Evaluation of the Pharmacophoric Role of the O–O Bond in Synthetic Antileishmanial Compounds: Comparison between 1,2-Dioxanes and Tetrahydropyrans

Leishmaniases are neglected diseases that can be treated with a limited drug arsenal; the development of new molecules is therefore a priority. Recent evidence indicates that endoperoxides, including artemisinin and its derivatives, possess antileishmanial activity. Here, 1,2-dioxanes were synthesized with their corresponding tetrahydropyrans lacking the peroxide bridge, to ascertain if this group is a key pharmacophoric requirement for the antileishmanial bioactivity. Newly synthesized compounds were examined in vitro, and their mechanism of action was preliminarily investigated. Three endoperoxides and their corresponding tetrahydropyrans effectively inhibited the growth of Leishmania donovani promastigotes and amastigotes, and iron did not play a significant role in their activation. Further, reactive oxygen species were produced in both endoperoxide- and tetrahydropyran-treated promastigotes. In conclusion, the peroxide group proved not to be crucial for the antileishmanial bioactivity of endoperoxides, under the tested conditions. Our findings reveal the potential of both 1,2-dioxanes and tetrahydropyrans as lead compounds for novel therapies against Leishmania.


X-ray crystallography
The X-ray intensity data were measured on a Bruker Apex II CCD diffractometer. Cell dimensions, and the orientation matrix were initially determined from a least-squares refinement on reflections measured in three sets of 20 exposures, collected in three different ω regions, and eventually refined against all data. A full sphere of reciprocal space was scanned by 0.3° ω steps. The software SMART 1 was used for collecting frames of data, indexing reflections and determination of lattice parameters.
The collected frames were then processed for integration by the SAINT program, 1 and an empirical absorption correction was applied using SADABS. 2 The structures were solved by direct methods (SIR 2014) 3 and subsequent Fourier syntheses and refined by full-matrix least-squares on F 2 (SHELXTL), 4 using anisotropic thermal parameters for all non-hydrogen atoms. The aromatic, methylene, methine and methyl hydrogen atoms were placed in calculated positions, refined with isotropic thermal parameters U(H) = 1.2 Ueq(C) or U(H) = 1.5 Ueq(C methyl ) and allowed to ride on their carrier carbons.
Molecular drawings were generated using Mercury. 5 Figure S1. Determination of the relative stereochemistry of 2-methoxy tetrahydropyran 9b through Xray crystallographic analysis (thermal ellipsoids are drawn at 30% of the probability level).

Cis-stereopreference in tetrahydropyrans 9
The favored cis relative stereochemistry established by X-ray crystallographic analysis for product 9b was extended to the other tetrahydropyrans 9a and 9c obtained using the same stereoselective synthetic approach (Scheme S1).
In Scheme S2 we present a hypothesis which could explain the cis-stereopreference in the construction of tetrahydropyrans 9.

Susceptibility test on Leishmania promastigotes with 10% FBS in the HOMEM assay medium
As the 20% FBS that is employed in the assay medium is a very nutrient-and antioxidant-rich environment, which could potentially prevent a stronger activation of endoperoxides 2 in comparison to the non-peroxidic analogs 3, we performed additional susceptibility tests for selected compounds by employing L. donovani cultures in HOMEM 10% FBS and we compared the results with those obtained by employing 20% FBS.
We observed a slight reduction of the IC 50 of all the tested compounds with 10% FBS in the assay medium as compared to 20% FBS. However, the bioactivities of endoperoxides 2 and tetrahydropyrans 3 remained comparable, suggesting a not significant pharmacophoric role of the O-O bond also under these bioassay-conditions. The antioxidant-rich environment (ie 20% FBS) does not appear to play a significant role in blocking the activation of endoperoxides. We can speculate that the increased antileishmanial activity of both peroxides and tetrahydropyrans in the presence of 10% FBS can be caused by a decreased parasite vitality with lower FBS concentration.

Cytotoxicity in THP-1 cell line
We performed additional cytotoxicity tests employing THP-1 cell line. We also calculated the corresponding selectivity indexes, as the ratio between CC 50 of THP-1 cell line and IC 50 on amastigotes of L. donovani.
We found that three compounds 3a, 3d and 2d showed a slightly higher cytotoxic effect on THP-1 cells than on VERO cells. On the other hand, compounds 2a, 3b and 2b showed a lower cytotoxic effect on THP-1 cells than on VERO cells; thus showing a not significant variance of selectivity index.

Effect of iron chelator DFO on bioactivity of 2b and 3b
As for compounds 2a and 3a, we evaluated the bioactivity of tetrahydropyran 3b and endoperoxide 2b

Effect of the iron chelator DFP on bioactivity of 2a and 3a
To investigate the role of iron in the activation of compounds 2a and 3a, we also tested these compounds against promastigotes of L. donovani in the presence of the iron chelator Deferiprone (DFP), which is a more lipophilic iron-chelator than DFO. IC 50 (DFP) = 219 M; CC 50 (DFP) = 143 M. Also in this case, we observed a not significant variation of the IC 50 values when the concentration of the iron-chelator was modified. At the highest concentration of the iron-chelator, the lowest IC 50 was recorded, confirming the activity of these compounds even in the absence of low molecular weight iron-species.
3.4 7.5 S11 As shown in isobolograms depicted in Figure S3, we observed a not significant variation of the IC 50 values when the concentration of the iron-chelator increased. FIC values for both compounds are > 0.5 and < 4, therefore no antagonism or synergism was observed.

Calculated LogP
The LogP values of the compounds tested on L. donovani promastigotes were calculated using the software ChemDraw Professional 15.0.