Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian <i>Eudistoma</i> sp. as active. Novel heterocyclic alkaloids eudistidines A (<b>1</b>) and B (<b>2</b>) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (<b>1</b>) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)­pyrimidin-2-amine (<b>3</b>) and 4-methoxy-phenylglyoxal (<b>4</b>), while eudistidine B (<b>2</b>) was synthesized in a similar fashion with glyoxylic acid (<b>5</b>) in place of <b>4</b>. Naturally occurring eudistidine A (<b>1</b>) effectively inhibited CH1/C-TAD binding with an IC₅₀ of 75 μM, and synthetic <b>1</b> had similar activity. The eudistidine A (<b>1</b>) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein–protein binding events

Screening and Biological Effects of Marine Pyrroloiminoquinone Alkaloids: Potential Inhibitors of the HIF-1α/p300 Interaction

Inhibition of the hypoxia-inducible factor 1α (HIF-1α) pathway by disrupting its association with the transcriptional coactivator p300 inhibits angiogenesis and tumor development. Development of HIF-1α/p300 inhibitors has been hampered by preclinical toxicity; therefore, we aimed to identify novel HIF-1α/p300 inhibitors. Using a cell-free assay designed to test compounds that block HIF-1α/p300 binding, 170 298 crude natural product extracts and prefractionated samples were screened, identifying 25 active extracts. One of these extracts, originating from the marine sponge <i>Latrunculia</i> sp., afforded six pyrroloiminoquinone alkaloids that were identified as positive hits (IC₅₀ values: 1–35 μM). Luciferase assays confirmed inhibition of HIF-1α transcriptional activity by discorhabdin B (<b>1</b>) and its dimer (<b>2</b>), 3-dihydrodiscorhabdin C (<b>3</b>), makaluvamine F (<b>5</b>), discorhabdin H (<b>8</b>), discorhabdin L (<b>9</b>), and discorhabdin W (<b>11</b>) in HCT 116 colon cancer cells (0.1–10 μM, <i>p</i> < 0.05). Except for <b>11</b>, all of these compounds also reduced HIF-1α transcriptional activity in LNCaP prostate cancer cells (0.1–10 μM, <i>p</i> < 0.05). These effects occurred at noncytotoxic concentrations (<50% cell death) under hypoxic conditions. At the downstream HIF-1α target level, compound <b>8</b> (0.5 μM) significantly decreased VEGF secretion in LNCaP cells (<i>p</i> < 0.05). In COLO 205 colon cancer cells no activity was shown in the luciferase or cytotoxicity assays. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein–protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription

Characterization and Synthesis of Eudistidine C, a Bioactive Marine Alkaloid with an Intriguing Molecular Scaffold

An extract of <i>Eudistoma</i> sp. provided eudistidine C (<b>1</b>), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (<b>1</b>) is a racemic natural product composed of a tetracyclic core structure further elaborated with a <i>p</i>-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (<b>1</b>) was accomplished in high yield by treating eudistidine A (<b>2</b>) with 4­(2-amino-1<i>H</i>-imidazol-5-yl)­phenol (<b>4</b>) in DMSO. Synthesis of eudistidine C (<b>1</b>) confirmed the proposed structure and provided material for further biological characterization. Treatment of <b>2</b> with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (<b>5</b>–<b>10</b>) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(<i>R</i>)-eudistidine C (<b>1a</b>) and (−)-(<i>S</i>)-eudistidine C (<b>1b</b>). The absolute configuration of these enantiomers was assigned by ECD analysis. (−)-(<i>S</i>)-Eudistidine C (<b>1b</b>) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds <b>1</b>, <b>2</b>, and <b>6</b>–<b>10</b> exhibited significant antimalarial activity against <i>Plasmodium falciparum</i>