Discovery of MK-1220: A macrocyclic inhibitor of hepatitis C virus NS3/4A Protease with Improved Preclinical Plasma Exposure

Article abstract of DOI:10.1021/ml1002426

The discovery of MK-1220 is reported along with the development of a series of HCV NS3/4A protease inhibitors containing a P2 to P4 macrocyclic constraint with improved preclinical pharmacokinetics. Optimization of the P2 heterocycle substitution pattern as well as the P3 amino acid led to compounds with greatly improved plasma exposure following oral dosing in both rats and dogs while maintaining excellent enzyme potency and cellular activity. These studies led to the identification of MK-1220.

Full text of DOI:10.1021/ml1002426

LETTER
pubs.acs.org/acsmedchemlett
Discovery of MK-1220: A Macrocyclic Inhibitor of Hepatitis C Virus
NS3/4A Protease with Improved Preclinical Plasma Exposure
Michael T. Rudd,*,† John A. McCauley,† John W. Butcher,† Joseph J. Romano,† Charles J. McIntyre,†
Kevin T. Nguyen,† Kevin F. Gilbert,† Kimberly J. Bush,† M. Katharine Holloway,‡ John Swestock,† Bang-Lin Wan,†
Steven S. Carroll,§ Jillian M. DiMuzio,§ Donald J. Graham,§ Steven W. Ludmerer,§ Mark W. Stahlhut,§
Christine M. Fandozzi,|| Nicole Trainor,|| David B. Olsen,§ Joseph P. Vacca,† and Nigel J. Liverton†
†Departments of Medicinal Chemistry, ‡Molecular Systems, §Antiviral Research, and Drug Metabolism, Merck Research Laboratories,
West Point, Pennsylvania 19486, United States
S Supporting Information
b
ABSTRACT: The discovery of MK-1220 is reported along with
the development of a series of HCV NS3/4A protease inhibi-
tors containing a P2 to P4 macrocyclic constraint with im-
proved preclinical pharmacokinetics. Optimization of the P2
heterocycle substitution pattern as well as the P3 amino acid led
to compounds with greatly improved plasma exposure follow-
ing oral dosing in both rats and dogs while maintaining
excellent enzyme potency and cellular activity. These studies
led to the identification of MK-1220.
KEYWORDS: Hepatitis C, NS3/4A, HCV, macrocycle, ring-closing metathesis
epatitis C virus (HCV) is a chronic infection that affects
an estimated 170-200 million people worldwide.1 The
inhibitors (danoprevir (ITMN-191),18,19 TMC435350,20-22
BI201335,23 and vaniprevir24 (Figure 1)) have also advanced into
late stage trials.
H
positive RNA strand flaviviridae family virus replicates pri-
marily in the liver, and while the disease progresses over of a
number of years, a significant fraction of those infected
develop serious liver disease, including cirrhosis and hepa-
tocellular carcinoma.2 HCV is also the most common in-
dication for liver transplantation.3 HCV displays a high
degree of genetic heterogeneity, with genotypes 1, 2, and 3
accounting for more than 90% of the infections in the
developed world and with genotype 1 being predominant
(∼70%) in the U.S., Europe, and Japan. The current standard
of care treatment for chronic HCV infection is combination
therapy with pegylated Interferon-R and Ribavirin4-6 for up
to 48 weeks. Sustained viral response (SVR) rates vary by
genotype, with ∼45% of HCV genotype 1-infected patients
and ∼80% of genotype 2- and 3-infected patients achieving
SVR. The limited efficacy, coupled with a number of serious
side-effects, limits the number of patients who complete
treatment.7,8
Efforts toward improved HCV treatment include the devel-
opment of direct antivirals which inhibit key steps in the viral
replication process.9 One of the more promising targets is the serine
protease NS3/4A,10-14 with clinical proof of concept established by
Boehringer-Ingelheim with BILN-2061 (Figure 1).15 Subsequently,
both Vertex (VX-950, telaprevir)16 and Schering-Plough (SCH-
503034, boceprevir)17 have moved R-keto-amide HCV NS3/4A
inhibitors into phase 3 clinical trials, while a number of noncovalent
Beginning in 2005,24-27 our program focused on a series of
macrocyclic inhibitors of HCV NS3/4A protease which contain a
linker from P2 to P4, in contrast to the P1 to P3 linker present in
BILN-2061 and several other known inhibitors (Figure 1). In
2008, we disclosed the general strategy for the discovery of this
series and the subsequent optimization of genotype 1b potency
and liver exposure, which led to compound 126 and vaniprevir
(MK-7009) (Figure 1).24 Compound 1 has picomolar binding
affinity for HCV NS3/4A protease (1b Ki = 0.04 nM), good
cellular potency (1b replicon IC50 = 4.5 nM), and excellent liver
exposure in rat following a 5 mg/kg oral dose ([liver]4h = 18,600
nM).26 Obtaining high liver exposure may be critical to the
success of any anti-HCV compound because the primary HCV
replication site is in hepatocytes.28 However, high systemic
exposure would also be desirable given the evidence of extra-
hepatic replication sites,28 and unfortunately, compound 1 does
not achieve significant plasma levels following oral dosing in
preclinical species (rat plasma AUC = 0.36 μM h; 5 mg/kg,
3
PO).26 With a goal of finding compounds in this series, with high
liver exposure and improved plasma exposure following oral
dosing in preclinical species while maintaining excellent potency,
Received: October 8, 2010
Accepted: December 24, 2010
Published: January 12, 2011
r
2011 American Chemical Society
207
dx.doi.org/10.1021/ml1002426 ACS Med. Chem. Lett. 2011, 2, 207–212
|

ACS Medicinal Chemistry Letters
LETTER
Figure 1. Macrocycle HCV NS3/4A protease inhibitors.
Scheme 1. Preparation of 6-Substituted and Unsubstituted Isoquinoline Derivativesa
a Reagents and conditions: (a) t-BuOK, DMSO, then HCl, EtOH. (b) HCl, dioxane. (c) HATU, DIEA, DMF. (d) vinylSnBu3, Pd(PPh3)4. (e) Zhan 1b,
DCM. (f) Pd/C, H2. (g) LiOH, THF, EtOH. (h) 16, HATU, DIEA, DMF.
we decided to further explore the effect of substituents on the P2
isoquinoline group as well as various P3 and P4 groups. These
efforts, which ultimately led to the discovery of MK-1220, are
described herein.
desired acyl-sulfonamides 18a-h after hydrolysis and coupling
with 16. 5-Trifluoromethoxy analogue 18i was prepared analo-
gously to the compounds in Scheme 1 using the corresponding
trifluoromethoxy isoquinoline, and P3-cyclohexyl analogue 19
was prepared analogously to 18c using 6b.
The synthesis of the various P2-isoquinoline analogues was
carried out using the modular approach detailed in Scheme 1. N-Boc
hydroxyproline 4 was reacted with either 6-substituted or unsub-
stituted bromo-chloroisoquinolines 2 and 3, which, following
esterification, gave 5 and 6. Boc-deprotection, followed by
coupling with acids 6a-k, gave 7a-i, 7k and 8a, 8c, 8j, which
could be vinylated under Stille conditions and then cyclized using
the Zhan 1b metathesis catalyst29 to give 9a-i, 9k and 10a, 10c,
and 10j. The resulting double bond could alternatively be left
intact or saturated under standard conditions to give 11a and b,
11d, 11f, 11i, 11k, and 12a, 12c, and 12j. The targeted
compounds 1, 13b and c, 13e, 13g-i, 14a, 14b, 14d, 14f, 14i,
14k, and 15a, 15c, and 15j were then prepared via hydrolysis of
the ester and coupling with amine 16.30 5-Substituted analogues
(see Supporting Information Scheme 2 for general scheme and
experimental details) were prepared by a regioselective halogena-
tion/cross-coupling sequence from 11a, which provided the
Compounds were evaluated in a genotype 1b enzyme binding
assay,31 and cellular activity was determined using the genotype
1b replicon system in the presence of 10% fetal bovine serum
(FBS) and 50% normal human serum (NHS).32,33 The plasma
and liver pharmacokinetics of lead HCV protease compounds
have been characterized and shown to result from the complex
interplay of absorption, uptake transport, efflux transport, pro-
tein binding, and metabolism. Since fully characterizing each of
these individual components in vitro may or may not translate
into accurate in vivo predictions, the team took an empirical
approach and differentiated potential lead compounds using
initial pharmacokinetic analysis focused on plasma AUC and
liver concentration at 4 h after a 5 mg/kg oral dose in rats.
As Table 1 illustrates, the effect on rat exposure in both liver
and plasma of various P3 groups can be dramatic. Simple
substitution the t-Bu of compound 126 for a cyclohexyl group
208
dx.doi.org/10.1021/ml1002426 |ACS Med. Chem. Lett. 2011, 2, 207–212

ACS Medicinal Chemistry Letters
LETTER
Table 1.
Table 2.
1b Replicon
IC50 (nM)b
rat PO
rat
1b Ki
(nM)a FBS
10%
50%
plasma AUC [liver] @
compd
R
NHS
(μM h)c
4 h (μM)d
3
14a
18a
18b
18c
18d
18e
18f
H
0.18
0.04
0.03
0.04
0.37
0.14
0.03
0.25
8.7
2
46
170
56
0.27
0.28
1.4
13.4
5.8
5-Br
5-I
2.3
2.7
6
24.5
33.2
15.2
12.5
12.6
5-OMe
5-OEt
5-Et
35
17.7
6.9
500
90
2.5
2
2.1
5-SMe
5-CF3
34
1.8
18g
18h
18i
21
410
>1000
4400
113
a Single or double bond at styryl position. b NS3/4A protease time-
resolved fluorescence assay. c Cell-based replicon assay. d 5 mg/kg dosed
orally in PEG400 (n = 2); AUC calculated based on 4 h experiment.
e 5 mg/kg dosed orally in PEG400 (n = 2); liver levels after 4 h.
5-SO2Me 0.11
280
50
3
5-OCF3
0.75
1.6
8.8
15a
6-OMe
0.04
9.6
3.0
a NS3/4A protease time-resolved fluorescence assay. b Cell-based repli-
con assay. c 5 mg/kg dosed orally in PEG400 (n = 2); AUC calculated
based on 4 h experiment. d 5 mg/kg dosed orally in PEG400 (n = 2); liver
levels after 4 h.
(13b) led to a 2-fold improvement in rat plasma AUC and liver
concentration, while reduction of the styryl-olefin (14b) led to a
nearly 10-fold improvement in rat plasma AUC (3.1 μM h).
3
the other 5-substituted derivatives tested in the standard rat PK
experiment showed good to excellent plasma exposure, with
most of the analogues losing significant replicon potency in the
presence of 50% NHS (18d and e, 18 g-i). In contrast,
thiomethyl analogue 18f displayed excellent in vitro and replicon
potency (gt 1b Ki = 0.03 nM; 1b replicon IC50 = 2 nM) and a
more modest shift in the presence of 50% NHS (IC50 = 34 nM);
however, the rat plasma AUC following a 5 mg/kg PO dose was
Unfortunately, while 14b had improved rat plasma exposure, the
genotype 1b replicon IC50 in the presence of 50% NHS was shifted by
greater than 10-fold, which may be due to the increased lipophilicity of
the cyclohexyl group. Introduction of a cyclopentyl P3 group (13c)
led to even greater plasma exposure (AUC = 4.2 μM h) with a
3
reduced loss in 50% NHS genotype 1b replicon potency. Given these
dramatic effects, a number of other macrocycles with various cyclic P3
groups were prepared. Smaller groups, such as cyclobutyl (14d), led
to much reduced potency, while larger groups, such as indane (13e)
or benzyl (13g), resulted in compounds with reduced potency and/
or moderate rat plasma and liver exposure. Aromatic P3 groups (14f,
13h) resulted in compounds with poor potency. In a striking example
of the unpredictable nature of the pharmacokinetics seen in this series,
simple substitution of oxygen for carbon in the cyclohexyl ring
(compare 13b/14b to 13i/14i) resulted in potent compounds which
had almost no rat liver exposure (<0.2 μM @ 4 h) and moderate
plasma levels. This type of result emphasizes the potential benefit of
screening a large number of compounds for rat oral plasma and liver
exposure due to the unpredictable pharmacokinetics of this series of
compounds.26
only moderate (1.8 μM h). The compound with the most
3
balanced profile was the 5-methoxyisoquinoline 18c, with good
gt 1b replicon IC50 (2.7 nM (10% FBS); 35 nM (50% NHS)) and
17.7 μM h plasma AUC following an oral 5 mg/kg dose in rat,
3
which is a nearly 100-fold improvement in plasma AUC com-
pared to the unsubstituted analogue 14a. A similar plasma
exposure enhancing effect was also seen with the 6-methoxyiso-
quinoline 15a (9.6 μM h), although this compound did have a
3
larger 50% NHS replicon shift and lower liver exposure.
While a number of the most promising analogues (14b,
13c, 18c, 15a) possessed the improved rat plasma exposure
we desired, all lost replicon potency in the presence of 50%
NHS to a degree that they were less potent than the targeted
potency of parent compound 1 (gt 1b replicon IC50 = 14 nM
(50% NHS)). We then investigated whether the plasma
enhancing effects of P3 cycloalkyl groups and P2-isoquino-
line substituents could be combined to produce a compound
which maintained high rat plasma while improving replicon
potency (Table 3). 5-Methoxyisoquinoline analogue 19
showed reduced rat plasma exposure compared to 18c, while
6-methoxy analogue (15c) showed an additive effect of the
Next, we explored the effect of 5- or 6-substitution on the P2-
isoquinoline ring, which in general led to increased plasma
exposure with variable effects on potency (Table 2). Interestingly,
while the 5-iodo derivative (18b) possessed a moderate 20-fold
shift in the replicon assay from 10% FBS to 50% NHS and good
plasma exposure (AUC = 1.4 μM h), the 5-bromo analogue
3
(18a) had a much larger shift in replicon IC50 and only moderate
plasma and liver exposure, thus demonstrating that lipophilicity
is not necessarily directly coupled to plasma/liver exposure. All
209
dx.doi.org/10.1021/ml1002426 |ACS Med. Chem. Lett. 2011, 2, 207–212

ACS Medicinal Chemistry Letters
LETTER
Table 3.
Replicon IC50 (nM)b
1b 50% NHS
compd
1b Ki (nM)a
1b 10% FBS
2a 10% FBS
rat PO plasma AUC (μM h)c
rat [liver] @ 4 h (μM)d
3
19
0.05
0.03
0.02
0.02
3.0
2.0
4.0
2.0
60.0
80.0
11.0
10.0
6.0
-
2.5
18.1
11.8
0.18
13.2
8.0
15c
15j (MK-1220)
14k
5.0
11.0
23.0
16.0
a NS3/4A protease time-resolved fluorescence assay. b Cell-based replicon assay. c 5 mg/kg dosed orally in PEG400 (n = 2); AUC calculated based on 4 h
experiment. d 5 mg/kg dosed orally in PEG400 (n = 2); liver levels after 4 h.
Table 4. Pharmacokinetic Parameters for 15j (MK-1220)
species
Cla (mL/(min kg))
T1/2a (h)
%F
POb Cmax (μM)
POb AUC (μM h)
POb [liver] (μM)
POb [liver] (μM)
3
rat
3.4
3.6
8.3
5.7
2.5
1.3
37%
41%
9%
4.4
4.0
0.3
12.1
12.4
1.2
23 @ 4 h
54 @ 2 h
9.9 @ 24 h
dog
10.3 @ 26 h
monkey
a IV: Rat (n = 4) and dog (n = 2) at 2 mg/kg and monkey (n = 3) at 1 mg/kg; DMSO. b PO (5 mg/kg, n = 3 in rat and n = 2 in dog and monkey; 10%
Tween).
P2-methoxy and the P3-cycloalkyl groups with a rat plasma AUC of
and bioavailability, and excellent exposure in rat liver even at
18.1 μM h. This compound still possessed a 40-fold shift in replicon
24 h (9.9 μM; 5 mg/kg). Plasma and liver exposure in dog are
3
IC50 in the presence of 50% NHS; however, as we demonstrated in
the development of MK-7009 (vaniprevir),24 incorporation of
longer and/or substituted P2-P4 linkers can both improve potency
and decrease the 50% NHS replicon shift. In this case, addition of
the gem-dimethyl group into the P2-P4 linker led to a compound
(15j, MK-1220)34 with excellent genotype 1 and 2 biochemical
and replicon potency and excellent rat plasma (plasma AUC =
similar to those in rat, with good plasma AUC (12.4 μM h)
3
and liver levels at both 2 h and 26 h (54 μM, 10.3 μM)
following a 5 mg/kg oral dose. In contrast, plasma exposure
in rhesus monkey is more limited (Table 4). Of particular note is
that the 24 h liver concentration following a 5 mg/kg oral dose in
both rats and dogs is ∼1000-fold greater than the 50% NHS-shifted
replicon IC50 (11 nM). Following an IV dose to bile-duct cannulated
animals, 15j isprimarilyexcreted into the bile as a mixture of parent
and oxidative metabolites in rat and dog. Compound 15j also has
no significant activity versus other serine proteases (>50,000-fold
versus trypsin and chymotrypsin) or in a broad Panlabs screen
(>15,000-fold selectivity). Given the excellent potency, favor-
able preclinical pharmacokinetic properties, and clean off-
target profile, 15j was determined to be the optimal compound
in this series and was selected for clinical development.
11.8 μM h) and liver exposure (23 μM @ 4 h). The dramatic effect
3
of the 6-methoxy group can be seen with the contrastingly low
plasma exposure seen with related P2-unsubstituted analogue 14k
(plasma AUC = 0.18 μM h).
3
Given the excellent potency and initial high plasma and
liver exposure following oral dosing in rat, 15j was profiled
further (Table 4). Compound 15j demonstrates low IV
clearance in rat, dog, and rhesus monkey, moderate half-life
210
dx.doi.org/10.1021/ml1002426 |ACS Med. Chem. Lett. 2011, 2, 207–212

ACS Medicinal Chemistry Letters
LETTER
In summary, optimization for potency and rapid screening for rat
plasma exposure (following oral dosing) in the P2-isoquinoline
series of the P2-P4 macrocyclic HCV NS3/4A protease inhibi-
tors has led to the discovery of several key structural features:
notably the plasma exposure enhancing effects of P2-methoxy
isoquinolines and P3-cycloalkyl groups. Within this optimized
series, MK-1220 (15j) emerged as a candidate suitable for
development.
(14) Avolio, S.; Summa, V. Advances in the development of macro-
cyclic inhibitors of hepatitis C virus NS3-4A protease. Curr. Top. Med.
Chem. (Sharjah, United Arab Emirates) 2010, 10, 1403–1422.
(15) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger,
G.; et al. An NS3 protease inhibitor with antiviral effects in humans infected
with hepatitis C virus. Nature (London, U. K.) 2003, 426, 186–189.
(16) Gentile, I.; Viola, C.; Borgia, F.; Castaldo, G.; Borgia, G.
Telaprevir: a promising protease inhibitor for the treatment of hepatitis
C virus infection. Curr. Med. Chem. 2009, 16, 1115–1121.
(17) Campas, C.; Pandian, R.; Bolos, J.; Castaner, R. Boceprevir:
NS3 protease inhibitor treatment of chronic hepatitis C. Drugs Future
2009, 34, 697–707.
(18) Seiwert, S. D.; Andrews, S. W.; Jiang, Y.; Serebryany, V.; Tan,
H.; et al. Preclinical characteristics of the hepatitis C virus NS3/4A
protease inhibitor ITMN-191 (R7227). Antimicrob. Agents Chemother.
2008, 52, 4432–4441.
(19) Rajagopalan, R.; Misialek, S.; Stevens, S. K.; Myszka, D. G.;
Brandhuber, B. J.; et al. Inhibition and Binding Kinetics of the Hepatitis
C Virus NS3 Protease Inhibitor ITMN-191 Reveals Tight Binding and
Slow Dissociative Behavior. Biochemistry 2009, 48, 2559–2568.
(20) Raboisson, P.; de Kock, H.; Rosenquist, A.; Nilsson, M.;
Salvador-Oden, L.; et al. Structure-activity relationship study on a novel
series of cyclopentane-containing macrocyclic inhibitors of the hepatitis
C virus NS3/4A protease leading to the discovery of TMC435350.
Bioorg. Med. Chem. Lett. 2008, 18, 4853–4858.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectra and HRMS
S
b
data for selected compounds, full experimental details for the
synthesis of MK-1220, and general scheme and structures for
5-substituted isoquinoline analogues. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 215-652-9727. Fax: 215-652-3971. E-mail: michael_rudd@
merck.com.
(21) Lin, T.-I.; Lenz, O.; Fanning, G.; Verbinnen, T.; Delouvroy, F.;
et al. In vitro activity and preclinical profile of TMC435350, a potent
hepatitis C virus protease inhibitor. Antimicrob. Agents Chemother. 2009,
53, 1377–1385.
(22) Lenz, O.; Verbinnen, T.; Lin, T.-I.; Vijgen, L.; Cummings,
M. D.; et al. In vitro resistance profile of the hepatitis C virus NS3/4A
protease inhibitor TMC435. Antimicrob. Agents Chemother. 2010, 54,
1878–1887.
(23) Llinas-Brunet, M.; Bailey, M. D.; Goudreau, N.; Bhardwaj,
P. K.; Bordeleau, J.; et al. Discovery of a Potent and Selective Non-
covalent Linear Inhibitor of the Hepatitis C Virus NS3 Protease (BI
201335). J. Med. Chem. 2010, 53, 6466–6476.
(24) McCauley, J. A.; McIntyre, C. J.; Rudd, M. T.; Nguyen, K. T.;
Romano, J. J.; et al. Discovery of Vaniprevir (MK-7009), a Macrocyclic
Hepatitis C Virus NS3/4A Protease Inhibitor. J. Med. Chem. 2010, 53,
2443–2463.
(25) Holloway, M. K.; Liverton, N. J.; Ludmerer, S. W.; McCauley,
J. A.; Olsen, D. B. et al. Preparation of macrocyclic compounds as HCV
NS3 protease inhibitors. In PCT Int. Appl.; Merck & Co., Inc.: USA, WO
2006/119061, 2006.
(26) Liverton, N. J.; Holloway, M. K.; McCauley, J. A.; Rudd, M. T.;
Butcher, J. W.; et al. Molecular Modeling Based Approach to Potent P2-
P4Macrocyclic Inhibitors of Hepatitis C NS3/4A Protease. J. Am. Chem.
Soc. 2008, 130, 4607–4609.
(27) Liverton, N. J.; Carroll, S. S.; Di Muzio, J.; Fandozzi, C.;
Graham, D. J.; et al. MK-7009, a potent and selective inhibitor of
hepatitis C virus NS3/4A protease. Antimicrob. Agents Chemother. 2009,
54, 305–311.
(28) Lindenbach, B. D.; Rice, C. M. Unravelling hepatitis C virus
replication from genome to function. Nature (London, U. K.) 2005, 436,
933–938.
’ ACKNOWLEDGMENT
We thank Dr. Charles W. Ross, III and Ms. Joan S. Murphy for
high-resolution mass spectral data and Sandor L. Varga for
interpretation of NMR spectra.
’ REFERENCES
(1) Hepatitic C-global prevalence (update). WHO Wkly. Epidemiol.
Rec. 1999, 74, 425-427.
(2) Liang, T. J.; Heller, T. Pathogenesis of hepatitis C-associated
hepatocellular carcinoma. Gastroenterology 2004, 127, S62–S71.
(3) Brown, R. S. Hepatitis C and liver transplantation. Nature
(London, U. K.) 2005, 436, 973–978.
(4) Hadziyannis, S. J.; Sette, H., Jr.; Morgan, T. R.; Balan, V.; Diago,
M.; et al. Peginterferon-a2a and ribavirin combination therapy in chronic
hepatitis C: A randomized study of treatment duration and ribavirin
dose. Ann. Intern. Med. 2004, 140, 346–355.
(5) Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi, V. K.;
Shiffman, M.; et al. Peginterferon alfa-2b plus ribavirin compared with
interferon a-2b plus ribavirin for initial treatment of chronic hepatitis C:
a randomized trial. Lancet 2001, 358, 958–965.
(6) McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.; Shiffman, M. L.;
Lee, W. M.; et al. Interferon alfa-2b alone or in combination with
ribavirin as initial treatment for chronic hepatitis C. N. Engl. J. Med. 1998,
339, 1485–1492.
(7) Aspinall, R. J.; Pockros, P. J. Review article: The management of
side-effects during therapy for hepatitis C. Aliment. Pharmacol. Ther.
2004, 20, 917–929.
(8) Fried Michael, W. Side effects of therapy of hepatitis C and their
management. Hepatology 2002, 36, S237–244.
(9) Gordon, C. P.; Keller, P. A. Control of Hepatitis C: A Medicinal
Chemistry Perspective. J. Med. Chem. 2005, 48, 1–20.
(29) The Zhan 1b catalyst is available from Strem, catalog # 44-0082;
CAS# 918870-76-5.
(10) Chen Kevin, X.; Njoroge, F. G. A review of HCV protease
inhibitors. Curr. Opin. Invest. Drugs 2009, 10, 821–837.
(11) Venkatraman, S.; Njoroge, F. G. Macrocyclic inhibitors of HCV
NS3 protease. Expert Opin. Ther. Pat. 2009, 19, 1277–1303.
(12) Thomson, J. A.; Perni, R. B. Hepatitis C virus NS3.4A protease
inhibitors: countering viral subversion in vitro and showing promise in
the clinic. Curr. Opin. Drug Discovery Dev. 2006, 9, 606–617.
(13) Chen, S.-H.; Tan, S.-L. Discovery of small-molecule inhibitors
of HCV NS3-4A protease as potential therapeutic agents against HCV
infection. Curr. Med. Chem. 2005, 12, 2317–2342.
(30) Wang, X. A.; Sun, L.-Q.; Sit, S.-Y.; Sin, N.; Scola, P. M. et al.
Hepatitis C Virus Inhibitors. In U.S. Patent 6,995,174, 2006.
(31) Mao, S.-S.; DiMuzio, J.; McHale, C.; Burlein, C.; Olsen, D.; et
al. A time-resolved, internally quenched fluorescence assay to character-
ize inhibition of hepatitis C virus nonstructural protein 3-4A protease at
low enzyme concentrations. Anal. Biochem. 2008, 373, 1–8.
(32) Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.;
Altamura, S.; et al. Characterization of Resistance to Non-obligate
Chain-terminating Ribonucleoside Analogs That Inhibit Hepatitis C
Virus Replication in Vitro. J. Biol. Chem. 2003, 278, 49164–49170.
211
dx.doi.org/10.1021/ml1002426 |ACS Med. Chem. Lett. 2011, 2, 207–212

ACS Medicinal Chemistry Letters
LETTER
(33) Lohmann, V.; Korner, F.; Koch, J. O.; Herian, U.; Theilmann,
L.; et al. Replication of subgenomic hepatitis C virus RNAs in a
hepatoma cell line. Science 1999, 285, 110–113.
(34) Holloway, M. K.; Liverton, N. J.; Ludmerer, S. W.; McCauley,
J. A.; Olsen, D. B. et al. Macrocyclic peptides as HCV NS3 Protease
Inhibitors. In PCT Int. Appl.; Merck & Co., Inc.: USA, WO 2007/
016441, 2007.
212
dx.doi.org/10.1021/ml1002426 |ACS Med. Chem. Lett. 2011, 2, 207–212