Synthesis, antiprotozoal activity and cytotoxicity in U-937 macrophages of triclosan–hydrazone hybrids

The synthesis and biological activities (cytotoxicity, leishmanicidal, and trypanocidal) of 11 triclosan–hydrazone hybrids are described herein. The structure of the products was elucidated by spectral data (NMR, IR) and mass spectrometric analyses. The synthesized compounds were evaluated against amastigotes forms of L. (V) panamensis, which is the most prevalent Leishmania species in Colombia, and against Trypanosoma cruzi, which is the major pathogenic species to Chagas disease in humans. In addition, the cytotoxic activity of the synthesized compounds was evaluated against human U-937 macrophages. Hydrazone hybrids were obtained as E-synperiplanar and E-antiperiplanar conformers. Nine of them were active against L. (V) panamensis (5a–5d, 5f–5j) and eight of them against T. cruzi (5a, 5c, 5d, 5f–5j), with EC50 values lower than 40 µM. The compounds 5c, 5e, and 5h exhibit the best selectivity index against both L. (V) panamensis and T. cruzi, with values ranging from 5.90 to 16.55, thus showing potential as starting compounds for the eventual development of drugs against these parasites. The presence of hydroxy or methoxy groups in positions 2 and 4 of the aromatic ring of the benzylidene moiety increases both activity and cytotoxicity. There is no clear relationship between the antiprotozoal activity and the methylation pattern of the hydroxy groups, since in some cases methylation decreases the activity (5d vs. 5g) while in other cases the activity is increased (5c vs. 5f and 5i vs. 5j).


Introduction
Protozoal diseases are a cause of mortality in various developing countries of tropical and subtropical regions. For leishmania and Chagas-endemic countries these diseases cause significant health problems affecting more than one billion people worldwide (WHO 2002(WHO , 2013Alvar et al. 2012;Nouvellet et al. 2015). This situation is aggravated by increasing treatment failures with available drugs (Bhutta et al. 2014). Chagas disease (American trypanosomiasis) and leishmaniasis are parasitic diseases caused by the parasitic protozoan Trypanosoma cruzi (T. cruzi) and Leishmania species, respectively.
The Leishmaniasis involves a wide spectrum of clinical manifestations in which L. (V) panamensis is one of the most prevalent Leishmania species involved in human cases of cutaneous leishmaniasis in Colombia (Alvar et al. 2012). Chagas disease (also named American trypanosomiasis) is produced by the protozoan parasite T. cruzi that is transmitted to the mammalian host through the bite of triatomine bugs belonging to Triatoma, Rhodnius, and Panstrongylus genus (Nouvellet et al. 2015). Current treatments for cutaneous leishmaniasis are based on pentavalent antimonials (meglumine antimoniate and sodium stibogluconate). For the treatment of Chagas disease nitroaromatic compounds (benznidazole and nifurtimox) are usually employed. However, these treatments are not without significant side effects, particularly in patients undergoing high-dose and long-term treatments. In addition, the development of drug resistance has significantly increased the health problems associated with these diseases (Chatelain and Ioset 2011;Den Boer et al. 2011;Keenan and Chaplin 2015).
Triclosan is an uncompetitive inhibitor of purified enoylacyl carrier protein reductase (ENR), which has demonstrated in vitro inhibitory activity against Plasmodium falciparum (Kapoor et al. 2004;McLeod et al. 2001;Surolia and Surolia 2001;Perozzo et al. 2002). A previous study showed that triclosan has in vitro anti-leishmanial activity against axenic amastigotes of L. panamensis with an effective concentration (EC 50 ) of 39 µM.
In recent years a promising strategy has emerged based on hybrid molecules, which bear in their structures two distinct pharmacophores having, for example, anti-protozoal, antiinflammatory, anti-fungal, or anti-cancer activity, thus showing a dual mode of action (Keith et al. 2005;Meunier 2008). These hybrid molecules may display dual activity, but do not necessarily act on the same biological target (Opsenica et al. 2008;Roth et al. 2004;Walsh et al. 2007).
In the search for new therapeutic alternatives to treat cutaneous leishmaniasis and Chagas disease a number of triclosan-hydrazone hybrids have been designed and synthesized. Their leishmanicidal and trypanocidal activities, as well as their cytotoxicity in U-937 macrophages, have been evaluated in vitro (Fig. 2).

General remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power 300 W, temperature control by IR sensor, fixed . 1 H and 13 C NMR spectra were recorded on a Varian instrument operating at 500 and 125 MHz, respectively. The signals of the deuterated solvent (CDCl 3 ) were used as reference (the singlet at δ = 7.27 ppm for 1 H NMR and the triplet centered at δ = 77.00 ppm for 13 C NMR). Carbon atom types (C, CH, CH 2 , CH 3 ) were determined by using the DEPT or APT pulse sequence. Signals were assigned using two-dimensional heteronuclear correlations (COSY and HSQC). 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 for 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 into 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 was set to U c = 10 V. For 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. 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).
Synthetic procedure for ethyl 2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)acetate Triclosan 1 (6.0 g, 0.021 mol), potassium hydroxide (1.7 g, 0.03 mol), and acetonitrile (20 mL) were placed in a 50 mL flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated under microwave irradiation to reflux for a period of 5 min. Then, ethyl bromoacetate (2.4 mL, 3.67 g, 0.022 mol) was added to the reaction mixture, which was then refluxed for 30 min (200 W). 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 ester 2 with 75% yield (0.016 mol, 5.9 g). Monitoring of the reaction progress and product purification were carried out by TLC. Synthetic procedure for 2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)acetohydrazide Hydrazine monohydrate (3 mL of a 80% solution) was added to a solution of 2 (5 g, 0.013 mol) in ethanol (15 mL). The reaction mixture was submitted to microwave irradiation and maintained under reflux for 30 min. Then, the reaction mixture was poured on ice and the resulting precipitate was filtered out, affording the title compound 3 in 83% yield (3.90 g, 0.011 mmol).

Synthetic procedure for hydrazones
A triclosan-carbohydrazide 3 (0.5 g, 1.38 mmol) solution in methanol (2 mL) was sonicated for 2 min and then benzaldehyde 4 (1.38 mmol) and acetic acid (0.1 mL) were added dropwise to the reaction mixture. Upon completion of the reaction (5-10 min), the product was filtered, sequentially washed with water (20 mL) and ethyl ether (5 mL), dried in vacuo and recrystallized from ethanol, affording the corresponding hydrazones in yields ranging from 42 to 93%.

Biological activity assays
The compounds were subjected to in vitro evaluation as regards their cytotoxicity, leishmanicidal, and trypanocidal activity against U-937 human cells and intracellular amastigotes of L. (V) panamensis and T. cruzi, respectively.

In vitro cytotoxicity
The cytotoxic activity of the compounds was assessed based on the viability of the human promonocytic cell line U-937 (ATCC CRL-1593.2 TM ) evaluated by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay following the methodology described elsewhere (Pulido et al. 2012). Briefly, cells grown in tissue flasks were harvested and washed with phosphate buffered saline (PBS) by centrifuging. Cells were counted and adjusted at 1 × 10 6 cells/mL of RPMI-1640 supplemented with complete 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). One hundred µL was dispensed into each well of a 96-well cell-culture plate and then 100 mL of RPMI-1640 and the corresponding concentrations of the compounds were added, starting at 200 µg/mL in duplicate. Plates were incubated at 37°C, 5% CO 2 for 72 h in the presence of compounds. The effect of compounds was determined by measuring the activity of the mitochondrial dehydrogenase by adding 10 µL/well of MTT solution (0.5 mg/mL) and incubated at 37°C for 3 h. The reaction was stopped by adding 100 µL/well of 50% isopropanol solution with 10% sodium dodecyl sulfate and 30 min incubation. The cell viability was determined based on the quantity of formazan produced according to the intensity of color (absorbance) registered as optical densities (O.D.) obtained at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader-Thermo Scientific, USA). Cells cultured in the absence of compounds were used as control of viability (100% viability), while amphotericin B (AmB) was used as non-cytotoxic and cytotoxic drug control, respectively. Assays were conducted in two independent runs with three replicates per each concentration tested.

In vitro anti-leishmanial activity
The activity of the compounds was evaluated on intracellular amastigotes of L. (V) panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140-EGFP) (Taylor et al. 2011). The effect of each compound was determined according to the inhibition of the infection evidenced by the decrease of the infected cells and parasite inside the cells. Briefly, U-937 human cells at a concentration of 3 × 10 5 cells/mL in RPMI 1640 and 0.1 μg/mL of phorbol-12-myristate-13-acetate (PMA) were dispensed into each well of a 24-well cell culture plate and then infected with 5 days old promastigotes in a 15:1 parasites per cell ratio. Plates were incubated at 34°C, 5% CO 2 for 3 h and cells were washed two times with PBS to eliminate internalized parasites. One mL of fresh RPMI 1640 supplemented with 10% FBS and 1% antibiotics was added into each well, cells were incubated again to guarantee multiplication of intracellular parasites. After 24 h of infection, culture medium was replaced by fresh culture medium containing each compound at four-fold dilutions (100-25-6.25 and 1.56 μg/mL) and plates were incubated at 37°C, 5% CO 2 . After 72 h, inhibition of the infection was determined. Shortly, cells were removed from the bottom plate with a trypsin/EDTA (250 mg) solution, recovered cells were centrifuged at 1100 rpm for 10 min at 4°C, the supernatant was discarded and cells were washed with 1 mL of cold PBS by centrifuging at 1100 rpm for 10 min at 4°C. The supernatant was discarded and cells were suspended in 500 μL of PBS and analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, USA). All determinations for each compounds and standard drugs were carried out in triplicate, in two independent experiments (Pulido et al. 2012). Activity of the tested compounds was carried out in parallel with infection progress in culture medium alone and in culture medium with AmB as antileishmanial drugs.

In vitro anti-trypanosomal activity
Compounds were tested on intracellular amastigotes of T. cruzi, Tulahuen strain transfected with β-galactosidase gene (donated by Dr. F. S. Buckner, University of Washington) (Buckner et al. 1996). The activity was determined according to the ability of the compounds to reduce the infection of U-937 cells by T. cruzi. In this case, 100 μL of U-937 human cells at a concentration of 2.5 × 10 5 cells/mL in RPMI-1640, 10% SFB, and 0.1 μg/mL of PMA were placed in each well of 96-well plates and then infected with phase growth epimastigotes in 5:1 (parasites per cell) ratio and incubated at 34°C, 5% CO 2 . After 24 h of incubation four-fold dilutions of each compound (100-25-6.25 and 1.56 mg/mL) were added to infected cells. After 72 h of incubation, the effect of all compounds on viability of intracellular amastigotes was determined by measuring the β-galactosidase activity by spectrophotometry adding 100 μM CPRG and 0.1% nonidet P-40 to each well. After 3 h of incubation, plates were read at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA) and intensity of color (absorbance) was registered as O.D. Infected cells exposed to benznidazol (BNZ) were used as control for antitrypanosomal activity while infected cells incubated in culture medium alone were used as control for infection. Non-specific absorbance was corrected by subtracting the O.D. from the blank. Determinations were done by triplicate in at least two independent experiments (Buckner et al. 1996;Insuasty et al. 2015).

Statistical analysis
Cytotoxicity was determined according to viability and mortality percentages obtained for each isolated experiment (compounds, amphotericin B, Benznidazole, 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 and calculated by Probit analysis (Finney 1978). Percentage of viability was calculated by Eq. 1, where the O.D. of control corresponds to 100% of viability. In turn, mortality percentage corresponds to 100%-%viability: The degree of toxicity was graded according to the LC 50 value using the following scale: high cytotoxicity: LC 50 < 200 μM; moderate cytotoxicity: LC 50 > 200-<400 μM and potential non-cytotoxicity: LC 50 > 400 μM.
Anti-leishmanial activity was determined according to the percentage of infection (amount of parasites in infected cells) obtained for each experimental condition by flow cytometry. The 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 amount of parasites in the infected cells was determined by analysis of mean fluorescence intensity (MFI) in fluorescent parasites (Pulido et al. 2012). The parasite inhibition was calculated by Eq. 2, where the MFI of control corresponds to 100% of parasites. In turn, inhibition percentage corresponds to 100%-%Parasites. Results of anti-leishmanial activity were expressed as EC 50 determined by the Probit method (Finney 1978): Similarly, anti-trypanosomal activity was determined according to the amount of parasites in infected cells obtained for each experimental condition by colorimetry.
The parasite inhibition was calculated by Eq. 3, where the O.D. of unexposed parasites corresponds to 100% of parasites. In turn, percentage of inhibition corresponds to 100%-% Parasites. Results of anti-trypanosomal activity were also expressed as EC 50 determined by the Probit method (Finney 1978): The anti-leishmanial and anti-trypanosomal activities were graded according to the EC 50 value using the following scale: high activity: EC 50 < 40 μM, moderate activity: EC 50 > 40-<80 μM; and potential non-activity: EC 50 > 80 μM.
The selectivity index (SI), was calculated by dividing the cytotoxic activity and the anti-leishmanial or antitrypanosomal activity using the following formula: SI = LC 50 /EC 50 .

Results and discussion
Chemistry Microwave-assisted Williamson etherification of triclosan 1 with ethyl bromoacetate gave rise to ester 2 in 75% yield (Otero et al. 2014). Nucleophilic reaction of hydrazine hydrate on compound 2 gave rise to acylhydrazide 3 in 83% yield. Coupling compound 3 with a number of aldehydes in alcoholic medium provided hydrazones 5a-6h in 42-93% yields (Coa et al. 2015). This synthetic strategy involves ultrasound-assisted or microwave-assisted reactions which allows to achieve the compounds with shorter reaction times than the conventional heating methods. In addition, the products were obtained in very good to excellent yields and without appreciable by-product formation (Scheme 1).
Integration values of these signals showed a~1:1 ratio between the two stereoisomers when compounds were dissolved in DMSO-d 6 . However, a~2:1 ratio was obtained when 1 H-NMR spectra of hydrazones were taken in acetone-d 6 . This result shows the presence of an equilibrium system that is dependent on the polarity of the solvent (Rahman et al. 2005). NOESY experiment showed coupling between C(=O)-NHand -N=CH, and between -N=CH and Ar-O-CH 2 − hydrogens. These couplings can be explained for the presence of E-antiperiplanar isomer, which is depicted in Fig. 3 (Basilio et al. 2013). Therefore, the other constituent of the isomeric mixture must be the E-synperiplanar isomer (see Fig. 3).

Biological activities
The effect of hydrazones on cell growth and viability was assessed in human macrophages (U-937 cells) (Pulido et al. 2012), which are the host cells for L. (V) panamensis and T. cruzi parasites. On the other hand, the antiparasite activity of these compounds was tested on intracellular amastigotes of L. (V.) panamensis (Taylor et al. 2011) and T. cruzi (Buckner et al. 1996;Insuasty et al. 2015) according to the ability of these compounds to reduce the amount of parasite inside infected macrophages. Results are summarized in Table 1.
In general, the anti-leishmanial and anti-trypanosomal activity of the hydrazones were higher than their cytotoxicity. Thus, the SI values calculated for these compounds were >1. Compounds 5c, 5e, and 5h showed the best SI with values from 5.90 to 16.55 (Table 1). Amphotericin B has very high SI values. Benznidazole exhibited a SI of 17.0. Although several hybrid compounds showed better activity than benznidazole, the SI of these compounds is affected by their cytotoxicity. These results suggest that biological activity of the triclosan-hydrazone hybrids is selective, being more active against T. cruzi parasites than U-937 cells.
On a structure-activity relationship basis, it is worth noting the synergistic effect of the parent subunits in the hybrids in comparison with the unlinked cases. For example, triclosan is by itself less potent than their hybrids 5a, 5d, 5f, 5g, 5i, and 5j. The presence of hydroxy or methoxy groups in positions 2 and 4 on the benzylidene moiety increases both the activity and cytotoxicity (5d and 5g vs. 5b, 5c, and 5e), which could be explained by a better molecular recognition ability towards target bioreceptors. There is not a clear relationship between the antiprotozoal activity and the methylation of the hydroxy groups, since in some cases methylation decreases the activity (5d vs. 5g) while in other cases an increase in activity is measured (5c vs. 5f and 5i vs. 5j). Furthermore, increasing the number of hydroxy groups decreases the activity (5a vs. 5b and 5h; 5a vs. 5d and 5k). The biological activity of these compounds could be explained by their action as iron chelators (Walcourt et al. 2004;Coa et al. 2015) or/and as alkylating Scheme 1 Synthetic pathway to triclosan-hydrazone hybrids agents (Michael acceptor) ). Studies in vitro have shown that chelating agents are able to inhibit parasite growth and proliferation by deprivation of iron, which is an essential nutrient for cell growth and division (Richardson et al. 1995). An electrophilic-conjugated system could be generated from o-hydroxybenzylidene-Nacylhydrazone framework due to the ability of this system to be converted into an electrophilic quinone methide intermediate through a pericyclic rearrangement (Ifa et al. 2000). The generation of such a system would allow conjugate addition of nucleophilic amino acid residues such as those found in Leishmania cysteine proteases (Mottram et al. 2004).

Conclusions
The synthesis, cytotoxicity, and activity against L. (V) panamensis and T. cruzi amastigotes of 11 triclosan-hydrazone hybrids are reported. Hydrazones were obtained as two E-synperiplanar and E-antiperiplanar conformers. Nine of them were active against L. (V) panamensis (5a-5d, 5f-5j) and eight of them were active against T. cruzi (5a, 5c, 5d, 5f-5j), with EC 50 values lower than 40 µM. Compounds 5c, 5e, and 5h showed the best SI against both L. panamensis and T. cruzi, with values between 5.90 and 16.55. These results suggest that these compounds have potential as templates for drug development against protozoal diseases. The presence of hydroxy or methoxy groups in positions 2 and 4 on the benzylidene moiety increases both activity and cytotoxicity. There is no clear relationship between the antiprotozoal activity and the methylation pattern of the hydroxy groups, since in some cases methylation decreases the activity (5d vs. 5g), while in other cases the activity is increased (5c vs. 5f and 5i vs. 5j). The mechanism of action of these compounds needs to be adressed and will be the objective of further studies.