In vitro anti-leishmanial and anti-trypanosomal activity of hydrazones , pyrazoles , pyrazolo [ 1 , 5-a ] pyrimidines and pyrazolo [ 3 , 4-b ] pyridine , synthesized from 6-substituted-3-formylchromones

Led by the biological and pharmacological relevance of the 3formylchromone derivatives and its interesting chemistry, in this work we present the synthesis of a series of pyrazoles (4a-c), hydrazones (5a-c), pyrazolo[1,5a]pyrimidines (6a, 6b) and one pyrazolo[3,4-b]pyridine (7) and the report on their in vitro anti-leishmanial and anti-trypanosomal activity. Chemical results showed that the formation of regioisomer 7 may arise from an imine intermediary that undergoes 1,4addition by attack of C-4' from the pyrazole. To the best of our knowledge, this is the first report regarding formation of pyrazolo[3,4-b]-pyridines by intramolecular attack of an sp2 carbon atom. The in vitro studies were performed against strains of Leishmania mexicana (bel 21) and Tripanosoma cruzi (DM28). Compounds 5a and 5b showed activity at micromolar level and good selectivity index (SI) with IC50 values of 6.3 (SI = 3.4) and 15 (SI = 1.9) mM for L. Mexicana and 4.1 (SI = 5.2) and 10 (SI = 3) mM for T. cruzi respectively. From the above-mentioned, compounds 5a and 5b may be considered for further chemical modifications in order to increase their activity as potential antiparasitic agents.


Introduction
The research of compounds with antiparasitic properties is a matter of great importance.Chaga's disease is present mostly in countries of South America with about 5-6 million individuals infected and 25 million at risk [1].Leishmaniasis on the other hand, is a growing public health problem, with an annual incidence of about 1.3 million cases [2].
The pyrazolo [3,4-b]pyridine 7 was obtained in two steps: the reaction of 1b with 3 produced a precipitate which was then refluxed using AcOH as solvent in presence of I 2 (Scheme 1).The formation of regioisomer 8 may arise from an imine intermediary (Scheme 2) that undergoes 1,4-addition at C-2 by attack of C-4' from the pyrazole instead of the nitrogen atom N-2'.To the best of our knowledge, this is the first report regarding formation of pyrazolo [3,4-b]-pyridines by intramolecular attack of an sp 2 carbon atom.
All reactions were monitored by TLC and obtained in good yields (80-95%).The compounds were fully characterized using NMR, IR and MS methods; all physical constants and spectroscopic data were in accordance with those reported in the literature [11,13,14,19].

Antileishmanial and anti-trypanosomal activity
The nitrogenated compounds 4a-c, 5a-c, 6a, 6b and 7 were tested for leishmanicidal and trypanocidal activities.Primary cultures of human dermal fibroblast were used as reference.The selectivity action of the compound over the parasite was calculated using the selectivity index (SI), which is expressed as the ratio of IC 50 on fibroblast to the IC 50 of the corresponding parasite strain.
As observed in Table 1, all tested compound showed good to moderate activity.
Compound 5a exhibited the strongest antiparasitic activity against both parasites L. mexicana and T. cruzi with IC 50 values of 6.3 ± 0.7 M and 4.1 ± 0.3 M respectively.Compound 5a also displayed the highest SI with values of 3.4 for L. mexicana and 5.2 for T. cruzi.Therefore, 5a was the most effective compound tested against both parasites.Compounds 5b and 5c also exhibited good antiparasitic activity since their IC 50 values were relatively low.When 5b was tested against L. Mexicana, the IC 50 was 15 ± 3 M having a SI value of 1.9, while against T. cruzi the IC 50 value was 10 ± 1 M with a SI value of 3. 5c showed activity only for T.cruzi (IC 50 = 13 ± 1 M) with SI value of 2. Compounds 4b and 7 exhibited moderate activity against L. mexicana (IC 50 = 25 ± 2 and 35 ± 2 M, respectively) but in the case of compound 7 the selectivity was poor.
We compared the antiparasitic results action of 4a-c, 5a-c, 6a-6b and 7 with their antilarvae properties [11], and found that the most active compounds against L. mexicana and T. cruzi were also among the most toxic to A. salina.We also evaluated the correlation between leishmanicidal and trypanocidal activities of the synthesized compounds, using a linear regression method.There was a significant association (p<0.05) between both antiparasitic effects.The correlation coefficient was 0.71, indicating a moderately strong relationship.So the most active compounds could be evaluated in a wider range of parasite strains.
The strongest antiparasitic activity was found in the series 5a-c, which has a hydrazone-type structure bearing a 2,4-dinitrophenyl moiety.The hydrazone nucleus has been found to be important in the leishmanicidal activity [15].The presence of the nitro groups could also contribute to the activity since they are able to play a role in antiprotozoal substances [16][17][18].
From the above-mentioned, compounds 5a and 5b may be considered for further chemical modifications, in order to increase their activity and selectivity as potential antiparasitic agents.

Table 1:
In vitro leishmanicidal and trypanocidal activities of synthesized compounds against L. mexicana and T. cruzi.
IC 50 : concentration of the compound that induces 50% growth inhibition in 48 h.Values are expressed as the mean ± SD; SI: selectivity index, is defined as the ratio of IC 50 on fibroblasts to the IC 50 on the corresponding parasite; ND: not determinated.

Antileishmanial and antitrypanosomal activity
Epimastigotes of T. cruzi and promastigotes of L. mexicana were grown at 26°C in liver infusion tryptose (LIT) medium with penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% heat-inactivated FBS.A modified MTT assay was used to determine the compounds IC 50 on each parasite strain [20].Briefly, 199 μL per well of exponential growing parasites at a dilution of 2×10 6 cells per mL, were trasfered into a 96-well flat-bottom plate, and compounds or solvents (1 µL compound or DMSO) were added to evaluate different concentrations.Plates were incubated at 26°C for 48h and 5 days for L. mexicana promastigotes or T. cruzi epimastigotes respectively.Then, parasites were seeded, the medium was removed, and the cells were incubated with MTT solution in PBS (0.2 mg/ml for L. mexicana or 0.5 mg/ml for T. cruzi), F-formazan crystals were dissolved by addition of 100 µL DMSO after plates were centrifuged and MTT solution was removed.The following equation was used to calculate the percentage of growth inhibition (%GI): A p is the absorbance of compound-treated cells, A c is the absorbance of solventtreated cells, and A b is the absorbance of culture medium.The IC 50 value was calculated applying a sigmoidal analysis when %GI and the corresponding concentration were plotted using the Origin 8.0 ® software (OriginLab Corporation).For each condition evaluated at least three independent assays were performed in triplicate.

Conclusions
 All synthesized compounds exhibited good to moderate antiparasitic activities against strains of L. mexicana and T. cruzi.
 Compounds 5a and 5b showed the strongest activities with good selectivity indexes.The results suggest that these molecules are suitable candidates for further chemical modification and evaluation as potential anti-leishmanial and antitrypanosomal agents.
 The good correlation exhibited for the results between both parasite lines indicates that the most active compounds 5a and 5b, could be used in a wider range of parasite strains.
 Finally, the comparison of the antiparasitic results with the previous BSLA study allowed us to conclude that this anti-larvae test may be a good and inexpensive tool in predicting trypanocidal action.