Versatile Photorearrangement of Photocycloadducts from 5-Fluoro-1,3-dimethyluracil and Naphthalene

Direct UV-irradiation of 5-fluoro-1,3-dimethyluracil (5-FDMU) and naphthalene (1) with a 500 W high-pressure mercury lamp in a degassed Pyrex tube (λ > 300 nm) predominantly afforded benzopyrimidobarrelene derivative (2) through 1,4-addition, while irradiation in the presence of piperylene in singlet excited states preferentially afforded naphthocyclobuta-pyrimidine derivative (3) via 1,2-addition. Upon 254 nm light-irradiation of 2 gave rise to the formation of benzopyrimidosemibullvalene (4) in fair yields. The reaction pathway for the formation of 4 is reasonably explained in the terms of di-π-methane rearrangement. Adduct 3 was newly converted to the corresponding barrelene derivative (2) by long-wave-length irradiation in the presence of a triplet sensitizer

Preclinical investigation of potential use of thymidine phosphorylase-targeting tracer for diagnosis of nonalcoholic steatohepatitis

Introduction: Although liver biopsy is the gold standard for the diagnosis of nonalcoholic steatohepatitis (NASH), it has several problems including high invasiveness and sampling errors. Therefore, the development of alternative methods to overcome these disadvantages is strongly required. In this study, we evaluated the potential use of our tracer targeting thymidine phosphorylase (TYMP),5-[I-123]iodo-6-[(2-iminoimidazolidinyl)methylluracil ([I-123]IIMU) for the diagnosis of NASH. Methods: The mice used as the NASH model (hereafter, NASH mice) were prepared by feeding a methionine- and choline-deficient diet for 4 weeks. A control group was similarly given a control diet. The expression levels of the TYMP gene and protein in the liver were examined by real-time reverse-transcription polymerase chain reaction and western blot analyses. The localizations of [I-125]IIMU and the TYMP protein in the liver were examined by autoradiography and immunohistochemical staining, respectively. Finally, the mice were injected with [I-123] IIMU and single-photon emission tomography (SPECT) imaging was conducted. Results: The hepatic expression levels of TYMP were significantly lower in the NASH mice than in the control mice at both mRNA and protein levels, suggesting that a decrease in TYMP level could be an indicator of NASH. [I-125] IIMU was uniformly distributed in the liver of the control mice, whereas it showed a patchy distribution in that of the NASH mice. The localization of [ I-125]IIMU was visually consistent with that of the TYMP protein in the liver of the control and NASH mice. SPECT analysis indicated that the hepatic accumulation of [I-123]IIMU in the NASH mice was significantly lower than that in the control mice ISUV (g/ml): 4.14 +/- 0.87 (Control) vs 2.31 = 029 (NASH)]. Conclusions: [I-12(3)]IIMU may provide a noninvasive means for imaging TYMP expression in the liver and may be applicable to the diagnosis of NASH. (C) 2019 Published by Elsevier Inc

Isolation and identification of new vasodilative substances in diesel exhaust particles

Goal, Scope, and Background: We recently developed a new isolation method for diesel exhaust particles (DEP), involving successive extraction with H2O, sodium bicarbonate, and sodium hydroxide, in which the sodium hydroxide extract was found to consist of phenolic components. Analysis of the extract revealed that vasodilative active nitrophenols are in DEP in significantly higher concentration than those estimated by an earlier method involving a combination of solvent extraction and repeated chromatography. These findings indicated that our new procedure offers a simple, efficient and reliable method for the isolation and identification of bioactive substances in DEP. This encouraged us to extend our work to investigating new vasodilatory substances in the sodium bicarbonate extract. Materials and Methods: DEP were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). GC-MS analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). Results: DEP dissolved in 1-butanol was successively extracted with water, sodium bicarbonate, and then aqueous sodium hydroxide. The sodium bicarbonate extract was neutralized and the resulting mixture of acidic components was subjected to reverse-phase (RP) column chromatography followed by RP-HPLC with fractions assayed for vasodilative activity. This led to the identification of terephthalic acid, p-hydroxybenzoic acid, isophthalic acid, phthalic acid, 3-hydroxy-4-nitrobenzoic acid, 4-hydroxy-3-nitrophenol, and 1,4,5-naphthalene tricarboxylic acid as components of DEP. Discussion: The sodium bicarbonate extract was rich in the acidic components. Repeated reverse-phase chromatography resulted in the successful isolation of several acidic substances including the new vasodilative materials, 4-hydroxy-3-nitrobenzoic acid, and 3-hydroxy-4-nitrobenzoic acid. Conclusion: Our new fractionation method for DEP has made possible the isolation of new vasodilative compounds from the sodium bicarbonate extract

Radiolabeled uracil derivative as a novel SPECT probe for thymidine phosphorylase : suppressed accumulation into tumor cells by target gene knockdown

Objectives: We developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU), as a novel SPECT probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP, and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of 125I-labeled IIMU (125I-IIMU) in the tumor cells. Methods: TP-specific siRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 hours after transfection, respectively. The cellular uptake level of 125I-IIMU was also evaluated 72 hours after transfection. The results were compared after normalization with the corresponding negative controls. Results: After TP- and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA significantly decreased to 41% and 29%, respectively, as compared with the negative control (p < 0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34% and 30%, respectively (p < 0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of 125I-IIMU significantly decreased to 66% (p < 0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of 125I-IIMU. Conclusion: siRNA-mediated TP knockdown significantly decreased the cellular uptake level of 125I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP-specific and directly corresponds to TP expression levels

Relationship between biodistribution of a novel thymidine phosphorylase (TP) imaging probe and TP expression levels in normal mice

Objective: Thymidine phosphorylase (TP) is a key enzyme in the pyrimidine nucleoside salvage pathway and its expression is upregulated in a wide variety of solid tumors. In mice, we previously observed high and specific accumulation levels of our TP imaging probe, radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) not only in high-TP-expressing tumors, but also in the liver and small intestine. To clarify the reason for the high accumulation levels of radioiodinated IIMU in the liver and small intestine, we investigated the expression levels of TP in mice in comparison with the biodistribution of radioiodinated IIMU (123I-IIMU). Methods: BALB/cCrSlc mice were injected with 123I-IIMU, and the radioactivity levels [%ID/g (normalized to a mouse of 25 g body weight)] in the tissues of interest were determined 0.5, 1, 3 and 24 h after the injection (n = 5, each time point). To determine the expression levels of TP, BALB/cCrSlc and ddy mice (n = 3/each strain) were euthanized, and the heart, liver, lung, spleen, kidney, stomach, small intestine, large intestine and brain were collected. The mRNA and protein expression levels of TP in these organs were examined by quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. Results: In BALB/cCrSlc mice administered 123I-IIMU, markedly high radioactivity levels were observed in the liver [1.568 ± 0.237 (%ID/g)] and small intestine [0.506 ± 0.082 (%ID/g)], whereas those in the other tissues were fairly low [<0.010 ± 0.003 (%ID/g)] 30 min after the injection. The highest expression levels of TP mRNA were also observed in the liver and small intestine among the tissues tested. Immunoblotting showed intense immunoreactive bands of the TP protein for the liver and small intestine, whereas no notable bands were detected for other tissues. Similar expression profiles of TP mRNA and protein were observed in ddy mice. Conclusion: We confirmed TP expression in various tissues of mice at the mRNA and protein levels: high TP expression levels were observed in the liver and small intestine. These high TP expression levels are consistent with the high accumulation levels of 123I-IIMU in these tissues. Our results may provide important information about the physiological accumulation of 123I-IIMU, which may be useful for the clinical diagnostic imaging of TP

Radiolabeled uracil derivative as a novel SPECT probe for thymidine phosphorylase

Objectives: We developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU), as a novel SPECT probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP, and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of 125I-labeled IIMU (125I-IIMU) in the tumor cells. Methods: TP-specific siRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 hours after transfection, respectively. The cellular uptake level of 125I-IIMU was also evaluated 72 hours after transfection. The results were compared after normalization with the corresponding negative controls. Results: After TP- and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA significantly decreased to 41% and 29%, respectively, as compared with the negative control (p < 0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34% and 30%, respectively (p < 0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of 125I-IIMU significantly decreased to 66% (p < 0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of 125I-IIMU. Conclusion: siRNA-mediated TP knockdown significantly decreased the cellular uptake level of 125I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP-specific and directly corresponds to TP expression levels