Evaluation of the Anti- Leishmania mexicana and - Trypanosoma brucei Activity and Mode of Action of 4,4'-(Arylmethylene)bis(3-methyl-1-phenyl-1 H-pyrazol-5-ol)

Abstract

Trypanosomiasis and leishmaniasis are neglected infections caused by trypanosomatid parasites. The first-line treatments have many adverse effects, high costs, and are prone to resistance development, hence the necessity for new chemotherapeutic options. In line with this, twenty five 4,4′-(arylmethylene)bis(1H-pyrazol-5-ols) derivatives were synthesized and evaluated in vitro for their anti-trypanosomatid activity. Ten and five compounds from this series showed IC50 ≤ 10 µM against the promastigote and the bloodstream stage of Leishmania mexicana and Trypanosoma brucei brucei, respectively. Overall, derivatives with pyrazole rings substituted with electron-withdrawing groups proved more active than those with electron-donating groups. The hits proved moderately selective towards L. mexicana and T. brucei (selectivity index, SI, compared to murine macrophages = 5−26). The exception was one derivative displaying an SI (>111−189) against T. brucei that surpassed, by >6-fold, the selectivity of the clinical drug nifurtimox (SI = 13−28.5). Despite sharing a common scaffold, the hits differed in their mechanism of action, with halogenated derivatives inducing a rapid and marked intracellular oxidative milieu in infective T. brucei. Notably, most of the hits presented better absorption, distribution, metabolism, and excretion (ADME) properties than the reference drugs. Several of the bioactive molecules herein identified represent a promising starting point for further improvement of their trypanosomatid potency and selectivity.

Keywords: 4,4′-(arylmethylene)bis(1H-pyrazol-5-ols); ADME; Leishmania; Trypanosoma; drug-like; redox biosensor.

Scheme 1

Synthesis of 4,4′-(arylmethylene)bis(1H-pyrazol-5-ols) 2a–y.

Figure 1

Intracellular thiol-redox perturbations produced by hit compounds in infective T. brucei: (A) Exponentially growing bloodstream T. b. brucei expressing the hGrx-roGFP2 biosensor was incubated for 1 h in the absence (UT—untreated) or presence of DMSO 1% v/v (DMSO) or hit compounds added at their corresponding IC₅₀ in DMSO 1% v/v (see Table 3). Treatment with diamide 250 μM for 20 min was included as positive oxidation control (DIA). The solid bars correspond to samples subjected to the treatments described above and incubated for additional 20 min with DTT 1 mM as reducing control condition. The % biosensor oxidation is expressed relative to control conditions, inducing full biosensor oxidation (DIA) and reduction (DMSO + DTT). The dotted line denotes the cut-off value for the mean oxidation achieved in the DMSO sample. ns—not statistically significant (p < 0.05). Further methodological details about the assay and analysis can be found in Section 2.2.5. (B) Substituents included in the aromatic ring of the hit compounds subjected to redox assays.

Figure 2

Fluorometric analyses of T. brucei’s hit compounds: (A) Emission spectra (λ_ex = 488 nm) of the recombinant biosensor (5 µM, roGFP2) in 1% v/v DMSO. Inset: zoom-in of the emission spectra (λ_em = 500–550 nm) of the hit compounds (50 µM) acquired under the same conditions. (B) Bars represent the percentage of biosensor oxidation based on the fluorescence emission of roGFP2. The oxidized (ox) and reduced (red) forms of the recombinant biosensor (5 µM) were incubated for 1 h with 50 μM 2r, 2w, or 2x dissolved in 1% v/v DMSO (for further technical details see Section 2.2.6).