Synthesis of uracil nucleotide analogs with a modified, acyclic ribose moiety as P2Y 2 receptor antagonists Roland Sauer, Ali El-Tayeb   , Marko Kaulich, Christa E. Müller * PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany article info Article history: Received 29 March 2009 Revised 16 May 2009 Accepted 23 May 2009 Available online 30 May 2009 Keywords: P2Y 2 receptors UTP, UDP, UMP analogs Nucleotide analogs Acyclic ribose modification Phosphonates abstract A series of new uracil nucleotide analogs (monophosphates, triphosphates, and phosphonates) was syn- thesized, in which the ribose moiety was replaced by acyclic chains, including branched or linear alkyl or dialkylether linkers. 1-x-Bromoalkyluracil derivatives (2) were converted to the corresponding alcohols by treatment with sodium hydroxide and subsequently phosphorylated using phosphorus oxychloride followed by hydrolysis to yield the monophosphates, or by coupling with diphosphate to form the tri- phosphates. Reaction of 2 with triethyl phosphite followed by deprotection with trimethylsilyl bromide led to the x-phosphonylalkyluracil derivatives. These products could be further phosphorylated by con- verting them into their imidazolides and subsequent treatment with diphosphate yielding the corre- sponding UTP analogs. Nucleoside analogs with an oxygen atom in the 2 0 -position, which are more similar to the natural ribosides, were synthesized from silylated uracil and trimethylsilyl iodide-treated 1,3-dioxolane, or 1,3-dioxane, respectively, and subsequently phosphorylated by standard procedures. The nucleotide analogs were investigated in a functional assay at NG108-15 cells, a neuroblastoma  gli- oma hybrid cell line which expresses the UTP- and ATP-activated nucleotide receptor subtype P2Y 2 . The acyclic nucleotide analogs were generally weaker ligands than UTP, and—in contrast to UTP—they were antagonistic. The most potent compound was diphosphoric 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)pentylphosphonic anhydride (5c) with an IC 50 value of 92 lM showing that the replacement of the a-phosphate by phosphonate, which leads to enhanced stability, was well tolerated. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The pyrimidine nucleotides UTP and UDP have been recognized as important signalling molecules activating G protein-coupled membrane receptors (GPCRs) of the P2 (nucleotide) receptor fam- ily. 1–3 P2Y receptors are currently subdivided into eight members, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 . 3 While the human P2Y 1 , P2Y 11 , P2Y 12 , and P2Y 13 receptors are activated exclu- sively by adenine nucleotides, the human P2Y subtypes P2Y 4, 6, 14 are only activated by uracil nucleotides. The human P2Y 2 receptor re- sponds to ATP and UTP. 2,3 Species differences exist, for example, in contrast to the human P2Y 4 receptor (a UTP receptor), the rat ortho- logue is potently activated by UTP and ATP. 2–4 P2Y receptors have been found to be implicated in a variety of physiological functions and pathophysiological conditions. Therefore P2Y receptors are of interest as drug targets. Irreversible P2Y 12 antagonists are already in use as antithrombotic drugs, for example, clopidogrel (I). 5 The ATP analogue AR-C69931 MX (cangrelor, II) was studied in clinical trials as a competitive P2Y 12 antagonist for antithrombotic therapy; 6 further P2Y 12 antagonists are currently evaluated. 7 Another P2Y receptor subtype which is of considerable interest as a new drug tar- get, is the P2Y 2 receptor subtype. 3,8–11 P2Y 2 agonists are being devel- oped for the treatment of cystic fibrosis (INS37217, denufosol, III) and dry eye syndrome (INS 365, diquafosol, IV), see Figure 1. 12–14 However, III and IV are moderately potent and weakly selective P2Y 2 agonists. P2Y 2 receptor antagonists possess therapeutic poten- tial for the treatment of inflammatory conditions, pain, coronary vasospastic disorders, and neurodegeneration. 2,3 However, potent and selective P2Y 2 antagonists are currently not available. 3 In previous studies, we modified the uracil base and the phos- phate residue of UDP and UTP. 15,16 The resulting compounds were agonists at P2Y 2 receptors. In the present study we modified the ri- bose part of UTP with the goal to develop novel P2Y 2 receptor li- gands. We selected UTP over ATP, as a lead structure due to the expected higher selectivity of pyrimidine nucleotides versus the other P2 receptor subtypes that are activated by the adenine nucle- otides ATP or ADP. Furthermore, the degradation products of ATP analogs (the nucleoside adenosine and the nucleobase adenine) may interact with adenosine (P1) or adenine (P0) receptors and thus lead to unwanted side-effects. 3 Our goal was to investigate the consequences of replacing the ribose moiety in uracil nucleo- tides by various acyclic ribose-mimetic structures. 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.05.062 * Corresponding author. Tel.: +49 228 73 2301; fax: +49 228 73 2567. E-mail address: christa.mueller@uni-bonn.de (C.E. Müller).   A.E.-T. is on leave from the University of Al-Azhar, Assiut, Egypt. Bioorganic & Medicinal Chemistry 17 (2009) 5071–5079 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc