Evaluation of naphthoquinones identified the acetylated isolapachol as a potent and selective antiplasmodium agent

Figures & data

Figure 1. Structures of naphthoquinones investigated here as anti-P. falciparum compounds.

Table 1. In vitro antiparasitic activity and cytotoxicity.

Compounds	P. falciparum W2 strain IC50 ± SD (μM)^a	Splenocytes CC50 ± SD(μM)^b	SI^c
lapachol	80.4 ± 20	26.2 ± 3.5	0.3
Compound 1	2.3 ± 0.1	9.07 ± 0.9	3
α-lapachone	15.8 ± 3.5	13.9 ± 0.8	0.8
Compound 2	19.4 ± 1.7	21.4 ± 1.0	1.1
β-lapachone	20.5 ± 1.0	3.0 ± 0.5	0.1
Compound 3	15.8 ± 2.0	13.7 ± 0.5	0.8
mefloquine	0.04 ± 0.01	5.2 ± 0.4	131

^aAntiparasitic activity was determined in erythrocytic P. falciparum after 48 h incubation with the compounds.

^bCytotoxicity was determined in splenocytes after 24 h incubation with the compounds.

^cSI: selectivity index, calculated from the CC₅₀/IC₅₀. SD: standard deviation of two experiments.

Figure 2. Antiparasitic activity of compound 1 in the cell-cycle development of blood-stage P. falciparum. Erythrocytes were infected with W2 strain P. falciparum (synchronized to ring stage) and incubated with compound 1 at 2.3 and 4.7 µM during one cycle. Parasites were analyzed by optical microscopy in different times. Significance in reference to % of parasitemia: *p < 0.05; ***p < 0.001.

Table 2. Effects on the hematin formation and hemolysis.

	Hematin formation^a		Hemolysis^b
Compounds	% inhibition at 2.0 mM	IC50 ± SD (mM)^b	% hemolysis (200 µM)	CC50 ± SD (µM)
lapachol	38.6 ± 3.2	>2.0	5.6	>200
(1)	89.6 ± 1.0	0.85 ± 0.2	6.3	>200
chloroquine	61.0 ± 0.4	0.86 ± 0.30	–	–
mefloquine	–	–	36.4	>200

^aInhibitory activity was determined after 24 h incubation with the compounds.

^bHemolysis was determined in uninfected erythrocytes after 1 h incubation with the compounds. SD: standard deviation of one experiment.

Figure 3. TEM analysis of P. falciparum. Parasites were treated with 2.3 µM of compound 1 and incubated for 24 h. Panels A and B show untreated parasites, highlighting the presence of hemozoin crystals inside the digestive vacuoles. Panel C and D show treated parasites, where is possible to observe the hemozoin crystal absence. Panel E exhibits the number of parasites containing hemozoin crystals.

Figure 4. DNA fragmentation in W2 strain P. falciparum incubated with compound 1. Parasites were incubated with 0.05% DMSO (untreated), DNAse (positive control) or compound 1 and then analyzed by flow cytometry by using to the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling assay (TUNEL). One experiment was performed, in triplicate. *p < 0.001 in comparison to untreated cells.

Figure 5. (A) Blood parasitemia and (B) survival in P. berghei-infected mice. Swiss Webster mice (n = 5/group) were infected with P. berghei and then treated daily for four consecutive days. Compound 1 was given by intraperitoneal injection, chloroquine by oral gavage. Values represent the means ± SEM of one experiment (n = 5). (A) *p < 0.05; **p < 0.01; ***p < 0.001 in comparison to vehicle group. (B) According to the Log-rank (Mantel Cox) analysis, there is no statistically significance between vehicle and compound 1.

Table 3. Antimalarial activity in P. berghei-infected mice.

		% parasitemia reduction^a
Compounds	Dose (mg/kg)/ route	Day 5	6	7	8	Survival (days)^b
Vehicle	Saline / i.p.	–	–	–	–	19
1	10 / i.p.	16	4.1	0	15	20
1	50 / i.p.	61	59	52	39	24
1	100 / i.p.	79	71	79	65	23
1	200 / p.o.	80	38	63	49	24
Chloroquine	50 / p.o.	100	100	100	100	>30

^aDetermined as [(vehicle − treated)/vehicle] × 100.

^bDays post-infection. i.p.: intraperitoneal; p.o.: oral administration.

Figure 6. Frequency of micronucleated polychromatic erythrocytes (NPCE) observed in Swiss Webster mouse bone marrow. Swiss mice (n = 5/group) received intraperitoneal injection of saline (untreated), cyclosphophamide (50 mg/kg) and compound 1 (100 mg/kg). After 18 to 24 h of treatment, the percentage of NPCE was determined relatively to 8000 polychromatic erythrocytes (PCE) per group. Values represent the means ± SEM of one experiment (n = 5/group). ANOVA, ***p < 0.001 in comparison to untreated group.