Structures of the N-alkylated analogues of levamisole synthesized and tested in this study.

<p><i>^a</i>Standard conditions applied for all compounds except the <i>N</i>-methylated analogue <b>7a</b>, which was furnished by treatment with methyltriflate in diethylether for 2 h at room temperature as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045405#pone.0045405-Cabeza1" target="_blank">[50]</a>.</p

Commercially obtained compounds that were tested in this study.

<p>Commercially obtained compounds that were tested in this study.</p

The effect of anti-VEGF and various concentrations of N-methyllevamisole (7a) and suramin (5) recorded in the in vitro angiogenesis assay performed with HUVECs growing on a fibroblast monolayer.

<p>The images show HUVECs visualized by immunostaining for CD31 after treatment with: <i>A)</i> 1 mM <i>N</i>-methyllevamisole triflate (7a); <i>B)</i> 0.5 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>C)</i> 0.25 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>D)</i> 0.13 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>E)</i> 0.06 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>F)</i> 0.03 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>G)</i> 0.02 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>H)</i> 0.1 % DMSO (the control was diluted 1∶1000 corresponding the concentration of DMSO present in A); <i>I)</i> 5 μg/mL goat anti-recombinant human VEGF; <i>J)</i> 12 μM suramin (<b>5</b>); <i>K)</i> 1.5 μM suramin (<b>5</b>); <i>L)</i> medium (control); <i>M)</i> 10 mM BIS-TRIS; <i>N)</i> 10 mM BICINE; <i>O)</i> 10 mM <i>N</i>-methylimidazole; <i>P)</i> 1 mM <i>N</i>-methylimidazole.</p

GRID calculated Molecular Interaction Fields (MIFs) for levamisole.

<p>The depicted conformation corresponds to the global energy minimum conformation of levamisole. <i>A)</i> Methyl probe, contour level –1 kcal/mol; <i>B)</i> amide nitrogen probe, contour level –5 kcal/mol; <i>C)</i> carbonyl oxygen probe, contour level –1 kcal/mol.</p

Results of in vitro angiogenesis inhibition of levamisole and its derivatives in comparison with suramin (5), vehicle (DMSO), and medium alone, as observed by HUVEC number and morphology.

a<p>Percent of the area covered by HUVEC as observed by CD31 staining.</p>b<p><i>Clusters</i> refer to morphologies where several cells that form round or elongated clusters; <i>Cords</i> refer to morphologies where single to a few cells form cord-like structures without forming a network.</p>c<p>(++++)  =  clusters with area <4%; (+++)  =  cords with area <4%; (++)  =  clusters with area >4%; (+)  =  intermediate morphologies and varying area percentages.</p