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Synthesis, structural characterisation and cytotoxicity of new iron(III) complexes with pyrazolyl thiosemicabazones
Polyhedron 34 (2012) 1–12
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Synthesis, structural characterisation and cytotoxicity of new iron(III) complexes with pyrazolyl thiosemicabazones Nitis Chandra Saha a,⇑, Chinmoy Biswas a,c, Atanu Ghorai b, Utpal Ghosh b, Saikat Kumar Seth d,e, Tanusree Kar d a
Department of Chemistry, University of Kalyani, Kalyani 741235, Nadia, West Bengal, India Department of Biochemistry & Biophysics, University of Kalyani, Kalyani 741235, Nadia, West Bengal, India Department of Chemistry, Rishi Bankim Chandra College, Naihati, 743165, 24 PGS (N), West Bengal, India d Department of Materials Science, Indian Association for the Cultivation of Science, Kolkata-700032, India e Department of Physics, M.G. Mahavidyalaya, Bhupatinagar, Midnapore (East) 721425, West Bengal, India b c
a r t i c l e
i n f o
Article history: Received 19 June 2011 Accepted 24 October 2011 Available online 9 November 2011 Keywords: Synthesis Iron(III) Pyrazolylthiosemicarbazone X-ray crystallography Cytotoxicity
a b s t r a c t New iron(III) complexes of 5-methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZNMe2) (I) and 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (III), [Fe(MPZNMe2)2]NO3H2O (II) and [Fe(MPZNEt2)2]NO3H2O (IV), respectively, have been synthesised for the first time and physico-chemically characterised by magnetic measurements (polycrystalline state), electronic and IR spectra. Both are cationic complexes containing two monodeprotonated tridentate ligands with NNS donor sites and an anionic counterpart; the complex species behave as 1:1 electrolytes in MeOH. IR spectra (4000–200 cm 1) indicate coordination to iron(III) centre via the pyrazolyl (tertiary) ring nitrogen, azomethine nitrogen and thiolato sulfur atom. X-ray crystallographic data of (II) (monoclinic, C2/c) triclinic) have authenticated the FeN4S2 octahedral coordination in both complex ions, as and (IV) (P1, envisaged from the spectral data. In both species, the two azomethine nitrogen atoms are trans to each other, while the pyrazolyl ring nitrogen and the thiolato sulfurs atoms are in cis positions. We further observed that both the ligands and their corresponding complexes were capable of killing HeLa cells in culture, but the cytotoxicity of the complexes was found to be greater than their corresponding ligands. Complex (II) does not have any role in cell-cycle progression, but complex (IV) arrests the M-phase to prevent cell-cycle progression. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction A number of heterocyclic thiosemicarbazones and their metal ion complexes have attracted considerable attention in chemistry and biology owing to their potentially beneficial biological activities (antitumoral, antibacterial, antimalarial, antiviral, etc.) over the years and such activities have often been related to their chelation phenomenon with one or more essential trace metal ions [1–10]. It is documented that iron(III) complexes of thiosemicarbazones have been shown to be more active in cell destruction, as well as in the inhibition of DNA synthesis, than the uncomplexed thiosemicarbazones [11]. The success in therapeutic applications of several heterocyclic thiosemicarbazones [3,12–14] for removing excess iron from iron-loaded mice through chelation therapy is also remarkable. These findings have initiated further research on this specific area. In continuation of our earlier reports on iron(III) complexes and related publications [15–20] on metal ion complexes of ⇑ Corresponding author. Fax: +91 33 25828282. E-mail address: [email protected] (N.C. Saha). 0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2011.10.033
pyrazolyl thiosemicarbazones, the present communication intends to report the synthesis, structural characterisation and cytotoxic activities of two new iron(III) complexes with the title ligands. 2. Experimental Hoechst dye, propidium iodide and RNase A were purchased from Sigma Chemicals (USA). Culture media MEM and serum were purchased from HiMedia, India. All other reagents were of AR grade/molecular biology grade and were obtained from commercial sources and used without further purification. Spectrograde solvents were used for spectral and conductance measurements. 2.1. Preparation of HMPZNMe2 (I) and HMPZNEt2 (III) Both the title ligands, 5-methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZNMe2) and 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (Fig. 1) have been synthesized by a method similar to that reported earlier [21], but appropriately modified, followed by the final transamination [22]
2
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
Fig. 1. Schematic structural formulation of the ligands (I) and (III).
each reaction. Aqueous NH3 was added dropwise until the solution was strongly ammoniacal. The resulting precipitate of Fe2O3xH2O was filtered off and washed with H2O until Cl free. The precipitate was then dissolved in a minimum quantity of dilute HNO3 and the resulting solution was added to a hot 1.05 mmol EtOH solution of HMPZNMe2 (0.2215 g) for (II) and HMPZNEt2 (0.251 g) for (IV). The resulting dark brown solution, in each case, was heated to reflux for about 30 min, then the solvent was allowed to evaporate at room temperature. The dark brown solids thus obtained were filtered off, washed with cold ethanol and dried over anhydrous CaCl2. The yield, in each case was 75–80%. Brown black (II) and dark brown (IV) needle shaped crystals were found to be suitable for X-ray diffraction.
of the S-methyldithiocarbazate of 5-methyl-3-formylpyrazole (HMPZSMe) with dimethylamine for HMPZNMe2 and diethylamine for HMPZNEt2. For both the cases, white crystallised products (from ethanol) were obtained in a yield of ca. 60–70%. HMPZNMe2 (M.P. 182–184 °C). Anal. Calc. for C8H13N5S: C, 45.5; H, 6.2; N, 33.2. Found: C, 45.5; H, 6.0; N, 32.7%. 1H NMR d(DMSO-d6): 2.25 (3H, s), 6.24 (1H, s), 8.06 (1H, s), 10.79 (1H, s), 3.26 (6H, s). m/z: 211 (M+, 82%). For HMPzNEt2 (M.P. 160–162 °C). Anal. Calc. for C10H17N5S: C, 50.2; H, 7.1; N, 29.3. Found: C, 50.3; H, 7.1; N, 29.5%. 1H NMR d(CHCl3-d): 2.29 (3H, s), 6.22 (1H, s), 7.39 (1H, s), 10.71 (1H, s), 3.80 (4H, m), 1.28 (6H, t). m/z: 239 (M+, 86%). 2.2. Synthesis of [Fe(MPZNMe2)2]NO3H2O (II) and [Fe(MPZNEt2)2] NO3. H2O (IV)
2.3. Physical measurements
0.17 g, 1.05 mmol anhydrous FeCl3 was dissolved in aqueous EtOH (10 cm3) containing 2 N HCl (0.5 cm3) in a small beaker for
Elemental analyses (C, H and N) were done with a Perkin-Elmer 2400 CHNS/O analyser and the iron content of the complexes
Table 1 Crystal data and structure refinement parameters for C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1(III), and C20H34Fe1N11O4S2(IV). Crystal data
(I)
(II)
(III)
(IV)
Empirical formula Formula weight T (K) k (Å) Crystal system Unit cell dimensions Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) DCalc (mg/m3) Absorption coefficient (mm F(0 0 0) Crystal size (mm) h ranges (°) Limiting indices
C8H13N5S1 211.29 150(2) 0.71073 monoclinic
C32H56Fe2N23O13S4 1210.94 150(2) 0.71073 monoclinic
C10H17N5S1 239.35 150(2) 0.71073 orthorhombic
C20H34Fe1N11O4S2 612.55 150(2) 0.71073 triclinic
P21 11.986(3) 7.3218(18) 12.172(3) 90 100.768(5) 90 1049.4(4) 4, 1.337 0.278 448 0.32 0.26 0.22 1.70 to 25.96 14 6 h 6 14 86k68 14 6 l 6 14 9642 3683 [Rint = 0.0532] 99.2 full-matrix least squares on F2 3173 3683/259 1.105 R1 = 0.0407 wR2 = 0.0913 R1 = 0.0549 wR2 = 0.0981 0.289 and 0.395
C2/c 26.7009(11) 13.4698(4) 17.4483(9) 90 125.542(2) 90 5106.2(4) 4, 1.575 0.813 2516 0.28 0.23 0.18 1.78 to 25.00 31 6 h 6 31 16 6 k 6 15 20 6 l 6 20 23 676 4498 [Rint = 0.0549] 100.0 full-matrix least squares on F2 3619 4498/351 1.074 R1 = 0.0554 wR2 = 0.1623 R1 = 0.0702 wR2 = 0.1742 1.101 and 1.375
Pccn 21.929(3) 9.6397(10) 12.0354(13) 90 90 90 2544.2(5) 8, 1.250 0.238 1024 0.42 0.34 0.22 1.86 to 24.99 26 6 h 6 26, 11 6 k 6 11, 13 6 l 6 14 22 210 2246 [Rint = 0.0251] 100.0 full-matrix least squares on F2 2069 2246/148 1.069 R1 = 0.0313 wR2 = 0.0834 R1 = 0.0338 wR2 = 0.0852 0.259 and 0.234
P1 13.145(2) 13.662(2) 8.531(3) 104.86(3) 95.08(3) 86.56(2) 1473.9(6) 2, 1.380 0.699 642 0.30 0.28 0.24 3.02 to 25.00 15 6 h 6 15 16 6 k 6 15 0 6 l 6 10 5186 5186[Rint = 0.0000] 99.8 full-matrix least squares on F2 2272 5186/349 0.914 R1 = 0.0704 wR2 = 0.1450 R1 = 0.1827 wR2 = 0.1846 0.979 and 0.473
1
)
Reflections collected Unique reflections Completeness to theta (%) Refinement method Observed reflections [I > 2r (I)] Data/parameters Goodness-of-fit (GOF) on F2 Final R indices [I > 2r (I)] R indices (all data) Largest difference peak, hole (e Å
3
)
3
N.C. Saha et al. / Polyhedron 34 (2012) 1–12 Table 2 Selected bond lengths (Å), bond angles and torsion angles (°) of C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1 (III) and C20H34Fe1N11O4S2 (IV). (I)
(II)
(III)
Moiety A
Moiety B
Ligand A
1.360(4) 1.360(3) 1.364(4) 1.292(4) 1.381(3) 1.361(4) 1.351(3) 1.687(3)
1.973(4) 1.927(4) 2.2186(12) 1.358(4) 1.360(3) 1.360(4) 1.286(4) 1.379(3) 1.358(4) 1.352(4) 1.694(3)
1.956(4) 1.925(4) 2.2237(13) 1.354(6) 1.345(5) 1.341(6) 1.292(6) 1.371(5) 1.331(6) 1.340(6) 1.760(5)
Bond angles N2–Fe–N3 N2–Fe–S1 N3–Fe–S1 N1–N2–C4 N2–C4–C5 N3–C5–C4 C5–N3–N4 N3–N4–C6 N5–C6–N4 N4–C6–S1 N5–C6–S1
103.5(2) 122.9(3) 130.5(3) 115.9(2) 120.2(2) 114.7(2) 122.0(2) 123.3(2)
80.53(15) 165.39(11) 85.03(11) 103.4(2) 122.0(3) 130.7(3) 116.0(2) 119.9(2) 114.3(2) 122.4(2) 123.3(2)
80.66(15) 164.90(11) 85.01(11) 106.1(4) 113.9(4) 114.2(4) 118.5(4) 111.6(4) 117.8(4) 123.3(3) 118.9(4)
Torsion angles N1–N2–C4–C5 N2–C4–C5–N3 N4–N3–C5–C4 C6–N4–N3–C5 N3–N4–C6–N5 N3–N4–C6–S1 S1–C6–N5–C7
179.8(3) 1.7(5) 2.3(5) 179.1(3) 177.3(2) 3.3(4) 177.3(2)
180.0(3) 0.9(5) 1.0(5) 175.3(3) 172.5(2) 6.7(4) 176.7(2)
174.9(4) 5.0(6) 176.4(4) 178.8(4) 179.0(4) 1.5(6) 176.7(4)
Bond lengths Fe1–N2 Fe1–N3 Fe1–S1 N1–C2 N2–N1 N2–C4 N3–C5 N4–N3 N4–C6 N5–C6 S1–C6
(IV)
Ligand B
Ligand A
Ligand B
1.352(6) 1.350(5) 1.346(6) 1.300(6) 1.382(5) 1.329(6) 1.344(6) 1.748(5)
1.353(2) 1.346(2) 1.347(2) 1.284(2) 1.370(2) 1.375(2) 1.339(2) 1.683(2)
1.986(6) 1.931(6) 2.233(2) 1.363(1) 1.334(8) 1.334(9) 1.269(9) 1.381(8) 1.319(1) 1.371(1) 1.756(9)
1.962(6) 1.928(6) 2.220(3) 1.344(1) 1.361(8) 1.348(9) 1.300(9) 1.372(8) 1.318(1) 1.379(1) 1.729(9)
106.2(4) 114.1(4) 113.6(4) 118.8(4) 111.5(3) 118.2(4) 123.7(3) 118.1(3)
104.22(11) 122.28(13) 130.64(13) 117.44(12) 119.29(12) 114.63(12) 121.51(11) 123.87(11)
79.9(3) 163.9(2) 84.8(2) 106.6(6) 114.3(7) 114.4(7) 118.5(7) 112.6(7) 115.1(8) 123.1(7) 121.8(7)
80.0(3) 163.9(2) 84.5(2) 105.9(6) 113.3(7) 115.1(7) 119.1(7) 110.9(7) 117.0(8) 124.7(7) 118.3(7)
175.5(4) 1.2(6) 178.8(4) 175.8(4) 176.3(4) 0.6(5) 173.6(4)
177.7(2) 4.1(2) 3.1(2) 165.9(2) 172.0(2) 8.3(2) 169.3(2)
175.9(7) 0.7(1) 177.4(7) 176.4(9) 176.3(8) 1.0(1) 2.2(1)
179.7(6) 0.5(1) 179.9(7) 179.5(8) 178.6(8) 0.7(1) 9.8(1)
Bond angles involving both ligands (A and B) around the Fe atom in complexes(II) and (IV) (II) (IV) N3B–Fe1–N2A N2A–Fe1–S1B N3B–Fe1–S1A N2B–Fe1–N2A
100.55(15) 88.62(11) 93.86(11) 89.34(15)
100.7(3) 91.0(2) 94.7(2) 87.7(3)
N3A–Fe1–N2B N2B–Fe1–S1A N3A–Fe1–S1B S1B–Fe1–S1A
(II)
(IV)
98.71(15) 90.63(11) 95.71(12) 95.08(5)
100.5(3) 90.4(2) 95.1(2) 95.2(1)
Table 3 Analytical data and other pertinent physico-chemical properties of the complexes. Complex (colour)
Elemental analyses Found (calc.)
Conductivity at 30 °C in MeOH (X
%C
%H
%N
%Fe
[Fe(MPzNMe2)2]NO3H2O (Brown black) [Fe(MPzNEt2)2]NO3H2O (Dark brown)
34.2 (34.5) 39.0 (39.2)
5.2 (5.1) 5.1 (5.2)
27.3 (27.7) 25.4 (25.2)
9.9 (10.0) 9.0 (9.1)
was determined titrimetrically. The molar conductance of the complexes in methanol solutions was measured with a Systronics 304 digital conductivity meter. Magnetic susceptibilities were measured in the polycrystalline state on a PAR 155 sample vibrating magnetometer. 1H NMR spectra for the ligands were recorded in DMSO-d6/CDCl3 with a Bruker AM 300L (300 MHz)
Table 4 IR assignments (cm
1
cm2 mol
1
)
leff (BM) at 298 K
110
1.95
106
2.05
superconducting FT NMR spectrometer. Mass spectroscopy of the ligands was done with a Jeol JMS-D300 mass spectrometer. The electronic spectra and the diffuse reflectance spectra were recorded on a Hitachi U-3501 spectrophotometer. IR spectra (4000–200 cm 1) were recorded on a Jasco FT/IR-420 spectrophotometer with KBr pellets.
1
) for the ligands and the complexes pertaining to the coordination sites.
Compound
m(CH@N) (azomethine)
m(C@N) (pyrazole)
m(C@S)
HMPzNMe2 (I) [Fe(MPzNMe2)2]NO3H2O (II) HMPzNEt2 (III) [Fe(MPzNEt2)2]NO3H2O (IV)
1611 1598 1620 1600
1503 1520 1510 1525
864 785 865 780
m(Fe–N) (azomethine)
m(Fe–N) (pyrazole)
m(Fe–S)
482
287
375
478
280
381
4
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
2.4. Structure determination Single crystal X-ray diffraction intensity data of the free ligand 5methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZ NMe2) (I), which was crystallized with two independent molecules per asymmetric unit, its metal complex [Fe(MPZNMe2)2]NO3H2O (II), which has been represented crystallographically as 2[Fe(MPZNMe2)2](NO3)34H2O, another free ligand 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (III) and its metal complex [Fe(MPZNEt2)2]NO3H2O (IV) were collected at 150(2) K using a Bruker APEX-II CCD diffractometer equipped with graphite monochromated Mo Ka radiation (k = 0.71073 Å). Data reduction was carried out using the program Bruker SAINT [23]. Because of the very small values of the absorption coefficients, no absorption correction was applied. The structures of the compounds were solved by direct methods and refined by the full-matrix least-square technique on F2 with anisotropic thermal parameters to describe the thermal motions of all non-hydrogen atoms using the programs SHELXS 97 and SHELXL 97 [24]. All calculations were carried out using the WinGX system Ver-1.64 [25]. In the complexes delu, simu and isor constraints were applied for a few atoms. In the free ligands (I) and (III), the hydrogen atoms were located from a difference Fourier map and were treated as riding, whereas in the complexes, the hydrogen atoms were located at geometrically calculated positions and were treated by a mixture of independent and constraint refinement. The refinements were based on F2 for all reflections except those with non-reliable F2 values. In compound (II), one of the nitrate anions is disordered over two positions with occupancies of 1:2.5, and the water molecules of crystallization are also disordered, for which the H-atoms could not be located. The crystal of compound (IV) diffracted weakly, less than half of the reflections were considered to be observed, and the atoms in the –CH2CH3 substituents and the nitrate anion all undergo considerable thermal motion. A summary of the crystal data and relevant refinement parameters are given in Table 1. Selected bond-lengths, bond angles and torsion angles of both the ligands and the complexes are given in Table 2. 2.5. Effect of the synthesized product in cultured HeLa cells 2.5.1. Cell killing activity The human cervical cancer cell line HeLa was obtained from the National Centre for Cell Sciences, Pune, India. HeLa cells were grown in MEM supplemented with 10% bovine serum (complete medium) at 37 °C in a humidified atmosphere containing 5% CO2 medium [26]. After inoculation in a fresh medium, the HeLa cells were incubated overnight and then treated with different concentrations of the synthesized products under investigation (0–50 lM) for 13 h. After the treatment, the cells were harvested by trypsinization and suspended in PBS buffer. After washing twice, the cells were incubated with 0.1% tryphan blue for 5 min at room temperature and counted using a hemocytometer under a light microscope. The dead cells appeared to be blue, whereas the viable cells were colourless. Each experiment was repeated four times and the mean of the percentage viable cells at each dose was compared with the mean of the untreated control using one way ANOVA with a post hoc test such as the Tukey’s test and Dunnett’s test. The number of viable cells in the untreated control was considered as 100% and the viable cell number at different doses were normalized accordingly. 2.5.2. Induction of apoptosis as detected by nuclear fragmentation Detection of nuclear fragmentation was done as reported earlier [27]. In summary, after 24 h, the treatment cells were harvested in PBS buffer and fixed with 70% ethanol for 45 min at 4 °C. Then the cells were suspended in PBS buffer, stained with Hoechst (5 lM), incubated for 5 min in the dark, and washed twice with PBS. The
Fig. 2. (a) An ORTEP diagram of the two independent molecules of the free ligand (I) with the atom numbering scheme. Thermal ellipsoids are drawn at the 30% probability level. (b) Packing diagram for (I), viewed down the b-axis. Hydrogen bonds are indicated by dotted lines.
cells were examined by a fluorescence microscope (Carl Zeiss) using the appropriate filter. Apoptotic cells were distinguished by nuclear fragmentation and chromatin condensation. The apoptotic cells were randomly counted and the percentage of the apoptotic cells was calculated at each dose. The mean percentage of apoptotic cells with a standard deviation was calculated from four independent experiments. 2.5.3. Cell cycle analysis After 18 h treatment, the cells were trypsinized, washed twice with cold phosphate buffered saline (PBS), fixed with 70% ethanol in PBS for 2 h at 4 °C, and then stained with a propidium iodide (PI) solution (10 lg/ml) containing DNase-free RNase (100 lg/ml) and 0.1% (v/v) Triton X-100 in the dark for 30 min at room temperature before flow cytometric analysis. The samples were detected with FACS Calibur (BD Bioscience, USA). A minimum of 20,000 cells, analyzed in each sample, served to determine the percentages of cells in each phase of the cell cycle using ModFit LT software [28]. 3. Results and discussion 3.1. Spectroscopic characterisation of HMPZNMe2 (I) and HMPZ NEt2 (III) The title ligands, HMPZNMe2 and HMPZNEt2, have been characterised by elemental analyses (C, H and N), IR, 1H NMR and mass spectra. The characteristic IR bands (cm 1) at 3300–3200 (mNH), 1615–1611 (mCH@N), 1510–1505 (mC@N), 1040–1020 (mN–NPZ) and 867–865 (mC@S) are in good agreement with the structure in Fig. 1(a). Both the ligand molecules have a proton adjacent to the thiocarbonyl group and consequently can exhibit thione–thiol
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
tautomerism. The IR spectra of the free ligands do not show any (mSH) band in the region 2600–2200 cm 1, indicating that the thiol tautomer is absent in the solid state [29,30], but exhibit (mNH) bands in the region 3300–3200 cm 1, indicating the existence of only the thioketo tautomer Fig. 1(a). The 1H NMR spectra of the free ligands in DMSO-d6 give singlets at d 2.48–2.41 (3H), d 6.20– 6.17 (1H) assigned to C5–CH3 and C4–H of the pyrazole rings, respectively. One-proton singlets at d 8.09–7.29, ascribed to CH@N, indicate the existence of hydrogen bonded stabilized forms of the ligands as shown in Fig. 1(b). The 1H NMR spectra also show signal at d 13.40–13.10, indicating the presence of a hydrogen bonded proton [31]. The mass fragmentation data are compatible with the proposed molecular formulae. The strongest molecular ion peak appears at m/z 211 (M+ 58%) for HMPZNMe2 and m/z 239 (M+ 86%) for HMPzNEt2.
3.2. Characterisation of the Fe(III) complexes Both the Fe(III) complexes give satisfactory C, H, N and Fe analyses and conform to the compositions [Fe(MPzNMe2)2]NO3H2O and [Fe(MPzNEt2)2]NO3H2O. The molar conductance values in MeOH (30 °C) classify them as 1:1 electrolytes [32] and the complexes are paramagnetic (at 25 °C), as expected for a low spin d5 ion (Table 3).
5
3.2.1. IR spectra The characteristic IR bands (4000–200 cm 1) for the free ligands, when compared with those of its Fe(III) complexes, provide meaningful information regarding the bonding sites of the primary ligand molecules (Table 4) A negative shift in the m(CH@N) bands (1620–1611 cm 1) in the spectra of the free ligands to a lower value (1600–1598 cm 1) in their complexes are consistent with coordination of the azomethine nitrogen to the central Fe(III) ion. The IR bands at 482-478 cm 1 in the complexes are assignable to m(Fe–N) [32]. Intense bands at 865 cm 1 in the free ligand spectra, assigned to m(C@S), have been found to shift to lower frequency regions (785–780 cm 1) in the complexes, indicating the coordination of the thiol sulfur to Fe(III); moreover, the appearance of new bands in the region (381–375 cm 1) in the complexes are assignable to m(Fe–S) [33]. IR bands at 1510–1503 and 615– 597 cm 1 in the free ligand spectra, assignable to m(C@N) (pyrazole ring) and the in-plane deformation of the pyrazole ring, have been shifted to the higher frequency region, indicating that the tertiary ring nitrogen atom (2N) is a possible bonding site [34–36]. The appearance of bands around 287–280 cm 1 in the complexes are clearly assignable to m(Fe-N) (pyrazole ring) [33]. 3.2.2. Electronic spectra The diffuse reflectance spectral data of the iron(III) complexes appear as three main bands in the regions 19 802–19 760,
Fig. 3. (a) An ORTEP diagram of complex (II) with the atom numbering scheme. (b) One-dimensional chain propagating along the (1 0 0) direction. (c) Packing diagram of (II) displaying the two-dimensional framework.
6
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
16 393–16 050 and 10 830–10 753 cm 1. The absorption bands can be tentatively assigned to a 2T2 ground state for iron(III). The intense bands at ca. 19 802–19 760 cm 1 and ca. 16 393–16 050 cm 1 may be assigned to CT transitions arising from the d ? p⁄ transition. The d–d transition is located at ca. 10,830–10,753 cm 1, which might correspond to a 2T2 ? 2T1 transition [37,38]. The electronic spectra of the complexes in methanol exhibit three bands in the regions 20 800–20 325, 16 313–16 100 and 10 910– 10 858 cm 1. The bands which are found between 20 800 and 16 100 cm 1 are likely due to d ? p⁄ metal to ligand bands as well as a sulfur to Fe(III) transition [39,40]. The bands at ca. 10 910– 10 858 cm 1 can be assigned to d-d transitions of spin paired d5 species with a distorted octahedral structure [41,42]. 3.3. Structural description The molecular views [43] of the free ligand HMPZNMe2 (I), its metal complex 2[Fe(MPZNMe2)2](NO3)34H2O (II), the other free ligand HMPZNEt2 (III) and its metal complex [Fe(MPZNEt2)2]NO3H2O (IV), together with the atom numbering scheme, are shown in Figs. 2a, 3a, 4a and 5a, respectively. The crystallographic asymmetric units in the ligands (I) and (III) comprise of HMPZNMe2 and HMPZNEt2 molecular moieties, respectively, while that in the complexes comprises of a [Fe(MPZNMe2)2]+ cation for (II) and a [Fe(MPZNEt2)2]+ cation for (IV), NO3- counter ions and solvent water of crystallization. In both complexes (II) and (IV), the Fe(III) centre has a distorted octahedral coordination where a pair of mono-deprotonated ligands, (MPZNMe2 ) and (MPZNEt2 ), respectively, coordinate the Fe(III) ion through the thiolato sulfurs (S1A, S1B), pyrazolyl (tertiary) ring nitrogens (N2A, N2B) and the hydrazinic chain nitrogens (N3A,
N3B). Due to this octahedral coordination four five-membered chelate rings [Fe–S(1A)–C(6A)–N(4A)–N(3A), Fe–S(1B)–C(6B)–N(4B)– N(3B), Fe–N(2A)–C(4A)–C(5A)–N(3A), and Fe–N(2B)–C(4B)–C (5B)–N(3B)] were formed. In both the complex species, the two azomethine nitrogen atoms [N(3A) and N(3B)] coordinate the Fe ion in a trans orientation, while the pyrazolyl ring nitrogen atoms [N(2A) and N(2B)] and the thiol sulfur atoms [S(1A) and S(1B)] are in ciscoordination. This coordination is analogous to that observed in the crystal structures of similar metal complexes [15–18]. The bond length and bond angles for the non-hydrogen atoms of the compounds are given in Table 2. The bond lengths of the hydrazinic, pyrazolyl and thiol sulfur atoms to the Fe(III) ion agree well with the corresponding values reported earlier [15,16]. As expected, the S(1)–C(6) double bond distances of 1.687(3) and 1.694(3) Å in moiety A and B, respectively in the free ligand (I) are lengthened to 1.760(5) and 1.748(5) Å in complex (II), with a shortening of the N4–C6 bond from 1.361(4) and 1.358(4) Å to 1.331(6) and 1.329(6) Å, respectively (Table 2). A similar distortion has been observed in (III) and (IV), where the S(1)–C(6) double bond distance of 1.683(2) Å in the free ligand (III) is lengthened to 1.756(9) and 1.729(9) Å in molecules A and B, respectively in complex (IV), with a shortening of the N4–C6 bond from 1.375(2) Å in the free ligand (III) to 1.319(1) [molecule A] and 1.318(1) Å [molecule B] in the Fe(III) complex (IV) (Table 2). These bond distances do not vary significantly in the structures despite the different intermolecular interaction patterns observed in the ligand and the metal complex [18]. The S(1)–C(6), C(6)–N(4) and C(5)–N(3) bond distances in (II) and (IV) show partial double bond character. In (II) and (IV), the partial double bond character of C(4)–C(5) in both molecules (A and B) favour the conjugation of the delocalized p-electrons of the
Fig. 4. (a) An ORTEP diagram of free ligand (III) with the atom numbering scheme. The thermal ellipsoids are drawn at the 30% probability level. (b) One-dimensional zigzag chain propagating along the (0 0 1) direction. (c) Packing diagram of (III) viewed along the (0 0 1) direction.
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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Fig. 5. (a) An ORTEP diagram of complex (IV) with the atom numbering scheme. (b) One-dimensional chain propagating along the (0 0 1) direction. Hydrogen atoms not involved in hydrogen bonding are shown as broken-off bonds. (c) Formation of the R66(24) dimeric ring in complex (IV). (d) Formation of the supramolecular assembly generated through the water-nitrate cluster (colour code: N, blue; O, red; H, white). Hydrogen atoms not involved in hydrogen bonding have been omitted for the sake of clarity. (Colour online)
heterocyclic ring with the thiosemicarbazone skeleton. This conjugation results in near co-planarity of the pyrazolyl ring with the thiosemicarbazone skeleton. The pyrazole ring is therefore restricted with respect to the thiosemicarbazone moiety due to coordination of the pyrazolyl nitrogen atom.
The significant closing of the N(3)–C(5)–C(4) and N(2)–C(4)– C(5) angles in both complexes compared to the corresponding ligands, by about 15–16° and 8–9°, respectively, as indicated by Table 2, may be attributed to geometrical necessity for coordination. The N–Fe1–N, N–Fe1–S and S–Fe1–S angular distributions
Scheme 1. Reaction mechanism showing the rotation about the azomethine double bond.
8
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
indicate that the coordination polyhedron is distorted. This distortion in the metal ion coordination polyhedron results from steric interactions, and the maximum distortion from an ideal octahedral geometry occurs for the N(3B)–Fe1–N(2B) [80.66(15)°] and N(3B)– Fe1–N(2A) [100.5(3)°] angles in (II) and those for (IV) are 80.0(3)° and 100.7(3)°, respectively. A similar distortion has been observed in the literature [44]. Some selected torsion angles of the free ligands (I) and (III) and the metal complexes (II) and (IV) are given in Table 2, which may establish the molecular conformation. The ligand molecules HMPZNMe2 (I) and HMPZNEt2 (III), and the tridentate ligands MPZNMe2 and MPZNEt2 in complexes (II) and (IV), respectively are practically planar. In the ligand molecule (I), the C(6A), C(2A), N(4B) and C(6B) atoms have the largest deviations in opposite directions [C(6A): +0.0678(31) Å, C(2A): 0.0497(32) Å; N(4B): +0.0551(28) Å, C(6B): 0.0205(27) Å], whereas in ligand (III), the C(2) and N(4) atoms have the largest deviations in opposite directions [C(2): +0.3819(15) Å, N(4): 0.4693(13) Å] from the least square mean plane through S(1)–C(6)–N(4)–N(3)–C(5)–C(4)–N(2)– N(1)–C(2)–C(3). In complex (II), the C(2A) and N(2A) atoms in ligand A and the N(4B) and C(4B) atoms in ligand B have the largest deviations in opposite directions [C(2A): +0.1508(61) Å, N(2A): 0.0942(47) Å; N(4B): +0.1032(36) Å, C(4B): 0.1074(43) Å] whereas those in (IV) are for N(4A) and N(1A), and C(5B) and N(1B) [N(4A): +0.0582(84) Å, N(1A): 0.1762(68) Å, C(5B): +0.0284(79) Å, N(1B): 0.1038(63) Å] in ligand A and B, respectively from the least square mean planes through Fe1–S(1)–C (6)–N(4)–N(3)–C(5)–C(4)–N(2)–N(1)–C(2)–C(3). The coordinating ligand A and B of both complexes are orthogonal to each other; the dihedral angle between them is 86.52(5)° in (II) and 89.75(6)° in (IV). The three individual rings (of A and B), namely the pyrazolyl and two five membered chelate rings, are individually almost planar, with a small dihedral angle between them. In (II), the maximum dihedral angle of 8.32(18)° is between the pyrazolyl ring C(2A)–C(3A)–C(4A)–N(1A)–N(2A) and the chelate ring Fe1– N(2A)–C(4A)–C(5A)–N(3A) in ligand A, and that of 5.19(7)° is between the two chelate rings Fe1–N(2B)–C(4B)–C(5B)–N(3B) and Fe1–S(1B)–C(6B)–N(4B)–N(3B) in ligand B. In (IV), the maximum dihedral angle of 8.47(21)° is between the pyrazolyl ring C(2A)– C(3A)–C(4A)–N(1A)–N(2A) and the chelate ring Fe1–S(1A)–C(6A)– N(4A)–N(3A) in ligand A, and that of 3.42(16)° is between the two
chelate rings Fe1–N(2B)–C(4B)–C(5B)–N(3B) and Fe1–S(1B)– C(6B)–N(4B)–N(3B) in ligand B. It is known [2,45–49] that many thiosemicarbazone ligands exist in solution as two conformers, E and Z. In the present crystallographic study, the Z-form, as indicated by the C(4)–C(5)–N(3)–N(4) torsion angles of 2.3(5)° and 1.0(5)°, was obtained in the solid state for the free ligand (I). In complex (II), the coordinating ligand exists as E conformers [the C(4)–C(5)–N(3)–N(4) torsion angle is 176.4(4)° in ligand A and 178.8(4)° in ligand B], as necessitated by the requirements of coordination. A similar observation was observed for (III) and (IV), where the C(4)–C(5)–N(3)–N(4) torsion angle in (III) is 3.1(2)° whereas those in (IV) are 177.4(7)° and 179.9(7)°. Therefore, the Z-conformers of the ligand, which could be present in solution, must be transformed to the E-form before complexation. A possible mechanism for this transformation, leading to an unusual rotation about the azomethine (C@N) double bond, is shown in Scheme 1. The feasibility of this mechanism is due to the electropositive character of the azomethine carbon, C(5), as reported earlier [50,51]. The intermediate conversion from a double bond to a single bond allows the rotation of the thiosemicarbazone moiety to bring the sulfur atom, S(1), to the cis position with respect to the pyrazolyl nitrogen, N(2), in order to permit complex formation. The crystal structures adopted by (I) and (II) are quite different. The structures of the ligand and the complex includes a combination of N–H S, N–H N, N–H O and O–H O hydrogen bonding interactions (Table 5). It is convenient to consider the substructures generated by each type of hydrogen bonds as acting individually, and then the combination of substructures build a three dimensional framework. In ligand (I), the pyrazole ring nitrogen N(1A) acts as a donor to the hydrazinic chain nitrogen N(3B) in the molecules at (x, y, z) and (1 x, ½ y, 1 z). Another pyrazole nitrogen N(1B) of moiety B acts as a donor to N(3A) at (x, y, z) and (1 x, 1/2 + y, z). The thiolato sulfur atom of both the moieties acts as an acceptor of N(1B) and C(1A) in the molecule at (1 x, 1/2 + y, z) and (1 x, ½ + y,1 z), respectively. The packing view of (I) is displayed in Fig. 2b. In complex (II), the pyrazole ring nitrogen of both moieties act as a donor to the oxygen atom of a nitrate anion, thus forming a 1D chain which propagates along the (1 0 0) direction (Fig. 3b). The packing view of the title complex
Table 5 Hydrogen bonding geometry of C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1 (III) and C20H34Fe1N11O4S2 (IV) (Å, °). D–H A
d(D–H)
d(H A)
d(D A)
D–H A
Symmetry
Compound (I) N1A–H1A N3B N1B–H1B S1A N1B–H1B N3A C7B–H7B3 N3A C1A–H1A3 S1B
0.86 0.86 0.86 0.96 0.96
2.20 2.72 2.44 2.57 2.83
2.996(3) 3.363(3) 3.214(3) 3.480(4) 3.637(4)
153 133 150 157 142
1 1 1 x, 1
Compound (II) N1A–H1A O3 N1B–H1B O4 O1W–H1W1 O2 O1W–H1W1 O3 O1W–H2W1 N4B C1A–H1A1 O1 C7B–H7B2 O2
0.86 0.86 0.87 0.87 0.86 0.96 0.96
1.86 1.96 1.85 2.15 2.17 2.47 2.45
2.710(9) 2.797(7) 2.627(10) 2.927(11) 2.928(6) 3.388(11) 3.319(11)
168 164 148 148 148 159 150
x, 1
½ x, ½ + y, ½ z ½ x, ½ + y, ½ z ½ x, ½ y, 1 z x, 1 y, ½ + z 1 x, y, ½ z
Compound (III) N1–H1 S1 N1–H1 N3
0.86 0.86
2.79 2.13
3.448(1) 2.910(2)
135 151
½ ½
x, y, x, y,
½+z ½+z
Compound (IV) N1A–H1A O1W N1B–H1B O1 O1W–H1W1 O3 O1W–H2W1 O2
0.86 0.86 0.82 0.83
1.95 2.03 2.15 1.99
2.803(9) 2.862(10) 2.966(11) 2.815(12)
170 164 178 168
2
x, 1
y, 2
z
2
x, 1
y, 1
z
x, ½ + y, 1 z x, ½ + y, z x, ½ + y, z 1 + y, z x, ½ + y, 1 z y, ½ + z
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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are bonded to O1 and O1W, respectively, and this generates a supramolecular synthon which is formed through a hydrogen bonded water–nitrate cluster (Fig. 5d).
3.4. Cytotoxicity 3.4.1. Comparison of the cell killing effect between the ligands and their corresponding complexes The percentage of viable HeLa cells was decreased dose-dependently with the increase in concentration of the ligand or its complex, but the complex showed a significantly greater killing effect than the corresponding ligand. For example, Fig. 6A shows the % viability after treatment with (I) (solid square) and (II) (hollow square). Although there was no appreciable difference in % viability produced by (I) and (II) at 5 lM concentration, there was a significant difference in % viability at and above 20 lM concentration, as shown in Fig. 6A. The difference in % viability by (I) and (II) was increased with concentrations above 20 lM and reached a maximum at 50 lM. Only 7% cells were viable after 50 lM of (II) treatment where as 57% cells were viable at the same concentration of (I). This data indicates that the metal complex (II) has a greater cell killing effect than (I) (ligand). Similarly, both (III) and (IV) induced cell death dose-dependently, but complex (IV) shows a greater cell killing effect than its ligand (III). Cell death by (IV) was found to be significantly (p = 0.000) greater than (III) at and above 4 lM (IV) killed about 95% cells at 10 lM whereas (III) killed about 20% at the same concentration, implicating that (IV) was observed to be more effective in inducing cell death than (III). This result is reflected in a cell morphology study, as shown later. Furthermore, LD50 of (II) and (IV) was calculated to be at 28 and 8 lM, respectively from the graph. The reason for the higher cell killing effect of the complexes over their corresponding ligands is not really known. Possibly, bulkier groups (–CH2CH3 for (IV) and –CH3 for (II)) may play a role to potentiate its cytotoxic effect.
Fig. 6. Cell viability. Each square represents the mean ± SD of the% Cell viability obtained from four independent experiments and p values calculated at each concentration of the compound with respect to the untreated control are shown above the respective point. Solid and hollow squares represent the ligand and its complex, respectively.
displayed in Fig. 3c is generated through several intermolecular interactions (Table 5). The amino N(1) atom of the pyrazolyl ring of the free ligand (III) in the molecule at (x, y, z) acts as a donor to the thiolato sulfur atom S(1) at ½ x, y, ½ + z, forming a one-dimensional zigzag chain which propagates along the (0 0 1) direction (Fig. 4b). The packing diagram of the free ligand (III) is shown in Fig. 4c, where the pyrazolyl nitrogen atom plays a role as a donor to the thiolato sulfur and hydrazinic chain nitrogen. In complex (IV), the pyrazolyl N(1B) atom in ligand B in the molecule at (x, y, z) acts as a donor to the O1 atom of the nitrate anion. Another amino N(1A) atom of the pyrazolyl ring in ligand A acts as a donor to the solvent water atom O1W in the molecule at x + 2, y + 1, z + 2. Again, O1W acts as a donor to the oxygen atom O2 of the nitrate anion via H2W1 at x + 2, y + 1, z + 1, forming a one dimensional chain propagating along the (0 0 1) direction (Fig. 5b). Additional reinforcement between these molecules combine to form a dimer with a characteristic R66(24) ring (Fig. 5c). The solvent water O1W acts as a donor to both O3 and O2 atoms of the nitrate anion via H1W1 and H2W1, respectively, forming a water–nitrate cluster through O–H O hydrogen bonding. The amino N(1A) and N(1B) atoms of the pyrazolyl ring act as a donor and
Fig. 7. Cell morphology as photographed under a light microscope (Carl Zeiss) with the appropriate attachment. (a) Untreated control HeLa cells and (b) and (c) are HeLa cells treated with 50 lM of (I) and (II), respectively. (d) and (e) Represent cells treated with 10 lM of (III) and (IV), respectively.
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Fig. 8. Cell cycle distribution of HeLa cells treated with different concentrations of (I) and (II) in a typical experiment after analysis with ModFit LT software. DNA content (PI) staining is shown in the X-axis (FL2-A) in all the figures. A, represents the untreated control HeLa; B, C, D, and E represents HeLa cells treated with 5, 20, 35 and 50 lM of (I), respectively; F, G, H, and I shows HeLa cells treated with same concentrations of (II), respectively, as mentioned for (I).
The cell morphology as seen under a light microscope revealed that cells got damaged with the increase in concentration of the compounds. A typical picture of untreated HeLa cells (Fig. 7A), HeLa treated with 50 lM of (I) and (II) is shown in Fig. 7B and C, respectively. The cells are observed to be healthy (long attached) in Fig. 7A. In Fig. 7B the cells are less damaged (smooth and round shaped) than for Fig. 7C where the cells are short, wrinkled and irregular in shape. Most of the cells were healthy at 10 lM of (III), as shown in Fig. 7D, whereas almost all the cells are dead (nuclear portion that looks darker and a small patch is tending to separate from the cytoplasm that is a lighter big circular patch) at the same concentration of (IV), as shown in Fig. 7E. Therefore, the greater cytotoxicity of the complexes over their ligands was also reflected from our morphological studies. 3.4.2. Detection of nuclear fragmentation or apoptosis Nuclear fragmentation is one of the hallmarks of apoptotic cell death, a special type of cell death [26,27]. We have monitored nuclear fragmentation of HeLa cells after treatment with various doses (0–50 lM) of the synthesized products. We observed only 1–2% apoptotic cells after treatment with the products and this percentage is within the background level. Therefore, this data revealed that although the species (I), (II), (III) and (IV) exhibit a cell-killing effect, they were not capable of inducing apoptosis. This data implicates that the compounds under investigation put stress on the cells resulting in shrinkage and small irregular shapes, but it could not induce apoptosis.
3.4.3. Cell cycle analysis The cell cycle alterations in HeLa cells induced by (I) and (II) are shown in Fig. 8A–I whereas those by (III) and (IV) are shown in Fig. 9A–K, after analysis by MODFIT LT software. The cell cycle phase distribution is graphically shown in Fig. 10A [for (I) and (II)] and Fig. 10B [for (III) and (IV)]. Each bar of a particular pattern represented as average (denoted as ‘avg’ in the figure) values of % G0/G1 cells, % S cells and % G2/M cells with standard deviations after treatment with (I), (II), (III) and (IV). In the figure, ‘L’ designates ligand treatment i.e., (I) and (III), and ‘C’ is for complex treatment i.e., (II) and (IV). The change of cell population at each dose in a particular phase with respect to the untreated control was evaluated to be statistically significant using Dunnett’s test. There was a dose-dependent decrease in % G0/G1 cells and a dose-dependent increase in % G2/M cells after treatment with (II), indicating that this complex might be arresting cells at the M phase and thereby prevent cell cycle progression from the M to G0 phase up to 35 lM concentration, in comparison with the control. On the contrary, there was a significant increase in the S phase at and above 20 lM (I), but there was no appreciable change of cell population in the G0/G1 and G2/M phases with respect to the untreated control. This implies that (I) arrested cells in the S phase so that no cells moved onto the subsequent G2/M or G0/G1 phases. Therefore, (II) was capable of arresting cells at the G2/M phase while (I) can block the cell cycle progression at the S phase. On the other hand, there was consistently an increase of the S-phase population in a dosedependent manner of the ligand-(III) treated cells, implicating that
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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Fig. 9. Cell cycle distribution of HeLa cells treated with different concentration of (III) and (IV) in a typical experiment after analysis with ModFit LT software. DNA content (PI) staining is shown in the X-axis (FL2-H) in all the figures. A, represents untreated control HeLa; B, C, D, E, and F represents HeLa cells treated with 1, 2, 4, 8 and 10 lM of (III), respectively; G, H, I, J, and K shows HeLa cells treated with same concentrations of (IV), respectively, as mentioned for (III).
Fig. 10. Effect of the compounds on the cell cycle distribution in HeLa cells by fluorescence-activated cell sorting (FACS). (A) Cells treated with different concentrations of (I) and (II); (B) cells treated with different concentrations of (III) and (IV).
(III) arrested cells at the S-phase, thereby inhibiting cells to move onto the next G2/M phase. However, there was a dose-dependent increase of the G2/M cell population of complex-(IV) treated cells up to 4 lM concentration, indicating that (IV) might be arresting
cells in the G2/M phase. Above 4 lM of (IV), the majority of the cells were dying, as shown from the cell viability assay, and hence there was no appreciable cell population was found in the G2/M phase at 8 or 10 lM concentrations of (IV).
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Acknowledgement The author, NCS, is grateful to the UGC (Government of India) for financial support. Appendix A. Supplementary data CCDC 760586, 760587, 760584 and 760585 contains the supplementary crystallographic data for compounds (I), (II), (III) and (IV). These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected]. References [1] D.L. Klayman, J.F. Bartosevich, T.S. Griffin, C.J. Mason, J.P. Scovill, J. Med. Chem. 22 (1979) 855. [2] D.X. West, S.B. Padhye, P.B. Sonawane, Struct. Bond. 76 (1991) 1. [3] A.E. Liberta, D.X. West, Biometals 5 (1992) 121. [4] F.A. French, E.J. Blanz Jr., J. Med. Chem. 13 (1970) 1117. [5] T.S. Lobana, R. Sharma, G. Bawa, S. Khanna, Coord. Chem. Rev. 253 (2009) 977. [6] W.E. Antholine, S.H. Smith, J.C. Drach, D.L. Klayman, Antimicrob. Agents Chemother. 19 (1981) 682. [7] A.S. Dobek, D.L. Klayman, E.T. Dickson, J.P. Scovill, E.C. Tramont, Antimicrob. Agents Chemother. 18 (1980) 27. [8] D.X. West, A.E. Liberta, S.B. Padhye, R.C. Chikate, P.B.V. Sonawane, A.S. Kumbhar, R.G. Yerande, Coord. Chem. Rev. 123 (1993) 49. [9] R. Maccari, R. Ottanã, F. Monforte, M.G. Vigorita, Antimicrob. Agents Chemother. 46 (2002) 29. [10] H. Beraldo, D. Gambino, Mini Rev. Med. Chem. 4 (2004) 31. [11] L.A. Saryan, E. Ankel, C. Krishnamurti, W. Antholini, D.H. Petering, Biochem. Pharmacol. 30 (1981) 1595. [12] R. Raina, T.S. Srivastava, Inorg. Chim. Acta 67 (1982) 83. [13] R. Raina, T.S. Srivastava, Ind. J. Chem. 22A (1983) 701. [14] H. Beraldo, L. Tosi, Inorg. Chim. Acta 75 (1983) 249. [15] N.C. Saha, A. Saha, R.J. Butcher, S. Chaudhuri, N. Saha, Inorg. Chim. Acta 339 (2002) 348. [16] N.C. Saha, R.J. Butcher, S. Chaudhuri, N. Saha, Polyhedron 22 (2003) 375. [17] N.C. Saha, R.J. Butcher, S. Chaudhuri, N. Saha, Polyhedron 21 (2002) 779. [18] N.C. Saha, R.J. Butcher, S. Chaudhuri, N. Saha, Polyhedron 24 (2005) 1015. [19] N.C. Saha, N. Saha, S. Chaudhuri, Struct. Chem. 18 (2007) 245.
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Synthesis, structural characterisation and cytotoxicity of new iron(III) complexes with pyrazolyl thiosemicabazones Nitis Chandra Saha a,⇑, Chinmoy Biswas a,c, Atanu Ghorai b, Utpal Ghosh b, Saikat Kumar Seth d,e, Tanusree Kar d a
Department of Chemistry, University of Kalyani, Kalyani 741235, Nadia, West Bengal, India Department of Biochemistry & Biophysics, University of Kalyani, Kalyani 741235, Nadia, West Bengal, India Department of Chemistry, Rishi Bankim Chandra College, Naihati, 743165, 24 PGS (N), West Bengal, India d Department of Materials Science, Indian Association for the Cultivation of Science, Kolkata-700032, India e Department of Physics, M.G. Mahavidyalaya, Bhupatinagar, Midnapore (East) 721425, West Bengal, India b c
a r t i c l e
i n f o
Article history: Received 19 June 2011 Accepted 24 October 2011 Available online 9 November 2011 Keywords: Synthesis Iron(III) Pyrazolylthiosemicarbazone X-ray crystallography Cytotoxicity
a b s t r a c t New iron(III) complexes of 5-methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZNMe2) (I) and 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (III), [Fe(MPZNMe2)2]NO3H2O (II) and [Fe(MPZNEt2)2]NO3H2O (IV), respectively, have been synthesised for the first time and physico-chemically characterised by magnetic measurements (polycrystalline state), electronic and IR spectra. Both are cationic complexes containing two monodeprotonated tridentate ligands with NNS donor sites and an anionic counterpart; the complex species behave as 1:1 electrolytes in MeOH. IR spectra (4000–200 cm 1) indicate coordination to iron(III) centre via the pyrazolyl (tertiary) ring nitrogen, azomethine nitrogen and thiolato sulfur atom. X-ray crystallographic data of (II) (monoclinic, C2/c) triclinic) have authenticated the FeN4S2 octahedral coordination in both complex ions, as and (IV) (P1, envisaged from the spectral data. In both species, the two azomethine nitrogen atoms are trans to each other, while the pyrazolyl ring nitrogen and the thiolato sulfurs atoms are in cis positions. We further observed that both the ligands and their corresponding complexes were capable of killing HeLa cells in culture, but the cytotoxicity of the complexes was found to be greater than their corresponding ligands. Complex (II) does not have any role in cell-cycle progression, but complex (IV) arrests the M-phase to prevent cell-cycle progression. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction A number of heterocyclic thiosemicarbazones and their metal ion complexes have attracted considerable attention in chemistry and biology owing to their potentially beneficial biological activities (antitumoral, antibacterial, antimalarial, antiviral, etc.) over the years and such activities have often been related to their chelation phenomenon with one or more essential trace metal ions [1–10]. It is documented that iron(III) complexes of thiosemicarbazones have been shown to be more active in cell destruction, as well as in the inhibition of DNA synthesis, than the uncomplexed thiosemicarbazones [11]. The success in therapeutic applications of several heterocyclic thiosemicarbazones [3,12–14] for removing excess iron from iron-loaded mice through chelation therapy is also remarkable. These findings have initiated further research on this specific area. In continuation of our earlier reports on iron(III) complexes and related publications [15–20] on metal ion complexes of ⇑ Corresponding author. Fax: +91 33 25828282. E-mail address: [email protected] (N.C. Saha). 0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2011.10.033
pyrazolyl thiosemicarbazones, the present communication intends to report the synthesis, structural characterisation and cytotoxic activities of two new iron(III) complexes with the title ligands. 2. Experimental Hoechst dye, propidium iodide and RNase A were purchased from Sigma Chemicals (USA). Culture media MEM and serum were purchased from HiMedia, India. All other reagents were of AR grade/molecular biology grade and were obtained from commercial sources and used without further purification. Spectrograde solvents were used for spectral and conductance measurements. 2.1. Preparation of HMPZNMe2 (I) and HMPZNEt2 (III) Both the title ligands, 5-methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZNMe2) and 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (Fig. 1) have been synthesized by a method similar to that reported earlier [21], but appropriately modified, followed by the final transamination [22]
2
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
Fig. 1. Schematic structural formulation of the ligands (I) and (III).
each reaction. Aqueous NH3 was added dropwise until the solution was strongly ammoniacal. The resulting precipitate of Fe2O3xH2O was filtered off and washed with H2O until Cl free. The precipitate was then dissolved in a minimum quantity of dilute HNO3 and the resulting solution was added to a hot 1.05 mmol EtOH solution of HMPZNMe2 (0.2215 g) for (II) and HMPZNEt2 (0.251 g) for (IV). The resulting dark brown solution, in each case, was heated to reflux for about 30 min, then the solvent was allowed to evaporate at room temperature. The dark brown solids thus obtained were filtered off, washed with cold ethanol and dried over anhydrous CaCl2. The yield, in each case was 75–80%. Brown black (II) and dark brown (IV) needle shaped crystals were found to be suitable for X-ray diffraction.
of the S-methyldithiocarbazate of 5-methyl-3-formylpyrazole (HMPZSMe) with dimethylamine for HMPZNMe2 and diethylamine for HMPZNEt2. For both the cases, white crystallised products (from ethanol) were obtained in a yield of ca. 60–70%. HMPZNMe2 (M.P. 182–184 °C). Anal. Calc. for C8H13N5S: C, 45.5; H, 6.2; N, 33.2. Found: C, 45.5; H, 6.0; N, 32.7%. 1H NMR d(DMSO-d6): 2.25 (3H, s), 6.24 (1H, s), 8.06 (1H, s), 10.79 (1H, s), 3.26 (6H, s). m/z: 211 (M+, 82%). For HMPzNEt2 (M.P. 160–162 °C). Anal. Calc. for C10H17N5S: C, 50.2; H, 7.1; N, 29.3. Found: C, 50.3; H, 7.1; N, 29.5%. 1H NMR d(CHCl3-d): 2.29 (3H, s), 6.22 (1H, s), 7.39 (1H, s), 10.71 (1H, s), 3.80 (4H, m), 1.28 (6H, t). m/z: 239 (M+, 86%). 2.2. Synthesis of [Fe(MPZNMe2)2]NO3H2O (II) and [Fe(MPZNEt2)2] NO3. H2O (IV)
2.3. Physical measurements
0.17 g, 1.05 mmol anhydrous FeCl3 was dissolved in aqueous EtOH (10 cm3) containing 2 N HCl (0.5 cm3) in a small beaker for
Elemental analyses (C, H and N) were done with a Perkin-Elmer 2400 CHNS/O analyser and the iron content of the complexes
Table 1 Crystal data and structure refinement parameters for C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1(III), and C20H34Fe1N11O4S2(IV). Crystal data
(I)
(II)
(III)
(IV)
Empirical formula Formula weight T (K) k (Å) Crystal system Unit cell dimensions Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) DCalc (mg/m3) Absorption coefficient (mm F(0 0 0) Crystal size (mm) h ranges (°) Limiting indices
C8H13N5S1 211.29 150(2) 0.71073 monoclinic
C32H56Fe2N23O13S4 1210.94 150(2) 0.71073 monoclinic
C10H17N5S1 239.35 150(2) 0.71073 orthorhombic
C20H34Fe1N11O4S2 612.55 150(2) 0.71073 triclinic
P21 11.986(3) 7.3218(18) 12.172(3) 90 100.768(5) 90 1049.4(4) 4, 1.337 0.278 448 0.32 0.26 0.22 1.70 to 25.96 14 6 h 6 14 86k68 14 6 l 6 14 9642 3683 [Rint = 0.0532] 99.2 full-matrix least squares on F2 3173 3683/259 1.105 R1 = 0.0407 wR2 = 0.0913 R1 = 0.0549 wR2 = 0.0981 0.289 and 0.395
C2/c 26.7009(11) 13.4698(4) 17.4483(9) 90 125.542(2) 90 5106.2(4) 4, 1.575 0.813 2516 0.28 0.23 0.18 1.78 to 25.00 31 6 h 6 31 16 6 k 6 15 20 6 l 6 20 23 676 4498 [Rint = 0.0549] 100.0 full-matrix least squares on F2 3619 4498/351 1.074 R1 = 0.0554 wR2 = 0.1623 R1 = 0.0702 wR2 = 0.1742 1.101 and 1.375
Pccn 21.929(3) 9.6397(10) 12.0354(13) 90 90 90 2544.2(5) 8, 1.250 0.238 1024 0.42 0.34 0.22 1.86 to 24.99 26 6 h 6 26, 11 6 k 6 11, 13 6 l 6 14 22 210 2246 [Rint = 0.0251] 100.0 full-matrix least squares on F2 2069 2246/148 1.069 R1 = 0.0313 wR2 = 0.0834 R1 = 0.0338 wR2 = 0.0852 0.259 and 0.234
P1 13.145(2) 13.662(2) 8.531(3) 104.86(3) 95.08(3) 86.56(2) 1473.9(6) 2, 1.380 0.699 642 0.30 0.28 0.24 3.02 to 25.00 15 6 h 6 15 16 6 k 6 15 0 6 l 6 10 5186 5186[Rint = 0.0000] 99.8 full-matrix least squares on F2 2272 5186/349 0.914 R1 = 0.0704 wR2 = 0.1450 R1 = 0.1827 wR2 = 0.1846 0.979 and 0.473
1
)
Reflections collected Unique reflections Completeness to theta (%) Refinement method Observed reflections [I > 2r (I)] Data/parameters Goodness-of-fit (GOF) on F2 Final R indices [I > 2r (I)] R indices (all data) Largest difference peak, hole (e Å
3
)
3
N.C. Saha et al. / Polyhedron 34 (2012) 1–12 Table 2 Selected bond lengths (Å), bond angles and torsion angles (°) of C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1 (III) and C20H34Fe1N11O4S2 (IV). (I)
(II)
(III)
Moiety A
Moiety B
Ligand A
1.360(4) 1.360(3) 1.364(4) 1.292(4) 1.381(3) 1.361(4) 1.351(3) 1.687(3)
1.973(4) 1.927(4) 2.2186(12) 1.358(4) 1.360(3) 1.360(4) 1.286(4) 1.379(3) 1.358(4) 1.352(4) 1.694(3)
1.956(4) 1.925(4) 2.2237(13) 1.354(6) 1.345(5) 1.341(6) 1.292(6) 1.371(5) 1.331(6) 1.340(6) 1.760(5)
Bond angles N2–Fe–N3 N2–Fe–S1 N3–Fe–S1 N1–N2–C4 N2–C4–C5 N3–C5–C4 C5–N3–N4 N3–N4–C6 N5–C6–N4 N4–C6–S1 N5–C6–S1
103.5(2) 122.9(3) 130.5(3) 115.9(2) 120.2(2) 114.7(2) 122.0(2) 123.3(2)
80.53(15) 165.39(11) 85.03(11) 103.4(2) 122.0(3) 130.7(3) 116.0(2) 119.9(2) 114.3(2) 122.4(2) 123.3(2)
80.66(15) 164.90(11) 85.01(11) 106.1(4) 113.9(4) 114.2(4) 118.5(4) 111.6(4) 117.8(4) 123.3(3) 118.9(4)
Torsion angles N1–N2–C4–C5 N2–C4–C5–N3 N4–N3–C5–C4 C6–N4–N3–C5 N3–N4–C6–N5 N3–N4–C6–S1 S1–C6–N5–C7
179.8(3) 1.7(5) 2.3(5) 179.1(3) 177.3(2) 3.3(4) 177.3(2)
180.0(3) 0.9(5) 1.0(5) 175.3(3) 172.5(2) 6.7(4) 176.7(2)
174.9(4) 5.0(6) 176.4(4) 178.8(4) 179.0(4) 1.5(6) 176.7(4)
Bond lengths Fe1–N2 Fe1–N3 Fe1–S1 N1–C2 N2–N1 N2–C4 N3–C5 N4–N3 N4–C6 N5–C6 S1–C6
(IV)
Ligand B
Ligand A
Ligand B
1.352(6) 1.350(5) 1.346(6) 1.300(6) 1.382(5) 1.329(6) 1.344(6) 1.748(5)
1.353(2) 1.346(2) 1.347(2) 1.284(2) 1.370(2) 1.375(2) 1.339(2) 1.683(2)
1.986(6) 1.931(6) 2.233(2) 1.363(1) 1.334(8) 1.334(9) 1.269(9) 1.381(8) 1.319(1) 1.371(1) 1.756(9)
1.962(6) 1.928(6) 2.220(3) 1.344(1) 1.361(8) 1.348(9) 1.300(9) 1.372(8) 1.318(1) 1.379(1) 1.729(9)
106.2(4) 114.1(4) 113.6(4) 118.8(4) 111.5(3) 118.2(4) 123.7(3) 118.1(3)
104.22(11) 122.28(13) 130.64(13) 117.44(12) 119.29(12) 114.63(12) 121.51(11) 123.87(11)
79.9(3) 163.9(2) 84.8(2) 106.6(6) 114.3(7) 114.4(7) 118.5(7) 112.6(7) 115.1(8) 123.1(7) 121.8(7)
80.0(3) 163.9(2) 84.5(2) 105.9(6) 113.3(7) 115.1(7) 119.1(7) 110.9(7) 117.0(8) 124.7(7) 118.3(7)
175.5(4) 1.2(6) 178.8(4) 175.8(4) 176.3(4) 0.6(5) 173.6(4)
177.7(2) 4.1(2) 3.1(2) 165.9(2) 172.0(2) 8.3(2) 169.3(2)
175.9(7) 0.7(1) 177.4(7) 176.4(9) 176.3(8) 1.0(1) 2.2(1)
179.7(6) 0.5(1) 179.9(7) 179.5(8) 178.6(8) 0.7(1) 9.8(1)
Bond angles involving both ligands (A and B) around the Fe atom in complexes(II) and (IV) (II) (IV) N3B–Fe1–N2A N2A–Fe1–S1B N3B–Fe1–S1A N2B–Fe1–N2A
100.55(15) 88.62(11) 93.86(11) 89.34(15)
100.7(3) 91.0(2) 94.7(2) 87.7(3)
N3A–Fe1–N2B N2B–Fe1–S1A N3A–Fe1–S1B S1B–Fe1–S1A
(II)
(IV)
98.71(15) 90.63(11) 95.71(12) 95.08(5)
100.5(3) 90.4(2) 95.1(2) 95.2(1)
Table 3 Analytical data and other pertinent physico-chemical properties of the complexes. Complex (colour)
Elemental analyses Found (calc.)
Conductivity at 30 °C in MeOH (X
%C
%H
%N
%Fe
[Fe(MPzNMe2)2]NO3H2O (Brown black) [Fe(MPzNEt2)2]NO3H2O (Dark brown)
34.2 (34.5) 39.0 (39.2)
5.2 (5.1) 5.1 (5.2)
27.3 (27.7) 25.4 (25.2)
9.9 (10.0) 9.0 (9.1)
was determined titrimetrically. The molar conductance of the complexes in methanol solutions was measured with a Systronics 304 digital conductivity meter. Magnetic susceptibilities were measured in the polycrystalline state on a PAR 155 sample vibrating magnetometer. 1H NMR spectra for the ligands were recorded in DMSO-d6/CDCl3 with a Bruker AM 300L (300 MHz)
Table 4 IR assignments (cm
1
cm2 mol
1
)
leff (BM) at 298 K
110
1.95
106
2.05
superconducting FT NMR spectrometer. Mass spectroscopy of the ligands was done with a Jeol JMS-D300 mass spectrometer. The electronic spectra and the diffuse reflectance spectra were recorded on a Hitachi U-3501 spectrophotometer. IR spectra (4000–200 cm 1) were recorded on a Jasco FT/IR-420 spectrophotometer with KBr pellets.
1
) for the ligands and the complexes pertaining to the coordination sites.
Compound
m(CH@N) (azomethine)
m(C@N) (pyrazole)
m(C@S)
HMPzNMe2 (I) [Fe(MPzNMe2)2]NO3H2O (II) HMPzNEt2 (III) [Fe(MPzNEt2)2]NO3H2O (IV)
1611 1598 1620 1600
1503 1520 1510 1525
864 785 865 780
m(Fe–N) (azomethine)
m(Fe–N) (pyrazole)
m(Fe–S)
482
287
375
478
280
381
4
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
2.4. Structure determination Single crystal X-ray diffraction intensity data of the free ligand 5methyl-3-formylpyrazole-N(4)-dimethylthiosemicarbazone (HMPZ NMe2) (I), which was crystallized with two independent molecules per asymmetric unit, its metal complex [Fe(MPZNMe2)2]NO3H2O (II), which has been represented crystallographically as 2[Fe(MPZNMe2)2](NO3)34H2O, another free ligand 5-methyl-3-formylpyrazole-N(4)-diethylthiosemicarbazone (HMPZNEt2) (III) and its metal complex [Fe(MPZNEt2)2]NO3H2O (IV) were collected at 150(2) K using a Bruker APEX-II CCD diffractometer equipped with graphite monochromated Mo Ka radiation (k = 0.71073 Å). Data reduction was carried out using the program Bruker SAINT [23]. Because of the very small values of the absorption coefficients, no absorption correction was applied. The structures of the compounds were solved by direct methods and refined by the full-matrix least-square technique on F2 with anisotropic thermal parameters to describe the thermal motions of all non-hydrogen atoms using the programs SHELXS 97 and SHELXL 97 [24]. All calculations were carried out using the WinGX system Ver-1.64 [25]. In the complexes delu, simu and isor constraints were applied for a few atoms. In the free ligands (I) and (III), the hydrogen atoms were located from a difference Fourier map and were treated as riding, whereas in the complexes, the hydrogen atoms were located at geometrically calculated positions and were treated by a mixture of independent and constraint refinement. The refinements were based on F2 for all reflections except those with non-reliable F2 values. In compound (II), one of the nitrate anions is disordered over two positions with occupancies of 1:2.5, and the water molecules of crystallization are also disordered, for which the H-atoms could not be located. The crystal of compound (IV) diffracted weakly, less than half of the reflections were considered to be observed, and the atoms in the –CH2CH3 substituents and the nitrate anion all undergo considerable thermal motion. A summary of the crystal data and relevant refinement parameters are given in Table 1. Selected bond-lengths, bond angles and torsion angles of both the ligands and the complexes are given in Table 2. 2.5. Effect of the synthesized product in cultured HeLa cells 2.5.1. Cell killing activity The human cervical cancer cell line HeLa was obtained from the National Centre for Cell Sciences, Pune, India. HeLa cells were grown in MEM supplemented with 10% bovine serum (complete medium) at 37 °C in a humidified atmosphere containing 5% CO2 medium [26]. After inoculation in a fresh medium, the HeLa cells were incubated overnight and then treated with different concentrations of the synthesized products under investigation (0–50 lM) for 13 h. After the treatment, the cells were harvested by trypsinization and suspended in PBS buffer. After washing twice, the cells were incubated with 0.1% tryphan blue for 5 min at room temperature and counted using a hemocytometer under a light microscope. The dead cells appeared to be blue, whereas the viable cells were colourless. Each experiment was repeated four times and the mean of the percentage viable cells at each dose was compared with the mean of the untreated control using one way ANOVA with a post hoc test such as the Tukey’s test and Dunnett’s test. The number of viable cells in the untreated control was considered as 100% and the viable cell number at different doses were normalized accordingly. 2.5.2. Induction of apoptosis as detected by nuclear fragmentation Detection of nuclear fragmentation was done as reported earlier [27]. In summary, after 24 h, the treatment cells were harvested in PBS buffer and fixed with 70% ethanol for 45 min at 4 °C. Then the cells were suspended in PBS buffer, stained with Hoechst (5 lM), incubated for 5 min in the dark, and washed twice with PBS. The
Fig. 2. (a) An ORTEP diagram of the two independent molecules of the free ligand (I) with the atom numbering scheme. Thermal ellipsoids are drawn at the 30% probability level. (b) Packing diagram for (I), viewed down the b-axis. Hydrogen bonds are indicated by dotted lines.
cells were examined by a fluorescence microscope (Carl Zeiss) using the appropriate filter. Apoptotic cells were distinguished by nuclear fragmentation and chromatin condensation. The apoptotic cells were randomly counted and the percentage of the apoptotic cells was calculated at each dose. The mean percentage of apoptotic cells with a standard deviation was calculated from four independent experiments. 2.5.3. Cell cycle analysis After 18 h treatment, the cells were trypsinized, washed twice with cold phosphate buffered saline (PBS), fixed with 70% ethanol in PBS for 2 h at 4 °C, and then stained with a propidium iodide (PI) solution (10 lg/ml) containing DNase-free RNase (100 lg/ml) and 0.1% (v/v) Triton X-100 in the dark for 30 min at room temperature before flow cytometric analysis. The samples were detected with FACS Calibur (BD Bioscience, USA). A minimum of 20,000 cells, analyzed in each sample, served to determine the percentages of cells in each phase of the cell cycle using ModFit LT software [28]. 3. Results and discussion 3.1. Spectroscopic characterisation of HMPZNMe2 (I) and HMPZ NEt2 (III) The title ligands, HMPZNMe2 and HMPZNEt2, have been characterised by elemental analyses (C, H and N), IR, 1H NMR and mass spectra. The characteristic IR bands (cm 1) at 3300–3200 (mNH), 1615–1611 (mCH@N), 1510–1505 (mC@N), 1040–1020 (mN–NPZ) and 867–865 (mC@S) are in good agreement with the structure in Fig. 1(a). Both the ligand molecules have a proton adjacent to the thiocarbonyl group and consequently can exhibit thione–thiol
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
tautomerism. The IR spectra of the free ligands do not show any (mSH) band in the region 2600–2200 cm 1, indicating that the thiol tautomer is absent in the solid state [29,30], but exhibit (mNH) bands in the region 3300–3200 cm 1, indicating the existence of only the thioketo tautomer Fig. 1(a). The 1H NMR spectra of the free ligands in DMSO-d6 give singlets at d 2.48–2.41 (3H), d 6.20– 6.17 (1H) assigned to C5–CH3 and C4–H of the pyrazole rings, respectively. One-proton singlets at d 8.09–7.29, ascribed to CH@N, indicate the existence of hydrogen bonded stabilized forms of the ligands as shown in Fig. 1(b). The 1H NMR spectra also show signal at d 13.40–13.10, indicating the presence of a hydrogen bonded proton [31]. The mass fragmentation data are compatible with the proposed molecular formulae. The strongest molecular ion peak appears at m/z 211 (M+ 58%) for HMPZNMe2 and m/z 239 (M+ 86%) for HMPzNEt2.
3.2. Characterisation of the Fe(III) complexes Both the Fe(III) complexes give satisfactory C, H, N and Fe analyses and conform to the compositions [Fe(MPzNMe2)2]NO3H2O and [Fe(MPzNEt2)2]NO3H2O. The molar conductance values in MeOH (30 °C) classify them as 1:1 electrolytes [32] and the complexes are paramagnetic (at 25 °C), as expected for a low spin d5 ion (Table 3).
5
3.2.1. IR spectra The characteristic IR bands (4000–200 cm 1) for the free ligands, when compared with those of its Fe(III) complexes, provide meaningful information regarding the bonding sites of the primary ligand molecules (Table 4) A negative shift in the m(CH@N) bands (1620–1611 cm 1) in the spectra of the free ligands to a lower value (1600–1598 cm 1) in their complexes are consistent with coordination of the azomethine nitrogen to the central Fe(III) ion. The IR bands at 482-478 cm 1 in the complexes are assignable to m(Fe–N) [32]. Intense bands at 865 cm 1 in the free ligand spectra, assigned to m(C@S), have been found to shift to lower frequency regions (785–780 cm 1) in the complexes, indicating the coordination of the thiol sulfur to Fe(III); moreover, the appearance of new bands in the region (381–375 cm 1) in the complexes are assignable to m(Fe–S) [33]. IR bands at 1510–1503 and 615– 597 cm 1 in the free ligand spectra, assignable to m(C@N) (pyrazole ring) and the in-plane deformation of the pyrazole ring, have been shifted to the higher frequency region, indicating that the tertiary ring nitrogen atom (2N) is a possible bonding site [34–36]. The appearance of bands around 287–280 cm 1 in the complexes are clearly assignable to m(Fe-N) (pyrazole ring) [33]. 3.2.2. Electronic spectra The diffuse reflectance spectral data of the iron(III) complexes appear as three main bands in the regions 19 802–19 760,
Fig. 3. (a) An ORTEP diagram of complex (II) with the atom numbering scheme. (b) One-dimensional chain propagating along the (1 0 0) direction. (c) Packing diagram of (II) displaying the two-dimensional framework.
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16 393–16 050 and 10 830–10 753 cm 1. The absorption bands can be tentatively assigned to a 2T2 ground state for iron(III). The intense bands at ca. 19 802–19 760 cm 1 and ca. 16 393–16 050 cm 1 may be assigned to CT transitions arising from the d ? p⁄ transition. The d–d transition is located at ca. 10,830–10,753 cm 1, which might correspond to a 2T2 ? 2T1 transition [37,38]. The electronic spectra of the complexes in methanol exhibit three bands in the regions 20 800–20 325, 16 313–16 100 and 10 910– 10 858 cm 1. The bands which are found between 20 800 and 16 100 cm 1 are likely due to d ? p⁄ metal to ligand bands as well as a sulfur to Fe(III) transition [39,40]. The bands at ca. 10 910– 10 858 cm 1 can be assigned to d-d transitions of spin paired d5 species with a distorted octahedral structure [41,42]. 3.3. Structural description The molecular views [43] of the free ligand HMPZNMe2 (I), its metal complex 2[Fe(MPZNMe2)2](NO3)34H2O (II), the other free ligand HMPZNEt2 (III) and its metal complex [Fe(MPZNEt2)2]NO3H2O (IV), together with the atom numbering scheme, are shown in Figs. 2a, 3a, 4a and 5a, respectively. The crystallographic asymmetric units in the ligands (I) and (III) comprise of HMPZNMe2 and HMPZNEt2 molecular moieties, respectively, while that in the complexes comprises of a [Fe(MPZNMe2)2]+ cation for (II) and a [Fe(MPZNEt2)2]+ cation for (IV), NO3- counter ions and solvent water of crystallization. In both complexes (II) and (IV), the Fe(III) centre has a distorted octahedral coordination where a pair of mono-deprotonated ligands, (MPZNMe2 ) and (MPZNEt2 ), respectively, coordinate the Fe(III) ion through the thiolato sulfurs (S1A, S1B), pyrazolyl (tertiary) ring nitrogens (N2A, N2B) and the hydrazinic chain nitrogens (N3A,
N3B). Due to this octahedral coordination four five-membered chelate rings [Fe–S(1A)–C(6A)–N(4A)–N(3A), Fe–S(1B)–C(6B)–N(4B)– N(3B), Fe–N(2A)–C(4A)–C(5A)–N(3A), and Fe–N(2B)–C(4B)–C (5B)–N(3B)] were formed. In both the complex species, the two azomethine nitrogen atoms [N(3A) and N(3B)] coordinate the Fe ion in a trans orientation, while the pyrazolyl ring nitrogen atoms [N(2A) and N(2B)] and the thiol sulfur atoms [S(1A) and S(1B)] are in ciscoordination. This coordination is analogous to that observed in the crystal structures of similar metal complexes [15–18]. The bond length and bond angles for the non-hydrogen atoms of the compounds are given in Table 2. The bond lengths of the hydrazinic, pyrazolyl and thiol sulfur atoms to the Fe(III) ion agree well with the corresponding values reported earlier [15,16]. As expected, the S(1)–C(6) double bond distances of 1.687(3) and 1.694(3) Å in moiety A and B, respectively in the free ligand (I) are lengthened to 1.760(5) and 1.748(5) Å in complex (II), with a shortening of the N4–C6 bond from 1.361(4) and 1.358(4) Å to 1.331(6) and 1.329(6) Å, respectively (Table 2). A similar distortion has been observed in (III) and (IV), where the S(1)–C(6) double bond distance of 1.683(2) Å in the free ligand (III) is lengthened to 1.756(9) and 1.729(9) Å in molecules A and B, respectively in complex (IV), with a shortening of the N4–C6 bond from 1.375(2) Å in the free ligand (III) to 1.319(1) [molecule A] and 1.318(1) Å [molecule B] in the Fe(III) complex (IV) (Table 2). These bond distances do not vary significantly in the structures despite the different intermolecular interaction patterns observed in the ligand and the metal complex [18]. The S(1)–C(6), C(6)–N(4) and C(5)–N(3) bond distances in (II) and (IV) show partial double bond character. In (II) and (IV), the partial double bond character of C(4)–C(5) in both molecules (A and B) favour the conjugation of the delocalized p-electrons of the
Fig. 4. (a) An ORTEP diagram of free ligand (III) with the atom numbering scheme. The thermal ellipsoids are drawn at the 30% probability level. (b) One-dimensional zigzag chain propagating along the (0 0 1) direction. (c) Packing diagram of (III) viewed along the (0 0 1) direction.
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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Fig. 5. (a) An ORTEP diagram of complex (IV) with the atom numbering scheme. (b) One-dimensional chain propagating along the (0 0 1) direction. Hydrogen atoms not involved in hydrogen bonding are shown as broken-off bonds. (c) Formation of the R66(24) dimeric ring in complex (IV). (d) Formation of the supramolecular assembly generated through the water-nitrate cluster (colour code: N, blue; O, red; H, white). Hydrogen atoms not involved in hydrogen bonding have been omitted for the sake of clarity. (Colour online)
heterocyclic ring with the thiosemicarbazone skeleton. This conjugation results in near co-planarity of the pyrazolyl ring with the thiosemicarbazone skeleton. The pyrazole ring is therefore restricted with respect to the thiosemicarbazone moiety due to coordination of the pyrazolyl nitrogen atom.
The significant closing of the N(3)–C(5)–C(4) and N(2)–C(4)– C(5) angles in both complexes compared to the corresponding ligands, by about 15–16° and 8–9°, respectively, as indicated by Table 2, may be attributed to geometrical necessity for coordination. The N–Fe1–N, N–Fe1–S and S–Fe1–S angular distributions
Scheme 1. Reaction mechanism showing the rotation about the azomethine double bond.
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N.C. Saha et al. / Polyhedron 34 (2012) 1–12
indicate that the coordination polyhedron is distorted. This distortion in the metal ion coordination polyhedron results from steric interactions, and the maximum distortion from an ideal octahedral geometry occurs for the N(3B)–Fe1–N(2B) [80.66(15)°] and N(3B)– Fe1–N(2A) [100.5(3)°] angles in (II) and those for (IV) are 80.0(3)° and 100.7(3)°, respectively. A similar distortion has been observed in the literature [44]. Some selected torsion angles of the free ligands (I) and (III) and the metal complexes (II) and (IV) are given in Table 2, which may establish the molecular conformation. The ligand molecules HMPZNMe2 (I) and HMPZNEt2 (III), and the tridentate ligands MPZNMe2 and MPZNEt2 in complexes (II) and (IV), respectively are practically planar. In the ligand molecule (I), the C(6A), C(2A), N(4B) and C(6B) atoms have the largest deviations in opposite directions [C(6A): +0.0678(31) Å, C(2A): 0.0497(32) Å; N(4B): +0.0551(28) Å, C(6B): 0.0205(27) Å], whereas in ligand (III), the C(2) and N(4) atoms have the largest deviations in opposite directions [C(2): +0.3819(15) Å, N(4): 0.4693(13) Å] from the least square mean plane through S(1)–C(6)–N(4)–N(3)–C(5)–C(4)–N(2)– N(1)–C(2)–C(3). In complex (II), the C(2A) and N(2A) atoms in ligand A and the N(4B) and C(4B) atoms in ligand B have the largest deviations in opposite directions [C(2A): +0.1508(61) Å, N(2A): 0.0942(47) Å; N(4B): +0.1032(36) Å, C(4B): 0.1074(43) Å] whereas those in (IV) are for N(4A) and N(1A), and C(5B) and N(1B) [N(4A): +0.0582(84) Å, N(1A): 0.1762(68) Å, C(5B): +0.0284(79) Å, N(1B): 0.1038(63) Å] in ligand A and B, respectively from the least square mean planes through Fe1–S(1)–C (6)–N(4)–N(3)–C(5)–C(4)–N(2)–N(1)–C(2)–C(3). The coordinating ligand A and B of both complexes are orthogonal to each other; the dihedral angle between them is 86.52(5)° in (II) and 89.75(6)° in (IV). The three individual rings (of A and B), namely the pyrazolyl and two five membered chelate rings, are individually almost planar, with a small dihedral angle between them. In (II), the maximum dihedral angle of 8.32(18)° is between the pyrazolyl ring C(2A)–C(3A)–C(4A)–N(1A)–N(2A) and the chelate ring Fe1– N(2A)–C(4A)–C(5A)–N(3A) in ligand A, and that of 5.19(7)° is between the two chelate rings Fe1–N(2B)–C(4B)–C(5B)–N(3B) and Fe1–S(1B)–C(6B)–N(4B)–N(3B) in ligand B. In (IV), the maximum dihedral angle of 8.47(21)° is between the pyrazolyl ring C(2A)– C(3A)–C(4A)–N(1A)–N(2A) and the chelate ring Fe1–S(1A)–C(6A)– N(4A)–N(3A) in ligand A, and that of 3.42(16)° is between the two
chelate rings Fe1–N(2B)–C(4B)–C(5B)–N(3B) and Fe1–S(1B)– C(6B)–N(4B)–N(3B) in ligand B. It is known [2,45–49] that many thiosemicarbazone ligands exist in solution as two conformers, E and Z. In the present crystallographic study, the Z-form, as indicated by the C(4)–C(5)–N(3)–N(4) torsion angles of 2.3(5)° and 1.0(5)°, was obtained in the solid state for the free ligand (I). In complex (II), the coordinating ligand exists as E conformers [the C(4)–C(5)–N(3)–N(4) torsion angle is 176.4(4)° in ligand A and 178.8(4)° in ligand B], as necessitated by the requirements of coordination. A similar observation was observed for (III) and (IV), where the C(4)–C(5)–N(3)–N(4) torsion angle in (III) is 3.1(2)° whereas those in (IV) are 177.4(7)° and 179.9(7)°. Therefore, the Z-conformers of the ligand, which could be present in solution, must be transformed to the E-form before complexation. A possible mechanism for this transformation, leading to an unusual rotation about the azomethine (C@N) double bond, is shown in Scheme 1. The feasibility of this mechanism is due to the electropositive character of the azomethine carbon, C(5), as reported earlier [50,51]. The intermediate conversion from a double bond to a single bond allows the rotation of the thiosemicarbazone moiety to bring the sulfur atom, S(1), to the cis position with respect to the pyrazolyl nitrogen, N(2), in order to permit complex formation. The crystal structures adopted by (I) and (II) are quite different. The structures of the ligand and the complex includes a combination of N–H S, N–H N, N–H O and O–H O hydrogen bonding interactions (Table 5). It is convenient to consider the substructures generated by each type of hydrogen bonds as acting individually, and then the combination of substructures build a three dimensional framework. In ligand (I), the pyrazole ring nitrogen N(1A) acts as a donor to the hydrazinic chain nitrogen N(3B) in the molecules at (x, y, z) and (1 x, ½ y, 1 z). Another pyrazole nitrogen N(1B) of moiety B acts as a donor to N(3A) at (x, y, z) and (1 x, 1/2 + y, z). The thiolato sulfur atom of both the moieties acts as an acceptor of N(1B) and C(1A) in the molecule at (1 x, 1/2 + y, z) and (1 x, ½ + y,1 z), respectively. The packing view of (I) is displayed in Fig. 2b. In complex (II), the pyrazole ring nitrogen of both moieties act as a donor to the oxygen atom of a nitrate anion, thus forming a 1D chain which propagates along the (1 0 0) direction (Fig. 3b). The packing view of the title complex
Table 5 Hydrogen bonding geometry of C8H13N5S1 (I), C32H56Fe2N23O13S4 (II), C10H17N5S1 (III) and C20H34Fe1N11O4S2 (IV) (Å, °). D–H A
d(D–H)
d(H A)
d(D A)
D–H A
Symmetry
Compound (I) N1A–H1A N3B N1B–H1B S1A N1B–H1B N3A C7B–H7B3 N3A C1A–H1A3 S1B
0.86 0.86 0.86 0.96 0.96
2.20 2.72 2.44 2.57 2.83
2.996(3) 3.363(3) 3.214(3) 3.480(4) 3.637(4)
153 133 150 157 142
1 1 1 x, 1
Compound (II) N1A–H1A O3 N1B–H1B O4 O1W–H1W1 O2 O1W–H1W1 O3 O1W–H2W1 N4B C1A–H1A1 O1 C7B–H7B2 O2
0.86 0.86 0.87 0.87 0.86 0.96 0.96
1.86 1.96 1.85 2.15 2.17 2.47 2.45
2.710(9) 2.797(7) 2.627(10) 2.927(11) 2.928(6) 3.388(11) 3.319(11)
168 164 148 148 148 159 150
x, 1
½ x, ½ + y, ½ z ½ x, ½ + y, ½ z ½ x, ½ y, 1 z x, 1 y, ½ + z 1 x, y, ½ z
Compound (III) N1–H1 S1 N1–H1 N3
0.86 0.86
2.79 2.13
3.448(1) 2.910(2)
135 151
½ ½
x, y, x, y,
½+z ½+z
Compound (IV) N1A–H1A O1W N1B–H1B O1 O1W–H1W1 O3 O1W–H2W1 O2
0.86 0.86 0.82 0.83
1.95 2.03 2.15 1.99
2.803(9) 2.862(10) 2.966(11) 2.815(12)
170 164 178 168
2
x, 1
y, 2
z
2
x, 1
y, 1
z
x, ½ + y, 1 z x, ½ + y, z x, ½ + y, z 1 + y, z x, ½ + y, 1 z y, ½ + z
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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are bonded to O1 and O1W, respectively, and this generates a supramolecular synthon which is formed through a hydrogen bonded water–nitrate cluster (Fig. 5d).
3.4. Cytotoxicity 3.4.1. Comparison of the cell killing effect between the ligands and their corresponding complexes The percentage of viable HeLa cells was decreased dose-dependently with the increase in concentration of the ligand or its complex, but the complex showed a significantly greater killing effect than the corresponding ligand. For example, Fig. 6A shows the % viability after treatment with (I) (solid square) and (II) (hollow square). Although there was no appreciable difference in % viability produced by (I) and (II) at 5 lM concentration, there was a significant difference in % viability at and above 20 lM concentration, as shown in Fig. 6A. The difference in % viability by (I) and (II) was increased with concentrations above 20 lM and reached a maximum at 50 lM. Only 7% cells were viable after 50 lM of (II) treatment where as 57% cells were viable at the same concentration of (I). This data indicates that the metal complex (II) has a greater cell killing effect than (I) (ligand). Similarly, both (III) and (IV) induced cell death dose-dependently, but complex (IV) shows a greater cell killing effect than its ligand (III). Cell death by (IV) was found to be significantly (p = 0.000) greater than (III) at and above 4 lM (IV) killed about 95% cells at 10 lM whereas (III) killed about 20% at the same concentration, implicating that (IV) was observed to be more effective in inducing cell death than (III). This result is reflected in a cell morphology study, as shown later. Furthermore, LD50 of (II) and (IV) was calculated to be at 28 and 8 lM, respectively from the graph. The reason for the higher cell killing effect of the complexes over their corresponding ligands is not really known. Possibly, bulkier groups (–CH2CH3 for (IV) and –CH3 for (II)) may play a role to potentiate its cytotoxic effect.
Fig. 6. Cell viability. Each square represents the mean ± SD of the% Cell viability obtained from four independent experiments and p values calculated at each concentration of the compound with respect to the untreated control are shown above the respective point. Solid and hollow squares represent the ligand and its complex, respectively.
displayed in Fig. 3c is generated through several intermolecular interactions (Table 5). The amino N(1) atom of the pyrazolyl ring of the free ligand (III) in the molecule at (x, y, z) acts as a donor to the thiolato sulfur atom S(1) at ½ x, y, ½ + z, forming a one-dimensional zigzag chain which propagates along the (0 0 1) direction (Fig. 4b). The packing diagram of the free ligand (III) is shown in Fig. 4c, where the pyrazolyl nitrogen atom plays a role as a donor to the thiolato sulfur and hydrazinic chain nitrogen. In complex (IV), the pyrazolyl N(1B) atom in ligand B in the molecule at (x, y, z) acts as a donor to the O1 atom of the nitrate anion. Another amino N(1A) atom of the pyrazolyl ring in ligand A acts as a donor to the solvent water atom O1W in the molecule at x + 2, y + 1, z + 2. Again, O1W acts as a donor to the oxygen atom O2 of the nitrate anion via H2W1 at x + 2, y + 1, z + 1, forming a one dimensional chain propagating along the (0 0 1) direction (Fig. 5b). Additional reinforcement between these molecules combine to form a dimer with a characteristic R66(24) ring (Fig. 5c). The solvent water O1W acts as a donor to both O3 and O2 atoms of the nitrate anion via H1W1 and H2W1, respectively, forming a water–nitrate cluster through O–H O hydrogen bonding. The amino N(1A) and N(1B) atoms of the pyrazolyl ring act as a donor and
Fig. 7. Cell morphology as photographed under a light microscope (Carl Zeiss) with the appropriate attachment. (a) Untreated control HeLa cells and (b) and (c) are HeLa cells treated with 50 lM of (I) and (II), respectively. (d) and (e) Represent cells treated with 10 lM of (III) and (IV), respectively.
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Fig. 8. Cell cycle distribution of HeLa cells treated with different concentrations of (I) and (II) in a typical experiment after analysis with ModFit LT software. DNA content (PI) staining is shown in the X-axis (FL2-A) in all the figures. A, represents the untreated control HeLa; B, C, D, and E represents HeLa cells treated with 5, 20, 35 and 50 lM of (I), respectively; F, G, H, and I shows HeLa cells treated with same concentrations of (II), respectively, as mentioned for (I).
The cell morphology as seen under a light microscope revealed that cells got damaged with the increase in concentration of the compounds. A typical picture of untreated HeLa cells (Fig. 7A), HeLa treated with 50 lM of (I) and (II) is shown in Fig. 7B and C, respectively. The cells are observed to be healthy (long attached) in Fig. 7A. In Fig. 7B the cells are less damaged (smooth and round shaped) than for Fig. 7C where the cells are short, wrinkled and irregular in shape. Most of the cells were healthy at 10 lM of (III), as shown in Fig. 7D, whereas almost all the cells are dead (nuclear portion that looks darker and a small patch is tending to separate from the cytoplasm that is a lighter big circular patch) at the same concentration of (IV), as shown in Fig. 7E. Therefore, the greater cytotoxicity of the complexes over their ligands was also reflected from our morphological studies. 3.4.2. Detection of nuclear fragmentation or apoptosis Nuclear fragmentation is one of the hallmarks of apoptotic cell death, a special type of cell death [26,27]. We have monitored nuclear fragmentation of HeLa cells after treatment with various doses (0–50 lM) of the synthesized products. We observed only 1–2% apoptotic cells after treatment with the products and this percentage is within the background level. Therefore, this data revealed that although the species (I), (II), (III) and (IV) exhibit a cell-killing effect, they were not capable of inducing apoptosis. This data implicates that the compounds under investigation put stress on the cells resulting in shrinkage and small irregular shapes, but it could not induce apoptosis.
3.4.3. Cell cycle analysis The cell cycle alterations in HeLa cells induced by (I) and (II) are shown in Fig. 8A–I whereas those by (III) and (IV) are shown in Fig. 9A–K, after analysis by MODFIT LT software. The cell cycle phase distribution is graphically shown in Fig. 10A [for (I) and (II)] and Fig. 10B [for (III) and (IV)]. Each bar of a particular pattern represented as average (denoted as ‘avg’ in the figure) values of % G0/G1 cells, % S cells and % G2/M cells with standard deviations after treatment with (I), (II), (III) and (IV). In the figure, ‘L’ designates ligand treatment i.e., (I) and (III), and ‘C’ is for complex treatment i.e., (II) and (IV). The change of cell population at each dose in a particular phase with respect to the untreated control was evaluated to be statistically significant using Dunnett’s test. There was a dose-dependent decrease in % G0/G1 cells and a dose-dependent increase in % G2/M cells after treatment with (II), indicating that this complex might be arresting cells at the M phase and thereby prevent cell cycle progression from the M to G0 phase up to 35 lM concentration, in comparison with the control. On the contrary, there was a significant increase in the S phase at and above 20 lM (I), but there was no appreciable change of cell population in the G0/G1 and G2/M phases with respect to the untreated control. This implies that (I) arrested cells in the S phase so that no cells moved onto the subsequent G2/M or G0/G1 phases. Therefore, (II) was capable of arresting cells at the G2/M phase while (I) can block the cell cycle progression at the S phase. On the other hand, there was consistently an increase of the S-phase population in a dosedependent manner of the ligand-(III) treated cells, implicating that
N.C. Saha et al. / Polyhedron 34 (2012) 1–12
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Fig. 9. Cell cycle distribution of HeLa cells treated with different concentration of (III) and (IV) in a typical experiment after analysis with ModFit LT software. DNA content (PI) staining is shown in the X-axis (FL2-H) in all the figures. A, represents untreated control HeLa; B, C, D, E, and F represents HeLa cells treated with 1, 2, 4, 8 and 10 lM of (III), respectively; G, H, I, J, and K shows HeLa cells treated with same concentrations of (IV), respectively, as mentioned for (III).
Fig. 10. Effect of the compounds on the cell cycle distribution in HeLa cells by fluorescence-activated cell sorting (FACS). (A) Cells treated with different concentrations of (I) and (II); (B) cells treated with different concentrations of (III) and (IV).
(III) arrested cells at the S-phase, thereby inhibiting cells to move onto the next G2/M phase. However, there was a dose-dependent increase of the G2/M cell population of complex-(IV) treated cells up to 4 lM concentration, indicating that (IV) might be arresting
cells in the G2/M phase. Above 4 lM of (IV), the majority of the cells were dying, as shown from the cell viability assay, and hence there was no appreciable cell population was found in the G2/M phase at 8 or 10 lM concentrations of (IV).
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