Synthesis, Leishmanicidal and Cytotoxic Activity of Triclosan-Chalcone, Triclosan-Chromone and Triclosan-Coumarin Hybrids

Twelve hybrids derived from triclosan were obtained via Williamson etherification of O-triclosan alkyl bromide plus chalcone and O-coumarin or O-chromone alkyl bromide plus triclosan, respectively. Structures of the products were elucidated by spectroscopic analysis. The synthesized compounds were evaluated for antileishmanial activity against L. (V) panamensis amastigotes. Cytotoxic activity was also evaluated against mammalian U-937 cells. Compounds 7–9 and 17, were active against Leishmania parasites (EC50 = 9.4; 10.2; 13.5 and 27.5 µg/mL, respectively) and showed no toxicity toward mammalian cells (>200 µg/mL). They are potential candidates for antileishmanial drug development. Compounds 25–27, were active and cytotoxic. Further studies using other cell types are needed in order to discriminate whether the toxicity shown by these compounds is against tumor or non-tumor cells. The results indicate that compounds containing small alkyl chains show better selectivity indices. Moreover, Michael acceptor moieties may modify both the leishmanicidal activity and cytotoxicity. Further studies are required to evaluate if the in vitro activity against Leishmania panamensis demonstrated here is also observed in vivo.


Introduction
Leishmaniasis is a group of diseases caused by protozoan parasites of the genus Leishmania, which infect and replicate inside macrophages of the vertebrate host. The diseases is a major health problem because is present in 98 countries and three territories worldwide, affecting mostly low-income people in rural areas of tropical and subtropical countries. Approximately 0.7 to 1.2 million cutaneous leishmaniasis (CL) cases occur annually. Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica and Peru, together account for 70% to 75% of global estimated CL incidence [1]. Leishmania (Viannia) panamensis is one of the most important causal agent of CL in Central and South America [2]. The different forms of leishmaniasis require expensive treatments, and the currently used medicines, pentavalent antimonials, pentamidine isothianate and miltefosine, show high toxicity and therefore severe side effects and therefore there is an urgent need for new drugs [3]. However, due to the lack of interest shown by the pharmaceutical industry to develop drugs against neglected diseases, it is necessary to join forces to develop new and better drugs to manage the disease and help patients to improve their quality of life [4].
Triclosan is an uncompetitive inhibitor of purified enoyl-acyl carrier protein reductase (ENR), which has demonstrated inhibitory activity in vitro against Plasmodium falciparum [5][6][7][8]. A previous study showed that triclosan and quinoline-triclosan hybrids with shorter spacers, that is three and five methylene units, have in vitro activity against axenic and intracellular amastigotes with effective concentration (EC 50 ) below of 24 µg/mL) of Leishmania panamensis [9]. Similarly, antileishmanial activity of several chalcones has been reported [10][11][12]. The most promising of this class of compounds is licochalcone A, an oxygenated chalcone isolated from the roots of the Chinese plant Glycyrrhiza spp., which inhibits the fumarate reductase, a selective target present in the mitochondria of the parasite [13].
The combination of two pharmacological agents into a single molecule, called hybrid molecule, is an emerging strategy in medicinal chemistry and drug discovery research [28,29]. These hybrid molecules may display dual activity but do not necessarily act on the same biological target [30][31][32][33][34].
In this work, four chalcone-triclosan, chromone-triclosan, and coumarin-triclosan hybrids (Schemes 1 and 2) were synthesized and their cytotoxic and leishmanicidal activities determined in the search for new therapeutic alternatives for the treatment of leishmaniasis.
The effective concentration against the intracellular forms of L. (V) panamensis was also measured for those compounds showing percentages of inhibition higher than 50% at 20 µg/mL (Table 1). Thus, compounds 10, 16, 18, 19 and 28 were not evaluated. The most active compounds for intracellular parasites were 25, 26 and 7 with EC 50 of 2.7, 7.5 and 9.4 µg/mL, respectively, followed by compounds 8, 9 and 27 with EC 50 values of 10.2, 13.5 and 16.0 µg/mL, respectively ( Table 1). All compounds showed leishmanicidal activity higher than cytotoxicity and therefore, selectivity indexes higher than 1. The best SI´s were observed for compounds 7-9 and 17 with values higher than of 21.3, 19.6, 14.8 and 7.3, respectively. Although compound 25 showed better activity than meglumine antimoniate, its SI is affected by the high cytotoxicity.
On a structure-activity relationship basis, it is interesting to note the synergistic effect of the parent subunits in the hybrids in comparison with the unlinked cases. For example, chalcone 6 and triclosan are less potent and more cytotoxic individually than their hybrids 7-9. This phenomenon can also be observed in compounds 25-27 in which an increase in activity is evident in the hybrids at the expense of cytotoxicity compared to the individual units. The length of the alkyl linker also plays a pivotal role, with an inverse correlation between increased length and activity for the evaluated cases.
One possible mechanisms of action for these compounds may be formulated in terms of conjugated addition of nucleophilic amino acid residues present in biomolecules of the natural receptors, in a Michael type mechanism. This mechanism has been reported for other α,β-unsaturated compounds such as lactones and cinnamic acid esters [42][43][44]. In this regard, the low reactivity shown by triclosan-coumarin hybrids could be rationalized in terms of steric hindrance and electronic deactivation caused by the methyl group at the β-position.

General Remarks
The syntheses were carried out in a MW domestic oven adapted for the use of a reflux condenser and magnetic stirrer, at constant power (400 W). NMR spectra were recorded as CDCl 3 solutions on an AMX 300 instrument (Bruker, Billerica, MA, USA) operating at 300 MHz for 1H and 75 MHz for 1 C. Chemical shifts (δ) are expressed in ppm with the solvent peak as reference and TMS as an internal standard; coupling constants (J) are given in Hertz (Hz). High resolution mass spectra were recorded using electrospray ionization mass spectrometry (ESI-MS). A QTOF Premier instrument with an orthogonal Z-spray-electrospray interface (Waters, Manchester, UK) was used operating in the W-mode. The drying and cone gas was nitrogen set to flow rates of 300 and 30 L/h, respectively. Methanol sample solutions (ca. 1 × 10 −5 M) were directly introduced to the ESI spectrometer at a flow rate of 10 µL/min. A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone voltage set to Uc = 10 V. For the accurate mass measurements, a 2 mg/L standard solution of leucine enkephalin was introduced via the lock spray needle at a cone voltage set to 85 V and a flow rate of 30 μL/min. IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer, Waltham, MA, USA) in KBr disks. Elemental analysis were recorded on TruSpec Micro Series equipment (LECO Corporation, St. Joseph, MI, USA). Commercially available reagents were used as received. Silica gel 60 (0.063-0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column chromatography, and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin layer chromatography (TLC).

General Procedure for the Synthesis of Bromoalkyl Derivatives
Triclosan, chromone or coumarin (1 mmol, 1 eq.), potassium hydroxide (3 eq.) and acetonitrile (10 mL), were placed in a 50 mL flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated to reflux for a period of 5 min, under microwave irradiation. Then, 1,ω-dibromoalkane (1.1 eq.) was added to the reaction mixture which was refluxed for 30 min. The crude reaction mixture was concentrated on a rotatory evaporator, and the residue was purified by column chromatography over silica gel eluting with hexane and a mixture of hexane-ethyl acetate (9:1 ratio) to obtain the bromoalkyl derivatives in yields ranging between 60%-85%. Monitoring of the reaction progress and product purification was carried out by TLC.

General Procedure for the Synthesis of Triclosan-Chalcone Hybrids
Chalcone (1.1 eq.), potassium hydroxide (2 eq.) and acetonitrile (10 mL), were placed in a 50 mL flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated to reflux for a period of 5 min, under microwave irradiation. Then, bromoalkyltriclosan (100 mg, 1 eq.) was added to the reaction mixture which was then refluxed for 30 min. The crude reaction mixture was concentrated on a rotatory evaporator, and the residue was purified by column chromatography over silica gel eluting with hexane-ethyl acetate (9:1 ratio) to obtain the triclosan-chalcone hybrids in yields between 35%-60%. Monitoring of the reaction progress and product purification was carried out by TLC.

General Procedure for the Synthesis of Triclosan-Coumarin and Triclosan-Chromone Hybrids
Triclosan (1.1 eq.), potassium hydroxide (2 eq.) and acetonitrile (10 mL), were placed in a 50 mL flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated to reflux for a period of 5 min under microwave irradiation. Then, bromoalkylcoumarin or bromoalkylchromone (1 eq.) was added to the reaction mixture which was refluxed for 30 min. The crude reaction mixture was concentrated on a rotatory evaporator, and the residue was purified by column chromatography over silica gel eluting with hexane-ethyl acetate (4:1 and then 3:2 ratio) to obtain the triclosan-chalcone hybrids in yields between 50%-80%. Monitoring of the reaction progress and product purification was carried out by TLC.  (17). Yield 53%

Biological Activity Assays
The compounds were subjected to in vitro cytotoxic activity on mammalian cells and leishmanicidal activity on intracellular amastigotes of L. panamensis.

In Vitro Cytotoxic Activity in Mammalian Cells
The cytotoxic activity of the compounds was assessed based on the viability of the human promonocytic cell line U937 (ATCC CRL-1593.2 TM ) evaluated by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay [41]. In brief, cells were grown in 96-well cell-culture dishes at a concentration of 100,000 cells/mL in RPMI-1640 supplemented with 10% FBS and the corresponding concentrations of the compounds, starting at 200 µg/mL in duplicate. The cells were incubated at 37 °C with 5% CO 2 for 72 h in the presence of the compounds, and then the effect was determined using the MTT assay, incubating at 37 °C for 3 h. The effect of the compounds was determined by measuring the activity of the mitochondrial dehydrogenase by adding 10 µL/well of MTT solution (0.5 mg/mL) and incubating at 37 °C for 3 h. The reaction was stopped by adding a 50% isopropanol solution with 10% sodium dodecyl sulfate for 30 min. Cell viability was determined based on the quantity of formazan produced, according to the optical density (O.D) obtained at 570 nm in a Bio-Rad (Hercules, CA, USA) ELISA instrument. Cultured cells in the absence of extracts were used as viability controls, while meglumine antimoniate and amphotericin B were used as cytotoxicity controls. Assays were performed twice with three replicates per each concentration tested.

Activity against Intracellular Amastigotes
The leishmanicidal activity of compounds was evaluated on intracellular amastigotes of L. panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140pIR-GFP) [42]. Effect of each compound was determined according to the inhibition of the infection evidenced by both decrease of the infected cells and decrease of intracellular parasite load. Briefly, U-937 human cells at a concentration of 3 × 10 5 cells/mL in RPMI 1640 and 0.1 μg/mL of PMA (phorbol-12-myristate-13-acetate) were infected with promastigotes in stationary phase growth in a 15:1 parasites per cell ratio and incubated at 34 °C and 5% CO 2 for 3 h. Cells were washed two times with phosphate buffer solution (PBS) to eliminate not internalized parasites. Fresh RPMI 1640 1 mL was added and cells were incubated during 24 h to guarantee multiplication of intracellular parasites.
After 24 h of infection, the culture medium was replaced by fresh culture medium containing each compound at concentrations of 20 μg/mL. After 72 h, the inhibition of the infection progress was determined. Cells were removed from the bottom plate with a trypsin/EDTA (250 mg) solution. Recovered cells were centrifuged at 1100 rpm during 10 min at 4 °C, the supernatant was discarded and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm during 10 min at 4 °C. Supernatant was discarded and cells were suspended in 500 μL of PBS and analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, USA) counting 20.000 events. All determinations for each compound and standards were carried out in triplicate, in two isolated experiments [41,42]. Activity of tested compounds was carried out in parallel with infection progress in culture medium alone and in culture medium with amphotericin B 0.05 μg/mL and meglumine antimoniate (10.0 μg/mL) as control drugs. Compounds that showed growing inhibition percentages higher than 50% were then evaluated at four additional concentrations to determine the 50% effective concentration (EC 50 ). The infected cells were exposed against each concentration of synthesized compounds during 72 h, then, cells were removed and tested by flow cytometry as described before.

Statistical Analysis
Cytotoxicity was determined according to viability and mortality percentages obtained for each isolated experiment (compounds, amphotericin B, meglumine antimoniate and culture medium alone). The results were expressed as 50 lethal concentrations (LC 50 ), that corresponds to the concentration necessary to eliminate 50% of cells, calculated by Probit analysis [45]. Percentage of viability was calculated by Equation (1) The degree of toxicity was established according to the LC 50 value using the following scale: highly toxic: LC 50 < 50 μg/mL, toxic: LC 50 > 50 to < 100 μg/mL; moderately toxic: LC 50 > 100 to < 200 μg/mL and potentially non-toxic: LC 50 > 200 μg/mL.
Antileishmanial activity was determined according to percentage of infected cells and parasite load obtained for each experimental condition by the cytometer. Percentage of infected cells was determined as the number of positive events by double fluorescence (green for parasites and red for cells) using dotplot analysis. On the other hand, the parasitic load was determined by analysis of mean fluorescence intensity (MFI) [41].

Conclusions
The design, synthesis, and antileishmanial screening of twelve triclosan derivatives are reported. Several of the synthetic compounds have potential as templates for drugs development. Owing to the high leishmanicidal activity and the low cytotoxicity we consider that compounds 7-9 and 17 are good candidates. Studies on an animal model of leishmaniasis are needed to confirm the results observed in vitro. On the other hand, compounds 25-27 that were active against Leishmania parasite but toxic for mammalian cells still have potential to be considered as candidates for antileishmanial drug development. However, more studies on toxicity using other cell lines are needed to discriminate whether the toxicity shown by these compounds is against tumor or non-tumor cells. The results indicate that compounds containing small alkyl chains show better selectivity indices. Moreover, Michael acceptor moieties may modify both the leishmanicidal activity and cytotoxicity. The mechanism of action of these promising compounds also needs to be addressed.