Dual inhibition of HCV and HIV by ring-expanded nucleosides containing the 5:7-fused imidazo[4,5-e][1,3]diazepine ring system. in vitro results and implications

Article abstract of DOI:10.1016/j.bmcl.2013.12.121

Examples of ring-expanded nucleosides (RENs), represented by general structures 1 and 2, exhibited dual anti-HCV and anti-HIV activities in both cell culture systems and the respective target enzyme assays, including HCV NTPase/helicase and human RNA helicase DDX3. Since HCV is a leading co-infection in late stage HIV AIDS patients, often leading to liver cirrhosis and death, the observed dual inhibition of HCV and HIV by the target nucleoside analogues has potentially beneficial implications in treating HIV patients infected with HCV.

Full text of DOI:10.1016/j.bmcl.2013.12.121

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1154–1157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dual inhibition of HCV and HIV by ring-expanded nucleosides
containing the 5:7-fused imidazo[4,5-e][1,3]diazepine ring system. In
vitro results and implications
Ning Zhang a, , Peng Zhang a, , Andrea Baier b, Lucyna Cova c,à, Ramachandra S. Hosmane a, ,§
⇑
a Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
b Department of Molecular Biology, John Paul II Catholic University of Lublin, Lublin, Poland
c INSERM U871, Molecular Physiopathology & New Treatments of Viral Hepatitis, 151 Cours A. Thomas, 69003 Lyon Cedex 03, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Examples of ring-expanded nucleosides (RENs), represented by general structures 1 and 2, exhibited dual
anti-HCV and anti-HIV activities in both cell culture systems and the respective target enzyme assays,
including HCV NTPase/helicase and human RNA helicase DDX3. Since HCV is a leading co-infection in late
stage HIV AIDS patients, often leading to liver cirrhosis and death, the observed dual inhibition of HCV
and HIV by the target nucleoside analogues has potentially beneficial implications in treating HIV
patients infected with HCV.
Received 11 November 2013
Revised 27 December 2013
Accepted 30 December 2013
Available online 8 January 2014
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Ring-expanded nucleosides
Imidazo[4,5-e][1,3]diazepines
Organic synthesis
Antiviral compounds
In vitro screening
Hepatitis C virus (HCV)
Human immunodeficiency virus (HIV)
Dual inhibitors of HCV and HIV
Inhibition of HCV NTPase/helicase
Inhibition of RNA helicase DDX3
Highly active antiretroviral therapy (HAART), employing a
three-drug regimen acting on different stages of the viral life
cycle, has dramatically increased the survival rate of the HIV-in-
fected individuals, and has transformed Acquired Immunodefi-
had tested positive for antibodies to HCV.13 Out of the HIV oppor-
tunistic infections, HCV in particular has lately taken the center
stage, causing alarms in the AIDS research community for many
reasons, including (a) the vastly successful HAART therapy is
considerably less effective with HIV patients co-infected with
HCV,14 (b) the protease inhibitors used in the HAART therapy
exert a significant degree of extra strain on the liver that is al-
ready stressed by HCV.14,15 This results in dramatic exacerbation
of HCV and its accelerated progress to liver cirrhosis and death.
Thus, patients on HAART therapy are even at more risk for liver
diseases,14,15 (c) the approved anti-HCV therapy with a combina-
ciency Syndrome (AIDS) into a controllable chronic illness.1,2
A
fateful outcome of the chronic HIV condition, however, is the pro-
gressively weakening immune system since HIV primarily infects
the CD4 lymphocytes which help the body fight infections.3–5
This makes HIV patients vulnerable to opportunistic co-infec-
tions, including but not limited to that caused by the Hepatitis
C virus (HCV).6–9 The end-stage liver diseases caused by hepatitis
viral infection is now one of the major causes of death (>50%) in
HIV patients in the Western World.10–12 In a recent study explor-
ing the cause of death in HIV patients, a vast majority of the dead
tion of
a-interferon and ribavirin was shown to decrease the
potency of anti-HIV therapy because of the suspected molecular
interaction of ribavirin with the reverse transcriptase inhibitors
used in HAART, resulting in the latter’s diminished effective-
ness.16 It is also not yet clear how the recently approved protease
inhibitors for HCV treatment, including Victrelis (boceprevir),17
Incivek (telaprevir),17 and Olysio (simeprevir)17 would affect
disease progression of HIV patients infected with HCV. For these
reasons, mutually compatible anti-HCV and anti-HIV drugs are
needed to combat HCV co-infection in HIV patients. These drugs
should neither exacerbate the clinical manifestations of the
⇑
Corresponding author.
E-mail address: hosmane@umbc.edu (R.S. Hosmane).
Present address: Pharmaceutics International Inc. (pii), 10819 Gilroy Road, Hunt
Valley, MD 21031, USA.
à
Present address: Universite Lyon 1, INSERM U1052, Centre de Recherche en
Cancerologie de Lyon (CRCL), 151 Cours A. Thomas, 69003 Lyon Cedex 03, France.
§
Retired.
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.121

N. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1154–1157
1155
Me
Me
Me
co-infection nor diminish the efficacy or effectiveness of the ther-
apy used for treatment of the individual infection.
O
N
N
Me
Cl
N
p-Tol-O
O
H3C(H2C)3O
H3C(H2C)3O
We have recently reported18 the in vitro anti-HIV activity of a
series of ring-expanded nucleosides (RENs) containing imi-
dazo[4,5-e][1,3]diazepine ring system, represented by general
structural formulas 1 and 2. Out of the many compounds screened
for inhibition of HIV-1 replication in virus-infected T cell line (MT4
cells), compounds 1f and 2e were identified as the lead compounds
with low micromolar IC50’s. Both compounds contained the long
(18-carbon) alkyl chain at the C-6 position of the heterocycle,
and differed from one another in their stereochemical configura-
tion at the anomeric junction and the lack of 20-OH group in 2e.
Neither compound was significantly toxic in ex vivo cell culture
or in vivo in mice.18 Both compounds strongly inhibited cellular
RNA helicase DDX3, which the virus is believed to exploit for its
replication as it lacked its own helicase.19 Helicases are capable
of unwinding duplex RNA and DNA structures by disrupting the
hydrogen bonds that keep the two strands together.20,21 This
unwinding activity, which is normally accompanied by simulta-
neous hydrolysis of an NTP (ATP or GTP),22 is essential for the virus
replication.
H
CHCl3
+
N
H
p-TOl=C(O)C6H4-CH3-p
p-Tol-O
3
4
O
NH2 NO3
O
O
HN
N
N
N
N
N
H3C(H2C)3O
RHN
NH2
RHN
NaOMe/MeOH
RT/24 h
H3C(H2C)3O
H
H
O
O
O
p-Tol
O
O
OH
OH
O-Tol-p
2
5
62%
a; R=(CH ) CH3
b; R=(CH ) CH3
92%
94%
2 7
2 9
H
O
H
OH
HO
O
O
O
N
N
N
N
N
O
R-HN
HO
OH
O
HN
NH
NHR
2
N
a; R=(CH ) CH3
b; R=(CH ) CH3
2 7
2 9
Several years ago, we had also reported23 that a wide variety of
RENs, containing both the imidazo[4,5-e][1,3]diazepine and imi-
dazo[4,5-e][1,2,4]triazepine ring systems, potently inhibited the
virus-encoded NTPases/helicases of several RNA viruses belonging
to the Flaviviridae family, including but not limited to the West
Nile Virus (WNV), Hepatitis C Virus (HCV), and the Japanese
Encephalitis Virus (JEV). Subsequently, we had discovered24 that
compounds represented by general structural formulas 1 and 2
were especially effective in inhibition of the WNV NTPase/helicase.
To our surprise and delight, these RENs failed to inhibit a truncated
Scheme 1.
Syntheses of a majority of the compounds necessary for our
present study, listed under general structural formulas 1 and 2,
were carried out by us earlier for the mentioned HIV and WNV
studies18,24 and so will not be repeated here. Synthesis of the
two new compounds used in this study, 2a and 2b, is outlined in
Scheme 1. Butyl imidazole-4,5-dicarboxylate (3) was glycosylated
form of the human helicase Suv3( 1–159), which we had included in
D
the study in order to assess their selectivity and toxicity.24 Because
of the perceived serious threat of WNV epidemic in North America
in the early 2000’s, our research efforts during that time, along
with many other laboratories in US and Canada, were largely fo-
cused on WNV. In light of the promising results of 1 and 2 with
HIV inhibition, coupled with the increasing problem of HCV co-
infection in HIV patients as described above, and not to mention
that HCV belongs to the same viral family as WNV, against which
1 and 2 had already exhibited potent antiviral activity, it was only
logical to extend our investigation of these RENs to HCV, as elabo-
rated in this Letter.
with
2-deoxy-3,5-di-O-p-toluoyl-b-
D
-erythropentofuaranosyl
sugar,
chloride (4),24 generated in situ from the corresponding
a
in the presence of N,N,N0,N0-tetramethyl-1,8-naphthalenediamine
(proton sponge) to obtain butyl 1-(20-deoxy-30,50-di-O-p-toluoyl-
a-D-erythropentofuranosyl)-4,5-imidazoledi-carboxylate (5) in
62% yield. Condensation of the latter with (N-octyl)- and
(N-decyl)guanidinium nitrates, freshly generated from the reaction
of 3,5-dimethylpyrazole-1-carboxamidine nitrate with N-octyl-
and N-decylamine, respectively,24 catalyzed by sodium methoxide
in methanol, afforded 2a and 2b in 92% and 94%, respectively. Both
compounds were fully characterized by spectroscopic and
microanalytical data.25
O
The NTPase/helicase of HCV was expressed in Escherichia coli
and purified by affinity chromatography.25 The homogeneity of
the enzyme preparation was verified by Coomassie Blue staining.
In order to monitor the inhibitory potential of RENs toward
helicase activity of HCV NTPase/helicase, radiolabelled partially
double-stranded (ds) DNA was used as substrate.25 Out of the 11
compounds listed under 1 and 2 that were biochemically screened,
only 1f and 2e showed promising anti-helicase activity of HCV
NTPase/helicase as shown in Table 1. This is remarkably consistent
with what we had earlier observed with anti-HIV activity.18 A long
alky chain of a minimum of 18 carbon atoms, attached at the 6-po-
sition of the heterocycle, appears to be necessary for both anti-HIV
and anti-HCV activities. We then repeated our above experiment of
HCV NTPase/helicase with 1f and 2e using a double-stranded RNA
substrate, and found that the compounds were equally effective as
with a DNA substrate within a margin of experimental error. All
other compounds listed under 1 and 2 failed to show activity even
N
N
N
(a) n=7
(b) n=9
(c) n=11
(d) n=13
(e) n=15
(f) n=17
NH(CH2)nCH3
NH
O
O
HO
OH
HO
1 (β-anomer)
H
O
HO
N
N
(a) n=7
O
HO
(b) n=9
O
(c) n=13
(d) n=15
(e) n=17
NH
N
NH(H2C)nCH3
2 (α-anomer)
up to 300 lg/mL, although some of them (e.g., 1c and 1d) had ear-
lier exhibited excellent activity against WNV NTPase/helicase.24
In light of the above encouraging anti-enzymic activity, both 1f
and 2e were further screened for in vitro anti-viral activity against

1156
N. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1154–1157
Table 1
w
Inhibitory effect of RENs 1f and 2e against HCV NTPase/helicase1 as well as against the whole virus in tissue culture system assessed by Primary Replicon Assay
w
Compound no.
Inhibition of helicase activity1 of HCV NTPase/helicase
Antiviral activity HCV RNAb
control
%
Toxicity b-actin RNAc
control
%
SId toxicity/antiviral
activity
a
IC50
(
lg/mL)
1f
2e
IFN-
6
0.5
9
1.3
10 0.1
10
98
99
108
3
2
4
10.89
9.9
11.3
5.5 0.4
—
a
(REF1)
1
(10 IU/mL)
Ribavirin (REF2)
—
89 10
12
1
0.14
1
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%.
w
The antiviral activity is based on a primary replicon assay26,27 in Huh7 ET cell line.
a
The term IC50 is defined as the concentration of REN required for 50% inhibition of enzyme activity.
HCV RNA-derived LUC activity is used as an indirect measure of HCV RNA levels.
b-Actin RNA level is used as a positive control for cellular RNA in order to compute cytotoxicity.
b
c
d
Selectivity index (SI) is represented as a ratio of the levels of b-actin RNA/HCV RNA.
Figure 1. Anti-HCV activity of 1f and 2e was assayed in BM 4-5 cell line carrying
subgenomic HCV replicon in Huh7 human hepatoma cells described by Guo
et al.25,32
Figure 2. The cytotoxicity was determined using the Neutral Red Dye assay.30,31
the whole virus as well as cellular toxicity in a Primary Replicon
assay.26,27 The results collected in Table 1 reveal that both activity
and toxicity profiles of 1f and 2e are closely comparable to that of
replicon) was used, which is a relevant model for studying the
effect and mechanism of action of antiviral agents. Dose response
curves were generated and IC50 values were computed. Com-
interferon-
a
, one of the reference compounds normally used for
pounds 1f and 2e were found to have IC50 values of ꢂ16
lM and
the assay. However, the two compounds showed significantly
higher antiviral activity and much lower toxicity as compared with
those of ribavirin, the second reference compound used for the as-
18 M, respectively. They were also found to exhibit low toxicity,
l
if any, at the concentration levels tested, employing Neutral Red
Dye Assay30,31 (Fig. 2).
say. A combination therapy with IFN
a
and ribavirin has long been
Finally, in order to verify if the observed activities of 1f and 2e
are not solely the result of simple, long-chain alkylamines present
at their 6-position, we separately tested two long-chain alkyl-
amines, CH3(CH2)17NH2 and CH3(CH2)13NH2 for their anti-HCV
and anti-HIV activities in vitro, and found them totally inactive
against both viruses. This suggests that the entire structural frame-
work of 1f and 2e, and not simply the long alkyl chains attached at
their 6-position, is necessary for the observed antiviral activities.
In conclusion, we have discovered two nucleoside analogues 1f
and 2e that show promising in vitro antiviral activity against both
HCV and HIV. The long-chain alkyl moiety of a minimum of 18-car-
bon length attached to the 6-position of each is a requirement for
activity. At this time, it is not known if a longer alkyl chain would
further enhance each activity or what the threshold of chain length
is for optimum antiviral activity. In light of HCV being a major
co-infection in HIV patients in the Western World, RENs might rep-
resent a new concept in the treatment of both viruses, and merit
further exploration. It should also be noted that while vast major-
ity of current HCV research is targeted to the NS5 region of HCV
encoding its polymerase RdRp (RNA-dependent RNA polymerase),
our target is the relatively little explored NS3 region of HCV that
encodes its NTPase/helicase. Nonetheless, a distinct possibility that
compounds 1f and 2e could also undergo viral and/or host kinase-
mediated in vivo phosphorylation to their corresponding 50-tri-
phosphates, and the subsequent RdRp-catalyzed incorporation into
viral genome, followed by chain-termination, cannot be ruled out
at this point.
the FDA-approved treatment for HCV.28,29
Since the viral NTPase activity is often coupled to its helicase
activity as mentioned earlier,22 an important question to address
was whether compounds 1f and 2e also inhibited the NTPase activ-
ity of HCV NTPase/helicase. This would give further insight into
mechanism of activity since the compounds could simply be
binding to the NTP binding site of HCV NTPase/helicase to cause
interference in NTP hydrolysis that is required for the viral helicase
activity. The HCV NTPase activity was assessed25 by a standard
assay that determines the amount of Pi released from [c-
32P]ATP
during the enzyme-mediated hydrolysis. The ATPase activity was
measured as a function of increasing concentrations of the inhibi-
tors. However, when investigated at ATP concentration equal to the
KM value of the enzyme (105 l
M for HCV NTPase/helicase),23
neither of the two compounds inhibited the hydrolytic activity of
the enzyme even up to the concentration of 0.7 mM. The succes-
sive lowering of ATP concentration down to value corresponding
to 1 ꢀ 10ꢁ5 of KM also did not lead to any observable inhibition
of the ATPase activity. These results suggest that these inhibitors
did not act via the ATP (NTP)-binding site of HCV NTPase/helicase
as ATP-competitive inhibitors.
As anti-HCV activity was detected in the primary replicon assay
as mentioned above, more thorough investigations were then
made on promising RENs 1f and 2e in order to determine their
IC50 values (see Fig. 1). The BM 4-5 cell line (a human hepatoma
Huh7 cell line harboring an HCV genotype 1b subgenomic

N. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1154–1157
1157
11. Lichterfeld, M.; Haas, S.; Fischer, H.-P.; Voigt, E.; Rockstroh Jurgen, K.; Spengler,
U. J. Gastroenterol. Hepatol. 2005, 20, 739.
Acknowledgments
12. Duclos-Vallee, J.-C.; Vittecoq, D.; Teicher, E.; Feray, C.; Roque-Afonso, A.-M.;
Lombes, A.; Jardel, C.; Gigou, M.; Dussaix, E.; Sebagh, M.; Guettier, C.; Azoulay,
D.; Adam, R.; Ichai, P.; Saliba, F.; Roche, B.; Castaing, D.; Bismuth, H.; Samuel, D.
J. Hepatol. 2005, 42, 341.
13. Mohsen, A. H.; Murad, S.; Easterbrook, P. J. HIV Med. 2005, 6, 206.
14. Pineda, J. A.; Macias, J. J. Antimicrob. Chemother. 2005, 55, 417.
15. Macias, J.; Castellano, V.; Merchante, N.; Palacios, R. B.; Mira, J. A.; Saez, C.;
Garcia-Garcia, J. A.; Lozano, F.; Gomez-Mateos, J. M.; Pineda, J. A. AIDS (London,
United Kingdom) 2004, 18, 767.
We are grateful to Dr. Christoph Seeger for providing Huh7
BM4-5 cell line and to Dr. Ralf Bartenschlager for Huh7 cells carry-
ing HCV-Luciferase replicon (RepLuc). This research was supported
in part by Grants (#1R01 GM087738-01A1 and #1 R21 AI071802)
from the National Institute of General Medical Sciences and the
National Institute of Allergy & Infectious Diseases, respectively, of
the National Institutes of Health.
16. Filippini, P.; Coppola, N.; Scolastico, C.; Liorre, G.; Nocera, R.; Sagnelli, E.;
Piccinino, F. Arch. Virol. 2000, 145, 937.
17. See FDA News Release: May 13, 2011 (Victrelis); May 23, 2011 (Incivek);
November 22, 2013 (Olysio).
Supplementary data
18. Yedavalli, V. S. R. K.; Zhang, N.; Cai, H.; Zhang, P.; Starost, M. F.; Hosmane, R. S.;
Jeang, K.-T. J. Med. Chem. 2008, 51, 5043.
19. Jeang, K.-T.; Yedavalli, V. S. R. K. Nucleic Acids Res. 2006, 34, 4198.
20. Kim, J. L.; Morgenstern, K. A.; Griffith, J. P.; Dwyer, M. D.; Thomson, J. A.;
Murcko, M. A.; Lin, C.; Caron, P. R. Structure (London) 1998, 6, 89.
21. Hodgman, T. C. Nature 1988, 333, 22.
Supplementary data (organic synthetic procedures, spectral and
microanalytical data as well as experimental procedures used for
biochemical and biological assays) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.12.121.
22. Galinari, P.; Brennan, D.; Nardi, C.; Brunetti, M.; Tomei, L.; Steinkühler, C.; De
Francesco, R. J. Virol. 1998, 72, 6758.
23. Zhang, N.; Chen, H.-M.; Koch, V.; Schmitz, H.; Liao, C.-L.; Bretner, M.; Bhadti, V.
S.; Fattom, A. I.; Naso, R. B.; Hosmane, R. S.; Borowski, P. J. Med. Chem. 2003, 46,
4149.
References and notes
24. Zhang, N.; Chen, H.-M.; Koch, V.; Schmitz, H.; Liao, C.-L.; Fattom, A. I.; Naso, R.
B.; Kalicharran, K.; Borowski, P.; Hosmane, R. S. J. Med. Chem. 2003, 46, 4776.
25. Experimental: See Supplementary data for details.
26. Buckwold, V. E.; Wei, J.; Huang, Z.; Huang, C.; Nalca, A.; Wells, J.; Russell, J.;
Collins, B.; Ptak, R.; Lang, W.; Scribner, C.; Blanchett, D.; Alessi, T.; Langecker, P.
Antiviral Res. 2007, 73, 118. for details of HCV assay used, see AACF website:
http://niaid-aacf.org/protocols/HCV.htm.
27. Krieger, N.; Lohmann, V.; Bartenschlager, R. J. Virol. 2001, 75, 4614.
28. Moreno-Otero, R. J. Viral Hepat. 2005, 12, 10.
29. Teo, M.; Hayes, P. Br. Med. Bull. 2004, 70, 51.
1. Piacenti, F. J. Pharmacotherapy 2006, 26, 1111.
2. Yeni, P. J. Hepatol. 2006, 44, S100.
3. Roeling, J.; Schmid, H.; Fischereder, M.; Draenert, R.; Goebel, F. D. Clin. Infect.
Dis. 2006, 42, 1488.
4. Manfredi, R. Expert Rev. Anti-Infect. 2004, 2, 821.
5. Battegay, M. Expert Opin. Investig. Drugs 2001, 10, 175.
6. Streeck, H.; Rockstroh, J. K. Expert Rev. Clin. Immunol. 2006, 2, 811.
7. Jones, R.; Dunning, J.; Nelson, M. Int. J. Clin. Pract. 2005, 59, 1082.
8. Balestra, E.; Aquaro, S.; Perno, C. F. J. Biol. Regul. Homeost. Agents 2003, 17, 138.
9. Antonucci, G.; Goletti, D.; Lanini, S.; Girardi, E.; Loiacono, O. Cell Death Differ.
2003, 10, S25.
10. Merchante, N.; Giron-Gonzalez Jose, A.; Gonzalez-Serrano, M.; Torre-Cisneros,
J.; Garcia-Garcia Jose, A.; Arizcorreta, A.; Ruiz-Morales, J.; Cano-Lliteras, P.;
Lozano, F.; Martinez-Sierra, C.; Macias, J.; Pineda Juan, A. AIDS (London, United
Kingdom) 2006, 20, 49.
30. Babich, H.; Borenfreund, E. Toxicol. In Vitro 1987, 1, 3.
31. Babich, H.; Borenfreund, E. In In Vitro Methods of Toxicology; Watson, R. R., Ed.;
CRC Press: Boca Raton, Florida, 1992; p 237. Chapter 17.
32. Guo, J. T.; Bichko, V. V.; Seeger, C. J. Virol. 2001, 75, 8516.