Gold(i) thiolates containing amino acid moieties. Cytotoxicity and structure-activity relationship studies

Article abstract of DOI:10.1039/c4dt02299h

Several gold(i) complexes containing a thiolate ligand functionalised with several amino acid or peptide moieties of the type [Au(SPyCOR)(PPh₂R′)] (where R = OH, amino acid or dipeptide and R′ = Ph or Py) were prepared. These thiolate gold comp

Full text of DOI:10.1039/c4dt02299h

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Gold(I) thiolates containing amino acid moieties.
Cytotoxicity and structure–activity relationship
studies
Cite this: Dalton Trans., 2014, 43,
17054
Alejandro Gutiérrez,a Lucia Gracia-Fleta,b Isabel Marzo,b Carlos Cativiela,c
Antonio Lagunaa and M. Concepción Gimeno*a
Several gold(I) complexes containing a thiolate ligand functionalised with several amino acid or peptide
moieties of the type [Au(SPyCOR)(PPh2R’)] (where R = OH, amino acid or dipeptide and R’ = Ph or Py)
were prepared. These thiolate gold complexes bearing biological molecules possess potential use as anti-
tumor agents. Cytotoxicity assays in different tumour cell lines such as A549 (lung carcinoma), Jurkat
(T-cell leukaemia) and MiaPaca2 (pancreatic carcinoma) revealed that the complexes exhibit good anti-
proliferative activity, with IC50 values in the low micromolar range. Several structural modifications such as
in the type of phosphine, number of metal atoms and amino acid (type, stereochemistry and functionali-
sation) were carried out in order to establish the structure–activity relationship in this family of complexes,
which has led to the design of new and more potent cytotoxic complexes. Observations of different cel-
lular events after addition of the complexes indicated the possible mechanism of action or the biological
targets of this type of new gold(I) drug.
Received 28th July 2014,
Accepted 17th September 2014
DOI: 10.1039/c4dt02299h
www.rsc.org/dalton
dal, anti-HIV, or in the treatment of asthma or parasitic
diseases, among others, have been prepared.3 In addition to
Introduction
Metal-based drugs are an important class of compounds in the excellent antiproliferative activities displayed by some of
medicinal chemistry employed clinically for the treatment of these gold(I) and gold(III) compounds, they have attracted a lot
different diseases.1 The most representative example is cispla- of attention because it seems that these species exert their
tin,2 a Pt complex used successfully in testicular, ovarian and therapeutic effect by a different mechanism, and over different
other types of cancer. Although today this anticancer drug targets in comparison with Pt drugs.
remains one of the most effective chemotherapeutic agents in
For this purpose, different approaches have been employed
clinical use, some drawbacks such as the lack of activity in order to prepare new gold complexes with antitumor activi-
against other types of cancer, development of resistance and ties, including the synthesis of (a) auranofin analogues
undesired toxic side-effects make necessary the design of new (a gold(I) phosphine complex with a thioglucose ligand),4
compounds that overcome these limitations and possess a (b) gold(I) phosphine or diphosphine complexes,5 (c) gold(I)-
good pharmaceutical profile, as potency or selectivity. The use carbene derivatives,6 and (d) gold(III) compounds (Fig. 1).7
of different metal compounds with different biological pro-
perties and targets has emerged as an alternative strategy.
Taking into account all these facts and in view of the impor-
tance of gold(I) thiolate complexes, we believe it would be
Gold compounds have been employed since the last century interesting to prepare these gold-thiolate derivatives bearing in
for the treatment of rheumatoid arthritis and in the past few the skeleton different key biological molecules such as amino
decades some gold(I) and gold(III) complexes with promising acids and peptides. Although gold thiolate derivatives contain-
biological activities such as anticancer, antimicrobial, fungici- ing carbohydrates or DNA bases are well known, only a few
examples containing cysteine or glutathione species have been
reported.8
aDepartamento de Química Inorgánica, Instituto Síntesis Química y Catálisis
Homogénea (ISQCH), CSIC-Universidad de Zaragoza, E-50009 Zaragoza, Spain.
E-mail: gimeno@unizar.es
bDepartamento de Química Orgánica, Instituto Síntesis Química y Catálisis
Homogénea (ISQCH), CSIC-Universidad de Zaragoza, E-50009 Zaragoza, Spain
cDepartamento de Bioquímica y Biología Celular, Universidad de Zaragoza, E-50009
Zaragoza, Spain
In order to introduce the amino acid or peptide moieties in
the structure we chose the nicotinic acid thiolate, a biological
active molecule.9 Starting from the complex [Au(SPyCOOH)-
(PR3)], functionalisation of the carboxylic group with amino
acid methyl esters or oligopeptides was carried out. The latter
is an important class of biomolecules that constitute proteins
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Further studies have been carried out over a selection of
these complexes in order to know the mechanism of action. In
the last few years, some selenium containing enzymes like TrX
or GR, located at mitochondria, have been postulated as poss-
ible targets of gold(I) complexes, which seem to induce cell
death via apoptosis by the mitochondrial (intrinsic)
pathway.5a,7d,15 To confirm this, several studies on ROS pro-
duction, loss of mitochondrial membrane potential, cell and
nuclei morphology and TrX inhibition have been performed.
Results and discussion
Synthesis and spectroscopic characterization
Fig. 1 Examples of gold(I) and gold(III) complexes of types (a)–(d) with
anticancer activity.
We have previously reported the preparation of several gold(I)
complexes (1–6) obtained by coupling different amino esters
(glycine, alanine, valine, phenylalanine, methionine or
proline) with the free carboxylic acid of the complex [Au-
(SPyCOOH)(PPh3)] (see Scheme 1).14 This compound was pre-
pared by Nomiya et al. some years ago, who reported its anti-
microbial activity.16 The basic hydrolysis of the ester
complexes allowed access to the corresponding acid species
(7–12). Finally, coupling of isopropylamine to these com-
pounds afforded the corresponding amide derivatives (13–18).
In the present work, we report the biological activity of the
previously described complexes 1–18 and, in addition, we have
introduced different structural modifications in these families
of complexes with the purpose of obtaining new derivatives
with better activity, in order to establish some structure–
activity relationships (see Fig. 2).
and possess other different roles in the organism.10 We think
that the introduction of amino acids and peptides in our com-
plexes might decrease the undesired toxic side-effects (they are
biocompatible ligands) and they could serve as good carriers
to deliver the gold atom to the biological target.11 The major
problem for cancer chemotherapeutics is crossing the cell
membranes, and a strategy to overcome this is to make use of
the peptide-based delivery systems, which can transport small
peptides and peptide-like drugs to the cells. These peptide
transporters are over-expressed in some types of tumours, these
could be targeted by the gold-peptide compounds providing
them with more internalization and subsequent selectivity.12
Several studies on the biological activity of gold compounds
have revealed that the cytotoxicity is due mainly to the gold(I)
atom, and the ligands bonded to gold are important as carriers
to reach the biological target.3 Phosphine ligands are impor-
tant in the resulting potency of the final complex, and the thio-
late ligand could participate in ligand-exchange reactions with
other biomolecules in the organism.13 Taking this into
account, we have rationally designed these complexes consid-
ering these ideas and introducing more biologically compati-
ble fragments in the molecule with the aim of facilitating the
gold atom to easily reach its biological target.
In a previous study, we reported the synthesis of several
gold(I) phosphine complexes with nicotinic acid thiolate func-
tionalised with amino acids.14 Three families of complexes
(ester, acid and amide) were prepared. Here we report on the
antiproliferative activity studies, tested by the MTT viability
assay in different tumour human cell lines, which have shown
that the gold(I) complexes prepared possess good antiprolifera-
tive activity in the low micromolar range. Following these
results, we have introduced different structural modifications
in these families of complexes, such as (1) the type of phos-
phine, (2) the charge and number of gold atoms per molecule,
and (3) the number, type, stereochemistry or functionalisation
of the coupled amino acid, in order to evaluate changes in the
biological activity and thus can establish a structure–activity
relationship. This has led to the synthesis of the most active
complex of these families.
Scheme 1 Preparation of complexes 1–18 of the ester, acid and amide
families. (i) HCl·H-AA-OMe, PyBOP, DIPEA, MeCN, (ii) LiOH, MeOH and
(iii) iPrNH2, PyBOP, DIPEA, MeCN.
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35.9 ppm, a value similar to those observed for other thiolate
gold(I) phosphine complexes (35–37 ppm). The IR spectra
present, among others, absorptions for the free carboxylic acid
at around 3400–3049 (br, s), which is no longer observed for
complex 20, and for the latter the bands belonging to the new
amide bond at 3280 (br, w) and 1637 (s), and the ester at
1736 (s) appear. The mass spectra (ESI+) present for both com-
pounds the protonated molecular peak [M + H]+ (19) or with a
sodium cation [M + Na]+ (20), together with the association
fragment [M + AuPPh2Py]+ observed for 20.
Fig. 2 Selected modifications introduced in our complexes.
Synthesis of
a dinuclear gold derivative. The reaction
between the oxonium salt, [O(AuPPh3)3]BF4, and 2-mercapto
nicotinic acid yielded the desired dinuclear gold(I) complex
[Au2(SPyCOOH)(PPh3)2]BF4 (21). In this reaction, stoichio-
metric amounts of the oxonium salt must be used (2/3 equiv.)
to avoid mixtures due to the very reactive [AuPPh3]+ fragment
that could bind to other donor atoms of the molecules such as
N or O (Scheme 3).
In complex 21 two gold(I) atoms are bonded to the sulfur
centre, and it presents great interest because on the one hand,
we could expect an increase in the cytotoxicity of the final
complex due to the introduction of another gold(I) atom in the
molecule, and on the other hand, this compound is charged
and presents high lipophilicity that could help the complex to
cross the cell membranes and/or accumulate in the
mitochondria.
Synthesis of the complexes with a different type of phos-
phine. The complex [Au(SPyCOOH)(PPh2Py)] (19) can be
readily prepared by reaction of [AuCl(PPh2Py)] with 2-mercap-
toniconitic acid in acetone in the presence of K2CO3, yielding
the pure product in high yield. This complex presents a car-
boxylic moiety on the thiolate ligand that can be used for coup-
ling amino acid esters such as L-alanine methyl ester, to give
the derivative [Au(SPyCOAlaOMe)(PPh2Py)] (20) that was
obtained in the pure form by chromatography (see Scheme 2).
Complexes 19 and 20 are analogous to complexes
[Au(SPyCOOH)(PPh3)] and 2 reported previously,14 except for
the type of phosphine ligand bonded to the gold atom, for
which the 2-diphenylphosphino pyridine ligand is employed
instead of triphenylphosphine. Both phosphines exhibit
similar electronic and steric properties, although PPh2Py pos-
sesses a pyridine ring, which could further bind to other
metals, interact with other biomolecules or alter the lipophili-
city of the complex.
The 1H NMR spectrum shows all the resonances expected
for the molecule, in a similar way to that observed for the com-
pound [Au(SPyCOOH)(PPh3)], except for the aromatic protons
of the triphenylphosphine that integrate for 30 instead of
15 H. The protons of the pyridine rings appear slightly down-
field in relation to the resonances observed for the mono-
nuclear derivative. The 31P{1H} NMR spectrum presents a
unique resonance at 33.3 ppm for the equivalent gold(I) phos-
phine fragments, about 3–4 ppm more shielded than the
mononuclear complex, a feature observed for similar dinuclear
gold(I) complexes. In the 19F{1H} NMR spectrum a signal at
−153.9 ppm is observed and corresponds to the counter anion
tetrafluoroborate. The 13C{1H} NMR and IR spectra are essen-
tially the same as the mononuclear analogue, while the mass
spectra (ESI+) presents the molecular peak [M]+.
Complexes 19 and 20, as all the complexes reported in this
work, were fully characterized according to the usual tech-
niques (1H, 31P{1H}, 13C{1H}-NMR and IR spectroscopy, MS
spectrometry and elemental analysis). The 1H and 13C{1H}
NMR spectra show the expected resonances from all the
protons and carbon atoms that are essentially the same
observed for [Au(SPyCOOH)(PPh3)] and 2, and will not be dis-
cussed further here. The only exception are the signals arising
at the pyridine of the phosphine ligand, for which a correct
assignment could be done by carrying out 2D-NMR experi-
1
1
ments (1H–1H COSY, H–13C HSQC and H–13C HMBC), since
these resonances and those of the pyridine belonging to the
thiolate unit appear at similar chemical shift. The 31P{1H}
NMR spectra present a unique resonance for both complexes
corresponding to the phosphorus of the 2-diphenylphosphino
pyridine ligand, in both cases the chemical shift is observed at
Change in the coupled amino acid. In addition to the
different types of L-amino acids employed previously (Gly, Ala,
Val, Phe, Met and Pro) in the preparation of complexes 1–18,
we have made several modifications in the coupling process,
such as in the amino acid (lysine), in the use of a dipeptide
Scheme 2 Synthesis of the complexes 19 and 20 with a different phos-
phine coordinated to Au(I). (i) [AuCl(PPh2Py)], K2CO3, acetone, (ii)
HCl·H-Ala-OMe, PyBOP, DIPEA, MeCN.
Scheme 3 Synthesis of the dinuclear complex 21.
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(H-Gly-Pro-OMe), in the functionalisation of the amino acid receptors in the organism, and have influence on the cytotox-
with a tertiary amide, or by a change in the stereochemistry icity or biodistribution of the final complex.
using D-enantiomers (Ala, Phe), as summarised in Scheme 4.
All the complexes were prepared as discussed earlier, except
The amino ester derivatives [Au(SPyCOLys(Boc)OMe)(PPh3)] in the case of complex 23, where the removal of the Boc group
(22) and [Au(SPyCOGlyProOMe)(PPh3)] (24) can be readily pre- was carried out employing standard conditions (TFA), carefully
pared by the coupling of the lysine ester or a dipeptide [HGly- to avoid the loss of the integrity of the Au–S and Au–P bonds,
ProOMe] (previously synthesized in solution by standard as was observed when 3 M HCl/AcOEt was employed.
peptide chemistry), in a similar way to that reported for the
The 1H and 13C{1H} NMR spectra show all the signals
synthesis of compounds 1–6. The desired complexes were corresponding to the coupled amino esters, dipeptide or
obtained in good yields and in a pure form after chromato- amine, similar to that observed in the spectra of complexes
graphic purification, confirming the generality of this pro- 1–18, and are in agreement with the proposed formulation.
cedure for the incorporation of thiolate gold fragments into The 31P{1H} NMR spectra show in all cases a singlet in the
more complex molecules. Boc group deprotection in the lysine range of 35–37 ppm, which are also in agreement with a phos-
side chain afforded the complex [Au(SPyCOLys(H)OMe)(PPh3)] phorus atom trans to a sulfur centre. In the mass spectra
(23), with the free ε-amino group in the side chain. This (ESI+) the molecular peaks, as the protonated species [M + H]+,
complex may be interesting under physiological conditions appear in all the cases. In addition the species generated in
because it is a water-soluble species with a basic group in the the experiment [M + AuPPh3]+ were observed. The D-enantio-
molecule. Coupling of diethylamine through the free car- mers (26–28) show exactly the same physical properties as
boxylic acid of complex 2 gave the compound [Au(SPyCOAla- their L-enantiomers (2, 4 and 8). To confirm the stereochemi-
NEt2)(PPh3)] (25), in which we have a tertiary amide instead of cal configuration of the chiral centre (Cα amino acid), the
the secondary amide in the analogue 14. Furthermore, we measurement of the optical rotation angle was carried out,
carried out a change in the stereochemistry of the coupled obtaining approximately opposite values to that observed for
amino ester. The use of enantiomerically pure D alanine or the L-analogues.
phenylalanine amino esters yielded complexes [Au(SPyCO(D)-
Synthesis of the hybrid complex of 6 and 21. As we will
AlaOMe)(PPh3)] (26) or [Au(SPyCO(D)PheOMe)(PPh3)] (27) that comment in the next section, complexes 6 and 21 were found
are the corresponding enantiomers of previously described to be the most cytotoxic against different tumor cell lines.
2 or 4. Finally, hydrolysis of complex 26 gave the amino acid These derivatives contain firstly proline as the coupled amino
derivative [Au(SPyCO(D)AlaOH)(PPh3)] (28), corresponding to ester (6), and secondly two gold atoms bonded to the thiolate
D-alanine, which is the analogue of 8.
unit in 21. According to the values obtained and the structure–
The different structural modifications introduced in the activity relationship observed, we decided to prepare the
molecule could influence the activity and/or the mechanism of hybrid complex between these two candidates. The reaction
action of the resulting complexes, and might determine the between complex 6 and [Au(OTf)(PPh3)], generated in situ,
interactions of the complexes with different biomolecules or afforded the desired compound [Au2(SPyCOProOMe)(PPh3)2]-
OTf (29), conjugating the most important characteristics of
both complexes mentioned above (Scheme 5).
This complex presents the following SAR: functionalization
as esters, containing a rigid and conformationally restricted
amino acid and more than one gold(I) atom per molecule. The
1H NMR spectrum of complex 29 appears as a mixture of rota-
mers (ratio 1 : 0.8), showing similar signals to those observed
for 6, with the exception of the protons of the pyridine (the
ortho and para protons appear slightly downfield while the
meta protons appear upfield), and the aromatic protons of
PPh3 that integrate for 30 H instead of 15 H (Fig. 3). The ratio
between both rotamers can be calculated in the integral of the
resonance for CαH.
Scheme 4 Synthesis of the complexes 22–28. (i) HCl·H-Lys(Boc)-OMe,
PyBOP, DIPEA, MeCN, (ii) TFA–DCM (1 : 1 v/v), (iii) HCl·H-Gly-Pro-OMe,
PyBOP, DIPEA, MeCN, (iv) HCl·H-Gly-OMe, PyBOP, DIPEA; LiOH, KHSO4;
HNEt2, PyBOP, DIPEA; MeCN, (v) HCl-H-(D)-Aa-OMe, PyBOP, DIPEA,
MeCN, (vi) LiOH, KHSO4.
Scheme 5 Synthesis of complex 29.
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some of the complexes was also evaluated. Compounds are
soluble in DMSO and in the DMSO–water mixtures used in the
tests, which contain a small amount of DMSO (concentration
is always <0.5%). We did not observe any precipitation of the
complexes while performing the tests. Their colourless
D6-DMSO solutions are very stable at room temperature, as
shown in the 31P{1H} NMR spectra in which the signals
remain the same for weeks.
Cells were exposed to different concentrations of each com-
pound for a total of 24 h. Using the colorimetric MTT viability
assay,17 the IC50 values (final concentration <0.5% DMSO)
were calculated from dose–response curves obtained by non-
linear regression analysis. IC50 values are concentrations of
drugs required to inhibit tumour cell proliferation by 50%
compared to control cells treated with DMSO alone.
The IC50 values for the ester (1–6), acid (7–12) and amide
(13–18) complexes are collected in Table 1. Cytotoxicity values
of cisplatin, measured under the same conditions, are
included too and used for comparison purposes. It should be
noted that the values measured at 24 h can be considerably
greater than other values reported in the studies in the biblio-
graphy at 48 h or 72 h. In order to evaluate the cytotoxicity of
the new complexes prepared, the cytotoxicity of the precursor
compound, [Au(SPyCOOH)(PPh3)], was also determined.
As can be observed, all the complexes synthesized were
active against all the different tumour cell lines at low concen-
trations (low micromolar range). The starting material
[Au(SPyCOOH)(PPh3)], with known antimicrobial activity, pos-
sesses also cytotoxic activity. Complexes 1–18 exhibit good
antiproliferative activities, with IC50 values ranging from 7.4 to
30.5 μM in A549 cells, 2.4 to 7.7 μM in Jurkat cells, and 8.2 to
27.2 μM in MiaPaca2. The Jurkat cell line was the most sensi-
tive to our compounds, while A549 or MiaPaca2 showed more
resistance to the complexes. Importantly, the compounds
showed some selectivity for leukaemia cells against non-
tumour cells (Jurkat vs. R69) but this difference was not seen
in the case of solid tumours.
Fig. 3 1H NMR spectrum of complex 29, showing the presence of
rotamers.
In the 13C{1H} NMR spectrum the complex also appears as
a mixture of rotamers, and the resonances do not present any
significant variation in relation to 6 and 21 (Fig. 4). The
31P{1H} NMR spectrum shows one resonance at 33.0 ppm,
with a chemical shift similar to 22, and consequently the two
[AuPPh3]+ fragments are equivalents. The 19F{1H} NMR spec-
trum shows one resonance at −78.0 ppm corresponding to the
triflate counter anion. The mass spectrum (ESI+) presents the
molecular peak [M]+.
Biological evaluation
Cytotoxic activity. The cytotoxic activity of the previously
synthesised complexes (1–18), and the new complexes pre-
pared including all the structural modifications (19–29) were
tested against three different human tumour cell lines: A549
(lung carcinoma), Jurkat (T-cell leukaemia) and MiaPaca2
(pancreatic carcinoma). The sensitivity of non-tumor R69 (lym-
phoid cell line) and 293 T (embryonic kidney fibroblasts) to
The family of ester complexes (1–6) displayed better
cytotoxicity values than the precursor complex in all the cell
lines, except in the MiaPaca2 where both showed similar
potency. Particularly, the proline derivative 6 was the most
potent of this family of complexes. The acid complexes (7–12)
were more active than the precursor complex only in the A549
cell line. The other cell lines were less active, except the
proline 12, alanine 8 and glycine 7 derivatives in each case. In
comparison with the ester compounds, the acid complexes
were less active. The amide complexes (13–18) were more
active than the precursor only in the Jurkat cell line. The other
cell lines were less active than the precursor complex and also
than the ester and acid analogues.
These data indicate that the coupling of amino esters to the
precursor gold-thiolate complex affords more cytotoxic com-
pounds. The functionalisation as acids or amides decreases
the activity of the complexes. Finally, the type of amino acid
coupled has an influence on the antiproliferative activity of the
complex; the best results have been achieved mainly with the
Fig. 4 Compounds 6 and 21 induce apoptosis and necrosis in Jurkat
cells and non-apoptotical cell death in A549 cells. Nuclei were stained
with Hoechst 333248 and cells were photographed under UV light.
Arrows point to apoptotic nuclei showing condensed and fragmented
chromatin.
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Table 1 IC50 (μM) of complexes 1–18 after 24 h of incubation
Compounds
A549
MiaPaca2
Jurkat
293T
R69
Cisplatin
[Au(SPyCOOH)(PPh3)]
1
2
3
4
5
6
105 0.90
15.5 0.92
11.5 0.55
13.7 0.71
10.9 0.40
8.9 0.36
8.2 0.41
7.4 0.34
14.7 0.88
7.7 0.22
14.7 0.20
15.9 0.50
14.1 0.55
14.3 0.61
28.3 1.02
19.1 0.67
14.4 0.60
18.8 0.71
19.4 0.62
30.5 0.82
71 0.80
9.2 0.28
9.7 0.22
11.0 0.20
10.2 0.25
12.3 0.37
12.8 0.32
9.4 0.19
8.2 0.13
10.7 0.16
12.3 0.32
11.5 0.29
14.5 0.26
11.6 0.20
27.2 0.65
8.1 0.23
12.5 0.32
14.1 0.29
15.2 0.33
19.2 0.36
7.4 0.10
4.6 0.08
3.8 0.07
4.0 0.07
3.3 0.05
4.0 0.08
4.1 0.06
2.4 0.04
7.6 0.11
3.7 0.06
4.3 0.08
6.7 0.13
3.6 0.06
7.5 0.07
3.9 0.06
3.9 0.07
3.8 0.06
3.7 0.06
5.3 0.11
7.7 0.15
14.0 0.20
4.6 0.13
4.2 0.08
2.7 0.07
10.7 0.31
5.5 0.16
3.7 0.07
10.0 0.19
35.2 0.53
12.8 0.27
11.3 0.29
65.5 1.12
49.3 1.53
3.0 0.08
14.4 0.42
8.1 0.28
7.6 0.26
14.6 0.57
13.6 0.40
5.8 0.16
65 0.92
16 0.64
8.6 0.33
2.2 0.08
19.0 0.60
14.0 0.59
9.6 0.36
4.0 0.13
25.9 1.04
6.1 0.18
6.5 0.25
33.1 1.13
28.4 1.16
4.0 0.11
4.9 0.16
1.4 0.04
5.2 0.15
6.8 0.23
3.0 0.09
4.0 0.16
7
8
9
10
11
12
13
14
15
16
17
18
incorporation of the proline moiety, although glycine or changing the triphenylphosphine for 2-diphenylphosphino
alanine provided very active compounds too. pyridine was not significant, probably because both phos-
Compared with the reference drug cisplatin, our complexes phines have similar electronic and steric properties.
(1–18) exhibit much better antiproliferative activities in vitro in
The coordination of an additional [AuPPh3]+ fragment
all the cell lines.
afforded a more potent complex. The dinuclear derivative 21
The IC50 values of the modified derivatives (19–29) are showed better antiproliferative activity than the precursor
listed in Table 2. The modified complexes were also active complex in all cell lines. This is in agreement with the fact
against the different human tumor cell lines at low concen- that the cytotoxic activity of the complex is mainly due to the
trations (low micromolar range), with IC50 values measured gold(I) atom and the thiolate ligand first, and later the phos-
after 24 h of incubation, ranging from 6.9 to 33.5 μM in A549 phine ligands are lost in exchange reactions with other bio-
cells, 3.3 to 8.6 μM in Jurkat cells and 7.5 to 29.3 μM in molecules in the organism.
MiaPaca2. The Jurkat cell line was more sensitive to our com-
The coupling of a different type of amino ester (lysine)
plexes than A549 or MiaPaca2. The values compared with cis- afforded compound 22 that exhibited good antiproliferative
platin are considerably lower for our complexes in the toughest activity, in the same range as the most potent compounds of
cell lines, A549 or MiaPaca2, and present similar values in the the ester family. The removal of the protective group to yield a
Jurkat cell line.
water-soluble complex and with a basic group in the side
The complex 19 or 20 with different types of phosphine co- chain (23) decreased dramatically the potency of the com-
ordinated to gold(I) afforded slightly more cytotoxic complexes pound. This could be explained because the free amino group
in the R69 (non-tumor) and Jurkat cell lines, respectively. In is a powerful nucleophile that could react with other bio-
the other cell lines the complexes were slightly less active than molecules in the organism, resulting in a shorter life-time of
their analogues with triphenylphosphine. Then, the effect of the complex, or may prevent the gold(I) to reach its target.
Table 2 IC50 (μM) of complexes 19–29 after 24 h of incubation
Compounds
A549
MiaPaca2
Jurkat
5.2 0.07
3.8 0.04
3.3 0.04
3.4 0.06
36.5 0.77
8.6 0.14
NT
293T
R69
4.9 0.18
19
20
21
22
23
24
25
26
NT
18.2 0.51
17.4 0.48
7.5 0.19
13.1 0.26
29.3 0.70
22.5 0.67
>50
17.1 0.39
15.1 0.27
16.1 0.47
14.0 0.28
12.0 0.19
7.8 0.19
3.5 0.11
>25
17.9 0.45
8.3 0.19
7.7 0.24
>25
15.7 0.66
6.9 0.21
8.3 0.39
32.5 1.24
18.7 0.64
33.5 1.31
16.5 0.92
18.3 0.75
17.8 0.83
3.2 0.13
1.8 0.05
2.8 0.08
10.4 0.35
15.4 0.60
1.9 0.05
3.0 0.08
1.2 0.02
8.8 0.28
4.2 0.05
3.6 0.06
4.6 0.10
27
Racemic mixture
4.5 0.10
of 2 and 27
28
29
>50
4.1 0.11
>50
1.2 0.04
NT
0.9 0.07
>25
4.5 0.14
10.7 0.29
0.8 0.02
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The complexes that present a dipeptide, tertiary amide or D-
Dalton Transactions
amino esters in their structures were less cytotoxic than their
respective analogues in all the cases. Then, the increment in
the proportion of amino acids in the molecule does not
increase the potency of the complex, since the optimal biologi-
cal activity is achieved with one single amino acid coupled.
The interaction with biomolecules, such as proteins or
enzymes, is a very important process that determines the trans-
port, mode of action and activity of the compound. The ability
to form hydrogen bonds or the stereospecific recognition
seems to have a great influence on the final cytotoxicity of the
complex, and we can see experimentally that the optimal
activity is shown with molecules that can establish hydrogen
bonds (like secondary amides) and natural amino acids with
the L-configuration.
In view of these data, better cytotoxicity was achieved by the
compounds functionalised as esters, with two gold(I) atoms
per molecule and with conformationally-restricted or rigid
amino acids, as in the case of proline. Following this struc-
ture–activity relationship observed, complex 29 was prepared.
Complex 29 showed excellent antiproliferative activity, with
IC50 values in the low micromolar range even in the submicro-
molar range as in the case of the Jurkat cell line. This com-
pound is the most potent among all complexes prepared in
these series, and it is significantly more active than the precur-
sors 6 and 21 in all the cell lines. Furthermore, complex 29 is
much more active than cisplatin in all studied cell lines. This
result confirms our hypothesis and provides access to the
design and preparation of more potent and selective gold(I)
compounds of this type.
Fig. 5 Annexin V-PE labeling and 7-AAD staining assays. Data indicate
that our complexes induced cell death both by apoptosis and necrosis.
Antioxidants such as NAC and GSH protect the cells from cytotoxic
effects of 6 and/or 21.
that thiols like N-acetyl cysteine (NAC) or glutathione (GSH) are
employed by the organism as antioxidants and chemoprotec-
tive agents,18 and high levels of this type of compound have
been observed in certain types of cancer cells that developed
resistance to chemotherapeutic agents. The addition of NAC
and GSH protected A549 from cell death induced by our com-
pounds, but only GSH inhibited cell death in Jurkat cells.
These results indicate that 6 and 21 activate different mecha-
nisms of cell death in Jurkat and A549 cells.
Depolarization of mitochondria and ROS production. It is well-
known that mitochondria plays the main role in the apoptotic
process19 because it can start or amplify the cell signals that
result in apoptosis, by coordinating caspase activation through
the release of apoptogenic factors such as cytochrome c. More-
over, in the last few years several studies indicate that gold
compounds might exert their cytotoxic effect at the mitochon-
dria.20 For all these reasons, the determination of the mito-
chondrial membrane potential was performed. This parameter
is closely connected with the correct performance of mito-
chondria and is very important because the existing potential
gradient makes possible vital cell events like oxidative phos-
phorylation (ATP generation). Complexes 6 and 21 caused a
strong decrease in the mitochondrial membrane potential
(MMP). Although in all cases our complexes induced mito-
chondrial toxicity, the effect was more marked in Jurkat than
in A549 cells, and complex 6 was more effective than 21. With
the addition of GSH, the potential gradient recovery was
achieved, specifically in Jurkat cells, an effect that did not
occur after addition of NAC (Fig. 6).
Determination of the mechanism and type of death
Cell and nuclear morphological changes. In order to deter-
mine the mechanism of molecular death (apoptosis or necro-
sis) and potential targets of our compounds, several biological
studies were performed.
We carried out the analysis of the cell death and nuclear
morphology in A549 and Jurkat cells in the presence of the
selected compounds 6 and 21. Nuclear staining with Hoechst
333248, a membrane-permeable dye, which binds to nucleic
acids, did not show chromatin condensation and fragmenta-
tion in A549 cells, typical features of apoptosis, so in this case
our complexes induced non-apoptotic cell death, while in
Jurkat ones cell death proceeds, both by apoptosis and necro-
sis (Fig. 4).
Cell death was also evaluated by annexin V-PE (phycoery-
thrin-conjugated recombinant annexin V) labelling and sub-
sequent quantification of fluorescent cells by flow cytometry.
The loss of membrane asymmetry and phosphatidylserine (PS)
exposition is a typical feature observed in apoptosis and
annexin V is able to bind to PS exposed in the outer cell mem-
brane. The data show that a significant percentage of cells
leads to apoptosis, especially in A549 cells with complex 6
(Fig. 5). However, the staining with 7-AAD, which is not able to
cross the cell membrane, revealed that a great percentage of
cells lost the cell membrane integrity, suggesting that cell
death proceeds, by both apoptosis and necrosis. It is known
Reactive Oxygen Species (ROS) have attracted great attention
because of their relationship with many diseases like cancer.21
These radicals, whose main source is mitochondria, are able
to damage proteins, enzymes or DNA in an irreversible way
leading to cell death. In addition to this, some chemothera-
peutic agents exert their cytotoxic effect by ROS production
and the deregulation of the cellular redox state inducing oxi-
dative stress. The generation of ROS by our compounds was
analysed by flow cytometry after staining with 2-hydroxy-
17060 | Dalton Trans., 2014, 43, 17054–17066
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chased from Aldrich, Fluka and Bachem and used without
further purification. C, H, and N analyses were carried out
with a Perkin-Elmer 2400 microanalyser. Mass spectra were
recorded on a Bruker Esquire 3000 Plus, with the electrospray
(ESI) technique and on a Bruker Microflex (MALDI-TOF). 1H,
13C{H} and 19F NMR, including 2D experiments, were per-
formed at room temperature on a Bruker Avance 400 spectro-
meter (1H, 400 MHz, 13C, 100.6 MHz) or on a Bruker Avance II
300 spectrometer (1H, 300 MHz, 13C, 75.5 MHz), with chemical
shifts (δ, ppm) reported relative to the solvent peaks of the
deuterated solvent. The RPMI 1640 (Roswell Park Memorial
Institute) cell culture medium and fetal bovine serum (FBS)
were purchased from LONZA Co. MTT (3-(4,5-dimethyl-thiazol-
2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from
Sigma Chemical. MTT was dissolved (5 mg ml−1) in phosphate
buffer at pH 7.2. Fluorescence intensity measurements were
carried out on a PTI QM-4/206 SE Spectrofluorometer (PTI,
Birmingham, NJ) with right angle detection of fluorescence
using a 1 cm path length quartz cuvette.
Fig. 6 Determination of the mitochondrial membrane potential and
ROS formation after addition of complexes 6 and 21.
Starting materials
ethidium. The assay revealed that higher levels of these radicals
are generated in Jurkat cells than in A549 cells and complex 21
induced the generation of higher levels of ROS than 6. The ROS
levels produced showed good correlation with the loss of mito-
chondrial membrane potential. These data suggest that the
damage to the mitochondria could lead to an increase in the
production of ROS that in final instance triggers cell death.
Thioredoxin reductase (TrX) inhibition assay. Recently, some
selenol-containing enzymes like TrX have been postulated as
possible targets for gold(I) compounds,5a,7d,15 due to the high
affinity of gold for selenium. In fact, some gold(I) complexes
inhibited strongly this type of enzyme even in the presence of
other thiol-containing enzymes.7e The TrX system plays a key
role in the cellular redox state (reducing oxidised proteins) and
others, and inhibition of this enzyme is thought to cause apop-
tosis and cell death, which could explain the mechanism of
action of some gold(I) complexes. The inhibition of the TrX
assay revealed that complexes 6 and 21 at concentrations near
IC50 values are potent inhibitors of this enzyme, by decreasing
the enzyme activity at least in 50%. Complex 21 inhibited
more efficiently the TrX than 6 (65% against 50% of total inhi-
bition, respectively). The inhibition of TrX by our complexes
would prevent the removal of ROS and may be the reason for
the high levels of ROS observed in the previous assay.
[AuCl(PPh3)],22 [AuCl(PPh2Py)]22 and [O(AuPPh3)3]BF4
were
23
prepared according to published procedures. All other reagents
were commercially available.
Synthesis
Compounds 1–18 were synthesised as reported elsewhere.14
[Au(SPyCOOH)(PPh2Py)] (19). To a suspension of 2-mercap-
tonicotinic acid (0.155 g, 1 mmol) in acetone (10 ml) was
added an excess of K2CO3 (0.276 g, 2 mmol) and [AuCl-
(PPh2Py)] (0.496 g, 1 mmol). The mixture was stirred for 24 h
at room temperature. The solution was concentrated to ca.
5 ml and addition of hexane (20 ml) afforded 19 as a white
solid (0.456 g, 74.2%). 1H-NMR (CDCl3, 400 MHz δ (ppm),
J (Hz)): 8.79 (d, 1H, J = 4.4, H12), 8.50 (dd, 1H, J = 8.0 and 2.0,
H1), 8.48 (dd, 1H, J = 4.6 and 2.0, H3), 7.95 (“t”, 1H, J = 7.6,
H9), 7.80 (m, 1H, H10), 7.72 (m, 4H, ArH), 7.50 (m, 6H, ArH),
7.39 (m, 1H, H11), 7.11 (dd, 1H, J = 8.0 and 4.6, H2). 31P-NMR
(CDCl3, 400 MHz,
δ
(ppm)): 35.9. 13C-NMR (d6-DMSO,
400 MHz, δ (ppm), J (Hz)): 171.1 (COOH), 163.3 (C, Py), 154.2
(d, C, J = 78.7, C8), 151.3 (d, CH, J = 15.2, C12), 144.9 (CH, C1),
140.9 (C, C13), 137.3 (d, CH, J = 10.4, C10), 134.3 (d, CH, J =
14.0, C5), 130.9 (d, CH, J = 31.7, C9), 131.8 (CH, C7), 129.7 (C,
C4), 129.3 (d, CH, J = 11.3, C6), 133.7 (CH, C3), 125.7 (CH,
C11), 117.7 (CH, C2). MS (ESI+) m/z: [M + H]+ = 615.0 (calcd),
615.0 (found). IR (cm−1): 3400–3049 (br, COOH), 1570, 1480
and 1435 (w, Ar), 1099 and 1067 (s, C–O), 741, 710 and 690
(w, Ar). Anal. Calcd for C23H18AuN2O2PS (614.41): C, 44.96; H,
2.95; N, 4.56; S, 5.22. Found: C, 45.07; H, 3.01; N, 4.65; S, 5.40.
Experimental section
Materials and methods
General procedure for the synthesis of complexes 20, 22, 24, 26
and 27
All manipulations were routinely carried out under Ar using
common Schlenk techniques. Solvents were purified by stan-
dard procedures immediately prior to use. 2-Mercaptonicotinic To a suspension of [Au(SPyCOOH)(PPh3)] or [Au(SPyCOOH)-
acid, 2-diphenylphosphino-pyridine, L-H-Lys(Boc)-OMe·HCl, (PPh2Py)] (1 mmol) in acetonitrile (6 mL) was added DIPEA
L-H-Pro-OMe·HCl, L-H-Gly-OMe·HCl, L-H-Ala-OMe·HCl, D-H-Ala- (2.2 mmol). The mixture was stirred for 5 min at room temp-
OMe·HCl, D/L-H-Ala-OMe·HCl and diethylamine were pur- erature and then PyBOP was added (1.2 mmol), and the result-
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Dalton Transactions
ing solution was stirred for an additional 45 min. To this solu- 4.8, H2), 4.86 (“dt”, 1H, J = 7.6 and 5.3, Cα,LysH), 4.61 (m, 1H,
tion was added dropwise at 0 °C a solution of the corres- NHBoc), 3.76 (s, 3H, OCH3), 3.12 (m, 2H, Cε,LysH2), 1.98 and
ponding amino acid or dipeptide methyl ester (1.5 mmol) in 1.90 (m, 2H, diastereotopic protons, Cβ,LysH2), 1.59 (m, 2H,
acetonitrile (4 mL) and DIPEA (1.5 mmol). After the addition Cγ,LysH2), 1.53 (m, 2H, Cδ,LysH2), 1.42 (s, 9H, CBocH3). 31P-NMR
the mixture was stirred for 48 h at room temperature. The (CDCl3, 300 MHz, δ (ppm)): 37.6. 13C-NMR (CDCl3, 300 MHz,
acetonitrile was evaporated and dichloromethane (40 mL) was δ (ppm), J (Hz)): 172.7 (COOMe), 166.4 (CONHLys), 164.9 (C,
added. This solution was washed with a saturated NaHCO3 Py), 155.9 (NHCBoc), 149.1 (CH, C1), 139.2 (CH, C3), 134.3 (d,
solution in water (3 × 15 mL) and a saturated solution of NaCl CH, J = 13.9, C5), 131.7 (d, CH, J = 2.4, C7), 130.0 (C, C4), 129.1
(3 × 15 mL). The organic phase was dried over anhydrous (CH, J = 11.5, C6), 118.8 (CH, C2), 53.0 (Cα,LysH), 52.3 (OCH3),
MgSO4, filtered off and evaporated to dryness. The complexes 40.3 (Cε,LysH2), 32.0 (Cβ,LysH2), 29.4 (Cδ,LysH2), 28.4 (CBocH3),
were purified by column chromatography on silica gel using as 22.8 (Cγ,LysH2). MS (ESI+) m/z: [M + H]+ = 856.2 (calcd), 856.3
the eluent a mixture of acetone–hexane 3 : 7.
(found). IR (cm−1): 3266 (br, w, CONH), 1739 (s, COOMe), 1703
[Au(SPyCOAlaOMe)(PPh2Py)] (20). Yield. 67.3%. 1H-NMR (s, OCONH), 1641 (s, CONH), 1571, 1520, 1480 and 1435 (w,
(CDCl3, 400 MHz δ (ppm), J(Hz)): 9.15 (d, 1H, J = 6.8, Ar), 1100 and 1070 (s, C–O), 746, 710 and 691 (w, Ar). Anal.
CONHAla), 8.65 (dd, 1H, J = 4.8 and 0.8, H12), 8.22 (d, 1H, J = Calcd for C36H41AuN3O5PS (855.73): C, 50.53; H, 4.83; N, 4.91;
3.2, H1), 8.14 (dd, 1H, J = 8.0 and 2.0, H3), 7.90 (“t”, 1H, J = S, 3.75. Found: C, 50.61; H, 4.89; N, 4.99; S, 3.79. TLC Rf: 0.5
7.7, H9), 7.56 (m, 4H, Ar), 7.64 (m, 1H, H11), 7.36 (m, 6H, ArH), (acetone–hexane 1 : 1). [α]D: +21.18 (c = 1.0 g ml−1, CHCl3).
7.27 (m, 1H, H10), 6.88 (dd, 1H, J = 8.0 and 5.0, H2), 4.70 (“q”,
[Au(SPyCOLys(H)OMe)(PPh3)] (23). To a 1 : 1 (v/v) CH2Cl2–
1H, J = 7.1, Cα,AlaH), 3.63 (s, 3H, OCH3), 1.45 (d, 3H, J = 7.2, TFA solution (12 ml) was added complex 23 (0.296 g,
Cβ,AlaH3). 31P-NMR (CDCl3, 400 MHz, δ (ppm)): 36,1. 13C-NMR 0.346 mmol). The mixture was stirred at room temperature for
(CDCl3, 400 MHz, δ (ppm), J (Hz)): 173.4 (COOMe), 166.0 15 minutes until completion by thin-layer chromatography
(CONHAla), 165.5 (C, Py), 151.2 (d, CH, J = 14.7, C12), 148.6 (the reaction was complete when the starting compound was
(CH, C1), 147.8 (C, C8), 139.5 (CH, C3), 136.5 (d, CH, J = 11.3, strongly retained at the origin of TLC, Rf = 0, acetone–hexane
C9), 134.7 (d, CH, J = 13.8, C5), 132.2 (CH, C11), 131.7 (d, CH, 1 : 1). Then, the solvent was evaporated and the residue was
J = 2.4, C7), 130.0 (d, C, J = 86.9, C4), 129.0 (d, CH, J = 11.7, C6), dissolved in water (40 ml). The resulting clear solution was
126.8 (C, Py), 125.2 (d, CH, J = 2.5, C10), 118.5 (CH, C2), 52.3 washed with CH2Cl2 (1 × 20 ml). K2CO3 was added carefully to
(OCH3), 49.1 (Cα,AlaH), 18.2 (Cβ,AlaH3). MS (ESI+) m/z: [M + H]+ = the aqueous phase until slightly basic pH (11–12). At this
700.1 (calcd), 700.0 (found). IR (cm−1): 3280 (br, w, CONH), point, the white suspension was extracted with CH2Cl2 (3 ×
1736 (s, COOMe), 1637 (s, CONH), 1569, 1519, 1480 and 1434 20 ml). The organic phase was dried over anhydrous MgSO4,
(w, Ar), 1099 and 1069 (s, C–O), 742, 711 and 691 (w, Ar). Anal. filtered off, and the solvent was evaporated, affording 23 as a
Calcd for C27H25AuN3O3PS (699.51): C, 46.36; H, 3.60; N, 6.01; white solid (0.089 g, 34.1%). 1H-NMR (CDCl3, 300 MHz,
S, 4.58. Found: C, 46.44; H, 3.68; N, 6.10; S, 4.69. TLC Rf: 0.5 δ (ppm), J (Hz)): 9.01 (d, 1H, J = 7.2, CONHLys), 8.31 (dd, 1H,
(acetone–hexane 1 : 1). [α]D: + 35.69 (c = 1.0 g ml−1, CHCl3).
J = 4.8 and 1.8, H1), 8.22 (dd, 1H, J = 7.8 and 1.8, H3), 7.56 (m,
[Au2(SPyCOOH)(PPh3)2]BF4 (21). To a suspension of 2-mer- 15H, ArH), 7.00 (dd, 1H, J = 7.8 and 4.8, H2), 4.86 (“dt”, 1H, J =
captonicotinic acid (0.016 g, 0.1 mmol) in CH2Cl2 (10 ml) was 7.6 and 5.4, Cα,LysH), 3.76 (s, 3H, OCH3), 2.71 (t, 2H, J = 6.5,
added [O(AuPPh3)3]BF4 (0.098 g, 0.066 mmol). The mixture Cε,LysH2), 1.99 (m, 2H, Cβ,LysH2), 1.59 (m, 2H, Cγ,LysH2), 1.52 (m,
was stirred for 2 h at room temperature. Then, the solution 2H, Cδ,LysH2). 31P-NMR (CDCl3, 300 MHz, δ (ppm)): 37.6.
was concentrated to ca. 5 ml and addition of hexane (20 ml) 13C-NMR (CDCl3, 300 MHz, δ (ppm), J (Hz)): 172.8 (COOMe),
afforded 21 as an orange solid (0.110 g, 95.0%). 1H-NMR 166.4 (CONHLys), 149.1 (CH, C1), 139.1 (CH, C3), 134.3 (d, CH,
(CDCl3, 400 MHz, δ ppm), J (Hz)): 8.43 (d, 1H, J = 3.2, H1), 8.17 J = 13.9, C5), 131.6 (d, CH, J = 2.1, C7), 130.2 (C, C4), 129.1
(d, 1H, J = 7.6, H3), 7.40 (m, 30H, ArH), 7.19 (dd, 1H, J = 7.6 (CH, J = 11.6, C6), 118.8 (CH, C2), 53.2 (Cα,LysH), 52.2 (OCH3),
and 4.8, H2). 31P-NMR (CDCl3, 400 MHz, δ (ppm)): 33.4. 41.7 (Cε,LysH2), 32.0 (Cβ,LysH2), 26.4 (Cδ,LysH2), 22.8 (Cγ,LysH2).
13C-NMR (CDCl3, 400 MHz, δ (ppm), J (Hz)): 168.5 (COOH), MS (ESI+) m/z: [M + H]+ = 756.2 (calcd), 756.2 (found).
157.3 (C, Py). 150.0 (CH, C1), 139.9 (CH, C3), 134.5 (d, CH, J = IR (cm−1): 3260 (br, w, CONH), 2961 and 2941 (br, w, NH2),
13.4, C5), 132.2 (CH, C7), 129.4 (d, CH, J = 11.9, C6), 128.1 (d, 1739 (s, COOMe), 1634 (s, CONH), 1571, 1525, 1480 and 1435
C, J = 60.4, C4), 122.2 (CH, C2). 19F-NMR (CDCl3, 400 MHz, (w, Ar), 1100 and 1071 (s, C–O), 746, 710 and 691 (w, Ar). Anal.
δ ppm)): −153.9. MS (ESI+) m/z: [M]+ = 1072.1 (calcd), 1072.3 Calcd for C31H33AuN3O3PS (755.62): C, 49.28; H, 4.40; N, 5.56;
(found). IR (cm−1): 3300–3053 (br, COOH), 1713 (s, COOH), S, 4.24. Found: C, 49.41; H, 4.51; N, 5.61; S, 4.32. [α]D: +9.64
1571, 1480 and 1435 (w, Ar), 1098 and 1051 (s, C–O), 744, 726, (c = 1.0 g ml−1, CHCl3).
and 710 (w, Ar). Anal. Calcd for C42H34Au2BNO2P2SF4
(1159.48): C, 43.51; H, 2.96; N, 1.21; S, 2.77. Found: C, 43.65; 2 mmol) in acetonitrile (10 ml) was added DIPEA (0.753 ml,
H, 2.99; N, 1.26; S, 2.80. 4.4 mmol). The mixture was stirred for 5 min at room tempera-
[BocGlyProOMe]. To a solution of Boc-Gly-OH (0.350 g,
1
[Au(SPyCOLys(Boc)OMe)(PPh3)] (22). Yield. 60.7%. H-NMR ture, and then PyBOP (1.25 g, 2.4 mmol) was added, and the
(CDCl3, 300 MHz, δ (ppm), J (Hz)): 9.07 (d, 1H, J = 7.6, resulting mixture was stirred for an additional 45 min. At this
CONHLys), 8.33 (dd, 1H, J = 4.8 and 1.8, H1), 8.26 (dd, 1H, J = point, a solution of HCl·H-Pro-OMe (0.497 g, 3 mmol) and
7.6 and 1.8, H3), 7.58 (m, 15H, ArH), 7.02 (dd, 1H, J = 7.6 and DIPEA (0.515 ml, 3 mmol) in acetonitrile was added dropwise
17062 | Dalton Trans., 2014, 43, 17054–17066
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and at 0 °C. After the addition, the mixture was stirred for 48 h chromatography using as an eluent acetone–hexane 1 : 1,
at room temperature. Acetonitrile was evaporated and CH2Cl2 affording 25 as a green solid (0.106 g, 22.7%). 1H-NMR (CDCl3,
was added (40 ml). This solution was washed with a saturated 300 MHz, δ (ppm), J (Hz)): 9.14 (d, 1H, J = 7.2, CONHAla), 8.28
NaHCO3 solution in water (3 × 15 mL) and a saturated solution (dd, 1H, J = 4.8 and 1.8, H1), 8.16 (dd, 1H, J = 7.6 and 1.8, H3),
of NaCl (3 × 15 mL). The organic phase was dried over anhy- 7.54 (m, 15H, ArH), 6.96 (dd, 1H, J = 7.6 and 4.8, H2), 5.12 (“q”,
drous MgSO4, filtered off and evaporated to dryness. The crude 1H, J = 6.8, Cα,AlaH), 3.54 (m, 2H, diastereotopic protons,
of the reaction was purified by column chromatography of
CNEt H2), 3.29 (m, 2H, diastereotopic protons, CNEt H2), 1.49 (d,
2
2
silica gel using as an eluent a mixture of ethyl acetate–pentane 3H, J = 6.8, Cβ,AlaH3), 1.26 (m, 3H, CNEt H3) and 1.13 (t, 3H, J =
2
(1 : 3), affording the dipeptide as a colourless oil (0.436 g, 7.1, CNEt H3). 31P-NMR (CDCl3, 300 MHz, δ (ppm)): 37.6.
2
76.2%). 1H-NMR (CDCl3, 400 MHz, δ (ppm), J (Hz)): rotamer 13C-NMR (CDCl3, 300 MHz, δ (ppm), J (Hz)): 165.1 (CONEt2),
mixture (ratio: 1 : 0.2): 5.35 (m, 1H, CONHGly), 4.41 (dd, 1H, J = 164.8 (CONHAla), 163.4 (C, Py), 147.5 (CH, C1), 138.6 (CH, C3),
8.8 and 3.6, Cα,ProH), 3.88 and 3.81 (dd, ABX system, 2H, dia- 134.3 (d, CH, J = 14.0, C5), 131.6 (d, CH, J = 2.0, C7), 129.1 (d,
stereotopic protons, J = 17.2 and 4.8, J = 17.2 and 4.4, Cα,GlyH2), CH, J = 11.5, C6), 118.3 (CH, C2), 46.3 (Cα,AlaH), 41.7 and 40.2
3.66 and 3.62 (s, 3H, OCH3), 3.49 and 3.38 (m, 2H, diastereoto- (CNEt H2), 19.2 (Cβ,AlaH3), 14.6 and 12.9 (CNEt H3). MS (ESI+) m/z:
2
2
pic protons, Cδ,ProH2), 2.11 and 1.89 (m, 2H, diastereotopic [M + H]+ = 740.2 (calcd), 740.2 (found). Anal. Calcd for
protons, Cβ,ProH2), 1.94 (m, 2H, Cγ,ProH2), 1.33 (s, 9H, CBocH3). C31H33AuN3O2PS (739.62): C, 50.34; H, 4.50; N, 5.68; S, 4.34.
[HGlyProOMe]. The dipeptide BocProGlyOMe (0.436 g, Found: C, 50.46; H, 4.59; N, 5.70; S, 4.39. TLC Rf: 0.3 (acetone–
1.52 mmol) was dissolved in a solution 3 M HCl–AcOEt (6 ml). hexane 1 : 1).
The solution was stirred at room temperature for 3 h until
[Au(SPyCO(D)AlaOMe)(PPh3)] (26). Yield: 67.7%. 1H-NMR
completion by thin-layer chromatography. The solvent was (CDCl3, 400 MHz, δ (ppm), J (Hz)): 9.63 (d, 1H, J = 5.2,
evaporated, and the residue was dissolved in water and freeze- CONHAla), 8.38 (dd, 1H, J = 7.6 and 2.0, H1), 8.19 (dd, 1H, J =
dried, affording the desired dipeptide as a white solid (0.322 g, 5.2 and 2.0, H3), 7.63–7.47 (m, 15H, ArH), 6.98 (dd, 1H, J = 7.6
95.3%). 1H-NMR (CDCl3, 400 MHz, δ (ppm), J (Hz)): rotamer and 5.2, H2), 4.80 (“q”, 1H, J = 7.2, Cα,AlaH), 3.76 (s, 3H, OCH3),
mixture (ratio 1 : 0.2): 8.25 (m, 3H, NH3), 4.55 (m, 1H, Cα,ProH), 1.57 (d, 3H, J = 7.2, Cβ,AlaH3). 31P-NMR (CDCl3, 400 MHz,
4.10 (m, 2H, Cα,GlyH2), 3.77 (A) and 3.72 (B) (s, 3H, OCH3), 3.61 δ (ppm)): 37.6. 13C-NMR (CDCl3, 400 MHz, δ (ppm), J (Hz)):
(m, 2H, diastereotopic protons, Cδ,ProH2), 2.25 and 1.99 (m, 173.3 (COOMe), 165.9 (CONHAla), 165.6 (C, Py), 148.4 (CH, C1),
2H, diastereotopic protons, Cβ,ProH2), 1.99 (m, 2H, Cγ,ProH2).
139.4 (CH, C3), 134.2 (d, CH, J = 14.1, C5), 131.5 (d, CH, J =
[Au(SPyCOGlyProOMe)(PPh3)] (24). Yield: 60.3%. 1H-NMR 2.3, C7), 130.4 (C, Py), 129.7 (d, C, J = 55.2, C4), 129.1 (d, CH,
(CDCl3, 400 MHz, δ (ppm), J (Hz)): rotamer mixture ratio: J = 11.5, C6), 118.3 (CH, C2), 52.4 (OCH3), 49.1 (Cα,AlaH), 18.1
1 : 0.3; 9.06 (“t”, 1H, J = 3.9, CONHGly), 8.31 (dd, 1H, J = 4.8 and (Cβ,AlaH3). MS (ESI+) m/z: [M + H]+ = 699.1 (calcd), 699.0
2.0, H1), 8.14 (dd, 1H, J = 7.6 and 2.0, H3), 7.54 (m, 15H, ArH), (found). IR (cm−1): 3273 (br, w, NH), 1739 (s, COOMe), 1639 (s,
6.99 (dd, 1H, J = 7.6 and 4.8, H2), 4.58 (A) and 4.51 (B) (dd, 1H, CONH), 1570, 1523, 1479 and 1434 (w, Ar), 1098 and 1069 (s,
J = 8.6 and 3.4 and J = 8.0 and 2.8, Cα,ProH), 4.37 and 4.26 (dd, C–O), 744, 709 and 690 (w, Ar). Anal. Calcd for
ABX system, 2H, diastereotopic protons, J = 17.8 and 4.6, J = C28H26AuN2O3PS (698.52): C, 48.14; H, 3.75; N, 4.01; S, 4.59.
17.8 and 4.2, Cα,GlyH2), 3.71 (s, 3H, OCH3), 3.55 (m, 2H, diastereo- Found: C, 48.07; H, 3.81; N, 4.09; S, 4.32. TLC Rf: 0.5 (acetone–
topic protons, Cδ,ProH2), 2.19 and 2.04 (m, 2H, diastereotopic hexane 1 : 1). [α]D: −35.83 (c = 1.0 g ml−1, CHCl3).
protons, Cβ,ProH2), 2.08 (m, 2H, Cγ,ProH2). 31P-NMR (CDCl3,
[Au(SPyCO(D/L)AlaOMe)(PPh3)] (racemic mixture). Yield:
400 MHz, δ (ppm)): 37.5. 13C-NMR (CDCl3, 400 MHz, δ (ppm), 68.5%. The data are the same as those for complex 27, except
J (Hz)): 172.4 (COOMe), 167.0 (CONPro), 165.1 (CONHGly), 149.2 for the angle of polarised light. [α]D: +0.04 (c = 1.0 g ml−1
(CH, C1), 138.5 (CH, C3), 134.3 (d, CH, J = 13.9, C5), 131.7 (CH, CHCl3).
,
C7), 130.2 (d, C, J = 74.7, C4), 129.2 (d, CH, J = 11.6, C6), 118.6
[Au(SPyCO(D)PheOMe)(PPh3)] (27). Yield: 62.0%. 1H-NMR
(CH, C2), 58.9 (Cα,ProH), 52.3 (OCH3), 46.1 (Cδ,ProH2), 43.2 (CDCl3, 400 MHz, δ (ppm), J (Hz)): rotamer mixture (ratio
(Cα,GlyH2), 29.1 (Cβ,ProH2), 24.6 (Cγ,ProH2). MS (ESI+) m/z: 1 : 0.8): 9.33 (d, 1H, J = 6.8, CONHPhe), 8.27–8.25 (m, 2H, H1
[M + H]+ = 782.1 (calcd), 782.1 (found). IR (cm−1): 3205 (br, w, and H3), 7.64–7.48 (m, 15H, ArH), 7.33–7.18 (m, 5H, ArPheH),
NH), 1736 (s, COOMe), 1628 (s, CONH), 1571, 1523, 1481, 1455 6.97 (m, 1H, H2), 5.06 (A) and 3.75 (B) (“dd”, 1H, J = 13.6 and
and 1435 (w, Ar), 1100 and 1073 (s, C–O), 749, 710 and 692 (w, Ar). 6.8 and J = 8.0 and 5.2, Cα,PheH), 3.72 (A) and 3.69 (B) (s, 3H,
Anal. Calcd for C32H31AuN3O4PS (781.61): C, 49.17; H, 4.00; N, OCH3), 3.28 and 3.22 (A) and 3.10 and 2.86 (B) (dd, 2H, diastereo-
5.38; S, 4.10. Found: C, 49.29; H, 4.07; N, 5.41; S, 4.19. TLC Rf: 0.4 topic protons, J = 13.8 and 6.0 and J = 14.0 and 6.8; and J =
(acetone–hexane 1 : 1). [α]D: −21.36 (c = 1.0 g ml−1, CHCl3).
13.6 and 5.2 and J = 13.6 and 8.0, Cβ,PheH2). 31P-NMR (CDCl3,
[Au(SPyCOAlaNEt2)(PPh3)] (25). To a solution of complex 8 400 MHz, δ (ppm)): 37.4. 13C-NMR (CDCl3, 400 MHz, δ (ppm),
(0.433 g, 0.63 mmol) in acetonitrile (10 mL) was added DIPEA J (Hz)): rotamer mixture: 172.0 (COOMe), 166.1 (CONHPhe),
(0.237 ml, 1.39 mmol). The mixture was stirred at room temp- 165.8 (C, Py), 148.4 (CH, C1), 139.4 (CH, C3), 136.4 (C, C8),
erature for 15 min and PyBOP was added (0.393 g, 0.76 mmol). 134. (d, CH, J = 13.9, C5), 131.7 (d, CH, J = 2.2, C7), 130.1 (d, C,
After 15 min diethylamine (0.072 ml, 0.69 mmol) was added J = 64.0, C4), 129.5 and 129.2 (CH, C10), 129.1 (d, CH, J = 11.5,
and then the mixture was stirred for 36 h. The solvent was C6), 128.6 and 128.4 (CH, C9), 126.9 and 126.8 (CH, C11),
evaporated and the crude was purified by silica gel column 118.3 (CH, C2), 55.8 (A) and 55.0 (B) (Cα,PheH), 52.1 (A) and
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52.0 (B) (OCH3), 41.1 (A) and 38.2 (B) (Cβ,PheH2). MS (ESI+) m/z: 11.9, C5), 127.6 (d, C, J = 61.4, C4), 123.1 and 122.6 (CH, C2),
[M + H]+ = 775.1 (calcd), 775.1 (found). IR (cm−1): 3281 (br, w, 118.8 (C, CF3), 60.3 and 58.7 (Cα,ProH), 52.4 and 52.1 (OCH3),
CONH), 1736 (s, COOMe), 1640 (s, CONH), 1571, 1518, 1495, 48.5 and 46.4 (Cδ,ProH2), 31.1 and 29.4 (Cβ,ProH2), 24.7 and 22.8
1480, 1453 and 1435 (w, Ar), 1100 and 1071 (s, C–O), 745 and (Cγ,ProH2). HRMS (ESI+) m/z: [M]+
= 1183.1795 (calcd),
709 (w, Ar). Anal. Calcd for C34H30AuN2O3PS (774.62): C, 52.72; 1183.1859 (found). IR (cm−1): 1739 (s, COOMe), 1631 (s, CON),
H, 3.90; N, 3.62; 4.14. Found: C, 52.81; H, 3.68; N, 3.78; S, 4.01. 1570, 1545, 1479 and 1435 (w, Ar), 1097 and 1093 (s, COOMe),
TLC Rf: 0.5 (acetone–hexane 1 : 1). [α]D: −4.65 (c = 1.0 g ml−1
,
746 and 709 (w, Ar).
CHCl3).
Cell culture
[Au(SPyCO(D)AlaOH)(PPh3)] (28). To
a
suspension of
complex 2 (0.704 g, 1 mmol) in methanol (20 mL) was added Jurkat (leukaemia), R69 (lymphoid) and MiaPaca2 (pancreatic
LiOH (0.480 g, 20 mmol). The mixture was stirred at room carcinoma) cell lines were maintained in RPMI 1640, while
temperature for a period between 24 and 48 h. The reaction is A549 (lung carcinoma) and 293 T cells (kidney embryonic
followed by thin layer chromatography and is complete when fibroblasts) were grown in DMEM (Dulbecco’s Modified
the starting compound (Rf = 0.4 (acetone–hexane 1 : 1)) is Eagle’s Medium). Both media were supplemented with 5%
strongly retained at the origin of the thin layer chromatography fetal bovine serum (FBS), 200 U ml−1 penicillin, 100 μg ml−1
(Rf = 0 (acetone–hexane 1 : 1)). At this point the methanol is streptomycin and 2 mM L-glutamine. The medium for A549
evaporated and the product dissolved in water. The resulting cells was also supplemented with 2.2 g l−1 Na2CO3, 100 μg
white solution is acidified dropwise with a saturated solution ml−1 pyruvate and 5 ml non-essential amino acids (Invitrogen).
of KHSO4 until a slightly acidic pH (3–4). Then the solution Cultures were maintained under a humidified atmosphere of
was extracted three times with dichloromethane and the 95% air/5% CO2 at 37 °C.
organic phase was dried over anhydrous MgSO4, filtered off
and evaporated to yield the desired complex 28 as a white
Cytotoxicity assay by MTT
solid (0.521 g, 76.2%). 1H-NMR (CDCl3, 400 MHz, δ (ppm), The MTT assay was used to determine the cell viability as an
J (Hz)): 9.25 (d, 1H, J = 5.6, CONHGly), 8.33 (dd, 1H, J = 4.6 and indicator for the cell sensitivity to the complexes.
2.0, H1), 8.24 (dd, 1H, J = 7.6 and 2.0, H3), 7.59–7.42 (m, 15H,
Exponentially growing cells were seeded at a density of
ArH), 7.02 (dd, 1H, J = 7.6 and 4.6, H2), 4.65 (“q”, 1H, J = 6.8, approximately 1 × 105 cells ml−1 (A549, MiaPaca2) or 3 × 105
Cα,AlaH), 1.59 (d, 3H, J = 6.8, Cβ,AlaH3). 31P-NMR (CDCl3, cells ml−1 (Jurkat), in a 96-well flat-bottomed microplate and
400 MHz, δ (ppm)): 37.2. 13C-NMR (CDCl3, 400 MHz, δ (ppm), 24 h later they were incubated for 24 h with the compounds.
J (Hz)): 173.6 (COOH), 168.3 (CONHAla), 164.6 (C, Py), 149.6 The complexes were dissolved in DMSO (concentration 0.1 M)
(CH, C1), 139.1 (CH, C3), 134.2 (d, CH, J = 13.8, C5), 131.7 (d, and tested in concentrations ranging from 0.5 to 100 μM
CH, J = 1.3, C7), 129.6 (C, Py), 129.3 (d, C, J = 57.7, C4), 129.2 (dilutions with 1 μl of the stock solution for 100 μM) and in
(CH, J = 11.5, C6), 119.1 (CH, C2), 50.5 (Cα,AlaH), 16.9 (Cβ,AlaH3). quadruplicate. Cells were incubated with our compounds for
MS (ESI+) m/z: [M + H]+ = 685.1 (calcd), 685.0 (found). 24 h at 37 °C. 10 μl of MTT (5 mg ml−1) was added and plates
IR (cm−1): 3400–2900 (br, s, COOH), 1731 (s, COOH), 1637 (s, were incubated for 1–3 h at 37 °C. Finally, 100 µl per well
CONH), 1570, 1519, 1479 and 1434 (w, Ar), 1098 and 1069 (s, iPrOH (0.05 M HCl) was added. The optical density was
C–O) 743 and 709 (w, Ar). Anal. Calcd for C27H24AuN2O3PS measured at 570 nm using a 96-well multiscanner autoreader
(684.50): C, 47.38; H, 3.53; N, 4.09; S, 4.68. Found: C, 47.60; H, (ELISA). The IC50 was calculated by non-linear regression ana-
3.90; N, 3.78; S, 4.72. [α]D: −5.2 (c = 1.0 g ml−1, CHCl3).
[Au2(SPyCOProOMe)(PPh3)2]OTf (29). To
lysis using the Origin software (Origin Software, Electronic
solution of Arts, Redwood City, California, USA).
a
complex 6 (0.072 g, 0.1 mmol) in CH2Cl2 (5 ml) was added
freshly prepared [Au(OTf)(PPh3)] (0.061 g, 0.1 mmol). The
Cell death analysis
mixture was stirred for 2 h at room temperature. Then, the Apoptosis/necrosis hallmarks were analysed by simultaneously
resulting yellow solution was filtered on Celite. The solution measuring the mitochondrial membrane potential (ΔΨm),
was concentrated to ca. 5 ml and addition of hexane (20 ml) exposure of phosphatidylserine (PS). Concentrations of the
afforded 29 as an orange solid (0.135 g, 92.1%). 1H-NMR complexes near IC50 values (6 μM in A549 cells, 2 μM Jurkat)
(CDCl3, 300 MHz, δ (ppm), J (Hz)): rotamer mixture (ratio were employed. In order to evaluate the chemoprotective
1 : 0.8): 8.56 (m, 1H, H1), 7.73 (dd, 1H, J = 7.5 and 1.5, H3), effects of antioxidants, N-acetylcysteine (10 mM) and gluta-
7.42 (m, 30H, ArH), 7.36 (m, 1H, H3), 4.57 (A) and 4.43 (B) (dd, thione (2 mM) were added and incubated with our compounds
1H, J = 8.1 and 3.6, and J = 8.4 and 2.7, Cα,ProH), 3.67 (m, 2H, (10 μM A549 cells, 2 μM Jurkat cells) for 24 h. In brief, 2.5 ×
Cδ,ProH2), 3.61 (A) and 3.49 (B) (s, 3H, OCH3), 2.28 (m, 2H, 105 cells in 200 μl were incubated in ABB (140 mM NaCl,
Cβ,ProH2), 1.92 (m, 2H, Cγ,ProH2). 31P-NMR (CDCl3, 300 MHz, 2.5 mM CaCl2, 10 mM Hepes/NaOH, pH 7.4), with either 5 nM
δ (ppm)): 33.0. 19F-NMR (CDCl3, 300 MHz, δ (ppm)): −77.98. DiOC6 (3) or 60 nM tetramethylrhodamine (TMRE, both from
13C-NMR (CDCl3, 300 MHz, δ (ppm), J (Hz)): rotamer mixture: Molecular Probes) at 37 °C for 10 min. Annexin V-PE or
172.0 and 171.9 (COOMe), 166.4 and 166.2 (CONPro), 156.0 (C, Annexin V-FITC (Invitrogen) at a concentration of 0.5 μg ml−1
Py), 151.4 and 150.9 (CH, C1), 137.9 and 136.7 (CH, C3), 133.9 or 7-AAD (0.5 mg μl−1) was added to samples and incubated at
(d, CH, J = 13.7, C5), 132.3 (d, CH, J = 2.4, C7), 129.4 (d, CH, J = 37 °C for an additional 15 min. In all cases, cells from each
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well were diluted to 1 ml ABB to be analyzed by flow cytometry mine structure–activity relationships (SAR). All the compounds
(FACScan, BD Bioscience, Spain). were tested against diverse tumor cell lines, showing good
To additionally assess cell viability after treatment with the cytotoxicity, with IC50 values in the low micromolar range. The
compounds, 2.5 × 105 cells were harvested and incubated in SAR established for this new bioconjugated family of com-
200 μl of PBS containing 50 ng μl−1 of 7-amino-actinomycin D pounds allowed us to prepare the complex [Au2(SPyCO-
(7-AAD, Inmunostep). When analyzed simultaneously for ProOMe)(PPh3)2]OTf (29), the most potent of the series,
either PS exposure or ΔΨm, 7-AAD was added to the samples in confirming our hypothesis. Moreover, some important cellular
ABB.
events like changes in cell and nucleus morphology, loss of
the mitochondrial membrane potential, production of ROS
and inhibition of TrX were observed after addition of the com-
plexes 6 and 21. Further biological studies and refinement of
our rational design might allow us shortly to prepare more
cytotoxic and selective complexes.
Nuclear morphology
The morphology of nuclei after the treatment with the
different compounds was analysed by staining cells with
Hoechst 33342 (Molecular Probes) at 25 mg ml−1. Cells were
visualized in a fluorescence microscope (Nikon Eclipse 50i)
and the ACT software was used for the acquisition of the
images.
Acknowledgements
Intracellular ROS quantification
The authors thank the Ministerio de Economía y Competitivi-
dad (MEC/FEDER CTQ2013-48635-C2-1-P, CTQ2010-17436 and
SAF2010-14920) and DGA-FSE (E77, E40 and B16) for financial
support. A.G. thanks the CSIC for a JAE-predoc fellowship. LGF
was the recipient of an AECC fellowship.
Oxidative stress was analysed by intracellular staining with the
fluorescent probe 2-hydroxyethidium (2-HE, Molecular
Probes). After 16 h of culture in the presence of compounds 6
and 21, cells were incubated with 2 μM 2-HE at 37 °C for
15 min. The red fluorescence produced by reduction of 2-HE
to ethidium was quantified in a flow cytometer.
Notes and references
Thioredoxin reductase inhibition assay
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17066 | Dalton Trans., 2014, 43, 17054–17066
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