Eserine

Physostigmine (Eserine), CijHjjOjNa. The alkaloid is best prepared in the laboratory by Salway s process. Methods of manufaetiu e are described by Chemnitius and by Sehwyzer. ... 

Ellis, Krayer and Plachte have shown that the degradation products of physostigmine, eseroline and eserine-brown do not inhibit choline-esterase, but rubreserine and eserine-blue are inhibitors in vitro, having... 

A number of synthetic approaches to the pyrrolo[2,3-6]indole ring system, present in tlie physostigmine [i.e., eserine (22)] molecule, which involve indole Grignard reagents as intermediates, have been described. 

Physostigmine (eserine) is a carbamate found in the calabar bean (Physostigma benenosum), which acts as an anticholinesterase. It was used in West Africa in witchcraft trials by ordeal. It has also been used in human medicine. Insecticidal carbamates are structurally related to it and also act as anticholinesterases (Ballantyne and Marrs 1992). 

Julian, P.L. PM, J. (1935) Studies in the Indole Series. V. The Complete Synthesis of Physostigmine (Eserine). Journal of the American Chemical Society, 57, 755-757. 

Wunberg, J.B.P.A. Speckamp, W.N. (1978) New Total Synthesis of dl-Physostigmine (dl-Eserine) Via Regioselective NaBH4-reduction of Imides. Tetrahedron, 34, 2399-2404. 

Longo, V. G. Silvestrini, B. (1957). Action of eserine and amphetamine on the electrical activity of the rabbit brain. J. Pharmacol. Exp. Ther. 120, 160-70. 

Feldberg W., Sherwood S. L. (1954). Behaviour of cats after intraventricular injections of eserine and DFO. J. Physiol. 125, 488-500. 

The reactions of AChE-biotests results in the red-brown product (Hettchet pigment). The reaction of AChE-biotests on inhibitors can be estimated visually. The residual activity of AChE in biotests after the action of different concentrations of eserine (physostigmine) and proserine (neostigmine) is seen in Fig. 3. 

These systems have been used in many bioanalytical applications. A Prospekt system coupled with MS quantitated eserine N-oxide, a cholinesterase inhibitor, in human plasma for low level (4.5 mg) oral administration pharmacokinetic studies (Pruvost et al. 2000). After conditioning of the SPE cartridge (PLRP-S, Spark) with methanol (5 mL/min, 0.5 min) and water (5 mL/min, 0.5 min), a volume of 250 jj.L plasma plus internal standard was injected and washed (water, 1 mL/min, 3 min). The analytes were flushed out with 80 20 ammonium acetate (20 mM, pH 3.5 adjusted with formic acid) and acetonitrile (0.3 mL/min) and separated on a Zobax SB-CN column (150 x 2.1 mm inner diameter, 5 jim). A calibration range of 25 pg/mL to 12.5 ng/mL was achieved with a run time of 10.5 min. 

Pruvost A. et al., 2000. Fully automated determination of eserine N-oxide in human plasma using online solid-phase extraction with liquid chromatography coupled with electrospray ionization tandem mass spectrometry. J Mass Spectrom 35 625. 

To test this possibility, they first examined the effect of phosphorofluoridates on isolated rabbit s intestine. On such a preparation the action of drugs, like acetylcholine, which act directly on the muscle differs characteristically from the action of those, like eserine, which act by inhibition of cholinesterase activity. The directly acting drugs produce an immediate contraction which proceeds rapidly to a maximum, and after the drug has been washed out the muscle again quickly relaxes. The contraction produced by cholinesterase-inhibiting drugs, such as... 

The cholinesterase-inhibiting activity of the phosphorofluoridates was compared quantitatively with that of eserine sulphate thus. To 0-2 ml. of heparinized human plasma was added 05 ml. of a solution containing either eserine or the phosphorofluoridate in varying concentrations then the mixture was kept at room temperature for 10 min. before 1 /tg. of acetylcholine in 1 c.c. saline solution was added. After 5 min. at room temperature, the mixture was made up to 10 ml. with frog saline containing eserine 1/100,000, which at once stopped the action of any cholinesterase not yet inactivated. The solution was then assayed for acetylcholine on the frog rectus-muscle preparation. 

Results. Both the dimethyl and di-isopropyl ester were found to inhibit the cholinesterase activity of human plasma, and their action was stronger than that of eserine. Of the two esters, the di-isopropyl had a more powerful cholinesterase-inhibiting action than the dimethyl ester. An accurate quantitative comparison was made of the action of the di-isopropyl ester with that of eserine sulphate. Under the conditions of Adrian s experiment, the ester at 1/80 million had about the same cholinesterase-inhibiting action as eserine sulphate at 1/14 million, i.e. the di-isopropyl ester was about 5 times as active as eserine sulphate when compared weight for weight, and about 3 times as active when compared in molar solution. 

Poisons. These were the phosphorofluoridates prepared by the Cambridge team of chemists (pp. 2-7), and eserine. Since the phosphorofluoridates slowly hydrolyse in water, stock solutions of these and of eserine were prepared in ethylene glycol monoethyl ether, and... 

Fig. 12. Progress curve of inhibition of horse-serum cholinesterase by eserine and by di -isopropyl phosphorofluoridate in the absence of a substrate at pH 7-4 and 20°. x---x, 5x 10 8 M eserine O—O.ca. 3 x 10-10 M di-isopropyl phosphorofluoridate.

With lower concentrations, the inhibition produced varied with the time of incubation. Fig. 12 shows the inhibition of cholinesterase by di-tsopropyl phosphorofluoridate, and by a comparable amount of eserine, after varying times. The action of eserine reaches a maximum within 5 min., while the inhibition by phosphorofluoridate is initially less rapid, but is progressive and ultimately more complete. The latter effect suggests an irreversible inactivation of the enzyme rather than an equilibrium. 

Reversibility. It is known that the effect of eserine on cholinesterase can be completely reversed by prolonged dialysis against water. On the other hand, it proved impossible to obtain any reversal of the poisoning by the phosphorofluoridate esters (see table below). The enzyme solution (5 ml.) was treated with the inhibitor for 15 min. at 38° 1 ml. was used at once for activity estimation, and the remainder dialysed against running water for 24 hr. in the case of the eserine experiment, 36 hr. in the others. It was clear that the combination between the phosphorofluoridate esters and the enzyme is much firmer than that between eserine and the enzyme. 

Effect of dialysis against water on activity of cholinesterase poisoned with eserine and phosphorofluoridate... 

Under the conditions used, the enzyme was less sensitive to phosphorofluoridate than in the manometric experiments. With an acetylcholine concentration of 0-0045m, 50 per cent inhibition was produced by 2 x 10 7m eserine or 3-5 x 10 8m di-isopropyl phosphorofluoridate. When the substrate concentration was varied over the range 0-0004-0-06 m, the percentage inhibition by the phosphorofluoridate (compared with a standard having a similar substrate concentration but no inhibitor) remained more or less constant. On the other hand, the inhibition due to eserine decreased when the acetylcholine concentration was raised. The difference in the behaviour of... 

Fig. 13. Effect of substrate concentration on inhibition of horse-serum cholinesterase.1 Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 7 m eserine 0—O, 5 x 10 8 m di-isopropyl phosphorofluoridate.

Fig. 13 shows the results obtained with 2 x 10 7m eserine and 5x 10 8m di-isopropyl phosphorofluoridate. Eserine behaves like a... 

Fig. 14. Inhibition of horse-serum cholinesterase by various compounds. Incubated for 15 min. at 20° before addition of 2 mg. of acetylcholine chloride. A, di-isopropyl phosphorofluoridate B, di ec.-butyl phosphorofluoridate C, eserine D, diphenyl phosphorofluoridate E, dithioethyl phosphorofluoridate F, tetramethylphosphorodiamidic fluoride O, diethyl ALmethylphosphor-amidate.

Inhibitory power of eserine, phosphorofluoridates and related compounds on horse-serum cholinesterase... 

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