Activity of Pt(II) and Ru(III) Triazolopyrimidine Complexes Against Parasites of the Genus Leishmania, Trypanosomas and Phytomonas

The synthesis and characterization of two Pt(II) Complexes with the isomeric ligands 4,5-dihydro-5-oxo- [1,2,4]triazolo-[ 1,5-a]pyrimidine (5HtpO) and 4,7-dihydro-7-oxo-[ 1,2,4]-triazolo-[ 1,5-a]pyrimidine (7HtpO) are described, as well as a Ru(III) complex with 7HtpO. The crystal structure of cis-[PtCl2(7HtpO)2].2H2O has been solved by X-ray diffraction analysis. In vitro activity of the new isolated complexes against the epimastigote form of T. cruzi, procyclic form of T. b. brucei and promastigote form of L. donnovani and P. characias has also been studied. The three complexes markedly affect the growth of the parasites and none of them shows cytotoxicity against macrophage of the J774.2 line at the heaviest dosages used.


INTRODUCTION
The members of the family Trypanosomatidae are the etiological agents of numerous diseases which afflict not only humans but also animals and plants. Examples include American trypanosomiasis, or Chagas' disease, which is caused by Trypanosoma cruzi, African trypanosomiasis, caused by two strains of Trypanosoma brucei, '-I Leishmania donovani, inflicting visceral leishmaniasis or Kala-azar and Ph)'tomonas characias, which attacks the lactiferous plant Euphorbia chctracias, resulting in defoliation, deformation, chlorosis and atrophy, t3 Even in the 21st century, despite the development of efficient drugs against these protozoan parasites, the eradication of such diseases remains seriously deficient. The administration of these drugs entails a number of drawbacks, such as variable efficacy, normally long treatment often with toxic sideeffects, parenteral administration for some diseases and the appearance of resistant Stl'ains. t4 Another problem is that the development of new drugs requires heavy financial investment from the industry and the areas most in need of these drugs are economically depressed, and therefore the industry takes little interest in developing these drugs. The search for new compounds is, therefore, urgent.
Choosing [1,2,4]triazolo [1,5-a]pyrimidine derivatives for our studies is based on the fact that these ligands could be considered as mimetic of purines. Different researchers have studied them as chemotherapeutic agents, finding different interesting properties, such as the growth inhibition of some microorganisms. With regard to their antitumoural activity, studies have been made for various cell lines such as the breast cancer MDA/MB-231 .8 Their potential in the treatment ofneurodegenerative disorders such as Parkinson's disease has also been tested. 9 The coordination chemistry of these ligands has been extensively studied in recent years.
They have, as potential donors, three endocyclic nitrogen atoms at positions 1, 3 and 4, N(3) being the preferred metal binding site in the monodentate mode (see Figure forthe numbering scheme used for these ligands). Another possibility found in several cases is the bridging N(3)-N(4) mode giving dinuclear metal complexes. -'' 3 We have shown, in previous works, the biological activity of some metal complexes of [1,2,4]triazolo[1,5-a]pyrimidine derivatives. In this way, we have recently tested the effect of several metal complexes of 4,7-dihydro-5-methyl-7-oxo [1,2,4]-triazolo-[1,5-a]pyrimidine (HmtpO),-, against" Phytomonas Staheli (promastigote form), Trypanosoma cruziand Leishmania donovani. 4 ome ofthem proved capable of inhibiting the growth of these flagellates at dosages of50 IaM after 24 h of activity, inhibiting the synthesis of macromolecules (DNA, RNA and proteins) by the parasites and inducing severe damage in their u trastructure. 4) With this new work, we seek to advance one step further in the development of effective chemotherapy against members of the family Trypanosomatidae by testing three newly synthesized metal complexes (two of Pt(II) and one of Ru(III)) oftriazolopyrimidine derivatives on the epimastigote forms of T. cruzi, procyclic forms of T. b. brucei and promastigote forms of L. donovani and P. characias, determining the effect of these ActiviO' of Pt(ll) and Ru(llI) Triazolopyrimidine Complexes Against Parasites of the Genus Leishmania, Trypanosomas and Phytomonas complexes on the in vitro growth of the parasites as well as the possible toxicity of these treatments at macrophage level.
The three metal complexes assayed were dissolved in DMSO, and tested at 1, 20, 50 and 100 tM in culture medium with 0.01% DMSO final concentration. Controls and assays were prepared including 0.01% DMSO. Assays were performed in Roux flasks with a final volume of 10 ml and an original density of lx I0 cells/ml. The cultures were maintained for 24, 48 and 72 h at 28C. For each time and dosage tested, five replicates and corresponding controls were quantified using a Neubauer chamber.

RESULTS AND DISCUSSION NMR spectra
The H-NMR spectra of DMSO-d6 solutions of both platinum complexes, if recorded immediately after dissolving the compounds, display a set of signals of a majoritary species, presurniblely the same present at the solid state. The positions (in ppm.) and assignments of the signals are as follows: Compound 1:6.10 (d, H(6)), 8.08 (d, H(5)), 8 If the DMSO-d6 solutions ofthe complexes are left for several hours and spectra are taken again, we can see that the intensity of the signals assigned to the Pt complexes decrease at the same time that new signals appear and grow, at positions identical to those of the free ligands. This indicate us that the complexes are unstable in DMSO solution, the solvent probablydisplacing the triazolopyrimidineligand fi'om the coordination sphere of the metal atom. This decomposition is almost complete after 24 hours.

Crystal structure of
The molecular structure of 1, depicted in Figure 1, is almost identical to that of the analogous compound with the iigand HmtpO 2 which differs of 7HtpO by the presence of a methyl group at position 5. Both compounds are not, however, crystallographically isostructural.
The platinum atom is in a typical square-planar environment (see distances and angles in Table 2), coordinated by two chloride anions and two N3-bonded 7HtpO ligands in a cis disposition. The relative orientation of the ligands is head-head, stabilized by the presence of a water molecule (OIW) which acts as acceptor of hydrogen bonds of the N4-H groups of both iigands. The second water molecule also contributes to the stabilization of the complex by the formation of hydrogen bonds with O 1W and one ofthe chloride ligands. The three triazolopyrimidine derivatives markedly affect the growth of the epimastigotes of T. cruzi, procyclic forms T. b. brucei and promastigotes of L. donovani cultured in vitro. The results are reflected in Tables 3, 4 and 5. This inhibition is highest for compound 1. On the other hand, in the case of the growth of P. characias cells, 3 proved the most inhibitory, whereas and 2 did not cause any significant growth inhibition, apparently due to the metabolic differences between the promastigote forms of P. characias and the other three trypanosomatids.
None of these derivatives presented cytotoxicity against rnacrophages ofthe J774.2 line (Table 6)  The results are expressed in percentages of cell-growth inhibition (n=5). Significant differences between control and treatment cells were found according to the Newman-Keuls test. * P< 0.05. ** P< 0.025.