Synthesis and bioactivity evaluation of novel nuciferine derivatives with antihyperuricemia and nephroprotective effects.

Hyperuricemia is a common metabolic disease with a series of complications. Nuciferine, a typical aporphine alkaloid natural compound extracted from the leaves of Nelumbo nucifera Gaertn., was confirmed to have an antihyperuricemia effect. In the present study, 30 novel nuciferine derivatives were designed and synthesized. The effects of all derivatives on the regulation of URAT1 were studied in a uric acid-induced HK-2 cell model with benzbromarone as a positive control. The results indicated that Compound 1j showed the optimal URAT1 inhibitory activity through repressing PI3K/Akt pathway in HK-2 cells and the inhibitory effect was similar to that of benzbromarone. In addition, in vivo experiments demonstrated that Compound 1j could reduce uric acid levels and ameliorate kidney damage in hyperuricemic mice. On the one hand, Compound 1j could inhibit the expression of URAT1 and GLUT9 to increase the uric acid excretion index. On the other hand, Compound 1j could regulate the TLR4/IκBα/NF-κB signaling pathway to reduce the levels of inflammatory cytokines, thereby alleviating kidney damage. Meanwhile, a molecular docking assay revealed the potential molecular binding power (-9.79 kcal/mol) between Compound 1j and URAT1, which was more tightly bound than the lead compound nuciferine (-7.44 kcal/mol). Based on these results, Compound 1j may be a future drug for the development of new potential antihyperuricemia and nephroprotective drug candidates.

Design, synthesis and bioactive evaluation of geniposide derivatives for antihyperuricemic and nephroprotective effects.

Hyperuricemia is a principal factor mediating gout and kidney damage, and xanthine oxidase (XOD) is a key enzyme in the pathogenesis of hyperuricemia. In this context, a series of geniposide derivatives were designed and synthesized, and antihyperuricemic and nephroprotective effects of all derivatives was evaluated in vitro and in vivo. Compound 2e emerged as the most potent XOD inhibitor, with an IC50 value of 6.67 ± 0.46 µM. Simultaneously, cell viability, ROS generation, and SOD levels assay showed that compound 2e could repair the damage of HKC cells by inhibiting the oxidative stress response. The results of the study indicated compound 2e significantly decreased uric acid levels by inhibiting the XOD activity, and repaired kidney damage by inhibiting the expression of TLR4/TLR2/MyD88/NF-κB and NALP3/ASC/caspase-1 signaling pathways. Enzyme inhibition kinetics suggested that compound 2e functioned via reversible mixed competitive inhibition. Moreover, a molecular docking study was performed to gain insight into the binding mode of compound 2e with XOD. These results suggest that geniposide derivatives were potential to be developed into a novel medicine to reveal healthy benefits in natural prevention and reduction risk of hyperuricemia and kidney damage.

Design, synthesis and in vitro anticancer research of novel tetrandrine and fangchinoline derivatives.

Cancer treatment is one of the major public health issues in the world. Tetrandrine (Tet) and fangchinoline (d-Tet) are two bis-benzyl isoquinoline alkaloids extracted from Stephania tetrandra S. Moore, and their antitumor activities have been confirmed. However, the effective dose of Tet and d-Tet were much higher than that of the positive control and failed to meet clinical standards. Therefore, in this study, as a continuation of our previous work to study and develop high-efficiency and low-toxic anti-tumor lead compounds, twenty new Tet and d-Tet derivatives were designed, synthesized and evaluated as antitumor agents against six cancer cell lines (H460, H520, HeLa, HepG-2, MCF-7, SW480 cell lines) and BEAS-2B normal cells by CCK-8 analysis. Ten derivatives showed better cytotoxic effects than the parent fangchinoline, of which 4g showed the strongest cell growth inhibitory activity with an IC50 value of 0.59 µM against A549 cells. Subsequently, the antitumor mechanism of 4g was studied by flow cytometry, Hoechst 33258, JC-1 staining, cell scratch, transwell migration, and Western blotting assays. These results showed that compound 4g could inhibit A549 cell proliferation by arresting the G2/M cell cycle and inhibiting cell migration and invasion by reducing MMP-2 and MMP-9 expression. Meanwhile, 4g could induce apoptosis of A549 cells through the intrinsic pathway regulated by mitochondria. In addition, compound 4g inhibited the phosphorylation of PI3K, Akt and mTOR, suggesting a correlation between blocking the PI3K/Akt/mTOR pathway and the above antitumor activities. These results suggest that compound 4g may be a future drug for the development of new potential drug candidates against lung cancer.

Design, synthesis and in vitro evaluation of fangchinoline derivatives as potential anticancer agents.

The isolation and modification of natural products play an important role in the synthesis of anti-tumor drugs for the treatment of cancer. The present study was designed to evaluate the effects of fangchinoline derivatives against cancer cells. In vitro cytotoxicity of all derivatives against five cancer cell lines (A549, Hela, HepG-2, MCF-7 and MDA-MB-231 cell lines) and HL-7702 normal cells was assessed using the CCK-8 assay, and the results showed that most of the synthesized compounds displayed better cytotoxic effects on all the tested cells compared to that of the parent fangchinoline. In particular, compound 3i had the strongest inhibitory effect on cell proliferation, with an IC50 value of 0.61 µM against A549 cells. Compared with fangchinoline and HCPT (hydroxycamptothecine), the anti-proliferative activity of compound 3i was significantly increased. More interestingly, compound 3i had slight toxic side effects on normal cells, with an IC50 value of 27.53 µM. Moreover, the cell viability and cell cycle assays revealed that compound 3i inhibited A549 cell proliferation and arrested A549 cells at the G2/M-phase. The apoptosis-inducing effects of compound 3i and the associated molecular mechanisms were assessed using flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR and Western blot analysis. These results suggested that compound 3i induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that compound 3i is a promising candidate for future development as an anti-tumor drug.

Design, synthesis and biological activities of tetrandrine and fangchinoline derivatives as antitumer agents.

The isolation and modification of natural products is always a very important resources to anti-tumor drugs. Therefore, a novel series of tetrandrine and fangchinoline derivatives were designed and synthesized, and their antiproliferative activities against HepG2, MCF-7 cells were evaluated and described. From the activity result obtained, high to very high activity in vitro has been found, one of the tested compounds (compound 5d) exhibited the most significant cytotoxic effects. Compound 5d increased 29.2, 7.37 times anti-proliferative activity for HepG2 cells and MCF-7 cells compared to sunitinib (IC50=16.06µM and 25.41µM). Finally flow cytometry determined that compound 5d could indeed inhibit the proliferation of HepG2 cells via inducing apoptosis.

Guaranteeing Global Stability for Neuro-Adaptive Control of Unknown Pure-Feedback Nonaffine Systems via Barrier Functions.

Most existing approximation-based adaptive control (AAC) approaches for unknown pure-feedback nonaffine systems retain a dilemma that all closed-loop signals are semiglobally uniformly bounded (SGUB) rather than globally uniformly bounded (GUB). To achieve the GUB stability result, this article presents a neuro-adaptive backstepping control approach by blending the mean value theorem (MVT), the barrier Lyapunov functions (BLFs), and the technique of neural approximation. Specifically, we first resort the MVT to acquire the intermediate and actual control inputs from the nonaffine structures directly. Then, neural networks (NNs) are adopted to approximate the unknown nonlinear functions, in which the compact sets for maintaining the approximation capabilities of NNs are predetermined actively through the BLFs. It is shown that, with the developed neuro-adaptive control scheme, global stability of the resulting closed-loop system is ensured. Simulations are conducted to verify and clarify the developed approach.

A review on the balancing design of micro drills.

In recent years, micro-hole drilling with a diameter of less than 1 mm has been widely applied in electronic information, semiconductor, metal processing, and other fields. Compared with conventional drilling, the engineer problems that micro drills are more prone to suffer failure in advance have restricted further development of mechanical micro drilling. In this paper, the main substrate materials of micro drills were introduced. And two important technical means to improve properties of tool material, namely, grain refinement and tool coating, were also introduced, which are current main research directions of micro drills from the perspective of materials. The failure mechanisms of micro drills were briefly analyzed, mainly tool wear and drill breakage. In the structure of micro drills, cutting edges and chip flutes are directly related to tool wear and drill breakage, respectively. So the structural optimization and design of micro drills, especially for key structures such as cutting edges and chip flutes, have to face great challenges. Based on the above, two pairs of requirements for micro drills were proposed, that is, the balance between chip evacuation and drill stiffness and the balance between cutting resistance and tool wear. So some innovative schemes and related researches of micro drills regarding cutting edges and chip flutes were reviewed. Finally, a summary of micro drill design and existing problems and challenges is proposed.

Adaptive Approximation-Based Tracking Control for a Class of Unknown High-Order Nonlinear Systems With Unknown Powers.

In this article, the problem of adaptive tracking control is tackled for a class of high-order nonlinear systems. In contrast to existing results, the considered system contains not only unknown nonlinear functions but also unknown rational powers. By utilizing the fuzzy approximation approach together with the barrier Lyapunov functions (BLFs), we present a new adaptive tracking control strategy. Remarkably, the BLFs are employed to determine a priori the compact set for maintaining the validity of fuzzy approximation. The primary advantage of this article is that the developed controller is independent of the powers and can be capable of ensuring global stability. Finally, two illustrative examples are given to verify the effectiveness of the theoretical findings.

Synthesis and biological evaluation of geniposide derivatives as inhibitors of hyperuricemia, inflammatory and fibrosis.

Hyperuricemia is a metabolic disease caused by abnormal purine metabolism in the body. Long-term high levels of uric acid in the body will lead to gout and kidney disease. Xanthine oxidase (XOD) is a key enzyme in the pathogenesis of hyperuricemia. In this context, a series of geniposide derivatives were designed, synthesized and evaluated as xanthine oxidase inhibitors. Most of these compounds exhibited potent XOD inhibitory activities in vitro, and representatives 6a, 6c, 6g and 6j were found to be the most potent inhibitors against the enzyme with IC50 values of 2.15 ± 1.03, 1.37± 0.26, 4.14± 0.79 and 1.86± 0.13 µM, which were 33.03-158.37 fold more active than geniposide, respectively. Compounds 6a, 6c, 6g and 6j were evaluated in hyperuricemia mice, and the results demonstrated that compound 6c showed the strongest anti-hyperuricemia and renal protective activity in vivo. Subsequently, the molecular mechanism of compound 6c was studied in this investigation. In vitro cell experiments showed that compound 6c inhibited the inflammation of HK-2 cells under high uric acid conditions by inhibiting the expressions of TGF-ß, TNF-α and IL-1ß, and reduced the cell fibrosis by decreasing the expressions of α-SMA and Collagen I. The results of the mice experiments indicated that compound 6c efficiently decreased the level of serum uric acid (SUA) in hyperuricemia mice by inhibiting the XOD activity. Moreover, compound 6c effectively reduced the urate accumulation in the kidney and simultaneously decreased inflammation by regulating the expression of the TLR4/IκBα/NF-κB signaling pathway. In addition, consistent with cell experiments, compound 6c also reduced renal fibrosis in hyperuricemia mice, which may be due to compound 6c inhibiting the expression of inflammatory factor TGF-ß. Furthermore, a molecular docking study was performed to gain insight into the binding mode of compound 6c with XOD. These results suggest that compound 6c has the potential to be developed into a novel medicine to reduce blood uric acid and treat renal diseases caused by hyperuricemia.

Role of the Hippo signaling pathway in safflower yellow pigment treatment of paraquat-induced pulmonary fibrosis.

OBJECTIVE: To elucidate the molecular mechanisms by which safflower yellow (SY) mediates therapeutic effects in rats with paraquat intoxication-induced pulmonary fibrosis. METHODS: Rats received combinations of paraquat, SY, and SB431542, a transforming growth factor (TGF)-ß1 receptor antagonist. Survival over 28 days was assessed by Kaplan-Meier analysis. Rat tissue and serum samples were assessed by hematoxylin and eosin staining, Masson's Trichrome staining, immunoblotting, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and transmission electron microscopy. RESULTS: Survival rates were higher in SY and SB431542 groups (treatment and paraquat) than in the exposure group (paraquat alone). In the exposure group, serum TGF-ß1 levels increased between days 3 and 14; mammalian STE20-like (MST) levels increased between days 3 and 7; TGF-ß1 and Smad3 levels increased between days 3 and 14; and Yap and connective tissue growth factor levels increased between days 3 and 28. TGF-ß1 levels were lower in SY and SB431542 groups than in the exposure group. Pathology scores were higher in exposure, SY, and SB431542 groups than in the control group throughout the experiment. CONCLUSIONS: In rats with paraquat intoxication-induced pulmonary fibrosis, Hippo signaling could be activated by the MST-Yap pathway; SY and SB431542 could alleviate pulmonary fibrosis via Hippo signaling.

[ITS sequence identification of Radix Bupleuri].

OBJECTIVE: To provide the basis of molecular authentication of Radix Bupleuri by the comparison of the internal transcribed spacer (ITS) sequences of five kinds of Radix Bupleuri in common use. METHOD: Firstly, total DNA of five kinds of Radix Bupleuri was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR product was directly sequenced after purification. RESULT: The length of ITS1 and ITS2 sequence was 214-220 bp and 230-231 bp respectively. There were distinct variation sites between the ITS sequences of the five kinds of Radix Bupleuri. CONCLUSION: ITS sequence may be the evidence for the molecular authentication of Radix Bupleuri.

Composition and potential anticancer activities of essential oils obtained from myrrh and frankincense.

The present study aimed to investigate the composition and potential anticancer activities of essential oils obtained from two species, myrrh and frankincense, by hydrodistillation. Using gas chromatography-mass spectrometry (GC-MS), 76 and 99 components were identified in the myrrh and frankincense essential oils, respectively, with the most abundant components, 2-Cyclohexen-1-one, 4-ethynyl-4-hydroxy-3,5,5-trimethyl- and n-Octylacetate, accounting for 12.01 and 34.66%, respectively. The effects of the two essential oils, independently and as a mixture, on five tumor cell lines, MCF-7, HS-1, HepG2, HeLa and A549, were investigated using the MTT assay. The results indicated that the MCF-7 and HS-1 cell lines showed increased sensitivity to the myrrh and frankincense essential oils compared with the remaining cell lines. In addition, the anticancer effects of myrrh were markedly increased compared with those of frankincense, however, no significant synergistic effects were identified. The flow cytometry results indicated that apoptosis may be a major contributor to the biological efficacy of MCF-7 cells.

Neuroprotection by small molecule activators of the nerve growth factor receptor.

There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. However, the blood-brain barrier presents a major hurdle in the use of peptide therapeutics. The goal of this study was to identify small molecule, cell-permeable nerve growth factor (NGF) activators. Combinatorial libraries of asterriquinones (>300) and mono-indolyl-quinones (>60) were screened using a 96-well enzyme-linked immunosorbent assay that detects phosphorylated TrkA, the NGF receptor. The libraries were also screened for dose-dependent cytotoxicity. From these screens, we generated quantitative structure-activity relationship models for activity and toxicity, and then we selected two compounds, 2-(6-chloro-1H-indol-3-yl)-5-(2-cyclopropyl-1H-indol-3-yl)-3,6-dihydroxy-[1,4]benzoquinone (1H5) and 2,5-dimethoxy-3-(7-fluoro-1H-indol-3-yl)-[1,4]-benzoquinone (5E5), for further study based on high activity and low toxicity. Compound 1H5 (30 microM) is an asterriquinone that is a moderate TrkA activator (50% the activity of 100 ng/ml NGF), and it shows little toxicity at concentrations up to 100 microM. 1H5 can protect differentiated PC12 neurons from apoptotic cell death induced by NGF withdrawal. Compound 5E5 (30 microM) is a mono-indolyl-quinone that is a very strong activator of TrkA (>200% the activity of 100 ng/ml NGF), and it is nontoxic at concentrations up to 10 microM. Activation of TrkA can be detected at 1 microM 5E5, and 3 to 10 microM 5E5 activates TrkA and extracellular signal-regulated kinase as strongly as a maximal dose of NGF (100 ng/ml). A combination of a low dose of 5E5 (1 microM) with a submaximal dose of NGF (10 ng/ml) promotes neuronal differentiation of PC12 cells. These compounds represent a new class of TrkA activators that could have potential utility in the treatment of neurodegenerative diseases.

Parallel synthesis of indolylquinones and their cell-based insulin mimicry.

A synthetic route to bis-indolyldihydroxybenzoquinones was adapted for parallel organic synthesis. The route involves selective conjugate addition of an indole to dichlorobenzoquinone promoted by Brønsted acid, followed by a Lewis acid-promoted conjugate addition of a second indole and a final hydrolysis. Methods for high-throughput purification of the products of this synthesis were also developed. Using these methods, we prepared a library whose structures are based on asterriquinone natural products, which have a wide range of biological activities. In this report, the activities of the library members in activation of the insulin receptor on mammalian cells were examined. Novel compounds were discovered that fall outside earlier developed structure-activity relationships for insulin mimics, supporting the value of systematic investigation (inspired by Nature) for the discovery of novel biologically active molecules.

Methyl scanning: total synthesis of demethylasterriquinone B1 and derivatives for identification of sites of interaction with and isolation of its receptor(s).

The principle of methyl scanning is proposed for determination of the sites of interaction between biologically active small molecules and their macromolecular target(s). It involves the systematic preparation of a family of methylated derivatives of a compound and their biological testing. As a functional assay, the method can identify the regions of a molecule that are important (and unimportant) for biological activity against even unknown targets, and thus provides an excellent complement to structural biology. Methyl scanning was applied to demethylasterriquinone B1, a small-molecule mimetic of insulin. A new, optimal total synthesis of this natural product was developed that enables the family of methyl scan derivatives to be concisely prepared for evaluation in a cellular assay. The results of this experiment were used to design a biotin-demethylasterriquinone conjugate for use as an affinity reagent. This compound was prepared in tens of milligram quantities in a four-step synthesis.