Identification of thieno[3,2-b]pyrroles as allosteric inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2006.05.012

Thieno[3,2-b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently blocks subg

Full text of DOI:10.1016/j.bmcl.2006.05.012

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4026–4030
Identification of thieno[3,2-b]pyrroles as allosteric inhibitors
of hepatitis C virus NS5B polymerase
*
`
Jesus M. Ontoria, Jose I. Martın Hernando, Savina Malancona, Barbara Attenni,
Ian Stansfield, Immacolata Conte, Caterina Ercolani, Jo¨rg Habermann, Simona Ponzi,
Marcello Di Filippo, Uwe Koch, Michael Rowley and Frank Narjes
Department of Medicinal Chemistry, IRBM-MRL Rome, Via Pontina, Km 30.600, 00040 Pomezia (Rome), Italy
Received 9 February 2006; revised 2 May 2006; accepted 3 May 2006
Available online 22 May 2006
Abstract—Thieno[3,2-b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which
show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently
blocks subgenomic HCV RNA replication in HUH-7 cells with an EC50 of 2.9 lM.
Ó 2006 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is a major world health prob-
lem that affects an estimated 170 million individuals
worldwide.1 Despite considerable reduction of the inci-
dence of new infections due to the improvements in
blood screening in many countries,2 HCV has become
the leading cause of liver transplantation worldwide
and results in up to 10,000 deaths annually in the U.S.
alone.3 Current treatments based on interferon-a are
effective in only a fraction of the patient population
who are infected with HCV genotype 1 that predomi-
nates in industrialized nations.4 These treatments are
also poorly tolerated and expensive, adding urgency to
the need for novel therapeutic agents to combat HCV.
that bind at the active site of NS5B have been found
to show potency in the submicromolar range.7 Con-
versely, allosteric inhibition by small-molecule inhibitors
of NS5B has emerged as a bona fide route toward inhi-
bition of subgenomic HCV RNA replication, and a
number of structurally diverse inhibitor classes have
now been identified.8
In initial SAR studies on small-molecule inhibitors with
a benzimidazole core we and others identified benzimid-
azoles such as 1a as inhibitors of the NS5B polymerase.9
This molecule possesses a benzimidazole core with
pendant aryl functionality at C2, cycloalkyl functional-
ity at N1, and a carboxylic acid group at C5. All three
groups were identified as essential for the activity of this
series of inhibitors. Replacement of the central core by
the more lipophilic indole ring led to 2a which displays
nanomolar potency against NS5B polymerase and
shows efficacy in the replicon assay.9a
The virally encoded HCV non-structural proteins are
attractive targets for antiviral therapy as they have been
shown to play an essential role in the replication of the
virus.5 In particular, NS5B RNA-dependent RNA poly-
merase has emerged as an especially attractive target for
drug discovery efforts.6 The absence of a functional
counterpart to NS5B in mammalian cells, where DNA
is usually the template during DNA transcription, may
favor the development of selective and nontoxic inhibi-
tors. Toward this goal several series of NS5B inhibitors
that show activity in the replicon assay have been
reported.7,8 Chain terminating nucleoside analogues
R1
HO2C
HO2C
N
N
N
R
2a R1: H
2b R1: CH2CONMe2
1a R: H
1b R: OMe
1
O
N
2c R : CH2CO
Keywords: HCV NS5B polymerase; Allosteric inhibitors; Thieno[3,2-b]-
pyrrole.
1a
1b
2a
2b
2c
IC50 6.5 μM
EC50 >50 μM
IC50 0.048 μM EC50 6.7 μM
IC50 0.059 μM EC50 1.5 μM
IC50 0.026 μM EC50 0.8 μM
IC50 11.2 μM EC50 >50 μM
*
Corresponding author. Tel.: +39 06 91093520; fax: +39 06
91093654; e-mail: jesus_ontoria@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.012

J. M. Ontoria et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4026–4030
Table 1. SAR in the central core: NS5B enzyme inhibition (IC50
4027
12
)
Introduction of an acetamide side chain at the position N1
of the indole ring, as in 2b, led to a 5-fold improvement in
cell-based activity over the N-unsubstituted indole 2a.9a
Benzimidazole inhibitors were shown by us10,11 and
others6b to bind to the thumb domain of NS5B and repli-
cons resistant to this class of inhibitors harbor a single es-
cape mutation of Pro495, located in the upper thumb
region, to either Leu or Ala.11 This was confirmed by
the crystal structure of NS5B complexed with indole 2c,
where it occupies a well-defined lipophilic site with the in-
dole ring stacked against the side chain of Pro495, the
cyclohexyl and phenyl ring substituents filling two closely
spaced pockets, and the carboxylate substituent forms a
salt bridge with the side chain of Arg503.10 Binding of
the inhibitors to the site in the thumb domain prevents
interactions with loops extending from the finger domain.
It is hypothesized that this locks the polymerase in an
unproductive conformation.10
Compound
IC50 (lM)a
HO2C
N
N
O
3
77.3
13.8
17.2
na
S
N
HO2C
HO2C
HO2C
N
4
5
6
7
8
N
S
N
N
N
N
H
In parallel to the work done in the indole series, we also
explored the replacement of the 6,5-bicyclic core of
benzimidazole 1a with other scaffolds, and present here
the initial results of this work. Trisubstituted imidazole 3
(Table 1), where the 6-membered ring is replaced by a
double bond, shows a 7-fold decrease in activity with re-
spect to benzimidazole 1b.12 This loss in potency can be
explained by two factors: the higher flexibility of the
acid moiety with respect to 1b, that would weaken a
critical hydrogen bond interaction with the protein,
and the absence of the carbons of the benzo moiety,
which engage in a lipophilic interaction in the binding
pocket.10 Next, we focused our work on the study of
other bicyclic systems exploring the effect of the ring size
and position of the heteroatoms. Introduction of a
5-membered ring, as in thieno[2,3-d]imidazole 4, led to
2-fold loss in enzymatic activity which may be due to
a slightly different orientation of the carboxylic acid.
Similar potency was observed in the regioisomeric thi-
eno[2,3-d]imidazole 5. Interestingly, introduction of a
pyrrole ring as in 6 led to a complete loss of activity
probably due to the basic character (calcd pKa 9.6) of
the 3,4-dihydropyrrolo[2,3-d]imidazole 6 compared with
benzimidazole 1a (calcd pKa 5.4). The replacement of
the benzimidazole core by fused 6,6-bicyclic scaffolds
gave quinoline 7 which showed similar potency to our
lead compound 1a. By contrast, the isomeric quinoline
8, with the carboxylic acid differently positioned in
space, was not active. An overlay of quinoline 7 with
benzimidazole 1a showed a good overlap between the
three key functional groups which we believe to be
important for activity (see Fig. 1). Quinoline 8 presented
the same good overlap of the cyclohexyl and phenyl
groups, but the acid is in a different position, possibly
explaining the difference in potency. Finally, indolizine
9, with a 6,5-bicyclic scaffold, displayed a 70-fold in-
crease in potency with respect to the benzimidazole 1a.
This is a result of a less polar core favoring interaction
at a lipophilic site of the enzyme as was observed previ-
ously for indole 2a.9a
HO2C
HO2C
N
4.8
N
N
na
HO2C
9
0.094
a na means IC50 > 50 lM.
insufficient potency in the case of compounds 3–8,
whereas indolizine 9, which is only 2-fold less active than
2a, was found to be unstable in the medium used for the
cell-based assay.
A first hint of cell-based activity was observed when we
made and tested the 5-azaindole 10a (Table 2). This
compound showed activity in the low micromolar range
on the enzyme, but measurable inhibition of the replicon
(EC50 = 48.2 lM). The loss of potency with respect to 2a
was thought to be due to the position and the basicity
of the newly introduced nitrogen (calcd pKa 8.6) in a
lipophilic environment. Conversely, the less basic
7-azaindole 11a (calcd pKa 5.4) is 12-fold more active
on the enzyme, confirming the hypothesis, but only
3-fold more active in the replicon assay with respect to
10a.
We have found that the introduction of a N,N-dimethyl-
acetamide chain in indole 2b improved cell-based poten-
cy, probably by modulating the physicochemical
properties of the molecule. For example, it was noted
Unfortunately, all the compounds from Table 1 were
not active up to 100 lM concentration in a HCV
RNA replication assay. This can be explained by

4028
J. M. Ontoria et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4026–4030
As shown in thieno[2,3-d]imidazole 4, replacement of
the phenyl ring by a thiophene in the bicyclic scaffold
is tolerated, and consequently this was also applied to
the indole scaffold. Thieno[2,3-b]pyrrole 12a is a potent
inhibitor of NS5B polymerase, being only 3-fold less
active than indole 2a. Interestingly, the regioisomeric
thieno[3,2-b]pyrrole 13a is nearly as potent as indole
2a. Due to the bigger length of the C-S versus the
C–C bond, the spatial orientation of the carboxylic acid
is different between the two regioisomers and also in
comparison to 2a. The overlap is better for the more
active thieno[3,2-b]pyrrole 13a (Fig. 1). Compound 13a
showed a 170-fold loss in potency in the cell-based assay
(EC50 = 11.2 lM) with respect to the enzyme assay. As
shown previously, introduction of the N,N-dimethylace-
tamide group resulted in a less lipophilic compound 13b
(log D 1.6 vs 3.4 for 13a) and a 4-fold improvement in
replicon activity (EC50 = 2.9 lM). For the less active
thieno[2,3-b]pyrrole 12a a 2-fold gain in the replicon
assay was seen with the acetamide group. In order to
determine the mechanism of inhibition of this series of
inhibitors, 13a was tested against NS5B bearing the
P495L mutation. Compound 13a lost around two orders
of magnitude in potency (IC50 = 4 lM), showing to bind
at the same binding site as indole 2c.9a,10
Figure 1. Overlapping of quinolines 7 (orange) and 8 (green) with
indole 2a (gray) (left) and of thienopyrroles 12a (orange) and 13a
(green) with indole 2a (gray) (right).
Table 2. Trisubstituted azaindoles and thienopyrroles: NS5B enzyme
12
inhibition (IC50) and cell-based efficacy (EC50
)
Compounda
IC50
(lM)
EC50
(lM)
log Dc,13
b
R
HO2C
N
10
11
12
13
a 1.9
b 2.2
48.2
na
2.5
0.8
N
N
Some compounds described in Table 1 were accessed
by synthetic procedures described in the literature.14
Indolizine 9 was prepared as outlined in Scheme 1.
Alkylation of 2-methylpyridine-5-carboxylic acid 14
in the substituent in position 2 was followed by
hydrogenation of the cyclohexene ring and in situ
esterification to give the intermediate 15. Reaction of
compound 15 with 2-bromoacetophenone followed by
treatment with NaHCO3 led to the formation of the
indolizine system.15 Final hydrolysis of the ester with
KOH led to compound 9.
R
HO2C
N
a 0.15
b 0.32
16.8
9.8
3.3
1.1
R
S
N
HO2C
a 0.12
b 0.23
19.5
9.5
nd
nd
Compound 11b was synthesized starting from commer-
cially available pyridine 16. Esterification of the carbox-
ylic group with AcCl/MeOH led to concomitant
deprotection of the amide in position 2. Subsequent reg-
ioselective bromination afforded the intermediate 17.
Construction of the azaindole system was done by Sono-
gashira reaction with 1-phenyl-2-(trimethylsilyl)acety-
lene and subsequent ring closure in the presence of Pd
catalyst.16 Subsequent bromination with NBS in the
position 2 followed by N-alkylation with tert-butyl
bromoacetate gave the intermediate 18. Reaction of 18
with phenylboronic acid under Suzuki-type cross-
coupling conditions led to the introduction of the
substituent at the position C2 as described in Scheme
2. Elaboration of the N1 chain by selective ester
deprotection and amide coupling, followed by
R
N
HO2C
a 0.066 11.2
b 0.058 2.9
3.4
1.6
S
a a R: H, b R: CH2CONMe2.
b na means EC50 > 50 lM.
c nd means not determined.
that the log D decreased by two units (compare 2b log D
2.1 with 2a calcd log D 4.2).13 This same behavior was
observed for the 7-azaindole series, where the acetamide
11b (log D 1.1), despite the 2-fold loss in intrinsic poten-
cy, is nearly 2-fold more active in the cell-based assay
with respect to 11a (log D 3.3). However, this was not
the case for the 5-azaindole scaffold, where the introduc-
tion of the acetamide lowered the log D from 2.5 for 10a
to 0.8 for 10b, but not the basicity of the azaindole
scaffold (calcd pKa 7.6), with the consequence that no
improvement in replicon activity was noted for 10b with
respect to 10a.
HO2C
MeO2C
a, b
N
N
c, d, e
9
14
15
Scheme 1. Reagents and conditions: (a) n-BuLi, 3-bromocyclohexene,
THF, À78 °C; (b) H2, Pd/C; TMSCHN2 (30% over two steps); (c)
2-bromoacetophenone, acetone, reflux; (d) NaHCO3; (e) KOH (50%
over three steps).

J. M. Ontoria et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4026–4030
4029
HO2C
N
NHAc
MeO2C
N
NH2
Br
Acknowledgments
a, b
c, d, e
The authors thank Monica Bisbocci and Nadia Gennari
for biological testing; and Fabio Bonelli, Anna Alfieri,
and Vincenzo Pucci for analytical work.
16
17
O
O
MeO2C
N
N
f, g, h, i
Br
11b
References and notes
1. Cohen, J. Science 1999, 285, 26.
18
2. EASL International Consensus Conference on Hepatitis
C, Consensus Statement. J. Hepatol. 1999, 30, 956.
3. Steedman, S. A.; Younossi, Z. M. J. Clin. Gastroenterol.
2000, 30, 125.
4. Dymock, B. W. Emerging Drugs 2001, 6, 13.
5. Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C.
M. J. Virol. 2000, 74, 2046.
Scheme 2. Reagents and conditions: (a) AcCl, MeOH, reflux (64%);
(b) Br2, CHCl3, rt (28%); (c) C6H11CꢀCTMS, Pd(dppf)Cl2, LiCl,
t
Na2CO3, DMF, 110 °C (63%); (d) BrCH2CO2 Bu, NaH, DMF, 60 °C;
(e) NBS, DCM, rt (48% over two steps); (f) C6H5B(OH)2, Pd(PPh3)4,
K3PO4, toluene, 110 °C (61%); (g) TFA, DCM (100%); (h) Me2N-
HÆHCl, HATU, DIPEA, DMF, RT (100%); (i) BBr3, DCM (42%).
6. (a) Wu, J. Z.; Hong, Z. Curr. Drug Targets–Infect. Dis.
2003, 3, 207; (b) LaPlante, S.; Jakalian, A.; Aubry, N.;
Bousquet, Y.; Ferland, J.-M.; Gillard, J.; Lefebvre, S.;
Poirier, M.; Tsantrizos, Y. S.; Kukolj, G.; Beaulieu, P. L.
Angew. Chem., Int. Ed. 2004, 43, 4306; (c) Tan, S.-L.; He,
Y.; Huang, Y.; Gale, M., Jr. Curr. Opin. Pharmacol. 2004,
4, 465; (d) Beaulieu, P. L.; Tsantrizos, Y. S. Curr. Opin.
Investig. Drugs 2004, 5, 838; (e) Gordon, C. P.; Keller, P.
A. J. Med. Chem. 2005, 48, 1; (f) Condon, S. M.; LaPorte,
M. G.; Herbertz, T. Curr. Med. Chem.–Anti-Infective
Agents 2005, 4, 99; (g) De Francesco, R.; Migliaccio, G.
Nature 2005, 436, 953.
7. Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.;
Stahlhut, M. W.; Eldrup, A. B.; Bhat, B.; Hall, D.; Simcoe,
A. L.; LaFemina, R.; Rutkowski, C. A.; Bohdan, W.; Yang,
Z.; Migliaccio, G.; De Francesco, R.; Kuo, L. C.; MacCoss,
M.; Olsen, D. B. J. Biol. Chem. 2003, 278, 11979.
8. (a) Dhanak, D.; Duffy, K. J.; Johnston, V. K.; Lin-
Goerke, J.; Darcy, M.; Shaw, A. N.; Gu, B.; Silverman,
C.; Gates, A. T.; Nonnemacher, M. R.; Earnshaw, D. L.;
Casper, D. J.; Kaura, A.; Baker, A.; Greenwood, C.;
Gutshall, L. L.; Maley, D.; DelVecchio, A.; Macarron, R.;
Hofmann, G. A.; Alnoah, Z.; Cheng, H.-Y.; Chan, G.;
Khandekar, S.; Keenan, R. M. J. Biol. Chem. 2002, 277,
38322; (b) Chan, L.; Das, S. K.; Reddy, T. J.; Poisson, C.;
Proulx, M.; Pereira, O.; Courchesne, M.; Roy, C.; Wang,
W.; Siddiqui, A.; Yannopoulos, C. G.; Nguyen-Ba, N.;
Labrecque, D.; Bethell, R.; Hamel, M.; Courtemanche-
Asselin, P.; L’Heureux, L.; David, M.; Nicolas, O.;
Brunette, S.; Bilimoria, D.; Bedard, J. Bioorg. Med. Chem.
Lett. 2004, 14, 793; (c) Chan, L.; Pereira, O.; Reddy, T. J.;
Das, S. K.; Poisson, C.; Courchesne, M.; Proulx, M.;
Siddiqui, A.; Yannopoulos, C. G.; Nguyen-Ba, N.; Roy,
C.; Nasturica, D.; Moinet, C.; Bethell, R.; Hamel, M.;
L’Heureux, L.; David, M.; Nicolas, O.; Courtemanche-
Asselin, P.; Brunette, S.; Bilimoria, D.; Bedard, J. Bioorg.
Med. Chem. Lett. 2004, 14, 797; (d) Beaulieu, P. L.;
Bousquet, Y.; Gauthier, J.; Gillard, J.; Marquis, M.;
McKercher, G.; Pellerin, C.; Valois, S.; Kukolj, G. J. Med.
Chem. 2004, 47, 6884; for a recent review see (e) Koch, U.;
Narjes, F. Infect. Disorders–Drug Targets 2006, 6, 31.
9. (a) Harper, S.; Pacini, B.; Avolio, S.; Di Filippo, M.;
Migliaccio, G.; Laufer, R.; De Francesco, R.; Rowley, M.;
Narjes, F. J. Med. Chem. 2005, 48, 1314; (b) Harper, S.;
Avolio, S.; Pacini, B.; Di Filippo, M.; Altamura, S.;
hydrolysis of the methyl ester in the presence of BBr3,
gave the trisubstituted azaindole 11b.
Compound 13b was prepared as described in Scheme 3.
The thieno[3,2-b]pyrrole core was synthesized by
condensation of methyl 5-methyl-4-nitrothiophene-2-
carboxylate 19 with benzaldehyde followed by ring-
closure with triethyl phosphite as described in the
literature.17 Introduction of the cyclohexyl ring was
performed by condensation of intermediate 20 with
cyclohexanone and reduction with triethylsilane giving
precursor 21. Introduction of the N,N-dimethylacet-
amide chain at the position N1 was done as described
in the literature.9a Subsequent hydrolysis of the ester
group afforded compound 13b.
In summary, we have described here the SAR performed
in the central core of our initial NS5B polymerase
inhibitor lead 1a. This work led to the discovery of a
novel series of thieno[3,2-b]pyrroles that are potent
allosteric inhibitors of the HCV NS5B polymerase.
Introduction of a polar substituent in the position N1
led to compound 13b which efficiently blocks subgenom-
ic HCV RNA replication in HUH-7 cells at low
micromolar concentration. Further optimization of this
new series of inhibitors will be described in the near
future.
H
N
NO2
a, b
MeO2C
MeO2C
c, d
S
S
20
19
H
N
e, f, g, h
MeO2C
13b
S
21
`
Tomei, L.; Pavonessa, G.; Di Marco, S.; Carfı, A.;
Scheme 3. Reagents and conditions: (a) PhCHO, pyrrolidine, MeOH,
reflux; (b) (EtO)3P, MW (55% over two steps); (c) cyclohexanone,
H3PO4, Ac2O, AcOH, 80 °C (70%); (d) Et3SiH, TFA, rt (100%); (e)
Giuliano, C.; Padron, J.; Bonelli, F.; Migliaccio, G.; De
Francesco, R.; Laufer, R.; Rowley, M.; Narjes, F. J. Med.
Chem. 2005, 48, 4547; (c) Beaulieu, P. L.; Bos, M.;
Bousquet, Y.; Fazal, G.; Gauthier, J.; Gillard, J.; Goulet,
S.; LaPlante, S.; Poupart, M.-A.; Lefebvre, S.; McKer-
t
BrCH2CO2 Bu, NaH, DMF, rt; (f) TFA, DCM (95% over two steps);
(g) Me2NHÆHCl, HATU, DIPEA, DMF, rt; (h) NaOH 2 N, MeOH/
THF (40% over two steps).

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J. M. Ontoria et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4026–4030
cher, G.; Pellerin, C.; Austel, V.; Kukolj, G. Bioorg. Med.
of the procedure described in Lohmann, V.; Korner, F.;
Koch, J.; Henan, U.; Theilmann, L.; Bartenschlager, R.
Science 1999, 285, 110. IC50 and EC50 values are the
average of at least two independent determinations. Cell-
based data (EC50) were measured in the presence of 10%
fetal calf serum.
Chem. Lett. 2004, 14, 119; (d) Beaulieu, P. L.; Bos, M.;
Bousquet, Y.; DeRoy, P.; Fazal, G.; Gauthier, J.; Gillard,
J.; Goulet, S.; McKercher, G.; Poupart, M.-A.; Valois, S.;
Kukolj, G. Bioorg. Med. Chem. Lett. 2004, 14, 967.
10. Di Marco, S.; Volpari, C.; Tomei, L.; Altamura, S.;
Harper, S.; Narjes, F.; Koch, U.; Rowley, M.; De
Francesco, R.; Migliaccio, G.; Carfi, A. J. Biol. Chem.
2005, 280, 29765.
11. Tomei, L.; Altamura, S.; Bartholomew, L.; Biroccio, A.;
Ceccacci, A.; Pacini, L.; Narjes, F.; Gennari, N.; Bisbocci,
M.; Incitti, I.; Orsatti, L.; Harper, S.; Stansfield, I.;
Rowley, M.; De Francesco, R.; Migliaccio, G. J. Virol.
2003, 77, 13225.
13. The partition coefficient of the compounds between 1-
octanol and buffer, log D, was determined at pH 7.4 using
a modification of the procedure described in Hitzel, L.;
Watt, A. P.; Locker, K. L. Pharm. Res. 2000, 17, 1389.
log D values are the average of at least two independent
determinations.
14. Iddon, B.; Khan, N.; Lim, B. L. J. Chem. Soc., Perkin
Trans. 1 1987, 1457.
12. The compounds described herein were assessed for activity
(IC50) against the purified BK strain of NS5BDC55
enzyme in the presence of heterogenic template RNA
cells using a modification of the procedure described in
Ref. 11. Inhibition of the replication of subgenomic HCV
RNA was measured in HUH-7 cells using a modification
15. Chai, W.; Kwok, A.; Wong, V.; Carruthers, N. I.; Wu, J.
Synlett 2003, 2086.
16. Ujjainwalla, F.; Walsh, T. F. Tetrahedron Lett. 2001, 42,
6441.
17. Srinivasan, K.; Srinivasan, K. G.; Balasubramanian, K.
K.; Swaminathan, S. Synthesis 1973, 5, 313.