Antiprotozoal Activity of ,-Unsaturated -Lactones: Promising Compounds for the Development of New Therapeutic Alternatives

The parasite resistance and side effects of drugs used to treat protozoal diseases have led to the search for new therapies, both natural and synthetic. Studies have shown that various �,�-unsaturated �lactones displayed high antiprotozoal activity and thus are promising compounds for new drug discovery and development. These compounds and their activity are examined in this review.


INTRODUCTION
Protozoal diseases, particularly malaria, leishmaniasis and Chagas disease, represent major causes of mortality in various tropical and subtropical regions. These diseases remain significant health problems in many developing countries, and this situation is compounded by increasing treatment failures using current drugs.
Malaria causes more than 300 million acute illnesses and at least one million deaths annually. Resistance of the malaria parasites, Plasmodium spp., to drugs such as chloroquine (and, more lately, quinine) occurs with increasing frequency [1,2]. This resistance underlies the necessity of developing new agents for malaria chemotherapy, with new modes of action to replace current ineffective drugs.
Leishmaniasis is a major health problem that affects approximately 12 million people worldwide, with 2 million new cases diagnosed every year [3]. The causative agents of this disease are parasites of the genus Leishmania, which infect and replicate in macrophages of the vertebrate host. Recently, a dramatic increase in the number of cases of leishmaniasis has been observed in patients with compromised T-cell function, such as those infected with the human immunodeficiency virus [4]. The chemotherapy of leishmaniasis has been based on pentavalent antimonials, sodium stibogluconate (pentostam) and meglumine antimonite (glucantime). These drugs contain multiple uncharacterized molecular structures with variable efficacies and toxicities, they are associated with moderate and severe side effects [5-7], prone to induce resistance [8,9] and require parenteral administration over a long period [10]. Second-line drugs, such as Amphotericin B and its lipid formulations, are either more toxic and expensive for routine use in developing countries.
Trypanosoma cruzi is a protozoa that causes Chagas disease (American trypanosomiasis); it is an obligate intracellular protozoan parasite that causes acute and chronic infection in several mammalian species including humans. This illness affects approximately 16 to 18 million people in tropical and sub-tropical Americas leading to the death of approximately 400.000 people per year [11]. Nifurtimox and benznidazole, the drugs currently in use against this disease, present several side effects and have limited efficacy [12]. Gentian violet, another compound for the prevention of Chagas disease by blood transfusion [13], leads to purple colouring of the blood and staining of patients' tissues. The use of gentian violet is limited due to its toxicity and other side effects such as alteration of skin color, mucous membranes and urine [14].
The development of new, effective, non-toxic and less expensive drugs is required to contribute to the world-wide control of these diseases.
In an effort to discover new compounds for infectious diseases treatment, several α,βunsaturated δ-lactones were evaluated and found to have high antiprotozoal activity. Our finding are summarizes in this review.
Argentilactone exhibited in vitro activity against various strains of Leishmania ssp. at 10 µg/ml. BALB/c mice infected with Leishmania amazonensis were treated four weeks after infection with argentilactone by oral or subcutaneous routes for 14 days at 25 mg/kg daily. The reference drug, Nmethylglucamine antimonate, was administered by subcutaneous injections at l00 mg/kg for 14 days. Under these conditions, argentilactone showed the same efficacy as the reference drug, reducing by 96 % the parasite loads in the lesion and by 50% the parasite burden in spleen [36]. Other studies showed that 16 exhibited cytotoxic activity against P-388 mouse leukemia cells (IC 50 = 21.4µM) [32] and both enantiomers of argentilactone displayed antiproliferative activity against several human cancer cell lines [37]. Two argentilactone epoxides derivatives, 17 and 18 (Fig 2) showed in vitro activity against Leishmania amazonensis (IC 50 = 22.3 and 44.6 mg/mL, respectively), epoxide (19) displayed no activity [38].
Lactones (20) and (21) (Fig 2) were isolated from dichloromethane extracts of both the roots and the leaves of Raimondia cf. monoica. These compounds showed in vitro leishmanicidal activities against Leishmania panamensis with ED 50 of 1.9 and 0.42 µg/mL, respectively, and cytotoxicity on U-937 cells with LD 50 of 2.1 and 1.0 µg/mL, respectively [34].
Passifloricin A was isolated from Passiflora foetida resin [39], its structure was revised as a consequence of a stereospecific synthesis [40]. Several natural, hemisynthetic and synthetic passifloricins were tested against Leishmania panamensis amastigotes [41,42] (Fig 2). High leishmanicidal activity as well as toxicity were observed. Passifloricin A (22) showed activity (EC 50 ) and cytotoxicity (LC 50 ) of 0.36 and 2.3, respectively. Change in the C-12 configuration decreases both the activity and cytotoxicity (22 vs 23) (see Table  1  In general, most active compounds are those with two hydroxyl groups in the side chain (29, 32, 33, 35, 39 and 40). Compounds with hydroxyl group in side chains have a tendency to increase activity and decrease cytotoxicity when the hydroxyl group is farther from the closure of the lactone (31, 34 and 36-38). However, a comparison of the biological profiles of these compounds indicate there is no tendency to increase activity against L. panamensis according to the number of hydroxyl groups. Several aliphatic and aromatic lactones and two dimers were synthesized to establish a relationship between structure and activity (see Fig 2) [43]. These compounds were active in vitro against intracellular amastigotes of Leishmania panamensis (see Table 1).
Change in the stereochemistry of the lactone closure has no significant effect on the biological activity (50 vs 51). However, shortening the length of the side chain decreases it (51 vs 53). Lactam 54 exhibited lower leishmanicidal activity (3.4 µg/mL) than compound rac-50 (0.8 µg/mL), but it displayed less cytotoxicity (45.1 µg/mL vs 3.5 µg/mL) and the selectivity index (SI) was  almost three times. Regarding changes in the lactone ring, the reduction of the ring size (compound 55) leads to a considerable decrease in cytotoxicity (3.5 µg/mL vs 33.9 µg/mL, rac-50 vs 55), but its activity is three times lower (0.8 µg/mL vs 2.8 µg/mL compounds rac-50 vs 55). However, the γlactone shows a higher SI than δ-lactone. Not only the presence of an α,β-unsaturated lactone, but also a less polar portion which can be a long aliphatic chain (compound rac-50) or an aromatic ring (compounds 59,63) appears essential for leishmanicidal activity.
The change of the aliphatic side chain (compound rac-50) for an aromatic ring (compounds 59-63) led to an increase in cytotoxicity and a decrease in activity. Additionally, functionalization causes change in biological activity, which is affected by the relative position in the aromatic ring (59 vs 60 and 61), although cytotoxicity is still high. On the other hand, a large molecule such as dimer 58 is less active, even though it has two lactone rings, probably due to steric hindrance of the interaction with a putative receptor, but dimer 57, which also has two lactone rings, displays a significant activity, even though it is smaller than compound 58. However, dimer 57 has a similar activity to compound rac-50, but improved cytotoxicity.
Perhaps, the improved activity of 57 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 58, otherwise activity will be compromised. Activity against Leishmania amastigotes practically disappears in the Michael adducts (compounds 49, 52 and 56). Both, the increased hydrophilicity (60 vs 62) and the reduction of the exocyclic double bond (63 vs rac-6) lead to a decrease in activity (Fig 2).
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. Synthesis of analogs 23 to 53 allowed identification of the pharmacophoric groups responsible for the high antileishmanial activity shown by passifloricin A (22) against the Leishmania panamensis amastigotes (see Fig 3).
In conclusion, most of the compounds had a significant activity against L. panamensis amastigotes, exhibiting an EC 50 lower than 5.0 µg/mL ( Table 1)

TRYPANOCIDAL ACTIVITY
Cryptofolione (64) was isolated from the fruits of Cryptocarya alba (see Fig 4). In the trypanocidal test, this compound reduced the number of Trypanosoma cruzi trypomastigotes by 77 % at a concentration of 250 µg/mL. Under the same conditions, gentian violet lysed 100 % of the parasites. At the lowest concentration tested, 25 µg/mL this compound showed a moderate effect on T. cruzi amastigotes infecting mice peritoneal macrophages at the cytotoxic IC 50 . As the trypanocidal effect and cytotoxicity values are similar, the compound presented little selectivity. However, this compound showed a moderate effect against Leishmania spp promastigotes (< 70 % of lysis) [44].
Results showed three factors increasing trypanocidal activity: when the endocyclic double bond is removed (ent-6 vs 69 and 71, IC 50 = 0.21 and 0.19 mM, respectively), when the exocyclic double bond in the molecule remains (ent-6 vs 70 IC 50 = 0.91 mM) and by the presence of electron withdrawing groups in the aromatic ring (ent-6 vs 74, 75, IC 50 = 0.12 and 0.09 mM, respectively). Compounds with electron-rich aromatic rings such as 72 (IC 50 = 2.39 mM) and 73 (IC 50 = 6.27 mM) were significantly less potent when compared to ent-6. Finally, substitution of the aromatic group by cyclohexyl group in analogue 76 (IC 50 = 0.22 mM) as well the use of isogoniothalamin oxide (67, IC 50 = 0.25 mM) or its enantiomer 68 (IC 50 = 0.26 mM) provided lower IC 50 values than ent-6 but were still not as effective as derivatives 74 and 75 (Table 3). However, epoxides 67 and 68 presented higher activity than their corresponding stereoisomers 65 (IC 50 = 0.41 mM) and 66 (IC 50 = 1.50 mM). These results allowed the identification of the pharmacophoric groups in goniothalamin (6) (Fig 3)

CONCLUDING REMARKS
In this review, we have presented the antiprotozoal activity of several α,βunsaturated δ-lactones. The results shows that the α,β-unsaturated-δ-lactone moiety is essential for biological activity. This is due to the fact that this unit is an excellent potential Michael acceptor for nucleophilic amino acid residues (cysteine, lysine, serine or threonine) and N7 of guanine present in biomolecules of the natural receptors interacting with these compounds [47,48]. Several compounds showed high antiprotozoal activity, but cytotoxic properties were also observed. The synthesis of structural and functional lactones analogs is needed to obtain compounds with high selectivity index for validation as potential antiparasitic drugs.