Synthesis and biological evaluation of new potent and selective HCV NS5A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.089

NS5A inhibitors are a new class of direct-acting antiviral agents which display very potent anti-HCV activity in vitro and in humans. Rationally designed modifications to the central biphenyl linkage of a known NS5A series led to selection of several comp

Full text of DOI:10.1016/j.bmcl.2012.03.089

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3488–3491
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of new potent and selective HCV
NS5A inhibitors
Junxing Shi a, Longhu Zhou b,c, Franck Amblard b,c, Drew R. Bobeck a, Hongwang Zhang b,c, Peng Liu b,c
,
Lavanya Bondada b,c, Tamara R. McBrayer a, Phillip M. Tharnish a, Tony Whitaker a, Steven J. Coats a,
Raymond F. Schinazi b,c,
⇑
a RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
b Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
c Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
NS5A inhibitors are a new class of direct-acting antiviral agents which display very potent anti-HCV
activity in vitro and in humans. Rationally designed modifications to the central biphenyl linkage of a
known NS5A series led to selection of several compounds that were synthesized and evaluated in a
HCV genotype 1b replicon. The straight triphenyl linked compound 11a showed similar anti-HCV activity
to the clinical compound BMS-790052 and a superior cytotoxicity profile in three different cell lines, with
an EC50 value of 26 pM and a therapeutic index of over four million in an HCV replicon assay. This tri-
phenyl analog warrants further preclinical evaluation as an anti-HCV agent.
Received 20 February 2012
Revised 16 March 2012
Accepted 22 March 2012
Available online 29 March 2012
Keywords:
HCV
NS5A inhibitor
Antiviral
Ó 2012 Published by Elsevier Ltd.
Hepatitis C virus (HCV) is an important pathogen affecting
nearly 170 million people worldwide.1 HCV infections become
chronic in about 50% of cases,2 and about 20% of these chronic per-
sons develop liver cirrhosis that can lead to hepatocellular carci-
pi electron interaction capabilities with the NS5A protein, and the
length of these molecules is quite different, leaving room for us to
modify and optimize these molecules. We hypothesized that the
two phenyl rings of BMS-790052 act only as a core linker between
the two substituted imidazolylpyrrolidine regions. Thus, we envis-
aged that changing the length and/or the geometry of this central
biphenyl linkage might lead to a more potent compound showing,
perhaps, less cytotoxicity. Therefore, a series of bis-imidazolylpyrr-
olidine compounds with phenyl, phenoxyphenyl, triphenyl, pyr-
idinyldiphenyl, triazole containing, and tetraphenyl linkages have
been synthesized and evaluated for their anti-HCV activity and
cytotoxicity in different cell lines.
noma.3 Current therapies, interferon-alpha (IFN-
a) and ribavirin,
even when used with the newly approved HCV protease inhibitors
Incivek and Victrelis, have limited efficacy and serious side-ef-
fects.4 Therefore, there is a need for more effective and safer small
molecule anti-HCV agents.
In our continuing efforts to identify more effective direct-acting
antiviral agents (DAA), a relatively new target, the non-structural
protein NS5A, has emerged as an attractive objective.5 Currently
in the clinic, the two most common targets for DAA with HCV
are the non-structural proteins NS3 and NS5B.6 Among the recently
discovered NS5A inhibitors, BMS-790052 showed a median effec-
tive antiviral concentration (EC50) in vitro, in the picomolar range
and demonstrated in clinical trials, a reduction in HCV RNA of over
3log10 IU/mL at 24 h following a single dose of 10 mg (Fig. 1).7
We carefully studied the SAR of some known NS5A inhibitors,7,8
including BMS-790052 and earlier hits BMS-858, BMS-824, and
BMS-665. For the BMS-790052 and BMS-665 series of compounds
we found that many of the compounds were symmetrical or al-
most symmetrical around a central core, this core had only/mainly
Phenyl, phenoxyphenyl, phenylthiopheneyl, and phenylben-
zenesulfonamide linked compounds 5a–d were prepared in four
steps by adapting a reported procedure9 as depicted in Scheme 1.
O
H
NH
O
N
MeO
N
N
N
N
N
H
OMe
O
HN
O
HCV 1b replicon EC50 = 9 pM
⇑
Corresponding author. Tel.: +1 404 728 7711; fax: +1 404 417 1535.
E-mail address: rschina@emory.edu (R.F. Schinazi).
Figure 1. Chemical structure of BMS-790052.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.03.089

J. Shi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3488–3491
3489
Boc
N
Boc
N
moacetophenone, 6a (Fig. 2) via esterification and cyclization,
analogous to the preparation of compounds 3 in Scheme 1. Suzuki
coupling of the boronate 8 with various dihalogenated arenes
(1,4-diiodobenzene, 2,5-dibromopyridine, 1,3-dibromobenzene,
2,5-dibromothiophene, or 4,40-dibromobiphenyl) resulted in the
formation of tricyclic and tetracyclic linked compounds 9a–e in
excellent yields. After Boc deprotection with HCl and coupling with
O
O
a
O O
O
O O
O
X
X
BrH2C
CH2Br
1
2
H
N
N
d
b
N
R
N
X
R
N-methoxycarbonyl-L-valine, compounds 11a–e were obtained in
N
N
H
fair overall yields (Scheme 2). Tricyclic analog 13 (Table 1) with
the pyridine ring shifted to the terminal core position relative to
11b was prepared by the sequence outlined in Scheme 2 utilizing
boronate 7d with pyridyl bromide 12 (Fig. 2) in place of the dibro-
mide starting at step b.11
As shown in Scheme 3, the triazolodiphenyl linked compound
17 was prepared from azido 7c and acetylene 14 by using Cu(I)-
catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition
(CuAAC).12 The iodo derivative 7b, prepared from the correspond-
ing bromoketone 6b (Fig. 2), was reacted with trimethylsilylacety-
lene under Sonogashira coupling conditions13 then desilylated
under basic conditions to give the acetylene derivative 14
(Scheme 3). The cycloaddition reaction of azido compound 7c (pre-
pared analogously to Scheme 1) and acetylene 14 resulted in the
formation of triazole intermediate 15 in 67% yield. After N-Boc
deprotection with 6 N HCl and coupling with N-methoxycar-
3 (R = Boc)
4 (R = H)
c
5a: X =
5b: X =
O
O
H
NH
O
N
MeO
N
N
NH
S
5c: X =
5d: X =
X
O
N
N
O
N
OMe
O
HN
H
5a-d
O
S
Scheme 1. Synthesis of 5a–d. Reagents and conditions: (a) N-Boc-
Et3N, rt, 2 h, 51–100%; (b) NH4OAc, toluene, 95–100 °C, 14 h, 51–73%; (c) 6 N HCl,
MeOH, 50 °C, 4 h, 85–99%; (d) HOBt, EDAC, N-(methoxycarbonyl)-L-valine, MeCN,
L-proline, MeCN,
DIPEA, rt, 14 h, 64–95%.
Di bromoketones 1a and 1c,d were prepared by bromination of the
corresponding diketone while 1b was prepared by Friedel–Crafts
acylation of the corresponding diphenylether with bromoacetyl
bonyl-L-valine, the triazolo-diphenyl compound 17 was obtained
in good overall yield (Scheme 3). The related triazole 18 (Table 1)
was prepared by Scheme 3 and started from the Boc’ed azide 7c
and involved Boc removal (70%) and acylation with N-(methoxy-
chloride. Reaction of 1a–d with N-Boc-L-proline to give the diesters
2a–d in good to excellent yield. The esters 2a–d were then refluxed
in toluene with ammonium acetate to form imidazoles 3a–d. Fol-
lowing Boc deprotection with 6 N HCl and coupling with N-
carbonyl)-L-valine (95%). A CuAAC reaction of 1,4-diethynylben-
zene with the resulting azide gave 18 in a 40% isolated yield.
All synthesized NS5A inhibitors were evaluated for inhibition of
HCV RNA replication in Huh7 cells containing a subgenomic HCV
1b replicon. Cytotoxicity in Huh7 cells was determined simulta-
neously with anti-HCV activity by extraction and amplification of
both HCV RNA and ribosomal RNA (rRNA).14 Cytotoxicity was
determined in peripheral blood mononuclear (PBM), CEM (a hu-
man-T-cell-derived cell line), and Vero (kidney epithelial cells from
the African green monkey) cells.15 The results are summarized in
Table 1.
(methoxycarbonyl)-L-valine in presence of N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride (EDAC), the target
compounds 5a–d were obtained in fair to excellent overall yields.
The tricyclic and tetracyclic linked analogs were prepared as de-
scribed in Scheme 2. The key intermediate boronate 8 was pre-
pared from the bromo derivative 7a by a palladium catalyzed
cross-coupling reaction with bis(pinacolato)diboron,10 while the
bromide 7a was prepared from commercially available 2,40-dibro-
First, it is noteworthy that compound 5a which possess only
one phenyl ring as a linker showed a complete loss of cytotoxicity,
but also significantly less potency against HCV replication with a
EC50 >1 nM. This suggested that the length of the linker could have
a marked effect on both anti-HCV activity as well as cytotoxicity.
Furthermore, increase of the linker’s size to three straight aromatic
rings (compounds 11a and 11b) did not severely impact the anti-
HCV activity, while for compound 11a, no cytotoxicity was ob-
served up to 100 lM in PBM, CEM and Vero cells. It is interesting
that 13 which has the pyridine ring shifted to the outside aryl ring
of the core has similar cyctotoxicity but 10-fold less anti-HCV
activity when compared to its similar pyridyl analog 11b. However,
the four straight aromatic ring (11e) seems to be problematic with
a 37-fold loss of activity at the EC50 level when compared to the
biphenyl derivative BMS-790052.
Introduction of torsion angle by incorporation of an oxygen
atom (5b), a sulfonamide (5c), a 1,3-substituted phenyl (11c), a
1,3-triazole (17) or a thiophene ring (11d) between the two phenyl
ring of the linker led to a substantial decrease of activity (16- to
15,000-fold). With a 2-phenylthiophene core (5d) a torsion angle
was also introduced and accompanied by an almost complete loss
of anti-HCV activity. Compound 18 which contains five aromatic
rings in its core was less potent but gave a similar profile to its
shorter triazole analog 17 but with significantly reduced
cytotoxicity.
Scheme 2. Synthesis of 11a–e. Reagents and conditions: (a) Pd(PPh3)4, bis(pina-
colato) diboron, KOAc, 1,4-dioxane, 80 °C, 16 h, 84%; (b) Pd(PPh3)4, NaHCO3,
1,2-dimethoxyethane, H2O, dihalide (1,4-diiodobenzene, 2,5-dibromopyridine,
1,3-dibromobenzene, 2,5-dibromothiophene, or 4,40-diiodobiphenyl), 80 °C, 14 h,
80–99%; (c) 6 N HCl, MeOH, 50 °C, 4 h, 86–95%; (d) HOBt, EDAC, N-(methoxycar-
From this SAR study we can conclude that: (i) the length of the
straight central core linkage is optimal at 2–3 phenyl or pyridyl
bonyl)-L-valine, MeCN, DIPEA, rt, 14 h, 28–59%.

3490
J. Shi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3488–3491
O
H
H
N
Br
N
N
Boc
N
X
Br
Boc
N
N
X
N
6a-c
7a-d
12
O
B
a: X = Br; b: X = I; c: X = N3; 7d: X =
O
Figure 2. Chemical structures for 6a–c, 7a–d and 12.
Table 1
In vitro anti-HCV activity and cytotoxicity data for compounds 5a–d, 11a–e, 13, 17 and 18
Compd
X
Anti-HCVa (pM)
EC90
Cytotoxicity CC50
CEM
(lM)
EC50
>1000
PBM
>100
Vero
>100
5a
5b
>1000
>100
16
30,000
90,000
16
15
19
H
N
O
S
5c
O
100,000
>100,000
13
48
>100
S
5d
90,600
>100,000
93
99
11a
11b
26
26
90
68
>100
>100
2.3
>100
3.9
6.5
11c
11d
800
100
2900
300
18
3.0
12
>100
>100
26
11e
13
240
200
1000
700
>100
>100
31
>100
29
5.5
N
17
200
600
900
18
5.0
35
N
N
N
18
1633
23
46
19
>100
9.6
>100
21
N
N
N
BMS-790052
6.6
a
All dose–response determinations were calculated from at least four concentrations. All concentrations were run in triplicate and reported as mean values.

J. Shi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3488–3491
3491
pounds 11a and 11b retain potent anti-HCV activity in the low
picomolar range and, in the case of compound 11a, have signifi-
cantly less cytotoxicity in vitro versus BMS-790052, with a thera-
peutic index CC50/EC50 over four million. Thus, this compound
warrants further preclinical investigation as a potential anti-HCV
agent.
H
N
a
H
N
N
Boc
N
Boc
I
N
N
14
7b
H
N
N
Acknowledgments
N
N
d
N
R
b
R
N
N
We thank Melissa D. Johns for excellent technical assistance.
R.F.S. is supported by CFAR NIH Grant 2P30-AI-050409 and by
the Department of Veterans Affairs. R.F.S. is a founder and major
shareholder of RFS Pharma, LLC. All other authors have no compet-
ing interests.
H
N
N
15 (R = Boc)
16 (R = H)
c
N
N
N
References and notes
H
N
O
N
O
NH
N
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rings (11a, 11b and BMS-790052), shorter (5a) or longer (11e) than
this range caused markedly reduced antiviral activity; (ii) for the
three aryl core linkage, pyridyl is tolerated as the middle aryl ring
(11b) while a ten fold loss of anti-HCV activity is observed with
pyridyl at the outer aryl position (13); (iii) straight linked com-
pounds (11a, 11b and BMS-790052) have a superior anti-HCV ef-
fect relative to the non-linear linked compounds (5b–d, 11c, 11d,
17 and 18); and (iv) for the three aryl core linkage, the linkage
composed completely of phenyl rings rather than containing a pyr-
idyl ring results in an improved cytotoxicity profile (11a vs 11b
and 13).
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Tharnish, P.; Choi, Y.; Chong, Y.; Choo, H.; Chu, C. K.; Otto, M. J.; Schinazi, R. F.
Antimicrob. Agents Chemother. 2002, 46, 3854.
In summary, 12 HCV inhibitors with different lengths and an-
gles at the central aromatic linkage were synthesized and evalu-
ated for anti-HCV activity and cytotoxicity in cell based assays.
This study revealed that the straight tricyclic aromatic linked com-