ABSTRACT
Malaria remains one of the most serious global infectious diseases. An important target for antimalarial chemotherapy is the enzyme dihydroorotate dehydrogenase from Plasmodium falciparum (PfDHODH), which is responsible for the conversion of dihydroorotate to orotate in the de novo pyrimidine biosynthetic pathway. In this study, we have designed and synthesized fifteen 7-arylpyrazolo[1,5-a]pyrimidine derivatives using ring bioisosteric replacement and molecular hybridization of functional groups based on the highly active 5-methyl-N-(naphthalen-2-yl)-2-(trifluoromethyl)- [1,2,4]triazolo[1,5-a]pyrimidin-7-amine. The compounds were tested against Plasmodium falciparum, as antimalarials in mice with P. berghei, and as inhibitors of PfDHODH. Thirteen compounds were found to be active against P. falciparum, with IC50 values ranging from 1.2 ± 0.3 to 92 ± 26 µM in the anti-HRP2 and hypoxanthine assays. Four compounds showed the highest selective index (SI), which is a ratio between cytotoxicity and activity in vitro. The inhibition of PfDHODH showed that compound 30 (R2 = CH3; R5 = CF3; Ar = 7-ß-naphthyl) displayed higher and selective inhibitory activity, with IC50 = 0.16 ± 0.01 µM, followed by 25 (R2 = CH3; R5 = CH3; Ar = 7-ß-Naphthyl) and 19 (R2 = CF3; R5 = CF3; Ar = 7-ß-naphthyl), with IC50 = 4 ± 1 µM and 6 ± 1 µM, respectively. The trifluoromethyl group at the 2- or 5-positions of the pyrazolo[1,5-a]pyrimidine ring led to increased drug activity. The docking results agreed with the values obtained from enzymatic assays.
Subject(s)
Antimalarials/pharmacology , Enzyme Inhibitors/pharmacology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Animals , Antimalarials/chemistry , Antimalarials/metabolism , Antimalarials/toxicity , Cell Line , Dihydroorotate Dehydrogenase , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/toxicity , Humans , Mice , Molecular Docking Simulation , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Protein Conformation , Pyrimidines/metabolism , Pyrimidines/toxicityABSTRACT
BACKGROUND: Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. METHODS: Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (HssLDH) or plasmodial lactate dehydrogenase (PfLDH) enzymes, and, a ß-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. RESULTS: All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC(50)) values below 1 µΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of PfLDH; specificity between the residues involved in H-bonds of the PfLDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of PfLDH. CQAn33 and CQAn37 inhibited ß-haematin formation with either a similar or a 2-fold higher IC(50) value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. CONCLUSIONS: The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than PfLDH inhibition. The ß-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.
Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Chloroquine/analogs & derivatives , Chloroquine/pharmacology , Drug Resistance , Plasmodium falciparum/drug effects , Antimalarials/toxicity , Cell Survival/drug effects , Chloroquine/toxicity , Hepatocytes/drug effects , Hepatocytes/physiology , Inhibitory Concentration 50 , Molecular Docking Simulation , Parasitic Sensitivity Tests , Structure-Activity RelationshipABSTRACT
A series of novel 6-thiopurine derivates containing 1,2,3-triazole were synthesized and their in vivo antimalarial activity and in vitro antileishmanial activity were examined. The compounds 10, 11, 12 and 14 presented higher values of inhibition of parasite multiplication than chloroquine. For antileishmanial activity, the compound 14 showed activity against the three species of Leishmania tested. None of compounds showed cytotoxicity against mammalian cells.
