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Ψ-1,4-naphthoquinones (Ψ-NQ) are non-quinoid compounds in which aromaticity-found in 1,4-naphthoquinones-is broken by the introduction of an angular methyl at C-4a or -8a. This series was designed to act as prodrugs of 1,4-naphthoquinones in an oxidative environment. Furthermore, from a medicinal chemistry point of view, the loss of planarity of the scaffold might lead to an improved solubility and circumvent the bad reputation of quinones in the pharmaceutical industry. In this work, we illustrated the concept by the synthesis of Ψ -plasmodione regioisomers as prodrugs of the antimalarial plasmodione. The presence of a chiral center introduces a new degree of freedom to be controlled by enantioselectivity and regioselectivity of the cycloaddition in the Diels-Alder reaction. The first strategy that was followed was based on the use of a chiral enantiopure sulfoxide to govern the stereoselective formation of (+)Ψ-NQ or (-)Ψ-NQ, depending on the chirality of the sulfoxide ( R or S ). New sulfinylquinones were synthesized but were found to be ineffective in undergoing cycloaddition with different dienes under a wide range of conditions (thermal, Lewis acid). The second strategy was based on the use of boronic acid-substituted benzoquinones as auxiliaries to control the regioselectivity. Using this methodology to prepare the (±)Ψ-NQ racemates, promising results (very fast cycloaddition time: ~2 h) were obtained with boronic acid-based quinones 25 and 27 in the presence of 1-methoxy-1,3-butadiene, to generate the 4a- and the 8a-Ψ-plasmodione regioisomers 1 and 2 (synthesized in six steps with a total yield of 10.5% and 4.1%, respectively. As the expected prodrug effect can only be revealed if the molecule undergoes an oxidation of the angular methyl, e.g., in blood-feeding parasites that digest hemoglobin from the host, the antimalarial and the antischistosomal properties of both (±)Ψ-NQ regioisomers were determined in drug assays with Plasmodium falciparum and Schistosoma mansoni . Metabolic studies under quasi-physiological conditions and LC-MS analyses were undertaken to reveal the generation of plasmodione from both the 4a- and the 8a-Ψ-plasmodione regioisomers.

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Investigations regarding the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives revealed 3-phenoxymethyl menadiones as a novel anti-schistosomal chemical series. These newly synthesized compounds (1-7) and their difluoromethylmenadione counterparts (8, 9) were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol methyl ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione derivative (7) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compounds tested was active in vivo, suggesting that the limited bioavailability may compromise compound activity. Therefore, future studies will be directed toward improving pharmacokinetic properties and bioavailability., (© 2015 FEBS.)

The homodimeric flavoenzyme glutathione reductase catalyzes NADPH-dependent glutathione disulfide reduction. This reaction is important for keeping the redox homeostasis in human cells and in the human pathogen Plasmodium falciparum. Different types of NADPH-dependent disulfide reductase inhibitors were designed in various chemical series to evaluate the impact of each inhibition mode on the propagation of the parasites. Against malaria parasites in cultures the most potent and specific effects were observed for redox-active agents acting as subversive substrates for both glutathione reductases of the Plasmodium-infected red blood cells. In their oxidized form, these redox-active compounds are reduced by NADPH-dependent flavoenzyme-catalyzed reactions in the cytosol of infected erythrocytes. In their reduced forms, these compounds can reduce molecular oxygen to reactive oxygen species, or reduce oxidants like methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Furthermore, studies on a fluorinated suicide-substrate of the human glutathione reductase indicate that the glutathione reductase-catalyzed bioactivation of 3-benzylnaphthoquinones to the corresponding reduced 3-benzoyl metabolites is essential for the observed antimalarial activity. In conclusion, the antimalarial lead naphthoquinones are suggested to perturb the major redox equilibria of the targeted cells. These effects result in developmental arrest of the parasite and contribute to the removal of the parasitized erythrocytes by macrophages.

Improving the solubility of polysubstituted 1,4-naphthoquinone derivatives was achieved by introducing nitrogen in two different positions of the naphthoquinone core, at C-5 and at C-8 of menadione through a two-step, straightforward synthesis based on the regioselective hetero-Diels-Alder reaction. The antimalarial and the antischistosomal activities of these polysubstituted aza-1,4-naphthoquinone derivatives were evaluated and led to the selection of distinct compounds for antimalarial versus antischistosomal action. The Ag(II)-assisted oxidative radical decarboxylation of the phenyl acetic acids using AgNO(3) and ammonium peroxodisulfate was modified to generate the 3-picolinyl-menadione with improved pharmacokinetic parameters, high antimalarial effects and capacity to inhibit the formation of β-hematin.

Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage.

Various aza-analogues of 1,4-naphthoquinone and menadione were prepared and tested as inhibitors and substrates of the plasmodial thioredoxin and glutathione reductases as well as the human glutathione reductase. The replacement of one to two carbons at the phenyl ring of the 1,4-naphthoquinone core by one to two nitrogen atoms led to an increased oxidant character of the molecules in accordance with both the redox potential values and the substrate efficiencies. Compared to the 1,4-naphthoquinone and menadione, the quinoline-5,8-dione 1 and both quinoxaline-5,8-diones 5 and 6 behaved as the most efficient subversive substrates of the three NADPH-dependent disulfide reductases tested. Modulation of these parameters was observed by alkylation of the aza-naphthoquinone core.

Glutathione reductase is an important housekeeping enzyme for redox homeostasis both in human cells and in the causative agent of tropical malaria, Plasmodium falciparum. Glutathione reductase inhibitors were shown to have anticancer and antimalarial activity per se and to contribute to the reversal of drug resistance. The development of menadione chemistry has led to the selection of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called M(5), as a potent reversible and uncompetitive inhibitor of both human and P. falciparum glutathione reductases. Here we describe the synthesis and kinetic characterization of a fluoromethyl-M(5) analogue that acts as a mechanism-based inhibitor of both enzymes. In the course of enzymatic catalysis, the suicide substrate is activated by one- or two-electron reduction, and then a highly reactive quinone methide is generated upon elimination of the fluorine. Accordingly the human enzyme was found to be irreversibly inactivated with a k(inact) value of 0.4 +/- 0.2 min(-1). The crystal structure of the alkylated enzyme was solved at 1.7 A resolution. It showed the inhibitor to bind covalently to the active site Cys58 and to interact noncovalently with His467', Arg347, Arg37, and Tyr114. On the basis of the crystal structure of the inactivated human enzyme and stopped-flow kinetic studies with two- and four-electron-reduced forms of the unreacted P. falciparum enzyme, a mechanism is proposed which explains naphthoquinone reduction at the flavin of glutathione reductase.

Plasmodium parasites are exposed to elevated fluxes of reactive oxygen species during intraerythrocytic life. The most important antioxidative systems are based on the glutathione reductases of the malarial parasite Plasmodium falciparum and the host erythrocyte. The development of menadione chemistry has led to the selection of the carboxylic acid 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl] hexanoic acid M(5) as an inhibitor of the parasitic enzyme. As reported here, revisiting the mechanism of M(5) action revealed an uncompetitive inhibition type with respect to both NADPH and glutathione disulfide. Masking the polarity of the acidic function of M(5) by ester or amide bonds improved antiplasmodial activity. Bioisosteric replacement of the carboxylic function by tetrazole to increase bioavailability and to maintain comparable acidity led to improved antimalarial properties as well, but only with the cyanoethyl-protected tetrazoles. Using computed ab initio quantum methods, detailed analyses of the electronic profiles and the molecular properties evidenced the similarity of M(5) and the bioisoteric tetrazole T(4). The potential binding site of these molecules is discussed in light of the recently solved crystallographic structure of P. falciparum enzyme.

Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.

Solid- and solution-phase parallel syntheses of 1,4-naphthoquinones (1,4-NQ) are described. A library of 1360 amides was constructed from the combination of 12 newly synthesised 1,4-NQ carboxylic acid and 120 amines, and was screened for inhibition of trypanothione reductase (TR) from Trypanosoma cruzi. The most active hits from a primary screening were re-synthesised and confirmed. This approach proves that it is possible to design potent and highly specific TcTR inhibitors deriving from menadione, juglone and plumbagin.