Sara Ginsburg

Molecular complementariness as basis for reactivation of alkyl phosphate-inhibited enzyme

Abstract The rate of reactivation of alkyl phosphate-inhibited acetylcholinesterase by a number of pyridine aldoximes and related compounds was measured. These data can be readily interpreted on the basis of molecular complementariness. They illustrate the promoting effect of a quaternary structure by contrasting the much lower activity of tertiary compounds and by the marked salt effect. The importance of spatial relationships, conformation, is very well illustrated by comparison with the very potent reversible competitive inhibitor neostigmine. Some data are given for the enzymes serum esterase and chymotrypsin.

Activity-structure relationships in the reactivation of diethylphosphoryl acetylcholinesterase by phenyl-1-methyl pyridinium ketoximes

Abstract The effect of structure on the activity of phenyl-1-methyl pyridinium ketoximes and bis quaternary derivatives in reactivating diethylphosphoryl acetylcholinesterase was investigated. The inhibited enzyme was reactivated in the presence of acetylcholine, with a pH stat to maintain the pH and record the amount of ester hydrolyzed vs. time during the reactivation. A graphical technique was devised to obtain the pseudo first-order rate constant from this record. The configurations of the 4-derivatives were assigned on the basis of the Beckmann rearrangement. The anti -2-derivative is 20 times more active than the syn , whereas the anti -4-derivative is 6 times less active than the syn . The remarkable increase in activity that occurs in the bis-4-quatemary compounds occurs with both configurations. Compared to the aldoximes, introducing a phenyl group has a modest negative effect on the activity in the 4-derivatives, but the effect is more pronounced in the 2-derivatives.

Reactivation of alkylphosphate inhibited acetylcholinesterase by bis quaternary derivatives of 2-PAM and 4-PAM☆

Abstract Symmetrical and unsymmetrical bis-quaternary derivatives of 2-PAM and 4-PAM were synthesized and studied as reactivators in vitro of acetylcholinesterase inhibited by TEPP and DFP. Certain of the 2-PAM-derivatives and all of the 4-PAM-derivatives were far superior to the parent compound in the sense of producing reactivations at lower concentrations. Despite the fact that 2-PAM is much more active than 4-PAM, the bis-quaternary derivatives of the latter were considerably more active in regard to DFP inhibition, but only slightly more active in regard to TEPP inhibition. The best compound with regard to DFP inhibition was the unsymmetrical bisquaternary salt of 4-PAM and pyridine with a pentamethylene linking chain. In general, the addition of extra binding features in these series produced better reactivators.

The reaction of acetylcholinesterase with O-dimethylcarbamyl esters of quaternary quinolinium compounds

Abstract The dimethylcarbamate esters of some mono- and diquaternary quinolinols were prepared and their anticholinesterase properties investigated by using acetyl-cholinesterase from electric eel. The I 50 values in the steady state of equal rates of carbamylation and decarbamylation were measured and the values of the second-order rate constants for carbamylation were evaluated. Compounds containing the dimethyl-carbamyl function substituted in the 5 and 7 positions were of the same order of activity as neostigmine to which they are structurally related. When substituted in the 3 position, the dimethylcarbamyl derivative was about as active as pyridostigmine which is structurally similar. It was concluded that the structure of the leaving group is important in determining the activity of these compounds and the p K a value of the leaving group appears to be relatively unimportant.

Studies on new, centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimers disease, and other memory disorders.

A study in vitro of new short-acting, non-depolarizing neuromuscular blocking agents.

Abstract Presently available short-acting, depolarizing myoneural blocking agents have significant side effects such as increased intraocular tension, cardiac arrhythmias desensitization of the postjunctional membrane, skeletal muscle injury and muscle pains. Non-depolarizing blocking drugs do not produce profound blocks of short duration. A program was evolved to synthesize compounds producing a non-depolarizing block at low concentrations with a brief action span requiring no antagonists. Several series of mono- and diquaternary esters of alkyl aminoethanols were prepared and studied in vitro . The rates of spontaneous hydrolysis and the hydrolysis catalyzed by acetylcholinesterase and plasmacholinesterase were measured. In addition, the anticholinesterase effects of these agents were evaluated. The myoneural blocking properties were studied with intracellular recordings in frog sciatic nerve-sartorius muscle preparations. Activity-structure relationships are reported. Several compounds warrant further study including investigations in vivo .

The reaction of acetylcholinesterase (AChe) with some quaternary hydroxy aminophenols

Abstract A series of mono- and diquaternary hydroxyl derivatives of quinolinol, isoquinolinol, and stilbazole were synthesized. The dimethylcarbamyl and diethylphosphoryl analogs of these hydroxyl compounds are potent anticholinesterase agents. The anticholinesterase properties of the hydroxyl analogs were evaluated by measuring the values of their competitive and noncompetitive binding constants for the reaction with electric eel acetylcholinesterase. The p K a values were also measured. When compared to the nonhydroxylated reference compounds, the addition of the OH group was found to increase binding of the undissociated species to free AChe (competitive component). These derivatives are generally more poorly bound to the acetyl enzyme (noncompetitive component), and the diquaternary compounds are less potent than their monoquaternary analogs. The values of the binding constants for the reaction of the undissociated forms of the inhibitor with the enzyme were found to correlate qualitatively with the second-order rate constants for carbamylation of the enzyme by the dimethylcarbamyl analogs, but not with the constants for phosphorylation by the diethylphosphoryl compounds. The latter compounds have been shown to correlate well with the p K a value of the quaternary hydroxyl derivatives.

The pharmacology of new short-acting nondepolarizing ester neuromuscular blocking agents: clinical implications.

OR over 15 years, a need has been recogF nized in anesthesia for a short-acting nondepolarizing neuromuscular blocking agent.l-4 Such an agent might well eliminate many or all of the clinical problems currently experienced with succinylcholine.3~4 To this end, we have designed, synthesized, and pharmacologically evaluated more than 100 diester diquaternary ammonium compounds,- of which some in the current series may merit clinical trial, in view of the results obtained in experimental animals.

The reaction of acetylcholinesterase with methanesulfonyl esters of quaternary quinolinium compounds

The methanesulfonyl esters of quaternary and diquaternary quinolinium compounds were synthesized and studied as inhibitors of purified eel acetylcholinesterase. Some derivatives produced only reversible inhibition of the enzyme; the values of their binding constants were measured. For those compounds producing irreversible (acid-transferring) inhibition, the values of the binding constants, first-order and second-order rate constants were measured. It was shown that the values of the second-order rate constants cannot be correlated with the pKa value of the leaving group (unlike the previously studied diethylphosphoryl quinolinium compounds). The values of the binding constants for the reaction of the quaternary hydroxyl analogs with the enzyme were compared with the values measured for the respective methanesulfonyl compounds. The data indicate that, depending on position, the methanesulfonyl group has little effect on or is detrimental to non-covalent binding between enzyme and inhibittor. The potency of these compounds is generally less than their respective dimethylcarbamyl and diethylphosphoryl analogues. However, several decamethylene bridged diquaternary compounds are the most potent of the methanesulfonate anticholinesterase agents.

Action of thiamine and its analog on neuromuscular transmission in the cat

Abstract The action of thiamine and a number of its analogs on tibialis twitch response to peroneal nerve stimulation, respiration, and arterial pressure, as well as their effect on the neuromuscular block produced by d -tubocurarine and decamethonium, were studied in anesthetized cats. It was found that thiamine, pyrithiamine, and pyrithiamine analogs in which there is a hydroxy group on the pyridinium ring caused a decrease in the twitch without initial potentiation, depressed respiration, and lowered arterial pressure. These compounds also antagonized both d -tubocurarine and decamethonium. On the other hand, methyl thiazolium iodide, methyl pyridinium iodide, and the analogs of thiamine and pyrithiamine in which there is no hydroxy group on the ring bearing the quarternary nitrogen, produced an increase in the twitch with muscular fasciculation, and with larger doses, a decrease in the twitch. Changes in respiratory movements generally paralleled those of the twitch. Arterial pressure was usually elevated. These compounds antagonized d -tubocurarine and potentiated decamethonium. The thiazolium fragment, at the dose level studied, increased the twitch only slightly. It also antagonized d -tubocurarine and potentiated decamethonium. The pyrimidine fragment seemed to have no effect on the twitch. It appears that the activity of thiamine and pyrithiamine on the neuromuscular junction is related to the quarternary nitrogen, the “pyrimidyl” coupling, and the presence of a hydroxy group on the onium-bearing ring. The antagonistic action of these compounds against df -tubocurarine and decamethonium also depends on the same structural components.