Synthesis and Stability of Boratriazaroles

We describe the synthesis and stability analysis of novel boratriazaroles that can be viewed as bioisosteres of imidazoles or pyrazoles. These heterocycles could conveniently be obtained by condensing a boronic acid and amidrazone <b>1</b> in various solvents. A detailed stability analysis of selected compounds at different pH values as a function of time led to the identification of steric hindrance around the boron atom as a key element for stabilization

Spiroindolines induce coiling (A) and inhibit pharyngeal pumping (B) in <i>C. elegans</i>.

<p>Wild-type (N2) animals treated with SYN351 (shown) or SYN876 grow slowly and display a coiling, “loopy” posture during locomotion (right panel) compared to controls (left panel). Pharyngeal pumping rate was determined for four wild-type (N2) animals at each concentration of SYN876, by manually counting the contractions of the posterior bulb of the pharynx over one minute.</p

The relationship between insecticidal activity against lepidopteran larvae and potency in displacing [3H]-SYN876 from head membranes of <i>L. sericata</i>.

<p>The number of compounds in each category of biological activity is shown above the column. Structures of the analogues used and data for the correlation are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone.0034712.s003" target="_blank">tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone.0034712.s004" target="_blank">S3</a>. The category of biological activity was determined by the lowest EC₈₀ against any of the species tested.</p

The effect of two Spiroindolines and vesamicol on the survival of parental and mutant <i>C. elegans</i> larvae to adulthood.

<p>Black lines represent sensitivity of the wild-type strain N2. Coloured lines represent sensitivity of the mutant alleles selected for resistance to SYN351; <i>cb28</i> (magenta), <i>cb29</i> (red), <i>cb30</i> (green), <i>cb34</i> (cyan). Solid symbols represent the means, and bars represent the maximum and minimum values for three replicate experiments. The amino acid change for each mutant allele is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone-0034712-g008" target="_blank">Figure 8</a>.</p

Binding of Spiroindolines in insect tissues.

<p><b>A</b> - Saturation isotherm for the binding of [³H]-SYN876 in the blowfly <i>L. sericata</i>. Data from two independent experiments is combined and analyzed by fitting to a single site binding model (y = Bmax * x/(Kd+x)). The line represents the theoretical curve for the indicated values of Kd and Bmax. <b>B</b> - Two way correlation plots comparing the pharmacology of [³H]-SYN876 displacement between insect orders. <i>S. littoralis</i>,(Lepidoptera); <i>L. Sericata,</i> (Diptera); and <i>Locusta migratoria,</i> (Orthoptera). Data for the 24 analogues used is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone.0034712.s004" target="_blank">table S3</a>.</p

Field efficacy of SYN876 at different application rates, expressed as percent insect control compared with untreated plots.

<p>Pest and application rates are given on the x axis. Each point represents data from an independent trial and is an average of multiple assessments. Full details are given in online methods.</p

<i>cha-1(p1152)</i> is hypersensitive to SYN876.

<p>Staged L1 hermaphrodite larvae were plated onto agar culture medium (∼50 animals/well of a 24 well microplate) containing SYN876 added as a concentrated solution in dimethylsulphoxide (DMSO) to give the indicated final concentrations. The final concentration of DMSO in all assays was 1%. Micrographs show growth after 3 days.</p

From hit to lead: selected structures of Spiroindolines.

<p>a) <b>1</b>: insecticidal hit identified through high-throughput screening; b) <b>SYN351</b>: lead compound used in studies of resistance in <i>C.elegans</i> and <i>D.melanogaster</i> (prepared according to path b in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone-0034712-g002" target="_blank">Figure 2</a>); c) <b>SYN876</b>: lead compound tested in the field for insecticidal activity (prepared according to path a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone-0034712-g002" target="_blank">Figure 2</a>) and used as radioligand ([³H]- <b>SYN876</b> prepared according to path d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034712#pone-0034712-g002" target="_blank">Figure 2</a>).</p

General structure of Spiroindolines and major synthetic routes:

<p>a) Fischer Indole synthesis with flexible variation of R₁; b) alternative Fischer Indole synthesis allowing convergent variation at R₃; c) alternative, novel synthetic pathway through intramolecular Heck reaction allowing broad variation at R₂; and d) functionalisation of piperidine ring (X₁, X₂ = H, ³H, F). Detailed methods are provided in on-line materials.</p

Sublethal doses of SYN876 suppress aldicarb effects.

<p>Wild-type <i>C. elegans</i> was exposed to the indicated compound concentrations. Worms exposed to 1 mM aldicarb are hypercontracted and exhibit little movement (lower left panel). In the presence of both 1 mM aldicarb and 1 µg.ml^-1 SYN876 the worms are much less hypercontracted and exhibit fairly normal sinusoidal locomotion (arrow, lower right panel). Worms exposed to 1 mM aldicarb and <u>></u>2.5 µg/ml SYN876 exhibit movement and growth defects similar to those induced by SYN876 alone (uncoordinated movement and coiling without hypercontraction; not shown).</p