Synthesis of apoptosis-inducing iminophosphorane organogold(III) complexes and study of their interactions with biomolecular targets

Article abstract of DOI:10.1021/ic801925k

New stable cationic organogold(lll) complexes containing the "pincer" iminophosphorane ligand (2-C₆H₄- PPh₂=NPh) have been prepared by reaction of the previously described [Au{κ²-C,N-C₆H₄(P

Full text of DOI:10.1021/ic801925k

Inorg. Chem. 2009, 48, 1577-1587
Synthesis of Apoptosis-Inducing Iminophosphorane Organogold(III)
Complexes and Study of Their Interactions with Biomolecular Targets
Neha Shaik,† Alberto Mart´ınez,† Idline Augustin,† Hugh Giovinazzo,‡ Armando Varela-Ram´ırez,‡
Mercedes Sanau´,§ Renato J. Aguilera,‡ and Mar´ıa Contel*,†
Department of Chemistry, Brooklyn College and The Graduate Center, The City UniVersity of New
York, Brooklyn, New York 11210, Department of Biological Sciences, The UniVersity of Texas at
El Paso, El Paso, Texas 79968, and Departamento de Qu´ımica Inorga´nica, UniVersidad de
Valencia, Burjassot, Valencia 46100, Spain
Received October 9, 2008
New stable cationic organogold(III) complexes containing the “pincer” iminophosphorane ligand (2-C6H4-PPh2dNPh)
have been prepared by reaction of the previously described [Au{κ2-C,N-C6H4(PPh2dN(C6H5)-2}Cl2] 1 and a
combination of sodium or silver salts and appropriate ligands. The presence of the P atom in the PR3 fragment has
been used as a “spectroscopic marker” to study the in vitro stability (and oxidation state) of the new organogold
complexes in solvents like dimethyl sulfoxide and water. Compounds with dithiocarbamato ligands and water-
soluble phosphines of the general type [Au{κ2-C,N-C6H4(PPh2dN(C6H5)-2}(S2CN-R2)]PF6 (R ) Me 2; Bz 3) and
[Au{κ2-C,N-C6H4(PPh2dN(C6H5)-2}(PR3)nCl]PF6 (PR3 ) P{Cp(m-C6H4-SO3Na)2} n ) 1 4, n ) 2 TPA {1,3,5-
triaza-7-phosphaadamantane} 5) have been synthesized and characterized in solution and in the solid state (the
crystal structure of 2 has been determined by X-ray diffraction studies). Complexes 1-5 have been tested as
potential anticancer agents, and their cytotoxicity properties were evaluated in vitro against HeLa human cervical
carcinoma and Jurkat-T acute lymphoblastic leukemia cells. Compounds 2 and 3 are quite cytotoxic for these two
cell lines. There is a preferential induction of apoptosis in HeLa cells after treatment with 1-5. However in the
case of the more cytotoxic complex (2), cell death is activated because of both apoptosis and necrosis. The
interactions of 1-5 with Calf Thymus DNA have been evaluated by Thermal Denaturation methods. All these
complexes show no or little (electrostatic) interaction with DNA. The interaction of 2 with two model proteins
(cytochrome c and thioredoxin reductase) has been analyzed by spectroscopic methods (vis-UV and fluorescence).
Compound 2 manifests a high reactivity toward both proteins. The mechanistic implications of these results are
discussed here.
Introduction
by some clinical problems related to its use in curative
therapy, such as normal tissue toxicity and the frequent
occurrence of initial and acquired resistance to the treat-
ment.1-8 One promising family of non-platinum anticancer
agents may be found in gold complexes. Attention was
directed toward gold compounds9 for two reasons: (1)
gold(III) centers are isoelectronic to Pt(II) compounds and
The discovery of the antiproliferative properties of plati-
num(II) complexes was serendipitous.1 The subsequent
successful development of antitumor platinum drugs has
paved the way for studying other metal-based chemothera-
peutic compounds.2 The high effectiveness of cisplatin in
the treatment of several types of tumors is severely hindered
(3) Reedijk, J. Platinum Met. ReV. 2008, 52 (1), 2.
(4) Lippert, B. Cisplatin: chemistry and biochemistry of a leading
anticancer drug; Wiley-VCH: Zurich, 1999.
* To whom correspondence should be addressed. E-mail: mariacontel@
brooklyn.cuny.edu. Phone: +1-7189515000 x2833. Fax: +1-7189514607.
†
The City University of New York.
The University of Texas at El Paso.
Universidad de Valencia.
(5) Lippert, B. Coord. Chem. ReV. 1999, 182, 263.
(6) Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99, 2467.
(7) Dong, Y.; Berners-Price, S. J.; Thorburn, D. R.; Antalis, T.; Dickinson,
J.; Hurst, T.; Qiu, L.; Khoo, S. K.; Parsons, P. C. Biochem. Pharmacol.
1997, 53, 1673.
‡
§
(1) Kelland, L. Nat. ReV. 2007, 7, 573.
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197.
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10.1021/ic801925k CCC: $40.75  2009 American Chemical Society
Inorganic Chemistry, Vol. 48, No. 4, 2009 1577
Published on Web 01/15/2009

Shaik et al.
Chart 1. Relevant Examples of Gold(III) Compounds That Display Anti-Tumor Propertiesa
a
(a) gold (III) with a chelating N,N,N ligand;22-24 (b) gold(III) with a pincer C,N ligand;31-36 (c) dinuclear gold(III) compound with a combination of
a C,N,C pincer and diphosphine ligands;37 (d) gold(III) with dithiocarbamato ligands.27-29
adopt square-planar configurations similar to that of cisplatin,
and (2) gold(I) compounds are well-known pharmaceuti-
cals,9,10 some of which are currently being used to treat
rheumatoid arthritis.11 During the past few years there has
been a renewed interest in the application of gold(I) and
gold(III) compounds in cancer chemotherapy driven by a
few novel compounds, endowed with improved stability and
with encouraging pharmacological properties.12-20 More-
over, gold(I) compounds are nowadays being designed
specifically as mitochondria-targeted chemotherapeutics.21
Three main types of gold(III) complexes have shown
activity, and these are the following: (a) coordination
compounds (with N-polydentate22-24 (Chart 1a), macrocyclic
ligands,25,26 or dithiocarbamato ligands with S-donor atoms
(Chart 1d)27-29); (b) organometallic complexes with an
N-C30 (e.g., DAMP ) o-C6H4CH2NMe2, Chart 1b)31-36 or
CNC-pincer backbone;37 or (c) gold(III) complexes contain-
ing bioligands (e.g., naturally occurring amino acids).38-41
Furthermore, dinuclear gold(III) complexes with bipyridyl
ligands42,43 and multinuclear gold (III) compounds with
CNC-backbones and chelating phosphines (Figure 1c)37 have
been reported recently.
While the mechanism of action for platinum relies on
covalent binding to DNA purine bases, it seems that gold(III)
compounds behave in a different way. There is a large body
of evidence to believe that DNA does not represent the
primary target for many gold(III) complexes. There is none
or a very weak interaction between most gold(III) complexes
and calf thymus DNA. Recently it has been reported that
some gold(III) compounds promote apoptosis via mitochon-
drial pathways,44-46 and it has been assessed that selective
modification of surface protein residues by gold(III) com-
pounds could be the molecular basis for their biological
effects on the basis of the reaction of gold(III) compounds
with a few model proteins.14,42,47
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B. K.; Messori, L. J. Inorg. Biochem. 2008, 102, 564.
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phorane group (2-C6H4-PPh2)NPh) with different ligands
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Price, S.; Filipovska, A. J. Am. Chem. Soc. 2008, 138, 12570.
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1578 Inorganic Chemistry, Vol. 48, No. 4, 2009

Organogold(III) Iminophosphorane Complexes
reduced pressure to about 2 mL. Addition of 20 mL of Et2O
afforded 2 and 3 as yellow solids that were filtered and dried under
vacuum and used without further purification. 2: Yield: 0.224 g,
81%. Anal. Calcd for C27H25AuF6N2P2S2 (814.55): C, 39.81; H,
3.09; N, 3.44; S, 7.87; found: C, 39.90; H, 2.73; N, 3.77; S, 8.45.
that have allowed us to tune the lipophilicity/hydrophilicity
of the resulting compounds. The main advantage of the
iminophosphorane ligand is that it provides a C,N-backbone
that stabilizes the resulting square-planar cycloaurated com-
plexes. An extra advantage is that the P atom in the PR3
fragment can be used as a “spectroscopic marker” to study
the in vitro stability (and oxidation state) by 31P NMR. We
and others48,49 reported on the synthesis, characterization,
and catalytic activity (C-C and C-O bond formations) of
neutral [Au{κ2-C,N-C6H4(PPh2dN(C6H5)-2}Cl2] 1. This
compound displayed moderated cytotoxicity on a P388
murine leukemia cell line49 due mainly to lack of solubility
in biologically relevant solvents. We have prepared cationic
compounds soluble in dimethyl sulfoxide (DMSO), mixtures
of DMSO/water, or water that have also displayed cytotox-
icity against HeLa human cervical carcinoma and Jurkat-T
acute lymphoblastic leukemia cells with preferential induc-
tion of apoptosis. We present here the results of the
interactions of these compounds with DNA and two model
proteins and the mechanistic implications derived from these
data.
MS(ESI+) [m/z, (%)]: 669 [M]+. IR: ν(N-CSS) ) 1562 cm-1
;
ν(C-S) 986 cm-1
.
31P{1H} NMR (d6-acetone) δ ) 65.7 (s); -144.1
1
(sept). H NMR (d6-acetone) δ ) 3.45 (s, 3H, Me), 3.62 (s, 3H,
Me); 7.06 (dt, 2H, H2+H6, NAr, 3JH-H ) 7.9, 4JH-H ) 4), 7.15 (td,
3
4
1H, H4, NAr, JH-H ) 7.8, JH-H ) 1.2), 7.26 (br t, 2H, H3+H5,
NAr, 3JH-H ) 7.9), 7.49 (dd, 1H, H3′, C6H4, 3JH-H ) 7.9, 3JP-H
)
3), 7.60-7.78 (m, 10H, Hp+Ho+Hm, PPh2), 7.89 (td, 1H, H4′, C6H4,
4
3
3JH-H ) 8 Hz, JP-H ) 2), 7.95 (dd, 1H, H5′, C6H4, JH-H ) 12,
5JP-H ) 1.5), 7.98 (br dd, 1H, H6′, C6H4, JH-H ) 12). 13C{1H}
3
NMR (d6-acetone) δ ) 40.03 (Me), 41.40 (Me), 124.90 (d, Ci, PPh2,
3
1JPC ) 92.5), 126.49 (br, C4, NAr), 128.71 (d, C6, NAr, JPC
)
5.5), 129.18 (s, C5, NAr), 129.19 (d, C4′, C6H4, 3JPC ) 13.5), 129.86
(d, Cm, PPh2, 3JPC ) 12.3), 130.22 (d, C3′, C6H4, 2JPC ) 12.8), 132.34
3
2
(d, C6′, C6H4, JPC ) 19.16); 134.02 (d, Co, PPh2, JPC ) 10.50);
4
4
134.62 (d, C5′, C6H4, JPC ) 2.54); 134.83 (d, Cp, PPh2, JPC
)
1
2.4); 137.48 (d, C2′, C6H4, JPC ) 128.8); 143.22 (br d, C1, NAr);
2
148.59 (d, C1, C6H4, JPC ) 17.2), 194.97 (NCS2). C3, NAr (not
seen). 31P{1H} NMR (d6-DMSO) δ ) 64.9 (s); -144.1 (sept). H
1
NMR (d6-DMSO) δ ) 3.20 (s, 3H, Me), 3.49 (s, 3H, Me); 6.97
(br d, 2H, H2+H6, NAr, 3JH-H ) 8), 7.05 (br t, 1H, H4, NAr, 3JH-H
Experimental Section
3
) 12), 7.19 (br t, 2H, H3+H5, NAr, JH-H ) 8), 7.43 (br dd, 1H,
H3′, C6H4, JH-H ) 8, 3JP-H ) 4), 7.58 (m, 1H, H5′, C6H4), 7.65-7.78
3
1. Synthesis and Characterization of the Gold(III)
complexes. Solvents were purified by use of a PureSolv purification
unit from Innovative Technology, Inc.; all other chemicals were
used as received. Elemental analyses were carried out by Atlantic
Microlab, Inc. (U.S.). Infrared spectra (4000-400 cm-1) were
recorded on a Nicolet 380 FT-IR infrared spectrophotometer on
(m, 8H, Hp+Ho, PPh2), 7.85 (m, 5H, Hm,, PPh2; H4′, C6H4; H6′,
C6H4).
3: Yield: 0.231 g, 60%. Anal. Calcd for C39H33AuF6N2P2S2
(966.73): C, 48.45; H, 3.44; N, 2.90; S, 6.63; found: C, 47.96; H,
3,31; N, 2.68; S, 6.44. MS(MALDI+) [m/z, (%)]:821.78 [M]+. IR:
1
KBr pellets. The H, 13C{1H}, and 31P{1H} NMR spectra were
ν(N-CSS) ) 1533 cm-1; ν(C-S) 983 cm-1
.
31P{1H} NMR (d6-
recorded in d6-acetone, d6-DMSO, or D2O solutions at 25 °C on a
Bruker 400 and spectrometer (δ, ppm; J, Hz); 1H and 13C{1H} were
referenced using the solvent signal as internal standard while
31P{1H} was externally referenced to H3PO4 (85%). The mass
spectra (electrospray ionization, ESI) were recorded from acetone
or water solutions by the mass spectrometry facility of the
University of California Riverside (U.S.). Compound 1 was
prepared as previously reported.48 The preparation of phosphine
P{Cp(m-C6H4-SO3Na)2} will be reported elsewhere. Calf Thymus
DNA, cytochrome c for heart horse, thioredoxin reductase from
Escherichia coli, DL-dithiothreitol (DTT), buffers, and solvents were
purchased from Sigma-Aldrich. Spectrophotometric studies and
thermal denaturation experiments were performed on an Agilent
8453 diode-array spectrophotometer equipped with a HP 89090
Peltier temperature control accessory.
1
acetone) δ ) 66.2 (s); -144.1 (sept). H NMR (d6-acetone) δ )
5.08 (d, 4H, CH2, 2JH-H ) 36), 7.1 (td, br, 2H, H2+H6, NAr, 3JH-H
3
) 8), 7.15 (br, 1H, H4, NAr, JH-H ) 8), 7.30-7.80 (m, 15H,
3
Hp+Ho+Hm, PPh2, C6H5 Bz), 7.9 (td, br, 1H, H4′, C6H4, JH-H
)
8), 8.0 (q, 2H, H5′+H6′, C6H4). 13C{1H} NMR (d6-acetone) δ )
54.37 (Ph-CH2), 55.97 (Ph-CH2), 124.84 (d, Ci, PPh2, 1JPC ) 93.6),
3
126.64 (d, br, C6, NAr), 128.31 (d, C6, NAr, JPC ) 3.59), 128.86
3
(br, C5, NAr), 128.88 (d, C4′, C6H4, JPC ) 10.65); 129.14 (d, Cm,
3
PPh2, JPC ) 7.29), 129.24 (C4, Bz); 129.88 (d, C3′, C6H4,2JPC
2
)13.01); 130.28 (d, Co, PPh2, JPC ) 11.98); 132.47 (C6′, C6H4,
3JPC ) 13.53); 133.54 (C3,C5 Bz), 133.64, (C2, C6, Bz), 134.71 (d,
Cp, PPh2, 4JPC ) 2.9), 134.90 (d, C3, NAr, 2JPC ) 2.64); 137.33 (d,
1
C2′, C6H4, JPC ) 130.3), 143.17 (br d, C1, NAr), 146.96 (Ci, Bz),
2
148.26 (d, C1, C6H4, JPC ) 20.5), 198.96 (NCS2).31P{1H} NMR
1
(d6-DMSO) δ ) 65.6 (s); -144.1 (sept). H NMR (d6-DMSO) δ
1.1. [Au{K2-C,N-C6H4(PPh2dN(C6H5)-2}(S2CN-R2)]PF6 (R
) Me 2; Bz 3). To a solution of 1 (0.261 g, 0.34 mmol) in 40 mL
of MeOH was added Na[Me2NCS2] (0.657 g, 0.34 mmol) for the
preparation of 2 or Na[Bz2NCS2] (0.118 g, 0.34 mmol) for the
preparation of 3. The resulting mixture was stirred at room
temperature (RT) for 2 h, and a solution of NaPF6 (0.08 g, 0.5
mmol) in 7 mL of MeOH was added. The resulting suspension
was stirred at RT for 30 min and then filtered through celite (to
remove the NaCl formed). The resulting yellow solution was
concentrated under vacuum to dryness. The yellow residue was
extracted with 20 mL of acetone, and the solvent was reduced under
2
) 5.04 (d, 4H, CH2, JH-H ) 36), 6.97 (d, br, 2H, H2+H6, NAr,
3
3JH-H ) 8), 7.05 (t, br, 1H, H4, NAr, JH-H ) 8), 7.18 (t, br, 2H,
H3+H5, NAr, 3JH-H ) 12); 7.25 (d, br, 2H, 3JH-H ) 8), 7.30-7.48
(m, 10H, C6H5 Bz), 7.58 (m, 1H, H5′, C6H4), 7.64-7.75 (m, 8H,
Ho+Hm, PPh2), 7.82-7.91 (m, 6H, Hm,, PPh2; H4′, C6H4; H4′, C6H4;
H6′, C6H4).
1.2. [Au{K2-C,N-C6H4(PPh2dN(C6H5)-2}(PR3)nCl]PF6 (PR3
) P{Cp(m-C6H4-SO3Na)2} 4 n ) 1, TPA 5 n ) 2). 4: To a
solution of 1 (0.217 g, 0.35 mmol) in 20 mL of dry acetonitrile
was added AgPF6 (0.097 g, 0.385 mmol) in 5 mL of acetonitrile,
and the flask protected from light exposure. The reaction mixture
was stirred at RT for 30 min, and it was subsequently filtered
through celite (to remove AgCl). To the resulting yellow filtrate
was added P{Cp(m-C6H4-SO3Na)2} (0.136 g, 0.31 mmol) in a
mixture of 2 mL of acetonitrile and 0.5 mL of H2O at 0 °C. The
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tallics 2007, 26, 4604.
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Chim. Acta 2007, 360, 1310.
Inorganic Chemistry, Vol. 48, No. 4, 2009 1579

Shaik et al.
reaction mixture was stirred at 0 °C for 15 min and at RT for another
15 min. All solvents were subsequently removed under reduced
pressure. The oily yellow residue was dissolved in 2 mL of
acetonitrile, and by addition of Et2O a yellow solid precipitated
which was filtered and washed with 2 portions of Et2O (10 mL).
The solid was dried under vacuum to afford pure 4. Yield: 0.217
g, 59%. Anal. Calcd for 4·8H2O: C41H52AuClF6NNa2O14P3S2
(1332.31.): C, 36.96; H, 3.93; N, 1.05; found: C 36.47; H, 3.82; N,
0.61; MS(ESI+) [m/z, (%)]: 590.07 [M - PR3]+. 31P{1H} NMR
(d6-DMSO) δ ) 57.47 (s), 45.84 (s), -144.1 (sept). 1H NMR (d6-
DMSO) δ ) 1.3-1.8 (br m, 9H, Cp), 6.97 (m, 4H, H2+H6, NAr;
H4, m-C6H4-SO3Na), 7.13 (m, 5H, H4 + H3 + H5 NAr; H3,
m-C6H4-SO3Na), 7.35 (m, 1H, H3′, C6H4),7.43 (m, 1H, H5′, C6H4),
7.50 (d, 4H, H2, m-C6H4-SO3Na, 3JP-H ) 12), 7.54-7.77 (m, 3H,
H5′, C6H4; Ho, PPh2), 7.72-8.08 (br m, 8H, Hp + Hm,, PPh2; H4′,
C6H4; H6′, C6H4; H1, m-C6H4-SO3Na). 13C{1H} NMR (d6-dmso)
Logan, UT). After overnight incubation, to allow cell attachment,
they were exposed for 22 h to several concentrations of chemical
compounds. Floating cells were collected in a ice-cold tube and
placed on ice, while attached cells were treated for 15 min with
0.25% of trypsin solution (Invitrogen, Carlsbad, CA), diluted in
serum free DMEM, and incubated at 37 °C. Cells from each
individual well, including both those harvested by trypsinization
and those floating, were centrifuged at 1,400 rpm for 5 min at 4
°C. The media was then removed and cells resuspended in 500 µL
of staining solution, containing 2 µg/mL propidium iodide dissolved
in FACS buffer (PBS, 0.5 mM EDTA, 2% heat inactivated fetal
bovine serum, and 0.1% sodium azide), incubated in the dark at
room temperature for 15 min, and analyzed by flow cytometry,
using Cytomic FC 500 (Beckman-Coulter, Miami, FL). The data
were analyzed using CXP software (Beckman-Coulter).
3. Apoptosis Assay. Adherent HeLa cells (American type Culture
Collection, Manassas, VA) were seeded in 24 well plate format at
100,000 cells/well using 1 mL DMEM media (HyClone, Logan,
UT) supplemented with antibiotics and 10% heat-inactivated
newborn calf serum (also referred as complete media). Cells were
exposed for 8 or 16 h to IC50 concentrations of the chemical
compounds as determined by the cytotoxicity assays. Floating and
attached cells were collected from tissue culture plates as described
above. Cells from each individual well were centrifuged and
washed, first with cold complete media, and second with cold PBS.
Treated cells were then resuspended in staining solution and
immediately analyzed by flow cytometry. Prior to data acquisition,
the flow cytometer was set up and calibrated utilizing unstained,
single- (PI or Annexin V-FITC) and double- (Annexin V-FITC plus
PI) stained cells.
Staining Protocol for Apoptosis Assay. After incubation with
chemical compound, cells were transferred to ice-cold 5 mL flow-
cytometry tubes and washed twice, first with ice-cold complete
media and second with ice-cold PBS. The staining procedure was
performed with cells resuspended in 100 µL binding buffer (0.1 M
HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl2) containing 1 µL of
25 µg/mL Annexin V-FITC (Beckman Coulter, Miami, FL) and 5
µL of 250 µg/mL PI. After incubation for 15 min on ice in the
dark, cell suspensions were added to 400 µL of ice-cold binding
buffer, gently homogenized, and analyzed by flow cytometry. For
each sample, 10,000 individual events were collected and analyzed
using CXP software (Beckman Coulter, Miami, FL). After the
exposure of cells to chemical compounds and trypsinization, all
subsequent procedures were carried out on ice or at 4 °C to arrest
or slow down progression of cell damage, from viability to stages
of apoptosis and necrosis.
4. Interactions with DNA. Melting curves were recorded in
media containing 50 mM NaClO4 and 5 mM Tris/HCl buffer (pH
) 7.29). The absorbance at 260 nm was monitored for solutions
of Calf Thymus DNA (35 µM) before and after incubation with a
solution of the drug under study (17.5 µM in Tris/HCl buffer) for
1 h at room temperature. The temperature was increased by 0.5
°C/min between 65 and 82 °C and by 3 °C/min between 25 and 65
°C and between 82 and 97 °C.
2
δ ) 26.48 (d, JP-C ) 19.10 Hz, Cb Cp), 30.2 (br, Cc Cp), 34.2
(vbr, Ca Cp), 121.9 (br, C4, NAr), 124.45 (d, Ci, PPh2, 1JPC ) 92.1),
assignment of the peaks due to the other arylic carbons becomes
very difficult because of the overlapping of broad signals of all the
aryl groups and couplings in this molecule, 126.00 (s), 128,39 (d),
128.49 (s), 128.63, 128.99 (br), 129.41 (vbr, m), 129.92 (m), 130.54
(d, JPC ) 12.37); 130.93 (m) 131.2 (br), 134.14 (d, JPC ) 10.24);
134.30 (br), 135.29 (br), 143.6 (br), 149.65 (s, C1, C6H4), 151.1
(brd), 152.9 (brd).
5: To a solution of 1 (0.155 g, 0.25 mmol) in 10 mL of dry
acetonitrile was added AgPF6 (0.069 g, 0.29 mmol) in 4 mL of
acetonitrile, and the flask protected from light exposure. The
reaction mixture was stirred at RT for 30 min, and it was
subsequently filtered through celite (to remove AgCl). To the
resulting yellow filtrate PTA (0.035 g, 0.22 mmol) in a mixture of
1 mL of acetonitrile and 1 mL of H2O was added at 0 °C. The
reaction mixture was stirred at 0 °C for 15 min and at RT for another
15 min. All solvents were subsequently removed under reduced
pressure. The white off residue was dissolved in 2 mL of
acetonitrile, and by addition of Et2O (10 mL) a pale yellow solid
precipitated which was filtered and dried under vacuum to afford
pure 5. Yield: 0.062 g, 54%. Anal. Calcd for C36H37AuClF6N7P4
(1038.03): C, 41.65; H, 3.59; N, 9.45; found: C, 41.15; H, 4.17; N,
9.14; MS(ESI+) [m/z, (%)]: 509 [Au(TPA)2]+. 31P{1H} NMR (d6-
dmso) δ ) 7.44 (s, PA); -56.6 (s + vbr, PB + Pc), -144.1 (sept,
PD). 1H NMR (d6-dmso) δ ) 4.07 and 4.58 (AB system, 6H,
N-CH2-N), 4.32 (s, 6H, NCH2P), 4.41 (t, 6H, 2JH-H ) 12), 5.07
(d, 6H, 2JP-H ) 8, NCH2P), 6.77 (br d, 2H, H2+H6, NAr, 2JH-H
)
2
8), 6.83 (br t, 1H, H4, NAr, JH-H ) 8), 7.16 (br t, 2H, H3+H5,
NAr, 2JH-H ) 8), 7.58-7.85 (m, 10H, H3′+H5′,C6H4, Hp+Ho, PPh2),
7.85-8.09 (m, 5H, Hm,, PPh2; H4′, C6H4; H6′, C6H4). 13C{1H} NMR
1
(d6-dmso) δ ) 50.88 (d, JP-C ) 16.0 Hz, P-CH2-N), 53.02 (d,
1JPC ) 31.2, P-CH-N), 71.25 (d, 3JP-C ) 7.4, N-CH2-N), 72.13
(d, 3JPC ) 10.2, N-CH2-N), 120.91 (br, C4, NAr), 125.51 (d, C6,
NAr, 3JPC ) 11.39), 129.61 (s, C5, NAr), 129.84 (d, Cm, PPh2, 3JPC
) 11.7 Hz), 130.06 (s, Cp, PPh2), 130.45 (s, C5′, C6H4), 133.07 (br
2
d, C1, NAr), 133.36 (d, Co, PPh2, JPC ) 9.68 Hz), 133.61 (s, C4′,
1
C6H4), 133.44 (br dd, C3′, C6H4), 136.31 (dm, C2′, C6H4, JPC
)
122.7), 148.23 (s, C1, C6H4). C3, NAr (not seen).
5. Interactions with Proteins. UV-visible Absorption
Spectra and Fluorescence Studies. Electronic spectra of each
selected protein (horse heart cytochrome c and thioredoxin reduc-
tase) at 5 µM were recorded in buffer (0.1 mol % DMSO) consisting
of 50 mM NaClO4 and 5 µM Tris/HCl (pH ) 7.29). Subsequently
solutions of cytochrome c (5 mM) and gold compound 2 in different
concentrations (5, 15, and 25 µM) were prepared in 3000 µL of
buffer (0.1% DMSO) and recorded at 25.9 °C and after incubation
during 24 h at 37 °C (see Figure 6 in section 3.2). Solutions of
2. Cytotoxicity Assay. Adherent HeLa-GFP human cervical
carcinoma cells (Montoya et al.)57 and Jurkat-GFP acute lympho-
blastic leukemia cells (to be described in a subsequent publication)
were seeded in 24 well plate format, 100,000 cells/well using 1
mL of DMEM media (HyClone, Logan, UT) supplemented with
antibiotics and 10% heat-inactivated newborn calf serum (HyClone,
(50) Parish, R. V.; Howe, B. P.; Wright, J. P.; Mack, J.; Pritchard, R. G.
Inorg. Chem. 1996, 35, 1659.
1580 Inorganic Chemistry, Vol. 48, No. 4, 2009

Organogold(III) Iminophosphorane Complexes
Scheme 1. Preparation of Organogold(III) Compounds with
Dithiocarbamato Ligands
cytochrome c (5 µM) and DTT in different concentrations (5, 15,
and 25 µM) were prepared in 3000 µL of buffer (0.1% DMSO)
and recorded at 25.9 °C.62
Fluorescence spectra were recorded on a XFLUORSAFIREII
spectrofluorimeter at 25.9 °C and excitation wavelength 285 nm
(emission wavelength start: 310 nm; emission wavelength end: 700
nm). Fluorescence spectra of thioredoxin reductase (from E. coli)
at 20 µM in 100 µL of buffer (10 mM phosphate, 20 mM NaCl,
pH ) 7.4) 0.1% in DMSO was recorded. We recorded spectra of
gold compound 2 in higher concentrations (200 µM and 600 µM)
in the same conditions (buffer, 0.1% of DMSO). Subsequently,
solutions of thioredoxin reductase (20 µM) and gold compound 2
in different concentrations (10, 30, 60, 100, 200, and 600 µM) were
prepared in 100 µL of buffer (0.1% DMSO) and recorded at 25.9
°C (see Figure 7 in section 3.2).
Figure 1. Molecular drawing of the cation in compound [Au{κ2-
C,N-C6H4(PPh2dN(C6H5)-2}(S2CN-Me2)]PF6 2 with the atomic numbering
scheme.
Table 1. Selected Bond Lengths [A] and Angles [deg] for 2
Au(1)-C(12)
Au(1)-N(1)
2.031(9)
2.053(7)
2.305(2)
2.376(2)
1.721(9)
1.613(8)
85.9(3)
C(12)-Au(1)-S(2)
N(1)-Au(1)-S(2)
S(1)-Au(1)-S(2)
C(1)-S(1)-Au(1)
N(1)-P(1)-C(11)
N(1)-P(1)-C(31)
C(11)-P(1)-C(31)
C(2)-N(1)-P(1)
174.0(2)
100.0(2)
75.31(9)
87.2(3)
102.6(4)
111.7(4)
112.0(4)
123.6(6)
Au(1)-S(1)
Au(1)-S(2)
S(1)-C(1)
P(1)-N(1)
C(12)-Au(1)-N(1)
C(12)-Au(1)-S(1)
N(1)-Au(1)-S(1)
98.8(2)
174.8(2)
Results
1. Chemistry. The cycloaurated compound [Au{κ2-
C,N-C6H4(PPh2dN(C6H5)-2}Cl2] 1 was prepared as de-
scribed previously by the greener transmetalation process
with [Au{C6H4(PPh2dN(C6H5))-2}(PPh3)] and K[AuCl4] that
avoids the use of more toxic organomercurial reagents.48 By
reaction of 1 and sodium dithiocarbamato salts and addition
of 1 equiv of NaPF6 (Scheme 1) cationic compounds with
dithiocarbamato ligands of the general type [Au{κ2-
C,N-C6H4(PPh2dN(C6H5)-2}(S2CN-R2)]PF6 (R ) Me 2; Bz
3) were synthesized. These bright yellow cationic compounds
are soluble in DMSO and in mixtures of DMSO/H2O.
The 31P NMR spectra of the complexes allow us to asses
their stability in solution, the oxidation state of the metal,
and the binding mode of the iminophosphorane ligand.48
Cyclometalallated gold(III) complexes 2 and 3 have distinct
31P NMR chemical shifts in d6-acetone δ 65.7 (2), 66.2 (3)
ppm consistent with C,N-coordination of the iminophospho-
rane ligand to the gold (III) center in a square-planar
arrangement (δ in CDCl3 65.4 ppm (1)) completed by the
two S donor atoms of the dithiocarbamato ligands. The
chemical shift due to the PF6- as an anion is visible for the
spectra of both compounds at -144.1 ppm (sept). The
cationic character of these species can be also inferred from
the slight downfield shift of the 31P signal with respect to
compound 1. Chemical shifts of the 31P signals in d6-DMSO
solutions are very similar: δ 64.9 (2), 65.6 (3) ppm and
-144.0 (sept) ppm for the PF6- anion for both compounds.
More importantly the spectra remained the same over time.
Solutions of 2 and 3 in d6-DMSO at RT do not show any
changes in the chemical shifts even after 3 months (see 31P
NMR spectra in Supporting Information). 2 and 3 are thus
quite stable in solution and do not decompose to Au(I)
complexes or metallic gold.
For these complexes, in which both ligands are chelated,
signals from the R groups of the dithiocarbamato ligands
are non-equivalent (two signals for the two methyl groups
1
in the H and 13C NMR spectra of 2). In the case of the Bz
derivative (3) this non-equivalence is only apparent in the
13C NMR spectrum. This reflects the absence of a plane of
symmetry between the two different ligands (iminophos-
phorane and dithiocarbamato).
The crystal structure of 2 has been elucidated by X-ray
diffraction studies. The structure of the gold(III) cation is
shown in Figure 1 while selected bond lenghts and angles
are presented in Table 1.
The AuIII ion is four-coordinated in a square-planar
geometry as expected. The Au-C and Au-N bond lengths
are 2.053(7) and 2.031(9) Å, similar to those found for
neutral compound 1 (both distances are 2.035(4) Å).49 The
AuIII-S bond lengths for the S atoms of the dithiocarbamato
ligands are 2.305(2) and 2.376(2) Å, which reflects the higher
trans-influence of C versus N (longer Au-S(2) distance).
A similar trans influence was reported for the dithiocarbam-
ate complexes of the C,N-pincer damp (o-C6H4CH2NMe2)
ligand.50 For instance for the compound [Au(dmtc)(damp)]B-
Ph4 (dmtc ) S2CN-Me2) the distance Au-S(1) was 2.179(9)
and Au-S(2) 2.340(9) Å.50 In our case the Au-S(1) bond
(51) 31P{1H}NMR (D2O): δ [AuClP{Cp(m-C6H4-SO3Na)2]: 45.1 (s) ppm
(to be reported elsewhere).
Inorganic Chemistry, Vol. 48, No. 4, 2009 1581

Shaik et al.
Scheme 2. Preparation of Cycloaurated Compounds with
) -144.1 ppm), and a minor compound with a δP ) 25.8
ppm was always obtained. 4 and 6 are hydrolyzed in water
as it can be seen in their 31P NMR spectra (D2O). New major
broad signals appear at δPA ) 56.2 and δPB ) 47.7 ppm
(4) and δPB ) 45.5 and δPA ) 10 ppm (6). In the case of
compound 6 the chemical shift of PA indicates that the
iminophosphorane ligand decoordinates from the metallic
center. In DMSO a new signal appears for 4 at δP ) 33.5
ppm over time. After 12 days the signals that can be assigned
to 4 still remained (ca. 60%). The signals at δP ) 33.5 ppm
(4) and δP ) 25.8 ppm (6) can be assigned to Au(III)-PR3
derivatives (with loss of the iminophosphorane ligand) and
that for 6 constitute the only impurity in their preparation.
The more basic, less hydrophobic phosphine P{Cp(m-
C6H4-SO3Na)2} seems to stabilize more the resulting
gold(III) complexes. Reduction to Au(I)-PR3 species is not
proposed since we did not find signals that could be assigned
to compounds of the type AuCl(PR3).51,52
Water-Soluble Phosphines Containing Sulfonated Groups
Scheme 3. Reaction of Compound 1 with the PTA Ligand
The reaction in the same conditions of 1 with PTA (1,3,5-
triaza-7-phosphaadamantane) afforded a totally different
compound regardless of the mol ratio of phosphine employed
(Scheme 3). The iminophosphorane ligand is only coordi-
nated through the C-atom, and the incorporation of two PTA
phosphines is evident from microanalysis and spectroscopic
data. The structure described in Scheme 3 is the most
plausible one. The coordination of the iminophosphorane
ligand through the carbon atom only is evident from the
chemical shift in the 31P NMR (d6-DMSO) for the phospho-
rus atom from the N-PPh2 fragment (δPA ) 7.4 ppm) and
from the high-field displacement of the signals due to the
protons of the N-phenyl ring (see Experimental Section). The
31P NMR (CDCl3) chemical shift for N-PPh2 in [Au{C6H4-
(PPh2dN(C6H5))-2}(PPh3)] and [Hg{C6H4(PPh2dN(C6H5))-
2}Cl] are 8.8 and 8.6 ppm, respectively.48 The 1H NMR and
13C{1H} NMR spectra (in d6-DMSO) shows clearly two PTA
phosphines that are coordinated to the gold(III) center
differently. The protons that can be assigned to one coor-
dinated PTA ligand appear as a singlet at 4.32 (6H, NCH2P)
and an AB system at 4.07 and 4.58 ppm (6H, JA-B ) 16 Hz,
NCH2N). A similar pattern is found for some Pt(II) with
phosphines in trans and some gold compounds containing
PTA ligands.52 The lack of P-H coupling for the NCH2P
protons is not uncommon in some PTA metal-transition
complexes.53 Signals that can be assigned to the protons of
another coordinated PTA ligand appear at 4.41 ppm (br t,
JH-H ) 12 Hz, N-CH2-N) and 5.07 (d, JH-P ) 8 Hz,
NCH2P). The ratio of PTA ligands to iminophosphorane
ligand is thus 2:1. The proton spectrum is in accordance with
the structure shown in Scheme 3. The 13C{1H} NMR
spectrum (see Experimental Section) is also in accordance
with two phosphines coordinated to the gold(III) center (one
in cis and one in trans) as two doublets can be observed for
length is similar to those found recently for [Au(ESDT)Br2]
-
(ESDT ) CH3CH2O(CO)CH2N(CH3)CS2 ) of 2.302(2) and
2.319(2) Å.43
The coordination geometry around the gold atom is slightly
distorted from square-planarity, with the C(12)-Au(1)-N(1)
angle of 85.9(3)° suggesting a rigid “bite” angle. The
C(12)-Au(1)-S(1) also deviates (98.8(2)°). The five-
membered metallocyclic ring is puckered, with deviations
of -0.1566 [N(1)] and 0.14 [P(1)] from the least-squares
plane. The coordination plane for gold and the metallocyclic
plane are slightly twisted to give an angle of 5.66° between
them. The metallocyclic ring is essentially more planar than
that for related compound 1.49 The N-bonded phenyl ring is
twisted 80.7° from the metallocycle plane.
To improve the solubility of the compounds in water new
complexes the type [Au{κ2-C,N-C6H4(PPh2dN(C6H5)-
2}(PR3)nCl]PF6 (PR3 ) P{Cp(m-C6H4-SO3Na)2} n ) 1 4,
n ) 2 PTA 5) were prepared by reaction of 1 with 1 equiv
of AgPF6 and subsequent addition of 1 equiv of water soluble
phosphine (Schemes 2 and 3). Compound 4 (yellow solid)
resulted totally soluble in water while compound 5 (off white
solid) resulted only soluble in DMSO and in mixtures
DMSO-water. The 31P NMR spectra in d6-DMSO of com-
pound 4 shows clearly the cyclometalation for the gold(III)
center (δPA ) 57.5 (4)), the incorporation of the phosphine
(δPB ) 45.8 (4)) and its cationic nature as the signal for the
-
anion PF6 can be observed at δPC ) -144.1 ppm.
(52) 31P{1H} NMR (D2O): δ [AuCl(TPPS)]: 33.4 (s) ppm. Sanz, S.; Jones,
L. A.; Mohr, F.; Laguna, M. Organometallics 2007, 26, 952.
(53) Miranda, S.; Vergara, E.; Mohr, F.; de Vos, D.; Cerrada, E.; Media,
A.; Laguna, M. Inorg. Chem. 2008, 47, 5641.
Our attempts to prepare [Au{κ2-C,N-C6H4(PPh2dN(C6H5)-
2}(PR3)nCl]PF6 6 with the less basic and commercially
available phosphine TPPTS P{(4-C6H4-SO3Na)3} were not
successful, and we got a mixture of two compounds. The
major one was compound 6 (δPA ) 60.9, δPB ) 40.2, δPC
(54) 31P{1H} NMR (d6-DMSO): δ [AuCl(PTA)]: -51.4 (s) ppm. Assefa,
Z.; McBurnett, G.; Staples, R. J.; Fickler, J. P., Jr.; Assman, B.;
Angermaler, K.; Schmidbaur, H. Inorg. Chem. 1995, 34, 75.
1582 Inorganic Chemistry, Vol. 48, No. 4, 2009

Organogold(III) Iminophosphorane Complexes
Table 2. IC50 Values of Iminophosphorane Gold(III) Compounds,
the methylene groups of each phosphine (higher JP-C
coupling constants should correspond to the methylene
groups of the phosphine in trans PC). However, the 31P NMR
spectrum results are more complex. The signal corresponding
to PA appears, as mentioned before at 7.4 ppm while the
PF6- signal appears at -144.1 ppm (sept). There is a signal
at -56.7 ppm that appears as a singlet and that overlaps a
very broad signal in the same region. The ratio of these
signals PB and PC with respect to PA is 2:1. We believe that
there is not an exchange between a cis and trans configu-
ration for these systems since these phosphines are quite
rigid. However, there may be an equilibrium and coordina-
tion-decoordination of one of the phosphines and coordina-
tion-decoordination of a molecule of DMSO. This would
explain the broad signal that coalesces at RT in the 31P NMR.
The signals do not coalesce at RT in the 1H NMR spectrum.
Unfortunately compound 5 is only soluble in DMSO and
partly soluble in H2O (like [AuCl(PTA)] 54) and is totally
insoluble in acetone or MeOH, solvents in which we could
have performed a low temperature 31P NMR to observe the
two distinct signals that correspond to PB and PC. Similar
31P chemical shifts due to the PTA ligand in square-planar
Pt(II) complexes with thionate ligands of the type
[Pt(SR2)(P)2] have been reported recently.53
Compound 5 gives rise to a new compound in DMSO
solution after 4 days (40%) with signals at δPA ) 31.8 (s),
δPB ) -11.5 (s), and δPC ) -53.2 (s) ppm that might
correspond to a situation of partial coordination of the N of
the N-Ph fragment and displacement of the chloride. The
ratio of 5: new compound remains the same over time. Again,
signals that could be assign to Au(I) complexes were not
observed.54 Overall we saw that these complexes are not as
stable as the dithiocarbamato derivatives in solution.
2. Cytotoxic Properties. 2.1. Cytotoxicity. The cytotoxic
properties of gold(III) 1-5 compounds were analyzed in vitro
according to a procedure described by Montoya et al.55
utilizing two human cell lines, HeLa cervical carcinoma and
Jurkat T-cell acute lymphoblastic leukemia cells, that express
the Green Fluorescence Protein (GFP) primarily in the
nucleus. Before use, all tested compounds were dissolved
in DMSO, except for 4 which was dissolved in water, and
dilutions of each compound were then added to the cells in
normal growth medium. The final solvent concentration
(0.1%) had no discernible effect on cell killing. All the tested
complexes have proven, by 31P NMR studies, to be stable
in DMSO over 24 h or more (see above). Cisplatin was used
as a positive control as the cytotoxity assays were performed
under different conditions as those previously reported (e.g.,
Shekan et al.,56 and Alley et al.57). The procedure reported
here measured the cytotoxicity of the tested compounds after
Compared to Cisplatin
IC50(µM)a
cell lines
cisplatin
1
2
3
4
5
HeLa-GPF
Jurkat-GFP
14.9
31
33.7
35
6.2
1
7.75
2.5
109
52.83
35
40
a IC50 is defined as the concentration of drug required to disrupt the
plasma membrane of 50% of cell population, compared to untreated cells,
after 22 h of incubation. Cells with compromised plasma membrane were
monitored using Propidium iodide (PI) and flow cytometry. Cisplatin was
used as reference compound.
22 h instead of after 2457 or, most commonly, 72 h56 of
exposure to the chemicals, and the results are shown in Table
2. As cell death was strongly induced after 8 h (see Figures
2 and 3), incubation for prolonged periods (>24 h) of time
were not deemed necessary and would make dead cell
detection impractical as cell lysis would likely eliminate
many cells from the viability counts.
It is apparent that some of the gold(III) compounds exhibit
strong cytotoxicity toward the HeLa cell line. Remarkably,
the dithiocarbamate derivatives 2 and 3 appear to be much
more potent than cisplatin or 1, with IC50 values 4-low lower
than that for 1 or half of the value of the reference drug.
Similar values were obtained for this cell line with different
gold(III) coordination compounds containing a variety of
dithiocarbamate ligands [(DMDT)AuX2], [(ESDT)AuX2]
(DMDT ) N,N-dimethyldithiocarbamate; ESDT ) ethyl-
sarcosinedithiocarbamate; X ) Cl, Br).28 The IC50 values
for these complexes were between 2.1-8.2 µM (measured
after 24 h by the method described by Alley et al.57). The
cytotoxicity of this type of gold(III) complexes was also
studied with other cell lines like HL60.27 In this case, drug
sensitivity profiles of HL60 cells toward the gold compounds
(studied after 3, 6, 8, and 24 h incubation) showed complexes
were more cytotoxic after longer incubation periods (IC50
values decreased over time).27 The cytotoxicity of water
soluble 4 was much lower for the HeLa-GPF cell lines, and
the cytotoxicity of 5 was comparable to that of 1 and,
therefore, lower than that for cisplatin. Remarkably while
Jurkat-GPF cells were more resistant to the effects of cisplatin
(IC50 ) 31 µM), compounds 2 and 3 were quite cytotoxic
for this cell line (IC50 ) 1 (2) and 2.5 (3) µM). This is in
accordance to the cytotoxicity of some gold(III)12-20 and,
more recently copper(II) compounds,58 against cisplatin
resistant cancer cell lines. Compounds 1 and 5 had a
cytotoxicity comparable to cisplatin. 4 again displayed the
lowest cytotoxicity but still was more cytotoxic for Jurkat-
GFP cells than for HeLa-GPF cells.
2.2. Apoptosis Studies. To gain some insight of the type
of cell death that the more cytotoxic gold complexes induce
in the cancer cell lines, we performed some apoptosis assays
with the HeLa cells (without GFP) with complexes 1-5 (see
experimental for details). As cells may undergo programmed
cell death (apoptosis) or necrosis, the mode of death mediated
by our compounds was investigated. In early stages of
(55) Montoya, J.; Varela-Ramirez, A.; Estrada, A.; Martinez, L. E.; Garza,
K.; Aguilera, R. J. Biochem. Biophys. Res. Commun. 2004, 325, 1517.
(56) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
J. Natl. Cancer. Inst. 1990, 82 (13), 1107.
(57) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski,
M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.;
Boyd, M. R. Cancer Res. 1988, 48, 589.
(58) Giovagnini, L.; Sitran, S.; Montpoli, M.; Caparrotta, L.; Corsini, M.;
Rosani, C.; Zanello, P.; Dou, Q. P.; Fregona, D. Inorg. Chem. 2008,
47, 6336.
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Shaik et al.
Figure 2. Apoptosis of HeLa (no GFP-expressing) cells induced by 1 (33.75 µM) measured by using two-color flow cytometric analysis, at two different
times of incubation.
Figure 3. Apoptosis of HeLa (no GFP-expressing) cells induced by 2 (6.25 µM) measured by using two-color flow cytometry, at two different incubation
times.
apoptosis, one of the significant biochemical features is loss
of plasma membrane phospholipid asymmetry because of
translocation of phosphatidylserine (PS) from cytoplasmic
to extracellular side. This characteristic allows detection of
externalized PS by the specific binding of Annexin V (FITC-
conjugated). Initiation of cell death will eventually result in
the permeabilization of the cell membrane, allowing PI to
stain DNA within the nucleus. As shown in Figure 3, each
histogram is divided into four quadrants with the left top
quadrant detecting necrotic cells without Annexin-V FITC
signal. The right top quadrant shows cells with compromised
membranes that are permeable to PI and stained with
Annexin V-FITC, which is indicative of late apoptosis. The
left bottom quadrant shows live cells that have intact
membranes (not stained) while the right bottom quadrant
represents cells that were stained (bound) with Annexin
V-FITC, which is indicative of early apoptosis.
to undergo apoptosis (either early or late) as visualized by
the Annexin V-FITC fluorescence. As mentioned earlier,
necrosis almost doubled from 8.9% at 8 h to 16.5% by 16 h.
In Figure 3, we can see that upon treatment with 6.25 µM
of 2, 41% and 35% of the cells were found to undergo
apoptosis by 8 and 16 h, respectively. As in the previous
experiments with 1, necrosis was found to increase from
18.1% at 8 h to 29.4% by 16 h. These studies were also
performed with compounds 3-5, and Figure 4 shows the
preferential induction of apoptosis in HeLa cells after
treatment with 1-5. Remarkably for compound 2, necrosis
is an important cell death pathway. These results are similar
to those found by Fregona and Bindoli with other gold(III)-
dithiocarbamato complexes.26,44,45 They found (by studying
PARP cleavage) that these complexes were able to induce
cell death in a dose-dependent way, and they hypothesized
that their compounds triggered cell death by activating not
only apoptotic pathways but also other death mechanisms
such as necrosis. This is in contrast to what Che and Chiu
found with a gold(III) porphyrin complex.59 In this case the
gold compound induced cytotoxicity through an apoptotic
Using fluorescein-labeled Annexin V, we have quantified
the percentage of apoptotic cells by flow cytometry. As
shown in Figure 2, upon treatment with 1 (33.75 µM) for 8
or 16 h, 34.2% of the cells (8 h) or 24.9% (16 h) were found
1584 Inorganic Chemistry, Vol. 48, No. 4, 2009

Organogold(III) Iminophosphorane Complexes
Figure 5. Melting curves for free CT DNA and CT DNA after incubation
with complexes 1, 2, 4, and 5 for 1 h in 5 mM tris/NaClO4 buffer at pH
7.39 and r ) 0.5.
Figure 4. Preferential induction of apoptosis in HeLa cells after treatment
with the various organogold(III) complexes. IC50 was the concentration used
for each organogold compound and cisplatin. The total percent of cells
reacting with Annexin V-FITC is expressed as the sum of both percentage
of early and late stage of apoptosis. Each bar represents average and standard
deviation results from triplicate samples. The concentration of DMSO was
0.1%. White and black bars represent percentages of apoptotic and necrotic
cells, respectively.
Table 3. Changes in the Tm of CT DNA after Incubation with
Complexes 1, 2, 4, and 5 for 1 h in 5 mM tris/NaClO4 Buffer at pH
7.39 and r ) 0.5
complex
∆T (Tm DNA/Complex-Tm DNA) °C
1
2
3
4
-0.30
0.00
0.30
0.05
way only, as demonstrated by laser confocal and flow
cytometry studies.26,59
3. Mechanistic Studies. 3.1. Reactions with Calf
Thymus DNA. To either prove or rule out the possibility of
a DNA-drug interaction, some Thermal Denaturation experi-
ments were carried out. The melting technique is a sensitive
and easy tool to detect even slight DNA conformational
changes. It is known that a destabilizing interaction with the
double helix (typically, covalent) is observed as a decrease
in the Tm, while a stabilizing interaction (usually by
intercalation or by electrostatic attraction) induces an increase
of the Tm. Bearing that in mind, Calf Thymus DNA was
incubated for 1 h with each drug at a DNA/drug 2:1 ratio.
The results are summarized in Table 3 and Figure 5. Under
solution conditions, compound 3 was not completely soluble,
and its interaction with DNA could not be studied by this
technique.
None of the complexes were able to modify the melting
temperature of a solution of calf thymus DNA beyond the
experimental error. This fact suggests that either they do not
interact with DNA or the interaction is so weak that it can
not be detected by this technique, indicating that the
mechanism for which the complexes are cytotoxic are not
likely related to DNA damage.
Figure 6. UV-vis absorption spectra of the titration of 5 µM of cytochrome
c with Gold-Derivative 2 after incubation at 37 °C during 24 h. The mol
ratios studied were 1:1, 1:3, and 1:5.
several groups have investigated their interaction with model
proteins such as ubiquitin, cytochrome c, and thioredoxin
reductase. Indeed, Messori et al. reported significant spectral
changes in gold(III) binuclear oxo compounds as character-
ized by UV-vis spectroscopy that were indicative of their
interaction with these proteins.14,42 In our case, incubation
for 24 h at 37 °C of compound 2 (the most cytotoxic and
solution stable of the compounds) with the protein cyto-
chrome c did reveal changes in the representative protein
spectral bands (Figure 6). There was a significant decrease
in the intensity of the Soret band (409 nm) of the hemo group
in the protein when treated with increasing amounts of the
gold complex. This effect suggests protein-drug60 interaction
that is not related to a redox-process (via initial reduction of
2 to gold(I) and subsequent reduction of the Fe(III) of the
(59) Wang, Y.; He, Q.-Y.; Sun, R. W.-Y.; Che, C.-M.; Chiu, J.-F. Eur.
J. Pharmacol. 2007, 554, 113.
(60) Similar spectroscopic changes for interactions of metals with serum
proteins have been reported. Timerbaev, A. R.; Hartinger, C. G.;
Aleksenko, S. S.; Keppler, B. K. Chem. ReV 2006, 106, 2224; and
references therein.
3.2. Reactions with Representative Proteins. As men-
tioned earlier, gold(III) complexes have been suggested to
exert cytotoxicity by inducing apoptosis.26,44-46,58 Since the
binding of these complexes to DNA is generally weak,
Inorganic Chemistry, Vol. 48, No. 4, 2009 1585

Shaik et al.
our results, we hypothesize that compound 2 interacts with
thioredoxin reductase.
Discussion
The last five years have been very important in the field
of gold compounds as potential antitumor agents.12-21
Several gold(III) and organogold(III) derivatives have dis-
played encouraging in vitro pharmacological properties. The
search for new biomolecular targets has attracted great
interest lately since it seems that DNA is not a primary target
for most gold(III) complexes.14-19 Recent reports favor the
idea that the cytotoxic effects of gold (III) compounds are
primarily a consequence of protein interactions and mito-
chondrial damage.13,27-29,42-45 Thus, the search for new gold
compounds and, more importantly, studies to understand the
mechanism of action of gold compounds becomes extremely
relevant. For instance, direct inhibition of thioredoxin re-
ductase that may activate apoptosis after triggering mito-
chondrial cyt c release has been recently reported.44 We
report here a series of cationic organogold(III) compounds
with a C,N backbone that stabilizes the resulting square-
planar cycloaurated complexes. Besides, the P atom in the
PR3 fragment can be used as a “spectroscopic marker” to
study the in vitro stability (and oxidation state) by 31P NMR.
All cationic compounds exhibit a high solubility in DMSO
and a reasonable solubility within a 99:1 water/DMSO or
buffer/DMSO environment. 4 is completely soluble in water.
We have used 31P NMR spectroscopy to establish their
stability and oxidation state in solutions of DMSO or water.
While compounds with dithiocarbamato ligands [Au{κ2-
C,N-C6H4(PPh2dN(C6H5)-2}(S2CN-R2)]PF6 (R ) Me 2; Bz
3) result extremely stable in DMSO or DMSO/water solu-
tions new complexes with water-soluble phosphines [Au{κ2-
C,N-C6H4(PPh2dN(C6H5)-2}(PR3)nCl]PF6 (PR3 ) P{Cp(m-
C6H4-SO3Na)2} n ) 1 4, n ) 2 PTA 5) give rise to solvation
products with total or partial decoordination of the N atom
of the iminophosphorane ligand and/or decomposition to
Au(III)-PR3 species over time. The cytotoxic properties of
these compounds were evaluated in vitro against HeLa and
Jurkat cells. Compounds 2 and 3 resulted more cytotoxic
than parent neutral compound 1, and their IC50 values (HeLa)
where comparable to those reported for other gold(III)
dithiocarbamato derivatives of the type [(DMDT)AuX2],
[(ESDT)AuX2] (DMDT ) N,N-dimethyldithiocarbamate;
ESDT ) ethylsarcosinedithiocarbamate; X ) Cl, Br).29 The
cytotoxicity of 4 and 5 was significantly lower (4) or
comparable (5) to that of parent compound 1 supporting the
idea that the stability afforded by the C,N-backbone is an
important consideration. The fact that the organogold(III)
derivatives with dithiocarbamato ligands have very similar
cytotoxicity to dithiocarbamato compounds with halides is
of interest since it will allow a possibility to further
functionalize and tune electronic and steric factors and
lipohilicity/hydrophilicity of dithiocarbamato compounds by
inclusion of some other ligands similar to the one described
here (iminophosphorane). The apoptosis studies that we have
carried out have shown that our compounds trigger cell death
by activating not only apoptotic pathways (major) but also
Figure 7. Fluorescence spectra of the titration of 20 µM of thioredoxin
reductase with 2. Excitation wavelength 285 nm. The mol ratios studied
were 1:0.5; 1:1.5, 1:3; 1:5, 1:10, 1:30. In the figure the colored lines
correspond to different concentrations of gold compound with respect to
20 µM of protein (10, 30, 60, 100, 200, and 600 µM). We have also included
the spectra of the TR alone (0 µM in gold compound), as well as 2 alone
(200, 600 µM alone).
heme group by reoxidation to gold(III)).61 On the other hand,
the bands of the gold compound (200-300 nm) could not
be studied as a consequence of an overlapping with the
DMSO used to dissolve it and some other bands of the
cytochrome c.
We also studied the interaction of 2 with thioredoxin
reductase. In this case the characteristic bands for this protein
are overlapped by the bands of the gold compound in the
range of 200-300 nm when absorption experiments were
carried out. As an alternative approach we monitored the
interaction of thioredoxin reductase using fluorescence
spectroscopy.
As shown in Figure 7, thioredoxin reductase displays
typical fluorescence emission spectra with a maximum at
349 nm that is indicative of tryptophan fluorescence. Titration
of the protein with compound 2 resulted in an immediate
and significant reduction in tryptophan (trp 53) fluorescence
emission intensity. Incubation at 37 °C was not necessary.
The spectra revealed changes immediately after addition of
2 at room temperature. Above mol:mol ratios of 1:5, the
fluorescence emission maxima shifted toward higher wave-
lengths to reach correspondence with the spectra of com-
pound 2. This spectral behavior resembles that of compound
imidazolium[trans-tetrachlorobis(imidazol)ruthenate(III)] when
incubated at 37 °C with human serum albumin (HAS).62 In
this case there is also a quenching of the tryptophan
fluorescence from HSA. The authors claimed this quenching
was due to the fact that tryptophan residues in the Ru-im-
HAS system are considered to be brought to a more
hydrophilic environment as a result of the ruthenium bindings
in the proximity of the tryptophan residue. On the basis of
(61) We performed a titration of cytochrome c with dithiothreitol DTT (in
mol ratios protein/DTT 1:1, 1:3, and 1:5, concentration of protein 5
µm) in buffer containing 0.1 mol % DMSO and the vis-UV spectra
shows a shift of the Soret band of the hemo group at 409 to 415 nm
but also two characteristic bands at 521 and 550 nm consistent with
the reduction of Fe(III) to Fe(II) that we do not see in the experiment
of titration of cytochrome c with 2.
(62) Trynda-Lemiesz, L.; Keppler, B. K.; Kozlowski, H. J. Inorg. Biochem.
1999, 73, 123.
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Organogold(III) Iminophosphorane Complexes
other death mechanisms such as necrosis like in the case of
the dithiocarbamato halide complexes. This effect is more
marked with complex 2. To gain more information on the
mechanistic aspects of the cytotoxic compounds 1-5 we
have carried out spectrophotometric studies (UV-vis) on
their interaction with DNA. None of the complexes were
able to modify the melting temperature of a solution of calf
thymus DNA. This fact suggests that either they do not
interact with DNA or the interaction is very weak and
electrostatic in nature. This is in accordance to what has been
reported for most gold(III) compounds.14 The reactivity of
the more cytotoxic compound 2 toward model biomolecules
was studied spectroscopically. By UV-vis, we could assess
a slow interaction with cytochrome c. By fluorescence
spectroscopy, we have been able to confirm that compound
2 interacts with protein thioredoxin reductase, and that at
high concentrations, it likely induces an irreversible dena-
turation. This in accordance to the behavior exhibited by
recently reported gold(III) compounds with bipyridil42,43 and
dithiocarbamato ligands44,45 and supports the idea that
mitochondrial proteins may be the target of gold(III)
compounds that subsequently activate apoptosis. Fregona et
al. found that the gold(III) dithiocarbamato complexes of the
type [Au(SS)X2] (SS ) DMDT: N,N-dimethyldithiocarbam-
ate; ESDT ) ethylsarcosinedithiocarbamate; X ) Cl, Br)
inhibited thioredoxin reductase activity and hypothesized a
model suggesting that deregulation of the thioredoxin re-
ductase/thioredoxin redox system is a major mechanism
involved in the anticancer activity of those gold(III) com-
pounds.44
demonstrated that compound 2 interacts with proteins like
cytochrome c and thioredoxin reductase. The use of 31P NMR
spectroscopy as a simple characterization tool to asses
stability and oxidation state of the gold compounds in
solution has been demonstrated. Further modification of the
iminophosphorane ligand is currently underway to prepare
new gold(III) compounds with higher hydrophilicity and
improved pharmacological properties. More detailed studies
on mitochondrial damage and interaction of these compounds
with the proteins (by different techniques) are also underway.
Acknowledgment. We thank Brooklyn College (start-up
budget M.C.), NSF (#DBI 0353887), and NIH RCMI Grant
(2G112RR08124) at University of Texas at El Paso for
financial support and the Arnold and Ruth T. Kaufman
Chemistry Fund at Brooklyn College for a summer research
undergraduate award (N.S.). We thank Prof. Iban Ubarretx-
ena-Belandia (Mt. Sinai Hospital, New York) for useful
discussions and assistance with the fluorescence measure-
ments and Prof. Istva´n T. Horva´th (Eo¨tvo¨s University,
Budapest, Hungary) for the generous donation of the water-
soluble phosphine P{Cp(m-C6H4-SO3Na)2}. This paper is
dedicated to Maria Antonia Casas Gurpegui (grandmother
of M.C. and supporter of her scientific career) who died of
cancer in 2006.
Supporting Information Available: Selected 31P{1H} NMR
spectra of compounds 2-6 in different solvents. This material is
available free of charge via the Internet at http://pubs.acs.org. Tables
of thermal parameters and observed and calculated structure factors
(crystal structure of compound 2) have been deposited at the Cambridge
Crystallographic Data Center. Any request for this material should
quote a full literature citation and the reference number CCDC 704381
and may be obtained from The Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, U.K. (fax: +441233336033; email: deposit@
ccdc.cam.ac.uk or www: http://ccdc.cam.ac.uk).
Conclusions
The present study gives support to previous reports that
the cytotoxicity displayed by gold(III) compounds is not
related to DNA-damage. We have demonstrated that our
compounds do not interact with DNA. We have also
IC801925K
Inorganic Chemistry, Vol. 48, No. 4, 2009 1587