Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel P4-benzoxaborole moieties

Article abstract of DOI:10.1016/j.bmcl.2011.02.006

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure-activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.

Full text of DOI:10.1016/j.bmcl.2011.02.006

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2048–2054
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel
P4-benzoxaborole moieties
Xianfeng Li a, , Suoming Zhang a, Yong-Kang Zhang a, Yang Liu a, Charles Z. Ding a, Yasheen Zhou a,
⇑
b
Jacob J. Plattner a, Stephen J. Baker a, Wei Bu a, Liang Liu a, Wieslaw M. Kazmierski b, , Maosheng Duan ,
Richard M. Grimes b, Lois L. Wright b, Gary K. Smith b, Richard L. Jarvest c, 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:
We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease
inhibitors. Structure–activity relationships were investigated, leading to the identification of compounds
5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-trun-
cated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting
that further reduction of molecular weight and polar surface area could result in improved drug-like
properties of this novel series.
Received 12 January 2011
Revised 1 February 2011
Accepted 2 February 2011
Available online 23 February 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Synthesis and SAR
Structure–activity relationships
Benzoxaborole
HCV NS3 protease
Acyclic inhibitors
Hepatitis C virus (HCV) infection is the principal cause of
chronic liver disease that can lead to cirrhosis, carcinoma and liver
failure.1 More than 200 million people worldwide are chronically
infected by this virus. Currently, the most effective treatment for
HCV infection is based on a combination therapy of injectable
HCV NS3 serine protease plays a critical role in the HCV replica-
tion by cleaving downstream sites (with the assistance of the
cofactor NS4A) along the HCV viral polyprotein to produce func-
tional proteins. Recently, NS3/4A protease inhibitors have emerged
as a promising treatment for HCV infection.5 There are two distinct
classes of NS3 protease inhibitors in clinical development. The first
class is comprised of serine-trap inhibitors, exemplified by VX-950
(telaprevir)6 and SCH-503034 (boceprevir).7 The second class is
represented by reversible noncovalent inhibitors such as macrocy-
clic inhibitors BILN-2061 (ciluprevir),8 ITMN-191 (danoprevir),9
TMC-43535010 and MK-7009 (vaniprevir).11 Due to concern over
cardiac issues in animals treated with macrocyclic BILN-2061,12
newer acyclic inhibitors have recently been developed exemplified
by BI-20133513 and BMS-650032.14 However, a rapid development
of viral resistance has been observed for patients treated with HCV
NS3 protease inhibitors.15 Therefore, the discovery of new NS3 pro-
tease inhibitors with novel binding paradigm and thus potentially
differentiated resistance profile is highly desirable.
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 addition, this treatment regimen
cures only less than 50% of patients infected with genotype-1
which is the predominant genotype (while genotype 1a is most
abundant in the US, the majority of sequences in Europe and Japan
are from genotype 1b).3 Limited efficacy and adverse side effects of
current treatment, and high prevalence of infection worldwide
highlight an urgent need for more effective, convenient, and
well-tolerated treatments.4
As part of our ongoing efforts to discover new HCV NS3 protease
inhibitors,16–19 we have recently identified a novel series of P4-
benzoxaborole based macrocyclic NS3 protease inhibitors as exem-
plified by compound 1 (Fig. 1).19 These macrocyclic inhibitors are
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.006

X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
2049
N
F
O
N
O
S
O
O
N
O
O
O
O
S
O
H
N
O
O
NH
H
N
N
S
O
O
N
O
H
N
O
N
H
O
ITMN-191
(danoprevir)
TMC-435350
P2*
O
O
N
Cl
P1'
N
O
Cl
O
O
P1
O
S
O
O
P4
P3
O
H
N
NH
O
S
N
O
O
H
NH
N
P2
N
O
O
O
O
B
N
H
N
H
O
HO
N
H
O
P4 benzoxaborole-based
macrocyclic inhibitor 1
BMS-650032
Figure 1. Selected known NS3 serine protease inhibitors.
very potent in the HCV NS3 protease and cell-based replicon as-
says. However, these macrocyclic compounds had high molecular
weight (MW) and polar surface area (PSA), which may limit their
oral absorption and bioavailability. In parallel, we also pursued
an acyclic series with the plan to potentially, if needed, rebalance
their physicochemical properties (MW and PSA) in order to im-
prove their in vivo absorption and bioavailability. In this Letter,
we detail the SAR in this acyclic series by exploring various linkers
and substitutions around the benzoxaborole moiety as well as the
P2⁄ groups.
Benzoxaboroles 2a–d and 2g were prepared as described in our
previous publication.19 Benzoxaboroles 2e–f and 2h–i were pre-
pared according to procedures described in Supplementary data.
Our initial medicinal chemistry efforts examined the acyclic
series with P2⁄ isoquinoline group, which was successfully demon-
strated by Bristol-Myers Squibb14 and our group19 to support high
potency of NS3/4A inhibitors in two different series. Introduction
of P4-benzoxaborole groups in this P2⁄ isoquinoline based acyclic
series to explore SAR was carried out according to Scheme 1. Cou-
pling of amine 320 with Boc-protected
L-tert-leucine and subse-
Figure 2 lists functionalized benzoxaboroles 2a–i that were
used to make P4-benzoxaborole acyclic inhibitors 5a–i, Table 1.
quent Boc removal afforded amine 4. The carbamate-linked
compound (5a) was prepared by reaction of 4 with benzoxaborole
2a in the presence of CDI. The urea-linked compounds (5b, 5d–i)
were prepared via the isocyanide reaction with amino benzoxabo-
roles (2b, 2d–i), as described previously.19 Amide 5c was prepared
by HATU/DIEA—mediated coupling of 4 with benzoxaborole car-
boxylic acid 2c.
A shorter amine-based linker-truncated compound 5j was syn-
thesized according to Scheme 2. Reductive amination of 3,3-di-
methyl-2-oxobutanoic acid 6 with 2b gave the benzoxaborole-
substituted amino acid 7. Coupling of 7 with amine 3 in presence
of HATU and DIEA afforded diastereomers, subsequently separated
by the prep-HPLC. The active diastereomer in the enzyme assay
was assumed to be 5j, although the absolute stereochemistry has
not been determined.
The inhibitory activity of the P4-benzoxaborole based inhibitors
were evaluated by FRET NS3/4A 1a protease domain assays.21a,b
Cellular activity was determined using 1a and 1b HCV replicon as-
say.22 Results are reported in Table 1.
Compound 5a with a carbamate linker exhibited nanomolar
potency against NS3 1a enzyme with IC50 value of 1.3 nM. It dis-
played cellular activity in the replicon 1a and 1b assay with EC50
O
HO
HO
HO
OH
NH2
B
B
B
OH
NH2
NH2
O
O
O
2a
2b
OH
2c
O
B
HO
O
B
HO
HO
B
NH2
NH2
O
2d
2g
2e
2f
2i
HO
HO
B
NH2
F
B
NH2
Cl
B
O
O
O
2h
Figure 2. Functionalized benzoxaboroles prepared.

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X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
Table 1
In vitro activity of acyclic inhibitors 5a–j with various linkers and substitutions
O
N
Cl
O
O
O
O
H
N
S
N
H
N
H
O
R
N
O
5a-j
a,b
c
Compd
R group
NS3/4A 1a IC50 (nM)
Replicon EC50 (nM)
1a
60
1b
3.9
1
0.6a
1.3a
HO
O
B
5a
170
160
23
O
O
O
HO
H
N
2.2a
4.6b
2.0b
24
91
25
B
5b
5c
5d
O
O
HO
B
290
130
O
O
HO
B
N
H
O
OH
O
B
H
N
5e
16b
65
38
81
O
O
B
H
N
4.0b
1.7a
500
31
HO
HO
5f
O
O
H
N
B
5g
4.3
O
F
HO
H
N
B
5h
1.3b
47
34
O
O
O
Cl
HO
H
N
B
5i
26b
280
110
130
34
O
HO
B
5jd
4.5b
O
a
b
c
FRET assay as described in Ref. 21a.
FRET assay as described in Ref. 21b.
Replicon assay as described in Ref. 22.
d
The active diastereomer, and the absolute stereochemistry not determined. The other diastereomer showed IC50 of 350 nM in the same enzyme assay.

X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
2051
O
O
O
N
Cl
N
O
O
O
a,b
O
Cl
H
N
S
O
O
O
N
H
H
N
S
N
N
O
H
H2N
N
O
H
O
3
4
e
c
d
5c
5b, 5d-i
L-tert-leucine, HATU, DIEA, DCM, overnight, 70%; (b) 4 N HCl, dioxane, 100%; (c) 2a, CDI, DCM, DMF, rt, 48 h, 19%; (d) triphosgene,
5a
Scheme 1. Reagents and conditions: (a) Boc-
TEA, THF, rt, 4 h; then 2b, 2d–i, rt, overnight, 10–50%; (e) 2c, HATU, DIEA, anhydrous DMF, rt, overnight, 41%.
O
N
OH
Cl
OH
HO
H
N
O
O
S
O
O
b
O
a
B
O
O
H
N
O
N
H
N
HO
H
N
O
6
7
B
O
O
5j,
S-isomer
Scheme 2. Reagents and conditions: (a) 2b, acetic acid, 4 h, then NaBH(OAc)3, overnight, 13%; (b) 3, HATU, DIEA, DCM, overnight, 19%.
values of 170 and 23 nM, respectively. Replacement of the carba-
mate linker in 5a with urea in 5b resulted in similar enzyme
(IC50 = 2.2 nM) and replicon potency (replicon 1a, EC50 = 160 nM;
replicon 1b, EC50 = 24 nM). Extending the urea linker in 5d re-
tained potency. On the other hand, replacing the urea linker with
an amide in 5c was detrimental to potency in both enzymatic
and replicon assay. Compounds 5e and 5f are regioisomers of
5b. Isomer 5f was found to be less active than 5b in the enzyme
and replicon assay. Interestingly, while 5e was 8-fold less potent
than 5b in the enzyme assay, it was about equipotent to 5b in
the replicon assay. We hypothesized that the ability of the ben-
zoxaborole BOH group to form an intramolecular H-bonding with
the amide hydrogen may have resulted in improved cell perme-
ability23 and thus replicon potency. The differential activity of
various P4-benzoxaborole regioisomers contrasts the results ob-
served in our P10-benzoxaborole series,18 where very little differ-
ence in potency is observed within acylsulfamoyl benzoxaborole
regioisomers. This result underlines special structural role played
by the P4-benzoxaborole moiety. A limited exploration of substi-
tution around the benzoxaborole ring was also conducted. The
most effective substitution was para-F atom in the phenyl ring,
which improved replicon potency fivefold in compound 5g (1a,
EC50 = 31 nM; 1b, EC50 = 4.3 nM). These results were very encour-
aging considering that the fluoro-containing inhibitor 5g was
equipotent to the macrocyclic compound 1 in the replicon assay.
Gem-dimethyl benzoxaborole ring substitution in 5i was detri-
mental to potency, potentially due to disfavorable interactions
with the enzyme. Finally, compound 5j, which connects P3 moi-
ety directly to the benzoxaborole ring was found about equipo-
tent to other linker-containing unsubstituted benzoxaborole
compounds, such as 5a, 5b and 5d.
Encouraged by benefits of the fluoro substituent in 5g, we
decided to explore the impact of other P2⁄ groups on the activity
of the fluoro-containing acyclic series (Table 2). Compound 12 with
P2⁄-isoindoline group and 17 with P2⁄-quinoline group were pre-
pared according to Schemes 3 and 4, respectively.
As shown in Scheme 3, acid 8 with P2⁄-isoindoline group was
prepared using our previously published procedure.24 Subsequent
reaction of acid 8 with (1R,2S)-ethyl-1-amino-2-vinylcyclopropan-
ecarboxylate 9, followed by hydrolysis afforded acid 10. Reaction of
10 with cyclopropanesulfonamide, followed by Boc removal gave
amine 11. The urea-linked P4-benzoxaborole compound 12 was
prepared via the isocyanide reaction with amino benzoxaborole
2g, as described.19
P2⁄-quinoline-based compound 17 was synthesized as de-
scribed in Scheme 4. To that end, acid 13 was prepared according
to a literature procedure.25 Reaction of 13 with (1R,2S)-1-amino-
N-(cyclopropylsulfonyl)-2-vinylcyclopropane carboxamide 14 fol-
lowed by Boc removal afforded amine 15. Coupling of amine 15
with Boc-protected L-tert-leucine and subsequent Boc removal
gave amine 16. Subsequently, urea-linked compound 17 was pre-
pared via the isocyanide reaction with amino benzoxaborole 2g,
as described.19
The P2⁄-isoindoline-based compound 12 was equipotent to 5b
in the enzyme assay, but was 10-fold less potent in the replicon as-
say. This observation is consistent with the result obtained from
our previous macrocyclic series.19 Compound 17, on the other
hand, exhibited 3–4-fold improvement in replicon 1a assay.

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X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
Table 2
In vitro activity of acyclic inhibitors with different P2⁄ groups
P2*
O
O
O
O
H
N
S
N
H
N
HO
H
N
H
N
O
B
O
O
O
F
P2⁄ group
c Log P
NS3/4A 1a IC50 (nM)
Replicon EC50 (nM)
1b
a,b
c
Compd
1a
F
12
3.88
1.1a
360
56
N
O
O
5g
17
5.22
6.85
1.7a
31
4.3
N
Cl
N
3.1b
8.1
8.6
O
N
S
a
b
c
FRET assay as described in Ref. 21a.
FRET assay as described in Ref. 21b.
Replicon assay as described in Ref. 22.
P2*
O
P2*
O
O
O
H
N
OH
OH
a,b
OEt
H2N
N
N
H
+
H
O
O
O
N
O
N
O
O
O
O
8
9
10
F
c,d
*
P2*
O
P2
=
O
O
O
N
O
H
N
S
e
N
H
12
N
O
H2N
O
11
Scheme 3. Reagents and conditions: (a) EDC, HOBt, NMM, rt, overnight, 84%; (b) LiOH, THF, water, rt, 48 h, 90%; (c) cyclopropanesulfonamide, HATU, DIEA, DMAP, DBU, DMF,
rt, overnight, 85%; (d) TFA, DCM, rt, 2 h, 85%; (e) triphosgene, TEA, THF, ꢀ40 °C, 1 h; then 2g, rt, 24 h, 30%.
Despite a limited set of compounds, we note an apparent correla-
tion between the cell-based potency (especially replicon 1a) and
c Log P values of these compounds, suggesting that increased lipo-
philicity might improve compound’s cell permeability.
We previously reported that the benzoxaborole-bearing macro-
cyclic HCV protease inhibitors showed very low oral exposure in
rats.18,19 Subsequent portal vein sampling and analysis suggested
that poor oral bioavailability was primarily caused by low com-

X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
2053
P2*
O
P2*
O
O
O
O
O
O
O
S
H
N
S
OH
N
H2N
N
a,b
H
H
N
+
N
H
O
O
O
O
13
14
15
c,d
*
N
P2
=
P2*
O
O
N
S
O
O
O
H
N
S
e
N
H
17
N
O
H2N
O
16
Scheme 4. Reagents and conditions: (a) EDC, HOBt, NMM, rt, overnight, 74%; (b) 4 N HCl, dioxane, overnight, quant.; (c) Boc-
(d) 4 N HCl, dioxane, overnight, quant.; (e) triphosgene, TEA, THF, 4 h; then 2g, rt, 24 h, 44%.
L-tert-leucine, HATU, DIEA, DCM, overnight, 70%;
pound absorption, possibly reflecting compounds’ unfavorable
physicochemical properties, such as high MW and PSA. The acyclic
inhibitors described herein have somewhat reduced molecular
weight, but have PSA values similar to their corresponding macro-
cyclic counterparts. Consequently, many inhibitors in this acyclic
benzoxaborole series still exhibited low oral exposure in rats, as
exemplified by 5b, Table 3. However, compared to 5b, the linker
truncated compound 5j exhibited improved absorption (4.9%)
and oral bioavailability (4.3%) in rats, potentially due to its more
drug-like properties, such as reduced MW (by 43) and PSA (by
29). This result was further corroborated by finding out that the
permeability of 5j in the parallel artificial membrane permeability
assay (PAMPA) was 10-fold greater than 5b. Therefore, these re-
sults suggest that further reduction of MW and PSA in benzoxabo-
role-based compounds may be an effective strategy towards drug-
like boron-containing HCV protease inhibitors.
In conclusion, we synthesized and evaluated a new series of
acyclic P4-benzoxaboroles-based NS3 protease inhibitors. Com-
pounds in this series exhibited nanomolar potencies in enzymatic
and cell-based replicon assays. The fluoro-containing acyclic inhib-
itor 5g is characterized by cell-based potency comparable to the
macrocyclic compound 1. Furthermore, compared to 5g, com-
pound 17 exhibited further 3–4-fold improved potency in replicon
1a assay. While most compounds in this class had low oral avail-
Table 3
Physicochemical properties and PK parameters of selected inhibitors in male Sprague–Dawley ratsa
O
O
O
N
N
Cl
Cl
O
O
O
O O
O
O
H
N
S
H
N
S
N
N
H
H
N
N
HO
HO
H
N
H
N
H
N
O
O
B
B
O
O
O
O
O
5b
5j
Parameter
Compd
5b
5j
MW
c log P
823
5.2
780
5.6
m log D
PSA
PAMPA
CL (mL/h/kg), iv
4.5
214
7.2
476
0.041
0.92
0.42
4.0
185
73
2927
0.075
4.9
AUC0-inf (h lg/mL), po
% Absorptionb
%Fc
4.3
a
b
c
Compounds were dosed orally at a dose of 5 mg/kg (n = 3) and intravenously at a dose of 1 mg/kg (n = 3).
Calculated from portal vein drug concentrations after oral administration as compared to that after IV administration.
Calculated from jugular vein drug concentrations after oral administration as compared to that after IV administration.

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X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2048–2054
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Acknowledgments
16. Li, X.; Zhang, Y.-K.; Liu, Y.; Ding, C. Z.; Li, Q.; Zhou, Y.; Plattner, J. J.; Baker, S. J.;
Qian, X.; Fan, D.; Liao, L.; Ni, Z.-J.; White, G. V.; Mordaunt, J. E.; Lazarides, L. X.;
Slater, M. J.; Jarvest, R. L.; Thommes, P.; Ellis, M.; Edge, C. M.; Hubbard, J. A.;
Somers, D.; Rowland, P.; Nassau, P.; McDowell, B.; Skarzynski, T. J.; Kazmierski,
W. M.; Grimes, R. M.; Wright, L. L.; Smith, G. K.; Zou, W.; Wright, J.; Pennicott,
L. E. Bioorg. Med. Chem. Lett. 2010, 20, 3550.
17. Li, X.; Zhang, Y.-K.; Liu, Y.; Ding, C. Z.; Zhou, Y.; Li, Q.; Plattner, J. J.; Baker, S. J.;
Zhang, S.; Kazmierski, W. M.; Wright, L. L.; Smith, G. K.; Grimes, R. M.; Crosby,
R. M.; Creech, K. L.; Carballo, L. H.; Slater, M. J.; Jarvest, R. L.; Thommes, P.;
Hubbard, J. A.; Convery, M. A.; Nassau, P. M.; McDowell, W.; Skarzynski, T. J.;
Qian, X.; Fan, D.; Liao, L.; Ni, Z.-J.; Pennicott, L. E.; Zou, W.; Wright, J. Bioorg.
Med. Chem. Lett. 2010, 20, 5695.
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
intermediates.
Supplementary data
18. Li, X.; Zhang, Y.-K.; Liu, Y.; Zhang, S.; Ding, C. Z.; Zhou, Y.; Plattner, J. J.; Baker, S.
J.; Liu, L.; Bu, W.; Kazmierski, W. M.; Wright, L. L.; Smith, G. K.; Jarvest, R. L.;
Duan, M.; Ji, J.-J.; Cooper, J. P.; Tallant, M. D.; Crosby, R. M.; Creech, K.; Ni, Z.-J.;
Zou, W.; Wright, J. Bioorg. Med. Chem. Lett. 2010, 20, 7493.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.006.
19. Ding, C. Z.; Zhang, Y.-K.; Li, X.; Liu, Y.; Zhang, S.; Zhou, Y.; Plattner, J. J.; Baker, S.
J.; Liu, L.; Duan, M.; Jarvest, R. L.; Ji, J.; Kazmierski, W. M.; Tallant, M. D.; Wright,
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lM
NS4A, 0.5 peptide substrate (Ac-DE-Dap(QXL520)-EE-Abu- -[COO]-AS-
lM
w
C(5-FAMsp)-NH2 is the FRET substrate purchased from Anaspec Inc. San Jose,
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of 490 ex and 520 em were used on a Molecular Devices plate reader to
measure initial rates; (b) Compounds were assayed in the fluorescence
enzymatic assay using HCV NS3/4A 1a protease domain. Conditions: 1 nM
enzyme (1a domain), 2
lM NS4A, 5 lM peptide substrate (Ac-DE-D(EDANS)-
EE-Abu- -[COO]-AS-K(DABCYL)-NH2 is the FRET substrate purchased from
w
Anaspec Inc. San Jose, CA) in 50 mM HEPES, 20% sucrose, 5 mM DTT, and 0.05%
NP-40. Wavelengths of 355 ex and 495 em were used on a Molecular Devices
plate reader to measure initial rates.
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