Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization

Article abstract of DOI:10.1016/j.bmc.2011.09.019

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K⁺ channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase-a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.

Full text of DOI:10.1016/j.bmc.2011.09.019

Bioorganic & Medicinal Chemistry 19 (2011) 6203–6209
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Formulating a new basis for the treatment against botulinum neurotoxin
intoxication: 3,4-Diaminopyridine prodrug design and characterization
Joseph S. Zakhari a,b, Isao Kinoyama a, Mark S. Hixon c, Antonia Di Mola a,
Daniel Globisch a, Kim D. Janda a,b,
⇑
a Departments of Chemistry, Immunology and Microbial Sciences, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b Worm Institute of Medical Research (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
c Discovery Biology, Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2011
Accepted 8 September 2011
Available online 14 September 2011
Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neuro-
toxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces
the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of produc-
tion and transport, the use of these agents as potential bioterrorist weapons has become of utmost
concern. No small molecule therapies against BoNT intoxication have been approved to date. However,
3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an
effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require
cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action
potential post botulinum intoxication by blocking K+ channels. Unfortunately, 3,4-DAP displays toxicity
largely due to blood–brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic
enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug
is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stig-
mines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a sec-
ondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP,
which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive
inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junc-
tions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization
of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the
prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while
selectively inhibiting AChE over butyrylcholinesterase—a plasma-based high activity esterase. Future
in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication
as well as other neuromuscular disorders.
Keywords:
Botulinum neurotoxins
Acetylcholinesterase inhibitors
3,4-DAP
Prodrug
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
potential bioterorrist weapons has become of concern. As such,
the US Centers for Disease Control (CDC) have classified BoNTs as
Botulinum neurotoxins (BoNTs) produced from the Gram posi-
tive bacterium Clostridium botulinum are some of the deadliest tox-
ins known to man1 with lethal doses as low as 1 ng/kg body weight
being 10 million times more toxic than cyanide.2 Intoxication by
BoNT produces generalized muscle weakness, and in severe cases
flaccid paralysis leading to impaired respiration and autonomic
function. In many cases rapid intubation and mechanical respira-
tion are required to prevent greater risk and even death.3 BoNT
intoxication is primarily caused by the consumption of poisoned
canned food products; however, the use of these agents as
one of the six highest-risk agents for bioterrorism (class A).
The induction of neuromuscular paralysis by BoNTs requires
three biochemical steps. First, BoNT protein binds to gangliosides
on the presynaptic cholinergic nerve terminal through interactions
with the heavy chain. These interactions allow subsequent endocy-
tosis into the neuron through several possible mechanisms
involving synaptotagmins I and II (BoNT/B and BoNT/G) and SV2
(BoNT/A).4 The toxin is then translocated into the cytosol where
its light chain (LC), a metalloprotease, binds to, and cleaves soluble
N-ethylmaleimide-sensitive factor attachment protein receptor
proteins (SNAREs).5 This action halts the release of acetylcholine
(ACh) at the neuromuscular junction, leading to the cessation of
neurotransmission.
⇑
Corresponding author. Tel.: +1 858 784 2515; fax: +1 858 784 2595.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.019

6204
J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
Currently, the only approved therapies against BoNT intoxica-
(BChE), a highly active esterase with largely overlapping substrate
specificity that must be avoided for therapeutic efficacy.19 Although
obtaining selectivity for AChE over BChE is a challenging task, the
successof the stigmines suggeststhat prodrug-type molecules could
be similarly designed with sufficient selectivity towards AChE over
BChE.
tion include pre-exposure prophylaxis with a vaccine and post-
exposure administration of sera containing anti-BoNT antibodies.6
Upon cellular intoxication, however, it is imperative to provide fast
acting neuro-modulatory drugs to recover neurotransmission
through ACh release, to at least restore partial muscle function.
Thus, a potential small molecule pharmacological treatment could
provide many benefits over these antibody-based approaches.
Most small molecule research efforts have targeted the metallo-
proteolytic properties of the BoNT LC protease, however, no small
molecule therapeutics have been approved to date.7 Yet, clinically
approved cholinergic enhancing drugs have been implemented to
treat similar neurological disorders including Lambert-Eaton
myasthenic syndrome (LEMS),8 multiple sclerosis (MS),9 and
Alzheimer’s disease.10
Of these classes of drugs, aminopyridines, in particular 3,4-
diaminopyridine (3,4-DAP), have shown promise due to its agonistic
effect on neurotransmitter release through Kv channel blockade.11
The aminopyridines, 3,4-DAP along with 4-aminopyridine (4-AP)
facilitate recovery of neuromuscular action potential post botulism
intoxication by reversibly blocking voltage-dependent K+ chan-
nels.12 This action promotes Ca2+ influx, driving signal transduction
and ACh release at the synapse. The mechanism by which
aminopyridines inactivate the Kv channel is unknown; however,
using molecular modeling Caballero and colleagues hypothesized
that two putative receptor sites found within the tetrameric
channel are important in this overall process.13 It has also been
hypothesized that once aminopyridines cross the membrane, the
molecules become protonated intracellularly, thereby allowing
the molecule to interact with receptors within the pore of the
channel.
From the structure–activity relationship found within the stig-
mines, carbamate conjugates of 3,4-DAP could act as potential
pseudo-substrate inhibitors of AChE thereby providing the en-
hanced benefit of selectively unmasking 3,4-DAP proximal to its
site of action. Also of note, carbamate inhibitors are competitive
with respect to ACh meaning that inhibition of AChE and liberation
of 3,4-DAP will occur in those synaptic junctions most depleted
(and in need) of ACh. AChE is homeostatically occupied by ACh
(Km = 0.4 mM) within the neuromuscular junction.20 Interestingly,
studies by Smart and McCammon have found that the deactivation
of AChE increases ACh lifetime in the synaptic cleft from 200 to
900 l
s.21 Acetyl choline depletion could produce up to a 25-fold
inhibition selectivity for AChE at BoNT/A intoxicated neuromuscu-
lar junctions over undamaged junctions. Therefore, there could be
a therapeutically relevant directed inhibition of AChE and 3,4-DAP
liberation in those synaptic clefts most depleted of ACh by BoNT
intoxication.
Herein, we describe our studies regarding the design, synthesis
and in vitro study of 3,4-DAP-based dual-acting prodrugs against
BoNT intoxication.
2. Materials and methods
2.1. Materials
Recently, our laboratory initiated studies toward deciphering
the pKa/hydrogen-bonding properties of 3,4-DAP in conjunction
with the Kv channel.14 By synthesizing structural analogues, we
hoped to discover more potent, yet, less toxic forms of the pharma-
cophore for treatment against BoNT intoxication. Previous reports
indicate that 3,4-DAP and 4-AP, when administered at high doses,
demonstrate toxicity issues such as seizures due to the molecules’
ability to penetrate the blood–brain barrier (BBB). As such, the
development of new compounds that alleviate BBB penetration
without hampering the necessary molecular dynamics between
the compound and the Kv channel would provide a path forward
for treating BoNT/A intoxication. Exploring this possibility, a series
of compounds were synthesized, and 3,4,5-triaminopyridine and
3,4-diamino-1-(prop-2-ynl)pyridinium were found to exhibit ther-
apeutic potential, as characterized by paralysis reversal in mouse
phrenic nerve-hemidiaphragm assays post BoNT/A induced paraly-
sis. Yet, a shortcoming with these molecules is that they possessed
modest plasma half-lives of 0.31 h and 1.2 h, respectively. Drug
candidates with limited plasma lifetimes would require repeated
dose regimen or a continuous iv infusion to systemically provide
paralytic extinction.
Acetylthiocholine iodide (AChI), S-Butyrylthiocholine iodide
(BChI), 5,50-Dithiobis(2-nitrobenzoic acid) (DTNB), and sera from
clotted mouse blood were purchased from Sigma Aldrich (St. Louis,
MO). 100 mM stocks of AChI, BChI, and DTNB were prepared in
100 mM sodium phosphate pH 8.0. All other chemicals used were
of analytical grade and highest chromatographic purity available.
AChE purified from Electrophorus electricus (Electric eel)—Type
V-S and BChE from equine serum were purchased from Sigma Al-
drich and reconstituted in 1% gelatin/100 mM sodium phosphate
pH 8.0 at 500 U/mL each.
2.2. Enzyme inhibition assays
The activities of class I, II, and III inhibitors were measured
according to the method of Ellman et al.22 using AChI or BChI as
the respective substrates for each enzyme. In brief, 100
lM AChI
or BChI was added to 100 mM sodium phosphate pH 8.0 containing
360
to 2
l
l
M DTNB and a range of prodrug concentrations from 500
M. To initiate the reaction, 0.5 U of enzyme was added to
l per well in Costar 3904
lM
each sample in a final volume of 200
l
96 well plates (Corning Inc., Corning, NY). Enzyme rates were mea-
sured at 37 °C over the linear range using a Spectromax 250 spec-
trophotometer with 412 nm absorbance readings. (All reagent
stocks were prepared in 100 mM sodium phosphate pH 8.0 unless
stated otherwise).
In order to determine the kinetic mechanism of inhibition,
inhibitors and substrate(s) were run in varying concentrations
bracketing their Kis and Kms, respectively.
While many research programs have focused on inhibition of
the BoNT LC protease itself, therapies directed toward the inhibi-
tion of acetylcholinesterase (AChE) post BoNT intoxication remain
sparse.15
The stigmines, a class of carbamate-based AChE inhibitors, pro-
vide effective treatment of multiple sclerosis,16 myasthenia gravis,17
and Alzheimer’s disease.18 Mechanistically, carbamates produce
reversible covalent inhibition via transesterification between the
carbamoyl-moiety and the active site serine. Hydrolysis of the
serine-carbamate intermediate is considerably slower (seconds to
tens of minutes) than that of the acetyl-serine adduct formed during
substrate hydrolysis. Clinically approved stigmines such as
Rivastigmine (Exelon) are characterized as pseudo-substrate
inhibitors of both AChE and serum-based butyrylcholinesterase
2.3. Enzyme-specific drug release assays
To determine enzyme-mediated drug release, 500
prodrug was incubated in 100 mM sodium phosphate pH 8.0
containing 1.0 U enzyme (100 L total volume) for 3 h at 37 °C.
lM of
l

J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
6205
Post incubation, samples were quenched with 17.4 M glacial acetic
acid and analyzed by analytical reverse-phase HPLC using a Vydac
C18 218TP54 column (Western Analytical Products, Lake Elsinore,
CA) monitored at 254 and 320 nm. A linear gradient of 0.5–50%
(v/v) of solvent B (0.09% v/v TFA/acetonitrile) in solvent A (0.1%
v/v TFA/water) from 5 to 15 min, then 50–90% (v/v) B over 5 min
was used for all compounds. Peak area and linear dynamic range
used to calculate 3,4-DAP was by use of authentic standards. Pro-
drugs demonstrating greater 3,4-DAP release by AChE over BChE
were further analyzed for half-life in mouse sera.
were calculated by non-linear regression analysis using GraphPad
Prism v.5.0b software (GraphPad Software Inc., CA).
3. Results and discussion
3.1. Design of prodrugs
Based on previous reports of SAR and plasma stability, three
prodrug models were designed with the hope of discovering dual
action AChE inhibitors providing the selective release of 3,4-DAP
at the site of intoxication. Class I molecules were designed from
substrate specificity studies and modeled such that the anionic
binding site of AChE would be occupied by 3,4-DAP while the ac-
tive site would be occupied by an ester (Fig. 1). Upon ester cata-
lyzed hydrolysis, the prodrug would degrade to release 3,4-DAP
into the synaptic cleft to act on nearby Kv channels (Fig. 1). Class
II prodrugs, on the other hand, were designed to contain a carbam-
oyl scissile linker like the stigmines. Lastly, class III prodrugs were
designed to test whether non-specific esterases/peptidases in sera
would allow the release of 3,4-DAP slowly over time.
2.4. Sera half-life assays
Fifteen microliters of a 10 mM prodrug stock was added to
85
the following fixed time intervals of 0, 5, 10, 20, 40, 60, and
120 min. Upon extraction with 300 L acetonitrile at 0 °C, precipi-
lL sera, pre-warmed to 37 °C. Samples were then incubated at
l
tated proteins were removed by centrifugation (20,817 rcf) and the
liquid phase was subsequently analyzed by analytical reverse-
phase HPLC as described above. Stability of prodrug was deter-
mined based on presence of breakdown product 3,4-DAP. Half-life
values were calculated using the 1-hour endpoint samples.
3.2. Synthesis of class I prodrugs
The synthesis of class I prodrugs followed one of two paths.
Class Ia prodrugs (6a–i) began from 4-hydroxybenzaldehyde (1),
which was treated with various alkyl acid chlorides affording
compounds 2a–i, (Scheme 1). Subsequent reduction with NaBH4
2.5. Statistical analysis
The prodrug concentration producing the 50% AChE and BChE
activity inhibition (IC50), along with inhibition constant Ki values
Anionic Locus
Catalytic Locus
HO
a
O
δ+
N
δ−
N
H
O
O
H
O
O
R
HN
O
NH2
N
CO2
O
N
NH2
NH2
+
N
N
H
O
O
O
NH2
Anionic Locus
Catalytic Locus
b
O
HO
δ+
N
δ−
O
N
R'
HN
H
H
O
HN R''
N
O
CH3
R' =
H
O
O
O
O
R''
=
H
O
Figure 1. (a) Proposed mechanism of class I, 3,4-DAP prodrugs. (b) Representative carbamate and amide class II and III inhibitors and AChE active site mechanism of action.

6206
J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
H
H
HO
a
b
O
O
O
O
O
R
OH
O
R
1
2a-i
3a-i
O
O
c
d
HN
N
O
NO2
O
R
HN
N
O
NH2
O
O
N3
O
O
R
NO2
N
4
5a-i
6a-i
6a
6b R = Et
6c
6d R = i-Pr
6e R = sec-Bu
6f
6g R = Ph
6h
6i R = Bn
R = Me
R = c-Pen
R = Pr
R = (3-OMe)Ph
Scheme 1. Synthesis of class Ia prodrugs. Reagents and conditions: (a) RCOCl, Et3N, THF, 0 °C–rt, 3 h; (b) NaBH4, THF, 0 °C–rt, 0.5–2.5 h; (c) benzene, reflux, 0.5–1 h; (d)
SnCl2ꢀ2H2O, MeCN, reflux, 1 h.
resulted in benzyl alcohols 3a–i. Reaction of benzoyl azide 4,23 and
subsequently its Curtius rearrangement; the resulting isocyanate
was trapped by alcohols 3a–i to provide carbamates 5a–i. Finally,
reduction of the nitro group with stannous chloride afforded DAP
prodrugs 6a–i. Here yields ranged from 4% to 32% over four steps.
The general synthesis of class Ib prodrugs followed a similar se-
quence. For compound 10, 1,2-phenylenedimethanol (7) was trea-
ted with isobutyryl chloride affording 8, (Scheme 2a). Next, alcohol
8 underwent reaction with 4 providing carbamate 9. The nitro moi-
ety was subsequently reduced yielding 10 in an overall yield of 9%
over three steps.
isobutyryl chloride. Conversion to nitrocarbamate 13, vide supra,
followed by its reduction, (Scheme 2b), afforded 14 in 12% yield
over three steps.
3.3. Synthesis of class II prodrugs
The construction of the class II prodrugs began with the reac-
tion of commercially available 3,4-DAP (15) and methyl chlorofor-
mate to provide carbamates 16 and 17 in one pot, (Scheme 3),
which upon separation resulted in yields of 10% and 22%, respec-
tively. The 3,4-DAP prodrug 19, in which the carbamate is located
at the 4-position, was prepared via palladium-catalyzed hydroge-
nation of the known precursor 18,24 with a yield of 36%.
On the other hand, the synthesis of carbamate 14 began from
ethylene glycol (11), which was converted to ester 12 using
O
O
O
O
NH2
HN
N
O
NO2
HN
N
a
b
c
OH
OH
O
OH
O
O
O
O
7
8
9
10
O
O
O
O
O
a
b
c
HN
N
O
NO2
HN
N
O
OH
OH
O
O
NH2
O
HO
11
12
13
14
Scheme 2. Synthesis of class Ib prodrugs 10 (Top)a and 14 (Bottom)b. aReagents and conditions: (a) isobutyryl chloride, Et3N, CH2Cl2, rt, 16 h; (b) 4, benzene, reflux, 0.5 h; (c)
SnCl2ꢀ2H2O, MeCN, reflux, 1.5 h. bReagents and conditions: (a) isobutyryl chloride, Et3N, CH2Cl2, rt, 1 h; (b) benzene, reflux, 20 min; (c) H2 (50 psi), Pd/C, EtOAc, rt, 14 h.

J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
6207
3.4. Synthesis of class III prodrugs
Diamide 22 was prepared in one step from 3,4-DAP (15) using
acetyl chloride.
The synthesis of class III prodrugs are depicted in Scheme 4 and
Scheme 5. Amide 21 was obtained via palladium-catalyzed hydro-
genation of the known aminopyridine 20,25 (Scheme 4), 68% yield.
The synthesis of amides 25 and 27 proceeded from a common,
commercially available starting material, 3-nitro-4-aminopyridine
(23). In pursuit of 25, amide bond formation with benzoyl chloride
O
NH2
N
NH2
N
H
N
HN
N
O
NH2
O
a
N
H
O
+
O
O
15
16
17
O
O
HN
O
NO2
HN
N
O
NH2
b
N
18
19
Scheme 3. Synthesis of class II prodrugs. Reagents and conditions: (a) ClCO2Me, DMA, rt, 5 h; (b) H2 (50 psi), Pd/C, EtOAc, rt, 48 h.
O
O
O
HN
N
HN
N
HN
N
NH2
N
H
N
NO2
NH2
NH2
a
b
O
20
21
15
22
O
O
NH2
NH
N
NH
N
NO2
NO2
NH2
c
d
N
23
24
25
e
O
O
NH
NH
N
NO2
NH2
f
N
26
27
Scheme 4. Synthesis of class III prodrugs 21, 22, 25, and 27. Reagents and conditions: (a) H2 (60 psi), Pd/C, MeOH, rt, 16 h; (b) AcCl, DMA, rt, 14 h; (c) BzCl, Et3N, CH2Cl2, 0 °C–
rt, 14 h; (d) H2 (50 psi), Pd/C, EtOAc, rt, 60 h; (e) cyclopentanecarbonyl chloride, Et3N, CH2Cl2, 0 °C–rt, 60 h; (f) H2 (50 psi), Pd/C, EtOAc, rt, 13 h.

6208
J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
O
O
O
O
O
O
a
b
c
OH
Cl
O
O
O
28
29
30
O
O
O
O
O
O
d
N
NH
N
NH
NH2
NO2
31
32
Scheme 5. Synthesis of class III prodrug 32. Reagents and conditions: (a) iPrOH, reflux, 14 h, then 150 °C, 3 h; (b) (COCl)2, CH2Cl2, DMF, rt, 0.5 h; (c) 23, Et3N, CH2Cl2, rt, 15 h;
(d) H2 (50 psi), Pd/C, EtOAc, rt, 3 h.
Table 1
yielded 24, followed by reduction of the nitro group to provide 25,
with a yield of 18% over two steps. Synthesis of 27 began with the
In vitro inhibition profiles of 6b, 6d, and 32
Compd
Kia
amide union between 23 and cyclopentanecarbonyl chloride, fol-
lowed by reduction to provide 27 with a yield of 48% over two
steps.
The final class III prodrug, 32, started from commercially avail-
able succinic anhydride (28), which was treated with isopropyl
alcohol to form the ester 29, followed by conversion to acyl chlo-
ride 30 (Scheme 5). The reaction of 30 with 23 in the presence of
base provided 31, which in turn was reduced to the desired pri-
mary amine 32 via palladium-catalyzed hydrogenation. The overall
yield over this four-step synthesis was 17%.
AChE
BChE
6b
6d
32
5.7 0.7
3.9 0.6
3.2 0.6
25
42
3
5
62 11
a
Calculated Ki values in lM. All data are represented as average S.E.M.
Table 2
In vitro kinetic profiles of 6b, 6d, and 32
Drug releasea
BChE
3.5. Inhibition, release profile, and stability of prodrugs
Compd
AChE
Sera half-lifeb
From screens of candidate compounds against AChE and BChE,
only compounds 6b and 6d, of the substrate-like class I prodrugs,
demonstrated moderate selectivity of AChE over BChE, as well as
AChE-dependent release of 3,4-DAP (Tables 1 and 2). Both com-
pounds bound to AChE 100-fold more tightly than Km for ACh. Pro-
gress curves of substrate consumption in the presence of the
inhibitors were essentially linear indicating no significant buildup
of acyl-enzyme intermediates (which would give time-dependent
inhibition). While inhibition was relatively potent, turnover and
release of 3,4-DAP was modest (Table 2). Incubation studies with
AChE and BChE at saturating levels of 6b and 6d gave the expected
zero order kinetics with apparent kcats that were approximately
10ꢁ4 slower than the natural substrates ACh and BCh. Given the
higher concentration of 3,4-DAP release by AChE, these two pro-
drugs were further tested for their respective half-lives in mouse
sera. Unfortunately compounds 6b and 6d were unstable to incu-
bation in sera, with half-lives of just 1.2 and 1.9 min, respectively.
The extremely short half-lives of these molecules are due to
unidentified esterases of the labile esters found within the prodrug
molecules. The structural similarity of all class I substrate-like pro-
drugs meant that no other compounds from this class were likely
to be stable in mouse sera.
6b
6d
32
145
105
3.4
82
82
5.8
1.2
1.9
165
a
Concentration of 3,4-DAP in
Calculated half-life values in min.
l
M post 3 h enzymatic hydrolysis.
b
(Supplementary data Table S2). Simple methyl carbamates of 3,4-
DAP are not recognized by the cholinergic esterases as stigmine-
like substrates. As such, these carbamates were not analyzed for
half-lives in sera.
Amide 21 from the class III prodrugs demonstrated competitive
inhibition of AChE (32
amide bond did not undergo internal release upon incubation with
AChE for 3 h. Amide 32, while potently inhibiting AChE (3 M Ki)
also demonstrated negligible release of 3,4-DAP over the 3 h incu-
bation period (Table 2). The greater stability of the amide-conju-
gated 3,4-DAP gave this prodrug an appreciable half-life of 2.8 h
in mouse sera, providing a path forward for in vivo analysis, as well
as elucidating the core functionality necessary in the design and
synthesis of additional 3,4-DAP prodrugs.
lM Ki). Not surprisingly, the less labile
l
The carbamates most representative of our intended dual action
strategy, compounds 16, 17, and 19 from class II prodrugs all pro-
4. Conclusion
duced moderate to poor inhibition profiles (IC50s: 36–660
failed as pseudosubstrates to generate 3,4-DAP enzymatically
lM) but
The design and synthesis of small molecule inhibitors of BoNT
have been of great interest over the past decade.26 While most

J. S. Zakhari et al. / Bioorg. Med. Chem. 19 (2011) 6203–6209
6209
efforts have focused on the direct inhibition of the BoNT protease,
Acknowledgments
few have been designed to symptomatically relieve neuromuscular
paralysis by blocking voltage-dependent potassium channels. This
report took an alternative approach by investigating the possibility
of dual action toward cholinergic enhancement at BoNT damaged
synapses. We attempted to mask the Kv channel blocker 3,4-DAP
as a carbamate or amide conjugated pseudo-substrate inhibitor
of AChE thus allowing its directed delivery to afflicted synapses.
The combined effect of the prodrug is to enhance the action poten-
tial for the neuron pre-synaptically as well as increase the lifetime
of ACh post-synaptically. Although simple methylcarbamate con-
jugates to 3,4-DAP were not recognized by AChE as pseudo-sub-
strates, the carbamoyl-linked 4-benzylalcohol-phenol esters 6b
and 6d were. These compounds bound relatively tightly to AChE
This project was supported by a postdoctoral fellowship from
the German Academic Exchange Service (DAAD) to D.G., as well
as The Skaggs Institute for Chemical Biology, and federal funds
from the National Institute of Allergy and Infectious Diseases, Na-
tional Institutes of Health, Department of Health and Human Ser-
vices, under award number AI080671 (K.D.J.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.019.
(low lM) but were sluggish in their turnover by AChE having only
References and notes
10ꢁ4 the kcat of ACh. The esters within these compounds rendered
them unstable to mouse sera.
1. Johnson, E. A.; Bradshaw, M. Toxicon 2001, 39, 1703.
2. Schantz, E. J.; Johnson, E. A. Microbiol. Rev. 1992, 56, 80.
3. Turton, K.; Chaddock, J. A.; Acharya, K. R. Trends Biochem. Sci. 2002, 27, 552.
4. Dong, M.; Yeh, F.; Tepp, W. H.; Dean, C.; Johnson, E. A.; Janz, R.; Chapman, E. R.
Science 2006, 312, 592.
5. Simpson, L. L. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 167.
6. Arnon, S. S.; Schechter, R.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.;
Ascher, M. S.; Eitzen, E.; Fine, A. D.; Hauer, J.; Layton, M.; Lillibridge, S.;
Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T. M.; Russell, P. K.; Swerdlow, D.
L.; Tonat, K. JAMA 2001, 285, 1059.
7. (a) Dickerson, T. J.; Janda, K. D. ACS Chem. Biol. 2006, 1, 359; (b) Capkova, K.;
Salzameda, N. T.; Janda, K. D. Toxicon 2009, 54, 575.
8. (a) McEvoy, K. M.; Windebank, A. J.; Daube, J. R.; Low, P. A. N. Engl. J. Med. 1989,
321, 1567; (b) Oh, S. J.; Claussen, G. G.; Hatanaka, Y.; Morgan, M. B. Muscle
Nerve 2009, 40, 795.
Alternatively, amide conjugation of 3,4-DAP exemplified by 32,
resulted in compounds that inhibited AChE selectively over BChE
with good potency but did not liberate 3,4-DAP through the in-
tended internal release mechanism. The greater stability of the
amide bond gave 32 a reasonable half-life (2.8 h) for the release
of 3,4-DAP in mouse sera. Although such compounds lack the dual
action originally intended, masking 3,4-DAP for controlled release
holds therapeutic promise. On its own, 3,4-DAP generates muscle
action potential and muscle contraction in vivo, yet its ability to
cross the BBB and produce seizures has questioned the potential
therapeutic relevance of this molecule.27 As such, the design of
prodrugs of 3,4-DAP that limit toxicity would be pertinent as a
means to effectively treat neuro-paralytic disorders including
BoNT induced paralysis.
9. Schwid, S. R.; Petrie, M. D.; McDermott, M. P.; Tierney, D. S.; Mason, D. H.;
Goodman, A. D. Neurology 1997, 48, 817.
10. (a) Davidson, M.; Zemishlany, Z.; Mohs, R. C.; Horvath, T. B.; Powchik, P.; Blass,
J. P.; Davis, K. L. Biol. Psychiatry. 1988, 23, 485; (b) Youdim, M. B.; Buccafusco, J.
J. J. Neural Transm. 2005, 112, 519.
Three classes of prodrugs were designed, synthesized, and eval-
uated in vitro with the goal of elucidating structures that act as
pseudosubstrates for AChE and release 3,4-DAP at affected neu-
rons, via enzyme mediated catalysis. Localized 3,4-DAP would act
to increase vesicular ACh release at intoxicated junctions, allowing
the recovery of neuromuscular transmission while reduced acetyl-
cholinesterase activity via AChE inhibition will allow the limited
ACh present to persist. We anticipate that continual 3,4-DAP in
circulation, released slowly overtime, would nullify the previous
inefficacies of concentrated, repeated dosing, as well as BBB
penetration.
Of the 19 compounds synthesized, two demonstrated inhibition
and selectivity between enzyme classes, as well as a drug-release
profile necessary for half-life determination in mouse sera (6b
and 6d). Unfortunately, further testing revealed that these two
class I prodrugs were unstable in sera limiting their usefulness
in vivo. Prodrug 32 on the other hand, demonstrated inhibition
and selectivity for AChE, but did not act as a pseudosubstrate for
the enzyme. Regardless, 32 was tested for half-life in sera to deter-
mine if unidentified esterases/peptidases present could degrade
the prodrug while in circulation, ultimately releasing 3,4-DAP over
time. Prodrug 32 in fact had the best half-life of 2.76 h. The stabil-
ity of 32 in sera may possibly be due to its amide bond, which
would not be readily cleaved within the active site of AChE, but
may be hydrolyzed by non-specific peptidases in sera. Modeling
studies may help to clarify the chemical properties necessary for
binding and release via AChE. In sum, 32 could be used as a lead
structure to elucidate more potent inhibitors. However, future
in vivo experiments will be necessary to validate prodrug 32 as
an effective treatment for BoNT intoxication and other neuromus-
cular disorders.
11. (a) Flet, L.; Polard, E.; Guillard, O.; Leray, E.; Allain, H.; Javaudin, L.; Edan, G. J.
Neurol. 2010, 257, 937; (b) Ionno, M.; Moyer, M.; Pollarine, J.; van Lunteren, E.
Respir. Physiol. Neurobiol. 2008, 160, 45; (c) Smith, D. T.; Shi, R.; Borgens, R. B.;
McBride, J. M.; Jackson, K.; Byrn, S. R. Eur. J. Med. Chem. 2005, 40, 908.
12. (a) Adler, M.; Capacio, B.; Deshpande, S. S. Toxicon 2000, 38, 1381; (b) Adler, M.;
Macdonald, D. A.; Sellin, L. C.; Parker, G. W. Toxicon 1996, 34, 237.
13. Caballero, N. A.; Melendez, F. J.; Nino, A.; Munoz-Caro, C. J. Mol. Model. 2007, 13,
579.
14. Mayorov, A. V.; Willis, B.; Di Mola, A.; Adler, D.; Borgia, J.; Jackson, O.; Wang, J.;
Luo, Y.; Tang, L.; Knapp, R. J.; Natarajan, C.; Goodnough, M. C.; Zilberberg, N.;
Simpson, L. L.; Janda, K. D. ACS Chem. Biol. 2010, 5, 1183.
15. Chiou, C. Y. Arch. Int. Pharmacodyn. Ther. 1973, 201, 170.
16. Judge, S. I.; Bever, C. T., Jr. Pharmacol. Ther. 2006, 111, 224.
17. Henze, T. Fortschr Neurol Psychiatr 1996, 64, 110.
18. (a) Verheijen, J. C.; Wiig, K. A.; Du, S.; Connors, S. L.; Martin, A. N.; Ferreira, J. P.;
Slepnev, V. I.; Kochendorfer, U. Bioorg. Med. Chem. Lett. 2009, 19, 3243; (b)
Zheng, H.; Youdim, M. B.; Fridkin, M. J. Med. Chem. 2009, 52, 4095; (c) Darvesh,
S.; Darvesh, K. V.; McDonald, R. S.; Mataija, D.; Walsh, R.; Mothana, S.;
Lockridge, O.; Martin, E. J. Med. Chem. 2008, 51, 4200; (d) Toda, N.; Tago, K.;
Marumoto, S.; Takami, K.; Ori, M.; Yamada, N.; Koyama, K.; Naruto, S.; Abe, K.;
Yamazaki, R.; Hara, T.; Aoyagi, A.; Abe, Y.; Kaneko, T.; Kogen, H. Bioorg. Med.
Chem. 2003, 11, 1935.
19. Bar-On, P.; Millard, C. B.; Harel, M.; Dvir, H.; Enz, A.; Sussman, J. L.; Silman, I.
Biochemistry 2002, 41, 3555.
20. Colquhoun, D. Neuromuscular Transmission: Basic and Applied Aspects; Elsevier:
New York, 1990. Chapter 7; (b) Shen, T.; Tai, K.; Henchman, R. H.; McCammon,
J. A. Acc Chem Res. 2002, 35, 332.
21. Smart, J. L.; McCammon, J. A. Biophys. J. 1998, 75, 1679.
22. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
23. Holt, J.; Andreassen, T.; Bakke, J. M.; Fiksdahl, A. J. Heterocycl. Chem. 2005, 42,
259.
24. Bakke, J. M.; Gautun, H. S. H.; Svensen, H. J. Heterocycl. Chem. 2003, 40, 585.
25. Bakke, J. M.; Riha, J. J. Heterocycl. Chem. 1999, 36, 1143.
26. Willis, B.; Eubanks, L. M.; Dickerson, T. J.; Janda, K. D. Angew. Chem., Int. Ed.
2008, 47, 8360.
27. (a) Uchiyama, T.; Lemeignan, M.; Lechat, P. Jpn. J. Pharmacol. 1985, 38, 329; (b)
Plewa, M. C.; Martin, T. G.; Menegazzi, J. J.; Seaberg, D. C.; Wolfson, A. B. Ann.
Emerg. Med. 1994, 23, 499; (c) Damsma, G.; Biessels, P. T.; Westerink, B. H.; De
Vries, J. B.; Horn, A. S. Eur. J. Pharmacol. 1988, 145, 15.