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Synthesis, structure, spectroscopic properties and biological activity of mixed diorganotin(IV) complexes containing pyridine-2-carbaldehyde thiosemicarbazonato and diphenyldithiophosphinato ligands
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 76 (1999) 277–284
Synthesis, structure, spectroscopic properties and biological activity of mixed diorganotin(IV) complexes containing pyridine-2-carbaldehyde thiosemicarbazonato and diphenyldithiophosphinato ligands a b a ˜ ´ C. Rodrıguez-Arguelles ´ ´ Sanchez ´ ¨ Jose´ S. Casas a,*, Alfonso Castineiras , Marıa , Agustın , a ´ ´ Lopez , Silvana Pinelli c, Paolo Lunghi c, Paolo Ciancianaini c, Jose´ Sordo a, Antonia Vazquez Antonio Bonati c, Pierpaolo Dall’Aglio c, Roberto Albertini c a
´ ´ Departamento de Quımica Inorganica, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain b ´ ´ Departamento de Quımica Inorganica, Universidade de Vigo, 36200 Vigo, Spain c Istituto di Patologia Speciale Medica and Centro di Ricerca Interuniversitario per la diagnosi, terapia e prognosi dei tumori humani, Universita` di Parma, 43100 Parma, Italy Received 3 June 1999; received in revised form 17 August 1999; accepted 7 September 1999
Abstract Reaction of the title ligands (HPyTSC and HS(S)PPh2, respectively) with R2SnO (RsMe, Et, Bu) in ethanol (EtOH) afforded the complexes [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnR2(PyTSC)(S2PPh2)] (RsEt (2), Bu (3)). The structures of 1 and 2 were determined by single-crystal X-ray diffractometry. In both these complexes the tin atom is coordinated to an N,N,S-dentatethiosemicarbazonate ligand, an anisobidentate dithiophosphinato ligand and the two R groups. The coordination polyhedrons can be described as distorted pentagonal bipyramids. A comparative study of the IR spectra of 1, 2 and 3 indicates that the butyl complex has a similar structure. Multinuclear (1H, 13 C, 31P and 119Sn) NMR data suggest that the structures of 1 and 2 probably remain in CDCl3 (or DMSO-d6) solution but compound 3 partially decomposes in these media. Preliminary results on the effects of the complexes on the proliferation and differentiation of FLC, CEM, U937, K562 and TOM-1 leukaemia cells, and on the clonogenic activity of K562 cells are also described. q1999 Elsevier Science Inc. All rights reserved. Keywords: Diorganotin(IV) complexes; Pyridine-2-carbaldehyde thiosemicarbazonato ligands; Diphenyldithiophosphinato ligands
1. Introduction The possibility of synergic coupling of pyridine-2-carbaldehyde thiosemicarbazone (HPyTSC) with diorganotin(IV) compounds to obtain a product with greater antiproliferative activity than either [1,2] was explored in previous work on the mixed complex [SnMe2(PyTSC)(OAc)]PHOAc, which inhibited proliferation of Friend erythroleukaemia cells (FLC) by about 80% at moderate dosages without suppressing DMSO-induced cell differentiation [3]. It was hypothesized that hydrolysis of the complex after crossing the cell membrane removed the acetate coligand, leaving [SnMe2(PyTSC)]q (aq), a species capable of reaching the biological target. Suspecting that the high affinity of AcOy for the organotin cation might have limited hydrolysis of the above complex, * Corresponding author. Fax: q34-81-594912; e-mail: qiscasas@ uscmail.usc.es
and hence its efficacy, we have now prepared complexes [SnR2(PyTSC)(S2PPh2)] (Rsmethyl, ethyl and butyl), reasoning that the dithiophosphinato ligand must be replaced with water more easily than AcOy due to the conflict between its ‘soft’ character and the ‘borderline’ behaviour of the R2Sn2q cation [4] and to the greater steric demands of the disulphur ligand. We describe here the synthesis and structural characterisation of these new complexes, and report preliminary results on their effects on the proliferation and differentiation of FLC, CEM, U937, K562 and TOM-1 leukaemia cells [5–8].
2. Experimental Thiosemicarbazide (Merck), pyridine-2-carbaldehyde (Merck), dimethyltindichloride (Alfa), diethyltindichloride (Aldrich), dibutyltinoxide (Aldrich) and diphenyldithio-
0162-0134/99/$ - see front matter q1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 8 5 - 3
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phosphinic acid (Aldrich) were used as supplied. The diorganotin(IV) oxides and the thiosemicarbazone ligand were obtained by the procedures described in Refs. [9,10], respectively. HPyTSC 1H NMR data, d (ppm): in DMSO-d6 8.37 (s, br, 1H) [N(1)H2], 8.10 (s, br, 1H) [N(1)H2], 11.56 (s, br, 1H) [N(2)H], 8.07 (s, 1H) [C(2)H], 8.26 (d, 1H) [C(4)H], 7.79 (t, 1H) [C(5)H], 7.34 (dd, 1H) [C(6)H], 8.33 (d, 1H) [C(7)H]; in CDCl3 6.35 (s, vbr, 1H) [N(1)H2], 9.09 (s, br, 1H) [N(2)H], 7.87 (s, 1H) [C(2)H], 7.85 (d, 1H) [C(4)H], 7.76 (td, 1H) [C(5)H], 7.33 (dd, 1H) [C(6)H], 8.65 (d, 1H) [C(7)H]. 2.1. Preparation of the complexes 2.1.1. [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) A solution of HPyTSC (0.22 g, 1.2 mmol) in ethanol (35 ml) was added to a suspension of SnMe2O (0.20 g, 1.2 mmol) and HS(S)PPh2 (0.30 g, 1.2 mmol) in 55 ml of the same solvent.The mixture was stirred for 7 days at room temperature, and a yellow–green crystalline solid was filtered out. M.p. 1608C. Anal. Found: C, 44.1; H, 4.8; N, 9.1. Calc. for C23H29N4POS3Sn: C, 44.3; H, 4.7; N, 9.0%. IR spectrum (cmy1): 3457w, 3287s, 3160s, n(N–H); 1593m, n(C_N); 796m, n(C_S); 646m, nas(P–S); 557s, nsym(P–S); 557s, nas(Sn–C); 515w, nsym(Sn–C). 1H NMR data, d (ppm): in DMSO-d6 7.92 (s, br, 2H) [N(1)H2], 8.75 (s, 1H) [C(2)H], 7.90 (overlapped) [C(4)H], 8.13 (td, 1H) [C(5)H], 7.67 (dd, 1H) [C(6)H], 8.97 (d, 1H) [C(7)H], 7.94 (m, 4H) [PPh2], 7.24 (m, 6H) [PPh2], 0.96 (s, 6H, 2 Js96/92 Hz) [Sn–R], 1.05 (t,3H) [EtOH], 3.44 (q, 2H) [EtOH], 4.34 (t, 1H) [EtOH]; in CDCl3 5.44 (s, br, 2H) [N(1)H2], 8.33 (s, 1H) [C(2)H], 7.47 (d, 1H) [C(4)H], 7.86 (td, 1H) [C(5)H], 7.43 (m, 1H) [C(6)H], 9.25 (d, 1H) [C(7)H], 8.15 (m, 4H) [PPh2], 7.37 (m, 6H) [PPh2], 1.42 (s, 6H, 2Js101/98 Hz) [Sn–R], 1.25 (t, 3H) [EtOH], 3.72 (q, 2H) [EtOH]. 2.1.2. [SnEt2(PyTSC)(S2PPh2)] (2) To a solution of HPyTSC (0.22 g, 1.2 mmol) in 25 ml of absolute ethanol was added a suspension of HS(S)PPh2 (0.30 g, 1.2 mmol) and SnEt2O (0.24 g, 1.2 mmol) in the same solvent (60 ml). After stirring for 5 days, the yellow solid formed was filtered out and vacuum dried. M.p. 1888C. Anal. Found: C, 45.2; H, 4.4; N, 8.9. Calc. for C23H27N4PS3Sn: C, 44.5; H, 4.4; N, 9.0%. IR spectrum (cmy1): 3397s, 3276s, 3241s, n(N–H); 1599m, n(C_N); 783w, n(C_S); 636m, nas(P–S); 560m, nsym(P–S); 530m, nas(Sn–C); 490m, nsym(Sn–C). 1H NMR data, d (ppm): in DMSO-d6 7.82 (s, br, 2H) [N(1)H2], 8.75 (s, 1H) [C(2)H], 7.89 (d, 1H) [C(4)H], 8.15 (td, 1H) [C(5)H], 7.68 (m,1H) [C(6)H], 9.01 (d, 1H) [C(7)H], 7.97 (m, 4H) [PPh2], 7.29 (m, 6H) [PPh2], 1.68 (m, 2H) [Sn–R], 1.57 (m, 2H) [Sn–R], 0.93 (t, 6H, 3Js166 Hz) [Sn–R]; in CDCl3 5.43 (s, br, 2H) [N(1)H2], 8.36 (s, 1H) [C(2)H], 7.47 (d, 1H) [C(4)H], 7.87 (td, 1H) [C(5)H], 7.49 (m, overlapped) [C(6)H], 9.22 (d, 1H) [C(7)H], 8.05 (m,
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4H) [PPh2], 7.49 (m, overlapped) [PPh2], 2.15 (m, 2H) [Sn–R], 1.86 (m, 2H) [Sn–R], 1.13 (t, 6H, 3Js175/168 Hz) [Sn–R]. 2.1.3. [SnBu2(PyTSC)(S2PPh2)] (3) A solution of HPyTSC (0.18 g, 1.0 mmol) in 25 ml of ethanol was added to a stirred suspension of SnBu2O (0.25 g, 1.0 mmol) and HS(S)PPh2 (0.25 g, 1.0 mmol) in 50 ml of the same solvent. The mixture was stirred for 3 days, and a yellow solid was filtered out. M.p. 1078C(d). Anal. Found: C, 47.9; H, 5.1; N, 8.0. Calc. for C27H35N4PS3Sn: C, 49.0; H, 5.3; N, 8.5%. IR spectrum (cmy1): 3424m, 3285m, 3145m, n(N–H); 1595m, n(C_N); 775w, n(C_S); 650m, nas(P– S); 550m, nsym(P–S); 615w, nas(Sn–C). 1H NMR data, d (ppm): in CDCl3 5.37 (s, br, 2H) [N(1)H2], 8.85 (s, 1H) [C(2)H], 7.50 (d, 1H) [C(4)H], 7.89 (td, 1H) [C(5)H], 7.43 (t, 1H) [C(6)H], 9.22 (d, 1H) [C(7)H], 8.05 (m, 4H) [PPh2], 7.35 (m) [PPh2], 2.12 (t, 4H) [Sn–R], 1,68 (q, 4H) [Sn–R], 0.99 (sx, 4H) [Sn–R], 0.62 (t, 6H) [Sn– R]. 2.2. Physical measurements Elemental analyses were performed with a Carlo-Erba ¨ 1108 analyser. Melting points were determined with a Buchi apparatus. IR spectra were recorded in KBr pellets or Nujol mulls on a Bruker IFS-66V spectrometer. 1H, 13C, 31P and 119 Sn NMR spectra were recorded at room temperature on Bruker WM250, AMX 300 or AMX 500 instruments and were referred to TMS (1H, 13C), external 85% H3PO4 (31P) and external neat Sn(CH3)4 (119Sn). 2.3. X-ray crystallography Crystals of 1 and 2 suitable for X-ray diffraction were mounted on a glass fibre and transferred to an Enraf-Nonius CAD4 diffractometer. Accurate unit cell parameters and an orientation matrix were determined by least-squares from the setting angles of a set of well-centred reflections (SET4 [11]) in the range 5.30–13.328 (1) or 8.09–11.608 (2). Reduced cell calculations did not indicate higher lattice symmetry [12]. Crystal data and details of data collection and refinement are given in Table 1. Data were corrected for Lp effects and for the observed linear decay of ten reference reflections. An empirical absorption correction (DIFABS [13]) was applied for both compounds. The cell constants for 1 suggested the space group to be either orthorhombic or monoclinic. The structures were solved for the monoclinic space group P21/c (No. 14) by automated Patterson (1) or direct methods (2) and subsequent difference Fourier techniques (SHELXS86 [14]), and successful refinement (confirming the space group) was performed on F (1; SDP/VAX [15]) or F2 (2; SHELXL97 [16]) by a full-matrix leastsquares procedure using anisotropic displacement parameters. Hydrogen atoms were included in the refinement in calculated positions riding on their carrier atoms. Neutral
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Table 1 Crystallographic data of [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnEt2(PyTSC)(S2PPh2)] (2)
2.4. Biological studies
Compound
1
2
Empirical formula Formula weight ˚ Wavelength (A) Crystal size (mm) Crystal shape Crystal system Space group ˚ a (A) ˚ b (A) ˚ c (A) a (8) b (8) g (8) ˚ 3) V (A Z, Dcalc (Mg/m3) F(000) u Range (8) Temperature (K) hmin/hmax kmin/kmax lmin/lmax m (mmy1) Max./min. transmissions Refl. collected/unique Data/parameters Final R Final wR2 GOOF Max./min. Dr (e/A3)
C23H29N4OPS3Sn 623.34 0.71073 0.60=0.30=0.10 plate monoclinic P21/c (No. 14) 20.628(8) 9.727(3) 13.477(3) 90.00(–) 90.08(2) 90.00(–) 2704(1) 4, 1.531 1264 3.01–27.03 213 y1/12 0/26 y17/17 1.259 0.885/0.519 6994/6188 4832/303 0.045 0.047 1.144 0.117/0.042
C23H27N4PS3Sn 605.35 0.71073 0.80=0.40=0.20 prism monoclinic P21/c (No. 14) 12.722(4) 14.963(3) 14.198(4) 90.00(–) 93.01(2) 90.00(–) 2698(1) 4, 1.490 1224 3.08–29.98 293 0/17 0/21 y19/19 1.257 0.787/0.433 8137/7824 7824/291 0.088 0.125 0.965 0.699/y1.591
Stock solutions of HPyTSC in dimethyl sulphoxide were prepared and stored at room temperature. Cells (K562, U937, CEM, FLC and TOM-1) were grown at 378C under 5% CO2 in RPMI-1640 supplemented with 100 mg/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine (Gibco/BRL) and 5% (FLC), 10% (K562, U937 and CEM cells) or 20% (TOM-1) of fetal calf serum. Cells were seeded at 2=105/ml in the presence of the above-mentioned compounds added to culture medium prior to cell seeding. Cell mortality evaluated by the Trypan Blue dye exclusion method was 5% or less in all experiments. The degree of differentiation was determined as the percentage of benzidine-positive (Bq) cells [20].
atom scattering factors and anomalous dispersion corrections were taken from the International Tables for X-ray Crystallography [17]. Geometrical calculations were performed and illustrations obtained with the SHELXL97 [16], ZORTEP [18] and PLATON98 [19] packages.
2.4.1. Clonogenic assays K562 cells were plated in 35 mm dishes (Falcon, Becton Dickinson) in 1 ml a-MEM containing 2.3% methylcellulose, 30% FCS, 2% L-glutamine 200 mM and 8% Iscove’s modified Dulbecco’s medium (IMDM, Sigma-Aldrich) at a concentration of 500 cells/dish and incubated at 378C in 5% CO2 humidified atmosphere. The number of colonies CFUAML (colony forming unit of acute myeloid leukaemia), formed after treating the cells with low doses of the compounds for 16 h, was determined at day 14 using light microscopy.
2.4.2. Morphological assessment For light microscopy, cytospin smears were prepared using a Cytospin Centrifuge (Shandon, Cytospin 2) and stained with May–Grunwald–Giemsa stain.
Fig. 1. An ORTEP plot showing the molecular structure of [SnEt2(PyTSC)(S2PPh2)] (2) and the numbering scheme.
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J.S. Casas et al. / Journal of Inorganic Biochemistry 76 (1999) 277–284 Table 3 ˚ and angles (8) in [SnEt2(PyTSC)(S2PPh2)] Bond lengths (A)
3. Results and discussion 3.1. Crystal structures of [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnEt2(PyTSC)(S2PPh2)] (2) The molecular structure of 2 is shown in Fig. 1 together with numbering scheme. Complex 1 differs only slightly in structure (see selected distances and angles in Tables 2 and 3). In both complexes the metal atom is coordinated to a tridentate thiosemicarbazonato ligand, an anisobidentate dithiophosphinato ligand and the two organic groups. The TSC ligand is N,N,S-bound, as in [SnMe2(PyTSC)(OAc)]PHOAc [3], but the Sn–S and Sn–N(Py) bond lengths are longer in the dithiophosphinato complexes, probably due to the greater steric demands created by replacement of the two oxygens by two sulfurs in the equatorial plane. Curiously, the Sn–N3 bond length is equal to (in 2) or shorter than (in 1) its value in the OAcy complex, suggesting that this bond is more stable than the Sn–S and Sn–N(Py) bonds. Both the Sn–S (dithiophosphinate) bonds are longer than the Sn–S (TSC) bond suggesting that, as expected, the former ligand is the more weakly bound. Nevertheless, all Sn– S distances are less than the sum of the van der Waals radii ˚ [21], though the longest (3.403(1) in 1 and (4.0 A) 3.302(3) in 2) probably indicates a secondary bond. The coordination number of the tin atom can thus be considered as 6q1. The apical Sn–C bonds are slightly longer than in the acetate complex [3], and the C–Sn–C angles slightly narrower (154.1 and 155.88 as against 156.08), bending away from the TSC bite and toward the empty equatorial area between N(4) and S(3). The equatorial kernel (Sn, N(3), N(4), S(1), S(2), S(3)) is virtually planar in 2, the rms deviation from the least-squares plane being 0.0702 and the ˚ (N(4)). The distortion is greater maximum deviation 0.09 A ˚ from the best fitting plane. in 1, in which S(1) lies 0.12 A Table 2 ˚ and angles (8) in [SnMe2(PyTSC)(S2PPh2)]PEtOH a Bond lengths (A) Sn–S(1) Sn–S(2) Sn–S(3) Sn–N(3) Sn–N(4) Sn–C(8) N(1)–H(1)∆O(1)i
2.519(1) 2.715(1) 3.403(1) 2.364(4) 2.578(4) 2.131(7) 2.8731
Sn–C(9) S(2)–P S(3)–P P–C(10) P–C(16)
2.112(6) 2.015(2) 1.963(2) 1.815(7) 1.808(6)
O(1)–H(101)∆N(2)ii
2.8731
S(1)–Sn–S(2) S(1)–Sn–N(3) S(1)–Sn–N(4) S(1)–Sn–C(8) S(1)–Sn–C(9) S(2)–Sn–N(3) S(2)–Sn–N(4) S(2)–Sn–C(8) S(2)–Sn–C(9) N(3)–Sn–N(4) N(3)–Sn–C(8)
75.39(4) 73.6(2) 139.5(2) 99.4(2) 106.3(2) 148.6(2) 145.0(2) 92.9(2) 90.9(2) 66.4(1) 97.0(3)
N(4)–Sn–C(8) N(3)–Sn–C(9) N(4)–Sn–C(9) S(2)–P–S(3) S(2)–P–C(10) S(2)–P–C(16) S(3)–P–C(10) S(3)–P–C(16) C(8)–Sn–C(9) C(10)–P–C(16)
80.1(2) 93.1(2) 82.3(2) 116.0(1) 107.5(2) 107.4(3) 110.6(2) 111.1(2) 154.1(2) 103.4(3)
a
isyx, y1/2qy, 1/2yz; iisyx, 1yy, yz.
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Sn–S(1) Sn–S(2) Sn–S(3) Sn–N(3) Sn–N(4) Sn–C(8)
2.548(3) 2.722(3) 3.302(3) 2.438(8) 2.717(8) 2.122(10)
Sn–C(10) P–C(12) P–C(18) S(3)–P S(2)–P
2.135(9) 1.811(11) 1.809(11) 1.977(4) 2.027(4)
S(1)–Sn–S(2) S(1)–Sn–N(3) S(1)–Sn–N(4) S(1)–Sn–C(8) S(1)–Sn–C(10) N(4)–Sn–C(8) N(4)–Sn–C(10) C(8)–Sn–C(10) S(2)–P–S(3) S(2)–P–C(12) S(2)–P–C(18)
80.45(9) 73.18(19) 137.58(18) 101.1(3) 102.13(5) 81.1(3) 77.5(3) 156.0(4) 113.93(18) 107.1(4) 109.3(4)
S(2)–Sn–N(3) S(2)–Sn–N(4) S(2)–Sn–C(8) S(2)–Sn–C(10) N(3)–Sn–N(4) N(3)–Sn–C(8) N(3)–Sn–C(10) S(3)–P–C(12) S(3)–P–C(18) C(12)–P–C(18)
153.62(19) 141.87(18) 96.1(3) 94.0(3) 64.5(2) 89.3(4) 91.2(3) 110.2(4) 111.5(4) 104.3(5)
As in [SnMe2(PyTSC)(OAc)]PHOAc [3], the coordination sphere around the metal can be described as a distorted pentagonal bipyramid in which one of the equatorial positions is ‘almost’ vacant, except that now most of the metal–ligand bonds are more relaxed, especially the tin–dithiophosphinato bonds. This difference suggests that, as hoped, Ph2PS2y may be a better leaving group than AcOy when the complex is dissolved in water. The PyTSCy ligand, which adopts the E configuration with respect to the azomethine C–N bond, is almost planar (especially in 1), with the N(3) atom showing the greatest displacement from the least-squares plane through S(1), ˚ C(1), N(1), N(2), N(3) and N(4) (about 0.03 and 0.06 A in 1 and 2, respectively). It is also practically co-planar with the equatorial kernel (S(1), N(3), N(4), S(2), S(3)), the dihedral angle between the two planes being about 68 in 1 and 18 in 2. The phenyl rings of the dithiophosphinato ligand are planar and almost mutually orthogonal (dihedral angle about 928 in 1 and 838 in 2). The EtOH molecule of [SnMe2(PyTSC)(S2PPh2)]P EtOH is held in the lattice by two hydrogen bonds. In one (O(1)–H(101)∆N(2)ii) the hydrogen atom is provided by the EtOH hydroxyl group, and in the other (N(1)– H(1A)∆O(1)i) by N(1) (for structural parameters and symmetry operations see Table 2). Thus, EtOH links the [SnMe2(PyTSC)(S2PPh2)] molecules in chains, whereas in [SnEt2(PyTSC)(S2PPh2)] intermolecular N(1)–H∆N(2)i ˚ N(1)∆N(2)is hydrogen bonds (H∆N(2)is2.29 A, i ˚ 3.138(11) A, N(1)–H∆N(2) s168.0o; isyxq1, yy, yzq1) leads to the same kind of dimeric arrangement as is found in many other metal thiosemicarbazonates. 3.2. IR spectra The significant bands of the thiosemicarbazone moiety (see Section 2) in the dimethyl- and diethyltin complexes are close to those of other compounds in which, as in these
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175.89
175.51
CDCl3
145.09
144.74
143.56 143.32
145.11
142.73 143.81
d[C(2)]
EtOH signals: in DMSO-d6, 18.54 and 56.02; in CDCl3, 18.42 and 58.42. s, singlet; d, doublet. c Organometallic fragment: Sn–C8–C9–C10–C11.
b
a
[SnBu2(PyTSC)(S2PPh2)]
174.30
CDCl3
DMSO-d6
175.93
DMSO-d6 DMSO-d6
HPyTSC [SnMe2(PyTSC)(S2PPh2)]PEtOH a
[SnEt2(PyTSC)(S2PPh2)]
178.58 173.41
Solvent
Compound
d[C(1)]
148.53
148.35
147.28
147.71
153.36 146.16
d[C(3)]
126.03
125.55
126.25
125.77
120.29 126.65
d[C(4)]
Table 4 C, 31P and 119Sn NMR data (d, ppm; J, Hz) of the thiosemicarbazone ligand and the complexes
13
138.99
138.57
140.20
138.58
136.58 140.24
d[C(5)]
125.68
125.21
126.01
125.28
124.16 126.20
d[C(6)]
150.59
150.32
148.18 147.86
150.00
149.37 147.51
d[C(7)]
145.29d b(Ci) 130.21d(Co) 128.31d(Cp) 127.00d(Cm) 141.80d(Ci) 130.52d(Co) 129.88s(Cp) 127.83d(Cm) 144.58d(Ci) 130.08d(Co) 128.55s(Cp) 127.05d(Cm) 140.98d(Ci) 130.47d(Co) 129.98s(Cp) 127.84d(Cm) 141.38d(Ci) 131.12d(Co) 130.92d(Cp) 128.30d(Cm)
d[PPh]
28.27(C9) 27.90(C8) 26.25(C10) 13.95(C11)
57/56 805/770
54 789/764
9.80(C9) c 26.77(C8)
10.86(C9) 35.56(C8)
857/822
802/765
J
n
25.07
13.98
d[Sn–R]
59.49 57.50 56.17
55.84
–
56.18
62.79
d[31P]
y142.4 y230.4 y249.1
y242.0
–
y271.0
–
d[119Sn]
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cases, tridentate coordination has been proved by X-ray diffraction studies [3,22]. Similarly, the positions of nas(P–S) and nsym(P–S) (see Section 2) are as expected for an anisobidentate dithiophospinato ligand [23,24]. The IR pattern of the dibutyl derivative is similar to those of the dimethyl and diethyl compounds, suggesting that all three have roughly similar structures, although the high value of nas(P–S)– nsym(P–S) for the dibutyl complex (100 cmy1) suggests that the dithiophosphinato ligand is almost monodentate in this complex [23,24]. 3.3. NMR studies NMR results are collected in Section 2 (1H NMR data) and in Table 4. The spectra of some of the compounds were recorded in both DMSO-d6 and CDCl3 in order to study the effect of the solvent. Although HPyTSC is poorly soluble in chloroform, a 1H NMR spectrum was recorded using 600 transients and was interpreted on the basis of the spectrum of HPyTSC in DMSO-d6 [3] (one of the NH2 signals is masked by the CDCl3 signal). All the protons except C(7)–H are more shielded in CDCl3 than in DMSO-d6 (Section 2). The same pattern is seen in the dimethyl- and diethyltin complexes; although the solvent slightly affects the positions of the peaks it does not alter the trends exhibited, and its effect will be ignored in the following. The changes in the spectrum of HPyTSC upon deprotonation and complexation are as expected [3]. The presence of the dithiophosphinato ligand is shown by two groups of peaks, one located around 8.0 ppm and corresponding to the ortho protons of the phenyl groups, and the other appearing around 7.3 ppm and assigned to the meta and para protons. Both appear as multiplets due to coupling with the phosphorus atom. In the organometallic moieties the signal of the a-protons is located at significantly lower field than in other recently studied thiosemicarbazonates [25,26], probably due to the presence of the dithiophosphinato ligand. In the spectrum of [SnEt2(PyTSC)(S2PPh2)] these protons appear as two multiplets, both of which integrate to two protons. This splitting is not observed in the 13C NMR spectrum of this compound (see below) or in the 1H NMR spectra of the other complexes, which rules out the possibility of its being due to coupling with the phosphorus atom. It is possible that it may be due to the almost orthogonal Ph2PS2y phenyl rings having dissimilar anisotropic effects above and below the equatorial coordination plane. The 2J(1H–119Sn) value of [SnMe2(PyTSC)(S2PPh2)]PEtOH is larger than in the pyridoxal thiosemicarbazonate [25]; when substituted in Eq. (2) of Lockhart and Manders’ work [27], the value obtained for the C–Sn–C angle (1558) is very close to the value measured in the solid state by X-ray diffraction (1548). EtOH bands were also observable in the spectrum of this complex. In Table 4 are listed the 13C NMR data of the compounds. Since the solubility of HPyTSC in CDCl3 was insufficient for 13 C NMR spectrometry, the spectra of the methyl and ethyl
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derivatives in DMSO-d6 were also recorded; as can be seen, the solvent has quite a small effect on the chemical shifts of the thiosemicarbazone and dithiophosphinato carbons of these complexes. The changes in the ligand signals upon complexation are similar to those described for [Cd(PyTSC)2] [22], except that the C(2) signal shifts to higher frequencies in the tin complexes but not in the cadmium(II) complex. This may be due to differences in the relative magnitudes of two opposite effects: shielding due to conformational changes in PyTSC under deprotonation and metallation, and deshielding due to the inductive effect of the N(3)–M bond. If so, the tin complexes probably have a strong N(3)–Sn bond in solution, as was suggested by the X-ray data for the solid state. It may therefore be expected that the [SnR2(PyTSC)]q moiety will have remained intact in the biological experiments. The signals of the dithiophosphinato phenyl carbons are located between 142 and 127 ppm, most of them appearing as doublets due to coupling with the phosphorus atom. The coordination of this ligand is shown by the deshielding of Ci relative to its environment in diphenyldithiophosphinic acid [28]. Structurally more significant are the data for the organometallic moiety. In the dimethyltin compound, the methyl carbon is strongly deshielded in comparison with its environment in [SnMe2(L)] (Lspyridoxal thiosemicarbazone) [25]. This fact and the large value of 1J(13C–119Sn) are attributable to the presence of sulfur atoms and are indicative of a coordination number six or higher [29], showing that the solid state molecular structure remains intact in solution. If the coupling constant is substituted in the equation that Lockhart et al. proposed for methyltin(IV) derivatives [30], the value obtained for the C–Sn–C angle (1528) is, like that obtained from 2J(1H–119Sn), close to the solid state value (see above). In [SnEt2(PyTSC)(S2PPh2)] too, Ca is unusually deshielded and the 13C–119Sn coupling constants are very large. The values of the C–Sn–C angle calculated using 1 J(13C–119Sn) in Eq. (2) of Ref. [31] are 158 and 1558 in CDCl3 and DMSO-d6 respectively. The good agreement with the value obtained in the X-ray study (1568) suggests that, as in the case of the dimethyltin(IV) compound, the solid state molecular structure remains intact in solution. We were not able to obtain the value of 1J for the butyl derivative, but other spectroscopic data (vide infra) suggest that this compound does not remain intact in solution. The proton decoupled 31P spectra of the methyl and ethyl derivatives each present a single peak that lies at higher frequencies than the signal of diphenyldithiophosphinic acid [32]. The spectrum of the butyl derivative shows three peaks (Table 4). The signal located at 57.50 ppm is attributable to the presence of [SnBu2(S2PPh2)2] as a decomposition product [33], and shows that this compound is not totally stable in solution. Table 4 also includes the 119Sn chemical shifts. All are indicative of coordination numbers of six [34]. As in the case of the 31P data, the spectrum of the butyl derivative shows
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Fig. 2. Effects of HPyTSC and the complexes (means of triplicate experiments). Cells were counted on day 4.
Fig. 3. Effects of HPyTSC and the complexes on DMSO-induced differentiation (means of triplicate experiments).
Fig. 4. Inhibition of the clonogenic activity of K562 cells.
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three peaks, the most intense being located at y249 ppm. The peak located at y142 ppm, which corresponds to [SnBu2(S2PPh2)2] [33], confirms the lowest stability of this compound. 3.4. Biological activity Both HPyTSC and its complexes inhibited the proliferation of all the cell lines used (Fig. 2). In particular, the dibutyl derivative was active at a concentration of 0.2 mg/ml, ten times lower than was required by the other compounds. The dimethyl complex was less active than [SnMe2(PyTSC)(OAc)]PHOAc against FLC cells [3], possibly because in DMSO solution 1 maintains the same structure as in the solid state but the mixed PyTSC/OAc complex undergoes prototropic changes due to the presence of the acetic acid solvate [3]. Induction of the differentiation of FLC and K562 cells was assayed using concentrations of the compounds which inhibited cell proliferation by at least 40%. The ligand and the three complexes induced significant differentiation of K562 cells but not of FLC cells (Fig. 3). The clonogenic activity of K562 cells fell sharply after treatment with HPyTSC (by 88%) or 1 (by 70%) (Fig. 4), although not after treatment with 2 or 3 (data not shown). Acknowledgements This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Xunta de Galicia (Spain) under project XUGA 20308B97. M.C. R.-A. is grateful for a grant from the University of Vigo, Spain References [1] K.C. Agrawal, A.C. Sartorelli, Progr. Med. Chem. 15 (1978) 321 and Refs. therein. [2] Y. Arakawa, Main Group Met. Chem. 17 (1994) 225 and Refs. therein. ¨ [3] J.S. Casas, M.S. Garcia Tasende, C. Maichle Mossmer, M.C. ´ ´ ´ ´ ¨ Rodrıguez Arguelles, A. Sanchez, J. Sordo, A. Vazquez Lopez, S. Pinelli, P. Lunghi, R. Albertini, J. Inorg. Biochem. 62 (1996) 41. ˜ A. Macıas, ´ A. Castineiras, ˜ ´ [4] M.V. Castano, A. Sanchez, E. Garcia Martinez, J. Sordo, S. Hiller, E.E. Castellano, J. Chem. Soc., Dalton Trans. (1990) 1001.
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[5] L.C. Andersson, K. Nilsson, C.G. Gahmberg, Int. J. Cancer 23 (1979) 143. [6] E. Foley, H. Lazarus, S. Farber, B.G. Uzman, B.A. Boone, R.E. McCarthy, Cancer 18 (1965) 522. [7] Y. Yamaguchi, M. Kanatani, T. Kasukabe, Y. Honma, Exp. Hematol. 24 (1996) 682. [8] G. Battistini, A. Marziali, R. Albertini, D. Habetswallner, D. Bulgarini, E.M. Coccia, G. Fiorucci, G. Romeo, R. Orsatti, V. Testa, E. Affabris, G. Peschle, G.B. Rossi, J. Biol. Chem. 266 (1991) 528. [9] Y. Maeda, R. Okawara, J. Organomet. Chem. 10 (1967) 247. [10] F.E. Andersen, C.J. Duca, J.V. Scudi, J. Am. Chem. Soc. 73 (1951) 4967. [11] J.L. de Boer, A.J.M. Duisenberg, Acta Crystallogr., Sect. A 40 (1984) 410. [12] A.L. Spek, J. Appl. Crystallogr. 21 (1988) 578. [13] N. Walker, D. Stuart, Acta Crystallogr., Sect. A 39 (1983) 158. ¨ Gottingen, ¨ 1986. [14] G.M. Sheldrick, SHELXS86, Universitat [15] B.A. Frenz and Associates Inc. & Enraf-Nonius, VAX/SDP Structure Determination Package, version 2.2, College Station, TX, USA, 1986. ¨ Gottingen, ¨ [16] G.M. Sheldrick, SHELXL97, Universitat 1997. [17] A.J.C. Wilson, International Tables for Crystallography, vol. C, Kluwer, Dordrecht, 1992. ¨ Heidelberg, 1997. [18] L. Zsolnai, ZORTEP, Universitat [19] A.L. Spek, Acta Crystallogr., Sect. A 46 (1990) C34. [20] S.M. Orkin, F. Harosi, P. Leder, Proc. Natl. Acad. Sci. USA 72 (1975) 98. [21] A. Bondi, J. Phys. Chem. 68 (1964) 441. ˜ M.S. Garcia Tasende, I. Martinez Santa[22] J.S. Casas, M.V. Castano, ´ marta, A. Sanchez, J. Sordo, E.E. Castellano, J. Zukerman-Schpector, J. Chem. Res. (S) (1992) 324; M (1992) 2626. [23] I. Haiduc, I. Silaghi-Dumitrescu, R. Grecu, R. Constantinescu, L. Silaghi-Dumitrescu, J. Mol. Struct. 114 (1984) 467. [24] D.C. Silvestru, I. Haiduc, J. Organomet. Chem. 365 (1989) 83. ´ ¨ ´ [25] J.S. Casas, M.C. Rodrıguez Arguelles, U. Russo, A. Sanchez, J. Sordo, ´ ´ A. Vazquez Lopez, S. Pinelli, P. Lunghi, A. Bonati, R. Albertini, J. Inorg. Biochem. 69 (1998) 283. ´ ´ ´ [26] J.S. Casas, A. Sanchez, J. Sordo, A. Vazquez Lopez, E.E. Castellano, ´ ¨ J. Zukerman-Schpector, M.C. Rodrıguez Arguelles, U. Russo, Inorg. Chim. Acta 216 (1994) 169. [27] T.P. Lockhart, W.P. Manders, Inorg. Chem. 25 (1986) 892. ´ ´ [28] E. M. Vazquez Lopez, Ph.D. Thesis, University of Santiago de Compostela, Santiago de Compostela, Spain, 1993. [29] K. Pihlaja, E. Kleinpeter, Carbon-13 NMR Chemical Shifts in Structural and Stereochemical Analysis, VCH, New York, 1994, p. 332. [30] T.P. Lockhart, W.P. Manders, J.J. Puckerman, J. Am. Chem. Soc. 107 (1985) 4546. [31] W.F. Howard, R.W. Crecely, W.H. Nelson, Inorg. Chem. 24 (1985) 2204. ´ ´ ´ [32] J.S. Casas, A. Sanchez, J. Sordo, E.M. Vazquez Lopez, Polyhedron 11 (1992) 2889. ´ ´ [33] A. Vazquez Lopez, Ph.D. Thesis, University of Santiago de Compostela, Santiago de Compostela, Spain, 1996. ´ ´ A. Lycka, J. Organomet. Chem. [34] J. Holecek, M. Nadvornik, K. Handlır, 315 (1986) 299.
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Synthesis, structure, spectroscopic properties and biological activity of mixed diorganotin(IV) complexes containing pyridine-2-carbaldehyde thiosemicarbazonato and diphenyldithiophosphinato ligands a b a ˜ ´ C. Rodrıguez-Arguelles ´ ´ Sanchez ´ ¨ Jose´ S. Casas a,*, Alfonso Castineiras , Marıa , Agustın , a ´ ´ Lopez , Silvana Pinelli c, Paolo Lunghi c, Paolo Ciancianaini c, Jose´ Sordo a, Antonia Vazquez Antonio Bonati c, Pierpaolo Dall’Aglio c, Roberto Albertini c a
´ ´ Departamento de Quımica Inorganica, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain b ´ ´ Departamento de Quımica Inorganica, Universidade de Vigo, 36200 Vigo, Spain c Istituto di Patologia Speciale Medica and Centro di Ricerca Interuniversitario per la diagnosi, terapia e prognosi dei tumori humani, Universita` di Parma, 43100 Parma, Italy Received 3 June 1999; received in revised form 17 August 1999; accepted 7 September 1999
Abstract Reaction of the title ligands (HPyTSC and HS(S)PPh2, respectively) with R2SnO (RsMe, Et, Bu) in ethanol (EtOH) afforded the complexes [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnR2(PyTSC)(S2PPh2)] (RsEt (2), Bu (3)). The structures of 1 and 2 were determined by single-crystal X-ray diffractometry. In both these complexes the tin atom is coordinated to an N,N,S-dentatethiosemicarbazonate ligand, an anisobidentate dithiophosphinato ligand and the two R groups. The coordination polyhedrons can be described as distorted pentagonal bipyramids. A comparative study of the IR spectra of 1, 2 and 3 indicates that the butyl complex has a similar structure. Multinuclear (1H, 13 C, 31P and 119Sn) NMR data suggest that the structures of 1 and 2 probably remain in CDCl3 (or DMSO-d6) solution but compound 3 partially decomposes in these media. Preliminary results on the effects of the complexes on the proliferation and differentiation of FLC, CEM, U937, K562 and TOM-1 leukaemia cells, and on the clonogenic activity of K562 cells are also described. q1999 Elsevier Science Inc. All rights reserved. Keywords: Diorganotin(IV) complexes; Pyridine-2-carbaldehyde thiosemicarbazonato ligands; Diphenyldithiophosphinato ligands
1. Introduction The possibility of synergic coupling of pyridine-2-carbaldehyde thiosemicarbazone (HPyTSC) with diorganotin(IV) compounds to obtain a product with greater antiproliferative activity than either [1,2] was explored in previous work on the mixed complex [SnMe2(PyTSC)(OAc)]PHOAc, which inhibited proliferation of Friend erythroleukaemia cells (FLC) by about 80% at moderate dosages without suppressing DMSO-induced cell differentiation [3]. It was hypothesized that hydrolysis of the complex after crossing the cell membrane removed the acetate coligand, leaving [SnMe2(PyTSC)]q (aq), a species capable of reaching the biological target. Suspecting that the high affinity of AcOy for the organotin cation might have limited hydrolysis of the above complex, * Corresponding author. Fax: q34-81-594912; e-mail: qiscasas@ uscmail.usc.es
and hence its efficacy, we have now prepared complexes [SnR2(PyTSC)(S2PPh2)] (Rsmethyl, ethyl and butyl), reasoning that the dithiophosphinato ligand must be replaced with water more easily than AcOy due to the conflict between its ‘soft’ character and the ‘borderline’ behaviour of the R2Sn2q cation [4] and to the greater steric demands of the disulphur ligand. We describe here the synthesis and structural characterisation of these new complexes, and report preliminary results on their effects on the proliferation and differentiation of FLC, CEM, U937, K562 and TOM-1 leukaemia cells [5–8].
2. Experimental Thiosemicarbazide (Merck), pyridine-2-carbaldehyde (Merck), dimethyltindichloride (Alfa), diethyltindichloride (Aldrich), dibutyltinoxide (Aldrich) and diphenyldithio-
0162-0134/99/$ - see front matter q1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 8 5 - 3
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phosphinic acid (Aldrich) were used as supplied. The diorganotin(IV) oxides and the thiosemicarbazone ligand were obtained by the procedures described in Refs. [9,10], respectively. HPyTSC 1H NMR data, d (ppm): in DMSO-d6 8.37 (s, br, 1H) [N(1)H2], 8.10 (s, br, 1H) [N(1)H2], 11.56 (s, br, 1H) [N(2)H], 8.07 (s, 1H) [C(2)H], 8.26 (d, 1H) [C(4)H], 7.79 (t, 1H) [C(5)H], 7.34 (dd, 1H) [C(6)H], 8.33 (d, 1H) [C(7)H]; in CDCl3 6.35 (s, vbr, 1H) [N(1)H2], 9.09 (s, br, 1H) [N(2)H], 7.87 (s, 1H) [C(2)H], 7.85 (d, 1H) [C(4)H], 7.76 (td, 1H) [C(5)H], 7.33 (dd, 1H) [C(6)H], 8.65 (d, 1H) [C(7)H]. 2.1. Preparation of the complexes 2.1.1. [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) A solution of HPyTSC (0.22 g, 1.2 mmol) in ethanol (35 ml) was added to a suspension of SnMe2O (0.20 g, 1.2 mmol) and HS(S)PPh2 (0.30 g, 1.2 mmol) in 55 ml of the same solvent.The mixture was stirred for 7 days at room temperature, and a yellow–green crystalline solid was filtered out. M.p. 1608C. Anal. Found: C, 44.1; H, 4.8; N, 9.1. Calc. for C23H29N4POS3Sn: C, 44.3; H, 4.7; N, 9.0%. IR spectrum (cmy1): 3457w, 3287s, 3160s, n(N–H); 1593m, n(C_N); 796m, n(C_S); 646m, nas(P–S); 557s, nsym(P–S); 557s, nas(Sn–C); 515w, nsym(Sn–C). 1H NMR data, d (ppm): in DMSO-d6 7.92 (s, br, 2H) [N(1)H2], 8.75 (s, 1H) [C(2)H], 7.90 (overlapped) [C(4)H], 8.13 (td, 1H) [C(5)H], 7.67 (dd, 1H) [C(6)H], 8.97 (d, 1H) [C(7)H], 7.94 (m, 4H) [PPh2], 7.24 (m, 6H) [PPh2], 0.96 (s, 6H, 2 Js96/92 Hz) [Sn–R], 1.05 (t,3H) [EtOH], 3.44 (q, 2H) [EtOH], 4.34 (t, 1H) [EtOH]; in CDCl3 5.44 (s, br, 2H) [N(1)H2], 8.33 (s, 1H) [C(2)H], 7.47 (d, 1H) [C(4)H], 7.86 (td, 1H) [C(5)H], 7.43 (m, 1H) [C(6)H], 9.25 (d, 1H) [C(7)H], 8.15 (m, 4H) [PPh2], 7.37 (m, 6H) [PPh2], 1.42 (s, 6H, 2Js101/98 Hz) [Sn–R], 1.25 (t, 3H) [EtOH], 3.72 (q, 2H) [EtOH]. 2.1.2. [SnEt2(PyTSC)(S2PPh2)] (2) To a solution of HPyTSC (0.22 g, 1.2 mmol) in 25 ml of absolute ethanol was added a suspension of HS(S)PPh2 (0.30 g, 1.2 mmol) and SnEt2O (0.24 g, 1.2 mmol) in the same solvent (60 ml). After stirring for 5 days, the yellow solid formed was filtered out and vacuum dried. M.p. 1888C. Anal. Found: C, 45.2; H, 4.4; N, 8.9. Calc. for C23H27N4PS3Sn: C, 44.5; H, 4.4; N, 9.0%. IR spectrum (cmy1): 3397s, 3276s, 3241s, n(N–H); 1599m, n(C_N); 783w, n(C_S); 636m, nas(P–S); 560m, nsym(P–S); 530m, nas(Sn–C); 490m, nsym(Sn–C). 1H NMR data, d (ppm): in DMSO-d6 7.82 (s, br, 2H) [N(1)H2], 8.75 (s, 1H) [C(2)H], 7.89 (d, 1H) [C(4)H], 8.15 (td, 1H) [C(5)H], 7.68 (m,1H) [C(6)H], 9.01 (d, 1H) [C(7)H], 7.97 (m, 4H) [PPh2], 7.29 (m, 6H) [PPh2], 1.68 (m, 2H) [Sn–R], 1.57 (m, 2H) [Sn–R], 0.93 (t, 6H, 3Js166 Hz) [Sn–R]; in CDCl3 5.43 (s, br, 2H) [N(1)H2], 8.36 (s, 1H) [C(2)H], 7.47 (d, 1H) [C(4)H], 7.87 (td, 1H) [C(5)H], 7.49 (m, overlapped) [C(6)H], 9.22 (d, 1H) [C(7)H], 8.05 (m,
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4H) [PPh2], 7.49 (m, overlapped) [PPh2], 2.15 (m, 2H) [Sn–R], 1.86 (m, 2H) [Sn–R], 1.13 (t, 6H, 3Js175/168 Hz) [Sn–R]. 2.1.3. [SnBu2(PyTSC)(S2PPh2)] (3) A solution of HPyTSC (0.18 g, 1.0 mmol) in 25 ml of ethanol was added to a stirred suspension of SnBu2O (0.25 g, 1.0 mmol) and HS(S)PPh2 (0.25 g, 1.0 mmol) in 50 ml of the same solvent. The mixture was stirred for 3 days, and a yellow solid was filtered out. M.p. 1078C(d). Anal. Found: C, 47.9; H, 5.1; N, 8.0. Calc. for C27H35N4PS3Sn: C, 49.0; H, 5.3; N, 8.5%. IR spectrum (cmy1): 3424m, 3285m, 3145m, n(N–H); 1595m, n(C_N); 775w, n(C_S); 650m, nas(P– S); 550m, nsym(P–S); 615w, nas(Sn–C). 1H NMR data, d (ppm): in CDCl3 5.37 (s, br, 2H) [N(1)H2], 8.85 (s, 1H) [C(2)H], 7.50 (d, 1H) [C(4)H], 7.89 (td, 1H) [C(5)H], 7.43 (t, 1H) [C(6)H], 9.22 (d, 1H) [C(7)H], 8.05 (m, 4H) [PPh2], 7.35 (m) [PPh2], 2.12 (t, 4H) [Sn–R], 1,68 (q, 4H) [Sn–R], 0.99 (sx, 4H) [Sn–R], 0.62 (t, 6H) [Sn– R]. 2.2. Physical measurements Elemental analyses were performed with a Carlo-Erba ¨ 1108 analyser. Melting points were determined with a Buchi apparatus. IR spectra were recorded in KBr pellets or Nujol mulls on a Bruker IFS-66V spectrometer. 1H, 13C, 31P and 119 Sn NMR spectra were recorded at room temperature on Bruker WM250, AMX 300 or AMX 500 instruments and were referred to TMS (1H, 13C), external 85% H3PO4 (31P) and external neat Sn(CH3)4 (119Sn). 2.3. X-ray crystallography Crystals of 1 and 2 suitable for X-ray diffraction were mounted on a glass fibre and transferred to an Enraf-Nonius CAD4 diffractometer. Accurate unit cell parameters and an orientation matrix were determined by least-squares from the setting angles of a set of well-centred reflections (SET4 [11]) in the range 5.30–13.328 (1) or 8.09–11.608 (2). Reduced cell calculations did not indicate higher lattice symmetry [12]. Crystal data and details of data collection and refinement are given in Table 1. Data were corrected for Lp effects and for the observed linear decay of ten reference reflections. An empirical absorption correction (DIFABS [13]) was applied for both compounds. The cell constants for 1 suggested the space group to be either orthorhombic or monoclinic. The structures were solved for the monoclinic space group P21/c (No. 14) by automated Patterson (1) or direct methods (2) and subsequent difference Fourier techniques (SHELXS86 [14]), and successful refinement (confirming the space group) was performed on F (1; SDP/VAX [15]) or F2 (2; SHELXL97 [16]) by a full-matrix leastsquares procedure using anisotropic displacement parameters. Hydrogen atoms were included in the refinement in calculated positions riding on their carrier atoms. Neutral
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Table 1 Crystallographic data of [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnEt2(PyTSC)(S2PPh2)] (2)
2.4. Biological studies
Compound
1
2
Empirical formula Formula weight ˚ Wavelength (A) Crystal size (mm) Crystal shape Crystal system Space group ˚ a (A) ˚ b (A) ˚ c (A) a (8) b (8) g (8) ˚ 3) V (A Z, Dcalc (Mg/m3) F(000) u Range (8) Temperature (K) hmin/hmax kmin/kmax lmin/lmax m (mmy1) Max./min. transmissions Refl. collected/unique Data/parameters Final R Final wR2 GOOF Max./min. Dr (e/A3)
C23H29N4OPS3Sn 623.34 0.71073 0.60=0.30=0.10 plate monoclinic P21/c (No. 14) 20.628(8) 9.727(3) 13.477(3) 90.00(–) 90.08(2) 90.00(–) 2704(1) 4, 1.531 1264 3.01–27.03 213 y1/12 0/26 y17/17 1.259 0.885/0.519 6994/6188 4832/303 0.045 0.047 1.144 0.117/0.042
C23H27N4PS3Sn 605.35 0.71073 0.80=0.40=0.20 prism monoclinic P21/c (No. 14) 12.722(4) 14.963(3) 14.198(4) 90.00(–) 93.01(2) 90.00(–) 2698(1) 4, 1.490 1224 3.08–29.98 293 0/17 0/21 y19/19 1.257 0.787/0.433 8137/7824 7824/291 0.088 0.125 0.965 0.699/y1.591
Stock solutions of HPyTSC in dimethyl sulphoxide were prepared and stored at room temperature. Cells (K562, U937, CEM, FLC and TOM-1) were grown at 378C under 5% CO2 in RPMI-1640 supplemented with 100 mg/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine (Gibco/BRL) and 5% (FLC), 10% (K562, U937 and CEM cells) or 20% (TOM-1) of fetal calf serum. Cells were seeded at 2=105/ml in the presence of the above-mentioned compounds added to culture medium prior to cell seeding. Cell mortality evaluated by the Trypan Blue dye exclusion method was 5% or less in all experiments. The degree of differentiation was determined as the percentage of benzidine-positive (Bq) cells [20].
atom scattering factors and anomalous dispersion corrections were taken from the International Tables for X-ray Crystallography [17]. Geometrical calculations were performed and illustrations obtained with the SHELXL97 [16], ZORTEP [18] and PLATON98 [19] packages.
2.4.1. Clonogenic assays K562 cells were plated in 35 mm dishes (Falcon, Becton Dickinson) in 1 ml a-MEM containing 2.3% methylcellulose, 30% FCS, 2% L-glutamine 200 mM and 8% Iscove’s modified Dulbecco’s medium (IMDM, Sigma-Aldrich) at a concentration of 500 cells/dish and incubated at 378C in 5% CO2 humidified atmosphere. The number of colonies CFUAML (colony forming unit of acute myeloid leukaemia), formed after treating the cells with low doses of the compounds for 16 h, was determined at day 14 using light microscopy.
2.4.2. Morphological assessment For light microscopy, cytospin smears were prepared using a Cytospin Centrifuge (Shandon, Cytospin 2) and stained with May–Grunwald–Giemsa stain.
Fig. 1. An ORTEP plot showing the molecular structure of [SnEt2(PyTSC)(S2PPh2)] (2) and the numbering scheme.
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J.S. Casas et al. / Journal of Inorganic Biochemistry 76 (1999) 277–284 Table 3 ˚ and angles (8) in [SnEt2(PyTSC)(S2PPh2)] Bond lengths (A)
3. Results and discussion 3.1. Crystal structures of [SnMe2(PyTSC)(S2PPh2)]PEtOH (1) and [SnEt2(PyTSC)(S2PPh2)] (2) The molecular structure of 2 is shown in Fig. 1 together with numbering scheme. Complex 1 differs only slightly in structure (see selected distances and angles in Tables 2 and 3). In both complexes the metal atom is coordinated to a tridentate thiosemicarbazonato ligand, an anisobidentate dithiophosphinato ligand and the two organic groups. The TSC ligand is N,N,S-bound, as in [SnMe2(PyTSC)(OAc)]PHOAc [3], but the Sn–S and Sn–N(Py) bond lengths are longer in the dithiophosphinato complexes, probably due to the greater steric demands created by replacement of the two oxygens by two sulfurs in the equatorial plane. Curiously, the Sn–N3 bond length is equal to (in 2) or shorter than (in 1) its value in the OAcy complex, suggesting that this bond is more stable than the Sn–S and Sn–N(Py) bonds. Both the Sn–S (dithiophosphinate) bonds are longer than the Sn–S (TSC) bond suggesting that, as expected, the former ligand is the more weakly bound. Nevertheless, all Sn– S distances are less than the sum of the van der Waals radii ˚ [21], though the longest (3.403(1) in 1 and (4.0 A) 3.302(3) in 2) probably indicates a secondary bond. The coordination number of the tin atom can thus be considered as 6q1. The apical Sn–C bonds are slightly longer than in the acetate complex [3], and the C–Sn–C angles slightly narrower (154.1 and 155.88 as against 156.08), bending away from the TSC bite and toward the empty equatorial area between N(4) and S(3). The equatorial kernel (Sn, N(3), N(4), S(1), S(2), S(3)) is virtually planar in 2, the rms deviation from the least-squares plane being 0.0702 and the ˚ (N(4)). The distortion is greater maximum deviation 0.09 A ˚ from the best fitting plane. in 1, in which S(1) lies 0.12 A Table 2 ˚ and angles (8) in [SnMe2(PyTSC)(S2PPh2)]PEtOH a Bond lengths (A) Sn–S(1) Sn–S(2) Sn–S(3) Sn–N(3) Sn–N(4) Sn–C(8) N(1)–H(1)∆O(1)i
2.519(1) 2.715(1) 3.403(1) 2.364(4) 2.578(4) 2.131(7) 2.8731
Sn–C(9) S(2)–P S(3)–P P–C(10) P–C(16)
2.112(6) 2.015(2) 1.963(2) 1.815(7) 1.808(6)
O(1)–H(101)∆N(2)ii
2.8731
S(1)–Sn–S(2) S(1)–Sn–N(3) S(1)–Sn–N(4) S(1)–Sn–C(8) S(1)–Sn–C(9) S(2)–Sn–N(3) S(2)–Sn–N(4) S(2)–Sn–C(8) S(2)–Sn–C(9) N(3)–Sn–N(4) N(3)–Sn–C(8)
75.39(4) 73.6(2) 139.5(2) 99.4(2) 106.3(2) 148.6(2) 145.0(2) 92.9(2) 90.9(2) 66.4(1) 97.0(3)
N(4)–Sn–C(8) N(3)–Sn–C(9) N(4)–Sn–C(9) S(2)–P–S(3) S(2)–P–C(10) S(2)–P–C(16) S(3)–P–C(10) S(3)–P–C(16) C(8)–Sn–C(9) C(10)–P–C(16)
80.1(2) 93.1(2) 82.3(2) 116.0(1) 107.5(2) 107.4(3) 110.6(2) 111.1(2) 154.1(2) 103.4(3)
a
isyx, y1/2qy, 1/2yz; iisyx, 1yy, yz.
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Sn–S(1) Sn–S(2) Sn–S(3) Sn–N(3) Sn–N(4) Sn–C(8)
2.548(3) 2.722(3) 3.302(3) 2.438(8) 2.717(8) 2.122(10)
Sn–C(10) P–C(12) P–C(18) S(3)–P S(2)–P
2.135(9) 1.811(11) 1.809(11) 1.977(4) 2.027(4)
S(1)–Sn–S(2) S(1)–Sn–N(3) S(1)–Sn–N(4) S(1)–Sn–C(8) S(1)–Sn–C(10) N(4)–Sn–C(8) N(4)–Sn–C(10) C(8)–Sn–C(10) S(2)–P–S(3) S(2)–P–C(12) S(2)–P–C(18)
80.45(9) 73.18(19) 137.58(18) 101.1(3) 102.13(5) 81.1(3) 77.5(3) 156.0(4) 113.93(18) 107.1(4) 109.3(4)
S(2)–Sn–N(3) S(2)–Sn–N(4) S(2)–Sn–C(8) S(2)–Sn–C(10) N(3)–Sn–N(4) N(3)–Sn–C(8) N(3)–Sn–C(10) S(3)–P–C(12) S(3)–P–C(18) C(12)–P–C(18)
153.62(19) 141.87(18) 96.1(3) 94.0(3) 64.5(2) 89.3(4) 91.2(3) 110.2(4) 111.5(4) 104.3(5)
As in [SnMe2(PyTSC)(OAc)]PHOAc [3], the coordination sphere around the metal can be described as a distorted pentagonal bipyramid in which one of the equatorial positions is ‘almost’ vacant, except that now most of the metal–ligand bonds are more relaxed, especially the tin–dithiophosphinato bonds. This difference suggests that, as hoped, Ph2PS2y may be a better leaving group than AcOy when the complex is dissolved in water. The PyTSCy ligand, which adopts the E configuration with respect to the azomethine C–N bond, is almost planar (especially in 1), with the N(3) atom showing the greatest displacement from the least-squares plane through S(1), ˚ C(1), N(1), N(2), N(3) and N(4) (about 0.03 and 0.06 A in 1 and 2, respectively). It is also practically co-planar with the equatorial kernel (S(1), N(3), N(4), S(2), S(3)), the dihedral angle between the two planes being about 68 in 1 and 18 in 2. The phenyl rings of the dithiophosphinato ligand are planar and almost mutually orthogonal (dihedral angle about 928 in 1 and 838 in 2). The EtOH molecule of [SnMe2(PyTSC)(S2PPh2)]P EtOH is held in the lattice by two hydrogen bonds. In one (O(1)–H(101)∆N(2)ii) the hydrogen atom is provided by the EtOH hydroxyl group, and in the other (N(1)– H(1A)∆O(1)i) by N(1) (for structural parameters and symmetry operations see Table 2). Thus, EtOH links the [SnMe2(PyTSC)(S2PPh2)] molecules in chains, whereas in [SnEt2(PyTSC)(S2PPh2)] intermolecular N(1)–H∆N(2)i ˚ N(1)∆N(2)is hydrogen bonds (H∆N(2)is2.29 A, i ˚ 3.138(11) A, N(1)–H∆N(2) s168.0o; isyxq1, yy, yzq1) leads to the same kind of dimeric arrangement as is found in many other metal thiosemicarbazonates. 3.2. IR spectra The significant bands of the thiosemicarbazone moiety (see Section 2) in the dimethyl- and diethyltin complexes are close to those of other compounds in which, as in these
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175.89
175.51
CDCl3
145.09
144.74
143.56 143.32
145.11
142.73 143.81
d[C(2)]
EtOH signals: in DMSO-d6, 18.54 and 56.02; in CDCl3, 18.42 and 58.42. s, singlet; d, doublet. c Organometallic fragment: Sn–C8–C9–C10–C11.
b
a
[SnBu2(PyTSC)(S2PPh2)]
174.30
CDCl3
DMSO-d6
175.93
DMSO-d6 DMSO-d6
HPyTSC [SnMe2(PyTSC)(S2PPh2)]PEtOH a
[SnEt2(PyTSC)(S2PPh2)]
178.58 173.41
Solvent
Compound
d[C(1)]
148.53
148.35
147.28
147.71
153.36 146.16
d[C(3)]
126.03
125.55
126.25
125.77
120.29 126.65
d[C(4)]
Table 4 C, 31P and 119Sn NMR data (d, ppm; J, Hz) of the thiosemicarbazone ligand and the complexes
13
138.99
138.57
140.20
138.58
136.58 140.24
d[C(5)]
125.68
125.21
126.01
125.28
124.16 126.20
d[C(6)]
150.59
150.32
148.18 147.86
150.00
149.37 147.51
d[C(7)]
145.29d b(Ci) 130.21d(Co) 128.31d(Cp) 127.00d(Cm) 141.80d(Ci) 130.52d(Co) 129.88s(Cp) 127.83d(Cm) 144.58d(Ci) 130.08d(Co) 128.55s(Cp) 127.05d(Cm) 140.98d(Ci) 130.47d(Co) 129.98s(Cp) 127.84d(Cm) 141.38d(Ci) 131.12d(Co) 130.92d(Cp) 128.30d(Cm)
d[PPh]
28.27(C9) 27.90(C8) 26.25(C10) 13.95(C11)
57/56 805/770
54 789/764
9.80(C9) c 26.77(C8)
10.86(C9) 35.56(C8)
857/822
802/765
J
n
25.07
13.98
d[Sn–R]
59.49 57.50 56.17
55.84
–
56.18
62.79
d[31P]
y142.4 y230.4 y249.1
y242.0
–
y271.0
–
d[119Sn]
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cases, tridentate coordination has been proved by X-ray diffraction studies [3,22]. Similarly, the positions of nas(P–S) and nsym(P–S) (see Section 2) are as expected for an anisobidentate dithiophospinato ligand [23,24]. The IR pattern of the dibutyl derivative is similar to those of the dimethyl and diethyl compounds, suggesting that all three have roughly similar structures, although the high value of nas(P–S)– nsym(P–S) for the dibutyl complex (100 cmy1) suggests that the dithiophosphinato ligand is almost monodentate in this complex [23,24]. 3.3. NMR studies NMR results are collected in Section 2 (1H NMR data) and in Table 4. The spectra of some of the compounds were recorded in both DMSO-d6 and CDCl3 in order to study the effect of the solvent. Although HPyTSC is poorly soluble in chloroform, a 1H NMR spectrum was recorded using 600 transients and was interpreted on the basis of the spectrum of HPyTSC in DMSO-d6 [3] (one of the NH2 signals is masked by the CDCl3 signal). All the protons except C(7)–H are more shielded in CDCl3 than in DMSO-d6 (Section 2). The same pattern is seen in the dimethyl- and diethyltin complexes; although the solvent slightly affects the positions of the peaks it does not alter the trends exhibited, and its effect will be ignored in the following. The changes in the spectrum of HPyTSC upon deprotonation and complexation are as expected [3]. The presence of the dithiophosphinato ligand is shown by two groups of peaks, one located around 8.0 ppm and corresponding to the ortho protons of the phenyl groups, and the other appearing around 7.3 ppm and assigned to the meta and para protons. Both appear as multiplets due to coupling with the phosphorus atom. In the organometallic moieties the signal of the a-protons is located at significantly lower field than in other recently studied thiosemicarbazonates [25,26], probably due to the presence of the dithiophosphinato ligand. In the spectrum of [SnEt2(PyTSC)(S2PPh2)] these protons appear as two multiplets, both of which integrate to two protons. This splitting is not observed in the 13C NMR spectrum of this compound (see below) or in the 1H NMR spectra of the other complexes, which rules out the possibility of its being due to coupling with the phosphorus atom. It is possible that it may be due to the almost orthogonal Ph2PS2y phenyl rings having dissimilar anisotropic effects above and below the equatorial coordination plane. The 2J(1H–119Sn) value of [SnMe2(PyTSC)(S2PPh2)]PEtOH is larger than in the pyridoxal thiosemicarbazonate [25]; when substituted in Eq. (2) of Lockhart and Manders’ work [27], the value obtained for the C–Sn–C angle (1558) is very close to the value measured in the solid state by X-ray diffraction (1548). EtOH bands were also observable in the spectrum of this complex. In Table 4 are listed the 13C NMR data of the compounds. Since the solubility of HPyTSC in CDCl3 was insufficient for 13 C NMR spectrometry, the spectra of the methyl and ethyl
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derivatives in DMSO-d6 were also recorded; as can be seen, the solvent has quite a small effect on the chemical shifts of the thiosemicarbazone and dithiophosphinato carbons of these complexes. The changes in the ligand signals upon complexation are similar to those described for [Cd(PyTSC)2] [22], except that the C(2) signal shifts to higher frequencies in the tin complexes but not in the cadmium(II) complex. This may be due to differences in the relative magnitudes of two opposite effects: shielding due to conformational changes in PyTSC under deprotonation and metallation, and deshielding due to the inductive effect of the N(3)–M bond. If so, the tin complexes probably have a strong N(3)–Sn bond in solution, as was suggested by the X-ray data for the solid state. It may therefore be expected that the [SnR2(PyTSC)]q moiety will have remained intact in the biological experiments. The signals of the dithiophosphinato phenyl carbons are located between 142 and 127 ppm, most of them appearing as doublets due to coupling with the phosphorus atom. The coordination of this ligand is shown by the deshielding of Ci relative to its environment in diphenyldithiophosphinic acid [28]. Structurally more significant are the data for the organometallic moiety. In the dimethyltin compound, the methyl carbon is strongly deshielded in comparison with its environment in [SnMe2(L)] (Lspyridoxal thiosemicarbazone) [25]. This fact and the large value of 1J(13C–119Sn) are attributable to the presence of sulfur atoms and are indicative of a coordination number six or higher [29], showing that the solid state molecular structure remains intact in solution. If the coupling constant is substituted in the equation that Lockhart et al. proposed for methyltin(IV) derivatives [30], the value obtained for the C–Sn–C angle (1528) is, like that obtained from 2J(1H–119Sn), close to the solid state value (see above). In [SnEt2(PyTSC)(S2PPh2)] too, Ca is unusually deshielded and the 13C–119Sn coupling constants are very large. The values of the C–Sn–C angle calculated using 1 J(13C–119Sn) in Eq. (2) of Ref. [31] are 158 and 1558 in CDCl3 and DMSO-d6 respectively. The good agreement with the value obtained in the X-ray study (1568) suggests that, as in the case of the dimethyltin(IV) compound, the solid state molecular structure remains intact in solution. We were not able to obtain the value of 1J for the butyl derivative, but other spectroscopic data (vide infra) suggest that this compound does not remain intact in solution. The proton decoupled 31P spectra of the methyl and ethyl derivatives each present a single peak that lies at higher frequencies than the signal of diphenyldithiophosphinic acid [32]. The spectrum of the butyl derivative shows three peaks (Table 4). The signal located at 57.50 ppm is attributable to the presence of [SnBu2(S2PPh2)2] as a decomposition product [33], and shows that this compound is not totally stable in solution. Table 4 also includes the 119Sn chemical shifts. All are indicative of coordination numbers of six [34]. As in the case of the 31P data, the spectrum of the butyl derivative shows
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Fig. 2. Effects of HPyTSC and the complexes (means of triplicate experiments). Cells were counted on day 4.
Fig. 3. Effects of HPyTSC and the complexes on DMSO-induced differentiation (means of triplicate experiments).
Fig. 4. Inhibition of the clonogenic activity of K562 cells.
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three peaks, the most intense being located at y249 ppm. The peak located at y142 ppm, which corresponds to [SnBu2(S2PPh2)2] [33], confirms the lowest stability of this compound. 3.4. Biological activity Both HPyTSC and its complexes inhibited the proliferation of all the cell lines used (Fig. 2). In particular, the dibutyl derivative was active at a concentration of 0.2 mg/ml, ten times lower than was required by the other compounds. The dimethyl complex was less active than [SnMe2(PyTSC)(OAc)]PHOAc against FLC cells [3], possibly because in DMSO solution 1 maintains the same structure as in the solid state but the mixed PyTSC/OAc complex undergoes prototropic changes due to the presence of the acetic acid solvate [3]. Induction of the differentiation of FLC and K562 cells was assayed using concentrations of the compounds which inhibited cell proliferation by at least 40%. The ligand and the three complexes induced significant differentiation of K562 cells but not of FLC cells (Fig. 3). The clonogenic activity of K562 cells fell sharply after treatment with HPyTSC (by 88%) or 1 (by 70%) (Fig. 4), although not after treatment with 2 or 3 (data not shown). Acknowledgements This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Xunta de Galicia (Spain) under project XUGA 20308B97. M.C. R.-A. is grateful for a grant from the University of Vigo, Spain References [1] K.C. Agrawal, A.C. Sartorelli, Progr. Med. Chem. 15 (1978) 321 and Refs. therein. [2] Y. Arakawa, Main Group Met. Chem. 17 (1994) 225 and Refs. therein. ¨ [3] J.S. Casas, M.S. Garcia Tasende, C. Maichle Mossmer, M.C. ´ ´ ´ ´ ¨ Rodrıguez Arguelles, A. Sanchez, J. Sordo, A. Vazquez Lopez, S. Pinelli, P. Lunghi, R. Albertini, J. Inorg. Biochem. 62 (1996) 41. ˜ A. Macıas, ´ A. Castineiras, ˜ ´ [4] M.V. Castano, A. Sanchez, E. Garcia Martinez, J. Sordo, S. Hiller, E.E. Castellano, J. Chem. Soc., Dalton Trans. (1990) 1001.
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[5] L.C. Andersson, K. Nilsson, C.G. Gahmberg, Int. J. Cancer 23 (1979) 143. [6] E. Foley, H. Lazarus, S. Farber, B.G. Uzman, B.A. Boone, R.E. McCarthy, Cancer 18 (1965) 522. [7] Y. Yamaguchi, M. Kanatani, T. Kasukabe, Y. Honma, Exp. Hematol. 24 (1996) 682. [8] G. Battistini, A. Marziali, R. Albertini, D. Habetswallner, D. Bulgarini, E.M. Coccia, G. Fiorucci, G. Romeo, R. Orsatti, V. Testa, E. Affabris, G. Peschle, G.B. Rossi, J. Biol. Chem. 266 (1991) 528. [9] Y. Maeda, R. Okawara, J. Organomet. Chem. 10 (1967) 247. [10] F.E. Andersen, C.J. Duca, J.V. Scudi, J. Am. Chem. Soc. 73 (1951) 4967. [11] J.L. de Boer, A.J.M. Duisenberg, Acta Crystallogr., Sect. A 40 (1984) 410. [12] A.L. Spek, J. Appl. Crystallogr. 21 (1988) 578. [13] N. Walker, D. Stuart, Acta Crystallogr., Sect. A 39 (1983) 158. ¨ Gottingen, ¨ 1986. [14] G.M. Sheldrick, SHELXS86, Universitat [15] B.A. Frenz and Associates Inc. & Enraf-Nonius, VAX/SDP Structure Determination Package, version 2.2, College Station, TX, USA, 1986. ¨ Gottingen, ¨ [16] G.M. Sheldrick, SHELXL97, Universitat 1997. [17] A.J.C. Wilson, International Tables for Crystallography, vol. C, Kluwer, Dordrecht, 1992. ¨ Heidelberg, 1997. [18] L. Zsolnai, ZORTEP, Universitat [19] A.L. Spek, Acta Crystallogr., Sect. A 46 (1990) C34. [20] S.M. Orkin, F. Harosi, P. Leder, Proc. Natl. Acad. Sci. USA 72 (1975) 98. [21] A. Bondi, J. Phys. Chem. 68 (1964) 441. ˜ M.S. Garcia Tasende, I. Martinez Santa[22] J.S. Casas, M.V. Castano, ´ marta, A. Sanchez, J. Sordo, E.E. Castellano, J. Zukerman-Schpector, J. Chem. Res. (S) (1992) 324; M (1992) 2626. [23] I. Haiduc, I. Silaghi-Dumitrescu, R. Grecu, R. Constantinescu, L. Silaghi-Dumitrescu, J. Mol. Struct. 114 (1984) 467. [24] D.C. Silvestru, I. Haiduc, J. Organomet. Chem. 365 (1989) 83. ´ ¨ ´ [25] J.S. Casas, M.C. Rodrıguez Arguelles, U. Russo, A. Sanchez, J. Sordo, ´ ´ A. Vazquez Lopez, S. Pinelli, P. Lunghi, A. Bonati, R. Albertini, J. Inorg. Biochem. 69 (1998) 283. ´ ´ ´ [26] J.S. Casas, A. Sanchez, J. Sordo, A. Vazquez Lopez, E.E. Castellano, ´ ¨ J. Zukerman-Schpector, M.C. Rodrıguez Arguelles, U. Russo, Inorg. Chim. Acta 216 (1994) 169. [27] T.P. Lockhart, W.P. Manders, Inorg. Chem. 25 (1986) 892. ´ ´ [28] E. M. Vazquez Lopez, Ph.D. Thesis, University of Santiago de Compostela, Santiago de Compostela, Spain, 1993. [29] K. Pihlaja, E. Kleinpeter, Carbon-13 NMR Chemical Shifts in Structural and Stereochemical Analysis, VCH, New York, 1994, p. 332. [30] T.P. Lockhart, W.P. Manders, J.J. Puckerman, J. Am. Chem. Soc. 107 (1985) 4546. [31] W.F. Howard, R.W. Crecely, W.H. Nelson, Inorg. Chem. 24 (1985) 2204. ´ ´ ´ [32] J.S. Casas, A. Sanchez, J. Sordo, E.M. Vazquez Lopez, Polyhedron 11 (1992) 2889. ´ ´ [33] A. Vazquez Lopez, Ph.D. Thesis, University of Santiago de Compostela, Santiago de Compostela, Spain, 1996. ´ ´ A. Lycka, J. Organomet. Chem. [34] J. Holecek, M. Nadvornik, K. Handlır, 315 (1986) 299.
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