Antiplasmodial activity, structure-activity relationship and studies on the action of novel benzimidazole derivatives

Abstract

Malaria cases and deaths keep being excessively high every year. Some inroads gained in the last two decades have been eroded especially due to the surge of resistance to most antimalarials. The search for new molecules that can replace the ones currently in use cannot stop. In this report, the synthesis of benzimidazole derivatives guided by structure-activity parameters is presented. Thirty-six molecules obtained are analyzed according to their activity against P. falciparum HB3 strain based on the type of substituent on rings A and B, their electron donor/withdrawing, as well as their dimension/spatial properties. There is a preference for electron donating groups on ring A, such as Me in position 5, or better, 5, 6-diMe. Ring B must be of the pyridine type such as picolinamide, other modifications are generally not favorable. Two molecules, 1 and 33 displayed antiplasmodial activity in the high nanomolar range against the chloroquine sensitive strain, with selectivity indexes above 10. Activity results of 1, 12 and 16 on a chloroquine resistance strain indicated an activity close to chloroquine for compound 1. Analysis of some of their effect on the parasites seem to suggest that 1 and 33 affect only the parasite and use a route other than interference with hemozoin biocrystallization, the route used by chloroquine and most antimalarials.

Figure 1

Procedure for the synthesis of 2-amidobenzimidazole derivatives.

Figure 2

General structure of 2-amidobenzimidazole derivatives.

Figure 3

Membrane integrity tests. BZ 1, 5, 16, 26 and 33 were tested in triplicates in two independent experiments. (a) Incorporation of propidium iodide (PI) was analyzed through fluorometry after 3 h incubation with the compounds. (b) Hemolysis was measured by light absorbance 24 h after exposure to the compounds. One-way ANOVA with Turkey´s comparison was used for analysis of significance. ****p < 0.001.

Figure 4

Effect of selected benzimidazole derivatives on ROS production. Five potent compounds were incubated with P. falciparum-infected cultures and the production of reactive oxygen species was measured through fluorometry at 0.5, 6 and 9 h after their addition at 1 µM. As positive control, a sample containing hydrogen peroxide at 200 μM was added. Samples were measured in triplicates. One-way ANOVA with Turkey´s comparison was used to measure significance with respect to the untreated control at each different time point. n = 2. (a) BZ 1, (b) BZ 5, (c) BZ 16, (d) BZ 26 and (e) BZ 33. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 5

Changes to mitochondrial membrane potential caused by antiplasmodial compounds. Fluorometry was used to measure the effects on the mitochondria of P. falciparum parasites caused by treatment with compounds that show activity against their growth in culture. A membrane disruptor (CCCP) was added as a positive control and untreated cultures were used as the negative one. All compounds were tested at 1 µM in triplicates, n = 3. (a) BZ 1, (b) BZ 5, (c) BZ 16, (d) BZ 26 and (e) BZ 33. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 6

Influence in the biocrystallization of hemozoin in P. falciparum. Compounds 1, 16 and 33 were added to Pf cultures and the amount of hemozoin produced was analyzed after 36 h. Mefloquine (MQ) was used as a positive control. The experiment was performed in triplicates and analyzed with one-way ANOVA. *p < 0.05.