Organometallic titanocene-gold compounds as potential chemotherapeutics in renal cancer. Study of their protein kinase inhibitory properties

Article abstract of DOI:10.1021/om500965k

Early-late transition metal TiAu₂ compounds [(η-C₅H₅)₂Ti{OC(O)CH₂PPh₂AuCl}₂] (3) and new [(η-C₅H₅)₂Ti{OC(O)-4-C₆H₄PPh₂

Full text of DOI:10.1021/om500965k

Article
pubs.acs.org/Organometallics
Organometallic Titanocene−Gold Compounds as Potential
Chemotherapeutics in Renal Cancer. Study of their Protein Kinase
Inhibitory Properties
Jacob Fernandez-Gallardo,† Benelita T. Elie,† Florian J. Sulzmaier,‡,⊥ Mercedes Sanau,§ Joe W. Ramos,*,‡
́
́
and María Contel*,†,‡
†Department of Chemistry, Brooklyn College and The Graduate Center, The City University of New York, Brooklyn, New York
11210, United States
‡Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Manoa, Honolulu, Hawaii 96813, United
States
§Departamento de Química Inorgan
́
ica, Universidad de Valencia, Burjassot, Valencia, 46100, Spain
S
* Supporting Information
ABSTRACT: Early−late transition metal TiAu2 compounds [(η-C5H5)2Ti{OC(O)CH2PPh2AuCl}2] (3) and new [(η-
C5H5)2Ti{OC(O)-4-C6H4PPh2AuCl}2] (5) were evaluated as potential anticancer agents in vitro against renal and prostate
cancer cell lines. The compounds were significantly more effective than monometallic titanocene dichloride and gold(I)
[{HOC(O)RPPh2}AuCl] (R = −CH2− 6, −4-C6H4− 7) derivatives in renal cancer cell lines, indicating a synergistic effect of the
resulting heterometallic species. The activity on renal cancer cell lines (for 5 in the nanomolar range) was considerably higher
than that of cisplatin and highly active titanocene Y. Initial mechanistic studies in Caki-1 cells in vitro coupled with studies of their
inhibitory properties on a panel of 35 kinases of oncological interest indicate that these compounds inhibit protein kinases of the
AKT and MAPKAPK families with a higher selectivity toward MAPKAPK3 (IC50 3 = 91 nM, IC50 5 = 117 nM). The selectivity
of the compounds in vitro against renal cancer cell lines when compared to a nontumorigenic human embryonic kidney cell line
(HEK-293T) and the favorable preliminary toxicity profile on C57black6 mice indicate that these compounds (especially 5) are
excellent candidates for further development as potential renal cancer chemotherapeutics.
antitumor activity in in vitro and in vivo experimental models
even against cisplatin-resistant cells and tumors generally
difficult to treat.12,13 However, the efficacy of Cp2TiCl2 in
phase II clinical trials in patients with metastatic renal cell
carcinoma14 or metastatic breast cancer was too low to be
pursued.15
During the past years there has been a renewed interest in
the potential of more stable titanocene complexes as anticancer
agents.16 The most promising candidates have been com-
pounds described by the group of M. Tacke in Dublin.17−22
Substituted titanocenes such as Titanocene Y (Chart 1) have
INTRODUCTION
■
Cisplatin and the follow-on drugs carboplatin (paraplatin) and
oxaliplatin (eloxatin) are used to treat 40−80% of cancer
patients alone or in combination chemotherapy.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 have
been reported in the past two decades.3−8 Metallocene
dihalides (Cp2MCl2, Cp = cyclopentadienyl, M = Ti, V, Nb,
Mo, Re) were the first organometallic compounds with
antitumor properties to be identified.9,10 Titanocene dichloride
(Cp2TiCl2, Chart 1) was the first organometallic complex to
enter clinical trials in 1993.11 Cp2TiCl2 exhibited considerable
Received: September 21, 2014
Published: October 30, 2014
© 2014 American Chemical Society
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metals with multiple biological targets or by the improved
chemicophysical properties of the resulting heterometallic
compound. Early−late transition metal complexes have been
the subject of research due to their potential in catalysis.38 We
and others have reported on early−late transition metal
compounds based on titanocene fragments (Ru−Ti39 and
Ti−Au40,41 in Chart 2). These compounds showed a cytotoxic
effect on human ovarian39−41 and prostate41 cancer cell lines
and were more active than their Ti or M (Ru or Au) separate
monometallic fragments.
Chart 1. Selected Organometallic Titanium Derivatives with
Relevant Antitumor Properties
It has been proposed that titanocene dichloride hydrolyzes at
pH 7, liberating the Cp rings42 and binding to transferrin to get
transported into the tumor cells and released by a nonredox
mode of action (different from that of iron).43 Thus, the
heterometallic compounds previously described39−41 could
potentially break into monometallic species in physiological
media or in vivo before reaching the tumors. We hypothesized
that incorporating the second metal to a ligand strongly bound
to the titanium(IV) center would ensure that heterometallic
Ti−M species remain after the Ti−Cp hydrolysis takes place
under physiological pH conditions. Since Ti−O bonds are
considerably stronger (ΔHf 298 = 662(16) kJ/mol) than Ti−C
(ΔHf 298 = 439 kJ/mol) or Ti−Cl (ΔHf 298 = 494 kJ/mol)
bonds, we envisioned a carboxylate as the ideal group to bind
titanium(IV) centers. Indeed, a compound in which gold−
diphenylphosphinoacetate fragments had been coordinated to a
titanocene moiety to generate a TiAu2 heterometallic species44
was described in 2000 (3 in Scheme 1).
We report here on the synthesis and characterization of a
related compound with a more rigid structure (5 in Scheme 1)
as well as the complete characterization of 3 (including its
crystal structure). The stability of these compounds over time
in DMSO-d6 (dimethyl sulfoxide) and PBS (phosphate buffer
saline) solution has been monitored by NMR and UV−vis
spectroscopy, respectively. We describe preliminary biological
data on their in vitro activity against human renal (A498, UO31,
Caki-1) and prostate (PC3, DU145) cancer cell lines and a
nontumorigenic human embryonic kidney cell line (HEK-
293T). The compounds induced cell death, and the type of cell
death (apoptosis versus necrosis) has been investigated. The
compounds were also tested for their possible interactions with
plasmid (pBR322) DNA used as a model nucleic acid. More
importantly, we present a study of the inhibitory effects of some
titanocene−gold TiAu2compounds (1, 3) and titanocene
dichloride against a panel of 34 protein kinases of oncological
interest and verify activity in cancer cell lines against these
targets. In addition, we include preliminary toxicity data of
compounds 3 and 5 on C57black6 mice.
shown activity in vivo against human breast18 and epidermoid
carcinoma19 xenografts in mice.
The in vivo studies on human renal cancer cells (Caki-1) in
mice with Titanocene Y,20 Titanocene Y*,21 and recently
described water-soluble Titanocene T22 (Chart 1) have shown
significant tumor inhibition, which may lead to clinical tests
against metastatic renal cancer.
Another promising family of metallodrugs for cancer
chemotherapy is that of gold complexes. In particular, a
number of gold compounds have overcome cisplatin resistance
to specific cancer cells,23,24 which makes them attractive
potential therapeutics. In addition, it has been found that
DNA is not the primary target for most gold compounds,25
reinforcing the idea that their mode of action is different than
that of cisplatin. The inhibition of mitochondrial enzymes and
of the proteasome for gold compounds has been reported.26,27
Histone deacetylases, mTor, cathepsin cysteine proteases, and
PKC and cyclin-dependent kinases have been proposed as
possible biochemical targets for some of the gold(III) and
gold(I) complexes.28−31 Gold(I)−thiolate compounds (such as
aurothiomalate) inhibit the protein kinase PKCi,32,33 while
auranofin is known to inhibit IkB kinase (IKK).34 More
recently, gold compounds have been found to efficiently inhibit
PARP-1 (poly(ADP-ribose) polymerase-1).35−37
Within the frame of exploring compounds that may
overcome cisplatin resistance, there has been a growing interest
in heterometallic complexes as potential anticancer agents.37
The hypothesis is that the incorporation of two different
cytotoxic metals in the same molecule may improve their
activity as antitumor agents due to interaction of the different
Chart 2. Selected Heterometallic Titanocene−Ruthenium and Titanocene−Gold Derivatives with Antiproliferative Activities
against Human Ovarian39,40 and Prostate41 Cancer Cell Lines
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a
Scheme 1. Preparation of Heterometallic Titanocene−Gold Complexes [(η-C5H5)2Ti{OC(O)RPPh2AuCl}2]
a
R = −CH2− 3;44 −4-C6H4− 5; (i) CH2Cl2 RT, 6 h; (ii) CHCl3 RT, 1.5 h; (iii) CH2Cl2 RT.
Figure 1. Two ORTEP views of the molecular structure of 3 showing the labeling scheme. Labeling for hydrogen and carbon atoms is omitted for
clarity. A drawing of the molecular structure containing carbon atoms labeled is provided in the SI.
yields. Addition of 2 equiv of [AuCl(tht)] to titanocenes 244
RESULTS AND DISCUSSION
1. Synthesis and Characterization. The synthesis of
■
and 4 affords the heterometallic complexes [(η-C5H5)2Ti{OC-
(O)RPPh2AuCl}2] (R = −CH2− 3;44 −4-C6H4− 5) in
heterometallic compound TiAu2 3 has been performed
following the procedure previously described.44 The reaction
moderate to high yields. Compounds 3 and 5 are obtained as
of [Cp2TiMe2] and 2 equiv of acid [PPh2-CH2-CO2H]
generates the titanocene species with phosphino acetate ligands
[(η-C5H5)2Ti{OC(O)CH2PPh2}2] (R = −CH2− 244) with
concomitant elimination of methane (Scheme 1).
Remarkably, we were able to prepare new titanocene
complex [(η-C5H5)2Ti{OC(O)-4-C4H6PPh2}2], 4, starting
from commercially available [Cp2TiCl2] and the sodium salt
[PPh2-4-C6H4-CO2Na], which eliminates the need for using
[Cp2TiMe2]. The reaction to obtain 2 starting from [Cp2TiCl2]
and the corresponding sodium salt [PPh2-CH2-CO2Na] was
unsuccessful. Both 2 and 4 are obtained as yellow solids in high
pale yellow, air- and moisture-stable solids, which can be kept at
low temperatures (fridge) for months. Titanocenes 2 and 4 and
the heterometallic compounds 3 and 5 are less acidic than
titanocene dichloride (Experimental Section). The compounds
are soluble in DMSO/H2O (1:99) mixtures at micromolar
concentrations, which is relevant for subsequent biological
testing. The structures in Scheme 1 have been proposed based
on published44 spectroscopic and analytical results for
compounds 2 and 3, crystallographic data for 2,44 and on our
own results for 4 and 5 (see Experimental Section).
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Table 1. Selected Structural Parameters of Complex 3 Obtained from X-ray Single-Crystal Diffraction Studies (Bond Lengths in
Å and Angles in deg)
Ti(1)−O(1)
O(1)−C(2)
O(2)−C(2)
C(2)−C(1)
1.962(10)
1.302(17)
1.169(17)
1.56(2)
Ti(1)−O(3)
O(3)−C(4)
O(4)−C(4)
C(4)−C(3)
1.957(10)
1.310(17)
1.188(17)
1.51(2)
Au(2)−P(2)
Au(2)−Cl(2)
Ti(1)−O(1)
O(1)−C(2)
O(2)−C(2)
C(2)−C(1)
2.226(4)
2.279(4)
1.962(10)
1.302(17)
1.169(17)
1.56(2)
Au(1)−P(1)
Au(1)−Cl(1)
Ti(1)−O(3)
O(3)−C(4)
O(4)−C(4)
C(4)−C(3)
2.222(4)
2.287(4)
1.957(10)
1.310(17)
1.188(17)
1.51(2)
Au(2)−P(2)
Au(2)−Cl(2)
2.226(4)
2.279(4)
2.381(2)
2.385(2)
2.069
Au(1)−P(1)
Au(1)−Cl(1)
average C−C in Cp ring C(21)−C(25)
average C−C in Cp ring C(11)−C(15)
2.222(4)
2.287(4)
1.386(3)
1.392(3)
average Ti(1)−C(21−25)
average Ti(1)−C(11−15)
distance Ti−Z (centroid of Cp)
Ti(1)−O(3)−C(4)
O(1)−Ti(1)−O(3)
Ti(1)−O(1)−C(2)
C15−Ti1−C21
O(1)−C(2)−O(2)
O(2)−C(2)−C(1)
C(1)−C(2)−P(2)
141.0(9)
95.8(4)
C23−Ti1−C12
171.9(7)
O(3)−C(4)−O(4)
O(4)−C(4)−C(3)
C(3)−C(4)−P(1)
P(2)−Au(2)−Cl(2)
P(1)−Au(1)−Cl(1)
123.0(13)
122.6(14)
111.5(11)
173.71(19)
173.15(17)
137.2(9)
100.9(9)
126.5(15)
122.1(14)
112.8(11)
The crystal structure of 3 has been determined by an X-ray
diffraction study confirming that there are two molecules of
gold(I)−chloride−phosphino acetate coordinated to the
titanocene moiety through one of the oxygen atoms of the
carboxylate ligand. Figure 1 depicts two different views of the
molecule in compound 3, and selected bond distances and
angles are collected in Table 1 (crystallographic details can be
found in the SI).
d6/D2O solution in the amount necessary to perform NMR
spectroscopy. The vis−UV of compounds 3 and 5 (micromolar
concentration) in 1:99 DMSO/PBS solution do not change
much over time (24 h, Figures S21 and S.23 in the SI). Mass
spectrometry also indicates the presence of species containing
both titanium and gold in 1% DMSO/PBS solution after 24 h
(Figures S24−S33 in the SI). We used micromolar solutions of
3 and 5 in DMSO/media or DMSO/PBS (1:99) for
subsequent biological testing.
2. Biological Activity. 2.1. Cytotoxicity and Cell Death.
The cytotoxicity of the heterometallic complexes [(η-C5H5)2Ti-
{OC(O)RPPh2AuCl}2] (R = −CH2− 3;44 −4-C6H4− 5) and
monometallic gold(I) complexes [HOC(O)RPPh2AuCl] (R =
−CH2− 6;45 −4-C6H4− 7 in Figure 2) was assayed by
monitoring their ability to inhibit cell growth using the XTT
assay (see the Experimental Section).
The structure of titanocene attached to the two phosphine−
acetate ligands is very similar to that of [(η-C5H5)2Ti{OC(O)-
CH2PPh2}PdCl2].44 As in the Pd structure, the monodentate
carboxylate coordination is also indicated by the significant
differences in C−O bond lengths depending on whether the
oxygen is coordinated to titanium [1.962(10); 1.957(10) Å] or
not [3.460(11), 3.486(11) Å]. The titanium has a distorted
tetrahedral environment comprising two cyclopentadienyl
rings, the Z−Ti−Z angle being 132.89° and the O(1)−Ti−
O(3) angle 95.8(4)°. The gold atoms are in a quasi linear
arrangement (P(1)−Au(1)−Cl(1) 173.15(17)° and P(2)−
Au(2)−Cl(2) 173.71(19)°). The distances Au−P and Au−Cl
(Table 1) are almost identical to the distances found in
complex [{HOC(O)CH2PPh2}AuCl] (6),45 of 2.220(2) and
2.270(3) Å, respectively, and are in the range of many other
gold−phosphino chloride derivatives described in the literature.
There is no contact Au−Au, as the distance found is 3.693 Å.
The stability of the compounds has been evaluated by
Figure 2. Structures of monometallic [{HOC(O)RPPh2}AuCl] (R =
−CH2− 6;45 −4-C6H4− 7).
1
31P{1H} and H NMR spectroscopy in DMSO-d6 and by vis−
The cytotoxic activity of the compounds was determined as
described in the Experimental Section in the human renal
cancer A498, UO31, and Caki-1 cell lines and in the human
prostate cancer PC3 and DU-145 cell lines, in comparison to
cisplatin, titanocene dichloride (Chart 1), and Titanocene Y46
(Chart 1). The results are summarized in Table 2.
UV spectroscopy in DMSO/PBS solution over time (see SI).
As reported for titanocene dichloride, compounds 3 and 5 lose
the cyclopentadienyl groups in DMSO (most plausibly being
replaced by OH groups).42 We studied a solution of 3 in 50:50
1
DMSO-d6/D2O by 31P{1H} and H NMR spectroscopy, and
We had previously reported41 on the cytotoxic activity of
[TiCl2{η5-C5H4PPh2(AuCl)}2] (1) in human ovarian (IC50 24
h = 2.44 μM) and prostate cancer cell line DU145 (IC50 24 h =
27.26 μM), and we did a preliminary evaluation of 1 in human
prostate cancer cell line PC3, human renal cancer cell line
we observed that 3 is stable for 24 h with a half-live of 48 h,
indicating that the compound is more stable in D2O than in
DMSO (see Figures S16 and S17 in the SI). Titanocene
dichloride is also known to hydrolyze much faster in DMSO
than in water.42 Compound 5 precipitated in a 50:50 DMSO-
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Table 2. IC50 (μM) of Heterometallic TiAu2 Compounds 3 and 5, Monometallic Au 6 and 7, Cisplatin, Titanocene Dichloride,
a
and Titanocene Y in Human Cell Lines
A498
UO31
Caki-1
HEK-293T
24 0.73 (24 h) 51.7 4.2 (24 h) 38.6 5.5 (24 h)
35.5 1.4 (72 h)
58 11.0 (24 h) 27.3 2.7 (24 h) 33.8 2.1 (24 h) 36.5 3.3 (24 h) 38.2 2.2 (24 h) 34.7 1.8 (24 h)
43 4.9 (72 h) 5.2 1.6 (72 h) 23.0 4.1 (72 h) 9.7 2.5 (72 h) 30.2 4.2 (72 h) 31.3 3.1 (72 h)
6.8 0.2 (24 h) 10.3 4.1 (24 h) 39 4.1 (24 h) 51 4.2 (24 h) 33.4 1.8 (24 h)
0.3 0.06 (72 h) 1.0 0.29 (72 h) 20.1 1.6 (72 h) 37.7 7.1 (72 h) 6.6 1.8 (72 h)
PC3
DU145
[Cp2Ti{OC(O)CH2PPh2AuCl}2], 3
[HOC(O)CH2PPh2AuCl], 6
8.7 1.7 (24 h)
6.3 1.1 (24 h) 6.0 1.8 (24 h)
3.2 0.38 (72 h) 1.4 0.1 (72 h) 2.2 0.99 (72 h) 6.9 2.4 (72 h) 27.1 4 (72 h)
[Cp2Ti{OC(O)-4-C6H4PPh2AuCl}2], 5 28 3.4 (24 h)
6.9 2.2 (72 h)
[HOC(O)-4-C6H4PPh2AuCl], 7
cisplatin
36.1 6.3 (24 h) 38.3 4.1 (24 h) 28.4 + 5.9 (24 h) 34.2 3.7 (24 h) >200 (24 h)
32 2.7 (24 h)
21 2.5 (72 h)
1.2 0.8 (72 h) 19.2 2.9 (72 h) 31 0.9 (72 h) 78 18.1 (72 h) 39 5.7 (72 h)
74.7 6.0 (24 h) >100 (24 h)
68.8 0.14 (24 h) 64.4 7.9 (24 h) 92 18 (24 h)
3.2 0.13 (72 h) 14 2.3 (72 h)
44.5 0.33 (24 h)
12.1 3.9 (72 h)
>200 (24 h)
37.2 4.6 (72 h) 8.9 2.7 (72 h) 29 4.1 (72 h)
[Cp2TiCl2] titanocene dichloride
Titanocene Y
>200 (24 h)
>200 (72 h)
>200 (24 h)
>200 (24 h)
>200 (72 h)
>200 (24 h)
>200 (24 h)
>200 (72 h)
>200 (24 h)
>200 (24 h)
>200 (7 2 h)
>200 (24 h)
>200 (24 h)
>200 (72 h)
>200 (24 h)
>200 (72 h)
>200 (24 h)
>29.6 2.8 (72 h) >200 (72 h)
29.4 4.2 (72 h) >200 (72 h)
58.1 11.2 (72 h) 55.2 7.9 (72 h)
a
All compounds were dissolved in 1% of DMSO and diluted with water before addition to cell culture medium for a 24 or 72 h incubation period.
Cisplatin and titanocene dichloride were dissolved in H2O. Data are expressed as mean SD (n = 3).
Figure 3. Selectivity of TiAu2 compounds 3 and 5 between normal cells and cancer cells in vitro. The effects on cell viability of 3, 5, or cisplatin on
UO31 and HEK-293T. UO31 and HEK-293T cells were seeded into a 96-well plate, and different concentrations of 3, 5, or cisplatin were then
added to the cells after 24 h. The cell viability was measured by the XTT assay after the cells were treated with the compounds for 72 h.
A498, and nontumorigenic human MCF10A breast cancer cell
line (IC50 PC3 = 18.95 μM; IC50 A498 = 16.26 μM; IC50
MCF10A = 9.64 μM; XTT assay 20 h). However, the fact that
1 decomposes over time to species [AuClPPh2Cp], which are
known to be cytotoxic but also poorly selective (as we observed
when testing 1 on MCF10A), led us to focus attention on the
study of heterometallic species 3 and 5 (Table 2). The
heterometallic compounds 3 and 5 are more cytotoxic on renal
cancer cell lines than on prostate cancer cell lines. In the case of
prostate cancer cell lines the compounds are more toxic than
cisplatin after 24 h, but only compound 5 is more toxic on
DU145 after 72 h. They are also more toxic than titanocene
dichloride and more toxic than Titanocene Y in these cell lines.
The heterometallic compounds are considerably more toxic
to the renal cancer cell lines at both 24 and 72 h than cisplatin,
titanocene dichloride, and even Titanocene Y. In addition,
heterometallic compounds 3 and 5 are more toxic than the
monometallic gold compounds on A498, UO31, and Caki-1
(both at 24 and 72 h), while being considerably less toxic to the
nontumorigenic human embryonic kidney cell line (HEK-
293T). This selectivity (see Figure 3) is especially pronounced
for compound 5 on UO31 cells with an IC50 value in the
nanomolar range.
synergistic effect of the heterometallic complexes in their in
vitro activity on renal cancer cell lines.
The trend for the monometallic complexes is not that clear. 6
is more toxic to prostate cell lines than to A498. 7 is more toxic
to UO31 than to PC3 and DU145 cell lines and in general is
poorly cytotoxic to prostate cell lines. Both 6 and 7 are more
toxic to UO31 and Caki-1 with respect to the other cell lines
studied.
In order to gain some insight into the type of cell death that
the heterometallic complexes induce in the cancer cell lines, we
performed cell death assays on Caki-1 cells with complexes 3
and 5 dissolved in 1% DMSO (see Experimental Section for
details) and using 1% DMSO alone in media and staurosporine
as controls. As cells may die through programmed cell death
(apoptosis) or necrosis, the mode of death mediated by our
compounds was investigated.
In early stages of apoptosis, one of the significant
biochemical features is loss of plasma membrane phospholipid
asymmetry, due to translocation of phosphatidylserine (PS)
from the cytoplasmic to extracellular side. This characteristic
allows detection of externalized PS by the specific binding of
annexin V (FITC-conjugated). Apoptotic cell death will
eventually result in the permeabilization of the cell membrane,
allowing propidium iodide (PI) to stain DNA within the
nucleus. Alternatively, necrotic cells are immediately permeable
and stain positive for PI and PS with no intervening PS positive
only step. As shown in Figure 3, each histogram is divided into
four quadrants with the left top quadrant detecting necrotic
We studied the effect of the combination of monometallic
compound 6 and titanocene dichloride in renal cancer cell lines
at 24 h, which in all cases gave IC50 values > 100 μM (data not
shown). This fact supports the idea that there is indeed a
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Figure 4. Cell death assays on Caki-1 cells induced by 3 and 5 (10 μM) measured by using two-color flow cytometric analysis, after 6 h of
incubation. 1% DMSO is vehicle alone control, and staurosporine is a known inducer of apoptosis as positive control.
Figure 5. Electrophoresis mobility shift assays for cisplatin, titanocene dichloride, heterometallic TiAu2 compounds 3 and 5, and monometallic 6 and
7 (see Experimental Section for details). DNA refers to untreated plasmid pBR322. Letters a, b, c, and d correspond to metal/DNAbp ratios of 0.25,
0.5, 1.0, and 2.0, respectively.
cells without an annexin V-FITC signal. The right top quadrant
shows cells with compromised membranes that are permeable
to PI and stained with annexin V-FITC, which is indicative of
necrosis and late apoptosis. The left bottom quadrant shows
live cells that have intact membranes (not stained), while the
right bottom quadrant represents cells that were stained
(bound) with annexin V-FITC, which is indicative of early
apoptosis. After incubation during 6 h with 10 μM of
compounds 3 and 5, necrosis can be clearly proposed for 3
(Figure 4). Compound 5 (with slower action on cancer cells,
see in vitro IC50 values at 24 and 72 h) shows a pattern in
accordance with apoptosis (compared to staurosporine, an
apoptotic agent). Experiments at shorter (3) and longer (3 and
5) times are collected in the SI (Figures S34−S36), affording
the same results. We are currently investigating the precise
mechanism of cell death of these compounds. The induction of
both apoptosis and necrosis are known for gold com-
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pounds,47,48 while titanocenes such as Titanocenes C, X, and Y
are known to induce apoptosis in different cancer cell lines.13
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.49 However, a
variety of platinum compounds act as DNA intercalators upon
coordination to the appropriate ancillary ligands.50 DNA was
believed to be the target for titanocene dichloride.42a Titanium
accumulates in the cells in nuclear heterochromatin and, to a
minor extent, in the nucleolus and ribosomes.51 Titanium−
DNA adducts were detected in A2780 cells treated with
Cp2TiCl2, and this compound also inhibited DNA and RNA
synthesis.52 However, most recent reports on titanocene
dichloride indicate that at physiological pH it neither binds
strongly to DNA nor suppresses DNA-processing enzymes.53
Titanocene Y has been recently shown to interact weekly with
DNA.54 As commented before, most gold-based compounds do
not display a strong interaction with DNA.25
Figure 6. Electrophoresis mobility shift assays for titanocene
dichloride and compounds 3, 5, 6, and 7 at a metal/DNAbp ratio of
2.0 (see Experimental Section for details). DNA refers to untreated
plasmid pBR322. Lanes a, b, and c correspond to pH 6, 7, and 8,
respectively.
from the serine/threonine family (see Experimental Section for
details). The graph in Figure 7 shows the inhibitory activity on
30 nonlipid kinases for titanocene chloride and compounds 1
and 3 at concentrations of 10 μM (see Experimental Section for
details).
Thus, we performed agarose gel electrophoresis studies to
unravel the effects of the heterometallic compounds 3 and 5,
monometallic gold(I) derivatives 6 and 7, titanocene dichloride,
and cisplatin on plasmid (pBR322) DNA (Figure 5). 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 metal−DNA binding.
Generally, the larger the retardation of supercoiled DNA
(CCC, form I), the greater the DNA unwinding produced by
the drug.55 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 a−d for cisplatin
in Figure 5). Treatment with increasing amounts of
monometallic Au(I) compounds 6 and 7 or heterometallic
TiAu2 derivatives 3 and 5 does not affect the mobility of the
faster-running supercoiled form (form I) even at the highest
molar ratios (d). This is also in accordance with previously
reported results on a titanocene−gold(I) phosphine derivative,
[(η5-C5H5)-(μ-η5:k1-C5H4PPh2)TiCl2]2Au]PF6 (Chart 2),
which did not interact with plasmid DNA.40 We had found
previously that titanocene dichloride does not interact with CT-
DNA at physiological pH by CD spectroscopic studies.41
Compounds of the type [TiCl2{η5-C5H4 PPh2(AuCl)}2] (1)
displayed a stronger interaction with CT-DNA at pH 7 than
titanocene dichloride.41 However, the interaction was shown to
be electrostatic in nature in accordance with the data for most
gold compounds that show no or a weak interaction with
DNA.25
The graph in Figure 8 shows the IC50 values (values at which
50% of the enzymatic activity is inhibited) on the four lipid PI3
kinases for titanocene dichloride. Compound 3 was inactive
against the PI3 kinases even at values of 100 μM, while the IC50
values for compound 1 ranged from 39.8 to 114.5 μM and thus
are not plotted in Figure 8. From this preliminary screening we
narrowed down the panel of 30 kinases to those for which the
compounds show an inhibitory effect of at least 50%.
Titanocene dichloride did not inhibit the enzymatic activity
to levels of 50% or below for any of these 30 kinases even at
concentrations of 100 μM. The IC50 values of 1 and 3 were
subsequently calculated for the kinases in which they had an
inhibitory effect of at least 50% (AKT1, AKT2, and AKT3 for 1
and 3; ERK5/MAPK7 for 1; and MAPKAPK2, MAPKAPK3,
MAPKAPK5/PRAK, and PKCtheta for 3).
Compound 1 inhibited ERK5/MAPK7 in the micromolar
range (IC50 = 3.95 μM), and the IC50 for compound 3 on
PKCtheta was 11.6 μM. Figure 9 shows the IC50 values below
1500 nM for 1 and/or 3 in specific kinases. From these studies
we found that compounds 1 and 3 inhibit AKT protein kinases
in the micromolar and nanomolar range (342 to 1425 nM).
The AKT3 protein kinase is more effectively inhibited than
AKT1 or AKT2 for both compounds (3.5 and 2.5 times,
respectively).
In conclusion the inhibitory effects of titanocene dichloride
and the heterometallic TiAu2 compounds 1 and 3 on protein
kinases involved in cancer are very different. While titanocene
dichloride inhibits exclusively protein kinases of the PI3 kinase
family at nanomolar concentrations, compounds 1 (based on
gold(I) fragments attached directly to the Cp rings of the
titanocene, [TiCl2{η5-C5H4PPh2(AuCl)}2]) and 3 (based on
titanocene attached to the gold(I) fragments through acetate
groups [(η-C5H5)2Ti{OC(O)CH2PPh2AuCl}2]) inhibit in a
similar manner protein kinases from the family AKT (especially
AKT3). In addition, compound 3 inhibits protein kinases of the
MAPKAPK families with IC50 in the nanomolar range for
MAPKAPK2 (712 nM) and MAPKAPK3 (91 nM).
Compound 5 [(η-C5H5)2Ti{OC(O)-4-C6H4PPh2AuCl}2],
closely related to 3, was tested against the three kinases for
which 3 was most active, confirming that this type of TiAu2
compound inhibits AKT3 (IC50 = 355 nM), MAPKAPK2 (IC50
= 317 nM), and MAPKAPK3 (IC50 = 117 nM) very efficiently.
In a separate experiment, we confirmed that compounds 3 and
The study of the interaction of these compounds at a more
acidic or basic pH shows that there is no significant change with
respect to neutral pH, and all the compounds do not interact
with plasmid (pBR322) DNA (Figure 6).
2.2. Protein Kinase Inhibition Studies and Initial
Mechanistic Insights. Protein kinases have an important role
in oncogenesis and tumor progression, and they have therefore
received increasing attention as targets for anticancer drugs,
including recent and relevant examples of organometallic
complexes (Meggers and co-workers).55−57 In order to gain
some mechanistic understanding of the mode of action of
titanocene−gold derivatives, we tested the previously described
compounds [TiCl2{η5-C5H4PPh2(AuCl)}2], 1, [(η-C5H5)2Ti-
{OC(O)CH2PPh2AuCl}2], 3, and titanocene dichloride
[Cp2TiCl2] against a panel of 34 kinases of oncological interest
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Figure 7. Inhibition of enzymatic activity on a panel of 30 kinases (cell-free assay using purified recombinant kinases) by titanocene dichloride
[Cp2TiCl2] and heterometallic TiAu2 compounds 1 (Chart 2) and 3 (Scheme 1). The y-axis shows the 30 selected protein kinases, while the x-axis
displays the enzymatic activity (0 to 100%) in the presence of titanocene dichloride and compounds 1 and 3.
Figure 9. IC50 (nM values) for TiAu2 compounds 1 and 3 against
selected protein kinases of the AKT and MAKPAPK families. The IC50
were calculated only for compounds that showed an inhibitory effect
of at least 50% when tested at 10 μM concentration (see Figure 7).
Figure 8. IC50 (nM values) for titanocene dichloride [Cp2TiCl2]
against PI3 kinases. Compounds 1 and 3 were poor inhibitors of these
lipid PI3 kinases.
performed with titanocene derivatives. A COMPARE analysis
on some gold(III) and gold(I) complexes showed some protein
kinases as likely biochemical targets.58 Thus, kinase mTOR is a
likely target for Au(I) compounds such as auranofin. For
5 do not inhibit the protein kinase mTOR/FRAP1 at
concentrations of 1 μM.
There are a very limited number of studies on the inhibition
of specific kinases by gold compounds, and to the best of our
knowledge there are no reports on studies of this type
gold(III) derivatives with nitrogen ligands PKC and cyclic
protein kinases (CDKs) appear to be plausible targets.58 More
detailed studies were performed for Au(I) compounds
aurothioglucose and aurothiomalate, which resulted to be
potent inhibitors of PKCι-par6 in vitro (IC50 ca. 1 μM).59,60
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We have shown here the inhibitory properties of TiAu2 on
AKT protein kinases. Importantly, specific TiAu2 derivatives
such as 3 and 5 also inhibit MAPKAPK2 and MAPKAPK3 with
a higher specificity toward MAPKAPK3. Compounds 3 and 5
have an IC50 value 5 times lower than the most potent
MAPKAPK3 kinase inhibitors found among 158 commercially
available small molecules that have been screened across 234
human kinases.61
To further investigate the ability of these compounds to
impair MAPKAPK2/3 activity in cancer cells in vitro, we
examined the ability of the heterometallic compounds 3 and 5
to inhibit IL6 secretion, which can be activated by
MAPKAPK2/3. We found that the compounds significantly
reduced secretion of IL-6 in Caki renal cancer cells, which may
support the hypothesis that the compounds inhibit activity of
MAPKAPK2/3 in these cells.62 However, other effects such as
mitochondrial damage cannot be excluded, and more detailed
mechanistic studies are under way.
been reported in response to treatment with chemotherapeu-
tics. The compounds can therefore be well tolerated in mice
and will be used in subsequent in vivo analyses.
CONCLUSIONS
■
In conclusion, we have demonstrated that early−late transition
metal TiAu2 compounds of the type [(η-C5H5)2Ti{OC(O)-
RPPh2AuCl}2] (R = −CH2− 3, −4-C6H4− 5) display
significant cytotoxicity against human renal cancer cell lines in
vitro. The convenient high-yield two-step synthesis of 5 starting
from commercially available titanocene dichloride is described.
The compounds have been significantly more effective than
monometallic titanocene dichloride and gold(I) [{HOC(O)-
RPPh2}AuCl] (R = −CH2− 6, −4-C6H4− 7) derivatives in
renal cancer cell lines, indicating a synergistic effect of the
resulting heterometallic species. The activity on renal cancer
cell lines (for 5 in the nanomolar range) has been considerably
higher than that of cisplatin and highly active Titanocene Y.
The cell death induced by the compounds has been studied,
indicating apoptosis for compound 5. The lack of interaction of
the compounds with plasmid (pBR322) DNA indicates that
other biomolecular targets may be implicated in the cell death
pathways. The study of their inhibitory properties on a panel of
35 kinases of oncological interest shows that these compounds
inhibit protein kinases of the AKT and MAPKAPK families
with a higher selectivity toward MAPKAPK3 (IC50 3 = 91 nM,
IC50 5 = 117 nM). These values make compounds 3 and 5 the
most potent MAPKAP3 kinase inhibitors reported so far. In
addition, the inhibition of secretion of IL6 in Caki-1 cells
observed for 3 and 5 may support the hypothesis that these
compounds inhibit activity of MAPKAP2/3 in these cells.
Inhibition of MAPKAPK2/3 is therefore likely to mediate in
part the antitumor activity of the compounds. We also report
here for the first time that titanocene dichloride inhibits PI3
kinases.
This study will undoubtedly help in the design of related
compounds (titanocenes and titanocene−gold) with even
higher target specificity by rational modification of the ligand
scaffolds.
The selectivity of 3 and 5 in vitro against renal cancer cell
lines when compared to a nontumorigenic human embryonic
kidney cell line (HEK-293T) and the favorable preliminary
toxicity profile on C57black6 mice indicate that these
compounds (especially 5) can be excellent candidates for
further development as potential renal cancer chemotherapeu-
tics.
Figure 10. Compounds 3 and 5 may affect downstream targets of
MAPKAP2/3 in renal cancer cells. Inhibition of IL6 secretion over the
course of 6 h in Caki-1 cells treated with compound 3 or 5 (10 μM).
Analysis was done using a capture ELISA approach (human IL-6
ELISA kit).
2.3. Preliminary Toxicity Data on C57black6 Mice. The
preliminary toxicity testing of compounds 3 and 5 was
performed in C57BL/6 female mice 6 to 8 weeks of age (see
Experimental Section for details). The lethal dose for
compounds 3 and 5 is 15 mg/kg/day, as the mice died within
less than 24 h following injection.
The maximum tolerated dose (MTD) was determined by
observing the progression of the mice treated at doses below
the lethal dose. Body weights, changes in behavior, and signs of
distress were recorded. The dose at which neither debilitating
effects nor signs of distress were observed was set as the MTD.
MTD for compounds 3 and 5 is 10 mg/kg/day. The MTD
dose was then confirmed by treating a cohort of three mice per
compound and one control group every other day for 14 days
with the aforementioned MTD dose. One group of mice was
treated with the solvent (negative control). During the trial the
mice did not exhibit any notable sings of distress.
EXPERIMENTAL SECTION
■
General Procedures. All compounds involving titanocene frag-
ments were prepared and handled with rigorous exclusion of air and
moisture under a nitrogen atmosphere by using standard nitrogen/
vacuum manifold and Schlenk techniques. Solvents were purified by
use of a PureSolv purification unit from Innovative Technology, Inc.
Titanocene dichloride and 4-(diphenylphosphino)benzoic acid were
purchased from Aldrich and used without further purification.
Diphenylphosphinoacetic acid,68 complexes 1,41 2,44 3,44 and 6,45
[AuCl(tht)],69 and Titanocene Y46 were prepared as previously
reported. NMR spectra were recorded in a Bruker AV400 (1H NMR at
400 MHz, 13C NMR at 100.6 MHz, and 31P NMR at 161.9 MHz).
Chemical shifts (δ) are given in ppm using CDCl3 as the solvent,
1
unless otherwise stated. H and 13C NMR resonances were measured
Necropsy indicated no notable change in liver or kidney size
and appearance. We observed that the spleens of mice treated
with compounds 3 and 5 were slightly smaller than control
spleens. Enlargement63 and shrinkage64−67 of the spleen have
relative to solvent peaks considering tetramethylsilane = 0 ppm, and
31P{1H} NMR was externally referenced to H3PO4 (85%). Coupling
constants J are given in hertz. IR spectra (4000−250 cm−1) were
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recorded on a Nicolet 6700 Fourier transform infrared spectropho-
tometer on KBr pellets. Elemental analyses were performed by Atlantic
Microlab Inc. (US). Mass (MS) spectra (electrospray ionization, ESI)
were performed on an Waters XEVO triple quadrupole analyzer and
on a Waters Q-Tof Ultima analyzer. The pH was measured in an
OAKTON pH conductivity meter in 1:99 DMSO/H2O solutions.
UV−visible spectra have been recorded using a PerkinElmer Lambda
20 Bio spectrophotometer. X-ray collection was performed at room
temperature on a Kappa CCD diffractometer using graphite-
monochromated Mo Kα radiation (λ = 0.710 73 Å). Electrophoresis
experiments were carried out in a Bio-Rad Mini subcell GT horizontal
electrophoresis system connected to a Bio-Rad Power Pac 300 power
supply. Photographs of the gels were taken with an Alpha Innotech
FluorChem 8900 camera. Protein kinase inhibition studies were
performed by Reaction Biology Corporation.70
Complex 7 was isolated as a white solid in 66% yield (0.168 g). Anal.
Calcd for C19H15AuClO2P (538.71): C, 42.36; H, 2.81. Found: C,
42.54; H, 2.98. 31P{1H} NMR (CDCl3): δ 33.22. 1H NMR (CDCl3): δ
11.17 (1H, s), 8.19 (2H, dd, 3JHH = 8.3 Hz, 4JHH = 1.9 Hz,), 7.57 (13H,
m). 13C{1H} NMR (CDCl3): δ 169.89 (s, CO), 135.53 (d, JPC
=
=
59.6 Hz, 4-C6H4), 134.29 (d, JPC = 13.9 Hz, 2′-C6H4), 134.07 (d, JPC
13.9 Hz, 2-C6H5), 132.43 (d, JPC = 2.5 Hz, 1-C6H5), 132.19 (s, 4′-
C6H4), 130.57 (d, JPC = 11.9 Hz, 3-C6H4), 129.50 (d, JPC = 12.0 Hz,
3′-C6H5), 127.77 (d, JPC = 62.6 Hz, 1′-C6H5). IR (cm−1): 1682 vs
(νasym CO2), 1377 s (νsym CO2), 342 ms (ν AuCl).
X-ray Crystallography. A single crystal of 3 (see details in Table
S1 in the SI) was mounted on a glass fiber in a random orientation.
Data collection was performed at RT on a Kappa CCD diffractometer
using graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å).
Space group assignments were based on systematic absences, E
statistics, and successful refinement of the structures. The structure
was solved by direct methods with the aid of successive difference
Fourier maps and was refined using the SHELXTL 6.1 software
package. All non-hydrogen atoms were refined anisotropically.
Hydrogen atoms were assigned to ideal positions and refined using
a riding model. Details of the crystallographic data are given in Table
S1 (SI). These data can be obtained free of charge from the
Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/
data_request/cif. (CCDC 1008332) or in the SI. Crystals of 3 (yellow
prisms with approximate dimensions 0.25 × 0.23 × 0.22 mm) were
obtained from a solution of 3 in CH2Cl2 by slow diffusion of Et2O at
RT.
Cell Culture. Human renal cell carcinoma lines A498, Caki-1, and
UO31, as well as the human prostate carcinoma cell lines DU145 and
PC3, were newly obtained for these studies from the American Type
Culture Collection (ATCC) (Manassas, VA, USA) and cultured in
Roswell Park Memorial Institute (RPMI-1640) (Mediatech Inc.,
Manassas, VA, USA) media containing 10% fetal bovine serum (FBS,
Life Technologies, Grand Island, NY, USA), 1% minimum essential
media (MEM) nonessential amino acids (NEAA, Mediatech), and 1%
penicillin−streptomycin (PenStrep, Mediatech). HEK-293 cells were
newly purchased from ATCC and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Mediatech) supplemented with 10% FBS,
1% NEAA, and 1% PenStrep. All cells were cultured at 37 °C and 5%
CO2 in a humidified incubator.
Cell Viability Assay. Cells were seeded at a concentration of 5000
cells/90 μL per well of either RPMI or DMEM without phenol red
and without antibiotics, supplemented with 10% FBS and 2 mM L-
glutamine into tissue culture grade 96-well flat bottom microplates
(Thermo Scientific BioLite microwell plates, Fisher Scientific,
Waltham, MA, USA) and grown for 24 h at 37 °C in a humidified
incubator. Afterward, the intermediate dilutions of the compounds
were added to the wells (10 μL) to obtain a final concentration
ranging from 0.1 to 200 μM, and the cells were incubated for 24 or 72
h. Following 24 or 72 h drug exposure, 50 μL per well of 2,3-bis(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) (Roche Diagnostics, Indianapolis, IN, USA) labeling mixture
was added to the cells at a final concentration of 0.3 mg/mL and
incubated for 4 h at 37 °C in a humidified incubator. The optical
absorbance of each well (96-well plates) was quantified using EnVision
multilabel plate readers (PerkinElmer, Waltham, MA, USA) at 450 nm
wavelength. The percentage of surviving cells was calculated from the
ratio of absorbance of treated to untreated cells. The IC50 value was
calculated as the concentration reducing the proliferation of the cells
by 50% and is presented as a mean ( SE) of at least two independent
experiments each with triplicates.
Annexin V/PI Assay. Confluent Caki-1 cells were treated with
either 10 μM 3 or 5, 0.1% DMSO, or 5 μM staurosporine for 6 h
(Figure 4) or for 1 h, 12 h, or 24 h (S34−S36). After incubation, cells
were trypsinized with 0.25% trypsin without EDTA (ethylenediami-
netetracaetic acid, Life Technologies) and stained for extracellular
phosphatidylserine expression using FITC conjugated annexin V to
label early apoptotic cells and costained with propidium iodide to
identify necrotic cells according to the manufacturer’s instructions for
the dyes (BD Biosciences, San Jose, CA, USA). Stained cells were
Ph2P-4-C6H4-COONa. A ethanolic solution of NaOH (1.6 mL, 1 M)
was added to a solution of 4-(diphenylphosphino)benzoic acid (0.5 g,
1.63 mmol) in 20 mL of ethanol and stirred for 30 min at room
temperature. The ethanol was then removed under reduced pressure
to give rise to a white solid, which was washed with diethyl ether (3 ×
15 mL) and isolated in 96% yield (0.514 g). 31P{1H} NMR (CDCl3):
1
δ −6.00. H NMR (CDCl3): δ 7.09 (14H, m). NMR (CDCl3): δ
176.60 (s, CO), 140.37 (s, 1-C6H4), 136.85 (d, JPC = 11.6 Hz, 3-
C6H4), 133.76 (d, JPC = 20.6 Hz, 1-C6H5), 132.82 (s, 4-C6H5), 133.64
(s, 4-C6H4), 128.60 (m, 2-C6H4, 3-C6H5, 2-C6H5). IR (cm−1): 1583 m,
1536 m (νasym CO2), 1377.24 s, 1306 m (νsym CO2).
[(η-C5H5)2Ti{OC(O)-4-C6H4-PPh2}2] (4). Titanocene dichloride
(0.095 g, 0.38 mmol) and p-Ph2P-C6H4-COONa were dissolved in 6
mL of chloroform, giving rise to a red suspension, which was stirred at
room temperature. After 1 h, the suspension became orange. The
solvent was removed under vacuum, and the residue dissolved in
dichloromethane (8 mL) and extracted to yield a yellow solid (0.249 g,
83%), which was characterized as 4. Anal. Calcd for C48H38O4P2Ti
(788.64): C, 73.10; H, 4.86. Found: C, 73.35; H, 4.90. 31P{1H} NMR
1
(CDCl3): δ −5.32. H NMR (CDCl3): δ 7.96 (4H, m), 7.37−7.30
(24H, m), 6.63 (10H, s). 13C{1H} NMR (CDCl3): δ 171.73 (s, C
O), 142.36 (d, JPC = 13.5 Hz, 1-C6H4), 136.56 (d, JPC = 10.8 Hz, 3-
C6H4), 133.87 (d, JPC = 19.8 Hz, 1-C6H5), 133.70 (s, 4-C6H5), 133.32
(d, JPC = 19.0 Hz, 4-C6H4), 129.68 (d, JPC = 6.7 Hz, 2-C6H4), 129.05
(s, 3-C6H5), 128.66 (d, JPC = 7.1 Hz, 2-C6H5), 118.59 (s, C5H5). IR
(cm−1): 3090 m (Cp), 1632 s, 1592 m (νasym CO2), 1338 s, 1305 vs
(νsym CO2), 1134 m (Cp), 820 m (Cp). pH of 4 (5 × 10−5 M in 1:99
DMSO/H2O) = 5.32. pH of 2 (5 × 10−5 M in 1:99 DMSO/H2O) =
5.52.
[(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCl}2] (5). Complex 4 (0.213 g,
0.27 mmol) and [AuCl(tht)] (0.175 g, 0.54 mmol) were dissolved in
dichloromethane (12 mL) to yield an orange solution, which was
stirred for 20 min at room temperature. The dark brown solution was
then concentrated to ca. 2, and 20 mL of diethyl ether was added to
precipitate complex 5. The heterometallic complex was then isolated
by filtration and washed two more times with 20 mL of a mixture of
dichloromethane/diethyl ether (1:9). Complex 5 was isolated as a pale
yellow solid in 77% yield (0.265 g). Anal. Calcd for
C48H38Au2Cl2O4P2Ti (1253.48): C, 45.99; H, 3.06. Found: C, 45.76;
1
H, 3.31. 31P{1H} NMR (CDCl3): δ 32.99. H NMR (CDCl3): δ 8.08
(4H, m), 7.55 (24H, m), 6.66 (10H, s). 13C{1H} NMR (CDCl3): δ
170.35 (s, CO), 136.83 (d, JPC = 2.4 Hz, 1-C6H4), 134.22 (d, JPC
13.8 Hz, 3-C6H4), 133.96 (d, JPC = 13.9 Hz, 1-C6H5), 132.80 (d, JPC
60.3 Hz, 4-C6H5), 132.39 (d, JPC = 2.5 Hz, 4-C6H4), 130.26 (d, JPC
12.0 Hz, 2-C6H4), 129.46 (d, JPC = 12.0 Hz, 2-C6H5), 128.13 (d, JPC
=
=
=
=
62.5 Hz, 3-C6H5), 119.0 (s, C5H5). IR (cm−1): 3180 w (Cp), 1643 vs
(νasym CO2), 1558 s (Cp), 1330s, 1292 vs (νsym CO2), 1100 m (Cp),
823 s (Cp), 328 ms (ν AuCl). pH of 5 (5 × 10−5 M in 1:99 DMSO/
H2O) = 5.10. For comparison, the pH of previously described
compound 344 (5 × 10−5 M in 1:99 DMSO/H2O) = 6.12.
[AuCl(P(Ph2)-4-C6H4-COOH)] (7). 4-(Diphenylphosphino)benzoic
acid (0.144 g, 0.47 mmol) and [AuCl(tht)] (0.151 g, 0.47 mmol) were
dissolved in dichloromethane (10 mL) and stirred for 30 min at room
temperature. The solvent was removed under reduced pressure to
yield a white crude, which was washed with diethyl ether (3 × 10 mL).
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analyzed by flow cytometry using Accuri C6 (BD Biosciences) and
Accuri C6 analyzing software.
three mice per compound and one control group every other day for
14 days with the aforementioned MTD dose. One group of mice was
treated with the solvent (negative control). During the trial the mice
did not exhibit any notable sings of distress.
Interaction of Compounds 3 and 5−7, Titanocene Di-
chloride, and Cisplatin with Plasmid (pBR322) DNA by
Electrophoresis (Mobility Shift Assay). Aliquots of 10 μL of
plasmid (pBR322) DNA (20 μg/mL) in buffer (5 mM Tris/HCl, 50
mM NaClO4, pH = 7.39) were incubated with different concentrations
of the compounds (3, 5−7, and titanocene dichloride) (in the range
0.25 and 4.0 metal complex/DNAbp (bp = base pairs)) at 37 °C for
20 h in the dark. Samples of free DNA and cisplatin-DNA were
prepared as controls. After the incubation period, the samples were
loaded onto the 1% agarose gel. The samples were separated by
electrophoresis for 1.5 h at 80 V in Tris-acetate/EDTA buffer.
Afterward, the gel was stained for 30 min with a solution of GelRed
nucleic acid stain.
Kinase Inhibition Studies. In vitro profiling of 34 selected
member kinase panel was performed at Reaction Biology Corporation
using the “HotSpot” assay platform. Briefly, specific kinase/substrate
pairs along with required cofactors70 were prepared in reaction buffer:
20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA (ethylene glycol
tetracetic acid), 0.02% Brij35, 0.02 mg/mL BSA (bovine serum
albumin), 0.1 mM Na3VO4, 2 mM DTT (dithioethreitol), 1% DMSO.
Compounds were delivered into the reaction, followed ∼20 min later
by addition of a mixture of ATP (Sigma) and 33P-ATP (PerkinElmer)
to a final concentration 10 μM. Reactions were carried out at 25 °C for
120 min, followed by spotting of the reactions onto P81 ion exchange
filter paper (Whatman). Unbound phosphate was removed by
extensive washing of filters in 0.75% phosphoric acid. After subtraction
of background derived from control reactions containing inactive
enzyme, kinase activity data were expressed as the percent remaining
kinase activity in test samples compared to vehicle (dimethyl
sulfoxide) reactions. IC50 values and curve fits were obtained using
Prism (GraphPad Software, La Jolla, CA, USA).
ASSOCIATED CONTENT
* Supporting Information
■
S
Table with the crystal data and structure refinement for
complex 3. CIF file for the X-ray crystal structure of compound
3. Ortep view of the crystal structure of 3 including labeled
1
carbon atoms. H, 31P{1H}, and 13C{1H} NMR spectra of new
compounds 4, 5, and 7. Stability of compounds 3 and 5 in d6-
DMSO solution and in d6-DMSO/D2O (50:50) over time
assessed by 31P{1H} and 1H NMR spectroscopy. UV−vis
spectra of compounds 3 and 5 in CH2Cl2, DMSO, and (1%
DMSO) PBS solution over time. Mass spectra of 3 and 5 in
(1% DMSO) PBS solution over time. Cell death experiments
(annexin V/PI assay) for compound 3 at 1 and 12 h and
compound 5 at 12 and 24 h. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Phone: 1-808-564-5843. Fax: 1-808-586-2970. E-mail:
jramos@cc.hawaii.edu.
■
*Phone: 1-7189515000, x2833. Fax: 1-718-951-4607. E-mail:
mariacontel@brooklyn.cuny.edu.
Present Address
⊥Moores Cancer Center, University of California, San Diego,
La Jolla, California 92093, United States.
Concentrations of Cytokine IL-6. The concentrations of
cytokine IL-6 secreted were determined from cell supernatants
collected after 6 h of incubation with compound 3 or 5 by an
ELISA kit (human IL-6 ELISA kit) according to the manufacturer’s
instructions (Thermo Fisher Scientific, Rockford, IL, USA). Optical
density was measured using a microplate reader (PerkinElmer) at 450
nm wavelength. Concentrations of the cytokine were determined by
interpolation from the standard curves using Prism (GraphPad
Software).
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Determination of Lethal and Maximum Tolerated Doses (LD
and MTD) in Mice. The preliminary toxicity testing of compounds 3
and 5 was performed in C57BL/6 female mice 6 to 8 weeks of age,
maintained in accordance with institutional guidelines at the
University of Hawaii Cancer Center (UHCC) governing the care of
laboratory animals (IACUC number: A3423-01). To determine the
lethal dose, mice were treated for five consecutive days at dosages
ranging from 5 to 20 mg/kg/day. We used one mouse per dose. Mice
were weighed every 48 h and sacrificed 24 h after the last dose. The
compounds were administered in a solution of 0.5% DMSO and 99.5%
normal saline (0.9% NaCl) (G-Biosciences, St. Louis, MO, USA) once
daily by subcutaneous injection. In order to determine the maximum
tolerated dose, the animals were monitored by trained individuals for
pain and distress as appropriate for the species, condition, and
procedure at the UH vivarium by the veterinarian staff and person
doing the in vivo studies (B.T.E.). The maximum tolerated dose was
determined by observing the progression of the mice treated at doses
below the lethal dose. Body weights, changes in behavior, and signs of
distress were recorded. The dose at which neither debilitating effects
nor signs of distress was observed and set as the MTD. More
specifically the signs of distress monitored were (1) decreased food
and water consumption; (2) weight loss (more than 20% loss in body
weight or dropping to or below 18 g) was consistent with significant
distress and mice exhibiting such weight loss were euthanized; (3)
abnormal posture/positioning (e.g., head-pressing, hunched back); (4)
unkempt appearance (erected, matted, or dull haircoat); (5) self-
mutilation, gnawing at limbs; (6) excessive self-imposed isolation/
hiding. The MTD dose was then confirmed by treating a cohort of
■
We thank the National Cancer Institute (NCI) for grant
1SC1CA182844 (M.C.) and the National General Medical
Sciences Institute (NGMSI) for grant RO1GM088266-A1
(J.W.R.). M.C. is very grateful to Mr. and Mrs. Leonard and
Claire Tow and the Tow Foundation for a travel fellowship to
perform some experiments at the University of Hawaii Cancer
Center. We thank Dr. Monica Carreira for her assistance with
the synthesis of compound 1. We thank Dr. Chris Farrar
(University of Hawaii Cancer Center) for assistance with the
flow cytometry experiments and Prof. Wei Jia and Dr. Guoxiang
Xie (University of Hawaii Cancer Center) for performing the
MS spectra of compounds 3 and 5.
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