Synthesis and Antileishmanial Activity of 1,2,4,5-Tetraoxanes against Leishmania donovani

A chemically diverse range of novel tetraoxanes was synthesized and evaluated in vitro against intramacrophage amastigote forms of Leishmania donovani. All 15 tested tetraoxanes displayed activity, with IC50 values ranging from 2 to 45 µm. The most active tetraoxane, compound LC140, exhibited an IC50 value of 2.52 ± 0.65 µm on L. donovani intramacrophage amastigotes, with a selectivity index of 13.5. This compound reduced the liver parasite burden of L. donovani-infected mice by 37% after an intraperitoneal treatment at 10 mg/kg/day for five consecutive days, whereas miltefosine, an antileishmanial drug in use, reduced it by 66%. These results provide a relevant basis for the development of further tetraoxanes as effective, safe, and cheap drugs against leishmaniasis.


. General methods and analytical techniques
Commercial reagents were used as purchased. When required, solvents were dried following standard procedures. 1 1 H and 13 C-NMR spectra were recorded on a 400 MHz NMR spectrometer Bruker Avance III 400. 1 H-NMR-chemical shifts are referred to the residual signal of CDCl3 (δH 7.27) and 13 C-NMR-chemical shifts to the CDCl3 signal (δC 77.0), or using TMS as internal standard. Thin-layer chromatography was carried out on silica gel 60 F254 plates (AL TLC 20x20). Column chromatography was performed on Silica Gel 60 (0.04 -0.063 mm). IR spectra were recorded on a Tensor 27 FT/IR spectrometer in the 600-3800 cm -1 range. Melting points (°C) were obtained on a SMP3 Melting Point Apparattus and are uncorrected.

S.1.2. Preparation of intermediate building blocks
The synthetic approach followed to the preparation of intermediate blocks for the synthesis of final target compounds is illustrated in Schemes S3-S5. Synthetic procedures for each compound prepared are also provided in this section.
The mixture was stirred until complete dissolution of the suspended material. Dimethyl sulphate (110 mmol) was then added in small portions, keeping an alkaline medium through addition of aqueous sodium hydroxide. The final mixture was refluxed for 1 h, then cooled, and finally left in ice bath for 48h. Colourless needles of the desired compound were filtered and dried (51% yield); m.p. 220-221º C. 1

S.1.3. Synthetic route to trioxolanes
The synthetic approach followed to trioxolanes is illustrated in Scheme S6. Synthetic procedures for the preparation of each compound are also provided in this section (as described in literature 6 ).

S.1.4. Synthetic route to tetraoxanes
The synthetic approach followed to tetraoxanes is depicted in Schemes S7 to S11. Synthetic  To a solution of LC138 (4 mmol) in methanol (15 mL) was added a solution of potassium hydroxide (20 mmol) in water (6 mL). The mixture was refluxed for 6 hours. Then the solution was allowed to cool to room temperature and concentrated under reduced pressure.
The crude was taken up in water (50 ml) and washed with dichloromethane (30 ml to give product.

S3.2. In vitro antileishmanial evaluation on intramacrophage amastigotes.
The determination of the cytotoxicity as presented above, was necessary to use the highest drug concentrations to be studied on the intramacrophage amastigote model. Miltefosine was used as the reference drug.

S4.1. Animal and housing
The animal phase of these studies was conducted at Animex platform of University Paris-Saclay which is committed to the highest standards of laboratory animal welfare and is subject to legislation under the agreement number C 92-019-01. All procedures involving animals were conducted humanely and were performed by or under the direction of trained and experienced personnel. The protocol was reviewed and approved by the Institutional  days, blood samples were collected for a biochemical analysis using an Integra1 800 apparatus (Roche Diagnostic, Paris, France). Current nephrotoxicity was monitored via creatinine assay, and hepatic toxicity via AST (aspartate amino transferase) and ALT (alanine amino transferase) assays. U-Rank test was used for statistical analysis.

S4.3. In vivo antileishmanial evaluation
The selected compounds from in vitro and in vivo acute toxicity analyses were