Structure‐Reactivity Relationships as Probes to Acetylcholinesterase Inhibition Mechanisms by Aryl Carbamates. I. Steady‐State Kinetics

Abstract

From enzyme kinetics, 4-nitrophenyl-N-substituted carbamates 1 are characterized as pseudo-substrate inhibitors of acetylcholinesterase. However, the activity of the carbamyl enzyme does not recover in the presence of a competitive inhibitor, edrophonium. Therefore, carbamates 1 should be called as the "pseudopseudo-substrate" inhibitors of the enzyme. Moreover, the -logKi, logk c , and logk i values are linearly correlated with Taft-Ingold equation, log (k/k o ) = r*s* + d E s . A three-step AChE inhibition mechanism by carbamates 1 is proposed. The first step is the pre-equilibrium protonations of carbamates 1 with r* value of -1.4 from pK a-s*-correlation. The second step is the enzyme-carbamates 1 tetrahedral intermediate formation from nucleophilic attack of the active site Ser200 on the protonated carbamates 1. The r* value for the -logKi-s*-Es-correlation indicates that the true r* value for the second step is 0.5 [= -0.9 -(-1.4)]. The d value of 0.56 for the -logKi-s*-Es-correlation indicates that carbamates 1 with bulky substituents retarded the formation of enzyme-inhibitor tetrahedral intermediates. The third step (k c step) is the carbamylation step and is the carbamyl enzyme conjugate formation from the enzyme-carbamates 1 tetrahedral intermediate. The r* value of 0.21 for the logkc-correlation indicates that the transition state for the carbamylation step is more negative charge than the enzyme-carbamates 1 tetrahedral intermediate. Moreover, the kc step is insensitive to substituent effects since there is a cancellation of electronic demands for bond-making and bond-breaking components, like SN2 reactions. The d value of 0.00 for the logkc-correlation indicates that the kc step is independent of substituent steric effect. Therefore, the product of this step carbamyl enzyme conjugate is as crowded as the enzyme-carbamates 1 tetrahedral intermediate and is likely bound to the leaving group, p-nitrophenol.

Quantitative structure-activity relationships for the pre-steady state acetylcholinesterase inhibition by carbamates

“…(4)) [15], and AChE inhibitions by carbamates 1 show TaftIngold type correlation (Eq. (5)) [21,38,39]. Cholesterol esterase inhibitions by substituted phenyl-Nbutylcarbamates correlate with Hammett equation (4) [19,[26][27][28], and cholesterol esterase inhibitions by carbamates 1 correlate with Taft-Ingold equation (6) [19,28,29].…”

“…In the presence of substrate, the pseudosubstrate inhibition mechanisms of AChE by carbamates 1 (Figure 2) have been proposed (Scheme 1) [15,21]. Since the inhibition follows first-order kinetics over the observed time period for the steady-state kinetics, the rate of EI hydrolysis must be significantly slower than the rate of EI' formation (k c k d ) [16][17][18][19].…”

“…(1), the k app values are the first-order rate constants that can be calculated according to Hosie's method [17][18][19]21,27]. The bimolecular rate constant, the third criterion for the pseudosubstrate inhibitors as proposed by Abeles and Maycock [20], the enzyme does not protect from inhibition by carbamates 1 in the presence of a reversible inhibitor, edrophonium ( Figure 2) [5,6].…”

“…Edrophonium is excluded from accessing the enzyme active site by the carbamyl moiety of the carbamyl enzyme, which stretches along the active site gorge and/or due to a conformational change for His440 and Phe330 of the carbamyl enzyme [7,14]. Therefore, like rivastigmine ( Figure 2) [14], carbamates 1 should be characterized as the active-directed pseudosubstrate inhibitors of AChE [21,38,39]. According to pre-steady state kinetics, formation of the enzyme-inhibitor tetrahedral intermediates is further divided into two steps: (1) formation of the noncovalent enzyme-inhibitor complexes (E · I) with the dissociation constant (K S ) and (2) formation of the covalent enzyme-inhibitor tetrahedral intermediates with the equilibrium constant (k 2 /k −2 ) (Scheme 2) (Eq.…”

Synthesis of enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate and endo‐2‐norbornyl‐N‐n‐butylcarbamate for stereoselective inhibition of acetylcholinesterase

“…2) are also potential drugs for the treatment of AD. 15 Since rivastigmine 16 and physostigmine are carbamates, the inhibition mechanism of AChE by carbamates [17][18][19][20][21][22][23] plays important roles for treatment of AD.…”