title: Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.
abstract: OBJECTIVE
  To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism.
  
  
  METHODS
  The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm.
  
  
  RESULTS
  Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm).
  
  
  CONCLUSIONS
  Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).
authors.affiliation: Phytochemistry and Organic Synthesis Laboratory, Federal University of Rio Grande do Sul, Porto Alegre, Brazil; Plasmodium Molecular and Cellular Biology Laboratory, Department of Physiology, São Paulo University, São Paulo, Brazil.
authors.forename: Gloria N S
authors.initials: GN
authors.lastname: Silva
authors.forename: Desirée C
authors.initials: DC
authors.lastname: Schuck
authors.forename: Laura N
authors.initials: LN
authors.lastname: Cruz
authors.forename: Miriam S
authors.initials: MS
authors.lastname: Moraes
authors.forename: Myna
authors.initials: M
authors.lastname: Nakabashi
authors.forename: Grace
authors.initials: G
authors.lastname: Gosmann
authors.forename: Célia R S
authors.initials: CR
authors.lastname: Garcia
authors.forename: Simone C B
authors.initials: SC
authors.lastname: Gnoatto
mesh_terms: D000962:Antimalarials / Q000138:chemical synthesis / Q000494:pharmacology*; D005434:Flow Cytometry; D057809:HEK293 Cells / Q000187:drug effects; D006420:Hemeproteins / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D053978:Pentacyclic Triterpenes; D010963:Plasmodium falciparum / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014315:Triterpenes / Q000138:chemical synthesis / Q000494:pharmacology*; D053058:Trophozoites / Q000187:drug effects; D000094062:Betulinic Acid
chemical_list: D000962:Antimalarials; D006420:Hemeproteins; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; C020753:hemozoin; D000094062:Betulinic Acid
keywords: Betulinic acid; Plasmodium falciparum; acide bétulinique; action mechanism; derivados del triterpeno; dérivés triterpéniques; malaria; mecanismo de acción; mécanisme d'action; paludisme; piperazina; piperazine; pipérazine; triterpene derivative; Ácido betulínico