Phenoxyacetohydrazones against Trypanosoma cruzi

Herein, we reported the design, synthesis, antitrypanosomal and cytotoxic evaluation of a new phenoxyacetohydrazones series. All derivatives were assayed against bloodstream trypomastigote forms of T. cruzi (Y strain) and intracellular amastigotes using the model of L-929 cells infected with trypomastigotes of the Tulahuen strain. Compound (E)-N′-(3.4-dihydroxybenzylidene)-2-phenoxyacetohydrazide (11) showed activity against trypomastigotes (IC50/24 h = 10.3 µM) equivalent to that of benznidazole and with selectivity index (SI) = 46. Against infected cultures, (E)-N′-((5-nitrofuran-2-yl) methylene)-2-phenoxyacetohydrazide (19) was active at the nanomolar range (IC50/96 h = 40 nM), being about 38-fold more active than the standard drug and with SI equal to 2500. Thus, derivatives 11 and 19 could be considered a good prototypes for the development of new candidates for Chagas disease therapy.


Introduction
Chagas disease is a parasitic illness resulting from infection by the hemoflagellate parasite Trypanosoma cruzi. It is endemic in 21 countries in Latin America and affects 6-7 million people worldwide, causing 10,000 deaths per year [1,2]. It is estimated that 752,000 working days per year are lost due to premature deaths and U$ 1.2 billion in lost productivity in seven southernmost American countries. Brazilian absenteeism of workers affected by Chagas disease represented an estimated minimum of $5.6 million per year. Due to the migration of T. cruzi-infected people to other continents, Chagas disease is also reported in nonendemic regions, such as the European continent [3][4][5].
The treatment of Chagas disease is restricted to the nitro derivatives nifurtimox and benznidazole and both are effective in reducing the duration and clinical severity of the disease. The available chemotherapy is unsatisfactory as a result of severe side effects and restricted efficacy in the chronic phase of the disease [6][7][8].
The use N-acylhydrazones (NAH) plays a strategic role in medicinal chemistry since the imine vicinal to the amide group allows the insertion of different substituents to create a library of bioactive compounds, which can assay against different targets as described by some come cysteinyl proteases inhibitors [9][10][11][12]. In this context, compound 1 displayed an excellent cruzain inhibitory activity (IC 50 = 200 nM) and a considerable in vitro potency against T. cruzi (IC 50 = 16.2 μM) [13]. Our group reported compound 2 containing a hydrazone-cathecol subunit which demonstrated an important profile over T. cruzi, possibly interfering in the oxidative metabolism [14].
In our effort to develop new potent trypanocidal compounds, we constructed a new class of phenoxyacetohydrazones designed by molecular hybridization between a potent cruzain inhibitor represented by the 2phenoxyacetamide derivative (1) (IC 50 = 200 nM) [13] and the 1,3,4-thiadiazole-2-arylhydrazone derivative (2) (IC 50 = 5.3 μM) [14]. The design concept of these compounds generates a NAH subunit with the aim to potentialize the cruzain inhibitor framework of (1) and explore the introduction of a cathecol moiety a potential radical scavenger group [15]. The planned compounds were synthesized with substituents with different stereoelectronic properties attached to the phenyl subunit A (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) (Fig. 1) and heterocycles derivatives (17)(18)(19). The variations of substituents in the phenyl subunit A were introduced in order to assess how the electronic effect of the substituents could influence the antiparasitic profile of this new series. Derivatives substituted in the para position (4-9) were planned due to the different lipophilic and electronic effects on the ring in order to allow the evaluation of the different contributions to the trypanocidal activity (Table 1). Derivative (10) was planned due to the ability of the o-hydroxybenzylidene N-acylhydrazone framework to form an electrophilic quinone methide intermediate that could interact with nucleophilic sites in target enzymes of T. cruzi, e.g., cysteine proteases [10]. Derivatives (12, 15, and 16) were planned due to the recognized ability of these derivatives to have antiprotozoal activity [16]. Both intracellular amastigotes and circulating trypomastigotes forms of T. cruzi are relevant in drug development assays since they are present in the vertebrate host during the acute and chronic phases of Chagas' disease. Then, we have elected two types of in vitro assays, the direct effect on bloodstream trypomastigote form after 24 h of treatment and the effect on the intracellular proliferation of amastigotes after 96 h. Benznidazole was used as a positive control against T. cruzi and the cytotoxicity was determined in mouse peritoneal macrophages (24 h) and mouse fibroblast L929 cells (96 h).

Crystallography
Compound 3 was recrystallized from ethanol solution by slow evaporation at room temperature. Atomic coordinates, bond lengths and angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Center, deposit number 2050640. The angle between the two phenyl rings is 69.33(8)°. The geometry of the exo-C = N bond is (E), while the H and O1 atoms in the H1-N1-C8=O1 unit have a cis arrangement, which allows the formation of centrosymmetric dimers, formed from pairs of classical N1-H1---O1 hydrogen bonds. Additional contacts between molecules are generated from C-H---O hydrogen bonds and π(C = N)---π(Ph) stacking interactions.
In addition, to expand this study, another strain of T. cruzi was employed, Tulahuen (DTU VI), considering its susceptibility to nitro derivatives. All phenoxyacetohydrazones were also evaluated on intracellular amastigote forms for Tulahuen strain ( Table 1). The most active phenoxyacetohydrazone was 5-nitrofurane derivative (19), which showed an IC 50 /96 h = 40 nM, thirty-eight times more potent than benznidazole. The para substituted compounds 4-9 and 17 and the disubstituted ones, 12-14 and 16, presented only moderated activity. The trisubstituted derivative (15) was inactive and o-OH derivative (10), planned to increase the interaction with nucleophilic sites in the cruzain, presented moderate activity (IC 50 /96 h = 22.7 μM) ( Table 1). Although the intracellular location of the amastigote form difficult the access of a given drug, in this study we observed that the best results were observed in amastigotes forms. The distinct behavior of the Tulahuen and Y parasites could be also due to intrinsic differences between both strains and to the time of treatment of the standardized protocols [23], requiring the assay with the intracellular form 96 h of contact with the drug.
The cytotoxic results of the most active compounds 11 for bloodstream trypomastigotes and 19 for intracellular amastigotes were determined and the selectivity indexes (SI) (LC 50 for cytotoxicity divided by IC 50 for antitrypanosomal activity) were 46 and 2500 respectively. In this context, the two compounds with SI ≥ 50 were considered good candidates for subsequent studies on antitrypanosomal activity in a murine model [23].

Conclusions
We described the synthesis, antitrypanosomal and cytotoxic evaluation of new phenoxyacetohydrazones against both infective forms of T. cruzi, bloodstream trypomastigotes, and intracellular amastigotes. The synthetic methodology practiced was reproducible, with global yields ranging from 43% to 74%. The assay against trypomastigote form of T. cruzi reported compound 11 (IC 50 /24 h = 10.3 μM) equivalent to benznidazole with the selectivity index (SI) = 46. The assay against T. cruzi-infected cultures presented compound 19 (IC 50 /96 h = 40 nM) with the selectivity index (SI) = 2,500. Then phenoxyacetohydrazones derivatives 11 and 19 furnishes supporting data for forwarding in vivo studies of these compounds in pertinent animal models for Chagas disease.

General experimental details
Melting points were determined on a Buchi (B-545). The gas chromatography coupled to the mass detector (GC-MS) was performed using the Agilent chromatograph model 6890 with Agilent masses model 5973 at 70 eV. The fragmentation and molecular ion values were expressed in terms of mass/charge (m/z). The Agilent column was used DB-5MS (5% diphenyl: 95% dimethylpolysiloxane). The chromatographic analyzes were performed by split injection method by using a ratio of 10:1 and temperature ramp of 50°C to 325°C in the rate 10°C/min for 35 min with an initial flow rate of 0.5 mL/min. The infrared spectrums (IR) were obtained in a spectrophotometer from the Thermo Scientific brand, model Nicolet 6700 FT-IR by ATR (Attenuated Total Reflectance). The absorption values were reported in wavelength (λ) whose unit is cm −1 . 1 H NMR and 13 C NMR spectra were recorded at room temperature on Bruker Avance 500 and Bruker Avance 400 spectrometers operating at 500 and 400 MHz ( 1 H)/125 and 100 MHz ( 13 C), respectively. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as an internal standard and coupling constant (J) values are given in Hertz (Hz). The chromatographic purity of the final products was determined by a Shimadzu (VP) apparatus with model LC-20ADXR pumps, DGU-20A5R degasser, CBM20A controller, and SPD-M20A model photodiode array detector (DAD). Data acquisition and control were performed using Shimadzu Labsolutions. Chromatographic analyzes were monitored by scanning from 225 nm to 489 nm. In the analyzes, the mobile phase used as eluent (A) water, pH 5.8 adjusted with 0.025 Mol/L ammonium acetate, and eluent (B) methanol, isocratic elution for 70 min at 30°C; the flow of the mobile phase was 1 mL/min and the volume injected was 1000 μL. The separation was obtained on a Supelcosil LC 18-3 reverse phase column, 200 × 4.6 mm, with a particle diameter of 5 μm. The progress of all reactions was monitored by TLC (Thin Layer Chromatography). which was performed on 2.0-6.0 cm aluminum sheets precoated with silica gel 60 (HF254. Merck) to a thickness of 0.25 mm. The developed chromatograms were viewed under ultraviolet light (254 and 365 nm).
General procedures for the preparation of methyl phenoxyacetate (22) To a suspension of 20 (6.68 g; 0.071 mol; 1.01 eq.) and potassium carbonate (11.61 g; 0.084 mol; 1.2 eq.) in 100 mL of acetonitrile was added slowly 21 (10.71 g; 0.07 mol; 1.0 eq.). The reaction mixture was stirred under reflux for 4 h and after the complete reaction was cooled and added distilled water until solubilized the mixture. The solution was extracted with ethyl acetate (3 × 50 mL) and the organic phase was dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo providing the ester used in the next step. General procedure for the preparation of 2phenoxyacetohydrazide (23) To an ethanolic solution of 22 (9.97 g; 0.06 mol; 1.0 eq.) was added slowly hydrazine hydrate 80% (aq.) (15 g; 0.3 mol; 5.0 eq.) and the reaction mixture was stirred for 3 h under reflux. The mixture was cooled, the precipitate was collected by vacuum filtration, washed with cold ethanol, and dried under vacuum.
(E)-N′-Benzylidene-2-phenoxyacetohydrazide (3)  Experimental procedure for biological activity The use of animals in our trial was performed in accordance with the Brazilian Law 11.794/2008 and regulations of the National Council of Animal Experimentation Control under the license L038/2018 from the Ethics Committee for Animal Use of the Oswaldo Cruz Institute (CEUA/IOC). The assays were carried out using the bloodstream trypomastigotes of Y strain obtained from infected mice at peak parasitemia [27]. The stock solutions of the phenoxyacetohydrazones and benznidazole were prepared in dimethyl sulfoxide, with the final concentration of the solvent never exceeding 1%, which has no deleterious effect on the parasite. The compounds were diluted, in series 1:2, in decreasing concentrations, with an initial concentration of 1 mM. The assays were performed with the parasites (5 × 10 6 cells/mL) incubated for 24 h at 37°C and 5% CO 2 atmosphere [19,28]. Untreated and benznidazole-treated parasites were used as controls. The parasites were quantified in Neubauer's chamber. The results were analyzed by plotting % lysis of T. cruzi against the concentration of the test compound and the activity of compounds was expressed as the IC 50 /24 h corresponding to the concentration that led to 50% lysis of the parasite and is summarized in Table 1. Next, to evaluate the effects of the compounds on intracellular parasites, L929 fibroblasts (ATCC® CCL-1™) were infected with trypomastigotes of Tulahuen strain, expressing Escherichia coli β-galactosidase as reporter gene according to the method described previously [23]. Briefly, for the bioassay, 4000 L929 cells were added to each well of a 96-well microtiter plate. After overnight incubation, 40,000 trypomastigotes were added to the cells and incubated for 2 h. The culture was maintained for 24 h to establish the infection and then it was treated with the compounds at serial decreasing dilutions for a further 96 h at 37°C. After this period the chlorophenol red-β-D-galactopyranoside (CPRG) reagent (Roche) was added in Nonidet P40 solution (Sigma-Aldrich) and the plate was incubated for another 18 h and the absorbance was measured at 570 nm. Controls with uninfected cells, untreated infected cells, and infected cells treated with benznidazole at 3.8 μM (positive control) or DMSO 1% were used. The results were expressed as the percentage of T. cruzi growth inhibition in compoundtested cells as compared to the infected cells and untreated cells. The IC 50 values were calculated by linear interpolation. Quadruplicates were run on the same plate, and the experiments were repeated at least once. The active compounds were tested in vitro for the determination of cellular toxicity against uninfected L-929 cells and primary cultures of peritoneal macrophages obtained from Albino Swiss mice, using the AlamarBlue® dye [23]. The cells were exposed to compounds at increasing concentrations starting at IC 50 value for T. cruzi. After 24 or 96 h of incubation, the AlamarBlue® was added and the absorbance at 570 and 600 nm was measured after 4-6 h for L-929 cells and after 2 h for peritoneal macrophages. The cell viability was expressed as the percentage of difference in the reduction between treated and untreated cells being the LC 50 value, corresponding to the concentration that leads to lysis of 50% of the mammalian cells [29]. The selectivity index (SI) was determined based on the ratio of the LC 50 value in the host cell and the IC 50 value of the parasite. Quadruplicates were run on the same plate, and the experiments were repeated at least twice.

Statistical analysis
The results of IC 50 /96 h are presented as mean and standard deviation and were analyzed by Kruskal-Wallis followed by comparison with the Tukey test using GraphPad Prism 3.06 software. The significance level was p < 0.05.
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