Inhibitory effect of a novel thiazolidinedione derivative on hepatitis B virus entry.

The development of novel antivirals to treat hepatitis B virus (HBV) infection is still needed because currently available drugs do not completely eradicate chronic HBV in some patients. Recently, troglitazone and ciglitazone, classified among the compounds including the thiazolidinedione (TZD) moiety, were found to inhibit HBV infection, but these compounds are not clinically available. In this study, we synthesized 11 TZD derivatives, compounds 1-11, and examined the effect of each compound on HBV infection in HepG2 cells expressing NTCP (HepG2/NTCP cells). Among the derivatives, (Z)-5-((4'-(naphthalen-1-yl)-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (compound 6) showed the highest antiviral activity, with an IC50 value of 0.3 µM and a selectivity index (SI) of 85, but compound 6 did not affect HCV infection. Treatment with compound 6 inhibited HBV infection in primary human hepatocytes (PHHs) but did not inhibit viral replication in HepG2.2.15 cells or HBV DNA-transfected Huh7 cells. Moreover, treatment with compound 6 significantly impaired hepatitis delta virus (HDV) infection and inhibited a step in HBV particle internalization but did not inhibit attachment of the preS1 lipopeptide or viral particles to the cell surface. These findings suggest that compound 6 interferes with HBV infection via inhibition of the internalization process.

Quest for a potent antimalarial drug lead: Synthesis and evaluation of 6,7-dimethoxyquinazoline-2,4-diamines.

Quinazolines have long been known to exert varied pharmacologic activities that make them suitable for use in treating hypertension, viral infections, tumors, and malaria. Since 2014, we have synthesized approximately 150 different 6,7-dimethoxyquinazoline-2,4-diamines and evaluated their antimalarial activity via structure-activity relationship studies. Here, we summarize the results and report the discovery of 6,7-dimethoxy-N4-(1-phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (20, SSJ-717), which exhibits high antimalarial activity as a promising antimalarial drug lead.

Rapid Access to Dispirocyclic Scaffolds Enabled by Diastereoselective Intramolecular Double Functionalization of Benzene Rings.

Here we describe the diastereoselective synthesis of (5r,8r)-1,9-diazadispiro[4.2.48 .25 ]tetradecatrienes via domino double spirocyclization of N-arylamide derivatives. This reaction can serve as a fast way to synthesize diazadispirocycles, which are found in the core structures of bioactive natural products. Product diversification via Suzuki-Miyaura cross coupling and application to the synthesis of 1-oxa-9-azadispiro[4.2.48 .25 ]tetradecatrienes were also conducted.

In vitro and in vivo characterization of anti-malarial acylphenoxazine derivatives prepared from basic blue 3.

BACKGROUND: Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated. METHODS: Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model. RESULTS: All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, Plasmodium berghei NK65 strain. CONCLUSIONS: The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.

Growth Inhibition of Human Breast and Prostate Cancer Cells by Cinnamic Acid Derivatives and Their Mechanism of Action.

Cancer is the leading cause of death and there is a particularly pressing need to develop effective treatments for breast and prostate cancer. In the current study, we show the inhibitory effects of cinnamic acid derivatives, including caffeic acid phenethyl ester (CAPE, 1), on the growth of breast and prostate cancer cells. Among the compounds examined, 3,4,5-trihydroxycinnamic acid decyl ester (6) showed the most potent inhibition of cancer cell growth by the induction of apoptosis. Compound 6 could be a new anti-cancer agent for use against breast and prostate cancer.

Food polyphenols targeting peptidyl prolyl cis/trans isomerase Pin1.

We searched for inhibitors against prolyl isomerase Pin1 in order to develop functional foods to prevent and cure various Pin1 related diseases such as cancer, diabetes, cardiovascular disease, Alzheimers's disease, and so on. We created a polyphenol library consisting of ingredients in healthy foods and beverages, since polyphenols like epigallocatechin gallate (EGCG) in green tea and 974B in brown algae had been identified as its Pin1 inhibitors. Several polyphenols such as EGCG derivatives, caffeic acid derivatives and tannic acid inhibited Pin1 activity. These results provide a first step in development of the functional foods and beverage targeting Pin1 and its related diseases.

Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress.

Several cinnamic acid derivatives have been reported to exhibit antiviral activity. In this study, we prepared 17 synthetic cinnamic acid derivatives and screened them to identify an effective antiviral compound against hepatitis C virus (HCV). Compound 6, one of two hit compounds, suppressed the viral replications of genotypes 1b, 2a, 3a, and 4a with EC50 values of 1.5-8.1 µM and SI values of 16.2-94.2. The effect of compound 6 on the phosphorylation of Tyr705 in signal transducer and activator of transcription 3 (STAT3) was investigated because a cinnamic acid derivative AG490 was reported to suppress HCV replication and the activity of Janus kinase (JAK) 2. Compound 6 potently suppressed HCV replication, but it did not inhibit the JAK1/2-dependent phosphorylation of STAT3 Tyr705 at the same concentration. Furthermore, a pan-JAK inhibitor tofacitinib potently impaired phosphorylation of STAT3 Tyr 705, but it did not inhibit HCV replication in the replicon cells and HCV-infected cells at the same concentration, supporting the notion that the phosphorylated state of STAT3 Tyr705 is not necessarily correlated with HCV replication. The production of reactive oxygen species (ROS) was induced by treatment with compound 6, whereas N-acetyl-cysteine restored HCV replication and impaired ROS production in the replicon cells treated with compound 6. These data suggest that compound 6 inhibits HCV replication via the induction of oxidative stress.

Involvement of FKBP6 in hepatitis C virus replication.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

Inhibitory effects of hydroxylated cinnamoyl esters on lipid absorption and accumulation.

Obesity is a risk factor associated with several lifestyle-related diseases, for example, diabetes, high blood pressure, hyperlipidemia and cancer. Caffeic acid 2-phenylethyl ester (CAPE, 1), a naturally-occurring compound found in various plants and propolis, which exhibits anti-inflammatory, immunomodulatory and cytotoxic activities and inhibits 3T3-L1 differentiation to adipocytes. As part of our efforts to moderate lifestyle-related diseases, we synthesized analogs of 1 and studied their effects on pancreatic lipase activities, lipid absorption, and 3T3-L1 differentiation. We found that catechols 1-4 show inhibitory activities against pancreatic lipase in a dose-dependent manner in vitro. Compounds 1-3 proved to be more potent inhibitors of pancreatic lipase than 5, 6, 8, and 9, which have one hydroxyl group, respectively. Compound 7 has three aromatic hydroxyl groups and restrains greater lipase inhibitory activity than the other compounds. In addition, 7 and 3 significantly suppress a rise in blood triglyceride (TG) levels in mice given corn oil orally. Furthermore, 2 and 3 are more potent at preventing 3T3-L1 differentiation (lipid accumulation) than 1, while 7 is more potent than 3, 8, and 9 in these assays. Compounds 2, 3, and 7 inhibit lipid absorption and accumulation, with new compound 7 being the most potent. These results indicate that 7 may have potential benefits as a health agent with anti-obesity properties.

PBDE: structure-activity studies for the inhibition of hepatitis C virus NS3 helicase.

The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

Identification and biochemical characterization of halisulfate 3 and suvanine as novel inhibitors of hepatitis C virus NS3 helicase from a marine sponge.

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase.

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

Psammaplin A inhibits hepatitis C virus NS3 helicase.

Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose-response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC50 values of 17 and 32 µM, respectively. Psammaplin A has no influence on the apparent Km value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC50 values of 6.1 and 6.3 µM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3.

Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Inhibition of hepatitis C virus NS3 helicase by manoalide.

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC(50) values of 15 and 70 µM, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K(m) value (0.31 mM) of NS3 ATPase activity, suggesting that 1 acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by 1. Manoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

Analyses of biologically active steroids: antitumor active OSW-1 and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.

Naturally occurring constituents of biological or pharmaceutical interest often exist in the form of glycosides or conjugates. Mass spectral investigations of these compounds require soft ionization techniques if information on molecular mass, sugar sequence, or conjugate content is desired. In this study, matrix-assisted laser desorption/ionization (MALDI) quadrupole ion trap (QIT) time-of-flight tandem mass spectrometry (TOF-MS(n)) was used to identify both OSW-1, an acetylated cholestane diglycoside showing antitumor activity, and the cardiotonic steroid, bufotoxin. Each molecular-related ion was identified, and subsequent collision-induced dissociation experiments in which a molecular-related ion was selected as a precursor ion produced the characteristic product ions that are essential for structural elucidation. OSW-1 and its analogue with a modified side chain, thienyl OSW-1, were synthesized, and bufotoxins, i.e., marinobufotoxin and its homologue, marinobufagin 3-pimeloylarginine ester, were isolated from toad venom. On MALDI-TOF-MS, sodium-adduct [M+Na](+) ions were observed in the steroid glycosides, although protonated [M+H](+) ions were relatively more abundant than sodium-adduct [M+Na](+) ions in the bufotoxins. On the basis of tandem MS results, we propose key fragmentation pathways. The sugar moiety or side chain from the precursor ion was eliminated in OSW-1. However, characteristic product ions originating from the cleavage of the side chain with an ester formation were observed in the bufotoxins. Post-source decay (PSD) on MALDI-TOF-MS is also described when evaluating alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as a matrix to obtain useful ions required for the identification of compound.

Analysis of antitumor active OSW-1 and its analogues by liquid chromatography coupled with electrospray and atmospheric pressure chemical ionization quadrupole mass spectrometry.

Three cholestane glycosides including OSW-1 with antitumor activity and two new analogues with modified steroidal side chains, thienyl OSW-1 and silylated thienyl OSW-1, were synthesized. Analyses were performed using optimized, reversed-phase liquid chromatography (LC) with electrospray ionization and atmospheric pressure chemical ionization quadrupole mass spectrometry (MS). The ionization mode and polarity, cone voltage, and chromatographic conditions were evaluated. The optimum LC/MS conditions to obtain valuable ions, indispensable for identifying the structures, are described. The key fragmentation pathways, which will be useful for confirming the detailed structures of steroidal glycosides, are also proposed.

Separation of stereoisomers of some terpene derivatives by capillary gas chromatography-mass spectrometry and high-performance liquid chromatography using beta-cyclodextrin derivative columns.

Gas chromatographic separations of the stereoisomers of menthol derivatives, important intermediates in the synthesis of physiologically active natural products, were carried out on several substituted beta-cyclodextrin (CD) columns, including per-O-methyl-beta-cyclodextrin (PME-beta-CD), heptakis(2,3-di-O-acetyl-6-tert-butyldimethylsilyl)-beta-CD (DIAC-6-TBDS-beta-CD), and heptakis(2,3-di-O-methyl-6-tert-butyldimethylsilyl)-beta-CD (DIME-6-TBDS-beta-CD) as chiral stationary phases (CSPs). With the DIME-6-TBDS-beta-CD column, a separation of the Z- and E-isomers of methylidenementhol was accomplished; no separation was achieved with the other columns. The stereoisomers of methylidenementhol and the corresponding tert-butyldimethylsilyl (TBS) ether were separated on both the beta-CD and the heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TME-beta-CD) columns by high-performance liquid chromatography (HPLC) with a mobile phase involving acetonitrile and H(2)O. For the separation of the Z- and E-isomers of methylidenementhol, the TME-beta-CD column was superior. In contrast, the beta-CD column was preferable in the case of the corresponding TBS ether.

Analyses of anandamide and endocannabinoid-like compounds using collision-induced dissociation in fast atom bombardment ionization-mass spectrometry and gas chromatography/chemical ionization-mass spectrometry.

The utility of the collision-induced dissociation (CID) of two different forms of precursor cations generated by the fast atom bombardment (FAB) ionization of N-arachidonylethanolamine (anandamide) and a series of endocannabinoid-like compounds, such as N-oleoylethanolamine, N-palmitoylethanolamine, N-stearoylethanolamine, N-linoleoylethanolamine, N-oleoylpropanolamine, and N-palmitoylpropanolamine, as a method of providing general information on their characterizations was examined. The CID spectra of lithium-adduct [M+Li]+ ions of the amines with unsaturated hydrocarbon chains were rich in structurally informative charge-site-remote (CSR) fragmentation patterns that provide information on the locations of double bonds in hydrocarbon chains. On the other hand, the CID reactions of [M+H]+ ions produced acylium ions that are derived from the cleavage of amide bonds, thus providing information on the size of the hydrocarbon chains, although CSR fragmentations were not observed. These compounds without derivatization were analyzed using gas chromatography/chemical ionization-mass spectrometry (GC/CI-MS) with a polyethylene glycol phased column with fused silica capillary pre-tubing. Identifiable molecular-related [M+H]+ ions were observed.