Discovering novel anti-HCV compounds with inhibitory activities toward HCV NS3/4A protease.

AIM: To discover novel hepatitis C virus (HCV) inhibitors and elucidate the mechanism of action of the active compounds. METHODS: HCV subgenomic replicon-based luciferase reporter cell line was used to screen 1200 synthetic compounds with novel structures. Huh7.5.1 cell line stably transfected with HCV NS3/4A protease reporter was established to investigate the anti-HCV mechanism of the active compounds. The active compounds were further examined in an in vitro HCV infection assay to confirm their anti-HCV activity. RESULTS: After two-round screening in the anti-HCV replicon assay, some 2,4-diaminoquinazoline derivatives and carboxamide analogues were found to possess anti-HCV replicon activities (the IC50 values were less than 5 µmol/L). Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 µmol/L, respectively. Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 µmol/L, respectively. CONCLUSION: Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

Natural product triptolide mediates cancer cell death by triggering CDK7-dependent degradation of RNA polymerase II.

Triptolide is a bioactive ingredient in traditional Chinese medicine that exhibits diverse biologic properties, including anticancer properties. Among its many putative targets, this compound has been reported to bind to XPB, the largest subunit of general transcription factor TFIIH, and to cause degradation of the largest subunit Rpb1 of RNA polymerase II (RNAPII). In this study, we clarify multiple important questions concerning the significance and basis for triptolide action at this core target. Triptolide decreased Rpb1 levels in cancer cells in a manner that was correlated tightly with its cytotoxic activity. Compound exposure blocked RNAPII at promoters and decreased chromatin-bound RNAPII, both upstream and within all genes that were examined, also leading to Ser-5 hyperphosphorylation and increased ubiqutination within the Rbp1 carboxy-terminal domain. Notably, cotreatment with inhibitors of the proteasome or the cyclin-dependent kinase CDK7 inhibitors abolished the ability of triptolide to ablate Rpb1. Together, our results show that triptolide triggers a CDK7-mediated degradation of RNAPII that may offer an explanation to many of its therapeutic properties, including its robust and promising anticancer properties.

[Diterpene constituents of Tripterygium willfordii (II)].

In order to study the constituents and pharmacology of Tripterygium plants (Tripterygium willfordii Hook.f), a variety of chromatography methods were used. Four compounds were isolated from Tripterygium plant and their structures were elucidated by UV, IR, MS, HR-MS, 1H NMR, 13C NMR and 2D-NMR techniques. The isolated compounds were named as triptonide (1), neo-triptetraolide (2), 2alpha-hydroxytriptonide (3), and 15-hydroxytriptonide (4), separately. Compounds 3, 4 belong to new diterpenoids, which can inhibit the growth of K562 cells (leukemia cells) and HL60 cells (acute myeloid leukemia cells).

Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α. RESULTS: Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide. CONCLUSIONS: The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.

[Vitro antitumor activity and synthesis of the key intermediate of bakuchiol].

The in vitro antitumor activity of bakuchiol was exploited, compared with tamoxifen. The result of biological activities showed that bakuchiol could inhibit human breast cancer and the IC50 values were 2.89 x 10(-5) mol L(-1) and 8.29 x 10(-3) mol L(-1) against the cells line T-47D and MDA-MB-231 respectively. On the other hand, the key intermediate to synthesize bakuchiol was obtained by the method of Ireland-Claisen rearrangement. Comparing with traditional Claisen rearrangement, the reaction conditions are milder and the reaction reagents are safer.

[Chemical constituents in root of Hemerocallis fulva].

OBJECTIVE: To study the chemical constituents of the root of Hemerocallis fulva. METHOD: Compounds were isolated by repeated silica gel, Sephadex LH-20, MCI gel, etc. column chromatography and their structures were determined by spectral analyses and physicochemical properties. RESULT: From the n-butanol fraction of the EtOH extract of the roots of H. fulva, 9 glycosides were isolated and identified as sweroside (1), laganin (2), picraquassioside C (3), puerarin (4), 3'-methoxypuerarin (5), 7-hydro xylnaphthalide-O-beta-D-glucopyranoside (6), orcinol-3-O-beta-glucopyranoside (7), HN saponin F (8), hederagenin-3-0-beta D-glucopyranosyl-(1-3)-alpha-L-arabinopyranoside-28-O-beta-D-glucopyranosyl ester (9). CONCLUSION: Compounds 1-9 were isolated from the genus Hemerocallis for the first time.

(5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo.

A series of triptolide analogs have been successfully synthesized. In the present study we demonstrated one of them, (5R)-5-hydroxytriptolide (LLDT-8), showed low cytotoxicity and relative high immunosuppressive activities as compared with its parent compound triptolide in vitro. The CC50 values of triptolide and LLDT-8 were 2.1+/-0.3 and 256.6+/-73.8 nM, respectively. LLDT-8 significantly inhibited the proliferation of splenocytes induced by concanavalin A (ConA), lipopolysaccharide (LPS), or mixed lymphocyte reaction (MLR), and the IC50 values were 131.7+/-32.4, 171.5+/-17.3, and 38.8+/-5.1 nM, respectively. LLDT-8 (25, 50, 100 nM) dose-dependently reduced the production of Th1 type cytokines (IFN-gamma, IL-2) and inflammatory cytokines (TNF-alpha, IL-6) in vitro. Administration of LLDT-8 (at the low dose of 0.4 microg/kg, i.p.; 40 microg/kg, p.o.) intensively suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) reactions. Treatment with LLDT-8 (40 microg/kg, i.p. and p.o.) also markedly inhibited the sheep red blood cell (SRBC)-induced antibody production in BLAB/c mice. Most importantly, comparing with triptolide, LLDT-8 significantly reduced toxicity, with a 122-fold lower cytotoxicity in vitro and 10-fold lower acute toxicity in vivo. The results suggested that LLDT-8 had immunosuppressive activities in both cellular and humoral immune responses. LLDT-8 might be a potential therapeutic agent for immune-related diseases.

[Diterpenes constituents of Tripterygium wilfordii].

AIM: To study the chemical constituents of Tripterygium wilfordii Hook. F. METHODS: Various column chromatographies with silica gel were used for the isolation and purification. The structures of compounds were established on the basis of its IR, MS, UV, 1H NMR, 13C NMR and HRMS, 1H-1H COSY, 1H-13C COSY and NOESY. RESULTS: Four diterpenoids were isolated: 16-hydroxytriptolide (I), triptolidenol (II), tripdiolide (III), 2-epitripdiolide (IV). CONCLUSION: Compound IV is a new diterpenoid.

[Isolation and structure identification of chemical constituents from Anabasis brevifolia].

AIM: To study the chemical consitituents of the n-butanol-extracts of Anabasis salsa and Various chromatographic techniques were used to the chloroform-extract of Anabasis brevifolia. METHODS: separate and purify the constituents. Their physico-chemical properties and spectral data were used to elucidate their structures. RESULTS: Five compounds were isolated and identified as 2-O-beta-D-glucopyranosyloxy-4,6-dimethoxy phenylenthanone (1), 2-O-(2)-beta-D-glucopyranosyloxy-4, 6-dimethoxy phenylenthanone (2), 3-methyl-but-2-enoic acid-[2-(4-methoxy phenyl)-ethyl]-amide (3), 5,6,7,2'-tetramethoxy isoflavonoid (4), 2'-hydroxy-5,6,7-trimethoxyisoflavonoid (5). CONCLUSION: Compounds 2, 3, and 5 are new compounds. And the others were isolated from Anabasis L. for the first time.

Analysis of triptolide-regulated gene expression in Jurkat cells by complementary DNA microarray.

AIM: To investigate the global gene expression profile changes in Jurkat cells after triptolide treatment in order to find the possible triptolide targets. METHODS: Jurkat cells were treated with or without triptolide 10 microg/L for 2 h. Total RNA were isolated and used as templates for reverse transcriptional labeling of fluorescent cDNA probes. High density DNA microarray chips with a set of 13,872 human genes/Ests were used to generate the expression profile of triptolide-treated or untreated control Jurkat cells by hybridizing with fluorescent labeled probes. Array image was acquired and analyzed with array analyzing software GeneSpring. RESULTS: Triptolide significantly suppressed expression of 117 genes in Jurkat cells. Among these 117 genes, 30 % were Ests or genes without known functions, 13 % were transcription factors, 9 % were signal transduction pathway regulators, and 9 % were DNA binding proteins. Notably, the expression of mitogen-activated protein kinase kinase kinase kinase 5 (MAP kinase 5) and phosphoinositide-3-kinase (PI-3 kinase) was inhibited more than 100-fold. Moreover, the expression of genes involved in lipid transportation and metabolism was down-regulated by triptolide. CONCLUSION: High-density microarray provided an effective approach to identify drug targeting molecules. It is suggested that the widely known immune suppressive and antitumor effects of triptolide were mediated at least in part by suppression of MAP kinase and PI-3 kinase gene expression.

[Studies on the chemical constituents of Lycianthes biflora].

AIM: To study the chemical constituents of Lycianthes biflora. METHODS: Column chromatography was used to separate the chemical constituents. IR, MS, 1HNMR, 13CNMR and 2D-NMR technique were used to determine the structures of the isolated constituents. RESULTS: Five compounds were isolated from this plant. Their structures were identified to be bifloride A (1), N-trans-cinnamoyltyramine (2), liquiritigenin (3), N-trans-p-coumaroyloctopamine (4), 1-O-beta-D-glucopyranosyl-2-N-2'-hydroxypalmitoyl-sphinga-4- trans-8-trans-dienine (5). CONCLUSION: Compounds 1 and 2 are new compounds, the others were isolated from this plant for the first time. Compound 2 showed inhibitory effects on P-388.

[Isolation and structure of wilfordlonging from Tripterygium wilfordii Hook. f].

AIM: To study the chemical composition of Tripterygirm wilfordii Hook. f. METHODS: Colunm chromatography was used to separate the chemical constituents. UV, IR, MS, HRMS, 1HNMR, 13CNMR (COM and OFR), 1H-1H COSY, 1H-13C COSY, NOESY and COLOC spectra were used to determine the structures of the isolated constituents. RESULTS: Two sesquiterpene alkaloids were isolated and their structures were elucidated as wilforgine and wilfordlongine on the basis of spectral evidence. CONCLUSION: Wilfordclonine is a new sesquiterpene alkaloid.