Total Synthesis of Denbinobin

A total synthesis of denbinobin (1) in seven steps with an overall yield of 10% is reported. This synthesis used an FeCl3-assisted cyclization of stilbene to form a phenanthrene. The poor yields of the decarboxylation and methoxylation steps were improved upon to become essentially quantitative. This scalable methodology was carried out using ordinary laboratory reagents

Additional file 1: of Novel microtubule inhibitor MPT0B098 inhibits hypoxia-induced epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma

Supplemental materials. (DOCX 739 kb

<i>N</i>‑Methylpropargylamine-Conjugated Hydroxamic Acids as Dual Inhibitors of Monoamine Oxidase A and Histone Deacetylase for Glioma Treatment

Glioma treatment remains a challenge with a low survival rate due to the lack of effective therapeutics. Monoamine oxidase A (MAO A) plays a role in glioma development, and MAO A inhibitors reduce glioma growth. Histone deacetylase (HDAC) inhibition has emerged as a promising therapy for various malignancies including gliomas. We have synthesized and evaluated N-methylpropargylamine-conjugated hydroxamic acids as dual inhibitors of MAO A and HDAC. Compounds display potent MAO A inhibition with IC50 from 0.03 to <0.0001 μM and inhibit HDAC isoforms and cell growth in the micromolar to nanomolar IC50 range. These selective MAO A inhibitors increase histone H3 and α-tubulin acetylation and induce cell death via nonapoptotic mechanisms. Treatment with 15 reduced tumor size, reduced MAO A activity in brain and tumor tissues, and prolonged the survival. This first report on dual inhibitors of MAO A and HDAC establishes the basis of translational research for an improved treatment of glioma

<i>N</i>‑Methylpropargylamine-Conjugated Hydroxamic Acids as Dual Inhibitors of Monoamine Oxidase A and Histone Deacetylase for Glioma Treatment

Glioma treatment remains a challenge with a low survival rate due to the lack of effective therapeutics. Monoamine oxidase A (MAO A) plays a role in glioma development, and MAO A inhibitors reduce glioma growth. Histone deacetylase (HDAC) inhibition has emerged as a promising therapy for various malignancies including gliomas. We have synthesized and evaluated N-methylpropargylamine-conjugated hydroxamic acids as dual inhibitors of MAO A and HDAC. Compounds display potent MAO A inhibition with IC50 from 0.03 to <0.0001 μM and inhibit HDAC isoforms and cell growth in the micromolar to nanomolar IC50 range. These selective MAO A inhibitors increase histone H3 and α-tubulin acetylation and induce cell death via nonapoptotic mechanisms. Treatment with 15 reduced tumor size, reduced MAO A activity in brain and tumor tissues, and prolonged the survival. This first report on dual inhibitors of MAO A and HDAC establishes the basis of translational research for an improved treatment of glioma

DKG blocks HIF-1α degradation, rather than inducing its transcription or translation.

<p>(<b>A, B</b>) DKG increases HIF-1α abundance in both normoxic and hypoxic conditions. MDA-MB-231 (A) and MCF7 (B) cells were cultured in normoxia (21% O₂) or hypoxia (1% O₂) for 2-, 6- or 24-hour in the presence or absence of DKG (5 mM). (<b>C</b>) DKG reduces mTOR signaling. MDA-MB-231 cells were treated with DKG in the presence or absence of Gln. The signals of pT389-p70S6K and pT37/46-4EBP1 were used to as surrogate markers for mTOR activation. (<b>D</b>) DKG fails to further elevate HIF-1α protein abundance in cells pre-treated with MG132. MDA-MB-231 cells were pre-treated with a proteasome inhibitor, MG132 (5 µM, 4-hour) to block the proteasomal degradation. The cells were then continuously treated with MG132 and vehicle or DKG to assess HIF-1α abundance. (<b>E</b>) DKG, like DMOG or DFO, converges on proteasome-mediated degradation of HIF-1α. MDA-MB-231 cells treated with MG132 were co-treated with DKG (5 mM), DMOG (0.1 mM) or DFO (100 µM) for 6-hour and assessed for HIF-1α abundance. (<b>F</b>) Inhibiting PHD does not further increase the level of HIF-1α protein induced by DKG. MDA-MB-231 cells were treated with DKG (5 mM), DMOG (0.1 mM) or both for the indicated time periods under normoxia. (<b>E</b>–<b>F</b>) Numbers in <i>italic</i> represent the relative levels of HIF-1α protein. The level in untreated cells was set to 1. (<b>A</b>, <b>C</b>–<b>F</b>) A representative Western image from 3 independent experiments is shown. <i>Italic numbers</i> indicate the relative protein level after normalization with the level in the untreated cells set as 1.</p

Intermediary Metabolite Precursor Dimethyl-2-Ketoglutarate Stabilizes Hypoxia-Inducible Factor-1α by Inhibiting Prolyl-4-Hydroxylase PHD2

<div><p>Hypoxia-inducible factor 1α (HIF-1α), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable precursor of a key metabolic intermediate, α-ketoglutarate (α-KG), known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1α by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1α abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1α stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO), an iron chelator, or dimethyloxalyglycine (DMOG), an established PHD inhibitor, but was not recapitulated with other α-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an α-KG precursor to increase HIF-1α abundance and activity. We propose that DKG acts as a potent HIF-1α activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1α pathway to tumor biology.</p></div

2-Amino and 2‘-Aminocombretastatin Derivatives as Potent Antimitotic Agents

A novel series of 2-amino and 2‘-aminocombretastatin derivatives were synthesized and evaluated for antitumor activity. Several compounds had excellent antiproliferative activity as inhibitors of tubulin polymerization. Compounds 11, 20, and 21 with IC50 values of 1.6, 1.7, and 1.8 μM, respectively, exhibited more potent inhibition of tubulin polymerization than colchicine and approximately as active as combretastatin A-4. They also displayed antiproliferative activity with an IC50 values ranging from 11 to 44 nM in a variety of human cell lines from different organs. Structure activity relationship information suggests that the NH2 substituent at the 2-position of either ring A or ring B in combretastatin molecular skeleton may play an important role in the bioactivity of this series of compounds

DKG activates HIF-1α downstream signaling.

<p>(<b>A, B, C</b>) DKG induces HIF-1α target gene expression. <i>GLUT1 (A)</i>, <i>PDK1 (B) and CAIX (C)</i> mRNA abundance was assessed in MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (5 mM, 6- or 16-hour) by quantitative PCR. _*: <i>p</i><0.05; _**: <i>p</i><0.01 (DKG-treated versus. untreated); #: <i>p</i><0.05; ##: <i>p</i><0.01 (shHIF-1α versus. shCON); n = 3. (<b>D</b>) CAIX is induced by DKG in a HIF-1α-dpenendent manner. CAIX protein level in the MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (5 mM, 6- or 24-hour) was examined by Western blot analyses. A representative Western image from 3 independent experiments is shown. (<b>E</b>) DKG mediates a HIF-1α-dependent increase of <i>p21</i> mRNA abundance. <i>p21</i> mRNA abundance was measured in MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (10 mM, 48-hour) by quantitative PCR. (<b>F</b>) HIF-1α activates <i>p21-Luc</i> reporter. <i>p21-Luc</i> reporter activity was quantitated by luciferase assays in HEK293 cells co-transfected with a combination of HIF-1α expression construct, <i>p21-Luc</i>, and renilla control reporter. (<b>G</b>) DKG inhibits cell proliferation. Cell proliferation was determined by measuring the activity of acid phosphatase (ACP) in the MDA-MB-231 cells treated with DKG (10 mM, 72-hour). (<b>H</b>) DKG does not induce apoptosis/cell death. Annexin V/PI double staining was performed to quantitate apoptosis/cell death in MDA-MB-231 cells treated with DKG (10 mM, 24-hour). (<b>I</b>) DKG pre-treatment renders doxorubicin resistance. MDA-MB-231 cells were pre-treated with DKG (10 mM, 7-day) and then treated with increasing doses of doxorubicin (0–400 nM). Cell proliferation was measured by ACP assay. _*: <i>p</i><0.05; _**: <i>p</i><0.01; n = 3.</p

DKG stabilizes HIF-1α by inhibiting HIF-1α proline hydroxylation and degradation.

<p>(<b>A</b>) DKG increases HIF-1α protein stability. MDA-MB-231 cells were pretreated with DKG (5 mM, 2-hour), then treated with CHX (20 µg/ml) in the presence or absence of DKG (5 mM) for an additional time period as indicated (<i>upper panel</i>). The relative abundance of HIF-1α is shown (<i>lower panel</i>). _*: <i>p</i><0.05; n = 3. (<b>B</b>) DKG blocks HIF-1α degradation mediated by PHD2. MDA-MB-231 cells were transfected with control siRNA or siRNAs targeting PHD1, PHD2 or PHD3 before treating with DKG (5 mM, 2-hour) (upper panel). <i>Italic numbers</i> represent the relative quantitation of protein levels. The level in the untreated sample from each pair of siCON-transfected, untreated cells was set as 1. The extent of respective knockdown of PHD1, 2 or 3 was assessed with semi-quantitative nested RT-PCR, followed by agarose gel electrophoresis (<i>lower panel</i>). (<b>C</b>) DKG increases HIF-1α stability through its oxygen-dependent degradation domain (ODD). ODD-luciferase activity was assayed in MDA-MB-231 cells co-transfected with ODD-luciferase reporter and control renilla luciferase reporter and treated with increasing doses of DKG for 6-hour. DMOG-treated cells serve a positive control. _*: <i>p</i><0.05; n = 3. (<b>D</b>) Hydroxylation at Pro564 of HIF-1α is inhibited by DKG. MDA-MB-231 cells were treated with the proteasome inhibitor, MG132, combined with the indicated chemicals to assess the level of HIF-1α hydroxylated at Pro564. The high-molecular weight, smeared species are the ubiquitylated HIF-1α. (<b>A</b>, <b>B</b>, <b>D</b>) A representative Western image from 3 independent experiments is shown.</p