Original article Synthesis of pyrazolo[1,5-a][1,3,5]triazine derivatives as inhibitors of thymidine phosphorylase Lingyi Sun, Hriday Bera, Wai Keung Chui * Department of Pharmacy, Faculty of Science, National University of Singapore,18 Science Drive 4, Singapore 117543, Singapore article info Article history: Received 28 November 2012 Received in revised form 21 March 2013 Accepted 28 March 2013 Available online 10 April 2013 Keywords: Pyrazolo[1,5-a][1,3,5]triazines Thymidine phosphorylase Enzyme inhibitor abstract Thymidine phosphorylase (TP) is an enzyme that promotes tumor growth and metastasis and therefore is an attractive druggable target. Using a reported TP inhibitor, 7-deazaxanthine (7DX), as the lead compound; this study was set up to evaluate whether pyrazolo[1,5-a][1,3,5]triazin-2,4-diones and pyr- azolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones would exhibit TP inhibitory activity. The pyrazolo[1,5-a][1,3,5] triazine nucleus was constructed using a reaction that annulated the 1,3,5-triazine ring onto a pyrazole scaffold. Among the 52 compounds synthesized and tested, it was found that 1,3-dihydro-pyrazolo[1,5-a] [1,3,5]triazin-2-thioxo-4-ones exhibited various extent of inhibitory activity against TP. The best com- pound 17p, which bears a para-substituted pentafluorosulfur group, showed an IC 50 value of 0.04 mM, which was around 800 times more potent than the 7DX (IC 50 ¼ 32 mM) under the same bioassay con- ditions. The results of the study suggested that a substituent with þs and þp properties inserted at position 4 of a phenyl ring that is attached to position 8 of the pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4- one scaffold would give excellent TP inhibitory action. In addition, 17p was found to be a non-competitive inhibitor thus suggested that it might interact with TP at a position different from the substrate binding site. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Thymidine phosphorylase (TP) is an enzyme that catalyzes the reverse phosphorolysis of pyrimidine nucleosides. It plays a key role in maintaining the balance of the nucleotide pool and con- trolling nucleic acid homeostasis by ensuring an ample supply of deoxyribonucleoside triphosphates (dNTPs) for DNA replication and repair [1]. However, besides pyrimidine nucleosides, TP can also induce the phosphorolysis of several nucleoside analog therapeutic agents thus reduce their bioavailability [2]. Therefore, it has been proposed that a co-administration of a nucleoside analog thera- peutic agent together with a TP inhibitor might improve the bio- logical efficacy of the therapeutic agent. TP has been also found to be associated with tumor angiogenesis [3] and metastasis [4,5], and it can promote tumor growth by preventing apoptosis as well [6]. Therefore, in addition to improving the biological efficacy of nucleoside analog therapeutic agents, the clinical implication of inhibiting TP has broadened to include controlling the develop- ment of tumor. Structurally, most of the active TP inhibitors are derivatives of pyrimidine and its analogs which were designed to interact with the thymidine binding site. The best inhibitor found to date is 5- chloro-6-[1-(2-iminopyrrolidinyl)methyl] uracil hydrochloride (TPI) [7] which exhibits strong inhibitory activity as denoted by a reported IC 50 value of 0.035 mM [8]. Besides pyrimidines, some purine analogs such as 7-deazaxanthine (7DX) [9] (Fig. 1) has been found to demonstrate inhibitory activity against TP too. However, 7DX (IC 50 value ¼ 40 mM [9]) is not as potent as TPI, and further structural modifications may lead to more potent purine-like TP inhibitors. In addition, some multisubstrate inhibitors designed for interacting with both the thymidine and phosphate binding sites have been reported in the literature [10e12]. However, these multisubstrate inhibitors showed weaker inhibition against TP, and they inhibited the enzyme in its opened conformation. The pyrazolo[1,5-a][1,3,5]triazine heterocyclic system is recog- nized as an analog of purine in which the 9-N atom is translocated to position 5 of the bicyclic ring system. This heterocycle has been substituted for purine in the area of nucleoside chemistry and many biologically active agents have been developed [13]. * Corresponding author. Postal Address: Department of Pharmacy, Faculty of Science, Block S4,18 Science Drive 4, Singapore 117543, Singapore. Tel.: þ65 6516 2933; fax: þ65 6779 1554. E-mail addresses: g0801101@nus.edu.sg (L. Sun), g0701228@nus.edu.sg (H. Bera), phacwk@nus.edu.sg (W.K. Chui). Contents lists available at SciVerse ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.03.063 European Journal of Medicinal Chemistry 65 (2013) 1e11