Regioselective Ring Opening Reactions of Pyridine N‑Oxide Analogues by Magnesium Hydride Complexes

The stoichiometric reactions of phosphinimino-amino (PIA)-supported magnesium hydride complex <b>1</b>, [L₁MgH]₂ (L₁ = (2,6-^<i>i</i>Pr₂-C₆H₃)­NC­(Me)­CHP­(Cy₂)­N­(2,6-Me₂-C₆H₃)), with pyridine <i>N</i>-oxide and 2-phenylpyridine <i>N</i>-oxide afforded 2,4-pentadiene-1-oximate complex <b>2</b> and 5-phenyl-2,4-pentadiene-1-oximate complex <b>3</b>, respectively. The reaction of <b>1</b> with 2-methylpyridine <i>N</i>-oxide showed a unique regioselectivity to produce 2,4-hexadiene-1-oximate <b>4a</b> in toluene and 3,5-hexadiene-2-oximate <b>4b</b> in THF, respectively. Treatment of β-diketiminato (BDI)-supported magnesium hydride complex <b>5</b>, [L₂MgH]₂ (L₂ = (2,6-^<i>i</i>Pr₂-C₆H₃)­NC­(Me)­CHC­(Me)­N­(2,6-^<i>i</i>Pr₂-C₆H₃)), with quinoline <i>N</i>-oxide gave 1,2-dihydroquinoline type product <b>6</b>, while treatment of complex <b>5</b> with 2-methylpyridine <i>N</i>-oxide either in toluene or THF afforded 1-methyl-2,4-pentadiene-1-oximate complex <b>7</b> as the only product. All these complexes were fully characterized by NMR spectroscopy and X-ray diffraction analyses, and mechanism researches were conducted to understand the ring-opening reaction of pyridine <i>N</i>-oxide

Hyaluronan Polymer Length, Grafting Density, and Surface Poly(ethylene glycol) Coating Influence <i>in Vivo</i> Circulation and Tumor Targeting of Hyaluronan-Grafted Liposomes

Hyaluronan-grafted liposomes (HA-liposomes) preferentially target CD44-overexpressing tumor cells <i>in vitro via</i> receptor-mediated endocytosis. We investigated the pharmacokinetics and biodistribution of HA-liposomes with various sizes of HA (MW 5–8, 50–60, and 175–350 kDa) in mice. Incorporation of negatively charged HA on the liposome surface compromised its blood circulation time, which led to decreased tumor accumulation in CD44+ human breast cancer MDA-MB-231 xenografts compared to PEGylated liposomes (PEG-5000). Clearance of HA-liposomes was HA polymer length-dependent; high MW (175–350 kDa, highest ligand binding affinity) HA-liposomes displayed faster clearance compared to low MW (5–8, 50–60 kDa) HA-liposomes or PEGylated liposomes. Surface HA ligand density can also affect clearance of HA-liposomes. Thus, HA is not an effective stealth coating material. When dual coating of PEG and HA was used, the PEG-HA-liposomes displayed similar blood circulation time and tumor accumulation to that of the PEGylated liposomes; however, the PEG-HA-liposomes displayed better cellular internalization capability <i>in vivo</i>. Tumor histology showed that PEG-HA-liposomes had a more direct association with CD44+ cancer cells, while PEGylated liposomes located predominantly in the tumor periphery, with less association with CD44+ cells. Flow cytometry analysis of <i>ex vivo</i> tumor cells showed that PEG-HA-liposomes had significantly higher tumor cell internalization compared to PEGylated liposomes. This study demonstrates that a long blood circulation time is critical for active tumor targeting. Furthermore, the use of the tumor-targeting ligand HA does not increase total tumor accumulation of actively targeted liposomes in solid tumors; however, it can enhance intracellular delivery

Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

<div><p>BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1<em>H</em>-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity <em>in vitro</em>. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels <em>in vitro</em> and <em>ex vivo</em>. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.</p> </div

Plasma pharmacokinetics of BPR0L075 in CD-1 mice after administration of drug at 50 mg/kg intraperitoneally.

<p>(A) LC-MS/MS analysis of BPR0L075 with chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.1 and 2.8 min, respectively. (B) The plasma concentration-time curve following BPR0L075 treatment.</p

Ligand-Free Magnesium Catalyst System: Immortal Polymerization of l‑Lactide with High Catalyst Efficiency and Structure of Active Intermediates

A simple, inexpensive, and convenient catalyst system consisting of supporting ligand-free Mg^<i>n</i>Bu₂ in combination with an alcohol, isopropanol (^<i>i</i>PrOH), benzyl methanol (PhCH₂OH), diphenylmethanol (Ph₂CHOH), or triphenylmethanol (Ph₃COH), generates a convenient catalyst system to promote the polymerization of l-LA. In particular, the binary system Mg^<i>n</i>Bu₂/Ph₂CHOH demonstrates an unprecedentedly high activity in the presence of a large excess amount of Ph₂CHOH with the [OH]₀/[Mg]₀ ratio varying from 2 to 500, producing up to 500 polylactide (PLA) chains per Mg center and thus showing a typical nature of immortal polymerization. The molecular weights of the obtained PLAs with a broad range of monomer-to-metal ratios ([l-LA]₀/[Mg]₀ = 200–5000) are rather accurately controlled by the Ph₂CHOH loading, relative to [Mg]₀, while the molecular weight distributions remain nearly constant with polydispersity index (PDI) = 1.08–1.18. Moreover, the active polymerization intermediate has been isolated from the stoichiometric reaction between Mg^<i>n</i>Bu₂ and Ph₂CHOH and structurally characterized as a tetranuclear complex, Mg₄(Ph₂CHO)₈(THF)₂ (<b>1</b>). Complex <b>1</b> remains the tetranuclear structure in solution or in the presence of excess Ph₂CHOH as determined by 2D DOSY. On the basis of structural information about the active intermediates and polymerization kinetics, a coordination–insertion polymerization mechanism is proposed

Histological validation of temporary vascular shutdown in MCF7-luc-GFP-mCherry mammary fat pad tumors with respect to BPR0L075 administration.

<p>(A) Tumor tissues from control mouse 4 hours after vehicle treatment showing H&E staining (original magnification ×100), mCherry fluorescence signals (×100), and luciferase expression stained with anti-luciferase antibody (red) with DAPI (blue) (magnification ×400); (B) Tumor sections from four control tumors showing vascular extent based on anti-CD31 (green) and perfusion marker Hoechst 33342 (blue); and (C) corresponding tumor sections from four tumors in mice treated with BPR0L075 (50 mg/kg i.p.) at different time points (magnification ×400). (D) Whole mount H&E section 24 hrs after administering BPR0L075 showing about 70% necrotic fraction.</p

Magnesium and Zinc Complexes Supported by <i>N</i>,<i>O</i>-Bidentate Pyridyl Functionalized Alkoxy Ligands: Synthesis and Immortal ROP of ε-CL and l-LA

The <i>N</i>,<i>O</i>-bidentate pyridyl functionalized alkoxy ligands 2-(6-methyl-2-pyridinyl)-1,1-dimethyl-1-ethanol (<b>L¹–H</b>) and 2-(6-methyl-2-pyridinyl)-1,1-diphenyl-1-ethanol (<b>L²–H</b>) have been prepared by treatment of acetone and benzophenone with monolithiated 2,6-lutidine. Deprotonolysis of the ligands <b>L¹–H</b> and <b>L²–H</b> with 1 equiv of Mg^<i>n</i>Bu₂ and ZnEt₂ in toluene by releasing butane and ethane, respectively, gave the corresponding dimeric metal-monoalkyl complexes [L¹Mg^<i>n</i>Bu]₂ (<b>1</b>), [L²Mg^<i>n</i>Bu]₂ (<b>2</b>), [L¹ZnEt]₂ (<b>3</b>), and [L²ZnEt]₂ (<b>4</b>). Complexes <b>1</b>–<b>4</b> were characterized by ¹H and ¹³C NMR spectroscopy analysis, and the molecular structures of <b>1</b>, <b>3</b>, and <b>4</b> were further confirmed by X-ray diffraction analysis. The investigation of the catalytic behavior of these complexes toward ε-caprolactone (ε-CL) and l-lactide (l-LA) polymerizations showed that the Mg-based complexes gave higher activity than those attached to zinc metal, probably owing to the greater ionic character of the magnesium metal. Remarkably, the magnesium complex <b>2</b> exhibited a striking “immortal” nature in the presence of primary alcohols where up to 500 PCL chains grew from each Mg active center when benzyl alcohol was employed, while, in particular, in the presence of triethanolamine, complex <b>2</b> also displayed an immortal mode for the polymerization of l-LA

Magnesium and Zinc Complexes Supported by <i>N</i>,<i>O</i>-Bidentate Pyridyl Functionalized Alkoxy Ligands: Synthesis and Immortal ROP of ε-CL and l-LA

The <i>N</i>,<i>O</i>-bidentate pyridyl functionalized alkoxy ligands 2-(6-methyl-2-pyridinyl)-1,1-dimethyl-1-ethanol (<b>L¹–H</b>) and 2-(6-methyl-2-pyridinyl)-1,1-diphenyl-1-ethanol (<b>L²–H</b>) have been prepared by treatment of acetone and benzophenone with monolithiated 2,6-lutidine. Deprotonolysis of the ligands <b>L¹–H</b> and <b>L²–H</b> with 1 equiv of Mg^<i>n</i>Bu₂ and ZnEt₂ in toluene by releasing butane and ethane, respectively, gave the corresponding dimeric metal-monoalkyl complexes [L¹Mg^<i>n</i>Bu]₂ (<b>1</b>), [L²Mg^<i>n</i>Bu]₂ (<b>2</b>), [L¹ZnEt]₂ (<b>3</b>), and [L²ZnEt]₂ (<b>4</b>). Complexes <b>1</b>–<b>4</b> were characterized by ¹H and ¹³C NMR spectroscopy analysis, and the molecular structures of <b>1</b>, <b>3</b>, and <b>4</b> were further confirmed by X-ray diffraction analysis. The investigation of the catalytic behavior of these complexes toward ε-caprolactone (ε-CL) and l-lactide (l-LA) polymerizations showed that the Mg-based complexes gave higher activity than those attached to zinc metal, probably owing to the greater ionic character of the magnesium metal. Remarkably, the magnesium complex <b>2</b> exhibited a striking “immortal” nature in the presence of primary alcohols where up to 500 PCL chains grew from each Mg active center when benzyl alcohol was employed, while, in particular, in the presence of triethanolamine, complex <b>2</b> also displayed an immortal mode for the polymerization of l-LA

Fluorescence images of mammary breast tumor response to administration of BPR0L075 at successive time points.

<p>Sequential images of a single nude mouse with orthotopic MCF7-<i>lacZ</i> and MCF7-luc-GFP-mCherry breast tumors growing in the front right (blue circle) and left mammary fat pad, respectively. Fluorescent signal (mCherry) increased over 24 hours following administration of carrier vehicle (A), but signals diminished 4, 6, and 24 hours after injection of BPR0L075 (B); (C) Variation in normalized fluorescent signal intensity for the group of three MCF7-luc-GFP-mCherry tumors in response to vehicle injection; (D) Variation in normalized fluorescent signal intensity for the group of six MCF7-luc-GFP-mCherry tumors in response to injection of BPR0L075. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.0001; ⁺ only three tumors observed at this time point.</p

Induction of late-stage apoptosis and necrosis by EDC-Herceptin conjugate treatment.

<p>(<b>a</b>) OS187 cells were treated with sham, wt Herceptin (wild-type Herceptin), or EDC conjugate (EDC-Herceptin conjugate) for 48 or 72 h and then stained with annexin v and PI to determine the necrosis and apoptosis under flow cytometry. Necrosis control, freeze cells in −80°C for 5 min, and then thaw cells at 37°C. Serum starving, serum starving overnight as apoptosis control. (<b>b</b>) SKBR3 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023270#pone-0023270-g003" target="_blank">Figure 3</a>, followed by PI, annexin v staining, and flow cytometry analysis.</p