Synthesis and antiviral activity of 7-O-(omega-substituted)-alkyl-3-O-methylquercetin derivatives.

3',4'-Di-O-benzyl-3-O-methylquercetin (2), the precursor in the synthesis of an important antivirally active flavone 3-O-methylquercetin (1), was regioselectively alkylated at the 7-OH position by a series of 1,omega-dihaloalkanes and omega-bromoalkanols. Dimerization of the flavone had to be avoided by applying strict reaction conditions. Subsequent debenzylation was carried out by catalytic transfer hydrogenolysis, affording quantitatively the 7-O-(omega-haloalkyl)-3-O-methylquercetin (11-14) and 7-O-(omega-hydroxyalkyl)-3-O-methylquercetin derivatives (15, 16). All compounds were tested for their antiviral activity against poliomyelitis- and HIV-viruses.

Quality assessment in in vivo NMR spectroscopy: VI. Multicentre quantification of MRS test signals.

In this paper the results are presented of a joint European quantitative data-analysis study on a series of in vivo NMR time-domain test signals. The purpose of this study was to investigate, whether the quantitative MRS results from the various European biomedical research-centres can be made more comparable and reproducible. From the results of the study it can be concluded that in case of heavily overlapping NMR lines the quantifications should be done by some form of model function fitting and that, whenever possible, prior knowledge on the quantitative parameters should be incorporated into the quantification algorithms.

Complementary use of T2-weighted and postcontrast T1- and T2*-weighted imaging to distinguish sites of reversible and irreversible brain damage in focal ischemic lesions in the rat brain.

The evolution of a photochemically induced cortical infarct was monitored using T2-, postcontrast (GdDOTA) T1-, and postcontrast (DyDTPA-BMA) T2*-weighted NMR imaging techniques. Data acquired with these different NMR imaging types were compared, both qualitatively and quantitatively. The T2*-weighted NMR images after spordiamide injection (DyDTPA-BMA) were perfusion-weighted images that allowed the differentiation between several infarct-related areas in terms of different degrees of perfusion deficiency. No quantitative information on cerebral blood flow (CBF) was obtained. A clear distinction was made between areas with a complete lack of CBF located in the core of the lesion and temporary CBF insufficiencies in the rim surrounding this core. Concomitant observations on T2-weighted and postcontrast T1-weighted images revealed the same temporary rim characterized by an increased water content, and an intact blood-brain barrier (BBB), as well as by reduced perfusion. This rim appeared within the first hours after infarct induction, reached a maximum 24 h later, and lasted between 3-5 days, when its size gradually decreased until complete disappearance. These observations suggest the existence of an area at risk. Only on postcontrast T1-weighted images, the core of the lesion remained visible during the whole experimental period (10 days) and reflected in all likelihood the irreversibly damaged ischemic central core. The combined application of different NMR imaging techniques when studying focal cerebral infarctions in the rat brain allowed us to distinguish, in terms of NMR characteristics, zones of reversible from irreversible brain damage and to estimate the severity of the damage. This might offer an appropriate experimental setup for the screening of cerebroprotective compounds.

Complementary use of T2- and postcontrast T1-weighted NMR images for the sequential monitoring of focal ischemic lesions in the rat brain.

The noninvasive nature of NMR imaging enables serial studies on a single animal. In 12 male Wistar rats, the dynamic progression of a photochemically induced (Rose Bengal) infarct was studied starting immediately after induction and up to 10 days. The results demonstrated that both T2- and postcontrast T1-weighted NMR images are required to discern the time dependent dynamics of the ischemic process. The ischemic lesion was already visible on T2-weighted images within 30 min after the induction. Twenty-four hours after the insult, both area and intensity reached maximum values. Hereafter and up to day 10, both parameters decreased. Postcontrast T1- weighted images revealed a blood-brain barrier (BBB) rupture immediately after the induction which persisted until 10 days after the insult. The application of contrast agents such as Gd-DOTA or Gd-HP-DO3A also allowed the detection of 10-day-old lesions which were not always discernable on T2-weighted images. The penetration of both contrast agents in the affected area proceeded slower the first 5 days after the insult while at day 10, maximum contrast enhancement was reached almost immediately after administration of the contrast agent. At 24 hr after the insult, the discrepancy between the lesion area as determined on T2-weighted images and on postcontrast T1-weighted images was maximal. At this stage, the lesion was characterized by central core with a leaky BBB surrounded by a reversible zone which appeared enhanced on T2-weighted images.

Noninvasive in vivo 13C-NMR spectroscopy in the rat to study the pharmacokinetics of 13C-labeled xenobiotics.

Noninvasive NMR methodology has been developed to enable monitoring of 13C-labeled xenobiotics in the rat in vivo. 2,2-Dichloro-1-(2-chlorophenyl)-1-(4-chlorophenyl)-[3-13C]-propane can be detected in the liver of intact rats by in vivo 13C surface coil NMR spectroscopy after ip administration of the compound. The experiments were performed at 1.9 and 9.4 Tesla. The intrahepatic changes of the signal intensity of the labeled compound were followed as a function of time. In the days following administration, the concentration decreased and dropped to values below the detection limit after 12 days. The study demonstrates the feasibility of studies on pharmacokinetics of 13C-labeled compounds in the rat using noninvasive, in vivo surface coil NMR spectroscopy in animals. The sensitivity allows the detection of a single dose of the drug of 200 mg/kg, but can be improved.

Proton relaxation enhancement by means of serum albumin and poly-L-lysine labeled with DTPA-Gd3+: relaxivities as a function of molecular weight and conjugation efficiency.

A series of poly-L-lysine chains, with molecular weight ranging from 3300 up to 102,000 Da, were labeled with DTPA-Gd3+. No significant differences in longitudinal and transversal relaxivity, could be demonstrated as a function of the chain length. The R1 and R2 relaxivities were respectively 2.5 and 5 times superior to those of plain DTPA-Gd3+ (at 2.4 T). Bovine serum albumin was also labeled in a way that a wide (DTPA-Gd3+)/BSA range (3-39) was obtained. The longitudinal relaxivity, of these paramagnetically labeled albumins, increased with increasing (DTPA-Gd3+)/BSA ratios. This effect was most pronounced at very low (DTPA-Gd3+)/BSA ratios.

Noninvasive in vivo 13C-NMR spectroscopy of a 13C-labeled xenobiotic in the rat.

This study demonstrates that the xenobiotic product, 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloro-3-13C-propane can be monitored in the liver of an intact animal by in vivo 13C surface coil NMR spectroscopy after intraperitoneal administration. The carbon-13 label could be detected after a single dose of only 200 mg/kg of the product. The intrahepatic changes of the signal intensity of the labeled product were monitored as a function of time. No signals corresponding to metabolites could be detected.

High resolution NMR imaging: Gd-DTPA labeled enzyme as a probe for permeability studies in polyacrylamide gels.

The penetration of horse liver alcohol dehydrogenase (HLAD) molecules into polyacrylamide gel beads, which are used to immobilize the enzyme, was studied. HLAD was labeled with gadolinium diethylene-triamine-pentaacetic acid (Gd-DTPA), using the N-hydroxy-succinimide active ester of DTPA as a chelating agent. The HLAD-(Gd-DTPA)27 has a 3.7-fold larger longitudinal (R1) and a 14-fold larger transversal relaxivity (R2) (at 2.4 T) than the plain Gd-DTPA. A series of dry polyacrylamide gel beads, with total monomer concentration ranging from 5% to 30% were synthesized and swollen in a buffered solution of HLAD-(Gd-DTPA)27. The gel beads were examined with high resolution NMR imaging. The T1- and T2-weighted images revealed that the permeability for the labeled HLAD decreased with increasing total monomer concentration of the gel beads. These imaging results correlate fairly well with the enzymatic reactivities measured for the same range of gel beads but swollen in a solution of non labeled HLAD and NAD+ (nicotinamide adenine dinucleotide). It is concluded that Gd-labeling can be used to monitor the distribution of weakly concentrated, water soluble products in a solid matrix.

Plant antiviral agents, VI. Isolation of antiviral phenolic glucosides from Populus cultivar Beaupre by droplet counter-current chromatography.

Bioassay-guided isolation of the 80% EtOH extract of the leaves of Populus cultivar Beaupré by means of dccc afforded four known phenolic glucosides. Salicin and salireposide were shown to be, at least partially, responsible for the antiviral activity of the extract against poliomyelitis and Semliki forest viruses.

Identification of urinary metabolites of penticainide in the rat, dog, baboon and man.

[14C]-Penticainide, 2-[2-(diisopropylamino) ethyl]-4-methyl-2-(2-pyridyl)pentanamide, a new antiarrhythmic agent, was administered as a single oral dose to rats, dogs, baboons (30 mg kg-1) and to healthy, informed volunteers (300 mg). Excretion of radioactivity was followed for 3 days in urine and faeces. In man, about 95% of the administered radioactivity was eliminated in the urine and levels ranging from 56 to 86% were observed in animals. The radioactivity that did not appear in the urine was almost quantitatively recovered in the faeces. Metabolites in urine were isolated by thin-layer chromatography and identified by mass spectrometry and nuclear magnetic resonance. In addition to the unchanged drug, nine metabolites and an artifact compound resulting from the partial degradation of one metabolite, were identified among the thirteen radioactive compounds detected. The major metabolites resulted from N-dealkylation of the diisopropyl moiety, oxidation of the isobutyl side-chain and hydrolytic cleavage of the amide. Comparison of the excretion and metabolic patterns of animals with those of man revealed that the dog should be a most suitable model for predicting the pharmacological and toxicological effects of penticainide in man.

Plant antiviral agents. III. Isolation of alkaloids from Clivia miniata Regel (Amaryllidaceae).

Lycorine (1), one of the main alkaloids of the Amaryllidaceae family, was found to be responsible for the pronounced antiviral activity of the crude extracts from the roots and leaves of Clivia miniata Regel. It was shown that the inhibitory activity of lycorine on the cytopathogenic effect caused by a DNA and several RNA viruses on VERO cells was not virucidal. Poliomyelitis virus inhibition occurred at lycorine concentrations as low as 1 microgram/ml, whereas concentrations exceeding 25 micrograms/ml were found to be cytotoxic. Clivimine (2), clivonine (3) and cliviamartine (4) were also isolated and characterized, but these alkaloids exhibited no antiviral properties.

Isolation and identification of 2-oxo-5-guanidinovaleric acid in urine of patients with hyperargininaemia by chromatography and gas chromatography/mass spectrometry.

An unknown guanidino-positive peak has been identified in urines of three sisters affected with hyperargininaemia. Identification was made on the basis of its similarity with the liquid and thin-layer chromatographic characteristics of enzymatically synthesized 2-oxo-5-guanidinovaleric acid. Identification was also made by combined gas chromatography-mass spectrometry of the unknown compound peak. The synthesis of enzymatically formed 2-oxo-5-guanidinovaleric acid was controlled by nuclear magnetic resonance and combined gas chromatography-mass spectrometry.

Isolation and identification of polyprenols from bovine thyroid gland.

The presence of polyprenols in bovine thyroid was demonstrated. After preparative isolation, the structure was elucidated by chemical and spectroscopic techniques. The main polyprenol homologue has a molecular weight of 1380 corresponding to the presence of 20 isoprene units. From NMR studies it appears that 18 units have the cis configuration and that the 2 others are trans isoprene units. The dolichol content amounts to 0.2 mg/g wet weight. About 5% was found in the esterified form.