Bao-Xiang Zhao

Synthesis and structure-activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against A549 lung cancer cells.

A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives were synthesized and the effects of all the compounds on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells. The study on structure-activity relationships and prediction of lipophilicities of compounds showed that compounds with LogP values in the range of 4.12-6.80 had inhibitory effects on the growth of A549 cells, and among of them the hydrazone derived from salicylaldehyde had much more inhibitory effects.

A new rhodamine B-based lysosomal pH fluorescent indicator

We designed and synthesized a new pH fluorescent probe, RCE, based on structural changes of rhodamine dye at different pH values. The probe exhibits high selectivity, high sensitivity and quick response to acidic pH, as well as low cytotoxicity, excellent photostability, reversibility and cell membrane permeability. Fluorescence intensity at 584 nm was increased more than 150-fold within pH range 7.51-3.53. This probe has pKa value 4.71, which is valuable for studying acidic organelles. Because of its long absorption and emission wavelengths, RCE can avoid associated cell damage. The probe can selectively stain lysosomes and monitor lysosomal pH changes in living cells.

An activator of mTOR inhibits oxLDL-induced autophagy and apoptosis in vascular endothelial cells and restricts atherosclerosis in apolipoprotein E -/- mice

Oxidized low-density lipoprotein (oxLDL) inhibits mammalian target of rapamycin (mTOR) and induces autophagy and apoptosis in vascular endothelial cells (VECs) that play very critical roles for the cardiovascular homostasis. We recently defined 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as a new activator of mTOR. Therefore, we hypothesized that 3BDO had a protective role in VECs and thus stabilized atherosclerotic lesions in apolipoprotein E-/- (apoE-/-) mice. Our results showed that oxLDL inhibited the activity of mTOR and increased the protein level of autophagy-related 13 (ATG13) and its dephosphorylation, thus inducing autophagy in human umbilical vein endothelial cells (HUVECs). All of these effects were strongly inhibited by 3BDO. In vivo experiments confirmed that 3BDO activated mTOR and decreased the protein level of ATG13 in the plaque endothelium of apoE-/- mice. Importantly, 3BDO did not affect the activity of mTOR and autophagy in macrophage cell line RAW246.7 and vascular smooth muscle cells of apoE-/- mice, but suppressed plaque endothelial cell death and restricted atherosclerosis development in the mice. 3BDO protected VECs by activating mTOR and thus stabilized atherosclerotic lesions in apoE-/- mice.

A simple and effective coumarin-based fluorescent probe for cysteine

Acrylic acid 3-acetyl-2-oxo-2 H-chromen-7-yl ester (ACA) was rationally designed and synthesized as a simple and effective fluorescent probe for sensing cysteine with high selectivity and naked-eye detection. The probe can detect cysteine by fluorescence spectrometry with a detection limit of 0.657 μM and can be used with calf serum and in live cell imaging. The conjugate addition/cyclization sequence mechanism of the reaction between ACA and cysteine was confirmed by ESI-MS and fluorescence spectra.

Synthesis of novel oxime-containing pyrazole derivatives and discovery of regulators for apoptosis and autophagy in A549 lung cancer cells

A series of novel oxime-containing pyrazole derivatives were synthesized by the reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and 2-bromo-1-phenylethanone followed by the reaction with hydroxylamine hydrochloride. The structures were determined by IR, (1)H NMR, HRMS, and X-ray analysis. A dose- and time-dependent inhibition of proliferation was observed in A549 lung cancer cell after compound treatment. Inhibition of growth was mainly attributed to the autophagy induction.

A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system

In this study, a novel ratiometric pH probe RNL based on fluorescence resonance energy transfer (FRET) was well developed. It was fabricated by integrating the naphthalimide moiety as an FRET donor with the rhodamine moiety as an FRET acceptor. Meanwhile, 4-(2-aminoethyl)morpholine, which was a lysosome-locating group, was introduced. The sensing mechanism was the integration of PET and FRET processes and the comprehensive effect led to the simultaneous intensity enhancement of naphthalimide and rhodamine along with the pH value decrease. With a pKa of 4.82, the fluorescence intensity ratio (I529/I580) of the probe changed significantly within the pH range from 4.50 to 5.50. The probe showed excellent selectivity among various metal cations, amino acids and ATP. Moreover, RNL has been successfully applied in HeLa cells, and the results demonstrated that it could be used to detect lysosomal pH changes. The probe could also selectively stain lysosome in HeLa cells. Besides, the probe exhibited low cytotoxicity and satisfactory photostability in living HeLa cells.

A rhodamine B-based lysosomal pH probe

A novel rhodamine B-based fluorescent probe (RML) for lysosomal pH was developed by integrating a 4-(2-aminoethyl)morpholine moiety, which is a lysosome-targetable group, into a rhodamine B fluorophore, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. The probe responded to acidic pH at low concentration in a short amount of time. In addition, RML showed good membrane permeability and brilliant selectivity among various amino acids and metal cations. RML exhibited an 80-fold increase in fluorescence intensity at 583 nm throughout the pH range of 7.40-4.00 with a pKa of 5.16, which indicates that RML is valuable for studying intracellular acidic organelles. Moreover, RML has been successfully applied in HeLa cells, and the results demonstrated that RML could selectively stain lysosomes in living HeLa cells. Note that RML could be used to detect the pH increase in lysosomes induced by bafilomycin A1 within HeLa cells.

A new fluorescent pH probe for extremely acidic conditions.

A novel turn-off fluorescent probe based on coumarin and imidazole moiety for extremely acidic conditions was designed and developed. The probe with pKa=2.1 is able to respond to very low pH value (below 3.5) with high sensitivity relying on fluorescence quenching at 460 nm in fluorescence spectra or the ratios of absorbance maximum at 380 nm to that at 450 nm in UV-vis spectra. It can quantitatively detect pH value based on equilibrium equation, pH=pKa-log[(Ix-Ib)/(Ia-Ix)]. It had very short response time that was less than 1 min, good reversibility and nearly no interference from common metal ions. Moreover, using (1)H NMR analysis and theoretical calculation of molecular orbital, we verified that a two-step protonation process of two N atoms of the probe leaded to photoinduced electron transfer (PET), which was actually the mechanism of the fluorescence quenching phenomenon under strongly acidic conditions. Furthermore, the probe was also applied to imaging strong acidity in bacteria, E.coli and had good effect. This work illustrates that the new probe could be a practical and ideal pH indicator for strongly acidic conditions with good biological significance.

Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells

MTOR, a central regulator of autophagy, is involved in cancer and cardiovascular and neurological diseases. Modulating the MTOR signaling balance could be of great significance for numerous diseases. No chemical activators of MTOR have been found, and the urgent challenge is to find novel MTOR downstream components. In previous studies, we found a chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)–dihydrofuran-2(3H)-one (3BDO), that inhibited autophagy in human umbilical vein endothelial cells (HUVECs) and neuronal cells. Here, we found that 3BDO activated MTOR by targeting FKBP1A (FK506-binding protein 1A, 12 kDa). We next used 3BDO to detect novel factors downstream of the MTOR signaling pathway. Activation of MTOR by 3BDO increased the phosphorylation of TIA1 (TIA1 cytotoxic granule-associated RNA binding protein/T-cell-restricted intracellular antigen-1). Finally, we used gene microarray, RNA interference, RNA-ChIP assay, bioinformatics, luciferase reporter assay, and other assays and found that 3BDO greatly decreased the level of a long noncoding RNA (lncRNA) derived from the 3′ untranslated region (3′UTR) of TGFB2, known as FLJ11812. TIA1 was responsible for processing FLJ11812. Further experiments results showed that FLJ11812 could bind with MIR4459 targeting ATG13 (autophagy-related 13), and ATG13 protein level was decreased along with 3BDO-decreased FLJ11812 level. Here, we provide a new activator of MTOR, and our findings highlight the role of the lncRNA in autophagy.

Progress of the synthesis of condensed pyrazole derivatives (from 2010 to mid-2013)

Condensed pyrazole derivatives are important heterocyclic compounds due to their excellent biological activities and have been widely applied in pharmaceutical and agromedical fields. In recent years, numerous condensed pyrazole derivatives have been synthesized and advanced to clinic studies with various biological activities. In this review, we summarized the reported synthesis methods of condensed pyrazole derivatives from 2010 until now. All compounds are divided into three parts according to the rings connected to pyrazole-ring, i.e. [5, 5], [5,F 6], and [5, 7]-condensed pyrazole derivatives. The biological activities and applications in pharmaceutical fields are briefly introduced to offer an orientation for the design and synthesis of condensed pyrazole derivatives with good biological activities.