Daniel Cordova

New and selective ryanodine receptor activators for insect control

Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety. Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera. This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle. The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors.

Insect ryanodine receptors: molecular targets for novel pest control chemicals

Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. They are located on the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons and many other cell types. Ryanodine, a plant alkaloid and an important ligand used to characterize and purify the receptor, has served as a natural botanical insecticide, but attempts to generate synthetic commercial analogues of ryanodine have proved unsuccessful. Recently two classes of synthetic chemicals have emerged resulting in commercial insecticides that target insect RyRs. The phthalic acid diamide class has yielded flubendiamide, the first synthetic ryanodine receptor insecticide to be commercialized. Shortly after the discovery of the phthalic diamides, the anthranilic diamides were discovered. This class has produced the insecticides Rynaxypyr® and Cyazypyr™. Here we review the structure and functions of insect RyRs and address the modes of action of phthalic acid diamides and anthranilic diamides on insect ryanodine receptors. Particularly intersting is the inherent selectivity both chemical classes exhibit for insect RyRs over their mammalian counterparts. The future prospects for RyRs as a commercially-validated target site for insect control chemicals are also considered.

Discovery and mode of action of afoxolaner, a new isoxazoline parasiticide for dogs

Afoxolaner is an isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration. In vitro membrane feeding assay data and in vivo pharmacokinetic studies in dogs established an afoxolaner blood concentration of 0.1-0.2 μg/ml to be effective against both fleas (Ctenocephalides felis) and ticks (Dermacentor variabilis). Pharmacokinetic profiles in dogs following a 2.5mg/kg oral dosage demonstrated uniform and predictable afoxolaner plasma concentrations above threshold levels required for efficacy for more than one month. Dose ranging and a 5-month multi-dose experimental study in dogs, established that the 2.5mg/kg oral dosage was highly effective against fleas and ticks, and produced predictable and reproducible pharmacokinetics following repeated dosing. Mode of action studies showed that afoxolaner blocked native and expressed insect GABA-gated chloride channels with nanomolar potency. Afoxolaner has comparable potency between wild type channels and channels possessing the A302S (resistance-to-dieldrin) mutation. Lack of cyclodiene cross-resistance for afoxolaner was confirmed in comparative Drosophila toxicity studies, and it is concluded that afoxolaner blocked GABA-gated chloride channels via a site distinct from the cyclodienes.

Discovery of cyantraniliprole, a potent and selective anthranilic diamide ryanodine receptor activator with cross-spectrum insecticidal activity

Anthranilic diamides are an exceptionally active class of insect control chemistry that selectively activates insect ryanodine receptors causing mortality from uncontrolled release of calcium ion stores in muscle cells. Work in this area led to the successful commercialization of chlorantraniliprole for control of Lepidoptera and other insect pests at very low application rates. In search of lower logP analogs with improved plant systemic properties, exploration of cyano-substituted anthranilic diamides culminated in the discovery of a second product candidate, cyantraniliprole, having excellent activity against a wide range of pests from multiple insect orders. Here we report on the chemistry, biology and structure-activity trends for a series of cyanoanthranilic diamides from which cyantraniliprole was selected for commercial development.

Effects of [3H]‐BIDN, a novel bicyclic dinitrile radioligand for GABA‐gated chloride channels of insects and vertebrates

The radiolabelled bicyclic dinitrile, [3H]‐3,3‐bis‐trifluoromethyl‐bicyclo[2.2.1]heptane‐2,2‐dicarbonitrile ([3H]‐BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ‐aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35±3 nM) and dieldrin (IC50, 93±7 nM), displaced [3H]‐BIDN from rootworm membranes. When tested at 100 μM, 1‐(4‐ethynylphenyl)‐4‐n‐propyl‐2,6,7‐trioxabicyclo[2.2.2]octane(EBOB), 4‐t‐butyl‐2,6,7‐trioxa‐1‐phosphabicyclo[2.2.2]octane‐1‐thione (TBPS), 1‐phenyl‐4‐t‐butyl‐2,6,7‐trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]‐BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [3H]‐BIDN from rootworm membranes by a competitive mechanism. Rat brain membranes were also shown to possess a population of saturable, specific [3H]‐BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]‐BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. Functional GABA‐gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABAB receptors were unaffected. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA‐derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo‐oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA‐gated Cl− channels to which a number of insecticidally‐active molecules bind.

4-Azolylphenyl isoxazoline insecticides acting at the GABA gated chloride channel.

Isoxazoline insecticides have been shown to be potent blockers of insect GABA receptors with excellent activity on a broad pest range, including Lepidoptera and Hemiptera. Herein we report on the synthesis, biological activity and mode-of-action for a class of 4-heterocyclic aryl isoxazoline insecticides.

Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides.

Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.

In vitro neuropharmacological evaluation of piperovatine, an isobutylamide from Piper piscatorum (Piperaceae)

Piperovatine, a sialogogic, piscicidal, and buccal local anesthesia producing isobutyl amide from the amazonian piscicidal and toothache-relieving plant, Piper piscatorum Trelease et Yuncker (Piperaceae), was evaluated for its ability to induce changes in neuronal intracellular calcium concentration. Ratiometric calcium imaging of Periplaneta americana neuronal cell cultures upon piperovatine application revealed that this compound induced dramatic increases in intracellular calcium concentration. Calcium flux was not affected by co-application of the muscarinic acetylcholine receptor antagonist, atropine, indicating that the parasympathomimetic system was not involved in piperovatines sialogogic actions. Calcium flux was, however, totally eliminated by co-application of the voltage-gated sodium channel blocker, tetrodotoxin (TTX). This, in conjunction with the repetitive calcium spikes observed in the assay and previous radioligand binding studies on the chemical class, strongly suggest that activation of voltage-gated sodium channels characterizes piperovatines mode of action

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (γ-aminobutyric a acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN ([3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1] heptane-2,2-dicarbonitrile), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.

Discovery, synthesis, and evaluation of N-substituted amino-2(5H)-oxazolones as novel insecticides activating nicotinic acetylcholine receptors.

N-Substituted amino-2(5H)-oxazolones A are a novel class of insecticides acting as nicotinic acetylcholine receptor (nAChR) agonists and show potent activity against hemipteran insect species. Here we report the discovery and preparation of this class of chemistry. Our efforts in SAR elucidation, biological activity evaluation, as well as mode-of-action studies are also presented.