Synthesis and preliminary evaluation of 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones as angiogenesis inhibitors.

Sunitinib (Sutent®) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treatment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours. A key structural motif retained throughout medicinal chemistry efforts during sunitinib's development was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib predecessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is not essential for activity. This Letter describes the synthesis and structure-activity relationships of another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple novel process, wherein commercially available substituted arylacetic acids activated with an amide coupling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 compounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologically relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at 10µg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogenesis inhibitors.

The antiangiogenic properties of sulfated ß-cyclodextrins in anticancer formulations incorporating 5-fluorouracil.

Sulfated ß-cyclodextrins (S-ß-CDs) are useful excipients for improving the solubility of drugs. One such formulation incorporating 5-fluorouracil (5-FU), termed FD(S), showed improved efficacy over 5-FU alone in orthotopic carcinoma xenograft models. S-ß-CDs have heparin-like anticoagulant properties, which may have contributed toward the improved antitumor effect of FD(S). S-ß-CDs have also been reported to modify a number of processes involved in angiogenesis. Although the anticoagulant nature of S-ß-CDs was established, the antiangiogenic properties of S-ß-CDs within FD(S) were unknown. The effect of S-ß-CD and FD(S) on the proliferation and migration of endothelial cells in live-cell kinetic assays, and the reorganization of human umbilical vein endothelial cells into tubule structures in vitro was assessed. The effects of S-ß-CD on angiogenesis in vitro were validated ex vivo using the rat aorta ring assay and the chick embryo chorioallantoic membrane assay. S-ß-CD does not alter proliferative endothelial cell sensitivity to 5-FU cytotoxicity. S-ß-CD alone and within FD(S) significantly inhibited angiogenesis by impeding endothelial cell migration, resulting in the inhibition of tubule formation and hence new vasculature. In addition to the cytotoxic action of the drug 5-FU, therapeutic inhibition of angiogenesis by S-ß-CDs within FD(S) could potentially limit local invasion and metastases. This has important implications for the exploitation of S-ß-CDs for drug formulation improvements or for drug delivery of anticancer biologics.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids.

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion and liquid chromatography to purify components of soybean xylem sap that have pro-angiogenic activity. Using high resolution accurate mass spectrometry and nuclear magnetic resonance spectroscopy, the structure of two pro-angiogenic molecules (FK1 and FK2) were identified as erythro-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (eGGCE), and threo-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (tGGCE). These two molecules, which are coniferyl neolignan stereoisomers, promoted in vitro angiogenesis in the µM to nM range. Independently sourced samples of eGGCE and tGGCE exhibited comparable pro-angiogenic activity to the soybean derived molecules. The cellular mode of action of these molecules was investigated by studying their effect on endothelial cell proliferation, migration, tube formation and adhesion to the extracellular matrix (ECM) components, fibronectin and vitronectin. They were found to enhance endothelial cell proliferation and endothelial cell tube formation on Matrigel, but did not affect endothelial cell migration or adhesion to fibronectin and vitronectin. Thus, this study has identified two coniferyl neolignan stereoisomers, eGGCE and tGGCE, as pro-angiogenic molecules, with eGGCE being less active than tGGCE.

Antiangiogenic platinum through glycan targeting.

Heparan sulfate is identified as a ligand receptor for polynuclear platinum anti-cancer agents through sulfate cluster binding. We present a new biological role for platinum and coordination compounds and a new target for metal-based drugs while presenting a new chemotype for heparanase and growth factor inhibition through modulation (metalloshielding) of their interactions. Masking of extracellular (ECM)-resident heparan sulfate (HS) through metalloshielding results in very effective inhibition of physiologically critical HS functions including enzyme (heparanase, HPSE) and protein growth factor recognition. The interaction of the highly cationic polynuclear platinum complexes (PPCs) with the highly sulfated pentasaccharide Fondaparinux (FPX, in this case as a model HS-like substrate) results in inhibition of its cleavage by the HS-related enzyme heparanase. Binding of the fibroblast growth factor FGF-2 to HS is also inhibited with consequences for downstream signalling events as measured by a reduction in accumulation of phospho-S6 ribosomal protein in human colon tumor HCT-116 cells. The end-point of inhibition of HPSE activity and growth factor growth factor signaling is the prevention of cell invasion and angiogenesis. Finally these events culminate in inhibition of HCT-116 cell invasion at sub-cytotoxic concentrations and the process of angiogenesis. A competition assay shows that Fondaparinux can sequester the 8+ TriplatinNC from bound DNA, emphasising the strength of PPC-HS interactions. Altering the profile of platinum agents from cytotoxic to anti-metastatic has profound implications for future directions in the development of platinum-based chemotherapeutics.

Lipo-chitin oligosaccharides, plant symbiosis signalling molecules that modulate mammalian angiogenesis in vitro.

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.

Eotaxin selectively binds heparin. An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo.

An important feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo. These interactions play an important role in modulating the action of chemokines by facilitating the formation of stable chemokine gradients within the vascular endothelium and directing leukocyte migration, by protecting chemokines from proteolysis, by inducing chemokine oligomerization, and by facilitating transcytosis. Despite the importance of eotaxin in eosinophil differentiation and recruitment being well established, little is known about the interaction between eotaxin and GAGs and the functional consequences of such an interaction. Here we report that eotaxin binds selectively to immobilized heparin with high affinity (K(d) = 1.23 x 10(-8) M), but not to heparan sulfate or a range of other GAGs. The interaction of eotaxin with heparin does not promote eotaxin oligomerization but protects eotaxin from proteolysis directly by plasmin and indirectly by cathepsin G and elastase. In vivo, co-administration of eotaxin and heparin is able to significantly enhance eotaxin-mediated eosinophil recruitment in a mouse air-pouch model. Furthermore, when heparin is co-administered with eotaxin at a concentration that does not normally result in eosinophil infiltration, eosinophil recruitment occurs. In contrast, heparin does not enhance eotaxin-mediated eosinophil chemotaxis in vitro, suggesting protease protection or haptotactic gradient formation as the mechanism by which heparin enhances eotaxin action in vivo. These results suggest a role for mast cell-derived heparin in the recruitment of eosinophils, reinforcing Th2 polarization of inflammatory responses.

C8c-C15 monoseco-analogues of the phenanthroquinolizidine alkaloids julandine and cryptopleurine exhibiting potent anti-angiogenic properties.

Four enantiomerically pure monoseco-analogues, 5, 7, 9, and 11, of the phenanthroquinolizidine alkaloid julandine (1) and four of congener cryptopleurine (2), viz. compounds 6, 8, 10, and 12, have been prepared and subjected to preliminary biological evaluation. These analogues show dramatically reduced cytotoxicity compared with the parent system 2 but they are, nevertheless, potent anti-angiogenic agents.

Use of sulfated linked cyclitols as heparan sulfate mimetics to probe the heparin/heparan sulfate binding specificity of proteins.

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.

Convergent synthesis and preliminary biological evaluations of the stilbenolignan (+/-)-aiphanol and various congeners.

Treatment of an equimolar mixture of stilbene 7 and cinnamyl alcohol 8 with silver carbonate in acetone-benzene afforded a ca. 2:1:2:1 mixture of the stilbenolignan (+/-)-aiphanol (1) and congeners 2-4 each of which show significant anti-angiogenic and COX-2 inhibitory properties.