1. Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase 1 (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoural immune resistance. 1 Many human tumours constitutively express the enzyme. 2 IDO1 inhibition has accordingly been an active area of research in drug development. 3 Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalysing the first step of tryptophan degradation, is as well expressed in many tumours, where it prevents their rejection by means of locally depleting tryptophan. 4 The complementary role of tryptophan catabolites in this process was demonstrated by others. 5 We therefore set out to develop new, improved TDO inhibitors using as the starting point the only, unoptimised series previously known in the literature. 6 Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase 1 (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoural immune resistance. 1 Many human tumours constitutively express the enzyme. 2 IDO1 inhibition has accordingly been an active area of research in drug development. 3 Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalysing the first step of tryptophan degradation, is as well expressed in many tumours, where it prevents their rejection by means of locally depleting tryptophan. 4 The complementary role of tryptophan catabolites in this process was demonstrated by others. 5 We therefore set out to develop new, improved TDO inhibitors using as the starting point the only, unoptimised series previously known in the literature. 6 3-ALKENYL INDOLES AS TRYPTOPHAN 2,3-DIOXYGENASE INHIBITORS FOR THE ENHANCEMENT OF CANCER IMMUNOTHERAPY Eduard Dolušić, a Luc Pilotte, b Laurence Moineaux, a Pierre Larrieu, b Vincent Stroobant, b Didier Colau, b Lionel Pochet, a Etienne De Plaen, b Catherine Uyttenhove, b Benoît Van den Eynde, b Johan Wouters, a Bernard Masereel, a Steve Lanners a and Raphaël Frédérick a 1 ) Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, B-5000 Namur, Belgium 2 ) Ludwig Institute for Cancer Research, Brussels Branch, and de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium eduard.dolusic@unamur.be 1. Introduction 2. Synthesis and SAR 7 Scheme 1. Synthetic schemes for (2-pyridin-3-yl)vinylarenes Table 1. TDO inhibitory potency of analogues 3 - 41. IC 50 values tested in cells transfected with mouse TDO (mTDO) Scheme 2. Synthetic schemes for modifications of the side chain Scheme 3. Synthetic schemes for linker modifications comp.Ar mTDO IC 50 / [  M] comp.R mTDO IC 50 / [  M] comp.R mTDO IC 50 / [  M] comp.R mTDO IC 50 / [  M] 30 6 112 1-Me, 6-F >200225-Cl20326-Br>200 31132-Me>200235-Br40336-Me>200 4 142-Ph>200245-Me>200346-OMe>200 5 154-F10255-OMe>200356-OH>200 6 164-Cl>40265-CN>200366-CO 2 Me>200 718174-Br>200275-NO 2 >200377-F50 - 100 8>200184-CN>200285-CO 2 Me>200387-Cl>200 9 194-NO 2 >200295-CO 2 H>200397-Br>20 10>200204-CO 2 Me>200306-F1407-Me>200 11>200215-F5316-Cl20417-OMe>200 Table 2. TDO inhibitory potency of indole derivatives with different side chains (tested as above) comp.RR'R'' mTDO IC 50 / [  M] comp.RR'R'' mTDO IC 50 / [  M] comp.RR'R'' mTDO IC 50 / [  M] 3HH149F3-F-PhH1058F H2 30FH150F3-Cl-PhH>20059FCO 2 MeH2 42HH2051F3-Br-PhH>20060HCO 2 HH18 43FH352F3-OMe-PhH1061FCO 2 HH3 44HH2053F3-CN-PhH162FCH 2 OHH80 45FH354F3-NO 2 -PhH364FPhCN>80 46HMeH>8055-transHCNH1365FCO 2 EtCN>80 47FPhH4056-transFCNH366FCO 2 HCN>80 48FH>20057HH1067FCO 2 H >80 Table 3. TDO inhibitory potency of indole derivatives with different linkers (tested as above) comp.XR mTDO IC 50 / [  M] comp.XR mTDO IC 50 / [  M] 3173CN>80 688074CN44 6965710 55-transCN1375>80 55-cisCN>8076>80 71CN>8077>80 72CN>8078>80 Table 5. Oral bioavailability of 30 and 58 in mice (administr. 160 mg/kg/day) 8 30 58 = LM 10 Figure 1. View of 58 docked inside the TDO binding cleft 7 Table 4. Exp. solubility and stability for 30, 58 and 61 7 Figure 2. Reversal of tumoural immune resistance by systemic inhibition of TDO 8 3. Docking, physicochemistry and in vivo properties 1) Munn, D. H. and Mellor, A. L., J. Clin. Invest. 2007, 117, 1147-1154; Katz, J. B., et al, Immunol. Rev. 2008, 222, 206-221; Prendergast, G. C., Oncogene 2008, 27, 3889-3900. 2) Uyttenhove, C., et al, Nat. Med. 2003, 9, 1269-1274. 3) Macchiarulo, A., et al, Amino Acids 2009, 37, 219-229; Liu, X., et al, Blood 2010, 115, 3520-3530; Röhrig, U. F., et al, J. Med. Chem. 2010, 53, 1172-1189; Dolušić, E., et al, Bioorg. Med. Chem. 2011, 19, 1550-1561. 4) Van den Eynde, B., et al, WO2010008427, 2010; 5) Opitz, C. A., et al, Nature 2011, 478, 197-203; 6) Madge, D. G., et al, Bioorg. Med. Chem. Lett. 1996, 6, 857-860. 7) Dolušić, E., et al, J. Med. Chem. 2011, 54, 5320-5334; Moineaux, L., et al, Eur. J. Med. Chem. 2012, 54, 95-102; 8) Pilotte, L., et al, Proc. Natl. Acad. Sci. USA 2012, 109, 2497 – 2502. This work was supported in part by FNRS-Télévie (7.4.543.07). 4. 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