Design, synthesis and biological activity of pyrazolo[1,5-a]pyrimidin-7(4H)-ones as novel Kv7/KCNQ potassium channel activators.

Voltage-gated Kv7/KCNQ/M-potassium channels play a pivotal role in controlling neuronal excitability. Genetic reduction of KCNQ channel activity as a result of mutations causes various human diseases such as epilepsy and arrhythmia. Therefore, discovery of small molecules that activate KCNQ channels is an important strategy for clinical intervention of membrane excitability related disorders. In this study, a series of pyrazolo[1,5-a]pyrimidin-7(4H)-ones (PPOs) have been found to be novel activators (openers) of KCNQ2/3 potassium channels through high-throughput screening by using atomic absorption rubidium efflux assay. Based on structure-activity relationship (SAR), the substituted PPOs have been optimized. The 5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one (17) was identified as a novel, potent, and selective KCNQ2/3 potassium channel opener by patch-clamp recording assay.

ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation.

INTRODUCTION: The present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers. METHODS: We investigated the effects of ABL-N on the induction of apoptosis in human breast cancer cells and further examined the underlying mechanisms. Moreover, tumor growth inhibition of ABL-N was done in xenograft models. RESULTS: ABL-N induced the activation of caspase-3 in estrogen receptor (ER)-negative cell lines MDA-MB-231 and MDA-MB-468, as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP). Pretreatment of cells with pan-caspase inhibitor z-VAD-fmk or caspase-3-specific inhibitor z-DEVD-fmk inhibited ABL-N-induced apoptosis. ABL-N treatment also resulted in an increase in the expression of pro-apoptotic members (Bax and Bad) with a concomitant decrease in Bcl-2. Furthermore, c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) were activated in the apoptosis induced by ABL-N and JNK-specific inhibitor SP600125 and JNK small interfering RNA (siRNA) antagonized ABL-N-mediated apoptosis. However, the p38-specific inhibitor SB203580 had no effect upon these processes. Moreover, neither of the caspase inhibitors prevented ABL-N-induced JNK activation, indicating that JNK is upstream of caspases in ABL-N-initiated apoptosis. Additionally, in a nude mice xenograft experiment, ABL-N significantly inhibited the tumor growth of MDA-MB-231 cells. CONCLUSIONS: ABL-N induces apoptosis in breast cancer cells through the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment causes a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.

1,6-O,O-diacetylbritannilactones inhibits IkappaB kinase beta-dependent NF-kappaB activation.

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1,6-O,O-diacetylbritannilactone (ABLOO). Electrophoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-kappaB (NF-kappaB) p65. The expressions of IkappaBalpha, pIkappaBalpha, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IkappaB kinase alpha/beta (IKKalpha/beta) and NF-kappaB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-gamma-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-gamma were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKKbeta down to based level, but not IKKalpha, corresponding with decreased in IkappaBalpha degradation and phosphorylation induced by LPS/IFN-gamma, resulting in the suppression of NF-kappaB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases.

Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response.

To investigate the mechanism of action by which a new anti-inflammatory active compound, 1-O-acetylbritannilactone (ABL) isolated from Inula britannica-F., inhibits inflammatory responses in vascular smooth muscle cells (VSMCs). Enzyme immunoassay was used to measure the levels of prostandin E(2) (PGE(2)) production. Immunocytochemistry staining and Western blot analysis were performed to detect the nuclear translocation of nuclear factor-kappaB (NF-kappaB) p65 and the expression of IkappaB-alpha, pIkappaB-alpha and cyclooxygenase-2 (COX-2). Electrophoretic mobility shift assays (EMSA) were used to detect DNA-binding activity of NF-kappaB in VSMCs. ABL (5, 10, 20 micrommol/l) had several concentration-dependent effects, including inhibition of lipopolysaccharide (LPS)-induced PGE(2) production and COX-2 expression, and blockade of NF-kappaB activation and translocation. These effects were owing to reductions in IkappaB-alpha phosphorylation and degradation induced by LPS. In addition, ABL directly inhibited the binding of active NF-kappaB to specific DNA cis-element. These results indicate that ABL is a potent inhibitor of LPS-stimulated VSMC inflammatory responses through blockade of NF-kappaB activity and inhibition of inflammatory gene COX-2 expression.

[Contents comparison of resveratrol and polydatin in the wild Polygonum cuspidatum plant and its tissue cultures].

OBJECTIVE: To compare the contents of resveratrol and polydatin in some materials of Polygonum cuspidatum from various sources, so to screen and obtain the suitable cultures for the following metabolism regulation study. METHOD: RP-HPLC method was applied to simultaneously assay resveratrol and polydatin in different samples. RESULT: By the modified methods of extraction and determination, large amount of materials were screened. The results indicated that the contents of resveratrol and polydatin in root and rhizome were evidently higher than those in the leave and stems. The content of polydatin in the seedlings cultured indoor for three months was 1.27% and showed a 1.25-time increse than that in the wild plants, while the content of resveratrol (0.401%) approached that in the wild plants. Both of resveratrol and polydatin could be examined from different tissue cultures of P. cuspidatum, such as the sterile seedlings, callus, suspended cells and hairy roots, and the levels of them were closely related to the growth speed, physiological status and developmental phase. Hairy roots had the highest potentiality in several tested cultures and the increase rate of dry weight was 8.29 when cultured in vitro for 30 days, and showed a 8.4-fold and a 192.8-fold increase compared with those of natural roots and suspended cells, respectively. The content of polydatin in the hairy roots was up to 0.037% and that of resveratrol was 0.007%. CONCLUSION: The established analysis method is rapid, simple and accurate, especially adapted to the simultaneous determination of resveratrol and polydatin in massive biological samples. Hairy-root cultures have the superiority among the tested materials of P. cuspidatum and are suitable for the large-scale biomass and consistent production of efficient constituents.

Simple analysis of four bisphosphonates simultaneously by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing agent.

Volatile organic amine was used as the mobile phase addictive during the separation of four bisphosphonates (alendronate, pamidronate, zoledronic acid and etidronate). An isocratic liquid chromatography method with evaporative light-scattering detection (ELSD) was developed for these bisphosphonates which are not retained on non-polar column and lack chromophore for detection. The analytes have sufficiently separated from each other on a Phenomenex C18 column. The effects of mobile phase composition and instrumental parameters of ELSD were studied. This newly developed method enables direct measurement for analysis of bisphosphonates without the need of derivatization. This developed method provides high separation and specificity to bisphosphonate analysis. In quantitative analysis, the method showed satisfactory precision (less than 2.8%) and accuracy (higher than 94.4%), good linearity (r=0.9991-0.9997) and sufficient sensitivity (15-18 microg/ml). It can be easily and conveniently adopted for the routine quality control analysis.

[Study of chemical constituents of the essential oil from Inula britannica L. by GC-MS].

OBJECTIVE: The chemical components of the essential oil from Inula britannica L., which were collected from Yuncheng area of Shanxi province, were analyzed by GC-MS. METHODS: The essential oil was extracted from Inula britannica L. by steam distillation, the components were separated with the capillary chromatographic columns, the amount of the components from the essential oil was determined by normalization method. The components separated were identified by data search system. The chromatographic conditions were as follows: DB-5 (30m x 0.25mm, 0.25 microm) capillary column; High purity helium was used as carrier gas, and the flow rate was 1.0 ml/min; Column temperature: 70 degrees C keeping 2min, from 70 degrees C to 230 degrees C at rising rate 10 degrees C/min and keeping 10 min; Split ratio 15:1; Injector temperature 250 degrees C. RESULTS: 62 peaks were identified representing 68.4% of the total contents. Main component was 1-Benzoxepin-3-ol,2,3,4,5-tetrahydro-(4. 276% ). CONCLUSION: The method is so reliable, stable, and good reproducible that can be applied to the analysis of the essential oil from Inula britannica L.

[Preparation and determination of 1-O-acetylbritannilactone in Inula Britannica L].

To prepare reference substance for quality control of Inula Britannica L., 1-O-acetylbritannilactone was extracted and separated from chloroform extraction of Inula Britannica L. Chemical structure of the 1-O-acetylbritannilactone product was elucidated by ultraviolet spectrometry (UV), infrared spectroscopy (IR), nuclear magnetic resonance (NMR) and mass spectrometry (MS), and the results were in agreement with the reference. The purity of the 1-O-acetylbritannilactone product was 99.5%, which satisfies the need of reference substances of traditional Chinese medicines. A method of high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) is described for the determination of 1-O-acetylbritannilactone in Inula Britannica L. The chromatographic conditions include Hypersil ODS-2 column, a mixture of methanol-water (52: 48, v/v) with the flow rate of 1.0 mL/min used as mobile phase. The temperature of drift tube of the ELSD was 90 degrees C. Flow rate of carrier gas was 2.5 L/min. Linear range of 1-O-acetylbritannilactone was 1.37 - 8.21 microLg (r = 0.999 8). The average recovery of 1-O-acetylbritannilactone was 100.2% with the relative standard deviation (RSD) of 1.3% (n = 6). The method is simple, accurate, time saving and reproducible.

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression.

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.