Letters in Peptide Science, 9: 101–109, 2002. KLUWER/ESCOM © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 101 Novel synthetic RGD analogs incorporating salicylic acid derivatives show antiplatelet activity in vitro Yiannis M. Sarigiannis 1 , George P. Stavropoulos 1∗ , Maria T. Liakopoulou-Kyriakides 2 & Pantelis E. Makris 3 1 Department of Chemistry, University of Patras, Patras, Greece; 2 Department of Chemical Engineering, Ar- istotle University of Thessaloniki, Thessaloniki, Greece; 3 Haemostasis and Thrombosis Unit, AHEPA Hospital, Thessaloniki, Greece ( ∗ Author for correspondence, e-mail: G.Stavropoulos@chemistry.upatras.gr, Fax: +302610 993127) Received 29 October 2002; Accepted 6 December 2002 Key words: GpIIb/IIIa inhibitors, inhibitory activity, platelet aggregation, RGD analogs, salicylic acid derivatives Summary Continuing our investigational efforts for more active GpIIb/IIIa inhibitors we have synthesized four novel RGD (Arg-Gly-Asp) analogs incorporating salicylic acid derivatives at their N-terminal amino group using the solid phase synthesis. The synthesized compounds 5-Cl-2-HO-C 6 H 3 -CO-Arg-Gly-Asp(OBzl)-NH 2 and 5-Br-2-HO- C 6 H 3 -CO-Arg-Gly-Asp(OBzl)-NH 2 were tested for their inhibitory activity on human platelet aggregation in vitro and found to be potent inhibitors of the platelet aggregation with 75 and 67% inhibitory activity respectively. In order to confirm our results flow cytometry with monoclonal antibodies against GpIb, GpIIb/IIIa, GpIIIa and GMP140 receptors was used. Abbreviations: AcOH, acetic acid; ADP, adenosine diphospate; Bzl, benzyl; BuOH, butanol; 2- CLTR, 2-chlorotrityl chloride resin; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DIC, N,N- diisopropylcarbodiimide; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; EDT, 1,2-ethanedithiol; ES-MS, electrospray mass spectrometry; Et 2 O, diethylether; Fmoc, 9-fluorenylmethyloxycarbonyl; FT-IR, Fourier transform infrared; GpIIb/IIIa, glycoprotein IIb/IIIa; HOBt, 1-hydroxybenzotriazole; MeCN, acetonitrile; PyBOP, (benzotriazol-1-yloxy)-tris(pyrrolidino) phosphonium hexafluorophosphate; RP-HPLC, reversed phase HPLC; tBu, tert-butyl, TFA, trifluoroacetic acid, TFE, trifluoroethanol. Introduction The development of new antithrombotic agents has been stimulated by clinical needs as long as the ma- jor cause of death in developed western countries are the cardiovascular diseases, mainly unstable angina, stroke and acute myocardial infraction. Two classes of antithrombotic agents – anticoagulants and anti- platelet agents – are used in antithrombotic treatment. Anticoagulants inhibit thrombin generation and fibrin formation while the antiplatelet agents block plate- let aggregation. Aspirin, coumarins, low-molecular weight heparins and direct inhibitors of thrombin have been used as antithombotic agents for many years with remarkable effectiveness even they have some their limitations like bleeding, lab monitoring, etc. The de- velopment of novel antithrombotic compounds might lead to a further reduction of cardiovascular mortal- ity and simultaneously reduced bleeding side effects. Antagonists of platelet glycoprotein IIb/IIIa represent a therapeutic approach in inhibiting platelet aggrega- tion, thus providing a powerful form of antithrombotic therapy [1–3]. The binding of fibrinogen by glycoprotein IIb/IIIa (GpIIb/IIIa) on the surface of activated platelets is the final common pathway for platelet aggregation. This binding is mediated in part by Arg-Gly-Asp (RGD) sequences located on fibrinogen and other proteins of