Synthesis and anti-hepatocellular carcinoma activity of aminopyridinol–sorafenib hybrids

Sorafenib is recommended as the primary therapeutic drug for patients with hepatocellular carcinoma. To discover a new compound that avoids low response rates and toxic side effects that occur in sorafenib therapy, we designed and synthesized new hybrid compounds of sorafenib and 2,4,5-trimethylpyridin-3-ols. Compound 6 was selected as the best of 24 hybrids that inhibit each of the four Raf kinases. The anti-proliferative activity of 6 in HepG2, Hep3B, and Huh7 cell lines was slightly lower than that of sorafenib. However, in H6c7 and CCD841 normal epithelial cell lines, the cytotoxicity of 6 was much lower than that of sorafenib. In addition, similar to sorafenib, compound 6 inhibited spheroid forming ability of Hep3B cells in vitro and tumour growth in a xenograft tumour model of the chick chorioallantoic membrane implanted with Huh7 cells. Compound 6 may be a promising candidate targeting hepatocellular carcinoma with low toxic side effects on normal cells.     

Action of 6-amino-3-pyridinols as novel antioxidants against free radicals and oxidative stress in solution,plasma, and cultured cells

Free radical-mediated lipid peroxidation has been implicated in the pathogenesis of various diseases. Lipid peroxidation products are cytotoxic and they modify proteins and DNA bases, leading eventually to degenerative disorders. Various synthetic antioxidants have been developed and assessed for their capacity to inhibit lipid peroxidation and oxidative stress induced by free radicals. In this study, the capacity of novel 6-amino-2,4,5-trimethyl-3-pyridinols for scavenging peroxyl radicals, inhibiting plasma lipid peroxidation in vitro, and preventing cytotoxicity induced by glutamate, 6-hydroxydopamine, 1-methyl-4-phenylpyridium (MPP⁺ ), and hydroperoxyoctadecadienoic acid was assessed. It was found that they exerted higher reactivity toward peroxyl radicals and more potent activity for inhibiting the above oxidative stress than α-tocopherol, the most potent natural antioxidant, except against the cytotoxicity induced by MPP⁺. These results suggest that the novel 6-amino-3-pyridinols may be potent antioxidants against oxidative stress.     

香桃木叶片粗多酚抗氧化活性的研究

以香桃木叶片为材料,60%乙醇提取获得粗多酚样品,并对样品的油脂抗氧化性和自由基清除能力进行研究。结果表明,未经纯化的香桃木粗多酚对油脂抗氧化效果优于BHT和V_C等人工合成抗氧化剂;在清除自由基的实验中,相同浓度的香桃木粗多酚对DPPH自由基的清除率为V_E的5倍,对羟自由基的清除效果也略优于V_C。由此可以预见香桃木粗多酚在精细加工、经纯化后抗氧化效果会更加显著。     

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog,Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT₁ receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.     

Novel series of 3-amino-N-(4-aryl-1,1-dioxothian-4-yl)butanamides as potent and selective dipeptidyl peptidase IV inhibitors

A series of novel 3-amino-N-(4-aryl-1,1-dioxothian-4-yl)butanamides were investigated as dipeptidyl peptidase IV (DPP-4) inhibitors. Introduction of a 4-phenylthiazol-2-yl group showed highly potent DPP-4 inhibitory activity. Among various derivatives, (3R)-3-amino-N-(4-(4-phenylthiazol-2-yl)-tetrahydro-2H-thiopyran-4-yl)-4-(2,4,5-trifluorophenyl)butanamide 1,1-dioxide (30) reduced blood glucose excursion in an oral glucose tolerance test by oral administration.     

2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor agents selectively active in securin−/− cells

A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure–activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.     

From the covalent linkage of drugs to novel inhibitors of ribonucleotide reductase: Synthesis and biological evaluation of valproic esters of 3′-C-methyladenosine

We synthesized a series of serum-stable covalently linked drugs derived from 3′-C-methyladenosine (3′-Me-Ado) and valproic acid (VPA), which are ribonucleotide reductase (RR) and histone deacetylase (HDAC) inhibitors, respectively. While the combination of free VPA and 3′-Me-Ado resulted in a clear synergistic apoptotic effect, the conjugates had lost their HDAC inhibitory effect as well as the corresponding apoptotic activity. Two of the analogs, 2′,5′-bis-O-valproyl-3′-C-methyladenosine (A160) and 5′-O-valproyl-3′-C-methyladenosine (A167), showed promising cytotoxic activities against human hematological and solid cancer cell lines. A167 was less potent than A160 but had interesting features as an RR inhibitor. It inhibited RR activity by competing with ATP as an allosteric effector and concomitantly reduced the intracellular deoxyribonucleoside triphosphate (dNTP) pools. A167 represents a novel lead compound, which in contrast to previously used RR nucleoside analogs does not require intracellular kinases for its activity and therefore holds promise against drug resistant tumors with downregulated nucleoside kinases.     

The chemopreventive effect of Ginkgo biloba and Silybum marianum extracts on hepatocarcinogenesis in rats

Background/objective

This study was designed to evaluate the potential chemopreventive activities of Ginkgo biloba extract (EGb) and Silybum marianum extract (silymarin) against hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in rats.

Methods

Rats were divided into 6 groups. Group 1 served as normal control rats. Group 2 animals were intragastrically administrated NDEA at a dose of 10 mg/kg five times a week for 12 weeks to induce hepatocellular carcinoma (HCC). Groups 3 and 4 animals were pretreated with silymarin and EGb respectively. Groups 5 and 6 animals were posttreated with silymarin and EGb respectively. The investigated parameters in serum are alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) and vascular endothelial growth factor (VEGF). The investigated parameters in liver tissue are malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and comet assay parameters.

Results

In NDEA group, MDA level was elevated with subsequent decrease in GSH level and SOD, GPx and GR activities. In addition, NDEA group revealed a significant increase in serum ALT, AST and GGT activities and VEGF level. Furthermore, NDEA administrated animals showed a marked increase in comet assay parameters. These biochemical alterations induced by NDEA were confirmed by the histopathological examination of rat livers intoxicated with NDEA that showed an obvious cellular damage and well differentiated HCC. In contrast, silymarin+NDEA treated groups (3&;5) and EGb+NDEA treated groups (4&;6) showed a significant decrease in MDA level and a significant increase in GSH content and SOD, GPx and GR activities compared to NDEA group. Silymarin and EGb also beneficially down-regulated the increase in serum ALT, AST, GGT activities and VEGF level induced by NDEA. In addition, silymarin and EGb significantly decreased comet assay parameters. Histopathological examination of rat livers treated with either silymarin or EGb exhibited an improvement in the liver architecture compared to NDEA group.

Conclusions

The obtained findings suggested that silymarin and EGb may have beneficial chemopreventive roles against hepatocarcinogenesis through their antioxidant, antiangiogenic and antigenotoxic activities.     

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C₁₈ columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3′-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.     

Protective effects of 6-ureido/thioureido-2,4,5-trimethylpyridin-3-ols against 4-hydroxynonenal-induced cell death in adult retinal pigment epithelial-19 cells

Dysfunction or progressive degeneration of retinal pigment epithelium (RPE) contributes in the initial pathogenesis of age-related macular degeneration (AMD) causing irreversible vision loss, which makes RPE the prime target of the disease. The present study aimed to identify compounds to protect 4-hydroxynonenal (4-HNE)-induced RPE cell death by inhibiting NADPH oxidase 4 (NOX4) activity, not just as free radical scavengers, using ARPE-19, a human adult retinal pigment epithelial cell line, as a RPE representative. Novel thirty-two 6-ureido/thioureido-2,4,5-trimethylpyridin-3-ol derivatives 17 were synthesized and tested. We found that there was a strong correlation between level of protective effect of compounds 17 against 4-HNE-induced APRE-19 cell death and that of inhibitory activity against 4-HNE-induced superoxide production, and that most of the compounds 17 showed minimal DPPH radical scavenging activity. Compound 17–28 showed the best protective activity against 4-HNE-induced superoxide production (79.5% inhibition) and cell death (85.1% recovery) at 10?μM concentration, which was better than that of VAS2870, a NOX2/4 inhibitor. In addition, compound 17–28 blocked 4-HNE-induced apoptosis of ARPE-19 cells in a concentration-dependent manner. The results indicate that compound 17–28 may be a lead compound to develop AMD therapeutics.     

Novel VEGFR-2 kinase inhibitors identified by the back-to-front approach

We report a novel VEGFR-2 inhibitor, developed by the back-to-front approach. Docking experiments indicated that the 3-chloromethylphenylurea motif of the lead compound occupied the back pocket of VEGFR-2 kinase. An attempt was made to enhance the binding affinity of 1 by expanding the structure to access the front pocket using a triazole linker. A library of 1,4-(disubstituted)-1H-1,2,3-triazoles were screened in silico, and one compound (VH02) was identified with an IC₅₀ against VEGFR-2 of 0.56 μM. VH02 showed antiangiogenic effects, inhibiting tube formation in HUVEC cells (EA.hy926) at 0.3 μM, 13 times lower than its cytotoxic dose. These enzymatic and cellular activities suggest that VH02 has potential as a lead for further optimization.     

The antiangiogenic activity of Kushecarpin D,a novel flavonoid isolated from Sophora flavescens Ait

Aims

Kushecarpin D (KD) is a novel flavonoid isolated from the traditional Chinese herbal medicine Kushen (the dried root of Sophora flavescens Ait). As part of our continuous effort to explore Chinese traditional medicinal herbs and to identify novel natural anticancer products, the antiangiogenic properties of KD were examined in vitro using a human umbilical vein endothelial cell line (ECV304).

Main methods

The SRB and Trypan Blue exclusion assays were used to evaluate the effect of KD on cell proliferation. The antiangiogenic activities of KD were evaluated through studies of cell migration, cell adhesion, and tube formation. DCFH-DA and DHE fluorescent assays were used to detect the reactive oxygen species (ROS) levels. Catalase activity was detected using the colorimetric ammonium molybdate method. Cell cycle and apoptosis were measured using flow cytometry and the Hoechst 33258 staining assay.

Key findings

The results indicated that KD showed antiangiogenic activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. ROS levels were down-regulated and catalase activity was up-regulated after treatment with KD. The cell cycle was arrested at the G2/M phase, while no apoptosis was observed using the Hoechst 33258 staining assay or following the flow cytometric analysis of the sub-G1 proportion.

Significance

The antiangiogenic properties of KD, in combination with its anti-proliferative effect and ability to induce cell cycle arrest without inducing apoptosis, make it a good candidate for development as antitumor agent. However, further studies are essential to elucidate its mechanism of action.     

Genetic control of 3-hydroxy- and 3-deoxy- flavonoids in Zea mays

Genetic strains of maize have been derived that have strong concentrations of 3-deoxyanthocyanins, flavan-4-ols and C-glycosyl-flavones in silks and cob. The gene Pr controls the B-ring hydroxylation pattern of both 3-deoxy- and 3-hydroxy-flavonoids in these plants. Cob phlobaphenes are red in Pr plants and orange in pr plants, corresponding to the presence of the probable precursors luteoforol and aniforol.     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.     

Accumulation of dodecyltriphenylphosphonium in mitochondria induces their swelling and ROS-dependent growth inhibition in yeast

Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C₁₂-TPP), in the yeast Saccharomyces cerevisiae. First, we found that C₁₂-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C₁₂-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C₁₂-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.     

Carbazole alkaloids with antiangiogenic activities from Clausena sanki

Two new carbazole alkaloids 1 and 2, and eleven known congeners 3–13 were isolated and identified from Clausena sanki for the first time. Their structures were elucidated on the basis of extensive UV, IR, MS, NMR spectroscopic data and comparison with literatures. The compounds 1–13 were evaluated by MTT assay to determine whether they decreased VEGF-mediated cell proliferation in HUVECs with Axitinib as positive control. Among them, compounds 1, 2, 6, 8, and 13 (μM) exhibited moderate antiangiogenic activities, which inhibited VEGF-induced HUVEC proliferation in vitro with IC₅₀ values of 12.1 (C.I. 8.2–15.2), 58.1 (C.I. 56.3–63.4), 13.7 (C.I. 9.2–15.4), 16.0 (C.I. 9.5–16.4), and 63.2 (C.I. 57.8–65.7) μM, respectively. Moreover, the antiangiogenic activities of compounds 1–13 were evidenced in vivo in the zebrafish embryo model. As a result, compounds 1, 2, 6, 8, and 13 showed effectively suppress angiogenesis. These research results may guide the search for new natural products with antiangiogenic attributes.     

Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin)

Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding.     

The origin of the generalized anomeric effect: possibility of CH/n and CH/π hydrogen bonds

Ab initio MO calculations were carried out at the MP4/6-311++G(3df,3pd)//MP2/6-311++G(3df,3pd) level to investigate the conformational Gibbs energy of a series of methyl ethers CH₃O-CH₂-X (X = OH, OCH₃, F, Cl, Br, CN, CCH, C₆H₅, CHO). It was found that the Gibbs energy of the gauche conformers is lower in every case than that of the corresponding anti conformers. In the more stable gauche conformers, the interatomic distance between X and the hydrogen atom was shorter than the sum of the van der Waals radii. The natural bonding orbital (NBO) charges of group X were more negative in the gauche conformers than in the anti conformers. We suggest that the CH/n and CH/π hydrogen bonds play an important role in stabilizing the gauche conformation of these compounds.     

Antiangiogenic properties of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol/one analogs and their derivatives

In the past half century research efforts have defined a critical role for angiogenesis in tumor growth and metastasis. We previously reported that inhibition of a novel target, ENOX1, by a (Z)-2-benzylindol-3-ylmethylene) quinuclidin-3-ol, suppressed tumor angiogenesis. The present study was undertaken in order to establish structure-activity relationships for quinuclidine analogs. The angiogenesis inhibiting activity of a series of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ols (1a-1k), (Z)-2-benzylindol-3-ylmethylene)quinuclidin-3-ones (2a-2h), (Z)-(±)-2-(1H/N-methyl-indol-3-ylmethylene)quinuclidin-3-ols (3a-3b), and substituted (Z)-(±)-2-(N-benzenesulfonylindol-3-yl-methylene)quinuclidin-3-ols and their derivatives (4a-4d) that incorporate a variety of substituents in both the indole and N-benzyl moieties was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) subjected to in vitro cell migration scratch assays, tubule formation in Matrigel, cell viability and proliferation assays. In total, 25 different analogs were evaluated. Based on in vitro cell migration scratch assays, eight analogs were identified as potent angiogenesis inhibitors at 10 μM, a concentration that was determined to be nontoxic by colony formation assay. In addition, this approach identified a potent antiangiogenic ENOX1 inhibitor, analog 4b.