Rhodium-Catalyzed Relay Carbenoid Functionalization of Aromatic C–H Bonds toward Fused Heteroarenes

A rhodium-catalyzed annulation between ethyl benzimidates and α- aroyl sulfur ylides was developed, affording a series of pyrano­[4,3,2-<i>ij</i>]­isoquinoline derivatives in moderate to good yields with good functional group compatibility. The procedure featured dual <i>ortho</i>-C–H functionalization and dual cyclization in one pot. The optoelectronic properties of those fused heteroarenes were tested by UV/vis and fluorescence spectrometers

BMD of femora and lumbar vertebrae in the OVX model and experiment groups.

<p>(A) BMD in OVX and sham-operated groups. Three-month-old virgin female Sprague-Dawley rats were ovariectomized or sham-operated. They were euthanized at 16 weeks after surgery, and their femora and lumbar vertebrae were dissected free of soft tissues and stored in 75% ethanol for the BMD assay. (B) BMD in different experimental groups after 60-day continuous administration. The ovariectomized rats were divided into two groups and intravenously injected with saline or NUCB2^1–83 (50 nmol) once a day. After 60 days, their femora and lumbar vertebrae were collected for the BMD assay. Data represented the mean±SEM(*P<0.05). Number of bone samples used is showed within the parentheses.</p

The effect of NUCB2^1–83 on mineralization measured by Alizarin Red staining.

<p>MC3T3-E1 cells were cultured for 15 days with or without NUCB2^1–83 and the mineralized matrices were stained by Alizarin-red staining method (A) NUCB2^1–83 (B) control group just with conditional medium (C) The mineralized nodules were observed by microscope <i>(magnification 100×).</i></p

The effect of NUCB2^1–83 on osteoclast differentiation quantified by measuring TRAP activity.

<p>RAW 264.7 cells were cultured in the presence of RANKL (50 ng/mL) for 5 days with or without NUCB2^1–83. The cells were fixed and incubated in 10 mM citrate buffer (pH 4.6) containing 10 mM sodium tratrate and 5 mM p-nitrophenylphosphate for 1 h followed by transferring into new well plates containing an equal volume of 0.1 N NaOH. TRAP activity was measured at λ = 405 nm and expressed as percent of that of untreated control. Data represented the mean±SEM(*P<0.05). Number of samples used is showed within the parentheses.</p

The effect of NUCB2^1–83 on osteoclastogenesis of RAW 264.7 measured by TRAP staining.

<p>RAW 264.7 cells were cultured in the presence of RANKL (50 ng/mL) for 5 days with or without NUCB2^1–83 and stained for TRAP using leukocyte acid phosphatase kit. TRAP-positive multinucleated cells were visualized under light microscopy. (A,B) control group just with α-MEM (C,D) RANKL (E,F) RANKL and NUCB2^1–83. B, D, F were taken at 1000× magnification.</p

Expression of NUCB2^1–83 mRNA in MC3T3-E1 cells and differentiated RAW 264.7 cells.

<p>The precise quantitative data were obtained by using Real-Time Quantitative PCR. The results were normalized to beta-actin, which served as a control to verify the amount of samples. Data represented the mean±SEM. Number of samples used is showed within the parentheses.</p

The effect of NUCB2^1–83 and its mutants on the ALP activity of MC3T3-E1.

<p>MC3T3-E1 cells were treated with test samples for six days in the presence of rhBMP-2 (1 µg/mL). The concentrations of NUCB2^1–83 and its mutants were 10 nmol/mL. Data represented the mean±SEM. Number of samples used is showed within the parentheses. Statistical analysis according to one-way ANOVA was followed by Dunnett's test for BMP-2 vs. test samples (**P<0.01, ***P<0.001).</p