Synthesis of 1-[(O,O-diphenyl phosphonyl)arylmethyleneamino carbonylmethyl]uracils

A new series of compounds, 1-[(O,O-diphenyl phosphonyl)arylmethylene aminocarbonylmethyl]uracils, were synthesized in yield of 54.6-72.0% with DCC/BtOH as the coupling reagent, and their biological activities are being tested.

Benign perfluoroalkylation of uracils and uracil nucleosides via visible light-induced photoredox catalysis

In this work, an efficient and facile method for the preparation of 5-perfluoroalkylation uracils and uracil nucleosides through visible-light-mediated reaction has been developed. The reaction processes in high efficiency under mild reaction conditions and show broad substrate scope by employing commercial available perfluoroalkyl sources, thus demonstrates high potent application in life and medicinal science.

Biochemical Characterization of Uracil-DNA Glycosylase from Pyrococcus furiosus

We report the characterization of a uracil-DNA glycosylase（UDG） from the hyperthermophilic archaea Pyrococcus furiosus（P, furiosus）. P. furiosus UDG（PfUDG） has high sequence similarity to the families IV and V UDGs（thermostable UDG family and PaUDG-b family）. PfUDG excises uracil from various DNA substrates with the following order： U/T=U/C〉U/G=U/AP=U/-〉U/U=U/I=U/A. The optimal temperature and pH value for uracil exci- sion by PfUDG are 70 ℃ and 9.0, respectively. The removal of U is inhibited by the divalent ions of Fe, Ca, Zn, Cu, Co, Ni and Mn, as well as a high concentration of NaC1. The phosphorothioates near uracil strongly inhibit the exci- sion of uracil by PfUDG. Interestingly, pfuDNA（Pyrococcusfuriosus DNA） polymerase, which tightly binds the ura- cil-carrying oligonucleotide, does not inhibit the excision by Pfl.IDG, suggesting PfUDG in vivo functions as the re- pair enzyme to excise uracil damage in genome.

Regiospecific Addition of Uracil to Acrylates Catalyzed by Alkaline Protease from Bacillus subtilis

Michael addition reactions of uracil to acrylates were catalyzed by an alkaline protease from Bacillus subtilis in dimethyl sulfoxide at 55 ℃ for 72 h. The adducts were determined by TLC, IR and 1H NMR.

Synthesis of Novel Nucleoside Analog (3R)-2,3-Dideoxy-3- (N-hydroxy-N-methylamino)-L-arabinofuranosyl Uracil

The synthesis of novel nucleoside analog (3R)-2,3-dideoxy-3-(N-hydroxy-N- methylamino)-L-arabinofuranosyl uracil was studied. A twelve-step synthetic route, started from L-ascorbic acid, was designed, and the final product was obtained in 20.8% yield.

The facile synthetic method of 5-hydroxymethyluracil

One practical and industrial procedure for preparation of 5-hydroxymethyluracil has been developed. This method has the advantage of facile operation, low cost and stable yield.

A Novel Photoproduct of Uracil in Phosphate-Buffered Saline

The photolysis of uracil in phosphate-buffered saline (PBS, pH 8.0) under the irradiation pf medium pressure mercury lamp (MPML) leads to the production of a novel compound C4H5N2O6P. The composition and structure of the compound has been identified by elemental analysis, EI-MS, UV, IR, H-1, C-13, P-31-NMR.

Efficient Utilization of 6-Aminouracil to Synthesize Fused and Related Heterocyclic Compounds and Their Evaluation as Prostate Cytotoxic Agents with Cathepsin B Inhibition

6-aminouracil 1 was utilized to introduce different heterocyclic rings at C-6 position through various synthetic strategies. The synthesized compounds bear rings that are either directly attached to the uracil back bone as in compounds 6, 12a-c and 15, or attached through an amino bridge as compounds 3a-c, 5a, b, 7a, b, 9 and 10, or through an imino bridge as compound 18. Also, compounds 4, 8, 11a-c, 14, 16 and 17 bearing biologically active side chains were synthesized. In addition to, compounds 13, 19, 20, 21 and 22 bear fused rings to the uracil backbone. All synthesized compounds were evaluated for their anticancer activity against prostate PC3 cell line using in-vitro sulforhodamine-B (SRB) method, from which compounds 3a, c, 4, 5a, b, 6, 7a, b, 11a-c, 12a, b, 17 and 20 were the most active. These active compounds were further evaluated for their ability to inhibit cathepsin B enzyme by using enzyme-linked immunosorbent assay, which revealed that compounds 5a, b, 7a, 11a, 12a and 17 exhibited more than 50% inhibition of cathepsin B. Among which the phenyl thiourea derivative 17 was the most active exhibiting 82.3% inhibition, while the reference doxorubicin exerted 18.7% inhibition.

Feasibility and Efficacy Study of Biweekly Irinotecan Combined with Oral Tegafur/Uracil in Advanced Colorectal Cancer

Background: We evaluated the feasibility and efficacy of irinotecan (CPT-11) plus tegafur/uracil (UFT) combination chemotherapy in patients with advanced colorectal cancer. Patients and Methods: PK parameters were concurrently measured to confirm the presence of drug interactions in this treatment schedule. CPT-11 was administered intravenously at the dose of 150 mg/m2 on days 1, 15. UFT was administered at the dose of 375 mg/m2/day (B.I.D.) on days 3 - 7, 10 - 14, 17 - 21, 24 - 28 repeated every 5 weeks. Results: 31 patients were enrolled. PK parameters for CPT-11, FT, 5-FU and uracil are available from 5 patients. The overall response rate was 16.1%. The median time to treatment discontinuation was 3.9 months. There was no significant difference in PK parameters of CPT-11 between day 1 and day 15 and of UFT between day 3 and day 10. Conclusion: CPT-11 plus UFT combination chemotherapy exhibited a tolerable toxicity profile with acceptable efficacy. Pharmacokinetic analysis showed that there were no drug interactions in this treatment schedule.

A Case of Advanced Multiple Hepatocellular Carcinomas with Portal Vein Tumor Thrombosis Successfully Treated by Oral Tegafur/Uracil

A case of advanced multiple hepatocellular carcinomas (HCC) with portal vein tumor thrombosis successfully treated by oral tegafur/uracil is reported. A 69-year-old Japanese woman with advanced HCC with tumor thrombosis underwent transcatheter arterial infusion chemotherapy in April 2001. However, 1 year later, the patient experieced a recurrence with advanced multiple HCC with portal vein tumor thrombosis and ascites. Treatment with oral tegafur/uracil was started in May 2002 and resulted in the partial response of liver tumors and the complete improvement of ascites. She remained in good health for about 6 years. This case strongly suggests that oral tegafur/uracil is an effective treatment for some cases of advanced HCC with portal vein tumor thrombosis.

A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter(pAOX1).However,it is difficult to use P.pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination(HR)efficiency and the limitation of handy selective markers,especially in the condition of multistep biosynthetic pathways.Hence,we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker(HiEE-ReSM)to facilitate cell factory in P.pastoris.Firstly,we improved the HR rates of P.pastoris through knocking out the non-homologous-end-joining gene(Δku70)and overexpressing HR-related proteins(RAD52 and RAD59),resulting in higher positive rate compared to the basal strain,achieved 97%.Then,we used the uracil biosynthetic genes PpURA3 as the reverse screening marker,which can improve the recycling efficiency of marker.Meanwhile,the HR rate is still 100%in uracil auxotrophic yeast.Specially,we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter(scFUR4)to increase the uptake of exogenous uracil from medium.Meanwhile,we explored the optimal concentration of uracil(90 mg/L)for strain growth.In the end,the HiEE-ReSM system has been applied for the inositol production(250 mg/L)derived from methanol in P.pastoris.The systems will contribute to P.pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon(C1)bio-utilization.

Identification of the Decay Pathway of Photoexcited Nucleobases

The identification of the decay pathway of the nucleobase uracil after being photoexcited by ultraviolet light has been a long-standing problem.Various theoretical models have been proposed but yet to be verified.Here,we propose an experimental scheme to test the theoretical models of gas phase uracil decay mechanism by a combination of ultrafast x-ray spectroscopy,x-ray diffraction,and electron diffraction methods.Incorporating the signatures of multiple probing methods,we demonstrate an approach that can identify the dominant mechanism of the geometric and electronic relaxation of the photoexcited uracil molecule among several candidate models.

Rapid Analysis of Polar Components in Ophiocordyceps sinensis by Conventional Liquid Chromatography System

Objective To develop a rapid high-performance liquid chromatography coupled with diode array detection （HPLC-DAD） method for the simultaneous determination of six polar compounds in Ophiocordyceps sinensis. Methods A poroshell SB Aq column （50 mm × 4.6 mm, 2.7 μm） and gradient elution were used; The detection wavelength of compounds was set at 260 nm. The chromatographic peaks of the six investigated compounds in sample were identified by comparing their retention times with reference compounds. Results All calibration curves showed good linearity （r〉 0.999） within the tested ranges. The intra- and inter-day precisions of the six analytes were less than 0.8% and 2.1%, respectively, and the recoveries of the six analytes were between 95% and 103%. The validated method was successfully applied to the determination of six polar compounds in O. sinensis samples. Conclusion The poroshell SB Aq column is suitable for the rapid analysis of polar components in Chinese materia medica on conventional HPLC system and the developed HPLC method is also helpful to the quality control of O. sinensis.

Pharmacokinetics and the bystander effect in CD：：UPRT/5-FC bi-gene therapy of glioma

Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides （F-Nuctd）. For the C6 mixture, only the 5-FC peak was detected. In vivo serial ^19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD：：UPRT cells, the survival rate was （79.55±0.88）% （P 〈0.01）. As the C6-CD：：UPRT ratio increased, the survival rate of the cells decreased. ^19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD：：UPRT in the mixture increased. Conclusions ^19F-MRS studies indicated that C6-CD：：UPRT cells could effectively express CD and UPRT enzymes. The CD：：UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD：：UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and ^19F-MRS can monitor transferred CD：：UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.

Synthesis and Herbicidal Activities of Novel Uracil Derivatives Containing Pyrimidinyl Moiety

In order to find novel protoporphyrinogen oxidase inhibitors with high efficacy, broad-spectrum activity, and safety to crops, nine title compounds（4a-4i） were designed and synthesized by introducing pyrimidine moiety into the uracil skeleton with commercially herbicide butafenacil as the lead compound. Their structures were con- firmed by 1H NMR, IR, mass spectroscopy and elemental analysis. The bioassay results indicate that most of com- pounds 4 tested exhibit good to excellent herbicidal activities against B. campestris, A. retroflexus, E. crusgalli and D. sanguinalis in pre-emergence treatment at a dose of 1.5 kg/ha（1 ha=10^4 m^2）, for example, compound 4i showed 100% inhibition against the four plants tested in pre-emergence treatment at a dose of 1.5 kg/ha. So, this type of skeleton can be used as a valuable lead compound for the further development of a pre-emergent herbicide.

Synthesis and Biological Evaluation of Novel Uracil and 5-Fluorouracil-l-yl Acetic Acid-Colchicine Conjugate

A series of novel uracil and 5-fluorouracil-l-yl-acetic acid-colchicine derivatives（6a--6n） was synthe- sized via coupling and 5-fluorouracil（5-FU） with C-10 analogues of colchicine. The antitumor activities of the target compounds against human hepatocellular earcinoma（BEL7402） cells, human ovary carcinoma（A2780） cells, human lung adenocarcinoma（A549） cells and human breast carcinoma（MCF7） cells were tested in vitro, and the structure-activity relationship（SAR） of the compounds was also studied. The bioassay results demonstrate that most of the tested compounds display significant activity and particularly, compounds 6a, 6e, 6h and 61 show more potent cytotoxic activities than 5-fluorouracil and colchicine. The results show that the new derivatives of colchicine are potential suppressors on human cancer.