Synthesis, structural characterization and study of antioxidant and anti-PrPSc properties of flavonoids and their rhenium(I)-tricarbonyl complexes

Abstract

This study aims at the synthesis and initial biological evaluation of novel rhenium-tricarbonyl complexes of 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,7,4΄-trihydroxyflavone (resokaempferol), 5,7-dihydroxyflavone (chrysin) and 4΄,5,7-trihydroxyflavonone (naringenin) as neuroprotective and anti-PrP agents. Resokaempferol was synthesized from 2,2΄,4-trihydroxychalcone by H₂O₂/NaOH. The rhenium-tricarbonyl complexes of the type fac-[Re(CO)₃(Fl)(sol)] were synthesized by reacting the precursor fac-[Re(CO)₃(sol)₃]⁺ with an equimolar amount of the flavonoids (Fl) quercetin, resokaempferol, chrysin and naringenin and the solvent (sol) was methanol or water. The respective Re-flavonoid complexes were purified by semi-preparative HPLC and characterized by spectroscopic methods. Furthermore, the structure of Re-chrysin was elucidated by X-ray crystallography. Initial screening of the neuroprotective properties of these compounds included the in vitro assessment of the antioxidant properties by the DPPH assay as well as the anti-lipid peroxidation of linoleic acid in the presence of AAPH and their ability to inhibit soybean lipoxygenase. From the above studies, it was concluded that the complexes' properties are mainly correlated with the structural characteristics and the presence of the flavonoids. The flavonoids and their respective Re-complexes were also tested in vitro for their ability to inhibit the formation and aggregation of the amyloid-like abnormal prion protein, PrP^Sc, by employing the real-time quaking-induced conversion assay with recombinant PrP seeded with cerebrospinal fluid from patients with Creutzfeldt-Jakob disease. All the compounds blocked de novo abnormal PrP formation and aggregation.

Keywords: Anti-oxidant; Flavonoids; Lipid peroxidation; Lipoxygenase; Prion diseases; Rhenium complexes.

Fig. 1

Structure of the Re–tricarbonyl–flavonoid complexes

Scheme 1

Synthesis of resokaempferol

Fig. 2

¹H-NMR comparison of top: quercetin (gray)/Re–quercetin (black); bottom: chrysin (gray)/Re–chrysin (black)

Fig. 3

Structure of [Re(CO)₃(chrysin)(MeOH)], 2(MeOH). Ellipsoids were drawn with 50% probability. Selected bond lengths (Å): Re1—O1 = 2.124 (5); Re1—O2 = 2.092 (5); Re1—O8 = 2.185 (6); Re1—C16 = 1.925 (8); Re1—C17 = 1.921 (9); Re1—C18 = 1.913 (9). Selected angles (.^o): O1—Re1—O2 = 84.2 (2); O1—Re1—O8 = 81.4 (2); O2—Re1—O8 = 84.4 (2); O2—Re1—C16 = 176.5 (3); O1—Re1—C17 = 174.0 (3); O8—Re1—C18 = 177.3 (3)

Fig. 4

RT-QuIC assays were performed for each compound for 80 h. Thioflavin T emission was recorded every 30 min and expressed as relative fluorescence units (RFU). Each reaction consisted of the compound (diluted in DMSO), cerebrospinal fluid (CSF) from healthy controls or patients with sporadic Creutzfeldt–Jakob disease (sCJD) diluted in Phosphate Buffered Saline (PBS) containing 170 mM sodium chloride, 1 mM EDTA, 10 μM Thioflavin-T and 0.1 mg/mL recombinant PrP. For all the compounds except Chrysine two different final concentrations (1 and 20 μM) were tested. The higher concentration of the compounds (20 μΜ) was also added to control reactions, in which CSF from healthy individuals that did not contain the pathogenic PrP^Sc isoform was used as the seed. Furthermore, DMSO only control reactions (DMSO), in which DMSO with a final concentration corresponding to the one obtained with the higher compound concentration and CSF from sCJD patients were used as positive control reactions