AROMATIC THIOSEMICARBAZONES ARE INHIBITORS OF TRYPTOPHAN 2,3-DIOXYGENASE (TDO), AN EMERGING TARGET FOR CANCER TREATMENT Introduction and aim of the work. Indoleamine 2,3-dioxygenase (IDO; EC ) is a heme dioxygenase that catalyzes the first and rate-limiting step of the kynurenine pathway of tryptophan catabolism. The resulting local tryptophan depletion and catabolite formation are important causes of peripheral immune tolerance contributing to tumoral immune resistance. IDO inhibition is thus an active area of research in drug development. 1 Recently, our group has shown that tryptophan 2,3-dioxygenase (TDO; EC ), a structurally unrelated hepatic enzyme catalyzing the same reaction, is also expressed in many tumors. This expression similarly prevents tumor rejection. 2 Very recently, we published a structure-activity study on a series of 3-ethenylindoles as TDO inhibitors. 3 The best compound, LM10 (58), was chosen for preclinical evaluation in mice in order to decipher the exact role of TDO in cancer immunosuppression. Thiosemicarbazones are pharmacologically interesting compounds exhibiting a spectrum of biological activities. 4 Compounds of this class, such as marboran (an antiviral used against pox viruses) and triapine (a ribonucleotide reductase inhibitor used in anti-cancer treatment), have been advanced into clinical trials. Their biological actions have generally been attributed to metal chelating properties 5. We set out to synthesize a range of thiosemicarbazones and investigate their inhibitory potencies on IDO and TDO in cellular assays. References. [1] (a) Uyttenhove, C. et al, Nat. Med. 2003, 9, ; (b) Macchiarulo, A. et al, Amino Acids 2009, 37, ; (c) Röhrig, U. et al, J. Med. Chem. 2010, 53, ; (d) Dolušić, E. et al, Bioorg. Med. Chem. 2011, 19, ; (e), Dolušić, E. et al, Eur. J. Med. Chem. 2011, 46, [2] Van den Eynde, B. et al, 2010, WO [3] Dolušić, E. et al, J. Med. Chem. 2011, doi: /jm [4] (a) Beraldo, H. et al, Mini-Rev. Med. Chem. 2004, 4, 31–39; (b) Duffy, K.J. et al, J. Med. Chem. 2002, 45, [5] Yu, Y. et al, J. Med. Chem. 2009, 52, [6] Husain, K. et al, Eur. J. Med. Chem. 2008, 43, [7] Rizal, R. M. et al, Acta Cryst. 2008, E64, o919–o920. [8] Madge et al, Bioorg. Med. Chem. Lett. 1996, 6, This work was supported by FNRS-Télévie and by the Walloon Region (grant ‘CANTOL’ n° 5678). Eduard Dolušić, a Sara Modaffari, a Pierre Larrieu, b Christelle Vancraeynest, a Luc Pilotte, b Didier Colau, b Vincent Stroobant, b Johan Wouters, a Bernard Masereel, a Benoît Van den Eynde b and Raphaël Frédérick a a Drug Design and Discovery Center, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium ; b Ludwig Institute for Cancer Research, Université Catholique de Louvain, 74 Avenue Hippocrate, 1200 Brussels, Belgium Synthesis. The compounds were synthesized by a condensation of an appropriate (hetero)aromatic aldehyde with the corresponding thiosemicarbazide in ethanol, either by conventional 6,7 or microwave heating. If the required aldehyde was not commercially available, it was prepared by a Vilsmeier-Haack formylation of the parent heterocycle. The double bond geometry in the final products was assumed to be (E), as suggested both by literature 7 and our own NOESY experiments. Biological evaluation Results and conclusion. The influence of various aromatic scaffolds was first appraised (Table 1). Only the indole ring (entry 1) provided a TDO inhibitor of a modest potency while being devoid of any IDO inhibitory activity. We thus decided to explore indole carbaldehyde thiosemicarbazones as potential selective TDO inhibitors. Substitution of the indole core was next evaluated (Table 2). The results revealed a very narrow SAR at this part of the molecule. Only a fluorine atom in certain positions seemed to be tolerated, the best compound (entry 16) being the 6-fluoro derivative, analogously to LM10 and the literature compound 680C91. 8 Introducing a substituent on the N-4 atom caused a drop in potency (Table 3, entries 20 and 21). A methyl substituent on the  -C atom of the thiosemicarbazone chain (Table 3, entry 22), had a similar effect. Finally, methylating the N-2 atom provided the most potent compounds (Table 3, entries 24 and 25). 6-F derivative 25 is equipotent to LM10, suggesting this series could yield effective pharmacological tools for anti-cancer treatment with a novel mechanism of action. LM10 (58), IC 50 = 2  M in a cellular TDO assay 3 Table 1 Table 2 Table 3