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.