Diversity-oriented synthesis and activity evaluation of substituted bicyclic lactams as anti-malarial against Plasmodium falciparum

Abstract

Background: Malaria remains the world's most important devastating parasitic disease. Of the five species of Plasmodium known to infect and cause human malaria, Plasmodium falciparum is the most virulent and responsible for majority of the deaths caused by this disease. Mainstream drug therapy targets the asexual blood stage of the malaria parasite, as the disease symptoms are mainly associated with this stage. The prevalence of malaria parasite strains resistance to existing anti-malarial drugs has made the control of malaria even more challenging and hence the development of a new class of drugs is inevitable.

Methods: Screening against different drug resistant and sensitive strains of P. falciparum was performed for few bicyclic lactam-based motifs, exhibiting a broad spectrum of activity with low toxicity generated via a focussed library obtained from diversity oriented synthesis (DOS). The synthesis and screening was followed by an in vitro assessment of the possible cytotoxic effect of this class of compounds on malaria parasite.

Results: The central scaffold a chiral bicyclic lactam (A) and (A') which were synthesized from (R)-phenylalaninol, levulinic acid and 3-(2-nitrophenyl) levulinic acid respectively. The DOS library was generated from A and from A', by either direct substitution with o-nitrobenzylbromide at the carbon α- to the amide functionality or by conversion to fused pyrroloquinolines. Upon screening this diverse library for their anti-malarial activity, a dinitro/diamine substituted bicyclic lactam was found to demonstrate exceptional activity of >85% inhibition at 50 μM concentration across different strains of P. falciparum with no toxicity against mammalian cells. Also, loss of mitochondrial membrane potential, mitochondrial functionality and apoptosis was observed in parasite treated with diamine-substituted bicyclic lactams.

Conclusions: This study unveils a DOS-mediated exploration of small molecules with novel structural motifs that culminates in identifying a potential lead molecule against malaria. In vitro investigations further reveal their cytocidal effect on malaria parasite growth. It is not the first time that DOS has been used as a strategy to identify therapeutic leads against malaria, but this study establishes the direct implications of DOS in scouting novel motifs with anti-malarial activity.

Figure 1

Overview of the synthesis of substituted chiral bicyclic lactams as potential anti-malarials. (A) The scheme depicts the synthesis of chiral bicyclic lactams from phenylalaninol/levulinic acid and 3-(2-nitrophenyl)levulinic acid. The final compounds were derived either via enolization and alkylation with o-nitrobenzylbromide and subsequent hydrogenation or by lewis acid based ring opening and subsequent cyclization. (B) Screening of synthesized compounds for P. falciparum growth-inhibitory activities at four different concentrations of 1 μM, 10 μM, 25 μM, and 50 μM. Bar graph indicates compounds C and D as the most potent inhibitor of P. falciparum growth. Three independent assays were performed in duplicates. The error bars show the standard errors of the means.

Figure 2

Effect of compounds C and D on parasite growth. (A) Percent parasite growth curve showing the effect of compounds C and D on P. falciparum growth at different concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 25 μM, and 50 μM. Three independent experiments were performed in duplicates. (B) Comparison of parasite intra-erythrocytic maturation in presence and absence of compounds C and D. Light microscopy Giemsa-stained images of P. falciparum-infected RBCs at 12, 26, 48, and 54 hpi incubated with and without compounds. Bars indicate relative percent frequency of parasite ring, trophozoite and schizont stages in the 12, 26, 48, and 54 hpi. Healthy trophozoites are observed 26 hpi, either in presence or absence of compounds. At 48 hpi, healthy new rings are observed in untreated control but, are >70% reduced in the presence of 50 μM of compound C and compound D with parasite growth arrested as early and late trophozoites.

Figure 3

Growth-inhibitory activities of compounds across different Plasmodium falciparum strains and on viability of COS-7 cells. (A) Bar graph showing the growth-inhibitory effect of dintro-substituted compound C at 50 μM concentration on sorbitol synchronized P. falciparum drug-sensitive clones 3D7, HB3 and drug-resistant clones Dd2. (B) Bar graph showing the growth inhibitory effect of diamine-substituted compound D at 50 μM concentration on sorbitol synchronized P. falciparum drug-sensitive clones 3D7, HB3 and drug-resistant clones Dd2. Three independent assays were performed in duplicates. The error bars show the standard errors of the means. (C) Bar graph showing percentage survival of COS-7 cells after 24 hours of treatment with compounds C and D. Three independent assays were performed in duplicates. The error bars show the standard errors of the means.

Figure 4

Loss of mitochondrial membrane potential and apoptosis like cell-death in compound D-treated Plasmodium falciparum parasites. (A) (i) Bar graph showing reduction in ratio of JC-1 (red)/JC-1 (green) in parasite population after treatment with compound D. Two independent experiments were performed in duplicates. The error bars show the standard errors of the means. (ii) Fluorescent images of JC-1-stained parasites showing aggregated JC-1 (red) in the mitochondria and monomeric JC-1 (green) in the cytoplasm after treatment with solvent alone or compound D. Parasite nuclei were stained with DAPI (blue). (B) (i) Bar graph showing percentage of TUNEL-positive parasites after treatment with compound D as compared to solvent alone (control). Two independent assays were performed in duplicates. The error bars show the standard errors of the means. (ii) Fluorescent images of parasites stained with TMR Red (TUNEL staining) showing DNA fragmentation after treatment with compound D.