Potent inhibitors of hepatitis C core dimerization as new leads for anti-hepatitis C agents

Article abstract of DOI:10.1016/j.bmcl.2011.03.014

New indoline alkaloid-type compounds which inhibit HCV production by infected hepatoma cells have been identified. These compounds, dimeric-type compounds of previously known inhibitors, display double digit nanomolar IC ₅₀ and EC₅₀

Full text of DOI:10.1016/j.bmcl.2011.03.014

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2198–2202
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent inhibitors of hepatitis C core dimerization as new leads
for anti-hepatitis C agents
Feng Ni a, Smitha Kota b, Virginia Takahashi b, A. Donny Strosberg b,c, , John K. Snyder a,
⇑
⇑
a Department of Chemistry and the Center for Chemical Methodology and Library Development (CMLD-BU), Boston University, 590 Commonwealth Ave,
Boston, MA 02215, United States
b The Scripps Research Institute, Department of Infectology, 130 Scripps Way #3C1, Jupiter, FL 33458, United States
c University of Paris-Denis-Diderot-P7, Institut Cochin, 22 rue Mechain, Paris 75014, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New indoline alkaloid-type compounds which inhibit HCV production by infected hepatoma cells have
been identified. These compounds, dimeric-type compounds of previously known inhibitors, display dou-
Received 9 January 2011
Revised 1 March 2011
Accepted 3 March 2011
ble digit nanomolar IC50 and EC50 values, with cytotoxicity CC50 indexes higher than 36
ing ample therapeutic windows for further development of HCV drugs.
lM, thus provid-
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus
Protein–protein interaction
Core protein
Inverse electron demand Diels–Alder
Dimeric inhibitors
Hepatitis C virus (HCV),1 which infects 130–170 million people
worldwide,2 is the main cause of liver disease in humans. This sin-
gle-strand, positive RNA virus encodes ten proteins3 all of which
are essential for viral infection and propagation. Currently, the only
treatment of HCV infection is a 48-week regimen, a combination of
interferon and ribavirin, that cures less than half the people in-
fected, depending on viral genotype and strain.4 Several of the viral
proteins have been targeted for drug development, with the viral
NS3 protease5 and NS5B polymerase6 key among them, and several
candidates have advanced significantly in the drug pipeline.7
Core, the HCV capsid protein, is the most conserved of the ten
viral proteins across all HCV genotypes.8 Core is responsible for
nucleocapsid formation, recruitment of HCV replicase proteins to
lipid droplets, and assembly of the viral particles in infected cells.
As such, core is an interesting target for HCV drug development
distinct from the HCV protease and polymerase enzymes, from
which resistant mutants have already emerged.9 With combination
therapies likely to evolve for HCV treatment,10 inhibitors of core
dimerization, and hence nucleocapsid formation, could play a key
role.
with other HCV proteins, including NS3 helicase.12 Screening of a
small molecule library led to the identification of 1 as an initial
hit for further elaboration (Fig. 1)13 An additional focused library
was prepared, which revealed three new core dimerization inhibi-
tors as racemates (2–4) with IC50’s in the single digit lM range. All
four inhibitors were also found to inhibit HCV production in in-
fected Huh-7.5 hepatoma cells; only 4 showed levels of cytotoxic-
ity comparable to activity against core dimerization. We now
report a successful search for more potent inhibitors of core
O
O
N
MeO2C
N
N
MeO
N
CO2Me
N
CO2Me
CO2Me
CO2Me
N
1
2
Me
OMe
O
N
O
N
OMe
N
N
OMe
OMe
CO2Me
CO2Me
We have been interested in the discovery of new, small mole-
cule inhibitors of core dimerization as lead structures for anti-
HCV agents. To this end, we have reported new assays to screen
for inhibitors of core dimerization11 and of interactions of core
N
N
CO2Me
CO2Me
3
4
⇑
Corresponding authors. Tel.: +1 617 353 2621; fax: +1 617 353 6466 (K.S.).
E-mail address: jsnyder@bu.edu (J.K. Snyder).
Figure 1. Previously reported inhibitors of core dimerization.11
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.014

F. Ni et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2198–2202
2199
protein dimerization based on the structures of 2–4, which are also
effective in blocking HCV proliferation in infected cells.
difficult to separate. Nonetheless, if such a mixture would show
improved activity over the original monomer, this would suggest
that attachment of the second heterocyclic subunit could be a via-
ble approach to preparing compounds with improved activity.
Attempts to dimerize 2 through metathesis gave disappoint-
ingly low yields unless 20 mol % of Grubbs II catalyst was em-
ployed (Scheme 1). Under optimal conditions, a 62% yield of
dimer 5 was obtained as a mixture of E- and Z-isomers of the race-
mate and the meso diastereomer, along with 6 (15%), resulting
from metathesis with the benzylidene ligand of the catalyst.
Due to the difficulty in purification, the poor yields in the subse-
quent non-chemoselective hydrogenation of the olefinic double
Since inhibition of core dimerization requires blockage of a pro-
tein–protein interaction (PPI)14 our initial approach to improve on
the activity of 1–4 was to prepare a dimer of the most active inhib-
itor 2 since dimerization of inhibitors of PPI’s have been shown to
be an effective strategy to improve activity.15–17 The hope was that
a second binding site or ‘hot spot’ might be found on the surface of
core for the second heterocycle to occupy that would magnify the
dimerization inhibition. It was also understood that since 2 was
racemic, dimerization of 2 would produce a mixture of enantio-
mers and as well as the meso diastereomer which would be likely
O
N
O
N
N
CO2Me
N
Grubbs II
N
CO2Me
CO2Me
N
5
CO2Me
MeO2C
MeO2C
N
2
N
N
O
O
N
N
Cat. (mol%) T (oC) Time (h) Solvent Yield 5
CO2Me
N
CO2Me
5
5
20
30
50
50
14
2
3
DCM
toluene
toluene
5%
7%
62%
6
Ph
Scheme 1. Dimerization of 2 through metathesis.
CO2Me
1) NaOH, MeOH↑↓
4 h (95%)
2) (COCl)2 (2.6 eq)
DMF (cat.), DCM,
rt, 1 h
CO2Me
N
O
i) NaH (1.5 eq), THF,
rt, 30 min
O
ii)
Br
Br
N
H
(0.6 eq), 50 oC, 38 h
3) 9 (1.6 eq), TEA (2.4 eq)
DMAP (cat.), DCM, rt
3 h (90%, 2 steps)
8
7
N
(60%)
MeO2C
NHBn
N
O
N
N
Bn
O
N
CO2Me
CO2Me
N
N
N
9
N
CO2Me
N
N
CO2Me
CO2Me
MeO2C
N
O
O
PhCl ↑↓, 8 h
11
(85%)
CO2Me
10
CO2Me
MeO2C
MeO2C
N
N
N
N
N
N
N
Bn
N
O
O
Scheme 2. Preparation of dimer 11 with an ether linkage between the monomeric subunits.

2200
F. Ni et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2198–2202
Table 1
Summary of bioactivities
CO2Me
CO2Me
a
b
c
d
Compd
IC50
T1-EC50
M)
T2-EC50
M)
CC50
M)
MW c log P
(l
M)
(l
(l
(l
i) NaH (1.5 eq), THF,
rt, 30 min
N
( )n
1e
9.3
1.4
2.0
2.0
14.8
2.3
4.9
22.2
3.2
0.7
3.0
2.5
0.735
29.9
1.1
56.2
0.071
>320
127.2
>320
5.3
>100
>36
513
485
465
575
960
930
958
702
653
––
4.7
6
4 steps
2e
ii)
N
H
3e
4.6
5.3
10.2
10.5
10.1
7.9
4.4
––
( )n
Br
Br
4e
3.8
(0.6 eq), 50 oC, 38 h
7
N
11
14
15
20
21
0.098
2.9
0.72
0.092
0.341
0.48
0.088
0.09
1.4
26.2
0.072
MeO2C
>36
>100
150
n = 1: 12 (56%)
n = 3: 13 (71%)
BILN2061f N/A
5.3
a
O
N
O
Values are means of three experiments.
Values are means of three experiments. T1 corresponds to initial 72 h culture of
b
N
cells in presence of inhibitors. See Refs. 11, 13.
c
N
N
Values are means of three experiments. T2 corresponds to second 72 h culture
of fresh cells, infection resulting from virus secreted in T1. See Refs. 11, 13.
CO2Me
CO2Me
d
Values are means of three experiments. 50% Cytotoxic concn versus Huh-7.5
N
N
N
CO2Me
CO2Me
hepatoma cells.
e
Data from Ref. 13.
14
15
f
Known inhibitor of the HCV NS3/NS4A protease20
.
(81%, 4 steps)
(83%, 4 steps)
MeO2C
MeO2C
MeO2C
N
conversion to the bis-acid chloride, then acylation of triazine 9 as
previously described in the preparation of monomer 213 all pro-
ceeded uneventfully, yielding dimer 10 (86% over three steps).
Double inverse electron demand Diels–Alder cycloadditions in
refluxing chlorobenzene produced the target dimer 11. Beginning
with the 3- and 5-carbon tethers, 1,3-dibromopropane and 1,5-
dibromopentane, dimers 14 and 15 were similarly prepared in
45% and 59% overall yields, respectively (five steps each beginning
with 7, Scheme 3). In each of the tethering reactions with the
respective dibromides, it was imperative to keep the number of
equivalents of dibromide relatively low (0.6 equiv) to avoid mono-
alkylations (vide supra). Dimers 11, 14, and 15 were also mixtures
of enantiomers and the meso diastereomer.
MeO2C
N
N
N
O
N
O
Scheme 3. Preparation of dimers 14 and 15 with all-carbon tethers linking the
monomeric subunits.
bond of 5, as well as the relatively poor yield of 5 without the use of
a large amount of catalyst, an alternative approach was explored.
Dimer 11 was ultimately prepared by first linking two indolyl-
propionate esters 7 with dibromoethyl ether, producing the sym-
metric dimer 8 (60%, Scheme 2). Basic hydrolysis of the esters,
CO2Me
1) LIOH 95 eq), rt
THF/H2O, 4 h
CO2Me
i) NaH (1.5 eq), THF,
rt, 30 min
2) (COCl)2 (2.6 eq)
N
DMF (cat.), DCM,
rt, 1 h
ii)
Br
Br
N
H
(1.6 eq), 50 oC, 14 h
3) 9 (1.6 eq), TEA (2.4 eq)
DMAP (cat.), DCM, rt
3 h (90%, 3 steps)
16
7
(80%)
Br
O
Bn
O
Bn
N
N
N
N
N
NaN3 (1.2 eq), DMF
N
PhCl ↑↓, 12 h
(99%)
50 oC, 10 h
CO2Me
CO2Me
MeO2C
N
(98%)
N
18
CO2Me
Br
17
Br
Me
O
Bn
N
O
N
20: R =
Bn
N
OMe
R
N
CO2Me
N
CO2Me
CO2Me
Cu (0.8 eq), CuSO4 (0.2 eq)
tBuOH/H2O/DCM (1:1:1)
80 oC, 10 min
N
CO2Me
21: R = -CH2NHAc
R
(both 98%)
N
19
N
N
N3
Scheme 4. Preparation of tethered bis-heterocycles 20 and 21 via click dipolar addition chemistry.

F. Ni et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2198–2202
2201
In the core dimerization assay, all three dimers were active,
with dimeric mixture 11 having an IC50 value of 98 nM, more than
an order of magnitude greater than that observed for the monomer
2 (Table 1). None of the dimers showed significant cytotoxicity
the meso diastereomer contamination (Scheme 4). To this end,
alkylation of 7 with 1,5-dibromopentane (1.6 equiv) produced
the monobromide 16 in 80% yield, with only a small amount of
tethered dimer 13 (9%). Conversion to the corresponding acid chlo-
ride and attachment of triazine 9 proceeded smoothly under stan-
dard conditions, as did the cycloaddition to give 18. No unwanted
chemistry of this primary alkyl bromide disrupted the strategy.
Azide displacement to 19, then Cu-catalyzed dipolar cycloaddi-
tion18 with appropriate alkynes produced racemic triazoles 20
and 21, with the cycloadditions both occurring in 98% yield.
Both 20 and 21 proved to be excellent inhibitors of core dimer-
ization, with sub-micromolar IC50’s (92 and 341 nM, respectively);
the activity of 20 was comparable to that of dimer 11. Equally
(CC50’s >36
infected with HCV 2a strain J6/JFH-1 were treated with increasing
concentrations (0.001–100 M) of the dimers to assess their effect
lM). Whole cell assays with Huh-7.5 hepatoma cells
l
on HCV propagation as previously described.11,13 The known NS3/
NS4A protease inhibitor BILN2061 was included as a positive con-
trol. The EC50’s were calculated at an early (T1) and late (T2) stage.
Sensitivity to the nature of the tether linking the dimers is readily
apparent, with 14 emerging as the best inhibitor (EC50 T1: 88 nM,
T2: 735 nM). Dimeric mixture 15 was also quite active in early
stage inhibition of HCV infectivity (T1 EC50 90 nM), but with a dra-
important, the cytotoxicities of 20 and 21 were in the high
range, and the whole cell activity of 20 remained in the single digit
lM region. The main drawback to be addressed is that the most
lM
matic loss of activity at late stage (T2 EC50 29.9 lM). The main
drawbacks with these dimers are their high MW and c log P values
(Table 1).
effective of these new tethered bis-heterocycles (20, Fig. 2) also
has the highest MW (702) and the highest c log P value (7.9).
In conclusion, several dimers of the previously reported core
dimerization inhibitor 2 have been prepared, and been shown to
be more effective core dimerization and HCV inhibitors than the
original lead compound, though with cellular activity that declines
with increasing incubation time, perhaps due to compound insta-
bility in the cellular assays. All compounds were stable upon stor-
age. Using click chemistry,19 two racemic tethered bis-heterocycles
(20 and 21) were also prepared and shown to be even more effec-
tive inhibitors of core dimerization as well as inhibitors of HCV
production in infected hepatoma cells. Further studies to (i) probe
the nature of the interaction of these inhibitors with core, (iii) re-
solve and screen the enantiomers of 20 and 21; and (iii) prepare
an additional focused library of analogues of 20 and 21 are under-
way. Completion of these studies should allow for a better SAR
understanding.
With the validation of tethering a second heterocyclic unit to
the basic active scaffold as an approach to enhanced activity
against core dimerization as well as inhibition of HCV production
in infected cells, two dimer ‘mimics’ were then prepared in an ef-
fort to discover inhibitors equally potent to 11 and 14, but without
Acknowledgments
The Scripps-Florida group thanks Professor Weissmann and Dr
T. Tellinghuisen (Department of Infectology, The Scripps Research
Institute-Florida) for helpful discussions and assistance with the
HCV studies, the State of Florida for start-up funds, and the NIH
(1X01MH085709-01 and 1R21NSO66411 (ADS) for grant support.
The Boston University group thanks Professors John Porco and Aar-
on Beeler for helpful discussions, and NIGMS CMLD initiative (P50
GM067041) for financial support. We (CMLD-BU) are also grateful
to the National Science Foundation for supporting the purchase of
the NMR (CHE 0619339) and HRMS (CHE 0443618) spectrometers
used in this work.
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