Discovery of novel potent and selective dipeptide hepatitis C virus NS3/4A serine protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.124

Starting from the previously reported HCV NS3/4A protease inhibitor BILN 2061 (1), we have used a fast-follower approach to identify a novel series of HCV NS3/4A protease inhibitors in which (i) the P3 amino moiety and its capping group have been truncated, (ii) a sulfonamide is introduced in the P1 cyclopropyl amino acid, (iii) the position 8 of the quinoline is substituted with a methyl or halo group, and (iv) the ring size of the macrocycle has been reduced to 14 atoms. SAR analysis performed with a limited set of compounds led to the identification of N-{17-[8-chloro-2-(4-isopropylthiazol-2-yl)-7-methoxyquinolin-4-yloxy]-2,14-dioxo-3,15-diazatricyclo [13.3.0.0 [Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000, 81, 1631; 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, 1-19]]octadec-7-ene-4-carbonyl}(1-methylcyclopropyl)(1-methylcyclopropyl)sulfonamide 19l an extremely potent (K_i = 0.20 nM, EC₅₀ = 3.7 nM), selective, and orally bioavailable dipeptide NS3/4A protease inhibitor, which has features attractive for further preclinical development.

Full text of DOI:10.1016/j.bmcl.2008.07.124

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5095–5100
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel potent and selective dipeptide hepatitis C virus NS3/4A
serine protease inhibitors
a
a
a
a
Pierre Raboisson a, , Tse-I Lin , Herman de Kock , Sandrine Vendeville , Wim Van de Vreken ,
David McGowan a, Abdellah Tahri a, Lili Hu a, Oliver Lenz a, Frederic Delouvroy a, Dominique Surleraux a,
Piet Wigerinck a, Magnus Nilsson b, Åsa Rosenquist b, Bertil Samuelsson b, Kenneth Simmen a
*
a Tibotec BVBA, Gen. De Wittelaan L11 B3, B-2800 Mechelen, Belgium
b Medivir AB, P.O. 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:
Starting from the previously reported HCV NS3/4A protease inhibitor BILN 2061 (1), we have used a fast-
follower approach to identify a novel series of HCV NS3/4A protease inhibitors in which (i) the P3 amino
moiety and its capping group have been truncated, (ii) a sulfonamide is introduced in the P1 cyclopropyl
amino acid, (iii) the position 8 of the quinoline is substituted with a methyl or halo group, and (iv) the
ring size of the macrocycle has been reduced to 14 atoms. SAR analysis performed with a limited set
of compounds led to the identification of N-{17-[8-chloro-2-(4-isopropylthiazol-2-yl)-7-methoxyquino-
lin-4-yloxy]-2,14-dioxo-3,15-diazatricyclo [13.3.0.0 [Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000,
81, 1631; 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, 1–19]]octadec-7-ene-4-carbonyl}(1-methylcyclopropyl)(1-methylcyclopropyl)sulfon-
amide 19l an extremely potent (Ki = 0.20 nM, EC50 = 3.7 nM), selective, and orally bioavailable dipeptide
NS3/4A protease inhibitor, which has features attractive for further preclinical development.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 25 June 2008
Revised 28 July 2008
Accepted 30 July 2008
Available online 3 August 2008
Keywords:
Hepatitis C
NS3
Macrocycle
Protease
An estimated 3% of the global human population is infected by
hepatitis C virus (HCV),1 an infection that often leads to cirrhosis,
hepatocellular carcinoma, and liver failure in later life. The current
standard of care therapy is based upon the use of pegylated inter-
peptidic character of this first series of NS3/4A inhibitors was de-
creased by successive N-terminal truncations leading to the discov-
ery of potent and selective tripeptide inhibitors.9 Further,
improvement of pharmaceutical properties was achieved by the
design of macrocyclic b-strand mimic scaffolds that culminated
with the discovery of the potent, highly selective, and orally bio-
available drug BILN 2061 (1).11 This inhibitor is characterized by
three unnatural amino acid residues (P1, P2, and P3), oriented in
a macrocycle as shown in Chart 1. Oral administration of BILN
feron-a in combination with ribavirin, leading to a sustained viro-
logical response (SVR) in approximately 50% of patients infected
with genotype 1 virus.2 Moreover, this limited efficacy is often
associated with side effects leading to discontinuation of treat-
ment.3 In this context, there is an urgent need for more effective,
convenient, and well-tolerated therapies. The HCV non-structural
protein 3 (NS3) serine protease has been shown to be essential
for viral replication.4 Together with RNA-dependent RNA polymer-
ase (RdRp) NS5B, NS3/4A protease are the most promising targets
in the search for next generation anti-HCV drugs.5 As such, multi-
ple antiviral agents targeting NS3/4A protease activity are cur-
rently under clinical development.6
On the basis of the initial finding that N-terminal cleavage prod-
ucts of NS3/4A protease peptide substrates are competitive inhib-
itors of this enzyme,7,8 preliminary SAR studies allowed the
identification of nanomolar substrate-based hexapeptide inhibi-
tors.7,9 Since it is well documented that polypeptides are unlikely
to permeate cells resulting in limited oral bioavailability,10 the
H
N
P2
N
N
S
O
N
O
P1
OH
O
H
N
O
O
P3 capping
group
O
O
N
H
1
(BILN 2061)
P3
* Corresponding author. Tel.: +32 15 44 42 62; fax: +32 1540 1257.
E-mail address: praboiss@tibbe.jnj.com (P. Raboisson).
Chart 1. NS3/4A protease inhibitor 1 (BILN 2061) with the P1, P2 and P3 moieties.11
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.124

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P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5095–5100
2061 to patients infected with the hepatitis C genotype 1 virus re-
sulted in an impressive reduction of viral RNA levels, establishing
the first proof-of-concept for HCV NS3/4A protease inhibitors as
therapeutic agents.12 Further development of this compound was
discontinued due to cardiotoxicity findings in primates given
supratherapeutic doses for several weeks.13 Although the mecha-
nism by which BILN 2061 exerts cardiotoxic effects has not been
published, a number of companies have initiated fast-follower pro-
grams with the rationale that the toxicity observed with the pio-
neer drug should be compound specific. As a result, several
groups have recently reported on macrocyclic HCV NS3/4A prote-
ase inhibitors entering phase 1 clinical trials.6,14 In this letter, we
report on our efforts to design a novel series of NS3/4A protease
inhibitors obtained by further truncation of the N-terminal part
of previously reported macrocyclic series (e.g., BILN 2061, 1).11
Although the optimization of the N-terminal cleavage products
of NS3/4A protease peptide substrates into tripeptide macrocyclic
b-strand mimics (e.g., BILN 2061) is well documented,9,11 the
importance of the P3 amino moiety and its capping group in the
overall potency and pharmaceutical properties of this series of
inhibitors have never been reported. Previously, however, the suc-
cessful truncation of P3 capping groups has been reported for an-
other series of macrocyclic HCV NS3/4A inhibitors incorporating
a cyclopentane dicarboxylic acid as a mimic of proline in the P2 po-
sition.15 Previous structural studies have indicated that the rigid
carbamate functionality of the capping group is responsible of
the proper positioning of the N-terminal alkyl group into the shal-
low S4 binding pocket of the enzyme,16 and that both the amino
and carbonyl groups of the P3 residue are involved in hydrogen
bonding to the protein backbone.16–18 Given the remarkable po-
tency of BILN-2061, we predicted that the truncation of the P3 ami-
no moiety and its capping group should lead to a compound which
retains enough intrinsic potency to be further optimized into a no-
vel series of drug-like NS3/4A protease inhibitors.
of the alkenylcarbonyl moiety using a standard peptide coupling
procedure with HATU led to the esters 13a and 13b in 97% and
93% isolated yields, respectively. The regioselective hydrolysis of
the p-nitrophenyl moiety of 13a and 13b was performed with lith-
ium hydroxide at low temperature to afford alcohols 14a and 14b,
respectively (91% and 87% yield, respectively). Introduction of the
P2 quinoline moieties 7a–h onto the proline central scaffold of
14a and 14b was achieved by a Mitsunobu reaction,23 which pro-
vided the definitive configuration of the chiral center at position
4 of the proline via a second epimerization leading to non-cyclic
intermediates 15a–k. Subsequently, the 14- and 15-membered
macrocyclic ring esters 16a–k were obtained in good yields (68–
87%) by ring-closing olefin metathesis of the open precursors
15a–k using Hoveyda–Grubbs 1st generation catalyst. Interest-
ingly, previous cyclization studies performed with intermediate
15a in refluxing dichloromethane did not produce the desired
product 16a with satisfactory yields.25 Instead, a complex mixture
of cis and trans olefins was observed in these reaction conditions
with low overall yields (<15%).
In the present study, the fact that 16a was obtained as the major
product in good yield (71%) with only traces of the trans derivative
is most likely due to the elevated temperature used, which may al-
low the open structures 15 to adopt the preorganized conforma-
tion needed for the cyclization to the thermodynamically more
stable ruthenium complex, thereby giving access to the cis olefin
macrocycle. Thus, we suggest that the temperature used in the
present study is high enough to overcome the rotational barrier
of the proline amide in position 1 leading to a rapid equilibrium be-
tween the cis and trans rotamers. This hypothesis is in accordance
with previous studies performed with similar diolefin substrates,
showing the coalescence of resonances obtained at elevated tem-
perature (67 and 77 °C) in 1H NMR spectroscopy experiments.25
Hydrolysis of the ethyl esters 16a–j provided the corresponding
acids 17a–j in quantitative yields.
The synthesis of the target products 19a–l (Table 1) is outlined
in Schemes 1 and 2. Preparation of 2-substituted quinolines 7a–f
was carried out either from commercially available m-anisidine
4d to generate the 8 non-substituted quinolines 7a and 7d, or from
the 2-substituted 3-methoxyanilines 4a–c obtained via a Curtius
rearrangement19 from the corresponding 3-methoxybenzoic acids
2a–c, as depicted in Scheme 1. Treatment of 3-methoxyanilines
4a–d with boron trichloride followed by the addition of acetoni-
trile and aluminum chloride afforded the corresponding ketones
5a–d via an aromatic electrophilic substitution in 40–73% yield.20
Finally, acylation of the anilines 5a–d followed by the subsequent
ring closure and aromatization of intermediates 6a–f led to the de-
sired quinolin-4-ol derivatives 7a–f (58-88%).20
Finally, treatment of acids 17a–j with carbonyldiimidazole led
to the oxazolidinediones 18a–j, that were subsequently opened
with sulfonamides to complete the synthesis of the target products
19a–l. Reduction of the double bond in the macrocycle of 19c was
performed with 2,4,6-triisopropylbenzenesulfonohydrazide and
triethylamine at 80 °C in methanol to give 19m (Chart 2) in only
6% yield after purification (reaction conditions non-optimized).
The new macrocyclic derivatives 19a–m prepared in the pres-
ent study were first tested for their inhibition of the full-length
NS3 protease activity in a biochemical assay in presence of a trun-
cated NS4A cofactor, as described earlier.26 The inhibition con-
stants (Ki values)27 were determined and are listed in Table 1.
The cell-based activities were measured in the Huh7-Rep cell line
containing the subgenomic bicistronic replicon clone ET with a
luciferase reporter read out.28 Inhibitory activities (EC50’s) were
calculated as the concentration of compound that caused a 50%
reduction in luciferase signals compared to that of the untreated
control (Chart 3).
The truncation of the terminal P3 amino moiety and its capping
group of BILN 2061 provided the dipeptide proline amide 17a, in
which the P3 amino acid has been replaced by an acyl group link-
ing the P2 proline with the P1 cyclopropyl amino acid. Although,
this modification led to a dramatic decrease in intrinsic potency,
17a still retained significant potency on the enzyme with a Ki of
46 nM. This observation prompted us to further investigate struc-
ture–activity relationships (SAR) of this shorter and less peptidic
series of NS3/4A protease inhibitors. Indeed, we predicted that
the decrease in intrinsic potency due to the cleavage of the P2
NH-capping group moiety could be compensated by further opti-
mizations. Since recent studies on NS3/4A protease inhibitors have
demonstrated the utility of acylsulfonamides as valuable bioisos-
teres of the carboxylic acid,29 we explored whether the introduc-
The other 4-hydroxyquinolines 7g and 7h were prepared fol-
lowing the procedures reported in the literature.11,21
The target macrocyclic products 19a–l were efficiently prepared
in a 10-step procedure from the N-Boc-trans-4-hydroxy-L-proline
8, 7-octenoic acid, or 8-nonenoic acid, (2R,3S)-3-vinyl-2-amino-2-
cyclopropylcarboxylic acid ethyl ester 9, and the 4-hydroxyquino-
lines 7a–h as depicted in Scheme 2. This synthesis represents an
adaptation of the literature procedure reported for the synthesis
of BILN 2061.11,21,22 Given the high cost of cis-hydroxyproline, we
have utilized a tandem of epimerization sequence involving two
successive Mitsunobu reactions,23 which allowed the use of the
readily available N-Boc-trans-4-hydroxy-L-proline as a starting
material for the P2 core.
The P1 cyclopropyl amino acid 924 was coupled to the P2 pro-
line core 8 using a standard HATU-mediated peptide coupling pro-
cedure in 97% yield. Inversion of the configuration at the position 4
of intermediate 10 via a Mitsunobu reaction23 was achieved using
p-nitrobenzoic acid to give ester 11 in 78% yield. Subsequent
unmasking of the BOC protecting group followed by the introduction

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5095–5100
5097
Table 1
HCV inhibition and metabolic stability
R2
N
R3
O
O
O
N
H
O
N
R1
O
R4
( )n
Compound
R1
R2
R3
H
R4
n
Ns3-Ki (nm)a
HUH7-Rep EC50 (
l
M)b
HLM stability (% metb.)c
28.5
H
N
H
N
O
1
–OH
2
0.3
0.00163
N
O
S
H
N
17a
17g
–OH
–OH
H
H
H
H
2
2
46
18
1.98
5.04
—
—
N
N
S
S
H
O O
N S
N
19a
19b
19c
19d
19e
H
H
H
H
H
H
2
2
2
2
2
0.9
0.204
91.5
N
N
H
S
O O
N S
H
0.5
0.00397
0.0180
0.0168
0.00437
—
S
H
O O
N S
N
H
0.65
1.0
98
H
S
O O
N S
N
H
100
H
S
S
O O
N S
N
CH3
0.50
98.4
H
O O
N S
19f
N
CH3
H
H
2
2
2.8
0.00584
0.00190
—
H
S
O O
N
19g
N S
Br
0.10
100
H
S
O
O
N S
19h
H
H
1
0.40
0.0239
82.4
N
H
S
(continued on next page)

5098
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5095–5100
Table 1 (continued)
Compound
R1
R2
R3
R4
H
n
Ns3-Ki (nm)a
HUH7-Rep EC50 (
l
M)b
HLM stability (% metb.)c
50.2
O O
N S
19i
19j
N
CH3
1
0.20
0.00355
H
S
O O
N S
CH3
H
H
H
1
1
1
0.40
0.40
0.20
0.0141
0.0117
0.00376
28.8
N
H
S
O
O
O
N S
19k
N
Cl
—
H
S
O
N S
19l
Cl
42.6
N
H
S
a
Inhibition of the full-length HCV NS3/4A protease26 measured by the inhibition constants (Ki values).27
Inhibition of HCV replication in Huh-7-Rep cells containing the subgenomic bicistronic replicon clone ET with a luciferase reporter read out for macrocyclic HCV NS3/4A
b
protease inhibitors 1, 17a, 17g, 19a–l measured by 50% effective concentration (EC50).28
c
Human liver microsomes (HLMs) stability measured by the percentage of metabolized product after 15 min at 37 °C in the presence of 5 lM of tested compound.
tion of this moiety into the acid P1 residue could also positively
impact the potency of the resulting compound 19a. To our satisfac-
tion, a substantial increase in intrinsic potency was observed for
the cyclopropylsulfonamide analogue 19a (Ki = 0.9 nM) compared
to the original acid derivative 17a (Ki = 46 nM). A similar beneficial
effect on the replicon cell-based assay was observed
of subtle changes in three different parts of the macrocyclic inhib-
itor: (i) P1 cyclopropylsulfonamide, (ii) P2 quinoline moiety, and
(iii) the macrocycle (ring size and saturation of the double bond).
Since we have found with a similar macrocyclic series (data pub-
lished elsewhere) that the incorporation of a 2-thiazole in position
2 of the quinoline provided a compound which exhibits better Caco
permeability (Papp values) compared to the 2-isopro-
pylaminothiazol-4-yl present in compounds BILN 2061, 17a, and
19a, we decided to perform this study on the 2-thiazolylquinoline
series. Although the first compounds synthesized 17g and 19c
were found equipotent compared to the corresponding isopropyla-
minothiazole analogues 17a and 19a, the metabolic stability was
still very poor (98% metabolized). The saturation of the double
bond in the macrocycle of 19c was found to be highly detrimental
for the potency of the resulting compound 19m (19c and 19m,
EC50 = 18 and 65.9 nM, respectively). This significant loss in po-
tency might be due to the unfavorable increased flexibility of
19m leading to an entropy penalty to adopt the proper bioactive
conformation.30 Interestingly, the introduction of either a methyl
(19e and 19f, EC50 = 4.3 and 5.8 nM, respectively) or a bromo sub-
stituent (19g, EC50 = 1.9 nM) in the position 8 of the quinoline led
to derivatives that were approximately fourfold more potent in
the replicon assay, providing single digit nanomolar NS3/4A inhib-
itors. This improvement in replicon activity of the 8-substituted
derivatives 19e–g is not fully understood at this point and might
be explained by more favorable overall physicochemical properties
leading to improved cell permeability. However, the metabolic sta-
bility of 19e–g remained very poor. Since the parent drug candi-
date BILN 2061 (1) was found metabolically stable, we
hypothesized that the low stability observed in the present series
might be attributed to the more flexible scaffold generated by
the cleavage of the P3-NH-capping group moiety, known to con-
strain the inhibitor in a b-strand mimic conformation.9,11 To verify
this hypothesis, we synthesized a series of more rigid 14-mem-
(EC50 = 1.98 lM and 20.4 nM, respectively, for 17a and 19a), mak-
ing this series of P3-truncated macrocyclic inhibitors interesting
candidates for further optimization. However, despite the dramat-
ically improved potency in both enzymatic and cell-based assays,
the very low metabolic stability of 19a, measured in vitro using hu-
man liver microsome (HLM) preparations, rapidly emerged as a
major drawback for this novel series. In fact, 19a was found to be
91.5% metabolized after 15 min, whereas BILN 2061 was only
28.5% metabolized under the same conditions. Therefore, we
decided to explore the effect on both the potency and HLM stability
R3
O
R3
R3
H
MeO
MeO
MeO
MeO
iii
NH2
iv, v
N
O
OH
i, ii
O
2a-c
R3
4a-d
R3
3a-c
R3
H
MeO
vi
N
R2
MeO
R2
N
NH2
O
vii
O
5a-d
6a-f
7a-f OH
O
with 2a-5a: R3 = Me
2b-5b: R3 = Cl
2c-5c: R3 = Br
6a, 7a, R3 = H
N
6b, 7b, R3 = CH3
R2:
S
6c, 7c, R3 = Br
4d-5d: R3 = H
6d, 7d, R3 = H
N
6e, 7e, R3 = CH3
R2:
S
6f, 7f, R3 = Cl
bered macrocycles listed in Table
1 (19h–l). Following this
approach, we were gratified to see that metabolic stability of 19h
was significantly better (82.4% metabolized) than the correspond-
ing 15-membered analogue 19c (98% metabolized). In addition, the
replicon activity was not affected throughout this ring constriction
Scheme 1. Reactions and conditions: (i) TEA, diphenylphosphorylazide (dppa),
toluene, 100 °C; (ii) t-BuOH, toluene, 100 °C; (iii) TFA, CH2Cl2, 20 °C; (iv) BCl3,
xylene, 0 °C; (v) CH3CN, AlCl3, CH2Cl2, 0 °C to 70 °C; (vi) R2COOH, POCl3, pyridine,
À20 to 0 °C; (vii) tBuOK, tBuOH, 80 °C.

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5095–5100
5099
NO2
O
O
OH
O2N
ii
O2N
OH
O
O
O
H2N
O
O
O
O
N
iii
iv
i
v
O
H
O
N
O
H
+
N
N
HN
O
N
O
O
O
H
H
O
OH
O
N
N
O
O
N
O
O
O
O
O
O
8
9
10
O
O
11
12
R3
R3
( )n
13a,b
R3
N
O
R2
O
O
N
O
R2
O
O
O
R2
O
N
H
O
HO
O
N
H
O
O
H
N
N
OH
viii
vi
ix
N
O
H
N
vii
O
O
N
O
N
O
O
N
O
O
( )n
( )
n 14a,b
O
( )n
( )n
R3
R3
15a-j
16a-j
R2
O
N
R2
O
N
17a-j
O
O
N
R2
R3
H
n
Cpd #
R2
R3
n
R1
O
O
Cpd #
H
N
O
O
H
N
15f-18f
15g-18g
15h-18h
15i-18i
15j-18j
CH3
H
H
CH3
Cl
2
N
15a-18a
2
x
2
1
1
1
N
N
N
O
S
O
O
( )n
N
O
( )n
S
15b-18b
H
H
2
S
18a-j
19a-l
(see Table 1)
15c-18c
15d-18d
15e-18e
2
2
N
13a,14a: n = 1
13b,14b: n = 2
CH 3
S
Br
2
Scheme 2. Reactions and conditions: (i) HATU, diisopropylethylamine, DMF, 0–20 °C; (ii) p-nitrobenzoic acid, PPh3, DIAD, THF, À20 to 20 °C; (iii) TFA, CH2Cl2, 20 °C; (iv)
7-octenoic acid (for n = 1) or 8-nonenoic acid (for n = 2) HATU, diisopropylethylamine, DMF, 0– 20 °C; (v) LiOH, H2O/THF, 0 °C; (vi) 7a-h, PPh3, DIAD, THF, -20 °C to room
temperature; (vii) Hoveyda-Grubbs 1st generation catalyst, dichloroethane, 70 °C; (viii) LiOH, H2O/MeOH/THF, 20 °C; (ix) CDI, THF, reflux; (x) cyclopropylsulfonamide or
methylcyclopropylsulfonamide, DBU, THF, 50 °C.
(EC50 = 23.4 and 18 nM, respectively). This beneficial effect was
even more pronounced for the 8-methyl quinoline derivative 19i
(50.2% metabolized). Eventually, introduction of a methyl group in
the P1 cyclopropyl sulfonamide moiety led to a compound that was
only 28.8% metabolized in the HLM assay. Similarly, the
8-chloroquinoline derivative 19l turned out to be stable (42.6%
metabolized) and very potent (EC50 = 3.76 nM) inhibitor of the HCV
NS3/4A protease. Together with the good permeability data observed
with 19l in Caco-2 cells (Papp apical to basolateral=11.1 Â 10À6 cm/s),
we selected this compound as the lead for this novel series of P3 trun-
cated derivatives. It is noteworthy that all the compounds tested in
this study, including 19l, did not exhibit any significant cytotoxicity
toward both MT4-LTR-Luc and Huh7-CMV-Luc cell lines
were compared to an oral administration of 10 mg/kg of 19l in a
50% PEG400 solution in which 2.5% vitamin E-TPGS was added.
The compound levels were quantifiable up to 8 h post administra-
tion. As shown in Figure 1 and Table 2, the mean maximum con-
centration (Cmax = 715 ng/mL) in the plasma was achieved at
0.75 h post-dose (Tmax), indicating a rapid absorption of 19l. Given
that viral replication of HCV is known to occur primarily in hepa-
tocytes, achieving high drug concentrations in the liver is believed
to be critical for the clinical success of HCV inhibitors. In this re-
spect, 19l was found to be well distributed in the liver with a very
high concentration observed after 8 h (3655 ng/g and 7665 ng/mg
after IV and oral administration, respectively). In contrast, the
heart exposure was found to be very low (15.1 ng/g and 35.8 ng/
mg after IV and oral administration, respectively), revealing a
200-fold higher concentration in the liver relative to the heart
(CC50 > 20 lM). To verify if the promising in vitro parameters (Caco-
2 permeability and improved HLM stability) reflected the in vivo situ-
ation, the pharmacokinetic (PK) properties of 19l were investigated
and the data are summarized in Table 2 and Fig. 1.
tissue. Furthermore, the mean half-life of 19l in the plasma (t1/2 (2–8h)
)
was found to be 2.73 h after oral administration. The oral bioavail-
The plasma kinetics, oral bioavailability together with heart and
liver-plasma tissue distribution in male SD rats were determined
after a single intravenous (IV) administration of 2 mg/kg of 19l
using 20% hydroxypropyl b-cyclodextrine as vehicle. These data
O
N
N
R
O
S
N
S
MeO
N
O
N
O O
S
H
O
N
N
OH
O
H
7g: R = H
7h: R =
i-PrNH
Chart 2. Quinoline reported in the literature.11,21
Chart 3. 19m (Ki = 1.9 nM, EC50 = 65.9 nM).

5100
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5095–5100
3. Fried, M. W. Hepatology 2002, 36, S237.
Table 2
4. Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000, 81, 1631.
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Res. 2003, 58, 1; (b) Griffith, R. C.; Lou, L.; Roberts, C. D.; Schm, U. Annu. Rep.
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Dev. 2004, 7, 446; (d) Goudreau, N.; Llinàs-Brunet, M. Expert Opin. Invest. Drugs
2005, 14, 1129.
Mean plasma and tissue levels (n = 2) together with some basic pharmacokinetic
parameters of macrocycle 19l after a single intravenous administration of 2 mg/kg in
hydroxypropyl-b-cyclodextrine and oral administration of 10 mg/kg in vitamin
E-TPGS in the male SD rat
Intravenous (n = 2)
Oral (n = 2)
6. Soriano, V.; Madejon, A.; Vispo, E.; Labarga, P.; Garcia-Samaniego, J.; Martin-
Carbonero, L.; Sheldon, J.; Bottecchia, M.; Tuma, P.; Barreiro, P. Expert Opin.
Emerg. Drugs 2008, 13, 1.
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Maurice, R.; Poirier, M.; Poupart, M.-A.; Thibeault, D.; Wernic, D.; Lamarre, D.
Bioorg. Med. Chem. Lett. 1998, 8, 1713.
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A.; De Francesco, R. Biochemistry 1998, 37, 8899.
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Rancourt, J.; Goudreau, N. J. Med. Chem. 2004, 47, 6584.
10. Lipinski, C. A.; Lombardo, F.; Domony, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
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11. Llinàs-Brunet, M.; Bailey, M. D.; Bolger, G.; Brochu, C.; Faucher, A.-M.; Ferland,
J. M.; Garneau, M.; Ghiro, E.; Gorys, V.; Maître, Grand.; Ted Halmos, C.; Lapeyre-
Paquette, N.; Liard, F.; Poirier, M.; Rhéaume, M.; Tsantrizos, Y. S.; Lamarre, D. J.
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12. Lamarre, D.; Anderson, P.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.; Bös,
M.; Cameron, D.; Cartier, M.; Cordingley, M.; Faucher, A.-M.; Goudreau, N.;
Kawai, S.; Kukolj, G.; Lagacé, 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.; Llinàs-Brunet, M.
Nature 2003, 426, 186.
Concentrations (ng/mL)
0.12 h
0.33 h
0.5 h
1 h
2 h
4 h
1500
708
—
449
290
139
53
—
—
698
693
632
363
133
715
0.75
2.73
3067
3607
—
8 h
Cmax (ng/ml)
Tmax (h)
T1/2 (2–8 h) (h)
AUC(0–8h) (ng h/mL)
AUC0-inf (ng h/mL)
Vdss (l/kg)
Cl (l/h/kg)
F (%)
Heart (8 h) (ng/g)
Liver (8 h) (ng/g)
—
—
2.49
1961
2154
2.75
3.94
—
31
72.7
7665
15.1
3655
13. 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.
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Herman de Kock, Gerben van’t Klooster, Asa Rosenquist, Michael Edluna,
Magnus Nilsson, Lotta Vrang, Bertil Samuelsson. 58th Annual Meeting of the
American Association for Study of Liver Diseases (AASLD), Nov 6th, 2007,
Boston (MA).
15. (a) Johansson, P.-O.; Bäck, M.; Kvarnström, I.; Vrang, L.; Hamelink, E.; Hallberg,
A.; Rosenquist, A.; Samuelsson, B. Bioorg. Med. Chem. 2006, 14, 5136; (b) 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.
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Halmos, T.; Llinàs-Brunet, M. World Patent WO2000059929A1, 2000.
21. Tsantrizos, Y. S.; Cameron, D.; Faucher, A. -M.; Ghiro, E.; Goudreau, N.; Halmos,
T.; Llinàs-Brunet, M.; U.S. Patent 6,608,027B1, 2003.
oral (10 mg/kg)
intravenous (2 mg/kg)
1000
100
10
0
2
4
6
8
Time (h)
Figure 1. Mean plasma concentrations (n = 3) of 19l after a single intravenous
administration of 2 mg/kg in hydroxypropyl-b-cyclodextrine and oral administra-
tion of 10 mg/kg in vitamin E-TPGS in the male SD rat.
ability (F) and oral AUC are, respectively, 31% and 3607 ng.h/mL. A
promising PK profile was also observed in Beagle dog (n = 3) after a
single intravenous (IV) administration of 1 mg/kg of 19l character-
ized by a high AUC (1322 ng h/mL) and low clearance (0.73 l/h/kg).
The observed oral bioavailability (F) after single oral administra-
tion of 4 mg/kg was 21%, with a Cmax at 498 ng/mL, a half-life
(T1/2) of 4.0 h, and an AUC of 928 ng h/mL.
In conclusion, we have reported here a novel series of potent
and non-cytotoxic P3-truncated macrocyclic HCV NS3/4A protease
inhibitors. Structure–activity relationships studies performed with
a very limited set of compounds (<20) in parallel on the potency
and HLM stability led to the identification of N-{17-[8-chloro-2-
(4-isopropylthiazol-2-yl)-7-methoxy-quinolin-4-yloxy]-2,14-di-
oxo-3,15-diazatricyclo [13.3.0.04,6]octadec-7-ene-4-carbonyl}-(1-
methylcyclo propyl)-(1-methylcyclopropyl)sulfonamide 19l, a po-
22. Faucher, A.-M.; Bailey, M. D.; Beaulieu, P. L.; Brochu, C.; Duceppe, J.-S.; Ferland,
J.-S.; Ghiro, E.; Gorys, V.; Halmos, T.; Kawai, S. H.; Poirier, M.; Simoneau, B.;
Tsantrizos, Y. S.; Llinàs-Brunet, M. Org. Lett. 2004, 6, 2901.
23. Mitsunobu, O. Synthesis 1981, 1.
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Goudreau, N.; Halmos, T.; Poupart, M. -A.; Rancourt, J.; Tsantrizos, Y. S.;
Wernic, D. M.; Simoneau, B. World Patent WO00/09543, 2000.
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26. Poliakov, A.; Hubatsch, I.; Schuman, C. F.; Stenberg, G.; Danielsson, U. H. Protein
Expr. Purif. 2002, 25, 363.
27. The enzyme inhibition assays were performed using the recombinant full
length genotype 1 NS3 enzyme following the literature procedure reported by
Johansson, P.-O.; Bäck, M.; Kvarnström, I.; Jansson, K.; Vrang, L.; Hamelink, E.;
Hallberg, A.; Rosenquist, Å.; Samuelsson, B. Bioorg. Med. Chem. 2006, 14, 5136.
28. Lohmann, V.; Hoffmann, S.; Herian, U.; Penin, F.; Bartenschlager, R. J. Virol.
2003, 77, 3007.
29. (a) Johansson, A.; Poliakov, A.; Akerblom, E.; Wiklund, K.; Lindeberg, G.;
Winiwarter, S.; Danielson, UH.; Samuelsson, B.; Hallberg, A. Bioorg. Med. Chem.
2003, 11, 2551; (b) Örtqvist, P.; Peterson, S. D.; Åkerblom, E.; Gossas, T.; Sabnis,
Y. A.; Fransson, R.; Lindeberg, G.; Danielson, U. H.; Karlén, A.; Sandström, A.
Bioorg. Med. Chem. 2007, 15, 1448; (c) Rönn, R.; Sabnis, Y. A.; Gossas, T.;
Åkerblom, E.; Danielson, U. H.; Hallberg, A.; Johansson, A. Bioorg. Med. Chem.
2006, 14, 544.
tent (Ki = 0.2 nM, EC50 = 3.76 nM), non-cytotoxic (CC50 > 20 lM),
and metabolically stable HCV inhibitor. Moreover, 19l exhibits
advantageous drug metabolism and pharmacokinetic properties
in rat and dog, with a high liver exposure after oral administration,
but limited heart exposure observed in rat. Further preclinical
exploration of this novel inhibitor series is warranted.
References and notes
30. Goudreau, N.; Brochu, C.; Cameron, D. R.; Duceppe, J.-S.; Faucher, A.-M.;
Ferland, J. M.; Grand-Maître, C.; Poirier, M.; Simoneau, B.; Tsantrizos, Y. S. J. Org.
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