β-Nitrostyrenes as Potential Anti-leishmanial Agents

Abstract

Development of new therapeutic approach to treat leishmaniasis has become a priority. In the present study, the antileishmanial effect of β-nitrostyrenes was investigated against in vitro promastigotes and amastigotes. A series of β-nitrostyrenes have been synthesized by using Henry reaction and were evaluated for their antimicrobial activities by broth microdilution assay and in vitro antileishmanial activities against Leishmania donovani promastigotes by following standard guidelines. The most active compounds were futher evaluated for their in vitro antileishmanial activities against intracellular amastigotes. Among the tested β-nitrostyrenes, compounds 7, 8, 9, 12, and 17 exhibited potential activities (MICs range, 0.25-8 μg/mL) against clinically significant human pathogenic fungi. However, the microbactericidal concentrations (MBCs) and the microfungicidal concentrations (MFCs) were found to be either similar or only two-fold greater than the MICs. Anti-leishmanial results demonstrated that compounds 9, 12, 14, and 18 were found to be most active among the tested samples and exhibited 50% inhibitory concentration (IC50) by 23.40 ± 0.71, 37.83 ± 3.74, 40.50 ± 1.47, 55.66 ± 2.84 nM against L. donovani promastigotes and 30.5 ± 3.42, 21.46 ± 0.96, 26.43 ± 2.71, and 61.63 ± 8.02 nM respectively against intracellular L. donovani promastigotes amastigotes respectively which are comparable with standard AmB (19.60 ± 1.71 nM against promastigotes and 27.83 ± 3.26 nM against amastigotes). Compounds 9, 12, 14, and 18 were found to have potent in vitro leishmanicidal activity against L. donovani and found to be non-toxic against mammalian macrophages even at a concentration of 25 μM. Nitric oxide (NO) estimation studies reveals that these compounds are moderately inducing NO levels.

Keywords: antimicrobicidal; leishmanicidal; macrophages; promastigotes; β-nitrostyrenes.

Figure 1

Naturally occurring β-nitrostyrenes.

Scheme 1

Synthesis.

Figure 2

Estimation of IC₅₀ and IC₉₀ of Compounds 9, 12, 14, and 18 against promastigotes. Parasites (2 × 10⁶ cells/mL) were incubated with serial four-fold dilutions of test compounds (starting at 25 μM) for 72 h and viability was determined microscopically, as described in methods. Each point or bar corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments.

Figure 3

(A) Leishmanicidal effects of treated promastigotes with compounds 9, 12, 14, and 18 revealed no reversion of growth. Each point or bar corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments. (B) Analysis of cellular morphology of promastigotes treated with compounds 9, 12, 14, 18 and ampotericin B. Exponential-phase promastigotes (2 × 10⁶ cells/mL) were incubated with 1μM of compounds 9, 12, 14, 18 for 72 h and analyzed by light microscopy (400 magnification), as described in Methods.

Figure 4

Effects of β-nitrostyrenes (9, 12, 14, and 18) compounds on L. donovani intracellular amastigote forms. After infection with promastigotes, peritoneal macrophages were incubated with serial four-fold dilutions of β-nitrostyrenes compounds (starting at 25000-0 nM) for 48 h and infection (A) as a percentage of control, (B) as number of amastigotes per 100 macrophages) was determined microscopically, as described in Methods. Each point corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments.

Figure 5

β-Nitrostyrenes (9, 12, 14, and 18) induced nitric oxide production in ex vivo model. Macrophages isolated from peritoneal cavity of BALB/c mice, infected with promastigotes and stimulated with β-nitrostyrenes (9, 12, 14, and 18) compounds at 37°C in a CO₂ incubator with serial four-fold dilutions of test compounds (starting at 25–0 μM) for 48 h. Nitrite generation was determined by Gries methods, as described in Methods. Each point corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments.

Figure 6

Absence of adverse toxicity of test compounds 9, 12, 14, and 18 on macrophage cell line RAW264.7. The cell lines were incubated for 48 h at 37°C in a CO₂ incubator with increasing concentrations of test compounds or AmB and viability was ascertained. Each point or bar corresponds to the mean ± SE of triplicate samples and is representative of one of three independent experiments.

Figure 7

Diversity oriented synthesis of natural products inspired β-nitrostyrene library.

Figure 8

Structure-activity relationship of β-nitrostyrenes.