Improving the drug-likeness of inspiring natural products - evaluation of the antiparasitic activity against Trypanosoma cruzi through semi-synthetic and simplified analogues of licarin A

Abstract

Neolignan licarin A (1) was isolated from leaves of Nectandra oppositifolia (Lauraceae) and displayed activity against trypomastigote forms of the etiologic agent of American trypanosomiasis, Trypanosoma cruzi. Aiming for the establishment of SAR, five different compounds (1a - 1e) were prepared and tested against T. cruzi. The 2-allyl derivative of licarin A (1d) exhibited higher activity against trypomastigotes of T. cruzi (IC₅₀ = 5.0 μM and SI = 9.0), while its heterocyclic derivative 1e displayed IC₅₀ of 10.5 μM and reduced toxicity against NCTC cells (SI > 19.0). However, these compounds presented limited oral bioavailability estimation (<85%, Papp <1.0 × 10^-6 cm/s) in parallel artificial membrane permeability assays (PAMPA) due to excessive lipophilicity. Based on these results, different simplified structures of licarin A were designed: vanillin (2), vanillyl alcohol (3), isoeugenol (4), and eugenol (5), as well as its corresponding methyl (a), acetyl (b), O-allyl (c), and C-allyl (d) analogues. Vanillin (2) and its acetyl derivative (2b) displayed expressive activity against intracellular amastigotes of T. cruzi with IC₅₀ values of 5.5 and 5.6 μM, respectively, and reduced toxicity against NCTC cells (CC₅₀ > 200 μM). In addition, these simplified analogues showed a better permeability profile (Papp > 1.0 × 10^-6 cm/s) on PAMPA models, resulting in improved drug-likeness. Vanillyl alcohol acetyl derivative (3b) and isoeugenol methyl derivative (4a) displayed activity against the extracellular forms of T. cruzi (trypomastigotes) with IC₅₀ values of 5.1 and 8.8 μM respectively. Based on these results, compounds with higher selectivity index against extracellular forms of the parasite (1d, 1e, 3d, and 4a) were selected for a mechanism of action study. After a short incubation period (1 h) all compounds increased the reactive oxygen species (ROS) levels of trypomastigotes, suggesting cellular oxidative stress. The ATP levels were increased after two hours of incubation, possibly involving a high energy expenditure of the parasite to control the homeostasis. Except for compound 4a, all compounds induced hyperpolarization of mitochondrial membrane potential, demonstrating a mitochondrial imbalance. Considering the unique mitochondria apparatus of T. cruzi and the lethal alterations induced by structurally based on licarin A, these compounds are interesting hits for future drug discovery studies in Chagas disease.

Figure 1

Licarin A (1) and semi-synthetic derivatives 1a – 1e.

Figure 2

Simplified analogues (2a-d – 5a-d) of licarin A (1).

Figure 3

Molecular simplification approach to design the analogues 2–5 of licarin A (1). Note that licarin is comprised by a vanillin-like subunit (red – part A) and an isoeugenol-like subunit (blue – part B), which was explored in the search for the pharmacophore in licarin A.

Figure 4

Possible pharmacophore fragment on licarin A (1) structure (blue) suggested by the activity observed to isoeugenol derivatives 4a and 4d, and the auxophore moieties (magenta).

Figure 5

Plasma membrane permeability analysis on T. cruzi trypomastigotes with the probe Sytox Green treated with compounds 1d, 1e, 3b and 4a at the respective IC₅₀ values. As positive (C+) and negative (C−) controls were used trypomastigotes treated with TX-100 at 0.5% (maximum permeabilization) and untreated T. cruzi parasites (minimum permeabilization), respectively. One representative experiment of two assays is shown. **p < 0.005.

Figure 6

Evaluation of reactive oxygen species (ROS) generation in T. cruzi trypomastigotes treated with compounds 1d, 1e, 3b and 4a for 1 h (A) and 2 h (B). The H₂DCFDA fluorescent probe was analyzed spectrofluorimetrically (excitation 485 nm and emission 520 nm). Untreated trypomastigotes and treated with azide (10 mM) were used to achieve minimal and maximal ROS production, negative (C−) and positive control (C+), respectively. One representative experiment of two assays is shown. **p  <  0.001.

Figure 7

Evaluation of ATP production in T. cruzi trypomastigotes treated by 1 h (A) and 2 h (B) with the compounds 1d, 1e, 3b and 4a (IC₅₀ values). Untreated trypomastigotes, negative control (C−) and treated with CCCP (100 µM), positive control (C+) were used as controls of minimal and maximal depolarization. One representative experiment of two assays is shown. ***p < 0.0001.

Figure 8

Mitochondrial membrane potential analysis in T. cruzi trypomastigotes treated with compounds 1d, 1e, 3b and 4a for two hours labeled with JC-1 probe (0.2 μM). The fluorescence was measured in a flow cytometer (ATTUNE). Minimum (untreated – negative control, C−) and maximum (treated with CCCP- 100 µg/mL - positive control, C+) alterations in the mitochondrial membrane potential were obtained. Fluorescence was quantified by calculating the ratio between the channels BL2/BL1. One representative experiment of two assays is shown.