Targeting the Liver Stage of Malaria Parasites - American Chemical ...

Journal of Medicinal Chemistrytime-consuming, although automated image acquisition andtreatment protocols can be used, particularly in high-throughputapproaches, to facilitate data collection and analysis. 12,16 Flowcytometry enables measuring infection in cell lines infected withfluorescent transgenic parasites. 18 Furthermore, this techniquecan be used to distinguish cell invasion from parasite development19 (Figure 2B). Invasion rates can be measured by determiningthe percentage of fluorescence-positive cells at early timepoints after sporozoite addition. 12,16,19−21 At later time points,fluorescence intensity is an effective measure of parasite copynumbers in cells, i.e., development. 12,15,19−22 Fast sample acquisitionenables using flow cytometry for medium-to-highthroughput analyses. Finally, luminescence provides a valuablealternative for hepatic infection measurements. 23,24 Luciferaseexpressingparasites can be used to assess infection both in vitroand in vivo. In vitro, addition of luciferase’s substrate, luciferin, tocell lysates results in the emission of an amount of light thatis proportional to the parasite load in the lysate 23 (Figure 2C).A dye such as AlamarBlue can be employed to assess cell confluencyprior to lysis. The speed and simplicity of luciferase-basedinfection measurement make it well-suited for high-throughputstudies where large numbers of samples need to be analyzed.In vivo, infection can be quantified by injecting luciferin intoanesthetized, infected mice and imaging the bioluminescentsignal emitted by the parasites in the liver 23,24 (Figure 2D).One important technical challenge faced when measuringinfection is the low infectivity levels that can be obtainedexperimentally. In fact, in vitro invasion rates of cells by sporozoitesare low, rarely above 4% and often below 1%. An increasein signal-to-noise ratio as a result of the parasite’s intracellularmultiplication facilitates detection and quantification of infection.Nevertheless, infection can be measured by a varietyof established methods with high sensitivity. Microscopy andflow cytometry techniques, for example, enable quantificationof invasion by single parasites. 19,25The array of methods outlined above provides several alternativesto assess Plasmodium LS infection. While each hasadvantages and drawbacks, they can all provide valuable informationabout compound efficacy and guide researchers in thedevelopment of novel, efficient compounds to act on this crucial■stage of the parasite’s lifecycle. 26SMALL MOLECULES TARGETING LIVER-STAGE OFMALARIA8-Aminoquinolines. In the 1930s, a series of experimentsby Shute and co-workers with pamaquine, 1, demonstrated thatadministration of 60 mg/day po (per os) of this compoundacted as a true causal prophylactic for P. falciparum and P. vivaxinfections, 27 in contrast with later reports showing that pamaquineonly delayed the development of the exoerythrocyticforms that cause relapses. 28,29 Methemoglobinemia was regularlyfound in treated patients, which hampered the clinical use ofpamaquine. 28 On the other hand, Alving et al. showed in 1948that pentaquine, 2, had plasmodicidal activity identical to thatof pamaquine at doses that produced only mild reactions toindividuals. Also, it was 80- to 128-fold more potent thanquinine, suppressing fever and clearing parasitemia in a maximumof 6 days when administered alone in a 60 mg/dayoral regimen. This dosage of pentaquine, coadministrated withquinine in moderately infected individuals, resulted in radicalcure in 97% of the cases, significantly higher than when individualswere only treated with suppressive drugs (33%). 30 Higherdoses of pentaquine were required for severely infected individuals,Perspectivebut they also resulted in serious toxic effects, which did notwarrant safety in prophylaxis or prolonged treatment of malaria. 30From 1947 until 1951, both the synthesis of new 8-aminoquinolinesand their evaluation in infected human volunteerswere guided by results of assessments of curative and prophylacticactivities in monkeys either actively infected or recentlychallenged with sporozoites of P. cynomolgi and by appraisal ofsubacute toxicity in noninfected monkeys. This process led tothe identification of primaquine (PQ), 3. The exact mechanismby which PQ effectively eliminates Plasmodium hypnozoitesand gametocytes is still unknown, but it is thought that themitochondrial metabolism is impaired by interference with theelectron transport chain. 31−33 Presently, PQ is still the onlyavailable active drug against both gametocytes, which are responsiblefor the transmission of parasites between human andmosquito, and all the exoerythrocytic forms of Plasmodium,including the latent hypnozoites of P. ovale and P. vivax. A totalof 200 mg of PQ leads to radical cure, and the usually adoptedregimen of 15 mg/day po over 14 days is generally welltolerated.34 However, the use of PQ has been curtailed by twofactors: (i) low oral bioavailability due to rapid oxidative deaminationto carboxyprimaquine; 35,36 (ii) hemolytic anemia afterinduction of methemoglobinemia, which is highest in individualswith deficiency in glucose 6-phosphate dehydrogenase. 34,37,38In the years following the discovery of PQ, hundreds of 8-aminoquinolines were assayed for their antimalarial activity inRhesus monkeys and mouse models. 39,40 In 1983, Schmidtdisclosed 34 compounds found to display activity equal to orhigher than PQ, and several SARs were drawn. Like PQ, allthe active compounds contained the 6-OMe substituent, 24 ofthem had an additional substitution on the quinoline ring, and10 had two substitutions. Methyl substitutions at C2 or C4had a favorable impact on activity and resulted in compoundsthat were less toxic than PQ. Alkoxy, fluoro, and meta- orpara-substituted phenoxy groups at C5 also improved activity.Regarding the substituents at C8 most of the 34 compoundscarried a branched alkyl side chain near the 8-amino group andhad four or five carbons in length. 39 The structures of thosecompounds can be found in Table 1. From analysis of Table 1it is also noteworthy that there is the apparent disconnectionbetween the high potency in vivo of 8-aminoquinolines andtheir modest in vitro activity, e.g. ED 50 of 0.38 mg/kg in vivoand IC 50 of 2 μM in vitro for PQ. 21 Biotransformation of8-aminoquinolines seems to be necessary for their toxicity aswell as efficacy, 34 a reason why compounds from this classhave consistently displayed higher activities in vivo rather thanin vitro.On the other hand, aromatic substituents at C2 generallydecreased activity and branching at the C8 side chain had greaterimpact on activity for terminal secondary/tertiary amines.Further SAR analysis by Schmidt includes the detrimental effecton activity of hydroxyl groups and cyclic substituents, eitheraromatic or aliphatic, at the C8 side chain. 39 Moreover, Davidsonet al. showed that 6- and 7-aminoquinolines were devoid of997dx.doi.org/10.1021/jm201095h | J. Med. Chem. 2012, 55, 995−1012