Potent and Selective Inhibitors of Trypanosoma cruzi Triosephosphate Isomerase with Concomitant Inhibition of Cruzipain: Inhibition of Parasite Growth through Multitarget Activity Elena Aguilera, [a] Javier Varela, [a] Estefanía Birriel, [a] Elva Serna, [b] Susana Torres, [b] Gloria Yaluff, [b] Ninfa Vera de Bilbao, [b] Beatriz Aguirre-López, [c] Nallely Cabrera, [c] Selma Díaz Mazariegos, [c] Marieta Tuena de Gómez-Puyou, [c] Armando Gómez-Puyou, [c] Ruy PØrez-Montfort, [c] Lucia Minini, [d] Alicia Merlino, [d] Hugo Cerecetto, [a, e] Mercedes Gonzµlez, [a] and Guzmµn Alvarez* [a, f] Dedicated to the memory of Armando Gómez-Puyou—an exemplary scientist and friend. Introduction Chagas disease is caused by the parasite Trypanosoma cruzi. It remains the major parasitic disease in Latin America, despite recent advances in the control of its vector-borne and transfu- sion-mediated transmission. [1] Moreover, migration of infected people has spread the disease to non-endemic areas, present- ing a new worldwide challenge. [2] The chemotherapy regime employed to control the parasitic infection employs old and nonspecific drugs, such as Nifurtimox and Benznidazole, and requires long-term treatment that can give rise to severe side effects. [3] Although Nifurtimox and Benznidazole are able to eliminate patent parasitemia and decrease serological titers in acute and early chronic infections, they are not active against all T. cruzi strains, exhibit low efficiency in long-term chronic in- fections, and are mutagenic. [4] Unfortunately, due to a perceived deficit in potential revenue, most pharmaceutical companies have neglected this disease despite the urgent need for new drugs. A variety of molecular targets has been identified for design- ing new drugs, among which are metabolites formed during sterol biosynthesis, glycolysis, and DNA synthesis. [5, 6] An impor- tant characteristic of T. cruzi is its dependence on glycolysis as an energy source for cellular survival. [7] Thus, enzymes of this Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic in- hibitor with an IC 50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cru- zipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these deriv- atives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an im- portant feature in the research and development of future drugs for neglected diseases. [a] E. Aguilera, J. Varela, E. Birriel, Dr. H. Cerecetto, Dr. M. Gonzµlez, Dr. G. Alvarez Grupo de Química Medicinal, Facultad de Ciencias Universidad de la Repfflblica, Iguµ 4225, Montevideo, 11600 (Uruguay) [b] E. Serna, S. Torres, G. Yaluff, Dr. N. V. deBilbao Departamento de Medicina Tropical Instituto de Investigaciones en Ciencias de la Salud Universidad Nacional de Asunción, Asunción, 2511 (Paraguay) [c] B. Aguirre-López, N. Cabrera, S. Díaz Mazariegos, Dr. M. T. de Gómez-Puyou, Dr. A. Gómez-Puyou, Dr. R. PØrez-Montfort Departamento de Bioquímica y Biología Estructural Instituto de Fisiología Celular Universidad Nacional Autónoma de MØxico, MØxico DF, 04510 (MØxico) [d] L. Minini, Dr. A. Merlino Laboratorio de Química Teórica y Computacional, Facultad de Ciencias Universidad de la Repfflblica, Iguµ 4225, Montevideo, 11600 (Uruguay) [e] Dr. H. Cerecetto rea de Radiofarmacia, Centro de Investigaciones Nucleares Facultad de Ciencias, Universidad de la Repfflblica Iguµ 4225, Montevideo, 11600 (Uruguay) [f] Dr. G. Alvarez Laboratorio de MolØculas Bioactivas Centro Universitario Regional Litoral Norte Universidad de la Repfflblica, Rute 3 km 363, Paysandffl, 60000 (Uruguay) E-mail : guzmanalvarezlqo@gmail.com Supporting information and ORCID(s) from the author(s) for this article are available on the WWW under http://dx.doi.org/10.1002/cmdc.201500385. This article is part of a Special Issue on Polypharmacology and Multitarget Drugs. To view the complete issue, visit: http://onlinelibrary.wiley.com/doi/10.1002/cmdc.v11.12/issuetoc. ChemMedChem 2016, 11, 1328 – 1338 # 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1328 Full Papers DOI: 10.1002/cmdc.201500385