Leishmanicidal Activity of Aliphatic and Aromatic Lactones: Correlation Structure-Activity

Several aliphatic and aromatic lactones and two dimers were synthesized using the sequence: allylation - esterification - metathesis. These compounds were active in vitro against intracellular amastigotes of Leishmania panamensis. The structure-activity relationship showed the importance of the aliphatic side chain to enhance the biological activity and to obtain lower cytotoxicity. It was also observed that a decrease in the size of the lactone ring increases the selectivity index.


Introduction
Protozoan parasites are responsible for some of the most common and devastating diseases affecting humans and animals [1]. Among these diseases is leishmaniasis, which causes more deaths after malaria [2], the reason why it has become a priority for the World Health Organization (WHO) [2]. Classical antiparasite drugs are highly toxic and are largely no longer effective because of the emergence of resistance in the parasites, thus becoming a major public health problem [3]. Therefore, it is necessary to search for new active-low-toxicity molecules which may be obtained from the structural modification of natural compounds through organic synthesis.
Aiming at exploring the structure-activity relationships of the products, we previously reported some structural analogues of the mentioned compounds above [10]. However, in spite of the significant leishmanicidal activity against L. panamensis of some of these synthetic analogues, high cytotoxicity [21] was also observed, as well as the fact that the activity does not depend on the stereochemistry of the lactone ring [10]. Based on these results other racemic lactones analogues were synthesized by introducing aromatic rings in the chain and changing the type of lactone ring. In this paper we describe the synthesis these new analogues and their activity against Leishmania panamensis.
Following similar synthetic strategies, we synthesized the following set of lactones ( Figure 2). Lactones 13 and 14 have been synthesized with similar strategies and reported in the literature [27,28]

Synthesis of compounds 9, 15 and the Michael adduct 16
Lactone 9 was prepared by treating hexadecanal with vinyl magnesium bromide, followed by the esterification and metathesis. In the same way, compound 15 was prepared following the general methodology previously described, but using decanediol as starting material [29]. Finally, compound 16 was obtained by treatment of lactone 8 with MeOH and Et 3 N. The synthesis of the remaining compounds was achieved in yields ranging 70% for the allylation (vinylation)-esterification-ring closing metathesis sequence.

Leishmanicidal Activity Studies
The leishmanicidal activity of synthetic compounds as well as glucantime, which was used as control drug, was evaluated following the method reported in the literature [21,30]. The results are shown in Table 1 and may provide some insights as to structure-activity relationships. Not only the presence of an α,β-unsaturated lactone, but also a less polar portion which can be a long aliphatic chain (compound 8) or an aromatic ring (compounds 10,14) appears essential for leishmanicidal activity. The change of the aliphatic side chain (compound 8) for an aromatic ring (compounds 10-14) led to an increase in cytotoxicity and a decrease in activity; however, this ring should not be directly attached to the lactone (6, 13 vs. 14). Additionally, functionalization causes change in biological activity, which is affected by the relative position in the aromatic ring (10 vs 11 and 12), although cytotoxicity is high still. On the other hand, a large molecule such as dimer 7 is less active, even though it has two lactone rings, probably due to steric hindrance of the interaction with a putative receptor, but dimer 15, which also has two lactone rings, displays a significant activity, even though it is smaller than compound 7. However, dimer 15 has a similar activity to compound 8, but improved cytotoxicity. Perhaps, the improved activity of 15 is related to the lipophilic central chain joining the lactone rings. However, the central chain cannot be as large as in the case of dimeric molecule 7, otherwise activity will be compromised.
Regarding changes in the lactone ring, the reduction of the ring size (compound 9) leads to a considerable decrease in cytotoxicity (3.5 vs 33.9, 8 vs 9), but its activity is three times lower (2.8 vs 0.8 compound 8). However, surprisingly, the γ-lactone shows a high level of leishmanicidal activity and its cytotoxicity is lower than for the δ-lactone. In Michael adduct 16 both cytotoxicity and activity decrease significantly, which confirms the importance of the α,β-unsaturated carbonyl system in this type of compounds.
In general, most of the compounds have significant activity in L. panamensis amastigotes, exhibiting an EC 50 of less than 10.0 mg/mL (Table 1). With regard to selectivity index (SI = LC 50 /EC 50 ) compound 9 (SI = 12.1) would be the most promising, because of its simple structure and low LC 50 (33.9). These results are comparable with the previously reported leishmanicidal activity of lactams [7].

General
NMR spectra were recorded as CDCl 3 solutions on a Bruker AMX 300 instrument operating at 300 MHz for 1 H and 75 MHz for 13 C. Chemical shifts (δ) are expressed in ppm with the solvent peak as reference; coupling constants (J) are given in Hertz (Hz). Multiplicity assignments of 13 C signals were made by means of the DEPT pulse sequence. High resolution mass spectra were run in the electron impact mode (EIMS 70 eV). IR data were measured with oily films on KBr pellets (solids) on a Perkin-Elmer RXI-2000 (FT-IR). Experiments which required an inert atmosphere were carried out under dry N 2 in flame-dried glassware. Et 2 O and THF were freshly distilled from sodium/benzophenone and transferred via syringe. Dichloromethane was freshly distilled from CaH 2 . Commercially available reagents were used as received. Unless otherwise indicated, "work-up" means pouring the reaction mixture into brine, followed by extraction with the solvent indicated in parenthesis. If the reaction medium was acidic (basic), an additional wash with 5% aq NaHCO 3 (aq NH 4 Cl) was performed. Washing with brine, drying over anhydrous Na 2 SO 4 and elimination of the solvent under reduced pressure were followed by chromatography on a silica gel column (60-200 µm) with the indicated eluent.

Bioassays
Leishmanicidal activity and cytotoxicity were measured according to previously reported procedures [21].

Conclusions
A number of aliphatic and aromatic lactones as well as two dimers were synthesized and evaluated against amastigotes of Leishmania panamensis. The analysis of the results showed that the lactones with an aliphatic side chain are more active and less cytotoxic than lactones with aromatic side chains. We also found that reducing the size of the lactone ring results in increased activity and selectivity index. Furthermore, it confirms the importance of the α,β-unsaturated lactone moiety for biological activity in this area.