Palladium-Catalyzed Cyclocarbonylation of Pyridinylated Vinylogous Amides and Ureas to Generate Ring-Fused Pyridopyrimidinones

As part of a program aimed at generating new heterocyclic frameworks for medicinal chemistry exploration, an efficient approach to the assembly of novel ring-fused pyridopyrimidinones was undertaken. Specifically, a collection of 11<i>H</i>-pyrido­[2,1-<i>b</i>]­quinazoline-1,11­(2<i>H</i>)-diones and 2,3-dihydropyrido­[1,2-<i>a</i>]­pyrrolo­[3,4-<i>d</i>]­pyrimidine-1,10-diones was generated via a palladium-catalyzed, pyridine-directed, cyclocarbonylation of 2-pyridyl-linked vinylogous amides and ureas in yields of up to 90%

Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p

A Pan-GTPase Inhibitor as a Molecular Probe

<div><p>Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.</p></div

Lack of covalent bond formation between GTPases and compound 1.

<p>Rab7 (A), Cdc42 (B) or Ras (C) was incubated with or without compound <b>1</b> overnight followed by extensive washing. Binding of BODIPY^-FL GTP to GTPases was indistinguishable whether the enzymes had been treated with compound <b>1</b> or not.</p

Compound 1 inhibited EGFR degradation in SCC-12F cells.

<p>A. Immunoblots of EGFR at different time points after treatment with <b>1</b> at different concentrations or DMSO. Actin served as a loading control. Each experiment included duplicate samples and was repeated in triplicate. B. Time course of EGFR degradation. The ratio of EGFR to actin was quantified at different time points and compared to time zero when ligand EGF was added.</p

Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p

Compound 1 structure and dose-response curves.

<p>(A) Structure of <b>1</b> and sites of functionalization. Shaded areas are modified regions for SAR. (B) General structure of compound <b>1</b> and analogues. (C) The concentration dependence of compound <b>1</b> on BODIPY^- FL GTP binding to eight GTPases. (D) The concentration dependence of compound <b>1</b> on BODIPY^-FL GDP binding to eight GTPases. Data shown in (C) are representative of at least three independent sets of measurements with each set conducted in duplicate. All the data points from each set were used to fit the sigmoidal dose-response equation. Percent response was calculated as the ratio of (sample MCF–negative control MCF) / (positive control MCF–negative control MCF). For the positive control, DMSO instead of the compound was added; for the negative control, the compound was replaced with GTP at a concentration 5000 fold greater than BODIPY^-FL GTP. MCF stands for median channel fluorescence.</p

Development of (<i>E</i>)‑2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro‑<i>N</i>‑phenylbenzamide, ML336: Novel 2‑Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit <b>1</b> with promising cellular antiviral activity (EC₅₀ = 0.8 μM), limited cytotoxic liability (CC₅₀ > 50 μM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 μM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound <b>45</b>, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC₅₀ = 0.02–0.04 μM, CC₅₀ > 50 μM) while limiting in vitro viral replication (EC₉₀ = 0.17 μM). Brain exposure was observed in mice with <b>45</b>. Significant protection was observed in VEEV-infected mice at 5 mg kg^–1 day^–1 and viral replication appeared to be inhibited through interference of viral nonstructural proteins

Preincubation of compound 1 with GTPases had no effects on EC50s.EC50

<p>The incubation time was 2 h before the addition of BODIPY^-FL GTP. The experiments with and without incubation were carried out in parallel on the same day, in duplicates and repeated. Cdc42_1 was purified in house while Cdc42_2 was purchased from Cytoskeleton. Rab7_1 and Rab7_2 were from two separate preparations purified in house.</p><p>Preincubation of compound 1 with GTPases had no effects on EC50s.EC50</p

Compound 1 inhibited EGFR degradation in Rab7-expressing HeLa cells.

<p>A. Immunoblots of EGFR at different time points after treatment with <b>1</b> or DMSO. B. Time course of EGFR degradation. Data were processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134317#pone.0134317.g007" target="_blank">Fig 7</a>. The experiments were replicated three times.</p