Synthesis and biological evaluation of 2-thiopyrimidine derivatives

Various 2-thiopyrimidine derivatives have been synthesized by an efficient, one-pot reaction of functionalized amines with either 4-isothiocyanato-4-methyl-2-pentanone or 3-isothiocyanatobutanal. All the synthesized compounds were fully characterized by elemental analysis (CHN), FT-IR, (1)H NMR, and mass spectral data. One of the compounds, 7,7,8a-trimethyl-hexahydro-thiazolo[3,2-c]pyrimidine-5-thione (17) showed good anti-inflammatory (37.4% at 100 mg/kg p.o.) and analgesic activity (75% at 100 mg/kg p.o.). 7-(1-Mercapto-3,3,4a-trimethyl-4,4a,5,9b-tetrahydro-3H-pyrido[4,3-b]indol-7-yl)-3,3,4a-trimethyl-3,4,4a,5-tetrahydro-benzo[4,5]imidazo[1,2-c]pyrimidine-1-thiol (3) showed moderate activity against CDK-1 (IC(50)=5 microM). The other compounds showed moderate anti-inflammatory (5-20%), analgesic (25-75%) and protein kinase (CDK-5, GSK-3) inhibitory activities (IC(50)> 10 microM).     

Synthesis of oligodeoxynucleotides containing 2-thiopyrimidine residues--a new protection scheme.

A method is described for the incorporation of 2'-deoxy-2-thiouridine (dS2U) and 2'-deoxy-2-thiothymidine (dS2T) into oligodeoxynucleotides at predetermined positions. This requires N3 or O4-acylation of dS2U and dS2T with toluoyl chloride. These base-protected thiopyrimidines are completely stable toward the aqueous iodine oxidation reagent used in the phosphoramidite DNA synthesis method. The toluoyl protecting group is removed during the standard post-synthetic ammonia treatment. This novel protection strategy allows dS2U and dS2T to be efficiently incorporated into oligodeoxynucleotides at predetermined sites without the usual problem of desulfurization and decomposition. Several 14-mers containing the Eco-RI recognition site (dGGCGGAAXXCCGCC and dGGCGGAAXXCGCGG, where X represents dT, dS2U or dS2T) have been synthesized and characterized by base composition, thermal denaturation, CD spectroscopy and endonuclease substrate activity.     

Reduction of nitrofuran derivatives by xanthine oxidase and microsomes: Isolation and identification of reduction products

An investigation was carried out to identify the reduction products of nitrofurazone and AF-2 (2-furyl)-3-(5-nitro-2-furyl)acrylamide by milk xanthine oxidase, rat liver xanthine oxidase, and rat liver microsomes. Data obtained from mass spectrometry and other methods indicated that the ethyl acetate-extractable major product of each nitrofuran derivative should be the corresponding amine derivative or the equivalent compound. This conclusion was further confirmed by an examination of stoichiometry. The reduced nitrofurazone was finally identified as 5-amino-2-furfural semicarbazone by comparative studies with the authentic specimen. The reduced AF-2 was tentatively identified as 2-(2-furyl)-3-(5-oxo-2-pyrrolin-2-yl)acrylamide. A reduction pathway for this conversion is postulated.     

Binding to opioid receptors of enkephalin derivatives taking alpha-helical conformation and its dimer.

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the delta-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the mu-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-Gly-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)2-OH (2) showed 51- and 96-fold decreases in affinities for delta- and mu-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to alpha-helical conformation, which might not be compatible with topological requirements of delta- and mu-receptors. A dimer, Tyr-Gly-Gly-Leu-Phe-(Lys-Aib-Leu-Aib)2-Lys(X)-Aib-OCH3 (X = Tyr-Gly-Gly-Phe-Leu-, (4], showed 2.5- and 3.0-fold increases in affinities respectively for delta- and mu-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between delta- and mu-receptors in bovine brain membranes.     

Properties of matrix-bound dimer and monomer derivatives of immobilized creatine kinase from rabbit skeletal muscle.

Dimeric creatine kinase (EC 2.7.3.2) from rabbit skeletal muscle can be immobilized via a single subunit to CNBr-activated Sepharose 4B and subsequently treated with guanidine hydrochloride followed by renaturation to yield a catalytically active matrix-bound subunit derivative. The importance of the intact dimeric structure in the activation of the enzyme by acetate was demonstrated. Immobilization did not appear to alter the pH optimum of the enzyme, and the kinetic parameters fot the matrix-bound derivatives were generally similar to those for the soluble enzyme, but the matrix-bound derivatives showed higher thermal stability and greater resistance to denaturation than did the soluble enzyme. The rates of reaction of thiol groups of the matrix-bound derivatives with iodoacetamide in the absence and in the presence of combinations of substrates were similar to those of the soluble enzyme. Studies with 5,5'-dithiobis-(2-nitrobenzoic acid) and with iodoacetamide revealed the presence of an additional reactive thiol group in the matrix-bound subunit derivative, which is presumably masked in the dimeric derivatives.     

Cellular and chemical reduction products of misonidazole

Misonidazole is readily reduced by zinc dust in aqueous solution in the presence of ammonium chloride. High pressure liquid chromatographic (HPLC) separation of the reduction mixture revealed the presence of three products. These were identified as the hydroxylamine, amine and the hydrazo derivative of misonidazole. There is evidence that the azoxy derivative was an intermediate in the reduction process. When the reduction was carried out in dilute solution (0.1 mg/ml), the hydroxylamine was the only product. In concentrated solution (20 mg/ml), the hydrazo derivative was the major product. When misonidazole was reduced with hydrogen using palladium as catalyst, the amine was the only detectable product. Of the three products, only the hydroxylamine was found to bind covalently to bovine albumin. In Chinese hamster ovary (CHO) cells under hypoxic conditions the amine was confirmed as one of the metabolites. There was no evidence for the presence of detectable amounts of the hydroxylamine in the cell extracts. These studies suggest that the hydroxylamine is probably the reactive reduction metabolite responsible for the in vivo and in vitro binding of misonidazole to cellular macromolecules.     

Dark transition of 1st level reduction products of porphyrins to 2d level

Transition of the products of the first restoration of porphyrines to the second one in the darkness and practically in the absence of a reducer and oxygen has been studied. Its cause is the disproportioning of dyhydroform into tetrahydro- and the initial pigment. Regeneration of porphyrines under these conditions is also shown. The rate of both reactions significantly increases in the light.     

Synthesis of 2,3-dihydroinosine derivatives by reduction using BH3-THF.

A novel reductive method for the chemical modification of nucleosides is described. Reaction of inosine derivatives with boran-THF resulted in the regioselective reduction of purine ring to afford the corresponding 2,3-dihydroinosine derivatives in moderate yields.     

Intermediate reduction products of porphyrins and their structure

It was shown on the basis of NMR-H spectra that the addition of proton to dianions of Zn-octaethylchlorine, Zn-octaethylporphin, and Zn-monoasaethioporphyrin results in the formation of products with the structure of alpha-dihydroflorine, alpha-florine, and gamma-florine respectively. This indicates that electron density greatly increases when the anions are formed in the centres which had a decreased density in the initial molecule.     

Ploidy reduction usingp-Fluorophenylalanine of fusion products ofSaccharomyces cerevisiae

Fusion of yeast protoplasts was induced in the presence of polyethylene glycol and Ca++ ions. Two auxotrophic complementingSaccharomyces cerevisiae mutant strains were used in fusion experiments. One diploid and several polyploid fusion products were selected by complementation in minimal medium. The assessment of ploidy has been based on the DNA content of the parental cells and fusion products, assayed with the diphenylamine method. Treating the fusion product cells withp-Fluorophenylalanine (p-FPA), parentalhis andleu markers could not be recovered. Instead, a strong reduction of polyploid fusion product cell DNA content was evident. The analysis of meiotic products after hybridizing one fusion product with a prototrophicSaccharomyces cerevisiae standard strain led to the recovery of thehis parental marker. Preliminary evidence thatp-Fluorophenylalanine could be used as a diploidizing agent towards polyploid strains ofSaccharomyces cerevisiae is reported.     

Mutagenic activities of hydroperoxythymine derivatives, products of radiation and oxidation reactions.

By using a bacterial test system, it has been shown that hydroperoxy derivatives of thymine and thymidine produced by ionizing radiation, near-UV radiation, and certain oxidation reactions are highly mutagenic. Considering that hydroperoxy derivatives of biomolecules have been implicated widely as likely candidates causing mutagenesis, carcinogenesis, and aging, it would be advantageous to screen these compounds when they can be isolated in pure state in order to assess their potential hazards to human health. The findings from these assays would provide information to further our understanding of the mechanism of their mutagenic action.     

Chromous ion reduction of mammalian cytochrome oxidase and some of its derivatives.

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 X 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.     

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC₅₀ = 0.1–10 μM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC₅₀ = 0.1 μM) and BVDV (50, 51, and 53 with EC₅₀ = 1.5, 0.8, and 1.0 μM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.