Janet L. Rideout

Structure–activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y1 while uridine containing dinucleotides have some level of agonist response on P2Y2 and P2Y6. The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

An enzymic synthesis of purine d-Arabinonucleosides

Abstract A method is described for the synthesis of purine d -arabinonucleosides that uses purine bases and 2,2′-anhydro-(1-β- d -arabinofuranosylcytosine), AraC-an, as the starting materials. AraC-an was chosen as the precursor to the d -arabinosyl donor, because it is more readily available than any of the products that may be sequentially derived from it, namely, 1-β- d -arabinofuranosylcytosine (AraC), 1-β- d -arabinofuranosyluracil (AraU), and α- d -arabinofuranosyl-1-phosphate (Ara f 1-P), a d -arabinofuranosyl donor. Four reactions were involved in the overall process; ( a ) AraC-an was nonenzymically hydrolyzed at alkaline pH to AraC which was then ( b ) deaminated by cytidine deaminase to AraU, a nucleoside, ( c ) phosphorylyzed by uridine phosphorylase to Ara f 1-P, and ( d ) this ester caused to react with a purine base to afford a purine d -arabinonucleoside, the reaction being catalyzed by purine nucleoside phosphorylase. All four reactions occurred in situ , the first and second being performed sequentially, whereas the third and fourth were combined in a single step. The three enzyme catalysts were purified from Escherichia coli . The efficiency of the method is exemplified by the synthesis of the d -arabinonucleosides of 2,6-diaminopurine and adenine; the overall yields, based on AraC-an, were 60 and 80%, respectively.

2-amino-9-(3-azido-2,3-dideoxy-β-d-erythro-pentofuranosyl)-6-substituted-9H-purines: Synthesis and anti-HIV activity

A series of 2-amino-9-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)-6- substituted-9H-purines was synthesized and tested for the ability to protect MT4 cells from the cytopathic effect of HIV-1IIIB. These compounds were prepared by a combination of chemical and enzymatic reactions. Some of the nucleoside analogs with 6-alkoxy, 6-alkylamino, or 6-arylamino substituents were active against HIV-1IIIB. Their IC50 values were in the range of 2-60 microM. In contrast, analogs with 6-thio, 6-alkylthio, 6-methyl, or 6-carbonitrile substituents did not protect cells from the cytopathic effect of HIV infection.

Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species

Ribonucleosides of some pyrazolo [3,4-d] pyrimidines have been shown to be potent anticoccidial agents. To investigate their interactions with adenosine kinase, this enzyme was purified by affinity chromatography from the sporulated oocysts of 3 avian coccidia, Eimeria tenella, E. acervulina and E. brunetti as well as from chicken liver. Comparative studies revealed several differences among the enzymes. Magnesium appeared not to be inhibitor of the E. tenella enzyme but did inhibit the enzymes from the other three sources. ATP in excess of the magnesium concentration strongly inhibited the E. brunetti enzyme but had only a small effect on the other enzymes. The chicken liver enzyme utilized a broader variety of triphosphate donors than did any of the enzymes from Eimeria species. ATP, dATP, GTP, dGTP and ITP was the best substrates. Studies with pyrazolo [3,4-d] pyrimidine nucleosides revealed two groups of enzymes with similar inhibitor specificities, the chicken liver and E. Acervulina vs. the E. tenella and E. brunetti enzyme. This grouping roughly correlates with the in vivo anticoccidial specificity of these compounds. Substrate specificity studies using two 4-substituted pyrazolo [3,4-d] pyrimidine ribonucleosides (ethylthio- and cinnamylthio-), which have shown potent anticoccidial activity in vivo, revealed that each served as a substrate for the enzymes from E. tenella and E. acervulina. The E. tenella enzyme was the more efficient at the phosphorylation of those compounds. However, only the ethylthio- compound was detectably phosphorylated by the enzyme from E. brunetti. In contrast to the inhibitor specificity, the substrate activities of these nucleosides do not correlate well with their in vivo anticoccidial activity.

Synthesis of 2-amino-9-(3'-azido-2',3'-dideoxy-beta-D-erythro-pentofuranosyl)-6-methoxy-9H-purine (AzddMAP) and AzddGuo

Abstract A novel synthesis of AzddMAP and AzddGuo is described starting from 1, 2-O-isopropylidene-alpha-D-xylofuranose. The key step in this synthesis involves the replacement of a diphenyl carbamoyl protecting group in the purine 6-position by methanol.

Inhibition of lymphocyte-mediated cytolysis by 2-fluoroadenosine--evidence for two discrete mechanisms of drug action.

Abstract 2-Fluoroadenosine (F-Ado) is a potent, irreversible inhibitor of lymphocyte-mediated cytolysis (LMC) in vitro: the irreversibility of this inhibition has been attributed to the metabolism of F-Ado to 2-fluoroadenosine t′-triphosphate (F-ATP) and 2-fluoroadenosine 3′, 5′-monophosphate (F-cAMP) within the cytotoxic lymphocytes [T. P. Zimmerman, J. L. Rideout, G. Wolberg, G.S. Duncan and G. B. Elion, J. biol. Chem.251, 6757 (1976)]. The present study was undertaken to define better the biochemical events intrinsic to the inhibition of LMC by F-Ado. Several purine ribonucleosides, which are themselves non-inhibitory towar LMC, have been found to inhibit the metabolism of F-Ado to F-ATP and F-cAMP by the cytotoxic lymphocytes. The reduction in F-cAMP formation caused by these ribonucleosides was counterbalanced by their augmentation of the elevation of lymphocytic cyclic AMP (cAMP) caused by F-Ado. While interference with the metabolism of F-Ado had little or no effect on the immediate inhibitory activity of F-Ado toward LMC, prevention of the cellular formation of F-ATP and F-cAMP did allow most of the inhibitory activity of F-Ado to be reversed after washing the lymphocytes free of exogenous F-Ado. The relative efficacy of these ribonucleosides in allowing reversibility of the inhibitory activity of F-Ado toward LMC followed the same order as did their efficacy in preventing the metabolism of F-Ado by the cytotoxic lymphocytes: 8-aza-adenosine > inosine > guanosine. Cytotoxic lymphocytes which had been preloaded with nucleotides of F-Ado (via prior incubation with F-Ado and subsequent washout of residual extracellular drug) exhibited increased inhibition of their cytolytic activity upon subsequent incubation with an inhibitor (Ro 20-1724) of cAMP phosphodiesterase. Under these latter experimental conditions, Ro 20-1724 caused a 2- to 3-fold elevation of F-cAMP in the cytotoxic lymphocytes but did not raise cAMP above control levels. These results suggest that F-Ado can inhibit LMC by either of two distinct mechanisms: (1) an extracellular mechanism, wherein F-Ado binds reversibly to an adenosine receptor present on the plasma membrane of the cytotoxic lymphocytes and reversibly activates a functionally associated adenylate cyclase, thereby causing an elevation of cellular cAMP; and (2) an intracellular mechanism, wherein F-Ado is metabolized irreversibly (during the 1- to 2-hr experimental period) by the cytotoxic lymphocytes to F-cAMP which, by reason of its ability to activate cAMP-dependent protein kinase, mimics the effect of elevated cellular levels of cAMP.

SYNTHESIS OF ()-CIS-1-2-(HYDROXYMETHYL)-1,3-OXATHIOLAN-5-YL CYTOSINE AND ITS ()-TRANS ISOMER

Abstract The title compounds were synthesized by the formation of 2-[(benzyloxy)methyl]-1, 3-oxathiolan-5-one and subsequent DIBALH reduction, acetylation, coupling with N-(1, 2-dihydro-2-oxo-4-pyrimidinyl)-2-ethylhexanamide and deprotection.

Synthesis of 1-(3-Azido-2,3-Dideoxy-Beta-D-Ribo-Hexofuranosyl)Thymine

Abstract The nucleoside derivative 1-(3-azido-2,3-dideoxy-beta-D-ribo-hexofuranosyl)thymine has been synthesized from 3-0-benzyl-1,2-0-isopropylidene-alpha-D-glucofuranose-5,6-carbonate in an overall yield of 16%. The key step in the synthesis involves the selective deacetylation of a nucleoside derivative having a cyclic carbonate moiety.