Peter H. Doukas

Inhibition of High‐Affinity Choline Uptake and Acetylcholine Synthesis by Quinuclidinyl and Hemicholinium Derivatives

Abstract: Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high‐affinity, sodium‐ and temperature‐dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure‐activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC‐3, acetylsecohemicholinium, and HC‐15 are potent inhibitors of HAChU, HC‐3 being the most potent (I50= 6.1 × 10−8M). In the present study, the most potent quinuclidinyl derivative was the N‐methyl‐3‐quinuclidinone (I50= 5.6 × 10−7M). This compound had approximately 100‐fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3‐hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N‐functional group from a methyl to an allyl in the alcohol led to a 10‐fold increase in activity. However, removal of the quaternizing N‐methyl group yielding the tertiary amine, 3‐quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2‐benzylidene‐3‐quinuclidinone derivatives studied, only the m‐chloro derivative significantly reduced HAChU.

In vivo protection against soman toxicity by known inhibitors of acetylcholine synthesis in vitro

Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of administration, dose-response relationships, and time to effect.

3-carbamyl-N-allylquinuclidinium bromide: Effects on cholinergic activity and protection against soman

3-Carbamyl-N-allylquinuclidinium bromide (CAB) was synthesized and evaluated for its pharmacological effects on cholinergic activity and for protection in vivo against soman toxicity in guinea pigs. This carbamylated derivative of N-allyl-3-quinuclidinol (NAQ), a potent inhibitor of high-affinity choline uptake, demonstrated stereospecific alterations of cholinergic function as well as protection against soman. The R-isomer, but not the S-isomer, of CAB inhibited erythrocyte acetylcholinesterase (AChE) and plasma pseudocholinesterase (pChE) in a concentration-response manner (IC50 = 25 and 29 microM, respectively). The R-isomer of CAB was also a more potent inhibitor of high-affinity choline uptake (IC50 = 4.8 microM) than S-CAB (IC50 = 63 microM). When R-CAB (10 mumol/kg, i.m.) was administered to guinea pigs 30 min prior to soman in conjunction with atropine (16 mg/kg, i.m.) given 1 min post-soman, the compound significantly reduced lethality up to 5 LD50S. This represents enhanced protection when compared to NAQ (up to 100 mumol/kg); the S-isomer of CAB failed to protect against soman intoxication. The results demonstrate that reversible inhibition of AChE with suppression of acetylcholine synthesis into a single compound, CAB, enhances the protection against organophosphates.