Select β- and γ-branched 1-alkylpyrazol-4-yl methylcarbamates exhibit high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase

Abstract

The widespread emergence of pyrethroid-resistant Anopheles gambiae has intensified the need to find new contact mosquitocides for indoor residual spraying and insecticide treated nets. With the goal of developing new species-selective and resistance-breaking acetylcholinesterase (AChE)-inhibiting mosquitocides, in this report we revisit the effects of carbamate substitution on aryl carbamates, and variation of the 1-alkyl group on pyrazol-4-yl methylcarbamates. Compared to aryl methylcarbamates, aryl dimethylcarbamates were found to have lower selectivity for An. gambiae AChE (AgAChE) over human AChE (hAChE), but improved tarsal contact toxicity to G3 strain An. gambiae. Molecular modeling studies suggest the lower species-selectivity of the aryl dimethylcarbamates can be attributed to a less flexible acyl pocket in AgAChE relative to hAChE. The improved tarsal contact toxicity of the aryl dimethylcarbamates relative to the corresponding methylcarbamates is attributed to a range of complementary phenomena. With respect to the pyrazol-4-yl methylcarbamates, the previously observed low An. gambiae-selectivity of compounds bearing α-branched 1-alkyl groups was improved by employing β- and γ-branched 1-alkyl groups. Compounds 22a (cyclopentylmethyl), 21a (cyclobutylmethyl), and 26a (3-methylbutyl) offer 250-fold, 120-fold, and 96-fold selectivity, respectively, for inhibition of AgAChE vs. hAChE. Molecular modeling studies suggests the high species-selectivity of these compounds can be attributed to the greater mobility of the W84 side chain in the choline-binding site of AgAChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC₅₀ = 269 μg/mL) and low cross-resistance to Akron strain (LC₅₀ = 948 μg/mL), which bears the G119S resistance mutation.

Fig. 1

Representative compounds investigated as An. gambiae-selective (1a–3a, 9,10,12,13) or resistance-breaking (5a, 6–8, 11) acetylcholinesterase inhibitors. Propoxur (4a) is approved for IRS but has modest selectivity for An. gambiae [9] and is not resistance-breaking [11].

Fig. 2

A) Comparison of the structures of rivastigmine, dimethylcarbamate 4b, and methylcarbamate 4a. The transferrable ethylmethylcarbamoyl portion of rivastigmine is shown in bold blue; its CH₂ unit is indicated with an arrow. B) Overlay of select residues in ethylmethylcarbamoyl T. californica AChE (gray PDB ID: 1GQR), apo hAChE (green, PDB ID: 4EY4, 2.16 Å) and apo G119S AgAChE (magenta, PDB ID: 6ARX, 2.30 Å). The ethylmethylcarbamoyl group of rivastigmine (gray spheres) is covalently attached to the catalytic serine of TcAChE (S200); the phenolic leaving group is not shown. Note that AgAChE residue numberings begin with the start of the conserved catalytic subunit, and do not include the 161-amino acid N-terminal domain. Thus, D1 is D162 in full- length AgAChE, and F399 is F560 in full- length AgAChE.

Fig. 3

Comparison of hAChE k_i, AgAChE k_i, Ag/h inhibition selectivity, and G3 strain tarsal contact toxicity within three isomeric series a)–c), varying the branch point of the 1-alkyl group from α- to β- to γ-branched to unbranched. Data are taken from Table 2. Note there is minimal variation in CLogP within each isomeric series; calculated topological polar surface area (tPSA) is 53.9 Ų for all structures (ChemDraw Professional 16.0).

Fig. 4

A) Covalent difluoromethyl ketone inhibitor 34, and An. gambiae-selective inhibitor 22a. B) Superposition of the choline-binding sites of hAChE (PDB ID: 1EY4, gray sticks), and apo G119S AgAChE (PDB ID: 6ARX, magenta ribbon, magenta sticks). Note the canonical orientation of the indole rings of W86 (human) and W84 (apo G119S AgAChE). An alternate conformation of the indole of W84 is shown in aquamarine sticks; this movement expands the hydrophobic binding pocket of AgAChE. C) A view of the tetrahedral intermediate of AgAChE-selective inhibitor 22a with G119S AgAChE, focusing on the cyclopentyl ring of the inhibitor. The surface of the hydrophobic binding pocket of AgAChE is colored gray, except for the surface defined by the indole ring of the W84 in its alternate conformation, which is painted aquamarine. Note that AgAChE residue numberings begin with the start of the conserved catalytic subunit, and do not include the 161-amino acid N-terminal domain. Thus, W84 is W245 in full- length AgAChE.