Synthesis, Spectroscopic Characterization, Structural Studies, and In Vitro Antitumor Activities of Pyridine-3-carbaldehyde Thiosemicarbazone Derivatives

Facultad de Ingenieŕıa y Arquitectura, Universidad de Lima, Av. Javier Prado Este 4600, Lima 33, Peru Laboratorio de Investigación y Desarrollo, Facultad de Ciencias y Filosof́ıa, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 31, Peru Facultad de Ciencias Quı́micas y Farmacéuticas, Universidad de Chile, CEDENNA, Olivos 1007, Casilla 233, Independencia, 8330492 Santiago, Chile Facultad de Ciencias Fı́sicas y Matemáticas, Universidad de Chile, Av. Beauchef 850, 8320198 Santiago, Chile Fakultät für Chemie und Mineralogie, Universität Leipzig, Johannisallee 29, D-04103 Leipzig, Germany

iosemicarbazones usually react as chelating ligands with metal ions by bonding through the thiocarbonyl sulfur and the azomethine nitrogen atoms [15][16][17]. In addition to this, thiosemicarbazones and the corresponding coordination compounds have been extensively investigated for their antriproliferative activity against different human tumor cell lines. It has been shown that the mechanism of antitumoral action of α-(N)-heterocyclic thiosemicarbazones is due to its ability to inhibit the enzyme ribonucleotide diphosphate reductase, which catalyzes the conversion of ribonucleotides into deoxyribonucleotides during the DNA syntheses [18,19].
A variety of heterocyclic thiosemicarbazones also proved to be cytotoxic against several tumor cell lines.
As a part of our efforts towards the synthesis and structural characterization of new materials containing biorelevant pyridinyl thiosemicarbazones and the understanding of their cytotoxic activity against different human tumor cell lines, the present work describes the synthesis and spectral characterization of eight new pyridine-3-carbaldehyde thiosemicarbazone derivatives. Compounds 1-8 were tested for their in vitro antiproliferative activity against six human tumor cell lines: H460 (lung large cell carcinoma), HuTu80 (duodenum adenocarcinoma), DU145 (prostate carcinoma), MCF-7 (breast adenocarcinoma), M-14 (amelanotic melanoma), and HT-29 (colon adenocarcinoma).

Chemicals and Instrumentation.
All reagents and solvents were purchased from Sigma-Aldrich of analytical grade and were used without further purification. e tested human tumor cell lines were H460 (lung large cell carcinoma), HuTu80 (duodenum adenocarcinoma), DU145 (prostate carcinoma), MCF-7 (breast adenocarcinoma), M-14 (amelanotic melanoma), and HT-29 (colon adenocarcinoma), while the tested non-tumor cell line consisted of BALB/3T3 mouse embryonic fibroblast cells. Both the human tumor cell lines and the nontumor cells were obtained from the American Type Culture Collection or from the National Cancer Institute. Cytotoxicity screening was performed using the sulforhodamine B (SRB) colorimetric assay [27].
Melting points were determined on a Büchi melting point B-545 apparatus. Elemental analyses were determined on an Elementar Vario EL analyzer. ESI-MS spectra were recorded on a Waters-Quattro Premier XE ™ tandem quadrupole mass spectrometer and MicrOTOF Bruker Daltonics mass spectrometer, using methanol as the sample dissolution medium. e Infrared (IR) spectra were recorded using a Nicolet iS10 Fourier Transform Infrared (FT-IR) spectrometer equipped with an attenuated total reflectance accessory using a diamond crystal. e measurements were obtained in absorbance mode, recorded for 32 scans at a resolution of 4 cm −1 . All the measurements were carried out with an automatic baseline correction. e UV-VIS spectra were recorded on a ermo Scientific Evolution 201 spectrophotometer. e 1 H (300 MHz or 400 MHz), 13

Crystal Structure Determination.
Data were collected at 180 K using a STOE StadiVari diffractometer equipped with a copper X-ray microsource (Cu Kα radiation) and a Dectris Pilatus 300 K detector. All data were corrected for Lorentz and polarization effects; absorption effects were corrected based on numerical absorption corrections. In addition, a scaling correction was performed using Stoe X-Area software [28]. e structure of 7 was solved by direct methods (ShelxS) and refined using the full-matrix leastsquares method against F 2 (ShelxL) [29]. Diagrams of the molecular structure showing thermal ellipsoids with 50% probability were generated using Diamond3 software [30].

Biological Activity.
Cell Culture. BALB/3T3 cells were maintained in Dul-becco´s modified Eagle´s medium (DMEM) supplemented with 10% calf serum and 50 μg/mL gentamycin. H460, HuTu80, and DU145 were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum and 50 μg/ mL gentamycin. MCF-7 and HT-29 were maintained in RPMI 1640 supplemented with 7.5% fetal bovine serum and 50 μg/mL gentamycin. Cells were grown at 37°C in a 5% CO 2 humidified environment. Assessment of Cytotoxicity. In vitro cytotoxic activity of the prepared compounds was tested using the sulforhodamine B (SRB) assay [27]. Briefly, cells were seeded onto 96-well plates at a density of 3000-5000 cells per well and incubated at 37°C, 5% CO 2 , 95%, air and 95% relative humidity, with their corresponding growth medium for 24 h to allow for cell attachment. Solutions of the pyridine-3-carbaldehyde thiosemicarbazone derivatives in DMSO at different concentrations (1.95, 7.81, 31.25, and 125 μg/mL) and solutions of 5-fluorouracyl in DMSO at different concentrations (0.061, 0.244, 0.977, and 3.91 μg/mL) were added to the different cell lines and incubated for 48 h at 37°C in 5% CO 2 humidified atmosphere. After 48 h, cells were treated with trichloroacetic acid (TCA), washed, dried, and stained with a solution of 0.4% sulforhodamine B in 1% acetic acid for 20 minutes. Excess stain was washed out four times with 1% acetic acid. After complete drying, the bound dye was solubilized with 10 mM Tris buffer (pH 10.5) and color intensity was measured on an automated plate reader at a wavelength of 510 nm. e IC 50 value was defined as the concentration of test sample resulting in a 50% reduction of absorbance as compared with untreated controls, i.e., 50% reduction in the growth of the cells, and was determined by linear regression analysis.
All the new synthesized compounds were obtained in good yields (56-90%) and were satisfactorily characterized by elemental analysis, ESI-Mass, UV-Vis (ultraviolet-visible), FT-IR, and ( 1 H, 13 C, 19 F) nuclear magnetic resonance spectroscopy. e isolated compounds are soluble in common organic solvents, such as dichloromethane, chloroform, acetone, dimethylformamide, and dimethylsulphoxide. e mass and spectroscopic data obtained for all the thiosemicarbazone derivatives are in agreement with the proposed structures and are given as Supplementary Material.

Infrared Spectra.
e corresponding FT-IR spectra of the studied compounds are given as Supplementary Material (Figures S1-S8). In the FT-IR spectra of all eight compounds, the broad bands observed in the ranges of 3457-3225 and 3158-3078 cm −1 were assigned to the ](NH 2 ) and ](NHCS) vibrations, respectively [31,32]. e strong and medium absorption bands at 1587-1683 cm −1 were attributed to the (CH=N) stretching vibrations of the imine group, which is in agreement with the vibrations found for other thiosemicarbazone derivatives [31]. e strong bands of the pyridine C=N group were observed at 1593-1525 cm −1 , while the bands in the 1096-1015 cm −1 region were assigned to the ](N-N) vibrations. In all FT-IR spectra, a peak around 2500 cm −1 attributed to the SH group was not observed; the medium bands which are in 867-804 cm −1 range were ascribed to (C=S) stretching vibrations, indicating that the studied compounds are present in the thione form [33,34].

NMR Spectra.
e NMR spectra of the compounds were recorded in DMSO-d 6 solution in order to confirm the presence of the functional groups and proposed molecular formulas. e 1 H resonances were assigned on the basis of chemical shifts, multiplicities, and coupling constants and, in some cases, by 2D NMR data. e 1 H-NMR, 13 C NMR, and 19 F NMR spectra of 1 are shown in Figures 1-3.
All 1 H-NMR spectra of compounds 1-8 showed a singlet in the region δ � 11.79 − 11.49 for the �N-NH proton [35]. ese results are similar to the chemical shifts found for compound 4-phenyl-1-benzaldehyde thiosemicarbazone (δ �11.83), which exists in the E isomeric form [23]. e signal of the imine-CH�N proton appeared as a singlet at δ � 8.57 − 7.97 [36]. e NH 2 protons of the thioamide group showed broad peaks at δ � 8.45 − 8.08 [23]. On the other hand, the resonance lines of the protons corresponding to the pyridine ring were observed at δ � 9.02 − 7.05, in agreement with the chemical shifts found for other compounds derived from pyridine-2-carbaldehyde thiosemicarbazone [33,37]. For compounds 2, 5, and 8, the aromatic proton signals of the phenyl fragment bound to the pyridine ring were affected by the presence of the fluoro, methoxy, and chloro substituents in the C-4′, C-3′,4′, and C-3′,5′positions, respectively. ese signals are deshielded for the protons in   e aromatic carbons of the phenyl group in all synthesized thiosemicarbazones were observed at δ = 162.41−110.29 [37].

Description of the Crystal Structure of Compound 7.
Good quality crystals of 7, suitable for single crystal X-ray diffraction analysis, were obtained by slow evaporation from a concentrated reaction mixture in methanol. For the other compounds, only microcrystalline solids were obtained from acetone. 7 crystallizes with two different crystal shapes and different unit cells. Its molecular structure together with the atomic numbering scheme is shown in Figure 4. Crystal data and refinement results for both types of crystals are summarized in Table 1, and selected bond lengths, bond angles, and torsion angles are given in Table 2. e values in the right column of Table 1 belong to the plate-like crystals, which correspond to the major fraction (>90%) of crystals, while the values in the left column belong to the minor fraction of rod-like crystals. e bond lengths, bond angles, and torsion angles for 7, shown in Table 2, are similar for both crystal shapes. As reported in Table 1    e ORTEP diagram ( Figure 4) reveals that 7 exists in the E conformation regarding the N2-N3 bond, as evidenced by the C7-N2-N3-C8 torsion angle of 177.0(4).
On the other hand, 5 (X � 3, 4-dimethoxyphenyl) showed a more acceptable cytotoxicity (IC 50 � 40.34-43.04 μM) than 8 (X � 3, 5-dichorophenyl), with IC 50 values of >384.36 μM against the HuTu80 and DU145 cell lines. ese results indicate that the presence of the methoxy substituent groups in the C-3 and C-4 positions of the benzene ring enhance the antitumor activity against these specific cell lines.
Compound 1 tested in vitro against the amelanotic melanoma cell line (M-14) (IC 50 = 3.36 μM) showed to be more cytotoxic as compared to pyridine-2-carbaldehyde thiosemicarbazone (IC 50 => 100 μM) assayed in vitro against the mouse metastatic skin melanoma (B16F10) cell line [44]. Nevertheless, 1 (IC 50 = 11.25 μM) tested in vitro against the colon adenocarcinoma (HT-29) was slightly less active than pyridine-2-carbaldehyde thiosemicarbazone (IC 50 = 8.6 μM) and 2-acetylpyridine-N(4)-orthochlorophenyl thiosemicarbazone (IC 50 = 6.96 μM) assayed in vitro against the colon cancer (CT26.WT) and HT-29 cell lines, respectively [44,45]. On the other hand, 3-aminepyridinecarbaldehyde thiosemicarbazone and 3-phenyl-1-pyridin-2-ylprop-2-en-1one thiosemicarbazone tested in vitro against the large cell lung cancer (NCI-H460) [46] and the human breast carcinoma (MDA-MB 231) cell line [10], respectively, were found to be about two times more cytotoxic than 1 tested in vitro against the H460 and MCF-7 cell lines with IC 50 values of 10.9 and 6.27 μM, respectively. However, 1 (IC 50 = 6.27 μM) was more active than 4-phenyl-1-(quinoline-2-carbaldehyde) thiosemicarbazone (IC 50 => μM) [47] and diacetylpyridine bis( 4 N-tolylthiosemicarbazone) (IC 50 => 100 μM) assayed on the MCF-7 cell line [41]. In addition, 1 (IC 50 = 10.90 μM) tested on the H460 cell line showed higher cytotoxicity than 2acetyl-pyridine thiosemicarbazone (IC 50 = 14.34 and 15.68 μM) and 4-methyl-1-(2-acetyl-pyridine) thiosemicarbazone (IC 50 = 11.10 and 15.53 μM) assayed against the NCI-H460 and MSTO-211H lung carcinoma cell lines, respectively [48]. e selectivity indexes which represent the ratio of the IC 50 values of the compounds on non-tumor cell line to those in the tumor cell line were calculated in order to determine if the pyridine-3-carbaldehyde thiosemicarbazone derivatives (1-8) were more cytotoxic on tumor cell lines compared with the 3T3 non-tumor cell line, and the results have been summarized in Table 4. Considering the low IC 50 values obtained for the tested compounds against the tumor and non-tumor cell lines, 1 displayed the highest selectivity index (SI = 1.82) against M-14 cell line as compared to those indexes observed for the other tested thiosemicarbazones and the 5-fluorouracile chemotherapeutic agent. is value means that compound 1 is 1.82 times more cytotoxic to the tumor cell line as compared to the 3T3 non-tumor cell line. is value can be considered as an acceptable selectivity index with respect to the highest selectivity index (2.81) found for the essential oil from T. erecta leaves against HT-29 tumor cell line and VT79 normal hamster lung fibroblast cells [49]. On the other hand, 6 displayed the highest selectivity indexes of 2.49 and 1.54 against HuTu80 and MCF-7 cell lines, respectively, with respect to the other tested thiosemicarbazone derivatives. However, the IC 50 values were very high (>200 μM) for both tumor cell lines. us, it becomes evident that the studied thiosemicarbazones have a different activity depending on the substituents present in the respective structures. However, 2, 3, and 6, even though the most innocuous compounds against the 3T3 normal cells do not present antitumor activity against the studied cell lines.

Conclusions
In this study, eight new pyridine-3-carbaldehyde thiosemicarbazone derivatives with different substituents were synthesized, characterized, and investigated for their antitumor activities. Only 7 gave good quality crystals for single crystal X-ray diffraction studies. e crystal structure of 7 exhibits an E conformation about the N2-N3 bond.
is conformation was also assessed from solution 1 H NMR studies. e results of the cytotoxic assays and the selectivity indexes calculated demonstrated that compound 1 with IC 50 value of 3.36 μM and selectivity index of 1.82 has significant bioactivity towards amelanotic melanoma (M14) human tumor cell line.
erefore, 1 is a promising candidate as a pharmacological agent, since it presents significant activity and is more innocuous than the 5-fluorouracile anticancer drug against 3T3 mouse embryo fibroblast normal cells. Further studies are required to evaluate the mechanism of action for the anticancer activity of 1.

Data Availability
e data used to support the findings of this study are included within the article. ese data will be available when the researchers request it. Crystallographic data for the structural analyses have been deposited with the Cambridge Crystallographic Data Centre, numbers CCDC 1986928 and 1986929 for both forms of 7. Copies of this information can be obtained free of charge via http://www.ccdc.cam.ac.uk/ conts/retrieving.html, or from the Cambridge Crystallographic Data Centre (CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax (+44) 1223-336-033; e-mail: deposit@ ccdc.cam.ac.uk).