Novel imino thioether complexes of platinum(II): Synthesis, structural investigation, and biological activity

Article abstract of DOI:10.1021/ic3024452

The reactions of the nitrile complexes cis- and trans-[PtCl ₂(NCR)₂] (R = Me, Et, CH₂Ph, Ph) with an excess of ethanethiol, EtSH, in the presence of a catalytic amount of n-BuLi in tetrahydrofuran (THF), afforded in good yield the bis-imino thioether derivatives cis-[PtCl₂{E-N(H)=C(SEt)R}₂] (R = Me (1), Et (2), CH₂Ph (3), Ph (4)) and trans-[PtCl₂{E-N(H)=C(SEt)R} ₂] (R = Me (5), Et (6), CH₂Ph (7), Ph (8)). The imino thioether ligands assumed the E configuration corresponding to a cis addition of the thiol to the nitrile triple bond. The spectroscopic properties of these complexes have been reported along with the molecular structures of 1, 2, and 7 as established by X-ray crystallography which indicated that these compounds exhibit square-planar coordination geometry around the platinum center. Four N-H?Cl intermolecular contacts (N-H?Cl ca. 2.5-2.7 ?) between each chlorine atom and the N-H proton of the imino thioether ligand gave rise to dimers Pt₂Cl₄L₄ (L = imino thioether) formed by two PtCl₂L₂ units. The cytotoxic properties of these new platinum(II) complexes were evaluated against various human cancer cell lines. Among all derivatives, trans-[PtCl₂{E-N(H)= C(SEt)CH₂Ph}₂] showed the greatest in vitro cytotoxic activity being able to decrease cancer cell viability roughly 3-fold more effectively than cisplatin.

Full text of DOI:10.1021/ic3024452

Article
pubs.acs.org/IC
Novel Imino Thioether Complexes of Platinum(II): Synthesis,
Structural Investigation, and Biological Activity
Paolo Sgarbossa,† Silvia Mazzega Sbovata,† Roberta Bertani,†,∥ Mirto Mozzon,† Franco Benetollo,‡
Cristina Marzano,§ Valentina Gandin,§ and Rino A. Michelin*,†
†Department of Industrial Engineering, University of Padua, Via F. Marzolo, 9, 35131 Padua, Italy
‡ICIS-CNR, Corso Stati Uniti, 4-35100 Padua, Italy
§Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, I-35131 Padova, Italy
∥C.N.R.-ISTM, Istituto di Scienze e Tecnologie Molecolari, Via F. Marzolo 1, I- 35131 Padova, Italy
S
* Supporting Information
ABSTRACT: The reactions of the nitrile complexes cis- and trans-
[PtCl2(NCR)2] (R = Me, Et, CH2Ph, Ph) with an excess of ethanethiol,
EtSH, in the presence of a catalytic amount of n-BuLi in tetrahydrofuran
(THF), afforded in good yield the bis-imino thioether derivatives cis-
[PtCl2{E-N(H)C(SEt)R}2] (R = Me (1), Et (2), CH2Ph (3), Ph (4))
and trans-[PtCl2{E-N(H)C(SEt)R}2] (R = Me (5), Et (6), CH2Ph (7),
Ph (8)). The imino thioether ligands assumed the E configuration
corresponding to a cis addition of the thiol to the nitrile triple bond. The
spectroscopic properties of these complexes have been reported along with
the molecular structures of 1, 2, and 7 as established by X-ray
crystallography which indicated that these compounds exhibit square-planar
coordination geometry around the platinum center. Four N−H···Cl
intermolecular contacts (N−H···Cl ca. 2.5−2.7 Å) between each chlorine
atom and the N−H proton of the imino thioether ligand gave rise to
“dimers” Pt2Cl4L4 (L = imino thioether) formed by two PtCl2L2 units. The cytotoxic properties of these new platinum(II)
complexes were evaluated against various human cancer cell lines. Among all derivatives, trans-[PtCl2{E-N(H)
C(SEt)CH2Ph}2] showed the greatest in vitro cytotoxic activity being able to decrease cancer cell viability roughly 3-fold
more effectively than cisplatin.
either Z or E corresponding to the trans or cis addition of the
protic nucleophile along the CN triple bond, respectively.
Most of the biological studies reported by Coluccia and
Natile have been concerned with the trans-configured
iminoether Pt(II) species.8 On the other hand, some of us
previously reported that also the cis-configured benzylimi-
noether complex cis-[PtCl2{E-N(H)C(OMe)CH2Ph}2] pos-
sesses a significant in vitro and in vivo cytotoxic activity.6e
Recently, we have also reported that Pt(II) amidine complexes
of the type cis- and trans-[PtCl2{amidine}2], derived by the
addition of primary and secondary amines to the dibenzonitrile
complex cis- and trans-[PtCl2(NCPh)2], were endowed with
significant antitumor activity.7e It is worthwhile noting that,
among this type of complexes, the benzamidine Pt(II) species
trans-[PtCl2{N(H)C(NMe2)Ph}2] appeared as the most
effective derivative in the biological assays. It has been proposed
that amidine ligands behave as carrier ligands which are
retained within the cell, in agreement with the superbasic
properties recently stated.9 In general, trans platinum
1. INTRODUCTION
In the past ten years the addition reactions of nucleophiles and
electrophiles to organonitrile ligands coordinated to electron-
withdrawing transition metal ions have experienced a rapid
growth.1 The RCN metal-promoted and/or catalytic con-
version into other organic ligands as a consequence of chemical
processes such as insertion, coupling, and nucleophilic or
electrophilic attack has been reviewed in a number of articles2
and is of current interest in view of its importance in synthetic
chemistry3 and catalysis,2a as well as for its biological
implications. Interestingly, a class of relevant biologically active
Pt(II)-based drugs has been prepared from the synthetically
useful organonitrile Pt(II) complexes cis- and trans-
[PtCl2(NCR)2] (R = Me, Ph) by taking advantage of their
ability to undergo nucleophilic addition of alcohols4 and
amines5 at the CN triple bond affording iminoether6 and
amidine7 derivatives, respectively (Scheme 1). The Pt(II)-
mediated alcohol-nitrile4 and amine-nitrile7 coupling reactions
have been broadly explored and established using nitrile
complexes of the type cis- and trans-[PtCl2(NCR)2] (R =
alkyl, aryl). The configuration of the reaction products can be
Received: November 8, 2012
Published: May 6, 2013
© 2013 American Chemical Society
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Article
and ototoxicity of cisplatin.29 It was recently demonstrated that
the DNA platination rate of a trans antitumor drug was
dramatically enhanced by thioether binding and a detailed
study carried out on the interaction between trans-PtCl2(E-
iminoether)2 with thioether indicated that coordination to
DNA of platinum-protein adducts was highly feasible for trans-
configuration platinum complexes.30
Scheme 1. Configuration of Iminoether and Amidine
Complexes
In this context, owing to our interest in the investigation of
reactivity of Pt(II) coordinated organonitriles and in the design
of novel biologically active metal drugs, we have focused our
attention on the utilization of S-protic nucleophiles such as
thiols in the metal-promoted addition reactions to coordinated
nitrile ligands. In the present article, we wish to report
preliminary results on the synthesis and characterization of the
novel iminothioether Pt(II) complexes some of which display
high levels of cytotoxic activity against both cisplatin sensitive
and resistant cell lines.
It is noteworthy that, to our knowledge, only one example
has been reported of metal-promoted addition reactions of S-
protic nucleophiles such as thiols and thiophenols to the
coordinated CN group in the cationic cyanobenzyl cis-[Pt-(o-
CH2C6H4CN)(PPh3)2]2(BF4)2 complex to afford the corre-
sponding imino thioether derivatives.31
complexes are attracting great attention10 because some classes
of compounds (mononuclear, polynuclear,11 Pt(IV) deriva-
tives12) showed a better cytotoxic activity and ability to
overcome cisplatin resistance due to their distinct cellular
pharmacological properties with respect to cisplatin based also
on different interactions with DNA.13 It seems that trans
platinum compounds form interstrand cross-links stabilizing
DNA double helix, whereas cisplatin has an intrastrand cross-
link pattern.14 Detailed studies concerning the speciation of
trans complexes in water solution, the influence of the spectator
ligands, and the role of the leaving group have been carried out
by the Navarro-Ranninger’s research team in particular for trans
complexes bearing aliphatic amines15 and by Farrell for
mononuclear trans platinum complexes with a heterocyclic
planar system in the coordination sphere.16 Quite recently it
was shown that irradiation at 365 nm can be used to activate
trans-Pt(IV) diamine complexes improving the selective
interaction with biomolecules.17
Expanding the investigation of new ligands and/or new
complexes having the ability to selectively impact the life cycle
of cancerous cells remains an important goal of the
bioinorganic chemistry of anticancer metallodrugs. Conse-
quently, it is not surprising that a wide range of ligands and
metal complexes have been designed and are currently under
investigation.18
Platinum is known to have a large affinity for intracellular
sulfur nucleophiles19 and some mechanisms of platinum drug
resistance are associated with drug inactivation by sulfur
molecules.20 Thiols and thioethers such as cysteine, glutathione,
and methionine decrease DNA platination.21 Glutathione and
metallothionein are overproduced in cell lines resistant to
cisplatin.22 While the fixation of thiols on platinum is
irreversible, thioether adducts can be displaced by thiourea
and N7 atom of guanine moieties.23 This reversibility is
considered to be involved in cisplatin induced nephrotoxicity,24
but the formation of adducts with the methionine residues of
proteins might act as a reservoir for the drug21d,25 and/or
activate it.26 Glutathione, thiosulfate, D-methionine, diethyldi-
thiocarbamate (DDTC), aminothiazole,27 and N,S-chelated
ligands28 have been studied and tested to reduce the nephro-
2. EXPERIMENTAL SECTION
2.1. Materials and Methods. Reagents were obtained from
commercial sources and used as supplied. All solvents were reagent
grade and were distilled prior to use. Deuterated solvents were
purchased from Cambridge Isotope Laboratories (CIL) and stored
under molecular sieves. The infrared spectra were taken on a Perkin-
Elmer Spectrum 100 FT IR Spectrophotometer (CsI films); the
frequencies are given in cm−1. 1H, 13C, and 15N NMR solution spectra
were obtained at 298 K (unless otherwise stated) on a Bruker Avance-
400 spectrometer (9.4 T field) operating at 400.13, 100.61, and 40.56
MHz, respectively, and using a Bruker AvanceIII-200 spectrometer
1
operating at 200.12 and 50.32 MHz for H and 13C, respectively; δ
1
values (parts per million, ppm) are relative to Me4Si for H and 13C.
Suitable integral values for the proton spectra were obtained with a
prescan delay of 10 s. The assignments of the proton resonances were
performed by standard chemical shift correlations, as well as by COSY,
TOCSY, and NOESY experiments. In the phase-sensitive NOESY
measurements the presence of intense cross-peaks, in phase with the
diagonal, indicates a chemical exchange between the correlated nuclei
(EXSY).32 The 13C resonances were attributed through 2D-
heterocorrelated COSY experiments: heteronuclear multiple quantum
correlation (HMQC) with bilinear rotation decoupling33 and
quadrature along F1 achieved using the time proportional phase
increment method34 for the hydrogen-bonded carbon atoms,
heteronuclear multiple bond correlation (HMBC)35 for the quaternary
ones. The purity of compounds 1−7 was stated to be higher than 98%
by elemental analyses which were performed by the Microanalysis
Laboratory of the Department of Chemical Sciences, University of
Padova. Electrospray ionization mass spectrometry (ESI-MS) analyses
were performed using a LCQ-Duo (Thermo-Finnigan, San Jose, CA,
U.S.A.) operating in positive ion mode. Instrumental parameters:
capillary voltage 10 V, spray voltage 4.5 kV; capillary temperature 200
°C; mass scan range from 150 to 2000 amu; N2 was used as sheath gas;
the He pressure inside the trap was kept constant. The pressure
directly read by an ion gauge (in the absence of the N2 stream) was
1.33 × 10−5 Torr. The collision-induced dissociation experiments were
performed by applying a supplementary RF voltage (tickle voltage) to
the end-caps of the ion trap in the range 0−80% of its maximum value
(5 V peak to peak). Sample solutions were prepared by dissolving the
compounds (1 mg) in CH2Cl2 (500 μL) and then diluted 1:1000 with
CH3CN (unless otherwise stated). Sample solutions were directly
infused into the ESI source by a syringe pump at 8 μL/min flow rate.
The formation of ionic species reported in the experimental section 2.2
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and discussed in the text was confirmed by MS/MS experiments and
isotope pattern analysis.
2.2.5. trans-[PtCl2{E-N(H)C(SEt)Me}2] (5). A solution of
CH3CH2SH (8.62 mmol; ρ = 0.839 g/mL) in THF (30 mL) was
treated with BuLin (0.17 mmol, 1.6 M) at −10 °C. Then, complex
trans-[PtCl2(NCMe)2] (0.3 g, 0.86 mmol) was added, and the reaction
mixture was stirred for 1 h at low temperature (−10 °C) and then
reacted at room temperature for additional 2 h. The solution was then
concentrated to a small volume (5 mL) and treated with n-hexane to
afford a pale yellow solid. The product was collected and dried under
vacuum. Yield 70%. Anal. Calcd for C8H18Cl2N2S2Pt (5, M = 472.36):
C, 20.34; H, 3.84; N, 5.93; S, 13.58. Found: C, 20.30; H, 3.88; N, 5.90;
S, 13.55. IR (λmax, KBr, cm−1): 3399 νas(N−H), 3196 νs(N−H);
2.2. Preparation of the Complexes. cis- and trans-
[PtCl2(NCMe)2] were synthesized as described in the literature.36
cis-Complexes 1−4 were prepared by a similar procedure which is
described for 1, while trans-complexes were prepared according to the
procedure described for 5.
2.2.1. cis-[PtCl2{E-N(H)C(SEt)Me}2] (1). A solution of CH3CH2SH
(8.62 mmol; ρ = 0.839 g/mL) in THF (30 mL) was treated with BuLin
(0.17 mmol, 1.6 M) at room temperature. Then, complex cis-
[PtCl2(NCMe)2] (0.3 g, 0.86 mmol) was added, and the reaction
mixture was stirred for 1 day at room temperature. The solution was
then concentrated to a small volume (5 mL) and treated with n-hexane
to afford a pale yellow solid. The product was collected and dried
under vacuum. Yield: 73%. Anal. Calcd for C8H18Cl2N2S2Pt (1, M =
472.36): C, 20.34; H, 3.84; N, 5.93; S, 13.58. Found: C, 20.32; H,
3.90; N, 5.88; S, 13.63. IR (λmax, KBr, cm−1): 3456 νas(N−H), 3222
νs(N−H); 1594ν(CN); 705 ν(C−S). IR (λmax, PE, cm−1): 333 and
326 ν(Pt−Cl). 1H NMR (200 MHz, CDCl3, t = triplet, q =
1
1594ν(CN); 702 ν(C−S). IR (λmax, PE, cm−1): 344 ν(Pt−Cl). H
NMR (200 MHz, CDCl3, t = triplet, q = quadruplet, s = singlet): δ
3
3
1.29 (t, JH−H = 7.4 Hz, SCH2CH3, 6H); 3.00 (q, JH−H = 7.4 Hz,
SCH2CH3, 4H); 2.89 (s, NCCH3, 6H); 9.14 (s, NH, 2H). 13C {1H}
NMR (50 MHz, CDCl3, s = singlet): δ 12.86 (s, SCH2CH3); 25.02 (s,
SCH2CH3); 27.03 (s, NCCH3); 178.03 (s, CN). ESI-MS (fragments
were based on 195Pt; m/z (rel.ab.%)): 967 ([2 M + Na]+, 20); 1439
([3 M + Na]+, 25); 950 [Pt2C20H48N4S6Na]+, 100); 966
[Pt2C20H48N4S6K]+, 25); 401 [PtC8H19N2S2]+, 17); 934
[Pt2C19H44N4S6Na]+, 40).
3
quadruplet, s = singlet): δ 1.25 (t, JH−H = 7.4 Hz, SCH2CH3, 6H);
3
3.04 (q, JH−H = 7.4 Hz, SCH2CH3, 4H); 2.76 (s, NCCH3, 6H); 9.27
(s, NH, 2H). 13C {1H} NMR (50 MHz, CDCl3, s = singlet): δ 13.08
(s, SCH2CH3); 25.51 (s, SCH2CH3); 27.75 (s, NCCH3); 178.71 (s,
CN). ESI-MS (fragments were based on 195Pt; m/z (rel.ab.%)): 495
([M + Na]+, 27); 511 ([M + K]+, 5); 967 ([2 M + Na]+, 100); 983 ([2
M + K]+, 8); 1439 ([3 M + Na]+, 4).
2.2.6. trans-[PtCl2{E-N(H)C(SEt)Et}2] (6). Yield 80%. Anal. Calcd
for C10H22Cl2N2S2Pt (6, M = 500.42): C, 24.00; H, 4.43; N, 5.60; S,
12.82. Found: C, 23.93; H, 4.47; N, 5.58; S, 12.80. IR (λmax, KBr,
cm−1): 3433 νas(N−H); 3257 νs(N−H); 1601 ν(CN); 702 ν(C−
S). IR (λmax, PE, cm−1): 338 ν(Pt−Cl). 1H NMR (200 MHz, CD2Cl2, t
3
= triplet, q = quadruplet, s = singlet): δ 1.28 (t, JH−H = 7.4 Hz,
2.2.2. cis-[PtCl2{E-N(H)C(SEt)Et}2] (2). Yield 76%. Anal. Calcd for
C10H22Cl2N2S2Pt (2, M = 500.42): C, 24.00; H, 4.43; N, 5.60; S,
12.82. Found: C, 23.93; H, 4.47; N, 5.58; S, 12.80. IR (λmax, KBr,
cm−1): 3434 νas(N−H); 3228 νs(N−H); 1583 ν(CN); 678 ν(C−
S). IR (λmax, PE, cm−1): 337 and 326 ν(Pt−Cl). 1H NMR (200 MHz,
CD2Cl2, t = triplet, q = quadruplet, s = singlet): δ 1.24 (t, 3JH−H = 7.4
3
SCH2CH3, 6H); 1.45 (t, JH−H = 7.6 Hz, NCCH2CH3, 6H); 2.71 (q,
3JH−H = 7.4 Hz, SCH2CH3, 4H); 3.36 (q, 3JH−H = 7.6 Hz, NCCH2CH3,
4H); 9.02 (s, NH, 2H). 13C {1H} NMR (50 MHz, CD2Cl2, s =
singlet): δ 12.76 (s, SCH2CH3); 11.70 (s, NCCH2CH3); 23.90 (s,
SCH2CH3); 33.75 (s, NCCH2CH3); 182.22 (s, CN). ESI-MS
(fragments were based on 195Pt; m/z (rel.ab.%)): 1006
[Pt2C24H57N4S6Na]+, 100); 1506 [Pt3C34H77N6S8Na]+, 45).
3
Hz, SCH2CH3, 6H); 1.41 (t, JH−H = 7.6 Hz, NCCH2CH3, 6H); 3.05
3
3
(q, JH−H = 7.4 Hz, SCH2CH3, 4H); 3.30 (q, JH−H = 7.6 Hz,
NCCH2CH3, 4H); 9.15 (s, NH, 2H). 13C {1H} NMR (50 MHz,
CD2Cl2, s = singlet): δ 13.11 (s, SCH2CH3); 12.35 (s, NCCH2CH3);
25.04 (s, SCH2CH3); 35.07 (s, NCCH2CH3); 184.71 (s, CN). ESI-
MS (fragments were based on 195Pt; m/z (rel.ab.%)): 523 ([M + Na]+,
5); 1023 ([2 M + Na]+, 55); 1523 ([3 M + Na]+, 15); 506 [M − Cl +
CH3CN]+, 3); 1006 [Pt2C24H57N4S6Na]+, 100); 1506
[Pt3C34H77N6S8Na]+, 45).
2.2.7. trans-[PtCl2{E-N(H)C(SEt)CH2Ph}2] (7). Yield 76%. Anal.
Calcd for C20H26Cl2N2S2Pt (7, M = 624.56): C, 38.46; H, 4.20; N,
4.49; S, 10.27. Found: C, 38.48; H, 4.22; N, 4.45; S, 10.20. IR (λmax
,
KBr, cm−1): 3447 νas(N−H); 3246 νs(N−H); 1589 ν(CN); 707
1
ν(C−S). IR (λmax, PE, cm−1): 337 ν(Pt−Cl). H NMR (200 MHz,
CD2Cl2, t = triplet, q = quadruplet, s = singlet, m = multiplet): δ 1.24
3
3
(t, JH−H = 7.4 Hz, SCH2CH3, 6H); 2.99 (q, JH−H = 7.4 Hz,
SCH2CH3, 4H); 4.88 (s, CH2Ph, 4H); 9.48 (s, NH, 2H); 7.62 (m, Ph,
o-H, 4H); 7.32 (m, Ph, m-H, 4H); 7.28 (m, Ph, p-H, 2H). 13C {1H}
NMR (50 MHz, CD2Cl2, s = singlet): δ 12.77 (s, SCH2CH3); 24.97 (s,
SCH2CH3); 46.61 (s, CH2Ph,); 129.80 (s, Ph, o-C); 128.48 (s, Ph, m-
C); 127.34 (s, Ph, p-C); 134.91 (s, Ph,Cj); 181.23 (s, CN). ESI-MS
(fragments were based on 195Pt; m/z (rel.ab.%)): 647 ([M + Na]+,
20); 589 ([M − Cl]+, 30); 552 ([M − HCl − Cl]+, 35); 630 ([M − Cl
+ CH3CN]+, 12); 1255 ([Pt2C42H54Cl2N6S4Na]+, 35); 1271
([Pt2C42H54Cl2N6S4K]+, 15); 1313 ([C42H54Cl3N6S4Pt2Na2]+, 10);
1879 ([Pt3C62H79Cl4N8S4Na]+, 70); 1895 ([Pt3C62H79Cl4N8S4K]+,
20). ESI-MS (fragments were based on 195Pt; m/z (rel.ab.%), 300 μM
in DMF/water physiological solution 1/1): 647 ([M + Na]+, 45); 589
([M − Cl]+, 50); 552 ([M − HCl − Cl]+, 100); 1319 ([2M − Cl +
DMF]+, 30).
2.2.3. cis-[PtCl2{E-N(H)C(SEt)CH2Ph}2] (3). Yield 77%. Anal.
Calcd for C20H26Cl2N2S2Pt (3, M = 624.56): C, 38.46; H, 4.20; N,
4.49; S, 10.27. Found: C, 38.38; H, 4.18; N, 4.42; S, 10.19. IR (λmax
,
KBr, cm−1): 3436 νas(N−H); 3239 νs(N−H); 1568 ν(CN); 703
1
ν(C−S). IR (λmax, PE, cm−1): 326 and 319 ν(Pt−Cl). H NMR (200
MHz, CD2Cl2, t = triplet, q = quadruplet, s = singlet, m = multiplet): δ
3
3
1.24 (t, JH−H = 7.4 Hz, SCH2CH3, 6H); 3.03 (q, JH−H = 7.4 Hz,
SCH2CH3, 4H); 4.45 (s, CH2Ph, 4H); 9.38 (s, NH, 2H), 7.26−7.30
(m, Ph, 10H). 13C {1H} NMR (50 MHz, CD2Cl2, s = singlet): δ 13.02
(s, SCH2CH3); 25.83 (s, SCH2CH3); 47.11 (s, CH2Ph,); 127.89 (s, Ph,
o-C); 128.93 (s, Ph, m-C); 130.12 (s, Ph, p-C); 134.56 (s, Ph,Cj);
183.02 (s, CN). ESI-MS (fragments were based on 195Pt; m/z
(rel.ab.%)): 647 ([M + Na]+, 51); 1271([2 M + Na]+, 100); 1895 ([3
M + Na]+, 20); 1208 (2 M + Na − CH3CH2SH]+, 20).
2.2.8. trans-[PtCl2{E-N(H)C(SEt)Ph}2] (8). Yield 30%. Anal. Calcd
C18H22Cl2N2S2Pt (8, M = 596.50): C, 36.24; H, 3.72; N, 4.70; S,
10.75. Found: C, 36.35; H, 3.79; N, 4.61; S, 10.68. IR (λmax, KBr,
cm−1): 3399 νas(N−H); 1586 ν(CN); 695 ν(C−S). IR (λmax, PE,
2.2.4. cis-[PtCl2{E-N(H)C(SEt)Ph}2] (4). Yield 77%. Anal. Calcd
C18H22Cl2N2S2Pt (4, M = 596.50): C, 36.24; H, 3.72; N, 4.70; S,
10.75. Found: C, 36.28; H, 3.75; N, 4.68; S, 10.72. IR (λmax, KBr,
cm−1): 3467 νas(N−H); 3253 νs(N−H); 1583 ν(CN); 714 ν(C−
S). IR (λmax, PE, cm−1): 344 and 325 ν(Pt−Cl). 1H NMR (200 MHz,
CD2Cl2, t = triplet, q = quadruplet, s = singlet, m = multiplet): δ 1.22
1
cm−1): 317 ν(Pt−Cl). H NMR (200 MHz, CD2Cl23, t = triplet, q =
quadruplet, s = singlet, m = multiplet): δ 1.20 (t, JH−H = 7.4 Hz,
3
SCH2CH3, 6H); 2.97 (q, JH−H = 7.4 Hz, SCH2CH3, 4H); 9.67 (s,
3
3
NH, 2H), 7.51−7.73 (m, Ph, 6H); 8.06−8.08 (m, Ph, 4H). 13C {1H}
NMR (50 MHz, CD2Cl2, s = singlet): δ 12.96 (s, SCH2CH3); 28.42 (s,
SCH2CH3); 128.29 (s, Ph, o-C); 128.97 (s, Ph, m-C); 129.39 (s, Ph, p-
C); 135.22 (s, Ph,Cj); 187.38 (s, CN). ESI-MS (fragments were
based on 195Pt; m/z (rel.ab.%)): 619 ([M + Na]+, 15); 1215 ([2 M +
Na]+, 100); 1811 ([3 M + Na]+, 35); 523 [M − H − 2HCl]+, 65); 561
[M -Cl]+, 25); 1199 [2 M + Na − CH4]+, 70); 1795 [3 M + Na −
CH4]+, 40); 1773 [3 M − CH3]+, 15).
(t, JH−H = 7.4 Hz, SCH2CH3, 6H); 2.53 (q, JH−H = 7.4 Hz,
SCH2CH3, 4H); 8.12 (s, NH, 2H), 7.45−7.69 (m, Ph, 6H); 7.99−8.04
(m, Ph, 4H). 13C {1H} NMR (50 MHz, CD2Cl2, s = singlet): δ 12.50
(s, SCH2CH3); 28.40 (s, SCH2CH3); 128.21 (s, Ph, o-C); 128.79 (s,
Ph, m-C); 132.28 (s, Ph, p-C); 136.79 (s, Ph,Cj); 178.56 (s, CN).
ESI-MS (fragments were based on 195Pt; m/z (rel.ab.%)): 619 ([M +
Na]+, 100); 635 ([M + K]+, 28); 1215 ([2 M + Na]+, 80); 1231 ([2 M
+ K]+, 30); 561 ([M − Cl]+, 50); 525 ([M − Cl − HCl]+, 85).
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The reaction of trans-[PtCl2(NCPh)2] with CH3CH2SH gave rise
to a mixture of E,E (8), E,Z and Z,Z atropisomers, as previously
observed in the case of the reaction of trans-[PtCl2(NCPh)2] with
MeNH2 and Me2NH,5b together with a small amount (ca.10%) of a
cationic species. The NMR spectra of the mixture obtained by the
reaction of trans-[PtCl2(NCPh)2] with CH3CH2SH showed the
presence of trans-[PtCl2{EZ-N(H)C(SEt)Ph}2] (ca. 30%) [Z
Table 1. Crystallographic Data for 1, 2, and 7
1
2
7
empirical
formula
C8H18N2Cl2S2Pt
C10H22N2Cl2S2Pt
C20H26N2Cl2S2Pt
fw
472.36
293(2)
0.71073
500.41
624. 54
293(2)
0.71073
triclinic
T, K
λ (Å)
293(2)
3
system: 1H, δ 1.36 (t, JH−H = 7.4 Hz, SCH2CH3); 3.50 (q,
0.71073
monoclinic
P21/n
SCH2CH3); 9.57 (s, NH, NOE correlation with phenyl protons),
8.12 (o-Ph); 7.45 (m-Ph); 7.74 (p-Ph); 13C {1H}: δ 12.33
(SCH2CH3); 28.36 (SCH2CH3); 128.87 (o-C); 128.01 (m-C);
crystal-system triclinic
space-group
a (Å)
P1
̅
P1
̅
8.797(2)
9.277(2)
10.069(2)
106.68(2)
104.19(2)
93.56(2)
755.3(3)
2
12.989(3)
14.486(3)
18.594(3)
13.337(3)
14.836(3)
13.275(3)
94.14(2)
109.02(3)
104.16(3)
2374.5(9)
4
1
3
131.02 (p-C); 186.70 (CN); E system: H, δ 1.34 (t, JH−H = 7.4
Hz, SCH2CH3); 3.10 (q, SCH2CH3); 9.32 (s, NH, NOE correlation
with SCH2 protons); 13C {1H}: δ 12.70 (SCH2CH3); 26.77
(SCH2CH3); 178.90(CN)] and of trans-[PtCl2{ZZ-N(H)C-
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
3
(SEt)Ph}2] (ca. 30%) [1H, δ 1.41 (t, JH−H = 7.4 Hz, SCH2CH3);
94.17(3)
3.16 (q, SCH2CH3); 9.38 (s, NH, NOE correlation with SCH2
protons), 8.38 (o-Ph); 7.59 (m-Ph); 7.67 (p-Ph); 13C {1H}: δ 12.74
(SCH2CH3); 26.08 (SCH2CH3); 129.01 (o-C); 128.00 (m-C); 132.00
(p-C); 178.70 (CN)]. A cationic species originated by the
substitution of a chlorine atom with CH3CH2SH was also observed.
The presence of broad signals of the thioethereal SCH2CH3 moiety at
δ 1.52 (δ 13C 12.55) and 3.50 (δ 13C 28.27) and of the entering
CH3CH2SH system at δ 1.75 and 2.51 (δ 13C 27.4) indicated in
solution the occurrence of a slow exchange (on the NMR time scale)
within the coordination sphere of the cationic species [PtCl2{ N(H)
C(SEt)Ph}(HSCH2CH3]Cl (δ 13C 187.4, CN).
3
́
V (Å )
3489(1)
8
Z
ρcalc, g-cm−3
2.077
1.905
8.572
1.747
μ(Mo−Kα),
9.893
6.038
mm−1
a
R(F2)
0.025
0.061
1.202
0.063
0.063
1.352
0.056
0.129
1.330
b
Rw(F2)
goodness of
fit
F(000)
448
1920
3−25
6695
1216
3−25
8719
2.3. X-ray Structure Determinations. White crystals of 1, 2, and
7 were obtained by slow diffusion at −4 °C of diethyl ether into
solutions of the complexes in CH2Cl2. Crystals were lodged in a
Lindemann glass capillary and centered on a four circle Philips
PW1100 diffractometer using graphite monochromated MoKα
radiation (0.71073 Å), following the standard procedures at room
temperature. All intensities were corrected for Lorentz polarization
and absorption.37 The structures were solved by standard direct
methods.38 Refinement was carried out by full-matrix least-squares
procedures (based on F2o) using anisotropic temperature factors for all
non-hydrogen atoms. Hydrogen atoms were placed in calculated
position with isotropic thermal parameters (1.2 Uequiv of the parent
carbon atom). Structure refinement and final geometrical calculations
were carried out with the SHELXL-9739 program, implemented in the
WinGX package.40 Crystallographic data and selected bond lengths
and angles have been reported in Table 1 and 2, respectively.
2.4. Biological Assays. Platinum(II) imino thioether derivatives
were dissolved in dimethylformamide (DMF) just before the
experiment and a calculated amount of drug solution was added to
the growth medium containing cells to a final solvent concentration of
0.15% which had no discernible effect on cell killing. MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), cisplatin,
transplatin, and doxorubicin were obtained from Sigma Chemical
Co, St.Louis, U.S.A.
θ range/deg
3−27
no. reflections 3729
collected
no. observed
3500
5238
7285
[I ≥ 2σ(I)]
a
b
R = ∑||Fo| − |Fc||/∑|Fo|. Rw=[∑{w(|Fo|2 − |Fc|2)2}/∑{w(|
Fo|2)2}]1/2
.
Table 2. Selected Bond Lenghts (Å) and Angles (deg) in the
Coordination Metal Sphere for cis-[PtCl2{E-N(H)
C(SEt)Me}2] (1), cis-[PtCl2{E-N(H)C(SEt)Et}2] (2), and
trans-[PtCl2{E-N(H)C(SEt)CH2Ph}2] (7)
1
2
7
Pt(1)−Cl(1)
Pt(1)−Cl(2)
Pt(2)−Cl(3)
Pt(2)−Cl(4)
Pt(1)−N(1)
Pt(1)−N(2)
Pt(2)−N(3)
Pt(2)−N(4)
Cl(1)−Pt(1)−Cl(2)
Cl(1)−Pt(1)−N(1)
Cl(1)−Pt(1)−N(2)
N(1)−Pt(1)−Cl(2)
N(1)−Pt(1)−N(2)
N(2)−Pt(1)−Cl(2)
Cl(3)−Pt(2)−Cl(4)
Cl(3−Pt(2)−N(3)
Cl(3)−Pt(2)−N(4)
N(3)−Pt(2)−Cl(4)
N(3)−Pt(2)−N(4)
N(4)−Pt(2)−Cl(4)
2.309(2)
2.307(2)
2.302(4)
2.314(5)
2.314(4)
2.307(4)
2.000(12)
2.020(12)
2.017(99)
1.997(12)
90.0(2)
2.304(3)
2.305(3)
2.301(3)
2.306(3)
2.018(10)
2.006(10)
2.025(9)
2.019(8)
179.7(1)
87.0(3)
2.017(5)
2.010(4)
91.3(1)
177.3(1)
90.1(1)
91.1(1)
87.6(2)
177.9(1)
2.4.1. Cell Cultures. Human breast (MCF-7) and lung (A549)
carcinoma along with melanoma (A375) cell lines were obtained by
ATCC, Rockville, MD. 2008 and its cisplatin resistant variant, C13*,
are human ovarian cancer cell lines, and they were kindly provided by
Prof. G. Marverti (Dept. of Biomedical Science of Modena University,
Italy). A431 and A431/Pt are sensible and resistant human cervical
carcinoma cells, respectively, kindly provided by Prof. F. Zunino
(Division of Experimental Oncology B, Istituto Nazionale dei Tumori,
Milan, Italy). LoVo human colon-carcinoma cell line and its derivative
multidrug-resistant subline (LoVo MDR) were kindly provided by
Prof. F. Majone (Dept. of Biology of Padova University, Italy). Cell
lines were maintained in the logarithmic phase at 37 °C in a 5% carbon
dioxide atmosphere using the following culture media containing 10%
fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units·mL−1
penicillin and 50 μg·mL−1 streptomycin) and 2 mM L-glutamine: (i)
RPMI-1640 medium (Euroclone) for MCF-7, 2008, C13*, A431 and
A431/Pt; (ii) F-12 HAM’S (Sigma Chemical Co) for A549, LoVo and
LoVo MDR cells (LoVo MDR culture medium also contained 0.1
177.1(2)
92.4(4)
93.0(3)
89.2(4)
93.1(3)
88.4(5)
177.7(4)
86.8(3)
177.5(4)
91.5(2)
177.8(1)
88.9(3)
89.5(4)
177.1(4)
178.9(4)
88.6(5)
90.8(3)
90.5(3)
176.5(4)
90.0(3)
90.4(4)
μg·mL−1 doxorubicin); (iii) D-MEM medium (Euroclone) for A375
cells.
2.4.2. Cytotoxicity Assay. The growth inhibitory effect toward
tumor cell lines was evaluated by means of MTT (tetrazolium salt
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Scheme 2. Synthesis of the Imino Thioether Complexes cis-[PtCl2{E-N(H)C(SEt)R}2]
reduction) assay.41 Briefly, between 3 and 8 × 103 cells, dependent
3. RESULTS AND DISCUSSION
upon the growth characteristics of the cell line, were seeded in 96-well
microplates in growth medium (100 μL) and then incubated at 37 °C
in a 5% carbon dioxide atmosphere. After 24 h, the medium was
removed and replaced with fresh media containing the compound to
be studied at the appropriate concentration. Triplicate cultures were
established for each treatment. After 48 h, each well was treated with
10 μL of a 5 mg·mL−1 MTT saline solution, and after 5 h of
incubation, 100 μL of a sodium dodecylsulfate (SDS) solution in HCl
(0.01 M) was added. After overnight incubation in the dark at 37 °C in
a 5% carbon dioxide atmosphere, the inhibition of cell growth induced
by tested compounds was detected by measuring the absorbance of
each well at 570 nm using a Bio-Rad 680 microplate reader (Milan,
Italy). Mean absorbance for each drug dose was expressed as a
percentage of the control untreated well absorbance and plotted vs
drug concentration. IC50 values represent the drug concentrations that
reduced the mean absorbance at 570 nm to 50% of those in the
untreated control wells.
3.1. Synthesis and Characterization. By reaction of the
dinitrile complexes cis-[PtCl2(NCR)2] (R = Me, Et, CH2Ph,
Ph) in THF with a 10-fold excess of EtSH in the presence of of
LiBun at room temperature for 24 h, the imino thioether
derivatives cis-[PtCl2{E-N(H)C(SEt)R}2] (R = Me (1), Et
(2), CH2Ph (3), Ph (4)) have been prepared in good yields,
where both the imino thioether ligands had the E configuration
(Scheme 2) as can be readily determined on the basis of NMR
data and also observed in the X-ray structures carried out for 1
and 2 (see below).
In fact, the 1H NMR spectra showed a downfield shift of the
chemical shifts of the protons of the R groups being closer to
platinum, while this did not occur when they are far from the
metal center as in the Z configuration. This feature had been
already reported in the study of complexes of similar structure
such as iminoether cis- and trans-[PtCl2{E-N(H)C(OMe)-
R}2]7d (R = Me, Ph, CH2Ph) and amidine cis- and trans-
[PtCl2{E-N(H)C(NMe2)R}2]7c (R = Me, Ph, CH2Ph)
derivatives. Thus, the chemical shifts of the methyl and
methylene R protons of complexes 1−3 were observed at δ
2.76, 3.30, and 4.45, respectively. On the other hand, the
−SCH2 protons of the thiolate group were found in the range
3.03−2.53 ppm, the lowest value being observed for 4. From a
synthetic point of view, there was no evidence of formation of
cis derivatives with a Z arrangement of the imino thioether
ligand, even carrying out the reactions at room temperature in
shorter reaction times (30 min), which, as proposed for the
corresponding reactions of alcohols and amines, should favor
the formation of this type of configuration.4b Complexes 1−4
2.4.3. Cellular Uptake. 2008 and C13* cells (2 × 106) were seeded
in 75 cm2 flasks in growth medium (20 mL). After 24 h, the medium
was replaced, and the cells were incubated for different times (6, 12,
and 24 h) with IC50 doses of the tested complexes. Cell monolayers
were washed twice with cold PBS and harvested. Samples were
subjected to three freezing/thawing cycles at −80 °C and then
vigorously vortexed. Aliquots were removed for the determination of
protein content by the BioRad protein assay (BioRad). The samples
were treated with 1 mL of highly pure nitric acid ([Pt] ≤ 0.01 ng kg−1,
Trace SELECT Ultra, Sigma Chemical Co.) and transferred into a
microwave Teflon vessel. Subsequently, samples were submitted to the
standard procedure using a speed wave MWS-3 Berghof instrument
(Eningen, Germany). After cooling, each mineralized sample was
analyzed for platinum by using a Varian AA Duo graphite furnace
atomic absorption spectrometer (Varian, Palo Alto, CA) at a
wavelength of 324.7 nm. The calibration curve was obtained using
known concentrations of standard solutions purchased from Sigma
Chemical Co.
2.4.4. Caspase-3 Activity. Caspase-3 activity was detected with the
ApoAlert Caspase-3 Fluorescent Assay Kit (Clontech, Mountain View,
CA) according to the producer’s recommended procedures. 1 × 106
2008 cells were collected following 12 or 24 h of incubation of tested
compounds (at concentrations corresponding to IC50 values) and
lysed on ice in 50 μL of lysis buffer for 10 min. Subsequently, cell
lysates were then treated with 50 μL of reaction buffer containing
dithiothreitol (DTT) and 5 μL of caspase-3 substrate solution (Asp-
Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin [DEVD-AFC],
Clontech). Fluorescence was determined on a Perkin-Elmer 550
spectrofluorometer (excitation 440 nm, emission 505 nm). Caspase-3
activity was expressed as the increase in AFC-emitted fluorescence.
2.5. Statistical Analysis. All the values are the means SD of not
less than five measurements. Multiple comparisons were made by
ANOVA followed by Tukey−Kramer multiple comparison test (**P <
0.01; *P < 0.05), using GraphPad Software.
1
were characterized by microanalysis, IR, H and 13C NMR
spectroscopies. The IR spectra showed the N−H stretching
bands in the range 3222−3467 cm−1, while the CN
stretching appeared in the range 1568−1594 cm−1 at lower
values compared to those found for amidine (1609−1628
cm−1) and iminoether (1630−1660 cm−1) complexes.6e,7b Two
bands typical of the cis geometry were observed for the Pt−Cl
absorptions in the expected range of 319−337 cm−1.36 The 1H
NMR spectra exhibited the N−H signal as a broad singlet in
the range δ 8.12−9.38. NOESY experiments confirmed the E
conformation of the complexes showing correlations between
the NH and SCH2CH3 signals. In the 13C NMR spectra, the
resonances of the CN carbons fell in the range δ 178−184 as
expected for Pt(II) imino derivatives and downfield shifted with
respect to iminoether (170−176 ppm)4,6 and amidine (165−
169 ppm)6e ligands. The resonances of the −SCH2 methylene
carbons were observed in the range δ 25−28.
The imino thioether derivatives trans-[PtCl2{E-N(H)
C(SEt)R}2] (R = Me (5), Et (6), CH2Ph (7), Ph (8)) were
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Scheme 3. Synthesis of the Imino Thioether Complexes trans-[PtCl2{E-N(H)C(SEt)R}2]
prepared by reaction of the corresponding complexes trans-
[PtCl2(NCR)2] in THF with a 10-fold excess of EtSH in the
presence of LiBun at low temperature for 1 h and then reacted
at room temperature for additional 2 h (see Scheme 3).
Complexes 5−8 contained both imino thioether moieties in E
configuration.
Pt(II) imino derivatives. The resonances of the −SCH2
methylene carbons were observed in the range δ 24−28.
The characterization of compounds 1−8 included also the
ESI-MS spectra. The ESI spectra of the cis-complexes 1−4 were
characterized by the presence of the molecular ionic species
cationized with Na+ and K+ together with the dimeric species
[2M+Na]+ which represented the base peak for 1 and 3. Similar
behavior has been previously observed for a series of platinum
amidine complexes.42 Retrosynthetic processes with loss of
CH3CH2SH and subsequent addition in multinuclear species
has been observed for 1−3. In the case of 4 the loss of chlorine
atoms might be favored by the formation of an orthometalated
species because of the presence of the phenyl ring close to the
metal center in the E arrangement. The most relevant feature in
the ESI-MS spectra of 5, 7, and 8 was the presence of abundant
dinuclear and trinuclear species retaining intact the imino
thioether ligands. A striking feature of the iminoether
complexes in the solid state, as shown by the X-ray
determination reported below for 1, 2, and 7, and previously
observed for iminoether and amidine complexes in either cis- or
trans-configuration, is that individual molecules are connected
to each other through intermolecular hydrogen bonds forming
dimeric or oligomeric species that could play a role in the
concentration dependence of their hydrolysis rate in the
biological tests.43 This aspect has been previously investigated
as for the stability of infusion solutions of carboplatin and
oxaliplatin.44 It is noteworthy that in the ESI-MS spectra of 5
dissolved in DMF (300 μM) and then diluted 1/1 with
CH3CN, H2O, or physiological water solution, respectively
(Figure 2), the base peak was at m/z 967 corresponding to the
[2M+Na]+ ionic species together with the [M+Na]+ ionic
species with increased abundance in the presence of Cl− (15%
in DMF/CH3CN, 25% in DMF/water and 35% in DMF/
physiological water solution). The presence in the ESI MS
spectrum of 7 (300 μM), in DMF/physiological water solution
(1/1) of the ionic species at m/z 589 corresponding to chlorine
loss with retaining of the N donor ligands, was in agreement
with the proposed mechanism explaining the antitumor activity
of trans-PtL2Cl2 complexes and their interaction with biological
The chemical shifts of the methyl and methylene R protons
of complexes 5−7 were observed at δ 2.89, 3.36, and 4.88,
respectively. On the other hand, the −SCH2 protons of the
thiolate group were found in the range 2.71−3.00 ppm.
1
Complexes 5−8 were characterized by microanalysis, IR, H
and 13C NMR spectroscopies. The IR spectra showed the N−H
stretching bands in the range 3196−3447 cm−1, while the C
N stretching appeared in the range 1586−1601 cm−1 at lower
values compared to those found for amidine and iminoether
complexes.6e,7b One band typical of the trans geometry was
observed for the Pt−Cl absorptions in the expected range of
1
317−344 cm−1. The H NMR spectra exhibited the N−H
signal as a broad singlet in the range δ 9.02−9.67. NOESY
experiments confirmed the E conformation of the complexes
showing correlations between the NH and the SCH2CH3
1
signals. In Figure 1 the H-1H NOESY spectrum of 7 has
been reported. In the 13C NMR spectra, the resonances of the
CN carbons fell in the range δ 178−187 as expected for
substrates.45 The H NMR spectra, recorded in water free
1
CD2Cl2 (Figure 3) confirmed the dependence on association
processes from concentration showing a highfield shift of the
PtNH proton (involved in N−H···Cl intermolecular bonds)
from 8.93 ppm for a 18.0 mM solution to 8.08 ppm for a 0.35
mM solution. The solution association equilibria and the
speciation in water solution of trans-iminothioether compounds
to understand the nature of the platinum species present in
culture media are under investigation. It is noteworthy that also
interaction between DNA and dinuclear platinum species has
been demonstrated.46
Figure 1. Portion of 400 MHz NOESY Spectrum of 7 showing the
correlation peak between Pt−NH and −SCH2- protons.
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Figure 2. ESI mass spectra of 5 dissolved in DMF (300 μM) and then diluted 1/1 with CH3CN (a), H2O (b), or physiological water solution (c).
3.2. X-ray Crystal Structures. 3.2.1. X-ray Structure of
Compound (1). Compound 1 exhibits square-planar coordina-
tion geometry around the platinum center, with the chlorine
ligands in cis positions. Four N−H···Cl intermolecular contacts
(average N−H···Cl 2.665(2) Å, Table 3) between each chlorine
atom and the N−H proton of the imino thioether ligand (L)
shows that the complex consisted of one dimer Pt2Cl4L4
formed by two centrosymmetrically related PtCl2L2 units
(Figure 4). The Pt···Pt contact distance of 3.438(1) Å was
not indicative of a metal−metal interaction (as already observed
in other Pt(II) complexes5b,7b,31 such as cis-[PtCl2{Z-N(H)
C(OCH3)CH2C6H5}2],6e trans-[PtCl2{E-N(H)C(OCH3)-
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1
Figure 3. Portion of 400 MHz H NMR Spectrum of 7 in CD2Cl2 at 0.35 mM (a) and 18.0 mM (b) concentration showing the Pt−NH peak.
CH2−C6H4-p-F}2],6f and cis-[PtCl2{E-N(H)C(N(CH3)2)-
Table 3. Intermolecular Hydrogen Bond Interactions (Å)
and Angles (deg)
CH3}2]).47 The Pt−Cl (average 2.308(2) Å) and Pt−N
(average 2.013(5) Å) bond distances as well as the Cl(1)−
1 (cis)
2 (cis)
7 (trans)
Pt−Cl(2) (91.3(1)°), N(1)−Pt−N(2) (87.6(2)°), N(1)−Pt-
Cl(2) (90.1(1)°) and N(2)−Pt-Cl(1) (91.1(1)°) angles have
normal values for Pt(II) square planar complexes.48 The two
iminothioether ligands had E configuration and comparable
geometric parameters. The N(1)−C(1) [1.279(6) Å] and
N(2)−C(5) [1.280(6) Å] bond distances were in agreement
with a double-bond character and a sp2 hybridization of the N
atom.4b,49 The C(sp2)−S and C(sp3)−S bond distances were
1.736(6) Å and 1.803(7) Å, respectively (averaged values). The
difference (less than 0.1 Å) could be explained on the basis of a
partial double-bond character of C−S [typical values: C(sp3)−
S, 1.82; C(sp2)S, 1.60 Å]50 as indicated also by the values of
the N(1)−C(1)−S(1), N(1)−C(1)−C(2), and S(1)−C(1)−
C(2) angles of 125.3(4)°, 121.8(5)°, and 112.9(4)°,
respectively. The bond distance average of 1.736(6) Å found
for C(1)−S(1) and C(5)−S(2) fell halfway between those of
single and double carbon−sulfur bonds. This could indicate
that whenever a sulfur atom is bound to a carbon atom engaged
in a double bond, a lone pair of the sulfur participates in the π
system. It is worthwhile to note that the NC bond distances
in the imino thioether ligands (N(1)−C(1) 1.279(7) Å and
N(2)−C(5) 1.280(6) Å) were comparable to those observed in
the iminoether derivatives such as cis-[PtCl2{Z-N(H)C-
(OCH3)CH2C6H5}2]6e (1.281(6) Å) while they were shorter
compared with NC distance in amidine derivatives such as
cis-[PtCl2{E-N(H)C(N(CH3)2)CH3}2]48a (1.304(4) Å).
The two NCS planes of the imino thioethereal ligands in
each unit of the dimer formed with the coordination plane
dihedral angles of 70° and 80°, and of 85° each other, while
within the dimeric system the corresponding NCS planes were
exactly parallel.
Cl(1)···H−N(1)′
Cl(2)···H−N(4)
Cl(2)···H−N(4)
2.595(2) 157.2(3)
2.720(2) 139.8(8)
2.6682) 157.8(7)
Cl(2)···H−N(2)′
Cl(3)···H−N(2)
Cl(3)···H−N(1)
2.736(2) 154.3(3)
2.619(2) 155.1(8)
2.707(2) 147.4(8)
′ at 1−x, −y, 1−z
Cl(1)···H−N(3)
Cl(1)···H−N(3)
2.681(2) 143.5(9)
2.5262) 150.2(8)
Cl(4)···H−N(1)
Cl(4)···H−N(2)
2.592(2) 148.8(8)
2.750(2) 137.9(7)
Figure 4. Perspective ORTEP drawing of a dimer of 1 with the atom
labeling scheme. H atoms involved in the N−H···Cl bridges were set
in calculated positions (′ at 1−x, −y, 1−z); (with the exception of the
Pt−NH, the H atoms have been omitted for clarity).
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3.2.2. X-ray Structure of Compound (2). Compound 2
which differs from 1 only in an ethyl substituent to C(6)
(instead of methyl) had the same square planar coordination
geometry around the metal ion with the same cis position of the
imino thioether ligands (Figure 5). Also in this case the
values of the N−C−S, N−C−C, and S−C−C angles of
126(2)°, 120(2)°, and 114(2)°, respectively. The NC bond
distances in the imino thioether ligands (average 1.30(2) Å)
were comparable to those observed in 1. The two NCS planes
of the imino thioethereal ligands in each unit of the dimer
formed with the coordination plane dihedral angles of 60° and
90°, and were almost perpendicular each other, whereas the
corresponding NCS planes were not parallel in the dimer
forming dihedral angles of 70° and 30°, respectively.
3.2.3. X-ray Structure of Compound (7). Compound 7
presented a square planar coordination geometry around the
metal center with the ligands in trans positions. The unit was
formed by two independent PtCl2L2 (L = N(H)C(SEt)-
CH2Ph) moieties, where the two Pt-square planes were parallel
but rotated each other of 95° (Figure 6). The Pt−Cl and Pt−N
bond distances were again comparable to those of compound 1
and 2 and was evidenced by the presence of a dimer due to the
occurrence of four intermolecular Cl···H−N hydrogen bonds
with lengths comparable to those of 1 and 2 (see Table 3). The
Pt···Pt contact distance of 3.428(1) Å indicated the absence of
metal−metal interaction. The imino thioether moieties had E
configuration and comparable geometrical parameters. Some
relevant bond distances in the iminothioether ligands (average
Pt−N 2.017(9) Å, CN 1.28(2) Å and C−CH2 1.49(2) Å)
resembled the values observed in the iminoethereal ligands of
cis-[PtCl2{Z-N(H)C(OCH3)CH2C6H5}2]6e (average Pt−N
2.006(4) Å, CN 1.281(6) Å, C−CH2 1.499(7) Å). Again
the C(sp2)−S bond distance average of 1.75(2) Å could be
explained on the basis of a partial double-bond character of C−
S as indicated also by the average values of the N−C−S, N−C−
C, and S−C−C angles of 123(1)°, 123(1)°, and 112(1)°,
respectively. The NC bond distances in the imino thioether
ligands (average 1.28(2) Å) were comparable to those observed
in 1 and 2. Both NCS planes of the imino thioether ligands in
the Pt(1) unit formed with the coordination plane dihedral
angles of 60° and of 80° in the Pt(2) unit, the corresponding
NCS planes in the dimer being almost perpendicular. Within
each unit the phenyl ring planes were almost perpendicular
with respect the corresponding NCS planes, and despite the
presence of several aromatic moieties, the inspection of the
Figure 5. ORTEP drawing of a dimer of compound 2 (with the
exception of the Pt−NH, the H atoms have been omitted for clarity).
structure consisted of dimers Pt2Cl4L4 (L = N(H)C(SEt)-
CH2CH3) formed by two PtCl2L2 units intermolecularly held
together by four N−H···Cl hydrogen bonds involving the
chlorines and the imino thioether NH protons of two adjacent
molecules. The Pt···Pt contact distance of 3.258(1) Å indicated
a degree of metal−metal interaction. The Pt−Cl and Pt−N
bond distances (Table 2) were comparable to those of 1 as well
the N−H···Cl intermolecular contacts (Table 3). The imino
thioether moieties had E configuration and comparable
geometrical parameters. Again the C(sp2)−S bond distance
average of 1.73(2) Å could be explained on the basis of a partial
double-bond character of C−S as indicated also by the average
Figure 6. ORTEP drawing of a dimer of compound 7 (with the exception of the Pt−NH, the H atoms have been omitted for clarity).
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Table 4. Cytotoxic Activity of the Platinum(II) Imino Thioether Complexes in Human Cancer Lines Derived from Solid
Tumors
a
b
IC50 (μM) SD
compound
MCF-7
A549
A375
A431
A431/Pt
92
46.3 1.1
R.F.
2008
C13*
R.F.
LoVo
2
LoVo MDR R.F.
5
97.0 1.2
43.0 1.6
7.0 0.5
>100
86
39
2
2
88
34
3
2
93.2 1.9
43.0 0.8
7.1 1.4
>100
2
1.0
1.1
0.9
97.2 1.6
47.2 1.6
88
43
2
2
0.9
0.9
1.1
91
98.8 1.6
51.8 1.4
5.8 0.7
>100
1.1
1.3
0.8
6
44.1 1.0
6.9 1.1
>100
7
5.1 1.1
>100
6.5 1.0
>100
6
2
7
2
8.1 1.1
>100
transplatin
>100
>100
cisplatin
23
2
27.2 1.5
21
2
17.2 1.1
41.3 1.2
2.6
12.7 1.7
89.2 1.5
7.0
16.5 1.9
a
IC50 values were calculated by PL model (P < 0.05). Cells (3-8 × 104 ml−1) were treated for 48 h with increasing concentrations of tested
b
compounds dissolved in DMF. Cytotoxicity was assessed by MTT test. The RF is defined as IC50 resistant/parent line. S.D. = standard deviation.
Figure 7. Intracellular accumulation of platinum complexes. 2008 (A) and C13* (B) cells were incubated with IC50 doses of complex 7 and cisplatin
for 6, 12, and 24 h. Intracellular platinum accumulation was detected by GF-AAS analysis. Error bars indicate the standard deviation. Key: *, p <
0.05; **, p < 0.01 compared to the control, °, p < 0.05 compared to cisplatin-sensitive cells.
packing diagram did not reveal the presence of π staking
interactions.
and A431/Pt IC50 values) for 5 and cisplatin, respectively.
Similarly, the complex trans-[PtCl2{E-N(H)C(SEt)Et}2] (6)
showed across the various cancer cell lines a cytotoxic activity
lower than that of cisplatin, the mean IC50 (μM) values being
respectively 43.2 and 20.0 (excluding C13* and A431/Pt IC50
values) for 6 and cisplatin, respectively. Among all derivatives
the complex trans-[PtCl2{E-N(H)C(SEt)CH2Ph}2] (7)
showed the greatest in vitro antitumor activity being able to
decrease cell viability roughly 3-fold more effectively than
cisplatin (mean IC50 (μM) value 6.5).
Cytotoxic activity of imino thioethers was also evaluated onto
additional human cancer cell lines, including two cisplatin-
resistant cell lines (C13* ovarian adenocarcinoma and A431/Pt
cervical squamous carcinoma cells) and a multidrug resistant
(MDR) phenotype (LoVo MDR colon adenocarcinoma cells).
Cross-resistance profiles were evaluated by means of the
resistance factor (R.F.) which is defined as the ratio between
IC50 values calculated for the resistant cells and those obtained
with the sensitive ones. Although cisplatin resistance is
multifactorial, the main molecular mechanisms involved in
C13* and A431/Pt cell resistance have almost been defined. In
particular, in human ovarian adenocarcinoma C13* cells,
cisplatin resistance has been correlated to reduced cell drug
uptake, high cellular thioredoxin reductase and glutathione
3.3. Biological Assays. 3.3.1. Cytotoxicity Assays. The
newly synthesized platinum(II) imino thioether derivatives
have been evaluated for their cytotoxic activity toward a panel
of nine human tumor cell lines derived from solid tumors and
including examples of ovarian (2008 and C13*), cervical (A431
and A431/Pt), lung (A549), colon (LoVo and loVo MDR), and
breast (MCF-7) cancers and melanoma (A375). Cytotoxicity
has been evaluated by means of MTT tests after 48 h of
treatment with increasing concentrations of the tested
compounds. For comparison purposes, the cytotoxicity of
cisplatin, the most widely used anticancer metallodrug, and of
transplatin were evaluated in the same experimental conditions.
IC50 values, calculated from dose-survival curves, are shown in
Table 4. The cis complexes 1−4 were not tested for their
biological activity, because of their very low solubility in the
experimental conditions. All the trans-imino thiother complexes
appeared to be more effective than transplatin, which, as well
documented,6a was found to be scarcely effective. Among Pt(II)
trans-derivatives, the complex trans-[PtCl2{E-N(H)C(SEt)-
Me}2] (5) was the least active agent, showing an antiprolifer-
ative activity roughly 5-fold lower than that of cisplatin, the
mean IC50 (μM) values being 93.2 and 20.0 (excluding C13*
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levels, and enhanced repair of DNA damage.51 In human
squamous cervical carcinoma A431/Pt cells, resistance is due to
defect in drug uptake and to decreased levels of proteins
involved in DNA mismatch repair (MSH2), causing an
increased tolerance to cisplatin-induced DNA damage. As
shown in Table 4, all derivatives exhibited a different cross-
resistance profile than that of cisplatin, possessing a quite
similar cytotoxic potency both on cisplatin sensitive and
resistant cell lines. The R.F. for all derivatives, calculated on
the A431-A431/Pt and 2008-C13* cell pairs, were about 2 and
7 times lower than those of cisplatin, respectively, clearly
revealing no cross-resistance phenomena and thus supporting
the hypothesis that these Pt(II) imino thioether complexes
have different cytotoxic mechanisms compared to cisplatin.
Tested on LoVo MDR colon cancer cells, suitably selected for
their resistance to doxorubicin and thus retaining the MDR
phenotype, all derivatives yielded R.F. values roughly 28 times
lower than that obtained with doxorubicin (R.F.: 30.7). Given
that in LoVo MDR cells doxorubicin-resistance is mainly
associated with an overexpression of the multispecific drug
transporters, such as the 170 kDa P-glycoprotein,52 the ability
of Pt(II) imino thioether complexes to overcome multidrug
resistance phenomena in this cell line may suggest that these
agents are not potential MDR substrates.
Figure 8. Induction of caspase-3 activity. 2008 cells were incubated for
24 h with complex 7 or cisplatin and then processed for caspase-3
activity detection as described in the Experimental Section. Data are
the mean values of at least three independent experiments. Error bars
indicate standard deviation.
CH2Ph}2] treated cells, protease activity was four times
enhanced with respect to control cells.
3.3.2. Cellular Uptake. It is well-known that cellular uptake
is an important factor influencing drug efficacy. Moreover, since
one of the main mechanisms controlling cisplatin resistance is
cellular uptake, uptake experiments were performed in human
ovarian adenocarcinoma cells, sensitive (2008) and resistant
(C13*) to cisplatin. Cancer cells were treated for 6, 12, and 24
h with IC50 doses of complex 7, the most promising Pt(II)
imino thioether derivative, and cisplatin. The intracellular
platinum accumulation was quantified by means of GF-AAS
analysis, and the results, expressed as μg of metal mg−1 of
cellular proteins, are summarized in Figure 7. Cellular uptake of
complex 7 was time dependent, both in sensitive as well as in
cisplatin resistant cells. There was, however, a marked
difference between sensitive and resistant cells as far as the
discrimination between cisplatin and complex 7 is concerned.
In 2008 sensitive cells the amount of internalized Pt amount
was very similar for both cisplatin and Pt(II) imino thioether
derivative 7 (Figure 7A). In resistant C13* cells (Figure 7B)
the cellular uptake was remarkably decreased for cisplatin but
not for compound 7, which was internalized with the same
efficacy in 2008 and C13* cells. These data can well justify the
lack of cross-resistance between compound 7 and cisplatin in
ovarian C13* cells.
3.3.3. Caspase-3 Activation. To investigate if the decrease
in cell viability induced by trans-[PtCl2{E-N(H)C(SEt)-
CH2Ph}2] could be due to apoptosis, caspase-3 activity was
assayed. Caspase-3, a downstream caspase, playing a pivotal role
in the terminal execution phase of apoptosis induced by diverse
stimuli, including anticancer metallodrugs.53 The cleavage of
various substrates contributes to the typical morphological and
biochemical features observed in apoptosis. Because of the
diversity of its substrates, caspase-3 is thought to be a general
mediator of physiological and stress-induced apoptosis.
Caspase-3 activity was assayed in 2008 human ovarian
carcinoma cells treated for 24 h with IC50 of compound 7, or
cisplatin, the first line chemotherapeutic drug against ovarian
cancer. Figure 8 shows that trans-[PtCl2{E-N(H)C(SEt)-
CH2Ph}2] markedly stimulated caspase-3 activity, similarly to
cisplatin. In particular, in trans-[PtCl2{E-N(H)C(SEt)-
4. CONCLUSIONS
In this paper we reported the high yield synthesis of novel
biologically active imino thioether complexes of the type cis-
and trans-[PtCl2{E-N(H)C(SEt)R}2] by addition of
CH3CH2SH to coordinated nitrile ligands in cis- and trans-
[PtCl2(NCR)2] (R = Me, Et, CH2Ph, Ph). These complexes
complete a series of structurally similar Pt(II)-imino derivatives
bearing amidine or iminoether ligands of the type cis- and trans-
[PtCl2{E-N(H)C(NR′R″)R}2] and cis- and trans-[PtCl2{E-
N(H)C(OR′)R}2], respectively, which have been also found
to exhibit cytotoxic activity higher than cisplatin.6a−d,7e It is
worthwhile noting that the nature of the heteroatom affects the
biological activity of these imino-Pt(II) complexes. Generally,
the S- and O-derivatives show higher activity compared to the
N-analogues. The cytotoxicity of the new imino thioether
products has been studied in different cell lines derived from
solid tumors and endowed with different sensitivity to cisplatin.
The compound trans-[PtCl2{E-N(H)C(SEt)CH2Ph}2] (7)
proved to be the most active cytotoxic agent and exhibited a
biological activity similar to that shown by trans-[PtCl2{E-
N(H)C(OMe)Me}2] and trans-[PtCl2(NH3){E-N(H)C-
(OMe)Me}] previously reported by Natile et al.6a We also
noticed that the presence of a benzyl group increases the
cytoxicity of this type of complexes. In fact, 7 shows the highest
biological activity within the series of imino thioethers
prepared. Moreover, the cellular uptake of compound 7 is
similar into cisplatin-sensitive and -resistant cells. This indicates
that, somehow, 7 can easily escape the resistance mechanisms
that allow for a decreased uptake and/or increased export of
platinum drugs in resistant cells. In conclusion, it appears that
the imino thioether platinum complexes are good candidates
for further investigation in the field of cytotoxic drugs and
therefore a wider series of imino thioether derivatives will be
developed. In particular, a synthetic strategy for the preparation
of complexes bearing cyclic imino thioethers, which are of
relevant interest for biomedical applications,54 will be
investigated.
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Inorganic Chemistry
Article
Sbovata, S.; Bettio, F.; Michelin, R. A.; Seraglia, R.; Kiss, T.; Venzo, A.;
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ASSOCIATED CONTENT
* Supporting Information
Cystallographic data in CIF format. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Fax: +39 049 8275525. Phone: +39 049 8275522. E-mail:
rino.michelin@unipd.it.
■
̀
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the University of Padova (PRAT Project
2011, code CPDA114144, SA 31/10/2012, and
CPDA121121973/12) and MIUR (PRIN 2010 project, code
2010L9SH3K) for financial support and the Inter-University
Consortium for Research on the Chemistry of Metal Ions in
Biological Systems (CIRCMSB, Bari, Italy).
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