Toonanoronoids A-E, five new limonoids from Toona ciliata var. yunnanensis.

Five new B-seco-limonoids, namely toonanoronoids A-E (1-5), in conjunction with three previously reported compounds, were isolated from the EtOAc extract of the twigs and leaves of Toona ciliata var. yunnanensis. Their structures were elucidated through comprehensive spectroscopic and X-ray crystallographic analysis. The cytotoxic activities of new compounds against five human tumor cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW480) were screened, Compounds 4 and 5 exerted inhibition toward two tumor cell lines (HL-60, SW-480) with IC50 values between 1.7 and 5.9 µM.

AKT2 is involved in the IL­17A­mediated promotion of differentiation and calcification of murine preosteoblastic MC3T3­E1 cells.

Interleukin (IL)­17A exhibits pleiotropic biological activities and serves a role in the progression of periodontitis. However, data describing the association between IL­17 and osteogenesis are not conclusive. It was previously demonstrated that RAC­ß serine/threonine protein kinase (AKT2)­specific knockdown in MC3T3­E1 cells weakened osteogenic effects. The role of AKT2 in the regulation of IL­17A for osteoblast differentiation and calcification remains unclear. The MTT method was adopted in the present study to assess cell proliferation; cell cycle distribution was analyzed by flow cytometry. Following osteogenic induction treatment, the involvement of phosphatidylinositol 3­kinase (PI3K) and phosphorylated­PI3K was evaluated by western blotting. The effects of IL­17A on osteogenesis­associated markers, including Runt­related transcription factor 2 (Runx­2), alkaline phosphatase (ALP) and osteocalcin (OCN) were evaluated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis. An ALP activity assay and Alizarin Red S staining were used to assess the differentiation and calcification functions. AKT2 knockdown inhibited MC3T3­E1 cell proliferation, inducing significantly increased G0/G1 cell counts, and reduced S and G2/M cell numbers. IL­17A exerted no significant effects. The protein levels of p­PI3K, gene expression levels of IL­17A, Runx­2, ALP and OCN, and relative ALP activity and calcification areas were increased in the induction group, and these effects were markedly promoted by treatment with IL­17A. AKT2 knockdown in MC3T3­E1 cells resulted in reduced IL­17A­induced differentiation and calcification, although it was not completely inhibited. The results of the present study suggested that AKT2 signaling was required for MC3T3­E1 cell proliferation. IL­17A promoted osteoblast differentiation and calcification in a partly AKT2­dependent manner in MC3T3­E1 cells in vitro, possibly reflecting compensation by other signaling pathways. The results of the present study may offer novel perspectives to guide the clinical strategy for the prevention and treatment of periodontitis.

Effect of 15-Deoxy-Δ12,14-prostaglandin J2Nanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model.

BACKGROUND: 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect. METHODS: The study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05. RESULTS: Application of l5d-PGJ2-NC (100 µg/ml) in the local bone defect significantly decreased IL-6, IL-1ß, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05). CONCLUSION: Stable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1ß, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.

Th17 and Th1 Lymphocytes Are Correlated with Chronic Periodontitis.

T cells are involved in the homeostasis of periodontal tissues and mediate bone loss in periodontitis, but the involvement of T-helper cells in chronic periodontitis (CP) in a Chinese population is still unclear. This study aimed to assess the distribution of peripheral and local T helper (Th17) and Th1 in CP. Sixty-eight patients with CP and 43 healthy controls were recruited from April 2012 to July 2014 at the Department of Stomatology, People's Hospital of Xinjiang Uygur Autonomous Region (China). The proportions of Th17 (CD3(+)CD4(+)IL-17(+)) and Th1 (CD3(+)CD4(+)IFN-γ(+)) T-cells in peripheral blood samples were assessed by flow cytometry. Immunohistochemistry was used to quantify interleukin-17 (IL-17) and interferon-gamma (IFN-γ) protein levels in gingival biopsy samples. mRNA levels of IL-17, IFN-γ RORγt, and T-bet in gingival biopsy samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The proportions of circulating Th17 cells and Th1 cells were both more abundant in CP patients than in controls (Th17: 1.05% ± 0.87% vs. 0.62% ± 0.49%, P < 0.01; Th1: 13.93% ± 7.94% vs. 8.22% ± 4.50%, P < 0.001). Positive correlations were obtained between the proportion of circulating Th17 cells and probing depth (PD) (r = 0.320, P = 0.001) and between the proportion of circulating Th1 cells and PD (r = 0.372, P < 0.001). IL-17 and IFN-γ protein levels in gingival biopsy samples were markedly increased in CP compared to controls (both P < 0.05). Relative IFN-γ, IL-17A, and T-bet mRNA levels in CP biopsies were higher compared to controls (all P < 0.05). These results suggest that elevated peripheral and local Th17 and Th1 cells might be involved in the pathogenesis of CP.

Cytokine levels in plasma and gingival crevicular fluid in chronic periodontitis.

PURPOSE: To evaluate the Th1/Th2/Th17 cytokine levels in plasma and gingival crevicular fluid (GCF) from chronic periodontitis patients and healthy controls. METHODS: The concentration of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, TNF, and IFN-γ were determined using a flow cytometric multiplex immunoassay (CBA), and was compared between the periodontitis group and the healthy group. Spearman rho coefficient was used to correlate cytokines in GCF in the periodontitis group and the healthy group, respectively. RESULTS: Comparisons of two groups of Th1/Th2/Th17 cytokine levels in plasma and GCF showed no statistically significant differences (P > 0.05), except Th17 (IL-17) level in plasma that was higher in the periodontitis group than the healthy group (P < 0.05). A stronger correlation between IL-17/IL-4 and IL-17/IL-10 was observed in periodontitis patients than in healthy controls.

Structure-activity relationships and discovery of a G protein biased µ opioid receptor ligand, [(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine (TRV130), for the treatment of acute severe pain.

The concept of "ligand bias" at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical µ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in ß-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased µ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased µ opioid receptor agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain.

PI3K/AKT pathway involvement in the osteogenic effects of osteoclast culture supernatants on preosteoblast cells.

This study aimed to investigate the ability of osteoclasts during bone resorption activities to regulate the differentiation and calcification of osteoblast precursor cells. The bone resorption model was established using in vitro cortical bone slices and mouse RAW264.7 cells, which were differentiated into osteoclasts by stimulation with the receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and scanning electron microscopy (SEM) were used to detect osteoclast differentiation. The osteoblast precursor cell line MC3T3-E1 was cultured with the bone resorption supernatant (BRS). Involvement of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in osteogenesis was evaluated by Western blotting, RT-PCR, and ELISA analysis of markers of the early (runt-related transcription factor-2 and alkaline phosphatase) and late (osteocalcin [OCN]) stages of osteogenesis, and Alizarin Red S staining of matrix mineralization. TRAP staining, RT-PCR, and SEM analysis demonstrated the successful establishment of the bone resorption model. Osteoclast BRS effectively increased the differentiation and calcification of MC3T3-E1 cells. Western blot analysis indicated that the BRS enhanced AKT and p-AKT expression levels in MC3T3-E1 cells. Following AKT2 knockdown and treatment with the PI3K/AKT pathway inhibitor LY294002, the expression of OCN in MC3T3-E1 cells was decreased (p<0.05), as was the calcification area (p<0.05). The data obtained in this study indicated that the osteoclast bone resorption medium promoted the differentiation and calcification of MC3T3-E1 cells and that the PI3K/AKT pathway played a role in this process.

A G protein-biased ligand at the µ-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine.

The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in ß-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the µ-opioid receptor (MOR) to G proteins, but not ß-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less ß-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.

Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors.

Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

(Z)-2-(1,3-Thia-zolidin-2-yl-idene)cyan-amide.

In the title compound, C(4)H(5)N(3)S, the tdihydrothiazole ring is almost planar, the maximum and minimum deviations being 0.188 (2) Šand 0.042 (3) Å, respectively. The crystal structure involves intermolecular N-H⋯N hydrogen bonds.

Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily.

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase.

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.

A new 4-(2-methylquinolin-4-ylmethyl)phenyl P1' group for the beta-amino hydroxamic acid derived TACE inhibitors.

A new P1' group for TACE inhibitors was identified by eliminating the oxygen atom in the linker of the original 4-(2-methylquinolin-4-ylmethoxy)phenyl P1' group. Incorporation of this 4-(2-methylquinolin-4-ylmethyl)phenyl group onto different beta-aminohydroxamic acid cores provided compound 18, which demonstrated potent porcine TACE (p-TACE) and human whole blood activity, excellent PK properties, and good selectivity against a variety of MMPs.

Discovery of novel hydantoins as selective non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE).

A series of novel hydantoins was designed and synthesized as structural alternatives to hydroxamate inhibitors of TACE. 5-Mono- and di-substituted hydantoins exhibited activity with IC50 values of 11-60 nM against porcine TACE in vitro and excellent selectivity against other MMPs.

[The correlation between polymorphism at position -511C/T in the promoter region of interleukin 1B and the severity of coronary heart disease].

OBJECTIVE: To investigate the correlation between polymorphism at position -511C/T in the promoter region of interleukin 1B (IL1B) and the severity of coronary heart disease (CHD). METHODS: The polymerase chain reaction-restriction fragment length polymorphism technique was applied to analyze the polymorphisms of IL1B -511C/T in 127 patients with CHD and 152 controls. And the serum level of lipoproteins was detected by enzymology method. RESULTS: The distribution of IL1B -511C/T polymorphism between acute coronary syndrome (ACS) patients and controls was significantly different (chi-square test=5.72, P<0.01). CT and TT genotype carriers were in increased risk of ACS with more double ratio to CC genotype (OR=2.56, 95%CI=1.17-5.59). In CHD group, total cholesterol and low density lipoprotein-cholesterol levels of patients with CT and TT genotypes (6.09+/-0.97 mmol/L and 3.97+/-0.92 mmol/L) were significantly higher than those of patients with CC genotype (5.12+/-0.56 mmol/L and 2.87+/-0.71 mmol/L, P<0.05). CONCLUSION: The polymorphism at position -511C/T in IL1B is associated with the severity of CHD, and the DNA variation at this position may affect the secretion of IL1B, and aggravate the reaction of inflammation and dyslipoidemia.

Synthesis and structure-activity relationship of a novel sulfone series of TNF-alpha converting enzyme inhibitors.

Replacement of the amide functionality in IM491 (N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]5-piperidinecarboxamide) with a sulfonyl group led to a new series of alpha,beta-cyclic and beta,beta-cyclic gamma-sulfonyl hydroxamic acids, which were potent TNF-alpha converting enzyme (TACE) inhibitors. Among them, inhibitor 4b (N-hydroxy-(4S,5S)-1-methyl-5-[[4-(2-methyl-4-quinolinylmethoxy)phenyl]sulfonylmethyl]-4-pyrrolidinecarboxamide) exhibited IC50 values of < 1 nM and 180 nM in porcine TACE (pTACE) and cell assays, respectively, with excellent selectivity over MMP-1, -2, -9 and -13 and was orally bioavailable with an F value of 46% in mice.

Principles of mucin architecture: structural studies on synthetic glycopeptides bearing clustered mono-, di-, tri-, and hexasaccharide glycodomains.

The structural characteristics of a mucin glycopeptide motif derived from the N-terminal fragment STTAV of the cell surface glycoprotein CD43 have been investigated by NMR. In this study, a series of molecules prepared by total synthesis were examined, consisting of the peptide itself, three glycopeptides having clustered sites of alpha-O-glycosylation on the serine and threonine side chains with the Tn, TF, and STF carbohydrate antigens, respectively, and one with the beta-O-linked TF antigen. Additionally, a glycopeptide having the sequence SSSAVAV, triglycosylated with the Le(y) epitope, was investigated. NMR data for the tri-STF-STTAV glycopeptide were used to solve the structure of this construct through restrained molecular dynamics calculations. The calculations revealed a defined conformation for the glycopeptide core rooted in the interaction of the peptide and the first N-acetylgalactosamine residue. The similarity of the NMR data for each of the alpha-O-linked glycopeptides demonstrates that this structure persists for each construct and that the mode of attachment of the first sugar and the peptide is paramount in establishing the organization of the core. The core provides a common framework on which a variety of glycans may be displayed. Remarkably, while there is a profound organizational effect on the peptide backbone with the alpha-linked glycans, attachment via a beta-linkage has little apparent consequence.

The Total Synthesis of Eleutherobin: A Surprise Ending.

An "sp2 -sp3 Stille coupling" of the vinyl triflate 1 and the stannyl compound 2 is a key step toward the completion of the total synthesis of eleutherobin, a natural product exhibiting taxol-like cytotoxic activity.