Identificação de potenciais novos antimaláricos a partir de produtos naturais de plantas amazônicas, e seus derivados: estudos in vitro, in vivo e de mecanismo de ação

Abstract

Increasing resistance of Plasmodium spp. to the currently available antimalarials has led to the search of new drugs for the treatment of malaria. The Amazon region has one of the greatest biodiversities on the planet with large pharmacological potential for exploration. In this work, the aim was to identify natural products from traditionally used antimalarial plants from the Amazon and derivatives having antimalarial potential through in vitro, in vivo and mechanism studies. 73 extracts and fractions of antimalarial plants were tested for in vitro antimalarial activity against P. falciparum K1 strain. The most active were the 16 extracts and fractions of Picrolemma sprucei (IC50 0.01-3.7 μg/mL), 5 extracts and fractions of Aspidosperma vargasii (IC50 0.14-4.38 μg/mL) and 4 fractions of A. desmanthum (IC50 0.18-0.20 μg/mL). The remaining plants in general exhibited inactive or partially active extracts. 27 substances were tested for in vitro antimalarial activity: indole alkaloids ellipticine (9) (IC50 0.35 μM) and olivacine (10) (IC50 0.35 μM), 5 alkaloids from A. ulei (IC50 16.7 to > 100 μM), the synthetic indole alkaloid cryptolepine triflate (11a, IC50 0.8 μM) and a synthetic analog of cryptolepine (11b, IC50 0.08 μM), semi-synthetic derivatives of 9 (IC50 0.55-17 μM), 4 limonoids (IC50 7.0- 20.7 μM) and a semi-synthetic limonoid 6α-hydroxy-deacetylgedunin" (IC50 5.0 μM), 4- nerolidylcatechol (1) (IC50 0.68 μM) and its O-monobenzyl (4, IC50 7.05 μM), O,O-dibenzoyl (5, IC50 28.73 μM) and O,O-diacetyl (12, IC50 4.85 μM) derivatives. 9 substances were evaluated for possible in vivo antimalarial activity in mice infected with P. berghei. 1 exhibited low in vivo antimalarial activity with maximal inhibition of parasitemia of 63% at an oral dose of 600 mg/kg/day. Derivatives 5 and 12 were more active than the natural product 1 and 12 exhibited the highest suppression of parasitemia (62%) at a dose of 200 mg/kg/day. The limonoids 6α- acetoxygedunin (23) and 7-deacetoxy-7-oxogedunin (24) exhibited maximal in vivo inhibitions of 65.7 and 40.3%, respectively, at oral doses of 100 mg/kg/day. Alkaloids 9 and 10 exhibit excellent in vivo antimalarial activity evidenced by 100 and 97% suppressions of parasitemia, respectively, at oral doses of 50 mg/kg/day and were the most active substances in this assay. 5 substances were selected for studies on the antimalarial mechanism of action. The alkaloids 9-11a and derivatives 4 and 12 were evaluated for potential inhibition of the formation of hemozoin. These substances all inhibited the polymerization of heme. 4 exhibited the lowest activity. Inhibition of the formation of hemozoin is one of the mechanisms of antimalarial action proposed for these substances. The effects of 12 and 4 on the isoprenoid biosynthetic pathway in P. falciparum in vitro were also studied using incorporation of radiolabeled metabolite geranylgeranylpyrophosphate. 4 and 12 exhibited an important inhibitory effect on the biosynthesis of dolichol 12 (45 and 42%, respectively), and 12 also exhibited inhibitory effects on the biosynthesis of ubiquinone 8 (36%) and menaquinone 4 (41%). It is possible to conclude that the índole alkaloids ellipticine and olivacine and derivatives of 4- nerolidylcatechol exhibited potential for continued studies based on the antimalarial potential demonstrated in the present work.