Title : Synthesis , biological evaluation and bioavailability prediction of novel furoxan derivatives as leishmanicidal compounds

Four novel furoxan derivatives were synthesized, characterized and evaluated for in vitro activity against promastigote form of Leishmania amazonensis and Leishmania infantum, and amastigote form of L. infantum. Compound 7 presented low cytotoxicity, being 30 times less toxic than pentamidine. All compounds were more active against L. infantum than L. amazonensis promastigote form, with IC50 reaching values around 8 μM. Regarding amastigote form of L. infantum, the compounds (3b and 7) have exhibited IC50 values around 60 μM. The in silico prediction of ADME properties has shown that all compounds demonstrated drugability according to ‘Lipinski rules’ and presented intestinal absorption ranging from 67 to 70%. In conclusion, the compounds presented here have demonstrated to be interesting prototypes against the visceral form of leishmaniasis.


INTRODUCTION:
Leishmaniasis is a neglected tropical disease caused by more than 20 species of the protozoan parasite Leishmania [1] and transmitted to humans by the bite of the female sanfly of the genera Phlebotomus, which is the only vector responsible for transmitting the disease [2].
There are three forms of leishmaniasis: cutaneous, visceral and mucocutaneous.
Afghanistan, Algeria, Brazil and Colombia are among the countries with the majority of cutaneous leishmaniasis cases.Only in 2015, Brazil has reported more than one thousand new cases of visceral leishmanisis [3].
The increasing drug resistance in leishmaniasis treatment and the lack of recent costeffective drugs are important issues to be considered in order to discover new drugs.
Therefore, there is a high urgency for new compounds active against leishmaniasis.In the present study, four furoxan derivatives were evaluated against promastigote and amastigote forms of L. amazonensis and L. infantum, which are responsible for cutaneous and visceral leishmaniasis, respectively.
Chemical shifts were expressed in parts per million (ppm) relative to tetramethylsilane.

Animals
Adult male Swiss albino mice (20-35 g) were used in the experiments.They were housed in single-sex cages under a 12 h light:12 h dark cycle (lights on at 06:00) in a controlled-temperature room (22  2 °C).The mice had free access to food and water.
Groups of two animals were used in each test group.The experiments were performed after the protocol was approved by the local Institutional Ethics Committee (protocol number CEUA/FCF/Car n° 53/2012).All experiments were performed in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals.

Cytotoxicity using murine macrophages
To access the cytotoxicity thioglycolate-stimulated mice were used to collect peritoneal macrophages.Murine peritoneal macrophages were seeded in 96 well flat-bottom plates (TPP ® ) at a density of 3x10 5 cells/well (100 μL/well) in RPMI-1640 medium supplemented with 10% heat inactivated FBS, 25 mM HEPES and 2 mM l-glutamine and incubated for 4 h at 37 °C in a 5% CO2-air mixture.The medium was removed and then new medium was added to the cells which were treated with different concentrations of compounds and pentamidine (Sigma-Aldrich ® ).Cells without drugs were used as negative control.After that, plates were incubated for 24 h at 37 °C in a 5% CO2-air mixture.
Subsequently, the MTT colorimetric assay was carried out as described above.Absorbance was read in a 96-well plate reader (Robonik ® ) at 590 nm.The drug concentration corresponding to 50% of cell growth inhibition was expressed as the inhibitory concentration (CC50) [11].

Amastigotes
Murine peritoneal macrophages were plated at 3x10 5 cells/well on coverslips (13 mm diameter) previously arranged in a 24-well plate in RPMI-1640 medium supplemented with 10% inactivated FBS, and allowed to adhere for 4 h at 37 °C in 5% CO2.Adherent macrophages were infected with promastigotes in the end of the exponential growth phase using a ratio of 5:1 for L. amazonensis per cell at 37 °C in 5% CO2 for 4 h.After that time, the non-internalized parasites were removed by washing, and infected cultures were incubated in RPMI-1640 medium for 24 h at 37 °C in 5% CO2 to parasite multiplication.
Then infected cells were treated with different concentrations of the compounds and pentamidine (Sigma-Aldrich ® ) for 24 h.However, for L. infantum macrophages were infected with promastigotes in the end of the exponential growth phase using was used at a ratio of 10:1 per cell at 37 °C in 5% CO2 for 18 h.After that time, infected cells were treated with different concentrations of the compounds and pentamidine (Sigma-Aldrich ® ) for 24 h.The cells were then fixed in a methanol solution and stained with Giemsa.The number of amastigotes/100 cells and percent infected cells were determined.The concentration that caused a 50% decrease of growth inhibition compared to the control was determined by regression analysis and expressed as the inhibitory concentration (IC50) in μM [11].

In silico prediction of ADME properties
In order to evaluate the human absorption of the compounds, the drugability through 'Lipinsk rule', Log P, water solubility and human absorption were predicted using the website pKCSM [12].

Chemistry:
The synthetic routes for the preparation of compounds 3a, 3b, 7, 11 are summarized in Fig. 1 to 3.

Leishmanicidal activity:
The first step of the biological evaluation was the IC50 determination of the compounds against promastigote forms of Leishmania amazonensis and Leishmania infantum.The intermediates 2a, 2b, 6 and 10 were previously evaluated against the promastigote form of L. amazonensis with an IC50 range from 0.79 to 4.29 µM and selective index range from 0.38 to 3.61, suggesting a relative cytotoxicity of these molecules [8].
Regarding L. amazonensis promastigote form, the compounds presented IC50 values of 2.7 to 19 times higher than pentamidine; however, the CC50 has demonstrated that the cytotoxicity of the new molecules is expressive lower than pentamidine, also demonstrated by the SI.Compound 7 is the less toxic among the molecules, with an outstanding CC50 more than 30 times higher than pentamidine.During the evaluation against promastigote form of L. infantum, all compounds presented an IC50 values lower than pentamidine, reaching values around 8 µM.Compound 3b and 7 have exhibited selective index superior to 15, therefore, these compounds were selected to further studies involving amastigote form of L. infantum.Interestingly, both compounds (3b and 7) have exhibited related IC50 values around 60 µM.

In silico prediction of ADME properties
Lipinski rule, LogP, water solubility and intestinal absorption were parameters predicted of the compounds and presented in Table 2.For the Lipinski rule evaluation, the molecular weight, hydrogen bond donors and acceptors and LogP were analyzed, and the compounds were classified in "yes", as the compounds which follow the rules, and "no" as the compounds that do not follow the rules.Based on these parameters, all compounds exhibited drugability through Lipinski rules.
Intestinal absorption is a parameter that results from LogP and water solubility.The results presented here demonstrates that the predicted absorption of the compounds is close, varying from 67 to 70%.The compounds with higher absorption presented a higher water solubility, as observed in compounds 7 and 11.

CONCLUSION
The new series of furoxan derivatives (compounds 3a, 3b, 7 and 11) were synthesized and characterized using analytical methods.The compounds presented CC50 values superior to that of pentamidine.The CC50 of compound 7 is 30 times higher than the control drug.All compounds have demonstrated to be more active against L. infantum than L. amazonensis, which leaded to an evaluation against amastigote form of L. infantum and resulted in IC50 three times higher than pentamidine.The in silico study has demonstrated that the compounds exhibits drug-like properties.The results presented here demonstrate that the compounds showed low toxicity and interesting activity against L. infantum, therefore they are interesting prototypes against the visceral form of leishmaniasis.

AKNOWLEGMENTS
This study was supported by FAPESP, CAPES and CNPq.

Figure 1 .
Figure 1.Reagents and conditions to obtain compounds 3a and 3b.

Figure 2 .
Figure 2. Reagents and conditions to obtain compound 7.

Figure 3 .
Figure 3. Reagents and conditions to obtain compound 11.