Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole

Article abstract of DOI:10.1016/j.bmcl.2012.11.095

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a h

Full text of DOI:10.1016/j.bmcl.2012.11.095

Bioorganic & Medicinal Chemistry Letters xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of potent broad-spectrum arenavirus inhibitors
derived from benzimidazole
⇑
Dongcheng Dai , James R. Burgeson, Dima N. Gharaibeh, Amy L. Moore, Ryan A. Larson ,
Natasha R. Cerruti, Sean M. Amberg, Tove’ C. Bolken, Dennis E. Hruby
SIGA Technologies, Inc., 4575 SW Research Way, Suite 230, Corvallis, OR 97333, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against
lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-
throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core
which led to a potent compound series. Herein, we report SAR studies which involved structural modi-
fications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many
analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in
this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from
other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ulti-
mately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig
model showing superior protection compared to ribavirin treatment.
Keywords:
Lassa fever
Lassa virus
Entry inhibitor
Arenavirus
Broad-spectrum inhibitor
SAR
Antiviral
Ó 2012 Elsevier Ltd. All rights reserved.
Viral hemorrhagic fever is a serious illness characterized by
extensive vascular damage and bleeding diathesis, fever, and mul-
tiple organ involvement. Many different viruses can cause this syn-
drome, each with its own animal reservoir, mode of transmission,
fatality rate, and clinical outcome in humans. They are distributed
throughout four virus families, the Arenaviridae, Bunyaviridae,
Filoviridae, and Flaviviridae,1 a fifth virus family (Rhabdoviridae)
has recently been implicated in hemorrhagic fever as well.2 Several
of these viruses generate high morbidity and mortality and can be
highly infectious by aerosol dissemination, promoting concern
regarding their potential weaponization.3 The arenaviruses are a
diverse family of enveloped RNA viruses found worldwide and
are phylogenetically grouped into Old World and New World lin-
eages.4 Arenavirus infection in rodents, the natural host animal,
is usually chronic and asymptomatic. Five distinct arenaviruses
cause severe hemorrhagic fever in humans and are classified as
Category A pathogens.5 These include the Old World arenavirus
Lassa and four New World arenaviruses Machupo (MACV), Junín
(JUNV), Guanarito, and Sabiá viruses. Lassa virus is the most prom-
inent pathogen in this group based upon the global estimate of a
half-million cases annually, current endemic status in West Africa,
and a history of weaponization research.6 The current prevention
and treatment options for hemorrhagic fever viruses are limited.
The use of intravenous ribavirin has shown some promise against
Lassa fever in high-risk patients if intervention is performed suffi-
ciently early in the disease,7 although anemia is an important side
effect that often limits dosing.8 Thus, there remains an unmet need
to develop a safe and effective therapeutic to treat arenaviruses.
Our initial approach involved the use of high-throughput
screening (HTS) against lentivirus-based pseudotypes incorporat-
ing LASV GP.9 This pseudotype viral assay was utilized as a surro-
gate due to the fact that work with live LASV requires a Biosafety
Level 4 (BSL-4) facility. The viral pseudotype incorporates the het-
erologous envelope protein and provides a platform to assess the
function of that protein with respect to mediating entry into the
target host cell. The envelope GP functions to attach virus to spe-
cific receptors on the host cell and also to drive fusion between
the cell and viral membrane lipid bilayers.10 The preliminary
screen identified benzimidazole derivative ST-37 ( Fig. 1) which
O
H
N
N
N
ST-37
⇑
Corresponding author. . Tel.: +1 541 766 3758; fax: +1 541 753 9999.
O
E-mail address: ddai@siga.com (D. Dai).
Present address: Millennium: The Takeda Oncology Company, Cambridge, MA
02139, USA
Figure 1. Initial hit discovered via high-throughput screening.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.095
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2
D. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
The benzimidazole derivatives were readily accessible from
1-fluoro-2,4-dinitrobenzene (2), as shown in Scheme 1.14 The first
step involved an aromatic substitution of the corresponding aniline
compound to install the first point of diversity as Ar1 Following
hydrogenation of the nitro moieties, intermediate 4 was cyclized
using a mixture of formic acid and 4 N HCl. With cyclized interme-
diate 5 in hand, the final step was performed in which the amino
functionality was reductively alkylated with the corresponding
aldehyde to introduce the second point of diversity as Ar2 and af-
ford the final compounds (6a–o, 7a–y). Compounds 13–15 were
synthesized through alkylation, sulfonation or acylation of the
products of Scheme 1. In the case of 16, the corresponding acid,
as opposed to the aldehyde, was reacted with intermediate 5 to
form the amide-linked analog.15 The alternate methylamino linker
positioning of 17 could be achieved through the alternate use of
1-fluoro-2,5-dinitrobenzene as the starting material in Scheme 1.
For the synthesis of compound 12 containing an inverted linker
portion, brominated starting material 8 was advanced to interme-
diate 10 using a similar sequence as outlined above with alternate
reagents (Scheme 2). The bromo group of 10 was transformed into
a nitrile through a palladium-catalyzed cyanation reaction fol-
lowed by reduction with DIBAL to aldehyde 11. Finally, reductive
alkylation conditions were utilized to install Ar2 and form com-
pound 12.
The initial SAR studies performed on this benzimidazole scaf-
fold probed the spatial and electronic properties of the phenyl
rings (Ar1 and Ar2) and their appended substituents. To start, benz-
imidazole analogs were investigated by keeping Ar2 as a para-alkyl
substituted phenyl ring (as in ST-193) and modifications to Ar1
were made to determine the types of substituents and ring types
tolerated in this region of the molecule (Table 1). When an unsub-
stituted phenyl ring was placed at the Ar1 position (6a), there was a
nearly fourfold decrease in activity against LASV GP pseudotypes
compared to ST-193. This discovery alluded to the possibility of a
hydrophobic pocket in this region or even a key hydrogen bond
interaction. Similar potency was observed when the methoxy
group of ST-193 was replaced with an ethyl group (6b) indicating
that a hydrogen bond accepting alkoxy group may not be required
in this region of the molecule. For analogs appended with larger
alkyl and alkoxy substituents in the 4-position (6c–f), the observed
antiviral potency was fairly consistent with EC50 values ranging
from 4 to 9 nM. A large decrease in potency was found for analogs
with phenyl and naphthyl ring substitution at the 4-position of Ar1
with alkyl or alkoxy linkages (6g–j). It appears that the presumed
hydrophobic pocket associated with Ar1 positioning may not be
large enough to accommodate these groups with very large steric
bulk. Attempts to replace the Ar1 phenyl ring with more polar
and ionizable nitrogen containing rings (6k–n) were not well toler-
ated with respect to antiviral activity. The exception was 6l which
H
N
N
N
ST-193
O
Figure 2. Benchmark LASV GP pseudotype inhibitor molecule.
exhibited potent activity against LASV GP pseudotype.11 The selec-
tivity of ST-37 for virus entry mediated by the LASV GP relative to
non-arenavirus envelope-mediated entry is 2500-fold. In addition
to blocking virus entry, cell-cell fusion mediated by either the LASV
or JUNV GP is inhibited; another indicator of fusion activation,
shedding of the GP1 subunit, is also inhibited by the compound.12
Genetic determinants of resistance have been mapped by several
methods: (1) virus passaging in the presence of compound to select
resistant virus; this method used a nonpathogenic arenavirus,
Tacaribe virus, so that it could be done under BSL-2 containment;
(2) engineering of chimeric envelopes between a sensitive GP
(LASV) and an insensitive arenavirus GP (lymphocytic choriomen-
ingitis virus); and (3) site-directed mutagenesis. These methods
have identified several amino acids that can modulate sensitivity
by varying degree, all mapping to a fragment of about 30 residues
of the GP2 subunit of the envelope glycoprotein11; this region in-
cludes the transmembrane domain and the membrane-proximal
ectodomain. Another compound series described by Whitby et al.
also inhibits arenavirus entry and may function similarly.13
A benchmark molecule ST-193, closely related to the HTS hit
(ST-37), was discovered early in the medicinal chemistry cam-
paign; it possessed an EC50 value of 1.4 nM in the LASV pseudotype
viral assay (Fig. 2). While ST-193 displayed very potent antiviral
activity, the remaining properties, most notably PK properties,
had not yet been examined. Therefore, a SAR study was designed
to probe the chemical space around this particular molecule to un-
cover related analogs that may offer advantageous drug-like prop-
erties for preclinical development. In this Letter we will discuss
studies which involved modifications of the appended phenyl rings
and methylamino linker moiety. In a separate Letter, we will report
the SAR study of the core structure. It should be noted that all of
the antiviral data that is reported herein was derived from lentivi-
rus-based pseudotypes for which descriptions and methods have
previously been disclosed.11 In addition, all of the EC50 values are
averages (geometric mean) of at least three experiments unless
otherwise noted.
H2N
NO2
F
O2N
a
NH2
b
c
NO2
NO2
2
NH
3
4
NH
Ar1
Ar1
H
N
Ar2
N
N
H2N
N
d
N
5
Ar1
Ar1
6a-o, 7a-y
Scheme 1. Reagents and conditions: (a) H2N–Ar1, Cs2CO3, THF. (b) H2, Pd/C, EtOAc. (c) HCO2H, 4 N HCl, 90 °C. (d) Ar2CHO, Na(OAc)3BH, DCM.
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D. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
3
Br
NO2
NH
Br
N
N
Br
NO2
F
a
b,c
d,e
10
8
9
O
O
O
N
H
NH
f
N
N
12
11
N
O
O
Scheme 2. Reagents and conditions: (a) 4-ethoxyaniline, TEA, EtOH, 55 °C, 8 h, then rt, 18 h (89%); (b) conc. HCl, EtOH, iron dust, 60 °C, 1 h, then 9, NH4Cl, H2O, 60 °C, 3 h
(91%); (c) Formamidine acetate, 2-methoxyethanol, reflux, 2 h (99%); (d) Zn(CN)2, Zn dust, Pd(dppf)Cl2, poly(methoxyhydro)siloxane, DMA, water, 100 °C, 3 h (88%); (e) 1.5 M
DIBAL in toluene, DCM, À78 °C to rt, 30 min (44%); (f) 4-ethylaniline, MeOH, 60 °C, 1 h, then NaBH4, rt, 30 min (31%).
possessed a modest fourfold reduction in potency compared to
ST-193. A cyclohexane ring was placed at Ar1 (6o) and this analog
showed a dramatic decrease in activity when compared to the
unsubstituted phenyl ring analog 6a. Because of the structural flex-
ibility of this saturated ring, it was postulated that this substitution
may be disfavored due to the preferred orientation at the site of
biological interaction. A small set of compounds was prepared with
the Ar1 phenyl ring replaced by alkyl groups (i.e., methyl, ethyl, or
n-butyl). While the analog containing n-butyl had submicromolar
antiviral activity, the other compounds had micromolar potency
tained a furyl ring in place of the Ar2 phenyl ring and this com-
pound possessed single digit micromolar activity against Lassa.
Since we did not have an analog containing an unsubstituted phe-
nyl ring for Ar2, it was difficult to determine if the change in ring
type or lack of substitution had the most detrimental effect on po-
tency. Additionally, a set of two compounds was prepared in which
the Ar2 phenyl ring was replaced with a hydrogen atom or an ethyl
group. In each case, the compound was rendered completely inac-
tive at the highest concentration tested (50 lM). In general, polar
phenyl ring substituents (i.e., amino, carboxylic acid) were not tol-
erated in this region of the molecule with regards to antiviral po-
tency. As a result of this study, it was determined that an alkyl
group (ethyl or i-propyl) in the para-position of Ar2 would be uti-
lized when expanding the SAR analysis to probe other portions of
the parent molecule.
The methylamino linker portion was the next region of the scaf-
fold to be explored and a diverse array of analogs were designed
and tested in the pseudotype assay (Table 3). Methylation of the
secondary amine (13) resulted in a fortyfold decrease in potency
versus the parent molecule (ST-193). Conversion of the amino moi-
ety to a sulfonamide (14) or acetamide group (15) resulted in a
large decrease in antiviral activity compared to ST-193. The evi-
dence from these three analogs led us to believe that there may
be a key hydrogen bond interaction requiring the presence of this
secondary methylamino linker. When the methylamino group was
transformed into an amide moiety (16), the result was complete
or were completely inactive at a concentration of 50 lM. Overall
for Ar1, good antiviral potency was observed for compounds
containing a phenyl ring substituted with either medium-sized al-
kyl or alkoxy substituents in the para-position. Additionally, a
para-alkoxy substituted pyridyl ring of the type contained in 6l
may offer a path forward for introducing an ionizable ring with
the potential to enhance solubility and other drug-like properties.
The phenyl ring of the left hand portion of the molecule (Ar2)
was probed with a variety of substituents arranged in various sub-
stitution patterns to determine the impact on antiviral potency
(Table 2). Alternate rings away from benzene were also explored
for Ar2 in this study. For analogs 7a–f, a nearly fourfold improve-
ment in potency was observed for alkyl substitution in the para-
position when compared to alkoxy substitution, regardless of steric
bulk. A similar trend to that previously described for Ar1 variation
involving additional appended rings was also observed for Ar2 sub-
stitution (7h–j) with a large decrease in pseudotyped LASV antivi-
ral activity. Attempts to introduce a basic nitrogen moiety capable
of adding hydrophilic character, as either dimethylamino (7k and
7l) or contained within a six-membered saturated ring (7m–o), re-
sulted in a large reduction in antiviral activity. The addition of an
acetic acid moiety (7p) resulted in a compound with micromolar
potency. Based upon the reduced activity of 7q–t, it was evident
that mono-alkoxy substitution at the para-position was preferred
over alternate methoxy group substitution patterns. Replacement
of the phenyl ring with a pyridyl or pyrimidyl ring (7v–x) greatly
increased the observed pseudotyped LASV EC50 value. In contrast
with Ar1, we were not able to find a nitrogen containing heterocy-
cle to place at Ar2 that possessed similar potency compared to the
parent compound (ST-193). An analog (7y) was tested which con-
inactivity at a concentration of 50 lM. It should be noted that
the parent compound of 16, sharing the same substituents and cus-
tomary methylamino linker, had an EC50 value of 3.6 nM. It is pos-
sible that the trigonal planar orientation of the amide-linked
molecule prohibits the preferred geometry at the site of biological
interaction leading to the dramatic reduction in activity. It was evi-
dent from analog 17 that relocating the linker portion from the 5-
position to the 6-position of the bicyclic core was disfavored with a
twentyfold decrease in potency compared to the parent compound
(6f). It was interesting to find that reversal of the methylamino lin-
ker (12) did not have a large effect on activity with a pseudotyped
LASV EC50 value of 11 nM representing a modest, sevenfold de-
crease in potency compared to the corresponding analog with
the usual methylamino linkage (compound and data not shown).
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D. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
Table 1
Table 2
Antiviral potency of Ar1 analogs
Antiviral potency of Ar2 analogs
Ar2
R
H
N
N
N
H
N
N
N
Ar1
6a-o
7a-y
Compound
Ar1
R
LASV EC50 (nM)
O
R
6a
6b
6c
6d
ST-193
6e
6f
Phenyl
iPr
iPr
iPr
iPr
iPr
iPr
iPr
5.2
1.9
4.4
4.0
1.4
5.6
8.6
4-Ethylphenyl
4-n-Butylphenyl
4-t-Butylphenyl
4-Methoxyphenyl
4-n-Butoxyphenyl
4-t-Butoxyphenyl
Compound
R
Ar2
LASV EC50 (nM)
7a
7b
7c
7d
7e
7f
Me
Me
Me
Me
Me
Me
Me
4-Ethylphenyl
1.2
1.5
1.1
4.0
6.0
5.2
47
4-n-Butylphenyl
4-t-Butylphenyl
4-Ethoxyphenyl
4-n-Butoxyphenyl
4-t-Butoxyphenyl
4-Chlorophenyl
6g
6h
iPr
iPr
120
130
7g
7h
7i
Me
Me
46
O
O
O
380
6i
iPr
iPr
Et
43
540
32
7j
Me
Me
69
6j
O
N
O
N
7k
3300
6k
6l
O
N
N
7l
Me
Me
Me
Me
270
1400
Et
5.8
81
O
N
N
N
7m
7n
7o
6m
Et
O
N
N
O
790
6n
6o
Et
450
320
N
O
N
N
16,000
Cyclohexyl
Me
HO
O
7p
Et
1300
To assess the spectrum of antiviral activity, additional arenavi-
rus potency data was gathered for some of the aforementioned
analogs which included pseudotypes of envelopes for MACV, JUNV,
Sabiá virus, and Guanarito virus (Table 4). Overall, these com-
pounds retained nanomolar in vitro activity against these Old
World and New World arenaviruses.
7q
7r
7s
7t
Me
Me
Me
Me
Me
3,4-Dimethoxyphenyl
3-Methoxyphenyl
2,3-Dimethoxyphenyl
2-Methoxyphenyl
2-Hydroxyphenyl
700
35
160
460
310
7u
With ST-193 having a favorable activity profile, this molecule
was advanced to pharmacokinetic studies followed by evaluation
in a lethal LASV infection in the strain 13 guinea pig model.16 This
most practical small animal disease model was used to test the le-
vel of protection offered by ST-193 and allowed direct comparison
to ribavirin, the current standard of care. ST-193 was evaluated at
low dose (25 mg/kg/day) and high dose (80 mg/kg/day), while
ribavirin was delivered at a dose of 25 mg/kg/day. ST-193 treated
guinea pigs displayed significantly reduced viral loads (2- to
3-log reduction in plasma titers) and an overall survival rate of
63% (10/16, with five survivors in each dose group) compared with
0% for the ribavirin (0/8) and vehicle (0/7) groups. This study
N
7v
Et
Et
240
350
N
7w
N
N
7x
7y
Et
450
Me
2-Furyl
1200
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D. Dai et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
5
Table 3
Modifications of the methylamino linker
Cmpd
Structure
LASV EC50 (nM)
Cmpd
Structure
LASV EC50 (nM)
NH
N
N
O
N
N
N
12
11
15
380
O
O
O
HN
N
N
N
N
N
13
57
16
>50,000
O
O
N
N
N
H
O
N
N
S
O
14
110
17
180
N
O
O
Table 4
Broad-spectrum arenavirus activity
Compound
LASV EC50 (nM)
MACV EC50 (nM)
JUNV EC50 (nM)
Sabiá EC50 (nM)
Guanarito EC50 (nM)
ST-37
ST-193
14
11
45
3.1
40
12
7.7a
0.62
16
110
21
510
43
5.6
1.4
110
11
0.44
n.d.
n.d.
12
5.0a
N.d. = not determined.
a
Average value from only two experiments.
served to validate the chemical scaffold and the LASV pseudotype
viral assay that was used for HTS and compound optimization.
In summary, given the potent HTS hit ST-37 (11 nM), it was pos-
sible to develop focused SAR studies and discover a number of po-
tent inhibitors in the LASV GP pseudotype assay. We were able to
uncover many potent analogs with single digit nanomolar LASV
antiviral activity. In addition, compounds of this structure type
may offer the added value of broad-spectrum activity against a
variety of other Category A arenaviruses as displayed by ST-193
and related analogs. Ultimately these studies provide important
support for further development of this compound series.
Acknowledgements
We gratefully acknowledge the financial support from NIH
grants 5R44AI056525 and 1R01AI093387. We thank Kayla Kickner
and Tracy Tan for technical support. We also thank Ricerca Biosci-
ences for synthesizing the majority of the custom synthesized
compounds used in this SAR study.
References and Notes
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