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Diorganotin(IV) complexes of pyridoxal thiosemicarbazone: Synthesis, spectroscopic properties and biological activity
Journal of Inorganic Biochemistry 69 (1998) 283±292
Diorganotin(IV) complexes of pyridoxal thiosemicarbazone: Synthesis, spectroscopic properties and biological activity Jose S. Casas a,*, Marõa C. Rodrõguez-Arg uelles b, Umberto Russo c, Agustõn S anchez a, pez a, Silvana Pinelli d, Paolo Lunghi d, azquez-Lo Jose Sordo a, Antonia V Antonio Bonati d, Roberto Albertini d a
d
Departamento de Quõmica Inorg anica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain b Departamento de Quõmica Inorg anica, Universidade de Vigo, 36200 Vigo, Spain c Dipartimento di Chimica Inorganica, Metallorganica ed Analitica, Universit a di Padova, 35131 Padova, Italy Istituto di Patologia Speciale Medica and Centro di Ricerca Interuniversitario per la diagnosi, terapia e prognosi dei tumori humani, Universit a di Parma, 43100 Parma, Italy Received 22 September 1997; received in revised form 3 December 1997; accepted 11 December 1997
Abstract The complexes [SnR2 (L)] (R Me, Et, Bu, Ph; H2 L pyridoxal thiosemicarbazone) have been prepared and characterized. In the light of the spectral properties of the complexes in the solid state (IR, mass, M ossbauer) the bideprotonated thiosemicarbazonato anion is O(phenolic)-, N(3)-, S-bonded to the tin atom which probably has trigonal bipyramidal coordination with N(3) atom and R groups occupying equatorial positions. NMR ( 1 H, 13 C and 119 Sn) data in CDCl3 or DMSO-d6 suggest that this coordinative picture remains in these solutions. The ethyl, butyl and phenyl derivatives suppress proliferation of Friend erithroleukaemia cells (FLC). Of the pyridoxal thiosemicarbazone complexes so far evaluated, [SnBu2 (L)] and [SnPh2 (L)] showed the lowest thresholds for inhibition of FLC proliferation. The eects of these compounds on DMSO-induced dierentiation of FLC, DNA synthesis and reverse transcriptase were also assayed. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Diorganotine complexes; Pyridoxal thiosemicarbazone complexes; Biological activity studies
1. Introduction Thiosemicarbazones were the ®rst antiviral substances to be synthesized and have a wide range of pharmacological applications [1]. The in¯uence of certain metals on the biological activity of these compounds and their intrinsic chemical interest as multidentate ligands has prompted a considerable increase in the study of their coordination behaviour [2]. As part of our research on the thiosemicarbazonates of main group elements [3] we recently became interested in the structures and pharmacological properties of organotin thiosemicarbazonates [4]. In biological assays against Friend erithroleukaemia cells (FLC), organotin derivatives of both pyridine-2-carbaldehyde thiosemicarbazone and 2,6-diacetylpyridine bis(thiosemicarba-
*
Corresponding author. Tel.: +34 9 81 594636; fax: +34 9 81 594912; e-mail: [email protected]. 0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 0 0 0 0 4 - X
zone) suppressed cell proliferation but they have dierent eects on DMSO-induced FLC dierentiation [4c]. To investigate the extent to which the in¯uence of the organotin substituent on biological activity depends on the thiosemicarbazone ligand, we have now prepared the complexes [SnR2 (L)] (R Me, Et, Bu, Ph) where H2 L pyridoxal thiosemicarbazone (I) a ligand which complexes with other metals [5] showed biological activity. This paper describes their synthesis, characterization and biological activity
284
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
Induction of Erythroid Dierentiation. FLC were seeded at 1 105 /ml in culture medium to which complexes had been added previously. Cell mortality evaluated by the trypan blue exclusion method never exceeded 5%. The degree of dierentiation was determined by evaluating the percentage of benzidine positive (B ) cells [8]. Reverse Transcriptase (RT) Assay. This assay was performed in a ®nal volume of 100 ll containing 20 mM tris HCl (pH 7.8), 60 mM NaCl, l mM MnCl2 , 5 mM dithiothreitol, 5 ll/ml poly r(A), 0.5 lg/ml oligo d(T), 2 lM TTP and 3 H-TTP (80 Ci/mmol, 20 lCi/ ml). Clari®ed supernatants were ultracentrifuged at 200,000x g at 4 C, then diluted in 100 ll of TNE (10 mM tris HCl pH 7.4, 100 mM NaCl, l mM EDTA) containing of 0.1% TRITON-X. Samples ranging from 5 to 30 ll of this solution were added to the reaction mixture. Enzymatic activity was expressed as c.p.m. per 106 cells incorporated into acid insoluble material. DNA Synthesis. Cells were grown to con¯uence in 60 mm diameter plastic culture dishes containing RPMI 1640 with 5% FCS. The cells were treated with the compounds, then seeded in complete medium microwell plates at 2 105 /well. 3 H-Thymidine (6.7 Ci/mmol, 1 lCi/ml) was added to the cultures at various times thereafter. 3 H-Thymidine incorporation into DNA was determined 4 h after exposure. The cells were washed three times with PBS, ®xed with ice-cold 10% trichloroacetic acid, and after an additional wash and ®ltration, the radioactivity associated with acid insoluble material was determined by liquid scintillation spectrometry.
2. Experimental 2.1. Chemical studies Thiosemicarbazide (Merck), pyridoxal hydrochloride (Aldrich), dimethyltin(IV) chloride (Alfa), diethyltin(IV) chloride (Aldrich), dibutyltin(IV) oxide (Aldrich) and diphenyltin(IV) chloride (Aldrich) were used as supplied. The ligand (H2 L, I) and the diorganotin(IV) oxides other than Bu2 SnO were obtained by the procedures described in Refs. [6] and [7], respectively. Preparation of the Complexes. The ligand and the appropriate diorganotin(IV) oxide were suspended in 100 ml of benzene (caution) and re¯uxed for several days in a round botton ¯ask ®tted with a Dean Stark moisture trap. After cooling, the solid residue was removed by ®ltration and the solution was slowly concentrated until a solid formed which was ®ltered o and vacuum dried. The speci®c conditions of the syntheses and the elemental analyses and physical properties of the compounds are listed in Table 1. Measurements. Elemental analyses were performed with a Carlo-Erba 1108 analyser. Melting points were determined with a B uchi apparatus. IR spectra were recorded in KBr pellets or Nujol mulls on a Bruker IFS66V spectrometer. Mass spectra were obtained using a Kratos MS50TC spectrometer connected to a DS90 data system and operating in electron impact (EI) mode (direct insertion probe, 70 eV, 250 C); all fragments were identi®ed using DS90 software. The M ossbauer spectra were recorded at 80 K in a constant acceleration apparatus and d was referred to r.t. SnO2 . 1 H and 13 C NMR spectra were recorded at room temperature on Bruker WM250, AMX300 or AMX500 instruments and were referred to the solvents signals (for 1 H, 7.27 in CDCl3 and 2.50 in DMSO-d6 ; for 13 C 77.0 in CDCl3 and 39.50 ppm in DMSO-d6 ). 119 Sn NMR spectra were recorded at room temperature on a Bruker AMX500 and were referred to external neat Sn CH3 4 .
3. Discussion Mass spectra. Table 2 lists the principal ions detected in the EI mass spectra. The molecular ion appears in all cases, but is very weak for the ethyl and butyl compounds; the most stable of the complexes under EI at the working temperature is [SnMe2 (L)]. The fragmentation patterns of the dialkyltin compounds are similar to those reported in other cases [4a]. IR spectra. Table 3 lists the main bands for the ligand and its complexes, along with their assignment following Refs. [5,6,9±12]. The m(NH2 ), m(OH) bands, show the non-coordination of the NH2 group and the presence of the 3155 cmÿ1 H2 L band in the spectra of the com-
2.2. Biological studies Stock solutions of the ligand and the complexes were prepared in DMSO (0.4%) and stored at room temperature. Cells. Clone 745A Friend erythroleukaemia cells (FLC) were grown in RPMI 1640 supplemented with 5% fetal calf serum (FCS) and antibiotics.
Table 1 Synthesis conditions, analytical data and some physical properties of [SnR2 (L)] Reactants(g)
M.P. ( C)
Compound
Tin compound
Ligand
Me2 SnO (0.50) Et2 SnO (0.32) Bu2 SnO (0.59) Ph2 SnO (0.42)
0.73 0.40 0.58 0.29
C11 H16 N4 O2 SSn C13 H20 N4 O2 SSn C17 H28 N4 O2 SSn C21 H20 N4 O2 SSn
[SnMe2 (L)] [SnEt2 (L)] [SnBu2 (L)] [SnPh2 (L)]
213 177 126 207
Colour
Yellow Yellow Yellow Yellow
Analysis % found(calc.) C
H
N
33.9(34.1) 37.8(37.6) 43.6(43.3) 49.1(49.1)
4.1(4.2) 5.0(4.9) 6.4(6.0) 4.6(4.2)
14.2(14.4) 13.2(13.5) 11.7(11.9) 10.1(10.9)
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
285
Table 2 Main metallated peaks in the IE mass spectra of compounds m/z
Intensity(rel)
Assignment
m/z
Intensity(rel)
Assignment
[SnMe2 (L)] 388 373 358 149 135 120
24 100 45 17 30 15
[M] [M-Me] [M-2Me] [SnMe2 ] [SnMe] [Sn]
[SnBu2 (L)] 471 415 358 341 209 119
4 100 26 9 15 16
[M] [M-Bu] [M-2Bu] [C9 H7 N3 O2 SSn] [CH4 N3 SSn] [Sn]
[SnEt2 (L)] 418 388 359 328 211 179 120
3 100 20 27 62 19 11
[M] [M-Et] [M-2Et] [M-2Et-CH2 OH] [CH4 N3 SSn] [SnEt2 ] [Sn]
[SnPh2 (L)] 512 435 358 197 120 78
10 23 2 17 11 100
[M] [M-Ph] [M-2Ph] [SnPh] [Sn] [C5 H5 N]
Table 3 Main IR bands (cmÿ1 ) of H2 L and the complexes
a
Assignment
H2 L
[SnMe2 (L)]
[SnEt2 (L)]
[SnBu2 (L)]
m(NH2 ) + m(OH)
3440 m 3240 s 3155 s, b 1600 s 1535 s 1526 s 1505 s 1460 m 1423 m 1298 m 1260 m 1230 m 1100 s 1040 m 920 m
3441 w 3277 s 3163 m, b 1620 m 1531 m
3441 w 3298 m 3148 m, b 1616 m 1528 m
± 3316 w 3142 m 1620 sh 1522 w
3439 m 3242 m 3155 s 1613 m 1528 m
1500 sh 1468 s 1412 m 1339 m 1262 m 1207 m 1153 m 1016 m 891 m
1510 sh 1466 s, sh 1396 m 1298 w 1265 m 1211 m 1169 m 1022 m 893 w
1483 s, b 1404 m 1304 m 1261 m 1201 m 1163 m 1020 m 885 w
1491 s 1427 m 1368 m 1298 m 1261 m 1198 m 1170 w 1040 m 918 w 883 w
m(NH) m(C@N) + m(C@C) m(C@N) Ring m(C±O) phenyl d(NCS)
m(C@S) a
[SnPh2 (L)]
Abbreviations: m medium, w weak, s strong, b broad, sh shoulder.
plexes, albeit with less intensity, suggests that in H2 L this band is not a pure m(N±H) band. Other changes in the IR spectrum of H2 L upon coordination are similar to those occurring upon the formation of complexes in which X-ray diraction studies show this ligand [5,9b,9e] or related thiosemicarbazones [13] to be O-, N(3)- and S- coordinated. O-coordination is shown by the permanence or slight shift to higher wavenumbers of m(C±O), N(3)-coordination by the slight shift undergone by the band contributed to by m(C@N) and also by the elimination of bands from the 1500±1400 cmÿ1 region [5] and S-coordination by the shifts to lower wavenumbers undergone by the m(C@S) band. Similar O,N,S coordination of salicylaldehyde thiosemicarbazone [4a] to diorganotin(IV) ions leads to C±
Sn±C angles close to 130 in the latter; the non linearity of the C±Sn±C fragment in the [SnR2 (L)] complexes is con®rmed by the M ossbauer data (vide infra). M ossbauer data. All M ossbauer spectra show a single quadrupole split doublet with a narrow linewidth, consistent with there being just a single tin site in all these compounds. Both the isomer shift and the quadrupole splitting (Table 4) are typical of diorganotin(lV) derivatives. The d value slightly increases on going from the methyl to the butyl derivative, as expected, and is lower for the phenyl complex because of the electronegativity of the Ph groups. For all the compounds DEq is quite small, intermediate between values typical of tetraand penta-coordination. Comparison with data for compounds studied by X-ray diraction suggests trigon-
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J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
Table 4 M ossbauer parameters of compounds at 80.0 K Compound
da (mm/s)
DEq (mm/s)
C (mm/s)
[SnMe2 (L)] [SnEt2 (L)] [SnBu2 (L)] [SnPh2 (L)]
1.30 1.34 1.41 1.12
2.44 2.52 2.42 1.91
0.84 0.89 0.86 0.84
a
Relative to room temperature SnO2 .
al bipyramidal coordination, with N(3) atom and R groups occuping equatorial positions. Point charge calculations [14] performed ignoring the in¯uence of the thiosemicarbazone on the distribution of the bonding electrons among the hybrid tin orbitals but taking into account the C±Sn±C bond angle found experimentally in similar compounds [4a,14] aorded greatly overestimated values of DEq (3.28 and 2.70 mmsÿ1 for the alkyl and the phenyl derivatives, respectively), whereas calculations performed assuming ideal trigonal bipyramidal C±Sn±C angle of 120 but taking the eects of the ligand into account by giving the bonding atoms appropriate p.q.s. values (ÿ0.13 mm.sÿ1 for O(phenolic), 0.2 for N(3) and )0.6 for S) gave values of 2.38 and 2.03 mm sÿ1 in good agreement with those found experimentally. All this puts in clear evidence that the in¯uence of this type of ligands, and specially the coordination via anionic sulfur, cannot be neglected at least in the simpli®ed mainframe of the point charge model. Its eect is in fact much larger than that due to the deviations of alkyl or aryl groups from their ideal positions. NMR spectra. NMR signals were identi®ed using previously published data [3f] and, when necessary, HMQC and HMBC experiments. Table 5 list 1 H NMR data in DMSO-d6 and CDCl3 . In DMSO-d6 bideprotonation of the ligand upon complexation suppresses the N(2)H and C(7)OH signals that appear as broad singlets at 11.61 and 9.64 ppm, respectively in the spectrum of H2 L. The two very broad singlets for N(l)H2 at 8.33 and 8.10 ppm in the free ligand spectrum merge in those of the complexes like a single basal ligand as a consequence of structural and electronic changes upon complexation (reduction of C(l)±N(l) bond order due to thione to-thiol evolution [3f,4a], and the breaking of the intramolecular N(l)H. . .N(3) hydrogen bond when the ligand conformation changes from Z to E [3e,15]). The sharp singlet for C(2)H at 8.56 ppm in the H2 L spectrum shifts down®eld in those of the complexes, in which it is ¯anked by two satellites corresponding to 3 J( 1 H119 Sn) values, Table 5; this is indicative of the coordination of N(3) to the organometallic moiety. Similarly, the shielding of C(5)H may be attributed to coordination through the phenolic oxygen. The positions of the C(8)OH and C(8)H2 signals are unaected by complexation, so coordination through C(8)OH may be ruled out. The ligand is too poorly soluble in CDCl3 for NMR spectroscopy. For the complexes, the 1 H NMR data in
CDCl3 follow the same trend as in DMSO-d6 . There are no peaks corresponding to N(2)H and C(7)OH, N(l)H2 appears as a single signal (at higher ®eld than in DMSOd6 ) and C(2)H, which like C(5)H is more deshielded than in DMSO, is ¯anked by two satellites. The C(8)OH signal was only identi®ed in the [SnBu2 (L)] spectrum in which the C(8)OH and C(8)H2 signals lay respectively slightly up®eld and slightly down®eld of their positions in the spectrum run in DMSO-d6 . For [SnMe2 (L)] the 1 H chemical shift of the organometallic moiety and the value of 2 J( 1 H- 119 Sn) are very 0 close to those found for the compound [SnMe2 L ] 0 (L salicylaldehyde thiosemicarbazone) [4a]. Together with the C±Sn±C angle of 123 obtained upon substituting the 2 J value in the Lockhart±Manders equation [16], this con®rm that [SnMe2 (L)] must have the same trigonal bipyramidal arrangement as was reported for 0 [SnMe2 L ]. Both H2 L and [SnPh2 (L)] are too poorly soluble in CDC13 for 13 C and 119 Sn NMR spectroscopy. The 13 C and 119 Sn NMR spectra of the other compounds in CDCl3 and those of all the compounds in DMSOd6 are summarized in Table 6. [SnMe2 (L)] was too insoluble in CDCl3 for conventional 13 C spectroscopy but it was possible to measure its 13 C chemical shifts by means of an HMBC experiment; and both HMQC and HMBC (Fig. 1), experiments were performed for [SnBu2 (L)]. The C(1) signal appears at higher ®eld in the spectra of the complexes than in that of H2 L, and its position is independent of the solvent.0 This behaviour, which is similar to that of [SnR2 L ] [4a], suggests thione-tothiol evolution in the ligand, an increase in the C(1)N(2) bond order [17] and the possible coordination of the tin atom to the thiolic sulphur atom. C(2), C(6) and C(7) are strongly deshielded by complexation, especially in CDCl3 , suggesting coordination of the organometallic fragment to the phenolic oxygen atom. The C(3) and C(5) signal appear at higher ®eld in the spectra of complexes than in that of the free ligand and are only slightly aected by the solvent. The chemical shifts of the other ligand carbons undergo no signi®cant changes upon complexation. The similarity between the 13 C chemical shifts of the 0 organometallic moiety in [SnMe2 (L)] and [SnMe2 L ] [4a] reinforce the conclusions of the proton study about the coordination sphere around the metallic atom. The proton coupled 119 Sn spectrum of [SnMe2 (L)] in DMSO-d6 shows two kinds of coupling, one corresponding to the usual septuplet 2 J( 119 Sn-CH3 ) and a second, corresponding to 3 J( 1 H- 119 Sn), that splits the septuplet in doublets and con®rms the interaction of the tin atom with N(3). The 119 Sn chemical shift of this compound in CDC1 3 , ÿ102.9 ppm, is close to that 0 found in [SnMe2 L ] [4a] and is in the accepted range of 90 to ÿ330 ppm for pentacoordination around the tin atom in methyltin compounds [18]. In CDCl3 the 119 Sn chemical shifts of the other compounds appear at progressively higher ®eld as the aliphatic chain lengthens but this sequence is not followed in DMSOd6 possibly because of the interaction between DMSO
)
5.10s, br(2)
CDCl3
b
a
CDCl3
5.27s, br(2)
b
)
)
)
5.15s, br(2)
CDCl3
DMSO-d6
)
7.15s, br(2)
)
5.10s, br(2)
CDCl3
DMSO-d6
)
7.07s, br(2)
DMSO-d6
a
9.07s(1)
8.72s(1)
9.07s(1)
8.83s(1)
9.03s(1)
8.78s(1)
9.02s(1)
8.75s(1)
8.56s(1)
d[C(2)H]
7.74s(1)
7.36s(1)
7.69s(1)
7.62s(1)
7.74s(1)
7.57s(1)
7.74s(1)
7.59s(1)
7.97s(1)
d[C(5)H]
)
)
)
)
)
)
)
)
9.64s, vbr(1)
d[C(7)OH]
4.50d(2) J 5.0
4.49d(2) J 4.8 4.73s(2)
4.56d(2) J 5.3 4.49d(2) J 5.0 4.73s(2)
d[C(8)H2 ]
5.27t(1) J 5.0 3.2s, br(2)
4.49d(2) J 5.1 4.69s(2)
5.17s, vbr(1) 4.71s(2)
5.28t(1) J 5.1
5.25t(1) J 5.1 )
5.26t(1) J 5.3 5.26t(1) J 5.0 )
d[C(8)OH]
2.68s(3)
2.45s(3)
2.42s(3)
2.28s(3)
2.45s(3)
2.29s(3)
2.42s(3)
2.27s(3)
2.39s(3)
d[C(9)H3 ]
Ha 1.32m(4) Hb 1.15t(6) Ha 1.50m(4) Hb 1.31t(6) Ha 1.40m(4) Hb 1.55m(4) Hc 1.25m(4) Hd 0.79t(6) Ha 1.57m(4) Hb 1.66m(4) Hc 1.35m(4) Hd 0.89t(6) Ho 7.60m(4) Hm;p 7.27m(6) Ho 7.87m(4) Hm;p 7.41m(6)
0.88s(6)
0.70s(6)
)
d(R-Sn)
J
)
)
83.0 79.7 72.7 69.6 140.3 134.3 135.2 129.2
2
J
40.9
41.4
35.7
31.1
35.5
28.3
37.8
33.0
3
Numbering scheme for ligand see I. Abbrevations: s singlet, d doublet, t triplet, m multiplet, b broad, v very. In parentheses relative number of protons calculated by integration. Overlapped Hm;p .
[SnPh2 (L)]
[SnBu2 (L)]
[SnEt2 (L)]
)
DMSO-d6
[SnMe2 (L)]
11.61s, vbr(1)
8.33s, vbr(1) 8.10s, vbr(1) 7.12s, br(1)
DMSO-d6
d[N(2)H]
H2 L
d[N(1)H2 ]
Solvent
Compound
Table 5 t 1 H NMR data (d, ppm; J, Hz) of the ligand and complexes
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292 287
[SnPh2 (L)]
[SnBu2 (L)]
167.70
152.29
155.92
169.91
CDCl3
DMSO-d6
153.16
169.04
DMSO-d6
156.61
169.32
CDCl3
[SnEt2 (L)]
142.17 152.84 156.67 152.85
178.01 169.04 169.15 169.35
DMSO-d6 DMSO-d6 CDCl3 DMSO-d6
d[C(2)]
H2 L [SnMe2 (L)]
d[C(1)]
Sn NMR data of the ligand and complexes
Solvent
119
Compound
Table 6 13 C, and
119.16
118.26
118.23
117.68
121.48 118.35 117.54 118.37
d[C(3)]
132.69
132.11
132.56
131.16
132.80 132.50 131.05 132.63
d[C(4)]
135.24
135.01
134.64
135.41
139.10 134.53 135.67 134.25
d[C(5)]
150.35
154.21
152.10
155.20
147.03 152.27 155.14 152.20
d[C(6)]
157.52
158.79
157.77
159.10
148.97 157.35 158.37 158.06
d[C(7)]
59.09
60.17
59.08
60.99
58.91 59.01 60.98 59.15
d[C(8)]
19.80
19.21
19.29
19.75
19.12 19.27 19.71 19.44
d[C(9)] ) 7.69 5.82 Ca 19.95 Cb 10.00 Ca 17.72 Cb 9.90 Ca 26.16 Cb 27.24 Cc 25.82 Cd 18.52 Ca 25.89 Cb 27.42 Cc 26.47 Cd 13.61 Ci 149.05 Co 134.50 Cm 128.14 Cp 128.50
d[R]
J
556.4/533.0 30.5 87.5
605.3/574.8 33.6 89.5
644.9 42.7
n
ÿ323.6
ÿ123.1
ÿ147.7
ÿ119.6
) ÿ158.2 ÿ102.9 ÿ170.8
d119 Sn]
288 J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
289
with trigonal bipyramidal coordination where the N(3) atom and R groups probably occupy equatorial positions. 4. Biological activity
Fig. 1. 2D 1 H-13 C HMBC spectrum at 500, 137 MHz of [SnBu2 (L)] in CDCl3 solution showing the correlation peak patterns.
and the organometallic fragments modi®es when ÿR changes. In [SnPh2 (L)] the nucleus is more shielded than might be expected given the large capability of the phenyl group for electron withdrawal [4a,19]. To sum up, both solid state and solution studies on [SnR2 (L)] suggest the presence of O(phenolic)-, N(3)-, S-bonded thiosemicarbazonato anions and tin atoms
FLC proliferation. The ligand had no inhibitory eect on FLC proliferation even at concentrations of around 30 lg/ml. Inhibition was total for the butyl and phenyl complexes, 75% for the ethyl complex, and zero for the methyl complex (Fig. 2). Inhibition by the ethyl, butyl and phenyl complexes was maintained down to concentrations of 1, 0.065 and 0.22 lg/ml, respectively (data not shown). DMSO-induced FLC dierentiation. As shown in Fig. 3, [SnEt2 (L)] which had a marked inhibitory eect on cell proliferation, it did not aect cell dierentiation. On the contrary, the phenyl and butyl complexes inhibited cell dierentiation at a dose that did not inhibit cell proliferation. DNA synthesis. In keeping with the results on FLC proliferation, the butyl and phenyl complexes totally inhibited synthesis of DNA (Fig. 4). However, the ethyl complex had no eect on DNA synthesis although it inhibited FLC proliferation, while the methyl complex depressed DNA synthesis in spite of not aecting FLC proliferation. Eects on reverse transcriptase. H2 L inhibited reverse transcriptase activity by 50% at a concentration of 2 lg/
Fig. 2. Eects on FLC growth. Cells were counted at days 3th and 4th. Each condition was performed in duplicate.
290
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
Fig. 3. Eects on FLC DMSO-induced dierentiation. Each condition was performed in duplicate.
ml, while the methyl and ethyl complexes had no eect at this concentration and the butyl complex caused total inhibition (Fig. 5). The phenyl complex also caused a strong reduction of the RT activity. In this case the in-
hibition was not total although this complex totally inhibited cell proliferation at the same dosis. General conclusions. The above results are in keeping with those obtained with other thiosemicarbazones in
Fig. 4. Eects on DNA synthesis in growing FLC. Each value represents the mean of three experiments.
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
291
Fig. 5. Eects on reverse transcriptase activity in growing FLC. Each value represents the mean of three experiments.
that complexation increased the biological activity of the ligand [4c,5,20]. Together with results for the copper and cobalt complexes of the same ligand [5], they allow the following conclusions. (a) Pyridoxal thiosemicarbazone has no activity even at high concentrations (and regardless of whether it is dissolved in DMSO or ethanol/water). (b) Of the pyridoxal thiosemicarbazone complexes so far evaluated, those with the lowest thresholds for inhibition of FLC proliferation are [SnBu2 (L)] and [SnPh2 (L)]. (c) On the contrary of [Cu(H2 L)(OH)2 Cl]Cl and [fCu(HL)(H2 O)g2 ]Cl2 á H2 O [5], the tin complexes do not enhance DMSO-induced FLC dierentiation. (d) The strong dependence of the results for the SnR2 complexes on the identity of R suggests the desirability of carrying out further assays to determine the mechanistic basis of this dependence. Acknowledgements We thank Mrs. Cristina Everti for technical assistance. Finantial support by the Xunta de Galicia, Spain, (Project XUGA 2038B97), the Associazione Italiana per la Ricerca sul Cancro and CNR, Italy, is acknowledged. MCRA is grateful for a grant from the University of Vigo, Spain.
[3]
[4]
[5] [6] [7] [8] [9]
References [1] C.J. Pfau, Handb. Exp. Pharmacol. 61 (1982) 147. [2] (a) M.J.M. Campbell, Coord. Chem. Rev. 15 (1975) 279; (b) S. Padhye, G.B. Kauman, Coord. Chem. Rev. 63 (1985) 127; (c) D.X. West, S.B. Padhye, P.B. Sonawane, Structure and Bonding 76 (1991) 4; (d) D.X. West, A.E. Liberta, S.B. Padhye, R.C.
[10] [11]
Chitake, P.B. Sonawane, A.S. Kumbhar, R.G. Yerande, Coord. Chem. Rev. 123 (1993) 49. (a) A. Macõas, M.C. Rodrõguez-Arg uelles, M.I. Su arez, A. S anchez, J.S. Casas, J. Sordo. J. Chem. Soc., Dalton Trans. (1989) 1787; (b) J. Zukerman-Schpector, M.C. RodrõguezArg uelles, M.I. Su arez, A. S anchez, J.S. Casas, J. Sordo, J. Coord. Chem. 24 (1991) 177; (c) J.S. Casas, M.V. Casta~ no, M.S. Garcõa-Tasende, I. Martõnez-Santamarta, A. S anchez, J. Sordo, E.E. Castellano, J. Zukerman-Schpector, J. Chem. Res. 5 (1992) 324; (d) J.S. Casas, E.E. Castellano, A. Macõas, M.C. RodrõguezArg uelles, A. S anchez, J. Sordo, J. Chem. Soc., Dalton Trans. (1993) 353; (e) J.S. Casas, M.V. Casta~ no, M.C. RodrõguezArg uelles, A. S anchez, J. Sordo, J. Chem. Soc., Dalton Trans. (1993) 1253; (f) J.S. Casas, E.E. Castellano, M.C. RodrõguezArg uelles, A. S anchez, J. Sordo, J. Zukerman-Schpector, Inorg. Chim. Acta 260 (1997) 183. (a) J.S. Casas, A. S anchez, J. Sordo, A. V azquez-L opez, E.E. Castellano, J. Zukerman-Schpector, M.C. Rodrõguez-Arg uelles, U. Russo, Inorg. Chim. Acta 216 (1994) 169; (b) J.S. Casas, A. Casti~ neiras, A. S anchez, J. Sordo, A. V azquez-L opez, M.C. Rodrõguez-Arg uelles, U. Russo, Inorg. Chim. Acta 221 (1994) 61; (c) J.S. Casas, M.S. Garcõa-Tasende, C. Maichle-M ossmer, M.C. Rodrõguez-Arg uelles, A. S anchez, J. Sordo, A. V azquez-L opez, S. Pinelli, P. Lunghi, R. Albertini, J. Inorg. Biochem. 62 (1996) 4l. M. Belicchi Ferrari, G. Gasparri Fava, P. Tarasconi, R. Albertini, S. Pinelli, R. Starcich, J. Inorg. Biochem. 53 (1994) 13. M. Ferrari Belicchi, G. Fava Gasparri, C. Pelizzi, P. Tarasconi, G. Tosi, J. Chem. Soc. Dalton Trans. 2455 (1986). R.S. Tobias, Y. Ogrins, B.A. Nevett, Inorg. Chem. 1 (1962) 636. S.M. Orkin, F. Harosi, P. Leder, Proc. Natl. Acad. Sci. USA 72 (1975) 98. (a) M. Belicchi Ferrari, G. Gasparri Fava, C. Pelizzi, P. Tarasconi, G. Tosi, J. Chem. Soc., Dalton Trans. 227 (1987); (b) M. Belicchi Ferrari, G. Gasparri Fava, M. Lanfranchi, C. Pelizzi, P. Tarasconi, J. Chem. Soc., Dalton Trans. 1951 (1991); (c) M. Belicchi Ferrari, G. Gasparri Fava, C. Pelizzi, P. Tarasconi, J. Chem. Soc., Dalton Trans. 2153 (1992); (d) M. Belicchi Ferrari, G. Gasparri Fava, G. Pelosi, M.C. Rodrõguez-Arg uelles, P. Tarasconi, J. Chem. Soc., Dalton Trans. 3035 (1995). A. Syamal, M.R. Maurya, Transition Met. Chem. 11 (1986) 255. N.S. Gupta, M. Mohan, N.K. Jha, W.E. Antholine, Inorg. Chim. Acta 184 (1991) 13.
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[12] C. Colonna, A. Cosse-Barbi, A. Massat, R. Ben Abdelmoumene, J.P. Doucet, Bull. Soc., Chim. Belg. 102 (1993) 411. [13] G. Singh, P.A. Kumar, T.R. Rao, J. Chem. Res. (S) 123 (1994). [14] R.V. Parish, Structure and Bonding in tin compounds, M ossbauer spectroscopy applied to Inorganic Chemistry, in: G.J. Long (Ed.), Plenum Press, New York, 1984, ch. 16, p. 327. [15] D. Chattopadhyay, S.K. Mazumdar, T. Banerjee, S. Ghosh, T.C.W. Mak, Acta Crystallogr., Section C 44 (1988) 1025. [16] T.P. Lockhart, W.F. Manders, Inorg. Chem. 25 (1986) 892.
[17] A.M. Brodie, H.D. Holden, J. Lewis, M.J. Taylor, J. Chem. Soc., Dalton Trans. 633 (1986). [18] J. Otera, J. Organomet. Chem. 221 (1981) 57. [19] P.J. Smith, A.P. Tupciauskas, Annu. Rep. NMR Spectrosc. 8 (1978) 291. [20] M.C. Rodrõguez-Arguelles, M. Belicchi Ferrari, G. Gasparri Fava, C. Pelizzi, P. Tarasconi, R. Albertini, P.P. Dall'Aglio, P. Lunghi, S. Pinelli, J. Inorg. Biochem. 58 (1995) 157.
Diorganotin(IV) complexes of pyridoxal thiosemicarbazone: Synthesis, spectroscopic properties and biological activity Jose S. Casas a,*, Marõa C. Rodrõguez-Arg uelles b, Umberto Russo c, Agustõn S anchez a, pez a, Silvana Pinelli d, Paolo Lunghi d, azquez-Lo Jose Sordo a, Antonia V Antonio Bonati d, Roberto Albertini d a
d
Departamento de Quõmica Inorg anica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain b Departamento de Quõmica Inorg anica, Universidade de Vigo, 36200 Vigo, Spain c Dipartimento di Chimica Inorganica, Metallorganica ed Analitica, Universit a di Padova, 35131 Padova, Italy Istituto di Patologia Speciale Medica and Centro di Ricerca Interuniversitario per la diagnosi, terapia e prognosi dei tumori humani, Universit a di Parma, 43100 Parma, Italy Received 22 September 1997; received in revised form 3 December 1997; accepted 11 December 1997
Abstract The complexes [SnR2 (L)] (R Me, Et, Bu, Ph; H2 L pyridoxal thiosemicarbazone) have been prepared and characterized. In the light of the spectral properties of the complexes in the solid state (IR, mass, M ossbauer) the bideprotonated thiosemicarbazonato anion is O(phenolic)-, N(3)-, S-bonded to the tin atom which probably has trigonal bipyramidal coordination with N(3) atom and R groups occupying equatorial positions. NMR ( 1 H, 13 C and 119 Sn) data in CDCl3 or DMSO-d6 suggest that this coordinative picture remains in these solutions. The ethyl, butyl and phenyl derivatives suppress proliferation of Friend erithroleukaemia cells (FLC). Of the pyridoxal thiosemicarbazone complexes so far evaluated, [SnBu2 (L)] and [SnPh2 (L)] showed the lowest thresholds for inhibition of FLC proliferation. The eects of these compounds on DMSO-induced dierentiation of FLC, DNA synthesis and reverse transcriptase were also assayed. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Diorganotine complexes; Pyridoxal thiosemicarbazone complexes; Biological activity studies
1. Introduction Thiosemicarbazones were the ®rst antiviral substances to be synthesized and have a wide range of pharmacological applications [1]. The in¯uence of certain metals on the biological activity of these compounds and their intrinsic chemical interest as multidentate ligands has prompted a considerable increase in the study of their coordination behaviour [2]. As part of our research on the thiosemicarbazonates of main group elements [3] we recently became interested in the structures and pharmacological properties of organotin thiosemicarbazonates [4]. In biological assays against Friend erithroleukaemia cells (FLC), organotin derivatives of both pyridine-2-carbaldehyde thiosemicarbazone and 2,6-diacetylpyridine bis(thiosemicarba-
*
Corresponding author. Tel.: +34 9 81 594636; fax: +34 9 81 594912; e-mail: [email protected]. 0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 0 0 0 0 4 - X
zone) suppressed cell proliferation but they have dierent eects on DMSO-induced FLC dierentiation [4c]. To investigate the extent to which the in¯uence of the organotin substituent on biological activity depends on the thiosemicarbazone ligand, we have now prepared the complexes [SnR2 (L)] (R Me, Et, Bu, Ph) where H2 L pyridoxal thiosemicarbazone (I) a ligand which complexes with other metals [5] showed biological activity. This paper describes their synthesis, characterization and biological activity
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Induction of Erythroid Dierentiation. FLC were seeded at 1 105 /ml in culture medium to which complexes had been added previously. Cell mortality evaluated by the trypan blue exclusion method never exceeded 5%. The degree of dierentiation was determined by evaluating the percentage of benzidine positive (B ) cells [8]. Reverse Transcriptase (RT) Assay. This assay was performed in a ®nal volume of 100 ll containing 20 mM tris HCl (pH 7.8), 60 mM NaCl, l mM MnCl2 , 5 mM dithiothreitol, 5 ll/ml poly r(A), 0.5 lg/ml oligo d(T), 2 lM TTP and 3 H-TTP (80 Ci/mmol, 20 lCi/ ml). Clari®ed supernatants were ultracentrifuged at 200,000x g at 4 C, then diluted in 100 ll of TNE (10 mM tris HCl pH 7.4, 100 mM NaCl, l mM EDTA) containing of 0.1% TRITON-X. Samples ranging from 5 to 30 ll of this solution were added to the reaction mixture. Enzymatic activity was expressed as c.p.m. per 106 cells incorporated into acid insoluble material. DNA Synthesis. Cells were grown to con¯uence in 60 mm diameter plastic culture dishes containing RPMI 1640 with 5% FCS. The cells were treated with the compounds, then seeded in complete medium microwell plates at 2 105 /well. 3 H-Thymidine (6.7 Ci/mmol, 1 lCi/ml) was added to the cultures at various times thereafter. 3 H-Thymidine incorporation into DNA was determined 4 h after exposure. The cells were washed three times with PBS, ®xed with ice-cold 10% trichloroacetic acid, and after an additional wash and ®ltration, the radioactivity associated with acid insoluble material was determined by liquid scintillation spectrometry.
2. Experimental 2.1. Chemical studies Thiosemicarbazide (Merck), pyridoxal hydrochloride (Aldrich), dimethyltin(IV) chloride (Alfa), diethyltin(IV) chloride (Aldrich), dibutyltin(IV) oxide (Aldrich) and diphenyltin(IV) chloride (Aldrich) were used as supplied. The ligand (H2 L, I) and the diorganotin(IV) oxides other than Bu2 SnO were obtained by the procedures described in Refs. [6] and [7], respectively. Preparation of the Complexes. The ligand and the appropriate diorganotin(IV) oxide were suspended in 100 ml of benzene (caution) and re¯uxed for several days in a round botton ¯ask ®tted with a Dean Stark moisture trap. After cooling, the solid residue was removed by ®ltration and the solution was slowly concentrated until a solid formed which was ®ltered o and vacuum dried. The speci®c conditions of the syntheses and the elemental analyses and physical properties of the compounds are listed in Table 1. Measurements. Elemental analyses were performed with a Carlo-Erba 1108 analyser. Melting points were determined with a B uchi apparatus. IR spectra were recorded in KBr pellets or Nujol mulls on a Bruker IFS66V spectrometer. Mass spectra were obtained using a Kratos MS50TC spectrometer connected to a DS90 data system and operating in electron impact (EI) mode (direct insertion probe, 70 eV, 250 C); all fragments were identi®ed using DS90 software. The M ossbauer spectra were recorded at 80 K in a constant acceleration apparatus and d was referred to r.t. SnO2 . 1 H and 13 C NMR spectra were recorded at room temperature on Bruker WM250, AMX300 or AMX500 instruments and were referred to the solvents signals (for 1 H, 7.27 in CDCl3 and 2.50 in DMSO-d6 ; for 13 C 77.0 in CDCl3 and 39.50 ppm in DMSO-d6 ). 119 Sn NMR spectra were recorded at room temperature on a Bruker AMX500 and were referred to external neat Sn CH3 4 .
3. Discussion Mass spectra. Table 2 lists the principal ions detected in the EI mass spectra. The molecular ion appears in all cases, but is very weak for the ethyl and butyl compounds; the most stable of the complexes under EI at the working temperature is [SnMe2 (L)]. The fragmentation patterns of the dialkyltin compounds are similar to those reported in other cases [4a]. IR spectra. Table 3 lists the main bands for the ligand and its complexes, along with their assignment following Refs. [5,6,9±12]. The m(NH2 ), m(OH) bands, show the non-coordination of the NH2 group and the presence of the 3155 cmÿ1 H2 L band in the spectra of the com-
2.2. Biological studies Stock solutions of the ligand and the complexes were prepared in DMSO (0.4%) and stored at room temperature. Cells. Clone 745A Friend erythroleukaemia cells (FLC) were grown in RPMI 1640 supplemented with 5% fetal calf serum (FCS) and antibiotics.
Table 1 Synthesis conditions, analytical data and some physical properties of [SnR2 (L)] Reactants(g)
M.P. ( C)
Compound
Tin compound
Ligand
Me2 SnO (0.50) Et2 SnO (0.32) Bu2 SnO (0.59) Ph2 SnO (0.42)
0.73 0.40 0.58 0.29
C11 H16 N4 O2 SSn C13 H20 N4 O2 SSn C17 H28 N4 O2 SSn C21 H20 N4 O2 SSn
[SnMe2 (L)] [SnEt2 (L)] [SnBu2 (L)] [SnPh2 (L)]
213 177 126 207
Colour
Yellow Yellow Yellow Yellow
Analysis % found(calc.) C
H
N
33.9(34.1) 37.8(37.6) 43.6(43.3) 49.1(49.1)
4.1(4.2) 5.0(4.9) 6.4(6.0) 4.6(4.2)
14.2(14.4) 13.2(13.5) 11.7(11.9) 10.1(10.9)
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
285
Table 2 Main metallated peaks in the IE mass spectra of compounds m/z
Intensity(rel)
Assignment
m/z
Intensity(rel)
Assignment
[SnMe2 (L)] 388 373 358 149 135 120
24 100 45 17 30 15
[M] [M-Me] [M-2Me] [SnMe2 ] [SnMe] [Sn]
[SnBu2 (L)] 471 415 358 341 209 119
4 100 26 9 15 16
[M] [M-Bu] [M-2Bu] [C9 H7 N3 O2 SSn] [CH4 N3 SSn] [Sn]
[SnEt2 (L)] 418 388 359 328 211 179 120
3 100 20 27 62 19 11
[M] [M-Et] [M-2Et] [M-2Et-CH2 OH] [CH4 N3 SSn] [SnEt2 ] [Sn]
[SnPh2 (L)] 512 435 358 197 120 78
10 23 2 17 11 100
[M] [M-Ph] [M-2Ph] [SnPh] [Sn] [C5 H5 N]
Table 3 Main IR bands (cmÿ1 ) of H2 L and the complexes
a
Assignment
H2 L
[SnMe2 (L)]
[SnEt2 (L)]
[SnBu2 (L)]
m(NH2 ) + m(OH)
3440 m 3240 s 3155 s, b 1600 s 1535 s 1526 s 1505 s 1460 m 1423 m 1298 m 1260 m 1230 m 1100 s 1040 m 920 m
3441 w 3277 s 3163 m, b 1620 m 1531 m
3441 w 3298 m 3148 m, b 1616 m 1528 m
± 3316 w 3142 m 1620 sh 1522 w
3439 m 3242 m 3155 s 1613 m 1528 m
1500 sh 1468 s 1412 m 1339 m 1262 m 1207 m 1153 m 1016 m 891 m
1510 sh 1466 s, sh 1396 m 1298 w 1265 m 1211 m 1169 m 1022 m 893 w
1483 s, b 1404 m 1304 m 1261 m 1201 m 1163 m 1020 m 885 w
1491 s 1427 m 1368 m 1298 m 1261 m 1198 m 1170 w 1040 m 918 w 883 w
m(NH) m(C@N) + m(C@C) m(C@N) Ring m(C±O) phenyl d(NCS)
m(C@S) a
[SnPh2 (L)]
Abbreviations: m medium, w weak, s strong, b broad, sh shoulder.
plexes, albeit with less intensity, suggests that in H2 L this band is not a pure m(N±H) band. Other changes in the IR spectrum of H2 L upon coordination are similar to those occurring upon the formation of complexes in which X-ray diraction studies show this ligand [5,9b,9e] or related thiosemicarbazones [13] to be O-, N(3)- and S- coordinated. O-coordination is shown by the permanence or slight shift to higher wavenumbers of m(C±O), N(3)-coordination by the slight shift undergone by the band contributed to by m(C@N) and also by the elimination of bands from the 1500±1400 cmÿ1 region [5] and S-coordination by the shifts to lower wavenumbers undergone by the m(C@S) band. Similar O,N,S coordination of salicylaldehyde thiosemicarbazone [4a] to diorganotin(IV) ions leads to C±
Sn±C angles close to 130 in the latter; the non linearity of the C±Sn±C fragment in the [SnR2 (L)] complexes is con®rmed by the M ossbauer data (vide infra). M ossbauer data. All M ossbauer spectra show a single quadrupole split doublet with a narrow linewidth, consistent with there being just a single tin site in all these compounds. Both the isomer shift and the quadrupole splitting (Table 4) are typical of diorganotin(lV) derivatives. The d value slightly increases on going from the methyl to the butyl derivative, as expected, and is lower for the phenyl complex because of the electronegativity of the Ph groups. For all the compounds DEq is quite small, intermediate between values typical of tetraand penta-coordination. Comparison with data for compounds studied by X-ray diraction suggests trigon-
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J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
Table 4 M ossbauer parameters of compounds at 80.0 K Compound
da (mm/s)
DEq (mm/s)
C (mm/s)
[SnMe2 (L)] [SnEt2 (L)] [SnBu2 (L)] [SnPh2 (L)]
1.30 1.34 1.41 1.12
2.44 2.52 2.42 1.91
0.84 0.89 0.86 0.84
a
Relative to room temperature SnO2 .
al bipyramidal coordination, with N(3) atom and R groups occuping equatorial positions. Point charge calculations [14] performed ignoring the in¯uence of the thiosemicarbazone on the distribution of the bonding electrons among the hybrid tin orbitals but taking into account the C±Sn±C bond angle found experimentally in similar compounds [4a,14] aorded greatly overestimated values of DEq (3.28 and 2.70 mmsÿ1 for the alkyl and the phenyl derivatives, respectively), whereas calculations performed assuming ideal trigonal bipyramidal C±Sn±C angle of 120 but taking the eects of the ligand into account by giving the bonding atoms appropriate p.q.s. values (ÿ0.13 mm.sÿ1 for O(phenolic), 0.2 for N(3) and )0.6 for S) gave values of 2.38 and 2.03 mm sÿ1 in good agreement with those found experimentally. All this puts in clear evidence that the in¯uence of this type of ligands, and specially the coordination via anionic sulfur, cannot be neglected at least in the simpli®ed mainframe of the point charge model. Its eect is in fact much larger than that due to the deviations of alkyl or aryl groups from their ideal positions. NMR spectra. NMR signals were identi®ed using previously published data [3f] and, when necessary, HMQC and HMBC experiments. Table 5 list 1 H NMR data in DMSO-d6 and CDCl3 . In DMSO-d6 bideprotonation of the ligand upon complexation suppresses the N(2)H and C(7)OH signals that appear as broad singlets at 11.61 and 9.64 ppm, respectively in the spectrum of H2 L. The two very broad singlets for N(l)H2 at 8.33 and 8.10 ppm in the free ligand spectrum merge in those of the complexes like a single basal ligand as a consequence of structural and electronic changes upon complexation (reduction of C(l)±N(l) bond order due to thione to-thiol evolution [3f,4a], and the breaking of the intramolecular N(l)H. . .N(3) hydrogen bond when the ligand conformation changes from Z to E [3e,15]). The sharp singlet for C(2)H at 8.56 ppm in the H2 L spectrum shifts down®eld in those of the complexes, in which it is ¯anked by two satellites corresponding to 3 J( 1 H119 Sn) values, Table 5; this is indicative of the coordination of N(3) to the organometallic moiety. Similarly, the shielding of C(5)H may be attributed to coordination through the phenolic oxygen. The positions of the C(8)OH and C(8)H2 signals are unaected by complexation, so coordination through C(8)OH may be ruled out. The ligand is too poorly soluble in CDCl3 for NMR spectroscopy. For the complexes, the 1 H NMR data in
CDCl3 follow the same trend as in DMSO-d6 . There are no peaks corresponding to N(2)H and C(7)OH, N(l)H2 appears as a single signal (at higher ®eld than in DMSOd6 ) and C(2)H, which like C(5)H is more deshielded than in DMSO, is ¯anked by two satellites. The C(8)OH signal was only identi®ed in the [SnBu2 (L)] spectrum in which the C(8)OH and C(8)H2 signals lay respectively slightly up®eld and slightly down®eld of their positions in the spectrum run in DMSO-d6 . For [SnMe2 (L)] the 1 H chemical shift of the organometallic moiety and the value of 2 J( 1 H- 119 Sn) are very 0 close to those found for the compound [SnMe2 L ] 0 (L salicylaldehyde thiosemicarbazone) [4a]. Together with the C±Sn±C angle of 123 obtained upon substituting the 2 J value in the Lockhart±Manders equation [16], this con®rm that [SnMe2 (L)] must have the same trigonal bipyramidal arrangement as was reported for 0 [SnMe2 L ]. Both H2 L and [SnPh2 (L)] are too poorly soluble in CDC13 for 13 C and 119 Sn NMR spectroscopy. The 13 C and 119 Sn NMR spectra of the other compounds in CDCl3 and those of all the compounds in DMSOd6 are summarized in Table 6. [SnMe2 (L)] was too insoluble in CDCl3 for conventional 13 C spectroscopy but it was possible to measure its 13 C chemical shifts by means of an HMBC experiment; and both HMQC and HMBC (Fig. 1), experiments were performed for [SnBu2 (L)]. The C(1) signal appears at higher ®eld in the spectra of the complexes than in that of H2 L, and its position is independent of the solvent.0 This behaviour, which is similar to that of [SnR2 L ] [4a], suggests thione-tothiol evolution in the ligand, an increase in the C(1)N(2) bond order [17] and the possible coordination of the tin atom to the thiolic sulphur atom. C(2), C(6) and C(7) are strongly deshielded by complexation, especially in CDCl3 , suggesting coordination of the organometallic fragment to the phenolic oxygen atom. The C(3) and C(5) signal appear at higher ®eld in the spectra of complexes than in that of the free ligand and are only slightly aected by the solvent. The chemical shifts of the other ligand carbons undergo no signi®cant changes upon complexation. The similarity between the 13 C chemical shifts of the 0 organometallic moiety in [SnMe2 (L)] and [SnMe2 L ] [4a] reinforce the conclusions of the proton study about the coordination sphere around the metallic atom. The proton coupled 119 Sn spectrum of [SnMe2 (L)] in DMSO-d6 shows two kinds of coupling, one corresponding to the usual septuplet 2 J( 119 Sn-CH3 ) and a second, corresponding to 3 J( 1 H- 119 Sn), that splits the septuplet in doublets and con®rms the interaction of the tin atom with N(3). The 119 Sn chemical shift of this compound in CDC1 3 , ÿ102.9 ppm, is close to that 0 found in [SnMe2 L ] [4a] and is in the accepted range of 90 to ÿ330 ppm for pentacoordination around the tin atom in methyltin compounds [18]. In CDCl3 the 119 Sn chemical shifts of the other compounds appear at progressively higher ®eld as the aliphatic chain lengthens but this sequence is not followed in DMSOd6 possibly because of the interaction between DMSO
)
5.10s, br(2)
CDCl3
b
a
CDCl3
5.27s, br(2)
b
)
)
)
5.15s, br(2)
CDCl3
DMSO-d6
)
7.15s, br(2)
)
5.10s, br(2)
CDCl3
DMSO-d6
)
7.07s, br(2)
DMSO-d6
a
9.07s(1)
8.72s(1)
9.07s(1)
8.83s(1)
9.03s(1)
8.78s(1)
9.02s(1)
8.75s(1)
8.56s(1)
d[C(2)H]
7.74s(1)
7.36s(1)
7.69s(1)
7.62s(1)
7.74s(1)
7.57s(1)
7.74s(1)
7.59s(1)
7.97s(1)
d[C(5)H]
)
)
)
)
)
)
)
)
9.64s, vbr(1)
d[C(7)OH]
4.50d(2) J 5.0
4.49d(2) J 4.8 4.73s(2)
4.56d(2) J 5.3 4.49d(2) J 5.0 4.73s(2)
d[C(8)H2 ]
5.27t(1) J 5.0 3.2s, br(2)
4.49d(2) J 5.1 4.69s(2)
5.17s, vbr(1) 4.71s(2)
5.28t(1) J 5.1
5.25t(1) J 5.1 )
5.26t(1) J 5.3 5.26t(1) J 5.0 )
d[C(8)OH]
2.68s(3)
2.45s(3)
2.42s(3)
2.28s(3)
2.45s(3)
2.29s(3)
2.42s(3)
2.27s(3)
2.39s(3)
d[C(9)H3 ]
Ha 1.32m(4) Hb 1.15t(6) Ha 1.50m(4) Hb 1.31t(6) Ha 1.40m(4) Hb 1.55m(4) Hc 1.25m(4) Hd 0.79t(6) Ha 1.57m(4) Hb 1.66m(4) Hc 1.35m(4) Hd 0.89t(6) Ho 7.60m(4) Hm;p 7.27m(6) Ho 7.87m(4) Hm;p 7.41m(6)
0.88s(6)
0.70s(6)
)
d(R-Sn)
J
)
)
83.0 79.7 72.7 69.6 140.3 134.3 135.2 129.2
2
J
40.9
41.4
35.7
31.1
35.5
28.3
37.8
33.0
3
Numbering scheme for ligand see I. Abbrevations: s singlet, d doublet, t triplet, m multiplet, b broad, v very. In parentheses relative number of protons calculated by integration. Overlapped Hm;p .
[SnPh2 (L)]
[SnBu2 (L)]
[SnEt2 (L)]
)
DMSO-d6
[SnMe2 (L)]
11.61s, vbr(1)
8.33s, vbr(1) 8.10s, vbr(1) 7.12s, br(1)
DMSO-d6
d[N(2)H]
H2 L
d[N(1)H2 ]
Solvent
Compound
Table 5 t 1 H NMR data (d, ppm; J, Hz) of the ligand and complexes
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292 287
[SnPh2 (L)]
[SnBu2 (L)]
167.70
152.29
155.92
169.91
CDCl3
DMSO-d6
153.16
169.04
DMSO-d6
156.61
169.32
CDCl3
[SnEt2 (L)]
142.17 152.84 156.67 152.85
178.01 169.04 169.15 169.35
DMSO-d6 DMSO-d6 CDCl3 DMSO-d6
d[C(2)]
H2 L [SnMe2 (L)]
d[C(1)]
Sn NMR data of the ligand and complexes
Solvent
119
Compound
Table 6 13 C, and
119.16
118.26
118.23
117.68
121.48 118.35 117.54 118.37
d[C(3)]
132.69
132.11
132.56
131.16
132.80 132.50 131.05 132.63
d[C(4)]
135.24
135.01
134.64
135.41
139.10 134.53 135.67 134.25
d[C(5)]
150.35
154.21
152.10
155.20
147.03 152.27 155.14 152.20
d[C(6)]
157.52
158.79
157.77
159.10
148.97 157.35 158.37 158.06
d[C(7)]
59.09
60.17
59.08
60.99
58.91 59.01 60.98 59.15
d[C(8)]
19.80
19.21
19.29
19.75
19.12 19.27 19.71 19.44
d[C(9)] ) 7.69 5.82 Ca 19.95 Cb 10.00 Ca 17.72 Cb 9.90 Ca 26.16 Cb 27.24 Cc 25.82 Cd 18.52 Ca 25.89 Cb 27.42 Cc 26.47 Cd 13.61 Ci 149.05 Co 134.50 Cm 128.14 Cp 128.50
d[R]
J
556.4/533.0 30.5 87.5
605.3/574.8 33.6 89.5
644.9 42.7
n
ÿ323.6
ÿ123.1
ÿ147.7
ÿ119.6
) ÿ158.2 ÿ102.9 ÿ170.8
d119 Sn]
288 J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
289
with trigonal bipyramidal coordination where the N(3) atom and R groups probably occupy equatorial positions. 4. Biological activity
Fig. 1. 2D 1 H-13 C HMBC spectrum at 500, 137 MHz of [SnBu2 (L)] in CDCl3 solution showing the correlation peak patterns.
and the organometallic fragments modi®es when ÿR changes. In [SnPh2 (L)] the nucleus is more shielded than might be expected given the large capability of the phenyl group for electron withdrawal [4a,19]. To sum up, both solid state and solution studies on [SnR2 (L)] suggest the presence of O(phenolic)-, N(3)-, S-bonded thiosemicarbazonato anions and tin atoms
FLC proliferation. The ligand had no inhibitory eect on FLC proliferation even at concentrations of around 30 lg/ml. Inhibition was total for the butyl and phenyl complexes, 75% for the ethyl complex, and zero for the methyl complex (Fig. 2). Inhibition by the ethyl, butyl and phenyl complexes was maintained down to concentrations of 1, 0.065 and 0.22 lg/ml, respectively (data not shown). DMSO-induced FLC dierentiation. As shown in Fig. 3, [SnEt2 (L)] which had a marked inhibitory eect on cell proliferation, it did not aect cell dierentiation. On the contrary, the phenyl and butyl complexes inhibited cell dierentiation at a dose that did not inhibit cell proliferation. DNA synthesis. In keeping with the results on FLC proliferation, the butyl and phenyl complexes totally inhibited synthesis of DNA (Fig. 4). However, the ethyl complex had no eect on DNA synthesis although it inhibited FLC proliferation, while the methyl complex depressed DNA synthesis in spite of not aecting FLC proliferation. Eects on reverse transcriptase. H2 L inhibited reverse transcriptase activity by 50% at a concentration of 2 lg/
Fig. 2. Eects on FLC growth. Cells were counted at days 3th and 4th. Each condition was performed in duplicate.
290
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
Fig. 3. Eects on FLC DMSO-induced dierentiation. Each condition was performed in duplicate.
ml, while the methyl and ethyl complexes had no eect at this concentration and the butyl complex caused total inhibition (Fig. 5). The phenyl complex also caused a strong reduction of the RT activity. In this case the in-
hibition was not total although this complex totally inhibited cell proliferation at the same dosis. General conclusions. The above results are in keeping with those obtained with other thiosemicarbazones in
Fig. 4. Eects on DNA synthesis in growing FLC. Each value represents the mean of three experiments.
J.S. Casas et al. / J. Inorg. Biochem. 69 (1998) 283±292
291
Fig. 5. Eects on reverse transcriptase activity in growing FLC. Each value represents the mean of three experiments.
that complexation increased the biological activity of the ligand [4c,5,20]. Together with results for the copper and cobalt complexes of the same ligand [5], they allow the following conclusions. (a) Pyridoxal thiosemicarbazone has no activity even at high concentrations (and regardless of whether it is dissolved in DMSO or ethanol/water). (b) Of the pyridoxal thiosemicarbazone complexes so far evaluated, those with the lowest thresholds for inhibition of FLC proliferation are [SnBu2 (L)] and [SnPh2 (L)]. (c) On the contrary of [Cu(H2 L)(OH)2 Cl]Cl and [fCu(HL)(H2 O)g2 ]Cl2 á H2 O [5], the tin complexes do not enhance DMSO-induced FLC dierentiation. (d) The strong dependence of the results for the SnR2 complexes on the identity of R suggests the desirability of carrying out further assays to determine the mechanistic basis of this dependence. Acknowledgements We thank Mrs. Cristina Everti for technical assistance. Finantial support by the Xunta de Galicia, Spain, (Project XUGA 2038B97), the Associazione Italiana per la Ricerca sul Cancro and CNR, Italy, is acknowledged. MCRA is grateful for a grant from the University of Vigo, Spain.
[3]
[4]
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