Parahydrogen-induced 13C hyperpolarizer using a flow guide for magnetic field cycling to evoke 1H-13C spin order transfer toward metabolic MRI.

OBJECTIVE: The pair-wise addition of parahydrogen, the singlet form of molecular hydrogen, to unsaturated precursors evokes the hyperpolarization of two parahydrogen-derived 1H nuclear spins through a process known as parahydrogen-induced polarization (PHIP). Subsequent spin order transfer (SOT) from the 1H to the surrounding 13C nuclear spins via magnetic field cycling (MFC) results in substantial signal enhancement in 13C magnetic resonance imaging (MRI). Here, we report the development of a unique PHIP 13C hyperpolarizer system using a flow guide for MFC. METHODS: The optimal MFC scheme for 1H to 13C spin order transfer was quantum-chemically simulated using the J-coupling values of 13C-labeled metabolic tracers. The flow guide system was three-dimensionally designed based on the simulated MFC scheme and pre-measured magnetic field distribution in a zero-field chamber. RESULTS: The system efficiently transfers the spin order of hyperpolarized 1H to a particular 13C spin when the parahydrogenated tracer passes through the flow guide at a designated flow rate. The 13C MRI signal is enhanced more than 40,000 times in 13C-labeled pyruvate and fumarate, compared to the thermal equilibrium level at 1.5 T, was achieved for conducting in vivo metabolic MRI of mice. CONCLUSION: A fully automated PHIP-based 13C polarizer was developed using a unique flow guide to conduct the MFC for 1H to 13C SOT. SIGNIFICANCE: The PHIP hyperpolarizer with a flow guide can conduct efficient 1H-13C SOT without a MFC magnetic field sweep system and offers a cost-effective alternative to conventional dynamic nuclear polarization.

Novel Approach to the Construction of Fused Indolizine Scaffolds: Synthesis of Rosettacin and the Aromathecin Family of Compounds.

Camptothecin-like compounds are actively employed as anticancer drugs in clinical treatments. The aromathecin family of compounds, which contains the same indazolidine core structure as the camptothecin family of compounds, is also expected to display promising anticancer activity. Therefore, the development of a suitable and scalable synthetic method of aromathecin synthesis is of great research interest. In this study, we report the development of a new synthetic approach for constructing the pentacyclic scaffold of the aromathecin family by forming the indolizidine moiety after synthesizing the isoquinolone moiety. Thermal cyclization of 2-alkynylbenzaldehyde oxime to the isoquinoline N-oxide, followed by a Reissert-Henze-type reaction, forms the key strategy in this isoquinolone synthesis. Under the optimum reaction conditions for the Reissert-Henze-type reaction step, microwave irradiation-assisted heating of the purified N-oxide in acetic anhydride at 50 °C reduced the formation of the 4-acetoxyisoquinoline byproduct to deliver the desired isoquinolone at a 73% yield after just 3.5 h. The eight-step sequence employed afforded rosettacin (simplest member of the aromathecin family) at a 23.8% overall yield. The synthesis of rosettacin analogs was achieved by applying the developed strategy and may be generally applicable to the production of other fused indolizidine compounds.

Study on the Use of Ozone Water as a Chemical Decontamination Agent for Antineoplastic Drugs in Clinical Settings.

The exposure of healthcare workers to antineoplastic drugs in hospitals has been recognized to be harmful. To minimize the risk of exposure, the removal of these drugs from work environments, such as compounding facilities, has been recommended. In our previous paper, the degradation and inactivation efficacy of ozone water, which is being introduced into Japanese hospitals as a chemical decontamination agent, was reported for its effects on typical antineoplastic drugs (gemcitabine, irinotecan, paclitaxel). This article aims to further investigate the efficacy of ozone water for eight antineoplastic drugs to clarify its application limitations. A small amount (medicinal ingredient: typically ca. 1.5 µmol) of formulation containing 5-fluorouracil, pemetrexed, cisplatin, oxaliplatin, cyclophosphamide, ifosfamide, doxorubicin, or docetaxel was mixed with 50 mL of ozone water (~8 mg/L), and the resulting solutions were analyzed by high-performance liquid chromatography over time to observe the degradation. Consequently, the ozonation was overall effective for the degradation of the drugs, however this varied depending on the chemical structures of the drugs and additives in their formulations. In addition, after the parent drugs were completely degraded by the ozonation, the degradation mixtures were subjected to 1H nuclear magnetic resonance spectroscopy and evaluated for mutagenicity against Salmonella typhimurium strains and cytotoxicity against human cancer cells. The degradation mixtures of cisplatin and ifosfamide were mutagenic while those of the other drugs were non-mutagenic. Further, the ozonation resulted in clear decreases of cytotoxicity for 5-fluorouracil, oxaliplatin, and doxorubicin, but increases of cytotoxicity for pemetrexed, cisplatin, cyclophosphamide, and ifosfamide. These results suggest that the ozone water should be restrictedly used according to the situation of contamination in clinical settings because the ozonation enhances toxicity depending on the drug even if degradation is achieved.

Degradation and inactivation efficacy of ozone water for antineoplastic drugs in hospital settings.

PURPOSE: Occupational exposure to antineoplastic drugs in hospital settings is recognized to be hazardous, and as such environmental decontamination including degradation and inactivation of such drugs is recommended. To data, although various agents such as oxidants have been reported to be useful for decontamination, simpler, safer, and more convenient methods are required. In this study, the degradation and inactivation efficacy of ozone water, which has newly been introduced for decontamination of antineoplastic drugs in spills, was investigated for formulations of gemcitabine, irinotecan, and paclitaxel. METHODS: Antineoplastic formulations (medicinal ingredient: ∼1.5 µmol) were mixed with 50 mL of ozone water (>4 mg/L). The reactions were monitored by high-performance liquid chromatography, and the degradation mixtures were analyzed by 1H nuclear magnetic resonance spectroscopy in order to obtain the structural information of the degradation products. The formulations of gemcitabine and irinotecan and those degradation mixtures were evaluated for their mutagenicity using the Ames test and cytotoxicity against human cancer cells. RESULTS: gemcitabine and irinotecan were found to be readily degraded by the ozone treatment, and their active sites were suggested to be degraded. In contrast, paclitaxel was hard to be decomposed, possibly owing to the consumption of ozone by the polyoxyethylene castor oil added as a pharmaceutical additive of the formulation. No significant mutagenic changes of Salmonella typhimurium strains used for the Ames test were observed for the samples within the concentration ranges examined. The ozone treatment showed obvious increases in cell viability for gemcitabine formulation, and mild increases for irinotecan formulation. CONCLUSIONS: Ozone water was shown to be effective as a decomposition agent for the antineoplastic drug formulations examined, although the efficacy depends on the chemical structures of the drugs and the pharmaceutical additives. It was also suggested that ozone treatment has a tendency to decrease the toxicity of the antineoplastic drug formulations. As such, further studies are required in order to clarify the effects and application limitations of ozone water.

Total synthesis of pyrrolo[2,3-c]quinoline alkaloid: trigonoine B.

The first total synthesis of the pyrrolo[2,3-c]quinoline alkaloid trigonoine B (1) was accomplished via a six-step sequence involving the construction of an N-substituted 4-aminopyrrolo[2,3-c]quinoline framework via electrocyclization of 2-(pyrrol-3-yl)benzene containing a carbodiimide moiety as a 2-azahexatriene system. The employed six-step sequence afforded trigonoine B (1) in 9.2% overall yield. The described route could be employed for the preparation of various N-substituted 4-aminopyrroloquinolines with various biological activities.

Effects of Carbazole Derivatives on Neurite Outgrowth and Hydrogen Peroxide-Induced Cytotoxicity in Neuro2a Cells.

Many studies have demonstrated that oxidative stress plays an important role in several ailments including neurodegenerative diseases and cerebral ischemic injury. Previously we synthesized some carbazole compounds that have anti-oxidant ability in vitro. In this present study, we found that one of these 22 carbazole compounds, compound 13 (3-ethoxy-1-hydroxy-8- methoxy-2-methylcarbazole-5-carbaldehyde), had the ability to protect neuro2a cells from hydrogen peroxide-induced cell death. It is well known that neurite loss is one of the cardinal features of neuronal injury. Our present study revealed that compound 13 had the ability to induce neurite outgrowth through the PI3K/Akt signaling pathway in neuro2a cells. These findings suggest that compound 13 might exert a neurotrophic effect and thus be a useful therapy for the treatment of brain injury.

Long-range heteronuclear J-coupling constants in esters: Implications for 13C metabolic MRI by side-arm parahydrogen-induced polarization.

Side-arm parahydrogen induced polarization (PHIP-SAH) presents a cost-effective method for hyperpolarization of 13C metabolites (e.g. acetate, pyruvate) for metabolic MRI. The timing and efficiency of typical spin order transfer methods including magnetic field cycling and tailored RF pulse sequences crucially depends on the heteronuclear J coupling network between nascent parahydrogen protons and 13C, post-parahydrogenation of the target compound. In this work, heteronuclear nJHC (1 < n ≤ 5) couplings of acetate and pyruvate esters pertinent for PHIP-SAH were investigated experimentally using selective HSQMBC-based pulse sequences and numerically using DFT simulations. The CLIP-HSQMBC technique was used to quantify 2/3-bond JHC couplings, and 4/5-bond JHC ≲ 0.5 Hz were estimated by the sel-HSQMBC-TOCSY approach. Experimental and numerical (DFT-simulated) nJHC couplings were strongly correlated (P < 0.001). Implications for 13C hyperpolarization by magnetic field cycling, and PH-INEPT and ESOTHERIC type spin order transfer methods for PHIP-SAH were assessed, and the influence of direct nascent parahydrogen proton to 13C coupling when compared with indirect homonuclear TOCSY-type transfer through intermediate (non-nascent parahydrogen) protons was studied by the density matrix approach.

Concise synthesis and antiproliferative activity evaluation of ellipticine quinone and its analogs.

We developed a concise protocol for the synthesis of ellipticine quinone from the appropriate 3-iodoindole-2-carbaldehydes in four steps. The key step is the construction of carbazole-1,4-quinone through tandem Ring-Closing Metathesis (RCM) and dehydrogenation under oxygen atmosphere. Therefore, the ellipticine quinone analogs possessing substitution at the 8- and/or 9-positions were synthesized using this method. In total, 14 compounds were evaluated for antiproliferative activity against HCT-116 and HL-60 cell lines; 9-nitroellipticine quinone was found to have superior activity compared to calothrixin B.

Ni-Pd Catalyzed Cyclization of Sulfanyl 1,6-Diynes: Synthesis of 1'-Homonucleoside Analogues.

The Ni-Pd catalyzed addition-cyclization of sulfanyl 1,6-diynes 2-9 with nucleobases is described. The reactions of N-tethered 1,6-diynes with N3-benzoylthymine, N4,N4-bis(Boc)cytosine, N3-benzoyluracil and N6,N6-bis(Boc)adenine exclusively afforded the pyrrolylmethyl and furylmethyl nucleotides in good yields. Deprotection of nucleobases was completed by treatment with acids or bases. Furthermore, the reactions of pyrroles and furans with nucleophiles such as alkoxides and amines underwent detosylation and conversion to the alkoxymethyl- and arylaminomethyl-pyrroles and furans in good yields.

Concise synthesis of carbazole-1,4-quinones and evaluation of their antiproliferative activity against HCT-116 and HL-60 cells.

We report a convenient synthesis of carbazole-1,4-quinone alkaloid koeniginequinones A and B using a tandem ring-closing metathesis with the dehydrogenation reaction sequence under an O2 atmosphere as an important step. Using this method, carbazole-1,4-quinones substituted at the 5-, 6-, 7-, and/or 8-positions have been synthesized. Moreover, 24 compounds, including koeniginequinones A and B, have been evaluated for their antiproliferative activity against HCT-116 and HL-60 cells, and the 6-nitro analog exhibited the most potent activity against both tumor cell types.

Synthesis of Azepines via a [6 + 1] Annulation of Ynenitriles with Reformatsky Reagents.

A protocol for the direct synthesis of azepines using a hafnium(III)-catalyzed [6 + 1] annulation of N-tethered ynenitriles with Reformatsky reagents is reported. A broad range of 3-amino-2,7-dihydro-1H-azepine-4-carboxylates 4aa-4he were obtained in high yields and with excellent functional group tolerance. The copper-mediated reactions of isolable Blaise intermediates (enamino esters 3), uniquely underwent 5-endo cyclization to afford the ß-2,5-dihydropyrrolyl α,ß-unsaturated esters 5aa-5fc, which exhibit anticancer activity.

Antiproliferative activity of O4-benzo[c]phenanthridine alkaloids against HCT-116 and HL-60 tumor cells.

The O4-benzo[c]phenanthridine alkaloids exhibit potent antiproliferative activity against cancer cells, which is derived from their ability to inhibit of topoisomerase I and II. It has been reported that in the alkaloids a cationic quaternary ammonium atom, which results in resonance effects between ring A and B, is necessary for increased antiproliferative activity. These findings indicate the role of their substituents at ring A on inhibition of tumor cell proliferation. In the present study, we systematically assessed the cytotoxic activities of naturally occurring alkaloids and their derivatives containing various ring A substituents against two tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. Among the cationic iminium alkaloids, which displayed more potent activity than the corresponding neutral derivatives, and the 7,8-oxygenated benzo[c]phenanthridine alkaloids, chelerythrine and NK109, exhibited stronger antiproliferative activity than the 8,9- and 9,10-oxygenated alkaloids. The activity of cationic iminium alkaloids could be correlated with the bond lengths of their ring A substituents and the electrostatic potentials of their ammonium molecules by DFT calculation.

Antioxidant effects of the highly-substituted carbazole alkaloids and their related carbazoles.

Antioxidant activities of 3-oxygenated and 3,4-dioxygenated carbazole alkaloids and their related carbazoles were comprehensively evaluated. In all assay systems, the 3,8-dihydroxycarbazoles carbazomadurin A (2) and B (3), and their synthetic precursors 2a and 3a exhibited higher antioxidant activities than the 3-monohydroxycarbazoles carazostatin (1), and the synthetic precursors 4a and 4b of carquinostatin A (4). In particular, 2a and 3a exhibited strong scavenging activities due to the reducing ability of formyl group at the C-5 position of carbazoles. The results suggest that these compounds could serve as useful clues for designing and developing novel antioxidants.

Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells.

9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC50 values of these analogs were indicated over 10 µM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity.

Synthesis and antimalarial activity of calothrixins A and B, and their N-alkyl derivatives.

We synthesized calothrixin B using our developed biomimetic method and derived N-alkyl-calothrixins A and B. The in vitro antimalarial activity of the calothrixin derivatives, including calothrixins A and B, against the Plasmodium falciparum FCR-3 strain was evaluated. All test compounds exhibited antimalarial activity over a concentration range of 6.4×10(-6)-1.2×10(-7) M.

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca2+ pathway in a lipid raft-dependent manner.

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G(i/o)-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca(2+)-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP(3) formation and intracellular Ca(2+) mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca(2+) ionophore and the activation of a typical G(q)-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G(q)/phospholipase C activation and intracellular Ca(2+) mobilization in a lipid raft microdomain-dependent manner.

Cloning and functional identification of novel endothelin receptor type A isoforms in pituitary.

Mammalian endothelin (ET) receptors, termed ET(A)R and ET(B)R, are derived from two intron-containing genes and the functional splice variants of ET(B)R but not ET(A)R have been identified. Here, we report about the isolation of cDNAs of ET(A)R transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed ET(A)R-C13. In this splice variant, the C-terminal 382S-426N sequence of ET(A)R was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to G(q/11) and G(s) proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for ET(A)R-C13 were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant ET(A)R and ET(A)R-C13 were highly comparable, but the coupling of ET(A)R-C13 to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the potency for agonist. Furthermore, the efficacy of ET(A)R-C13 to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel ET(A) splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types.

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM.

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1.

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.

A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors.

To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.