Characterization of lassa virus cell entry inhibitors: Determination of the active enantiomer by asymmetric synthesis

Article abstract of DOI:10.1016/j.bmcl.2009.04.098

The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypi

Full text of DOI:10.1016/j.bmcl.2009.04.098

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3771–3774
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Characterization of lassa virus cell entry inhibitors: Determination of the
active enantiomer by asymmetric synthesis
Landon R. Whitby a, Andrew M. Lee b, Stefan Kunz d, Michael B. A. Oldstone b,c, Dale L. Boger a,
*
a Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
b Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
c Department of Infectology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
d Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne CH-1011, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry
inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination
of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the
more potent (–)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer.
The absolute configuration of (–)-3 was established by asymmetric synthesis of the active inhibitor (–)-
(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of
the prototypical inhibitors.
Received 30 March 2009
Revised 20 April 2009
Accepted 21 April 2009
Available online 3 May 2009
Keywords:
Viral entry inhibitors
Lassa fever virus
Ó 2009 Elsevier Ltd. All rights reserved.
A serious threat to public health in areas of Africa and South
America is a group of arenaviruses that cause severe viral hemor-
rhagic fevers in humans with rates of fatality among hospitalized
patients in the range of 15–35%.1 These arenaviruses, which
include the Old World Lassa virus2,3 (LASV) and the New World
arenaviruses Junin2 (JUNV), Guanarito4 (GTOV), and Machupo4
(MACV), have been classified as Category A pathogens by the Cen-
ters for Disease Control (CDC) due to the extremely high mortality
rates and because there currently exists limited therapeutic
options to combat these human pathogens.5 The need for effective
treatments against these arenaviruses is substantial not only in the
viral endemic regions, but also worldwide as international air traf-
fic has facilitated the spread of arenaviral hemorrhagic fever cases
into metropolitan areas.6,7 In order to facilitate the discovery of
small molecule therapeutics to treat these hemorrhagic fevers, a
high-throughput screen utilizing pseudotyped virion particles
bearing the glycoproteins (GPs) of these arenaviruses was devel-
oped and utilized to screen a series of synthetic combinatorial
libraries for inhibitors of cellular infection.8 Emerging from these
screening efforts was a series of 4-acyl-1,6-dialkylpiperazin-2-
ones9 that were found to be the first potent and selective inhibitors
of hemorrhagic arenavirus cellular entry (Fig. 1A).8 These small
molecules proved to be potent viral entry inhibitors against both
Old World and New World hemorrhagic arenaviruses not only
using pseudotyped virion particles, but they also effectively inhib-
ited infection of human and primate cells with live hemorrhagic
fever viruses with IC50 values of 500–800 nM (conducted by
CDC).8 Characterization of their mechanism of inhibition revealed
that compounds 1–3 selectively block the late endosomal pH-
dependent fusion mediated by the arenavirus GPs, exhibiting IC50
values of 200–350 nM in a GP-mediated cell–cell fusion assay
and defining a new stage at which to inhibit viral entry (Fig. 1B).8
Compounds 1–3 emerged from the initial screen of our
library10–13 (ca. 95,000 compounds) as remarkably potent and ro-
bust inhibitors already possessing drug-like characteristics. This
trisubstituted piperazinone scaffold is clearly well-suited in the
spatial and configurational display of the aryl substituents neces-
sary for interaction with the protein target which mediates the
inhibitory activity of the compounds. Evidence for this can be
found in the well-defined structure–activity relationship (SAR)
exhibited by the 150-membered piperazinone library from which
the lead compounds emerged. Analysis of the trends indicated that
key elements important for activity included the closely related 2-
indole, 2-benzofuran, or 2-benzothiophene aryl substituent
attached to N4 of the piperazinone core. These bicyclic aryl substit-
uents proved more potent than the corresponding 2-pyrrole, 2-fur-
an, 2-thiophene, or phenyl aryl substituents defining a clear
pattern of activity. Similarly, although not as widely explored,
the activity was observed most often or significantly with a phen-
ethyl C6 substituent (PhCH2CH2 > PhCH2 > Et) and the nature of the
aryl substitution at N1 significantly modulated this activity. Impor-
tant in this work was the number of active inhibitors that arose
from the piperazinone sublibrary during the primary screening.
Only compounds 1–3 were further characterized in the initial stud-
ies that subsequently focused on defining the scope and selectivity
* Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784 7550.
E-mail address: boger@scripps.edu (D.L. Boger).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.098

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L. R. Whitby et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3771–3774
O
A
A
O
S
O
N
N
N
N
X
O
O
N
O
O
N
O
N
N
O
N
16H9
16H8
O
O
O
Cl
Cl
3
O
N
(16G8)
X = O, 1 (17C8)
X = S, 2 (17C9)
OMe
Me
N
NH
NH
N
N
O
B
16D7
17C7
F3C
OMe
N
O
O
N
N
O
NH
O
O
17A8
16F7
O
F3C
OMe
O
N
N
O
N
N
N
O
Me
O
16D8
17D2
F3C
Me
O
O
N
N
O
O
N
MeO
O
16F8
17D1
OMe
Me
B
Compound IC50 (µM)
17C8 (1)
17C9 (2)
16G8 (3)
16H8
1
0.5
0.8
0.8
2.1
2.4
1.7
1.2
1.8
1.6
1.2
0.6
0.4
16H9
16D7
Figure 1. (A) Lead compounds. (B) Mechanism of inhibition of hemorrhagic
arenaviruses by compounds 1–3. The compounds inhibit infection by blocking late
endosomal pH-dependent membrane fusion. RNP = ribonucleoparticle.
17C7
16F7
17A8
of their activity and the underlying mechanism.8 However, 10
additional compounds were identified that displayed significant
activity comparable to compounds 1–3 in the LASV pseudotype
assay and they have now been characterized (IC50 determination,
Fig. 2). The evaluation of these additional structures reveals that
a consistently potent combination of aryl substituents is C6 benzyl
combined with the benzofuran at N4 and a para-substituted
(Me = ClꢀOMe) benzyl group on N1 (see 16G8 (3), 16H8, and
16F8). 3-Trifluoromethyl substitution on the N1 benzyl ring or
replacement of the benzofuran with a benzothiophene (16H8 vs
16H9) or indole (16F7 vs 16F8) all proved slightly less active.
Another potent combination contains the C6 phenethyl substituent
and a para-substituted N1 benzyl substituent. Unlike the C6 benzyl
series, however, there is substantial flexibility at the N4 position for
16D8
17D2
16F8
17D1
Figure 2. (A) Additional LASV entry inhibitors. (B) Inhibition of LASV pseudotype
infection of permissive human A549 lung epithelial cells, see Ref. 8.
this series, with the benzothiophene, benzofuran, indole,
p-methoxyphenyl and tolyl groups all leading to active
compounds. Direct comparison of these N4 groups reveals that

L. R. Whitby et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3771–3774
3773
Me
Me
1. TBSCl
4
2. p-tolualdehyde
∗
NaBH4
TBSO HN
BocN
N
O
HO
NH2
5
6
1. Boc-Gly
EDCI
2. Bu4NF
Me
ChiralCel OD
10% i-PrOH/hexanes
α = 1.38
Me
Me
Me
1. TosCl
Pyr
HO
N
O
TosO
N
O
2. TFA
BocN
N
BocN
N
O
8
7
BocHN
TFA•H2N
O
[α]24D −48
(c 0.3, CHCl3)
[α]24D +52
(c 0.3, CHCl3)
DIPEA
Δ
Me
Me
1. 4N HCl/dioxane
EDCI
O
N
N
2. benzofuran-2-
carboxylic acid
EDCI
O
HN
N
O
O
9
3
(S)-
[α]24D −30
Me
Me
(c = 0.1, CH2Cl2)
O
O
Scheme 2. Synthesis of (S)-3 (lassamycin-1).
N
N
N
N
O
O
O
O
-3
optimization libraries is based on the use of
the starting material for 3 (Scheme 2). The synthesis was accom-
plished starting with silylation of -phenylalaninol (5) using TBSCl
L-phenylalaninol as
(−)
-3
(+)
[α]24D +34
[α]24D −36
L
(c 0.3, CHCl3)
IC50 = 3.0 μM
(c 0.2, CHCl3)
IC50 = 0.2 μM
(lassamycin-1)
(imidazole, CH2Cl2) to give the O-silyl ether followed by a stepwise
reductive amination14 with p-tolualdehyde entailing pre-forma-
tion of the imine and subsequent NaBH4 reduction (MeOH) to give
intermediate 6. Compound 6 was then coupled to Boc-glycine to
generate the tertiary amide (EDCI, HOAt, 2,6-lutidine, DMF) and
was followed by TBS removal effected by treatment with Bu4NF
(THF) to give 7. Formation of the tosylate of 7 was accomplished
upon treatment of 7 with tosyl chloride (TosCl) and pyridine
(CH2Cl2) and subsequent Boc deprotection with TFA (1:1 TFA/
CH2Cl2) gave the cyclization precursor 8. Warming 8 at 50 °C in
DMF in the presence of diisopropylethylamine (DIPEA) resulted
in cyclization to the piperazinone 9, which was then coupled to
the benzofuran-2-carboxylic acid (EDCI, HOAt, 2,6-lutidine, DMF)
to furnish (S)-3. The optical rotation of (S)-3 was measured
Scheme 1. Synthesis and characterization of both enantiomers of 16G8, (–)-3
(lassamycin-1) and (+)-3.
benzothiophene, benzofuran, and indole exhibit very comparable
activity (compare 17C9 (2), 17C8 (1), and 17C7) and p-methoxy-
phenyl>tolyl (compare 17D1 and 17D2). Several of these addition-
ally characterized compounds proved to be equally (16H8) or even
more potent (16F8, 17D1) than the original lead compounds in
Figure 1A. These additional inhibitors of LASV GP-mediated cellular
infection represent exciting additional lead structures available to
pursue as we move forward in optimization of these compounds
for in vivo examination.
(½a 2D4
ꢁ
ꢂ 30 (c 0.1, CH2Cl2), >98% ee) and found to match that of
Among the first and most important questions yet to be
addressed in the examination of this initial set of hemorrhagic are-
navirus entry inhibitors was the activities of the individual enanti-
omers of the compounds. To address this question and after
unsuccessful efforts to resolve 1–3 directly, we found that the pen-
ultimate Boc-protected precursor to 3, but not those leading to 1–
2, could be resolved into its enantiomers using chiral HPLC
(Scheme 1). N-Boc deprotection of the individual enantiomers of
4 (4 N HCl/dioxane) and subsequent acylation of the free amine
with benzofuran-2-carboxylic acid (EDCI, HOAt, 2,6-lutidine,
DMF) gave (+)-3 and (–)-3 (Scheme 1). Examination of the two
enantiomers of 3 in the LASV pseudotype assay revealed a consid-
erable difference (15-fold) in their inhibitory activity, with (–)-3
being slightly more potent than the racemic mixture ( )-3.
Having established that there is a substantial difference in the
activity of the enantiomers of 3, we undertook the assignment of
the absolute configuration of the active (–)-3 by synthesis of (S)-
3 and subsequent comparison. An attractive approach to (S)-3 that
could be generalized to the preparation of subsequent lead
Me
O
O
N
NH
N
N
O
O
O
10
inactive
11
IC50 = 3.1 μM
Me
Me
N
N
HN
N
O
12
O
9
IC50 = 10 μM
inactive
Figure 3. Partial structures of (S)-3 (lassamycin-1).

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L. R. Whitby et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3771–3774
the active inhibitor (–)-3 to which we can now assign its absolute
configuration as (S).
the molecule with a methylene group resulted in a significant loss
in activity (15-fold and 50-fold) and that further removal of either
the N1 or N4 aryl substituents completely abolished activity.
We also conducted a modest deletion scan of (S)-3 that would
enable us to confirm the importance of the aryl substituents and
define that of the carbonyl groups attached to the N1 and N4
centers (Fig. 3). We synthesized two partial structures in which
the C2 carbonyl oxygen (11) and N1 aryl substituent (10) were
successively removed and the two partial structures in which the
N4-substituent carbonyl oxygen (12) and the N4 substituent (9)
were successively removed.15 Evaluation of these partial structures
in the LASV pseudotype assay revealed that both carbonyl to meth-
ylene modifications resulted in a substantial loss of activity, with
Acknowledgments
We gratefully acknowledge the financial support of the National
Institutes of Health (CA78045 to D.L.B.; AI55540 to M.B.A.O.). Lan-
don R. Whitby is a Skaggs Fellow and Andrew M. Lee was sup-
ported by
(AI072994).
a Kirschstein National Research Service Award
11 showing
a
15-fold loss of activity compared to (S)-3
References and notes
(IC50 = 200 nM) while compound 12 showed a 50-fold loss, demon-
strating that the carbonyl oxygens are key elements in the potent
inhibitory activity of (S)-3. The removal of the aryl substituents
at either N1 (10) or N4 (9) gave completely inactive compounds
and further underscored the critical nature of each of the aryl
subunits towards activity.
Ten additional trisubstituted piperazinones were characterized
as inhibitors of LASV GP-mediated cellular infection representing
the first set of small, drug-like molecules with potent and robust
activity that are available for further investigation and optimiza-
tion against hemorrhagic arenavirus infections. Resolution of the
enantiomers of the initially reported inhibitor 3 revealed that
(–)-3 (IC50 = 200 nM) is 15-fold more potent than (+)-3 and slightly
more potent than ( )-3. Development of an enantioselective route
to (S)-3 and comparison of its optical rotation with that of the
active enantiomer allowed the assignment of the absolute configu-
ration of (–)-3 as (S). In the process, an effective synthetic route
was developed to obtain the optically active trisubstituted piperaz-
inones starting from an enantiopure amino alcohol (such as phe-
nylalaninol), which can be obtained from readily available
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optically active
a-amino acids. This route can now be utilized to
access a variety of optically active 4-acyl-1,6-dialkylpiperazin-2-
ones bearing a natural or unnatural amino acid side chain as the
substituent on the C6 chiral center. We also conducted a limited
deletion scan of (S)-3 probing the importance of the two carbonyl
groups in the molecule as well as the N1 and N4 aryl substituents.
Results of this scan revealed that replacement of either carbonyl in
15. The synthesis of 9 is shown in Scheme 2 and 12 was synthesized from 9 by
reductive amination with benzofuran-2-carboxaldehyde (NaBH(OAc)3,
ClCH2CH2Cl). The synthesis of 10 and 11 started from commercially available
(S)-N1-Boc-2-benzylpiperazine (Anaspec). Coupling at N4 with benzofuran-2-
carboxylic acid (EDCI, HOAt, 2,6-lutidine, DMF) followed by Boc deprotection
(HCl, dioxane) gave 10. Compound 11 was prepared from 10 by reductive
amination with p-tolualdehyde (NaBH(OAc)3, ClCH2CH2Cl).