Synthesis and kinetic analysis of some phosphonate analogs of cyclophostin as inhibitors of human acetylcholinesterase

Abstract

Two new monocyclic analogs of the natural AChE inhibitor cyclophostin and two exocyclic enol phosphates were synthesized. The potencies and mechanisms of inhibition of the bicyclic and monocyclic enol phosphonates and the exocyclic enol phosphates toward human AChE are examined. One diastereoisomer of the bicyclic phosphonate exhibits an IC(50) of 3 microM. Potency is only preserved when the cyclic enol phosphonate is intact and conjugated to an ester. Kinetic analysis indicates both a binding and a slow inactivation step for all active compounds. Mass spectrometric analysis indicates that the active site Ser is indeed phosphorylated by the bicyclic phosphonate.

Fig. 1

Structures of Some Organophosphate Inhibitors of AChE.

Fig. 2

Structures of Cyclophostin and Selected Phosphonate Analogs.

Fig. 3. Inhibition AChE by (2a) and (2b)

34 nM human AChE was incubated with inhibitor for 30 min, then assayed. See Experimental section for additional details. (2a) Circles and solid line and (2b) squares and dashed line. The lines are not fits, but rather meant to guide the eye

Fig. 4. Irreversibility of Inhibition by (2a)

E, activity of enzyme incubated in buffer for 30 min and subsequently desalted. E + I, activity of enzyme treated with inhibitor for 30 min but not desalted. The average activity for three trials was 0.2 % of initial activity. E + I + desalt, activity of enzyme incubated with inhibitor for 30 min and subsequently purified by desalting gel. See Experimental section for additional details.

Fig. 5. Kinetics of AChE Inactivation by Cyclic Organophosphonates

Left column: plots of percent residual activity vs. incubation time for the indicated inhibitors. Right column: k_obs derived from global analysis vs. [I]. Data were collected and processed as described in Experimental. For (2a), [I] = 0.5, 1, 2, 4, 8, 16 uM; (2b), [I] = 5, 10, 20, 40, 80 uM; (9) [I] = 5, 10, 20, 40, 80 uM; (15) [I] = 5, 10, 20, 40, 80, 160 uM; DIFP [I] = 2, 4, 8, 16 uM.

Fig. 6. Oxime Reactivation of AChE

After 30 min incubation of human AChE with (2), pralidoxime was introduced and enzyme activity monitored. E+P, enzyme and pralidoxime control. E+I, enzyme and inhibitor control. E+I+P, enzyme and inhibitor and pralidoxime. Conditions are given in the text. All rates are expressed relative to the enzyme control at the same time point in the experiment.

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