Synthesis of β‑Substituted Cyclic Enones via Phosphonium Salt-Activated, Palladium-Catalyzed Cross-Coupling of Cyclic 1,3-Diones

Phosphonium salt-activated, Pd-catalyzed Suzuki–Miyaura and Sonogashira cross-coupling reactions of cyclic 1,3-diones in the synthesis of β-substituted cyclic enones are described. These transformations exhibit good isolated yield and high generality with respect to both substrates and coupling partners. Extension of the substrate scope to cyclic 1,3-dione equivalents, such as 2-cyanocyclohexanone (<b>4</b>), is also briefly examined

A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity

<div><p>Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules.</p></div

ALDH1A1 is differentially expressed in cancer cell lines and its activity can be indirectly visualized by BAA fluorescence.

<p>(<b>A</b>) Protein extracts from pancreatic cancer cell lines MIA PaCa-2 (lane 1) and PANC-1 (lane 2), colon cancer cell lines HT-29 (lane 3) and SW480 (lane 4) and glioma cell lines LN-18 (lane 5) and LN-229 (lane 6) were analyzed by immunoblot for ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1 expression. β-Actin was used as loading control. The corresponding recombinant protein was loaded for reference (lane 7). (<b>B</b>) Representative fluorescent images of MIA PaCa-2 and HT-29 cells incubated with 500 nM of BAAA substrate and treated with 22 μM DEAB or DMSO for 30 minutes. Green fluorescent images indicate intracellular BAA and blue fluorescent images indicate nuclei staining.</p

Assay optimization.

<p>S:B and Z’ factor heat map according to variation in assay parameters. Green represents best assay conditions. Parameters were calculated using signal from control DMSO wells and inhibitor DEAB (4.6 μM final) wells with n = 32.</p

Assay automation.

<p><b>(A)</b> DEAB dose response curves of 1,000 cells/well MIA PaCa-2 cells incubated with 500 nM of BAAA substrate for 30 min. R1 to R4 represent four different independent replicates of the fully automated assay. R5 and R6 represent two different independent replicates of the semi-automated assay. Data is represented as mean +/- SD, n = 2. (<b>B</b>) Semi- and automated cellular BAA intensity measurements provide similar IC₅₀ values for active compounds. Correlation plot of Log IC₅₀ values of 11 active compounds in the semi-automated (x-axis) and automated (y-axis) assays. Inactive compounds are not included. (<b>C</b>) Correlation plot of Log IC₅₀ values of 288 active compounds in MIA-PaCa-2 (x-axis) and HT-29 (y-axis) cells. Inactive compounds are not included. (<b>D, E</b>) Correlation plot of Log IC₅₀ values for 288 active compounds in enzymatic ALDH1A1 assay (y-axis) and ALDEFLUOR assay (x-axis) in MIA-PaCa-2 (D) and HT-29 (E) cells. Inactive compounds are not included.</p

Assay miniaturization to 1,536-well format.

<p><b>(A)</b> DEAB dose response curves of 1,000 MIA PaCa-2 cells/well incubated with the indicated concentration of BAAA substrate for 60 min. <b>(B)</b> DEAB dose response curves of indicated number of MIA PaCa-2 cells/well incubated with 500 nM of BAAA substrate for 60 min. <b>(C)</b> DEAB dose response curves of 1,000 MIA PaCa-2 cells/well incubated with the indicated concentration of BAAA substrate for 60 min. Data are represented as mean +/- SD, n = 3, and normalized to DMSO (100% activity) and 4.6 μM DEAB (0% activity).</p

Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1)

Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog <b>64</b> (NCT-501) is also highlighted

Structure-Activity Relationship for compound 8.

<p><b>(A)</b> Structure of compound <b>8</b>. The 2,9-diazaspiro[5.5]undecane spirocyclic core, the diphenylmethyl group and the 3,5-dimethylisoxazole moiety are highlighted in yellow, red and blue, respectively. <b>(B)</b> U87-MG cells treated with the indicated analogs were analyzed by immunoblot for GRP78 expression. GAPDH levels were used as housekeeping controls. Data are represented as GRP78/GAPDH ratio for each compound and normalized to the GRP78/GAPDH ratio for 20 μM thapsigargin. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s003" target="_blank">S3 Fig</a> for blot images. <b>(C)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated analog for 16 h. XBP-1 mRNA splicing was monitored by RT-PCR using XBP-1-specific primers, which amplify a spliced or unspliced fragment of 304 or 326 bp, respectively. <b>(D)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 24 h. CHOP and housekeeping GAPDH mRNA levels were monitored by qRT-PCR. Data is plotted relative to the DMSO treated sample set to 1. Error bars indicate the SD of three replicates. <b>(E)</b> U87-MG cells treated with the indicated compounds were tested for their ability to form colonies. Data are represented as the number of colonies, normalized to vehicle DMSO. Error bars indicate SE with n = 3. Note that <b>8e</b> is an inactive control. Analogs <b>8a</b> and <b>8d</b> were tested only at 20 μM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s007" target="_blank">S7 Fig</a> for representative images.</p

Secondary assays identify <i>bona fide</i> ERSR inducers with cytotoxicity activity.

<p><b>(A)</b> U87-MG cells treated with the indicated compounds were analyzed by immunoblot for GRP78 expression. GAPDH levels were used as housekeeping controls. Data are represented as GRP78/GAPDH ratio for each compound and normalized to the GRP78/GAPDH ratio for 20 μM thapsigargin. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s003" target="_blank">S3 Fig</a> for blot images. <b>(B)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 16 h. XBP-1 mRNA splicing was monitored by RT-PCR using XBP-1-specific primers, which amplify a spliced or unspliced fragment of 304 or 326 bp, respectively. <b>(C)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 24 h. CHOP and housekeeping GAPDH mRNA levels were monitored by qRT-PCR. Data is plotted relative to the DMSO treated sample set to 1. Error bars indicate the SD of three replicates. <b>(D)</b> U87-MG cells treated with the indicated compounds were tested for their ability to form colonies. Data are presented as the number of colonies, normalized to vehicle DMSO. Error bars indicate SE with n = 3. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s005" target="_blank">S5 Fig</a> for representative images.</p

Chemical structure and AC50 (μM) of hit compounds in qHTS assays.

<p>For Ca2+ mobilization, values obtained in + EGTA conditions are displayed. Active compounds are considered those with a high quality CRC and efficacy. Efficacy cutoffs are as follow: grp78-luciferase and UPRE-luciferase, >40%; Caspase 3/7 and Ca2+ mobilization, >30%; all cell viability assays, >50%.</p