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

Article abstract of DOI:10.1016/j.bmcl.2012.11.093

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.

Full text of DOI:10.1016/j.bmcl.2012.11.093

Bioorganic & Medicinal Chemistry Letters 23 (2013) 750–756
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 and related heterocycles
James R. Burgeson, Amy L. Moore, Dima N. Gharaibeh, Ryan A. Larson , Natasha R. Cerruti,
⇑
Sean M. Amberg, Dennis E. Hruby, Dongcheng Dai
SIGA Technologies, Inc., 4575 SW Research Way, Suite 230, Corvallis, OR 97333, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 1 December 2012
A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our
group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl
rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These
changes included the introduction of various substituents to the bicyclic benzimidazole ring system along
with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring
systems were found to retain antiviral potency compared to the benzimidazole series from which we
derived our lead compound, ST-193. In fact, 21h, built on an imidazopyridine core, possessed a near ten-
fold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benz-
imidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs
discovered during this study.
Keywords:
Lassa fever
Lassa virus
Entry inhibitor
Arenavirus
Broad-spectrum inhibitor
SAR
Antiviral
Ó 2012 Elsevier Ltd. All rights reserved.
During the previous medicinal chemistry campaign, the focus of
the SAR studies encompassed substitutions and replacements of
the appended phenyl rings along with modifications of the methyl-
amino linker.1 Lead compound ST-193 was discovered during this
initial campaign featuring the benzimidazole core ring system
(Fig. 1). To further the analysis of this scaffold, changes to the core
bicyclic ring system were explored. As with the previous SAR
study, EC50 values were derived from lentiviral pseudotype assays
generated from the Lassa virus envelope glycoprotein (LASV GP)
gene.2 The single digit nanomolar potency of ST-193 was used as
a benchmark to compare all subsequent analogs in this series.3
The use of this pseudotype LASV assay was validated by the dem-
onstration of ST-193-mediated protection from lethal disease in a
small animal model.4
H
N
N
N
ST-193
O
Figure 1. Lead compound from benzimidazole series.
4-ethylbenzaldehyde to form 20d. Compound 20f was obtained
through acid hydrolysis of the nitrile-containing analog.
In Scheme 1,
a representative example is shown which
Boc-protected pyridine 4 was utilized as the starting material
for compound 21e which contained an imidazopyridine core with
a bridgehead-nitrogen (Scheme 2). The starting material was
deprotected and cyclized6 with chloroacetaldehyde to form inter-
mediate 6. After the installation of an iodo moiety (7), a Suzuki
coupling was performed to install the ethoxy substituted phenyl
ring (8). Finally, the 4-ethylbenzylamine portion was attached
through a Buchwald–Hartwig cross coupling reaction to form 21e.
Another imidazopyridine series was synthesized which more
closely resembled the original benzimidazole series as in ST-193
(Scheme 3). The chloro group of pyridine 9 was substituted with
an aniline derivative to install the first point of diversity as Ar.1
Intermediate 10 was reduced via hydrogenation and cyclized with
formic acid to form bicyclic intermediate 12. The methylamino
describes the incorporation of substituents at the 2-position of
the benzimidazole core during the cyclization step. Intermediate
1, for which a synthesis has previously been described,1 was
cyclized with methyl 2,2,2-trichloroacetimidate to install a trichlo-
romethyl group at the 2-position and form the bicyclic ring.5
Following the transformation of halogenated compound 2 to nitrile
intermediate 3, the material was reductively alkylated with
⇑
Corresponding author. Tel.: +1 541 766 3758; fax: +1 541 753 9999.
E-mail address: ddai@siga.com (D. Dai).
Present address: Millennium: The Takeda Oncology Company, Cambridge, MA
02139, United States.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.093

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 750–756
751
H2N
H2N
H2N
N
N
N
N
NH2
CCl3
CN
b
c
a
NH
3
1
2
O
O
O
H
N
H
N
O
N
N
N
CN
d
N
20f
NH2
20d
O
O
Scheme 1. Reagents and conditions: (a) methyl 2,2,2-trichloroacetimidate, AcOH, rt, 4 h (40%); (b) 30% NH4OH, EtOH, 0 °C for 1 h, then 100 °C for 2 h (10%); (c) 4-
ethylbenzaldehyde, MeOH, DCM, rt, 1 h, then NaBH4, rt, 1.5 h (57%); (d) concd H2SO4, DCM, ꢀ10 °C to rt, 16 h (42%).
Cl
N
Cl
N
Cl
N
b
c
a
NH2
NHBoc
N
5
6
4
H
Cl
N
Cl
N
N
N
N
e
d
N
8
21e
7
N
I
O
O
Scheme 2. Reagents and conditions: (a) 4 N HCl in 1,4-dioxane, rt, 18 h (99%); (b) 50% aq chloroacetaldehyde, NaHCO3, EtOH, reflux, 4 h, then rt, 16 h (89%); (c) N-
iodosuccinimide, DMF, rt, 6 h (50%); (d) Pd(PPh3)4, 4-ethoxyphenylboronic acid, Na2CO3, 3:1 dioxane/water, 90 °C, 3 h (70%); (e) 4-ethylbenzylamine, Pd2(dba)3, ( )BINAP,
NaOtBu, toluene, reflux 18 h (27%).
H2N
O2N
O2N
NO2
Cl
a
NH2
b
c
NO2
N
N
N
9
NH
10
11
NH
Ar1
Ar1
H
Ar2
N
N
H2N
12
N
d
21h, 22c-d
N
N
N
N
Ar1
Ar1
Scheme 3. Reagents and conditions: (a) H2N–Ar1, TEA, ACN or H2N–Ar1, Cs2CO3, THF, rt, 18 h; (b) H2, Pd/C, EtOAc, 18 h; (c) Formic acid, reflux, 3 h; (d) Ar2CHO, Na(OAc)3BH,
DCM, rt, 3 h.
linked Ar2 portion was attached through a reductive alkylation
with the appropriate aldehyde to form the final compounds (21h
and 22c–d).
Some of the analogs that were designed required a number of
additional steps in order to synthesize the corresponding Ar1 and
Ar2 starting materials to be attached to the core. An example of this
is illustrated in Scheme 4 which outlines the synthetic scheme
used to install an aminomethyl group as part of the Ar2 group.
The carboxylic acid starting material (13) was brominated and
esterified to form intermediate 14. The bromide was substituted
with a cyano group and the methyl ester moiety was saponified
to form 15. Intermediate 16 was isolated through reduction of
the nitrile and carboxylic acid groups with LAH followed by Boc-
protection of the aminomethyl group. The primary alcohol moiety
was oxidized with the Dess–Martin reagent to form the key alde-
hyde intermediate (17). Intermediate 18 was prepared in a similar

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J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 750–756
O
O
O
a,b
c,d
e
O
OH
13
OH
15
14
Br
CN
H2N
N
N
g
N
N
H
N
O
H2
N
Br
N
N
N
N
O
O
OH
16
NHBoc
18
19
H
f
h,i
N
22q
17
NHBoc
O
Scheme 4. Reagents and conditions: (a) Br2, HNO3 (fuming), AgNO3, AcOH, water, rt, 3 h (99%); (b) concd H2SO4, MeOH, reflux, 18 h (58%); (c) CuCN, NMP, reflux, 5 h (99%);
(d) 1 N NaOH, MeOH, rt, 18 h (56%); (e) 1 M LiAlH4 in THF, THF, rt, 18 h, then Boc2O, rt, 18 h (47%); (f) Dess–Martin periodinane, DCM, rt, 18 h (99%); (g) Benzophenone imine,
Pd2(dba)3, ( )BINAP, KOtBu, toluene, 106 °C, 64 h (34%); (h) 19, MeOH, rt, 3 h, then NaBH4, rt, 1 h; (i) 4 N HCl in 1,4-dioxane, THF, rt, 18 h (22% combined yield for final two
steps).
logs with a cyano or carboxylic acid in the 2-position had similar
potency compared to parent compound 20a. Simple amino (20g)
and hydroxyl (20h) analogs showed a large decrease in pseudo-
Table 1
Probing 2- and 7-positions of the benzimidazole scaffold
typed LASV antiviral activity. Overall, the carboxylic acid group
H
N
substitution at the 2-position showed the most promise and of-
fered the potential to improve the solubility profile of a lead com-
pound in this benzimidazole series while retaining potency. As for
the 7-position, only a few substitutions were attempted which in-
cluded a methyl ester group (20i), carboxylic acid (20j) and pri-
mary amino moiety (20k). In all cases there was a dramatic
reduction in antiviral potency.
N
N
R
R'
20a-k
O
More substantial structural modifications were explored by
changing the nitrogen atom positioning of the original benzimid-
azole core. Additional compounds included aza analogs to deter-
mine the effect of adding more nitrogen character to the core
ring system. All of these results are summarized in Table 2. During
this endeavor, indole, indazole, benzotriazole, imidazopyridine,
pyrrolopyridine, and triazolopyridine analogs were tested. For
the indole derivatives, two regioisomers (21a and 21b) were syn-
thesized and the resulting activity was found to be quite different
with a 46-fold and 9-fold decrease in potency compared to 20a,
respectively. Indazole derivative 21c was nearly 100-fold less po-
tent than the corresponding parent benzimidazole compound,
ST-193. The benzotriazole analog (21d) and the bridgehead nitro-
gen containing imidazopyridyl compound 21e were more compa-
rable to parent compound 20a with only a modest increase in
pseudotyped LASV EC50 values. Our most potent analog was dis-
covered with imidazopyridine analog 21h which possessed sub-
nanomolar activity (0.16 nM). The aza analog (21i) of this molecule
(21h) resulted in a decrease in potency similar to results observed
for the same modification to the benzimidazole core (21d com-
pared to 20a). Overall, a significant decrease in antiviral activity
was observed when the nitrogen at the 3-position of the original
benzimidazole scaffold was absent (21a, 21c, and 21f). The identi-
fication of the imidazopyridine scaffold of 21h offered the potential
for an alternate lead series of compounds with improved potency.
After discovering the potency of 21h derived from the 3H-imi-
dazopyridine core, a focused SAR study was performed around this
scaffold (Table 3). This study repeated some of the more favorable
substitutions attempted with the benzimidazole core along with
novel modifications to ultimately improve drug-like properties.
Installation of a polar functionality at the 2-position of the bicyclic
core was replicated on this scaffold utilizing a carboxamide (22a) or
carboxylic acid (22b) moiety. The results of this study were compa-
rable to those observed with the corresponding benzimidazole
Compound
R
R0
LASV EC50 (nM)
20aa
20b
20c
20d
20e
20f
20g
20hb
20i
H
H
H
H
H
H
H
H
H
1.5
1.9
170
1.6
1.5
9.2
29
1800
220
>50,000
620
Me
n-Pr
CN
CO2H
CONH2
NH2
OH
H
CO2Me
CO2H
NH2
20j
20k
H
H
a
Compound included for comparison purposes.
One of two possible tautomers for this analog.
b
fashion as previously reported for ST-193 analogs.1 The bromo
group of 18 was transformed into an amino moiety through a
Buchwald-Hartwig coupling and then reductively alkylated with
aldehyde 17. Compound 22q was isolated following deprotection
of the Boc group with HCl.
Substitution of the benzimidazole core, namely the 2- and 7-
positions, was explored in this particular SAR study and the LASV
pseudotype antiviral activity was determined (Table 1). A variety
of substituents were installed at the 2-position which widely var-
ied in electronic properties as well as hydrogen bond donating/
accepting capabilities. While a methyl group (20b) was tolerated
at this position based upon potency compared to 20a, the addition
of a propyl group (20c) resulted in a nearly 100-fold reduction in
antiviral activity. This observation led us to believe that more focus
should be placed on smaller groups when substituting at the 2-
postion. Groups with electron withdrawing capability, such as cy-
ano (20d), carboxylic acid (20e), and carboxamide (20f) were also
explored at the 2-position. It was interesting to discover that ana-

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 750–756
753
Table 2
Nitrogen substitution within the bicyclic ring system
Compound
Structure
LASV EC50 (nM)
Compound
Structure
LASV EC50 (nM)
HN
HN
N
N
N
21a
69
21f
25
O
O
HN
N
H
N
N
HN
N
21b
13
21g
6.8
O
O
HN
N
HN
N
N
N
21c
130
21h
0.16
N
O
O
HN
HN
N
N
N
N
N
N
N
21d
6.1
21i
0.87
O
O
HN
N
N
21e
9.3
O

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J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 750–756
Table 3
Exploration of the imidazopyridine scaffold
Compound Structure
LASV EC50 (nM) Compound Structure
LASV EC50 (nM)
H
N
H
N
O
N
N
N
N
NH2
N
N
22a
4.4
22j
13
O
O
OH
H
H
N
N
N
O
N
N
N
N
OH
N
22b
0.16
22k
80
O
N
O
H
N
H
N
N
N
N
N
N
N
22c
9.0
22l
11
N
O
NH
O
H
H
N
N
N
N
N
N
N
N
22d
22e
22f
0.98
22m
22n
22o
250
1500
58
N
N
O
O
H
N
N
H
N
N
N
N
N
N
NH2
65
O
O
H
N
H
N
N
N
N
N
N
N
210
NH2
O
O

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755
Table 3 (continued)
Compound Structure
LASV EC50 (nM) Compound Structure
LASV EC50 (nM)
N
N
N
N
H
N
N
N
N
22g
4300
22p
5.9
N
O
O
NH2
H
N
N
H
N
N
N
N
22h
1600
22q
26
O
N
N
O
H
N
N
N
N
22i
19
OH
Table 4
Broad-spectrum arenavirus activity
Compound
LASV EC50 (nM)
MACV EC50 (nM)
JUNV EC50 (nM)
Sabiá EC50 (nM)
Guanarito EC50 (nM)
ST-193
21h
21b
1.4
0.16
13
3.1
28
40
81
0.62
7.9
12a
14
21
54
3.9
93
0.44
3.5
n.d.
7.7
21i
0.87
n.d. = not determined.
a
Average value from only two experiments.
analogs as far as potency differences are concerned. Pyridyl ring
replacement (22c and 22d) of the Ar1 phenyl ring also mirrored re-
sults seen with the benzimidazole core with more favorable nitro-
gen positioning at the 2-position of the Ar1 heterocycle. Additional
modifications included changes to the methylamino linker portion
by increasing the methylene chain length (22e and 22f) and trans-
formation into an imine group (22g) which resulted in a significant
reduction in antiviral activity. The addition of a methylene linker
connecting Ar1 (22h) had a dramatic effect with a 1000-fold reduc-
tion in potency. Various polar functionalities (22i–l) were substi-
tuted at the para-position of the Ar1 phenyl ring which resulted in
at least a 70-fold reduction in potency derived from the LASV
pseudotype assay. An alternative approach was explored with polar
amino moieties installed at various positions of the Ar1 and Ar2
rings while retaining the customary alkoxy and alkyl groups,
respectively. These nitrogen-based groups included dimethylamino
(22m and 22p) and aminomethyl (22n, 22o, and 22q) functional-
ities and were better tolerated when appended to the Ar2 phenyl
ring. Overall, potential solubility enhancement of this imidazopyri-
dine scaffold showed promise with compounds derived from
carboxylic acid placement at the 2-position of the core and ioniz-
able pyridyl ring incorporation at Ar1. Both of these compounds re-
tained most of the antiviral activity observed with the parent
molecule (21h). To assess the impact of the modifications on drug-
likeness, ClogP values of the lead molecules were calculated.7 It
was shown that the modifications provided more favorable hydro-
philicity, especially when compared with the original lead 20a.
The more potent LASV pseudotype inhibitors were tested
against additional arenaviruses which included Machupo (MACV),
Junín (JUNV), Sabiá virus, and Guanarito virus. A small set of these
newer analogs, along with ST-193, are shown in Table 4. The trend
of favorable broad-spectrum arenavirus activity was observed even
for these compounds which contained alternate core scaffolds
away from benzimidazole.
In summary, the SAR study of substitutions on the benzimid-
azole scaffold led to a number of analogs with potent LASV pseud-
otype activity. Some of these analogs may offer improved drug-like
characteristics as they contain polar/ionizable moieties. The study
involving modifications through various nitrogen incorporation
into the core bicyclic system uncovered many alternate scaffolds

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J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 750–756
that offer similar and even enhanced LASV pseudotype activity
when compared to the benzimidazole series. The subnanomolar
analogs have the potential to outperform ST-193 in the LASV gui-
nea pig disease model which already showed favorable protection
from death compared to ribavirin, the current method of treatment
for Lassa hemorrhagic fever. These alternate scaffolds have value
not only as backup compound series but also for securing addi-
tional intellectual property space.
References and notes
1. Dai, D.; Burgeson, J. R.; Gharaibeh, D. N.; Moore, A. L.; Larson, R. A.; Cerruti, N. R.;
Amberg, S. M.; Bolken, T. C.; Hruby, D. E. Bioorg. Med. Chem. Lett. 2012. http://
dx.doi.org/10.1016/j.bmcl.2012.11.095.
2. Larson, R. A.; Dai, D.; Hosack, V. T.; Tan, Y.; Bolken, T. C.; Hruby, D. E.; Amberg, S.
M. J. Virol. 2008, 82, 10768.
3. The reported EC50 values are averages (geometric mean) of at least three
experiments unless otherwise noted.
4. Cashman, K. A.; Smith, M. A.; Twenhafel, N. A.; Larson, R. A.; Jones, K. F.; Allen, R.
D., III; Dai, D.; Chinsangaram, J.; Bolken, T. C.; Hruby, D. E.; Amberg, S. M.;
Hensley, L. E.; Guttieri, M. C. Antiviral Res. 2011, 90, 70.
Acknowledgements
5. Bonfanti, J-F.; Muller, P.; Fortin, J. M. C., Doublet, F. M. M. PCT Application
WO2006136563, December 28, 2006.
6. Mosby, W. L. Heterocyclic Systems with Bridgehead Nitrogen Atoms In The
Chemistry of Heterocyclic Compounds, A Series of Monographs; Weissberger, A.,
Ed.; Interscience Publishers: New York, 1961; Part Two, p 749.
7. The ClogP values were calculated with Pipeline Pilot of Accelrys, Inc. (San Diego,
CA). The values for compounds 20a, 21h, 21i, 22b and 21d are 5.70, 4.84, 4.93,
4.88 and 4.22, respectively.
We gratefully acknowledge the financial support from NIH
Grants 5R44AI056525 and 1R01AI093387. We thank Kayla Kickner,
Jordan Boutilier, and Keith McKinley for technical support. We also
thank Adesis, Inc. for synthesizing the majority of the compounds
used for this SAR study.