Octahydro-Protoberberine and Protoemetine-Type Alkaloids from the Stems of Alangium salviifolium and Their Cytotoxicity.

Two octahydro-protoberberine alkaloids, alangiifoliumines A (1) and B (2), and two new protoemetine derivatives, alangiifoliumines C (3) and D (4), together with 11 known compounds, have been isolated from the stems of Alangium salviifolium. While the structures of these compounds were elucidated by spectroscopic methods, the absolute configurations of the new alkaloids were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism spectra calculations on selected stereoisomers. Compounds 1 and 2 represent the first 5,8,8a,9,12,12a,13,13a-octahydro-protoberberine derivatives, in which the aromatic ring D was reduced to cyclohexene. All the compounds isolated were evaluated for their cytotoxic activity against three human cancer cell lines: A-549, HeLa, and SKOV-3. Alkaloids 1, 3, and 6-14 exhibited inhibitory effects against all three human cancer cell lines, with half-maximal inhibitory concentration (IC50) values in the range of 3 nM to 9.4 µM.

Flabellipparicine, a Flabelliformide-Apparicine-Type Bisindole Alkaloid from Tabernaemontana divaricata.

Four new monoterpenoid bisindole alkaloids, flabellipparicine (1), 19,20-dihydrovobparicine (2), 10'-demethoxy-19,20-dihydrovobatensine D (3), and 3'-(2-oxopropyl)ervahanine A (4), and 10 known monoterpenoid indole alkaloids were isolated from the stems of Tabernaemontana divaricata. All structures were elucidated based on spectroscopic methods, and the absolute configuration of 1 was established using conformational analysis and TDDFT-ECD calculation of selected stereoisomers. Compound 1 represents the first flabelliformide-apparicine-type bisindole alkaloid, in which the flabelliformide-like unit connects to the apparicine-like unit with a C-3-C-22' bond and an N-1-C-16' bond to form an uncommon five-membered ring between the two monomers. All alkaloids were evaluated for their cytotoxicity against two human cancer cell lines, MCF-7 and A-549. Compounds 2, 4, and 14 exhibited cytotoxicity against MCF-7 and A-549 with IC50 values in the range of 2 nM to 8 µM.

Three New Cytotoxic Monoterpenoid Bisindole Alkaloids from Tabernaemontana bufalina.

Three new bisindole alkaloids, 3'-(2-oxopropyl)-19,20-dihydrotabernamine (1: ), 3'-(2-oxopropyl)-ervahanine B (2: ), 19,20-dihydrovobparicine (3: ), and 20 known compounds were isolated from the aerial parts of Tabernaemontana bufalina. The structures of these alkaloids were elucidated using spectroscopic methods. The absolute configurations of 1: -3: were determined by the circular dichroic exciton chirality method. Compounds 1: -23: were screened for their cytotoxicity against two human cancer cell lines, A-549 and MCF-7. Ten compounds (1: -3, 10, 14, 16, 17, 19, 22: , and 23: ) exhibited inhibitory effects against the two human cancer cells with IC50 values of 1.19 ~ 6.13 µM.

Catalytic Asymmetric Cascade Vinylogous Mukaiyama 1,6-Michael/Michael Addition of 2-Silyloxyfurans with Azoalkenes: Direct Approach to Fused Butyrolactones.

An unprecedented cascade vinylogous Mukaiyama 1,6-MA/MA of 2-silyloxyfurans and azoalkenes was realized with a Cu(II)/(t)Bu-Box complex. An array of fused butyrolactones containing multiple stereocenters was generally obtained in good yield (up to 90% yield) with exclusive diastereoselectivity (>20:1 dr) and excellent enantioselectivity (up to 99% ee). Carbon isotope effects measured by (13)C NMR revealed a stepwise mechanism for this annulation process.

Toward a biomarker of oxidative stress: a fluorescent probe for exogenous and endogenous malondialdehyde in living cells.

Malondialdehyde (MDA) is a significant biomarker of oxidative stress. Variations of MDA level in biological systems often represent pathological changes that are related with many types of diseases. Although a variety of techniques have been developed for MDA detection, the probing of this biomarker in living cells remains unexplored. Herein, we report a turn-on fluorescent probe, MDAP-1, with a synergistic photoinduced electron transfer (PET)-hydrogen bonding mechanism, which for the first time realizes MDA sensing under physiological conditions with excellent sensitivity and specificity. The probe responds to MDA with a fluorescence enhancement factor (FEF) of up to >170-fold and a large Stokes shift (∼180 nm). Further biological evaluations show that MDAP-1 is able to detect both endogenous and exogenous MDA in living cells. It can be used to track the generation of MDA under oxidative stress, as stimulated by H2O2. We believe the results of this work will be helpful to the studies of MDA-related biological events and the elucidation of the underlying pathological mechanism in the future.

Metabolic engineering of an industrial polyoxin producer for the targeted overproduction of designer nucleoside antibiotics.

Polyoxin and nikkomycin are naturally occurring peptidyl nucleoside antibiotics with potent antifungal bioactivity. Both exhibit similar structural features, having a nucleoside skeleton and one or two peptidyl moieties. Combining the refactoring of the polyoxin producer Streptomyces aureochromogenes with import of the hydroxypyridylhomothreonine pathway of nikkomycin allows the targeted production of three designer nucleoside antibiotics designated as nikkoxin E, F, and G. These structures were determined by NMR and/or high resolution mass spectrometry. Remarkably, the introduction of an extra copy of the nikS gene encoding an ATP-dependent ligase significantly enhanced the production of the designer antibiotics. Moreover, all three nikkoxins displayed improved bioactivity against several pathogenic fungi as compared with the naturally-occurring antibiotics. These data provide a feasible model for high efficiency generation of nucleoside antibiotics related to polyoxins and nikkomycins in a polyoxin cell factory via synthetic biology strategy.

HMDO-promoted peptide and protein synthesis in ionic liquids.

Hexamethyldisiloxane (HMDO) has been developed to efficiently promote the metal-free direct coupling of an amino function of one cysteine-free peptide or protein and a C-terminal thioester of the second peptide in ionic liquids. The amide-coupling reaction proceeds smoothly under mild conditions to afford the corresponding products in good to excellent yields (63-94%). Peptide couplings were also achieved using in-situ-generated thioesters by the thioesterification of oxo esters.

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics.

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

Aesculetin-induced apoptosis through a ROS-mediated mitochondrial dysfunction pathway in human cervical cancer cells.

Aesculetin (1) is an important coumarin found in various plant materials. It has been shown to have antiproliferative effects on several types of human cancer cells, but its effect on cervical cancer cells in vitro is unknown. In this study, we investigated that the cytotoxic effect of 1 on a non-cancer cell line (293) was smaller than on a tumor cell line (HeLa). This is the first report showing the possible mechanism of antiproliferative effect of 1 for the prevention of cervical cancer in cell culture models. It was found that 1 inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and the accumulation of cells in the sub-G1 phase. Treatment with compound 1 decreased the cell growth in a dose-dependent manner with an IC(50) value of 37.8 microM. Aesculetin-induced apoptosis was correlated with mitochondrial dysfunction (DeltaPsi(m)), leading to the release of cytochrome c from the mitochondria to the cytosol, as well as the proteolytic activation of caspases in HeLa cells. These results indicate that 1 induces apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway.

Spiroalanfurantones A-D, Four Eudesmanolide-Furan Sesquiterpene Adducts with a Pentacyclic 6/6/5/5/5 Skeleton from Inula helenium.

Spiroalanfurantones A-D (1-4), four eudesmanolide-furan sesquiterpene adducts with an unprecedented pentacyclic 6/6/5/5/5 skeleton, were isolated from the roots of Inula helenium. Their structures were elucidated by spectroscopic data analysis and single-crystal X-ray diffraction analysis. The plausible biosynthetic pathway for 1-4 is presented. Bioassay showed that compounds 1 and 2 significantly inhibited nitric oxide production in lipopolysaccharide-induced RAW264.7 macrophages with IC50 values of 17.3 and 9.5 µM, respectively.

Contraction of basal filopodia controls periodic feather branching via Notch and FGF signaling.

Branching morphogenesis is a general mechanism that increases the surface area of an organ. In chicken feathers, the flat epithelial sheath at the base of the follicle is transformed into periodic branches. How exactly the keratinocytes are organized into this pattern remains unclear. Here we show that in the feather follicle, the pre-branch basal keratinocytes have extensive filopodia, which contract and smooth out after branching. Manipulating the filopodia via small GTPases RhoA/Cdc42 also regulates branch formation. These basal filopodia help interpret the proximal-distal FGF gradient in the follicle. Furthermore, the topological arrangement of cell adhesion via E-Cadherin re-distribution controls the branching process. Periodic activation of Notch signaling drives the differential cell adhesion and contraction of basal filopodia, which occurs only below an FGF signaling threshold. Our results suggest a coordinated adjustment of cell shape and adhesion orchestrates feather branching, which is regulated by Notch and FGF signaling.

Apoptosis of Hela cells induced by extract from Cremanthodium humile.

Cremanthodium humile (C. humile) is a traditional herbal medicine for treatment of inflammation. Based on initial screening results, the purpose of this study was to evaluate the cytotoxic effect on four human cancer cell lines and one non-cancer cell line (293), then to determine the possible mechanisms of cell death elicited by the extract of C. humile on Hela cells. We have found the ether extract of C. humile (CH-EE) strongly decreased the survival rate of the four human tumor cell lines: Hela, A549, HepG2 and SW480. The cytotoxic effect of CH-EE on 293 was smaller than on tumor cell lines. Flow cytometry assays and nuclear staining showed that CH-EE induced apoptosis in Hela cells. This process was accompanied by the collapse of mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3/7 and -9. Furthermore, CH-EE generated reactive oxygen species (ROS) in Hela cells. These results indicate that CH-EE induces apoptosis in Hela cells through a ROS-mediated mitochondrial dysfunction pathway.

Palladium-catalyzed intermolecular amination of unactivated C(sp3)-H bonds via a cleavable directing group.

Palladium-catalyzed intermolecular amination of unactivated C(sp3)-H bonds was developed. Using NFSI as both the amino source and the oxidant, this protocol operates under mild conditions with excellent terminal selectivity and a broad substrate scope. Moreover, the directing group can be easily removed to produce 1,2-amino alcohols.

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana.

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.

Steroidal Ammonium Compounds as New Neuromuscular Blocking Agents.

Neuromuscular blocking agents are widely used as an anesthesia auxiliary in surgery, which induce relaxation of skeletal muscles by blocking signal transmission at the neuromuscular junction. Many neuromuscular blocking agents s were developed over the past decades, but none of them fully meets the needs of the clinic by various reasons. In this study, a series of quaternary ammonium steroidal neuromuscular blocking agents were synthesized and evaluated on isolated mouse phrenic nerve-hemidiaphragms for their bioactivities. The initial separation of mono- and bis-quaternary ammonium compounds turned out to be very challenging on regular silica gel chromatography. Therefore, a facile purification method, in which the silica gel was pretreated with methanolic sodium bromide solution, was finally achieved. Compounds 3g (0.36 µm) and 4g (0.37 µm) exhibited excellent neuromuscular blocking activities, which were about sixfold to sevenfold higher in potency than that of rocuronium (2.50 µm). In addition, other bis-quaternized compounds also showed good potencies close to that of rocuronium. Furthermore, the preliminary structure-activity relationship of this series was also elucidated. Benzyl group was found to be a promising quaternary group in this series.

[Synthesis of a novel L-nucleoside, beta-L-D4A and its inhibition on the replication of hepatitis B virus in vitro].

AIM: Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome. METHODS: beta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated. RESULTS: The synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10. CONCLUSION: beta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.

N-2-Hydroxybenzaldehyde acylhydrazone-Fe(III) complex: synthesis, crystal structure and its efficient and selective N-methylation.

N-Methyl-N'-2-hydroxybenzaldehyde acylhydrazones have been chemospecifically synthesized in good yield by N-methylation of the Fe(iii) complexes of N-2-hydroxybenzaldehyde acylhydrazones with methyl iodide in tetrahydrofuran. The reaction proceeds with the exclusive formation of the N-methyl derivative without any concurrent O-methylation side reactions. In addition, the N-methylation reaction occurred simultaneously with a complete deprotection step (elimination of the metal ion). As a result, the N-methyl product was obtained in excellent purity without time-consuming chromatographic workup. A free N-2-hydroxybenzaldehyde acylhydrazone ligand could not be methylated under the same conditions.

Organocatalytic asymmetric desymmetrization: efficient construction of spirocyclic oxindoles bearing a unique all-carbon quaternary stereogenic center via sulfa-Michael addition.

An unprecedented enantioselective desymmetrization of spiro cyclohexadienone oxindoles has been developed successfully via organocatalyzed asymmetric SMA, which provides facile access to spirocyclic oxindoles bearing a unique all-carbon quaternary stereogenic center with excellent levels of stereoselectivity.

Bioactive phenolics from the fruits of Livistona chinensis.

This study investigated the antioxidant and cytotoxic activity of the phenolics isolated from the fruits of Livistona chinensis. Four new compounds, 1-{ω-isoferul[6- (4-hydroxybutyl)pentadecanoic acid]}-glycerol (1), E-[6'-(5″-hydroxypentyl)tricosyl]-4-hydroxy-3-methoxycinnamate (2), 2-(3'-hydroxy-5'-methoxyphenyl)-3-hydroxylmethyl-7-methoxy-2,3-dihydrobenzofuran-5- carboxylic acid (3), 7-hydroxy-5,4'-dimethoxy-2-arylbenzofuran (4), together with eleven known phenolics (5-15), were isolated and identified. Among these compounds, 1-4, 5-O-caffeoylshikimic acid (5), caffeic acid (7), and 3-O-caffeoylshikimic acid (8) showed potent antioxidant activity. 1-5, and 8 showed potent antiproliferative activities with IC(50) values among 5-150 µM against HepG2 human liver cancer, HL-60 human myeloid leukemia, K562 human myeloid leukemia, and CNE-1 human nasopharyngeal carcinoma cell lines. On the basis of these findings, it could be proposed that the fruits of L. chinensis may serve as attractive mines of powerful anticancer and antioxidant agents for various purposes.

Synthesis and anticonvulsant activity of 1-(8-(benzyloxy)quinolin-2-yl)-6-substituted-4,6-diazaspiro[2,4]heptane-5,7-diones.

In the present study on the development of new anticonvulsants, 16 new1-(8-(benzyloxy)quinolin-2-yl-6-substituted-4,6-diazaspiro[2,4]heptane-5,7-diones were synthesized and tested for anticonvulsant activity using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test. Two compounds 8e and 8j showed promising anticonvulsant activities in both models employed for anticonvulsant evaluation. The most active compound 8e showed the MES-induced seizures with ED(50) value of 8.6 mg/kg and TD(50) value of 365.3 mg/kg after intraperitoneally injection to mice, which provided compound 8e with a protective index (TD(50)/ED(50)) of 26.8 in the MES test.