Discovery of novel P3-oxo inhibitor of hepatitis C virus NS3/4A serine protease

Article abstract of DOI:10.1016/j.bmcl.2012.02.039

A novel series of P3 oxo-modified macrocyclic hepatitis C virus NS3/4A serine protease inhibitor was designed, synthesized and biologically evaluated. The hydroxy-substituted inhibitor 10 demonstrated high potency in genotype 1a and 1b replicon and in the panel of HCV protease mutants. Interestingly, the t-butyl carbonate analog 9c, while not the most potent one in this series, exhibited a virtually flat potency profile in the panel of HCV protease mutants, thus providing opportunity for further optimization.

Full text of DOI:10.1016/j.bmcl.2012.02.039

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2993–2996
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel P3-oxo inhibitor of hepatitis C virus NS3/4A serine protease
Maosheng Duan a, , Wieslaw Kazmierski a, , Renae Crosby a, Margaret Gartland a, Jinjing Ji a, Matt Tallant a,
Amy Wang a, Robert Hamatake a, Lois Wright a, Min Wu a, Yong-Kang Zhang b, Charles Z. Ding b,
Xianfeng Li b, Yang Liu b, Suoming Zhang b, Yasheen Zhou b, Jacob J. Plattner b, Stephen J. Baker b
⇑
⇑
a Infectious Disease CEDD, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA
b Anacor Pharmaceuticals Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
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 oxo-modified macrocyclic hepatitis C virus NS3/4A serine protease inhibitor was
designed, synthesized and biologically evaluated. The hydroxy-substituted inhibitor 10 demonstrated
high potency in genotype 1a and 1b replicon and in the panel of HCV protease mutants. Interestingly,
the t-butyl carbonate analog 9c, while not the most potent one in this series, exhibited a virtually flat
potency profile in the panel of HCV protease mutants, thus providing opportunity for further
optimization.
Received 7 December 2011
Revised 11 February 2012
Accepted 13 February 2012
Available online 22 February 2012
Keywords:
HCV
Ó 2012 Elsevier Ltd. All rights reserved.
HCV protease
HCV protease inhibitor
Inhibitor
Hepatitis C virus (HCV) is the known causative pathogen for viral
hepatitis. It has been estimated that 3% of the human population
worldwide is infected with HCV.1 If left untreated, HCV infection
can lead to liver cirrhosis, hepatocellular carcinoma and subsequent
liver failure.2 Until the recent approval of boceprevir and telaprevir,3
the standard of care (SOC) to treat HCV has involved a combination
in the P4-P3 linker region, we substituted the P3-nitrogen with
oxygen, resulting in until now unreported, novel P3-oxo series, Fig-
ure 1. Herein, we wish to report the synthesis, structure–activity
relationship (SAR), mutant resistance profile and pharmacokinetic
properties of key compounds discovered in our work.
The synthesis began with preparation of ethyl 2-hydroxy-8-
nonenoate 2 (Scheme 1). Commercially available 7-bromo-1-hep-
tene was treated with Mg in THF, and freshly generated Grignard
reagent reacted with ethyl oxoacetate to give 2-hydroxyl ester 2.
Saponification of 2 followed by a HATU coupling of the resulting
hydroxyl acid 3 with 47 afforded 5. Using tert-butylisocyanate,
the free hydroxy group in 5 was derivatized to carbamate in 6a,
setting the stage to ring-closing metathesis. Macrocycle 7a under-
went hydrolysis with LiOH, and resulting acid 8a was treated with
CDI at elevated temperature, followed by addition of cyclopro-
panesulfonamide and DBU to furnish acylsulfonamide 9a, Scheme
1. Compounds 9b–d were prepared using the same synthetic se-
quence except for derivatization of the free OH in 5 with different
functional groups, which yielded respective final compounds,
Scheme 2. The free hydroxyl analog 10 was obtained by heating
9c with NaOMe in MeOH. Finally, carbamate 11 was obtained by
reacting 10 with CDI, Scheme 2.
Antiviral activities of the compounds were evaluated by FRET
enzyme assay with NS3/4A 1a protease domain8 and 1a and 1b
HCV Huh7 replicon assays.9 Results are summarized in Table 1.10
Although the hydroxy 10 and imidazolyl carbamate 11 compounds
were somewhat less potent than ITMN-191 in the protease enzyme
assay, they were essentially equipotent to ITMN-191 in both the
gt1a and gt1b replicon assays, potentially reflecting improved
of pegylated interferon-alpha (IFN-a) and the nucleoside analog
ribavirin. Effective clearance of HCV could be achieved in only less
than 50% of the most prevalent genotype (gt) 1 infections, and the
regimen often caused significant side-effects, such as flu-like symp-
toms and fatigue. Due to a heavy dosing regimen associated with the
use of boceprevir and telaprevir, there is a continued need to devel-
op more potent HCV Protease Inhibitors (PIs). Combined with im-
proved pharmacokinetic properties, such compounds would
enable lower pill burden compared to boceprevir and telaprevir, less
frequent drug dosing, and as such would likely enhance patient
compliance and thus improve treatment effectiveness. In addition,
emergence of resistance to PIs also necessitates that the next gener-
ation PIs possess greater potency against clinically-relevant HCV
protease mutants. To this end, new generation of protease inhibi-
tors, such as ITMN-1914 and TMC-4353505 offer promise. Our
own laboratories have also recently disclosed novel P4-urea based
HCV protease inhibitor 1, which was highly potent against the wt
and mutants raised against HCV PIs.6a,b To further explore the SAR
⇑
Corresponding authors. Address: HD Biosciences (China) Co., Ltd. Tel.: +86 21
51163719 (M.D.); tel.: +1 919 483 9462 (W.K).
E-mail addresses: duanmaosheng@hdbiosciences.com (M. Duan), wieslaw.
m.kazmierski@gsk.com (W. Kazmierski).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.039

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M. Duan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2993–2996
F
O
ITMN-191
N
O
O
O
N
O
O
X
NH
HN
O
O
S
O
P2*
O
HN
O
O
O
N
N
O
NH
HN
O
HN
S O
X
TMC 435350
O
P3
1
O
O
O
O
NH
S
X
N
N
O
O
H
O
N
O
NH
O
O
S O
R
O
N
O
HN
X=
N
S
P3-oxo series
Figure 1. ITMN-191, TMC 435350, urea-based PI 1 and the new P3-oxo-series.
OH
OH
b
a
OH
Br
O
O
O
3
2
O
O
N
N
N
N
N
O
S
N
O
S
S
d
O
O
c
O
H
N
O
O
O
O
N
O
H
H
N
O
N
O
O
O
N
N
H
31
O
O
O
2HCl
NH
HO
5
4
6a
O
O
O
N
N
N
N
N
N
N
N
g
S
S
S
e
f
O
O
O
O
O
O
O
S
H
H
H
N
N
N
OH
O
N
O
N
H
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
8a
9a
7a
Scheme 1. Reagents and conditions: (a) Mg, I2, THF, rt, then ethyl oxoacetate, À78 °C-rt, 4 h, 13%; (b) LiOH, THF/H2O, rt, overnight, then 1 N HCl, quant.; (c) 3, HATU, iPr2NEt,
DMF, rt, 45%; (d) tert-butylisocyanate, CH2Cl2, 40 °C, overnight, 36%; (e) Zhan-1b, ClCH2CH2Cl, 70 °C, overnight, 50%; (f) LiOH, THF/tBuOH/H2O, rt, overnight, then, 1 N HCl,
80%; (g) CDI, THF, 40 °C, 1.5 h, then cyclopropanesulfonamide, DBU, rt, overnight, 15%.
membrane transport of these compounds. Carbamate 9a exhibited
somewhat less attractive potency in the enzyme and gt 1a assays,
but was equipotent to ITMN-191 in the gt1b replicon assay.
Interestingly, compounds with no H-bond donor in the P4 region,
9b–9c, were substantially less potent in both assays, Table1. It
may be worth noting that the imidazole in 11 can be protonated

M. Duan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2993–2996
2995
O
N
N
N
R
O
N
S
O
O
O
O
O
S
N
H
N
N
N
O
Et
O
O
a
b
R
O
9c
10
11
9b
9d
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, 50 °C, 2 h, 40%; (b) CDI, THF, 60 °C, 1.5 h, 11%.
stable gt1a and gt1b replicon assays, consistent with its moderate
potency in the wt transient replicon.
Table 1
Anti-HCV activities of analogs and ITMN-191
Compounds in this series had low permeability in the parallel
artificial membrane permeability assay (PAMPA), as exemplified
by 11 (Papp = 12 nM/s at pH 7.05), and perhaps for that reason
exhibited only low to moderate bioavailability in the rat PK model
(F = 12%). Compound 11 was also characterized by high clearance
in rat PK model (Cl = 53.7 mL/min/kg).
Compound
R
NS3/4A 1a IC50 (nM)
Replicon EC50 (nM)
1a 1b
O
9a
9b
9c
31.0
107.2
195.0
5.0
251.2
50.1
0.8
N
H
O
In summary, we described novel P3-oxo inhibitors of HCV NS3/
4A serine protease. Novel hydroxy 10 and carbonyl-imidazole 11
compounds were found to be essentially equipotent to danoprevir
(ITMN-191) in the wt gt1a and gt 1b replicon assays (Table 1) and
had a noticeably improved viral mutant profiles EC50(mutant)/
EC50(wt), Table 2. The tert-butyl-carbonate 9c, which was less po-
tent in both stable replicon assays (Table 1), has a near-flat potency
profile in the transient mutant assay (Table 2), and as such is an
attractive lead molecule for further potency optimization towards
HCV protease inhibitors with high potency across all HCV protease
mutants. Further PK improvement will also be necessary for this
class to be further developable. This can potentially be accom-
plished by reducing the polar surface area PSA in this class (PSA
of 11 is 201), which should improve compound permeability and
bioavailability. The biological properties of compounds in this
class, especially of 9c, 10 and 11 warrant further optimization.
These efforts will be described in due course.
79.4
39.8
O
O
O
O
9d
10
56.2
6.8
10.0
1.0
3.2
0.8
N
O
H
N
O
O
11
3.2
0.4
0.8
1.0
0.5
1.1
N
O
ITMN-191
ITMN-191 lit.4a NS3/4A 1a IC50 = 0.2 nM, replicon 1b EC50 = 1.8 nM.
Table 2
Genotype 1b transient replicon activity of 9a–d, 10, 11 and ITMN-191a
Compound
1b transient replicon EC50 (nM)
A156S A156T A156V D168A D168V R155K
References and notes
wt
1.2
63.1
9a
9b
9c
9d
10
11
2.5
29.4
63.1 118.6
84.6
397.2
70.8
90.2
2.6
322.5
434.4
73.7
657.7
3.2
462.2 1071.5
323.6
1. White, P. W.; Llinas-Brunet, M.; Bos, M. Prog. Med. Chem. 2006, 44, 65.
2. (a) Cohen, J. Science 1999, 285, 26; (b) Alter, M. J.; Kruszon-Mran, D.; Nainan, O.
V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.; Margolis, H. S. N. Engl. J.
Med. 1999, 341, 566.
514.0
88.1
693.5 3981.0
131.8
170.5
7.1
1.3
0.5
0.1
9.7
0.1
0.1
1.6
2152.8 3981.1 1484.5
3. (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T.
J.; Perelson, A. S. Science 1998, 282, 103; (b) Rosen, H. R.; Gretch, D. R. Mol. Med.
Today 1999, 5, 393; (c) Di Bisceglie, A. M.; Mchutchison, J.; Rice, C. M.
Hepatology 2002, 35, 224.
4. (a) Seiwert, S. D.; Andrews, S. W.; Jiang, Y.; Serebryany, V.; Tan, H.; Kossen, K.;
Rajagopalan, P. T.; Misialek, S.; Stevens, S. K.; Stoycheva, A.; Hong, J.; Lim, S. R.;
Qin, X.; Rieger, R.; Condroski, K. R.; Zhang, H.; Do, M. G.; Lemieux, C.; Hingorani,
G. P.; Hartley, D. P.; Josey, J. A.; Pan, L.; Beigelman, L.; Blatt, L. M. Antimicrob.
Agents Chemother. 2008, 52, 4432; (b) WO 2007015824.
62.8
70.0
48.5
45.7
151.0
24.5
60.3
47.9
80.8
2.8
3.9
ITMN-191
6.8
8.3
a
Literature data for ITMN-191 EC50 (nM): wt = 1.5, A156T = 4, D168V = 12,
R155K = 180 (Seiwert et al., poster #938, EASL 2009, Copenhagen, April 22–26,
2009).
5. Raboisson, P.; de Kock, H.; Rosenquist, A.; Nilsson, M.; Salvador-Oden, L.; Lin, T.
I.; Roue, N.; Ivanov, V.; Wähling, H.; Wickström, K.; Hamelink, E.; Edlund, M;
Vrang, L.; Vendeville, S.; Van de Vreken, W.; McGowan, D.; Tahri, A.; Hu, L.;
Boutton, C.; Lenz, O.; Delouvroy, F.; Pille, G.; Surleraux, D., Wigerinck, P.;
Samuelsson, B.; Simmen, K. Bioorg. Med. Chem. Lett. 2008, 18, 4853.
6. (a) Cooper, J. P.; Duan, M.; Grimes, R. M.; Kazmierski, W. M.; Tallant, M. D. WO
2010088394.; (b) Kazmierski, W. M.; Hamatake, R.; Duan, M.; Wright, L. L.;
Smith, G. K.; Jarvest, R. L.; Ji, J.-J.; Cooper, J. P.; Tallant, M. D.; Crosby, R. M.;
Creech, K.; Wang, A.; Li, X.; Zhang, S.; Liu, Y.; Zhang, Y.-K.; Liu, L.; Ding, C. Z.;
Zhou, Y.; Plattner, J. J.; Baker, S. J.; Bu, W.; Liu, L. J. Med. Chem., accepted for
publication. doi:10.1021/jm201278q.
under physiological condition, resulting in potential H-bond
donating capability of this moiety.
Compounds were also evaluated in the wild type (wt) and mu-
tant genotype 1b transient replicon assays,11 Table 2. Compounds
10 and 11 were essentially equipotent to danoprevir (ITMN-191).
Carbamates 9a, 9b, 9d and to a lesser degree 9c were less potent,
particularly against mutants A156V, D168A and D169V.
The ratio of EC50(mutant)/EC50(wt) may be relevant in com-
pound dose consideration to provide sufficient coverage for all
relevant mutants. Danoprevir (ITMN-191), 9a, 9b, 9d, 10 and 11
exhibited relatively high mutant/wt ratios for some mutants, espe-
cially for D168A, D168V and R155K, Table 2. On the other hand, the
t-butyl-carbonate compound 9c had a near-flat profile in this assay.
Compound 9c had only modest potency in the wt enzyme and
7. See patent for preparation: Cooper, J. P.; Duan, M.; Grimes, R. M.; Kazmierski,
W. M.; Tallant, M. D. WO 2010088394.
8. (a) Compounds were assayed in the fluorescence enzymatic assay using HCV NS3/
4A 1a protease domain: Conditions: 0.75 nM enzyme (1a domain), 2
lM NS4A,
0.5 peptide substrate (Ac-DE-Dap(QXL520)-EE-Abu- -[COO]-AS-C(5-
lM
w
FAMsp)-NH2 is the FRET substrate purchased from Anaspec Inc., San Jose, CA)
in 50 mM HEPES, 20% sucrose, 5 mM DTT, and 0.05% NP-40. Wavelengths of
490 ex and 520 em were used on a Molecular Devices plate reader to measure

2996
M. Duan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2993–2996
initial rates.; (b) Compounds were assayed in the fluorescence enzymatic assay
using HCV NS3/4A 1a protease domain: Conditions: 1 nM enzyme (1a domain),
M NS4A, 5 M peptide substrate (Ac-DE-D(EDANS)-EE-Abu- -[COO]-AS-
immediately using a Gene Pulser system (Bio-Rad), settings: 270 V, 950 lF
with a capacitance extender. Transfer the electroporated cells from the cuvette
into a 50 mL Corning tube containing appropriate amount of medium; then,
2
l
l
w
K(DABCYL)-NH2 is the FRET substrate purchased from Anaspec Inc., San Jose,
CA) in 50 mM HEPES, 20% sucrose, 5 mM DTT, and 0.05% NP-40. Wavelengths of
355 ex and 495 em were used on a Molecular Devices plate reader to measure
initial rates.
transfer 190
column 12 add medium only. Finally, add 10
column 1 to 10. Place the plates in a 37 °C humidified incubator at atmosphere
of 5% CO2 in air. After 72 h, aspirate out the media and add 25 L of Bright-Glo
Luciferase substrate (Promega) to each well. Read luminescence on Perkin
l
L into 96-well assay plates from column 1 to column 11. To
l
L compounds to each well from
l
9. Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilman, L.; Batenschlager, R.
Science 1999, 285, 110.
10. The P3-oxo stereochemistry results in 1:1 ratio of diastereomers.
11. Add wild type 1b RNA or mutation RNA into 400uL of ET cured cells, then
Elmer Envision Multilabel Reader. Generate IC50 values, by plotting
inhibition against the compound concentrations.
% of
transfer into
a cuvette with a gap width of 0.4 cm and electroporate