Aims: Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. Results: We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure–activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. Innovation: The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. Conclusion: This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations. Antioxid. Redox Signal. 22, 1337–1351.

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Previously, we presented the chemical design of a promising series of antimalarial agents, 3-[substituted-benzyl]-menadiones, with potent in vitro and in vivo activities. Ongoing studies on the mode of action of antimalarial 3-[substituted-benzyl]-menadiones revealed that these agents disturb the redox balance of the parasitized erythrocyte by acting as redox cyclers-a strategy that is broadly recognized for the development of new antimalarial agents. Here we report a detailed parasitological characterization of the in vitro activity profile of the lead compound 3-[4-(trifluoromethyl)benzyl]-menadione 1c (henceforth called plasmodione) against intraerythrocytic stages of the human malaria parasite Plasmodium falciparum. We show that plasmodione acts rapidly against asexual blood stages, thereby disrupting the clinically relevant intraerythrocytic life cycle of the parasite, and furthermore has potent activity against early gametocytes. The lead's antiplasmodial activity was unaffected by the most common mechanisms of resistance to clinically used antimalarials. Moreover, plasmodione has a low potential to induce drug resistance and a high killing speed, as observed by culturing parasites under continuous drug pressure. Drug interactions with licensed antimalarial drugs were also established using the fixed-ratio isobologram method. Initial toxicological profiling suggests that plasmodione is a safe agent for possible human use. Our studies identify plasmodione as a promising antimalarial lead compound and strongly support the future development of redox-active benzylmenadiones as antimalarial agents.M alaria remains one of the most severe infectious diseases that disproportionately affects the public health and economic welfare of the world's poorest communities. The causative agents of malaria are protozoan parasites of the genus Plasmodium. Among the five Plasmodium species that can cause malaria in humans (Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi), P. falciparum is the most virulent and is responsible for severe clinical outcomes and most of the deaths associated with malaria. Until the development of an effective vaccine, chemotherapy remains a major frontline strategy for the control and possible future elimination of malaria. The emergence and rapid spread of drug-resistant parasites undermine efforts at reducing the global disease burden and emphasize the need for drugs with novel chemical entities that exploit new molecular targets while overcoming established drug resistance mechanisms. Ideally, such drugs should quickly kill the pathogenic asexual blood stages of P. falciparum and, in addition, be effective against other developmental stages, in particular the sexual stages that transmit the infection from the human to the mosquito host (1, 2).Previously, we presented the chemical design and synthesis of a novel class of compounds, the 3-[substituted-benzyl]-menadiones, that exhibit potent activities against P. falciparum blood stages in vitro and moderate activities in v...

A platform of synthetic methodologies has been established to access a focused library of polysubstituted 3‐benzylmenadione derivatives functionalized on the aromatic ring of the naphthoquinone core. Two main routes were explored: 1) The naphthol route, starting from either an α‐tetralone or a propiophenone, and 2) the regioselective Diels–Alder reaction, starting from various dienes and two 2‐bromo‐5(or 6)‐methyl‐1,4‐benzoquinones. 6‐Substituted 2‐methylnaphthols were synthesized by using a xanthate‐mediated free‐radical addition/cyclization sequence for the construction of the 6‐substituted menadione subunit. Furthermore, an efficient and simple new pathway that allows the formation of 6‐ or 7‐substituted 3‐(substituted‐benzyl)menadione regioisomers from a common commercial scaffold has also been developed by the naphthol route, advantageous with regard to step economy. Our synthetic methodologies exemplified by 34 compounds have allowed structure–activity relationships to be deduced for use as the basis for the development of new antimalarial redox‐active polysubstituted benzylmenadione derivatives.

Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas. Resistance to antimalarial drugs has spread all over the world in the past 50 years, thus new drugs are urgently needed. Plasmodione (benzylmenadione series) has been identified as a potent antimalarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages. To investigate its metabolism, a series of plasmodione-based tools, including a fully 13C-labelled lead drug and putative metabolites, have been designed and synthesized for drug metabolism investigation. Furthermore, with the help of UHPLC-MS/MS, two of the drug metabolites have been identified from urine of drug-treated mice.

Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.

With the aim of increasing the structural diversity on the early antimalarial drug plasmodione, an efficient and versatile procedure to prepare a series of biaryl- and N-arylalkylamines as plasmodione analogues is described. Using the naturally occurring and commercially available menadione as starting material, a 2-step sequence using a Kochi-Anderson reaction and subsequent Pd-catalyzed Suzuki-Miyaura coupling was developed to prepare three representative biphenyl derivatives in good yields for antimalarial evaluation. In addition, synthetic methodologies to afford 3-benzylmenadione derivatives bearing a terminal -N(Me)2 or -N(Et)2 in different positions (ortho, meta and para) on the aryl ring of the benzylic chain of plasmodione were investigated through reductive amination was used as the optimal route to prepare these protonable N-arylalkylamine privileged scaffolds. The antimalarial activities were evaluated and discussed in light of their physicochemical properties. Among the newly synthesized compounds, the para-position of the substituent remains the most favourable position on the benzyl chain and the carbamate -NHBoc was found active both in vitro (42 nM versus 29 nM for plasmodione) and in vivo in Plasmodium berghei-infected mice. The measured acido-basic features of these new molecules support the cytosol-food vacuole shuttling properties of non-protonable plasmodione derivatives essential for redox-cycling. These findings may be useful in antimalarial drug optimization.

A library of title compounds is envisaged where the derivatives are functionalized at the aromatic part of the naphthoquinone scaffold.