Synthesis and biological evaluations of P4-benzoxaborole-substituted macrocyclic inhibitors of HCV NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2010.10.071

We disclose here a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. These inhibitors are potent against HCV NS3 protease, their anti-HCV replicon potencies are largely impacted by substitutions on benzoxaborole ring system and P2 groups. P2 2-thiazole-isoquinoline provides best replicon potency. The in vitro SAR studies and in vivo PK evaluations of selected compounds are described herein.

Full text of DOI:10.1016/j.bmcl.2010.10.071

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7317–7322
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluations of P4-benzoxaborole-substituted
macrocyclic inhibitors of HCV NS3 protease
Charles Z. Ding a, , Yong-Kang Zhang a, Xianfeng Li a, Yang Liu a, Suoming Zhang a, Yasheen Zhou a,
⇑
Jacob J. Plattner a, Stephen J. Baker a, Liang Liu a, Maosheng Duan b, Richard L. Jarvest c, Jingjing Ji b,
Wieslaw M. Kazmierski b, Matthew D. Tallant b, Lois L. Wright b, Gary K. Smith b, Renae M. Crosby b,
Amy A. Wang 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:
We disclose here a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. These
inhibitors are potent against HCV NS3 protease, their anti-HCV replicon potencies are largely impacted
Received 7 September 2010
Revised 11 October 2010
Accepted 14 October 2010
Available online 21 October 2010
*
*
by substitutions on benzoxaborole ring system and P2 groups. P2 2-thiazole-isoquinoline provides best
replicon potency. The in vitro SAR studies and in vivo PK evaluations of selected compounds are described
herein.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HCV
HCV protease inhibitors
Benzoxaborole
Synthesis and biological evaluations
HCV NS3 protease inhibitors
Infection with Hepatitis C Virus (HCV) is a major cause of human
liver disease throughout the world, affecting over 200 million
individuals. In the US alone, an estimated 4.5 million Americans
are chronically infected. HCV infection is responsible for 40–60%
of all chronic liver disease cases and 30% of all liver transplants.
The current standard of care for HCV infection is a combination
early proof of concept in suppressing the virus and are currently
undergoing Phase III clinical trials.5 Newer protease inhibitors with
improved potency, different mode of binding interaction with the
protease enzyme and pharmacokinetic properties have emerged.
These inhibitors, such as danoprevir (ITMN-191),6 TMC-435,7
BMS-7913258 (Fig. 1), and vaniprevir (MK-7009)9 are in clinical
development representing structural diversity set of promising
HCV protease inhibitors.
This initial excitement about the potential novel HCV treat-
ments has been somewhat dampened by a quick emergence of
enzyme resistance to these agents.10 The opportunity for other
chemical classes of HCV protease inhibitors with better resistance
profile still exists. In our search for novel HCV protease inhibitors,
we considered benzoxaborole as the P4 moiety, the fourth amino-
acid residue from carboxyl terminus. Benzoxaboroles (core struc-
ture shown in compound 2 in Scheme 1) are a chemical class of
organoboron compounds that have excellent physicochemical
and biological properties.11 They are metabolically stable, and ex-
hibit good water solubility. Docking studies of compound 4 to
the enzyme active site suggest that benzoxaborole moiety with
suitable orientation and linkage can potentially interact with ac-
tive site polar aminoacid residues: Ser122, Arg123, Arg155 and
of injectable pegylated interferon-a (PEG IFN-a) plus oral ribavirin,
which is effective in only about 50% of genotype-1 patients achiev-
ing sustained viral response.1 This protocol has been associated
with side effects including neuropsychiatric events, flu-like
symptoms and hematological toxicities.2 Therefore, there has been
tremendous interest in the development of more effective thera-
peutics in treating HCV infection. One of the validated targets is
HCV NS3/4A serine protease.3
Extensive efforts in the discovery of HCV NS3 protease inhibi-
tors have resulted in a number of drug candidates at various stages
of clinical development.4 The two most advanced compounds,
VX-950 (telaprevir) and SCH-503034 (boceprevir), provided an
⇑
Corresponding author.
E-mail address: charles.ding@gmail.com (C.Z. Ding).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.071

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C. Z. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7317–7322
F
F
O
O
O
O
N
O
N
O
O
O
O
O
O
S
O
S
H
N
NH
H
N
NH
HO
N
N
B
O
O
O
O
O
O
N
B
N
O
O
H
H
HO
OH
6
5
O
O
B
OH
F
B
b
HO
12
HO
F
11
b
O
O
O
N
N
O
O
O
O
O
O
O
S
O
S
a
H
N
NH
H
NH
N
N
N
O
O
O
O
H2N
N
H
O
ITMN191
1
R2
O
R2
R2
R1
R1
R1
b
O
B
O
B
NH2
CO2H
B
HO
2: R1=R2=H
21: R1=F; R2=H
HO
HO
25:
NH2
1
2
1
2
26:
R =R =H
R =R =H
F
O
O
1
2
22:
R =F; R =CH3
N
c
O
O
O
S
R2
H
N
NH
NO2
N
O
R1
X
O
O
HO
d
O
O
B
O
N
H
B
N
O
H
HO
R1
R2
4: X=NH;R1=R2=H
7: X=CH2NH;R1=R2=H
1
2
3:
R =R =H
1
2
8:
9:
X=NH;R =F; R =H
1
2
X=NH;R =F; R =CH3
10: X=absent, R1=R2=H
Scheme 1. General syntheses of benzoxaborole-substituted macrocyclic HCV inhibitors. Reagents and conditions: (a) TFA, DCM, rt basic work-up, 83%; (b) triphosgene, TEA,
THF, À40 °C; then 2, 12, 21 and 22, 15–30%; for 10, HATU, DIEA, 26, DMF, rt 50%; (c) p-O2N-Ph-OCOCl, 25, ACN, rt 90%; (d) amine 1, ACN, 60 °C; 50%.
Asp168 of HCV NS3/4A serine protease. These additional interac-
tions may provide better resistance profile than the known inhib-
itors that do not have. Syntheses of the target compound 4 and its
analogs as well as their biological evaluations are reported herein.
P4-Benzoxaborole-substituted macrocyclic compounds based
on ITMN-191 scaffold were prepared using a general scheme as
OH
Br
O
Br
a
b
B
H
OH
O
F
F
F
O
B
R
R
18a: R=H
19a: R=H
17
O
OTf
O
O
OH
O
O
18b: R=CH3
a
19b: R=CH3
b
O
O
c
14
15
c, d
H
13
H
OH
OH
H
H2N
F
O2N
B
B
d
O
O
OH
OH
F
HO
O
B
B
e
R
R
O
O
21: R=H
22: R=CH3
20a: R=H
20b: R = CH3
12
16
Scheme 3. Preparation of substituted 6-aminobenzoxaboroles. Reagents and
conditions: (a) for 18a: NaBH4, MeOH; for 18b: MeMgBr, THF, 0 °C, ꢀ93%; (b)
B(iPrO)3, n-BuLi, THF, À70 °C, 40–47%; (c) fuming HNO3, À45 °C, 80–90%; (d) Raney
Ni, hydrazine monohydrate, MeOH, rt, 70–80%.
Scheme 2. Preparation of 6-hydroxybenzoxaborole. Reagents and conditions: (a)
Tf2O, pyridine, À78 °C to rt; (b) (pinB)2, PdCl2(dppf), KOAc, dioxane, 80 °C; (c)
NaBH4, MeOH, rt; (d) HCl; (e) BBr3, CH2Cl2, À78 °C to rt.

C. Z. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7317–7322
7319
HO
25 to its p-nitrophenyl carbamate 3, followed by reaction of it with
macrocyclic amine 1 in somewhat higher (50%) yield. The amide 10
was prepared in 50% by coupling of 6-benzoxaborole carboxylic
acid 26 with macrocyclic amine 1 in the presence of HATU, DIEA
in DMF at room temperature.
Br
CN
Br
HO
CN
CN
a
b
B
O
OHC
22
23
24
c
d
The functionalized benzoxaborole compounds necessary for the
preparation of benzoxaborole-substituted macrocyclic HCV inhibi-
tors were prepared as follows. The 6-aminobenzoxaborole 2
(R1@NH2, R@R2@H) was prepared according to a published proce-
dure.14 Both 5- and 6-hydroxybenzoxaboroles (11 and 12) were
prepared in the same fashion starting from separate starting mate-
rial 5 or 4-methoxysalicylaldehyde, as illustrated for 6-hydrox-
ybenzoxaborole in Scheme 2. 4-Methoxysalicylaldehyde was
converted to its triflate 14 by treatment with triflic anhydride in
the presence of pyridine at low temperature. Boronation was
accomplished by pinacol diborate in the presence of a palladium
catalyst to provide boronobenzaldehyde 15. Reduction with
sodium borohydride and acidic work up gave 6-methoxybenzoxab-
orole 16. Cleavage of the methoxy group by BBr3 provided
6-hydroxybenzoxaborole 12.
HO
HO
COOH
B
B
NH2
O
O
25
26
Scheme 4. Preparation of amine 25 and acid 26. Reagents and conditions: (a)
NaBH4, MeOH, 0 °C, 99%; (b) B(iPrO)3, BuLi, THF, À78 °C, 50%; (c) LAH, THF, rt, 46%;
(d) concn HCl, reflux, 85%
shown in Scheme 1. ITMN-191 was prepared by following a patent
procedure.12 The P4 BOC group was removed by treatment with
TFA in dichloromethane to give amine 1. The coupling of the amine
to 6-aminobenzoxaboroles (2, 21 and 22) providing urea com-
pounds (4, 8 and 9) was accomplished via an isocyanide interme-
diate, which was prepared by treatment of compound 1 with
triphosgene in the presence of triethylamine at low temperature.
The intermediate was not isolated, 6-aminobenzoxaboroles were
added in situ. This reaction sequence provided urea compounds
in modest 30% isolated yield.13 The carbamate-linked P4-benzox-
aborole-substituted macrocyclic compounds (5 and 6) were gener-
ated going through the same isocyanate intermediate reacting with
hydroxybenzoxaboroles instead (11 and 12). The synthesis of urea
compound 7 involves conversion of 6-aminomethylbenzoxaborole
6-Amino-5-fluorobenzoxaboroles 21 and 22 were prepared as
shown in Scheme
3 from 2-bromo-5-fluorobenzaldehyde 17.
Reduction with sodium borohydride or addition of methyl magne-
sium bromide to 17 produced benzyl alcohol 18a or 18b in high
yield. Halogen-metal exchange reaction and deprotonation were
accomplished using two equivalents of n-butyllithium, the result-
ing dianion was reacted with triisopropyl borate and acidic
work-up providing benzoxaboroles 19a and 19b. Nitration of both
compounds with fuming nitric acid produced 6-nitro derivatives
O
N
O
N
S
N
Cl
Cl
Cl
isoquinoline
quinoline
27a
27b
P2*
HO
O
O
O
O
O
O
S
O
S
NH
H
N
NH
a
H
N
N
O
N
O
O
O
O
O
N
O
N
O
H
H
28
29a: P2*=isoquinoline
29b:
P2*=quinoline
P2*
O
P2*
O
b
O
O
O
O
O
S
O
S
H
N
NH
H
NH
N
N
N
O
c
O
O
O
R'
N
H
H2N
30a: P2*=isoquinoline
30b
O
: P2*=quinoline
31: P2*=isoquinoline, R'=
O
B
HO
33:
P2*=quinoline, R'=
N
H
H
32:
P2*=isoquinoline, R'=
O
N
B
34: P2*=quinoline, R'=
HO
O
Scheme 5. Preparation of isoquinoline- and quinoline-based HCV inhibitors. Reagents and conditions: (a) KOtBu, DMF, rt; then, 27a or 27b, 60–70%; (b) HCl(g), dioxane, rt,
ꢀ85%; (e) triphosgene, Et3N, CH2Cl2, À40 °C, then, compound 2 or 25, 20–30%.

7320
C. Z. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7317–7322
Table 1
20a and 20b with high regio-selectivity and yield. Raney nickel
reduction of the two nitro compounds provided 6-amino-7-fluor-
obenzoxaboroles 21 and 22.
HCV protease NS3/4 1a IC50 and replicon EC50 values for ITMN191-based P4-
benzoxaborole-substituted macrocyclic inhibitors as compared to ITMN191
F
6-Aminomethylbenzoxaboroles 25 and benzoxaborole-6-car-
boxylic acid 2615 were prepared as shown in Scheme 4. 2-Bromo-
4-cyanobenzaldehyde was converted to 6-cyanobenzoxaborole 24
by following the same sequence of reactions as shown in Scheme 3
for preparation of benzoxoborole 19a and 19b. The amin-
omethylbenzoxaborole 25 was obtained from reduction of com-
pound 24 using lithium aluminium hydride, while compound 26
was obtained from hydrolysis of compound 24 with concn HCl.
Inhibitors with P2* groups other than isoindoline (ITMN-191
scaffold) were prepared as described in Scheme 5 through hetero-
aryl displacement reactions. Prerequisite chloro-isoquinoline 27a
and chloro-quinoline 27b were prepared by following the pub-
lished procedures.16 The other coupling partner hydroxymacrocy-
cle 28 was also prepared according to a published reference
procedure.17 Heteroaryl displacement reaction of compound 28
with either 27a or 27b was achieved in good yield by treatment
of 28 with slightly more than two equivalents of potassium
tert-butoxide in DMF at room temperature. Both products 29a
and 29b were treated with HCl in dioxane to produce aminomac-
rocycles 30a and 30b. Linking of either 30a or 30b with both
6-aminobenzoxaborole 2 and 6-aminomethylbenzoxaborole 25
was accomplished by the same reaction sequence as described in
Scheme 1. Compounds 31 and 32 are products of amine 30a cou-
pled to 6-aminobenzoxaborole 2 and aminomethylbezoxaborole
25; while 33 and 34 are products of amines 30b coupled with
the same benzoxaboroles.
O
N
O
O
S
O
O
H
N
NH
N
O
O
R
N
H
Compds
R
NS3/4 IC50 (nM)a
HCV replicon
EC50 (nM)
1a
1b
O
ITMN191
0.4
0.4
1.0
1.1
3.7
O
O
O
O
4
159
B
N
H
HO
O
5
6
1.6
2.4
661
15.0
B
O
O
HO
HO
B
O
3548
172
O
O
Biological evaluations of the compounds were done both using
HCV protease and replicon assays. Results are shown in Table 1.
The protease inhibitory IC50’s were determined using a FRET assay
with HCV NS3/4A 1a protease domain.18 The replicon EC50’s were
determined using a replicon luciferase cell-based assay.19 The ini-
tially designed compound 4 proved equipotent than danoprevir
(ITMN-191) with enzymatic activity IC50 of 0.4 nM. While 4 was
potent in the genotype 1b assay (EC50 3.7 nM), the P4 structural
H
N
7
8
0.4
160
35
0.9
1.5
B
HO
O
F
O
O
<0.2
B
N
H
HO
F
O
O
9
0.4
1.0
20
1.2
B
N
H
HO
N
O
F
O
N
O
S
O
O
N
10
201
3.2
B
O
O
O
O
OH
S
H
N
O
O
O
NH
H
N
N
a
O
FRET assay with HCV NS3 1a protease domain. Values are means of duplicate or
triplicate experiments; errors are usually within 10%.
S
O
O
N
H
O
N
H
N
O
O
change turned out to be detrimental to compound’s potency
against genotype 1a replicon activity (EC50 159 nM). Encouraged
by this result, we set out to further explore the linker SAR. Chang-
ing the urea linker to its corresponding carbamate (compound 5),
the potency dropped off in both enzymatic or replicon assays.
Compound 6 is a regioisomer of compound 5, its potency was even
worse. However, homologation of the urea linker (in compound 7)
restored enzyme activity and, compared to lead compound 4, im-
proved replicon EC50 by 4 fold for genotype 1b, although similar
for genotype 1a. The amide linker in compound 10 provided simi-
lar potency or slightly inferior to compound 4. Next, we explored
substitutions on benzoxaborole ring system by improving lipophil-
icity, reducing polar surface area (PSA) of the ring while maintain-
ing the oxaborole functionality, aimed to improve replicon potency
for genotype 1a. We kept the urea linker for this exploration be-
cause it showed best potency and ease of synthesis. Compound 8
ITMN-191
danoprevir
TMC-435
O
O
N
Cl
O
S
NH
O
O
H
N
N
O
O
O
N
H
O
BMS-791325
Figure 1. Representative HCV protease inhibitors in clinical development.

C. Z. Ding et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7317–7322
7321
Table 2
HCV protease NS3/4 1a IC50 and replicon EC50 values for isoquinoline and quinoline P2* inhibitors as compared to TMC435
O
O
N
O
N
O
S
N
Cl
O
O
O
O
O
S
O
S
H
N
NH
H
NH
N
N
N
O
O
O
O
O
O
O
O
B
B
Y
N
Y
N
H
H
HO
HO
31: Y=NH
32: Y=CH2NH
33: Y=NH
34: Y=CH2NH
Compds
NS3/4 IC50 (nM)
HCV replicon EC50 (nM)
1a
1b
TMC435
31
10.0
0.6
0.4
5.6
4.1
2.3
1.4
3.9
3.9
1.2
0.5
60.3
50.1
12.5
4.2
32
33
34
is a fluoro derivative of compound 4 with excellent enzyme
potency and improvement in both genotype 1a and 1b replicon
activity by 2 to 3-fold. The compound 9 with both methyl and flu-
oro substitutions on benzoxaborole ring improved further cellular
potency in genotype 1a. Preliminary SAR in this series strongly
suggests that it should be possible to further optimize cellular po-
tency by further modifications in the benzoxaborole ring.
However, benzoxaborole-substituted macrocylic inhibitors 4, 7
and 34 displayed minimal to undetectable level of absorption
and oral bioavailability. We noticed good water solubility of these
inhibitors compared to ITMN191 when the PK samples were made,
however, their permeability and absorption are almost certainly
limited by their high molecular weight and high polar surface area
(PSA).
*
We decided to explore the impact of P2 groups on the anti-HCV
In summary, we have designed and synthesized a series of
P4-benzoxaborole-substituted macrocyclic HCV protease inhibi-
tors. We suggest that the benzoxaborole moiety can be a useful
moiety towards developing compounds retaining potency against
resistant enzymes.20a These compounds exhibited potent inhibi-
tory activity against HCV NS3/4 protease. Their cellular replicon
potencies were impacted by substitutions on the benzoxaborole
potency of P4-benzoxoborole substituted macrocyclic compounds.
We picked representative isoquinoline and quinolone groups from
other classes of HCV protease inhibitors, namely BMS-791325 and
TMC-435. The in vitro profiles of these compounds are summarized
in Table 2. Interestingly, compounds 31 and 32 with isoquinoline
P2* have similar enzyme and replicon potency profile to isoindo-
*
*
*
line P2 . The more bulky, elaborated quinoline P2 -containing
compounds 33 and 34 showed better replicon potency, although
enzymatic potency fell off. We believe that some of the differences
between the enzyme and replicon potency may originate from fac-
tors that differentiate both assays, such as different transcellular
transport.
ring system and P2 groups, but even a limited exploration with
compounds 8 and 9 suggest that further potency optimization
should be possible. These compounds had high polar surface area
(PSA), which may initially limit their oral absorption and bioavail-
ability. However, our results with a related series suggest that
relatively simple structural changes can bring these molecules
back into more drug-like parameters.20b
Selected compounds were evaluated in rats for their pharmaco-
kinetic parameters, blood samples from both jugular and portal
vein were drawn and drug concentrations were measured. The re-
sults are shown in Table 3. The oral absorption was calculated from
the portal vein drug concentrations and oral bioavailability was
calculated from jugular vein drug concentrations as compared to
the drug concentrations after IV administration. ITMN-191 exhibits
calculated 17.4% absorption and 20% oral bioavailability in rats.
References and notes
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Physicochemical properties and oral PK parameters of selected benzoxaborole-
macrocyclic inhibitors
Compds
MW
c log P
PSA
Oral PK in ratsa
5. (a) Peese, K. Drug Discovery Today 2010, 15, 406; (b) Chen, K. X.; Njoroge, F. G.
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% Absorption
% BA
20
0.5
0.7
<LOD
6. Seiwert, S. D.; Andrews, S. W.; Jiang, Y.; Serebryany, V.; Tan, H.; Kossen, K.;
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ITMN191
732
807
821
939
5.6
4.5
4.7
7.7
181
213
213
226
17.4
0.9
0.6
4
7
34
<LOD
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a
% Absorption was calculated from portal vein drug concentration, while % BA
was from jugular vein.

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Wickstrom, K.; Hamelink, E.; Edlund, M.; Vring, L.; Vendeville, S.; Van de
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18. 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
10. Rong, L.; Dahari, H.; Ribeiro, R. M.; Perelson, A. S. Sci. Transl. Med. 2010, 2, 30.
11. Selected references on benzoxaboroles and their properties (a) Rock, F. L.; Mao,
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V.; Akama, T.; Baker, S. J.; Plattner, J. J.; Shapiro, L.; Martinis, S. A.; Benkovic, S.
J.; Cusack, S.; Alley, M. R. K. Science 2007, 316, 1759; (b) Baker, S. J.; Zhang, Y.-
K.; Akama, T.; Lau, A.; Zhou, H.; Hernandez, V.; Mao, W.; Alley, M. R. K.;
Sanders, V.; Plattner, J. J. J. Med. Chem. 2006, 49, 4447; (c) Akama, T.; Baker, S. J.;
Zhang, Y.-K.; Hernandez, V.; Zhou, H.; Sanders, V.; Freund, Y.; Kimura, R.;
Maples, K. R.; Plattner, J. J. Bioorg. Med. Chem. Lett. 2009, 19, 2129.
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13. Description of synthesis of a representative compound 4: A solution of compound
1 (65 mg, 0.103 mmol) and Et3N (26 mg, 0.258 mmol) in 6 mL of THF was
cooled to À40 to À50 °C. Triphosgene (11.3 mg, 0.038 mmol) was added. The
reaction mixture was stirred at À40 °C for 1 h and 6-aminobenzo[c]-
[1,2]oxaborol-1(3H)-ol (11.3 mg, 0.103 mmol) was added. Then the reaction
mixture was stirred at À40 °C for another 1 h and allowed to warm to room
temperature for 24 h. The reaction mixture was added 1 N HCl (20 mL) and
extracted with CH2Cl2 (15 mL Â 3). The combined organic layers was washed
with brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by prep-HPLC to give the product as a white solid (25 mg,
30%). 1H NMR (300 MHz, CD3OD): d 8.20 (s, 1H), 7.40 (d, 1H), 7.15 (m, 1H), 7.18
(m, 1H), 7.05 (m, 1H), 6.75–7.00 (m, 2H), 6.71 (m, 1H), 5.72 (m, 1H), 5.55 (m,
1H), 5.03 (m, 1H), 4.93–5.03 (m, 1H), 4.45–4.77 (m, 6H), 4.36 (m, 1H), 3.90 (d,
1H), 3.60 (m, 1 H), 2.89–2.91 (m, 1H), 2.65 (m, 1H), 2.41–2.56 (m, 2H), 1.72–
2.00 (m, 3H), 0.95–1.58 (m, 16H). LC-MS: 807 (M+H)+. Purity on HPLC: 96.6%
(214 nm), 98.1% (254 nm).
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 initial rates. Compounds were also assayed for activity in the HCV
genotype 1a and 1b subgenomic (NS3-NS5B) replicon model systems. The
genotype 1b ET cell line is stably transfected with RNA transcripts harboring a
I389luc-ubi-neo/NS3-3’/ET replicon with firefly luciferase-ubiquitin-neomycin
phosphotransferase fusion protein and EMCV-IRES driven NS3-NS5B
polyprotein containing cell culture adaptive mutations. The genotype 1a
replicon is a stable cell line licensed from Apath LLC, modified to contain the
firefly luciferase gene. The cells were grown in DMEM supplemented with 10%
fetal calf serum (SAFC Biosciences, Lenexa, KS), 1Â GlutaMax-1, penicillin
(100 IU/mL)/streptomycin (100
500 g/mL geneticin (all from Life Technologies, Bethesda, MD). The cells were
plated at 5 Â 103 cells/well in 384-well plates containing compounds. The final
concentration of compounds ranged from 170 pM to 10 M, with a final DMSO
lg/mL), 1Â nonessential amino acids, and
l
l
concentration of 1%. Luciferase activity was measured after 48 h by adding
Steady-Glo (Promega, Madison, WI). Percent inhibition at each compound dose
was calculated relative to the no compound control. EC50s were determined
from an 11-point dose response curve and 3-fold serial dilutions for each
compound, using a standard four parameter logistic fit equation.
19. (a) Pietschmann, T.; Lohmann, V.; Kaul, A.; Krieger, N.; Rinck, G.; Rutter, G.;
Strand, D.; Bartenschlager, R. J. Virol. 2002, 76, 4008; (b) Lohmann, V.; Korner,
F.; Koch, J.-O.; Herian, U.; Theilman, L.; Batenschlager, R. Science 1999, 285, 110.
20. (a) Preliminary results of co-crystal structure of compound
4 with HCV
protease enzyme did not show specific ligand–protein interactions. Co-crystal
structures with other ligands were unknown; (b) Publication of the series is
pending.