Discovery of highly potent small molecule Hepatitis C Virus entry inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.019

Novel, highly potent small molecule HCV entry inhibitors are reported. The SAR exploration of a hit molecule identified from screening of a compound library led to the identification of highly potent compounds with IC ₅₀ values of <5 nM in the tissue culture HCV infectious assay.

Full text of DOI:10.1016/j.bmcl.2011.09.019

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6852–6855
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of highly potent small molecule Hepatitis C Virus entry inhibitors
Gopi Kumar Mittapalli, Andrew Jackson, Fang Zhao, Haekyung Lee, Stephine Chow, Jeffrey McKelvy,
⇑
⇑
Flossie Wong-Staal , James E. Macdonald
iTherX Pharmaceuticals, Inc., PO Box 910530, San Diego, CA 92191-0530, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel, highly potent small molecule HCV entry inhibitors are reported. The SAR exploration of a hit mol-
ecule identified from screening of a compound library led to the identification of highly potent com-
pounds with IC50 values of <5 nM in the tissue culture HCV infectious assay.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 August 2011
Revised 1 September 2011
Accepted 6 September 2011
Available online 16 September 2011
Keywords:
Hepatitis C Virus
Entry inhibitors
Tetrahydrocarbazoles
HCVcc
Pharmacokinetics
Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, li-
ver cirrhosis, and hepatocellular carcinoma and is a leading indica-
tion for liver transplantation.1 The U.S. Centers for Disease Control
and Prevention (CDC) estimates the world wide prevalence of Hep-
atitis C Virus infection at ꢀ3%, or 170 million individuals, and in
the United States it estimates 3.2 million infected individuals.2
The current standard of care is a combination therapy with pegy-
lated interferon and the nucleoside analog ribavirin. However,
the treatment has adverse side effects and moderate efficacy.3
The anti-HCV drugs currently in development mostly target the
viral enzymes, protease and polymerase that are essential for viral
replication. More recently, two protease inhibitors, Incivek™ and
Victrelis™ were approved by the FDA to be used in combination
with pegylated interferon and ribavirin. The single agent studies
have shown the rapid emergence of resistant mutants to these
new inhibitors, suggesting cocktails of HCV specific antivirals tar-
geting multiple steps in the infection cycle will likely be required
for rapid viral clearance and to overcome resistant mutants.4–6
We have taken a different approach by focusing our efforts on
the discovery and development of small molecule HCV entry inhib-
itors7,8 that prevent entry of HCV into the host cell. The viral entry
is a critical step in the life cycle of HCV, and inhibitors of viral entry
can add significantly to a drug cocktail targeting multi-steps of the
viral life cycle. The entry inhibitors can protect cured cells from re-
infection and preventing viral spread could help stop amplification
of resistant mutants. The HCV entry into hepatocytes requires at
least five cellular co-factors, CD-81,9 claudin,10,11 occludin,12 scav-
enger receptor B113 and the more recently discovered epidermal
growth factor receptor 1 and 2.14 We have recently reported15
a
highly potent HCV inhibitor ITX 5061 (Fig. 1), which blocks viral
entry by binding to the scavenger receptor B1 (SR-B1). This first-
in-class drug is currently in Phase Ib clinical trials (Fig. 1).
Here, we report the synthesis and SAR of novel small molecule
HCV entry inhibitors that belong to a distinct chemical class from
ITX 5061. A library of compounds, comprising small molecules
and natural products, was screened using the HCV pseudo particle
(HCVpp) entry assay16–18 to identify compounds that were nearly
equally effective against genotype 1a and 2b. The identified hits
were further validated in
a HCV2aCH-Rluc infectious assay
(HCVcc-2a).19–21 One of the hits identified is the compound Rim-
cazole22 (1) that showed an IC50 of 1.2
lM in HCVcc-2a entry assay
(Fig. 2).
The initial SAR exploration at the 2- and 3-positions of the car-
bazole (not shown) to improve the potency and drug-like proper-
ties, suggested that the methyl ester at the 3-position improved
the potency by several fold. The compound 2 with a methyl ester
at the 3-position of the carbazole showed an IC50 of 230 nM in
HCVcc-2a entry assay, whereas its corresponding ethyl ester (3)
and isopropyl ester (4) were less potent (Fig. 2). It was also ob-
served that the tetrahydrocarbazoles are generally more potent
than their corresponding carbazoles. This led us to focus our efforts
on 6,9-disubstituted tetrahydrocarbazole series.
The synthesis of 6,9-disubstituted tetrahydrocarbazoles is
shown in Scheme 1. The Fischer indole condensation of 4-hydraz-
inobenzoic acid with cyclohexanone gave tetrahydrocarbazole-6-
carboxylic acid which was subsequently esterified to obtain the
⇑
Corresponding authors. Tel.: +1 858 824 1114; fax: +1 858 824 1112.
E-mail addresses: fwongstaal@itherx.com (F. Wong-Staal), 2jemacd@gmail.com
(J.E. Macdonald).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.019

G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6852–6855
6853
O
O
O
O
S
O
O
N
N
O
N
N
H
H
O
O
R
N
R =
N
ITX 5061 (Phase Ib)
O
O
Figure 1. Stucture of HCV entry inhibitor ITX 5061.
N
N
N
N
NH
O
O
NH
6
(25)
7
(40)
8
9
(65)
10
(50)
3
R
1 R = H
IC50 = 1.2 µM
(150)
2
2 R = COOCH3
3 R = COOC2H5
4 R = COOiC3H7
IC50 = 230 nM
IC50 = 600 nM
IC50 = 630 nM
N
O
S
O
O
O
S
S
O
O
O
S
O
O
NH
O
O
S
N
N
N
N
NH
O
S
N
N
N
O
O
O
12
(15)
13
(30)
14
(15)
16
(300)
17
(50)
11
(150)
15
(5)
Figure 2. The structure of hit compound (1) identified from screening of a library.
NH
NH
NH
NH
O
O
O
N
N
N
N
N
N
N
N
i
ii
HO
O
O
MeO
OMe MeO
N
OMe
OMe
N
H
N
NHNH2
19
(15)
18
(50)
20
(1.5)
21
(15)
5
X
X = Cl or NHBoc
v
NH
O
NH
N
iii (X=Cl)
or
N
N
N
N
N
N
N
Compds 11-16
O
iv (X=NHBoc)
N
MeO
OMe
OMe
O
Cl
MeO
O
O
25
(0.6)
23
(30)
24
(1.5)
22
(30)
O
6
N
9
O
O
O
O
N
S
O
R
N
N
N
N
N
N
Compds 6-10 and
N
Cl
F3C
17-34
30
(50)
26
(1.5)
27
(1.5)
28
(1.5)
29
(5)
Scheme 1. Reagents and conditions: (i) (a) cyclohexanone, AcOH, reflux, overnight;
(b) MeOH, H2SO4, reflux, overnight; (ii) NaH, 1-bromo-3-chloro propane, DMF, 3 h;
or NaH, DMF, BocNH-(CH2)3-OTs, rt, 18 h; (iii) n-Bu4NHþ4 Iꢁ, 2° amine, DMF, rt, 2–
3 days; (or) K2CO3, DMF, imidazole or 2-methyl imidazole or purine, n-Bu4NHþ4 I, rt,
18 h; (or) 2-oxo pyrimidines, K2CO3, 80 °C, overnight; (or) phenyl sulfinate, DMF,
80 °C, overnight; (iv) (a) TFA, CH2Cl2, 0 °C-rt, 3 h; (b) 2-chloro pyrimidines, DMF,
DIEA, 80 °C, overnight; (or) aryl sulfonyl chloride or isopropyl sulfonyl chloride,
CH2Cl2, Et3N, rt, overnight; (v) NaH, DMF, 3-chloropropyl sulfonamides, rt, 18 h; (or)
NaH, DMF, 3-chloropropyl-N,N-dimethylamide, rt, 18 h.
NH
S
N
N
N
O
F
N
N
N
N
O
N
33
(50)
F
31
32
(310)
34
(15)
(170)
Figure 3. SAR exploration of groups at 9-position. The values within parenthesis
indicate IC50 in nM are from HCV2aCH-Rluc assay. The IC50 values are mean of three
experiments.
compound 5. The alkylation of compound 5 with 1-bromo-3-
chloro-propane followed by chloro displacement with various
nucleophiles afforded compounds 6–10 and 25–33. The alkylation
with 3-chloropropyl sulfonamides or 3-chloropropyl-N,N-dimeth-
ylamide yielded the compounds 11–16. The 2-amino pyrimidine
analogs (18–24) were prepared by the alkylation of compound 5
with BocNH-(CH2)3-OTs, deprotection of the Boc group and subse-
quent reaction with 2-chloro pyrimidines. The compounds 17 and
34 were prepared by the reaction of methyl 9-(3-aminopropyl)-
2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate (intermediate of
the 2-amino pyrimidine compounds) with isopropylsulfonyl chlo-
ride or 3,5-difluorophenylsulfonyl chloride in the presence of
Et3N. The synthesized compounds were characterized by 1H NMR
and mass spectra23 and were tested in the HCV2aCH-Rluc infec-
tious assay.15
sulted in modest increase in the potency. The morpholine (8), 2-
oxo-piperiazine (9) and piperidine analogues (10) are less potent
than the 2,6-dimethyl morpholine analogue (6). The sulfonamide
and carboxamide analogues (11–16) were prepared to investigate
the impact of basic amine group on potency. The smaller N,N-dialkyl
sulfonamides showed higher potency thanlarger N,N-dialkylsulfon-
amides. Of these, the dimethyl sulfonamide compound 15 showed
excellent potency with an IC50 of 5 nM. The corresponding dimethyl
carboxamide (16) and the reverse sulfonamide (17) analogs were
prepared and found to be less potent than compound 15. It was ob-
served that aromatic heterocycles were also active at this position.
The compound with a 4,6-dimethoxy-2-amino pyrimidine (20) is
the most potent with an IC50 of 1.5 nM, whereas its corresponding
mono methoxy (19) or unsubstituted pyrimidine (18) are less po-
tent. The dimethoxy triazine (21) analog is less potent than its corre-
sponding pyrimidine 20. The N-methylated and 2-oxo analogues of
To understand the SAR, we explored alternatives to the 2,6-di-
methyl piperazine with a methyl ester group at 6-position of tetra-
hydrocarbazole (Fig. 3). The replacement of 2,6-dimethyl
piperazine with 2,6-dimethyl morpholine as exemplified by 6 re-

6854
G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6852–6855
O
42
43
44
45
R = COOH
IC50 = NA
R
R = COOiC3H7
R = CONHCH3
R = CON(CH3)2
IC50 = 10 nM
IC50 = 10 nM
IC50 = 30 nM
IC50 = 50 nM
IC50 = 150 nM
O
N
N
R
R =
NH
46 R = CON(C2H5)2
47 R = CONHiC3H7
N
N
O
2
O
N
N
NH
N
N
N
N
N
N
N
N
N
Figure 5. Isopropyl and amide derivatives of compound 20. NA: not active; the IC50
values are from the HCV2aCH-Rluc assay.
MeO
OMe
OMe MeO
OMe
MeO
OMe MeO
38
(50)
36
(300)
37
(15)
35
(30)
Table 1
PK profile of compounds 43, 44 and 45 in rata
Compd
AUCall
(uG/L h)
Cmax
(uG/L)
T1/2 (h)
Vz (L/Kg)
Cl (L/h/Kg)
%F
19
0.8
0.6
HN
N
HN
N
O
O
S
N
43
44
45
896
45
41
547
51
51
1.24
0.47
0.55
17.56
1.33
2.05
4.31
3.49
2.7
41
39
(5)
40
(5)
(>1000)
a
iv Dose 2 mg/kg; oral dose 20 mg/kg.
Figure 4. Exploration of chain length and conformationally-constrained linkers.
The values within parenthesis indicate IC50 in nM are from the HCV2aCH-Rluc
assay.
the series of compounds. Compound 20 is rapidly degraded in rat
plasma likely by plasma esterases and exclusively forms the
metabolite 42, which is confirmed by LC–MS and by preparation
of an authentic sample. The metabolite, carboxylic acid (42), was
tested and found to be completely inactive in HCVcc-2a entry assay
(Fig. 5). The corresponding isopropyl ester (43) and amide deri-
vates (44–47) were prepared to improve plasma stability and were
tested for both the potency and plasma stability. The amide deriv-
atives are very stable in rat plasma, whereas the isopropyl ester
analogue is slightly more stable than the methyl ester. In the
HCVcc-2a entry assay, although both the isopropyl ester (43) and
amide analogues (44 and 45) are less potent than the methyl ester
analogue (20), they are reasonably active (Fig. 5).
We consequently investigated the in vivo PK for compounds 43,
44 and 45. All the compounds showed poor PK properties (Table 1).
Of these, compound 44 showed 19% oral bioavailability. However,
the compound also has high clearance, shorter half-life and poor
exposure in rats, which signify that the class of compounds has
to be further investigated to improve the PK properties and to find
bioisosteres of the methyl ester group (Table 1).
We have also investigated the mechanism of action of the series
with one of the more advanced compounds.24 Based on binding
competition and resistance profiles, this compound class appears
to be mechanistically similar to ITX 506115 and targets one of the
cellular co-factors required for virus entry, the scavenger receptor
B-1 (SR-B1).
In conclusion, we have identified highly potent small molecule
HCV entry inhibitors through SAR exploration of a hit molecule
identified from screening of a library. Further identification of bio-
isosteric groups of the methyl ester, SAR exploration on tetrahydro
ring and optimization of PK properties of this series will be the
subject of future publications.
O
O
O
i
ii
O
O
O
N
N
N
H
5
OC2H5
N
C2H5O
HN
R
39 R = H
40 R = CH3
R
Scheme 2. Reagents and conditions: (i) NaH, 3-chloropropionaldehyde diethylac-
etal, n-Bu4NHþ4 Iꢁ, DMF, 50 °C, 24 h; (ii) (a) AcOH, aq HCl, 0 °C to rt, 3 h; (b) o-
phenylene diamine, DMF, 150 °C, overnight.
compound 20 were prepared to examine the necessity of an H-bond
donor. The N-methylatedanalogue(24) is as potent as compound20.
The 4,6-disubstituted-2-oxo pyrimidine analogs (25–29) showed
equal or better potency when compared to the corresponding 4,6-
disubstituted-2-amino pyrimidines. The other aromatic heterocy-
cles/aromatics such as imidazoles (31, 32), purine (33), phenyl sul-
fone (30) or difluoro phenyl sulfonamides (34) are also active but
less potent thanpyrimidineanalogues. The SARsuggests thatthe po-
sition has wide latitude for a variety of groups and in general disub-
stituted 2-amino/2-oxo pyrimidines and dimethyl sulfonamide
groups are best for high potency.
The length of side chain was then investigated by preparing 2-
carbon and 4-carbon linker analogues as exemplified by 35 and 41,
which were found to be less potent than the 3-carbon linker ana-
logues. The introduction of conformational constraint (36–40) into
the linker also resulted in decreased potency, suggesting both flex-
ibility and a 3-carbon linker are optimal for the potency (Fig. 4).
The potency of benzimdazole analogues (39 and 40) was only re-
duced by 3-folds. Compounds 35–38 and 41 were prepared simi-
larly to compounds 20 and 15, respectively. The benzimidazole
analogues 39 and 40 were prepared by the alkylation of compound
5 with 3-bromo-1,1-diethoxypropane and subsequent hydrolysis
followed by the reaction of resulting aldehyde with 1,2-diamino
benzene (Fig. 4 and Scheme 2).
Acknowledgments
We would like to thank Dr. Rene Rijnbrand for advice and dis-
cussion. We also thank Dr. Danielle Vellucci for proof reading of
the manuscript.
References and notes
We subsequently examined plasma stability of one of the most
potent compounds 20 due to the presence of methyl ester group in
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