Antitrypanosomal and Antiplasmodial Activity of

1. Aim of the study: • In vitro evaluation of new series of bis(2-aminoimidazolines) and their bisguanidine • counterparts4 against T. b. rhodesiense (STIB900) and P. falciparum (K1). • 2) Evaluation of the selectivity index (SI = IC50 mammalian L6-cells/ IC50 parasite). • 3) Study of the mechanism of action: • - DNA binding at AT-sites (DTm). • - Ferriprotoporphyrin IX binding inhibition test (FBIT).5 Table 2. Inhibition ofFPIX biomineralisation a The percentage of inhibition at 1 mg/mL (highest concentration tested) is given when the IC50 could not be determined. b Taken from ref. 4. a Gua = guanidinium, Imi = 2-aminoimidazolinium, (Boc)Gua = N,N’-di(tert-butoxycarbonyl)guanidine, (Boc)Imi = N,N’-di(tert-butoxycarbonyl)imidazolidin-2-yl; bT. brucei rhodesiense STIB900 strain. Control: melarsoprol, IC50 = 0.0055 mM; cP. falciparum K1 strain. Control: chloroquine, IC50 = 0.278 mM; d Data taken from ref. 1; e Data taken from: Arafa et al.J. Med. Chem. 2005, 48, 5480. Antitrypanosomal and Antiplasmodial Activity of Bis(2-aminoimidazoline) and Bisguanidine Derivatives Christophe Dardonvillea (dardonville@iqm.csic.es), Lidia Nietoa, Fernando Rodríguezb, Isabel Rozasb, Marcel Kaiserc, Reto Brunc, Binh Nguyend, W. David Wilsond, Rory Nelson Garcíae a Instituto de Química Médica, CSIC, Juan de la Cierva 3, E–28006 Madrid, Spain; b Centre for Synthesis and Chemical Biology, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland; c Swiss Tropical Institute, Socinstrasse, 57, CH-4002 Basel, Switzerland; d Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA; e Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Av. complutense s/n, E-28040 Madrid, Spain. Antecedents:Recent findings by our group have shown that bisguanidine and especially bis(2-aminoimidazoline)diphenyl compounds displayed potent antitrypanosomal activity in vitro1 and vivo2 against T. b. rhodesiense, the causative agent of acute human African trypanosomiasis (HAT). In addition, a correlation between antitrypanosomal activity and DNA binding affinity was observed, suggesting a possible mechanism of action for these compounds.2 We also showed that this kind of molecules entered into trypanosomes via different transporters in addition to P2, indicating that parasites that have lost the P2 transporter in selection of resistance to other drugs will not show cross-resistance to this class of molecules. Others have described excellent antiplasmodial activity of related aromatic dicationic structures such as pentamidine or DB289.3 This prompted us to test our compounds against P. falciparum as well. • In vitro antiprotozoal activity[Table 1]: • Antitrypanosomal activity: 9 new compounds with IC50 < 100 nM. • Best anti-T. brucei agent in vitro: bis(2-aminoimidazoline)fluorene 13b. • Antiplasmodial activity: 20 dicationic compounds with IC50 < 50 nM. • Best antimalarial compound in vitro: 1b and 13b. • Two cations are essential for good activity (data not shown) • Boc-protected derivatives (1d, 1e, 6e) show excellent in vitro antimalarial activity. • 2) Selectivity index (safety profile)[Table 1]: • Higher selectivity for the 2-aminoimidazolinium cation vs guanidinium. 3) Possible mechanism of action[Table 2]: • DNA binding: -Significant binding to A-T sites for several compounds: 1a, 1b, 5a, 12a, 12b, 9a, 9b, 13b. - DTm (4,4’-Imi) > DTm (4,4’-Gua) in all cases. • FBIT: - Some compounds show FPIX biomineralization inhibition similar to quinine - There is no correlation between FPIX inhibition and antimalarial activity. Table 1. In vitro antitrypanosomal, antiplasmodial and DNA binding activity of diphenyl dicationic compounds. Conclusions: • Bis(2-aminoimidazoline) compounds are promising anti-T. brucei and antimalarial agents with better safety profile vs bisguanidinecounterparts. • Binding to DNA minor groove possibly plays a role in the antiprotozoal activity. • Inhibition of heme biosynthesis may be responsible to some extent for the observed antiplasmodial activity. • In vivo assays are ongoing. Acknowledgements: This work was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (RB), PIE grant 200680I121 from the CSIC (CD, LN), the “Programa Nacional de Biomedicina” SAF2006-04698 grant from the Spanish “Ministerio de Educación y Ciencia” (RNG), Cycle III HEA PRTLI grant by means of the CSCB in Ireland,  and by a grant of the Consejeria de Educacion Cultura y Deporte de la Comunidad Autonoma de La Rioja, Spain (F.R.). References:[1] Dardonville, C.; Brun, R. J Med Chem 2004, 47, 2296. [2] Dardonville, C.; Barrett, M. P.; Brun, R.; Kaiser, M.; Tanious, F.; Wilson, W. D. J Med Chem 2006, 49, 3748. [3] Soeiro, M. N. C.; De Souza, E. M.; Stephens, C. E.; Boykin, D. W. Expert Opin Inv Drug 2005, 14, 957. [4] Rodriguez, F.; Rozas, I.; Ortega, J.E.; Meana, J.J.; Callado, L.F. J. Med. Chem., 2007 (submitted). [5] Deharo, E.; Garcia, R. N.; Oporto, P.; Gimenez, A.; Sauvain, M.; Jullian, V.; Ginsburg, H. Exp Parasitol 2002, 100, 252.