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Quantitative Structure-Activity Analysis of Acetylcholinesterase Inhibition by Oxono and Thiono Analogues of Organophosphorus Compounds Donald M. Maxwell* and Karen M. Brecht United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425 Received June 27, 1991
A comparison of the bimolecular rate constants (hi) for inhibition of electric eel acetylcholinesterase (AChE) by the oxono (i.e., P=O) and thiono (i.e., P=S) analogues of parathion, methylparathion, leptophos, fonofos, sarin, and soman revealed that the oxono/thiono ratios of ki values varied from 14 for soman to 1240 for parathion. Analysis of the relative importance of the dissociation equilibrium constant and the phosphorylation rate constant in producing this variation in kivalues indicated that the oxono analogues had phosphorylation rate constant values that varied in a narrow range from 8- to 14-fold greater than their thiono counterparts, while the oxono/thiono ratios for dissociation constants varied widely from 1for soman to 82 for fonofos. The lower affmities of thiono analogues for AChE probably resulted from differences in the hydrophobic binding of oxono and thiono analogues to the active site of AChE, inasmuch as the hydrophobicities (i.e., octanol/water partition coefficients) of thiono organophosphorus compounds were much greater than the hydrophobicities of their oxono analogues. Quantitative structure-activity analysis indicated that the hydrophobic effects of oxono and thiono moieties correlated with log ki for AChE inhibition to a greater extent (r2 = 0.79) than their electronic effects (r2I0.48). These observations suggest that the differences in hydrophobicity of oxono and thiono analogues of organophosphorus compounds may be as important as their electronic differences in determining their effectiveness as AChE inhibitors. Introduction The poor reactivity of phosphothioates and phosphonothioaks for acetylchoheskrase (AChE)’in to their oxono analogues has been well documented (1-3).
* Author to whom correspondence should be addressed.
The low reactivity of thiono organophosphorus compounds for mammalian AChE provides a safety factor for agricultural of pesticides, such as or while the rapid metabolic oxidation of these thiono (i-e., Abbreviations: AChE, acetylcholinesterase;Pr’, isopropyl; Pin, pinacolyl.
This article not subject to U.S. Copyright. Published 1992 by the American Chemical Society
AChE Inhibition by Oxono and Thiono Analogues Table I. Structures of Oxono and Thiono Analogues of Organophosphorus Compounds 0,s
II
A,-PP-X
I
A2
oxono/thiono analogues paraoxon/ parathion
A,
Et0 methylparaoxon/methylparathion Me0 fonofos oxon/fonofos Et leptophos oxon/leptophos Ph Me sarin/ thiosarin soman/ thiosoman Me a
A2
X"
Et0 OPh-4-NO2 Me0 OPh-4-N02 Et0 SPh Me0 OPh-4-Br-2,5-C12 Pr'O F Pin0 F
Leaving group.
P=S) pesticides to their oxono (i.e., P=O) analogues in insects, but not mammals, produces a beneficial selective toxicity. Although oxono organophosphorus inhibitors of AChE are consistently better inhibitors than their thiono analogues, the ratios of their reactivities with AChE vary considerably. The bimolecular rate constant for inhibition of AChE by paraoxon was reported to be 10000-fold greater than that of its thiono analogue ( l ) ,while the rate constant for inhibition of AChE by soman was only 3-fold greater than the rate constant for its thiono analogue (2). The usual explanation for the greater inhibition of AChE by oxono organophosphorus inhibitors is that oxygen is more electronegative than sulfur, which results in a correspondingly greater reduction in the electron density around phosphorus (4). The greater reduction of the electron density around phosphorus produced by oxygen vs sulfur enhances the electrophilicity of phosphorus and thereby increases its reactivity toward nucleophiles, such as water or the active site serine of AChE. This explanation appears to adequately explain the 10-fold greater hydrolysis rate of paraoxon vs parathion and methylparaoxon vs methylparathion (5), but it was inadequate to explain the 10000-fold difference in AChE reactivities of paraoxon vs parathion (1,6) or to account for the tremendous variation in the effect of oxono vs thiono analogues for AChE inhibition by alkyl methylphosphonofluoridates (2). To address these unresolved questions concerning the differences in AChE inhibition by oxono and thiono organophosphorus compounds, we performed a quantitative structure-activity analysis of the oxono and thiono analogues of a heterogeneous group of organophosphate and organophosphonate inhibitors (see Table I). The in vitro activity of these inhibitors was evaluated by measurement of their bimolecular rate constants for AChE inhibition, as well as their dissociation and phosphorylation constants, which had not been previously measured for any thiono organophosphorus compounds. A variety of electronic, hydrophobic, and steric properties for the substituents of these organophosphorus compounds were used as independent variables for quantitative structure-activity analysis.
Materials and Methods Chemicals. Parathion, paraoxon, methylparathion, methylparaoxon, leptophos, leptophos oxon, fonofos, and fonofos oxon were obtained from the Pesticides and Chemicals Repository, U.S. Environmental Protection Agency (Research Triangle Park, NC). Soman, thiosoman, sarin, and thiosarin were obtained from the Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD). The purity of all organophosphorus compounds was >98.9% as determined by 31Pnuclear magnetic resonance spectroscopy. Caution: All of the aboue-mentioned organophosphorus compounds are hazardous and should be
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 61 handled carefully (7).Acetylthiocholine iodide, 5,5'-dithiobis(Znitrobenzoic acid), and electric eel AChE (type V-S) were purchased from Sigma Chemical Co. (St. Louis, MO). Determination of Kinetic Constants for AChE Inhibition. The bimolecular rate constants (ki), dissociation equilibrium constant (&), and phosphorylation rate constants (k ) for inhibition of AChE by organophosphorus compounds, wiere ki = k /Kd, were determined by the method of Wang and Murphy (8). dectric eel AChE dissolved in 0.1 M phosphate buffer (pH 8.0) containing 0.1% bovine serum albumin was incubated a t 5 OC with a small volume of organophosphorus inhibitor dissolved in ethanol. The concentration of AChE used for inhibition studies was 0.3 pM except for organophosphorus compounds with ki > 10' M-l min-l, where a 10-fold reduction in enzyme concentration was necessary. In order to satisfy the experimental requirements for pseudo-firsborder inhibition kinetics, inhibitor concentrations were a t least 10-fold greater than enzyme concentrations. For inhibitors with adequate aqueous solubility, k, and Kd were determined from first-order inhibition kinetics using inhibitor concentrations from
