Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high‐affinity binding site for [3H]IMI with KD values of 1–2 nM and Bmax values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α‐bungarotoxin (α‐BGT)‐agarose affinity column are known to be α‐subunit homooligomers. This study uses 1 ‐ [N ‐ (6 ‐ chloro ‐ 3 ‐ pyridylmethyl) ‐ N ‐ ethyl]amino ‐ 1 ‐ amino‐2‐nitroethene (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2–3 nM) to develop a neonicotinoid‐agarose affinity column. The procedure—introduction of Triton‐solubilized Drosophila or Musca head membranes into this neonicotinoid‐based column, elution with IMI, and analysis by lithium dodecyl sulfate‐polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α‐BGT‐agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I‐α‐BGT‐4‐azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.
1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidine (1), the N-desnitro metabolite of the major insecticide imidacloprid, is known to have similar potency to that of (-)-nicotine as an inhibitor of [3H](-)-nicotine binding at the rat recombinant alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR); IC50 values in the present study are 3.8 nM for (-)-nicotine, 6.0 nM for 1, and 155 nM for imidacloprid. Synthesis of new analogues of 1, modified only in the heterocyclic moiety (five-, six-, or seven-membered rings with NH, S, O, and CH2 substituents), gave compounds varying from 4-fold higher potency (2-iminothiazole analogue 10) to >6000-fold less active than (-)-nicotine. Other potent N-[(6-chloro-3-pyridinyl)methyl] compounds are those in which the heterocyclic imine is replaced with pyrrolidine (19) (IC50 9 nM) or trimethylammonium (22) (IC50 18 nM). A novel conversion of (-)-nicotine to its 6-chloro analogue increased the potency 2-fold. These 6-chloro-3-pyridinyl compounds are of interest as novel nAChR probes and potential metabolites of candidate insecticides.
The insect nicotinic acetylcholine receptor (nAChR) is the target for
the major insecticide imidacloprid
(IMI) and for the first candidate photoaffinity probe described here.
Addition to 1-[(6-chloro-3-pyridinyl)methyl]-4,5-dihydro-2-nitromethylene-1H-imidazolidine
(the nitromethylene analog of the
nitroimine IMI) of formaldehyde and any one of several primary amines
is known to give hexahydro-8-nitroimidazo[1,2-c]pyrimidine derivatives.
These imidazopyrimidines with a wide range of N-substituents were found to inhibit [3H]IMI binding to
the Drosophila or Musca nAChR by 50%
(IC50)
at 0.7−38 nM. Esterification of the
N-(2-hydroxyethyl) derivative with
2-azido-5-(trimethylstannyl)benzoic acid and then iododestannylation using Na125I and
chloramine-T provide the candidate
photoaffinity probe
6-[2-(2-azido-5-[125I]iodobenzoyl)ethyl]-1-[(6-chloro-3-pyridinyl)methyl]-1,2,3,5,6,7-hexahydro-8-nitroimidazo[1,2-c]pyrimidine.
This compound (unlabeled) has an IC50 of 8 nM for
[3H]IMI binding in Drosophila head membranes, and the
125I-labeled photoaffinity probe labels only a
66
kDa protein(s) at a specific site inhibited by (−)-nicotine,
consistent with the insecticide-binding subunit
of the nAChR.
SUMMARYImidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [3H] imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB2H4 (in model studies) or NaB3H4 in absolute ethanol to 2-chloro-5-pyridinylmethanol which was transformed to 2 -chloro-5-chloromethylpyridine on refluxing with thionyl chloride. Coupling with 4,5-dihydro-N-nitro-lH-imidazol-2-amine then gave [ 2H2] imidacloprid incorporating about 95% of the deuterium or [3H2]imidacloprid (25 Ci/mmol) in 80% radiochemical yield. In studies not detailed here [3H] imidacloprid was found to undergo high affinity, specific and saturable binding to a site in insect brain.
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