Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: From anti-pestivirus to anti-hepatitis C virus activity

Article abstract of DOI:10.1016/j.bmcl.2006.10.039

A novel class of inhibitors of the hepatitis C virus [substituted 2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridines] is described. Introduction of a fluorine in position 2 of the 2-phenyl substituent of the lead anti-pestivirus compound 1 (5-[(4-bromophenyl)m

Full text of DOI:10.1016/j.bmcl.2006.10.039

Bioorganic & Medicinal Chemistry Letters 17 (2007) 390–393
Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines:
From anti-pestivirus to anti-hepatitis C virus activity
Gerhard Puerstinger,a,* Jan Paeshuyse,b Erik De Clercqb and Johan Neytsb
aInstitut fu¨r Pharmazie, Abteilung Pharmazeutische Chemie, Universita¨t Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 7 September 2006; revised 15 October 2006; accepted 16 October 2006
Available online 19 October 2006
Abstract—A novel class of inhibitors of the hepatitis C virus [substituted 2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridines] is
described. Introduction of a fluorine in position 2 of the 2-phenyl substituent of the lead anti-pestivirus compound 1 (5-[(4-brom-
ophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) resulted in an analogue with selective activity against HCV in the subgenomic
replicon system.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is an enveloped single stranded
(+) RNA virus that belongs to the separate genus
Hepacivirus of the family Flaviviridae.1 HCV causes
acute and chronic liver disease, including chronic hepa-
titis, cirrhosis, and hepatocellular carcinoma.2 World-
wide more than 170 million people are chronically
infected with HCV3 and are thus at increased risk of
developing serious life-threatening liver disease. Current
standard therapy for chronic hepatitis C consists of
pegylated IFN-a 2a in combination with ribavirin.4
The bovine viral diarrhea virus (BVDV) is the prototype
of the Pestivirus genus of the Flaviviridae family. BVDV
is a major pathogen of cattle and causes a range of clin-
ical manifestations varying from subclinical symptoms
to death.5 For the United States alone this translates
roughly into a loss of $10 to 40 million per million
calvings.6
To further understand the structure–activity relation-
ships within this class of compounds and to further opti-
mize the antiviral activities we have synthesized
additional analogues with modifications in position 2
of the imidazo[4,5-c]pyridine ringsystem, keeping the
5-(4-bromobenzyl) substituent as a constant. In a first
effort, we have introduced substituents into the 2-phenyl
(compounds 2–17, Table 1).9 With respect to anti-
BVDV activity, most substituents were tolerated, result-
ing in analogues with similar or slightly reduced activity,
with the exception of the 3,5-dibromo analogue 17,
which turned out to be totally inactive against BVDV.
Surprisingly, the 2-fluoro analogue 8 exhibited potent
and selective activity against HCV in the replicon
system.12
In a second set of analogues (Table 2) the 2-phenyl ring
was replaced by ethyl (compound 18), naphthyl or a
(substituted) heterocycle (compounds 19–25), or a linker
was introduced between the central imidazo[4,5-c]pyri-
dine and the 2-phenyl substituent (compounds 26–32).
The 2-ethyl analogue 18 showed reduced anti-BVDV
activity and was inactive against HCV. The 2-naphthyl
analogue 20 was more active than its 1-naphthyl isomer
19 against BVDV, but was also somewhat more cytotox-
ic. All heterocyclic analogues exhibited anti-BVDV
activity, whereas only the 2-pyridyl analogue 21 exhibit-
ed anti-HCV activity, being more cytostatic than the 2-
(2-fluorophenyl) analogue 8. Introduction of a linker
resulted in compounds that were inactive against BVDV
with the exception of the 2-(E)-styryl analogue 32. None
We have recently reported on 5-benzyl-2-phenyl-5H-
imidazo[4,5-c]pyridines as a new class of anti-pestiviral
compounds.7,8 Modification of the substitution pattern
on the 5-benzyl substituent resulted in an analogue (1)
with high activity and selectivity against BVDV, but this
analogue was inactive against HCV.
Keywords: Imidazo[4,5-c]pyridines; BVDV inhibitor; HCV inhibitor;
Antiviral.
*
Corresponding author. Tel.: +43 512 507 5260; fax +43 512 507
2940; e-mail: Gerhard.Puerstinger@uibk.ac.at
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.039

G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 390–393
391
Table 1. Structure, anti-BVDV activity, anti-HCV activity, and cytotoxic/cytostatic activity of compounds 1–17
N
N
N
R
Br
Compound
R
BVDV
HCV
SIb
EC50 (lM)
CC50 (lM)
SIb
a
a
a
a
EC50 (lM)
CC50 (lM)
1
2
H
0.070 0.02c
0.061 0.009
0.06 0.04
0.4 0.2
83 20
>33
23
1186
>541
372
>38
24
38
21
n.a.
n.a.
n.a.
2
2-CH3
3-CH3
4-CH3
2-Cl
3
>11
8.2
11
13
4
21
53
5
0.33 0.05
0.15 0.08
0.7 0.1
>33
>33
>33
>33
>33
>33
>33
>33
>33
24
>100
>220
>47
25
83
43
>125
17
2
6
3-Cl
4-Cl
>2
n.a.
52
7
13
8
2-F
3-F
0.12 0.01
0.04 0.01
0.17 0.06
0.5 0.2
>275
>825
>194
>66
1.0 0.6
>44
31
52 16
44
37
9
n.a.
n.a.
2
10
11
12
13
14
15
16
17
4-F
2-Br
21
>113
18
36
3-Br
0.11 0.02
0.05 0.02
0.14 0.01
0.4 0.1
>300
>660
171
>113
28
79
n.a.
2
3-OCH3
3-N(CH3)2
3-I
>79
5.7
n.a.
2
>33
>33
>33
>83
>72
12
>116
>96
3-CF3
3,5-Br2
0.5 0.1
23
>96
>5
n.a.
>33
n.a.
The EC50 (50% effective concentration) is the concentration of compound that offered 50% protection of the cells against virus-induced cytopathic
effect or the 50% reduction of luciferase signal in case of HCV.
The CC50 (50% cytostatic concentration) is the concentration that inhibited the proliferation of exponentially growing cells by 50%. n.a., not active.
a Data (for active compounds) are mean values SD for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).
c The here reported value does not significantly differ from the reported value.8
Table 2. Structure, anti-BVDV activity, anti-HCV activity, and cytotoxic/cytostatic activity of compounds 18–32
N
R
N
N
Br
Compound
R
BVDV
HCV
a
a
a
a
EC50 (lM)
CC50 (lM)
SIb
EC50 (lM)
CC50 (lM)
SIb
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Ethyl
0.38 0.09
0.3 0.2
0.030 0.009
0.10 0.02
0.16 0.01
0.47 0.07
0.04 0.02
0.6 0.4
>33
>33
23
>87
77
>92
9.7
92
12
n.a.
n.a.
2
1-Naphthyl
2-Naphthyl
2-Pyridyl
7
10
259
96
5. 7
0.35 0.3
109
14
9.7
5.2 1.9
126
38
15
3-Pyridyl
4-Pyridyl
>33
>33
>33
>33
>33
16
>206
>70
>825
>55
n.a.
n.a.
8
n.a.
3
2-Thienyl
5-Bromo-2-thienyl
Benzyl
>35
>18
79
35
18
n.a.
n.a.
>2
3
>132
7.6
12
2-Phenylethyl
3-Phenyl-1-propyl
4-Phenyl-1-butyl
Phenoxymethyl
Phenylthiomethyl
(E)-styryl
>16
3
2.5
7.4
1
22
5
2
>5
>8
>25
n.a.
4
>2.2
25
2.2
35
n.a.
1.5
2
>33
25
n.a.
88
4.9
1.7
9.7
2.5
0.16 0.08
14
>2
The EC50 (50% effective concentration) is the concentration of compound that offered 50% protection of the cells against virus-induced cytopathic
effect or the 50% reduction of luciferase signal in case of HCV.
The CC50 (50% cytostatic concentration) is the concentration that inhibited the proliferation of exponentially growing cells by 50%. n.a., not active.
a Data (for active compounds) are mean values SD for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).

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G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 390–393
Table 3. Structure, anti-BVDV activity, anti-HCV activity, and cytotoxic/cytostatic activity of compounds 33 and 34
F
N
N
N
R
Compound
R
BVDV
HCV
a
a
a
a
EC50 (lM)
CC50 (lM)
SIb
EC50 (lM)
CC50 (lM)
SIb
1.5
74
33
34
Cl
I
0.11 0.05
0.07 0.02
>33
>33
>300
>478
44
68
58
0.79
The EC50 (50% effective concentration) is the concentration of compound that offered 50% protection of the cells against virus-induced cytopathic
effect or the 50% reduction of luciferase signal in case of HCV.
The CC50 (50% cytostatic concentration) is the concentration that inhibited the proliferation of exponentially growing cells by 50%.
a Data (for active compounds) are mean values SD for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).
of these analogues exhibited significant anti-HCV
activity.
VIRGIL, the European Network of Excellence on Anti-
viral Drug Resistance (Grant LSHM-CT-2004-503359
from the Priority 1 ‘Life Sciences, Genomics and
Biotechnology’).
In an attempt to further prove the importance of the 2-
fluoro substituent in compound 8 on HCV activity, two
5-(4-halobenzyl) analogues (33 and 34) were prepared
(Table 3). Both of these proved active against BVDV,
but only the iodo analogue 34 was active against
HCV. The size of the substituent at position 4 of the
5-benzyl may thus be a critical component of the anti-
HCV activity of this class of compounds.
References and notes
1. Bartenschlager, R.; Frese, M.; Pietschmann, T. Adv.Virus
Res. 2004, 63, 71.
2. Pawlotsky, J. M. Trends Microbiol. 2004, 12, 96.
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5. Lindberg, A. L. Vet. Q. 2003, 25, 1.
In summary, introduction of a fluorine in position 2 of
the 2-phenyl substituent of the lead compound 1 result-
ed in analogues with activity against hepatitis C virus.
Interestingly, the structure–activity relationships for this
class of compounds against BVDV and HCV are rather
different. We show here, for the first time, that the
appropriate modification of a scaffold of non-nucleoside
inhibitors of pestiviruses may result in compounds with
activity against the related HCV. We have thus identi-
fied, to the best of our knowledge for the first time,
non-nucleoside compounds that exhibit potent activity
against both pestiviruses and HCV. We recently re-
vealed that imidazo[4,5-c]pyridines inhibit pestivirus
replication by targeting the viral polymerase in the close
neighborhood of residue F224.8 Since (i) the loop do-
main of the BVDV RdRp that carries the F224 residue
is absent in the HCV RdRp and (ii) the conformation
of the finger domain of the RdRp polymerase is different
from that of the BVDV polymerase,13 it is conceivable
that the precise molecular mechanism by which these
compounds inhibit the replication of BVDV and HCV,
respectively, is different. This will be subject of further
investigation.
6. Houe, H. Biologicals 2003, 31, 137.
7. Puerstinger, G.; Paeshuyse, J.; Herdewijn, P.; Rozenski, J.;
De Clercq, E.; Neyts, J. Bioorg. Med. Chem. Lett. 2006,
16, 5345.
8. Paeshuyse, J.; Leyssen, P.; Mabery, E.; Boddeker, N.;
Vrancken, R.; Froeyen, M.; Ansari, I. H.; Dutartre, H.;
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Canard, B.; Koenen, F.; Kerkhofs, P.; Donis, R. O.;
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9. All new compounds were fully characterized by melting
point, NMR, MS, and HRMS spectra. The syntheses of
the intermediate 2-substituted imidazo[4,5-c]pyridines
were performed in analogy to published procedures.7,10,11
The intermediates for compounds 2–12, 14–24, 26, and
33–34 were prepared by reaction of 3,4-diaminopyridine
with the corresponding carboxylic acid in polyphosphoric
acid (4 h at 190 ꢁC, with an exception in the case of
propionic acid, where the temperature of the reaction
mixture was kept at 150 ꢁC in the first hour, before heating
to 190 ꢁC for 3 h). The cyclizations with 3-methoxybenzoic
acid and 5-bromothiophene-2-carboxylic acid were per-
formed in refluxing POCl3. The intermediates for ana-
logues 27–32 were obtained by reacting 3,4-
diaminopyridine with 3 equiv of the corresponding car-
boxylic acids at 160 ꢁC for 20 h (neat), followed by
washing with diisopropyl ether to remove the excess acid
and recrystallization from an appropriate solvent mixture
(e.g., diisopropyl ether/ethyl acetate). The final benzyla-
Acknowledgments
´
We thank Katrien Geerts and Geoffrey Ferir for excel-
lent technical assistance. This work was supported by

G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 390–393
393
tions were performed as described before.7 Compound 8:
recrystallized from a mixture of diisopropyl ether and
ethyl acetate; off-white powder; mp 156 ꢁC; yield: 53%; 1H
incubation at 37 ꢁC, cell culture medium was removed and
luciferase activity was determined using the Steady-Glo
luciferase assay system (Promega, Leiden, The Nether-
lands); the luciferase signal was measured using a Lumi-
noskan Ascent (Thermo, Vantaa, Finland). The 50%
effective concentration (EC50) was defined as the concen-
tration of compound that reduced the luciferase signal by
50%. Cytostatic assay. MDBK or Huh 5-2 cells were
seeded at a density of 5 · 103 cells per well of a 96-well
plate in MEM-FCS; 24 h later, serial dilutions of the test
compounds were added. Cells were allowed to proliferate
for 3 days at 37 ꢁC, after which the cell number was
determined by means of the MTS/PMS method (Prome-
ga). The percentage cell growth was calculated as follows:
NMR (200 MHz, DMSO-d6)
d 9.18 (d, 1H, H4,
J = 1.6 Hz), 8.35–8.26 (m, 1H, arom. H), 8.20 (dd, 1H,
H6, J = 6.8, 1.6 Hz), 7.78 (d, 1H, H7, J = 6.8 Hz), 7.64–
7.58 (AA’BB’, 2H, arom. H), 7.52–7.24 (m, 5H, arom. H),
5.66 (s, 2H, CH2); HRMS (ESI+): m/z calcd for
C19H14BrFN3 (M+H)+ 382.0355; found 382.0358.
10. Robertson, D. W.; Beedle, E. E.; Krushinski, J. H.;
Pollock, G. D.; Wilson, H.; Wyss, V. L.; Hayes, J. S. J.
Med. Chem. 1985, 28, 717.
11. Buckle, D. R.; Foster, K. A.; Taylor, J. F.; Tedder, J. M.;
Thody, V. E.; Webster, R. A. B.; Bermudez, J.; Markwell,
R. E.; Smith, S. A. J. Med. Chem. 1987, 30, 2216.
(ODtreated)/(ODcontrol);
in
which
(ODtreated) = the
12. Anti-HCV assay in Huh 5-2 cells. Huh 5-2 cells were
seeded at a density of 5 · 103 per well in a 96-well white
view plate (Packard, Canberra, Canada) in complete
DMEM supplemented with 250 lg/ml G418. Following
incubation for 24 h at 37 ꢁC (5% CO2) medium was
removed and threefold serial dilutions in complete
DMEM (without G418) of the test compounds were
added in a total volume of 100 ll. After 4 days of
OD490 nm of cells treated with a certain dilution of
compound, (ODcontrol) = the OD490 nm of cells left untreat-
ed. The 50% cytostatic concentration (CC50) was defined
as the concentration that inhibited the proliferation of
exponentially growing cells by 50% and was calculated
using logarithmic interpolation.
13. Choi, K. H.; Gallei, H.; Becher, P.; Rossmann, M. G.
Structure 2006, 14, 1107.