Synthesis, Leishmanicidal, Trypanocidal, Antiproliferative Assay and Apoptotic Induction of (2-Phenoxypyridin-3-yl)naphthalene-1(2H)-one Derivatives

The coexistence of leishmaniasis, Chagas disease, and neoplasia in endemic areas has been extensively documented. The use of common drugs in the treatment of these pathologies invites us to search for new molecules with these characteristics. In this research, we report 16 synthetic chalcone derivatives that were investigated for leishmanicidal and trypanocidal activities as well as for antiproliferative potential on eight human cancers and two nontumor cell lines. The final compounds 8–23 were obtained using the classical base-catalyzed Claisen–Schmidt condensation. The most potent compounds as parasiticidal were found to be 22 and 23, while compounds 18 and 22 showed the best antiproliferative activity and therapeutic index against CCRF-CEM, K562, A549, and U2OS cancer cell lines and non-toxic VERO, BMDM, MRC-5, and BJ cells. In the case of K562 and the corresponding drug-resistant K562-TAX cell lines, the antiproliferative activity has shown a more significant difference for compound 19 having 10.3 times higher activity against the K562-TAX than K562 cell line. Flow cytometry analysis using K562 and A549 cell lines cultured with compounds 18 and 22 confirmed the induction of apoptosis in treated cells after 24 h. Based on the structural analysis, these chalcones represent new compounds potentially useful for Leishmania, Trypanosoma cruzi, and some cancer treatments.


Introduction
Leishmaniasis is a group of diseases caused by protozoan parasites from more than 20 Leishmania species. These parasites are transmitted to humans by the bite of an infected open chain in which the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl system. The wide variety of biological and pharmacological activities reported for these compounds include antioxidant, anticancer, anti-Alzheimer's, anti-inflammatory, immunomodulatory, antiulcer, antibacterial, antimicrobial, immunosuppressive, tyrosinase inhibitor, estrogenic, as well as anti protozoan activity, including trypanocidal, leishmanicidal, and antimalarial [38][39][40].

Chemistry
Our synthetic plan towards (2-phenoxypyridin-3-yl)naphthalen-1(2H)-one was based on a two-step, first procedure-attachment of phenol to pyridine ring at position 2, and finally the classical Claisen-Schmidt condensation base-catalyzed in methanol at room temperature (Scheme 1). This reaction has been widely used for the synthesis of chalcones, typically with good to excellent yields. In the required key starting materials, 2phenoxypyridine-3-carbaldehyde 3-6 were prepared using a modification of the procedure previously reported [47], the reaction of 2-chloro-3-pyridinecarboxaldehyde 1 with phenols 2a-d in the presence of anhydrous potassium hydroxide in dry N,N-dimethylformamide at 60 • C in 46-65% yields. The structures of 3-6 were elucidated by infrared (IR), 1 H-, and 13 C-NMR spectral analyses. The IR spectra of the compounds show one characteristic intense stretching band between 1686 and 1680 cm −1 , confirming the presence of C=O. In the 1 H-NMR spectra, the characteristic chemical shift of the proton of aldehyde was found around 10.51-10.54 ppm as a singlet(s), and the pyridine protons 4-6 around 7.81, 8.23, and 8.32 ppm as a double doublet (dd) with a J = 7.8, 7.2, and 1.8 Hz, respectively. The structures were confirmed further by 13 C-NMR spectra, the chemical shift of the C=O was found to be a signal between 188.8 and 188.5 ppm, and the pyridine carbons 4-6 as a signal around 119, 138, and 153 ppm, respectively. The final compounds 8-23 were synthesized through aldol condensation of Claisen-Schmidt between compounds 3-6 and several methoxys substituted 1-tetralones 7a-d, using potassium hydroxide in methanol at room temperature. These conditions were found to be satisfactory for the synthesis with a yield between 41 and 96%. Theoretically, E and Z geometric isomers can be equally formed during the reaction. However, only (E) isomers were obtained, which were confirmed by the singlet in the 1 H-NMR spectra between 7.87 and 7.95 assigned to the protons in the Hβ position. Perhaps due to diamagnetic anisotropy of carbonyl group, where vinyl proton of (E) isomer gives a signal with a greater chemical shift than the vinyl proton of (Z) isomer, an observation that has been noted previously [48,49]. The aliphatic signals expected at upfield shifts were found as t from 2.91 to 3.06 ppm. Based on their chemical shifts, multiplicity and J were assigned the rest of the protons. The 13 C-NMR spectrum of the same compounds exhibits signals between 120 and 127 ppm for Cβ, 135-139 ppm for Cα, and 186-187 ppm for C=O, which were also confirmed by DEPT 135 • (distortionless enhancement by polarization transfer) (see Supplementary Materials, NMR spectra). The infrared (IR) spectra of the compounds show one characteristic intense stretching band between 1660 and 1699 cm −1 , confirming the presence of α,β unsaturated C=O. The product formation was further substantiated by its mass spectra, all the compounds gave satisfactory elemental analysis. Scheme 1. Synthesis of (E)-3,4-dihydro-2-[(2-phenoxypyridin-3-yl)methylene]naphthalen-1(2H)-one analogs 8-23.

Preliminary Antiparasitic Activity: MTT Studies
The obtained de novo chalcones 8-23 were evaluated on L. braziliensis promastigotes and T. cruzi epimastigotes proliferation, and the cytotoxicity assays on mouse bone marrowderived macrophages (BMDM), and VERO cells derived from the kidney of an African green monkey. The concentration-response data for the most active derivatives were fitted by a nonlinear regression model and the concentration that induces 50% inhibition was calculated as the effective concentration EC 50 on L. braziliensis promastigotes and T. cruzi epimastigotes.
Cl An analysis of the structure-activity relationship for the evaluated chalcones 8-23 as leishmanicidal and trypanocidal, permitted us to establish the following remarks. Replacement of the chlorine atom 21-23 by hydrogen, fluorine, and bromine atom on position four of phenoxy on position two of pyridine, decreases the leishmanicidal and trypanocidal activities. The most notable differences in activity were observed when a chlorine atom on position 4 on phenoxy and a methoxy group on position 7 of the tetralone are present, however, when the methoxy group is on position 5 or 6 a marginal loss of biological activity was observed. The replacement of the methoxy group by a hydrogen atom in the tetralon system represents a significant loss of parasiticidal activity.

Antiproliferative Activity
Compounds 8-23 were tested in vitro for their antiproliferative activity on eight cancer cell lines: CCRF-CEM, CEM-DNR, K562, K562-TAX, A549, HCT116, HCT116p53−/−, U2OS, and on the two non-malignant cell lines MRC-5 and BJ. Antiproliferative activities are presented in (Table 2) and are expressed as IC 50 values. The tested compounds showed IC 50 values of between 3.91 ± 0.14 µM and more than 50 µM. The acute lymphoblastic leukemia CCRF-CEM cell line was the most sensitive to tested chalcones, particularly compounds 8, 9, 11-13, and 15 (IC 50 in the range of 4.15 ± 0.74 to 9.13 ± 1.18 µM) bearing H or F on phenoxy and H or OMe groups on position 5 or 7 of tetralone, and the compounds 16, 18, 20, and 22 (IC 50 in the range of 5.23 ± 0.30 to 8.7 ± 2.19 µM) bearing Br or Cl on phenoxy and H or OMe groups on position 6 of tetralone, what implies a correlation between the kind of halogen and position of the OMe group on tetralone and antiproliferative activity. All the compounds were less active against their daunorubicin-resistant CEM-DNR counterparts. In the case of K562 and the corresponding drug-resistant K562-TAX cell lines, the antiproliferative activities showed a more significant difference for compounds 8, 19, and 23 having 2.5, 10.3, and 3.1 times higher antiproliferative activity K562-TAX than K562 cell line (20.0 ± 2.16 µM vs. > 50 µM, 4.17 ± 2.93 µM vs. 42.95 ± 5.1 µM, and 16.05 ± 4.44 µM vs. > 50 µM). On the other hand, compounds 14, 18, and 22 were 6.8, 4.1, and 5.5 times higher cytotoxicity against K562 than K562-TAX cell line (5.74 ± 3.2 µM vs. > 39.25 ± 9.37 µM, 4.33 ± 0.19 µM vs. 17.62 ± 7.09 µM, and 3.91 ± 0.14 µM vs. > 21.44 ± 4.08 µM). These results indicate that for the resistance, other mechanisms than P-glycoprotein are responsible, which is common for both cell lines.
Except for compounds 18 and 22 (9.64 ± 1.71 µM, and 9.74 ± 1.28 µM) all other compounds were less active against the human lung adenocarcinoma A549. Antiproliferative activity of compounds 8-23 tested against HCT116 and HCT116p53−/− were similar. The most efficient was compound 22 with IC 50 of (12.95 ± 3.61 µM) in the case of HCT116. Except for compounds 18 and 22 (7.27 ± 0.68 µM, and 8.1 ± 1.71 µM), all other compounds were less active against the human U2OS cell line derived from osteosarcoma. In contrast, no effect was observed against the non-tumor lines BJ and MRC-5 when compounds 8-23 were evaluated (IC 50 > 50 µM).
In general, the compounds were substantially less toxic on non-cancer cell lines MRC-5 and BJ than against the eight cancer cell lines evaluated: CCRF-CEM, CEM-DNR, K562, K562-TAX, A549, HCT116, HCT116p53−/−, U2OS, the favorable therapeutic index (TI) is expressed as the ratio between the average IC 50 value of non-cancer cell line and the IC 50 value of a given cancer cell line.
The highest TI value among all tested compounds was observed for compound 8 (CCRF-CEM 12). Compound 22 also showed high TI (CCRF-CEM 9.6), against the same cell line as TI (CCRF-CEM 8.4) was calculated for compound 18. Therefore, we can infer that the antiproliferative effect of this class of compounds is related to the type of halogen atoms and the position of the methoxy group in the tetralone nucleus. The detailed mechanism of action will be the subject of our future investigation.

Annexin V/Propidium Iodide (PI) Labeling
Cell death was assessed after 24 h incubation with compounds 18 and 22. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide (PI) [50]. Early apoptosis was defined by annexin V-FITC expression and, necrosis by PI expression, late apoptosis by coexpression of annexin V/PI. Doxorubicin and quercetin were used as positive controls [51][52][53][54][55][56]. The results of a typical flow cytometry analysis of both compounds are illustrated in Figures 2 and 3. An increase in early apoptosis and late apoptosis was observed; necrosis was less predominant. The results are summarized in Tables S1 and S2 (Supplementary Materials). The expression of annexin V was different depending on the compound used.  In the 24 h-treated K562 cell line, the maximum effect was reached at the concentration range of 10 µM. At 10 µM, annexin V-FITC expression was higher than 50% in cells treated with compounds 18 and 22 (10 µM). On the other hand, annexin V-FITC fluorescence augmented significantly when the A549 cell line was treated with compounds 18 and 22, the maximum effect was observed at 25 µM in the cell line. The maximum effect of doxorubicin was recorded at 1 µM; however, it generated a marked increase in cell necrosis. Tables S1 and S2 show the influence of the compounds on PI expression. It can be concluded that the compounds did not induce necrosis of the cell lines tested.

Chemistry
Reactions were monitored by thin-layer chromatography (TLC) carried out on aluminum sheets precoated with silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). Compounds were visualized with UV light. Column chromatography was performed on Merck silica gel 60 (40-63 µm) as a stationary phase. The 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Ascen TM 600 (600 MHz/150.89 MHz) spectrometer (Bruker Bioscience, Billerica, MA, United States) using CDCl 3 as the solvent, and are reported in ppm downfield from the residual CHCl 3 (δ 7.25 ppm for 1 H-NMR and 77.0 ppm for 13 C-NMR). Spin multiples are given as singlet(s), doublet (d), double doublet (dd), and multiplet (m), where coupling constant (J) values were estimated in Hertz. A Thomas TM Hoover Capillary Melting Point Apparatus (Thomas Scientific, Seattle, WA, USA) was used to determine the melting points (mp) and are uncorrected. Infrared (IR) spectra were determined as KBr pellets on a Shimadzu TM IR-470 spectrophotometer (Shimadzu Co., Kyoto, Japan) and are expressed in cm −1 . A Perkin Elmer TM 2400 CHN elemental analyzer (Perkin Elmer, Inc., Waltham, MA, USA) was used to obtain the elemental analyses, and the results were within ±0.4% of the predicted values. Exact molecular masses were determined on a Agilent 5977E GC/MSD spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). Solvents were purchased from different chemical suppliers and were dried and distilled under a nitrogen atmosphere.

General Procedure for the Synthesis and Characterization of Compounds 3-6
Phenol 2a-d (3.0 mmol), powdered KOH (4.0 mmol), and dry N,N-dimethylformamide 5 mL, were combined in a 20 mL round-bottom flask fitted with a reflux condenser. The mixture was heated to 60 • C and allowed to stir for approximately 2 h. Subsequently, the suitable 2-chloropyridine-3-carbaldehyde 1 (2.0 mmol) was added and the reaction mixture was stirred and heated at 105 • C for 2 h plus. The reaction was quenched by the addition of water 10 mL. The organic phase was washed three times with saturated, aqueous K 2 CO 3 5 mL, and then separated and dried with anhydrous Na 2 SO 4 , which was collected by filtration. Solvents were removed under reduced pressure to produce a solid. The product was purified by recrystallization from ethanol:water (3:1).

Culture and Maintenance of Parasite
Promastigotes of Leishmania (V.) braziliensis strain MHOM/CO/87/UA301 were isolated from footpad lesions in Balb/C mice previously infected. In the process of maintenance and differentiation of the parasites, the LTI medium (tryptose 15 g/L, yeast extract 5 g/L, liver extract 2 g/L, hemin-NaOH 0.02 g/L, glucose 4 g/L, NaCl 9 g/L, KCl 0.4 g/L Na 2 HPO 4 , 7.5 g/L at pH 7.4) supplemented with 10% fetal calf serum and maintained at 29 • C was used.
T. cruzi epimastigotes MHOM/VE/92/YBM venezuelan strain were used to test the anti T. cruzi in vitro studies. The strain was maintained in a modified LIT medium with 10% fetal bovine serum (FBS). Mouse bone marrow was used to obtain BMDM macrophages. Mouse fibroblast-conditioned medium L-929 was used for the differentiation and maintenance of BMDM macrophages [57].

Leishmanicidal Activity
We evaluated through a colorimetric method the effect of compounds 8-23 on the promastigotes of L. brasiliensis [59]. Briefly, 96-well plates were used, where 2 × 10 6 par-asites/mL were seeded. A concentration of 50 µM was used for each compound and an incubation time of 96 h at 29 • C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 1 µg/mL was added and incubated in darkness for 4 h, acidic isopropanol (4N) was added and the plate was read at 570 nm in a spectrophotometer Synergy HT (Biotek). Miltefosine was used as a reference drug. The EC 50 calculation of the selected compound was performed using growth curves. Direct counting was used to monitor the proliferation of parasites, three independent experiments were performed for each concentration of the compounds evaluated. The in vitro evaluations of compounds toxicity by MTT in VERO and BMDM cells follow a methodology described above with some modifications [60]. The selectivity index was calculated with SI = CC 50 /EC 50 , where CC 50 was the maximum concentration range of compounds 10, 21-23 used to evaluate cytotoxicity in VERO and BMDM cells.

Trypanocidal Activity
The trypanocidal activity of compounds 8-23 was evaluated on the viability of T. cruzi epimastigotes through a colorimetric method adapted with minor modifications [57]. 2 × 10 6 parasites/mL were seeded in a 96-well plate, adding 50 µM of each derivative dissolved in DMSO (final concentration remained below 1%). The plate was incubated for 96 h at 29 • C. Then, 1 µg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added and incubated in darkness for 4 h. After this time, acidic isopropanol (4N) was added and the plate was read at 570 nm in a spectrophotometer Synergy HT (Biotek). Benznidazole was used as a reference drug. The in vitro evaluations of compounds toxicity by MTT in VERO and BMDM cells follow a methodology described above with some modifications [60]. Cells were counted in suspension and seeded at 20 × 10 3 cells/well. The test was carried out in triplicate on 96-well microplates in different concentrations: 50 µM, 100 µM, 200 µM and 300 µM, untreated cells and reference drug (benznidazole) controls.
3.2.6. Apoptosis Assay K562, A549 cell lines were treated as described by the manufacturer (Santa Cruz Biotechnology), washed, and resuspended in annexin V binding buffer (0.01 M HEPES, 0.14 M NaCl, and 2.5 mM CaCl 2 ) with the subsequent incubation of annexinV-FITC and then PI. An Epics XL cytometer (Beckman Coulter, Indianapolis, IN, USA) was used for the analysis. Untreated cells were the negative controls, and doxorubicin (Dox) (1 µM), and quercetin (QC) (50 µM) were the positive controls [64][65][66]. Early apoptosis refers to annexin V expression, late apoptosis, the coexpression of annexin V and PI, and necrosis the sole expression of PI. Normal fresh lymphocytes were not affected by the compounds, up to 5 µM. The experiments were performed as described before [64][65][66]. To avoid the unwanted effects of DMSO, the concentrations of the compounds were never higher than 100 µM. Since the IC50 values of the two most active derivatives 18 and 22 in both cell lines were: 3.91 ± 0.14 µM to 4.33 ± 0.19 µM for K562 cells and 9.64 ± 1.71 µM to 9.74 ± 1.28 µM for A549 cells, the experiments were carried out at 5, 10, and 25 µM, Figures 2 and 3.

Conclusions
In conclusion, the 16 compounds were synthesized by two-step synthesis, involving nucleophilic aromatic substitution and the classical Claisen-Schmidt condensation basecatalyzed in methanol at room temperature. All the title compounds were obtained in good yields and characterized by spectroscopic technique. Two compounds showed in vitro activities against promastigotes of Leishmania (V.) braziliensis strain MHOM/CO/87/UA301 and epimastigotes of T. cruzi MHOM/VE/92/YBM Venezuelan strain. The most active were compounds 22 and 23 exerting micromolar parasiticide effects and also good selectivity with low toxicity to BMDM and VERO cells.
Ten compounds showed in vitro antiproliferative activities against a broad panel of cancer cell lines with an IC 50 < 10 µM. The most active were compounds 18 and 22 exerting micromolar antiproliferative effects and also good selectivity to proliferating cancer cell lines with low toxicity to non-malignant MRC-5 or BJ fibroblasts. Unfortunately, except for compound 19, low cytotoxicity was observed against multidrug-resistant cancer cell lines (CEM-DNR, K562-TAX), suggesting these results that for resistance is responsible other mechanisms than P-glycoprotein. Flow cytometry analysis confirmed the induction of apoptosis by parts of compounds 18 and 22 in treated cells after 24 h. Therefore, we can infer that the antiparasitic and antiproliferative effects of this class of compounds are related to the type of halogen atoms and the position of the methoxy group in the tetralone nucleus.