker of 36, which would place its axial H-2′
farther away from H-1′, preventing effective coupling
between the two protons.
The conclusive configurational assignments were

4780 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Zhang et al.
Sch em e 5
Sch em e 6
finally made by two-dimensional nuclear Overhauser
and exchange spectroscopy (NOESY). The energy-
minimized structures of 30 and 36 by molecular model-
ing43 studies clearly indicated the proximity of the
imidazole H-2 of 36 with the sugar H-1′ (internuclear
distance ) 2.52 Å). By contrast, in the â-anomer 30, the
imidazole H-2 was considerably farther away from the
H-1′ (distance ) 3.79 Å) to exhibit any significant NOE.
These predictions were borne out by the observed NOE
correlations between H-2 and H-1′ in the R-anomer 36,
whereas no measurable correlation existed between the
same set of protons in the corresponding â-anomer 30.
The parent heterocyclic aglycon 38 (Scheme 6) of
nucleoside 16 or 30 was prepared by deglycosylation of
30 by treatment with acetic acid. The 1-aralkyl deriva-
tive 39 of 38 was synthesized by condensation of the
corresponding imidazole diester 37 with N-dodecylguani-
dine. The imidazole diester 37, in turn, was prepared
by alkylation of 2 with p-methoxybenzyl chloride, em-
ploying potassium carbonate in acetonitrile.
Resu lts a n d Discu ssion
The NTPase/helicase from the West Nile virus was
chosen for the intended enzyme inhibition studies by a
range of ring-expanded nucleosides/nucleotides. To as-
sess the selectivity of the RENs-mediated inhibition, an
enzyme from human source, namely, the NH2-termi-
nally truncated form of Suv3 protein [Suv3(∆1-159)], was
also included in the study. The latter protein, which is
commonly being referred to as putative RNA NTPase/
helicase to date, displays considerable homologies with
ATP- and RNA-binding sites (domains I and II) with

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4781
a
Ta ble 1. Inhibitory (IC50
)
or Activating (ED200) Effect of
inhibitor required for 50% inhibition of the enzyme
activity: (a) The inhibition of the helicase activity by
RENs results from a mechanism that could only be
speculated about. There is probably more than one
target for RENs within the substrate-enzyme complex.
Moreover, the inhibition appeared to be ATP-indepen-
dent. Under these conditions one can only roughly and
unreliably estimate the Ki. (b) Many of the RENs were
previously screened in tissue culture systems, and the
results were presented as IC50.46 To maintain the
conformity as well as facile comparison, IC50 was
considered the preferred unit for expression of activity.
(c) The most potent RENs are currently being tested in
vivo as antiviral agents, and therefore, it would be more
convenient to compare the in vitro and in vivo data
using either the IC50 or IC90 values.
Ring-Expanded Nucleoside (REN) Analogues against the
Helicase Activity of the West Nile Virus (WNV) NTPase/
Helicase, Using a DNA Substrateb
compound no.
WNV IC50 (µg/mL)a
Suv3IC50 (µg/mL)a
6-14
15
16
17
18
300 to >500
ND
30-100
>500
>500
300
>500
>500
>500
ND
3-5
250
>500
>500
1-3
3-10
250
19
5.0
20
activator: ED200 ) 300
21-24
>500
3-10
3-10
>500
5.0
30
36
38
39
a
The term IC50 is defined as the concentration of REN required
for 50% inhibition of enzyme activity. The term ED200 reflects the
effective dose of the compound yielding 200% activity. The helicase
activity was determined as a function of increasing concentrations
of the compounds in the presence of ATP adjusted to the Km values
of 9.5 µM and 4.2 µM for WNV and Suv3(∆1-159) NTPase/helicase,
respectively, and 4.7 pM of DNA substrate (concentration of
nucleotide base). The substrate and released strand were sepa-
rated in TBE polyacrylamide gel and visualized by exposition of
dried gel onto X-ray film for 20 h. The parts of the gels corre-
sponding to the released strand were excised, and the 32P
radioactivity was quantified as described under Biochemical
Methods in the Experimental Section. The inhibitory potential of
the compounds was expressed as the inhibitor concentration at
which 50% of the unwinding activity was measured. The helicase
activity of the enzyme measured in the absence of the compounds
was referred to as 100%. The results presented are representative
The RENs of Scheme 1, compounds 6-24, contained
substituents at position-6, incorporating judicious in-
cremental variations of length and bulk with alkyl or
aralkyl groups. The results obtained for 6-14 and 21-
24 were heterologous, with IC50 values ranging any-
where from 70 µg/mL to >500 µg/mL, indicating that
they were, by and large, inactive by conventional
standards. A notable exception, however, occurred with
compound 20 containing an N6-phenyl substituent,
which exerted an activating effect on the WNV helicase
activity. We have recently reported about a similar
observation made with imidazo[4,5-d][pyridazine nu-
cleosides.20 On the other hand, RENs 16-19 containing
an alkyl chain with 12 or more carbon atoms exhibited
IC50 values ranging 1-10 µg/mL, with the only excep-
tion of 18. Even more encouraging was the fact that they
were all inactive against the human enzyme Suv3 (IC50
> 300 µg/mL), suggesting that the compounds are likely
to be selective for the viral NTPase/helicase.
b
of three independent experiments. ND ) not determined For
the assessment of selectivity, the truncated version of human
helicase Suv3(∆1-159) has also been included in the enzyme inhibi-
tion studies.
the viral NTPase/helicase.44 The WNV NTPase/helicase
was isolated and purified from the cell culture medium
harvested from virus-infected Vero E6 cells according
to our own procedure reported previously.45 The NH2-
terminally truncated version of Suv3 NTPase/helicase
was expressed in E. coli via PCR amplification of the
appropriate human Suv3 cDNA coding for SuV3 protein
with truncated 159 amino acids from the amino termi-
nus, followed by cloning into NcoI and BamHI sites of
pQE60 expression vector. The homogenity of final
enzyme preparations was ascertained by SDS/polyacry-
lamide gel electrophoresis with Coomassie Blue stain-
ing.
Th e Helica se Rea ction . To monitor the inhibitory
potential of RENs toward WNV helicase reaction, we
prepared the necessary DNA and RNA substrates
consisting of two annealed DNA or RNA oligonucle-
otides. The unwinding activity was assessed by moni-
toring the release of the shorter labeled strand of the
DNA or RNA duplex. Interestingly, the efficacy of
unwinding was not affected by the nature of the
substrate used. The helicase activity was calibrated with
a DNA or RNA substrate that was unwound at an ATP
concentration equal to the Km value determined for the
NTPase reaction.
The favorable activity/selectivity profile of REN 16
against WNV helicase prompted us to test its 2′-deoxy
analogue 30 (see Schemes 3 and 4). REN 30 also
exhibited comparable antihelicase activity profile against
WNV (IC50 3-10 µg/mL), but unlike 16, it also inhibited
the human enzyme Suv3 with an IC50 value of 3-5 µg/
mL, suggesting that it would be less discriminatory
between viral and human NTPase/helicase.
The above results with 30 led us to explore the role
of R/â-configuration at the base-sugar junction, the
anomeric carbon, in biological activity. So, we synthe-
sized and screened compound 36 (Scheme 5), which is
the R-anomer of 30. Compound 36, like its â-anomeric
counterpart 30, exhibited potent and selective antihe-
licase activity against WNV NTPase/helicase (IC50 3-10
µg/mL)
The varied results obtained with different sugar
substitutions and their anomeric configurations left us
to be confronted with one important final question
concerning the modulation of WNV helicase reaction by
RENs. Is the sugar or the sugarlike moiety necessary
at all for the antihelicase activity of RENs? To address
that question, we synthesized the parent heterocycle of
30 (i.e., 38) (see Scheme 6). But compound 38 was found
to be completely devoid of activity. We then synthesized
an analogue of 38 that contained a p-methoxybenzyl
group attached to the imidazole ring (i.e., 39) in place
of the sugar moiety. Surprisingly, compound 39 turned
out to be both potent and selective against the helicase
The target compounds of Schemes 1-6 were screened
against the helicase activity of WNV, and the results
are summarized in Table 1. Although the inhibition
constant Ki is the normal parameter used to express the
enzyme inhibition, there are several reasons for pre-
senting our results as IC50, the concentration of the

4782 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Zhang et al.
a
Ta ble 2. Inhibitory Activity (IC50
)
of Ring-Expanded
reasonable to consider an analogous mechanism for the
RENs as well. It is also well established that the groove
binding is greatly dependent and highly sensitive to the
mode of substituents on the binder as well as the
bounded. Thus, various RENs with different substitu-
tions or topological structural features are expected to
differently modulate their interactions with a DNA or
an RNA substrate. Different modulations could poten-
tially result in either the more stable or the less stable
double-helix, thus accounting for the observed inhibitory
or activating effect of different RENs on the unwinding
activity of viral helicase. Corroborating the notion of
possible DNA and/or RNA binding of RENs is our
qualitative observation that compound 30 forms a tight
complex with a DNA substrate that was completely
stable in the presence of 0.5% sodium dodecyl sulfate
(SDS). Although rigorous investigation on this subject
is yet to be carried out, it was unmistakable to notice
that the migration of the DNA substrate in the TBE-
polyacrylamide gel electrophoresis was severely hin-
dered in the presence of 30 whether in the presence or
absence of the enzyme.
Nucleoside (REN) Analogues against the Helicase Activity of
the West Nile Virus (WNV) NTPase/Helicase, Using an RNA
Substrateb
compound no.
WNV IC50 (µg/mL)
300
Suv3 IC50 (µg/mL)
15
16
17
18
19
20
30
36
38
39
500
150
30-100
30
10
>500
)500
100
30
activator: ED200 ) 100
>500
>500
150
>500
>500
1.5
100-200
>500
1-3
a
The helicase activity was determined as described in the
legend to Table 1 and under Biochemical Methods in the Experi-
mental Section with the exception that 4.7 pM RNA duplex
(concentration of nucleotide base) was used as a substrate. The
term IC50 is defined as the concentration of REN required for 50%
inhibition of enzyme activity. The results presented are represen-
tative of three independent experiments For the assessment of
selectivity, the truncated version of human helicase Suv3(∆1-159)
has also been included in the enzyme inhibition studies.
b
activity of WNV NTPase/helicase with an IC50 value of
5 µg/mL. These results suggest that a simple aralkyl
group in place of a sugar moiety would not only suffice
but also may be preferred for an excellent activity/
selectivity profile.
Th e NTP a se Rea ction . We then focused our atten-
tion on the NTPase reaction mediated by the viral
enzymes. Our goal in this regard was 2-fold: (a) To
investigate if the compounds of Table 1 inhibit the viral
NTPase reaction as well, and (b) if they do, is the
inhibition of the NTPase reaction coupled to the ob-
served inhibition of the unwinding (helicase) reaction?
Finally, the above biochemical results were all ob-
tained using a DNA substrate. So, it was important to
consider if the results would be any different with an
RNA substrate. The data obtained with an RNA sub-
strate for a few selected RENs are collected in Table 2.
As is evident, the results in some cases were signifi-
cantly different from those obtained from a DNA
substrate. A case in point is REN 30, which is 2-6 times
more potent in inhibiting the WNV helicase activity
with an RNA substrate as compared to a DNA sub-
strate. In contrast, compound 16, the ribose counterpart
of 30, was more active with a DNA substrate rather
than an RNA substrate. Furthermore, compound 16 was
found to be more selective toward WNV NTPase/helicase
in comparison to the human enzyme Suv3 when a DNA
substrate was employed, whereas the trend was re-
versed with 30 which was highly selective toward the
viral enzyme only when an RNA substrate was em-
ployed.
The above antihelicase activity data raise an impor-
tant biochemical question about the mechanism of
activity of RENs. There appears to be no conspicuous
or single trend in activity. As a matter of fact, consider-
able differences exist in activity between the ribose and
deoxyribose sugar analogues, between analogues con-
taining R and â-anomeric configurations, between those
with or without substitutions at the imidazole ring, and
between those with different length, bulk, and the type
of substituents on the heterocyclic ring. Although specu-
lation on the exact mechanism of action of RENs is not
possible without further extensive studies, one likely
mechanism involves their potential interactions with the
DNA or RNA substrate of WNV helicase. This is based
on a number of documented reports demonstrating the
noncovalent interactions of analogues of nucleobases,
nucleosides, and nucleotides,47,48 in particular as binders
to major or minor grooves of duplex DNA or RNA. In
light of their structural similarity to purine bases, it is
The viral NTPase activity was assessed by a standard
assay that determines the amount of 33Pi released from
a [γ-33P] ATP during the enzyme-mediated hydrolysis.
The ATPase activity was measured as a function of
increasing concentrations of the RENs investigated
under the helicase tests described above. However,
when investigated at ATP concentrations equal to the
Km values determined for the ATPase reaction of each
of the viral NTPase/helicase [9.5 µM for WNV and 4.2
µM for Suv3 (∆1-159)], none of the compounds inhibited
the hydrolytic activity of the enzymes even up to the
concentration of 0.5 mM. The successive lowering of
concentration down to values corresponding to 1 × 10-5
of Km of each enzyme also did not lead to any significant
inhibition of the ATPase activity.
Interestingly, at concentrations >500 µM of RENs and
>10 × Km of ATP, an activation of the ATPase activity
was observed. We had earlier made a similar observa-
tion on the activating effect of ATPase activity with
ribavirin,26,45 a nucleoside containing a 1,2,4-triazole
ring with a carboxamide side chain, which is known to
interact with nucleotide-binding sites of various en-
zymes.49 The well-documented ATP binding studies of
Porter et al.29,30 suggest strongly the existence of a
second nucleoside binding site within WNV NTPase/
helicase. The first binding site is known to be equipped
with Walker motifs [the short amino acid sequences
interacting with â- and γ-phosphorus atoms of nucleo-
side-5′-triphosphate (Walker A), and with Mg2+ or Mn2+
that are chelated by the â- and γ-phosphorus atoms
(Walker B)] and is known to be involved in the hydroly-
sis of NTPs. The speculation is that this site possesses
significantly higher affinity for nucleoside-5′-triphos-
phates as compared to the second binding site, which
has a much lower affinity and selectivity, and could be

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4783
molecular sieves (type 3 Å); methylene chloride was distilled
from calcium hydride and was stored over molecular sieves
(type 3 Å); dimethylformamide was dried over calcium oxide
and then distilled under reduced pressure from calcium
hydride and was subsequently stored over molecular sieves
(type 3 Å). Starting materials were purchased from either
Aldrich Chemical Co. or Lancaster. All solvents were reagent
grade and were purchased from VWR Scientific. All yields
reported are for dry compounds that require no further
purification for use in other reactions.
Gen er a l Meth od for th e Syn th esis of (Com m er cia lly
Un a va ila ble) Su bstitu ted Gu a n id in es. A mixture of ap-
propriate amine (4 mmol) and 3,5-dimethylpyrazole-1-carboxa-
midine nitrate (5)36 (0.830 g, 4 mmol) was refluxed in methanol
(15 mL) for 2 h. The solution was rotary evaporated to dryness,
and the residue was dried over P2O5 in vacuo. It was directly
employed in the condensation reaction with 3 without further
purification.
Gen er a l Met h od for t h e Syn t h esis of Nu cleosid es
6-24. Hydrochloride, sulfate, or nitrate salt of guanidine (4
mmol) was added to 4 mL of 2.3 M sodium methoxide solution
resulting from sodium (0.75 g) dissolved in 15 mL of absolute
methanol. The mixture was stirred in an ice bath for 30 min.
The precipitated sodium chloride was removed by filtration,
and the filtrate was poured into a methanolic solution (20 mL)
of methyl 1-(2′,3′,5′-tri-O-benzoyl-â-D-ribofuranosyl)imidazole-
4,5-dicarboxylate (3)50 (1 mmol), prepared by Vorbru¨ggen
ribosylation32,33 of imidazole-4,5-dicarboxylate.34,35 The mixture
was stirred at room temperature for 24-48 h and was
monitored by frequent TLC analysis to check for the comple-
tion of reaction. The reaction mixture was filtered if necessary,
and the clear filtrate was neutralized with 2 M hydrochloric
acid. The resulting precipitate, if formed, was filtered and
washed with water and methanol to give product. In cases
where no precipitate formed, the solution was extracted with
an appropriate organic solvent and the combined extracts were
dried and evaporated to obtain the product. The latter was
either recrystallized from an appropriate solvent or purified
by flash chromatography on a silica gel column. The spectral
and analytical data, along with solvent of recrystallization and/
or solvent of elution for chromatography are collected here
below.
4,5-Dih yd r o-8H-6-(N-m eth yla m in o)-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (6). Yield 93%,
Rf ) 0.09 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], mp 210 °C (dec); 1H NMR (DMSO-d6): δ 10.63 (brs, 1H,
NH, exchangeable with D2O), 8.54 (s, 1H, imidazole), 7.01 (brs,
1H, NH, exchangeable with D2O), 6.36 (d, J ) 2.6 Hz, 1H, 1′-
H), 5.47 (d, J ) 5.1 Hz, 1H, OH, exchangeable with D2O), 5.16
(t, J ) 4.9 Hz, 1H, OH, exchangeable with D2O), 5.07 (d, J )
5.5 Hz, 1H, OH, exchangeable with D2O), 4.06 (m, 2H, 2′- and
3′- H), 3.90 (m, 1H, 4′-H), 3.73 (dd, J 1 ) 12.1 Hz, J 2 ) 4.0 Hz,
1H, 5′-H1), 3.58 (dd, J 1 ) 11.7 Hz, J 2 ) 4.0 Hz, 1H, 5′-H2),
2.75 (d, J ) 4.0 Hz, 3H, CH3). Anal. (C12H15N5O6‚H2O) C, H,
N.
occupied by a nucleotide, nucleoside, or even an aglycon
of nucleoside. This second binding site fulfills a rather
regulatory function with respect to the ATPase and/or
helicase activity.
The above REN-mediated inhibition and/or activation
results, obtained separately against the helicase and
ATPase activities of WNV, prompt us to corroborate the
speculation about the existence of an allosteric binding
site on WNV NTPase/helicase where nucleoside/nucle-
otide-type molecules bind. Occupation of this site is
largely responsible for modulation of WNV NTPase and/
or helicase. It remains to be resolved as to whether this
is a common site for both helicase and NTPase or
whether there are two independent allosteric sites for
the two enzymes. Most of the RENs that inhibit the viral
helicase activity seem to exert their effect by binding
to the major or minor groove of DNA or RNA substrate
of helicase. However, at high concentrations of ATP,
they seem to occupy the allosteric site and cause the
enzyme activation instead. The presence of this allos-
teric binding site for RENs might also explain the
seemingly uncoupled NTPase and helicase activities of
the viral NTPase/helicase.
Con clu sion
Ring-expanded nucleosides modulate the activities of
the West Nile virus. The specific modulation explored
in this study concerns the helicase and NTPase activi-
ties of the virus. The type of modulation exerted by
RENs can either be an inhibition or activation. The
mechanism of action of most of the ring-expanded ribo-,
2′-deoxyribo-, and R- and â-anomeric nucleoside ana-
logues, as well as their heterocyclic aglycons, involves
inhibition or activation of the viral helicase activity. In
the case of inhibition, the mechanism may involve the
interaction of RENs with a DNA or an RNA substrate
of the helicase through binding to the major or minor
groove of the double-helix. In the case of activation, the
mechanism may involve an allosteric binding site that
can be occupied by nucleoside/nucleotide-type molecules
including, but not limited to RENs. The occupation of
this allosteric site is dependent upon the high level of
ATP (NTP) concentration in the reaction mixture. Many
of the RENs tested were remarkably selective for the
viral NTPase/helicase over the truncated human form
of the enzyme Suv3, suggesting that the compounds
may not be toxic to the host. In that regard, RENs may
represent a starting point for the development of highly
selective inhibitors of WNV NTPase/helicase.
4,5-Dih ydr o-8H-6-(N,N-dim eth ylam in o)-1-(â-D-r ibofu r a-
n osyl)im idazo[4,5-e][1,3]diazepin e-4,8-dion e (7). Yield 44%,
Rf ) 0.17 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
Exp er im en ta l Section
(A) Ch em ica l Meth od s. The 1H and 13C NMR spectra were
recorded on a General Electric QE-300 NMR spectrometer
1
operating at 300 MHz for H and 75 MHz for 13C. The chemical
1
methanol (5:1), and methanol, mp 215 °C; H NMR (DMSO-
shift data are reported with reference to Me4Si (internal
standard) for 1H and 13C NMR. The data are reported in the
following format: Chemical shift, multiplicity (s ) singlet, d
) doublet, dt ) double triplet, dd ) double doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad, coupling constants,
integration, and assignment). Elemental Microanalyses were
performed by Atlantic Microlab, Inc., Norcross, GA. The mass
spectra were recorded at the Mass Spectral Facility, Depart-
ment of Biochemistry, Michigan State University. Thin-layer
chromatography was performed on Merck Kieselgel 60 GF254
plates (0.2 mm thickness). Melting points were determined on
a Thomas-Hoover capillary melting point apparatus and are
uncorrected. Dry solvents were prepared as follows: Methanol
was distilled from calcium hydride and was stored over
d6): δ 8.27 (s, 1H, imidazole), 6.47 (d, J ) 3.3 Hz, 1H, 1′-H),
5.81 (d, J ) 4.0 Hz, 1H, OH, exchangeable with D2O), 5.17 (t,
J ) 4.9 Hz, 1H, OH, exchangeable with D2O), 5.07 (d, J ) 4.4
Hz, 1H, OH, exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′-
H), 3.91 (m, 1H, 4′-H), 3.76 (brs, 1H, NH, exchangeable with
D2O), 3.70 (td, J 1 ) 12.6 Hz, J 2 ) 3.8 Hz, 1H, 5′-H1), 3.58 (td,
J 1 ) 12.3 Hz, J 2 ) 3.8 Hz, 1H, 5′-H2), 3.02 (s, 6H, 2 CH3). Anal.
(C13H17N5O6) C, H, N.
4,5-Dih ydr o-8H-6-(N-eth yl)am in o-1-(â-D-r ibofu r an osyl)-
im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (8). Yield 90%, Rf
) 0.13 [chloroform:methanol:ammonium hydroxide (2:1:0.3)],
purified by silica gel flash chromatography, eluting succes-
sively with chloroform-methanol (30:1), chloroform-methanol

4784 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Zhang et al.
1
(10:1), and methanol-water (4:1), mp 219 °C (dec); H NMR
chloroform-methanol (30:1), chloroform-methanol (10:1), chlo-
roform-methanol (5:1), and methanol, mp 227 °C (dec); 1H
NMR (DMSO-d6) δ 10.46 (brs, 1H, NH, exchangeable with
D2O), 8.55 (s, 1H, imidazole), 7.08 (brs, 1H, NH, exchangeable
with D2O), 6.36 (d, J ) 3.3 Hz, 1H, 1′-H), 5.47 (m, 1H, OH,
exchangeable with D2O), 5.16 (m, 1H, OH, exchangeable with
D2O), 5.08 (m, 1H, OH, exchangeable with D2O), 4.06 (m, 2H,
2′- and 3′- H), 3.91 (m, 1H, 4′-H), 3.72 (m, 1H, 5′-H1), 3.60 (m,
1H, 5′-H2), 3.22 (m, 2H, CH2) 1.47 (m, 2H, CH2), 1.26 (m, 8H,
CH2CH2CH2CH2) 0.85 (s, 3H, CH3). Anal. (C18H27N5O6•0.5H2O)
C, H, N.
(DMSO-d6): δ 10.48 (brs, 1H, NH, exchangeable with D2O),
8.55 (s, 1H, imidazole), 7.07 (brs, 1H, NH, exchangeable with
D2O), 6.36 (d, J ) 2.9 Hz, 1H, 1′-H), 5.50 (d, J ) 4.8 Hz, 1H,
OH, exchangeable with D2O), 5.18 (t, J ) 4.8 Hz, 1H, OH,
exchangeable with D2O), 5.08 (d, J ) 5.1 Hz, 1H, OH,
exchangeable with D2O), 4.06 (m, 2H, 2′- and 3′- H), 3.90 (m,
1H, 4′-H), 3.73 (dd, J 1 ) 11.7 Hz, J 2 ) 4.4 Hz, 1H, 5′-H1), 3.58
(dd, J 1 ) 11.9 Hz, J 2 ) 4.2 Hz, 1H, 5′-H2), 3.24 (m, 2H, CH2);
1.09 (t, J ) 7.1 Hz, 3H, CH3). Anal. (C13H17N5O6) C, H, N.
4,5-Dih yd r o-8H-6-(N-p r op yl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (9). Yield 47%,
Rf ) 0.25 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting with
chloroform-methanol (30:1), chloroform-methanol (10:1), chlo-
roform-methanol (5:1), and methanol, mp 180 °C (dec); 1H
NMR (DMSO-d6): δ 10.53 (brs, 1H, NH, exchangeable with
D2O), 8.55 (s, 1H, imidazole), 7.17 (brs, 1H, NH, exchangeable
with D2O), 6.36 (d, J ) 2.6 Hz, 1H, 1′-H), 5.50 (s, 1H, OH,
exchangeable with D2O), 5.18 (s, 1H, OH, exchangeable with
D2O), 5.08 (s, 1H, OH, exchangeable with D2O), 4.06 (m, 2H,
2′- and 3′- H), 3.90 (m, 1H, 4′-H), 3.73 (d, J ) 10.6 Hz, 1H,
5′-H1), 3.58 (d, J ) 11.7 Hz, 1H, 5′-H2), 3.17 (m, 2H, CH2)
1.48 (m, 2H, CH2), 0.88 (t, J ) 7.3 Hz, 3H, CH3). Anal.
(C14H19N5O6•H2O). C, H, N.
4,5-Dih ydr o-8H-6-(N-bu tyl)am in o-1-(â-D-r ibofu r an osyl)-
im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (10). Yield 71%, Rf
) 0.29 [chloroform:methanol:ammonium hydroxide (2:1:0.3)],
purified by silica gel flash chromatography, eluting with
chloroform-methanol (30:1), chloroform-methanol (10:1), chlo-
roform-methanol (5:1), and methanol, mp >250 °C; 1H NMR
(DMSO-d6): δ 10.43 (brs, 1H, NH, exchangeable with D2O),
8.55 (s, 1H, imidazole), 7.08 (brs, 1H, NH, exchangeable with
D2O), 6.33 (d, J ) 1.5 Hz, 1H, 1′-H), 5.49 (s, 1H, OH, exchange-
able with D2O), 5.18 (s, 1H, OH, exchangeable with D2O), 5.07
(s, 1H, OH, exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′-
H), 3.90 (m, 1H, 4′-H), 3.71 (m, 1H, 5′-H1), 3.60 (m, 1H, 5′-H2),
3.20 (m, 2H, CH2) 1.44 (m, 2H, CH2), 1.27 (m, 2H, CH2), 0.85
(t, J ) 6.6 Hz, 3H, CH3). Anal. (C15H21N5O6) C, H, N.
4,5-Dih yd r o-8H-6-(N-p en tyl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (11). Yield 51%,
Rf ) 0.33 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting with
chloroform-methanol (30:1), chloroform-methanol (10:1), chlo-
roform-methanol (5:1), and methanol, mp >250 °C; 1H NMR
(DMSO-d6) δ 10.44 (brs, 1H, NH, exchangeable with D2O), 8.55
(s, 1H, imidazole), 7.07 (brs, 1H, NH, exchangeable with D2O),
6.36 (d, J ) 2.6 Hz,1H, 1′-H), 5.49 (d, J ) 4.8 Hz, 1H, OH,
exchangeable with D2O), 5.18 (t, J ) 4.9 Hz, 1H, OH,
exchangeable with D2O), 5.08 (d, J ) 5.5 Hz, 1H, OH,
exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′- H), 3.90 (m,
1H, 4′-H), 3.73 (ddd, J 1 ) 12.0 Hz, J 2 ) 4.9 Hz, J 3 ) 3.0 Hz,
1H, 5′-H1), 3.58 (ddd, J 1 ) 12.2 Hz, J 2 ) 4.5 Hz, J 3 ) 3.2 Hz,
1H, 5′-H2), 3.21 (m, 2H, CH2) 1.48 (m, 2H, CH2), 1.27 (m,
4,5-Dih ydr o-8H-6-(N-octyl)am in o-1-(â-D-r ibofu r an osyl)-
im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (14). Yield 29%, Rf
) 0.37 [chloroform:methanol:ammonium hydroxide (2:1:0.3)],
purified by silica gel flash chromatography, eluting succes-
sively with chloroform-methanol (30:1), chloroform-methanol
(10:1), chloroform-methano (5:1), and methanol, mp 187 °C
1
(dec); H NMR (DMSO- d6): δ 10.49 (brs, 1H, NH, exchange-
able with D2O), 8.55 (s, 1H, imidazole), 7.12 (brs, 1H, NH,
exchangeable with D2O), 6.36 (d, J ) 1.8 Hz, 1H, 1′-H), 5.49
(m, 1H, OH, exchangeable with D2O), 5.18 (m, 1H, OH,
exchangeable with D2O), 5.08 (m, 1H, OH, exchangeable with
D2O), 4.06 (m, 2H, 2′- and 3′- H), 3.90 (m, 1H, 4′-H), 3.73 (d,
J ) 12.5 Hz, 1H, 5′-H1), 3.58 (d, J ) 12.1 Hz, 1H, 5′-H2), 3.21-
(m, 2H, CH2), 1.47 (m, 2H, CH2), 1.24 (m,10H, (CH2)5), 0.84
(t, J ) 6.4 Hz, 3H, CH3). Anal. (C19H29N5O6•2/3H2O) C, H, N.
4,5-Dih ydr o-8H-6-(N-decyl)am in o-1-(â-D-r ibofu r an osyl)-
im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (15). Yield 45%, Rf
) 0.38 [chloroform:methanol:ammonium hydroxide (2:1:0.3)],
purified by silica gel flash chromatography, eluting succes-
sively with chloroform-methanol (30:1), chloroform-methanol
(10:1), and methanol, mp 185 °C (dec); 1H NMR (DMSO-d6) δ
10.45 (brs, 1H, NH, exchangeable with D2O), 8.54 (s, 1H,
imidazole), 7.15 (brs, 1H, NH, exchangeable with D2O), 6.36
(d, J ) 2.2 Hz, 1H, 1′-H), 5.50 (m, 1H, OH, exchangeable with
D2O), 5.18 (m, 1H, OH, exchangeable with D2O), 5.08 (m, 1H,
OH, exchangeable with D2O), 4.06 (m, 2H, 2′- and 3′- H), 3.90
(m, 1H, 4′-H), 3.73 (d, J ) 11.7 Hz, 1H, 5′-H1), 3.58 (d, J )
12.5 Hz, 1H, 5′-H2), 3.20 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.22
(m, 14H, (CH2)7), 0.83 (t, J ) 6.6 Hz, 3H, CH3). Anal.
(C21H33N5O6•H2O) C, H, N.
4,5-Dih yd r o-8H-6-(N-d od ecyl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (16). Yield 89%,
Rf ) 0.41 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
methanol (10:1), and methanol, mp 194 °C (dec); 1H NMR
(DMSO-d6): δ 10.33 (brs, 1H, NH, exchangeable with D2O),
8.55 (s, 1H, imidazole), 7.08 (brs, 1H, NH, exchangeable with
D2O), 6.36 (d, J ) 2.2 Hz, 1H, 1′-H), 5.50 (d, J ) 3.1 Hz, 1H,
OH, exchangeable with D2O), 5.18 (t, J ) 4.8 Hz, 1H, 5′-OH,
exchangeable with D2O), 5.08 (d, J ) 3.7 Hz, 1H, OH,
exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′- H), 3.90 (m,
1H, 4′-H), 3.73 (dd, J 1 ) 8.4 Hz, J 2 ) 4.0 Hz, 1H, 5′-H1), 3.58
(dd, J 1 ) 9.0 Hz, J 2 ) 4.3 Hz, 1H, 5′-H2), 3.20 (m, 2H, CH2),
1.47 (m, 2H, CH2), 1.22 (m,18H,(CH2)9), 0.83 (t, J ) 6.2 Hz,
3H, CH3). Anal. (C23H37N5O6•0.5H2O) C, H, N.
4,5-Dih yd r o-8H-6-(N-tetr a d ecyl)a m in o-1-(â-D-r ibofu r a -
n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (17). Yield
75%, Rf ) 0.42 [chloroform:methanol:ammonium hydroxide (2:
1:0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
methanol (10:1), and methanol, mp 185 °C (dec); 1H NMR
(DMSO-d6) δ 10.38 (brs, 1H, NH, exchangeable with D2O), 8.54
(s, 1H, imidazole), 7.07 (brs, 1H, NH, exchangeable with D2O),
6.36 (d, J ) 2.2 Hz, 1H, 1′-H), 5.49 (d, J ) 4.4 Hz, 1H, OH,
exchangeable with D2O), 5.17 (t, J ) 4.9 Hz, 1H, 5′-OH,
exchangeable with D2O), 5.07 (d, J ) 4.8 Hz, 1H, OH,
exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′- H), 3.90 (m,
1H, 4′-H), 3.73 (dt, J 1 ) 12.1 Hz, J 2 ) 3.4 Hz, 1H, 5′-H1), 3.58
(dt, J 1 ) 12.2 Hz, J 2 ) 3.8 Hz, 1H, 5′-H2), 3.20 (m, 2H, CH2),
1.47 (m, 2H, CH2), 1.22 (m, 22H, (CH2)11), 0.84 (t, J ) 6.6 Hz,
3H, CH3). Anal. (C25H41N5O6•0.75H2O) C, H, N.
4H, CH2CH2) 0.86 (t,
J
) 6.8 Hz, 3H, CH3). Anal.
(C16H23N5O6•0.5H2O) C, H, N.
4,5-Dih ydr o-8H-6-(N-h exyl)am in o-1-(â-D-r ibofu r an osyl)-
im id a zo[4,5-e][1,3] d ia zep in e-4,8-d ion e (12). Yield: 36%,
Rf ) 0.35 [Chloroform:Methanol :Ammonium hydroxide (2:1:
0.3)]; mp > 250 °C; 1H NMR (DMSO-d6): δ 10.47 (brs, 1H,
NH, exchangeable with D2O), 8.55 (s, 1H, imidazole), 7.08 (brs,
1H, NH, exchangeable with D2O), 6.36 (d, J ) 2.2 Hz, 1H, 1′-
H), 5.50 (d, J ) 5.1 Hz, 1H, OH, exchangeable with D2O), 5.18
(t, J ) 4.4 Hz, 1H, 5′-OH, exchangeable with D2O), 5.08 (d, J
) 4.4 Hz, 1H, OH, exchangeable with D2O), 4.05 (m, 2H, 2′-
and 3′- H), 3.90 (m, 1H, 4′-H), 3.73 (dd, J 1 ) 11.2 Hz, J 2 ) 3.9
Hz, 1H, 5′-H1), 3.57 (dd, J 1 ) 12.1 Hz, J 2 ) 3.3 Hz, 1H, 5′-H2),
3.19 (m, 2H, -CH2- group), 1.43 (m, 2H, -CH2- group), 1.22
(m, 6H, -(CH2)3- chain), 0.86 (t, J ) 5.9 Hz, 3H, CH3- group).
Anal. (C17H25N5O6•0.5H2O) C, H, N.
4,5-Dih yd r o-8H-6-(N-h ep tyl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (13). Yield 36%,
Rf ) 0.36 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting with

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4785
4,5-Dih yd r o-8H-6-(N-h exa d ecyl)a m in o-1-(â-D-r ibofu r a -
n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (18). Yield
44%, Rf ) 0.45 [chloroform:methanol:ammonium hydroxide (2:
1:0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
methanol (10:1), and methanol, mp 191 °C (dec); 1H NMR
(DMSO-d6) δ 10.41 (brs, 1H, NH, exchangeable with D2O), 8.55
(s, 1H, imidazole), 7.07 (brs, 1H, NH, exchangeable with D2O),
6.34 (d, J ) 2.6 Hz, 1H, 1′-H), 5.49 (d, J ) 4.8 Hz, 1H, OH,
exchangeable with D2O), 5.17 (t, J ) 4.9 Hz, 1H, 5′-OH,
exchangeable with D2O), 5.07 (d, J ) 5.1 Hz, 1H, OH,
exchangeable with D2O), 4.06 (m, 2H, 2′- and 3′- H), 3.90 (m,
1H, 4′-H), 3.73 (dt, J 1 ) 12.1 Hz, J 2 ) 4.2 Hz, 1H, 5′-H1), 3.58
(dt, J 1 ) 12.8 Hz, J 2 ) 3.9 Hz, 1H, 5′-H2), 3.20 (m, 2H, CH2),
1.47 (m, 2H, CH2), 1.23 (m, 26H, (CH2)13), 0.84 (t, J ) 6.6 Hz,
3H, CH3). Anal. (C27H45N5O6•0.5H2O) C, H, N.
4,5-Dih yd r o-8H-6-(N-octa d ecyl)a m in o-1-(â-D-r ibofu r a -
n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (19). Yield:
53%, Rf ) 0.50 [chloroform:methanol:ammonium hydroxide (2:
1: 0.3), mp 149 °C (dec); 1H NMR (DMSO-d6) δ 10.42 (brs,
1H, NH, exchangeable with D2O), 8.56 (s, 1H, imidazole), 7.08
(brs, 1H, NH, exchangeable with D2O), 6.36 (d, J ) 2.6 Hz,
1H, 1′-H), 5.49 (d, J ) 4.0 Hz, 1H, OH, exchangeable with
D2O), 5.18 (t, J ) 4.9 Hz, 1H, 5′-OH, exchangeable with D2O),
5.08 (d, J ) 4.8 Hz, 1H, OH, exchangeable with D2O), 4.05
(m, 2H, 2′- and 3′- H), 3.90 (m, 1H, 4′-H), 3.71 (dt, J 1 ) 11.9
Hz, J 2 ) 3.8 Hz, 1H, 5′-H1), 3.58 (dt, J 1 ) 12.3 Hz, J 2 ) 3.7
Hz, 1H, 5′-H2), 3.20 (m, 2H, CH2), 1.47 (m, 2H, CH2), 1.22 (m,
26H, (CH2)15), 0.83 (t, J ) 6.6 Hz, 3H, CH3). Anal. (C29H49N5O6)
C, H, N.
4,5-Dih yd r o-8H-6-(N-p h en yl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (20). Yield 42%,
Rf ) 0.19 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
methanol (10:1), chloroform-methano (5:1), and methanol, mp
201 °C (dec); 1H NMR (DMSO-d6) δ 10.58 (brs, 1H, NH,
exchangeable with D2O), 9.21 (brs, 1H, NH, exchangeable with
D2O), 8.62 (s, 1H, imidazole), 7.58 (t, J ) 7.7 Hz, 2H, Phenyl),
7.35 (t, J ) 7.7 Hz, 2H, Phenyl), 7.12 (t, J ) 7.0 Hz, 1H,
Phenyl), 6.32 (d, J ) 2.9 Hz, 1H, 1′-H), 5.54 (d, J ) 5.1 Hz,
1H, OH, exchangeable with D2O), 5.19 (t, J ) 4.9 Hz, 1H, 5′-
OH, exchangeable with D2O), 5.09 (d, J ) 5.5 Hz, 1H, OH,
exchangeable with D2O), 4.08 (m, 2H, 2′- and 3′- H), 3.92 (m,
1H, 4′-H), 3.74 (dd, J 1 ) 9.2 Hz, J 2 ) 2.9 Hz, 1H, 5′-H1),
3.59 (dd, J 1 ) 9.2 Hz, J 2 ) 2.9 Hz, 1H, 5′-H2). Anal.
(C17H17N5O6•1.25H2O) C, H, N.
4.06 (m, 2H, 2′- and 3′- H), 3.91 (m, 1H, 4′-H), 3.73 (dd, J 1
12.8 Hz, J 2 ) 2.6 Hz, 1H, 5′-H1), 3.58 (dd, J 1 ) 12.6 Hz, J 2
2.8 Hz, 1H, 5′-H2), 3.47 (m, 2H, CH2), 2.76 (m, 2H, CH2). Anal.
(C19H21N5O6•2H2O) C, H, N.
)
)
4,5-Dih yd r o-8H-6-(N-(3-p h en ylp r op yl))a m in o-1-(â-D-r i-
bofu r a n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (23).
Yield 28%, Rf ) 0.27 [chloroform:methanol:ammonium hydrox-
ide (2:1:0.3)], purified by silica gel flash chromatography,
eluting successively with chloroform-methanol (30:1), chlo-
roform-methanol (10:1), chloroform-methano (5:1), and metha-
nol, mp 178 °C (dec); 1H NMR (DMSO-d6) δ 10.35 (brs, 1H,
NH, exchangeable with D2O), 8.54 (s, 1H, imidazole), 7.22 (m,
6H, Phenyl + NH, exchangeable with D2O), 6.36 (d, J ) 2.3
Hz, 1H, 1′-H), 5.51 (d, J ) 2.2 Hz, 1H, OH, exchangeable with
D2O), 5.18 (t, J ) 6.2 Hz, 1H, 5′-OH, exchangeable with D2O),
5.09 (d, J ) 3.7 Hz, 1H, OH, exchangeable with D2O), 4.05
(m, 2H, 2′- and 3′- H), 3.90 (m, 1H, 4′-H), 3.72 (dd, J 1 ) 10.9
Hz, J 2 ) 2.4 Hz, 1H, 5′-H1), 3.57 (d, J 1 ) 12.1 Hz, J 2 ) 2.6 Hz,
1H, 5′-H2), 3.22 (m, 2H, CH2), 2.62 (m, 2H, CH2), 1.79 (m, 2H,
CH2). Anal. (C20H23N5O6•0.5H2O) C, H, N.
4,5-Dih yd r o-8H -6-(N-(4-p h en ylb u t yl))a m in o-1-(â-D-r i-
bofu r a n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (24).
Yield 46%, Rf ) 0.29 [chloroform:methanol:ammonium hydrox-
ide (2:1:0.3)], purified by silica gel flash chromatography,
eluting successively with chloroform-methanol (30:1), chlo-
roform-methanol (10:1), chloroform-methano (5:1), and metha-
nol, mp 178 °C (dec); 1H NMR (DMSO-d6) δ 10.45 (brs, 1H,
NH, exchangeable with D2O), 8.55 (s, 1H, imidazole), 7.20 (m,
6H, Ph + NH exchangeable with D2O), 6.36 (d, J ) 1.5 Hz,
1H, 1′-H), 5.49 (s, 1H, OH, exchangeable with D2O), 5.18 (t, J
) 2.9 Hz, 1H, OH, exchangeable with D2O), 5.08 (d, J ) 4.4
Hz, 1H, OH, exchangeable with D2O), 4.05 (m, 2H, 2′- and 3′-
H), 3.91 (m, 1H, 4′-H), 3.71 (m, 1H, 5′-H1), 3.58 (d, J ) 10.6
Hz, 1H, 5′-H2), 3.23 (m, 2H, CH2), 2.58 (m, 2H, CH2), 1.53 (m,
4H, CH2CH2). Anal. (C21H25N5O6•H2O) C, H, N.
4,5-Dih yd r o-8H-6-(N-d od ecyla m in o)-1-[(3′,5′-O-(1,1,3,3-
tetr a isop r op yld isiloxa n -1,3-d iyl))-â-D-r ibofu r a n osyl]im i-
d a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (27). Nucleoside 17 (208
mg, 0.43 mmol) dissolved in dry pyridine (5 mL), and 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (0.14 mL, 0.45 mmol)
was added. After stirring overnight at room temperature, a
TLC analysis showed the completion of reaction. The reaction
mixture was rotary evaporated to dryness, and the residue was
washed with methanol and dried under vacuum to obtain 27
as a solid (91% yield), Rf ) 0.16 [chloroform:methanol (30:1)],
1
mp 200-202 °C; H NMR (DMSO-d6): δ 10.39 (brs, 1H, NH,
exchangeable with D2O), 8.22 (s, 1H, imidazole), 7.06 (brs, 1H,
NH, exchangeable with D2O), 6.27 (s, 1H, 1′-H), 5.79 (d, J )
4.8 Hz, 1H, 2′-OH, exchangeable with D2O), 4.21 (d, J ) 13.6
Hz, 2H, 5′-H), 4.10 (m, 2H, 3′-H, 4′-H), 3.93 (d, J ) 12.8 Hz,
1H, 2′-H ), 3.21 (m, 2H, CH2 ), 1.47 (m, 2H, CH2 ), 1.22 (s,
10H, 5 CH2) 1.00 (m, 28H, 4 (CH3)2CH), 0.83 (t, J ) 6.6 Hz,
3H, CH3). Anal. (C35H63N5O7Si2) C, H, N.
4,5-Dih yd r o-8H-6-(N-ben zyl)a m in o-1-(â-D-r ibofu r a n o-
syl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (21). Yield 44%,
Rf ) 0.22 [chloroform:methanol:ammonium hydroxide (2:1:
0.3)], purified by silica gel flash chromatography, eluting
successively with chloroform-methanol (30:1), chloroform-
methanol (10:1), chloroform-methano (5:1), and methanol, mp
>250 °C; 1H NMR (DMSO-d6) δ 10.60 (brs, 1H, NH, exchange-
able with D2O), 8.58 (s, 1H, imidazole), 7.47 (brs, 1H, NH,
exchangeable with D2O), 7.33 (m, 5H, Phenyl), 6.36 (d, J )
2.2 Hz, 1H, 1′-H), 5.50 (s, 1H, OH, exchangeable with D2O),
5.19 (m, 1H, OH, exchangeable with D2O), 5.08 (m, 1H, OH,
exchangeable with D2O), 4.47 (m, 2H, CH2), 4.06 (m, 2H, 2′-
and 3′- H), 3.90 (m, 1H, 4′-H), 3.73 (dd, J 1 ) 21.1 Hz, J 2 ) 8.3
Hz, 1H, 5′- H1), 3.58 (dd, J 1 ) 18.1 Hz, J 2 ) 6.1 Hz, 1H, 5′-
H2). Anal. (C18H19N5O6•0.5H2O). C, H, N.
4,5-Dih yd r o-8H -6-(N-d od ecyla m in o)-1-[(2′-O-p h en ox-
yt h ioca r b on yl-3′,5′-O-(1,1,3,3-t et r a isop r op yld isiloxa n -
1,3-d iyl))-â-D-r ibofu r a n osyl]im id a zo[4,5-e][1,3]d ia zep in e-
4,8-d ion e (28). A mixture of 27 (177 mg, 0.25 mmol) and
DMAP (62 mg, 0.51 mmol) in dry acetonitrile (4 mL) was
stirred for 30 min in an ice-bath. Phenoxythiocarbonyl chloride
(51µL, 0.37 mmol) was then added dropwise to the reaction
mixture. The mixture was stirred at ambient temperature for
19 h. The reaction mixture was directly loaded on to a silica
gel column, and the column was eluted successively with
chloroform and chloroform: methanol (30:1). The appropriate
UV-absorbing fractions were pooled and evaporated to give 28
as a colorless syrup (208 mg, 99%), Rf ) 0.28 [chloroform:
methanol (30:1). The syrup was dried over KOH pellets in a
vacuum oven overnight. 1H NMR (CDCl3) δ 10.69 (brs, 1H,
NH, exchangeable with D2O), 8.35 (s, 1H, imidazole), 8.16 (brs,
1H, NH, exchangeable with D2O), 7.35 (m, 5H, phenoxy), 6.21
(d, J ) 4.4 Hz, 1H, 1′-H), 4.67 (m, 1H, 2′-H), 4.33 (m, 2H, 3′-
H, 4′-H), 4.22 (dd, J 1 ) 9.5 Hz, J 2 ) 1.5 Hz, 1H, 5′-H1), 4.08
(dd, J 1 ) 13.9 Hz, J 2 ) 2.2 Hz 1H, 5′-H2) 3.41 (m, 2H, CH2),
4,5-Dih yd r o-8H -6-(N-(2-p h en ylet h yl))a m in o-1-(â-D-r i-
bofu r a n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (22).
Yield 34%, Rf ) 0.24 [chloroform:methanol:ammonium hy-
droxide (2:1:0.3)], purified by silica gel flash chromatography,
eluting successively with chloroform-methanol (30:1), chlo-
roform-methanol (10:1), chloroform-methano (5:1), and metha-
1
nol, mp >250 °C; H NMR (DMSO- d6) δ 10.57 (brs, 1H, NH,
exchangeable with D2O), 8.56 (s, 1H, imidazole), 7.30 (m, 5H,
Phenyl), 7.11 (brs, 1H, NH, exchangeable with D2O), 6.36 (d,
J
) 2.6 Hz, 1H, 1′-H), 5.51 (d, J ) 4.0 Hz, 1H, OH,
exchangeable with D2O), 5.19 (m, 1H, OH, exchangeable with
D2O), 5.08 (d, J ) 3.3 Hz, 1H, OH, exchangeable with D2O),

4786 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Zhang et al.
1.59 (m, 2H, CH2), 1.24 (s, 18H, (CH2)9) 1.07 (m, 28H, 4 (CH3)2-
CH), 0.87 (t, J ) 6.6 Hz, 3H, CH3). Anal. (C42H67N5O8SSi2) C,
H, N.
(d, J ) 3.3 Hz, 2H, aromatic), 7.24 (m, 4H, aromatic), 6.57
(dd, J 1 ) 7.5 Hz, J 2 ) 5.7 Hz 1H, 1′-H), 5.61 (m, 1H, 3′-H),
4.68 (m, 2H, 5′-H), 4.64 (m, 1H, 4′-H), 4.31 (m, 4H, CH2), 2.98
(m, J 1 ) 14.5 Hz, J 2 ) 5.7 Hz J 3 ) 2.0 Hz, 1H, 2′-H1), 2.48 (m,
1H, 2′-H2), 2.44 (s, 3H, p-CH3Ph), 2.40 (s, 3H, p-CH3Ph), 1.72
(m, 4H, CH2), 1.43 (m, 4H, CH2), 0.97 (t, J ) 3.7 Hz, 3H, CH3),
0.93 (t, J ) 3.7 Hz, 3H, CH3). Anal. (C34H40N2O9): C, H, N.
4,5-Dih yd r o-8H-6-(N-d od ecyla m in o)-1-[(2′-d eoxy-3′,5′-
O-(1,1,3,3-tetr a isop r op yld isiloxa n -1,3-d iyl))-â-D-er yth r o-
p en t ofu r a n osyl]im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e
(29). A mixture of 28 (331 mg, 0.39 mmol) and AIBN (15 mg)
in dry toluene (30 mL) was purged with argon for 30 min.
Tributyltin hydride (0.30 mL, 1.08 mmol) was added quickly
to the reaction flask. It was refluxed for 24 h under argon
protection. The solvent was evaporated and the pure product
was obtained as a colorless liquid by silica gel column chro-
matography, eluting with a mixture of chloroform-methanol
(60:1). The product was dried over KOH pellets in a vacuum
oven overnight. Yield 209 mg (77%), Rf ) 0.26 [chloroform:
methanol (30:1)]; 1H NMR (CDCl3): δ 10.74 (brs, 1H, NH,
exchangeable with D2O), 8.31 (s, 1H, imidazole), 8.15 (brs, 1H,
NH, exchangeable with D2O), 6.71 (d, J ) 7.0 Hz, 1H, 1′-H),
4.52 (m, 1H, 4′-H), 4.21 (d, J ) 13.2 Hz, 1H, 5′-H1), 4.06 (dd,
J 1 ) 13.2 Hz, J 2 ) 2.6 Hz, 1H, 5′-H2), 3.85 (d, J ) 8.4 Hz, 1H,
Bu tyl 1-(2′-Deoxy-3′,5′-d i-O-p-tolu oyl-R-D-er yth r op en to-
fu r a n osyl)-4,5-im id a zoled ica r boxyla te (35). 2-Deoxy-3,5-
di-O- p-toluoyl-R-D-erythropentofuaranosyl chloride (32) (778
mg, 2.0 mmol) was dissolved in anhydrous CHCl3 (10 mL), and
the solution was slowly added via a syringe pump over a period
of 2 h (generating 34 in situ) to a solution of butyl 4,5-
imidazoledicarboxylate (31) (0.537 g, 2.0 mmol) and proton
sponge (0.429 g, 2 mmol) in CHCl3 (10 mL). The reaction
mixture was stirred overnight. The reaction mixture was
rotary evaporated, and the residue was purified by silica gel
flash chromatography, eluting with chloroform. A syrup (0.761
g) was obtained after evaporation of the chloroform eluent. It
was dried over P2O5 in vacuo overnight. Yield 62%, Rf ) 0.25
(chloroform-methanol, 60:1); 1H NMR (CDCl3): δ 7.99 (s, 1H,
imidazole), 7.95 (t, J ) 4.0 Hz, 2H, aromatic), 7.62 (d, J ) 4.0
Hz, 2H, aromatic), 7.24 (m, 4H, aromatic), 6.59 (d, J ) 6.6
Hz, 1′-H), 5.65 (d, J ) 6.2 Hz, 1H, 3′-H), 4.90 (m, 1H, 4′-H),
4.62 (m, 2H, 5′-H), 4.31 (m, 4H, CH2), 3.03 (m, 1H, 2′-H1), 2.59
(m, 1H, 2′-H2), 2.44 (s, 3H, p-CH3Ph), 2.38 (s, 3H, p-CH3Ph),
1.72 (m, 4H, CH2), 1.43 (m, 4H, CH2), 0.95 (m, 6H, CH3). Anal.
(C34H40N2O9) C, H, N.
3′-H), 3.44 (m, 2H, CH2), 2.78 (m, 1H, 2′-H1), 2.44 (dd, J 1
)
13.2 Hz, J 2 ) 7.0 Hz, 1H, 2′-H2), 1.60 (m, 2H, CH2), 1.23 (m,
18H, (CH2)9) 1.02 (m, 28H, 4 (CH3)2CH), 0.88 (t, J ) 6.6 Hz,
3H, CH3). Anal. (C35H63N5O6Si2•0.75CH3OH) C, H, N.
4,5-Dih yd r o-8H -6-(N-d od ecyla m in o)-1-[(2′-d eoxy-â-D-
er yth r op en tofu r a n osyl] im id a zo[4,5-e][1,3]d ia zep in e-4,8-
d ion e (30). Meth od A: Compound 29 (0.133 g, 0.19 mmol)
was dissolved in anhydrous THF (3 mL) and cooled to 0 °C.
TBAF in THF (1 M, 0.38 mL) was added dropwise to the
solution. After 40 min, the starting material disappeared and
a new spot corresponding to the deoxynucleoside appeared as
monitored by TLC. Methanol (2 mL) was added to the reaction
mixture and was stirred overnight. Pure 30 (72 mg, 77%) was
obtained by flash chromatography over silica gel by successive
elutions with a mixture of chloroform-methanol, 30:1, chlo-
roform-methanol, 10:1 and methanol, mp > 250 °C. 1H NMR
(DMSO-d6): δ 10.50 (brs, 1H, NH, exchangeable with D2O),
8.45 (s, 1H, imidazole), 7.14 (brs, 1H, NH, exchangeable with
D2O), 6.62 (t, J ) 5.9 Hz, 1H, 1′-H), 5.27 (s, 1H, OH,
exchangeable with D2O), 5.08 (s, 1H, OH, exchangeable with
D2O), 4.25 (m, 1H, 4′-H), 3.84 (m, 1H, 3′-H1), 3.60 (m, 2H, 5′-
H), 3.21 (m, 2H, CH2), 2.42 (m, 1H, 2′-H1), 2.19 (m, 1H, 2′-H2),
1.46 (m, 2H, CH2), 1.22 (m,18H, (CH2)9), 0.83 (t, J ) 6.4 Hz,
3H, CH3). Anal. (C23H37N5O5‚0.5H2O) C, H, N.
Meth od B: A solution of dodecylamine (2.144 g, 11.34 mmol)
and 3,5-dimethylpyrazole-1-carboxamidine nitrate (5) (2.350
g, 11.34 mmol) in methanol (30 mL) was refluxed for 0.5 h.
Solvent was evaporated under vacuum, and the residue was
dried in a desiccator over P2O5 in a vacuum overnight. Without
further purification, the residue was dissolved in anhydrous
methanol (13 mL) in an ice-bath, and a solution of sodium
methoxide (5.95 mL, 25wt % in methanol) was added. The
reaction mixture was stirred for 0.5 h at 0 °C, and was directly
filtered into a solution of 33 (2.284 g, 3.78 mmol) in anhydrous
methanol (60 mL). The mixture was stirred overnight, the sol-
vent was evaporated, and the residue was purified by silica gel
flash chromatography, eluting successively with chloroform:
methanol (30:1), chloroform:methanol (15:1), and methanol.
Evaporation of the appropriate UV-absorbing fractions yielded
30 (1.3 g, 74%). The spectral data of this compound were
superimposable with those of 30 obtained by Method A above.
4,5-Dih yd r o-8H -6-(N-d od ecyla m in o)-1-(2′-d eoxy-R-D-
er yth r op en tofu r a n osyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-
d ion e (36). A mixture of dodecylamine (1.419 g, 7.5 mmol)
and 3,5-dimethylpyrazole-1-carboxamidine nitrate36 (1.554 g,
7.5 mmol) was refluxed in methanol (20 mL) for 2 h. The
solvent was evaporated under vacuum, and the residue was
dried in a desiccator over P2O5 in vacuo. Without further
purification, it was dissolved in anhydrous methanol (10.0 mL),
and the solution was cooled to 0 °C. A solution of sodium
methoxide (25 wt % in methanol, 3.94 mL) was added, and
the reaction mixture was kept in an ice-bath for 0.5 h. The
reaction mixture was directly filtered into a solution of 35
(1.534 g, 2.5 mmol) in anhydrous methanol (40 mL). The
reaction mixture was stirred overnight, and the solvent was
rotary evaporated. The residue was purified by silica gel flash
chromatography, eluting successively with chloroform-metha-
nol (30:1), chloroform:methanol (15:1), and methanol. Yield
35%, Rf ) 0.21 [chloroform:methanol:ammonium hydroxide (4:
1
1:0.1)], mp > 250 °C; H NMR (DMSO-d6) δ 10.42 (brs, 1H,
NH, exchangeable with D2O), 8.19 (s, 1H, imidazole), 7.05 (brs,
1H, NH, exchangeable with D2O), 6.62 (d, J ) 6.2 Hz, 1H, 1′-
H), 5.10 (d, J ) 2.6 H, 1H, OH, exchangeable with D2O), 4.87
(t, J ) 5.7 Hz, 1H, 5′-OH, exchangeable with D2O), 4.25 (m,
2H, 3′,4′-H), 3.43 (t, J ) 4.9 Hz, 2H, 5′-H1,2), 3.21 (m, 2H, CH2),
2.64 (m, 1H, 2′-H1), 2.01 (d, J ) 14.3 Hz, 1H, 2′-H2), 1.47 (m,
2H, CH2), 1.23 (m,18H, (CH2)9), 0.84 (t, J ) 6.2 Hz, 3H, CH3).
Anal. (C23H37N5O5‚0.75H2O) C, H, N.
Meth yl 1-(p-Meth oxyben zyl)im id a zole-4,5-d ica r boxy-
la te (37). A mixture of methyl 4,5-imidazoledicarboxylate
(2)34,35 (921 mg, 5 mmol) and potassium carbonate (907 mg,
6.5 mmol) was stirred at room temperature in anhydrous CH3-
CN (30 mL), followed by the dropwise addition of p-methoxy-
benzyl chloride (0.84 mL, 6.0 mmol). The reaction mixture was
stirred for 24 h and was then heated to 50 °C for an additional
72 h. The solvent was evaporated, and the residue was purified
by silica gel flash chromatography, eluting with a mixture of
chloroform-methanol (30:1). Evaporation of the appropriate
UV-absorbing fractions yielded 37 as a solid (1.426 g, 94%),
Rf ) 0.26 (chloroform-methanol, 30:1), mp 66-67.5 °C; 1H
NMR (DMSO-d6) δ 8.12 (s, 1H, imidazole), 7.13 (d, J ) 8.8
Hz, 2H, phenyl), 6.89 (d, J ) 8.4 Hz, 2H, phenyl), 5.31 (s, 2H,
CH2Ph), 3.75 (s, 3H, CH3OCO), 3.74 (s, 3H, CH3OCO), 3.71
(s, 3H, p-CH3OPh). Anal. (C15H16N2O5) C, H, N.
Bu tyl 1-(2′-Deoxy-3′,5′-d i-O-p-tolu oyl-â-D-er yth r op en to-
fu r a n osyl)-4,5-im id a zoled ica boxyla te (33). To a mixture
of butyl 4,5-imidazoledicarboxylate (31)51,52(1.59 g, 5.93 mmol)
and NaH (268 mg, 6.70 mmol) in anhydrous acetonitrile (30
mL), was added portionwise pure 2- deoxy-3,5-di-O- p-toluoyl-
R-D-erythropentofuaranosyl chloride (32) (1.538 g, 3.96 mmol)
during a 2 h period under nitrogen atmosphere. The reaction
mixture was stirred for an additional 1 h. The mixture was
filtered in vacuo, and the filtrate was evaporated to dryness
to obtain a syrup. The product was purified by silica gel flash
chromatography, eluting with chloroform. Yield 2.34 g (95%),
4,5-Dih yd r o-8H-6-(N-d od ecyla m in o)im id a zo[4,5-e][1,3]-
d ia zep in e-4,8-d ion e (38). Compound 30 (232 mg) was dis-
solved in glacial acetic acid (45 mL) and stirred overnight at
1
Rf ) 0.25 (chloroform-methanol, 60:1); H NMR (CDCl3): δ
7.99 (s, 1H, imidazole), 7.94 (d, J ) 2.6 Hz, 2H, aromatic), 7.84

Inhibition of NTPase/ Helicase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4787
room temperature. The solid (114 mg) that precipitated from
the solution was filtered and dried over KOH pellets under
(30% w/w), collected by centrifugation, solubilized with TGT
buffer, and applied to a hydroxyapatite (HA-Ultrogel) column
preequilibrated with TGT buffer. The column was washed with
10 mL of TGT buffer, and then with 2 mL of TGT buffer
containing 1 M KCl, and again with 5 mL of TGT buffer. The
NTPase/helicase was eluted with 1 mL of TGT buffer contain-
ing 50 mM KH2PO4, precipitated with PEG and dissolved in
TGT buffer.
vacuum. Yield, 66%, mp >250 °C. 1H NMR (DMSO-d6):
δ
11.94 (brs, 1H, NH, exchangeable with D2O), 10.42 (brs, 1H,
NH, exchangeable with D2O), 8.00 (s, 1H, imidazole), 7.01 (brs,
1H, NH, exchangeable with D2O), 3.24 (m, 2H, CH2 group),
1.47 (m, 2H, CH2 group), 1.23 (m, 18 H, (CH2)9 chain), 0.84 (t,
J ) 6.6 Hz, 3H, CH3 group); HRMS (FAB) calcd for C18H29N5O2
(MH+) m/z 348.2399, found, 348.2401. Anal. (C18H29N5O2‚
0.5H2O) C, H, N.
The final preparations of the enzymes were homogeneous,
as demonstrated by Coomassie Blue staining of SDS/polyacry-
lamide gels (data not shown).
4,5-Dih yd r o-8H-6-(N-d od ecyla m in o)-1-(p-m eth oxyben -
zyl)im id a zo[4,5-e][1,3]d ia zep in e-4,8-d ion e (39). A mixture
of dodecylamine (0.851 g, 4.5 mmol) and 3,5-dimethylpyrazole-
1-carboxamidine nitrate (933 g, 4.5 mmol) was refluxed in
methanol (15 mL) for 2 h. The solvent was evaporated under
vacuum, and the residue was dried in a desiccator over P2O5
in a vacuum for two nights. Without purification, it was
dissolved in anhydrous methanol (10.0 mL) and cooled to 0°C,
and then a solution of sodium methoxide (25wt % in methanol,
2.27 mL) was added dropwise. The reaction mixture was kept
in an ice-bath for 0.5 h. The reaction mixture was directly
filtered into a solution of methyl 1-(p-methoxybenzyl)imida-
zole-4,5-dicarboxylate (37) (0.456 g) in anhydrous methanol
(20 mL), and the solution was continued to stir at ambient
temperature for 2 days. After evaporation of the solvent, the
residue was purified by flash silica gel chromatography, eluting
successively with a mixture of chloroform-methanol (30:1) and
chloroform-methanol (10:1). After evaporation of the ap-
propriate UV-absorbing fractions, compound 39 was collected
as a solid (0.667 g, 95%), Rf ) 0.30 (chloroform:methanol, 10:
1), Rf ) 0.79 (chloroform:methanol:ammonium hydroxide, 4:1:
ATP a se a n d Helica se Assa ys. The ATPase activity of the
NTPase/helicases was determined as described previously.25,26
Briefly, assays were performed with 2 pmol of WNV or 0.2
pmol of Suv3(∆1-159) NTPase/helicase. The enzymes were
incubated in a reaction mixture (final volume 25 µL) containing
20 mM Tris/HCl, pH 7.5, 2 mM MgCl2, 1 mM â-mercaptoet-
hanol, 10% glycerol, 0.01% Triton X-100, 0.1 mg/mL BSA, 25
nCi [γ-33P]ATP, and ATP adjusted to concentrations indicated
in the legends to tables. The reaction was conducted for 30
min at 30 °C and terminated by addition of 0.5 mL of activated
charcoal (2 mg/mL). Following centrifugation at 10 000 × g
for 10 min, 100 µL aliquots of the supernatant were removed
and subjected to scintillation counting.
Helicase activity was tested with 2 pmol of WNV or 0.2 pmol
of Suv3(∆1-159) NTPase/helicase. Unwinding of the partially
hybridized DNA or RNA substrate (4.7 pM of nucleotide base)
was monitored in a reaction mixture (final volume 25 mL)
containing 20 mM Tris/HCl, pH 7.5, 2 mM MgCl2, 1 mM
â-mercaptoethanol, 10% glycerol, 0.01% Triton X 100, 0.1 mg/
mL BSA and ATP at concentrations indicated in legends. The
reaction was conducted for 30 min at 30 °C and stopped by
addition of 5 mL of 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 containing
0.1% SDS [Borowski et al.45]. 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 was counted.
Alternatively, the films were scanned, and the radioactivity
associated with the released strand and with the non-unwound
substrate was quantified with GelImage software (Amersham
Pharmacia Biotech). The assays were carried out with the
same activity of the enzyme, determined with the DNA
substrate under conditions described above
1
0.1), mp 274-276 °C; H NMR (DMSO-d6): δ 10.39 (brs, 1H,
NH, exchangeable with D2O), 8.22 (s, 1H, imidazole), 7.18 (d,
J ) 8.4 Hz, 2H, phenyl), 7.01 (brs, 1H, NH, exchangeable with
D2O), 6.87 (d, J ) 8.8 Hz, 2H, phenyl), 5.53 (s, 2H, CH2Ph),
3.70 (s, 3H, p-CH3OPh), 3.17 (m, 2H, CH2), 1.44 (m, 2H, CH2),
1.22 (m,18H, (CH2)9), 0.83 (t, J ) 6.4 Hz, 3H, CH3). Anal.
(C26H37N5O3) C, H, N.
(B) Bioch em ica l Meth od s. Ma ter ia ls. DNA oligonucle-
otides were prepared by Dr. M. Schreiber (Bernhard Nocht
Institute). RNA oligonucleotides were purchased from HHMI
Biopolymer/Keck Foundation, Biotechnology Resource Labora-
tory, Yale University School of Medicine (New Haven, CT.,
USA). [γ-32P]ATP (220 Tbq/mmol) and [γ-33P]ATP (110 Tbq/
mmol) were from Hartman Analytic. All other chemicals were
obtained from Sigma.
Su bstr a tes for Helica se Rea ction s. The RNA substrate
for the helicase assays consisted of two partially hybridized
oligonucleotides with sequences as reported by Gallinari et al.13
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 5’-end labeled with
[γ-32P]ATP, using T4 polynucleotide kinase (MBI, Fermentas)
as recommended by the manufacturer. For the annealing
reaction, the labeled oligonucleotide was combined at a molar
ratio of 1:10 with the template strand (40-mer), denatured for
5 min at 96 °C, and slowly renatured as elsewhere de-
scribed.26,45 The duplex DNA was electrophoresed on a 15%
native TBE-polyacrylamide gel, visualized by autoradiography,
and extracted as described previously.45 The amount of DNA
or RNA duplex used as substrates was determined by the
ethidium bromide fluorescent quantitation method.56
Oth er Assa ys. Protein concentrations of preparations of the
NTPase/helicases were determined on SDS/polyacrylamide
gels as described previously.57 Kinetic parameters were de-
termined by nonlinear regression analysis using ENZFITTER
(BioSoft) and Sigma Plot (J andel Corp.)
Sou r ces a n d P u r ifica tion of Su v3(∆1-159) a n d WNV
NTP a se/Helica ses. NH2-ter m in a lly tr u n ca ted Su v3 NT-
P a se/h elica se (Su v3(∆1-159)) was expressed in E. coli as
follows: a 1881 bp fragment of the human Suv3 cDNA coding
for Suv3 protein truncated 159 aa from the amino terminus
was amplified by PCR using the following primers: forward
5′-CAT GCC ATG GCG CCA TTT TTC TTG AGA CAT GCC-
3′; reverse 5′-CTG GGA TCC GTC CGA ATC AGG TTC CTT
C-3′ (purchased from SIGMA), and the pKK plasmid as a
template (Dmochowska et al.53) The resulting fragment was
cloned into NcoI and BamHI sites of the pQE60 expression
vector (Qiagen). Sequences of both strands were verified, using
an ABI Prism 377DNA Sequencer. The protein was purified
according to the method established for the HCV enzyme.54,55,58
WNV NTP a se/h elica se was purified from the cell culture
medium of virus-infected Vero E6 cells as described previously
by Borowski et al.,45 with some modifications. Briefly, the
concentrated cell culture medium was mixed with 10 mL of
Reactive Red 120 agarose (Sigma) equilibrated with TGT
buffer for 4 h at 4 °C. The matrix was collected by sedimenta-
tion, 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 membrane to a
final volume of 2 mL, and subjected to gel exclusion chroma-
tography on a Superdex-200 column. Fractions expressing
ATPase and helicase activities were chromatographed again
on Reactive Red 120 agarose (5 mL) as described above. The
salt-eluted protein was precipitated with poly(ethylene glycol)
Ack n ow led gm en t . This paper is dedicated to
R.S.H.’s Ph.D. mentor, advisor, and friend Dr. Stewart
W. Schneller, Dean of Science and Mathematics, Auburn
University, AL, on the occasion of his 61st birthday. The
research was supported in part by a grant (#9RO1

4788 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
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