Discovery of novel, potent and bioavailable proline-urea based macrocyclic HCV NS3/4A protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.004

A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved PK profiles, ultimately leading to compound 13Bh, an extremely potent (K_i = 0.1 nM, EC₅₀ = 4.5 nM) and selective (CC₅₀ (Huh-7 cells) > 50 μM) inhibitor, displaying an excellent PK profile in rats characterized by an oral bioavailability of 54% and a high liver exposure after oral administration.

Full text of DOI:10.1016/j.bmcl.2008.10.004

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6189–6193
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel, potent and bioavailable proline-urea based macrocyclic
HCV NS3/4A protease inhibitors
Sandrine Vendeville a, Magnus Nilsson b, Herman de Kock a, Tse-I Lin a, Dmitry Antonov b, Björn Classon b,
Susana Ayesa b, Vladimir Ivanov b, Per-Ola Johansson b, Pia Kahnberg b, Anders Eneroth b,
Kristina Wikstrom b, Lotta Vrang b, Michael Edlund b, Stefan Lindström b, Wim Van de Vreken a,
David McGowan a, Abdellah Tahri a, Lili Hu a, Oliver Lenz a, Frederic Delouvroy a, Marleen Van Dooren a,
Natalie Kindermans a, Dominique Surleraux a, Piet Wigerinck a, Åsa Rosenquist b, Bertil Samuelsson b,
Kenneth Simmen a, Pierre Raboisson a,
*
a Tibotec BVBA, Gen. De Wittelaan L11 B3, B-2800 Mechelen, Belgium
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:
A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea
or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of
the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid
moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved
PK profiles, ultimately leading to compound 13Bh, an extremely potent (Ki = 0.1 nM, EC50 = 4.5 nM) and
Received 28 August 2008
Revised 29 September 2008
Accepted 1 October 2008
Available online 5 October 2008
selective (CC50 (Huh-7 cells) > 50
an oral bioavailability of 54% and a high liver exposure after oral administration.
lM) inhibitor, displaying an excellent PK profile in rats characterized by
Keywords:
Hepatitis C
Serine protease
Macrocycle
NS3
Ó 2008 Elsevier Ltd. All rights reserved.
An estimated 170 million people are chronically infected by
HCV, a major cause of acute hepatitis and chronic liver disease
worldwide.1 Only around 20% of newly infected individuals spon-
taneously clear the virus from their body, while in the majority
of cases HCV persists as a chronic infection. Untreated HCV infec-
tion can ultimately progress to cirrhosis, hepatocellular carcinoma
and liver failure, and is the major indication for liver transplants in
western countries.2 The current standard of care therapy consists
Currently there are several NS3/4A protease inhibitors in clini-
cal trials, which can be divided into two main classes in Figure 1:
(i) the a-ketoamide electrophilic trap-containing inhibitors, exam-
ples being VX-950 (1, Telaprevir)6 and SCH-503034 (2, Bocepre-
vir)7 and (ii) macrocyclic inhibitors exemplified by BILN-2061
(3).8,9 Macrocyclic NS3/4A inhibitors are characterized by a hydro-
carbon bridge linking the P1 side-chain to the P3 moiety, leading to
a b-strand mimetic conformation. Other important features are the
of pegylated interferon-
a
and the antiviral drug ribavirin.3 This
treatment, indirectly targeting the virus, suffers from several draw-
backs,4 such as limited efficacy on genotype 1 infected patients, ad-
verse events and poor tolerability often leading to treatment
discontinuations. Consequently there is a unmet medical need for
new therapies, with a better efficacy and an improved adverse
event profile.
The virally encoded NS3/4A serine protease, which cleaves the
viral polyprotein has been shown to be an essential component
of the HCV life cycle.5 Numerous medicinal chemistry programs
have been initiated to find potent and selective inhibitors of this
target.
P2
N
N
O
H
N
H
N
N
O
H
N
H
N
N
H
O
O
N
O
S
O
O
O
N
O
1
(VX-950)
O
H
HN
O
N
O
N
O
OH
NH
N
O
P1
O
N
H
NH
P3
O
O
NH2
3
2
O
(BILN 2061)
(SCH 503034)
* Corresponding author. Tel.: +32 473 55 44 57; fax: +32 1540 1257.
E-mail address: praboiss@tibbe.jnj.com (P. Raboisson).
Figure 1. Structures of NS3/4A reference inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.004

6190
S. Vendeville et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6189–6193
P1 carboxylic acid binding to the active site oxyanion hole, the P2
quinoline and the P3-capping group.10
N
O
N
O
S
Although SAR on this class of inhibitors have been extensively
studied,11 little is known about the variations of the P3-capping
group in the macrocyclic series, whose presence was thought to
be essential to obtain a good enzymatic and cellular potency. We
have recently shown in a related macrocyclic series that adequate
combinations of P3-truncated molecules with optimized P2 moie-
ties and a P1 sulfonamide could lead to extremely potent and bio-
available compounds.12 Now we report on our efforts to transfer
our previous findings into a new series of P3-truncated inhibitors,
leading to derivatives with an excellent antiviral and pharmacoki-
netic (PK) profile. Structure–activity relationships and PK proper-
ties of the most promising compounds are reported.
The synthesis of the target proline-urea and carbamate prod-
ucts 13 is outlined in Schemes 1 and 2. The P2 quinoline substitu-
ents 4A–E were introduced at the first step of the synthesis, in very
good yields (75–100%), via a nucleophilic aromatic substitution of
the corresponding 4-chloroquinolines 5A–E by Boc-trans-4-hydro-
R2
O
N
O
R2
OH
O
N
i
O
O
+
N
ii
N
OH
O
O
X
( )n
O
O
O
S
NH
N
O
O
O
N
OH
R3
O
4F
H
O
6F
13Fa,h
Scheme 2. Reactions and conditions: (i) DIAD, pyridyldiphenylphosphine, THF,
À15 °C to rt; (ii) LiOH, THF/MeOH/H2O, rt.
(68–87%) by ring-closing olefin metathesis of the open precursors
10 using Hoveyda–Grubbs 1st generation catalyst.13 Hydrolysis
of the ethyl esters 11 provided the corresponding acids 12 in quan-
titative yields. Finally, in situ activation of the carboxylic acids 12
to the non-isolated oxazolidinone intermediates followed by reac-
tion with cyclopropylsulfonamide or methylcyclopropylsulfona-
mide, led to the desired target products 13. Noteworthy the low
yield observed in this last reaction (30% yield after recrystalliza-
tion) could be improved by protecting the NH of the urea with a
paramethoxybenzyl group, which can be introduced at the urea
formation stage. Although this pathway involved one more step
at the end of the synthesis (removal of the paramethoxybenzyl
group in TFA), overall yields were much higher with 95% for the
reaction with sulfonamide and almost quantitative yield for the
deprotection.
xy-L-proline. The P1 cyclopropyl amino acid was then coupled to
the P2 proline cores 6A–E using a standard HATU-mediated pep-
tide coupling procedure. After removal of the Boc protecting group
under standard conditions (TFA/DCM), the proline intermediates
8A–E were either treated with phosgene, to afford the correspond-
ing chloroimidate derivatives, which were subsequently reacted
with an alkenamine in the presence of sodium bicarbonate to af-
ford ureas 10Aa,b,d,e,f, 10Ba,b,e,f, 10Ca, 10Da and 10Ea in good
yields (i.e., 80%). Alternatively a pre-activated paranitrophenylcar-
bonate hydroxyalkene was used to give the corresponding carba-
mate derivatives 10Ac and 10Ag. Then, the 14- and 15-
membered macrocyclic ring esters 11 were obtained in good yields
Alternatively the more electron-rich 8-methylquinoline deriva-
tive 4F was introduced via a Mitsunobu reaction14 with Boc-cis-hy-
droxy-
L
-proline to yield the intermediate 6F in 85% yield. The
R1
R1
R2
R2
O
N
O
O
N
O
OH
R1
R2
O
O
N
H2N
O
O
N
+
ii
O
OH
R4
iii
O
O
O
N
O
N
4A-E, R4 = OH
5A-E, R4 = Cl
NH
O
O
i
OH
O
O
O
6A-E
7A-E
R1
R1
R1
R2
O
N
O
R2
iv
R2
O
N
O
O
N
O
H
( )n
X
O
O
vii
H
9a-h
O
O
N
HN
N
N
NH
O
X
( )n
X
( )n
NH
v or vi
O
O
O
O
O
O
8A-E
10Aa-g; 10Ba,b,e,f
10Ca; 10Da; 10Ea
11Aa-g; 11Ba,b,e,f
11Ca; 11Da; 11Ea
n = 5
X
NH
NMe
O
n = 4
X
NH
NMe
O
a
b
c
d
h
e
f
g
R3 = H
R1
R1
R2
R2
O
N
O
O
N
O
NNHBoc
R3 = Me NH
R2
S
R1
R2
N
O
O
O
ix, x
viii
N
S
N
N
N
N
S
O
NH
O
X
X
O
S
NH
O
R3
H
A
B
D
E
( )n
( )n
O
O
N
OH
R1
Cl
H
C
F
12Aa-g; 12Ba,b,e,f
12Ca; 12Da; 12Ea
13Aa-g; 13Ba,b,e,f,h
13Ca,h; 13Da; 13Ea
Me
Scheme 1. Reactions and conditions: (i) POCl3 reflux, 95% yield; (ii) DMSO, KOt-Bu, then 4-chloroquinoline derivative 5A–E, rt; (iii) HATU, diisopropylethylamine, DMF, 0–
20 °C; (iv) TFA, CH2Cl2, 20 °C; (v) phosgene 1.9 M in toluene, then 9a,b,d,e,f, NaHCO3, DCM; (vi) a—hydroxyalkenyl derivative 9c,g, 4-nitrophenylchloroformate, pyridine
(3 equiv), DCM, 0 °C to rt, 73%; b—triethylamine, DCM, rt, 12 h; (vii) Hoveyda–Grubbs 1st generation catalyst, dichloroethane, 70 °C, 20 h; (viii) LiOH, THF/MeOH/H2O, rt, 48 h;
(ix) CDI, THF, reflux; (x) cyclopropylsulfonamide or methylcyclopropylsulfonamide, DBU, THF, 50 °C.

S. Vendeville et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6189–6193
6191
cleavage of the methylester to obtain the carboxylic acid 6F was
subsequently achieved under classical conditions. Compounds
13Fa and 13Fh were then obtained using the procedure depicted
in Scheme 1.
The compounds described herein were tested in an enzymatic
assay against the full length NS3/4A protease as described earlier15
and their potency, expressed as rough Ki value,16,17 is listed in Ta-
ble 1. The cell-based anti-HCV activities (EC50) were determined in
the Huh-7 replicon cell line containing the subgenomic bicistronic
HCV replicon clone ET with a luciferase read out18 and compared to
the toxicity measured in MT4 and Huh-7 cell lines.
O
N
O
O
N
O
O
N
O
O
O
O
H
H
H
O
O
N
N
N
O
O
N
O
N
N
S
O
OH
O
OH
N
H
N
N
O
O
O
O
N
H
O
N
H
14
11 nM
77 nM
12Ad
600 nM
> 10,000 nM
12Ab
10 nM
4500 nM
IC50
EC50
Figure 2. NS3/4A inhibitors.
Although the replacement of the C-
a in the known inhibitor
1411 by a nitrogen as exemplified by compound 12Ad resulted in
a decrease in both enzymatic and cellular potency (Ki = 600 nM,
Pharmacokinetic parameters of compounds 13Ba, 13Be and 13Bf,
obtained after a single intravenous (2 mg/kg) or an oral (10 mg/
kg) administration using 50% PEG-400 in water as vehicle are sum-
marized in Table 2. After oral administration, compounds 13Ba and
13Bf exhibited promising oral bioavailabilities of 20% and 37%,
respectively, together with good Cmax values (resp. 0.95
0.73 M, respectively), unlike compound 13Be whose bioavailabil-
ity was very poor (1.3%) caused by limited absorption. These values
are in line with results from the Caco-2 assay that was used as a
prediction tool for absorption rate. Indeed with a Papp (A–B) of
3 Â 10À6 cm/s, 13Be was predicted to be poorly absorbed.
EC50 > 10 lM), previous work from our laboratory on trisubstituted
cyclopentane dicarboxylic acid as a novel P2 mimetic12 prompted
us to synthesize derivative 12Ab (Fig. 2). To our satisfaction, this
strategy was indeed applicable to this new proline-urea series,
and completely restored the enzymatic potency (compare 14 to
12Ab). Thus, shortening the P3-capping group from Boc-hydrazine
to a N-methyl moiety in combination with a carboxylic acid isoste-
re provided 12Ab with a similar enzymatic potency to the refer-
ence 14, however with a decreased cellular potency.
lM and
l
Preliminary analysis performed on a limited subset of N-substi-
tuted prolines bearing a 2-phenylquinoline (compounds 13Aa–c,e–
g) revealed that both the nature of the substitution, carbamate or
urea and the ring size dramatically influence the potency of the
compounds. Although the N-methylurea 13Ab in the 15-mem-
bered macrocycle series was found much less potent than the cor-
responding carbamate 13Ac, the opposite observation was made in
the 14-membered series (compare 13Af with 13Ag). However, the
NH proline-ureas 13Aa and 13Ae were constantly found to be the
most potent compounds in the cell-based assay in both the 14- and
15-membered ring series with EC50 values of 14 and 15 nM,
respectively. Noteworthy, a high shift between the enzymatic po-
tency and replicon activity was observed with the two carbamates
13Ac and 13Ag, which could be attributed to a lower cell mem-
brane permeability. The same trend was observed with the isopro-
pylthiazole quinolines 13Ba,b,e,f, with the NH-carbamate 13Ba
and 13Be being the most active compounds of this subseries.
Having three different potent series in hand, we decided to eval-
uate their in vivo PK properties in male Sprague–Dawley rats in or-
der to further prioritize and optimize these subseries.
Table 2
Mean plasma levels (n = 2) and pharmacokinetic parameters of macrocycles 13Ba,
13Bf and 13Be after a single intravenous (2 mg/kg) and oral administration (10 mg/
kg) in male Sprague–Dawley rats
Compound
13Ba
13Bf
13Be
iv (2 mg/kg, n = 2)
Cl (L/h/kg)
Vz (L/kg)
0.55
1.46
7.09
1.65
4.50
1.65
0.94
1.60
3.00
AUC (
l
M h)
Oral (10 mg/kg, n = 2)
AUC (
l
M h)
6.97
0.95
1.25
5.4
3.12
0.73
1.25
2.55
37
0.19
0.026
2
Cmax (lM)
Tmax (h)
T1/2
F (%)
3.8
20
1.3
Liver/plasma ratio
29*
12**
9.5*
*
At 6 h.
At 8 h.
**
Table 1
Effect of the macrocycle size and carbamate/urea substitution on enzymatic and cellular potency
N
O
N
O
N
S
Structure
X
Compound
Ki (nM)
EC50 (nM)
Compound
Ki (nM)
EC50 (nM)
R1
O
O
O
H
O O
S
NMe
O
NH
13Ab
13Ac
13Aa
10
2.4
0.24
4500
460
15
13Bb
—
13Ba
12
—
0.2
700
—
5
N
O
O
N
N
H
X
O
R1
O
H
N
O O
S
NMe
O
NH
13Af
13Ag
13Ae
0.4
0.35
1.5
44
62
14
13Bf
—
13Be
0.45
—
0.5
26
—
5
N
N
H
X
O

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S. Vendeville et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6189–6193
Values obtained for compounds 13Ba and 13Bf, just higher than
group on the sulfonamide moiety. Enzymatic and cellular potency,
Caco-2 permeability and metabolic stability in rat liver micro-
somes of the new derivatives were evaluated and are displayed
in Table 3.
10 Â 10À6 cm/s, suggested a better absorption. The 15-membered
ring compound 13Ba had a low clearance associated with a low
volume of distribution, and a 4-fold higher AUC after IV dosing
compared to the 14-membered ring NH compound 13Be.
Removal of the isopropyl group of compound 13Ba led to the
less lipophilic analogue 13Da, with a 2log difference in calculated
AlogP value. This drop of lipophilicity impacted the Caco-2 perme-
ability which became very low (0.4 Â 10À6 cm/s). Replacement of
the 2-thiazole moiety by its isomer 4-thiazole 13Ea led to an
important decrease of potency (EC50 = 98 nM). Interestingly, the
introduction of a methyl or chlorine on the position 8 of the quin-
oline provided compounds with a slightly better potency and a sig-
nificantly improved metabolic stability in RLM. Thus compounds
13Fa and 13Ca were only 23% and 45% metabolized in rat liver
microsomes. Furthermore, the introduction of a methyl on the P1
sulfonamide moiety also further enhanced metabolic stability
leading to the very stable compound 13Ch, which was only 21%
metabolized in RLM. The permeability for these more lipophilic
analogues remained high to very high in Caco-2 experiments. From
this list of highly potent, permeable and stable analogues, 13Fh
and 13Bh mainly differing by their lipophilicity were chosen for
rat PK evaluation. Compound 13Fh was found to have a moderate
Since HCV replicates in hepatocytes, achieving a high concen-
tration in the liver is of prime importance for an anti-HCV drug.
Thus, the ratios between liver and plasma concentrations were cal-
culated at 6 or 8 h post-dose and showed that all three compounds
had good liver exposure. The liver to plasma ratios were about 10
for the two 14-membered ring molecules, and 30 for the 15-mem-
bered ring inhibitor. Taken together with the good bioavailability
and high potency, these ratios led us to select the 15-membered
ring NH series for further optimization. Our challenge was mainly
the optimization of the permeability of our lead compound 13Ba to
increase in vivo exposure, while maintaining the other parameters
(i.e., EC50 < 10 nM and low clearance). Based on promising overall
properties of the lead compound 13Ba, we focused our efforts on
the 7-methoxyquinoline moiety substituted with a thiazole as P2
substituent and embarked on subtle changes such as variations
on the thiazole ring, introduction of a small lipophilic substituent
at the position 8 of the quinoline, and introduction of a methyl
Table 3
Potency and in vitro ADME parameters of lead optimization compounds
R1
R2
O
N
O
O
O
O
H
N
N
O
S
N
H
R3
HN
O
Compound
R1
H
R2
R3
H
Ki (nM)
EC50 (nM)
5
Papp (AB)a  10À6 cm/s
RLMb (% metabolized)
93
AlogP
Fc (%)
13Ba
0.28
10
4.13
20
N
S
N
N
N
13Da
H
H
0.3
11
0.4
—
2.72
—
S
13Ea
13Bh
H
H
H
—
98
—
—
2.74
4.34
—
S
Me
0.1
4.5
30
89
54
S
13Fa
13Ca
13Fh
Me
Cl
H
0.05
0.1
1.7
2.2
6.8
4
9
22
—
23
45
—
4.62
4.80
4.82
5.00
—
N
N
N
N
S
H
—
S
S
S
Me
Cl
Me
Me
0.2
11
—
13Ch
0.1
29
21
a
A–B apparent permeability coefficient (Papp) measured in Caco-2 cells.
Rat liver microsomes (RLM) stability measured by the % of metabolized product after 15 min at 37 °C in the presence of 5 lM of test compound using 1 mg of protein/mL.
Bioavailability after a single intravenous (2 mg/kg) and oral administration (10 mg/kg) in male Sprague–Dawley rats.
b
c

S. Vendeville et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6189–6193
6193
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(0.38 lM) and the clearance remained low but was not better
(Cl = 0.78 L/h/kg) despite a better in vitro metabolic stability. Over-
all compound 13Bh was found to possess the desired properties in
terms of permeability (highest Papp (AB) value), potency (single di-
git nanomolar) and metabolic stability (slightly better than the
lead). These properties translated in excellent PK parameters: a
very good Cmax (1.71 lM), low volume of distribution, low clear-
ance (0.59 L/h/kg) and excellent bioavailability (54%) associated
with a very good liver exposure (the liver/plasma ratio at 6 h after
oral dosing is 105).
In conclusion, we have developed a new series of P3-truncated
proline-urea based macrocyclic NS3/4A protease inhibitors. Early
rat PK evaluation and a good correlation between in vivo and
in vitro permeability allowed us to select, with a limited set of
derivatives, the promising compound 13Bh which combines a high
potency (EC50 = 4.5 nM) and no cytotoxicity (selectivity index-
Huh-7 cells > 10,000) with excellent PK properties and high liver
exposure. These data warrant further characterization of this novel
preclinical candidate.
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References and notes
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