Complexation of the anti-Trypanosoma cruzi Drug Benznidazole Improves Solubility and Efficacy Jean Jerley Nogueira Silva, † Wander Roge ´rio Pavanelli, ‡ Fredy R. Salazar Gutierrez, ‡ Francisco Chagas Alves Lima, † Albe ´rico Borges Ferreira da Silva, † Joa ˜o Santana Silva, ‡ and Douglas Wagner Franco* ,† Departamento de Quı ´mica e Fı ´sica Molecular, Instituto de Quı ´mica de Sa ˜o Carlos, UniVersidade de Sa ˜o Paulo (USP), Sa ˜o Carlos, SP, Brazil, Departamento de Bioquı ´mica e Imunologia, Faculdade de Medicina de Ribeira ˜o Preto, UniVersidade de Sa ˜o Paulo (USP), Ribeira ˜o Preto, SP, Brazil ReceiVed October 17, 2007 The ruthenium complex, trans-[Ru(Bz)(NH 3 ) 4 SO 2 ](CF 3 SO 3 ) 2 1, Bz ) benznidazole (N-benzyl-2-(2-nitro- 1H-imidazol-1-yl)acetamide), is more hydrosoluble and more active (IC 50try/1 h ) 79 ( 3 μM) than free benznidazole 2 (IC 50try/1 h > 1 mM). 1 also exhibits low acute toxicity in vitro (IC 50macrophages > 1 mM) and in vivo (400 μmol/kg < LD 50 < 600 μmol/kg) and the formation of hydroxylamine is more favorable in 1 than in 2 by 9.6 kcal/mol. In murine acute models of Chagas’ disease, 1 was more active than 2 even when only one dose was administrated. Moreover, 1 at a thousand-fold smaller concentration than the considered optimal dose for 2 (385 μmol/kg/day ) 100 mg/kg/day), proved to be sufficient to protect all infected mice, eliminating the amastigotes in their hearts and skeletal muscles as observed in H&E micrographics. Introduction American trypanosomiasis or Chagas’ disease (ChD) a remains a significant public health problem and is still one of the major causes of morbidity and mortality from cardiovascular diseases in Latin America despite nearly one century of research. 1 The American Red Cross has recently published a study showing that one in 4655 blood donors in the United States was confirmed as positive for T. cruzi antibodies. 2 Furthermore, actions for preventing transmission of T. cruzi infection by organ transplant or blood transfusion have also been taken in nonen- demic areas such as the European countries, 3 thus showing that this disease is a cause for concern to developed countries as well. 3 Despite the progress achieved in the study of T. cruzi’s biochemistry and physiology in which several enzymes have been identified as potential new targets, a definitive chemo- therapy for this parasitic infection remains undeveloped. 4,5 The compounds benznidazole 2 (Bz), (N-benzyl-2-(2-nitro-1H- imidazol-1-yl)acetamide) and gentian violet 3 (Gv), (N-[4-(bis[4- (dimethylamino)-phenyl]methylene]-2,5-cyclohaxadien-1-ylidene]- N-methyl-methanaminium chloride) are only clinically used for the treatment of ChD and blood sterilization, respectively. However, the Bz treatment is unsatisfactory and exhibit undesir- able side effects such as anorexia, dermatotoxicity, digestive disorders, peripheral polyneuropathy, genotoxicity, and allergic dermopathy attributed to the redox damage to the host’s tissue. 6–10 If administrated in the acute phase, 2 could cure 50-70% of the patients, but a very low cure rate (8-20%) was reported for chronically infected patients even when treated for more than 10 years. 6 Furthermore, several strains of T. cruzi (e.g., Y, Colombiana, CS-28, and VL-10) are naturally Bz- resistant. 11 2 is claimed to act through the formation of free radicals and/or electrophilic metabolites. 9 The nitro group in 2 is reduced to an amino group through the nitroreductases, and this process begins with a reaction catalyzed by NADPH- cytochrome P-450 reductase, producing a nitro anion radical (R-NO 2 •- ), 9 which is involved in its trypanocidal effect by covalent bonding to macromolecules such as the DNA parasite. 9 More recently, efforts have been devoted to the synthesis and evaluation of platinum, palladium, rhenium, copper, gold, and ruthenium derivatives as trypanocidal drugs. 12–14 However, these metal complexes have limited pharmacological application due to their low hydrosolubility and toxic effects. 12–15 An interesting series of ruthenium complexes, 14,15 type [Ru(H 2 O) 2 L 2 ](PF 6 ) 2 , [RuL 2 Cl 2 ], [RuL 3 Cl 3 ] · 2CH 3 OH, [RuL 2 (H 2 O)Cl 3 ] · 2H 2 O, and [Ru(bpy)L 2 ](PF 6 ) 2 ,L ) clotrimazole (Ctz) or ketoconazole (Ktz) less toxic than the ones mentioned above, has been used in the rational design of new antiparasitic agents. This species can block specific lipid biosynthesis pathways by, e.g., inhibiting the enzyme cytochrome P450 14R-demethylase involved in the production of ergoesterol, which are essential for the parasite. 12,14,15 According to the authors, 14,15 these ruthenium complexes are able to inhibit 70% of proliferation of epimastigote forms of T. cruzi. 12 Furthermore, the ruthenium complexes [Ru(Ctz) 2 (H 2 O) 2 ]- (PF 6 ) 2 and [Ru(Ktz) 2 (H 2 O)Cl 3 ] acting in synergism with anti- fungal drugs have proved to be more active than the corre- sponding free ligands. 14,15 Following this approach, the [Ru(NH 3 ) 4 L] n+ moiety has been successfully tested as NO carrier in vitro and in vivo 16,17 because the NO production has been ascribed as a responsible for the * To whom correspondence should be addressed. Phone: +55 16 3373 9976. Fax: +55 16 3373 9976. E-mail: douglas@iqsc.usp.br. Address: Av. Trabalhador Sa ˜o-Carlense, 400 CP 780, 13560-970 Sa ˜o Carlos, SP, Brazil. This paper is based on part of the J. J. N. Silva, Ph.D. Thesis and is presented as the Brazilian patent of invention PI 0704577-8. † Departamento de Quı ´mica e Fı ´sica Molecular, Instituto de Quı ´mica de Sa ˜o Carlos, Universidade de Sa ˜o Paulo. ‡ Departamento de Bioquı ´mica e Imunologia, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo. a Abbreviations: BT, bloodstream trypomastigote forms of Trypanosoma cruzi; Bz, benznidazole; ChD, Chagas’ disease; DFT, density functional theory; E aq , total energy in aqueous phase; E s , solvation energy; ΔE NO2 , difference in reduction potential of NO 2 moiety between 1 and 2; GAP ) ELUMO - E HOMO ; IC 50try/1 h , inhibitory concentration on trypomastigote forms after 1 h incubation; IC 50try/4 h , inhibitory concentration on trypomastigote forms after 4 h incubation; IC 50try/24 h , inhibitory concentration on trypo- mastigote forms after 24 h incubation; MLCT, metal-to-ligand charge- transfer transition; MO, molecular orbital; NBO, natural bond orbital analysis; PBS, phosphate-buffered saline; Protocol A, treatment for 15 consecutive days; Protocol B, treatment only on the fifth, sixth, and seventh days preceding the parasitaemic peak; S aq , water solubility; SCE, aqueous saturated calomel electrode; % TA, percentage of trypanocidal activity; T. cruzi, Trypanosoma cruzi. J. Med. Chem. 2008, 51, 4104–4114 4104 10.1021/jm701306r CCC: $40.75  2008 American Chemical Society Published on Web 06/21/2008