Design, Synthesis, Formulation, and Bioevaluation of Trisubstituted Triazines as Highly Selective mTOR Inhibitors for the Treatment of Human Breast Cancer.

The aberrant activation of the PI3K/mTOR signaling pathway is implicated in various human cancers. Thus, the development of inhibitors targeting mTOR has attracted considerable attention. In this study, we used a structure-based drug design strategy to discover a highly potent and kinase-selective mTOR inhibitor 24 (PT-88), which demonstrated an mTOR inhibitory IC50 value of 1.2 nM without obvious inhibition against another 195 kinases from the kinase profiling screening. PT-88 displayed selective inhibition against MCF-7 cells (IC50: 0.74 µM) with high biosafety against normal cells, in which autophagy induced by mTOR inhibition was implicated. After successful encapsulation in a lipodisc formulation, PT-88 demonstrated favorable pharmacokinetic and biosafety profiles and exerted a large antitumor effect in an MCF-7 subcutaneous bearing nude mice model. Our study shows the discovery of a highly selective mTOR inhibitor using a structure-based drug discovery strategy and provides a promising antitumor candidate for future study and development.

Synthesis and bioevaluation of Scutellarein-Tertramethylpyrazine hybrid molecules for the treatment of ischemic stroke.

Ischemic stroke caused by insufficient blood supply to the brain may produce a sequence of cascade reactions, leading to oxidative stress and ultimately inducing nerve cell damage. Therefore, hybrid molecules with multiple therapeutic effects have irreplaceable advantages for the treatment of ischemic stroke. Based on the previous works, two types of Scutellarein and Tertramethylpyrazine hybrid molecules were designed and synthesized according to the PepT 1-based design. After systematic research, all synthesized hybrid molecules exhibited more excellent neuroprotective effect and antiplatelet activity compared to the original drugs. Among them, the selected compound 1e with superior neuroprotective and antiplatelet effects could significantly enhance the permeability on the Caco-2 monolayer membrane and inhibit the Gly-Sar uptake on Caco-2 cells. Meanwhile, the result of intestinal perfusion has also confirmed that the absorption of the selected compound 1e is indeed increased. Further, the selected compound 1e significantly reduce the cerebral infarction volume of middle cerebral artery occlusion/reperfusion rats. Especially, the cerebral infarction volume of the high-dose 1e group reduced to one fourth of the model group. Meanwhile, results of hematoxylin-eosin staining also indicated that the damage in the hippocampus CA1 region was significantly alleviated after treatment with the compound 1e. Accordingly, molecular hybridization strategy is one of the simple and feasible ways to improve the therapeutic effect of single targeted drug.

Metabolomic Insights into the Mechanisms of Ganoderic Acid: Protection against α-Amanitin-Induced Liver Injury.

α-Amanitin is a representative toxin found in the Amanita genus of mushrooms, and the consumption of mushrooms containing α-Amanitin can lead to severe liver damage. In this study, we conduct toxicological experiments to validate the protective effects of Ganoderic acid A against α-amanitin-induced liver damage. By establishing animal models with different durations of Ganoderic acid A treatment and conducting a metabolomic analysis of the serum samples, we further confirmed the differences in serum metabolites between the AMA+GA and AMA groups. The analysis of differential serum metabolites after the Ganoderic acid A intervention suggests that Ganoderic acid A may intervene in α-amanitin-induced liver damage by participating in the regulation of retinol metabolism, tyrosine and tryptophan biosynthesis, fatty acid biosynthesis, sphingosine biosynthesis, spermidine and spermine biosynthesis, and branched-chain amino acid metabolism. This provides initial insights into the protective intervention mechanisms of GA against α-amanitin-induced liver damage and offers new avenues for the development of therapeutic drugs for α-Amanitin poisoning.

Design, synthesis and bioevaluation of PI3Kα-selective inhibitors as potential colorectal cancer drugs.

The dysregulation of the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin signaling pathway has been implicated in various human cancers, and isoform-selective inhibitors targeting PI3Kα have received significant interest in recent years. In this study, we have designed and synthesized three series of substituted benzoxazole derivatives based on the clinical candidate TAK-117 (8a). A detailed structure-activity relationship (SAR) study has identified the optimal compound 18a bearing a quinoxaline scaffold. Compared to the control 8a, 18a exhibited 4.4-fold more potent inhibitory activity against PI3Kα (IC50: 2.5 vs 11 nM) and better isoform-selective profiles over other PI3Ks. In addition, 18a showed a 1.5-fold more potent antiproliferative effect against HCT-116 cell lines (IC50: 3.79 vs 5.80 µM) and a better selectivity over the normal tissue cells. The potential antitumor mechanism and in vitro metabolic stability of 18a were also investigated. Notably, pharmacokinetic assays indicated that 18a had a higher plasma exposure, a higher maximum concentration and shorter elimination time compared to 8a.

Virtual screening of novel mTOR inhibitors for the potential treatment of human colorectal cancer.

The abnormal activation of the mTOR pathway is closely related to the occurrence and progression of cancer, especially colorectal cancer. In this study, a rational virtual screening strategy has been established and MT-5, a novel mTOR inhibitor with a quinoline scaffold, was obtained from the ChemDiv database. MT-5 showed potent kinase inhibitory activity (IC50: 8.90 µM) and antiproliferative effects against various cancer cell lines, especially HCT-116 cells (IC50: 4.61 µM), and this was 2.2-fold more potent than that of the cisplatin control (IC50: 9.99 µM). Western blot, cell migration, cycle arrest, and apoptosis assays were performed with HCT-116 cells to investigate the potential anticancer mechanism of MT-5. Metabolic stability results in vitro indicated that MT-5 exhibited good stability profiles in artificial gastrointestinal fluids, rat plasma, and liver microsomes. In addition, the key contribution of the residues around the binding pocket of MT-5 in binding to the mTOR protein was also investigated from a computational perspective.

In vivo anti-hyperuricemia and anti-gouty arthritis effects of the ethanol extract from Amomumvillosum Lour.

The incidence of hyperuricemia and gout has been increasing year by year, and it is showing a younger trend. However, the first-line drugs currently used for hyperuricemia and gouty arthritis have serious side effects that limit their clinical application. Amomum villosum Lour. has been widely used in China for thousands of years as a traditional medical and edible plant, and previous screening showed that the ethanol extract of Amomum villosum Lour. could effectively inhibit the activity of xanthine oxidase. Based on this discovery, this paper had achieved in-depth mechanism research. The results showed that the ethanol extract of Amomum villosum Lour. could treat hyperuricemia by reducing the production of uric acid via inhibition of xanthine oxidase and increasing the excretion of uric acid via regulation of urate transporters. Meanwhile, the extract also showed a certain protective effect on hepatic and renal damage caused by hyperuricemia. With the formation of extensive uric acid, gouty arthritis will be induced by the deposition of monosodium urate in the joint. The extract could also relieve the inflammation by reducing the expression of inflammatory cytokines. In conclusion, the extract deserves focused research and development as a potential medicine, health care product or supplemented food for the prevention and treatment of hyperuricemia and gouty arthritis.

Design, synthesis and bioevaluation of 1,2,4-thiadiazolidine-3,5-dione derivatives as potential GSK-3ß inhibitors for the treatment of Alzheimer's disease.

Tideglusib is a non-competitive GSK-3ß inhibitor which contain 1,2,4-thiadiazolidine-3,5-dione moiety, and now mainly used for progressive supranuclear palsy due to the lack of some primary cognitive endpoints and secondary endpoints in a phase IIb trail for Alzheimer's disease. Additionally, insufficient evidence exists to support that there are obvious covalent bonds between Tideglusib and GSK-3ß. Targeted covalent inhibition strategy could improve the binding efficiency, selectivity and duration of kinase inhibitors. Based on the above premise, two series of targeted compounds with acryloyl warheads were designed and synthesized. The kinase inhibitory activity of the selected compound 10a with better neuroprotective effect improved 2.7 fold than that of Tideglusib. After the preliminary screening of GSK-3ß inhibition and neuroprotective activity, the mechanism action of the selected compound 10a was investigated in vitro and in vivo. The results confirmed that 10a with excellent selectivity among the whole tested kinases could significantly reduce the expressions of APP and p-Tau via increasing the level of p-GSK-3ß. The pharmacodynamic assay in vivo showed that 10a could markedly improve the learning and memory functions in AD mice induced by AlCl3 combined with d-galactose. At the same time, the damage of hippocampal neurons in AD mice was obviously reduced. Accordingly, the introduction of acryloyl warheads could increase the GSK-3ß inhibitory activity of 1,2,4-thiadiazolidine-3,5-dione derivatives, and the selected compound 10a deserves further research as an effective GSK-3ß inhibitor for the potential treatment of AD.

Discovery of 2-(isoxazol-5-yl)phenyl 3,4-dihydroxybenzoate as a potential inhibitor for the Wnt/ß-catenin pathway.

Carnosic acid could disrupt the ß-catenin/BCL9 protein-protein interaction and inhibit ß-catenin dependent transcription, thereby reduce the incidence of colorectal cancer induced by abnormal activation of Wnt/ß-catenin signalling pathway. However, its activity was weak (IC50 for SW480: 28.2 ± 2.05 µM) and total synthesis was difficult. During the structural simplification of natural products, S0 was revealed to be the basic pharmacophore of carnosic acid. Subsequent structural optimization of S0 led to the discovery of S11 as a possible anticancer agent with prominent proliferation inhibition effect (IC50 for SW480: 9.56 ± 0.91 µM) and best selectivity index (SI = 3.0) against Wnt hyperactive cancer cells. Futher mechanism investigation through TOP/FOP dual luciferase reporter assay, immunofluorescence, co-immunoprecipitation, microscale thermophoresis, downstream oncoprotein expression and cell apoptosis showed that compound S11 could significantly inhibit the proliferation of SW480 cells via obvioudsly decreasing the nucleus translocation of ß-catenin and effectively disrupting ß-catenin/BCL9 protein-protein interaction. Additionally, cell migration, molecule docking, in vitro stability and solubility assays were also conducted. Overall, S11 was worthy of in-depth study as a potential inhibitor for the Wnt/ß-catenin pathway and its discovery also proved that the structural simplification of natural products was still one of the effective methods to find new lead compounds or candidate drugs.

Cobalt-promoted synthesis of sulfurated oxindoles via radical annulation of N-arylacrylamides with disulfides.

An efficient radical annulation of N-arylacrylamides with disulfides is developed for the synthesis of sulfurated oxindoles. The reaction occurs in a facile manner using CoBr2 as both an initiator and a promoter for the first time and (NH4)2S2O8 as the oxidant. By controlling the CoBr2/(NH4)2S2O8 ratio, a wide range of sulfurated and brominated/sulfurated oxindoles are selectively prepared in good to excellent yields. The present protocol is simple and highly atom economical, and can tolerate a broad range of substrates.

Iron-Catalyzed Radical Annulation of Unsaturated Carboxylic Acids with Disulfides for the Synthesis of γ-Lactones.

An efficient aerobic iron-catalyzed annulation of unsaturated carboxylic acids with disulfides has been developed. This procedure proceeds using FeCl3 as the catalyst and KI as an iodine source under an air atmosphere, which provides practical access to a wide range of substituted γ-lactone derivatives. The disclosed method is quite simple, highly atom-economic, environmentally friendly, and tolerates a broad substrate scope.

Rhizoma Bletillae polysaccharide elicits hemostatic effects in platelet-rich plasma by activating adenosine diphosphate receptor signaling pathway.

Rhizoma Bletillae, the tubes of Bletilla striata, has been traditionally used in China as a hemostatic agent. In preliminary studies, the major active fraction responsible for its hemostatic effect have been confirmed to be Rhizoma Bletillae polysaccharide (RBp), but the hemostatic mechanism of action of RBp is still unknown.The main aim of this study was to clarify its mechanism of hemostatic effect. RBp was prepared by 80 % ethanol precipitation of the water extract of Rhizoma Bletillae followed by the Sevag method to remove proteins. The average molecular weight (Mw) of the crude RBp maintained at a range of 30.06-200 KDa. The hemostatic effects of RBp were evaluated by testing its effect on the platelet aggregation of rat platelet-rich plasma (PRP). PRP was dealt with different concentrations of RBp and platelet aggregation was measured by the turbidimetric method. The hemostatic mechanism of RBp was investigated by examining its effect on platelet shape, platelet secretion, and activation of related receptors (P2Y1, P2Y12 and TXA2) by electron microscopy and the turbidimetric method. RBp significantly enhanced the platelet aggregations at concentrations of 50-200 mg/L in a concentration-dependent manner. The inhibitory rate of platelet aggregation was significantly increased by apyrase and Ro31-8220 in a concentration-dependent manner, while RBp-induced platelet aggregation was completely inhibited by P2Y1, P2Y12 and the PKC receptor antagonists. However, the aggregation was not sensitive to TXA2. RBp, the active ingredients of Rhizoma Bletillae responsible for its hemostatic effect, could significantly accelerate the platelet aggregation and shape change. The hemostatic mechanism may involve activation of the P2Y1, P2Y12, and PKC receptors in the adenosine diphosphate (ADP) receptor signaling pathway.

Hybrid molecules of scutellarein and tertramethylpyrazine's active metabolites for ischemic stroke.

A series of hybrid molecules of scutellarein and tertramethylpyrazine's active metabolites have been synthesized. Compared to the original compound, these prepared compounds exhibited higher water solubility, more appropriate logP and better stability. Importantly, compounds 11b, 11d and 11e showed improved neuroprotective activity against the H2O2-induced cell death in PC12 cells, and better antithrombosis activity. The optimized compound 11b was further evaluated by cerebral ischemia/ reperfusion in the middle cerebral artery occlusion (MCAO) model, the results showed that the compound could significantly reduce the infarct area and decrease the neuronal cell damage in CA1 pyramidal neurons. Overall, we demonstrated that the twin drug strategy could be applied in the development of agents for the treatment of ischemic stroke.

Organocatalytic Asymmetric α-Sulfenylation of 2-Substituted Indolin-3-ones: A Strategy for the Synthesis of Chiral 2,2-Disubstituted Indole-3-ones with S- and N-Containing Heteroquaternary Carbon Stereocenter.

An organocatalytic asymmetric α-sulfenylation of 2-substituted indolin-3-ones with N-(alkylthio or arylthio)succinimides has been developed for the first time using Cinchona-derived squaramide as the catalyst. Various chiral 2,2-disubstituted indole-3-ones with S- and N-containing heteroquaternary carbon stereocenters were obtained with up to 98% yield and 99% ee.

In vivo and in vitro anti-inflammatory effects of ethanol fraction from Periploca forrestii Schltr.

OBJECTIVE: To determine the anti-inflflammatory effects of an ethanol fraction of Periploca forrestii Schltr. (EFPF) and to investigate the potential mechanisms underlying in vivo and in vitro models. METHODS: The antiinflflammatory effects of EFPF were evaluated using the xylene-induced mouse ear edema and carrageenan-induced rat paw edema models in vivo. In vitro, RAW264.7 cells were exposed to 0-800 µg/mL EFPF and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then cells were treated with different concentrations of EFPF (100-400 µg/mL) and stimulated with lipopolysaccharide (LPS, 1 µg/mL) for 24 h. The supernatant was analyzed for nitric oxide (NO) using the Griess reagent, and the levels of inflflammatory mediators and cytokines were determined using enzyme-linked immunosorbent assays for prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin (IL) 6, and IL-10. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK were examined by Western blot. RESULTS: Compared with the control group, EFPF signifificantly reduced mouse ear edema and rat paw edema rate (P<0.05 or P<0.01). Compared with the LPS group, EFPF signifificantly inhibited the LPS-stimulated production of NO, PGE2, TNF-α and IL-6 (P<0.05 or P<0.01), and increased the IL-10 production (P<0.05). EFPF also signifificantly inhibited LPS-induced protein expressions of iNOS and COX-2, suppressed the phosphorylation and degradation of inhibitor of NF-κB-α, decreased p65 level, and inhibited the phosphorylation of p38, ERK1/2 and JNK P<0.05 or P<0.01). CONCLUSION: EFPF exerted anti-inflflammatory effect by reducing protein expressions of iNOS and COX-2 and the production of the inflflammation factors, including TNF-α, IL-6, NO and PGE2, mainly through inhibition of LPS-mediated stimulation of NF-κB and MAPK signaling pathways.

[Quality evaluation of bletillae rhizoma based on hemostatic biopotency].

This dissertation is to determine the biopotency of hemostat which processed in different places by establishing a bioassay method of Bletillae Rhizoma based on the thrombin time. Contrast test is the main methodology. Specifically, the reference substance of Bletillae Rhizoma is determined by comparing with the control substance of vitamin K1 using thrombin time, which is calibrated the Bletillae Rhizoma. The hemostatic biopotency is calculated by using the method of "parallel line assay method based on quantitative responses" (3.3) from different processed products. It indicates that there is a strong linear correlation between Bletillae Rhizoma and control drugs (Y = 66.332-23.913X, R2 = 0.995 3). The hemostatic biopotency of Bletillae Rhizoma from different processed products ranged between 821.93-1 187.53 U x g(-1) shown in the paper, and all of them can meet the requirements of the test. The methodology has an appropriate instrument precision (RSD 3.8%), intermediate precision (RSD 4.6%), repeatability (RSD 3.2%) and stability (RSD 3.7%). Therefore, it can be turned out that the methodology which established in the dissertation is good at determinating the hemostatic biopotency of Bletillae Rhizoma and it is reliable, simple and repeatable.