ACS Medicinal Chemistry Letters
Letter
ing ortho-analogues, the ortho-position was found to
accommodate a larger group such as benzyloxy (10b vs 10l).
Therefore, we decided to explore the 2-benzyloxy group
further. Substituted 2-(aryloxy)benzylpiperazine derivatives
were prepared according to Scheme 4. Thus, alkylation of
salicylaldehyde by variously substituted benzyl bromide was
followed by reductive amination (Scheme 4). The majority of
the analogues did not differ significantly in potency with the
notable exception of the 4′-carboxylic ester 2, which exhibited
dramatically enhanced antiviral activity (IC50 = 0.02 μM, Table
4).
Importantly, 2 did not inhibit VSV G or Lassa fever virus GP-
dependent infection or reduce the growth or ATP content of
target cells. The marked increase in activity of 2 was a surprise
as other closely related analogues such as 3′-ester (11e) and 4′-
amides (11i and 11j) were >100-fold less active. The activity of
11l was also <1% of 2 in the cell-free assay for antiviral activity,
thus discounting the possibility that the product of esterase-
mediated hydrolysis of 2 is the active species in the cell. The
addition of similarly sized ethynyl group in the same position as
the 4′-carboxylic acid methyl ester (3, see below) also enhanced
antiviral activity, suggesting that hydrophobic and/or van der
Waals interactions of the methyl ester group with the target are
beneficial. The antiviral activities of 1 and 2 were confirmed
using an EboV growth assay on Vero cells.8
antiviral compounds target NPC1 and interfere with NPC1
binding to cleaved EboV GP that is required for infection.
In summary, we have identified adamantane dipeptides by
high-throughput screening and hit-to-lead optimization that
target the lysosome membrane protein NPC1 and interfere
with its function as a receptor for EboV GP. In this regard, the
chemical basis of the anti-EboV activity of the adamantane
dipeptides may be analogous to that of the Food and Drug
Administration (FDA)-approved drug maraviroc, which inhibits
human immunodeficiency virus (HIV) infection by targeting
the gp120 receptor CCR5.15 Adamantane dipeptides are a new
class of antiviral compounds.
ASSOCIATED CONTENT
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S
* Supporting Information
Experimental protocols for synthesis and characterization of
compounds, cell culture, virus infection, cell membrane
purification, virus GP binding, and photoactivation and click
chemistry. This material is available free of charge via the
AUTHOR INFORMATION
■
Corresponding Author
Functional studies strongly suggest that NPC1 is the target of
the antiviral compounds.8 We developed derivatives of 2
suitable to test this hypothesis. On the basis of the SAR
described above, we chose the 5-position of the (2-aryloxy)
benzylpiperazine for attachment of an azide for photoactivation
by ultraviolet light and the 4′-position acetylene for an alkyne
for conjugation to Alexa Fluor 488 azide by click chemistry.
Toward these goals, compound 3 was prepared according to
Scheme 5. First, 5-nitrosalicylaldehyde was alkylated by 4-
ethynylbenzyl bromide, prepared from the corresponding
alcohol, to give the benzyl ether 12.12 The piperazine diamide
intermediate 9 was then reductively alkylated by 12 to give 13.
Reduction of the nitro group was followed by diazotization in
the presence of sodium azide to obtain the desired bait 3.13,14
Gratifyingly, 3 retained potent anti-EboV activity (IC50 < 0.5
μM) and was not cytotoxic.
Late endosomal/lysosomal membranes from Chinese
hamster ovary fibroblasts lacking NPC1 (CHONull) and
Chinese hamster ovary fibroblasts expressing NPC1
(CHONPC1) cells incubated with 3 were exposed to UV light,
and proteins cross-linked to 3 were labeled with Alexa Fluor
488 azide by click chemistry and analyzed by immunoblot
probed with Alexa Fluor 488 antibody. A number of proteins
covalently linked to 3 were identified in both CHONull and
CHONPC1 membranes (Figure 3A, left). The marked increase in
labeling of >150 kDa species in CHONPC1 correlated with
expression of NPC1 (Figure 3A, right), and immunoprecipi-
tation using NPC1 antibodies (Figure 3B, right) pulled down a
single protein corresponding to NPC1 that was detected using
Alexa Fluor 388 antibodies (Figure 3B, left). Because cleaved
EboV GP is a ligand for NPC1 and NPC1 is required for
infection,8,9 we reasoned that the antiviral compounds interfere
with NPC1 receptor function. Consistent with this hypothesis,
we found that these compounds inhibit binding of cleaved
EboV GP to membranes expressing NPC1 in a concentration-
dependent manner that is closely correlated with antiviral
activity (2 > 6 > 1, Figure 4A, and 2 > 3 > 5b, Figure 4B).
Taken together, these findings strongly suggest that the
Funding
The research was supported by NIH U54 AI057159 (NERCE/
BEID). J.M. was supported by PIDS-Sanofi-Pasteur and NIH
K08AI079381. B.G. was supported by Stena Foundation. M.C.
was supported by Fonds de la Recherche en Sante du Quebec.
́ ́
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Daniel Ory for CHOnull and CHONPC1 cell lines,
Bryden Considine for technical support, and Su Chiang for
thoughtful review of the manuscript.
ABBREVIATIONS
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CHONull, Chinese hamster ovary fibroblasts lacking NPC1;
CHONPC1, Chinese hamster ovary fibroblasts expressing NPC1;
EboV, Ebola virus; FDA, Food and Drug Administration; GP,
glycoprotein; HIV, human immunodeficiency virus; IC50, half-
maximal inhibitory concentration; LE/LY, late endosome/
lysosome; NPC1, Neimann-Pick C1; SAR, structure−activity
relationship; VSV, vesicular stomatitis virus
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dx.doi.org/10.1021/ml300370k | ACS Med. Chem. Lett. 2013, 4, 239−243