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 P2Y 1 while uridine containing dinucleotides have some level of agonist response on P2Y 2 and P2Y 6 . 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.

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Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.

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Several aromatic mono-and diamidines were found to block cell fusion induced by respiratory syncytial virus. The best inhibitors were able to achieve complete suppression of syncytium formation at a concentration of 1.0 AM. Inhibition occurred in respiratory syncytial virus-infected HEp-2 and CV-1 cells, but the same inhibitors were ineffective in preventing fusion induced by parainfluenza virus type 3. The fusion inhibitors did not reduce single-cycle virus yields, but did reduce multiple-cycle yields. In addition, the active compounds caused a significant retardation of respiratory syncytial virus penetration. The mechanism by which amidines interfere with respiratory syncytial virus-host cell interactions is unknown, but parallels can be drawn between antiviral activity and the ability of the compounds to inhibit certain trypsin-like proteases.Respiratory syncytial (RS) virus is a common respiratory pathogen which causes, almost without exception, a well-defined epidemic of respiratory illness every year. Early studies clearly defined the threat of RS virus to pediatric populations, and more recent studies demonstrated the susceptibility to infection of geriatric populations (1). Attempts to develop effective vaccines for RS virus have been unsuccessful (3,14). In addition, reinfections are a common event, suggesting that naturally acquired immunity does not provide long-lasting protection.Recently, we have reported that bis(5-amidino-2-benzimidazolyl)methane possesses a significant suppressive effect on the cytopathology and yield of RS virus (5). Since bis(5-amidino-2-benzimidazolyl)methane is the most potent reversible synthetic inhibitor of trypsin so far reported (20), we have examined other inhibitors of trypsin-like enzymes for their influence on RS virus-host cell interactions. In addition, homologs and isosters of the active compounds were included to define the specificity of the inhibitory process. This study has identified several other amidines which modulate the interaction of RS virus with the host cell. MATERIALS AND METHODSVirus and cell cultures. HEp-2 and CV-1 cells were propagated in Eagle minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). In experiments involving protease inhibitors, HEp-2 cells were used between passages 370 and 376.The A2 strain of RS virus, obtained from R. M. Chanock, was grown in suspensions of HEp-2 cells.After virus absorption, the infected cells were seeded in 250-ml plastic culture flasks containing MEM plus 10% FBS. When viral cytopathic effects were maximal (72 to 96 h), the flasks were frozen at -70°C. Typical lysates contained 107 to 10W 50% tissue culture infective doses per ml.All virus stocks and cell lines were determined to be free of mycoplasma contamination by a modification of the fluorochrome technique of Chen (2). Viral titrations. Virus yields were quantitated by calculating 50% endpoints by the method of Reed and Muench (18). Briefly, 0.05 ml of virus diluted in MEM plus 10% FBS was added to microtiter plates (Linbro S...

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