Journal of Inorganic Biochemistry 142 (2015) 1–7 Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio 2,6-Bis(2,6-diethylphenyliminomethyl)pyridine coordination compounds with cobalt(II), nickel(II), copper(II), and zinc(II): synthesis, spectroscopic characterization, X-ray study and in vitro cytotoxicity Pablo Martinez-Bulit a, Ariadna Garza-Ortíz a,1, Edgar Mijangos b,2, Lidia Barrón-Sosa a,c, Francisco Sánchez-Bartéz a,c, Isabel Gracia-Mora a,c, Angelina Flores-Parra b, Rosalinda Contreras b, Jan Reedijk d,e, Norah Barba-Behrens a,⁎ a Departamento de Química Inorgánica, Facultad de Química, Universidad Nacional Autónoma de México, C.U., Coyoacán, México D.F. 04510, Mexico Departamento de Química, CINVESTAV, AP 14-740, México D.F. 07000, Mexico c Unidad de Experimentación Animal, Facultad de Química, Universidad Nacional Autónoma de México, C.U., Coyoacán, México D.F. 04510, Mexico d Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands e Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia b a r t i c l e i n f o Article history: Received 14 March 2014 Received in revised form 12 September 2014 Accepted 12 September 2014 Available online 22 September 2014 Keywords: Heptacoordinated compounds 2,6-bis(arylimino)pyridine derivatives Cytotoxicity Cobalt(II) Copper(II) Zinc(II) a b s t r a c t Coordination compounds with cobalt(II), nickel(II), copper(II) and zinc(II) and the ligand 2,6-bis(2,6diethylphenyliminomethyl)pyridine (L) were synthesized and fully characterized by IR and UV–Vis-NIR spectroscopy, elemental analysis, magnetic susceptibility and X-ray diffraction for two representative cases. These novel compounds were designed to study their activity as anti-proliferative drugs against different human cancer cell lines. The tridentate ligand forms heptacoordinated compounds from nitrate metallic salts, where the nitrate acts in a chelating form to complete the seven coordination positions. In vitro cell growth inhibition was measured for CoII, CuII and ZnII complexes, as well as for the free ligand. Upon coordination, the IC50 value of the transition-metal compounds is improved compared to the free ligand. The copper(II) and zinc(II) compounds are the most promising candidates for further in vitro and in vivo studies. The activity against colon and prostate cell lines merits further research, in views of the limited therapeutic options for such cancer types. © 2014 Elsevier Inc. All rights reserved. 1. Introduction 2,6-Bis(arylimino)pyridine ligands, members of the Schiff base family, have been widely studied over the last decades due to their diverse chemical properties [1–5]. The possibility to expeditiously fine-tune their steric and electronic properties, as well as the donor atoms nature and number, make these ligands attractive for coordination chemistry studies and their applications on different fields such as catalysis, medicinal inorganic chemistry and magnetic devices [1,5–9]. Moreover, it has been shown that changes in the ligand structure can lead to a change in the chemical properties of the coordination compounds [10]. Studies regarding the catalytic applications of Schiff base-metal complexes have shown that there are various synthetic routes for the ⁎ Corresponding author. Tel.: +52 55 5622 3810. E-mail address: norah@unam.mx (N. Barba-Behrens). 1 Present address: Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco, Calzada del Hueso 1100, Villa Quietud, México D.F. 04960, México. 2 Present address: Department of Chemistry — Ångström Laboratory, Uppsala Universitet, P.O. Box 523, 75120 Uppsala, Sweden. http://dx.doi.org/10.1016/j.jinorgbio.2014.09.007 0162-0134/© 2014 Elsevier Inc. All rights reserved. complex formation, where the metallic precursor and the solvent can play important roles [11]. This fact broadens the diversity of Schiff base-metal catalysts and confers them with a unique flexibility during their syntheses. The chemical structure of 2,6-bis(arylimino)pyridine ligands allows strong and stable bonding with several metallic atoms, where the large conjugated system helps to stabilize several oxidation states as well as free radicals [12]. Coordination complexes with this type of ligands and mainly FeII and CoII have been investigated as olefin polymerization catalysts, and their activity was surprisingly high given their chemical structure [3–5,13–17]. Other studies have shown the cytotoxic effect of RuII/III complexes [6,7], as well as the anti-inflammatory properties of organotin(IV) compounds [18]. Finally, there are reports of the synthesis of 2,6-bis(arylimino)pyridine complexes with VIII, CrII, III, MnII, FeIII, NiII, CuII, ZnII, PdII, with diverse applications, but mainly focused on catalysis [19–25]. Since the discovery of the cisplatin antiproliferative properties, almost 50 years ago [26], a large number of metal complexes have been tested as anti-cancer drugs in an effort to find compounds with higher selectivity and less side effects than those commercially available 2 P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 [27–29]. The aim of the present study is to provide a new perspective on this issue, by designing coordination compounds with first-row transition metals and ligands that show unusual chemical characteristics. The selection of the metal ions is based on their presence in the human body as trace elements. In this work, the tridentate ligand 2,6-bis(2,6-diethylphenyliminomethyl)pyridine (L) coordination behavior was studied in the presence of four transition metals (CoII, NiII, CuII, and ZnII). The complexes were characterized by UV–Visible (UV–Vis)-near IR (NIR) and IR spectroscopy, elemental analysis, magnetic susceptibility, and single crystal X-ray diffraction in case good quality monocrystals could be obtained. The biological activity was assessed through a human cancer cell-growth inhibitory assay and the IC50 values were calculated and the results are analyzed and discussed in order to formulate structure activity relationships. 2. Experimental section 2.1. Physical measurements IR spectra in the range 4000–400 cm− 1 were obtained on a PerkinElmer FT-IR 1605 Spectrum spectrophotometer with the sample prepared as a KBr pellet, at 298 K. Electronic spectra were recorded using a Cary 5000 Varian Spectrophotometer by the diffuse reflectance method, in the 40,000–5000 cm−1 range at 298 K. Elemental analyses were carried out with a Fisons EA 1108 analyzer. Magnetic susceptibility measurements at ambient temperature were recorded on a JohnsonMatthey MSB (magnetic susceptibility balance) type MK II 13,094– 3002, using the Gouy method. 1H and 13C NMR spectra were recorded using a JEOL 400 MHz spectrometer. The chemical shifts were referenced to TMS (tetramethylsilane). EPR spectra of powder samples were recorded with a Bruker Elexsys E500 spectrometer using the Xband (9.45 GHz) microwave frequency operating at 100 kHz. g values were calculated using the microwave frequency and measuring the magnetic field H. Table 1 Crystallographic data of compounds 1 and 4. Compound 1 4 Chemical formula [Co(C27H31N3) (NO3)2]·CH3CN 621.55 [Zn(C27H31N3) (NO3)2]·CH3CN 627.99 0.30 × 0.15 × 0.15 Orange Monoclinic P21/n 0.30 × 0.23 × 0.20 Yellow Monoclinic P21/c 9.5104 (1) 12.3747 (2) 26.2443 (4) 90.0 92.145 (1) 90.0 3086.48 (8) 4 1.338 0.61 1300 293 (2) 3.1–27.5 −12 ≤ h ≤ 12 −16 ≤ k ≤ 16 −34 ≤ l ≤ 34 30,930 6993 5378 [I N 3σ(I)] 0.045 441 0.041 0.109 1.04 0.001 0.55 −0.30 14.6379 (2) 13.6490 (3) 16.2178 (3) 90.0 105.171 (2) 90.0 3094.82 (7) 4 1.348 0.84 1312 173 (2) 3.3–27.5 −18 ≤ h ≤ 18 −17 ≤ k ≤ 17 −21 ≤ l ≤ 20 46,745 7047 5663 [I N 3σ(I)] 0.035 502 0.037 0.104 1.04 0.001 0.39 −0.33 Formula weight (g mol−1) Crystal size (mm) Crystal color Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Formula units Z Dcalc (g/cm3) μ (mm−1) F(000) Temp (K) θ range (°) Index range Reflections measured Independent reflections Reflections Rint Parameters R Rw S Maximum Δ/σ Δρ Maximum (e/Å3) Δρ Minimum (e/Å3) X X X 2 D 2 E X 2 F o ; R1 ¼ jj F o j−j F c jj= j F o j; wR2  F o − F o = X   1  X  2 =2 2 2 2 2 ¼ w F o −F c = w Fo : R int ¼ 2.2. X-ray crystal structure determination X-ray diffraction data were collected on a Bruker Kappa CCD area detector diffractometer using Mo-Kα (λ = 0.71073 Å) radiation at 293(2) and 173(2) K. The samples were mounted on MicroMounts (MiTeGen company) [30] with paratone-N oil. Data collection, determination of unit cell and integration of frames were carried out using Suite Collect software [31]. Intensities were measured using φ + ω scans. Structure solution and refinement were carried out with the program SHELXL-97 [32]. All crystallographic software was used under WinGX program [33]. All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were placed geometrically and allowed to ride on their respective atoms. The crystallographic details are summarized in Table 1. Selected bond lengths and angles are listed in Table 2. Crystallographic data for structures 1 and 4 have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication CCDC 991489 and 991490. Copies of the structures can be obtained free of charge www.ccdc.cam.ac.uk/ data_request/cif. 2.3. Reagents The chemicals and analytical reagents were purchased from various commercial sources and were used without further purification unless otherwise stated. Co(NO3)2 · 6H2O, Ni(NO3)2 · 6H2O, Cu(NO3)2 · 3H2O, Zn(NO3)2 · 6H2O and solvents from J. T. Baker. 2,6-pyridinedimethanol and 2,6-diethylaniline from Aldrich. Dry solvents, when required, were prepared using standard procedures. Table 2 Selected bond lengths (Å) and bond angles (°) for compounds 1 and 4. Bond 1 4 M–N1 M–N8 M–N10 M–O31 M–O33 M–O35 M–O37 C2–N1 C9–N10 N1–M–N8 N1–M–N10 N8–M–N10 N1–M–O31 N1–M–O35 N8–M–O31 N8–M–O33 N8–M–O35 N8–M–O37 N10–M–O31 N10–M–O35 O31–M–O33 O31–M–O35 O33–M–O37 O35–M–O37 2.250(1) 2.061(1) 2.280(1) 2.117(1) 2.312(1) 2.167(2) 2.191(1) 1.278(3) 1.277(2) 75.30(6) 149.90(6) 74.61(6) 117.14(6) 87.44(6) 139.85(6) 86.02(6) 142.47(6) 87.82(6) 86.50(6) 117.27(6) 57.28(5) 77.68(6) 173.56(6) 58.36(6) 2.313(2) 2.021(1) 2.260(2) 1.989(1) 2.749(2) 1.999(1) 2.695(2) 1.263(2) 1.273(3) 74.72(6) 151.08(6) 76.36(6) 100.44(6) 93.78(6) 135.64(6) 84.68(6) 138.49(6) 86.90(5) 99.98(6) 108.13(6) 51.10(6) 85.24(6) 171.42(6) 52.31(5) P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 2.4. Synthesis 2.4.1. 2,6-bis(2,6-diethylphenyliminomethyl)pyridine (L) Ligand L [6] and 2,6-pyridinedicarboxaldehyde [34] were synthesized as reported. 2,6-diethylaniline (1.53 g, 10 mmol) was added to a solution of 2,6pyridinedicarboxaldehyde (0.68 g, 5 mmol) in absolute methanol. The resulting mixture was refluxed for 6 h over molecular sieves (4 Å) and filtered while hot. Upon cooling, a pale yellow solid was obtained (1.72 g, 87%). IR (KBr, cm−1): 3434 (w, b), 3064 (m), 3017 (w), 2964– 2870 (s), 1643 (s), 1587 (m), 1564 (m), 1458 (s), 1337 (m), 1189 (s), 854 (m), 792 (m), 759 (m). NMR (400 MHz, CDCl3, δ ppm, s = singlet, d = doublet, t = triplet, m = complex pattern) 1H: 8.4013 (d, 2H; H4, H6), 8.3960 (s, 2H; H2, H9), 7.9979 (t, 1H; H5), 7.1341–7.0686 (m, 6H, H13A, H14A, H15A, H23A, H24A, H25A), 2.5412 (dd, 8H, 2H17A, 2H19A, 2H27A, 2H29A) and 1.1667 (t, 12H, 3H18A, 3H20A, 3H28A, 3H30A). 13C: 162.8956 (C3, C7), 154.5375 (C2, C9), 149.6053 (C5) 137.4468 (C11A, C21A), 132.7822 (C12A, C16A, C22A, C26A), 126.3741 (C13A, C15A, C23A, C25A), 124.4624 (C4, C6), 122.7418 (C14A, C24A), 24.7853 (C17A, C19A, C27A, C29A), 14.6761 (C18A, C20A, C28A, C30A). UV–Vis: 25750 cm−1. Anal. for C27H31N3: Calc. (%): C, 81.57; N, 10.57; H, 7.86. Found (%): C, 81.82; N, 10.63; H, 7.69. 2.4.2. Synthesis of 2,6-bis(2,6-diethylphenyliminomethyl)pyridinebis (nitrato)cobalt(II) [Co(L)(NO3)2] (1). General synthetic procedure for compounds 1–4 A solution of Co(NO3)2 · 6H2O (87 mg, 0.3 mmol) in acetonitrile (30 mL) was added to a solution of L (119 mg, 0.3 mmol) in acetonitrile (30 mL). The mixture was refluxed 4 h and filtered while hot. After 2 days, brownish crystals suitable for X-ray diffraction (XRD) studies were isolated (62.6 mg, 36%). IR (KBr, cm−1): 3412 (w, b), 3072 (w) 2968–2876 (m), 1627 (w), 1586 (m), 1469 (s, b), 1384 (vs), 1288 (s), 1162 (w), 1026 (w), 808 (w), 762 (w), 745 (w). μeff at room temperature (RT): 4.99 BM. Anal. for [Co(C27H31N3)(NO3)2]: Calc. (%): C, 55.86; N, 12.06; H, 5.38. Found (%): C, 55.94; N, 12.19; H, 5.27. The isolated crystals had non-coordinated acetonitrile crystallization molecules, as shown in the XRD (vide infra), which were lost upon standing at room temperature (RT). 2.4.3. Synthesis of 2,6-bis(2,6-diethylphenyliminomethyl)pyridinebis (nitrato)nickel(II) [Ni(L)(NO3)2] · 3H2O (2) Ni(NO3)2 · 6H2O (87 mg, 0.3 mmol); L (119 mg, 0.3 mmol). After one week, from the acetonitrile solution a brown microcrystalline solid was filtered and washed with cold CHCl3 (73.6 mg, 39%). IR (KBr, cm−1): 3435 (w, b), 3071 (w) 2967–2876 (m), 1616 (w), 1584 (m), 1470 (s, b), 1384 (vs), 1297 (s), 1163 (w), 1015 (w), 806 (w), 759 (w), 744 (w). μeff (at RT): 3.32 BM. Anal. for [Ni(C27H31N3)(NO3)2] · 3H2O Calc. (%): C, 51.13; N, 11.04; H, 5.88. Found (%): C, 50.63; N, 11.51; H, 5.13. The small deviation of %H could be due to loss of some water during analysis, but the IR spectra are mutually almost indistinguishable. 2.4.4. Synthesis of 2,6-bis(2,6-diethylphenyliminomethyl)pyridinebis (nitrato)copper(II) [Cu(L)(NO3)2] (3) Cu(NO3)2 · 3H2O (72 mg, 0.3 mmol); L (119 mg, 0.3 mmol). The evaporation of acetonitrile afforded a microcrystalline deep-green solid (162 mg, 92%). IR (KBr, cm−1): 3387 (w, b), 3071 (w) 2967– 2875 (m), 1627 (w), 1589 (m), 1462 (s, b), 1384 (vs), 1288 (s), 1162 (w), 1019 (w), 808 (w), 761 (w). μeff (at RT): 1.72 BM. Anal. for [Cu(C27H31N3)(NO3)2]: Calc. (%): C, 55.42; N, 11.97; H, 5.34. Found (%): C, 55.50; N, 10.99; H, 5.26. The low experimental value for %N is not understood, but given the other characterization there is no doubt on the composition. 3 2.4.5. Synthesis of 2,6-bis(2,6-diethylphenyliminomethyl)pyridinebis (nitrato)zinc(II) [Zn(L)(NO3)2] (4) Zn(NO3)2 · 6H2O (89 mg, 0.3 mmol); L (119 mg, 0.3 mmol). After 2 days, the acetonitrile solution afforded yellow crystals suitable for Xray diffraction study (99 mg, 56%). IR (KBr, cm−1): 3456 (w, b), 3074 (w) 2969–2876 (m), 1639 (w), 1591 (m), 1469 (s, b), 1384 (vs), 1283 (s), 1163 (w), 1010 (w), 811 (w), 763 (w), 745 (w). Anal. for [Zn(C27H31N3)(NO3)2]: Calc. (%): C, 55.25; N, 11.93; H, 5.32. Found (%): C, 55.09; N, 11.92; H, 5.18. The isolated crystals had acetonitrile crystallization molecules in the lattice, which were lost upon standing at RT. 2.5. Measurements of cell growth inhibition Details of measuring cell growth inhibition are described elsewhere [35]. Human cancer cell lines: cervix (HeLa), colon (HTC-15), breast (MCF-7) and prostate (PC-3) were acquired from ATCC (American Tissue Culture Collection) and maintained in incubation at 310 K and 5% CO2 with RPMI medium (GIBCO®, Invitrogen corporation) supplemented with 10% BFS (GIBCO®, Invitrogen corporation), 1% L-glutamine and 1% penicillin/streptomycin. Experiments were performed with cells within at least five passages from each other. All cells were split when around 80–95% confluence was reached using 0.25% trypsin/EDTA. The EDTA/ trypsin solution was used to recover the cells from the culture stock and seeded in a 96 wells plate, where the coordination compound is added with fresh culture medium, as detailed below. In the in vitro growth inhibition assay, 2 × 104 cells/well were plated in a 96-well microplate with D-MEM supplemented with 10% BFS, and allowed to attach, incubating at 37 °C and 5% CO2 for 24 h. At the end of incubation time, the medium was put under vacuum and the cells were exposed to drugs in five different concentrations (0, 10, 20, 30 and 40 μg/mL) for 48 h under the conditions mentioned above. Cell growth was determined according to the sulforhodamine B assay (SRB assay), described by Skehan [36,37]. Absorbance was measured at 564 nm (microplate reader Bio-Rad 550) and the percentage cell growth for each concentration of drug was calculated as: percentage growth = 100x × [T/C], where T is the absorbance of treated wells and C is the absorbance of untreated wells. The 50% growth inhibition parameter (IC50) was computed with a log probit analysis by maximum likelihood employing the software SPSS 20 [38]. The variability of the in vitro cytotoxicity test depends on the cell line used and the serum applied. With the same batch of cell lines and the same batch of serum the interexperimental CV (coefficient of variation) is 1–11%, depending on the cell line and the intra-experimental CV is 2–4%. These values may be higher when using other batches of cell lines and/or serum. 3. Results and discussion 3.1. Spectroscopic characterization Ligand L was synthesized by condensation of one equivalent of 2,6pyridinedicarboxaldehyde and two equivalents of 2,6-diethylaniline in methanol with high yield. The ligand was fully characterized by spectroscopy and comparison with related systems [6,7] (see Fig. 1, for numbering). Reaction of the ligand and the nitrates of CoII, NiII, CuII, and ZnII, in acetonitrile, resulted in the corresponding coordination compounds [M(L)(NO3)2] (1–4). In the coordination compounds the ligand bands ν(C = N) imine (1643; 1458 cm−1) are shifted (1616–1639; 1462– 1470 cm−1), [6,7] indicative on the metal coordination through the nitrogen atoms. New bands at 1384, 1288 (ν3), 1015 (ν1) and 810 (ν2) cm−1 corresponding to coordinated nitrate ligands were observed in all complexes, indicating a bidentate coordination mode [39–41]. Bands corresponding to aryl and pyridine rings out of plane vibrations (792 and 759 cm−1) are shifted to higher vibrational energies in the coordination compounds. All relevant infrared spectra are shown in the supplementary information (Figs. S1–S5). 4 P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 Fig. 1. Numbering of C and N for ligand L. The electronic spectra of this family of coordination compounds are complex and show several d–d transitions in compounds 1 and 2, preceding the strong charge transfer band (CT) and are consistent with the heptacoordinated crystal field splitting [42]. Composed bands centered in 7576, 13,084, and a shoulder at 16,384 cm−1, at the edge of a CT (charge transfer) band, are observed for cobalt(II) compound (1). For the nickel(II) compound (2), bands centered in 9669, 12,458, 15,034, and a shoulder at 20,000 cm−1 are observed. In both spectra, a broad charge-transfer band is observed in the 25,000 cm−1 region, indicative of a large electronic delocalized system, where the metal ion also participates as a member of the conjugated rings. In the case of the spectrum for the copper(II) compound (3) an unresolved broad band is seen, from which the d–d transition is not clearly visible. In the zinc(II) compound (4) the π → π* transition is shifted to lower energy. The electronic spectra are shown in the supplementary information, Figs. S6–S9. 3.2. Magnetic susceptibility Effective magnetic moments for compounds 1–3 at ambient temperature are shown in the experimental section. All the results are close to predicted values within the expected range for the metal center in 2+ oxidation state, with a high spin configuration for cobalt(II). The EPR spectrum of copper(II) compound (3) at RT did not show any hyperfine interactions between the ligand L and the metal ion. The observed pseudo-isotropic g-value is consistent with CuII: g = 2.11. The EPR spectrum is shown in the supplementary information (Fig. S10). 3.3. Crystal structures Cobalt(II) compound 1 crystallizes in the P21/n space group with four coordination entities in the asymmetric unit. A heptacoordinated metallic center with a distorted pentagonal bipyramidal (PBP) geometry is present (Fig. 2). The equatorial plane around the CoII center is formed by the pyridine nitrogen atom N8 and the four oxygen atoms from the nitrato ligands, while the imine nitrogen atoms N1 and N10 occupy the axial positions A bidentate coordination of the nitrato ligand has been observed in other CoII compounds [43]. The ligand chelate angles produce a distortion of the axial nitrogen N1–Co–N10 angle [149.90(6)°]. Only one oxygen atom from each chelating nitrato ligand is in the equatorial plane. Possible explanations for this twisted arrangement of the nitrato ligands are O31–O35 repulsion and possible weak cooperative intermolecular hydrogen bonds generating dimers (see Fig. S12) The Co–N distance for the pyridine nitrogen of [2.061(1) Å] is slightly shorter than the Co–N distance for the imine nitrogen atoms Fig. 2. Coordination sphere in the cobalt(II) compound 1 and used ligand atomic numbering (lattice acetonitrile has been left out for clarity). The ORTEP diagram can be seen in Fig. S11. [2.250(1) and 2.280(1) Å]. The M\N1 and M\N10 coordination bonds do not have identical bond lengths, the origin for this small difference is that in the crystal packing the molecules are slightly asymmetric oriented, dictated by the space group; see also Fig. S13. The Co–O bond lengths vary from 2.117(1) to 2.312(1) Å. The aniline rings are almost perpendicular to the pyridine ring, which is the result of the steric bulk of the ethyl groups. A projection of the structure depicting this orientation is given in Fig. S11. Selected angles and bond distances are presented in Table 2. Intermolecular interactions in compound 1 are formed by π-stacking interactions and weak O⋯HC bonds. The orientation of the ethyl substituents also appears to play a role in this interaction, allowing the π conjugated systems to efficiently overlap. This is confirmed by the stacking distance between the planes (3.46 Å); the opposite ring, on N1, where the ethyl groups are not correctly oriented, is unable to interact in this way with other molecules. This stacking also facilitates the weak hydrogen bonding between one of the coordinated nitrato oxygen atoms and a hydrogen atom of the aromatic ring. This bond perhaps also stabilizes the PBP's equatorial plane distortion. The other contribution to the structure distortion is made by a hydrogen bond between the coordinated oxygen atom (O35) and a carbon atom (C5) from the pyridine ring (see Fig. S14). Intramolecular interactions in the cobalt(II) compound 1 are of great interest due to their distribution. Four weak hydrogen bonds between a methylene from the substituent groups and the equatorial nitrato oxygen atoms (O33, O37) are disposed in a square-type arrangement (Fig. 3). All these hydrogen bonds can be classified as weak electrostatic interactions, according to Steiner [44]. The bond distances are large and the orientation of the involved atoms is far from optimal. Finally, the lattice acetonitrile molecules are weakly interacting with coordination compound molecules as can be seen in Figure S15 and in the 3D arrangement (see Figure S16). The involved H⋯A and D⋯A distances are the following: H9⋯N39, 2.961 Å; H41A⋯O37, 2.606 Å; H41A⋯N39, 2.738 Å; C41⋯O31, 3.230 Å;C9⋯N39, 3.112 Å; C41⋯N39, 3.464 Å. P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 Fig. 3. Intramolecular H-bonding in the crystalline structure of the cobalt(II) compound 1. H27⋯O33 2.63; H17⋯O33 2.38; H19⋯O37 2.71; H29⋯O37 2.77 Å. The zinc(II) compound (4) crystallizes in the P21/c space group. The metal ion also presents a heptacoordination, with the three L nitrogen atoms and two oxygen atom of each nitrate coordinated towards it (Fig. 4). As in the cobalt(II) compound (1), the Zn–N distance is relatively shorter for the pyridine nitrogen, N8, (2.021 Å) compared to the imine nitrogen atoms, N1 and N10, (2.313 and 2.260 Å, respectively), as shown in Fig. S18. The nitrate ligands are coordinated also in a bidentate form, with asymmetric bonds. The oxygen atoms O31 and O35 are at distance to the ZnII center of 1.989(1) and 1.999(1) Å respectively, while oxygen atoms O33 and O37 distance to the metallic center is 2.749(2) and 2.695(2) Å respectively. This long distance is still shorter than the sum of zinc(II) and oxygen van der Waals radii [45]. Similar heptacoordinated zinc(II) structures have been reported [46,47]. Compared to the Co(II) compound the nitrate ligands are more asymmetric, which can be ascribed to the smaller size of Zn(II) compared to Co(II). Studying the crystalline packing, several weak hydrogen bond interactions can be seen. Four intramolecular hydrogen bonds are present in the structure, in a similar arrangement than in the cobalt(II) structure (see Fig. S19). Fig. 4. Coordination sphere in the zinc(II) compound 4. and used numbering of the coordinating atoms. The ORTEP diagram can be seen in Fig. S17. 5 These weak hydrogen bonds involve the imine nitrogen atoms, one oxygen atom from each nitrate ion and four carbon atoms from the ethyl moieties as donor atoms. This arrangement apparently helps to minimize the energy of the structure and stabilize the crystalline lattice. Intermolecular hydrogen bonds are established between backbone hydrogen atoms and the oxygen atoms from the nitrato groups (see Fig. S20). Two different π-stacking interactions were observed; one involves the pyridine ring and a phenyl ring (Fig. 5), and the other one is established between two phenyl rings (Fig. 6). The distance between the aromatic planes and the position of the rings are within the expected range for this kind of systems [48], as can be seen on Fig. 6. These π-stacking interactions, in combination with weak hydrogen bond interactions stabilize a supramolecular 3D arrangement (see Fig. S21). It is important to note that, although the structure of compounds 1 and 4 is similar, the supramolecular arrangement in the crystal lattice differs significantly between the two structures, which is ascribed to difference in size of the cation, resulting in a different space group. Both crystalline structures of compounds 1 and 4 exhibit a dynamic disorder on the phenyl substituted rings. In the cobalt(II) compound 1 this disorder is located only in one of the rings, where all the atoms have two possible positions; in the zinc(II) compound 4 both rings exhibit this type of disorder, even though the data collection was done at low temperature. Based on the X-ray structure determination, spectroscopic data, elemental analyses, and magnetic susceptibility measurements, the proposed structure for the other 2 compounds is shown in Fig. 7. The number of seven-coordinated transition metal compounds found in the literature is relatively low [49], but the role that these type of compounds play as reaction intermediates in substitution reactions or active sites in enzymes [50–52] is seen as of great importance. 3.4. Cancer cell growth inhibition In solution all coordination compounds appeared to have the coordinated nitrate anions exchanged by water molecules, which is in agreement with their conductivity measurements (in CH3CN/H2O). The metal ions remain coordinated to the ligand, as deduced from their electronic spectra in solution. Of course we cannot exclude that in real water (about 55 M), water or other biomolecules present in mM concentrations, like amino acids, may also bind. To explore the biological activity of 3 of the metal compounds and the free ligand presented in this research work, against cancer cell line proliferation, the in vitro cytotoxicity via the SRB assay was performed. The cytotoxicity of this family of 2,6-bis(arylimino)pyridine compounds, with cisplatin as reference compound, was studied in the following human cancer cell lines: HCT-15 (colon adenocarcinoma), PC-3 (prostate adenocarcinoma), MCF-7 (breast adenocarcinoma) and HeLa (cervical uterine adenocarcinoma). The IC50 value represents the amount of drug needed to inhibit 50% of the cancer cell growth, and it was obtained after 48 h of exposition (Table 3). The nickel(II) compound, 3, was not investigated in this work, as previous studies in our group had shown poor antiproliferative activity for compounds with this metal center and related ligands [53]. The ligand L shows the lowest cytotoxic effect. In contrast, 1, 3 and 4, show an important increment when compared with the free L, which underlines the effect of the metal center in the cytotoxic activity. The most active compounds showed IC50 values ranging from 25–53 μM. These results are seen as promising, considering other first-row transition metal-based compound values determined by our group [53,54]. Of course it cannot be totally excluded that under high dilution conditions, where the tests are done, some further decomposition may have occurred. The copper(II) and zinc(II) compounds are the most promising candidates for further in vitro and in vivo studies. 6 P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 Fig. 5. π-Stacking interactions in zinc(II) compound 4. a: 3.515 Å; b: 3.446 Å. The interaction is established between the central pyridine ring and a phenyl ring of a next unit. Hydrogen atoms have been omitted for clarity. 4. Conclusions Acknowledgments Four new transition metal nitrate compounds with a new tridentate 2,6-bis(arylimino)pyridine ligand are reported. For the cobalt(II) and zinc(II) compounds a heptacoordinated geometry was determined in the 3D solid state and a similar structure is proposed for the nickel(II) and copper(II) compounds. The in vitro cytotoxic activity of compounds 1, 3 and 4, and ligand L was assessed, showing a moderate, yet promising activity against the studied cell lines; the coordination compounds proved to be more active than the free ligand, especially in the case of the copper(II) compound 3. Upon coordination, the IC50 values of the transition-metal compounds are improved compared to the free ligand under the same conditions and selectivity was observed towards prostate and colon adenocarcinomas. The activity against these cell lines merits further research, in views of the limited therapeutic options for such cancer types. We are grateful to DGAPA (grant IN 222713) and CONACyT (CB2012 178851) for the financial support. EM thanks the post-doctoral fellowship provided by the CONACyT grant 60894-CB. AGO expresses gratitude to CONACyT and UAM-Xochimilco for postdoctoral fellowships. We thank Carlos Camacho-Camacho for his valuable contribution to the discussion of this work. One of us (JR) thanks the DSF Program of King Saud University for support. Appendix A. Supplementary data Selected intramolecular and intermolecular interactions in the crystalline structures of cobalt(II) and zinc(II) compounds can be consulted in the SI. Infrared, electronic and EPR spectra are also shown in the SI. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinorgbio.2014.09.007. Fig. 6. π-Stacking interactions in zinc(II) compound 4. Two phenyl rings symmetrically equivalent are stacked with a distance of 3.733 Å between the aromatic planes. Hydrogen atoms were omitted for clarity. P. Martinez-Bulit et al. / Journal of Inorganic Biochemistry 142 (2015) 1–7 Fig. 7. Proposed structures for heptacoordinated nickel(II) and copper(II) bis(arylimino) pyridine complexes. Table 3 Cell-growth inhibitory assay results. IC50 value after 48 h of incubation. Compound L1 [Co(L1)(NO3)2] (1) [Cu(L1)(NO3)2] (3) [Zn(L1)(NO3)2] (4) Cisplatin Cell line/IC50 (μM) (Confidence interval) HCT-15 PC-3 MCF-7 HeLa 65.1 (51.1–92.5) 45.6 (37.2–38.5) 25.2 (15.2–33.7) 36.8 (27.2–45.1) b33.2 130.2 (81.3–284.7) 99.9 (64.2–201.4) 38.9 (26.2–59.2) 53.5 (36.4–86.9) 38.4 (21.8–56.8) 169.0 (91.2–672.1) 407.1 (167.7–3576.7) 73.9 (45.5–176.5) 394.1 (163.6–3385.6) 27.3 (9.4–45.9) 68.6 (56.2–84.8) 79.4 (66.0–98.9) 44.341 (33.5–55.9) 29.8 (17.6–40.4) b33.2 References [1] V.C. Gibson, C. Redshaw, G.A. Solan, Chem. Rev. 107 (2007) 1745–1776. [2] B.L. Small, M. Brookhart, A.M.A. Bennett, J. Am. Chem. Soc. 120 (1998) 4049–4050. [3] I.E. Soshnikov, N.V. Semikolenova, A.N. Bushmelev, K.P. Bryliakov, O.V. Lyakin, C. Redshaw, V.A. Zakharov, E.P. Talsi, Organometallics 28 (2009) 6003–6013. 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