Synthesis and antiviral evaluation of a novel series of homoserine-based inhibitors of the hepatitis C virus NS3/4A serine protease

Article abstract of DOI:10.1016/j.bmcl.2015.07.020

Abstract We disclose here the synthesis of a series of macrocyclic HCV protease inhibitors, where the homoserine linked together the quinoline P2′ motif and the macrocyclic moiety. These compounds exhibit potent inhibitory activity against HCV NS3/4A protease and replicon cell based assay. Their enzymatic and antiviral activities are modulated by substitutions on the quinoline P2′ at position 8 by methyl and halogens and by small heterocycles at position 2. The in vitro structure activity relationship (SAR) studies and in vivo pharmacokinetic (PK) evaluations of selected compounds are described herein.

Full text of DOI:10.1016/j.bmcl.2015.07.020

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiviral evaluation of a novel series of homoserine-
based inhibitors of the hepatitis C virus NS3/4A serine protease
a
a,
François-René Alexandre a, , Guillaume Brandt , Catherine Caillet , Dominique Chaves a,
,
⇑
Thierry Convard a, Michel Derock a, , Damien Gloux a, , Yann Griffon a, , Lisa Lallos b, , Frédéric Leroy a,
Michel Liuzzi b, , Anna-Giulia Loi b, , Laure Moulat a, , Chiara Musiu b, , Christophe Parsy a,
Houcine Rahali a, Virginie Roques a, , Maria Seifer b, , David Standring b, , Dominique Surleraux a,
,
a IDENIX, an MSD Company, 1682 rue de la Valsière, Cap Gamma, BP 50001, 34189 MONTPELLIER Cedex 4, France
b IDENIX, an MSD Company, 320 Bent Street—4th Floor, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We disclose here the synthesis of a series of macrocyclic HCV protease inhibitors, where the homoserine
linked together the quinoline P20 motif and the macrocyclic moiety. These compounds exhibit potent
inhibitory activity against HCV NS3/4A protease and replicon cell based assay. Their enzymatic and
antiviral activities are modulated by substitutions on the quinoline P20 at position 8 by methyl and
halogens and by small heterocycles at position 2. The in vitro structure activity relationship (SAR) studies
and in vivo pharmacokinetic (PK) evaluations of selected compounds are described herein.
Ó 2015 Published by Elsevier Ltd.
Received 3 June 2015
Revised 6 July 2015
Accepted 7 July 2015
Available online xxxx
Keywords:
Hepatitis C
HCV NS3/4A protease inhibitors
Macrocycle
Synthesis and biological evaluation
Hepatitis C Virus (HCV) infection is a chronic disease affecting
around 3% of the world’s population.1 Initially asymptomatic, this
infection can ultimately lead to liver cirrhosis, fibrosis and hepato-
cellular carcinoma. In developed countries, HCV is the main cause
of liver transplantation. At the time of this study, the previous
standard of care therapy combined a subcutaneous injection of a
cleaved to produce structural and non-structural proteins constitu-
tive of the virus. The HCV Serine Protease NS3/4A enzyme is
responsible for the cleavage of four out of the five protein from
the polyprotein occurring in the nonstructural region and is essen-
tial for viral replication. To date, two classes of protease inhibitors
have been reported: serine-trap based covalent compounds and
non covalent macrocyclic inhibitors. The first class, the serine trap
inhibitors such as VX-950 (telaprevir) and SCH-503034 (bocepre-
modified interferon-a with an oral administration of Ribavirin.
However this treatment was inconvenient, poorly tolerated and
induced a sustained viral response (SVR) in 42–82% of patients
depending of the genotypes.2 In this context development of oral,
effective and well tolerated therapies became a major stake for
academic and industrial researchers. Quite recently, progress in
the understandings of the HCV organization, virus life cycle and
development of HCV replicons cell-based assays has conducted
number of research groups to work on rational drug design of
specific inhibitors of the virus life cycle.3 This recent therapeutic
progress, with the development of new direct antiviral agent
(DAA) combinations, are transforming chronic HCV into a curable
disease.4 Replication of HCV occurs predominantly in hepatocytes,
the genome is translated into a polyprotein that is subsequently
vir) are constructed around an
a-ketoamide that covalently and
reversibly binds the SER139 involved in the catalytic triad of the
HCV Chymotrypsin-like protease.5 The second class emerged from
the discovery that the N-terminal cleavage product of a substrate
derived from the NS5A/B cleavage site is a competitive inhibitor
of the NS3 protease.6 Truncation of this hexapeptide leads to a
tetrapeptide and to a rigid macrocyclic scaffold yielding the non-
covalent macrocyclic BILN-2061 (ciluprevir), which was the first
molecule for which clinical proof of concept was demonstrated
(Chart 1).7 In only two days of monotherapy, it reduces the viral
RNA by 2–3log10 IU. However, BILN-2061 had to be discontinued
in phase II clinical trials due to cardiac toxicities in animal studies.
Although the mechanism of this cardiac toxicity has not been
reported, it is believed to be compound specific and several groups
have now entered clinical trials with macrocycle inspired by this
pioneer drug leading to the recent approval of TMC435
⇑
Corresponding author. Tel.: +33 (0)499522252; fax: +33 (0)499522250.
E-mail address: francois-rene.alexandre@merck.com (F.-R. Alexandre).
Former Idenix employees.
http://dx.doi.org/10.1016/j.bmcl.2015.07.020
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Alexandre, F.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.020

2
F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Chiral
Chiral
HN
N
NH
O
N
H
N
NH
O
O
O
H
N
O
O
NH
O
O
NH2
N
HN
O
O
O
N
Telaprevir (VX-950)
Boceprevir (SCH 503034)
Chiral
HN
S
P2'
N
O
N
O
OH
P1
P2
N
HN
O
O
O
O
O
N
H
P4
P3
Ciluprevir (BILN 2061)
Chart 1. NS3/4A Protein Inhibitor reported in the literature.
(simeprevir)8 and MK-7009 (vaniprevir)9 as part of DAA
combination therapy as recommended by the Food and Drug
Administration (FDA).10
We present in this Letter our work to develop a novel series of
non covalent macrocyclic inhibitors based on a homoserine P2
scaffold linking together the quinoline P20 motif and the macro-
cyclic moiety.
In a precedent Letter, we reported a bioisosteric replacement of
the proline present in BILN-2061 by a 2,4-disubstituted azetidine
motif.11 Continuing this work of designing new P2 motif, we
turned our attention to less rigid inhibitors where the central pro-
line or azetidine P2 rings would be open to afford ‘seco-analogs’.
The advantages would be the suppression of a chiral center
compared to the proline motif while maintaining the key
interactions of the macrocycle with the catalytic site.
NS3/4A protease using the HiLyte Fluor™ 610 peptide from
AnaSpec, Inc. The IC50 values were determined from the 50% inhi-
bition versus concentration data using a sigmoidal non-linear
regression analysis based on four parameters with SymyxÒAssay
Explorer software. The cell based activities were measured using
Huh-7 cells containing HCV Con1 subgenomic replicon (GS4.1
cells) monitored for expression of the NS5A protein by enzyme-
linked immunosorbent assay (ELISA) to measure the cell based
activities (EC50). The EC50 values were determined from the 50%
inhibition versus concentration data as described above for the
protease assay. The CC50 were calculated as the concentration that
caused a 50% of cells death versus the concentration data com-
pared to the blank control. We were pleased to observe that both
molecules retained good enzymatic activities, as compared to
BILN-2061, with the 14 membered ring 8a being the most active
In order to evaluate the interest of the homoserine-P2 series, we
originally synthesized target products 8a–b having ring sizes of 14
and 15 atoms and bearing a simple 7-methoxy-2-phenylquinoline.
The synthesis is outlined in Scheme 1 and started from protected
homoserine 1.12 The quinoline moiety was introduced via a
Mistunobu reaction in good yield to afford compound 2. After
removing the carbamate protection, the unsymmetrical ureas were
obtained by reaction of the amine function of 3 with 1,10-car-
bonyldiimidazole (CDI) followed by the reaction with N-methyl
hex-5-en-1-amine and N-methyl hept-6-en-1-amine to give 4a
and 4b, respectively.13 Subsequent saponification of the ester
groups to acids and TBTU mediated coupling with (1R,2S)-ethyl-
1-amino-2-vinylcyclopropane-carboxylate (vinyl ACCA)14 afforded
dienes 6a–b. Ring closing metathesis (RCM) using Hoveyda–
Grubb’s 1st generation catalyst gave 14 and 15 membered rings
macrocycles 7a and 7b. In the later case, 7b was obtained contam-
inated with 15% of epimer at the vinylic position of vinyl ACCA, as
already observed by Zeng et al.15 Saponification of ester group,
activation with CDI and reaction with cyclopropylsulfonamine
gave the expected compounds 8a–b.
with an IC50 of 0.0198 lM (Table 1). We decided to further evaluate
this homoserine series with a 14 membered ring. Since the P20
heterocycles are known to be a key modulator of activities and
overall PK profiles of macrocyclic protease inhibitors, we initiated
a SAR analysis of the P20 quinoline at position 2 and 8 (Chart 2).16
Except isopropylaminothiazole quinoline 9a which was com-
mercially available, 2-thiazoles substituted quinolines 9b–f were
synthesized using described methods.16,17
Introduction of pyrazoles at position 2 of the quinolines was
realized according to Scheme 2. A concise synthesis of 2-pyra-
zolyl-4-hydroxy quinolines 9g–l was realized in three or four steps
from anilines 10a–b. First step consisted in a cyclization reaction of
malonic acid in the presence of phosphorus oxychloride to yield
the 2,4-dichloroquinolines 11a–b. In a first route, regioselective
Suzuki–Miyaura reaction at position 2 afforded the quinolines
12g–i in 56–76% yields. Eventually, hydrolysis of the chlorine at
position 4 by KOH in DMSO gave the 4-hydroxyquinolines 9g–i
in 38–96% yields. In a second approach, the 4 position was
protected in 50% yield with the reaction of para-methoxybenzyl
(PMB) alcohol giving a mixture of major 4-OPMB and minor
2-OPMB adducts, which were easily separable by chromatography
on silica gel. N-Branched pyrazoles were then introduced via a SNAr
The target molecules were evaluated in a biochemical FRET
assay for their inhibitory capacity on the full length 1b Con1 HCV
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F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
MeO
3
MeO
OH
N
N
i
ii
O
BocHN
O
OBn
O
OBn
H2N
HCl
OBn
BocHN
O
1
2
3
O
MeO
MeO
MeO
N
N
N
O
O
O
O
O
O
iv
v
OBn
N
N
iii
O
N
H
N
H
OH
N
N
H
O
NH
O
CO2Et
n
n
n
4a: n=1
4b: n=2
5a: n=1
5b: n=2
6a: n=1
6b: n=2
MeO
MeO
N
N
N
O
O
O
O
S
H
N
O
CO2Et
N
H
vi
vii, viii
HN
O
HN
HN
O
O
N
O
n
n
7a: n=1
7b: n=2
8a: n=1
8b: n=2
Scheme 1. Reagents and conditions: (i) 7-methoxy-2-phenylquinolin-4-ol, PPh3, DIAD, THF, 0 °C–RT; (ii) 4 M HCl in dioxane, RT, 67% from 1; (iii) (a) CDI, DIPEA, DCM,
(b) N-methylhex-5-en-1-amine tosylate salt (for 4a), reflux, 72% or N-methyl-hept-6-en-1-amine tosylate salt (for 4b), reflux, 69%; (iv) LiOHꢀH2O, H2O/THF; (v) (1R,2S)-ethyl
1-amino-2-vinyl-cyclopropanecarboxylate, TBTU, DIPEA, DCM, 69% (6a, 2 steps) and 65% (6b, 2steps); (vi) Hoveyda–Grubbs 1st generation catalyst, DCM, reflux, 23% (7a) and
33% (7b); (vii) LiOHꢀH2O, H2O/THF,; (viii) (a) CDI, DCM, 80 °C, (b) cyclopropylsulfonamide, DBU, 15% (8a, 3 steps) and 11% (8b, 3 steps).
Table 1
reaction in moderate yields. Deprotection of PMB either using
cerium trichloride/sodium iodide or under catalytic transfer
ydrogenation conditions afforded 9j–l in good yields.
HCV protease enzymatic inhibition and cell-based inhibition
a
b
c
Entry
Compound
IC50
(lM)
EC50
(lM)
CC50 (lM)
In order to easily introduce chemical diversity, an alternative
synthetic pathway was established where the quinoline part is
introduced at a late stage of the synthesis after the key RCM step
(Scheme 3). First step is the formation of the unsymmetrical urea
1
2
3
BILN-2061
8a
8b
0.0009
0.0198
0.0410
0.0028
8.2
14.6
21
>30
53
a
Biochemical FRET assay for inhibition capacity on the full length (1b) Con1 HCV
NS3/4A protease.
motif. Commercially available (S)-a-amino-c-butyrolactone
b
Inhibition of HCV replication Huh-7 cells containing HCV Con1 subgenomic
hydrobromide 14 was treated with diisopropylethylamine
(DIPEA) and CDI to afford imidazole activated urea which under-
went nucleophilic displacement with N-methylhexenyl amine to
afford urea 15. It should be noted that the activation of intermedi-
ate imidazole as imidazolium as mentioned in the literature was
not necessary since simple heating in dichloromethane afforded
expected product.13 The butyrolactone was then opened in the
presence of sodium hydroxide, water and ethanol, then the hydro-
xyl moiety was protected as a tert-butyldimethylsilyl group to give
acid 17. TBTU mediated coupling with vinyl ACCA afforded diene
18. The RCM step was performed using Hoveyda–Grubb’s 1st gen-
eration catalyst in 1,2-dichloroethane at 70 °C, with a substrate
concentration of 5 mM, giving the macrocycle in 50% yield on a
multi grams scale. In this case no detectable formation of trans-iso-
mer was observed. Quinolines 9a–l were then introduced via a
replicon (GS4.1 cells) with a luciferase read-out. EC50 values were determined from
the 50% inhibition versus concentration data.
CC50’s were calculated as the concentration that caused a 50% of cells death
versus concentration data.
c
H
N
R1
N
N
9b R1=H
9c R1=F
S
MeO
N
MeO
N
S
9d R1=Cl
9e R1=Br
9f R1=Me
OH
9a
OH
Chart 2. Quinolines 9a–f.
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F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
MeO
MeO
N
R2
MeO
N
R2
iii
ii
R1
R1
OH
Cl
MeO
N
Cl
MeO
NH2
12g-i
9g-i
i
R1
R1
R2
Cl
MeO
N
Cl
N
10a R1= Me
10b R1= Cl
v
11a R1= Me
iv
then vi
11b R1= Cl
OPMB
OH
9j-l
13j-l
F
F
F
N
N
N
N
N
R2:
R2:
N
12g, 9g
12h, 9h
12i, 9i
F
F
F
N
N
N
N
R1= Me
R1= Cl
13j, 9j
13k, 9k
13l, 9l
Scheme 2. Reagents and conditions: (i) malonic acid, POCl3, reflux, 43–74%; (ii) R2-B(OH)2, Na2CO3 aq, Pd(OAc)2, PPh3, THF, 60 °C, 50–75%; (iii) KOH, DMSO, 100 °C, 36–66%;
(iv) p-methoxybenzylalcohol, NaH, 15-crown-5, DMF, 38–50%; (v) pyrazole, NaH, DMF, 0–90 °C, 19–93%; (vi) CeCl3ꢀ7H2O, NaI, CH3CN, 85 °C or HCO2H, Pd/C 10%, EtOH, reflux,
93–96%.
OH
OH
O
OTBDMS
O
O
O
O
OH
O
O
i
ii
N
H
iii
N
O
N
H
N
NH
O
NH2
BrH
N
15
16
14
17
OH
OTBDMS
OTBDMS
CO2Et
H
CO2Et
CO2Et
H
N
H
N
N
iv
v
vi
HN
HN
HN
O
O
O
N
20
O
N
N
O
O
19
18
R1
R1
R1
MeO
MeO
MeO
N
N
N
R2
R2
R2
O
O
O
O
R3
O
S
O
N
H
H
H
N
CO2H
H
CO2Et
vii
viii
ix
N
N
HN
HN
HN
O
O
O
O
N
O
N
N
O
21a-l
23a-n
22a-l
Scheme 3. Reagents and conditions: (i) (a) DIPEA, CDI, DCM, RT; (b) N-methylhex-5-en-1-amine tosylate salt, reflux, 95% from 14; (ii) NaOH, H2O/EtOH, 95%; (iii) TBDMSCl,
Et3N, DCM, 87%; (iv) (1R,2S)-ethyl 1-amino-2-vinylcyclopropane-carboxylate, TBTU, DIPEA, DCM, 76%; (v) Hoveyda–Grubbs 1st generation catalyst, DCE, 70 °C, 50%;
(vi) TBAF, THF, RT, 44%; (vii) quinolines 9a–l, PPh3, DIAD, THF, 0 °C–RT, 26–84%; (viii) LiOHꢀH2O, H2O/THF, 40–92%; (ix) (a) CDI, DCM, 80 °C, (b) cyclopropylsulfonamide or
methylcyclopropylsulfonamide, DBU, 11–33%.
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F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 2
HCV protease enzymatic inhibition and Cells based inhibition of quinoline analogs
R1
R2
R3
IC50
(lM)
EC50
(l
M)
CC50
37.4
(lM)
RLM Clid
(l
L/min/mg)
a
b
c
Entry
Compound
NH
1
23a
H
Me
0.0012
1.0400
—
N
N
S
2
3
4
5
6
7
8
23b
23c
23d
23e
23f
H
Me
H
0.0009
0.0096
0.0080
0.0017
0.0017
0.0006
0.0023
0.0440
1.4900
0.5550
0.0765
0.0287
0.0205
0.0850
6.5
154.9
—
S
N
F
>75
57.1
40.4
37.4
28.3
31.9
S
N
F
Me
H
—
S
N
Cl
Cl
Br
Me
36.6
20.7
16.5
9.3
S
N
Me
Me
Me
S
N
23g
23h
S
N
S
N
N
9
23i
23j
23k
Me
Cl
Me
Me
Me
0.0020
0.0011
0.0030
0.0500
0.0434
0.4850
25.6
31.0
36.7
21
—
N
N
10
10
N
Me
—
N
11
12
23l
Me
Me
Me
Me
0.0014
0.0010
0.1450
0.4400
62.3
34.5
—
—
N
N
F
F
F
23m
N
N
F
F
F
13
14
23n
23o
Me
Cl
Me
Me
0.0018
0.0011
0.0350
0.0153
45.8
48.8
14.4
—
N
N
F
F
F
N
N
a
Biochemical FRET assay for inhibition capacity on the full length (1b) Con1 HCV NS3/4A protease.
Inhibition of HCV replication Huh-7 cells containing HCV Con1 subgenomic replicon (GS4.1 cells) with a luciferase read-out. EC50 values were determined from the 50%
b
inhibition versus concentration data.
c
CC50’s were calculated as the concentration that caused a 50% of cells death versus concentration data.
RLM Cli = Rat Liver Microsomes intrinsic clearance after 2 h of incubation.
d
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F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Mitsunobu reaction in good yields. The resulting esters were
converted to acids 22 then activated with CDI to form an oxazolidi-
none intermediate which underwent a nucleophilic attack of
cyclopropyl sulfonamide to afford macrocycles 23a–n as described
in Scheme 3.
The new macrocyclic compounds 23a–n were tested in enzy-
matic and cell-based assays and results are reported in Table 2.
All compounds demonstrated excellent inhibition of the NS3/4A
explain the difference in enzymatic activity compared to the bro-
mine as studied by Lu et al.18 As a result the above calculated inter-
action distances (between halogen or sulfur atom of the thiazole
ring and carbonyl of V78) for 23d (R = F, right orientation) were
longer than for 23g and 23f, giving a weaker inhibitor, this was
confirmed in the enzymatic assay (Table 2) with 23d being, respec-
tively, 13.3 and 4.7 fold less active than 23g and 23f. For 23b
(R1 = H, left orientation) and 23h (R1 = Me, left orientation), the
same interactions were observed as for the halogeno-series (except
protein with IC50 ranging from 0.010 to 0.006 lM. In contrast,
much larger shifts between tested compounds were observed in
the cellular assay. The 2-isopropylamino substitution (23a, entry
1) led to an important loss in replicon activity as compared to
BILN-2061. We attributed it to a decrease in lipophilicity due to
the combination of basic isopropylamine moiety and the absence
of the large P4 lipophilic moiety possessed by BILN2061 (calculated
AlogP = 3.7 for 23a as compared to 5.8 for BILN-2061).
Replacement of this substitution by a 4-isopropylthiazole (entry
2) led to a 23 fold more active compound 23b. This improvement
prompted us to further evaluate (entries 2–8) the 8-position of
the quinoline, that is known to modulate the cellular potency
and metabolic stability.11,17 In both assays, the best substituents
were Br, Cl, Me, respectively (entries 7, 6 and 8). Fluorine substitu-
tion (23d, entry 4) led to a loss in activity (10 fold difference
compared to 23g) and hydrogen to an increased cellular toxicity.
Docking studies of the inhibitors (23b, 23d, 23f, 23h and 23g) in
the NS3/4A protein confirmed the data observed in vitro and
showed that two orientations of the P20 quinolines were possible
around the bound that links quinoline to P2 area (C4-O). Either
thiazole ring was on left hand side (left orientation) as shown on
Figure 1, or was on the right hand side (right orientation), both
poses of the quinolines maintaining the key interactions with the
protein. The most potent molecule 23g (R1 = Br, left orientation)
showed two interactions with the carbonyl of V78, one halogen
bond interaction (d = 3.27 Å) and one with the sulfur atom of the
thiazole ring (d = 5.01 Å) (Fig. 1). Other interactions are: one
the halogen bond interaction), nevertheless an additional r–p
interaction was observed for 23b between thiazole ring and back-
bone hydrogen of D81, which could explain its good enzymatic
potency.
We then compared the homoserine P2 scaffold (23d) with a
proline based scaffold bearing the same quinoline (PDB: 4U01).
Very similar binding mode was observed at the macrocycle, P3
and P1 regions as well as at the P2 area as shown on Figure 2.
The
r–p interaction of C2 methylene on homoserine P2 with H57
is maintained compared to the proline scaffold (d = 2.88 and
2.77 Å, respectively).
Compounds were also assessed for their rat liver microsome
metabolic stability and results are reported in Table 2. Moderate
stability was shown for all the series, higher intrinsic clearance
was observed for R1 = H (23b) while R3 = Me (23f) showed the best
stability profile compared to R3 = H (23e). Cyclopropyl and methyl-
cyclopropyl at R3 were also compared in a limited set of com-
pounds (23c vs 23d and 23e vs 23f): methylcyclopropyl afforded
a gain of around 2 fold in replicon activity and had no effect in
the enzymatic assay. The methylcyclopropyl R3 afforded a similar
metabolic stability in rat liver microsomes as shown in Table 2
(23e vs 23f), in rat hepatocytes assays (S9 and cryopreserved)
and in liver microsomes of dog and human, with the exception of
cynomolgus monkey liver microsomes that is almost 4 fold higher
clearance compared to the cyclopropyl sulfonamide, as shown in
Table 3.
cation–
p
interaction between thiazole and R155 and two cation–
At this stage of the project we decided to evaluate the in vivo
properties in male Sprague–Drawley rats of compound 23f as a
representative of the series. Pharmacokinetic parameters obtained
p
interactions between the aromatic part of the quinoline and
H57. Same interactions were observed for 23f (R1 = Cl, left orienta-
tion) but with slightly longer distances calculated that could
Figure 2. Overlapping of inhibitor 23d docked in 1bCon1 wild type NS3/4a with
proline urea inhibitor in yellow (PDB 4U01), showing the interactions of P2
scaffolds with H57.
Figure 1. Inhibitor 23g docked in 1bCon1 wild type NS3/4a showing the
interactions of P20 quinoline with V78, R155 and H57.
Please cite this article in press as: Alexandre, F.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.020

F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
7
Table 3
Mean clearance (n = 2) observed in different species liver microsomes and cryopreserved hepatocytes
Compd Rat liver S9a
min/mg)
(
lL/
Rat cryopreserved hepatocytesb
L/min/mg)
Dog liver microsomesb
L/min/mg)
Cynomologus monkey liver
Human liver microsomesb
L/min/mg)
(l
(l
microsomesb
l
L/min/mg)
(l
23f
23e
7.6
8.9
12.6
19.1
19.5
25
479.8
124.4
214.6
262.9
a
Rat liver fraction S9 mean clearance after 2 h of incubation.
Mean clearance after 2 h of incubation.
b
Table 4
Male Sprague–Dawley rats pharmacokinetics (formulated in 2.5% Vitamin E-TPGS/10.5% PEG-400/80% phosphate buffer pH = 9.2)
Compound
Route
Dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
AUCt (ngꢀh/mL)
AUCinf (ngꢀh/mL)
t1/2 (h)
kel (1/h)
Cl (L/h/kg)
Vd (L/kg)
F (%)
23f
po
iv
10
2
798
2673
0.75
0.11
2059
1525
2353
1506
2.53
1.25
0.28
0.57
4.345
1.325
16.04
2.42
27
—
O
ii
R1
S
H2N
O
26a-c
O
O
i
R1
BOC
S
BOC
S
N
H
N
H
O
O
O
O
v, ii
S
iii, iv
BOC
S
O
H2N
N
H
24
25a-d
O
O
R1=CH2OBn
27
28
Scheme 4. Reagents and conditions: (i) n-BuLi, ꢁ78 °C, THF, then NFSI for 25a (R1 = F) EtI for 25b (R1 = Et), TsCN for 25c (R1 = CN) and BnOCH2Br for 25d (R1 = CH2OBn), 30–
50%; (ii) TFA, DCM, 25–90%; (iii) H2, Pd/C 10% cartridge, H-Cube flow reactor, 50 °C, 25d, 70%; (iv) PCC, DCM, 66%; (v) Bestmann–Ohira reagent, K2CO3, MeOH, 0 °C, 85%.
R2
HN
R2
N
R2
N
O
S
O
O
S
O
O
S
O
N+
BOC
N
ii
i
BOC
+
H2N
N
N
30-a-b
29
32-a-b
31a-b
Scheme 5. Reagents and conditions: (i) 30a (R2 = CN) or 30b (R2 = ethynyl), DCM, RT, 77–94%; (ii) SCX-2 resin, CH3CN, 99%.
activity. N-1-Branched pyrazoles 23i gave EC50 of 0.05 lM, replace-
Cl
Cl
MeO
MeO
ment of isopropyl by trifluoromethyl at position 3 of the pyrazole
(23i vs 23n) was likely to improve permeability and allowed a
slight gain in replicon activity (23n). At last, combination of the
R1 = Cl with CF3-pyrazole afforded 23o, the most active compound
of the series in the replicon assay.
While exploring the SAR on the quinoline part, a study around
the sulfonamide moiety, which occupies a lipophilic pocket in
the P10 region, was undertaken. Modified sulfonamides were syn-
thesized according to Scheme 4. N-Boc protected 24 was deproto-
nated using n-BuLi at low temperature in THF and submitted to
different electrophiles to furnish compounds 25a–d. tert-
Butyloxycarbonyl (Boc) deprotection gave sulfonamides 26a–c.
Ethynyl derivative 28 was synthesized from 25d in a benzy-
loxymethoxy (BOM) deprotection, oxidation and Seyfert–Gilbert
reaction sequence.
N
N
N
N
N
S
S
O
R
O
O
O
O
O
S
H
N
N
H
H
N
OH
i
HN
O
HN
O
O
O
N
23o-w
22d
Scheme 6. Reagents and conditions: (i) (a) CDI, DCM, 80 °C, (b) cyclopropylsulfon-
amide, DBU, 11–33%.
Sulfamides were either commercially available or synthesized
according to Scheme 5.19 Sulfonamides and sulfamides were cou-
pled to the macrocycle 22d via carboxylic activation with CDI
and nucleophilic displacement (Scheme 6).
Antiviral activities of these compounds were then assessed and
results are reported in Table 5. Molecules with small substituents
23p–t and 23x retained enzymatic activities where larger groups
of 23u–w led to a strong decrease. A modeling study showed that
only small substituents can accommodate this lipophilic pocket
after intravenous (2 mg/kg) and oral (10 mg/kg) administration are
summarized in Table 4. After oral administration, 23f exhibited an
oral bioavailability of 27% and a reasonable PK profile.
Based on the metabolic stability, the combination of R1 being a
methyl and R3 being a methylcyclopropyl moiety were chosen to
continue the SAR at R2.
The replacement of a thiazole by various pyrazoles was then
investigated. 3 and 4-branched pyrazoles retained good enzymatic
activities (entries 10–12) but were detrimental for the replicon
Please cite this article in press as: Alexandre, F.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.020

8
F.-R. Alexandre et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 5
References and notes
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a
b
c
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IC50
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(l
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(lM)
1
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23q
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49.3
>75
CN
4
5
6
7
23s
23t
0.0007
0.0021
0.1650
0.1498
0.0289
0.0845
>0.75
26.3
47.8
37.6
26.4
N
N
23u
23v
F
F
N
O
>0.75
NC
8
9
23w
23x
0.2640
0.0338
>0.75
>0.75
28.3
21.2
N
N
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b
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c
CC50’s were calculated as the concentration that caused a 50% of cells death
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defined by G41, S42, F43, V55, H57, G58 and S139. In the replicon
assay results, only 23s showed similar activity to reference com-
pound 23f. However none of these sulfonamide modifications
brought advantages as compared to cyclopropylsulfonamide or
methylcyclopropylsulfonamide.
In summary, we have synthesized a series of macrocyclic HCV
protease inhibitors that exhibited potent inhibitory activity against
HCV both in enzymatic and cellular assays, demonstrating that the
central proline P2 ring scaffold can be replaced by an homoserine
backbone. Compound 23f showed a good bioavailability and rea-
sonable PK profile in rat. Although the series was not pursued,
the SAR around P20 quinolines was applied and further investigated
in other series and led to the discovery of the clinical candidate
IDX320.20
19. Winum, J.-Y.; Toupet, L.; Barragan, V.; Dewynter, G.; Montero, J.-L. Org. Lett.
2001, 3, 2243.
20. Parsy, C.; Alexandre, F.-R.; Bidau, V.; Bonnaterre, F.; Brandt, G.; Caillet, C.;
Cappelle, S.; Chaves, D.; Convard, T.; Derock, M.; Gloux, D.; Griffon, Y.; Lallos, L.;
Leroy, F.; Liuzzi, M.; Loi, A.-G.; Moulat, L.; Musiu, C.; Rahali, H.; Roques, V.;
Savin, S.; Seifer, M.; Standring, D.; Surleraux, D. Bioorg. Med. Chem. Lett. 2015,
submitted for publication.
Acknowledgment
We thank Sylvie Cappelle for her help and assistance in the
preparation of the manuscript.
Please cite this article in press as: Alexandre, F.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.020