1. Indoleamine 2,3-Dioxygenase Inhibitory Activity of Derivatives of Marine Alkaloid Tsitsikammamine A Dr. Eduard Dolušić Drug Design and Discovery Center Department of Pharmacy University of Namur (FUNDP), Belgium

2. Indoleamine 2,3-Dioxygenase (IDO) Dr. Eduard Dolušić JFB 2011 Liège 2 R. Schwarcz, Curr. Opin. Pharmacol. 2004, 4, 12.  monomeric (~45 kDa)  protoheme IX prosthetic group  extrahepatic  other substrates: serotonin, melatonin, tryptamine…

3. Interest in IDO as a drug target Dr. Eduard Dolušić JFB 2011 Liège 3  many human tumors constitutively express IDO Uyttenhove, C. et al, Nat Med 2003, 9, 1269.  IDO expression in placenta is required for immune tolerance of fetus by the mother Munn, D. H. et al, Science 1998, 281, 1191. Katz, J. B. et al, Imm. Rev. 2008, 222, 206.  cancer immunotherapy shows limited efficacy in vivo; IDO plays a crucial role Munn, D. H.; Mellor, A. L. J. Clin. Invest. 2007, 117, 1147. Prendergast, G. C. Oncogene 2008, 27, 3889.

4. Sugimoto, H. et al, PNAS 2006, 103, 2611. Dr. Eduard Dolušić JFB 2011 Liège 4 IDO inhibitors - background  inhibitors (lit.): IC 50 = 4.8-7.7  M Kumar et al, JMC 2008, 51, 4968. K i = 61–70 nM Kumar et al, JMC 2008,51, 1706. K i = 200 nM Carr et al, JMC 2008,51, 2634. IC 50 =59–86 nM Yue et al, JMC 2009,52, 7364. IC 50 = 50  M Röhrig et al, JMC 2010, 53, 1172. IC 50 ~100  M clinical trials

5. Dr. Eduard Dolušić JFB 2011 Liège 5 IDO inhibitors discovered by virtual screening Dolušić, E. et al; Bioorg. Med. Chem. 2011, 19, 1550. Dolušić, E. et al; EJMC 2011, doi:10.1016/j.ejmech.2011.02.049.

6. Dr. Eduard Dolušić JFB 2011 Liège 6 Dolušić, E. et al; Bioorg. Med. Chem. 2011, 19, 1550. Dolušić, E. et al; EJMC 2011, doi:10.1016/j.ejmech.2011.02.049. Work on another (HTS) series in progress. IDO inhibitors discovered by virtual screening

7. Dr. Eduard Dolušić JFB 2011 Liège 7 Marine pyrroloquinoline alkaloids: wakayin and tsitsikammamines Legentil, L. et al; JMC 2006, 49, 2979. Delfourne, E.; Anti Canc. Agents Med. Chem. 2008, 8, 910.  wakayin (1): isolated in 1991 from the ascidian Clavelina species  reported to have diverse bioactivities including cytotoxicity and topoisomerase I inhibition  tsitsikammamines A (2) and B (3): isolated in 1996 from a Latrunculiidae sponge  similar bioactivity profile to wakayin  pyrazolic derivatives of general (a) and (b) structures also exhibit topoisomerase I/II inhibitory activity; some of them are cytotoxic

8. Dr. Eduard Dolušić JFB 2011 Liège 8 2010: two novel regioisomeric series of tsitsikammamine A analogues - synthesis Rives, A. et al; Eur. J. Med. Chem. 2010, 45, 343.  synthesized on the basis of the Michael addition chemistry a)abs. EtOH, rt, 2h; b) TFA, CH 2 Cl 2, rt, 4h; c) MnO 2, CH 2 Cl 2, rt, overnight; d) abs. EtOH, 4Å MS, , 3h a)1M NaOH, dioxane, rt, overnight; b) BBr 3, CH 2 Cl 2, N 2, 4h

9. Dr. Eduard Dolušić JFB 2011 Liège 9 Rives, A. et al; Eur. J. Med. Chem. 2010, 45, 343. 2010: two novel regioisomeric series of tsitsikammamine A analogues - synthesis a)abs. EtOH, rt, 2h; b) TFA, CH 2 Cl 2, rt, 4h; c) MnO 2, CH 2 Cl 2, rt, overnight; d) 1M NaOH, dioxane, rt, overnight; e) BBr 3, CH 2 Cl 2, N 2, 4h

10. Dr. Eduard Dolušić JFB 2011 Liège 10 Rives, A. et al; Eur. J. Med. Chem. 2010, 45, 343.  products were evaluated in an MTT colorimetric antiproliferative assay against several cancer and normal (fibroblast) cell lines  a cytotoxic synthetic intermediate 8b with an unknown mechanism of action was identified 2010: two novel regioisomeric series of tsitsikammamine A analogues – pharmacological evaluation

11. Dr. Eduard Dolušić JFB 2011 Liège 11 IDO inhibitory activity of tsitsikammamine derivatives

12.  several tsitsikammamine A analogues display (sub)micromolar potencies in an IDO enzymatic test Conclusion  a number of derivatives are also reasonably active in an IDO cellular test without being toxic  this class of compounds is a potential source of leads for the development of new anticancer compounds with a novel pharmacological profile (manuscript in preparation) Dr. Eduard Dolušić JFB 2011 Liège 12

13. Dr. Eduard Dolušić JFB 2011 Liège 13 D 3 C, FUNDP Namur Eduard Dolušić Raphaël Frédérick Bernard Masereel Sara Modaffari Laurence Moineaux Christelle Vancraeynest Johan Wouters Ludwig Institute for Cancer Research Brussels Branch Didier Colau Pierre Larrieu Luc Pilotte Vincent Stroobant Benoît Van den Eynde Euroscreen SA Sébastien Blanc Delphine Colette Graeme Fraser Université Paul Sabatier Laboratoire SPMIB Toulouse, France Evelyne Delfourne Arnaud Rives ULB Brussels Lab. de Toxicologie Robert Kiss Benjamin Le Calvé Collaborators €€€ FNRS (Belgium) Biowin (CANTOL: convention n5678) Thank you for your attention!