Three series of 4-anilino-1H-pyrazolo[3,4-b]pyridine-5-carboxylic esters were synthesized as part of a program to study potential anti-Leishmania drugs. These compounds were obtained by a condensation reaction of 4-chloro-1H-pyrazolo[3,4-b]pyridine with several aniline derivatives. Some of them were also obtained by an alternative pathway involving a Mannich-type reaction. The hydrophobic parameter, log P, was determined by shake-flask methodology, and using the Hansch-Fujita addictive hydrophobic fragmental constants. These compounds were tested against promastigote forms of Leishmania amazonensis. The very promising results showed the 3'-diethylaminomethyl-substituted compounds as the most active [IC50 = 0.39 (21) and 0.12 microM (22)]. Molecular modeling, using semiempirical AM1 method, predicted the most active compounds through the low-energy conformers superimposition on amodiaquine structure. QSAR equations, derived from the IC50 values against L. amazonensis, showed the hydrophobic (log P) and Sterimol steric (L and B2) parameters as most significant contributions on biological activity.

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH(3); MI-4-OCH(3) and MI-4-NO(2)) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO(2) showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO(2). A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO(2) enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas' disease.

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) Visceral leishmaniasis (VL or kala-azar) is the most severe form of leishmaniasis. Approximately 500,000 new cases of human VL occur annually and the disease is mainly found in Brazil, East Africa and on the Indian subcontinent. American visceral leishmaniasis (AVL), caused by Leishmania chagasi, is a major health problem in many parts of Brazil. Traditionally, the disease was confined to rural and peri-urban areas, but in the last few years the number of human cases of AVL in metropolitan regions is dramatically increasing (Silva et al. 2001). It is important to diagnose AVL as early as possible, because the disease is in most cases fatal if left untreated. The principal clinical symptoms of AVL are an enlarged spleen and a prolonged irregular fever (WHO 1996). Other signs and symptoms are loss of weight, pallor, enlarged liver, enlarged lymph nodes, anaemia, cough and diarrhoea. These signs and symptoms may mimic those of malaria, typhoid, tuberculosis, schistosomiasis and other diseases. The clinical suspicion may be confirmed directly by the detection of parasites in patient material or by culture. However, sample retrieval is inconvenient for the patient and parasite isolation by culture is time-consuming, expensive and difficult.Because of the above-mentioned limitations of direct diagnostic methods, a number of indirect immunological tests have been developed. These tests should meet several prerequisites in order to be of practical use. They should be sensitive and specific, cheap (in particular for use in developing countries) and easy to perform under harsh field conditions (Veeken 1999). The direct agglutination test (DAT) remains the first line diagnostic tool in many developing countries as it is a simple test with a high sensitivity, specificity and reproducibility, easyto-perform and not requiring specialised equipment (Zijlstra et al. 1991, Boelaert et al. 1999a,b, Schallig et al. 2001. The introduction of a freeze-dried antigen makes the DAT very suitable for use under remote field conditions as a cold chain for storage of antigen is not required (Meredith et al. 1995, Zijlstra et al. 1997, Schallig et al. 2001. A limitation of the DAT is the relatively long incubation time (18 h) and the fact that serial dilutions of blood or serum must be made. A dipstick, rK39 dipstick, based on the cloned antigen of a 39 amino acid repeat that is part of a 230 kDa protein encoded by a kinesin-like gene of L. chagasi (Burns et al. 1993), may circu...