Chemical and biological effects of substitution of the 2-position of ring-expanded ('fat') nucleosides containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system: The role of electronic and steric factors on glycosidic bond stability and anti-HCV a

Article abstract of DOI:10.1016/j.bmc.2007.04.043

The attempted removal of the aralkyl group of 2-bromo-1-p-methoxybenzyl-6-octylimidazo[4,5-e][1,3]diazepine (ZP-33) with trifluoroacetic acid resulted in replacement of the bromo group with a carbonyl at position-2 in addition to the desired deprotection

Full text of DOI:10.1016/j.bmc.2007.04.043

Bioorganic & Medicinal Chemistry 15 (2007) 4933–4945
Chemical and biological effects of substitution of the 2-position
of ring-expanded (‘fat’) nucleosides containing the
imidazo[4,5-e][1,3]diazepine-4,8-dione ring system: The role
of electronic and steric factors on glycosidic bond stability
and anti-HCV activity
Peng Zhang,a Ning Zhang,a Victor E. Buckwoldb,ꢀ and Ramachandra S. Hosmanea,*
aLaboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
bAntimicrobial Acquisition and Coordinating Facility (AACF), Southern Research Institute,
431 Aviation Way, Frederick, MD 21701, USA
Received 16 January 2007; revised 17 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—The attempted removal of the aralkyl group of 2-bromo-1-p-methoxybenzyl-6-octylimidazo[4,5-e][1,3]diazepine (ZP-33)
with trifluoroacetic acid resulted in replacement of the bromo group with a carbonyl at position-2 in addition to the desired depro-
tection at the 1-position. 20-Deoxynucleosides of 2-bromo-substituted-imidazole-4,5-diesters (ZP-35 and ZP-103) were synthesized
by direct glycosylation of the corresponding heterocycles. The attempted ring-closure of the above nucleosides resulted in deglyco-
sylation to form the starting heterocycles. The 2-phenyl derivatives of the above nucleosides (ZP-45 and ZP-73) were successfully
prepared by Suzuki coupling with the appropriate phenylboronic acids, but the attempted ring-closure with guanidines to form the
desired 5,7-fused ring-expanded nucleosides (RENs) resulted once again in the formation of the corresponding heterocyclic aglycons
(ZP-64 and ZP-75). The first successful 2-substituted REN (ZP-110) was synthesized by replacing the 2-deoxyribose sugar moiety
with a ribosyl group and replacing the bromo group with a p-methoxyphenyl substituent at the 2-position. A number of imidazole
riboside diester precursors containing a substituted phenyl group at the 2-position were synthesized in order to prepare analogues of
ZP-110. The substituents on the phenyl ring included a variety of electron-donating or electron-withdrawing groups operating
through inductive and/or resonance effects. However, the final ring-closure of the diesters with guanidines in order to prepare RENs
was successful only in a limited number of cases, including the ones containing a p-fluorophenyl (ZP-121), a m-methoxyphenyl (ZP-
122), or an unsubstituted phenyl (NZ-53) at the 2-position. Deglycosylation and incomplete ring-closure of the intermediates were
the major problems encountered with most other cases. The stability of glycosidic bonds was found to be dependent on several fac-
tors including, but not limited to, the electron-donating inductive effect of the 2-phenyl substituents and the temperature of the reac-
tion medium. The three target RENs ZP-110, ZP-121, and ZP-122 were screened for in vitro anti-HCV activity, employing an HCV
RNA replicon assay. While ZP-121 was inactive, the other two compounds showed only weak anti-HCV activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
against NTPases/helicases of a family of Flaviviridae,
which includes, but not limited to, the hepatitis C virus
(HCV)1 and the West Nile virus (WNV)1,2 Both WNV3–5
and HCV6–11 are among the current list of deadly viruses
eliciting global response on human health. Helicases are
capable of unwinding duplex RNA and DNA structures
by disrupting the hydrogen bonds that keep the two
strands together.12,13 This unwinding activity is essential
for the virus replication, and requires the hydrolysis of
a molecule of nucleoside-50-triphosphate (NTP), which
is catalyzed by the viral NTPase.1,2 While most of
the active anti-Flaviviridae compounds were nucleoside
We have recently reported1,2 the in vitro inhibitory
activity of a number of ring-expanded nucleosides
Keywords: Synthesis; Ring-expanded nucleoside analogues; Imi-
dazo[4,5-e][1,3]diazepines; 2-Substituents; Effects on glycosidic bond
stability; Anti-HCV activity.
*
Corresponding author. Tel.: +1 410 455 2520; fax: +1 410 455
1148; e-mail: hosmane@umbc.edu
ꢀ
Current address: Veracity Biotechnology, LLC, 401 Rosemont
Avenue, Frederick, MD 21701, USA.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.043

4934
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
analogues bearing a ribosyl or 2-deoxyribosyl sugar moi-
ety at the 1-position of the heterocycle, there were also a
few active compounds that simply possessed an aralkyl
moiety in place of a sugar. This latter observation evoked
our interest in exploring the structure–activity relation-
ships (SAR) in various heterocyclic aglycons of the above
nucleosides with an aralkyl moiety substituting for the su-
gar.14 This initiative led us to the mapping of a general
structure I bearing a few key features required for anti-
HCV activity, including a bulky substituent such as a
bromo or phenyl group at position-2, an aralkyl substitu-
ent at position-1, and an aminoalkyl chain at position-
6.14 The observed enhancement of antiviral activity of
heterocyclic aglycons by bulky substituents at the 2-posi-
tion raised another issue about the significance of substi-
tuting this position in the original ring-expanded
nucleoside analogues (RENs), most of which contained
no substituents at position-2. So, the present work is an
attempt to introduce various hydrophobic and hydro-
philic substituents at position-2 of the title RENs as well
as to explore the effects of such substituents on the ob-
served anti-HCV activity.
O
O
O
H
N
N
N
TFA
N
N
NHR
O
NHR
Br
CH3CN
N
H
NH
NH
O
H2C
ZP-60; R = (CH2)7CH3
ZP-58; R = (CH2)11CH3
OCH3
ZP-33; R = (CH2)7CH3
ZP-54; R = (CH2)11CH3
O
N
N
NHR
Br
N
H
NH
O
Scheme 1.
In view of the above result, we resorted to an alternative
approach to synthesize the desired nucleoside analogues.
The strategy involved glycosylation of an appropriate
2-bromoimidazole precursor before condensation with
guanidine. Thus, 2-bromoimidazole-4,5-dicarboxylic
acid esters, ZP-32 and ZP-72,14 were glycosylated using
standard procedures2 to obtain the respective nucleoside
analogues ZP-35 and ZP-103, along with the corre-
sponding a anomers as minor products in miniscule
amounts (Scheme 3). However, all attempts at ring-clo-
sure of ZP-35 and ZP-103 with substituted guanidines
to prepare the target nucleoside analogues resulted in
intractable mixtures of products as suggested by their
O
4
8
5
3
N
NH
6
2
R'
NHR
N
7
N
1
O
R''
1
I
TLC and NMR spectral analyses. In order to simplify
and facilitate the product analysis of the complex reac-
tion mixture that also contained the anticipated second-
ary products arising from the removal of the toluoyl
protecting groups with the excess guanidine used, we
decided to carry out the sugar deprotection with a hin-
dered base before condensation with guanidine. Surpris-
ingly, the treatment of ZP-35 and ZP-103 with
t-butylamine in MeOH resulted in deglycosylation to
form the starting heterocycles ZP-32 and ZP-72, respec-
tively, even at room temperature. In view of the ex-
pected greater nucleophilicity of guanidines as
compared with t-butylamine, it is safe to conclude that
the original ring-closure reactions of ZP-35 and ZP-
103 with excess guanidines must have also resulted in
the deglycosylated products.
2. Results and discussion
The compound with the most promising anti-HCV
activity in the heterocyclic series with the structural skel-
eton I was the 2-bromo-1-p-methoxybenzyl-6-octyl
derivative (ZP-33). Therefore, the most convenient and
facile method to prepare the corresponding nucleoside
analogue appeared to be the deprotection of the aralkyl
group of ZP-33 with a reagent such as trifluoroacetic
acid or by catalytic hydrogenation, followed by glyco-
sylation. However, while catalytic hydrogenation of
ZP-33 did not yield the desired compound, the treat-
ment with trifluoroacetic acid in acetonitrile, on the
other hand, resulted in the formation of ZP-60 (see
Scheme 1), containing a carbonyl group in place of the
bromo substituent at the 2-position, as evidenced by
analytical and spectroscopic data. An analogous result
was obtained with the N6-dodecyl analogue ZP-54,
which formed the corresponding carbonyl compound
ZP-58.
The observed deglycosylation was tentatively attributed
to the electron-withdrawing inductive effect of the bro-
mo group. As our SAR model I described earlier calls
for a hydrophobic group at position-2, we decided to re-
place the bromo group with a phenyl substituent, which
should be less electron-withdrawing, but more bulky
and hydrophobic than the bromo group. To that end,
ZP-35 was converted into two 2-phenyl (ZP-45) and
2-p-methoxyphenyl (ZP-73) analogues via Suzuki cou-
pling15
Suspecting the presence of adventitious water in the
reaction medium, we repeated the reaction with an
extensively dried reagent as well as the solvent, but ob-
tained the same result as before, suggesting that the
reaction mechanism for the formation of ZP-60 and
ZP-58 from their respective precursors ZP-33 and ZP-
54 does not involve water and that the carbonyl oxygen
in the products must be coming from the trifluoroacetic
acid employed in the reaction. A tentative mechanism
for the product formation is outlined in Scheme 2.
with the appropriate phenylboronic acids
(Scheme 4). Compound ZP-45 indeed underwent
smooth sugar deprotection with t-butylamine in metha-
nol to form the expected nucleoside ZP-52. Unfortu-
nately, however, the attempted condensations of a
substituted guanidine with either the sugar unprotected
(ZP-52) or the sugar-protected (ZP-45 and ZP-73)

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4935
O
CF3COO
O
O
H
N
N
N
N
N
H
NHR
Br
NHR
Br
NH
N
NH
O
H2C
H2C
OCH3
OCH3
O
O
O
H
H
N
N
N
N
NHR
CF3COO
NHR
Br
N
NH
N
NH
CF3COO
O
H2C
OCH3
OCH3
O
O
O
H
N
H
N
N
N
NHR
CF3COO
CF3COO
CF3COO
NHR
H
N
H
NH
N
NH
O
O
O
O
O
H
H
N
N
- (CF3CO)2O
N
N
F3C
O
NHR
O
NHR
N
H
O
NH
N
H
NH
F3C
O
O
Scheme 2.
nucleosides only produced the corresponding deglycosy-
lated products ZP-64, ZP-75, and ZP-109. Apparently,
the glycosidic bonds in the expected nucleoside products
are still too weak to withstand the experimental reaction
conditions employed for ring-closures of nucleoside pre-
cursors ZP-45 and ZP-73 with guanidine.
completely characterized by 1H and 13C NMR analyses,
coupled with mass spectral and microanalytical data.
Encouraged by the above result, we set out to synthesize
several other ribonucleosides containing the title 5:7-
fused heterocyclic system with a variety of substituted
phenyl and other substituents at the 2-position. The nec-
essary diester nucleoside precursors for guanidine ring-
closures were successfully prepared, as before, from
ZP-115 or ZP-77, using Suzuki coupling.15 These
precursors include (Scheme 6) 2-phenyl (ZP-78), 2-p-
F-phenyl (ZP-118), 2-m-methoxyphenyl (ZP-119),
As it is well known that glycosidic bonds in 20-deoxynu-
cleosides/-tides (DNA) are nearly two orders of magni-
tude less stable than their ribose counterparts
(RNA),16 our best hope for preparing the target
2-substituted 5:7-fused nucleoside analogues was to re-
place their 20-deoxyribose moiety with a ribosyl sugar
at position-1. Besides, since the target Flaviviridae are
RNA viruses, synthesis of ribose nucleoside analogues
as potential inhibitors would only be logical, notwith-
standing the potent in vitro anti-HCV/WNV activities
of a few 20-deoxyribose analogues of RENs.1,2 To this
end, we synthesized the required diester precursors in
the ribose series, namely ZP-115 and ZP-77, by stan-
2-p-phenylphenyl
(ZP-123),
2-p-tert-butylphenyl
(ZP-124), 2-m-nitrophenyl (ZP-129), 2-p-trifluorometh-
ylphenyl (ZP-132 and ZP-175), and 2-thienyl (ZP-128)
derivatives. However, the attempted ring-closures of
these precursors with guanidine or substituted guani-
dines were successful only in a limited number of cases,
in addition to the mentioned p-methoxyphenyl (ZP-110),
which included p-fluorophenyl (ZP-121), m-methoxy-
phenyl (ZP-122), and unsubstituted phenyl (NZ-53)
derivatives. While the reaction of ZP-108 with guani-
dine yielded the cleanest product (ZP-110), all other
reactions were accompanied with one or more side-
products, which often included the ring-closed or ring-
open heterocyclic aglycons and/or ring-open nucleosides
as outlined in Scheme 7 for the reactions of ZP-136 and
ZP-119 with N-methylguanidine and guanidine, respec-
tively. While individual components in the reaction mix-
ture of products formed from the reaction of ZP-136
with N-methylguanidine were identified mainly through
dard Vorbruggen ribosylation17–19 of ZP-32 and ZP-
¨
72, respectively (Scheme 5). Whereas an attempted
condensation reaction of ZP-115 with N-octylguanidine
resulted in the formation of the deglycosylated,
ring-closed heterocycle (ZP-16), the conversion of the
2-bromo group of ZP-77 into the corresponding p-meth-
oxyphenyl derivative (ZP-108) via Suzuki coupling15
proceeded with no problem. The ring-closure of ZP-
108 with guanidine provided the desired ZP-110, repre-
senting our first successful synthesis of a 2-substituted
ring-expanded nucleoside. The compound was

4936
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
O
TolO
H
Cl
OTol
O
O
N
Anhy. CH3CN
OR
OR
Br
N
H
N2, rt, 2 h, portionwise
addition
ZP-32; R = CH3
ZP-72; R = (CH2)3CH3
N
N
t
-butylamine/
MeOH
O
OR
N
N
Br
O
TolO
O
O
OR
O
TolO
OR
N
N
+
Br
OTol
OR
OTol
β-anomer
Major
O
α-anomer
Minor
ZP-35; R = CH3
ZP-103; R = (CH2)3CH3
t
-butylamine/
MeOH
NH
NHR
H2N
O
O
OR
N
N
NH
N
N
N
Br
O
NHR
Br
O
OR
HO
HO
O
O
OH
OH
Scheme 3.
high resolution mass spectral data, those produced from
the reaction of ZP-119 with guanidine were isolated and
the products were fully characterized by NMR as well as
elemental microanalytical and/or high resolution mass
spectral data. Our experience with these reactions sug-
gests that several factors, including the type of substitu-
ents attached to the phenyl ring, the temperature of the
reaction medium, the molar ratio of reactants used, the
reaction time, as well as the reaction work-up procedure,
affect the type, number, and yield of the products
formed. Especially important are the contributions of
electron-donating substituents at the p-position of the
phenyl ring, through resonance and inductive effects,
which seem to enhance the stability of the glycosidic
bond toward hydrolytic and nucleophilic cleavage. An-
other important factor is the temperature of the reaction
medium, the higher temperature leading to deglycosyla-
tion, while the ice-cold temperature yields un-ring-closed
intermediates. The excess base facilitates the final ring-
closure, but it also adversely affects the stability of the
glycosidic bond, especially if the base is also a good
nucleophile.

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4937
O
O
O
R
N
N
O
N
N
Br
O
O
TolO
O
O
O
TolO
O
Suzuki Coupling
OTol
OTol
R-PhB(OH)2/
(Ph3P)4Pd/
Na2CO3
ZP-45; R = H
ZP-73; R =
ZP-35
p
-OCH3
NH
NaOMe/
MeOH
t
-butylamine/
MeOH
H2N
N
H
NH
O
H2N
N
O
H
R
O
N
N
N
N
NHR'
R
NaOMe/
MeOH
O
HO
O
N
H
O
NH
O
OH
ZP-52; R = H
ZP-64; R = H; R' = CH2CH3
ZP-75; R = -OCH3; R' = CH2CH3
p
Scheme 4.
Finally, the three ring-expanded nucleosides ZP-110,
ZP-121, and ZP-122 were screened for anti-HCV activ-
ity in a primary HCV RNA replicon assay20,21 through
contractual arrangements with the National Institute of
Allergy and Infectious Diseases (NIAID) at its Antimi-
crobial Acquisition and Coordinating Facility (AACF)
in Birmingham, Alabama. The results are collected in
Table 1. Out of the three compounds tested, the one
with p-F substituent is completely inactive, while the
other two with a methoxy substituent in the m- and
p-position, are only weakly active, with the meta
preferred to para position.
tures leading to deglycosylation, while ice-cold tempera-
tures resulted in ring-open intermediates. Two of the
three target compounds screened showed only marginal
activity against HCV in vitro. The structure–activity
relationships (SAR) on the tested compounds as well
as on the whole class of 2-substituted RENs with the ti-
tle ring system still remain inconclusive for the following
reasons: (a) the number of compounds tested are far too
limited to draw any meaningful inference, and (b) none
of the three tested compounds possesses the long alkyl
chain at position-6, which may still be required for activ-
ity as has been the case with all of the RENs that have so
far exhibited potent activity against Flaviviridae. None-
theless, the outlined research is a good exercise for
exploring the problems involved in substitution of the
2-position of nucleosides bearing the target 5:7-fused
ring system, while also providing directions for future
rational designs of 2-substituents and sugar moieties
that do not adversely affect the glycosidic bond stability
of target RENs.
3. Conclusion
Substitution of position-2 of ring-expanded nucleosides
containing the title imidazo[4,5-e][1,3]diazepine-4,8-
dione ring system turned out to be challenging. The
attachment of the bromo or the phenyl group at this po-
sition leads to an unstable and fragile glycosidic bond,
often producing the corresponding aglycons either as
sole or by-products. Deglycosylation is especially more
pronounced in 20-deoxyribonucleosides as compared to
their ribose counterparts. Exchange of the bromo group
with phenyl considerably enhances the glycosidic bond
stability. The stability is further improved with an elec-
tron-donating group on the phenyl ring, in particular,
when located at a position capable of imparting reso-
nance effect (e.g., para vs meta). Another key factor that
governs the outcome of the final ring-closure reaction is
the temperature of the medium, with elevated tempera-
4. Experimental
4.1. General
1H and 13C NMR spectra were recorded on a General
Electric QE-300 NMR spectrometer or on an Oxford
AS400 NMR spectrometer operating at 300/400 MHz
for 1H and 75/100 MHz for 13C. The Chemical shift data
are reported with reference to Me4Si (internal standard)
1
for H and 13C NMR spectra. The data are reported in

4938
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
O
O
OAc
O
O
BzO
OR1
OR1
N
N
CH3CN
Br
OR1
OR1
N
OBzOBz
O
BzO
Br
O
H
N
N
H
(HMDS)
(TMSCl)
(TFMSA)
Si
Si
OBz
BzO
Cl
Si
ZP-32; R1 = CH3
ZP-72; R1 = (CH2)3CH3
ZP-115; R1 = CH3
ZP-77; R1 = (CH2)3CH3
O
F3C
S
OH
O
NH
H2N NHC8H17
H3CO
B(OH)2
NaOMe/MeOH
R1=CH3
R1=(CH2)3CH3
Pd(PPh3)4, Na2CO3
O
O
OButyl
OButyl
N
N
N
N
H3CO
NHC8H17
Br
N
NH
O
O
O
Na
BzO
m/z 413, 415
OBz
OBz
NH
ZP-108
H2N NH2 1/2 H2CO3
NaOMe/MeOH
O
N
N
N
H3CO
NH2
NH
O
O
HO
OH
OH
ZP-110
Scheme 5.
the following format: Chemical shift, multiplicity (s, sin-
glet; d, doublet; dt, double triplet; dd, double doublet; t,
triplet; q, quartet; m, multiplet; br, broad; coupling con-
stants, integration, and assignment). Elemental Microa-
nalyses were performed by Atlantic Microlab, Inc.,
Norcross, Georgia. The mass spectra were recorded at
the Mass Spectral Facility, Department of Biochemistry,
Michigan State University, or Mass Spectrometry Anal-
ysis Laboratory, Department of Chemistry and Bio-
chemistry, University of Maryland, College Park or
Mass Spectrometry Facility, Department of Chemistry
and Biochemistry, University of Maryland, Baltimore
County. 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
over calcium oxide and then distilled under reduced
pressure from calcium hydride, and was subsequently
˚
stored over molecular sieves (type 3 A); acetonitrile
was distilled from calcium hydride and was stored over
˚
molecular sieves (type 3 A). Starting materials were pur-
chased from Aldrich Chemical Co., Acros or Lancaster.
All solvents are of reagent grade and were purchased
from VWR Scientific or Fisher Scientific. All yields re-
ported are for dry compounds that require no further
purification for use in other reactions.
4.2. 6-N-Dodecylamino-1,3,4,5-tetrahydroimidazo[4,5-e]-
[1,3]diazepine-2,4,8-trione (ZP-58)
To a suspension of ZP-54 (0.275 g, 0.5 mmol) in 30 mL
of dry acetonitrile, anhyd TFA (3 mL) was added
slowly. The clear solution was stirred at 50 ꢁC for
72 h. The reaction mixture was filtered to give white so-
lid. Yield: 0.15 g (70%). Rf = 0.75 (chloroform/metha-
nol/ammonium hydroxide, 2:1:0.3), mp >220 ꢁC, 1H
NMR (DMSO-d6): d 11.2, 6.82 (2br, NH, exchangeable
˚
molecular sieves (type 3 A); methylene chloride was dis-
tilled from calcium hydride and was stored over molec-
˚
ular sieves (type 3 A); dimethylformamide was dried

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4939
O
O
O
NH
NHR'
O
N
R
N
N
salt
R
OR1
OR1
N
N
R-Ph-B(OH)2
OR1
OR1
N
N
NHR'
H2N
Br
NH
Pd (PPh3)4,
Na2CO3
NaOMe/MeOH
O
O
O
O
O
BzO
BzO
OBz
HO
OBz
OH
OBz
OBz
HO
ZP-115; R1 = CH3
ZP-77; R1 = (CH2)3CH3
ZP-78; R = H, R1= (CH2)3CH3
ZP-108; R = p-OCH3, R1= (CH2)3CH3
ZP-118; R = p-F, R1= (CH2)3CH3
ZP-119; R = m-OCH3, R1= (CH2)3CH3
ZP-123; R = p-Ph, R1= CH3
ZP-110; R = p-OCH3; R' = H
ZP-121; R = p-F; R' = H
ZP-122; R = m-OCH3; R' = H
NZ-53; R = H; R' = CH3
ZP-124; R = p-tert-butyl, R1= CH3
ZP-128; 2-Thienyl, R1= CH3
ZP-129; R = m-NO2, R1= CH3
ZP-132; R = p-CF3, R1=CH3
ZP-136; R = p-CH3, R1= CH3
ZP-175; R = p-CF3, R1= (CH2)3CH3
Scheme 6.
with D2O), 5.08 (br, 2H, D2O exchangeable), 3.16 (m,
2H, NCH2), 1.19, 1.42 (2m, 20H, C10H20), 0.83 (t, 3H,
CH3). 13C NMR (75 MHz, DMSO-d6): d 202.6, 183.1,
166.4, 158, 123.1, 99.7, 81.3, 31.1, 29, 28.8, 28.5, 22,
13.7. MS (FAB) m/z 364.2; HRMS (FAB) calcd for
C18H29N5O3 (MH+) m/z 364.2349, found, 364.2364.
were combined and evaporated to give 1.39 g white
foam. (91%). 1H NMR (DMSO-d6): d 7.1–7.9 (4m,
8H, 2C6H4), 6.42 (dd, J1 = 6.2 Hz, J2 = 2.6 Hz, 1H, 10-
H), 5.45 (m, 1H, 30-H), 4.6–4.7 (m, 1H, 40-H), 4.35–4.4
(m, 2H, 50,500-H), 3.71, 3.73 (2s, 6H, OCH3), 2.6–2.8
(2m, 2H, 20,200-H), 2.28, 2.32 ( 2s, 6H, 2 PhMe). Anal.
(C28H27BrN2O9Æ2 H2O) C, H, N.
4.3. 6-N-Octylamino-1,3,4,5-tetrahydroimidazo[4,5-e]-
[1,3]diazepine-2,4,8-trione (ZP-60)
4.5. 2-Bromo-1-(2-deoxy-3,5-di-O-p-toluoyl-b/a-D-ery-
thropentofuranosyl)-4,5- imidazoledicarboxylic Acid
Dibutyl Ester (ZP-103)
ZP-33 (0.17 g, 0.35 mmol) was suspended in 20 mL of
dry CH3CN. TFA (2 mL) was added to give a clear solu-
tion. The solution was heated at 45 ꢁC for 48 h. Some
white solid precipitate out, which was collected by filtra-
tion and dried. Yield: 15 mg (13%), mp 215 ꢁC (dec),
Rf = 0.47 (chloroform/methanol/ammonium hydroxide,
1-a-Chloro-2-Deoxy-D-ribofuranosyl-bis(p-toluate) (ZP-3)
(505 mg, 1.25 mmol) was slowly added portion-wise into
a solution of ZP-72 (434 mg, 1.25 mmol) and sodium
hydride (50 mg, 1.25 mmol) in anhyd acetonitrile
(20 mL) in a period of 2 h. The reaction mixture was
stirred overnight. It was evaporated and purified by
flash chromatography eluting with chloroform. Appro-
priate fractions were collected and evaporated to give
two products, one as a clear syrup (major product, b
anomer) and the other as a purple syrup (minor product,
a anomer). The physicochemical data of the two prod-
ucts are as follows: The b anomer: Yield: 51%, 1H
NMR (CDCl3) d 7.93 (d, J = 7.6 Hz, 4H, Ar–H), 7.28,
7.20 (2d, J = 8.0 Hz, 8.4, 4H, Ar–H), 6.38 (dd,
J1 = 8.44 Hz, J2 = 6.04 Hz, 1H, 10-H), 5.57, (m, 1H, 30-
H), 4.70 (m, 1H, 40-H), 4.53 (m, 2H, 50,500-H), 4.3 (m,
4H, OCH2 of butyl), 3.08 (m, 1H, 20-H), 2.63 (ddd,
J1 = 14.2 Hz, J2 = 5.96 Hz, J3 = 2.28 Hz, 1H, 200-H),
2.42, 2.39 (2s, 6H, 2PhCH3), 1.69, 1.35 (2m, 8H,
(CH2)2 of butyl), 0.94, 0.88 (2 t, J = 7.3 Hz, 6H,
2CH3). NOE: no effect between 10-H and 40-H. 13C
NMR (75 MHz, CDCl3) d 166.07, 165.58, 160.88,
160.56, 144.37, 143.88, 129.66, 129.64, 129.15, 129.0,
126.59, 126.19, 87.46, 82.21, 77.35, 77.03, 76.71, 73.79,
66.66, 65.15, 63.49, 38.07, 30.56, 30.03, 21.54, 18.91,
13.59, 13.51. Anal. (C34H39BrN2O9 ꢀ 1:5H2O) C, H, N.
1
2:1:0.3). H NMR (DMSO-d6): d 11.25 (br, 1H, NH,
D2O exchangeable), 6.92 (br, 1 H, NH, D2O exchange-
able), 4.80 (br, 2H, D2O exchangeable), 3.20 (m, 2H,
NCH2), 1.47, 1.26 (2m, 12H, (CH2)6), 0.85 (t, 3H,
CH3). 13C NMR (75 MHz, DMSO-d6): d 223.8, 184.7,
163.7, 142.7, 133.0, 87.2, 38.2, 28.3, 26.0, 21.7, 13.5.
MS (FAB) calcd for C14H21N5O3 (MH+) m/z 308.2,
found, 308.1.
4.4. 2-Bromo-1-(2-deoxy-3,5-di-O-p-toluoyl-b-D-erythro
pentofuranosyl)imidazole-4,5-dicarboxylic Acid Dimethyl
Ester (ZP-35)
To a mixture of ZP-3214 (0.65 g, 2.48 mmol) and proton
sponge (0.53 g, 2.48 mmol), 20 mL dry acetonitrile was
added. Then ZP-3 (1-a-chloro-2-deoxy-D-ribofurano-
syl-3,5-bis(p-toluate), chlorosugar)2 (1.00 g, 2.48 mmol)
was added portion-wise over a period of 2 h under nitro-
gen. The reaction mixture was kept stirring for another
3 h after addition. After filtration, the reaction mixture
was evaporated to give a syrup. The syrup was dissolved
in 10 mL chloroform and adsorbed onto flash silica gel.
Flash chromatography was performed using chloro-
form/ ethanol (100:1). The appropriate fractions
(Rf = 0.14 in the same solvent system used for elution)
1
The a anomer: Yield (trace), H NMR (CDCl3) d 7.93,
7.82, 7.25, 7.21 (4d, J = 7.8-8.0 Hz, 8H, Ar–H), 6.34 (t,
J = 5.2 Hz, 1H, 10-H), 5.56, (m, 1H, 30-H), 4.83 (q,

4940
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
O
N
N
NHMe
H2N
O
N
O
HO
O
ZP-181P1
m/z 448 (MH+)
OH
HO
O
N
O
O
N
NHMe
NH
HCl
OR1
OR1
N
N
H2N
O
CH3
ZP-181P2
N
H2N
N
m/z 316 (MH+)
H
H
O
O
BzO
O
NaOMe/MeOH
N
N
NHMe
NH
OBz
BzO
N
O
HO
O
ZP-136; R1 = CH3
ZP-181P3
m/z 416 (MH+)
OH
O
HO
N
N
NHMe
N
H
NH
ZP-181P4
m/z 284 (MH+)
O
O
O
MeO
N
N
N
NH2
NH
O
HO
OH
O
ZP-122
Anal. C, H, N)
HO
NH
H2N NH2
MeO
O
O
(
OR1
OR1
N
N
MeO
Carbonate Salt
N
N
O
NH2
NaOMe, Dry MeOH
N
NH
BzO
H
OBz
O
O
OBz
ZP-122fc
(HRMS)
ZP-119; R1= (CH2)3CH3
MeO
HO
OMe
OMe
N
N
O
O
OH
ZP-122P1
HO
(
Anal. C, H, N)
Scheme 7.
Table 1. In vitro Anti-HCV Activity in a primary (single dose) HCV RNA replicon assay of three ring-expanded nucleosides containing the target
imidazo[4,5-e][1,3]diazepine-4,8-dione ring system
Compound ID
Substituent at
the 2-position
Antiviral Activity % Inhibitiona
Cytotoxicity % Cell Controlb
ZP-110
ZP-121
p-OMe-Ph
p-F-Ph
9.5
0
109.8
108.6
97.2
ZP-122
Alpha-IFN (Ref.)
m-OMe-Ph
NA
31.8
96.1
119.6
a Antiviral activity is assessed with an HCV RNA replicon containing stable luciferase (LUC) reporter. The extent of LUC expression, which is
directly proportional to HCV RNA levels, is used as an indirect measure of HCV replication.20
b Compound cytotoxicity is assessed as the percent viable cells relative to the untreated cell controls.
J = 4.0 Hz, 1H, 40-H), 4.55 (m, 2H, 50,500-H), 4.29 (m,
4H, OCH2 of Butyl), 3.13, 2.87 (2m, 2H, 20,200-H),
2.42, 2.39 (2s, 6H, 2PhCH3), 1.66, 1.35 (2m, 8H,
(CH2)2 of Butyl), 0.92, 0.89 (2 t, J = 7.2 Hz, 6H,
2CH3). NOE: effect between 10-H and 40-H. 13C NMR
(100 MHz, CDCl3) d 166.31, 160.99, 160.73, 144.34,

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4941
132.8, 129.9, 129.7, 129.32, 129.16, 127.2, 126.2, 118.11,
90.0, 83,81, 77.55, 77.43, 77.23, 76.92, 73.96, 67.1, 65.31,
64.1, 39.77, 30.73, 30.26, 21.73, 19.05, 13.75. Anal.
(C34H39BrN2O9) C, H, N.
The appropriate fractions were combined and evapo-
rated to obtain the product. The latter was recrystallized
from an appropriate solvent if necessary. The necessary
ring-closure precursor, the spectral and analytical data
of the products, along with solvent of recrystallization
and/or solvent of elution for chromatography, are col-
lected as below.
4.6. 1-(2-Deoxy-3,5-di-O-p-toluoyl-b-D-erythro-pentofur-
anosyl)-2-phenylimidazo-4,5-dicarboxylic Acid Dimethyl
Ester (ZP-45)
4.8.1. 6-N-Ethylamino-7,8-dihydro-2-phenyl-1H-imidazo-
[4,5-e][1,3]diazepine-4,8-dione (ZP-64). Ring-closure pre-
cursor: ZP-45 condensed with ethylguanidine sulfate.
Yield: 12.9%, purified by silica gel flash chromatogra-
phy, eluting with chloroform/methanol (60:1, 30:1;
10:1; 5:1; 1:1) and methanol, mp >220 ꢁC. Rf = 0.75
(chloroform/methanol/ammonium hydroxide, 2:1:0.3).
1H NMR (DMSO-d6) d 13.78, 10.49, 7.04 (3br, 3H,
NH, exchangeable with D2O), 8.18, 7.48 (2m, 5H, Ar–
H), 3.34 (m, 2H, NHCH2), 1.12 (t, J = 6.6 Hz, 3H,
CH3). Anal. (C14H13N5O2) C, H, N.
A 25 mL round-bottomed flask was charged with 1.56 g
of ZP-35 (1.7 mmol), 0.22 g (1.8 mmol) of phenylboron-
ic acid, 0.57 g of sodium carbonate (5.4 mmol dissolved
in 2.7 mL water), 5.2 mL benzene, and 0.52 mL ethanol.
The flask was covered with aluminum foil and a trace
amount of tetrakis(triphenylphosphino) palladium was
added. The reaction was heated to reflux for 60 h and
the solvent was evaporated. The residue was dissolved
in 100 mL of dichloromethane and washed by water
(3 · 50 mL). The extract was dried over anhyd Na2SO4,
filtered, and evaporated to give a brown foam, which
was purified by column chromatography, eluting with
chloroform/methanol (60:1). The appropriate fraction
(Rf = 0.18 (chloroform/methanol, 60:1)) was pooled
and evaporated to give some white foam, which was
dried in vacuo overnight. Yield: 0.75 g (71.4%). 1H
NMR (DMSO-d6) d 7.75, 7.48, 7.25 (3m, 15H, Ar–H),
6.66 (t, J = 6.6 Hz, 1H, 10-H), 5.3 (m, 1H, 30-H), 4.56–
4.28 (2m, 3H, 40,50,500-H), 3.75, 3.7 (2s, 6H, OCH3),
2.68-3.08 (m, 2H, 20,200-H), 2.32 (s, 6H, 2 PhCH3). Anal.
(C34H32N2O9) C, H, N.
4.8.2. 6-N-Ethylamino-7,8-dihydro-2-p-methoxyphenyl-
1H-imidazo[4,5-e][1,3]diazepine-4,8-dione (ZP-75). Ring-
closure precursor: ZP-73, condensed with ethylguani-
dine sulfate, isolated by silica gel flash chromatography,
eluting with a gradient of chloroform/methanol (30:1 to
1
10:1). Yield: 4%, H NMR (DMSO-d6) d 13.59, 10.47,
4.58 (3br, 3H, NH, exchangeable with D2O), 8.16,
7.04, (2m, 4H, Ar–H), 3.82 (s, 3H, OCH3), 3.15 (q,
J = 7.3 Hz, 2H, NHCH2), 1.18 (t, 3H, CH3).
4.8.3. 7,8-Dihydro-2-p-methoxyphenyl-6-N-methylamino-
1H-imidazo[4,5-e][1,3]diazepine-4,8-dione (ZP-109). Ring-
closure precursor: ZP-73, condensed with methylguani-
dine hydrochloride. Yield: 5%, purified by silica gel flash
chromatography, eluting with mixtures of chloroform/
methanol (60:1, 30:1; 10:1; 5:1; 1:1) and methanol.
Rf = 0.52 (chloroform/methanol/ammonium hydroxide,
4.7. 1-(2-Deoxy-3,5-di-O-p-toluoyl-b-D-erythro-pentofur-
anosyl)-2-(p-methoxylphenyl)imidazole-4,5-dicarboxylic
Acid Dimethyl Ester (ZP-73)
Reaction conditions and work-up are the same as in ZP-
45 using p-methoxy-phenylboronic acid instead. Yield:
64%, Rf = 0.29 (chloroform/methanol, 60:1), purified
by silica gel flash chromatography, eluting with chloro-
form/methanol (60:1). 1H NMR (DMSO-d6) d 7.81,
7.62, 7.51, 7,29, 7.04 (5m, 12H, Ar–H), 6.64 (t,
J = 3.7 Hz, 1H, 10-H), 5.39 (m, 1H, 30-H), 4.57, 4.41
(2m, 3H, 40,50,500-H), 3.81, 3.80, 3.77 (3s, 9H, OCH3),
3.48, 3.41 (2m, 2H, 20,200-H), 2.39 (s, 6H, phenyl-CH3).
Anal. (C32H32N2O10Æ0.5 H2O) C, H, N.
1
2:1:0.3). Yield: H NMR (DMSO-d6) d 13.58, 10.62,
6.95 (3br, 3H, NH, exchangeable with D2O), 8.15, 7.06
(2m, 5H, Ar–H), 2.81 (s, 3H, OCH3), 2.80 (s, 3H,
NHCH3). Anal. (C14H13N5O3Æ0.75 H2O) C, H, N.
4.8.4. 1-(2-Deoxy-b-D-erythropentofuranosyl)-2-phenyl-
imidazole-4,5-dicarboxylic Acid Dimethyl Ester (ZP-
52). t-Butylamine (1.5 mL) was added to a solution of
ZP-45 (0.22 g, 0.36 mmol) in 10 mL methanol. The reac-
tion mixture was stirred at room temperature for 20 h
and the solvent was evaporated. The residue was ad-
sorbed onto silica gel and purified by flash chromatogra-
phy, eluting with chloroform/methanol (30:1). The
appropriate fractions were pooled and evaporated to
4.8. General method for ring-closure condensation reac-
tions with substituted guanidine
Hydrochloride, carbonate, sulfate/hemisulfate, or ni-
trate salt of a substituted guanidine (4 mmol) was sus-
pended in anhydrous methanol (6.0 mL) and cooled to
0 ꢁC. A solution of sodium methoxide (25 wt%,
2.1 mL, 9.2 mmol) was added. The resulting mixture
was stirred in an ice bath for 30 min. The precipitated
sodium chloride was removed by filtration, and the fil-
trate was poured into a methanolic solution (20 mL)
of ring-closure precursor (1 mmol). The mixture was
stirred at room temperature for 16–72 h, and was mon-
itored by frequent TLC analysis to check for the com-
pletion of reaction. The reaction mixture was filtered if
necessary, and the clear solution was evaporated and
purified by flash chromatography on a silica gel column.
1
give a white foam. Yield: 103 mg, (76.3%), H NMR
(DMSO-d6) d 7.59–7.36 (m, 5H, Ar–H), 5.95 (t,
J = 7.3 Hz, 1H, 10-H), 4.64, 5.24 (2m, 2H, OH,
exchangeable with D2O), 4.11 (m, 1H, 30-H), 3.75–3.84
(2s, 6H, OCH3), 3.15–3.48 (m, 3H, 40,50,500-H), 2.12–
2.34 (m, 2H, 20,200-H). Anal. (C18H20N2O7Æ0.5 H2O) C,
H, N.
4.8.5. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-bromo-
imidazole-4,5-dicarboxylic Acid Dibutyl Ester (ZP-77).
To a mixture of ZP-72 (1.74 g, 5 mmol) and 1-acetyl-
2,3,5-tri-O-benzoyl-b-D-ribofuranose (2.55 g, 5 mmol),

4942
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
25 mL acetonitrile was added and the solution was
placed in an ice bath. HMDS (1,1,1,3,3,3-hexamethyldi-
silazane) (3.55 mL, 16.5 mmol), TMSCl (trimethylsilyl
chloride) (2.29 mL, 18 mmol), and TFMSA (trifluorom-
ethanesulfonic acid) (1.63 mL, 18 mmol) were added
successively into the solution with caution. The reaction
was completed in 1.5 hrs. The solvent and extra reactant
were removed by evaporation, and the residue was dis-
solved in 40 mL CHCl3. The solution was washed with
a saturated solution of NaHCO3 (2·) and water (2·)
successively. The organic layer was adsorbed onto flash
silica gel and applied to a flash chromatography column
made in CHCl3. The column was eluted with a mixture
of chloroform/methanol, 100:1. The appropriate frac-
tions were collected, and evaporated to obtain two prod-
ucts, the second of which corresponded to ZP-77 as
determined by its 1H NMR. Rf = 0.21 (chloroform).
Yield: 25%, 1H NMR (DMSO-d6): d 8.11-7.27 (5m,
15H, 3phenyl), 6.48 (m, 1H, 10-H), 6.02 (m, 1H, 20-H),
5.76 (m, 1H, 30-H), 4.90, 4.74, 4.56 (3m, 3H, 40,50,500-
H), 4.18 (m, 4H, 2OCH2), 1.61, 1.51, 1.33, 1.22 (4m,
8H, 4CH2), 0.89, 0.77 (2 t, J = 7.0 Hz, 6H, 2CH3). Anal.
(C39H39BrN12O11) C, H, N, Br.
Rf = 0.38 (chloroform/methanol/ammonium hydroxide,
2:1:0.3). H NMR (DMSO-d6) d 10.70, 7.6 (2br, 2H,
1
NH, exchangeable with D2O), 7.70, 7.09 (2m, 4H, Ar–
H), 6.49 (br, 1H, NH, exchangeable with D2O),
5.81(d, J = 5.5 Hz, 1H, 10-H), 5.36, 4.99 (2d, J =
5.9 Hz, 2H, 20-OH, 30-OH, exchangeable with D2O),
4.75 (t, J = 6.1 Hz, 1H, 50-OH, exchangeable with
D2O), 4.47 (t, J = 6.0 Hz, 1H, 20-H), 3.83 (s, 3H,
OCH3), 3.75, 3.63, 3.54, 3.47 (4m, 4H, 30,40,50,500-H).
13C NMR (300 MHz, DMSO) d 160.8, 148.7, 131.6,
121.4, 116.6, 113.9, 91.4, 85.1, 71.8, 69.1, 61.5, 55.3.
Anal. (C18H19N5O:70:25H2O) C, H, N. MS m/z 440.2,
418.2, 286.2, 156.6; HRMS (FAB) calcd for
C18H20N5O7 (MH+) m/z 418.1363, found, 418.1349.
4.9. General method for suzuki coupling reactions with
substituted boronic acids
A 25 mL round-bottomed flask was charged with 1 mol
of ZP-77 or ZP-115, 1 mmol of substituted boronic
acid, 0.32 g of sodium carbonate (3 mmol dissolved in
1.5 mL water), 3 mL benzene, and 0.3 mL ethanol.
The flask was covered with aluminum foil and a trace
amount of tetrakis(triphenylphosphino)palladium was
added. The reaction mixture was heated to reflux for
48–60 h and the solvent was evaporated. The residue
was dissolved in 100 mL of dichloromethane or chloro-
form and washed with water (3 · 50 mL). The extract
was dried over anhyd Na2SO4, filtered and evaporated
to dryness. The residue was purified by column chroma-
tography or by recrystallization from an appropriate
solvent. The spectral and analytical data, along with sol-
vent of recrystallization and/or solvent of elution for
chromatography, are collected as below.
4.8.6. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-bromo-
imidazole-4,5-dicarboxylic Acid Dimethyl Ester (ZP-
115). Experimental and work-up are the same as in
1
ZP-77 using ZP-32 as reactant. Yield: 30%, H NMR
(DMSO-d6): d 8.07, 7.92, 7.56, 7.42, 7.36 (5m, 15H, 3
phenyl), 6.40 (d, J = 6 Hz, 1H, 10-H), 6.17 (t, J = 6 Hz,
1H, 20-H), 5.87 (t, J = 6 Hz, 1H, 30-H), 5.81, 4.67 (2m,
3H, 40,50,500-H), 3.90 (t, J = 3 Hz, 6H, 2CH3). MS
(FAB) m/z 728.9, 730.9 (MH+).
4.8.7. 2-Bromo-7,8-dihydro-6-N-octylamino-1H-imi-
dazo[4,5-e][1,3]diazepine-4,8-dione Sodium Salt (ZP-
16). Ring-closure precursor: ZP-115, condensed with
octylguanidine hemisulfate, isolated by silica gel flash
chromatography eluting with chloroform/methanol
(60:1, 30:1; 10:1; 5:1; 1:1) and methanol. 1H NMR
(DMSO-d6) d 3.14, 1.43, 1.26, 0.85 (NH-C8H17); MS
(FAB) 416, 418 (MH+).
4.9.1. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-pheny-
limidazole-4,5-dicarboxylic Acid Dibutyl Ester (ZP-78).
Reactant: ZP-77, coupling with phenylboronic acid,
eluting with chloroform/methanol, 100:1. Yield: 0.92 g
(53.5%), Rf = 0.14 (chloroform), purified by silica gel
flash chromatography, eluting with chloroform. 1H
NMR (CDCl3): d 8.14–7.18 (4m, 20H, 4 Ar–H), 6.31,
6.24, 5.85, 4.72, 4.55, (5m, 6H, 10,20,30,40,50,500-H), 4.32
(m, 4H, OCH2), 1.71, 1.42 (2m, 8H, CH2 of butyl
group), 0.95 (2t, 6H, CH3 of butyl group). Anal.
(C45H44N2O11ꢀ0:5H2O) C, H, N.
4.8.8. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-methoxy-
phenylimidazole-4,5-dicarboxylic Acid Dibutyl Ester
(ZP-108). Reaction conditions and work-up are the
same as in ZP-73 except using ZP-77 as the reactant.
Yield: 74.8%, Rf = 0.1 (chloroform), purified by silica
4.9.2. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-flu-
orophenylimidazole-4,5-dicarboxylic Acid Dimethyl Ester
(ZP-118). Reactant: ZP-115, coupling with p-fluor-
ophenylboronic acid, triturating with MeOH. Yield:
1
gel flash chromatography, eluting with chloroform. H
NMR (CDCl3) d 8.06–7.37 (6m, 19H, Ar–H), 6.90
(m, 1H, 10-H), 6.22 (m, 1H, 20-H), 5.87 (m, 1H, 30-H),
4.62-4.32 (2m, 3H, 40,50,500-H), 3.82 (s, 3H, OCH3),
1.71, 1.41 (2m, 8H, (CH2)2 of 2 butyl), 0.94 (m, 6H,
CH3 of Butyl). Anal. (C46H46N2O12Æ 2H2O) C, H, N.
1
30%, H NMR (CDCl3) d 8.04, 7.80, 7.57, 7.40, 7.28,
7.26, 7.07 (7m, 19H, Ar–H), 6.30, 6.14, 5.81, 4.70,
4.61, 4.54 (6m, 6H, 10,20,30,40,50,500-H), 3.96, 3.88 (2s,
6H, 2 OMe). Anal. (C39H31FN2O11) C, H, N.
4.8.9. 6-N-Amino-7,8-dihydro-2-p-methoxyphenyl-1-b-D-
ribofuranosylimidazo[4,5-e][1,3]diazepine-4,8-dione (ZP-
110). Ring-closure precursor: ZP-108 condensed with
guanidine carbonate (see General procedure for ring-
closure reactions described above), isolated by silica
gel flash chromatography, eluting with chloroform/
methanol (20:1, 5:1, 1:1) and methanol. The appropriate
fractions were collected and evaporated. Yield: 49%,
4.9.3. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-m-methoxy-
phenylimidazole-4,5-dicarboxylic Acid Dibutyl Ester
(ZP-119). Reactant: ZP-77, coupling with m-meth-
oxyphenylboronic acid, triturating with MeOH. Yield:
36%, H NMR (CDCl3) d 8.05, 7.84, 7.54, 7.40, 7.33,
7.16, 7.02 (7m, 19H, Ar–H), 6.27, 5.85, 4.72, 4.58 (4m,
6H, 10,20,30,40,50,500-H), 4.32 (m, 4H, CH2 of 2 Butyl),
1

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4943
1.71, 1.42 (2m, 8H, (CH2)2 of 2 Butyl), 0.95 (m, 6H, CH3
of Butyl). Anal. (C46H46N2O12Æ0.5 H2O) C, H, N.
(CDCl3) d 7.94, 7.73, 7.51, 7.36, 7.11 (5m, 19H, Ar–
H), 6.01 (d, 1H, 10-H), 5.61 (m, 1H, 20-H), 5.33 (m,
1H, 30-H), 4.85, 4.53 (2m, 3H, 40,50,500-H), 3.94, 3.85
(2s, 6H, OCH3), 2.27 (s, 3H, CH3). Anal.
(C40H34N2O11Æ2.5 H2O) C, H, N.
4.9.4. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-phenyl-
phenylimidazole-4,5-dicarboxylic Acid Dimethyl Ester
(ZP-123). Reactant: ZP-115, coupling with p-phen-
ylphenylboronic acid, triturating with MeOH. Yield:
4.9.10. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-tri-
fluoromethylphenylimidazole-4,5-dicarboxylic Acid Dibu-
tyl Ester (ZP-175). Reactant: ZP-77, coupling with
p-trifluoromethylphenylboronic acid, product was
recrystallized from MeOH. Yield: 40%, 1H NMR
(CDCl3) d 8.05, 7.90, 7.53, 7.40, 7.38 (5m, 19H, Ar–
H), 6.41 (d, J = 7.3 Hz, 1H, 10-H), 6.17 (t, J = 6 Hz,
1H, 20-H), 5.87 (t, J = 6, 1H, 30-H), 4.80, 4.58 (2m,
3H, 40,50,500-H), 4.27 (m, 4H, 2 OCH2), 1.71, 1.39 (m,
8H, 4 CH2), 0.93 (2 t, J = 7.3 Hz, 6H, CH3). Anal.
(C46H43F3N2O11Æ3.5 H2O) C, H, N.
1
44.7%, H NMR (CDCl3) d 8.55, 7.81, 7.67, 7.58, 7.47,
7.36 (6m, 24H, Ar–H), 6.35 (t, J = 6 Hz, 1H, 10-H),
6.28 (d, J = 3 Hz, 1H, 20-H), 5.87 (d, J = 6.6 Hz, 1H,
30-H), 4.73, 4.63, 4.56 (3m, 3H, 40,50,500-H), 3.97, 3.94
(2s, 6H, OCH3). 13C NMR (100 MHz, DMSO) d
163.9, 163.7, 149.6, 142.3, 138.9, 132.5, 132.2, 129.6,
128.8, 127.9, 127.4, 127.3, 88.6, 78.9, 75.6, 73.1, 69.0,
52.2, 51.3. Anal. (C45H36N2O11Æ3.5 H2O) C, H, N.
4.9.5. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-t-butyl-
phenylimidazole-4,5-dicarboxylic Acid Dimethyl Ester
(ZP-124). Reactant: ZP-115, coupling with p-t-buty-
lphenylenebisboronic acid, eluting with chloroform/
4.10. General method for ring-closure condensation reac-
tions with guanidine
1
MeOH, 100:1. Yield: 77.9%, H NMR (CDCl3) d 8.03,
7.82, 7.50, 7.39 (4m, 19H, Ar–H), 6.33 (t, J = 6 Hz,
1H, 10-H), 5.85 (d, J = 6 Hz, 1H, 20-H), 5.23 (dd,
J1 = J2 = 3 Hz, 1H, 30-H), 4.74 – 4.48 (m, 3H, 40,50,500-
H), 3.97, 3.92 (2s, 6H, OCH3), 1.31 (s, 9H, 3 CH3). Anal.
(C43H40N2O11Æ2 H2O) C, H, N.
Carbonate/Hydrochloride salt of guanidine (4 mmol)
was suspended in anhydrous methanol (6.0 mL) and
cooled to 0 ꢁC. A solution of sodium methoxide
(25 wt%, 2.1 mL, 9.2 mmol) was added. The resulting
mixture was stirred in an ice bath for 30 min. The pre-
cipitated sodium salt was removed by filtration, and
the filtrate was poured into a methanolic solution
(20 mL) of ring-closure precursor (1 mmol). The mixture
was stirred at room temperature for 16–72 h, and was
monitored by frequent TLC analysis to check for the
completion of reaction. The reaction mixture was fil-
tered if necessary, and the clear solution was evaporated
and purified by flash chromatography on a silica gel col-
umn, eluting with a mixture of chloroform/methanol
(20:1, 5:1, 1:1) and methanol, successively. The appro-
priate fractions were combined and evaporated to ob-
tain the product. The latter was recrystallized from an
appropriate solvent if necessary. The spectral and ana-
lytical data, along with solvent of recrystallization and/
or solvent of elution for chromatography are collected
as below.
4.9.6. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-thieny-
limidazole-4,5-dicarboxylic Acid Dimethyl Ester (ZP-
128). Reactant: ZP-115, coupling with thienylboronic
acid, eluting with chloroform. Yield: 38%, 1H NMR
(CDCl3) d 8.05, 7.82, 7.55, 7.39, 7.26 (5m, 18H, Ar–
H), 6.34 (d, J = 5.5 Hz, 1H, 10-H), 6.27 (t, J = 6.4 Hz,
1H, 20-H), 5.82 (t, J = 6.9 Hz, 1H, 30-H), 4.71, 4.62,
4.58 (3m, 3H, 40,50,500-H), 3.96, 3.92 (2s, 6H, OCH3).
Anal. (C37H30N2O11SÆ2 H2O) C, H, N.
4.9.7. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-m-nitro-
phenylimidazole-4,5-dicarboxylic Acid Dimethyl Ester
(ZP-129). Reactant: ZP-115, coupling with 3-nitro-
phenylboronic acid, eluting with chloroform. Yield:
1
83%, H NMR (CDCl3) d 8.12, 7.97, 7.52, 7.48, 7.40
(5m, 19H, Ar–H), 6.46 (d, J = 6 Hz, 1H, 10-H), 6.23 (t,
J = 6.3 Hz, 1H, 20-H), 5.93 (m, 1H, 30-H), 4.85, 4.74,
4.60 (m, 3H, 40,50,500-H), 3.97, 3.96 (2s, 6H, OCH3).
Anal. (C39H31N3O13Æ1.5 H2O) C, H, N.
4.10.1. 6-N-Amino-7,8-dihydro-2-p-fluorophenyl-1-b-D-
ribofuranosylimidazo[4,5-e][1,3]diazepine-4,8-dione (ZP-
121). Ring-closure precursor: ZP-118, condensed with
guanidine carbonate, isolated by silica gel flash chroma-
tography (for details, see General Procedure above). The
appropriate fractions were collected and evaporated to
dryness. The residue was recrystallized from ethyl ace-
tate. Yield: 50 mg (10%), Rf = 0.24 (chloroform/metha-
4.9.8.
1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-p-
trifluoromethylphenylimidazole-4,5-dicarboxylic Acid Di-
methyl Ester (ZP-132). Reactant: ZP-115, coupling with
p-trifluoromethylphenylboronic acid, product was
recrystallized from MeOH. Yield: 23.8%, 1H NMR
(CDCl3) d 8.05, 7.80, 7.73, 7.64, 7.55, 7.41, 7.33, 7.29
(8m, 19H, Ar–H), 6.30 (t, J = 7.3 Hz, 1H, 10-H), 6.12
(d, J = 6 Hz, 1H, 20-H), 5.85 (t, J = 6 Hz, 1H, 30-H),
4.72, 4.62, 4.59 (3m, 3H, 40H,50,500-H), 3.98, 3.94
(2s, 6H, OCH3). Anal. (C40F3H31N2O11Æ0.5 H2O) C,
H, N.
nol/ammonium
hydroxide = 2:1:0.3).
1H
NMR
(DMSO-d6) d 10.81, 8.06 (2br, 2H, NH, exchangeable
with D2O), 7.82, 7.38 (2m, 4H, Ar–H), 6.86 (br, 1H,
NH, exchangeable with D2O), 5.85 (d, J = 6 Hz, 1H,
10-H), 5.35, 4.99 (2d, J = 5.2 Hz, 2H, 20-OH, 30-OH,
exchangeable with D2O ), 4.75 (d, J = 5.6 Hz, 1H, 50-
OH, exchangeable with D2O ), 4.47 (t, J = 6.0 Hz, 1H,
20-H), 3.7, 3.65, 3.51, 3.45 (4m, 4H, 30,40,50,500-H). 13C
NMR (100 MHz, DMSO-d6) d 164.47, 162.15, 151.61,
149.21, 132.73, 132.64, 125.96, 115.68, 115.46, 91.09,
4.9.9. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-tolyl-
imidazole-4,5-dicarboxylic Acid Dimethyl Ester (ZP-
136). Reactant: ZP-115, coupling with tolylboronic acid,
eluting with chloroform. Yield: 47.5%, 1H NMR
85.16,
71.99,
68.99,
61.45,
48.48.
Anal.
(C17H16FN5O6 ꢀ 2H2O) C, H, N. MS (ESI) m/z 274.08,

4944
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
306.11, 406.13; HRMS (ESI) calcd for C17H17FN5O6
of chloroform/methanol (20:1, 5:1, 1:1) and methanol.
The fractions eluted with methanol were collected and
evaporated to obtain a yellow solid. The H NMR data
(MH+) m/z 406.11629, found, 406.11693.
1
4.10.2. 6-N-Amino-7,8-dihydro-2-m-methoxyphenyl-1-b-D-
ribofuranosylimidazo[4,5-e][1,3]diazepine-4,8-dione (ZP-
122). Ring-closure precursor: ZP-119, condensed with
guanidine carbonate, isolated by silica gel flash chroma-
tography (for details, see General Procedure above). The
appropriate fractions were collected and evaporated to
obtain a solid. Yield: 15%, 1H NMR (DMSO-d6) d
10.72, 6.65 (2br, 1H, NH, exchangeable with D2O),
7.45, 7.32, 7.12 (3m, 4H, Ar–H), 5.81(d, J = 5.6 Hz,
1H, 10-H), 5.37, 4.97 (2d, J = 6 Hz, 2H, 20-OH, 30-OH,
exchangeable with D2O), 4.7 (t, J = 6 Hz, 1H, 50-OH,
exchangeable with D2O ), 4.54 (m, 1H, 20-H), 3.82 (s,
3H, OCH3), 3.76, 3.63, 3.5, 3.4 (4m, 4H, 30,40,50,500-H).
13C NMR (100 MHz, DMSO-d6) d 159.08, 149.05,
130.44, 129.81, 122.55, 116.47, 115.24, 91.5, 85.13,
72.16, 69.59, 61.78, 55.37. Anal. (C18H19N5O7Æ0.5
H2O) C, H, N.
suggested that it is a mixture. MS (FAB) of the reaction
mixture exhibited the following ions: m/z 284.1, 316.1,
416.2, and 448.2. HRMS (ES) calcd for C19H22N5O6
and C20H26N5O7 (MH+): m/z 416.15701 and
448.18322, found, 416.15923, 448.18029.
4.11. Primary HCV RNA replicon assay
The cell line Huh7 ET (luc-ubi-neo/ET), which con-
tains an HCV RNA replicon with a stable luciferase
(LUC) reporter, was used for the assay.The HCV
RNA replicon ET contains the 50 NTR (IRES) of
HCV (50) which drives the production of a firefly lucif-
erase (Luc), ubiquitin (Ubiq), and neomycin phospho-
transferase (Neo) fusion protein. Ubiquitin cleavage
releases the LUC and Neo genes. The EMCV IRES
element (E-I) controls the translation of the HCV
structural proteins NS3-NS5. The NS3 protein cleaves
the HCV polyprotein to release the mature NS3,
NS4A, NS4B, NS5A, and NS5B proteins that are re-
quired for HCV replication. At the 30 end of the rep-
licon is the 30 NTR of HCV. The LUC reporter is
used as an indirect measure of HCV replication. The
activity of the LUC reporter is directly proportional
to HCV RNA levels and positive control antiviral
compounds behave comparably using either LUC or
RNA endpoints.
4.10.3. 6-N-Amino-7,8-dihydro-2-m-methoxyphenylimi-
da-zo[4,5-e][1,3]diazepine-4,8-dione (ZP-122fc). H NMR
1
(DMSO-d6) d 7.70 (s, 1H, Ar–H), 7.69 (d, J = 6.8 Hz,
1H, Ar–H), 7.22 (t, J = 8 Hz,1H, Ar–H), 6.76 (d,
J = 8.4 Hz, 1H, Ar–H), 3.80 (s, 3H, OMe). MS m/z
286.08; HRMS (ES) calcd for C13H12N5O3 (MH+) m/z
286.09401, found, 286.09339.
4.10.4. 2-(m-Methoxyphenyl)-1-b-D-ribofuranosylimida-
zole-4,5-dicarboxylic Acid Dimethyl Ester (ZP-122P1).
1H NMR (DMSO-d6) d 7.442, 7.326, 7.127 (3m, 4H, Ar–
H), 5.746 (d, 1H, J = 6 Hz, 10-H), 5.647, 5.157 (2d, 2H,
J = 6.8 Hz, J = 6 Hz, 20,30-OH, exchangeable with
D2O), 4.832 (t, 1H, J = 6 Hz, 50-H, exchangeable with
D2O), 4.491 (q, 1H, J = 6.4 Hz, 20-H), 3.849, 3.822,
3.797 (3s, 9H, OCH3), 3.673, 3.441, 3.374 (3m, 4H,
30,40,50,500-H). Anal. (C19H22N2O9Æ1.5 H2O) C, H, N.
In the primary assay, the effect of RENs was examined
in triplicate at a single high-test concentration of
20 lM on HCV RNA-derived LUC activity and cyto-
toxicity. Human interferon alpha-2b is included in each
run as a positive control compound. Subconfluent cul-
tures of the ET line are plated out into 96-well plates
that are dedicated for the analysis of cell numbers (cyto-
toxicity) or antiviral activity and the next day drugs are
added to the appropriate wells. Cells are processed 72 hr
later when the cells are still subconfluent. Compound
cytotoxicity is assessed as the percent viable cells relative
to the untreated cell controls. The results are collected in
Table 1 in the text.
4.10.5.
7,8-Dihydro-6-N-methylamino-2-phenyl-1-b-D-
ribofuranosylimidazo[4,5-e][1,3]diazepine-4,8-dione (NZ-
53). Ring-closure precursor: ZP-78, condensed with
methylguanidine, isolated by silica gel flash chromatog-
raphy, eluting with mixtures of chloroform/methanol
(20:1, 5:1, 1:1) and methanol. The appropriate fractions
were collected and evaporated to obtain a solid. Yield:
1
53%, H NMR (DMSO-d6) d 10.77, 7.1 (br, 2H, NH,
Acknowledgments
exchangeable with D2O), 7.72, 7.5 (m, 5H, Ar–H),
5.82(d, J = 5.7 Hz, 1H, 10-H), 5.33, 4.99 (2d, J = 6 Hz,
2H, 20-OH, 30-OH, exchangeable with D2O), 4.77 (t,
J = 6 Hz, 1H, 50-OH, exchangeable with D2O), 4.35
(m, 1H, 20-H), 3.64, 3.47 (2m, 4H, 30,40,50,500-H), 2.75
(s, 3H, NCH3). 13C NMR (75 MHz, DMSO-d6) d 162,
159, 147, 130.2, 130, 129.5, 128, 91, 85, 72, 70, 62, 29.
Anal. (C18H19N5O6Æ3.5 H2O) C, H, N. MS m/z 402.0,
424.0; HRMS (ES) calcd for C18H20N6O5 (MH+) m/z
402.1414, found, 402.1398.
The research was supported in part by grants (# 5 RO1
AI55452 & #1 R21 AI071802) from the National Insti-
tute of Allergy and Infectious Diseases (NIAID) of the
National Institutes of Health, Bethesda, Maryland,
and an unrestricted grant from Nabi Biopharmaceuti-
cals, Rockville, Maryland. We sincerely thank Dr.
Christopher Tseng, the Program Officer of Antiviral Re-
search and Antimicrobial Chemistry of the Virology
Branch of the National Institute of Allergy and Infec-
tious Diseases (NIAID), Bethesda, Maryland, for his
support and encouragement throughout the course of
this work. We also acknowledge the continual assistance
provided by Dr. Cecil Kwong, the coordinator of
NIAID’s Antimicrobial Acquisition and Coordinating
4.10.6. Reaction of ZP-136 with N-methylguanidine to
produce mixture of products (ZP-181). ZP-136 was con-
densed with N-methylguanidine using the general proce-
dure described above. The products were isolated by
silica gel flash chromatography, eluting with mixtures

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4933–4945
4945
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