New phenoxyacetohydrazones against Trypanosoma cruzi

Camila Capelini Instituto Militar de Engenharia Kátia de Souza Instituto Militar de Engenharia Juliana Barbosa Fiocruz Minas: Fundacao Oswaldo Cruz Instituto Rene Rachou Kelly Salomão FIOCRUZ: Fundacao Oswaldo Cruz Policarpo Junior Fiocruz Minas: Fundacao Oswaldo Cruz Instituto Rene Rachou Silvane Murta Fiocruz Minas: Fundacao Oswaldo Cruz Instituto Rene Rachou Solange Wardell CHEMSOL James Wardell University of Aberdeen Edson da Silva FIOCRUZ: Fundacao Oswaldo Cruz Samir Aquino Carvalho (  samircar@gmail.com ) FIOCRUZ: Fundacao Oswaldo Cruz https://orcid.org/0000-0002-8294-2901


Introduction
Chagas disease is a parasitic illness resulting from infection by the hemo agellate parasite Trypanosoma cruzi. It is endemic in 21 countries in Latin America and affects 6 to 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 loss of $5·6 million per year. Due to the migration of T. cruzi-infected people to other continents, Chagas disease is also reported in non-endemic 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, being the second most used in the Brazilian context. The available chemotherapy is unsatisfactory as a result of severe side effects and restricted e cacy in the chronic phase of the disease [6][7][8].
The N-acylhydrazone (NAH) framework has a special advantage in medicinal chemistry, the presence of an imine vicinal group to the amide is unique characteristic to be explored by the insertion of groups linked to its surroundings, allowing the modulation of multiple targets described in bioactive compounds such as cysteinyl protease inhibitors [9][10][11][12]. Compound 1 showed 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 a pharmacophoric character over T. cruzi, possibly interfering in the oxidative metabolism [14].
The HPLC and the ¹H NMR analysis was consistent with only one geometric isomer at the imine bond level, which X-ray diffraction data indicated as having the relative con guration (E) (Fig. 2), according to previous results [17,24]. Phenoxyacetohydrazones NAH exists in equilibrium between the two stable conformers synperiplanar (sp) and antiperiplanar (ap) in DMSO solution. It is described that the signal from the imine hydrogen being more down shield matches the ap conformer [25].

Crystallography
The 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 Centre, deposit number 2050640. The angle between the two phenyl rings is 69.33(8) o . 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.

Antitrypanosomal activity
We started our analysis by evaluating the effect of the compounds against bloodstream trypomastigotes of T. cruzi (Y strain), a representative of Discrete Typing Unit (DTU) II. The in vitro screening using trypomastigote revealed 11, with an 3,4-dihydroxyphenyl group attached to the imine unit, as the most active compound with an IC 50 /24 h= 10.3 µM, equivalent to benznidazole, the reference drug. In order to evaluate the contribution of its hydroxyl groups at C-3 and at C-4, we compared its activity with different analogues ; 3,4-OCH 3 (12); 3-OCH 3 , 4-OH (13); 3-OH, 4-OCH 3 (14) and -O-CH 2 -O-(16)]. We observed an important decrease in the activity, indicating that the two hydroxyl groups in the catechol subunit present a pharmacophoric characteristic against T. cruzi.
Among the phenyl substituted derivatives, the most active four derivatives presented a hydroxyl group (9, 10, 11 and 13), three of them in the para position. An attractive biopro le was also identi ed for 5nitrofurane derivative (19), which showed an IC 50 /24 h= 19.11 µM. The para substituted derivatives (4 -9) presenting different electronic and lipophilic properties and the trisubstituted derivative (15) showed no trypanocidal activity, except for the 4-OH derivative (9) with an IC 50 /24 h= 53.3 µM (Table 1).
Additionally, to expand this study, another strain of T. cruzi was employed, Tulahuen (DTU VI), considered susceptible to nitro derivatives. All phenoxyacetohydrazones were also evaluated on intracellular amastigote forms for Tulahuen strain ( Table 1). The most active phenoxyacetohydrazone was 5nitrofurane derivative (19), which showed an IC 50 /96 h= 100nM, fteen 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 di cults 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 [19], 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 1000 respectively. In this context, the two compounds with SI ≥50 were considered as good candidates for subsequent studies on antitrypanosomal activity in a murine model [19,26].

Conclusions
We described here in 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 43-74%. 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. cruziinfected cultures presented compound 19 (IC 50 /96 h = 100 nM) with the selectivity index (SI) = 1000. Then phenoxyacetohydrazones derivatives 11 and 19 furnishes supporting data for forward 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) and are uncorrected. 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/load (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 10:1 and temperature ramp of 50°C to 325°C in the rate 10°C/min for 35 min with an initial ow rate of 0.5 ml/min. The spectra in the infrared (IV) region were obtained in a spectrophotometer from the Thermo Scienti c brand, model Nicolet 6700 FTIR by ATR (Attenuated Total Re ectance). The absorption values were reported in wave number (u) whose unit is cm -1 . 1 H NMR and 13 C NMR spectra were recorded at room temperature on a Bruker Avance 500 and BrukerAvance 400 spectrometers operating at 500 and 400MHz ( 1 H)/125 and 100MHz ( 13 C), respectively. Chemical shifts (δ) are reported in parts per million (ppm) down eld from tetramethylsilane (TMS) used as an internal standard and coupling constant (J) values are given in Hertz (Hz). The chromatographic purity of the nal 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 minutes at 30 °C; the ow 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 x 4.6 mm, with a particle diameter of 5µm. 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 265 nm).
General procedures for the preparation of methyl phenoxyacetate (22) To a suspension of 20 (0.07 mol; 1.0 eq.) and potassium carbonate (1.2 eq.) in 100 mL of acetonitrile was added slowly 21 (1.0 eq.). The reaction mixture was stirred under re ux for 4 h and after complete reaction was cooled and added distilled water until solubilized the mixture. The solution was extracted with ethyl acetate (3 x 50 mL) and the organic phase was dried over anhydrous Na 2 SO 4 , ltered and concentrated in vacuo providing the ester used in the next step. General procedure for the preparation of 2-phenoxyacetohydrazide (23) To an ethanolic solution of 22 (0.06 mol; 1.0 eq.) was added slowly hydrazine hydrate 80% (aq.) (5.0 eq.) and the reaction mixture was stirred for 3 h under re ux. The mixture was cooled, the precipitate was General procedure for the preparation of NAH (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) To a solution of 23 (2.41 mmol; 1.0 eq.) in 30 mL of water, were added the corresponding aromatic aldehydes (2.43 mmol; 1.01 eq.) and hydrochloric acid in catalytic quantity. After the complete reaction, the mixture was cooled e the precipitate was collected by vaccum ltration and recrystallized in distilled water. The products were obtained in yields that varied 50-86%. 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 [16]. The stock solutions of the phenoxyacetohydrazones and Benznidazole were prepared in dimethyl sulfoxide, with the nal 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 x 10 6 cells/ml) incubated for 24 h at 37°C and 5% CO 2 atmosphere [17,18]. Untreated and Benznidazole-treated parasites were used as controls. The parasites were quanti ed in Neubauer's chamber. The results were analyzed by plotting % lysis of T. cruzi against the concentration of the test compound and activity of compounds was expressed as the IC 50 /24 h corresponded to the concentration that led to 50% lysis of the parasite and are summarized in Table 1. Next, to evaluate the effects of the compounds on intracellular parasites, L929 broblasts (ATCC® CCL-1™) were infected with trypomastigotes of Tulahuen strain, expressing Escherichia coli β-galactosidase as reporter gene according to the method described previously [19]. Brie y, for the bioassay, 4,000 L929 cells were added to each well of a 96-well microtiter plate. After an overnight incubation, 40,000 trypomastigotes were added to the cells and incubated for 2 h. The culture was maintained for 48 h to establish the infection and then it was treated with the compounds at serial decreasing dilutions for 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 compound-tested cells as compared to the infected cells and untreated cells. The IC 50 values were calculated by linear interpolation. Quadruplicates were run in 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 [19]. 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 [20]. 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 in the same plate, and the experiments were repeated at least twice.