Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2010.10.007

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1′ region was synthesized and

Full text of DOI:10.1016/j.bmcl.2010.10.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7493–7497
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV
NS3 protease
Xianfeng Li a, , Yong-Kang Zhang a, Yang Liu a, Suoming Zhang a, Charles Z. Ding a, Yasheen Zhou a,
⇑
b
Jacob J. Plattner a, Stephen J. Baker a, Liang Liu a, Wei Bu a, Wieslaw M. Kazmierski b, , Lois L. Wright ,
Gary K. Smith b, Richard L. Jarvest c, Maosheng Duan b, Jing-Jing Ji b, Joel P. Cooper b, Matthew D. Tallant b,
Renae M. Crosby b, Katrina Creech b, Zhi-Jie Ni d, Wuxin Zou e, Jon Wright e
⇑
a Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
b GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA
c GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK
d Acme Bioscience, Inc., 3941 E. Bayshore Road, Palo Alto, CA 94303, USA
e BioDuro LLC, Building E, No. 29, Life Science Park Road, Beijing 102206, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically vali-
dated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole
moiety at the P10 region was synthesized and evaluated. The resulting P1–P3 and P2–P4 macrocyclic
inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the
cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 September 2010
Revised 28 September 2010
Accepted 1 October 2010
Available online 30 October 2010
Keywords:
Hepatitis C virus
HCV NS3
Protease inhibitor
Macrocyclic inhibitor
Benzoxaborole
Hepatitis C virus (HCV) is a major cause of chronic liver disease
that can lead to cirrhosis, carcinoma and liver failure. It is esti-
mated that over 200 million people are chronically infected with
this virus and it is the leading cause of liver transplants.1 The cur-
rent standard treatment for HCV infection is based on a combina-
VX-950 (telaprevir)5 and SCH-503034 (boceprevir),6 currently in
Phase III clinical trials. The second class is represented by revers-
ible noncovalent inhibitors such as BILN-2061 (ciluprevir, Fig. 1),
the first compound in its class to achieve clinical proof of concept.7
Although its development was halted due to cardiac issues in
animals, BILN-2061 prompted extensive investigation on further
optimization of the peptide framework and P1 carboxylic acid re-
gion. To replace the P1 carboxylic acid, many different groups
(e.g., tetrazole, acylcyanamide, acysulfonamide, phosphonate)
have been studied.8 Among them, cyclopropyl acylsulfonamide ap-
pears to be the preferred replacement for the P1 carboxylic acid.9
Further application of this strategy led to the discovery of a
number of clinical candidates including ITMN-191 (danoprevir),10
TMC-435350 (medivir)11 and MK-7009 (vaniprevir),12 currently
in advanced clinical trials. However, rapid emergence of drug-
resistance has recently been observed for HCV NS3 protease inhib-
itors.13 Therefore, there remains a need for the discovery of new
HCV NS3 protease inhibitors with novel binding properties.
tion therapy of injectable pegylated interferon-
a (PEG IFN-a) and
antiviral drug ribavirin. This treatment, indirectly targeting the
virus, is associated with significant side effects often leading to
treatment discontinuation in certain patient populations.2 In addi-
tion, approximately 50% of genotype-1 (the predominant genotype
in the US) patients do not respond to this treatment regimen. Giv-
ing the high prevalence of the disease infection worldwide, there is
an enormous unmet medical need for new therapies against HCV
infection.3
HCV NS3/4A protease inhibitors have emerged as a promising
potential treatment for HCV infection.4 Two major classes of NS3
protease inhibitors have been developed. The first class is com-
prised of serine-trap inhibitors. The most advanced drugs are
As part of our continued efforts to discover novel HCV NS3
protease inhibitors,14 we envisioned that acylsulfamoyl benzoxab-
orole could be used to replace the cyclopropyl acylsulfonamide
moiety (Fig. 2). Benzoxaboroles15,16 are organoboron compounds
that have emerged as a new class of potential therapeutic agents.
⇑
Corresponding authors. Tel.: +1 650 543 7587; fax: +1 650 543 7660 (X.L.); tel.:
+1 919 483 9462; fax: +1 919 315 6923 (W.M.K.).
E-mail addresses: xli@anacor.com (X. Li), wieslaw.m.kazmierski@gsk.com (W.M.
Kazmierski).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.007

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X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7493–7497
HN
N
F
S
N
O
O
O
N
O
P1
O
O O
S
H
O
H
N
H
N
N
N
O
N
O
H
N
OH
O
O
O
N
O
O
O
H
BILN-2061 (ciluprevir)
ITMN-191 (danoprevir)
N
O
N
S
O
N
O
O
N S
O
O
O
O
H
O
O
O
H
N
N
HN
H
N
S
O
O
N
O
H
N
O
O
TMC-435350 (medivir)
MK-7009 (vaniprevir)
Figure 1. Selected reversible noncovalent inhibitors of HCV NS3 protease in advanced clinical trials.
F
P2*
O
N
OH
1
P1'
O
S
O
O
7
4
O
O
P1
6
OH
B
S
O
O
2
N
H
N
O
B
H
P4
O
5
N
O
N
H
N
3
H
P3
P2
O
O
acylsulfamoyl
benzoxaborole
O
Figure 2. Hypothetical P1–P3 macrocyclic inhibitor in which an acylsulfamoyl
benzoxaborole is used to replace the cyclopropyl acylsulfonamide in danoprevir.
Compounds containing a benzoxaborole moiety have been shown
to interact with a variety of biological targets and also exhibit good
drug properties.15 Studies of a hypothetical benzoxaborole inhibi-
tor derived from danoprevir (ITMN-191) docked into HCV NS3 pro-
tease suggest the benzoxaborole moiety can potentially form polar
interactions with Thr 42, and positively charged Lys 136 (Fig. 3).17
Our strategy was to scan for new potential benzoxaborole interac-
tions with the protease by exploring the impact of two distinct
macrocyclic series and different regioisomers of acylsulfamoyl
benzoxaboroles on the inhibitory potency.
Figure 3. Modeling of
a hypothetical benzoxaborole inhibitor derived from
danoprevir with HCV NS3 protease. Potential polar interactions for P10-benzoxab-
orole include main chain and side chain of Thr 42, and positively charged Lys 136.
The Boc group in danoprevir was replaced with
cyclopentyl carbamate in compound 17.
a chemically more-stable
Boronate 4 or 5, an important intermediate towards the 6-acy-
lsulfamoyl benzoxaboroles, was prepared according to Scheme 1.
Reaction of bromide 1 with pinacol diborane in the presence of pal-
ladium catalyst afforded boronate 2. Bromination of 2 with NBS
and AIBN gave benzyl bromide 3. Subsequently, treatment of 3
with sodium acetate in glacial acetic acid gave the corresponding
benzyl acetate 4. Reaction of 4 with sodium hydroxide followed
by acid treatment resulted in 6-sulfamoyl benzoxaborole 5. We
found that either boronate 4 or 5 could be coupled to HCV NS3
inhibitor scaffolds to form the targeted acylsulfamoyl benzoxabo-
role inhibitors.18
the boronate 15, an intermediate towards 4-acylsulfamoyl benzox-
aboroles, was prepared according to Scheme 3. The sulfonamides 7
and 12 used in the synthesis were made from the starting sulfonyl
chlorides 6 and 11, respectively.
With these key intermediates in hand, we set out to investigate
the P1–P3/P2–P4 macrocyclic series and also explore the impact of
regioisomers of acylsulfamoyl benzoxaboroles. The P1–P3 macro-
cyclic inhibitors 17–18 were synthesized according to Scheme 4.
Initially one of our targeted compounds was the hypothetical P1–P3
macrocyclic inhibitor in which 6-acylsulfamoyl benzoxaborole is
Similarly, towards the 5-acylsulfamoyl benzoxaboroles, the bor-
onate intermediate 10 was prepared according to Scheme 2, while

X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7493–7497
7495
F
F
O
B
O
O
O
B
O
O
O
O
S
O
O
b
S
Br
a
S
O
Br
N
H2N
N
H2N
O
H2N
O
O
OH
O O
S
H
O
O
H
N
H
N
B
a,b
OH
N
O
3
H
N
H
N
1
2
O
O
N
N
O
O
O
O
O
O
O
B
O
O
S
O O
OH
d,e
c
S
B
16
17
O
OAc
H2N
H2N
O
F
4
5
O
N
O
Scheme 1. Reagents and conditions: (a) pinacol diborane, PdCl2(dppf), KOAc,
dioxane, reflux, N2, 86%; (b) NBS, AIBN, CCl4, reflux; (c) NaOAc, glacial acetic acid,
reflux, N2, 23% in two steps; (d) 4 N NaOH, rt, 16 h; (e) 6 N HCl, 40 °C, THF, 16 h,
100% in two steps.
O O
S
H
O
H
N
c,d
N
H
N
O
OH
N
O
B
O
O
O
18
O
O
O
O
S
S
O O
S
Scheme 4. Reagents and conditions: (a) 4, HATU, DIEA, DMAP, DBU, anhydrous
DMF; (b) i-BuB(OH)2, 1 N HCl, hexane–MeOH (1:1), 28% in two steps; (c) 10, HATU,
DIEA, DMAP, DBU, anhydrous DMF; (d) i-BuB(OH)2, 1 N HCl, hexane–MeOH (1:1),
7% in two steps.
H2N
b
a
H2N
Cl
O
B
O
Br
Br
6
7
8
O
O
O
O
The P2–P4 macrocyclic inhibitors 20–22 were synthesized
according to Scheme 5. The starting macrocyclic acid 19, derived
from MK-7009, was made according to a reported procedure.12
Acid 19 was converted to the corresponding acylsulfonamides by
reaction with boronate 4, 10 or 15 as described previously, fol-
lowed by acid treatment to give isomeric benzoxaboroles 20–22.19
These compounds were evaluated for enzymatic potency in
FRET assay with NS3/4A 1a protease domain.20 The cellular activity
was determined using 1a and 1b HCV replicon assays.21 As shown
in Table 1, P1–P3 macrocyclic compounds 17–18 and P2–P4 mac-
rocyclic compounds 20–22 inhibited NS3 1a enzyme with IC50 val-
ues in the sub-nanomolar range (IC50 = 0.3–0.8 nM). They were
equipotent against NS3 1a enzyme, compared to danoprevir. These
benzoxaboroles exhibited low nanomolar potency against replicon
S
S
H2N
H2N
d
c
O
O
B
O
B
O
Br
AcO
9
10
Scheme 2. Reagents and conditions: (a) NH4OH, dioxane, 86%; (b) pinacol diborane,
PdCl2(dppf), KOAc, dioxane, reflux, N2, 67%; (c) NBS, AIBN, CCl4, reflux; (d) NaOAc,
glacial acetic acid, reflux, N2, 26% in two steps.
O
O
S
O
O
S
O
O
H2N
b
a
S
Cl
H2N
B
1b (EC50 = 8.0–20 nM), approaching that of danoprevir (EC50
=
O
O
Br
11
Br
1.1 nM). However, a higher shift between the enzyme potency
IC50 and replicon activity EC50 (especially for replicon 1a) was
observed for these benzoxaborole inhibitors than that observed
for danoprevir, which could be attributed to their poor cell
membrane permeability.
12
13
O
O
O
O
S
S
H2N
H 2
N
d
c
Interestingly, the regioisomers of acylsulfamoyl benzoxaboroles
appear to have a minimal influence on the inhibitor activity. For
the P1–P3 macrocyclic series, 6-acylsulfamoyl benzoxaborole 17
is equipotent in the enzyme and replicon 1a and 1b assay, com-
pared to 5-acylsulfamoyl analog 18. For the P2–P4 macrocyclic
series, very little difference in potency was observed with 6-,
5- or 4-acylsulfamoyl benzoxaboroles (20–22) in the enzyme and
replicon assay. These results demonstrate that regioisomers of
acylsulfamoyl benzoxaboroles are well tolerated, consistent with
the shallow enzyme binding pocket that is able to incorporate a
variety of diverse inhibitors.
B
Br
AcO
B
O
O
O
O
14
15
Scheme 3. Reagents and conditions: (a) NH4OH, dioxane, 100%; (b) pinacol
diborane, PdCl2(dppf), KOAc, dioxane, reflux, N2, 73%; (c) NBS, AIBN, CCl4, reflux;
(d) NaOAc, glacial acetic acid, reflux, N2, 21% in two steps.
used to replace the cyclopropyl acylsulfonamide in danoprevir
(structure shown in Fig. 2). During the synthesis, we found P4-
Boc group did not survive the acid treatment in the final step
and thus it was replaced with a chemically more-stable cyclopen-
tyl carbamate. The P1–P3 macrocyclic acid 16, derived from dano-
previr, was made according to a published procedure.10 Acid 16
was converted to the corresponding acylsulfonamides using boro-
nate 4 or 10 in the presence of HATU and DIEA. The removal of
pinacol/acetate groups and spontaneous formation of benzoxabo-
role ring was accomplished by acid treatment in the presence of
isobutyl boronic acid and 1 N HCl to afford the desired product
17 and 18, respectively.
Our results in acylsulfamoyl benzoxaboroles show that the
P1–P3 macrocyclic inhibitors are equipotent in the enzyme and
replicon assay compared to the P2–P4 macrocyclic inhibitors,
suggesting that these two macrocyclization strategies are equally
effective in enhancing inhibitor activity. Further in vivo PK evalua-
tion was undertaken to prioritize these two potent sub-series,
which is shown in Table 2.
Despite increased solubility relative to danoprevir, both the
P1–P3 macrocyclic inhibitor 17 and the P2–P4 macrocyclic inhibi-
tor 22 displayed undetectable to low oral exposures in rats. Portal
vein sampling evidenced very low absorption of 17 and 22. By
contrast, danoprevir exhibited calculated 17.4% absorption and

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X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7493–7497
O
O
N
OH
N
O
O O
S
B
O
O
O
O
O
O
O
a,b
OH
NH
HN
N
HN
N
H
N
H
N
O
O
O
O
O
N
19
20
O
O O
S
NH
O
O
O
c,d
OH
B
O
HN
N
H
N
O
O
O
21
O
N
O O
S
NH
O
O
O
e,f
HN
N
H
N
O
B
O
OH
O
O
22
Scheme 5. Reagents and conditions: (a) 4, HATU, DIEA, DMAP, DBU, anhydrous DMF; (b) i-BuB(OH)2, 1 N HCl, hexane–MeOH (1:1), 40% in two steps; (c) 10, HATU, DIEA,
DMAP, DBU, anhydrous DMF; (d) i-BuB(OH)2, 1 N HCl, hexane–MeOH (1:1), 25% in two steps; (e) 15, HATU, DIEA, DMAP, DBU, anhydrous DMF; (f) i-BuB(OH)2, 1 N HCl,
hexane–MeOH (1:1), 19% in two steps.
Table 1
caused by their limited absorption. We believe that the introduc-
tion of an unsubstituted benzoxaborole moiety results in unbal-
anced physicochemical properties, such as high molecular weight
In vitro activity of acylsulfamoyl benzoxaborole inhibitors against HCV NS3/4A 1a,
replicon 1a and 1b
Compounds
NS3/4A 1a
IC50 (nM)
HCV replicon 1a
EC50 (nM)
HCV replicon 1b
EC50 (nM)
(MW) and high polar surface area (PSA), of the final molecule
(Table 2). Further rebalancing of MW and PSA with carefully
redesigned benzoxaborole moieties could improve the permeabil-
ity of the final inhibitors, contributing to simultaneous increase
of potency in the replicon assay as well as improvement of bio-
availability in the in vivo PK assays. In fact, we applied this strategy
to another benzoxaborole-based series, resulting in more drug-like
properties in these molecules (manuscript in preparation).
a
b
b
Danoprevir
0.4
0.8
0.8
0.4
0.6
0.3
1.0
66
70
78
66
1.1
8.0
12
20
15
17
18
20
21
22
52
12
a
FRET assay with HCV NS3 1a protease domain in the buffer containing 20%
In summary, we have designed and explored synthetic routes
towards novel acylsulfamoyl benzoxaborole-based HCV NS3 prote-
ase inhibitors. Interestingly, the resulting, unoptimized P1–P3 and
P2–P4 macrocyclic inhibitors were equipotent in an enzyme assay
and somewhat less potent in replicon assays, compared to dano-
previr. Further optimization of the benzoxaborole moiety may al-
low to rebalance the physicochemical properties (e.g., MW, PSA)
of the resulting compounds and improve their membrane absorp-
tion, potency and bioavailability. In addition, it will be interesting
to examine the HCV protease inhibitor resistance profiles of these
compounds due to the anticipated additional interactions of P10
benzoxaborole with the enzyme active site as shown in Figure 3.
sucrose, as described in Ref. 20.
b
Replicon assay performed as described in Ref. 21.
Table 2
Physicochemical properties and PK parameters of selected inhibitors in male Sprague-
Dawley ratsa
Parameter
Compound
Danoprevir
17
22
MW
c Log P
PSA
732
5.6
836
5.4
210
848
6.3
210
4922
0.014
1.6
181
643
1.75
17.4
20.0
CL (mL/h/kg), iv
6640
b
AUC0-inf (h lg/mL), po
% Absorptionc
b
b
Acknowledgments
%Fd
1.2
a
The authors acknowledge Dazhong Fan, Xuelei Qian, Liang Liao,
Dianjun Chen, Xiantao Dong, Peng Liang, Jianbin Xiao, Guanghui Li,
Shuanghui Wang, Chong Li, Gang Lü, Baojuan Zhao and Xinming
Zhou for technical assistance and timely supply of key inter-
mediates.
Compounds were dosed orally at a dose of 5 mg/kg (n = 3) and intravenously at
a dose of 1 mg/kg (n = 3).
b
Below the limit of detection (0.5 ng/mL) and no PK data generated.
Calculated from portal vein drug concentrations after oral administration as
c
compared to that after IV administration.
d
Calculated from jugular vein drug concentrations after oral administration as
compared to that after IV administration.
References and notes
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20% oral bioavailability in rats. These results suggest that the poor
oral bioavailability of the benzoxaborole-containing compounds is

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7497
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18. Experimental procedure for the preparation of compound 4: To a mixture of
3-bromo-4-methylbenzenesulfonamide
1 (5.0 g, 20 mmol), 2,4,4,5,5-penta-
methyl-1,3,2-dioxaborolane (7.62 g, 30 mmol), and KOAc (7.85 g, 80 mmol) in
80 mL of dioxane was added PdCl2(dppf) (740 mg, 0.91 mmol). After degassed
three times with nitrogen, the reaction mixture was heated up to reflux for
16 h under nitrogen. Subsequently the mixture was cooled to room
temperature and filtered through Celite. The filtrate was concentrated and
the residue was purified by ISCO CombiFlash silica chromatography eluted
with 0–40% ethyl acetate in hexane to give 5.1 g of compound 2 as a white solid
(yield 86%). 1H NMR (300 MHz, CDCl3): d 8.26 (s, 1H), 7.81 (d, 1H),7.25 (d, 1H),
5.15 (s, 2H), 2.57 (s, 3H), 1.32 (s, 12H). To a solution of boronate 2 (5.1 g,
17.2 mmol) in 170 mL of CCl4 were added NBS (6.09 g, 34.2 mmol) and AIBN
(68 mg). The mixture was heated to reflux for 16 h under N2 atmosphere.
Subsequently, the mixture was cooled to room temperature and diluted with
ethyl acetate and washed with brine. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated under vacuum to give a yellow
syrup 3. To a solution of this crude syrup in 80 mL of HOAc was added NaOAc
(5.46 g, 66.6 mmol). The mixture was heated to reflux under nitrogen for 16 h.
Then the mixture was evaporated to dry and diluted in ethyl acetate. The
organic layer was washed with saturated NaHCO3, brine and dried over
anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was
purified by ISCO CombiFlash silica chromatography eluted with 0–50% ethyl
acetate in hexane to give 1.4 g of compound 4 as a white solid (yield 23% in two
steps). 1H NMR (300 MHz, CDCl3): d 8.37 (s, 1H), 7.97 (d, 1H), 7.52 (d, 1H), 5.42
(s, 2H), 4.82 (s, 2H), 2.12 (s, 3H), 1.34 (s, 12H).
19. Experimental procedure for the synthesis of compound 20: To
solution of acid 19 (78 mg, 0.12 mmol), HATU (52 mg, 0.14 mmol) in 1 mL of
anhydrous DMF was added DIEA (90 L, 0.50 mmol). The reaction mixture was
a stirred
l
stirred at room temperature for 1 h. Subsequently a solution of 4 (178 mg,
0.50 mmol), DMAP (61 mg, 0.5 mmol), and DBU (76 mg, 0.5 mmol) in 2 mL of
anhydrous DMF was added. The reaction mixture was stirred at room
temperature for three days. The mixture was diluted with ethyl acetate, and
washed with aqueous NaOAc buffer, 5% NaHCO3 and brine. The organic layer
was dried over Na2SO4, filtered and concentrated to give the coupling product
as a yellow oil. MS m/z 990.4 [M+1]+, 988.5 [Mꢀ1]ꢀ (calcd MS 989.5). To a
stirred solution of this coupling product in 5 mL of hexane and 5 mL of MeOH
were added isobutyl boronic acid (40 mg, 0.39 mmol) and HCl (1 mL, 6 N),
respectively. The reaction was stirred at room temperature for 16 h. The
mixture was concentrated in vacuo, diluted with ethyl acetate and washed
with brine. The organic layer was dried over Na2SO4, filtered and concentrated
in vacuo. The crude residue was purified on a reversed-phase column eluted
with ACN and H2O. The pure fractions were collected and ACN was removed in
vacuo. The aqueous solution was extracted with ethyl acetate three times. The
organic layer was dried over Na2SO4, filtered and concentrated to give
compound 20 as a white solid (36 mg, yield 40%). MS m/z 848.4 [M+1]+,
846.4 [Mꢀ1]ꢀ (calcd MS 847.3). 1H NMR (300 MHz, CDCl3): d 11.0 (1H, s), 9.60
(1H, s), 8.46 (1H, s), 7.82 (1H, m), 7.76 (1H, b), 7.42 (1H, d), 7.07 (1H, d), 7.15
(1H, t), 7.07 (1H, d), 5.76 (1H, d), 5.00–5.50 (5H, m), 4.93 (1H, d), 4.70 (2H, m),
4.36–4.64 (4H, m), 4.21 (1H, m), 3.88 (1H, d), 3.07 (1H, d), 2.81 (1H, m), 1.60–
2.40 (7H, m), 1.10–1.50 (6H, m), 1.06 (9H, m), 0.78 (3H, s), 0.60 (3H, s).
20. Compounds were assayed in the fluorescence enzymatic assay using HCV NS3/
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4A 1a protease domain. Conditions: 0.75 nM enzyme (1a domain), 2
lM NS4A,
16. (a) Bérubé, M.; Dowlut, M.; Hall, D. G. J. Org. Chem. 2008, 73, 6471; (b)
0.5 M peptide substrate (Ac-DE-Dap(QXL520)-EE-Abu- -[COO]AS-C(5-FAMsp)-
l
w
_
´
´
´
Adamczyk-Wozniak, A.; Cyranski, M. K.; Zubrowska, A.; Sporzynski, A. J.
Organomet. Chem. 2009, 694, 3533.
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 initial rates.
21. Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilman, L.; Batenschlager, R.
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17. The crystal structure of HCV NS3-4A complexed with a ketoamide inhibitor
SCH446211 (PDB code 2FM2) was selected for the docking analysis. The bound
inhibitor was deleted and hydrogen atoms added to the protein structure. The