6-Substituted and 5,6-disubstituted derivatives of uridine: stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity.

Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, 1H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo). Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F substituents capable of formation of an additional hydrogen bond with the enzyme. The 5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil.

Uridine phosphorylase from Hymenolepis diminuta (Cestoda): kinetics and inhibition by pyrimidine nucleoside analogs.

A single pyrimidine nucleoside phosphorylase was found in the cytoplasmic extract from Hymenolepis diminuta. This enzyme preferentially cleaves uridine and, to a much lesser extent, thymidine. Its presence directly indicates the existence of pyrimidine nucleoside salvage pathway in this parasite. Detailed kinetic studies in the phosphorolytic and synthetic direction pointed to the sequential mechanism of these reactions. For phosphorolysis, Kurd = 33 microM and Kp = 806 microM. For synthesis of uridine, Kura = 204 microM and K1-P-rib. = 50 microM. Over six times higher K(m) for uracil than for uridine indicates that phosphorolysis is the favoured reaction in this tapeworm. Well known inhibitors of mammalian uridine phosphorylase: 2,2'-anhydro-5-ethyluridine and 1-(1,3-dihydroxy-2-propoxymethyl)-5-benzyluracil (DHPBU), both with Ki = 0.07 microM were potent competitive inhibitors of the enzyme from H. diminuta. The newly synthesized 2,3'-anhydro-5-ethyluridine (K. Felczak, unpublished) showed only moderate inhibitory activity (Ki = 14 microM) similarly as 1-(1,3-dihydroxy-2-propoxy-methyl)-5-benzyluracil. The same order of Ki values obtained for the investigated inhibitors vs uridine phosphorylase, irrespective whether the enzyme was isolated from rat intestinal mucosa (Drabikowska et al., 1987, Biochem. Pharmacol. 36, 4125-4128) or H. diminuta may point to a great similarity between binding sites on the parasite and the host enzyme.

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

Inhibitor properties of some 5-substituted uracil acyclonucleosides, and 2,2'-anhydrouridines versus uridine phosphorylase from E. coli and mammalian sources.

Two series of 5-substituted uracil N(1)-acyclonucleosides, each with a different acyclic chain, were examined as inhibitors of uridine phosphorylase from rat intestinal mucosa, and several against the enzyme from Ehrlich ascites cells. In addition, several 5-substituted analogues of 2,2'-anhydrouridine were tested for their inhibitory effects vs a highly purified uridine phosphorylase from Escherichia coli. The results are compared with previously published data for inhibition of the E. coli enzyme by the acyclonucleosides, and of the rat enzyme by the anhydrouridines. In all instances, the inhibitors were active only vs the uridine, but not thymidine, phosphorylase from E. coli, and inhibition was competitive with respect to uridine as substrate. In general, with one or two exceptions, inhibitory effects were more pronounced against the enzyme from mammalian sources. Amongst the acyclonucleoside analogues, the most effective inhibitor of the enzyme from the rat and Ehrlich ascites cells exhibited a Ki = 0.1 microM, comparable to that reported with the Sarcoma-180 enzyme, whereas the Ki for inhibition of the E. coli enzyme was 0.7 microM. By contrast, another effective inhibitor of the bacterial enzyme was 7-fold less potent against the mammalian enzyme. The 2,2'-anhydrouridines were 10- to 30-fold more effective against the rat, as compared to the E. coli, enzyme. The overall quantitative data provide a reasonably good basis for the further design of potent inhibitors for possible use in chemotherapy.

Acyclonucleoside analogues consisting of 5- and 5,6-substituted uracils and different acyclic chains: inhibitory properties vs purified E. coli uridine phosphorylase.

Synthetic procedures are described for the preparation of a variety of pyrimidine acyclonucleoside analogues, in which the aglycones are 5- and 5,6-substituted uracils, and the ribose moiety is replaced by different acyclic chains. These were examined as potential inhibitors of purified E. coli uridine phosphorylase. None of the compounds was a substrate for uridine phosphorylase, or either a substrate or inhibitor of E. coli thymidine phosphorylase. Kinetic measurements were employed to determine inhibition constants, Ki, for inhibition of uridine phosphorylase. One of the more effective of these was 1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil, with Ki = 2.7 microM. The same compound was a reasonably good inhibitor of the reverse, synthetic, reaction, with Ki values of 19 microM vs uracil as the variable substrate, and 15 microM vs alpha-D-ribose-1-phosphate as the variable substrate. For one of the analogues, which was a racemate, 1-(2',3'-dihydroxypropyl)-5,6-tetramethyleneuracil, it was shown that only one of the enantiomers (R) was an inhibitor, the (S) enantiomer being totally inactive. For several of the analogues, the corresponding isomeric N(3)-acyclonucleosides were inactive as inhibitors. The results for several of the good inhibitors were compared with those of other observers for inhibition of uridine phosphorylase from mammalian sources. Preliminary measurements with several of our analogues demonstrated that some of them were indeed one to two orders of magnitude more effective against the enzyme from mammalian sources.

Selective syntheses of 6-substituted pyrimidine nucleosides and their interactions with uridine phosphorylase.

Several 6-substituted pyrimidine nucleosides have been prepared by condensation of an N(3)-benzyl or C(4)-methylthio pyrimidine with the appropriately blocked sugar, followed by the use of mild conditions at room temperature for removal of blocking groups. The substrate properties of these nucleosides, which are in the fixed syn conformation, have been examined in the uridine phosphorylase system.

Purification and properties of adenosine kinase from rat liver: separation from deoxyadenosine kinase activity.

Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS-polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 mumol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2'-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (approximately 1.5%) under optimal conditions (pH 7.5) vs 2'-deoxy-adenosine, the lowest yet reported for such a preparation, with a Km of 670 microM, as compared to 0.3 microM for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a Ki of 70 microM. Competitive inhibition was also exhibited by cordycepin (3'-deoxyadenosine) with a Ki of 150 microM. A more potent competitive inhibitor was tubercidin, the Ki for which was 1.9 microM.

Studies on the mechanism of resistance of Pseudomonas aeruginosa to neomycin. II. Correlation between neomycin resistance and hemoprotein concentration.

The cells of a newly isolated neomycin sensitive and neomycin resistant strains of Pseudomonas aeruginosa contained similar level of catalase activity. The difference spectrum of the cells revealed the presence of cytochrome c-554 and cytochrome b-560-. The presence of both cytochromes in the same molecular ratio in the cells and subcellular fractions indicates that they are tightly bound to the cytoplasmic membrane. The neomycin resistant strain contained five times less cytochromes than the sensitive strain. The lower cytochrome level in the neomycin resistant cells was found to be independent of the presence or absence of neomycin in the medium. The difference spectra of the cells and particulate fractions did not reveal any cytochromes of alpha-type.

Studies on the mechanism of antiviral action of 1-(beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin).

Syntheses are described for the 5'-phosphates of the 2'- and 3'-O-methylribavirins (2a and 2b) and the methyl ester of ribavirin 5'-phosphate (3). The 5'-phosphate of 1-beta-(D-ribofuranosyl)-1,2,4-triazole (2d) was obtained via the 3-carboxyl derivative of ribavirin 5'-phosphate (2c). Compounds 2a, 2b, and 2d were inactive as inactive as inhibitors of IMP dehydrogenase under conditions where the parent ribavirin 5'-phosphate (2) was an effective inhibitor. Weak inhibitory activity was exhibited by 3 (Ki approximately 200 muM) and 2c (K1 approximately 70 muM). Under conditions where ribavirin (1) is effectively phosphorylated by rat liver nucleoside kinase, the 2'- and 3'-O-methylribavirins (1a and 1b), the 3-carboxylate of ribavirin (1c), and the riboside of 1,2,4-triazole (1d) were totally inactive. The overall results are fully consistent with the lack of antiviral activity of 1a and 1b, while the specificity of ribavirin as an antiviral agent is further underlined by the behavior of the methyl ester 3.

Disappearance of the cyanide-insensitive pathway of oxidation in mitochondria of MI-1 mutant of Neurospora crassa in vitro.

Oxidation of exogenous NADH in mitochondria isolated from wild type and mi-1 mutant of Neurospora crassa decreases rapidly in vitro. In mi-1 mutant mitochondria the inactivation concerns the alternate pathway of oxidation whereas in the wild type it involves an unknown component of the respiratory chain. The activity of the primary NADH dehydrogenase is constant within the time of the experiments (2-4 h). NADH oxidase is not inactivated if oxygen is removed from the incubation medium by nitrogen bubbling. Succinate oxidase does not show any remarkable changes in activity within 2-3 h. In fresh mitochondria of the mi-1 mutant reduced ubiquinone is completely reoxidized by cytochrome oxidase but only 80% reoxidized by the alternate oxidase. In aged mitochondria of the mi-1 mutant in the presence of cyanide, ubiquinone is reduced to the level characteristic for fresh mitochondria in which respiration is completely inhibited by cyanide plus salicylhydroxamic acid. In these mitochondria the reoxidation of the reduced ubiquinone proceeds only via the cytochrome pathway. It is supposed that a labile component(s) of the respiratory chain present in the mi-1 mutant and the wild type mitochondria may, in mi-1 mutant, act as an alternate oxidase.

The respiratory chain of a newly isolated Methylomonas Pl1.

1. Whole cells of Methylomonas Pl1 contained ubiquinone, identified as ubiquinone-8. No naphthaquinone was detected. Ubiquinone was located predominantly in the particulate fraction, which also contained most of the NADH oxidase activity. 2. Aerobic incubation of cells with formaldehyde or methanol resulted in about 20% reduction of ubiquinone, irrespective of the presence or absence of dinitrophenol. On inhibition of the respiration by cyanide, ubiquinone became partly reduced by endogenous substrates (15--25%), and a further reduction occurred only in the presence of formaldehyde (up to 60%). When endogenous substrates were completely exhausted, then 44 and 23% of ubiquinone was reduced by formaldehyde or methanol respectively. 3. The difference spectra at room and liquid-N2 temperatures revealed the presence of cytochrome b and two cytochromes c (c-552.5 and c-549) all tightly bound to the membrane. Cytochrome c-552.5 was also found in the soluble fraction. 4. Redox changes of cytochromes b and c, with methanol or formaldehyde as substrates, respond to the aerobic and anaerobic states of the cell and to KCN inhibition in a manner characteristic of the electron carriers of the respiratory chain. 5. The merging point for electron transport from NADH dehydrogenase and formaldehyde dehydrogenase is suggested to be at the level of ubiquinone.

Oxidation processes and ubiquinone localization in the branched respiratory system of mi-1 mutant of Neurospora crassa.

1. Stimulation of succinate oxidation in mi-1 mitochondria by Mg2+ and Pi is abolished on uncoupling, which points to the energy-linked activation of succinate oxidation. 2. Mitochondria exhibited respiratory control with succinate and NADH when the cyanide-insensitive oxidation was inhibited by salicylhydroxamic acid (SHAM). SHAM lowered the oxidation rate with NADH and succinate to the same level, though the NADH oxidation rate was 2.5 times as high as with succinate. 3. Despite the high stimulation of succinate oxidation via the SHAM-sensitive pathway in the active and controlled state of mitochondria, the redox state of UQ in all metabolic states remains unchanged. On inhibition of the cyanide-insensitive pathway, UQ reduction is greatly increased only in the controlled and active state. With NADH as a substrate, UQ does not respond to the metabolic states of mitochondria. 4. The redox changes of cytochrome c parallel those of UQ. 5. Branching of the respiratory chain in mi-1 mitochondria is discussed.

Ubiquinone function in Neurospora crassa.

Mitochondria of cytoplasmic respiratory mutants [mi-1] (poky) and [mi-4] contain about a fourfold molar excess of ubiquinone as compared to the wild-type strain of Neurospora crassa. In the wild type and [mi-1] cultures the concentration of ubiquinone remains constant during the exponential and stationary phase of growth. In [mi-4] cultures it markedly decreases in the stationary phase. The reduction of ubiquinone by substrates is approximately the same in the three strains tested and amounts 60 to 70% of total ubiquinone present in mitochondria, independent of its absolute amount. The reduction of ubiquinone on addition of substrates is accompanied by the similar reduction of cytochrome c. These indicate that mitochondrial ubiquinone and cytochrome c are involved in processes of oxidation in Neurospora and that ubiquinone belongs mainly if not entirely to the cytochrome system of electron transport in these strains.