Potent triazolyl-proline-based inhibitors of HCV NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2008.04.012

The design and synthesis of tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole is described. Replacement of the P2 quinoline with a triazole moiety provided a versatile handle which could be expediently modified to generate a diverse series of inhibitors. Further refinement by the incorporation of an aryl-substituted triazole and replacement of the P1 acid with an acyl sulfonamide ultimately provided inhibitors with interesting cellular activity.

Full text of DOI:10.1016/j.bmcl.2008.04.012

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3400–3404
Potent triazolyl-proline-based inhibitors of HCV NS3 protease
Julie Naud, Christopher Lemke, Nathalie Goudreau, Eric Beaulieu,
*
`
Peter D. White, Montse Llinas-Brunet and Pat Forgione
Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 rue Cunard, Laval, QC, Canada H7S 2G5
Received 29 February 2008; revised 3 April 2008; accepted 7 April 2008
Available online 10 April 2008
Abstract—The design and synthesis of tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole is
described. Replacement of the P2 quinoline with a triazole moiety provided a versatile handle which could be expediently modified
to generate a diverse series of inhibitors. Further refinement by the incorporation of an aryl-substituted triazole and replacement of
the P1 acid with an acyl sulfonamide ultimately provided inhibitors with interesting cellular activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection is a major cause of
chronic liver disease that can lead to cirrhosis and
hepatocellular carcinoma. It is estimated that nearly
200 million individuals worldwide are currently in-
fected with HCV and it is the leading cause of liver
transplants.1 The current standard course of therapy,
pegylated interferon-a in combination with ribavarin,
suffers from low response rates and severe side effects.2
Thus, a specific and broadly effective therapeutic agent
for the treatment of HCV remains a critical unmet
medical need.
Effectively, 1H NMR studies demonstrated that the aryl
group is positioned above the catalytic triad pair (Asp81
and His57) in the bound conformation, partially shield-
ing the active site from solvent.7 In our continuing ef-
forts to discover novel NS3 protease inhibitors, we
envisioned employing an azide (2) as a useful handle
to evaluate substituted triazoles at the C4-position of
proline (3). This would provide a diverse class of inhib-
itors that could mimic the effect of the aryl-substituted
quinoline and allow us to map the large S2 surface bind-
ing site on the enzyme.
HCV is a member of the Flaviviridae family of envel-
oped, single-stranded, positive-sense RNA viruses. The
9.6-kilobase genome of HCV encodes a single polypro-
tein that is post-translationally processed into at least
10 structural and nonstructural (NS) proteins by a com-
bination of both host and viral proteases.3 The NS3 pro-
tease has been shown to be essential for viral replication4
and the first antiviral proof of principle studies in man
with BILN2061 demonstrated that the NS3 protease is
a relevant clinical target.5
Introduction of the azide substituent (2a, Scheme 2)
was straightforward via displacement of the corre-
MeO
N
N3
O
O
O
H
O
O
N
N
H
O
N
OH
N
N
H
O
N
OH
O
O
H
O
O
2
We reported previously the discovery of compound 1
containing a 7-methoxy-2-phenyl-4-oxo-quinoline on
the P2 proline (Scheme 1).6 We have demonstrated that
the aryl group on the 2-position of the quinoline con-
tributed substantially to the potency of the inhibitor.
P3
P2
P1
R
N
IC50 = 14 nM
EC50 = 550 nM
1
N
N
O
O
H
N
N
O
N
OH
H
O
O
Keywords: HCV NS3 protease; Protease; Hepatitis C; Inhibitors.
*
3
Corresponding author. Tel.: +1 450 682 4640; fax: +1 450 682
8434; e-mail: pforgione@lav.boehringer-ingelheim.com
Scheme 1. General strategy for P2 optimization.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.012

J. Naud et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3400–3404
3401
N
R
N
N
O
O
b, c
H
O
O
N
N
H
N
O
O
N
N
H
N
O
OH
O
O
H
O
O
R = S-Brosylate 4
R = -N3 2a
a
3a
R
IC50 59 uM
Scheme 2. Initial lead for optimization. Reagents and conditions: (a) NaN3, THF, 70 °C, 14 h (83%); (b) prop-2-yn-1-oic acid, DME, 100 °C; (c)
LiOH (10 equiv), THF, MeOH, H2O (34%, 2 steps).
sponding brosylate (4) with inversion of stereochemis-
try to yield the desired azide. The unsubstituted tria-
zole was then prepared by thermal cycloaddition of
2a with prop-2-yn-1-oic acid, followed by hydrolysis
of the P1-ester with concomitant decarboxylation to
afford triazole 3a that constituted the initial point of
diversity for SAR.
To evaluate this hypothesis, we employed a synthetic
strategy that provided both the C4- and C5-arylated
inhibitors (Scheme 3). Thermal cycloaddition of azide
(2a) with phenylacetylene yielded a 1:1 mixture of the
two regioisomers, which were separated and hydrolyzed
for independent testing.
As predicted, the C4-phenyltriazole (3b, Scheme 4) was
indeed the most potent of the two isomers and provided
an 84-fold improvement in intrinsic potency over the
unsubstituted triazole (3a).9 Although the C5-arylated
inhibitor (3c) also led to an improvement in potency
(4-fold) over 3a, we focused all further efforts on C4-
substituted analogues.
In order to gain insight on the binding conformation of
these series of inhibitors and to guide the SAR, com-
pound 3a was docked and minimized into the active site
of the NS3 protease (Fig. 1).6,7 The docking was based
on previously reported structures of related analogues
in complex with the NS3 protease. In this model, the
P1, P2, and P3 moieties of 3a, including the C-terminal
acid, were docked to invoke the known intermolecular
hydrogen bond network with the NS3 protease enzyme.
Importantly, the model indicated that the triazole sub-
stituent adopts a pseudo axial conformation on the P2
proline ring which induces a large, flat lipophilic surface
in the protein defined by the side chains of Asp168 and
Arg155.7 Although the exact orientation of the triazole
ring could not be unambiguously established from this
docking protocol, it was speculated that C4-substitution
would provide a basis for further exploration of this
binding area.
In an effort to further understand the binding mode of
these inhibitors, transfer nOe NMR experiments were
performed on 3b in the presence of the NS3 protease
(Fig. 2).7 This study was consistent with the model
which was obtained and confirmed that the P2 substitu-
ent occupies a pseudo axial conformation on the proline
ring in both the free and bound states. Further, the C5–
H of the triazole ring of 3b has specific nOe’s with the
d-hydrogens of the proline ring indicating the ring is
pointing toward the cyclopentyl carbamate in the bound
state. This conformation is likely due to the increased
interactions of p–p stacking of the phenyl group with
Arg155.
In an effort to further improve the intrinsic potency, we
evaluated the effect of the C4-substituent of the triazole
(Table 1). Replacement of the phenyl group with a
cyclohexyl 3d or with a 2-pyridyl 3e substituent led to
losses in potency. Increasing the size of the substituent
to a bi-phenyl 3f or naphthyl group 3g was tolerated.
Introduction of a p-chloro group 3h on the second aryl
ring had no significant effect, however, a p-methoxy
group 3i led to modest improvement in potency which
was improved further by the preparation of the related
dihydrobenzofuran 3j.
C-terminal acyl sulfonamides have been disclosed10 and
further evaluated11 as excellent bioisosteres for the C-
terminal carboxylic acids in NS3 protease inhibitors.
We were delighted to observe that a similar effect was
observed in the triazole series, leading to a further
improvement in potency of 260-fold over the corre-
sponding acid (Scheme 5). In addition, compound 5a
demonstrated sub-micromolar potency in the replicon
cell-based assay.12
Figure 1. Model of 3a docked into the NS3 protease active site.

3402
J. Naud et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3400–3404
N
4
N
N
1
O
O
H
N3
N
N
O
N
OH
H
O
O
O
5
O
H
a
N
N
3b
N
O
O
+
H
O
O
N
2a
N
1
N
O
O
H
N
N
N
O
OH
H
O
O
3c
Scheme 3. Synthesis of 1,4- and 1,5-disubstituted triazoles.8 Reagents and conditions: (a) i—phenylacetylene, DME, 100 °C, 5 h; ii—LiOH (20
equiv), THF, MeOH, H2O (55%, 2 steps).
N
5
N
4
N
4
5
N
N
N
1
N
N
1
N
1
O
O
H
N
N
N
O
OH
H
O
O
3b
IC50 = 0.7 uM
3c
3a
84-fold
4-fold
IC50 = 14 uM
IC50 = 59 uM
Scheme 4. Effect of C4 versus C5 substitution.
At this point, a regioselective synthesis of 1,4-disubsti-
tuted triazoles was implemented to facilitate SAR and
avoid the need for regioisomer separation. This was
achieved by the copper catalyzed cycloaddition chem-
istry previously reported for the regiospecific synthesis
of 1,4-disubstituted triazoles13 (see Scheme 6).
nOe’s
β
δ
Further modifications were evaluated toward improv-
ing cellular activity and a variety of substituted phen-
yls were prepared (Table 2). Introduction of a
dihydrobenzofuran 5b improved the cellular activity
3b
Figure 2. Transfer nOe of 3b in the presence of NS3 protease.
N
N
N
N
260 x
N
N
O
O
O
O
O
O
H
H
N
N
S
N
N
N
O
N
N
H
O
OH
H
H
O
O
O
O
5a
3b
IC50 = 2.7 nM
IC50 = 700 nM
EC50 = 430 nM
Scheme 5. Introduction of P1 acyl sulfonamide.

J. Naud et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3400–3404
Table 2. EC50 SAR of C4-substituted phenyltriazoles
3403
a,b
Table 1. Triazole C4 SAR
R
R
4
N
N
N
N
N
N
1
O
O
O
O
O
O
H
H
N
N
S
N
N
O
N
H
N
O
N
OH
H
H
O
O
O
O
Compound
R
IC50 (nM)
700
Compound
R
EC50 (nM)
3b
5a
430
230
O
5b
5c
3d
3e
3f
4300
3700
860
O
O
190
270
N
N
5d
5e
SO2Me
>7300
S
5f
1220
680
3g
890
Cl
5g
Cl
O
O
3h
3i
3j
790
360
130
Cl
5h
5i
220
170
Cl
Br
5j
210
2-fold over 5a, which was improved slightly when the
corresponding acetal 5c was introduced. Introduction
of a NMe2 5d group at the para-position led again
to a 2-fold increase in cellular activity while an elec-
tron withdrawing sulfone 5e resulted in complete loss
of cellular activity. Replacement of phenyl 5a with a
thiophene 5f led to a 3-fold loss in potency. The m-
Cl-substituted phenyl 5i was more potent than the
corresponding isomers 5g and 5h. Interestingly, m,m-
disubstitution with Cl led to a further increase in cel-
Cl
Br
5k
5l
140
150
Cl
Br
a All IC50 values for this set of inhibitors was <3 nM.
b EC50 values were obtained employing a previously described assay.12
R
N
4
N3
N
N
1
O
O
O
O
H
O
O
O
O
N
N
S
a
H
N
O
N
N
N
S
H
H
O
N
N
O
O
H
H
O
O
6
5
14
Scheme 6. Regioselective synthesis of 1,4-disubstituted triazoles.
BuOH, 23 °C, yields: 5–50%.
Reagents and conditions: (a) acetylene (1.2 equiv), CuSO4, ascorbic acid, t-

3404
J. Naud et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3400–3404
Br
Br
Br
Br
O
N
N
N
N
N
N
O
O
O
O
O
O
O
H
H
N
N
S
N
N
S
O
N
N
N
N
N
H
H
H
H
H
O
O
O
O
7
6
EC50 = 77 nM
EC50 = 105 nM
Scheme 7. Capping group and P10 modifications.
Narjes,H.; Poupart,M. A.; Rancourt,J.; Sentjens,R. E.; St.
George, R.; Simoneau, B.; Steinmann, G.; Thibeault, D.;
`
Tsantrizos, Y. S.; Weldon, S. M.; Yong, C. L.; Llinas-
Brunet, M. Nature, 2003, 426, 186.
lular activity 5k. The same exercise was performed
employing di-Br 5l in place of di-Cl which led to
our two most potent inhibitors.
`
6. Llinas-Brunet, M.; Bailey, M. D.; Ghiro, E.; Gorys, V.;
Further improvement in the cellular activity was
achieved by modification of the capping group from a
carbamate 5l to the corresponding urea 6 (Scheme 7).6
Also, the introduction of a methyl-substituted cyclopro-
pane on the acyl sulfonamide 715 improved cellular po-
tency over 5l and provided an inhibitor with a
promising profile for further exploration.
Halmos, T.; Poirier, M.; Rancourt, J.; Goudreau, N.
J. Med. Chem. 2004, 47, 6584.
7. Goudreau, N.; Cameron, D. R.; Bonneau, P.; Gorys, V.;
`
Plouffe, C.; Poirier, M.; Lamarre, D.; Llinas-Brunet, M.
J. Med. Chem. 2004, 47, 123.
8. Representative thermal cycloaddition: phenylacetylene
(1.3 equiv) was added to a solution of triazole (1.0 equiv)
in DME (0.28 M in triazole) in a sealed tube and was
heated at 100 °C for 5 h. The reaction was cooled,
concentrated, and diluted with THF/MeOH/H2 O (2:1:1,
0.05 M in triazole) and LiOH was added (20 equiv). The
reaction mixture was stirred for 14 h, concentrated and
purified by preparative HPLC (CH3CN/H2 O 0:0.06%
TFA) followed by lyophilization to yield the desired
compounds. Typical yields, 10–35%.
In summary, we have disclosed potent inhibitors of the
HCV NS3 protease which contains novel P2 triazole
substituents. These inhibitors exhibited good levels of
cellular activity while providing a diverse series for fur-
ther optimization.
9. Miao, Z.; Sun, Y.; Nakajima, S.; Tang, D.; Wang, Z.; Or,
Y. S. WO 2004/113365, 2004.
10. (a) Campbell, J. A.; Good, A. WO 2002/060926 2002; (b)
Acknowledgments
`
Bailey, M. D.; Forgione, P.; Llinas-Brunet, M.; Poupart,
M.-A. WO 2007/009227.
11. Ro¨nn, T.; Gossas, T.; Sabnis, Y. A.; Daoud, H.; Akerb-
We thank Sylvain Bordeleau and Michael Little for ana-
lytical support. We would also like to thank Dr. Lisette
´
˚
´
Lagace, Lyne Lamarre, Diane Thibeault, and Ewald
lom, E.; Danielson, U. H.; Sandstro¨m, A. Bioorg. Med.
Chem. 2007, 15, 4057, (and references therein) .
12. Beaulieu, P. L.; Gillard, J.; Bykowski, D.; Brochu, C.;
Welchner for EC50 and IC50 determinations and Franc¸-
ois Bilodeau for proofreading the manuscript. Finally,
we thank Drs. Michael Bo¨s and Michael Cordingley
for their guidance and support of this work.
´
Dansereau, N.; Duceppe, J.-S.; Hache, B.; Jakalian,
A.; Lagace, L.; LaPlante, S.; McKercher, G.; Moreau,
´
E.; Perreault, S.; Stammers, T.; Thauvette, T.; War-
rington, J.; Kukolj, G. Bioorg. Med. Chem. Lett. 2006,
16, 4987.
References and notes
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Kawai, S. H.; Kukolj, G.; Lagace, L.; LaPlante, S. R.;
14. Typical experimental procedure: azide 2b (0.05 mmol) was
dissolved in t-BuOH (0.05 M) followed by the addition of
copper sulfate pentahydrate (0.1 M in H2O, 0.1 equiv),
ascorbic acid (0.1 M in H2O, 0.8 equiv) and aceteylene (1.2
equiv). The resulting mixture was stirred at 23 °C for 14 h,
concentrated, and purified by preparative HPLC (CH3CN/
H2O containing 0.06% TFA) followed by lyophilization
yielding the desired compounds, typical yields 5–50%.
15. Ripka, A.; Campbell, J. A.; D’Andrea, S.; Good, A.; Li,
J.; McPhee, F.; Tu, Y. WO 2004/043339, 2004.