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A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) was synthesized and tested in vitro and in vivo. Compared with 2-PAM, the most promising cyclic amidine-oxime (i.e., 12e) showed comparable or greater reactivation of OP-inactivated AChE and OP-inactivated BChE. To the best of our knowledge, this is the first report of a nonquaternary oxime that has, comparable to 2-PAM, in vitro potency for reactivation of Sarin (GB)-inhibited AChE and BChE. Amidine-oximes were tested in vitro, and reactivation rates for OP-inactivated butyrylcholinesterase (BChE) were greater than those for 2-PAM or MINA. Amidine-oxime reactivation rates for OP-inactivated acetylcholinesterase (AChE) were lower compared to 2-PAM but greater compared with MINA. Amidine-oximes were tested in vivo for protection against the toxicity of nerve agent model compounds. (i.e., a model of Sarin). Post-treatment (i.e., 5 min after OP exposure, i.p,) with amidine oximes 7a-c and 12a, 12c, 12e, 12f, and 15b (145 μmol/kg, i.p.) protected 100% of the mice challenged with the sarin model compound. Even at 25% of the initial dose of amidine-oxime (i.e., a dose of 36 μmol/kg, i.p.), 7b and 12e protected 100% of the animals challenged with the sarin nerve agent model compound that caused lethality in 6/11 animals without amidine-oxime.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 μmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 μmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat.

We have prepared a series of alpha-ketothiohydroximic acid thioesters and evaluated them in vitro with respect to their ability to reactivate (diisopropylphosphoryl)acetylcholinesterase. The compounds conform to the general formula RC(=O)C(=NOH)S(CH2)nNR2'.HCl, where R = CH3, C6H5, 4-CH3OC6H4, 4-NO2C6H4; n = 2, 3; and R' = CH3, C2H5, or i-C3h7. We also prepared 4-BrC6H4C(=NOH)S(CH2)2N(C2H5)2.HCl and 4-CH3OC6H4C(=O)C(=NOH)S(CH2)2N(C2H5)2.CH3I for comparison. The alpha-ketothiohydroximates exhibit oxime acid dissociation constants (pKa) in the range 6.9 to 8.4, bracketing the value of pKa = 7.9, believed to be optimal for acetylcholinesterase reactivation. The compounds are also good nucleophiles; bimolecular rate constants (kn) for reaction with p-nitrophenyl acetate follow the expression log (kn) = 6.7 - 0.69(14 - pKa = The reactivation of (diisopropylphosphoryl)acetylcholinesterase is highly dependent on the alpha-ketothiohydroximate structure: 4-h incubation of inhibited enzyme at pH 7.6, 25 degrees C, with 1 x 10(-3) M 4-CH3OC6H4C(=O)C(=NOH)S(CH2)3N(CH3)2.HCl gives no detectable restoration of activity, whereas 4-CH3OC6H4C(=O)C(=NOH)S(CH2)2N(C2H5)2.HCl restores inhibited enzyme activity to 58% of control under identical conditions. With alpha-ketothiohydroximate in excess over inhibited enzyme, the kinetics of reactivation are governed by an equilibrium constant (Kr) for binding alpha-ketothiohydroximate to the inhibited enzyme and a nucleophilic displacement rate constant (kr) for attack on phosphorus.