Synthesis of Heterocyclic Triads by Pd-Catalyzed Cross-Couplings and Evaluation of Their Cell-Specific Toxicity Profile

Two complementary approaches for the preparation of linked 5-membered heterocycles were developed. The Pd-catalyzed Suzuki–Miyaura cross-coupling with halogenated furan, thiophene, and selenophene led to higher overall yields, but C,H-bond activation was a more efficient strategy for the coupling at C(2) of oxazoles. Potency and selectivity of the final hydroxymethyl products in renal (A498), lung (NCI-H226), kidney (CAKI-1), and breast (MDA-MB-468, MCF7) carcinoma cell lines were determined

The chemical names and structures of the identified compounds.

<p>The chemical names and structures of the identified compounds.</p

Mouse ES-derived motor neuron based assays identify phosphatase inhibitors as BoNT/A antagonists.

<p>Four phosphatase inhibitors identified as potential BoNT inhibitors in imaging based assays were analyzed in secondary screens using mES-MNs to confirm their protective activities against BoNT/A. mES-MNs were incubated with 20 μM compound for 30 minutes and then treated with 250 pM BoNT/A for 4 hrs. BoNT/A activity was measured by Western blot analysis and band densitometry. GAPDH and neuron specific β-III tubulin (Tuj1) served as loading controls. Error bars represent standard error of means (n = 3). ★★, ★ Value significant at 99% and 95% confidence level, respectively, compared to DMSO+Toxin control conditions (Student’s <i>t-</i>test).</p

Efficacies of the phosphatase inhibitors against BoNT/A were validated using human ES-MNs.

<p>Four phosphatase inhibitors were evaluated for their ability to protect SNAP-25 in hES-MNs. Cells were treated with increasing concentrations (0.1–20 μM) of inhibitors for 30 minutes and then incubated with 500 pM BoNT/A for 4 hrs (A). Cell lysates were then prepared and analyzed by Western blotting using SNAP-25 antibodies. Bafilomycin was used as a control compound and GAPDH served as a loading control. Efficacies of the inhibitors (20 μM) against 500 pM BoNT/A 30 min (B) and 60 min (C) post-intoxication. The values are given as mean±SEM from at least three independent experiments. ★★, ★ Value significant at 99% and 95% confidence level, respectively, compared to DMSO+Toxin control conditions (Student’s <i>t-</i>test).</p

Phosphatase inhibitors act on host cellular processes.

<p>A well-characterized HPLC-based LC inhibition assay was utilized to determine whether phosphatase inhibitors directly inhibit BoNT/A LC activity <i>in vitro</i>. MV150 is a previously identified LC inhibitor and used as a positive control. The values were calculated from three independent assays.</p><p>Phosphatase inhibitors act on host cellular processes.</p