(Arene)Ru(II) complexes of epidermal growth factor receptor inhibiting tyrphostins with enhanced selectivity and cytotoxicity in cancer cells

Article abstract of DOI:10.1016/j.ejmech.2010.01.040

Ru(η⁶-arene) complexes of epidermal growth factor receptor (EGFR) inhibiting tyrphostins 1a and 1b were prepared, characterized and tested for DNA interaction and bioactivity in four human tumor cell lines. The intrinsic cytotoxicity and cell line selectivity of o-hydroxyanisol 1a was greatly enhanced in its Ru(η⁶-p-cymene) complex 2a and in its Ru(η⁶-toluene) complex 3a. Complex 2a was particularly efficacious against multi-drug resistant EGFR(+) MCF-7/Topo breast carcinoma cells and also against mTOR-dependent EGFR(-) HL-60 leukemia cells. Complex 3a showed enhanced activity only against 518A2 melanoma cells and HL-60 cells, which are both known to express the mTOR protein. DNA was strongly metallated (ca. 1.7-2%) by all new Ru complexes without undergoing topological changes. Apparently, by complexation to Ru fragments tyrphostin derivatives can address additional biological targets in a manner instrumental to antitumoral strategies.

Full text of DOI:10.1016/j.ejmech.2010.01.040

European Journal of Medicinal Chemistry 45 (2010) 1972–1975
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
(Arene)Ru(II) complexes of epidermal growth factor receptor inhibiting
tyrphostins with enhanced selectivity and cytotoxicity in cancer cells
*
B. Biersack, M. Zoldakova, K. Effenberger, R. Schobert
Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany
a r t i c l e i n f o
a b s t r a c t
Ru(h
6-arene) complexes of epidermal growth factor receptor (EGFR) inhibiting tyrphostins 1a and 1b
Article history:
Received 9 November 2009
Received in revised form
15 January 2010
Accepted 18 January 2010
Available online 28 January 2010
were prepared, characterized and tested for DNA interaction and bioactivity in four human tumor cell
lines. The intrinsic cytotoxicity and cell line selectivity of o-hydroxyanisol 1a was greatly enhanced in its
Ru(h h
6-p-cymene) complex 2a and in its Ru( 6-toluene) complex 3a. Complex 2a was particularly effi-
cacious against multi-drug resistant EGFR(þ) MCF-7/Topo breast carcinoma cells and also against mTOR-
dependent EGFR(ꢀ) HL-60 leukemia cells. Complex 3a showed enhanced activity only against 518A2
melanoma cells and HL-60 cells, which are both known to express the mTOR protein. DNA was strongly
metallated (ca. 1.7–2%) by all new Ru complexes without undergoing topological changes. Apparently, by
complexation to Ru fragments tyrphostin derivatives can address additional biological targets in
a manner instrumental to antitumoral strategies.
Keywords:
Ruthenium complexes
Tyrphostin
Anticancer drugs
Multi-drug resistance
EGFR inhibitor
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
epidermal growth factor receptors (EGFR) and tested them for in
vitro anticancer activity (Fig. 1). EGFR, the products of c-erbB proto-
Ruthenium complexes are attracting considerable interest as
potential anticancer agents [1]. Two classical coordination
compounds of Ru(III) with anti-metastatic properties, NAMI-A [2]
and KP1019 [1], are currently undergoing clinical trials. Their
preferential accumulation in cancer cells and low toxicity are
thought to originate from their ability to bind to the iron trans-
porter transferrin. Receptors for this protein are overexpressed in
tumor cells [3]. Other mechanisms were also discussed [4]. In
addition, (arene)Ru(II) complexes were devised that contain the
metal in the biologically active yet ligand-stabilized oxidation state
þII and which also offer opportunities for efficacy optimization by
modifying the arene ring. Noteworthy examples are complexes
with pta (¼1,3,5-triaza-7-phosphaadamantane) ligands [5] and
a series of [(arene)Ru(en)Cl]þ complexes that were efficacious even
in cisplatin-resistant cancer cell lines [6]. More recently, the first
(arene)Ru(II) complexes with bioactive natural product ligands
such as CDK-inhibiting paullones were published [7] as were
staurosporin mimics containing (arene)Ru(II) fragments [8].
oncogenes, are dimeric transmembrane proteins which trigger
cancer-relevant downstream effectors such as Src kinase, PI3 kinase
or Ras protein [10] and upregulate DNA repair mechanisms [11]. On
the other hand, Ru complexes such as NAMI-A are also known to
interfere with membrane localized proteins, e.g., protein kinase C
and Ras [12]. Finally, the clinical benefit of combination regimes of
metal complexes and small inhibitors or antibodies for EGFR was
already demonstrated for various tumors [13,14].
2. Results and discussion
2.1. Chemistry
The tyrphostin derivatives 1a and 1b were obtained from
Knoevenagel reactions of 3-(cyanomethyl)pyridine and the corre-
sponding aryl aldehyde in hot ethanol in the presence of a little
piperidine [15]. The products 1 were then reacted with the
respective [Ru(arene)Cl2]2 complex in CH2Cl2/CH3OH mixtures to
give the dichloridoruthenium complexes 2a,b and 3a as light
brown solids in high yields (Scheme 1). They were characterized by
NMR, IR and mass spectrometry. The 1H and 13C NMR spectra of
complexes 2a and 2b (in CDCl3) and of 3a (in DMF-d7) showed
downfield shifts for the protons and carbons at positions 2 and 6 of
the pyridine ring when compared with the free ligands 1 due to the
deshielding effect of the central metal.
Following a similar approach we now prepared the first (are-
ne)Ru(II) complexes of compounds 1a and 1b (RG 13022 [9]), two
established inhibitors of the tyrosine kinase activity of the
* Corresponding author. Tel.: þ49 (0)921 552679; fax: þ49 (0)921 552671.
E-mail address: rainer.schobert@uni-bayreuth.de (R. Schobert).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.040

B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 1972–1975
1973
Table 1
Inhibitory concentrations IC50
a
[m
M] of compounds 1, 2 and 3a when applied to cells
of human 518A2 melanoma, HL-60 leukemia, Kb-V1/Vbl cervix carcinoma and MCF-
7/Topo breast carcinoma.
518A2
(24 h/48 h)
HL-60
(24 h/48 h)
Kb-V1/Vbl
(24 h/48 h)
MCF-7/Topo
24 h/48 h
1a
94/16 ꢁ 3.4 11 ꢁ 2.4/9 ꢁ 1.4
9 ꢁ 2.4/6 ꢁ 1.4
50/1.5 ꢁ 0.3
1b 45 ꢁ 1.5/50 ꢁ 6.2 33 ꢁ 4.2/32 ꢁ 3.3 46 ꢁ 5.6/20 ꢁ 4.8 48 ꢁ 5.8/11 ꢁ 3.8
2a 31 ꢁ 2.3/13 ꢁ 4.2
2 ꢁ 1.1/0.8 ꢁ 0.2
50/3 ꢁ 07
50/0.2 ꢁ 0.14
2b
–/60 ꢁ 1.7
–/95 ꢁ 5 59 ꢁ 3.1/31 ꢁ 4.7 49 ꢁ 3.9/29 ꢁ 3.4
3a 3 ꢁ 0.3/2.2 ꢁ 0.2 1.3 ꢁ 0.02/1 ꢁ 0.2 20 ꢁ 4.6/7 ꢁ 1.5 75 ꢁ 13.7/7 ꢁ 2.7
Fig. 1. EGFR inhibitors of the tyrphostin group.
a
Values are derived from concentration–response curves obtained by measuring
the percentage of grown cells relative to untreated controls after the indicated time
of incubation using an MTT assay.
2.2. Biological evaluation
The cytotoxicities of the tyrphostins 1a,b, of their p-cymene
complexes 2a,b and of the toluene complex 3a were evaluated by
means of MTT assays with cells of human 518A2 melanoma, HL-60
leukemia, KB-V1/Vbl cervix carcinoma and MCF-7/Topo breast
carcinoma (Table 1). The nature of both organic ligands, the tyr-
beneficially mediating the uptake or efflux of complex 2a, or the
specific metallation of certain bionucleophiles. In calcein-AM and
mitoxantron assays no significant differences were observed in the
interaction of the test compounds with the ABC-transporters of the
resistant cells.
phostin and the
h
6-bound arene, had a distinct influence on the
anticancer activity of the new complexes. The 1,2-dimethoxyphenyl
derivative RG 13022 (1b) and its Ru(p-cymene) complex 2b were
far less efficacious against all cell lines than the o-hydroxyanisyl
analogues 1a, 2a, and 3a. While coordination of 1b to a ruthenium
fragment afforded a complex 2b of diminished cytotoxicity, ligation
of the hydroxyanisol 1a led to complexes 2a and 3a with signifi-
The binding affinities of the complexes for various forms of
DNA were slightly different. According to ICP-OES analytics, the
metal content of salmon sperm DNA was about 2% when treated
for 24 h with 50 mM of the p-cymene complexes 2 and ca. 1.7%
upon exposure to the toluene complex 3a. This is ca. ten times the
metallation degree typically achieved with platinum complexes
[20]. However, in electrophoretic mobility shift assays (EMSA)
with pBR322 plasmid DNA the new complexes did not give rise to
any bandshift effects. This means they either did not bind to this
DNA form or, if bound, did not cause topological changes. This
resembles reports by Reedijk et al. for ruthenium polypyridyl
complexes [21]. Insofar, these Ru(II) complexes differ sharply
from cisplatin, and it is possible that DNA-binding contributes to
the cytotoxicity of the active Ru complexes 2a and 3a in certain
cancer cells.
cantly enhanced activity. The nature of the h
6-arene ligand also had
an influence, apparent from the different cell line specificities of
these complexes. The p-cymene complex 2a was most cytotoxic in
the multi-drug resistant MCF-7/Topo cancer cells at an IC50 (48 h) of
ca. 0.2 mM, which is a seventh of that of the free ligand 1a. A similar
increase in activity was observed for the toluene complex 3a
against 518A2 melanoma cells. In HL-60 cells both complexes 2a
and 3a showed the same high activity (IC50 (48 h) ca 1 mM),
exceeding that of the uncomplexed tyrphostin 1a by a factor of
nine. Unspecific ruthenation is an unlikely reason for this activity
boost as ruthenium complex 2b was virtually inactive in these cells.
An alternative rationale is based on the fact that HL-60 cells, while
lacking endogenous EGFR [16], depend on Akt/mTOR signaling [17].
Possibly, tyrphostin 1a and its complexes 2a and 3a interfere with
this pathway by EGFR-independent mechanisms. Like HL-60 cells,
518A2 melanoma cells express mTOR and respond to rapamycin,
the classical inhibitor of this protein. This matches with the
observation that 518A2 and HL-60 cells responded much faster to
2a and 3a than the multi-drug resistant cells. Only upon 48 h of
incubation complex 2a surpassed tyrphostin 1a in growth inhibi-
tion of both MDR(þ) cells which are derived from parent lines
possessing EGFR [18,19]. Complex 3a remained inferior to 1a even
after 48 h. Both EGFR-inhibiting tyrphostins 1a and 1b used in this
study are generally more cytotoxic in these multi-drug resistant
cells than in 518A2 and HL-60 cells. The extraordinary increase
in efficacy of complex 2a against MCF-7/Topo cells
[IC50(48 h) ¼ 200 nM] when compared with that of the parent
Their uptake by 518A2 melanoma cells correlated with their
sperm DNA-binding but not with their cytotoxicity in these cells.
The Ru content of 518A2 cells was found by ICP-MS to be
1.43 ꢁ 0.01
m
g/L upon 24 h exposure to 10
mM cymene complex 2a
but only 1.22 ꢁ 0.01
mg/L when treated with toluene complex 3a.
This difference is possibly a consequence of the more hydrophobic
character of the cymene when compared with the toluene ligand.
Apparently, the cellular complex concentration alone is not decisive
for their cytotoxic potency.
3. Conclusion
Two new DNA-binding (h
6-arene)Ru(II) complexes 2a and 3a
bearing a tyrphostin ligand were prepared and found to have
enhanced cytotoxicities and cancer cell line specificities when
compared with the free tyrphostin. The (p-cymene)Ru complex 2a
was particularly active in multi-drug resistant and EGFR(þ) cancer
cells, e.g., MCF-7/Topo breast carcinoma cells. The (toluene)Ru
complex 3a displayed a specific anti-melanoma activity which is
possibly based on interference with mTOR signaling and obviously
independent from EGFR inhibition. Unlike cytotoxic platinum
compounds the new ruthenium complexes bound strongly to DNA
without altering its topology. These findings open up new possi-
bilities for the development of selective and multi-drug resistance
breaking organometallic compounds consisting of a ligand specific
for a certain biochemical target and a Ru fragment that confers
selectivity to the whole complex for another target in a synergistic
manner. The detailed mechanism of action of the new complexes is
under investigation.
tyrphostin 1a [IC50(48 h) ¼ 1.5
mM] is nevertheless remarkable and
possibly due to synergisms originating from the cymene ligand
Cl
R1O
N
R1O
N
Cl
Ru
CN
CN
MeO
MeO
R2
1a: R1 = H
1b: R1 = Me
2a: R1 = H; R2 = i-Pr
2b: R1 = Me; R2 = i-Pr
3a: R1 = R2 = H
Scheme 1. Reagents and conditions: [Ru(arene)Cl2]2, CH2Cl2/CH3OH, 3 h, r.t., 87–91%.

1974
B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 1972–1975
4. Experimental protocols
(1H, s), 9.69 (1H, s); 13C NMR (75.5 MHz, DMF-d7)
d 18.4, 55.9, 80.0,
81.5, 87.4, 100.5, 103.1, 112.2, 115.7, 117.8, 123.8, 124.4, 126.8, 131.8,
134.9,145.9,147.6,151.5,152.6,155.2; MS (EI) m/z 252 (90), 223 (24),
205 (26), 91 (100).
Melting points were determined with a Gallenkamp apparatus
and are uncorrected. IR spectra were recorded on a Perkin–Elmer
One FT-IR spectrophotometer. Magnetic resonance (NMR) spectra
were recorded under conditions as indicated on a Bruker Avance
4.2. Biological studies
300 spectrometer. Chemical shifts (d) are given in parts per million
downfield from TMS as internal standard. Mass spectra were
recorded using a Varian MAT 311A (EI). Elemental analyses were
carried out with a Perkin–Elmer 2400 CHN elemental analyser.
Satisfactory microanalyses (C, ꢁ0.2; H, ꢁ0.1) were obtained for the
new complexes. [Ru(toluene)Cl2]2 was prepared according to
a procedure for the preparation of [Ru(benzene)Cl2]2 [22]. Tyr-
phostin derivatives 1a and 1b were prepared according to a litera-
ture procedure [15].
4.2.1. Cytotoxicity assays
HL-60 [23], KB-V1/Vbl and MCF-7/Topo cells were obtained
from the German National Resource Center for Biological Material
(DSMZ), Braunschweig. HL-60 cells were incubated in RPMI (Ros-
well Park Memorial Institute) media 1640 with 10% FBS (fetal
bovine serum), 0.55% antibiotic–antimycotic and 0.3% gentamycin
(all Gibco). KB-V1 cells were cultured in D-MEM (Dulbecco’s
modified eagle medium, Invitrogen, Carlsbad, CA) with 10% FBS,
0.55% antibiotic–antimycotic, 0.34 mM vinblastine and 0.3% genta-
mycin. MCF-7/Topo cells were grown in E-MEM (Eagle’s minimum
essential medium, Sigma–Aldrich) supplemented with 5% FBS and
0.55 mM topotecane. 518A2 cells [24,25] were obtained from the
department of oncology and hematology of the Martin-Luther-
University, Halle, and cultured like the KB-V1 cells.
Cultured cancer cells were grown in the presence of the test
compounds in 96-well plates. After 24 h and 48 h treatment, the
cell growth rates were determined by an assay with MTT [3-(4,5-
4.1. Chemistry
4.1.1. Dichlorido(p-cymene)[3-(10-cyano-20-(300-hydroxy-400-
methoxyphenyl)(Z)-ethenyl)pyridine]ruthenium(II) 2a
A mixture of compound 1a (58 mg, 0.23 mmol), CH2Cl2 (5 mL),
a
few drops of methanol and [Ru(p-cymene)Cl2]2 (70 mg,
0.11 mmol) was stirred at room temperature for 3 h. A mixture of
ethyl acetate and n-hexane (50 mL, 1:4) was added and the
resulting suspension was stirred for 5 min. The precipitate was
collected, washed with n-hexane and dried in vacuum. Yield:
119 mg (0.21 mmol, 91%); yellow solid of m.p. 190 ꢂC (dec.); nmax
(ATR)/cmꢀ1 3236, 2949, 2207, 1602, 1573, 1535, 1437, 1374, 1320,
1310, 1269, 1211, 1192, 1137, 1033, 937, 890, 873, 821, 794; 1H NMR
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
as
described in detail elsewhere [26]. These experiments were carried
out in triplicate; the percentage of viable cells quoted was calcu-
lated as the mean ꢁ S.D. with respect to the controls set to 100%.
(300 MHz, CDCl3/CD3OD)
d
1.27 (6H, d, J ¼ 6.9 Hz), 2.04 (3H, s), 2.8–
4.2.2. DNA-binding via ICP-OES
Salmon sperm DNA (10 mg/mL) was incubated with Ru-
complexes at a final concentration of 50 mM in TE buffer (Tris–
3.0 (1H, m), 3.90 (3H, s), 5.29 (2H, d, J ¼ 6.0 Hz), 5.50 (2H, d,
J ¼ 6.0 Hz), 6.89 (1H, d, J ¼ 8.5 Hz), 7.3–7.4 (2H, m), 7.47 (2H, s), 7.9–
8.0 (1H, m), 8.89 (1H, d, J ¼ 5.6 Hz), 9.20 (1H, s); 13C NMR
HCl, EDTA, pH 8.0) supplemented with 10 mM NaClO4 at 37 ꢂC for
24 h. The stock solutions (10 mM) of the complexes were prepared
in DMSO and freshly diluted in TE buffer with 10 mM NaClO4 before
using, while the DMSO content in every sample was 0.5%. DMSO
was used as negative control. To precipitate the DNA, equal volumes
of ethanol were added and incubated at ꢀ20 ꢂC overnight.
Following centrifugation at 12,000 g for 10 min the supernatants
were discarded, the pellets were washed twice with 70% ice-cold
ethanol, and finally DNA was lyophilized. The Ru content of the
DNA samples was ascertained with a Varian ‘‘Vista Pro’’ ICP-OES.
(75.5 MHz, CDCl3/CD3OD)
d 17.9, 22.0, 30.6, 55.7, 81.9, 82.8, 97.4,
102.5, 103.4, 111.0, 115.3, 123.6, 124.3, 125.9, 132.2, 134.8, 145.8,
146.1, 150.6, 151.4, 153.8; MS (EI) m/z 252 (19), 233 (4), 223 (6), 205
(5), 134 (26), 119 (100), 115 (42), 105 (31), 91 (22).
4.1.2. Dichlorido(p-cymene)[3-(10-cyano-20-(300,400-
dimethoxyphenyl)(Z)-ethenyl)pyridine]ruthenium(II) 2b
Analogously to complex 2a, compound 2b was obtained from 1b
(61 mg, 0.23 mmol) and [Ru(p-cymene)Cl2]2 (70 mg, 0.11 mmol).
Yield: 112 mg (0.20 mmol, 86%); yellow solid of m.p. 120 ꢂC (dec.);
nmax (ATR)/cmꢀ1 2961, 2868, 2208, 1588, 1567, 1515, 1465, 1428,
1369, 1327, 1276, 1243,1166, 1147, 1038, 1020, 907, 852, 803, 689; 1H
4.2.3. Cellular uptake
518A2 cells (0.5 ꢃ106/mL) were seeded into 6-well plates and
NMR (300 MHz, CDCl3)
d
1.30 (6H, d, J ¼ 7.0 Hz), 2.10 (3H, s), 2.9–3.1
grown overnight. The medium was replaced with medium con-
(1H, m), 3.92 (3H, s), 3.93 (3H, s), 5.25 (2H, d, J ¼ 6.0 Hz), 5.46 (2H,
d, J ¼ 6.0 Hz), 6.90 (1H, d, J ¼ 8.5 Hz), 7.2–7.5 (2H, m), 7.52 (1H, s),
7.65 (1H, s), 7.8–7.9 (1H, m), 8.89 (1H, d, J ¼ 5.6 Hz), 9.23 (1H, s); 13C
taining 10 mM ruthenium complex (2a or 3a; 0.1% DMF w/v) and
cells were incubated for 24 h. Trypsinated cells were centrifuged
and washed twice with PBS. The cell pellets were weighed and
resuspended in 10 mL of water. The Ru content was measured with
a Varian ICP-MS. Experiments were carried out in triplicate.
NMR (75.5 MHz, CDCl3)
d 18.2, 22.3, 30.7, 56.0, 82.3, 82.7, 97.1,
102.8, 103.7, 111.0, 111.1, 124.6, 125.6, 125.9, 132.1, 134.7, 145.6, 149.0,
151.5, 152.1, 154.1; MS (EI) m/z 577 (6), 306 (14), 266 (100), 179 (36),
119 (26).
Acknowledgment
4.1.3. Dichlorido(toluene)[3-(10-cyano-20-(300-hydroxy-400-
methoxyphenyl)(Z)-ethenyl)pyridine]ruthenium(II) 3a
We thank the Deutsche Forschungsgemeinschaft for financial
support (grant Scho 402/8-2).
Analogously to cymene complex 2a, the complex 3a was
obtained from 1a (58 mg, 0.23 mmol) and [Ru(toluene)Cl2]2
(60 mg, 0.11 mmol). The crude precipitate was collected, washed
with CH2Cl2 and dried. Yield: 101 mg (0.20 mmol, 87%); yellow
solid of m.p. 220 ꢂC (dec.); nmax (ATR)/cmꢀ1 3259, 3025, 2210, 1599,
1590, 1571, 1528, 1507, 1437, 1375, 1269, 1211, 1193, 1134, 1024, 857,
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