Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: Synthesis, crystallography, modeling, kinetic and cellular based studies

Article abstract of DOI:10.1016/j.bmc.2012.12.001

Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportun

Full text of DOI:10.1016/j.bmc.2012.12.001

Bioorganic & Medicinal Chemistry 21 (2013) 1344–1348
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of adamantane hydroxamates as botulinum neurotoxin
inhibitors: Synthesis, crystallography, modeling, kinetic and cellular
based studies
c
d
Peter Šilhár a,b, Nicholas R. Silvaggi , Sabine Pellett , Katerina Capková a,b, Eric A. Johnson d, Karen N. Allen c,
ˇ
ˇ
Kim D. Janda a,b,e,
⇑
a Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
b Department of Immunology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
c Department of Chemistry, Boston University, Boston, MA 02215, United States
d Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, United States
e Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for
the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based
and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease
(LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective
post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxa-
mates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of com-
pounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic
studies on these structures revealed a competitive inhibition model, with a Ki = 27 nM, which makes
these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported
to date.
Received 18 October 2012
Revised 1 December 2012
Accepted 3 December 2012
Available online 20 December 2012
Keywords:
Botulinum neurotoxin
Protease inhibitor
Adamantane derivatives
Small molecule inhibitor
Zinc-dependent metalloprotease
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ety of variety of maladies, including strabismus, migraines, and
even facial wrinkles.5 However, the potential use of BoNT in a biot-
Botulinum neurotoxins (BoNTs) produced by the anaerobic
spore-forming bacterium Clostridium botulinum are the most
lethal human poison.1 Serotype A (BoNT/A) is the most potent of
the identified serotypes with an estimated lethality of ꢀ1 ng/kg.2
There are seven BoNT serotypes (A–G) and while they differ by
up to 70% at the amino acid level all consist of heavy and light
chain subunits. Upon cellular internalization of the holotoxin (via
binding of heavy chain to cell surface receptors) the light chain
(LC), a 50 kDa Zn(II)-dependent metalloprotease, is released. Toxic-
ity from BoNT poisoning results from the site-specific cleavage of
the synaptosomal-associated protein 25 (SNAP-25) by the metallo-
protease, preventing acetylcholine-containing vesicles from fusing
with the presynaptic neuromuscular junction.3 The consequence of
protease cleavage of SNAP-25 is inhibition of acetylcholine release,
which leads to flaccid paralysis and eventually to death caused typ-
ically by heart or respiratory failure.4
errorist attack remains imminent and the Center for Disease Con-
trol (CDC) classifies this agent as ‘category A’, placing it among
the six highest-priority agents.
Currently, there are no approved pharmacological treatments
for BoNT intoxication. Although an effective vaccine is available
O
O
Cl
Cl
OH
OH
N
H
N
H
OH
Cl
Cl
X
, Ki = 300 nM
XI
, Ki = 160 nM
O
O
OH
OH
N
N
H
H
O
N
Bn
N
[(CH2)7NH2]2
HO
Despite their potentially lethal toxicity, BoNTs have emerged as
an extremely valuable therapeutic tool for the treatment of a vari-
3a
, Ki = 460 nM
AHP
, Ki = 760 nM
Figure 1. Examples of some of the most active inhibitors of BoNT/A protease: X,11
3a,10 XI,9 AHP.12
⇑
Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.001

P. Šilhár et al. / Bioorg. Med. Chem. 21 (2013) 1344–1348
1345
for immuno-prophylaxis,6 vaccine approaches cannot reverse the
2. Results and discussion
effects after the toxin has reached its target inside the cell. A small
molecule pharmacological intervention, especially one that would
be effective against the etiological agent responsible for BoNT
intoxication, the light chain protease, would be highly desirable
and could obviate vaccine deficiencies. Most research efforts have
been focused on the BoNT/A protease, since this serotype is the
most toxic to humans with the longest lasting cellular effect.7
Indeed, a number of small molecule, non-peptidic inhibitors of
BoNT/A LC have been reported over past two decades,6,8 however,
potency is still lacking (Fig. 1). Recently, we communicated a
logical attempt to improve the potency of our best BoNT/A LC
inhibitor X based upon crystallographic analysis and computa-
tional modeling.9 The resulting structure, XI, displayed an almost
twofold lower inhibition constant than the parent X. The research
described herein was directed again using crystallographic and
modeling studies, but now to a new scaffold: the adamantane
hydroxamate 3a10 (Fig. 1). A series of 19 derivatives were prepared
with improved potency of about 17-fold for the best two new
compounds.
2.1. Crystallography and modeling studies
The X-ray crystal structure of the complex between the BoNT/A
LC and 1-adamantyl N-hydroxyacetamide (3a) was determined to
2.5 Å resolution (PDB ID 4HEV, Fig. 2A and S1, Table S1). As ob-
served in other structures of BoNT/A LC complexes with hydroxa-
mate inhibitors,13 the hydroxamate moiety is liganding the Zn2+
ion in a bidentate fashion with the carbonyl and hydroxyl oxygen
atoms (2.1 and 2.2 Å, respectively). The hydroxamate nitrogen
makes a hydrogen bonding interaction with the main-chain car-
bonyl of Phe163, part of a b-strand that forms one wall of the active
site. The adamantyl group, like the phenyl rings of the previously-
characterized cinnamyl hydroxamates (e.g., X),13 occupies a largely
hydrophobic pocket comprised of the side chains of Ile161, Phe163,
Phe194, and Phe369. In structures of BoNT/A LC with no ligands
bound, complexed with L-arginine hydroxamate or with arginine-
containing peptides,14 Phe369 is rotated out of the active site
and Asp370 is rotated in, indicating that 3a induces the same
Figure 2. Stereo view of the adamantyl hydroxamate inhibitor 3a bound within the BoNT/A LC active site (A). Both the 2|Fo| À |Fc| and simulated annealing composite omit
maps are shown as magenta and green mesh, respectively, contoured at 1.1 . The active sites after refinement of two substituted adamantane hydroxamates (3i and 3f) are
r
shown in B and C, respectively. The hydroxymethyl and chlorine groups are placed near enough to Arg363 to form favorable non-covalent interactions (2.6 and 3.0 Å,
respectively).

1346
P. Šilhár et al. / Bioorg. Med. Chem. 21 (2013) 1344–1348
O
polar-to-hydrophobic change observed for other small, nonpolar li-
gands.13 While the 250 and 60/70 loops are both largely disordered
in the structure, it is clear that there is unoccupied space in the
hydrophobic pocket that could be exploited to improve the affinity
and potency of the 3a scaffold.
OH
Cl
a
O
O
R = H
OH
OMe
OH
1f
O
b
c
In order to improve the binding and thus enhance the potency
of our lead structure 3a, we decided to perform preliminary mod-
eling studies involving substitution upon the adamantane scaffold
(Fig. 2B and C). Probing the active site with 3a, two favorable inter-
actions emerged as a promising platform to build a structure–
activity relationship study. Thus, the first interaction identified in-
volved positioning on the core structure of an appropriate polar
substituent that could interact with Arg363. The second interaction
we viewed to be favorable consisted of a non-polar/aromatic sub-
stituent and the hydrophobic pocket comprising three phenylala-
nines (F163, F194 and F369). Therefore, our proposed studies
consisted of a series of derivatives stemming from the parent mol-
ecule 3a, which was focused upon exploring interactions with the
Arg363 within the enzyme’s active site (Scheme 2), as well as
interfacing with the ‘tris-Phe’ hydrophobic pocket (Scheme 3). Hav-
ing established this rationale, all the substituents were introduced
in position 3 relative to the acetohydroxamic moiety and were pre-
pared to contain either varying electronegativity and/or steric
requirements. Based on these constraints we anticipated that a
halogen atom should favorably interact with the Arg363 residue,
while carboxyl (COOH), if ionized to its carboxylate form could
generate a stable salt bridge. To further probe this reasoning hy-
droxyl (3b and 3i), ether (3c) and acetate (3d) were hypothesized
to make hydrogen bond donor–acceptor interactions within the
Arg363 side pocket. On the other hand, introduction of an aromatic
OH
Br
1g
R = H
O
1c
R
OH
1a, R = H
1b, R = OH
d
O
OAc
1d
OH
1h
COOH
R = OH
e
R = OH
Scheme 1. Synthesis of (3-functionalized-1-adamantyl)acetic acids. Reagents and
conditions: (a) tBuCl, H2SO4 (98%), CCl4, 10–20 °C, 2 h; (b) tBuBr, H2SO4 (98%), CCl4,
10–20 °C, 2 h; (c) MeONa, AgNO3, MeOH, rt, 16 h; (d) AcONa, AcOH, Ac2O, 100 °C,
16 h; (e) HCO2H (98%), H2SO4 (98%), CCl4, 20 °C, 2 h.
1-3
R
O
O
O
O
R
OH
R
Bn
H
a
b
c
d
e
f
N
H
OH
N
H
OH
a or b
e
OMe
OAc
F
R
3
2
1b, c, d, f, g, h
c
Cl
2g
2e
Br
g
h
i
R = Br
R = F
CO2H
CH2OH
d
2h
2i
R = CO2H
R = CH2OH
substituent would lead to a
idues inside the hydrophobic pocket.
p
–
p
stacking with one or more Phe res-
Scheme 2. Synthesis of (3-functionalized-1-adamantyl)acetohydroxamic deriva-
tives. Reagents and conditions: (a) NH2–OBnÁHCl (1.5 equiv), EDC (1.3 equiv),
iPr2NEt (3 equiv), CH2Cl2, rt, 8–12 h; (b) NH2–OBnÁHCl (1.5 equiv), PyBOP
i
(1.5 equiv), Pr2NEt (3 equiv), CH2Cl2, rt, 8–12 h; (c) AgF (1.3 equiv), THF, rt, 16 h;
(d) BH3ÁMe2S, THF, 0?20 °C, 12 h; (e) H2(1 bar), Pd/C, EtOH, rt, overnight.
2.2. Chemistry
The synthesis of (3-halogen-1-adamantyl)acetic acids was initi-
ated from the commercially available and easily accessible 1-ada-
mantylacetic acid (1a). Several methods have been reported for
the halogenation of an adamantane,15 and, although chlorination
has been somewhat problematic, because of contamination with
other chlorinated by-products, there existed a facile and efficient
procedure for mono-chlorination of adamantane at the tertiary
carbon.16 In accordance with this previous work minor changes in-
In the next iteration of compounds, we utilized the versatile
intermediate 1g for introduction of aromatic substituents to the
position 3 of 1-adamantyl scaffold (Scheme 3). Thus, the 3-aryl-
1-adamantyl acids 1j–p were readily prepared under Friedel–
Crafts conditions (AlCl3 catalysis). Interestingly, this adamantyla-
tion of an aromatic residue could be also catalyzed by use of iron
powder, although usually higher reaction temperatures were re-
quired.19 Apart from direct alkylation of bromobenzene, the p-
bromophenyl acid 1p was also prepared by bromination of 1j. In
addition, the bromo-functionality in 1p could be further utilized
for diversification of our small library by cross-coupling reactions.
Such an option is represented by the biphenyl derivative 1s,
(Scheme 3, reaction h). Unfortunately, nitrobenzene did not react
with adamantyl bromide; therefore, the nitrophenyl- (1q) along
with the dinitrophenyl- (1r) derivatives were prepared by nitration
of 1j. Finally, using standard conditions the (3-aryl-1-adaman-
tyl)acetic acids 1j–s were transformed to the corresponding
hydroxamates 3j–s via activated acyl species (either acyl imidaz-
ole20 or chloride; Scheme 3).
t
cluded the switch from two components; BuOH for cation gener-
ation and gaseous HCl as source of anion/nucleophile, to simply
tBuCl as a singular reagent (Scheme 1, reaction a). Using this same
t
rationale, the BuBr led to the 3-bromo derivative 1g in excellent
yield. The 3-fluoroadamantyl derivative 2e, was made from 2g by
simple substitution with AgF (Scheme 2, reaction c). Next, (3-hy-
droxy-1-adamantyl)acetic acid (1b) was examined for preparation
of 3-methoxy- (1c), 3-acetoxy- (1d), and 3-carboxyl- (1h) adaman-
tyl derivatives. Unfortunately, methylation of the 3-hydroxyl group
did not proceed either by reaction with MeI nor Me2SO4.
Therefore, acid 1c was prepared by nucleophilic (SN1) reaction of
bromide 1g with MeONa in the presence of AgNO3. To complete the
series of polar substituents, acetylation could be achieved with
Ac2O/AcONa granting 1d, while carboxylation required recourse to
the Koch–Haaf reaction,17 yielding 1h. Finally, 3-hydroxymethyl
derivative 2i was obtained by reduction of the carboxyl group from
intermediate 2h, (Scheme 2, reaction d). Subsequently, all adaman-
tylacetic acids 1b, c, d, f, g, h were converted to the corresponding
protected hydroxamate derivatives (2), by coupling with O-ben-
zylhydroxylamine.18 The removal of the benzyl protective group
under standard hydrogenation conditions afforded the (3-function-
alized-1-adamantyl)acetohydroxamic products (3), (Scheme 2).
2.3. Kinetic analysis
Having prepared the adamantane hydroxamates, all compounds
were subjected into screening for inhibitory activity of BoNT/A LC
(1–425aa). Towards this end we choose the well-established and
rapid assay utilizing FRET (fluorescence resonance energy transfer)
substrate, SNAPtide™,21 which is
a truncated and modified
sequence of the native BoNT/A LC substrate, SNAP-25. This 13aa
peptide contains a fluorophore/quencher pair separated by the

P. Šilhár et al. / Bioorg. Med. Chem. 21 (2013) 1344–1348
1347
O
O
O
O
OH
Ar
1, 3
Ar
N
H
OH
OH
OH
Phenyl
j
k
l
a
b or c
d or e
2-Naphthyl
Ar
Br
2-Tetrahydronaphthyl
4-Me-Phenyl
m
n
o
f
4-MeO-Phenyl
4-Cl-Phenyl
1g
1j-p
3j-s
1a
g
4-Br-Phenyl
p
q
r
4-NO2-Phenyl
2,4-(NO2)2-Phenyl
4-Ph-Phenyl
h
s
Scheme 3. Synthesis of (3-aryl-1-adamantyl)acetic and acetohydroxamic acids. Reagents and conditions: (a) tBuBr, H2SO4 (98%), CCl4,15 °C, 2 h; (b) ArH, AlCl3 (1.2 equiv), 80–
120 °C, 3–6 h; (c) ArH, Fe (powder), 110–150 °C, 5–12 h; (d) (i) CDI (1.5 equiv), dioxane, 80 °C, 1.5 h; (ii) NH2OH/H2O (ꢀ20 equiv), 5 °C–rt, overnight; (e) (i) SOCl2 (3 equiv),
toluene, 80–110 °C, 1 h; (ii) NH2OH/H2O (ꢀ20 equiv), 5 °C–rt, 4 h; (f) reaction 1j?1p: Br2 (1.3 equiv), CHCl3, Fe (powder), 55 °C, 24 h; (g) reaction 1j?1q, 1r: HNO3 (65%),
AcOH, (CF3CO2)O, 5 °C, 2 d; (h) reaction 1p?1s: PhB(OH)2 (1.5 equiv), Pd(PPh3) (5%), K2CO3(3 equiv), toluene, microwave, 120 °C, 90 min.
Table 1
IC50 values of prepared (3-substituted-1-adamantyl)acetohydroxamic derivatives
IC50 (Ki)b/
lM
Entry
Compound
Substituent
IC50 (Ki)b/
lM
a
a
Entry
Compound
Substituent
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
OH
OMe
OAc
F
Cl
Br
COOH
CH2OH
1.0 (0.46)
23
5.7
24
3.1
1.4
1.4
> 500
3.4
10
11
12
13
14
15
16
17
18
19
3j
3k
3l
3m
3n
3o
3p
3q
3r
Ph
0.5 (0.75)
0.3
1.0
0.25
0.25 (0.13)
0.03 (0.027)
0.04 (0.027)
0.5
0.4
0.2
2-Naphthyl
2-THNc
4-Me-Ph
4-MeO-Ph
4-Cl-Ph
4-Br-Ph
4-NO2-Ph
2,4-(NO2)2-Ph
4-Ph-Ph
3s
a
b
c
Determined with 5 lM [SNAPtide] (+0.01% Triton X-100) versus BoNT/A LC (1-425aa).
Determined with SNAP-66mer (141–206aa) versus BoNT/A LC (1-425aa).
2-THN = 5,6,7,8-tetrahydronaphthalen-2-yl.
cleavage site; thus producing fluorescence in the presence of an ac-
tive BoNT/A protease. Preliminary IC50 values from this assay are
summarized in Table 1. Unfortunately, introduction of a polar func-
tionality into position 3 of 1-adamantyl core did not improve bind-
ing affinity. In fact, the inhibitory potencies of compounds 3b–i
were considerably lower than the parent 3a, with exception of
chloro- (3f) and bromo- (3g) derivatives where the IC50’s were
comparable with the lead molecule (Table 1, entries 2–9). We were
especially surprised to see no activity for the 3-carboxyl derivative
(3h), which we hypothesized could form an ionic interaction with
Arg363 (vide supra). Without further crystallographic data we can
only speculate that conformation of residues within the enzyme’s
active site have been altered in a way which does not allow forma-
tion of these sought after interactions. Such high flexibility of ac-
tive site residues has been documented previously for BoNT/A
protease.13 Gratifyingly, better results were achieved by attaching
an aromatic moiety on the adamantyl skeleton (Table 1, entries
10–19). The most profound improvement in activity was seen with
p-chloro- (3o), and p-bromophenyl (3p) derivatives with ꢀ 30-fold
lower IC50 values (Table 1, entries 15 and 16).
While SNAPtide proved suitable for high-throughput screening
and for crude IC50 determinations it is inadequate for finer kinetic
analysis such as the evaluation of a kinetic mechanism of inhibi-
tion and Ki determinations.10 Therefore, the inhibition profiles of
the most potent compounds were examined in more detail using
a LC/MS-based assay that employs a substrate consisting of the
66 amino acid long C-terminal domain of SNAP-25 (141–
206aa).10 To determine the mechanism of inhibition as well as Ki,
concentrations of the inhibitor and the substrate were varied. As
expected, obtained data were most consistent with the competi-
tive inhibition model (Supplementary data). The inhibition con-
stants, Ki were determined by a non-linear least squares global
fit of a competitive inhibition model to the initial rates of product
formation for matrixes of substrate and inhibitor concentrations
bracketing KM and Ki, respectively. These results shown that
hydroxamates 3o and 3p with Ki = 27 4 nM and 27 2 nM,
respectively, are some of the most potent small molecule inhibitors
of BoNT/A LC reported to date.
2.4. Biology
As another measure of an LC/A inhibitor profile, a cell-based as-
say, which monitors intracellular cleavage of SNAP-25 can be used.
Accordingly, the cellular efficacy of three of the improved inhibi-
tors found in our study (3n, 3p, and 3q) were investigated using
primary rat spinal cord (RSC) cells and stem cell derived neurons,
respectively (Supplementary data). In the first assay, BoNT/A was
added to the cells in stimulation medium to allow its entry fol-
lowed by washing and addition of inhibitors. By exposing cells to
the toxin prior to inhibitor addition, secondary effects due to cellu-
lar changes that would inhibit BoNT/A uptake can be excluded.
In the second assay, the inhibitors were premixed with BoNT/A
(ꢀ1 pM) and immediately added to stem cell derived neurons.
Unfortunately, all three compounds tested showed significant
cytotoxicity at concentration ꢀ20
lM as judged by a low signal
as seen on the Western blot, and did not protect neuronal cells
from BoNT/A induced cleavage of SNAP-25 at lower concentrations
(Fig. 3, and Supplementary data).
Having established that these compounds did provide enzy-
matic inhibition of recombinant protease in vitro, yet, a lack of pro-
tection in cells; we surmise is likely due to an inability of the
compounds to properly enter the cell cytosol, such hypothesis will
be tested in future studies.

1348
P. Šilhár et al. / Bioorg. Med. Chem. 21 (2013) 1344–1348
Figure 3. Western blot analysis and graphical representation of SNAP-25 cleavage by BoNT/A and stem cell derived neurons (inhibitors and BoNT/A toxin were premixed
upon addition to the cells).
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In summary, a research approach incorporating computational
modeling, SAR studies, compound validation, and kinetic charac-
terization was used to successfully improve lead adamantane
structure 3a, and afforded (3-substituted-1-adamantyl) acetohy-
droxamic derivatives. The most potent of these BoNT/A LC inhibi-
tors, 3o and 3p, have Ki values approximately 30 nM, which is
17-fold lower than the parent lead molecule. These findings in fact,
represent the most potent small molecule, non-peptidic BoNT/A LC
inhibitors reported to date. Unfortunately, when examined in vitro
in cell-based assays, these compounds displayed significant cyto-
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toxicity at ꢀ20
lM and provided no protection against BoNT/A
intoxication at lower concentrations. We propose that the discrep-
ancy between very high activity (low nanomolar) against recombi-
nant LC/A and no apparent activity on the cells (at low micromolar)
is due to inefficient cellular uptake and future research will exam-
ine this hypothesis in due course.
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Acknowledgments
The authors gratefully acknowledge support of this project by
the National Institute of Allergy and Infectious Diseases, National
Institute of Health and the Department of Health and Human Ser-
vices under contract number AI080671.
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References and notes
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