There is significant interest in development of new antiplatelet agents as antithrombotic drugs that act directly and reversibly, avoiding documented drawbacks of the current drug of choice, clopidogrel. A synthetic ATP analog, which is a potent antagonist of one of the ADP- dependent platelet P2Y receptors, is in clinical testing as an antithrombotic drug. Diadenosine P1,P4-tetraphosphate (Ap4A) and its phosphonate and thiophosphonate analogs such as P Ap(S)pCHClp2(S)A (""""""""Avathrom"""""""") inhibit platelet aggregation in vitro, and show antithrombotic activity in vivo, but at high doses. Existing data suggest P2Y1 targeting, but do not rule out P2Y12 inhibition as well. We have discovered new, highly efficient methods for synthesis of dinucleoside tetraphosphates and tetraphosphonates that will enable the efficient preparation of modified Ap4A derivatives for the first time. One method is based on a new reagent class - stable but reactive bis-imidazolides of pyrophosphate and methylenebisphosphonates - and inexpensive starting materials, resulting in symmetric products in high yield. The other method, suitable also for unsymmetric analogs, relies on an efficient reaction between nucleoside-5'-metatriphosphates or -5'-metatriphosphonates and nucleoside monophosphates. We propose to exploit the new synthetic methods to prepare symmetric and unsymmetric adenine-modified Ap4A analogs, with modifications based on existing SAR data of ATP and ADP analogs, with the aim to create potent antagonists of platelet P2Y1 and/or P2Y12 receptors, and, possibly, a unique class of antiplatelet agents which targets both receptors. The antiplatelet potency and platelet shape change activity will be determined, and antagonist/agonist properties of the new compounds toward P2Y1, P2Y12, and P2X1 receptors will be measured. We will also measure the stability of selected new analogs in rat, dog, and human plasma. Our immediate goals are to validate and further develop breakthrough methods for synthesis of bis-nucleoside polyphosphates, to determine if the class of Ap4A analogs targets P2Y1, P2Y12, or even better, both P2Y1 and P2Y12 platelet receptors, and to demonstrate the therapeutic potential and plasma stability of novel Ap4A analogs. Our long range goals are to discover novel compounds and methods for treatment of arterial thrombosis, and more particularly, a fast and reversibly acting antiplatelet agent targeting platelet P2Y1, or better, both P2Y1 and P2Y12 receptors, to complement existing antiplatelet therapeutics which mainly target the platelet P2Y12 receptor. ? ? This project will result in an effective antithrombotic drug that will be used to treat arterial thrombosis. The candidate drug will directly and reversibly inhibit one or both of important receptors involved in platelet aggregation, and will not have the drawbacks of slow and variable action of current drugs such as clopidogrel. The new drug will complement related drugs under development for arterial thrombosis. ? ? ?
