Novel peptidomimetic hepatitis C virus NS3/4A protease inhibitors spanning the P2-P1′ region

Article abstract of DOI:10.1021/ml400217r

Herein, novel hepatitis C virus NS3/4A protease inhibitors based on a P2 pyrimidinyloxyphenylglycine in combination with various regioisomers of an aryl acyl sulfonamide functionality in P1 are presented. The P1′ 4-(trifluoromethyl)phenyl side chain was shown to be particularly beneficial in terms of inhibitory potency. Several inhibitors with K_i-values in the nanomolar range were developed and included identification of promising P3-truncated inhibitors spanning from P2-P1′. Of several different P2 capping groups that were evaluated, a preference for the sterically congested Boc group was revealed. The inhibitors were found to retain inhibitory potencies for A156T, D168V, and R155K variants of the protease. Furthermore, in vitro pharmacokinetic profiling showed several beneficial effects on metabolic stability as well as on apparent intestinal permeability from both P3 truncation and the use of the P1′ 4-(trifluoromethyl)phenyl side chain.

Full text of DOI:10.1021/ml400217r

Letter
pubs.acs.org/acsmedchemlett
Novel Peptidomimetic Hepatitis C Virus NS3/4A Protease Inhibitors
Spanning the P2−P1′ Region
Anna K. Lampa,† Sara M. Bergman,† Sofia S. Gustafsson,‡ Hiba Alogheli,† Eva B. Åkerblom,†
Gunnar G. Lindeberg,† Richard M. Svensson,§,∥ Per Artursson,§,∥ U. Helena Danielson,‡ Anders Karlen,†
́
and Anja Sandstrom*,†
̈
†Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala,
Sweden
‡Department of Chemistry−BMC, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
§Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden
∥The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Uppsala University, a Node of the Chemical
Biology Consortium Sweden (CBCS), Box 580, SE-751 23 Uppsala, Sweden
S
* Supporting Information
ABSTRACT: Herein, novel hepatitis C virus NS3/4A protease inhibitors based on a P2
pyrimidinyloxyphenylglycine in combination with various regioisomers of an aryl acyl sulfonamide
functionality in P1 are presented. The P1′ 4-(trifluoromethyl)phenyl side chain was shown to be
particularly beneficial in terms of inhibitory potency. Several inhibitors with Ki-values in the
nanomolar range were developed and included identification of promising P3-truncated inhibitors
spanning from P2−P1′. Of several different P2 capping groups that were evaluated, a preference for
the sterically congested Boc group was revealed. The inhibitors were found to retain inhibitory
potencies for A156T, D168V, and R155K variants of the protease. Furthermore, in vitro
pharmacokinetic profiling showed several beneficial effects on metabolic stability as well as on
apparent intestinal permeability from both P3 truncation and the use of the P1′ 4-
(trifluoromethyl)phenyl side chain.
KEYWORDS: HCV, NS3, protease inhibitors, peptidomimetics, phenylglycine
epatitis C is an inflammatory disease that affects around
H2.2−3.0% of the world’s population and is caused by the
blood-borne hepatitis C virus (HCV).1 The infection resides
mainly in hepatocytes and becomes chronic in approximately
80% of infected individuals. Progression of the disease often
develops into various stage of liver disease, such as cirrhosis or
liver cancer, within a time period of 30 years.2 There are several
potential targets for anti-HCV therapy, of which the protease
part of the bifunctional nonstructural protein 3 (NS3) is one of
the most well-studied.3 Proof-of-concept, that inhibition of the
NS3/4A protease leads to reduction in plasma HCV RNA loads
in infected patients, was established with ciluprevir (BILN
2061, Figure 1).4 Thereafter, several NS3/4A protease
Figure 1. Selection of HCV NS3/4A protease inhibitors that have
entered clinical trials.5
inhibitors have been developed and entered into clinical trials,
some of which are shown in Figure 1.5 Two of the them,
Incivek (Vertex) and Victrelis (Merck and Co.), have recently
been launched on the market and are now used in combination
with the standard therapy of pegylated interferon-α and
ribavirin for treatment of HCV genotype 1.6
The above-mentioned inhibitors of HCV NS3/4A (as
exemplified in Figure 1) share some structural similarities.
They are peptide-based and share a proline or a proline mimic
in the P2 position. Although they possess excellent antiviral
capacities, the emergence of resistant strains of the protease is
disquieting.6,7 The mutations that have emerged in in vitro and
Special Issue: HCV Therapies
Received: June 7, 2013
Accepted: August 2, 2013
© XXXX American Chemical Society
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a
in vivo studies of the viral genome, under a selective pressure of
HCV NS3/4A protease inhibitors, are often related to amino
acids close to the S2 position of the protease, thus accounting
for cross-resistance issues.6−9 We have therefore searched for
alternatives to the commonly employed P2 proline residue in
HCV NS3/4A protease inhibitors. A promising alternative is
the use of a phenylglycine as a P2 moiety.10,11 In addition, P2
phenylglycine-based inhibitors have been shown to retain
activity against mutated strains of the protease.11−13 Further
development and optimization are still necessary in order to
improve antiviral activities. During our initial studies, we
discovered that a 2-phenyl-6-methoxypyrimidine based P2
substituent was beneficial in combination with a vinylated
phenylglycine, an aromatic P1 moiety, and alkenylic P1′
substituents (1, Figure 2). We also discovered that a P3
truncated inhibitor (2) showed reasonable inhibitory potency
(Ki = 190 nM, Figure 2)
Scheme 1. Synthetic Route
a
Reagents: (a) 4-chloro-6-methoxy-2-phenylpyrimidine, KOtBu,
DMSO, 64 °C (87%); (b) 2,4,6-trivinylcycloboroxane pyridine
complex, Pd(OAc)2, [(tBu)3PH]BF4, K2CO3, DME, H2O, microwave
irradiation at 100 °C for 15 min (82%).
P1−P1′ building blocks, a microwave-assisted carbonylative
approach including bromoaniline, trans-bis(acetato)bis[o-(di-o-
tolylphosphino)benzyl]-dipalladium(II) (Herrmann’s pallada-
cycle), [(tBu)3PH]BF4, 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), and Mo(CO)6 as a solid carbon monoxide source
was applied (Scheme 2).16 Using this method, the P1−P1′
a
Scheme 2. Synthetic Route to P1−P1′ Building Blocks
Figure 2. P3−P1′ spanning HCV NS3/4A protease inhibitor (1), its
P3 truncated analogue (2), and a proline-based HCV NS3/4A
protease inhibitor (3).14
a
Reagents: (a) Herrmann’s palladacycle, [(tBu)3PH]BF4, Mo(CO)6,
We have investigated the use of an aromatic P1 moiety in
proline-based inhibitors previously (e.g., 3 in Figure 2).14
Because the aromatic P1 enables synthesis of regioisomers, it
was appealing to us to utilize this property to examine how acyl
sulfonamides at different positions around the aromatic P1
would influence inhibitory potency for phenylglycine-based
inhibitors. Furthermore, we felt prompted to investigate the
possibility to develop smaller, less peptide-like but more drug-
like inhibitors with only one α-amino acid and therefore
designed P3 truncated inhibitors (exemplified by 2). Thus, we
herein present the synthesis and biochemical evaluation of a
series of P3−P1′ and P2−P1′ spanning HCV NS3/4A protease
inhibitors comprising a vinylated hydroxy phenylglycine
substituted with a 2-phenyl-6-methoxy-pyrimidine, combined
with an ortho (o-), meta (m-), or para (p-)-regioisomer of a P1
aryl acyl sulfonamide with either the pent-4-enyl side chain or
the 4-(trifluoromethyl)phenyl side chain in P1′-position. For
the P3 truncated inhibitors, a scan of various P2 N-capping
groups was also performed to establish any important inhibitor-
protease interactions at the S3−S2 border. Since we are aiming
for inhibitors with a unique binding mode, the ability to inhibit
known drug resistant enzyme variants was evaluated, as well as
their in vitro pharmacokinetic properties.
DBU, 1,4-dioxane, microwave irradiation at 140 °C for 25 min (7,
50%; 8, 64%; 9, 62%); (b) CDI, DBU, THF, 60 °C → rt (10, 71%; 11,
69%); (c) 4.0 M HCl in 1,4-dioxane (quantitative).
building blocks as o-, m- and p-isomers 7−9 could be
synthesized in only 25 min, without need for amine protection.
The P1−P1′ building blocks containing a pent-4-enyl
sulfonamide substituent were synthesized in the standard way
by coupling of Boc-protected m- and p-aminobenzoic acids with
pent-4-ene sulfonamide using carbonyldiimidazole (CDI) in
dry THF with DBU as base (Scheme 2).
Amide bond formation between the P2 building block 6 and
the poorly nucleophilic P1−P1′ moieties 7−11 was employed
using HATU and N,N-diisopropylethylamine (DIEA) in dry
CH2Cl2 at 45 °C and gave P2−P1′spanning compounds 12−16
in reasonable yields. The P3−P1′ spanning analogues 17L−
21D were obtained after standard HATU promoted couplings
of P3 Boc-L-tLeu and RP-HPLC purification. Only the L-Phg
isomers could be isolated for compounds 17 and 18 (Scheme
3).
To evaluate the effect of the N-capping group, compounds
13−15 were N-deprotected and reacted with t-butylisocyanate,
acetyl chloride, 4-morpholinecarbonyl-chloride, nicotinoyl-
chloride hydrochloride, or cyclopentyl chloroformate, giving
compounds 23−29 (Scheme 4).
For compounds 1, 2, and 12−29, Ki values were determined
in a biochemical inhibition assay using full length NS3 protein
of genotype 1a (see Supporting Information) and a 16 amino
acid peptide corresponding to the activating region of NS4a
(Tables 1 and 2).
The synthesis of the P2 building block 6 is described in
Scheme 1. Compound 6 was synthesized from a 3-bromo, 4-
hydroxy-phenylglycine derivative via a substitution reaction
followed by a Suzuki reaction to give the partially racemic
compound 6. The racemization is a known side-effect during
Suzuki couplings with phenylglycine.15
In a previous study, we found that a 4-(trifluoromethyl)-
benzene sulfonamide was beneficial as a P1′ substituent in
combination with an aromatic P1.14 For the synthesis of the
B
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a
a
Scheme 3. Synthetic Route
Table 1. Inhibition Constants for NS3/4A Protease
a
Reagents: (a) HATU, DIEA, CH2Cl2, 45 °C (12, 83%; 13, 50%; 14,
56%; 15, 74%; 16, 73%); (b) 4.0 M HCl in 1,4-dioxane; (c) HATU,
DIEA, DMF, rt (17L, 31%; 18L, 29%; 19L, 16%; 19D, 5%; 20L, 34%;
20D, 10%; 21L, 22%; 21D, 12%).
a
Scheme 4. Synthetic Route
a
a
Reagents: (a) 4.0 M HCl in dioxane (100%); (b) CH2Cl2 or THF,
Ki values of P3−P1′ spanning inhibitors were measured with full-
Et3N/DMF, DIEA, t-butylisocyanate (23, 55%), acetyl chloride (24,
34%), 4-morpholinecarbonylchloride (25, 17%), nicotinoylchloride ×
HCl (26, 39%), or cyclopentyl chloro-formate (27, 48%; 28, 56%; 29,
51%).
length NS3/4A protein from genotype 1a.
less potent than the o-compounds (1L, Ki = 26 nM; 1D, Ki =
33 nM). Moreover, no apparent difference was seen between
the stereoisomers of these inhibitors. Noteworthy, a compar-
ison with the proline-based aromatic P1 inhibitor 3 (Figure 2)
reveals that the aromatic P1 moiety is better suited for
combination with phenylglycine than with proline. Both the 4-
(trifluoromethyl)phenyl and the pent-4-enyl P1′side chain
combined with a P2 2-(4-((6-methoxy-2-phenylpyrimidin-4-
yl)oxy)-3-vinylphenylglycine (compounds 17L, 1L, and 1D)
gives inhibitors with ca. 10-fold better inhibitory potency than
to the corresponding proline inhibitor 3 (Figure 2).
A further evaluation of the N-capping group of the P3
truncated m-isomer 13 showed that removal of the Boc-
protecting group (compound 22, Ki = 1200 nM) caused a 20-
fold impairment in the inhibitory activity. The replacement of
the Boc-group with an N-acetyl capping group reduced the
inhibitory potency 14-fold (24, Ki = 700 nM) possibly as a
result of the reduced bulk. The larger 4-morpholinecarbonyl
group (compound 25, Ki = 590 nM) was slightly better than
the N-acetyl capping group but was still 12 times less potent
compared to inhibitors bearing the Boc-group. The introduc-
tion of an N-nicotinoyl capping group (compound 26, Ki = 240
nM) gave a 2-fold improvement compared to the 4-
morpholinecarbonyl capping group but was still 4 times less
favorable than the Boc-group. The N-t-butyl carbamoyl (23 Ki
= 140 nM) and the N-cyclopentyloxy carbonyl (27, Ki = 72
nM) capping groups proved to be the best alternatives to the
Boc-group. Thus, the steric bulk of the capping group is clearly
of importance. Indeed, exchange of the Boc group on the P3
truncated p-compound 14 (Ki = 46 nM) by a cyclopentyl
carbamate gave the equipotent inhibitor 28 (Ki = 55 nM).
Gratifyingly, the use of a P1′ 4-(trifluoromethyl)phenyl side
chain was shown to be beneficial in terms of inhibitory potency
giving several inhibitors in the nanomolar range (13, 14, 17−
19L, 19D, 27, and 28, Ki ≈ 20−70 nM, Tables 1 and 2). The
aromatic P1′ side chain was especially advantageous for the
truncated inhibitors 13 and 14 (Ki = 58 and 46 nM) as
compared to their P1′ pent-4-enylic counterparts 15 and 16 (Ki
≈ 200 nM). Biochemical evaluation of truncated inhibitors
comprising P1 regioisomers 12 (o-, Ki = 240 nM), 13 (m-, Ki =
58 nM), and 14 (p-, Ki = 46 nM), with a 4-(trifluoromethyl)-
phenyl P1′ substituent, revealed a preference for the m- and p-
isomers.
The same comparison of the P1′ pent-4-enyl regioisomers 15
(m-, Ki = 210 nM) and 16 (p-, Ki = 220 nM) reveals that these
are equipotent to o-compound 2 (Ki = 190 nM), although with
a slightly decreased inhibitory potency compared to the P1′ 4-
(trifluoromethyl)-phenyl containing inhibitors. Coupling of
12−14 with the P3 Boc-L-tLeu gave P3−P1′ spanning
inhibitors 17L (o-, Ki = 45 nM), 18L (m-, Ki = 24 nM), 19L
(p-, Ki = 30 nM), and 19D (p-, Ki = 30 nM). Again, a slight
preference was shown for the m- and p-isomers, although the
contribution to inhibitory potency for the m- and p-isomers
from the P3 substituent was only 1.5- and 2.5-fold, respectively,
as compared with 5-fold for the o-isomer. The same increase in
potency was noted for the pent-4-enyl containing inhibitors
after coupling with the P3 Boc-L-tLeu. Here, it was revealed
that the m-compounds (20L, Ki = 88 nM; 20D, Ki = 100 nM)
and p-compounds (21L, Ki = 96 nM; 21D, Ki = 110 nM) were
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a
Table 2. Inhibition Constants for NS3/4A Protease
Table 3. Inhibition Constants and Vitality Values Evaluated
with A156T, D168V, and R155K Variants of NS3
A156T
D168V
R155K
Ki SD
(nM)
Ki SD
(nM)
Ki SD
(nM)
a
a
a
compd
V
V
V
13
110
53
1.6
1.9
180
110
1.3
1.9
580
210
1.8
1.6
18L
a
Vitality values (V) were calculated using the equation V = [Ki × (kcat/
12
Km)]variant/[Ki × (kcat/Km)]wild‑type
.
protease differs among compounds that have entered clinical
trials. For example, Incivek (Figure 1) is favored by the D168V
substitution17 (V = 0.4) but highly affected by the A156T
substitution (V = 480).12 Ciluprevir is also highly affected by
both the A156T (V = 1600) and D168V (V = 3200)
substitutions.12 Herein, the vitality values for compounds 13
and 18L shows that the inhibitors have the capacity to retain
most of their inhibitory activity on the A156T, D168V, and
R155K variants of the protease (vitality values around 1).
Compound 18L is equally affected by the A156T, D168V, and
R155K substitutions (V = 1.9, 1.9, and 1.6), while compound
13, lacking a P3 substituent, is slightly less affected by the
D168V substitution (V = 1.3) than the A156T and R155K (V =
1.6 and 1.8) substitutions.
Compounds 1 (racemate), 13, and 18L were also tested for
EC50-values in a replicon Huh-7 cell line containing
subgenomic HCV RNA genotype 1b replicon with firefly
luciferase.18 Compound 1 and the P2−P1′ inhibitor 13 showed
EC50 values of 12.5 and 27.5 μM (CC50 ≥ 50 μM; in Huh-7
cells), respectively, whereas the P3-comprising inhibitor 18L
had a EC50 value of >50 μM (CC50 ≥ 50 μM). In analogy with
acyl sulfonamide based NS3/4A protease inhibitors of
submicromolar potency in general there is a significant
discrimination between cellular and enzymatic potencies.19
Clearly, there is room for optimization of the overall potencies,
down to low or subnanomolar, for these type of inhibitors.
Compounds 12 (o), 13 (m), and 14 (p) were modeled into
the binding site of the full-length NS3/4A protein after removal
of the cocrystallized macrocyclic inhibitor.20 The modeling
showed that compound 13 (m) finds key interactions with
amino acid backbone of the protease (R155 and A157) and that
the P1 carbonyl interacts with the oxyanion hole (G137 and
S139) (Figure 3). A possible π stacking interaction is seen
between the 4-(trifluoromethyl)phenyl substituent and Q41.
This type of interaction is seen for the original cocrystallized
macrocyclic inhibitor, as well as for a prime side extended α-
keto amide inhibitor.20,21 Additionally, hydrogen bond
interaction with the helicase (Q526) is captured by compound
13 (m), in analogy with the cocrystallized structure used in the
molecular modeling. The p-regioisomer 14 showed a similar
binding mode in the protein active site. However, the o-
regioisomer 12 did not fit well in the protein active site. In the
docking suggested conformations, several of the significant
interactions with the protein were missing, supporting the
preference of m- and p-isomers observed for this type of
inhibitors.
a
Ki values of truncated inhibitors were measured with full-length NS3/
4A protein from genotype 1a.
Substitution of Boc by a cyclopentyl carbamate on m-
compound 15 (Ki = 210 nM) gave compound 29 (Ki = 130
nM) with a slightly improved potency.
Additionally, compounds 13 and 18L were evaluated toward
the drug resistant A156T, D168V, and R155K variants of the
protease (Table 3). Vitality values were calculated to normalize
the inhibitory effects of the inhibitors with respect to the effects
of amino acid substitutions on catalytic efficiency (kcat/Km) of
the enzyme variants. A vitality value less than 1 indicates a more
efficient inhibitor against the mutated variant compared to the
wild-type enzyme, whereas a value larger than 1 shows that the
inhibitor is less efficient against the mutated virus.12 The
possibility to endure antiviral activity on mutated strains of the
To further address the potential of this series of inhibitors
and the P3 truncated inhibitors in particular, drug-like
properties were evaluated for the various regioisomers of the
most potent P1′ 4-(trifluoromethyl)phenyl containing inhib-
itors with the Boc capping group (12−14), using in vitro
pharmacokinetic profiling (Table 4). One inhibitor each of the
D
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susceptible for oxidative metabolism in microsomes compared
to its aromatic counterparts.
In summary, truncated phenylglycine based HCV NS3/4A
protease inhibitors of nanomolar potency have been discovered.
The aryl acyl sulfonamide moiety in P1 position was preferably
combined with a 4-(trifluoromethyl)phenyl P1′ side chain in m-
and p-positions, and the sterically congested Boc group turned
out to be the preferred P2 capping group. In general, the P2−
P1′spanning inhibitors comprising the Boc and 4-
(trifluoromethyl)phenyl groups exhibited very low risk for
high first pass metabolism, good apparent intestinal perme-
ability, and moderate solubility under in vitro settings. Further
optimization should be devoted to improving the cell-based
inhibitory potency. This could probably be achieved by further
optimization of the inhibitory potency against the protease and
by improving solubility, while maintaining good cell-perme-
ability and metabolic stability. Special attention should be
dedicated to drug resistance variants in the lead optimization
process since the inhibitors displayed promising inhibition of
the A156T, D168V, and R155K variants of the protease.
Figure 3. Compound 13 (m) docked in the binding site of the full-
length NS3/4A protein (PDB code: 4A92).20 The protease domain is
shown in gray; the helicase domain is shown in green. Hydrogen
bonds are highlighted as red dashed lines.
corresponding urea- (23), P3 t-leucine- (18L), and the P1′
pent-4-enyl (2) analogues were included for comparison.
Although the predicted log D values were in the same range,
the compounds showed relatively large variation in solubility.
Whereas the inhibitor comprising the P1′ pent-4-enyl side
chain (2) showed excellent solubility (>100 μM), the
corresponding P1′ 4-(trifluoromethyl)phenyl based inhibitors
exhibited lower solubility. The ortho analogue (12) displayed a
poor solubility of 5 μM, while the p-, P3-comprising-, and the
urea analogue showed acceptable (>20 μM) solubilities in the
range of 30−40 μM.
In analogy, several of the inhibitors with the higher aromatic
ring count penetrated the intestinal epithelial cells very well
(Papp = (4−9) × 10−6 cm/s, compounds 12, 14, and 18L). The
m-isomers showed moderate permeability, and when an
additional hydrogen bond donor is introduced as in the urea
group, this appears to have a negative impact on the transport
over cell membranes as shown from the decreased Papp value
(0.4 × 10−6 cm/s) of compounds 23 as compared to the
corresponding carbamate 13 (Papp value = 1.0 × 10−6 cm/s),
although the difference is not statistically significant. Gratify-
ingly, all the truncated inhibitors with a P1′ 4-(trifluoromethyl)-
phenyl side chain (12−14 and 23) showed a very low risk for
high oxidative metabolism in microsomes (Clint = 7−25 μL/
min/mg). These compounds clearly benefitted from the
removal of the P3 amino acids since the P3−P1′ spanning
inhibitor 18L showed a significantly higher intrinsic clearance
value (80 μL/min/mg). Additionally, the inhibitor with a P1′
pent-4-enyl side chain (2, Clint = 66 μL/min/mg) was more
ASSOCIATED CONTENT
* Supporting Information
All experimental details such as synthetic procedures and
characterization of compounds 4−29, enzymatic inhibition
assays, molecular modeling procedures, in silico prediction, and
in vitro pharmacokinetic assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(A.S.) Tel: +46 18 471 42 85. Fax: +46 18 471 4474. E-mail:
anja.sandstrom@orgfarm.uu.se.
■
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the Swedish Research
Council (Grants 9478 and 21386 and a grant to the Chemical
Biology Consortium Sweden). We gratefully acknowledge
support from Knut and Alice Wallenberg’s foundation and
Science for Life Laboratory. We also thank Simulations Plus for
providing access to the ADMET Predictor software and Dr
Aleh Yahorau, Department of Pharmaceutical Biosciences,
Uppsala University, for help with HRMS analyzes and Medivir
Table 4. In Vitro and in Silico Determined Properties
in vitro
in silico
a
b
c
d
e
compd
solubility (μM) pH 7.4
Caco-2 permeability Papp (10−6 cm/s) a−b
Clint (μL/min/mg)
t1/2 (min)
pKa
log D7.4
12
13
14
18L
23
2
5
4.0
16
12
25
80
7
88
4.8
4.1
4.4
4.3
4.2
5.8
4.4
3.8
3.9
4.5
4.1
4.0
f
n.d.
1.0 0.1
5.7 0.5
9.7 0.9
0.36 0.3
1.0 0.7
117
55
37
43
17
31
194
21
101
66
a
b
c
d
e
Papp = apparent permeability coefficient. a−b = apical to basolateral. Clint = in vitro intrinsic clearance. t1/2 = in vitro half-life. Acidic pKa (acyl
f
sulfonamide). n.d. = not determined.
E
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(15) Prieto, M.; Mayor, S.; Rodriguez, K.; Lloyd-Williams, P.; Giralt,
E. Racemization in Suzuki couplings: a quantitative study using 4-
hydroxyphenylglycine and tyrosine derivatives as probe molecules. J.
Org. Chem. 2007, 72, 1047−1050.
AB, Huddinge, Sweden for EC50-determinations and financial
support.
ABBREVIATIONS
■
(16) Wu, X. Y.; Ronn, R.; Gossas, T.; Larhed, M. Easy-to-execute
̈
carbonylations: Microwave synthesis of acyl sulfonamides using
Mo(CO)6 as a solid carbon monoxide source. J. Org. Chem. 2005,
70, 3094−3098.
HCV, hepatitis C virus; NS, nonstructural; Phg, phenylglycine;
Herrmann’s palladacycle, trans-bis(acetato)bis[o-(di-o-tolyl-
phosphino)-benzyl]-dipalladium(II); DBU, 1,8-diazabicyclo-
[5.4.0]undec-7-ene; HATU, N-[(dimethylamino)-1H-1,2,3-tri-
azolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium
hexa-fluorophosphate N-oxide; CDI, carbonyldiimidazole;
DIEA, N,N-diisopropylethylamine; Clint, intrinsic clearance;
(17) Lin, C.; Gates, C. A.; Rao, B. G.; Brennan, D. L.; Fulghum, J. R.;
Luong, Y.-P.; Frantz, J. D.; Lin, K.; Ma, S.; Wei, Y.-Y.; Perni, R. B.;
Kwong, A. D. In vitro studies of cross-resistance mutations against two
Hepatitis C Virus serine protease inhibitors, VX-950 and BILN 2061. J.
Biol. Chem. 2005, 280, 36784−36791.
P
app, apparent permeability coefficient
(18) Lohmann, V.; Korner, F.; Koch, J. O.; Herian, U.; Theilmann,
L.; Bartenschlager, R. Replication of subgenomic hepatitis C virus
RNAs in a hepatoma cell line. Science 1999, 285, 110−113.
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