Synthesis and in vitro anti-hepatitis B and C virus activities of ring-expanded ('fat') nucleobase analogues containing the imidazo[4,5-e][1,3] diazepine-4,8-dione ring system

Article abstract of DOI:10.1016/j.bmcl.2005.09.015

As part of our structure-activity relationship studies, we report here the synthesis and in vitro anti-HBV and anti-HCV activities of a number of ring-expanded ('fat') nucleobases containing the imidazo[4,5-e][1,3]diazepine-4, 8-dione ring system. One of

Full text of DOI:10.1016/j.bmcl.2005.09.015

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5397–5401
Synthesis and in vitro anti-hepatitis B and C virus activities of
ring-expanded (ÔfatÕ) nucleobase analogues containing the
imidazo[4,5-e][1,3]diazepine-4,8-dione ring system
Peng Zhang,a Ning Zhang,a Brent E. Korbab and Ramachandra S. Hosmanea,*
aLaboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA
bDivision of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, MD 20850, USA
Received 13 August 2005; revised 1 September 2005; accepted 1 September 2005
Available online 5 October 2005
Abstract—As part of our structure–activity relationship studies, we report here the synthesis and in vitro anti-HBV and anti-HCV
activities of a number of ring-expanded (ÔfatÕ) nucleobases containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system. One of
the compounds, ZP-88, exhibited a good activity/toxicity profile against HBV by inhibition of the synthesis of extracellular virion
release (EC50 = 1.7 lM, CC50 = 286 lM, SI = 168) and intracellular HBV replication intermediates (EC50 = 8.4 lM,
CC50 = 286 lM, SI = 34) in cultured human hepatoblastoma 2.2.15 cells. By contrast, most of the compounds tested against
HCV had only marginal activity/toxicity profile, although that was still better than that of the reference compound ribavirin.
Ó 2005 Elsevier Ltd. All rights reserved.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) are
two of the most dreadful viruses currently challenging
the global human health.1,2 HBV has infected over
two billion people worldwide and 1.5 million in U.S.,
and the infection from HCV stands at >175 and 5 mil-
lions, respectively.3–5 Although both cause chronic liver
diseases, including liver cirrhosis and hepatocellular car-
cinoma,6,7 they belong to two totally different viral
families. While HBV is a double-stranded DNA virus
classified under Hepadnaviridae,8,9 HCV has a single-
stranded, positive-sense RNA genome and is classified
under Flaviviridae.10 A recombinant vaccine is available
for treatment of human HBV infection, but no such vac-
cine exists as yet against HCV. Furthermore, while there
are a few, effective, FDA-approved oraltherapies, such
as Lamivudine (3TC)11 and Adefovir Dipivoxil,12 avail-
able for HBV infection, the clinical benefits of the exist-
ing treatment options against HCV infection are limited,
and include a far less effective treatment with a combina-
tion therapy including interferon-a and a non-selective
and/or toxic drug ribavirin.13,14 Although several potent
inhibitors of HCV replication have recently been report-
ed,15–18 their clinical efficacy and safety have yet to be
proven. Both HBV and HCV transmission occurs pri-
marily through blood and blood-related products,19,20
and is most efficient via percutaneous mode (needles),21
although HCV is also common in sexually promiscuous
individuals.22 In addition, both HBV and HCV are the
major co-infections in HIV patients and are the frequent
causes of the end-stage liver disease associated with
death of HIV patients.23 We report herein the latest re-
sults of our continued structure–activity relationship
studies against HBV24 and HCV25 based on ring-ex-
panded heterocycles and nucleosides, containing the title
imidazo[4,5-e][1,3]diazepine-4,8-dione ring system.
A number of ring-expanded (ÔfatÕ) heterocycles and nucle-
oside analogues containing the title ring system I have
been synthesized in this laboratory in recent years and
were biologically screened in vitro against both HBV24
and HCV.25 In the case of HBV, the compounds were spe-
cifically tested for inhibition of replication in cultured hu-
man hepatoblastoma 2.2.15 cells,24 whereas screening
against HCV involved the assessment of inhibition of
the viral NTPase/helicase,25 a crucial enzyme involved
in replication. A number of compounds exhibited potent
activities against both viruses with IC50 values in the
micromolar range, and little or low toxicity to the host
cells (HBV)24 or the host enzyme (HCV).25
Keywords: HBV; HCV; Inhibition; Ring-expanded nucleobases;
Imidazo[4,5-e][1,3]diazepines.
*
Corresponding author. Tel.: +1 410 455 2520; fax: +1 410 455
1148; e-mail: hosmane@umbc.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.015

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5397–5401
O
the mechanism of viral inhibition by ring-expanded mol-
5
3
4
ecules in general. To this end, we set out to synthesize
and screen a number of judiciously chosen heterocycles
bearing the structural skeleton I. In our previous studies,
most of the compounds screened contained substitutions
at positions 1 and 6 of the general structure I,25,26 while
position 2 remained largely unexplored. As it is not yet
known if a hydrophobic or a hydrophilic substituent at
position 2 would enhance the antiviral activity, we intu-
itively chose to put a bromo substituent at this position.
This was mainly for two reasons: one, bromine atom is
reasonably large and polarizable and thus can act as a
quick, albeit approximate, dimensional probe of enzyme
(or receptor) active site, and two, it can be easily manip-
ulated into either hydrophobic or hydrophilic substitu-
ents for further structure–activity relationship (SAR)
NH
N
N
2
6
R'
NHR
1
N
7
8
R"
O
I
An interesting outcome of the above investigations was
that a number of ring-expanded heterocyclic aglycons,
in addition to the corresponding nucleoside analogues,
were active against Flaviviridae.25,26 This is what kindled
our interest in the present work using ring-expanded
heteocycles. We reasoned that the biological outcome
with the aglycons alone is likely to shed more light on
Cl
O
O
N
N
H CO
3
NBS/AIBN
Anhy. CH CN
OR
OR
OR
OR
Br
K
2
CO
/
An
hy.
CH
C
N
3
3
N
H
N
H
3
O
O
ZP-32; R = CH
ZP-43; R = CH
3
3
ZP-72; R = (CH ) CH
ZP-70; R = (CH ) CH
2 3
3
2 3
3
O
N
N
OR
OR
Br
Cl
O
O
Cl
H C
2
K
2
C
O
/ A
nhy
. C
H
3
CN
3
OCH
K
2
C
O
/ A
nhy
. C
H
3
CN
3
3
ZP-34; R = CH
3
ZP-74; R = (CH ) CH
2 3
3
O
NH
2
O
N
O(CH ) CH
N
N
2 3
3
OR
OR
RHN
NH
Br
Br
N
O(CH ) CH
2 3
3
NaOMe, Dry MeOH
O
O
O
O
ZP-82
ZP-44; R = CH
3
N
N
N
NHR
NH
Br
NH
2
NaOMe,
Dry MeOH
NH
2
O
RHN
O
NH
RHN
H C
2
NH
OCH
3
NaOMe, Dry MeOH
ZP-31; R = H
ZP-97; R = CH
3
N
N
O
ZP-30; R = CH CH
Br
NHR
2
3
N
NH
ZP-33; R =(CH ) CH
N
N
2 7
3
3
Br
O
NHR
ZP-81; R =(CH ) CH
2 9
N
NH
ZP-54; R = (CH ) CH
2 11
3
3
3
O
ZP-61; R = (CH ) CH
2 13
O
ZP-62; R = (CH ) CH
2 15
ZP-88; R =(CH ) CH
2 7
3
ZP-46; R =(CH ) CH
2 7
3
ZP-76; R = Benzyl
Scheme 1.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5397–5401
5399
studies. Therefore, keeping the 2-bromo group as a com-
mon functionality, we synthesized a series of compounds
with structural variations at the 1- and 6-positions, as
outlined in Scheme 1. The synthesis is straightforward
and involved condensation of the appropriate 1-substi-
tuted-2-bromoimidazole-3,4-dicarboxylic acid esters
with a variety of guanidine derivatives. The required 2-
bromo derivatives of imidazole 4,5-diesters were pre-
pared by bromination of the latter with N-bromosuccin-
imide (NBS) in the presence of a free radical initiator
Table 1. Anti-HBV activity of a ring-expanded heterocycle in vitroa
O
O
N
N
C8 H17
Br
N
H
N
NH
ZP-88
Antiviral activity EC90(lM)c
Antiviral activity EC50 (lM)c
Toxicity CC50 (lM)
Selectivity index (SI)f
CC50/EC50
b
Compound ID
Viriond
1.7 0.2
0.048 0.005
HBV RIe
Viriond
6.9 0.7
0.151 0.013
HBV RIe
Viriond
HBV RIe
ZP-88
3TC (Ref.)
8.4 0.8
0.251 0.029
28 2.2
0.648 0.062
286 20
2159 78
168
44,979
34
8602
a Appropriate concentrations of the test compounds were added daily for nine days to the HBV producing 2.2.15 cells. Culture medium was collected
daily and tested for extracellular (virion) HBV DNA at days 0, 3, 6, and 9. Cells were lysed 24 h after day 9 for the analysis of intracellular HBV
replication intermediates (HBV RI). HBV virion DNA and intracellular HBV DNA RI levels in the cells were measured by blot hybridization
methods (Southern and dot blot) and [P]32 labeled HBV-specific probes.
b CC50, is the drug concentration at which a twofold reduction of neutral red dye uptake from the average value in the untreated cultures was
observed.
c EC90and EC50 are concentrations which give 90% and 50% inhibition of viral replication in the cell cultures, respectively. Values presented
( standard deviation) were calculated by linear regression analysis using data combined from all treated cultures; standard deviations (SD) were
calculated by using the standard error of regression generated from the linear regression analysis.
d Extracellular HBV virion DNA.
e Intracellular HBV DNA replicative intermediates.
f Selectivity index was calculated as CC50/EC50 ratio.
Table 2. Anti-HCV activity of ring-expanded heterocycles in vitroa
O
N
N
N
Br
NHR
NH
O
H3CO
Compound ID
No. of carbons present
at position-6 n
Antiviral activity
HCV RNAb % control
Toxicity b-actin
Selectivity index SId
toxicity/antiviral activity
RNAc % control
ZP-31 (R = H)
0
62
33
34
1
3
4
89
48
65
69
32
47
34
3
4
2
9
3
1
3
7
1
2
8
6
4
1
1.435
1.453
1.912
2.155
0.604
1
ZP-97 (R = CH3)
ZP-30 (R = C2H5)
ZP-33 (R = C8H17
1
2
)
8
10
32 13
ZP-81 (R = C10H21
ZP-54 (R = C12H25
ZP-61 (R = C14H29
ZP-62 (R = C16H33
)
)
)
)
53
47
64
6
7
4
12
14
0.532
0.043
1.255
2.10
16
Benzyl group
70 10
ZP-76 (R = Benzyl)
ZP-46
ZP-88
55
29
78
10
1
7
5
1
69
61
83
108
12
8
8
1.06
Interferon-a (10 IU/mL)
Ribavirin
Ref. 1
Ref. 2
11.3
89 10
0.42
a The antiviral activity is based on a primary assay employing 10 lM concentrations of the test compound for determination of both antiviral activity
and toxicity. The assay was performed using an Huh7 ET cell line, which contains the HCV RNA replicon with a stable luciferase (LUC) reporter.
b HCV RNA-derived LUC activity is used as an indirect measure of HCV RNA levels.
c b-Actin RNA level is used as a positive control for cellular RNA in order to compute cytotoxicity.
d Selectivity index (SI) is represented as a ratio of the levels of b-actin RNA/HCV RNA.

5400
P. Zhang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5397–5401
azobisisobutyro-nitrile (AIBN), employing a literature
procedure.27 The necessary guanidine derivatives for
condensation, when not commercially available, were
synthesized by reaction of 3,5-dimethylpyrazole-1-car-
boxamidine nitrate with the appropriately substituted
amine in methanol at reflux, using the procedure of
Scott et al.28 All new intermediates and final products
were fully characterized by spectroscopic and microana-
lytical data.29,30
tance provided by Dr. Cecil Kwong, the coordinator
of NIAIDÕs Antimicrobial Acquisition and Coordinat-
ing Facility (AACF) at the Southern Research Insti-
tute, Birmingham, Alabama.
Supplementary data
Experimental procedures as well as physical, spectral
and analytical data (six pages) for the key compounds
reported in this article can be found in the supplementa-
ry data associated with the online version of this journal.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2005.
09.015.
The target compounds were screened against HBV and
HCV through contractual arrangements with the
National Institute of Allergy and Infectious Diseases
(NIAID), employing standard protocols, published on
NIAID-AACF website.31 Anti-HBV activity and toxici-
ty against confluent 2.2.15 cells were determined by the
published procedure of Korba and Gerin.32 Anti-HCV
activity and toxicity were assessed by the HCV RNA
Replicon assay of Krieger et al.33 The biological screen-
ing results for anti-HBV and anti-HCV activities of the
target compounds are collected in Tables 1 and 2,
respectively. Out of the several compounds tested, only
ZP-88 exhibited promising anti-HBV activity/toxicity
profiles as shown. The compound was also found to
be active against the 3TC-resistant mutant L180M
(EC50 = 12 lM). On the other hand, all of the heterocy-
cles tested had at least some activity against HCV.
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The research was supported in part by a grant
(#9RO1 AI55452) from the National Institute of
Allergy and Infectious Diseases (NIAID) of the
National Institutes of Health, Bethesda, Maryland,
and an unrestricted grant from Nabi Biopharmaceuti-
cals, Rockville, Maryland. We sincerely thank Dr.
Christopher Tseng, the Program Officer of Antiviral
Research 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
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