Development of 1,3,6-Tribenzoylated Glucose as an Antiausterity Agent Targeting Tumor Microenvironment.

One aspect of cancer-specific environments, nutrient starvation, is a factor in cancer cell resistance to treatment with chemotherapeutic agents and development of malignancy. Our newly synthesized novel glucose derivative ß-1,3,6-O-tribenzoyl-D-glucose (3) showed preferential cytotoxicity against PANC-1 human pancreatic cancer cells as well as HT-29 human colon cancer cells depending on low nutritional environment. The amount of ester functionalization in 3 is important. None of the mono- and tetrabenzoylated D-glucose analog showed cytotoxicity, and dibenzoylated D-glucoses showed only limited cytotoxicity. Fluorescence imaging with double staining of Hoechst 33342 and propidium iodide clearly showed that 3 actually causes cell death in a nutrient deprived medium. We thus demonstrate that an inexpensive natural product, D-glucose, is a unique template for attachment of acyl moieties to target tolerance to nutrient starvation. We expect these compounds will lead to additional compounds to treat refractory cancers by diversification of chemically modified glucose.

Advances in reaction-based synthetic fluorescent probes for studying the role of zinc and copper ions in living systems.

Recently, the behavior of essential trace metal elements in living organisms has attracted more and more attention as their dynamics have been found to be tightly regulated by metallothionines, transporters, etc. As the physiological and/or pathological roles of such metal elements are critical, there have been many non-invasive methods developed to determine their cellular functions, mainly by small molecule fluorescent probes. In this review, we focus on probes that detect intracellular zinc and monovalent copper. Both zinc and copper act not only as tightly bound cofactors of enzymes and proteins but also as signaling factors as labile or loosely bound species. Many fluorescent probes that detect mobile zinc or monovalent copper are recognition-based probes, whose detection is hindered by the abundance of intracellular chelators such as glutathione which interfere with the interaction between probe and metal. In contrast, reaction-based probes release fluorophores triggered by zinc or copper and avoid interference from such intracellular chelators, allowing the detection of even low concentrations of such metals. Here, we summarize the current status of the cumulative effort to develop such reaction-based probes and discuss the strategies adopted to overcome their shortcomings.

Extracts of Musa basjoo induce growth inhibition and changes in the protein expression of cell cycle control molecules in human colorectal cancer cell lines.

Musa basjoo (MB) is a species of the banana plant belonging to the genus Musa that has been used as a folk medicine. However, evidence-based biological activities and the molecular mechanism of action of MB are unknown. Thus, the aim of the present study was to examine whether the crude dried leaf extracts of MB inhibit the growth of colorectal (HT29 and HCT116) and other types (HepG2, MCF-7 and PC-3) of human cancer cell lines. Crude extracts of MB inhibited the growth of cells with IC50 values of 136 µg/ml (acetone extract, HT29), 51 µg/ml (acetone extract, HCT116), 45 µg/ml (acetone extract, HepG2), 40 µg/ml (acetone extract, MCF-7), 29 µg/ml (acetone extract, PC-3), 175 µg/ml (methanol extract, HT29), 137 µg/ml (methanol extract, HCT116), 102 µg/ml (methanol extract, HepG2), 85 µg/ml (methanol extract, MCF-7), and 85 µg/ml (methanol extract, PC-3) in colony formation assays, and 126 µg/ml (acetone extract, HT29), 68 µg/ml (acetone extract, HCT116), 260 µg/ml (methanol extract, HT29), and 216 µg/ml (methanol extract, HCT116) in MTT assays. Thin layer chromatography analysis revealed the potential existence of aromatic compounds in the acetone extract of MB. Flow cytometric analysis indicated that the percentage of cells in G1 increased, and this was associated with a concomitant decrease of cells in the S and/or G2-M phases of the cell cycle. When colorectal cancer cells were treated with acetone extract of MB, there was a marked decrease in the levels of expression of the cyclin D1, cyclin E, cdk2 and cdk4 proteins and a marked increase in the levels of the expression of the p21CIP1, p27KIP1, and p53 proteins, but those of apoptosis-associated protein PARP did not change. There was a tendency for acetone extract of MB to inhibit xenograft tumor growth in mice. Collectively, the crude extracts of MB contain active components that exert growth inhibition of human cancer cells. This is the first systematic study of the anticancer activity of MB and may broaden insights into the possible clinical approach of specific herbal medicines.

Development of antitumor biguanides targeting energy metabolism and stress responses in the tumor microenvironment.

To develop antitumor drugs capable of targeting energy metabolism in the tumor microenvironment, we produced a series of potent new biguanide derivatives via structural modification of the arylbiguanide scaffold. We then conducted biological screening using hypoxia inducible factor (HIF)-1- and unfolded protein response (UPR)-dependent reporter assays and selective cytotoxicity assay under low glucose conditions. Homologation studies of aryl-(CH2)n-biguanides (n = 0-6) yielded highly potent derivatives with an appropriate alkylene linker length (n = 5, 6). The o-chlorophenyl derivative 7l (n = 5) indicated the most potent inhibitory effects on HIF-1- and UPR-mediated transcriptional activation (IC50; 1.0 ± 0.1 µM, 7.5 ± 0.1 µM, respectively) and exhibited selective cytotoxicity toward HT29 cells under low glucose condition (IC50; 1.9 ± 0.1 µM). Additionally, the protein expression of HIF-1α induced by hypoxia and of GRP78 and GRP94 induced by glucose starvation was markedly suppressed by the biguanides, thereby inhibiting angiogenesis. Metabolic flux and fluorescence-activated cell sorting analyses of tumor cells revealed that the biguanides strongly inhibited oxidative phosphorylation and activated compensative glycolysis in the presence of glucose, whereas both were strongly suppressed in the absence of glucose, resulting in cellular energy depletion and apoptosis. These findings suggest that the pleiotropic effects of these biguanides may contribute to more selective and effective killing of cancer cells due to the suppression of various stress adaptation systems in the tumor microenvironment.

Total Synthesis of Antiausterity Agent (±)-Uvaridacol L by Regioselective Axial Diacylation of a myo-Inositol Orthoester.

The antiausterity natural product (±)-uvaridacol L was synthesized for the first time in seven steps from myo-inositol. The key reaction of this synthesis, axial selective dibenzoylation of myo-inositol orthoformate, was achieved using a catalytic amount of tetrabutylammonium fluoride (TBAF). The preferential cytotoxicity of racemic uvaridacol L against cancer cell lines able to adapt to nutrient deprivation was also evaluated under nutrient deprived conditions. Morphological evaluation was also carried out.

A fluorogenic probe using a catalytic reaction for the detection of trace intracellular zinc.

Labile zinc plays various roles in cells at low concentrations which most fluorescent probes are not able to detect. Here we report a cephem-based probe which coordinates to zinc and zinc-bound water cleaves the scaffold and releases the fluorophore. In addition, the zinc is recycled and reacts with multiple probes, amplifying the signal. This signal amplification system is useful for the detection of intracellular zinc at low concentrations and has potential for further development of probes with a similar molecular design.

Hydrogen sulfide increases copper-dependent neurotoxicity via intracellular copper accumulation.

Copper (Cu) is an essential trace element and acts as a redox cofactor for many enzymes; however, excess Cu is toxic to cells. Hydrogen sulfide (H2S) is a well-known toxic gaseous molecule, but it has various biological effects such as neuromodulation and vasodilation. H2S was recently demonstrated to be involved in the detoxification of heavy metals, including zinc and cadmium, suggesting that H2S helps to maintain the homeostasis of heavy metals in cells. However, it is unclear how H2S impacts cellular Cu dynamics. In this study, we examined the effects of H2S on Cu cytotoxicity. Human neuroblastoma SH-SY5Y cells were exposed to CuSO4 in the presence of the H2S donor NaHS. CuSO4 alone slightly induced cell injury, whereas the combination of CuSO4 and NaHS (Cu/NaHS) increased Cu cytotoxicity. The Cu chelator bathocuproinedisulfonic acid mitigated Cu/NaHS-induced cytotoxicity. Compared with CuSO4 alone, Cu/NaHS markedly promoted ROS generation, mitochondrial dysfunction, and a decrease in ATP production. In addition, reporter assay using the metal responsive element (MRE)-driven reporter plasmid revealed that Cu/NaHS augmented Cu-dependent MRE activation. The amount of intracellular Cu was significantly higher in cells treated with Cu/NaHS than in those treated with CuSO4 alone. Moreover, Cu/NaHS markedly suppressed the level of the Cu exporter ATP7A, but not ATP7B, protein, whereas the combination did not affect that of the Cu importer CTR1 protein. Taken together, we conclude that the marked decrease in the ATP7A protein level by Cu/NaHS promotes intracellular Cu accumulation and leads to increased Cu cytotoxicity.

Effects of 2-(2-Chlorophenyl)ethylbiguanide on ERAD Component Expression in HT-29 Cells Under a Serum- and Glucose-Deprived Condition.

We recently characterized the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition and found that 2-Cl-Phen attenuated ATF4 and GRP78, typical downstream targets of the unfolded protein response (UPR), together with c-Myc protein expression in a transcriptional and posttranscriptional manner. In the current study, we focused on the expression of ER-associated protein degradation (ERAD) components after treatment with 2-Cl-Phen under a serum- and glucose-deprived condition. Among nine ER-localizing factors regulating protein quality control within the ER, the amounts of Herp, GRP78, GRP94, and OS9 proteins were significantly downregulated by treatment with 2-Cl-Phen. In particular, replacement of the culture medium with the serum- and glucose-deprived medium induced the expression of Herp protein at the early phase. This increase in Herp protein was accompanied by an increase in its mRNA, and its induction was significantly dampened by 2-Cl-Phen. However, cotreatment with a proteasome inhibitor, MG132, restored Herp expression only to a limited extent. Taken together, these results show that 2-Cl-Phen changed the expression of several ERAD components, especially by transcriptional inhibition of Herp induction by 2-Cl-Phen when it occurred at an early phase, and this finding provides new insights into understanding the mechanisms of 2-Cl-Phen-mediated cytotoxicity.

Elucidating the rapid action of 2-(2-chlorophenyl)ethylbiguanide on HT-29 cells under a serum- and glucose-deprived condition.

We recently demonstrated the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition. In that study, we showed that the ATF6 arm of the ER stress pathway and c-Myc expression were downregulated 12 h after the treatment with 2-Cl-Phen. Through characterization of intracellular events at the early phase of the 2-Cl-Phen treatment before noticeable morphological changes, we found rapid fluctuations in the c-Myc and ATF4 proteins but not in their mRNAs in 2-Cl-Phen-treated HT-29 cells under the serum- and glucose-deprived condition. The 2-Cl-Phen-mediated downregulation of ATF4 protein was not paralleled by the phosphorylation status of PERK and eIF2α. Reduction of c-Myc expression by 2-Cl-Phen was more profound than that of ATF4 expression, and phosphorylated c-Myc was downregulated within 2 h. Pharmacological studies on the expression of c-Myc and ATF4 proteins showed that this decrease was mediated through proteasomal degradation but not by autophagy. Interestingly, treatment with lithium chloride, which is a well-known inhibitor of GSK3ß, partially recovered the expression of ATF4 protein, but its effect on the level of total c-Myc protein was negligible. Treatment with 2-Cl-Phen increased the expression of phosphorylated AMPK, but Compound C, an AMPK inhibitor, did not influence the expression of c-Myc protein in HT-29 cells. Finally, we observed that 2-Cl-Phen partially attenuated the gene expression of integrin subunit α1 (ITGA1), a downstream target of c-Myc. Taken together, these results show that 2-Cl-Phen rapidly downregulated the expression of c-Myc in addition to ER stress responses in a post-translational manner. Further elucidation and improvement of this multi-target-directed compound will provide new insights for developing therapeutic strategies against cancer.

Hydrogen sulfide ameliorates zinc-induced cell death in neuroblastoma SH-SY5Y cells.

Previous reports have demonstrated that excess zinc (Zn2+) released from nerve terminals following cerebral ischemia causes brain injury. Therefore, the disturbance of Zn2+ homeostasis in the brain is thought to be closely linked to neurotoxicity. Recently, hydrogen sulfide (H2S), a gaseous mediator, has been reported to ameliorate ischemic brain injury. However, its mechanism is not fully understood. In this study, we examined whether sodium hydrogen sulfide (NaHS), an H2S donor, protects against Zn2+ cytotoxicity using human neuroblastoma SH-SY5Y cells. NaHS dose-dependently prevented cell death caused by Zn2+ exposure. Treatment of cells with NaHS just before Zn2+ exposure exerted the most potent protection. Zn2+ induced loss of intracellular NAD+ and ATP and mitochondrial dysfunctions, resulting in cytotoxicity associated with failure of energy production; however, NaHS prevented these Zn2+-induced events. In addition, NaHS suppressed Zn2+-dependent activation of metal-responsive transcription factor-1 and induction of metallothionein gene expression. Zn2+ imaging with the Zn2+-specific fluorescent indicator FluoZin-3 revealed that NaHS abolished the elevation of intracellular Zn2+ levels after Zn2+ exposure. These results suggest that entry of Zn2+ into cells was suppressed by NaHS. The measurement of H2S derived from NaHS by o-fluorinated-azido-capped rhodamine (Rho-N3F2), a reaction-based H2S probe, revealed that H2S levels in aqueous solutions were markedly reduced in the presence of Zn2+. This finding suggests the possibility that H2S reacts directly with Zn2+ and decreases extracellular Zn2+ levels. Taken together, we conclude that the protection of NaHS against Zn2+ cytotoxicity is exerted by inhibiting entry of Zn2+ into SH-SY5Ycells.

Bismuth-rhodamine: a new red light-excitable photosensitizer.

Bismuth-rhodamine (BiR) was developed as a new photosensitizer scaffold, and its photophysical properties were evaluated. BiR showed significant red-shifted absorption and emission compared with other xanthene-based photosensitizers, together with an efficient quantum yield for the generation of 1O2. BiR showed efficient cell-permeability as well as photo-triggered generation of 1O2 in cells.

A universal fluorogenic switch for Fe(ii) ion based on N-oxide chemistry permits the visualization of intracellular redox equilibrium shift towards labile iron in hypoxic tumor cells.

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe2+. In this study, a universal fluorogenic switching system is established, which is responsive to Fe2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe2+ to generate various fluorescent probes of Fe2+-CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.

In vivo retinal and choroidal hypoxia imaging using a novel activatable hypoxia-selective near-infrared fluorescent probe.

PURPOSE: Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models. METHODS: The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection. RESULT: Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity. CONCLUSIONS: GPU-327 identified hypoxic area both in vivo and in vitro.

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells.

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.

Solution-phase synthesis and biological evaluation of triostin A and its analogues.

Triostin A is a biosynthetic precursor of echinomycin which is one of the most potent hypoxia inducible factor 1 (HIF-1) inhibitors. An improved solution-phase synthesis of triostin A on a preparative scale has been achieved in 17.5% total yield in 13 steps. New analogues of triostin A with various aromatic chromophores, oxidized intra-peptide disulfide bridges and diastereoisomeric cyclic depsipeptide cores were also successfully synthesized. All analogues had a significant inhibitory effect on HIF-1 transcriptional activation in hypoxia and cytotoxicity on MCF-7 cells, with the exception of the derivatives containing a naphthalene chromophore or a thiosulfonate bridge. For the first time, triostin A, echinomycin and the thiosulfinate analogue of triostin A have been revealed to inhibit not only DNA binding of HIF-1 but also HIF-1α protein accumulation in MCF-7 cells. Furthermore, the thiosulfinate analogue and triostin A exhibited a hypoxia-selective cytotoxicity on MCF-7 cells. The improved solution-phase synthetic procedure described herein will contribute to the development of diverse bicyclic depsipeptide drug candidates with the potential to act as novel anti-cancer agents targeting hypoxic tumor microenvironments.

A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors.

Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase. The NanoLuc reporter, which contained a portion of the spliced region of XBP1 upstream of NanoLuc, was highly sensitive and compatible with several types of cell lines. We found that NanoLuc was secreted into the extracellular space independent of the ER-Golgi pathway. The NanoLuc activity of an aliquot of culture medium from the neuroblastoma-spinal neuron hybrid cell line NSC-34 reflected the toxic stimuli-induced elevation of intracellular activity well. Using this technique, we evaluated the effects of several genetic and pathological factors associated with the onset and progression of amyotrophic lateral sclerosis (ALS) on NanoLuc reporter activity. Under our experimental conditions, inhibition of ER-Golgi transport by the overexpression of mutant Sar1 activated luciferase activity, whereas the co-expression of mutant SOD1 or the C-terminal fragment of TDP-43 (TDP-25) did not. The addition of homocysteine elevated the reporter activity; however, we did not observe any synergistic effect due to the overexpression of the mutant genes described above. Taken together, these data show that our analytical procedure is highly sensitive and convenient for screening useful compounds that modulate IRE1-XBP1 signaling pathways as well as for estimating IRE1 activation in several pathophysiological diseases.

A new class of high-contrast Fe(II) selective fluorescent probes based on spirocyclized scaffolds for visualization of intracellular labile iron delivered by transferrin.

Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe(2+)-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe(2+)-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe(2+), compared with the first generation N-oxide based Fe(2+) probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe(2+) but also the endogenous Fe(2+) levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.

Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment.

Adaptive cellular responses resulting from multiple microenvironmental stresses, such as hypoxia and nutrient deprivation, are potential novel drug targets for cancer treatment. Accordingly, we focused on developing anticancer agents targeting the tumor microenvironment (TME). In this study, to search for selective antitumor agents blocking adaptive responses in the TME, thirteen new compounds, designed and synthesized on the basis of the arylmethylbiguanide scaffold of phenformin, were used in structure activity relationship studies of inhibition of hypoxia inducible factor (HIF)-1 and unfolded protein response (UPR) activation and of selective cytotoxicity under glucose-deprived stress conditions, using HT29 cells. We conducted luciferase reporter assays using stable cell lines expressing either an HIF-1-responsive reporter gene or a glucose-regulated protein 78 promoter-reporter gene, which were induced by hypoxia and glucose deprivation stress, respectively, to screen for TME-targeting antitumor drugs. The guanidine analog (compound 2), obtained by bioisosteric replacement of the biguanide group, had activities comparable with those of phenformin (compound 1). Introduction of various substituents on the phenyl ring significantly affected the activities. In particular, the o-methylphenyl analog compound 7 and the o-chlorophenyl analog compound 12 showed considerably more potent inhibitory effects on HIF-1 and UPR activation than did phenformin, and excellent selective cytotoxicity under glucose deprivation. These compounds, therefore, represent an improvement over phenformin. They also suppressed HIF-1- and UPR-related protein expression and secretion of vascular endothelial growth factor-A. Moreover, these compounds exhibited significant antiangiogenic effects in the chick chorioallantoic membrane assay. Our structural development studies of biguanide derivatives provided promising candidates for a novel anticancer agent targeting the TME for selective cancer therapy, to be subjected to further in vivo study.

Facile one-pot synthesis of [1, 2, 3]triazolo[1, 5-a]pyridines from 2-acylpyridines by copper(II)-catalyzed oxidative N-N bond formation.

An efficient and simple method for the synthesis of various [1, 2, 3]triazolo[1, 5-a]pyridines has been established. The method involves a copper(II)-catalyzed oxidative N-N bond formation that uses atmospheric oxygen as the terminal oxidant following hydrazonation in one pot. The use of ethyl acetate as the solvent dramatically promotes the oxidative N-N bond-formation reaction and enables the application of oxidative cyclization in the efficient one-pot reaction. A mechanism for the reaction was proposed on the basis of the results of a spectroscopic study.

FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes.

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.