Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: Further optimization of anti-hepatitis C virus activity

Article abstract of DOI:10.1016/j.bmcl.2007.07.015

Substituted 5-benzyl-2-phenyl-5H-imidazo[4,5-c]pyridines represent a novel class of compounds with activity against pestiviruses and the hepatitis C virus (HCV). Several series of analogues with modifications of the substituents in positions 2 and 5 were prepared. These efforts resulted in the discovery of several compounds with potent antiviral activity of which 2-(2,3-difluorophenyl)-5-[4-(trifluoromethyl)benzyl]-5H-imidazo[4,5-c]pyridine (46) was most potent against HCV (EC₅₀ of 0.10 μM and a selectivity index of 1080).

Full text of DOI:10.1016/j.bmcl.2007.07.015

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5111–5114
Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines:
Further optimization of anti-hepatitis C virus activity
Gerhard Puerstinger,a,* Jan Paeshuyse,b Susanne Heinrich,a Joachim Mohr,a
Nicole Schraffl,a 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 19 May 2007; revised 4 July 2007; accepted 5 July 2007
Available online 13 July 2007
Abstract—Substituted 5-benzyl-2-phenyl-5H-imidazo[4,5-c]pyridines represent a novel class of compounds with activity against pes-
tiviruses and the hepatitis C virus (HCV). Several series of analogues with modifications of the substituents in positions 2 and 5 were
prepared. These efforts resulted in the discovery of several compounds with potent antiviral activity of which 2-(2,3-difluorophenyl)-
5-[4-(trifluoromethyl)benzyl]-5H-imidazo[4,5-c]pyridine (46) was most potent against HCV (EC50 of 0.10 lM and a selectivity index
of 1080).
Ó 2007 Elsevier Ltd. All rights reserved.
Worldwide more than 170 million people are chronically
infected with the hepatitis C virus and thus at increased
risk of developing serious life-threatening liver disease
(including chronic hepatitis, cirrhosis and hepatocellular
carcinoma). Current standard therapy for chronic hepa-
titis C consists of the combination of pegylated IFN-a
2a in combination with ribavirin. However, this therapy
is only effective in 50–60% of the patients and associated
with serious side-effects.1,2 There is, therefore, an urgent
need for potent and non-toxic drugs for the treatment of
HCV infections.
synthesized6 several sets of substituted 5-benzyl-2-phe-
nyl-5H-imidazo[4,5-c]pyridines.
First we have introduced additional substituents start-
ing from the lead compound 1. An additional fluorine
on the 2-phenyl was well tolerated with respect to
anti-BVDV activity,7 but only the 2,3-difluoro ana-
logue 2 was active against HCV in a subgenomic
HCV replicon system8 with slightly improved activity
and selectivity, compared to the lead compound (com-
pounds 2–5, Table 1). The 2,3,6-trifluoro analogue 6
was less active than the lead compound 1 both against
BVDV and HCV. Introduction of a chlorine in posi-
tion 3 of the 2-phenyl resulted in an analogue (7) with
excellent anti-BVDV but slightly reduced anti-HCV
activity. Introducing a trifluoromethyl group in the
same position resulted in reduced antiviral activity
(compound 8). Interestingly, an additional fluorine
in position 2 of the 5-benzyl improved anti-BVDV
activity but was detrimental for anti-HCV activity
(compound 9).
We recently reported on the identification of a class of
imidazo[4,5-c]pyridine analogues with potent anti-pesti-
virus activity.3,4 Pestiviruses belong, together with the
flaviviruses and HCV, to the family of the Flaviviridae.
Introduction of a fluorine atom in position 2 of the
phenyl of 5-(4-bromobenzyl)-2-phenyl-5H-imidazo[4,5-
c]pyridine resulted in a compound with not only anti-
pesti (bovine viral diarrhoea virus (BVDV)) but also
anti-HCV activity (compound 1, Table 1).5 To further
understand the structure–activity relationships for
HCV within this class of compounds and to identify
analogues with improved anti-HCV activity, we have
In the second series the substituent on the 5-benzyl res-
idue was modified (Table 2). In general, these modifica-
tions did not affect the anti-BVDV activities with the
exception of 4-carboxy analogue 19. The unsubstituted
analogue 10 and all three methyl-substituted compounds
(11–13) proved inactive against HCV. All other ana-
logues prepared were substituted in position 4 of the
benzyl. From these the 4-isopropyl (14), the 4-tert-butyl
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 Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.015

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G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5111–5114
Table 1. Structure, anti-BVDV activity, anti-HCV activity and cytotoxic/cytostatic activity of compounds 1–9
F
N
N
N
R1
R2
Br
Compound
R1
R2
BVDV
HCV
SIb
>275
EC50 (lM)
CC50 (lM)
SIb
a
a
a
a
EC50 (lM)
CC50 (lM)
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
F
0.12 0.01
0.24 0.12
0.50 0.28
0.08 0.04
0.48 0.27
0.53 0.10
0.12 0.04
>33
>104
>104
>104
>104
>104
>104
>104
>104
3.0 1.6
0.60 0.2
45 22
75
136 42
>55 27
65
45
3-F
4-F
5-F
6-F
3,6-F2
3-Cl
3-CF3
H
>433
>208
>1300
>217
>196
>867
>17
>92
1.4
115
80
1.5
75
69
1.07
1.1
77
1.2 1.3
47 11
>52
36 1.7
>125
52
30
>2.38
n.a.
6
0.050 0.02
2
>2080
a Data (for active compounds) are mean values standard deviation for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).
Table 2. Structure, anti-BVDV activity, anti-HCV activity and cytotoxic/cytostatic activity of compounds 10–28
F
N
N
N
R
Compound
R
BVDV
HCV
SIb
EC50 (lM)
CC50 (lM)
SIb
a
a
a
a
EC50 (lM)
CC50 (lM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
2-CH3
0.29 0.09
0.29 0.08
0.25 0.11
0.20 0.10
0.36 0.17
0.68 0.07
0.10 0.04
0.25 0.09
0.27 0.10
>52
>104
>104
>104
75
>179
>359
>416
>520
208
>162
95
162
101
85
n.a.
1.06
n.a.
n.a.
125
53
3-CH3
4-CH3
>85
>91
91
4-Isopropyl
4-tert-Butyl
4-Ph
0.080 0.009
0.30 0.2
0.60 0.08
0.20 0.1
>152
10
16
6
6
61
40
89
400
10
4-CF3
4-CN
>104
>104
>104
>104
>104
>104
>104
83
>416
>385
>5
43 30
>152
>144
>150
>144
121
215
n.a.
n.a.
n.a.
n.a.
n.a.
1.53
130
24
4-COOH
4-CH2OH
4-CH2OCH3
4-F
21
4
>144
>150
0.69 0.14
0.67 0.24
0.25 0.14
0.11 0.04
0.18 0.08
0.40 0.13
0.52 0.08
0.16 0.08
0.60 0.20
>151
>155
>416
>945
454
>144
>121
84
4-OCH3
4-OCF3
4-Benzyloxy
4-SCF3
4-NO2
129
26 1.5
0.20 0.03
1.0 0.8
0.30 0.2
115
>104
>104
>104
>104
>260
>200
>650
>173
24
2
40 10
>144
148
133
>1.25
1.38
4-NH2
107
a Data (for active compounds) are mean values standard deviation for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).
(15), the 4-trifluoromethoxy (24) and the 4-benzyloxy
analogue (25) had the best antiviral activity. The 4-phe-
nyl analogue (16) was active but also more toxic with a
selectivity index of only 9. All other 4-substituted ana-
logues proved more or less inactive.
Replacing the 4-bromophenyl part of the 5-(4-bromo-
benzyl) substituent by 2- or 3-pyridyl (29 and 30)
resulted in analogues without anti-HCV activity and
reduced anti-BVDV activity. The 5-cyclohexylmethyl
analogue 31 and the 5-(2-phenoxyethyl) analogue 32

G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5111–5114
5113
Table 3. Structure, anti-BVDV activity, anti-HCV activity and cytotoxic/cytostatic activity of compounds 29–32
F
N
N
N
R
Compound
R
BVDV
HCV
SIb
EC50 (lM)
CC50 (lM)
SIb
a
a
a
a
EC50 (lM)
CC50 (lM)
29
30
31
32
2-Pyridyl
3-Pyridyl
4.5 2.3
2.3 1.2
>104
>104
>104
>104
>23
>44
>164
>164
84
>164
>164
97
n.a.
n.a.
1.15
2
Cyclohexyl
Phenoxymethyl
0.61 0.05
0.28 0.08
>170
>371
48 27
96 33
a Data (for active compounds) are mean values standard deviation for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).
were active against BVDV but devoid of activity against
HCV (Table 3).
methyl)benzyl) resulted in compounds with potent
anti-HCV activity (33, 34, 46 and 47, respectively). An
additional methyl or methoxy function in position 3 of
the 2-phenyl resulted in compounds with reduced activ-
ity against HCV (36, 37, 48 and 49). Similar to the
results obtained with fluorine substitution, an additional
chlorine in position 4, 5 or 6 of the 2-(2-fluorophenyl)
Combining the above findings resulted in analogues with
further improved activity against HCV (Table 4). In par-
ticular the combination of 2-(3-halo-2-fluorophenyl)
with 5-(4-(trifluoromethoxy)benzyl) or 5-(4-(trifluoro-
Table 4. Structure, anti-BVDV activity, anti-HCV activity and cytotoxic/cytostatic activity of compounds 33–56
R1
N
N
N
R2
R3
Compound
R1
R2
R3
BVDV
HCV
SIb
EC50 (lM)
CC50 (lM)
SIb
a
a
a
a
EC50 (lM)
CC50 (lM)
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
F
3-F
3-Cl
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
CF3
0.20 0.02
0.38 0.19
0.49 0.21
0.32 0.11
0.56 0.24
1.7 0.8
>100
>104
>104
78
>500
>274
>212
246
0.20 0.03
0.20 0.16
0.30 0.08
5.2 0.3
53 14
81
>119
405
>595
93
F
F
3-Br
28
2
F
3-CH3
3-OCH3
4-Cl
40 21
>120
>119
>119
>125
58
7.6
>2.27
>1.56
>15
>250
44
F
>104
>104
>104
>104
>104
92
>186
>62
F
76 1.7
7.7 5.7
0.50 0.15
1.3 0.6
36
F
5-Cl
0.32 0.12
0.29 0.09
0.27 0.12
3.9 1.7
>325
>359
>385
23
F
5-CH3
5-OCH3
6-Cl
F
F
73
62
2
F
6-OCH3
H
0.69 0.14
5.7 4.8
91
75
132
13
43 14
1.4
1.23
1.27
1080
403
4.4
2
CF3
OCH3
F
39 4.5
11 1.8
0.10 0.013
0.30 0.17
16 0.8
27
48 3.2
14 1.7
108
121
70
H
3-F
0.050 0.03
0.72 0.19
0.44 0.22
0.38 0.15
0.37 0.10
0.23 0.10
0.80 0.16
0.72 0.06
0.83 0.08
0.18 0.14
0.14 0.07
0.71 0.26
26
520
>104
>104
>104
75
>144
>236
>274
203
F
3-Cl
CF3
CF3
F
3-CH3
3-OCH3
5-CH3
3-F
F
CF3
CF3
55
39
F
>104
>104
>104
89
>452
>130
>144
107
1.3 1.03
0.30 0.069
0.70 0.21
1.6 1.11
4.94
30
F
Isopropyl
Isopropyl
Isopropyl
Cl
33
47
110
67
F
3-Cl
F
5-CH3
3-Cl
3-Cl
45
30
28
F
>104
>104
68
>578
>743
96
6
50
F
I
tert-Butyl
0.43 0.24
0
22
25
F
3-Cl
0.36
71
a Data (for active compounds) are mean values standard deviation for 4–6 independent experiments.
b In vitro selectivity index (CC50/EC50).

5114
G. Puerstinger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5111–5114
substituent resulted in analogues with reduced anti-
HCV activity (38, 39 and 42). Introduction of a methyl
group in position 5 of the 2-phenyl was tolerated in view
of anti-HCV activity (40, 50), whereas a methoxy group in
the same position resulted in reduced activity against this
virus (41). Analogues with an isopropyl group in position
4 of the benzyl (51–53) were less active than the corre-
sponding 4-trifluoromethoxy or 4-trifluoromethyl ana-
logues, as were the analogues with chlorine, iodine or
tert-butyl (54–56). Substituting the fluorine in position 2
of the phenyl in compound 24 by trifluoromethyl or meth-
oxy resulted in analogues with reduced activity (44 and 45,
respectively). Again, most of the modifications were well
tolerated with respect to anti-BVDV activity.
from the Priority 1 ‘Life Sciences, Genomics and
Biotechnology’).
References and notes
1. Zoulim, F.; Chevallier, M.; Maynard, M.; Trepo, C. Rev.
Med. Virol. 2003, 13, 57.
2. Craxi, A.; Licata, A. Semin. Liver Dis. 2003, 23, 35.
3. Puerstinger, G.; Paeshuyse, J.; Herdewijn, P.; Rozenski, J.;
De Clercq, E.; Neyts, J. Bioorg. Med. Chem. Lett. 2006, 16,
5345.
4. Paeshuyse, J.; Leyssen, P.; Mabery, E.; Boddeker, N.;
Vrancken, R.; Froeyen, M.; Ansari, I. H.; Dutartre, H.;
Rozenski, J.; Gil, L. H. V. G.; Letellier, C.; Lanford, R.;
Canard, B.; Koenen, F.; Kerkhofs, P.; Donis, R. O.;
Herdewijn, P.; Watson, J.; De Clercq, E.; Puerstinger, G.;
Neyts, J. J. Virol. 2006, 80, 149.
This class of compounds was shown to target HCV
polymerase. The exact molecular mechanism by which
these compounds inhibit HCV replication will be pub-
lished elsewhere. We reported earlier on the fact that
the anti-pestivirus activity of this class of compounds
can be explained by an interaction at the tip of the fin-
gerdomain of the pestivirus polymerase.4
5. Puerstinger, G.; Paeshuyse, J.; De Clercq, E.; Neyts, J.
Bioorg. Med. Chem. Lett. 2007, 17, 390.
6. All compounds were synthesized as described before3,5 by a
two-step route, starting from 3,4-diaminopyridine and a
substituted benzoic acid, followed by alkylation of the
intermediate substituted 2-phenyl-imidazo[4,5-c]pyridine.
Synthesis of and analytical data for compound 46: prepared
from 2-(2,3-difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine
and 4-(trifluoromethyl)benzyl bromide; recrystallized from
a mixture of diisopropyl ether and ethyl acetate to give a
colourless powder; yield: 58%; mp 209 °C; 1H NMR
(200 MHz, DMSO-d6): d 9.27 (d, 1H, imidazopyridine-
H4, J = 1.4 Hz), 8.25 (dd, 1H, imidazopyridine-H6, J = 6.8,
1.4 Hz), 8.17–8.09 (m, 1H, phenyl-H6), 7.86–7.76 (m, 3H,
arom. H), 7.66–7.60 (AA0BB0, 2H, benzyl-H), 7.55–7.42 (m,
1H, phenyl-H), 7.36–7.24 (m, 1H, phenyl-H), 5.82 (s, 2H,
CH2); MS (CI): 390.1 [M+ +1, 44%]. Anal. calcd for
C20H12F5N3: C, 61.70; H, 3.11; N, 10.79. Found: C, 61.63;
H, 3.24; N, 10.72.
In summary, we have prepared several sets of analogues
of the lead compound 1 with modifications both at the
2-phenyl and 5-benzyl position. Whereas many modifi-
cations were well tolerated with respect to anti-BVDV
activity, only few modifications resulted in analogues
with anti-HCV activity. Combination of these findings
allowed us to synthesize analogues (e.g., 34 and 46) with
potent and selective activity on HCV replication.
Acknowledgments
7. For a detailed procedure regarding the anti-BVDV screen-
ing, see Ref. 4.
8. Paeshuyse, J.; Kaul, A.; De Clercq, E.; Rosenwirth, B.;
Dumont, J.-M.; Scalfaro, P.; Bartenschlager, R.; Neyts, J.
Hepatology 2006, 43, 761.
´
We thank Katrien Geerts and Geoffrey Ferir for excel-
lent technical assistance. This work was supported by
VIRGIL, the European Network of Excellence on Anti-
viral Drug Resistance (Grant LSHM-CT-2004-503359