New small molecule inhibitors of hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2009.10.070

New small molecule inhibitors of HCV were discovered by screening a small library of indoline alkaloid-type compounds. An automated assay format was employed which allowed identification of dimerization inhibitors of core, the capsid protein of the virus. These compounds were subsequently shown to block production of infectious virus in hepatoma cells.

Full text of DOI:10.1016/j.bmcl.2009.10.070

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6926–6930
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New small molecule inhibitors of hepatitis C virus
Wanguo Wei a, Cuifang Cai a, Smitha Kota b, Virginia Takahashi b, Feng Ni a, A. Donny Strosberg b,
,
*
John K. Snyder a,
*
a Department of Chemistry and the Center for Chemical Methodology for 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
a r t i c l e i n f o
a b s t r a c t
Article history:
New small molecule inhibitors of HCV were discovered by screening a small library of indoline alkaloid-
type compounds. An automated assay format was employed which allowed identification of dimerization
inhibitors of core, the capsid protein of the virus. These compounds were subsequently shown to block
production of infectious virus in hepatoma cells.
Received 22 September 2009
Revised 14 October 2009
Accepted 15 October 2009
Available online 21 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus
Core protein
Inverse electron demand Diels–Alder
reaction
Indoline
Protein–protein interaction
Hepatitis C, the leading cause of liver disease, affects over 170
million people worldwide.1 Over three million Americans are in-
fected by the HCV virus,2 which is particularly lethal for AIDS pa-
tients, of whom increasing numbers are co-infected with HIV and
HCV.3 There is no vaccine, and the only available treatment, based
on a combination of interferon and ribavirin, cures less than half of
the patients.4 Until recently, the main focus of efforts to develop
HCV inhibitors has been on the viral NS3 protease and NS5B poly-
merase enzymes, but escape mutants have already been reported.5
The failure to find other new anti-HCV drugs is mainly linked to the
dearth of novel targets. In the virus, the core protein is essential for
nucleocapsid formation and release of the viral particle from the
infected cell. Although HCV is extremely susceptible to mutations,
core is the most conserved of all 10 viral proteins, and thus repre-
sents an excellent, hitherto-unexplored target for novel anti-HCV
drug development.
We recently reported new bioassays based on the inhibition of
core dimerization, and hence the inhibition of nucleocapsid forma-
tion, with the goal of discovering small molecule inhibitors of this
protein–protein interaction.6 The effectiveness of core fragments
and other peptides in inhibiting core dimerization and viral pro-
duction was also demonstrated, thereby validating the assays as
an approach for the discovery of leads that inhibit hepatitis C.7
We now report the identification of four, small molecule inhibitors
of core dimerization revealed through the screening of the CMLD-
BU small molecule libraries.
As part of our effort to create diverse, small molecule libraries
rich in stereochemistry, a library of indoline alkaloid-type com-
pounds was prepared using inverse electron demand Diels–Alder
chemistry. The initial library (7, 132 members) originated from
the intramolecular cycloaddition of tryptophan derivatives8 with
tethered 1,2,4-triazines following protocols already reported by
our group (Scheme 1).9
The cycloaddition precursors 3 were prepared by SNAr displace-
ments following BOC-deprotection of L-tryptophan derivatives 1
on triazine 2.10 In situ trifluoroacetylation of the nitrogen linker,
accomplished by treatment with trifluoroacetic anhydride, lowers
the LUMO of the heteroaromatic azadiene and thereby enables
the cycloaddition to proceed immediately after acylation under
the reaction conditions without isolation of intermediate 4. Fol-
lowing deacylation upon chromatography on silica gel, the cyc-
loadducts 6, which resemble truncated Aspidosperma-type
alkaloids, were obtained in good to excellent yields. The two newly
generated two stereogenic centers at C1 and C9 were formed with
complete stereoselectivity controlled by chirality of the tryptophan
dienophile, as previously noted.9 The relative stereochemistry of
the cycloadducts was confirmed by NOE studies as previously de-
scribed.9 The overall yields of scaffolds 6 ranged from 42% to 64%
(three steps) on a 2 g scale (Chart 1). Diversification of scaffolds 6
then proceeded by acylation (17 commercially available acid
chlorides) and sulfonylation (10 commercially available sulfonyl
* Corresponding authors. Tel.: +1 617 353 2621; fax: +1 617 353 6466 (J.K.S.).
E-mail address: jsnyder@bu.edu (J.K. Snyder).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.070

W. Wei et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6926–6930
CO2Me
6927
TFAA,i-Pr2NEt
1,2-Cl2Ph, 120 ºC
2 h
CO2Me
SMe
N
NH
R2
1) TFA
NHBOC
R2
N
N
N
N
N
+
2) MeOH ↑↓,
i-Pr2NEt,
12 h
N
CO2Me
R1
CO2Me
N
3
R1
CO2Me
CO2Me
2
1
O
N
MeO2C
N
O
N
CF3
MeO2C
R2
R2
CF3
SiO2
N
N
(-N2)
N
CO2Me
N
CO2Me
R1
N
4
CO2Me
R1
5
CO2Me
O
R3
N
X
MeO2C
R2
R3COCl,
PS-DMAP
or
N
H
MeO2C
14
N
R2
N
1
CO2Me
CO2Me
7: X = C, SO
i) R3SO2Cl, Et3N
ii) PS-Trisamine
CO2Me
N
9
N
R1
CO2Me
6
R1
Scheme 1. Preparation of library 7 (homochiral).
H
N
H
N
H
N
MeO2C
MeO2C
MeO2C
MeO2C
N
N
N
N
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
N
N
N
Me
Bn
H
6a (42%)
6b (64%)
6c (60%)
H
N
H
N
H
N
MeO2C
MeO2C
MeO
N
N
CO2Me
CO2Me
6e (50%)
CO2Me
CO2Me
CO2Me
CO2Me
6d (54%)
N
N
Me
N
6f (45%)
Chart 1. Scaffolds 6 for library preparation (overall yields in parentheses).
O
O
O
O
O
O
O
MeO
Cl
Cl
O
Cl
Cl
Cl MeO2C
O
Cl
O
O
O
O
Cl
Cl
Cl
Cl
EtO
O
Cl
S
Cl
Br
O
O
O
BnO
Cl
Cl
Cl
O
Cl
N
Cl
S
O
O
O
O
O
S
O
O
O
O
O
O
S
S
Cl
Cl
S
Cl
Cl
S
Cl
S
O
O
N
O
CH3
MeO
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
Cl
NC
Chart 2. Acylating and sulfonylating reagents for diversification of scaffolds 6.
Cl
Cl
Cl
Cl
H3C
F3C
O2N
Br

6928
W. Wei et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6926–6930
chlorides) of N14, producing sublibrary 7 (Chart 2). Reactions were
run on a 39 mg/scaffold scale, and the library members were puri-
fied by mass-directed HPLC, then plated for storage. Of the 162
reactions attempted, 132 produced sufficient quantities (>40 mg)
for inclusion in the library. All library members were stable to stor-
age in the solid state, as well as in DMSO solution over several
months.
O
CO2H
Cl
(COCl)2, CH2Cl2
cat. DMF
rt, 1 hr
N
NR1
8a-e R1
10
PS-DMAP
(>95%)
X
N
NHR2
N
This library was subjected to the core dimerization inhibition
assay as a component of a 2240-membered CMLD-BU collection.
R2NH2, DIPEA
CH2Cl2
N
N
N
N
One member of this library (see below) showed single digit
lM
CO2Me
100%
IC50 activity that was validated by resynthesis and resubmission
to the assay, and confirmed in a secondary Alpha screen.7
A second generation indoline library with an expanded D-ring
was subsequently prepared exploiting the intramolecular inverse
electron demand Diels–Alder chemistry beginning with five N-
alkylated indolylpropionic acids 8a–e and triazine 2. The indolyl-
propionic acids were prepared by straightforward pathways
(Scheme 2). For N-methylindolylpropionic acid 8a, permethylation
of indolylpropionic acid, followed by basic methyl ester hydrolysis
with acid work-up gave 8a in 87% yield over three steps. For the
remaining acids 8b–e, methyl ester 9 formation, then N-alkylation,
followed by ester hydrolysis all proceeded uneventfully in good
yields.
With the five indolylpropionic acids in hand, the second subli-
brary again formed through an intramolecular inverse electron de-
mand Diels–Alder reaction of an indolyl subunit with a tethered
triazine, was pursued (Scheme 3). Conversion of the propionic
acids to the acid chlorides 10a–e, then amidation of 10 with
aminotriazines derived from 2 produced the tethered diene/dieno-
CO2Me
CO2Me
CO2Me
12
X = SMe (2)
Cl2
X =Cl (11)
O
R2
O
NR2
N
o-Cl2C6H4
120 ºC, 2 hr
N
N
N
N
CO2Me
100%
N
N
14
13
CO2Me
MeO2C
R1
CO2Me
R1
R1 = -CH3, -CH2CH=CH2, -CH2CH=C(CH3)2, -CH2Ph, -CH2C≡ CCH3
Scheme 3. Preparation of d-lactam sublibrary 14 (racemic).
O
OMe
O
N
MeO2C
N
N
MeO
CO2Me
N
N
CO2Me
CO2Me
N
Me
CO2H
CO2H
CO2Me
1) MeI (xs), KOH
2) KOH/H2O
3) HCl (aq)
15: 9.3 μM
16: 2.0 μM
N
H
N
CH3
(87%, 3 steps)
OMe
OMe
8a
O
N
MeOH,
SOCl2
(95%)
O
CO2Me
1) KH, R1-X
2) KOH/H2O
3) HCl (aq)
CO2H
N
N
N
OMe
CO2Me
CO2Me
CO2Me
N
N
H
CO2Me
N
9
(78-92%, 3 steps)
N
R1
8b-e
R1 = -CH2CH=CH2 (84%), -CH2CH=C(CH3)2 (78%),
-CH2Ph (92%), -CH2C≡ CCH3 (85%)
17: 1.4 μM
18: 2.0 μM
Figure 1. Library members with single digit
lM activities (IC50’s) inhibiting core
Scheme 2. Preparation of indolylpropionic acids 8.
dimerization.
H2N
OCH3
H2N
O
H2N
H2N
H2N
H2N
H2N
N
H2N
H2N
H2N
CO2CH3
H2N
CO2CH3
H2N
H2N
OCH3
H2N
O
H2N
O
O
H2N
H2N
OCH3
OCH3
OCH3
CF3
NO2
Br
H2N
H2N
H2N
H2N
OCH3
H2N
Cl
Chart 3. Primary amines used in the preparation of d-lactam sublibrary 14.

W. Wei et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6926–6930
6929
Table 1
Summary of activities
previously for core-derived peptides which inhibited core dimer-
ization.6 Cytotoxicity was also examined with hepatoma Huh7-5
cells (Table 1). The compounds were studied in the same cells in-
fected with HCV 2a strain J6/JFH-1.6 Real-time reverse-transcrip-
tase PCR was done on RNA extracted and purified from the cells
J6/JFH-1c
T2–EC50 (lM)
a
b
Compound
CC50
(
lM)
IC50
(lM)
HCV2a
T1–EC50
(lM)
BILN2061
5.3
N/A
9.3
2.0
1.4
2.0
0.072
14.8
4.9
2.3
3.8
0.071
22.2
0.7
3.2
3.0
15
16
17
18
>320
>320
127.2
5.3
treated with increasing concentrations (0.001–100
the compounds 15–18. The EC50’s were calculated at an early
(T1) and late (T2) stage, and varied from 2.3 to 20.8 M at T1,
lM) of each of
l
and from 0.7 to 36.3 lM. For comparative purposes, data for
a
b
c
BILN2061 a known inhibitor of the NS3/NS4A protease,11 is
included.
50% Cytotoxic conc versus Huh7-5 hepatoma cells average of triplicate runs.
See Figure 2.
EC50’s average of triplicate runs.
From this work, three relatively non-toxic compounds have
been identified (15–17) which could be the basis for the develop-
ment of new anti-HCV drugs, or which could be used in combina-
tion with compounds acting generally, such as interferon, or
specifically on other HCV targets. Compound 18, while of compara-
ble activity against core dimerization, was also cytotoxic. All four
of these compounds not only inhibited core dimerization, but they
also were shown to be effective in inhibiting HCV 2a strain J6/JFH-1
proliferation in isolated hepatoma cells, with the relatively non-
phile pairs 13. Intramolecular cycloadditions by heating in
1,2-dichlorobenzene yielded sublibrary 14 quantitatively.
Twenty-two primary amines (Chart 3) were employed in the SNAr
displacements on the chlorotriazine, producing 22 aminotriazines
12. All amidations and cycloadditions were accomplished
smoothly, resulting in a 110-membered, racemic sublibrary 14;
all members were stable to storage in DMSO.
toxic 16 and 17 having EC50’s in the single digit
lM range. These
Screening of the sublibrary 14 for inhibition of core dimeriza-
tion, performed in duplicate, revealed three additional ‘hits’ with
IC50 activities in the single digit lM range (Fig. 1 and Fig. 2). The
original ‘hit’ 15 from the initial library is homochiral. The other
three ‘hits’ (16–18) were from the second generation, d-lactam
sublibrary and are racemic. The activities of all four inhibitors were
validated by resynthesis and resubmission to the assay.
Subsequent to the identification of these four hits as core
dimerization inhibitors, all four compounds were evaluated for
their effect on production of HCV hepatoma cells, as done
two compounds are the two most promising candidates discovered
to date in our screening for core dimerization inhibitors. Work con-
tinues (Scripps-Florida) to study the binding of these lead com-
pounds with core protein, and to design more potent inhibitors
(CMLD-BU) based on these lead structures. Considerable recent
progress has been made in identifying small molecules that inhibit
protein–protein interactions with the potential of becoming thera-
peutic targets.12 Future work will determine whether 15–18 or re-
lated analogue(s) fall into this emerging class of promising
compounds.
Figure 2. Dose–response analyses of selected compound 15 analogues. Core106 ALPHA screen assay was used for the dose–response analyses. The compounds were dosed
from 0 to 100 M. The IC50 were calculated using a non-linear regression ‘Log (inhibitor) versus response’ with four points for each concentration.
l

6930
W. Wei et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6926–6930
2. http://www.cdc.gov/hepatitis/Statistics.htm#section1.
Acknowledgments
3. McGovern, B. H. J. Acquir. Immune Defic. Syndr. 2007, 45, S47.
4. (a) Simmonds, P. J. Gen. Virol. 2004, 85, 3173; (b) Cristina, J.; del Pilar Moreno,
M.; Moratorio, G. Virus Res. 2007, 127, 185.
The Scripps-Florida workers thank Prof. Weissmann (Depart-
ment of Infectology, The Scripps Research Institute-Scripps Florida)
for helpful discussions, Dr. T. Tellinghuisen (Department of Infec-
tology, The Scripps Research Institute-Scripps Florida) for help
with the HCV studies, Pierre Baillargeon and Lina Deluca (Depart-
ment of Lead Identification, The Scripps Research Institute-Scripps
Florida) for help with formatting the CMLD-BU library, Peter Chase
(Department of Lead Identification, The Scripps Research Institute-
Scripps Florida) for his support at the HTS facility, and the Factor
Foundation (ADS) for generous support. This is manuscript
#20,383 of The Scripps Research Institute. The Boston University
group thanks Professors John Porco and Aaron Beeler for helpful
discussions, and NIGMS CMLD initiative (P50 GM067041 and P41
GM086180) 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.
5. (a) Courcambeck, J.; Bouzidi, M.; Perbost, R.; Jouirou, B.; Amrani, N.; Cacoub, P.;
Pepe, G.; Sabatier, J. M.; Halfon, P. Antivir. Ther. 2006, 11, 847; (b) DeFrancesco,
R.; Carfi, A. Adv. Drug Delivery Rev. 2007, 59, 1242.
6. Kota, S.; Scampavia, L.; Spicer, T.; Beeler, A. B.; Takahashi, V.; Coito, C.; Snyder, J.
K.; Porco, J. A., Jr.; Hodder, P.; Strosberg, A. D. Assay Drug Dev. Tech., in press.
7. Kota, S.; Coito, C.; Mousseau, G.; Lavergne, J.-P.; Strosberg, A. D. J. Gen. Virol.
2009, 90, 1319.
8. The N-alkylated tryptophan derivatives were prepared by reaction of the N-
BOC tryptophan methyl ester with the appropriate alkyl halide after treatment
with NaH at À30 to À78 °C in THF. No racemization was observed under these
conditions.
9. Benson, S. C.; Lee, L.; Yang, L.; Snyder, J. K. Tetrahedron 1999, 62, 471.
10. Benson, S. C.; Gross, J. L.; Snyder, J. K. J. Org. Chem. 1990, 55, 3257.
11. Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieum, P.; Bolger, G.; Bonneau, P.;
Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A. M.;
Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagacé, L.; LaPlante, S. R.; Narjes, H.;
Poupart, M.-A.; Rancourt, J.; Sentjens, R. E.; St. George, R.; Simoneau, B.;
Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C.-L.;
Llinàs-Brunet, M. Nature 2003, 426, 186.
12. For some reviews on small molecule inhibitors of protein–protein interactions:
(a) Arkin, M. R.; Wells, J. A. Nat. Rev. Drug Disc. 2004, 3, 301; (b) Fry, D. C.
Biopolymers 2006, 84, 535; (c) Dudkina, A. S.; Lindsley, C. W. Curr. Top. Med.
Chem. 2007, 7, 952; (d) Blazer, L. L.; Neubig, R. R. Neuropsychopharm. Rev. 2009,
34, 126.
References and notes
1. Giannini, C.; Brechot, C. Cell Death Differ. 2003, 10, S27.