Structure/Antileishmanial Activity Relationship Study of Naphthoquinones and Dependency of the Mode of Action on the Substitution Patterns

Abstract

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMMΦ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC₅₀ 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC₅₀ 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC₅₀ of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group peri to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity in vitro against the clinically relevant intracellular amastigote with an IC₅₀ of 1.1 µM. With IC₅₀ values mostly in the range of 1–3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.

Key words

Leishmania major - antileishmanial - naphthoquinones - alkannin/shikonin derivatives

References

• 1 Ashford R W, Desjour P, DeRaadt P. Estimation of population at risk of infection and number of cases of leishmaniasis.  Parasitol Today. 1992;  8 104-105
• 2 Croft S L, Coombs G H. Leishmaniasis – current chemotherapy and recent advances in the search for novel drugs.  Trends Parasitol. 2003;  19 502-508
• 3 Kedzierski L, Sakthianandeswaren A, Curtis J M, Andrews P C, Junk P C, Kedzierska K. Leishmaniasis: current treatment and prospects for new drugs and vaccines.  Curr Med Chem. 2009;  16 599-614
• 4 Chen M, Christensen S B, Theander G, Kharazami A. Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani.  Antimicrob Agents Chemother. 1994;  38 1339-1344
• 5 Akedengue B, Ngou-Milama E, Laurens A, Hocquemiller R. Recent advances in the fight against leishmaniasis with natural products.  Parasite. 1999;  6 3-8
• 6 Chan-Bacab M J, Pena-Rodriguez L M. Plant natural products with leishmanicidal activity.  Nat Prod Rep. 2001;  18 674-688
• 7 Rocha L G, Almeida J R, Macêdo R O, Barbosa-Filho J M. A review of natural products with antileishmanial activity.  Phytomedicine. 2005;  12 514-535
• 8 Hudson A T, Randall A W, Fry M, Ginger C D, Hill B, Latter V S, McHardy N, Williams R B. Novel anti-malarial hydroxynaphthoquinones with potent broad spectrum anti-protozoal activity.  Parasitology. 1985;  90 45-55
• 9 Croft S L, Evans A T, Neal R A. The activity of plumbagin and other electron carriers against Leishmania donovani and Leishmania mexicana amazonensis.  Ann Trop Med Parasitol. 1985;  79 651-653
• 10 Babula P, Adam V, Havel L, Kizek R. Noteworthy secondary metabolites naphthoquinones – their occurrence, pharmacological properties and analysis.  Curr Pharm Anal. 2009;  5 47-68
• 11 Kayser O, Kiderlen A F, Laatsch H, Croft S L. In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones.  Acta Trop. 2000;  76 131-138
• 12 Papageorgiou V P, Assimopoulou A N, Couladouros E A, Hepworth D, Nicolaou K C. Chemistry and biology of alkannins, shikonins and related naphthazarin natural products.  Angew Chem Int Ed. 1999;  38 270-301
• 13 Papageorgiou V P, Assimopoulou A N, Ballis A C. Alkannins and shikonins: a new class of wound healing agents.  Curr Med Chem. 2008;  15 3248-3267
• 14 Yousefi R, Ghaffarifar F, Asl A D. The effect of Alkanna tincturia and Peganum harmala extracts on Leishmania major (MRHO/IR/75/ER) in vitro.  Iran J Parasitol. 2009;  4 40-47
• 15 Chakraborty A K, Majumder H K. Mode of action of pentavalent antimonials: specific inhibition of type I DNA topoisomerase of Leishmania donovani.  Biochem Biophys Res Commun. 1988;  152 605-611
• 16 Plyta Z F, Li T, Papageorgiou V P, Mellidis A S, Assimopoulou A N, Pitsinos E N, Couladouros E A. Inhibition of topoisomerase I of naphthaquinone derivatives.  Bioorg Med Chem Lett. 1998;  8 3385-3390
• 17 Assimopoulou Α Ν, Ganzera M, Stuppner Η, Papageorgiou V P. Simultaneous determination of monomeric and oligomeric alkannins and shikonins by high-performance liquid chromatography-diode array detection-mass spectrometry.  Biomed Chromatogr. 2008;  22 173-190
• 18 Papageorgiou V P. 13C-NMR spectra of some naturally occurring hydroxynaphthoquinones.  Planta Med. 1980;  40 305-307
• 19 Papageorgiou V P. 1H-NMR spectra of naturally occurring isohexenylnaphthazarin pigments.  Planta Med. 1979;  37 185-187
• 20 Spyros A, Assimopoulou A N, Papageorgiou V P. Structure determination of oligomeric alkannin and shikonin derivatives.  Biomed Chromatogr. 2005;  19 498-505
• 21 Kram D, Thäle C, Kolodziej H, Kiderlen A F. Intracellular parasite kill: flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation.  J Immunol Methods. 2008;  333 79-88
• 22 Öllinger K, Brunmark A. Effect of hydroxyl substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes.  J Biol Chem. 1991;  226 21496-21503
• 23 Inbaraj J J, Chignell C F. Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes.  Chem Res Toxicol. 2004;  17 55-62
• 24 Assimopoulou Α N, Papageorgiou V P. Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents, hydroxynaphthoquinones.  Phytother Res. 2005;  19 141-147
• 25 Gao D, Kakuma M, Oka S, Sugino K, Sakurai H. Reaction of β-alkannin (shikonin) with reactive oxygen species: detection of β-alkannin free radicals.  Bioorg Med Chem. 2000;  8 2561-2569
• 26 Ordoudi S A, Tsermentseli S K, Nenadis N, Assimopoulou A N, Tsimidou M Z, Papageorgiou V P. Structure-radical scavenging activity relationship of alkannin/shikonin derivatives.  Food Chem. 2011;  124 171-176
• 27 Gao D, Hiromura M, Yasui H, Sakurai H. Direct reaction between shikonin and thiols induces apoptosis in HL60 cells.  Biol Pharm Bull. 2002;  25 827-832
• 28 Mukerjee T. One-electron reduction of juglone (5-hydroxy-1,4-naphthoquinone): a pulse radiolysis study.  Radiat Phys Chem. 1987;  29 455-462
• 29 Klaus V, Hartmann T, Gambini J, Graf P, Stahl W, Hartwig A, Klotz L O. 1,4-Naphthoquinones as inducers of oxidative damage and stress signalling in HaCaT human keratinocytes.  Arch Biochem Biophys. 2010;  496 93-100
• 30 Valko M, Rhodes C J, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer.  Chem Biol Interact. 2006;  160 1-40
• 31 Gant T W, Rao D N R, Mason R, Cohen G M. Redox cycling and sulfhydryl arylation: their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes.  Chem Biol Interact. 1988;  65 157-173
• 32 Takahashi N, Schreiber J, Fischer V, Mason R P. Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study.  Arch Biochem Biophys. 1987;  252 41-48
• 33 Kot M, Karcz W, Zaborska W. 5-Hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) influence on jack bean urease activity: elucidation of the difference in inhibition activity.  Bioorg Chem. 2010;  38 132-137