Potential anticancer heterometallic Fe-Au and Fe-Pd agents: Initial mechanistic insights

Article abstract of DOI:10.1021/jm4007615

A series of gold(III) and palladium(II) heterometallic complexes with new iminophosphorane ligands derived from ferrocenylphosphanes [{Cp-P(Ph ₂) N-Ph}₂Fe] (1), [{Cp-P(Ph₂) N-CH ₂-2-NC₅H₄}₂Fe] (2), and [{Cp-P(Ph₂) N-CH₂-2-NC₅H₄}Fe(Cp)] (3) have been synthesized and structurally characterized. Ligands 2 and 3 afford stable coordination complexes [AuCl₂(3)]ClO₄, [{AuCl ₂}₂(2)](ClO₄)₂, [PdCl ₂(3)], and [{PdCl₂}₂(2)]. The complexes have been evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), in human breast cancer cells (MCF7) and in a nontumorigenic human embryonic kidney cell line (HEK-293T). The highly cytotoxic trimetallic derivatives M₂Fe (M = Au, Pd) are more cytotoxic to cancer cells than their corresponding monometallic fragments. Moreover, these complexes were significantly more cytotoxic than cisplatin in the resistant A2780R and the MCF7 cell lines. Studies of the interactions of the trimetallic compounds with DNA and the zinc-finger protein PARP-1 indicate that they exert anticancer effects in vitro based on different mechanisms of actions with respect to cisplatin.

Full text of DOI:10.1021/jm4007615

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Article
Potential Anticancer Heterometallic Fe-Au
and Fe-Pd Agents: Initial Mechanistic Insights
Nicholas Lease, Vadim Vasilevski, Monica Carreira, Andreia de
Almeida, Mercedes Sanau, Angela Casini, and Maria Contel
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4007615 • Publication Date (Web): 20 Jun 2013
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Potential Anticancer Heterometallic FeꢀAu and FeꢀPd
Agents: Initial Mechanistic Insights
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Nicholas Lease,a Vadim Vasilevski,a Monica Carreira,a Andreia de Almeida,b Mercedes Sanaú,c
Pipsa Hirva,d Angela Casini,b* and Maria Contela*
aDepartment of Chemistry, Brooklyn College and The Graduate Center, The City University of
b
New York, Brooklyn, NY, 11210, US. Pharmacokinetics, Toxicology and Targeting, Research
Institute of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands.
cDepartamento de Química Inorgánica, Universidad de Valencia, Burjassot, Valencia, 46100, Spain.
dDepartment of Chemistry, University of Eastern Finland, Joensuu Campus, FIꢀ80101 Joensuu,
Finland.
KEYWORDS: Gold and palladium complexes, ferrocenyl phosphaneꢀiminophosphorane ligands,
cytotoxicity, DNA, zincꢀfinger proteins.
ABSTRACT
A series of gold(III) and palladium(II) heterometallic complexes with new iminophosphorane
ligands derived from ferrocenylꢀphosphanes [{CpꢀP(Ph2)=NꢀPh}2Fe] (1), [{CpꢀP(Ph2)=NꢀCH2ꢀ2ꢀ
NC5H4}2Fe] (2) and [{CpꢀP(Ph2)=NꢀCH2ꢀ2ꢀNC5H4}Fe(Cp)] (3) have been synthesized and
structurally characterized. Ligands 2 and 3 afford stable coordination complexes [AuCl2(3)]ClO4,
[{AuCl2}2(2)](ClO4)2, [PdCl2(3)] and [{PdCl2}2(2)]. The complexes have been evaluated for their
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antripoliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin
(A2780S/R), in human breast cancer cells (MCF7) and in a nonꢀtumorigenic human embryonic
kidney cell line (HEKꢀ293T). The highly cytotoxic trimetallic derivatives M2Fe (M = Au, Pd) are
more cytotoxic to cancer cells than their corresponding monometallic fragments. Moreover, these
complexes were significantly more cytotoxic than cisplatin in the resistant A2780R and the MCF7
cell lines. Studies of the interactions of the trimetallic compounds with DNA and the zincꢀfinger
protein PARPꢀ1 indicate that they exert anticancer effects in vitro based on different mechanisms of
actions with respect to cisplatin.
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INTRODUCTION
Cisplatin and the followꢀon drugs carboplatin (paraplatinTM) and oxaliplatin (eloxatinTM) are used
to treat 40ꢀ80% of cancer patients alone or in combination chemotherpay.1 However, their
effectiveness is still hindered by clinical problems, including acquired or intrinsic resistance, a
limited spectrum of activity, and high toxicity leading to side effects.1,2 Promising anticancer
activities of a variety of other metal complexes (including organometallic compounds) have been
reported in the past two decades.3ꢀ8 Recent progress in this field has brought a better understanding
on the mode of action for some of these derivatives.9 In particular, a number of gold compounds
have overcome cisplatin resistance to specific cancer cells10 which makes them attractive potential
therapeutics. In addition, it has been found that DNA is not the primary target for most gold
compounds reinforcing the idea that their mode of action is different with respect to cisplatin.10,11
This has prompted the search for alternative biomolecular targets not only for gold but for other
nonꢀplatinum complexes10ꢀ13 and a revision of the mode of action of anticancer platinum compounds
themselves.14 Thus, multiple biological pathways have been proposed for nonꢀplatinum derivatives
including the inhibition of mitochondrial enzymes and of the proteasome for gold compounds.15ꢀ17
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Recently, some of us also reported on the potent inhibition of the zinc finger protein polyꢀ
(adenosine diphosphate (ADP)ꢀribose) polymerase 1 (PARPꢀ1) by Au(I) and Au(III) complexes.18,19
PARPs are essential proteins involved in cancer resistance to chemotherapies, and play a key role in
DNA repair by detecting DNA strand breaks and catalysing poly(ADPꢀribosylation).20,21
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Within this frame, there has been a growing interest in heterometallic complexes as potential
anticancer agents.22ꢀ26 The hypothesis is that the incorporation of two different cytotoxic metals in
the same molecule may improve their activity as antiꢀtumor agents due to interaction of the different
metals with multiple biological targets or by the improved chemicophysical properties of the
resulting heterometallic compound. The anticancer properties of certain molecules may be increased
by the introduction of the organometallic ferrocene motif in the framework due to its low toxicity,
high lipophilicity and unique electrochemical behavior.27ꢀ29 Thus, several examples of
heterometallic complexes incorporating ferrocene and a second cytotoxic metal (Au(I),30ꢀ36
Pt(II),30,37ꢀ42 Pd(II),40,43ꢀ45 Ru(II),46ꢀ50 Rh(I)40,51,52 Ir(I),40 and Cu(I)53,54) appeared in the literature. In
most cases the antiproliferative properties of the resulting compounds improved with respect of that
of the corresponding ferroceneꢀbased ligand, although synergistic effects of the effects of the two
different metals were evaluated only in one case.50
Some of us have reported that nonꢀtoxic iminophosphorane or iminophosphane (IM) compounds
serve as stabilizing (C,Nꢀ or N,N) chelating ligands in the preparation of anticancer organometallic
and coordination d8 metal complexes.55ꢀ58 Organogold(III) complexes containing iminophosphorane
(C,NꢀIM) ligands of the type PPh3=NPh displayed a high cytotoxicity in vitro against human
ovarian cancer and leukemia cell lines (in some cases with IC50 values in the nanomolar range)55,56
while being less toxic to normal Tꢀlymphocytes by a nonꢀcisplatin mode of action (involving
mitochondrial production of reactive oxygen species).56 More recently, we have reported on the
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cytotoxicity of coordination and organometallic Pt(II) and Pd(II) complexes with waterꢀsoluble
iminophosphorane ligands.57,58 Interestingly, we observed that some of these Pt and Pd compounds
were more cytotoxic than cisplatin to leukemia cell lines (both TꢀJurkat and cisplatinꢀresistant
Jurkat shꢀBak) by a mode of action different to that of cisplatin.57,58
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Within this frame, we aimed to explore the biological properties of related IM complexes
containing Au(III) and Pd(II) centers incorporating the ferrocene motif, and we report here on the
synthesis of new iminophosphorane ligands derived from ferrocenylꢀphosphines [{CpꢀP(Ph2)=Nꢀ
Ph}2Fe] (1), [{CpꢀP(Ph2)=NꢀCH2ꢀ2ꢀNC5H4}2Fe] (2) and [{CpꢀP(Ph2)=NꢀCH2ꢀ2ꢀNC5H4}Fe(Cp)]
(3). While ligand 1 becomes easily protonated while reacted with Au(III) derivatives, ligands 2 and
3 incorporating pyridyl groups serve as an excellent precursors to trimetallic M2Fe and dimetallic
MFe derivatives (M = Au, Pd). The antiproliferative properties of the organometallic ironꢀbased
ligands and the heterometallic complexes have been evaluated in human ovarian cancer cells
sensitive and resistant to cisplatin (A2780S/R), in human breast cancer cells (MCF7) and
nontumorigenic human embryonic kidney cell line (HEKꢀ293T).
The compounds were also tested for their possible interactions with plasmid (pBR322) DNA used
as a model nucleic acid, and for their reactivity with the transport protein serum albumin (HSA).
Moreover, the most promising compounds out of the cytotoxicity screening, the trimetallic M2Fe
complexes, were tested for PARPꢀ1 inhibition directly on the purified protein. The obtained results
have been discussed in relation to the compounds possible mechanisms of anticancer action.
RESULTS AND DISCUSSION
1. Synthesis and Characterization
Ligands 1 and 2 derived from 1,1'ꢀBisꢀdiphenylphosphinoꢀferrocene (DPPF) can be conveniently
prepared in moderate yields by reaction of commercial DPPF and phenyl azide (1) or 2ꢀ
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methylpyridine azide (2) by the Staundinger59 method (Scheme 1). Ligand 3 can be prepared in an
analogous way in high yields by reacting diphenylphosphinoꢀferrocene (MPPF)60 and
methylpyridine azide. The ligands hydrolyze quickly to amines and the corresponding phosphine
oxides at air in solution or as solids due to moisture. They can be conveniently stored in dry
glassware under nitrogen for months.
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Scheme 1. Preparation of iminophosphorane ligands 1ꢀ3 via the Staudinger59 method.
Ligands 2 and 3 serve as excellent precursors to bimetallic or trimetallic derivatives incorporating
Au(III) and Pd(II) centers (Scheme 2). The reaction of ligand 1 to obtain a dinuclear derivative Feꢀ
Au with the Au coordinated to the 2N of the iminophosphorane fragments did not proceed as
expected. The reaction of 1 with an in situ solution of [AuCl2(CH3CN)2](ClO4) led to the
protonation of the two nitrogen from the iminophosphorane ligand and the obtention of a cationic
salt. This compound can indeed be prepared and formulated as [{CpꢀP(Ph2)=NHꢀPh}2Fe](AuCl4)2 4
by reaction of 1 with 2 equivalents of HAuCl4x3H2O (see experimental). The reaction of ligand 1
with one equivalent of [PdCl2(COD)] resulted in mixtures of coordination and cyclometallated
compounds which could not be separated.
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Scheme 2. Preparation of new trimetallic FeꢀM2 (M = Au (5), Pd (6)) and bimetallic FeꢀM (M = Au (4, 7);
Pd (8)). i) 2K[AuCl4]:4AgClO4 in dry CH3CN; ii) 2HAuCl4x3H2O in CH3CN; iii) 2[PdCl2(COD)] in dry
CH2Cl2; iv) K[AuCl4]:2AgClO4 in dry CH3CN; v) [PdCl2(COD)] in CH2Cl2. All reactions carried out at RT
and under inert atmosphere of N2 or Ar.
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The incorporation of one or two pyridyl group in the iminophosphorane ligands 2 and 3 allows for
the preparation of coordination dimetallic (7, 8) and trimetallic (5, 6) Au(III) and Pd(II) complexes.
While ligands 1ꢀ3 are air sensitive the new heterometallic complexes are obtained as air stable
solids. The bimetallic complex FeꢀAu 7 (Scheme 2) is a cationic derivative while the trimetallic
compound FeꢀAu2 (5) and the protonated salt 4 are a dicationic species (3 electrolytes) as
demonstrated by the conductivity measurements (see Experimental). Both the trimetallic and
bimetallic complexes containing Pd(II) centers are neutral species. The presence of only one signal
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in the P{1H} NMR spectra of 5 and 7 suggests a symmetric structure (further confirmed by DFT
calculations).
Figure 1. Previously reported IMꢀPPh3 ligand 9,61 gold(III) derivative 1061 and new Pd(II) complex
11 evaluated for their antiproliferative properties (Table 3) for comparison purposes with the new
IM ligands containing ferrocenylꢀphosphanes (1ꢀ3) and the new IM heterometallic complexes (4ꢀ8).
In order to make comparisons about the antiproliferative properties of the new ligands and
heterometallic complexes and that of Au(III) and Pd(II) monometallic analogues we prepared
previously described ligand Ph3P=NꢀCH2ꢀ2ꢀNC5H4 (IMꢀPPh3) 961 and monometallic complexes
[(Ph3P=NꢀCH2ꢀ2ꢀNC5H4)AuCl2]ClO4 1061 and [(Ph3P=NꢀCH2ꢀ2ꢀNC5H4)PdCl2] 11 (figure 1).
The stability of the compounds in solution can be easily ascertained by 31P{1H} NMR spectroscopy
in deuterated solvents (see supporting information). Most compounds are very stable in CD3CN
solution (weeks). The stability in d6ꢀDMSO solution was also evaluated. While ligands 2 and 3 (1 is
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insoluble in d6ꢀDMSO) have half lives of minutes (giving phosphane oxide immediately) the
metallic complexes 4ꢀ11 have halfꢀlives of weeks (4, 5) or days (6, 7, 11) or several hours (8, 10).
The most stable complexes are the protonated ligand (4), the trimetallic complexes M2Fe (M = Au
5, Pd 6) and gold dimetallic (7) species. All the palladium complexes and the gold (10) with the
Ph3P=NꢀCH2ꢀ2ꢀNC5H4 (IMꢀPPh3) ligand (9) decompose to cyclometalled species (in the case of
ligand 9 the metallation occurs at the aryl group of the PPh3 phosphane while in the case of the
ferrocenylꢀphosphane derivatives the metalation occurs both at the aryl ring of the PPh2 fragment
and at the cyclopentadienyl of the ferrocene fragment). The stability of 10 in DMSOꢀH2O is very
poor, and, therefore, the incorporation of ferrocene fragments to the iminophosphorane ligand was
advantageous to improve the stability of the related gold complexes. In all cases the decomposition
products contain the ligand still bound to the metallic fragments. Of note, all complexes are soluble
in mixtures DMSO:H2O (1:99 ratio) in micromolar concentrations (relevant for subsequent
biological studies).
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It is clear that there is an advantage to use the new heterometallic complexes in biological studies as
opposed to use the iminophosphorane ligands derived from ferrocenyl phosphanes (1ꢀ3) in
combination with metallic salts (K[AuCl4] and [PdCl2(COD)]). The new metallic complexes,
particularly the trinuclear derivatives 5 and 6, have a much higher stability in DMSO and
DMSO:H2O solution than the parent ligands 1ꢀ3.
In order to further characterize the stability of the compounds, in physiologicalꢀtype conditions, we
recorded the UVꢀvisible spectra of the metal complexes in PBS solution over time. Representative
spectra of compound 5 and 10 are reported in Figure S22 in the supporting information. As it can be
seen in the case of 5, the compound’s absorption spectrum is almost unaltered even after 12 h
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incubation at room temperature, whereas the absorption rapidly decreases with time in the case of
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the mononuclear gold compound 10.
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The structure of compounds 8 and 11 have also been determined by Xꢀray diffraction analysis
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(Figure 2). Selected bond lengths and angles for both compounds are collected in Table 1.
Figure 2. Molecular structure of the compounds 8 and 11 with the atomic numbering scheme.
The geometry about the Pd(II) centers is pseudoꢀsquare planar with the N(2)ꢀPd(1)ꢀN(1) angle of
79.51(17)o (8) and 80.87(12)o (11) suggesting a rigid ‘bite’ angle of the chelating ligand. The Pd
centers are on an almost ideal plane with negligible deviations from the leastꢀsquares plane. The
main distances PdꢀN(1) or PdꢀN(iminic) and PdꢀN(2) found for 8 and 11 are similar to that in
related iminophosphorane complexes like [PdCl2(TPA=NꢀC(O)ꢀ2ꢀNC5H4)],57
C6H4(PPh2=NC6H4Meꢀ4’)ꢀ2}{ꢀꢀOAc)}]2,62 [Pd{k2ꢀC,NꢀC6H4(PPh2=NC6H4Meꢀ4’)ꢀ2}(tmeda)]
ClO4,62 [Pd(C6H4CH2NMe2)(Ph3PNC(O)ꢀ2ꢀNC5H4)]ClO4,63
[Pd{k2ꢀC,Nꢀ
and
[PtCl(PMe2Ph){(N(pꢀ
Tol)=PPh2)2CHCH3}]Cl.64 The distances P=N are shorter than in compounds which incorporate a
carbonyl group bonded to the iminic nitrogen (due to a smaller delocalized charge density as
compared to the CO group).57,58
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Table 1. Selected Structural Parameters of complexes 8 and 11 obtained from Xꢀray single crystal diffraction
studies. Bond lengths in Å and angles in °.
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11
P(1)ꢀN(1)
1.608(4)
P(1)ꢀN(1)
1.610(3)
1.463(5)
2.038(3)
2.029(3)
2.3081(11)
2.2895(11)
80.87(12)
90.13(4)
173.45(9)
175.94(9)
95.61(9)
93.50(9)
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N(1)ꢀC(1)
Pd(1)ꢀN(1)
Pd(1)ꢀN(2)
Pd(1)ꢀCl(1)
1.469(7)
2.041(4)
2.025(5)
N(1)ꢀC(1)
Pd(1)ꢀN(1)
Pd(1)ꢀN(2)
Pd(1)ꢀCl(1)
2.2938(15)
2.2845(16)
79.51(17)
90.08(7)
172.28(13)
174.06(14)
97.63(13)
92.84(13)
2.035(6) ꢀ 2.051(7)
Pd(1)ꢀCl(2)
Pd(1)ꢀCl(2)
N(2)ꢀPd(1)ꢀN(1)
Cl(2)ꢀPd(1)ꢀCl(1)
N(1)ꢀPd(1)ꢀCl(1)
N(2)ꢀPd(1)ꢀCl(2)
N(1)ꢀPd(1)ꢀCl(2)
N(2)ꢀPd(1)ꢀCl(1)
Fe(1)ꢀC
N(2)ꢀPd(1)ꢀN(1)
Cl(2)ꢀPd(1)ꢀCl(1)
N(1)ꢀPd(1)ꢀCl(1)
N(2)ꢀPd(1)ꢀCl(2)
N(1)ꢀPd(1)ꢀCl(2)
N(2)ꢀPd(1)ꢀCl(1)
Because of the difficulties of obtaining experimental crystal structures for the gold and
palladium complexes 5ꢀ7, we used computational DFT methods to verify if the suggested
molecular structures would be favourable. The initial geometry for the models was taken either
from the crystal structure of 8 and 11 or modelled using resembling structures obtained from the
Cambridge Crystallographic Data Base. Figure 3 shows the energetically most favourable
structures for the complexes, the freely optimized ligand structures can be found in the
supplementary material (Figure S1).
In all complexes, the coordination of the metal atoms (Au, Pd) was slightly twisted square
planar. The most important difference between the freely optimized ligand structures and
corresponding complexes was the torsional behaviour of the nitrogen atoms and also of the
ferrocene rings. In separately optimized ligands, the minimum energy conformation of the NꢀCꢀ
CꢀN(py) torsion was near 180 degrees, while obviously the complexation of the nitrogens
resulted in cisꢀconfomation. Also, for the doubly substituted free ligand 2 the ferrocene rings
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optimized in staggered conformation instead of the effectively eclipsed one obtained for the
metallic complexes FeꢀAu2 5 and FeꢀPd2 6 (see Figures 3 and S1). For these compounds the
energetically most stable structures were obtained when the substituents were located at the 1
and 3’ carbon atoms of the ferrocene rings.
10
11
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Figure 3. Optimized structures for FeM and FeM2 compounds 5ꢀ8 and monometallic analogues
10 and 11. Hydrogens have been omitted for clarity.
It should be noted, that we tested the effect of the initial substitution sites (1,2’) also for
compounds 5 and 6. In the case of the cationic FeꢀAu2 complex 5 the ferrocene rings turned into
a staggered conformation resulting effectively again into 1,3’ substitution. In the case of the Feꢀ
Pd2 complex 6, the optimization resulted in a more “closed” structure, which had only a slightly
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higher total energy. It could be concluded therefore, that both structures are feasible for the Feꢀ
5
6
Pd2 compound 6 (see Figure S2 in the supplementary material).
7
8
9
10
11
12
13
14
15
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17
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20
21
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23
24
25
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60
Table 2. Selected geometrical and electronic properties of the FeM and FeM2 compounds 5ꢀ8, 10 and 11.
Gibbs free energies refer to formation reactions, where the corresponding metal ions react with chloride
ions and the corresponding free ligands. GAP is the energy difference of the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).
5
6
7
8
10
11
d(P=N)
1.650
2.256
1.616
2.285
1.659
2.258
1.623
2.283
1.654
2.258
ꢀ1811
1.618
2.283
ꢀ708
d(MꢀCl)
ꢁrG [kJmolꢀ1]
GAP [eV]
ν(P=N) [cmꢀ1]a
ꢀ1797b
3.34
ꢀ701b
4.55
ꢀ1824
2.91
ꢀ711
4.35
4.08
4.58
948b
953b
948
957
942
951
(930)
(930)
(940)
(923)
(940)
(929)
ν(MꢀCl) [cmꢀ1]a
371;384 343;358
(359 br) (331;351)
374;385
(382 br)
345;357
370;384
(384 br)
344;357
(331;349)
(331;347)
aIR spectra were simulated in aqueous solvent; (experimental values obtained from nujol mulls between
polyethylene sheets). bFor the doubly substituted complexes, the reaction energies were scaled per metal atom.
Table 2 lists the selected bond distances and electronic properties for the compounds. The
calculated IR spectra are also compared for selected stretching modes in Table 2. The trend in
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the shifts for the ν(MꢀCl) values is clear: the Au complexes 5, 7 and 10 exhibited larger
wavenumbers for the asymmetric and symmetric MꢀCl stretch than the corresponding palladium
complexes 6, 8 and 11 (see more details in the supporting information section Figures S3ꢀS8).
10
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This correlates very well in the optimized MꢀCl distances, since the AuꢀCl distances were about
0.03 Å shorter than the PdꢀCl distances. The ν(P=N) values in the complexes were found to be
very similar. The same trends are seen also in the experimental frequencies, even though the
computational absolute values are slightly overestimated, which is typical for the applied DFT
method.65
The formation energies of the compounds were also investigated (see Table 2 and supporting
information for details) and it was found that all the studied complexes are readily formed. The
larger HOMOꢀLUMO gap, on the other hand, indicates that palladium complexes 6 and 8 are
more stable and thus less reactive than the gold analogues 5 and 7. This is in accordance with our
previous observation for gold(III) coordination compounds containing iminophosphorane
ligands.56 Interestingly, the gold complex 10 does not show similar reduction in the HOMOꢀ
LUMO gap, indicating that the ferrocene motif can increase the reactivity of the compounds and
that the ferrocene moiety has a larger effect on gold complexes that on palladium ones.
Experimentally 10 is the most unstable compound of the series most likely due to a
cyclometallation process in DMSO. However, this cyclometallation process was not in the scope
of our computational studies, and therefore the energetics could not reveal this particular detail
of the relative stability.
2. Biological Activity
2.1. Antiproliferative Studies
The antiproliferative properties of the new ligands and heterometallic complexes and of ligand
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Ph3P=NꢀCH2ꢀ2ꢀNC5H4 (IMꢀPPh3) 961 and monometallic complexes [(Ph3P=NꢀCH2ꢀ2ꢀ
NC5H4)AuCl2]ClO4 1061 and [(Ph3P=NꢀCH2ꢀ2ꢀNC5H4)PdCl2] 11 (figure 1) were assayed by
monitoring their ability to inhibit cell growth using the MTT assay (see Experimental Section).
Cytotoxic activity of the compounds was determined as described in the Experimental Section in
the human ovarian cancer A2780 cell line, and its cisplatin resistant variant (A2780cisR), as well
as in the human breast cancer cell line MCF7, in comparison to cisplatin. The results are
summarized in Table 3. In general the ferroceneꢀbased ligands are poorly cytotoxic in all tested
cell lines and this may be caused by their poor solubility in aqueous solution. Interestingly,
ligands 1ꢀ3 incorporating the ferrocenylꢀphosphanes are considerably more cytotoxic than the
purely organic ligand 9 containing triphenylphosphane. This is in agreement with the enhanced
antripoliferative properties for a vast majority of derivatives in which an aryl group is substituted
by the ferrocene motif.20,21 Concerning the dimetallic complexes derived from ligand 3 (based
on MPPF), the Pd(II) derivative 8 is markedly more cytotoxic than 3 and of the Au(III)
derivative 7. Moreover, such dimetallic complexes are less active than their monometallic IMꢀ
PPh3 analogues 10 (Au) and 11 (Pd) on the ovarian cancer cell lines A2780S/R. It must be noted,
that the reduced effect of the gold compound 10 may also be due to its reduced stability in
aqueous medium as explained in the previous section. Concerning the trimetallic M2Fe (M = Au
5, Pd 6) compounds with ligand 2 (based on DPPF), these complexex exhibit important cytotoxic
effects in the low micromolar range in all the studied cell lines. Notably, they are also
significantly more cytotoxic than cisplatin towards the resistant A2780R and MCF7 cell lines.
The trimetallic compounds also resulted to be significantly more cytotoxic than ligands 1 and 2
and more effective than the monometallic Au and Pd derivatives 10 and 11. In order to assess the
compounds selectivity for cancerous cells with respect to normal cell lines, the most cytotoxic
3
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heterometallic compounds were also screened for their antiproliferative effects on the nonꢀ
tumorigenic human embryonic kidney cells HEK293T. In most cases the cytotoxicity is
comparable for the cancerous and normal cell lines.
10
11
12
13
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Table 3. IC50 (ꢁM) of ligands 1-3 and 9, DPPF oxide and metal complexes 4ꢀ8, 10-11 and cisplatin in
human cell lines.a All compounds were dissolved in 1% of DMSO and diluted with water before addition
to cell culture medium for a 74 h incubation period. Cisplatin was dissolved in H2O.
A2780
40.3 ±5.2
5.3±0.8
A2780cisR
32.7 ±6.0
13.1±4.0
30.3 ±1.6
1.3 ±1.0
2.4±0.5
1.8±0.7
25.2±4.3
11.9±2.5
>50
MCF7
ꢀ
HEK-293T
45 ±5.0
10.5±4.0
>50
1
15.0±6.0
ꢀ
2
3
36.7 ±6.0
1.4 ±0.8
3.0±0.8
3.5±1.5
3.0±0.6
40.0±7.0
10.2±3.7
>100
1.2±0.6
4.0±0.8
5.6±0.3
32.2±5.0
15.0±3.2
>100
4
2.7±0.8
2.0±0.4
30.1±5.0
17.4±4
>100
5
6
7
8
9
19.0±1.1
7.1±1.7
8.0±2.4
6.2±0.3
30.0 ±3.8
25.0 ±4.3
>50
30.5±1.5
45.0±5.0
>50
10
11
DDPFꢀoxide
cisplatinb
113 ±72
1.9 ±0.6
85 ±38
35 ±7.0
20.0±3.0
11.0±2.9
aData are expressed as mean ± SD (n =4). bRef. 18.
Only the in the case of monometallic Pd compound 11 is markedly (ca. 7.5ꢀfold) more toxic on
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the cancerous cell lines (IC50 6ꢀ7 ꢀM) than on the nonꢀcancerous HEK293T cells (IC50 ca. 45
ꢀM). Finally, compound 4 was among the most cytotoxic in all the tested cell lines including the
7
8
9
ꢀ
HEK293T cells, and this may be attributed to the presence of the AuCl4 anion in the compound
10
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with a highly oxidative character leading to marked and unselective cell death. Such type of
goldꢀbased ionic compound being highly toxic also in the nonꢀtumorigenic cell line will not be
certainly selected for further biological screening.
2.2. Reactivity with biomolecules
Interactions with plasmid DNA
Since DNA replication is a key event for cell division, it is among critically important targets
in cancer chemotherapy. Most cytotoxic platinum drugs form strong covalent bonds with the
DNA bases.66 However, a variety of platinum compounds act as DNA intercalators upon
coordination to the appropriate ancillary ligands.67 There are also reports on palladium
derivatives interacting with DNA in covalent68,69 and nonꢀcovalent fashions.70,71 Conversely,
most goldꢀbased compounds do not display a strong interaction with DNA.10,11
Thus, we performed agarose gel electrophoresis studies to unravel the effects of the new
heterometallic compounds 5ꢀ8 and 10, 11 on plasmid (pBR322) DNA (Fig. 4). This plasmid has
two main forms: OC (open circular or relaxed form, Form II) and CCC (covalently closed or
supercoiled form, Form I). Changes in electrophoretic mobility of both forms are usually taken
as evidence of metalꢀDNA binding. Generally, the larger the retardation of supercoiled DNA
(CCC, Form I), the greater the DNA unwinding produced by the drug.72 Binding of cisplatin to
plasmid DNA, for instance results in a decrease in mobility of the CCC form and an increase in
mobility of the OC form (see lanes aꢀd for cisplatin in Fig. 4).
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Molecular
Marker
DNA
Cisplatin
Pd2-Fe (6)
Pd –Pd (8)
a
b
c
d
a
b
c
d
a
b
c
d
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OC
CCC
Au2-Fe (5)
Au-Fe (7)
a
b
c
d
a
b
c
d
OC
CCC
Fig. 4. Electrophoresis mobility shift assays for cisplatin and compounds 5-8 and 10, 11 (see
Experimental for details). DNA refers to untreated plasmid pBR322. a, b, c and d correspond to
metal/DNAbp ratios of 0.25, 0.5, 1.0 and 2.0 respectively.
Treatment with increasing amounts of Au(III) compounds with IM ligands containing ferrocene
5, and 7 and the Au(III) compound with an IMꢀPPh3 ligand 10 do not affect the mobility of the
fasterꢀrunning supercoiled form (Form I) even at the highest molar ratios (d). This is also in
accordance with previously reported results on organogold(III) compounds containing
iminophosphorane ligands did not interact with DNA.55
Conversely, the Pd(II) compounds with IM ligands containing ferrocene 8 or triphenylphosphine
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9
11 have a similar behaviour to that of a coordination iminophosphorane palladium complex
described by us [PdCl2(TPA=NꢀC(O)ꢀ2ꢀNC5H4)].56 In fact, these compounds significantly
modify the electrophoretic mobility and the retardation of the fasterꢀrunning supercoiled form
(Form I or CCC) and an increased electrophoretic mobility of form II (OC) was clearly observed
for 6, 8 and 11 at lower ratios than those for cisplatin. Moreover, the Pd complexes 8 and 11 are
able to induce a coalescence of the two plasmid forms, an effect that has already been described
for cisplatin, but at higher drug/DNAbp ratios.73 The presence of a coalescence point indicates a
strong unwinding of the pBR322 plasmid DNA for compounds 8 and 11. The trimetallic FeꢀPd2
compound 6 (with ligand 2 derived from DPPF) also shows a coalescence point at the lowest
metal/DNA ratio (0.25, line a), most likely followed by a change of conformation of plasmid
DNA from negative supercoil to positive supercoil at the highest metal compound/DNAbp ratios.
Overall, our results indicates an interaction of the new iminophosphorane palladium complexes
mono and heterometallic (FeꢀPd) with DNA stronger than that observed for cisplatin.
10
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Inhibition of PARP-1
As previously mentioned the zinc finger protein PARPꢀ1 has been recently identified as a
possible taget for Au(III) complexes in vitro.18,19 Information on the reactivity of the gold
complexes with the PARPꢀ1 zincꢀfinger domain was also obtained by highꢀresolution mass
spectrometry, and an excellent correlation between PARPꢀ1 inhibition in protein extracts and the
ability of the complexes to bind to the zinc finger motif (in competition with zinc) was
established.18 Thus, in order to further investigate the mechanisms of action of the new
heteronuclear complexes, we tested the mononuclear compounds 5 and 6 for their PARPꢀ1
inhibition properties in vitro as reported in the Experimental section. The obtained preliminary
results showed that the trinuclear AuꢀFe compound 5 is a potent PARPꢀ1 inhibitor with an IC50 =
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1± 0.5 ꢀM, while the trinuclear PdꢀFe analogue 6 is much less effective with an IC50 = 17± 0.8
ꢀM.
7
8
9
Interactions with HSA
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Human serum albumin (HSA) is the most abundant carrier protein in plasma and is able to bind a
variety of substrates including metal cations, hormones and most therapeutic drugs. It has been
demonstrated that the distribution, the free concentration and the metabolism of various drugs
can be significantly altered as a result of their binding to the protein.74 HSA possesses three
fluorophores, these being tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues, with
Trp214 being the major contributor to the intrinsic fluorescence of HSA. This Trp fluorescence
is sensitive to the environment and binding of substrates, as well as changes in conformation that
can result in quenching (either dynamic or static). Thus, the fluorescence spectra of HSA in the
presence of increasing amounts of the representative compounds 5, 7 and 8 and cisplatin were
recorded in the range of 300ꢀ450 nm upon excitation of the tryptophan residue at 295 nm (Fig.
5). The compounds caused a concentration dependent quenching of fluorescence without
changing the emission maximum or shape of the peaks (5, 7, 8), as seen in Fig. 5 (A) for
compound 7. All this data indicates an interaction of the heterometallic compounds with HSA.
The fluorescence data was analyzed by the SternꢀVolmer equation. While a linear SternꢀVolmer
plot is indicative of a single quenching mechanism, either dynamic or static, the positive
deviation observed in the plots of F0/F versus [Q] of our compounds (Fig. 5) suggests the
presence of different binding sites in the protein.75 Of note, a similar behavior was observed in
the case of coordination iminophosphorane complexes of d8 metals for which we also reported a
concentration dependent fluorescence quenching.57,58 In this graph higher quenching by the
iminophosphorane complexes was observed compared to that of cisplatin under the chosen
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conditions, most likely due to the faster reactivity of our compounds with HSA compared to
cisplatin.
7
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9
350000
A
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300000
250000
200000
150000
100000
50000
0
300
350
400
450
Wavelength (nm)
B
12
10
8
5
7
8
6
4
Cisplatin
2
0
-2E-05 1.4E-18 2E-05
4E-05
6E-05
8E-05 0.0001
[Q] (M)
Figure 5. (a) Fluorescence titration curve of HSA with compound 7. Arrow indicates the increase of
quencher concentration (10ꢀ100 ꢀM). (b) SternꢀVolmer plot for HSA fluorescence quenching observed
with compounds 5, 7, 8 and cisplatin.
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In the case of [MCl2(TPA=NꢀC(O)ꢀ2ꢀNC5H4)] (M = Pd, Pt) isothermal titration calorimetry
(ITC)57 showed two different binding interactions which explained the lack of linearity observed
in the fluorescence quenching studies, as the SternꢀVolmer method assumes all binding sites to
be equivalent. We believe that a similar reactivity takes place for the iminophosphorane
compounds described here.
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CONCLUSIONS
In conclusion we have prepared a series of coordination iminophosphorane complexes of
gold(III) and palladium(II) derived from ferrocenyl phosphanes. The new compounds are
markedly more stable than the ferrocenyl phosphane ligands in DMSO or aqueous solution, and
more stable than their monometallic analogues derived from PPh3 [(AuCl2(Ph3P=NꢀCH2ꢀ2ꢀ
NC5H4)]ClO4 10 and [PdCl2{NꢀCH2ꢀ2ꢀNC5H4}] 11. The most stable trimetallic M2Fe (M = Au,
Pd) derivatives (5 and 6) exhibit important cytotoxic effects in the low micromolar range in all
the cancer cells studied and are more cytotoxic than their corresponding monometallic fragments
(ligand 2 containing ferrocene and monometallic analogues 10 and 11) also indicating a possible
synergistic antiproliferative effect of the two different metals. Importantly, these complexes were
significantly more cytotoxic than cisplatin in the resistant A2780R and MCF7 cell lines
supporting the idea of different mechanisms of action than cisplatin.
It is worth mentioning that heterometallic trinuclear complexes have already been described by
some of us to be more stable than their mononuclear and dinuclear precursors, including goldꢀ
titanium complexes.24,25 Moreover, improved biological properties of multinuclear complexes
with respect to mononuclear compounds have been reported,26 which could be related to
favorable modulation of the stability, solubility, and/or lipophilicity of the organometallic
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scaffolds. Therefore, the results described herein support the idea that there is an advantage in
synthesizing heteronuclear complexes as anticancer agents.
7
8
9
When the reactivity of the new heteronuclear complexes was tested with plasmid DNA the Pd(II)
derivatives showed strong interactions with nucleic acids, while the Au(III) analogues were
practically ineffective. Instead, the Au(III) complexes resulted to be good inhibitors of the zincꢀ
finger protein PARPꢀ1, a possible target enzyme for anticancer metal compounds, while the
Pd(II) complexes were markedly less potent. In general, although further studies are necessary to
validate the mechanisms of biological action of this new series of heteronuclear metal
compounds, our results support the idea of different reactivity of the complexes with the
investigated biomolecules depending on the metal ion and not only on the ligand set.
10
11
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EXPERIMENTAL
All manipulations involving airꢀfree syntheses were performed using standard Schlenkꢀline
techniques under a nitrogen atmosphere or in a gloveꢀbox MBraun MOD System. Solvents were
purified by use of a PureSolv purification unit from Innovative Technology, Inc. The substrates
[(CpꢀPPh2)2Fe] 1,1'ꢀBisꢀdiphenylphosphinoꢀferrocene (DPPF), K[AuCl4] and [PdCl2(COD)]
were purchased from Strem chemicals and used without further purification. [(CpꢀPPh2)Fe(Cp)]
diphenylphosphinoꢀferrocene (MPPF),60 (Ph3P
=
NꢀCH2ꢀ2ꢀNC5H4)61
and [(Ph3P=NꢀCH2ꢀ2ꢀ
NC5H4)AuCl2]ClO461 were prepared by reported methods. Purity of the compounds is based on
elemental analysis and in all cases is ≥95%. Elemental analyses were performed on a Perkin
Elmer 2400 CHNS/O Analyzer, Series II. NMR spectra were recorded in a Bruker AV400 (1H
13
31
NMR at 400MHz, C NMR at 100.6 MHz, P NMR at 161.9 MHz). Chemical shifts (δ) are
given in ppm using CDCl3 or d6ꢀDMSO as solvent, unless otherwise stated. 1H and 13C chemical
shifts were measured relative to solvent peaks considering TMS = 0 ppm; 31P{1H} was
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externally referenced to H3PO4 (85%). Infrared spectra (4000ꢀ250 cmꢀ1) were recorded on a
Nicolet 6700 FTꢀIR spectrophotometer from nujol mulls between polyethylene sheets. Mass
spectra (ESI) were performed on an Agilent Analyzer or a Bruker Analyzer. Conductivity was
measured in an OAKTON pH/conductivity meter in CH3CN solutions (10ꢀ3M). Xꢀray collection
was performed at room temperature on a Kappa CCD diffractometer using graphite
monochromated MoꢀKα radiation (λ=0.71073 Å). Electrophoresis experiments were carried out
in a BioꢀRad Mini subꢀcell GT horizontal electrophoresis system connected to a BioꢀRad Power
Pac 300 power supply. Photographs of the gels were taken with an Alpha Innotech FluorChem
8900 camera. Fluorescence intensity measurements were carried out on a PTI QMꢀ4/206 SE
Spectrofluorometer (PTI, Birmingham, NJ) with right angle detection of fluorescence using a 1
cm path length quartz cuvette. Circular Dichroism spectra were recorded using a Chirascan CD
Spectrometer equipped with a thermostated cuvette holder. UVꢀvisible spectra have been
recorded using a on a PerkinꢀElmer Lambda 20 Bio spectrophotometer.
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Synthesis
[{Cp-P(Ph2)=N-Ph}2Fe] (1). A solution of DPPF (0.554 g, 1.0 mmol) in 15 mL of degassed
CH2Cl2 and a solution of phenyl azide (0.238 g, 2.0 mmol) in 5 mL of degassed CH2Cl2 were
mixed and refluxed for 3 hours and 40 minutes under nitrogen. After cooling the mixture to RT
the solvent was evaporated to ca 2 mL and by addition of 6 mL of dry Et2O and 4 mL of dry nꢀ
hexane a precipitate formed which was separated by filtration under nitrogen. 1 was obtained as
dark orange solid that was used without further purification. Yield: 0.483 g (61.3%). Anal. Calcd
for [C46H38FeN2P2].0.6CH2Cl2 (787.57): C, 71.07; H, 5.02; N, 3.56. Found: C, 71.22; H, 4.90; N,
3.46. MS (ESI+) [m/z]: 737.1 [M + H]. 31P{1H} NMR (CDCl3): δ 2.7. 1HꢀNMR (CDCl3): δ 4.04
(4H, s, Cp), δ 4.53 (4H, s, Cp), δ 6.66 (2H, t, J = 6.9 Hz, Hpara, NAr), δ 6.75 (4H, d, J = 7.4 Hz,
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Hortho, NAr), δ 7.02 (4H, t, J = 7.2 Hz, Hmeta, NAr), δ 7.43 (8H, d, J = 5.5 Hz, Hmeta, PPh2), δ 7.51
5
6
13
(4H, d, J = 6.8 Hz, Hpara, PPh2), δ 7.64 (8H, dd, J = 11.8, 7.8 Hz, Hortho, PPh2). C{1H} NMR
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8
9
(CDCl3) : δ 73.8 (d, J = 9.4 Hz, Cp), δ 74.4 (d, J = 11.3 Hz, Cp), δ 117.3 (s, Cpara, NAr), δ 123.4
(d, J = 17.9 Hz, Cortho, NAr), δ 128.4 (d, J = 12.1 Hz, Cmeta, PPh2), δ 128.6 (s, Cmeta, NAr), δ 131.5
(s, Cpara, PPh2), δ 132.1 (d, J = 9.7 Hz, Cortho, PPh2), δ 132.6 (s, PC), δ 151.1 (s, NC). IR (cmꢀ1): ν
1264 (P=N).
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[{Cp-P(Ph2)=N-CH2-2-NC5H4}2Fe] (2). A solution of DPPF (0.886 g, 1.6 mmol) in 15 mL of
degassed CH2Cl2 and a solution of 2ꢀmethylpyridine azide (0.968 g, 3.7 mmol) in 5 mL of
degassed CH2Cl2 were mixed for 3.5 h at RT under nitrogen. The solvent was then evaporated to
ca 2 mL and by addition of 6 mL of dry Et2O a precipitate formed which was separated by
filtration under nitrogen. 2 was obtained as brown solid that was used without further
purification. Yield: 0.577 g (66.7%). Anal. Calcd for C46H40FeN4P2 (766.6334): C, 72.07; H,
31
5.26; N, 7.31. Found: C, 71.81 ; H, 5.14 ; N, 7.12. MS (ESI+) [m/z]: 767.2151 [M+H]. P{1H}
(CDCl3): δ 11.9. 1H NMR (CDCl3): δ 4.18 (4H, s, Cp), δ 4.48 (4H, s, Cp), δ 4.52 (4H, s, CH2), δ
7.07 (2H, t, J = 6.0 Hz, NCHCH), δ 7.38 (8H, d, J = 5.9 Hz, Hmeta), δ 7.47 (4H, d, J = 6.8 Hz,
Hpara), δ 7.57 (8H, dd, J = 11.5, 7.5 Hz, Hortho), δ 7.69 (2H, t, J = 7.0 Hz, NC(CH2)CHCH), δ 8.00
13
(2H, d, J = 7.3 Hz, NC(CH2)CH), δ 8.44 (2H, d, J = 4.4 Hz, NCH). C{1H} NMR (CDCl3): δ
51.3 (s, CH2), δ 73.6 (d, J = 9.4 Hz, Cp), δ 120.8 (s, NCHCH), δ 121.4 (s, NC(CH2)CH), δ 128.1
(d, J = 10.5 Hz, Cmeta), δ 131.1 (s, Cpara), δ 132.0 (d, J = 9.0 Hz, Cortho), δ 136.3 (s,
NC(CH2)CHCH), δ 148.2 (s, NCH) IR (cmꢀ1): ν 1257 (P=N).
[{Cp-P(Ph2)=N-CH2-2-NC5H4}Fe(Cp)] (3). A solution of [(CpꢀPPh2)Fe(Cp)] (0.738 g, 2 mmol)
in 10 mL of degassed CH2Cl2 and a solution of 2ꢀmethylpyridine azide (0.2968 g, 2 mmol) in 5
mL of degassed CH2Cl2 were mixed for 3 hours and 30 minutes at RT under nitrogen. The
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solvent was then evaporated to ca 2 mL and by addition of 10 mL of dry Et2O a precipitate
5
6
formed which was separated by filtration under nitrogen. 3 was obtained as brown solid that was
7
8
9
used without further purification. Yield: 0.745
g
(76.8%). Anal. Calcd for
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[C28H25FeN2P]0.1CH2Cl2 (484.83): C, 69.61; H, 5.24; N, 5.78. Found: C, 69.52; H, 5.23; N,
5.77. MS (ESI+) [m/z]: 477.1 [M+H]. 31P{1H} NMR (CDCl3): δ 13.1. 1H NMR (CDCl3): δ 4.16
(5H, s, Cp), δ 4.33 (2H, d, J = 1.6 Hz, Cp), δ 4.45 (2H, d, J = 1.6 Hz, Cp), δ 4.64 (2H, d, J = 18.0
Hz, CH2), δ 7.10 (1H, t, J = 6.0 Hz, NCHCH), δ 7.40ꢀ7.46 (4H, m, Hmeta), δ 7.50 (2H, dd, J =
7.2, 1.5 Hz, Hpara), δ 7.67ꢀ7.71 (4H, m, Hortho), δ 7.72ꢀ7.74 (1H, m, NC(CH2)CHCH), δ 8.06 (1H,
d, J = 7.9 Hz, NC(CH2)CH), δ 8.46 (1H, d, J = 4.2 Hz, NCH). 13C{1H} NMR (CDCl3): δ 51.4 (s,
CH2), δ 69.6 (s, Cp), δ 70.98 (d, J = 8.7 Hz, Cp), δ 72.7 (d, J = 10.9 Hz, Cp), δ 120.7 (s,
NCHCH), δ 121.6 (s, NC(CH2)CH), δ 128.1 (d, J = 11.6 Hz, Cmeta), δ 131.1 (s, Cpara), 132.2 (d, J
= 9.1 Hz, Cortho), δ 136.3 (s, NC(CH2)CHCH), δ 148.1 (s, NCH). IR (cmꢀ1): ν 1303 (P=N).
[{Cp-P(Ph2)=NH-Ph}2Fe](AuCl4)2 4. A solution of H[AuCl4] (0.158 g, 0.4 mmol) in CH3CN (7
mL) was added over a solution of 1 (0.1472 g, 0.2 mmol) in degassed dry CH2Cl2. After 20 min
stirring at RT the reaction mixture was concentrated under vacuum to ca. 2 mL. The addition of
8 mL of Et2O afforded dark brown oil, which was washed with water (2 x 5 mL). 4 was obtained
as
a
light brown solid. Yield: 0.2053
g
(66.5%). Anal. Calcd for
[C46H40Au2Cl8FeN2P2]1.5CH2Cl2 (1543.56): C, 36.96; H, 2.81; N, 1.81. Found: C, 36.75; H,
2.75; N, 1.82. MS (ESI+) [m/z]: 737.1 [MꢀH]+. 31P{1H} NMR (d6ꢀDMSO): δ 36.3. 1HꢀNMR (d6ꢀ
DMSO): δ 4.58 (4H, s, Cp), δ 4.73 (4H, s, Cp), δ 6.89 (4H, d, J = 7.8 Hz, Hortho, NAr), δ 7.03
(2H, t, J = 7.2 Hz, Hpara, NAr), δ 7.20 (4H, t, J = 7.4 Hz, Hmeta, NAr), δ 7.78ꢀ7.84 (8H, m, Hmeta
,
13
PPh2), δ 7.88ꢀ7.91 (8H, m, Hortho, PPh2), δ 7.95 (4H, t, J = 6.8 Hz, Hpara, PPh2). C{1H} NMR
(d6ꢀDMSO): δ 76.2 (d, J = 13.4 Hz, Cp), δ 77.0 (d, J = 11.1 Hz, Cp), δ 121.9 (d, J = 5.7 Hz,
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Cortho, NAr), δ 124.7 (s, Cpara, NAr), δ 130.1 (s, Cmeta, NAr), δ 130.7 (d, J = 13.7 Hz, Cmeta, PPh2),
δ 133.4 (d, J = 11.5 Hz, Cortho, PPh2), δ 136.0 (s, Cpara, PPh2). IR (cmꢀ1): ν 1282, 963 (P=N).
7
Conductivity Λ (MeCN) = 221.3 ꢀS/cm (2 ions 1+/1ꢀ).
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[({Cp-P(Ph2)=N-CH2-2-NC5H4}AuCl2)2Fe](ClO4)2 (5). To a solution of K[AuCl4] (0.113 g; 0.3
mmol) in CH3CN (10 mL), AgClO4 (0.136 g, 0.66 mmol) was added. The resulting yellow
reaction mixture was stirred at RT (protected from the light) during 30 min and subsequently
filtered through a celite pad (to remove the AgCl formed). To the resulting yellow solution 2
(0.113 g, 0.14 mmol) was added. KClO4 precipitated immediately in the reaction media and after
1 h stirring at RT the reaction mixture was filtered through a celite pad. The resulting brown
solution was concentrated under vacuum to ca. 2 mL. The addition of 2 mL of EtOH and 8 mL
of Et2O afforded 5 as a black solid that was filtered, dried under vacuum and used without
further
purification.
Yield:
0.1088
g
(48.2%).
Anal.
Calcd
for
[C46H40Au2Cl6FeN4O8P2]1.5C4H10O (1612.46): C, 38.73; H, 3.44; N, 3.47. Found: C, 39.06; H,
3.10; N, 3.69. MS (ESI+) [m/z]: only ligand observed. 31P{1H} NMR (d6ꢀDMSO): δ 41.7 ppm.
1H NMR (d6ꢀDMSO): δ 4.31 (4H, d, J = 10.4 Hz, CH2), δ 4.69 (4H, s, Cp), δ 4.88 (4H, s, Cp), δ
7.44 (2H, d, J = 7.7 Hz, NCHCH), δ 7.48 (2H, s, NC(CH2)CH), δ 7.67ꢀ7.77 (12H, m, Hmeta
+
Hpara), δ 7.83ꢀ7.87 (8H, m, Hortho), δ 7.87ꢀ7.90 (2H, m, NC(CH2)CHCH), 8.51 (2H, d, J = 5.1 Hz,
13
NCH). C{1H} NMR (d6ꢀDMSO): δ 46.1 (s, CH2), δ 75.4 (d, J = 13.5 Hz, Cp), δ 76.8 (d, J =
11.0 Hz, Cp), δ 123.5 (s, NC(CH2)CH), δ 124.1 (s, NCHCH), δ 130.3 (d, J = 13.2 Hz, Cmeta), δ
133.2 (d, J = 11.5 Hz, Cortho), δ 135.9 (s, Cpara), δ 139.6 (s, NC(CH2)CHCH), δ 149.4 (s, NCH), δ
ꢀ
156.2 (s, NC). IR (cmꢀ1): ν 382 (AuꢀCl); 1071 (v br) and 623 (ClO4 ); 1291, 940 (P=N).
Conductivity Λ (MeCN) = 412.8 ꢀS/cm (3 ions, 12+/2ꢀ).
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[({Cp-P(Ph2)=N-CH2-2-NC5H4}PdCl2)2Fe] (6). A solution of [PdCl2(COD)] (0.074 g, 0.26
mmol) in 5 mL of degassed dry CH2Cl2 was added over a solution of 2 (0.0858 g, 0.13 mmol) in
degassed dry CH2Cl2. The reaction mixture was stirred for one hour at RT after which the
solvent was reduced to ca 2 mL. Addition of Et2O (15 mL) afforded 6 as a yellow solid that was
collected by filtration and dried under vacuum. Yield: 0.072 g (49.5%). Anal. Calcd for
C46H40Cl4FeN4P2Pd2 (1121.29): C, 49.27; H, 3.60; N, 5.00. Found: C, 49.56; H, 3.83; N, 3.27.
MS (ESI+) [m/z]: 1084.9214 [MꢀCl]+. 31P{1H} NMR (CDCl3): δ 40.8 ppm. 1H NMR (CDCl3): δ
4.20 (4H, s, Cp), δ 4.35 (4H, d, J = 10.6 Hz, CH2), δ 5.00 (4H, s, Cp), δ 7.23 (1H, d, J = 6.7 Hz,
NC(CH2)CH), δ 7.39 (1H, t, J = 5.9 Hz, NCHCH), δ 7.57 (4H, d, J = 4.4 Hz, Hmeta), δ 7.61ꢀ7.67
(2H, m, Hpara), δ 7.74 (4H, dd, J = 12.4, 7.5 Hz, Hortho), δ 7.78 (1H, t, J = 8.4 Hz,
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NC(CH2)CHCH), δ 9.16 (1H, d, J = 5.3 Hz, NCH). C{1H} NMR (CDCl3): δ 58.7 (s, CH2), δ
73.9 (s, Cp), δ 76.9 (s, Cp), δ 119.7 (s, NC(CH2)CH), δ 123.5 (s, NCHCH), δ 129.0 (d, J = 12.7
Hz, Cmeta), δ 133.0 (d, J = 10.1 Hz, Cortho), δ 133.3 (s, Cpara), δ 138.9 (s, NC(CH2)CHCH), δ 150.7
(s, NCH), δ 164.4 (d, J = 12.1 Hz, NC(CH2)). IR (cmꢀ1): ν 1287, 923 (P=N); 349, 331 (PdꢀCl).
Conductivity Λ (MeCN) = 24.6 ꢀs/cm (neutral).
[{(Cp-P(Ph2)=N-CH2-2-NC5H4)AuCl2}Fe(Cp)]ClO4 (7). To a solution of K[AuCl4] (0.113 g;
0.3 mmol) in CH3CN (10 mL), AgClO4 (0.068 g, 0.33 mmol) was added. The resulting yellow
reaction mixture was stirred at RT (protected from the light) during 30 min and subsequently
filtered through a celite pad (to remove the AgCl formed). To the resulting yellow solution 3
(0.071 g, 0.15 mmol) was added. KClO4 precipitated immediately in the reaction media and after
1 h stirring at RT the reaction mixture was filtered through a celite pad. The solvent was reduced
to ca. 2 mL and a precipitate was obtained by addition of 2 mL of EtOH and 8 mL of Et2O. The
precipitate was filtered off and dried under vacuum to afford 7 as a dark brown solid. Yield:
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0.100 g (71.8%). Anal. Calcd for [C28H25FeN2PCl3Au]CH2Cl2 (928.58): C, 37.51; H, 2.93; N,
3.02. Found: C, 37.82; H, 2.96; N, 3.12. MS (ESI+) [m/z]: 707.0361[(M ꢀ Cl) – ClO4]2+. 31P{1H}
7
8
9
1
NMR (d6ꢀDMSO): δ 43.1. HꢀNMR (DMSO): δ 4.26 (5H, s, Cp), δ 4.39 (2H, s, CH2), δ 4.71
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(2H, s, Cp), δ 4.90 (2H, s, Cp), δ 7.42 (1H, s, NCHCH), δ 7.44ꢀ7.47 (6H, m, Hmeta + Hpara), δ
7.49 (1H, s NC(CH2)CH), δ 7.65ꢀ7.74 (4H, m, Hortho), δ 7.90 (1H, s, NC(CH2)CHCH), δ 8.62
(1H, d, J = 4.4 Hz, NCH). 13C{1H} NMR (d6ꢀDMSO): δ 45.6 (s, CH2), δ 71.0 (s, Cp), δ 73.6 (s,
Cp), δ 74.6 (s, Cp), δ 123.1 (s, NC(CH2)CH), δ 124.0 (s, NCHCH), δ 128.8 (d, J = 12.4 Hz,
Cmeta), δ 131.3 (d, J = 10.9 Hz, Cortho), δ 131.8 (s, Cpara), δ 138.1 (NC(CH2)CHCH), δ 148.9 (s,
ꢀ
NCH). IR (cmꢀ1): ν 384 (AuꢀCl); 1084 (v br) and 624 (ClO4 ); 1289, 940 (P=N). Conductivity Λ
(MeCN) =190.9 ꢀs (2 ions 1+/1ꢀ).
[{(Cp-P(Ph2)=N-CH2-2-NC5H4)PdCl2}Fe(Cp)] (8). A solution of [PdCl2(COD)] (0.057 g, 0.20
mmol) in 5 mL of degassed dry CH2Cl2 was added over a solution of 3 (0.103, 0.20 mmol) in
degassed dry CH2Cl2. The reaction mixture was stirred for one hour at RT after which the
solvent was reduced to ca 2 mL. Addition of Et2O (15 mL) afforded 6 as a yellow solid that was
collected by filtration and dried under vacuum. Yield: 0.0806 g (61.8%). Anal. Calcd for
C28H25FeN2PCl2Pd (653.66): C, 51.44; H, 3.86; N, 4.29. Found: C, 50.94; H, 3.96; N, 4.50. MS
31
1
(ESI+) [m/z]: 653.1 [M]. P{1H} NMR (CDCl3): δ 41.0 ppm. H NMR (CDCl3): 4.03 (5H, s,
Cp), δ 4.19 (2H, d, J = 8.1 Hz, CH2), δ 4.34 (2H, s, Cp), δ 4.59 (2H, s, Cp), δ 7.11 (1H, d, J = 7.6
Hz, NC(CH2)CH), 7.26 (1H, t, J = 6.5 Hz, NCHCH), δ 7.58ꢀ7.64 (4H, m, Hmeta), 7.64ꢀ7.70 (2H,
m, Hpara), δ 7.74 (1H, t, J = 7.1 Hz, NC(CH2)CHCH), δ 8.13 (4H, dd, J = 12.7, 7.1 Hz, Hortho), δ
9.17 (1H, d, J = 5.3 Hz, NCH). 13C{1H} NMR (CDCl3): δ 58.1 (s, CH2), δ 70.2 (s, Cp), δ 72.4 (d,
J = 9.8 Hz, Cp), δ 73.7 (d, J = 11.3 Hz, Cp), δ 119.0 (s, NC(CH2)CH), δ 122.7 (s, NCHCH), δ
128.5 (d, J = 13.0 Hz, Cmeta), δ 133.0 (d, J = 10.1 Hz, Cortho), δ 138.1 (s, NC(CH2)CHCH), δ
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150.1 (s, NCH). IR (cmꢀ1): ν 1286, 929 (P=N); 347, 331 (PdꢀCl). Conductivity Λ (MeCN) = 17.1
ꢀS/cm (neutral).
7
8
9
[(Ph3P=N-CH2-2-NC5H4)PdCl2] (11). A solution of [PdCl2(COD)] (0.057 g, 0.2 mmol) in 7 mL
of degassed dry CH2Cl2 was added over a solution of (Ph3P=NꢀCH2ꢀ2ꢀNC5H4) 9 (0.074 g, 0.2
mmol) in degassed dry CH2Cl2. The reaction mixture was stirred for one hour at RT after which
the solvent was reduced to ca 2 mL. Addition of Et2O (8 mL) afforded 11 as a yellow solid that
was collected by filtration and dried under vacuum. Yield: 0.0675 g (61.8%). Anal. Calcd for
C24H21N2PCl2Pd (545.74): C, 52.82; H, 3.88; N, 5.13. Found: C, 52.57; H, 3.94; N, 5.17. MS
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11
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1
(ESI+) [m/z]: 510.2 [MꢀCl]+. 31P{1H} NMR (CDCl3): δ 36.0 ppm. H NMR (CDCl3): δ 4.20
(2H, d, J = 5.9 Hz, CH2), δ 7.06 (1H, d, J = 7.7 Hz, NC(CH2)CH), δ 7.26 (1H, t, J = 6.4 Hz,
NCHCH), δ 7.57 (6H, td, J = 7.5, 3.3 Hz, Hmeta), δ 7.64 (3H, td, J = 7.2, 1.3 Hz, Hpara), δ 7.71
(1H, td, J = 7.7, 1.4 Hz, NC(CH2)CHCH), δ 7.94 (6H, dd, J = 12.3, 7.2 Hz, Hortho), δ 9.22 (1H, d,
J = 5.9 Hz, NCH). 13C{1H} NMR (CDCl3): δ 58.2 (s, CH2), δ 119.4 (s, NC(CH2)CH), δ 122.8 (s,
NCHCH), δ 128.7 (d, J = 12.6 Hz, Cmeta), δ 132.9 (d, J = 2.8 Hz, Cpara), δ 133.9 (d, J = 9.8 Hz,
Cortho), δ 138.0 (s, NC(CH2)CHCH), δ 150.7 (s, NCH), δ 165.0 (d, J = 17.7 Hz, NCNCH). IR
(cmꢀ1): ν 1287, 930 (P=N), 351, 331 (PdꢀCl). Conductivity Λ (MeCN) = 13.3 ꢀS/cm (neutral).
X-Ray crystallography
Single crystals of 8 and 11 (see details in Table S1 in SI) were mounted on a glass fiber in a
random orientation. Data collection was performed at RT on a Kappa CCD diffractometer using
graphite monochromated MoꢀKα radiation (λ=0.71073 Å). Space group assignments were based
on systematic absences, E statistics and successful refinement of the structures. The structures
were solved by direct methods with the aid of successive difference Fourier maps and were
refined using the SHELXTL 6.1 software package. All nonꢀhydrogen atoms were refined
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