Water-soluble Cp ruthenium complex containing 1,3,5-triaza-7- phosphaadamantane and 8-thiotheophylline derivatives: Synthesis, characterization, and antiproliferative activity

Article abstract of DOI:10.1021/ic101466u

The new water-soluble ruthenium(II) mononuclear complexes [RuCp(X)(PTA)(L)] (X = 8-thio-theophyllinate (TTH- ), L = PTA (1), L = PPh₃ (7)); (X = 8-methylthio-theophyllinate (8-MTT-), L = PTA (2), L = PPh₃ (8)), (X = 8-benzylthio-theo

Full text of DOI:10.1021/ic101466u

Inorg. Chem. 2011, 50, 873–882 873
DOI: 10.1021/ic101466u
Water-Soluble Cp Ruthenium Complex Containing
1,3,5-Triaza-7-phosphaadamantane and 8-Thiotheophylline Derivatives:
Synthesis, Characterization, and Antiproliferative Activity
Lazhar Hajji,† Cristobal Saraiba-Bello,† Antonio Romerosa,*,† Gaspar Segovia-Torrente,† Manuel Serrano-Ruiz,†
Paola Bergamini,‡,§ and Alessandro Canella||
† ꢀ
´
ꢀ
´
´
Area de Quımica Inorganica, Facultad de Ciencias Experimentales, Universidad de Almerıa, 04071 Almerıa,
‡
ꢁ
Spain, Dipartimento di Chimica dell’Universita di Ferrara, via L. Borsari 46, 44100 Ferrara, Italia,
§Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Italia, and
||
ꢁ
Dipartimento di Biochimica e Biologia Molecolare dell’Universita di Ferrara, via L. Borsari 46,
44100 Ferrara, Italia
Received July 22, 2010
The new water-soluble ruthenium(II) mononuclear complexes [RuCp(X)(PTA)(L)] (X = 8-thio-theophyllinate (TTH-),
L = PTA (1), L = PPh3 (7)); (X = 8-methylthio-theophyllinate (8-MTT-), L = PTA (2), L = PPh3 (8)), (X = 8-benzylthio-
theophyllinate (8-BzTT-), L = PTA (3), L = PPh3 (9)) and binuclear complexes [{RuCp(PTA)(L)}2-μ-(Y-κN7,N07)]
(Y = bis(S-8-thiotheophyllinate)methane (MBTT2-), L = PTA (4), L = PPh3 (10)), (Y = 1,2-bis(S-8-thiotheophyllina-
te)ethane (EBTT2-), L=PTA (5), L=PPh3 (11)), (Y=1,3-bis(S-8-thiotheophyllinate)propane (PBTT2-); L=PTA (6),
L=PPh3 (12)) have been synthesized and characterized by NMR, IR spectroscopy and elemental analysis. The single
crystal X-ray structure of [RuCp(8-MTT-κS)(PTA)2] (2) was also obtained. The antiproliferative activity of the
complexes on cisplatin-sensitive T2 and cisplatin-resistant SKOV3 cell lines has been evaluated.
Introduction
Nevertheless, platinum-based drugs still present a few draw-
backs like activity restricted to a limited number of tumors,
severe side effects, drug resistance,4 and so forth. Therefore
the search for new metal-based drugs is still a lively field.
Ruthenium complexes are promising anticancer agents5 and
two of them, NAMI-A6 and KP10197 are currently under
clinical trials, for the treatment of metastatic and colorectal
cancers, respectively.
The cytotoxicity of ruthenium compounds, as for cisplatin,
correlates with their ability for DNA binding, which is based
on the formation of metal containing active species mainly by
aquation processes. It is known that Ru(III) complexes are
Transition metal complexes designed to bind to DNA have
been extensively studied in the last few decades.1 The dis-
covery of the anticancer properties of cisplatin2 in the 1960s
by Rosenberg proved that transition metal complexes could
be toxic for tumor cells. As a consequence, a huge number of
metal compounds of wide structural diversity have been
tested and some of them found to be therapeutic agents
for cancer treatment. Still now cisplatin and its parent
analogues are among the most widely used chemotherapeutic
agents.3
*To whom correspondence should be addressed. E-mail: romerosa@
ual.es.
(4) Stordal, B.; Davey, M. IUBMB Life 2007, 59, 696. Pabla, N.; Dong, Z.
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10690–10701. (b) Foxon, S. P.; Phillips, T.; Gill, M. R.; Towrie, M.; Parker, A. W.;
Webb, M.; Thomas, J. A. Angew. Chem., Int. Ed. 2007, 46, 3686. (c) Neves, A.;
Lanznaster, M.; Bortoluzzi, A. J.; Peralta, R. A.; Casellato, A.; Castellano, E. E.;
Herrald, P.; Riley, M. J.; Schenk, G. J. Am. Chem. Soc. 2007, 129, 7486.
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Zhu, F.; Frasca, D. R. Chem. Rev. 1999, 99, 2511. (f) Zeglis, B. M.; Pierre, V. C.;
Barton, J. K. Chem. Commun. 2007, 4565. (g) Reedijk, J. Proc. Natl. Acad. Sci.
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r
2011 American Chemical Society
Published on Web 01/12/2011
pubs.acs.org/IC

874 Inorganic Chemistry, Vol. 50, No. 3, 2011
Hajji et al.
Chart 1
relatively inert toward ligand substitution and therefore their
anticancer activity depends on the ease of reduction to more
labile Ru(II) complexes.8 As a consequence, organometallic
ruthenium(II) compounds attracted the attention of drug
designers in their search for novel antiproliferative agents.
The use of arene ligands, which stabilize ruthenium in the þ2
oxidation state, initiated a new phase in the search for
ruthenium anticancer drugs.
PR13 = PR23 = PTA, mPTA) (PTA = 1,3,5-triaza-7-phos-
phaadamantane; mPTA = N-methyl-1,3,5-triaza-7-phos-
phaadamantane).16 The DNA binding activity of these
complexes was found dependent on the ligand bonded to
the metal, the ones containing chloride being active agents
while the iodide analogues are inactive. The DNA bind-
ing activity was also dependent on which phosphine is
coordinated to the metal, PTA or mPTA. Therefore the
interaction of the biologically active [RuCpX(PR1 )(PR2 )]
Additionally, proteins, peptides, and also some small
molecules can play significant roles in the anticancer activ-
ity and resistance to drugs.9 Among biomolecules, sulfur-
containing proteins are of special interest for their abundance
and their high affinity for platinum and ruthenium (e.g.,
albumin).10 An important example of this family of proteins
is the copper transporter protein CTR1 that appears to
be involved in platinum-drug transport through the cell
membrane.11 The S-donor molecules seem to play a relevant
role in the anticancer activity of platinum complexes of trans
geometry acting with a different mechanism with respect to
traditional cisplatin complexes.12 Furthermore, it is possible
to exploit sulfur oxidation as a mechanism for the activation
of Ru(II) arene thiolate pro-drugs as well as for the reactiva-
tion of metabolites of Ru(II) arene anticancer drugs,13 which
are sometimes controlled by metal complexation to the S
atom of proteins and enzymes.14 Thiolate oxygenation seems
to activate cytotoxic half-sandwich [(η6-arene)Ru(en)(SR)]þ
complexes (en = ethylenediamine; arene = p-cymene, hex-
amethylbenzene; SR = alkyl or aryl thiolate) toward DNA
binding and the properties of Ru-S bonds depend on the
extent of S oxygenation, on the nature of the thiolate
substituent and of the arene group.15
3
3
(X = halogens) species with DNA can be favored and
maximized by an appropriate combination of hydro and
liposoluble phosphines and by the adequate choice of the
anionic ligand X.
Looking for biologically active metal complexes, a few
years ago we studied a group of Pt-phosphines complexes
bearing the deprotonated form of the 8-thiotheophylline
derivatives b-f depicted in Chart 1 as anionic ligands.17
We showed that Pt complexes containing PPh3 and the N7
coordinated anionic thiopurinic ligand 8-MTT- show a
remarkable antiproliferative activity on some human cancer
cell lines. Following the hypothesis that such activity could be
improved by modulating the lipo-hydrophilicity of the co-
ordinated phosphines, we tested the PTA analogues and also
a series of PTA/PPh3 dinuclear Pt complexes of the
bis-thiopurines bis(S-8-thiotheophylline)methane (MBTTH2;
Chart 1, d), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2;
Chart 1, e), and 1,3-bis(S-8-thiotheophylline)propane
(PBTTH2; Chart 1, f). We found that the platinum com-
plexes containing both PTA and PPh3, a hydrophilic and a
lipophilic phosphine, display on model tumoral cell lines
T2 and SKOV3 an antiproliferative activity better than the
analogues containing a single phosphine type, probably
because of a more favorable balance between lipophilicity
and hydrophilicity of the entire complex.18 Referring to such
promising results obtained on platinum, as the next step we
planned to extend the study of the favorable combination to
ruthenium complexes of thiopurinic and phosphinic PTA
and PPh3 ligands. It is reasonable to suppose that the sub-
stitution of the chloride ligand in [RuCpCl(PR1 )(PR2 )]
We have recently published the synthesis and interaction
with DNA of the Ru(II) piano-stool complexes [RuCpX-
(PR1 )(PR2 )] (X=Cl, I; PR1 =PPh3; PR2 =PTA, mPTA;
3
3
3
3
(8) Clarke, M. J. Coord. Chem. Rev. 2003, 236, 209.
(9) (a) Jung, Y. W.; Lippard, S. J. Chem. Rev. 2007, 107, 1387. (b) Kuo,
M. T.; Chen, H. H.W.; Song, I. S.; Savaraj, N.; Ishikawa, T. Cancer Metastasis
Rev. 2007, 26, 71. (c) Martin, L. P.; Hamilton, T. C.; Schilder, R. J. Clin. Cancer
Res. 2008, 14, 1291.
3
3
complexes (PR1 =PPh3; PR2 =PTA) by a thiopurine could
(10) (a) Ivanov, A. I.; Christodoulou, J.; Parkinson, J. A.; Barnham, K. J.;
Tucker, A.; Woodrow, J.; Sadler, P. J. J. Biol. Chem. 1998, 273, 14721.
(b) Montero, E. I.; Benedetti, B. T.; Mangrum, J. B.; Oehlsen, M. J.; Qu, Y.;
Farrell, N. P. Dalton Trans. 2007, 4938.
(11) Arnesano, F.; Scintilla, S.; Natile, G. Angew. Chem., Int. Ed. 2007,
46, 9062.
3
3
enhance the antiproliferative activity of the parent com-
plexes. Moreover, this study could provide additional infor-
mation on the interaction of ruthenium complexes with
purines.
(12) Li, C.; Li, Z.; Sletten, E.; Arnesano, F.; Losacco, M.; Natile, G.; Liu,
Y. Angew. Chem., Int. Ed. 2009, 48, 8497.
(13) (a) Charles, R. L.; Schroder, E.; May, G.; Free, P.; Gaffney, P. R. J.;
Wait, R.; Begum, S.; Heads, R. J.; Eaton, P. Mol. Cell. Proteomics 2007, 6,
1473. (b) Jacob, C.; Knight, I.; Winyard, P. G. Biol. Chem. 2006, 387, 1385–1397.
(14) (a) Maret, W. Antioxid. Redox Signaling 2006, 8, 1419–1441. (b) Dey,
A.; Jeffrey, S. P.; Darensbourg, M.; Hodgson, K. O.; Hedman, B.; Solomon, E. I.
Inorg. Chem. 2007, 46, 4989–4996.
(16) Romerosa, A.; Campos-Malpartida, T.; Lidrissi, C.; Saoud, M.;
Serrano-Ruiz, M.; Peruzzini, M.; Garrido-Cardenas, J. A.; Garcia-Maroto,
F. Inorg. Chem. 2006, 45, 1289–1298.
(17) Romerosa, A.; Bergamini, P.; Bertolasi, V.; Canella, A.; Cattabriga,
~
M.; Gavioli, R.; Manas, S.; Mantovani, N.; Pellacani, L. Inorg. Chem. 2004,
43, 905–913.
(15) Sriskandakumar, T.; Petzold, H.; Bruijnincx, P. C. A.; Habtemariam,
A.; Sadler, P. J.; Kennepohl, P. J. Am. Chem. Soc. 2009, 131, 13355–13361.
(18) Bergamini, P.; Bertolasi, V.; Marvellli, L.; Canella, A.; Gavioli, R.;
~
Mantovani, N.; Manas, S.; Romerosa, A. Inorg. Chem. 2007, 46, 4267–4276.

Article
In this paper we present the synthesis and characteriza-
Inorganic Chemistry, Vol. 50, No. 3, 2011 875
41.75; H, 5.33; N, 20.10; S, 4.47%; calcd. C, 41.67; H, 5.25; N,
20.25; S, 4.64%.
tion of a new family of water-soluble neutral mononuclear
ruthenium complexes [RuCp(X)(PR1 )(PR2 )] (X = 8-thio-
Synthesis of [RuCp(8-MTT-KS)(PTA)2] (2). Complex 2 was
prepared in the same way as 1 but using the 8-MTTH (0.079 g,
0.35 mmol) as the purine derivate. The product was obtained as
a yellow powder. Good quality yellow crystals for X-ray dif-
fraction were obtained by slow evaporation from an EtOH
solution of 2.
3
3
teophyllinate (TTH-; Chart 1, a), 8-methyl-thio-teophylli-
nate (8-MTT-; Chart 1, b), 8-benzyl-thio-theophyllinate (8-
BzTT-; Chart 1, c) and binuclear ruthenium complexes
[{RuCp(L)(L0)}2-μ-(Y-κN7,N07)] (Y = bis-thiopurines-bis-
(S-8-thiotheophyllinate)methane (MBTT2-; Chart 1, d), 1,
2-bis(S-8-thiotheophyllinate)ethane (EBTT2-; Chart 1, e),
1,3-bis(S-8-thiotheophyllinate)propane (PBTT2-; Chart 1, f)
with two coordinated PTA ((PR13 = PR23 = PTA) or one
Yield: 0.190 g, 75%. S25(mg/cm3): 32. Log P: -1.41. Ele-
mental analysis for C25H38N10O2P2RuS (705.72): Found C,
42.64; H, 5.52; N, 19.68; S, 4.40%; calcd. C, 42.55; H, 5.43; N,
19.85; S, 4.54%.
PPh3 and one PTA (PR1 = PPh3; PR23 = PTA) and their
Synthesis of [RuCp(8-BzTT-KS)(PTA)2] (3). The procedure
used for the synthesis of the complex 3 was similar to that
described for 1. The 8-BzTTH (0.105 g, 0.35 mmol) was dis-
solved into 10 mL of aqueous KOH solution (0.035 M), stirred
for 15 min, and [RuClCp(PTA)2] (0.180 g, 0.35 mmol) was
added. The product was obtained as a yellow powder.
Yield: 0.220 g, 78%. S25(mg/cm3): 38. Log P: -0.97. Ele-
mental analysis for C31H42N10O2P2RuS (781.81): Found C,
47.62; H, 5.52; N, 17.76; S, 3.91%; calcd. C, 47.62; H, 5.41; N,
17.92; S, 4.10%.
Synthesis of [{RuCp(PTA)2}2(μ-MBTT-KN7,N07)] (4). The
bis-thiopurine MBTTH2 (15.0 mg, 0.034 mmol) was introduced
into a 10 mL deoxygenated aqueous KOH solution (0.0068 M).
The mixture was stirred until complete dissolution of MBTTH2
and then [RuClCp(PTA)2] (35.5 mg, 0.068 mmol) was added.
The mixture was refluxed for 4 h, cooled, filtered, and concen-
trated by evaporation to 2 mL. The obtained precipitate was
filtered, washed with EtOH (2 ꢀ 1 mL) and Et2O (2 ꢀ 2 mL), and
dried under vacuum.
3
antiproliferative activity on cisplatin-sensitive T2 and cispla-
tin-resistant SKOV3 model cell lines.
Experimental Section
General Procedures. All chemicals were reagent grade and
were used as received by commercial suppliers unless otherwise
stated. The solvents were all degassed and distilled according to
standard procedures.19 All reactions and manipulations were
routinely performed under a dry nitrogen atmosphere by using
standard Schlenk-tube techniques. The compounds PTA,
[RuClCp(PTA)2], and [RuClCp(PPh3)(PTA)] were prepared
as described in the literature.16,20,21 Ligands 8-thiotheophylline
(8-TTH2), 8-methylthiotheophylline (8-MTTH), 8-benzylthi-
otheophylline (8-BzTTH), bis(S-8-thiotheophylline)methane
(MBTTH2), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2),
1,3-bis(S-8-thiotheophylline)propane (PBTTH2) have been
prepared following the procedure described by literature
methods.22,23 1H, 31P{1H} NMR, and 13C{1H} NMR spectra
were recorded on a Bruker DRX300 spectrometer operating at
300.13 MHz (1H), 121.49 MHz (31P), and 75.47 MHz (13C),
respectively. Peak positions are relative to tetramethylsilane and
were calibrated against the residual solvent resonance (1H) or
the deuterated solvent multiplet (13C). Chemical shifts for 31P-
{1H} NMR were measured relative to external 85% H3PO4 with
downfield values taken as positive. The 1H,1H-2D COSY NMR
experiments were routinely conducted on the Bruker DRX300
instruments in the absolute magnitude mode using a 45 or 90°
pulse after the incremental delay. Infrared spectra were recorded
on KBr discs using an FT-IR ATI Mattson Infinity Series.
Elemental analysis (C, H, N, S) were performed on a Fisons
Instruments EA 1108 elemental analyzer.
Yield: 43.0 mg, 85%. S25(mg/cm3): 57. Log P: -1.55. Ele-
mental analysis for C49H72N20O4P4Ru2S2 (1395.40): Found C,
42.30; H, 5.25; N, 19.91; S, 4.42%; calcd. C, 42.30; H, 5.25; N,
19.91; S, 4.42%.
Synthesis of [{RuCp(PTA)2}2(μ-EBTT-KN7,N07)] (5). The
synthesis of complex 5 was carried out by a similar procedure
to that described for 4. In this case, the ligand EBTTH2 (15.0 mg,
0.032 mmol) was dissolved into a deoxygenated aqueous KOH
solution (10 mL, 0.0064 M) and [RuClCp(PTA)2] (33.5 mg,
0.064 mmol) was added. The product was separated as a yellow
powder.
Yield: 30 mg, 62.0%. S25(mg/cm3): 40. Log P: -1.47. Ele-
mental analysis for C50H74N20O4P4Ru2S2 (1409.42): Found C,
43.10; H, 5.51; N, 19.50; S, 4.38%; calcd. C, 42.61; H, 5.29; N,
19.88; S, 4.55%.
Synthesis of [{RuCp(PTA)2}2(μ-PBTT-KN7,N07)] (6). The
bis-thiopurine PBTTH2 (15.0 mg, 0.031 mmol) dissolved in 10 mL
deoxygenated aqueous KOH solution (0.0062 M) reacted with
[RuClCp(PTA)2] (32.5 mg, 0.062 mmol) to give, after the same
work up as above, complex 6 as a yellow powder.
Yield: 33 mg, 70%. S25(mg/cm3): 36. Log P: -1.42. Elemental
analysis for C51H76N20O4P4Ru2S2 (1423.45): Found C, 43.10;
H, 5.51; N, 19.50; S, 4.38%; calcd. C, 43.03; H, 5.38; N, 19.68; S,
4.51%.
Synthesis of [RuCp(8-TTH-KS)(PTA)2] (1). The ligand
8-TTH2 (0.074 g, 0.35 mmol) was dissolved into 10 mL of
degassed aqueous KOH solution (0.035 M) and stirred for
15 min. The complex [RuClCp(PTA)2] (0.180 g, 0.35 mmol) was
added, and the resulting solution kept at refluxing temperature
for 4 h. The obtained solution was concentrated under reduced
pressure until 1 mL. The yellow precipitate obtained was filtered
and washed with EtOH (2 ꢀ 2 mL) and Et2O (2 ꢀ 2 mL), and
vacuum-dried.
Yield: 0.170 g, 68%. S25(mg/cm3): 18. Log P: -1.29. Ele-
mental analysis for C24H36N10O2P2RuS (691.69): Found C,
Synthesis of [RuCp(8-TTH-KS)(PPh3)(PTA)] (7). The com-
plex [RuClCp(PPh3)(PTA)] (0.150 g, 0.24 mmol) was added to a
solution of the ligand 8-TTH2 (0.051 g, 0.24 mmol) in 10 mL of
ethanolic KOH (0.024 M). The mixture refluxed and stirred for
4 h, cooled down to room temperature, and concentrated under
reduced pressure to 1 mL. The resulting yellow precipitate was
washed with EtOH (2 ꢀ 2 mL) and Et2O (2 ꢀ 2 mL), and
vacuum-dried.
(19) Purification of Laboratory Chemicals, 3rd ed.; Perrin, D. D.; Armarego,
W. L. F., Eds.; Butterworths and Heinemann: Oxford, 1988.
(20) Aqueous-Phase Organometallic Catalysis; Cornils, B.; Herrmann,
W. A., Eds.; Wiley-VCH: Weinheim, Germany, 1998.
ꢀ
(21) Joo, F.; Kovacs, J.; Katho, A.; Benyei, A. C.; Decuir, T.; Darensboug,
D. J. Inorg. Synth. 1998, 32, 2.
ꢀ
~
~
(22) Romerosa, A.; Lopez-Magana, C.; Goeta, A. E.; Manas, S.; Saoud,
M.; Benbdelouahab, F. B.; El Guemmout, F. Inorg. Chim. Acta 2003, 353,
99–106.
Yield: 0.160 g, 84%. S25(mg/cm3): 0.8. Log P: 0.96. Elemental
analysis for C36H39N7O2P2RuS (796.82): Found C, 54.35; H,
5.05; N, 12.25; S, 4.02%; calcd. C, 54.26; H, 4.93; N, 12.30; S,
4.02%.
ꢀ
~
(23) (a) Romerosa, A.; Lopez-Magana, C.; Saoud, M.; Colacio, E.;
ꢀ
Suarez-Varela, J. Inorg. Chim. Acta 2000, 307, 125–130. (b) Romerosa, A.;
ꢀ
~
~
Lopez-Magana, C.; Saoud, M.; Manas, S. Eur. J. Inorg. Chem. 2003, 348–355.
Synthesis of [RuCp(8-MTT-KS)(PPh3)(PTA)] (8). The yellow
complex 8 was obtained by the procedure described above for 7.
ꢀ
~
~
(c) Romerosa, A.; Lopez-Magana, C.; Manas, S.; Saoud, M.; Goeta, A. E. Inorg.
Chim. Acta 2003, 353, 145–150.

876 Inorganic Chemistry, Vol. 50, No. 3, 2011
Hajji et al.
The 8-MTTH (0.055 g, 0.24 mmol) was used as the purine
derivate and reacted with [RuClCp(PPh3)(PTA)] (0.150 g,
0.24 mmol) in 10 mL of an ethanolic solution of KOH (0.024 M).
Yield: 0.140 g, 70%. S25(mg/cm3): 1.1. Log P: 1.26. Elemental
analysis for C37H41N7O2P2RuS (810.85): Found C, 54.87; H,
5.17; N, 12.02; S, 3.87%; calcd. C, 54.81; H, 5.10; N, 12.09; S,
3.95%.
Table 1. Crystallographic Data for 2 4H2O
3
2 4H2O
3
formula
Mr
space group
cryst system
a/A
b/A
c/A
R/deg
β/deg
C25H46N10O6P2RuS
769.72
P1
triclinic
10.6102(5)
11.9397(6)
12.7843(7)
99.4780(10)
90.3920(10)
90.4700(10)
1597.33(14)
2
Synthesis of [RuCp(8-BzTT-KS)(PPh3)(PTA)] (9). The com-
plex 9 was obtained as a yellow powder by the procedure
described above for 7. The ligand 8-BzTTH (0.072 g, 0.24 mmol)
was reacted with [RuClCp(PPh3)(PTA)] (0.150 g, 0.24 mmol) in
10 mL of an ethanolic solution of KOH (0.024 M).
Powder yield: 0.150 g, 70%. S25(mg/cm3): 1.3. Log P: 1.54.
Elemental analysis for C43H45N7O2P2RuS (886.95): Found C,
58.32; H, 5.16; N, 10.87; S, 3.48%; calcd. C, 58.23; H, 5.11; N,
11.05; S, 3.62%.
γ/deg
V/A3
Z
Dc/g cm-3
F(000)
1.600
792
7.13
8423
5223
0.0564
4436
M(Mo KR)/cm-1
measd reflctns
unique reflctns
Rint
Synthesis of [{RuCp(PPh3)(PTA)}2(μ-MBTT-KN7,N07)] (10).
The ligand MBTTH2 (10.0 mg, 0.022 mmol) was dissolved in
10 mL of a KOH ethanolic solution (0.0044 M) and reacted with
[RuClCp(PPh3)(PTA)] (28.4 mg, 0.044 mmol). After refluxing
for 4 h the resulting solution was filtered at room temperature
and concentrated under reduced pressure to 2 mL. The pro-
duced precipitate was filtered, washed with Et2O (2 ꢀ 2 mL), and
finally dried under vacuum.
obsd reflctns [I g 2σ(I)]
θmin-θmax/deg
hkl ranges
R(F2) (obsd reflctns)
wR(F2) (all reflctns)
no. of variables
goodnes of fit
1.61-24.41
-12, 12; -13, 13; -11, 14
0.0423
0.1620
436
Yield: 33.4 mg, 88.4%. S25(mg/cm3): 0.8. Log P: 0.63. Ele-
mental analysis for C73H88N14O4P4Ru2S2 (1615.74): Found C,
54.35; H, 5.58; N, 12.02; S, 3.82%; calcd. C, 54.27; H, 5.49; N,
12.14; S, 3.97%.
1.095
-0.898, 0.800
F
min, Fmax/e A-3
least-squares methods with SHEL-XTL25 and refined by least-
squares procedures on F2 and final geometrical calculations and
graphical manipulations were carried our with the SHELXS-
XTL package.25 A disordered solvent molecule H2O was found
and refined isotropically. All the non-hydrogen non-disordered
atoms were refined with anisotropic atomic displacement para-
meters. All hydrogen atoms, except for disordered water mole-
cules, were included in calculated positions and refined using a
riding model.
Synthesis of [{RuCp(PPh3)(PTA)}2(μ-EBTT-KN7,N07)] (11).
The same procedure described above for 10 was used. The
purine derivative EBTTH2 (10.4 mg, 0.022 mmol) was reacted
with [RuClCp(PTA)2] (28.4 mg, 0.044 mmol) to give 11 as a
yellow powder.
Yield: 29.6 mg, 78%. S25(mg/cm3): 0.8. Log P: 0.78. Elemen-
tal analysis for C74H90N14O4P4Ru2S2 (1629.77): Found C,
54.57; H, 5.64; N, 11.92; S, 3.86%; calcd. C, 54.53; H, 5.57; N,
12.03; S, 3.94%.
Growth Inhibition Assays. Cell growth inhibition assays were
carried out using the cisplatin-sensitive T2 human cell line and
the cisplatin-resistant SKOV3 cell line. T2 is a cell hybrid
obtained by the fusion of the human lymphoblastoid line 174
(B lymphocyte transformed by the Epstein-Barr virus) with the
CEM human cancer line (leukemia T) while SKOV3 is derived
from a human ovarian tumor. The cells were seeded in triplicate
in 96-well trays at a density of 50 ꢀ 103 in 50 μL of AIM-V
medium for T2, 25 ꢀ 103 in 50 μL of AIM-V medium for
SKOV3. Stock solutions (10 mM) of the Ruthenium complexes
1-12 were made in dimethylsulfoxide (DMSO) and diluted
in AIM-V medium to give a final concentration of 2, 10, and
50 μM. Cisplatin was employed as a control for the cisplatin-
sensitive T2 cell line and for the cisplatin-resistant SKOV3.
Untreated cells were placed in every plate as a negative control.
The cells were exposed to the compounds for 72 h, and then
25 μL of a 4,5-dimethylthiozol-2-yl)2,5-diphenyltetrazolium
bromide solution (12 mM) were added. After 2 h of incubation,
100 μL of lysing buffer (50% DMF þ 20% SDS, pH 4.7) were
added to convert 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazo-
lium bromide into a brown colored formazane. After additional
18 h the solution absorbance, proportional to the number of live
cells, was measured by a spectrophotometer and converted in %
of growth inhibition.26
Synthesis of [{RuCp(PPh3)(PTA)}2(μ-PBTT-KN7,N07)] (12).
The purine derivative PBTTH2 (10.5 mg, 0.022 mmol) was
reacted with [RuClCp(PTA)2] (28.4 mg, 0.044 mmol) to give
12 as a yellow powder by using a similar procedure as that
described for 10.
Yield: 26 mg, 67.7%. S25(mg/cm3): 0.6. Log P: 1.12. Elemen-
tal analysis for C75H92N14O4P4Ru2S2 (1643.79): Found C,
54.87; H, 5.75; N, 11.77; S, 3.77%; calcd. C, 54.80; H, 5.64; N,
11.93; S, 3.90%.
Stability Tests of the Complexes with O2 and H2O. The
obtained ruthenium complexes were air stable for months in
the solid state and for 2 days in solution. In a standard pro-
cedure, 0.01 g of 1-6, 7-10 and 12, and 11 were introduced into
a 5 mm NMR tube and dissolved respectively in D2O, CDCl3,
and DMSO-d6 (1.0 mL). The solution was cooled to 0 °C and
then dry O2 was bubbled throughout the solution for 2 min
via a long syringe needle. 31P{1H} NMR monitoring showed no
change within 2 days at room temperature. No decomposition
was also observed after 2 days at 40 °C as well. Introduction of
50 μL of D2O in the CDCl3 and DMSO-d6 solutions did not
produced any significant change in the starting complexes after
2 days at 40 °C.
X-ray Structure Determinations. Data of compounds 2 4H2O
3
Octanol-Water Partition Coefficient Determination. The
octanol-water partition coefficient of the ruthenium complexes
was obtained by a slow-stirring method that provides accurate
Log P results over a wide range of concentration values.27
was collected on
(XDIFRACT service of the University of Almerı
a
Bruker APEX CCD diffractometer
´
a) using gra-
phite monochromated Mo KR radiation (λ=0.7107 A) at 150 K.
The crystal parameters and other experimental details of the
data collections are summarized in Table 1. The structures were
solved by direct methods SIR9224 and refined by full-matrix
(25) Sheldrick, G. M. SHELXTL, version 6.14; Bruker-AXS: Madison, WI,
2003.
(24) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla,
(26) Hansen, M. B.; Nielsen, S. E.; Berg, K. J. Immunol. Methods 1989,
119, 203.
M. C.; Polidori, G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435.

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 877
Scheme 1. Synthesis of Complex 1
Chart 2. Structure of [RuCp(8-MTT-κS)(PTA)2]
The procedure was adapted to the solubility properties of the
complexes. The aqua-soluble ruthenium complexes 1-6 were
previously dissolved in 15 mL of distilled water presaturated
with 1-octanol while water insoluble complexes 7-12 in 15 mL
of octanol previously saturated with distilled water. The final
complex concentration of the resulting solutions lay in the range
between 10-4 and 10-3 M. Into a 40 mL container with a
magnetic stir bar was introduced initially the water phase and
then by a syringe the octanol one so that the solution did not
emulsify. The container was closed with a silicone septum and
stirred slowly at 25 ( 1 °C. Samples were taken from the octanol
and water phases with a syringe through the septum. Samples of
each phase were taken periodically until the concentrations in
both phases stabilized. Concentrations of the complex in each
phase were measured using UV-vis spectroscopy.
presence of coordinated PTA is also supported, in the
31P{1H} NMR spectrum, by a singlet at -22.97 ppm
whose chemical shift is similar to that observed for the
PTA ligand in [RuClCp(PTA)2] (-23.6 ppm).28
Results and Discussions
Complex 2 was obtained from the reaction between
8-MTTH, KOH, and [RuClCp(PTA)2] in water. The
spectroscopic data suggested that the structure of 2 was
similar to other previously reported 8-MTT- metal com-
plexes. Its IR spectrum showed no ν(N-H) absorption
band but absorptions for ν(C6dO þ C2dO) (1677, 1628
cm-1) and ν(CdC þ CdN) (1519 cm-1) in the range
found for 8-MTT- complexes in which the purine is
bonded to the metal by the imidazolic N7 atom.17,18,22,23a
1H, 13C, and 31P NMR spectra of 2 in CDCl3 showed a
single pattern, excluding the formation of isomers. The
1H NMR displayed the expected signals for a Cp, two
PTA, and an 8-MTT-. It is important to point out that
the singlet at 2.75 ppm for S-CH3 is close to that reported
for cis-[PtCl(8-MTT-κN7)(PPh3)2] (2.70 ppm).17 The 13C-
{1H} NMR was also in agreement with the proposed
composition for 2. The signal for the S-CH3 arises at 14.15
ppm that is close to that for 8-MTTH (14.00 ppm).22,23
The 31P{1H} NMR spectrum showed a single signal at
-25.19 ppm due to phosphorus-coordinated PTA. There-
fore, the spectroscopic data for 2 are similar to the
corresponding found in previously reported 8-MTT-
complexes where the thiopurine is invariably coordinated
by the imidazolic N7. This fact suggests that in 2 the
8-MTT- is also coordinated by the N7 atom to the metal
atom of the moiety {RuCp(PTA)2}þ.
Mononuclear Complexes Containing Monothiopurines:
Synthesis of 1, 2, 3 and 7, 8, 9. The reaction of 8-TTH2 with
KOH and [RuClCp(PTA)2] in water at refluxing tem-
perature gave complex [RuCp(8-TTH-κS)(PTA)2] (1) as a
microcrystalline yellow powder (Scheme 1). The elemen-
tal analysis, IR, and NMR analysis support the proposed
structure for 1, in which a ruthenium atom is coordinated
to a η5-Cp, two PTA ligands, and to a 8-TTH- purine
molecule acting as an anionic monodentate ligand
through its deprotonated sulfur atom.
The presence of the S-coordinated thiopurine is evi-
denced by the characteristic IR spectrum absorption for
ν(C6dO þ C2dO) at 1690 and 1636 cm-1, for ν(CdC þ
CdN) at 1536 cm-1 and for ν(N-H) at 3419 cm-1. The
1H NMR spectrum in D2O displays two singlets at
3.15 ppm and 3.32 ppm that are ascribable to the N1-
CH3(8-TTH) and N3-CH3(8-TTH) for a coordinate TTH-.
The N7-H signal was not observed. The lack of N7-H
signal is probably due to rapid deuterium exchange in
D2O. Unfortunately 1 is not soluble in any aprotic solvent
like CDCl3, where this exchange process does not occur.
The characteristic NCH2P(PTA) and NCH2N(PTA) signals
are observed in the range 3.84-4.18 ppm and 4.38-4.55
ppm, respectively. The singlet of the Cp ligand is found
at 4.82 ppm. The 13C{1H} NMR spectrum is in agree-
ment with the proposed composition: signals for N1-
CH3(8-TTH) and N3-CH3(8-TTH) thiopurine methyl groups
and for the Cp ligand are observed at 27.97 ppm, 30.19
ppm, and 79.49 ppm, respectively. The chemical shift for
the C8-S carbon atom arises at 158.89 ppm, at higher
field than that found for 8-TTH2 in the same solvent
(164.83 ppm).22,23 Unfortunately the chemical shift of
C8-S has not been reported for analogue complexes. The
Surprisingly, the crystal structure of 2 obtained by
single crystal X-ray crystallography showed that in this
complex the MTT- ligand is coordinated to the metal by
the S atom instead of the expected N7 imidazolic atom
(Chart 2). The ruthenium-coordinated S atom is still
bonded both to C8 and CH3 acting therefore as a co-
ordinated thio-ethereal group, where sulfur is triply
bonded. The ligand MTT- acts as a monodentate anionic
(27) (a) Mannhold, R.; Poda, G. I.; Ostermann, C.; Tetko, I. G. J. Pharm.
Sci. 2009, 98, 861–893. (b) Lozano, H. R.; Martínez, F. Braz. J. Pharm. Sci.
(28) Akbayeva, D. N.; Gonsalvi, L.; Oberhauser, W.; Peruzzini, M.;
ꢁ
€
2006, 42, 601–613. (c) Ropel, L.; Belveze, L. S.; Aki, S. N. V. K.; Stadtherr,
Vizza, F.; Bruggeller, P.; Romerosa, A.; Sava, G.; Bergamo, A. Chem.
M. A.; Brennecke, J. F. Green. Chem. 2005, 7, 83–90.
Commun. 2003, 264.

878 Inorganic Chemistry, Vol. 50, No. 3, 2011
Scheme 2. Synthesis of 2 and 3
Hajji et al.
Scheme 3. Synthesis of 7, 8, and 9
S-donor ligand, and this unusual coordination mode is
not stabilized by any chelate formation.
metal by the N7. Nevertheless, a similar wrong conclusion
was previously drawn for complex 2, later corrected
by the crystal structure evidence, which unequivocally
showed that 8-MTT- was S-coordinated. The similarity
of reactants and reaction conditions in the formation of 2
and 3, differing only for the sulfur substituent (Me and
Bz), supports the hypothesis that also in complex 3 the
thiopurine coordination occurs through the S atom.
To obtain the mixed-phosphines analogues we re-
peated the above described reactions of 8-TTH2,
8-MTTH, and 8-BzTTH using the mixed phosphines
precursor [RuClCp(PTA)(PPh3)]. Complexes [RuCp(8-
TTH)(PPh3)(PTA)] (7), [RuCp(8-MTT)(PPh3)(PTA)]
(8), and [RuCp(8-BzTT)(PPh3)(PTA)] (9) were obtained
from each reaction as single products in good yields
(Scheme 3).
Although it is reasonable to ascribe the observed com-
plete regioselectivity for S versus N7 coordination to
the preference of the soft acid {PTA-Ru(II)} for the soft
S-donor, it must be noticed that the same behavior was
not observed with {PTA-Pt(II)} (also classified as a soft
acid), which was reported to form exclusively Pt-N7
bonds with MTT-.17,18
The complex [RuCp(8-BzTT)(PTA)2] (3) was obtained
by a procedure similar to that used for 2. The reaction
of 8-BzTTH with KOH and [RuClCp(PTA)2] in water
under an inert atmosphere led to 3 (Scheme 2). The IR
spectrum showed the typical bands of a coordinated
thiopurine and the lack of ν(N7H) absorption. Also in
this case, the NMR analysis showed a single pattern for
every observed nucleus, indicating the complete regios-
The IR spectra of the complexes are in agreement with
the proposed structures, the IR spectrum of 7 being the
only one in which a ν(NH) absorption was observed at
3250 cm-1. Additionally, the 1H NMR spectrum for 7 in
CDCl3 showed a singlet at 9.91 ppm which can only be
assigned to the N7-H proton of 8-TTH-, suggesting that
the coordination site is the deprotonated S atom.23b The
1H NMR spectra of 8 and 9 are similar to those observed
for the analogues 2 and 3, supporting similar structures.
The 13C{1H} NMR spectra of 7, 8, and 9 are similar to
those of 1, 2, and 3, respectively. The 31P{1H} NMR
spectra of 7, 8, and 9 show two doublets due to coordi-
nated PTA (-34.89 for complex 7, -38.53 for 8 and
-37.57 for complex 9) and PPh3 (51.44 ppm, 48.94 ppm
and 48.80 ppm for 7, 8, and 9) coupled each other with
1
electivity of the coordination process. The H NMR is
constituted by the characteristic signals for PTA, Cp, and
the thiopurine. The chemical shift for the S-CH2-Ph
signal (5.03 ppm) is between that found in [PdCl(8-
BzTT-κN7)(PPh3)2] (4.15 ppm) and [Pd(8-BzTT-κN7)2-
(PPh3)2] (5.20 ppm), the only reported metal complexes of
BzTT-, and close to that for free 8-BzTTH (4.49 ppm).23c
In 13C{1H} NMR, the chemical shifts of S-CH2-Ph and
C8-S carbon atoms are at 37.18 ppm and 150.50 ppm,
close to the corresponding observed for [PdCl(8-BzTT)-
(PPh3)2] (36.30 ppm; 150.2 ppm) and [Pd(8-BzTT)2-
(PPh3)2] (40.80 ppm; 148.4 ppm).23c In the 31P{1H}
NMR a singlet comes at -25.88 ppm, close to those
observed for [RuClCp(PTA)2] (-23.6 ppm), 1 (-22.29
ppm), and 2 (-25.19 ppm). The spectroscopic properties
of 3 are similar to those found for parent and analogue
complexes in which the 8-BzTT- ligand is bonded to the
2
2JPP = 40.52 Hz in complex 7, JPP = 38.42 Hz in com-
2
plex 8, and JPP = 37.71 Hz in complex 9. The data for
the starting complex [RuClCp(PTA)(PPh3)] are similar

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 879
Chart 3
[-34.96 and 48.16 ppm (2JPP=34.7 Hz)] showing that the
phosphorus environment is not much affected by the
nature of the anionic ligand. The proposed structure for
7, 8, and 9 is similar to that found for complex 2 where the
thiopurinic anionic ligand is S-coordinated to the metal,
which completes its coordination sphere binding to a η5-
Cp, one PTA, and one PPh3 (Scheme 3).
The obtained mononuclear products 1- 3 and 7-9 are
air stable for months in the solid state, while solutions in
water (complexes 1-3) or CDCl3 (complexes 7-9) are
stable for more than 2 days.
Binuclear Complexes Containing Bis-Thiopurines: Syn-
thesis of 4, 5, 6 and 10, 11, 12. The reactions of MBTTH2,
EBTTH2, and PBTTH2 (d-f, Chart 1) with 2 equiv of
KOH in H2O and 2 equiv of [RuClCp(PTA)2] gave the
binuclear complexes 4, 5, and 6, while the same ligands
with 2 equiv of KOH and 2 equiv of [RuClCp(PTA)-
(PPh3)] in EtOH produced 10, 11, and 12. (Chart 3). The
obtained binuclear products are air stable in solid state
for months as well as solutions of 4- 6 in water, of 10 and
12 in CDCl3 and of 11 in DMSO are air stable for more
than 2 days.
Moreover, in the 13C{1H} NMR spectra of 4 and 10 the
chemical shifts of S-CH2-S and C8 signals (4: δ S-CH2-S=
38.07, C8 = 153.83 ppm; 10: δ S-CH2-S = 33.95, C8 =
151.85 ppm) are very close to those found in cis-[{Pt-
(PTA)2}2(μ-Cl)(μ-MBTT-κN7,N70)]Cl (δ S-CH2-S =
35.85, C8 = 150.52 ppm) and trans-[{PdCl(PPh3)2}2(μ-
MBTT-κN7,N70)] (δ S-CH2-S=36.90, C8=150.30 ppm).
The comparative analysis of the 1H NMR spectra of the
complexes 5 and 11 was very useful for the right assign-
ment of their signals that was only possible as both
complexes are soluble in DMSO-d6. In complexes 5 and
11 the four S-(CH2)2-S protons are chemically different
and arise as multiplets (5: 3.17, 3.39, 3.41, 3.44 ppm; 11:
2.72, 2.95, 3.06, 3.62 ppm), while a broad singlet was
observed for this group in the few known EBTT2- metal
complexes trans-[{PdCl(PPh3)2}2(μ-EBTT-κN7,N07)] (δ
S-(CH2)2-S: 2.87 ppm)22 and cis-[{Pt(PTA)2}2(μ-Cl)(μ-
EBTT-κN7,N70)]Cl (δ S-(CH2)2-S: 1.92 ppm).18 All the
S-(CH2)2-S protons present an individual signal and
the large range of their chemical shifts suggest that the
coordination of the ligand to the metal has a large
influence on this group. Therefore, it is more likely that
the coordination of the EBTT- ligand occurs through the
S atom, directly bonded to -CH2-CH2-, than through the
quite distant N7. In the 13C{1H} NMR spectra of 5 and 11
the chemical shift of the S-(CH2)2-S and C8 signals (5: δ
S-(CH2)2-S=34.74, C8=151.68 ppm; 11: δ S-(CH2)2-S=
32.38, C8 = 150.67 ppm) are similar to those of free
EBTTH2 (δ S-CH2-S=32.00, C8=148.10 ppm) and of
the reported complexes cis-[{Pt(PTA)2}2(μ-Cl)(μ-N,N-
EBTT)]Cl (δ S-(CH2)2-S=35.85, C8=150.52 ppm) and
trans-[{PdCl(PPh3)2}2(μ-EBTT-κN7,N07)] (δ S-(CH2)2-S =
32.70, C8=150.90 ppm).
For every product, the elemental analysis þwas in agree-
ment with a proportion of two {CpRuLL0} (L=PPh3,
PTA; L0 = PTA) units to one bis-thiopurine molecule.
The 31P{1H} NMR spectra of complexes 4, 5, and 6 show
a singlet at -22.16 ppm, -22.41 ppm, and -25.34 ppm,
respectively, similar to those found for the monothiopur-
ine Cp-PTA ruthenium complexes 1-3. Complexes 10,
11, and 12 display two doublets reciprocally coupled due
to PTA and PPh3 bonded to the same metal nucleus
2
[10: -34.96 ppm, 51.36 ppm (d, JPP = 40.58 Hz);
2
11: -37.78 ppm, 50.17 ppm (d, JPP = 38.71 Hz);
12: -38.53 ppm, 48.21 ppm (d, 2JPP =38.52 Hz)]. The
1H NMR signals of 4, 5, and 6, and 10, 11, and 12 were
assigned by the use of 1H, 1H-2D COSY NMR and were
found to be similar to those of the corresponding mono-
nuclear analogues 1-3 and 7-9, plus the signals of the
linking CH2 chain of the bis-thiopurines. The MBTTH2
S-CH2-S group in 10 was found as a broad singlet at 4.62
ppm, which is in the range to that found for the complexes
cis-[{PtCl(PPh3)2}2(μ-MBTT-κN7,N70)] (5.0 ppm), cis-
[{Pt(PTA)2}2(μ-Cl)(μ-MBTT-κN7,N70)]Cl (4.21 ppm),18
and trans-[{PdCl(PPh3)2}2(μ-MBTT-κN7,N70)] (4.15ppm).22
In complexes 6 and 12 the S-(CH2)3-S 1H NMR signals
arise as multiplets at distinct chemical shifts (6: δ -CH2-=
1.56 ppm, δ S-CH2- = 2.95, 4.64 ppm; 12: δ -CH2- =
2.20 ppm, δ S-CH2-=3.22, 4.48 ppm) quite different from
those of the free PBTTH2 (δ -CH2-=2.17, S-CH2-=3.40
ppm) and of coordinated PBTT2- in cis-[{Pt(PTA)2}2(μ-
Cl)(μ-PBTT-κN7,N07)]Cl (δ -CH2-=2.03, S-CH2-=3.39
ppm)22 and trans-[{PdCl(PPh3)2}2(μ-PBTT-κN7,N07)]
(δ -CH2-=2.56, S-CH2-=3.72 ppm).18 This fact suggests
once more that the S atom is the coordination point of the
PBTT- ligand in 6 and 12. The compounds 6 and 12
display a similar 13C{1H} NMR spectra except for the
PPh3 signals. In particular, the chemical shifts of the
bridging -CH2- and of C8 carbon atoms are similar in 6
1
Nevertheless, in the H NMR spectrum of 4, it was not
possible to locate the S-CH2-S hydrogen atoms, whose
signal is probably overlapped to the H2O residue signal.

880 Inorganic Chemistry, Vol. 50, No. 3, 2011
Hajji et al.
ꢀ
Table 2. Selected Bond Distances (A) and Angles (deg) for 2
Ru1-P1
2.2665(12)
2.2722(13)
Ru1-P2
Ru1-S1t
2.3515(12)
1.865(5)
1.808(5)
1.792(5)
1.308(7)
1.376(6)
1.856(10)
94.17(5)
90.18(4)
89.32(4)
113.00(17)
109.72(18)
122.9(4)
116.8(4)
Ru1-Cp(centroid)
S1t-C9t
S1t-C8t
C8t-N7t
C8t-N9t
P1P-C3P
P1-Ru1-P2
P1-Ru1-S1t
P2-Ru1-S1t
C8t-S1t-Ru1
C9t-S1t-Ru1
S1t-C8t-N7t
S1t-C8t-N9t
Figure 1. ORTEP view of compound 2 with atom numbering scheme
showing 50% probability thermal ellipsoids. For the sake of clarity the H
atoms were omitted.
2 with respect to [RuClCp(PTA)2] is only able to be
attributed to the effect of the entire 8-MTT- ligand as a
whole.
(δ -CH2-, S-CH2-, C8=28.30, 45.87, 149.73 ppm) and 12
(δ -CH2-, S-CH2-, C8=30.23, 44.11, 152.06 ppm). If these
values are compared with those found for free PBTTH2
(δ -CH2-, S-CH2-, C8=29.20, 30.20, 148.20 ppm) and for
the reported complexes cis-[{Pt(PTA)2}2(μ-Cl)(μ-PBTT-
κN7,N07)]Cl18 (δ -CH2-, S-CH2-, C8 = 32.00, 32.20,
151.86 ppm) and trans-[{PdCl(PPh3)2}2(μ-PBTT-κN7,
N07)]22 (δ -CH2-, S-CH2-, C8=28.60, 30.40, 151.00 ppm),
a substantial invariance is found for δ -CH2- and δ C8,
while the S-CH2- chemical shift found in 6 (45.87 ppm)
and 12 (44.11 ppm) is about 14 ppm shifted with respect to
the same signal in free PBTTH2 (30.20 ppm) and in Pt/Pd-
PBTT2- complexes (32.20 and 30.40 ppm, respectively).
This remarkable difference, in our opinion, is another
NMR-based evidence supporting the hypothesis of S-co-
ordination versus N-coordination.
Because of the poor quality of the crystals resulting
from many recrystallization attempts, it was not possible
to obtain any X-ray structure for binuclear complexes
containing bis-8-thio-theophyllines. However, the spec-
troscopic evidence support the structures reported in
Chart 3 as the most probable for complexes 4-6 and
10-12, where each S of the bis-8-thio-theophylline deriv-
ative is bonded to a Ru, which completes its coordination
geometry with an η5Cp and two phosphines.
The Ru-Cpcentroid distance is 1.865 A that is larger
than that observed in the starting complex [RuCpCl-
(PTA)2] (Ru-Cpcentroid = 1.844 A)30 while the bond
lengths between the ruthenium atom and both PTA
phosphorus atoms (Ru-P1 = 2.266(1) A; Ru-P2 =
2.272(1) A) are different to those observed in the starting
complex: respectively 2.258(3) A and 2.247(3) A. Surpris-
ingly, the 8-MTT- anion is not coordinated by the
purine-N7 atom, which was the expected coordination
position as observed in most of the until now known
crystal structures of purine-metal complexes, but by the
purine-thio-ether-S atom.17,22,23 Although there are sev-
eral examples of bonds between Ru atoms and thioether S
atoms, there are only three examples of ruthenium com-
plexes containing nitrogen-thioether ligands,31 but none
of them contains the ligand exclusively coordinated by
sulfur. Additionally, there are no examples of complexes
containing thioether-purines or other thioethers bearing
NH groups, competing for coordination on the metal or
stabilizing the S-thio-ether coordination by the formation
of a chelate ring. This unexpected coordination mode
could be because the S bonded compound is lower in
energy than the N bonded, for steric reasons. Preliminary
theoretical studies on the interaction between thio-pur-
ines and {CpRuPTA} moieties support this idea; never-
theless an extensive theoretical and experimental study is
in progress, which will be presented in a future paper.
The distance Ru-S1 (2.3515(12) A) in 2 is shorter
Crystal Structure of [RuCp(8-MTT-KS)(PTA)2] 4H2O
3
(2 4H2O). Single crystals good for X-ray determination
3
of 2 were obtained by slow evaporation from its solution
in EtOH. An ORTEP29 view is displayed in Figure 1, the
crystallographic data are given in Table 1, and a list of
selected bond distances and angles appears in Table 2.
The asymmetric unit contains four disordered H2O
molecule and one chiral neutral [RuCp(8-MTT-κS)-
(PTA)2] molecule (Figure 1). The metal is coordinated
with a slightly distorted pseudo-octahedral geometry to
one η5-Cp, two PTA ligands bonded by the P atom, and
one 8-MTT- ligand through the S atom. The distortion of
the coordination geometry in 2 (P1-Ru-P2=94.17(5)°;
P1-Ru-S1t = 90.18(4)°; P2-Ru-S1t = 89.32(4)°) is
shorter than that in the starting complex [RuClCp(PTA)2]
(P1-Ru-P2=96.85°; P1-Ru-Cl=91.61(7)°; P2-Ru-
Cl=86.46(7)°).30 The atomic radius of S and Cl are nearly
similar and therefore the shorter geometrical distortion in
than that found in [Ru(N,N-dps)2(N,S-c)][PF6]2 C3H6O
3
(Ru-S = 2.370(1) A),31b its isomer [Ru(N,N-dps)2(N,S-
dps)](PF6)2 H2O (Ru-S(3)=2.424(2) A), and [Ru(dps-
3
N,N0)2pmprs](PF6)2 C2H3N (Ru1-S34 = 2.3581(13)
3
A)31a (dps = di-2-pyridyl sulfide; c = 2-pyridymethyl
2-pyridyl sulfide; pmprs =4-methylpyrimidin-2-yl 2-pyr-
idylmethyl sulfide). Until now, the crystal structure of
[Ru(8-MTT-κN7,O6)2(PPh3)2]32 is the only known ruthe-
nium complex containing a 8-MTT- ligand coordinated
ꢁ
(31) (a) Baradello, L.; Lo Schiavo, S.; Nicolo, F.; Lanza, S.; Alibrandi, G.;
Tresoldi, G.; Nicolo, F.; Lanza, S. Acta Crystallogr., Sect.C: Cryst. Struct.
Commun. 2005, C61, m169–m172. (b) Baradello, L.; Lo Schiavo, S.; Nicolo, F.;
Lanza, S.; Alibrandi, G.; Tresoldi, G. Eur. J. Inorg. Chem. 2004, 3358.
(c) Scopelliti, R.; Bruno, G.; Donato, C.; Tresoldi, G. Inor. Chim. Acta 2001,
313, 43–55.
(29) Johnson, C. K. ORTEP II. Report ORNL-5138; Oak Ridge National
Laboratory: Oak Ridge, TN, 1976.
(30) Frost, B. J.; Mebi, C. A. Organometallics 2004, 23, 5317–5323.
~
(32) Manas Carpio, S. Ph.D. Dissertation, University of Almerıa, Almerıa,
´ ´
Spain, 2003.

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 881
Figure 2. Hydrogen bond network produced by the interaction of the disordered H2O molecule, the N7, O2, O6, and N1 atoms of 2.
to a Ru atom. In this complex the ligand 8-MTT- is
coordinated to the metal by O6 and the deprotonated N7
atom. The C8-S1 and S1-CH3 distances in this complex
Table 3. Estimated IC50 on cisplatin-Sensitive T2 Cell Line, cisplatin-Resistant
SKOV3, and Partition Coefficient Log P of 1-3, 7-8, [RuClCp(PTA)2], and
[RuClCp(PPh3)(PTA)]
complex
T2
SKOV3
Log P
(C8-S1 = 1.755(4) A; S1-CH3 = 1.770(5) A) are sig-
nificantly shorter (ΔC8-S=0.037 A; ΔS-CH3 =0.038
A) than those for 2 (S1t-C8t = 1.792(5) A; S1t-C9t =
1.808(5) A). Additionally, the bond lengths between C8
and the imidazolic nitrogen atoms N7 and N9 in 2 are
quite different (C8t-N7t = 1.308(7) A; C8t-N9t =
1.376(6) A) in comparison with the distance C8-N7
and C8-N9 in the complex [Ru(8MTT-κN7,O6)2-
(PPh3)2] (C8-N7 = 1.338(4) A; C8-N9 = 1.377(5) A)
and the other known MTT- metals complexes and N7
coordinate purine metal complexes.17 The crystal struc-
ture of MBTTH2 is the only one known for a free 8-thio-
teophilline derivative.22 The bond C8-S and S-CH2-
distances in both sides of the molecule are the same
(S1A-C8A=1.749(2) A; S1B-C8B=1.748(2) A; S1A-
C10 = 1.807(2) A; S1B-C10 = 1.816(2) A) and quite
similar to those bond lengths in 2. Nevertheless, the C8-
N7 and C8-N9 (C8A-N7A=1.353(3) A; C8A-N9A=
1.339(2) A; C8B-N7B = 1.359(2) A; C8B-N9B =
1.345(2) A) are rather different to those found for 2. It
is important to point out that the average distance
between C8-N7 and C8-N9 in 2 (1.342(7) A) is the same
than that found in MBTTH2 (1.346(3) A) but different
than that for [Ru(8MTT-κN7,O6)2(PPh3)2] (1.357(5) A).
This fact suggests that the electrons in the skeleton of
complex 2 are mainly located between the N7-C8-
S-Ru atoms, with a particular high-density charge on
the N7-C8 and on the S-Ru bond. Therefore the
electrons coming from the deprotonation of the N7 atom
are finally located on the S8 atom, which is the donor site
of the ligand.
[RuClCp(PTA)2]
1
2
3
[RuClCp(PPh3)(PTA)]
7
8
9
>50 μM
>50 μM
>50 μM
>50 μM
10-50 μM
∼50 μM
∼50 μM
2-10 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
-1.85
-1.29
-1.41
-0.97
0.75
0.96
1.26
1.54
Cell Growth Inhibition. The mononuclear complexes
1-3 and 7-9 have been tested for cell growth inhibition
activity on two human cancer cell lines: the cisplatin-
sensitive T2 (results reported in Table 3) and the cisplatin-
resistant SKOV3 (results reported in Table 3). In the tests
the growth inhibition activity values of the precursors
[RuClCp(PPh3)(PTA)] and [RuClCp(PTA)2] were ac-
quired for comparison. Each complex was dissolved in
DMSO and diluted in AIM-V medium to obtain the final
concentrations of 50, 10, and 2 μM solutions. The che-
mical stability of the complexes in water/DMSO solu-
tions was verified by 31P{1H} NMR over 2 weeks. The
percentage of growth inhibition at the three doses for each
complex allowed us to estimate the IC50, the concentra-
tion reducing to 50% the cell growth of both cell lines
(Table 3).
Tests with the cisplatin-sensitive cells T2 show that
complexes containing one PTA and one PPh3 (7-9 and
the precursor [RuClCp(PPh3)(PTA)]) display a signifi-
cantly better activity than those containing two PTA
(1-3), whose activity is very low, including the precursor
[RuClCp(PTA)2]). This fact has been observed before
with PTA-Pt complexes, and it is probably related to the
difficulty for hydrophilic PTA complexes in crossing the
lipophilic membranes of the cell and of the nucleus to
reach their target, the nucleic acids. To give support to
this hypothesis, we determined the partition coefficient
Log P for each complex, and we found a good correlation
between Log P and the antiproliferative activity. The data
in Table 3 indicate that the highest value for Log P was
found for complex 9, which present also the highest
cell growth inhibition activity, comparable to cisplatin
The purine is planar, the distance between the purine
plane and the S, C9t, P1, and P2 being 0.243, 0.08, 0.134,
and 0.160 A, respectively. The purine and Cp are almost
parallel as the angle between them is 4.14°. The MTT-
disposition avoids repulsive interactions with the rest of
the molecule, as the distances among the purine and the
others ligands in the complex are longer than 3 A. A 3D
network through extensive hydrogen bonding involving
the disordered H2O molecule, the N7, O2, O6, and N1
atoms forms the crystal lattice structure (Figure 2).

882 Inorganic Chemistry, Vol. 50, No. 3, 2011
Hajji et al.
(IC50=2-10 μM). The found data support the hypoth-
esis that the liphophilic/hydrophilic balance could be a
determining factor for the biological activity trend inside
the group of the Ru complexes examined in this paper.
Tests with the cisplatin-resistant SKOV3 cell line show
that all the tested complexes are inactive like cisplatin
(IC50=> 50 μM) on the Pt-resistant cell line. This obser-
vation supports the hypothesis that complex 9, as active
as cisplatin on Pt-sensitive cells and as inactive as cisplatin
on Pt resistant cell line, is likely operating by the same
mechanism as classical Pt containing drugs.
The antiproliferative activity of the Ru complexes 1-12 on
model tumoral cell lines (T2 and SKOV3) has been tested.
It was found that complex 9 shows an activity comparable
with cisplatin on T2 and no activity on SKOV3, indicat-
ing that its antiproliferative activity is likely to be related
to a cisplatin-like mechanism. While the other complexes
showed a poor bioactivity, the notable result of 9 is
probably due to the most favorable hydrophilicity/
lipophilicity balance, reached with its combination of
ligands, associating one hydrophilic PTA with lipophilic
groups like PPh3 and the thiopurine substituent CH2Ph.
The values for dinuclear complexes 4-6 and 10-12 are
not reported because preliminary tests indicated poor
activity and therefore were abandoned.
Acknowledgment. Funding is provided by Junta de
Andalucıa through PAI (research teams FQM-317)
´
and Excellence Projects FQM-03092, MCYT (Spain) by
project CTQ2009-10538 and the COST Action CM0802
(WG2, WG3, WG4).
Conclusion
Two groups of new organometallic Ru(II) complexes have
been prepared and characterized: the first is composed by
mononuclear neutral complexes where Ru is bonded to Cp,
to two phosphines (two PTA or one PTA and one PPh3), and
to the deprotonated form of one of the thiopurines a-c,
depicted in Chart 1. The second group includes binuclear
complexes with a bis-thiopurine (d-f in Chart 1), in its dianionic
form, connecting two {CpRu(PR3)2} (PR3 = PTA, PPh3)
groups. The crystal structure of complex 2 showed that the
thiopurinate is S-coordinated. This is the first example of a
single S-coordinate thioether-amine and S-coordinated thio-
purinate, invariably found N7-coordinated even with soft
metals like Pt, Pd, and Au. All the reactions producing
complexes 1-12 occur with complete regioselectivity, and
the spectroscopic data support the hypothesis that all the
isolated products contain exclusively S-coordinated thiopurines.
Supporting Information Available: Further details about the
synthesis for compounds 1-12. This material is available free of
charge via the Internet at http://pubs.acs.org. Crystallographic
data for the structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publications nos. CCDC 785135. Copies of the data can be
obtained, free of charge on applications to CCDC, 12 Union
Road, Cambridge CB2 1EZ, U.K., (fax: þ44 1223 336033 or
e-mail: deposit@ccdc.cam.ac.uk).
Note Added after ASAP Publication. This paper was
published on the Web on January 12, 2011, with the
author name Canella misspelled. The corrected version
was reposted on January 31, 2011.