Microbiological synthesis of 5-ethyl- and (E)-5-(2-bromovinyl)-2′-deoxyuridine

Summary The title compounds were prepared by an enzymatic transdeoxyribosylation from 2 dGuo or 2 dThd to the respective heterocyclic bases, 5-ethyluracil and (E)-5-(2-bromovinyl)uracil, using the whole bacterial cells ofEscherichia coli as a biocatalyst.     

Reaction of Azide Ion with 1-(2,3-Anhydrolyxofuranosyl)Uracil. Isolation of 1-(2-Amino-2-deoxy-β-D-Xylo-Furanosyl)uracil and 1-(3-Amino-3-deoxy-β-D-arabinofuranosyl)uracil

Abstract

The reaction of the 2′,3′-lyxoepoxide (1) with ammonium azide gives two products; namely, the 3′-arabino azide (2a) and in low yield 2′-xylo azide (3a). After debenzoylation and reduction the resulting mixture of amines was resolved by chromatography on a weak cation exchanger, Amberlite IRC-50, and afforded crystalline 1-(3-amino-3-deoxy-β-D-arabinofuranosyl)uracil (2c) and 1-(2-amino-2-deoxy-β-D-xylofuranosyl)uracil (3c) in the ratio of 4:1.     

Synthesis and Antiviral Activity Assay of Novel (E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine

Abstract

(E)-3′,5′-diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Photobromination of 1,5-Anhydro-2,3-O-isopropylidene-β-d-ribofuranose and Synthesis of (5R) and (5S)-(5-2H1)-d-Riboses

The photobromination of 1,5-anhydro-2,3-O-isopropylidene-β-d-ribofuranose gave the corresponding (5S)-5-bromo compound. The reduction of the bromide with triphenyltindeuteride gave (5S)-(5-²H₁)-1,5-anhydro-2,3-O-isopropylidene-β-d-ribofuranose, with a chiral purity of 76% at C-5, which was converted to (5R)- and (5S)-(5-²H₁)-d-riboses and other ribofuranose derivatives.     

Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine and 5-(hydroxymethyl) uracil in Smokers

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54±2.07 vs. 6.70±1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16±0.35 vs. 1.35±0.50 nmol/mmol creatinine, p=0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r_p=0.44), BMI (r_p=-0.27) and nicotine derivatives (r_p=0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r_p=0.36), nicotine derivatives (r_p=0.29), BMI (r_p=-0.24) on the other.     

Therapeutic Implications of The Unusually Long Half-Life of (E)-5–(2–bromovinyl)uracil (Bvura) In Vivo

Abstract

The long half-life of BVUra in the plasma permits, on the one hand, the de novo formation of the antiviral drug (E)-5–(2-bromovinyl)-2′-deoxyuridine (BVdUrd) by a pentosyl transfer, and, on the other hand, the inhibition of the degradation of pyrimidine bases such as 5-fluorouracil (FUra).     

Derivatives of 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-phenyluracil and 5-Benzyluracil. Synthesis and Biological Properties

Abstract

A number of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil and -cytosine nucleosides substituted at the 5 position with a nitrophenyl or nitrobenzyl group were synthesized from 5-phenyl- and 5-benzyluracil via condensation of the fluorinated sugar, followed by nitration. The corresponding amino analogues were also prepared by reduction of the nitro nucleosides. The uracil nucleosides were converted into the corresponding cytosine nucleosides by way of the triazole intermediates. None of these nucleosides exhibited significant activity against herpes simplex virus type 1 in Vero cells. However, cytosine nucleosides containing the o-nitrophenyl, p-nitrophenyl, p-nitrobenzyl or p-aminobenzyl substituent were found to be toxic (even at 1 μM) to uninfected Vero cells, although they were essentially nontoxic in HL-60 cells. The 5′-monophosphates of the uracil nucleosides were inhibitors of the reaction catalyzed by purified Ehrlich ascites carcinoma thymidylate synthase, the 5-phenyluracil nucleotides causing a strong inhibition, competitive vs dUMP, described by the K_i value of 0.01 μM.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

High-performance liquid chromatographic determination of (S)- and (R)-propanolol in human plasma and urine with a chiral β-cyclodextrin bonded phase

The determination of propanolol enantiomers in microsamples of human plasma and urine by HPLC using a chiral stationary phase is described. After extraction from 200 μl of plasma or urine with racemic alprenolol as internal standard (I.S.), the enantiomers are separated on a β-cyclodextrin column with a polar organic mobile phase and determined by fluorescence detection. The retention times of I.S. and propranolol enantiomers are about 12–13 min and 16–18 min, respectively. Peak resolutions are 1.4 for I.S. and 2.2 for propranol. The use of alprenolol as I.S. improves significantly the coefficients of variation (C.V.: 0.6–4.2%). Sensitivity is approximately 1.5 ng/ml per propranolol enantiomer. The assay is applied to pharmacokinetic studies of racemic propranolol in human biological fluids. The (S)-propranolol levels are always higher than the (R)-antipode concentrations in plasma and urine.     

2′-(E)-O-p-coumaroylgalactaric acid and 2′-(E)-O-feruloylgalactaric acid in citrus

2′-(E)-O-p-Coumaroyl- and 2′-(E)-O-feruloylgalactaric acids, hitherto unknown in nature, have been isolated and identified from orange peel.     

Synthesis of 1-[3-deoxy-β-D-psicofuranosyl]uracil and related compounds

D-Fructose affords on treatment with cyanamide 2-amino-beta-D-fructofuro[2',3':3,4]-oxazoline which is not isolated but transformed directly by the reaction with ethyl propiolate into O(2,3)-anhydro-2-[beta-D-fructofuranosyl]uracil. This compound is benzoylated to the 1',4',6'-tri-O-benzoyl derivative by the action of benzoyl cyanide and triethylamine. On treatment with hydrogen chloride/dimethylformamide, the latter intermediate is converted to the 1-[1,4,6-tri-O-benzoyl-3-chloro-3-deoxy-beta-D-psicofuranosyl]uracil 1',4',5'-tribenzoate from which the title nucleoside derivative is obtained by methanolysis.     

High-performance liquid chromatographic determination of (−)-β-d-2,6-diaminopurine dioxolane and its metabolite,dioxolane guanosine,using ultraviolet and on-line radiochemical detection

(−)-β-d-2,6-Diaminopurine dioxolane (DAPD) and its metabolite dioxolane guanosine (DXG) have potent activity against hepatitis B virus and HIV, in vitro. A reversed-phase HPLC analytical method using UV and on-line radiochemical detection for the determination of DAPD and DXG in monkey serum and urine is described in this report. Retention times for DXG, DAPD and internal standard (2′,3′-didehydro-2′ deoxythymidine, D4T) were 5.0, 6.0 and 13.0 min, respectively. The extraction recovery was greater than 97% for DAPD and 94% for DXG. The limit of quantitation for UV detection was 100 ng/ml and 125 ng/ml for DXG and DAPD in monkey serum. The standard curves were linear from 0.1 μg/ml to 5 μg/ml for DXG and 0.125 μg/ml to 5 μg/ml for DAPD. For radiochemical detection, calibration curves of standard solutions of DAPD and DXG were linear in the range of 3500 Bq to 32 000 Bq and 7500 Bq to 60 000 Bq. The intra- and inter-day relative standard deviations were less than 7.2% using UV and less than 8.6% using on-line radiochemical detection. The HPLC method was applied to serum and urine samples collected from a male rhesus monkey that was administered 33.3 mg/kg DAPD with 200 μgCi of [³H]DAPD intravenously.     

Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).     

Mass fragmentographic determination of prostaglandin F2α in human and rabbit urine

Analysis of prostaglandin F_2α (PGF_2α) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF_2α analysis in human urine was developed using [3,3,4,4-²H₄]PGF_2α as an internal standard and carrier. PGF_2α was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatograph—mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 ± 2.6 (S.D.) ng/ml or 4.1 ± 1.0 ng PGF_2α per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 ± 2.7 (S.D.) ng/ml or 3.7 ± 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF_2α originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF_2α in rabbit urine.     

Synthesis of 1-(4-Keto-2, 3-O-isopropylidene-α-L-rhamnopyranosyl) uracil

Abstract

Synthesis of 1-(2, 3, 4-tri-0-acetyl-α-L-rhamnopyranosyl) uracil (3), 1-(α-L-rhamnopyranosyl) uracil (4), 1-(2, 3-0-isopropylidene-α-L-rhamnosyl) uracil (5), and 1-(2, 3-0-isopropylidene-4-keto-α-L-rhamnopyranosyl) uracil (6) are reported. Oxidation of (5) to (6) was effected using pyridinium chlorochromate in presence of molecular sieves.     

Synthesis and screening of (E)-1-(β-D-galactopyranosyl)-4-(aryl)but-3-ene-2-one against Mycobacterium tuberculosis

A series of galactose-derived aryl enones were synthesised and screened against Mycobacterium tuberculosis H₃₇Rv. Preliminary results were promising with MIC values in the range 1.56-12.5 μg/mL.     

Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

(E)-β-Famesene(EβF) synthase catalyses the production of EβF,which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies.Many plants possess EβF synthase genes and can release EβF to repel aphids.In order to effectively recruit the plant-derived EβF synthase genes for aphid control,by using chloroplast transit peptide(CTP) of the small subunit of Rubisco(rbcS) from wheat(Triticum aestivum L.),we targeted AαβFS1,an EβF synthase gene from sweet wormwood(Artemisia annua L),to the chloroplast of tobacco to generate CTP + AαβFS1 transgenic lines.The CTP +AαβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues,4-12 fold higher than the AαβFS1 transgenic lines without chloroplast targeting.Furthermore,aphid/parasitoid behavioral bioassays demonstrated that the CTP + AαβFS1 transgenic tobacco showed enhanced repellence to green peach aphid(Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae,thus affecting aphid infestation at two trophic levels.These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.     

Validation and application of an LC–ESI-MS method for simultaneous determination of astilbin and its major metabolite 3′-O-methylastilbin in rat plasma

We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C₁₈ column (150 mm × 2.0 mm I.D., 5 μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r > 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.