Structure-activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

Article abstract of DOI:10.1016/j.bmcl.2008.07.088

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]- 13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0^4,6]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamid e (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K_i = 0.36 nM) and viral replication (replicon EC₅₀ = 7.8 nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.

Full text of DOI:10.1016/j.bmcl.2008.07.088

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4853–4858
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study on a novel series
of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus
NS3/4A protease leading to the discovery of TMC435350
a
b
b
b
Pierre Raboisson a, , Herman de Kock , Åsa Rosenquist , Magnus Nilsson , Lourdes Salvador-Oden ,
Tse-I Lin a, Natalie Roue b, Vladimir Ivanov b, Horst Wähling b, Kristina Wickström b, Elizabeth Hamelink b,
Michael Edlund b, Lotta Vrang b, Sandrine Vendeville a, Wim Van de Vreken a, David McGowan a,
Abdellah Tahri a, Lili Hu a, Carlo Boutton a, Oliver Lenz a, Frederic Delouvroy a, Geert Pille a,
*
Dominique Surleraux a, Piet Wigerinck a, Bertil Samuelsson b, Kenneth Simmen a,
a Tibotec BVBA, Gen. De Wittelaan L11 B3, B-2800 Mechelen, Belgium & Tibotec Pharmaceuticals Ltd, Eastgate Village, EastGate, Little Island, Co Cork, Ireland
*
b Medivir AB, PO Box 1086, SE-141 22 Huddinge, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification
Received 25 June 2008
Accepted 19 July 2008
Available online 24 July 2008
of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-
diazatricyclo [13.3.0.04,6]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as
a
potent inhibitor of HCV NS3/4A protease (Ki = 0.36 nM) and viral replication (replicon EC50 = 7.8 nM).
TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo
pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C
Macrocycle
Protease
NS3
The virally encoded serine protease NS3/4A is essential to the
life cycle of the hepatitis C virus (HCV), an important human path-
ogen causing chronic hepatitis, liver cirrhosis, and ultimately hepa-
tocellular carcinoma.1 The search for potent and selective HCV
NS3/4A protease inhibitors is a major focus in the pharmaceutical
industry of ultimately delivering more efficacious and better-toler-
ated drugs to treat HCV-infected patients. Indeed, the current ther-
tinued due to cardiotoxicity issues in primates given suprathera-
peutic doses for several weeks.6 Although the mechanism by
which BILN 2061 (2)5 elicits cardiovascular damage was not re-
ported, the toxicity observed with this pioneer drug is believed
to be compound specific. As a result, we and others have recently
presented data on macrocyclic HCV NS3/4A protease inhibitors
entering into clinical development.7 We now report on our efforts
to optimize a novel series of cyclopentane-containing macrocyclic
inhibitors of the HCV NS3/4A protease, which has led to the iden-
tification of the clinical candidate N-[17-[2-(4-isopropylthiazole-2-
yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-
3,13-diazatricyclo[13.3.0.04,6]octadec-7-ene-4-carbonyl](cyclopro-
pyl)sulfonamide (TMC435350, 32c, Table 1), which is currently
being evaluated in clinical trials (Table 2).
apies based upon the combination of pegylated interferon-
a with
ribavirin provide a sustained virological response (SVR) in around
50% of patients infected with genotype 1, and are often associated
with tolerability issues, highlighting an urgent therapeutic need.2
To date, the most advanced NS3/4A protease inhibitors VX-950
(1, Chart 1)3 and SCH 503034 (4)4 belong to the covalent reversible
serine-trap inhibitors incorporating a reactive electrophilic
a-
ketoamide center at the cleavage site that targets the catalytic
Ser139 of the active site of the enzyme via a fully reversible mech-
anism. However, the first clinical proof-of-concept for HCV NS3/4A
protease inhibitors as therapeutic agents was established with the
non-covalent macrocyclic b-strand mimic BILN 2061 (2, Chart 1).5
Subsequently, the clinical development of BILN 2061 was discon-
The structure of BILN 2061 (2)5 is characterized by a 15-mem-
bered macrocycle incorporating three unnatural amino acid resi-
dues (P1, P2, P3), as shown in Chart 1. BILN 20615 was obtained
following successive truncations of the N-terminal cleavage prod-
ucts of the NS5A/5B substrate peptide, leading to a tetrapeptide
inhibitor 3a,8 which was further optimized to improve its pharma-
ceutical properties resulting in 2.5 Recently, the replacement of the
P2 N-acyl-(4R)-hydroxyproline of 3a8 with a trisubstituted cyclo-
pentane dicarbonyl moiety has been reported, leading to the
* Corresponding authors. Tel.: +32 15 44 42 62; fax: +32 15 40 1257 (P. Raboisson).
E-mail address: praboiss@tibbe.jnj.com (P. Raboisson).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.088

4854
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4853–4858
H
N
P2
N
N
S
O
N
O
P1
OH
O
N
H
N
H
N
O
O
N
H
H
N
H
N
O
O
N
O
O
P3 capping
group
O
O
O
N
O
1
N
H
(VX-950)
P3
O
2
(BILN 2061)
N
N
O
HN
O
O
H
O
N
N
N
H
H
N
O
O
H
N
X
H
N
H
N
O
O
O
H
N
O
O
O
O
O
NH2
O
O
S
OH
O
N
N
O
H
4
3a, (X = N)
(SCH 503034)
5
3b, (X = CH)
Chart 1. NS3/4A protease inhibitors reported in the literature: 1 (VX-950);3 2 (BILN 2061)5 with the P1 (in green), P2 (in blue), and P3 (in brown) moieties; 3a8, 3b;9 4 (SCH
503034)4 and 5.10
discovery of a novel series of potent HCV NS3/4A protease inhibi-
tors exemplified by 3b8 (Chart 1). Encouraged by the enzymatic po-
tency of 3b (Ki = 22 nM), we decided to explore whether the
cyclopentane moiety could also be used in the rigidified macrocy-
clic series as a surrogate for the P2 pyrrolidine of 2.5 Initial struc-
ture–activity relationships (SAR) led to the macrocyclic inhibitor
510 (Chart 1), a potent and specific HCV NS3/4A protease inhibitor
(Ki = 0.41 nM) exhibiting promising cell-based HCV replicon activ-
ity (EC50 = 9 nM).10 Although 510 showed promising in vitro Caco-2
permeability data (A–B apparent permeability coefficient
Papp = 3.8 Â 10À6 cm/s) and medium intrinsic clearance in both hu-
and aluminum chloride afforded the corresponding ketones 9a–e
in 40–73% yield.13 Finally, acylation of the anilines 9a–e followed
by a subsequent treatment of intermediates 10a–g with potassium
tert-butoxide led to the desired quinolin-4-ols 11a–g in 58–88%
yield via a tandem of ring closure and aromatization reactions.13
The isopropylaminothiazolequinoline 11h14 (Chart 2) was syn-
thesized according to a literature procedure, and the isopropylpy-
razolylquinoline 19 was synthesized following an adaptation of the
literature procedure13 as depicted in Scheme 2. Diethyl ethoxym-
ethylenemalonate (12) was condensed with the methoxyaniline
8a, then cyclized at elevated temperature (230 °C) to provide the
ester 13 in 58% yield. Saponification of 13, followed by the subse-
quent decarboxylation led to the quinolin-4-ol 14 (96% yield),
which was converted to the corresponding 4-chloroderivative 15
by treatment with phosphorus oxychloride. Oxidation with meta-
chloroperbenzoic acid provided the N-oxide derivative 16 in a
non-optimized yield of only 18%. Subsequent displacement of the
4-chloro group with sodium benzylalcoholate followed by the
treatment of intermediate 17 with phosphorus oxychloride led to
the key 2-chloro intermediate in 90% yield, which was ultimately
displaced with the commercially available 3-isopropylpyrazole at
elevated temperature (155 °C) leading to the desired quinoline
19 in 95% yield. The target macrocyclic products 31a–h (Table 1)
were efficiently prepared in a 7-step procedure either from the lac-
tone acid 259,15 or from its tert-butyl ester 209 as depicted in
Scheme 3.10 These two synthetic pathways differ in the order of
introduction of the P1 and P3 moieties on the P2 cyclopentane cen-
tral scaffold. In the first case, the tert-butyl protected lactone 20 is
opened by treatment with lithium hydroxide, followed by the pep-
tidic coupling of the in situ generated acid with the P1 vinylcyclo-
propyl aminoacid ester 21, leading to 22 in 89% isolated yield.
Introduction of the P2 quinoline moieties 11a and 11g onto the
cyclopentylalcohol 22 was achieved by a Mitsunobu reaction,16
which provided the proper configuration of the chiral center at po-
sition 3 of the cyclopentane, leading to intermediates 23a and 23b
in 63% and 68% yield, respectively. Subsequent deprotection of the
man and rat liver microsomes (Clint = 46 lL/min/mg), the inhibitor
was found to be poorly absorbed in rat after oral administration of
15 mg/kg (F = 2.5%), and was rapidly eliminated (clear-
ance = 2.79 L/h/kg). Further studies revealed that the observed
poor PK profile was attributed to a very high excretion of the par-
ent drug 510 in the bile. Indeed, 95% of the product was found un-
changed in the bile 1 h after iv administration of 1 mg/kg of 5.10
Since the P2 quinoline has been reported in the literature to be a
key moiety regulating the overall PK profile of macrocyclic HCV
protease inhibitors,5 we decided to expand the SAR analysis to this
P2 heterocyclic moiety by (i) replacing the 2-phenyl group with
different 5- and 6-member heterocycles and (ii) adding a small
sized substituent at the 8-position. In the present work we report
on the SAR analysis of this novel cyclopentane series, leading to
the drug candidate TMC435350 (32c).
The synthesis of the target products 32a–h and 37 (Table 1) is
outlined in Schemes 1–4. Preparation of 2-substituted quinolines
11a–g was carried out either from commercially available m-anisi-
dine 8d to generate the 8-non-substituted quinolines 11a, 11b, and
11g, or from the 2-substituted 3-methoxyanilines 8a–c obtained
via a Curtius rearrangement11 from the corresponding 3-methoxy-
benzoic acids 6a–c, as depicted in Scheme 1. The 2-ethyl-3-meth-
oxy aniline 8e was synthesized following a literature procedure.12
Electrophilic aromatic substitution of 3-methoxyanilines 8a–e
with boron trichloride followed by the addition of acetonitrile

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4853–4858
4855
Table 1
Table 2
Optimization of the positions 2 and 8 of the quinoline
Mean plasma levels (n = 2) together with some basic pharmacokinetic parameters of
macrocycles 32c, 32g and 32h after a single intravenous (2 mg base-eq./kg) and oral
administration (20 mg base-eq./kg) in the male Sprague–Dawley rats
R2
N
Compound
R8
32c
0.505
32g
2.5
4.6
1.05
23.52
32h
1.4
O
Iv (2 mg/kg, n = 2)
Cl (L/h/kg)
Vdss (L/kg)
O
0.49
5.21
0.70
1.36
O
O
H
N
O
AUC (
lM h)
O
63.51
ND3
S
Liver/plasma ratio
N
H
N
O
Oral (10 mg/kg, n = 2)
AUC ( M h)
l
2.79
0.73
3.0
1.30
0.31
1.5
1.4
0.30
2.0
Cmax M)
(l
Tmax (h)
T1/2
F (%)
Liver/plasma ratio
2.8
2.2
5.8
Compound R2
R8
NS3/4A
Kia (nM) EC50 (nM) min/mg)
HUH7-Rep
Clint (l
L/ Papp A–Bd
c
11
25
31
b
(cm/s)
321
442
ND3
Liver/plasma ratios were determined 1at 6 h, 2at 8 h, 3at 24 h (at this timepoint the
compound was no longer quantifiable in the blood or in the liver).
5
H
H
H
H
0.41
1.4
9
17
11
17
46
15
38
16
3.8
H
N
R4
O
R4
R4
37
1.4
1.6
13
H
N
N
MeO
MeO
MeO
O
MeO
NH2
S
OH
i, ii
iv,v
iii
O
N
N
N
N
N
N
32a
32b
0.20
0.84
6a-c
8a-e
7a-c
S
R4
R4
R4
H
MeO
vi
N
R3
MeO
R3
N
NH2
O
vii
O
S
S
S
S
S
OH
9a-e
10a-g
11a-g
O
32c
32d
32e
32f
32g
Me 0.36
7.8
<6
—
8.4
—
R3:
N
R4:
H
R4:
Cpd#
Cpd#
6a-9a: Me
6b-9b:
6c-9c: Cl
8d, 9d:
8e, 9e Et
10a, 11a
S
F
10b, 11b
10c, 11c
10d, 11d
10e, 11e
10f, 11f
H
Et
F
3.1
66
57
Me
Et
F
H
N
S
Cl
0.55
0.10
—
—
N
10g, 11g
H
Scheme 1. Reactions and conditions: (i) TEA, diphenylphosphorylazide (dppa),
toluene, 100 °C; (ii) tert-BuOH, toluene, 100 °C; (iii) TFA, CH2Cl2, 20 °C; (iv) BCl3,
xylene, 0 °C; (v) CH3CN, AlCl3, CH2Cl2, 0–70 °C; (vi) R3COCl, dioxane, rt; (vii) tert-
BuOK, tert-BuOH, 100 °C.
Cl
2.9
9.0
5.8
Me 0.16
Me 0.30
9.7
6.8
<6
<6
15
12
N
N
O
O
O
O
N
O
N
O
O
(i)
(ii)
(iii)
(vi)
O
+
8a
32h
N
12
N
OH O
13
OH
14
Inhibition of the full-length HCV NS3/4A protease20 measured by the inhibition
a
O
O
constants (Ki values).21
N+
O
N+
O
b
O
Inhibition of HCV replication in Huh-7-Rep cells (luciferase assay) for macro-
(iv)
(v)
N
cyclic inhibitors 32a–h and 37 measured by 50% effective concentration (EC50).22
c
Intrinsic clearance in human liver microsomes (HLM).
A–B apparent permeability coefficient (Papp) measured in Caco-2 cells.
Cl
N
15
d
Cl
O
O
16
(vii)
17
O
Cl
N
N
tert-butyl acid group followed by the introduction of the P3 alke-
nylamine 2617 led to the non-cyclic intermediates 24a and 24h.
Alternatively, the other open derivatives 24b–g were prepared
from the lactone acid 25, which was submitted to a tandem of cou-
pling reactions first with the N-methylhex-5-enamine (26)17 (68%
yield), then with the P1 aminoacid 218,14 to provide 28 in 60% yield.
This approach was better suited to explore the SAR on the P2 quin-
oline moiety because the final products could be obtained in only
O
OH
18
19
Scheme 2. Reactions and conditions: (i) a—Et2O, rt; b—diphenyl ether, 230 °C; (ii)
a—NaOH, H2O, reflux; b—diphenylether 250 °C; (iii) POCl3, reflux; (iv) metachlor-
operbenzoic acid, CHCl3, rt; (v) NaH, benzylalcohol, 0 °C to rt; (vi) POCl3, À78 °C to
reflux; (vii) 3-isopropylpyrazole, 155 °C.

4856
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4853–4858
R8
R2
O
N
O
OH
H2N
O
O
R8
O
N
O
R2
O
O
O
O
H
+
i
N
ii
O
O
O
20
O
O
O
H
N
22
21
O
O
O
O
iii
23a,b
N
vii
H
N
O
O
OH
O
H
N
vi
O
24a-h
O
O
N
O
O
H
O
iv
v
N
N
O
O
+
27
O
25
O
OH
28
O
26
R3
R4
R4
R4
R4
R3
N
O
R3
N
O
O
N
O
R3
O
O
N
O
O
O O
HN
O
O
O
O
viii
ix
O
x
H
H
H
S
N
N
N
O
N
N
N
N
O
O
O
N
O
O
O
O
OH
32a-h
(see Table 1)
29a-h
R3 R4
30a-h
R4
31a-h
R4
R3
Cpd#
R3
Cpd#
Cpd#
N
N
H
24g-31g
24a-31a
N
N
Me
H
25a
S
S
H
24b-31b
24c-31c
24d-31d
24e-31e
24f-31f
Me
Et
F
H
25b
N
N
N
24h-31h
Me
S
Cl
Scheme 3. Reactions and conditions: (i) a—LiOH, dioxane/water, 0 °C; b—(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid ethyl ester hydrochloride, diisopropyleth-
ylamine, HATU, DMF, 0 °C; (ii) 11a or 11g, PPh3, DIAD, THF, 0 °C; (iii) a—triethylsilane, TFA, CH2Cl2, rt; b—25, diisopropylethylamine, HATU, DMF, 0 °C to rt; (iv) HATU,
diisopropylethylamine, DMF, 0 °C to rt; (v) a—LiOH, THF/MeOH/water, 0 °C; b—21, HATU, diisopropylethylamine, DMF, 0 °C to rt; (vi) 11b–f or 19, PPh3, DIAD, THF, À15 °C to
rt; (vii) Hoveyda-Grubbs 1st generation catalyst,19 dichloroethane, 70 °C; (viii) LiOH, THF/MeOH/water, rt; (ix) CDI, THF, reflux; (x) cyclopropylsulfonamide, DBU, THF, 50 °C.
four steps from a common intermediate 28, considering that the
intermediates 31a–h were not isolated.
function on the P2 quinoline, which can hamper the Mitsunobu
coupling reaction. As an alternative synthesis, the cyclopentanol
28 was converted to a brosylate intermediate 33 in 80% yield,
which was directly cyclized to the corresponding macrocycle 34
via a ring-closing olefin metathesis18 (Scheme 4). The subsequent
displacement of the brosylate activating group of 34 with the
quinoline 11h in the presence of a base, led to the ester 35, which
was then converted to the final product 37, following a procedure
similar to the one reported in Scheme 3 for the synthesis of
32a–h.
The new macrocyclic derivatives 32a–h and 37 (Table 1) pre-
pared in the present study were first tested in a biochemical assay
for their inhibitory capacities on the full-length HCV NS3/4A prote-
ase incorporating the central part of the cofactor NS4A as described
earlier.20 The inhibition constants (Ki values)21 were determined,
and are listed in Table 1. The cell-based activities (EC50) were mea-
sured in the Huh7-Rep cell line containing the subgenomic bicis-
tronic replicon clone ET with a luciferase readout.22 EC50’s were
calculated as the concentration of compound that caused a 50%
Mitsunobu coupling reaction16 between 28 and quinolines 11b–g
provided the open derivatives 24b–g in 56–72% yield. The macro-
cyclic ring esters 29a–h were obtained in satisfactory yields (60–
83%) by ring-closing olefin metathesis10,18 of the open precursors
24a–h, using the Hoveyda-Grubbs 1st generation catalyst.19 In this
reaction, the cis derivatives 29a–h were obtained as the major
products together with traces of the trans isomer. Hydrolysis of
the ethyl esters 29a–h, followed by the activation of the so-formed
acids 30a–h with carbonyldiimidazole afforded the corresponding
oxazolidinediones 31a–h, which were readily opened with cyclo-
propylsulfonamide in the presence of DBU to afford the final target
products 32a–h in 40–78% yield. NMR analysis confirmed that the
stereochemistry of the different chiral centers was perfectly
retained throughout the synthesis. Attempt to use the procedures
reported in Scheme
3 for the preparation of the isopro-
pylaminothiazolequinoline derivative 37 led to very low overall
yield, probably due to the presence of the unprotected amidine

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4853–4858
4857
meable (Caco-2 Papp A–B = 5.8 Â 10À6 cm/s). Finally, the bioisosteric
replacement of the isopropylthiazole with an isopropylpyrazole
and an isopropylpyridyl led to two compounds, 32g and 32h,
respectively, with similar potency and stability/permeability
profile.
Based on these promising in vitro data, the three lead com-
pounds 32c, 32g, and 32h were selected for rat PK analysis.
The plasma kinetics, oral bioavailability together with liver tis-
sue distribution in male Sprague–Dawley rats were determined
after a single oral administration of 20 mg/kg of macrocycles 32c,
32g, and 32h using 50% PEG-400 in water as vehicle. These data
were analyzed and compared to those obtained after a single intra-
venous (iv) administration of 2 mg/kg in 20% hydroxypropyl-b-
cyclodextrine. The compound levels were quantifiable up to 8 h
Br
Br
S
O
O
OH
S
O
O
O
O
O
iii
N
i
O
ii
N
H
N
H
N
H
N
N
O
O
O
O
O
O
O
O
O
O
33
28
34
NH
S
NH
S
NH
S
N
post-administration. The mean maximum concentrations (Cmax
)
N
N
N
O
in the plasma were all achieved between 1.5 and 3.0 h post-dose
(Tmax), indicating a rapid (for 32g) to medium–slow (for 32c and
32h) rate of absorption. Given that viral replication of HCV is re-
ported to occur primarily in hepatocytes, achieving high drug con-
centrations in the liver is believed to be critical for the clinical
success of HCV inhibitors. In this respect, 32c and 32g were found
to be well distributed with the high concentration observed in the
liver (liver/plasma ratio after oral administration is 32 and 44,
respectively). Since the residual concentration of 32h in the blood
and in the liver was below the detection limit after 24 h the liver/
plasma ratio could not be estimated. The mean half-life (T1/2) was
found similar for 32c and 32g (2.8 and 2.2 h, respectively), but was
significantly higher for 32h (5.8 h). More differences were ob-
served for the clearance (Cl), volume of distribution (Vdss), and area
under the curve (AUC) parameters. While 32c exhibits a low clear-
ance (Cl = 0.505 L/h/kg) associated with a low Vdss (0.490 L/kg) and
N
O
O
N
O
O
O
v
iv
O
O
O O
S
O
H
N
O
H
N
N
O
HN
N
O
O
N
N
O
O
O
O
O
35
36
37
Scheme 4. Reactions and conditions: (i) 2,2,2-diazobicyclooctane, 4-BrPhSO2Cl,
toluene, CH2Cl2, rt; (ii) Hoveyda-Grubbs 1st generation catalyst,19 dichloromethane,
rt; (iii) 10h, N-methylpyrrolidinone, Cs2CO3, 60 °C; (iv) a—LiOH, THF/MeOH/water,
rt; b—CDI, THF, reflux; (v) cyclopropylsulfonamide, DBU, THF, 50 °C.
high Cmax (0.73 lM) and high AUC (2.79 lM h), 32g and 32h,
respectively, exhibit a 3 and 5 times higher clearance, associated
with a significantly lower Cmax and AUC (Table 1). An excellent
PK profile for 32c was also observed in Beagle dog. The oral bio-
availability (F) after single oral administration of 6.5 mg/kg of
S
MeO
N
N
N
H
32c was found as high as 100%, with a high Cmax (4.72 lM) and
OH
AUC (14,986 ng h/mL) combined with a medium-long half-life
(T1/2 = 5.1 h). Moreover, a negligible attenuation (ꢀ2-fold) of po-
tency for 32c was observed in the presence of human serum albu-
Chart 2. Quinoline 11h.
min (40 lg/mL). A detailed biological profile of 32c will be
reduction in luciferase signals compared to that of the untreated
control. Since the main drawback for the initial lead 510 was the
poor PK profile, the apparent permeability coefficient in Caco-2
cells (Papp A–B) and the stability in human liver microsomes, mea-
sured by the intrinsic clearance (HLM Clint), were evaluated for the
most active derivatives listed in Table 1. The introduction of the
BILN 2061 (2)5 2-isopropylaminothiazol-4-yl moiety in the posi-
tion 2 of the quinoline led to 37, a potent (EC50 = 17 nM) derivative
with relative low permeability (A–B Papp = 1.4 Â 10À6 cm/s) deriva-
tive. Although similar low permeability was also observed with the
thiazol-2-yl derivative 32a (A–B Papp = 1.6 Â 10À6 cms/s) a high
Papp value was observed for the 4-isopropyl analog 32b (A–B
published in due course.
In summary, we report here on the successful replacement of
the pyrrolidine ring from the proline-containing macrocycle (e.g.,
2)5 with a cyclopentane surrogate leading to a novel series of
HCV NS3/4A protease inhibitors combining high potency in the
HCV replicon with good rat and dog pharmacokinetic profiles. Alto-
gether, these results prompted the selection of 32c (TMC435350)
for further preclinical and clinical development as an HCV drug
candidate.
References and notes
P
app = 13 Â 10À6 cm/s). Further optimization of the potency and
1. Lauer, G. M.; Walker, B. D. N. Engl. J. Med. 2001, 345, 41.
2. (a) Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos, G.; Gonçales,
F. L., Jr.; Häussinger, D.; Diago, M.; Carosi, G.; Dhumeaux, D.; Craxi, A.; Lin, A.;
Hoffman, J.; Yu, J. N. Engl. J. Med. 2002, 347, 975; (b) Scott, L. J.; Perry, C. M.
Drugs 2002, 62, 507.
pharmaceutical properties of 32b was achieved by the introduction
of either a methyl or a chloro substituent in position 8 of the quin-
oline leading to compounds 32c and 32f, respectively, as shown in
Table 1.
These two compounds exhibit an improved potency
(EC50 = 7.8 nM and 2.9 nM, respectively, for 32c and 32f) compared
to 32b (EC50 = 17 nM) and an improved stability in HLM
3. Lin, C.; Kwong, A. D.; Perni, R. B. Infect. Disord.: Drug Targets 2006, 6, 3.
4. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu,
Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.;
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.;
Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.;
Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay,
A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, Y.; Brisson, J.-M.;
Prelusky, D.; Korfmacher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.;
Bradley, P.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge,
F. G. J. Med. Chem. 2006, 49, 6074.
(Clint < 6 lL/min/mg for 32c and 9.0 lL/min/mg for 32f vs 16 lL/
min/mg for 32b). However, although the methyl derivative 32c
exhibits a similar permeability compared to the parent molecule
32b (Caco-2 Papp A–B = 8.4 Â 10À6 cm/s and 13 Â 10À6 cm/s, respec-
tively), the 8-chloro derivative 32f was found significantly less per-
5. Llinàs-Brunet, M.; Bailey, M. D.; Bolger, G.; Brochu, C.; Faucher, A.-M.; Ferland,
J. M.; Garneau, M.; Ghiro, E.; Gorys, V.; Grand-Maître, C.; Halmos, T.; Lapeyre-

4858
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4853–4858
Paquette, N.; Liard, F.; Poirier, M.; Rhéaume, M.; Tsantrizos, Y. S.; Lamarre, D. J.
Med. Chem. 2004, 47, 1605.
6. Hinrichsen, H.; Benhamou, Y.; Wedemeyer, H.; Reiser, M.; Sentjens, R. E.;
Calleja, J. L.; Forns, X.; Erhardt, A.; Crönlein, J.; Chaves, R. L.; Yong, C.-L.; Nehmiz,
G.; Steinmann, G. G. Gastroenterology 2004, 127, 1347.
7. (a) Vincent, Soriano; Antonio, Madejon; Eugenia, Vispo; Pablo, Labarga; Javier,
Garcia-Samaniego; Luz, Martin-Carbonero; Julie, Sheldon; Marcelle,
Bottecchia; Paula, Tuma; Pablo, Barreiro Expert Opin. Emerg. Drugs 2008, 13,
1; (b) Simmen, Kenneth; Lenz, Oliver; Lin, Tse-I; Fanning, Greg; Raboisson,
Pierre; de Kock, Herman; van’t Klooster, Gerben; Rosenquist, Asa; Edluna,
Michael; Nilsson, Magnus; Vrang, Lotta; Samuelsson, Bertil. 58th Annual
Meeting of the American Association for Study of Liver Diseases (AASLD),
November 6th, 2007, Boston, MA.
12. Soll, R. M.; Guinosso, C.; Asselin, A. J. Org. Chem. 1988, 53, 2844.
13. Tsantrizos, Y. S.; Cameron, D. R.; Faucher, A.-M.; Ghiro, E.; Goudreau, N.;
Halmos, T.; Llinàs-Brunet, M. World Patent WO2000059929A1, 2000.
14. Llinas-Brunet, M.; Bailey, M. D.; Cameron, D.; Faucher, A.-M.; Ghiro, E.;
Goudreau, N.; Halmos, T.; Poupart, M.-A.; Rancourt, J.; Tsantrizos, Y. S.; Wernic,
D. M.; Simoneau, B. WO2000009543A2, 2000.
15. Bartlett, P. A.; Green, F. R., III J. Am. Chem. Soc. 1978, 100, 4858.
16. Mitsunobu, O. Synthesis 1981, 1.
17. Meyer, C.; Piva, O.; Pete, J.-P. Tetrahedron 2000, 56, 4479.
18. Poirier, M.; Aubry, N.; Boucher, C.; Ferland, J.-M.; LaPlante, S.; Tsantrizos, Y. S. J.
Org. Chem. 2005, 70, 10765.
19. (a) Harrity, J. P. A.; La, D. S.; Visser, M. S.; Hoveyda, A. H. J. Am. Chem. Soc. 1998,
120, 2343; (b) Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H.
J. Am. Chem. Soc. 1999, 121, 791.
20. Poliakov, A.; Hubatsch, I.; Schuman, C. F.; Stenberg, G.; Danielsson, U. H. Protein
Expr. Purif. 2002, 25, 363.
8. Llinàs-Brunet, M.; Bailey, M. D.; Ghiro, E.; Gorys, V.; Halmos, T.; Poirier, M.;
Rancourt, J.; Goudreau, N. J. Med. Chem. 2004, 47, 6584.
9. Johansson, P.-O.; Bäck, M.; Kvarnström, I.; Vrang, L.; Hamelink, E.; Hallberg, A.;
Rosenquist, Å.; Samuelsson, B. Bioorg. Med. Chem. 2006, 14, 5136.
10. Bäck, M.; Johansson, P.-O.; Wångsell, F.; Thorstensson, F.; Kvarnström, I.; Ayesa
Alvarez, S.; Wähling, H.; Pelcman, M.; Jansson, K.; Lindström, S.; Wallberg, H.;
Classon, B.; Rydergård, C.; Vrang, L.; Hamelink, E.; Hallberg, A.; Rosenquist, Å.;
Samuelsson, B. Bioorg. Med. Chem. 2007, 15, 7184.
21. The enzyme inhibition assays were performed by Professor Pei Zhen Tao at
the department of virology, Institute of Medicinal Technology, Beijing, China
using the recombinant full-length NS3 enzyme following
a literature
procedure.8
22. Lohmann, V.; Hoffmann, S.; Herian, U.; Penin, F.; Bartenschlager, R. J. Virol.
2003, 77, 3007.
11. Lebel, H.; Leogane, O. Org. Lett. 2006, 8, 5717.