Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 427–434
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Doubly end-on azido bridged mixed-valence cobalt trinuclear complex: Spectral study, VTM, inhibitory effect and antimycobacterial activity on human carcinoma and tuberculosis cells Amitabha Datta a,⇑, Kuheli Das b, Chandana Sen b, Nirmal Kumar Karan b, Jui-Hsien Huang c,⇑, Chia-Her Lin d, Eugenio Garribba e, Chittaranjan Sinha b,⇑, Tulin Askun f, Pinar Celikboyun f, Sandeep B. Mane g a
Department of Physics, Faculty of Sciences and Arts, University of Balikesir, Cagis Campus, 10145 Balikesir, Turkey Department of Chemistry, Inorganic Chemistry Section, Jadavpur University, Kolkata 700032, India c Department of Chemistry, National Changhua University of Education, Changhua 50058, Taiwan d Department of Chemistry, Chung-Yuan Christian University, Chun-Li 320, Taiwan e Dipartimento di Chimica and Centro Interdisciplinare per lo Sviluppo della Ricerca Biotecnologica e per lo Studio della Biodiversità della Sardegna, Università di Sassari, Via Vienna 2, I-07100 Sassari, Italy f Department of Biology, Faculty of Sciences and Arts, University of Balikesir, Cagis Campus, 10145 Balikesir, Turkey g Institute of Chemistry, Academia Sinica, Nangang - 11529, Taipei, Taiwan b
II
III
Co /Co -(2-[{[2-(dimethylamino)
ethyl]imino}methyl]6-methoxyphenol). Doubly azido-bridged Co trinuclear complex. III It is noteworthy that the Co atoms are diamagnetic in nature where as the central CoII atom behaves paramagnetically. The complex decreases the cell population growth on human carcinoma cells. Displays efficacy on the mycobacterial strains of human tuberculosis cells.
g r a p h i c a l a b s t r a c t Doubly azido-bridged trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is synthesized where L = NMe2CH2CH2N@CH(OH)(OMe)C6H3 in moderate yield. The Co atoms adopt a distorted octahedral geometry. Complex 1 inhibits the cell growth of human carcinoma cells and exhibits anti-mycobacterial activity on human tuberculosis cells.
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Article history: Received 28 June 2014 Received in revised form 7 April 2015 Accepted 9 April 2015 Available online 15 April 2015
a b s t r a c t Doubly end-on azido-bridged mixed-valence trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is afforded by employing a potential monoanionic tetradentate-N2O2 Schiff base precursor (2-[{[2(dimethylamino)ethyl]imino}methyl]-6-methoxyphenol; HL). Single crystal X-ray structure reveals that in 1, the adjacent CoII and CoIII ions are linked by double end-on azido bridges and thus the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. Complex 1 is subjected on
⇑ Corresponding authors. Fax: +91 33 2413 7121. E-mail addresses:
[email protected] (A. Datta),
[email protected] (J.-H. Huang),
[email protected] (C. Sinha). http://dx.doi.org/10.1016/j.saa.2015.04.014 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
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different spectral analysis such as IR, UV–vis, emission and EPR spectroscopy. On variable temperature magnetic study, we observe that during cooling, the vMT values decrease smoothly until 15 K and then reaches to the value 1.56 cm3 K mol1 at 2 K. Complex 1 inhibits the cell growth on human lung carcinoma (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human heptacellular (PLC5 cells) carcinoma cells. Complex 1 exhibits anti-mycobacterial activity and considerable efficacy on Mycobacterium tuberculosis H37Rv ATCC 27294 and H37Ra ATCC 25177 strains. Ó 2015 Elsevier B.V. All rights reserved.
Keywords: Co complex Crystal structure EPR VTM Inhibitory effect Anti-mycobacterial activity
Introduction Schiff base ligands have been extensively studied in coordination chemistry mainly due to their facile syntheses, easily tunable steric and electronic properties as well good solubility in common solvents [1]. Transition metal complexes with oxygen and nitrogen donor Schiff bases are of particular interest [2,3] because of their ability to possess unusual configurations, be structurally labile. Any pseudohalide ligand can provide the most interesting electron exchange pathway due to its structural versatility ranging from a terminal monodentate to bridging ligand [4]. Among them, azide ion is the most efficient ligand as regards the superexchange pathways between paramagnetic centers [5,6]. The versatility and efficiency of azido ligand lies in its functionality as a bridging bi-, tri-, and tetradentate ligand [6]. The different types of binding modes of azide ion are shown in Scheme 1. Depending upon the steric and electronic demand of the co-ligand, the azido ion has been shown to be able to link metal ions in various bridging modes, l1,1 (endon, EO) [7–9], l1,3 (end-to-end, EE) [10], etc. to give birth to a variety of cobalt(II) complexes [11] using Schiff bases or other polydentate N-donor organic ligands as auxiliary ligands. In symmetric bi-bridged end-on azido dimers, the interaction is strongly ferromagnetic [12–14] while with one or more symmetric end-to-end azido bridges the interaction is strongly antiferromagnetic [15,16]. Complexes with asymmetric [17] end-to-end azido bridges are usually weakly antiferromagnetic [13] whereas complexes with asymmetric end-on azido bridges are rare and show weak or moderately strong ferromagnetic interaction [18]. The control of tumor cell escalation by inhibition of the cell cycle and initiation of apoptosis could assist a therapeutic strategy for treatment of cancer affected cells [19]. Programmed cell death plays a crucial role in the regulation of cellular homeostasis [20]. MarínHernández and co-workers [21] earlier discussed that some mixed chelate transition metal-based drugs have potentiality regarding antitumor activity than cisplatin in in vivo and in vitro studies of different tumor cells. Tuberculosis quite infectious and chronically bacterial disease, which is caused by primarily Mycobacterium tuberculosis and rarely Mycobacterium bovis and Mycobacterium africanum, effects lung and even can spread to the other organs [22]. Millions of children die every year from this disease [23]. Mycobacteria resist many of chemicals, disinfectants antibiotics and chemo-therapeuticals [24]. Herein we report the synthesis and X-ray structure of one symmetric l1,1 doubly azido bridged tricobalt complex, [Co3(L)2
(N3)6(CH3OH)2] (1) incorporating with a tetradentate-N2O2 Schiffbase precursor, (HL, Scheme 2) [25]. Besides spectral and magnetic study [26], we focus our attention mainly on the inhibitory and anti-mycobacterial effect of complex 1 among different human carcinoma and tuberculosis cells. The result from an MTT, [3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide], assay demonstrates that complex 1 decreases the cell population growth of A549, COLO 205, HT-29 and PLC5 cells. The anti-mycobacterial activity on M. tuberculosis H37Rv ATCC 27294 and M. tuberculosis H37Ra ATCC 25177 strains are responded well. Before some articles are published on mixed [27] or same valence [28,29] cobalt system alongwith azido-bridging [30,28]. However, we first report the inhibitory and anti-mycobacterial effect on human carcinoma and tuberculosis cells with a mixed-valence cobalt derivative. Experimental Materials All chemicals and solvents used for the synthesis were of reagent grade, obtained commercially and used as received. O-vanillin, 2-dimethylaminoethyliamine and sodium azide were purchased from Aldrich Chemicals. Hydrated cobalt(II) trifluoroacetate was prepared by the treatment of basic cobalt(II) carbonate, CoCO3Co(OH)2 (AR grade, E. Merck), with 60% trifluoroacetic acid (AR grade, E. Merck), followed by slow evaporation on a steam bath. It was then filtered through a fine glass frit and stored in a CaCl2 desiccators. Caution! Azido compounds of metal ions are potentially explosive especially in presence of organic ligands. Though no difficulties have been encountered during the preparation and characterization of our complexes, only a small amount of materials should be prepared and handled with care. Physical techniques Elemental analyses were performed on a Heraeus CHN-OS Rapid Elemental Analyzer. Infrared spectra were recorded on a Perkin-Elmer 883-Infrared spectrophotometer in the range 4000– 400 cm1 as KBr pellets. Electronic spectra were measured on a Hitachi U 3400 (UV–Vis–NIR) spectrophotometer in methanol. Emission spectra were examined by LS 55 Perkin–Elmer spectrofluorometer at room temperature (298 K) in CH3CN solution H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
μ−1,1
μ−1,3
μ−1,1,3
μ−1,1,1
μ−1,1,3,3
Scheme 1. Different modes of azido bridging.
N NMe2 OH OMe Scheme 2. Synthesis of the ligand, HL.
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under degassed condition. EPR spectra were recorded from 0 to 10000 Gauss in the temperature range 77–298 K with an X-band (9.15 GHz) Varian E-9 spectrometer. The EPR parameters reported in the text were obtained by simulating the spectra with the computer program Bruker WinEPR SimFonia [31]. In all the simulations, second-order effects were taken into account, the ratio Lorentzian/Gaussian, affecting the line shape, was set to 1 and the line width used for x, y, z axes was 22, 25 and 30 Gauss, respectively. Magnetization curves versus temperature or applied field were measured using a Cryogenic SX-600 SQUID magnetometer on 27.2 mg of a polycrystalline sample. Zero-field cooled (ZFC) and field cooled (FC) were performed between 2.5 and 300 K. The sample was placed in a gelatin capsule and data were corrected for the diamagnetism and sample holder. Ac susceptibility measurements were carried out in a Quantum Design PPMS (Physical Properties Measurement System) model 6000 with frequencies ranging between 10 Hz and 10 kHz and constant magnetic field of 12 Oe. Synthesis of Schiff base ligand and complex Preparation of Schiff base ligand, HL The tetradentate Schiff base ligand, HL, is prepared [25] by condensation of o-vanillin (0.152 g, 1 mmol) with 2-dimethylaminoethylamine (0.109 ml, 1 mmol) in methanol (25 ml). After 2 h reflux, the pale yellow solution is cooled down to room temperature. The solvent is removed under reduced pressure, and the Schiff-base ligand is obtained as a light-yellow liquid that is used without further purification. 1H NMR (TMS, CDCL3) d: 12.80 (1H, s, H-1), 9.90 (1H, s, H-6,60 ), 6.46–6.86 (3H, d & t, Ar-H), 3.86 (3H, s, H-6), 3.45 (2H, t, J = 4.5 Hz, H-7), 3.72 (2H, t, J = 3.4 Hz, H-8), 2.29 (6H, s, H-9) ppm (see ESI, Fig. S1). Synthesis of [Co3(L)2(N3)6(CH3OH)2] To a 20 mL methanolic solution of Co(CF3COO)26H2O (0.393 g, 1 mmol), the Schiff base ligand (1 mmol) is added which results a brown solution. After that an aqueous solution (10 ml) of sodium azide (0.130 g, 2 mmol) is added drop wise and the resulting solution is kept under boiling for 10 min. On cooling and slow evaporation of the brown solution, the dark brown rectangular shaped single crystals of the complex (1) are separated out after 3 days. The crystals are filtered off and washed with water. A dark brown crystal of 1 is selected for X-ray data collection. Yield: 64%. Anal. Calc. for C13H21Co1.50N11O3: C, 33.38; H, 4.53; N, 32.93. Found: C, 33.59; H, 4.62; N, 32.72%. X-ray crystallography Details concerning crystal data, data collection characteristics and structure refinement are summarized in Table 1. The single crystal X-ray diffraction measurement was carried out on a Bruker APEXII CCD diffractometer, fine focus sealed tube equipped with graphite monochromatic Mo-Ka radiation (k = 0.71073 Å). The x: 2h scan technique was applied within a h range of 1.45–28.84°. No significant crystal decay was observed. Data were corrected for absorption empirically by means of w scans. A total of 24967 reflections were collected, from which 4856 independent [R(int) = 0.0694] reflections were measured. The intensity data were corrected for Lorentz and polarization effects and an empirical absorption correction was also employed using the SAINT program [32]. The structures were solved with the direct method of SHELXS-97 [33] and refined on F2 by full-matrix least-square techniques using the SHELXTL-97 [34] program. The non-hydrogen atoms of the complexes were refined with anisotropic temperature parameters. The hydrogen atom positions were calculated and they were constrained to idealized geometries and treated as riding where the H atom
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Table 1 Crystallographic data of 1. Empirical formula Formula weight Temperature Wavelength (Å) Crystal system Space group a, Å b, Å c, Å a, deg b, deg c, deg Volume, Å3 Z Density, Mg/m3 Absorption coefficient, mm1 F(0 0 0) Crystal size, mm3 h range for data collection Reflections collected Independent reflections Data/restraints/parameters Final R indices [I > 2r(I)] R indices (all data) Goodness-of-fit on F2 Largest diff. peak and hole, e Å3
C13H21Co1.50N11O3 467.81 150(2) 0.71073 Monoclinic C2/c 29.9652(13) 8.2241(4) 16.2286(7) 90 110.457(3) 90 3747.1(3) 8 1.658 1.386 1924 0.32 0.28 0.24 1.4528.84 24,967 4856 [R(int) = 0.0694] 4856/0/262 R1 = 0.0562, wR2 = 0.1444 R1 = 0.0898, wR2 = 0.1616 1.039 1.285 and 0.888
displacement parameter was calculated from the equivalent isotropic displacement parameter of the bound atom. Cell culture Human lung carcinoma cells (A549 cells) were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan). Human colorectal carcinoma cells (COLO 205 and HT-29 cells) were provided by Dr. Min-Hsiung Pan (National Kaohsiung Marine University, Kaohsiung, Taiwan). Human hepatocellular carcinoma cells (PLC5 cells) were obtained from the American Type Culture Collection (ATCC, Bethesda, MD, USA). A549 cells were grown in a medium consisting of 90% RPMI 1640 with 10% fetal bovine serum supplemented with 0.1 mM nonessential amino acid, 2 mM L-glutamine, 1 mM sodium pyruvate and 100 U/mL penicillin–streptomycin. COLO 205 cells and HT-29 cells were grown in 90% RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 lg/mL streptomycin. PLC5 cells were grown in 90% Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 units/mL penicillin and 100 lg/mL streptomycin. Human cancer cells were grown at 37 °C in a humidified incubator with a 5% CO2 atmosphere. Cell viability by MTT assay The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma Chemical Co., St. Louis, MO, USA) assay was performed according to the method of Mosmann [35]. Human colorectal carcinoma cells were plated into 96-well microtiter plates at a density of 1 104 cells/well. After 24 h, culture medium was replaced by 200 lL (0–100 lM) of compound, and the cells were incubated for 24 and 48 h. The final concentration of solvent was less than 0.1% in cell culture medium. Culture medium was removed and replaced by 90 lL fresh culture medium. Ten microliters of sterile filtered MTT solution (5 mg/mL) in phosphate buffered saline (PBS, pH = 7.4) was added to each well, thereby reaching a final concentration of 0.5 mg MTT/mL. After 5 h, the remaining unconsumed dye was removed, and the
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insoluble formazan crystals were dissolved in 200 lL/well DMSO and measured spectrophotometrically in a VersaMax tunable micro plate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. The relative cell viability (%) related to control wells containing cell culture medium without samples was calculated by A570 nm [sample] A570 nm [control] 100. Inoculum preparation For the cultivation of mycobacteria, the MGIT (Mycobacteria Growth Indicator Tube), a fluorescent compound is embedded in silicone on the bottom, then 4 mL of modified Middle brook 7H9 Broth base are added to the mixture. After that 0.5 mL of OADC enrichment, (an oleic acid, albumin, dextrose and catalase) and PANTA (Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin) antibiotic mixture to prevent the growth of any non-mycobacteria (0.1 mL) are added to this medium. Oleic acid plays an important role in metabolism of mycobacteria; Albumin acts as a protective agent; Dextrose is an energy source; Catalase destroys toxic peroxides. Tubes are incubated at 37 °C. For positive control, MGIT tubes are prepared by inoculating bacteria. An un-inoculated MGIT tubes are used as a negative control. Blood Agar is used for checking the growth of other bacteria. Tubes reading daily starts on the second day of incubation using a Micro MGIT fluorescence reader which has a long wave UV light [36]. To prepare inoculums from a positive BACTEC MGIT tube, the positive tubes (day 1 or day 2 positive) are used directly as inoculums. The positive tubes between day 3 and day 5 are diluted as 1:4 ratio by sterile saline. Inoculums, prepared from a MGIT 7 mL positive tube (from day 1 to day 5), ranges between 0.8 105 and 3.2 105 CFU/mL. Each assay is performed according to the MGIT manual fluorometric susceptibility test procedure recommended by the manufacturer [36,37]. Anti-mycobacterial susceptibility assay The activity of complex 1 against M. tuberculosis strains was tested using the Microplate Presto Blue Assay (MPBA) by the method described Collins & Franzblau [38] and modified by Jimenez-Arellanes et al. [39]. 128 lL of compound and 72 lL of 7H9 broth were transferred in the first column; then 100 lL of 7H9 broth was transferred from the column 2 to column 8. Column 9 and 10 were positive and negative control respectively. 100 lL of mixture of broth and compound were transferred from column 1 to column 2 then mixed by pipettes three times and go on the same to provide serial 1:2 dilutions. 100 lL of excess medium was discarded from the wells in column 8. Afterwards, 20 lL of M. tuberculosis inoculum was added to the wells column 1 to 8 and positive control columns. Negative control columns were not inoculated with bacteria. Positive control columns include 7H9 broth and bacteria; and negative control columns were include 7H9 broth and compound. Final test concentrations ranges were 64–1 lg/mL in the mixture. Microplates were inoculated with the bacterial suspension (20 lL) per wells except for the negative control and incubated at 37 °C for 6 days. Presto blue (15 lL, Life Technologies) was then added to the bacterial growth control wells (without compound) and the microplates were incubated at 37 °C for an additional 24 h. If the dye turned from blue to pink (indicating positive bacterial growth); then Presto blue solution was added to the other wells to determine the MIC values. All tests were performed in triplicate. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of sample that prevents a color change to pink. The minimal bactericidal concentration (MBC) was corresponded to the minimum compound concentration which is not cause a color change in the cultures when re-incubated in fresh medium [40,41]. Streptomycin (STR), ethambutol (EMB), and isoniazid (INH) were used as standard drugs.
Results and discussion Crystal structure Complex 1 features a doubly end-on azido bridged mixed-valence cobalt trinuclear unit as depicted in Fig. 1. The necessary bond lengths and angles are summarized in Table 2. The solid crystallizes in the monoclinic unit cell of C2/c space group with the tricobalt complex situates in a special position of 0, y, 1/4 such that only half of the tricobalt complex exists in an asymmetric unit. The full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. There are geometrically same two cobalt centers (CoII and CoIII), consisting with a N4O2 donor set for Co1 (CoII) and N5O donor set for Co3 (CoIII). In the trimeric unit, the cobalt atoms are held together by doubly end-on azide bridges. The coordination environment in the central Co1 atom can be described as a distorted (4 + 2) (NNNN + OO) octahedron. It is coordinated by two cis methanol molecules and four bridging azide ligands. The four atoms constructing the basal plane of the octahedron are the two nitrogen (N6 and N6#1) atoms from two bridging azides and the two oxygen (O2 and O1) atoms from two coordinated methanol molecules. The axial cite is occupied two bridging N atoms, subjecting the longer axial distance [2.215(4) Å] than equatorial bond lengths [2.098(3) and 2.099(3) Å]. The Co1 atom lies not in the basal plane but slightly out of it at a distance of 0.08 Å. The deviation from the octahedral geometry is indicated by the bond angles involving trans positions [169.05(13)° and 170.87(18)°], differs from 180°. In this structure, the Co1 Co3 distance is 3.2326(6) Å, and the Co1–N9–Co3 and Co1–N6–Co3 angles are 99.79(15) and 105.29(14)° taking Co1N9, Co1N6, Co3–N9 and Co3–N6 distances 2.215(4), 2.099(3), 2.008(3) and 1.966(3) Å, respectively. The Co3 atom at the flank is also in distorted octahedral coordination geometry. It is coordinated with one terminal and two cis bridging azide ligands and the tridentate ONN ligand via the amine and imine nitrogen atoms, as well phenolato oxygen atom. The extent of distortion at this cobalt atom is less than that at the central cobalt atom as evidenced by the three larger quasilinear bond angles, which are 173.86(14), 174.99(15), and 176.78(13)°. The bonding distances and angles between the tridentate ONN ligand to this cobalt atom are entirely normal with respect to end-on azido bridged cobalt compound in the literature [27]. The Co–N distance from the terminal azide is short [1.938(4) Å] compared with those from the bridging azide [1.966(3) and 2.008(3) Å]. These Co-N terminal and bridging bond distances are well comparable with previous literature [28,42], [1.942(5) and 1.954(6) Å]. One of bridging azide ligands is noticeably bent with a short N–N–N bond angle of 170.0(7)°, compared with those of 174.6(5) and 179.1(5)° at the other two azide ligands.
Fig. 1. Perspective view of complex 1. Ellipsoids are drawn from 40% probability level. Hydrogen atoms have been omitted for clarity.
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Table 2 Selected bond lengths (Å) and angles (°) of 1. Co(1)–O(3) Co(3)–N(1) Co(3)–N(6) Co(1)–N(9)–Co(3) N(1)–Co(3)–N(6) O(2)–Co(3)–N(2) N(5)–N(4)–N(3)
Fluorescence Intensity
500
2.098(3) 1.889(4) 1.966(3) 99.79(15) 173.86(14) 176.78(13) 174.6(5)
Co(1)–N(6) Co(3)–O(2) Co(3)–N(9) Co(1)–N(6)–Co(3) N(3)–Co(3)–N(9) N(8)–N(7)–N(6) N(11)–N(10)–N(9)
2.099(3) 1.895(3) 2.008(3) 105.29(14) 174.99(15) 170.0(7) 179.1(5)
Co(1)–N(9) Co(3)–N(3) Co(3)–N(2)
2.215(4) 1.938(4) 2.020(3)
337 nm
400
HL 300
λexc= 282 nm
200
100
410 nm 435 nm
λexc= 345 nm 0 300 350
complex 400
450
500
550
Wavelength (nm) Fig. 2. Emission spectra of HL (in MeOH) and Co-complex (in DMF).
Fig. 4. Temperature dependence of the dc vMT for complex 1 measured in a constant magnetic field of 1 T. The solid line represents the theoretical fitting results (see text).
Fig. 3. EPR spectra of complex 1 recorded at 80 K on a: (a) polycrystalline powder and (b) DMF solution. Diphenylpicrylhydrazyl (dpph) is the standard field marker (g = 2.0036).
The CoIII–CoII distance, 3.2326(6) Å is slightly longer than a similar trinuclear system, 3.105(1) Å [28], whereas lower than those (3.7121(16) and 3.828(9) Å) found in case of CoII–CoII system [29]. An intramolecular hydrogen bond exists between the proton on a coordinated methanol molecule and the phenolato oxygen atom of the tridentate ligand with the H O contact distance of 2.611(4) Å and O–H O contact angle of 111°. Infrared and electronic spectra In free ligand (HL), FTIR bands at 3436 and 1660 cm1 correspond to m(O–H) and m(C@N) stretching frequencies respectively. The m(C@N) band is shifted to 1632 cm1 for 1 which indicates the coordination of azomethine-N with metal ion. In the spectra,
Fig. 5. Molar magnetization in NlB units of complex 1 measured at 2 K as a function of magnetic field. The solid line represents the theoretical fitting results (see text).
well resolved strong band at 2042 cm1 and a bifurcated peak at 2068 cm1 are obtained, which indicates to mN@N stretching frequency of the azide group represented as bridging and terminal ligands respectively [43]. The ligand coordination to the cobalt center is substantiated by two bands appearing at 426 and 327 cm1 attributable to mCo–N and mCo–O, respectively. The UV–visible spectra have been generated for HL in methanol and complex 1 in DMF solution. In ligand, the maximum absorption bands are observed at 225 (e = 4365 M1 cm1) and 282 nm 1 1 (e = 2452 M cm ) which may be p ? p⁄ and n ? p⁄ transition. On complexation, the absorption band is shifted to 266 (e = 927 M1 cm1) and 345 nm (e = 394 M1 cm1) in 1 alongwith
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a broad dd transition band at 517 nm (e = 59 M1 cm1) due to 4 T1g(F) ? 4T1g(P), supporting the presence of octahedral cobalt complex [44]. The band at 652 nm (e = 15 M1 cm1) is probably due to the 4T1g ? 4A2g transition (see ESI, Fig. S2).
benzene (UR = 0.42) [45]. The quantum yield of the complex is very negligible, may be due to the paramagnetic quenching. EPR spectra EPR spectra on the polycrystalline sample of 1 were recorded from 80 to 300 K. These can be interpreted postulating that the external ions are Co(III) with a diamagnetic and EPR-silent t62g, whereas the central ion is a paramagnetic Co(II) which can be investigated by EPR spectroscopy. The spectrum at 300 K is not resolved, but the resolution increases at lower temperatures and that measured at 80 K is shown in Fig. 3a. This behavior is compatible and explainable with the short spin–lattice relaxation time for high-spin Co(II) complexes. At liquid nitrogen temperature, no hyperfine structure arising from an electron-nucleus interaction for the 59Co isotope (100% abundance, I = 7/2) is observed, but resonances at g 4.50, 2.31 and 2.04 are detected. The value of g
Emission spectra Emission spectroscopy studies of the ligand and Co-complex, are carried out in methanol and DMF, respectively and the spectra is shown in Fig. 2. When the ligand solution is excited at 282 nm the emission band is observed at 337 nm whereas a weak emission band appears at 410 and 435 nm for the complex on excitation at 345 nm. Longer wavelength of excitation (MLCT/d–d bands) does not show any emission. The fluorescence quantum yield of the ligand (UL: 0.06) and complex (Ucomplex,: 0.003) are determined using carbazole as reference with known quantum yield value in 120 100
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120
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Concentration ( μ M) Fig. 6. Effect of 1 on the cell viability of A549 cells (A), COLO 205 cells (B), COLO HT-29 cells (C), PLC5 cells (D) and NIH3T3 cells (E). Cells were treated with 100 lM compounds for 24 and 48 h. The reported values are the means ± SD (n = 3). ⁄p < 0.05 is significantly different from that of the control.
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A. Datta et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 427–434 Table 3 Anti-mycobacterial activity (MIC and MBC, lg/mL) of complex 1. Complex
1 ETH
The minimal inhibitory concentration (MIC)
The minimal bactericidal concentration (MBC)
Mycobacterium tuberculosis RA
Clinical isolate 1 (7/16)
Clinical isolate 2 (16/6)
Mycobacterium tuberculosis RV
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
8 1.87
32 1.87
8 3.74
32 7.48
8 3.74
32 3.74
8 1.87
32 >7.48
larger than 4.0 is typical of high-spin Co(II) complexes [46], and has been measured for other hexacoordinated Co(II) complexes with tetragonal symmetry [47–50]. The spectrum can be associated with the Kramer doublet ±1/2 arising from the S = 3/2 ground state [51], which must present two signals approximately at g1 = g2. Similar behavior was observed for others Co(III)/Co(II)/Co(III) complexes with the Co(II) ion exhibiting a high-spin configuration [52,53]. The EPR spectrum in a frozen DMF glass is characterized by comparable pattern (Fig. 3b), indicating that the arrangement of the solid state is retained in the solution, in agreement with the previous observations for other similar trinuclear cobalt species [53]. Magnetic moments The magnetic susceptibility vM of complex 1 was measured on polycrystalline samples between 2 K and 300 K. The thermal dependence of the vMT product is shown in Fig. 4. At room temperature, the value of vMT is 3 cm3 K mol1, larger than the spin-only value for S = 3/2 with g = 2 (1.875 cm3 K mol1), as expected for Co(II) systems with significant orbital contributions of the distorted octahedral Co(II) [54]. Upon cooling, the vMT values decrease smoothly until approximately 15 K and then more abruptly to the value 1.56 cm3 K mol1 at 2 K. These magnetic data are consistent with a mixed valence state CoIII(S = 0)–CoII(S = 3/2)– CoIII(S = 0) of complex 1. In order to interpret the magnetic properties of 1, the following general Hamiltonian was used with S = 3/2 and two parameters for the g-value (g||, g\).
^ ¼ D ^S2 1 SðS þ 1Þ þ Eð^S2 ^S2 Þ þ gbH S H z x y 3
ð1Þ
Taking into account rhombic distortions of the crystal field in the Hamiltonian, the low temperature magnetic behavior of the system is better resolved in comparison with only axial distortions. The solid line in Fig. 4 shows the best fit found with D = 61.4 cm1, E/D = 0.33, g|| = 2.7 and g\ = 1.95, comparable with other octahedral Co(II) systems [55,56]. The molar magnetization was measured as a function of the applied magnetic field at 2 K (Fig. 5). As well established, the combined effects of distorted octahedral crystal field and spin–orbit coupling produces six Kramer´s doublets, where only the ground-state Kramer´s doublet is thermally populated at low temperature [54]. Therefore the Co(II) ion can be described by an effective spin 1/2. The solid line in Fig. 5 represents the theoretical magnetization curve for a system in a ground state with effective spin S0 = 1/2 and effective g value of 4.43. Effects on cell-population growth in human carcinoma cells Fig. 6 shows the effects of complex 1 on cell population growth in human cancer cells [including human lung (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human hepatocellular (PLC5 cells) carcinoma cells]. To assess the inhibitory effect of the complex on the growth of human cancer cells, the cells are cultured for 24 and 48 h with or without test compound (0–100 lM), and population growth is determined by MTT assay. The results
Fig. 7. Antimycobacterial activity of compound 1 and a standard drug against Mycobacterium spp.
from an MTT assay demonstrate that 1 decreases the cell population growth of A549 cells, COLO 205 cells, HT-29 cells, and PLC5 cells. Moreover, the 50% inhibitory concentration (IC50), as determined by MTT assay after 48 h of incubation, show the highest activity for complex 1 with an IC50 value of 46.00 ± 1.09 lM. It is further tested by an in vivo model to justify whether it is effective for the prevention of human cancer. Fig. 6 also shows the effect of complex 1 on cell population growth in human fibroblasts cells (NIH3T3). The investigation regarding the apoptotic properties of 1 on human fibroblasts cells (NIH3T3) suggests that a mitochondria-mediated pathway is induced by this complex. Anti-mycobacterial activity In the anti-mycobacterial assay, complex 1 was tested against M. tuberculosis H37Rv ATCC 27294, M. tuberculosis H37Ra ATCC 25177 strains alongwith two clinical strains (strain 1 and strain 2). The results are depicted in Table 3 and Fig. 7. M. tuberculosis H37Rv and M. tuberculosis H37Ra were a drug susceptibility profiles consisting well-known indicator organisms that they used to for the drug sensitivity tests [37]. The results showed that compound 1 exhibited anti-mycobacterial activity against all the tested M. tuberculosis strains, with MICs at 8 lg/mL and MBC were in the range of 32 lg/mL. This complex showed a bactericidal activity and killed the bacteria. Trias et al. [57] showed that Mycobacteriumcheionae developed a pore-forming protein as a mycobacterial porin. On the study of permeability of Mycobacteriumsmegmatis, Trias & Benz [58] reported that mycobacterial porin formed an important hydrophilic structure which small molecules pass through the pores and diffuse into the cytoplasm. In this study, we reported that complex 1 showed a considerable efficacy on the mycobacterial strains and compared the effect with a known drug ETH (Ethambutol). The mycobacteria cell wall includes a large amount of complex lipids, lipopolysaccharides and mycolic acids. This constitution makes the cell wall a
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hydrophobic strong barrier against antimicrobial agents [59,60]. Recently, the studies on the effect of pyridine based inhibitors [61,62] against M. tuberculosis are being attracted a lot. Conclusion A monoanionic tetradentate-N2O2 Schiff base precursor (HL), is used to afford a mixed-valence trinuclear cobalt complex [Co3(L)2(N3)6(CH3OH)2] (1). In solid state, it shows that there are geometrically same two cobalt centers (CoII and CoIII) and the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. On investigating VTM study, it shows the value of vMT is 3 cm3 K mol1 at room temperature, larger than the spin-only value for S = 3/2 with g = 2 (1.875 cm3 K mol1) and the related data is well consistent with a mixed valence CoIII(S = 0)–CoII(S = 3/2)–CoIII(S = 0) system. Complex 1 restrains the cell growth of different human carcinoma cells (A549, COLO 205, HT-29 and PLC5) and shows efficaciousness on mycobacterial strains (H37Rv and H37Ra), involved in tuberculosis. Future work will explore the mechanistic pathway and modification that corresponds to several applications among mixed-valence metal complexes of newly designed organic precursors. Acknowledgements Amitabha Datta would like to thank the Scientific & Technological Research Council of Turkey (TÜBITAK) for the grant (2221 – Fellowship for Visiting Scientist). Kuheli Das and Chittaranjan Sinha would like to thank DST (Department of Science and Technology, New Delhi, India) for the grant (SR/S1/ IC-31/2008). Jui-Hsien Huang thanks the National Changhua University of Education for providing the X-ray diffractometer facility. Appendix A. Supplementary data Crystallographic data for structural analysis has been deposited to the Cambridge Crystallographic Data Center, bearing CCDC – 993646. Copies of this information may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 IEZ, UK (fax: +44 1223 336 033; e-mail
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