Asymmetric synthesis of host-directed inhibitors of myxoviruses

Article abstract of DOI:10.3762/bjoc.9.23

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC₅₀ values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

Full text of DOI:10.3762/bjoc.9.23

Asymmetric synthesis of host-directed
inhibitors of myxoviruses
Terry W. Moore1, Kasinath Sana1, Dan Yan2,3, Pahk Thepchatri1,
John M. Ndungu1, Manohar T. Saindane1, Mark A. Lockwood1,
Michael G. Natchus1, Dennis C. Liotta1,4, Richard K. Plemper2,3,5,
James P. Snyder1,4 and Aiming Sun*1,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 197–203.
1Emory Institute for Drug Development, Emory University, 1515
Dickey Drive, Atlanta, GA 30322, USA, 2Department of Pediatrics,
Emory University School of Medicine, 2015 Uppergate Drive, Atlanta,
GA 30322, USA, 3Children’s Healthcare of Atlanta, 2015 Uppergate
Drive, Atlanta, GA 30322, USA, 4Department of Chemistry, Emory
University, 1515 Dickey Drive, Atlanta, GA 30322, USA and
5Department of Microbiology & Immunology, Emory University School
of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA
doi:10.3762/bjoc.9.23
Received: 26 September 2012
Accepted: 07 January 2013
Published: 30 January 2013
This article is part of the Thematic Series "Synthetic probes for the study
of biological function".
Email:
Guest Editor: J. Aubé
Aiming Sun* - asun2@emory.edu
© 2013 Moore et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
§ Phone: 404-712-8680; Fax: 404-727-6689
Keywords:
asymmetric synthesis; benzimidazole; host-directed; myxovirus; small
molecule inhibitor
Abstract
High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity
against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we
sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent
antiviral activity against a panel of myxovirus family members (EC50 values in the low nanomolar range) and much improved
aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in
an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent
than the corresponding (R)-isomers.
Introduction
Myxoviruses are divided into two evolutionarily distinct yet includes respiratory syncytial virus (RSV), measles virus
related families: the orthomyxoviridae, which is composed (MeV), human parainfluenza virus (HPIV) and others [1].
largely of the influenza viruses, and the paramyxoviridae, which Because myxoviruses are responsible for the majority of human
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Beilstein J. Org. Chem. 2013, 9, 197–203.
morbidity and mortality cases due to viral respiratory illness
globally, a therapeutic strategy that targets these viruses could
have a substantial impact on human health [2-5]. Although
antivirals typically seek to disable viral proteins, cellular host
proteins can also be targeted, as is the case with selzentry,
which inactivates the coreceptor (CCR5) for human immunode-
ficiency virus (HIV) entry [6]. The former approach is likely to
yield compounds with a narrow spectrum of antiviral activities,
and such inhibitors will certainly face the inevitable challenge
of resistance [7,8]. Our research group has been actively
engaged in the identification of small molecule inhibitors
against myxoviruses in recent years [8-12], with a particular
focus on the development of agents that target host-cell proteins
enabling viral reproduction. Advantages of this strategy include
a vastly expanded list of potential targets; a broader spectrum of
activity, because many of the relevant host proteins are shared
among related viruses; and, in principle, less susceptibility to
the development of resistance.
Figure 1: Structure of first-generation lead compound 1.
Results and Discussion
Design. We previously reported a series of compounds with
antiviral activity against a number of myxoviruses [8,12,14].
Structure–activity relationship (SAR) studies suggested both the
2-chloro-4-methylanilide and the central α-thiopropionamide to
be moieties that confer good activity. Relatively unexplored in
our previous work was the importance of the p-methoxyphenyl
ring as well as the influence of the stereochemistry at the chiral
center. In the current work, we examine the replacement of the
p-methoxyphenyl ring with basic moieties that may increase
aqueous solubility while maintaining activity, and we also
developed synthetic routes to produce each enantiomer of these
compounds.
Using high-throughput screening, in combination with counter-
screening for detecting a broadened viral target spectrum that
extends to other pathogens of the myxovirus families, our
research group has been successful in identifying small-mole-
cule antiviral hits resident in host cells [13]. One such molecule
recently described is benzimidazole 1 [14] (Figure 1). Although
the compound is active in vitro against a number of different Synthesis. The compounds were prepared by modifications of
para- and orthomyxoviruses, 1 has poor water solubility our previously reported routes. Briefly, nitroanilines 7 were
(<15 μg/mL), which may contribute to its low oral bioavail- obtained by one of two routes: ortho-nitrobenzaldehyde (2) was
ability [15]. Additionally, it was shown that the methyl group at treated with N-methylpiperazine (3) in the presence of sodium
the stereogenic center alpha to the carbonyl is important for bio- triacetoxyborohydride to give nitrobenzene 4, which was
logical activity [8,12,14]. Compound 1 was previously prepared reduced under hydrogenation conditions to provide aniline 5.
as the racemate, but subsequently separated into enantiomers by o-Fluoronitrobenzene (6) was coupled with the previously
chiral HPLC. To enable large-scale preparation of the pure formed aniline under SNAr conditions to furnish anilino
isomers for further pharmacokinetic and animal studies, we nitrobenzene 7a (Scheme 1). Alternatively, meta- and para-
present here an asymmetric synthesis of 1 and its congeners nitrophenylethanols 8 were combined with o-fluoronitroben-
with improved aqueous solubility and antiviral potency.
zene (6) to deliver o-nitroanilines 9. The hydroxy groups of 9
Scheme 1: Synthesis of anilino nitrobenzene 7a.
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Beilstein J. Org. Chem. 2013, 9, 197–203.
were activated as the p-nitrobenzenesulfonates 10 and displaced aniline (16) by using HATU (O-(7-azabenzotriazol-1-yl)-
with morpholine to give o-nitroanilines 7b and 7c (Scheme 2). N,N,N',N'-tetramethyluronium hexafluorophosphate), a reagent
Hydrogenation was used to reduce o-nitroanilines 7 followed by known to limit racemization in peptide coupling reactions [16].
cyclization using thiocarbonyldiimidazole to yield benzothia- The enantiomeric excesses of the starting materials were ca.
zoles 11. In the case of the racemates, these were combined 95%. No erosion of stereochemistry in the products 12 was
with α-bromopropionamide 12 to afford racemic 14 (Scheme 3). observed as determined by chiral HPLC. Recrystallization of 12
from ethanol did not increase the enantiomeric excess of the
α-bromoamide. Interestingly, the final product can be achieved
in both high yield and ee by adding the preformed potassium
salt of the 2-mercaptobenzimidazole 11 to (S)- or (R)-α-bromo-
propionamides 12. Using a substoichiometric amount of potas-
sium carbonate (0.9 equiv) in warm methanol, followed by
removal of solvents under vacuum, provided a convenient route
to the potassium salt of 11. The salt was suspended in anhy-
drous DMF and added dropwise to a cold (0 °C) solution of the
previously formed bromides, to give the desired products 1 and
14 in high yield (80–90%) with little to no loss in enantiomeric
excess. Isolating the potassium salt proved necessary, as
attempts at preforming the potassium salt in dry DMF, fol-
lowed by addition to the chiral α-bromoamide, led to ee’s of ca.
60%. Although both chiral acids are readily available from
commercial sources, their enantiomeric excesses seem to be
limited to ca. 95%. For these studies, ee’s >90% were suffi-
Scheme 2: Preparation of morpholinyl-o-nitroanilines.
cient; if material with higher enantiopurity is needed, we note
that one recrystallization of (S)-1 from ethyl ether increases the
To address the issue of stereochemical control, we developed enantiomeric excess from 94% to 97.4%.
two strategies for preparing the compounds enantioselectively.
The first approach utilized commercially available (R)- or (S)-2- In an alternative approach that provides material with high
bromopropionic acid (15) from the chiral pool in a two-step enantiomeric excess (>98% ee), we utilized the Mitsunobu reac-
sequence (Scheme 4a). The first step involved amide bond for- tion of 2-mercaptobenzimidazoles with an amide obtained from
mation of 2-bromopropionic acid (15) with 2-chloro-4-methyl- (L)-lactic acid (17). Using one equivalent each of 2-mercapto-
Scheme 3: Asymmetric synthesis of (R)- and (S)-isomers by using two different approaches.
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Beilstein J. Org. Chem. 2013, 9, 197–203.
Scheme 4: Preparation of chiral propionamides by HATU-mediated coupling (a) or thionyl chloride-mediated coupling (b).
benzimidazole and α-hydroxyamide 13 (prepared from thionyl
chloride-mediated coupling of (L)-lactic acid (17) and 2-chloro-
4-methylaniline (16) [17] (Scheme 4b)) in the presence of a
slight excess of triphenylphosphine and diethyl azodicarboxy-
late, we acquired the desired products 14 in good yield and ee.
This methodology is attractive because of the high ee that can
be obtained from the inexpensive and readily available (L)-
lactic acid (17). However, the enantiomeric (D)-lactic acid is
substantially more expensive, and as seen below, the products
arising from (L)-lactic acid are less active, making this route
less attractive than the route utilizing chiral α-bromopropion-
amides 12.
Crystal structures of (R)- and (S)-1 obtained by crystallization
from ethyl ether allowed us to unambiguously assign the
absolute configuration of each enantiomer. Shown in Figure 2
are the (S)-enantiomer (left, magenta) and (R)-enantiomer
(right, cyan) of 1. It is interesting to note that the hydrogen-
Figure 2: Renderings of crystal structures of (S)- (left, magenta) and
(R)- (right, cyan) enantiomers of 1.
bond formed between the amide N–H and the unsubstituted improve solubility compared with the parent. Several com-
benzimidazole nitrogen in these crystal structures results in pounds exhibited moderate antiviral activities but poor to
pseudo seven-membered rings. Whether this conformation is moderate aqueous solubilities (i.e., 1, 18b, 18d, and 18f,
biologically relevant is unknown.
Table 1). Since 14a, 14b, and 14c showed good antiviral activi-
ties, as well as moderate aqueous solubilities, we decided to
examine the broader antiviral activities of these compounds and
Structure–activity relationships
A small set of compounds was synthesized based on variations to determine what, if any, effect the stereocenter present in each
of 1 by replacing the p-methoxyphenyl group with other substi- of these compounds may cause.
tuted phenyl rings or heterocyclic rings. The compounds were
initially assayed in two screening assays: (1) a measles virus We were motivated by the results of these two assays to more
cytopathic effect (CPE) reduction assay [12] and (2) a solu- completely characterize the antiviral activities and solubility
bility assay based on laser nephelometry [18]. Unfortunately, parameters of the most promising compounds. We assayed the
compounds with the highest aqueous solubilities (>100 µg/mL) compounds in three additional biological assays: (1) a firefly
had the poorest antiviral activity (i.e., 18a and 18e, Table 1). To luciferase minireplicon assay whose output is driven by infec-
explore the possibility of increasing solubility by salt formation, tion with influenza A/sw/Texas/2009 (WSN); (2) an assay using
the L-tartaric acid salt and benzenesulfonic acid salts of the a renilla luciferase reporter embedded as an additional transcrip-
most active compound 18f were synthesized (Figure 3) and tion unit in the genome of a measles virus (MeV) recombinant;
subjected to solubility testing. However, these salts failed to and (3) a colorimetric assay that measures reduction of MTT
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Table 1: In vitro screening of analogues of 1.
Compd. MeVa (CPE, µM)
Aqueous solubilityb
(µg/mL at pH 7.4)
Compd.
MeVa (CPE, µM)
Aqueous solubilityb
(µg/mL at pH 7.4)
1
3.1
>75
>0.29
>75
4.9
<15
140
19
18f-tartrate
NDc
ND
<15
<15
<15
20
18a
18b
18c
18d
18e
18f
18f-benzene sulfonate
18g
14a
14b
14c
0.05d
0.179
0.20
0.6
<15
22
15
>75
0.27
120
<15
25
a50% inhibitory concentrations were calculated by using the variable-slope (four parameters) nonlinear regression-fitting algorithm embedded in the
Prism 5 software package (GraphPad Software). Values represent averages of four experiments; highest concentration assessed, 75 μM.
bDetermined through laser nephelometry; cND. Not determined. dVirus yield reduction assay was used.
Figure 3: L-Tartaric acid salt (18f-tartrate) and benzenesulfonic acid salt (18f-benzenesulfonate) of 18f.
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Analysis of the data reveals several trends. First, with the
as a surrogate for general cytotoxicity, reported here as CC50. exception of a single case in which enantiomer potencies are
Additionally, the aqueous solubilities of the compounds were similar (14c, ΔEC50 < 3-fold), six other comparisons reveal the
measured by using laser nephelometry at pH 3.0, 5.0, and 7.4 (S)-enantiomer to be more active than the (R)-form by 7 to 110-
(Table 2).
fold. Although we do not know the identity of the specific bio-
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Beilstein J. Org. Chem. 2013, 9, 197–203.
Table 2: Antiviral potencies and solubilities for 1 and analogues.
Compd.
R
EC50 (nM)a
Solubility (µg/mL)b
CC50 (nM)c
MeV
WSN
pH 3.0
pH 5.0
pH 7.4
1
31
37
880
13
5
160
>50000
>50000
>50000
(S)-1
(R)-1
<15
<15
<15
14b
(S)-14b
(R)-14b
13
11
210
0.3
1
110
>50000
>50000
>50000
158
33
15
14c
(S)-14c
(R)-14c
110
180
530
43
36
120
NDd
ND
ND
212
51
92
25
20
14a
(S)-14ae
(R)-14ae
41
68
470
8
4
120
ND
25000
ND
>300
a50% effective concentrations were calculated by using the variable-slope (four parameters) nonlinear regression-fitting algorithm embedded in the
Prism 5 software package (GraphPad Software). Values represent averages of four experiments; highest concentration assessed 75 μM.
bDetermined through laser nephelometry. cDetermined through MTT assay, highest concentration assessed 50 μM. dND: Not determined. eAssayed
as a 1:1 mixture of atropisomers. Details of the separation of the atropisomers will be discussed elsewhere.
logical target(s), the enantio-discrimination implies to us that Among the compounds surveyed, (S)-14b is the most potent
the molecules bind in a well-defined binding pocket that is able compound, with EC50 values of 1–11 nM against the two
to accommodate the S-enantiomer more readily than the (R)- viruses. Because the CC50 values of the compounds are greater
enantiomer.
than 50 µM, the upper limit of the assay, we assume that the
compounds are not generally cytotoxic, giving selectivity
We note that the activity trends for the (S)- and (R)-enan- indices (CC50/EC50) for the active enantiomers of at least
tiomers against the measles and WSN influenza strain are quali- 103–104.
tatively similar. However, for antiviral potency differences
between the racemate and the (S)-enantiomer, the attenuation is We have also assayed the most active compounds in a human
indistinguishable under the testing conditions. Accordingly, we parainfluenza viral (HPIV) titer assay based on plaque assay
attribute the assay discrepancy to the inherent variability in the titration. The values for (S)-1, (S)-14a, (S)-14b, and (S)-14c are
assay system.
80, 13, 80, and 11 nM, respectively. These data further corrobo-
rate the broad-spectrum activity of these compounds.
While the compounds appear to be active against both influenza
and measles virus, they are somewhat more active against the Lastly, the solubilities of the compounds have been improved
influenza virus strain (WSN) than against the measles virus. relative to that of 1. At all pH values examined, the aqueous
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basic amine functionalities have improved solubilities relative
to 1, particularly at acidic pH values, but also at pH 7.4.
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Conclusion
We have extended our previously published work on host-
directed inhibitors of myxovirus replication by preparing
analogues that positively address the poor aqueous solubility of
1 (JMN3-003) while simultaneously improving its potency. The
compounds presented here furnish EC50 values as low as 1 nM
with aqueous solubilities ranging from 15–25 µg/mL at pH 7.4
to >300 µg/mL at pH 3.0. Additionally, we have developed two
complementary methods for the synthesis of each of the enan-
tiomers of the compounds discussed and have unequivocally
demonstrated that the (S)-enantiomer is more active in this
series than the (R)-enantiomer. Further work from our labora-
tories regarding the in vivo efficacy of these compounds is
underway.
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Supporting Information
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Contains detailed synthetic procedures and characterization
data for molecules described herein, a more detailed
description of the laser nephelometry assay, and data tables
for the crystal structures of (S)-1 and (R)-1.
14.Sun, A.; Ndungu, J. M.; Krumm, S. A.; Yoon, J.-J.; Thepatchri, P.;
Natchus, M.; Plemper, R. K.; Snyder, J. P. ACS Med. Chem. Lett.
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Supporting Information File 1
15.Pharmacokinetics Study of JMN3-003. Unpublished results.
16.Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397–4398.
doi:10.1021/ja00063a082
Detailed synthetic procedures and characterization data.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-23-S1.pdf]
17.Nair, V. P.; Mustafa, S. M.; Mohler, M. L.; Yang, J.; Kirkovsky, L. I.;
Dalton, J. T.; Miller, D. D. Tetrahedron Lett. 2005, 46, 4821–4823.
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Acknowledgments
This work was supported, in part, by Public Health Service
Grants AI071002 and AI085328 (to R. K. P.) from the NIH/
NIAID and by Public Health Service Grant HG003918-02 (to
J.P.S.) from the NIH. We also thank Deborah Culver for solu-
bility testing and Dr. John Bacsa for helpful discussions of
crystal structures.
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