Structure-Activity Study on a Novel Series of Macrocyclic Inhibitors of the Hepatitis C Virus NS3 Protease Leading to the Discovery of BILN 2061

Article abstract of DOI:10.1021/jm0342414

From the discovery of competitive hexapeptide inhibitors, potent and selective HCV NS3 protease macrocyclic inhibitors have been identified. Structure-activity relationship studies were performed focusing on optimizing the N-terminal carbamate and the aromatic substituent on the (4R) -hydroxyproline moiety. Inhibitors meeting the potency criteria in the cell-based assay and with improved oral bioavailability in rats were identified. BILN 2061 was selected as the best compound, the first NS3 protease inhibitor reported with antiviral activity in man.

Full text of DOI:10.1021/jm0342414

J . Med. Chem. 2004, 47, 1605-1608
1605
enzyme perhaps the best understood HCV enzyme.8 The
essentiality of this enzyme for viral replication has been
demonstrated by the nonproductive infection following
liver inoculation of chimpanzees with a genomic HCV
RNA mutated in the NS3 protease active site.9
Str u ctu r e-Activity Stu d y on a Novel
Ser ies of Ma cr ocyclic In h ibitor s of th e
Hep a titis C Vir u s NS3 P r otea se Lea d in g
to th e Discover y of BILN 2061
Early structure-activity studies were fueled by the
discovery of N-terminal product inhibition of NS3 pro-
tease by us10 and others.11 The interaction between the
NS3 protease and the C-terminal carboxylic acid
hexapeptide inhibitor is characterized by a series of
weak contacts that extend from the P1 to the P6
position. A cysteine is required at the P1 position, and
the interaction relies on high charge density especially
at the P6 position. Early structure-activity studies led
to the identification of potent hexapeptides12,13 (1)
followed by tetrapeptides14,15 (2, 3). More recently,
macrocyclic tripeptide inhibitors were identified, as
exemplified by 4 and 516 (Table 1).
Montse Llina`s-Brunet,*,† Murray D. Bailey,†
Gordon Bolger,‡ Christian Brochu,†
Anne-Marie Faucher,† J ean Marie Ferland,†
Michel Garneau,‡ Elise Ghiro,† Vida Gorys,†
Chantal Grand-Maˆıtre,† Ted Halmos,†
Nicole Lapeyre-Paquette,‡ Francine Liard,‡
Martin Poirier,† Manon Rhe´aume,‡
Youla S. Tsantrizos,† and Daniel Lamarre‡
Departments of Chemistry and Biological Sciences,
Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street,
Laval, Que´bec H7S 2G5, Canada
Received November 20, 2003
Ta ble 1. Peptidomimetic Inhibitors of the NS3 Protease
Abstr a ct: From the discovery of competitive hexapeptide
inhibitors, potent and selective HCV NS3 protease macrocyclic
inhibitors have been identified. Structure-activity relationship
studies were performed focusing on optimizing the N-terminal
carbamate and the aromatic substituent on the (4R)-hydroxy-
proline moiety. Inhibitors meeting the potency criteria in the
cell-based assay and with improved oral bioavailability in rats
were identified. BILN 2061 was selected as the best compound,
the first NS3 protease inhibitor reported with antiviral activity
in man.
In tr od u ction . Hepatitis C virus (HCV) is the leading
cause of chronic liver disease worldwide.1,2 It is esti-
mated that 3% of the world population is infected with
this virus.3 Currently, the most effective therapy con-
sists of using PEGylated R-interferon in combination
with ribavirin, giving a sustained virological response
of ∼50% in genotype 1 infected patients.4 The existing
therapies are associated with considerable side effects
and lead to discontinuation of treatment in certain
patient populations. The limited efficacy and side effects
of current therapies, linked to the high prevalence of
infection worldwide, clearly highlight the need for new
therapeutics.
HCV is a small enveloped positive stranded RNA
virus that encodes a polyprotein of ∼3000 amino acids.
The polyprotein consists of four structural and six
nonstructural (NS) proteins.5 The structural proteins
are processed by host peptidases, whereas the NS
proteins are processed by two virally encoded proteases,
the NS2/3 and NS3 proteases. The NS2/3 protease is
responsible for the cleavage between NS2 and NS3,
whereas the NS3 protease is responsible for the release
of the remaining nonstructural proteins.6 The NS3
protease, located at the N-terminal 180 amino acids of
the NS3 protein, is a chymotrypsin/trypsin-like serine
protease.7 The function and structure of the NS3 pro-
tease have been studied in great detail, making this
Macrocyclic inhibitor 5 is a selective inhibitor of HCV
RNA replication in the cell-based assay (HCV replicon)
with an EC50 of 77 nM. Compound 5 suffered from an
unfavorable pharmacokinetic profile when studied in
rats. It is cleared very rapidly from rat plasma (half-
life T1/2 ) 0.4 h) and is poorly bioavailable (F ) 2%), as
reflected by the low plasma concentration (area under
the plasma concentration-time curve, AUC0-∞ ) 0.2
µM‚h) following a single oral dose of 25 mg/kg in rats.16
The main goal of this study was to identify selective NS3
protease inhibitors with low nanomolar cell-based po-
tency (target EC50 < 10 nM) and with an adequate
pharmacokinetic profile for the selection of an orally
bioavailable HCV protease inhibitor for further develop-
ment. This communication describes the optimization
of 5 leading to the discovery of BILN 2061.17
* To
whom
correspondence
should
be
addressed.
Phone: 450-682-4640. Fax: 450-682-4189. E-mail: mllinas@
lav.boehringer-ingelheim.com.
† Department of Chemistry.
‡ Department of Biological Sciences.
10.1021/jm0342414 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

1606 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Letters
pound 5.23 However, when evaluated in the cell-based
replicon assay, a larger range of EC50 values were
observed. Compound 6, containing a 5-methyl-1,3,4-
oxadiazol-2-yl group, was the least potent analogue in
this series, showing an EC50 of 200 nM. All other
heterocycle-containing compounds shown in Table 2
displayed an improved cell-based potency over 5 and
with similar activities in both enzymatic and cellular
assays. Pyrrole (7) and pyrazole (8, 9) derivatives
produced very potent inhibitors, giving rise to com-
pounds with EC50 values below 10 nM. Interestingly,
two regioisomers, pyrazole derivatives 9 (3.0 nM) and
10 (22 nM) demonstrated the lowest and the highest
IC50 of this series of compounds with a similar range of
inhibitory activities when evaluated in the cell-based
replicon assay. Among the best heterocycles identified
were the 2-amino-4-thiazolyl derivatives. Compounds
13-15 are low-nanomolar inhibitors of the NS3 protease
in both enzymatic and cellular assays. The potency of
these inhibitors was independent of the substituent on
the amino group. From this study, several novel five-
membered ring heterocycles were identified as 2-quino-
line substituents that produced inhibitors with the
target cellular potency.
Ta ble 2. Macrocyclic Inhibitors with Different Five-Membered
Ring Heterocycles at the C2 Position of the Quinoline
We then evaluated alternative capping groups. The
stability of the tert-butyl carbamate capping group was
a concern because it is well documented that the tert-
butyl carbamate is not stable under acidic conditions.24
Early SAR studies conducted on a related series of
acyclic inhibitors (manuscript in preparation) had iden-
tified carbamates arising from cyclic alcohols, as well
as several ureas, which are well tolerated and impart
chemical stability. On the basis of these studies, cy-
clobutyl (16) and cyclopentyl (17) carbamates as well
as R-methylneopentyl urea (18) were introduced in
combination with the 2-(2-acetylamino-4-thiazolyl)-
quinoline present in 15. Results are summarized in
Table 3.
Resu lts. Studies on the tripeptide macrocyclic inhibi-
tor series demonstrated that the 15-membered hetero-
cycle is one of the optimal ring sizes.18 The C-terminal
carboxylic acid is maintained because it contributes
considerably to the potency and specificity of the inhibi-
tors.19 Therefore, SAR studies focused on two pharma-
cophores: the novel tricyclic substituent on the (4R)-
hydroxyproline moiety and the left-hand-side carbamate
group (capping group).
Ta ble 3. Evaluation of Different Capping Groups
The aromatic substituent in our previously reported
acyclic inhibitors has been extensively studied.12,20-22
Combined efforts between medicinal chemistry and
structural research led to the identification of 2-phenyl-
7-methoxy-4-quinolinoxy as the optimal group.14 In
particular, when combined with the optimal P1 residue
(1R,2S)-1-amino-2-vinylcyclopropylcarboxylic acid,15 this
substituent produced very potent inhibitors of the NS3
protease. In the macrocyclic inhibitor series, the addi-
tion of a C2-phenyl ring on the quinoline produced
inhibitors with a modest increase in enzymatic potency
(2- to 3-fold) but with greater than 15-fold difference in
the cell-based potency (EC50 of inhibitors 4 and 5). The
introduction of the 2-phenyl group on the quinoline
modulates the physicochemical properties and possibly
renders these compounds more cell-permeable. These
observations have prompted us to further investigate
substitution at the C2 position of the quinoline moiety.
The phenyl ring was replaced with different five-
membered ring aromatic heterocycles containing one,
two, or three heteroatoms (O, N, and S) as shown in
Table 2. Introduction of the various heterocycles shown
in Table 2 was well tolerated, producing inhibitors with
enzymatic potency comparable to that of starting com-
We were very pleased to find that introduction of
either the cyclobutyl or the cyclopentyl carbamate
improved considerably the cell-based potency. Indeed,
16 and 17 were ∼10-fold more potent than analogue 15
and are representative of a group of compounds with
subnanomolar potency in the HCV replicon assay. The
introduction of a urea capping group (inhibitor 18)
produced the most potent compound in the enzymatic
assay; however, considerable loss in potency was ob-
served in the cell-based assay. Perhaps the presence of
an additional polar (NH) group decreases cell perme-
ability.25

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1607
Ta ble 4. Combination of Cyclopentyl Carbamate Capping
Ta ble 5. Pharmacokinetic Parameters for BILN 2061 in Rats
Group with Optimal 2-Hetorocycle Quinoline
Following Oral and Intravenous Administration
oral, 20 mg/kg
iv, 5 mg/kg
Cmax
AUC0-∞
T1/2
Cl
F
(µM)
(µM‚h)
(h)
Vss (L/kg)
((mL/min)/kg)
(%)
2.5
12.5
1.3
1.0
13
42
concentration-time curve of 7.2 µM‚h. Increasing the
size of the alkyl group on the 2-aminothiazolyl deriva-
tive from ethyl to isopropyl generated BILN 2061 with
enzymatic and cell-based inhibitor potencies similar to
those of 24. BILN 2061 showed an improved pharma-
cokinetic profile (Table 5) with a much higher Cmax and
AUC0-∞ and an oral bioavailability of 42% in rats. On
the basis of its excellent in vitro potency and adequate
pharmacokinetic profile in rats, BILN 2061 was further
evaluated. A satisfactory pharmacokinetic profile in
higher species and adequate preclinical safety profile
were observed, supporting further evaluation in hu-
mans.17 When BILN 2061 was administered orally to
patients infected with HCV genotype 1 for 2 days, an
unprecedented (up to 3 log) decline in plasma viral load
was observed. This represented the first proof-of-concept
in man for an HCV NS3 protease inhibitor. A full
description of the biopharmaceutical profile of BILN
2061 has been reported elsewhere.17
All the inhibitors shown in this letter contain a
C-terminal carboxylic acid that renders them very
potent and specific for the NS3 protease.19 These
compounds do not significally inhibit human serine/
cysteine proteases such as human leukocyte elastase
and cathepsin S (IC50 > 30 µM).
Su m m a r y. Focused structure-activity studies on the
tripeptide macrocyclic inhibitor 5 led to the discovery
of BILN 2061, a very potent and specific inhibitor of the
HCV NS3 protease in both the enzymatic (IC50 ) 3.0
nM) and the cell-based replicon assays (EC50 ) 1.2 nM).
The compound shows good oral bioavailability in rats
(F ) 42%) with a low plasma clearance (Cl ) 13 (mL/
min)/kg). BILN 2061 was selected for further develop-
ment.
Syn t h esis. The synthesis of these compounds was
previously described in detail,28 and a general approach
is shown in Scheme 1. Appropriately, 2-substituted
4-hydroxy-7-methoxyquinolines 26 are coupled with a
preformed macrocyclic tripeptide via a Mitsunobu reac-
tion.29 The resulting methyl esters 27 were hydrolyzed
under basic conditions after appropriate adjustments of
the capping group.
Encouraged by the beneficial effect of introducing a
cyclopentyl carbamate capping group on derivative 15,
we decided to evaluate this capping group with other
quinoline derivatives. Table 4 shows the combination
of the cyclopentyl carbamate with pyrrole (19), pyrazole
(20, 21), and aminothiazole-containing derivatives (22-
24, BILN 2061). In all cases, the cell-based potency was
improved compared to the tert-butyl carbamate ana-
logue. Several compounds with low-nanomolar and
subnanomolar potency were identified, increasing the
pool of compounds with the target cellular potency.
We then evaluated the pharmacokinetic properties of
the best inhibitors in rats. Compounds were adminis-
tered either orally (20 mg/kg) or intravenously(5 mg/
kg).26 The oral dosing of compounds 8, 9, 13, 17, 20 (5
mg/kg), 21, and 22 resulted in undetectable levels in
the rat plasma. In contrast, the oral administration of
15 displayed low plasma concentrations (maximum
plasma concentration, Cmax ) 0.3 µM and AUC0-∞ ) 0.3
µM‚h) and an improved half-life (T1/2 ) 0.8 h) over 5
after intravenous dosing. The bioavailability of com-
pound 15 is 8%.
A short plasma half-life and a high clearance rate for
peptidomimetic compounds have been reported, an
example being the HIV protease inhibitors.27 In an
attempt to understand the poor oral bioavailability of
some of these compounds, the importance of the biliary
excretion to the clearance of 15 was investigated.
Following an intravenous bolus injection of 5 mg/kg in
rats,26 15 was efficiently excreted into the bile un-
changed with a recovery of greater than 67% of the dose
after 1 h. These results are consistent with a major
hepatobiliary elimination and would suggest a high liver
first-pass effect after oral dosing.
The synthesis of the various C2-substituted-4-hydroxy-
quinolines 26 was done on an individual basis and is
described in detail in the Supporting Information.
Sch em e 1. Inhibitor Assembly
Compound 24 showed higher plasma levels after oral
administration to rats with a maximum plasma con-
centration of 1 µM and an area under the plasma

1608 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Letters
(15) Rancourt, J .; Cameron, D.; Gorys, V.; Lamarre, D.; Poirier, M.;
Thibeault, D.; Llina`s-Brunet, M. Peptide-Based Inhibitors of the
Hepatitis C Virus NS3 Protease: Structure-Activity Relation-
ship at the C-Terminal Position. J . Med. Chem., in press.
(16) Tsantrizos, Y. S.; Bolger, G.; Bonneau, P.; Cameron, D. R.;
Goudreau, N.; Kukolj, G.; LaPlante, S. R.; Llina`s-Brunet, M.;
Nar, H.; Lamarre, D. Macrocyclic inhibitors of the NS3 protease
as potential therapeutic agents of hepatitis C virus infection.
Angew. Chem., Int. Ed. 2003, 42, 1356-1360.
(17) Lamarre, D.; Anderson, P.; Bailey, M.; Beaulieu, P.; Bolger, G.;
Bonneau, P.; Bo¨s, M.; Cameron, D.; Cartier, M.; Cordingley, M.;
Faucher, A.-M.; Goudreau, N.; Kawai, S.; Kukolj, G.; Lagace´,
L.; LaPlante, S.; Narjes, H.; Poupart, M.-A.; Rancourt, J .; St-
George, R.; Sentjens, R. E.; Simoneau, B.; Steinmann, G.;
Thibeault, D.; Tsantrizos, Y.; Weldon, A. M.; Yong, C.-L.; Llina`s-
Brunet, M. Discovery of BILN 2061sAn NS3 preotease inhibi-
tors with first antiviral proof-of-concept in humans infected with
hepatitis C virus. Nature 2003, 426, 186-189.
(18) Tsantrizos, Y. S.; et al. Manuscript in preparation.
(19) Llina`s-Brunet, M.; Bailey, M.; De´ziel, R.; Fazal, G.; Gorys, V.;
Goulet, S.; Halmos, T.; Maurice, R.; Poirier, M.; Poupart, M.-A.;
Rancourt, J .; Thibeault, D.; Wernic, D.; Lamarre, D. l. Studies
on the C-terminal of hexapeptide inhibitors of the hepatitis C
virus serine protease. Bioorg. Med. Chem. Lett. 1998, 8, 2719-
2724.
(20) Poupart, M.-A.; Cameron, D. R.; Chabot, C.; Ghiro, E.; Goudreau,
N.; Goulet, S.; Poirier, M.; Tsantrizos, Y. Solid-phase synthesis
of peptidomimetic inhibitors for the hepatitis C virus NS3
protease. J . Org. Chem. 2001, 66, 4743-4751.
Ack n ow led gm en t. The authors thank Dr. Lisette
Lagace´, Ms. Lyne Lamarre, Ms. Diane Thibeault, and
Ms. Andrea Gorys for EC50 and IC50 determinations. We
also thank the analytical chemistry department for
technical support and Ms. Christiane Yoakim and Dr.
J anice Kelland for proofreading the manuscript. Finally,
we thank Dr. Paul Anderson, Dr. Michael Bo¨s, and Dr.
Michael Cordingley for encouragement and support.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR, HPLC,
and HRMS data for all the compounds disclosed in this letter
and a detailed synthetic scheme for the various C2-substituted
4-hydroxyquinolines 26. This material is available free of
charge via the Internet at http://pubs.acs.org.
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