Figure 1.
Generic structures of compounds studied.
A) Levamisole and resonance forms of protonated levamisole; B) resonance forms of N-substituted analogues of levamisole.
Figure 2.
GRID calculated Molecular Interaction Fields (MIFs) for levamisole.
The depicted conformation corresponds to the global energy minimum conformation of levamisole. A) Methyl probe, contour level –1 kcal/mol; B) amide nitrogen probe, contour level –5 kcal/mol; C) carbonyl oxygen probe, contour level –1 kcal/mol.
Figure 3.
Commercially obtained compounds that were tested in this study.
Figure 4.
Structures of the N-alkylated analogues of levamisole synthesized and tested in this study.
aStandard conditions applied for all compounds except the N-methylated analogue 7a, which was furnished by treatment with methyltriflate in diethylether for 2 h at room temperature as previously described [50].
Figure 5.
The effect of levamisole and its analogues recorded in the in vitro angiogenesis assay performed with HUVECs growing on a fibroblast monolayer.
The images show HUVECs visualized by immunostaining for CD31 after treatment with: A) 1 mM levamisole (1); B) 1 mM tetramisole (2); C) 1 mM p-bromolevamisole (3); D) 1 mM compound 4; E) 1 mM N-methyllevamisole triflate (7a); F) 0.7 mM N-methyllevamisole trifluoroacetate (7b); G) 0.2 mM N-ethyllevamisole trifluoroacetate (8b); H) 0.7 mM N-ethyllevamisole iodide (8a); I) 0.6 mM N-isopropyllevamisole trifluoracetate (9); J) 0.3 mM N-butyllevamisole trifluoroacetate (10); K) 0.4 mM N-benzyllevamisole trifluoroacetate (11b); L) 0.7 mM N-benzyllevamisole bromide (11a); M) 0.1 mM N-cyclohexylmethylenelevamisole trifluoroacetate (12); N) 1 mM suramin (5); O) medium (control); P) 0.1 % DMSO (the control was diluted 1∶1000 corresponding the concentration of DMSO present when testing 1 mM of a compound diluted from a DMSO stock solution).
Figure 6.
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.
The images show HUVECs visualized by immunostaining for CD31 after treatment with: A) 1 mM N-methyllevamisole triflate (7a); B) 0.5 mM N-methyllevamisole triflate (7a); C) 0.25 mM N-methyllevamisole triflate (7a); D) 0.13 mM N-methyllevamisole triflate (7a); E) 0.06 mM N-methyllevamisole triflate (7a); F) 0.03 mM N-methyllevamisole triflate (7a); G) 0.02 mM N-methyllevamisole triflate (7a); H) 0.1 % DMSO (the control was diluted 1∶1000 corresponding the concentration of DMSO present in A); I) 5 μg/mL goat anti-recombinant human VEGF; J) 12 μM suramin (5); K) 1.5 μM suramin (5); L) medium (control); M) 10 mM BIS-TRIS; N) 10 mM BICINE; O) 10 mM N-methylimidazole; P) 1 mM N-methylimidazole.
Table 1.
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.
Table 2.
Inhibition of human placenta alkaline phosphatase (HPAP), bovine kidney tissue non-specific alkaline phosphatase (TNAP) and sirtuin 1 (SIRT1) by selected compounds.
Figure 7.
Test for activation of sirtuin 1 (SIRT1).
The ability of compound 7a to activate SIRT1 mediated deacetylation was tested in a commercially available fluorogenic assay. Resveratrol was used a positive control, although its relevance as a direct activator of sirtuin activity has been called into question recently [59]–[62]. The data represent two individual assays performed in duplicate.