Antileishmanial activity of synthetic analogs of the naturally occurring quinolone alkaloid N-methyl-8-methoxyflindersin

Abstract

Leishmaniasis is a neglected, parasitic tropical disease caused by an intracellular protozoan from the genus Leishmania. Quinoline alkaloids, secondary metabolites found in plants from the Rutaceae family, have antiparasitic activity against Leishmania sp. N-methyl-8-methoxyflindersin (1), isolated from the leaves of Raputia heptaphylla and also known as 7-methoxy-2,2-dimethyl-2H,5H,6H-pyran[3,2-c]quinolin-5-one, shows antiparasitic activity against Leishmania promastigotes and amastigotes. This study used in silico tools to identify synthetic quinoline alkaloids having structure similar to that of compound 1 and then tested these quinoline alkaloids for their in vitro antiparasitic activity against Leishmania (Viannia) panamensis, in vivo therapeutic response in hamsters suffering from experimental cutaneous leishmaniasis (CL), and ex vivo immunomodulatory potential in healthy donors' human peripheral blood (monocyte)-derived macrophages (hMDMs). Compounds 1 (natural), 2 (synthetic), and 8 (synthetic) were effective against intracellular promastigotes (9.9, 3.4, and 1.6 μg/mL medial effective concentration [EC50], respectively) and amastigotes (5.07, 7.94, and 1.91 μg/mL EC50, respectively). Compound 1 increased nitric oxide production in infected hMDMs and triggered necrosis-related ultrastructural alterations in intracellular amastigotes, while compound 2 stimulated oxidative breakdown in hMDMs and caused ultrastructural alterations in the parasite 4 h posttreatment, and compound 8 failed to induce macrophage modulation but selectively induced apoptosis of infected hMDMs and alterations in the intracellular parasite ultrastructure. In addition, synthetic compounds 2 and 8 improved the health of hamsters suffering from experimental CL, without evidence of treatment-associated adverse toxic effects. Therefore, synthetic compounds 2 and 8 are potential therapeutic candidates for topical treatment of CL.

Fig 1. Effect of treatment with compounds 2 and 8 on hamsters suffering from CL.

(a) Lesion size measurement (mm²) during treatment (pre- and posttreatment: weeks 4–12). Number of recovered hamsters showing improvement or lacking response to treatment with (b) compound 2 and (c) compound 8 (n = 6 for each treatment). CL, cutaneous leishmaniasis.

Fig 2. Histopathology of the skin of infected hamsters treated with compounds 2 and 8.

(a) Skin of cured hamsters treated with compound 2, (b) skin showing improvement, and skin of hamsters treated with compound 8, showing (c) cure or (d) improvement: hematoxylin/eosin method; 50–100 μm magnification.

Fig 3. LW (g) and serum ALT, creatinine, and BUN levels pre and posttreatment with compounds 2 and 8.

Data are presented as mean ± SD of (a) LW, (b) ALT, (c) creatinine, and (d) BUN in the serum of hamsters suffering from CL before (P0) and after (P8) treatment. Significant between-group differences (p < 0.05). The dotted area represents the reference values for each parameter. LW, live weight; ALT, alanine aminotransferase; BUN, blood urea nitrogen; SD, standard deviation; CL, cutaneous leishmaniasis.

Fig 4. Percentage hMDMs during early apoptosis (annexin V⁺ 7AAD^–). hMDMs treated with quinoline alkaloid–like compounds at EC₅₀, evaluated by flow cytometry.

(A) Dot plot of noninfected hMDMs: x axis, annexin V-PE; y axis, 7AAD. (1) M (uninfected or untreated hMDMs); (2) AmpB; (3) compound 1; (4) compound 2; (5) compound 8. (B) Bar plot of the percentage of events (cells) for (a) noninfected hMDMs and (b) Leishmania (Viannia) panamensis-infected hMDMs (IMs) treated with compounds 1, 2, and 8. *p < 0.05; **p < 0.01; ***p < 0.005. EC₅₀, medial effective concentration; hMDMs, human monocyte-derived macrophages.

Fig 5. ROS generation in Leishmania (Viannia) panamensis-infected hMDMs treated with quinoline alkaloid–like compounds at different times (h).

MFI. Comparing ROS induction by compounds in hMDMs. *p < 0.05; **p < 0.01; ***p < 0.005. ROS, reactive oxygen species; hMDMs, human monocyte-derived macrophages; MFI, medium fluorescent intensity; M, hMDMs; IM, infected hMDMs.

Fig 6. Inducing NO production in L. (V) panamensis-infected hMDM.

NO production kinetics in hMDMs infected with L. (V.) panamensis and treated with quinoline alkaloid–like compounds 1, 2, and 8 after 24, 48, and 72 h of treatment. *p < 0.05; **p < 0.01; ***p < 0.005. NO, nitric oxide; hMDMs, human monocyte-derived macrophages; M, uninfected or untreated hMDMs; LPS, lipopolysaccharide; IM, infected hMDMs.

Fig 7. Alterations in Leishmania (Viannia) panamensis-infected hMDMs’ ultrastructures.

Untreated infected hMDMs (a and b). Infected hMDMs treated with compounds (c and d) 1, (e and f) 2, and (g and h) 8. White arrows, mitochondria; white asterisks, double-membrane vacuoles (autophagosomes); white stars, electrodense bodies. Visualization scale = 5–10 μm; 10,000x magnification. hMDMs, human monocyte-derived macrophages; N, nucleus; PV, parasitophorous vacuole; TEM, transmission electron microscopy.

Fig 8. Ultrastructural alterations of Leishmania (Viannia) panamensis intracellular amastigotes.

Untreated hMDMs (a and b). hMDMs treated with compounds (c and d) 1, (e and f) 2, and (g and h) 8. White arrows, acidocalcisomes. Visualization scale = 2 μm; 10,000X magnification. hMDMs, human monocyte-derived macrophages; N, nucleus; F, flagellae; K, kinetoplast; V, vacuole; LV, lipid vacuole (electrodense vacuoles); M, mitochondria; TEM, transmission electron microscopy.